AU2014200458A1 - Agent for overcoming resistance to anti-cancer agent - Google Patents

Abstract

An agent comprising, as an active ingredient, a xan thine oxidase inhibitor such as 2-(3-cyano-4-(4 5 fluorophenoxy)phenyl]-4-hydroxythiazolo[5,4-d]pyrimidine potassium salt, 2-[3-cyano-4-(4 fluorophenoxy)phenyl]thiazolo[5,4-d]pyrimidine, 2-(3 cyano-4-phenoxyphenyl)-4-hydroxythiazolo[5,4 d]pyrimidine, 2-(3-cyano-4-phenoxyphenyl)-4 10 hydroxythiazolo[5,4-d]pyrimidine potassium salt, 2-(3 cyano-4-phenoxyphenyl)thiazolo[5,4-d]pyrimidine, TMX-67, and FYX-051, which can be used as an agent for overcoming anti-cancer agent resistance.

Description

SPECIFICATION AGENT FOR OVERCOMING RESISTANCE TO ANTI-CANCER AGENT 5 Technical field The present invention relates to an agent containing a xanthine oxidase inhibitor as an active ingredient for overcoming anti-cancer agent resistance. 10 Prior art Chemotherapy is indispensable for cancer treatment. On the other hand, cancer often develops resistance to chemotherapy. Once a cancer cell acquires resistance to a anti-cancer agent, the cell frequently shows resis 15 tance to another anti-cancer agent unused in treatment or multiple drugs having different action mechanisms or dif ferent structures, which is said to be multidrug resis tance. Resistance is an enormous problem in treatment, and multidrug resistance is a particularly serious prob 20 lem. It has been desired to develop an agent for overcom ing multidrug resistance. One mechanism of multidrug re sistance is overexpression of ABC transporters such as P glycoprotein (ABCB1) and MRP1 (ABCCl), which are local ized in cell membrane. The ABC transporters excrete in 25 tracellular substrates of various structures (such as agents, physiologically active substances) out of cells in an ATP-dependent manner. Therefore, overexpression of them decreases intracellular concentrations of the drugs to cause resistance to various drugs such as anti-cancer 30 agent. Breast Cancer Resistance Protein (BCRP/ABCG2) has recently been identified as an ABC transporter (Doyle LA et al.: Proc Natl Acad Sci USA., 95:15665-15670(1998); Allikmets R et al.: Cancer Res., 58:5337-5339(1998); and 35 Miyake K et al.: Cancer Res., 59:8-13(1999)).

-2 BCRP excretes SN-38, which is an active metabolite of irinotecan, or anti-cancer agents such as mitoxantrone and topotecan out of cells. Therefore, BCRP has been noted as a molecular target to overcome anti-cancer agent 5 resistance (cf., JP 2003-63989 A). It has been known that the compound having a con densed dicyclic heterocyclic ring represented by the be low-mentioned formula (I) or (II) has xanthine oxidase (XOD) inhibitory action (cf., WO 2005/121153 Al, WO 10 2003/042185 Al, WO 2007/004688 Al). Febuxostat (TMX-67) and 4-[5-pyridin-4-yl-lH [1,2,4]triazol-3-yl]pyridine-2-carbonitrile (FYX-051) have xanthine oxidase inhibitory action. They are avail able agents for treating hyperuricemia. 15 However, it has not been known that the compound represented by the below-mentioned formula (I) or (II), TMX-67 or the like has BCRP inhibitory action. Disclosure of invention 20 An object of the invention is to provide an agent for overcoming drug resistance or an agent for overcoming anti-cancer agent resistance. The present inventors have discovered that the com pound represented by the below-mentioned formula (I) or 25 (II), TMX-67 or the like, which has xanthine oxidase in hibitory action, also has BCRP inhibitory action. The present invention has been completed based on the discov ery. The present invention resides in an agent containing 30 a compound having the following formula (I) or a salt thereof as an active ingredient for overcoming drug re sistance: -3 (I) N R 2 wherein R 1 is C 2 -8 alkenyl, C6- 1 0 aryl, or heteroaryl 5 in which the aryl and heteroaryl can have a substituent selected from the group consisting of C 1

_

8 alkyl, CI- 8 al kyl substituted with halogen, Ci.

8 alkoxy, Ci_8 alkoxy substituted with halogen, C 2

-

8 alkoxycarbonyl, formyl, carboxyl, halogen, hydroxyl, nitro, cyano, amino, C6- 10 10 aryl, and C6- 10 aryloxy;

R

2 is cyano, nitro, formyl, carboxyl, carbamoyl, or

C

2

-

8 alkoxycarbonyl; X is oxygen, -N(R ),or -S(0)n-, and R 3 is a hydro gen, C1.

