WO2005081997A2

WO2005081997A2 - Isothiazole based protein kinase inhibitors

Info

Publication number: WO2005081997A2
Application number: PCT/US2005/005859
Authority: WO
Inventors: Congxin Liang
Original assignee: The Scripps Research Institute
Priority date: 2004-02-20
Filing date: 2005-02-22
Publication date: 2005-09-09
Also published as: WO2005082001A3; WO2005082001A2; WO2005081997A3

Abstract

Hydroxy containing isothiazole base derivatives have enhanced and unexpected drug properties as inhibitors of protein kinases and are useful in treating disorders related to abnormal protein kinase activities such as cancer.

Description

ISOTHIAZOLE BASED PROTEIN KINASE INHIBITORS

Description

Field of Invention: The invention relates to protein kinase inhibitors and to their use in treating disorders related to abnormal protein kinase activities such as cancer and inflammation. More particularly, the invention relates to isothiazole based compounds and their pharmaceutically acceptable salts employable as protein kinase inhibitors.

Background: Protein kinases are enzymes that catalyze the phosphorylation of hydroxyl groups of tyrosine, serine, and threonine residues of proteins. Many aspects of cell life (for example, cell growth, differentiation, proliferation, cell cycle and survival) depend on protein kinase activities. Furthermore, abnormal protein kinase activity has been related to a host of disorders such as cancer and inflammation. Therefore, considerable effort has been directed to identifying ways to modulate protein kinase activities. In particular, many attempts have been made to identify small molecules that act as protein kinase inhibitors.

Isothiazole based derivatives having activity as protein kinase inhibitors have been disclosed in Intemational Patent Application WO 09962890 and in US Patent No. 6235764. What is needed is a class of modified isothiazole based derivatives having both activity as protein kinase inhibitors and enhanced drug properties.

Summary: The invention is directed to hydroxy containing isothiazole derivatives and to their use as inhibitors of protein kinases. It is disclosed herein that hydroxy containing isothiazole derivatives have enhanced and unexpected drug properties that advantageously distinguish this class of compounds over known isothiazole derivatives having protein kinase inhibition activity. It is also disclosed herein that hydroxy containing isothiazole derivatives are useful in treating disorders related to abnormal protein kinase activities such as cancer.

This invention discloses that certain hydroxy compounds may have advantageous and unexpected properties that distinguish them from known compounds. They are therefore useful in treating disorders related to abnormal protein kinase activities such as cancer.

One aspect of the invention is directed to a compound represented by Formula (I):

In Formula I, X is O or S; R¹ is selected from the group consisting of (C1-C6) alkyl, (C3-C8) cycloalkyl, (C6-C10) arylalkyl, (C5-C9) heteroarylalkyl, substituted (C6-C10) arylalkyl, and substituted (C5-C9) heteroarylalkyl; R² and R³ are independently hydrogen or (C1-C6) alkyl; R⁴ is selected from the group consisting of hydrogen, (C1-C6) alkyl, and hydroxyl; n and m are independently 0, 1 , 2, or 3; p is independently 1 , 2, or 3; R⁵ is selected from the group consisting of hydroxyl, (C1- C6) alkoxy, (C3-C8) cycloalkoxy, substituted or unsubstituted O-(C6-C10) aryl, and NR⁶R⁷; R⁶ and R⁷ are independently selected from the group consisting of hydrogen, (C1-C6) alkyl, (C1-C6) hydroxyalkyl, (C1-C6) dihydroxyalkyl, (C1-C6) alkoxy , (C1-C6) alkyl carboxylic acid, (C1-C6) alkyl phosphoric acid, (C1-C6) alkyl sulfuric acid, (C1-C6) hydroxyalkyl carboxylic acid, (C1-C6) alkyl amide, (C3-C8) cycloalkyl, (C5-C8) heterocycloalkyl, (C6-C10) aryl, (C5-C9) heteroaryl, (C3-C8) cycloalkyl carboxylic acid; or R⁷ and R⁸ together with N forms a (C5-C8) heterocyclic ring either unsubstituted or substituted with one or more hydroxyls, ketones, ethers, and carboxylic acids; or NR⁶R⁷ may form a cyclic ring containing 0-3 additional heteroatoms selected from N, O, or S; or, a pharmaceutically acceptable salt, its tautomer, a pharmaceutically acceptable salt of its tautomer, prodrug thereof. It may also act as a prodrug. It should be understood that a compound of Formula (I) where R⁵ is OH may exist in its open-acid form or its cyclic-lactone form or the two forms may co-exist in solution or in vivo as illustrated in Figure I. Furthermore, all compounds of Formula (I) have at least one asymmetric center and the stereochemistry at the asymmetric center(s) is(are) either RS, R, or S. Preferred acid species of this embodiment are represented in Figure 2. Preferred amide species of this embodiment are represented in Figure 3.

