Pharmaceutical use of thiophene [3,2-d] pyrimidine-4-ketone compound

Abstract

Disclosed is use of a thieno[3,2-d]pyrimidin-4-one compound represented by general formula (I). Definition of each substituent is as stated in the description and claims, and the compound is used for preparing medicines for treating and/or preventing liver fibrosis and related diseases or preparing a DPP-4 inhibitor, or is used as a DPP-4 inhibitor. ##STR00001##

Claims

1. A method for treating liver fibrosis, comprising administering a thieno[3,2-d]pyrimidin-4-one compound represented by the following general formula (I): ##STR00055## or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof to a subject in need thereof, wherein: n is an integer from 1 to 3; each of R.sub.1 and R.sub.2 is independently H or COOR.sub.8, provided that R.sub.1 and R.sub.2 are different; R.sub.8 is H, or linear or branched C1-C6 alkyl; R.sub.3 is ##STR00056## which is a 3-7 membered nitrogen-containing heterocyclic radical, and is substituted by one amino; and each of R.sub.4 and R.sub.5 is independently H or cyano.

2. The method of claim 1, wherein: n is 1; each of R.sub.1 and R.sub.2 is independently H or COOR.sub.8, provided that R.sub.1 and R.sub.2 are different; R.sub.8 is H or methyl; R.sub.3 is ##STR00057## which is a 3-7 membered nitrogen-containing heterocyclic radical, and is substituted by one amino; and each of R.sub.4 and R.sub.5 is independently H or cyano, provided that R.sub.4 and R.sub.5 are different.

3. The method of claim 1, wherein n is 1; each of R.sub.1 and R.sub.2 is independently H or COOR.sub.8, provided that R.sub.1 and R.sub.2 are different; R.sub.8 is H, or linear or branched C1-C3 alkyl; R.sub.3 is pyrrolidinyl, piperidinyl, piperazinyl, or homopiperazinyl, and is substituted by one amino; and each of R.sub.4 and R.sub.5 is independently H or cyano, provided that R.sub.4 and R.sub.5 are different.

4. The method of claim 1, wherein the thieno[3,2-d]pyrimidin-4-one compound of general formula (I) is selected from the group consisting of: ##STR00058##

5. The method of claim 1, wherein the thieno[3,2-d]pyrimidin-4-one compound, pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof is administered through a pharmaceutically acceptable carrier.

6. The method of claim 5, wherein the pharmaceutically acceptable carrier is selected from the group consisting of diluent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, surfactant, adsorption carrier, and lubricant.

7. The method of claim 1, wherein the thieno[3,2-d]pyrimidin-4-one compound, pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof is formulated into a tablet, powder, pill, injection, capsule, film, suppository, ointment or electuary.

8. The method of claim 1, wherein the thieno[3,2-d]pyrimidin-4-one compound, pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof is introduced into the body by injection, spraying, nose drops, eye drops, penetration, absorption, physically or chemically mediated methods.

9. The method of claim 1, wherein the treating is: (1) down-regulating expression of α-SMA; (2) down-regulating expression of Col1α1 mRNA; (3) decreasing deposition of collagen in the liver; (4) reducing ALT level; (5) reducing AST level; (6) reducing content of hydroxyproline in liver tissue; or (7) inhibiting activation of hepatic stellate cell HSC.

Description

DESCRIPTION OF FIGURES

(1) FIG. 1 shows the results of the DPP-4 inhibitor in Example 3 of the present invention on inhibiting hepatic stellate cell activation.

(2) FIG. 2 shows the results of the DPP-4 inhibitor in Example 4 of the present invention on alleviating liver injury blood biochemical indexes, liver fibrosis-related gene expression and collagen characteristic amino acid indexes in CCl.sub.4-induced liver fibrosis model mice, wherein the bars from the left to the right represent model control group, DC291407 (20 mg/kg), DC291407 (60 mg/kg), OCA (20 mg/kg), OCA (40 mg/kg), WT, respectively.

(3) FIG. 3 shows the results of the DPP-4 inhibitor in Example 4 of the present invention on alleviating the pathological indexes of collagen deposition in liver pathological sections in CCl.sub.4-induced liver fibrosis model mice, wherein the bars from the left to the right represent model control group, DC291407 (20 mg/kg), DC291407 (60 mg/kg), OCA (20 mg/kg), OCA (40 mg/kg), WT, respectively.

(4) FIG. 4 shows the results of the DPP-4 inhibitor in Example 5 of the present invention on alleviating liver injury blood biochemical indexes and liver triglycerides in high fat-induced non-alcoholic fatty liver disease ob/ob mice model.

(5) FIG. 5 shows the results of the DPP-4 inhibitor in Example 5 of the present invention on improving liver steatosis and inflammatory pathological indexes in high fat-induced non-alcoholic fatty liver disease ob/ob mice model.

