TWI237029B

TWI237029B - Vasoactive intestinal peptide analogs

Info

Publication number: TWI237029B
Application number: TW90118676A
Authority: TW
Inventors: Anand C Burman; Sudhanand Prasad; Rama Mukherjee; Anu T Singh; Archna Mathur
Original assignee: Dabur Oncology Plc
Priority date: 2000-07-31
Filing date: 2001-07-31
Publication date: 2005-08-01
Also published as: AR028928A1; MY117747A

Abstract

The present invention encompasses novel analogs of vasoactive intestinal peptide (VIP), containing substitutions at appropriately selected amino acids. The invention particularly relates to the design and synthesis of novel biologically active VIP analogs containing alpha,alpha-dialkylated amino acids in a site-specific manner. Specifically, the invention relates to the synthesis of VIP peptide derivatives, which bind selectively to VIP receptors on target cells. The invention encompasses methods for the generation of these peptides, compositions containing the peptides and the pharmacological applications of these peptides especially in the treatment and prevention of cancer.

Description

1237029 五、發明說明（1) 發明之領域、本發明係包含新穎之腸血管肽（VIP)類似物，其包含位在被適當地選定之胺基酸處的取代。本發明特別是關於呈一種位置-專一性之方式來設計及合成新穎具有生物活性之包含有α，α-二烷基化胺基酸的VIP衍生物。特別地，本發明係關於VIP胜肽衍生物之合成，該胜肽衍生物可選擇性地結合位於標的細胞上之VIP受體。本發明包含用以產生此等胜肽的方法、包含該等胜肽之組成物以及該胜肽在特別是治療及預防癌症上之藥理學上應用。發明之背景腸血管肷被已知在調節涉及艘内怪定之細胞内及細胞外反應中扮演重要角色，且密切涉及所有主要的自覺性及非自覺性體内恆定系統（Schofl et al·，1994)。該胜肽之多重生物活性已導引大量的研究集中在以此等胜肽荷爾蒙做為治療藥物之研發。多重取代已被使用來避免醯胺鍵受蛋白質切割之可能性。這些包含使用非標準之胺基酸像是D-胺基酸、N-烷基及N-羥基胺基酸、α·氮胺基酸、硫代醯胺連接、胜肽模擬物及前型藥之設計以及在假胜肽連接規程下之醯胺鍵改質（Dutta，1993 ; Pasternak et al·，1999)。另一個方法則是藉由醯化或醯胺化來封阻胜肽之N-端或C-端。腸血管肽是一個具有28個胺基酸之神經胜肽，其最早是從豬的小腸内被分離出來（Said and Mutt，1970)。其係與分泌活素（secretin)、胜肽組織胺酸異亮胺酸（PHI)及升血糖激素具有高度的同源性。VIP之胺基酸序列為： 12370291237029 V. Description of the invention (1) Field of the invention The present invention includes a novel intestinal vascular peptide (VIP) analogue, which includes a substitution at an appropriately selected amino acid. In particular, the present invention relates to the design and synthesis of novel biologically active VIP derivatives containing α, α-dialkylated amino acids in a position-specific manner. In particular, the present invention relates to the synthesis of VIP peptide derivatives which can selectively bind VIP receptors located on target cells. The present invention includes a method for producing such peptides, a composition comprising the peptides, and a pharmacological application of the peptides, particularly for the treatment and prevention of cancer. BACKGROUND OF THE INVENTION Intestinal vasculature is known to play an important role in regulating intracellular and extracellular responses involving intra-shipment weirdness, and is closely related to all major conscious and non-conscious in vivo constant systems (Schofl et al., 1994 ). The multiple biological activities of this peptide have led a lot of research to focus on the development of these peptide hormones as therapeutic drugs. Multiple substitutions have been used to avoid the possibility that the amido bond is cleaved by the protein. These include the use of non-standard amino acids such as D-amino acids, N-alkyl and N-hydroxyamino acids, alpha nitrogen amino acids, thioamide linkages, peptide mimics, and prodrugs Design and modification of amido linkages under pseudopeptide ligation procedures (Dutta, 1993; Pasternak et al., 1999). Another method is to block the N-terminus or C-terminus of the peptide by tritiation or tritiation. Intestinal vascular peptide is a neuropeptide with 28 amino acids, which was first isolated from the small intestine of pigs (Said and Mutt, 1970). It is highly homologous to secretin, peptide histidine isoleucine (PHI), and glycemic hormone. The amino acid sequence of VIP is: 1237029

五、發明說明（2) His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號·· 1) VIP被已知可以在生物活體内展現多種生物活性，例如：autocrine、内分泌及paracrine功能（Said，1984)。其被已知可於腸胃道内刺激胰臟及膽汁分泌、肝臟的肝糖生成以及分泌胰島素及血糖激素（Kerrins and Said，1972 ; Domschke et al.，1977)。其於神經系統中之作用係做為一種神經傳遞物質及神經調節物質，可調控數種重要荷爾蒙之釋放及分泌（Said，1984)。於近年來，對VIP功能之關切已著重在中樞神經系統（CNS)的某些區域，以及其在腫瘤疾病之癌化進程及控制上所扮演的角色（Reubi，1995)。胜肽生長因子及調控荷爾蒙對數種惡性腫瘤在病因學及病理學上的重要性很早就已被認知。從流行病學及内分泌學的研究數據暗示：對像是胰臟之組織的正常生長負有責任之諸如VIP之神經胜肽亦可能引發其等之赘生性轉型 (transformation)之病狀（Sporn et al·，1980)。數項證據指出 VIP之作用係做為一種生長因子，且在癌細胞的持續性增生上扮演一種以主動分泌及共同分泌為主的角色（Said， 1984)。VIP對腫瘤生長之刺激作用可以直接經由其位於細胞膜之受體或者間接經由腫瘤細胞中其他生長因子所具有潛力之活性來調節（Scholar et al” 1991)。於神經膠質瘤中，VIP與相關之腦垂體腺苷酸環化酶活化多胜肽（PACAP) 的協同作用可做為上述論點之一個例證（Moody et al·， 1237029 五、發明說明（3) 1996) 〇 VIP之多重生理及藥學活性係藉由位於標的細胞表面可與G·蛋白質偶合之高親和性橫跨細胞膜受體來調節 (Reubi，1996)。VIP受體係經由活化腺苷酸環化酶而與細胞之作用子系統（effector system)偶合（Xia et al·，1996)。被發現在腫瘤細胞中高度過量表現之VIP受體係被認為是人類癌症的一個生物標記（Reubi，1996)。位於大部分之乳癌細胞、乳癌及***癌轉移、卵巢、結腸及胰臟腺癌、子宮内膜及鱗狀細胞瘤、非小細胞肺癌、淋巴瘤、神經膠質母細胞瘤、星狀細胞瘤、腦腫瘤及源自中腦之腫瘤的腫瘤細胞中之高親和性VIP受體已被鑑定出來。其中，神經内分泌腫瘤完全分化及未分化之腸胃胰臟瘤、嗜銘性細胞瘤、小細胞肺癌、神經囊胚瘤、腦垂體腺瘤以及與過量分泌病狀（如Verner-Morrison症狀）有關之腫瘤被發現會過量表現腸血管肷之受體（Tang et al·，1997a & b ; Moody et al·，1998 a & b ; Waschek et al·，1995 ; Oka et al·，1998))。這些發現暗示可以藉由設計相同的合成胜肽類似物來功能性調控 VIP活性做為用以診斷及治療這些癌症之新方法。本發明係關於使用擇定之受約制（constrained)胺基酸之胜肽類似物的抗腫瘤活性。這些新穎之VIP衍生物被發現可結合細胞膜上之VIP受體。上文所述之胜肽的抗腫瘤活性亦可以被測定。設計構形上受約制之具有生物活性的胜肽衍生物已成為用來研發以胜肽為基底之治療藥劑所廣泛使用的方法之 1237029V. Description of the invention (2) His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr -Leu-Asn-Ser-Ile-Leu-Asn-NH2 (sequence identification number · 1) VIP is known to exhibit a variety of biological activities in living organisms, such as autocrine, endocrine and paracrine functions (Said, 1984). It is known to stimulate pancreas and bile secretion in the gastrointestinal tract, hepatic glycogenesis in the liver, and secretion of insulin and blood glucose hormones (Kerrins and Said, 1972; Domschke et al., 1977). Its role in the nervous system is as a neurotransmitter and neuromodulator, which can regulate the release and secretion of several important hormones (Said, 1984). In recent years, concerns about VIP function have focused on certain areas of the central nervous system (CNS) and its role in the progression and control of cancerous disease (Reubi, 1995). The importance of peptide growth factor and hormone regulation in the etiology and pathology of several malignancies has long been recognized. Data from epidemiological and endocrinological studies suggest that neuropeptides such as VIP, which are responsible for the normal growth of tissues such as the pancreas, may also trigger their neoplastic transformation (Sporn et. al., 1980). Several evidences point to the role of VIP as a growth factor, and play a role of active secretion and co-secretion in the sustained growth of cancer cells (Said, 1984). The stimulating effect of VIP on tumor growth can be directly regulated by its receptor located on the cell membrane or indirectly by the potential activity of other growth factors in tumor cells (Scholar et al "1991). In glioma, VIP is related to The synergistic effect of pituitary adenylate cyclase-activated polycapeptide (PACAP) can be used as an example of the above argument (Moody et al., 1237029 V. Description of the Invention (3) 1996). Multiple physiological and pharmaceutical activities of VIP It is regulated by high affinity across cell membrane receptors located on the surface of the target cell, which can be coupled with G protein (Reubi, 1996). The VIP receptor system interacts with the cell by activating adenylate cyclase (effector) system) coupling (Xia et al., 1996). The VIP system that is found to be highly overexpressed in tumor cells is considered a biomarker for human cancer (Reubi, 1996). It is located in most breast cancer cells, breast cancer and prostate Cancer metastasis, ovarian, colon and pancreatic adenocarcinoma, endometrium and squamous cell tumor, non-small cell lung cancer, lymphoma, glioblastoma, stellate High affinity VIP receptors have been identified in cytomas, brain tumors, and tumor cells derived from mesencephalic tumors. Among them, neuroendocrine tumors are fully differentiated and undifferentiated gastrointestinal pancreatic tumors, homophilic cell tumors, Small cell lung cancer, neuroblastoma, pituitary adenoma, and tumors associated with excessive secretory conditions (such as Verner-Morrison symptoms) have been found to overexpress the intestinal vasculature receptors (Tang et al ·, 1997a & b Moody et al., 1998 a &b; Waschek et al., 1995; Oka et al., 1998)). These findings imply that VIP activity can be functionally regulated by designing the same synthetic peptide analogs as A novel method for diagnosing and treating these cancers. The present invention relates to the antitumor activity of selected peptide analogs of constrained amino acids. These novel VIP derivatives have been found to bind to cell membranes. VIP receptors. The antitumor activity of the peptides described above can also be determined. Designing a bioactive peptide derivative that is constrained in configuration has become a peptide-based therapeutic agent 1237029 widely used method of