In a preferred embodiment of this first aspect of the invention, the compound, salt, tautomer, or prodrug is represented by Formula (II):

In Formula II, R^5a is selected from the group consisting of hydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl, and substituted or unsubstituted (C6-C10) aryl. In a subgroup of this first embodiment, X is O; R¹ is selected from the group consisting of optionally substituted (C6-C10) arylmethylene and (C5-C9) heteroarylmethylene; R² and R³ are hydrogen; and R⁴ is selected from the group consisting of hydrogen and hydroxyl. Preferred species within this first embodiment are represented by the following structures:

In the second embodiment of the first aspect of the invention, R⁵ of Formula I is NR⁶R⁷ . In a subgroup of this second embodiment, X is O; R¹ is selected from the group consisting of optionally substituted (C6-C10) arylmethylene and (C5-C9) heteroarylmethylene; R² and R³ are hydrogen; and R⁴ is selected from the group consisting of hydrogen and hydroxyl. A first group of preferred species is represented by the following structures:

A second group of preferred species is represented by the following structures:

A third group of preferred species is represented by the following structures:

A fourth group of preferred species is represented by the following structures:

A fifth group of preferred species is represented by the following structures:

A sixth group of preferred species is represented by the following structures:

A seventh group of preferred species is represented by the following structures:

An eighth group of preferred species is represented by the following structures:

A ninth group of preferred species is represented by the following structures:

A tenth group of preferred species is represented by the following core structures:

Br r _Λ I H H F^H2NY°u _FH₂N θ

CORE II HO CORE III c H₂N^O r L _F H₂N^θ ^»ψ COR^"E IV^ °Φ& Ji_κ ^# o *X CORE V CORE VI

In the above core structure, R is selected from the group consisting of radical represented by the following structures:

NH COOH

Provisios may apply to any of the above subgroups or embodiments wherein any one or more of the other above described embodiments or species may be excluded from such subgroups or embodiments. Another aspect of the invention is directed to a method for the modulation of the catalytic activity of a protein kinase with a compound or salt of the first aspect of the invention. In a preferred mode, the protein kinase is selected from the group consisting of VEGF receptors and PDGF receptors.

Brief Description of the Drawings:

Figure 1 illustrates that the hydroxyl containing portion of the isothiazole based compound of the invention, and illustrates that this portion of the compound may exist in its open-acid form or its cyclic-lactone form or the two forms may co-exist in solution or in vivo. Figure 2 illustrates preferred compounds of the invention in acid or open-chain form. Figure 3 illustrates preferred compounds of the invention which are amides. Figure 4 illustrates a scheme used to synthesize the acid compounds in Figure 2. Figure 5 illustrates a method for completion of the synthesis of 1-10 which is representative of the acid compounds. Figure 6 illustrates a scheme used for the synthesis of compounds like 2-3.

Utility: The present invention provides compounds capable of regulating and/or modulating protein kinase activities of, but not limited to, VEGFR (Vascular Endothelial Growth Factor Receptor) and/or FGFR (Fibroblast Growth Factor Receptor). Thus, the present invention provides a therapeutic approach to the treatment of disorders related to the abnormal functioning of these kinases. Such disorders include, but not limited to, solid tumors such as glioblastoma, melanoma, and Kaposi's sarcoma, and ovarian, lung, prostate, pancreatic, colon and epidermoid carcinoma. In addition, VEGFR/FGFR inhibitors may also be used in the treatment of restenosis and diabetic retinopathy.

Furthermore, this invention relates to the inhibition of vasculogenesis and angiogenesis by receptor-mediated pathways, including the pathways comprising VEGF receptors, and/or FGF receptors. Thus the present invention provides therapeutic approaches to the treatment of cancer and other diseases which involve the uncontrolled formation of blood vessels.

Synthesis of Acid and Ester Compounds The compounds of this invention can be synthesized by following the published general procedures. But the following intermediates are specific to compounds of this invention and may be used in place of their respective counterparts in the published general procedures: (4 ^:?,6R)-t-butyl-6-(2-aminoethyl)- 2,2-dimethyl-1 ,3-dioxane-4-acetate; 4-amino-3-hydroxy-butanoic acid; 2-hydroxy-4- aminobutyric acid; and isoserine. These intermediates may be purchased from commercial sources (e.g. Fisher Scientific, Fairlawn, New Jersey and Sigma Aldrich, Milwaukee, Wisconsin). Another variation from the published general procedures is that in the synthesis of compounds using (4R,6R)-f-butyl-6-(2-aminoethyl)-2,2- dimethyl-1 ,3-dioxane-4-acetate, the protecting groups need to be removed from the final product. These variations from the published general procedures can be understood and carried out by those skilled in the art. The amides of Figure 3 can be readily synthesized from the acids of Figure 2. Thus, the compounds of the present invention can be synthesized by those skilled in the art.

The compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

Example 1: (3 ,5 ?)-7-{3-[3-(4-bromo-2,6-difluorobenzyloxy)-4- carbamoylisothiazol-5-yl]ureido}-3,5-dihydroxyheptanoic acid, sodium salt

The general synthetic sequence is depicted in Figure 4. The intermediates until isothiazole 1-6 were obtained according literature methods (Larson, E. R.; Noe, M. C. Gant, T. G. Isothiazole Derivatives Useful As Anticancer Agents. International publication number WO 99/62890; international publication date 12/09/1999. Larson, E. R.; Noe, M. O; Gant, T. G. Isothiazole Derivatives Useful As Anticancer Agents. United States patent US 6,235,764. Date of patent 5/22/2001 ) and are not further described in this report. However, the purification method for 1-5 was changed significantly and this process is therefore detailed in the experimental section. Accordingly, crude 1-5 was purified by crystallization from hot DMF, which proved more convenient than the tedious, repeated cent fugation procedure given in the patents cited above.

