Compound as WNT signaling inhibitor, composition, and use thereof

Abstract

The present invention relates to a compound having the structure of Formula I as inhibitor of WNT signal transduction pathways, as well as a composition comprising the compound. Further, the present invention relates to the use of the compound and the method of inhibiting the WNT signal transduction pathways. ##STR00001##

Claims

1. A compound, having the structure of Formula I: ##STR00145## or a physiologically acceptable salt thereof, wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4, and X.sub.6 are independently CR.sub.4 or N; X.sub.5, X.sub.7, and X.sub.8 are CR.sub.4; Y.sub.1 is hydrogen or C(R.sub.4).sub.3, each R.sub.4 is same or different; Y.sub.2 and Y.sub.3 are independently hydrogen, halogen or C(R.sub.3).sub.3, each R.sub.3 is same or different; R.sub.1 is selected from hydrogen, halogen, C.sub.1-6 alkyl, quinolinyl, ##STR00146## C.sub.6-30 aryl, 3 to 6 membered heterocycloalkyl containing 1-2 heteroatoms selected from N, O and S, and 5 or 6 membered heteroaryl containing 1-4 heteroatoms selected from N, O and S, wherein each of quinolinyl, ##STR00147## C.sub.6-30 aryl, 3 to 6 membered heterocycloalkyl, and 5 or 6 membered heteroaryl can be optionally substituted with one or two, and same or different R.sub.4; R.sub.2 is selected from halogen, C.sub.1-6 alkyl, quinolinyl, ##STR00148## C.sub.6-30 aryl, 3 to 6 membered heterocycloalkyl containing 1-2 heteroatoms selected from N, O and S, and 5 or 6 membered heteroaryl containing 1-4 heteroatoms selected from N, O and S, wherein each of quinolinyl; ##STR00149## C.sub.6-30 aryl, 3 to 6 membered heterocycloalkyl, and 5 or 6 membered heteroaryl can be optionally substituted with one or two, and same or different R.sub.4; wherein, if X.sub.6 is CR.sub.4, R.sub.2 is quinolinyl, ##STR00150## unsubstituted C.sub.6-30 aryl, 3 to 6 membered heterocycloalkyl containing 1-2 heteroatoms selected from N O and S or 5 or 6 membered heteroaryl containing 1-4 heteroatoms selected from N, O and S, wherein each of quinolinyl; ##STR00151## 3 to 6 membered heterocycloalkyl, or 5 or 6 membered heteroaryl can be optionally substituted with one or two, and same or different R.sub.4; each R.sub.3 is independently selected from hydrogen, halogen, cyano, C.sub.1-6 alkyl, and C.sub.1-6 alkoxy, wherein each of the C.sub.1-6 alkyl and C.sub.1-6 alkoxy can be optionally substituted with halo, amino, hydroxyl, C.sub.1-6 alkoxy or cyano; each R.sub.4 is independently selected from hydrogen, halogen, cyano, C.sub.1-6 alkoxy, S(O).sub.2R.sub.5, C(O)OR.sub.5, C(O)R.sub.5, C(O)NR.sub.6R.sub.7, C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl, wherein each of C.sub.1-6 alkoxy, S(O).sub.2R.sub.5, C(O)OR.sub.5, C(O)R.sub.5, C(O)NR.sub.6R.sub.7, C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl can be optionally substituted with halo, amino, hydroxyl, C.sub.1-6 alkoxy or cyano; R.sub.5, R.sub.6 and R.sub.7 are independently selected from hydrogen, C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl, in which each of the C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl can be optionally substituted with halo, amino, hydroxyl, C.sub.1-6 alkoxy or cyano.

2. The compound of claim 1, wherein the core structure of Formula I defined by X.sub.5, X.sub.6, X.sub.7 and X.sub.8 is selected from: ##STR00152##

3. The compound of claim 1, wherein the ring in Formula I defined by X.sub.1, X.sub.2, X.sub.3 and X.sub.4 is selected from: ##STR00153##

4. The compound of claim 2, wherein R.sub.1 and R.sub.2 are independently selected from hydrogen, fluorine, chlorine, methyl, ##STR00154## phenyl, morpholinyl, piperazinyl, and the 5 or 6 membered heteroaryl is selected from: ##STR00155##

5. The compound of claim 2, wherein each R.sub.4 is independently selected from hydrogen, chlorine, fluorine, cyano, CH.sub.3, CHF.sub.2, CF.sub.3, OCH.sub.3, COOCH.sub.3.

6. The compound of claim 2, wherein at least one atom in Formula I is at least one of corresponding isotope(s) selected from .sup.2H, .sup.3H, .sup.11C, .sup.13C, .sup.14C, .sup.15N, .sup.17O, .sup.18O, .sup.35S, .sup.18F, .sup.36Cl and .sup.123I.

7. The compound of claim 1, being selected from: ##STR00156## ##STR00157## ##STR00158## ##STR00159## ##STR00160## ##STR00161## ##STR00162## ##STR00163## ##STR00164## ##STR00165## ##STR00166## ##STR00167## ##STR00168## ##STR00169## ##STR00170## ##STR00171## ##STR00172## ##STR00173##

8. A pharmaceutical composition comprising the compound or physiologically acceptable salt thereof of claim 1.

9. The pharmaceutical composition of claim 8 that is an oral composition, an injectable composition or a suppository.

10. A method of inhibiting WNT secretion from a cell, comprising contacting the cell with an effective amount of the compound or physiologically acceptable salt thereof of claim 1.

11. A method of inhibiting WNT signaling in a cell, comprising contacting the cell with an effective amount of the compound or physiologically acceptable salt thereof of claim 1.

12. A method for treating colorectal cancer in a subject suffering therefrom, comprising administering to the subject a therapeutically effective amount of the compound or physiologically acceptable salt thereof of claim 1.

13. The method of claim 12, wherein the therapeutically effective amount is about 0.03 to 2.5 mg/kg per body weight at daily dosages.

14. The method of claim 13, wherein the therapeutically effective amount is about 0.5 mg to 1000 mg for humans.

15. The method of claim 12, wherein the compound is administrated enterally, orally, parenterally, topically or in a nasal or suppository form.

16. The compound of claim 1, having the core structure of: ##STR00174##

17. A method of inhibiting WNT secretion from a cell, comprising contacting the cell with an effective amount of the pharmaceutical composition of claim 8.

18. A method of inhibiting WNT signaling in a cell, comprising contacting the cell with an effective amount of the pharmaceutical composition of claim 8.

19. A method for treating colorectal cancer in a subject suffering therefrom, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 8.

Description

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(1) The present invention is further exemplified, but not limited, by the following and Examples that illustrate the preparation of the compounds of the invention.

