Pharmaceutical composition for treating cervical cancer and application thereof

Abstract

The invention provides a pharmaceutical composition for treating cervical cancer and application of the pharmaceutical composition. Specifically provided is a pharmaceutical composition comprising an active small molecule compound. The pharmaceutical composition provided by the invention has the following advantages and effects: (1) better tumor treatment effect; (2) good specific distribution characteristics; (3) the preparation is simple and convenient, and industrialization is easy.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a pharmaceutical composition for treating cervical cancer and application thereof. Background Art

[0002] Cervical cancer is the most common gynecological malignancy, and 80% of cervical cancer patients worldwide are distributed in developing regions. In clinical diagnosis and treatment, we found that a large number of patients were already in the middle and late stages of cervical tumors when they were diagnosed, and they had lost the opportunity for surgery when they were discovered. Traditional treatments for cervical cancer in clinical practice have always included surgical resection, chemotherapy, and radiotherapy. Domestic and foreign researchers have been committed to exploring more effective, safer, less invasive, and more specific clinical treatment methods and targeted therapeutic drugs for cervical cancer. Through effective clinical treatment, we can further benefit cervical cancer patients and improve the treatment effect, survival, and long-term quality of life of cervical cancer patients. Medicine is constantly developing. With the introduction of the concept of "precision medicine", all treatment methods for malignant tumors in clinical research have gradually entered the research field and gradually become diversified and combined. The standard treatment for patients with advanced, recurrent, or metastatic cervical cancer is palliative chemotherapy, radiotherapy, or targeted therapy. Although radiotherapy plays an important role in the treatment of cervical cancer, its side effects and radiotherapy resistance are clinical problems that need to be solved urgently.

[0003] Indocyanine green (ICG) is the only cyanine dye drug approved by the U.S. Food and Drug Administration. It is safe and non-toxic. The near-infrared fluorescence properties of indocyanine green enable it to achieve precise targeting in deep tissues. Further development of drugs with tumor suppressor activity can significantly improve patients' survival and quality of life, and has extremely high clinical research value. Summary of the invention

[0004] The present invention provides a pharmaceutical composition for treating cervical cancer and its application, and specifically provides an indocyanine green derivative and its application. The compound provided by the present invention has the following advantages: (1) good tumor treatment effect; (2) good specific distribution characteristics; (3) simple preparation and easy industrialization.

[0005] The present invention provides a pharmaceutical composition for treating cervical cancer, comprising a compound as shown in Formula I or a pharmaceutically acceptable salt thereof; .

[0006] Among them, R 1 and R 2 Independently H, C 1-3 Alkyl or C 1-3 Alkoxy; L 1is a single bond or a 5-6 membered heteroaryl; L 2 is a single bond, a 5-6 membered heteroalkyl group or a 5-6 membered heteroalkenyl group; Among them, in the 5-6 membered heteroaryl, 5-6 membered heteroalkyl and 5-6 membered heteroalkenyl, the heteroatom is one or more of N, O and S, and the number of heteroatoms is 1, 2 or 3.

[0007] In the present invention, the compound represented by formula I may be a compound represented by formula I-1;

[0008] Among them, L 1 and L 2 Independently as described herein.

[0009] In the present invention, the C 1-3 The alkyl group may be methyl, ethyl or n-propyl.

[0010] In the present invention, the C 1-3 The alkoxy group may be methoxy, ethoxy or n-propoxy.

[0011] In the present invention, the 5-6 membered heteroaryl group may be a pyridyl group or a pyrazolyl group.

[0012] In the present invention, the 5-6 membered heteroalkyl group may be a piperazinyl group or a piperidinyl group.

[0013] In the present invention, the 5-6 membered heteroalkenyl group may be a 1,2,3,6-tetrahydropyridyl group.

[0014] In the present invention, preferably, L 1 is a 5-6 membered heteroaryl group, L 2 It is a 5-6 membered heteroalkyl group.

[0015] In the present invention, preferably, the compound represented by formula I is any of the following compounds: , , .

[0016] In the present invention, preferably, the pharmaceutically acceptable salt of the compound represented by Formula I is a sodium salt, preferably a trisodium salt.

[0017] In the present invention, preferably, the pharmaceutically acceptable salt of the compound represented by formula I is any of the following compounds: , , .

[0018] In the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0019] In the present invention, the pharmaceutical composition preferably further comprises a polynucleotide CpG. Preferably, the polynucleotide CpG is an oligonucleotide with immunostimulatory function, which is used to increase the host's immune response to pathogens. Further preferably, the pharmaceutical composition is used to treat cervical cancer and / or breast cancer. The pharmaceutical composition is, for example, a photothermal therapy agent for treating tumors; the tumors are, for example, cervical cancer and / or breast cancer.

