Novel folic acid compound and application thereof

Abstract

The invention relates to the technical field of chemical synthesis of medicines, and particularly discloses a novel folic acid compound and application thereof, and the folic acid-containing compound has a structure as shown in a formula I or a formula II. The folic acid-containing compound can be used as a P-gp inhibitor, a chemotherapeutic drug sensitizer, a tumor metastasis inhibitor and a tumor multidrug resistance reversal agent. The folic acid-containing compound disclosed by the invention has good biological activity, excellent tumor multidrug resistance reversal activity and sensitization chemotherapy drug activity, the preparation method is simple, the yield is high, and the application prospect of folic acid and tetrahydroisoquinoline structures in the aspect of tumor multidrug resistance reversal agents is expanded. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemical synthesis, and more specifically, to a novel folic acid compound and application thereof. Background Art

[0002] As the course of chemotherapy increases, the multidrug resistance (MDR) exhibited by tumors has long troubled clinicians and directly threatens the majority of patients (Curr Med Chem. 2012, 19, 1946-2025.). Among them, the overexpression of P-glycoprotein (P-gp) in the ATP-binding (ABC) transporter family on the surface of tumor cell membranes is currently the most widely studied mechanism of tumor MDR production (Eur J Med Chem. 2016, 118, 219-229.). As a common transmembrane protein, P-gp is believed to be overexpressed on the surface of tumor cell membranes as chemotherapy proceeds. More than 200 chemotherapy drug molecules reported in clinical practice can become substrates of P-gp (Pharmacology Therapeutics. 2015, 149, 1-123.). The reason for the reduced clinical effects of these drugs is that they bind to P-gp overexpressed on the surface of tumor cell membranes and are pumped out of tumor cells, thereby reducing the drug concentration in the cells and leading to the failure of chemotherapy. At the same time, the increase in the concentration of extracellular chemotherapy drugs may lead to the generation of other side effects. Studies have shown that inhibiting P-gp can increase the accumulation of chemotherapy drugs in cells, increase the sensitivity of drug-resistant cells to chemotherapy drugs, exert the effects of chemotherapy drugs or induce tumor cell apoptosis, and thus reverse MDR. Therefore, finding and studying drugs that inhibit P-gp has become one of the research hotspots in the field of overcoming the MDR of malignant tumors, and it is also a difficult problem that needs to be solved urgently in the clinic and the market.

[0003] The development of new P-gp inhibitors has always been a hot topic in the research and development of new tumor drugs at home and abroad. Some P-gp inhibitors have entered clinical trials as breast cancer resistance reversal agents, such as Zosuquidar, Tariquidar and Elacridar, but public data show that they have toxicity to normal cells and tissues and abnormal pharmacokinetics in patients, which has led to the stagnation of related clinical trials. The reason is that P-gp is widely distributed and is very important for normal physiological and biochemical processes. Therefore, specific targeting of MDR tumors is a feature that needs to be considered in the development of new P-gp inhibitors, and it is also an important factor in determining the success of clinical trials or the final listing. Including P-gp inhibitors, the poor selectivity of drugs for tumor tissues or cells is the main reason for poor chemotherapy effects or serious toxic side effects such as damage to the body. Therefore, the research, development and application of targeted drugs are important directions for the effective clinical treatment of tumors. The development of a simple, stable and inexpensive targeted drug system is of great significance to the clinical application of P-gp inhibitors and ultimately to overcome tumor MDR.

[0004] Endowing small molecule P-gp inhibitors with tissue specificity for MDR breast cancer can not only effectively avoid adverse reactions caused by nonspecific effects on normal physiological and biochemical processes, but also improve the bioavailability of the inhibitors. Folate receptor (FOLR) is a transmembrane glycoprotein with a molecular weight of 30-40 kDa, which is expressed on the surface of various tumor cells and has extremely low expression levels in normal body cells. It is an ideal target for tumor cell recognition. FOLR has three subtypes: α, β, and γ, among which FOLR-β is highly expressed in breast cancer cells. Based on the specificity of FOLR binding to folic acid or folic acid analogs, it is expected that drug molecules can be targeted to tumors through the specific recognition effect of folic acid on FOLR. In order to obtain a tissue-specific P-gp inhibitor with stronger anti-MDR activity, the present invention proposes a novel folic acid compound and its application. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a novel folic acid compound and its application, which has good biological activity, and has both the targeting of folic acid to breast cancer and the reversal of MDR activity of novel phenylfuran P-gp inhibitors. The activity test results show that the compound has a significant effect of reversing the MDR of multidrug-resistant tumor cells, and its activity is equivalent to that of the positive control drug verapamil (VRP). It can be used for the development and utilization of drugs for multidrug resistance of malignant tumor cells caused by multidrug resistance-related proteins.