(6) FIG. 6 shows the results of the DPP-4 inhibitor in Example 6 of the present invention on alleviating liver injury blood biochemical indexes and collagen characteristic amino acid indexes in CCl.sub.4-induced liver fibrosis model mice.

(7) FIG. 7 shows the results of the DPP-4 inhibitor in Example 6 of the present invention on alleviating the pathological indexes of collagen deposition in liver in CCl.sub.4-induced liver fibrosis model.

DETAILED DESCRIPTION

(8) The inventors of the present application have conducted extensive and intensive research and have found that thieno[3,2-d]pyrimidin-4-one compounds can improve liver function, down-regulate the expression of α-SMA and Col1α1 mRNA, and reduce the deposition of collagen in the liver, and can be used to treat and/or prevent liver fibrosis and related diseases. On this basis, the present invention has been completed.

(9) Terms

(10) “Alkyl” refers to a straight-chain or branched saturated aliphatic hydrocarbon group, for example, “C.sub.1-8 alkyl” refers to a straight-chain and branched alkyl group including 1 to 8 carbon atoms, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, etc.

(11) “Cyclic hydrocarbyl” refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent. For example, “C.sub.3-8 cycloalkyl” refers to a cycloalkyl group containing 3 to 8 carbon atoms, which is classified as monocyclic cycloalkyl, and polycyclic cycloalkyl, wherein monocyclic cycloalkyl includes but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc. Polycyclic cycloalkyl includes spiro, fused, and bridged cycloalkyls.

(12) “Heterocyclic radical (or heterocyclyl)” refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent in which one or more ring atoms are selected from N, O or S.

(13) “Aryl” refers to an all-carbon monocyclic or fused polycyclic group (that is, a ring that shares adjacent pairs of carbon atoms), a polycyclic ring with a conjugated π-electron system (that is, a ring with adjacent pairs of carbon atoms), including but not limited to phenyl and naphthyl.

(14) “Heteroaryl” refers to a heteroaromatic system containing 1 to 4 heteroatoms including N, O and S, for example, a 5-7 membered heteroaryl refers to a heteroaromatic system containing 5-7 ring atoms, 5-10 membered heteroaryl refers to heteroaromatic system containing 5-10 ring atoms, including but not limited to furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc.

(15) “Alkoxy” refers to —O-(alkyl), where the definition of alkyl is as described above. For example, “C.sub.1-8 alkoxy” refers to an alkyloxy group containing 1-8 carbons, including but not limited to methoxy, ethoxy, propoxy, butoxy, etc.

(16) The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples usually follow conventional conditions (such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or according to the conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are percentages by weight and parts by weight.

(17) Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the contents described can be applied to the method of the present invention. The preferred embodiments and materials described herein are for demonstration purpose only.

Example 1 Preparation of DC291407

(18) Synthetic Route:

(19) ##STR00049## ##STR00050## ##STR00051##

(20) Compound 11-12 (100 mg) was synthesized by using the above route and then dissolved in DMF. 1.5 equivalents of methyl iodide and 2 equivalents of cesium carbonate were added and stirred at room temperature for 1 hour. The mixture was extracted with ethyl acetate, washed with water, and evaporated to dryness to obtain 100 mg of crude product 38-1.

(21) Compound 38-1 was dissolved in 40 ml of DCM and 15 ml TFA was added and stirred at room temperature for 4 hours. The solvent was removed by rotary evaporation and the residue was dissolved in 50 ml ethyl acetate, washed with saturated potassium carbonate solution and then saturated sodium chloride solution. The solvent was removed by rotary evaporation and the residue was separated by column chromatography (DCM: CH.sub.3OH═5:1) to obtain compound DC291407.

(22) .sup.1H-NMR (CDCl.sub.3-d.sub.3): δ=7.761(s, 1H), 7.610(d, 1H), 7.493(t, 1H), 7.320(t, 1H), 7180(d, 1H), 5.500(quartet, 2H), 3.895(s, 3H), 3.680(d,2H), 3.355(m, 1H), 3.010(m, 2H), 2.150(m, 1H), 1.894(m,2H), 1.644(m, 1H); LC-MS m/z 424.1 [M+H].sup.+.

Example 2 Preparation of DC291410

(23) Synthetic Route:

(24) ##STR00052## ##STR00053## ##STR00054##

(25) Compound 11-12 (100 mg, 0.237 mmol) was dissolved in 20 ml of ether hydrochloride, stirred at room temperature for 1 hour, and evaporated to dryness to obtain compound DC291410 (70 mg) with a yield of 80%, MS: 410.1 [M+H].sup.+.