五、發明說明（4) 一。於設計胜肽之新方法中，具有受約制構形特性之非標準化胺基酸可以被使用來主導多胜肽鏈之摺疊過程。cx，a-二烷基化胺基酸之構形特性已被詳加研究。這些胺基酸的嵌入嵌入這些胺基酸會限制分子内Φ、Ψ角度之旋轉，藉此來安定一個所欲之胜肽構形。α，α-二烷基化胺基酸、a-胺基-異丁酸（Aib)或a，a-二甲基甘胺酸之標準型成員已被展現可在其被嵌入一胜肽序列時誘發β·迴旋（β-turn)或螺旋構形（Prasad and Balaram，1984 ; Karle and Balaram，1990)。 α，α-二烷基化胺基酸之高度同源物[像是二乙基甘胺酸 (Deg)、二-η-丙基甘胺酸（Dpg)、二-η-丁基甘胺酸（Dbg)]以及α，α-二烷基化胺基酸之環化支鏈類似物像是[諸如1_胺基環戊烷羧酸（Ac5c)、1-胺基環己烷羧酸（Ac6c)、1-胺基環庚烷羧酸（Ac7c)以及1-胺基環辛烷羧酸（Ac8c)]的構形特性亦被顯示可誘發摺疊構形（Prasad etal·，1995 ; Karle etal·， 1995)。α，α-二烷基化胺基酸已被使用來設計具有高度潛力之趨化性胜狀類似物（Prasad et al·，1996)。本案申請人不知道有任何一種用以合成被包含於本發明中之新穎胜肽類似物的習知技術。本發明揭示供用來設計具有生物活性之胜肽衍生物之α，α-二烷基化胺基酸的構形特性，係以VIP做為考量之模式系統。參考文獻 Domschke, S. et al. Gastroenterology, 73, 478-480, 1977. Dutta, A.S. (193) Small Peptides: Chemistry, Biology and Clinical Studies, Elsevier, Pharmacochemistry Library, 19, pp 1237029 五、發明說明（5) 293-350.5. Description of the invention (4) In the new method of designing peptides, non-standardized amino acids with constrained conformation properties can be used to dominate the folding process of peptide chains. The configurational properties of cx, a-dialkylated amino acids have been studied in detail. Embedding of these amino acids Embedding these amino acids will restrict the rotation of the Φ and Ψ angles in the molecule, thereby stabilizing a desired peptide configuration. Standard members of α, α-dialkylated amino acids, a-amino-isobutyric acid (Aib), or a, a-dimethylglycine have been shown to be embedded in a peptide sequence Inducing β-turn or helical configuration (Prasad and Balaram, 1984; Karle and Balaram, 1990). Highly homologues of α, α-dialkylated amino acids [like diethyl glycine (Deg), di-η-propyl glycine (Dpg), di-η-butyl glycine Acid (Dbg)] and cyclic branched analogs of α, α-dialkylated amino acids like [such as 1-aminocyclopentanecarboxylic acid (Ac5c), 1-aminocyclohexanecarboxylic acid (Ac6c), 1-aminocycloheptanecarboxylic acid (Ac7c), and 1-aminocyclooctanecarboxylic acid (Ac8c)] were also shown to induce a folded configuration (Prasad etal ·, 1995; Karle etal., 1995). Alpha, alpha-dialkylated amino acids have been used to design chemotactically superior analogs with high potential (Prasad et al., 1996). The applicant of this case is not aware of any conventional technique for synthesizing the novel peptide analogs contained in the present invention. The present invention discloses a configuration system for designing an α, α-dialkylated amino acid of a peptide derivative having biological activity, and takes VIP as a model system for consideration. References Domschke, S. et al. Gastroenterology, 73, 478-480, 1977. Dutta, AS (193) Small Peptides: Chemistry, Biology and Clinical Studies, Elsevier, Pharmacochemistry Library, 19, pp 1237029 V. Description of the invention (5 ) 293-350.

Karle, LL. et al. (1995) J. Amen Chem. Soc. 117, 9632-9637. Karle, LL. and Balaram, P. (1990) Biochemistry 29, 6747-6756·Karle, LL. Et al. (1995) J. Amen Chem. Soc. 117, 9632-9637. Karle, LL. And Balaram, P. (1990) Biochemistry 29, 6747-6756 ·

Kerrins, C. and Said, S.I.( 1972) Proc. Soc. Exp. Biol. Med., 142, 1014-1017. Oka, H. et al.(1998) Am. J. Pathol., 153 (6), 1787-1796. Prasad, B.V.V and Balaram, P. (1984) CRC Crit. Rev. Biochem. 16, 3 07-347. Prasad, S et al. (1995) Biopolymers 35, 11-20. Prasad, S et al. (1996) mt. J. Peptide Protein Res. 48, 312-318. Reubi, J.C. et al. Cancer Res., 56 (8), 1922-193 1, 1996. Said, S. I. and Mutt, V. (1970)Science, 169, 1217-1218. Said, S.I. (1984)Peptides, 5, 143-150. Scholar E.M. Cancer 67(6): 1561-1569, 1991. Scofl, C. et al. (1994) Trends. Endocrinol. Metab. 5, 53-59. Sporn, M.B., and Todaro, G.J. (1980)N. Engi. J. Med., 303, 378-379· Stewart, J. and Yang, Y.D. (1969) Solid phase Peptide Synthesis, W.H. Freeman & Co. Tang, C. et al., (1997a) Gut, 40 (2), 267-271. Tang, C. et al., (1997b)Br. J. Cancer, 75 (10)1467-1473. 1237029Kerrins, C. and Said, SI (1972) Proc. Soc. Exp. Biol. Med., 142, 1014-1017. Oka, H. et al. (1998) Am. J. Pathol., 153 (6), 1787-1796. Prasad, BVV and Balaram, P. (1984) CRC Crit. Rev. Biochem. 16, 3 07-347. Prasad, S et al. (1995) Biopolymers 35, 11-20. Prasad, S et al (1996) mt. J. Peptide Protein Res. 48, 312-318. Reubi, JC et al. Cancer Res., 56 (8), 1922-193 1, 1996. Said, SI and Mutt, V. (1970 ) Science, 169, 1217-1218. Said, SI (1984) Peptides, 5, 143-150. Scholar EM Cancer 67 (6): 1561-1569, 1991. Scofl, C. et al. (1994) Trends. Endocrinol Metab. 5, 53-59. Sporn, MB, and Todaro, GJ (1980) N. Engi. J. Med., 303, 378-379 Stewart, J. and Yang, YD (1969) Solid phase Peptide Synthesis , WH Freeman & Co. Tang, C. et al., (1997a) Gut, 40 (2), 267-271. Tang, C. et al., (1997b) Br. J. Cancer, 75 (10) 1467-1473. 1237029

五、發明說明（6)V. Description of Invention (6)

Xia, M. et al., J. Clin. Immunol., 16 (1), 21-30, 1996. 下列縮寫以下列含義被使用於說明書及申請專利範圍之全文中： BOP 苯并三°坐-1·基-氧-參-(二甲基胺基)-鱗-六氟碟酸鹽 PyBOP 苯并三峻-l-基-氧-參-咐《洛咬基-鱗-六氣峨酸鹽 HBTU TBTU 0-苯并***-N，N，N’，N’-四甲基-正離子（uronium)-六氟磷酸鹽 2-(1Η-苯并***-1-基）-1，1，3,3-四甲基-正離子-四氟硼酸鹽 HOBt 1-羥基苯并*** DCC 二異丙基碳二亞胺 DIPCDI 二異丙基乙基胺 DIEA 二甲基甲醯胺 DMF 二氣甲烷 NMP N-甲基-2-吡咯啶酮 TFA 四氟醋酸於說明書及申請專利範圍之全文中，胺基酸是以其標準縮寫標示。除非另有於符號之前予以指明為D或DL並以一個連字符號將之分開，胺基酸表示為L-構形。發明之概要說明本發明包含具有下列通式之VIP拮抗劑，其中位於VIP 中之適當胺基酸已經一特定方式被取代為α，α·二烷基化胺基酸。本發明亦包含具有下列通式之拮抗劑之藥學上可接受的鹽類： His-Ser-Asp-Rl -Val-R2-Thi>Asp-Asn-Tyr-Thr-Arg-Leu- 1237029 五、發明說明（？） Arg-Lys-Gln-R3-Ala-Val-Lys-Val-Lys-Lys-Tyr-Leu- Asn-Ser-Ile-Leu-Asn-NH2其中 R1 是 Aib、Deg或 Ac5c、 R2是Phe或4-Cl-D-Phe、 R3是Met、Leu或Dpg或一個可水解之羧基保護基團；或該拮抗劑之藥學上可接受的鹽類。發明之詳細描述 /〆、本發明包含具有下列通式<VIP拮抗劑^"中位於VIP 中之適當胺基酸已經一特定方式被取代^為α，α-二烷基化胺基酸。本發明亦包含具有下列通式之拮抗劑之藥學上可接受的鹽類： His-Ser-Asp-Rl-Val-R2-Thr-Asp-Asn-Tyr-Thr-Arg-Leu- Arg-Lys-Gln-R3_Ala_Val-Lys-Val-Lys-Lys-Tyr-Leu- Asn-Ser-Ile-Leu-Asn-NH2其中 R1 是 Aib、Deg或 Ac5c、 R2是Phe或 4-CND-Phe、 R3是Met、Leu或Dpg或一個可水解之羧基保護基團；或該拮抗劑之藥學上可接受的鹽類。一種可水解之羧基保護基團為該等在水解時轉變為羧基基團，例如-CONH2、-COOMe等等。該"藥學上可接受之鹽類"之術語所包含之鹽類係指無毒性之本發明化合物的鹽類。代表性鹽類及酯類包含： 10 1237029Xia, M. et al., J. Clin. Immunol., 16 (1), 21-30, 1996. The following abbreviations are used throughout the specification and patent application in the following meanings: BOP Benzotri ° 1-yl-oxy-shen- (dimethylamino) -squat-hexafluorodisk salt PyBOP benzotriazine-l-yl-oxo-sat- HBTU TBTU 0-benzotriazole-N, N, N ', N'-tetramethyl-uronium-hexafluorophosphate 2- (1fluorene-benzotriazol-1-yl) -1, 1,3,3-tetramethyl-cation-tetrafluoroborate HOBt 1-hydroxybenzotriazole DCC diisopropylcarbodiimide DIPCDI diisopropylethylamine DIEA dimethylformamide DMF Dimethylmethane NMP N-methyl-2-pyrrolidone TFA tetrafluoroacetic acid is used throughout the specification and patent applications, and amino acids are indicated by their standard abbreviations. Unless otherwise indicated as D or DL before the symbol and separated by a hyphen, the amino acid is expressed in the L-configuration. SUMMARY OF THE INVENTION The present invention comprises a VIP antagonist having the general formula in which a suitable amino acid located in the VIP has been substituted with an α, α · dialkylated amino acid in a specific manner. The present invention also includes pharmaceutically acceptable salts of antagonists having the general formula: His-Ser-Asp-Rl-Val-R2-Thi > Asp-Asn-Tyr-Thr-Arg-Leu- 1237029 V. Invention Explanation (?) Arg-Lys-Gln-R3-Ala-Val-Lys-Val-Lys-Lys-Tyr-Leu- Asn-Ser-Ile-Leu-Asn-NH2 where R1 is Aib, Deg or Ac5c, and R2 is Phe or 4-Cl-D-Phe, R3 is Met, Leu or Dpg or a hydrolyzable carboxy protecting group; or a pharmaceutically acceptable salt of the antagonist. Detailed description of the invention / 〆 The present invention contains the appropriate amino acid having the following general formula < VIP antagonist ^ " in VIP which has been substituted in a specific way ^ as α, α-dialkylated amino acid . The present invention also includes pharmaceutically acceptable salts of antagonists having the general formula: His-Ser-Asp-Rl-Val-R2-Thr-Asp-Asn-Tyr-Thr-Arg-Leu- Arg-Lys- Gln-R3_Ala_Val-Lys-Val-Lys-Lys-Tyr-Leu- Asn-Ser-Ile-Leu-Asn-NH2 where R1 is Aib, Deg or Ac5c, R2 is Phe or 4-CND-Phe, R3 is Met, Leu or Dpg or a hydrolyzable carboxy protecting group; or a pharmaceutically acceptable salt of the antagonist. A hydrolyzable carboxyl protecting group is one which is converted into a carboxyl group upon hydrolysis, such as -CONH2, -COOMe, and the like. The term "pharmaceutically acceptable salts" includes salts that refer to salts of the compounds of the present invention which are non-toxic. Representative salts and esters include: 10 1237029

