Patent Application
20100267719
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 alpha-hydroxy-ω-(2-oxo-indolylidenemethyl-pyrrole-3′-carbonyl)amino alkanoic acid and amide derivatives and their pharmaceutically acceptable salts employable as protein kinase inhibitors.
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.
Several pyrrolyl-indolinone derivatives have demonstrated excellent activity as inhibitors of protein kinases (Ladd et al. FASEB J. 16, 681, 2002; Smolich et al. Blood, 97, 1413, 2001; Mendel et al. Clinical Cancer Res. 9, 327, 2003; Sun et al. J. Med. Chem. 46, 1116, 2003). The clinical utility of these compounds has been promising, but has been partially compromised due to the relatively poor aqueous solubility and/or other drug properties. What is needed is a class of modified pyrrolyl-indolinone derivatives having both inhibitory activity and enhanced drug properties.
The invention is directed to alpha-hydroxy-omega-(2-oxo-indolylidenemethyl-pyrrole-3′-carbonyl)amino alkanoic acid and amide derivatives and to their use as inhibitors of protein kinases. It is disclosed herein that alpha-hydroxy-ω-(2-oxo-indolylidenemethyl-pyrrole-3′-carbonyl) amino alkanoic acid and amide derivatives have enhanced and unexpected drug properties that advantageously distinguish this class of compounds over known pyrrolyl-indolinone derivatives having protein kinase inhibition activity and over their corresponding beta-hydroxy-ω-(2-oxo-indolylidenemethyl-pyrrole-3′-carbonyl)amino alkanoic acid and amide derivatives. It is also disclosed herein that alpha-hydroxy-ω-(2-oxo-indolylidenemethyl-pyrrole-3′-carbonyl)amino alkanoic acid and amide derivatives are 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), R1 is selected from the group consisting of hydrogen, halo, (C1-C6) alkyl, (C3-C8) cycloalkyl, (C1-C6) haloalkyl, hydroxy, (C1-C6) alkoxy, amino, (C1-C6) alkylamino, amide, sulfonamide, cyano, substituted or unsubstituted (C6-C10) aryl; R2 is selected from the group consisting of hydrogen, halo, (C1-C6) alkyl, (C3-C8) cycloalkyl, (C1-C6) haloalkyl, hydroxy, (C1-C6) alkoxy, (C2-C8) alkoxyalkyl, amino, (C1-C6) alkylamino, (C6-C10) arylamino; R3 is selected from the group consisting of hydrogen, (C1-C6) alkyl, (C6-C10) aryl, (C5-C10) heteroaryl, and amide; R4, R5 and R6 are independently selected from the group consisting of hydrogen and (C1-C6) alkyl; R7 is selected from the group consisting of hydroxy, (C1-C6) O-alkyl, (C3-C8) O-cycloalkyl, and NR8R9; where R8 and R9 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 phosphonic acid, (C1-C6) alkyl sulfonic acid, (C1-C6) hydroxyalkyl carboxylic acid, (C1-C6) alkyl amide, (C3-C8) cycloalkyl, (C5-C8) heterocycloalkyl, (C6-C8) aryl, (C5-C8) heteroaryl, (C3-C8) cycloalkyl carboxylic acid, or R8 and R9 together with N forms a (C5-C8) heterocyclic ring either unsubstituted or substituted with one or more hydroxyls, ketones, ethers, and carboxylic acids; and n is 1, 2, or 3. Alternatively, this aspect of the invention may be directed to a pharmaceutically acceptable salt, its tautomer, a pharmaceutically acceptable salt of its tautomer, or a prodrug of the compound of Formula (I). Preferred species of the invention include compounds represented by the following structures:
In the above structures, R2 is selected from the group consisting of hydrogen and fluoro. More particularly, a preferred stereoisomer is represented by the following structure:
A first subgenus of this aspect of the invention is represented by Formula (II):
In Formula (II), R19 is selected from the group consisting of hydrogen, (C1-C6) alkyl, and (C3-C8) cycloalkyl. In preferred species of this first subgenus, R1 and R2 are independently selected from the group consisting of hydrogen and fluoro; R3 and R4 are methyl; R5, R6, and R10 are hydrogen; and n is 1 or 2. Preferred species are represented by the following compounds:
A preferred chiral species is represented by the following compound:
A second subgenus of this aspect of the invention is directed to a compound according to Formula (III) or a salt, tautomer, or prodrug thereof:
