WO2012000421A1

WO2012000421A1 - Uses of gossypol derivatives in manufacture of antitumor medicaments

Info

Publication number: WO2012000421A1
Application number: PCT/CN2011/076482
Authority: WO
Inventors: 郭方; 姜标
Original assignee: 中国科学院上海生命科学研究院; 上海中科高等研究院
Priority date: 2010-07-02
Filing date: 2011-06-28
Publication date: 2012-01-05
Also published as: CN102311449A; CN102311449B

Abstract

Disclosed are gossypol derivatives, preparation methods and uses thereof in the manufacture of antitumor medicaments. Pharmaceutical compositions containing the said gossypol derivatives and preparation methods thereof are also disclosed.

Description

Application of gossypol derivatives in preparing antitumor drugs

The present invention belongs to the field of biomedicine; more specifically, the present invention relates to the use of gossypol derivatives for the preparation of antitumor drugs. Background technique

Malignant tumors are a serious threat to human health. Traditional chemotherapy and radiotherapy lack specificity, and they often bring more toxic side effects to patients.

Colorectal cancer is the third most common type of cancer in the United States and the third most common type of cancer that causes death. In 2009, there were approximately 146,970 new colon cancer patients in the United States, of which 49,920 died. The 5-year survival rate for patients with non-metastatic colon cancer is about 90%, but it is 68% after regional metastasis (such as lymph node metastasis) and only 10% after distant metastasis. Current strategies for clinical treatment of colon cancer include surgical removal of lesions, radiation, and chemotherapy. However, even after successful surgical removal of the lesion and adjuvant chemotherapy, the 5-year recurrence rate is still high. Therefore, further research and development of new drugs and treatment strategies for the treatment of colon cancer is particularly important and urgent. It has been reported that abnormal activation of Bcl-2 in early colon cancer will inhibit tumor cell apoptosis and promote tumorigenesis. Gossypol is a compound extracted from cottonseed and was originally used as a male contraceptive. It has been previously reported that gossypol achieves its pro-apoptotic function by binding to the BH3 domain of Bcl-2 and Bcl-xL in tumor cells. Therefore, gossypol can be considered as a candidate chemotherapy drug for clinical treatment of colon cancer.

Previous studies have indicated that oral gossypol has entered clinical trials for the treatment of hormone-independent metastatic breast cancer and adrenal cancer. However, due to the presence of two reactive aldehyde groups in the gossypol structure, it may cause toxic side effects such as liver damage and digestive tract damage in the body, making it unsuitable for clinical applications. A series of gossypol derivatives have been reported, such as AT-101, Apogossypol, TW-37, ABT-737 and WL-276, etc., which also have an activity of inhibiting the growth of cancer cells. In addition, experiments have confirmed that Apogossypol is less toxic to the liver and gastrointestinal tract than natural gossypol, and its efficacy is close to that of natural gossypol. Since water-soluble compounds generally have stronger pharmacokinetic properties than low-water-soluble similar compounds, they can produce greater efficacy at lower doses, so the water solubility of the compounds is an important factor in evaluating drug potency. index. However, Apogossypol is poorly water soluble, indicating that it is not a very suitable drug and needs further improvement. Therefore, it is necessary to develop a gossypol derivative having good water solubility and little toxic side effects as a potential candidate for treating colon cancer. Summary of the invention

It is an object of the present invention to provide a use of gossypol derivatives for the preparation of antitumor drugs.

In a first aspect of the invention, there is provided a compound of the formula I, an isomer thereof or a pharmaceutically acceptable salt thereof:

ONa

丫, o

*■ o

In a preferred embodiment, the compound has the structure shown below (6-AP A-sodium-cotton):

In another aspect of the invention, there is provided the use of a compound as hereinbefore described in the manufacture of a pharmaceutical composition for the prevention and treatment of tumors.

In a preferred embodiment, the tumor includes, but is not limited to, colon cancer, breast cancer, melanoma, lung cancer or prostate cancer.

In another preferred embodiment, the tumor is colon cancer.

In another aspect of the invention, there is provided the use of a compound as hereinbefore described in the manufacture of a pharmaceutical composition for reducing the expression of the anti-apoptotic proteins Bcl-2 and/or Bcl-xL.

In another aspect of the invention, there is provided a pharmaceutical composition comprising: an effective amount of said compound or a combination thereof; and a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is for use in the prevention and treatment of tumors.

In another preferred embodiment, the pharmaceutical composition further comprises: an effective amount of 5-fluorouracil.

In another aspect of the invention, a method of preparing a pharmaceutical composition is provided, the method comprising: mixing an effective amount of the compound or a combination thereof with a pharmaceutically acceptable carrier.

In another preferred embodiment, the method further comprises: mixing an effective amount of 5-fluorouracil with the compound or a combination thereof, and a pharmaceutically acceptable carrier. In another aspect of the invention, a kit comprising the compound; or a pharmaceutical composition is provided.

Other aspects of the invention will be apparent to those skilled in the art from this disclosure. DRAWINGS

Figure 1: Preparation and structure of gossypol derivatives.

Scheme 1 is a schematic flow diagram of the structure and preparation of compounds 3, 4, 5, 6, 8, 9.