[0006] In one aspect, the present invention provides a novel folic acid compound, which adopts the following technical scheme:

[0007] A novel folic acid compound, wherein the folic acid compound has a structure as shown in Formula I or Formula II:

[0008]

[0009] In the formula I, X is O, NH, methylene, etc., and n is an integer of 1-8.

[0010] Preferably, in the formula I, X is O or methylene, and n is 3, 4 or 5.

[0011] In the formula II, m is an integer of 0-8.

[0012] Preferably, in the formula II, m is 1 or 2 or 3 or 4 or 5.

[0013] On the other hand, the present invention provides an application of a novel folic acid compound, using the following technical solution:

[0014] Application of a novel folic acid compound in the preparation of P-gp inhibitors.

[0015] Application of a novel folic acid compound in the preparation of a chemotherapy drug sensitizer.

[0016] Application of a novel folic acid compound in the preparation of tumor metastasis inhibitors.

[0017] Application of a novel folic acid compound in the preparation of tumor multidrug resistance reversal agent.

[0018] Preferably, the novel folic acid compound is used in the preparation of an agent for reversing multidrug resistance of tumor cells caused by multidrug resistance-associated proteins.

[0019] Preferably, the novel folic acid compound is used in the preparation of an anti-breast cancer resistance reversal agent.

[0020] Preferably, the breast cancer cell line is a human breast cancer doxorubicin-resistant cell line (MCF-7 / ADR), and the novel folic acid compound can be used in combination with doxorubicin hydrochloride to effectively improve the efficacy of doxorubicin hydrochloride.

[0021] The novel folic acid compound of the present invention can be used in combination with anti-tumor drugs (clinical chemotherapy drugs) such as alkylating agents (such as cyclophosphamide or cisplatin), antimetabolites (such as 5-fluorouracil or hydroxyurea), topoisomerase inhibitors (such as camptothecin or topotecan), cell microtubule inhibitors (such as paclitaxel or vinblastine), DNA intercalating agents (such as doxorubicin or daunorubicin) and lysine kinase inhibitors (such as gefitinib). Through combined treatment with anti-tumor drugs, the sensitivity of multidrug-resistant tumor cells to anti-tumor drugs can be significantly enhanced, thereby improving the chemotherapy treatment effect.

[0022] In summary, the present invention has the following beneficial effects:

[0023] The novel folic acid compound of the present invention has good biological activity, and has both the targeting of folic acid to breast cancer and the reversal of MDR activity of novel phenylfuran P-gp inhibitors. Activity test results show that the compound has significant tumor multidrug resistance reversal activity and sensitization chemotherapy drug activity, and can be used to prepare P-gp inhibitors, chemotherapy drug sensitizers, tumor metastasis inhibitors, tumor multidrug resistance reversal agents and biological probes with good biological tolerance, which expands the application prospects of folic acid and tetrahydroisoquinoline structures in tumor multidrug resistance reversal agents.

[0024] The novel folic acid compound of the present invention has a simple preparation method, high yield, good selectivity for tumor tissues or cells, high biological safety, and no obvious toxicity to normal cells. Experiments have shown that the novel folic acid compound has a significant sensitizing effect on drug-resistant cancer cell lines when used alone at a concentration that has no cytotoxicity, and when used in combination with anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the target compound Ⅰ-1 prepared in Example 1 of the present invention.

[0026] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the target compound Ⅰ-2 prepared in Example 2 of the present invention.

[0027] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of the target compound Ⅱ-1 prepared in Example 3 of the present invention. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below with reference to the embodiments.

