TW200911757A

TW200911757A - Crystalline forms of histone deacetylase inhibitors

Info

Publication number: TW200911757A
Application number: TW097102083A
Authority: TW
Inventors: Patrick D Hayes; Paul K Isbester; Olga V Lapina; Bingidimi I Mobele; Grant J Palmer; Jonathon S Salsbury; Xufeng Sun; Luckner G Ulysse
Original assignee: Takeda San Diego Inc
Priority date: 2007-01-18
Filing date: 2008-01-18
Publication date: 2009-03-16
Also published as: CL2008000150A1; AR064971A1; WO2008089436A3; WO2008089436A2

Abstract

The application is related to polymorphs and salts of compounds having the formulae: N-(2-Aminophenyl)-4-((5, 6-dimethoxy-2H-indazol-2-yl)methyl)benzamide 4-((6-Acetamido-2H-indazol-2-yl)methyl)-N-(2-aminophenyl)benzamide 1-(4-(2-Aminophenylcarbamoyl)benzyl)-N-ethyl-1H-pyrazole-4-carboxamide Also provided are novel methods for the preparation of the polymorphs of Compound I, and kits and articles of manufacture of the compositions, and methods of using the compositions to treat various diseases.

Description

200911757 九、發明說明：【發明所屬之技術領域】本發明係關於可用以抑制組蛋白去乙醯基酶（HDAC)之化合物之固體形式（鹽及溶劑合物）且係關於其製備方法。本發明亦係關於包含此等鹽及溶劑合物之醫藥組合物、套組及製品’及其在抑制諸如HDAC1、HDAC2、HDACu HDAC8之I類HDAC中之使用方法。【先前技術】組蛋白為小型、帶正電荷之蛋白質，其與DNA緊密複合以在真核細胞中形成染色質。組蛋白富含鹼性胺基酸（在生理干pH值下帶正電荷），該等胺基酸接觸DNA之磷酸基 (在生理學PH值下帶負電荷）。大多數組蛋白係在細胞週期之S卩白奴期間合成，且新合成之組蛋白迅速進入核中以變得與DNA締合。藉由後轉澤添加甲基、乙醯基或磷酸基來酶促修飾一小部分組蛋白，從而中和側鏈之正電荷或將其轉化為負電荷應瞭解組蛋白之乙醯化及去乙醯化與引起細胞增生及/ 或分化之轉錄事件相關。一般而言，咸信組蛋白乙醯化與轉錄/舌化有關，而咸信組蛋白去乙醯化與基因抑制有關。越來越多的負責組蛋白去乙醯化的酵素（組蛋白去乙醯基酶（HDAC))已被鑑別且已顯示在細胞增生及分化中起到重要作用。咸彳g HDAC與多種不同疾病病況相關，該等疾病病況包括（但不限於）細胞增生疾病及病狀。因此認為抑制、、且蛋白去乙醯基酶之化合物為適用於預防、延遲進程及/ 128407.doc 200911757 制組蛋白去乙醯基酶之化合物為適用於預防、延遲進程及/ 或治療由HDAC引發之病狀的藥劑。為HDAC抑制劑之苯甲醯胺及幾醯胺化合物描述於鳩年7月13日申請之美國專利申請案第1 1/457,2⑼號中，其主張2005年7月14日中凊之美國臨時中請案第號的權益；該兩個揭示案均以全文引用的方式併入本文中。所合成之化合物可能不具有用於後續加工之最佳物理特徵；存在鑑別其他固體形式（其中鹽具有有利特性）之需要。【發明内容】BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to solid forms (salts and solvates) of compounds which can be used to inhibit histone deacetylase (HDAC) and relates to a process for the preparation thereof. The invention also relates to pharmaceutical compositions, kits and articles comprising such salts and solvates' and methods of use thereof for inhibiting Class I HDACs such as HDAC1, HDAC2, HDACu HDAC8. [Prior Art] Histones are small, positively charged proteins that bind tightly to DNA to form chromatin in eukaryotic cells. Histones are rich in basic amino acids (positively charged at physiological pH) which contact the phosphate group of the DNA (negatively charged at physiological pH). Most histone proteins are synthesized during the S. cerevisiae of the cell cycle, and newly synthesized histones rapidly enter the nucleus to become associated with DNA. Enzymatic modification of a small portion of histones by post-translational addition of methyl, acetyl or phosphoric acid groups to neutralize the positive charge of the side chain or convert it to a negative charge should be understood by the acetylation of histones and Acetylation is associated with transcriptional events that cause cell proliferation and/or differentiation. In general, the acetylation of histone histones is related to transcription/tongue, and the deacetylation of serotonin is associated with gene suppression. An increasing number of enzymes responsible for histone deacetylation (histone deacetylase (HDAC)) have been identified and have been shown to play an important role in cell proliferation and differentiation. Salty g-HDAC is associated with a variety of disease conditions including, but not limited to, cell proliferative diseases and conditions. Therefore, it is considered that the inhibitory, and protein-deacetylase-based compounds are suitable for the prevention, delay of the process and /128407.doc 200911757 for the preparation of histone deacetylases for the prevention, delay of progression and / or treatment by HDAC An agent that causes the condition. Benzalamine and a few guanamine compounds, which are HDAC inhibitors, are described in U.S. Patent Application Serial No. 1 1/457, 2, filed on Jul. 13, the entire disclosure of The rights to the No. 1 in the interim; both disclosures are incorporated herein by reference in their entirety. The compound synthesized may not have the best physical characteristics for subsequent processing; there is a need to identify other solid forms in which the salt has advantageous properties. [Summary of the Invention]

本發明係關於化合物卜„及m之結晶形及製備此等結晶形之方法以及包含s亥等結晶形中一或多個之組合物。化合物IThe present invention relates to a crystalline form of a compound and a crystalline form of the compound, and a composition comprising one or more of the crystalline forms such as shai.

在一實施例中，本發明係關於本文中稱作化合物I形式 A(或形式A)之化合物I游離鹼之多晶型物。形式a為二水合結晶形。形式A之特徵可為具有一或多種以下物理特徵（應注意組合物不需必要地展現所有此等特徵以表明形式A之存在）： (i) 包含在約8.64、19.95及18.17度2Θ(°2Θ)處之繞射峰的X射線粉末繞射圖案（CuKct); (ii) 如圖2中所示之iH NMR光譜； (iii) 如圖3中所示光譜； 128407.doc 200911757 (iv) 具有集令在約89°C之寬吸熱及在約i98°C之第二吸熱的示差掃描熱量測定圖譜；及 (v) 在iOOt以下顯示重量損失為約8.4%之tGa熱分析圖。在另一實施例中，本發明係關於本文中稱作形式B之化合物I之第二多晶型物。形式B為無水結晶形。形式B之特徵可為具有一或多種以下物理特徵（應注意組合物不需必要地展現所有此等特徵以表明形式B之存在）： ⑴包含在約17.21、20.4及16.02度2Θ(°2Θ)處之繞射峰的X射線粉末繞射圖案（CuKa); (11)如圖8中所示之1η NMR光譜； (in)如圖13中所示之ftir光譜； (1V)具有集中在約211 °C之吸熱的示差掃描熱量測定圖譜；及 (v)在120 C以下無重量損失之TGA熱分析圖。在另實施例中，本發明係關於化合物I之非晶形（化合物I形式非M )。形式非晶可由化學組成及X射線粉末繞射圖案（CuKa)(圖17)表徵。在另實施例中’本發明係關於化合物〗之反丁烯二酸鹽。基於其物理特性，反丁稀二酸鹽為結晶形。反丁稀一酸鹽之特徵可為具有一或多種以下物理特徵 (應庄思組合物不需必要地展現所有此等特徵以表明反丁烯二酸鹽之存在）： (0匕3在約5.39、η.24及度2θ(〇2θ)處之繞射峰 128407.doc 200911757 的X射線粉末繞射圖案（CuKa); (ii)如圖I9中所示之丨H NMR光譜； (ill)如圖20中所示之ftir光譜； (“）顯示在120。(：以下重量損失為2.5%之及在約127。〇之分解起始的TGA熱分析圖；及 (v) 具有集中在約128X：之吸熱的示差掃描熱量測定圖譜。在另一實施例中’本發明係關於化合物I之氫氯酸鹽。基於其物理特性，氫氯酸鹽為結晶形。氫氯酸鹽之特徵可為具有一或多種以下物理特徵（應注思組〇物不需必要地展現所有此等特徵以表明氫氣酸鹽之存在）： ⑴包含在約15.48、23.21及16.04度2Θ(°2Θ)處之繞射峰的X射線粉末繞射圖案（CuKa); (11)如圖25中所示之iH NMR光譜；In one embodiment, the invention relates to polymorphs of the free base of Compound I referred to herein as Compound I Form A (or Form A). Form a is a dihydrate crystal form. Form A may be characterized by one or more of the following physical characteristics (it is noted that the composition does not necessarily exhibit all of these features to indicate the presence of Form A): (i) is included at about 8.64, 19.95, and 18.17 degrees 2 Θ (°) X-ray powder diffraction pattern (CuKct) of the diffraction peak at 2Θ); (ii) iH NMR spectrum as shown in Fig. 2; (iii) spectrum as shown in Fig. 3; 128407.doc 200911757 (iv) A differential scanning calorimetry map having a broad endotherm at about 89 ° C and a second endotherm at about i98 ° C; and (v) a tGa thermogram showing a weight loss of about 8.4% below iOOt. In another embodiment, the invention relates to a second polymorph of Compound I, referred to herein as Form B. Form B is an anhydrous crystalline form. Form B may be characterized by one or more of the following physical characteristics (it is noted that the composition does not necessarily exhibit all of these features to indicate the presence of Form B): (1) is included in about 17.21, 20.4, and 16.02 degrees 2 Θ (° 2 Θ) X-ray powder diffraction pattern (CuKa) at the diffraction peak; (11) 1 η NMR spectrum as shown in FIG. 8; (in) ftir spectrum as shown in FIG. 13; (1 V) having concentration in about An endothermic differential scanning calorimetry map at 211 °C; and (v) a TGA thermogram with no weight loss below 120 C. In another embodiment, the invention relates to the amorphous form of Compound I (Formula I form is not M). Formal amorphous can be characterized by chemical composition and X-ray powder diffraction pattern (CuKa) (Figure 17). In another embodiment, the invention is directed to the compound of the fumarate. Based on its physical properties, the anti-succinic acid salt is crystalline. The anti-succinic acid salt may be characterized by having one or more of the following physical characteristics (should be necessary to exhibit all of these characteristics to indicate the presence of fumarate): (0匕3 in approx. 5.39, η.24 and degree 2θ (〇2θ) diffraction peak 128407.doc 200911757 X-ray powder diffraction pattern (CuKa); (ii) 丨H NMR spectrum as shown in Figure I9; (ill) The ftir spectrum is shown in Figure 20; (") is shown at 120. (: The following weight loss is 2.5% and at about 127. The TGA thermogram of the decomposition begins; and (v) has a focus on 128X: an endothermic differential scanning calorimetry map. In another embodiment, the present invention relates to a hydrochloride of Compound I. Hydrochloric acid is crystalline based on its physical properties. To have one or more of the following physical characteristics (should be necessary to show all of these characteristics to indicate the presence of hydrogen hydride): (1) Included at about 15.48, 23.21, and 16.04 degrees 2 Θ (° 2 Θ) An X-ray powder diffraction pattern (CuKa) of the diffraction peak; (11) an iH NMR spectrum as shown in FIG. 25;

(in)如圖26中所示之FTIR光譜； (iv)顯示在ucrc以下重量損失為41%之及在約16(rc 之分解起始的TGA熱分析圖；及 ⑺^有兩個集中在約95t^185t之吸熱事件的示差掃描熱量測定圖譜。實包例中，本發明係關於化合物〗之雙填酸鹽。基於其物理特性，雙磷酸鹽為結晶形。雙碟酸鹽之特徵可為具有一立4人 A夕種以下物理特徵（應注思、、且a物不需必要地 &寻w徵以表明磷酸鹽之存 128407.doc 10· 200911757 在）· (1) 包含在約17.80、25.80及21.88度2Θ(02Θ)處之繞射峰的X射線粉末繞射圖案（CuKa); (ii)如圖3 1中所示之iH nmr光譜； (in)顯不在180°C以下重量損失為2.0%之及在約127°C 之分解起始的TG A熱分析圖；及 (iv)具有集中在約179。(：之放熱事件及在256。(：之吸熱事件的示差掃描熱量測定圖譜。(in) FTIR spectrum as shown in Figure 26; (iv) shows a weight loss of 41% below ucrc and a TGA thermogram at about 16 (start of decomposition of rc; and (7)^ have two concentrated The differential scanning calorimetry map of the endothermic event of about 95t^185t. In the actual case, the invention relates to the double-filled acid salt of the compound. Based on its physical properties, the bisphosphonate is crystalline. In order to have the physical characteristics of a person with 4 persons A (should be in mind, and the a thing does not need to be & seek to indicate the deposit of phosphate 128407.doc 10· 200911757 in) · (1) X-ray powder diffraction pattern (CuKa) of diffraction peaks at about 17.80, 25.80, and 21.88 degrees 2 Θ (02 Θ); (ii) iH nmr spectrum as shown in Figure 31; (in) not at 180 °C The following weight loss is 2.0% and the TG A thermogram at the onset of decomposition at about 127 ° C; and (iv) has a concentration of about 179. (: the exothermic event and at 256. (: the end of the endothermic event) Scan the calorimetric map.

化合物IICompound II

在一實施例中’本發明係關於本文中稱作化合物π形式 Α或形式Α之化合物π之多晶型物。基於其物理特性，形式 A為結晶形。形式A之特徵可為具有一或多種以下物理特徵（應注意組合物不需必要地展現所有此等特徵以表明形式A之存在）： (1)包含在約2〇.92、17.54及2〇.36度20(。20)處之繞射峰的X射線粉末繞射圖案（CuKa); (ii) 如圖36中所示之1HNMR光譜； (iii) 如圖37中所示之FTIR光譜； (iv) 具有兩個集中在約77ΐ&179^之吸熱事件及在約 1 84 C之放熱事件的示差掃描熱量測定圖譜；及 128407.doc 200911757 (V)在1 00 C以下之溫度顯示重量損失為約2%的TGA熱分析圖。在一實施例中’本發明係關於化合物Η之丁二酸鹽。基於其物理特性，丁二酸鹽為結晶形。丁一酸鹽之特徵可為具有一或多種以下物理特徵（應注意組合物不需必要地展現所有此等特徵以表明丁二酸鹽之 ⑴包含在約22.17、27.13及20.94度2Θ(°2Θ)處之繞射峰的X射線粉末繞射圖案（CuKa); (11)如圖42中所示之iH NMR光譜； (U1)如圖43中所示之FTIR光譜； (W)顯不一個集中在約152。(：之吸熱事件及在212。(：之放熱事件的示差掃描熱量測定圖譜；及 (v)顯不在UiTC以下無重量損失及在約i5/rc之分解起始的TGA熱分析圖。在實施例中，本發明係關於化合物η之苯甲酸鹽。基In one embodiment, the invention relates to polymorphs of the compound π referred to herein as the compound π form ruthenium or the form ruthenium. Form A is crystalline based on its physical properties. Form A may be characterized by one or more of the following physical characteristics (it is noted that the composition does not necessarily exhibit all of these features to indicate the presence of Form A): (1) is included in about 2〇.92, 17.54, and 2〇 X-ray powder diffraction pattern (CuKa) of a diffraction peak at 36 degrees 20 (. 20); (ii) 1H NMR spectrum as shown in Fig. 36; (iii) FTIR spectrum as shown in Fig. 37; (iv) a differential scanning calorimetry map with two endothermic events centered at approximately 77ΐ&179^ and an exothermic event at approximately 1 84 C; and 128407.doc 200911757 (V) showing weight loss at temperatures below 1 000 C It is about 2% TGA thermogram. In one embodiment, the invention relates to the succinate salt of the compound hydrazine. Based on its physical properties, the succinate salt is crystalline. The butyrate salt may be characterized by one or more of the following physical characteristics (it is noted that the composition does not necessarily exhibit all of these characteristics to indicate that the succinate salt (1) is included in about 22.17, 27.13, and 20.94 degrees 2 Θ (° 2 Θ An X-ray powder diffraction pattern (CuKa) at the diffraction peak; (11) an iH NMR spectrum as shown in Fig. 42; (U1) an FTIR spectrum as shown in Fig. 43; (W) Concentrated on about 152. (: the endothermic event and at 212. (: the differential scanning calorimetry map of the exothermic event; and (v) no weight loss below UiTC and TGA heat at the beginning of the decomposition of about i5/rc In the examples, the present invention relates to a benzoate salt of the compound η.

苯甲酸鹽為結晶形。徵可為具有一或多種以下物理特徵（應注以表明苯甲酸鹽之 ⑴包含在約17.43、、25.9〇及23.93度20(。20)處之繞射 (ϋ) (iii) 峰的Χ射線粉末繞射圖案（CuKa); (i i)如圖 4 R tfo — 1The benzoate is crystalline. The sign may be one or more of the following physical characteristics (should be noted to indicate that the benzoate (1) contains diffraction (ϋ) (iii) peaks at about 17.43, 25.9 〇, and 23.93 degrees 20 (.20) Ray powder diffraction pattern (CuKa); (ii) Figure 4 R tfo - 1

(iii)如 128407.doc .12- 200911757 事件及在201 C之放熱、接著在216 °c之吸熱的示差掃描熱量測定圖譜；及 Ο)顯示在170°C以下重量損失為9 7%之及在約18(rc 之分解起始的TGA熱分析圖。在另一實施例中，本發明係關於化合物„之氫氣酸鹽。基於其物理特性，氫氯酸鹽為結晶形。氫氯酸鹽之特徵可為具有一或多種以下物理特徵（應注意組合物不需必要地展現所有此等特徵以表明氫氣酸鹽之存在）： ⑴包含在約19.83、23.〇9及21.89度20(。20)處之繞射峰的X射線粉末繞射圖案（CuKa); (ii) 如圖54中所示之iH NMR光譜； (iii) 如圖55中所示之fTIR光譜； (iv) 具有集中在約i97〇c之吸熱事件及在2〇8。匸之放熱事件的示差掃描熱量測定圖错；及 (v) 在160°C以下顯示無重量損失之TGA熱分析圖。在另一實施例中’本發明係關於化合物π之氫溴酸鹽（化合物II氫溴酸鹽）。基於其物理特性，氫漠酸鹽為結晶形。氫溴酸鹽之特徵可為具有一或多種以下物理特徵（應注意組合物不需必要地展現所有此等特徵以表明㈣酸鹽之存在）： 0)包含在約1丨.25、18.92及26.55度20(。20)處之繞射峰的Χ射線粉末繞射圖案（CuKa); (ii)如圖60中所示之1HNMR光譜； 128407.doc -13- 200911757 (111)如圖61中所示之FTIR光譜； (iv) 具有集中在約214它之吸熱事件接著在約224。〇之放熱的示差掃描熱量測定圖譜；及 (v) 在以下顯示無重量損失之τ〇Α熱分析圖。在另一實施例中，本發明係關於化合物π之龍膽酸鹽。基於其物理特性，龍膽酸鹽為結晶形。龍膽酸鹽之特徵可為具有一或多種以下物理特徵（應注忍組合物不需必要地展現所有此等特徵以表明龍膽酸鹽之存在）： ω 包含在約17.49、12.87及25.93度2Θ(°2Θ)處之繞射峰的X射線粉末繞射圖案（CuKa); (ii) 如圖66中所示之1η NMR光譜； (iii) 如圖67中所示之FTIR光譜； (W)具有集中在約丨27。(：、Mot及24〇t之吸熱事件的示差掃描熱量測定圖譜；及(iii) a differential scanning calorimetry map such as 128407.doc.12-200911757 and an exothermic heat at 201 C followed by an endotherm at 216 °c; and Ο) showing a weight loss of 97% below 170 °C In a further embodiment, the present invention relates to a hydrogen sulphate of the compound „. In accordance with its physical properties, the hydrochloride is crystalline. Hydrochloric acid hydrochloride It may be characterized by having one or more of the following physical characteristics (it is noted that the composition does not necessarily exhibit all of these features to indicate the presence of hydrogen hydride): (1) is included in about 19.83, 23. 9 and 21.89 degrees 20 (. 20) an X-ray powder diffraction pattern (CuKa) at the diffraction peak; (ii) an iH NMR spectrum as shown in Fig. 54; (iii) an fTIR spectrum as shown in Fig. 55; (iv) having a concentration An endothermic event at about i97〇c and a differential scanning calorimetry pattern at an exothermic event of 2〇8; and (v) a TGA thermogram showing no weight loss below 160°C. In another embodiment The present invention relates to a hydrobromide salt of a compound π (compound II hydrobromide). The hydrogen oxalate salt is crystalline. The hydrobromide salt may be characterized by one or more of the following physical characteristics (it is noted that the composition does not necessarily exhibit all of these characteristics to indicate the presence of the (iv) acid salt): 0) X-ray powder diffraction pattern (CuKa) of diffraction peaks at about 1丨25, 18.92, and 26.55 degrees 20 (.20); (ii) 1H NMR spectrum as shown in Fig. 60; 128407.doc -13 - 200911757 (111) FTIR spectrum as shown in Figure 61; (iv) differential scanning calorimetry map having an exothermic event centered at about 214 followed by an exotherm of about 224.; and (v) shown below In another embodiment, the present invention relates to a gentisate of the compound π. Based on its physical properties, the gentisate is crystalline. Having one or more of the following physical characteristics (the composition is not required to exhibit all of these features to indicate the presence of gentisate): ω is comprised around about 17.49, 12.87, and 25.93 degrees 2 Θ (° 2 Θ) X-ray powder diffraction pattern (CuKa) of the peak; (ii) 1η NMR light as shown in FIG. ; (Iii) FTIR spectrum shown in the FIG. 67; (W is) having a concentration of about Shu 27 (:, Mot and differential scanning calorimetry an endothermic event 24〇t the measured spectrum;. And

(V)在約6〇_17〇°C之間顯示重量損失為約5%之的TGA(V) shows a TGA with a weight loss of about 5% between about 6 〇 17 17 ° C

熱分析圖。化合物IIIThermal analysis chart. Compound III

A為結晶形。 128407.doc -14- 200911757 或多種以下物理特徵（應注意組A is crystalline. 128407.doc -14- 200911757 or a variety of physical characteristics (should note the group)

的X射線粉末繞射圖案（CuKc〇 ; 如圖72中所示之1H NMR光譜；如圖73中所示之FTIR光譜；形式A之特徵可為具有一或多種合物不f ⑴ 包含在約9.43、 (ϋ) (iii)X-ray powder diffraction pattern (CuKc〇; 1H NMR spectrum as shown in FIG. 72; FTIR spectrum as shown in FIG. 73; Form A may be characterized by having one or more compounds not f(1) included in 9.43, (ϋ) (iii)

定圖譜；及直至約230。(：均顯示無重量損失之TGA熱分析圖。在另一實施财’本發明係關於化合物m之龍膽酸鹽。基於其物理特性，龍膽酸鹽為結晶形。Set the map; and up to about 230. (: TGA thermograms showing no weight loss. In another embodiment, the present invention relates to gentisate of compound m. Based on its physical properties, gentisate is crystalline.

意組合物不需必要地展現所有此等特徵以表明龍膽酸鹽之存在）： ⑴包含在約 10.9、20.48、28.35及3〇.9〇度20(。20)處之繞射峰的X射線粉末繞射圖案（CuKo〇 ; (11)在120-1 90°C之間顯示3.2%之重量損失的TGA熱分析圖；及 (出）在約133°C之DSC吸熱事件。包含化合物I、II或ΙΠ之組合物本發明係關於包含化合物I、II或ΠΙ之組合物，其中化合物以上文所揭示之形式存在。注意化合物J、IU1III之其他結晶形及非晶形亦可存在於組合物中。在一變體中’組合物包含至少0.1〇/〇、〇 25%、〇.5%、 128407.doc •15· 200911757 l0/〇、5%、10%、25%、50%、75%、80%、85%、90%、 95%、97%或99%化合物I、II或III，其中大於0.1%、1%、 5%、10%、25%、50%、75%、80%、85%、90%、95%、 97%或99%之化合物I、II或III(以重量計）以上文所揭示之結晶形存在。組合物可視情況為醫藥組合物。醫藥組合物可視情況另外包括一或多種醫藥載劑。包含化合物I、II或III之套組及製品本發明亦提供包含包括化合物I、II或III之組合物之套組及其他製品’其中化合物I、II或ΠΙ以上文所揭示之形式存在。在一變體中，組合物包含至少O jo/o、0.25%、0.5%、 1%、5%、10%、25%、5 0%、75%、80%、85%、90%、 95%、97%或99%化合物I、Π或in，其中大於〇.1〇/0、p/0、 50/〇、10%、25%、50%、75%、80%、85%、90%、95%、 97%或99。/。之化合物I、Π或π：[(以重量計）以選自上文所揭不之群的形式存在於組合物中。在套組及製品中之組合物可視情況為醫藥組合物。醫藥組合物可視情況另外包括一或多種醫藥載劑。關於包括醫藥組合物之各上述實施例，可以任何方式調配醫藥組合物’其中至少—部分化合物卜π或则選自由任何上文所揭示之形式組成之群的形式存在。在對人類投與醫藥調配物之後，一部分化合物卜^或職情況以選自由上文所揭示之形式組成之群的形式存在一段時間。製造化合物I、ΙΙ&ΠΙ之結晶固體的方法亦提供各種方法用於製造所有所 - 吓’所揭不之形式。亦提供各 128407.doc • 16- 200911757 種方法用於製造包含化合物i、π或ΠΙ之結晶固體中之一或多個的醫藥組合物、套組及其他製品。使用化合物I、II及III之結晶固艘的方法亦提供使用包含一或多個上文所揭示之結晶固體的醫藥組合物、套組及其他製品以治療各種由HDAC引發之疾病的方法。在一實施例中’本發明係關於抑制HDAC之方法，其包含投與組合物，其中大於〇.1%、1%、5%、1()%、25%、 50%、75%、80%、85%、90%、95%、97% 或 99%之化合物 I、II或111(以重量計）以選自上文所揭示之群的形式存在於組合物中。組合物視情況包含至少〇丨％、〇 2 5 %、〇 . 5 %、 1%、5%、10%、25。/。、50。/。、75%、80%、85%、90%、 95%、97%或99%之化合物I。在另一實施例中，本發明係關於藉由投與化合物I、π或 III而用化合物I來抑制個體（例如人體）之hdAC的方法，其中當投與化合物時，大於〇.1〇/0、ρ/。、5%、ι〇〇/。、25%、 50%、75%、80%、85%、90%、95%、97%或 99%之化合物 I、II或111(以重量計）以選自上文所揭示之結晶固體群的形式存在於組合物中。組合物視情況包含至少〇.丨％、 0.25%、〇·5%、1%、5%、10%、25%、50%、75〇/〇、80%、 85%、90%、95%、97%或 99%之化合物 I、II或 in。在另一實施例中’本發明係關於藉由投與化合物I、„或 III而用化合物I、Η或m來抑制個體（例如人體）之hdac的方法，其中在已對人類投與化合物之後的一段時間内，大 128407.doc -17- 200911757 於 0.1%、1%、5%、10%、25%、50%、75%、80%、85%、 90%、95%、97%或99%之化合物I、II或III(以重量計）以選自上文所揭示之結晶固體群的形式存在於組合物中。組合物視情況包含至少 0.1%、0.25%、0.5%、1%、5%、10%、 25%、50%、75%、80%、85%、90% ' 95% ' 97% 或 99%之化合物I、II或III。在又一實施例中，本發明提供治療HDAC對其具有促進疾病病況之病理學及/或症狀學之活性的疾病病況的方法，其包含對個體（例如人體）投與組合物，其中當投藥時大於 0.1%、1%、5%、10%、25%、50%、75%、80%、 85%、90%、95%、97%或99%之化合物I、II或111(以重量計）以選自上文所揭示之結晶固體群的形式存在於組合物中。組合物視情況包含至少0.1%、0.25%、0.5%、1%、 5% ' 10%、2 5% ' 5 0% ' 75% ' 80%、85%、90%、95%、 97%或99%之化合物I、II或III。在又一實施例中，本發明提供治療HDAC對其具有促進疾病病況之病理學及/或症狀學之活性的疾病病況的方法，其包含使組合物存在於個體（例如人體）中，其中在已對人類投與組合物之後的一段時間内，大於0.1%、1%、 5% > 10%、25%、50%、75%、80%、85%、90%、95%、 97%或99%之化合物I、II或111(以重量計）以選自由上文所揭示之結晶固體組成之群的形式存在於組合物中。組合物視情況包含至少 0.1%、0.25%、0.5%、1%、5%、10%、 25% ' 50%、75% ' 80% ' 85%、90%、95%、97% 或 99%之 128407.doc 18- 200911757 化合物I、II或ΠΙ。在另實鈿例中，提供預防、延遲進程及/或治療由 HDAC引發之病狀的方法，該等病狀尤其為癌症：鱗狀細胞癌、星形細胞瘤、卡波西氏肉瘤（Kap〇si,s sarc〇ma)、神經膠母細胞瘤、非小細胞肺癌、膀胱癌、頭部及頸部癌、色素瘤、_巢癌、***癌、乳癌、小細胞肺癌、神經膠質瘤、結腸直腸癌、泌尿生殖器癌及胃腸癌；發炎性腸道疾病、+皮癬或移植排斥；關節炎、眼睛之退化疾病、多發性硬化症、肌萎縮性側索硬化、甲狀腺瘤或阿茲海默氏症（Alzhemier’s disease)、過度增生性皮膚病或發炎性皮膚病。在聲明化合物卜Π或m可以選自由上文所揭示之結晶形組成之群的形式存在於組合物中的各情況中，本發明意欲涵蓋僅存在一種形式之各化合物、存在兩種形式之各化合物（所有組合）及存在三種、四種或四種以上形式之各化合物（所有組合）的組合物。【實施方式】本發明係關於2006年7月13曰申請之美國專利申請案 1 1/457,260中揭示的HDAC抑制劑之多晶型物及鹽，且該美國專利申請案主張2005年7月14曰申請之美國臨時申請案60,699，139的權益。本發明尤其係關於化合物卜η或hi 之結晶固體以及包含化合物卜^或出之組合物，其中至少 -部分化合物以本文所述之固體形式之一存在於組合：中0 128407.doc •19· 200911757 本發月亦係關於諸多方法，其包括製備本文中鑑別之各多晶型及鹽的方法。本發月亦係關於组合物、包含組合物之套組及製品，其中·•亥等，、且α物包含化合物〗、Η或HI，其中至少一部分化合物以本文中繼別之多晶型及鹽形式之―存在於組合物中。本發明另外係關於製造醫藥組合物之方法及使用包含化〇物I 11或111之組合物的方法，其中至少一部分化合物以本文中鑑別之多晶型及鹽形式之一存在於組合物中。應瞭解，視如何製造包含給定化合物之組合物且接著在製造後如何儲存及操作組合物而定，將影響組合物之結晶 3里。因此，組合物可能不包含結晶含量或可包含較高濃度之結晶含量。另外應注意化合物可以一或多種不同多晶型存在於給定組合物中以及視情況亦以非晶形物質存在。此可能係由於 (a)兩種或兩種以上不同多晶型之物理混合；（b)具有自結晶條件產生之兩種或兩種以上不同多晶型；（c)具有轉化成另一多晶型之所有或一部分給定多晶型；（d)具有呈轉化成兩種或兩種以上多晶型之非晶態的所有或一部分化合物；以及由於許多其他原因。了發現，視如何製備包含化合物之組合物而定，該化合物在給定多晶型下之重量百分比可自〇%至i 〇〇%改變。在一態樣中，本發明係關於包含化合物〗之組合物，其中大於 0.1。/〇、0.5%、1〇/0、5%、10%、25%、5〇%、75%、 80%、85%、90%、95%、97% 或 99%或更多之化合 (以 128407.doc -20 - 200911757 重量計）以選自由以下組成之群的形式存在於組合物中：游離驗之形式A、B及非晶形；反丁締二酸鹽；填酸鹽；及氫氣酸鹽。在另一態樣中，本發明係關於包含化合物II之组合物，其中大於 0.1%、0.5%、1%、5%、1〇%、25%、50〇/〇、 75%、80%、85%、90%、95%、97% 或 99%或更多之化合物11(以重量計）以選自由以下組成之群的形式存在於組合物中：游離驗之形式A、丁二酸鹽、苯甲酸鹽、氫氣酸The composition of interest does not necessarily exhibit all of these features to indicate the presence of gentisate): (1) X comprising diffraction peaks at about 10.9, 20.48, 28.35, and 3〇.9〇20 (.20) Ray powder diffraction pattern (CuKo〇; (11) shows a TGA thermogram of 3.2% weight loss between 120-1 90 °C; and (out) DSC endothermic event at about 133 °C. Contains Compound I Compositions of II, II or oxime The present invention relates to compositions comprising Compounds I, II or oxime, wherein the compounds are present in the form disclosed above. Note that other crystal forms and amorphous forms of Compound J, IU1III may also be present in the composition. In a variant, the composition comprises at least 0.1〇/〇, 〇25%, 〇.5%, 128407.doc •15·200911757 l0/〇, 5%, 10%, 25%, 50%, 75 %, 80%, 85%, 90%, 95%, 97% or 99% of compounds I, II or III, of which greater than 0.1%, 1%, 5%, 10%, 25%, 50%, 75%, 80 %, 85%, 90%, 95%, 97% or 99% of the compound I, II or III (by weight) is present in the crystalline form disclosed above. The composition may optionally be a pharmaceutical composition. The pharmaceutical composition may be visualized. Happening Including one or more pharmaceutical carriers. Kits and articles comprising Compounds I, II or III The invention also provides kits and other articles comprising a composition comprising Compounds I, II or III 'wherein Compounds I, II or ΠΙ The form disclosed above exists. In one variation, the composition comprises at least Ojo/o, 0.25%, 0.5%, 1%, 5%, 10%, 25%, 50%, 75%, 80% , 85%, 90%, 95%, 97% or 99% of compound I, hydrazine or in, which is greater than 〇.1〇/0, p/0, 50/〇, 10%, 25%, 50%, 75% , 80%, 85%, 90%, 95%, 97% or 99% of the compound I, hydrazine or π: [(by weight) is present in the composition in a form selected from the group not disclosed above) The composition in the kit and the article may optionally be a pharmaceutical composition. The pharmaceutical composition may optionally include one or more pharmaceutical carriers. With regard to each of the above examples including pharmaceutical compositions, the pharmaceutical composition may be formulated in any manner. 'At least - part of the compound π or then selected from the group consisting of any of the forms disclosed above. After administration of the pharmaceutical formulation to humans, part of The compound or condition is present in a form selected from the group consisting of the forms disclosed above for a period of time. The method of making the crystalline solids of the compound I, ΙΙ & 亦 also provides various methods for the manufacture of all - No. Also available are 128407.doc • 16-200911757 methods for the manufacture of pharmaceutical compositions, kits and other articles comprising one or more of the crystalline solids of the compound i, π or cerium. The method of using the crystalline solids of Compounds I, II and III also provides a method of treating various diseases caused by HDAC using pharmaceutical compositions, kits and other articles comprising one or more of the crystalline solids disclosed above. In one embodiment, the invention relates to a method of inhibiting HDAC comprising administering a composition wherein greater than 0.1%, 1%, 5%, 1%, 25%, 50%, 75%, 80 %, 85%, 90%, 95%, 97% or 99% of the compound I, II or 111 (by weight) are present in the composition in a form selected from the group disclosed above. The composition comprises at least 〇丨%, 〇25%, 〇.5%, 1%, 5%, 10%, 25, as appropriate. /. 50. /. , 75%, 80%, 85%, 90%, 95%, 97% or 99% of Compound I. In another embodiment, the present invention relates to a method of inhibiting hdAC in an individual (eg, a human) with Compound I by administering Compound I, π, or III, wherein when the compound is administered, it is greater than 〇.1〇/ 0, ρ /. , 5%, ι〇〇/. 25%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or 99% of the compound I, II or 111 (by weight) selected from the group of crystalline solids disclosed above The form is present in the composition. The composition comprises at least 〇.丨%, 0.25%, 〇·5%, 1%, 5%, 10%, 25%, 50%, 75〇/〇, 80%, 85%, 90%, 95%, as the case may be. , 97% or 99% of the compounds I, II or in. In another embodiment, the invention relates to a method of inhibiting hdac in an individual (eg, a human) by administering Compound I, Η or m by administering Compound I, „ or III, wherein after administration of the compound to humans For a period of time, the big 128407.doc -17- 200911757 at 0.1%, 1%, 5%, 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or 99% of the compound I, II or III (by weight) is present in the composition in a form selected from the group of crystalline solids disclosed above. The composition optionally comprises at least 0.1%, 0.25%, 0.5%, 1% 5%, 10%, 25%, 50%, 75%, 80%, 85%, 90% '95% '97% or 99% of compound I, II or III. In yet another embodiment, the invention A method of treating a disease condition in which HDAC has activity to promote pathology and/or symptomology of a disease condition, comprising administering to a subject (eg, a human) a composition, wherein when administered, greater than 0.1%, 1%, 5 %, 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or 99% of the compound I, II or 111 (by weight) selected from the above The form of the crystalline solid group exists in the group In the composition, the composition comprises at least 0.1%, 0.25%, 0.5%, 1%, 5% '10%, 25% '50% '75% '80%, 85%, 90%, 95%, as the case may be. 97% or 99% of Compound I, II or III. In yet another embodiment, the invention provides a method of treating a disease condition in which HDAC has activity in promoting pathology and/or symptomology of a disease condition, comprising The composition is present in an individual (e.g., a human), wherein over a period of time after the composition has been administered to the human, greater than 0.1%, 1%, 5% > 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or 99% of the compound I, II or 111 (by weight) is present in the composition in the form of a group selected from the crystalline solids disclosed above. The composition optionally comprises at least 0.1%, 0.25%, 0.5%, 1%, 5%, 10%, 25% '50%, 75% '80% ' 85%, 90%, 95%, 97% or 99% 128407.doc 18- 200911757 Compound I, II or ΠΙ. In another example, methods for preventing, delaying, and/or treating a condition caused by HDAC, particularly cancers: squamous cells, are provided. Cancer, astrocytoma, Kaposi Sarcoma (Kap〇si, s sarc〇ma), glioblastoma, non-small cell lung cancer, bladder cancer, head and neck cancer, pigmented tumor, _ nest cancer, prostate cancer, breast cancer, small cell lung cancer, nerve Glioma, colorectal cancer, genitourinary cancer and gastrointestinal cancer; inflammatory bowel disease, + skin or transplant rejection; arthritis, degenerative diseases of the eye, multiple sclerosis, amyotrophic lateral sclerosis, thyroid tumor or Alzhemier's disease, hyperproliferative skin disease or inflammatory skin disease. In each case where it is stated that the compound di or m may be selected from the group consisting of the crystalline forms disclosed above, the present invention is intended to cover the presence of only one form of each compound, in the presence of two forms. Compounds (all combinations) and compositions in which three, four or more forms of each compound (all combinations) are present. [Embodiment] The present invention relates to polymorphs and salts of HDAC inhibitors disclosed in U.S. Patent Application Serial No. 1 1/457,260, the entire disclosure of which is incorporated herein by reference.权益 The right to apply for US Provisional Application 60, 699, 139. The invention relates in particular to crystalline solids of the compound η or hi and compositions comprising the compound, wherein at least one part of the compound is present in the combination in one of the solid forms described herein: 0 128407.doc • 19· 200911757 This Month is also directed to a number of methods including methods for preparing the various polymorphs and salts identified herein. The present month also relates to compositions, kits and articles comprising the compositions, wherein •·Hay et al., and the alpha compound comprises a compound, hydrazine or HI, at least a portion of which are relayed by the polymorph and The salt form is present in the composition. The invention further relates to a method of making a pharmaceutical composition and a method of using a composition comprising the chemical I 11 or 111, wherein at least a portion of the compound is present in the composition in one of the polymorphic forms and salt forms identified herein. It will be appreciated that depending on how the composition comprising the given compound is produced and then how the composition is stored and manipulated after manufacture, it will affect the crystallization of the composition. Thus, the composition may not contain a crystalline content or may contain a higher concentration of crystalline content. It should also be noted that the compound may be present in a given composition in one or more different polymorphic forms and, as the case may be, in the form of an amorphous material. This may be due to (a) physical mixing of two or more different polymorphs; (b) two or more different polymorphs with self-crystallization conditions; (c) with conversion to another All or a portion of the crystalline form is given a polymorph; (d) has all or a portion of the compound in an amorphous state that is converted to two or more polymorphs; and for a number of other reasons. It has been found that, depending on how the composition comprising the compound is prepared, the weight percentage of the compound under a given polymorphic form can vary from 〇% to i 〇〇%. In one aspect, the invention is directed to compositions comprising a compound wherein greater than 0.1. /〇, 0.5%, 1〇/0, 5%, 10%, 25%, 5%, 75%, 80%, 85%, 90%, 95%, 97% or 99% or more of the combination ( It is present in the composition in the form of a group selected from the group consisting of: 128407.doc -20 - 200911757 by weight: free form A, B and amorphous; transbutyrate; acid salt; and hydrogen Acid salt. In another aspect, the invention relates to a composition comprising Compound II, wherein greater than 0.1%, 0.5%, 1%, 5%, 1%, 25%, 50%/〇, 75%, 80%, 85%, 90%, 95%, 97% or 99% or more of the compound 11 (by weight) is present in the composition in a form selected from the group consisting of: free form A, succinate Benzoate, hydrogen acid

鹽、氫溴酸鹽及龍膽酸鹽。在另一態樣中’本發明係關於包含化合物m之組合物，其中大於 0.1〇/〇、0.5%、10/〇、50/〇、ι〇〇/。、25%、50%、 75%、80%、85〇/〇、90%、95〇/〇、97% 或 99%或更多之化合物111(以重莖什）以選自由游離鹼之形式八與龍膽酸鹽組成之群的形式存在於組合物中。定義當術語”結曰曰曰”用於本文中時係指含有可經水合及/或溶劑合之特^化合物且具有^夠結晶含量以藉由xRpD或其他繞射技術展現可辨別繞射圖樣的物質。藉由溶解於溶液中之化合物的直接結晶或在不同結晶條件下所得晶體之互變而獲得之結晶物質經常將具有含有用於結晶之溶劑的曰曰體，其被稱為結晶溶劑合物。此外，於其中進行結晶之^ 定溶劑系統及物理實施例（共同稱為結晶條件）可產生對於':晶條件而言之獨特物理及化學特性的結晶物質，、其般知因於化合物之化學部分在晶體内相對於彼此的定向 128407.doc -21- 200911757 及:或在結晶物質中化合物之特定多晶型的優勢。 2存在於組合物中之化合物的多晶m亦種1之呈非晶形固態之化合物，其子及/或句冬紝曰仏併您，'口日曰之副產物曰物質之晶體的降解產物。因此 rP田4» 丄丄 Θ々野S吾結 3 中時涵蓋組合物可包括非晶物含量；在其他方法中可藉由具有可辨別繞射圖樣之組合 :、質中結晶物質之存在。』非曰曰形物Salt, hydrobromide and gentisate. In another aspect, the invention relates to a composition comprising compound m, wherein greater than 0.1 〇/〇, 0.5%, 10/〇, 50/〇, ι〇〇/. , 25%, 50%, 75%, 80%, 85〇/〇, 90%, 95〇/〇, 97% or 99% or more of the compound 111 (in the form of a heavy stem) selected from the form of a free base The form of the group consisting of eight and gentisate is present in the composition. DEFINITIONS When the term "crust" is used herein, it is meant to include a compound that is hydratable and/or solvate and has a sufficient crystalline content to exhibit a discernible diffraction pattern by xRpD or other diffraction techniques. Substance. A crystalline material obtained by direct crystallization of a compound dissolved in a solution or intergranular transformation of crystals obtained under different crystallization conditions will often have a steroid containing a solvent for crystallization, which is called a crystalline solvate. In addition, the solvent system and physical examples in which crystallization is performed (collectively referred to as crystallization conditions) can produce crystalline materials having unique physical and chemical properties for ': crystal conditions, which are generally known as chemical compounds. Partial orientation within the crystal relative to each other 128407.doc -21- 200911757 and: or the advantage of a particular polymorphic form of the compound in the crystalline material. 2 a polycrystalline m of the compound present in the composition, which is also an amorphous solid compound, and its degradation product of the crystal of the by-product of the sputum . Therefore, the composition of the rP field 4» Θ々 Θ々 S 吾 3 3 3 可可可可可可可涵盖涵盖组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合组合Non-morphological

可藉由研磨或粉化物質來增加結晶物質之非晶物含量，其由繞射及其他譜線相對於研磨前之結晶物質拓寬而證明。足夠之研磨及/或粉化可相對於研磨前之結晶物質：寬該等線’拓寬程度為XRPD或其他晶體特定圖譜可變得不了辨別，使物質大體上為非晶形或準非晶形。預期連續研磨將增加非晶物含量且進—步&寬讀_ 樣，使得XRPD圖樣之界限變寬而不再能於雜訊上方予以辨別。當XRPD圖樣拓寬至不可辨別之界限時，可將物質視為不再為結晶物質且代之以完全非晶形。對於具有增加之非晶物含量且完全為非晶形物質的物質而言不應觀察到峰’其將表明研磨產生另一形式。當術語”非晶形”用於本文中時係指包含化合物之組合物’該化合4勿含有過少結晶含量之化合物以致不能產生藉由XRPD或其他繞射技術可辨別之圖#。破璃狀物質為_ 種類型之非晶形物質。非晶形物質並不具有真晶格且因此為玻璃狀而非真固體，技術上類似於極黏非結晶液體。可較佳將並非為真固體之玻璃描述為準固體非晶形物質。因 128407.doc •22· 200911757 此非晶形物質係指準固體玻璃狀物質。當術語’’寬"或"拓寬"用於本文中以描述包括xrpd、 NMR及IR光譜學及拉曼光譜學譜線之譜線時，其為關於基線光譜之線寬的相對術語。基線光譜通常為自給定組之物理及化學條件（包括溶劑組成及特性，諸如溫度及壓力）直接獲得之特定化合物的未經操作結晶形的基線光譜。舉例而口拓寬了用以描述相對於研磨前之物質，經研磨或粉化之包含結晶化合物之物質的XRPD光譜的譜線。在其中呈溶劑合物或水合物之組成分子、離子或原子並不迅速滾動的物質中，線拓寬表明化合物之化學部分定向之無規性增加，因此表明非晶物含量增加。當在經由不同結晶條件獲得之結晶物質之間作比較時，較寬譜線表明產生相對較寬譜線之物質具有較高含量之非晶形物質。當術語"約"用於本文中時係指實際值處於引用值之±5% 内之估計值。當分又”用以描述DS〇及熱及放熱時係指具有可區別峰位之重疊吸熱或放熱。當"一致’’用以描述iH NMR及質譜分析結果時意謂資料係如對於試樣之結構所預期一般。 "API"係指如本文中所用描述化合物之游離鹼形式的活性醫藥成份》 "ICso”意謂產生目標酶之5〇%抑制的抑制劑莫耳濃度。 "ec50"意謂產生目標作用之50%功效的抑制劑莫耳濃度。 128407.doc -23- 200911757 機酸形成之酸加成鹽：乙酸'丙酸、己酸、庚酸、環戊烷丙酸、乙醇酸、丙酮酸、乳酸、丙二酸、丁二酸、蘋果酸、順丁烯二酸、反丁烯二酸、酒石酸、檸檬酸、苯甲酸、鄰（4·經基节醯基）苯甲酸、肉桂酸、扁桃酸、甲續酸、乙磺酸、1，2-乙二石黃酸、2_經基乙磺酸、苯磺酸、對酸、硬脂酸、黏康酸及其颠似酸。The amorphous content of the crystalline material can be increased by grinding or chalking the material, as evidenced by the broadening of the diffraction and other spectral lines relative to the crystalline material prior to milling. Sufficient grinding and/or pulverization may be relative to the crystalline material prior to milling: the width of the lines 'expanded to an extent that the XRPD or other crystal specific map may become indistinguishable, making the material substantially amorphous or quasi-amorphous. Continuous grinding is expected to increase the amorphous content and advance & wide read so that the XRPD pattern is widened and can no longer be discerned above the noise. When the XRPD pattern is broadened to an indistinguishable boundary, the substance can be considered to be no longer crystalline and replaced by a completely amorphous form. For materials having an increased amorphous content and being completely amorphous, no peaks should be observed which would indicate that the milling produced another form. When the term "amorphous" is used herein, it is meant to mean a composition comprising a compound which does not contain a compound having too little crystalline content such that it cannot produce a pattern # discernible by XRPD or other diffraction techniques. The granulated material is an amorphous substance of the type _. Amorphous materials do not have a true crystal lattice and are therefore glassy rather than true solids, technically similar to very viscous non-crystalline liquids. Glass which is not a true solid can be preferably described as a quasi-solid amorphous material. Because 128407.doc •22· 200911757 This amorphous substance refers to a quasi-solid glassy substance. When the term ''wide' or 'quotation' is used herein to describe a line that includes xrpd, NMR, and IR spectroscopy and Raman spectroscopy lines, it is a relative term for the linewidth of the baseline spectrum. . The baseline spectrum is typically the baseline spectrum of the unprocessed crystalline form of a particular compound obtained directly from a given set of physical and chemical conditions, including solvent composition and characteristics, such as temperature and pressure. For example, the line of the XRPD spectrum for describing a substance containing a crystalline compound which is ground or pulverized with respect to the substance before grinding is broadened. In a substance in which a constituent molecule, ion or atom of a solvate or a hydrate does not rapidly roll, the line broadening indicates an increase in the randomness of the chemical moiety orientation of the compound, thus indicating an increase in the amorphous content. When comparing between crystalline materials obtained via different crystallization conditions, the broader line indicates that the material that produces the relatively broad spectrum has a higher content of amorphous material. When the term "about" is used herein, it is an estimate that the actual value is within ± 5% of the quoted value. When used to describe DS〇 and heat and exotherm, it refers to overlapping endotherms or exotherms with distinguishable peak positions. When "consistent' is used to describe iH NMR and mass spectrometry results, it means The structure is expected to be general. "API" means an active pharmaceutical ingredient in the form of a free base as described herein. "ICso" means an inhibitor molar concentration that produces 5% inhibition of the target enzyme. "ec50" means the molar concentration of the inhibitor that produces 50% of the effect of the target. 128407.doc -23- 200911757 Acid addition salt formed by acid: acetic acid 'propionic acid, caproic acid, heptanoic acid, cyclopentane propionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, apple Acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, o-(4·yl-based fluorenyl)benzoic acid, cinnamic acid, mandelic acid, methylation, ethanesulfonic acid, 1 , 2-ethane dilithenic acid, 2_ mercaptoethanesulfonic acid, benzenesulfonic acid, p-acid, stearic acid, muconic acid and its acid.

醫藥予上可接文之鹽"意謂本發明之化合物的鹽，其如 ,所定義為醫藥學上可接受且其具有所需藥理學活性。此等鹽包括以諸如氫氯酸、氫漠酸、硫酸、確酸'麟酸及其類似酸之無機酸形成之酸加成鹽；或以諸如以下各酸之；氣苯磺酸、2-萘磺酸、對曱笨磺酸、樟腦磺酸、4_甲基雙環[2.2.2]辛_2_烯+甲酸、葡糖庚酸、4,4，_亞甲基雙^-羥基-2-烯-1-曱酸）、3_苯基丙酸、三甲基乙酸、第三丁基乙酸、月桂基硫酸、葡萄糖酸、麩胺酸、羥基萘甲酸、水楊醫藥學上可接受之鹽亦包括鹼加成鹽，其可當所存在之酸性質子能夠與無機鹼或有機鹼反應時形成。可接受之無機鹼包括氫氧化鈉、碳酸鈉、氫氧化鉀、氫氧化鋁及氫氧化鈣。可接受之有機鹼包括乙醇胺、二乙醇胺、三乙醇胺、緩血酸胺、N-甲基葡萄胺及其類似驗。 ’’前藥"意謂活體内可經由代謝轉化成根據本發明之抑制劑的化合物。前藥本身可能亦具有或可能不具有1〇)八(：抑制活性。舉例而言，包含羥基之抑制劑可以酯形式投藥，該酯藉由活體内水解轉化成羥基化合物。可活體内轉化成經基化合物之合適酯包括乙酸酯、檸檬酸輯、乳酸^、、酉 128407.doc -24- 200911757 石酸醋、丙二酸醋、草酸醋、水揚酸酉旨、丙酸醋、丁二酸醋、反丁烯二酸酉旨、順丁稀二酸醋、亞甲基雙冬經基萃甲酸醋、龍膽酸醋、經乙績㈣、二-對甲苯甲醯基酒石酸醋、甲續酸醋、乙續酸醋、苯續酸醋、對子苯績酸醋、環己基胺基磺酸醋、奎尼酸醋(quinate)、胺基酸醋及其類似西曰。類似地，包含胺基之抑制劑可以酿胺形式投藥，該酿胺藉由活體内水解轉化成胺化合物。 f "異構體，•意謂具有相同分子式但在性質或其原子鍵結順序或其原子空間排列方面不同的任何化合物。將原子空間排列不同的異構體稱為"立體異構體"。將彼此不為鏡像之立體異構體稱為"非對映異構體"且將為非重疊鏡像之立體異構體稱為”對映異構體"或有時稱為"光學異構體"。將鍵結至四個不相同取代基之碳原子稱為"對掌性中心"。具有一個對掌性中心之化合物具有兩種對掌性相對之對映異構形式。將兩種對映異構形式之混合物稱為,，外消旋混合物"。具有一個以上對掌性中心之化合物具有個對映異構對，其中η為對掌性中心之數目。具有一個以上對掌性中心之化合物可呈個別非對映異構體或呈非對映異構體之混合物（稱為’’非對映異構混合物存在。當存在一個對掌性中心時，立體異構體可以該對掌性中心之絕對構型為特徵絕對構型係指連接於對掌性中心之取代基的空間排列。對映異構體可以其對掌性中心之絕對構型為特徵且由 Cahn、Ingold及prel〇g之尺_及3_定序規則來描述。在此項技術中熟知立體化學命名法之慣例、測定立體化學及分離 128407.doc •25· 200911757 S體異構體之方法（例如參見"Advanced 0rganic etmstry- « > March, Jerry, j〇hn WUey & s〇ns>The pharmaceutically acceptable salt of the present invention means a salt of a compound of the present invention, which is defined as pharmaceutically acceptable and which has the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrogen desert acid, sulfuric acid, acid-like acid and the like; or such as the following acids; gas benzenesulfonic acid, 2- Naphthalenesulfonic acid, p-sulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]oct-2-ene+carboxylic acid, glucoheptanoic acid, 4,4,_methylenebis-hydroxy- 2-ene-1-decanoic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicyl medicinally acceptable Salts also include base addition salts which can be formed when the acidic protons present are capable of reacting with an inorganic or organic base. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucosamine, and the like. ''Prodrug" means a compound that can be converted in vivo to an inhibitor according to the present invention by metabolism. The prodrug itself may or may not have 1 〇) VIII (inhibition activity. For example, an inhibitor containing a hydroxy group may be administered in the form of an ester which is converted into a hydroxy compound by in vivo hydrolysis. Suitable esters of the base compound include acetate, citric acid, lactic acid, 酉128407.doc -24- 200911757 vinegar, malonic acid vinegar, oxalic acid vinegar, salicylic acid, propionic acid vinegar, butyl Diacid vinegar, fumaric acid, cis-butanic acid vinegar, methylene bis-male-based extract of formic acid vinegar, gentian vinegar, y (b), di-p-toluamyl tartaric acid vinegar, A continuous acid vinegar, a continuous acid vinegar, a benzene vinegar, a pair of benzene vinegar, a cyclohexylamine sulfonate, a quinate, an amino acid vinegar and the like. The inhibitor containing an amine group can be administered in the form of an amine which is converted into an amine compound by in vivo hydrolysis. f "isomers, meaning the same molecular formula but in nature or its atomic bonding sequence or Any compound that differs in the arrangement of atoms in space. The atomic space is arranged differently. The conformation is called "stereoisomer". The stereoisomers that are not mirror images of each other are called "diastereomers" and the stereoisomers that are non-overlapping mirrors are called "pairs". Is a "optical isomer" or a carbon atom bonded to four different substituents The compound has two opposite enantiomeric forms of palmarity. A mixture of two enantiomeric forms is referred to as a racemic mixture ". A compound having more than one pair of palmar centers has a mapping Heterogeneous pair, where η is the number of palmar centers. Compounds with more than one palm center may be individual diastereomers or a mixture of diastereomers (referred to as ''non-optical) The presence of an isomeric mixture. When a pair of palmar centers exist, the stereoisomers can be characterized by the absolute configuration of the palm center. The absolute configuration refers to the spatial arrangement of the substituents attached to the palm center. Isomers can be characterized by their absolute configuration of the palm center It is described by Cahn, Ingold, and prel〇g's ruler and 3_sequencing rules. The practice of stereochemical nomenclature, stereochemistry and separation are well known in the art. 128407.doc •25· 200911757 S isomer Method (see, for example, "Advanced 0rganic etmstry- « > March, Jerry, j〇hn WUey &s〇ns>

New York, 1992) 〇 &療有效3： ”意謂當投與動物以治療疾病時足以實現疾病之該治療的量。療％、明本發明之化合物的任何投藥且包括：New York, 1992) 〇 & Therapeutic Effectiveness 3: ” means the amount of the treatment that is sufficient to achieve the disease when administered to an animal to treat the disease. Treatment, any formulation of a compound of the invention and includes:

⑴預防疾病在動物中發生，該動物可能易於患上該疾病仁尚未經歷或顯示該疾病之病理學或症狀學， ⑺抑制正經歷或顯示疾病之病理學或症狀學之動物的疾病（亦即’遏制病理學及/或症狀學之進一步發展），或 (3)改善正、經^員#疾病之病Μ或症狀|之動物的疾病（亦即，逆轉病理學及/或症狀學）。動物包括人類、非人類哺乳動物（例如狗、猶、兔、牛、馬、錦I、山羊、冑、鹿及其類似動物）及非哺乳動物（例如鳥及其類似動物）。製備化合物I、Π及m及其多晶型物及鹽 A•製備化合物1、11及工工1 為合成本發明之化合物卜ILaIII，可研發各種方法。在 2006年7月13日申請之美國專利申請案第1 1/457,260號中揭不可用以合成化合物之通用途徑，該令請案之揭示内容以全文引用的方式併入本文中。然而，應注意亦可由其他人可°又计之其他合成途徑來合成化合物I、π及in。在實例3、28及41中分別提供合成化合物工、11及111之結晶固體的代表性方法。本文中將結晶固體稱為對應化合物 128407.doc -26- 200911757 之形式A。 B•製備化合物I、II及III之多晶型物及鹽化合物之鹽或溶劑合物通常可自適當平衡離子與溶劑之溶液中直接沈澱。可使用熟習合成有機化學及醫藥調配之技術者已知且（例如）由J March，"以vanced Orgizm.c(1) Prevention of the disease occurs in an animal which may be susceptible to the pathology or symptomology of the disease that has not been experienced or manifested by the disease, (7) inhibition of the disease of the animal undergoing or showing the pathology or symptomology of the disease (ie 'Reducing the further development of pathology and/or symptomology, or (3) improving the disease of the animal (ie, reversing pathology and/or symptomology) of the disease or symptom of the disease. Animals include humans, non-human mammals (e.g., dogs, juveniles, rabbits, cows, horses, brocades, goats, donkeys, deer, and the like) and non-mammals (e.g., birds and the like). Preparation of Compound I, Hydrazine and M and Polymorphs and Salts A•Preparation of Compounds 1, 11 and 1 In order to synthesize the compound ILAIII of the present invention, various methods can be developed. A general approach that cannot be used to synthesize a compound is disclosed in U.S. Patent Application Serial No. 1 1/457,260, filed on Jan. However, it should be noted that compounds I, π and in can also be synthesized by other synthetic routes which others can take. Representative methods for synthesizing compound, 11 and 111 crystalline solids are provided in Examples 3, 28 and 41, respectively. The crystalline solid is referred to herein as Form A of the corresponding compound 128407.doc -26- 200911757. B. Preparation of Polymorphs and Salts of Compounds I, II and III Salts or solvates of the compounds are usually precipitated directly from a solution of a suitable counterion and a solvent. It is known to those skilled in the art of synthetic organic chemistry and pharmaceutical formulation and is, for example, by J March, "vanced Orgizm.c

Chemistry: Reacti〇ns，Mechanisms and Structure” 第 4 版 (New York: Wiley-Interscience，1992)所述之程序。在實例 1中描述可用以鐘別及製備中性化合物之多晶型物及穩定鹽的通用程序。該程序為多層的；對於各層，可取消不利於形成合適結晶固體之條件，且最終製程鑑別有利於形成具有有助於進一步純化或加工之有利特性的游離鹼化合物之鹽或固體的條件。應瞭解視固體沈澱之條件而定，所得固體可含有不同量之呈與非晶形相反之結晶形的固體。另外，所得組合物可含有結晶固體之不同多晶型的不同混合物。分別如藉由實例3、實例28及實例41中所述方法製備之化合物I、II及III可用作製備包括多晶型物、鹽及溶劑合物形式之所有其他結晶固體的起始物質。在實例7-10、20-24及26-27中描述可製備化合物！之纟士曰、曰白固體的特定程序。在實例3 1及3 2中描述可製備化合物π之結晶固體的特定程序。在實例45及46中描述可製備化合物m之結晶固體的特定程序。 128407.doc -27- 200911757 c.表徵化合物之鹽及溶劑合物包括化學及結構組成、繞射特性、轨胜热特性、溶解度及水分吸附及解吸附特性的固體之特定物理特徵订蚁遇用於鑑別此固體之存在。可使用各種分析方法獲得固於交卞口篮之所需物理特徵。此等分析方法包括（但不限於）χ射線粉末繞射 (XRPD)、示差掃描熱量測定（DSC)、熱解重量分析 (TGA)、偏光顯微術、衰減全反射傅裏葉變換紅外光^學 (ATR-FTIR)、溶液質子核磁共振（!H_NMR)、高效液相層析（HPLC)以及水分吸附及解吸附分析（MS/Ds)。實例2提供若干分析方法之詳細實驗條件，藉由該等分析方法可獲得化合物I、II及III之多晶型物及鹽的物理特徵。在實例 4、11-14及27中描述化合物ϊ之結晶固體的表徵。在實例μ 及33-37中描述化合物Π之結晶固體的表徵。在實例42及46 中描述化合物III之鹽的表徵。 D.化合物I之結晶固體Chemistry: Reacti〇ns, Mechanisms and Structure", 4th edition (New York: Wiley-Interscience, 1992). The polymorph and stable salts which can be used to prepare and prepare neutral compounds are described in Example 1. General procedure. The procedure is multi-layered; for each layer, conditions that are not conducive to the formation of a suitable crystalline solid can be eliminated, and the final process identification facilitates the formation of a salt or solid of a free base compound having advantageous properties for further purification or processing. Conditions It will be appreciated that depending on the conditions of solid precipitation, the resulting solid may contain varying amounts of solids in the opposite crystal form to the amorphous form. Additionally, the resulting composition may contain different mixtures of different polymorphs of crystalline solids. Compounds I, II and III as prepared by the methods described in Example 3, Example 28 and Example 41 can be used as starting materials for the preparation of all other crystalline solids in the form of polymorphs, salts and solvates. Specific procedures for the preparation of compounds! The gentleman's and white solids are described in 7-10, 20-24 and 26-27. Examples are described in Examples 3 1 and 3 2 Specific procedures for preparing crystalline solids of compound π. Specific procedures for preparing crystalline solids of compound m are described in Examples 45 and 46. 128407.doc -27- 200911757 c. Characterization of salts and solvates of compounds including chemical and structural compositions Specific physical characteristics of solids, diffraction characteristics, orbital thermal properties, solubility, and moisture adsorption and desorption characteristics are used to identify the presence of this solid. Various analytical methods can be used to obtain the solids required for the basket Physical characteristics. These analytical methods include, but are not limited to, X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), polarized light microscopy, attenuated total reflection Fourier transform Infrared light (ATR-FTIR), solution proton nuclear magnetic resonance (!H_NMR), high performance liquid chromatography (HPLC), and moisture adsorption and desorption analysis (MS/Ds). Example 2 provides detailed experimental conditions for several analytical methods. The physical characteristics of the polymorphs and salts of the compounds I, II and III can be obtained by these analytical methods. The characterization of the crystalline solids of the compound oxime is described in Examples 4, 11-14 and 27. Characterization of the crystalline solids of the compound oxime described in Examples μ and 33-37. Characterization of the salt of Compound III is described in Examples 42 and 46. D. Crystalline solid of Compound I

化合物I，N-(2-胺基苯基）_4·((5,6·二甲氧基_2H_吲唑·2_ 基）甲基）苯曱醯胺，具有下式：Compound I, N-(2-aminophenyl)_4·((5,6.dimethoxy_2H-indazole-2-yl)methyl)phenylamine, having the formula:

將藉由實例3中揭示之程序製備的化合物j之結晶固體指定為形式A。在夕aa型物/鹽篩檢期間起初鑑別化合物j之若干鹽、第二多晶型及非晶形。將第二多晶型指定為形式B，且將非 128407.doc -28- 200911757 晶形稱為形式非晶。由包括 iH NMR、HPLC、MS、XRPD、FTIR(ATR)、水溶性、DSC、TGA及水分吸附分析之分析方法表徵化合物I 之多晶型物及鹽。多晶型物及鹽亦經受熱應力以及漿料實驗以評估其物理形式轉化之傾向。將結晶固體之分析資料概括於表1中。表1.化合物I之多晶型物及鹽的分析結果固體溶劑 XRPD 結果 DSC分析藉由TGA所測之重量損失％ Stoich· (API:酸) 溶解度 (mg/mL) 在90% RH吸附時之水分吸附 % 起始 (°C) 峰值 (°C) 游離驗形式A N/A Xtal 79, 193 89 147(x) 198 8.4(二水合物） N/A <0.1 11.2(二水合物）游離鹼形式B MeCN* Xtal 137 147 0.0(在 140。。後分解） N/A <0.1 1.2% 游離鹼形式B 乙醇2B Xtal 198 211 ND N/A ND ND 反丁烯二酸鹽 MeCN Xtal 93 101 128 2.5 < 120°C (在127。。後分解） 1:1 <0.1 4.2%(通道水合物）雙磷酸鹽丙綱半 Xtal 166 179(x) 256 2.0 <180。。 2:1 - HC1 丙嗣半 Xtal 165 95 185 4.1 <130°C (在160°C後分解） 1:1(二水合物） 0.17 1乂6(二水合物及四水合物） *存在〜1.5莫耳比之丁二酸 Xtal .結晶The crystalline solid of compound j prepared by the procedure disclosed in Example 3 was designated as Form A. Several salts, second polymorphs, and amorphous forms of compound j were initially identified during the eve a-type/salt screening. The second polymorph is designated as Form B, and the non-128407.doc -28-200911757 crystal form is referred to as Form Amorphous. Polymorphs and salts of Compound I are characterized by analytical methods including iH NMR, HPLC, MS, XRPD, FTIR (ATR), water solubility, DSC, TGA, and moisture adsorption analysis. Polymorphs and salts are also subjected to thermal stress and slurry experiments to assess the propensity for their physical form to be converted. The analytical data of the crystalline solids are summarized in Table 1. Table 1. Analysis results of polymorphs and salts of Compound I Solid solvent XRPD results DSC analysis Weight loss by TGA % Stoich· (API: acid) Solubility (mg/mL) At 90% RH adsorption Moisture Adsorption % Start (°C) Peak (°C) Free Form AN/A Xtal 79, 193 89 147(x) 198 8.4 (Dihydrate) N/A <0.1 11.2 (Dihydrate) Free Base Form B MeCN* Xtal 137 147 0.0 (after decomposition at 140%) N/A <0.1 1.2% free base form B ethanol 2B Xtal 198 211 N/A ND ND fumarate MeCN Xtal 93 101 128 2.5 < 120 ° C (after 127 ° decomposition) 1:1 < 0.1 4.2% (channel hydrate) bisphosphonate propyl semi-Xtal 166 179 (x) 256 2.0 < 180. . 2:1 - HC1 propylene quinone Xtal 165 95 185 4.1 <130 ° C (decomposed after 160 ° C) 1:1 (dihydrate) 0.17 1 乂 6 (dihydrate and tetrahydrate) *present ~ 1.5 molar ratio of succinic acid Xtal. Crystallization

1·化合物I游離鹼之形式A 可藉由實例3中所述之合成途徑製備化合物I之游離鹼形式A(化合物I形式A)。藉由XRPD、MS、NMR、 HPLC、FTIR(ATR)、TGA、DSC、水分吸附-解吸附、熱 128407.doc -29- 200911757 應力測試、室溫漿料測試及水溶性研究測定形式A之物理特性（實例4)。化合物I形式A展現以下物理特徵，其可單獨或組合使用以鑑別該形式之存在。化合物I开> 式A為結晶。圖1顯示形式a之特徵XRpD光譜 (CuKct)。以±0_2。20表示主要X射線繞射線且將其相對強度概括於表4中（實例4)。此獨特組之xrPd峰位或其子集可用以鑑別化合物I形式A。一此子集包含在約8 64、19 % 及18.17±0,2 02θ之峰值。另一子集包含在約8 64、19 95、 1 18.17、20.77、23.03、22.32 及 30,15±0.2 02θ 之峰值。又一子集包含在約 8.64、12.33、19.95、18_17、、 23.03、22.32、26.48及 30.15土0.2 °2Θ之峰值。形式Α展現在403所/z之Μ+1峰值。此質量與化合物j之已知化學組成一致。形式A展現圖2中所示之特徵溶液iH NMR光譜。光譜與化合物I之已知化學結構一致。形式A展現具有滯留時間為1 0.4分鐘之主峰（92.4%曲線1. Compound A Form A of the free base The free base form of Compound I (Formula I Form A) can be prepared by the synthetic route described in Example 3. Determination of Form A by XRPD, MS, NMR, HPLC, FTIR (ATR), TGA, DSC, moisture adsorption-desorption, heat 128407.doc -29- 200911757 stress test, room temperature slurry test and water solubility study Characteristics (Example 4). Compound I Form A exhibits the following physical characteristics, which can be used alone or in combination to identify the presence of the form. Compound I is opened > Formula A is a crystal. Figure 1 shows the characteristic XRpD spectrum (CuKct) of Form a. The main X-rays are rayed at ±0_2.20 and their relative intensities are summarized in Table 4 (Example 4). The xrPd peak position of this unique set or a subset thereof can be used to identify Compound I Form A. This subset contains peaks at approximately 8 64, 19 % and 18.17 ± 0, 2 02θ. The other subset contains peaks at about 8 64, 19 95, 1 18.17, 20.77, 23.03, 22.32, and 30, 15 ± 0.2 02θ. A further subset contains peaks at about 8.64, 12.33, 19.95, 18_17, 23.03, 22.32, 26.48, and 30.15. The form Α is shown at Μ 403 / 峰值 peak +1 +1. This mass is consistent with the known chemical composition of compound j. Form A exhibits the characteristic solution iH NMR spectrum shown in Figure 2. The spectrum is consistent with the known chemical structure of Compound I. Form A exhibits a main peak with a residence time of 1 0.4 minutes (92.4% curve)

C 下面積（AUC))及滯留時間為12.3分鐘之次峰（6.0% ACU)的 HPLC層析圖。使用 Varian Pursuit XRs c 18(4.6x1 50 mm， 3 μ)管柱收集層析圖。形式Α展現圖3中所示之特徵ftIR(ATR)光譜圖。光譜與化合物I之結構一致。主帶（以波長倒數表示（以cm·1計之波數））定位於約 3398、3337、3253、3120、2938、2838、 1636、1610、1573、1521、1501、1458、1335、1304、 1267、1210、1 1774、1134、1017、998、943、902、 128407.doc -30- 200911757 844、821、783、745、711及仍Μ(捨入最近整數之值）。此獨特組之IR吸收帶或其子集可用以鑑別形式A。形式A為二水合物。形式A展現圖4中所示之tga熱分析圖。當形式A自6(TC加熱至loot時，TGA分析顯示8 5%之重量損失。此重量損失與水合作用之兩個水的損失一致。形式A展現圖5中所示之DSC迹線。DSC顯示在89。〇與 TG A中重篁損失一致之寬吸熱事件以及在約I%。匚之最終吸熱事件。亦觀察到15〇t附近可能之寬放熱事件。Ο% 分析表明形式A在此等轉變時經歷脫水、再結晶及最終熔融/分解。形式A在低相對濕度下易於形式轉化。水分吸附-解吸附研究（圖6)顯示視相對濕度而定，形式A在二水合物形式與無水形式之間轉化。在吸附起始時，形式A轉化為無水形式。在約60-70% RH下無水形式再轉化為二水合物形式。在解吸附後，觀察到以下滞後現象：直至相對濕度達到約 5% RH ’二水合物形式才再轉化回無水形式。藉由發現殘餘無水形式主要為非晶形。形式A不可溶於水，但當在室溫下暴露於水時易於形式轉化。使形式A之樣品在DI水中在室溫下平衡兩天（實例 15)。藉由HPLC面積百分比檢定未觀察到顯著溶解度（圖 34);然而，藉由XRPD將殘餘固體顯示為非晶形。在至多60°C之溫度在熱應力下形式A為穩定的。使形式 A之樣品在6(rc下經受熱應力2天。暴露之後，殘餘固體未展現形式轉化或降解之跡象（實例16)。 128407.doc -31 - 200911757 形式A可在反溶劑中穩定而避免形式轉化且在非極性反 /谷劑中傾向於形式轉化。在甲苯中及正庚院中在環境溫度下調漿形式A之樣品歷時兩天（實例丨7)。在曱苯中調聚之樣品未顯示形式轉化或降解之跡象。在正庚烧中調毁之樣品未顯示降解，但轉化為非晶形。The area under C (AUC) and the retention time were HPLC chromatograms of the sub-peak of 12.3 minutes (6.0% ACU). Chromatograms were collected using a Varian Pursuit XRs c 18 (4.6 x 1 50 mm, 3 μ) column. Form Α shows the characteristic ftIR (ATR) spectrum shown in Figure 3. The spectrum is consistent with the structure of Compound I. The main band (represented by the reciprocal of the wavelength (wavenumber in cm·1)) is positioned at approximately 3398, 3337, 3253, 3120, 2938, 2838, 1636, 1610, 1573, 1521, 1501, 1458, 1335, 1304, 1267 , 1210, 1 1774, 1134, 1017, 998, 943, 902, 128407.doc -30- 200911757 844, 821, 783, 745, 711 and still Μ (rounded the value of the nearest integer). This unique set of IR absorption bands or a subset thereof can be used to identify Form A. Form A is a dihydrate. Form A shows the tga thermogram shown in Figure 4. TGA analysis showed a weight loss of 85% when Form A was heated from 6 to TC. This weight loss is consistent with the loss of two waters for hydration. Form A exhibits the DSC trace shown in Figure 5. DSC showed a broad endothermic event consistent with heavy losses in TG A and a final endothermic event at about 1%. A wide exothermic event near 15 〇t was also observed. Ο% analysis indicates that Form A is These transformations undergo dehydration, recrystallization, and final melting/decomposition. Form A is readily converted in form at low relative humidity. Moisture adsorption-desorption studies (Figure 6) show that Form A is in the form of dihydrate depending on relative humidity. Conversion between the form and the anhydrous form. At the onset of adsorption, Form A is converted to the anhydrous form. The anhydrous form is reconverted to the dihydrate form at about 60-70% RH. After desorption, the following hysteresis is observed: Until the relative humidity reaches about 5% RH 'dihydrate form is converted back to the anhydrous form. It is found that the residual anhydrous form is mainly amorphous. Form A is insoluble in water, but is easy to form when exposed to water at room temperature. Transformation. Make form A The samples were equilibrated in DI water for two days at room temperature (Example 15). No significant solubility was observed by HPLC area percent determination (Figure 34); however, the residual solids were shown to be amorphous by XRPD. Up to 60 °C The temperature was stable under thermal stress for Form A. The sample of Form A was subjected to thermal stress for 2 days at 6 (rc. After exposure, the residual solids showed no signs of form conversion or degradation (Example 16). 128407.doc - 31 - 200911757 Form A can be stabilized in anti-solvents to avoid form conversion and tends to form conversion in non-polar anti-troreals. Samples of Form A are conditioned at ambient temperature for two days in toluene and in Zheng Gengyuan ( Example 丨 7). The sample telomerized in toluene showed no signs of form conversion or degradation. The sample conditioned in n-g- ing did not show degradation but was converted to amorphous.

