Salt type and crystal type of 4h-pyrazolo [1, 5-alpha] benzimidazole compound and preparation method and intermediate thereof

Abstract

Disclosed in the present invention are a salt type and crystal type of 4H-pyrazolo[1, 5-alpha]benzimidazole compound and the preparation method and intermediate thereof. The 4H-pyrazolo[1,5-alpha]benzimidazole compound has the structure of Compound 2. ##STR00001##

Claims

1. A compound 2, ##STR00029##

2. A method for inhibiting PARP activity in a subject thereof, comprising: administering an effective amount of compound 2 as defined in claim 1 to the subject.

3. A process for preparing compound 2 as defined in claim 1, comprising: ##STR00030## wherein, HA is maleic acid; R is isopropyl.

4. The process as defined in claim 3, wherein, the reaction solvent is an alcoholic solvent and/or a mixed solvent containing an alcoholic solvent and water.

5. The process as defined in claim 4, wherein, the volume ratio of the alcoholic solvent to water is 1:0.05-0.1.

6. The process as defined in claim 4, wherein, the alcoholic solvent is selected from methanol, ethanol and/or isopropanol.

7. The process as defined in claim 4, wherein, the amount of the reaction solvent is 5 to 20 times the weight of the compound (VII).

8. The process as defined in claim 7, wherein, the amount of the reaction solvent is 8 to 12 times the weight of the compound (VII).

9. The process as defined in claim 3, wherein, the mole ratio of the compound (VII) to the reagent HA is 1:0.5-2.

10. The process as defined in claim 9, wherein, the mole ratio of the compound (VII) to the reagent HA is 1:1.05-1.2.

11. The process as defined in claim 3, wherein, the reaction is conducted at 50 to 100 C.

12. The process as defined in claim 11, wherein, the reaction is conducted at 60 to 80 C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is the XRPD spectrum of Cu-K radiation of the crystal type A.

(2) FIG. 2 is the DSC pattern of the crystal type A.

(3) FIG. 3 is the TGA pattern of the crystal type A.

(4) FIG. 4 is the three-dimensional structure ellipsoid diagram of the single molecule of compound 3.

(5) FIG. 5 is the crystal cell packing diagram of the crystal type A in b-axis direction.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(6) For a better understanding of the contents of the present invention, the following examples further illustrate the present invention, but the present invention is not limited thereto.

Example 1: Preparation of Compound 3

(7) ##STR00015##

Step 1: Tert-butyl 4-(1-cyano-2-ethoxy-2-oxoethylidene)piperidine-1-carboxylate

(8) ##STR00016##

(9) A solution of tert-butyl 4-oxopiperidine-1-carboxylate (3 kg, 15.05 mol) in toluene (24 L) was heated to 95 C., and acetic acid (446 g, 7.43 mol), 2-cyanoacetate (1.68 kg, 14.85 mol), ammonium acetate (571 g, 7.41 mol) were added successively in one portion. It started to reflux to separate water when the external temperature rose to 130 C. and the internal temperature to 102 C., the complete consumption of the material was detected by HPLC when the internal temperature reached 114 C., which took about 3 hours. After the mixture was cooled to room temperature, the organic phase was washed with water (10 L), 10% aqueous Na.sub.2CO.sub.3 (8 L) and brine (5 L2) successively. The aqueous phases were combined and extracted with ethyl acetate (5 L2). The organic phases were combined and evaporated under reduced pressure to remove the solvent, giving 4.5 kg residue. The residue was mashed and purified with PE/EtOAc=10/1 (9 L). The white solid was collected by filtration to give the title compound (1.5 kg, yield 33.84%, purity 98.62%). (The filtrate was concentrated and further purified to give the title compound). 1H NMR (400 MHz, CHLOROFORM-d) ppm 1.38 (t, J=7.15 Hz, 3H), 1.50 (s, 9H), 2.79 (t, J=5.90 Hz, 2H), 3.15 (t, J=5.83 Hz, 2H), 3.56 (t, J=5.71 Hz, 2H), 3.63 (t, J=5.83 Hz, 2H), 4.31 (q, J=7.15 Hz, 2H). LCMS (ESI) m/z: 295 (M+1).

