Application of metformin analogue in preparation of antitumor drugs

Abstract

The invention belongs to the field of small molecule chemotherapeutic drugs, and particularly relates to application of a metformin analogue in preparation of antitumor drugs. The anti-tumor activities of the five metformin analogues (1b, 2b, 1c, 2c and 3c) are screened, the toxicity of the metformin analogues 2b, 2c and 3c to cancer cells is higher than that of metformin, the toxicity of the metformin analogue 3c to cancer cells is the strongest and is about 70-360 times that of metformin, and the metformin analogue 3c shows excellent anti-tumor activity and anti-tumor broad-spectrum performance, can be used for treating malignant tumors, and has a good application prospect. The invention provides a new direction and hope for developing novel antitumor drugs, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of small molecule chemotherapy drugs, and specifically relates to the use of metformin analogs in the preparation of anti-tumor drugs. Background Art

[0002] Metformin, as a classic oral hypoglycemic drug for the treatment of type 2 diabetes, has revealed its potential anti-tumor effects through research and clinical observations in recent years. Epidemiological studies have shown that patients treated with metformin have a significantly reduced risk of colorectal cancer and pancreatic cancer. In addition, a large number of experiments have shown that high-dose metformin can effectively inhibit the proliferation of a variety of tumor cells in vitro and in vivo, including colon cancer, pancreatic cancer, breast cancer, and leukemia. Currently, there are more than 50 clinical trials exploring the potential of metformin as a single drug or in combination with other drugs for the treatment of malignant tumors.

[0003] Mechanistic studies have shown that metformin inhibits oxidative phosphorylation by targeting mitochondrial complex I, regulates the AMPK / mTOR axis, reduces ATP production, leads to metabolic homeostasis imbalance, and ultimately induces tumor cell death. However, clinical trials have shown that the clinical dosage of metformin is large and its anti-tumor effect is limited, which is mainly due to the differences in physical and chemical properties caused by its molecular structure.

[0004] The strong alkalinity of metformin causes it to exist as a protonated cation under physiological conditions, which is not easily taken up by cells. Its cellular uptake mainly depends on membrane transporters, such as organic cation transporters, and therefore, high concentrations are required to exert anti-tumor effects. Existing studies mainly enhance the cellular uptake efficiency of metformin by increasing its hydrophobicity, such as phenformin, which is more lipophilic and exhibits better anti-tumor effects. However, the lactic acidosis and high metabolic risk caused by phenformin severely limit its clinical application.

[0005] Therefore, it is particularly urgent to optimize the molecular structure, efficacy and safety of metformin and develop a metformin analogue with excellent anti-tumor effect and good biosafety. This is not only of great significance in the field of tumor treatment, but also provides a new direction and hope for the development of new anti-tumor drugs. Summary of the invention

[0006] In view of the problems of the prior art, the present invention provides the use of metformin analogs in the preparation of anti-tumor drugs.

[0007] A compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a deuterated compound thereof, or a solvate thereof, or a crystal form thereof, for use in preparing an anti-tumor drug, wherein the structural formula of the compound is as shown in Formula I:

[0008]

[0009] Wherein, R1 is selected from H, C1-C5 alkyl;

[0010] R2 is selected from amino and guanidinyl.

[0011] Preferably, R1 is selected from C2-C4 alkyl.

[0012] Preferably, R1 is selected from C4 alkyl.

[0013] Preferably, R2 is selected from guanidino.

[0014] Preferably, the compound is one of the following structures:

[0015] Preferably, the tumor is at least one of lung cancer, breast cancer, stomach cancer, colon cancer, liver cancer, pancreatic cancer, prostate cancer, bladder cancer, ovarian cancer, cervical cancer, thyroid cancer, skin cancer, adenocarcinoma, and lymphoma.

[0016] Preferably, the tumor is at least one of lung cancer, breast cancer, and colon cancer.

[0017] Preferably, the lung cancer is at least one of lung adenocarcinoma, lung squamous cell carcinoma, small cell lung cancer, and large cell lung cancer.

[0018] The compounds and derivatives provided in the present invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

[0019] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for groups or terms in this document apply to the groups or terms throughout the specification; for terms that are not specifically defined in this document, the meaning that a person skilled in the art can give them should be given based on the disclosure and context.

[0020] The minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix C a -C b Alkyl refers to any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, "C1-C6 alkyl" refers to an alkyl group containing from 1 to 6 carbon atoms.