Preparation of 1-5: (4-Carbamoyl-3-hydroxyisothiazol-5-yl)-carbamic acid phenyl ester

Solid (3-benzyloxy-4-cyanoisothiazol-5-yl)-carbamic acid phenyl ester (1-4, 38.7g, 0.1 1 mol) was added slowly to concentrated sulfuric acid (75 mL) over 1 h and the mixture was stirred for additional 3 h. The viscous solution was diluted by slow addition of ice (150 g) followed by vigorous stirring for an additional 1 h. The precipitate was filtered and pressed on the filter without washing with water. The wet solid was dissolved in DMF (250 mL) under heating to 120 °C. The mixture was then cooled to room temperature and kept at ca. 5 °C for 3 h. The crystals were filtered and washed with acetonitrile (3x50 mL). The formed solid was then washed with aqueous NaHCO₃ solution (80 mL) and acetone (50 mL) and dried in vacuum to give (4-carbamoyl-3-hydroxyisothiazol-5-yl)-carbamic acid phenyl ester (15.0 g, 50 %) as a solid, which was used in the next step without further purification. ¹H NMR (300 MHz, DMSO-de/TMS): δ 8.08 (s, 1 H), 7.89 (s, 1 H), 7.45-7.40 (m, 2H), 7.31-7.27 (m, 3H), 7.00 (br s, 1 H). 4.00-3.70 (br, 1 H). ¹³C NMR (75 MHz, DMSO-d₆): δ 167.0, 164.8, 150.0, 130.0, 129.0, 126.0, 121.0, 1 17.4, 98.5. HMRS (LSIMS, nba): calcd for CnH₁₀O₄N₃S (MH⁺): 280.0392; found: 280.0383.

Preparation of 1-Na: (3 ?,5A?)-7-{3-[3-(4-bromo-2,6-difluorobenzyloxy)-4- carbamoylisothiazol-5-yl]ureido}-3,5-dihydroxyheptanoic acid, sodium salt

A solution of [3-(4-bromo-2,6-difluorobenzyloxy)-4-carbamoylisothiazol-5-yl]- carbamic acid phenyl ester (1-6, 2.55 g, 5.3 mmol) and (4R,6 ?)-[6-(2-aminoethyl)- 2,2-dimethyl-[1 ,3]dioxan-4-yl]-acetic acid te/f-butyl ester (1-7, 1.43 g, 5.3 mmol) in THF (50 mL) was stirred at 45 °C overnight. After cooling to room temperature, the mixture was filtered and the filtrate was concentrated. The residue was purified by column chromatography (silica gel, EtOAc:heptane = 1 :1 ) to give crude (4 ,6R)-[6- (2-{3-[3-(4-bromo-2,6-difluorobenzyloxy)-4-carbamoylisothiazol-5-yl]-ureido}-ethyl)- 2,2-dimethyl-[1 ,3]dioxan-4-yl]-acetic acid fe/τ-butyl ester (1-8, 1.5 g, 43%) as an oil. ¹H NMR (300 MHz, DMSO-de/TMS): δ 7.25 (d, J = 7.2 Hz, 2H), 7.12 (br, 2 H), 6.75 (br, 1 H), 6.05 (br, 1 H), 5.50 (s, 2H), 4.21-4.09 (m, 1 H), 4.00-3.90 (m, 1 H), 3.38-3.34 (m, 2H), 2.36-2.25 (m, 2H), 1.80-1.60 (m, 2H), 1.41 (s, 9H), 1.40-1.20 (m, 2 H), 1.39 (s, 3H), 1.29 (s, 3H). (4R,6R)-[6-(2-{3-[3-(4-Bromo-2,6-difluorobenzyloxy)-4- carbamoylisothiazol-5-yl]-ureido}-ethyl)-2,2-dimethyl-[1 ,3]dioxan-4-yl]-acetic acid tert- butyl ester (1-8, 0.5 g, 0.75 mmol) was added to MeOH (10 mL), coned aqueous HCl (0.5 mL) and THF (5 mL) and the mixture was stirred overnight at room temperature. The solvents were evaporated, the residue was extracted with EtOAc (50 mL), and the solution was dried over MgSO₄. After concentration, the solvent was evaporated and the residue was purified by column chromatography (CH₂CI₂:MeOH = 10:1) to give a mixture of (3R,5R)-7-{3-[3-(4-bromo-2,6-difluorobenzyloxy)-4- carbamoylisothiazol-5-yl]ureido}-3,5-dihydroxyheptanoic acid (1-9) and its methyl ester (0.17 g). This residue was stirred in a solution of NaOH (11 mg, 0.028 mmol) in MeOH (10 mL) and water (0.5 mL) for 3 h. The solvent was evaporated, MeOH (10 mL) was added and the mixture was concentrated. The residue (light yellow solid) was washed with /-PrOH (5 mL), filtered, washed with Et₂O (20 mL) and dried in vacuum. The obtained solid (0.2 g) was dissolved in MeOH (20 mL) and filtered through Celite filter agent. The solvent was evaporated and the residual solid was stirred with Et₂O (10 mL) and MeOH (1 mL) at room temperature overnight. The precipitate was filtered and dried in vacuum to give (3R,5R)-7-{3-[3-(4-bromo-2,6- difluorobenzyloxy)-4-carbamoylisothiazol-5-yl]ureido}-3,5-dihydroxyheptanoic acid, sodium salt (1-Na, 0.17 g, 38 %) as a white solid. Mp 207-208 °C (decomposition). ¹H NMR (300 MHz, CD₃OD/TMS), δ 7.29 (d, J = 7.2 Hz, 2H), 5.50 (s, 2H), 4.10 (m, 1 H), 3.86 (m, 1 H), 3.37-3.30 (m, 2H), 2.36-2.30 (m, 2H), 1.80-1.50 (m, 4H). ¹³C NMR (75 MHz, CD₃OD): D 180.3, 170.4, 167.0, 163.1 (dd, J =254, 8 Hz), 162.6, 156.1 , 124.4 (t, J = 13 Hz), 1 16.8 (d, J = 29 Hz), 113.0 (t, J = 19 Hz), 98.6, 69.1 , 68.7, 58.9, 45.6, 45.2, 38.3, 38.2. Example 2: (3R,5S)-6-{3-[3-(4-Bromo-2,6-difiuoro-benzyloxy)-4-carbamoyl- isothiazol-5-yl]-ureido}-3,5-dihydroxy-hexanoic acid tert-butyl ester.