Step 2: Tert-butyl 4-(1-cyano-2-ethoxy-2-oxoethyl)-4-methylpiperidine-1-carboxylate

(10) ##STR00017##

(11) A 3M solution of methyl magnesium bromide (5.66 L, 16.98 mol) was added dropwise to a mixture of cuprous iodide (1.29 kg, 6.78 mol) in anhydrous tetrahydrofuran (16 L) under nitrogen atmosphere at 50 to 40 C. (with an appropriate dropping speed keeping the inter temperature below or at 40 C.). Upon the completion of the addition, the mixture was stirred at 5 C. to 0 C. for 1 hour and cooled to 50 to 40 C., and a solution of tert-butyl 4-(1-cyano-2-ethoxy-2-oxoethylidene)piperidine-1-carboxylate (2 kg, 6.79 mol) in tetrahydrofuran (4 L) was added dropwise (with an appropriate dropping speed keeping the inter temperature at or below 40 C.). After the addition, the mixture was slowly warmed to room temperature and stirred for 15 hours. The mixture was cooled to 0-5 C. and quenched with saturated aqueous ammonium chloride solution (W/W=1:1) (2 L), and then filtered with diatomaceous earth and the filtrate was separated. The residue was washed with EtOAc (5 L2) and the combined organic layers were washed with saturated aqueous ammonium chloride (5 L2) and brine (5 L2) and evaporated to give the crude title compound (2.1 kg) as a yellow oil which was used directly in the next step without further purification.

Step 3: 2-(1-(tert-butoxycarbonyl)-4-methylpiperidin-4-yl)-2-cyanoacetic acid

(12) ##STR00018##

(13) A solution of sodium hydroxide (1.03 kg, 25.75 mol) in water (2.6 L) was added dropwise to a mixed solution of tert-butyl 4-(1-cyano-2-ethoxy-2-oxoethyl)-4-methylpiperidine-1-carboxylate (2 kg, crude, 6.44 mol) in THF/MeOH=10:1 (3.52 L) with an appropriate dropping speed keeping the inter temperature within 0-10 C. After the addition, the mixture was stirred at room temperature for 2 hours, TLC detected that the reaction was complete (the absorption of raw material & product was weak at 220 nm). A mixed solution of EtOAc (2 L)/tert-butyl methyl ether (4 L) was added and the mixture was stirred, then allowed to stand still to separate the aqueous layer. The organic phase was washed with water (1 L3). The aqueous layers were combined and extracted with tert-butyl methyl ether (1 L2), adjusted to pH 3-4 with 1N hydrochloric acid and extracted with DCM (5 L2). The combined dichloromethane layers were washed with brine (5 L2) and evaporated to give the crude title compound (1.5 kg) as a white solid, which was used directly in the next step without further purification.

Step 4: Tert-butyl 4-(cyanomethyl)-4-methylpiperidine-1-carboxylate

(14) ##STR00019##

(15) A mixture of 2-(1-(tert-butoxycarbonyl)-4-methylpiperidin-4-yl)-2-cyanoacetic acid (4 kg, crude, 14.17 mol) and Cu.sub.2O (405.45 g, 2.83 mol) in acetonitrile (20 mL) was stirred at 85 C. for 2 hours, TLC detected that the reaction was complete (the absorption of raw material & product was weak at 220 nm). After the mixture was cooled to room temperature, the insoluble materials were filtered off and the filtrate was evaporated to dryness. The residue was dissolved with ethyl acetate (20 L), washed sequentially with 0.5N hydrochloric acid (10 L2) and brine (20 L2), evaporated to dryness and the residue was mashed and purified with PE/EtOAc=10/1 (16 L). The white solid was collected by filtration to give the title compound (2.5 kg, yield 74.03%, purity: being detected after equivalent amount of internal standard was added as the absorption of the product was weak at 220 nm). 1H NMR (400 MHz, CHLOROFORM-d) ppm 1.16 (s, 3H), 1.43-1.54 (m, 13H), 2.31 (s, 2H), 3.23 (ddd, J=13.68, 8.78, 4.39 Hz, 2H), 3.52-3.70 (m, 2H). LCMS (ESI) m/z: 239 (M+1).