2.化合物I游離鹼之形式B 可自化合物I起始物質製備高純度（如由HPLC量測）之化合物I之形式B(化合物I形式B);特定言之，可藉由在乙醇 /0.5%甲苯（乙醇2B)中調漿化合物I起始物質來製備形式 B(實例26-27)。亦可藉由在其他有機溶劑中調漿化合物I游離驗來製備形式B ’該等其他有機溶劑包括（但不限於）乙醇、四氫呋喃、甲醇、EtOH/5% IPA/5% MeOH、EtOH/5% MeOH、EtOH/5% H20及EtOH/5%曱苯，其中用或未用形式B對反應混合物種晶（參見實例22-24)。另外，可藉由與丁二酸自所選溶劑（尤其MeCN)中共沈澱來製備高純度之形式B(實例7-10、18及20-21);且接著藉由在溶劑中調聚來移除丁二酸，該等溶劑包括（但不限於）水、EtOH及曱醇 (參見實例20及21)。藉由1H NMR、HPLC ' FTIR(ATR)、XRPD、水溶性、 TGA、DSC、水分吸附-解吸附、熱應力測試及漿料測試來表徵形式B。在實例11中報導藉由與丁二酸共沈澱而製備之形式B的表徵資料。在實例27中報導自乙醇2B漿料製備之形式B的表徵資料。形式B展現以下物理特徵’其可單獨或組合使用以鑑別 128407.doc -32- 200911757 該形式之存在。口物I开y式B為結晶。圖7顯示形式b之特徵xRpD光譜 ( )以〇’2 20表示主要X射線繞射線且將其相對強度概括於表29中。此獨特組之XRpD峰位或其子集可用以鑑別形式B。一此子集包含在約17 21、2〇 4及16侧2 2Θ之峰值。另—此子集包含在約17.21、20·4及26.3士0.2 〇2θ之峰值。另—子集包含在約Π.21、20.4、16.02、 26.3、11.05及21.92±〇2 〇2θ之峰值。又一子集包含在約 2〇·4 16,02、26.3、11.05、21.92、28.05 及 19.45±0·2 °2Θ之蜂值。化合物〗形式Β展現圖8中所示之特徵溶液1h nmr光譜。光譜與化合物I之已知化學結構一致。化合物ί形式B展現圖9中所示之HPLC層析圖（使用心如汕咖咖⑽^仙心^管柱收集^具有滞留時間為H)·4分鐘之主峰（97.7% AUC)及滯留時間為12 3分鐘2. Form I of Compound I Free Base B Form B of Compound I (Compound I Form B) of high purity (as determined by HPLC) can be prepared from the starting material of Compound I; in particular, by ethanol/0.5 Form B (Examples 26-27) was prepared by adjusting the starting material of Compound I in % toluene (ethanol 2B). Form B can also be prepared by slurrying Compound I in other organic solvents. These other organic solvents include, but are not limited to, ethanol, tetrahydrofuran, methanol, EtOH/5% IPA/5% MeOH, EtOH/5. % MeOH, EtOH / 5% H20 and EtOH / 5% terpene in which the reaction mixture was seeded with or without Form B (see Examples 22-24). Alternatively, high purity Form B (Examples 7-10, 18 and 20-21) can be prepared by coprecipitation with succinic acid from a solvent of choice (especially MeCN); and then moved by telomerization in a solvent. In addition to succinic acid, such solvents include, but are not limited to, water, EtOH, and sterol (see Examples 20 and 21). Form B was characterized by 1H NMR, HPLC 'FTIR (ATR), XRPD, water solubility, TGA, DSC, moisture adsorption-desorption, thermal stress testing, and slurry testing. Characterization data for Form B prepared by coprecipitation with succinic acid are reported in Example 11. Characterization data for Form B prepared from the ethanol 2B slurry was reported in Example 27. Form B exhibits the following physical characteristics 'which may be used alone or in combination to identify the presence of this form 128407.doc -32- 200911757. Oral material I open y formula B is crystal. Figure 7 shows the characteristic xRpD spectrum of Form b ( ) with 〇 '2 20 representing the main X-ray diffraction and summarizing its relative intensity in Table 29. The XRpD peak position of this unique set or a subset thereof can be used to identify Form B. This subset contains peaks at about 21 21, 2 〇 4, and 16 sides. In addition - this subset contains peaks at approximately 17.21, 20·4, and 26.3 ± 0.2 〇 2θ. The other subset contains peaks at about 2121.21, 20.4, 16.02, 26.3, 11.05, and 21.92±〇2 〇2θ. Another subset contains bee values at approximately 2〇·4 16,02, 26.3, 11.05, 21.92, 28.05, and 19.45±0·2 °2Θ. The compound Βform Β shows the 1h nmr spectrum of the characteristic solution shown in FIG. The spectrum is consistent with the known chemical structure of Compound I. Compound ί Form B exhibits the HPLC chromatogram shown in Figure 9 (using a heart such as 汕咖咖 (10) ^ 仙心 ^ pipe column collection ^ with a residence time of H) · 4 minutes of the main peak (97.7% AUC) and residence time For 12 3 minutes

(6.0% ACU)之次峰（2，1% Acu)的層析圖與對於化合物！ API先前觀察到之層析圖一致。化合物I形式B為無水且在約211艺下熔融/分解。形式b 展現圖9中所示之DSC迹線，其僅顯示在約2ιι^之單一吸熱事件（圖10)。應注意形式B之此溶融/分解溫度高於形式 A 〇含有幾乎1:1比率之丁二酸與API的形式B樣品之TGA分析在分解（在14(TC附近起始）以下未顯示重量損失，此與無水固體-致（圖15)。此樣品之水分吸附分析表明該物質為 128407.doc -33- 200911757 非吸濕性（圖16);在解吸附期間未觀察到滯後現象。在8〇 °C下將物質乾燥一小時後，殘餘物之xrPd分析得到與先前所得圖樣一致之圖樣。形式B不可溶於水且當在室溫下暴露於水時穩定而避免形式轉化。使形式B之樣品在DI水中在室溫下平衡兩天（實例15)。藉由HPLC面積百分比檢定未觀察到顯著溶解度（圖 34);且殘餘固體呈與先前所觀察相同之形式。形式B當在60。(：下經受熱應力兩天時大體上穩定（實例 16)。形式B顯示生成已知苯并咪唑雜質之最小降解（小於 1 °/〇)。殘餘固體保留與先前所觀察相同之形式。形式B可能不易於發生反溶劑引發之形式轉化（實例 ”)。在甲苯中及正庚烷中在環境溫度下調漿形◎之樣品歷時兩天。殘餘固體未顯示降解且保留與先前所觀察相同之形式。 3.化合物I之形式非晶可藉由在水中調漿形式A來製備化合物I形式非晶（實例 16)。暴露於高濕度之後由形式A可形成形式非晶（實例 4)亦觀察到形式非晶可由形式A在長久暴露於環境條件 :後形成。圖17顯示已在環境條件下儲存約兩個月的藉由 Λ例3之程序製備之—批化合物1的XRPD®樣。圖樣顯示非晶形物質之特徵暈輪。未進一步表徵形式非晶。 4·化合物I之反丁烯二酸鹽可在反丁烯二酸存在下自MeCN(1〇%水）使化合物【之反丁稀—酸鹽（化合物1反了烯二酸鹽）結晶（實例7、8及9)。 128407.doc -34- 200911757 由 XRPD、FTIR(ATR)、HPLC、(6.0% ACU) secondary peak (2,1% Acu) chromatogram versus compound! The chromatograms previously observed by the API are consistent. Compound I Form B is anhydrous and melts/decomposes at about 211 art. Form b shows the DSC trace shown in Figure 9, which shows only a single endothermic event at about 2 ι^ (Figure 10). It should be noted that the melting/decomposition temperature of Form B is higher than that of Form A. The TGA analysis of Form B sample containing almost a 1:1 ratio of succinic acid and API is not shown below in the decomposition (below 14 (starting near TC)). This is related to the anhydrous solid (Figure 15). The moisture adsorption analysis of this sample showed that the material was 128407.doc -33- 200911757 non-hygroscopic (Figure 16); no hysteresis was observed during desorption. After drying the material for one hour at 〇 ° C, the xrPd analysis of the residue gave a pattern consistent with the previously obtained pattern. Form B was insoluble in water and stable when exposed to water at room temperature to avoid form conversion. The samples were equilibrated in DI water for two days at room temperature (Example 15). No significant solubility was observed by HPLC area percent assay (Figure 34); and the residual solids were in the same form as previously observed. Form B when at 60 (: Substantially stable when subjected to thermal stress for two days (Example 16). Form B shows minimal degradation (less than 1 °/〇) of known benzimidazole impurities. The residual solid retains the same form as previously observed. Form B may not be easy Formal conversion initiated by anti-solvent (examples) occurred. The sample was adjusted to ambient temperature in toluene and n-heptane for two days at ambient temperature. The residual solids showed no degradation and retained the same form as previously observed. Form A of Compound I can be prepared by slurrying Form A in water to prepare Form I of Form Compound (Example 16). Form A can be formed from Form A after exposure to high humidity (Example 4) Form Amorphous is also observed. Form A can be formed after prolonged exposure to environmental conditions: Figure 17 shows an XRPD® sample of Batch 1 prepared by the procedure of Example 3, which has been stored under ambient conditions for about two months. The pattern shows an amorphous material. Characterized halo. Not further characterized as amorphous. 4. Compound I's fumarate can be compounded from MeCN (1% water) in the presence of fumaric acid. (Compound 1 reversed enedionate) crystallized (Examples 7, 8, and 9) 128407.doc -34- 200911757 by XRPD, FTIR (ATR), HPLC,

DSC 、 TGADSC, TGA

MSMS

lU NMR及水分吸附·解吸附分析來表徵實例9中製備之化合物 1反丁稀二酸鹽（實例12)。分析顯示化合物认丁稀二酸鹽展現以下特徵： ( % i 化合物I反丁烯二酸鹽為結晶且展現圖18中所示之特徵 XRPD光譜（CuKa)。以进2。29表示主要χ射線繞射線且將其相對強度概括於表1 5中。此獨特組之XRpD峰位或其子集可用以鑑別化合物I反丁烯二酸鹽。一此子集包含在約 5.39、 13.24及18.19士0.2。20之峰值。另一子集包含在約 5.39、 13.24、21.02、16.28及 18·19±〇.2。20之峰值。又一子集包含在約5.39、13.24、1〇.75、27.35及18.19±〇.2。20 之峰值。其他子集包含在約5.39、13_24及18.19±0.2。20之峰值及任何兩個其他來自剩餘39個峰值之峰值。化合物I反丁稀_一酸鹽展現圖19中所示之特徵溶液〖η NMR光譜。光譜與化合物I之已知化學結構及1:丨比率之反丁烯二酸鹽平衡離子與化合物I一致。化合物I反丁烯二酸鹽展現圖20中所示之特徵FTIR(ATR) 光譜。光譜與化合物I之結構及1:1比率之反丁烯二酸鹽平衡離子與化合物I API —致。以波長倒數（以cm·1計之波數）表示之主帶定位於約3339、283 6、2495、1850、1689、 1641、1569、1527、1500、1445 ' 1368、1338、1313、 1276、1257、1209、1178、1142、1006、969、903、 827、768、75 0及721 cm·1 (捨入最近整數之值）。此獨特組之IR吸收帶或其子集可用以鑑別化合物I反丁烯二酸鹽。 128407.doc -35· 200911757 化合物i反丁烯二酸鹽為水合固體。化合反丁烯二酸鹽展現圖21中所示之獄迹線。政分析顯示在12代之吸熱與可能由於水/溶劑損失而引起之先前額外事件。化合物I反丁烯二酸鹽展現圖22中所示之tga熱分析圖。TGA分析顯示在12(rc以下2_5%之重量損失及在約a? C之分解起始。化合物I反丁烯二酸鹽可為通道水合物。化合物丨反丁烯 f 二酸鹽之水分吸附分析顯示物質自20% RH至90% RH為潛 ? 在單水合物（圖23)。在解吸附期間未觀察到滯後現象，且在80 C下將物質乾燥一小時後，殘餘固體之XRpD分析得到與先前所得圖樣一致之圖樣，表明該物質可為通道水合物。化合物I反丁烯二酸鹽微溶於水且當暴露於水時易於形式轉化（實例15)。使化合物I反丁烯二酸鹽之樣品在〇1水中在室溫下平衡兩小時。藉由HPLC面積百分比檢定發現所 ( 得溶液含有0.03 mg/mL化合物I反丁烯二酸鹽（圖34)。溶解 V 度量測之後，對殘餘固體所得之XRPD光譜表明初始結晶固體變成非晶形。化合物I反丁烯二酸鹽在熱應力下傾向於降解（實例16)。在60°c下使化合物Ϊ反丁烯二酸鹽之樣品經受熱應力2天且藉由XRPD、MS及HPLC來分析殘餘固體。約6%試樣降解為環狀苯并咪唑雜質；未觀察到形式轉化。化合物I反丁烯二酸鹽在極性溶劑中易於形式轉化且在非極性溶劑中穩定（實例17)。在甲苯中及正庚烷中在環境 128407.doc • 36· 200911757 溫度下調漿化合物i反丁烯二酸鹽之樣品歷時兩天，且藉由XRPD、MS及HPLC分析殘餘物。在正庚烷中調漿之= 體未顯示形式轉化或降解。在甲苯中調漿之固體未顯示降解但初始結晶形轉化為不同之結晶形。 5.化合物I之氫氣酸鹽化合物I之氫氣酸鹽（化合物I氫氣酸鹽）可如實例7、8及9 中所述在氫氣酸存在下自丙酮結晶。由XRpD、 FTIR(ATR)、HPLC、DSC、TGA、MS、NMR及水分吸附-解吸附分析來表徵實例9中製備之化合物i氫氣酸鹽（實例 13)。分析結果顯示氫氯酸鹽展現以下物理特徵：化合物I氫氯酸鹽為半結晶且展現圖24中所示之XRpD光譜 (CuKa)。以±0.2 02θ表示主要X射線繞射線且將其相對強度概括於表16中。此獨特組之xrpd峰位或其子集可用以鑑別化合物I氫乳酸鹽。一此子集包含在約1 5.4 §、2 3.2 1 及16.04±0.2 。20之峰值。另一子集包含在約15.48、 23.21、18.82、20.92 及 16.04±0.2 °2Θ 之峰值。又一子华包含在約 15.48、23.21、18.82、20.92、5.90、22.32 及 16.04±0.2 02θ之峰值 ° 化合物I氫氯酸鹽展現圖2 5中所示之溶液1 η NMR光譜。光譜與化合物I之已知化學結構一致。化合物I氫氯酸鹽展現圖26中所示之特徵FTIR(ATR)光譜。光譜與化合物I之已知化學結構一致。以波長倒數（以 cm·1計之波數）表示之主帶定位於約3〇〇〇、2596、1671、 128407.doc -37- 200911757 1642、1614、1526、1494、1441、l361、1336、1314、 1272、1209、1174、1134、1010、998、92〇、9〇〇、862、 828、767、749、717及656 cm、捨入最近整數之值卜此獨特組之IR吸收帶或其子集可用以鑑別化合物j氫氣酸〇化合物I氫氯酸鹽為水合物。化合物^氫氣酸鹽展現圖27 中所示之TGA熱分析圖。化合物I氫氯酸鹽展現圖27中所示之TGA熱分析圖。TGA顯示在13 0 °C以下4.1 %之重量損失接著在> 160°C之分解。化合物I氫氣酸鹽展現圖28中所示之DSC迹線。DSC分析顯示在9 5 °C及18 5 °C之兩個吸熱事件。在13 0 °C以下之熱事件與脫水一致。水分吸附分析顯示化合物I氫氣酸鹽在吸附掃描期間為二水合物且在90% RH轉化為四水合物（圖29)。在解吸附後’至25。/。RH觀察到四水合物之滯後現象，表明四水合物形式為亞穩的。殘餘固體在8(TC乾燥一小時後顯示為非晶形，表明化合物I氫氣酸鹽並不易於轉化回穩定水合物形式。化合物I氫氣酸鹽微溶於水且暴露於水之後轉化成非晶形（參見實例1 5)。使化合物I氫氣酸鹽之樣品在DI水中在室溫下平衡兩小時。藉由HPLC面積百分比檢定來分析溶液且發現其含有0.17 mg/mL化合物I氫氣酸鹽（圖34)。對殘餘固體所得之XRPD光譜顯示初始結晶固體變成非晶形。化合物I氫氯酸鹽在高溫下易於降解（實例1 6)。在60°C下 128407.doc • 38· 200911757 使化合物i氫氣酸鹽之樣品經受熱應力兩天。殘餘固體顯示存在苯并味。坐降解產物；固體保留與先前所觀察相同之形式。化合物I氫氯酸鹽可能不易於發生溶劑引發之形式轉化 (實例17)。在甲苯中及正庚烷中在環境溫度下調漿化合物工氫氣酸鹽之樣品歷時兩天。殘餘固體未顯示存在降解產物且未顯示形式轉化。The lU NMR and moisture adsorption/desorption analysis were used to characterize the compound 1 transbutyrate produced in Example 9 (Example 12). Analysis shows that the compound succinic acid salt exhibits the following characteristics: (% i Compound I fumarate is crystalline and exhibits the characteristic XRPD spectrum (CuKa) shown in Figure 18. The main ray is represented by 2.29 The ray is circulated and its relative intensities are summarized in Table 15. The XRpD peak position of this unique set or a subset thereof can be used to identify the compound I fumarate. This subset is contained at about 5.39, 13.24 and 18.19 士. The peak of 0.220. The other subset contains peaks at about 5.39, 13.24, 21.02, 16.28, and 18.19±〇.2.20. The other subset is included in about 5.39, 13.24, 1.〇, 75, 27.35. And the peak of 18.19±〇.2.20. The other subsets contain peaks at about 5.39, 13_24, and 18.19±0.220, and any two other peaks from the remaining 39 peaks. Compound I anti-butylate The characteristic solution shown in Fig. 19 is shown as [η NMR spectrum. The spectrum is in agreement with the known chemical structure of compound I and the 1:2 ratio of the fumarate counterion to compound I. Compound I fumarate The characteristic FTIR (ATR) spectrum shown in Figure 20 is exhibited. The structure and 1:1 ratio of the spectrum to the compound I The fumarate counterion is the same as the compound I API. The main band represented by the reciprocal of the wavelength (the wave number in cm·1) is positioned at about 3339, 283 6, 2495, 1850, 1689, 1641, 1569. , 1527, 1500, 1445 '1368, 1338, 1313, 1276, 1257, 1209, 1178, 1142, 1006, 969, 903, 827, 768, 75 0 and 721 cm·1 (rounded to the nearest integer value). A unique set of IR absorption bands or a subset thereof can be used to identify compound I fumarate. 128407.doc -35· 200911757 Compound i fumarate is a hydrated solid. The prison line shown in Figure 21. Political analysis shows the endothermic heat in 12 generations and previous additional events that may be caused by water/solvent loss. Compound I fumarate exhibits the tga thermogram shown in Figure 22. TGA analysis showed a weight loss of 2_5% below 12 rc and a decomposition start at about a? C. Compound I fumarate can be a channel hydrate. The moisture of the compound 丨 antibutene f diacid salt Adsorption analysis showed that the material was latent from 20% RH to 90% RH in the monohydrate (Figure 23). During desorption Hysteresis was observed and after drying the material for one hour at 80 C, the XRpD analysis of the residual solid gave a pattern consistent with the previously obtained pattern, indicating that the material could be a channel hydrate. Compound I fumarate sparingly soluble It is easy to formally convert to water and when exposed to water (Example 15). A sample of Compound I fumarate was equilibrated in 〇1 water for two hours at room temperature. It was found by HPLC area percent determination (the solution contained 0.03 mg/mL of compound I fumarate (Fig. 34). After dissolution V measurement, the XRPD spectrum obtained for the residual solids showed that the initial crystalline solid became amorphous. Compound I fumarate tends to degrade under thermal stress (Example 16). The sample of the compound bismuth metamarate was subjected to thermal stress at 60 ° C for 2 days and by XRPD, MS and HPLC. The residual solids were analyzed. About 6% of the sample was degraded to cyclic benzimidazole impurities; no formal conversion was observed. Compound I fumarate was easily converted in a polar solvent and stabilized in a non-polar solvent (Example 17) The sample of the compound i fumarate was slurried in toluene and n-heptane at a temperature of 128407.doc • 36·200911757 for two days, and the residue was analyzed by XRPD, MS and HPLC. In the heptane, the slurry was not converted or degraded. The solids adjusted in toluene showed no degradation but the initial crystal form was converted into a different crystal form. 5. Hydrogenate salt of the compound I hydrogenate (Compound I hydrogen The salt can be crystallized from acetone in the presence of hydrogen acid as described in Examples 7, 8 and 9. Example 9 is characterized by XRpD, FTIR (ATR), HPLC, DSC, TGA, MS, NMR and moisture adsorption-desorption analysis. The compound i-hydrogen acid salt prepared in Example (Example 13). The analysis results show that the hydrochloride exhibits the following physical characteristics: Compound I hydrochloride is semi-crystalline and exhibits the XRpD spectrum (CuKa) shown in Figure 24. 0.2 02θ represents the main X-rays around the ray and its relative intensities are summarized in Table 16. The xrpd peak position of this unique set or a subset thereof can be used to identify the compound I hydrogen lactate. This subset is contained in about 1 5.4 §, 2 3.2 1 and 16.04 ± 0.2 The peak of 20. The other subset contains peaks at about 15.48, 23.21, 18.82, 20.92 and 16.04 ± 0.2 ° 2 。. Another sub-Hua is included in about 15.48, 23.21, 18.82, 20.92, Peaks of 5.90, 22.32 and 16.04 ± 0.2 02θ ° Compound I hydrochloride exhibits the solution 1 η NMR spectrum shown in Figure 25. The spectrum is consistent with the known chemical structure of Compound I. Characteristic FTIR (ATR) spectra shown in Figure 26. Spectra and Compounds The known chemical structure of I is consistent. The main band represented by the reciprocal of the wavelength (the wave number in cm·1) is located at about 3〇〇〇, 2596, 1671, 128407.doc -37- 200911757 1642, 1614, 1526, 1494, 1441, l361, 1336, 1314, 1272, 1209, 1174, 1134, 1010, 998, 92〇, 9〇〇, 862, 828, 767, 749, 717 and 656 cm, rounding the value of the nearest integer A unique set of IR absorption bands or a subset thereof can be used to identify compound j hydroquinone compound I hydrochloride as a hydrate. The compound ^hydrogenate exhibits the TGA thermogram shown in Figure 27. Compound I hydrochloride shows the TGA thermogram shown in Figure 27. TGA showed a 4.1% weight loss below 130 °C followed by a decomposition at > 160 °C. Compound I Hydrochloride shows the DSC trace shown in Figure 28. DSC analysis showed two endothermic events at 9 5 °C and 18 5 °C. Thermal events below 130 °C are consistent with dehydration. Moisture adsorption analysis showed that the compound I hydrogenate was dihydrate during the adsorption scan and converted to tetrahydrate at 90% RH (Figure 29). After desorption, 'to 25. /. The hysteresis of the tetrahydrate was observed by RH, indicating that the tetrahydrate form is metastable. The residual solid was shown to be amorphous after 8 hours of TC drying, indicating that the compound I hydrogenate was not readily converted back to the stable hydrate form. The compound I hydrogenate was slightly soluble in water and converted to amorphous after exposure to water ( See Example 1 5). A sample of Compound I Hydrochloride was equilibrated in DI water for two hours at room temperature. The solution was analyzed by HPLC area percent assay and found to contain 0.17 mg/mL Compound I Hydrochloride (Figure 34). The XRPD spectrum obtained from the residual solid showed that the initial crystalline solid became amorphous. Compound I hydrochloride was easily degraded at high temperature (Example 16). At 60 ° C, 128407.doc • 38· 200911757 The sample of the acid salt was subjected to thermal stress for two days. The residual solid showed the presence of a benzoic acid. The degradation product was taken; the solid remained in the same form as previously observed. Compound I hydrochloride may not be susceptible to solvent-induced conversion (Example 17) The sample of the compound hydrocollide was adjusted in toluene and n-heptane at ambient temperature for two days. The residual solid did not show the presence of degradation products and did not show shape. Transformation.

6.化合物I之磷酸II 化合物I之磷酸鹽（化合物Ϊ磷酸鹽）可如實例7及8中所述在磷酸存在下自丙酮（10%水）沈澱。由1H NMR、元素分析、MS、TG A及DSC進一步表徵在實例8中製備之物質（實例 14)。化合物I磷酸鹽為半結晶且展現圖30中所示之XRpD光譜 (CuKa)(參見實例14)。以士〇_2。20表示主要父射線繞射線且將其相對強度概括於表17中。此獨特組之Xrpd峰位或其子集可用以鑑別化合物I磷酸鹽。—此子集包含在約 17.80、25.80及21.88±0.2。29之峰值。另一子集包含在約 17.8〇、25.8〇'9.12、5.36及21.88±〇.2。20之峰值。化合物I填酸鹽展現圖31中所示之特徵溶液lH NMR光譜。光錯與化合物I之已知化學結構一致。化合物I磷酸鹽為雙鹽。元素組成分析與2:1比率之磷酸鹽平衡離子與化合物I API—致（表18)。 DSC資料顯示在179°C之放熱事件接著在256〇c之吸熱事件（圖32)且TGA資料顯示在i8(TC以下2〇%之重量損失（圖 128407.doc -39- 200911757 33)。 E.化合物II之結晶固鱧化合物II，4-((6-乙醢胺基-2H-吲唑-2-基）曱基）-N-(2-胺基苯基）苯甲醯胺，具有下式：6. Phosphate II of Compound I The phosphate of Compound I (compound ruthenium phosphate) can be precipitated from acetone (10% water) in the presence of phosphoric acid as described in Examples 7 and 8. The material prepared in Example 8 (Example 14) was further characterized by 1H NMR, elemental analysis, MS, TG A and DSC. The compound I phosphate was semi-crystalline and exhibited the XRpD spectrum (CuKa) shown in Figure 30 (see Example 14). The girth _2.20 indicates that the main parent ray is around the ray and its relative intensity is summarized in Table 17. The Xrpd peak position of this unique set or a subset thereof can be used to identify Compound I phosphate. - This subset contains peaks at approximately 17.80, 25.80 and 21.88 ± 0.29. Another subset contains peaks at approximately 17.8 〇, 25.8 〇 '9.12, 5.36, and 21.88 ± 〇.2. The compound I acid salt exhibits the characteristic solution lH NMR spectrum shown in Fig. 31. The optical error is consistent with the known chemical structure of Compound I. The compound I phosphate is a double salt. Elemental composition analysis and a 2:1 ratio of phosphate counterion to Compound I API (Table 18). DSC data showed an exothermic event at 179 °C followed by an endothermic event at 256 °C (Figure 32) and TGA data showed a weight loss of 2〇% below i8 (Figure 128407.doc -39- 200911757 33). . Crystalline solid compound II of compound II, 4-((6-acetamido-2H-indazol-2-yl)indolyl)-N-(2-aminophenyl)benzamide, The following formula:

之游離鹼為結晶且將其指定為形式A。另外，經由使用多種合適平衡離子在若干溶劑中之一系列結晶來鑑別化合物 II之五種結晶鹽。化合物II之五種結晶鹽為：苯甲酸鹽、龍膽酸鹽、氫氯酸鹽、丁二酸鹽及氫溴酸鹽。由1Η NMR、DSC、TGA、FTIR(ATR)、HPLC、水溶性及水分吸附-解吸附分析來表徵結晶固體。固體亦經受熱應力以及漿料實驗以評估其物理形式轉化之傾向。將結晶固體之分析資料概括於表2中。表2.化合物II之結晶固體的分析資料平衡離子溶劑 XRPD 結果 DSC TGA重量損失％ Stoich. (API: 酸）溶解度 (mg/mL) 在90% RH吸附時之水分吸附 % 起始 (°C) 峰值 (°C) 游離驗形式 A N/A xtal 172, 233 77 1.8 > <90°C N/A 7.0 5.3(單水合物） 179 184(x) 241 HC1 ACN 半 xtal 174, 275 197 0.0，低於 1:1 17 3.8 207(x) 128407.doc -40- 200911757 298 160°C HBr ACN 半 xtal 202, 214 0.0，低於 1:1 4.7 5.5 288 224(x) 160°C 296 苯甲 THF xtal 101, 109 9.7，低於 1:1 3.6 4.1(單水合物）酸鹽 276 116(x) 170°C 201⑻ 216 283 龍膽 THF 半 xtal 114 127 4.9(部分由 1:1 N/A 5.3(單水合物）酸鹽 160 於分解） 240 丁二酸鹽 ACN 半 xtal 140 152 212(x) 282 0.0(在 146°C 以上分解） 1:1 5.8 1.9 xtal :結晶The free base is crystalline and designated as Form A. In addition, the five crystalline salts of Compound II are identified by crystallization from one of several solvents using a plurality of suitable counterions. The five crystalline salts of Compound II are: benzoate, gentisate, hydrochloride, succinate and hydrobromide. Crystalline solids were characterized by 1 NMR, DSC, TGA, FTIR (ATR), HPLC, water solubility and moisture adsorption-desorption analysis. Solids are also subjected to thermal stress and slurry experiments to assess the propensity for their physical form to be converted. The analytical data of the crystalline solids are summarized in Table 2. Table 2. Analytical data for crystalline solids of compound II. Equilibrium ionic solvent XRPD results DSC TGA weight loss % Stoich. (API: acid) Solubility (mg/mL) Moisture adsorption % at 90% RH adsorption Start (°C) Peak (°C) free form AN/A xtal 172, 233 77 1.8 ><90°CN/A 7.0 5.3 (monohydrate) 179 184(x) 241 HC1 ACN half xtal 174, 275 197 0.0, low 1:1 17 3.8 207(x) 128407.doc -40- 200911757 298 160°C HBr ACN Semi-xtal 202, 214 0.0, less than 1:1 4.7 5.5 288 224(x) 160°C 296 Benzene THF xtal 101, 109 9.7, less than 1:1 3.6 4.1 (monohydrate) acid salt 276 116 (x) 170 ° C 201 (8) 216 283 gentian THF half xtal 114 127 4.9 (partially by 1:1 N/A 5.3 (single Hydrate) Acidate 160 Decomposed) 240 Succinate ACN Semi-xtal 140 152 212(x) 282 0.0 (decomposed above 146 ° C) 1:1 5.8 1.9 xtal : Crystallization

1.化合物II之游離鹼的形式A 可藉由如實例28中所揭示之程序來製備化合物II之游離鹼的形式A(化合物II形式A)。由XRPD、FTIR(ATR)、 HPLC、DSC、TGA、MS、NMR及水分吸附來表徵化合物（實例29)。分析顯示形式A展現以下特徵：化合物II形式A為結晶且展現圖3 5中所示之XRPD光譜 (CuKa)。以士0.2 02θ表示主要X射線繞射線且將其相對強度概括於表30中。此獨特組之XRPD峰位或其子集可用以鑑別形式A。一此子集包含在約20.92、17.54及20.36 °2Θ 之峰值。另一子集包含在約20.92、17.54、13.93、27.26及 20.36 。20之峰值。另一子集包含在約20.92、17.54、 24.97、22.72及20.3 6 °2Θ之峰值。又一子集包含在20.92、 17.54、13.93、14.54、16.23、22.20、22.72、24.97、 27.26及20.36之峰值。化合物II形式Α展現圖36中所示之1H NMR光譜。光譜與 128407.doc -41 - 200911757 化合物II游離驗之已知結構一致。化合物II形式A展現圖37中所示之FTIR(ATR)光譜。光譜與化合物II之已知化學結構一致。以波長倒數（以crn-1計之波數）表示之主要IR帶定位於約3620、3438 ' 3347、 3235、3116、3046、2836、1926、1903、1662、1635、 1610、1569、1525、1496、1480、1457、1420、1409、 1367、1306、1293、1 1269、1227、1187、1143、1122、 1035、1016、973、961、951、945、907 ' 864、818、 775、751、724、684及656 cm·1 (捨入最近整數之值）。此獨特組之IR吸收帶或其子集可用以鑑別化合物π之形式 Α 〇化合物II形式A展現圖38中所示之DSC迹線。DSC分析顯示在77°C及179°C之吸熱接著在184。(：之放熱。在241。(：觀察到最終事件且其表明在實驗期間形成之環狀雜質或其他降解產物。化合物II形式A展現圖39中之TGA熱分析圖。TGA在1 00 C以下顯示約2 %之重量損失’其可能歸因於殘餘溶劑或表面結合水。化合物II形式A為非吸濕性且自5% RH至90% RH為穩定單水合物。在圖40中顯示化合物II形式a之水分吸附_解吸附迹線。在解吸附時未觀察到滯後現象，且殘餘固體得到與先前所得圖樣一致之圖樣。此表明在環境實驗室條件下試樣可迅速轉變回單水合物。化合物II形式A可溶於水（實例38)。使化合物丨〗形式八之 128407.doc -42- 200911757 樣在DI水中在室溫下平衡約16小日夺。暴露之後，藉由肌C面積百分比檢定量㈣水含有7Q mg/mL。殘餘固體之結晶形保持不變且與起始物質一致。等於或低於6〇°C時化合物Η开以A為熱穩定的（實例39)。在’C下使化合物„形式A之樣品經受熱應力歷時兩天。殘餘固體未顯示降解或形式轉化之跡象。化合物Π形式A可能不傾向於發生溶劑引發之形式轉化 (實例40)在甲苯中及正庚燒中在環境溫度下調漿化合物 II形式A之樣品歷時兩天。殘餘固體未顯示降解或形式轉化之跡象。 2·化合物II之丁二酸鹽化合物II之丁二酸鹽（化合物„丁二酸鹽）可在如實例31及 32中所述之丁二酸存在下自乙腈結晶。由xRpD、 FTIR(ATR)、HPLC、DSC、TGA、Ms、lH 觀尺及水分吸附-解吸附分析來表徵由實例32製備之固體物質（實例Μ)。分析顯示化合物II 丁二酸鹽展現以下特徵：化合物II 丁二酸鹽為結晶且展現圖4丨中所示之XRPD光譜（CuKa)。以±0.2。20表示主要X射線繞射線且將其相對強度概括於表36中。此獨特組之XRpD峰位或其子集可用以鑑別化合物π 丁二酸鹽。一此子集包含在約2217、 27.Π及20.94±0·2。20之峰值。另一子集包含在約16〇9、 32.40、22.17、27.13及20.94±0.2。29之峰值。又一子集包含在約 11.37、13.83、18.88、16.09、32.40、22.17、27.13 及 20.94 土 0.2 °2Θ 之峰值。 128407.doc -43 - 200911757 化合物II丁二酸鹽展現圖42中所示之溶液NMR光譜。光譜與化合物I之已知化學結構及1:1比率之丁二酸平衡離子與化合物I 一致。化合物II 丁二酸鹽展現圖43中所示之FTIR(ATR)光譜。光譜與化合物I之已知化學結構及1:1比率之丁二酸平衡離子與化合物I一致。以波長倒數（以計之波數）表示之主要 FTIR 帶定位於約 3438、3347、3229、31 19、1727、 1662、1635、1611、1567、1544、1496、1457、1413、 1352、1327、1305、1266、1227、1187 ' 1140、1122、 1034 、 1017 、 944.25 、 907 、 862 、 818 、 798 、 776 、 751 、1. Form A of the free base of Compound II Form A (Compound II Form A) of the free base of Compound II can be prepared by the procedure as disclosed in Example 28. The compound was characterized by XRPD, FTIR (ATR), HPLC, DSC, TGA, MS, NMR and moisture adsorption (Example 29). Analysis shows that Form A exhibits the following characteristics: Compound II Form A is crystalline and exhibits the XRPD spectrum (CuKa) shown in Figure 35. The main X-rays are represented by ± 0.2 02θ and their relative intensities are summarized in Table 30. The XRPD peak position of this unique group or a subset thereof can be used to identify Form A. This subset contains peaks at approximately 20.92, 17.54, and 20.36 °2Θ. Another subset is included in approximately 20.92, 17.54, 13.93, 27.26, and 20.36. The peak of 20. Another subset contains peaks at about 20.92, 17.54, 24.97, 22.72, and 20.3 6 °2. A further subset includes peaks at 20.92, 17.54, 13.93, 14.54, 16.23, 22.20, 22.72, 24.97, 27.26 and 20.36. The compound II form oxime exhibits the 1H NMR spectrum shown in Figure 36. The spectra are consistent with the known structure of the compound II free test of 128407.doc -41 - 200911757. Compound II Form A exhibits the FTIR (ATR) spectrum shown in Figure 37. The spectrum is consistent with the known chemical structure of Compound II. The main IR bands, represented by the reciprocal of the wavelength (the number of waves in crn-1), are located at approximately 3620, 3438 ' 3347, 3235, 3116, 3046, 2836, 1926, 1903, 1662, 1635, 1610, 1569, 1525, 1496. , 1480, 1457, 1420, 1409, 1367, 1306, 1293, 1 1269, 1227, 1187, 1143, 1122, 1035, 1016, 973, 961, 951, 945, 907 '864, 818, 775, 751, 724, 684 and 656 cm·1 (rounded to the nearest integer value). This unique set of IR absorption bands or a subset thereof can be used to identify the form of the compound π Α Compound II Form A exhibits the DSC trace shown in Figure 38. DSC analysis showed an endotherm at 77 ° C and 179 ° C followed by 184. (: exotherm. At 241. (: The final event was observed and it indicates a ring-shaped impurity or other degradation product formed during the experiment. Compound II Form A exhibits the TGA thermogram in Figure 39. TGA is below 100 C Showing about 2% weight loss 'which may be due to residual solvent or surface bound water. Compound II Form A is non-hygroscopic and is stable from 5% RH to 90% RH. Compounds are shown in Figure 40. The moisture adsorption_desorption trace of Form II a. No hysteresis was observed at the time of desorption, and the residual solid obtained a pattern consistent with the previously obtained pattern. This indicates that the sample can be rapidly converted back to monohydrate under environmental laboratory conditions. Compound II Form A is soluble in water (Example 38). The compound 丨〗〖Formula 128407.doc -42- 200911757 The sample is equilibrated in DI water at room temperature for about 16 days. After exposure, by muscle Percentage of area C. (IV) Water contains 7Q mg/mL. The crystal form of the residual solid remains unchanged and consistent with the starting material. Compound is cleaved at or below 6 °C and is thermally stable (Example 39) Make a compound under 'C The sample of A was subjected to thermal stress for two days. The residual solids showed no signs of degradation or form conversion. The compound Π Form A may not be prone to solvent-induced form conversion (Example 40) in toluene and n-heptane at ambient temperature The sample of the slurry compound II Form A was down-regulated for two days. The residual solid showed no signs of degradation or form conversion. 2. The succinate salt of the compound II succinate compound II (the compound succinate) can be as Crystallization from acetonitrile in the presence of the succinic acid described in Examples 31 and 32. Characterization of Example 32 by xRpD, FTIR (ATR), HPLC, DSC, TGA, Ms, lH and moisture adsorption-desorption analysis. Solid material (Example Μ). Analysis showed that the compound II succinate exhibited the following characteristics: Compound II succinate was crystalline and exhibited the XRPD spectrum (CuKa) shown in Figure 4 。. The main X was represented by ± 0.220. The rays are rayed and their relative intensities are summarized in Table 36. The XRpD peak position of this unique set or a subset thereof can be used to identify the compound π succinate. This subset is contained at about 2217, 27. Π and 20.94 ± Peak of 0·2.20 The other subset contains peaks at about 16〇9, 32.40, 22.17, 27.13, and 20.94±0.229. The other subset contains about 11.37, 13.83, 18.88, 16.09, 32.40, 22.17, 27.13, and 20.94. The peak of 2Θ. 128407.doc -43 - 200911757 The compound II succinate exhibits the solution NMR spectrum shown in Figure 42. The known chemical structure of the spectrum and the compound I and the 1:1 ratio of succinic acid equilibrium ions and compounds I is consistent. Compound II succinate showed the FTIR (ATR) spectrum shown in Figure 43. The known chemical structure of the spectrum and the compound I and the 1:1 ratio of the succinic acid equilibrium ion are consistent with the compound I. The main FTIR bands, expressed in terms of wavelength reciprocal (in terms of wavenumbers), are located at approximately 3438, 3347, 3229, 31 19, 1727, 1662, 1635, 1611, 1567, 1544, 1496, 1457, 1413, 1352, 1327, 1305. , 1266, 1227, 1187 '1140, 1122, 1034, 1017, 944.25, 907, 862, 818, 798, 776, 751,

723、687及654 cm·1 (捨入最近整數之值）。此獨特組之IR 吸收帶或其子集可用以鑑別化合物π之丁二酸鹽。化合物π丁二酸鹽展現圖44中所示之DSC迹線。該迹線顯示在約15(TC之吸熱接著在約〗；^^之放熱。在282。〇觀察到最終事件，其表明在實驗期間形成之環狀雜質或其他降解產物。化合物II 丁二酸鹽展現圖45中所示之TGA熱分析圖。 TGA顯不在15〇。(：以下無重量損失且分解起始係在154它。化合物II丁二酸鹽為非吸濕性且自5%至9〇%相對濕度穩定。圖46顯示化合物„ 丁二酸鹽之水分吸附_解吸附迹線。在解吸附時未觀察到滯後現象；水分吸附後之殘餘固體得到與先前所得XRPD圖樣—致之圖樣，其表明在環境實驗室條件下固體可迅速轉變回無水形式。化合物II丁二酸鹽可溶於水（實例38)。使化合物Η丁二酸 128407.doc -44- 200911757 鹽之樣品在DI水中在室溫下平衡約丨6小時。暴露之後，藉由HPLC面積百分比檢定發現水含有5.8 mg/mL化合物II丁二酸鹽。殘餘固體之結晶形保持不變。化合物II 丁一酸鹽在熱應力下不易於形式轉化，但可能易於降解（實例39)。在60°C烘箱中使化合物II 丁二酸鹽之樣品經受熱應力歷時三天。藉由MS在殘餘固體中未偵測到降解產物。然而，HPLC分析表明純度降低約3%。化合物II 丁二酸鹽可能不易於發生固體形式之有機溶劑引發互變（實例40)。在曱苯中及正庚烷中在環境溫度下調漿化合物II 丁一酸鹽之樣品歷時三天。兩個漿料之殘餘固體均未顯示形式轉化或降解之跡象。 3.化合物II之苯甲酸鹽可藉由實例31及32中揭示之程序製備化合物η之苯甲酸鹽（化合物II苯曱酸鹽）。由XRPD、FTIR(ATR)、HPLC、 DSC、TGA、MS、4 NMR及水分吸附-解吸附分析來表徵實例3 2中製備之固體物質。分析顯示化合物π苯甲酸鹽展現以下特徵：化合物II苯曱酸鹽為結晶且展現圖47中所示之xrpd圖樣（CuKa)。以±0·2。20表示主要X射線繞射線且將其相對強度概括於表37中。此獨特組之XRPD峰位或其子集可用以鑑別笨甲酸鹽。—此子集包含在約17·43、25.90及 23·93±0.2 °2Θ之峰值。另一子集包含在約12.88、16.33、 Π.43 ' 2 5.90及23.93±0.2 02θ之峰值。又一子集包含在約 8.20、10.60、12.88、16.33、17.43、25.90、26.61 及 128407.doc -45- 200911757 23·93±〇·2。20之峰值。又一子集包含在約8.2〇、10.60、 12.88、16.33、17.43、22.46、25.90、9.07及 23.93±0.2 02θ 之峰值。化合物II苯曱酸鹽展現圖48中所示之1H NMR光譜。光譜與化合物II之已知化學結構及1:1比率之笨曱酸鹽平衡離子與化合物II 一致。化合物II苯曱酸鹽展現圖49中所示之FTIR(ATR)光譜。光譜與化合物II之已知化學結構及1:丨比率之苯曱酸鹽平衡離子與化合物II一致。以波長倒數（以cm·1計之波數）表示之主要 FTIR 帶定位於約 3400、3264、3114、3045、2836、 2560、1650、1635 ' 1583、1527、1484、1453、1421' 1386、1278、1172、1153、1131、1071、1021、1〇〇5、 926、911、853、843、809、751、706 及 683 cm·、捨入最近整數之值）。此獨特組之IR吸收帶或其子集可用以鑑別化合物II之苯甲酸鹽。化合物II苯曱酸鹽展現圖50中所示之DSC迹線e DSC分析顯示在約100°C之吸熱接著在！ 16〇c之放熱。另一在2〇〇 C之放熱接著在2 1 6°C之吸熱。在283。(：觀察到最終事件且可表明在實驗期間形成之環狀雜質或其他降解產物。化合物π苯甲酸鹽展現圖51中所示之TGA熱分析圖。當自60°C加熱至170T：時，TGA顯示總計9.6重量％之連續重量損失，其可能歸因於殘餘溶劑及/或降解。在i 8〇t：附近分解起始。自5% RH至90% RH，化合物π苯甲酸鹽為非吸濕性穩定 128407.doc -46- 200911757 單水合物。在圖52中顯示化合物π苯曱酸鹽之水分吸附-解吸附迹線。在解吸附時未觀察到滯後現象：水分吸附後之樣品的XRPD分析得到與起始物f —致之圖樣，其表明在環境實驗室條件下化合物Π笨甲酸鹽可迅速轉變回單水合物形式。化合物II苯曱酸鹽可溶於水（實例38)。使化合物^苯甲酸鹽之樣品在DI水中在室溫下平衡約16小時。暴露之後，發現水含有3.6 mg/mL化合物„苯甲酸鹽。殘餘固體之結晶形轉化為與化合物II游離鹼之形式A__致之結晶形。化合物II苯甲酸鹽在高溫下傾向於降解（實例39卜在6〇 C下使化合物II苯甲酸鹽之樣品經受熱應力歷時兩天；殘餘固體顯示存在環狀苯并咪唑降解雜質。未發生形式轉化。化合物II苯甲酸鹽未顯示有機溶劑引發之固體形式互變之傾向’但可傾向於在漿料下降解（實例40)。在甲苯及正庚院中在環境溫度下調漿化合物π苯甲酸鹽之樣品歷時兩天。調漿後殘餘固體中之任一者中未觀察到形式轉化。在殘餘固體中未發現常見降解雜質；然而，HPLC分析顯示在兩個殘餘固體中純度均降低約3%(hplc面積百分比檢定）。 4·化合物II之氫氣酸鹽可藉由實例31及32中揭示之程序製備化合物π之氫氣酸鹽（化合物II氫氯酸鹽）。由XrPD、FTIR(ATR)、HPLC、 DSC、TGA、MS、A NMR、水溶性及水分吸附來表徵實 128407.doc •47- 200911757 例32所製備之化合物。分析顯示化合物π氫氯酸鹽展現以下特徵’其可單獨或組合用以鑑別化合物π氫氯酸鹽之存在。化合物II氫氯酸鹽為結晶且展現圖53中所示之xrPD圖樣（CuKa)。以士0.2 °2Θ表示主要X射線繞射線且將其相對強度概括於表38中。此獨特組之xrpd峰位或其子集可用以鑑別苯甲酸鹽。一此子集包含在約19 83、23 〇9及 21_89 士 0.2。20之峰值。另一子集包含在約912、1716、 20.75、19.83、23.09 及 21·89±〇·2。20 之峰值。化合物II氫氯酸鹽展現圖54中所示之1η NMR。光譜與化合物11之已知結構一致。化合物II氫氣酸鹽展現圖55中所示之FTIR(ATR)光譜。光譜與化合物II之已知結構一致。以波長倒數（以cm-丨計之波數）表示之主要FTIR帶定位於約3〇86、3〇26、2969、723, 687, and 654 cm·1 (rounded to the nearest integer value). This unique set of IR absorption bands or a subset thereof can be used to identify the compound π succinate. The compound π succinate exhibits the DSC trace shown in Figure 44. The trace is shown to be at about 15 (the endothermic of TC followed by an exotherm; ^^. At 282. The final event is observed, indicating cyclic impurities or other degradation products formed during the experiment. Compound II Succinic acid The salt exhibits the TGA thermogram shown in Figure 45. The TGA is not at 15 Å. (There is no weight loss below and the decomposition is initiated at 154. Compound II succinate is non-hygroscopic and from 5% to 9〇% relative humidity is stable. Figure 46 shows the moisture adsorption_desorption trace of the compound „succinate. No hysteresis was observed during desorption; the residual solid after moisture adsorption was obtained from the previously obtained XRPD pattern. a pattern indicating that the solid can be rapidly converted back to the anhydrous form under environmental laboratory conditions. The compound II succinate is soluble in water (Example 38). The compound succinic acid 128407.doc -44- 200911757 salt sample is The DI water was equilibrated at room temperature for about 6 hours. After exposure, the water was found to contain 5.8 mg/mL of compound II succinate by HPLC area percent determination. The crystal form of the residual solid remained unchanged. Under thermal stress It is easy to form, but may be susceptible to degradation (Example 39). A sample of Compound II succinate was subjected to thermal stress for three days in an oven at 60 C. No degradation products were detected by MS in the residual solids. HPLC analysis showed a reduction in purity of about 3%. Compound II succinate may not be prone to organic solvent initiated interconversion in solid form (Example 40). Compound II is tempered at ambient temperature in toluene and n-heptane. The sample of the monoacid salt lasted for three days. The residual solids of the two slurries showed no signs of formal conversion or degradation. 3. The benzoate salt of Compound II can be prepared by the procedure disclosed in Examples 31 and 32. Benzoate (Compound II Benzoate). The solid material prepared in Example 32 was characterized by XRPD, FTIR (ATR), HPLC, DSC, TGA, MS, 4 NMR and moisture adsorption-desorption analysis. The compound π benzoate is shown to exhibit the following characteristics: Compound II benzoate is crystalline and exhibits the xrpd pattern (CuKa) shown in Figure 47. The main X-rays are rayed at ±0·2.20 and are opposite The strength is summarized in Table 37. The XRPD peak position of this unique group or a subset thereof can be used to identify the berylate. This subset contains peaks at about 17.43, 25.90, and 23.93 ± 0.2 °2. Another subset is included. 12.88, 16.33, Π.43 ' 2 5.90 and 23.93 ± 0.2 02θ peak. Another subset is included in about 8.20, 10.60, 12.88, 16.33, 17.43, 25.90, 26.61 and 128407.doc -45- 200911757 23·93± 〇·2. The peak of 20. A further subset contains peaks at about 8.2 〇, 10.60, 12.88, 16.33, 17.43, 22.46, 25.90, 9.07, and 23.93 ± 0.2 02θ. Compound II benzoate exhibited the 1H NMR spectrum shown in Figure 48. The spectrum and the known chemical structure of Compound II and the 1:1 ratio of the alum acid balance ion are consistent with Compound II. Compound II benzoate exhibited the FTIR (ATR) spectrum shown in Figure 49. The known chemical structure of the spectrum and the compound II and the benzoate balance of the 1: ratio are consistent with the compound II. The main FTIR band represented by the reciprocal of the wavelength (wavenumber in cm·1) is located at approximately 3400, 3264, 3114, 3045, 2836, 2560, 1650, 1635 '1583, 1527, 1484, 1453, 1421' 1386, 1278 , 1172, 1153, 1131, 1071, 1021, 1〇〇5, 926, 911, 853, 843, 809, 751, 706, and 683 cm·, rounding the value of the nearest integer). This unique set of IR absorption bands or a subset thereof can be used to identify the benzoate salt of Compound II. Compound II benzoate exhibits the DSC trace e DSC analysis shown in Figure 50 showing an endotherm at about 100 ° C followed by! The heat of 16〇c. The other is exothermic at 2 〇〇 C followed by an endotherm at 2 16 °C. At 283. (: The final event was observed and may indicate cyclic impurities or other degradation products formed during the experiment. The compound π benzoate exhibits the TGA thermogram shown in Figure 51. When heated from 60 ° C to 170 T: TGA shows a total weight loss of 9.6 wt%, which may be due to residual solvent and/or degradation. Decomposition initiation near i 8 〇 t: from 5% RH to 90% RH, compound π benzoate It is non-hygroscopic stable 128407.doc -46- 200911757 monohydrate. The moisture adsorption-desorption trace of compound π-benzoate is shown in Figure 52. No hysteresis is observed during desorption: after moisture adsorption XRPD analysis of the sample yielded a pattern consistent with the starting material f, which indicates that the compound oxime formate can be rapidly converted back to the monohydrate form under environmental laboratory conditions. The compound II benzoate is soluble in water ( Example 38) A sample of the compound benzoate was equilibrated in DI water at room temperature for about 16 hours. After the exposure, it was found that the water contained 3.6 mg/mL of the compound benzoate. The crystalline form of the residual solid was converted to Form A of the compound II free base A__ Crystalline form. Compound II benzoate tends to degrade at high temperatures (Example 39) The sample of compound II benzoate was subjected to thermal stress for two days at 6 ° C; residual solids showed degradation of cyclic benzimidazole Impurities. Formal conversion did not occur. Compound II benzoate did not show a tendency for organic solvents to initiate solid form interconversion 'but may tend to degrade under the slurry (Example 40). At toluene and gamma in the ambient temperature The sample of the slurry compound π benzoate was reduced for two days. No form conversion was observed in any of the residual solids after slurrying. No common degradation impurities were found in the residual solids; however, HPLC analysis showed two residues The purity in the solids was reduced by about 3% (hplc area percent assay). 4. Hydrogenate salt of compound II The hydrogen salt of compound π (compound II hydrochloride) was prepared by the procedure disclosed in Examples 31 and 32. The compounds prepared in Example 32 were characterized by XrPD, FTIR (ATR), HPLC, DSC, TGA, MS, A NMR, water solubility and moisture adsorption. The analysis showed that the compound π hydrochlorochloride The acid salt exhibits the following characteristics 'which can be used alone or in combination to identify the presence of the compound π hydrochloride. Compound II hydrochloride is crystalline and exhibits the xrPD pattern (CuKa) shown in Figure 53. ± 0.2 ° 2 Θ Representing primary X-rays around the ray and summarizing their relative intensities in Table 38. This unique set of xrpd peaks or a subset thereof can be used to identify benzoates. This subset is included at approximately 19 83, 23 〇 9 and 21_89 ± 0.2 peak. The other subset contains peaks at approximately 912, 1716, 20.75, 19.83, 23.09, and 21·89 ± 〇 · 2.20. Compound II hydrochloride showed the 1η NMR shown in Figure 54. The spectrum is identical to the known structure of Compound 11. The compound II hydrogenate exhibited the FTIR (ATR) spectrum shown in FIG. The spectrum is consistent with the known structure of Compound II. The main FTIR band, represented by the reciprocal of the wavelength (the number of waves in cm-丨), is located at approximately 3〇86, 3〇26, 2969,

近整數之值）。此獨特組之仪吸收帶或其子集可用以鑑別化合物II之氫氯酸鹽。 2850、2575、1672、1615、 1373、1314、1293、1272 ' 912、863、818、772、752、 1568、1528、1488、1442、 1192、1143、1020、949、 744、714 及 692 cm、捨入最化合物Π氫氯酸鹽展現圖56中所示之Dsc迹線。政分析顯示在197。(：之吸熱接著在2G8t之放熱。在2饥觀察到最終事件，其可表明在會聆s 仕貫驗期間形成之裱狀雜質或其他降解產物。所示之TGA熱分析圖化合物II氫氣酸鹽展現圖57中 128407.doc •48· 200911757 TGA顯示在l6(rc以了無重量損失及在16代至21代之間可能歸因於降解的4.9%之重量損失。化合物II氫氣酸鹽為無水且適度吸水^水分吸附分析（圖 58)顯示化合物Π氫氯酸鹽在9〇% RH獲得3 8重量。乂。水分吸附後之固體的XRPD分析得到與先前所得圖樣—致之圖樣。化〇物II氫氣酸鹽可溶於水。使化合物π氫氣酸鹽之樣品在DI水中在室溫下平衡約16小時（實例38)。暴露之後，藉由HPLC面積百分比檢定發現水含有17 mg/mL化合物π 氫氯酸鹽。在直至60 C之熱應力下化合物π氫氯酸鹽均穩定。在6〇 C下使化合物II氫氯酸鹽經受熱應力歷時兩天（實例39)。暴露之後’殘餘固體未顯示純度變化，且偵測到常見環狀苯并咪唑雜質。殘餘固體亦展現與先前所得相同之結晶形。化合物II氫氣酸鹽並不傾向於有機溶劑引發之固體形式互變。在甲苯及正庚烷中調漿化合物π氫氣酸鹽之樣品歷時兩天。調漿後殘餘固體中之任一者中未觀察到形式轉化。另外，未觀察到純度降低及存在常見環狀苯并咪唑降解雜質（實例40)。 5.化合物II之氩溴酸鹽可藉由實例3 1及32中揭示之程序製備化合物Π之氫溴酸鹽（化合物II氫溴酸鹽）。由XRPD、FTIR(ATR)、HPLC、 DSC、TGA、MS、4 NMR及水分吸附-解吸附分析來表徵 128407.doc 49· 200911757 在實例32中製備之固體物質(實例36)。分析顯示化合物η 氫溴酸鹽展現以下特徵’其可單獨或組合用以鑑別化合物 II氫溴酸鹽之存在。化合物π氫溴酸鹽為結晶且展現圖59中所示之xrpd圖樣（CuKa)。以±0·2。20表示主要X射線繞射線且將其相對強度概括於表39中。此獨特組之XRPD峰位或其子集可用以鑑別氫溴酸鹽。—此子集包含在約n.25、18.92及 26.55±0·2 °2Θ之峰值。另一子集包含在約1125、17 59、 18.92、22.19及26.55±〇.2。20之峰值。又一子集包含在約 8.67、11.25、Π.59、18.92、22·19、25 53及 26 55±〇 2 〇2θ 之峰值。化合物II氫溴酸鹽展現圖60中所示之lH NMR。光譜與化合物II之已知結構一致。Near integer value). This unique set of absorption bands or a subset thereof can be used to identify the hydrochloride salt of Compound II. 2850, 2575, 1672, 1615, 1373, 1314, 1293, 1272 '912, 863, 818, 772, 752, 1568, 1528, 1488, 1442, 1192, 1143, 1020, 949, 744, 714 and 692 cm The most compound hydrazine hydrochloride was shown to exhibit the Dsc trace shown in FIG. The political analysis is shown at 197. (: The endothermic heat is then exothermic at 2G8t. The final event is observed in 2 hunger, which can indicate the smear-like impurities or other degradation products formed during the inspection. TGA thermogram shown compound II hydrogen acid The salt is shown in Figure 57. 128407.doc • 48· 200911757 TGA is shown at l6 (rc with no weight loss and 4.9% weight loss between 16 and 21 generations possibly due to degradation. Compound II hydrogenate is Anhydrous and moderate water absorption. Moisture adsorption analysis (Fig. 58) showed that the compound hydrazine hydrochloride obtained 38 weight at 9 〇% RH. X The XRPD analysis of the solid after moisture adsorption gave a pattern from the previously obtained pattern. The hydrazine II hydrogenate was soluble in water. A sample of the compound π HCl was equilibrated in DI water at room temperature for about 16 hours (Example 38). After exposure, the water was found to contain 17 mg by HPLC area percent determination. mL compound π hydrochloride. Compound π hydrochloride was stable under thermal stress up to 60 C. Compound II hydrochloride was subjected to thermal stress at 6 ° C for two days (Example 39). 'Residual solids show no change in purity And the common cyclic benzimidazole impurity is detected. The residual solid also exhibits the same crystal form as previously obtained. The compound II hydrogenate does not tend to be an organic solvent-induced solid form interconversion. In toluene and n-heptane The sample of the pulverized compound π-hydrogenate lasted for two days. No form conversion was observed in any of the residual solids after slurrying. In addition, no decrease in purity and the presence of common cyclic benzimidazole degradation impurities were observed (Example 40 5. Argonate Bromide of Compound II The hydroquinone salt of the compound (Compound II hydrobromide) can be prepared by the procedures disclosed in Examples 31 and 32. By XRPD, FTIR (ATR), HPLC, DSC, TGA, MS, 4 NMR and moisture adsorption-desorption analysis to characterize 128407.doc 49· 200911757 The solid material prepared in Example 32 (Example 36). The analysis showed that the compound η hydrobromide exhibits the following characteristics Used alone or in combination to identify the presence of the compound II hydrobromide salt. The compound π hydrobromide salt crystallizes and exhibits the xrpd pattern (CuKa) shown in Figure 59. The main X-ray diffraction ray is represented by ±0.22. And compare it The degree is summarized in Table 39. The XRPD peak position of this unique set or a subset thereof can be used to identify the hydrobromide salt. This subset contains peaks at about n.25, 18.92, and 26.55±0·2 °2. A subset consists of peaks at approximately 1125, 17 59, 18.92, 22.19, and 26.55 ± 〇.2. 20. Another subset is included at approximately 8.67, 11.25, Π.59, 18.92, 22·19, 25 53 and 26 The peak value of 55 ± 〇 2 〇 2θ. Compound II hydrobromide salt exhibited the 1H NMR shown in Figure 60. The spectrum is consistent with the known structure of Compound II.

化合物II氫溴酸鹽展現圖61中所示之ftir(atr)光譜。光譜與化合物II之已知結構一致。以波長倒數（以計之波數）表示之主要FTIR帶定位於約3154、3〇94、2977、 2660、2597、1666、1603、1562、1523、1500、1480、 1461、1373、1305、1270、1197、1139、1〇35、1123、 951 、 939 、 913 、 868 、 828 、 812 、 762 、 751 、 742 、 696及 666 cm—1(捨入最近整數之值）。此獨特組之伙吸收帶或其子集可用以鑑別化合物II之氫溴酸鹽。化合物II氫溴酸鹽展現圖62中所示之DSC迹線。DSC分析顯示在約214°C之放熱接著在約224°C之放熱。在296。(：觀察到最終事件，其可表明在實驗期間形成之環狀雜質或 128407.doc •50· 200911757 其他降解產物。化合物π氫漠酸鹽展現圖63中所示之tga熱分析圖。直至在1 70 C以上TGA熱分析圖才顯示重量損失；當繼續加熱至220T：時在兩個步驟中重量損失總計3篇，其可能歸因於降解。化合㈣氫漠酸鹽為適度吸濕。水分吸附分析顯示該物質自5。/。RH至90% RH在吸附時獲得4 5重量％(圖64)。水分吸附後之m體的X R p D分析得到與先前所得_樣_致之圖樣。化合物II氫溴酸鹽可溶於水（實例38卜使化合物丨〗氫漠酸鹽之樣品在DI水中在室溫下平衡約16小時。暴露之後，發現水含有4.7 mg/mL化合物π氫溴酸鹽；殘餘固體已轉化為與化合物II形式A —致之結晶形。在至多60°C之熱應力下，化合物η氫溴酸鹽穩定。在6〇 C下使化合物Π氫溴酸鹽之樣品經受熱應力歷時兩天；未觀察到形式變化及純度降低（實例39)。化合物II氫溴酸鹽未顯示固體形式之有機溶劑引發互變的傾向（實例40)。在曱苯及正庚烷中調漿化合物η氫溴酸鹽之樣品歷時兩天。在來自兩個漿料之殘餘固體中均未觀察到形式轉化或降解之跡象。 6·化合物II之龍膽睃鹽可藉由實例3 1及32中揭示之程序製備化合物II之龍膽酸鹽（化合物II龍膽酸鹽）。由XRPD、FTIR(ATR)、HPLC、 DSC、TGA、MS、4 NMR及水分吸附-解吸附分析來表散在實例3 1中製備之固體物質（實例37)。分析顯示化合物π 128407.doc -51 - 200911757 龍膽酸鹽展現以下特徵，呈可置彳g 4、/ ，、了早獨或組合用以鑑別化合物 π龍膽酸鹽之存在。化合物II龍膽酸鹽為結晶且展饮兄圖65中所示之XRPD圖樣（CuKa)。以±0.2 02θ表示主亚γ匕μ * 要X射線繞射線且將其相對強度概括於表40中（實例37)。吐想4* λ J J此獨特組之XRPD峰位或其子集可用以鑑別龍膽酸鹽。_此此千集包含在約17.49、 12.87 及 25.93±0.2 〇2θ 之峰值〇 s -? ^ 之哗值另—子集包含在約9.06、 Π.49、19.56、12.87、25.93及26·67±〇 2，之峰值。化合物II龍膽酸鹽展現圖Μ中所示^Hnmr光譜。光譜與化合物II之已知結構及1 · 1比垄夕菇& 丹^ 1 半之龍膽酸鹽平衡離子與化合物II 一致。化合物II龍膽酸鹽展現圖67中所示之ftir(atr)光譜。光4與結構及1:1比率之龍膽酸鹽平衡離子與化合物Η 一致。以波長倒數（以cm 1計之波數）表示之主要FTIR帶定位於約 3260、1654、1635、1584、1550、1528、1504、Compound II hydrobromide salt exhibited the ftir (atr) spectrum shown in Figure 61. The spectrum is consistent with the known structure of Compound II. The main FTIR bands, expressed in terms of wavelength reciprocal (in terms of wavenumbers), are located at approximately 3154, 3〇94, 2977, 2660, 2597, 1666, 1603, 1562, 1523, 1500, 1480, 1461, 1373, 1305, 1270, 1197, 1139, 1〇35, 1123, 951, 939, 913, 868, 828, 812, 762, 751, 742, 696, and 666 cm-1 (rounding the value of the nearest integer). This unique group of absorption bands or a subset thereof can be used to identify the hydrobromide salt of Compound II. Compound II hydrobromide salt exhibited the DSC trace shown in Figure 62. The DSC analysis showed an exotherm at about 214 ° C followed by an exotherm at about 224 ° C. At 296. (: The final event was observed, which may indicate cyclic impurities formed during the experiment or other degradation products of 128407.doc • 50· 200911757. The compound π hydrogen oxalate exhibits the tga thermogram shown in Figure 63. The TGA thermal analysis chart above 1 70 C shows the weight loss; when heating to 220T: the total weight loss in two steps is 3, which may be attributed to degradation. Compound (4) Hydrogen oxalate is moderately hygroscopic. Adsorption analysis showed that the material obtained from 5 % RH to 90% RH was obtained by adsorption at 45 % by weight (Fig. 64). The XR p D analysis of the m body after moisture adsorption was obtained from the previously obtained sample. The compound II hydrobromide salt was soluble in water (Example 38) The sample of the compound hydrazine hydrochloride was equilibrated in DI water at room temperature for about 16 hours. After the exposure, it was found that the water contained 4.7 mg/mL of compound π hydrobromide. The residual solid has been converted to a crystalline form which is consistent with Form II of Compound II. The compound η hydrobromide is stable under thermal stress of up to 60 ° C. The compound is hydrobromate at 6 ° C. The sample was subjected to thermal stress for two days; no shape was observed Change and purity reduction (Example 39). Compound II hydrobromide salt did not show a tendency for organic solvents in solid form to initiate interconversion (Example 40). Samples of the compound η hydrobromide salt were slurried in toluene and n-heptane. It took two days. No signs of formal transformation or degradation were observed in the residual solids from the two slurries. 6. The gentiosulfonium salt of Compound II can be prepared by the procedure disclosed in Examples 31 and 32. Citrate (Compound II gentisate). The solid matter prepared in Example 31 was dispersed by XRPD, FTIR (ATR), HPLC, DSC, TGA, MS, 4 NMR and moisture adsorption-desorption analysis ( Example 37). Analysis shows compound π 128407.doc -51 - 200911757 gentisic acid exhibits the following characteristics, which can be placed in g 4 , / , , alone or in combination to identify the presence of the compound π gentisate. Compound II gentisate is crystalline and exhibits the XRPD pattern (CuKa) shown in Figure 65. The main sub-γ 匕 μ is represented by ± 0.2 02θ * X-rays are rayed and their relative intensities are summarized in Table 40. (Example 37). Speaking 4* λ JJ The XRPD peak of this unique group or its sub The set can be used to identify gentisate. _ This thousand set contains the peak value of 17s -? ^ at about 17.49, 12.87, and 25.93 ± 0.2 〇 2θ. The other subset is included in about 9.06, Π.49, 19.56. , peaks of 12.87, 25.93 and 26.67 ± 〇 2, the compound II gentisate exhibits the ^Hnmr spectrum shown in the figure 光谱. The spectrum and the known structure of the compound II and 1 · 1 ratio of the mushroom and the dan ^ 1 Half of the gentisate counterion is consistent with Compound II. Compound II gentisate exhibited the ftir (atr) spectrum shown in Figure 67. The light 4 and the structure and the 1:1 ratio of the gentisate counterion are consistent with the compound 。. The main FTIR band, represented by the reciprocal of the wavelength (wavenumber in cm 1 ), is located at approximately 3260, 1654, 1635, 1584, 1550, 1528, 1504,

1484、1443、1385、1302、1278、1241、1218、1153、 1133 、 1082 、 1034 、 1021 、 1〇〇6 、 940 、 911 、 876 、 854 、 842、810、799、788、753、725 及 680 cm、捨入最近整數之值）。此獨特組之IR吸收帶或其子集可用以鑑別化合物π 之龍膽酸鹽。化合物II龍膽酸鹽展現圖68中所示之DSC迹線。迹線顯示在127C、160°C及240。(：之吸熱。在240°C之最終事件可表明在實驗期間形成之環狀雜質或其他降解產物。化合物II龍膽酸鹽展現圖69中所示之TGA熱分析圖。 128407.doc -52· 200911757 TGA顯示在60°C與170°C之間幾乎5%之重量損失，其可能歸因於殘餘溶劑及/或降解。降解起始於約213°C。化合物II龍膽酸鹽為單水合物。水分吸附-解吸附顯示試樣在5°/。RH與90% RH之間為單水合物（圖70)。水分吸附後之殘餘固體的XRPD分析未顯示形式轉化。 F.化合物III之結晶固體化合物III，1-(4-(2-胺基苯基胺甲醯基）苄基VN-乙基-1H-吡唑-4-甲醯胺，具有下式：1484, 1443, 1385, 1302, 1278, 1241, 1218, 1153, 1133, 1082, 1034, 1021, 1〇〇6, 940, 911, 876, 854, 842, 810, 799, 788, 753, 725, and 680 Cm, rounds the value of the nearest integer). This unique set of IR absorption bands or a subset thereof can be used to identify the compound π gentisate. Compound II gentisate exhibited the DSC trace shown in Figure 68. Traces are shown at 127C, 160°C and 240. (: Endothermic. The final event at 240 ° C may indicate cyclic impurities or other degradation products formed during the experiment. Compound II gentisate exhibits a TGA thermogram shown in Figure 69. 128407.doc -52 · 200911757 TGA shows almost 5% weight loss between 60 ° C and 170 ° C, which may be due to residual solvents and / or degradation. Degradation starts at about 213 ° C. Compound II gentisate is a single Hydrate. Moisture adsorption-desorption showed that the sample was monohydrate between 5 ° / RH and 90% RH (Figure 70). XRPD analysis of residual solids after moisture adsorption showed no formal conversion. Crystalline solid compound III, 1-(4-(2-aminophenylamine-methane)benzyl VN-ethyl-1H-pyrazole-4-carboxamide, having the formula:

如藉由實例41中揭示之程序製備的化合物π之游離鹼經確定為結晶且指定為形式A。由1H NMR、DSC、TGA、 FTIR(ATR) ' HPLC '水溶性及水分吸附-解吸附分析來表徵化合物III之形式A。固體亦經受熱應力以及漿料實驗以評估其物理形式轉化之傾向。將結晶固體之分析資料概括於表3中。表3.化合物III之結晶固艘的物理表徵資料平衡離子溶劑 XRPD 結果 DSC分析藉由TGA所測之重量損失％ Stoich. (API: 酸）溶解度 (mg/mL) 在90% RH吸附時之水分吸附％起始值 ΓΟ (°C) 游離鹼 N/A Xtal 254 258 0.0 N/A <0.1 0.6 龍膽酸二0惡半 Xtal 125 133 〇·〇，在 120°c 以 1:1 0.7 4.1 鹽固體炫 255 下； 268 3.2 ’ 在 13〇°C 以上分解 128407.doc -53- 200911757The free base of the compound π prepared by the procedure disclosed in Example 41 was determined to be crystallized and designated as Form A. Form A of Compound III was characterized by 1H NMR, DSC, TGA, FTIR (ATR) 'HPLC' water solubility and moisture adsorption-desorption analysis. Solids are also subjected to thermal stress and slurry experiments to assess the propensity for their physical form to be converted. The analytical data of the crystalline solids are summarized in Table 3. Table 3. Physical Characterization of Crystalline Solids of Compound III. Equilibrium Ion Solvent XRPD Results DSC Analysis Weight Loss by TGA % Stoich. (API: Acid) Solubility (mg/mL) Moisture at 90% RH Adsorption Adsorption % Starting value ΓΟ (°C) Free base N/A Xtal 254 258 0.0 N/A <0.1 0.6 gentisic acid dioxin Xtal 125 133 〇·〇 at 120°c with 1:1 0.7 4.1 Salt solid dazzle 255; 268 3.2 ' Decompose above 13 ° ° C 128407.doc -53- 200911757

1.化合物III之形式A 可藉由實例41中所述之方法製備化合物III之游離鹼的形式 A(化合物 III形式 A)。由 XRPD、FTIR(ATR)、HPLC、 DSC、TGA、MS、4 NMR、水溶性及水分吸附-解吸附分析來表徵化合物。分析顯示化合物III形式A展現以下特徵，其可單獨或組合用以鑑別化合物III形式A之存在（實例 42) ° 化合物III形式A為結晶固體且展現圖71中所示之XRPD 光譜（CuKot)。以±0.2。20表示主要X射線繞射線且將其相對強度概括於表44中。此獨特組之XRPD峰位或其子集可用以鑑別化合物III形式A。一此子集包含在約9.43、4.73 及14.14士0.2。20之峰值。另一子集包含在約9.43、4.73、 18.89 ' 23.65及 14.14±0.2 02θ之峰值。化合物III形式Α展現圖72中所示之1H NMR。光譜與化合物III之已知結構一致。化合物III形式Α展現圖73中所示之FTIR(ATR)。光譜與化合物III之已知結構一致。以波長倒數（以cm'1計之波數）表示之主要FTIR帶定位於約3459、3371、3303、3106、 3053、2976、2938、2875、1642、1627、1576、1529、 1 507、1494、1456、1404、1 358、1304、1266、1216、 1138、1118、1055、1020、999、959、940、907、892、 864、837、761、743、728、721 及 670 cm-彳捨入最近整數之值）。此獨特組之IR帶或其子集可用以鑑別化合物II。化合物III形式A展現圖74中所示之DSC迹線。迹線顯示 128407.doc -54- 200911757 出現僅一個集中於約258。(：之吸熱事件。化合物III形式A展現圖75中所示之TGa熱分析圖。化合物III形式A之TGA熱分析圖顯示直至23〇艽均無重量損失Y 表明無水形式。化口物III形式A展現圖76中所示之水分吸附及解吸附迹線。迹線顯示該物質為非吸濕性且未觀察到潛在水合物形式。水分吸附後之殘餘固體未顯示形式變化。化合物III形式A為微溶。使化合物m形式A之樣品在〇工水中在室溫下平衡1 6小時。濾出殘餘物固體之後，發現上澄液含有<0.1 mg/mL之化合物出形式a(實例49)。在至夕60 C之熱應力下，化合物m形式a穩定。在6〇。〇下使化合物III形式A之樣品經受熱應力歷時兩天。暴露之後，殘餘固體未顯示降解之跡象且未觀察到形式轉化（實例 5 1)。化合物III形式A未顯示固體形式之有機溶劑引發互變的傾向（實例5 0)。在環境溫度下在甲苯中或正庚烷中調漿化合物III形式A之樣品歷時兩天。測試殘餘固體；未觀察到形式變化及純度降低。另外，未在殘餘物中偵測出常見環狀苯并咪唑降解雜質。 2·化合物III之龍膽酸鹽固體在初始鹽篩檢期間發現在龍膽酸存在下在二噁烷中結晶的化合物III之結晶固體。再次藉由實例46及47中所述之程序製備此結晶固體。後續分析顯示結晶固體為大部分起始物質與少量化合物III之龍膽酸鹽的混合物；本文中將該結 128407.doc -55- 200911757 晶固體稱作化合物III之龍膽酸鹽固體。由XRPD、 NMR、FTIR(ATR)、HPLC、DSC、TGA、MS及水分吸附- 解吸附分析來表徵該物質。分析顯示化合物III龍膽酸鹽展現以下特徵；其可單獨或組合用以鑑別龍膽酸鹽固體化合物III之存在。化合物III之龍膽酸鹽固體為結晶且展現圖77中所示之 XRPD光譜（CuKa)。以土0·2 °2Θ表示主要X射線繞射線且將其相對強度概括於表46中（實例46)。將XRPD圖樣與化合物III形式Α之XRPD圖樣相比較，且鑑別龍膽酸鹽所獨有之一組峰值。此獨特組之XRPD峰位包含在約10.9、 20.48、28.35及30.90。20之峰值，其可用以鑑別化合物III 之龍膽酸鹽固體。化合物III之龍膽酸鹽固體展現圖78中所示之1H NMR。光譜與化合物III之已知結構一致，且另外顯示化學位移及所觀察共振之差異，其係歸因於質子化物質之存在。積分顯示接近1:1比率之龍膽酸鹽離子與化合物III。化合物III之龍膽酸鹽固體展現圖79中所示之FTIR(ATR) 光譜。光譜與化合物ΠΙ之已知化學結構一致。以波長倒數 (以cnT1計之波數）表示之主要FTIR帶定位於約3296、 2975、1626、1575、1529、1485、1442、1375、1302、 1266 、 1216 、 1135' 1055 、 1020 、 999 、 940 、 906 、 888 、 866、840、798、743、727及670 cm·〗（捨入最近整數之值）。此組獨特帶可用以鑑別化合物III之龍膽酸鹽固體的存在。 128407.doc > 56- 200911757 化合物III之龍膽酸鹽固體展現圖81中所示之DSC迹線。 DSC顯示在約133°C之吸熱事件，其為化合物m之龍膽酸鹽固體所獨有。化合物III之龍膽酸鹽固體展現圖82中所示之TGA熱分析圖。TGA顯示在120°C與19〇t之間3.2%之重量損失，其為化合物III之龍膽酸鹽固體所獨有。在25 c下自0% RH至90% RH進行之水分吸附分析顯示龍膽酸鹽固體在60% RH吸附2.4%水（圖83)。在解吸附後未觀察到顯著滞後現象，表明在實驗期間該物質並未形成穩定水合物。水分吸附分析之後對殘餘固體進行XRpD分析且表明樣品並未轉化成替代結晶形。化合物III龍膽酸鹽微溶於水。在室溫下在水中調漿龍膽酸鹽固體之樣品歷時1 6小時。過濾殘餘固體後，發現上澄液含有0.73 mg/mL化合物III龍膽酸鹽。注意到化合物ΙΠ 龍膽酸鹽顯示比化合物出形成Α高之溶解度；然而，此溶解度增加可能歸因於雜質含量之差異或歸因於存在龍膽酸之pH值降低。化合物I、II及ΙΠ及其多晶型物及鹽之使用指示本發明亦係關於藉由投與呈化合物之任何醫藥學上可接受之形式的化合物ί、Π或m來改變、較佳降低在個體中 HDAC活性的方法，該等形式包括化合物之任何形式，包括（但不限於）多晶型物、溶劑合物、酯、互變異構體、對映異構體及醫藥學上可接受之鹽形式。作為實例且不構成限制，化合物I可以游離鹼之形式八及/或形式B的形式或以 128407.doc -57- 200911757 虱氯酸鹽、反丁烯二酸鹽或其混合物之形式進行投藥化合物II可以游離鹼之形式或以氫氣酸鹽、反丁稀二酸趟、氫溴酸鹽、苯甲酸鹽、雙三氟乙酸鹽、龍膽酸鹽或其混合物之形式進行投藥。化合物m可以化合物之游離鹼^形二進行投藥。呈本文中所揭示之任何結晶形的本發明化合物為hdac 抑制劑且可適用於治療、預防由HDAC引發之疾病及病狀。組蛋白為小型、帶正電荷之蛋白質，其與Dna緊密複合以在真核細胞中形成染色質。真核細胞中之dna與蛋白質(組蛋白)緊密複合以形成染色質。組蛋白為富含鹼性胺基酸（在生理學pH值下帶正電荷）之小型、帶正電荷蛋白質，其接觸DNA之磷酸基（在生理學pH值下帶負電荷）。存在五個主要類別之組蛋白H1、H2A、H2B'扪及則。、H2B、H3及H4之胺基酸序列在物種之間顯示顯著保守性’其中H1稍作改變且在-些情況下經例如H5之另一組蛋白置換。四對每一 Η2Α、Η2β、_Η4 一起形成盤形八聚體蛋白質核’ DNA(約14〇個驗基對）纏繞在其周圍以形成核小體。個別核小體經由與另一組蛋白分子締合之連接子DNA的短伸出物而連接以形成類似於珠串之結構，其本身配置於稱為螺線管之螺旋堆疊中。大夕數組蛋白係在細胞週期之㈣段期間合成，且新合白迅速進人核中以變得與譲締合。在其合成之拉内新DNA變得與組蛋白在核小體結構中締合。後轉譯添加甲基、乙醯基或碟酸基來酶促修飾-小 128407.doc -58- 200911757 部为組蛋白、更特定言之其胺基酸側鍵’從而中和側鍵之正電荷或將其轉化為負電荷。舉例而言，可使離胺酸及精胺酸基甲基化’可使離胺酸基乙醯化且可使絲胺酸基磷酸化。對於離胺酸而言，可例如用乙醯基轉移酶使_(cH2)4_ NH2側鏈乙醯化以得到醯胺_(CH2)4_NHC(=〇)CH3。自核小體核心延伸出之組蛋白的胺基末端之甲基化、乙醯化及碟酸化衫響染色質結構及基因表現。Spencer及Davie 1999. Gene 240:1 1 -1 2。組蛋白之乙醯化及去乙酿化與導致細胞增生及/或分化之轉錄事件相關。亦經由乙醯化引發轉錄因子之功能調節。有關組蛋白去乙醯化之最近回顧包括K〇uzaHdes等人 I"9，Curr. 〇pin· Genet. Dev. 9:1, 40-48 及 Pazin 等人，1997, 89··3 325-328。1. Form A of Compound III Form A (Compound III Form A) of the free base of Compound III can be prepared by the method described in Example 41. The compounds were characterized by XRPD, FTIR (ATR), HPLC, DSC, TGA, MS, 4 NMR, water solubility and moisture adsorption-desorption analysis. Analysis showed that Compound III Form A exhibited the following characteristics, either alone or in combination, to identify the presence of Compound III Form A (Example 42) ° Compound III Form A was a crystalline solid and exhibited the XRPD spectrum (CuKot) shown in Figure 71. The main X-rays are rayed at ±0.20 and their relative intensities are summarized in Table 44. The XRPD peak position of this unique set or a subset thereof can be used to identify Compound III Form A. This subset contains peaks at about 9.43, 4.73, and 14.14 ± 0.22. Another subset contains peaks at about 9.43, 4.73, 18.89 ' 23.65, and 14.14 ± 0.2 02θ. The compound III form oxime exhibited the 1H NMR shown in Figure 72. The spectrum is consistent with the known structure of Compound III. The compound III form oxime exhibits the FTIR (ATR) shown in Figure 73. The spectrum is consistent with the known structure of Compound III. The main FTIR band represented by the reciprocal of the wavelength (the wave number in cm'1) is located at approximately 3459, 3371, 3303, 3106, 3053, 2976, 2938, 2875, 1642, 1627, 1576, 1529, 1 507, 1494, 1456, 1404, 1 358, 1304, 1266, 1216, 1138, 1118, 1055, 1020, 999, 959, 940, 907, 892, 864, 837, 761, 743, 728, 721 and 670 cm-彳 rounded nearest The value of an integer). This unique set of IR bands or a subset thereof can be used to identify Compound II. Compound III Form A exhibits the DSC trace shown in Figure 74. Trace display 128407.doc -54- 200911757 appears only one focus on approximately 258. (: The endothermic event. Compound III Form A exhibits the TGa thermogram shown in Figure 75. The TGA thermogram of Compound III Form A shows no weight loss up to 23 Y Y indicates an anhydrous form. A shows the moisture adsorption and desorption traces shown in Figure 76. The traces show that the material is non-hygroscopic and no latent hydrate forms are observed. The residual solids after moisture adsorption show no change in form. To be slightly soluble, a sample of compound m Form A was equilibrated in plumbing water for 16 hours at room temperature. After the residue solid was filtered off, it was found that the supernatant contained <0.1 mg/mL of compound out form a (Example 49) Under the thermal stress of 60 C, the compound m form a is stable. The sample of Compound III Form A is subjected to thermal stress for two days at 6 。. After exposure, the residual solid shows no signs of degradation and Formal conversion was observed (Example 51). Compound III Form A did not show a tendency for the organic solvent in solid form to initiate interconversion (Example 50). Compound A Form A was pulverized in toluene or n-heptane at ambient temperature. Like The product lasted for two days. The residual solid was tested; no change in form and purity was observed. In addition, the common cyclic benzimidazole was not detected in the residue to degrade the impurities. 2. The gentisate solid of compound III was in the initial salt. A crystalline solid of Compound III crystallized in dioxane in the presence of gentisic acid was found during the screening. This crystalline solid was again prepared by the procedure described in Examples 46 and 47. Subsequent analysis showed that the crystalline solid was the majority of the starting a mixture of a substance and a small amount of gentisate of compound III; herein, the crystalline solid of 128407.doc -55-200911757 is referred to as the gentisate solid of compound III. By XRPD, NMR, FTIR (ATR), HPLC, The material was characterized by DSC, TGA, MS and moisture adsorption-desorption analysis. The analysis showed that the compound III gentisate exhibited the following characteristics; it can be used alone or in combination to identify the presence of the gentisate solid compound III. The gentisate solid was crystalline and exhibited the XRPD spectrum (CuKa) shown in Figure 77. The main X-rays were rayed at 0 2 ° 2 且 and their relative intensities are summarized in Table 46 (Example 46). The XRPD pattern is compared to the XRPD pattern of the compound III form of ruthenium and identifies one of the unique peaks of the gentisate group. The XRPD peak position of this unique group is comprised at peaks of about 10.9, 20.48, 28.35, and 30.90. It can be used to identify the gentisate solid of compound III. The gentisate solid of compound III exhibits the 1H NMR shown in Figure 78. The spectrum is consistent with the known structure of compound III, and additionally shows the chemical shift and the observed resonance. The difference is due to the presence of protonated substances. The integral shows a 1:1 ratio of gentisate ion to compound III. The gentisate solid of Compound III exhibited the FTIR (ATR) spectrum shown in Figure 79. The spectrum is consistent with the known chemical structure of the compound ΠΙ. The main FTIR band represented by the reciprocal of the wavelength (wavenumber in cnT1) is located at approximately 3296, 2975, 1626, 1575, 1529, 1485, 1442, 1375, 1302, 1266, 1216, 1135' 1055, 1020, 999, 940 , 906, 888, 866, 840, 798, 743, 727, and 670 cm· (round the value of the nearest integer). This unique set of bands can be used to identify the presence of the gentisate solid of Compound III. 128407.doc > 56- 200911757 The gentisate solid of Compound III exhibits the DSC trace shown in Figure 81. DSC showed an endothermic event at about 133 ° C, which is unique to the gentisate solid of compound m. The gentisate solid of Compound III exhibited the TGA thermogram shown in Figure 82. TGA showed a 3.2% weight loss between 120 ° C and 19 Torr, which is unique to the gentisate solid of Compound III. Moisture adsorption analysis from 0% RH to 90% RH at 25 c showed that gentisate solids adsorbed 2.4% water at 60% RH (Figure 83). No significant hysteresis was observed after desorption indicating that the material did not form a stable hydrate during the experiment. XRpD analysis of the residual solids after moisture adsorption analysis indicated that the sample was not converted to an alternative crystalline form. Compound III gentisate is slightly soluble in water. The sample of the citrate solid was slurried in water at room temperature for 16 hours. After filtering the residual solid, it was found that the supernatant contained 0.73 mg/mL of compound III gentisate. It is noted that the compound ΙΠ gentisate shows a higher solubility than the compound formation; however, this increase in solubility may be attributed to the difference in the impurity content or due to the decrease in the pH of the presence of gentisic acid. Use of Compounds I, II and Indole and Polymorphs and Salts It is also indicated that the present invention also relates to the modification, preferably reduction, by administering a compound ί, Π or m in any pharmaceutically acceptable form of the compound. A method of HDAC activity in an individual, including any form of the compound including, but not limited to, polymorphs, solvates, esters, tautomers, enantiomers, and pharmaceutically acceptable The salt form. By way of example and not limitation, Compound I can be administered as a free base in the form of VIII and/or Form B or as a compound in the form of 128407.doc-57-200911757 chlorate, fumarate or mixtures thereof. II can be administered in the form of a free base or in the form of a hydrogenate, a ruthenium dibutoxide, a hydrobromide, a benzoate, a bistrifluoroacetate, a gentisate or a mixture thereof. Compound m can be administered as a free base of the compound. The compounds of the invention in any of the crystalline forms disclosed herein are hdac inhibitors and are useful in the treatment or prevention of diseases and conditions caused by HDAC. Histones are small, positively charged proteins that bind tightly to Dna to form chromatin in eukaryotic cells. The dna in eukaryotic cells is tightly complexed with the protein (histone) to form chromatin. Histones are small, positively charged proteins rich in basic amino acids (positively charged at physiological pH) that contact the phosphate group of the DNA (negatively charged at physiological pH). There are five main categories of histones H1, H2A, H2B'扪 and then. The amino acid sequences of H2B, H3 and H4 show significant conservation between species 'where H1 is slightly altered and in some cases replaced by another set of proteins such as H5. Four pairs of each Η2Α, Η2β, _Η4 together form a disc-shaped octamer protein nucleus 'DNA (about 14 验 test pairs) wound around it to form nucleosomes. Individual nucleosomes are joined by short extensions of the linker DNA associated with another set of protein molecules to form a structure similar to a bead string, which itself is disposed in a spiral stack called a solenoid. The Daxi array protein is synthesized during the (4) segment of the cell cycle, and the new complex rapidly enters the nucleus to become associated with sputum. The new DNA in its synthesis becomes associated with histones in the nucleosome structure. Post-translationally add methyl, acetamino or disc acid groups to enzymatically modify - small 128407.doc -58- 200911757 is a histone, more specifically its amino acid side bond 'to neutralize the positive charge of the side bond Or convert it to a negative charge. For example, methylation of the lyophilic acid and the arginine group can be made to acetylate the lysine group and phosphorylate the leucine group. For the lyophilic acid, the _(cH2)4_NH2 side chain can be acetylated, for example, with an acetyltransferase to give the guanamine _(CH2)4_NHC(=〇)CH3. Methylation, acetylation, and acidification of the amino terminus of histones derived from the core of the nucleosome and the expression of the chromatin and gene expression. Spencer and Davie 1999. Gene 240: 1 1 -1 2. The acetylation and deacetylation of histones is associated with transcriptional events leading to cell proliferation and/or differentiation. Functional regulation of transcription factors is also triggered by acetylation. Recent reviews of histone deacetylation include K〇uza Hdes et al. I"9, Curr. 〇pin·Genet. Dev. 9:1, 40-48 and Pazin et al., 1997, 89·3 325-328 .