8 alkyl, or the group mentioned above for R 1 , or R1 15 and R 3 are combined to form morpholinyl, thiomorpholinyl, or piperazinyl, and n is an integer of 0 to 2; and Y is oxygen, sulfur, or NH. The invention also resides in an agent containing a compound having the following formula (II) or a salt 20 thereof as an active ingredient for overcoming drug re sistance: (II) R3 N

R'-X

N R 2 -4 wherein R 1 is C 6

-

1 0 aryl or heteroaryl in which the aryl and heteroaryl can have a substituent selected from the group consisting of C 1

-

8 alkyl, C 1

_

8 alkyl substituted with halogen, C 1

_

8 alkoxy, C 1 8 alkoxy substituted with Ci_ 5 8 alkoxy, C 2

-

8 alkoxycarbonyl, formyl, carboxyl, halogen, hydroxyl, nitro, cyano, amino, C6-10 aryl, and C 6

-

1 0 ary loxy; R2 is cyano, nitro, formyl, carboxyl, carbamoyl, or

C

2

-

8 alkoxycarbonyl; 10 R 3 is hydroxyl, amino, carboxyl, mercapto, OR 4 , or NHRS, and each of R 4 and Rs is C 1 8 alkyl or C1_ 8 alkyl substituted with a group or atom selected from the group consisting of halogen, hydroxyl, nitro, cyano, amino, C 6 10 aryl, and C6- 1 0 aryloxy; 15 X is oxygen, -N(R 6 )-, or -S(O)n-, and R 6 is a hydro gen, C1_8 alkyl, or the group mentioned above for R1, and n is an integer of 0 to 2; and Y is oxygen or sulfur. The invention also resides in an agent containing 20 Febuxostat or 4-[5-pyridin-4-yl-lH-[1,2,4]triazol-3 yllpyridine-2-carbonitrile as an active ingredient for overcoming drug resistance. The present invention further resides in an agent containing a compound having the above-mentioned formula 25 (I) or a salt thereof as an active ingredient for over coming anti-cancer agent resistance. The invention also resides in an agent containing a compound having the following formula (II) or a salt the reof as an active ingredient for overcoming anti-cancer 30 agent resistance. The invention furthermore resides in an agent con taining Febuxostat or 4-[5-pyridin-4-yl-1H [1,2,4]triazol-3-yl]pyridine-2-carbonitrile as an active ingredient for overcoming anti-cancer agent resistance.

-5 The invention also resides in a therapeutic composi tion against a cancer cell of multidrug resistance, wherein the composition comprises an anti-cancer agent and a compound selected from the group consisting of 2 5 (3-cyano-4-phenoxyphenyl)thiazolo[5,4-d]pyrimidine, 2-[3 cyano-4-(4-fluorophenoxy)phenyl]thiazolo[5,4 d]pyrimidine, 2-(3-cyano-4-phenoxyphenyl)-4 hydroxythiazolo[5,4-d]pyrimidine, potassium salt of 2-(3 cyano-4-phenoxyphenyl)-4-hydroxythiazolo[5,4 10 dlpyrimidine, and potassium salt of 2-(3-cyano-4-(4 fluorophenoxy)phenyl]-4-hydroxythiazolo[5,4-d]pyrimidine, Febuxostat and 4-[5-pyridin-4-yl-1H-[1,2,4]triazol-3 yl]pyridine-2-carbonitrile. 15 Best embodiments of invention The present invention is described below in more de tail. The compound described in the formula (I) can be prepared according to methods described in the above 20 mentioned Wo 2005/121153 Al, WO 2003/042185 Al, WO 2007/004688 Al and the below-mentioned reference exam ples. The compound described in the formula (I) preferably is a compound described below or a salt thereof. 25 (1-1) A compound having the above-mentioned formula (I) or a salt thereof, wherein R1 is phenyl, naphthyl, furyl, pyrrolyl, thienyl, piperidyl, pyrimidinyl, pyranyl, pyridyl, thiazolyl, imidazolyl, indolyl, or qui nolyl in which the phenyl, naphthyl, furyl, pyrrolyl, 30 thienyl, piperidyl, pyrimidinyl, pyranyl, pyridyl, thia zolyl, imidazolyl, indolyl, and quinolyl can have a sub stituent selected from the group consisting of C 1 8 alkyl,

C

1 8 alkyl substituted with halogen, C1_8 alkoxy, C 1

.

8 alkoxy substituted with halogen, C 2

-

8 alkoxycarbonyl, for- -6 myl, carboxyl, halogen, hydroxyl, nitro, cyano, amino, C6 10 aryl, and C 6

-

1 0 aryloxy (1-2) A compound having the above-mentioned formula (I) or a salt thereof, wherein R' is phenyl or pyridyl in 5 which the phenyl and pyridyl can have a substituent se lected from the group consisting of C 1 8 alkyl, C 1

.

8 alkyl substituted with halogen, CI- 8 alkoxy, C 1

_

8 alkoxy substi tuted with halogen, C 2

-

8 alkoxycarbonyl, formyl, carboxyl, halogen, hydroxyl, nitro, cyano, amino, C 6

-

1 0 aryl, and 10 C6- 1 0 aryloxy (1-3) A compound having the above-mentioned formula (I) or a salt thereof, wherein R' is phenyl or pyridyl in which the phenyl and pyridyl can have a substituent se lected from the group consisting 'Of C 1 8 alkyl, C 1 8 alkyl 15 substituted with halogen, C1_8 alkoxy, carboxyl, halogen, hydroxyl, nitro, cyano, and amino (1-4) A compound having the above-mentioned formula (I), a compound defined in one of (1-1) to (1-3), or a salt thereof, wherein R2 is cyano or nitro 20 (1-5) A compound having the above-mentioned formula (I), a compound defined in one of (1-1) to (1-3), or a salt thereof, wherein R2 is cyano (1-6) A compound having the above-mentioned formula (I), a compound defined in one of (1-1) to (1-5), or a 25 salt thereof, wherein X is oxygen, NH, or sulfur (1-7) A compound having the above-mentioned formula (I), a compound defined in one of (1-1) to (1-5), or a salt thereof, wherein X is oxygen or sulfur (1-8) A compound having the above-mentioned formula 30 (I), a compound defined in one of (1-1) to (1-7), or a salt thereof, wherein Y is sulfur or NH (1-9) A compound having the above-mentioned formula (I), a compound defined in one of (1-1) to (1-7), or a salt thereof, wherein Y is sulfur -7 (1-10) A compound having the above-mentioned formula (I) or a salt thereof, wherein R 1 is phenyl or pyridyl in which the phenyl and pyridyl can have a substituent se lected from the group consisting of C1_8 alkyl, C 1