Example 3: (3R,5S)-6-{3-[3-(4-Bromo-2,6-difluoro-benzyloxy)-4-carbamoyl- isothiazol-5-yl]-ureido}-3,5-dihydroxy-hexanoic acid.

The preparation of Examples 2 and 3 are outlined in Scheme 1 , below:

Scheme 1

Preparation of ((4R,6S)-6-Aminomethyl-2,2-dimethyl-[1 ,3]dioxan-4-yl)-acetic acid tert-butyl ester:

Triflic anhydride 1.4mL (2.36g, 8.345mmol) was dropwise added at -78 °C to a solution of 2,6-lutidine 1.35mL (11.63mmol) and f-Butyl (3R,5S)-6-hydroxy-3,5-O- isopropylidene-3,5-dihydroxyhexanoate 1.981g (7.609 mmol, obtained from Kaneka Corp.) in dichloromethane (anh., 50mL) over 3 minutes. The mixture was stirred at - 78 °C for 10 min, then placed on ice-slush bath and stirred at 0 °C for 45 min. The resulting pink mixture was transferred into ice-cooled solution of ammonia in methanol (7M solution, 200mL). The mixture was placed on ambient water bath and stirred at RT for 6 hours. The reaction mix was evaporated to dryness, the residue partitioned between ether (200mL) and aqueous potassium carbonate (6g in 200 mL of water), the aqueous phase re-extracted with ether (150mL). Combined organic extracts were dried (magnesium sulfate) and evaporated. The crude product was purified on a column of silica (125g) eluting with a mixture of chloroform-methanol- conc. aq. ammonia 100:10:1 (v/v) (1.5L) Y = 1.777g of a colorless liquid (90% th) ¹H (^dDMSO, 400MHz): 4.167(m, 1 H), 3.741 (m, 1 H), 2.484 (m, 2H), 2.384 (ddAB, J=15.2Hz, 5.1 Hz, 1 H), 2.201 (ddAB, J=15Hz, 7.8Hz, 1 H), 1.533 (br d, J=12.5Hz, 1H), 1.373 (s, 9H), 1.363 (s, 3H), 1.250 (br s, 2H), 1.223 (s, 3H)

Preparation of compound 2-1. ((4R,6S)-6-Aminomethyl-2,2-dimethyl-[1 ,3]dioxan-4- yl)-acetic acid tert-butyl ester (0.39 mmol) was added to a solution of compound 1-6 (100 mg, 0.2 mmol), and DIEA (0.3 mmol) in THF (5 mL). After the solution was stirred at 55 °C overnight, the solvent was removed via evaporation under reduced pressure. The resulting residue was subjected to flash chromatography (ISCO system, 25% - 60% ethyl acetate in hexane, 22 min.) to get the final product 2-1 (93 mg, 72%). LC-MS: single peak at 254 nm, MH⁺ calcd. for C₂₅H₃₁BrF₂N₄O₇S: 650, obtained: 650. ¹H-NMR (DMSO-d₆, 400 MHz), δ 11.06 (s, 1H), 8.32 (t, J = 5.6 Hz, 1H), 7.57 (m, 3H), 6.82 (s, 1H), 5.42 (s, 2H), 4.20 (m, 1H), 3.95 (m, 1H), 3.12 (m, 2H), 2.38 (dd, J = 4.8 Hz, J = 14.0 Hz, 1 H), 2.21 (dd, J - 4.8 Hz, J = 14.8 Hz, 1 H), 1.53 (dd, J = 2.8 Hz, J = 8.0 Hz, 1 H), 1.39 (s, 3H), 1.37 (s, 9H), 1.27 (s, 3H), 1.15 (dd, J = 7.2 Hz, J = 14.4 Hz, 1 H).

Synthesis of compound 2-2. Aqueous HCl (1.5 mL, 1.0M) was added to a solution of compound 2-1 (285 mg, 0.44 mmol) in MeOH (10 mL). A precipitation was observed immediately. After the suspension was stirred at 50 °C for 2.5h, the solution became clear, and LC-MS showed the reaction was complete. The MeOH was removed via evaporation under reduced pressure. The resulting aqueous solution was extracted using ethyl acetate (3x 50 mL). The combined organic solution was dried over Na₂SO , and the ethyl acetate was evaporated under vacuum to obtain the crude product (261 mg). This crude material was used directly in the next step without further purification. A small portion of this crude product was subjected to preparative HPLC to obtain the pure product 2-2. LC-MS: single peak at 254 nm, MH⁺ calcd. for C₂₂H₂₇BrF₂N₄O₇S: 610, obtained: 610. ¹H-NMR (DMSO- d₆, 400 MHz), δ 11.05 (s, 1 H), 8.30 (t, J = 5.6 Hz, 1 H), 7.57 (m, 3H), 6.81 (s, 1 H), 5.42 (s, 2H), 4.82 (d, J = 4.8 Hz, 1 H), 4.74 (d, J = 4.8 Hz, 1 H), 3.94 (m, 1 H), 3.65 (m, 1 H), 3.19 (m, 1 H), 3.04 (m, 1 H), 2.30 (dd, J = 4.2 Hz, J = 14.8 Hz, 1 H), 2.20 (dd, J = 8.0 Hz, J = 14.4 Hz, 1 H), 1.47 (t, J = 6.0 Hz, 2H), 1.36 (s, 9H).