長久X來已知組蛋白之乙酿化狀態與基因轉錄之間的相關性。調節組蛋白之乙醯化狀態之特定酵素、特定而言乙醯化酶（例如，組蛋白乙醯基轉移酶（ΗΑτ))及去乙酿基酶 (•蛋白去乙I基酶或HDAC)已在許多有機體中鑑別出且已與大量基因之調節相關聯，從而證實乙醯化與轉錄之間的聯繫。—般而言，咸信組蛋白乙醯化與轉錄活化有關，而咸信組蛋白去乙醯化與基因抑制有關。 •另J越來越多的組蛋白去乙酿基酶（hdac)。充當繫栓至啟動子且抑制轉錄之大型多蛋白質複合物的部 …如MAD、核受體及YY1之良好表徵轉錄抑制物與 HDAC複合物締合以發揮其抑制物功能。 128407.doc -59· 200911757 本發明之化合物可適用於治療或預防涉及細胞增生及分化之疾病病狀。對HDAC抑制劑之研究已顯示此等酵素在細胞增生及分化中起重要作用。咸信HDAC與多種不同疾病病況相關，包括（但不限於）細胞增生疾病及病狀（Marks， P.A.，Richon，V.M.，Breslow，R.及 Rifkind, R.A·，J. Natl. Cancer Inst. (Bethesda) 92，1210-1215, 2000)，諸如白血病 (Lin 等人，1998. Nature 391:811-814 ; Grignani 等人， 1998. Nature 391:815-818 ; Warrell等人，1998，J, Natl. Cancer Inst. 90:1621-1625 ; Gelmetti等人，1998, Mol. Cell Biol. 18:7185-7191 ; Wang等人，1998，PNAS 951 0860-1 0865)、黑色素瘤/鱗狀細胞癌（Giiienwater等人，1 998, Int. J· Cancer 75217-224 ; Saunders等人，1999, Cancer Res. 59:399-404)、乳癌、***癌、膀胱癌（Geimetti等人 ’ 1998, Mol. Cell Biol· 18:7185-7191 ; Wang等人， 1998，PNAS 951 0860-10865)、肺癌、卵巢癌、結腸癌 (Hassig等人 ’ 1997, Chem. Biol. 4:783-789 ; Archer等人， 1998，PNAS，956791-6796 ; Swendeman等人，1999，Proc. Amer. Assoc· Cancer Res. 40，摘要 #3836)及過度增生性皮膚病如癌及癌如期皮膚病變，以及發炎性皮膚病症。組蛋白去乙醯基酶抑制劑為在培養物中及帶有腫瘤之動物中之多種轉化細胞中生長停滯、分化或細胞凋亡性細胞死亡之有效誘導物（Histone deacetylase inhibitors as new cancer drugs，Marks, P.A·，Richon，V.M.，Breslow，R.及 Rifkind，R.A.，Current 〇pini〇ns in 〇nc〇i〇gy，2001年 11 月 128407.doc -60- 200911757 13 日(6) 477-83 ; Histone deacetylases and cancer: causes and therapies, Marks, P., Rifkind, R.A., Richon, V.M., Breslow, R., Miller, T·及 Kelly, W.K·，Nat. Rev. Cancer 2001 年 12月 1 曰（3):194.202)。另外’呈任何所揭示結晶形之本發明化合物亦可適用於治療或預防原蟲病（美國專利5,922,837)及牛皮癖（PCT公開案第 WO 02/26696號）。組合療法多種治療劑與根據本發明之HDAC抑制劑可具有治療加成或協同效應。此等治療劑可與HDAC抑制劑加成地或協同地組合以抑制不欲之細胞生長，諸如導致不欲之良性病狀或腫瘤生長之不當細胞生長。在個A施例中，提供一種治療細胞增生疾病病況之方法，其包含用根據本發明之化合物與一種抗增生劑組合治療細胞，其中在以抗增生劑治療細胞之前、同時及/或之後用^據本發明之化合物治療細胞，在本文中稱作組合療法。請注意，本文中將一種藥劑治療在另一種藥劑之前稱為順序療法，即使該㈣劑亦—起投與。請注意，組合療法意欲涵蓋藥劑彼此先或後投與(順序療法)以及藥劑同時可與HDAC抑制劑組合使用之治療劑的實例包括（但不限 :)抗癌劑、烷化劑、抗生素藥劑、抗代謝藥劑、激” 劑、植物來源藥劑及生物藥劑。素糸烧化劑為具有以,ρ I 友凡土取代氣離子能力的多官能化合物。 128407.doc -61 - 200911757 烷化劑之實例包括（但不限於）雙氣乙胺（氮芥類（nitrogen mustards) ’例如苯丁酸氮芥（chl〇rambucil)、環填醯胺 (cyclophosphamide)、異環磷醯胺（ifosfamide)、氮芥 (mechlorethamine)、美法余（melphalan)、尿嘲。定氮芬 (uracil mustard))、氮丙咬（aziridine)(例如售替派 (thiotepa))、烧基酮石黃酸酉旨（例如白消安（busuifan))、亞硝基脲（nitrosourea)(例如卡莫司汀（carrnustine)、洛莫司；丁 (lomustine)、鏈佐星（streptozocin))、非典型烧化劑（六甲蜜胺（altretamine)、達卡巴嗓（dacarbazine)及丙卡巴肼 (procarbazine))、始化合物（卡翻（carb〇piastjn)及順始 (cisplatin))。此等化合物與磷酸根、胺基、羥基、硫氫基、叛基及咪唑基反應。在生理條件下，此等藥物離子化且產生帶正電離子’附著於易受影響之核酸及蛋白質，導致細胞週期停滯及/或細胞死亡。包括HDAC抑制劑及烷化劑之組合療法可對癌症具有治療協同效應且減少此等化學治療劑相關之副作用。抗生素藥劑為一群以類似於抗生素之方式產生的天然產物之修飾藥物。抗生素藥劑之實例包括（但不限於）蒽環黴素（anthracycline)(例如阿黴素（d〇xorubicin)、道諾黴素 (daunorubicin)、表票比星（epirubicin)、黃膽素（idarubicin) 及蒽二酮（anthracenedione))、絲裂黴素 c(mit〇mycin c)、博萊黴素（bleomyCin)、放線菌素D(dactin〇mycin)、普卡黴素（plicatomycin)。此等抗生素藥劑藉由靶向不同細胞組份來干擾細。舉例而f ’ 一般咸#蒽環#素在轉錄活 128407.doc -62- 200911757 性DNA區域内干擾DNA拓撲異構酶Π之作用，導致DNA股分離。一般咸信博萊黴素螯合鐵且形成活化複合物，其接著結合DNA之鹼基，引起股分離及細胞死亡。包括HDAC 抑制劑及抗生素藥劑之組合療法可對癌症具有治療協同效應且減少與此等化學治療劑相關之副作用。抗代謝藥劑為干擾生理學及癌細胞增生所必需之代謝過程的一群藥物。活躍增生之癌細胞需要連續合成大量核酸、蛋白質、脂質及其他重要細胞成份。許多抗代謝物抑制嗓呤或嘧啶核苷之合成或抑制DNA複製之酵素。一些抗代謝物亦干擾核糖核苷及RNA之合成及/或胺基酸代謝以及蛋白質合成。藉由干擾重要細胞成份之合成，抗代謝物可延遲或停滯癌細胞生長。抗代謝藥劑之實例包括（但不限於）氟尿鳴啶（fluorouracU，5_FU)、氟尿苷（n〇xuridine， 5-FUdR)、甲胺嗓吟（meth〇trexate)、甲醯四氫葉酸 (leucovorin)、經基脲（hydr0XyUrea)、硫鳥嘌岭 (thioguamne，6-TG)、巯基。票呤（mercapt〇purine，6_Mp)、阿糖胞苷（Cytarabine)、喷司他丁（pent〇statin)、氟達拉賓雄酸鹽（fludarabine phosphate)、克拉屈濱（cladribine，2_ CDA)、天門冬醯胺酶及吉西他濱（gemcitabine)。包括 HDAC抑制劑及抗代謝藥劑之組合療法可對癌症具有治療協同效應且減少與此等化學治療劑相關之副作用。激素藥劑為調節其目標器官之生長及發育的一群藥物。大部分激素藥劑為性類固醇及其衍生物及其類似物，諸如 ***、雄激素及助孕素。此等激素藥劑可充#性類固醇 128407.doc •63· 200911757 文體之拮抗劑以下調重要基因之受體表現及轉錄。此等激素藥劑之實例為合成***（例如己烯雌酚 (diethylstibestrol))、抗***（例如他莫昔芬（tam〇xifen)、托瑞米芬（toremifene)、氟曱固醇（flu〇xymesier〇1)及雷洛昔芬（ral〇xifene))、抗雄激素（比卡魯胺（bicalutamide)、尼魯胺（nilutamide)、氟他胺（fiutamide))、芳香酶抑制劑（例如，胺魯米特（amin〇glutethimide)、安美達錠（anastr〇z〇le) 及四唑（tetraz〇ie))、明康唑（ketoconaz〇le)、戈舍瑞林乙酸酯（goserelin acetate)、亮丙立德（ieupr〇Hde)、曱地孕酮乙酸酯（megestrol acetate)及米非司酮（mifeprist〇ne)。包括 HDAC抑制劑及激素藥劑之組合療法可對癌症具有治療協同效應且減少與此等化學治療劑相關之副作用。植物來源藥劑為來源於植物或基於藥劑之分子結構經修飾之一群藥物。植物來源藥劑之實例包括（但不限於）長春 !匕屬生物驗（例如，長春新驗（vincristine)、長春驗 (vinblastine)、長春地辛（vindesine)、長春利定 (vinzolidine)及長春瑞賓（vin〇relbine))、鬼臼毒素 (podophyllotoxin)(例如，足葉乙甙（et〇p〇side，vp_16)及替尼泊武（teniposide ’ VM-26))、紫杉烷（例如紫杉醇 (paclitaxel)及多西他赛（doceuxei))。此等植物來源藥劑一般充當結合於微管蛋白且抑制有絲***之抗有絲***劑。咸信諸如足葉乙甙之鬼臼毒素藉由與拓撲異構酶π之相互作用來干擾DNA合成，引起DNA股分離。包括HDAc抑制劑及植物來源藥劑之組合療法可對癌症具有治療協同效應 128407.doc -64 - 200911757 且減V、與此專化學治療劑相關之副作用。生物樂劑為當單獨或與化學療法及/或放射療法組合使用時引«m;肖退之-群生物分子例包括(但不限於)免疫調節蛋白質，諸如細胞激：= 腫瘤抗原之單株抗體、腫瘤抑制基因及癌症疫苗。包括 H D A C抑制劑及生物藥劑之組合療法可對癌症具有治療協同效應，增強患者對致瘤信號之免疫反應，且減少與此化學治療劑相關之潛在副作用。細胞激素具有深遠的免疫調節活性。諸如介白素_2(匕_ 2、阿地白介素（aldesleukin))之一些細胞激素及干擾素具有經證明之抗腫瘤活性且已批准用於治療具有轉移性腎細胞癌及轉移性惡性黑色素瘤之患者。IL_2為τ細胞生長因子，其對於T細胞引發免疫反應為重要的。咸信IL_2對一二心者之選擇性抗腫瘤作用為區別自身與非自身之細胞引發免疫反應的結果。可結合HDAC抑制劑使用之介白素的實例包括（但不限於）介白素2(IL-2)及介白素4(IL-4) '介白素 12(IL-12)。干擾素包括2 3種以上具有重疊活性之相關次型，所有 IFN次型均在本發明範疇内。IFN已對許多實體及血液科惡性腫瘤顯示活性，後者似乎尤其敏感。可結合HDAC抑制劑使用之其他細胞激素包括對血細胞生成及免疫功能發揮深遠作用之彼等細胞激素。此等細胞激素之實例包括（但不限於）紅血球生成素、粒細胞_c(邦格司亭（filgrastin))及粒細胞、巨噬細胞-CSF(沙格司亭 128407.doc -65- 200911757 (5&^^111031；丨111))。可結合110八(^抑制劑使用此等細胞激素以減小化學療法誘導之骨髓細胞生成毒性。亦可結合HDAC抑制劑使用除細胞激素外之其他免疫調節藥劑以抑制異常細胞生長。此等免疫調節藥劑之實例包括（但不限於）卡介苗（bacillus Calmette-Guerin)、左旋β米口坐 (levamisole)及奥曲肽（octreotide)( —種模擬天然產生激素生長抑素之作用的長效八肽）。針對腫瘤抗原之單株抗體為針對由腫瘤表現之抗原、較佳腫瘤特異性抗原引發的抗體。舉例而言，針對在包括轉移性乳癌之一些***腫瘤中過度表現的人類表皮生長因子受體2(HER2)產生單株抗體HERCEPTIN®(曲妥珠單抗 (Trastruzumab))。HER2蛋白質之過度表現與侵襲性較強之疾病及較差臨床預後相關。HERCEPTIN®用作治療具有轉移性乳癌且腫瘤過度表現HER2蛋白質之患者的單一藥劑。包括HDAC抑制劑及HERCEPTIN®之組合療法可對腫瘤、尤其對轉移性癌症具有治療協同效應。針對腫瘤抗原之單株抗體的另一實例為RITUXAN®(利妥昔單抗（Rituximab))，其係針對淋巴瘤細胞上之CD20而產生且選擇性地耗盡正常及惡性CD20+前B細胞及成熟B細胞。RITUXAN®用作治療具有復發或難治性低級或濾泡性、CD20+、B細胞非霍奇金淋巴瘤（non-Hodgkin's lymphoma)之患者的單一藥劑。包括HDAC抑制劑及 RITUXAN®之組合療法不僅可對淋巴瘤而且對其他形式或類型之惡性腫瘤具有治療協同效應。 128407.doc -66- 200911757 腫瘤抑制基因為用以抑制細胞生長及***週期，由此預防瘤形成發展之基因。腫瘤抑制基因中之突變使得細胞忽略抑制仏號網路之一或多個分量，從而克服細胞週期檢查點且產生經控制細胞生長癌症之較高速率。腫瘤抑制基因之實例包括（但不限於）DPC_4、nim、NF_2、RB、p53、 WTl、BRCAl 及 BRCA2。 DPC-4與胰腺癌有關且參與抑制細胞***之細胞質途各·。NF-1編碼抑制Ras(一種細胞質抑制蛋白質）之蛋白質。NF-1與神經系統之神經纖維瘤及嗜鉻細胞瘤及骨髓白血病有關。NF-2編碼與神經系統之脊膜瘤、神經鞘瘤及室管膜瘤有關之核蛋白。RB編碼pRB蛋白質（一種為細胞週功之主要抑制劑的核蛋白）。RB與視網膜母細胞瘤以及骨癌' 膀胱癌、小細胞肺癌及乳癌有關^ p53編碼調節細胞 ***且可誘導細胞凋亡之p53蛋白質。p53之突變及/或無作用可見於多種癌症中。WT1與腎臟之威爾姆氏腫瘤 (Wilms tumor)有關。BRCA1與乳癌及印巢癌有關，且 BRCA2與乳癌有關。可將腫瘤抑制基因轉移至腫瘤細胞中，腫瘤抑制基因於其中發揮其腫瘤抑制功能。包括 HDAC抑制劑及腫瘤抑制物之組合療法可對患有各種形式癌症之患者具有治療協同效應。癌症疫苗為料身體對腫狀特異十生免疫反應的一群藥劑。大部分在進行研究與開發及臨床試驗之癌症疫苗為腫瘤相關抗原（TAA)。ΤΑA為存在於腫瘤細胞上且在正常細胞上相對不存在或減少之結構(亦即蛋白冑、酵素或碳水 128407.doc -67- 200911757 化合物）。由於對腫瘤細胞相當獨特，TAA提供免疫系統之目標以識別且引起其破壞。TAA之實例包括（但不限於）神經節苷脂（GM2)、***特異性抗原（pSA)、α胎蛋白 (AFP)、癌胚抗原（CEA)(由結腸癌及例如乳癌、肺癌、胃癌及胰腺癌之其他腺癌產生）、黑色素瘤相關抗原（mart_ 1、gplOO、MAGE 1,3酪胺酸酶）、乳頭狀瘤病毒£6及以月段、自體腫瘤細胞及異源腫瘤細胞之全細胞或部分/溶胞物。佐劑可用以加強對TAA之免疫反應。佐劑之實例包括 (但不限於）卡介苗（BCG)、内毒素脂多醣、匙孔螺血氰蛋白（GKLH)、介白素-2(IL-2)、粒細胞-巨噬細胞群落刺激因子（GM-CSF)及環鱗醯胺（Cytoxan)( 一種被認為當以低劑量給予時減小腫瘤誘導抑制之化學治療劑）。包含化合物I、II或III之醫藥組合物，其中存在化合物I、 II或III之結晶固體中之至少一者化合物I、II或III可用於各種醫藥組合物中，其中至少一部分化合物I、II或in以化合物之結晶形存在於組合物中。醫藥組合物應含有足量化合物I、11或ΙΠ以充分減小活體内 HDAC活性，以提供所需治療作用。此等醫藥組合物可包含在0.005%與100%(重量/重量）之間、視情況〇.ι_95%且視情況1 -95°/。之範圍内的存在於組合物中之化合物I、η或 III。在特定實施例中，醫藥組合物包含至少〇. 1 %、0 2 5 %、 0·5ο/〇、1%、5%、1〇。/0、25%、50%、75〇/〇、80%、85%、 128407.doc •68- 200911757 90%、95°/。、97%或99%化合物I、II或III，該化合物為本文中所揭示之結晶形。在另一實施例中，在醫藥組合物中化合物I、II或III之特定結晶形可占至少0.1%、1%、5%、 10% ' 25% > 50% > 75% ' 80% > 85% > 90% > 95% > 97%¾ 99%化合物I、π及IIt之總量（重量/重量）。除化合物I、II或III之外，醫藥組合物亦可包含一或多種並不不利地影響化合物】、Π4ΠΙ之用途之額外組份。舉例而言，除化合物I之外，醫藥組合物亦可包括習知醫藥载劑；賦形劑；稀釋劑；潤滑劑；黏合劑；濕潤劑；崩解劑；滑動劑；甜味劑；調味劑；乳化劑；增溶劑；ρΗ值緩衝劑；芳香劑；表面穩定劑；懸浮劑；及其他習知的醫藥學非活性藥劑。詳言之，醫藥組合物可包含乳糖、甘露糖醇、葡萄糖、蔗糖、磷酸二鈣'碳酸鎂、糖精鈉、羧甲基纖維素、硬脂酸鎂、硬脂酸鈣、交聯羧曱基纖維素鈉、滑石 '澱粉、天然膠（例如***明膠）、糖蜜、聚乙烯吡咯啶、纖維素及其衍生物、聚乙H各酮、交聯聚乙稀料 :乙酸酯、檸檬酸鈉、環糊精衍生物、脫水山梨醇單月桂酸酯、三乙醇胺乙酸鈉、三乙醇胺油酸酯、生物相容性聚 :物(諸如膠原蛋白、乙烯乙酸乙烯酯、聚酸酐、聚乙醇酸、聚原酸酯、聚乳酸及其他此等藥劑）。根據本發明之醫藥組合物可適於藉由多種途徑中之任一以緩ϋ舉例而s，根據本發明之醫藥組合物可視情況欸k釋放劑型經以下方式投與：經口、非經口、腹膜内、靜脈内、動脈内、局部、經皮、經舌下、肌肉内、經 128407.doc •69- 200911757 腸么頰、經鼻内、經脂質體、經由吸入、經***、經眼内、經由局部傳遞（例如藉由導管或血管内支架）、皮下、經脂肪内、經關節内或鞘内。在特定實施例中，經口、藉由吸入或藉由皮下、肌肉内、靜脈内或直接注射入腦脊髓液中來投與醫藥化合物。一般而言，本發明之醫藥組合物可製備成氣體、液體、半液體、凝膠或固體形式，且以適用於欲使用之投藥途徑的方式加以調配。視情況提供根據本發明之組合物以用於以單位劑型或多劑型對人類及動物投藥，該等單位劑型或多劑型諸如為錠劑、膠囊、丸劑、散劑、用於吸入者之乾粉、顆粒'無菌非經口溶液或懸浮液、口服溶液或懸浮液、油水乳化液、持續釋放調配物（諸如（但不限於）植入物及微囊密封傳遞系統），其含有合適量之化合物丨。製備此等劑型之方法在此項技術中已知且將對熟習此項技術者顯而易見；例如參見 Remingt〇n’s Pharmaceutical Sciences，第 19版（£邮抓，:Long-term X is related to the correlation between the brewing state of histones and gene transcription. Specific enzymes that regulate the acetylation status of histones, specifically acetamylase (eg, histone acetyltransferase (ΗΑτ)) and de-acetylase (• protein-de-I-based enzyme or HDAC) It has been identified in many organisms and has been associated with regulation of a large number of genes, confirming the link between acetylation and transcription. In general, the acetylation of histone histones is related to transcriptional activation, and the deacetylation of serotonin is related to gene suppression. • Another J more and more histones go to the enzyme (hdac). A well-characterized transcriptional inhibitor, such as MAD, nuclear receptor, and YY1, which binds to the promoter and inhibits transcription of large multiprotein complexes, associates with the HDAC complex to exert its inhibitor function. 128407.doc -59· 200911757 The compounds of the present invention are useful for treating or preventing diseases involving cell proliferation and differentiation. Studies on HDAC inhibitors have shown that these enzymes play an important role in cell proliferation and differentiation. HDAC is associated with a variety of disease conditions including, but not limited to, cell proliferative diseases and conditions (Marks, PA, Richon, VM, Breslow, R. and Rifkind, RA., J. Natl. Cancer Inst. (Bethesda) 92, 1210-1215, 2000), such as leukemia (Lin et al, 1998. Nature 391: 811-814; Grignani et al, 1998. Nature 391: 815-818; Warrell et al, 1998, J, Natl. Cancer Inst. 90: 1621-1625; Gelmetti et al., 1998, Mol. Cell Biol. 18:7185-7191; Wang et al., 1998, PNAS 951 0860-1 0865), melanoma/squamous cell carcinoma (Giiienwater et al. , 1 998, Int. J. Cancer 75217-224; Saunders et al, 1999, Cancer Res. 59: 399-404), breast cancer, prostate cancer, bladder cancer (Geimetti et al. '1998, Mol. Cell Biol 18: 7185-7191; Wang et al., 1998, PNAS 951 0860-10865), lung cancer, ovarian cancer, colon cancer (Hassig et al. 1997, Chem. Biol. 4: 783-789; Archer et al., 1998, PNAS, 956791 -6796; Swendeman et al, 1999, Proc. Amer. Assoc· Cancer Res. 40, Abstract #3836) and hyperproliferative skin diseases such as cancer and cancer Skin lesions and inflammatory skin disorders. Histone deacetylase inhibitors as new cancer drugs, which are effective inducers of growth arrest, differentiation or apoptotic cell death in culture and in a variety of transformed cells in tumor-bearing animals. Marks, PA·, Richon, VM, Breslow, R. and Rifkind, RA, Current 〇pini〇ns in 〇nc〇i〇gy, November 2001 128407.doc -60- 200911757 13 (6) 477-83 Histone deacetylases and cancer: causes and therapies, Marks, P., Rifkind, RA, Richon, VM, Breslow, R., Miller, T. and Kelly, WK·, Nat. Rev. Cancer December 1, 2001 曰( 3): 194.202). Further, the compounds of the present invention in any of the disclosed crystalline forms are also suitable for use in the treatment or prevention of protozoa (U.S. Patent No. 5,922,837) and psoriasis (PCT Publication No. WO 02/26696). Combination Therapy A variety of therapeutic agents can have therapeutic additive or synergistic effects with the HDAC inhibitors according to the present invention. Such therapeutic agents may be combined or synergistically combined with HDAC inhibitors to inhibit unwanted cell growth, such as inappropriate cell growth leading to undesirable benign conditions or tumor growth. In one embodiment, a method of treating a condition of a cell proliferative disorder comprising treating a cell in combination with a compound according to the invention and an anti-proliferative agent, wherein before, simultaneously with and/or after treatment of the cell with the anti-proliferative agent The treatment of cells according to the compounds of the invention is referred to herein as combination therapy. Please note that a drug treatment is referred to herein as a sequential therapy prior to another agent, even if the agent is administered. Please note that examples of combination therapies intended to encompass the first or subsequent administration of the agents to each other (sequential therapy) and agents that can be used in combination with HDAC inhibitors include, but are not limited to, anticancer agents, alkylating agents, antibiotic agents. , an antimetabolite, a stimulating agent, a plant-derived agent, and a biological agent. A bismuth-burning agent is a polyfunctional compound having the ability to replace a gas ion with ρ I 友凡土. 128407.doc -61 - 200911757 Example of an alkylating agent These include, but are not limited to, dioxetine (nitrogen mustards) such as chlorambucil, cyclophosphamide, ifosfamide, nitrogen mustard (mechlorethamine), melphalan, urinary uridine mustard, aziridine (eg thiotepa), ketone ketone citrate (eg white) Busuifan), nitrosourea (such as carnumustine, lovastatin; lomustine, streptozocin), atypical burning agent (hexamethylene melamine) Altretamine), dacarbazine And procarbazine, carb〇piastjn and cisplatin. These compounds react with phosphate, amine, hydroxyl, sulfhydryl, thiol and imidazolyl groups. Under physiological conditions, these drugs ionize and produce positively charged ions' attachment to susceptible nucleic acids and proteins, leading to cell cycle arrest and/or cell death. Combination therapy including HDAC inhibitors and alkylating agents can It has a therapeutic synergistic effect on cancer and reduces the side effects associated with such chemotherapeutic agents. Antibiotic agents are a group of modified drugs that produce natural products in a manner similar to antibiotics. Examples of antibiotic agents include, but are not limited to, anthracycline ( Anthracycline) (eg, doxorubicin (d〇xorubicin), daunorubicin, epirubicin, idarubicin, and anthracenedione), mitomycin c ( Mit〇mycin c), bleomyCin, dactin〇mycin, plicatomycin. These antibiotic agents interfere with fine by targeting different cell components. For example, f ‘ general salty#蒽环# prime interferes with DNA topoisomerase 在 in the transcriptional DNA region, causing DNA strand segregation. In general, bleomycin chelating iron and forming an activation complex, which in turn binds to the base of DNA, causing sequestration and cell death. Combination therapies including HDAC inhibitors and antibiotic agents can have a therapeutic synergistic effect on cancer and reduce the side effects associated with such chemotherapeutic agents. Antimetabolites are a group of drugs that interfere with the metabolic processes necessary for physiology and cancer cell proliferation. Actively proliferating cancer cells require continuous synthesis of large amounts of nucleic acids, proteins, lipids, and other important cellular components. Many antimetabolites inhibit the synthesis of purine or pyrimidine nucleosides or enzymes that inhibit DNA replication. Some anti-metabolites also interfere with the synthesis of ribonucleosides and RNA and/or amino acid metabolism as well as protein synthesis. By interfering with the synthesis of important cellular components, antimetabolites can delay or arrest cancer cell growth. Examples of antimetabolites include, but are not limited to, fluorouracU (5_FU), fluorouridine (5-FUdR), meth〇trexate, formazan tetrahydrofolate ( Leucovorin), hydrazine (hydr0XyUrea), thioguamne (6-TG), sulfhydryl. Mercapt〇purine (6_Mp), Cytarabine, pent〇statin, fludarabine phosphate, cladribine (2_CDA), Aspartate aglyase and gemcitabine. Combination therapies including HDAC inhibitors and antimetabolites can have a therapeutic synergistic effect on cancer and reduce the side effects associated with such chemotherapeutic agents. Hormone agents are a group of drugs that regulate the growth and development of their target organs. Most hormonal agents are sex steroids and their derivatives and analogs such as estrogens, androgens and progestins. These hormone agents can be used to supplement the sex steroids. 128407.doc •63· 200911757 Stylistic antagonists downregulate receptor expression and transcription of important genes. Examples of such hormonal agents are synthetic estrogens (e.g., diethylstibestrol), antiestrogens (e.g., tamoxifen, toremifene, flu〇xymesier〇). 1) and raloxifene (ral〇xifene), antiandrogens (bicalutamide, nilutamide, fiutamide), aromatase inhibitors (eg, amine Amin〇glutethimide, anastr〇z〇le and tetraz〇ie, ketoconaz〇le, goserelin acetate, bright propyl ieupr〇Hde, megestrol acetate and mifeprist〇ne. Combination therapies including HDAC inhibitors and hormonal agents have therapeutic synergistic effects on cancer and reduce the side effects associated with such chemotherapeutic agents. Plant-derived agents are a group of drugs that have been modified from plant or drug-based molecular structures. Examples of plant-derived agents include, but are not limited to, Changchun! Quercus bioassays (eg, vincristine, vinblastine, vindesine, vinzolidine, and vinorelbine) (vin〇relbine)), podophyllotoxin (eg, et 〇p〇side (vp_16) and teniposide 'VM-26), taxane (eg paclitaxel) Paclitaxel) and docetaxel (doceuxei)). These plant-derived agents generally act as anti-mitotic agents that bind to tubulin and inhibit mitosis. The podophyllotoxin, such as Etoposide, interferes with DNA synthesis by interacting with the topoisomerase π, causing DNA strand separation. Combination therapies including HDAc inhibitors and plant-derived agents can have a therapeutic synergistic effect on cancer 128407.doc -64 - 200911757 and reduce V, side effects associated with this specialty chemotherapeutic agent. Bio-music agents are used when used alone or in combination with chemotherapy and/or radiation therapy. Examples of biomolecules include, but are not limited to, immunomodulatory proteins, such as cells: = tumor antigens. Antibodies, tumor suppressor genes and cancer vaccines. Combination therapies including H D A C inhibitors and biologic agents can have a therapeutic synergistic effect on cancer, enhance the patient's immune response to tumorigenic signals, and reduce potential side effects associated with this chemical therapeutic agent. Cytokines have profound immunomodulatory activities. Some cytokines and interferons such as interleukin-2 (匕_2, aldesleukin) have proven antitumor activity and have been approved for the treatment of metastatic renal cell carcinoma and metastatic malignant melanoma. The patient. IL_2 is a tau cell growth factor that is important for eliciting an immune response by T cells. The selective anti-tumor effect of Xianxin IL_2 on a two-hearted person is the result of an immune response that distinguishes between self and non-self cells. Examples of interleukins that can be used in conjunction with HDAC inhibitors include, but are not limited to, interleukin 2 (IL-2) and interleukin 4 (IL-4) 'interleukin 12 (IL-12). Interferons include more than 23 related subtypes with overlapping activities, all of which are within the scope of the invention. IFN has been shown to be active in many solid and hematological malignancies, which appear to be particularly sensitive. Other cytokines that can be used in conjunction with HDAC inhibitors include those cytokines that exert profound effects on hematopoiesis and immune function. Examples of such cytokines include, but are not limited to, erythropoietin, granulocyte_c (filgrastin), and granulocytes, macrophages-CSF (sagstatin 128407.doc-65-200911757) (5&^^111031;丨111)). It can be combined with 110 VIII inhibitors to reduce the cytotoxicity induced by chemotherapy. It can also be combined with HDAC inhibitors to use other immunomodulatory agents other than cytokines to inhibit abnormal cell growth. Examples of modulators include, but are not limited to, bacillus Calmette-Guerin, levamisole, and octreotide (a long-acting octapeptide that mimics the action of the naturally occurring hormone somatostatin). Monoclonal antibodies against tumor antigens are antibodies raised against tumor-expressing antigens, preferably tumor-specific antigens. For example, for human epidermal growth factor receptor 2 that is overexpressed in some breast tumors including metastatic breast cancer (HER2) produces the monoclonal antibody HERCEPTIN® (Trastruzumab). The overexpression of HER2 protein is associated with aggressive disease and poor clinical prognosis. HERCEPTIN® is used to treat metastatic breast cancer with over-tumor A single agent for patients exhibiting HER2 protein. Combination therapy including HDAC inhibitor and HERCEPTIN® can be swollen In particular, there is a therapeutic synergistic effect on metastatic cancer. Another example of a monoclonal antibody against a tumor antigen is RITUXAN® (Rituximab), which is produced and selectively targeted to CD20 on lymphoma cells. Depletion of normal and malignant CD20+ pre-B cells and mature B cells. RITUXAN® is used to treat patients with relapsed or refractory low-grade or follicular, CD20+, B-cell non-Hodgkin's lymphoma Single agent. Combination therapy including HDAC inhibitor and RITUXAN® not only has a therapeutic synergistic effect on lymphoma but also on other forms or types of malignant tumors. 128407.doc -66- 200911757 Tumor suppressor gene is used to inhibit cell growth and division Cycles, thereby preventing the development of neoplastic formation. Mutations in tumor suppressor genes cause cells to ignore one or more components of the nickname network, thereby overcoming cell cycle checkpoints and producing a higher rate of cancer growth by controlled cells. Examples of tumor suppressor genes include, but are not limited to, DPC_4, nim, NF_2, RB, p53, WT1, BRCAl, and BRCA2. DPC-4 and pancreas NF-1 encodes a protein that inhibits Ras, a cytoplasmic inhibitory protein. NF-1 is involved in neurofibroma and pheochromocytoma and myeloid leukemia in the nervous system. NF-2 A nuclear protein encoding a neurotrophic meningioma, schwannomas, and ependymoma. RB encodes a pRB protein (a nuclear protein that is a major inhibitor of cell cycle function). RB is associated with retinoblastoma and bone cancer 'bladder cancer, small cell lung cancer, and breast cancer. p53 encodes a p53 protein that regulates cell division and induces apoptosis. Mutations and/or no effects of p53 can be found in a variety of cancers. WT1 is associated with the Wilms tumor of the kidney. BRCA1 is associated with breast cancer and nest cancer, and BRCA2 is associated with breast cancer. The tumor suppressor gene can be transferred to tumor cells in which the tumor suppressor gene exerts its tumor suppressing function. Combination therapies including HDAC inhibitors and tumor suppressors can have therapeutic synergistic effects in patients with various forms of cancer. The cancer vaccine is a group of drugs that are responsible for the body's specific immune response to the tumor. Most cancer vaccines that conduct research and development and clinical trials are tumor-associated antigens (TAAs). ΤΑA is a structure which is present on tumor cells and which is relatively absent or reduced on normal cells (i.e., peptone, enzyme or carbon water 128407.doc-67-200911757 compound). Because of its uniqueness to tumor cells, TAA provides the goal of the immune system to recognize and cause its destruction. Examples of TAA include, but are not limited to, ganglioside (GM2), prostate specific antigen (pSA), alpha fetoprotein (AFP), carcinoembryonic antigen (CEA) (from colon cancer and, for example, breast cancer, lung cancer, gastric cancer and Other adenocarcinomas of pancreatic cancer), melanoma-associated antigens (mart_1, gplOO, MAGE 1, 3 tyrosinase), papillomavirus £6, and monthly, autologous tumor cells and heterologous tumor cells Whole cells or parts/lysates. Adjuvants can be used to enhance the immune response to TAA. Examples of adjuvants include, but are not limited to, BCG, endotoxin lipopolysaccharide, keyhole snail glycoprotein (GKLH), interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF) and Cytoxan (a chemotherapeutic agent believed to reduce tumor-induced inhibition when administered at low doses). A pharmaceutical composition comprising Compound I, II or III, wherein at least one of the crystalline solids of Compound I, II or III is present, and Compounds I, II or III can be used in various pharmaceutical compositions, at least a portion of Compounds I, II or In is present in the composition in the form of a crystal of the compound. The pharmaceutical compositions should contain sufficient amounts of Compound I, 11 or oxime to substantially reduce HDAC activity in vivo to provide the desired therapeutic effect. These pharmaceutical compositions may be included between 0.005% and 100% (weight/weight), optionally ι_ι%, and optionally from 1 to 95 °/. Compounds I, η or III present in the composition within the range. In a particular embodiment, the pharmaceutical composition comprises at least 0.1%, 0 2 5 %, 0·5ο/〇, 1%, 5%, 1〇. /0, 25%, 50%, 75〇/〇, 80%, 85%, 128407.doc •68- 200911757 90%, 95°/. 97% or 99% of compound I, II or III, which is the crystalline form disclosed herein. In another embodiment, the specific crystalline form of Compound I, II or III in the pharmaceutical composition may comprise at least 0.1%, 1%, 5%, 10% '25% > 50% > 75% ' 80% > 85% > 90% > 95% > 97% 3⁄4 99% Total amount of compound I, π and IIt (weight/weight). In addition to the compound I, II or III, the pharmaceutical composition may also contain one or more additional components which do not adversely affect the use of the compound. For example, in addition to the compound I, the pharmaceutical composition may also include a conventional pharmaceutical carrier; an excipient; a diluent; a lubricant; a binder; a wetting agent; a disintegrant; a slip agent; a sweetener; Agent; emulsifier; solubilizer; pH value buffer; fragrance; surface stabilizer; suspension; and other conventional pharmaceutical inactive agents. In particular, the pharmaceutical composition may comprise lactose, mannitol, glucose, sucrose, dicalcium phosphate 'magnesium carbonate, sodium saccharin, carboxymethyl cellulose, magnesium stearate, calcium stearate, cross-linked carboxymethyl group. Cellulose sodium, talc' starch, natural gum (such as arabic gelatin), molasses, polyvinylpyrrolidine, cellulose and its derivatives, polyethylene ketone, cross-linked polyethylene: acetate, sodium citrate, Cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, biocompatible poly (such as collagen, ethylene vinyl acetate, polyanhydride, polyglycolic acid, poly Orthoester, polylactic acid and other such agents). The pharmaceutical composition according to the present invention may be suitably exemplified by any of a variety of routes, and the pharmaceutical composition according to the present invention may optionally be administered in the following manner: oral, parenteral , intraperitoneal, intravenous, intraarterial, topical, transdermal, sublingual, intramuscular, by 128407.doc •69- 200911757 intestinal cheek, intranasal, transliposome, via inhalation, transvaginal, transocular Internal, via local delivery (eg by catheter or intravascular stent), subcutaneous, intra fat, intra-articular or intrathecal. In a particular embodiment, the pharmaceutical compound is administered orally, by inhalation or by subcutaneous, intramuscular, intravenous or direct injection into the cerebrospinal fluid. In general, the pharmaceutical compositions of the present invention can be prepared in a gaseous, liquid, semi-liquid, gel or solid form and formulated in a manner suitable for the route of administration desired. The composition according to the present invention is optionally provided for administration to humans and animals in unit dosage form or in multiple dosage forms such as tablets, capsules, pills, powders, dry powders for inhalers, granules. 'Aseptic parenteral solutions or suspensions, oral solutions or suspensions, oil-water emulsions, sustained release formulations (such as, but not limited to, implants and microencapsulated delivery systems) containing a suitable amount of the compound hydrazine. Methods of preparing such dosage forms are known in the art and will be apparent to those skilled in the art; for example, see Remingt〇n's Pharmaceutical Sciences, 19th Edition (£ post,:

Mack Publishing Company，1995)。如本文中所用之單位劑量形式係指適用於人類及動物個體且如在此項技術中已知經個別包裝之物理上離散單位。各單位劑量含有足以產生所需治療作用的預定量之與醫藥載劑、媒劑或稀釋劑組合之化合物jΠΙ。單位劑量形式之實例包括安瓿及注射器及個別包裝之錠劑或膠囊。可分多份或多個投與單位劑量形式。多劑量形式為欲以隔離單位劑量形式投與之包裝於單一容器中之複數個相同單位 128407.doc -70- 200911757 劑量形式。多劑量形式之實例包括小瓶、錠劑或膠囊瓶或品脫或加侖瓶。因此，可將多劑量形式視作未經隔離於包裝中之多個單位劑量。一叙而言，在根據本發明之醫藥組合物中的化合物丨、η 或III之總量應足以提供所需治療作用。此量可作為單一每天劑置、欲以時間間隔投藥之每天多個劑量或作為連續釋放劑型來傳遞。當對患者投藥時，可以在i毫克/天與250 毫克/天之化合物I、II或III之間、視情況在2 5 〇^與2〇〇 mg化合物I、n或ΠΙ之間、視情況在2 5 〇^與15〇 mg化合物I、II或III之間及視情況在5 mg與100 mg化合物J、11或 hi之間（在各情況下以游離鹼形式之化合物I、Π或III之分子量計）的日劑量有利地使用化合物I、„或ΙΠ。可使用之特定劑量的量包括（但不限於）2.5 mg、5 mg、&25 mg、1() mg、20 mg、25 mg、50 mg、75 mg及 100 mg化合物 j、π 或in/天。可能需要每天一次投與化合物i。因此’本發明之醫藥組合物可呈單一劑型之形式，該單一劑型包含在1毫克/天與250毫克/天之間的化合物j、視情況在 2.5 mg與2〇0 mg之間的化合物I、η或in、視情況在2 5 mg 與150 mg之間的化合物I、II或m及視情況在5 mg與1〇〇 mg之間的化合物I、II或III。在特定實施例中，醫藥組合物包含 2.5 mg、5 mg、6.25 mg、10 mg、2〇 mg、25 mg、 5 0 mg、75 mg或 1 00 mg化合物 I。用於口服之調配物口服醫藥劑型可呈固體、凝膠或液體形式。固體劑型之 128407.doc 200911757 a例包括（但不限於）錠劑、膠囊、顆粒及散裝散劑。口服鏡劑之齡姓八 ^ 、疋實例包括可經腸溶包衣塗佈、糖包衣塗佈或專膜包衣塗佈之壓縮、可咀嚼***劑及錠劑。膠囊之實例包^硬質明膠膠囊或軟質明膠膠囊。可以非發泡或發泡形式提仏顆粒及散劑。每一者均可與熟習此項技術者已知之其他成份組合。在某些實施例中，以固體劑型、較佳膠囊或錠劑提供根據本發明之化合物。錠劑、丸劑、膠囊、片劑及其類似物可視情況含有—或多種以下成份或具有類似性質之化合物·黏合劑；稀釋劑；崩解劑；潤滑劑；滑動劑；甜味劑；及調味劑。可用之黏合劑的實例包括（但不限於）微晶纖維素、黃蓍膠葡萄糖溶液、***膠聚（acacia mucilage)、明膠溶液、蔗糖及澱粉糊。可用之潤滑劑的實例包括（但不限於）滑石、澱粉、硬脂酸鎂或硬脂酸鈣、石鬆粉及硬脂酸。可用之稀釋劑的實例包括（但不限於）乳糖、蔗糖、澱粉、高嶺土、鹽、甘露糖醇及磷酸二鈣。可用之滑動劑的實例包括（但不限於）膠狀二氧化矽。可用之崩解劑的實例包括（但不限於）交聯羧曱基纖維素鈉、經基乙酸澱粉鈉、褐藻酸、玉米殿粉、馬鈴薯殿粉、膨潤土、甲基纖維素、瓊脂及羧曱基纖維素。可用之著色劑的實例包括（但不限於）任何經批准認證之水溶性FD及C染料、其混合物；及懸浮於水合氡化紹上之 128407.doc •72· 200911757 水不溶性FD及C染料。可用之甜味劑的實例包括（但不限於）蔗糖、乳糖、甘露糖醇及人造甜味劑（諸如赛克拉美鈉（s〇dium cyciamate)及糖精）及許多噴霧乾燥香料。可用之調味劑的實例包括（但不限於）自諸如水果之植物中提取之天然香料及產生愉悅感覺之化合物的合成摻合物’諸如（但不限於）薄荷及水揚酸甲醋。可用之濕潤劑的實例包括（但不限於）單硬脂酸丙二醇酉旨、脫水山梨醇單油酸酿、單月桂酸二乙二醇醋及聚氧化乙烯月桂基醚。可用之止吐包衣的實例包括（但不限於）脂肪酸、脂肪、壌蟲膠氨化蟲膠（ammoniated shellac)及醜酸乙酸纖維素。可用之薄膜包衣的實例包括（但不限於）羥乙基纖維素、羧甲基纖維素鈉、聚乙二醇4〇〇〇及酞酸乙酸纖維素。若需要口服，則可視情況將化合物之鹽提供於保護其免欠胃之酸性環境影響之組合物中。舉例而言，可將組合物調配於在月中保持完整性且在腸中釋放活性化合物之腸溶包衣t。組合物亦可與抗酸劑或其他此種成份組合調配。當單位劑型為膠囊時，其可視情況另外包含諸如脂肪油之液體載劑。另外，單位劑型可視情況另外包含各種其他 G飾JI單位物理形式之物質，例如糖及其他腸溶藥劑之包衣。根據本發明之化合物亦可作為醜劑、懸浮液、糖漿、糯 128407.doc •73 - 200911757 米紙囊劑（wafer)、噴灑劑、口嚼錠或其類似物之組份來投藥。除活性化合物之外’糖漿亦可視情況包含作為甜味劑之蔗糖及特定防腐劑、染料及著色劑及香料。本發明之化合物亦可與並不削弱所需作用之其他活性物質混合，或與補充所需作用之物質混合，諸如抗酸劑、H2 阻斷劑及利尿劑。舉例而言，若化合物用於治療哮喘或高企壓，則其可分別與其他支氣管擴張藥及抗高血壓藥一起使用。 ^ 包含本發明之化合物的錠劑中可包括之醫藥學上可接受 =載劑的實例包括（但*限於）黏合劑、潤㈣、稀釋劑^ 崩解劑、I色劑、調味劑及濕潤劑。腸溶包衣錠劑由於腸溶包衣，因此抵抗胃酸作用且溶解或崩解於中性或驗性腸中。糖包衣錠劑可為經塗覆醫藥學上可接受之物質之不同層的壓縮錠劑。薄臈包衣錠劑可為已塗有聚合物或其他合適包衣之壓縮鍵劑。多重壓縮鍵劑可為由—個以上利用先刖提及之醫藥學上可接受之物質的壓縮循環製造之壓縮錠劑。著色劑亦可用於錠劑中。調味劑及甜味劑可用於鍵劑中，且尤其適用於形成咀嚼錠及***劑。、可用^夜體口服劑型的實例包括（但不限於）水溶液、乳液、m、溶液及/或自非發泡顆粒復水之懸浮液及自發泡顆粒復水之發泡製劑。可用之水溶液的實例包括(但不限於)酏劑及糖漿。如本文中所用’酏劑係指澄清、加甜的水醇製劑。可用於酏劑中之醫藥學上可接受之載劑的實例包括（但不限於）溶劑。 I28407.doc -74- 200911757 可用之溶劑的特定實例包括甘油、山梨糖醇、乙醇及糖桌士本文中所用，糖聚係指例如蔗糖之糖的濃縮水溶液。糖漿可視情況另外包含防腐劑。乳液係指-種液體以小球形式分餘整個另—液體中之兩相系統。乳液可視情況為水包油或油包水乳液。可用於札液中之醫樂學上可接受之載劑的實例包括(但不限於)非水液體、乳化劑及防腐劑。可用於欲經復水為液體口服劑型之非發泡顆粒的醫藥學上可接又之物I的實例包括稀釋劑、甜味劑及渴潤劑。 ’、可用於欲經復水為液體口服劑型之發泡顆粒的醫藥學上可接受之物質的實例包括有機酸及二氧化碳源。者色劑及調味劑可視情況用於所有上述劑型中。可用之防腐劑的特定實例包括甘油、對經基苯甲酸"旨及對羥基苯甲酸丙酯、苯甲酸、苯甲酸鈉及醇。可用於乳液中之非水液體的特定實例包括礦物油及棉杆油。可用之礼化劑的特定實例包括明勝、***夥、黃著膠、膨潤土及諸如嘴® >{卜7、I* . 洧如汆軋化乙烯脫水山梨醇單油酸酯之界面活性劑。一可用之懸浮劑的特定實例包括缓甲基纖維素鈉、果膠、黃箸膠、Mg贿及***膠。稀釋劑包括乳糖及簾糖。甜味劑包括薦糖、糖衆、甘油及諸如赛克拉美鈉及糖精之人造甜味劑。 128407.doc -75- 200911757 可用之濕潤劑的特定實例包括單硬脂酸丙二醇酯、單油凌脫水山4醇s日、單月桂酸二乙二醇g旨及聚氧化乙稀月桂基峻。可用之有機酸的特定實例包括檸檬酸及酒石酸。 °用於發&組合物中之三氧化碳源包括破酸氫納及碳酸鈉著色劑包括任何經批准認證之水溶性FD& c染料及其混合物。可用之調味劑的特定實例包括自諸如水果之植物中提取之天然香料及產生愉快味覺之化合物的合成摻合物。對於固體劑型而言，較佳將在（例如）碳酸丙二酯、植物油或甘油二酯中之溶液或懸浮液囊封於明膠膠囊中。在美國專利第4,328,245號、第4,409,239號及第4,410,545號中揭示此等溶液及其製備及封裝。對於液體劑型而言，例如在聚乙二醇中之溶液可以足量醫藥學上可接受之液體載劑 (例如水）稀釋以易於針對投藥進行量測。或者’可藉由將活性化合物或鹽溶解或分散於植物油、一醇、甘油三酯、丙二酵酯（例如碳酸丙二酯）及其他此等载劑中且將此等溶液或懸浮液囊封於硬質明膠膠囊或軟質明膠膠囊外殼中來製備液體或半固體口服調配物。其他適用之調配物包括在美國專利第Re 28,8 19號及第4,358,603 號中闡明之彼等調配物。可注射物、溶液及乳液本發明亦係關於設計成藉由一般特徵在於皮下、肌肉内或靜脈内注射之非經口投藥來投與本發明之化合物的組合 128407.doc •76· 200911757 物。可注射物可製備為任何習知縣.重、为士 t式’例如呈液體溶液武序液、適用於在注射前於液科士、〜次或呈乳液形式。J於液體中洛解或懸浮之固體形式 :與根據本發明之可注射物結合使狀賦括（但不限於）水、鹽水、右旋糖、甘油或乙醇。可^包合物亦可視情況包含少量無毒性辅所 /射、-且乳化劑、pH值緩衝劑、稃定劑々s = °濕潤劑或 “ n溶解度增強劑及其他此笼樂劑，諸如乙酸鈉、單月桂醚等早月桂I脫水山梨醇酯、三乙酸酯及環糊精。本文亦、¥ -油尽文亦A盍植入緩慢釋放或持續釋放統，使得保持怪定含量、 ’、菫之劑量（例如參見美國專利第 3,710,795號）。此等非經口相入私山丈、〜組合物中所含之活性化合物的百为比面度取決於其特定性質買^及化合物之活性及個體之需要。調配物之非經口投筚白把餘又樂包括靜脈内、皮下及肌肉内投藥。用於非經口投藥之製劑包括準 ’ ^ 干W用於注射之無菌溶液、無囷無水可溶性產品（諸如進從^ + 丰備在即將使用之前與溶劑組合的本文所述之來乾散齋丨，由紅+ ^ 欺劑包括皮下錠劑）、準備用於注射之無菌懸浮液、準備在即將使 a 月十彳更用之則與媒劑組合之無菌無水不溶性產品及無菌乳液。溶液可為水性或#水性；當靜脈内投藥時，合適載劑之實例包括（但不限於）生理鹽水或填酸鹽緩衝生理睡火A ^ ^ 1水（PBS)及含有諸如葡萄糖、聚乙-醇及聚丙—醇及其混合物之增稠劑及增溶劑的溶液。可視情況用於非經口製劑中之醫藥學上可接受之載劑的實例包括(但不限於)水性媒劑、非水性媒劑、抗菌劑、等 128407.doc •77· 200911757 張J、緩衝劑、抗氧化劑、局部麻醉劑、懸浮劑及分散劑、乳化劑、鉗合劑或螯合劑及其他醫藥學上可接受之物質。可視情況使用之水性媒劑的實例包括氣化鈉注射劑、林袼氏注射劑（Ringers Injection)、等張右旋糖注射劑、無菌水注射劑、右旋糖及乳酸化林格氏注射劑。可視情況使狀非水性非經口媒劑的實例包括植物來源之不揮發性油、棉籽油、玉米油、芝麻油及花生油。尤其當非經π製劑經包裝於多劑量容器中且因此設計成用於儲存且多個等分試樣欲經移料，可添加抑g或抑直菌濃度之抗菌劑至該等製劑中。可使用之抗菌劑的實例包括笨酚或甲酚、汞劑，、氯丁醇、對經基苯甲酸甲醋及對羥基苯甲酸丙酯、硫柳汞、氣化笨甲烴銨 (benzalkoniu, chl〇ride) ^ ^ ^ ^ (benzethoniu, chloride)。可用之等張劑的實例包括氯化納及右旋糖。可用之緩衝劑的實例包括磷酸鹽及擰檬酸 ^ j用之抗氧化劑的實例包括硫酸氣納。可用之片立β、用之局°卩麻醉劑的實例包括氫氯酸奴佛卡因（Pr〇C⑽e hydrochloride)。可用之懸浮劑及分實例包括羧甲基纖維素鈉、羥丙土 τ基纖維素及聚乙稀叫匕略咬嗣。彳用之乳化劑的實例包括聚山梨醇㈣(TW卿 80)。金屬離子之鉗合劑或f合劑包括edta。醫藥載劑亦可視情況包括 ^ 作水可混溶媒劑之乙醇、聚乙二醇及丙二醇及用於1^值即之虱礼化鈉、氫氯酸、檸 128407.doc •78· 200911757 樣酸或乳酸。可調節在非經口調配物中之抑制劑的濃度以使得注射劑投與足以產生所需藥理學作用之醫藥學有效量。如在此項技術中已知，欲使用之抑制劑及/或劑量的精確濃度將最終視患者或動物之年齡'重量及病狀而定。可將單位劑量非經口製劑包裝於安瓿、小瓶或具有針頭 f 之注射器中。如在此項技術中已知及實施，用於非經口投藥之所有制劑應無菌。 °將可注射物設計為局部及全身性投藥。通常將治療有效劑量调配為含有至少約01% w/w至約9〇% 或卯％ w/wu上、較佳大於1% w/以之 hdac抑制劑相對於所治療組織的艰度。可一次性或可分成多個欲以時間間隔投與之較小劑量來投與抑㈣卜應瞭解治療之精確劑量及持續時間將隨組合物非經口投與之位置、載劑及其他變數而變， σ吏用已知κ方案或藉由自活體内或活體外測試資料外推來以經驗確定該等其他變數。應注意濃度及劑量值亦可隨所治療個體之年齡而改變。應進—步瞭解對於任何特定個體而言’根據個體需要及投與或監督調配物投與之人員之專業判斷可能需要隨著時間調節特定給藥方案。因此，本文中闡明之濃度範圍意欲為例示性的且並不意欲限制所主張調配物之範疇或實務。、：c抑制劑可視情況以微米尺寸化或其他合適形式懸 :或可經衍生化以產生可溶性較高之活性產物或產生前、。所得混合物之形式視多種因素而定，包括預定投藥模 128407.doc -79- 200911757 。有效濃度足以式及化合物在所選載劑或媒劑中之溶解度改善疾病病況之症狀且可憑經驗判定。义凍乾散劑劑’其可針對投藥可將凍乾散劑調配亦可將本發明之化合物製備為凍乾散而復水為溶液、乳液及其他混合物。亦為固體或凝膠劑。可错由將化合物溶解於含有右旋糖或其他合適賦形劑之 :酸納緩衝溶液中來製備無菌康乾散劑。隨後無菌過滤溶液，接著在熟習此項技術者已知之標準條件下來乾提供所需调配物。簡言之，可視情況藉由通常在約中性pH值下將約1-鳩、較佳約5%至15%之右旋糖、山梨糖醇、果糖、玉米糖激、木糖醇、甘油、葡萄糖、薦糖或其他合適藥劑溶解於諸如檸檬酸鹽、錢納或磷酸鉀或熟f此項技術者已知之其他此類緩衝劑之合適緩衝劑中來製備凍乾散劑。接著，較佳在室溫以上、更佳在約3(rc_35〇c添加HDAC# 制劑至所得混合物中，且攪拌直至其溶解。藉由添加更多緩衝劑至所需濃度來稀釋所得混合物。所得混合物經無菌 ^;慮或處理以移除微粒且確保無菌性，且經分配於小瓶中用於涞乾。各小瓶可含有單一劑量或多劑量之抑制劑。局部投藥本發明之化合物亦可以局部混合物形式來投藥。局部混合物可用於局部及全身性投藥。所得混合物可為溶液、懸浮液、乳液或其類似物且經調配為乳膏、凝膠劑、軟膏、乳液、溶液、醜劑、洗劑、懸浮液、g了劑、糊劑、發泡 128407.doc -80 - 200911757 體、氣霧劑、灌洗劑、噴霧、栓劑、繃帶、表皮貼片或適用於局部投藥之任何其他調配物。 HDAC抑制劑可調配為諸如藉由吸入而局部施用之氣霧劑（參見美國專利第4,044，126號、第4,414,2〇9號及第 4,364,923號，其描述用於傳遞適用於治療發炎性疾病、尤八哮而之類固醇的氣務劑）。此等用於投與呼吸道之調配物可呈氣霧劑或溶液形式以用於噴霧器，或呈微細粉末形 ( 式以用於吹入’其單獨或與諸如乳糖之惰性載體組合。在此情況下，調配物之顆粒通常將具有小於5〇微米、較佳小於10微米之直徑。抑制劑亦可調配為呈凝膠劑、乳膏及洗劑之形式用於局部施用’ I#如用☆對皮膚及黏膜(諸如在眼睛中)之局部施用及用於對眼睛施用或用於腦池内或脊柱内施用。對於經 f傳遞以及對於投與眼睛或黏膜或對於吸人療法而言，涵蓋局部投藥。亦可投與單獨或與其他醫藥學上可接受之賦开夕劑組合的HDAC抑制劑之經鼻溶液。用於其他投藥途徑之調配物視所治療之疾病病況而定，亦可使用諸如局部施用、經皮貼片及經直腸投藥之其他投藥途徑。舉例而言，用於: 直腸投藥之醫藥劑型為用於全身作用之直腸栓劑、膠囊及錠劑。本文中所使用之直腸栓劑意謂用於插人直腸中、在體溫下溶融或軟化從而釋放一或多種藥理學上或治療上活性成份之固體。用於直腸栓劑中之醫藥學上可接受之為基劑或媒劑及升高㈣之藥劑。基劑之實例包括可可月匕 128407.doc •81 · 200911757 (可可油）、甘油明膠、聚乙二醇、（聚氧乙二醇）及脂肪酸之甘油單酯、甘油二酯及甘油三酯之適當混合物。可使用各種基劑之組合。升高栓劑熔點之藥劑包括鯨蠟及蠟。可藉由壓縮方法或藉由模製來製備直腸栓劑。直腸栓劑之典型重量為約2至3 gm。可使用相同的醫藥學上可接受之物質且藉由與用於口服調配物相同之方法製造用於直腸投藥之錠劑及膠囊。調配物之實例以下為可視情況用於本發明之化合物之口服、靜脈内及錠劑調配物的特定實例。注意此等調配物可視所用特定化合物及將要使用何種調配物之指示而改變。口服調配物本發明之化合物單水合擰檬酸氫氧化納調味劑水 10-100 mg 105 mg 18 mgMack Publishing Company, 1995). Unit dosage form as used herein refers to physically discrete units that are suitable for use in human and animal individuals and are individually packaged as is known in the art. Each unit dose contains a predetermined amount of a compound in combination with a pharmaceutical carrier, vehicle or diluent sufficient to produce the desired therapeutic effect. Examples of unit dosage forms include ampoules and syringes and individually packaged tablets or capsules. The unit dosage form can be administered in multiple or multiple doses. The multiple dose form is a plurality of identical units that are intended to be packaged in a single container in the form of a separate unit dose. 128407.doc -70- 200911757 Dosage form. Examples of multiple dosage forms include vials, lozenges or capsules or pints or gallons. Thus, multiple dose forms can be considered as multiple unit doses that are not isolated in the package. In general, the total amount of the compound 丨, η or III in the pharmaceutical composition according to the invention should be sufficient to provide the desired therapeutic effect. This amount can be delivered as a single daily dose, multiple doses per day to be administered at time intervals, or as a continuous release dosage form. When administered to a patient, between i mg/day and 250 mg/day of compound I, II or III, as appropriate, between 2 5 〇^ and 2 〇〇mg of compound I, n or 、, as appropriate Between 2 5 〇^ and 15 〇mg of compound I, II or III and optionally between 5 mg and 100 mg of compound J, 11 or hi (in each case in the form of the free base, compound I, hydrazine or III) The daily dose of the molecular weight meter advantageously uses the compound I, „ or ΙΠ. The specific doses that can be used include, but are not limited to, 2.5 mg, 5 mg, & 25 mg, 1 () mg, 20 mg, 25 Mg, 50 mg, 75 mg and 100 mg of compound j, π or in/day. It may be necessary to administer compound i once a day. Therefore, the pharmaceutical composition of the invention may be in the form of a single dosage form comprising 1 mg. Compound j between days and 250 mg/day, Compound I, η or in between 2.5 mg and 2.0 mg, as appropriate, between Compounds I and II, between 25 mg and 150 mg, as appropriate m and optionally between 5 mg and 1 mg of compound I, II or III. In a particular embodiment, the pharmaceutical composition comprises 2.5 mg, 5 mg, 6.25 m g, 10 mg, 2 mg, 25 mg, 50 mg, 75 mg or 100 mg of compound I. Oral formulations for oral administration can be in solid, gel or liquid form. Solid dosage form 128407.doc 200911757 a case includes, but is not limited to, tablets, capsules, granules and bulk powders. The age of the oral lens is 8^, examples of which include enteric coating, sugar coating or film coating. Coated compressed, chewable buccal and lozenge. Examples of capsules include hard gelatin capsules or soft gelatin capsules. Non-foaming or foaming forms can be used to extract granules and powders. Each can be familiar with this item. Combinations of other ingredients known to the skilled artisan. In certain embodiments, the compounds according to the invention are provided in solid dosage forms, preferably capsules or lozenges. Tablets, pills, capsules, tablets and the like may optionally contain - or a plurality of the following ingredients or compounds having similar properties; a binder; a diluent; a disintegrant; a lubricant; a slip agent; a sweetener; and a flavoring agent. Examples of useful binders include, but are not limited to, microcrystalline cellulose. Astragalus Sugar solution, acacia mucilage, gelatin solution, sucrose and starch paste. Examples of useful lubricants include, but are not limited to, talc, starch, magnesium stearate or calcium stearate, stone pine powder and hard Fatty acids Examples of useful diluents include, but are not limited to, lactose, sucrose, starch, kaolin, salt, mannitol, and dicalcium phosphate. Examples of useful slip agents include, but are not limited to, colloidal ceria. . Examples of useful disintegrants include, but are not limited to, croscarmellose sodium, sodium starch glycolate, alginic acid, corn powder, potato powder, bentonite, methylcellulose, agar, and carboxyhydrazine. Cellulose. Examples of useful coloring agents include, but are not limited to, any of the approved certified water soluble FD and C dyes, mixtures thereof; and water-insoluble FD and C dyes suspended in hydrated hydrazine. Examples of useful sweeteners include, but are not limited to, sucrose, lactose, mannitol, and artificial sweeteners (such as s〇dium cyciamate and saccharin) and a number of spray-dried flavors. Examples of flavoring agents which may be used include, but are not limited to, natural flavors extracted from plants such as fruits and synthetic blends of compounds which produce a pleasant sensation such as, but not limited to, peppermint and salicylate. Examples of humectants which may be used include, but are not limited to, propylene glycol monostearate, sorbitan monooleate, monolaurate diethylene glycol vinegar, and polyoxyethylene lauryl ether. Examples of useful antiemetic coatings include, but are not limited to, fatty acids, fats, ammoniated shellac, and ugly cellulose acetate. Examples of useful film coatings include, but are not limited to, hydroxyethyl cellulose, sodium carboxymethyl cellulose, polyethylene glycol 4, and cellulose acetate phthalate. If oral administration is desired, the salt of the compound can optionally be provided in a composition which protects against the acidic environmental effects of the stomach. For example, the composition can be formulated with an enteric coating t which maintains integrity throughout the month and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient. When the unit dosage form is a capsule, it may optionally contain a liquid carrier such as a fatty oil. In addition, the unit dosage form may optionally contain various other physical forms of the JI unit, such as sugar and other enteric coatings. The compound according to the present invention can also be administered as a component of a ugly agent, a suspension, a syrup, a sputum, a spray, a chewing tablet or the like. In addition to the active compound, the syrup may optionally contain sucrose as a sweetener and specific preservatives, dyes and colorants and perfumes. The compounds of the present invention may also be combined with other active substances which do not impair the desired action, or with substances which complement the desired action, such as antacids, H2 blockers and diuretics. For example, if the compound is used to treat asthma or high stress, it can be used with other bronchodilators and antihypertensives, respectively. ^ Examples of pharmaceutically acceptable = carrier which may be included in the tablet containing the compound of the present invention include (but are limited to) binders, moisturizing agents, diluents, disintegrating agents, coloring agents, flavoring agents, and moisturizing agents. Agent. Enteric coated tablets are resistant to gastric acid and dissolve or disintegrate in neutral or intestines due to enteric coating. The sugar-coated lozenge can be a compressed tablet of a different layer coated with a pharmaceutically acceptable material. The thin enamel coated lozenge can be a compression bond that has been coated with a polymer or other suitable coating. The multiple compression bond can be a compressed tablet manufactured from more than one compression cycle using the pharmaceutically acceptable materials mentioned above. Colorants can also be used in tablets. Flavoring and sweetening agents can be used in the bonding agent and are especially useful in forming chewable ingots and buccal formulations. Examples of usable oral dosage forms include, but are not limited to, aqueous solutions, emulsions, m, solutions and/or suspensions from reconstituted non-foamed granules and foamed preparations from reconstituted granules. Examples of useful aqueous solutions include, but are not limited to, tinctures and syrups. As used herein, "tanning agent" refers to a clarified, sweetened hydroalcoholic preparation. Examples of pharmaceutically acceptable carriers that can be used in the elixirs include, but are not limited to, solvents. Specific examples of the solvent which can be used include glycerin, sorbitol, ethanol and sugar. As used herein, sugar polymerization refers to a concentrated aqueous solution of sugar such as sucrose. The syrup may additionally contain a preservative as the case may be. Emulsion refers to a two-phase system in which a liquid is dispersed in the form of small spheres throughout the other liquid. The emulsion may optionally be an oil-in-water or water-in-oil emulsion. Examples of pharmaceutically acceptable carriers that can be used in the sap include, but are not limited to, non-aqueous liquids, emulsifiers, and preservatives. Examples of pharmaceutically acceptable non-foamable granules which can be used for reconstitution into a liquid oral dosage form include diluents, sweeteners, and cravings. Examples of pharmaceutically acceptable substances which can be used for the foamed granules to be reconstituted into liquid oral dosage forms include organic acids and carbon dioxide sources. The colorant and flavoring agent can be used in all of the above dosage forms as appropriate. Specific examples of usable preservatives include glycerin, p-benzoic acid " and propylparaben, benzoic acid, sodium benzoate and alcohol. Specific examples of non-aqueous liquids that can be used in the emulsion include mineral oil and cotton stalk oil. Specific examples of useful rituals include Mingsheng, Arabian, Yellow Gum, Bentonite, and surfactants such as Mouth® > {i 7, I*. For example, rolled ethylene sorbitan monooleate . Specific examples of a suspending agent that can be used include sodium hyaluronate, pectin, tragacanth, Mg bribe, and gum arabic. Diluents include lactose and curtain sugar. Sweeteners include sucrose, sugar, glycerin and artificial sweeteners such as sikaram and saccharin. 128407.doc -75- 200911757 Specific examples of usable humectants include propylene glycol monostearate, sulphate monosodium sulphate, monolauric acid diethylene glycol g, and polyethylene oxide lauric. Specific examples of useful organic acids include citric acid and tartaric acid. The carbon trioxide source used in the hair & composition comprises sodium hydrogencarbonate and sodium carbonate colorants including any of the approved certified water soluble FD& c dyes and mixtures thereof. Specific examples of usable flavoring agents include natural flavors extracted from plants such as fruits and synthetic blends of compounds which produce a pleasant taste. For solid dosage forms, a solution or suspension in, for example, propylene carbonate, vegetable oil or diglyceride is preferably encapsulated in a gelatin capsule. Such solutions, as well as their preparation and encapsulation, are disclosed in U.S. Patent Nos. 4,328,245, 4,409,239, and 4,410,545. For liquid dosage forms, solutions such as those in polyethylene glycol can be diluted with a sufficient amount of a pharmaceutically acceptable liquid carrier (e.g., water) for ease of administration for administration. Or 'by dissolving or dispersing the active compound or salt in vegetable oils, mono-, triglycerides, propylene glycol esters (such as propylene carbonate) and other such carriers and such solutions or suspensions The liquid or semi-solid oral formulation is prepared by sealing in a hard gelatin capsule or a soft gelatin capsule shell. Other suitable formulations include those formulated as set forth in U.S. Patent Nos. Res. 28,819, and 4,358,603. Injectables, Solutions, and Emulsions The present invention is also directed to compositions designed to administer a compound of the invention by parenteral administration which is generally characterized by subcutaneous, intramuscular or intravenous injection. 128407.doc • 76· 200911757. The injectable preparation can be prepared in any conventional county. The weight is in the form of a liquid solution, for example, in the form of a liquid solution, and is suitable for use in a liquid science before injection, in the form of a liquid, or in the form of an emulsion. Solid form in which J is either dissolved or suspended in a liquid: in combination with an injectable preparation according to the present invention, including but not limited to water, saline, dextrose, glycerol or ethanol. The inclusion compound may also optionally contain a small amount of non-toxic auxiliary/shot, and an emulsifier, pH buffer, sputum 々s = ° humectant or "n solubility enhancer and other such cages, such as Early laurel I sorbitan ester, triacetate and cyclodextrin such as sodium acetate and monolauryl ether. This article also applies to the slow release or sustained release system, so that the content is kept constant. ', 菫菫 dose (see, for example, U.S. Patent No. 3,710,795). These non-oral entanglements into the yam, the composition of the active compound contained in the composition of the specificity depends on its specific properties to buy ^ and compounds The activity and the needs of the individual. The non-oral administration of the formulation includes the intravenous, subcutaneous and intramuscular administration of the drug. The preparation for parenteral administration includes the sterile solution for injection. Innocent and anhydrous soluble products (such as 进从丰在与与与与与与与与与与与与与与与与与与与与与与与与与与与与与与与与与与与与与与与与与与与Suspension, preparation is about to make A month's use of a sterile anhydrous insoluble product and a sterile emulsion in combination with a vehicle. The solution may be aqueous or #aqueous; when administered intravenously, examples of suitable carriers include, but are not limited to, saline or Acid buffered physiological sleep fire A ^ ^ 1 water (PBS) and a solution containing a thickener and a solubilizing agent such as glucose, polyethyl alcohol and polypropylene-alcohol and mixtures thereof, as appropriate for use in parenteral preparations Examples of pharmaceutically acceptable carriers include, but are not limited to, aqueous vehicles, non-aqueous vehicles, antibacterial agents, etc. 128407.doc • 77· 200911757 J, buffers, antioxidants, local anesthetics, suspending agents And dispersing agents, emulsifiers, chelating agents or chelating agents and other pharmaceutically acceptable substances. Examples of aqueous vehicles which may be used as the case include sodium gas injection, Ringers injection, isotonic dextrose injection , sterile water injection, dextrose and lactated Ringer's injection. Examples of non-aqueous non-oral vehicles which may be used as the case include plant-derived fixed oil, cottonseed oil, corn , sesame oil and peanut oil. Especially when the non-π preparation is packaged in a multi-dose container and thus designed for storage and a plurality of aliquots are to be transferred, an antibacterial agent can be added to inhibit the concentration of g or inhibitor Among the preparations, examples of the antibacterial agent which can be used include phenol or cresol, amalgam, chlorobutanol, methylparaben and propylparaben, thimerosal, and gasified ammonium. (benzalkoniu, chl〇ride) ^ ^ ^ ^ (benzethoniu, chloride). Examples of useful isotonic agents include sodium chloride and dextrose. Examples of useful buffering agents include phosphate and citric acid. Examples of the antioxidant include sodium sulphate. An example of an available anesthetic agent for the use of a tablet, such as PrC(10)e hydrochloride. Suspending agents and examples which may be used include sodium carboxymethylcellulose, hydroxypropyl terphenyl cellulose, and polyethylene sulphate. Examples of emulsifiers for use include polysorbate (IV) (TW Qing 80). The metal ion chelating agent or f-mixing agent includes edta. The pharmaceutical carrier may also include ethanol, polyethylene glycol and propylene glycol as water miscible media, and can be used as a sodium salt, hydrochloric acid, and lemon in the 1^ value. 128407.doc •78· 200911757 Or lactic acid. The concentration of the inhibitor in the parenteral formulation can be adjusted such that the injection is administered in a pharmaceutically effective amount sufficient to produce the desired pharmacological effect. As is known in the art, the precise concentration of the inhibitor and/or dose to be used will ultimately depend on the age and weight of the patient or animal. Unit dose parenteral preparations can be packaged in ampoules, vials or syringes with needle f. All formulations for parenteral administration should be sterile, as is known and practiced in the art. ° Injectables are designed for topical and systemic administration. The therapeutically effective dose is typically formulated to contain at least about 01% w/w to about 9% or about w% w/wu, preferably greater than 1% w/ of the hdac inhibitor relative to the tissue being treated. May be administered once or divided into smaller doses to be administered at intervals of time. (4) It should be understood that the precise dose and duration of treatment will follow the position, carrier and other variables of the composition. Alternatively, σ吏 is empirically determined by known kappa schemes or by extrapolation from in vivo or in vitro test data. It should be noted that the concentration and dose values may also vary with the age of the individual being treated. It should be noted that for any particular individual, the professional judgment of a person who is dependent on the needs of the individual and who is administering or supervising the formulation may require adjustment of the particular dosage regimen over time. Therefore, the range of concentrations set forth herein is intended to be illustrative and not intended to limit the scope or practice of the claimed. The c-inhibitor may optionally be suspended in micron-sized or other suitable form: or may be derivatized to produce a more soluble active product or pre-production. The form of the resulting mixture will depend on a number of factors, including the intended dosage mode 128407.doc-79-200911757. The effective concentration is sufficient and the solubility of the compound in the selected carrier or vehicle improves the symptoms of the disease condition and can be determined empirically. The lyophilized powder can be formulated to administer the lyophilized powder. The compound of the present invention can also be prepared to be lyophilized and reconstituted into solutions, emulsions and other mixtures. Also a solid or gel. Sterile Kanggan powder can be prepared by dissolving the compound in a sodium acid buffer solution containing dextrose or other suitable excipient. The solution is then sterile filtered and then dried to provide the desired formulation under standard conditions known to those skilled in the art. Briefly, about 1 to about 1, preferably about 5% to about 15% of dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol are typically employed at about neutral pH. The lyophilized powder is prepared by dissolving, glucose, sucrose or other suitable agent in a suitable buffer such as citrate, quinine or potassium phosphate or other such buffer known to those skilled in the art. Next, the HDAC# formulation is preferably added to the resulting mixture at room temperature or higher, more preferably at about 3 (rc_35〇c, and stirred until it dissolves. The resulting mixture is diluted by adding more buffer to the desired concentration. The mixture is sterilized or treated to remove particulates and ensure sterility, and is dispensed into vials for drying. Each vial may contain a single dose or multiple doses of the inhibitor. Topical administration of the compounds of the invention may also be topical. The mixture is administered as a mixture. The topical mixture can be used for topical and systemic administration. The resulting mixture can be a solution, a suspension, an emulsion or the like and formulated into a cream, a gel, an ointment, an emulsion, a solution, an ugly agent, a wash. Agent, suspension, g, paste, foaming 128407.doc -80 - 200911757 Body, aerosol, douche, spray, suppository, bandage, epidermal patch or any other formulation suitable for topical administration The HDAC inhibitor can be formulated as an aerosol for topical application by inhalation (see U.S. Patent Nos. 4,044,126, 4,414, 2, 9 and 4,364,923, the disclosure of which is incorporated herein by reference. It is suitable for the treatment of inflammatory diseases, steroids of steroids. These formulations for administration to the respiratory tract may be in the form of an aerosol or a solution for use in a nebulizer, or in the form of a fine powder. For insufflation 'either alone or in combination with an inert carrier such as lactose. In this case, the particles of the formulation will typically have a diameter of less than 5 μm, preferably less than 10 μm. The inhibitor may also be formulated as a gel. In the form of a lotion, cream and lotion for topical application 'I# as applied ☆ to the skin and mucous membranes (such as in the eye) for topical application and for administration to the eye or for intracerebral or intraspinal application. f delivery and for administration to the eye or mucosa or for inhalation therapy, topical administration is also contemplated. Nasal solutions of HDAC inhibitors alone or in combination with other pharmaceutically acceptable ecstasy agents may also be administered. Formulations for other routes of administration may depend on the condition of the disease to be treated, and other routes of administration such as topical administration, transdermal patches, and rectal administration may also be used. For example, for: rectal administration of medicine Rectal suppositories, capsules and lozenges for systemic action. Rectal suppositories as used herein are meant to be inserted into the rectum, melted or softened at body temperature to release one or more pharmacologically or therapeutically active ingredients. Solid. It is a pharmaceutically acceptable base or vehicle for rectal suppositories and an agent for raising (4). Examples of bases include cocoa niobium 128407.doc •81 · 200911757 (cocoa butter), glycerin gelatin , polyethylene glycol, (polyoxyethylene glycol) and a suitable mixture of monoglycerides, diglycerides and triglycerides of fatty acids. Combinations of various bases can be used. Agents for raising the melting point of suppositories include cetyl wax and wax. Rectal suppositories can be prepared by compression or by molding. Typical weights of rectal suppositories are from about 2 to 3 gm. The same pharmaceutically acceptable substances can be used and the tablets and capsules for rectal administration can be made by the same method as used for oral formulations. Examples of Formulations The following are specific examples of oral, intravenous and lozenge formulations which may optionally be employed in the compounds of the present invention. Note that these formulations may vary depending on the particular compound used and the indication of which formulation to use. Oral formulation The compound of the invention monohydrate citric acid sodium hydroxide flavoring water 10-100 mg 105 mg 18 mg

適量補足至100 mL 靜脈内調配物本發明之化合物單水合右旋糖單水合擰檬酸氫氧化納 0.1-10 mg 適量補足以使得等張 1.05 mg 0.18 mg 注射用水Appropriate amount to 100 mL intravenous formulation Compound of the present invention Monohydrate dextrose Monohydrate citric acid Sodium hydroxide 0.1-10 mg Appropriate amount to make isotonic 1.05 mg 0.18 mg water for injection

適量補足至1.0 mL 鍵劑調配物本發明之化合物微晶纖維素硬脂酸膠狀二氧化矽 1% 73% 25% 1% 包含HDAC抑制劑之套組 128407.doc -82- 200911757 本發明亦係關於用其他製品。注意疾病t _DAC相關之疾病的套組及 …、病忍欲涵蓋HDAc對其具有促病理學及/或症狀學之活性有促進病狀之古性的所有病狀。在-實施例中，提供包含合物包含至少m ⑬興次月書之套組，該組组合物^ w 抑制劑。說財可指明欲投與 …：、儲存資訊、給藥量資訊及/或關於如可包含用於容的;^物亦可包含包裝材料。包裝材料组件，諸‘ 容器。套組亦可視情況包含額外， s如用於投與組合物 ,^ /王射态。套組可包含呈輩一或多劑量形式之組合物。該組合物含組合物與包裝材料之製品，用於容納組合物之M 劑;包裝材料可包含欲投與情況包含標籤，其指明奴投與、、且合物之疾病病況、關於如何投與組合物之^ 訊及/或 m田之說明。套組亦可視情況包含額外組件，诸如用於投與組合 n 初之^主射态。套組可包含呈單-或多篁开> 式之組合物。注意用於根據本發明之、备奴加v _ 及製°口中的包裝材料可形成複數個为隔容器，諸如分 j. ,, TS ^ 服4刀^泊封包。容器可呈如在此項技術中已知由醫筚學知开㈠…^ 晉梁學上可接受之材料製成之任何習 '狀或开> 式’例如紙盒或各飞^板|、破璃或塑膠瓶或罐、再在封衣（例如，用於容納旋劑士曙敎再充填，，以置於不同容發令勺捉縻進度自包裝壓出之個別劑量的 / 所用容器將視所涉及之精確劑型而定，例如習 128407.doc -83 - 200911757 知紙板盒-般將不用於容納液體懸浮液。在單—包裝中可 -起使用-個以上容器以銷售單一劑型為可行的。舉例而 =，錠劑可容納於瓶中，該瓶又容納於盒令。套組通常包括投與獨立組份之指導。當獨立組权佳以不同劑型（例如口服、局部、經皮及非經口）也味4丄興以不同劑量間隔投與時’或當開藥醫師黨I宗έ 合之個範份時，套組形尤具有利。根據本發明之套組的一特定實例為所謂的發泡包裝。發Appropriate amount to 1.0 mL of the key formulation The compound of the present invention microcrystalline cellulose stearic acid gelled cerium oxide 1% 73% 25% 1% The kit containing the HDAC inhibitor 128407.doc -82- 200911757 The present invention also It is about using other products. Note that the disease t _DAC-related disease sets and ..., the disease endurance covers all the conditions in which HDAc promotes pathology and/or symptomology and promotes the archaea of the disease. In an embodiment, a kit comprising at least m 13 Hz, the composition of the composition is provided. Saying money may indicate that you want to invest in...: , store information, dosing information, and/or as may be included for containment; Packaging materials components, ‘containers. The kit may also contain additional items as appropriate, such as for administration of the composition, ^ / Wang morphism. The kit can comprise a composition in one or more dosage forms. The composition comprises a composition of the composition and the packaging material for containing the M agent of the composition; the packaging material may include a label to be included, which indicates the disease condition of the slave, and the method of how to administer Description of the composition and/or m field. The set may also include additional components, such as for the purpose of casting a combination n. The kit may comprise a composition in the form of a single- or multi-opening > Note that the packaging materials used in the preparation of the slaves and the mouthpieces according to the present invention can be formed into a plurality of compartments, such as sub-j., TS^, 4-cylinders. The container may be in the form of any of the materials known in the art as known in the art (i), which can be made of materials that are acceptable for the study, such as a carton or a flying plate. , broken glass or plastic bottles or cans, and then in the seal (for example, to accommodate the refilling of the sputum, the individual doses / containers used to put the different doses to the spoon Depending on the precise dosage form involved, for example, 128128.doc -83 - 200911757 know that cardboard boxes will not be used to hold liquid suspensions. It is possible to use more than one container in a single package to sell a single dosage form. For example, the lozenge can be contained in a bottle, which in turn is contained in a box. The kit usually includes instructions for administering the independent component. When the independent group is in different dosage forms (eg oral, topical, transdermal) And non-oral) also tastes that when the group is administered at different dose intervals, or when the prescription is opened, the kit is particularly advantageous. The specificity of the kit according to the present invention. An example is a so-called blister pack.

泡包裝為包装I堂由& # I Q W ’、所无、知且正廣泛用於包裝醫藥單位劑 =鍵劑、膠囊及其類似物)。發泡包裝一般由以較佳透明覆蓋的相對較硬材料之薄片組成。在包裝製劑㈣嚢的膠泊中形成凹座。凹座具有欲包裝之個別錠寸及形狀或可具有容納欲包裝之多個錠劑及/ 3 -、尺寸及形狀。接著’相應地將錠劑或膠囊置於凹座中且對著塑膠箔在盥The blister pack is a package I &## I Q W ', none, know and is widely used for packaging pharmaceutical unit agents = key agents, capsules and the like). The blister pack typically consists of a sheet of relatively hard material that is preferably transparently covered. A recess is formed in the glue of the packaging agent (4). The recess has individual ingots and shapes to be packaged or may have a plurality of tablets and/or sizes and shapes for containing the packages to be packaged. Next, the corresponding tablet or capsule is placed in the recess and facing the plastic foil.