(2) TABLE-US-00001 Abbreviation Definition or Explanation DCM Dichloromethane DIEA N,N-Diisopropylethylamine DMF N,N-Dimethylformamide eq. equivalents TEA Triethylamine THF Tetrahydrofuran RT Room Temperature EA Ethyl acetate Pd.sub.2(dba).sub.3 Tris(dibenzylideneacetone)dipalladium(0) s-Phos 2-Dicyclohexylphosphino-2,6-dimethoxybiphenyl Pd(PPh.sub.3).sub.4 Tetrakis(triphenylphosphine)palladium

Example 1

N-(4-(2-methylpyridin-4-yl)benzyl)-6-(2-methylpyridin-4-yl)-2,7-naphthyridin-1-amine (Compound No. 1)

(3) ##STR00009##

(4) Step 1:

(5) ##STR00010##

(6) 2-Cyanoacetamide (50 g, 601.8 mmol) and ethyl acetoacetate (75 mL, 601.8 mmol) were dissolved in MeOH. KOH (37.0 g, 1.1 eq) was dissolved in MeOH, and added dropwise into the mixture, some white solid came out. The mixture was heated up to reflex at oil bath for 8 h, and then cooled down to RT. The solid was filtered and then re-dissolved into hot water, and then filtered again. 6N HCl was added into the filtration to neutralize till pH<7. The white solid was out again and filtered. The solid was further washed with MeOH, water and MeOH, and then dried by vacuum to get the final product 3-ethynyl-4-methylpyridine-2,6-diol (yield 41%).

(7) Step 2:

(8) ##STR00011##

(9) 3-ethynyl-4-methylpyridine-2,6-diol (28.0 g, 195.2 mmol) was dissolved in POCl.sub.3 (60.0 mL). The reaction mixture was sealed in a pressure tube and heated up to 180 C. for 6 h. After the reaction was cooled down to room temperature, the excessive POCl.sub.3 was removed under the vacuum. Slowly added crushed ice into the mixture, and the solid came out. Filtered the solid out and dried under the vacuum to get the final product 2,6-dichloro-4-methylpyridine-3-carbonitrile (yield 92%) without further purity.

(10) Step 3:

(11) ##STR00012##

(12) 2,6-dichloro-4-methylpyridine-3-carbonitrile (20.0 g, 107.5 mmol) in 200 mL of isopropyl alcohol was added N,N-dimethylformamide dimethylacetal (12.82 g, 107.5 mmol) and the reaction was stirred at 65 C. for 18 h. After cooling down the reaction to RT, the precipitate was collected by filtration and washed with 50 mL of isopropyl alcohol, and air dried to give the product 2,6-dichloro-4-((E)-2-(dimethylamino)vinyl)pyridine-3-carbonitrile (yield 26%) without further purification.

(13) Step 4:

(14) ##STR00013##

(15) 2,6-dichloro-4-((E)-2-(dimethylamino)vinyl)pyridine-3-carbonitrile (4.0 g, 16.6 mmol) was added with 20 mL concentrated HCl in a sealed tube. The reaction is stirred at 45 C. for 18 h. After cooling down the reaction to RT, ice water was added to the solution resulting heavy yellow slurry. The precipitate was collected by filtration, washed with cold water, ether and ethyl acetate, and dried under vacuum to get light yellow solid 6,8-dichloro-2,7-naphthyridin-1(2H)-one (yield 80%). MS m/z 215.0 (M+1). .sup.1HNMR (300 MHz, DMSO-d6): 11.75 (s, 1H), 7.76 (s, 1H), 7.50 (t, J=6.6 Hz, 1H), 6.52 (d, J=6.6 Hz, 1H).

(16) Step 5:

(17) ##STR00014##

(18) 6,8-dichloro-2,7-naphthyridin-1(2H)-one (3.0 g, 13.96 mmol) was dissolved in iPrOH (120 mL) to form a kind of suspension. The solution was cooled down to 0 C. in ice bath, and then hydrazine solution (5.6 g, 80%, 10 eq) was added dropwise. The mixture was stirred at RT for 15 minutes, and then heated in oil bath at 55 C. for overnight. After the reaction mixture was cooled down to RT, filtered to get the solid directly, and then the solid was washed with 70 mL MeOH and dried by vacuum. The product 6-chloro-8-hydrazinyl-2,7-naphthyridin-1(2H)-one (yield 98%) was used in the next step reaction directly without further purification.

(19) Step 6:

(20) ##STR00015##

(21) 6-chloro-8-hydrazinyl-2,7-naphthyridin-1(2H)-one (1.50 g, 7.12 mmol) was dissolved into MeCN (90 mL) to form a kind of suspension. 1N NaOH (17.80 mL, 2.5 eq) was added, and then equal amount of water (107.80 mL) was added into the mixture. The reaction mixture was heated at 50 C., stirred till becoming the clear solution. The solution was cooled down to 0 C. again, and NaOCl (11.05 g, 12% solution, 2.5 eq) was added dropwise, and then reaction was stirred at RT for overnight. After the reaction was done, the solution was cooled down to 0 C. and then added into 1N HCl to neutralize (pH 6). Precipitate was collected and the filtrate was extracted with 100 mL2 EA. The organic layer was combined and dried over Na.sub.2SO.sub.4 and evaporated to give additional crude product. The combined solid material 6-chloro-2,7-naphthyridin-1(2H)-one (yield 93%) was used in the next reaction without further purification. MS m/z 181.1 (M+1).

(22) Step 7:

(23) ##STR00016##

(24) 6-chloro-2,7-naphthyridin-1(2H)-one (400 mg, 2.2 mmol) was added in POCl.sub.3 (20.0 mL) in a pressure tube. The reaction mixture was heated up to 160 C. for 4 h to get a clear solution. The solution was cooled down to room temperature and poured in DCM, and added crushed ice slowly. Saturated NaHCO.sub.3 was added into the mixture to neutralize HCl generated in the reaction. Vacuum to remove DCM and the left water solution was extracted by 100 mL2 EA. The combined organic layers were washed with brine once, and dried by Na.sub.2SO.sub.4, and then evaporated under the vacuum to get the solid 1,6-dichloro-2,7-naphthyridine (yield 73%) to use in the next step reaction without further purifications. MS m/z 199.0 (M+1).

(25) Step 8:

(26) ##STR00017##

(27) (4-bromophenyl)methanamine (1.00 g, 5.37 mmol) and 2-methylpyridin-4-yl-4-boronic acid (883.30 mg, 6.45 mmol) were dissolved in BuOH (10.0 mL) and water (2.0 mL). K.sub.3PO.sub.4 (2.28 g, 10.75 mmol), Pd.sub.2(dba).sub.3 (120.20 mg, 0.27 mmol) and S-phos (220.70 mg, 0.54 mmol) were added in under N.sub.2. The reaction mixture was sealed in a pressure tube and heated up to 125 C. for 1 h. After cooling down the reaction to RT, the mixture was poured into the water and extracted by 100 mL3 EA. The combined organic layer was washed with brine, dried over Na.sub.2SO.sub.4, and concentrated under the vacuum to give the crude product. The solid was purified by silicone gel column with 10% MeOH (containing 2N NH.sub.3) in DCM to get the pure (4-(2-methylpyridin-4-yl)phenyl)methanamine (yield 89%). MS m/z 199.1 (M+1).