In preferred species of this second subgenus, R1 and R2 are independently selected from the group consisting of hydrogen, halo, cyano; R3, R4, R5 and R6 are independently hydrogen or (C1-C6))alkyl; n is 1 or 2; and R8 and R9 are 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 phosphonic acid, (C1-C6) alkyl sulfonic acid, (C1-C6) hydroxyalkyl carboxylic acid, (C1-C6) alkyl amide, (C3-C8) cycloalkyl, (C5-C8) heterocycloalkyl, (C6-C8) aryl, (C5-C8) heteroaryl, (C3-C8) cycloalkyl carboxylic acid, or R8 and R9 together with N forms a (C5-C8) heterocyclic ring either unsubstituted or substituted with one or more hydroxyls, ketones, ethers, and carboxylic acids. Preferred species of the second subgenus are represented by the following structures:
In a first subset of the second subgenus, n is 1. Preferred species within this first subset are represented by the following structures:
Preferred chiral species within the first subset of the second subgenus are represented by the following structures:
Further preferred chiral species within the first subset of the second subgenus are represented by the following structures:
In a second subset of the second subgenus, n is 2. Preferred species within this first subset are represented by the following structures:
Further preferred species of the first aspect of the invention are represented by the following structures:
In the above structures, R2 is selected from the group consisting of hydrogen and fluoro; and R7 is selected from the group consisting of hydroxyl or radicals represented by the following structures:
A second 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 represented by Formulas I-III, above. In a preferred mode of the second aspect of the invention, said protein kinase is selected from the group of receptors consisting of VEGF, PDGF, c-kit, Flt-3, Axl, and TrkA.
The present invention provides compounds capable of regulating and/or modulating protein kinase activities of, but not limited to, VEGFR and/or PDGFR. 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/PDGFR 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 PDGF receptors. Thus the present invention provides therapeutic approaches to the treatment of cancer and other diseases which involve the uncontrolled formation of blood vessels.
Examples 1-7: The Synthesis of Acid (1-3) and Amides (1-4) is Shown in
Compound 1-1 was prepared by following a literature procedure used for similar compounds (Li Sun, Chris Liang, et al; Discovery of 5-[5-Fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic Acid (2-Diethylaminoethyl)amide, a Novel Tyrosine Kinase Inhibitor Targeting Vascular Endothelial and Platelet-Derived Growth Factor Receptor Tyrosine Kinase. J. Med. Chem. 2003, 46, 1116-1119). Compound 1-1 and DIEA (di-isopropyl ethylamine) (3 equiv) were suspended in dry DMF at room temperature (
The general procedure for the synthesis of amides of Example 1: An amine (2 equiv) was added to a solution of the acid from Example 1, HATU (1.05 mmol), and DIEA (5 equiv) in DMF (5 mL). After the solution was stirred at 25° C. for 2 h, aqueous HCl (2 mL, 1N) was added. This solution was subjected to preparative HPLC to obtain the pure amide product, which was subsequently characterized by LC-MS and NMR spectroscopy.
Preparative HPLC gave 32 mg of the title compound (34%) from 90 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C24H27FN4O4: 455, obtained: 455.
Preparative HPLC gave 27 mg of the title compound (41%) from 61 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C24H27FN4O5: 471, obtained: 471.
Preparative HPLC gave 22 mg of the title compound (37%) from 61 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C22H25FN4O4: 429, obtained: 429.
Preparative HPLC gave 43 mg of the title compound (27%) from 140 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C24H29FN4O4: 457, obtained: 457.
Preparative HPLC gave 15 mg of the title compound (20%) from 81 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C20H21FN4O4: 401, obtained: 401.
Preparative HPLC gave 18 mg of the title compound (21%) from 81 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C24H27FN4O5: 471, obtained: 471.