形成方向相反之箔面處密封相對較硬材料之薄片。6士 m h A % ..^ 、，·σ果，按需要將錠劑或膠囊個別密封A sheet of relatively hard material is sealed at the foil faces that are formed in opposite directions. 6士 m h A % ..^,,·σ fruit, individually seal the tablets or capsules as needed

或共同密封於在塑膠％I W…片之間的凹座中。薄片之強度較仫為藉由手動施壓於座上藉此在凹座處之薄片中形而可自發泡包裝中移除錠劑或膠囊。經由該開口可接著移除錠劑或膠囊。套組之另-特定實施例為設計成按其所需用途之次序一二人一個分配日劑量之八刀兄15。分配器較佳配備有記憶輔助物以便進一步有助於對 ,,^ _ 了蜃去之順應性。此記憶輔助物之實例為4日示已分配之日齋丨旦缸里數目的機械計數器。此記憶輔助 128407.doc • 84 - 200911757 物之另一實例為與（例如）讀出最近已服用日劑量之日期及/ 或提醒人何時服用下一劑量的液晶讀數或可聽提醒信號耦合之電池供電微晶片記憶體。實例實例1.鑑別本發明之多晶型物及鹽本文中提供用以鑑別本發明之結晶固體的通用程序。 1.選擇合適溶劑對所關注化合物進行初始溶解度研究以鑑別用於鹽形成之合適單一溶劑。基於ICH方針（π類及ΙΠ類）；官能性、極性及/弗點之多樣性；以及製造適應性來選擇用於溶解度研九之/谷劑。所選溶劑為：乙腈（MeCN)、丙酮、1,4_二。惡烷 '丙酮、乙醇（EtOH)、異丙醇（IPA)、乙酸乙酯 (EtOAc)、乙酸異丙酯（iPAc)、曱基第三丁基醚（MTBE)、四氫呋喃（THF)、2-曱基-四氫呋喃（2-Me-THF)、曱基乙基酮（MEK)、庚烷、環己烷、甲苯、二氣曱烷（DcM)、乙酸 (AcOH)、二曱基甲醯胺（DMF)及甲醇（Me〇H)。 2·選擇合適平衡離子基於pKa值及國際調諧會議（Internati〇nalOr co-sealed in a recess between the plastic %I W... sheets. The strength of the sheet is reduced by the fact that the tablet or capsule can be removed from the blister pack by manually applying pressure to the seat thereby forming the sheet in the recess. The tablet or capsule can then be removed via the opening. Another particular embodiment of the kit is designed to dispense a daily dose of eight knives 15 in the order of its intended use. The dispenser is preferably equipped with a memory aid to further aid in the compliance of the pair. An example of this memory aid is a mechanical counter on the 4th day indicating the number of days in the pool. This memory assists 128407.doc • 84 - 200911757 Another example of a device is a battery coupled to, for example, the date on which the most recent daily dose has been taken and/or when the reminder is taken to take the next dose of liquid crystal reading or audible alert signal. Powered microchip memory. EXAMPLES Example 1. Identification of Polymorphs and Salts of the Invention A general procedure for identifying crystalline solids of the invention is provided herein. 1. Selection of a suitable solvent An initial solubility study of the compound of interest is performed to identify a suitable single solvent for salt formation. Based on the ICH guidelines (π and anthraquinones); the diversity of functionality, polarity and/or point; and manufacturing flexibility to select for the solubility. The solvent selected is: acetonitrile (MeCN), acetone, 1,4_2. Oxane' acetone, ethanol (EtOH), isopropanol (IPA), ethyl acetate (EtOAc), isopropyl acetate (iPAc), mercaptotributyl ether (MTBE), tetrahydrofuran (THF), 2- Mercapto-tetrahydrofuran (2-Me-THF), mercaptoethyl ketone (MEK), heptane, cyclohexane, toluene, dioxane (DcM), acetic acid (AcOH), dimercaptocarboxamide ( DMF) and methanol (Me〇H). 2. Select the right balance ion based on the pKa value and the International Tuning Conference (Internati〇nal

Conference onConference on

Harmonisation ’ ICH)類別選擇平衡離子。當所關注之化合物展現不良水溶性時，pKa評估係基於帶有相同可電離基團之類似結構片段的文獻資料（例如SciFinder文獻搜尋）。接著選擇具有可接受之安全概況且具有2-3 pKa單位差異的I類及II類平衡離子以確保完全的鹽篩檢。包括一或兩種 in類酸以確保至少n_12種平衡離子用於各篩檢，且選擇 128407.doc -85· 200911757 —/、有#乂同pKa值之平衡離子以拓寬發現結晶鹽形成之可能。針對I篩檢所選之平衡離子為龍膽酸、反丁烯二酸、丁酉义順丁缚—酸、L-酒石酸、對甲苯磺酸（p-TSA)、甲㉟西夂（MSA)、笨甲酸、氫氣酸（HC1)、氫演酸（HBr)、硫酸 (h2so4)及磷酸（H3p〇4)。 3·鐘別有利鹽形成之條件的初始鹽筛檢胃使用8種’合劑與u種平衡離子之組合在兩個複製％孔微里滴疋盤中進仃小規模（約i叫）鹽形成實驗。將所關注化。物之游離㈣等分試樣及相應溶劑及平衡離子饋入各孔 =且使其反應-定時間，-般在1-3小時之間。接著使溶劑在諸如氮或任何惰性氣體之惰性氣氛下緩慢蒸發通常 10-24小時之間。評估在兩板巾之錢物的結晶性及閲值溶解度以鐘別顯示雙折射率及與游離驗相比改良之水中溶解度的固體。藉由將少I顆粒分散於顯微鏡載玻片上之礦物油中且評估在正交偏振光下展現雙折射率之顆粒而對含固體孔進行 =二具有雙折射率及消光位不同之顆粒的殘餘固，、政為〜晶。具有-些展現雙折射率及消光位之顆粒而^具有大篁對於偏轉偏振光未觀察到之顆粒的殘餘固體經表徵為部分結晶。並不具有任何偏轉偏# # 轉偏振先之顆粒的殘之、!表徵為非晶形。產生固體之孔中溶劑與平衡離子㉟用於放大及“斤。不含固體之孔中平衡離子/ 命Μ之組合經表徵為不利於鹽形成且未經進—步研究。 128407.doc -86· 200911757 另外’因為鹽一般具有比相應中性物質較佳之水溶性，所以孔中殘餘物固體較之起始物質改良之水溶性可表明鹽已形成。遞增地添加去離子（DI)水至含有固體之孔中。$ 添加之後，在正交偏振光下觀察孔以藉由檢查不存在雙折射率來判定溶解是否已發生。含有水溶性改良之固體的孔中平衡離子/溶劑之組合繼續用於放大及進一步研究。 4.中度放大實驗在初始鹽篩檢中所得固體可為”偽陽性”，因為其可為結晶A PI、結晶平衡離子或A p丨與非鹽平衡離子之結晶混合物。又，已知若以不同方式攪拌沈澱混合物，例如使用^ 拌棒代替震盪，則產生非晶形固體之溶劑/平衡離子組合可產生結晶固體。因此，藉由例如XRpD、dsc及分析之其他分析法來證實鹽形成為謹慎的。產生為半結晶: 在初始鹽筛檢中具有改良溶解度之固體的溶劑/平衡離子組合經放大以進一步表徵。 ’但以10-17 mg規瓶中且視物質在溶 \ 放大程序類似於用於初始筛檢之程序模而非1.0 mg規模。將起始物質稱入小劑中之溶解度而^，使用適當量（2_1() mL)溶劑將固體溶解。在％境溫度下攪拌此等混合物以確保完全溶解。在起始固體已完全溶解之後，以量平衡離子裝載各小瓶。添加平衡離子之後，使反應混合物在攪拌下反應，通常隔夜。藉由過濾收集具有沈澱之樣品。在非反應性氣氛下在至溫將無沈殿之樣品濃縮至無水，且在真空中乾燥。首先由XRPD表徵在初始篩檢中鑑別之在溶劑/平衡離子 128407.doc -87· 200911757 組合下如此製備之固體且將所觀察圖樣與起始游離驗物質之圖獅。獨特圖樣可表明鹽形成或游離驗之新顆物理形式。顯不獨特結晶圖樣之彼等樣品進—步經4 N蠢分析以檢查鹽或初始游離驗之新賴物理形式的形成，且料由 MS及肌C分析以檢查穩定性。在此等中度放大樣品中，得到獨特結晶或半結晶咖圖樣且無可觀察降解之樣品繼續用於進一步放大及表徵。 5.沈澱條件之預放大最優化將在中度規模實驗中用於沈澱固體之類似程序用以產生較大樣品(50 mg)’但是在鹽沈殿期間將鹽形成溶劑用作平衡離子傳遞溶劑。使游離驗與相應溶劑混合以在環境溫度下製得溶H性添加传料域錢摔所得混合物以混合。使溶劑在溫和氮氣流下蒸發直至觀察到大體上 :殺且接著經由過遽來分離固體。若未觀察到沈殿，則將溶液蒸發至無水。在真空下在環境溫度乾燥所有固體。藉由XRPD來分析如此製備之所有固體。藉由η%、 TGA H NMRA凡素分析進—步分析顯示結晶圖樣之固體以證實鹽形成及檢查降解。通過之固體可繼續用於進一步放大及表徵。實例2.表徵本發明之多晶型物及鹽使用以了分析技術及其組合來測定使用由實例3、似 41中所揭不程序製備之化合物作為起始物質自鹽篩檢製備之口相的物理特性。溶劑及其他試劑具有acs或等級且按原樣使用。 I28407.doc •88- 200911757 1. 示差掃描熱量測定分析在 Mettler 822e DSC 或 Perkin Elmer DSC 6 上進行示差掃描熱量測定（DSC)分析。未經其他先前處理按原樣分析樣品。樣品在鋁盤中稱重，以穿孔蓋覆蓋且接著捲曲。分析條件為 30°C 至 300-350°C，以 l〇°C/min勻變。 2. 熱解重量分析在Mettler 85Γ SDTA/TGA上進行熱解重量分析（TGA)。未經其他先前處理按原樣分析樣品。在氧化鋁坩鍋中稱重樣品且以30°C至230°C及l〇°C /min之勻變率分析。 3. X射線粉末繞射使用Shimadzu XRD-6000系統來進行X射線粉末繞射 (XRPD)分析。未經其他先前處理按原樣分析XRPD樣品。將樣品置於Si歸零超微量樣品固持器上且使用以下條件來分析：The Harmonisation ’ ICH) category selects the counter ion. When the compound of interest exhibits poor water solubility, the pKa assessment is based on literature with similar structural fragments of the same ionizable group (eg, SciFinder literature search). Class I and Group II counterions with an acceptable safety profile and a 2-3 pKa unit difference are then selected to ensure complete salt screening. Including one or two in acids to ensure that at least n_12 counter ions are used for each screening, and 128274.doc -85· 200911757 —/, there are balanced ions with the same pKa value to broaden the possibility of crystal salt formation. . The counter ions selected for I screening are gentisic acid, fumaric acid, butyl succinyl-butyric acid, L-tartaric acid, p-toluenesulfonic acid (p-TSA), and A35 (MSA). Odoric acid, hydrogen acid (HC1), hydrogen acid (HBr), sulfuric acid (h2so4) and phosphoric acid (H3p〇4). 3. The initial salt screening test for favorable salt formation conditions. The use of 8 kinds of 'mixtures and u kinds of balanced ions in the two replicate % pore micro-drip trays into small scale (about i called) salt formation experiment. Will pay attention to. The free (iv) aliquot of the material and the corresponding solvent and counterion are fed into each well = and allowed to react - for a period of time, typically between 1-3 hours. The solvent is then slowly evaporated under an inert atmosphere such as nitrogen or any inert gas for typically between 10 and 24 hours. The crystallinity and the readout solubility of the money in the two sheets were evaluated to show the birefringence and the solid solubility in the water compared to the free test. Residues of particles with different birefringence and extinction sites by dispersing less I particles in mineral oil on a microscope slide and evaluating particles exhibiting birefringence under orthogonally polarized light Solid, and political is ~ crystal. Particles having a number of particles exhibiting birefringence and extinction sites and having a large amount of residual solids which are not observed for deflecting polarized light are characterized as partially crystallized. It does not have any residual deflection, and it is characterized by amorphous. The solvent and counterion ions 35 in the pores producing solids are used for amplification and "jin. The combination of counter ion/life in the pores without solids is characterized as not conducive to salt formation and has not been studied in advance. 128407.doc -86 · 200911757 In addition, because salt is generally more water soluble than the corresponding neutral substance, the water solubility of the residue solids in the pores compared to the starting material may indicate that the salt has formed. Add deionized (DI) water to the inclusion. In the pores of the solid. After the addition, the pores were observed under crossed polarized light to determine whether or not dissolution occurred by checking the absence of birefringence. The combination of counter ion/solvent in the well containing the water-soluble modified solid continued. For amplification and further research. 4. Medium-amplification experiments The solids obtained in the initial salt screening can be "false positive" because it can be a crystalline A PI, a crystalline counterion or a crystalline mixture of A p and non-salt equilibrium ions. Further, it is known that if the precipitation mixture is stirred in a different manner, for example, using a stir bar instead of shaking, a solvent/balance ion combination which produces an amorphous solid can produce a crystalline solid. Salt formation was demonstrated to be cautious by other assays such as XRpD, dsc, and analysis. Produced as semi-crystalline: The solvent/balance ion combination of solids with improved solubility in the initial salt screening was amplified for further characterization. In a 10-17 mg vial, the apparent substance in the solvent amplification procedure is similar to the procedure used for the initial screening rather than the 1.0 mg scale. The starting material is weighed into the solution in the small amount, and the appropriate amount is used ( 2_1 () mL) Solvent dissolves the solids. Stir the mixture at % temperature to ensure complete dissolution. After the starting solids have completely dissolved, load each vial with a balanced amount of ions. After adding the counterion, the reaction mixture is allowed to The reaction is stirred, usually overnight. The sample with the precipitate is collected by filtration. The sample without sedum is concentrated to anhydrous in a non-reactive atmosphere at room temperature, and dried in vacuum. First identified by XRPD in the initial screening. The solid prepared in this way under the combination of solvent/balance ion 128407.doc -87· 200911757 and the observed pattern and the initial free test material. The unique pattern can be A new physical form indicating the formation or freeness of the salt. The samples that are not uniquely crystallized are subjected to a 4 N stupid analysis to examine the formation of the new physical form of the salt or the initial free test, and are derived from MS and muscle. C analysis to check stability. In such moderately amplified samples, samples with unique crystalline or semi-crystalline coffee patterns and no observable degradation continue to be used for further amplification and characterization. 5. Pre-amplification optimization of precipitation conditions will be A similar procedure for precipitating solids in a medium scale experiment was used to generate a larger sample (50 mg)' but the salt forming solvent was used as a balanced ion transport solvent during the salt sump. The free test was mixed with the corresponding solvent at ambient temperature. The mixture obtained by dissolving H is added to the feed zone to be mixed. The solvent was allowed to evaporate under a gentle stream of nitrogen until substantially the residue was observed and then solids were separated by hydrazine. If no sediment is observed, the solution is evaporated to dryness. All solids were dried under vacuum at ambient temperature. All solids thus prepared were analyzed by XRPD. Further analysis by η%, TGA H NMRA phenanthrene showed the solid of the crystallographic pattern to confirm salt formation and to examine degradation. The solids passed through can continue to be used for further amplification and characterization. EXAMPLE 2. Characterization of Polymorphs and Salts of the Invention The analytical techniques and combinations thereof were used to determine the phase of the salt prepared by salt screening using the compound prepared by the procedure of Example 3, No. 41 as a starting material. Physical characteristics. Solvents and other reagents have an acs or equivalent and are used as received. I28407.doc •88- 200911757 1. Differential Scanning Calorimetry Analysis Differential Scanning Calorimetry (DSC) analysis was performed on a Mettler 822e DSC or Perkin Elmer DSC 6. Samples were analyzed as they were without other prior treatment. The sample was weighed in an aluminum pan, covered with a perforated lid and then crimped. The analytical conditions ranged from 30 ° C to 300-350 ° C and were ramped at l ° ° C / min. 2. Thermogravimetric analysis Thermogravimetric analysis (TGA) was performed on a Mettler 85 Γ SDTA/TGA. Samples were analyzed as they were without other prior treatment. The sample was weighed in an alumina crucible and analyzed at a ramp rate of 30 ° C to 230 ° C and 10 ° C / min. 3. X-ray powder diffraction X-ray powder diffraction (XRPD) analysis was performed using a Shimadzu XRD-6000 system. XRPD samples were analyzed as they were without other prior treatment. The sample was placed on a Si zero-zero ultra-micro sample holder and analyzed using the following conditions:

X射線管：Cu Κα，40 kV，40 mA 掃描狹縫發散缝 1.00 度散射縫 1.00 度接收縫 0.30 mi 掃描範圍： 3.0-45.0 度掃描模式：連續步長： 0.04° 掃描速率： 27min \ 4.衰減全反射傅裏葉變換紅外分析自配備有衰減全反射（ATR)附件之Thermo-Nicolet 128407.doc -89- 200911757X-ray tube: Cu Κα, 40 kV, 40 mA scanning slit divergence slit 1.00 degree scattering slit 1.00 degree receiving slit 0.30 mi Scanning range: 3.0-45.0 degrees Scanning mode: Continuous step: 0.04° Scanning rate: 27min \ 4. Attenuated Total Reflection Fourier Transform Infrared Analysis from Thermo-Nicolet equipped with Attenuated Total Reflection (ATR) Accessories 128407.doc -89- 200911757

Avatar 370儀器獲得衰減全反射傅裏葉變換紅外（ATR-FTIR)光譜。未經其他先前處理按原樣分析樣品。獲得環境實驗室條件背景之後，將樣品置於ATR上，以鐵砧壓縮且獲得光譜。 5. 水分吸附-解吸附分析在Hiden IGAsor;?水分吸附儀器中進行水分吸附及解吸附實驗。首先在0% RH及25°C下乾燥樣品直至達到平衡重量或歷時至多四小時。接著使樣品經受10% RH至90% RH 以10% RH為步進之等溫（25°C)吸附掃描。使樣品平衡至在各點歷時至多四小時之漸近重量。吸附之後，以-1 0% RH 為步進執行85% RH至0% RH(在25°C)之解吸附掃描，從而又允許至多四小時用於平衡至漸近重量。接著在8(TC將樣品乾燥兩小時且由XRPD分析殘餘固體。 6. 1H核磁共振波譜法在500 MHz Bruker AVANCE NMR質譜儀上獲得質子核磁共振"H NMR)光譜。將樣品（2-10 mg)溶解於含有0.05% 四曱基矽烷（TMS)之DMSO-i/6(0.8 mL)中用於内部參考。在500 MHz使用5 mm寬帶觀察（1H-X)Z梯度探測器獲得1Η NMR光譜。將具有20 ppm譜寬、1.0 s重複率及16-128瞬變之3 0度脈衝用於獲得光譜。 7. HPLC分析自Varian ProStar HPLC獲得高效液相層析圖。在水/乙腈（50:50)中製備樣品之溶液且使用以下條件來分析：管枉： Zorbax SB-C8(4.6 X 150 mm，3 μιη)管柱， 128407.doc -90- 200911757 或 Varian Pursuit XRs C18(4.6 χ 150 mm，3 μηι)管柱。管柱溫度：環境移動相A :水移動相B :乙腈梯度：The Avatar 370 instrument obtained an attenuated total reflection Fourier transform infrared (ATR-FTIR) spectrum. Samples were analyzed as they were without other prior treatment. After obtaining the environmental background of the environmental laboratory, the sample was placed on the ATR, compressed with an anvil and the spectrum was obtained. 5. Moisture Adsorption-Desorption Analysis Water adsorption and desorption experiments were performed in a Hiden IGAsor;? moisture adsorption instrument. The sample is first dried at 0% RH and 25 ° C until equilibrium weight is reached or for up to four hours. The sample was then subjected to an isothermal (25 ° C) adsorption scan of 10% RH to 90% RH in 10% RH steps. The sample was equilibrated to an asymptotic weight of up to four hours at each point. After adsorption, a desorption scan of 85% RH to 0% RH (at 25 °C) was performed in steps of -1 0% RH, allowing up to four hours for equilibration to asymptotic weight. The sample was then dried at 8 (TC) for two hours and the residual solids were analyzed by XRPD. 6. 1H NMR spectroscopy Proton nuclear magnetic resonance "H NMR spectroscopy spectra were obtained on a 500 MHz Bruker AVANCE NMR mass spectrometer. The sample (2-10 mg) was dissolved in DMSO-i/6 (0.8 mL) containing 0.05% tetradecyl decane (TMS) for internal reference. 1 NMR spectra were obtained at 500 MHz using a 5 mm wideband observation (1H-X) Z gradient detector. A 30 degree pulse with a 20 ppm spectral width, a 1.0 s repetition rate, and a 16-128 transient was used to obtain the spectrum. 7. HPLC analysis High performance liquid chromatograms were obtained from Varian ProStar HPLC. A sample solution was prepared in water/acetonitrile (50:50) and analyzed using the following conditions: Tube: Zorbax SB-C8 (4.6 X 150 mm, 3 μιη) column, 128407.doc -90- 200911757 or Varian Pursuit XRs C18 (4.6 χ 150 mm, 3 μηι) column. Column Temperature: Environment Mobile Phase A: Water Mobile Phase B: Acetonitrile Gradient:

Zorbax SB-C8 時間(min) Varian Pursuit XRs C18 時間(min) 移動相A % 移動相B % 0:00 0:00 90 10 11:00 11:00 30 70 16:00 16:00 0 100 18:00 16:30 90 10 23:00 22:00 90 10 流率： 2.5 mL/min(Zorbax 管柱）或 1.0 mL/min (Varian 管柱）偵測：在 254 nm之 UV 樣品稀釋劑 :水/乙腈（50:50) 分析時間： 22-23分鐘 8.有機揮發性雜質在配備有HP 7694頂空採樣器之Agilent HP 6890氣相層析儀中獲得氣相層析圖。將約1 〇〇 mg樣品稱入個別20 mL 頂空小瓶中且添加5 mL DMSO。接著密封小瓶且使用輕微震盪/渦流以確保樣品完全溶解。藉由將5.0 mL DMSO轉移至20 mL頂空小瓶中來製備空白樣品且接著密封。使用在 DMSO中之儲備溶液來製備標準。管柱： DB-1，60公尺χ 0.32毫米（内徑），3 μηι薄膜厚度，P/N : 123-1064，债測器·· FID ; 40 mL/min之氫氣流’ 450 mL/min之空氣流。 30 mL/min之補充氣(氦氣)流。載氣：氦 128407.doc -91 - 200911757 載流：烘箱溫度：注射器溫度偵測器溫度注射類型：分流：注射體積：分析時間： 2.2 mL/min 保持40°C等溫歷時5分鐘；以 5°C/min 勻變至 105°C ; 以 10°C/min 勻變至 165°C ; 以20°C/min勻變至245°C ;保持2分鐘 140。。 260〇C *** 25 mL/min (包括由頂空採樣器提供之流） 1 mL(頂空） 30 min 頂空採樣器條件：烘箱溫度：迴路溫度：輸送管溫度： GC循環時間：小瓶平衡時間：加壓時間：迴路填充時間：迴路平衡時間：注射時間：載流. 小瓶加壓：震盪： 80°C 100。。 110°C 40分鐘 20分鐘 0.13分鐘 0.06分鐘 0.06分鐘 0.20分鐘 20 mL/min 10.0 psi 2(高） 9.卡爾費雪（Karl Fisher)水分析在配備有 HYDRANAL-Coulomat AD 之 Metrohm 756 KF 電量計上進行分析。按原樣分析樣品。添加試樣（10-50 mg)至儀器中，且藉由電量滴定來測定水含量（微克）。實例3.製備N-(2-胺基苯基）-4-((5,6-二甲氧基-2H-吲唑-2-基）甲基）苯甲醯胺（化合物I)Zorbax SB-C8 Time (min) Varian Pursuit XRs C18 Time (min) Mobile phase A % Mobile phase B % 0:00 0:00 90 10 11:00 11:00 30 70 16:00 16:00 0 100 18: 00 16:30 90 10 23:00 22:00 90 10 Flow rate: 2.5 mL/min (Zorbax column) or 1.0 mL/min (Varian column) Detection: UV sample at 254 nm Thinner: water / Acetonitrile (50:50) Analysis time: 22-23 minutes 8. Organic volatile impurities A gas chromatogram was obtained on an Agilent HP 6890 gas chromatograph equipped with an HP 7694 headspace sampler. Approximately 1 〇〇 mg sample was weighed into individual 20 mL headspace vials and 5 mL of DMSO was added. The vial is then sealed and a slight vortex/vortex is used to ensure complete dissolution of the sample. A blank sample was prepared by transferring 5.0 mL DMSO into a 20 mL headspace vial and then sealed. Standards were prepared using stock solutions in DMSO. Column: DB-1, 60 m χ 0.32 mm (inside diameter), 3 μηι film thickness, P/N: 123-1064, debt detector · FID; 40 mL/min hydrogen flow '450 mL/min The flow of air. 30 mL/min of supplemental gas (helium) flow. Carrier gas: 氦128407.doc -91 - 200911757 Current carrying: Oven temperature: Syringe temperature detector Temperature Injection type: Split: Injection volume: Analysis time: 2.2 mL/min Maintain 40 °C isothermal duration for 5 minutes; to 5 °C / min to 105 ° C; 10 ° C / min to 165 ° C; 20 ° C / min to 245 ° C; hold 2 minutes 140. . 260〇C split 25 mL/min (including flow provided by headspace sampler) 1 mL (headspace) 30 min Headspace sampler conditions: oven temperature: loop temperature: duct temperature: GC cycle time: vial equilibration time : Pressurization time: Circuit fill time: Loop balance time: Injection time: Current carrying. Vial pressurization: Oscillation: 80 °C 100. . 110 ° C 40 minutes 20 minutes 0.13 minutes 0.06 minutes 0.06 minutes 0.20 minutes 20 mL / min 10.0 psi 2 (high) 9. Karl Fisher water analysis on a Metrohm 756 KF fuel gauge equipped with HYDRANAL-Coulomat AD analysis. The sample was analyzed as it was. A sample (10-50 mg) was added to the instrument and the water content (micrograms) was determined by coulometric titration. Example 3. Preparation of N-(2-aminophenyl)-4-((5,6-dimethoxy-2H-indazol-2-yl)methyl)benzamide (Compound I)

128407.doc -92 200911757 A. 5,6-二甲氧基-1H-吲唑128407.doc -92 200911757 A. 5,6-Dimethoxy-1H-carbazole

KOAc, Ac20 亞硝酸異戊酯 chci3 0°C-RT-60°CKOAc, Ac20 isoamyl nitrite chci3 0°C-RT-60°C

根據 Dennler 等人，”Synthesis 〇f indazoles using polyphosphoric acid-I” Tetrahedron, 22 (9):3131-3141 (1966)中所述之程序製備5,6-二曱氧基-1H-吲唑，該文獻以全文引用的方式併入本文中。特定而言，將4,5 -二甲氧基-2-甲基苯胺 1Α(16·72 g’ 1〇〇 mmol)與乙酸錦（Π.78 g， 120 mmol)在CHC13(100 mL)中之溶液冷卻至〇。〇。在冷卻後’緩t叉添加乙酸針（3 0.6 3 g ’ 3 0 0 mm ο 1)至反應混合物中。在添加完成後，移除冰浴且使反應升溫至環境溫度 (3 0 min)。將反應燒瓶置於預熱之60。(：油浴中，且接著添加亞硝酸異戍酯（23.43 g，200 mmol)。接著在保持在6〇。^ 之油洛溫度下將反應授摔16小時。將反應冷卻至環境溫度，以飽和NaHC〇3(pH = 8-9)中止反應且轉移至5〇〇虹分液漏斗中且將兩層分離，從而去除有機層。以額外 CHC13(3x50 mL)洗條水層。有機層經組合，以鹽水（ιχ3〇〇 mL)洗滌，經NaaSO4乾燥，過濾且經由旋轉蒸發濃縮成棕色固體。將粗棕色固體藉由以己院沖洗來純化且在真空中在室溫下乾燥以得到16.535 g (75%)呈棕色固體之卜（5,6_ 二甲氧基-1H-。引σ坐-1-基）乙自同13。NMR (400 MHz, OMSO-d6) δ ppm 2.68 (s, 3H), 3.82 (s, 3H), 3.85 (s, 3H), 7.31 (s, 1H), 7.79 (s, 1H), 8.25 (s, 1H)。ESI-MS: m/z 221.3 128407.doc -93- 200911757 (Μ + H)+。在攪拌下，在環境溫度下，以2N HCl/MeOH溶液（250 mL)溶解ib(16.535 g’ 75.1 mmol)。在30分鐘反應時間之 HPLC分析顯示無起始物質剩餘。經由旋轉蒸發器濃縮反應以移除MeOH，且接著使用飽和NaHC03水溶液鹼化 (pH==8-9)。藉由過濾分離產物’以水沖洗且在真空中在室溫下乾燥以得到棕色結晶固體，得到1 1.473 g (86%)5,6-二甲氧基-1H-吲唑 1C。4 NMR (400 MHz, DMSO-A) δ ppm 3-75 (s, 3H), 3.80 (s, 3H), 6.92 (s, 1H), 7.12 (s, 1H), 7.82 (s，1H)，12.69 (br. s., 1H)。ESI-MS: m/z 179.3 (M + H)+。 B. 4-((5,6-二曱氧基-2H-吲唑-2-基）甲基）苯甲酸甲酯 (1E)Preparation of 5,6-dimethoxy-1H-carbazole according to the procedure described in Dennler et al., "Synthesis 〇f indazoles using polyphosphoric acid-I" Tetrahedron, 22 (9): 3131-3141 (1966), The literature is incorporated herein by reference in its entirety. In particular, 4,5-dimethoxy-2-methylaniline 1 Α (16·72 g' 1 〇〇 mmol) and acetonitrile (Π.78 g, 120 mmol) in CHC13 (100 mL) The solution is cooled to 〇. Hey. After cooling, the acetic acid needle (3 0.6 3 g '3 0 0 mm ο 1) was added to the reaction mixture. After the addition was complete, the ice bath was removed and the reaction was allowed to warm to ambient (30 min). The reaction flask was placed in preheated 60. (: in an oil bath, and then isodecyl nitrite (23.43 g, 200 mmol) was added. The reaction was then allowed to fall for 16 hours while maintaining the temperature at 6 ° C. The reaction was cooled to ambient temperature to The reaction was stopped with saturated NaHC〇3 (pH = 8-9) and transferred to a 5 〇〇分分漏斗 funnel and the two layers were separated to remove the organic layer. The aqueous layer was washed with additional CHC 13 (3×50 mL). Combined, washed with brine (3 mL), dried over Na.sub.sub.sub.sub.sub.sub.sub.sub.ssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssss g (75%) is a brown solid (5,6-dimethoxy-1H-. sigma-l-yl). From the same 13. NMR (400 MHz, OMSO-d6) δ ppm 2.68 (s, 3H), 3.82 (s, 3H), 3.85 (s, 3H), 7.31 (s, 1H), 7.79 (s, 1H), 8.25 (s, 1H). ESI-MS: m/z 221.3 128407.doc - 93- 200911757 (Μ + H)+. Ib (16.535 g' 75.1 mmol) was dissolved in 2N HCl/MeOH (250 mL) at ambient temperature with stirring. HPLC analysis over 30 minutes showed no Starting material remaining. The reaction was concentrated with a rotary evaporator to remove MeOH and then basified (pH == 8-9) with saturated aqueous NaHC03. The product isolated by filtration was rinsed with water and dried at room temperature in vacuo to give a brown crystalline solid. , 1 1.473 g (86%) of 5,6-dimethoxy-1H-indazole 1C. 4 NMR (400 MHz, DMSO-A) δ ppm 3-75 (s, 3H), 3.80 (s, 3H ), 6.92 (s, 1H), 7.12 (s, 1H), 7.82 (s, 1H), 12.69 (br. s., 1H). ESI-MS: m/z 179.3 (M + H) + B. Methyl 4-((5,6-didecyloxy-2H-indazol-2-yl)methyl)benzoate (1E)

將1C(18.095 g ’ 101.5 mmol)與4-溴曱基苯曱酸甲醋 1D(23.26 g’ 101.5 mmol)在 DMF(100 mL)中之溶液置於經預熱之120 °C油浴中且藉由磁力攪拌劇烈攪拌2小時。 HPLC分析顯示在N-1區位異構物存在下反應為>9〇%完全。將反應冷卻至23°C且傾入EtOAc(400 mL)中，以水 (4x75 mL)及鹽水（75 mL)洗滌，接著乾燥（MgS04)且在真空中濃縮。使用具有10-90%於己烷中之EtOAc的ISCO系統 128407.doc -94- 200911757 藉由石夕膠急驟層析純化所得殘餘物以提供14.3 14 g (43ο/。；) 呈检色結晶固體之4-((5,6-二甲氧基-2Η-π弓丨嗤-2-基）甲基）苯甲酸甲酯 1Ε。4 NMR (400 MHz，DMSO-A) δ ppm 3.74 (s 3H), 3.77 (s，3H), 3.81 (s, 3H)，5.61 〇, 2H)，6.92 (s, lH)， 6.98 (s, 1H), 7.32 (d, J = 8.08 Hz, 2H), 7.91 (d, J = 8.34A solution of 1C (18.095 g '101.5 mmol) and 4-bromomercaptobenzoic acid methyl vinegar 1D (23.26 g '101.5 mmol) in DMF (100 mL) was placed in a preheated 120 ° C oil bath and Stir vigorously for 2 hours by magnetic stirring. HPLC analysis showed a reaction > 9 % in the presence of the N-1 positional isomer. The reaction was cooled to 23 <0>C and EtOAc (EtOAc) (EtOAc)EtOAc. The resulting residue was purified by flash chromatography eluting with EtOAc (EtOAc: EtOAc: EtOAc: EtOAc: 4-((5,6-Dimethoxy-2Η-π丨嗤丨嗤-2-yl)methyl)benzoic acid methyl ester 1 Ε. 4 NMR (400 MHz, DMSO-A) δ ppm 3.74 (s 3H), 3.77 (s, 3H), 3.81 (s, 3H), 5.61 〇, 2H), 6.92 (s, lH), 6.98 (s, 1H ), 7.32 (d, J = 8.08 Hz, 2H), 7.91 (d, J = 8.34

Hz, 2H), 8.22 (s，1H)。ESI-MS: tw/z 327.4 (M + H)+。 N-l 異構體 IE，NMR (400 MHz，DMSO〇 δ ppm 3.76-3.84 (m, 3H), 3.82 (d, J = 4.29 Hz, 6H), 5.70 (s, 2H), 7.17 (s, 1H), 7.29 (s, 1H), 7.26 (d, J = 8.34 Hz, 2H), 7.9l (s, 1H), 7.90 (d, J = 6.32 Hz, 2H), ESI-MS: m/z 327A (M + H)+。 C_ 4-((5,6-二甲氧基-2H-吲唑-2-基）甲基）苯甲酸（1F)Hz, 2H), 8.22 (s, 1H). ESI-MS: tw/z 327.4 (M + H)+. Nl isomer IE, NMR (400 MHz, DMSO 〇 δ ppm 3.76-3.84 (m, 3H), 3.82 (d, J = 4.29 Hz, 6H), 5.70 (s, 2H), 7.17 (s, 1H), 7.29 (s, 1H), 7.26 (d, J = 8.34 Hz, 2H), 7.9l (s, 1H), 7.90 (d, J = 6.32 Hz, 2H), ESI-MS: m/z 327A (M + H)+. C_ 4-((5,6-Dimethoxy-2H-indazol-2-yl)methyl)benzoic acid (1F)

\ 以LiOH水溶液（1 Μ，130 mL)處理4-((5,6-二甲氧基_2H- 吲唑-2-基）甲基）苯甲酸甲酯（lE，14·314g，43·85mnu)l) 在二°惡院（13 0 mL)中之溶液，且在23°C將反應磁力搜拌1 小時。HPLC分析顯示無起始物質剩餘。添加HC1水溶液（1 Μ，150 mL)且使用布赫納漏斗（Buchner funnel)藉由過渡來分離所得固體。用水將固體沖洗若干次且接著在真空中在室溫下乾燥固體以得到13.07 g (95%)呈淺橙色粉末之 IF ° !H NMR (400 MHz, DMSO-^6) δ ppm 3.74 (s, 3H), 3.76 (s, 3H), 5.59 (s, 2H), 6.92 (s, 1H), 6.97 (s, 1H), 7.30 128407.doc -95- 200911757 (d, J = 8.34 Hz, 2H), 7.88 (d, J = 8.08 Hz, 2H), 8.22 (s 1H), 12.93 (s, 1H)。ESI-MS: m/z 313.4 (M + H)+。 D. N-(2-胺基苯基）_4_((5_甲氧基_2H_n引唑_2_基）曱基）苯甲醢胺（1H)Treatment of methyl 4-((5,6-dimethoxy_2H-indazol-2-yl)methyl)benzoate (1E, 14·314g, 43·) in aqueous LiOH (1 Μ, 130 mL) 85mnu)l) A solution in a 2° hospital (130 mL) and magnetically mixed for 1 hour at 23 °C. HPLC analysis showed no starting material remaining. An aqueous solution of HCl (1 Torr, 150 mL) was added and the resulting solid was separated by a mixture using a Buchner funnel. The solid was rinsed several times with water and then the solid was dried at room temperature in vacuo to give 13.07 g (95%) of y s.H NMR (400 MHz, DMSO-^6) δ ppm 3.74 (s, 3H), 3.76 (s, 3H), 5.59 (s, 2H), 6.92 (s, 1H), 6.97 (s, 1H), 7.30 128407.doc -95- 200911757 (d, J = 8.34 Hz, 2H), 7.88 (d, J = 8.08 Hz, 2H), 8.22 (s 1H), 12.93 (s, 1H). ESI-MS: m/z 313.4 (M + H)+. D. N-(2-Aminophenyl)_4_((5-methoxy-2H_n- oxazol-2-yl)indolyl)benzamide (1H)

在500 mL圓底燒瓶中添加苯甲酸（1F，13 〇2 g，41/7 mmo卜 1.0 eq)、1，2_ 苯二胺（1G，13 53 g，125」酿〇卜 3_0 eq)、EDC(9_593 g，50.0 mmol，1.2 eq)及 H〇Bt(6.758 g，50.0 mmol，1.2 eq)。添加 DMF(200 mL)且在磁力攪拌下將混合物冷卻至〇°C。經10分鐘逐滴地緩慢添加N_甲基嗎琳（13.75 mL，125.1 mm〇l ’ 3.〇 eq)。當添加完成時，使反應混合物緩慢升溫至23〇C。在2小時之HPLC分析顯示無起始酸剩餘。在攪拌下將反應混合物傾入冰冷水（1.5 l) 中，且使用布赫納漏斗藉由過濾來分離所得固體。將固體以水及己烷洗滌若干次且接著在真空中乾燥以得到14 597 g (87/。）呈棕褐色固體之1H。iH NMR (4⑽ δ ppm 3.74 (s, 3H), 3.77 (s, 3H), 4.86 (br. s., 2H), 5.59 (s, 2H), 6.56 (t, J = 7.58 Hz, 1H), 6.75 (dd, J = 8.08, 1.26 Hz, 1H), 6.90-6.99 (m, 3H), 7.13 (d, J = 6.82 Hz, 1H), 7.34 (d, J = 8.34 Hz, 2H), 7.91 (d, J = 8.08 Hz, 2H), 8.22 (s, 1H) 9.60 (s，1H)。ESI-MS: m/z 403.4 (M + H)+。 128407.doc -96- 200911757 藉由XRPD發現固體1H為結晶（參見實例4);因此將其指定為形式A。將藉由上文概述之程序製備之物質用作起始物質以鑑別化合物I之其他多晶型及鹽。應瞭解形式A當藉由暴露於高相對濕度（實例4)或藉由長期暴露於周圍環境（實例5)而暴露於水時轉化成非晶形；因此，各批化合物I之多晶型組合物可視儲存史而改變。不論多晶型組合物如何，將用作起始物質之所製備物質均稱為化合物I起始物質、化合物I API或化合物I游離鹼。Add benzoic acid (1F, 13 〇 2 g, 41/7 mmo Bu 1.0 eq), 1,2-phenylenediamine (1G, 13 53 g, 125", 3_0 eq), EDC in a 500 mL round bottom flask (9_593 g, 50.0 mmol, 1.2 eq) and H〇Bt (6.758 g, 50.0 mmol, 1.2 eq). DMF (200 mL) was added and the mixture was cooled to 〇 ° C with magnetic stirring. N_Methylline (13.75 mL, 125.1 mm〇l ' 3.〇 eq) was slowly added dropwise over 10 minutes. When the addition was complete, the reaction mixture was slowly warmed to 23 °C. HPLC analysis at 2 hours showed no starting acid residue. The reaction mixture was poured into ice cold water (1.5 l) with stirring, and the obtained solid was separated by filtration using a Buchner funnel. The solid was washed several times with water and hexanes and then dried in vacuo to give <RTI ID=0.0> iH NMR (4(10) δ ppm 3.74 (s, 3H), 3.77 (s, 3H), 4.86 (br. s., 2H), 5.59 (s, 2H), 6.56 (t, J = 7.58 Hz, 1H), 6.75 (dd, J = 8.08, 1.26 Hz, 1H), 6.90-6.99 (m, 3H), 7.13 (d, J = 6.82 Hz, 1H), 7.34 (d, J = 8.34 Hz, 2H), 7.91 (d, J = 8.08 Hz, 2H), 8.22 (s, 1H) 9.60 (s, 1H). ESI-MS: m/z 403.4 (M + H) + 128407.doc -96- 200911757 The solid 1H was found by XRPD Crystallization (see Example 4); therefore designated as Form A. Substances prepared by the procedures outlined above are used as starting materials to identify other polymorphs and salts of Compound I. It should be understood that Form A Exposure to high relative humidity (Example 4) or exposure to water upon prolonged exposure to the surrounding environment (Example 5) converted to amorphous; therefore, the polymorphic composition of each batch of Compound I may vary depending on the history of storage. For the polymorphic composition, the materials to be used as starting materials are referred to as Compound I starting materials, Compound I API or Compound I free base.

實例4·表徵化合物I游離鹼之形式A 藉由 XRPD、MS、NMR、HPLC、FTIR(ATR)、 TGA、DSC及水分吸附分析來表徵根據實例3製備之化合物I游離驗之形式A(化合物I形式A)。將Varian Pursuit XRs Cl 8(4.6x150 mm，3 μ)管柱用於HPLC分析。藉由XRPD發現化合物I形式A為結晶固體。圖1顯示形式 A之特徵XRPD光譜（CuKct)。將主要X射線繞射線（以±〇 2 °2Θ表示）及其相對強度概括於表4中。表4·化合物I游離鹼之形式a的XRPD峰值（CuKc〇峰號 2Θ (度） d間距 (A) 強度 (計數） 1/1〇 1 7.23 12.21 57 3 2 8.12 10.88 85 5 3 8.64 10.23 1853 100 4 10.44 8.47 109 6 5 Γ 11.10 7.97 106 6 6 11.53 7.67 141 g 7 12.33 7.17 327 18 8 13.16 6.72 68 4 9 13.50 6.55 85 5 128407.doc •97· 200911757 峰號 2Θ (度） d間距 (A) 強度 (計數） 1/1〇 10 14.40 6.15 145 8 11 15.80 5.60 401 22 12 16.08 5.51 167 9 13 17.24 5.14 706 38 14 17.48 5.07 446 24 15 18.17 4.88 1386 75 16 19.56 4.53 233 13 17 19.95 4.45 1637 88 18 20.36 4.36 173 9 19 20.77 4.27 868 47 20 21.51 4.13 324 17 21 21.96 4.04 139 8 22 22.32 3.98 490 26 23 23.03 3.86 978 53 24 23.52 3.78 56 3 25 23.76 3.74 71 4 26 24.58 3.62 219 12 27 25.08 3.55 113 6 28 25.31 3.52 232 13 29 25.76 3.46 630 34 30 26.48 3.36 673 36 31 27.13 3.28 169 9 32 27.61 3.23 170 9 33 27.96 3.19 209 11 34 28.20 3.16 73 4 35 28.92 3.08 235 13 36 30.15 2.96 569 31 37 30.52 2.93 64 3 38 30.86 2.90 140 8 39 31.77 2.81 140 8 40 32.29 2.77 86 5 41 34.61 2.59 65 4 42 35.33 2.54 61 3 43 36.73 2.44 110 6 44 38.63 2.33 110 6 化合物I形式A得到圖2中所示之溶液^ NMR光譜。光譜與化合物I之已知化學結構一致。化合物I形式A之MS分析顯示在403 m/z之M+1奇，其與化合物I游離驗之已知化學組成一致。化合物I形式A之HPLC分析（未顯示光譜）顯示具有10.4分 128407.doc -98- 200911757 鐘滯留時間之主峰（92.4% AUC)及具有12.3分鐘滯留時間之小峰（6,0% ACU)。圖3顯示化合物I形式A之特徵FTIR(ATR)光譜。光譜與化合物I游離鹼之結構一致。主帶（以波長倒數表示（以cm·1 計之波數））定位於約 3398、3337、3253、3120、2938、 2838、1636、1610、1573、1521、1501、1458、1 335、 1304、1267、1210、1 1774、1134、1017、998、943、 902、844、821、783、745、711 及 679 cirf、捨入最近整數之值）。圖4為化合物I形式A之TGA熱分析圖。形式a當自6(TC加熱至100C時展現8.5°/。之重量損失，其與二水合物形式一致。圖5顯示化合物I形式Λ之特徵DSC熱分析圖。DSC顯示在89 °C與TGA中重量損失一致之寬吸熱事件以及在198 〇c 之最終吸熱事件。亦觀察到15 〇附近可能之寬放熱事件。此表明物質在此等轉變時經歷脫水、再結晶及最終熔融/分解。圖6顯示化合物I形式A之水分吸附-解吸附掃描。水分吸附實驗顯示視相對濕度而定，形式A在脫水形式與無水形式之間轉化。在吸附掃描開始時，形式A物質當暴露於〇% RH時變成無水。在高濕度（60_7〇% RH)時，無水物質再轉化成脫水形式。在解吸附後，觀察到自9〇% 1111至丨〇% rh 二水合物為穩定的。水分分析後之XRpD分析顯示殘餘物主要為非晶形。 128407.doc -99- 200911757 實例5·化合物Γ之形式非晶的XRpD表徵將一批由實例3中所述方法塑供備之化合物I游離驗儲存於環境條件巾歷時2㈣’且接著根據實例2巾所述程序由 XRPD進行分析。物質得到圖17中所示之XRPD(CuK, 樣，其具有非晶固體之特徵。將化合物〗之此非晶物質指定為形式非晶。 ' 實例6.化合物I游離鹼在有機溶劑中之溶解度評估化合物I游離鹼在環境溫度下在〗8種有機溶劑中之溶解度。溶劑為：乙腈（MeCN)、丙酮、二噁烷、乙醇 (EtOH)、異丙醇（IPA)、乙酸乙酯（Et〇Ac)、乙酸異丙酯 (IPAc)、甲基第三丁基趟（mtbE)、四氫〇夫喃（THF)、2-曱基-四氫呋喃（2-曱基-THF)、甲基乙基酮（MEK)、庚烧、環己烷（c-己烷）、曱苯、二氣曱烷（Dcm)、乙酸（Ac〇H)、二甲基甲醯胺（DMF)及曱醇（MeOH)。向各測試小瓶中裝載1 ·〇_2·0 mg之間的化合物I游離鹼且以0.25 mL之小份向各小瓶添加所關注之溶劑。在室溫下震盪小瓶5分鐘且目視檢查以評估溶解情況。若物質未溶解’則添加另_份溶劑。重複此程序直至達成完全溶解或總溶劑量達到5 mL。表5顯示此等丨8種溶劑中之溶解度結果。 128407.doc • 100· 200911757 表5.化合物I游離檢在環境溫度下之近似溶解度溶劑物質量(mg) 溶劑量 (mL) 濃度 (mg/rnL^ 加熱？ (Y/N) 可溶 ICH類別 MeCN 1.4 0.75 1.9 N 1 一 Y II 二噁烷 1.3 0.75 1.7 N Y II 丙嗣 2.1 0.75 2.8 N Υ III MTBE 1.6 5,00 — N 部分 III EtOH 2.5 4.00 0.6 N Υ ΤΤΤ EtOAc 1,5 4.00 0.4 N Υ τττ IPAc 2.2 5.00 — N ~部分 III IPA Γ 2.8 5.00 N 部分 III THF 1.7 0.75 2.3 N Υ II MEK 2.0 0.75 2.7 N Υ III DMF 1.6 0.25 —_ >6.4 N Υ ττ MeOH 1.5 1.00 1.5 N Υ IX II AcOH 2.0 0.25 >8.0 N γ ΤΤΤ c-己垸 2.3 5.00 N Ν 111 π 庚烷 1.7 5.00 … N Ν III DCM 2.6 0.75 3.5 N γ II 甲笨 2.5 5.00 — N —- . 部分 II z-iVLe-1 rlr 2 5.00 0.4 N Υ ΝΑ 實例7.化合物I之鹽的15 mg規模結晶將約HM7 mg根據實例3之方法製備的化合物丨游離鹼稱入各小瓶中且視溶解度而定使用適當量（對於MeCN而言1〇 mL，對於丙酮而言5 mL)溶劑在環境溫度下溶解固體。將此等混合物攪拌五分鐘以確保完全溶解。接著向各小瓶中裝載200-400卟之平衡離子在相當於1〇5當量各平衡離子之二噁烷中的0.126 Μ溶液。表6顯示添加至各小瓶中之化合物1及平衡離子之量。添加平衡離子之後，在環境溫度下授拌小瓶隔夜。過濾具有沈殿之樣品。在溫和氮氣流下在20。。將無沈澱之樣品濃縮至無水。在真空中在環境溫度下及30吋Hg下將所有固體均乾燥16小時。、皿又 128407.doc -101 - 200911757 表6. 15 mg規模化合物I之結晶固體的15 mg規模製備實驗化合物I量 (mg) 溶劑量 (mL) 平衡離子 (0.126 M) 平衡離子 (mL) 沈澱 XRPD 結果 15.0 MeCN 10.0 反丁烯二酸 0.31 蒸發 xtal 11.3 丙酉同 5.0 HC1 0.23 蒸發半 xtal 11.8 丙酮 5.0 磷酸 0.24 蒸發半 xtal xtal =結晶藉由XRPD發現所得固體為結晶或半結晶，且所觀察圖樣當與形式A之圖樣相比時為獨特的。藉由MS、NMR 及HPLC進一步分析固體以檢查穩定性。將資料概括於表7 中〇表7.以15 mg結晶作用回收之固體的物理表徵資料概括主要溶劑平衡離子冷卻概況 XRPD Stoichio/N MR MS 游離鹼之 HPLC 面積計數 (%) 游離鹼結晶一致一致 91.2 MeCN 反丁烯二酸蒸發結晶 1:1 一致 ND 丙酮 HC1 蒸發半 xtal 一致一致 97.0 丙嗣磷酸蒸發半 xtal ND 一致 94.9 ND未收集到資料 "一致”意謂資料與試樣結構一致（如預期）xtal =結晶實例8.化合物I之結晶固體的50 mg規模製備使用根據實例3之方法製備之化合物I游離鹼作為起始物質來製備結晶固體。使50 mg化合物I與每一相應溶劑（丙酮或MeCN)混合以在環境溫度下製得溶液。一次性添加於鹽形成溶劑中所攜帶之平衡離子且攪拌所得混合物1 5 min。在溫和氮氣流下蒸發溶劑直至觀察到大體上沈澱且接著經由過濾來分離固體。若未觀察到沈澱，則將溶液蒸 128407.doc -102- 200911757 發至無水。在真空下在環境溫度將所有固體均乾燥16小時。將此50 mg規模製備之實驗概括於表8中。表8.化合物I之結晶固體的50 mg規模製備實驗化合物I量 (mg) 溶劑溶劑量 (mL) 平衡離子 1.05 eq 之量(mL) 沈殿 50 MeCN 30 反丁烯二酸，0.1 Μ 在MeCN中(10%水) 1.305 蒸發至~4 mL，接著過濾 50 MeCN 30 丁二酸，0.25 Μ 在 MeCN 中(5%水） 0.525 蒸發至無水 50 丙嗣 20 磷酸，0.48 Μ在丙酮中 0.275 蒸發至~2 mL，接著過濾 50 丙酮 20 11(：1，0.92{4在丙酮中 0.145 蒸發至〜10mL，接著過濾對所得固體進行XRPD分析。資料顯示固體得到與化合物I形式A相比獨特之XRPD圖樣。藉由DSC、TGA、iNMR及元素分析進一步分析在反丁烯二酸、磷酸及氫氯酸存在下形成之形式A及固體。未對在丁二酸存在下形成之物質進行進一步研究。藉由1H NMR分析對於所有四種鹽均未觀察到降解。對於具有有機平衡離子之鹽而言，1H NMR分析及元素分析均顯示存在接近於1:1莫耳比之有機平衡離子與游離鹼。對於無機鹽而言，元素分析顯示氳氯酸鹽具有1:1莫耳比之平衡離子與游離鹼且磷酸鹽具有約略2:1莫耳比之平衡離子與游離鹼。將分析結果概括於表9中。將鹽之元素分析概括於表10及11中。 128407.doc -103- 200911757 表9.化合物I之結晶固體之物理表徵的比較概括形式A 反丁烯二酸鹽磷酸鹽氫氣酸鹽 XRPD 結晶結晶a 半結晶a 半結晶a DSC(°C) 89，147(x)， 198 115 > 128 179(x) ， 256 195 ^NMR 一致一致a 一致a 一致a 平衡離子:化合物I 之比率 1:1° 2:lb l:lb 藉由TGA所測之重量損失％ 8.4(二水合物） 1.4<100°C ' 2.9，有分解 2.0< 180°C 0.0(160°c之後分解） a鹽顯示與游離鹼相比之差異 b元素分析表10.無機平衡離子鹽之元素分析氫氣酸鹽磷酸鹽元素量測值理論值(1:1) 量測值理論值(2:1) C 63.9 62.9 43.8 46.2 Η 5.7 5.3 4.8 4.7 Ν 11.6 12.8 8.7 9.4 Cl 7.0 8.1 - - P - - 10.2 10.4 表11.反丁烯二酸鹽之元素分析結果反丁烯二酸鹽元素量測值理論值(1:1) C 59.9 62.5 Η 5.4 5.1 Ν 10.1 10.8 實例9.化合物I之結晶固體的200 mg規模製備用在MeCN中之反丁烯二酸、在MeCN中之丁二酸、在丙酮中之HC1及在丙酮中之磷酸進行結晶。在250 mL圓底燒瓶中，在環境溫度下將200 mg化合物I溶解於相應溶劑中。接著添加適當平衡離子且攪拌所得溶液10 min。若觀察到沈澱，則過濾反應混合物以收集固體。若未觀察到沈 128407.doc -104- 200911757 澱，則在環境溫度在溫和氮氣流下蒸發溶劑直至產生大量沈版接著過;慮反應混合物以收集固體。在真空下在環产溫度乾燥所有固體65小時。與HC1之反應未經溶劑蒸發即產生沈澱。與反丁烯二酸、丁二酸及磷酸之反應僅在將溶劑分別蒸發至降低1〇〇體積、5體積及5體積之後才產生沈澱。與磷酸之反應亦在燒瓶壁上產生大量薄膜狀物質。丙酮中之沈澱僅構成總物質之較小百分比。因此，用刮勺刮下燒瓶壁上之物質。再添加1.0 mL丙酮且攪拌所得漿料1〇分鐘，隨後過瀘。注意在使用丁二酸/ M e C N組合之另一放大結晶期間發現顯著高於預期（1.4比1之莫耳比）之平衡離子含量。在進一步研究後，證實在丁二酸存在下之整個此篩檢期間所產生之物質為游離鹼之替代結晶形（參見實例1〇)。將此結晶物質指定為化合物I之形式B (化合物I形式b )。以85%產率（2 1 8 mg)獲得化合物〗之反丁烯二酸鹽。以 6〇%產率（155 mg)獲得化合物〗之形式B。以77%產率（168 mg)獲得化合物I之氫氣酸鹽。以72%產率〇8〇 mg)獲得化合物I之磷酸鹽。與其中鹽呈半結晶或結晶物質而獲得之所有其他先前择晶相比’發現在此最終放大中之磷酸鹽呈非晶形物質而經分離。在MTBE中進一步調漿磷酸鹽16小時以求形成梦晶產物。經調漿物質之XRPD分析顯示其仍為非晶开^。表12概括200 mg規模結晶實驗之條件。 128407.doc •105- 200911757 表12.化合物I之結晶固體的200 mg規模結晶實驗溶劑量 (mL) 平衡離子 1.05 eq 之量 (mL) 沈殿回收率 (%) MeCN 90 反丁烯二酸，0.1 Μ 在 MeCN 中(10%水） 5.220 蒸發至〜20 mL，接著過濾。 85 MeCN 90 丁二酸， 0.25 Μ 在 MeCN 中 (5% 水） 2.089 蒸發至1.0 mL，接著過濾。 60 丙s同 50 HC1 > 1.0 Μ 在 MeCN 中 0.522 未經蒸發即過濾。 77 丙綱 50 磷酸，0.48 Μ在丙酮中 1.088 蒸發至〜1.0 mL且在燒瓶壁上觀察到黃色薄膜狀物質。將該物質刮落入溶液中且再添加1.0mL丙酮。擾拌所得漿料10分鐘且接著過濾。 72 使用包括 XRPD、DSC、TGA、FTIR(ATR)、NMR及 HPLC之方法表徵在此200 mg製備中回收之形式B、反丁烯二酸鹽及氫氣酸鹽固體。所回收之磷酸鹽固體為非晶形，且因此未經進一步分析。表13概括此等鹽之分析資料。在實例11-13中報導表徵實驗之細節。表13.化合物I之200 mg規模結晶固體的分析資料 isL -7. I .、办丸1 1 νοτ>ΐΛ I r\cr^ I Hi 丄 1 xjt>t 〇/ Γ^ττ χτλ/ττϊ 平衡離子溶劑 XRPD DSC rc) 藉由 TGA所測之重量損失％ HPLC % AUC *H NMR 化合物I: 酸在90% RH吸附時之水分吸附％反丁烯二酸鹽 MeCN xtal 101 > 128 2.5 < 120 。(:(在 127 °C後分解) 94 1:1 4.2%(通道水合物）形式B MeCN xtal 147 0.0(在 140 °C後分解) 94 1:1 1.2% H3P〇4 丙酮 amorp. ND ND ND ND ND HC1 丙g同半 xtal 95 ， 185 0.0(在 160 °C後分解) 93 1:1 17.6(二水合物及四水合物） 128407.doc •106- 200911757 ND =未測定 API =化合物I xtal = 結晶 amorp =非晶形實例10.證實形成形式B之其他實驗在” 丁二酸鹽”之放大結晶期間，發現平衡離子含量高於預期（1.4比1之莫耳比）。在水及MeCN中調漿樣品以移除額外平衡離子之後，由XRPD分析物質且得到與先前所得圖樣一致之圖樣，其中觀察到很小差異。由1H NMR進行之進一步分析顯示不存在平衡離子，表明在使用丁二酸之整個篩檢期間產生之物質為游離鹼之替代結晶形。此由實驗進一步證實，在該實驗中加熱游離鹼起始物質使其經過在 DSC中之第一吸熱事件且接著冷卻至室溫。XRPD分析產生與由用丁二酸結晶所得之圖樣一致之圖樣。Example 4 Characterization of Form I of Compound I Free Base A characterization of Compound I by Compound I prepared according to Example 3 by XRPD, MS, NMR, HPLC, FTIR (ATR), TGA, DSC and moisture adsorption analysis (Compound I Form A). A Varian Pursuit XRs Cl 8 (4.6 x 150 mm, 3 μ) column was used for HPLC analysis. Compound I Form A was found to be a crystalline solid by XRPD. Figure 1 shows the characteristic XRPD spectrum (CuKct) of Form A. The main X-ray diffraction (expressed as ± 〇 2 ° 2 )) and their relative intensities are summarized in Table 4. Table 4. XRPD peaks of Form a of Compound I free base (CuKc peak number 2Θ (degrees) d Spacing (A) Strength (count) 1/1〇1 7.23 12.21 57 3 2 8.12 10.88 85 5 3 8.64 10.23 1853 100 4 10.44 8.47 109 6 5 Γ 11.10 7.97 106 6 6 11.53 7.67 141 g 7 12.33 7.17 327 18 8 13.16 6.72 68 4 9 13.50 6.55 85 5 128407.doc •97· 200911757 Peak number 2Θ (degrees) d spacing (A) strength (count) 1/1〇10 14.40 6.15 145 8 11 15.80 5.60 401 22 12 16.08 5.51 167 9 13 17.24 5.14 706 38 14 17.48 5.07 446 24 15 18.17 4.88 1386 75 16 19.56 4.53 233 13 17 19.95 4.45 1637 88 18 20.36 4.36 173 9 19 20.77 4.27 868 47 20 21.51 4.13 324 17 21 21.96 4.04 139 8 22 22.32 3.98 490 26 23 23.03 3.86 978 53 24 23.52 3.78 56 3 25 23.76 3.74 71 4 26 24.58 3.62 219 12 27 25.08 3.55 113 6 28 25.31 3.52 232 13 29 25.76 3.46 630 34 30 26.48 3.36 673 36 31 27.13 3.28 169 9 32 27.61 3.23 170 9 33 27.96 3.19 209 11 34 28.20 3.16 73 4 35 28.92 3.08 235 13 36 30.15 2.96 569 31 37 30.52 2.93 64 3 38 30.86 2.90 140 8 39 31.77 2.81 140 8 40 32.29 2.77 86 5 41 34.61 2.59 65 4 42 35.33 2.54 61 3 43 36.73 2.44 110 6 44 38.63 2.33 110 6 Compound I Form A gives the solution NMR spectrum shown in Figure 2. The spectrum is consistent with the known chemical structure of Compound I. MS analysis of Compound I Form A showed an M+1 sing at 403 m/z which is consistent with the known chemical composition of Compound I. HPLC analysis of Compound I Form A (spectrum not shown) showed a major peak (92.4% AUC) with a residence time of 10.4 minutes 128407.doc -98-200911757 and a small peak with a 12.3 minute residence time (6,0% ACU). Figure 3 shows the characteristic FTIR (ATR) spectrum of Compound I Form A. The spectrum is consistent with the structure of Compound I free base. The main band (represented by the reciprocal of the wavelength (wavenumber in cm·1)) is positioned at approximately 3398, 3337, 3253, 3120, 2938, 2838, 1636, 1610, 1573, 1521, 1501, 1458, 1 335, 1304, 1267, 1210, 1 1774, 1134, 1017, 998, 943, 902, 844, 821, 783, 745, 711, and 679 cirf, rounding the value of the nearest integer). Figure 4 is a TGA thermogram of Compound I Form A. Form a exhibits a weight loss of 8.5 °/g from 6 (TC heated to 100 C, which is consistent with the dihydrate form. Figure 5 shows a characteristic DSC thermogram of the compound I form Λ. DSC shows at 89 ° C with TGA The wide endothermic event with consistent weight loss and the final endothermic event at 198 〇c. A wide exothermic event near 15 〇 was also observed. This indicates that the material undergoes dehydration, recrystallization and final melting/decomposition during these transitions. 6 shows the moisture adsorption-desorption scan of Compound I Form A. The moisture adsorption experiment shows that Form A is converted between the dehydrated form and the anhydrous form depending on the relative humidity. Form A substance is exposed to 〇% at the beginning of the adsorption scan. When RH becomes anhydrous. At high humidity (60_7〇% RH), the anhydrous substance is converted into a dehydrated form. After desorption, it is observed that the stability from 9〇% 1111 to 丨〇% rh dihydrate is stable. Subsequent XRpD analysis showed that the residue was predominantly amorphous. 128407.doc -99- 200911757 Example 5. Compound XRpD Characterization of Amorphous Compounds A batch of Compound I prepared by the method described in Example 3 was isolated. Stored in an environmental condition towel for 2 (four)' and then analyzed by XRPD according to the procedure described in Example 2. The material obtained the XRPD (CuK, as shown in Figure 17, which has the characteristics of an amorphous solid. The crystalline material was designated as amorphous in form. 'Example 6. Solubility of Compound I free base in organic solvent. Solubility of Compound I free base in 8 organic solvents at ambient temperature. Solvent: acetonitrile (MeCN), acetone , dioxane, ethanol (EtOH), isopropanol (IPA), ethyl acetate (Et〇Ac), isopropyl acetate (IPAc), methyl tert-butyl hydrazine (mtbE), tetrahydrofurfuran (THF), 2-mercapto-tetrahydrofuran (2-mercapto-THF), methyl ethyl ketone (MEK), heptane, cyclohexane (c-hexane), toluene, dioxane (Dcm ), acetic acid (Ac〇H), dimethylformamide (DMF) and decyl alcohol (MeOH). Each test vial was loaded with 1·〇_2·0 mg of Compound I free base and at 0.25 mL. Aliquots Add the solvent of interest to each vial. Shake the vial for 5 minutes at room temperature and visually inspect to assess dissolution. If the material is not dissolved, then Add another solvent. Repeat this procedure until complete dissolution or total solvent volume reaches 5 mL. Table 5 shows the solubility results for these 8 solvents. 128407.doc • 100· 200911757 Table 5. Compound I free detection Approximate Solubility at Ambient Temperature Solvent Mass (mg) Solvent Amount (mL) Concentration (mg/rnL^ Heating? (Y/N) Soluble ICH Class MeCN 1.4 0.75 1.9 N 1 Y 2 II Dioxane 1.3 0.75 1.7 NY II 嗣嗣 2.1 0.75 2.8 N Υ III MTBE 1.6 5,00 — N Part III EtOH 2.5 4.00 0.6 N Υ ΤΤΤ EtOAc 1,5 4.00 0.4 N Υ τττ IPAc 2.2 5.00 — N ~ Part III IPA Γ 2.8 5.00 N Part III THF 1.7 0.75 2.3 N Υ II MEK 2.0 0.75 2.7 N Υ III DMF 1.6 0.25 —_ > 6.4 N Υ ττ MeOH 1.5 1.00 1.5 N Υ IX II AcOH 2.0 0.25 > 8.0 N γ ΤΤΤ c-hexane 2.3 5.00 N Ν 111 π heptane 1.7 5.00 ... N Ν III DCM 2.6 0.75 3.5 N γ II methyl 5.2 5.00 — N —- . Part II z-iVLe-1 rlr 2 5.00 0.4 N Υ 实例 Example 7. 15 mg scale of the salt of Compound I Crystallization will be about HM7 mg prepared according to the method of Example 3. Shu free base was weighed into a vial and each may be used depending on the solubility of an appropriate amount (for MeCN 1〇 mL, acetone for the purposes 5 mL) dissolving the solid in a solvent at ambient temperature. These mixtures were stirred for five minutes to ensure complete dissolution. Next, each vial was loaded with a 0.126 Torr solution of 200-400 Torr of counter ion in dioxane equivalent to 1 〇 5 equivalents of each counter ion. Table 6 shows the amounts of Compound 1 and counterion added to each vial. After adding the counterion, the vial was mixed overnight at ambient temperature. Filter the sample with the temple. Under a gentle nitrogen flow at 20. . The precipitate-free sample was concentrated to anhydrous. All solids were dried under vacuum at ambient temperature and 30 Torr Hg for 16 hours. Table 128. 15 mg Scale Compound I Crystalline Solids 15 mg Scale Preparation Experimental Compound I Amount (mg) Solvent Amount (mL) Counterion Ion (0.126 M) Balanced Ion (mL) Precipitate XRPD result 15.0 MeCN 10.0 fumaric acid 0.31 evaporation xtal 11.3 propionan with 5.0 HC1 0.23 evaporation half xtal 11.8 acetone 5.0 phosphoric acid 0.24 evaporation half xtal xtal = crystallized The solid obtained by XRPD was crystallized or semi-crystalline, and the observed pattern It is unique when compared to the pattern of Form A. The solid was further analyzed by MS, NMR and HPLC to check the stability. The data are summarized in Table 7. Table 7. Physical characterization of solids recovered by 15 mg crystallization. Summary of main solvent equilibrium ion cooling profiles XRPD Stoichio/N MR MS Free base HPLC area count (%) Free base crystals consistent 91.2 MeCN fumaric acid evaporation crystallization 1:1 uniform ND acetone HC1 evaporation half xtal consistent 97.0 propionate phosphoric acid evaporation half xtal ND consistent 94.9 ND not collected data "consistent" means that the data is consistent with the sample structure (eg Expected) xtal = crystallization Example 8. 50 mg scale preparation of crystalline solid of compound I. A crystalline solid was prepared using the free base of compound I prepared according to the method of Example 3 as starting material. 50 mg of compound I and each corresponding solvent ( Acetone or MeCN) was mixed to prepare a solution at ambient temperature. The counterion carried in the salt forming solvent was added in one portion and the resulting mixture was stirred for 15 min. The solvent was evaporated under a gentle stream of nitrogen until substantial precipitation was observed and then via Filtration to separate the solids. If no precipitation is observed, the solution is steamed 128407.doc -102- 200911757 Water. All solids were dried under vacuum for 16 hours at ambient temperature. The experiments for this 50 mg scale preparation are summarized in Table 8. Table 8. 50 mg scale preparation of crystalline solids of Compound I. Solvent solvent amount (mL) Balance ion 1.05 eq amount (mL) Shen Dian 50 MeCN 30 Fumaric acid, 0.1 Μ In MeCN (10% water) 1.305 Evaporate to ~4 mL, then filter 50 MeCN 30 succinic acid , 0.25 Μ In MeCN (5% water) 0.525 Evaporate to anhydrous 50 嗣 20 phosphoric acid, 0.48 Μ 0.275 in acetone to evaporate to ~2 mL, then filter 50 acetone 20 11 (: 1, 0.92 {4 in acetone 0.145 Evaporation to ~10 mL, followed by XRPD analysis of the obtained solid by filtration. The data showed that the solid gave a unique XRPD pattern compared to Compound I Form A. Further analysis of the fumaric acid, phosphoric acid by DSC, TGA, iNMR and elemental analysis Form A and solid formed in the presence of hydrochloric acid. Further investigation of the substance formed in the presence of succinic acid was carried out. No degradation was observed for all four salts by 1H NMR analysis. Salt Both 1H NMR analysis and elemental analysis showed the presence of organic equilibrium ions and free bases close to 1:1 molar ratio. For inorganic salts, elemental analysis showed that chlorate had a 1:1 molar ratio of equilibrium ions. The free ion and the phosphate have a balance ion and a free base of about 2:1 molar ratio. The analysis results are summarized in Table 9. The elemental analysis of the salts is summarized in Tables 10 and 11. 128407.doc -103- 200911757 Table 9. Comparison of Physical Characterization of Crystalline Solids of Compound I General Form A Fumarate Phosphate Hydrogenate XRPD Crystalline Crystalline a Semicrystalline a Semicrystalline a DSC (°C) 89 , 147(x), 198 115 > 128 179(x) , 256 195 ^ NMR Consistent a Consistent a Consistent a Balanced ion: Ratio of compound I 1:1° 2: lb l: lb As measured by TGA Weight loss % 8.4 (dihydrate) 1.4 < 100 ° C ' 2.9, with decomposition 2.0 < 180 ° C 0.0 (decomposed after 160 ° c) a salt shows the difference compared with the free base b elemental analysis Table 10. Elemental Analysis of Inorganic Equilibrium Ion Salts Theoretical Values of Hydrogen Phosphate Phosphate Measurements (1:1) Measured Values (2:1) C 63.9 62.9 43.8 46.2 Η 5.7 5.3 4.8 4.7 Ν 11.6 12.8 8.7 9.4 Cl 7.0 8.1 - - P - - 10.2 10.4 Table 11. Elemental analysis of fumarate results Theoretical value of fumarate elemental value (1:1) C 59.9 62.5 Η 5.4 5.1 Ν 10.1 10.8 Example 9. Preparation of 200 mg on the crystalline solid of Compound I using fumaric acid in MeCN, succinic acid in MeCN, in acetone HC1 in acetone and crystallized from the phosphoric acid. In a 250 mL round bottom flask, 200 mg of Compound I was dissolved in the corresponding solvent at ambient temperature. The appropriate counterion was then added and the resulting solution was stirred for 10 min. If a precipitate is observed, the reaction mixture is filtered to collect a solid. If no sedimentation is observed, the solvent is evaporated at ambient temperature under a stream of mild nitrogen until a large amount of plate is produced. The reaction mixture is considered to collect solids. All solids were dried under vacuum at a ring temperature for 65 hours. The reaction with HC1 produces a precipitate without evaporation of the solvent. The reaction with fumaric acid, succinic acid and phosphoric acid only occurs after evaporation of the solvent to a reduction of 1 Torr, 5 vol and 5 vol. The reaction with phosphoric acid also produces a large amount of film-like substance on the walls of the flask. The precipitate in acetone constitutes only a small percentage of the total material. Therefore, the material on the wall of the flask was scraped off with a spatula. An additional 1.0 mL of acetone was added and the resulting slurry was stirred for 1 minute and then passed through. Note that the equilibrium ion content was significantly higher than expected (1.4 to 1 molar ratio) during another magnified crystallization using the succinic acid/M e C N combination. After further investigation, it was confirmed that the substance produced during the entire screening period in the presence of succinic acid was an alternative crystalline form of the free base (see Example 1). This crystalline substance is designated as Form B of Compound I (Compound I Form b). The compound fumarate was obtained in 85% yield (2 1 8 mg). Form B of the compound was obtained in 6% yield (155 mg). The hydrogenate salt of Compound I was obtained in 77% yield (168 mg). The phosphate of Compound I was obtained in 72% yield 〇8 〇 mg). The phosphate found in this final amplification was found to be amorphous and separated from all other previously selected crystals obtained in which the salt was a semicrystalline or crystalline material. The phosphate was further slurried in MTBE for 16 hours to form a dream crystal product. XRPD analysis of the tempered material showed that it was still amorphous. Table 12 summarizes the conditions for the 200 mg scale crystallization experiment. 128407.doc •105- 200911757 Table 12. 200 mg of Crystalline Solids of Compound I Crystallization Experimental Solvent Amount (mL) Balanced Ion 1.05 eq (mL) Separation Rate (%) MeCN 90 Fumaric Acid, 0.1蒸发 Evaporate to ~20 mL in MeCN (10% water) 5.220, then filter. 85 MeCN 90 Succinic acid, 0.25 Μ In MeCN (5% water) 2.089 Evaporate to 1.0 mL, then filter. 60 丙s with 50 HC1 > 1.0 Μ In MeCN 0.522 Filtered without evaporation. 77 Aminophene 50 Phosphoric acid, 0.48 Μ in acetone 1.088 Evaporated to ~1.0 mL and a yellow filmy material was observed on the wall of the flask. The material was scraped into the solution and an additional 1.0 mL of acetone was added. The resulting slurry was scrambled for 10 minutes and then filtered. 72 Form B, fumarate and hydrogenate solids recovered in this 200 mg preparation were characterized using methods including XRPD, DSC, TGA, FTIR (ATR), NMR, and HPLC. The recovered phosphate solids were amorphous and thus were not further analyzed. Table 13 summarizes the analytical data for these salts. Details of the characterization experiments are reported in Examples 11-13. Table 13. Analytical data for a 200 mg scale crystalline solid of Compound I isL -7. I., 丸1 1 νοτ> ΐΛ I r\cr^ I Hi 丄1 xjt>t 〇/ Γ^ττ χτλ/ττϊ Solvent XRPD DSC rc) % weight loss by TGA HPLC % AUC * H NMR Compound I: moisture adsorption of acid at 90% RH adsorption % fumarate MeCN xtal 101 > 128 2.5 < 120 . (: (decomposes after 127 °C) 94 1:1 4.2% (channel hydrate) Form B MeCN xtal 147 0.0 (decomposed after 140 °C) 94 1:1 1.2% H3P〇4 Acetone amorp. ND ND ND ND ND HC1 propylene g with the same half xtal 95 , 185 0.0 (decomposed after 160 ° C) 93 1:1 17.6 (dihydrate and tetrahydrate) 128407.doc •106- 200911757 ND = not determined API = compound I xtal = Crystal amorp = Amorphous Example 10. Other experiments demonstrating Form B formation During the enlarged crystallization of "succinate", the equilibrium ion content was found to be higher than expected (1.4 to 1 molar ratio). In water and MeCN After the sample was pulverized to remove additional counter ions, the material was analyzed by XRPD and a pattern consistent with the previously obtained pattern was obtained, with little difference observed. Further analysis by 1H NMR showed no equilibrium ions, indicating the use of dibutyl The material produced during the entire screening of the acid is an alternative crystalline form of the free base. This is further confirmed by experiments in which the free base starting material is heated to pass the first endothermic event in the DSC and then cooled to room temperature. XRPD analysis Acid crystals obtained from the pattern coincidence between patterns.

實例11.表徵藉由與丁二酸共沈澱所得之化合物I游離鹼之形式B 根據實例2中所述之程序，由XRPD、DSC、TGA、 FTIR(ATR)、NMR及水分吸附分析表徵藉由在丁二酸存在下200 mg規模結晶（實例9)而製備之化合物I形式B物質。藉由XRPD發現形式B物質為結晶固體。圖11顯示形式B 物質之特徵XRPD光譜（CuKa)。以士 0.2 °2Θ表示主要X射線繞射線且將其相對強度概括於表14中。Example 11. Characterization of Form B of Compound I Free Base by Co-precipitation with Succinic Acid According to the procedure described in Example 2, characterization by XRPD, DSC, TGA, FTIR (ATR), NMR and moisture adsorption analysis Compound I Form B material prepared by crystallizing on a 200 mg scale (Example 9) in the presence of succinic acid. Form B material was found to be a crystalline solid by XRPD. Figure 11 shows the characteristic XRPD spectrum (CuKa) of Form B material. The main X-rays are circulated by ± 0.2 ° 2 且 and their relative intensities are summarized in Table 14.

表14.在200 mg規模與丁二酸之共沈澱中所得化合物I 128407.doc •107· 200911757 游離鹼之形式B的XRPD峰值（CuKoi) 峰號 2Θ(°) d間距強度 I/I〇 1 10.28 8.60 100 3 2 10.53 8.40 501 16 3 11.04 8.01 664 22 4 11.52 7.67 113 4 5 13.15 6.73 434 14 6 13.98 6.33 262 9 7 14.76 6.00 250 8 8 16.01 5.53 1378 45 9 16.59 5.34 153 5 10 17.18 5.16 1533 50 11 17.60 5.04 1079 35 12 18.96 4.68 123 4 13 19.45 4.56 1042 34 14 20.05 4.42 3079 100 15 20.36 4.36 1305 42 16 20.52 4.32 959 31 17 20.85 4.26 1161 38 18 21.52 4.13 319 10 19 21.89 4.06 1448 47 20 22.20 4.00 213 7 21 23.09 3.85 581 19 22 23.44 3.79 475 15 23 23.72 3.75 877 28 24 24.48 3.63 323 10 25 25.46 3.50 309 10 26 25.80 3.45 144 5 27 26.20 3.40 2222 72 28 26.72 3.33 212 7 29 27.72 3.22 238 8 30 27.88 3.20 496 16 31 28.12 3.17 879 29 32 28.56 3.12 194 6 33 28.84 3.09 100 3 34 30.80 2.90 176 6 35 31.56 2.83 526 17 36 32.32 2.77 113 4 37 32.48 2.75 115 4 38 33.72 2.66 94 3 39 34.08 2.63 105 3 40 37.58 2.39 99 3 41 38.09 2.36 352 11 42 38.52 2.34 233 8 43 42.12 2.14 162 5 128407.doc -108- 200911757 圖12為形式B物質之姓他1ττ ΧΤΧίΓΤΛ , 、特徵H NMR光譜。光譜與化合之已知化學結構及幾乎卜 % 丁 i.i比率之丁一酸離子與化合物t — 致。圖13為形式B物質之特徵舰(ATR)光譜。光譜與結構及幾乎1:1比率之丁二酸平衡離子與化合物ι一致。以波長倒數（Ucm·1計之波數）表示之主帶定位於約3413、3344、 3254、2932、2838、1635、16〇2、1572、i524、15〇2、 / 1457、1336、1304、咖、⑵2、1177、1139、1〇17、 997 901、854、837、820、769、748、735、717及 680 cnT1(捨入最近整數之值）。圖14為形式B物質之特徵DSC掃描。DSC分析顯示在147 C之單一吸熱。圖15為形式B物質之特徵TGA熱分析圖。tga顯示在接近140°C起始之分解以下無重量損失。分析另外表明形式b 物質可為化合物I之無水形式。在丁二酸存在下結晶之形式B的水分吸附分析表明物質為非吸濕性（圖1 6)。在解吸附及XRPD分析期間未觀察到滯後現象；在8〇<t 下將物質乾燥一小時後，殘餘固體得到與先前所得圖樣一致之圖樣。實例12.表徵化合物I之反丁稀二酸盥根據實例2中所述之程序，由xRPD、DSC、TGA、 FTm(ATR)、MS、4 NMR表徵藉由在反丁烯二酸存在下 2〇〇 mg規模結晶丨（實例9)而製備的化合物ι之反丁稀二酸鹽 128407.doc •109- 200911757 (化合物i反丁烯二酸鹽）。化合物I反丁烯二酸鹽為如XRPD所示之結晶固體。圖1 8 顯示化合物I反丁烯二酸鹽之特徵XRPD光譜（CuKa)。以士0.2 °2Θ表示主要X射線繞射線且將其相對強度概括於表15中。表15·化合物I反丁烯二酸鹽之XRPD峰值（CuKa) 峰號 2Θ(度） d間距(人）強度 (計數） 1/1〇 1 5.00 17.66 275 14 2 5.39 16.37 1907 100 3 10.73 8.23 826 43 4 12.35 7.16 230 12 5 13.24 6.68 1744 91 6 14.12 6.27 198 10 7 14.55 6.08 1177 62 8 16.28 5.44 480 25 9 18.19 4.87 1304 68 10 18.68 4.75 175 9 11 19.37 4.58 789 41 12 19.80 4.48 320 17 13 20.24 4.38 476 25 14 21.02 4.22 1029 54 15 22.16 4.01 389 20 16 22.56 3.94 690 36 17 22.92 3.88 793 42 18 23.16 3.84 440 23 19 23.44 3.79 115 6 20 23.88 3.72 358 19 21 24.28 3.66 758 40 22 24.80 3.59 320 17 23 25.76 3.46 302 16 24 26.12 3.41 1018 53 25 26.65 3.34 1278 67 26 27.35 3.26 710 37 27 27.84 3.20 176 9 28 28.26 3.16 331 17 29 28.85 3.09 726 38 30 29.45 3.03 579 30 31 30.64 2.92 181 9 32 30.80 2.90 197 10 33 31.24 2.86 94 5 34 32.36 2.76 70 4 35 32.68 2.74 69 4 128407.doc -110- 200911757 峰號 2Θ(度） d間距(A) 強度 (計數） ------ 36 32.88 2.72 105 6' 37 34.72 2.58 67 ~~1 ' 38 37.22 2.41 64 —T— 39 37.57 2.39 70 40 38.03 2.36 116 41 38.72 2.32 104 ~~5· 42 39.99 2.52 131 ~7 ' NMR光譜（圖19)與化合物I之已知化學結構及1:1比率之反丁烯二酸平衡離子與化合物T一致。圖20為化合物I反丁烯二酸鹽之特徵ftIR(atR)光譜。光 { 譜顯示與化學結構之一致性且顯示1:1比率之反丁稀二酸平衡離子與化合物I。以波長倒數（以cm·1計之波數）表示之主帶定位於約 3339、2836、2495、1850、1689、1641、 1 569、1527、1500、1445、1368、1338、1313、1276、 1257 、 1209 、 1178 、 1142 、 1006 、 969 、 903 、 827 、 768 、 750及721 cm_1(捨入最近整數之值）。 DSC分析顯示在128°C之吸熱與可能歸因於水/溶劑損失之先前額外事件（圖21)。 ( TGA顯示在120°C以下2.5%之重量損失及在約127t之分解起始（圖22)。化合物Ϊ反丁烯二酸鹽之水分吸附分析顯示物質自2〇0/〇 RH至90% RH為潛在單水合物（圖23)。在解吸附期間未觀察到滯後現象’在80°C下將物質乾燥一小時後，殘餘固體之XRPD分析得到與先前所得圖樣一致之圖樣。水分吸附解吸附表明物質可為通道水合物。實例13.表徵化合物I氫氣酸鹽 128407.doc • 111 - 200911757 根據實例2中所述之程序，由XRPD、4 NMR、DSC、 TGA、FTIR(ATR)及水分吸附分析表徵藉由在氫氯酸存在下自丙酮進行200 mg規模結晶（實例9)而製備的化合物I之氫氯酸鹽（化合物I氫氣酸鹽）。藉由XRPD發現氫氣酸鹽為半結晶固體。圖24顯示化合物I氫氯酸鹽之特徵XRPD光譜（CuKoi)。以士 0.2。20表示主要X射線繞射線且將其相對強度概括於表1 6中。表16.化合物I之氫氣酸鹽的XRPD峰值（CuKa) 峰號 2Θ(度） d間距(A) 強度 (計數） 1/1〇 1 5.90 14.97 121 16 2 6.80 12.99 48 6 3 8.12 10.88 132 17 4 9.40 9.40 30 4 5 10.24 8.63 94 12 6 11.11 7.96 139 18 7 12.00 7.37 83 11 8 14.20 6.23 192 25 9 14.52 6.10 212 28 10 15.48 5.72 766 100 11 16.04 5.52 628 82 12 17.00 5.21 155 20 13 17.56 5.05 149 19 14 18.82 4.71 258 34 15 19.95 4.45 135 18 16 20.92 4.24 411 54 17 22.32 3.97 605 79 18 23.21 3.83 710 93 19 24.38 3.65 317 41 20 25.52 3.49 105 14 21 26.41 3.37 261 34 22 27.80 3.21 121 16 23 28.04 3.18 145 19 24 28.48 3.13 196 26 25 29.15 3.06 100 13 26 31.29 2.86 99 13 27 33.43 2.68 49 6 28 33.76 2.65 26 3 29 35.51 2.53 31 4 128407.doc -112- 200911757 峰號 2Θ(度） d間距(A) 強度 (計數） 1/1〇 30 36.03 2.49 44 6 31 36.64 2.45 37 5 32 37.Ϊ2 Γ 2.42 61 8 33 39.03 2.31 25 3 34 39.77 2.26 26 3 35 42.59 2.12 37 5 4 NMR光譜（圖25)與化合物I之已知化學結構一致。 FTIR(ATR)分析顯示與化合物I之已知結構一致的光譜 (圖26)。以波長倒數（以em-i計之波數）表示之主帶定位於約3000 、 2596 、 1671 、 1642 、 1614 、 1526 、 1494 、 1441 、 1361、1336、1314、1272、1209、1174、1134、1010、 998、920、900、862、828、767、749、717及 656 cm.1(捨入最近整數之值）。 TGA顯不在130 c以下4.1%之重量損失，接著>16〇〇c之分解（圖27)。 DSC分析顯示在Mt：及185。(：之兩個吸熱事件（圖28)。Table 14. Compound I obtained in coprecipitation of 200 mg scale with succinic acid 128 127.doc • 107· 200911757 XRPD peak of Form B of free base (CuKoi) Peak number 2 Θ (°) d Pitch strength I/I 〇 1 10.28 8.60 100 3 2 10.53 8.40 501 16 3 11.04 8.01 664 22 4 11.52 7.67 113 4 5 13.15 6.73 434 14 6 13.98 6.33 262 9 7 14.76 6.00 250 8 8 16.01 5.53 1378 45 9 16.59 5.34 153 5 10 17.18 5.16 1533 50 11 17.60 5.04 1079 35 12 18.96 4.68 123 4 13 19.45 4.56 1042 34 14 20.05 4.42 3079 100 15 20.36 4.36 1305 42 16 20.52 4.32 959 31 17 20.85 4.26 1161 38 18 21.52 4.13 319 10 19 21.89 4.06 1448 47 20 22.20 4.00 213 7 21 23.09 3.85 581 19 22 23.44 3.79 475 15 23 23.72 3.75 877 28 24 24.48 3.63 323 10 25 25.46 3.50 309 10 26 25.80 3.45 144 5 27 26.20 3.40 2222 72 28 26.72 3.33 212 7 29 27.72 3.22 238 8 30 27.88 3.20 496 16 31 28.12 3.17 879 29 32 28.56 3.12 194 6 33 28.84 3.09 100 3 34 30.80 2.90 176 6 35 31.56 2.83 526 17 36 32.32 2.77 113 4 37 32.48 2.75 115 4 38 33.72 2.66 94 3 39 34.08 2.63 105 3 40 37.58 2.39 99 3 41 38.09 2.36 352 11 42 38.52 2.34 233 8 43 42.12 2.14 162 5 128407.doc -108- 200911757 Figure 12 is the name of the substance of Form B. 1ττ ΧΤΧίΓΤΛ , , Characteristic H NMR spectrum. The known chemical structure of the spectrum and the combination and the butyric acid ion of the ratio of the tetrabutyl i.i. Figure 13 is a characteristic ship (ATR) spectrum of Form B material. The spectrum and structure and the almost 1:1 ratio of the succinic acid counterion are consistent with the compound ι. The main band represented by the reciprocal of the wavelength (wavenumber in Ucm·1) is positioned at about 3413, 3344, 3254, 2932, 2838, 1635, 16〇2, 1572, i524, 15〇2, /1457, 1336, 1304, Coffee, (2) 2, 1177, 1139, 1〇17, 997 901, 854, 837, 820, 769, 748, 735, 717, and 680 cnT1 (rounding the value of the nearest integer). Figure 14 is a characteristic DSC scan of Form B material. DSC analysis showed a single endotherm at 147 C. Figure 15 is a characteristic TGA thermogram of Form B material. Tga showed no weight loss below the initial decomposition at 140 °C. The analysis additionally indicates that the form b material can be the anhydrous form of Compound I. The moisture adsorption analysis of Form B crystallized in the presence of succinic acid showed that the material was non-hygroscopic (Fig. 16). No hysteresis was observed during desorption and XRPD analysis; after drying the material for one hour at 8 Torr <t, the residual solids gave a pattern consistent with the previously obtained pattern. Example 12. Characterization of Compound I of anti-succinic acid bismuth according to the procedure described in Example 2, characterized by xRPD, DSC, TGA, FTm (ATR), MS, 4 NMR by 2 in the presence of fumaric acid The compound ι, the anti-succinic acid salt prepared by 〇〇mg-scale crystallization of hydrazine (Example 9) 128407.doc • 109- 200911757 (Compound i fumarate). Compound I fumarate is a crystalline solid as indicated by XRPD. Figure 18 shows the characteristic XRPD spectrum (CuKa) of Compound I fumarate. The main X-ray diffraction rays are represented by ± 0.2 ° 2 且 and their relative intensities are summarized in Table 15. Table 15. XRPD peak (CuKa) of compound I fumarate peak number 2 Θ (degrees) d spacing (human) intensity (count) 1/1 〇 1 5.00 17.66 275 14 2 5.39 16.37 1907 100 3 10.73 8.23 826 43 4 12.35 7.16 230 12 5 13.24 6.68 1744 91 6 14.12 6.27 198 10 7 14.55 6.08 1177 62 8 16.28 5.44 480 25 9 18.19 4.87 1304 68 10 18.68 4.75 175 9 11 19.37 4.58 789 41 12 19.80 4.48 320 17 13 20.24 4.38 476 25 14 21.02 4.22 1029 54 15 22.16 4.01 389 20 16 22.56 3.94 690 36 17 22.92 3.88 793 42 18 23.16 3.84 440 23 19 23.44 3.79 115 6 20 23.88 3.72 358 19 21 24.28 3.66 758 40 22 24.80 3.59 320 17 23 25.76 3.46 302 16 24 26.12 3.41 1018 53 25 26.65 3.34 1278 67 26 27.35 3.26 710 37 27 27.84 3.20 176 9 28 28.26 3.16 331 17 29 28.85 3.09 726 38 30 29.45 3.03 579 30 31 30.64 2.92 181 9 32 30.80 2.90 197 10 33 31.24 2.86 94 5 34 32.36 2.76 70 4 35 32.68 2.74 69 4 128407.doc -110- 200911757 Peak number 2Θ (degrees) d Spacing (A) Intensity (counting) ------ 36 32.88 2.72 105 6' 37 34.72 2.58 67 ~ ~1 ' 38 37.22 2.41 64 —T— 39 37.57 2.39 70 40 38.03 2.36 116 41 38.72 2.32 104 ~~5· 42 39.99 2.52 131 ~7 'The NMR spectrum (Fig. 19) and the known chemical structure of compound I and the ratio of 1:1 The fumarate counterion is consistent with the compound T. Figure 20 is a characteristic ftIR (atR) spectrum of Compound I fumarate. The light {spectrum shows consistency with the chemical structure and shows a 1:1 ratio of the counter-succinic acid counterion to the compound I. The main band represented by the reciprocal of the wavelength (the wave number in cm·1) is positioned at about 3339, 2836, 2495, 1850, 1689, 1641, 1 569, 1527, 1500, 1445, 1368, 1338, 1313, 1276, 1257. , 1209 , 1178 , 1142 , 1006 , 969 , 903 , 827 , 768 , 750 , and 721 cm_1 (round the value of the nearest integer). DSC analysis showed an endothermic at 128 °C with previous additional events that may be attributed to water/solvent loss (Figure 21). (TGA shows a weight loss of 2.5% below 120 °C and a decomposition start at about 127t (Figure 22). The moisture adsorption analysis of the compound bismuthate shows that the material is from 2〇0/〇RH to 90% RH is a potential monohydrate (Fig. 23). No hysteresis was observed during desorption. After drying the material for one hour at 80 ° C, the XRPD analysis of the residual solids gave a pattern consistent with the previously obtained pattern. Adsorption indicates that the material can be a channel hydrate.Example 13. Characterization of Compound I Hydrogenate 128407.doc • 111 - 200911757 According to the procedure described in Example 2, by XRPD, 4 NMR, DSC, TGA, FTIR (ATR) and moisture Adsorption analysis Characterization of the hydrochloride salt of Compound I (Compound I Hydrogenate) prepared by a 200 mg scale crystallization from acetone in the presence of hydrochloric acid (Example 9). Hydrogen acid salt was found to be semi-crystalline by XRPD Solid. Figure 24 shows the characteristic XRPD spectrum (CuKoi) of Compound I hydrochloride. The main X-ray diffraction is represented by ±0.20 and its relative intensity is summarized in Table 16. Table 16. Hydrogen acid of Compound I Salt XRPD peak (CuKa) peak number 2Θ ( Degree) d Spacing (A) Intensity (counting) 1/1〇1 5.90 14.97 121 16 2 6.80 12.99 48 6 3 8.12 10.88 132 17 4 9.40 9.40 30 4 5 10.24 8.63 94 12 6 11.11 7.96 139 18 7 12.00 7.37 83 11 。。。。。。。。。。。。。。。 18 23.21 3.83 710 93 19 24.38 3.65 317 41 20 25.52 3.49 105 14 21 26.41 3.37 261 34 22 27.80 3.21 121 16 23 28.04 3.18 145 19 24 28.48 3.13 196 26 25 29.15 3.06 100 13 26 31.29 2.86 99 13 27 33.43 2.68 49 6 28 33.76 2.65 26 3 29 35.51 2.53 31 4 128407.doc -112- 200911757 Peak number 2Θ (degrees) d spacing (A) intensity (counting) 1/1〇30 36.03 2.49 44 6 31 36.64 2.45 37 5 32 37.Ϊ2 Γ 2.42 61 8 33 39.03 2.31 25 3 34 39.77 2.26 26 3 35 42.59 2.12 37 5 4 NMR spectrum (Fig. 25) is consistent with the known chemical structure of compound I. FTIR (ATR) analysis showed a spectrum consistent with the known structure of Compound I (Figure 26). The main band represented by the reciprocal of the wavelength (the wave number in em-i) is positioned at approximately 3000, 2596, 1671, 1642, 1614, 1526, 1494, 1441, 1361, 1336, 1314, 1272, 1209, 1174, 1134, 1010, 998, 920, 900, 862, 828, 767, 749, 717, and 656 cm.1 (rounded to the nearest integer value). The TGA showed no weight loss of 4.1% below 130 c, followed by decomposition of >16〇〇c (Figure 27). DSC analysis is shown at Mt: and 185. (: Two endothermic events (Figure 28).