[0020] The present invention provides the use of the above-mentioned pharmaceutical composition in preparing a drug for treating cervical cancer and / or breast cancer.

[0021] Terminology explanation: The term "pharmaceutically acceptable salt" refers to those salts which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and which are commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are amino salts formed with inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or with organic acids (acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid) or by using other methods known in the art (such as ion exchange). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydrogen iodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, dodecylsulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 alkyl) 4 - Salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates, as appropriate.

[0022] The term "excipient" includes inert diluents, dispersants and / or granulating agents, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Excipients, such as cocoa butter and suppository waxes, colorants, coatings, sweeteners, flavorings and fragrances may also be present in the composition.

[0023] In the present invention, the composition, compound or pharmaceutically acceptable salt thereof has one or more of the following advantages: (1) The pharmaceutical composition and compound provided by the present invention can be used as photothermal therapeutic agents, which are highly specific and only attack cancer cells without damaging normal cells.

[0024] (2) Immunophotothermal therapy drugs are relatively cheap, with low raw material costs, simple drug production process and short production cycle, which makes the drug composition obtained by the present invention relatively low in price and can reduce the economic burden on patients. These characteristics make immunophotothermal therapy an important choice for cancer treatment and have attracted much attention.

[0025] (3) It can be specifically distributed and expressed in the tumor, with good therapeutic effect and safety.

[0026] (4) It has good inhibitory effects on tumor cells, such as cervical cancer and breast cancer, and has good application prospects. DETAILED DESCRIPTION

[0027] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0028] Example 1: Preparation of indocyanine green derivative-1.

[0029]

[0030] Dissolve 7-bromo-4-chloroquinazoline (2 mmol, 0.48 g) in 4 mL of isopropanol, add compound 2-0, raise the temperature to 80°C, stir overnight, cool to room temperature, filter, wash the solid with methanol (10 mL), and concentrate the solid to obtain the title compound 2-1 (1.5 mmol).

[0031] LC-MS(ESI): m / z=447 .3[M+H] +

[0032] Compound 2-1 (1 mmol) and compound 1-(N-Boc-piperidin-4-yl)pyrazole-4-boronic acid pinacol ester (1.5 mmol) were dissolved in 1,4-dioxane (6 mL) and water (4 mL), and sodium carbonate (3 mmol), Pd(dppf)Cl 2 (0.1mmol), nitrogen was introduced three times, and the reaction was carried out at 100°C. After the reaction was completed, the mixture was cooled to room temperature, the reaction solution was removed by concentration under reduced pressure, DCM (25mL) was added, the mixture was filtered, concentrated by rotary evaporation, and purified by silica gel column to obtain compound 2-2 (0.75mmol).

[0033] LC-MS (ESI): m / z = 618.2 [M+H] +

[0034] Compound 2-2 (0.75 mmol) was dissolved in dichloromethane (10 mL), and a methanol solution of hydrochloric acid (5 mL) was added. After the reaction was complete in 1 hour, the mixture was concentrated. DMAP (2 mg) and DMF (2 mL) solvents were added, and L-tyrosine (0.8 mmol) and CDI (15 mg) were added. The mixture was reacted at room temperature for 5 hours under nitrogen protection. The reaction was completed after HPLC monitoring. 5 mL of water was added, the organic phase was concentrated, MTBE was added, and the compound 2-3 (0.5 mmol) was dried by centrifugation.

[0035] LC-MS (ESI): m / z = 679.3 [MH] -

[0036] Compound 2-3 (1 mmol) was added to a NaOH aqueous solution (2 ml of NaOH), and compound 2-4 (1.2 mmol) was added, and the temperature was raised to 70 ~ The reaction was carried out at 75°C. After the reaction was completed, the temperature was lowered to room temperature and freeze-dried to obtain derivative-1 (0.2 mmol) with a purity of 97.65%.

[0037] MS:[M-3Na] 3- =509.2.

[0038] 1 H NMR (400 MHz, Chloroform- d ) δ 9.0 (s, 1H), 8.8–8.5 (m, 4H), 8.3 (s, 1H), 8.2–8.0 (m, 4H), 7.8–7.7 (m, 3H), 7.6 – 7.4 (m, 6H), 7.3 (d, J=15.0 Hz, 1H), 7.1 (dt, J = 7.6, 1.0 Hz, 2H), 7.0 – 6.9 (m, 5H), 6.6 – 6.5 (m,1H), 4.5 – 4.5 (m, 2H), 4.4 (m, 4H), 4.1 – 3.9 (m, 3H), 3.6 – 3.4 m, 5H), 3.2– 3.0 (m, 5H), 2.8 – 2.6 (m, 5H), 2.2 (s, 2H), 2.1 – 1.7 (m, 12H), 1.7 – 1.6(m, 1H), 1.5 (m, 10H), 1.4 (s, 2H).