Scheme 2 is the structure and preparation procedure of Compound 7.

Wherein the numbers 1-7 represent the compounds 1-7; respectively; R represents a substituent of the corresponding compound.

Figure 2: Compound 7 inhibits cell growth and induces apoptosis with a time and dose dependent effect.

(A) CT26 cells were tested for cell proliferation after treatment with different concentrations of compound 7 for different periods of time. (B) CT26 cells were treated with different concentrations of compound 7 for various times, and apoptosis rate was measured by TUNEL kit, and treated with DMSO as a negative control (NC).

(C) Statistical chart of the TUNEL results for (B).

Figure 3: Compound 7 down-regulates its protein expression levels by binding to the BH3 domain of Bcl-2 and Bcl-xL. (A-B) Molecular docking simulation studies. Docking anti-apoptotic protein Bcl-2 (Protein Data Bank code 1YSW) (A) with Bcl-xL (PDB code 1BXL) (B) with compound 7 using software AutoDock Vina (http://vina.scripps.edu/) .

(C) CT26 cells were treated with 5 μΜ and 10 μΜ of compound 7 at different times and treated with DMSO as a negative control (CNC); cells were then harvested, lysed, and changes in Bcl-2 and Bcl-xL protein expression levels were detected, β-actin As an internal reference control.

Figure 4: Compound 7 in combination with the traditional anticancer drug 5-fluorouracil enhances its activity of inhibiting colon cancer growth in vitro and in vivo.

(A) Compound 7 can inhibit colon cancer growth in vivo. 5 x l0 ⁵ /0.1 ml of CT26 cells were injected subcutaneously into the right side of the mouse. After the tumor volume reached approximately 60 mm ³ , the mice were randomly divided into different groups of 4 each. CT26 tumor-bearing mice were intragastrically administered with compound 7 for 12 consecutive days at a dose of 10 μιηοΐ/kg body weight, and 10% alcohol was administered intragastrically as a negative control. Tumor length and width were measured every 2 days. Tumor volume was calculated as the formula length X width X width Χ 0.52. (Ρ < 0.01 is indicated by *).

(Β) Compound 7 combined with 5-fluorouracil significantly inhibited colon cancer growth in vitro. CT26 cells were treated with 10 μΜ of compound 7 and 25 μΜ of 5-fluorouracil or treated separately, and the cell viability was measured. DMSO treatment was used as a negative control (NCXP < 0.01 is indicated by *; P < 0.001 is indicated by **;) .

(C) CT26 tumor-bearing mice were grouped according to (A). The tumor-bearing mice were intragastrically administered with compound 7 at a dose of 10 μιηοΐ/kg body weight per day, using 10% ethanol as a control. Meanwhile, 5-fluorouracil was intraperitoneally injected once every two days at a dose of 10 mg/kg body weight to PBS. as comparison. Mouse tumors were measured every 2 days and the volume was calculated (Ρ < 0.01 with *; Ρ < 0.001 with **).

Figure 5: Comparison of the toxicity of Compound 7 with natural gossypol (Compound 1) in normal mice. 6-8 weeks old male BALB/c mice were divided into 3 groups, 6 rats in each group, respectively, according to the dose of 120 μιηηοΐ / kg body weight for 12 consecutive days, compound 7 or 1 was administered by gavage, and the control group was given 10% ethanol (NC; ).

(Α) Statistics on the survival of mice in different treatment groups.

(Β) Weighed all mice 12 days later and performed statistical analysis (Ρ < 0.01 in *).

(C) The small intestines of three groups of mice were taken, embedded in paraffin, sectioned with 5 μιη, stained with hematoxylin-eosin, and observed under the microscope. Detailed ways

Gossypol has not been clinically used for anti-tumor because of its low water solubility and side effects in the body. Treatment. In order to overcome the above disadvantages, the inventors conducted extensive and in-depth research, designed and synthesized some new gossypol derivatives, and analyzed their anticancer activities, and found a class of antitumor effects and water solubility. , Gossypol derivatives with low toxic side effects. The present invention has been completed on this basis. Compound

A compound having the structure shown in the formula (I):

R1-R4 are independently selected from OH, H or 0.

Relative to natural gossypol, due to a change in the position of the aldehyde group (from an aldehyde group to an R group), a water-soluble fragment is introduced to prepare a Schiff base derivative, so that the compound of the present invention (especially compound 7, compound 3) And the solubility of compounds 6-9) is significantly better than that of natural gossypol.

As a preferred mode of the present invention, the compound has the structure shown below:

The representation of "li" is well known to those skilled in the art and indicates that the bond at this position can be a single bond or a double bond.

The present invention also includes pharmaceutically acceptable salts, hydrates or precursors of the above compounds as long as they also have a tumor-preventing effect. The "pharmaceutically acceptable salt" means a salt formed by reacting a compound with an inorganic acid, an organic acid, an alkali metal or an alkaline earth metal. These salts include, but are not limited to, (1) salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid; (2) salts with organic acids such as acetic acid, oxalic acid, succinic acid, tartaric acid, Sulfonic acid, maleic acid, or arginine. Other salts include salts with alkali or alkaline earth metals (such as sodium, potassium, calcium or magnesium), esters, carbamates, or other conventional "pre- The form of the body drug.