Examples 8-11: The synthesis of acid (2-2) and amides (2-3) is shown in
To a solution of compound 1-2 (1.0 mmol) and DIEA (3 equiv) in DMF, the HCl salt of methyl 3-amino-2-hydroxypropionate (1.2 equiv, prepared by refluxing the isoserine in dry methanol with 1.2 equiv HCl) was added. After stirring at 25° C. for 2 h (at which time LC-MS showed the completion of the reaction), KOH in water (5 equiv) was added, and the stirring was continued until the hydrolysis was complete (monitored by LC-MS). The solvents were removed by evaporation under reduced pressure. Aqueous HCl (1N) was added to the residue, and the precipitate was collected by filtration, washed with water, and dried under high vacuum to obtain compound 2-2 (0.33 g, 85%). LC-MS: single peak at 254 nm, MH+ calcd. for C16H18FN3O6: 388, obtained: 388.
Examples 9-11: The general procedure for the synthesis of amides of Example 8: An amine (2 equiv) was added to a solution of the acid, HATU (1.05 mmol), and DIEA (5 equiv) in DMF (5 mL). After the solution was stirred at 25° C. for 2 h, aqueous HCl (2 mL, 1N) was added. This solution was subjected to preparative HPLC to obtain the pure amide product, which was subsequently characterized by LC-MS and NMR spectroscopy.
Preparative HPLC gave 50 mg of the title compound (72%) from 65 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C21H23FN4O4: 415, obtained: 415. 1H NMR (DMSO-d6, 400 MHz) δ 13.67 (s, 1H), 10.87 (s, 1H), 7.75 (dd, J=2.4 Hz, 9.6 Hz, 1H), 7.70 (s, 1H), 7.56 (t, J=6.0 Hz, 1H), 6.92 (m, 1H), 6.83 (dd, J=4.8 Hz, 8.4 Hz, 1H), 4.53 (t, J=5.6 Hz, 1H), 3.48-3.25 (m, 2H), 3.08 (s, 3H), 2.85 (s, 3H), 2.43 (s, 3H), 2.41 (s, 3H).
Preparative HPLC gave 14 mg of the title compound (18%) from 65 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C23H25FN4O5: 457, obtained: 457. 1H NMR (DMSO-d6, 400 MHz) δ 13.68 (s, 1H), 10.90 (s, 1H), 7.75 (dd, J=2.4 Hz, 9.6 Hz, 1H), 7.71 (s, 1H), 7.60 (t, J=6.0 Hz, 1H), 6.92 (m, 1H), 6.83 (dd, J=4.4 Hz, 8.4 Hz, 1H), 5.2 (b, 1H), 4.51 (t, J=6.0 Hz, 1H), 3.65-3.35 (m, 10H), 2.43 (s, 3H), 2.41 (s, 3H).
Preparative HPLC gave 16 mg of the title compound (18%) from 80 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C21H23FN4O5: 431, obtained: 431. 1H NMR (DMSO-d6, 400 MHz) δ 13.67 (s, 1H), 10.89 (s, 1H), 7.75 (dd, J=2.0 Hz, 9.2 Hz, 1H), 7.70 (s, 1H), 7.55 (t, J=5.6 Hz, 1H), 6.92 (m, 1H), 6.82 (dd, J=4.8 Hz, 8.8 Hz, 1H), 4.51 (t, J=6.0 Hz, 1H), 3.74 (s, 3H), 3.55-3.40 (m, 2H), 3.13 (s, 3H), 2.42 (s, 3H), 2.41 (s, 3H).
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.
A general scheme for synthesizing chiral species of the invention is outline below:
A mixture of 5-fluoro-1,3-dihydroindol-2-one (1.62 g, 10.2 mmol), 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (1.96 g, 10.7 mmol), pyrrolidine (12 drops) and absolute ethanol was heated to reflux for 3 hours. The mixture was cooled to 25° C. and the solids were collected by filtration. The solids were stirred with ethanol (30 mL) at 72° C. for 30 min. The mixture was cooled to 25° C. and the solids were collected again by filtration, washed with ethanol (6 mL), and dried under vacuum overnight to give an orange solid (Z)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (3.094 g, 96%). LC-ESIMS observed [M+H]+ 300.95 (calculated for C16H13FN2O3 300.09).