Synthesis of compound 2-3. Aqueous NaOH (2 mL, 1.0M) was added to a solution of compound 2-2 (93 mg, -0.44 mmol. Crude from the previous step) in MeOH (2 mL). The solution was stirred at 25 °C for 1.5h, and LC-MS showed the reaction was complete. This solution was directly subjected to preparative HPLC to obtain the pure product 2-3 (62 mg, 71 %). LC-MS: single peak at 254 nm, MH⁺ calcd. for C₁₈H₁₈BrF₂N₄O₇S: 554, obtained: 554. ¹H-NMR (DMSO-d₆, 400 MHz), δ 11.05 (s, 1 H), 8.30 (t, J = 5.6 Hz, 1 H), 7.57 (m, 3H), 6.81 (s, 1 H), 5.40 (s, 2H), 5.05 (s, 1 H), 4.15 (s, 1 H), 3.76 (m, 1 H), 3.64 (m, 1 H), 3.13 (m, 1 H), 2.99 (m, 1 H), 2.02 (dd, J = 4.0 Hz, J = 14.8 Hz, 1 H), 1.82 (dd, J = 8.4 Hz, J = 15.2 Hz, 1 H), 1.42 (m, 1 H), 1.34 (m, 1 H). Example 4: 3-hydroxy-butyric acid derivatives.

Following the above procedures and procedures published in patents such as WO 09962890 and US 6235764, the following 3-hydroxy-butyric acid derivatives 4a-d can be made using the commercially available 4-amino-3-hydroxy-butyric acid (e.g. Fisher Scientific, Fairlawn, New Jersey).

Example δ: 3-(4-Bromo-2,6-difluoro-benzyloxy)-5-[3-((2S,4R)-2,4-dihydroxy-6- oxo-6-pyrrolidin-1 -yl-hexyl)-ureido]-isothiazole-4-carboxylic acid amide

The general procedure for the synthesis of amides of Examples 1 and 3 is shown in Scheme 3 below:

Scheme 3

An amine (3 equiv) was added to a solution of the sodium salt of a free acid (1 equiv), EDC (5 equiv), HOBt (5 equiv), and DIEA (5 equiv) in DMF. After the solution was stirred at 25 °C overnight (stirred at 55 °C for a couple of hours if necessary), DMF was removed via evaporation under reduced pressure. The resulting residue was suspended in ethyl acetate, washed by saturated NaHCO₃ (3x) and brine (3x), and dried over Na₂SO4. The ethyl acetate was removed under vacuum to give the crude product. This crude material was subjected to preparative HPLC to give the final product amide, which was subsequently characterized by LC-MS and NMR spectroscopy.

Synthesis of 3-(4-Bromo-2,6-difluoro-benzyloxy)-5-[3-((2S,4 ?)-2,4-dihydroxy-6- oxo-6-pyrrolidin-1-yl-hexyl)-ureido]-isothiazole-4-carboxylic acid amide: An amount of 31 mg (92%) product was obtained after preparative HPLC from 32 mg (0.056 mmol) of the free acid sodium salt. LC-MS: single peak at 254 nm, MH⁺ calcd. for C₂₂H₂6BrF₂N₅O₆S: 607, obtained: 607. ¹H-NMR (DMSO-d₆, 400 MHz), δ 11.05 (s, 1 H), 8.31 (s, 1H), 7.57 (s, 2H), 7.55 (s, 1H), 6.81 (s, 1 H), 5.42 (s, 2H ), 4.82 (d, J = 4.0 Hz, 1 H ), 4.76 (d, J = 4.0 Hz, 1 H ), 4.02 (m, 1 H ), 3.68 (m, 1 H), 3.42 (m, 2H), 3.27 (m, 2H), 3,16 (m, 1H), 3.03 (m, 1 H), 2.33 (m, 2H), 1.82 (m, 2H), 1.73 (m, 2H), 1.50 (m, 2H).

Examples 6 and 7: Amide derivatives of Examples 1 and 3.

The following amide derivatives 6a-f, 7a-f of Examples 1 and 3 can be made by those skilled in the art following the above and/or known procedures.

Example 8: Amide derivatives of Example 4.

The following amide derivatives 8a-f of Example 4 can be made by those skilled in the art following known procedures.

Example 9: further amide derivatives of Example 4.

The following amide derivatives 9a-f of Example 4 can be made by those skilled in the art following known procedures.

Example A. 4-{3-[3-(4-Bromo-2,6-difluoro-benzyloxy)-4-carbamoyl-isothiazol-5- yl]-ureido}-2-hydroxy-butyric acid

Example B. 3-{3-[3-(4-Bromo-2,6-difluoro-benzyloxy)-4-carbamoyl-isothiazol-5- yl]-ureido}-2-hydroxy-propionic acid

Example C. Amide examples of Example A.

Example D. Amide examples of Example B

Example E: Amide examples of Example 3:

Synthesis of Amide Compounds The amide compounds of this invention can be readily synthesized by those skilled in the art starting from the acid compound disclosed herein.