The term "precursor of a compound" means a compound which is converted into a structure of the formula (I) by a metabolic or chemical reaction in a patient after administration by an appropriate method, or a structural formula ( I) A salt or solution of a compound of the structure shown.

The present invention also includes isomers and racemates of the above compounds as long as they also have a tumor controlling effect. The compound has one or more asymmetric centers. Therefore, these compounds may exist as racemic mixtures, individual enantiomers, individual diastereomers, diastereomeric mixtures, cis or trans isomers.

As a particularly preferred mode of the invention, the compound is 6-APA-sodium-cotton. It has been verified that the water solubility of the compound is significantly greater than that of natural gossypol, the toxic side effect is significantly lower than that of natural gossypol, and it has a very good anti-tumor effect.

It will be understood by those skilled in the art that upon knowledge of the structure of the compounds of the present invention, the compounds of the present invention can be obtained by a variety of methods well known in the art, using known starting materials, such as chemical synthesis or from organisms (such as animals or plants). The method of extraction, which is included in the present invention.

Synthetic chemical modification, protective functional group methodology (protection or deprotection) is very useful for the synthesis of compounds, and is well known in the art, such as R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3 ^rd Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and

Fieser ' ^y Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) are disclosed. use

Based on the novel findings of the present inventors, the present invention provides the use of a compound having the structure of the formula (I) or an isomer thereof, a racemate, a pharmaceutically acceptable salt, a hydrate or a precursor thereof, For the preparation of a drug (or composition) for preventing and treating tumors. In the examples of the present invention, the inhibitory effects of each compound on various tumor cell lines were specifically verified, and as a result, it was confirmed that each compound can exert an antitumor effect to varying degrees. In particular, 6-APA-sodium-cottonone (Compound 7) has a much improved water solubility compared to natural gossypol and has excellent antitumor activity. 6-APA-sodium-cottonone induces tumor cell apoptosis by inducing a decrease in the expression levels of Bcl-2 and Bcl-xL proteins in tumor cells and inhibiting the formation of dimers between Bcl-2 and Bcl-xL and its pro-apoptotic proteins. Die. In vivo experiments confirmed that 6-APA-sodium-cottonone can inhibit the growth of tumor cells in mouse colon cancer cells. In addition, 6-APA-sodium-cottonone has a less toxic side effect than natural gossypol, and when this compound is used in combination with 5-fluorouracil, it has a better inhibitory effect on colon cancer growth. Therefore, the prospect of 6-APA-sodium-cottonone for the treatment of tumors (especially colon cancer) is superior to that of natural gossypol, which has considerable clinical research and application value. combination

As used herein, the term "composition of the invention" is generally a pharmaceutical composition comprising a compound of the formula (I) or an isomer thereof, a racemate, a pharmaceutically acceptable salt, a hydrate or The precursor acts as an active ingredient for the prevention and treatment of tumors; and a pharmaceutically acceptable carrier or excipient.

In the present invention, the term "containing" means that the various ingredients can be used together in the mixture or composition of the present invention. Therefore, the terms "consisting mainly of" and "by .. and becoming" are included in the term "contains".

In the present invention, a "pharmaceutically acceptable" ingredient is a substance which is suitable for use in humans and/or animals without excessive adverse side effects (e.g., toxic, irritating, and allergic), i.e., having a reasonable benefit/risk ratio.

In the present invention, a "pharmaceutically acceptable carrier" is a compound of the present invention having a structure represented by the formula (I) or an isomer thereof, a racemate, a pharmaceutically acceptable salt, a hydrate or The pharmaceutically or foodly acceptable solvent, suspending agent or excipient that the precursor delivers to the animal or human. The carrier can be a liquid or a solid. Pharmaceutically acceptable carriers suitable for use in the present invention include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.

The present invention also provides a method of preparing a composition for preventing and treating tumors, which comprises using a compound having the structure represented by the formula (I). An effective amount of a compound of formula (I) can be combined with a pharmaceutically acceptable carrier to provide a composition of the present invention. The proportion by weight of active ingredient in the composition can be, for example, 0.0001 to 50% by weight; preferably 0.001- 20 wt %.

The pharmaceutical composition of the present invention may also be a traditional Chinese medicine or natural product extract containing the compound of the present invention having the structure represented by the formula (I) as an active ingredient, and extraction may be carried out by some known methods.

The dosage form of the pharmaceutical composition of the present invention may be various, as long as it is a dosage form capable of effectively bringing the active ingredient to the affected part of the mammal. From the standpoint of ease of preparation and administration, the preferred pharmaceutical composition is an injection or oral preparation. For example, it may be selected from the group consisting of: solutions, or suspensions, powders, granules, tablets, capsules. The compound having the structure of the formula (I) or an isomer thereof, a racemate, a pharmaceutically acceptable salt, a hydrate or a precursor thereof may be present in a suitable solid or liquid carrier or diluent. Various conventional carriers or excipients, such as fillers, flavoring agents, antioxidants, which are required for preparing different dosage forms, may be added to the pharmaceutical composition of the present invention. Perfumes, pigments, lubricants, glidants, wetting agents, emulsifiers, pH buffers, etc. These additives are well known to those skilled in the art.