Step 5: Tert-butyl 4-(1-cyano-2-oxoethyl)-4-methylpiperidine-1-carboxylate

(16) ##STR00020##

(17) 2M LDA (3.15 L, 6.3 mol) was added dropwise to a mixture of tert-butyl 4-(cyanomethyl)-4-methylpiperidine-1-carboxylate (1 kg, 4.2 mol) in THF (8 L) under nitrogen atmosphere at 60 to 50 C. After being stirred at 60 to 50 C. for 1 hour, the reaction solution was added with ethyl formate (622 g, 8.4 mol) dropwise. After the addition, the reaction solution was slowly heated to room temperature and stirred for 15 hours. After the reaction was complete, the reaction solution was cooled to 30 to 20 C., quenched with 1N aqueous hydrochloric acid (5 L) and the aqueous layer was extracted with EtOAc (2 L3). The combined organic layers were washed with 0.5N hydrochloric acid (5 L2) and brine (5 L2), evaporated to dryness and the residue was purified by being mashed with PE/EtOAc=10/1 (2 L). The white solid was collected by filtration to give the title compound (900 g, yield 80.46%, purity 100%). 1H NMR (400 MHz, CHLOROFORM-d) ppm 1.22 (s, 3H), 1.47-1.55 (m, 11H), 1.73-2.07 (m, 4H), 3.38-3.48 (m, 4H), 6.97 (s, 1H), 7.55-8.14 (m, 1H). LCMS (ESI) m/z: 289 (M+23).

Step 6: (2,6-dibromo-4-fluorophenyl)hydrazine hydrochloride

(18) ##STR00021##

(19) A solution of sodium nitrite (141 g, 2.05 mol) in water (1.8 L) solution was slowly added dropwise to a solution of 2,6-dibromo-4-fluoroaniline (500 g, 1.86 mol) in concentrated hydrochloric acid (1.8 L) at 5 to 0 C. After the addition, the reaction mixture was stirred at 5 to 0 C. for 40 minutes, and the mixture was added to a solution of stannous chloride dihydrate (629 g, 2.79 mol) in concentrated hydrochloric acid (2 L) dropwise at 10 to 5 C. with an appropriate dropping speed that kept the inter temperature at or below 5 C. The resultant mixture was slowly heated to about 20 C. and stirred for 12 hours. The solid was collected by filtration, washed with isopropanol (0.5 L4) and dried in vacuo to give the title compound (430 g, yield 72%, purity 97.75%) as an off-white solid which can be used in the next step without further purification. 1H NMR (400 MHz, DMSO-d6) ppm 2.37-2.68 (m, 1H), 6.94-7.28 (m, 1H), 7.80 (d, J=8.03 Hz, 2H), 10.13 (br. s., 3H).

Step 7: Tert-butyl 4-(5-amino-1-(2,6-dibromo-4-fluorophenyl)-1H-pyrazol-4-yl)-4-methylpiperidine-1-carboxylate

(20) ##STR00022##

(21) A mixture of potassium acetate (1.11 kg, 11.27 mol) and (2,6-dibromo-4-fluorophenyl)hydrazine hydrochloride (2.65 kg, 8.27 mol) in ethanol (25 L) was stirred at room temperature for 0.5 hours, then tert-butyl 4-(1-cyano-2-oxoethyl)-4-methylpiperidine-1-carboxylate (2 kg, 7.51 mol) was added and the mixture was stirred at 60 C. for 2 hours. After completion of the reaction, NaHCO.sub.3 (1.89 kg, 22.5 mol) was added portionwise to the mixture and stirred for another 15 hours at 80 to 90 C. After being cooled to room temperature, the resulting mixture was evaporated and the residue was quenched with water (20 L) and extracted with EtOAc (10 L2). The combined organic layer was washed with brine (10 L2), evaporated and the residue was mashed and purified with PE/EtOAc=10/1 (6 L). The white solid was collected by filtration to give the title compound (3.5 kg, yield 87.5%, purity 99.84%). 1H NMR (400 MHz, CHLOROFORM-d) ppm 1.32 (s, 3H), 1.48 (s, 9H), 1.57-1.63 (m, 2H), 2.03-2.14 (m, 2H), 3.30 (br. s., 4H), 3.67 (d, J=13.30 Hz, 2H), 7.41-7.52 (m, 3H).