[0021] "Alkyl" refers to a saturated hydrocarbon chain having a specified number of carbon atoms. For example, C1-C6 alkyl refers to a saturated hydrocarbon chain having 1 to 6 carbon atoms. The alkyl group can be straight or branched. The alkyl group can be optionally substituted with one or more substituents as defined herein. Alkyl includes methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl and tert-butyl), pentyl (n-pentyl, isopentyl and neopentyl).

[0022] "Amino" refers to a group that is attached to a linking site through a carbon chain or N, and the carbon chain contains at least one N.

[0023] "Guanidyl" refers to a group with the chemical formula -CN3H4, in which one C is connected to three N groups, one of which is connected to the N group with a double bond, and the remaining two N groups are connected to the C group with single bonds.

[0024] The term "pharmaceutically acceptable" means that a carrier, vehicle, diluent, excipient, and / or formed salt is generally chemically or physically compatible with the other ingredients that constitute a pharmaceutical dosage form and physiologically compatible with the receptor.

[0025] The terms "salt" and "pharmaceutically acceptable salt" refer to acidic and / or basic salts of the above-mentioned compounds or their stereoisomers, formed with inorganic and / or organic acids and bases, and also include zwitterionic salts (inner salts), and also include quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final separation and purification of the compound. It can also be obtained by mixing the above-mentioned compound, or its stereoisomer, with a certain amount of acid or base appropriately (e.g., equivalent). These salts may form a precipitate in the solution and be collected by filtering, or be recovered after solvent evaporation, or be obtained by freeze drying after reaction in an aqueous medium. The salt described in the present invention can be a hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compound.

[0026] In certain embodiments, one or more compounds of the present invention may be used in combination with each other. It is also possible to select the compounds of the present invention and any other active agent for use in combination to prepare a drug or pharmaceutical composition for regulating cell function or treating a disease. If a group of compounds is used, these compounds may be administered to a subject simultaneously, separately or sequentially.

[0027] The metformin analogs 2b, 2c and 3c of the present invention are more toxic to cancer cells than metformin, among which metformin analog 3c is the most toxic to cancer cells, about 70-360 times that of metformin, has excellent anti-tumor activity and broad anti-tumor spectrum, and can be used to treat malignant tumors.

[0028] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0029] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 H-NMR characterization of compound 1b.

[0031] Figure 2 C NMR characterization of compound 1b.

[0032] Figure 3 HRMS characterization of compound 1b.

[0033] Figure 4 H-NMR characterization of compound 2b.

[0034] Figure 5 C NMR characterization of compound 2b.

[0035] Figure 6 HRMS characterization of compound 2b.

[0036] Figure 7 H-NMR characterization of compound 1c.

[0037] Figure 8 C NMR characterization of compound 1c.

[0038] Fig. 9 HRMS characterization of compound 1c.

[0039] Fig.10 H-NMR characterization of compound 2c.

[0040] Fig.11 C NMR characterization of compound 2c.

[0041] Fig.12 HRMS characterization of compound 2c.

[0042] Fig.13 H-NMR characterization of compound 3c.

[0043] Fig.14 C NMR characterization of compound 3c.

[0044] Fig.15 HRMS characterization of compound 3c.

[0045] Fig.16 Cytotoxicity of biguanide analogs against tumor cells PC9 (A) and PC9-OR (B).

[0046] Fig.17Cytotoxicity of biguanide analogs with different numbers of guanidine groups on tumor cells PC9 (A) and PC9-OR (B).

[0047] Fig.18 Cytotoxicity of biguanide analogs containing different ester groups against tumor cells PC9 (C) and PC9-OR (D).

[0048] Fig.19 Cytotoxicity of biguanide analogs 2c and 3c against H441, H358 and H460 cells.

[0049] Fig. 20 Comparison of the cytotoxicity of biguanide analog 3c with metformin in breast cancer cell lines.

[0050] Fig.21 Comparison of the cytotoxicity of biguanide analog 3c with metformin in lung cancer cell lines.

[0051] Fig. 22 Comparison of the cytotoxicity of biguanide analog 3c with metformin in colon cancer cell lines. DETAILED DESCRIPTION

[0052] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.

[0053] Among them, dicyandiamide (DCD) was purchased from Sigma-Aldrich Reagent Company; concentrated hydrochloric acid was purchased from KESHI Reagent Company, and the remaining chemical reagents were purchased from Adamas-Beta Reagent Company.