[0029] Example 1

[0030] Preparation of (S)-16-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-1-(4-((2-(4-((4-(5-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)furan-2-yl)phenyl)carbamoyl)phenoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)-13-oxo-3,6,9-trioxa-12-azaheptadecan-17-oic acid (I-1)

[0031]

[0032] As shown in the following synthetic route of intermediate 5, N-hydroxysuccinimide (intermediate 2, 521 mg, 4.53 mmol, 1 eq) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 955.46 mg, 1.09 mL, 4.98 mmol, 1.1 eq) were added to a DMSO solution (40 mL) of acidic folic acid (starting material 1, 2 g, 4.53 mmol, 1 eq). The mixture was stirred for 16 h in the dark at 10° C. under nitrogen to obtain a DMSO solution (40 mL) of (2S)-2-[(4-{[(2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl]amino}phenyl)formamido]-5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoic acid (intermediate 3, 2.44 g, crude product), which was directly used in the next step.

[0033] To a DMSO solution (40 mL) of (2S)-2-[(4-{[(2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl]amino}phenyl)formamido]-5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoic acid (2.44 g, 4.53 mmol, 1 eq) was added 11-azido-3,6,9-trioxaundecan-1-amine (Intermediate 4, 988.95 mg, 899.05 μL, 4.53 mmol, 1 eq) and triethylamine (TEA, 458.52 mg, 629.83 μL, 4.53 mmol, 1 eq). The mixture was stirred at 10° C. under nitrogen atmosphere and protected from light for 16 h, and the mixture was purified by prep-HPLC (water (0.1% TFA)-ACN). The eluate was evaporated to remove the organic solvent and then lyophilized to a yellow solid to give (2S)-2-[(4-{[(2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl]amino}phenyl)formamido]-4-[(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethyl)carbamoyl]butanoic acid (Intermediate 5, 1.10 g, yield 37.8%).

[0034]

[0035] As shown in the synthetic route of the target compound I-1 below, under nitrogen protection, 5-bromofuran-2-carboxylic acid (intermediate 6, 8.89 g, 46.57 mmol), EDCI (10.71 g, 55.89 mmol), 1-hydroxybenzotriazole (HOBT, 7.55 g, 55.89 mmol) and TEA (6.13 g, 60.55 mmol) were added to a solution of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (9 g, 46.57 mmol) in dry DCM (150 mL). The mixture was stirred at 20° C. for 3 h. H 2 O (100 mL), and the aqueous phase was extracted with DCM (200 mL×3). The combined organic layers were washed with brine (100 mL) and 2 SO 4 The residue was chromatographed on silica gel (SiO 2 , DCM:MeOH=20:1) to give (5-bromofuran-2-yl)(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methanone (Intermediate 7, 15 g, yield 88.0%) as a white solid.

[0036] Intermediate 7 (10 g, 27.31 mmol), K 2 CO 3 (11.32 g, 81.92 mmol) and Pd(PPh 3 ) 4 (3.16 g, 2.73 mmol) in 1,4-dioxane (200 mL) and H 2 O (40 mL) was stirred at 85 ° C under nitrogen for 16 h, filtered and concentrated. The liquid was purified by silica gel column (PE: EA = 10: 1) to obtain (5-(4-aminophenyl) furan-2-yl)(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methanone (Intermediate 8, 15 g, yield 45.2%, purity 80.0%) as a yellow solid.

[0037] To intermediate 8 (6.50 g, 17.18 mmol) and EDCI (6.59 g, 34.35 mmol). The reaction mixture was stirred at 25 ° C for 16 h, the mixture was concentrated and filtered to give a filter cake of 4-(benzyloxy)-N-(4-(5-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)furan-2-yl)phenyl)benzamide (intermediate 9, 8.70 g, yield 86.1%) as a light white solid.

[0038] A solution of intermediate 9 (5 g, 8.49 mmol) in TFA (50 mL) was stirred at 50 °C for 16 h. DCM (100 mL) and H 2 O (50 mL), the aqueous phase was extracted with DCM (100 mL × 3). The combined organic layers were washed with brine (50 mL) and 2 SO 4 The crude product was purified by prep-Flash (water (0.1% TFA)-ACN) to give N-(4-(5-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)furan-2-yl)phenyl)-4-hydroxybenzamide (Intermediate 10, 6.50 g, yield 53.5%) as a pale white solid.

[0039] To a solution of intermediate 10 (1.0 g, 2.01 mmol) in DMF (10 mL) was added K 2 CO 3 (832 mg, 6.02 mmol). The mixture was stirred at 50 °C for 16 h, the reaction was filtered and concentrated under reduced pressure to give a residue purified by prep-HPLC (water (0.225% FA)-ACN). It was then lyophilized to give N-(4-(5-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)furan-2-yl)phenyl)-4-(2-(prop-2-yn-1-yloxy)ethoxy)benzamide (Intermediate 11, 915 mg, yield 78.56%) as a white solid.