五、發明說明（8) 醋酸鹽、抗壞血酸鹽、苯磺酸鹽、苯甲酸鹽、碳酸氩鹽、硫酸氫鹽、酒石酸氫鹽、硼酸鹽、樟腦磺酸鹽、碳酸鹽、檸檬酸鹽、二氫氣酸鹽、甲磺酸鹽、乙磺酸鹽、p-甲苯磺酸鹽、環己基胺基磺酸鹽、奎寧酸鹽、二乙胺四乙酸鹽、edisylate、estolate、乙讀醢酸鹽、反丁烤二酸鹽、葡萄糖醛酸鹽、脯胺醯胺酸鹽、甘油磷酸鹽、氫溴酸鹽、氩氣酸鹽、羥基萘甲酸鹽、乳酸鹽、乳糖酸鹽（lactobionate)、月桂酸鹽、蘋果酸鹽、順-丁烯二酸鹽、扁桃酸鹽、甲磺醯酸鹽、黏液酸鹽（mucate)、萘磺酸鹽（napsylate)、硝酸鹽、 η·甲基葡萄糖胺、油酸鹽、草酸鹽、Palmoates、雙羥萘酸鹽（pamoate、embonate)、棕棚酸鹽、泛酸鹽、過氣酸鹽、磷酸鹽/二磷酸鹽、多聚半乳糖醛酸鹽、柳酸鹽、硬脂酸鹽、琥珀酸鹽、硫酸鹽、胺基磺酸鹽、鹼式乙酸鹽（subacetate)、琥珀酸鹽、丹寧酸鹽、酒石酸鹽、甲苯磺醯酸鹽、三氟醋酸鹽以及戊酸鹽。其他鹽類包含鈣、鋰、鎂、鈉及鉀鹽；胺基酸（例如：離胺酸、精胺酸）之鹽類；胍、二乙醇胺或膽素；銨、取代銨鹽或鋁鹽。該等鹽類可以藉由常用之技術予以製備。較佳之本發明VIP受體拮抗劑係如下列： (Rl=Aib、R2=4-D_Cl-Phe、R3=Leu): His-Ser-Asp-Aib-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys- Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：2) 1237029 五、發明說明（9) (Rl=Deg、R2=4-D-Cl-Phe、R3=Leu): His-Ser-Asp-Deg-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys- Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：3) (Rl=Ac5c、R2=4-D-Cl-Phe、R3=Leu): His-Ser-Asp-Ac5c-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys- Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：4) (Rl=Aib、R2=Phe、R3=Met): His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：5) (Rl=Aib、R2=Phe、R3=Leu): His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gin-Leu-Ala-Val_Lys-Ly s-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：6) (Rl=Ac5c、R2=Phe、R3=Leu): His-Ser-Asp-Ac5c-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：7) (Rl=Deg、R2=Phe、R3=Leu): His-Ser-Asp-Deg-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu· Arg-Lys-Gin-Leu-Ala-Val_Lys-Ly s-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：8)V. Description of the invention (8) Acetate, ascorbate, benzenesulfonate, benzoate, argon carbonate, bisulfate, hydrogen tartrate, borate, camphor sulfonate, carbonate, citrate, Dihydrogenate, mesylate, ethanesulfonate, p-toluenesulfonate, cyclohexylaminosulfonate, quinine salt, diethylamine tetraacetate, edisylate, estolate, acetoate Salt, fumarate, glucuronide, proline glutamate, glycerol phosphate, hydrobromide, argonate, hydroxynaphthoate, lactate, lactobionate , Laurate, malate, cis-butenoate, mandelate, mesylate, mucate, napsylate, nitrate, η · methylglucose Amine, oleate, oxalate, Palmoates, pamoate, embonate, palmitate, pantothenate, peroxyacid, phosphate / bisphosphate, polygalacturonic acid Salt, salicylate, stearate, succinate, sulfate, aminosulfonate, subacetate, Succinate, tanninate, tartrate, tosylate, trifluoroacetate and valerate. Other salts include calcium, lithium, magnesium, sodium, and potassium salts; salts of amino acids (eg, lysine, arginine); guanidine, diethanolamine, or choline; ammonium, substituted ammonium, or aluminum salts. These salts can be prepared by commonly used techniques. Preferred VIP receptor antagonists of the present invention are as follows: (Rl = Aib, R2 = 4-D_Cl-Phe, R3 = Leu): His-Ser-Asp-Aib-Val-4-Cl-D-Phe-Thr -Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys- Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (Sequence ID: 2 ) 1237029 V. Description of the invention (9) (Rl = Deg, R2 = 4-D-Cl-Phe, R3 = Leu): His-Ser-Asp-Deg-Val-4-Cl-D-Phe-Thr-Asp -Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys- Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (Sequence ID: 3) ( (Rl = Ac5c, R2 = 4-D-Cl-Phe, R3 = Leu): His-Ser-Asp-Ac5c-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu -Arg-Lys-Gln-Leu-Ala-Val-Lys- Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (sequence identification number: 4) (Rl = Aib, R2 = Phe, R3 = Met): His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn -Ser-Ile-Leu-Asn-NH2 (sequence identification number: 5) (Rl = Aib, R2 = Phe, R3 = Leu): His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr -Thr-Arg-Leu-Arg-Lys-Gin-Leu-Ala-Val_Lys-Ly s-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (Serial Identification Number: 6) (Rl = Ac5c, R2 = Phe, R3 = Leu): His-Ser-Asp-Ac5c-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser- Ile-Leu-Asn-NH2 (sequence identification number: 7) (Rl = Deg, R2 = Phe, R3 = Leu): His-Ser-Asp-Deg-Val-Phe-Thr-Asp-Asn-Tyr-Thr- Arg-Leu · Arg-Lys-Gin-Leu-Ala-Val_Lys-Ly s-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (Serial Identification Number: 8)

12 123702912 1237029

五、發明說明（10) (Rl=Aib、R2=Phe、R3=Dpg): His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：9) (Rl=Aib、R2=4-Cl-D-Phe、R3=Dpg): His-Ser-Asp-Aib-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號·· 10) (Rl=Deg、R2=Phe、R3=Dpg): His-Ser-Asp-Deg-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：11) (Rl=Ac5c、R2=Phe、R3=Dpg): His-Ser-Asp-Ac5c-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：12) 本發明之新穎的化合物具有重要的藥學應用。其為具有潛力之抗腫瘤藥劑，且因此而具有治療多種人類癌症之潛力。適合之投藥途徑為該技藝中所已知，且其包含口服、肛門、穿過皮層、***、穿過黏膜或小腸投藥；非經消化道傳送，其包含肌肉内注射、皮下注射、脊髓内注射、以及腦脊髓膜内注射、直接心室内注射、靜脈内注射、腹腔内注射、鼻内注射或眼内注射。 13 1237029 五、發明說明（11) 適合供本發明使用之藥學組成物包含組成物，其中所包含之活性成份係呈一個可達到其所欲療效之有效數量。該’’一個有效數量"之術語係意指一種藥物或藥學藥劑可以引發所推設之一組織、系統、動物或人體產生生物或醫學反應。除了活性成份之外，這些藥學組成物可以包含適合之藥學上可接受的載劑賦形劑、稀釋劑、溶劑、調味劑、調色劑等等。該製備可以被配方呈任何一種形式，其包含但不限制為藥錠、袠糖衣之藥丸、膠囊、粉末、糖漿、懸浮液、淤漿、隨時間釋放配方、持續釋放配方、藥丸、顆粒、乳化劑、貼片、注射劑、溶液、脂質體及奈米粒子。實際之配方、投藥途徑以及劑量可以由每位醫師視該病患之情況做選擇。本發明胜肽之毒性及治療效力可以藉由在細胞培養或實驗動物體内進行標準之藥學方法予以測定。本發明所示例之新穎性胜肽類似物包含胺基酸，稱為 α，α-二院基化胺基酸，其已經被已知可在胜肽之骨架中引發高度專一性之約制（constraint)。本發明中所使用之α，α·二烷基化胺基酸是合成自對應之網。於一個本發明之較佳具體例中，該酮被轉變為對應之乙内醯脲，其可使用一種強酸或鹼（較佳是硫酸、氫氣酸、氫氧化納或碳酸鈉）予以水解，俾以產生上述之胺基酸。於一個本發明之較佳具體例中，6〇0/〇之硫酸被使用來做為水解試劑。將酮轉變為其等所適合之α，α-二烷基化胺 14 1237029V. Description of the invention (10) (Rl = Aib, R2 = Phe, R3 = Dpg): His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys -Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (sequence identification number: 9) (Rl = Aib, R2 = 4-Cl-D-Phe, R3 = Dpg): His-Ser-Asp-Aib-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys -Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (sequence identification number · 10) (Rl = Deg, R2 = Phe, R3 = Dpg): His-Ser-Asp-Deg-Val- Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (Sequence Identification Number: 11) (Rl = Ac5c, R2 = Phe, R3 = Dpg): His-Ser-Asp-Ac5c-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln- Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (sequence identification number: 12) The novel compound of the present invention has important pharmaceutical applications. It is a potential anti-tumor agent and therefore has the potential to treat a variety of human cancers. Suitable routes of administration are known in the art and include oral, anal, transdermal, vaginal, transmucosal, or small intestine administration; non-gastrointestinal delivery, which include intramuscular, subcutaneous, and spinal injection , And intracranial spinal injection, direct intraventricular injection, intravenous injection, intraperitoneal injection, intranasal injection or intraocular injection. 13 1237029 V. Description of the invention (11) The pharmaceutical composition suitable for use in the present invention includes the composition, and the active ingredient contained therein is in an effective amount to achieve the desired therapeutic effect. The term "'an effective amount" means that a drug or pharmaceutical agent can cause a biological or medical response of a putative tissue, system, animal or human body. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carrier excipients, diluents, solvents, flavors, colorants, and the like. The preparation can be formulated in any form, which includes but is not limited to tablets, capsules, sugar-coated pills, capsules, powders, syrups, suspensions, slurries, release formulations over time, sustained release formulations, pills, granules, emulsification Agents, patches, injections, solutions, liposomes and nano particles. The actual formulation, route of administration and dosage can be chosen by each physician depending on the condition of the patient. The toxicity and therapeutic efficacy of the peptides of the present invention can be determined by performing standard pharmaceutical methods in cell culture or experimental animals. The novel peptide analogs exemplified in the present invention include amino acids, known as α, α-diethylated amino acids, which have been known to trigger a highly specific restriction in the peptide backbone ( constraint). The α, α · dialkylated amino acid used in the present invention is synthesized from the corresponding net. In a preferred embodiment of the present invention, the ketone is converted into the corresponding hydantoin, which can be hydrolyzed using a strong acid or base (preferably sulfuric acid, hydrogen acid, sodium hydroxide or sodium carbonate). To produce the amino acids described above. In a preferred embodiment of the invention, 600/0 sulfuric acid is used as a hydrolysis reagent. Conversion of ketones to their suitable α, α-dialkylated amines 14 1237029

五、發明說明（12) 基酸係如實施例1所顯示。本發明之新穎性胜肽類似物係已藉由使用固相技術或者藉由溶液相方法與固相技術之組合或者藉由片斷縮合予以產生（Stewart and Young，1969)。於一個本發明之較佳具體例中，胜肽之合成是使用 Fmoc方法，於一半自動胜肽合成儀（CS Bio,536型）(CS Bio Co.，San Carlos，California，U. S. Α·)上使用最佳之支鍵保護。胜肽之組合是從C·端向N-端。羧基端被醯胺化之胜肽是使用Rink醯胺樹脂予以合成。第一個受Fmoc保護之胺基酸是經由與該固相擔體形成一個醯胺鍵來達成，其係藉由異丙基碳二亞胺（DIPCDI)及HOBt做為媒介。自動化合成之取代數量較佳是介於每公克樹脂在0.2及0.8毫莫耳之間。使用下列步驟來合成包含於本發明之VIP類似物： 15 12370295. Description of the invention (12) The basic acid system is as shown in Example 1. The novel peptide analogs of the present invention have been produced by using solid phase technology or by a combination of solution phase method and solid phase technology or by fragment condensation (Stewart and Young, 1969). In a preferred embodiment of the present invention, the peptide is synthesized using the Fmoc method on a semi-automatic peptide synthesizer (CS Bio, Model 536) (CS Bio Co., San Carlos, California, US Α ·). Use the best branch protection. The combination of peptides is from the C-terminus to the N-terminus. The peptides whose carboxyl end is aminated with amidine are synthesized using Rink amidine resin. The first Fmoc-protected amino acid was achieved by forming an amido bond with the solid support, which was mediated by isopropylcarbodiimide (DIPCDI) and HOBt. The amount of substitution by automated synthesis is preferably between 0.2 and 0.8 millimoles per gram of resin. The following steps were used to synthesize the VIP analogs included in the present invention: 15 1237029