[0054] Human lung adenocarcinoma cells H441, human small cell lung cancer cells H358, human large cell lung cancer cells H460 and human lung cancer cells PC9 were purchased from Nanjing Keygene Biotechnology Development Co., Ltd. Human lung cancer resistant cells PC9-OR (PC9 osimertinib resistant cell line) were induced by the drug osimertinib.

[0055] The metformin analogs of the present invention include small molecule monoguanidine compounds (1b, 2b) and small molecule biguanidine compounds (1c, 2c, 3c)

[0056] Example 1 Preparation of small molecule monoguanidine compound 1b

[0057] The structure of the small molecule monoguanidine compound 1b is shown in Formula II:

[0058]

[0059] The small molecule monoguanidine compound 1b of the present invention is synthesized, and the specific synthesis steps are as follows:

[0060]

[0061] 1a (5.40 mg, 39.37 mmol) and cyanamide (3.84 g, 84.08 mmol) were dissolved in 1.68 M hydrochloric acid aqueous solution (57 mL), and the resulting mixture was placed in a 90 ° C oil bath for one hour. After cooling to room temperature, the precipitated white solid was collected to obtain the target product 1b, with a mass of 580.00 mg and a yield of 6.80%.

[0062] 1 HNMR(400MHz,DMSO-d6)δ12.98(s,1H),10.48(s,1H),8.01-7.93(m,2H),7.80(s,4H),7.33(d,J=8.6Hz,2H)( Figure 1 ). 13 CNMR(101MHz,DMSO-d6)δ166.86,155.79,140.04,131.03,127.82,122.98( Figure 2 ). HRMS(ESI,M / Z):calcd for[(M+H) + ]:180.0768,found180.0767( Figure 3 ).

[0063] Example 2 Preparation of small molecule monoguanidine compound 2b

[0064] The structure of the small molecule monoguanidine compound 2b is shown in Formula II:

[0065]

[0066] The small molecule monoguanidine compound 2b of the present invention was synthesized. The specific synthesis steps are as follows:

[0067]

[0068] 2a (1.01 g, 6.15 mmol) and cyanamide (259.00 mg, 6.15 mmol) were dissolved in 1.10 M hydrochloric acid ethanol solution (5.50 mL), and the resulting mixture was placed in an 83 °C oil bath for four hours. The reaction solution was then spun dry, petroleum ether was added, and the precipitated solid was collected to obtain a crude product. The crude product was first slurried once with a mixed solvent of ethyl acetate and ethanol (ethyl acetate: ethanol = 7: 1), and then slurried once with anhydrous ethanol to obtain 2b. The mass was 189.00 mg, and the yield was 12.68%.

[0069] 1HNMR (400MHz, DMSO-d6) δ10.47(s,1H),7.98(d,J=8.6Hz,2H),7.81(s,4H),7.35(d,J=8.6Hz,2H),4.31(q,J=7.1Hz,2H),1.31(t,J=7.1Hz,3H)( Figure 4 ). 13 CNMR(101MHz,DMSO-d6)δ165.14,155.79,140.35,130.69,126.57,122.70,60.77,14.19( Figure 5 ). HRMS(ESI,M / Z):calcd for[(M+H) + ]:208.1081,found 208.0993( Figure 6 ).

[0070] Example 3 Preparation of small molecule biguanide compound 1c

[0071] The structure of the small molecule biguanide compound 1c is shown in IV:

[0072]

[0073] The small molecule biguanide compound 1c of the present invention is synthesized, and the specific synthesis steps are as follows:

[0074]

[0075] Compound 1a (549.00 mg, 4.00 mmol) and dicyandiamide (672.00 mg, 8.00 mmol) were dissolved in 0.3 M aqueous hydrochloric acid solution (12 mL). The obtained mixture was placed in an oil bath at 80 degrees Celsius for 5 hours, and then the reaction solution was spin-dried. The obtained residue was first washed with a mixed solvent of acetone and water (acetone: water = 1: 1), and then with a mixed solvent of dichloromethane and methanol (dichloromethane: methanol = 20: 1) to obtain 1c. The mass was 80.00 mg and the yield was 9.04%.

[0076] 1 HNMR(400MHz, DMSO-d6)δ12.69(s,1H),10.04(s,1H),7.85(d,J=8.7Hz,2H),7.65-7.25(m,6H),7.13(s,2H)( Figure 7 ). 13 CNMR(101MHz,DMSO-d6)δ167.02,161.59,154.33,143.32,130.24,124.66,119.18( Figure 8). HRMS(ESI,M / Z):calcd for[(M+H) + ]:222.0986,found 222.0916( Fig. 9 ).