化&物I氫氯酸鹽之水分吸附分析顯示物質在吸附掃描期間為二水合物，其在9〇% RH轉化為四水合物（圖29)。在解吸附後，至25% rH觀察到四水合物之滯後現象，表明升y式為亞穩的。在8G c將物質乾燥―小時後，XRPD分析 ,4 丁非aa圖樣’表明樣品並不易於轉化回穩^水合物形式。實例14. |徵化合物域暖鹽之雄酸里根據實例2中所# 4 + ,The moisture adsorption analysis of the & I hydrochloride was shown to be a dihydrate during the adsorption scan which was converted to tetrahydrate at 9% RH (Figure 29). After desorption, a hysteresis of tetrahydrate was observed up to 25% rH, indicating that the ascending y is metastable. After drying the material at 8 G c for an hour, XRPD analysis, 4 butyl non-aa pattern ' indicates that the sample is not readily converted back to the stable hydrate form. Example 14. The compound acid domain of warm salt in the male acid according to #2 + in Example 2

斤述之耘序，由XRPD、1H NMR、TG DSC及元素分柏矣从斤表徵精由在磷酸存在下自丙酮進行5〇規模製備（實例而制 g )I備的化合物I之磷酸鹽（化合物I碟酸鹽）。 128407.doc •113- 200911757 藉由XRPD發現磷酸鹽為半結晶固體。圖3〇顯示化合物I 墙酸鹽之特徵XRPD光譜（CuKa)。以±0.2。20表示主要X射線繞射線且將其相對強度概括於表17中。表17·化合物I之磷酸鹽的XRPD峰值（CuKa) 峰號 1 2Θ (度） d間距 (A) 強度 (計數） Ι/Ι〇 4.23 20.90 13 3 2 4.79 18.43 21 5 3 5.48 16.12 13 3 4 5 6.71 13.15 46 10 9.20 9.60 27 6 6 9.69 9.12 119 27 7 11.02 8.02 13 3 8 11.92 7.42 28 6 9 12.53 7.06 61 14 10 11 13.25 6.68 24 5 14.14 6.26 46 10 12 14.68 6.03 30 7 13 15.68 5.65 15 3 14 16.52 5.36 79 18 15 17.32 5.12 41 9 16 17.80 4.98 442 100 17 19.68 4.51 67 15 18 20.40 4.35 173 39 19 20.84 4.26 106 24 20 21.88 4.06 186 42 21 22.60 3.93 59 Π 22 23.68 3.75 36 1D 8 - 23 24.64 3.61 15 24 25 25.08 25 80 3.55 3 4S 72 16 26 26.60 3.35 202 38 46 9 ~~ 27 27.44 3.25 31 28 28.36 3.14 ' 41 7 9 29 28.68 3.11 26 --Τϊ-— ~~~6-- 30 30.88 2.89 31 31.39 2.85 Zi 1 Q 5 Ιο 4 H NMR分析表明光§晋與已知化學結構一致（圖3 1) DSC資料顯示在約179°C之放熱事件，接著在約256它吸熱事件（圖32)。 128407.doc •114· 200911757 TGA資料（圖33)顯示在180°C以下2.0%之重量損失。元素分析與2:1比率之磷酸鹽平衡離子與化合物I一致，表明磷酸鹽物質為化合物I之雙磷酸鹽。將元素组成概括於表18中。表18.化合物I之磷酸鹽的元素分析磷睃鹽元素量測值理論值(2:1) C 43.8 46.2 Η 4.8 4J Ν 8.7 9Λ Cl - — P 10.2 10.4 實例15 :化合物I之游離鹼及鹽的水溶性量測化合物I形式A(實例3)、形式B(實例9)、化合物1反丁烯二酸鹽（實例9)及化合物I氫氯酸鹽（實例9)之溶解度。秸由製備化合物I在1:1 MeCN:DI-H2〇中具有0.05、〇·1〇、0·20、〇,40 mg/mL·濃度之四個溶液來產生校正曲線。接著藉由HPLC來分析標準溶液以獲得峰面積計數；使用 Varian pursuit XRs C18(4 6xl5〇 mm，3 μηι)管柱。在去離子水（2.0 mL)中調漿形式A、形式B'反丁烯二酸鹽及氫氣酸鹽（20 mg)之樣品歷時24小時。離心漿料樣品。上澄溶液經過濾且藉由HpLC加以分析。對照校正曲線，基於面積計數來測定溶解度。由XRpD分析殘餘固化合物I形式A顯示在水中之零溶解度。藉由XRpD測得殘餘固體變成非晶形。化合物I形式B顯示在水中之零溶解度。殘餘固體以相同 128407.doc •115· 200911757 結晶形保留。化合物I反丁烯二酸鹽為微溶；發現上澄溶液含有0.03 mg/mL化合物I。藉由XRPD測得殘餘物固體變成非晶形。化合物I氫氣酸鹽為微溶；發現上澄溶液含有0.1 7 mg/mL化合物I。藉由XRPD測得殘餘物固體變成非晶形。圖34顯示對照HPLC校正曲線，化合物I之結晶固體的溶解度。實例16.化合物之形式A、形式B、反丁烯二酸鹽及氫氣酸鹽的熱應力研究為評估在熱應力下形式轉化及降解之傾向，對化合物I 形式A(實例3)、化合物I形式B(實例9)、化合物I反丁烯二酸鹽（實例9)及化合物I氳氣酸鹽（實例9)固體進行熱應力研究。將約10-15 mg各固體置於琥珀色小瓶中。將樣品置於60 °C烘箱中歷時七天。接著藉由XRPD、MS及HPLC來分析固體。將結果概括於表19中。表19.化合物I之形式A、形式B、反丁烯二酸鹽及氫氣酸鹽之熱應力研究預應力溫度 (7天）後應力 HPLC XRPD3 MSb HPLCc 預應力後應力形式A 92% 60°C 一致無降解 91% 反丁烯二酸鹽 94% 60°C 一致降解 88% 形式B 94% 60°C 一致最小降解 93% HC1 93% 60°C 一致最小降解 89% 128407.doc -116- 200911757 a與發起批次圖樣相比 b降解表明觀察到環狀雜質之質量峰（m/z = 385) e面積百分比檢定實例17.化合物之形式A、形式B、反丁烯二酸鹽及氫氣酸鹽的環境溫度漿料研究為評估固體形式之有機溶劑引發互變之傾向，在環境溫度下對化合物I形式A(實例3)、化合物I形式B(實例9)、化合物I反丁烯二酸鹽（實例9)及化合物I氫氣酸鹽（實例9)進行漿料研究。將約10-15 mg各固體置於琥珀色小瓶中且向每一者中添加0.5 mL正庚烷或甲苯及攪拌棒。用鐵氟龍（Teflon)蓋密封小瓶，且將漿料在環境溫度下攪拌七天。傾析溶劑。在溫和氮氣淨化下乾燥殘餘固體約1 ·〇小時。在室溫下在真空烘箱中（30吋Hg)進行進一步乾燥歷時1-2小時。接著藉由XRPD、MS及HPLC分析固體。將結果概括於表20中。表20.化合物I之形式A、形式B、反丁烯二酸鹽及氫氣酸鹽之環境溫度漿料實驗固體預漿料溶劑(7天)* 後漿料結果 HPLC XRPDa MSb HPLCc 形式A 93% 甲苯一致無降解 91% 反丁烯二酸鹽 94% 甲苯獨特，最小降解 92% 形式B 94% 甲苯一致無降解 94% HC1 93% 甲苯一致無降解 93% 形式A 93% 正庚烧非晶形無降解 91% 反丁烯二酸鹽 94% 正庚烧一致最小降解 93% 預漿料後漿料結果形式B 94% 正庚烷一致無降解 95% HC1 93% 正庚烧一致無降解 92% 128407.doc -117- 200911757 a與發起批次圖樣相比降解表明觀察到環狀雜質之質量峰=385) ^面積百分比檢定耘述 , , , , , , X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X Compound I disc salt). 128407.doc •113- 200911757 Phosphate was found to be a semi-crystalline solid by XRPD. Figure 3A shows the characteristic XRPD spectrum (CuKa) of the compound I wall salt. The main X-rays are rayed at ±0.20 and their relative intensities are summarized in Table 17. Table 17. XRPD peak (CuKa) of the phosphate of Compound I Peak number 1 2 Θ (degrees) d Spacing (A) Strength (count) Ι/Ι〇 4.23 20.90 13 3 2 4.79 18.43 21 5 3 5.48 16.12 13 3 4 5 6.71 13.15 46 10 9.20 9.60 27 6 6 9.69 9.12 119 27 7 11.02 8.02 13 3 8 11.92 7.42 28 6 9 12.53 7.06 61 14 10 11 13.25 6.68 24 5 14.14 6.26 46 10 12 14.68 6.03 30 7 13 15.68 5.65 15 3 14 16.52 5.36 79 18 15 17.32 5.12 41 9 16 17.80 4.98 442 100 17 19.68 4.51 67 15 18 20.40 4.35 173 39 19 20.84 4.26 106 24 20 21.88 4.06 186 42 21 22.60 3.93 59 Π 22 23.68 3.75 36 1D 8 - 23 24.64 3.61 15 24 25 25.08 25 80 3.55 3 4S 72 16 26 26.60 3.35 202 38 46 9 ~~ 27 27.44 3.25 31 28 28.36 3.14 ' 41 7 9 29 28.68 3.11 26 --Τϊ-- ~~~6-- 30 30.88 2.89 31 31.39 2.85 Zi 1 Q 5 Ιο 4 H NMR analysis indicated that the light cis was consistent with the known chemical structure (Fig. 31). The DSC data showed an exothermic event at about 179 °C, followed by an endothermic event at about 256 (Fig. 32). 128407.doc •114· 200911757 TGA data (Figure 33) shows a weight loss of 2.0% below 180 °C. Elemental analysis and a phosphate balance ion of a 2:1 ratio are consistent with Compound I, indicating that the phosphate material is the bisphosphonate of Compound I. The elemental composition is summarized in Table 18. Table 18. Elemental Analysis of Phosphate of Compound I. Theoretical Value of Phosphonium Salt Elemental Value (2:1) C 43.8 46.2 Η 4.8 4J Ν 8.7 9Λ Cl - — P 10.2 10.4 Example 15: Free Base and Salt of Compound I The solubility of Compound I Form A (Example 3), Form B (Example 9), Compound 1 fumarate (Example 9) and Compound I Hydrochloride (Example 9) was measured for water solubility. The straw was prepared from the preparation of Compound I in 1:1 MeCN:DI-H2〇 with four solutions of 0.05, 〇·1〇, 0·20, 〇, 40 mg/mL· concentration to produce a calibration curve. The standard solution was then analyzed by HPLC to obtain a peak area count; a Varian pursuit XRs C18 (4 6 x 15 〇 mm, 3 μηι) column was used. A sample of Form A, Form B' fumarate and Hydrogenate (20 mg) was slurried in deionized water (2.0 mL) for 24 hours. Centrifuge the slurry sample. The supernatant solution was filtered and analyzed by HpLC. The calibration curve was used to determine the solubility based on the area count. Analysis of residual solid compound Form A by XRpD showed zero solubility in water. The residual solid became amorphous by XRpD. Compound I Form B shows zero solubility in water. The residual solids are retained in the same crystalline form as 128407.doc •115· 200911757. Compound I fumarate was slightly soluble; it was found that the supernatant solution contained 0.03 mg/mL of Compound I. The residue solids were made amorphous by XRPD. Compound I hydrogenate was slightly soluble; it was found that the supernatant solution contained 0.17 mg/mL of Compound I. The residue solids were made amorphous by XRPD. Figure 34 shows the solubility of the crystalline solid of Compound I as a control HPLC calibration curve. Example 16. Thermal stress studies of Form A, Form B, fumarate and hydrogenate of compounds To evaluate the propensity for formal transformation and degradation under thermal stress, for Compound I Form A (Example 3), Compound I Thermal stress studies were performed on Form B (Example 9), Compound I fumarate (Example 9), and Compound I helium hydride (Example 9) solids. Approximately 10-15 mg of each solid was placed in an amber vial. The sample was placed in an oven at 60 °C for seven days. The solids were then analyzed by XRPD, MS and HPLC. The results are summarized in Table 19. Table 19. Thermal Stress Study of Form A, Form B, Fumarate and Hydrogenate of Compound I Prestressing Temperature (7 days) Post Stress HPLC XRPD3 MSb HPLCc Stress Form after Prestress A 92% 60 °C Consistent non-degradation 91% Fumarate 94% 60°C Consistent degradation 88% Form B 94% 60°C Consistent minimum degradation 93% HC1 93% 60°C Consistent minimum degradation 89% 128407.doc -116- 200911757 a degradation of b compared to the initiation batch pattern indicates that the mass peak of the ring-shaped impurity was observed (m/z = 385) e area percentage test Example 17. Compound Form A, Form B, fumarate and hydrogen acid Salt ambient temperature slurry studies to assess the tendency of solid forms of organic solvents to initiate interconversion, at ambient temperature for compound I Form A (Example 3), Compound I Form B (Example 9), Compound I fumaric acid Salt (Example 9) and Compound I Hydrochloride (Example 9) were subjected to slurry studies. Approximately 10-15 mg of each solid was placed in an amber vial and 0.5 mL of n-heptane or toluene and a stir bar were added to each. The vial was sealed with a Teflon cap and the slurry was stirred at ambient temperature for seven days. The solvent was decanted. The residual solid was dried under a gentle nitrogen purge for about 1 hour. Further drying was carried out in a vacuum oven (30 Torr Hg) at room temperature for 1-2 hours. The solid was then analyzed by XRPD, MS and HPLC. The results are summarized in Table 20. Table 20. Ambient temperature slurry of Form A, Form B, fumarate and hydrogenate of Compound I. Experimental solid pre-slurry solvent (7 days) * Post-slurry results HPLC XRPDa MSb HPLCc Form A 93% Toluene consistently degraded 91% fumarate 94% toluene unique, minimal degradation 92% Form B 94% toluene consistent without degradation 94% HC1 93% toluene consistent without degradation 93% Form A 93% n-g-burn amorphous Degradation 91% Fumarate 94% Mn-gum consistent minimum degradation 93% Pre-slurry slurry results Form B 94% n-heptane consistent non-degradation 95% HC1 93% n-g-burn consistent non-degradation 92% 128407 .doc -117- 200911757 a Degradation compared to the initiated batch pattern indicates that the mass peak of the ring-shaped impurity was observed = 385) ^ Area percentage test

實例18.與丁二酸共沈澱形式B 為重複在產生化合物之鹽筛檢中所觀察之結果，進行化合物I與丁二酸之共沈澱實驗。在環境溫度下將化合物1(500 mg，丨24歷〇1)溶解於 MeCN(l63 mL)中’接著添加i 〇5 eq丁二酸（5 22虹在 MeCN/5%水中之0.25 Μ溶液）。將混合物在環境溫度下攪拌3 0 min，且接著在溫和氮氣流下歷時〜丨6 h緩慢濃縮至無水以付到定量產率。藉由1H NMR分析經分離物質且顯示1丨：丨莫耳比之丁二酸與化合物I。亦進行XRPD分析且其與先前所觀察之形式 B與丁二酸混合物的圖樣一致。實例19.評估化合物I游離鹼及丁二酸之相對溶解度咸信可藉由在適當溶劑中調漿而自形式B/丁二酸混合物中選擇性地移除丁二酸。進行化合物I及丁二酸在多種溶劑中之快速溶解度研究以選擇用於進一步調漿及再結晶之適當溶劑。接著進行漿料實驗以求獲得化合物I之純形式 B。將測試物質（化合物I或丁二酸）置於小瓶中且以1 00 pL之小份添加溶劑。基於極性及官能性差異且基於其根據國際調諧會議（ICH)之分類來挑選溶劑，較佳為II類及III類溶劑。各溶劑添加之後，目視檢查小瓶之殘餘固體。 128407.doc -118- 200911757 表2 1顯示所用溶劑及其在環境溫度下溶解物質之能力。表22顯示化合物I及丁二酸兩者之初始溶解度。發現二噁烷、EtOH、THF及MeOH為丁二酸之良好溶劑及化合物I之不良溶劑。因此將此等溶劑用於漿料實驗（實例20)以自丁二酸與化合物I之混合物中移除丁二酸，力圖獲得化合物I 之純形式B。表21. 丁二酸之近似溶解度：溶劑物質量 (mg) 溶劑量 (mL) 濃度(mg/mL) 溫度可溶 MeCN 2.9 1.40 2.07 室溫是二噁烷 2.1 0.20 10.50 室溫是丙酮 2.0 0.30 6.67 室溫是 MTBE 2.1 2.00 1.05 室溫是 EtOH 3.0 0.40 7.50 室溫是 EtOAc 3.3 4.00 0.83 室溫部分 IPAC 3.4 4.00 0.85 室溫否 IPA 2.6 1.50 1.73 室温是 THF 3.1 0.50 6.20 室溫是 MEK 2.7 1.80 1.50 室溫是 DMF 2.5 0.10 25.00 室溫是 MeOH 2.7 0.20 13.50 室溫是 AcOH 2.2 4.00 0.55 室溫 c·己烧 2.4 4.00 0.60 室溫否庚烧 2.6 4.00 0.65 室溫否 DCM 1.8 4.00 0.45 室溫否甲苯 2.8 4.00 0.70 室溫否 2-Me-THF 2.5 2.00 1.25 室溫是表22.化合物I游離鹼相對於丁二酸之近似溶解度溶劑化合物I游離鹼丁二酸濃度(mg/mL) 可溶濃度(mg/mL) 可溶 MeCN 1.9 是 2.07 是二噁烧 1.7 是 10.50 是丙嗣 2.8 是 6.67 是 MTBE … 部分 1.05 是 128407.doc -119- 200911757Example 18. Coprecipitation with succinic acid Form B To repeat the results observed in the salt screening of the resulting compound, a coprecipitation experiment of Compound I with succinic acid was carried out. Compound 1 (500 mg, 丨24 〇1) was dissolved in MeCN (l63 mL) at ambient temperature' followed by the addition of i 〇5 eq succinic acid (5 22 0.25 Μ solution in MeCN/5% water) . The mixture was stirred at ambient temperature for 30 min and then slowly concentrated to dryness under a gentle stream of nitrogen to <~> The isolated material was analyzed by 1H NMR and showed 1 丨: 丨 molar ratio of succinic acid and Compound I. XRPD analysis was also performed and was consistent with the pattern of Form B previously observed with a mixture of succinic acid. Example 19. Evaluation of the relative solubility of Compound I free base and succinic acid The succinic acid can be selectively removed from the Form B/succinic acid mixture by slurrying in a suitable solvent. A rapid solubility study of Compound I and succinic acid in various solvents was carried out to select a suitable solvent for further slurrying and recrystallization. A slurry experiment was then carried out to obtain the pure form B of Compound I. The test substance (Compound I or Succinic Acid) was placed in a vial and the solvent was added in small portions of 100 pL. Solvents are selected based on their polarity and functional differences and based on their classification according to the International Tuning Conference (ICH), preferably Class II and Class III solvents. After each solvent was added, the residual solids of the vials were visually inspected. 128407.doc -118- 200911757 Table 2 1 shows the solvent used and its ability to dissolve substances at ambient temperature. Table 22 shows the initial solubility of both Compound I and succinic acid. Dioxane, EtOH, THF and MeOH were found to be good solvents for succinic acid and poor solvents for compound I. These solvents were therefore used in a slurry experiment (Example 20) to remove succinic acid from a mixture of succinic acid and Compound I in an effort to obtain pure Form B of Compound I. Table 21. Approximate Solubility of Succinic Acid: Solvent Mass (mg) Solvent Amount (mL) Concentration (mg/mL) Temperature Soluble MeCN 2.9 1.40 2.07 Room Temperature is Dioxane 2.1 0.20 10.50 Room Temperature is Acetone 2.0 0.30 6.67 Room temperature is MTBE 2.1 2.00 1.05 Room temperature is EtOH 3.0 0.40 7.50 Room temperature is EtOAc 3.3 4.00 0.83 Room temperature part IPAC 3.4 4.00 0.85 Room temperature No IPA 2.6 1.50 1.73 Room temperature is THF 3.1 0.50 6.20 Room temperature is MEK 2.7 1.80 1.50 Room temperature Is DMF 2.5 0.10 25.00 Room temperature is MeOH 2.7 0.20 13.50 Room temperature is AcOH 2.2 4.00 0.55 Room temperature c·hexane 2.4 4.00 0.60 Room temperature gh 2.6 4.00 0.65 Room temperature No DC 1.8 8.00 0.45 Room temperature No toluene 2.8 4.00 0.70 Room temperature No 2-Me-THF 2.5 2.00 1.25 Room temperature is Table 22. Approximate Solubility of Compound I Free Base vs. Succinic Acid Solvent Compound I Free Base Succinic Acid Concentration (mg/mL) Soluble Concentration (mg/mL ) MeCN 1.9 is 2.07 is dioxin 1.7 is 10.50 is 嗣 2.8 is 6.67 is MTBE ... Part 1.05 is 128407.doc -119- 200911757

EtOH 0.6 是 7.50 是 EtOAc 0.4 是 0.83 部分 IPAC — 部分 0.85 否 IPA --- 部分 1.73 是 THF 2.3 是 6.20 是 MEK 2.7 是 1.50 是 DMF >6.4 是 25.00 是 MeOH 1.5 是 13.50 是 AcOH >8.0 是 0.55 是 c-己烧 — 否 0.60 否庚烧 --- 否 0.65 否 DCM 3.5 是 0.45 否甲苯 --- 部分 0.70 否 2-Me-THF 0.4 否 1.25 是實例20.自共沈澱之化合物I形式B-丁二酸混合物製備純化合物I形式B的漿料實驗接著進行漿料實驗，力圖獲得化合物I之純形式B。將實例1 8中製備之化合物I 丁二酸鹽固體用作起始物質。基於初始溶解度資料，為漿料實驗選擇二噁烷、EtOH、THF、 MeOH、水、MeCN/10°/〇水及丙酮/10%水。向各小瓶中裝載〜30 mg測試物質及1 mL相應溶劑。在環境温度下攪拌所有漿料〜19 h，接著過濾。在真空下在環境溫度乾燥所得固體且藉由XRPD及1H NMR加以分析。分析結果顯示在所有情況下均獲得形式Β，其中水、 EtOH及甲醇提供最佳物質回收。在所有批次中均存在少量殘餘丁二酸。表2 3顯示所有實驗細節及結果。 128407.doc -120- 200911757 表23.自共沈激之化合物I形式B_ 丁二酸混合物製備純化合物I形式B的漿料實驗化合物1/丁二酸量(mg) 溶劑量 (mL) 溫度 CC) -------- 回收率 (%) 游離鹼形式重量％丁二酸 (*H NMR)* 33.3 二噁烷 1.00 3L /JBL 56.0 B 2 6 31.5 EtOH 1.00 金、、田主/孤 84.8 B 1.9 ~ 31.2 THF 1.00 它、、田主 /J0L 70.0 B 2.2 30.1 MeOH 1.00 含、、田 3L /JBL 74.7 B 1.4 32.4 水 1.00 室溫 88.6 B 1.5 30.0 MeCN/10% 水 1.00 玄、、田主/瓜 36.2 B 1.2 32.8 丙酮/10%水 1.00 金、、田 JL /JQL ------— 29.8 B 1.3 由於與溶劑（DMSO-心）峰之i3c衛星體重疊，故值可高於實際值。實例21. —爸形成純化合物丨形式b 為在不分離形式B/丁二酸混合物之情況下流線製備化合物I形式B ’ §平估水性毁料之前的蒸顧程序。在環境溫度下將藉由實例3之程序製備的化合物 mg，〇.75 mm〇i)溶解於MeCN(97 mL)中，接著將 ι·〇5 eqT 一酸添加至游離鹼溶液（丁二酸在MeCN/5%水中之〇 25 Μ 溶液3.13 mL)中。混合物在環境溫度下攪拌3〇 min，接著在減壓下在旋轉蒸發器上（浴溫為2〇_23 ^ )濃縮至〜5_7 mL。在環境溫度下攪拌殘餘物質丨〇_〗5 min以得到漿料。接著添加水（20 mL)至此漿料中，在減壓下進一步濃縮至〜15 mL以移除殘餘MeCN。在環境溫度下進一步攪拌漿料 1 ·5 h，固體藉由過濾分離，以水（5 mL)洗，在環境溫度在真空下乾燥〜16 h以得到290 mg化合物Ι(96·7%回收率）。此物質以XRPD分析顯示其與先前所觀察之化合物τ形式 128407.doc •121- 200911757 NMR分析，其與所觀察之 B之圖樣一致。此物質亦藉由1h 無丁二酸之化合物的結構—致。下自溶劑製備化合物ΪEtOH 0.6 is 7.50 is EtOAc 0.4 is 0.83 part IPAC — part 0.85 no IPA --- part 1.73 is THF 2.3 is 6.20 is MEK 2.7 is 1.50 is DMF >6.4 is 25.00 is MeOH 1.5 is 13.50 is AcOH >8.0 is 0.55 Yes c-hexane-No 0.60 No G-burning--- No 0.65 No DCM 3.5 is 0.45 No toluene--- Part 0.70 No 2-Me-THF 0.4 No 1.25 is Example 20. Self-precipitated Compound I Form B- Succinic acid mixture Preparation of pure compound I Form B slurry experiment Next, a slurry experiment was conducted in an attempt to obtain the pure form B of Compound I. The compound I succinate solid prepared in Example 18 was used as a starting material. Based on the initial solubility data, dioxane, EtOH, THF, MeOH, water, MeCN/10°/hydrophobic and acetone/10% water were selected for the slurry experiments. Each vial was loaded with ~30 mg test substance and 1 mL of the corresponding solvent. All slurries were stirred at ambient temperature for ~19 h, followed by filtration. The resulting solid was dried under vacuum at ambient temperature and was analyzed by XRPD and 1H NMR. The results of the analysis show that in all cases the form is obtained, with water, EtOH and methanol providing optimum material recovery. A small amount of residual succinic acid was present in all batches. Table 2 3 shows all experimental details and results. 128407.doc -120- 200911757 Table 23. Preparation of pure compound I Form B slurry from co-stimulated compound I Form B_ succinic acid mixture Test compound 1/succinic acid amount (mg) Solvent amount (mL) Temperature CC) - ------- Recovery (%) Free base form Weight % Succinic acid (*H NMR)* 33.3 Dioxane 1.00 3L / JBL 56.0 B 2 6 31.5 EtOH 1.00 Gold, Field Master / Lone 84.8 B 1.9 ~ 31.2 THF 1.00 It, Field Master / J0L 70.0 B 2.2 30.1 MeOH 1.00 Contains, Field 3L / JBL 74.7 B 1.4 32.4 Water 1.00 Room Temperature 88.6 B 1.5 30.0 MeCN/10% Water 1.00 Xuan, Tian Tian / Melon 36.2 B 1.2 32.8 Acetone/10% water 1.00 Gold, Honda JL / JQL ------ 29.8 B 1.3 The value may be higher than the actual value due to the overlap with the i3c satellite of the solvent (DMSO-heart) peak. Example 21. - Dad forms a pure compound 丨 Form b is a streamlined preparation of Compound I Form B' without the separation of Form B/succinic acid mixture. The compound mg, 〇.75 mm〇i) prepared by the procedure of Example 3 was dissolved in MeCN (97 mL) at ambient temperature, and then ι·〇5 eqT monoacid was added to the free base solution (succinic acid). In MeCN/5% water, 〇25 Μ solution 3.13 mL). The mixture was stirred at ambient temperature for 3 min, then concentrated to ~5_7 mL under reduced pressure on a rotary evaporator (bath. The residual material 搅拌_〗 5 min was stirred at ambient temperature to obtain a slurry. Water (20 mL) was then added to the slurry and further concentrated to ~15 mL under reduced pressure to remove residual MeCN. The slurry was further stirred at ambient temperature for 1.5 h, and the solid was separated by filtration, washed with water (5 mL), and dried at ambient temperature under vacuum for ~16 h to obtain 290 mg of compound hydrazine (96.7% recovery) ). This material was analyzed by XRPD to be consistent with the previously observed compound τ form 128407.doc • 121-200911757 NMR analysis, which is consistent with the pattern of B observed. This material is also obtained by the structure of a compound having no succinic acid for 1 hour. Preparation of compounds from solvents

實例22·在化合形式3晶種存在之純形式B 可能不必要之在形式B晶種更希望在不使用丁二酸以避免使用其他及試劑的情況下分離化合物丨游離鹼之形式B。存在下進行化合物I起始物質之漿料實驗。藉由實例3之方法製備化合物z起始物f。藉由化合㈣丁二酸物質在EtOH中調衆共沈殿來製借形式B晶種（實例 21)。漿料溶劑為二噁烷、Et〇H、THF、Me〇H及水。各小瓶中裝載化合物I起始物質（〜3〇 mg)、〜1〇重量％之形式B晶種（〜3 mg)，接著添加所選溶劑（〇 5 mL)。在環境溫度下將混合物攪拌16小時。所得固體藉由過濾分離在環境溫度在真空下乾燥，藉由XRPD分析。表Μ顯示所有實驗細節及結果。除水提供非晶形物質外，所有溶劑均提供化合物I之形式B。表24 :使用化合物I形式B晶種之漿料實驗化合物I量 (mg) 溶劑量 (mL) 晶種量 (mg) 溫度 (°C) 回收量 (mg) 回收率 (%) 游離蜍形式 31.2 二噁炫 0.50 3.3 室溫 23.2 67.2 B 29.2 EtOH 0.50 3.0 室溫 26.9 83.5 B 29.8 THF 0.50 3.0 室溫 21.1 64.3 B 29.4 MeOH 0.50 2.9 室溫 25.9 80.2 B 30.6 水 0.50 3.0 室溫 29.9 89.0 非晶形實例23.在不存在形式B晶種之情況下藉由調漿製備化合 128407.doc -122- 200911757Example 22. Pure Form B in the presence of Compound Form 3 seed crystals may be unnecessary. Form B seed crystals It is more desirable to isolate Form B of the free base of the compound 在 without the use of succinic acid to avoid the use of other reagents. A slurry experiment of the starting material of Compound I was carried out in the presence. Compound z starting material f was prepared by the method of Example 3. Form B seed crystals were prepared by compounding (iv) succinic acid species in EtOH to modulate the population (Example 21). The slurry solvent is dioxane, Et〇H, THF, Me〇H and water. Each vial was loaded with Compound I starting material (~3 mg), ~1% by weight of Form B seed crystals (~3 mg), followed by the addition of the selected solvent (〇 5 mL). The mixture was stirred at ambient temperature for 16 hours. The resulting solid was dried by filtration at ambient temperature under vacuum and analyzed by XRPD. The table shows all the experimental details and results. All solvents provide Form B of Compound I, except that water provides an amorphous material. Table 24: Slurry using Compound I Form B seed crystals Test compound amount I (mg) Solvent amount (mL) Seed crystal amount (mg) Temperature (°C) Recovery amount (mg) Recovery rate (%) Free hydrazine form 31.2 Dioxin 0.50 3.3 Room temperature 23.2 67.2 B 29.2 EtOH 0.50 3.0 Room temperature 26.9 83.5 B 29.8 THF 0.50 3.0 Room temperature 21.1 64.3 B 29.4 MeOH 0.50 2.9 Room temperature 25.9 80.2 B 30.6 Water 0.50 3.0 Room temperature 29.9 89.0 Amorphous Example 23. Preparation of compound by slurrying in the absence of Form B seed crystals 128407.doc -122- 200911757

物I之純形式B 在無形式B晶種之情況下，進行化合物I起始物質之漿料實驗以確定種晶對於在常見方法溶劑中促進轉化是否必要。將四種在利用晶種之漿料實驗（實例22)中提供化合物I之形式B的溶劑（二噁烷、EtOH、THF及MeOH)用於此研究中〇向各小瓶中裝載化合物I起始物質（〜3 0 mg)，接著添加所選溶劑（0.5 mL);在環境溫度下攪拌混合物1 6 h。立即在 EtOH中及在MeOH中獲得漿料，而起始物質完全溶解於二噁烷及THF中。在環境溫度下攪拌之後，最終在THF溶液中形成沈澱，然而二噁烷中之樣品保留溶液形式。將所得固體藉由過濾分離，在真空下在環境溫度乾燥且由XRPD分析。表25顯示所得固體之所有實驗細節及XRPD 結果。自EtOH、THF及MeOH獲得化合物游離鹼之形式B。表25:無化合物I游離鹼形式B晶種之漿料實驗Pure Form B of Compound I In the absence of Form B seed crystals, a slurry experiment of the starting material of Compound I was carried out to determine if seed crystals were necessary to facilitate conversion in a common process solvent. Four solvents (dioxane, EtOH, THF, and MeOH) that provided Form B of Compound I in a slurry experiment using a seed crystal (Example 22) were used in this study to start loading Compound I into each vial. Substance (~30 mg), then the solvent (0.5 mL) was added; the mixture was stirred at ambient temperature for 16 h. The slurry was immediately obtained in EtOH and in MeOH, and the starting material was completely dissolved in dioxane and THF. After stirring at ambient temperature, a precipitate eventually formed in the THF solution, however the sample in the dioxane remained in solution. The resulting solid was isolated by filtration, dried under vacuum at ambient temperature and analyzed by XRPD. Table 25 shows all experimental details and XRPD results for the resulting solid. Form B of the free base of the compound was obtained from EtOH, THF and MeOH. Table 25: Slurry test without Compound I free base form B seed crystal

化合物I量 (mg) 溶劑量 (mL) 溫度 (°C) 回收量 (mg) 回收率 (%) 游離鹼形式 32.0 二噁烷 0.5 室溫 n/a1 n/a1 n/a1 31.2 EtOH 0.5 室溫 27.5 88.1 B 31.8 THF1 0.5 室溫 21.7 68.2 B 31.9 MeOH 0.5 室溫 26.8 84.0 B 128407.doc • 123 · 1 起始物質溶解。Amount of Compound I (mg) Solvent Amount (mL) Temperature (°C) Recovery (mg) Recovery (%) Free base form 32.0 Dioxane 0.5 Room temperature n/a1 n/a1 n/a1 31.2 EtOH 0.5 Room temperature 27.5 88.1 B 31.8 THF1 0.5 Room temperature 21.7 68.2 B 31.9 MeOH 0.5 Room temperature 26.8 84.0 B 128407.doc • 123 · 1 The starting material is dissolved.

實例24.藉由在變性乙醇中調漿來製備化合物I形式B 進行漿料實驗以確定變性乙醇將化合物I起始物質轉化 200911757 成形式B之能力。測試若干以5% IPA/5% MeOH、5% MeOH、5%水及5%甲苯變性之乙醇等級。在0.5 mL所選溶劑中調漿約30 mg化合物I起始物質（由實例3之方法製備）。將此等漿料攪拌〜16小時，將固體藉由過濾分離，在真空下在環境溫度乾燥且由XRPD分析。表 26顯示所得固體之所有實驗細節及XRPD結果。所有所得固體得到與化合物I之形式B —致之XRPD圖樣。表26:在變性乙醇中之化合物I游離鹼的漿料實驗Example 24. Preparation of Compound I Form B by slurrying in denatured ethanol A slurry experiment was conducted to determine the ability of denatured ethanol to convert the Compound I starting material to Form 2009B757. Several ethanol grades denatured with 5% IPA/5% MeOH, 5% MeOH, 5% water, and 5% toluene were tested. Approximately 30 mg of the compound I starting material (prepared by the method of Example 3) was slurried in 0.5 mL of the selected solvent. The slurry was stirred for ~16 hours and the solid was isolated by filtration, dried under vacuum at ambient temperature and analyzed by XRPD. Table 26 shows all experimental details and XRPD results for the resulting solids. All of the resulting solids gave an XRPD pattern identical to Form B of Compound I. Table 26: Slurry test of compound I free base in denatured ethanol

化合物I量 (mg) 溶劑量 (mL) 溫度 (°C) 回收量 (mg) 回收率 (%) 形式 31.1 EtOH/5% IPA/5% MeOH 0.5 室溫 25.5 82.0 B 31.8 EtOH/5% MeOH 0.5 室溫 26.7 84.0 B 30.5 EtOH/5°/〇水 0.5 室溫 26.9 88.2 B 30.7 EtOH/5% 曱苯 0.5 室溫 27.2 88.6 B 實例25.在高溫下之漿料實驗起始在高溫下之漿料實驗以確定是否將產生其他穩定形式。在40°C下在EtOH或MeOH中調漿化合物I起始物質（由實例3之方法製備）歷時3小時。採用固體之等分試樣用於分析。接著以20 °C /小時之速率將漿料冷卻至環境溫度且攪拌隔夜，此後再次採用固體之等分試樣用於分析。在環境溫度下攪拌7天之後，將固體藉由過濾分離，在真空下在環境溫度乾燥。對固體進行XRPD分析以確定其物理形式。將實驗細節及XRPD結果顯示於表27中。 128407.doc -124- 200911757 所有經調漿之固體（3小時、24小時及7天）得到與化合物I 之形式B —致之XRPD圖樣。未鑑別到新穎多晶型。表27:在高溫下之化合物I游離鹼的漿料實驗Amount of Compound I (mg) Solvent Amount (mL) Temperature (°C) Recovery (mg) Recovery (%) Form 31.1 EtOH/5% IPA/5% MeOH 0.5 Room Temperature 25.5 82.0 B 31.8 EtOH/5% MeOH 0.5 Room temperature 26.7 84.0 B 30.5 EtOH/5°/〇水0.5 Room temperature 26.9 88.2 B 30.7 EtOH/5% Benzene 0.5 Room temperature 27.2 88.6 B Example 25. Slurry at high temperature Experimental slurry at high temperature Experiment to determine if other stable forms will be produced. The compound I starting material (prepared by the method of Example 3) was pulverized in EtOH or MeOH at 40 °C for 3 hours. An aliquot of the solid was used for the analysis. The slurry was then cooled to ambient temperature at a rate of 20 ° C / hour and stirred overnight, after which an aliquot of solid was again used for analysis. After stirring at ambient temperature for 7 days, the solid was separated by filtration and dried under vacuum at ambient temperature. The solid was subjected to XRPD analysis to determine its physical form. Experimental details and XRPD results are shown in Table 27. 128407.doc -124- 200911757 All of the pulverized solids (3 hours, 24 hours and 7 days) gave an XRPD pattern identical to Form B of Compound I. No novel polymorphs were identified. Table 27: Slurry test of compound I free base at elevated temperature

化合物I量 (mg) 溶劑量 (mL) 溫度(°c) 1號等分試樣， 40°C · 3 h(形式） 2號等分試樣，室溫，24 h(形式） 3號等分試樣，室溫，7天 (形式） 101.0 EtOH 1.0 40/室溫 B B B 101.6 MeOH 1.0 40/室溫 B B B 實例26.在EtOH/O.5%甲苯（”乙醇2B")中之化合物I形式B 的0.5 gm規模製備進行兩個使用乙醇2B之0.5 g規模漿料實驗以證實已先前提供形式B之篩檢實驗。在10或15體積變性乙醇/0.5%甲苯中調漿化合物I起始物質（0.5 g)且在環境溫度下攪拌〜19小時，接著過濾。將所得固體以乙醇/0.5%甲苯（1 mL)洗滌，在真空下在環境溫度乾燥〜16小時且由XRPD分析。固體均得到與形式B—致之 XRPD圖樣。表28顯示所有實驗細節及結果。表28 :在變性乙醇/0.5%曱苯中化合物I起始物質向形式 B之形式轉化Amount of Compound I (mg) Solvent Amount (mL) Temperature (°c) Aliquot No. 1, 40 °C · 3 h (Form) Aliquots No. 2, room temperature, 24 h (form) No. 3, etc. Fraction, room temperature, 7 days (form) 101.0 EtOH 1.0 40/room temperature BBB 101.6 MeOH 1.0 40/room temperature BBB Example 26. Compound I form in EtOH/O.5% toluene ("ethanol 2B") 0.5 gm scale preparation of B. Two 0.5 g scale slurry experiments using ethanol 2B were performed to confirm that the screening test of Form B was previously provided. The compound I starting material was adjusted in 10 or 15 volumes of denatured ethanol/0.5% toluene. (0.5 g) and stirred at ambient temperature for ~19 hours, followed by filtration. The obtained solid was washed with ethanol / 0.5% toluene (1 mL), dried under vacuum at ambient temperature for ~16 hours and analyzed by XRPD. XRPD pattern with Form B. Table 28 shows all experimental details and results. Table 28: Conversion of Compound I starting material to Form B in denatured ethanol / 0.5% guanidine

化合物I量 (mg) 溶劑量 (mL) 溫度 (°C) 回收量 (mg) 回收率 (%) 形式 505.2 EtOH/O.5% 曱苯 5.0 室溫 479.4 94.89 B 502.1 EtOH/O.5% 曱苯 7.5 室溫 473.5 94.30 B 實例27.在EtOH/O.5%甲苯乙醇2B”）中化合物I游離鹼形式B的5 gm規模製備藉由化合物I起始物質在以0.5%甲苯變性之乙醇（乙醇 128407.doc -125- 200911757 2B)中的形式轉化來進行化合物I游離鹼形式B之5 gm規模製備。經30 min逐份添加化合物I起始物質（由實例3之程序製備）至攪拌之乙醇2B(105 mL，15 vol.)中。在環境溫度下攪拌所得漿料〜19小時，接著過濾。將經分離固體以EtOH 2B(7 mL，1 vol.)洗滌且在真空下在環境溫度乾燥以得到 6.788 g形式Β(96·97°/〇回收率）。藉由XRPD分析所得固體且發現其為結晶。藉由GC進一步表徵此物質之有機揮發性雜質且顯示存在大量乙醇（〜15,000 ppm)及206 ppm甲苯。藉由1H NMR亦觀察到此量之乙醇。藉由OVI或1H NMR未偵測到甲醇及DMA。DSC顯示208 °C之單一吸熱事件。KF 顯示0.45重量°/。水。HPLC及1H NMR並未顯示降解跡象。在40°C下，在真空（30 mm Hg)下進一步乾燥此物質若干天，採用處理中樣品用於NMR分析。若干天之後，樣品並未損失大量乙醇，因此將溫度增至80°C且在此溫度下乾燥 5天。當觀察到乙醇含量為約5000 ppm時，自熱及真空中移除樣品以用於進一步表徵。根據實例2之程序，藉由XRPD、OVI、針對有機揮發性雜質之GC、4 NMR、HPLC、DSC及卡爾費雪分析此乾燥物質。對於HPLC分析而言，使用Varian Pursuit XRs C18(4.6X150 mm，3 μηι)管柱。經乾燥物質得到圖7中所示之XRPD圖樣（CuKa)。以±0.2 °2Θ表示主要X射線繞射線且將其相對強度概括於表29中。圖樣與化合物I游離鹼之形式Β —致。 128407.doc •126- 200911757 表29.自乙醇2B製備之化合物I形式B的XRPD峰值 (CuKa) 峰號 2Θ (度） d間距 (A) 強度 (計數） 1/1« 1 3.12 28.29 67 5 2 10.16 8.70 63 5 3 10.54 8.38 432 33 4 11.04 8.00 555 43 5 11.56 7.65 94 7 6 13.16 6.72 421 33 7 13.98 6.33 218 17 8 14.78 5.99 214 17 9 16.02 5.53 1117 86 10 16.60 5.34 153 12 11 17.21 5.15 1294 100 12 17.60 5.03 900 70 13 19.04 4.66 223 17 14 19.45 4.56 909 70 15 20.40 4.35 1174 91 16 20.88 4.25 1022 79 17 21.52 4.12 287 22 18 21.91 4.052 1098 85 19 23.12 3.84 419 32 20 23.65 3.76 792 61 21 24.52 3.63 260 20 22 25.40 3.50 250 19 23 26.29 3.39 704 54 24 27.36 3.26 73 6 25 28.05 3.18 761 59 26 28.60 3.12 215 17 27 29.72 3.00 57 4 28 30.15 2.96 54 4 29 30.84 2.90 136 11 30 31.18 2.87 76 6 31 32.36 2.76 75 6 32 32.72 2.73 43 3 33 33.89 2.64 101 8 34 35.64 2.52 42 3 35 37.61 2.39 88 7 36 39.82 2.26 48 4 37 41.20 2.19 43 3 38 41.68 2.16 42 3 39 43.89 2.06 45 3 40 44.66 2.03 76 6 128407.doc -127- 200911757 圖8顯不化合物！形式B之特徵巾nmr光譜，其與化合物 I游離鹼之已知結構一致。圖9顯示化合物1形式B之HPLC層析圖。層析圖包括具有 10.4分鐘滯留時間之主峰（97 7% Auc)及具有12 3分鐘 (6_0% ACU)滯留時間之次峰（2 1〇/〇 ACU)。層析圖與先前對化合物I游離鹼所觀察—致。圖10顯示开》式B之DSC迹線。迹線顯示在2 1 rc之單一吸熱事件。 KF顯示0.36重量％水。 OVI顯示5,300 ppm乙醇。亦藉由iH NMR觀察到此量之乙醇。HPLC及1HNMR並未顯示降解跡象。實例28.製備4-((6-乙酿胺基-2Η-β弓丨嗤-2-基）甲基）-N-(2-胺基苯基）苯曱醯胺（化合物Π)Amount of Compound I (mg) Solvent Amount (mL) Temperature (°C) Recovery (mg) Recovery (%) Form 505.2 EtOH/O.5% Benzene 5.0 Room Temperature 479.4 94.89 B 502.1 EtOH/O.5% 曱Benzene 7.5 room temperature 473.5 94.30 B Example 27. 5 gm scale of compound I free base form B in EtOH/O.5% toluene ethanol 2B") Preparation of starting material by compound I in ethanol denatured with 0.5% toluene ( Form conversion in ethanol 128407.doc -125- 200911757 2B) for 5 gm scale preparation of Compound I free base Form B. Compound I starting material (prepared by the procedure of Example 3) was added portionwise over 30 min to stirring Ethanol 2B (105 mL, 15 vol.). The resulting slurry was stirred at ambient temperature for ~19 h then filtered. The separated solid was washed with EtOH 2B (7 mL, 1 vol.) and under vacuum at ambient temperature Drying to give 6.788 g in the form of ruthenium (96.97 ° / oxime recovery). The obtained solid was analyzed by XRPD and found to be crystalline. The organic volatile impurities of this material were further characterized by GC and showed the presence of a large amount of ethanol (~15,000) Ppm) and 206 ppm toluene. This amount of ethanol was also observed by 1H NMR. By OVI or 1H NMR did not detect methanol and DMA. DSC showed a single endothermic event at 208 ° C. KF showed 0.45 wt ° / water. HPLC and 1H NMR did not show signs of degradation. At 40 ° C, under vacuum (30 mm The material was further dried under Hg for several days, using the sample in process for NMR analysis. After several days, the sample did not lose a large amount of ethanol, so the temperature was increased to 80 ° C and dried at this temperature for 5 days. When the ethanol content is about 5000 ppm, the sample is removed from the heat and vacuum for further characterization. According to the procedure of Example 2, by XRPD, OVI, GC for organic volatile impurities, 4 NMR, HPLC, DSC and Karl Fisher analyzed the dried material. For HPLC analysis, a Varian Pursuit XRs C18 (4.6 X 150 mm, 3 μηι) column was used. The dried material gave the XRPD pattern (CuKa) shown in Figure 7. ± 0.2 ° 2 Θ Indicates that the main X-rays are ray-irradiated and their relative intensities are summarized in Table 29. The pattern is in the form of a free base of Compound I. 128407.doc • 126- 200911757 Table 29. XRPD of Compound I Form B Prepared from Ethanol 2B Peak (CuKa) peak number 2Θ (degrees) D-pitch (A) Intensity (count) 1/1« 1 3.12 28.29 67 5 2 10.16 8.70 63 5 3 10.54 8.38 432 33 4 11.04 8.00 555 43 5 11.56 7.65 94 7 6 13.16 6.72 421 33 7 13.98 6.33 218 17 8 14.78 5.99 214 17 9 16.02 5.53 1117 86 10 16.60 5.34 153 12 11 17.21 5.15 1294 100 12 17.60 5.03 900 70 13 19.04 4.66 223 17 14 19.45 4.56 909 70 15 20.40 4.35 1174 91 16 20.88 4.25 1022 79 17 21.52 4.12 287 22 18 21.91 4.052 1098 85 19 23.12 3.84 419 32 20 23.65 3.76 792 61 21 24.52 3.63 260 20 22 25.40 3.50 250 19 23 26.29 3.39 704 54 24 27.36 3.26 73 6 25 28.05 3.18 761 59 26 28.60 3.12 215 17 27 29.72 3.00 57 4 28 30.15 2.96 54 4 29 30.84 2.90 136 11 30 31.18 2.87 76 6 31 32.36 2.76 75 6 32 32.72 2.73 43 3 33 33.89 2.64 101 8 34 35.64 2.52 42 3 35 37.61 2.39 88 7 36 39.82 2.26 48 4 37 41.20 2.19 43 3 38 41.68 2.16 42 3 39 43.89 2.06 45 3 40 44.66 2.03 76 6 128407.doc -127- 200911757 Figure 8 shows no compound! The characteristic sheet nmr spectrum of Form B is consistent with the known structure of Compound I free base. Figure 9 shows an HPLC chromatogram of Compound 1 Form B. The chromatogram included a major peak with a 10.4 minute residence time (97 7% Auc) and a secondary peak with a retention time of 12 3 minutes (6_0% ACU) (2 1 〇/〇 ACU). The chromatogram was observed as previously observed for the free base of Compound I. Figure 10 shows the DSC trace of the open type B. The trace shows a single endothermic event at 2 1 rc. KF showed 0.36 wt% water. OVI shows 5,300 ppm ethanol. This amount of ethanol was also observed by iH NMR. HPLC and 1H NMR did not show signs of degradation. Example 28. Preparation of 4-((6-ethylamino)-2Η-β-indol-2-yl)methyl)-N-(2-aminophenyl)phenylguanamine (Compound Π)

A. 4-((6-硝基_2H-吲唑-2-基）甲基）苯曱酸甲酯（2C)A. 4-((6-Nitro-2H-indazol-2-yl)methyl)benzoic acid methyl ester (2C)

將6-硝基吲唑（2A，20.0 g，122.5941 mmol)及4-溴曱基苯甲酸曱酯（2B，30.89 g，134.8535 mmol)在 DMF(122 mL) 中之溶液置於預熱之120°C油浴中，且藉由磁力攪拌劇烈 128407.doc -128- 200911757 攪拌3小時。HPLC分析顯示反應完全。將反應冷卻至η C ’、’二由旋轉瘵發器移除DMF且傾入冰水混合物（900 mL) 中。添加1 L EtOAc至所得褐黃色水/油/固體混合物中。使用布赫納漏斗來過濾且棄置經分離之未溶解固體。將濾液轉移至2 L分液漏斗且分離各層，丟棄水|。向有機層中添加來自布赫納漏斗過濾之原始固體。在攪拌下，加熱溶液以獲得適度沸騰，使得所有固體均溶解。在冷卻至環境溫度且於冰水浴中再冷卻後，沈澱出淺橙色結晶固體。藉由使用布赫納漏斗過濾將所得固體分離且以冷扮〇心洗蘇。經由旋轉蒸發器濃縮濾液且使用25〇 mL EtOAc重複再結晶。自兩個再結晶提供25.5 g (67%)呈淺絨毛橙色固體之 2C。4 NMR (400 MHz, DMSO〇 δ ppm 3.84 (S，3H) 5.88 (s, 2H) 7.45 (d, J = 8.08 Hz, 2H) 7.83 (dd, J = 9.35, 2.02 Hz, 1H) 7.96 (d, J = 8.08 Hz, 2H) 8.01 (d, J = 9.09 Hz, 1H) 8.64 (d, J = l.oi Hz, 1H) 8.82 (s, 1H) 〇 ESI-MS: m/z 3 12.3 (M+H)+。比率為約1:3(N1:N2)。N-l異構體之NMR資料為： NMR (400 MHz, DMSO-^6) δ ppm 3.82 (s, 3H), 5.96 (s, 2H), 7.35 (d, J = 8.34 Hz, 2H), 7.91 (d, J = 8.34 Hz, 2H), 7.96-8.07 (m，2H)，8.41 (d, J = l.oi Hz, 1 H)，8.86 (s, 1H)。A solution of 6-nitrocarbazole (2A, 20.0 g, 122.5941 mmol) and 4-bromomethyl benzoate (2B, 30.89 g, 134.8535 mmol) in DMF (122 mL) was placed in preheated 120 In a °C oil bath, stir vigorously for 3 hours by magnetic stirring at 128407.doc -128-200911757. HPLC analysis showed the reaction was complete. The reaction was cooled to η C ', and the DMF was removed by a rotary spinner and poured into a mixture of ice water (900 mL). 1 L of EtOAc was added to the resulting brown-yellow water/oil/solid mixture. The Buchner funnel was used to filter and discard the separated undissolved solids. The filtrate was transferred to a 2 L separatory funnel and the layers were separated and water was discarded. The original solid filtered from the Buchner funnel was added to the organic layer. With stirring, the solution was heated to obtain moderate boiling so that all solids dissolved. After cooling to ambient temperature and re-cooling in an ice water bath, a pale orange crystalline solid precipitated. The resulting solid was separated by filtration using a Buchner funnel and washed with cold. The filtrate was concentrated via a rotary evaporator and recrystallized repeatedly using 25 mL EtOAc. 25.5 g (67%) of 2C was obtained as a light yellowish orange solid from two recrystallizations. 4 NMR (400 MHz, DMSO 〇 δ ppm 3.84 (S, 3H) 5.88 (s, 2H) 7.45 (d, J = 8.08 Hz, 2H) 7.83 (dd, J = 9.35, 2.02 Hz, 1H) 7.96 (d, J = 8.08 Hz, 2H) 8.01 (d, J = 9.09 Hz, 1H) 8.64 (d, J = l.oi Hz, 1H) 8.82 (s, 1H) 〇ESI-MS: m/z 3 12.3 (M+ H)+ The ratio is about 1:3 (N1:N2). The NMR data for the Nl isomer are: NMR (400 MHz, DMSO-^6) δ ppm 3.82 (s, 3H), 5.96 (s, 2H) , 7.35 (d, J = 8.34 Hz, 2H), 7.91 (d, J = 8.34 Hz, 2H), 7.96-8.07 (m, 2H), 8.41 (d, J = l.oi Hz, 1 H), 8.86 (s, 1H).

B. 4-((6_胺基-2H-吲唑-2-基）甲基）苯甲酸甲酯（2d) 0 128407.doc -129- 200911757 向4-((6-硝基-2H-吲唑-2-基）曱基）苯甲酸甲酯（2C，25.5 g，91.917 mmol)與單水合肼（39.74 mL)在 MeOH(1638 mL) 中之混合物中添加阮尼鎳（Raney Nickel)(39.74 mL在H20 中之漿料）。在環境溫度下攪拌反應。在10分鐘時之HPLC 分析顯示反應完全，因此抽樣且經由石夕藻土過滤。以 MeOH(lxl〇〇 mL)洗滌矽藻土餅且經由旋轉蒸發來濃縮所得濾液且在真空中進一步乾燥以提供23.3 g (100%)中間物 2D。在未進一步純化之情況下，此物質具有欲使用之足夠純度。4 NMR (400 MHz，DMSO-d6) δ ppm 3.84 (s，3H)， 5.07 (s, 2H), 5.56 (s, 2H), 6.45 (s, 1H), 6.53 (dd, J = 8.84, 1.77 Hz, 1H), 7.35 (d, J = 8.34 Hz, 2H), 7.38 (d, J = 8.84 Hz，1H)，7.92 (d，J = 8.34 Hz, 2H)，8_20 (s, 1H)。ESI-MS: m/z 282.3 (M+H)+。 C. 4-((6-乙醯胺基-2H-吲唑-2-基）曱基）苯曱酸甲酯（2F)B. 4-((6-Amino-2H-indazol-2-yl)methyl)benzoic acid methyl ester (2d) 0 128407.doc -129- 200911757 to 4-((6-nitro-2H- Addition of Raney Nickel to a mixture of methyl carbazol-2-yl)hydrazino)benzoate (2C, 25.5 g, 91.917 mmol) and hydrazine monohydrate (39.74 mL) in MeOH (1638 mL) 39.74 mL of slurry in H20). The reaction was stirred at ambient temperature. HPLC analysis at 10 minutes showed complete reaction, so it was sampled and filtered through Shixia. The diatomaceous earth cake was washed with MeOH (1×1 mL) and the filtrate was concentrated by rotary evaporation and dried in vacuo to afford 23.3 g (100%) Intermediate 2D. This material has sufficient purity to be used without further purification. 4 NMR (400 MHz, DMSO-d6) δ ppm 3.84 (s, 3H), 5.07 (s, 2H), 5.56 (s, 2H), 6.45 (s, 1H), 6.53 (dd, J = 8.84, 1.77 Hz , 1H), 7.35 (d, J = 8.34 Hz, 2H), 7.38 (d, J = 8.84 Hz, 1H), 7.92 (d, J = 8.34 Hz, 2H), 8_20 (s, 1H). ESI-MS: m/z 282.3 (M+H)+. C. 4-((6-Acetylamino-2H-indazol-2-yl)indolyl)benzoic acid methyl ester (2F)

首先將4-((6-胺基-2H-吲唑-2-基）曱基）苯甲酸甲酯（2D， 23.04 g，81.917 mmol)懸浮於無水 DCM(819 mL)中，接著添加三乙胺（34.25 mL，245.751 mmol，3 eq)。在擾摔下’經由加料漏斗逐滴添加乙醯氣（2E，11.64 mL， 163.834 mmol ’ 2 eq)。在1小時之HPLC分析顯示反應完全。將呈澄清橙/黃色溶液之反應混合物轉移至2 L分液漏斗且以飽和NaHC03(2x700 mL)及鹽水（1x700 mL)洗滌有 128407.doc -130- 200911757 機層。以MgSCU乾燥有機層，過濾且經由旋轉蒸發來濃縮且在真空中乾燥以得到23 9 g (9〇%)橙/黃色發泡體。藉由分析型HPLC測得此物質為純的；因此其在未進一步純化之情況下使用。1H NMR (400 MHz, DMSO-d6) δ ppm 2.05 (s, 3H), 3.83 (s, 3H), 5.69 (s, 2H), 7.06 (dd, J = 8.97, 1.64 Hz, 1H), 7.38 (d, J = 8.34 Hz, 2H), 7.62 (d, J = 8.84 Hz, 1H), 7.93 (d, J = 8.34 Hz, 2H), 8.03 (s, 1H), 8.42 (s, 1H), 9.93 (s, 1H)。ESI-MS: m/z 324.3 (M+H)+。 D. 4-((6-乙酿胺引嗤_2_基）甲基）苯甲酸（2g)First, methyl 4-((6-amino-2H-indazol-2-yl)indenyl)benzoate (2D, 23.04 g, 81.917 mmol) was suspended in anhydrous DCM (819 mL). Amine (34.25 mL, 245.751 mmol, 3 eq). Ethylene gas (2E, 11.64 mL, 163.834 mmol ' 2 eq) was added dropwise via an addition funnel. HPLC analysis at 1 hour showed complete reaction. The reaction mixture in clear orange/yellow solution was transferred to a 2 L seperate funnel and washed with saturated NaHC03 (2 x 700 mL) and brine (1 x 700 mL) with a layer of 128407.doc-130-200911757. The organic layer was dried with MgSO.sub.sub.sub.sub.sub. This material was obtained by analytical HPLC to be pure; therefore it was used without further purification. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.05 (s, 3H), 3.83 (s, 3H), 5.69 (s, 2H), 7.06 (dd, J = 8.97, 1.64 Hz, 1H), 7.38 (d , J = 8.34 Hz, 2H), 7.62 (d, J = 8.84 Hz, 1H), 7.93 (d, J = 8.34 Hz, 2H), 8.03 (s, 1H), 8.42 (s, 1H), 9.93 (s , 1H). ESI-MS: m/z 324.3 (M+H)+. D. 4-((6-Ethylamine 嗤_2_yl)methyl)benzoic acid (2g)

以 LiOH水溶液（1 μ ’ 370 mL，5 eq)處理 2F(23.9 g， 73.914 mmol)在1，4-二噁烷（370 mL)中之溶液且在23°C將反應磁力攪拌30分鐘。HPLC分析顯示無起始物質剩餘。添加HC1水溶液（1 Μ，390 mL)且在經由旋轉蒸發器移除1，4-二11惡烧後’所需酸2G呈米色沈澱析出。藉由使用布赫納漏斗過濾將所得固體分離。將固體以水及***沖洗若干次且接著在真空中乾燥以得到18,4 g(80%)淺米色固體。藉由分析型HPLC測得此物質為純的；因此其在未進一步純化之情況下使用。4 NMR (400 MHz, DMSO-d6) δ ppm 2.05 (s， 3H), 5.67 (s, 2H), 7.05 (dd, J = 8.97, 1.64 Hz, 1H), 7.36 (d, J = 8.08 Hz, 2H), 7.62 (d, J = 8.84 Hz, 1H), 7.91 (d, J = 8.08 Hz，2H)，8.03 (s，1H)，8.42 (s，1H), 9.93 (s，1H)。ESI- 128407.doc .131 - 200911757 MS: m/z 310.3 (Μ + H)+。 E. 4-((6-乙醯胺基_211-吲唑-2-基）甲基）-Ν-(2·胺基苯基）苯甲醯胺（2Η)A solution of 2F (23.9 g, 73.914 mmol) in 1,4-dioxane (370 mL) was taken from aq. HPLC analysis showed no starting material remaining. An aqueous solution of HCl (1 Torr, 390 mL) was added and after removal of 1,4-di 11 methane via a rotary evaporator, the desired acid 2G precipitated in a beige color. The resulting solid was separated by filtration using a Buchner funnel. The solid was washed several times with water and diethyl ether and then dried in vacuo to afford &lt This material was obtained by analytical HPLC to be pure; therefore, it was used without further purification. 4 NMR (400 MHz, DMSO-d6) δ ppm 2.05 (s, 3H), 5.67 (s, 2H), 7.05 (dd, J = 8.97, 1.64 Hz, 1H), 7.36 (d, J = 8.08 Hz, 2H ), 7.62 (d, J = 8.84 Hz, 1H), 7.91 (d, J = 8.08 Hz, 2H), 8.03 (s, 1H), 8.42 (s, 1H), 9.93 (s, 1H). ESI-128407.doc .131 - 200911757 MS: m/z 310.3 (Μ + H)+. E. 4-((6-Ethylamino-211-oxazol-2-yl)methyl)-indole-(2·aminophenyl) benzamide (2Η)

向500 mL圓底燒瓶中添加苯甲酸2G(18.40 g，59.4853 mmol ’ 1.0 eq)、1，2-笨二胺（19.30 g，178.4556 mmol，3.0 eq)、EDC(17.11 g ’ 89.2276 mmo卜 1.5 eq)及 HOBt(12.06 g，89.2276 mmol ’ 1.5 eq)。添加DMF(238 mL)且在磁力攪拌下將混合物冷卻至0 °C。經10分鐘逐滴地緩慢添加N-甲基嗎琳（32_7 mL，297,4245 mmol，5.0 eq)。當添加完全時’使反應混合物緩慢升溫至2 3 C。在6 · 5小時之HPLC分析顯示反應完全。在攪拌下將反應混合物傾入水（丨8 L) 中’且藉由使用布赫納漏斗過遽來分離所得固體。將固體以水、己烷及***洗滌若干次’接著在真空中乾燥以得到 21.561 g (90%)呈淺黃色固體之 2H。丨H NMR (400 MHz DMSO-d6) δ ppm 2.06 (s, 3H) 4.89 (s, 2H) 5.68 (s, 2H) 6 58 (t, J = 7.45 Hz, 1H) 6.76 (d, J = 8.08 Hz, 1H) 6 96 (t J = 7.71 Hz, 1H) 7.06 (d，J = 8.84 Hz, 1H) 7.15 (d，J = 7 83 Hz 1H) 7.41 (d, J = 8.34 Hz, 2H) 7.63 (d, J = 9.09 Hz, lH) 7 94 (d，J = 8.08 Hz，2H) 8.04 (s，1H) 8.43 (s，1H) 9 63 (s iH) 9.93 (s，1H)。ESI-MS: m/z 400.4 (M + H)+。 128407.doc •132- 200911757 藉由XRPD發現固體2H為結晶（參見實例29);因此將其指定為化合物II游離鹼之形式A(或化合物I形式A)。由 HPLC、iH NMR、XRPD、TGA、DSC、FTIR(ATR)及水分吸附分析進一步表徵化合物II形式A。在實例29中報導分析結果。將由上文概述之程序製備之物質用作化合物II之其他多晶型物及鹽之所有後續實驗的起始物質。不論其多晶型組合物如何，本文中將起始物質稱為化合物II起始物質、化合物II API、化合物II游離鹼或化合物II。To a 500 mL round bottom flask was added 2 G of benzoic acid (18.40 g, 59.4853 mmol '1.0 eq), 1,2-phenylenediamine (19.30 g, 178.4556 mmol, 3.0 eq), EDC (17.11 g '89.2276 mmo, 1.5 eq And HOBt (12.06 g, 89.2276 mmol '1.5 eq). DMF (238 mL) was added and the mixture was cooled to 0 ° C under magnetic stirring. N-methylmethionine (32_7 mL, 297, 4245 mmol, 5.0 eq) was slowly added dropwise over 10 minutes. When the addition was complete, the reaction mixture was slowly warmed to 2 3 C. HPLC analysis at 6.5 hours showed complete reaction. The reaction mixture was poured into water (丨 8 L) with stirring and the obtained solid was separated by using a Buchner funnel. The solid was washed several times with water, hexanes and diethyl ether' then dried in vacuo to afford <RTI ID=0.0>>丨H NMR (400 MHz DMSO-d6) δ ppm 2.06 (s, 3H) 4.89 (s, 2H) 5.68 (s, 2H) 6 58 (t, J = 7.45 Hz, 1H) 6.76 (d, J = 8.08 Hz , 1H) 6 96 (t J = 7.71 Hz, 1H) 7.06 (d, J = 8.84 Hz, 1H) 7.15 (d, J = 7 83 Hz 1H) 7.41 (d, J = 8.34 Hz, 2H) 7.63 (d , J = 9.09 Hz, lH) 7 94 (d, J = 8.08 Hz, 2H) 8.04 (s, 1H) 8.43 (s, 1H) 9 63 (s iH) 9.93 (s, 1H). ESI-MS: m/z 400.4 (M + H)+. 128407.doc • 132- 200911757 Solid 2H was found to be crystalline by XRPD (see Example 29); thus it was designated as Form A (or Compound I Form A) of Compound II free base. Compound II Form A was further characterized by HPLC, iH NMR, XRPD, TGA, DSC, FTIR (ATR) and moisture adsorption analysis. The analysis results are reported in Example 29. The materials prepared by the procedures outlined above were used as starting materials for all subsequent experiments of other polymorphs and salts of Compound II. Regardless of its polymorphic composition, the starting materials are referred to herein as Compound II starting materials, Compound II API, Compound II free base or Compound II.