(Z)-5-(5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (3.094 g, 10.3 mmol) was suspended in DMF (15 mL), and stirred for 5 minutes. DIEA (2.7 mL, 15.5 mmol) was then added and the mixture was stirred for 10 minutes. HATU (3.91 g, 10.28 mmol) was added and the reaction mixture was stirred at 25° C. for completion. LC/MS detected the completion of the reaction. Most of the DMF was removed and the residue was suspended in ACN and stirred for another 40 minutes. The solid was collected by filtration, washed with ACN, and dried under high vacuum overnight. (Z)-3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl 5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxylate (3.97 g, 92%) was obtained. LC-ESIMS observed [M+H]+ 418.68 (calculated for C21H15FN6O3 418.12).
To (Z)-3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl 5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxylate (1.0 eq) DMF solution was added amine (1.2 eq), the reaction mixture was stirred at 25° C. for 2 h. LC/MS was applied to detect the completion of the reaction. Remove DMF under reduced pressure and the crude was precipitated with 5% diethylamine/methanol (3 mL) under sonication, the solid was collected by filtration and washed with 5% diethylamine/methanol (1 mL) twice.
To the (S)-isoserine (921.6 mg, 8.77 mmol) in methanol (20 mL) was added concentrated HCl (0.5 mL), and the mixture was refluxed overnight. The mixture was cooled to 25° C. and the solvent was removed under reduced pressure. The crude material was dried and used directly in the next step.
To (S)-methyl 3-amino-2-hydroxypropanoate hydrochloride (172.3 mg, 1.11 mmol) DMF solution was added DIEA (0.48 mL, 2.76 mmol) and the mixture was stirred at 25° C. for 20 minutes. (Z)-3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl 5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxylate (174.8 mg, 0.418 mmol) was added, and the mixture was stirred at 25° C. for the completion. The solvent was removed under reduced pressure to afford (S)-3-({5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}-amino)-2-hydroxypropanoic acid methyl ester (quantitative yield). The product was used in the next step with no purification. LC-ESIMS observed [M+H]+ 401.98 (calculated for C20H20FN3O5 401.15).
(S)-3-({5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}-amino)-2-hydroxypropanoic acid methyl ester (167 mg, 0.418 mmol) and LiOH.H2O (36 mg, 0.86 mmol) and methanol/water (10 ml/2 mL) was stirred at 25° C. overnight. Most of the solvent was removed under reduced pressure and excess 1N HCl was added to acidify the mixture. The orange solid was collected by filtration and washed with cold methanol to afford (S)-3-({5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}-amino)-2-hydroxypropanoic acid (yield 88%). LCESIMS observed [M+H]+ 387.96 (calculated for C19H18FN3O5 387.12); 1H NMR (400 MHz, DMSO-d6) δ 13.91 (s, 1H), 10.89 (s, 1H), 7.75 (dd, J=9.6 Hz, 2.4 Hz, 1H), 7.70 (s, 1H), 7.57 (t, J=6.2 Hz, 1H), 6.92 (td, J=9.2 Hz, 2.4 Hz, 1H), 6.85-6.82 (m, 1H), 4.17-4.14 (m, 1H), 3.64 (s, 1H), 3.55-3.49 (m, 1H), 3.45-3.39 (m, 1H), 2.43 (s, 3H), 2.41 (s, 3H).
Examples 13-17: The general procedure for the synthesis of amides: An amine (1.2 equiv) was added to a suspension of the (Z)-3H-[1,2,3]-triazolo[4,5-b]pyridin-3-yl 5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxylate (1.0 eq) in DMF. The mixture was stirred at 25° C. for 2 h and LC/MS was applied to detect the completion of the reaction. The final solution was removed to get the crude solid, which was precipitated in 5% diethylamine/methanol, the solid was collected by filtration and washed with 5% diethylamine/methanol to afford the pure amide product, which was subsequently characterized by LC-MS and NMR spectroscopy.