_

8 alkyl 5 substituted with halogen, C1_8 alkoxy, carboxyl, halogen, hydroxyl, nitro, cyano, and amino; R2 is cyanc or nitro; X is oxygen or sulfur; and Y is sulfur or NH (1-11) A compound having the above-mentioned formula (I) or a salt thereof, wherein R1 is phenyl or pyridyl in 10 which the phenyl and pyridyl can have a substituent se lected from the group consisting of Ci- 8 alkyl, C 1

_

8 alkyl substituted with halogen, C 1

_

8 alkoxy, carboxyl, halogen, hydroxyl, nitro, cyano, and amino; R2 is cyano or nitro; X is oxygen or sulfur; and Y is sulfur 15 (1-12) A compound having the above-mentioned formula (I) or a salt thereof, wherein R 1 is phenyl or pyridyl in which the phenyl and pyridyl can have a substituent se lected from the group consisting of Cig alkyl, Ci-8 alkyl substituted with halogen, C 1

_

8 alkoxy, carboxyl, halogen, 20 hydroxyl, nitro, cyano, and amino; R 2 is cyano or nitro; X is oxygen; and Y is sulfur (1-13) 2-(3-Cyano-4-phenoxyphenyl)thiazolo[5,4 d]pyrimidine and 2-[3-cyano-4-(4 fluorophenoxy)phenyl]thiazolo[5,4-d]pyrimidine (which are 25 particularly preferred) WO 2005/121153 Al discloses a process for prepara tion of the compound described in the formula (II). The compound described in the formula (II) prefera 30 bly is a compound described below or a salt thereof. (2-1) A compound having the above-mentioned formula (II) or a salt thereof, wherein R 1 is phenyl, naphthyl, furyl, pyrrolyl, thienyl, imidazolyl, pyrimidinyl, thia zolyl, pyridyl, indolyl, or quinolyl in which the phenyl, 35 naphthyl, furyl, pyrrolyl, thienyl, imidazolyl, pyrimid- -8 inyl, thiazolyl, pyridyl, indolyl, and quinolyl can have a substituent selected from the group consisting of C 1

_

8 alkyl, C 1

_

8 alkyl substituted with halogen, C 1

_

8 alkoxy,

C

2

-

8 alkoxycarbonyl, formyl, carboxyl, halogen, hydroxyl, 5 nitro, cyano, amino, C 6

-

1 0 aryl, and C6- 1 0 aryloxy (2-2) A compound having the above-mentioned formula (II) or a salt thereof, wherein R 1 is phenyl which can have a substituent selected from the group consisting of

C

1

_

8 alkyl, C 1

_

8 alkyl substituted with halogen, C 1 _8 10 alkoxy, C 2

-

8 alkoxycarbonyl, formyl, carboxyl, halogen, hydroxyl, nitro, cyano, amino, C 6

-

1 0 aryl, and C6- 1 0 ary loxy (2-3) A compound having the above-mentioned formula (II) or a salt thereof, wherein R1 is phenyl which can 15 have a substituent selected from the group consisting of