[0040] CuI (0.012 mg, 0.062 mmol) was added to a DMF solution (20 mL) of intermediate 11 (100 mg, 0.17 mmol) and intermediate 5 (100 mg, 0.16 mmol). The mixture was stirred for 3 h at 40 ° C under nitrogen, and the reaction solution was filtered. The residue was purified by prep-HPLC (water (0.1% FA)-ACN). It was then freeze-dried to give (S)-16-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-1-(4-((2-(4-((4-(5-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)furan-2-yl)phenyl)carbamoyl)phenoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)-13-oxo-3,6,9-trioxa-12-azaheptadecan-17-oic acid (target compound Ⅰ-1, 90 mg, yield 47.25%, purity: 95.9%) as a yellow solid.

[0041]

[0042] The physicochemical constants and high-resolution mass spectrometry data of the target compound Ⅰ-1 are listed in Table 1, and the nuclear magnetic resonance hydrogen spectrum results are listed in Table 2. In summary, the structure of the above target compound Ⅰ-1 is correct.

[0043] Example 2

[0044] The preparation method of Example 2 is the same as that of Example 1, except that intermediate 4 is replaced by 2-azidoethanol to obtain the corresponding target compound I-2.

[0045] The physicochemical constants and high-resolution mass spectrometry data of the target compound Ⅰ-2 are listed in Table 1, and the nuclear magnetic resonance hydrogen spectrum results are listed in Table 2. In summary, the structure of the above target compound Ⅰ-2 is correct.

[0046] Example 3

[0047] Preparation of (S)-2-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-5-(2-(4-(4-(4-(4-(5-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)furan-2-yl)phenyl)carbamoyl)phenoxy)butyl)-1H-1,2,3-triazol-1-yl)ethoxy)-5-oxopentanoic acid (II-1)

[0048]

[0049] As shown in the synthetic route of the target compound II-1, under the protection of inert gas, 2-azidoethanol (intermediate 12, 394 mg, 4.53 mmol), DMAP (277 mg, 2.27 mmol) and N, N'-dicyclohexylcarbodiimide (DCC, 1.03 g, 4.98 mmol) were added to a DMSO solution (40 mL) of folic acid (starting material 1, 2 g, 4.53 mmol). The reaction was stirred at 30 ° C for 16 h, and the mixture was filtered and concentrated. The reaction mixture was purified by prep-HPLC (water (10 mM NH 4 HCO 3 )-ACN) was used to purify the residue. After preparation, it was purified by HPLC, and the eluate was lyophilized to obtain compound (S)-2-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-5-(2-azidoethoxy)-5-oxopentanoic acid (Intermediate 3, 500 mg, 0.97 mmol, yield 10.81%) as a yellow solid.

[0050] To a solution of intermediate 3 (374 mg, 0.65 mol, 1.1 eq) in DMF (10 mL) were added N-(4-(5-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)furan-2-yl)phenyl)-4-(hex-5-yn-1-yloxy)benzamide (intermediate 4, 341.5 mg, 0.59 mmol, 1.0 eq), CuI (56 mg, 0.29 mol, 0.5 eq) and N,N-diisopropylethylamine (DIPEA, 228 mg, 1.76 mmol, 3 eq). The mixture was stirred at 25° C. under nitrogen for 16 h, filtered, and directly purified by Prep-HPLC. The residue was purified by prep-HPLC (water-(FA)-ACN) and then lyophilized to give (S)-2-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-5-(2-(4-(4-((4-((4-(5-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)furan-2-yl)phenyl)carbamoyl)phenoxy)phenoxy)butyl)-1H-1,2,3-triazol-1-yl)ethoxy)-5-oxopentanoic acid (target compound II-1, 20.5 mg, 0.019 mmol, yield 3.2%) as a yellow solid.

[0051]

[0052] The physicochemical constants and high-resolution mass spectrometry data of the target compound Ⅱ-1 are listed in Table 1, and the nuclear magnetic resonance hydrogen spectrum results are listed in Table 2. In summary, the structure of the above target compound Ⅱ-1 is correct.