(28) Step 9:

(29) ##STR00018##

(30) 1,6-dichloro-2,7-naphthyridine (160 mg, 0.80 mmol) and (4-(2-methylpyridin-4-yl)phenyl)methanamine (239.10 mg, 1.21 mmol) were dissolved in BuOH (5.0 mL) and heated up to 115 C. for overnight. After the reaction was cooled down to RT, the organic solvent was removed under the vacuum. The crude product was purified by silicone gel flash chromatography with EA/Hexane (1:1) to get the solid N-(4-(2-methylpyridin-4-yl)benzyl)-6-chloro-2,7-naphthyridin-1-amine (yield 90%). MS m/z 361.1 (M+1).

(31) Step 10:

(32) ##STR00019##

(33) N-(4-(2-methylpyridin-4-yl)benzyl)-6-chloro-2,7-naphthyridin-1-amine (50.00 mg, 0.14 mmol) and 2-methylpyridin-4-yl-4-boronic acid (56.90 mg, 0.42 mmol) were dissolved in BuOH (3.0 mL) and water (0.6 mL). K.sub.3PO.sub.4 (88.20 mg, 0.028 mmol), Pd.sub.2(dba).sub.3 (6.20 mg, 0.014 mmol) and S-phos (11.40 mg, 0.011 mmol) were added into the mixture under N.sub.2. The reaction was sealed in a pressure tube and heated up to 105 C. for overnight. After cooling down the reaction to RT, the mixture was poured in water and extracted by EA for three times. The combined organic layer was washed with brine, dried by Na.sub.2SO.sub.4, and concentrated under the vacuum. The crude product was further purified by prep-TLC with 5% MeOH in DCM to get the final product N-(4-(2-methylpyridin-4-yl)benzyl)-6-(2-methylpyridin-4-yl)-2,7-naphthyridin-1-amine (yield 70%). MS m/z 418.2 (M+1). .sup.1HNMR (300 MHz, CDCl.sub.3): 2.46 (s, 3H), 2.63 (s, 3H), 4.94 (d, J=5.10 Hz, 2H), 5.94 (br, 1H), 6.97 (d, J=5.70 Hz, 1H), 7.31 (d, J=4.20 Hz, 1H), 7.36 (s, 1H), 7.54 (d, J=8.10 Hz, 2H), 7.63 (d, J=8.40 Hz, 2H), 7.90 (s, 1H), 8.19 (d, J=6.00 Hz, 1H), 8.22 (s, 1H), 8.51 (m, 2H), 9.08 (s, 1H), 9.30 (s, 1H).

Example 2

N-(3-methyl-4-(2-methylpyridin-4-yl)benzyl)-6-(2-methylpyridin-4-yl)-2,7-naphthyridin-1-amine (Compound No. 2)

(34) ##STR00020##

(35) Step 1:

(36) ##STR00021##

(37) 6-chloro-2,7-naphthyridin-1(2H)-one (200 mg, 1.10 mmol) and 2-methylpyridin-4-yl-4-boronic acid (227.60 mg, 1.66 mmol) were dissolved in BuOH (5.0 mL) and water (1.0 mL). K.sub.3PO.sub.4 (705.20 g, 3.32 mmol), Pd.sub.2(dba).sub.3 (49.60 mg, 0.22 mmol) and S-phos (91.00 mg, 0.11 mmol) were added under N.sub.2. The reaction mixture in the pressure tube was heated up to 130 C. for 1 h. After cooling down the reaction to RT, poured the mixture into the water, extracted by EA for three times. The combined organic layer was washed with brine, dried over Na.sub.2SO.sub.4, concentrated under the vacuum to get the crude. The crude product was purified by column with 5% MeOH in DCM to get the final compound 6-(2-methylpyridin-4-yl)-2,7-naphthyridin-1(2H)-one (yield 61%). MS m/z 238.1 (M+1).

(38) Step 2:

(39) ##STR00022##

(40) 6-(2-methylpyridin-4-yl)-2,7-naphthyridin-1(2H)-one (150 mg, 0.63 mmol) was dissolved in POCl.sub.3(15.0 mL), the pressure tube was sealed and heated up to 160 C. for 4 h. After cooling down the reaction to RT, excessive POCl.sub.3 was removed under vacuum. Crushed ice was slowly added into the mixture, and then added into NaHCO.sub.3 to neutralize until pH 7.5. Extracted the solution by EA three times, the combined organic layer was washed with brine, dried over Na.sub.2SO.sub.4, and concentrated under vacuum. The crude was purified by column with EA/Hexane (1:1) to get the compound 1-chloro-6-(2-methylpyridin-4-yl)-2,7-naphthyridine (yield 55%). MS m/z 256.1 (M+1).

(41) Step 3:

(42) ##STR00023##

(43) 1-chloro-6-(2-methylpyridin-4-yl)-2,7-naphthyridine (10.00 mg, 0.039 mmol) and (3-methyl-4-(2-methylpyridin-4-yl)phenyl)methanamine (10.00 mg, 0.047 mmol) were dissolved in Toluene (1.0 mL). KO.sup.tBu (8.80 mg, 0.078 mmol), Pd(OAc).sub.2 (0.90 mg, 0.0039 mmol) and BINAP (4.90 mg, 0.0078 mmol) was added into the mixture under N.sub.2, The reaction was heated up to 100 C. for overnight. After cooling down the reaction to RT, poured the mixture into the water, extracted by EA for three times. The combined organic layer was washed with brine, dried over Na.sub.2SO.sub.4, then concentrated under vacuum. The crude product was purified by prep-TLC by EA/Hexane (4:1) to get N-(3-methyl-4-(2-methylpyridin-4-yl)benzyl)-6-(2-methylpyridin-4-yl)-2,7-naphthyridin-1-amine (8.8 mg, yield 52%). 1H NMR (300 MHz, CDCl3): 2.31 (s, 3H), 2.63 (s, 3H), 2.70 (s, 3H), 4.91 (d, J=5.10 Hz, 2H), 5.88 (br, 1H), 7.00 (d, J=5.40 Hz, 1H), 7.08 (d, J=5.10 Hz, 1H), 7.12 (s, 1H), 7.22 (d, J=7.50 Hz, 1H), 7.36 (m, 2H), 7.77 (d, J=4.50 Hz, 1H), 7.88 (s, 1H), 7.98 (s, 1H), 8.24 (d, J=6.00 Hz, 1H), 8.53 (d, J=4.80 Hz, 1H), 8.64 (d, J=5.40 Hz, 1H), 9.31 (s, 1H). MS m/z 432.2 (M+1).