Example 3 Treatment of Liver Fibrosis with DPP-4 Inhibitor

(26) Human hepatic stellate cell line LX-2 was used in cell function experiments. The hepatic stellate cell activation and inducement agent TGF-β was added to activate hepatic stellate cells thereby significantly increasing the expression of multiple extracellular matrixes. By comparing the expression levels of extracellular matrix in the TGF-β group and the cell group in which TGF-β and the compound to be tested were both added at the same time, the efficacy of the compound in alleviating liver fibrosis through hepatic stellate cells was characterized.

(27) The cell experiment procedure was as follows. The human hepatic stellate cell line LX-2 was selected and inoculated into the treated 24-well plate at 7.5×10.sup.4/well. After the cells were adhered overnight and they were subjected to starvation treatment for 12 hours, the cells were treated with 10 ng/mL TGF-β and the corresponding test compound for 24 hours and then protein samples were collected. The expression of extracellular matrix represented by α-smooth muscle myofilament (α-SMA) and fibronectin was detected by Western Blot.

(28) The compounds of the present invention were all high-activity inhibitors of DPP-4, among which two compounds, DC291407 and DC291410 concentration-dependently inhibited the activation of hepatic stellate cells HSC as the concentration increased, and the inhibitory activity was better than that of the positive control Alogliptin, as shown in FIG. 1. Such compounds have good application prospects in the treatment of liver fibrosis, and therefore have good commercial value.

(29) The efficacy of DC291407 was investigated in the cell function test, and it was found that it had an inhibitory effect on the activation of HSC (OCA (obeticholic acid) and GW4064 were used as positive controls). Subsequently, the inhibitory effect of DC291410, a metabolite of DC291407 on the activation of hepatic stellate cells, was evaluated on this cell system. The results showed that the inhibitory activity of DC291410 on HSC activation was better than that of the positive control Alogliptin.

Example 4 Test of In Vivo Pharmacological Activity on Liver Fibrosis

(30) In this experiment, CCl.sub.4 induced liver fibrosis model mice were used to investigate the effect of long-term oral administration of compound DC291407 on liver fibrosis in the model mice.

(31) Animal experiment: Male C57BL/6j mice were injected intraperitoneally with 2 mL/kg, 10% CCl.sub.4 (dissolved in olive oil) three times a week to induce liver fibrosis model. Two weeks after modeling, the blood was taken from the retro-ocular venous plexus of the mice, and serum ALT and AST indexes were detected. According to ALT, AST, body weight and other indexes, the mice were randomly divided into 5 groups, each with 10 mice. There were the model control group (vehicle), DC291407 low-dose group (20 mg/kg), DC291407 high-dose group (60 mg/kg), positive compound OCA low-dose group (20 mg/kg), OCA high-dose group (40 mg/kg), respectively, and mice were administered by oral gavage. DC291407 was administered twice a day, and the other groups were administered once a day. During the administration period, the animals' food intake and body weight were monitored. After 3 weeks of administration, the blood was taken from the retro-ocular venous plexus of the mice, and serum ALT and AST indexes were detected. After 6 weeks of administration, the blood was taken from the retro-ocular venous plexus of the mice and then the mice were killed by dislocation. The liver was taken and weighed, part of the liver was fixed with 4% paraformaldehyde, and part of the liver was frozen at −80° C. During the whole experiment, another 10 mice in the same cage were injected intraperitoneally with the same dose of olive oil as a normal control group (WT). This experiment reflected whether the compound had the effect of relieving liver fibrosis by detecting the levels of liver function indexes ALT and AST in serum, the expression of α-SMA and Col1α1 gene levels in the liver, the content of hydroxyproline (a characteristic amino acid of collagen) in the liver, and liver pathological changes (Sirius scarlet staining) and the like.

(32) The results of the study showed that no decrease in serum ALT and AST levels was observed after 3 weeks of administration of Compound DC291407 of the present invention, and the level of ALT and AST in the serum could be significantly reduced after 6 weeks of administration; the expression of α-SMA and Col1α1 mRNA in liver tissues was significantly down-regulated after 6 weeks of administration and the content of hydroxyproline in liver tissues was reduced (FIG. 2). In the quantitative analysis of liver pathological sections stained with Sirius scarlet, the test compound DC291407 could reduce the collagen content in the liver without statistical difference (FIG. 3).

(33) In summary, long-term administration of the test compound DC291407 significantly improved the liver function in CCl.sub.4-induced liver fibrosis mice, down-regulated the expression of α-SMA and Col1α1 mRNA, reduced the deposition of collagen in the liver, and had a certain alleviating effect on liver fibrosis.

Example 5 Test of In Vivo Pharmacological Activity on Liver Fibrosis

(34) In this experiment, the NAFLD model of ob/ob mice induced by high-fat diet was used to investigate the treatment effect of long-term oral administration of compound DC291407 on NAFLD.