C

1

_

8 alkyl, C 1 8 alkyl substituted with halogen, C 1

_

8 al koxy, C 2

-

8 alkoxycarbonyl, formyl, carboxyl, halogen, phenyl, and phenoxy (2-4) A compound having the above-mentioned formula 20 (II), a compound defined in one of (2-1) to (2-3), or a salt thereof, wherein R 2 is cyano or nitro (2-5) A compound having the above-mentioned formula (II), a compound defined in one of (2-1) to (2-3), or a salt thereof, wherein R 2 is cyano 25 (2-6) A compound having the above-mentioned formula (II), a compound defined in one of (2-1) to (2-5), or a salt thereof, wherein R 3 is hydroxyl (2-7) A compound having the above-mentioned formula (II), a compound defined in one of (2-1) to (2-6), or a 30 salt thereof, wherein the substitution position of R 3 is 4-position of condensed (dicyclic) heterocyclic ring (2-8) A compound having the above-mentioned formula (II), a compound defined in one of (2-1) to (2-7), or a salt thereof, wherein X is oxygen, NH, or sulfur -9 (2-9) A compound having the above-mentioned formula (II), a compound defined in one of (2-1) to (2-7), or a salt thereof, wherein X is oxygen (2-10) A compound having the above-mentioned formula 5 (II), a compound defined in one of (2-1) to (2-9), or a salt thereof, wherein Y is sulfur (2-12) 2-(3-cyano-4-phenoxyphenyl)-4 hydroxythiazolo[5,4-d]pyrimidine, potassium salt of 2-(3 cyano-4-phenoxyphenyl)-4-hydroxythiazolo[5,4 10 d]pyrimidine, and potassium salt of 2-[3-cyano-4-(4 fluorophenoxy)phenyl]-4-hydroxythiazolo[5,4-dlpyrimidine (which are particularly preferred active ingredients) The agent of the present invention for overcoming 15 resistance is intended for anti-cancer agents such as ir inotecan, SN-38, which is an active metabolite of iri notecan, mitoxantrone, topotecan, methotrexate, doxorubi cin, daunorubicin, etoposide, gefitinib, and imatinib. There is no specific limitation on kinds of cancers 20 targeted with the agent of the present invention for overcoming anti-cancer agent resistance, so long as the cancers overexpressing BCRP. Examples of cancers with re sistance include blood cancer (hematological malignancy), liver cancer, colon cancer, lung cancer, breast cancer, 25 ovarian cancer, cervical cancer, osteosarcoma, brain tu mor, pancreatic cancer, and prostate cancer. The pharmacological effects of the invention are de scribed below. 30 Below-described Example 1 is an experiment confirm ing an effect of compounds on BCRP-mediated methotrexate transport. As is evident from Table 1, it has been confirmed that each of the compound 1 (potassium salt of 2-[3 35 cyano-4-(4-fluorophenoxy)phenyl]-4-hydroxythiazolo[5,4- - 10 dipyrimidine), the compound 2 (2-[3-cyano-4-(4 fluorophenoxy)phenyl]thiazolo[5,4-d]pyrimidine), the com pound 3 (potassium salt of 2-(3-cyano-4-phenoxyphenyl)-4 hydroxythiazolo[5,4-d]pyrimidine), the compound 4 (2-(3 5 cyano-4-phenoxyphenyl)thiazolo[5,4-d]pyrimidine), TMX-67, and FYX-051 strongly inhibits BCRP-mediated methotrexate transport. Below described Example 2 is an experiment confirm ing an effect of compounds on anti-cancer agent resis 10 tance in BCRP-overexpressing cells (Flp-In-293/BCRP). Each of the compounds 1 and 2 reverses resistance of Flp-In-293/ABCG2 cells to SN-38 in a concentration dependent manner. The compound 1 and the compound 2 re versed the resistance by about 96% and about 90% respec 15 tively at the concentration of 5 pmol/L. Further, the compounds do not affect viability of Flp-In-293/ABCG2 cells up to the examined concentration of 10 pmol/L. Therefore, it has been confirmed that the compounds 1 and 2 exhibit effect of overcoming anti-cancer agent resis 20 tance in BCRP-overexpressing cells without exhibiting toxicity to the cells. In addition, in the experiments conducted in the same manner as in Example 2, the com pound 5 (2-(3-cyano-4-phenoxyphenyl)-4 hydroxythiazolo[5,4-d]pyrimidine), TMX-67, and FYX-051 do 25 not exhibit toxicity to the cells at the concentration of 10 pmol/L, and each of them reversed the resistance by about 96%, about 82% and about 39% respectively at the concentration of 5 pmol/L. On the other hand, among the known agents having 30 xanthine oxidase (XOD) inhibitory action, allopurinol and oxypurinol do not have an inhibitory action on BCRP mediated methotrexate transport, and do not exhibit an effect of overcoming resistance to SN-38. Further, Y-700 (1-[3-cyano-4-(2,2-dimethylpropoxy)phenyl]-lH-pyrazole-4- -: - 11 carboxylic acid) also does not exhibit an effect of over coming resistance to SN-38(Comparison example 1 and 2). As is described above, the agent having xanthine oxidase (XOD) inhibitory action, particularly the com 5 pound represented by the above-mentioned formula (I) or (II), TMX-67, or FYX-051 can be used as an active ingre dient for overcoming drug resistance, particularly as an active ingredient for overcoming anti-cancer agent resis tance. 10 The agent for overcoming drug resistance or the agent for overcoming anti-cancer agent resistance of the invention can be administered to human by ordinary ad ministration methods such as oral administration or par enteral administration. 15 The agent can be granulated in ordinary manners for the preparation of pharmaceuticals. For instance, the agent can be processed to give pellets, granule, powder, capsule, suspension, injection, suppository, and the like. 20 For the preparation of these pharmaceuticals, ordi nary additives such as vehicles, disintegrators, binders, lubricants, dyes, and diluents can be used. As the vehi cles, lactose, D-mannitol, crystalline cellulose and glu cose can be mentioned. Further, there can be mentioned 25 starch and carboxymethylcellulose calcium (CMC-Ca) as the disintegrators, magnesium stearate and talc as the lubri cants, and hydroxypropylcellulose (HPC), gelatin and po lyvinylpyrrolidone (PVP) as the binders. The agent of the invention can be administered to an 30 adult generally in an amount of 0.1 mg to 100 mg a day by parenteral administration and 1 mg to 2,000 mg a day by oral administration. The dosage can be adjusted in con sideration of age and conditions of the patient. A therapeutic agent (composition) comprising an 35 agent for overcoming anti-cancer agent resistance and a - 12 known anti-cancer agent can be prepared according to a preparation method of a conventional composition. The two agents can also separately be administered to a patient at the same time or at some intervals. 5 In case of hematological malignancy, solid tumor with metastasis and the like, the administration of an ti-cancer agent induces abrupt tumor necrosis resulting in the release of a large amount of the intracellular components of the tumor cells into blood. As a result, 10 hyperuricemia, hyperkalemia, or hypocalcemia occurs and these symptoms sometimes induce renal failure or cardiac arrest (tumor lysis syndrome) . The active ingredient of the invention has xanthine oxidase inhibitory action as well as effect of overcoming anti-cancer agent resis 15 tance. Therefore, the ingredient of the invention is also effective in treating hyperurcemia caused with tumor ly sis. The invention is further described by the following examples, but is not limited to these examples. 20 Examples Reference example 1 (1) 4-Chloro-N-(4-chloro-5-pyrimidinyl)-3-cyanobenzamide 4-Chloro-3-cyanobenzoic acid (7.01 g, 38.6 mmol) was 25 suspended in benzene (70 mL). Thionyl chloride (3.6 mL, 49.6 mmol) was added to the suspension, and the mixture was refluxed with heating for 4 hours. The reaction liq uid was condensed under reduced pressure. 5-Amino-4 chloropyrimidine (5.00 g, 38.6 mmol), dichloromethane (70 30 mL), and pyridine (3.6 mL, 44.5 mmol) were added to the obtained acid chloride. The mixture was stirred at room temperature for 7 hours. Chloroform (50 mL) and water (50 mL) was added to the reaction solution to filtrate crys tals. The obtained crystals were washed with chloroform 35 (20 mL) and water (20 mL), and air-dried to obtain 7.35 g - 13 (yield: 65%) of the subject compound as white crystals. Further, 0.62 g (yield: 8 %) of the subject compound was obtained from a mixed solution of mother liquid and wash ings as pale brown crystals (secondary crystals). The to 5 tal yield was 73%. mp: 189-190'C 'H NMR (CDCl 3 , 400MHz): 5=7.74 (lH,d,J=8Hz), 8.07 (1H,dd,J=2Hz,8Hz), 8.13 (1H,s), 8.23 (lH,d,J=2Hz), 8.83 (lH,s), 9.79 (lH,s). 10 (2) 2-(4-Chloro-3-cyanophenyl)thiazolo[5,4-d]pyrimidine The above-obtained 4-chloro-N-(4-chloro-5 pyrimidinyl)-3-cyanobenzamide (7.98 g, 27.2 mmol) and La wesson's reagent (8.25 g, 20.4 mmol) were suspended in 15 toluene (150 mL). The suspension was refluxed with heat ing for 8 hours, and cooled to room temperature to fil trate precipitated crystals. The crystals were washed with chloroform (75x2), and air-dried to obtain 7.25 g (yield: 98%) of the subject compound as pale yellow crys 20 tals. mp: 278-280'C (decomposition) 'H NMR (DMSO-ds, 400MHz): 5=7.99 (lH,d,J=9Hz), 8.47 (lH,dd,J=2Hz,9Hz), 8.70 (1H,d,J=2Hz), 9.20 (lH,s), 9.54 (1H,s). 25 (3) 2-[3-Cyano-4-(4-fluorophenoxy)phenyl]thiazolo[5,4 dlpyrimidine 4-Fluorophenol (383 mg, 3.42 mmol) was added to a suspension of 55% sodium hydride (150 mg, 3.44 mmol) and 30 dried DMSO (7 mL) . The mixture was stirred at 50*C for 30 minutes. The above-mentioned 2-(4-chloro-3 cyanophenyl)thiazolo[5,4-d]pyrimidine (776 mg, 2.85 mmol) was added to the reaction liquid. The mixture was stirred at 50 0 C for 4 hours, and cooled to room temperature. Wa 35 ter (35 mL) was added to the reaction liquid to filtrate - 14 precipitated crystals. The crystals were washed with wa ter (20 mL), and air-dried. The obtained crystals were purified by a silica gel column chromatography (chloro form), washed with ether (15 mL), and dried to obtain 701 5 mg (yield: 71%) of the subject compound as pale yellow crystals. mp: 175-177*C 1 H NMR (CDCl 3 , 400MHz): 5=6.94 (1H,d,J=9Hz), 7.1-7.2 (4H,m), 8.18 (1H,dd,J=2Hz,9Hz), 8.44 (1H,d,J=2Hz), 9.13 10 (1H,s), 9.35 (1H,s). IR(KBr)cm 1 : 2233, 1606, 1564, 1419, 1300, 1119, 1011, 916, 893, 847, 829, 777, 760, 758, 723, 702, 700, 650, 648, 597, 526, 496, 490. FAB-MS(m/e): 349(M+l) 15 Reference example 2 2-(3-Cyano-4-phenoxyphenyl)thiazolo[5,4,-d]pyrimidine Phenol (45 mg, 0.48 mmol) was added to a suspension of 55% sodium hydride (23 mg, 0.53 mmol) and dried DMSO 20 (1 mL). The mixture was stirred at room temperature for 30 minutes. The above-mentioned 2-(4-chloro-3 cyanophenyl)thiazolo[5,4-d]pyrimidine (120 mg, 0.44 mmol) was added to the reaction liquid. The mixture was stirred at 60'C for 4 hours, and cooled to room temperature. Wa 25 ter (5 mL) was added to the reaction liquid to filtrate precipitated crystals. The crystals were washed with wa ter (5 mL), ethanol (1 mL), and then ether (2 mL) in the order. The crystals were dried at room temperature under reduced pressure to obtain 100 mg (yield: 69%) of the 30 subject compound as pale brown crystals. mp: 154-156*C 1H NMR (DMSO-d 6 , 400MHz): 5=7.08 (1H,d,J=9Hz), 7.2-7.4 (3H,m), 7.5-7.6 (2H,m), 8.42 (1H,dd,J=2Hz,9Hz), 8.69 (1H,d,J=2Hz), 9.18 (1H,s), 9.52 (1H,s).