五、發明說明（η)表I 步驟試劑混合時間（分鐘）循環之數目 1. 二氮甲烷 1 2 2. 二甲基甲醯胺 1 1 3. 溶於二甲基甲醯胺之20%六氩吡啶 1 1 4. 溶於二曱基甲醯胺之20%六氩吡啶 29 1 5. 二甲基甲醯胺 1 3 6. 異丙酵 1 2 7· 二氯甲烷 1 2 8. 胺基酸可變 1 9. 二甲基甲醯胺 1 2 10. 停止或進行下一個循環被使用來合成羧基-醯胺化類似物之樹脂是為購自於 Calbioichem-Novabiochem Corp·，La Jolla, U.S.A 之 4-(2’，4’·二甲氧基苯基-Fmoo胺基甲基苯氧基甲基衍生的聚笨乙烤1 %二乙稀基苯（Ring Amide)樹脂（100-200篩孔）(0.47毫當量NH2/公克樹脂）。本發明亦提供一種用以製備一個具有通式⑴之胜肽類似物的固相合成法： His-Ser-Asp-Rl-Val-R2-Thr-Asp-Asn-Tyr-Thr-Arg-Leu- Arg-Lys-Gln-R3-Ala-Val-Lys-Val-Lys-Lys-Tyr-Leu-Asn- Ser-Ile-Leu-Asn-NH2其中 R1 是 Aib、Deg或 Ac5c、 R2是Phe或 4-Cl-D-Phe、 16 1237029V. Description of the invention (η) Table I Step reagent mixing time (minutes) Number of cycles 1. Diazomethane 1 2 2. Dimethylformamide 1 1 3. 20% soluble in dimethylformamide Argon pyridine 1 1 4. 20% hexaargyridine dissolved in dimethylformamide 29 1 5. Dimethylformamide 1 3 6. Isozyme 1 2 7 · Dichloromethane 1 2 8. Amine Acid variable 1 9. Dimethylformamide 1 2 10. Resin used to synthesize carboxy-fluorenated analogs to stop or proceed to the next cycle was purchased from Calbioichem-Novabiochem Corp., La Jolla, USA 4- (2 ', 4' · Dimethoxyphenyl-Fmoo Aminomethylphenoxymethyl Derived Polybenzyl Ethylene 1% Ring Amide) Resin (100-200 sieve Pores) (0.47 milliequivalent NH2 / g resin). The present invention also provides a solid-phase synthesis method for preparing a peptide analogue having the general formula ⑴: His-Ser-Asp-Rl-Val-R2-Thr- Asp-Asn-Tyr-Thr-Arg-Leu- Arg-Lys-Gln-R3-Ala-Val-Lys-Val-Lys-Lys-Tyr-Leu-Asn- Ser-Ile-Leu-Asn-NH2 where R1 is Aib, Deg or Ac5c, R2 is Phe or 4-Cl-D-Phe, 16 1237029

五、發明說明（14) R3 是 Met、Leu或 Dpg 該方法包含：在連續循環中將對應之受保護之αα•二烷基化胺基酸循序地裝載至一個固相樹脂之胺基端，在傳統溶劑及試劑之存在下偶合胺基酸來組裝一個胜肽·樹脂組裝體，移除保濩基團以及自樹脂切割出胜狀來產生一個粗製之胜肽類似物。於本發明之一個特佳具體例中，下列之化學分子部分於合成步驟中被使用來保護胜肽之反應性支鏈。 Ν端胺基基團係利用為9-第基甲氧基羰基（Fm〇c)基團來保護。三苯甲基（trt)或t-丁氧羰基（B〇c)為組織胺酸之咪嗤基團之較佳的保濩基團。絲胺酸、穌胺酸以及酷胺酸之經基基團較佳地係為t- 丁基（t-Bu)基團來予以保護。 (2’2,5’7’8-五甲基-色滿_6-硫酿基）(1>111(：)或（2,2,4,7-五甲基二氩苯并呋喃-5-磺醯基（pbf)為精胺酸之胍基基團之較佳的保濩基團。二本甲基保護基團係為護天冬醯胺及楚胺醯胺精胺酸及麩醯胺酸，而t-丁基（t-Bu)係為天冬胺酸及麩胺酸之較佳保護基團。色胺酸係為未受保護的或使用B〇c予以保護。離胺酸之支鏈較佳地係使用B〇c予以保護。於一個本發明之較佳具體例中，每一樹脂氮當量使用 2-8當量之受Fmoc保護的胺基酸。在固相合成胜肽中活化試劑被使用來偶合胺基酸為本技藝中所熟知。活化試劑包含 DCC、DIPCDI、DIEA、BOP、PyBOP、HBTU、TBTU 以及HOBt〇較佳地，DCC或DIPCDI/HOBt 或HBTU/HOBt 及DIEA被使用於偶合反應中做為活化試劑。 17 1237029 五、發明說明（15) 受保護之胺基酸若非於原位被活化即為呈預先活化之本技藝中所熟知的酯類（例如：NHS酯類、〇?印酯類等等）來被添加。Atherton, E· et al” 1988, J Chem· Soc. Perkin Trans· I· 2887，Bodansky，M.於"Peptides，Analysis， Synthesis and Biology (E. Gross, J. Meienhofer eds.)Vol. I, Academic Press，New York，1979，106 o 偶合反應於DMF、DCM、或NMP或者此等溶劑之混合物中被進行，且藉由Kaiser試驗（Kaiser et al·，Anal. Biochem·，34, 595-598, 1970)予以監測。任何一種未完全之. 反應係使用新鮮配製之活化試劑予以重新偶合。於完成本發明類似物之組裝後，使用步驟1-6移除胺基端之Fmoc基團，然後以甲醇沖洗該胜肽·樹脂並乾燥之。此時本發明類似物脫去保護且藉由於室溫下與三氟乙酸、結晶紛、乙二硫醇、硫代茴香醚以及去離子水反應歷時 1.5-5小時來切除樹脂擔體。藉由以冷的乾燥***沉澱、過滅、溶解及冷/東乾燥’產生粗製之胜肷。純化所產生之粗製的胜版係藉由製備式高壓液態層析儀（HPLC) ’於一製備式高壓液態層析儀（HpLC)系統 (Shimadzu Corporation，japan)上使用一 Lichr〇CART⑧C18(250· Times.10)逆相管柱 Darmstadt， Germany)，使用一配於於乙腈及水内之〇1〇/〇 TFA梯度。該洗提出來的分離部分於分析用HPLC系統（ShimadzuCorporation，Japan)上使用―個^ Lichr〇sphere⑧ wp-300 (300.X 4)逆相管柱予以再次分析。揮發掉乙腈，然後冷凍 18 1237029 五、發明說明（16) 乾燥該分離部分來製得純質之胜肽。每一個胜肽之鑑別是藉由電子喷灑式質譜儀予以確定。本發明之一個類似物可以藉由絕對固相技術、藉由部分固相/溶液相技術以及/或藉由片斷縮聚法來製成。用以合成本發明胜肽之較佳的半自動、循序式固相法被提供在本案下一個部分所討論之實施例。本發明將參照下列實施例做更進一步詳細的描述，對熟習本技藝之人士將瞭解到，這僅為闡釋而不應被解釋為限制性。在不偏離本發明之精義及範疇下，本發明之各種其他的修改會是有可能的。整基酸之合成 cx，a-二烷基化胺基酸係自適當之酮被合成。首先這些酮被轉變為其對應之乙内醯脲，其以一種強酸或鹼（較佳是硫酸、氫氣酸、氩氧化鈉或碳酸鈉）水解而產生個別之胺基酸。實施例1 環戊酮（0.1莫耳）、氰化鉀（KCN)(0.3莫耳）被溶解於300 毫升之50%水性甲醇中，然後該混合物在水浴上予以迴流，歷時4-6小時。接續地，該溶液被濃縮至其一半之體積且於一冰浴中被冷凍。以2N氫氣酸（HC1)酸化該冷凍之溶液。過濾且以冷水沖洗數次藉此所製得之沉澱物來移除殘餘之氰化物。接續地乾燥且自水性酒精溶液再結晶該固體。上文所述反應之產物的產率被發現為86%。藉此製得之5,5’-螺環戊烷乙内醯脲是以LR•光譜法（羰基團之特徵展 19 1237029 五、發明說明（17) 開帶（stretching bands)可分別在 1710-1740 cm·1 以及 1760-1780 cm·1被觀察到）予以鑑定。 5,5’-螺環戊烷乙内醯脲（0.05莫耳）被溶解於45毫升之 60%硫酸中且於150-160°C下予以迴流歷時約28小時。該反應混合物被冷卻至室溫且以水（150毫升）稀釋。過濾該稀釋之溶液來移除焦炭顆粒。於冰冷之水中冷凍該澄清液。該溶液被進一步濃縮及冷卻。製得耀眼之白色沉澱物。藉此製得之沉澱物被過濾且乾燥。該胺基酸（即1·胺基環戊烷羧酸（Ac5c))是以I.R.光譜法（羰基團之1610-1640 cm·1以及 NH3 +基團之3060-3090 cm·1)予以鑑定。實施例2 製備 Fmoc-Asn-(trt)-樹脂 Fmoc-Asn-(trt)-樹脂之一個典型製備係使用0.5公克之構自於Calbioichem -Novabiochem Corp.，La Jolla, U.S.A· 之4-(2’，4’-二甲氧基苯基-Fmoc·胺基甲基）苯氧基甲基-衍生的聚苯乙烯1%二乙烯苯（Rink Amide)樹脂（0.47 mM/g)(100-200篩孔）。首先允許該樹脂於二氣甲烷中膨脹 (兩次的25毫升歷時10分鐘）。於二甲基曱醯胺中沖洗一次，歷時1分鐘。自動化程序中所有的溶劑每次添加是以20 毫升之分量。樹脂上之Fmoc_保護基團係藉由依照合成製程之步驟3至7予以移除。脫去保護之Fmoc-基團是藉甴在一正Kaiser試驗中有藍色珠粒存在來予以檢測。對於在樹脂之自由胺基（NH2)基團上裝載第一個胺基酸而言，於胜肽合成儀之胺基酸容槽中稱量超過4倍重量之Fmoc· 20 1237029 五、發明說明（18)V. Description of the invention (14) R3 is Met, Leu or Dpg. The method includes: sequentially loading the corresponding protected αα • dialkylated amino acid to the amine end of a solid phase resin in a continuous cycle. Coupling amino acids in the presence of traditional solvents and reagents to assemble a peptide-resin assembly, removing the cymbals, and cleavage of the peptide from the resin to produce a crude peptide analog. In a particularly preferred embodiment of the present invention, the following chemical molecules are used in the synthetic steps to protect the reactive branched chain of the peptide. The N-terminal amine group is protected with a 9-th methoxycarbonyl (Fmoc) group. Trityl (trt) or t-butoxycarbonyl (Boc) is a preferred sulfonium protecting group of the imidino group of histamine. The serine, serine, and glutamic acid radicals are preferably protected as t-butyl (t-Bu) groups. (2'2,5'7'8-pentamethyl-chroman-6-sulfanyl) (1> 111 (:) or (2,2,4,7-pentamethyldiargonbenzofuran- 5-sulfofluorenyl (pbf) is a preferred guanidine group of arginine. The dimethyl protective group is aspartame and chloramine arginine and bran. Amines, and t-butyl (t-Bu) is a preferred protecting group for aspartic acid and glutamic acid. Tryptophan is either unprotected or protected with Boc. Ionamine The branched chain of the acid is preferably protected by using Boc. In a preferred embodiment of the present invention, 2 to 8 equivalents of Fmoc-protected amino acid are used for each resin nitrogen equivalent. It is synthesized in solid phase. Activation reagents in peptides are used to couple amino acids and are well known in the art. Activation reagents include DCC, DIPCDI, DIEA, BOP, PyBOP, HBTU, TBTU and HOBt. Preferably, DCC or DIPCDI / HOBt or HBTU / HOBt And DIEA is used as the activating reagent in the coupling reaction. 17 1237029 V. Description of the invention (15) If the protected amino acid is not activated in situ, it is an ester well known in the art (eg, pre-activated) : NHS esters, ? Esters, etc.) to be added. Atherton, E. et al "1988, J Chem. Soc. Perkin Trans. I. 2887, Bodansky, M. " Peptides, Analysis, Synthesis and Biology (E. Gross , J. Meienhofer eds.) Vol. I, Academic Press, New York, 1979, 106 o Coupling reactions were performed in DMF, DCM, or NMP or a mixture of these solvents, and Kaiser test (Kaiser et al. , Anal. Biochem., 34, 595-598, 1970). Any one is not complete. The reaction is re-coupled using a freshly prepared activating reagent. After the assembly of the analogue of the present invention is completed, use steps 1-6 The Fmoc group at the amine end was removed, and then the peptide · resin was washed with methanol and dried. At this time, the analog of the present invention was deprotected and reacted with trifluoroacetic acid, crystals, and ethylenedithiol at room temperature. The reaction of thioanisole and deionized water took 1.5-5 hours to remove the resin support. Precipitation, quenching, dissolution, and cold / dong drying with cold dry ether produced a crude tritium. Purification produced Crude Winning Plate Analytical Instrument (HPLC) 'A LichroCART⑧C18 (250 · Times.10) reverse phase column Darmstadt, Germany) was used on a preparative high pressure liquid chromatography (HpLC) system (Shimadzu Corporation, Japan) using a 〇0 / 〇TFA gradient in acetonitrile and water. The eluted fraction was reanalyzed on an analytical HPLC system (Shimadzu Corporation, Japan) using a Lichrosphere ™ wp-300 (300.X 4) reverse phase column. Evaporate the acetonitrile and freeze it. 18 1237029 V. Description of the invention (16) The separation is dried to obtain a pure peptide. The identification of each peptide was determined by an electron spray mass spectrometer. An analog of the present invention can be made by absolute solid phase technology, by partial solid phase / solution phase technology, and / or by fragment polycondensation. A preferred semi-automatic, sequential solid-phase method for synthesizing the peptides of the invention is provided in the examples discussed in the next section of this case. The present invention will be described in further detail with reference to the following examples. Those skilled in the art will appreciate that this is only an illustration and should not be construed as limiting. Various other modifications of the invention will be possible without departing from the spirit and scope of the invention. Synthesis of intact acids cx, a-dialkylated amino acids are synthesized from appropriate ketones. First these ketones are converted to their corresponding hydantoin, which is hydrolyzed with a strong acid or base (preferably sulfuric acid, hydrogen acid, sodium argon oxide or sodium carbonate) to produce individual amino acids. Example 1 Cyclopentanone (0.1 mol) and potassium cyanide (KCN) (0.3 mol) were dissolved in 300 ml of 50% aqueous methanol, and the mixture was refluxed on a water bath for 4-6 hours. Successively, the solution was concentrated to half its volume and frozen in an ice bath. The frozen solution was acidified with 2N hydrogen acid (HC1). The remaining cyanide was removed by filtering and rinsing the precipitate several times with cold water. The solid is successively dried and recrystallized from an aqueous alcohol solution. The yield of the reaction described above was found to be 86%. The 5,5'-spirocyclopentanehydantoin obtained in this way is based on LR spectrometry (Characteristics of Carbonyl Group 19 1237029) V. Description of the invention (17) The opening bands can be found in 1710- 1740 cm · 1 and 1760-1780 cm · 1 were observed). 5,5'-spirocyclopentanehydantoin (0.05 mol) was dissolved in 45 ml of 60% sulfuric acid and refluxed at 150-160 ° C for about 28 hours. The reaction mixture was cooled to room temperature and diluted with water (150 ml). The diluted solution was filtered to remove coke particles. The clear solution was frozen in ice-cold water. The solution was further concentrated and cooled. A dazzling white precipitate was obtained. The precipitate thus obtained was filtered and dried. The amino acid (ie, 1.aminocyclopentanecarboxylic acid (Ac5c)) was identified by I.R. spectroscopy (1610-1640 cm · 1 of carbonyl group and 3060-3090 cm · 1 of NH3 + group). Example 2 Preparation of Fmoc-Asn- (trt) -resin A typical preparation of Fmoc-Asn- (trt) -resin uses 0.5 g of 4- (2) from Calbioichem-Novabiochem Corp., La Jolla, USA. ', 4'-Dimethoxyphenyl-Fmoc · aminomethyl) phenoxymethyl-derived polystyrene 1% Rink Amide resin (0.47 mM / g) (100-200 Sieve). The resin was first allowed to swell in two-gas methane (25 ml twice for 10 minutes). Rinse once in dimethylamidamine for 1 minute. All solvents in the automated procedure are added in 20 ml portions. The Fmoc_ protecting group on the resin was removed by following steps 3 to 7 of the synthetic process. Deprotected Fmoc-groups were detected by the presence of blue beads in a positive Kaiser test. For loading the first amino acid on the free amine (NH2) group of the resin, weigh more than 4 times the weight of Fmoc · 20 1237029 in the amino acid tank of the peptide synthesizer. 5. Description of the invention (18)