The invention also provides a method of preventing and treating a tumor comprising the steps of: administering to a subject in need thereof an effective amount of a compound of formula (I). The amount of active ingredient administered is a therapeutically effective amount. When used topically, the safe and effective amount of the compound of the present invention is usually from about 0.1 ng to 100 mg/kg body weight; preferably from about 1 ng to 10 mg/kg body weight. Of course, specific doses should also take into account factors such as the route of administration, the health of the user, and the like, which are within the skill of the skilled physician.

Furthermore, the compounds of the invention may be used in combination with other active ingredients or therapeutic agents (e.g., other anti-tumor drugs, etc.). As a preferred embodiment of the present invention, the other active ingredient or therapeutic agent is 5-fluorouracil.

As a preferred mode of the present invention, the compound of the present invention and 5-fluorouracil are used in the preparation of a pharmaceutical composition in a molar ratio of 1: (1-20); preferably 1: ( 5-10); more preferably 1:8.

The invention also provides a kit comprising one or more compounds of the invention; or a pharmaceutical composition according to the invention. The kit may also contain instructions for use to guide the use of the drug. The main advantages of the invention are:

(1) For the first time, a new class of gossypol derivatives is disclosed which is more water soluble than natural gossypol and has a good antitumor effect.

(2) The compound of the present invention has low toxicity and is safe to use.

(3) The compound of the present invention can be produced by a synthetic method at a low cost. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Guide (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Percentages and parts are by weight unless otherwise stated.

I. Materials and methods

The reagents used were purchased from GL Biochem (Shanghai) Co., Ltd. unless otherwise stated.

1. Preparation of compounds

The summary process for compound preparation is shown in Figure 1. The method is described as follows:

Preparation of 6-APA- Na-cotton (Compound 7)

Take 39 mg of 6-APA (6-aminopenicillin alkanoic acid), 10 mg of sodium hydroxide, dissolved in 6 ml of methanol and isopropanol [1: 1 (v/v)] at 0 ° C, and the pH is close to 6 Add 48 mg of cotton ketone, keep at room temperature for 5 hours, filter by suction, rinse with isopropanol, and dry to obtain 64 mg of compound 7.

a compound 7

1H NMR (300 MHz, CD ₃ OD) δ 9.54 (s, 2H), 9.53 (s, 2H), 5.65 (d, J = 6 Hz, 2H) „ 5.66 (d, J = 5.7 Hz, 2H), 5.44 -5.46 (m, 4H), 4.28(br s, OH), 4.25 (br s, OH), 3.99 (m, 4H), 1.96(s, 6H), 1.98 (s, 6H), 1.66(s, 6H ), 1.66 (s, 6H), 1.59 (s, 12H), 1.44 (d, J = 6.9 Hz, 12H), 1.43 (d, J = 7.2 Hz, 12H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 187.3, 187.3, 185.4, 185.3, 172.8, 171.2, 171.1, 167.4, 167.2, 161.8, 161.5, 151.7, 151.6, 145.9, 145.8, 138.3, 138.3, 135.4, 135.4, 126.6, 126.5, 125.8, 125.7, 110.2, 73.3 , 70.7, 70.5, 66.8, 66.7, 64.5, 63.4, 31.2, 28.3, 26.3, 23.9, 19.3, 19.1, 19.0, 13.4, 13.2. Anal. Calcd for C ₄₆ H ₄₄ N ₄ Na ₂ 0 ₁₄ S2.3CH ₃ 0 Gu ₂ 0: C, 51.75; H, 5.50; N, 4.93. Found: C, 51.23; H, 5.06; N, 4.87. Preparation of sodium L-phenylalaninate-racenitol (compound 3)

Take 8 mg of sodium hydroxide, dissolve in 6 ml of methanol and isopropanol [1: 5 (v / v)], add 33 mg of L-phenylalanine, and heat the reaction to 60 ° C for 0.5 hour. After the pH was 8, 52 mg of racemic gossypol was added, and the reaction solution was kept at 65-70 ° C for 3 hours to obtain a yellow precipitate. The precipitate was filtered, washed with isopropyl alcohol, and dried to give the compound 3 of 55 mg.

Compound 3

1H NMR (300 MHz, CD ₃ OD) δ 9.45 (s, 2H), 9.26 (s, 2H), 7.50 (s, 2H), 7.49 (s, 2H), 7.23-7.00 (m, 20H), 4.19 ( Dd, J = 9.3, 3.6 Hz, 2H), 4.13 (dd, J = 9.3, 3.6 Hz, 2H), 3.74 (m, 4H), 3.39 (dd, J = 13.5, 3.6 Hz, 4H), 3.07 (dd , J = 13.5, 9.3 Hz, 2H), 2.97 (dd, J = 13.5, 9.3 Hz, 2H), 1.95 (s, 12H), 1.51 (d, J = 6.3 Hz, 12H), 1.49 (d, J = 6.9 Hz, 12H); ¹³ C NMR (75 MHz, CDCI3) δ 175.2, 175.2, 172.0, 171.9, 161.5, 149.5, 149.4, 146.9, 137.2, 131.5, 131.4, 129.4, 129.3, 129.1, 128.5, 128.2, 128.2, 128.0, 128.0, 127.2, 127.2, 126.3, 126.3 , 117.5, 117.4, 116.8, 116.7, 115.4, 103.5, 103.4, 67.2, 67.1, 63.4, 40.9, 40.5, 27.2, 23.9, 19.4, 19.1, 19.0. Preparation of 2-amino-2-indole-methyl acetate- (-) Gossypol (Compound 4)

Take 2-amino-2-indole-methyl acetate 41 mg, dissolve in methanol at room temperature, add (-) gossypol 52 mg, then the reaction solution is heated to 50-54 ° C, 2-3 hours, suction filtration to obtain 62 Mg of compound 4.