- 15 IR(KBr)cm 1 : 3037, 2227, 1605, 1587, 1560, 1525, 1504, 1470, 1369, 1365, 1257, 1238, 1190, 1171. FAB-MS(m/e): 331(M+1) 5 Example 1 Action on BCRP-mediated methotrexate transport (Testing method) In the experiment, plasma membrane was prepared from BCRP-expressing Sf9 cells. Its membrane vesicle was used 10 (Ishikawa et al.: Methods of Enzymol., 400:485-510 (2005)). The activity of methotrexate transport was measured using 96 wells-plate according to the following method. 15 Preparation and composition of reaction solution 50 pL of solution containing sucrose (250 mmol/L) and Tris/Hepes (10 mmol/L, pH: 7.4) 30 pL of solution containing ATP (3.33 mmol/L), creatine phosphate (33.3 mmol/L), and MgCl 2 (33.3 mmol/L) 20 (or solution containing creatine phosphate (33.3 mmol/L) and MgCl 2 (33.3 mmol/L)) 5 pL of creatine kinase (2 mg/mL) 2 pL of [3H]methotrexate (10 mmol/L, final concentra tion: 200 pmol/L) 25 3 pL of test compound 10 pL of membrane sample of BCRP-expressing Sf9 cells (total protein: 50 pg) Total: 100 pL 30 The reaction solution was incubated at 37*C for 20 minutes. 1 mL of ice-cooled solution containing sucrose (250 mmol/L), EDTA (2 mmol/L), and Tris/Hepes (10 mmol/L, pH: 7.4) was quickly added to the reaction solution to stop the reaction. Each 270 pL of the mixed solution was 35 poured into each well of MultiScreen