Asn-(trt)-OH，以及超過相同倍數重量之丁醇（HOBt)。二者被溶於二甲基甲醯胺中（A.C.S.等級）(J.T.Baker， Phillipsburg，New Jersey，U.S.A.)，然後於添加至合成儀之反應槽中的樹脂之前先以DIPCDI活化。丁醇（HOBt)被添加於所有的結合反應中，特別是在Arg、Asn、Gin、His之情況中。結合反應被進行所歷時之一段時間範圍是1·3小時。當Kaiser測試產生一個負結果時，第一個胺基酸之裝載完成且當該樹脂於抽真空下乾燥過夜之後被稱重時，該具有第一個胺基酸相連之樹脂有一足量之重量增加。實施例3Asn- (trt) -OH, and butanol (HOBt) in excess of the same multiple weight. Both were dissolved in dimethylformamide (A.C.S. grade) (J.T. Baker, Phillipsburg, New Jersey, U.S.A.) and then activated with DIPCDI before adding the resin to the synthesizer's reaction tank. Butanol (HOBt) is added to all binding reactions, especially in the case of Arg, Asn, Gin, His. The binding reaction was carried out over a period of time ranging from 1.3 hours. When the Kaiser test produces a negative result, the first amino acid is loaded and when the resin is weighed after drying overnight under vacuum, the resin with the first amino acid attached has a sufficient weight increase. Example 3

合成序列辨識編號：2 (Aib4· 4-Cl-D-Phe6,Leu17VVIPSynthetic sequence identification number: 2 (Aib4 · 4-Cl-D-Phe6, Leu17VVIP

His-Ser-Asp-Aib-4-Cl_D-Phe-Thr-Asp-Asn-Tvr-Thr-Arg-His-Ser-Asp-Aib-4-Cl_D-Phe-Thr-Asp-Asn-Tvr-Thr-Arg-

Leu-Arg-Lvs-Gln-Leu-Ala-Val_Lvs-Tvr-Leu-Asn-Ser-Ile-Leu-Arg-Lvs-Gln-Leu-Ala-Val_Lvs-Tvr-Leu-Asn-Ser-Ile-

Leu-Asn-NH? 合成羧基端醯胺化之（Aib4，4-Cl-D-Phe6，Leu17)-VIP是藉由使用所有裝載有如上文實施例2所製備之 Fmoc-Asn-(trt)-OH的樹脂來起始。其被引至合成循環内步驟1-10中的逐步脫去保護及結合步驟。於每一個結合反應中，一超過4倍重量之胺基酸、DIPCDI以及丁醇（HOBt)被使用之。於完成合成及移除N端Fmoc保護基團（合成循環之步驟1-6)，該胜狀-樹脂以甲醇沖洗兩次後，乾燥稱重製得 0.649公克。該胜肽-樹脂被引至一個包含有三氟乙酸及捕 21 1237029 五、發明說明（19) 捉劑結晶盼、結晶酚、乙二硫醇、硫代茴香醚以及水之混合物中於室溫下被連續攬拌，歷時3-5小時的一段時間。使用冷的乾燥***沉澱該胜肽來製得約33〇毫克之粗製的胜狀。該粗製的胜肷於一 C18製備式逆相高壓液態層析儀 (HPLC)逆相管柱（250 X 1〇)之一個0.P/。tFa溶於乙醯腈及水之梯度上被純化，係如本技藝中所描述。顯著之波峰被收集且冷凍乾燥後，在分析式高壓液態層析儀（HPLC)上被再次分析，然後被引至質譜分析。觀測到之分子量與計算之分子量之間有良好的相關性。此時再以所產生之純質胜肽進行生物-分析。實施例4 合成序列辨謐編號：5『Aib4l-VIP His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tvr-Thr-Arg-Leu- Arg-Lvs-Gln-Met-Ala-Val-Lvs-Lvs-Tvr-Leu-Asn-Ser-Ile- Leu-Asn-NH^ 一個得自實施例2之0.255公克之Fmoc- Asn-(trt)-Rink 醯胺樹脂被引至使用"本發明之詳細說明"所載述之方法進行固相合成。所有的偶合都是使用適當超過所需Fmoc-胺基酸莫耳數予以進行。使用為諸等熟習此項技藝人士所熟知之偶合試劑及添加物。於完成該胜肽之裝載後，自樹脂移除Fmoc基團係如上文所述。依據上文所述之方法切割、冷凍乾燥、純化及定性該胜肽。實施例5 合成序列辨識編號：9『Aib4,Dpg17l-VI£ 22 1237029Leu-Asn-NH? Synthesis of carboxyl-terminated amidated (Aib4,4-Cl-D-Phe6, Leu17) -VIP by using all loaded with Fmoc-Asn- (trt) prepared as in Example 2 above -OH resin to start. It is directed to the stepwise deprotection and binding steps in steps 1-10 in the synthesis cycle. In each binding reaction, more than 4 times the weight of amino acids, DIPCDI and butanol (HOBt) were used. After completion of the synthesis and removal of the N-terminal Fmoc protecting group (steps 1-6 of the synthesis cycle), the winner-resin was washed twice with methanol, and then dried and weighed to obtain 0.649 g. The peptide-resin was introduced into a mixture containing trifluoroacetic acid and carbon dioxide 21 1237029 V. Description of the invention (19) Catch crystals, crystalline phenol, ethylene dithiol, thioanisole and water at room temperature It was stirred continuously for a period of 3-5 hours. The peptide was precipitated with cold dry ether to obtain about 33.0 mg of crude peptide. The crude product was obtained in a C18 preparative reverse-phase high-pressure liquid chromatography (HPLC) reverse-phase column (250 X 10) at a rate of 0.1 p /. tFa was purified on a gradient of acetonitrile and water, as described in this technique. Significant peaks were collected and lyophilized, analyzed again on an analytical high pressure liquid chromatography (HPLC), and then directed to mass spectrometry. There is a good correlation between the observed molecular weight and the calculated molecular weight. Bio-analysis was then performed with the resulting pure peptide. Example 4 Synthetic sequence identification number: 5 "Aib4l-VIP His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tvr-Thr-Arg-Leu- Arg-Lvs-Gln-Met-Ala- Val-Lvs-Lvs-Tvr-Leu-Asn-Ser-Ile- Leu-Asn-NH ^ A 0.255 g Fmoc-Asn- (trt) -Rink amine resin obtained from Example 2 was introduced to use " The method described in the detailed description of the present invention performs solid phase synthesis. All couplings were performed using an appropriate number of moles of Fmoc-amino acid. Use coupling reagents and additives known to those skilled in the art. After loading of the peptide, the removal of the Fmoc group from the resin is as described above. The peptide was cut, freeze-dried, purified, and characterized according to the methods described above. Example 5 Synthetic sequence identification number: 9 "Aib4, Dpg17l-VI £ 22 1237029