To the THF/water (50 mL/50 mL) solution of (S)-isoserine (2.429 g, 23.12 mmol) was added K2CO3 (3.834 g, 27.74 mmol) and N-(Benzyloxycarbonyloxy)-succinimide (5.76 g, 23.11 mmol). The reaction mixture was stirred at 25° C. overnight. The reaction mixture was concentrated and diluted with EtOAc and acidified with excess HCl. The aqueous layer was extracted with EtOAc, and the combined organic layers were washed with dilute HCl, water, brine and dried over sodium sulfate. The solvent was removed under reduced pressure to afford (S)-3-(benzyloxycarbonyl)-2-hydroxypropanoic acid (5.11 g, 92%), which was used in the next step with no further purification. LC-ESIMS observed [M+H]+ 239.91 (calculated for C11H13NO5 239.08).
To (S)-3-(benzyloxycarbonyl)-2-hydroxypropanoic acid (377.8 mg, 1.58 mmol) in DMF (5 mL) was added dimethylamine hydrogen chloride (193.2 mg, 2.37 mmol) and DIEA (0.9 mL, 5.17 mmol). The mixture was then stirred for 5 min and EDC (454.3 mg, 2.37 mmol) and HOBt (320.3 mg, 2.37 mmol) were added. The reaction mixture was stirred at 25° C. overnight. DMF was removed under reduced pressure and the crude material was diluted with EtOAc and washed with saturated NaHCO3. The aqueous layer was extracted twice with EtOAc and the combined organic layers were washed with water, 1N HCl and dried over NaSO4. The solution was concentrated and the crude material was purified by flash chromatography with 0-20% MeOH/DCM to obtain the (S)-benzyl 3-(dimethylamino)-2-hydroxy-3-oxopropylcarbamate (349.2 mg, 83%). LC-ESIMS observed [M+H]+ 266.96 (calculated for C13H18N2O4 266.13).
To the degassed (S)-benzyl 3-(dimethylamino)-2-hydroxy-3-oxopropylcarbamate (256.6 mg, 0.964 mmol) in ethanol (10 mL) was added Pd/C (10%, 30 mg) under argon protection, and then the mixture was degassed. The hydrogen balloon was used to provide the H2 source. The reaction was stirred at 50° C. overnight. The mixture was filtered with Celite 521. The filtrate was evaporated to afford (S)-3-amino-2-hydroxy-N,N-dimethylpropanamide (125.2 mg, 98%). 1H NMR (400 MHz, CDCl3) δ 4.65 (t, J=5.4 Hz, 1H), 3.71-3.59 (m, 2H), 3.07 (s, 3H), 3.04 (s, 3H), 1.94 (broad s, 2H).
The title compound was obtained following the general procedure for amide synthesis (79%). LC-ESIMS observed [M+H]+ 414.97 (calculated for C21H23FN4O4414.17); 1H NMR (400 MHz, DMSO-d6) δ 13.68 (s, 1H), 10.89 (s, 1H), 7.76 (dd, J=9.6 Hz, 2.4 Hz, 1H), 7.71 (s, 1H), 7.59 (t, J=6.2 Hz, 1H), 6.92 (td, J=9.2 Hz, 2.4 Hz, 1H), 6.85-6.82 (m, 1H), 5.04 (d, J=7.6 Hz, 1H), 4.53 (q, J=6.2 Hz, 1H), 3.47-3.41 (m, 1H), 3.36-3.30 (m, 1H), 3.08 (s, 3H), 2.85 (s, 3H), 2.43 (s, 3H), 2.40 (s, 3H).
Similar method to synthesis of (S)-benzyl 3-(dimethylamino)-2-hydroxy-3-oxopropylcarbamate was applied and the title compound was obtained (yield 86%). LC-ESIMS observed [M+H]+ 408.96 (calculated for C15H20N2O5 308.96).
Similar method to synthesis of (S)-3-amino-2-hydroxy-N,N-dimethylpropanamide was applied and the title compound was obtained (yield 94%). 1H NMR (400 MHz, CDCl3) δ 4.36-4.34 (m, 1H), 3.75-3.54 (m, 8H), 3.50 (d, J=4.0 Hz, 1H), 2.96-2.79 (m, 2H), 1.94 (broad s, 2H).