TM

(Millipore), and - 16 sucked. The wells were washed four times with 200 pL of ice-cooled solution containing sucrose (250 mmol/L) and Tris/Hepes (10 mmol/L, pH: 7.4). The radioactivity trapped on a filter of each well was measured. The amount 5 of methotrexate transported into the membrane vesicle was calculated from the radioactivity. Further, the IC 50 value of the test compound [concen tration inhibiting methtrexate transport by 50%, inhibi tory concentration (pmol/L)] was obtained according to 10 the above-mentioned method. (Results) As is shown in Table 1, it was confirmed that each of the compound 1 to 4, TMX-67, and FYX-051 strongly in 15 hibits the BCRP-mediated methotrexate transport. Action on BCRP-mediated methotrexate transport TABLE 1 20 Test compound ICs 0 (pmol/L) Compound 1 0.46 Compound 2 1.1 Compound 3 0.23 Compound 4 0.66 TMX-67 0.25 FYX-051 0.77 Compound 1: Potassium salt of 2-[3-cyano-4-(4 fluorophenoxy)phenyl]-4-hydroxythiazolo[5,4-d]pyrimidine Compound 2: 2-[3-Cyano-4-(4 25 fluorophenoxy)phenyl]thiazolo[5,4-d]pyrimidine - 17 Compound 3 Potassium salt of 2-(3-cyano-4 phenoxyphenyl)-4-hydroxythiazolo[5,4-d]pyrimidine Compound 4: 2-(3-cyano-4 phenoxyphenyl)thiazolo[5,4-d]pyrimidine 5 Comparison example 1 Action on BCRP-mediated methotrexate transport (Testing method) Action on BCRP-mediated methotrexate transport was 10 tested with respect to allopurinol and oxypurinol in the same manner as in Example 1. (Results) Each of allopurinol and oxypurinol did not exhibit 15 an inhibitory action even at concentration 'of 100 pmol/L. Example 2 Influence on anti-cancer agent resistance in BCRP overexpressing cells (Flp-In-293/BCRP) 20 (Testing method) Flp-In-293/ABCG2 cells and Flp-In-293/Mock cells were used in the experiment (Wakabayashi et al.: J. Exp. Ther. Oncol., 5:205-222 (2006)). The cells were cultured under the atmosphere of 5% 25 CO 2 using DMEM containing FCS (10%), penicillin (100 U/mL), streptomycin (100 pg/mL), amphotericin B (250 ng/mL), hy gromycin B (100 pg/mL), and L-glutamine (2 mmol/L). The resistance of Flp-In-293 cells to an agent was profiled by counting living cells using MTT assay. In 30 more detail, the cells were seeded in 96-wells plate at the concentration of 2x10 3 cells/well, and cultured for 24 hours. SN-38 and the compound 1 (potassium salt of 2-[3 cyano-4-(4-fluorophenoxy)phenyl]-4-hydroxythiazolo[5,4 d]pyrimidine or compound 2 (2-[3-cyano-4-(4 35 fluorophenoxy)phenyl]thiazolo[5,4-d]pyrimidine) were add- - 18 added to the cells, and the cells were further incubated for 72 hours. The cells were treated with MTT (500 pg/mL), and were further incubated for 4.hours. Then the cells were treated with 100 pL of 10% SDS, and were incubated 5 overnight. The formed MTT-formazan (metabolite of MTT, which is formed by living cells) was measured at the wa velength of 570 nm and 630 nm. (Results) 10 Each of the compounds 1 and 2 reversed the resis tance of Flp-In-293/ABCG2 cells to SN-38 in a concentra tion-dependent manner. The compound 1 reversed the resis tance by about 96%, and the compound 2 reversed by about 90% at the concentration of 5 pmol/L. Further, the com 15 pounds did not affect viability of Flp-In-293/ABCG2 cells up to the examined concentration of 10 pmol/L. Therefore, it has been confirmed that the compounds 1 and 2 do not exhibit toxicity to the cells, and do ex hibit effect of overcoming anti-cancer agent resistance 20 in the BCRP-overexpressing cells without exhibiting tox icity to the cells. Example 3 and Comparison example 2 Influence on anti-cancer agent resistance in BCRP 25 overexpressing cells (Flp-In-293/BCRP) (Testing method) Influence on anti-cancer agent resistance in BCRP overexpressing cells (Flp-In-293/BCRP) was tested in the same manner as in Example 2 with respect to 2-(3-cyano-4 30 phenoxyphenyl)-4-hydroxythiazolo[5,4-d]pyrimidine (com pound 5), TMX-67, FYX-051, allopurinol, oxypurinol, and Y-700.