(22) ##STR00023##

Step 1: tert-butyl 4-(8-bromo-6-fluoro-4H-benzo[4,5]imidazo[1,2-b]pyrazol-3-yl)-4-methylpiperidine-1-carboxylate

(23) ##STR00024##

(24) A mixture of tert-butyl 4-(5-amino-1-(2,6-dibromo-4-fluorophenyl)-1H-pyrazol-4-yl)-4-methylpiperidine-1-carboxylate (2.1 kg, 3.95 mol), Pd.sub.2(dba).sub.3 (289.37 g, 0.316 mol). Xamphos (365.69 g, 0.632 mol) and cesium carbonate (2.57 kg, 7.9 mol) in DMF (16.8 L) was stirred at 125 to 135 C. for 5-6 hours under nitrogen atmosphere. After being cooled to room temperature, the resulting mixture was filtered through celite. The filtrate was diluted with EtOAc (20 L) and water (40 L) and stirred to portion. The aqueous phase was extracted with ethyl acetate (20 L2) and the organic phase was evaporated under reduced pressure to dryness to give the crude title compound (2.68 kg) which was used in the next step without further purification. LCMS (ESI) m/z: 451, 453 (M, M+2).

Step 2: Tert-butyl 4-(8-carbamoyl-6-fluoro-4H-benzo[4,5]imidazo[1,2-B]pyrazol-3-yl)-4 -methylpiperidine-1-carboxylate

(25) ##STR00025##

(26) A 10 L autoclave was charged with tert-butyl 4-(8-bromo-6-fluoro-4H-benzo[4,5]imidazo[1,2-B]pyrazol-3-yl)-4-methylpiperidine-1-carboxylate (535 g, 1.19 mol), HMDS (956.55 g, 5.93 mol), Pd(dppf)Cl.sub.2 (43.37 g, 0.0593 mol), Xantphos (34.29 g, 0.0593 mol), DIPEA (306.40 g, 2.37 mol) and DMF (5 L), and purged with carbon monoxide for 3 times, pressurized to 0.8 to 1 MPa, heated to 100 to 110 C. and stirred for 18-20 hours. After being cooled to room temperature, the mixture was filtered through celite. The filtrate was diluted with ethyl acetate (5 L) and water (15 L) and stirred to portion. The aqueous phase was extracted with ethyl acetate (5 L2), and the organic phase was washed with brine (5 L) and concentrated to dryness under reduced pressure to give the crude title compound (552 g, crude) which was used in the next step without further purification. LCMS (ESI) m/z: 416 (M+1).

Step 3: 6-fluoro-3-(4-methylpiperidin-4-yl)-4H-benzo[4,5]imidazo[1,2-b]pyrazol-8-carboxamide

(27) ##STR00026##

(28) A 4M HCl(g)/MeOH solution was added dropwise to a solution of tert-butyl 4-(8-carbamoyl-6-fluoro-4H-benzo[4,5]imidazo[1,2-B]pyrazol-3-yl)-4-methylpiperidine-1-carboxylate (2.7 kg, 6.5 mol) in MeOH (10 L) at 10 to 0 C. After the addition, the reaction solution was heated to 20-25 C. and stirred for 2-3 hours. After the reaction was completed, the resulting mixture was concentrated to 5 L and filtered to obtain solid. The solid was dispersed in water (12 L), and the mixture was cooled to 0 to 5 C., and 20% sodium hydroxide solution (1 L) was added dropwise slowly to pH 9-10, after the addition, the mixture was stirred at 0 to 5 C. for 1 hour, filtered and the filter cake was washed with water until it was neutral and dried to give the title compound (1.03 kg, yield 82%, purity 99.72%) as a pale yellow solid. 1H NMR (400 MHz, DMSO-d6) ppm 1.31 (s, 3H), 1.68-1.85 (m, 2H), 2.27 (d, J=14.81 Hz, 2H), 2.83 (t, J=9.79 Hz, 2H), 3.00-3.13 (m, 2H), 7.42 (dd, J=8.66, 2.51 Hz, 1H), 7.53 (dd, J=11.11, 2.57 Hz, 1H), 7.78 (s, 1H), 8.06 (s, 1H), 10.66 (s, 1H). LCMS (ESI) m/z: 316 (M+1).