[0053] Physicochemical constants and high-resolution mass spectrometry data of the novel folic acid compounds prepared in Examples 1-3

[0054] Table 1 Physicochemical constants and high-resolution mass spectrometry data of novel folic acid compounds

[0055]

[0056]

[0057] H NMR spectroscopy data of the novel folic acid compounds prepared in Examples 1-3

[0058] Table 2 H NMR spectrum data of new folic acid compounds

[0059]

[0060]

[0061] Study on the multidrug resistance reversal activity of the novel folic acid compounds prepared in Examples 1-3 on MCF-7 / ADR cells

[0062] Cell line: MCF-7 / ADR (human breast cancer cell line resistant to doxorubicin)

[0063] Sample test concentration: 5μM

[0064] Positive control drug: Verapamil

[0065] Test method: MTT (methyl thiazolyl tetrazolium) method was used to test the cell proliferation activity of the novel folic acid compound and doxorubicin in combination, as follows:

[0066] MCF-7 / ADR cells were cultured in RPMI 1640 medium containing 10% calf serum at 37°C and 5% CO. 2 The cells were cultured under saturated humidity. Cells in the logarithmic growth phase were taken and 1×10 5 The cells were inoculated at a density of / mL in a 96-well culture plate and 100 μL per well was incubated at 37°C and 5% CO 2 The cells were cultured under saturated humidity conditions, and blank control group, test compound group, and positive control group were set up. Doxorubicin and different test compounds were added to the test compound group; 5 μM verapamil was given to the positive control group; and an equal volume of PBS was given to the blank control group. The cells were cultured for another 48 hours, and the MTT working solution was added. After centrifugation after 4 hours, the culture solution was poured off, 100 μL DMSO was added to each well to dissolve, and then the optical density was read at a wavelength of 490 nm on an ELISA reader to calculate the effect of the test compound on the survival rate of the cells. The anti-proliferative activity IC value of doxorubicin on the MCF-7 / ADR cell line was calculated when it was used in combination with 5 μM of the test compound described in Formula I-1, Formula I-2, or Formula II-1. 50 (μM), the results are shown in Table 3. Combined with the biological activity results, structure-activity relationship analysis was performed.

[0067] Table 3 Results of the reversal activity of 5 μM novel folic acid compounds on multidrug resistance of MCF-7 / ADR cell lines

[0068]

[0069] Note: a represents the IC of doxorubicin combined with the test compound (5 μM) 50 (DOX). b Reversal fold = IC 50 (DOX) / IC 50 (P-gp inhibitor+DOX). c Indicates doxorubicin alone IC 50(DOX). All experiments were repeated three times and were independent results.

[0070] From the data shown in Table 3 above, it can be seen that the compounds represented by Formula I-1 and Formula I-2 generally have good activity in reversing multidrug resistance of tumor cells. Among them, the anti-multidrug resistant tumor proliferation activity of the compound of Formula I-1 combined with doxorubicin is the best, exceeding the activity of the control drug verapamil, and has good application prospects.

[0071] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A novel folic acid compound, characterized in that: The folic acid-containing compound has a structure as shown in Formula I or Formula II: In the formula I, X is one of O, NH, and methylene, and n is an integer of 1-8; In the formula II, m is an integer of 0-8.

2. The novel folic acid compound according to claim 1, characterized in that: In the formula I, X is O or methylene, and n is 3, 4 or 5.

3. The novel folic acid compound according to claim 1, characterized in that: In the formula II, m is 1, 2, 3, 4 or 5.

4. Use of the novel folic acid compound according to any one of claims 1 to 3 in the preparation of a P-gp inhibitor.

5. Use of the novel folic acid compound according to any one of claims 1 to 3 in the preparation of a chemotherapeutic drug sensitizer.

6. Use of the novel folic acid compound according to any one of claims 1 to 3 in the preparation of a tumor metastasis inhibitor.

7. Use of the novel folic acid compound according to any one of claims 1 to 3 in the preparation of a tumor multidrug resistance reversal agent.

8. The use of the novel folic acid compound according to claim 7, characterized in that: The novel folic acid compound is used in the preparation of a reversal agent for multidrug resistance of tumor cells caused by multidrug resistance-related proteins.

9. The use of the novel folic acid compound according to claim 7, characterized in that: The novel folic acid compound is used in the preparation of an anti-breast cancer resistance reversal agent.

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