- 19 (Results) (i) Degree of overcoming resistance to SN-38 The compound 5, TMX-67 and FYX-051 reversed the re sistance by about 96%, about 82%, and about 39% respec 5 tively at the concentration of 5 pmol/L. On the other hand, allopurinol and oxypurinol did not exhibit an ef fect of overcoming resistance to SN-38. Further, Y-700 also did not exhibit an effect of overcoming the resis tance in the concentration not exhibiting toxicity to the 10 cells. (ii) Toxicity to the cells The compound 5, TMX-67, FYX-051, allopurinol, and oxypurinol did not exhibit toxicity to the cells at the 15 concentration of 10 lmol/L. Y-700 exhibits toxicity to the cells at the concentration of 5 pmol/L or higher.

Claims (6)

1. An agent containing a compound having the fol lowing formula (I) or a salt thereof as an active ingre 5 dient for overcoming drug resistance: (I) N N N R2 wherein R 1 is C 2 - 8 alkenyl, C6-10 aryl, or heteroaryl 10 in which the aryl and heteroaryl can have a substituent selected from the group consisting of C 1 8 alkyl, Ci-g al kyl substituted with halogen, Cj_ 8 alkoxy, C 1 8 alkoxy substituted with halogen, C 2 - 8 alkoxycarbonyl, formyl, carboxyl, halogen, hydroxyl, nitro, cyano, amino, C 6 - 10 15 aryl, and C 6 - 1 0 aryloxy; R 2 is cyano, nitro, formyl, carboxyl, carbamoyl, or C 2 - 8 alkoxycarbonyl; X is oxygen, -N(R 3 )-, or -S(O)n-, and R3 is a hydro gen, C 1 _ 8 alkyl, or the group mentioned above for R , or R 20 and R 3 are combined to form morpholinyl, thiomorpholinyl, or piperazinyl, and n is an integer of 0 to 2; and Y is oxygen, sulfur, or NH. - 21 2. An agent containing a compound having the fol lowing formula (II) or a salt thereof as an active ingre dient for overcoming drug resistance: (II) R 3 NN N N R'-X - l ) --- Y N 5R2 wherein R 1 is C 6 - 1 0 aryl or heteroaryl in which the aryl and heteroaryl can have a substituent selected from the group consisting of C18 alkyl, C 1 _ 8 alkyl substituted 10 with halogen, C 1 _ 8 alkoxy, Ci_ 8 alkoxy substituted with Ci_ 8 alkoxy, C 2 - 8 alkoxycarbonyl, formyl, carboxyl, halogen, hydroxyl, nitro, cyano, amino, C 6 - 1 0 aryl, and C 6 - 1 0 ary loxy; R2 is cyano, nitro, formyl, carboxyl, carbamoyl, or 15 C 2 -8 alkoxycarbonyl; 3 4 R is hydroxyl, amino, carboxyl, mercapto, OR , or NHRS, and each of R 4 and R 5 is C1_8 alkyl or Ci_ 8 alkyl substituted with a group or atom selected from the group consisting of halogen, hydroxyl, nitro, cyano, amino, C 6 20 10 aryl, and C 6 -1 0 aryloxy; X is oxygen, -N(R6)-, or -S(0)n-, and R6 is a hydro gen, C 1 _ 8 alkyl, or the group mentioned above for R1, and n is an integer of 0 to 2; and Y is oxygen or sulfur. 25 - 22 3. An agent containing a compound selected from the group consisting of 2-(3-cyano-4 phenoxyphenyl)thiazolo[5,4-d]pyrimidine,

2-[3-cyano-4-(4 fluorophenoxy)phenyl]thiazolo[5,4-d]pyrimidine, 2-(3 5 cyano-4-phenoxyphenyl)-4-hydroxythiazolo[5,4 dipyrimidine, potassium salt of 2-(3-cyano-4 phenoxyphenyl)-4-hydroxythiazolo[5,4-d]pyrimidine, and potassium salt of 2 -[ 3 -cyano-4-(4-fluorophenoxy)phenyl]