五、發明說明（20) His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tvr-Thr-Arg-Leu- Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile- Leu-Asn-NHi 一個得自實施例2之0·255公克之Fmoc-Asn-(trt)-Rink 醯胺樹脂被引至使用“本發明之詳細說明”内所載述之方法進行固相合成。所有的偶合都是使用適當超過所需Fmoc-胺基酸莫耳數予以進行。使用為諸等熟習此項技藝人士所熟知之偶合試劑及添加物。於一個本發明之較佳具體例中，依據上文所述之序列使用適當受保護之胺基酸進行27 個偶合循環。於完成該胜肽之裝載後，自樹脂移除Fmoc 基團係如上文所述。依據上文所述之方法切割、冷;東乾燥、純化及定性該胜肽。實施例6 合成胜肽之細胞毒性被測試於6株人類腫瘤細胞株，其名稱為：PA1(印巢）、SW620(直腸）、HuTu80(十二指腸）、 L132(肺）、U87MG(神經膠質母細胞）、KB(口腔）、 MIAPaCa2(胰臟）、A549(非小細胞肺）以及HT-29(直腸）。該等腫瘤細胞於指數生長期被收取，然後再懸浮於培養液 (1.5 X 106細胞/毫升懸浮於含有10%胎牛血清（FBS)之 RPMI 1640)。添加150 μΐ之培養液至96凹槽之組織培養盤 (Nunc，Denmark)之凹槽内，繼之以30 μΐ之細胞懸浮液。該培養盤在培養箱（37°C，5% C02)内放置隔夜。添加20 μΐ之胜肷（100 ρΜ -ΙμΜ)至96凹槽培養盤之標記的凹槽内。每一個濃度以三重複被佈植於盤内。20 μΐ之培養液被添加至對 23 1237029 五、發明說明（21) 照之凹槽内，而以沒有細胞之凹槽做為空白組。確定每一個凹槽之總體積為200 μΐ，然後培養盤被留置於培養箱内 (37°C，5% C02)。於72小時培養之後，進行一種ΜΤΤ分析法，然後細胞毒性百分比係對比對照組之細胞而被計算出來。下列表格顯示在各種不同之細胞株所達到之細胞毒性的極大值。 His-Ser-Asp-Aib-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn_Ser-Ile-Leu-Asn-NH2(序列辨識編號：2) 24 1237029V. Description of the invention (20) His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tvr-Thr-Arg-Leu- Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr -Leu-Asn-Ser-Ile- Leu-Asn-NHi A Fmoc-Asn- (trt) -Rink amine resin from Example 2.255g was introduced into the "Detailed Description of the Invention" The method described performs solid phase synthesis. All couplings were carried out using an appropriate number of moles of Fmoc-amino acid. Use coupling reagents and additives well known to those skilled in the art. In a preferred embodiment of the present invention, 27 coupling cycles are performed using a suitably protected amino acid according to the sequence described above. After the loading of the peptide is completed, the Fmoc group is removed from the resin as described above. Cut, chill; dry, purify, and characterize the peptide according to the methods described above. Example 6 The cytotoxicity of synthetic peptides was tested on 6 human tumor cell lines, and their names were: PA1 (Indus nest), SW620 (rectum), HuTu80 (duodenum), L132 (lung), U87MG (gliablast) ), KB (oral), MIAPaCa2 (pancreas), A549 (non-small cell lung), and HT-29 (rectal). These tumor cells were collected during the exponential growth phase, and then resuspended in culture medium (1.5 X 106 cells / ml suspended in RPMI 1640 containing 10% fetal bovine serum (FBS)). Add 150 μΐ of the culture solution to the groove of a 96-well tissue culture plate (Nunc, Denmark), followed by 30 μΐ of the cell suspension. The plate was placed in an incubator (37 ° C, 5% CO2) overnight. Add 20 μΐ of tritium (100 pM-1 μM) to the marked grooves of a 96-well plate. Each concentration was planted in pans in triplicate. 20 μΐ of the culture medium was added to the groove of the photo of 23, 1237029 V. Description of the invention (21), and the groove without cells was used as a blank group. Make sure that the total volume of each groove is 200 μΐ, and then place the plate in the incubator (37 ° C, 5% C02). After 72 hours of incubation, an MTT assay was performed and the percentage of cytotoxicity was calculated compared to the cells of the control group. The following table shows the maximum cytotoxicity achieved in various cell lines. His-Ser-Asp-Aib-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu- Asn_Ser-Ile-Leu-Asn-NH2 (Serial Identification Number: 2) 24 1237029

五、發明說明（22) 細胞株於不同濃度下之細胞毒性百分比 ΙμΜ ΙΟΟηΜ ΙΟηΜ InM ΙΟΟρΜ PA1 16.5 ±3.4 18.9 ±4.2 28.9 ±5.5 30.0 ±6.7 16 士 3·3 SW620 18.5 i 5.1 23±3·8 30±4·5 28 ± 6.6 16.9 ±4.5 HuTu80 39 ±4.5 24 土 5·6 18 土 4.5 20 士 5·5 10 土 3·5 L132 15.9 土 7.5 18.9 ±5.0 30.9 土 7.0 28 士 4.5 18 士 2.3 U87MG 14 土 4.5 19.0 士 7·0 28.9 士 5·6 12 ±7.6 10.5 ±4.5 KB 43 ± 0.5 37 土 5·0 34 土 6·0 42 士 8.0 47 土 8.5 MIAPaCa2 36 士 0.5 32 士 4·5 35 ±3.5 31 土 5.0 20 ±6.5 A549 45 ± 5.5 41 士 6·0 21 士 5.5 19 土 4.5 16 土 5.5 HT29 38 ±5.5 30 ±5.8 25 士 4.5 25 土 4.5 26 ±5.5V. Description of the invention (22) Percent cytotoxicity of cell strains at different concentrations 1 μM 10OηΜ 1 10ηΜ InM ΙΟΟρΜ PA1 16.5 ± 3.4 18.9 ± 4.2 28.9 ± 5.5 30.0 ± 6.7 16 ± 3 SW620 18.5 i 5.1 23 ± 3 · 8 30 ± 4 · 5 28 ± 6.6 16.9 ± 4.5 HuTu80 39 ± 4.5 24 soil 5.6 18 soil 4.5 20 ± 5 5 10 soil 3.5 · 132 L 15.9 soil 7.5 18.9 ± 5.0 30.9 soil 7.0 28 ± 4.5 18 ± 2.3 U87MG 14 Soil 4.5 19.0 ± 7.0 28.9 ± 5.6 12 ± 7.6 10.5 ± 4.5 KB 43 ± 0.5 37 ± 5.0 34 ± 6.0 42 ± 8.0 47 ± 8.5 MIAPaCa2 36 ± 0.5 32 ± 4.5 35 ± 3.5 31 soil 5.0 20 ± 6.5 A549 45 ± 5.5 41 ± 6.0 21 ± 5.5 19 ± 4.5 16 ± 5.5 HT29 38 ± 5.5 30 ± 5.8 25 ± 4.5 25 ± 4.5 26 ± 5.5

His-Ser-Asp-Deg-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser_Ile-Leu-Asn-NH2(序列辨識編號：3) 細胞株於不同濃度下之細胞毒性百分比 ΙμΜ ΙΟΟηΜ ΙΟηΜ InM ΙΟΟρΜ ΡΑ1 22.1 ±4.5 23.5 ±5.2 22 ± 4.5 29 ±5.8 15 士 4.5 SW620 16 士 3.4 22 ± 7.3 27 ±5.5 29 ± 5.6 15.9 土 6.6 HuTu80 14.5 ±7.1 24 ± 7.8 28 ± 4.7 29 ± 6.2 14 土 7.8 L132 14 士 26 ± 6.5 29 ±6.7 23 i 3.5 14 士 5.6 U87MG 25 士 4.6 26 士 6.7 28 ±7.5 16 ±6.6 11 土 7.8 KB 14 土 3.4 18 士 8·5 22 ± 8.2 24.5 ± 9.5 13 ±8.5His-Ser-Asp-Deg-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr- Leu-Asn-Ser_Ile-Leu-Asn-NH2 (sequence identification number: 3) Percent cytotoxicity of the cell line at different concentrations 1 μM 10〇ηΜ ΙΟηΜ InM ΙΟΟρΜ ΡΑ1 22.1 ± 4.5 23.5 ± 5.2 22 ± 4.5 29 ± 5.8 15 ± 4.5 SW620 16 ± 3.4 22 ± 7.3 27 ± 5.5 29 ± 5.6 15.9 ± 6.6 HuTu80 14.5 ± 7.1 24 ± 7.8 28 ± 4.7 29 ± 6.2 14 ± 7.8 L132 14 ± 26 ± 6.5 29 ± 6.7 23 i 3.5 14 ± 5.6 U87MG 25 ± 4.6 26 ± 6.7 28 ± 7.5 16 ± 6.6 11 ± 7.8 KB 14 ± 3.4 18 ± 8.5 22 ± 8.2 24.5 ± 9.5 13 ± 8.5

His-Ser-Asp-Ac5c-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：4) 25 1237029 五、發明說明（23) 細胞株於不同濃度下之細胞毒性百分比 ΙμΜ ΙΟΟηΜ ΙΟηΜ InM ΙΟΟρΜ PA1 21 土 5·5 22.3 土 4·5 23 ±3.5 30 ±6.0 16.1 ±0.0 SW620 15 土 4.5 23 ± 6.7 28.5 土 4.5 31 土 6·5 16.9 ±5.5 HuTu80 15.5 ±6.5 27 ± 7.9 27 ±5.0 30 ±7.0 11 土 8·0 L132 13 ±4.5 28 ±5.5 30 ±5.5 27 士 4·5 11 土 8.0 U87MG 27 ±5.5 28 i 7.7 29 土 6·7 15 士 7.8 10 ±4.6 KB 27 土 4.3 26 ±5.6 27 ±7.8 30 ±7.8 13 土 8·0His-Ser-Asp-Ac5c-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr- Leu-Asn-Ser-Ile-Leu-Asn-NH2 (sequence identification number: 4) 25 1237029 V. Description of the invention (23) Percent cytotoxicity of cell lines at different concentrations ΙμΜ ΙΟΟηΜ ΙΟηΜ InM ΙΟΟρΜ PA1 21 土 5 · 5 22.3 Soil 4.55 23 ± 3.5 30 ± 6.0 16.1 ± 0.0 SW620 15 Soil 4.5 23 ± 6.7 28.5 Soil 4.5 31 Soil 6.5 16.9 ± 5.5 HuTu80 15.5 ± 6.5 27 ± 7.9 27 ± 5.0 30 ± 7.0 11 Soil 8.0 L132 13 ± 4.5 28 ± 5.5 30 ± 5.5 27 ± 4 · 5 11 Soil 8.0 U87MG 27 ± 5.5 28 i 7.7 29 Soil 6.7 15 ± 7.8 10 ± 4.6 KB 27 Soil 4.3 26 ± 5.6 27 ± 7.8 30 ± 7.8 13 Soil 8 · 0

His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：9) 細胞株於不同濃度下之細胞毒性百分比 ΙμΜ ΙΟΟηΜ ΙΟηΜ InM ΙΟΟρΜ ΡΑ1 15.1 ±2.3 17.4 土 3.2 25.6 土 4·5 27.8 土 4.3 15.5 土 3.3 SW620 16.7 土 4·1 21 土 3.6 27 士 3.4 28 ±5.0 15.9 士 4.5 HuTu80 14.6 ±5.1 23 ± 6.0 26 ±5.5 28·5 土 3.2 14.6 ±2.7 L132 13.6 ± 5.6 17.5 ±5.5 18.9 ±6.0 20 土 0·0 17 土 2.3 U87MG 12.5 ±6.5 18.9 ±6.5 26.7 ±3.5 13.7 ±3.6 11.5 ±5.0 KB 10.5 ±6.5 16·7 土 5.1 16.7 ±3.2 21·5 土 4.5 13 ±5.0His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser- Ile-Leu-Asn-NH2 (sequence identification number: 9) Percent cytotoxicity of cell strains at different concentrations 1 μM ΙΟΟΜΜ ΙΟηΜ InM ΙΟΟρΜ ΡΑ1 15.1 ± 2.3 17.4 ± 3.2 25.6 ± 4.5 27.8 ± 4.3 15.5 ± 3.3 SW620 16.7 4 · 1 21 soil 3.6 27 people 3.4 28 ± 5.0 15.9 people 4.5 HuTu80 14.6 ± 5.1 23 ± 6.0 26 ± 5.5 28 · 5 soil 3.2 14.6 ± 2.7 L132 13.6 ± 5.6 17.5 ± 5.5 18.9 ± 6.0 20 soil 0 · 0 17 soil 2.3 U87MG 12.5 ± 6.5 18.9 ± 6.5 26.7 ± 3.5 13.7 ± 3.6 11.5 ± 5.0 KB 10.5 ± 6.5 16 · 7 soil 5.1 16.7 ± 3.2 21 · 5 soil 4.5 13 ± 5.0

His-Ser-Asp-Aib-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：10) 26 1237029His-Ser-Asp-Aib-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr- Leu-Asn-Ser-Ile-Leu-Asn-NH2 (Serial Identification Number: 10) 26 1237029