[0077] Example 4 Preparation of small molecule biguanide compound 2c

[0078] The structure of the small molecule biguanide compound 2c is shown in Formula V:

[0079]

[0080] The small molecule biguanide compound 2c of the present invention is synthesized, and the specific synthesis steps are as follows:

[0081]

[0082] Compound 2a (2.02 g, 12.30 mmol) and dicyandiamide (1.04 g, 12.30 mmol) were dissolved in 1.1 M hydrochloric acid aqueous solution, and the resulting mixture was placed in an oil bath at 80 degrees Celsius for five hours. After the reaction solution was cooled to room temperature, the precipitated solid was collected to obtain product 2c. The mass was 1.15 g, and the yield was 32.86%.

[0083] 1 HNMR(400MHz,DMSO-d6)δ10.39-9.85(m,1H),7.87(dd,J=8.8,1.9Hz,2H),7.72-7 .26(m,6H),7.15(d,J=32.6Hz,2H),4.27(q,J=7.1Hz,2H),1.30(t,J=7.1Hz,3H)( Fig.10 ). 13 CNMR(101MHz,DMSO-d6)δ165.54,161.71,154.32,143.66,130.15,123.82,119.39,60.57,14.34( Fig.11 ). HRMS(ESI,M / Z):calcd for[(M+H) + ]:250.1299,found 250.1294( Fig.12 ).

[0084] Example 5 Preparation of small molecule biguanide compound 3c

[0085] The structure of the small molecule biguanide compound 3c is shown in Formula VI:

[0086]

[0087] The small molecule biguanide compound 3c of the present invention was synthesized, and the specific synthesis steps are as follows:

[0088]

[0089] Compound 3a (1.01 g, 6.15 mmol) and dicyandiamide (518.00 mg, 6.15 mmol) were dissolved in 1.1 M hydrochloric acid ethanol solution, and the resulting mixture was placed in an oil bath at 83 degrees Celsius for reaction for 3 hours. After the reaction solution was cooled to room temperature, the precipitated solid was collected to obtain 3c, with a mass of 130.00 mg and a yield of 7.92%.

[0090] 1 HNMR(400MHz,DMSO-d6)δ10.40-9.80(m,1H),7.93-7.81(m,2H),7.68-7.32(m,6H),7.28-7 .02(m,2H),4.23(t,J=6.5Hz,2H),1.75-1.59(m,2H),1.49-1.32(m,2H),0.97-0.88(m,3H)( Fig.13 ). 13 CNMR(101MHz,DMSO-d6)δ165.52,161.67,154.28,143.63,130.11,123.79,119.43,64.19,30.37,18.84,13.71( Fig.14 ). HRMS(ESI,M / Z):calcd for[(M+H) + ]:278.1612,found 278.1511( Fig.15 ).

[0091] The technical solution of the present invention is further illustrated by experiments below.

[0092] Experimental Example 1 Toxic Effects of Metformin Analogs on Lung Cancer Cells

[0093] 1. Experimental Methods

[0094] The CCK-8 method was used to compare the cytotoxicity of metformin analogs and metformin on human lung cancer cells PC9, human lung cancer resistant cells PC9-OR (PC9 osimertinib resistant cell line), human lung adenocarcinoma cells H441, human small cell lung cancer cells H358, and human large cell lung cancer cells H460.

[0095] 2. Experimental Results

[0096] The results are shown in Table 1. The IC 50The value was significantly lower than that of metformin, showing stronger cytotoxicity; Fig.16 Similar results were also shown. Both 2b and 2c showed certain cytotoxicity to PC9 / PC9-OR cells at the tested concentrations, and with the increase of the dosage, the IC 50 The value also decreased, and the cytotoxicity also increased. The results showed that the cytotoxicity of metformin analogs (2b, 2c, 3c) to PC9 / PC9-OR cells was significantly higher than that of metformin, among which the cytotoxicity of 3c was increased by about 80 times compared with metformin.

[0097] Table 1 Cytotoxicity IC50 of biguanide compounds (n=3)

[0098]

[0099] / : not tested; IC50: μM.