Example 3

6-(3-fluorophenyl)-N-((6-(2-methylpyridin-4-yl)pyridin-3-yl)methyl)isoquinolin-1-amine (Compound No. 3)

(44) ##STR00024##

(45) Step 1:

(46) ##STR00025##

(47) 6-bromoisoquinoline (1.80 g, 8.66 mmol) was dissolved in DCM (40 mL), after cooling down the reaction to 0 C. m-CPBA (2.30 g, 1.3 eq, 77% max) was added slowly in small portion. The reaction was warmed up to RT to become a kind of white suspension. In 4 hours, 100 mL DCM was added into the solution, and washed with saturated Na.sub.2CO.sub.3 solution, water and brine. The separated organic layer was dried over Na.sub.2SO.sub.4 and removed under the vacuum to get the yellow solid N-oxide 6-bromoisoquinoline without further purification (1.82 g, yield 93%).

(48) Step 2:

(49) ##STR00026##

(50) N-oxide 6-bromoisoquinoline (1.82 g, 8.12 mmol) was dissolved in dry DCM (80 mL), POCl.sub.3 (1.12 ml, 1.5 eq) was added dropwise at RT. The reaction was heated to 45 C. for 2 hours. After cooling down the reaction to RT, DCM and excessive POCl.sub.3 were removed under the vacuum. The crude was re-dissolved into 100 mL DCM and was washed by saturated Na.sub.2CO.sub.3, water and brine. The separated organic layer was dried over Na.sub.2SO.sub.4, and concentrated to give brown solid. The crude was purified by flash column using 2% MeOH in DCM to get the pale yellow solid 6-bromo-1-chloroisoquinoline (1.27 g, yield 65%). MS m/z 242.0 (M+1).

(51) Step 3:

(52) ##STR00027##

(53) (6-chloropyridin-3-yl)methanamine (300 mg, 2.1 mmol) and 2-methylpyridin-4-ylboronic acid (345 mg, 2.52 mmol) were dissolved in a pressure tube with n-butanol (10 mL) and water (2 mL). K.sub.3PO.sub.4 (893 mg, 4.2 mmol), Pd.sub.2(dba).sub.3 (96.3 mg, 0.105 mmol), and S-phos (86.4 mg, 0.21 mmol) were added under the nitrogen protection. The reaction was heated to 125 C. for 30 minutes and then cooled down to room temperature. The solution was pull in water and extracted by EA for three times. The combined organic layer was washed by brine and dried over Na.sub.2SO.sub.4, and concentrated under the vacuum. The crude was further purified by flash chromatography with 10% MeOH (containing 2N NH.sub.3) in DCM to get the pure (6-(2-methylpyridin-4-yl)pyridin-3-yl)methanamine (0.19 g, yield 45%). MS m/z 200.1 (M+1).

(54) Step 4:

(55) ##STR00028##

(56) 6-bromo-1-chloroisoquinoline (100 mg, 0.41 mmol) and (6-(2-methylpyridin-4-yl)pyridin-3-yl)methanamine (165 mg, 0.82 mmol) were dissolved in 0.5 mL n-BuOH in a sealed tube. The reaction was heat up to 160 C. for 6 h and cooled down to RT. The crude was purified by flash chromatography using 8% MeOH (containing 2N NH3) in DCM to get the pure 6-bromo-N-((6-(2-methylpyridin-4-yl)pyridin-3-yl)methyl)isoquinolin-1-amine (116 mg, 70%). MS m/z 405.2 (M+1).

(57) Step 5:

(58) ##STR00029##

(59) 6-bromo-N-((6-(2-methylpyridin-4-yl)pyridin-3-yl)methyl)isoquinolin-1-amine (20 mg, 0.05 mmol), 3-fluorophenylboronic acid (10.5 mg, 0.075 mmol), Na.sub.2CO.sub.3 (21 mg, 0.2 mmol) and Tetrakis(triphenylphosphine)palladium (5.8 mg, 0.005 mmol) were added in a pressure tube. Dioxane/water (3:1, 2 mL) was added into the tube and heated to 125 C. for 10 minutes. After cooling down the reaction to RT, the solution was diluted by 50 ml, water and extracted by EA for 3 times. The combined organic layer was dried over Na.sub.2SO.sub.4, and concentrated under the vacuum. The crude was further purified by flash chromatography with 10% MeOH (containing 2N NH3) in DCM to get the pure 6-(3-fluorophenyl)-N-((6-(2-methylpyridin-4-yl)pyridin-3-yl)methyl)isoquinolin-1-amine (15.8 mg, 75%). 1H NMR (400 MHz, CDCl3): 2.71 (s, 3H), 5.00 (d, 2H), 7.32-7.38 (m, 2H), 7.59-7.65 (m, 1H), 7.75-7.83 (m, 3H), 8.10 (d, J=8.4 Hz, 1H), 8.21 (d, J=8.8 Hz, 1H), 8.27-8.31 (m, 2H), 8.39 (s, 2H), 8.72 (d, J=8.8 Hz, 1H), 8.79 (d, J=6.0 Hz, 1H), 8.91 (d, J=1.6 Hz, 1H), 10.02 (s, 1H). MS m/z 421.2 (M+1).

Example 4

N-(4-(2-methylpyridin-4-yl)benzyl)-2-(2-methylpyridin-4-yl)-1,6-naphthyridin-5-amine (Compound No. 4)

(60) ##STR00030##

(61) Step 1:

(62) ##STR00031##

(63) 1,6-naphthyridin-5(6H)-one (2.9 g, 19.84 mmol) was dissolved in POCl.sub.3 (40 mL) and heated up to 100 C. for 24 h. After cooling down the reaction to room temperature, the excessive POCl.sub.3 was removed under the vacuum. Small amount crushed ice in saturated Na.sub.2CO.sub.3 solution was added slowly, and lots of bubbles and solid came out. The solid was filtered, and the solution was extracted by EA for 3 times. The combined organic layer was dried over Na.sub.2SO.sub.4, and concentrated under the vacuum. The combined solid was further dried under the vacuum to get 5-chloro-1,6-naphthyridine without further purification (2.6 g, yield 80%). MS m/z 165.1 (M+1).