實例29.表徵化合物II游離鹼之形式A 由 XRPD、FTIR(ATR)、DSC、TGA、NMR及水分吸附分析表徵實例28中製備之化合物II游離鹼之形式A(化合物II形式A)。藉由XRPD發現化合物II形式A為結晶。圖35顯示化合物 II之特徵XRPD光譜（CuKa)。以±0.2。20表示主要X射線繞射線且將其相對強度概括於表30中。表30.彳匕合物II游離鹼之形式Α的XRPD峰值（CuKa) 峰號 2Θ (度） d間距 (A) 強度 (計數） 1/1〇 1 4.39 20.10 25 4 2 4.99 17.68 37 6 3 11.32 7.81 86 13 4 12.65 6.99 79 12 5 13.94 6.35 253 39 6 14.54 6.09 200 31 7 15.16 5.84 32 5 8 16.23 5.46 155 24 9 17.08 5.19 124 19 10 17.54 5.05 539 83 11 18.98 4.67 39 6 128407.doc -133- 200911757 峰號 2Θ (度） d ~~ (A) 強度 (計數） 1/1〇 12 20.36 4.36~~~~ 465 72 13 20.92 4^4 648 1〇〇 14 22.20 4.00~~'~~ 349 54 15 22.73 3.91 ~~ 456 70 16 23.48 379 ~~~ 42 6 17 23.98 3/71 51 8 18 24.97 " 3l6~~~ 319 49 19 25.48 3.49~~~~ 19 3 20 26.64 3Μ 23 4 21 27.26 254 39 22 28.46 V ------- 3.13 107 17 23 29.16 ΪΜ~~~ 48 7 24 29.88 2.99 50 8 25 30.48 2Τ93 21 3 26 30.84 Ϊ9〇 ~~ 42 6 27 31.58 2^83~~~~~ 19 3 28 32.43 -- 丄/6 24 4 29 32.91 -- 丄/2 23 4 30 38.93 2.31 51 8 31 40.18 22 3 --- 圖36顯示化合物II形式A之特徵iH NMR光譜。光譜與化合物11之已知結構一致。圖37顯示化合物I之形式A的特徵FTIR(ATR)光譜。以波長倒數（以cm 1計之波數）表示之主帶定位於約362〇、 3438、3347、3235、3116、3046、2836、1926、1903、 1662、1635、1610、1569、1525 ' 1496、1480、1457、 1420、1409、1367、1306、1293、1 1269、1227、1187、 1143、1122、1035、1016、973、961、951、945、907、 864、818、775、751、724、084 及 650 cm、捨入最近整數之值）。 DSC分析顯示在77〇c&i79〇c之吸熱，接著在ι84^之放熱（圖38)。在24TC觀察到最終事件，其可表明在製備期間形成之環狀雜質或其他降解產物。 128407.doc -134- 200911757 TGA顯示在l〇〇t：以下約2%之重量損失（圖39)，其可能歸因於殘餘溶劑或表面結合水之損失。水刀及附分析顯示自RH至90% RH該物質為非吸濕性及穩定單水合物（圖40)。水分吸附後之xRPD分析得到與先4所得XRPD圖樣一致之圖樣。此表明在環境實驗室條件下該物質迅速轉變回單水合物。實例30·化合物II游離鹼之溶劑溶解度將根據實例28之程序製備之化合物„游離鹼用作鑑別及結晶實驗之起始物質。對化合物π進行此溶解度研究以鑑別用於鹽形成之合適單一溶劑。基於ICH分類選擇溶劑，較佳為II類及Ιπ類溶劑。亦基於其極性及官能性來選擇溶对J 所選’谷劑為.乙腈（MeCN)、丙綱、1，4-二°惡烧、乙醇 (EtOH)、異丙醇（IPA)、乙酸乙酯（Et〇Ac)、乙酸異丙酯 (IPAC)、甲基第三丁基醚（MTBE)、四氫呋喃（THF)、甲基乙基_ (MEK)、庚烷、環己烷、曱苯、二氣甲烷（DCM)、乙酸（AcOH)、二甲基曱醯胺（DMF)及甲醇（Me〇H)。向樣品小瓶中裝載1.〇_2·〇 mg之間的化合物π游離鹼且以〇·25 mL之小份向各小瓶中添加所關注之溶劑直至至多5.〇 mL °在室溫下震盪小瓶十分鐘且目視檢查以評估溶解情況’且接著加熱至55°c且保持在5yc十分鐘以測定較高溫度是否將改良溶解。表3 1顯示化合物Π游離鹼之溶解度結果。 128407.doc -135- 200911757 表31.化合物Π游離鹼之溶劑溶解度Example 29. Characterization of Form II of Compound II Free Base A Form A (Compound II Form A) of Compound II free base prepared in Example 28 was characterized by XRPD, FTIR (ATR), DSC, TGA, NMR and moisture adsorption analysis. Compound II Form A was found to be crystalline by XRPD. Figure 35 shows the characteristic XRPD spectrum (CuKa) of Compound II. The main X-ray diffraction is represented by ± 0.20 and the relative intensities are summarized in Table 30. Table 30. Form of chelate II free base XRPD peak of Α (CuKa) Peak number 2 Θ (degrees) d Spacing (A) Strength (count) 1/1 〇 1 4.39 20.10 25 4 2 4.99 17.68 37 6 3 11.32 7.81 86 13 4 12.65 6.99 79 12 5 13.94 6.35 253 39 6 14.54 6.09 200 31 7 15.16 5.84 32 5 8 16.23 5.46 155 24 9 17.08 5.19 124 19 10 17.54 5.05 539 83 11 18.98 4.67 39 6 128407.doc -133- 200911757 Peak number 2Θ (degrees) d ~~ (A) Intensity (count) 1/1〇12 20.36 4.36~~~~ 465 72 13 20.92 4^4 648 1〇〇14 22.20 4.00~~'~~ 349 54 15 22.73 3.91 ~~ 456 70 16 23.48 379 ~~~ 42 6 17 23.98 3/71 51 8 18 24.97 " 3l6~~~ 319 49 19 25.48 3.49~~~~ 19 3 20 26.64 3Μ 23 4 21 27.26 254 39 22 28.46 V ------- 3.13 107 17 23 29.16 ΪΜ~~~ 48 7 24 29.88 2.99 50 8 25 30.48 2Τ93 21 3 26 30.84 Ϊ9〇~~ 42 6 27 31.58 2^83~~~~~ 19 3 28 32.43 -- 丄/6 24 4 29 32.91 -- 丄/2 23 4 30 38.93 2.31 51 8 31 40.18 22 3 --- Figure 36 shows the characteristic iH NMR spectrum of Compound II Form A. The spectrum is identical to the known structure of Compound 11. Figure 37 shows the characteristic FTIR (ATR) spectrum of Form A of Compound I. The main band represented by the reciprocal of the wavelength (the wave number in cm 1 ) is positioned at about 362 〇, 3438, 3347, 3235, 3116, 3046, 2836, 1926, 1903, 1662, 1635, 1610, 1569, 1525 ' 1496, 1480, 1457, 1420, 1409, 1367, 1306, 1293, 1 1269, 1227, 1187, 1143, 1122, 1035, 1016, 973, 961, 951, 945, 907, 864, 818, 775, 751, 724, 084 And 650 cm, rounding the value of the nearest integer). DSC analysis showed an endotherm at 77 °c & i79〇c followed by an exotherm at ι84^ (Figure 38). A final event was observed at 24TC which may indicate cyclic impurities or other degradation products formed during preparation. 128407.doc -134- 200911757 TGA shows a weight loss of about 2% below l〇〇t: (Fig. 39), which may be due to the loss of residual solvent or surface bound water. The waterjet and analysis showed that the material was non-hygroscopic and stable monohydrate from RH to 90% RH (Figure 40). The xRPD analysis after moisture adsorption gave a pattern consistent with the XRPD pattern obtained in the first 4. This indicates that the substance is rapidly converted back to monohydrate under environmental laboratory conditions. Example 30. Solvent Solubility of Compound II Free Base The compound „free base prepared according to the procedure of Example 28 was used as a starting material for the identification and crystallization experiments. This solubility study was performed on compound π to identify a suitable single solvent for salt formation. The solvent is selected based on the ICH classification, preferably the type II and Ιπ-type solvents. The solvent selected according to the polarity and the functionality is selected as the glutamine (MeCN), the propyl group, and the 1,4-two Erosification, ethanol (EtOH), isopropanol (IPA), ethyl acetate (Et〇Ac), isopropyl acetate (IPAC), methyl tert-butyl ether (MTBE), tetrahydrofuran (THF), methyl Ethyl (MEK), heptane, cyclohexane, toluene, dihydromethane (DCM), acetic acid (AcOH), dimethyl decylamine (DMF) and methanol (Me〇H). Load the compound π free base between 1.〇_2·〇mg and add the solvent of interest to each vial in 0.25 mL aliquots until at most 5. 〇mL ° shake the vial for ten minutes at room temperature and Visually inspect to evaluate the dissolution 'and then heat to 55 ° C and hold at 5 yc for ten minutes to determine if the higher temperature is The dissolution is improved. Table 3 1 shows the solubility results of the compound hydrazine free base. 128407.doc -135- 200911757 Table 31. Solvent solubility of the compound hydrazine free base

溶劑物質量 (mg) 溶劑量 (mL) 濃度 (mg/mL) 溫度 (°C) 可溶 ICH類別 MeCN 1.6 0.50 3.2 55 是 '-— II 二噁烷 2.5 0.50 5.0 55 是 II 一丙_ 2.1 0.25 >8.4 室溫是 III MTBE 1.7 5.00 — n/a 否 III EtOH 1.5 0.75 2.0 55 是 ----- III EtOAc 1.4 2.00 0.7 55 是 III IPAC 1.3 3.00 0.4 55 是 ^lii ' IPA 1.7 1.25 1.4 55 是 III THF 2.5 0.25 >10.0 室溫是 ~' MEK 1.1 0.25 >4.4 室溫是 III DMF 1.7 0.25 >6.8 室溫是 II 一 MeOH ^ 2.1 0.50 4.2 55 是 II AcOH 2.2 0.25 >8.8 室溫是 ----- III c-己烷 1.2 5.00 n/a 否 II 庚烷 2.4 5.00 … n/a 否 III DCM 2.7 0.50 5.4 55 是 II 曱笨 2.0 5.00 … n/a 否 ---- II 溶解度資料顯示在室溫或55°C下觀察到大多數溶劑將物質溶解於小於1 mL之溶劑中，酯（乙酸乙酯及乙酸異丙酿）及異丙醇除外，其需要1-3 mL。發現為化合物Π之不良溶 el的彼專溶劑為曱基第三丁基謎及非極性溶劑（庚烧、環己烷及曱笨）。實例31.化合物Η之結晶固體的80 mg規模結晶將化合物II API溶解於相應溶劑中且在攪拌下加熱聚料 (對於THF而言25。(：，對於MeCN而言7(TC，對於丙酮而言 5〇°C且對於二噁烷而言9〇。(：）以達成完全溶解，且接著將 μ度調節至結晶溫度。一次性添加平衡離子且將所得混合物授拌分鐘。若適用’則以抓/小時之速率將反應混合物冷卻至環境溫度。在環境溫度下，使用溫和氮氣流將 128407.doc -136· 200911757 反應混合物蒸發至原始體積之一半。若觀察到沈澱，則過濾反應混合物以收集固體。若未觀察到沈澱，則使用溫和氮氣流將反應混合物蒸發至無水。在真空下在環境溫度乾燥所有固體隔夜。將實驗細節概括於表32中。表32.化合物II之結晶固體的80 mg規模結晶的實驗條件 API量 (mg) 溶劑溶劑量 (mL) 平衡離子 1.05當量之量(mL) 溫度 (°C) 沈澱 84 THF 15 苯曱酸，1.0M在 THF中 0.22 60 蒸發至無水 80 THF 15 龍膽酸，0.5 Μ在 THF中 0.42 25 蒸發至無水 79 MeCN 30 HCl’l.ON^ MeCN 中 0.21 25 過渡 80 丙酮 15 HC1，1.0M在丙酮中 0.21 25 蒸發至無水 80 MeCN 30 丁二酸，0.25 Μ 在 MeCN(5% 水)中 0.84 25 蒸發至無水 80 MeCN 30 ，1.01^1在 MeCN 中 0.21 25 過濾由XRPD、質譜分析、DSC及1H NMR來分析所有樣品。將結果概括於表33中。得到具有與形式Α相比獨特XRPD圖樣之固體的結晶溶劑為：苯曱酸/THF、龍膽酸/THF、HCl/MeCN、丁二酸/ MeCN及 HBr/MeCN。 MS分析顯示所有樣品均與化合物II之結構一致。質譜分析顯示所有五種鹽均無降解。 NMR分析顯示所有樣品均與化合物II之結構一致。另外，對龍膽酸鹽及丁二酸鹽進行之1H NMR分析顯示存在有機平衡離子（龍膽酸或丁二酸離子），具有約1:1莫耳比之平衡離子與化合物Π。 128407.doc -137- 200911757 對氫氯酸鹽及氫溴酸鹽候選物進行之元素分析顯示約 1:1莫耳比之平衡離子（氯離子或溴離子）與化合物II。在表 3 4中報導結果。表33.以80 mg規模結晶製備之化合物II鹽之鹽的分析資料平衡離子/溶劑 XRPD結果 MS結果 DSC 結果(°C) 1H NMR結果 HC1/THF 非晶形無降解 - 一致苯曱酸/THF 獨特無降解 112,167,180 ⑻， 200 < 1:1比率龍膽酸/THF 獨特無降解 127, 160, 240 1:1比率 HCl/MeCN 獨特無降解 198, 206, >300 一致丁二酸/MeCN 獨特無降解 152, 212 ⑻，282 〜1:1比率 HBr/MeCN 獨特無降解 210, 225,270 一致表34.氫氣酸鹽及氫溴酸鹽之元素分析資料量測值理論值(1:1 HC1) 量測值理論值(1:1 HBr) C 62.45 63.37 56.65 57.51 Η 4.91 5.09 4.36 4.62 Ν 15.75 16.07 14.26 14.58 Cl 8.43 8.13 - - Br - - 15.18 16.63 實例32.化合物II固體之100 mg規模結晶放大在苯曱酸、丁二酸、氫氯酸及氫溴酸存在下結晶之固體。向配備有熱電偶、磁力攪拌棒及冷凝器之三頸圓底燒瓶中裝載100-120 mg化合物II樣品，接著在加熱至60°C的同時逐份（1 mL)添加THF及MeCN以達成最小溶劑體積，直至觀察到完全溶解。接著使所得溶液冷卻至環境溫度，隨後逐滴添加在反應溶劑中之平衡離子。在室溫下攪拌反應混合物隔夜。 128407.doc -138- 200911757 使用氫氯酸及氫溴酸在MeCN中之1 Μ溶液的鹽形成得到沈澱出溶液之固體，其經由真空過濾而分離。獲得呈棕褐色固體的化合物II之氫溴酸鹽（124.5 mg，86%產率）及化合物II之氫氣酸鹽（42.9 mg，33%產率）。然而，對於苯曱酸或丁二酸未觀察到沈澱，且反應混合物均蒸發至無水以得到呈棕褐色固體之苯甲酸鹽（1 10.3 mg，70%產率）及丁二酸鹽（92.3 mg，67%產率）。此等反應之概括呈現於表35 中〇表35.化合物II之結晶鹽的100 mg規模製備樣品量 (mg) 溶劑溶劑量 (mL) 平衡離子所得質量 (mg) %產率 XRPD結果* 120.2 THF 10 苯曱酸 110.3 70 結晶-一致 120.0 MeCN 61 HBr 124.5 86 半結晶-一致 101.3 MeCN 33 HC1 42.9 33 半結晶-一致 106.6 MeCN 35 丁二酸 92.5 67 半結晶-'一致 *與在80 mg規模結晶期間產生之固體相比。根據實例2中所概述之程序，由包括XRPD、DSC、 TGA、FTIR(ATR)、NMR及水分吸附分析之方法分析所有鹽。發現分析結果與鹽篩檢期間之先前結晶一致。實例33.表徵化合物II之丁二酸鹽由 XRPD、4 NMR、FTIR(ATR)、DSC、TGA及水分吸附及解吸附分析來表徵實例32中製備之化合物II之丁二酸鹽（化合物II丁二酸鹽）。藉由XRPD發現丁二酸鹽為結晶固體。圖4 1顯示化合物 II 丁二酸鹽之特徵XRPD光譜（CuKcx)。以士 0.2 °2Θ表示主要 X射線繞射線且將其相對強度概括於表36中。 128407.doc •139- 200911757 表36·化合物ιι之丁二酸鹽的XRPD峰值（CuKa) 1 2 (度） 4.99^、~ 1 Π ---- d間距 (A) 強度 (計數） Γ ι/h 17.70 137 一.Τ'''''- X W · 1 ^ 8.49 48 3 〜 3 11.37 7.78 226 14~~~~ 4 12.63 一^ 5 1 a Q1 7.00 201 Ϊ3 ^ U.OJ 6.40 416 ----- 6 14.52 ^- 6.10 201 Is— 7 1 j.UU 5.90 197 8 9 16-09 1 η Λ(\^ 5.51 603 38 ~~ 1 / Λ\) 5.09 126 8 — 10 lo.UU 4.92 205 I3 ' ^ 11 18.44 4.81 525 33 ~~ 12 1 8.00 4.70 635 '40--' 13 20.28 4.38 619 39" 14 20.95 4.24 1129 7〇 —- 15 22.17 4.01 1603 100 — 16 23.16 3.84 130 _ "8--— 17 23.93 3.72 133 8 — 18 24.57 3.62 548 _ _ '3?— 19 25.68 3.47 52 3 — 9~~~~— 20 26.16 3.40 141 21 27.13 3.28 1209 75 — 22 28.08 3.17 243 .is^〜 23 28.76 3.10 264 .'16- 24 29.20 3.06 120 ""T — 25 29.60 3.02 63 4 ^ 26 31.62 2.83 112 7 ^ 27 32.40 2.76 306 19 28 38.29 2.35 73 5 '^ 1H NMR分析發現光譜與結構及1:1比率之丁二酸平衡離子與API—致（圖42)。 3229、 3119 、 1727 、 1662、 1635 > 1611、 1567、 1544、 1496、 1457 、 1413 、 1352、 1327、 1305、 1266 ' 1227、 FTIR(ATR)分析發現光譜與化合物II之已知結構及丨：1比率之丁二酸平衡離子與API—致（圖43)。以波長倒數（以cm_i 計之波數）表示之主要FTIR帶定位於約3438、3347、 128407.doc 140 200911757 1187、1140、1122、1034、1017、944.25、907、862、 818、798、776、751、723、687 及 654 cm·、捨入最近整數之值）。 DSC分析顯示在約150°C之吸熱，接著在約210°C之放熱。在282°C觀察到最終事件且可表明在實驗期間形成之環狀雜質或其他降解產物（圖44)。 TGA顯示在150°C以下無重量損失及在154°C之分解起始 (圖 45)。水分吸附分析顯示自5% RH至90% RH該物質為非吸濕性物質（圖46)。水分吸附後之XRPD分析得到與先前所得 XRPD圖樣一致之圖樣。實例34.表徵化合物II之苯甲酸鹽由 XRPD、4 NMR、FTIR(ATR)、DSC、TGA及水分吸附及解吸附分析來表徵實例32中製備之化合物II之苯曱酸鹽（化合物II苯甲酸鹽）。藉由XRPD發現苯曱酸鹽為結晶固體。圖47顯示丁二酸鹽之特徵XRPD光譜（CuKa)。以±0.2。20表示主要X射線繞射線且將其相對強度概括於表37中。表37.彳匕合物II之苯曱酸鹽的XRPD峰值（CuKa) 峰號 2Θ (度） d間距 (A) 強度 (計數） 1/1〇 1 7.38 11.97 51 3 2 8.20 10.77 302 20 3 9.07 9.74 257 17 4 10.60 8.34 184 12 5 12.44 7.11 51 3 6 12.88 6.87 756 51 7 16.33 5.42 314 21 128407.doc • 141 - 200911757 峰號 8 11互 ΊΓ 15 17Solvent mass (mg) Solvent amount (mL) Concentration (mg/mL) Temperature (°C) Soluble ICH class MeCN 1.6 0.50 3.2 55 Yes '-- II Dioxane 2.5 0.50 5.0 55 Yes II One C _ 2.1 0.25 >8.4 Room temperature is III MTBE 1.7 5.00 — n/a No III EtOH 1.5 0.75 2.0 55 Yes --- III EtOAc 1.4 2.00 0.7 55 Yes III IPAC 1.3 3.00 0.4 55 Yes ^lii ' IPA 1.7 1.25 1.4 55 Yes III THF 2.5 0.25 > 10.0 Room temperature is ~' MEK 1.1 0.25 > 4.4 Room temperature is III DMF 1.7 0.25 > 6.8 Room temperature is II - MeOH ^ 2.1 0.50 4.2 55 is II AcOH 2.2 0.25 > 8.8 Room temperature is ----- III c-hexane 1.2 5.00 n/a no II heptane 2.4 5.00 ... n/a no III DCM 2.7 0.50 5.4 55 Yes II 曱 2.0 2.0 5.00 ... n/a No---- II Solubility data It was shown that most solvents were observed to dissolve the material in less than 1 mL of solvent at room temperature or 55 ° C, with the exception of esters (ethyl acetate and isopropyl acetate) and isopropanol, which required 1-3 mL. The specific solvent which was found to be a poor solution of the compound el was a mercapto-tert-butyl ketone and a non-polar solvent (geptane, cyclohexane and oxime). Example 31. 80 mg scale crystallization of the crystalline solid of the compound 化合物 Compound II API was dissolved in the corresponding solvent and the polymer was heated with stirring (25 for THF (:, for MeCN 7 (TC for acetone) 5 〇 ° C and 9 对于 for dioxane. (:) to achieve complete dissolution, and then adjust the μ degree to the crystallization temperature. Add the counter ion once and stir the mixture for a few minutes. The reaction mixture was cooled to ambient temperature at a rate of capture/hour. The ambient reaction temperature was used to evaporate the 128407.doc-136.200911757 reaction mixture to one and a half of the original volume at ambient temperature. If precipitation was observed, the reaction mixture was filtered. The solid was collected. If no precipitate was observed, the mixture was evaporated to dryness using a gentle nitrogen stream. All solids were dried overnight at ambient temperature under vacuum. The experimental details are summarized in Table 32. Table 32. Crystalline solids of compound II Experimental conditions for 80 mg scale crystallization API amount (mg) Solvent solvent amount (mL) Balance ion 1.05 equivalent amount (mL) Temperature (°C) Precipitate 84 THF 15 Benzoic acid, 1.0 M Evaporation of 0.22 in THF to anhydrous 80 THF 15 gentisic acid, 0.5 Μ in THF 0.42 25 Evaporation to anhydrous 79 MeCN 30 HCl'l.ON^ MeCN 0.21 25 Transition 80 Acetone 15 HC1, 1.0 M in acetone 0.21 25 Evaporate to anhydrous 80 MeCN 30 succinic acid, 0.25 蒸发 in MeCN (5% water) 0.84 25 Evaporate to anhydrous 80 MeCN 30, 1.01^1 in MeCN 0.21 25 Filter by XRPD, mass spectrometry, DSC and 1H NMR All samples. The results are summarized in Table 33. The crystallization solvent to obtain a solid having a unique XRPD pattern compared to the form Α is: benzoic acid/THF, gentisic acid/THF, HCl/MeCN, succinic acid/MeCN and HBr/MeCN. MS analysis showed that all samples were consistent with the structure of compound II. Mass spectrometry showed no degradation of all five salts. NMR analysis showed that all samples were consistent with the structure of compound II. In addition, for gentisate and butyl 1H NMR analysis of the diacid salt revealed the presence of an organic counterion (gentocholic acid or succinic acid ion) with a counter ion of about 1:1 molar ratio with the compound hydrazine. 128407.doc -137- 200911757 For hydrochloric acid Salt and hydrobromide candidates Elemental analysis showed a balanced ion (chloride or bromide) of about 1:1 molar ratio with compound II. The results are reported in Table 34. Table 33. Analysis of the salt of the compound II salt prepared by crystallization on a 80 mg scale Data Balanced Ion/Solvent XRPD Results MS Results DSC Results (°C) 1H NMR Results HC1/THF Amorphous No Degradation - Uniform Benzoic Acid/THF Unique No Degradation 112,167,180 (8), 200 < 1:1 ratio gentisic acid/THF Unique non-degradable 127, 160, 240 1:1 ratio HCl/MeCN Unique non-degradable 198, 206, > 300 Uniform succinic acid / MeCN Unique non-degradable 152, 212 (8), 282 ~ 1:1 ratio HBr/MeCN Unique Degradation 210, 225, 270 Consistent Table 34. Elemental Analysis of Hydrogenate and Hydrobromide Data The theoretical value (1:1 HC1) The theoretical value (1:1 HBr) C 62.45 63.37 56.65 57.51 Η 4.91 5.09 4.36 4.62 Ν 15.75 16.07 14.26 14.58 Cl 8.43 8.13 - - Br - - 15.18 16.63 Example 32. Compound II solid 100 mg scale crystallisation solidified in the presence of benzoic acid, succinic acid, hydrochloric acid and hydrobromic acid . A 100-120 mg sample of Compound II was loaded into a three-necked round bottom flask equipped with a thermocouple, a magnetic stir bar and a condenser, followed by THF and MeCN in portions (1 mL) while heating to 60 ° C to achieve a minimum Solvent volume until complete dissolution was observed. The resulting solution is then cooled to ambient temperature and the counterion in the reaction solvent is then added dropwise. The reaction mixture was stirred overnight at room temperature. 128407.doc -138- 200911757 The salt formation of a solution of hydrochloric acid and hydrobromic acid in MeCN was used to obtain a solid which precipitated a solution which was separated by vacuum filtration. The hydrobromide salt of Compound II (124.5 mg, 86% yield) and the Hydrogen salt of Compound II (42.9 mg, 33% yield) were obtained as a brown solid. However, no precipitation was observed for benzoic acid or succinic acid, and the reaction mixture was evaporated to dryness to give benzoate as a tan solid (1 10.3 mg, 70% yield) and succinate (92.3 Mg, 67% yield). A summary of these reactions is presented in Table 35. Table 35. 100 mg of the crystalline salt of Compound II. Sample size (mg) Solvent solvent amount (mL) Balanced ion mass (mg) % yield XRPD result * 120.2 THF 10 Benzoic acid 110.3 70 Crystalline - Consistent 120.0 MeCN 61 HBr 124.5 86 Semi-crystalline - Consistent 101.3 MeCN 33 HC1 42.9 33 Semi-crystalline - Consistent 106.6 MeCN 35 Succinic acid 92.5 67 Semi-crystalline - 'consistent* with crystallization during 80 mg scale Compared to the solid produced. All salts were analyzed by a method including XRPD, DSC, TGA, FTIR (ATR), NMR, and moisture adsorption analysis according to the procedure outlined in Example 2. The results of the analysis were found to be consistent with previous crystallization during salt screening. Example 33. Succinate salt characterization of compound II The succinate salt of compound II prepared in Example 32 was characterized by XRPD, 4 NMR, FTIR (ATR), DSC, TGA and moisture adsorption and desorption analysis (Compound II) Diacid salt). The succinate salt was found to be a crystalline solid by XRPD. Figure 41 shows the characteristic XRPD spectrum (CuKcx) of the compound II succinate. The main X-rays are rayed at ± 0.2 °2 且 and their relative intensities are summarized in Table 36. 128407.doc •139- 200911757 Table 36·XRPD peak of compound ι succinate (CuKa) 1 2 (degrees) 4.99^,~ 1 Π ---- d spacing (A) Strength (counting) Γ ι/ h 17.70 137 I.Τ'''''- XW · 1 ^ 8.49 48 3 ~ 3 11.37 7.78 226 14~~~~ 4 12.63 1^ 5 1 a Q1 7.00 201 Ϊ3 ^ U.OJ 6.40 416 ---- - 6 14.52 ^- 6.10 201 Is— 7 1 j.UU 5.90 197 8 9 16-09 1 η Λ(\^ 5.51 603 38 ~~ 1 / Λ\) 5.09 126 8 — 10 lo.UU 4.92 205 I3 ' ^ 11 18.44 4.81 525 33 ~~ 12 1 8.00 4.70 635 '40--' 13 20.28 4.38 619 39" 14 20.95 4.24 1129 7〇—- 15 22.17 4.01 1603 100 — 16 23.16 3.84 130 _ "8--- 17 23.93 3.72 133 8 — 18 24.57 3.62 548 _ _ '3?— 19 25.68 3.47 52 3 — 9~~~~— 20 26.16 3.40 141 21 27.13 3.28 1209 75 — 22 28.08 3.17 243 .is^~ 23 28.76 3.10 264 .' 16- 24 29.20 3.06 120 ""T — 25 29.60 3.02 63 4 ^ 26 31.62 2.83 112 7 ^ 27 32.40 2.76 306 19 28 38.29 2.35 73 5 '^ 1H NMR analysis found spectrum and structure and 1:1 ratio Diacid balanced ion and API - (FIG. 42). 3229, 3119, 1727, 1662, 1635 > 1611, 1567, 1544, 1496, 1457, 1413, 1352, 1327, 1305, 1266 ' 1227, FTIR (ATR) analysis found the known structure of the spectrum and compound II and 丨: The 1 ratio of succinic acid counterion is related to the API (Figure 43). The main FTIR band represented by the reciprocal of the wavelength (wavenumber in cm_i) is located at approximately 3438, 3347, 128407.doc 140 200911757 1187, 1140, 1122, 1034, 1017, 944.25, 907, 862, 818, 798, 776, 751, 723, 687, and 654 cm·, round the value of the nearest integer). DSC analysis showed an endotherm at about 150 °C followed by an exotherm at about 210 °C. The final event was observed at 282 °C and may indicate cyclic impurities or other degradation products formed during the experiment (Figure 44). TGA showed no weight loss below 150 °C and decomposition initiation at 154 °C (Figure 45). Moisture adsorption analysis showed that the material was non-hygroscopic from 5% RH to 90% RH (Figure 46). XRPD analysis after moisture adsorption yielded a pattern consistent with the previously obtained XRPD pattern. Example 34. Characterization of the benzoate salt of Compound II The benzoate salt of Compound II prepared in Example 32 was characterized by XRPD, 4 NMR, FTIR (ATR), DSC, TGA, and moisture adsorption and desorption analysis. Formate). The benzoate was found to be a crystalline solid by XRPD. Figure 47 shows the characteristic XRPD spectrum (CuKa) of succinate. The main X-ray diffraction is represented by ± 0.20 and the relative intensities are summarized in Table 37. Table 37. XRPD peak (CuKa) of the phthalate salt of the chelate II peak number 2 Θ (degrees) d spacing (A) intensity (counting) 1/1 〇 1 7.38 11.97 51 3 2 8.20 10.77 302 20 3 9.07 9.74 257 17 4 10.60 8.34 184 12 5 12.44 7.11 51 3 6 12.88 6.87 756 51 7 16.33 5.42 314 21 128407.doc • 141 - 200911757 Peak number 8 11 interaction 15 17

20 ΤΓ互 ~23~ 14 ~25~互互互 ~29~ 10" 1Γ互 1T 2Θ(度） 17.43 18.18 19.20 19.48 20.50 21.38 22.08 22.46 23.48 23.93 24.59 25.44 25.90 26.61 26.92 27.84 28.16 28.81 29.30 30.24 30.64 31.87 32.88 34.94 38.71 39.3120 ΤΓ mutual ~23~ 14 ~25~ mutual mutual ~29~ 10" 1Γ mutual 1T 2Θ (degrees) 17.43 18.18 19.20 19.48 20.50 21.38 22.08 22.46 23.48 23.93 24.59 25.44 25.90 26.61 26.92 27.84 28.16 28.81 29.30 30.24 30.64 31.87 32.88 34.94 38.71 39.31

Η N M R分析發現光譜（圖4 8 )與化合物〗】之已知結構及略大於1:1比率之苯曱酸平衡離子與API一致。 \ FTIR(ATR)分析發現光譜（圖49)與化合物π之已知結構及略大於1:1比率之苯曱酸平衡離子與API一致。以波長倒數（以cm·1計之波數）表示之主要FTIR帶定位於約3400、 3264、3114、3045、2836、2560、1650、1635、1583、 1527、1484、1453、1421、1386、1278、1172 ' 1153、 1131、1071、1〇21、1005、926、911、853、843、809、 751、706及683 cm·1 (捨入最近整數之值）。 DSC分析顯示在約not：之吸熱’接著在約116°C之放 128407.doc -142· 200911757 熱。觀察到在約200。(：之另一放熱’接著在約2丨6之吸熱。在283T：觀察到最終事件，其可表明在實驗期間形成之環狀雜質或其他降解產物（圖50)。在約60 C至170°C之間，TGA分析顯示總計9.6重量。/〇之連續重量損失’其可能歸因於殘餘溶劑及/或降解。在18〇 °C附近分解起始（圖5 1)。水分吸附分析顯示自5% RH至90% RH該物質為非吸濕性及穩定單水合物（圖52)。水分吸附後殘餘固體之XRpD 分析得到與先前所進行之XRPD圖樣一致之圖樣。此表明在環境實驗室條件下物質迅速轉變回單水合物。實例35.表徵化合物II之氩氣酸鹽由 XRPD、^ NMR、FTIR(ATR)、DSC、TGA及水分吸附及解吸附分析來表徵藉由實例32之方法製備之化合物Π 之氫氯酸鹽（化合物II氫氯酸鹽）。藉由XRPD發現氫氯酸鹽為半結晶固體。圖53顯示氫氣酸鹽之特徵XRPD光譜（CuKa)。以±0.2 °2Θ表示主要X射線繞射線且將其相對強度概括於表38中。表38.化合物Π之氩氯酸鹽的XRPD峰值（CuKa) 峰號 2Θ (度） d間距 (A) 強度 (計數） 1/1〇 1 3.21 27.48 21 5 2 3.59 24.61 17 4 3 A ~~~ - 4.02 21.94 13 3 4 _ —— 4.69 18.82 13 3 5 5.86 15.08 16 4 6 6.80 12.99 25 6 7 7.23 12.22 15 4 8 7.64 11.56 14 3 9 8.44 10.47 16 4 128407.doc -143- 200911757Η N M R analysis found that the known structure of the spectrum (Fig. 48) and the compound] and the benzoic acid equilibrium ion slightly larger than the 1:1 ratio are consistent with the API. The FTIR (ATR) analysis revealed that the spectrum (Fig. 49) is consistent with the known structure of the compound π and the benzoic acid counterion of slightly larger than 1:1 ratio with the API. The main FTIR bands, represented by the reciprocal of the wavelength (wavenumber in cm·1), are located at approximately 3400, 3264, 3114, 3045, 2836, 2560, 1650, 1635, 1583, 1527, 1484, 1453, 1421, 1386, 1278. , 1172 ' 1153 , 1131 , 1071 , 1〇 21 , 1005 , 926 , 911 , 853 , 843 , 809 , 751 , 706 , and 683 cm · 1 (round the value of the nearest integer). The DSC analysis showed an endotherm at about not: then followed by a heat of about 128 ° C. 407 - 142 · 2009 11757. Observed at about 200. (The other exotherm' followed by an endotherm at about 2丨6. At 283T: a final event was observed, which may indicate ring-shaped impurities or other degradation products formed during the experiment (Figure 50). At about 60 C to 170 Between ° C, TGA analysis showed a total of 9.6 wt. / 〇 continuous weight loss 'which may be attributed to residual solvent and / or degradation. Decomposition initiation near 18 ° C (Figure 5 1). Water adsorption analysis shows The material is non-hygroscopic and stable monohydrate from 5% RH to 90% RH (Figure 52). XRpD analysis of residual solids after moisture adsorption yields a pattern consistent with the previously performed XRPD pattern. Substance conditions rapidly converted back to monohydrate. Example 35. Characterization of compound II argonate was characterized by XRPD, NMR, FTIR (ATR), DSC, TGA and moisture adsorption and desorption analysis by Example 32. Method for the preparation of the compound Π of the hydrochloride (Compound II hydrochloride). The hydrochloride was found to be a semi-crystalline solid by XRPD. Figure 53 shows the characteristic XRPD spectrum (CuKa) of the hydrogenate. ± 0.2 ° 2Θ indicates that the main X-rays ray around and make it relatively strong Summarized in Table 38. Table 38. XRPD peak (CuKa) of the compound argon chlorate peak number 2 Θ (degrees) d spacing (A) intensity (counting) 1/1 〇 1 3.21 27.48 21 5 2 3.59 24.61 17 4 3 A ~~~ - 4.02 21.94 13 3 4 _ —— 4.69 18.82 13 3 5 5.86 15.08 16 4 6 6.80 12.99 25 6 7 7.23 12.22 15 4 8 7.64 11.56 14 3 9 8.44 10.47 16 4 128407.doc -143- 200911757

NMR分析發現光譜與化合物π之結構一致（圖54) FTIR(ATR)分析發現光譜與化合物Π之結構一致（圖 55)。以波長倒數（以cm-4十之波數）表示之主要FTIR帶定位於約 3_、3026、2969、2850、2575、1672、1615、 1568、1528、1488、1442、1373、1314、1293、1272、 128407.doc -144· 200911757 1192、1143、1020、949、912、863、818、772、752、 744、714及692 cm、捨入最近整數之值）。 DSC分析顯示在197t之吸熱，接著在約2〇^C2放熱。在292°C觀察到最終事件且可表明在實驗期間形成之環狀雜質或其他降解產物（圖56)。 TGA顯示在16(rc以下無重量損失及在約i6〇 2i〇c>c之間約4.9%之重量損失，其可能歸因於降解（圖57)。水为吸附分析顯示物質為適度吸濕性物質，在9〇% RH 獲知3.8重量％(圖58)。水分吸附後殘餘固體之XRpD分析得到與先前所進行之XRPD圖樣一致之圖樣。實例36·表徵化合物η之氫溴酸鹽由 XRPD、NMR、FTIR(ATR)、DSC、TGA及水分吸附及解吸附分析來表徵藉由實例32之方法製備之化合物π 之氫溴酸鹽（化合物II氫溴酸鹽）。藉由XRPD發現氫溴酸鹽為結晶固體。圖59顯示氫溴酸鹽之特徵XRPD光譜（CuKa)。以土〇_2 °2Θ表示主要X射線繞射線且將其相對強度概括於表39中。表39.化合物II之氫溴酸鹽的XRPD峰值（CuKa) 峰號 2Θ (度） d間距 (A) 強度 (計數） m〇 1 8.67 10.19 82 9 2 -r— 10.80 ΪΪ25 8.19 X86 117 961 12 100 4 5 6 12.96 6.82 37 4 16.32 5.43 85 ] 9 17.16 5.16 106 11 7 17.59 5.04 343 36 8 1 18.52 4.79 92 10 9 18.92 4.69 954 99 128407.doc • 145 - 200911757 峰號 2Θ (度） d間距 (A) 強度 (計數） 1/1〇 10 19.44 4.56 196 20 11 19.76 4.49 196 20 12 20.12 4.41 155 16 13 21.52 4.13 29 3 14 22.19 4.00 407 42 15 22.84 3.89 161 17 16 23.80 3.74 118 12 17 24.16 3.68 80 8 18 24.56 3.62 241 25 19 24.88 3.58 167 17 20 25.53 3.49 294 31 21 25.96 3.43 247 26 22 26.55 3.35 530 55 23 27.92 3.19 89 9 24 28.46 3.13 293 30 25 28.95 3.08 278 29 26 29.52 3.02 80 8 27 30.22 2.95 103 11 28 31.64 2.83 172 18 29 32.54 2.75 44 5 30 33.40 2.68 43 4 31 34.12 2.63 82 9 32 34.48 2.60 57 6 33 34.80 2.58 64 7 34 35.28 2.54 94 10 35 36.12 2.48 50 5 36 36.52 2.46 66 7 37 37.58 2.39 43 4 38 39.95 2.25 40 4 39 40.92 2.20 66 7 40 41.16 2.19 40 4 41 41.56 2.17 51 5 42 42.11 2.14 31 3 43 43.99 2.06 30 3 44 44.49 2.03 45 5 NMR分析發現光譜與化合物II之結構一致（圖60)。 FTIR(ATR)分析發現光譜與化合物II之結構一致（圖 61)。以波長倒數（以cm·1計之波數）表示之主要FTIR帶定位於約 3154、3094、2977、2660、2597、1666、1603、 1562、1523、1500、1480、1461、1373、1305、1270、 128407.doc -146- 200911757 1197、1139、1035、1123、951、939、913、868 > 828 ' 812、762、751、742、696 及 666 cm_1(捨入最近整數之值）。 DSC分析顯示在約214 °C之吸熱，接著在約224 °C之放熱。在296。(：觀察到最終事件，其可表明在實驗期間形成之環狀雜質或其他降解產物（圖62)。直至170°C以上TGA才顯示重量損失；在兩個步驟中當繼續加熱至約2201時，重量損失總計3.8%。重量損失可能歸因於降解（圖63)。水分吸附分析顯示物質為適度吸濕性，在9〇% RH獲得 5_5重量％(圖64)。水分吸附後之Xrpd分析得到與先前所進行之XRPD圖樣一致的圖樣。附件1概括物理表徵結果。實例37.表徵化合物η之龍膽酸鹽由 XRPD、咕 NMR、FTIR(ATR)、DSC、TGA及水分吸附及解吸附分析來表徵藉由實例3丨之方法製備之化合物工工之龍膽酸鹽（化合物Π龍膽酸鹽）。藉由XRPD發現化合物II龍膽酸鹽為半結晶固體。圖65 顯示龍膽酸鹽之特徵XRPD光譜（CuKa)。以±〇 2。20表示主要X射線繞射線且將其相對強度概括於表4〇中。表4〇.化合物11之龍膽酸鹽的XRPD峰值（CuKa)NMR analysis revealed that the spectrum was consistent with the structure of the compound π (Fig. 54). FTIR (ATR) analysis revealed that the spectrum was consistent with the structure of the compound ( (Fig. 55). The main FTIR band represented by the reciprocal of the wavelength (in the range of cm-4 ten waves) is located at about 3_, 3026, 2969, 2850, 2575, 1672, 1615, 1568, 1528, 1488, 1442, 1373, 1314, 1293, 1272. 128407.doc -144· 200911757 1192, 1143, 1020, 949, 912, 863, 818, 772, 752, 744, 714 and 692 cm, rounding the value of the nearest integer). DSC analysis showed an endotherm at 197t followed by an exotherm at about 2〇C2. The final event was observed at 292 °C and may indicate cyclic impurities or other degradation products formed during the experiment (Figure 56). TGA showed no weight loss below 16 (rc below and about 4.9% weight loss between about i6 〇 2i 〇 c > c, which may be due to degradation (Figure 57). Water for adsorption analysis shows that the material is moderately hygroscopic The substance was found to be 3.8% by weight at 9 % RH (Fig. 58). XRpD analysis of residual solids after moisture adsorption gave a pattern consistent with the previously performed XRPD pattern. Example 36. Characterization of Compound η Hydrobromide by XRPD , NMR, FTIR (ATR), DSC, TGA, and moisture adsorption and desorption analysis to characterize the hydrobromide salt of the compound π prepared by the method of Example 32 (Compound II hydrobromide salt). Hydrobromination was found by XRPD. The acid salt is a crystalline solid. Figure 59 shows the characteristic XRPD spectrum (CuKa) of the hydrobromide salt. The main X-ray diffraction is indicated by soil 〇 2 ° 2 且 and its relative intensity is summarized in Table 39. Table 39. XRPD peak (CuKa) of hydrobromide peak number 2Θ (degrees) d spacing (A) intensity (counting) m〇1 8.67 10.19 82 9 2 -r- 10.80 ΪΪ25 8.19 X86 117 961 12 100 4 5 6 12.96 6.82 37 4 16.32 5.43 85 ] 9 17.16 5.16 106 11 7 17.59 5.04 343 36 8 1 18.52 4.7 9 92 10 9 18.92 4.69 954 99 128407.doc • 145 - 200911757 Peak number 2Θ (degrees) d spacing (A) intensity (counting) 1/1〇10 19.44 4.56 196 20 11 19.76 4.49 196 20 12 20.12 4.41 155 16 13 21.52 4.13 29 3 14 22.19 4.00 407 42 15 22.84 3.89 161 17 16 23.80 3.74 118 12 17 24.16 3.68 80 8 18 24.56 3.62 241 25 19 24.88 3.58 167 17 20 25.53 3.49 294 31 21 25.96 3.43 247 26 22 26.55 3.35 530 55 23 27.92 3.19 89 9 24 28.46 3.13 293 30 25 28.95 3.08 278 29 26 29.52 3.02 80 8 27 30.22 2.95 103 11 28 31.64 2.83 172 18 29 32.54 2.75 44 5 30 33.40 2.68 43 4 31 34.12 2.63 82 9 32 34.48 2.60 57 6 33 34.80 2.58 64 7 34 35.28 2.54 94 10 35 36.12 2.48 50 5 36 36.52 2.46 66 7 37 37.58 2.39 43 4 38 39.95 2.25 40 4 39 40.92 2.20 66 7 40 41.16 2.19 40 4 41 41.56 2.17 51 5 42 42.11 2.14 31 3 43 43.99 2.06 30 3 44 44.49 2.03 45 5 NMR analysis revealed that the spectrum was consistent with the structure of Compound II (Fig. 60). FTIR (ATR) analysis revealed that the spectrum was consistent with the structure of Compound II (Fig. 61). The main FTIR band represented by the reciprocal of the wavelength (the wave number in cm·1) is located at about 3154, 3094, 2977, 2660, 2597, 1666, 1603, 1562, 1523, 1500, 1480, 1461, 1373, 1305, 1270. 128407.doc -146- 200911757 1197, 1139, 1035, 1123, 951, 939, 913, 868 > 828 '812, 762, 751, 742, 696 and 666 cm_1 (rounding the value of the nearest integer). DSC analysis showed an endotherm at about 214 °C followed by an exotherm at about 224 °C. At 296. (: The final event was observed, which may indicate ring-shaped impurities or other degradation products formed during the experiment (Figure 62). TGA shows weight loss up to 170 °C; when heating continues to about 2201 in two steps The weight loss is 3.8% in total. The weight loss may be attributed to degradation (Fig. 63). The moisture adsorption analysis shows that the material is moderately hygroscopic, obtaining 5-5 wt% at 9〇% RH (Fig. 64). Xrpd analysis after moisture adsorption A pattern consistent with the previously performed XRPD pattern was obtained. Annex 1 summarizes the physical characterization results. Example 37. Characterization of compound η gentisate by XRPD, 咕NMR, FTIR (ATR), DSC, TGA and moisture adsorption and desorption Analysis to characterize the gentisate compound (compound gentisolic acid salt) prepared by the method of Example 3. The compound II gentisate was found to be a semi-crystalline solid by XRPD. Figure 65 shows gentisic acid The characteristic XRPD spectrum (CuKa) of the salt. The main X-rays are rayed by ± 〇 2.20 and their relative intensities are summarized in Table 4 。. Table 4 X. XRPD peak (CuKa) of the gentisate of compound 11.

128407.doc -147- 200911757 峰號 2Θ(°) d間距強度 Ι/Ι〇 7 12.52 7.06 136 10 8 12.87 6^87 707 54 9 16.96 5.22 85 7 10 17.49 5.07 1306 100 11 18.04 4.91 158 12 12 18.44 4.81 94 7 13 19.56 4.53 ' 343 26 14 20.60 4.31 135 10 15 21.48 4Λ3 264 20 16 22.45 Γ 3.96 349 27 17 23.6U 3.77 59 5 18 24.16 3.68 266 20 19 24.77 "Ϊ59 249 19 20 25.28 3S2 ' 90 7 21 25.93 3.43 ' 690 53 22 26.67 3.34 403 31 23 28.32 3.15 216 17 24 28,88 3.09 93 7 25 29.32 3.04 49 4 26 30.20 2.96 " 42 3 27 30.64 2.92 116 9 28 31.00 2.88 69 5 29 31.94 2.80 55 4 30 32.73 '2J3 50 4 H NMR分析發現光譜與結構及1:1比率之龍膽酸鹽平衡離子與API—致（圖66)。 FTIR(ATR)及1H NMR分析發現光譜與結構及1:丨比率之龍膽酸鹽平衡離子與API—致（圖67)。以波長倒數（以cm-1 計之波數）表示之主要FTIR帶定位於約3260、1654、 1635、1584、1550、1528、1504、1484、1443、1385、 1302、1278 ' 1241、1218、1153、1133、1082、1034、 1021、1006、940、911、876、854、842、810、799、 788、75 3、725及680 cm·1(捨入最近整數之值）。 DSC分析顯示在127°C、16〇°C及24〇°C之吸熱。最終事件係在240°C且可表明在實驗期間形成之環狀雜質或其他 128407.doc -148- 200911757 降解產物（圖68)。 TGA顯示在60X：與17(TC之間幾乎5%之重量損失，其可能歸因於殘餘溶劑及/或降解（圖69)。降解起始於約Μ]”。水分吸附分析顯示物質在5% RH與90% RH之間為單水合物（圖70)。水分吸附後殘餘固體之XRpD分析得到與先前所進行之XRPD圖樣一致的圖樣。實例38.化合物Π之多晶型物及鹽的水溶性以3〇 mg規模在DI水中對形式A及為化合物π所鑑別之各引導鹽進行溶解度量測。向配備有磁力攪拌棒之4打蘭 (dram)琥珀色小瓶中裝載各樣品且在環境溫度下在以水中調衆1 6 1 8小時。接著離心樣品以自上澄溶液分離固體且移除上澄液之丨mL部分且使其穿過注射過濾器。基於針對呈游離鹼形式之各化合物產生之校正資料，得到關於各鹽 /辰度之近似值且因此以移動相（丨：丨比率之MeCN/水）稀釋樣。使用HPLC分析來測定所得各鹽相對於游離鹼之溶解度。將各種鹽之溶解度概括於表41中。表41.化合物π之多晶型物及鹽的水溶性128407.doc -147- 200911757 Peak number 2Θ(°) d pitch strength Ι/Ι〇7 12.52 7.06 136 10 8 12.87 6^87 707 54 9 16.96 5.22 85 7 10 17.49 5.07 1306 100 11 18.04 4.91 158 12 12 18.44 4.81 94 7 13 19.56 4.53 ' 343 26 14 20.60 4.31 135 10 15 21.48 4Λ3 264 20 16 22.45 Γ 3.96 349 27 17 23.6U 3.77 59 5 18 24.16 3.68 266 20 19 24.77 "Ϊ59 249 19 20 25.28 3S2 ' 90 7 21 25.93 3.43 ' 690 53 22 26.67 3.34 403 31 23 28.32 3.15 216 17 24 28,88 3.09 93 7 25 29.32 3.04 49 4 26 30.20 2.96 " 42 3 27 30.64 2.92 116 9 28 31.00 2.88 69 5 29 31.94 2.80 55 4 30 32.73 '2J3 50 4 H NMR analysis revealed spectral and structure and 1:1 ratio of gentisate counterion to API (Figure 66). FTIR (ATR) and 1H NMR analysis revealed spectral and structure and 1: 丨 ratio of gentisate counterion and API (Figure 67). The main FTIR band, expressed in terms of wavelength reciprocal (wavenumber in cm-1), is located at approximately 3260, 1654, 1635, 1584, 1550, 1528, 1504, 1484, 1443, 1385, 1302, 1278 '1241, 1218, 1153 , 1133, 1082, 1034, 1021, 1006, 940, 911, 876, 854, 842, 810, 799, 788, 75 3, 725, and 680 cm·1 (rounding the value of the nearest integer). DSC analysis showed an endotherm at 127 ° C, 16 ° C and 24 ° C. The final event is at 240 ° C and may indicate cyclic impurities formed during the experiment or other degradation products of 128407.doc -148- 200911757 (Figure 68). TGA showed almost 5% weight loss between 60X: and 17 (TC, which may be attributed to residual solvent and/or degradation (Figure 69). Degradation starts at about Μ].) Moisture adsorption analysis shows that the material is at 5 Between % RH and 90% RH is a monohydrate (Figure 70). XRpD analysis of residual solids after moisture adsorption yields a pattern consistent with previously performed XRPD patterns. Example 38. Compounds of polymorphs and salts Water solubility The dissolution profile of Form A and each of the lead salts identified for compound π was measured in DI water on a 3 〇 mg scale. Each sample was loaded into a 4 dram amber vial equipped with a magnetic stir bar and The mixture was conditioned in water at ambient temperature for 1661 hours. The sample was then centrifuged to separate the solids from the supernatant and the aliquot of the supernatant was removed and passed through a syringe filter. The calibration data generated for each compound gave an approximation for each salt/density and was therefore diluted with the mobile phase (MeCN/water ratio of the ruthenium ratio). The solubility of the resulting salt relative to the free base was determined using HPLC analysis. Summary of solubility of various salts Table 41. Table 41. Compound π polymorphs and as much as the water-soluble salts thereof

固體游離鹼丁二酸鹽苯甲酸鹽 HC1 ~HBr-- 水溶性 (rag/mL、 7.0 5.8 3.6 17 ~4?7 ND =未測定 '—-J 實例39.化合物Π之結晶固體的熱應力研究實施熱應力以評估化合物II形式Α及其鹽轉化成替代择晶形及/或降解之傾向。將各約1 0 mg的化合物II之形式a、苯曱酸鹽、氫氣酸 128407.doc -149- 200911757 鹽、氫溴酸鹽及丁二酸鹽置於1打蘭琥珀色小瓶中。將小瓶置於烘箱中在60 °C下歷時兩天。接著由XRPD、MS及 HPLC分析固體。將結果概括於表42中。表42.化合物II之結晶固體的熱應力研究條件結晶固體天數 XRPD* MS HPLC %(AUC) 60。。形式A 2 一致一致 95 60V 氫溴酸鹽 2 一致一致 96 60°C 苯甲酸鹽 2 一致觀察到降解 - 60°C Hce鹽 2 一致一致 94 60°C 丁二酸鹽 3 一致一致 93 *與化合物I形式A相比。實例40.化合物II之結晶固體的環境溫度漿料研究在兩種反溶劑中進行漿料研究以評估化合物II形式A及其鹽轉化成替代結晶形及/或降解之傾向。將各約1 0 mg之形式A、苯甲酸鹽、氫氣酸鹽、氫溴酸鹽及丁二酸鹽置於裝配有攪拌棒之1打蘭琥珀色小瓶中。添加二分之一毫升溶劑（正庚烷、甲苯）至樣品小瓶中且將漿料在室溫下攪拌兩天。藉由過濾來移除溶劑且在室溫下在真空烘箱中（30吋Hg)進行進一步乾燥歷時1-2小時。接著由XRPD、MS及HPLC來分析固體。將結果概括於表43中。 128407.doc •150- 200911757 表43.化合物II固體之環境溫度漿料研究條件結晶鹽天數 XRPD* MS HPLC % (AUC) 曱苯(環境）形式A 2 一致一致 98 正庚烷(環境）一致一致 97 甲苯(環境）氫溴酸鹽 2 一致一致 98 正庚烷(環境）一致一致 98 曱苯(環境）苯曱酸鹽 2 一致一致 93 正庚烷(環境）一致一致 93 甲苯(環境） Hce 2 一致一致 95 正庚烷(環境）一致一致 94 甲苯(環境）丁二酸鹽 3 一致一致 95 正庚烷(環境）一致一致 94 *與形式A相比。實例41 :製備1-(4-(2-胺基苯基胺甲醯基）苄基）-N-乙基-1H-吡唑-4-甲醢胺（化合物III)Solid free base succinate benzoate HC1 ~HBr-- Water solubility (rag/mL, 7.0 5.8 3.6 17 ~4?7 ND = not determined'--J Example 39. Thermal stress of crystalline solids of compound Π Thermal stress was investigated to evaluate the tendency of the compound II form of ruthenium and its salts to be converted into alternative crystal forms and/or degradation. Each form of about 10 mg of compound II, a benzoate, hydrogen acid 128407.doc-149 - 200911757 Salt, hydrobromide and succinate were placed in 1 blue amber vials. The vials were placed in an oven at 60 °C for two days. The solids were then analyzed by XRPD, MS and HPLC. Summarized in Table 42. Table 42. Thermal Stress Study Conditions for Crystalline Solids of Compound II Crystalline Solid Days XRPD* MS HPLC % (AUC) 60. Form A 2 Consistent 95 60V Hydrobromide 2 Consistent 96 60° C benzoate 2 consistently observed degradation - 60 ° C Hce salt 2 consistent 94 60 ° C succinate 3 consistent 93 * compared to compound I Form A. Example 40. Crystalline solids of compound II Temperature slurry study for slurry research in two antisolvents to evaluate Form II Form A and its salt are converted to an alternative crystalline form and/or degradation tendency. Each form of about 10 mg of Form A, benzoate, hydrogenate, hydrobromide and succinate is placed in the assembly. 1 bar blue amber vial with stir bar. Add one-half ml of solvent (n-heptane, toluene) to the sample vial and stir the slurry for two days at room temperature. Remove the solvent by filtration and Further drying was carried out in a vacuum oven (30 吋Hg) for 1-2 hours at room temperature. The solids were then analyzed by XRPD, MS and HPLC. The results are summarized in Table 43. 128407.doc •150- 200911757 Table 43 .Compound II Solid Ambient Temperature Slurry Study Conditions Crystalline Salt Days XRPD* MS HPLC % (AUC) Toluene (Environment) Form A 2 Consistent 98 n-Heptane (Environment) Consistent 97 Toluene (Environment) Hydrobromide 2 Consistent 98 n-heptane (environment) Consistent 98 terpene (environment) benzoate 2 Consistent 93 n-heptane (environment) Consistent 93 Toluene (environment) Hce 2 Consistent 95 n-heptane (environment) Consistent 94 toluene (environment) Acid salt 3 Consistent 95 n-heptane (environment) Consistent 94 *Compared to Form A. Example 41: Preparation of 1-(4-(2-aminophenylaminomethane)benzyl)-N-B keto-1H-pyrazole-4-carboxamide (Compound III)

A· 1-(4-(甲氧羰基）苄基）-1Η-«比唑-4-曱酸（3C)A· 1-(4-(methoxycarbonyl)benzyl)-1Η-«bizozol-4-furoic acid (3C)

ho2cHo2c

3C3C

3B DMF, 80°C 96% 將1H-吡唑-4-曱酸3B(2.58 g，23 mmol)及4-溴甲基苯曱酸曱醋3A(5.27 g，23 mmol)在DMF(25 mL)中之溶液置於預熱之80°C油浴中且藉由磁力攪拌劇烈攪拌24小時。冷卻 128407.doc -151 - 200911757 反應且真空濃縮。將殘餘物溶解於水卩〇中以得到懸洋液。將所得固體藉由過濾分離，以水洗滌且在真空中在 23 C下乾燥以得到呈白色固體之3C(5 743 g，96%)。丨η NMR (400 MHz，DMSO-d6) § ppm 3 85 (s，3H)，5 47 (s 2Η), 7.36 (d, J = 8.08 Hz, 2H), 7.86 (s, 1H), 7.95 (d, J = 8.34 Hz, 2H), 8.44 (s, 1H), 12.42 (br. s., 1H) 〇 ESI-MS: m/z 261.3 (M + H)+。 Β· 4-((4-(乙基胺甲醯基）_1H_吡唑4_基）曱基）苯甲酸甲酯（3E)3B DMF, 80°C 96% 1H-pyrazole-4-decanoic acid 3B (2.58 g, 23 mmol) and 4-bromomethylbenzoic acid vinegar 3A (5.27 g, 23 mmol) in DMF (25 mL) The solution was placed in a preheated 80 ° C oil bath and stirred vigorously for 24 hours by magnetic stirring. Cooling 128407.doc -151 - 200911757 Reaction and concentration in vacuo. The residue was dissolved in hydrazine to obtain a suspension. The resulting solid was separated by EtOAc (EtOAc) elute丨η NMR (400 MHz, DMSO-d6) § ppm 3 85 (s, 3H), 5 47 (s 2Η), 7.36 (d, J = 8.08 Hz, 2H), 7.86 (s, 1H), 7.95 (d , J = 8.34 Hz, 2H), 8.44 (s, 1H), 12.42 (br. s., 1H) 〇ESI-MS: m/z 261.3 (M + H)+. Β· 4-((4-(ethylamine-methyl)-H-pyrazole-4-yl)methyl)benzoic acid methyl ester (3E)

3E EDC, HOBt NMM, DMF 0 °C,2h 100% 向200 mL圓底燒瓶中添加〇比。坐甲酸（3C，5.72 g，22 0 mmol ’ 1.0 eq)、乙胺氫氣酸鹽（3D，j.99 g，24.4 mmol， 1.1 eq)、EDC(4.67 g，24.4 mmol，1.1 eq)及 HOBt(3.29 g，24.4 mmol，1.1 eq)。添加DMF(5 8 mL)且在磁力攪拌下將混合物冷卻至〇°C。經10分鐘逐滴地緩慢添加N_曱基嗎琳（4.74 mL，88.5 mmol，4.0 eq)。當添加完全時，使反應混合物緩慢升溫至23 °C。在3小時之HPLC分析顯示反應完全。在攪拌下將反應混合物傾入冰水（800 mL)中，且藉由使用布赫納漏斗過濾來分離所得固體。將固體以水及己烧洗滌若干次且接著在真空中乾燥以得到6.36 g(l 〇〇〇/〇)呈白色固體之 3E。4 NMR (400 MHz，DMSO-D6) δ ppm 1.06 128407.doc -152- 200911757 (t, J = 7.20 Hz, 3H), 3.12-3.22 (m, 2H), 3.83 (s, 3H), 5.44 (s, 2H), 7.32 (d, J = 8.34 Hz, 2H), 7.87 (s, 1H), 7.93 (d, J = 8.34Hz,2H)，8.07(t，J = 4_93Hz，lH)，8.24(s，lH)°ESI-MS: m/z 288.3 (M + H)+。 C. 4-((4-(乙基胺甲醯基）-1Η-吡唑-1-基）甲基）苯甲酸（3F)3E EDC, HOBt NMM, DMF 0 °C, 2h 100% Add a ruthenium ratio to a 200 mL round bottom flask. Formic acid (3C, 5.72 g, 22 0 mmol '1.0 eq), ethylamine hydrochloride (3D, j.99 g, 24.4 mmol, 1.1 eq), EDC (4.67 g, 24.4 mmol, 1.1 eq) and HOBt ( 3.29 g, 24.4 mmol, 1.1 eq). DMF (5 8 mL) was added and the mixture was cooled to 〇 ° C under magnetic stirring. N_mercaptoline (4.74 mL, 88.5 mmol, 4.0 eq) was slowly added dropwise over 10 minutes. When the addition was complete, the reaction mixture was slowly warmed to 23 °C. HPLC analysis at 3 hours showed complete reaction. The reaction mixture was poured into ice water (800 mL) with stirring, and the obtained solid was separated by filtration using a Buchner funnel. The solid was washed several times with water and hexane and then dried in vacuo to give <RTI ID=0.0>> 4 NMR (400 MHz, DMSO-D6) δ ppm 1.06 128407.doc -152- 200911757 (t, J = 7.20 Hz, 3H), 3.12-3.22 (m, 2H), 3.83 (s, 3H), 5.44 (s , 2H), 7.32 (d, J = 8.34 Hz, 2H), 7.87 (s, 1H), 7.93 (d, J = 8.34Hz, 2H), 8.07 (t, J = 4_93Hz, lH), 8.24(s, lH) °ESI-MS: m/z 288.3 (M + H)+. C. 4-((4-(ethylamine-mercapto)-1Η-pyrazol-1-yl)methyl)benzoic acid (3F)

3E 3F3E 3F

以 LiOH水溶液（1 Μ，110 mL，5 eq)處理 3E(6.36 g， 22.0 mmol)在1,4-二噁烷（110 mL)中之溶液且在23°C下磁力授拌反應1小時。HPLC分析顯示無起始物質剩餘。添加 HC1水溶液（1 Μ，25 0 mL)且藉由使用布赫納漏斗過濾來分離所得白色固體。以水將固體沖洗若干次且接著在真空中乾燥固體以得到4.43 g(73%)呈白色固體之3F。4 NMR (400 MHz, DMSO-D6) δ ppm 1.06 (t, J = 7.20 Hz, 3H), 3.13-3.23 (m, 2H), 5.42 (s, 2H), 7.30 (d, J = 8.08 Hz, 2H), 7.86 (s, 1H), 7.91 (d, J - 8.08 Hz, 2H), 8.06 (t, J = 5.56 Hz, 1H)，8.24 (s, 1H)，12.97 (s, 1H)。ESI-MS: m/z 274.3 (M + H)+。ESI-MS: m/z 274.3 (M + H)+。 D. l-(4-(2·胺基苯基胺甲酿基）节基）_N-乙基-1H-"比也-4-甲醯胺（3H)A solution of 3E (6.36 g, 22.0 mmol) in 1,4-dioxane (110 mL) was taken from aqueous EtOAc (1 EtOAc, EtOAc (EtOAc) HPLC analysis showed no starting material remaining. An aqueous solution of HCl (1 Torr, 25 mL) was added and the obtained white solid was separated by filtration using a Buchner funnel. The solid was rinsed several times with water and then dried in vacuo to give 4.43 g (73%) of 3F as white solid. 4 NMR (400 MHz, DMSO-D6) δ ppm 1.06 (t, J = 7.20 Hz, 3H), 3.13-3.23 (m, 2H), 5.42 (s, 2H), 7.30 (d, J = 8.08 Hz, 2H ), 7.86 (s, 1H), 7.91 (d, J - 8.08 Hz, 2H), 8.06 (t, J = 5.56 Hz, 1H), 8.24 (s, 1H), 12.97 (s, 1H). ESI-MS: m/z 274.3 (M + H)+. ESI-MS: m/z 274.3 (M + H)+. D. l-(4-(2·Aminophenylamine) Alkyl)_N-Ethyl-1H-"Bis-4-pyridylamine (3H)

3G EDC, HOBt NMM, DMF 0弋,2卜 90%3G EDC, HOBt NMM, DMF 0弋, 2 Bu 90%

3H3H

nh2 128407.doc •153· 200911757 向100 mL圓底燒瓶中添加苯甲酸（3F，4.43 g，16.2 mmol ’ 1.0 eq)、1，2-苯二胺（3G，7.〇2 g，64 8 咖〇卜 4 〇 eq)、EDC(4.66 g，24·3 mmo卜 1.5 eq)及 HOBt(3.29 g， 24_3 mmol，1·5 eq)。添加DMF(65 mL)且在磁力攪拌下將混合物冷卻至。經10分鐘逐滴地緩慢添加N_曱基嗎啉 (8.9 mL，81.1 mmo卜5.〇 eq)。當添加完全時，使反應混合物緩慢升溫至23°C。在5小時之HPLC分析顯示反應完全。在攪拌下將反應混合物傾入冰水（1.〇 L)中，且藉由使用布赫納漏斗過濾來分離所得固體。將固體以水及己烧洗滌若干次且接著在真空中乾燥以得到5 24 g(90%)呈白色固體之3H。 NMR (400 MHz, OUSO-d6) δ ppm 1.08 (7, J = 7.2〇 Hz, 3H), 3.20 (m, 2H), 4.90 (s, 2H), 5.43 (s, 2H), 6.54-6.64 (m, 1H), 6.77 (dd, J = 7.83, 1.26 Hz, 1H), 6.87-7.05 (m, 1H), 7.15 (d, J = 7.58 Hz, 1H), 7.36 (d, J = 8.08 Hz, 2H), 7.88 (s, 1H), 7.96 (d, J = 8.08 Hz, 2H), 8.01-8.12 (m, 1H), 8.25 (s, 1H), 9.64 (s，1H)。ESI-MS: m/z 364.4 (M + H)+。將藉由上文概述之程序製備的物質用作所有後續實驗之起始物質以鑑別化合物III之其他多晶型物及鹽。各批起始物質之多晶型組合物可視製程或儲存條件而不同。不論其多晶型組合物如何’本文中將起始物質稱為化合物in起始物質、化合物III API、化合物III游離驗或化合物III。Nh2 128407.doc •153· 200911757 Add benzoic acid (3F, 4.43 g, 16.2 mmol ' 1.0 eq), 1,2-phenylenediamine (3G, 7. 2 g, 64 8 coffee) to a 100 mL round bottom flask 4 〇 eq), EDC (4.66 g, 24.3 mmo, 1.5 eq) and HOBt (3.29 g, 24_3 mmol, 1.5 eq). DMF (65 mL) was added and the mixture was cooled to magnetic stirring. N_Mercaptomorpholine (8.9 mL, 81.1 mmob 5. 〇 eq) was slowly added dropwise over 10 minutes. When the addition was complete, the reaction mixture was slowly warmed to 23 °C. HPLC analysis at 5 hours showed complete reaction. The reaction mixture was poured into ice water (1. 〇 L) with stirring, and the obtained solid was separated by filtration using a Buchner funnel. The solid was washed several times with water and hexane and then dried in vacuo to give 5 24 g (90%) of 3H as white solid. NMR (400 MHz, OUSO-d6) δ ppm 1.08 (7, J = 7.2〇Hz, 3H), 3.20 (m, 2H), 4.90 (s, 2H), 5.43 (s, 2H), 6.54-6.64 (m , 1H), 6.77 (dd, J = 7.83, 1.26 Hz, 1H), 6.87-7.05 (m, 1H), 7.15 (d, J = 7.58 Hz, 1H), 7.36 (d, J = 8.08 Hz, 2H) , 7.88 (s, 1H), 7.96 (d, J = 8.08 Hz, 2H), 8.01-8.12 (m, 1H), 8.25 (s, 1H), 9.64 (s, 1H). ESI-MS: m/z 364.4 (M + H) +. The materials prepared by the procedure outlined above were used as starting materials for all subsequent experiments to identify other polymorphs and salts of Compound III. The polymorphic composition of each batch of starting material will vary depending on the process or storage conditions. Regardless of its polymorphic composition, the starting materials are referred to herein as the compound in starting material, the compound III API, the compound III free test or the compound III.

實例42.表徵化合物III之游離鹼的形式A 由 XRPD、NMR、FTIR(ATR)、DSC、TGA及水分吸 128407.doc -154- 200911757 附及解吸附分析來表徵藉由實例4 1之方法製備的固體。藉由X射線粉末繞射分析測定所製備之固體為結晶（圖 71)。將結晶物質指定為化合物III之形式A。以±0.2。20表示主要X射線繞射線且將其相對強度概括於表44中。表44.化合物III形式A之XRPD峰值（CuKa) 峰號 2Θ (度） d間距 (A) 強度 (計數） 1/1〇 1 4.36 20.25 310 10 2 4.73 18.65 2737 84 3 9.08 9.73 286 9 4 9.43 9.37 3245 100 5 14.14 6.26 381 12 6 17.65 5.02 118 4 7 18.89 4.69 133 4 8 23.65 3.76 181 6 圖72顯示化合物III形式A之1H NMR光譜。光譜與化合物III之已知化學結構一致。圖73顯示化合物III形式Α之FTIR(ATR)光譜。光譜與化合物III之已知結構一致。以波長倒數（以cm·1計之波數）表示之主要FTIR帶定位於約3459、3371、3303、3106、 3053、2976、2938、2875、1642、1627、1576、1529、 1507、1494、1456、1404、1358、1304、1266、1216、 1138、1118、1055、1020、999、959、940 ' 907、892 ' 864、837、761、743、728、721 及 670 cm·^ 捨入最近整數之值）。 DSC迹線（圖74)顯示在258°C之單一吸熱事件。 TGA熱分析圖（圖75)顯示直至230°C才有重量損失。圖76顯示化合物III形式A之水分吸附及解吸附迹線。迹 128407.doc -155 - 200911757 線顯示物質為非吸濕性且未觀察到潛在水合物形式。水分吸附後之殘餘固體未顯示形式變化。實例43·化合物III游離鹼在所選溶劑中之溶解度進行化合物III游離鹼之初始溶解度研究以鑑別用於鹽形成之合適單一溶劑。將如實例3中製備之化合物m游離驗用作起始物質。基於ICH方針（II類及III類）；官能性、極性及沸點之多樣性；以及製造適應性來選擇用於溶解度研究之溶劑。所選測試溶劑為：乙腈（MeCN)、丙酮、烷、乙醇（EtOH)、異丙醇（IPA)、乙酸乙酯（Et〇Ac)、乙酸異丙酯（IPAc)、甲基第三丁基醚（MTBE)、四氫吱味 (THF)、甲基乙基酮（MEK)、庚烷、環己烷、甲苯、二氣甲烷（DCM)、乙酸（Ac0H)、二甲基甲醯胺（DMF)及甲醇 (MeOH)。向測試小瓶中各自裝載1·〇_2.0 mg之間的游離鹼且以 0.25 mL之小份向各小瓶中添加所關注之溶劑直至至多7 〇 mL。在室溫下將小瓶震盪十分鐘且目視檢查，且接著加熱至55°C以評估在高溫下之溶解度，平衡時間為十分鐘。溶解度師檢之結果呈現於表4 5中。溶解度研究之结果表明化合物III形式A在大多數所選溶劑中具有低溶解度。觀察到僅少數溶劑在室溫下或在5rc將〜1 mg API溶解於小於1 mL溶劑中’但二甲基甲醯胺、乙酸及甲醇除外。 128407.doc -156- 200911757 表45.化合物III之溶解度Example 42. Form A which characterizes the free base of Compound III was prepared by XRPD, NMR, FTIR (ATR), DSC, TGA, and moisture absorption 128407.doc-154-200911757 with desorption analysis to characterize by the method of Example 41 s solid type. The solid prepared was determined to be crystalline by X-ray powder diffraction analysis (Fig. 71). The crystalline material is designated as Form A of Compound III. The main X-rays are radiant at ±0.20 and their relative intensities are summarized in Table 44. Table 44. XRPD peaks (CuKa) for Compound III Form A Peak number 2 Θ (degrees) d Spacing (A) Strength (count) 1/1 〇 1 4.36 20.25 310 10 2 4.73 18.65 2737 84 3 9.08 9.73 286 9 4 9.43 9.37 3245 100 5 14.14 6.26 381 12 6 17.65 5.02 118 4 7 18.89 4.69 133 4 8 23.65 3.76 181 6 Figure 72 shows the 1H NMR spectrum of Compound III Form A. The spectrum is consistent with the known chemical structure of Compound III. Figure 73 shows the FTIR (ATR) spectrum of the compound III form ruthenium. The spectrum is consistent with the known structure of Compound III. The main FTIR bands, represented by the reciprocal of the wavelength (wavenumber in cm·1), are located at approximately 3459, 3371, 3303, 3106, 3053, 2976, 2938, 2875, 1642, 1627, 1576, 1529, 1507, 1494, 1456. , 1404, 1358, 1304, 1266, 1216, 1138, 1118, 1055, 1020, 999, 959, 940 '907, 892 '864, 837, 761, 743, 728, 721 and 670 cm·^ round the nearest integer value). The DSC trace (Figure 74) shows a single endothermic event at 258 °C. The TGA thermogram (Figure 75) shows a weight loss up to 230 °C. Figure 76 shows the moisture adsorption and desorption traces of Compound III Form A. Trace 128407.doc -155 - 200911757 The line shows that the material is non-hygroscopic and no latent hydrate forms are observed. The residual solids after moisture adsorption showed no change in form. Example 43. Solubility of Compound III Free Base in Selected Solvents An initial solubility study of Compound III free base was conducted to identify a suitable single solvent for salt formation. The compound m prepared as in Example 3 was freely used as a starting material. Solvents for solubility studies are selected based on ICH guidelines (Class II and III); diversity of functionality, polarity and boiling point; and manufacturing flexibility. Selected test solvents are: acetonitrile (MeCN), acetone, alkane, ethanol (EtOH), isopropanol (IPA), ethyl acetate (Et〇Ac), isopropyl acetate (IPAc), methyl tert-butyl Ether (MTBE), tetrahydroanthracene (THF), methyl ethyl ketone (MEK), heptane, cyclohexane, toluene, dihydromethane (DCM), acetic acid (Ac0H), dimethylformamide ( DMF) and methanol (MeOH). The test vials were each loaded with 1·〇_2.0 mg of free base and the solvent of interest was added to each vial in 0.25 mL aliquots up to 7 〇 mL. The vial was shaken for ten minutes at room temperature and visually inspected, and then heated to 55 ° C to evaluate the solubility at elevated temperatures for an equilibration time of ten minutes. The results of the solubility test are presented in Table 45. The results of the solubility study indicate that Compound III Form A has low solubility in most selected solvents. It was observed that only a few solvents dissolved ~1 mg of API in less than 1 mL of solvent at room temperature or at 5 rc, except for dimethylformamide, acetic acid and methanol. 128407.doc -156- 200911757 Table 45. Solubility of Compound III

溶劑物質量 (mg) 溶劑量 (mL) 濃度 (mg/mL) 溫度 (°C) 可溶 ICH類別 MeCN 1.3 3.50 0.4 55 是 II 二噁烷 1.1 1.50 0.7 55 是 II 丙明 1.5 1.25 1.2 室溫是 III MTBE 1.2 7.00 — 55 否 III EtOH 1.5 1.25 1.2 55 是 III EtOAc 2.1 7.00 — 55 否 III IPAC 1.8 7.00 55 否 III IPA 1.3 4.50 — 55 否 III THF 1.4 2.00 0.7 室溫是 II MEK 2.9 3.50 0.8 室溫是 III DMF 1.2 0.25 4.8 室溫是 II AcOH 1.4 0.25 5.6 55 是 III MeOH 1.4 0.75 1.9 室溫是 II c-己院 1.4 7.00 --- 55 否 II 庚烷 1.1 6.00 — 55 否 III DCM 2.0 7.00 — 55 否 II 甲苯 1.8 6.00 55 否 II 實例44.化合物III之龍膽酸鹽固體的15 mg規模製備將化合物III游離鹼用作起始物質。在6.5 mL二噁烷中將 13.3 mg化合物III加熱至80°C直至溶解。接著添加305 pL 龍膽酸（1.05當量，0.126 Μ在二噁烷中）。在80°C攪拌反應十分鐘，隨後以20°C /小時冷卻至室溫，且接著在室溫下攪拌隔夜。藉由在溫和N2流下蒸發溶劑來收集殘餘物固體，接著在真空下在45°C乾燥隔夜。接著由XRPD、4 NMR及MS來分析所得固體。XRPD分析證實殘餘固體為結晶且展現儘管類似於起始物質之 XRPD圖樣但含有若干表明潛在鹽形成之獨特峰的XRPD圖樣。1H NMR資料表明物質含有一莫耳當量平衡離子，且未表明存在雜質。MS分析顯示存在雜質；然而，MS實驗 128407.doc •157- 200911757 條件在較小程度上可為降解之原因。實例4S.化合物ΙΠ之龍膽酸鹽的⑽—規模製備將80.6 mg化合物ΠΙ游離鹼加熱至8〇。〇以於3i mL二噁烷中溶解，且向其中添加龍膽酸〇〇5當量，〇41河在二噁烷中）。在80 C下攪拌反應十分鐘，以2〇〇c /小時冷卻至環境溫度，且接著在室溫下繼續攪拌反應隔夜。藉由在溫和N2 流下蒸發溶劑來收集固體，且接著在真空下在45艺乾燥隔夜。XRPD分析表明在此製備中回收之物質與先前製備一致。實例46.表徵化合物m之龍膽酸鹽由XRPD、4 NMR及MS分析化合物ΠΙ之龍膽酸鹽固體 (實例44及45)。圖77顯示在80 mg規模結晶實驗（實例45)中所得之化合物ΠΙ之龍膽酸鹽固體的xrpd圖樣（CuKa)。此光譜與在15 mg規模結晶實驗（實例44)中所得之龍膽酸鹽固體之所得光譜一致。以±0.2。20表示主要X射線繞射線且將其相對強度概括於表46中。表46.化合物ΠΙ之龍膽酸鹽固體的XRPD峰值（CuKa) 峰號 2Θ(°) d間距強度 m〇 1 4.52 19.53 53 5 2 4.75 18.58 230 21 3 8.84 10.00 42 4 4 9.12 9.69 147 13 5 9.45 9.35 1105 100 6 10.90 8.11 148 13 7 14.17 6.24 157 14 8 15.80 5.60 111 10 9 16.96 5.22 55 5 10 17.64 5.02 746 68 128407.doc • 158- 200911757 峰號 2Θ(。） d間距強度 I/I〇 11 18.36 4.83 85 g 12 13 14 18.96 4.68 295 27 iy.2〇 4.62 192 17 iy.4〇 4.57 133 12 15 20.20 4.39 330 3〇 16 17 20.48 2Γ08 4.33 42Ϊ 299 501 27 45 18 19 20 21.48 4.13 93 8 22.20 4.00 46 4 3.94 143 13 21 23.18 3.83 191 17 22 23.75 3J4 57 5 23 24 24.73 3.60 621 56 25.23 3.53 510 46 25 25.76 3.46 176 「16 26 26.22 3.40 251 23 27 26.84 3.32 106 10 28 27.77 3.21 41 4 29 30 28.35 • ·— 3.15 172 16 30.90 2.89 206 19 31 31.86 2.81 44 4 5 32 33.00 2.71 59 33 34 33.24 2.69 50 5 33.60 2.67 43 4 35 37.65 2.39 73 7 36 38.90 2.31 34 3 37 40.28 2.24 42 4 38 40.48 2.23 33 3 39 43.09 2.10 33 3 使龍膽酸鹽固體之XRPD圖樣（CuKa)與化合物ΠΙ形式A 在堆疊圖（圖78)中相比，該堆疊圖比較（a)化合物ΙΠ形式A 之XRPD圖樣（CuKa)(圖71)(頂部）與（b)龍膽酸鹽固體之 XRPD圖樣（CuKcx)(圖77)(底部）；鑑別龍膽酸鹽所獨有之一組峰。此獨特組之XRPD峰位包含在約10.9、20.48、28 35 及30.90±0.2。20之峰，其可用以鑑別化合物m龍膽酸鹽。在圖79中顯示化合物in之龍膽酸鹽固體的nmr光譜。光譜與化合物III之已知結構一致，且另外顯示化學位移及所觀察共振之差異，其係歸因於質子化物質之存在。 128407.doc •159· 200911757 在圖80中顯示化合物in之龍膽酸鹽固體的FTIR(ATR)光譜。光譜與化合物III之已知化學結構一致，但與游離鹼之光谱相比顯示差異（圖75)。以波長倒數（以cm-1計之波數）表示之主要FTIR帶定位於約3296、2975、1626、1575、 1529、1485、1442、1375、1302、1266、1216、1135、 1055、1020、999、940、906、888、866、840、798、 743、727及670 cm·1 (捨入最近整數之值）。將此光譜與化合物III形式A之光譜相比，且發現在1485、1442及798之峰為化合物III龍膽酸鹽所獨有。圖81顯示龍膽酸鹽固體之DSC迹線。DSC顯示在約133°C 之吸熱事件，其為化合物III龍膽酸鹽所獨有。圖82顯示龍膽酸鹽固體之tGA熱分析圖。tga顯示在 120 C與190 C之間3.2%之重量損失，其可能歸因於殘餘溶劑、水抽失或分解。圖83顯示在25下0% RH至90% RH進行之水分吸附分析。固體在60% RH吸附2.4。/。水。在解吸附後未觀察到顯著滯後現象，表明在實驗期間該物質並未形成穩定水合物。水分吸附分析之後對殘餘固體進行XRPD分析且表明樣品並未轉化成替代結晶形。實例47:高溫下化合物ΙΠ在THF中之穩定性添加I4·4 mg化合物111至10.5 mL THF中且在攪拌同時將混合物加熱至64。(：。固體已溶解之後，再繼續加熱及攪拌 W分鐘，隨後以2〇t/小時冷卻至室溫。接著在室溫下將溶液攪拌隔夜。藉由在溫和N2流下蒸發溶劑來收集沈澱。 128407.doc 200911757 接著在真空下在45C將固體乾燥隔夜。藉由xrpd來分析固體且複歸與化合物ΙΠ形式A—致之結晶XrPd圖樣。實例48 :高溫下化合物hi在丙酮中之穩定性添加15.5 mg化合物ΙΠ至10.5 mL丙酮中且在授拌同時將混合物加熱至56t。固體已溶解之後，再繼續加熱及攪拌 1〇分鐘，隨後以20。〇/小時冷卻至室溫。接著在室溫下將溶液攪拌隔夜。藉由在溫和％流下蒸發溶劑來收集沈澱。接著在真空下在45。(：將固體乾燥隔夜。藉由XRpD來分析固體且複歸與化合物ΙΠ之形式A—致的結晶xrpD圖樣。實例49 :化合物ΙΠ之結晶固體的水溶性研究Solvent mass (mg) Solvent amount (mL) Concentration (mg/mL) Temperature (°C) Soluble ICH class MeCN 1.3 3.50 0.4 55 is II Dioxane 1.1 1.50 0.7 55 is II propylamine 1.5 1.25 1.2 Room temperature is III MTBE 1.2 7.00 — 55 No III EtOH 1.5 1.25 1.2 55 Yes III EtOAc 2.1 7.00 — 55 No III IPAC 1.8 7.00 55 No III IPA 1.3 4.50 — 55 No III THF 1.4 2.00 0.7 Room temperature is II MEK 2.9 3.50 0.8 Room temperature is III DMF 1.2 0.25 4.8 Room temperature is II AcOH 1.4 0.25 5.6 55 is III MeOH 1.4 0.75 1.9 Room temperature is II c-house 1.4 7.00 --- 55 No II heptane 1.1 6.00 — 55 No III DCM 2.0 7.00 — 55 No II Toluene 1.8 6.00 55 No II Example 44. 15 mg scale preparation of gentisate solids of compound III Compound III free base was used as starting material. 13.3 mg of Compound III was heated to 80 ° C in 6.5 mL of dioxane until dissolved. Then 305 pL of gentisic acid (1.05 equivalents, 0.126 hydrazine in dioxane) was added. The reaction was stirred at 80 ° C for ten minutes, then cooled to room temperature at 20 ° C / hour, and then stirred at room temperature overnight. The residue solids were collected by evaporating the solvent under a gentle N2 stream and then dried overnight at 45 ° C under vacuum. The resulting solid was then analyzed by XRPD, 4 NMR and MS. XRPD analysis confirmed that the residual solid was crystalline and exhibited an XRPD pattern containing several distinct peaks indicating potential salt formation despite analogy to the XRPD pattern of the starting material. 1H NMR data indicated that the material contained a molar equivalent of equilibrium ions and no impurity was indicated. MS analysis showed the presence of impurities; however, MS experiments 128407.doc • 157-200911757 conditions to a lesser extent may be the cause of degradation. Example 4S. (10)-scale preparation of the compound gentisanic acid salt 80.6 mg of the compound hydrazine free base was heated to 8 Torr. The hydrazine was dissolved in 3 μ mL of dioxane, and 5 equivalents of gentamic acid 〇〇 was added thereto, and 〇41 river was in dioxane). The reaction was stirred at 80 C for ten minutes, cooled to ambient temperature at 2 ° C / h, and then the reaction was stirred at room temperature overnight. The solids were collected by evaporating the solvent under a gentle stream of N2 and then dried overnight under vacuum at 45. XRPD analysis indicated that the material recovered in this preparation was consistent with previous preparations. Example 46. Characterization of gentisate of compound m The compound guanidine gentisate solid was analyzed by XRPD, 4 NMR and MS (Examples 44 and 45). Figure 77 shows the xrpd pattern (CuKa) of the gentisate solid of the compound obtained in the 80 mg scale crystallization experiment (Example 45). This spectrum is consistent with the spectrum obtained from the gentisate solids obtained in the 15 mg scale crystallization experiment (Example 44). The main X-rays are rayed at ±0.20 and their relative intensities are summarized in Table 46. Table 46. XRPD peak (CuKa) of the compound gentamic acid solid peak number 2 Θ (°) d spacing strength m 〇 1 4.52 19.53 53 5 2 4.75 18.58 230 21 3 8.84 10.00 42 4 4 9.12 9.69 147 13 5 9.45 9.35 1105 100 6 10.90 8.11 148 13 7 14.17 6.24 157 14 8 15.80 5.60 111 10 9 16.96 5.22 55 5 10 17.64 5.02 746 68 128407.doc • 158- 200911757 Peak number 2Θ (.) d-pitch strength I/I〇11 18.36 4.83 85 g 12 13 14 18.96 4.68 295 27 iy.2〇4.62 192 17 iy.4〇4.57 133 12 15 20.20 4.39 330 3〇16 17 20.48 2Γ08 4.33 42Ϊ 299 501 27 45 18 19 20 21.48 4.13 93 8 22.20 4.00 46 4 3.94 143 13 21 23.18 3.83 191 17 22 23.75 3J4 57 5 23 24 24.73 3.60 621 56 25.23 3.53 510 46 25 25.76 3.46 176 "16 26 26.22 3.40 251 23 27 26.84 3.32 106 10 28 27.77 3.21 41 4 29 30 28.35 • — 3.15 172 16 30.90 2.89 206 19 31 31.86 2.81 44 4 5 32 33.00 2.71 59 33 34 33.24 2.69 50 5 33.60 2.67 43 4 35 37.65 2.39 73 7 36 38.90 2.31 34 3 37 40.28 2.24 42 4 38 40.48 2.23 33 3 39 43.09 2.10 33 3 make The XRPD pattern (CuKa) of the cholate solids is compared to the compound ΠΙ form A in the stacked diagram (Fig. 78), which compares (a) the XRPD pattern (CuKa) of the compound ΙΠ form A (Fig. 71) (top) And (b) XRPD pattern of gentisate solid (CuKcx) (Fig. 77) (bottom); identify one of the unique peaks of gentisate. The XRPD peak position of this unique set contains peaks at about 10.9, 20.48, 28 35 and 30.90 ± 0.220, which can be used to identify the compound m gentisate. The nmr spectrum of the gentisate solid of the compound in is shown in FIG. The spectrum is consistent with the known structure of Compound III and additionally shows the difference in chemical shift and observed resonance due to the presence of protonated species. 128407.doc •159· 200911757 The FTIR (ATR) spectrum of the compound in gentisate solid is shown in FIG. The spectrum is consistent with the known chemical structure of Compound III, but shows a difference compared to the spectrum of the free base (Figure 75). The main FTIR band, expressed in terms of wavelength reciprocal (wavenumber in cm-1), is located at approximately 3296, 2975, 1626, 1575, 1529, 1485, 1442, 1375, 1302, 1266, 1216, 1135, 1055, 1020, 999. , 940, 906, 888, 866, 840, 798, 743, 727, and 670 cm·1 (rounded to the nearest integer value). This spectrum was compared to the spectrum of Compound III Form A, and the peaks at 1485, 1442, and 798 were found to be unique to Compound III gentisate. Figure 81 shows the DSC trace of gentisate solids. DSC showed an endothermic event at about 133 °C, which is unique to Compound III gentisate. Figure 82 shows a tGA thermogram of gentisate solids. Tga shows a 3.2% weight loss between 120 C and 190 C, which may be due to residual solvent, water loss or decomposition. Figure 83 shows the moisture adsorption analysis performed at 0% RH to 90% RH at 25. The solid adsorbed 2.4 at 60% RH. /. water. No significant hysteresis was observed after desorption indicating that the material did not form a stable hydrate during the experiment. XRPD analysis of the residual solids after moisture adsorption analysis indicated that the sample was not converted to an alternative crystalline form. Example 47: Stability of compound hydrazine in THF at elevated temperature I4·4 mg of compound 111 was added to 10.5 mL of THF and the mixture was heated to 64 while stirring. (: After the solid had dissolved, heating and stirring were continued for another w minutes, followed by cooling to room temperature at 2 ° t / hour. The solution was then stirred overnight at room temperature. The precipitate was collected by evaporating the solvent under a gentle stream of N 2 . 128407.doc 200911757 The solid was then dried overnight at 45 C under vacuum. The solid was analyzed by xrpd and the crystallographic XrPd pattern was reverted to the compound ΙΠ form A. Example 48: Stability of compound hi in acetone at elevated temperature 15.5 mg of the compound was poured into 10.5 mL of acetone and the mixture was heated to 56 t while being mixed. After the solid had dissolved, heating and stirring were continued for another 1 minute, followed by cooling to room temperature at 20 ° C / h. The solution was stirred overnight. The precipitate was collected by evaporating the solvent under a gentle % flow. Then under vacuum at 45. (: The solid was dried overnight. The solid was analyzed by XRpD and reverted to form A with the compound oxime. Crystalline xrpD pattern. Example 49: Water solubility study of crystalline solids of the compound

以30 mg規模在DI水中進行藉由實例41之方法製備之化合物III形式A及實例41中製備之龍膽酸鹽固體的溶解度量測。向配備有磁力攪拌棒之7 mL小瓶中裝載試樣，在環境溫度下在DI水中調漿16_18小時。接著離心樣品以自上澄洛液分離固體且移除上澄液之丨mL部分且使其穿過注射過濾器。基於針對游離驗產生之校正資料，得到關於各樣品 /辰度之近似值，且因此以移動相（丨·丨比率之MeCN/水）稀釋樣品。使用HPLC分析來測定各樣品之溶解度。顯示形式A 具有< 1 mg/mL之水溶性，且顯示龍膽酸鹽固體具有〇7 mg/mL之水溶性。實例50 :環境溫度漿料及熱應力研究將如實例4 1中所述製備之化合物m游離鹼用作起始物質。為進行漿料研究，將約10 mg化合物m游離鹼置於裝配 128407.doc -161 · 200911757 有攪拌棒之1打蘭琥珀色小瓶中。添加二分之一毫升溶劑 (正庚烷、曱苯）至樣品小瓶中且將漿料在室溫下攪拌兩天。藉由過濾移除溶劑且在室溫下在真空烘箱中（30吋Hg) 進行進一步乾燥歷時一至兩小時。接著由XRPD、MS及 HPLC分析固體。為進行熱應力研究，將約10 mg化合物111(游離鹼）置於1 打蘭琥珀色小瓶中。將樣品置於烘箱中在60°C下歷時兩天。接著由XRPD、MS及HPLC分析固體。表47詳述漿料及熱應力實驗之結果。表47.化合物III游離鹼之漿料及熱應力資料的概括條件起始物質天數 XRPD* MS HPLC (AUC, %) 曱苯(環境）化合物III 2 一致一致 98 正庚烷(環境）一致一致 97 60°C 一致一致 >99 *與起始物質相比生物學實例實例51 :檢定提供可用以評估本發明之化合物I、II及III及其鹽及多晶型物之生物學活性的檢定。注意亦可使用熟習此項技術者已知之其他檢定。 1.活體外酵素活性可活體外、活體内或在細胞株中檢定化合物作為HDAC 抑制劑之活性。另外，可篩檢根據本發明之化合物對一或多種HDAC之活性。下文提供對HDAC1、HDAC2、 128407.doc -162- 200911757 HDAC5、HDAC6 及 HDAC8 之活，14 的檢定。如下可得經純化HDAC1、HDAC2、HDAC6及HDAC8。對於HDAC 1而言，編碼人類酵素之全長序列之殘基1-482的DNA可藉由PCR而擴增且經選殖於The dissolution metric of the gentisate solid prepared in the form of Compound III Form A and Example 41 prepared by the method of Example 41 was carried out in DI water at a scale of 30 mg. The sample was loaded into a 7 mL vial equipped with a magnetic stir bar and slurried in DI water at ambient temperature for 16-18 hours. The sample was then centrifuged to separate the solids from the supernatant and the 丨mL fraction of the supernatant was removed and passed through an injection filter. Based on the calibration data generated for the free test, an approximation for each sample/length is obtained, and thus the sample is diluted with the mobile phase (MeCN/water of the 丨·丨 ratio). The solubility of each sample was determined using HPLC analysis. Display Form A has a water solubility of < 1 mg/mL and shows that the gentisate solid has a water solubility of 〇7 mg/mL. Example 50: Ambient temperature slurry and thermal stress study The compound m free base prepared as described in Example 41 was used as the starting material. For the slurry study, approximately 10 mg of the compound m free base was placed in a 1 blue amber vial with a stir bar of 128407.doc -161 · 200911757. One-half ml of solvent (n-heptane, toluene) was added to the sample vial and the slurry was stirred at room temperature for two days. The solvent was removed by filtration and further dried in a vacuum oven (30 吋 Hg) for one to two hours at room temperature. The solid was then analyzed by XRPD, MS and HPLC. For thermal stress studies, approximately 10 mg of Compound 111 (free base) was placed in a 1 blue amber vial. The sample was placed in an oven at 60 ° C for two days. The solid was then analyzed by XRPD, MS and HPLC. Table 47 details the results of the slurry and thermal stress experiments. Table 47. Summary of Slurry and Thermal Stress Data for Compound III Free Base Conditions Starting Material Days XRPD* MS HPLC (AUC, %) Toluene (Environment) Compound III 2 Consistent 98 n-Heptane (Environment) Consistent 97 60 °C Consistency >99*Comparative to the starting material Biological Example 51: The assay provides an assay that can be used to assess the biological activity of the compounds I, II and III of the invention and their salts and polymorphs. Note that other tests known to those skilled in the art may also be used. 1. In vitro enzyme activity The activity of a compound as an HDAC inhibitor can be assayed in vitro, in vivo or in a cell line. Alternatively, the activity of a compound according to the invention against one or more HDACs can be screened. The verification of HDAC1, HDAC2, 128407.doc -162- 200911757 HDAC5, HDAC6 and HDAC8, 14 is provided below. Purified HDAC1, HDAC2, HDAC6 and HDAC8 were obtained as follows. For HDAC 1, DNA encoding residues 1-482 of the full length sequence of human enzymes can be amplified by PCR and colonized.

pFastbac(Invitrogen)之BamHI/Xbal位點中，該pFastbac在N 端及C端處均併有Flag標籤。SEQ ID NO: 1(圖84)對應於具有N端以及N端Flag標籤及C端Flag標籤的HDAC1之殘基1-482外加作為選殖人工產物在N端***之額外Met殘基。 SEQ ID NO: 2(圖 85)為用以編碼 SEQ ID NO: 1 之 DNA 序列。對於HDAC2而言，編碼人類酵素之全長序列之殘基1-488的DNA可藉由PCR而擴增且經選殖於 pFastbac(Invitrogen)之 BamHI/Smal位點中，該pFastbac 在 C 端處併有6-組胺酸標籤。SEQ ID NO: 3(圖86)對應於具有C 端6-組胺酸標籤之殘基1-488外加四個作為選殖人工產物之額外殘基（在N端***之Met-Gly-Ser及在6-組胺酸標藏之前 ***的Gly殘基）。SEQ ID NO: 4(圖87)為用以編碼SEQ ID NO: 3之DNA序列。In the BamHI/Xbal site of pFastbac (Invitrogen), the pFastbac has a Flag tag at both the N-terminus and the C-terminus. SEQ ID NO: 1 (Fig. 84) corresponds to residues 1-482 of HDAC1 having an N-terminal and N-terminal Flag tag and a C-terminal Flag tag plus an additional Met residue inserted as a colonization artifact at the N-terminus. SEQ ID NO: 2 (Fig. 85) is the DNA sequence used to encode SEQ ID NO: 1. For HDAC2, DNA encoding residues 1-488 of the full length sequence of human enzyme can be amplified by PCR and cloned in the BamHI/Smal site of pFastbac (Invitrogen) at the C-terminus and There is a 6-histidine tag. SEQ ID NO: 3 (Figure 86) corresponds to residues 1-488 with a C-terminal 6-histidine tag plus four additional residues as a selection artifact (Met-Gly-Ser inserted at the N-terminus and The Gly residue inserted before the 6-histamine label). SEQ ID NO: 4 (Fig. 87) is the DNA sequence used to encode SEQ ID NO: 3.