[0100] Experimental Example 2: Structure-activity relationship study of metformin analogs' toxic effects on lung cancer cells

[0101] 1. Experimental Methods

[0102] The CCK-8 method was used to compare the cytotoxicity of metformin analogs with different structures on human lung cancer cells PC9 and human lung cancer resistant cells PC9-OR.

[0103] 2. Experimental Results

[0104] The effect of the number of guanidine groups is shown in the following table. Fig.17 AB shows the cytotoxicity IC of compounds 2b / 2c containing guanidine groups. 50 The cytotoxicity of the monoguanidine compound 2b and the biguanidine compound 2c was not significantly different, indicating that the guanidine group is a necessary functional group for its activity, but its number has little effect on its activity.

[0105] The effect of the length of the alkyl carbon chain in the ester group is as follows: Fig.18 AB shows the cytotoxicity IC values ​​of compounds 2c / 3c containing ester chains. 50 It is significantly higher than compound 1c without ester group. At the same time, the ester compound 3c with four carbon atoms alkyl chain has a cytotoxicity about 5 times higher than that of 2c with two carbon atoms, indicating that the ester group is a functional group necessary for its activity, and the longer the length of its alkyl carbon chain is within a certain range, the stronger its cytotoxicity is.

[0106] Experimental Example 3: Metformin analogs have a broad spectrum of inhibitory effects on lung cancer

[0107] 1. Experimental Methods

[0108] The CCK-8 method was used to compare the cytotoxicity of highly active metformin analogs against human lung adenocarcinoma cells H441, human non-small cell lung cancer cells H358, and human large cell lung cancer cells H460.

[0109] 2. Experimental Results

[0110] like Fig.19 As shown in AB, 2c and 3c showed excellent anti-tumor activity on three lung cancer cell lines, showing good broad-spectrum anti-tumor activity. At the same time, 3c with a long alkyl carbon chain showed better anti-tumor activity on three lung cancer cell lines, which also showed that improving lipophilicity can better improve the anti-tumor activity of metformin analogs.

[0111] Experimental Example 4: Metformin analog 3c has a broad spectrum of inhibitory effects on human and mouse lung cancer, breast cancer, and colon cancer

[0112] 1. Experimental Methods

[0113] The CCK-8 method was used to compare the cytotoxicity of the highly active metformin analog 3c and metformin on lung cancer cells: PC9 (human), LLC (mouse); colon cancer cells: HCT116 (human), CT26 (mouse); breast cancer cells: MDA-MB-231 (human), 4T1 (mouse).

[0114] 2. Experimental Results

[0115] like Figure 20-22 As shown in Table 2, metformin analog 3c exhibited excellent antitumor activity on six cancer cell lines, showing good broad-spectrum antitumor activity. At the same time, metformin analog 3c with a long alkyl carbon chain exhibited better antitumor activity than metformin.

[0116] Table 2 Metformin, 3c cytotoxicity IC50 (n=3)

[0117]

[0118] IC50: μM.

[0119] In summary, the metformin analogs 2b, 2c, and 3c of the present invention are more toxic to cancer cells than metformin, among which metformin analog 3c is the most toxic to cancer cells, has excellent anti-tumor activity and broad anti-tumor spectrum, and can be used to treat malignant tumors, especially for the treatment of lung cancer.

Claims

1. Use of a compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a deuterated compound thereof, or a solvate thereof, or a crystalline form thereof in the preparation of an anti-tumor drug, characterized in that: The structural formula of the compound is shown in Formula I: Wherein, R1 is selected from H, C1-C5 alkyl; R2 is selected from amino and guanidinyl.

2. The use according to claim 1, characterized in that R1 is selected from C2-C4 alkyl groups.

3. The use according to claim 2, characterized in that R1 is selected from C4 alkyl.

4. The use according to claim 1, characterized in that R2 is selected from guanidino.

5. The use according to claim 1, characterized in that: The compound is one of the following structures:

6. The use according to claim 1, characterized in that The tumor is at least one of lung cancer, breast cancer, stomach cancer, colon cancer, liver cancer, pancreatic cancer, prostate cancer, bladder cancer, ovarian cancer, cervical cancer, thyroid cancer, skin cancer, adenocarcinoma, and lymphoma.

7. The use according to claim 6, characterized in that The tumor is at least one of lung cancer, breast cancer, and colon cancer.

8. The use according to claim 7, characterized in that The lung cancer is at least one of lung adenocarcinoma, lung squamous cell carcinoma, small cell lung cancer, and large cell lung cancer.

Images