[0039] Example 2: Preparation of indocyanine green derivative-2.

[0040]

[0041] Compound 2-1 (1.2 mmol) and 6-(4-Boc-piperazin-1-yl)pyridine-3-boronic acid (1.8 mmol) were dissolved in 1,4-dioxane (5 mL) and water (3 mL), and potassium carbonate (3.0 mmol) and Pd(dppf)Cl were added. 2 (0.1mmol), nitrogen was introduced for replacement, and the reaction was carried out at 100°C. The reaction was monitored by LC-MS, and the temperature was lowered to room temperature, and the mixture was concentrated under reduced pressure. DCM (18mL) was added to the residue, and the mixture was filtered through diatomaceous earth, concentrated under reduced pressure, separated on silica gel (dichloromethane: methanol (v / v) = 90:10), redissolved in dichloromethane (10mL), and a methanol solution of hydrochloric acid (7mL) was added. The mixture was stirred for reaction for 1 hour, and concentrated to obtain compound 2-1A (0.75mmol, 62%).

[0042]

[0043] Compound 2-1A (0.75 mmol) was dissolved in dichloromethane (10 mL), and a methanol solution of hydrochloric acid (5 mL) was added. After the reaction was complete in 1 hour, the mixture was concentrated. DMAP (2 mg) and DMF (2 mL) solvents were added, and L-tyrosine (0.8 mmol) and CDI (15 mg) were added. The mixture was reacted at room temperature for 6 hours under nitrogen protection. The reaction was completed after HPLC monitoring. 6 mL of water was added, the organic phase was concentrated, MTBE was added, and the compound 2-2A (0.5 mmol) was dried by centrifugation.

[0044] LC-MS (ESI): m / z = 691.3 [MH] -

[0045] Compound 2-2A (1 mmol) was added to a NaOH aqueous solution (2 ml of NaOH), and compound 2-4 (1.2 mmol) was added and the temperature was raised to 70 ~ The reaction was carried out at 75°C. After the reaction was completed, the temperature was lowered to room temperature and freeze-dried to obtain derivative-2 (0.3 mmol) with a purity of 96.5%.

[0046] MS:[M-3Na] 3- =513.2.

[0047] 1 H NMR (400MHz, Chloroform- d ) δ 9.4 (s, 1H), 8.6 (m, 2H), 8.5 – 8.3 (m, 1H), 8.1 – 8.0 (m, 4H), 7.9 – 7.8 (m, 2H), 7.7 (dd, J = 7.5, 1.6 Hz, 2H),7.6 – 7.5 (m, 4H), 7.4 (dt, J = 7.5, 1.2 Hz, 2H), 7.3 (d, J = 15.0 Hz, 1H),7.1 (dt, J = 7.6, 1.0 Hz, 2H), 7.0 (m, 5H), 6.9 (d, J = 7.5 Hz, 1H), 6.7 (d, J = 7.5 Hz, 1H), 6.6 (dt, J = 8.0, 0.9 Hz, 1H), 6.5 (dt, J = 8.2, 0.9 Hz,1H), 4.5 – 4.3 (m, 4H), 4.1 – 4.0 (m, 3H), 3.7 – 3.6 (m, 9H), 3.1 – 3.0 (m,5H), 2.8 – 2.7 (m, 2H), 2.7 – 2.6 (m, 3H), 2.2 (s, 2H), 2.0 – 1.5 (m, 19H), 1.4 (s, 2H).

[0048] Effect test example 1: The SiHa cervical cancer subcutaneous tumor-bearing mouse model was established in BALB / c mice, and the average tumor volume reached about 300-400mm 3The group dosing started at 1h. The compositions 1, 2, and 3 obtained in Example 1 were diluted with 5% glucose solvent, and 0.05 mg / kg of derivatives 1, 2, and 3 were intravenously injected into mice bearing tumor xenografts. The blank control group was injected with 5% glucose solution, and then a whole-body imaging study was performed using a small animal in vivo imaging system. The imaging equipment contained a near-infrared fluorescence imaging system. The imaging was performed at 1h, 2h, 4h, 8h, and 24h, respectively. The tumor and background parts were circled with the same diameter, the fluorescence value was obtained, and the tumor-to-background ratio (TBR) was calculated. The larger the value, the better the specific absorption of the drug. The specific results are shown in the table below.