Exemplary Synthesis. An exemplary synthesis of an amide compound is illustrated in Scheme 4 shown below:

Scheme 4 An amine (3 equiv) is added to a solution of the sodium salt of a free acid (1 equiv), EDC (5 equiv), HOBt (5 equiv), and DIEA (5 equiv) in DMF. After the solution is stirred at 25 °C overnight (stirred at 55 °C for a couple of hours if necessary), DMF is removed via evaporation under reduced pressure. The resulting residue is suspended in ethyl acetate, washed by saturated NaHCO₃ (3x) and brine (3x), and dried over Na₂SO₄. The ethyl acetate is removed under vacuum to give the crude product. This crude material is then subjected to preparative HPLC to give the final product amide, which may subsequently be characterized by LC-MS and NMR spectroscopy.

Example 7. Further amide derivatives of Scheme 4.

Following the above procedure described in Scheme 4 or other known procedures and employing the compounds of Examples 1-Na and 2-3 as starting materials, the following amides can be made.

Example 8. Amide derivatives of Example 4.

Following the above procedure described in Scheme 4 or other known procedures and employing a monohydroxy compound corresponding to Example 2-3 as a starting material, the following compounds may be made:

Example 9: further amide derivatives of Example 4.

Examples 16 - 315: Still further amide examples are shown in the following table:

Ex# Core R Ex# Core R Ex# Core R 16 1 a 66 II a 116 III a 17 1 [ b 67 II b 117 III b 18 1 c 68 π c 118 III c 19 1 [ d 69 π d 119 III d 20 1 [ e 70 π e 120 πi e 21 1 [ f 71 π f 121 πi f 22 1 t g 72 π g 122 πi g 23 1 [ h 73 π li 123 III It 24 1 i 74 II i 124 III i 25 1 t j 75 π j 125 III j 26 1 [ k 76 π k 126 III k 27 1 [ 1 77 II 1 127 III 1 28 1 m 78 π m 128 III m 29 1 n 79 II n 129 III n 30 1 0 80 π 0 130 III 0 Ex# Core R Ex# Core R Ex# Core R 31 1 ^[ P 81 π P 131 III P 32 1i q 82 π q 132 III q 33 1 r 83 π r 133 III r 34 1 [ s 84 π s 134 III s 35 1 [ t 85 π t 135 III t 36 1 [ u 86 π u 136 m u 37 1 V 87 π V 137 III V 38 1 88 π w 138 HI w 39 1 X 89 π X 139 III x 40 1 [ y 90 π y 140 m y 41 1 z 91 π z 141 in z 42 1 aa 92 π aa 142 m aa 43 1 [ ab 93 π ab 143 πi ab 44 1 ac 94 π ac 144 III ac 45 1 [ ad 95 π ad 145 III ad 46 1 [ ae 96 π ae 146 in ae 47 1 [ af 97 π af 147 III af 48 1 [ ag 98 π ag 148 III ag 49 1 [ ah 99 π ah 149 m ah 50 1 ai 100 II ai 150 III ai 51 1 ^[ aj 101 π aj 151 III aj 52 ] [ ak 102 π ak 152 rπ ak 53 ] [ al 103 II al 153 III al 54 ] am 104 π am 154 in am 55 1 an 105 II an 155 πi an 56 1 [ ao 106 π ao 156 in ao

60 ] as 110 π as 160 III as 61 ] [ at 111 π at 161 in at 62 1 au 112 II au 162 III au 63 J av 113 II av 163 in av 64 ] [ aw 114 π aw 164 in aw 65 ] [ ax 115 π ax 165 m ax Ex# Core R Ex# Core R Ex# Core R 166 IV a 216 V a 266 VI a 167 rv b 217 V b 267 VI b 168 IV c 218 V c 268 VI c 169 rv d 219 V d 269 VI d 170 IV e 220 V e 270 VI e 171 IV f 221 V f 271 VI f 172 IV g 222 V g 272 VI g 173 IV h 223 V h 273 VI h 174 IV i 224 V i 274 VI i 175 rv j 225 V j 275 VI j 176 rv k 226 V k 276 VI k 177 IV 1 227 V 1 277 VI 1 178 IV m 228 V m 278 VI m 179 IV n 229 V n 279 VI n 180 rv 0 230 V 0 280 VI 0 181 IV P 231 V P 281 VI P 182 IV q 232 V q 282 VI q 183 IV r 233 V r 283 VI r 184 IV s 234 V s 284 VI s 185 rv t 235 V t 285 VI t 186 IV u 236 V u 286 VI u 187 rv V 237 V V 287 VI V 188 IV w 238 V w 288 VI w 189 IV X 239 V x 289 VI X 190 IV y 240 V y 290 VI y 191 IV z 241 V z 291 VI z 192 IV aa 242 V aa 292 VI aa 193 IV ab 243 V ab 293 VI ab 194 IV ac 244 V ac 294 VI ac 195 IV ad 245 V ad 295 VI ad 196 IV ae 246 V ae 296 VI ae 197 IV af 247 V af 297 VI af 198 rv ag 248 V ag 298 VI ag 199 IV ah 249 V ah 299 VI ah 200 IV ai 250 V ai 300 VI ai 201 rv aj 251 V aj 301 VI aj 202 IV ak 252 V ak 302 VI ak 203 IV al 253 V al 303 VI al 204 IV am 254 V am 304 VI am 205 rv an 255 V an 305 VI an 206 IV ao 256 V ao 306 VI ao 207 rv ap 257 V ap 307 VI ap 208 rv aq 258 V aq 308 VI aq 209 rv ar 259 V ar 309 VI ar 210 IV as 260 V as 310 VI as 211 rv at 261 V at 311 VI at Ex# Core R Ex# Core R Ex# Core R

212 IV au 262 V au 312 VI au 213 IV av 263 V av 313 VI av 214 IV aw 264 V aw 314 VI aw 215 IV ax 265 V ax 315 VI ax

In the above table, R is selected from the following radicals:

-_> 9 h i J K i

m n o p q

aa ab ac ad

ae af ag ah ai

^aj ak a' am an NH COOH ao _ap aq ^ar as

at au av aw ax

These amide examples 16 - 315 can be made by those skilled in the art following the above procedure and/or known procedures.