(64) Step 2:

(65) ##STR00032##

(66) 5-chloro-1,6-naphthyridine (1.5 g, 9.11 mmol) was dissolved in DCM (45 mL) and cooled down by ice bath, m-CPBA (3.7 g, 2 eq, 77% max) was added in small portion and slowly. The reaction was warmed up to RT and continued for 3 hours. 100 mL more DCM was added into the solution, and washed with saturated Na.sub.2CO.sub.3 solution, water and brine. The organic layer was dried over Na.sub.2SO.sub.4, and concentrated under the vacuum to get yellow solid N-oxide 5-chloro-1,6-naphthyridine without further purification (1.25 g, yield 76%).

(67) Step 3:

(68) ##STR00033##

(69) N-oxide 5-chloro-1,6-naphthyridine (1.2 g, 6.64 mmol) was dissolved in dry DCM (30 mL), Et3N (1.85 mL, 13.29 mmol) was added and followed by dropwise adding POCl.sub.3 (0.93 mL, 9.97 mmol) in 5 mL dry DCM. The reaction was heated to 48 C. for 2 hours. 100 mL more DCM was added into the solution, and washed with saturated Na.sub.2CO.sub.3 solution, water and brine. The organic layer was dried over Na.sub.2SO.sub.4, and concentrated under the vacuum to get the yellow solid. The crude was further purified by silicon column using EA/Hexane (1:4) to get white solid 2,5-dichloro-1,6-naphthyridine (0.6 g, yield 45%). MS m/z 199.0 (M+1)

(70) Step 4:

(71) ##STR00034##

(72) 2,5-dichloro-1,6-naphthyridine (200 mg, 1.0 mmol), 2-methylpyridin-4-yl-4-boronic acid (137 mg, 1.0 mmol), Na.sub.2CO.sub.3 (424 mg, 4.0 mmol) and Tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol) were added in a flask, dioxane 16 mL and water 4 mL were further added. The reaction was stirred very well and heated to 90 C. for 4 hours. After cooling down the reaction to RT, the solution was diluted by 100 mL water and extracted by EA for 3 times. The combined organic layer was dried over Na2SO4, and concentrated under the vacuum. The crude was further purified by flash chromatography with EA/Hexane (1:1) to get the solid 5-chloro-2-(2-methylpyridin-4-yl)-1,6-naphthyridine (143 mg, yield 56%). MS m/z 256.1 (M+1)

(73) Step 5:

(74) ##STR00035##

(75) 5-chloro-2-(2-methylpyridin-4-yl)-1,6-naphthyridine (20.00 mg, 0.078 mmol) and (4-(2-methylpyridin-4-yl)phenyl)methanamine (25 mg, 0.118 mmol) were dissolved in Toluene (2.0 mL). KO.sup.tBu (13.2 mg, 0.118 mmol), Pd(OAc).sub.2 (2.7 mg, 0.012 mmol) and BINAP (15.0 mg, 0.024 mmol) were added into the mixture under N.sub.2, The reaction was heated up to 100 C. for overnight. After cooling down the reaction to RT, poured the mixture into the water, extracted by EA for three times. The combined organic layer was washed with brine, dried over Na.sub.2SO.sub.4, then concentrated under vacuum. The crude product was purified by prep-TLC by 8% MeOH in DCM to N-(4-(2-methylpyridin-4-yl)benzyl)-2-(2-methylpyridin-4-yl)-1,6-naphthyridin-5-amine (31 mg, yield 61%). .sup.1H NMR (400 MHz, DMSO-d6): 9.12 (d, J=8.8 Hz, 1H), 8.77-8.83 (m, 2H), 8.49 (d, J=8.4 Hz, 1H), 8.40 (s, 1H), 8.31 (d, J=6.4 Hz, 1H), 8.21 (s, 1H), 8.11 (d, J=5.6 Hz, 1H), 8.06 (d, J=6.4 Hz, 1H), 7.99 (d, J=8.4 Hz, 2H), 7.65 (d, J=8.4 Hz, 2H), 7.23 (d, J=6.4 Hz, 1H), 5.76 (s, 1H), 4.93 (d, J=5.6 Hz, 2H), 2.72 (s, 6H). MS m/z 432.2 (M+1).

Example 5

N-(4-(2-methylpyridin-4-yl)benzyl)-2-phenylpyrido[4,3-b]pyrazin-5-amine (Compound No. 5)

(76) ##STR00036##

(77) Step 1:

(78) ##STR00037##

(79) To 20 mL of ethanol was added phenyl gloyoxal monohydrate (940 mg, 6.99 mmol) and 2-chloro-3,4-diaminopyridine (1000 mg, 6.99 mmol). The mixture was refluxed for overnight. After cooling down the reaction, the crude precipitated product was filtered and washed with 15 mL ethanol and dried under vacuum to get 5-chloro-2-phenylpyrido[3,4-b]pyrazine without further purification (1.28 g, yield 76%), MS m/z 241.0 (M+1); 1H NMR (300 MHz, DMSO-d6): 9.82 (s, 1H), 8.64 (d, J=6.0 Hz, 1H), 8.38-8.43 (m, 2H), 8.07 (d, J=6.0 Hz, 1H), 7.64-7.68 (m, 3H).

(80) Step 2:

(81) ##STR00038##

(82) N-(4-(2-methylpyridin-4-yl)benzyl)-2-phenylpyrido[3,4-b]pyrazin-5-amine (50 mg, 0.21 mmol) and (4-(2-methylpyridin-4-yl)phenyl)methanamine (42 mg, 0.21 mmol) were dissolved in Toluene (4.0 mL). KO.sup.tBu (24 mg, 0.21 mmol), Pd(OAc).sub.2 (4.5 mg, 0.021 mmol) and BINAP (26.4 mg, 0.042 mmol) was added into the mixture under N.sub.2. The reaction was heated up to 100 C. for overnight. After cooling down the reaction to RT, poured the mixture into the water, extracted by EA for three times. The combined organic layer was washed with brine, dried over Na.sub.2SO.sub.4, then concentrated under vacuum. The crude product was purified by flash chromatography using 7% MeOH in DCM to get N-(4-(2-methylpyridin-4-yl)benzyl)-2-phenylpyrido[4,3-b]pyrazin-5-amine (61 mg, yield 72%). MS m/z=404.2 (M+1); .sup.1H NMR (400 MHz, DMSO-d6) 9.53 (s, 1H), 8.77 (d, J=6.4 Hz, 1H), 8.35-8.39 (m, 2H), 8.21 (s, 1H), 8.11 (d, J=6.0 Hz, 1H), 8.07 (d, J=6.4 Hz, 1H), 7.96 (d, J=8.4 Hz, 2H), 7.60-7.65 (m, 5H), 7.14 (d, J=6.0 Hz, 1H), 5.76 (s, 1H), 4.90 (d, J=6.4 Hz, 2H), 2.71 (s, 3H).

(83) A person skilled in the art can clearly understand and know that the other compounds could be prepared by the same strategy as examples 1-5.