NMR NMR (300 MHz, CDC1 ₃ ) δ 9.64 (d, J = 7.8 Hz, IH), 9.60 (d, J = 7.5 Hz, IH), 8.30 (br s, OH), 7.95 (br s, NH), 7.64 (m, 2H), 7.52 (s, 2H), 7.08-7.36 (m, 6H), 5.53 (m, IH), 5.55 (m, IH), 5.37 (br s, OH), 5.32 (br s, OH), 3.76(s, 3H), 3.78 (s, 3H), 3.66 (m, 2H), 1.99 (s, 3H), 2.02 (s, 3H), 1.50 (d, J = 6.6 Hz, 12H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 173.5, 170.2, 161.2, 149.1, 147.1, 136.4, 132.2, 129.1, 128.0, 128.0, 125.0, 124.1, 123.0, 120.7, 118.8, 118.2, 116.0, 114.6, 111.7, 104.0 , 58.8, 58.7, 53.1, 27.5, 20.3, 20.3, 20.0. Preparation of (Rg, S, S) 2-amino-3methoxy-3-(4-p-nitro)propanol-(-) gossypol ( Compound 5)

Take (S, S) 2-amino-3methoxy-3-(4-p-nitro)propanol 39 mg, sodium hydroxide 6 mg, dissolved in 5 ml of anhydrous methanol at room temperature, and the pH is 8 Thereafter, 39 mg of (-) gossypol was added, and the reaction solution was kept at 50-54 ° C for 5 hours, and the solvent was evaporated to give 36 mg of Compound 5 from petroleum ether / acetone (4: 1).

Compound 5 ESI-MS [MH]": 933.3; 1H NMR (300 MHz, CDC1 ₃ ) δ 13.45 (m, NH), 9.31 (d, J = 9 Hz, 2H), 8.17 (d, J = 7.8 Hz, 4H) , 7.93 (br s, OH), 7.54 (s, 2H), 7.49 (d, J = 7.8 Hz, 4H), 5.36 (br s, OH), 4.62 (br s, OH), 3.86 (m, 2H) , 3.73 (m, 4H), 3.44 (m, 2H), 3.31 (s, 6H), 2.00 (s, 6H), 1.49 (d, J = 7.8 Hz, 12H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 172.9, 163.4, 149.5, 147.8, 146.6, 146.0, 131.9, 128.2, 128.0, 127.9, 127.9, 123.3, 123.3, 117.8, 117.1, 115.1, 103.6, 81.0, 68.1, 61.5, 56.8, 27.2, 19.4, 19.4, 18.8. Preparation of 6-APA-K-(+) gossypol (compound 6)

Take 43 mg of 6-APA, 11 mg of potassium hydroxide, dissolved in 6 ml of methanol and isopropanol [l: l(v/v)] at 0 ° C. When the pH is close to 6, add C +) gossypol 52 mg. , kept at room temperature for 5 hours, with a yellow precipitate, suction filtered, rinsed with isopropyl alcohol, and dried to give a yellow powder of 42 mg of Compound 6.

K compound 6

1H NMR (300 MHz, CD ₃ OD) δ 9.86 (s, 2H), 7.55 (s, 2H), 5.66 (br s, 2H), 5.41 (br s, 2H) 4.30 (br s, OH), 3.82 ( m, 2H), 2.03 (s, 6H), 1.73 (s, 6H), 1.58 (s, 6H), 1.44 (br d, 12H). Preparation of sodium L-isoleucine-racenitol (compound 8 )

Take 9 mg of sodium hydroxide, dissolve it in 6 ml of methanol and isopropanol [1: 5 (v/v)], add 29 mg of L-isoleucine, and heat the reaction solution to 60 ° C for 0.5 hour. After the pH was 9, 58 mg of racemic gossypol was added, and the reaction solution was kept at 65-70 ° C for 3 hours to obtain a yellow precipitate. The precipitate was filtered, washed with isopropyl alcohol, and dried to give 52 mg of Compound 8.