VEGFR Biochemical Assay

The compounds were assayed for biochemical activity by Upstate Ltd at Dundee, United Kingdom, according to the following procedure. In a final reaction volume of 25 μl, KDR (h) (5-10 mU) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 0.33 mg/ml myelin basic protein, 10 mM MgAcetate and [γ-³³P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required). The reaction is initiated by the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 5 μl of a 3% phosphoric acid solution. 10 μl of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.

Compounds of the present invention were tested in this assay and exhibited IC₅₀ between 1 - 5,000 nM.

Cellular Assay: HUVEC: VEGF induced proliferation

The compounds were assayed for cellular activity in the VEGF induced proliferation of HUVEC cells. HUVEC cells (Cambrex, CC-2517) were maintained in EGM (Cambrex, CC-3124) at 37°C and 5% CO₂. HUVEC cells were plated at a density 5000 cells/well (96 well plate) in EGM. Following cell attachment (1 hour) the EGM- medium was replaced by EBM (Cambrex, CC-3129) + 0.1% FBS (ATTC , 30-2020) and the cells were incubated for 20 hours at 37°C. The medium was replaced by EBM +1% FBS, the compounds were serial diluted in DMSO and added to the cells to a final concentration of 0 - 5,000 nM and 1% DMSO. Following a 1 hour pre- incubation at 37°C cells were stimulated with 10ng/ml VEGF (Sigma, V7259) and incubated for 45 hours at 37°C. Cell proliferation was measured by BrdU DNA incorporation for 4 hours and BrdU label was quantitated by ELISA (Roche kit, 16472229) using 1 M H₂SO₄ to stop the reaction. Absorbance was measured at 450nm using a reference wavelength at 690nm.

Detailed Description of Figures: Figure 1 shows that a compound like the first structure may exist in its open- acid form or its cyclic- lactone form or the two forms may co-exist in solution or in vivo.

Figure 2 shows the preferred compounds of the invention in acid or open- chain form. There are two variable regions on these compounds. The first variable region is found on the "left end" of the molecule, as shown, which is the substitution on the phenyl ring of the 3-benzyl ether. The second variable region is the side chain on the 5-urea functionality.

Figure 3 shows the preferred compounds of the invention which are all amides. Both these structures and those from Figure 2 show the same core isothiazole ring with its 3-benzyl ether, 4- carboxamide and 5-urea functionality. The three variable regions on the amide compounds are found in the substitution of the phenyl ring of the benzyl ether, the length of the side chain on the 5- urea functionality and the type of substitution on the amide of the side chain.

Figure 4 shows the scheme used to synthesize the acid compounds in Figure 2. The intermediates until isothiazole 1-6 were obtained according literature methods (Larson, E. R.; Noe, M. O; Gant, T. G. Isothiazole Derivatives Useful As Anticancer Agents. International publication number WO 99/62890; international publication date 12/09/1999. Larson, E. R.; Noe, M. C; Gant, T. G. Isothiazole Derivatives Useful As Anticancer Agents. United States patent US 6,235,764. Date of patent 5/22/2001) and are not further described in this report. However, the purification method for 1-5 was changed significantly and this process is therefore detailed in the experimental section. Accordingly, crude 1-5 was purified by crystallization from hot DMF, which proved more convenient than the tedious, repeated centrifugation procedure given in the patents cited above.

Figure 5 shows the completion of the synthesis of 1-10 which is representative of the acid compounds. Simple deprotection of the acetonide group is followed by neutralizing the carboxylic acid to give the desired sodium salt 1-10.

Figure 6 shows the scheme used for the synthesis of compounds like 2-3. This starts from compound 1-6 and substitutes a different primary amine for the urea formation than the earlier scheme in Figures 4 and 5.

Claims

What is claimed is: 1. A compound of Formula (I):

wherein: X is O or S; R is selected from the group consisting of (C1-C6) alkyl, (C3-C8) cycloalkyl, (C6-C10) arylalkyl, (C5-C9) heteroarylalkyl, substituted (C6-C10) arylalkyl, and substituted (C5-C9) heteroarylalkyl; R² and R³ are independently hydrogen or (C1-C6) alkyl; R⁴ is selected from the group consisting of hydrogen, (C1-C6) alkyl, and hydroxyl; n and m are independently 0, 1 , 2, or 3; p is independently 1 , 2, or 3; R⁵ is selected from the group consisting of hydroxyl, (C1-C6) alkoxy, (C3-C8) cycloalkoxy, substituted or unsubstituted O-(C6-C10) aryl, and NR⁶R⁷; R⁶ and R⁷ are independently selected from the group consisting of hydrogen, (C1-C6) alkyl, (C1-C6) hydroxyalkyl, (C1-C6) dihydroxyalkyl, (C1-C6) alkoxy , (C1- C6) alkyl carboxylic acid, (C1-C6) alkyl phosphoric acid, (C1-C6) alkyl sulfuric acid, (C1-C6) hydroxyalkyl carboxylic acid, (C1-C6) alkyl amide, (C3-C8) cycloalkyl, (C5- C8) heterocycloalkyl, (C6-C10) aryl, (C5-C9) heteroaryl, (C3-C8) cycloalkyl carboxylic acid; or R⁷ and R⁸ together with N forms a (C5-C8) heterocyclic ring either unsubstituted or substituted with one or more hydroxyls, ketones, ethers, and carboxylic acids; or NR⁶R⁷ may form a cyclic ring containing 0-3 additional heteroatoms selected from N, O, or S; or, a pharmaceutically acceptable salt, its tautomer, a pharmaceutically acceptable salt of its tautomer, or prodrug thereof.