(84) Compounds Table:

(85) TABLE-US-00002 No. Compound Structure Compound physical characterization 6 MS m/z = 404.2 (M + 1); 7 0 MS m/z = 403.2 (M + 1); 8 MS m/z = 437.2 (M + 1); 9 MS m/z = 421.2 (M + 1); .sup.1H NMR (400 MHz, DMSO-d6) 9.82 (s, 1H), 8.76 (d, J = 6.0 Hz, 1H), 8.39 (s, 1H), 8.17 (s, 1H), 7.95-8.18 (m, 6H), 7.58-7.66 (m, 3H), 7.35 (t, J = 8.0 Hz, 1H), 7.07 (d, J = 6.0 Hz, 1H), 5.77 (s, 1H), 4.92 (d, J = 6.0 Hz, 1H), 2.70 (s, 3H) 10 MS m/z = 422.2 (M + 1); 11 MS m/z = 475.2 (M + 1); 12 MS m/z = 436.2 (M + 1); 13 MS m/z = 405.2 (M + 1); 14 MS m/z = 418.2 (M + 1); .sup.1H NMR (300 MHz, CDCl.sub.3): 2.46 (s, 3H), 2.63 (s, 3H), 4.94 (d, J = 5.10 Hz, 2H), 5.94 (br, 1H), 6.97 (d, J = 5.70 Hz, 1H), 7.31 (d, J = 4.20 Hz, 1H), 7.36 (s, 1H), 7.54 (d, J = 8.10 Hz, 2H), 7.63 (d, J = 8.40 Hz, 2H), 7.90 (s, 1H), 8.19 (d, J = 6.00 Hz, 1H), 8.22 (s, 1H), 8.51 (m, 2H), 9.08 (s, 1H), 9.30 (s, 1H). 15 MS m/z = 418.2 (M + 1); 16 MS m/z = 428.2 (M + 1); .sup.1H NMR (300 MHz, CDCl.sub.3): 2.64 (s, 3H), 4.96 (d, J = 5.10 Hz, 2H), 5.99 (br, 1H), 7.31 (d, J = 5.10 Hz, 1H), 7.37 (s, 1H), 7.63 (m, 1H), 7.73 (m, 1H), 7.91 (s, 1H), 8.22 (d, J = 5.70 Hz, 1H), 8.33 (m, 1H), 8.44 (s, 1H), 8.53 (d, J = 5.10 Hz, 1H), 9.33 (s, 1H). 17 0 MS m/z = 428.2 (M + 1); 18 MS m/z = 420.2 (M + 1); 19 MS m/z = 417.2 (M + 1); 20 MS m/z = 326.1 (M + 1); .sup.1H NMR (300 MHz, CDCl.sub.3): 2.58 (s, 3H), 4.90 (d, J = 5.1 Hz, 2H), 5.96 (br, 1H), 6.91 (d, J = 6.0 Hz, 1H), 7.48-7.58 (m, 4H), 7.62 (d, J = 5.7 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 5.7 Hz, 1H), 8.40 (d, J = 5.1 Hz, 1H), 8.53 (d, J = 5.7 Hz, 1H), 9.50 (s, 1H). 21 MS m/z = 404.2 (M + 1); 22 MS m/z = 422.2 (M + 1); .sup.1H NMR (300 MHz, CDCl.sub.3): 2.64 (s, 3H), 4.96 (d, J = 5.40 Hz, 2H), 5.96 (br, 1H), 7.01 (d, J = 6.00 Hz, 1H), 7.31 (m, 1H), 7.37 (s, 1H), 7.56 (d, J = 8.10 Hz, 2H), 7.64 (d, J = 8.10 Hz, 2H), 7.88 (m, 1H), 7.99 (s, 1H), 8.25 (d, J = 6.00 Hz, 1H), 8.36 (d, J = 8.10 Hz, 1H), 9.32 (s, 1H). 23 MS m/z = 421.2 (M + 1); 24 MS m/z = 404.2 (M + 1); 25 MS m/z = 403.2 (M + 1); 26 MS m/z = 404.2 (M + 1); 27 0 MS m/z = 476.2 (M + 1); 28 MS m/z = 440.2 (M + 1); 1H NMR (300 MHz, CDCl3): 2.61 (s, 3H), 4.88 (d, J = 5.70 Hz, 2H), 5.98 (br, 1H), 6.92 (d, J = 5.7 Hz, 1H), 7.02 (s, 1H), 7.26 (m, 3H), 7.37 (t, J = 7.8 Hz, 1H), 7.68 (d, J = 5.4 Hz, 1H), 7.79 (s, 1H), 7.89 (s, 1H), 8.11 (d, J = 6.0 Hz, 1H), 8.17 (d, J = 5.1 Hz, 1H), 8.55 (d, J = 5.4 Hz, 1H), 9.26 (s, 1H). 29 MS m/z = 473.2 (M + 1); 30 MS m/z = 497.2 (M + 1); 31 MS m/z = 436.2 (M + 1); .sup.1H NMR (300 MHz, CDCl.sub.3): 2.63 (s, 3H), 2.70 (s, 3H), 4.96 (d, J = 5.70 Hz, 2H), 6.02 (br, 1H), 7.02 (d, J = 5.70 Hz, 1H), 7.34 (s, 1H), 7.45 (d, J = 7.80 Hz, 2H), 7.61 (s, 1H), 7.78 (d, J = 4.80 Hz, 2H), 7.88 (s, 1H), 7.98 (s, 1H), 8.22 (d, J = 5.70 Hz, 1H), 8.55 (d, J = 5.10 Hz, 2H), 8.64 (d, J = 5.10 Hz, 2H), 9.34 (s, 1H). 32 MS m/z = 423.2 (M + 1); 33 MS m/z = 461.2 (M + 1); .sup.1H NMR (300 MHz, CDCl.sub.3): 2.69 (s, 3H), 3.06 (t, 4H), 4.18 (t, 4H), 4.79 (d, J = 5.40 Hz, 2H), 5.85 (br, 1H), 6.76 (d, J = 8.70 Hz, 1H), 6.99 d, J = 6.00 Hz, 1H), 7.69 (q, 1H), 7.76 (q, 1H), 7.86 (s, 1H), 7.96 (s, 1H), 8.22 (d, J = 6.00 Hz, 1H), 8.31 (s, 1H), 8.63 (d, J = 5.40 Hz, 1H), 9.27 (s, 1H). 34 MS m/z = 405.2 (M + 1); 35 MS m/z = 405.2 (M + 1); .sup.1H NMR (300 MHz, CDCl.sub.3): 2.64 (s, 3H), 4.96 (d, J = 5.40 Hz, 2H), 5.96 (br, 1H), 7.05 (d, J = 5.70 Hz, 1H), 7.31 (m, 1H), 7.37 (s, 1H), 7.56 (d, J = 8.40 Hz, 2H), 7.64 (d, J = 8.40 Hz, 2H), 8.23 (d, J = 5.70 Hz, 1H), 8.54 (d, J = 5.40 Hz, 1H), 8.57 (s, 1H), 8.64 (d, J = 2.40 Hz, 1H), 8.67 (m, 1H), 9.32 (s, 1H), 9.71 (d, J = 1.50 Hz, 1H). 36 MS m/z = 405.2 (M + 1); 37 0 MS m/z = 412.2 (M + 1); 38 MS m/z = 425.2 (M + 1); 39 MS m/z = 460.2 (M + 1); .sup.1H NMR (300 MHz, CD.sub.3OD): 2.56 (s, 3H), 3.13 (t, 4H), 4.28 (t, 4H), 4.81 (s, 2H), 6.79 (d, J = 6.30 Hz, 1H), 6.99 (s, 1H), 7.47 (m, 2H), 7.51 (s, 1H), 7.55 (d, J = 6.60 Hz, 2H), 7.71 (d, J = 8.40 Hz, 2H), 8.38 (d, J = 5.40 Hz, 1H), 9.27 (s, 1H). 