Step 4: 6-fluoro-3-(1-isopropyl-4-methylpiperidin-4-yl)-4H-benzo[4,5]imidazo[1,2-b]pyrazol-8-carboxamide

(29) ##STR00027##

(30) A 10 L autoclave was charged with 6-fluoro-3-(4-methylpiperidin-4-yl)-4H-benzo[4,5]imidazo[1,2-b]pyrazol-8-formamide (505 g, 1.6 mol), 10% Pd/C (51 g), acetone (930.12 g, 16 mol) and NMP (5 L), and purged with hydrogen gas for 3 times, pressurized to 0.8 to 1 MPa, heated to 60 to 70 C. and stirred for 18-20 hours. After being cooled to room temperature, the mixture was filtered through celite. The filtrate was poured into water (20 L) and filtered with stirring. The filter cake was washed with water until it was neutral and dried to give the title compound as a pale yellow solid (405 g, yield 78.8%, purity 99.05%). 1H NMR (400 MHz, DMSO-d6) ppm 0.92 (d, J=6.53 Hz, 6H), 1.26 (s, 3H), 1.58-1.75 (m, 2H), 1.86-1.93 (m, 1H), 2.09-2.20 (m, 2H), 2.35 (t, J=7.72 Hz, 2H), 2.63-2.74 (m, 2H), 7.43 (dd, J=8.41, 2.64 Hz, 1H), 7.59 (dd, J=11.11, 2.57 Hz, 1H), 7.76 (s, 1H), 8.12 (s, 1H), 10.62 (s, 1H). LCMS (ESI) m/z: 358 (M+1).

Step 5: 6-fluoro-3-(1-isopropyl-4-methylpiperidin-4-yl)-4H-benzo[4,5]imidazo[1,2-b]pyrazol-8-formamide.maleate monohydrate

(31) ##STR00028##

(32) A solution of 6-fluoro-3-(1-isopropyl-4-methylpiperidin-4-yl)-4H-benzo[4,5]imidazo[1,2-b]pyrazol-8-formamide (0.404 kg, 1.13 mol) and maleic acid (0.137 kg, 1.18 mol) in 95% methanol (5.25 L) was heated to reflux for 2 hours and then filtered when hot. The filtrate was allowed to stand to portion and cool, then filtered to give a white crystal (420 g, yield 78.2%, purity 99.66%). .sup.1H NMR (400 MHz, DMSO-d6) ppm 0.63-1.70 (m, 10H), 1.81-2.32 (m, 3H), 2.82 (br. s., 1H), 3.11-3.36 (m, 4H), 6.04 (s, 2H), 7.52 (dd, J=8.28, 2.51 Hz, 1H), 7.62 (dd, J=11.04, 2.51 Hz, 1H), 7.77-7.98 (m, 1H), 8.16 (s, 1H), 8.91 (br. s., 1H), 10.54 (br. s., 1H), 12.20 (br. s., 1H). LCMS (ESI) m/z: 358 (M+1).

(33) Stability Test for Crystal Type A in Different Solvents

(34) A certain number of aliquots of 50 mg of Crystal type A were weighed out, and added with 0.3 to 0.4 mL single or mixed solvent as listed below respectively, and the mixture was stirred at 25 C. After being stirred for 3 days, the samples were centrifuged and the solid in all samples were collected. XRPD was used to determine the crystalline state. The results were shown in Table 2.

(35) TABLE-US-00002 TABLE 2 Stability test of Crystal type A in different solvents Appearance No. Solvent (3 days) Results 1 Methanol Suspension Crystal type A 2 Ethanol Suspension Crystal type A 3 Isopropanol Suspension Crystal type A 4 Acetone Suspension Crystal type A 5 Ethyl acetate Suspension Crystal type A 6 Methanol:water = 3:1 Suspension Crystal type A 7 Ethanol:water = 3:1 Suspension Crystal type A 8 Acetonitrile:water = 1:1 Suspension Crystal type A 9 Acetone:water = 1:2 Suspension Crystal type A 10 Isopropanol:water = 1:1 Suspension Crystal type A

(36) Solid Stability Test for Crystal Type A Under the Conditions of High Temperature, High Humidity and Strong Illumination

(37) A sample of Crystal type A (about 10 mg) was weighed out, placed at the bottom of a glass vial and spread into a thin layer. The sample placed at 60 C. and 92.5% relative humidity was sealed with aluminum foil at the vial mouth, and the aluminum foil was pricked to ensure that the sample was fully exposed to the ambient air; the sample placed under strong illumination (5Klux) was sealed with a screw cap. The samples placed under different conditions were sampled and tested on the 10th day. The test results were compared with the initial test results on the 0th day. The test results are shown in the following Table 3:

(38) TABLE-US-00003 TABLE 3 Solid stability test for Crystal type A Sampling Total time impurities Test conditions (Day) Appearance Content (%) (%) 0 White powder 98.9 0.10 60 C. (open) 10 White powder 98.5 0.11 92.5% RH (open) 10 White powder 99.5 0.10 Strong 10 White powder 99.4 0.11 illumination (sealed)