2. The compound, salt, tautomer, or prodrug according to claim 1 represented by Formula (II):

wherein:

R^5a is selected from the group consisting of hydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl, and substituted or unsubstituted (C6-C10) aryl.

3. The compound, salt, tautomer, or prodrug according to claim 2 represented by Formula (II): wherein: X is O; R¹ is selected from the group consisting of optionally substituted (C6-C10) arylmethylene and (C5-C9) heteroarylmethylene; R² and R³ are hydrogen; and R⁴ is selected from the group consisting of hydrogen and hydroxyl.

4. The compound, salt, tautomer, or prodrug according to claim 2 selected from the group represented by the following structures:

5. The compound, salt, tautomer, or prodrug according to claim 2 selected from the group represented by the following structures:

6. The compound, salt, tautomer, or prodrug according to claim 2 selected from the group represented by the following structures:

7. The compound, salt, tautomer, or prodrug according to claim 2 selected from the group represented by the following structures:

8. The compound, salt, tautomer, or prodrug according to claim 1 wherein R⁵ is NR⁶R⁷ .

9. The compound, salt, tautomer, or prodrug according to claim 8 wherein: X is O; R¹ is selected from the group consisting of optionally substituted (C6-C10) arylmethylene and (C5-C9) heteroarylmethylene; R² and R³ are hydrogen; and R⁴ is selected from the group consisting of hydrogen and hydroxyl.

10 . The compound, salt, tautomer, or prodrug according to claim 8 selected from the group represented by the following structures:

11 . The compound, salt, tautomer, or prodrug according to claim 8 selected from the group represented by the following structures:

12 . The compound, salt, tautomer, or prodrug according to claim 8 selected from the group represented by the following structures:

13 . The compound, salt, tautomer, or prodrug according to claim 8 selected from the group represented by the following structures:

14 . The compound, salt, tautomer, or prodrug according to claim 8 selected from the group represented by the following structures:

15 . The compound, salt, tautomer, or prodrug according to claim 8 selected from the group represented by the following structures:

16 . The compound, salt, tautomer, or prodrug according to claim 8 selected from the group represented by the following structures:

17 . The compound, salt, tautomer, or prodrug according to claim 8 selected from the group represented by the following structures:

18 . The compound, salt, tautomer, or prodrug according to claim 8 selected from the group represented by the following structures:

19. The compound, salt, tautomer, or prodrug according to claim 8 selected from the group represented by the following structures:

20. The compound, salt, tautomer, or prodrug according to claim 8 selected from the group represented by the following structures:

21. The compound, salt, tautomer, or prodrug according to claim 8 selected from the group represented by the following structures:

22. The compound, salt, tautomer, or prodrug according to claim 8 selected from the group represented by the following structures:

23 . The compound, salt, tautomer, or prodrug according to claim 8 selected from the group represented by the following structures:

wherein: R is selected from the group consisting of radical represented by the following structures: v_V v_/ >-_/ ^%0 I ^. nOHu ^ L ^ nO. NH

NNHH NN

N COOH

24. The compound, salt tautomer, or prodrug according to any of claims 1-23 with the following provisios: the compound, sal tautomer, or prodrug of cla m 2 is excluded or the compound, sal tautomer, or prodrug of cla m 3 is excluded or the compound, sal tautomer, or prodrug of cla m 4 is excluded or the compound, sal tautomer, or prodrug of cla m 5 is excluded or the compound, sal tautomer, or prodrug of cla m 6 is excluded or the compound, sal tautomer, or prodrug of cla m 7 is excluded or the compound, sal tautomer, or prodrug of cla m 8 is excluded or the compound, sal tautomer, or prodrug of cla m 9 is excluded or the compound, sal tautomer, or prodrug of cla m 10 is excluded or the compound, sal tautomer, or prodrug of cla m 11 is excluded or the compound, sal tautomer, or prodrug of cla m 12 is excluded or the compound, sal tautomer, or prodrug of cla m 13 is excluded or the compound, sal tautomer, or prodrug of cla m 14 is excluded or the compound, sal tautomer, or prodrug of cla m 15 is excluded or the compound, sal tautomer, or prodrug of cla m 16 is excluded or the compound, sal tautomer, or prodrug of cla m 17 is excluded or the compound, sal tautomer, or prodrug of cla m 18 is excluded or the compound, sal tautomer, or prodrug of cla m 19 is excluded or the compound, sal tautomer, or prodrug of cla m 20 is excluded or the compound, sal tautomer, or prodrug of cla m 21 is excluded or the compound, sal tautomer, or prodrug of cla m 22 is excluded or the compound, sal tautomer, or prodrug of cla m 23 is excluded.

25. A method for the modulation of the catalytic activity of a protein kinase with a compound or salt of any one of claims 1-24.

26 . The method of claim 25, wherein said protein kinase is selected from the group consisting of VEGF receptors, PDGF receptors.