五、發明說明（24) 細胞株於不同濃度下之細胞毒性百分比 ΙμΜ ΙΟΟηΜ ΙΟηΜ InM ΙΟΟρΜ PA1 23 土 6·5 25 ± 5.0 26 ±3.0 31 土 6.0 28 土 0.0 SW620 16 土 5·5 24 ±7.3 29 士 5.0 30 士 6.5 17 ±4.0 HuTu80 16 士 4.5 28 ± 8.0 28 ± 5.0 31 士 7·0 15 ±7.8 L132 17 ±5.0 29 ±5.5 31 士 6.0 28 土 6.0 15 ±7.0 U87MG 29 土 4.0 29 ±7.0 30.1 ±4.0 16 士 7.8 12 ±5.0 KB 30 ±5.0 27 土 6.0 26 ± 7.8 32 土 7.8 12 士 8.0 所有的參考文獻在此併入本案做為參考資料，其包含每一篇公開發表文獻中所列述之胺基酸序列。所有於上文所提到之公開發表文獻中揭露及參照之化合物皆在此併入本案做為參考資料，其包含該被引述於公開發表文獻中之期刊論文所揭露及參照之化合物。 27 1237029 五、發明說明（25) 序列表 <110>亞納德C.布爾門蘇德哈納德•帕拉薩德拉瑪•穆克海爵亞努T.辛弗亞爾契納·瑪士爾尼納·谷普塔 <120>腸血管肽類似物 <130> U012800-2 <140〉 <141〉 <150> 136/DEL/2000 <151〉 2000-02-18 <160〉 12 <170> Patentln Ver. 2.0 <210〉 1 <211> 28 <212〉 PRT <213〉 Sus barbatus <400〉 1 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Met Ala Val Lys Lys Tyr Leu Asn Ser He Leu Asn 20 25 28 1237029V. Description of the invention (24) Percent cytotoxicity of cell strains at different concentrations 1 μM ΙΟΟηΜ ΙΟηΜ InM ΙΟΟρΜ PA1 23 soil 6.5 25 ± 5.0 26 ± 3.0 31 soil 6.0 28 soil 0.0 SW620 16 soil 5.5 5 ± 24 29 ± 5.0 30 ± 6.5 17 ± 4.0 HuTu80 16 ± 4.5 28 ± 8.0 28 ± 5.0 31 ± 7.0 15 ± 7.8 L132 17 ± 5.0 29 ± 5.5 31 ± 6.0 28 ± 6.0 15 ± 7.0 U87MG 29 ± 4.0 29 ± 7.0 30.1 ± 4.0 16 ± 7.8 12 ± 5.0 KB 30 ± 5.0 27 ± 6.0 26 ± 7.8 32 ± 7.8 12 ± 8.0 All references are hereby incorporated into this case as reference material, which contains a description of each publicly published document Amino acid sequence. All of the compounds disclosed and referenced in the published literature mentioned above are incorporated herein as reference materials, which include the compounds disclosed and referenced in the journal articles cited in the published literature. 27 1237029 V. Description of the invention (25) Sequence list < 110 > Yannard C. Burmensud Hanad Parasdram Mukhail Yanu T. Simfer Alcina Mas Elna Gupta < 120 > Intestinal vascular peptide analogue < 130 > U012800-2 < 140> < 141> < 150 > 136 / DEL / 2000 < 151> 2000-02-18 < 160〉 12 < 170 > Patentln Ver. 2.0 < 210〉 1 < 211 > 28 < 212〉 PRT < 213〉 Sus barbatus < 400〉 1 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Met Ala Val Lys Lys Tyr Leu Asn Ser He Leu Asn 20 25 28 1237029

五、發明說明（26) <210> 2 <211〉 28 <212〉 PRT <213〉人工序列 <220〉 <223〉人工序列之描述：此胜肽係被合成地產生者。 <220〉 <221〉 MOD 一 RES <222〉（4) <223〉/產物=〇:-胺基異丁酸/標記=Aib <220〉 <221〉 MOD 一RES <222〉（6) <223> /產物=4-氣-D-***酸/標記4-Cl-D-Phe <400〉 2 His Ser Asp Xaa Val Xaa Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Asn Ser He Leu Asn 20 25 <210〉 3 <211〉 28 <212〉 PRT <213〉人工序列 <220〉 <223>人工序列之描述：此胜肽係被合成地產生者。 29 1237029 五、發明說明（27) <220〉 <221〉 MOD 一 RES <222〉（4) <223〉/產物=二-乙基甘胺酸/標記=Deg <220〉 <221〉 MOD 一 RES <222〉（6) <223〉/產物=4-氯-D-***酸/標記=4-Cl-D-Phe <400〉 3 His Ser Asp Xaa Val Xaa Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Asn Ser lie Leu Asn 20 25 <210〉 4 <211> 28 <212〉 PRT <213〉人工序列 <220〉 <223〉人工序列之描述：此胜肽係被合成地產生者。 <220〉 <221〉 MOD一 RES <222〉（4) <223> /產物=1-胺基環丙烷羧酸/標記=Ac5c <220> <221〉 MOD— RES <222〉（6) <223〉/產物=4-氣-D-***酸/標記=4-Cl-D-Phe 30 1237029V. Description of the invention (26) < 210 > 2 < 211〉 28 < 212〉 PRT < 213〉 Artificial sequence < 220〉 < 223〉 Description of artificial sequence: This peptide was produced synthetically . < 220〉 < 221〉 MOD-RES < 222〉 (4) < 223〉 / product = 〇: -aminoisobutyric acid / label = Aib < 220〉 < 221〉 MOD-RES < 222〉 (6) < 223 > / product = 4-gas-D-phenylalanine / label 4-Cl-D-Phe < 400〉 2 His Ser Asp Xaa Val Xaa Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Asn Ser He Leu Asn 20 25 < 210〉 3 < 211〉 28 < 212〉 PRT < 213〉 artificial sequence < 220> < 223 > Description: This peptide was produced synthetically. 29 1237029 V. Description of the invention (27) < 220〉 < 221〉 MOD-RES < 222〉 (4) < 223〉 / product = di-ethylglycine / label = Deg < 220〉 < 221〉 MOD-RES < 222〉 (6) < 223〉 / product = 4-chloro-D-phenylalanine / label = 4-Cl-D-Phe < 400〉 3 His Ser Asp Xaa Val Xaa Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Asn Ser lie Leu Asn 20 25 < 210〉 4 < 211 > 28 < 212〉 PRT < 213〉 artificial sequence < 220> < 223> Description of the artificial sequence: This peptide was produced synthetically. < 220〉 < 221〉 MOD-RES < 222〉 (4) < 223 > / product = 1-aminocyclopropanecarboxylic acid / label = Ac5c < 220 > < 221〉 MOD— RES < 222〉 (6) < 223〉 / product = 4-gas-D-phenylalanine / label = 4-Cl-D-Phe 30 1237029

五、發明說明（28) <400〉 4 His Ser Asp Xaa Val Xaa Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Leu Ala Val Lys Lys Tyr Leu Asn Ser He Leu Asn 20 25 <210〉 5 <211〉 28 <212〉 PRT <213〉人工序列 <220〉 <223〉人工序列之描述：此胜肽係被合成地產生者。 <220> <221〉 MOD一 RES <222〉（4) <223〉/產物胺基異丁酸/標記=Aib <400〉 5 His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Asn Ser He Leu Asn 20 25 <210〉 6 <211> 28 <212> PRT <213〉人工序列 <220> 31 1237029 五、發明說明（29) <223〉人工序列之描述：此胜肽係被合成地產生者。 <220〉 <221〉 MOD 一RES <222> (4) <223〉/產物胺基異丁酸/標記=Aib <400〉 6 His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Leu Ala Val Lys Lys Tyr Leu Asn Ser He Leu Asn 20 25 <210> 7 <211〉 28 <212〉 PRT <213>人工序列 <220> <223>人工序列之描述：此胜肽係被合成地產生者。 <220〉 <221〉 MOD 一 RES <222〉（4) <223> /產物=1-胺基環丙烧敌酸/標記=Ac5c <400〉 7 His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Leu Ala Val Lys Lys Tyr Leu Arg Ser He Leu Asn 20 25 32 1237029V. Description of the invention (28) < 400〉 4 His Ser Asp Xaa Val Xaa Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Leu Ala Val Lys Lys Tyr Leu Asn Ser He Leu Asn 20 25 < 210〉 5 < 211> 28 < 212> PRT < 213> Artificial sequence < 220> < 223 > Description of artificial sequence: This peptide was produced synthetically. < 220 > < 221〉 MOD-RES < 222〉 (4) < 223〉 / product aminoisobutyric acid / label = Aib < 400〉 5 His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Asn Ser He Leu Asn 20 25 < 210〉 6 < 211 > 28 < 212 > PRT < 213> Artificial sequence < 220 > 31 1237029 five 2. Description of the invention (29) < 223> Description of the artificial sequence: This peptide was produced synthetically. < 220〉 < 221〉 MOD-RES < 222 > (4) < 223〉 / product aminoisobutyric acid / label = Aib < 400〉 6 His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Leu Ala Val Lys Lys Tyr Leu Asn Ser He Leu Asn 20 25 < 210 > 7 < 211〉 28 < 212〉 PRT < 213 > Artificial Sequences < 220 > < 223 > Description of the artificial sequence: This peptide was produced synthetically. < 220〉 < 221〉 MOD-RES < 222〉 (4) < 223 > / product = 1-aminocyclopropanedioic acid / label = Ac5c < 400〉 7 His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Leu Ala Val Lys Lys Tyr Leu Arg Ser He Leu Asn 20 25 32 1237029

五、發明說明（30) <210〉 8 <211〉 28 <212〉 PRT <213>人工序列 <220〉 <223>人工序列之描述：此胜肽係被合成地產生者。 <220〉 <221〉 MOD-RES <222〉（4) <223〉/產物=二-乙基甘胺酸/標記=Deg <400> 8 His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Leu Ala Val Lys Lys Tyr Leu Asn Ser lie Leu Asn 20 25 <210〉 9 <211〉 28 <212〉 PRT <213>人工序列 <220> <223>人工序列之描述：此胜肽係被合成地產生者。 <220〉 <221〉 MOD 一 RES <222〉（4) <223> /產物胺基異丁酸/標記=Aib 33 1237029 五、發明說明（31) <220〉 <221〉 MOD 一RES <222〉（17) <223〉/產物=二-正丙基甘胺酸/標記=Dpg <400〉 9 His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Xaa Ala Val Lys Lys Tyr Leu Asn Ser lie Leu Asn 20 25 <210〉 10 <211> 28 <212〉 PRT <213〉人工序列 <220〉 <223>人工序列之描述：此胜肽係被合成地產生者。 <220〉 <221〉 MOD 一 RES <222〉（4) <223> /產物=〇;-胺基異丁酸/標記=Aib <220〉 <221> M0D.RES <222〉（6) <223〉/產物=4-氣-D-***酸/標記=4-Cl-D-Phe <220〉 <221〉 MOD 一 RES <222> (17) 34 1237029V. Description of the invention (30) < 210〉 8 < 211〉 28 < 212〉 PRT < 213 > Artificial sequence < 220〉 < 223 > Description of artificial sequence: This peptide was produced synthetically . < 220〉 < 221〉 MOD-RES < 222〉 (4) < 223〉 / product = di-ethylglycine / label = Deg < 400 > 8 His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Leu Ala Val Lys Lys Tyr Leu Asn Ser lie Leu Asn 20 25 < 210〉 9 < 211〉 28 < 212〉 PRT < 213 > Artificial Sequences < 220 > < 223 > Description of the artificial sequence: This peptide was produced synthetically. < 220〉 < 221〉 MOD-RES < 222〉 (4) < 223 > / product aminoisobutyric acid / label = Aib 33 1237029 V. Description of the invention (31) < 220> < 221〉 MOD-RES < 222〉 (17) < 223〉 / product = di-n-propylglycine / label = Dpg < 400〉 9 His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Xaa Ala Val Lys Lys Tyr Leu Asn Ser lie Leu Asn 20 25 < 210〉 10 < 211 > 28 < 212〉 PRT < 213〉 artificial sequence < 220〉 < 223 > description of artificial sequence : This peptide was produced synthetically. < 220〉 < 221〉 MOD-RES < 222〉 (4) < 223 > / product = 〇; -aminoisobutyric acid / label = Aib < 220〉 < 221 > M0D.RES < 222〉 (6) < 223〉 / product = 4-gas-D-phenylalanine / label = 4-Cl-D-Phe < 220〉 < 221〉 MOD-RES < 222 > (17) 34 1237029