對於HDAC6而言，編碼人類酵素之殘基73-845的DNA可藉由PCR而擴增且經選殖於pFastbac(Invitrogen)之Smal位點中，該pFastbac在C端處併有6-組胺酸標籤。SEQ ID NO: 5(圖88)對應於具有C端6-組胺酸標籤之殘基73-845外加三個作為選殖人工產物之額外殘基（在N端***之Met-Pro殘基對及在C端6x-組胺酸標籤前***之Gly殘基）。SEQ 128407.doc •163- 200911757 ID NO: 6(圖89)為用以編碼SEQ ID NO: 5之DNA序列。對於HDAC8而言，編碼對應於人類酵素之全部序列之殘基1-377的DNA可藉由PCR而擴增且經選殖於 pFastbac(Invitrogen)之 BamHI/Smal位點中，該pFastbac在 N端處併有6-組胺酸標籤。SEQ ID NO: 7(圖90)對應於具有 N端6-組胺酸標籤之殘基1-377外加兩個作為選殖人工產物之額外殘基（在6-組胺酸標籤之前及之後***的Met及 Pro)。SEQ ID NO: 8(圖 91)為用以編碼 SEQ ID NO: 7 之 DNA序列。可藉由使用Bac-to-Bac系統（In vitro gen)之轉位來產生併有HDAC構築體之重組桿狀病毒。可藉由感染草地黏蟲 (Spodoptera frugiperda)Sf9細胞來產生高效價病毒儲備物；可藉由在10L Wave生物反應器（Wave Biotech)中感染草地黏蟲Sf9或粉紋夜蛾（Trichoplusia ni)Hi5細胞 (Invitrogen)來進行重組蛋白質之表現。可藉由在ProBond樹脂（invitr〇gen)或針對HDAC1之抗 Flag M2親和凝膠（Sigma)上通過而自細胞提取物分離重組蛋白貝。部为純化之HDAC 1可接著藉由Mono Q管柱上之高壓液相層析進一步純化。除HDAC1及HDAC6外之HDAC 的部分純化提取物可接著藉由BioSep S3000凝膠過濾樹脂上之高壓液相層析經進一步純化。可在變性SDS-PAGE凝膠上測定HDAC蛋白質之純度。可接著將經純化HDAC濃縮至對於HDAC1而言0.6 mg/ml、對於HDAC2而言10 mg/ml、對於HDAC6而言〇.3 mg/mi且對於HDAC8而言3 128407.doc 200911757 mg/ml之最終濃度。蛋白質可在-78。。儲存於含有25 mM TRIS-HC1 pH 7.6 ' 150 mM NaCl ' 0.1 mM EDTA及 0.25 mM TCEP之緩衝液中或在-20°C在甘油（甘油最終濃度為 5 0%)存在下儲存。或者，HDAC6蛋白質可在-78°C儲存於含有 25 mM TRIS-HC1 pH 7.2、250 mM NaCl及 5%甘油之緩衝液中。如熟習此項技術者易於瞭解，應注意多種其他表現系統及宿主亦適用於HDAC表現。可使用白色或黑色384孔板型式在以下反應條件下測定化合物關於 HDAC1、HDAC2、HDAC5、HDAC6及 HDAC8 之抑制特性：25 mM Tris pH 8.0、100 mM NaCl、50 mM KC卜 0.1 mM EDTA、0.01% Brij35、0.1 mM TCEP、50 μΜ tBoc-Lys(Ac)-AMC、2% DMSO。可使用具有在 370 nm 之激發波長及在480 nm(對於白色板而言）或465 nm(對於黑色板而言）之發射的勞光板讀取器（Molecular Devices Gemini)藉由螢光強度來定量測定反應產物。檢定反應可如下起始：添加5 μΐ之150 μΜ tBoc-Lys(Ac)AMC至板之各孔中，接著添加5 μΐ含有6% DMSO 之抑制劑（對於各抑制劑而言，11個數據點之2倍連續稀釋）。可添加五微升HDAC1、HDAC2、HDAC5、HDAC6 或 HDAC8溶液以起始反應（最終酵素濃度為：對於HDAC1而言2.5 nM、對於HDAC2而言1 nM、對於HDAC6而言2.5 nM及對於HDAC8而言10 nM)。可接著在室溫下培育反應混合物60分鐘，且藉由添加5 μΐ之10 mM啡琳 128407.doc -165- 200911757 (phenanthroline)及4 mg/mL胰蛋白酶來中止反應及誘發（嗓啉之最終濃度為2.5 mM，且胰蛋白酶之最終濃度為工 mg/mL)。可在室溫下培育3〇分鐘之後量測所得反應混合物之螢光強度。對於隔夜預培育ICm測定（HDAC2-0N)而言，檢定反應可如下起始：添加5 pL含有6% DMSO之抑制劑（對於一抑制劑而言，11個數據點之2倍連續稀釋）至5 HDAC2酵素溶液中。接著將板密封’且在室溫下培育12小時。藉由添加150 μΜ uM tBoc-Lys(Ac)AMC來起始反應，且接著在室溫下培育60 min，藉由添加5 μι之1 〇 mM啡啉及4 mg/mL胰蛋白酶來中止反應及誘發（啡啉之最終濃度為2 5 mM’且胰蛋白酶之最終濃度為1 mg/mL)。可在室溫下培育3 0分鐘之後量測所得反應混合物之螢光強度。可使用具有在370 nm之激發波長及在480 nm(對於白色板而言）或465 nm(對於黑色板而言）之發射的螢光板讀取器 (Molecular Devices Gemini)藉由螢光強度來定量測定反應產物。可使用具有在370 nm之激發波長及在480 nm(對於白色板而言）或465 nm(對於黑色板而言）之發射的螢光板讀取器 (Molecular Devices Gemini)藉由螢光強度而經由螢光來定量測定HDAC活性。在基質添加之後測定螢光強度且在37 °C培育30分鐘之後再次測定。亦測定僅使用媒劑之空白樣品（無抑制）的螢光強度。HDAC活性與所觀察之螢光變化成線性比例（最終-初始）。 128407.doc -166- 200911757 將給定化合物濃度下之HDAC抑制百分比定義為： 100% X [l-(Fcompound/Fblank)] 其中FC()mp_d*在測試化合物之給定濃度下的所觀察螢光且Fblank為僅在媒劑存在下之所觀察螢光。藉由以下方程式之非線性最小平方曲隸合來計算測試化合物之“^❶值（產生5〇%抑制之化合物莫耳濃度的負對數）：抑制百分比=100%/(1 + (1〇11〇50/1〇1。8[1])) 知到抑制百分比相對於化合物濃度。藉由i 0之負pICM次方 (10 picso)來計算測試化合物之抑制濃度（IQ。）。 2·細胞生存力檢定根據供應商（美國典型菌種保藏中心（American TypeFor HDAC6, DNA encoding residues 73-845 of human enzymes can be amplified by PCR and housed in the Smal site of pFastbac (Invitrogen) with a 6-histamine at the C-terminus. Acid label. SEQ ID NO: 5 (Figure 88) corresponds to residues 73-845 with a C-terminal 6-histidine tag plus three additional residues as a selection artifact (Met-Pro residue pair inserted at the N-terminus) And a Gly residue inserted before the C-terminal 6x-histidine tag). SEQ 128407.doc • 163- 200911757 ID NO: 6 (Figure 89) is the DNA sequence used to encode SEQ ID NO: 5. For HDAC8, DNA encoding residues 1-377 corresponding to the entire sequence of human enzymes can be amplified by PCR and housed in the BamHI/Smal site of pFastbac (Invitrogen) at the N-terminus. There is a 6-histidine tag. SEQ ID NO: 7 (Figure 90) corresponds to residues 1-377 with an N-terminal 6-histidine tag plus two additional residues as a selection artifact (inserted before and after the 6-histidine tag) Met and Pro). SEQ ID NO: 8 (Fig. 91) is the DNA sequence used to encode SEQ ID NO: 7. Recombinant baculoviruses harboring HDAC constructs can be generated by translocation using the Bac-to-Bac system (In vitro). High-valent virulence stocks can be produced by infecting Spodoptera frugiperda Sf9 cells; Sf9 or Trichoplusia ni Hi5 can be infected in a 10L Wave bioreactor (Wave Biotech) Cells (Invitrogen) are used to perform the performance of recombinant proteins. Recombinant protein shells can be isolated from cell extracts by passage on ProBond resin (invitr〇gen) or anti-Fat M2 affinity gel (Sigma) against HDAC1. The purified HDAC 1 can then be further purified by high pressure liquid chromatography on a Mono Q column. A partially purified extract of HDAC other than HDAC1 and HDAC6 can then be further purified by high pressure liquid chromatography on a BioSep S3000 gel filtration resin. The purity of the HDAC protein can be determined on a denatured SDS-PAGE gel. Purified HDAC can then be concentrated to 0.6 mg/ml for HDAC1, 10 mg/ml for HDAC2, mg.3 mg/mi for HDAC6 and 3128407.doc 200911757 mg/ml for HDAC8 Final concentration. The protein is available at -78. . Store in buffer containing 25 mM TRIS-HC1 pH 7.6 '150 mM NaCl '0.1 mM EDTA and 0.25 mM TCEP or in the presence of glycerol (final glycerol concentration 50%) at -20 °C. Alternatively, the HDAC6 protein can be stored at -78 ° C in a buffer containing 25 mM TRIS-HC1 pH 7.2, 250 mM NaCl, and 5% glycerol. As is familiar to those skilled in the art, it should be noted that a variety of other performance systems and hosts are also suitable for HDAC performance. The inhibitory properties of the compounds for HDAC1, HDAC2, HDAC5, HDAC6 and HDAC8 can be determined using the white or black 384-well plate format under the following reaction conditions: 25 mM Tris pH 8.0, 100 mM NaCl, 50 mM KC, 0.1 mM EDTA, 0.01% Brij35, 0.1 mM TCEP, 50 μΜ tBoc-Lys(Ac)-AMC, 2% DMSO. Quantitative by fluorescence intensity using a gloss plate reader (Molecular Devices Gemini) with excitation at 370 nm and emission at 480 nm (for white plates) or 465 nm (for black plates) The reaction product was measured. The assay reaction can be initiated by adding 5 μΐ of 150 μΜ tBoc-Lys(Ac)AMC to each well of the plate followed by 5 μΐ of inhibitor containing 6% DMSO (11 data points for each inhibitor) 2 times serial dilution). Five microliters of HDAC1, HDAC2, HDAC5, HDAC6, or HDAC8 solution can be added to initiate the reaction (final enzyme concentration: 2.5 nM for HDAC1, 1 nM for HDAC2, 2.5 nM for HDAC6, and for HDAC8 for HDAC8 10 nM). The reaction mixture can then be incubated for 60 minutes at room temperature and quenched and induced by the addition of 5 mM 10 mM morphine 128407.doc-165-200911757 (phenanthroline) and 4 mg/mL trypsin (final of porphyrin) The concentration was 2.5 mM and the final concentration of trypsin was mg/mL). The fluorescence intensity of the resulting reaction mixture can be measured after incubation for 3 minutes at room temperature. For the overnight pre-incubation ICm assay (HDAC2-0N), the assay reaction can be initiated by adding 5 pL of inhibitor containing 6% DMSO (for one inhibitor, 2 replicates of 11 data points) to 5 HDAC2 enzyme solution. The plate was then sealed' and incubated for 12 hours at room temperature. The reaction was initiated by the addition of 150 μΜ uM tBoc-Lys(Ac)AMC and then incubated for 60 min at room temperature by adding 5 μM of 〇 morphine and 4 mg/mL trypsin to stop the reaction and Induced (final concentration of morpholino is 25 mM ' and the final concentration of trypsin is 1 mg/mL). The fluorescence intensity of the resulting reaction mixture can be measured after 30 minutes of incubation at room temperature. Quantitative by fluorescence intensity using a fluorescence plate reader (Molecular Devices Gemini) with excitation at 370 nm and emission at 480 nm (for white plates) or 465 nm (for black plates) The reaction product was measured. A fluorescent plate reader (Molecular Devices Gemini) having an excitation wavelength of 370 nm and an emission of 480 nm (for white plates) or 465 nm (for black plates) can be used by fluorescence intensity Fluorescence was used to quantitatively determine HDAC activity. Fluorescence intensity was measured after matrix addition and again after incubation at 37 °C for 30 minutes. The fluorescence intensity of the blank sample (without inhibition) using only the vehicle was also determined. The HDAC activity is linearly proportional to the observed fluorescence change (final-initial). 128407.doc -166- 200911757 The percentage of HDAC inhibition at a given compound concentration is defined as: 100% X [l-(Fcompound/Fblank)] where FC()mp_d* is observed at a given concentration of test compound Light and Fblank is the observed fluorescence only in the presence of the vehicle. The "^❶ value of the test compound (the negative logarithm of the molar concentration of the compound that produces 5〇% inhibition) is calculated by the nonlinear least squares curvature of the following equation: Percent inhibition = 100% / (1 + (1〇11) 〇50/1〇1.8[1])) The percent inhibition is known relative to the compound concentration. The inhibitory concentration (IQ) of the test compound is calculated by the negative pICM power of i 0 (10 picso). Survivability check according to supplier (American Type Culture Collection (American Type)

Culture Collection)(R〇ckville，MD))保持 A549、HL60 及 PC3腫瘤細胞株。以3,000-15,000個細胞/孔將細胞接種於 96孔微量組織培養板中且培養24小時，隨後添加化合物或 DMSO(二甲亞砜）媒劑。處理小時之後，經由用 Spectramax微板讀取器（Molecular Devices, San Diego，CA) 量測ODoo nm來測定代謝活性細胞所引起之四嗤鑌鹽 MTS(Promega, Madison, WI)之轉化。為產生濃度-反應曲線，以多種連續化合物稀釋物（最終DMSO濃度為0.5%) — 式兩份地處理細胞。藉由修正背景且對DMSO處理細胞標準化來測定每孔之活細胞百分比。使用XLfit4 Microsoft Excel曲線擬合軟體來計算細胞生存力之抑制的EC5〇值。 3·過度乙醯化組蛋白之西方墨點法（Western Blotting) 128407.doc -167- 200911757 將HL60細胞接種於1 mL 96深孔板中含有20%胎牛血清之伊氏DMEM(Iscove’ DMEM)中且與在DMSO(最終DMSO 濃度為0.5%)中經連續稀釋2.5倍之化合物一起培育。4 hr 後，以磷酸鹽緩衝鹽水將細胞洗滌三次。藉由將細胞溶解於 62_5 mM Tris-HCl(pH 7)、1% SDS、10%甘油中且在 95 °C加熱5分鐘接著使用板式配警報笛音波器（Misonix， Farmingdale, NY)進行超音波處理來製備全細胞溶胞物。藉由SDS-PAGE離析蛋白質且轉移至PVDF膜。以適當第一抗體探測膜，接著與二級IRDye 680或800 CW共軛抗體 (Li-Cor, Lincoln, NE) —起培育。使用識別乙酿化組蛋白 H3、乙醯化組蛋白H4之抗體（UBI，Lake Placid, NY)來分析組蛋白乙醯化。將PCNA用作蛋白質負載之對照物且用單株抗體（Santa Cruz Biotechnology, Santa Cruz, CA)對其债測。在Odyssey(Li-Cor)上掃描墨點且使用LiCor軟體來定量分別對應於PCNA、過度乙醯化組蛋白H3及H4之信號。使用XLfit4 Microsoft Excel曲線擬合軟體，藉由曲線擬合乙醯化蛋白質信號與PCNA蛋白質信號之比率來獲得組蛋白乙醯化EC5〇。 4. p21誘導檢定藉由將跨越nt-168至+51之小p21WAFl/Cipl啟動子片段* ***質體pGL3-basic(Promega, Madison, WI)中來改造啟動子-螢光素酶報導基因構築體P21-GL3。藉由使用 Effectene(Qiagen, Valencia, CA)以質體 pCIneo(Promega)共轉染 p21-GL3 接著在 G418(Invitrogen，Carlsbad, CA)存在下 128407.doc 168· 200911757 進行有限稀释及選擇來產生穩定轉染物HeLa/p21-GL3純系。將HeLa/p21-GL3細胞以25,000個/孔之密度接種於96 孔板中。在37°C在5% C02培育24小時之後，添加連續稀釋之化合物至培養基中’最終DMSO濃度為〇·5%。將細胞再培育40 hr且溶解於Glo溶解緩衝液（Promega)中。混合細胞溶胞物與相同體積之Bright Glow試劑（Promega)之後，在 Analyst光度計上量測螢光素酶活性。藉由與經DMSO處理之細胞相比來測定螢光素酶活性之相對增加。EC5Q表示一半最大誘導且如上加以計算。 5·活體内功效模型用藉由在0.5%羧曱基纖維素（CMC)中經口管飼之化合物或由10%乙醇、20%聚乙二醇-400、10%丙二醇及60%含有 0.5%羧甲基纖維素（CMC)之水組成之媒劑處理帶有自植入腫瘤細胞或片段生長之既定皮下腫瘤的無胸腺裸小鼠。使用數位測徑規來量測腫瘤體積。將抗腫瘤活性（％ T/c)表示為針對起始體積修正之後經治療腫瘤體積（T)對比對照物腫瘤體積（C) ; (T-T。）X 1 oo/(c-c〇)。 6·對hERG之作用實驗方法：細胞：將穩定表現hERG通道的Aviva之CHO 細胞株用於研究。將細胞於含有10% FBS、1%青黴素/鏈黴素及500 pg/mL G418之DMEM/F12中培養。在測試之前使用 Accumax(Innovative Cell Technologies)採集細胞。溶液：對於電生理學記錄，使用以下溶液。外部溶液 (mM) : 1.8 CaCl2 ; 1.0 MgCl2 ; 4 KC1 ; 137 NaCl ; 1〇葡萄 128407.doc -169- 200911757 糖；10 HEPES ;(以1 M NaOH調節至pH 7_4，容積滲透濃度-295 mOsm)。内部溶液含有（mM) : 130 KC1、1 MgCl2、5 EGTA、10 HEPES、5 ATP(以 KOH將 pH值調節為7.25 ;容積渗透濃度〜280 mOsm)。電生理學：使用ρχ 70 00A( Axon Instruments)以 A VIVA之 SealChip™技術來進行全細胞記錄。將細胞電壓箝制在保持電勢_8〇 mV且藉由首先達到-50 mV歷時300 msec之去極化步驟、達到+2〇 mv 歷時5 sec以活化通道之步驟來活化HERG電流，接著最終回至-5 0 mV歷時5 sec以移除鈍化且觀察去活化曳尾電流。將在-50 mV之第一步驟用作量測曳尾電流峰值振幅之基線。化合物處理及稀釋物：將所有化合物製備為在玻璃中之10 mM DMSO儲備物。藉由在室溫下劇烈渦流及超音波處理約1分鐘來混合儲備溶液。對於測試，使用外部溶液將化合物稀釋於玻璃小瓶中；錢用之前不超過3〇分鐘時製備稀釋物。在所有最終稀釋物中均存在等量之腦〇(().1%)。電生理學程序：達成全細胞組態之後，監測細胞90 W評估穩定性且以外部溶液絲Μ ”接著每 12 s及在整個程序期„上料壓方案應詩細胞。僅允許具有臨限值以上之印之°己錄參數（參見品質控制部分）且穩定之細胞進入藥物添加程序。將. 斤將含有0·1% DMS〇(媒劑）之外部》谷液應用於細胞以涂☆其給肥建立基線。使電流穩定3至5分鐘之後，應用測試化合物。在3個牛在3個步驟中添加化合物溶液且將細胞保持於測試溶液中直至化人板十& 化〇物之作用達到穩態或至多 128407.doc -170· 200911757 12 min。隨後添加陽性對照（1 μΜ西沙必利（Cisapride))。以外部溶液進行清洗直至電流恢復達到穩態。資料分析：使用 DataXpress、ClampHt(均由 Axon Instruments提供）及 Origin 7(Originiab Corporation)來分析資料。品質控制：報導中所包括之資料源自滿足所有以下標準之實驗：a-記錄參數：薄膜電阻Rm > 200 ΜΩ ;接取電阻 (Ra) < 15 ;曳尾電流振幅> 150 pA ;掃描週期< 1%/ 为名里’電流穩定性：在8個掃描之矣尾電流峰值振幅之間的差異及此等掃描之平均值，其不應超過0.2% ; b-藥理學參數：1 μΜ西沙必利阻斷> 95%。 7·調配物除非另有說明，否則根據以下配方之一來調配檢定中所用之化合物： a. 10%乙醇：20% PEG400:100/〇丙二醇：60%稀釋劑（在水中之 0.5% CMC)及 b. 在水中之0.5% CMC。實例52·檢定化合物I之形式A的活體外酵素活性根據實例51-1之檢定來評估化合物I之形式A對各種 HDAC酵素之活體外酵素活性且報導於表48中。表48:化合物I之形式A對HDAC之IC50 HDAC酵素 ic5〇 HDAC1 500-1000 nM HDAC2 500-1000 nM HDAC2-隔夜 < 100 nM HDAC5 > 1 μΜ HDAC6 1 > 1 μΜ HDAC8 > 1 μΜ 128407.doc • 171 - 200911757 實例53.化合物I之形式A對抑制細胞生存力之作用根據實例52-2之程序來評估化合物I之形式A對各種癌細胞類型之抑制作用。將結果概括於表49中。表49 :化合物I之形式A對細胞生存力之EC50 細胞株 ECs〇 DU145 > 1 μΜ HCT116 <500ηΜ HL60 500-1000 ηΜ MES-SA > 1 μΜ MES-SA/DX5 > 1 μΜ PC3 500-1000 ηΜ 實例54.化合物I之形式A之活體内功效（組蛋白乙醯化）根據實例53-3之程序來評估化合物I之形式A對組蛋白乙醯化之誘導作用。將結果概括於表50中。表50.化合物I之形式A對HDAC乙醯化之EC50 乙醢化蛋白質 ECs〇組蛋白Η3 8 1-10 μΜ 組蛋白Η4 8 1-10 μΜ 實例55.化合物I之形式A對p21誘導之作用根據實例51-4之程序來評估化合物I之形式A對p21誘導之作用。將結果概括於表5 1中。表51.化合物I之形式A對p21謗導之EC50 ρ21 ECso HELA/P21-LUC 40 1-10 μΜ 實例56.化合物I之形式A之活體内功效根據實例5 1 -5之檢定來評估小鼠模型中化合物I之形式A 對各種癌細胞之功效。將結果列表於表52中。 128407.doc -172- 200911757 表52.小鼠模型中化合物I之形式A對癌細胞之功效劑量 (毫克/公斤/天）細胞株給藥方案 T/C (%) 0.5 PC3 QD χ 15 33 1.5 PC3 QD x 15 27 2.5 PC3 QD χ 15 14 4 PC3 QD χ 15 1.5 2.5 22Rvl QD χ 14 52 4 22Rvl QD χ 14 47 0.5 DU 145 QD χ 15 41 4 DU 145 QD χ 15 30 4 LNCap-hr QD χ 14 6 1 PC3 5 ON/2 OFF χ 2 53 4 PC3 5 ON/2 OFF χ 2 8 6 PC3 5 ON/2 OFF χ 2 5 5 DU145 5 ON/2 OFF χ 3 6 10 DU 145 5 ON/2 OFF χ 3 17 15 DU 145 5 ON/2 OFF χ 3 6 25 HCT116 QD χ 15 47 50 HCT116 QD χ 15 - 10 HCT116 QD χ 15 56 5 A431 QD χ 15 34 10 A431 QD χ 15 23 6 A2780 QD χ 15 36 9 A2780 QD χ 15 22 14 A2780 QD χ 15 10 實例57.化合物I之形式A對hERG之作用根據實例5 1-6中之檢定來評估化合物I之形式A對hERG 之抑制作用。結果顯示在1 〇 μΜ抑制劑濃度下< 1 0%抑制。實例58.檢定化合物II之雙TFA鹽的活體外酵素活性根據實例5 1 -1之檢定來評估化合物II之雙TF Α鹽對各種 HDAC酵素之活體外酵素活性且報導於表53中。表53 :化合物II之雙TFA鹽對HDAC之IC50Culture Collection) (R〇ckville, MD)) maintained A549, HL60 and PC3 tumor cell lines. The cells were seeded at 3,000-15,000 cells/well in 96-well micro-tissue culture plates and cultured for 24 hours, followed by addition of a compound or DMSO (dimethyl sulfoxide) vehicle. After the treatment hours, the conversion of the tetrahydrous salt MTS (Promega, Madison, WI) caused by the metabolically active cells was determined by measuring ODoo nm with a Spectramax microplate reader (Molecular Devices, San Diego, CA). To generate a concentration-response curve, cells were treated in duplicate with multiple serial compound dilutions (final DMSO concentration of 0.5%). The percentage of viable cells per well was determined by correcting the background and normalizing the cells treated with DMSO. The XLfit4 Microsoft Excel curve-fitting software was used to calculate the EC5 〇 value for inhibition of cell viability. 3. Western Blotting of over-acetylated histones (Western Blotting) 128407.doc -167- 200911757 HL60 cells were seeded in 1 mL 96-deep well plates containing 20% fetal bovine serum in I. DMEM (Iscove' DMEM) And incubated with compounds that were serially diluted 2.5-fold in DMSO (final DMSO concentration 0.5%). After 4 hr, the cells were washed three times with phosphate buffered saline. The cells were sonicated by dissolving the cells in 62_5 mM Tris-HCl (pH 7), 1% SDS, 10% glycerol and heating at 95 °C for 5 minutes followed by a plate-type alarm beeper (Misonix, Farmingdale, NY). Treatment to prepare whole cell lysates. The protein was isolated by SDS-PAGE and transferred to a PVDF membrane. The membrane was probed with an appropriate first antibody and then incubated with secondary IRDye 680 or 800 CW conjugated antibody (Li-Cor, Lincoln, NE). Histone acetylation was analyzed using an antibody (UBI, Lake Placid, NY) that recognizes B-histone H3 and acetylated histone H4. PCNA was used as a control for protein loading and was tested with a monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Ink dots were scanned on Odyssey (Li-Cor) and LiCor software was used to quantify signals corresponding to PCNA, over-acetylated histones H3 and H4, respectively. Using the XLfit4 Microsoft Excel curve-fitting software, the group protein acetylated EC5〇 was obtained by curve fitting the ratio of the acetylated protein signal to the PCNA protein signal. 4. p21 induction assay to engineer the promoter-luciferase reporter gene by inserting a small p21WAF1/Cip1 promoter fragment* spanning nt-168 to +51 into the plastid pGL3-basic (Promega, Madison, WI) Body P21-GL3. Co-transfection of p21-GL3 with plastid pCIneo (Promega) using Effectene (Qiagen, Valencia, CA) followed by limiting dilution and selection in the presence of G418 (Invitrogen, Carlsbad, CA) 128407.doc 168· 200911757 to stabilize Transfectant HeLa/p21-GL3 pure line. HeLa/p21-GL3 cells were seeded at a density of 25,000 cells/well in 96-well plates. After incubation for 24 hours at 37 ° C in 5% CO 2 , serially diluted compounds were added to the medium to a final DMSO concentration of 〇·5%. The cells were incubated for an additional 40 hr and dissolved in Glo Dissolution Buffer (Promega). After mixing the cell lysate with the same volume of Bright Glow reagent (Promega), the luciferase activity was measured on an Analyst luminometer. The relative increase in luciferase activity was determined by comparison to DMSO treated cells. EC5Q represents one-half maximum induction and is calculated as above. 5. In vivo efficacy model with a compound that is orally gavaged in 0.5% carboxymethyl cellulose (CMC) or from 10% ethanol, 20% polyethylene glycol-400, 10% propylene glycol and 60% containing 0.5 A vehicle consisting of water of % carboxymethylcellulose (CMC) treats athymic nude mice bearing established subcutaneous tumors grown from tumor cells or fragments. The tumor volume was measured using a digital caliper. Antitumor activity (% T/c) is expressed as the treated tumor volume (T) after comparison with the control tumor volume (C); (T-T.) X 1 oo/(c-c〇). 6. Effect on hERG Experimental method: Cells: A CHO cell line of Aviva stably expressing hERG channels was used for the study. The cells were cultured in DMEM/F12 containing 10% FBS, 1% penicillin/streptomycin and 500 pg/mL G418. Cells were harvested using Accumax (Innovative Cell Technologies) prior to testing. Solution: For electrophysiological recording, the following solutions were used. External solution (mM): 1.8 CaCl2; 1.0 MgCl2; 4 KC1; 137 NaCl; 1〇 grape 128407.doc -169- 200911757 sugar; 10 HEPES; (adjusted to pH 7_4 with 1 M NaOH, volume osmotic concentration -295 mOsm) . The internal solution contained (mM): 130 KC1, 1 MgCl2, 5 EGTA, 10 HEPES, 5 ATP (pH adjusted to 7.25 with KOH; volume osmotic concentration ~ 280 mOsm). Electrophysiology: Whole cell recording was performed using A VIVA's SealChipTM technology using ρχ 70 00A (Axon Instruments). The cell voltage was clamped to maintain the potential _8 〇 mV and the HERG current was activated by the step of first reaching a depolarization step of -50 mV for 300 msec, reaching +2 〇mv for 5 sec to activate the channel, and finally returning to -50 mV for 5 sec to remove passivation and observe deactivation of the tail current. The first step at -50 mV is used as the baseline for measuring the peak amplitude of the tail current. Compound treatment and dilution: All compounds were prepared as 10 mM DMSO stock in glass. The stock solution was mixed by vigorous vortexing and ultrasonic treatment at room temperature for about 1 minute. For the test, the compound was diluted in a glass vial using an external solution; the dilution was prepared using no more than 3 minutes before. An equal amount of cerebral palsy (().1%) was present in all final dilutions. Electrophysiological procedure: After achieving a full-cell configuration, the cells were monitored for 90 W to assess stability and the external solution was sputum "and then every 12 s and throughout the program period." Only cells with a threshold above the threshold (see the Quality Control section) are allowed and the stable cells enter the drug addition procedure. The jin will contain 0.1% DMS 〇 (vehicle) outside the "solution" applied to the cells to coat ☆ its fertilizer to establish a baseline. After the current was stabilized for 3 to 5 minutes, the test compound was applied. In 3 cows, the compound solution was added in 3 steps and the cells were kept in the test solution until the effect of the human plate 10 & pupate reached steady state or at most 128407.doc -170· 200911757 12 min. A positive control (1 μC Cisapride) was then added. Wash with an external solution until the current returns to a steady state. Data Analysis: Data was analyzed using DataXpress, ClampHt (both supplied by Axon Instruments) and Origin 7 (Originiab Corporation). Quality Control: The information included in the report is derived from experiments that meet all of the following criteria: a-recording parameters: film resistance Rm > 200 Μ Ω; access resistance (Ra) <15; trailing current amplitude > 150 pA; Scan period < 1% / for name 'current stability: the difference between the peak amplitudes of the tail currents at 8 scans and the average of these scans, which should not exceed 0.2%; b- Pharmacological parameters: 1 μΜ cisapride blocked > 95%. 7. Formulations Unless otherwise stated, the compounds used in the assay are formulated according to one of the following formulations: a. 10% ethanol: 20% PEG 400: 100 / propylene glycol: 60% diluent (0.5% CMC in water) And b. 0.5% CMC in water. Example 52. Characterization of in vitro enzyme activity of Form A of Compound I The in vitro enzyme activity of Form A of Compound I against various HDAC enzymes was evaluated according to the assay of Example 51-1 and reported in Table 48. Table 48: Form I of Compound I vs. IC of HDAC HDAC Enzyme ic5 〇 HDAC1 500-1000 nM HDAC2 500-1000 nM HDAC2-overall <100 nM HDAC5 > 1 μΜ HDAC6 1 > 1 μΜ HDAC8 > 1 μΜ 128407 .doc • 171 - 200911757 Example 53. Effect of Form A of Compound I on Inhibition of Cell Viability The inhibitory effect of Form A of Compound I on various cancer cell types was evaluated according to the procedure of Example 52-2. The results are summarized in Table 49. Table 49: EC50 of Compound A Form A on Cell Viability ECs〇DU145 > 1 μΜ HCT116 <500ηΜ HL60 500-1000 ηΜ MES-SA > 1 μΜ MES-SA/DX5 > 1 μΜ PC3 500 -1000 η Μ Example 54. In vivo efficacy of Form A of Compound I (histone acetylation) The induction of histone acetylation by Form A of Compound I was evaluated according to the procedure of Example 53-3. The results are summarized in Table 50. Table 50. Form I of Compound I to EC50 of HDAC acetylated protein ECs 〇 histone Η 3 8 1-10 μΜ histone Η 4 8 1-10 μΜ Example 55. Effect of Form A of Compound I on p21 induction The effect of Form A of Compound I on p21 induction was evaluated according to the procedure of Example 51-4. The results are summarized in Table 51. Table 51. Form I of Compound I versus p21 EC EC50 ρ21 ECso HELA/P21-LUC 40 1-10 μΜ Example 56. In vivo efficacy of Form A of Compound I Evaluate mice according to the assay of Example 5 1 -5 The effect of Form A of Compound I on various cancer cells in the model. The results are listed in Table 52. 128407.doc -172- 200911757 Table 52. Efficacy dose of Form A of Compound I in cancer cells (mg/kg/day) Cell line dosage regimen T/C (%) 0.5 PC3 QD χ 15 33 1.5 PC3 QD x 15 27 2.5 PC3 QD χ 15 14 4 PC3 QD χ 15 1.5 2.5 22Rvl QD χ 14 52 4 22Rvl QD χ 14 47 0.5 DU 145 QD χ 15 41 4 DU 145 QD χ 15 30 4 LNCap-hr QD χ 14 6 1 PC3 5 ON/2 OFF χ 2 53 4 PC3 5 ON/2 OFF χ 2 8 6 PC3 5 ON/2 OFF χ 2 5 5 DU145 5 ON/2 OFF χ 3 6 10 DU 145 5 ON/2 OFF χ 3 17 15 DU 145 5 ON/2 OFF χ 3 6 25 HCT116 QD χ 15 47 50 HCT116 QD χ 15 - 10 HCT116 QD χ 15 56 5 A431 QD χ 15 34 10 A431 QD χ 15 23 6 A2780 QD χ 15 36 9 A2780 QD χ 15 22 14 A2780 QD χ 15 10 Example 57. Effect of Form A of Compound I on hERG The inhibition of hERG by Form A of Compound I was evaluated according to the assay in Examples 5 1-6. The results showed < 10% inhibition at 1 〇 μΜ inhibitor concentration. Example 58. In vitro enzyme activity assay for the double TFA salt of Compound II The in vitro enzyme activity of the double TF sulfonium salt of Compound II against various HDAC enzymes was evaluated according to the assay of Example 5 1 -1 and reported in Table 53. Table 53: IC50 of double TFA salt of compound II versus HDAC

HDAC酵素 IC5〇 HDAC1 500-1000 nM HDAC2 500-1000 nM HDAC2-隔夜 < 100 nM 128407.doc -173 - 200911757 實例59.化合物II之雙TFA鹽對細胞生存力之作用根據實例51-2之程序來評估化合物II之雙TFA鹽對各種癌細胞類型之抑制作用。將結果概括於表54中。表54 :化合物II之雙TFA鹽對細胞生存力之EC50 細胞株 ECso HCT116 500-1000 nM HL60 1-10 μΜ MES-SA 10-100 μΜ MES-SA/DX5 > 100 μΜ 實例60.化合物II之雙TFA鹽的活體内功效（組蛋白乙醯化）根據實例5 1 -3之程序來評估化合物II之雙TF A鹽對組蛋白乙醯化之誘導作用。將結果概括於表55中。表55 :化合物II之雙TFA鹽對誘導HDAC乙醯化之EC50 乙醯化蛋白質 ECso 組蛋白Η3 8 10-100 μΜ 組蛋白Η4 8 10-100 μΜ 實例61·化合物II之雙TFA鹽對p21誘導之作用根據實例5 1 -4之程序來評估化合物II之雙TFA鹽對p2 1誘導之作用。將結果概括於表56中。表56:化合物II之雙TFA鹽對p21誘導之EC50 p21 ECs〇 HELA/P21-LUC 40 1-10 μΜ 實例62.檢定化合物II之形式A的活體外酵素活性根據實例5 1 -1之檢定來評估化合物II之形式A(游離鹼）對各種HDAC酵素之活體外酵素活性且報導於表57中。 128407.doc -174- 200911757 表57.化合物II之形式A對HDAC之IC50 HDAC酵素 IC5〇 HDAC1 500-1000 nM HDAC2 500-1000 nM HDAC2-隔夜 < 100 nM HDAC5 >1 μΜ HDAC6 >1 μΜ HDAC8 >1 μΜ 實例63.化合物II之形式A對細胞生存力之作用根據實例5 1-2之程序來評估化合物II之形式A對各種癌細胞類型之抑制作用。將結果概括於表58中。表58 :化合物II之形式A對細胞生存力之EC50 細胞株 ECso HCT116 500-1000 nM HL60 500-1000 nM MES-SA > 1 μΜ MES-SA/DX5 > 1 μΜ 實例64.化合物II之形式A之活體内功效（組蛋白乙醯化）根據實例51-3之程序來評估化合物II之形式A對組蛋白乙醯化之誘導作用。將結果概括於表59中。表59 :化合物II之形式A對誘導HDAC乙醯化之EC50 乙醯化蛋白質 ECso 組蛋白Η3 8 1-10 μΜ 組蛋白Η4 8 1-10 μΜ 實例65.化合物II之形式A對p21誘導之作用根據實例51-4之程序來評估化合物II之形式A對p21誘導之作用。將結果概括於表60中。表60:化合物II之形式A對p21誘導之EC50 p21 ECso HELA/P21-LUC 40 1-10 μΜ 128407.doc -175- 200911757 實例66.化合物II之形式A之活體内功效根據實例5 1 -5之檢定來評估小鼠模型中化合物I之形式A 對各種癌細胞之功效。將結果列表於表6 1中。表61.小鼠模型中化合物II之形式A對癌細胞之功效鹽形式異種移植株劑量進度 T/C 游離驗 HCT116 65 mg/kg QD x 15 36% 游離鹼 A2780 65 mg/kg QD x 15 65% 游離驗 A2780 100 mg/kg QD x 15 38% 游離驗 HCT116 20 mg/kg BID x 15 54% 游離驗 HCT116 100 mg/kg QD x 14 40% 游離驗 PC3 50 mg/kg QD x 14 16% 游離驗 PC3 100 mg/kg QD x 14 7% 實例67.化合物II之形式A及雙TFA鹽對hERG之作用根據實例5 1-6中之檢定來評估化合物II之形式A及雙TFA 鹽對hERG之抑制作用。結果顯示在10 μΜ濃度之化合物II 之雙TFA鹽下<10%之抑制，及在10 μΜ濃度之化合物II之形式Α下為12%之抑制。實例68.檢定化合物III之形式A(游離鹼）的活體外酵素活性根據實例51-1之檢定來評估化合物III之形式A(游離鹼）對各種HDAC酵素之活體外酵素活性且報導於表62中。表62 :化合物III之形式A對HDAC之IC50HDAC enzyme IC5 〇 HDAC1 500-1000 nM HDAC2 500-1000 nM HDAC2-overnight < 100 nM 128407.doc -173 - 200911757 Example 59. Effect of Compound T's double TFA salt on cell viability According to the procedure of Example 51-2 The inhibitory effect of the double TFA salt of Compound II on various cancer cell types was evaluated. The results are summarized in Table 54. Table 54: EC50 cell line for cell viability of double TFA salt of Compound II ECso HCT116 500-1000 nM HL60 1-10 μΜ MES-SA 10-100 μΜ MES-SA/DX5 > 100 μΜ Example 60. Compound II In vivo efficacy of double TFA salt (histone acetylation) The induction of histone acetylation by the double TF A salt of Compound II was evaluated according to the procedure of Example 5 1 -3. The results are summarized in Table 55. Table 55: Double TFA salt of Compound II for induction of HDAC acetylation EC50 acetylated protein ECso histone Η3 8 10-100 μΜ histone Η4 8 10-100 μΜ Example 61·Compound II double TFA salt for p21 induction Effect The effect of the double TFA salt of Compound II on p2 1 induction was evaluated according to the procedure of Example 51-4. The results are summarized in Table 56. Table 56: Double TFA salt of Compound II versus p21 induced EC50 p21 ECs 〇 HELA/P21-LUC 40 1-10 μΜ Example 62. Characterization of in vitro enzyme activity of Form A of Compound II according to the assay of Example 5 1 -1 The in vitro enzyme activity of Form A (free base) of Compound II against various HDAC enzymes was evaluated and reported in Table 57. 128407.doc -174- 200911757 Table 57. Form II of Compound II IC50 to HDAC HDAC Enzyme IC5〇HDAC1 500-1000 nM HDAC2 500-1000 nM HDAC2-overnight<100 nM HDAC5 >1 μΜ HDAC6 >1 μΜ HDAC8 > 1 μΜ Example 63. Effect of Form A of Compound II on Cell Viability The inhibition of Form A of Compound II against various cancer cell types was evaluated according to the procedure of Example 5 1-2. The results are summarized in Table 58. Table 58: EC50 cell line for cell viability of Compound A Form ECS HCT116 500-1000 nM HL60 500-1000 nM MES-SA > 1 μΜ MES-SA/DX5 > 1 μΜ Example 64. Form of Compound II In vivo efficacy of A (histone acetylation) The induction of histone acetylation by Form A of Compound II was evaluated according to the procedure of Example 51-3. The results are summarized in Table 59. Table 59: Form II of Compound II for EC50 inducing HDAC acetamylation Ethylated protein ECso Histone Η3 8 1-10 μΜ histone Η4 8 1-10 μΜ Example 65. Effect of Form A of Compound II on p21 induction The effect of Form A of Compound II on p21 induction was evaluated according to the procedure of Example 51-4. The results are summarized in Table 60. Table 60: Form II of Compound II versus p21 induced EC50 p21 ECso HELA/P21-LUC 40 1-10 μΜ 128407.doc -175- 200911757 Example 66. In vivo efficacy of Form A of Compound II according to Example 5 1 -5 The assay was used to assess the efficacy of Form A of Compound I in various mouse cancer cells in a mouse model. The results are listed in Table 61. Table 61. Effect of Form A of Compound II on Cancer Cells in a Mouse Model Salt Form Xenograft Strain Progress T/C Free Test HCT116 65 mg/kg QD x 15 36% Free Base A2780 65 mg/kg QD x 15 65 % Free test A2780 100 mg/kg QD x 15 38% Free test HCT116 20 mg/kg BID x 15 54% Free test HCT116 100 mg/kg QD x 14 40% Free test PC3 50 mg/kg QD x 14 16% free PC3 100 mg/kg QD x 14 7% Example 67. Effect of Form A and Double TFA Salts of Compound II on hERG Form A and double TFA salts of Compound II were evaluated according to the assays in Examples 5-6 Inhibition. The results showed a <10% inhibition at a double TFA salt of Compound II at a concentration of 10 μΜ and a 12% inhibition at a concentration of Compound II at a concentration of 10 μΜ. Example 68. In vitro enzyme activity assay for Form A (free base) of Compound III The in vitro enzyme activity of Form A of Compound III (free base) against various HDAC enzymes was evaluated according to the assay of Example 51-1 and reported in Table 62. in. Table 62: IC50 of Form A of Compound III for HDAC

HDAC酵素 IC5〇 HDAC1 500-1000 nM HDAC2 > 1 μΜ HD AC2-隔伙 < 100 nM 實例69.化合物III之形式A對細胞生存力之作用根據實例51-2之程序來評估化合物III之形式A對各種癌細胞類型之抑制作用。將結果概括於表63中。 128407.doc -176- 200911757 表63 :化合物III之形式A對細胞生存力之EC50 細胞株 ECs〇 HCT116 500-1000 nM HL60 1-10 μΜ MES-SA 10-100 μΜ MES-SA/DX5 > 100 μΜ 實例70·化合物III之形式A之活體内功效（組蛋白乙醢化）根據實例5 1 -3之程序來評估化合物III之形式A對組蛋白乙醯化之誘導作用。將結果概括於表64中。表64 :化合物III之形式A對誘導HDAC乙醯化之EC50 乙醯化蛋白質 ECs〇組蛋白Η3 8 1-10 μΜ 組蛋白Η4 8 1-10 μΜ 實例71.化合物III之形式A對p21誘導之作用根據實例51-4之程序來評估化合物III之形式A對p21誘導之作用。將結果概括於表65中。表65 :化合物III之形式A對p21誘導之EC50 p21 ECs〇 HELA/P21-LUC_40 1-10 μΜ 實例72·化合物III之形式A的活體内功效根據實例5 1 -5之檢定來評估小鼠模型中化合物III之形式 A對各種癌細胞之功效。將結果列表於表66中。表66.小鼠模型中化合物III之形式A對癌細胞之功效鹽形式異種移植株劑量進度 T/C 游離驗 Α2780 100 mg/kg QD X 15 44% 實例73.化合物III之形式A及雙TFA鹽對hERG之作用HDAC enzyme IC5 〇 HDAC1 500-1000 nM HDAC2 > 1 μΜ HD AC2-isolated < 100 nM Example 69. Effect of Form A of Compound III on cell viability The form of Compound III was evaluated according to the procedure of Example 51-2. A inhibition of various cancer cell types. The results are summarized in Table 63. 128407.doc -176- 200911757 Table 63: Compounds of Compound III Form A Cell viability EC50 Cell line ECs〇HCT116 500-1000 nM HL60 1-10 μΜ MES-SA 10-100 μΜ MES-SA/DX5 > 100 μΜ Example 70. In vivo efficacy of Form A of Compound III (histone acetylation) The induction of histone acetylation by Form A of Compound III was evaluated according to the procedure of Example 5 1 -3. The results are summarized in Table 64. Table 64: Form A of Compound III EC50 for induction of HDAC acetylation ECs 〇 histone Η 3 8 1-10 μΜ histone Η 4 8 1-10 μΜ Example 71. Form A of Compound III for p21 induction Effect The effect of Form A of Compound III on p21 induction was evaluated according to the procedure of Example 51-4. The results are summarized in Table 65. Table 65: Form A of Compound III versus p21 induced EC50 p21 ECs 〇 HELA/P21-LUC_40 1-10 μΜ Example 72 · In vivo efficacy of Form A of Compound III The mouse model was evaluated according to the assay of Example 5 1 -5 The effect of Form A of Compound III on various cancer cells. The results are listed in Table 66. Table 66. Effect of Form A of Compound III on Cancer Cells in a Mouse Model Salt Form Xenograft Strain Schedule T/C Free Test 2780 100 mg/kg QD X 15 44% Example 73. Form A of Compound III and Double TFA The effect of salt on hERG

根據實例5 1 -6中之檢定來評估化合物III之形式A對hERG 128407.doc -177- 200911757 之抑制作用。η罢％ _丄〇1.項不在10 μΜ濃度之化合物III之形式A 下為20°/。之抑制。【圖式簡單說明】圖1為化合物I > π a 〈办式A的特徵X射線粉末繞射（XRPD)光譜。圖為化σ物1之形式A的溶液質子核磁共振"H NMR)光譜。圖3為化合物1之形式A的特徵衰減全反射傅裏葉變換紅外吸收(ATR-FTIK)光譜（4__6⑽波數㈣力）。圖4為化合物1之形式A的特徵熱解重量分析（TGA)熱分析圖。圖5為化合物1之形式A的特徵示差掃描熱量測定（DSC)熱分析圖。圖6為化合物1之形式A的水分吸附-解吸附曲線。圖7為藉由在乙醇2B中形式轉化而製備之化合物丨之形式 B的特徵XRPD光譜。圖8為藉由在乙醇2B中形式轉化而製備之化合物I之形式 B的溶液NMR光譜。圖9為藉由在乙醇2B中形式轉化而製備之化合物I之形式 B的HPLC層析圖。圖1〇為藉由在乙醇2B中形式轉化而製備之化合物I之形式B的特徵DSC熱分析圖。圖11為藉由與丁二酸在MeCN中共結晶而製備之化合物工之形式B的特徵XRPD光譜。 128407.doc •178- 200911757 圖12為藉由與丁二酸在MeCN中共結晶製備之化合物I之形式B的溶液hNMR光譜。圖13為藉由與丁二酸在MeCN中共結晶而製備之化合物I 之形式B的特徵ATR-FTIR光譜（4000-600波數（cm·1))。圖14為藉由與丁二酸在MeCN中共結晶製備之化合物I之形式B的特徵DSC熱分析圖。圖15為藉由與丁二酸在MeCN中共結晶而製備之化合物I 之形式B的特徵TGA熱分析圖。圖1 6為藉由與丁二酸在MeCN中共結晶而製備之化合物I 之形式B的水分吸附-解吸附曲線。圖17為化合物I之形式非晶的特徵XRPD光譜。圖18為化合物I之反丁烯二酸鹽之特徵XRPD光譜。圖19為化合物I之反丁烯二酸鹽之溶液1H NMR光譜。圖20為化合物I之反丁烯二酸鹽之特徵ATR-FTIR光譜 (4000-600 波數（cm·1))。圖21為化合物I之反丁烯二酸鹽之特徵DSC熱分析圖。圖22為化合物I之反丁烯二酸鹽之特徵TGA熱分析圖。圖23為化合物I之反丁烯二酸鹽之水分吸附-解吸附曲線。圖24為化合物I之氫氯酸鹽之特徵XRPD光譜。圖25為化合物I之氫氯酸鹽之溶液1H NMR光譜。圖26為化合物I之氫氣酸鹽之特徵ATR-FTIR光譜（4000-600 波數（cm·1))。圖27為化合物I之氫氯酸鹽之特徵TGA熱分析圖。 128407.doc -179- 200911757 圖28為化合物I之氫氯酸鹽之特徵DSC熱分析圖。圖29為化合物I之氫氯酸鹽之水分吸附-解吸附曲線。圖30為化合物I之磷酸鹽之特徵XRPD光譜。圖31為化合物I之磷酸鹽之溶液1H NMR光譜。圖32為化合物I之磷酸鹽之特徵DSC熱分析圖。圖33為化合物I之磷酸鹽之特徵TGA熱分析圖。圖34為所有化合物I固體之面積百分比檢定的高效液相層析（HPLC)校正曲線；將化合物I之結晶固體的水溶性繪製於曲線上。圖35為化合物II之形式A的特徵XRPD光譜。圖36為化合物II之形式A的溶液hNMR光譜。圖37為化合物II之形式A的特徵ATR-FTIR光譜（4000-600 波數（crrf i)。圖38為化合物II之形式A的特徵DSC熱分析圖。圖39為化合物II之形式A的特徵TGA熱分析圖。圖40為化合物II之形式A的水分吸附-解吸附曲線。圖41為化合物II之丁二酸鹽的特徵XRPD光譜。圖42為化合物II之丁二酸鹽的溶液1H NMR光譜。圖43為化合物II之丁二酸鹽的特徵ATR-FTIR光譜（4000-600 波數（cm—”）。圖44為化合物II之丁二酸鹽的特徵DSC熱分析圖。圖45為化合物II之丁二酸鹽的特徵TGA熱分析圖。圖46為化合物II之丁二酸鹽的水分吸附-解吸附曲線。圖47為化合物II之苯曱酸鹽的特徵XRPD光譜。 128407.doc •180· 200911757 圖48為化合物II之苯曱酸鹽的溶液1H NMR光譜。圖49為化合物II之苯曱酸鹽的特徵ATR-FTIR光譜（4000-600 波數（crrT1))。圖50為化合物II之苯曱酸鹽的特徵DSC熱分析圖。圖51為化合物II之苯甲酸鹽的特徵TGA熱分析圖。圖52為化合物II之苯曱酸鹽的水分吸附-解吸附曲線。圖53為化合物II之氫氯酸鹽的特徵XRPD光譜。圖54為化合物II之氫氯酸鹽的溶液1H NMR光譜。圖55為化合物II之氫氯酸鹽的特徵ATR-FTIR光譜（4000-600 波數（ctiT1))。圖56為化合物II之氫氯酸鹽的特徵DSC熱分析圖。圖57為化合物II之氫氯酸鹽的特徵TGA熱分析圖。圖5 8為化合物II之氫氯酸鹽的水分吸附-解吸附曲線。圖59為化合物II之氫溴酸鹽的特徵XRPD光譜。圖60為化合物II之氫溴酸鹽的溶液1H NMR光譜。圖61為化合物II之氫溴酸鹽的特徵ATR-FTIR光譜（4000-600 波數（cm·1))。圖62為化合物II之氫溴酸鹽的特徵DSC熱分析圖。圖63顯示化合物II之氫溴酸鹽的特徵TGA熱分析圖。圖64為化合物II之氫溴酸鹽的水分吸附-解吸附曲線。圖65為化合物II之龍膽酸鹽的特徵XRPD光譜。圖66為化合物II之龍膽酸鹽的溶液1H NMR光譜。圖67為化合物II之龍膽酸鹽的特徵ATR-FTIR光譜（4000-600波數（cnT1))。 128407.doc -181 - 200911757 圖68為化合物II之龍膽酸鹽的特徵DSC熱分析圖。圖69為化合物II之龍膽酸鹽的特徵TGA熱分析圖。圖70為化合物II之龍膽酸鹽的水分吸附-解吸附曲線。圖71為化合物III之形式A的特徵XRPD光譜。圖72為化合物III之形式A的溶液^NMR光譜。圖73為化合物III之形式A的特徵ATR-FTIR光譜（4000-600 波數（cnT1))。圖74為化合物III之形式A的特徵DSC熱分析圖。圖75為化合物III之形式A的特徵TGA熱分析圖。圖76為化合物III之形式A的水分吸附-解吸附曲線。圖77為化合物III之龍膽酸鹽固體的特徵XRPD光譜。圖78為（a)化合物III形式A(圖71)與（b)化合物III之龍膽酸鹽固體（圖77)的XRPD圖樣的疊置圖。圖79為化合物III之龍膽酸鹽固體的溶液1H NMR光譜。圖80為化合物III之龍膽酸鹽固體的特徵ATR-FTIR光譜 (4000-600 波數（cm—1))。圖81為化合物III之龍膽酸鹽固體的特徵DSC熱分析圖。圖82為化合物III之龍膽酸鹽固體的特徵TGA熱分析圖。圖83為化合物III之龍膽酸鹽固體的水分吸附-解吸附曲線。圖84說明HDAC1之殘基1-482外加在N端及C端之Flag標籤及在N端之Met殘基選殖人工產物（SEQ ID NO: 1)。圖85說明用以編碼SEQ ID NO: 1之DNA序列（SEQ ID NO: 2)。 128407.doc -182- 200911757 圖86說明HDAC2之殘基1-488外加在C端之6-組胺酸標籤及四個選殖人工產物殘基（在N端之Met-Gly-Ser片段及在6-組胺酸標籤前之Gly)(SEQ ID NO: 3)。圖87說明用以編碼SEQ ID NO: 3之DNA序列（SEQ ID NO: 4)。圖88說明HDAC6之殘基73-845外加在C端之6-組胺酸標籤及三個選殖人工產物殘基（在N端之Met-Pro對及在6-組胺酸標籤前之Gly)(SEQ ID NO: 5)。 / 圖89說明用以編碼SEQ ID NO: 5之DNA序列（SEQ ID NO: 6)。圖90說明HDAC8之殘基1-377外加在N端之6-組胺酸標籤及兩個選殖人工產物殘基（在6-組胺酸標籤之前的Met及之後的 Pro)(SEQ ID NO: 7)。圖91說明用以編碼SEQ ID NO: 7之DNA序列（SEQ ID NO: 8)。 128407.doc -183 - 200911757 序列表 <11〇>美商武田聖地牙哥公司 <120>組蛋白去乙醯基酶抑制劑之結晶形 <130> HDAC-5016 <140> 097102083 <141> 2008-01-18 <150> 60/885,616 <151> 2007-01-18 <160> 8 < 170> patentln version 3.3 <210> 1 <21卜 499 <212> PRT <213>人工 <220> <223> HDAC1之殘基1-482外加在N端及C端之Hag標籤及在N端之Met殘基選殖人工產物的胺基酸序列 <400> 1The inhibition of Form A of Compound III on hERG 128407.doc-177-200911757 was evaluated according to the assay in Example 5 1-6. η % % _ 丄〇 1. The term is not 20 ° / under the form A of the compound III at a concentration of 10 μΜ. Suppression. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a characteristic X-ray powder diffraction (XRPD) spectrum of the compound I > π a < The figure shows the solution proton nuclear magnetic resonance "H NMR) spectrum of Form A of Sigma 1 . Figure 3 is a characteristic attenuated total reflection Fourier transform infrared absorption (ATR-FTIK) spectrum of Form A of Compound 1 (4__6 (10) wave number (tetra) force). Figure 4 is a characteristic thermogravimetric analysis (TGA) thermogram of Form A of Compound 1. Figure 5 is a characteristic differential scanning calorimetry (DSC) thermogram of Form A of Compound 1. Figure 6 is a moisture adsorption-desorption curve of Form A of Compound 1. Figure 7 is a characteristic XRPD spectrum of Form B of a compound prepared by formal conversion in ethanol 2B. Figure 8 is a solution NMR spectrum of Form B of Compound I prepared by formal conversion in ethanol 2B. Figure 9 is an HPLC chromatogram of Form B of Compound I prepared by formal conversion in ethanol 2B. Figure 1 is a characteristic DSC thermogram of Form B of Compound I prepared by formal conversion in ethanol 2B. Figure 11 is a characteristic XRPD spectrum of Form B of a compound prepared by co-crystallization with succinic acid in MeCN. 128407.doc • 178- 200911757 Figure 12 is a solution h NMR spectrum of Form B of Compound I prepared by co-crystallization with succinic acid in MeCN. Figure 13 is a characteristic ATR-FTIR spectrum (4000-600 wave number (cm·1)) of Form B of Compound I prepared by co-crystallization with succinic acid in MeCN. Figure 14 is a characteristic DSC thermogram of Form B of Compound I prepared by co-crystallization with succinic acid in MeCN. Figure 15 is a characteristic TGA thermogram of Form B of Compound I prepared by co-crystallization with succinic acid in MeCN. Figure 16 is a moisture adsorption-desorption curve of Form B of Compound I prepared by co-crystallization with succinic acid in MeCN. Figure 17 is a characteristic XRPD spectrum of the amorphous form of Compound I. Figure 18 is a characteristic XRPD spectrum of the fumarate of Compound I. Figure 19 is a 1H NMR spectrum of the solution of the compound of the compound I. Figure 20 is a characteristic ATR-FTIR spectrum (4000-600 wave number (cm·1)) of the fumarate of Compound I. Figure 21 is a characteristic DSC thermogram of the fumarate salt of Compound I. Figure 22 is a characteristic TGA thermogram of the fumarate salt of Compound I. Figure 23 is a graph showing the moisture adsorption-desorption curve of the fumarate of Compound I. Figure 24 is a characteristic XRPD spectrum of the hydrochloride salt of Compound I. Figure 25 is a 1H NMR spectrum of a solution of the hydrochloride salt of Compound I. Figure 26 is a characteristic ATR-FTIR spectrum (4000-600 wave number (cm·1)) of the hydrogenate salt of Compound I. Figure 27 is a characteristic TGA thermogram of the hydrochloride salt of Compound I. 128407.doc -179- 200911757 Figure 28 is a characteristic DSC thermogram of the hydrochloride salt of Compound I. Figure 29 is a graph showing the moisture adsorption-desorption curve of the hydrochloride of Compound I. Figure 30 is a characteristic XRPD spectrum of the phosphate of Compound I. Figure 31 is a 1H NMR spectrum of a solution of the phosphate of Compound I. Figure 32 is a characteristic DSC thermogram of the phosphate of Compound I. Figure 33 is a characteristic TGA thermogram of the phosphate of Compound I. Figure 34 is a high performance liquid chromatography (HPLC) calibration curve for area percent determination of all Compound I solids; the water solubility of the crystalline solid of Compound I is plotted on the curve. Figure 35 is a characteristic XRPD spectrum of Form A of Compound II. Figure 36 is a solution h NMR spectrum of Form A of Compound II. Figure 37 is a characteristic ATR-FTIR spectrum (4000-600 wave number (crrf i) of Form A of Compound II. Figure 38 is a characteristic DSC thermogram of Form A of Compound II. Figure 39 is a characteristic of Form A of Compound II. TGA thermogram Fig. 40 is a moisture adsorption-desorption curve of Form A of Compound II. Figure 41 is a characteristic XRPD spectrum of the succinate salt of Compound II. Figure 42 is a solution of the succinate salt of Compound II 1H NMR Figure 43 is a characteristic ATR-FTIR spectrum (4000-600 wave number (cm-") of the succinate salt of Compound II. Figure 44 is a characteristic DSC thermogram of the succinate salt of Compound II. A characteristic TGA thermogram of the succinate salt of Compound II. Figure 46 is a moisture adsorption-desorption curve of the succinate salt of Compound II. Figure 47 is a characteristic XRPD spectrum of the benzoate salt of Compound II. • 180· 200911757 Figure 48 is a solution 1H NMR spectrum of the benzoate salt of Compound II. Figure 49 is a characteristic ATR-FTIR spectrum (4000-600 wave number (crrT1)) of the benzoate salt of Compound II. A characteristic DSC thermogram of the benzoate salt of Compound II. Figure 51 is a benzoate salt of Compound II. Fig. 52 is a water adsorption-desorption curve of the benzoate of the compound II. Fig. 53 is a characteristic XRPD spectrum of the hydrochloride of the compound II. Fig. 54 is a hydrochloride of the compound II. 1H NMR spectrum of the solution Fig. 55 is a characteristic ATR-FTIR spectrum (4000-600 wave number (ctiT1)) of the hydrochloride of the compound II. Fig. 56 is a characteristic DSC thermogram of the hydrochloride of the compound II. 57 is a characteristic TGA thermogram of the hydrochloride of Compound II. Figure 5 is a moisture adsorption-desorption curve of the hydrochloride of Compound II. Figure 59 is a characteristic XRPD spectrum of the hydrobromide salt of Compound II. Figure 60 is a 1H NMR spectrum of a solution of the hydrobromide salt of Compound II. Figure 61 is a characteristic ATR-FTIR spectrum (4000-600 wave number (cm·1)) of the hydrobromide salt of Compound II. Figure 62 is a compound II A characteristic DSC thermogram of the hydrobromide salt is shown in Figure 63. Figure 63 shows a characteristic TGA thermogram of the hydrobromide salt of Compound II. Figure 64 is a moisture adsorption-desorption curve of the hydrobromide salt of Compound II. Characteristic XRPD spectrum of gentisate of compound II. Figure 66 is a solution of compound II of gentisate 1H NMR Figure 67 is a characteristic ATR-FTIR spectrum (4000-600 wave number (cnT1)) of gentisate of Compound II. 128407.doc -181 - 200911757 Figure 68 is a characteristic DSC heat of gentisate of Compound II. Analysis chart. Figure 69 is a characteristic TGA thermogram of the gentisate salt of Compound II. Figure 70 is a graph showing the moisture adsorption-desorption curve of gentisate of Compound II. Figure 71 is a characteristic XRPD spectrum of Form A of Compound III. Figure 72 is a solution NMR spectrum of Form A of Compound III. Figure 73 is a characteristic ATR-FTIR spectrum (4000-600 wave number (cnT1)) of Form A of Compound III. Figure 74 is a characteristic DSC thermogram of Form A of Compound III. Figure 75 is a characteristic TGA thermogram of Form A of Compound III. Figure 76 is a moisture adsorption-desorption curve of Form A of Compound III. Figure 77 is a characteristic XRPD spectrum of the gentisate solid of Compound III. Figure 78 is an overlay of the XRPD pattern of (a) Compound III Form A (Figure 71) and (b) Compound III gentisate solid (Figure 77). Figure 79 is a solution 1H NMR spectrum of the gentisate solid of Compound III. Figure 80 is a characteristic ATR-FTIR spectrum (4000-600 wave number (cm-1)) of the gentisate solid of Compound III. Figure 81 is a characteristic DSC thermogram of the gentisate solid of Compound III. Figure 82 is a characteristic TGA thermogram of the gentisate solid of Compound III. Figure 83 is a moisture adsorption-desorption curve of the gentisate solid of Compound III. Figure 84 illustrates that residues 1-482 of HDAC1 are appended to the Flag tag at the N-terminus and C-terminus and the Met residue-selected artifact at the N-terminus (SEQ ID NO: 1). Figure 85 illustrates the DNA sequence (SEQ ID NO: 2) used to encode SEQ ID NO: 1. 128407.doc -182- 200911757 Figure 86 illustrates residues 1-488 of HDAC2 plus a 6-histidine tag at the C-terminus and four colonized artifact residues (Met-Gly-Ser fragment at the N-terminus and Gly) before 6-histidine tag (SEQ ID NO: 3). Figure 87 illustrates the DNA sequence (SEQ ID NO: 4) used to encode SEQ ID NO: 3. Figure 88 illustrates residues 73-845 of HDAC6 plus a 6-histidine tag at the C-terminus and three colonized artifact residues (Met-Pro pair at the N-terminus and Gly before the 6-histidine tag) (SEQ ID NO: 5). / Figure 89 illustrates the DNA sequence (SEQ ID NO: 6) used to encode SEQ ID NO: 5. Figure 90 illustrates residues 1-377 of HDAC8 plus a 6-histidine acid tag at the N-terminus and two colonized artifact residues (Met before and after the 6-histidine tag) and subsequent Pro (SEQ ID NO) : 7). Figure 91 illustrates the DNA sequence (SEQ ID NO: 8) used to encode SEQ ID NO: 7. 128407.doc -183 - 200911757 Sequence Listing <11〇> American Takeda San Diego Co., Ltd. <120> Histone Deacetylase Inhibitor Crystal Form <130> HDAC-5016 <140> 097102083 <141> 2008-01-18 <150> 60/885,616 <151> 2007-01-18 <160> 8 <170> patentln version 3.3 <210> 1 <21 499 <212>; PRT < 213 > Artificial <220><223> Residue 1-482 of HDAC1 plus the Hag tag at the N-terminus and C-terminus and the amino acid sequence of the Met residue-selected artifact at the N-terminus ;400> 1

Met Asp Tyr Lys Asp Asp Asp Asp Lys Met Ala Gin Thr Gin Gly Thr 15 10 15 A.rg Arg Lys Val Cys Tyr Tyr Tyr Asp Gly Asp Val Gly Asn Tyr Tyr 20 25 30Met Asp Tyr Lys Asp Asp Asp Asp Lys Met Ala Gin Thr Gin Gly Thr 15 10 15 A.rg Arg Lys Val Cys Tyr Tyr Tyr Asp Gly Asp Val Gly Asn Tyr Tyr 20 25 30

Tyr Gly Gin Gly His Pro Met Lys Pro His Arg lie Arg Met Thr His 35 40 45Tyr Gly Gin Gly His Pro Met Lys Pro His Arg lie Arg Met Thr His 35 40 45

Asn Leu Leu Leu Asn Tyr Gly Leu Tyr Arg Lys Met Glu lie Tyr Arg 50 55 60Asn Leu Leu Leu Asn Tyr Gly Leu Tyr Arg Lys Met Glu lie Tyr Arg 50 55 60

Pro His Lys Ala Asn Ala Glu Glu Met Thr Lys Tyr His Ser Asp Asp 65 70 75 80Pro His Lys Ala Asn Ala Glu Glu Met Thr Lys Tyr His Ser Asp Asp 65 70 75 80

Tyr lie Lys Phe Leu Arg Ser lie Arg Pro Asp Asn Met Ser Glu Tyr 85 90 95Tyr lie Lys Phe Leu Arg Ser lie Arg Pro Asp Asn Met Ser Glu Tyr 85 90 95

Scr Lys Gin Met Gin Arg Phe Asn Val Gly Glu Asp Cys Pro Val Phe 100 105 110Scr Lys Gin Met Gin Arg Phe Asn Val Gly Glu Asp Cys Pro Val Phe 100 105 110

Asp Gly Leu Phe Glu Phe Cys Gin Leu Ser Thr Gly Gly Ser Val Ala 115 120 125Asp Gly Leu Phe Glu Phe Cys Gin Leu Ser Thr Gly Gly Ser Val Ala 115 120 125

Ser Ala Val Lys Leu Asn Lys Gin Gin Thr Asp lie Ala Val Asn 丁rp 130 135 140Ser Ala Val Lys Leu Asn Lys Gin Gin Thr Asp lie Ala Val Asn Ding rp 130 135 140

Ala Gly Gly Leu His His Ala Lys Lys Ser Glu Ala Ser Gly Phe Cys )45 150 155 160Ala Gly Gly Leu His His Ala Lys Lys Ser Glu Ala Ser Gly Phe Cys )45 150 155 160

Tyr Val Asn Asp He Val Leu Ala lie Leu Glu Leu Leu Lys Tyr His 165 170 175Tyr Val Asn Asp He Val Leu Ala lie Leu Glu Leu Leu Lys Tyr His 165 170 175

Gin Arg Val Leu Tyr He Asp lie Asp lie His His Gly Asp Gly Val 180 185 190 128407.doc 200911757Gin Arg Val Leu Tyr He Asp lie Asp lie His His Gly Asp Gly Val 180 185 190 128407.doc 200911757

Glu Glu Ala Phe Tyr Thr Thr Asp Arg Val Met Thr Val Ser Phe His 195 200 205Glu Glu Ala Phe Tyr Thr Thr Asp Arg Val Met Thr Val Ser Phe His 195 200 205

Lys Tyr Gly Glu Tyr Phe Pro Gly Thr Gly Asp Leu Arg Asp lie Gly 210 215 220Lys Tyr Gly Glu Tyr Phe Pro Gly Thr Gly Asp Leu Arg Asp lie Gly 210 215 220

Ala Gly Lys Gly Lys Tyr Tyr Ala Val Asn Tyr Pro Leu Arg Asp Gly 225 230 235 240Ala Gly Lys Gly Lys Tyr Tyr Ala Val Asn Tyr Pro Leu Arg Asp Gly 225 230 235 240

Me Asp Asp Glu Ser Tyr Glu Ala lie Phe Lys Pro Val Met Ser Lys 245 250 255Me Asp Asp Glu Ser Tyr Glu Ala lie Phe Lys Pro Val Met Ser Lys 245 250 255

Val Met Glu Met Phe Gin Pro Ser Ala Val Val Leu Gin Cys Gly Ser 260 265 270Val Met Glu Met Phe Gin Pro Ser Ala Val Val Leu Gin Cys Gly Ser 260 265 270

Asp Ser Leu Ser Gly Asp Arg Leu Gly Cys Phe Asn Leu Thr lie Lys 275 280 285 lly His Ala Lys Cys Val Glu Phe Val Lys Ser Phe Asn Leu Pro Met 290 295 300Asp Ser Leu Ser Gly Asp Arg Leu Gly Cys Phe Asn Leu Thr lie Lys 275 280 285 lly His Ala Lys Cys Val Glu Phe Val Lys Ser Phe Asn Leu Pro Met 290 295 300

Leu Mel Leu Gly Gly Gly Gly Tyr Thr lie Arg Asn Val Ala Arg Cys 305 310 315 320Leu Mel Leu Gly Gly Gly Gly Tyr Thr lie Arg Asn Val Ala Arg Cys 305 310 315 320

Trp Thr Tyr Glu Thr Ala Val Ala Leu Asp Thr Glu lie Pro Asn Glu 325 330 335Trp Thr Tyr Glu Thr Ala Val Ala Leu Asp Thr Glu lie Pro Asn Glu 325 330 335

Leu Pro Tyr Asn Asp Tyr Phe Glu Tyr Phe Gly Pro Asp Phe Lys Leu 340 345 350Leu Pro Tyr Asn Asp Tyr Phe Glu Tyr Phe Gly Pro Asp Phe Lys Leu 340 345 350

His lie Ser Pro Ser Asn Met Thr Asn Gin Asn Thr Asn Glu Tyr Leu 355 360 365His lie Ser Pro Ser Asn Met Thr Asn Gin Asn Thr Asn Glu Tyr Leu 355 360 365

Glu Lys lie Lys Gin Arg Leu Phe Glu Asn Leu Arg Met Leu Pro His 370 375 380Glu Lys lie Lys Gin Arg Leu Phe Glu Asn Leu Arg Met Leu Pro His 370 375 380

Ala Pro Gly Val Gin Met Gin Ala lie Pro Glu Asp Ala He Pro Glu 385 390 395 400Ala Pro Gly Val Gin Met Gin Ala lie Pro Glu Asp Ala He Pro Glu 385 390 395 400

Glu Ser Gly Asp Glu Asp Glu Asp Asp Pro Asp Lys Arg lie Ser lie 405 410 415Glu Ser Gly Asp Glu Asp Glu Asp Asp Pro Asp Lys Arg lie Ser lie 405 410 415

Cys Ser Ser Asp Lys Arg lie Ala Cys Glu Glu Glu Phe Ser Asp Ser 420 425 430Cys Ser Ser Asp Lys Arg lie Ala Cys Glu Glu Glu Phe Ser Asp Ser 420 425 430

Glu Glu Glu Gly Glu Gly Gly Arg Lys Asn Ser Ser Asn Phe Lys Lys 435 440 445Glu Glu Glu Gly Glu Gly Gly Arg Lys Asn Ser Ser Asn Phe Lys Lys 435 440 445

Ala Lys Arg Val Lys Thr Glu Asp Glu Lys Glu Lys Asp Pro Glu Glu 450 455 460Ala Lys Arg Val Lys Thr Glu Asp Glu Lys Glu Lys Asp Pro Glu Glu 450 455 460

Lys Lys Glu Val Thr Glu Glu Glu Lys Thr Lys Glu Glu Lys Pro Glu 465 470 475 480Lys Lys Glu Val Thr Glu Glu Glu Lys Thr Lys Glu Glu Lys Pro Glu 465 470 475 480

Ala Lys Gly Val Lys Glu Glu Val Lys Leu Ala Asp Tyr Lys Asp Asp 485 490 495 -2- 128407.doc 200911757Ala Lys Gly Val Lys Glu Glu Val Lys Leu Ala Asp Tyr Lys Asp Asp 485 490 495 -2- 128407.doc 200911757

Asp Asp Lys <210> 2 <211> 1500 <212> DNA <213>人工 <220> <223>用以編碼HDAC1之殘基1-482及在N端及C端之Flag標籤的 DNA序列 <400〉 2 atggactaca aagacgacga cgacaaaatg gcgcagacgc agggcacccg gaggaaagtc 60 tgttactact acgacgggga tgttggaaat tactattatg gacaaggcca cccaatgaag 120 cctcaccgaa tccgcatgac tcataatttg ctgctcaact atggtctcta ccgaaaaatg 180 gaaatctatc gccctcacaa agccaatgct gaggagatga ccaagtacca cagcgatgac 240 tacattaaat tcttgcgctc catccgtcca gataacatgt cggagtacag caagcagatg 300 cagagattca acgttggtga ggactgtcca gtattcgatg gcctgtttga gttctgtcag 360 [ ttgtctactg gtggttctgt ggcaagtgct gtgaaactta ataagcagca gacggacatc 420 gcigtgaatt gggctggggg cctgcaccat gcaaagaagt ccgaggcatc tggcttctgt 480 tacgicaatg atalcgtctt ggccatcctg gaactgctaa agtatcacca gagggtgctg 540 tacattgaca ttgatattca ccatggtgac ggcgtggaag aggccttcta caccacggac 600 cgggtcatga ctgtgtcctt tcataagtat ggagagtact tcccaggaac tggggaccta 660 cgggatatcg gggctggcaa aggcaagtat tatgctgtta actacccgct ccgagacggg 720 attgatgacg agtcctatga ggccattttc aagccggtca tgtccaaagt aatggagatg 780 ttccagccta gtgcggtggt cttacagtgi ggctcagact ccctatctgg ggatcggtta 840 ggttgcttca atctaactat caaaggacac gccaagtgtg tggaatttgt caagagcttt 900 aacctgccta tgctgatgct gggaggcggt ggttacacca ttcgtaacgt tgcccggtgc 960 tggacatatg agacagctgt ggccctggat acggagatcc ctaatgagct tccatacaat 1020 i?actactttg aatactttgg accagatttc aagctccaca tcagtccttc caatatgact 1080 i aaccagaaca cgaatgagta cctggagaag atcaaacagc gactgtttga gaaccttaga 1】4〇 atgctgccgc acgcacctgg ggtccaaatg caggcgattc ctgaggacgc catccctgag 1200 gagagtggcg atgaggacga agacgaccct gacaagcgca tctcgatctg ctcctctgac 1260 aaacgaattg cctgtgagga agagttctcc gattctgaag aggagggaga ggggggccgc 1320 aagaactctt ccaacttcaa aaaagccaag agagtcaaaa cagaggatga aaaagagaaa 1380 gacccagagg agaagaaaga agtcaccgaa gaggagaaaa ccaaggagga gaagccagaa 1440 gccaaagggg tcaaggagga ggtcaagttg gccgactaca aagacgacga cgacaaatga 1500 <210> 3 <211> 498 <212> PRT <213>人工 <220> <223> HDAC2之殘基1-488外加在C端之6-組胺酸標籤及四個選殖人工產物殘基（在N端之Met-Gly-Ser片段及在6-組胺酸標籤前之Gly) 的胺基酸序列 128407.doc 200911757 <400〉 3Asp Asp Lys <210> 2 <211> 1500 <212> DNA <213>Labor<220><223> to encode residues 1-482 of HDAC1 and Flag at N-terminus and C-terminus DNA sequence & lt tag; 400> 2 atggactaca aagacgacga cgacaaaatg gcgcagacgc agggcacccg gaggaaagtc 60 tgttactact acgacgggga tgttggaaat tactattatg gacaaggcca cccaatgaag 120 cctcaccgaa tccgcatgac tcataatttg ctgctcaact atggtctcta ccgaaaaatg 180 gaaatctatc gccctcacaa agccaatgct gaggagatga ccaagtacca cagcgatgac 240 tacattaaat tcttgcgctc catccgtcca gataacatgt cggagtacag caagcagatg 300 cagagattca acgttggtga ggactgtcca gtattcgatg gcctgtttga gttctgtcag 360 [ttgtctactg gtggttctgt ggcaagtgct gtgaaactta ataagcagca gacggacatc 420 gcigtgaatt gggctggggg cctgcaccat gcaaagaagt ccgaggcatc tggcttctgt 480 tacgicaatg atalcgtctt ggccatcctg gaactgctaa agtatcacca gagggtgctg 540 tacattgaca ttgatattca ccatggtgac ggcgtggaag aggccttcta caccacggac 600 cgggtcatga ctgtgtcctt tcataagtat ggagagtact tcccaggaac tggggaccta 660 cgggatatcg gggctggcaa aggcaagtat tatgctgt ta actacccgct ccgagacggg 720 attgatgacg agtcctatga ggccattttc aagccggtca tgtccaaagt aatggagatg 780 ttccagccta gtgcggtggt cttacagtgi ggctcagact ccctatctgg ggatcggtta 840 ggttgcttca atctaactat caaaggacac gccaagtgtg tggaatttgt caagagcttt 900 aacctgccta tgctgatgct gggaggcggt ggttacacca ttcgtaacgt tgcccggtgc 960 tggacatatg agacagctgt ggccctggat acggagatcc ctaatgagct tccatacaat 1020 i? actactttg aatactttgg accagatttc aagctccaca tcagtccttc caatatgact 1080 i aaccagaaca cgaatgagta cctggagaag atcaaacagc gactgtttga gaaccttaga 1] 4〇atgctgccgc acgcacctgg ggtccaaatg caggcgattc ctgaggacgc catccctgag 1200 gagagtggcg atgaggacga agacgaccct gacaagcgca tctcgatctg ctcctctgac 1260 aaacgaattg cctgtgagga agagttctcc gattctgaag aggagggaga ggggggccgc 1320 aagaactctt ccaacttcaa aaaagccaag agagtcaaaa cagaggatga aaaagagaaa 1380 gacccagagg agaagaaaga agtcaccgaa gaggagaaaa ccaaggagga gaagccagaa 1440 gccaaagggg tcaaggagga ggtcaagttg gccgactaca aagacgacga cgacaaatga 1500 <210> 3 <211> 498 <212> PRT <213>Manual<220><223> Residue 1-48 of HDAC2 plus a 6-histidine acid tag at the C-terminus and four colonized artifact residues (Met-Gly-Ser at the N-terminus) Fragment and amino acid sequence of Gly) before 6-histidine tag 128407.doc 200911757 <400> 3

Met Gly Ser Met Ala Tyr Ser Gin Gly Gly Gly Lys Lys Lys Val Cys 15 10 15Met Gly Ser Met Ala Tyr Ser Gin Gly Gly Gly Lys Lys Lys Val Cys 15 10 15

Tyr Tyr Tyr Asp Gly Asp lie Gly Asn Tyr Tyr Tyr Gly Gin Gly His 20 25 30Tyr Tyr Tyr Asp Gly Asp lie Gly Asn Tyr Tyr Tyr Gly Gin Gly His 20 25 30

Pro Met Lys Pro His Arg lie Arg Met Thr His Asn Leu Leu Leu Asn 35 40 45Pro Met Lys Pro His Arg lie Arg Met Thr His Asn Leu Leu Leu Asn 35 40 45

Tyr Gly Leu Tyr Arg Lys Met Glu lie Tyr Arg Pro His Lys Ala Thr 50 55 60Tyr Gly Leu Tyr Arg Lys Met Glu lie Tyr Arg Pro His Lys Ala Thr 50 55 60

Ala Glu Glu Met Thr Lys Tyr His Ser Asp Glu Tyr lie Lys Phe Leu 65 70 75 80 \rg Ser lie Arg Pro Asp Asn Met Ser Glu Tyr Ser Lys Gin Met Gin 85 90 95Ala Glu Glu Met Thr Lys Tyr His Ser Asp Glu Tyr lie Lys Phe Leu 65 70 75 80 \rg Ser lie Arg Pro Asp Asn Met Ser Glu Tyr Ser Lys Gin Met Gin 85 90 95

Arg Phe Asn Val Gly Glu Asp Cys Pro Val Phe Asp Gly Leu Phe Glu 100 105 110Arg Phe Asn Val Gly Glu Asp Cys Pro Val Phe Asp Gly Leu Phe Glu 100 105 110

Phe Cys Gin Leu Ser Thr Gly Gly Ser Val Ala Gly Ala Val Lys Leu 115 120 125Phe Cys Gin Leu Ser Thr Gly Gly Ser Val Ala Gly Ala Val Lys Leu 115 120 125