[0049]

[0050] Effect test example 2: The test method is based on the biological test example of CN 116199685 A. Specifically, breast cancer cells BT474 were purchased from ATCC (the culture medium was Hybri-Care + 10% FBS), cultured at 37°C, 5% CO 2 Incubator. On the first day, cells in the exponential growth phase were collected, and the cell suspension was adjusted to the corresponding concentration with culture medium for plating, so that the number of cells / well was 2500. The next day, different concentrations of the example compound (DMSO dissolved) were added, and the cells were placed in the incubator for 4 days. After the culture was completed, according to the operating instructions of the CellTiter-Glo kit (Promega, G7573), 60 μL of CellTiter-Glo solution that had been melted and equilibrated to room temperature was added to each well, and the mixture was mixed with a microplate oscillator for 2 minutes. After being placed at room temperature for 10 minutes, the chemiluminescent signal value (RLU) was measured with an enzyme plate reader (PHERAstar FSX). The proliferation inhibition rate was calculated.

[0051] Derivative-1 inhibits active GI 50 = 0.009 μM, derivative-2 inhibits active GI 50 = 10.6 μM.

[0052] Calculation formula: Cell viability % = 100 - (RLU compound - RLU0) / (RLUcontrol - RLU0) × 100%, where RLU compound is the reading of the drug-treated group, and RLU control is the average value of the solvent control group.

[0053] The derivatives of the present application have good anti-cancer activity, such as breast cancer and cervical cancer.

[0054] Effect Test Example 3 Beagle Dog Experiment: Beagle dogs were used as test animals, and the drug concentration in plasma at different times after intravenous injection of the example compounds was determined by LC / MS / MS to study the pharmacokinetic behavior of the compounds of the present invention in beagle dogs and evaluate their pharmacokinetic characteristics.

[0055] 6 male beagle dogs. Fasting was performed at noon the day before administration for 12 hours, and food was resumed 6 hours after administration. A certain amount of Example 1-3 compounds were weighed respectively, and a completely clear solution with a final concentration of 1 mg / mL was prepared with a mixed solvent of DMSO:Solutol:Normal saline (5%:5%:90%). The dosage was 0.7 mg / kg, and the administration volume was 0.7 mL / kg. 1-3 mL of blood samples were collected from the forelimb vein of male beagle dogs every ten minutes before and after intravenous injection for the first hour, and every hour for 2-24 hours thereafter, and placed in an anticoagulant tube, and the plasma was quickly centrifuged to separate. After the plasma sample was thawed at room temperature, 60 µL was taken and added to 600 µL of acetonitrile containing the internal standard (40 ng / mL, terfenadine), vortexed for 2 minutes, centrifuged, and the supernatant was diluted 10 times with 80% acetonitrile water and injected with 2 µL for analysis.

[0056] The experimental conclusion is that the derivatives of the present application (such as derivative-1) have good metabolic stability and a large exposure amount, indicating good efficacy.

Claims

1. A pharmaceutical composition for treating cervical cancer, comprising a compound as shown in formula I or a pharmaceutically acceptable salt thereof; , in, R1 and R2 are independently H, C 1-3 Alkyl or C 1-3 Alkoxy; L1 is a single bond or a 5-6 membered heteroaryl group; L2 is a single bond, a 5-6 membered heteroalkyl group or a 5-6 membered heteroalkenyl group; Among them, in the 5-6 membered heteroaryl, 5-6 membered heteroalkyl and 5-6 membered heteroalkenyl, the heteroatom is one or more of N, O and S, and the number of heteroatoms is 1, 2 or 3.

2. The pharmaceutical composition according to claim 1, characterized in that The compound represented by formula I is the compound represented by formula I-1; ， Wherein, L1 and L2 are independently as described in claim 1.

3. The pharmaceutical composition according to claim 1, characterized in that The C 1-3 Alkyl is methyl, ethyl or n-propyl; and / or, the C 1-3 Alkoxy is methoxy, ethoxy or n-propoxy; and / or, the 5-6 membered heteroaryl group is pyridyl or pyrazolyl; and / or, the 5-6 membered heteroalkyl group is piperazinyl or piperidinyl; And / or, the 5-6 membered heteroalkenyl group is 1,2,3,6-tetrahydropyridinyl.

4. The pharmaceutical composition according to claim 1, characterized in that L1 is a 5-6 membered heteroaryl group, and L2 is a 5-6 membered heteroalkyl group.

5. The pharmaceutical composition according to claim 1, characterized in that The compound represented by formula I is any of the following compounds: , , .

6. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutically acceptable salt of the compound represented by formula I is sodium salt.

7. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutically acceptable salt of the compound represented by formula I is any of the following compounds , , .

8. The pharmaceutical composition according to any one of claims 1 to 7, characterized in that It also contains a pharmaceutically acceptable excipient.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition further comprises nucleotide CpG, which is an oligonucleotide with immunostimulatory function and is used to increase the host's immune response to pathogens. The pharmaceutical composition is used to treat cervical cancer and / or breast cancer.

10. Use of the pharmaceutical composition according to any one of claims 1 to 9 in the preparation of drugs for treating cervical cancer and / or breast cancer.

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