五、發明說明（32) <223> /產物=二-正丙基甘胺酸/標記=Dpg <400〉 10 His Ser Asp Xaa Val Xaa Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Xaa Ala Val Lys Lys Tyr Leu Asn Ser He Leu Asn 20 25 <210〉 11 <211〉 28 <212〉 PRT <213〉人工序列 <220〉 <223>人工序列之描述：此胜肽係被合成地產生者。 <220〉 <221〉 MOD 一 RES <222〉（4) <223> /產物=二-乙基甘胺酸/標記=Deg <220〉 <221〉MODJ^ES <222> (17) <223〉/產物=二-正丙基甘胺酸/標記=Dpg <400> 11 His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Xaa Ala Val Lys Lys Tyr Leu Asn Ser He Leu Asn 20 25 35 1237029 五、發明說明（33) <210> 12 <211〉 28 <212〉 PRT <213〉人工序列 <220〉 <223〉人工序列之描述：此胜狀係被合成地產生者。 <220〉 <221〉 MOD 一RES <222〉（4) <223〉/產物=1-胺基環丙烷羧酸/標記=Ac5c <220〉 <221〉 MOD-RES <222〉（17) <223〉/產物=二-正丙基甘胺酸/標記=Dpg <400〉 12 His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Xaa Ala Val Lys Lys Tyr Leu Asn Ser He Leu Asn 20 25 36V. Description of the invention (32) < 223 > / product = di-n-propylglycine / label = Dpg < 400〉 10 His Ser Asp Xaa Val Xaa Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Xaa Ala Val Lys Lys Tyr Leu Asn Ser He Leu Asn 20 25 < 210〉 11 < 211〉 28 < 212〉 PRT < 213〉 Artificial Sequence < 220〉 < 223 > Description of Artificial Sequence: This win Peptides are produced synthetically. < 220〉 < 221〉 MOD-RES < 222〉 (4) < 223 > / product = di-ethylglycine / label = Deg < 220〉 < 221〉 MODJ ^ ES < 222 > (17) < 223〉 / product = di-n-propylglycine / label = Dpg < 400 > 11 His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Xaa Ala Val Lys Lys Tyr Leu Asn Ser He Leu Asn 20 25 35 1237029 V. Description of the invention (33) < 210 > 12 < 211〉 28 < 212〉 PRT < 213〉 artificial sequence < 220〉 < 223〉 artificial Description of sequence: The winner is a synthetic producer. < 220〉 < 221〉 MOD-RES < 222〉 (4) < 223〉 / product = 1-aminocyclopropanecarboxylic acid / label = Ac5c < 220〉 < 221〉 MOD-RES < 222〉 (17) < 223〉 / product = di-n-propylglycine / label = Dpg < 400〉 12 His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin 15 10 15 Xaa Ala Val Lys Lys Tyr Leu Asn Ser He Leu Asn 20 25 36

Claims

、申請專利範圍第090118676號專利再審查案申請專利範圍修正本修正曰期：94年4月 1. 一種具有通式（I)之腸血管肽類似物： His-Ser-Asp-Rl-Val-R2-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-R3-Ala-Val-Lys-Val-Lys-Lys-Tyr-Leu-Asn_ Ser-Ile-Leu-Asn-NH2 (I) 其中 R1 是 Aib、Deg 或 Ac5c， R2是 Phe或 4-Cl-D-Phe， R3 是 Met、Leu 或 Dpg ; 其中Aib是oc-胺基酸-異丁酸，Deg是二乙基甘胺酸， Ac5c是1-胺基環戊烷羧酸，以及Dpg是二-η-丙基甘胺酸；或其一藥學上可接受的鹽類。 2. 如申請專利範圍第1項之腸血管肽類似物，其中R1是 Aib、R2是4-D-Cl-Phe，且R3是Leu，且該腸血管肽類似物是： His-Ser-Asp-Aib-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr- Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Sel、Ile-Leu-Asn-NH2(序列辨識編號：2) 或其一藥學上可接受的鹽類。 3. 如申請專利範圍第1項之腸血管肽類似物，其中R1是 Deg、R2是4-D-Cl-Phe，且R3是Leu，且該腸血管肽類似物是： 1237029 六、申請專利範圍 His-Ser-Asp-Deg-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：3) 或其一藥學上可接受的鹽類。 4·如申請專利範圍第i項之腸血管肽類似物，其中以是 Ac5c、R2是4-D-Cl-Phe，且R3是Leu，且該腸血管肽類似物是： His-Ser-Asp-Ac5c-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gin-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：4) 或其一藥學上可接受的鹽類。 5. 如申請專利範圍第1項之腸血管肽類似物，其中R1是 Aib、R2是Phe，且R3是Met，且該腸血管肽類似物是： His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：5) 或其一藥學上可接受的鹽類。 6. 如申請專利範圍第1項之腸血管肽類似物，其中R1是 Aib、R2是Phe，且R3是Leu，且該腸血管月太類似物是： His-Ser-Asp- Aib-Val-Phe-Thr，Asp- Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH^ (序列辨識編 *5虎.6) 或其一藥學上可接受的鹽類。 7. 如申請專利範圍第1項之腸血管肽類似物，其中R1是 38 1237029 f、申請專利範圍 Ac5c、R2是Phe，且R3是Leu，且該腸血管肽類似物是： His-Ser-Asp-Ac5c-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：7) 或其一藥學上可接受的鹽類。 8·如申請專利範圍第1項之腸血管肽類似物，其中R1是 Deg、R2是Phe，且R3是Leu，且該腸血管肽類似物是： His-Ser-Asp-Deg-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：8) 或其一藥學上可接受的鹽類。 9·如申請專利範圍第1項之腸血管肽類似物，其中R1是 Aib、R2是Phe，且R3是Dpg，且該腸血管肽類似物是： His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Se卜Ile-Leu-Asn-NH2(序列辨識編號：9) 或其一藥學上可接受的鹽類。 10.如申請專利範圍第1項之腸血管肽類似物，其中R1是 Aib、R2是4-Cl-D-Phe，且R3是Dpg，且該腸血管肽類似物是： His-Ser-Asp-Aib-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg~Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2(序列辨識編號：10) 或其一藥學上可接受的鹽類。 39 1237029 六、申請專利範圍 11 ·如申请專利範圍第1項之腸血管肽類似物，其中r 1是 Deg、R2是Phe，且R3是Dpg，且該腸血管肽類似物是： His-Ser-Asp-Deg-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg- Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn- Ser-Ile-Leu-Asn-NH2(序列辨識編號：u) 或其一藥學上可接受的鹽類。 12 ·如申凊專利範圍第1項之腸血管肽類似物，其中& 1是 Ac5c、R2是Phe，且R3是Dpg，且該腸血管肽類似物是： His.Ser-Asp-Ac5c-Val-Phe.Thr-Asp-Asn-Tyr-Thr.Arg- Leu-Arg-Lys-Gln-Opg-Ala-Val-Lys-Lys-Tyr-Leu-Asn- Ser-Ile-Leu-Asn-NH2 (序列辨識編號：12) 或其一藥學上可接受的鹽類。 13. —種用於治療哺乳動物之癌症的藥學組成物，其包含有一有效數量之一如申請專利範圍第丨項之腸血管肽類似物，以及一藥學上可接受之載劑。 14·如申請專利範圍第13項之藥學組成物，其進_步包含一化學治療用化合物。 15·—種如申請專利範圍第丨項之腸血管肽類似物於製造〆藥物的用途，該藥物係供用於治療哺乳動物體内的癌症。 16.如申請專利範圍第15項之用途，其中該藥物進—步包含一化學治療用化合物。 402. The scope of application for patent re-examination of the patent scope No. 090118676 The amendment of the scope of patent application Date of revision: April 1994 1. An enterovascular peptide analogue with general formula (I): His-Ser-Asp-Rl-Val- R2-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-R3-Ala-Val-Lys-Val-Lys-Lys-Tyr-Leu-Asn_ Ser-Ile-Leu-Asn-NH2 (I) where R1 is Aib, Deg or Ac5c, R2 is Phe or 4-Cl-D-Phe, R3 is Met, Leu or Dpg; where Aib is oc-amino acid-isobutyric acid and Deg is diethyl Glycine, Ac5c is 1-aminocyclopentanecarboxylic acid, and Dpg is di-n-propylglycine; or a pharmaceutically acceptable salt thereof. 2. The intestinal vascular peptide analogue of item 1 in the patent application scope, wherein R1 is Aib, R2 is 4-D-Cl-Phe, and R3 is Leu, and the intestinal vascular peptide analogue is: His-Ser-Asp -Aib-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr- Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Sel Ile-Leu-Asn-NH2 (sequence identification number: 2), or a pharmaceutically acceptable salt thereof. 3. For example, the intestinal vaso-peptide analogue of item 1 of the patent application scope, wherein R1 is Deg, R2 is 4-D-Cl-Phe, and R3 is Leu, and the intestinal vaso-peptide analogue is: 1237029 Range His-Ser-Asp-Deg-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr -Leu-Asn-Ser-Ile-Leu-Asn-NH2 (sequence identification number: 3) or a pharmaceutically acceptable salt thereof. 4. The intestinal vascular peptide analogue according to item i of the application, wherein Ac5c, R2 is 4-D-Cl-Phe, and R3 is Leu, and the intestinal vascular peptide analogue is: His-Ser-Asp -Ac5c-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gin-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser -Ile-Leu-Asn-NH2 (sequence identification number: 4) or a pharmaceutically acceptable salt thereof. 5. The intestinal vascular peptide analogue of item 1 of the patent application scope, wherein R1 is Aib, R2 is Phe, and R3 is Met, and the intestinal vascular peptide analogue is: His-Ser-Asp-Aib-Val-Phe -Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (Sequence ID : 5) or a pharmaceutically acceptable salt thereof. 6. The intestinal vascular peptide analogue of item 1 of the patent application scope, wherein R1 is Aib, R2 is Phe, and R3 is Leu, and the intestinal vascular analogue is: His-Ser-Asp- Aib-Val- Phe-Thr, Asp- Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH ^ (Sequence Identification Editor * 5 Tiger. 6) or one of its pharmaceutically acceptable salts. 7. For example, the intestinal vaso-peptide analogue of item 1 of the patent application range, wherein R1 is 38 1237029 f, the application range of Ac5c, R2 is Phe, and R3 is Leu, and the intestinal vaso-peptide analogue is: His-Ser- Asp-Ac5c-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu- Asn-NH2 (sequence identification number: 7) or a pharmaceutically acceptable salt thereof. 8. The intestinal vascular peptide analogue according to item 1 of the patent application scope, wherein R1 is Deg, R2 is Phe, and R3 is Leu, and the intestinal vascular peptide analogue is: His-Ser-Asp-Deg-Val-Phe -Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (Sequence ID : 8) or a pharmaceutically acceptable salt thereof. 9. The intestinal vascular peptide analogue of item 1 in the patent application scope, wherein R1 is Aib, R2 is Phe, and R3 is Dpg, and the intestinal vascular peptide analogue is: His-Ser-Asp-Aib-Val-Phe -Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Se Ile-Leu-Asn-NH2 (sequence identification number : 9) or a pharmaceutically acceptable salt thereof. 10. The intestinal vascular peptide analogue according to item 1 of the patent application scope, wherein R1 is Aib, R2 is 4-Cl-D-Phe, and R3 is Dpg, and the intestinal vascular peptide analogue is: His-Ser-Asp -Aib-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg ~ Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser -Ile-Leu-Asn-NH2 (sequence identification number: 10) or a pharmaceutically acceptable salt thereof. 39 1237029 VI. Application for Patent Scope 11 · If the intestinal vascular peptide analogue of item 1 of the patent application scope, where r 1 is Deg, R2 is Phe, and R3 is Dpg, and the intestinal vascular peptide analogue is: His-Ser -Asp-Deg-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg- Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn- Ser-Ile-Leu -Asn-NH2 (sequence identification number: u) or a pharmaceutically acceptable salt thereof. 12 · The enterovascular peptide analogue of item 1 of the patent application, wherein & 1 is Ac5c, R2 is Phe, and R3 is Dpg, and the enterovascular peptide analogue is: His.Ser-Asp-Ac5c- Val-Phe.Thr-Asp-Asn-Tyr-Thr.Arg- Leu-Arg-Lys-Gln-Opg-Ala-Val-Lys-Lys-Tyr-Leu-Asn- Ser-Ile-Leu-Asn-NH2 ( Sequence identification number: 12) or a pharmaceutically acceptable salt thereof. 13. A pharmaceutical composition for treating cancer in mammals, which comprises an effective amount of an enterovascular peptide analogue such as item 1 of the patent application scope, and a pharmaceutically acceptable carrier. 14. The pharmaceutical composition according to item 13 of the application, further comprising a chemical therapeutic compound. 15 · —The use of an intestinal vaso peptide analogue such as item 丨 in the scope of patent application for the manufacture of a medicament for the treatment of cancer in mammals. 16. The use as claimed in claim 15 wherein the drug further comprises a chemical therapeutic compound. 40