(35) Animal experiment: ob/ob obese mice were divided into 3 groups according to random blood glucose, random body weight, fasting blood glucose, and fasting body weight at the 8th week of age, with 9 mice in each group. There were the model control group (vehicle) and the DC291407 group (20 mg/kg), the positive compound Alogliptin group (20 mg/kg), respectively and mice were administered by oral gavage, twice a day. A group of 6 normal mice in the same cage was set up as a normal control group (WT). Except for the WT group, they were fed with normal feed, and the rest groups were fed with high-fat feed. The administration was continued for 8 weeks. During the administration, the animal's food intake and body weight were monitored. After the last administration, the animals were fasted for 6 hours and allowed to drink water freely. Then the body weight was measured, the blood was collected from the retro-ocular venous plexus, and liver was dissected. Part of the liver was placed in 4% paraformaldehyde, and the rest was stored in a refrigerator at −80° C. This experiment reflected whether the compound had the treating effect on NAFLD by detecting the levels of liver function indexes ALT and AST in the serum, the contents of TC and TG in the liver, the NAS score of liver pathology (HE staining) and the like.

(36) The results showed that DC291407 could significantly reduce the level of ALT and AST in serum and the content of TG in liver tissue after 8 weeks of administration, and its effect was equivalent to that of alogliptin (see FIG. 4); NAS score was performed on HE stained liver pathological sections and the results showed that DC291407 could improve steatosis, its effect was better than that of alogliptin, and had a certain alleviating effect on lobular inflammation, and the overall improvement effect on NAS score was equivalent to that of alogliptin (see FIG. 5).

(37) In summary, the long-term administration of DC291407 significantly improved the liver function of NAFLD model mice, reduced liver steatosis, and had a certain alleviating effect on lobular inflammation.

Example 6 Test of In Vivo Pharmacological Activity on Liver Fibrosis

(38) In this experiment, CCl.sub.4-induced liver fibrosis model mice were used to investigate the effect of long-term oral administration of compound DC291407 under multiple doses on liver fibrosis in the model mice, and comparison was conducted with the same dose of sitagliptin.

(39) Animal experiment: Male C57BL/6j mice were injected intraperitoneally with 2 mL/kg, 10% CCl.sub.4 (dissolved in olive oil) three times a week to induce liver fibrosis model. Two weeks after modeling, the blood was taken from the retro-ocular venous plexus of the mice, and serum ALT and AST indexes were detected. According to ALT, AST, body weight and other indexes, the mice were randomly divided into 8 groups with 10 mice in each group. There were the models control group (vehicle), DC291407 low-dose group (6 mg/kg), DC291407 middle-dose group (20 mg/kg), DC291407 high-dose group (60 mg/kg), sitagliptin low-dose group (6 mg/kg), Sitagliptin middle-dose group (20 mg/kg), sitagliptin high-dose group (60 mg/kg) and system positive compound OCA group (20 mg/kg). The mice were administered by oral gavage. The OCA group was administered once a day, and the other groups were administered twice a day. During the administration period, the animals' food intake and body weight were monitored. After 3 weeks of administration, the blood was taken from the retro-ocular venous plexus of the mice, and serum ALT and AST indexes were detected. After 6 weeks of administration, the blood was taken from the retro-ocular venous plexus of the mice and then the mice were killed by dislocation. The liver was taken and weighed, part of the liver was fixed with 4% paraformaldehyde, and part of the liver was frozen at −80° C. During the whole experiment, another 10 mice in the same cage were injected intraperitoneally with the same dose of olive oil as a normal control group (WT). This experiment reflected whether the compound had the effect of relieving liver fibrosis by detecting the levels of liver function indexes ALT and AST in serum, the content of hydroxyproline (a characteristic amino acid of collagen) in the liver, and liver pathological changes (Sirius scarlet staining) and the like.

(40) The results of the study showed that the low-dose and medium-dose groups of the compound DC291407 of the present invention could significantly reduce ALT and AST levels in serum after 6 weeks of administration of the compound DC291407, and the high-dose group could significantly reduce the ALT level in serum; the sitagliptin low-dose group had no effect on ALT and AST levels in serum, the middle-dose group significantly reduced the AST level in serum and the high-dose group significantly reduced ALT and AST levels in serum; and the DC291407 low-dose group reduced the content of hydroxyproline in liver tissues without statistics significance (FIG. 6). In the quantitative analysis of liver pathological sections stained with Sirius scarlet, the high, medium and low dose groups of the test compound DC291407 could reduce the collagen content in the liver without dose dependence; only the low dose group of sitagliptin could significantly reduce the collagen content in the liver (FIG. 7).

(41) In summary, long-term administration of the test compound DC291407 in each dose group significantly improved the liver function of CCl.sub.4-induced liver fibrosis mice, reduced the deposition of collagen in the liver, and had a certain alleviating effect on liver fibrosis, but no dose dependence was shown in this dose range. All documents mentioned in the present invention are incorporated by reference in this application, as each document is individually incorporated by reference. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.