The title compound was obtained following the general procedure for amide synthesis (75%). LC-ESIMS observed [M+H]+ 457.01 (calculated for C23H25FN4O5 456.18); 1H NMR (400 MHz, DMSO-d6) δ 13.68 (s, 1H), 10.89 (s, 1H), 7.76 (dd, J=9.6 Hz, 2.4 Hz, 1H), 7.71 (s, 1H), 7.59 (t, J=6.2 Hz, 1H), 6.92 (td, J=9.2 Hz, 2.4 Hz, 1H), 6.85-6.82 (m, 1H), 5.18 (d, J=8.8 Hz, 1H), 4.51 (q, J=6.0 Hz, 1H), 3.61-3.51 (m, 6H), 3.49-3.36 (m, 4H), 2.43 (s, 3H), 2.41 (s, 3H).
Sodium azide (5.487 g, 84.39 mmol) and ammonium chloride (2.257 g, 42.2 mmol) were added to a solution of methyl (2R)-glycidate (2.872 g, 28.13 mmol) in methanol (40 mL) and water (2 mL). After refluxing for 10 h, methanol was evaporated. The mixture was diluted in CHCl3, washed with 1N HCl (5 mL) and extracted. After drying over sodium sulfate, the organic phase was concentrated and purified by flash chromatography to give the (R)-methyl 3-azido-2-hydroxypropanoate (2.82 g, 69%). 1H NMR (400 MHz, CDCl3) δ 4.39-4.36 (m, 1H), 3.84 (s, 3H), 3.67-3.48 (m, 2H), 3.18 (d, J=4.0 Hz, 1H).
To a solution of (R)-methyl 3-azido-2-hydroxypropanoate (7.3 g, 50.3 mmol) in MeOH (150 mL) at 0° C. was added 1N NaOH (65 mL, 65 mmol). After being stirred at room temperature for 1 h, the mixture was acidified by 1N HCl and extracted with EtOAc. The organic layers were dried over sodium sulfate and concentrated in vacuo to give the acid as a white solid. The compound was used in the next step with no further purification.
Similar method to synthesis of (S)-benzyl 3-(dimethylamino)-2-hydroxy-3-oxopropylcarbamate was applied and the title compound was obtained (yield 93%). 1H NMR (400 MHz, CDCl3) δ 4.39-4.36 (m, 1H), 3.67-3.48 (m, 2H), 3.18 (d, J=4.0 Hz, 1H), 3.08 (s, 3H), 3.04 (s, 3H).
To the degassed (R)-3-azido-2-hydroxy-N,N-dimethylpropanamide (8.37 g, 46.6 mmol) in ethanol (150 mL) was added Pd/C (10%, 837 mg) under argon protection, and then the mixture was degassed. A hydrogen balloon was used to provide an H2 source. The reaction was stirred at 25° C. for 2 h, and TLC was applied to detect the completion of the reaction. The mixture was filtered with Celite 521. The filtrate was evaporated to afford the desired compound (5.38 g, 87%). 1H NMR (400 MHz, CDCl3) δ 4.65 (t, J=5.4 Hz, 1H), 3.71-3.59 (m, 2H), 3.07 (s, 3H), 3.04 (s, 3H).
The title compound was obtained following the general procedure for amide synthesis (yield 85%), LC-ESIMS observed [M+H]+ 414.97 (calculated for C21H23FN4O4414.17); 1H NMR (400 MHz, DMSO-d6) δ 13.67 (s, 1H), 10.89 (s, 1H), 7.76 (dd, J=9.6 Hz, 2.4 Hz, 1H), 7.71 (s, 1H), 7.59 (t, J=6.2 Hz, 1H), 6.92 (td, J=9.2 Hz, 2.4 Hz, 1H), 6.85-6.82 (m, 1H), 5.04 (d, J=7.6 Hz, 1H), 4.53 (q, J=6.2 Hz, 1H), 3.47-3.41 (m, 1H), 3.36-3.30 (m, 1H), 3.08 (s, 3H), 2.85 (s, 3H), 2.43 (s, 3H), 2.40 (s, 3H).