40 MS m/z = 443.2 (M + 1); 41 MS m/z = 439.2 (M + 1); 42 MS m/z = 494.2 (M + 1); 43 MS m/z = 426.2 (M + 1); 44 MS m/z = 435.2 (M + 1); 45 MS m/z = 464.2 (M + 1); 46 MS m/z = 361.2 (M + 1); 47 0 MS m/z = 341.1 (M + 1); .sup.1H NMR (300 MHz, CD.sub.3OD): 2.31 (s, 3H), 2.65 (s, 3H), 4.76 (s, 2H), 6.98 (m, 1H), 7.12 (d, J = 7.80 Hz, 2H), 7.28 (d, J = 8.10 Hz, 2H), 7.92 (m, 1H), 8.03 (m, 2H), 8.17 (s, 1H), 8.52 (d, J = 5.40 Hz, 1H), 9.56 (s, 1H). 48 MS m/z = 328.1 (M + 1); 49 MS m/z = 330.1 (M + 1); 50 MS m/z = 422.2 (M + 1); .sup.1H NMR (400 MHz, DMSO-d6) 8.96 (d, J = 8.4 Hz, 1H), 8.87 (s, 1H), 8.76 (d, J = 6.0 Hz, 1H), 802-8.37 (m, 8H), 7.61-7.67 (m, 1H), 7.42 (t, J = 8.0 Hz, 1H), 7.19 (d, J = 6.4 Hz, 1H), 5.76 (s, 1H), 4.93 (d, J = 5.6 Hz, 2H), 2.69 (s, 3H). 51 MS m/z = 419.2 (M + 1); 52 MS m/z = 422.2 (M + 1); 53 MS m/z = 422.2 (M + 1); 54 MS m/z = 472.2 (M + 1); 55 MS m/z = 433.2 (M + 1); 56 MS m/z = 405.2 (M + 1); 57 0 MS m/z = 423.2 (M + 1); 58 MS m/z = 403.2 (M + 1); 59 MS m/z = 437.2 (M + 1); 60 MS m/z = 402.2 (M + 1); 61 MS m/z = 417.2 (M + 1); 1H NMR (300 MHz, CDCl3): 2.45 (s, 3H), 2.64 (s, 3H), 4.94 (d, J = 5.10 Hz, 2H), 5.93 (br, 1H), 7.00 (d, J = 5.70 Hz, 1H), 7.32 (d, J = 5.10 Hz, 1H), 7.36 (s, 1H), 7.54 (d, J = 8.10 Hz, 2H), 7.63 (d, J = 8.10 Hz, 2H), 7.80 (m, 2H), 8.20 (d, J = 6.00 Hz, 1H), 8.21 (s, 1H), 8.53 (m, 2H), 9.10 (s, 1H), 9.31 (s, 1H). 62 MS m/z = 403.2 (M + 1); 63 MS m/z = 417.2 (M + 1); .sup.1H NMR (300 MHz, CDCl.sub.3): 2.63 (s, 3H), 2.65 (s, 3H), 4.93 (d, J = 5.10 Hz, 2H), 7.06 (d, J = 6.00 Hz, 1H), 7.30 (m, 2H), 7.37 (s, 1H), 7.55 (d, J = 8.10 Hz, 2H), 7.63 (d, J = 8.10 Hz, 2H), 7.67 (m, 1H), 7.88 (m, 3H), 8.07 (d, J = 6.00 Hz, 1H), 8.53 (d, J = 5.10 Hz, 1H), 8.82 (d, J = 2.40 Hz, 1H). 64 MS m/z = 416.2 (M + 1); 65 MS m/z = 417.2 (M + 1); 66 MS m/z = 403.2 (M + 1); 67 00 MS m/z = 404.2 (M + 1); 68 01 MS m/z = 404.2 (M + 1); 69 02 MS m/z = 405.2 (M + 1); .sup.1H NMR (400 MHz, DMSO-d6) 9.52 (d, J = 1.2 Hz, 1H), 8.92 (d, J = 2.0 Hz, 1H), 8.84-8.86 (m, 1H), 8.75- 8.82 (m, 4H), 8.56 (d, J = 8.8 Hz, 1H), 8.42 (s, 1H), 8.31 (d, J = 8.8 Hz, 2H), 8.12 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 6.8 Hz, 1H), 7.40 (d, J = 6.8 Hz, 1H), 5.76 (s, 1H), 5.00 (d, J = 5.6 Hz, 2H), 2.73 (s, 1H). 70 03 MS m/z = 419.2 (M + 1); 71 04 MS m/z = 418.2 (M + 1); 72 05 MS m/z = 435.2 (M + 1); 73 06 MS m/z = 432.2 (M + 1); 74 07 MS m/z = 405.2 (M + 1); 75 08 MS m/z = 422.2 (M + 1); 76 09 MS m/z = 423.2 (M + 1); 77 0 MS m/z = 436.2 (M + 1); 78 MS m/z = 440.2 (M + 1); 79 MS m/z = 419.2 (M + 1); 80 MS m/z = 420.2 (M + 1); 81 MS m/z = 433.2 (M + 1); 82 MS m/z = 437.2 (M + 1); 83 MS m/z = 420.2 (M + 1); 84 MS m/z = 426.2 (M + 1); 85 MS m/z = 426.2 (M + 1); 86 MS m/z = 426.2 (M + 1); 87 0 MS m/z = 453.2 (M + 1); 88 MS m/z = 393.1 (M + 1); 89 MS m/z = 407.2 (M + 1); 90 MS m/z = 395.1 (M + 1); 91 MS m/z = 409.2 (M + 1); 92 MS m/z = 407.2 (M + 1); 93 MS m/z = 410.2 (M + 1); 94 MS m/z = 394.1 (M + 1); 95 MS m/z = 433.2 (M + 1); 96 MS m/z = 433.2 (M + 1); .sup.1H NMR (300 MHz, CDCl3): 2.30 (s, 3H), 2.55 (s, 3H), 2.61 (s, 3H), 4.86 (d, J = 5.4 Hz, 2H), 5.98 (br, 1H), 6.94 (d, J = 5.7 Hz, 1H), 7.17 (m, 1H), 7.24 (s, 1H), 7.61 (s, 1H), 7.70 (d, J = 5.1 Hz, 1H), 7.79 (s, 1H), 7.89 (s, 1H), 8.14 (d, J = 6.0 Hz, 1H), 8.49 (d, J = 5.1 Hz, 1H), 8.56 (m, 2H), 9.25 (s, 1H). 97 0 MS m/z = 437.2 (M + 1); .sup.1H NMR (300 MHz, CDCl3): 2.31 (s, 3H), 2.61 (s, 3H), 4.90 (d, J = 5.4 Hz, 2H), 6.00 (br, 1H), 6.94 (d, J = 5.7 Hz, 1H), 7.18 (m, 1H), 7.24 (s, 1H), 7.63 (s, 1H), 7.70 (d, J = 5.1 Hz, 1H), 7.80 (s, 1H), 7.90 (s, 1H), 8.14 (d, J = 6.0 Hz, 1H), 8.33 (s, 1H), 8.50 (d, J = 5.1 Hz, 1H), 8.54 (m, 1H), 9.25 (s, 1H). 98 MS m/z = 437.2 (M + 1); 99 MS m/z = 419.2 (M + 1); 100 MS m/z = 423.2 (M + 1); 101 MS m/z = 469.2 (M + 1); 102 MS m/z = 425.2 (M + 1); 103 MS m/z = 450.2 (M + 1); 104 MS m/z = 434.2 (M + 1); 105 MS m/z = 453.2 (M + 1); 106 MS m/z = 438.2 (M + 1); 107 0 MS m/z = 443.2 (M + 1); .sup.1H NMR (300 MHz, CDCl3): 2.30 (s, 3H), 2.61 (s, 3H), 4.98 (d, J = 5.7 Hz, 2H), 6.00 (br, 1H), 7.03 (d, J = 5.70 Hz, 1H), 7.35 (s, 1H), 7.45 (d, J = 7.8 Hz, 2H), 7.62 (s, 1H), 7.79 (d, J = 5.1 Hz, 2H), 7.89 (s, 1H), 7.98 (s, 1H), 8.20 (d, J = 5.70 Hz, 1H), 8.56 (d, J = 5.10 Hz, 2H), 8.66 (d, J = 5.10 Hz, 2H), 9.30 (s, 1H). 108 MS m/z = 448.2 (M + 1); 109 MS m/z = 453.2 (M + 1); 110 MS m/z = 444.2 (M + 1); 111 MS m/z = 454.2 (M + 1);