1H NMR (300 MHz, CD ₃ OD) δ 9.78 (s, 2H), 7.54 (s, 2H), 3.85 (d, J = 7.5 Hz, 2H), 3.77 (m, 2H), 2.06 (m, 2H) , 2.02 (s, 6H), 1.65 (m, 2H), 1.50 (br d, 12H), 1.24 (m, 2H), 1.00 (2 br d, 6H), 0.94 (br t, 6H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 175.6, 175.5, 171.7, 161.8, 149.7, 147.1 131.6, 128.2, 127.5, 127.4, 117.9, 117.7, 117.0, 115.8, 103.8, 63.4, 38.9, 38.8, 27.2, 24.1, 24.1, 19.6 , 19.2, 15.3, 15.3, 10.8. Preparation of sodium L-isoleucine-(+) gossypol (compound 9)

Take 13 mg of sodium hydroxide, dissolve it in 6 ml of methanol and isopropanol [1: 5 (v/v)], add 42 mg of L-isoleucine, and heat the reaction solution to 60 ° C for 0.5 hour. After the pH was 9, 85 mg of (+) gossypol was added, and the reaction solution was kept at 65-70 ° C for 3 hours to obtain a yellow precipitate. The precipitate was filtered, washed with isopropyl alcohol, and dried to give 119 mg of Compound 9.

9

1H NMR (300 MHz, CD ₃ OD) δ 9.76 (s, 2H), 7.53 (s, 2H), 3.82 (d, J = 2.4 Hz, 2H), 3.75

(m, 2H), 2.03 (m, 2H), 2.03 (s, 6H), 1.50 (t, J = 6.9 Hz, 12H), 1.00 (d, J = 6.6 Hz, 6H), 0.93 (t, J = 7.2 Hz, 6H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 175.4, 171.8, 161.8, 149.7, 147.0, 131.7, 128.1, 127.3, 117.8, 116.9, 115.7, 103.7, 63.4, 38.8, 27.2, 24.6, 24.2 , 24.0, 19.5, 19.2, 15.3, 10.7.

2. Cell culture

Mouse colon cancer cell line CT26 (obtained from ATCC Bioresource Center, USA), breast cancer cell line 4T1 (obtained from ATCC Bioresource Center, USA) and melanoma cell line B16-F10 (obtained from ATCC Bioresource Center) 10% fetal bovine serum (HyClone;), 100 units/ml penicillin and 100 ug/mL streptomycin (Invitrogen, Carlsbad, CA) were added in RMPI-1640 medium (HyClone, Logan, UT). Human colon cancer cell line HCT116 (obtained from ATCC Bioresource Center of the United States) and SW620 (obtained from ATCC Bioresource Center of the United States), lung cancer cell line A549C obtained from ATCC Bioresource Center of the United States;), breast cancer cell line MDA-MB-435C Obtained from the ATCC Bioresources Center of the United States and MCF-7 (obtained from the ATCC Bioresources Center of the United States), the prostate cancer cell line PC-3 (obtained from the ATCC Bioresource Center of the United States), and added 10% fetal bovine serum with DMEM (HyClone). Penicillin and streptomycin were cultured in a 37 ° C, 5% CO ₂ incubator.

3. Cell viability assay

The synthesized compound was first dissolved in DMSO. Different types of tumor cell lines were seeded in 96-well plates at 5 X 10 ³ /100 μl per well. After adherence, different concentrations of the compound or combined with 5-fluorouracil (Wako Pure Chemical Industries, Osaka, Japan) were added for 72 hours with DMSO as a negative control. After the end of the treatment, 3-(4,5-methylazo)-2,5-diphenyltetrazolium salt (MTT, Sigma-Aldrich, St. Louis, MO) was added, and then protected from light at 37 ° C. Incubate for 4 hours. The resulting crystals were dissolved in DMSO and read on a microplate reader.

4. Cell proliferation ability detection

CT26 cells were seeded in 96-well plates at 2 x 10 ³ cells/μM per well. Cell proliferation ability was measured using a Cell Titer 96 Aqueous One Solution Cell Proliferation Kit (Promega Corporation, Madison, WI) after various times of treatment with different concentrations of Compound 7. See the kit instructions for the specific procedure of the experiment.

5. TUNEL apoptosis detection

After the CT26 cells were treated with different concentrations of Compound 7 for various times, the degree of apoptosis was measured using an In Situ Cell Death Detection Kit (Roche Applied Science, Mannheim, Germany). The cells were washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 1 hour, washed twice with PBS, and treated with permeable cell solution for 2 minutes on ice, washed twice with PBS, and The apoptosis detection reaction mixture was incubated at 37 ° C for 1 hour in the dark, the nuclei were stained with DAPI, and finally washed twice with PBS. The samples were analyzed under a fluorescence microscope, and five cells with positive visual field counts were randomly selected, and the percentage of positive cells was statistically analyzed.

6. Molecular docking simulation

The inventors used the software AutoDock Vina (http://vina.scripps.edu/) to anti-apoptotic protein

Bcl-2 (Protein Data Bank code 1YSW) and Bcl-xL (PDB code 1BXL) were docked with Compound 7.

7. Immunoblotting

After the cells were treated with compound 7, the cell lysate was electrophoresed on a 10% SDS-PAGE gel and then transferred to nitric acid. On a cellulose membrane (Amersham Bioscience, Buckinghamshire, UK). The membrane contains 5% (w/v) skim milk and 1%. (v/v) Tween-20 TBS solution was blocked at room temperature for 1 hour, then anti-Bcl-2 (Cell Signaling, Beverly, MA): Bcl-xL (Cell Signaling) and β-actin (Santa Cruz Biotechnology, Santa Cruz) , CA) antibodies were incubated overnight at 4 °C. After TBST was washed 3 times, the horseradish peroxidase (HRP)-labeled secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was incubated for 1 hour at room temperature, and after TBST was washed 3 times, the chemiluminescent substrate SuperSignal was used. West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL, USA) Color development, the specific procedure of the experiment can be found in the kit instructions.