Similar method to synthesis of (S)-benzyl 3-(dimethylamino)-2-hydroxy-3-oxopropylcarbamate was applied and the title compound was obtained (yield 90%), 1H NMR (400 MHz, CDCl3) δ 4.55 (t, J=5.2 Hz, 1H), 3.71-3.60 (m, 6H), 3.48-3.41 (m, 3H), 3.40-3.35 (m, 2H).
A similar method to synthesis of (R)-3-amino-2-hydroxy-N,N-dimethylpropanamide was used and the title compound was obtained in high yield (yield 95%). 1H NMR (400 MHz, CDCl3) δ 4.36-4.34 (m, 1H), 3.75-3.54 (m, 8H), 3.50 (d, J=4.0 Hz, 1H), 2.96-2.79 (m, 2H), 1.94 (broad s, 2H).
The title compound was obtained following the general procedure for amide synthesis (yield 75%). LC-ESIMS observed [M+H]+ 457.01 (calculated for C23H25FN4O5 456.18); 1H NMR (400 MHz, DMSO-d6) δ 13.68 (s, 1H), 10.89 (s, 1H), 7.76 (dd, J=9.6 Hz, 2.4 Hz, 1H), 7.71 (s, 1H), 7.59 (t, J=6.2 Hz, 1H), 6.92 (td, J=9.2 Hz, 2.4 Hz, 1H), 6.85-6.82 (m, 1H), 5.18 (d, J=6.4 Hz, 1H), 4.54-4.49 (m, 1H), 3.61-3.51 (m, 6H), 3.49-3.36 (m, 4H), 2.43 (s, 3H), 2.41 (s, 3H).
(R)-methyl 3-azido-2-hydroxypropanoate (505.4 mg, 3.48 mmol) and methylamine ethanol solution (15 mL) was sealed and stirred at 60° C. oil bath overnight. TLC analysis was applied to detect the reaction completion. The solvent was removed and the crude was purified by flash chromatography (0˜20% Methanol/DCM) to afford (R)-3-azido-2-hydroxy-N-methylpropanamide (385.2 mg, yield 77%), 1H NMR (400 MHz, CDCl3) δ 6.90-6.70 (broad s, 1H), 4.28-4.24 (m, 1H), 3.69-3.57 (m, 3H), 2.87 (d, J=5.6 Hz, 3H).
Similar method to synthesis of (R)-3-amino-2-hydroxy-N,N-dimethylpropanamide was used and the title compound was obtained (yield 98%). 1H NMR (400 MHz, CDCl3) δ 7.05 (broad s, 1H), 3.97 (t, J=5.6 Hz, 1H), 3.12-2.96 (m, 2H), 2.85 (d, J=5.2 Hz, 3H), 1.90 (broad, 2H).
The title compound was obtained following the general procedure for amide synthesis (yield 86%), LC-ESIMS observed [M+H]+ 400.96 (calculated for C20H21FN4O4400.15); 1H NMR (400 MHz, DMSO-d6) δ 13.69 (s, 1H), 10.89 (s, 1H), 7.87 (d, J=4.8 Hz, 1H), 7.76 (dd, J=9.6 Hz, 2.4 Hz, 1H), 7.71 (s, 1H), 7.52 (t, J=5.6 Hz, 1H), 6.95-6.90 (m, 1H), 6.85-6.82 (m, 1H), 5.83 (d, J=5.2 Hz, 1H), 4.07-4.03 (m, 1H), 3.57-3.51 (m, 1H), 3.37-3.30 (m, 1H), 2.62 (d, J=4.4 Hz, 3H) 2.45 (s, 3H), 2.42 (s, 3H).
Examples 18-217: Still further amide examples are shown in the following table:
In the above core structures, R2 is selected from the group consisting of hydrogen and fluoro; and R7 is selected from the group consisting of hydroxyl or radicals represented by the following structures:
In the above table, R7 is selected from the following radicals:
These amide examples 18-217 can be made by those skilled in the art following the above procedure and/or known procedures.
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.
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 10 ng/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 1M H2SO4 to stop the reaction. Absorbance was measured at 450 nm using a reference wavelength at 690 nm.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2006/020363 | 5/26/2006 | WO | 00 | 9/24/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60685144 | May 2005 | US | |
| 60754360 | Dec 2005 | US |