(39) Evaluation of In Vitro Activity

(40) Cell PARylation Analysis

(41) HCC1937 cells were seeded into a 965-well plate at 4104 cells/well and cultured in a 37 C. incubator overnight. After the cells were treated with the test compound for 30 minutes, they were treated with 1 mM hydrogen peroxide for 10 minutes. Cells were washed twice with 200 UL of precooled PBS and fixed with 100 ul of precooled methanol/acetone (7:3) for 30 minutes in an ice bath. After being air-dried, they were blocked with PBS-Tween-20 blocking solution (0.05%) with 5% nonfat dry milk dissolved at room temperature for 30 minutes. Cells and anti-PAR 10H antibody in a ratio of 1:100 were incubated in a blocking solution at room temperature for 1 hour and then washed with PBS-Tween-20 for three times, and added into a blocking solution containing goat anti-mouse fluorescein-5 (6) thiocyanate (FITC)-based secondary antibody and 1 g/mL DAPI to be incubated at room temperature in the dark for 1 hour. After being washed with PBS-Tween-20 for three times, the data was analyzed using a fluorescent microplate counter (Flexstation III, Molecular Device). PARP enzyme assay (according to the instruction of HT universal PARP1 colorimetric assay kit). Histones were packaged in a 96-well plate and incubated overnight at 4 C. After being washed with 200 UL PBST solution for three times, the plate was blocked with blocking solution, incubated for 30 minutes at room temperature and washed with PBST solution for three times. The compounds to be tested were added to the well plate and then 20 ml of diluted PARP1 (1 nM) or 20 ml of PARP2 (3 nM) was added to the reaction system and incubated for 1 or 2 hours. A mixed of streptavidin-HRP (1:50) (50 l) was added to the well plate and incubated at room temperature for 30 minutes, and washed with the PBST buffer for three times. 100 ml (HRP) (Chemiluminescent Substrate A and Substrate B (1:1)) was added to the well plate. Read immediately onto a microplate reader (Envision, PerkinElmer).

(42) Anti-Proliferation Test

(43) MDA-MB-436 and MDA-MB-231 cells were seeded in a 96-well plate at a density of 500 and 2000 cell per well, respectively, and cultured overnight. The medium was RPMI 1640 containing 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin. They were treated for 8 days after the compound to be tested was added. Cell viability was measured by CCK8 kit. Specifically, 10 UL CCK8 reagent was added to each well and incubated in a 5% CO2 incubator at 37 C. for 3 hours. After shaking for 10 minutes, the light absorbance (OD value) was measured with a Flexstation III (Molecular Device) at 450 nm.

(44) For the test of compound combinations (in combination with DNA damage drugs), the PF50 value was used to calculate the synergistic effect of the drug. PF50=[IC50 of the tested compound]/[IC50 of the compound at a fixed DNA damage drug concentration]. Temozolomide (TMZ) was used as DNA damage drug in this study.

(45) IC50 data of inhibition of MDA-MB-231/436 cell proliferation by Compound 1 and ABT888 when used singly and synergistically with TMZ are shown in Table 4 below:

(46) TABLE-US-00004 TABLE 4 In vitro screening test results of the compounds of the present invention MDA-MB-231 MDA-MB-436 IC50_N = IC50_N = IC50_Avg IC50_N = IC50_N= IC50_Avg Compound No. 1 (uM) 2 (uM) (uM) PF50 Compound No. 1 (uM) 2 (uM) (uM) PF50 ABT-888 44.04 36.99 40.51 ABT-888 0.444 0.167 0.305 ABT-888 25.46 35.96 30.71 1.32 ABT-888 0.026 0.010 0.018 17.14 combined combined with with 1uM 25uM TMZ TMZ TMZ 4.61 3.72 4.17 TMZ 80.18 141.93 111.06 Compound 1 6.21 5.44 5.83 Compound 1 0.089 0.053 0.071 Compound 1 3.96 5.75 4.86 1.20 Compound 1 0.009 0.005 0.007 9.84 combined combined with with 1uM 25uM TMZ TMZ TMZ 3.33 3.52 3.42 TMZ 152.3 61.66 106.98

CONCLUSION

(47) Compound 1 shows a strong inhibitory effect on BRAC mutant MDA-MB-436 cell line and shows good synergistic effect combined with TMZ.