8. In vivo anti-tumor activity test

Male BALB/c mice, 6-8 weeks old, were purchased from the Shanghai Experimental Animal Center. The breeding of the mice was based on the guidelines for the use of experimental animals by Shanghai Second Medical University. 5 x l0 ⁵ /0.1 ml of CT26 cells were injected subcutaneously into the right side of the mouse. After the tumor volume reached approximately 60 mm ³ , the mice were randomly divided into different groups of 4 each. Compound 7 was dissolved in 10% ethanol. Mice were administered intragastrically for 12 consecutive days at a dose of ΙΟ μιηοΙ/kg body weight for 10 days to give 10% ethanol as a control. 5-fluorouracil was administered intraperitoneally once every two days at a dose of 10 mg/kg body weight, and PBS was intraperitoneally injected as a negative control. Tumor length and width were measured every 2 days. Tumor volume was calculated as the formula length X width X width Χ 0.52.

9. In vivo toxicity test

Compound 7 and natural gossypol (Compound 1) were dissolved in 10% (v/v) ethanol. 6-8 weeks old male BALB/c mice were divided into 3 groups, 6 in each group, and administered with compound 7 or 1 for 12 consecutive days at a dose of 120 μιηηΐ/kg body weight, and the control group was administered with 10% ethanol. The survival of the mice was counted, and the body weight of all mice was weighed 12 days later and statistically analyzed. The small intestines of the three groups of mice were embedded in paraffin, sectioned with 5 μιη, and stained with hematoxylin-eosin, and observed under the microscope.

10. Statistical analysis

Student's t-test test was used to compare the differences between the two groups. P values less than 0.05 were considered statistically significant.

11. Example

Example 1. Cell viability assay

The present inventors first examined the killing effect of newly synthesized compounds (Fig. 1, Compounds 3-9; wherein Compound 8 is an optical racemate, and Compound 9 is a positively-rotating compound) on different tumor cell lines. Some human and mouse tumor cells were treated with different concentrations of compounds for 72 hours, and cell viability was measured by MTT assay.

The results showed that Compound 3-9 had a certain killing effect on tumor cells, especially Compound 7. Colon cancer human and mouse cell lines SW620, HCT1 16 and 7 CT26 sensitive compound, IC ₅₀ LD50 It is about 6 to 18 μΜ (Table 1). The breast cancer cell lines MDA-MB-435, MCF-7 and 4T 1 are also sensitive to compound 7, with IC _5Q values exceeding 28 μΜ. Compound 7 also had a significant killing effect on mouse melanoma cell line B 16-F 10 , human lung cancer cell line Α 549 and prostate cancer cell line PC-3 (Table 1). These results indicate that Compound 7 has a significant killing effect on colon cancer cell lines. Therefore, Compound 7 can be further studied as a drug candidate for the treatment of tumors, particularly colon cancer.

Table 1. Detection of cell proliferation activity

To further investigate whether the killing effect of Compound 7 on colon cancer cell lines has a time- and dose-dependent effect, the inventors treated CT26 cells at different time points with different concentrations of Compound 7, and tested the cell proliferation ability. The results showed that 20 μΜ of compound 7 significantly inhibited the growth of CT26 cells (Fig. 2Κ). After 24 hours of treatment with 20 μΜ of Compound 7, approximately 80% of the CT26 cells survived. After 24 hours of treatment, only about 60% of the cells survived; after 3 days of treatment, almost all of the cells died. 10 μΜ and 5 μΜ Compound 7 were also effective in inhibiting the growth of CT26 cells (Fig. 2Α). However, 2 μΜ of compound 7 had only a slight inhibitory effect on CT26 cells, and even after 120 h of treatment, more than 90% of the cells survived (Fig. 2A). In addition, at the same time point, high doses of Compound 7 were more potent against CT26 cells than low doses. These data indicate that Compound 7 has a time- and dose-dependent effect on the killing of CT26 cells. Example 2. Compound 7 induces apoptosis in CT26 cells

The inventors treated CT26 cells with different concentrations of Compound 7 for 24, 48 or 72 hours, and examined the apoptosis rate of tumor cells by TUNEL kit (Fig. 2B). The TUNEL statistics are shown in Figure 2C.

The above results indicate that Compound 7 can induce tumor cell apoptosis and induce tumor cell apoptosis with a time- and dose-dependent effect. Example 3. Compound 7 downregulates its protein expression level by binding to the BH3 domain of Bcl-2 and Bcl-xL. Previous studies have shown that gossypol can act as a mimetic of the BH3 domain to regulate the expression of Bcl-2 family proteins. The inventors simulated the docking of compound 7 with Bcl-2 and Bcl-xL by AutoDock Vina software. The results of the docking model showed that Compound 7 could bind to the BH3 domain of Bcl-2 and Bcl-xL (Fig. 3A), indicating that Compound 7 binds to Bcl-2 and Bcl-xL.