Asn Arg Gin Gin Thr Asp Met Ala Val Asn Trp Ala Gly Gly Leu His 130 135 140Asn Arg Gin Gin Thr Asp Met Ala Val Asn Trp Ala Gly Gly Leu His 130 135 140

His Ala Lys Lys Ser Glu Ala Ser Gly Phe Cys Tyr Val Asn Asp He 145 150 155 160His Ala Lys Lys Ser Glu Ala Ser Gly Phe Cys Tyr Val Asn Asp He 145 150 155 160

Val Leu Ala lie Leu Glu Leu Leu Lys Tyr His Gin Arg Val Leu Tyr 165 170 175 I le Asp lie Asp He His His Gly Asp Gly Val Glu Glu Ala Phe Tyr 180 185 190Val Leu Ala lie Leu Glu Leu Leu Lys Tyr His Gin Arg Val Leu Tyr 165 170 175 I le Asp lie Asp He His His Gly Asp Gly Val Glu Glu Ala Phe Tyr 180 185 190

Thr Thr Asp Arg Val Met Thr Val Ser Phe His Lys Tyr Giy Glu Tyr 195 200 205Thr Thr Asp Arg Val Met Thr Val Ser Phe His Lys Tyr Giy Glu Tyr 195 200 205

Phe Pro Gly Thr Gly Asp Leu Arg Asp He Gly Ala Gly Lys Gly Lys 210 215 220Phe Pro Gly Thr Gly Asp Leu Arg Asp He Gly Ala Gly Lys Gly Lys 210 215 220

Tyr Tyr Ala Val Asn Phe Pro Met Arg Asp Gly lie Asp Asp Glu Ser 225 230 235 240Tyr Tyr Ala Val Asn Phe Pro Met Arg Asp Gly lie Asp Asp Glu Ser 225 230 235 240

Tyr Giy Gin lie Phe Lys Pro lie lie Ser Lys Val Met Glu Met Tyr 245 250 255Tyr Giy Gin lie Phe Lys Pro lie lie Ser Lys Val Met Glu Met Tyr 245 250 255

Gin Pro Ser Ala Val Val Leu Gin Cys Gly Ala Asp Ser Leu Ser Gly 260 265 270Gin Pro Ser Ala Val Val Leu Gin Cys Gly Ala Asp Ser Leu Ser Gly 260 265 270

Asp Arg Leu Gly Cys Phe Asn Leu Thr Val Lys Gly His Ala Lys Cys 275 280 285 128407.doc 200911757Asp Arg Leu Gly Cys Phe Asn Leu Thr Val Lys Gly His Ala Lys Cys 275 280 285 128407.doc 200911757

Va 1 Glu Val Val Lys Thr Phe Asn Leu Pro Leu Leu Met Leu Gly Gly 290 295 300Va 1 Glu Val Val Lys Thr Phe Asn Leu Pro Leu Leu Met Leu Gly Gly 290 295 300

Gly Gly Tyr Thr He Arg Asn Val Ala Arg Cys Trp Thr Tyr Glu Thr 305 310 315 320Gly Gly Tyr Thr He Arg Asn Val Ala Arg Cys Trp Thr Tyr Glu Thr 305 310 315 320

Ala Val Ala Leu Asp Cys Glu lie Pro Asn Glu Leu Pro Tyr Asn Asp 325 330 335Ala Val Ala Leu Asp Cys Glu lie Pro Asn Glu Leu Pro Tyr Asn Asp 325 330 335

Tyr Phe Glu Tyr Phe Gly Pro Asp Phe Lys Leu His lie Ser Pro Ser 340 345 350Tyr Phe Glu Tyr Phe Gly Pro Asp Phe Lys Leu His lie Ser Pro Ser 340 345 350

Asn Met Thr Asn Gin Asn Thr Pro Glu 丁yr Met Glu Lys lie Lys Gin 355 360 365Asn Met Thr Asn Gin Asn Thr Pro Glu Dyr Met Glu Lys lie Lys Gin 355 360 365

Arg Leu Phe Glu Asn Leu Arg Met Leu Pro His Ala Pro Gly Val Gin 370 375 380Arg Leu Phe Glu Asn Leu Arg Met Leu Pro His Ala Pro Gly Val Gin 370 375 380

Met Gin Ala lie Pro Glu Asp Ala Val His Glu Asp Ser Gly Asp Glu 385 390 395 400Met Gin Ala lie Pro Glu Asp Ala Val His Glu Asp Ser Gly Asp Glu 385 390 395 400

Asp Gly Glu Asp Pro Asp Lys Arg lie Ser lie Arg Ala Ser Asp Lys 405 410 415Asp Gly Glu Asp Pro Asp Lys Arg lie Ser lie Arg Ala Ser Asp Lys 405 410 415

Arg He Ala Cys Asp Glu Glu Phe Ser Asp Ser Glu Asp Glu Gly Glu 420 425 430Arg He Ala Cys Asp Glu Glu Phe Ser Asp Ser Glu Asp Glu Gly Glu 420 425 430

Gly Gly Arg Arg Asn Val Ala Asp His Lys Lys Gly Ala Lys Lys Ala 435 440 445Gly Gly Arg Arg Asn Val Ala Asp His Lys Lys Gly Ala Lys Lys Ala 435 440 445

Arg lie Glu Glu Asp Lys Lys Glu Thr Glu Asp Lys Lys Thr Asp Val 450 455 460Arg lie Glu Glu Asp Lys Lys Glu Thr Glu Asp Lys Lys Thr Asp Val 450 455 460

Lys Glu Glu Asp Lys Ser Lys Asp Asn Ser Gly Glu Lys Thr Asp Thr 465 470 475 480Lys Glu Glu Asp Lys Ser Lys Asp Asn Ser Gly Glu Lys Thr Asp Thr 465 470 475 480

Lys Gly Thr Lys Ser Glu Gin Leu Ser Asn Pro Gly His His His His 485 490 495Lys Gly Thr Lys Ser Glu Gin Leu Ser Asn Pro Gly His His His His 485 490 495

His His <210> 4 <211> 1497 <212> DNA <213〉人工 <220>His His <210> 4 <211> 1497 <212> DNA <213>manual <220>

<223>用以編碼HDAC2之殘基1-488及在C端之6-組胺酸標籤的DNA <400> 4 atgggatcca tggcgtacag tcaaggaggc ggcaaaaaaa aagtctgcta ctactacgac 60 ggtgatattg gaaattatta ttatggacag ggtcatccca tgaagcctca tagaatccgc 120 atgacccata acttgctgtt aaattatggc ttatacagaa aaatggaaat atataggccc 180 cataaagcca ctgccgaaga aatgacaaaa tatcacagtg atgagtatat caaatttcta 240 128407.doc 200911757 cggtcaataa gaccagataa catgtctgag tatagtaage agatgeagag atttaatgtt 300 ggagaagatt gtccagtgtt tgatggactc tttgagtttt gtcagctctc aactggcggt 360 tcagttgctg gagctgtgaa gt taaaccga caacagactg atatggctgt taattgggct 420 ggaggattac atcatgctaa gaaatcagaa gcatcaggat tctgt taegt taatgatatt 480 gtgct tgcca tcct tgaat t actaaagtat catcagagag tet tatatat tgatatagat 540 at icatcatg gtgatggtgt tgaagaaget ttttatacaa cagatcgtgt aatgacggta 600 teat tccata aatatgggga atactttcct ggcacaggag acttgaggga tattggtgct 660 ggaaaaggca aatactatgc tgtcaatttt ccaatgagag atggtataga tgatgagtea 720 tatgggcaga tatttaagee tattatetea aaggtgatgg agatgtatca acctagtgct 780 gtggtattac agtgtggtgc agactcatta tctggtgata gactgggttg tttcaatcta 840 acagtcaaag gtcatgctaa atgtgtagaa gttgtaaaaa cttttaactt accattactg 900 atgettggag gaggtggcta cacaatccgt aatgttgctc gatgttggac atatgagact 960 icagtigccc Ugattgtga gattcccaat gagttgccat ataatgatta etttgagtat 1020 tttggaccag acttcaaact gcatat tagt ccttcaaaca tgacaaacca gaacactcca 1080 gaatatatgg aaaagataaa acagcgtttg tttgaaaatt tgcgcatgtt acctcalgca 1140 cctggtgtcc agatgcaagc tattccagaa gatgctgttc atgaagacag tggagatgaa 1200 gatggagaag atccagacaa gagaatttet attegageat cagacaagcg gatagettgt 1260 gatgaagaat tctcagaitc tgaggatgaa ggagaaggag gtcgaagaaa tgtggctgat 1320 cataagaaag gagcaaagaa agetagaatt gaagaagata agaaagaaac agaggacaaa 1380 aaaacagacg t taaggaaga agataaatcc aaggacaaca gtggtgaaaa aacagatacc 1440 aaaggaacca aatcagaaca gctcagcaac cccgggcatc accatcacca tcactaa 1497 <210> 5 <2]1> 782 <212> PRT <213>人工 <220> <223> HDAC6之殘基73-845外加在C端之6-組胺酸標籤及三個選殖人工產物殘基 (在N端之Met-Ριυ對及在6-組胺酸標籤前之Gly)的胺基酸序列 <400> 5<223> DNA <400>4 atgggatcca tggcgtacag tcaaggaggc ggcaaaaaaa aagtctgcta ctactacgac 60 ggtgatattg gaaattatta ttatggacag g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g aaatggaaat atataggccc 180 cataaagcca ctgccgaaga aatgacaaaa tatcacagtg atgagtatat caaatttcta 240 128407.doc 200911757 cggtcaataa gaccagataa catgtctgag tatagtaage agatgeagag atttaatgtt 300 ggagaagatt gtccagtgtt tgatggactc tttgagtttt gtcagctctc aactggcggt 360 tcagttgctg gagctgtgaa gt taaaccga caacagactg atatggctgt taattgggct 420 ggaggattac atcatgctaa gaaatcagaa gcatcaggat tctgt taegt taatgatatt 480 gtgct tgcca tcct tgaat t actaaagtat Catcagagag tet tatatat tgatatagat 540 at icatcatg gtgatggtgt tgaagaaget ttttatacaa cagatcgtgt aatgacggta 600 teat tccata aatatgggga atactttcct ggcacaggag acttgaggga tattggtgct 660 ggaaaaggca aatactatgc tgtcaatttt ccaatgagag atggtataga tgatgagtea 720 tatgggcaga tatttaagee tattatete a aaggtgatgg agatgtatca acctagtgct 780 gtggtattac agtgtggtgc agactcatta tctggtgata gactgggttg tttcaatcta 840 acagtcaaag gtcatgctaa atgtgtagaa gttgtaaaaa cttttaactt accattactg 900 atgettggag gaggtggcta cacaatccgt aatgttgctc gatgttggac atatgagact 960 icagtigccc Ugattgtga gattcccaat gagttgccat ataatgatta etttgagtat 1020 tttggaccag acttcaaact gcatat tagt ccttcaaaca tgacaaacca gaacactcca 1080 gaatatatgg aaaagataaa acagcgtttg tttgaaaatt tgcgcatgtt acctcalgca 1140 cctggtgtcc agatgcaagc tattccagaa gatgctgttc atgaagacag tggagatgaa 1200 gatggagaag atccagacaa gagaatttet attegageat cagacaagcg gatagettgt 1260 gatgaagaat tctcagaitc tgaggatgaa ggagaaggag gtcgaagaaa tgtggctgat 1320 cataagaaag gagcaaagaa agetagaatt gaagaagata agaaagaaac agaggacaaa 1380 aaaacagacg t taaggaaga agataaatcc aaggacaaca gtggtgaaaa aacagatacc 1440 aaaggaacca aatcagaaca gctcagcaac cccgggcatc accatcacca tcactaa 1497 < 210 > 5 < 2] 1> 782 <212> PRT < 213 > Labor <220><223> HDAC6 residues 73-845 plus amino acid sequence at the C-terminal 6-histidine tag and three colonized artifact residues (Met-Ριυ at the N-terminus and Gly before the 6-histidine tag) ;400> 5

Met Pro Gly Met Asp Leu Asn Leu Glu Ala Glu Ala Leu Ala Gly Thr 15 10 15Met Pro Gly Met Asp Leu Asn Leu Glu Ala Glu Ala Leu Ala Gly Thr 15 10 15

Gly Leu Val Leu Asp Glu Gin Leu Asn Glu Phe His Cys Leu Trp Asp 20 25 30Gly Leu Val Leu Asp Glu Gin Leu Asn Glu Phe His Cys Leu Trp Asp 20 25 30

Asp Ser Phe Pro Glu Gly Pro Glu Arg Leu His Ala He Lys Glu Gin 35 40 45Asp Ser Phe Pro Glu Gly Pro Glu Arg Leu His Ala He Lys Glu Gin 35 40 45

Leu I!e Gin Glu Gly Leu Leu Asp Arg Cys Val Ser Phe Gin Ala Arg 50 55 60Leu I!e Gin Glu Gly Leu Leu Asp Arg Cys Val Ser Phe Gin Ala Arg 50 55 60

Phe Ala Glu Lys Glu Glu Leu Met Leu Val His Ser Leu Glu Tyr lie 65 70 75 80 6- 128407.doc 200911757Phe Ala Glu Lys Glu Glu Leu Met Leu Val His Ser Leu Glu Tyr lie 65 70 75 80 6- 128407.doc 200911757

Asp Leu Met Glu Thr Thr Gin Tyr Met Asn Glu Gly Glu Leu Arg Val 85 90 95Asp Leu Met Glu Thr Thr Gin Tyr Met Asn Glu Gly Glu Leu Arg Val 85 90 95

Leu Ala Asp Thr Tyr Asp Ser Val Tyr Leu His Pro Asn Ser Tyr Ser 100 105 110Leu Ala Asp Thr Tyr Asp Ser Val Tyr Leu His Pro Asn Ser Tyr Ser 100 105 110

Cys Ala Cys Leu Ala Ser Gly Ser Val Leu Arg Leu Val Asp Ala Val 115 120 125Cys Ala Cys Leu Ala Ser Gly Ser Val Leu Arg Leu Val Asp Ala Val 115 120 125

Leu Gly Ala Glu He Arg Asn Gly Met Ala lie He Arg Pro Pro Gly 130 135 140Leu Gly Ala Glu He Arg Asn Gly Met Ala lie He Arg Pro Pro Gly 130 135 140

His His Ala Gin His Ser Leu Met Asp Gly Tyr Cys Met Phe Asn His 145 150 155 160His His Ala Gin His Ser Leu Met Asp Gly Tyr Cys Met Phe Asn His 145 150 155 160

Val Ala Val Ala Ala Arg Tyr Ala Gin Gin Lys His Arg lie Arg Arg 165 170 175Val Ala Val Ala Ala Arg Tyr Ala Gin Gin Lys His Arg lie Arg Arg 165 170 175

Val Leu lie Val Asp Trp Asp Val His His Gly Gin Gly Thr Gin Phe 180 185 190Val Leu lie Val Asp Trp Asp Val His His Gly Gin Gly Thr Gin Phe 180 185 190

Thr Phe Asp Gin Asp Pro Ser Val Leu Tyr Phe Ser lie His Arg Tyr 195 200 205Thr Phe Asp Gin Asp Pro Ser Val Leu Tyr Phe Ser lie His Arg Tyr 195 200 205

Glu Gin Gly Arg Phe Trp Pro His Leu Lys Ala Ser Asn Trp Ser Thr 210 215 220Glu Gin Gly Arg Phe Trp Pro His Leu Lys Ala Ser Asn Trp Ser Thr 210 215 220

Thr Gly Phe Gly Gin Gly Gin Gly Tyr Thr lie Asn Val Pro Trp Asn 225 230 235 240Thr Gly Ghe Gly Gin Gin Gin Gly Tyr Thr lie Asn Val Pro Trp Asn 225 230 235 240

Gin Val Gly Met Arg Asp Ala Asp Tyr lie Ala Ala Phe Leu His Val 245 250 255Gin Val Gly Met Arg Asp Ala Asp Tyr lie Ala Ala Phe Leu His Val 245 250 255

Leu Leu Pro Val Ala Leu Glu Phe Gin Pro Gin Leu Val Leu Val Ala 260 265 270Leu Leu Pro Val Ala Leu Glu Phe Gin Pro Gin Leu Val Leu Val Ala 260 265 270

Ala Gly Phe Asp Ala Leu Gin Gly Asp Pro Lys Gly Glu Met Ala Ala 275 280 285Ala Gly Phe Asp Ala Leu Gin Gly Asp Pro Lys Gly Glu Met Ala Ala 275 280 285

Thr Pro Ala Gly Phe Ala Gin Leu Thr His Leu Leu Met Gly Leu Ala 290 295 300Thr Pro Ala Gly Phe Ala Gin Leu Thr His Leu Leu Met Gly Leu Ala 290 295 300

Gly Gly Lys Leu lie Leu Ser Leu Glu Gly Gly Tyr Asn Leu Arg Ala 305 310 315 320Gly Gly Lys Leu lie Leu Ser Leu Glu Gly Gly Tyr Asn Leu Arg Ala 305 310 315 320

Leu Ala Glu Gly Val Ser Ala Ser Leu His Thr Leu Leu Gly Asp Pro 325 330 335Leu Ala Glu Gly Val Ser Ala Ser Leu His Thr Leu Leu Gly Asp Pro 325 330 335

Cys Pro Met Leu Glu Ser Pro Gly Ala Pro Cys Arg Ser Ala Gin Ala 340 345 350Cys Pro Met Leu Glu Ser Pro Gly Ala Pro Cys Arg Ser Ala Gin Ala 340 345 350

Ser Val Ser Cys Ala Leu Glu Ala Leu Glu Pro Phe Trp Glu Val Leu 355 360 365Ser Val Ser Cys Ala Leu Glu Ala Leu Glu Pro Phe Trp Glu Val Leu 355 360 365

Val Arg Ser Thr Glu Thr Val Glu Arg Asp Asn Met Glu Glu Asp Asn 128407.doc 200911757 370 375 380Val Arg Ser Thr Glu Thr Val Glu Arg Asp Asn Met Glu Glu Asp Asn 128407.doc 200911757 370 375 380

Val Glu Glu Ser Glu Glu Glu Gly Pro Trp Glu Pro Pro Val Leu Pro 385 390 395 400 lie Leu Thr Trp Pro Val Leu Gin Ser Arg Thr Gly Leu Val Tyr Asp 405 410 415Val Glu Glu Ser Glu Glu Glu Gly Pro Trp Glu Pro Pro Val Leu Pro 385 390 395 400 lie Leu Thr Trp Pro Val Leu Gin Ser Arg Thr Gly Leu Val Tyr Asp 405 410 415

Gin Asn Met Met Asn His Cys Asn Leu Trp Asp Ser His His Pro Glu 420 425 430Gin Asn Met Met Asn His Cys Asn Leu Trp Asp Ser His His Pro Glu 420 425 430

Val Pro Gin Arg lie Leu Arg lie Met Cys Arg Leu Glu Glu Leu Gly 435 440 445Val Pro Gin Arg lie Leu Arg lie Met Cys Arg Leu Glu Glu Leu Gly 435 440 445

Leu Ala Gly Arg Cys Leu Thr Leu Thr Pro Arg Pro Ala Thr Glu Ala 450 455 460Leu Ala Gly Arg Cys Leu Thr Leu Thr Pro Arg Pro Ala Thr Glu Ala 450 455 460

Glu Leu Leu Thr Cys His Ser Ala Glu Tyr Val Gly His Leu Arg Ala 465 470 475 480Glu Leu Leu Thr Cys His Ser Ala Glu Tyr Val Gly His Leu Arg Ala 465 470 475 480

Thr Glu Lys Met Lys Thr Arg Glu Leu His Arg Glu Ser Ser Asn Phe 485 490 495Thr Glu Lys Met Lys Thr Arg Glu Leu His Arg Glu Ser Ser Asn Phe 485 490 495

Asp Scr lie Tyr lie Cys Pro Ser Thr Phe Ala Cys Ala Gin Leu Ala 500 505 510Asp Scr lie Tyr lie Cys Pro Ser Thr Phe Ala Cys Ala Gin Leu Ala 500 505 510

Thr Gly Ala Ala Cys Arg Leu Val Glu Ala Va! Leu Ser Gly Glu Val 5] 5 520 525Thr Gly Ala Ala Cys Arg Leu Val Glu Ala Va! Leu Ser Gly Glu Val 5] 5 520 525

Leu Asn Gly Ala Ala Val Val Arg Pro Pro Gly His His Ala Glu Gin 530 535 540Leu Asn Gly Ala Ala Val Val Arg Pro Pro Gly His His Ala Glu Gin 530 535 540

Asp Ala Ala Cys Gly Phe Cys Phe Phe Asn Ser Val Ala Val Ala Ala 545 550 555 560Asp Ala Ala Cys Gly Phe Cys Phe Phe Asn Ser Val Ala Val Ala Ala 545 550 555 560

Arg His Ala Gin Thr lie Ser Gly His Ala Leu Arg lie Leu lie Val 565 570 575 \sp Trp Asp Val His His Gly Asn Gly Thr Gin His Met Phe Glu Asp 580 585 590Arg His Ala Gin Thr lie Ser Gly His Ala Leu Arg lie Leu lie Val 565 570 575 \sp Trp Asp Val His His Gly Asn Gly Thr Gin His Met Phe Glu Asp 580 585 590

Asp Pro Ser Val Leu Tyr Val Ser Leu His Arg Tyr Asp His Gly Thr 595 600 605Asp Pro Ser Val Leu Tyr Val Ser Leu His Arg Tyr Asp His Gly Thr 595 600 605

Phe Phe Pro Met Gly Asp Glu Gly Aia Ser Ser Gin lie Gly Arg Ala 610 615 620Phe Phe Pro Met Gly Asp Glu Gly Aia Ser Ser Gin lie Gly Arg Ala 610 615 620

Ala Gly Thr Gly Phe Thr Val Asn Val Ala Trp Asn Gly Pro Arg Met 625 630 635 640Ala Gly Thr Gly Phe Thr Val Asn Val Ala Trp Asn Gly Pro Arg Met 625 630 635 640

Gly Asp Ala Asp Tyr Leu Ala Ala Trp His Arg Leu Val Leu Pro lie 645 650 655Gly Asp Ala Asp Tyr Leu Ala Ala Trp His Arg Leu Val Leu Pro lie 645 650 655

Ala Tyr Glu Phe Asn Pro Glu Leu Val Leu Val Ser Ala Gly Phe Asp 660 665 670 128407.doc 200911757Ala Tyr Glu Phe Asn Pro Glu Leu Val Leu Val Ser Ala Gly Phe Asp 660 665 670 128407.doc 200911757

AlaAlaAr.CAspPro Leu Gly Gly Cys G!n Val Ser Pro Glu G.yAlaAlaAr.CAspPro Leu Gly Gly Cys G!n Val Ser Pro Glu G.y

Tyr Ala His Leu Thr His Leu Leu Met Gly Leu Ala Ser Gly Are Up 690 695 700 K lie Leu He Leu Glu Gly Gly Tyr Asn Leu Thr Ser He Ser Glu Ser 705 710 715 7¾Tyr Ala His Leu Thr His Leu Leu Met Gly Leu Ala Ser Gly Are Up 690 695 700 K lie Leu He Leu Glu Gly Gly Tyr Asn Leu Thr Ser He Ser Glu Ser 705 710 715 73⁄4

Met Ala Ala Cys Thr Arg Ser Leu Leu Gly Asp Pro Pro Pro Leu Leu 725 730 735Met Ala Ala Cys Thr Arg Ser Leu Leu Gly Asp Pro Pro Pro Leu Leu 725 730 735

Thr Leu Pro Arg Pro Pro Leu Ser Gly Ala Leu Ala Scr lie Thr Glu 740 745 750Thr Leu Pro Arg Pro Pro Leu Ser Gly Ala Leu Ala Scr lie Thr Glu 740 745 750

Thr lie Gin Val His Arg Arg Tyr Trp Arg Ser Leu Arg Val Met Lvs 755 760 765Thr lie Gin Val His Arg Arg Tyr Trp Arg Ser Leu Arg Val Met Lvs 755 760 765

Val Glu Asp Arg Glu Gly Pro Gly His His His His His His 770 775 780 <210> 6 <211> 2349 <212> DNA <213>人工 <220> <223>用以編碼HDAC6之殘基73-845及在C端之6-組胺酸標籤的DNA序列 <400〉 6 atgcccggga tggatctgaa ccttgaggct gaagcactgg ctggcactgg cttggtgttg 60 gatgagcagt taaatgaatt ccattgcctc tgggatgaca gcttcccgga aggccctgag 120 cggctccatg ccatcaagga gcaactgatc caggagggcc tcctagatcg ctgcgtgtcc 180 11 tcaggccc ggtttgctga aaaggaagag ctgatgttgg ttcacagcct agaatatatt 240 gatctgatgg aaacaaccca gtacatgaaV gagggagaac tccgtgtcct agcagacacc 300 tacgactcag tttatctgca tccgaactca tactcctgtg cctgcctggc ctcaggctct 360 gtcctcaggc tggtggatgc ggtcctgggg gctgagatcc ggaatggcat ggccatcatt 420 aggcctcctg gacatcacgc ccagcacagt cttatggatg gctattgcat gttcaaccac 480 gtggctgtgg cagcccgcta tgctcaacag aaacaccgca tccggagggt ccttatcgta 540 gattgggatg tgcaccacgg tcaaggaaca cagttcacci tcgaccagga ccccagtgtc 600 ctctatttct ccatccaccg ctacgagcag ggtaggttct ggccccacct gaaggccict 660 aactggtcca ccacaggtit cggccaaggc caaggatata ccatcaatgt gccttggaac 720 caggtgggga tgcgggaigc tgactacatt gctgct ttcc tgcacgtcct gctgccagtc 780 gccctcgagt tccagcctca gctggtcctg gtggctgctg gatttgatgc cctgcaaggg 840 gaccccaagg gtgagatggc cgccactccg gcagggttcg cccagctaac ccacctgctc 900 atgggtctgg caggaggcaa gctgatcctg tctctggagg gtggctacaa cctccgcgcc 960 ctggctgaag gcgtcagtgc t tcgctccac acccttctgg gagacccttg ccccatgctg 1020 gagicacctg gtgccccctg ccggagtgcc caggcttcag tttcctgtgc tctggaagcc 1080 cttgagccct tctgggaggt tcttgtgaga tcaactgaga ccgtggagag ggacaacatg 1140 128407.doc 200911757 gaggaggaca atgtagagga gagcgaggag gaaggaccct gggagccccc tgtgctccca 1200 atcctgacat ggccagtgct acagtctcgc acagggctgg tctatgacca aaatatgatg 1260 aatcactgca acttgtggga cagccaccac cctgaggtac cccagcgcat cttgcggatc 1320 atgtgccgtc tggaggagct gggccttgcc gggcgctgcc tcaccctgac accgcgccct 1380 gccacagagg ctgagctgct caccigtcac agtgctgagt acgtgggtca tctccgggcc 1440 acagagaaaa tgaaaacccg ggagctgcac cgtgagagtt ccaactttga ctccatctat 1500 atctgcccca gtaccttcgc ctgtgcacag cttgccactg gcgctgcctg ccgcctggtg 1560 gaggctgtgc tctcaggaga ggttctgaat ggtgctgctg tggtgcgtcc cccaggacac 1620 cacgcagagc aggaigcagc ttgcggtttt tgctttttca actctgtggc tgtggctgct 1680 cgccatgccc agactatcag tgggcatgcc ctacggatcc tgattgtgga ttgggatgtc 1740 caccacggta atggaactca gcacatgttt gaggatgacc ccagtgtgct atatgtgtcc 1800 ctgcaccgct atgatcatgg caccttcttc cccatggggg atgagggtgc cagcagccag 1860 ucggccggg ctgcgggcac aggcttcacc gtcaacgtgg catggaacgg gccccgcatg 1920 〆 ggtgaigctg actacctagc tgcctggcat cgcctggtgc itcccattgc ctacgagttt 1980 aacccagaac tggtgctggt ctcagctggc tttgatgctg cacgggggga tccgctgggg 2040 ggctgccagg tgtcacctga gggttatgcc cacctcaccc acctgctgat gggccttgcc 2100 agtggccgca tlatccttat cctagagggt ggctataacc tgacatccat ctcagagtcc 2160 atggctgcct gcactcgctc cctccttgga gacccaccac ccctgctgac cctgccacgg 2220 cccccactat caggggccct ggcctcaatc actgagacca tccaagtcca tcgcagatac 2280 tggcgcagct tacgggtcat gaaggtagaa gacagagaag gacccgggca tcaccatcac 2340 catcactaa 2349 <210> 7 <211> 385 <212> PRT <213>人工 <220> <2B> HDAC8之殘基卜377外加在N端之6-組胺酸標籤及兩個選殖人工 / 產物殘基（在6-組胺酸標籤之前的Met及之後的Pro)的胺基酸序列 <400> 7Val Glu Asp Arg Glu Gly Pro Gly His His His His His His 770 775 780 <210> 6 <211> 2349 <212> DNA <213>Labor<220><223> used to encode HDAC6 residues 73-845 DNA sequence and 6- histidine tag of the C-terminus < 400> 6 atgcccggga tggatctgaa ccttgaggct gaagcactgg ctggcactgg cttggtgttg 60 gatgagcagt taaatgaatt ccattgcctc tgggatgaca gcttcccgga aggccctgag 120 cggctccatg ccatcaagga gcaactgatc caggagggcc tcctagatcg ctgcgtgtcc 180 11 tcaggccc ggtttgctga aaaggaagag ctgatgttgg ttcacagcct agaatatatt 240 gatctgatgg aaacaaccca gtacatgaaV gagggagaac tccgtgtcct agcagacacc 300 tacgactcag tttatctgca tccgaactca tactcctgtg cctgcctggc ctcaggctct 360 gtcctcaggc tggtggatgc ggtcctgggg gctgagatcc ggaatggcat ggccatcatt 420 aggcctcctg gacatcacgc ccagcacagt cttatggatg gctattgcat gttcaaccac 480 gtggctgtgg cagcccgcta tgctcaacag aaacaccgca tccggagggt ccttatcgta 540 gattgggatg tgcaccacgg tcaaggaaca cagttcacci tcgaccagga ccccagtgtc 600 ctctatttct ccatccaccg ctacgagcag ggtaggttct gg ccccacct gaaggccict 660 aactggtcca ccacaggtit cggccaaggc caaggatata ccatcaatgt gccttggaac 720 caggtgggga tgcgggaigc tgactacatt gctgct ttcc tgcacgtcct gctgccagtc 780 gccctcgagt tccagcctca gctggtcctg gtggctgctg gatttgatgc cctgcaaggg 840 gaccccaagg gtgagatggc cgccactccg gcagggttcg cccagctaac ccacctgctc 900 atgggtctgg caggaggcaa gctgatcctg tctctggagg gtggctacaa cctccgcgcc 960 ctggctgaag gcgtcagtgc t tcgctccac acccttctgg gagacccttg ccccatgctg 1020 gagicacctg gtgccccctg ccggagtgcc caggcttcag tttcctgtgc tctggaagcc 1080 cttgagccct tctgggaggt tcttgtgaga tcaactgaga ccgtggagag ggacaacatg 1140 128407.doc 200911757 gaggaggaca atgtagagga gagcgaggag gaaggaccct gggagccccc tgtgctccca 1200 atcctgacat ggccagtgct acagtctcgc acagggctgg tctatgacca aaatatgatg 1260 aatcactgca acttgtggga cagccaccac cctgaggtac cccagcgcat cttgcggatc 1320 atgtgccgtc tggaggagct gggccttgcc gggcgctgcc tcaccctgac accgcgccct 1380 gccacagagg ctgagctgct caccigtcac agtgctgagt acgtgggtca tctccgggcc 1440 Acagagaaaa tgaaaacccg ggagc tgcac cgtgagagtt ccaactttga ctccatctat 1500 atctgcccca gtaccttcgc ctgtgcacag cttgccactg gcgctgcctg ccgcctggtg 1560 gaggctgtgc tctcaggaga ggttctgaat ggtgctgctg tggtgcgtcc cccaggacac 1620 cacgcagagc aggaigcagc ttgcggtttt tgctttttca actctgtggc tgtggctgct 1680 cgccatgccc agactatcag tgggcatgcc ctacggatcc tgattgtgga ttgggatgtc 1740 caccacggta atggaactca gcacatgttt gaggatgacc ccagtgtgct atatgtgtcc 1800 ctgcaccgct atgatcatgg caccttcttc cccatggggg atgagggtgc cagcagccag 1860 ucggccggg ctgcgggcac aggcttcacc gtcaacgtgg catggaacgg gccccgcatg 1920 〆ggtgaigctg actacctagc tgcctggcat cgcctggtgc itcccattgc ctacgagttt 1980 aacccagaac tggtgctggt ctcagctggc tttgatgctg cacgggggga tccgctgggg 2040 ggctgccagg tgtcacctga gggttatgcc cacctcaccc acctgctgat gggccttgcc 2100 agtggccgca tlatccttat cctagagggt ggctataacc tgacatccat ctcagagtcc 2160 atggctgcct gcactcgctc cctccttgga gacccaccac ccctgctgac cctgccacgg 2220 cccccactat caggggccct ggcctcaatc actgagacca tccaagtcca tcgcagatac 2280 tggcgcagct tacgggtcat gaaggtag Aa gacagagaag gacccgggca tcaccatcac 2340 catcactaa 2349 <210> 7 <211> 385 <212> PRT <213>Manual<220><2B> HDAC8 residue 377 plus N-terminal 6-histamine Amino acid sequence of the acid tag and two selected artificial/product residues (Met before the 6-histidine tag and subsequent Pro) <400>

Met His His His His His His Pro Met Glu GIu Pro Glu Glu Pro Ala 15 10 15Met His His His His His His Pro Met Glu GIu Pro Glu Glu Pro Ala 15 10 15

Asp Ser Gly Gin Ser Leu Val Pro Val Tyr lie Tyr Ser Pro Glu Tyr 2〇 25 30Asp Ser Gly Gin Ser Leu Val Pro Val Tyr lie Tyr Ser Pro Glu Tyr 2〇 25 30

Val Ser Met Cys Asp Ser Leu Ala Lys lie Pro Lys Arg Ala Ser Met 35 40 45Val Ser Met Cys Asp Ser Leu Ala Lys lie Pro Lys Arg Ala Ser Met 35 40 45

Val His Ser Leu lie Glu Ala Tyr Ala Leu His Lys Gin Met Arg lie 50 55 60Val His Ser Leu lie Glu Ala Tyr Ala Leu His Lys Gin Met Arg lie 50 55 60

Val Lys Pro Lys Val A,a Ser Met G,u G,u Met AU A,a Phe H,s Thr • 10· 128407.doc 200911757Val Lys Pro Lys Val A, a Ser Met G, u G, u Met AU A, a Phe H, s Thr • 10· 128407.doc 200911757

Asp Ala Tyr Leu Gin His Leu Gin Lys Val Ser Gin Glu Gly Asp Asp 85 90 95Asp Ala Tyr Leu Gin His Leu Gin Lys Val Ser Gin Glu Gly Asp Asp 85 90 95

Asp His Pro Asp Ser lie Glu Tyr Gly Leu Gly Tyr Asp Cys Pro Ala 100 105 110Asp His Pro Asp Ser lie Glu Tyr Gly Leu Gly Tyr Asp Cys Pro Ala 100 105 110

Thr Glu Gly lie Phe Asp Tyr Ala Ala Ala lie Gly Gly Ala Thr lie 115 120 125Thr Glu Gly lie Phe Asp Tyr Ala Ala Ala lie Gly Gly Ala Thr lie 115 120 125

Thr Ala Ala Gin Cys Leu lie Asp Gly Met Cys Lys Val Ala lie Asn 130 135 140Thr Ala Ala Gin Cys Leu lie Asp Gly Met Cys Lys Val Ala lie Asn 130 135 140

Trp Ser Gly Gly Trp His His Ala Lys Lys Asp Glu Ala Ser Gly Phe 145 150 155 160Trp Ser Gly Gly Trp His His Ala Lys Lys Asp Glu Ala Ser Gly Phe 145 150 155 160

Cys Tyr Leu Asn Asp Ala Va! Leu Gly lie Leu Arg Leu Arg Arg Lys 165 170 175 °he Glu Arg lie Leu Tyr Val Asp Leu Asp Leu His His Gly Asp Gly 180 185 190Cys Tyr Leu Asn Asp Ala Va! Leu Gly lie Leu Arg Leu Arg Arg Lys 165 170 175 °he Glu Arg lie Leu Tyr Val Asp Leu Asp Leu His His Gly Asp Gly 180 185 190

Val Glu Asp Ala Phe Ser Phe Thr Ser Lys Val Met Thr Val Ser Leu 195 200 205Val Glu Asp Ala Phe Ser Phe Thr Ser Lys Val Met Thr Val Ser Leu 195 200 205

His Lys Phe Ser Pro Gly Phe Phe Pro Gly Thr Gly Asp Val Ser Asp 210 215 220His Lys Phe Ser Pro Gly Phe Phe Pro Gly Thr Gly Asp Val Ser Asp 210 215 220

Val Gly Leu Gly Lys Gly Arg Tyr Tyr Ser Val Asn Val Pro He Gin 225 230 235 240Val Gly Leu Gly Lys Gly Arg Tyr Tyr Ser Val Asn Val Pro He Gin 225 230 235 240

Asp Gly lie Gin Asp Glu Lys Tyr Tyr Gin lie Cys Glu Ser Val Leu 245 250 255Asp Gly lie Gin Asp Glu Lys Tyr Tyr Gin lie Cys Glu Ser Val Leu 245 250 255

Lys Glu Val Tyr Gin Ala Phe Asn Pro Lys Ala Val Val Leu Gin Leu 260 265 270Lys Glu Val Tyr Gin Ala Phe Asn Pro Lys Ala Val Val Leu Gin Leu 260 265 270

Gly Ala Asp Thr lie Ala Gly Asp Pro Met Cys Ser Phe Asn Met Thr 275 280 285Gly Ala Asp Thr lie Ala Gly Asp Pro Met Cys Ser Phe Asn Met Thr 275 280 285

Pro Val Gly lie Gly Lys Cys Leu Lys Tyr lie Leu Gin 丁rp Gin Leu 290 295 300Pro Val Gly lie Gly Lys Cys Leu Lys Tyr lie Leu Gin Ding rp Gin Leu 290 295 300

Ala Thr Leu He Leu Gly Gly Gly Gly Tyr Asn Leu Ala Asn Thr Ala 305 310 325 320Ala Thr Leu He Leu Gly Gly Gly Gly Tyr Asn Leu Ala Asn Thr Ala 305 310 325 320

Arg Cys Trp Thr 丁yr Leu Thr Gly Val lie Leu Gly Lys Thr Leu Ser 325 330 335Arg Cys Trp Thr Dinger Leu Thr Gly Val lie Leu Gly Lys Thr Leu Ser 325 330 335

Ser Glu lie Pro Asp His Glu Phe Phe Thr Ala Tyr Gly Pro Asp Tyr 340 345 350Ser Glu lie Pro Asp His Glu Phe Phe Thr Ala Tyr Gly Pro Asp Tyr 340 345 350

Val Leu Glu lie Thr Pro Ser Cys Arg Pro Asp Arg Asn Glu Pro His 355 360 365Val Leu Glu lie Thr Pro Ser Cys Arg Pro Asp Arg Asn Glu Pro His 355 360 365

Arg lie Gin Gin He Leu Asn Tyr lie Lys Gly Asn Leu Lys His Val 370 375 380 -11 - 128407.doc 200911757Arg lie Gin Gin He Leu Asn Tyr lie Lys Gly Asn Leu Lys His Val 370 375 380 -11 - 128407.doc 200911757

Val 385 <210> 8 <2Π> 1158 <212> DNA <213> 乂工 <220〉 <223>用以編碼HDAC8之殘基1-377及在N端之6-組胺酸標籤 <400> 8 atgcaccatc accatcacca tcccatggag gagccggagg aaccggcgga cagtgggcag 60 tcgctggtcc cggtttatat ctatagtccc gagtatgtca gtatgtgtga ctccctggcc 120 aagatcccca aacgggccag tatggtgcat tctttgattg aagcatatgc actgcataag 180 cagatgagga tagttaagcc taaagtggcc tccatggagg agatggccgc cttccacact 240 gatgcttatc tgcagcatct ccagaaggtc agccaagagg gcgatgatga tcatccggac 300 tccatagaat atgggctagg t tatgactgc ccagccactg aagggatatt tgactatgca 360 gcagctatag gaggggctac gatcacagct gcccaatgcc tgattgacgg aatgtgcaaa 420 gtagcaatta actggtctgg agggtggcat catgcaaaga aagatgaagc atctggtttt 480 tgt tatctca atgatgctgt cctgggaata ttacgattgc gacggaaatt tgagcgtatt 540 ctctacgtgg atttggatct gcaccatgga gatggtgtag aagacgcatt cagt t tcacc 600 iccaaagtca tgaccgtgic cctgcacaaa ttctccccag gatttttccc aggaacaggt 660 gacgtgtctg atgttggcct agggaaggga cggtactaca gtgtaaatgt gcccattcag 720 gatggcatac aagatgaaaa atat taccag atctgtgaaa gtgtactaaa ggaagtatac 780 caagcctt ta atcccaaagc agtggtctta cagctgggag ctgacacaat agctggggat 840 cccatgigct cctttaacat gactccagtg ggaattggca agtgtcttaa gtacatcctt 900 caaiggcagt tggcaacact cattttggga ggaggaggct ataaccttgc caacacggct 960 cgatgctgga catacttgac cggggtcatc ctagggaaaa cactatcctc tgagatccca 1020 gatcatgagt ttttcacagc atatggtcct gattatgtgc tggaaatcac gccaagctgc 1080 cggccagacc gcaatgagcc ccaccgaatc caacaaatcc tcaactacat caaagggaat 1140 ctgaagcatg tggtctag 1158 的DNA序列 12- 128407.docVal 385 <210> 8 <2Π> 1158 <212> DNA <213> Completion <220><223> used to encode residues 1-377 of HDAC8 and 6-histamine at the N-terminus acid tag < 400 > 8 atgcaccatc accatcacca tcccatggag gagccggagg aaccggcgga cagtgggcag 60 tcgctggtcc cggtttatat ctatagtccc gagtatgtca gtatgtgtga ctccctggcc 120 aagatcccca aacgggccag tatggtgcat tctttgattg aagcatatgc actgcataag 180 cagatgagga tagttaagcc taaagtggcc tccatggagg agatggccgc cttccacact 240 gatgcttatc tgcagcatct ccagaaggtc agccaagagg gcgatgatga tcatccggac 300 tccatagaat atgggctagg t tatgactgc ccagccactg aagggatatt tgactatgca 360 gcagctatag gaggggctac gatcacagct gcccaatgcc tgattgacgg aatgtgcaaa 420 gtagcaatta actggtctgg agggtggcat catgcaaaga aagatgaagc atctggtttt 480 tgt tatctca atgatgctgt cctgggaata ttacgattgc gacggaaatt tgagcgtatt 540 ctctacgtgg atttggatct gcaccatgga gatggtgtag aagacgcatt cagt t tcacc 600 iccaaagtca tgaccgtgic cctgcacaaa ttctccccag gatttttccc aggaacaggt 660 gacgtgtctg atgttggcct agggaaggga cggtactaca gtgtaaatgt gcccattcag 720 gatggcatac aagatgaaaa atat taccag atctgtgaaa gtgtactaaa ggaagtatac 780 caagcctt ta atcccaaagc agtggtctta cagctgggag ctgacacaat agctggggat 840 cccatgigct cctttaacat gactccagtg ggaattggca agtgtcttaa gtacatcctt 900 caaiggcagt tggcaacact cattttggga ggaggaggct ataaccttgc caacacggct 960 cgatgctgga catacttgac cggggtcatc ctagggaaaa cactatcctc tgagatccca 1020 gatcatgagt ttttcacagc atatggtcct gattatgtgc tggaaatcac gccaagctgc 1080 cggccagacc gcaatgagcc ccaccgaatc Caacaaatcc tcaactacat caaagggaat 1140 ctgaagcatg tggtctag 1158 DNA sequence 12-128407.doc

Claims

200911757 十、申請專利範圍： 1. -種具有下式之化合物之形式A :200911757 X. Patent application scope: 1. Form A of a compound having the following formula:

自由以下所組成之群的或其中形式A之特徵在於選多種物理特性： ⑴ X射線粉末繞射圖案（CuKct)包含在約8 64、 19.95及18_17度2Θ(°2Θ)之繞射峰； (11)如圖2中所示之1η NMR光譜； (in)如圖3中所示之fTIr光譜； (1V)具有集中在約89。〇之寬吸熱及在約198。(：之第二吸熱的示差掃描熱量測定圖譜；及 ⑺顯示在以下重量損失為約8.4%的TGA熱分析圖。一種具有下式之化合物之形式B :The group consisting of or consisting of the following is characterized by a plurality of physical properties: (1) the X-ray powder diffraction pattern (CuKct) contains diffraction peaks at about 8 64, 19.95 and 18-17 degrees 2 Θ (° 2 Θ); 11) 1η NMR spectrum as shown in Fig. 2; (in) fTIr spectrum as shown in Fig. 3; (1V) having a concentration of about 89. The wide heat absorption of the 〇 is at about 198. (: the second endothermic differential scanning calorimetry map; and (7) a TGA thermogram showing a weight loss of about 8.4%. A form B of a compound having the formula:

組成之群的一或多其中形式B之特徵在於選自由以下種物理特性： ⑴ X射線粉末繞射圖案（CuKa)包含在約1721 20.4及16.02度2θ(〇2θ)之繞射峰； (ii) 如圖8中所示之1η NMR光譜； 128407.doc 200911757 (III) 如圖13中所示之FTIR光譜； (IV) 具有集中在約21之吸熱的示差掃描熱量測定圖譜；及 () 在120 C以下無重量損失之TGA熱分析圖。 3. 種具有下式之化合物之反丁烯二酸鹽： ~~〇One or more of the constituent groups wherein Form B is characterized by being selected from the following physical properties: (1) X-ray powder diffraction pattern (CuKa) comprising diffraction peaks at about 1721 20.4 and 16.02 degrees 2θ (〇 2θ); 1 NMR spectrum as shown in Figure 8; 128407.doc 200911757 (III) FTIR spectrum as shown in Figure 13; (IV) differential scanning calorimetry map with endotherms concentrated at about 21; and () TGA thermogram without weight loss below 120 C. 3. A compound of the formula: fumarate: ~~〇

其中的一或 ⑴ (Π) (iii) (iv) (v) 4. 一種具有下式之化合物之氫氯酸鹽：該反丁稀二酸鹽之特徵在於選自由以下組成之群多種物理特性： x射線粉末繞射圖案（CuKa)包含在約5 39、 i3.24及18.19度2Θ(°2Θ)之繞射峰；如圖19中所示之1H NMR光譜；如圖20中所示之ftir光譜；顯示在120°C以下重量損失為2.5%及在約12<Γ(：分解起始的TGA熱分析圖；及具有集中在約128°C吸熱的示差掃描熱量測定圖譜0One or (1) (Π) (iii) (iv) (v) 4. A hydrochloride salt of a compound having the formula: the anti-succinic acid salt is characterized by a plurality of physical properties selected from the group consisting of : The x-ray powder diffraction pattern (CuKa) contains diffraction peaks at about 5 39, i3.24, and 18.19 degrees 2 Θ (° 2 Θ); 1H NMR spectrum as shown in FIG. 19; Ftir spectrum; shows a weight loss of 2.5% below 120 ° C and a TGA thermogram at about 12 Γ (: decomposition initiation; and a differential scanning calorimetry map with endotherms concentrated at about 128 ° C.

其中該氫氣酸鹽之特徵在於選自由以下組成之群的一或多種物理特性： (i) x射線粉末繞射圖案（CuKa)包含在約15.48、 128407.doc 200911757 23.21及16.〇4度20(。20)之繞射峰； (Π) 如圖25中所示之1η NMR光譜； (iii) 如圖26中所示之FTIR ATR光譜； (iY)顯示在UOt以下重量損失為4.1%及在約16〇。〇分解起始的TGA熱分析圖；及 (v) 具有兩個集中在約95°C及185°C之吸熱事件的示差掃描熱量測定圖譜。一種具有下式之化合物之磷酸鹽：Wherein the hydrogen acid salt is characterized by one or more physical properties selected from the group consisting of: (i) an x-ray powder diffraction pattern (CuKa) comprised at about 15.48, 128407.doc 200911757 23.21 and 16. 4 degrees 20 (.20) diffraction peak; (Π) 1η NMR spectrum as shown in Figure 25; (iii) FTIR ATR spectrum as shown in Figure 26; (iY) shows a weight loss of 4.1% below UOt and At about 16 baht. T TGA thermal analysis of the initial decomposition; and (v) a differential scanning calorimetry map with two endothermic events centered at approximately 95 ° C and 185 ° C. a phosphate of a compound of the formula:

其中該磷酸鹽之特徵在於選自由以下組成之群的一或多種物理特性： ⑴ X射線粉末繞射圖案（CuKa)包含在約17 8〇、 25.80及21.88度2Θ(°2Θ)之繞射峰； (ii) 如圖31中所示之1H NMR光譜； (出）具有集中在約179°C之放熱事件及在256°c之吸熱事件的示差掃描熱量測定圖譜；及 (1V)顯示在180〇C以下重量損失為2·〇〇/。及在約i27°C 分解起始的TGA熱分析圖。 6. 一種具有下式之化合物之形式a :Wherein the phosphate is characterized by one or more physical properties selected from the group consisting of: (1) an X-ray powder diffraction pattern (CuKa) comprising diffraction peaks at about 17 8 , 25.80 and 21.88 degrees 2 Θ (° 2 Θ) (ii) 1H NMR spectrum as shown in Figure 31; (out) a differential scanning calorimetry map with an exothermic event centered at about 179 °C and an endothermic event at 256 °C; and (1V) shown at 180 The weight loss below 〇C is 2·〇〇/. And a TGA thermogram at the beginning of decomposition at about i27 °C. 6. A form of a compound having the formula:

Ο 128407.doc 200911757 其中形式A之特徵在於選自由以下組成〜坪的一或多種物理特性： ⑴ X射線粉末繞射圖案（CuKa)包含A & 0 仅約 20.92、 17.54及2〇.36度20(。20)之繞射峰； (ϋ) 如圖36中所示之1η NMR光譜； (iii) 如圖37中所示之FTIR光譜； (iv) 具有兩個集中在約77。(：及在179。〇之吸熱事件的示差掃描熱量測定圖譜；及 (v) 顯示在loot以下之溫度重量損失為約的 TGA熱分析圖。一種具有下式之化合物之丁二酸鹽： H f~\ \=/ •丁二酸鹽 ^NH NH2 0 其中該丁二酸鹽之特徵在於選自由以下組成之群的一或多種物理特性： ⑴ χ射線粉末繞射圖案（CuKa)包含在約2217、 27.13及2〇.94度20(。20)之繞射峰； (ϋ) 如圖42中所示之1H NMR光譜； (iii) 如圖43中所示之FTIR光譜； (iv) 具有集中在約152°C之吸熱事件及在212°C之放熱事件的示差掃描熱量測定圖譜；及 (v) 顯示在150¾以下無重量損失及在約154°C分解 128407.doc 200911757 起始的TGA熱分析圖。 8.種具有下式之化合物之苯曱酸鹽：Ο 128407.doc 200911757 wherein Form A is characterized by one or more physical properties selected from the group consisting of: (1) X-ray powder diffraction pattern (CuKa) comprising A & 0 only about 20.92, 17.54 and 2〇.36 degrees 20 (. 20) diffraction peak; (ϋ) 1 η NMR spectrum as shown in Fig. 36; (iii) FTIR spectrum as shown in Fig. 37; (iv) having two concentrations of about 77. (: and the differential scanning calorimetry map of the endothermic event at 179.) and (v) the TGA thermogram showing the temperature loss below about the loot. A succinate salt of the compound of the formula: H f~\ \=/ • succinate ^NH NH2 0 wherein the succinate is characterized by one or more physical properties selected from the group consisting of: (1) a x-ray powder diffraction pattern (CuKa) is included 2217, 27.13 and 2〇.94 degrees 20 (.20) diffraction peaks; (ϋ) 1H NMR spectrum as shown in Fig. 42; (iii) FTIR spectrum as shown in Fig. 43; (iv) An endothermic event focused on an endothermic event at about 152 ° C and an exothermic event at 212 ° C; and (v) shows no weight loss below 1503⁄4 and a decomposition at about 154 ° C. 128407.doc 200911757 Starting TGA Thermal analysis. 8. Benzoate of the compound of the formula:

苯甲酸鹽其中該苯甲酸鹽之特徵在於選自由以下組成之群的一或多種物理特性：Benzoate The benzoate is characterized by one or more physical properties selected from the group consisting of:

(ϋ) (iii) (iv) (v) χ射線粉末繞射圖案（CuKa)包含在約1743 25.9〇及23.93度20(。20)之繞射峰；如圖48中所示之iH NMR光譜；如圖49中所示之FTIR光譜；吸熱事件接著在約U6t之放熱事件，及在2〇1 °C之放熱事件及接著在216t之吸熱事件；及顯示在17(TC以下重量損失為97%及在約⑽。c 分解起始的TGA熱分析圖。 9. 一種具有下式之化合物之氫氯酸鹽··(ϋ) (iii) (iv) (v) The X-ray powder diffraction pattern (CuKa) contains diffraction peaks at about 1743 25.9 〇 and 23.93 degrees 20 (. 20); iH NMR spectrum as shown in Fig. 48 The FTIR spectrum is shown in Figure 49; the endothermic event is followed by an exothermic event at about U6t, and an exothermic event at 2〇1 °C followed by an endothermic event at 216t; and shown at 17 (weight loss below TC is 97) % and the TGA thermogram at the beginning of the decomposition of about (10).c 9. A hydrochloride of a compound of the formula

由以下組成之群的一其中該氫氣酸鹽之特徵在於選自或多種物理特性：One of the group consisting of: wherein the hydrogenate is characterized by one or more physical properties:

128407.doc 200911757 23.09及21.89度2Θ(°2Θ)之繞射峰； (II) 如圖54中所示之丨H NMR光譜； (III) 如圖55中所示之FTIR光譜； (iv) 具有集中在約之吸熱事件及在2〇8(>(：：之放熱事件的示差掃描熱量測定圖譜；及 (v) 顯不在I60°C以下無重量損失之TGA熱分析圖。 10. —種具有下式之化合物之氫溴酸鹽：128407.doc 200911757 23.09 and 21.89 degrees 2 Θ (° 2 Θ) diffraction peak; (II) 丨H NMR spectrum as shown in Figure 54; (III) FTIR spectrum as shown in Figure 55; (iv) Concentrate on the endothermic event and the TGA thermogram at 2〇8 (>::: the differential scanning calorimetry map of the exothermic event; and (v) no significant weight loss below I60 °C. a hydrobromide salt having a compound of the formula:

其中氫溴酸鹽之特徵在於選自由以下組成之群的一或多種物理特性： ⑴ X射線粉末繞射圖案（CuKa)包含在約u 25、 18.92及26.55度20(。20)之繞射峰； (11) 如圖60中所示之1η NMR光譜； (⑴）如圖61中所示之pTIR光譜； (iv) 具有集中在約214t之吸熱事件接著在約224；)(：之放熱事件的示差掃描熱量測定圖譜；及 (v) 顯示在170°C以下無重量損失之TGA熱分析圖。 11. 一種具有下式之化合物之龍膽酸鹽：Wherein the hydrobromide salt is characterized by one or more physical properties selected from the group consisting of: (1) the X-ray powder diffraction pattern (CuKa) comprising diffraction peaks at about u 25, 18.92 and 26.55 degrees 20 (. 20). (11) 1 η NMR spectrum as shown in FIG. 60; ((1)) pTIR spectrum as shown in FIG. 61; (iv) having an endothermic event concentrated at about 214 t followed by an exothermic event at about 224; a differential scanning calorimetry map; and (v) a TGA thermogram showing no weight loss below 170 ° C. 11. A gentisate having a compound of the formula:

128407.doc 200911757 由以下組成之群的一其中該龍膽酸鹽之特徵在於選自或多種物理特性： ⑴X射線粉末繞射圖案（CuKo〇包含在約a#、 12.87及25.93度20(。20)之繞射峰； ⑴）如圖66中所示之1H NMR光譜； (iii)如圖67中所示之fTIr光譜； (lV)示差掃描熱量測定圖譜具有集中在約127χ：、 160°C及24(TC之吸熱事件及在24(Γ(：《放熱事件；及 ^ Ο) 顯示在約60-17(TC之間重量指生炎认， i〜丨j里里損失為約5%的tga 熱分析圖。 12· —種具有下式之化合物之形式a :128407.doc 200911757 One of the group consisting of the gentisate is characterized by one or more physical properties: (1) X-ray powder diffraction pattern (CuKo〇 is included in about a#, 12.87, and 25.93 degrees 20 (.20) Diffraction peak; (1)) 1H NMR spectrum as shown in Fig. 66; (iii) fTIr spectrum as shown in Fig. 67; (lV) differential scanning calorimetry map having concentration of about 127 χ:, 160 ° C and 24 (TC's endothermic event and at 24 (Γ (: "Exothermic event; and ^ Ο) shown in about 60-17 (TC between the weight refers to the inflammation, i ~ 丨j loss of about 5% of the tga Thermal analysis diagram 12. A form of a compound having the formula a:

其中形式A之特徵在於自選由以下組成之群的一或多種物理特性： ⑴ X射線粉末繞射圖案（CuKa)包含在約9 43、4 73 及14.14度20(。20)之繞射峰； (ii) 如圖72中所示之1H NMR光譜； (iii) 如圖73中所示之FTIR光譜； (iv) 具有集中在約258°C之吸熱事件的示差掃描熱量測定圖譜；及 (v) 顯示至約230°C無重量損失之TGA熱分析圖。 128407.doc 200911757 種具有下式之化合物之龍膽酸鹽：Wherein Form A is characterized by one or more physical properties of the group consisting of: (1) an X-ray powder diffraction pattern (CuKa) comprising diffraction peaks at about 9 43 , 4 73 and 14.14 degrees 20 (. 20); (ii) a 1H NMR spectrum as shown in Figure 72; (iii) an FTIR spectrum as shown in Figure 73; (iv) a differential scanning calorimetry map having an endothermic event centered at about 258 °C; ) A TGA thermogram showing no weight loss to about 230 °C. 128407.doc 200911757 A gentisate of the compound having the formula:

•龍膽酸鹽其中至少一部分所存在之化合物的特徵在於選自由以下組成之群的一或多種物理特性： (‘） X射線私末繞射圖案（CuKa)包含在約1〇9、 20.48、28.35及3〇.9〇度20(。20)之繞射峰； ⑴）顯示在120_190。(：之間重量損失為3·2°/。的TGA熱分析圖；及 (iH) 在約133°C之DSC吸熱事件。 14_ 一種組合物’其包含具有下式之化合物：• The compound in which at least a portion of the gentisate is present is characterized by one or more physical properties selected from the group consisting of: (') the X-ray private diffraction pattern (CuKa) is comprised at about 1〇9, 20.48, The diffraction peaks of 28.35 and 3〇.9〇20 (.20); (1)) are shown at 120_190. (: TGA thermogram with a weight loss of 3.2 ° C.; and (iH) a DSC endothermic event at about 133 ° C. 14_ A composition 'which contains a compound having the formula:

其中至少一部分所存在之化合物的特徵在於選自由以下組成之群的一或多種物理特性： ⑴ X射線粉末繞射圖案（CuKa)包含在約8.64、 19.95及18.17度20(。20)之繞射峰； (η) 如圖2中所示之1η NMR光譜； (in)如圖3中所示之ftIR光譜； (iv) 具有集中在約89°C之寬吸熱及在約198°C之第二吸熱的示差掃描熱量測定圖譜；及 (v) 顯示在100°C以下重量損失為約8.4%的TGA熱分 128407.doc 200911757 析圖。 15.種組合物，《包含具有下式之化合物:At least a portion of the compounds present are characterized by one or more physical properties selected from the group consisting of: (1) X-ray powder diffraction pattern (CuKa) comprising diffraction at about 8.64, 19.95, and 18.17 degrees 20 (.20). (η) 1η NMR spectrum as shown in Figure 2; (in) ftIR spectrum as shown in Figure 3; (iv) having a broad endotherm concentrated at about 89 ° C and at about 198 ° C Two endothermic differential scanning calorimetry maps; and (v) TGA thermal fractions showing a weight loss of about 8.4% below 100 ° C. 128407.doc 200911757. 15. A composition comprising "a compound having the formula:

中至>_部分所存在之化合物的特徵在於選自由以下組成之群的一或多種物理特性： / * \ 父射線粉末繞射圖案（CuKa)包含在約17.21、 20.4及16.02度2Θ(°2Θ)之繞射峰； (II) 如圖8中所示之1η NMR光譜； (III) 如圖13中所示之FTIR光譜； ()具有集中在約211。(：吸熱的示差掃描熱量測定圖譜；及 (V)在120<t以下無重量損失之TGA熱分析圖。 16. —種組合物，其包含具有下式之化合物：〜0The compound present in the middle to >_ portion is characterized by one or more physical properties selected from the group consisting of: / * \ The parent ray powder diffraction pattern (CuKa) is comprised at about 17.21, 20.4, and 16.02 degrees 2 Θ (°) 2Θ) diffraction peak; (II) 1η NMR spectrum as shown in Fig. 8; (III) FTIR spectrum as shown in Fig. 13; () having a concentration of about 211. (: an endothermic differential scanning calorimetry map; and (V) a TGA thermogram without weight loss below 120 < t. 16. A composition comprising a compound having the formula: ~0

甘士至少一部分所存在之化合物的特徵在於選自由以下組成之群的一或多種物理特性： ⑴ X射線粉末繞射圖案（CuKa)包含在約5.39、 13_24及18.19度20(。20)之繞射峰； (II) 如圖19中所示之NMR光譜； (III) 如圖20中所示之ftir光譜； (lv)顯示在12(TC以下重量損失為2 5%及在約127t 128407.doc 200911757 分解起始的TGA熱分析圖；及 (v) *有集中在約12代吸熱的示差掃描熱量測定圖譜。 17. -種組合物’其包含具有下式之化合物： f i Ό 八 QThe compound in which at least a portion of the glycine is present is characterized by one or more physical properties selected from the group consisting of: (1) The X-ray powder diffraction pattern (CuKa) is comprised at about 5.39, 13-24, and 18.19 degrees 20 (20). (II) NMR spectrum as shown in Figure 19; (III) ftir spectrum as shown in Figure 20; (lv) shows at 12 (weight loss below TC is 2 5% and at about 127t 128407. Doc 200911757 Decomposition-initiated TGA thermogram; and (v) * There is a differential scanning calorimetry map focused on about 12 generations of endotherms. 17. - A composition comprising a compound of the formula: fi Ό 八 Q

HC1 /、中至v部分所存在之化合物的特徵在於選自由以下組成之群的一或多種物理特性： ⑴ X射線粉末繞射圖案（CuKa)包含在約15 48、23.21及16.〇4度20(。20)之繞射峰；如圖25中所示之1H NMR光譜；如圖26中所示之FTIR ATR光講；顯示在13(TC以下重量損失為41%及在約16〇。〇分解起始的TGA熱分析圖；及具有兩個集中在約95。〇及在185。〇之吸熱事件的示差掃描熱量測定圖譜。 18. -種組合物，其包含具有下式之化合物： V (ϋ)(iii)(iv)(v)The compound present in the HC1 /, intermediate to v portion is characterized by one or more physical properties selected from the group consisting of: (1) The X-ray powder diffraction pattern (CuKa) is comprised at about 15 48, 23.21, and 16.4 degrees. 20 (. 20) diffraction peak; 1H NMR spectrum as shown in Figure 25; FTIR ATR light diagram as shown in Figure 26; shown at 13 (weight loss below TC is 41% and at about 16 〇. a TGA thermogram of the initial decomposition of hydrazine; and a differential scanning calorimetry map having two endothermic events centered at about 95 Å and at 185. 18. A composition comprising a compound having the formula: V (ϋ)(iii)(iv)(v)

•雙磷酸鹽其中至少-部分所存在之化合物的特徵在於選自下組成之群的一或多種物理特性： ⑴ x射線粉末繞射圖案（CuKoc)包含在約17 8〇、 25.80及21.88度2Θ(°2Θ)之繞射峰； 128407.doc *10- 200911757 (ii) 如圖31中所示之1η NMR光譜； (iii) 具有集中在約179°C之放熱事件及在256°C之吸熱事件的示差掃描熱量測定圖譜；及 (iv) 顯不在18CTC以下重量損失為2.0%及在約127°C 分解起始的TGA熱分析圖。 19. 一種組合物，其包含具有下式之化合物：• The compound in which at least a portion of the bisphosphonate is present is characterized by one or more physical properties selected from the group consisting of: (1) The x-ray powder diffraction pattern (CuKoc) is comprised at about 17 8 , 25.80 and 21.88 degrees 2 . (°2Θ) diffraction peak; 128407.doc *10- 200911757 (ii) 1η NMR spectrum as shown in Fig. 31; (iii) having an exothermic event concentrated at about 179 ° C and an endothermic at 256 ° C A differential scanning calorimetry map of the event; and (iv) a TGA thermogram showing a weight loss of 2.0% below 18 CTC and a decomposition start at about 127 °C. 19. A composition comprising a compound having the formula:

其中至少一部分所存在之化合物的特徵在於選自由以下組成之群的一或多種物理特性： ⑴ X射線粉末繞射圖案（CuKot)包含在約2〇 92、 17.54及2〇.36度20(。20)之繞射峰； (ii)如圖36中所示之1η NMR光譜； (in)如圖37中所示之FTIR光譜；㈣具有兩個集中在約听及在179〇c之吸熱事件的示差掃描熱量測定圖譜；及 (V) 顯示在100 〇c以下之溫度重量指生又里里損失為約2%的 TGA熱分析圖。 20· —種組合物，其包含具有下式之化合物：At least a portion of the compounds present are characterized by one or more physical properties selected from the group consisting of: (1) The X-ray powder diffraction pattern (CuKot) is comprised at about 2〇92, 17.54, and 2〇.36 degrees 20 (. 20) the diffraction peak; (ii) the 1 η NMR spectrum as shown in Figure 36; (in) the FTIR spectrum as shown in Figure 37; (d) having two endothermic events centered on the hearing and at 179 °c The differential scanning calorimetry map; and (V) shows a TGA thermogram with a temperature loss of less than 100 〇c and a loss of about 2%. 20. A composition comprising a compound having the formula:

128407.doc 200911757 其中至少一部分所存在之化合物的特徵在於選自由以下組成之群的一或多種物理特性： )X射線粉末繞射圖案（CuKcc)包含在約22.17、 27.13及2〇.94度20(。20)之繞射峰； (II) 如圖42中所示之丨η NMR光譜； (III) 如圖43中所示之ftIR光譜； ()具有集中在約mt：之吸熱事件及在]。·^之放熱事件的示差掃描熱量測定圖譜；及 (v)顯示在以下無重量損失及在約154t分解起始的TGA熱分析圖。 21. —種組合物，其包含具有下式之化合物：128407.doc 200911757 wherein at least a portion of the compounds present are characterized by one or more physical properties selected from the group consisting of:) X-ray powder diffraction pattern (CuKcc) comprised at about 22.17, 27.13, and 2〇.94 degrees 20 (.20) diffraction peak; (II) 丨η NMR spectrum as shown in Fig. 42; (III) ftIR spectrum as shown in Fig. 43; () having an endothermic event concentrated at about mt: ]. · The differential scanning calorimetry map of the exothermic event; and (v) shows the TGA thermogram below without weight loss and at about 154 t decomposition. 21. A composition comprising a compound having the formula:

其中至少一部分所存在之化合物的特徵在於選自由以下組成之群的一或多種物理特性： (I) X射線粉末繞射圖案（CuKa)包含在約17.43、 25.9〇及23.93度20(。20)之繞射峰； (II) 如圖48中所示之iH NMR光譜； (in)如圖49中所示之FTIR光譜； (lV)示差掃描熱量測定圖譜具有兩個集中在約1 i〇°c 之吸熱事件接著在約116 °C之放熱事件，及在 201°C之放熱事件及接著在216〇c之吸熱事件；及 128407.doc (V) 200911757 顯不在17〇C以下重量損头发里谓天為9·7%及在約i80〇C 分解起始的TGA熱分析圖。 22. —種組合物，其包含具有下式之化合物··At least a portion of the compounds present are characterized by one or more physical properties selected from the group consisting of: (I) X-ray powder diffraction pattern (CuKa) comprised at about 17.43, 25.9 Å, and 23.93 degrees 20 (20). The diffraction peak; (II) the iH NMR spectrum as shown in Fig. 48; (in) the FTIR spectrum as shown in Fig. 49; (1V) the differential scanning calorimetry map has two concentrations at about 1 i〇 The endothermic event of c is followed by an exothermic event at about 116 °C, and an exothermic event at 201 °C followed by an endothermic event at 216 °C; and 128407.doc (V) 200911757 is not in the weight loss hair below 17〇C It is said that the day is 9.7% and the TGA thermogram is at the beginning of the decomposition of about i80〇C. 22. A composition comprising a compound having the formula:

•HC1 其中至少一部分所存在之化合物的特徵在於選自由以下組成之群的一或多種物理特性： ⑴X射線粉末繞射圖案（CuKa)包含在約19 83、 23.09及21.89度20(。20)之繞射蜂； (Π) (iii) (iv) (v) 如圖54中所示之iH NMR^ _ ; 如圖55中所示之FTIr光譜； 23. —種組合物，其包含具有下式之化合物：具有集中在約197°C之吸熱事件及在2〇8〇c之放熱事件的示差掃描熱量測定圖譜；及顯示在^。(：以下無重量損失之TGA熱分析圖。• HC1 wherein at least a portion of the compound present is characterized by one or more physical properties selected from the group consisting of: (1) X-ray powder diffraction pattern (CuKa) comprised at about 19 83, 23.09, and 21.89 degrees 20 (.20) (b) (iv) (iv) (v) iH NMR^ _ as shown in FIG. 54; FTIr spectrum as shown in FIG. 55; 23. a composition comprising the following formula Compound: a differential scanning calorimetry map having an endothermic event concentrated at about 197 ° C and an exothermic event at 2 〇 8 ° C; and shown at ^. (: The following TGA thermogram without weight loss.

NH NH2NH NH2

*HBr "”丨ΠΆ 视的将徵在於選自由以下組成之群的—或多種物理特性： ⑴ X射線粉末繞射圖案（CuKcx)包含在約u 25、 18·92及26.55度20(。2〇)之繞射峰； 128407.doc •13- 200911757 (11) 如圖60中所示之1H NMR光譜； (ill) 如圖61中所示之FTIR光譜； (iv) 具有集中在約214°C之吸熱事件、接著在約224 C之放熱事件的示差掃描熱量測定圖譜；及 (v) 顯示在1701以下無重量損失之TGA熱分析圖。 24. —種組合物，其包含具有下式之化合物：*HBr "" is characterized by a group selected from the group consisting of - or a plurality of physical properties: (1) The X-ray powder diffraction pattern (CuKcx) is contained at about u 25, 18.92, and 26.55 degrees 20 (. 2〇) diffraction peak; 128407.doc •13- 200911757 (11) 1H NMR spectrum as shown in Figure 60; (ill) FTIR spectrum as shown in Figure 61; (iv) with concentration at about 214 An endothermic event at °C, followed by a differential scanning calorimetry map at an exothermic event of about 224 C; and (v) a TGA thermogram showing no weight loss below 1701. 24. A composition comprising the following formula Compound:

其中至少一部分所存在之化合物的特徵在於選自由以下組成之群的一或多種物理特性： ⑴ X射線粉末繞射圖案（CuKot)包含在約17 49、 12.87及25.93度20(。2〇)之繞射峰； Ο) 如圖66中所示之1η NMR光譜； (⑴）如圖67中所示之FTIR光譜；〇V)示差掃描熱量測定圖譜具有集中在約127C>c、 16(TC及24(TC之吸熱事件及在“吖之放熱事件；及 (v) 顯不在約60-1 70°C之間重量損类炎& 谓天為約5%的TGA 熱分析圖。 25. —種組合物，其包含具有下式之化合物：At least a portion of the compounds present are characterized by one or more physical properties selected from the group consisting of: (1) the X-ray powder diffraction pattern (CuKot) is comprised at about 17 49, 12.87, and 25.93 degrees 20 (. 2 〇) Diffraction peak; Ο) 1 η NMR spectrum as shown in Fig. 66; ((1)) FTIR spectrum as shown in Fig. 67; 〇V) differential scanning calorimetry map having concentration at about 127 C > c, 16 (TC and 24 (the endothermic event of TC and the exothermic event of 吖; and (v) is not between about 60-1 70 °C. Weight loss inflammation & said the day is about 5% TGA thermogram. 25. a composition comprising a compound having the formula:

128407.doc -14- 200911757 其中至少-部分所存在之化合物的特徵在於選自由以下組成之群的一或多種物理特性： ⑴ X射線粉末繞射圖案（CuKa)包含在約9 43、4 73 及14.14度2Θ(°2Θ)之繞射峰； (ι〇如圖72中所示之1H NMR光譜； (出）如圖73中所示之FTIR光譜； (1V)具有集中在約258°C之吸熱事件的示差掃描熱量測定圖譜；及 (v)顯不至約230°C無重量損失之TGA熱分析圖。 26. —種組合物，其包含具有下式之化合物：128407.doc -14- 200911757 wherein at least a portion of the compound present is characterized by one or more physical properties selected from the group consisting of: (1) an X-ray powder diffraction pattern (CuKa) comprised at about 9 43 and 4 73 and 14.14 degrees 2 Θ (° 2 Θ) diffraction peak; ( 〇 1 1H NMR spectrum as shown in Figure 72; (out) FTIR spectrum as shown in Figure 73; (1V) has a concentration of about 258 ° C A differential scanning calorimetry map of the endothermic event; and (v) a TGA thermogram showing no weight loss of about 230 ° C. 26. A composition comprising a compound having the formula:

其中至少一部分所存在之化合物的特徵在於選自由以下組成之群的一或多種物理特性： ⑴ X射線粉末繞射圖案（CuKa)包含在約1 〇.9、 - 20.48、28.35 及 30.90 度 2Θ(°2Θ)之繞射峰； (11) 顯示在120-190°C之間重量損失為3.2°/。的TGA熱分析圖；及 (iii) 在約133。(：之DSC吸熱事件。 2 7.如晴求項14至2 6中任一項之組合物，其中該至少一部分在該組合物之0 · 1 %至99%(以重量計）之間。 2 8.如请求項14至2 6中任一項之組合物，其中該至少一部分大於該組合物之0.1 %(以重量計）。 128407.doc 200911757 2 9.如請求項14至2 6中任一項之組合物，其中該至少一部分大於該組合物之1 %(以重量計）。 3 0. T如請求項14至26中任一項之組合物，其中該至少一部分大於該組合物之5 %(以重量計）。 3 1.如請求項14至26中任一項之組合物，其中該至少一部分大於該組合物之2 5 %(以重量計）。 3 2.如請求項14至26中任一項之組合物，其中該至少一部分大於該組合物之50%(以重量計）。 33 .如請求項1 4至26中任一項之組合物，其中該至少一部分大於該組合物之75%(以重量計）。 34.如請求項14至26中任一項之組合物，其中該至少一部分大於該組合物之90%(以重量計）。 3 5.如請求項14至2 6中任一項之組合物，其中該至少一部分大於該組合物之95%(以重量計）。 3 6.如請求項14至26中任一項之組合物，其中該至少一部分大於該組合物之97%(以重量計）。 3 7.如請求項14至26中任一項之組合物，其中該至少一部分大於該組合物之99%(以重量計）。 3 8. —種醫藥組合物，其包含如請求項1至37中任一項之化合物或組合物作為活性成份。 3 9.如請求項3 8之醫藥組合物，其中該組合物為適於口服之固體調配物。 40.如請求項38之醫藥組合物，其中該組合物為適於口服之液體調配物。 128407.doc -16- 200911757 札如請求項38之醫藥組合物，其中該組合投藥之液體調配物。、於非經口 42_ -種醫藥組合物’其包含如請求項… 合：或組合物’其中該組合物適於藉由選自由以下^ :群的-種途徑來投藥：經口、非經口、腹膜内、靜: =脈内、經皮、經舌下、肌肉内、經直腸: 、經由吸入、經***、㈣内、經由 …遞、經皮下、經脂肪内、經關節内及經鞘内。 43. —種套組，其包含：如請求項⑴了中任一項之化合物或組合物，及之=書:其包含選自由以下組成之群的一或多種形式針L 投與該組合物之疾病病況'該組合物之存貝況、給藥量資訊及如何投與該組合物之說明。 h求項43之套組’其中該套組包含多劑量形式之該化合物或該組合物。 45. —種製品，其包含：如請求項1至37中任-項之化合物或組合物；及包裝材料。 46. 如請求項45之製品，其中該包裝材料包含-個用於容納戎組合物之容器。 47. 如請求項46之製品，其中該容器包含標籤，其指明由以下組成之群的—或多個部分：可投與該組合物之疾病病況、儲存資m、給藥量資訊及/或如何投與該組合物明0 128407.doc 200911757 48.:種::：項1至37中任-項之化合物或組合物的用性用於製備用以治療腿c具有造成病理及/或症狀活挫之疾病病況之藥物。 4 9,如清求項4 8之用挣，立士斗用迚其中该疾病病況為癌症。 50.如請求項49之用途，i中 ^ . 〃中該癌症係包含以下之群：鱗狀、、，田胞癌、星形細胞瘤、卡卞皮西氏肉瘤（Kaposi’s arcoma)、神經膠母細胞瘤、頭部及頸部癌…音鹿、''田胞肺癌、膀胱癌、小…Γ 即巢癌、***癌、乳癌、 J、，.胞肺癌、神經膠質姓癌、胃腸癌、腎声血，腸癌、泌尿生殖器 ^ 月癌、血液痛、非乘* λ Ηη ., 瓜非霍奇金淋巴瘤（non- H〇dgJans lymph〇ma)、巴瘤、夕發性骨髓瘤、白血病 (包括急性骨髓性白血病細貽ώ a十、又注月知性白血病、慢性淋巴 :血病)、骨髓發育不良症候群及間皮瘤。 51. 如明求項48之用途，其中誃 ,Ά ^ ^ 、 '"疾病病況係包含炎症、發炎腸m牛皮癬或移植排斥之群。 52. 如請求項48之用途，苴 53.如請求項48之用途 5 4 ·如請求項4 8之用途肌萎縮性側索硬化 mer’s disease)。 55. 如請求項48之用途病或發炎性皮膚病 56. 如請求項48至55中任— HDAC1。 a <用途’其中該HDAC為 ”中以疾病病況為關節炎。 /、中》亥疾病病況為眼睛退化疾病。其中該疾病病況為多發性硬化症、曱狀腺瘤或阿茲海默氏症（Alzhei-”中該疾病病況為過度增生性皮膚 128407.doc ‘18- 200911757 57. 如清求項、至55中任一項之用途’其中該HD AC為 HDAC2 〇 58. 如請求項48 至55中任一項之用途，其中該HDAC為 HDAC5。 59. 如請求，，、至55中任一項之用途’其中該HDAC為 HDAC6 〇 60. 如請求項4&. 至55中任一項之用途，其中該HDAC為 HDAC8。 61. 種如4求項1至37中任一項之化合物或組合物的用途，其係用於製備用於抑制HDAC1以治療癌症之藥物。 62_ 一種如請求項1至37中任一項之化合物或組合物的用途’其係用於製備用於抑制HDAC2以治療癌症之藥物。 63. 如請求項61或62之用途，其中該癌症為非霍奇金淋巴瘤。 64. 如請求項61或62之用途’其中該癌症為淋巴瘤。 65 ·如請求項61或62之用途，其中該癌症為多發性骨髓瘤。 66.如請求項61或62之用途，其中該癌症為白血病，包括条性骨髓性白血病、慢性骨髓性白血病、慢性淋巴細胞白血病。 128407.doc -19·At least a portion of the compounds present are characterized by one or more physical properties selected from the group consisting of: (1) the X-ray powder diffraction pattern (CuKa) is comprised at about 1 〇.9, - 20.48, 28.35, and 30.90 degrees 2 Θ ( The diffraction peak of °2Θ); (11) shows a weight loss of 3.2°/ between 120-190 °C. The TGA thermogram; and (iii) at approximately 133. The composition of any one of the items 14 to 26, wherein the at least a portion is between 0.1% and 99% by weight of the composition. The composition of any one of claims 14 to 26, wherein the at least a portion is greater than 0.1% by weight of the composition. 128407.doc 200911757 2 9. As claimed in claims 14 to 26 The composition of any one, wherein the at least a portion is greater than 1% by weight of the composition. The composition of any one of claims 14 to 26, wherein the at least a portion is greater than the composition The composition of any one of claims 14 to 26, wherein the at least a portion is greater than 25% by weight of the composition (by weight). The composition of any one of clauses 14 to 26, wherein the at least a portion is greater than 50% by weight of the composition. The composition of any one of claims 14 to 26, wherein the at least a portion is greater than The composition of any one of claims 14 to 26, wherein the at least a portion is greater than the The composition of any one of claims 14 to 26, wherein the at least a portion is greater than 95% by weight of the composition. The composition of any one of claims 14 to 26, wherein the at least a portion is greater than 97% by weight of the composition. The composition of any one of claims 14 to 26, wherein the at least A portion is greater than 99% by weight of the composition. 3 8. A pharmaceutical composition comprising the compound or composition of any one of claims 1 to 37 as an active ingredient. A pharmaceutical composition according to claim 38, wherein the composition is a solid formulation suitable for oral administration. The pharmaceutical composition according to claim 38, wherein the composition is a liquid formulation suitable for oral administration. 128407.doc -16- 200911757 The pharmaceutical composition of claim 38, wherein the combination is administered as a liquid formulation. In the case of a pharmaceutical composition, the composition comprises the following: By administering a drug selected from the following group: group: oral, non-oral Intraperitoneal, static: = intrapulmonary, percutaneous, sublingual, intramuscular, transrectal: through inhalation, transvaginal, (four), via, subcutaneous, trans fat, intra-articular and intrathecal 43. A kit comprising: a compound or composition according to any one of claims (1), and a book comprising: one or more forms of needles selected from the group consisting of: Disease condition of the article 'The preservation of the composition, information on the amount of administration and instructions on how to administer the composition. h The kit of claim 43 wherein the kit comprises the compound or the composition in multiple dose forms. 45. An article of manufacture comprising: a compound or composition of any one of claims 1 to 37; and a packaging material. 46. The article of claim 45, wherein the packaging material comprises a container for holding the enamel composition. 47. The article of claim 46, wherein the container comprises a label indicating a plurality or portions of the group consisting of: a disease condition, a storage amount, a dose amount information, and/or the composition of the composition How to administer the composition 0 128407.doc 200911757 48.: Seed::: The use of the compound or composition of any of items 1 to 37 for the preparation of a treatment for the treatment of leg c has pathology and/or symptoms A drug that is in a state of illness. 4 9, if the use of the item 4 8 earned, Li Shidou used to treat the disease as cancer. 50. The use of claim 49, wherein the cancer system comprises the following population: squamous,, cytocellular carcinoma, astrocytoma, Kaposi's arcoma, neurasine Blastoma, head and neck cancer... Yinlu, ''Field lung cancer, bladder cancer, small...Γ nest cancer, prostate cancer, breast cancer, J,, cell lung cancer, glial surname cancer, gastrointestinal cancer, Kidney sound, intestinal cancer, genitourinary tract ^ lunar cancer, blood pain, non-multiple * λ Η η., non-H〇dgJans lymph〇ma, gynecological, idiopathic myeloma, Leukemia (including acute myeloid leukemia, a, combined with monthly leukemia, chronic lymph: blood disease), myelodysplastic syndrome and mesothelioma. 51. The use of the item 48, wherein 誃 , Ά ^ ^ , '" the disease condition is a group comprising inflammation, inflamed intestinal m psoriasis or transplant rejection. 52. For the purposes of claim 48, 苴 53. Use of claim 48 5 4 • Use of claim 48 for mer’s disease. 55. Use of claim 48 Disease or inflammatory skin disease 56. As requested in items 48 to 55 - HDAC1. a <use' wherein the HDAC is "in the disease condition is arthritis. /," Zhong Hai disease condition is an eye degenerative disease. The disease condition is multiple sclerosis, sickle adenoma or Alzheimer's disease The condition of the disease (Alzhei-" is hyperproliferative skin 128407.doc '18- 200911757 57. The use of any of the items of 55, wherein the HD AC is HDAC2 〇58. The use of any one of 55, wherein the HDAC is HDAC 5. 59. The use of any of the claims, wherein, the HDAC is HDAC6 〇 60. as in any of claims 4 &. The use of the compound or composition of any one of the items 1 to 37, which is for the preparation of a medicament for inhibiting HDAC1 for treating cancer. Use of the compound or composition of any one of claims 1 to 37 for the preparation of a medicament for the inhibition of HDAC2 for the treatment of cancer. 63. The use of claim 61 or 62, wherein the cancer is non-Hodge Golden lymphoma. 64. As requested in claim 61 or 62 Wherein the cancer is lymphoma. 65. The use of claim 61 or 62, wherein the cancer is multiple myeloma. 66. The use of claim 61 or 62, wherein the cancer is leukemia, including strip bone marrow Leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia. 128407.doc -19·