4-hydroxythiazolo[5,4-d]pyrimidine as an active ingredi 10 ent for overcoming drug resistance. 4. An agent containing Febuxostat or 4-[S-pyridin 4 -yl-1H-[1, 2 ,4]triazol-3-yl]pyridine-2-carbonitrile as an active ingredient for overcoming drug resistance. 15 -23 5. An agent containing a compound having the fol lowing formula (I) or a salt thereof as an active ingre dient for overcoming anti-cancer agent resistance: (I) N R--X N N 5 R2 wherein R1 is C 2 -8 alkenyl, C 6 - 10 aryl, or heteroaryl in which the aryl and heteroaryl can have a substituent selected from the group consisting of Ci- 8 alkyl, Ci_ 8 al 10 kyl substituted with halogen, Ci_ 8 alkoxy, C 1 8 alkoxy substituted with halogen, C 2 - 8 alkoxycarbonyl, formyl, carboxyl, halogen, hydroxyl, nitro, cyano, amino, C 6 - 10 aryl, and C6- 10 aryloxy; R2 is cyano, nitro, formyl, carboxyl, carbamoyl, or 15 C 2 - 8 alkoxycarbonyl; X is oxygen, -N (R 3 ) -, or -S (0) -, and R 3 is a hydro gen, C 1 _ 8 alkyl, or the group mentioned above for R 1 , or R and R 3 are combined to form morpholinyl, thiomorpholinyl, or piperazinyl, and n is an integer of 0 to 2; and 20 Y is oxygen, sulfur, or NH. -24 6. An agent containing a compound having the fol lowing formula (II) or a salt thereof as an active ingre dient for overcoming anti-cancer agent resistance: (II) N R 1 -X/ N 5 R2 wherein R' is C6- 1 0 aryl or heteroaryl in which the aryl and heteroaryl can have a substituent selected from the group consisting of Ci_ 8 alkyl, C 1 _ 8 alkyl substituted 10 with halogen, Ci_ 8 alkoxy, C 1 _ 8 alkoxy substituted with C 1 _ 8 alkoxy, C 2 - 8 alkoxycarbonyl, formyl, carboxyl, halogen, hydroxyl, nitro, cyano, amino, C6- 1 0 aryl, and C6- 10 ary loxy; R2 is cyano, nitro, formyl, carboxyl, carbamoyl, or 15 C 2 - 8 alkoxycarbonyl; 3 4 R is hydroxyl, amino, carboxyl, mercapto, OR , or NHR , and each of R 4 and R 5 is C 1 _ 8 alkyl or C 1 8 alkyl substituted with a group or atom selected from the group consisting of halogen, hydroxyl, nitro, cyano, amino, C 6 20 10 aryl, and C6-10 aryloxy; X is oxygen, -N(R 6 )-, or -S(0),-, and R 6 is a hydro gen, C 1 - 8 alkyl, or the group mentioned above for R1, and n is an integer of 0 to 2; and Y is oxygen or sulfur. 25 - 25 7. An agent containing a compound selected from the group consisting of 2-(3-cyano-4 phenoxyphenyl)thiazolo[5,4-d]pyrimidine, 2-[3-cyano-4-(4 fluorophenoxy)phenyl]thiazolo[5,4-d]pyrimidine, 2-(3 5 cyano-4-phenoxyphenyl)-4-hydroxythiazolo[5,4 d]pyrimidine, potassium salt of 2-(3-cyano-4 phenoxyphenyl)-4-hydroxythiazolo[5,4-d]pyrimidine, and potassium salt of 2 -[ 3 -cyano-4-(4-fluorophenoxy)phenyl] 4-hydroxythiazolo[5,4-d]pyrimidine as an active ingredi 10 ent for overcoming anti-cancer agent resistance.

8. An agent containing Febuxostat or 4-[5-pyridin 4 -yl-1H-[1, 2 , 4 ]triazol-3-yl]pyridine-2-carbonitrile as an active ingredient for overcoming resistance to an anti 15 cancer agent.

9. An agent for overcoming resistance to an anti cancer agent defined in any one of claims 5 to 8, wherein overexpression of BCRP in a cancer cell is involved in 20 acquiring anti-cancer agent resistance.

10. A therapeutic composition against a cancer cell of multidrug resistance, wherein the composition com prises an anti-cancer agent and a compound selected from 25 the group consisting of 2-(3-cyano-4 phenoxyphenyl)thiazolo[5,4-d]pyrimidine, 2-[3-cyano-4-(4 fluorophenoxy)phenyl]thiazolo[5,4-d]pyrimidine, 2-(3 cyano-4-phenoxyphenyl)-4-hydroxythiazolo[5,4 d]pyrimidine, potassium salt of 2-(3-cyano-4 30 phenoxyphenyl)-4-hydroxythiazolo[5,4-d]pyrimidine, potas sium salt of 2 -[ 3 -cyano-4-(4-fluorophenoxy)phenyl]-4 hydroxythiazolo[5,4-dpyrimidine, Febuxostat, and 4-[5 pyridin-4-yl-lH-[1,2,4]triazol-3-yl]pyridine-2 carbonitrile. 35