Example 6

WNT Pathway Reporter Gene Assay

(86) Materials and Methods:

(87) NIH3T3 mouse fibroblast cells (American Type Culture Collection, Manassas, Va.) were transfected with a plasmid containing a luciferase gene driven by 5 copies of TCF elements. Stale cells selected with 1 g/mL of Zeocin (Gibco/Invitrogen, Carlsbad, Calif.) are cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, Calif.) supplemented with 10% FBS (Invitrogen), 50 unit/mL penicillin and 50 g/mL of streptomycin (Invitrogen) at 37 C. with 5% CO2 in air atmosphere. Suspension HEK293 cells (ATCC) were transfected with a plasmid containing full-length human WNT-3a cDNA sequence driven by a CMV promoter, and stable cells were selected in FreeStyle 293 medium (Invitrogen) supplemented with 100 ug/mL G418.

(88) The NIH3T3 TCF-Luc cells and 293 WNT3a cells were co-cultured in a 96-well plate with DMEM medium supplemented with 0.5% FBS. After 16 hours, the firefly luciferase activities are measured with the Steady-Glo Luciferase Assay System (Promega). The cells were treated with different concentrations of compounds of this invention during the co-culture. The IC50s were defined as the concentration when the compounds reduce the luminescence intensity by 50%. To normalize for cell quantity and viability, CellTiter Glo assay is next performed in a duplicate plate.

(89) All compounds presented in the patent have IC.sub.50<5 M in WNT pathway reporter gene assay. Selective examples were listed in the table below.

(90) TABLE-US-00003 Compound No. IC50 (M) 1 <0.003 2 <0.003 3 0.010 4 0.005 5 0.070 9 0.010 14 0.003 16 0.015 20 0.050 22 0.005 23 0.020 28 <0.003 33 0.050 35 <0.003 37 0.020 39 0.070 47 1.25 50 0.035 61 0.005 63 0.005 68 0.025 69 0.015 70 <0.003 75 0.005 84 0.015 96 0.001 97 0.001 104 0.005 107 0.008 109 0.002

Example 7

Mechanistic Studies of the WNT Pathway Inhibitors

(91) Compounds that inhibited the TCF reporter gene activity induced by the co-cultured Wnt-3a cells in the primary assay were followed up in a mechanistic study to identify the point of action of the compounds. Two different of activators were assessed, one with purified recombinant Wnt-3a protein (StemRD Inc., Burlingame, Calif.), the other with a GSK-3b inhibitor 6-bromoindirubin-3-oxime (StemRD Inc., Burlingame, Calif.).

(92) Results of such mechanistic studies showed that some of the active compounds in this invention inhibit WNT pathway activation at a point before the WNT-3a interaction with the receptors, as they did not inhibit the TCF reporter gene activation by recombinant WNT-3a protein. The candidates of such action include, but are not limited to wntless/evenness interrupted (Wls/Evi), porcupine (Porcn), and Vps35p.

Example 5

Effect of WNT Pathway Inhibitors on Cancer Cells

(93) Compounds that inhibit Wnt secretion and intracellular signal transduction are expected to inhibit proliferation of cancer cells that depend on autocrine Wnt signaling. The effect of the Wnt pathway inhibitors on cell proliferation in 2-D culture, anchorage independent growth and apoptosis resistance in cell lines known to require Wnt autocrine signaling. Compounds are evaluated by using standard assays on the Wnt dependent cell lines known in the published literature: PA-1 (ovarian teratocarcinoma cancer), MDA-MB-157 (breast cancer), Saos-2 (osteosarcoma) and SNU1076 (head and neck squamous carcinoma). Effects of the inhibitors are seen in these cell lines, further confirming the activities expected for the compounds.

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