Therefore, the inventors examined the changes in the expression levels of Bcl-2 and Bcl-xL proteins after treating CT26 cells with Compound 7. The results showed that Bcl-2 and Bcl-xL protein levels were significantly decreased after treatment of CT26 cells for 5 μΜ and 10 μΜ of compound 7 (Fig. 3C).

The above data indicate that Compound 7 can reduce the expression of anti-apoptotic proteins Bcl-2 and Bcl-xL in CT26 cells, consistent with the results of molecular simulation docking. Example 4. Compound 7 combined with 5-fluorouracil enhances its activity of inhibiting colon cancer growth in vitro and in vivo.

According to the results of previous studies by the present inventors, Compound 7 has an antitumor activity in vitro. The inventors gave CT26 tumor-bearing mice a compound dose of 10 μιηοΐ/kg body weight per day for 7 days, and the tumor volume of the mice in the administration group was significantly smaller than that of the control group (Ρ < 0.01) (Fig. 4Α). The present inventors selected 5-fluorouracil (5-FU) in combination with Compound 7 (abbreviated as "7" in the figure) to study the inhibitory effect of the combination on the growth of colon cancer. The results showed that ΙΟ μιηοΙ / liter of compound 7 combined with 25 μιηοΐ / liter of 5-fluorouracil significantly inhibited the growth of CT26 cells over compound 7 or 5-fluorouracil alone (Ρ < 0.01) (Fig. 4Β).

Therefore, the present inventors administered Compound 7 to a CT26 tumor-bearing mouse at a dose of 10 μm ηοΐ/kg body weight per day, with 10% ethanol as a control; and, at a dose of 10 mg/kg body weight, every two days by intraperitoneal injection. 5-fluorouracil once, with PBS as a control. The results showed that after 12 days, the tumor volume of the mice in the combination of Compound 7 and 5-FU was significantly smaller than that of Compound 7 or 5-FU alone (Ρ < 0.01) (Fig. 4C;).

The above results indicate that Compound 7 combined with 5-FU can be used as a new colon cancer chemotherapy strategy. Example 5. Comparison of in vivo toxicity of compound 7 with natural gossypol (Compound 1)

Previously, it was reported that apogossypol treated mice at a dose of 120 μιηοΐ/kg body weight was less toxic than the equivalent dose of natural gossypol. Therefore, the present inventors administered the compound 7 (abbreviated as "7" in the figure;) or the compound 1 (abbreviated as "1" in the figure) by the same dose at the same dose, and the control group was administered with 10% ethanol, and the observation was small. Rat survival and weight changes.

As a result, due to the accumulation of damage of Compound 1 in mice, on day 8, Compound 1 treated mice had one death; on Day 12, the second mouse died (Fig. 5Α;). However, all Compound 7 treated mice survived on day 12. In addition, the body weight of mice after 12 days of treatment with Compound 1 was significantly lower than that before treatment (Ρ < 0.01), while the body weight of mice treated with Compound 7 did not decrease significantly (Fig. 5Β).

Moreover, the mouse small intestine treated with Compound 1 showed significant damage compared with the control group, but the compound 7 treated group of mice There was no significant difference from the control group (Fig. 5C).

Therefore, Compound 7 is less toxic in vivo than Compound 1, indicating that it is an ideal antitumor drug for further clinical use. Example 6. Water solubility test of each compound

The solubility of natural gossypol (compounds 1 and 2) and each new compound (compounds 3-9) in 100 g of water at 37 ° C was measured to compare their water solubility.

As a result, it was found that the solubility of the compound 7 in 100 g of water at 37 ° C was 9.25 g, and the solubility of the natural gossypol under the same conditions was less than 0.001 g. Therefore, the water solubility of Compound 7 is significantly better than that of natural gossypol.

Compounds 3, 6 and 8-9 (both salts) also have good water solubility, greatly improved solubility, and are significantly superior to natural gossypol. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entirety as if they are individually incorporated by reference. In addition, it should be understood that various modifications and changes may be made to the present invention, and the equivalents of the scope of the invention.

Claims

Rights request

A compound of the formula I, an isomer thereof or a pharmaceutically acceptable salt thereof:

,

The compound according to claim 1, which has a structure as shown below:

a compound characterized in that R is selected from

,

6. Use of a compound according to any of claims 1-5 for the preparation of a pharmaceutical composition for controlling tumors.

7. The use according to claim 6, wherein the tumor comprises: colon cancer, breast cancer, melanoma, lung cancer or prostate cancer.

8. Use of a compound according to any of claims 1-5 for the manufacture of a medicament for reducing the expression of the anti-apoptotic proteins Bcl-2 and / or Bcl-xL.

9. A pharmaceutical composition comprising: an effective amount of a compound of any of claims 1-5, or a combination thereof; and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, further comprising: an effective amount of 5-fluorouracil.

11. A method of preparing a pharmaceutical composition, the method comprising: mixing an effective amount of a compound of any of claims 1-5, or a combination thereof, with a pharmaceutically acceptable carrier.

12. The method of claim 11, further comprising: mixing an effective amount of 5-fluorouracil with a compound of any of claims 1-3, or a combination thereof, and a pharmaceutically acceptable carrier.

A kit comprising the compound according to any one of claims 1 to 5; or the pharmaceutical composition according to any one of claims 9 to 10.