WO2012000421A1 - Utilisation de dérivés de gossypol lors de la préparation de médicaments antitumoraux - Google Patents

Utilisation de dérivés de gossypol lors de la préparation de médicaments antitumoraux Download PDF

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WO2012000421A1
WO2012000421A1 PCT/CN2011/076482 CN2011076482W WO2012000421A1 WO 2012000421 A1 WO2012000421 A1 WO 2012000421A1 CN 2011076482 W CN2011076482 W CN 2011076482W WO 2012000421 A1 WO2012000421 A1 WO 2012000421A1
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compound
gossypol
bcl
cells
pharmaceutical composition
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Chinese (zh)
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郭方
姜标
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中国科学院上海生命科学研究院
上海中科高等研究院
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Publication of WO2012000421A1 publication Critical patent/WO2012000421A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/04—Nitro compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
    • A61K31/431—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems containing further heterocyclic rings, e.g. ticarcillin, azlocillin, oxacillin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C251/00—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton
    • C07C251/02—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton containing imino groups
    • C07C251/24—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton containing imino groups having carbon atoms of imino groups bound to carbon atoms of six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/18—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D209/20—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals substituted additionally by nitrogen atoms, e.g. tryptophane
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D499/00—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
    • C07D499/04—Preparation
    • C07D499/10—Modification of an amino radical directly attached in position 6
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D499/00—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
    • C07D499/04—Preparation
    • C07D499/14—Preparation of salts
    • C07D499/16—Preparation of salts of alkali or alkaline earth metals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D499/00—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
    • C07D499/21—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring with a nitrogen atom directly attached in position 6 and a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2

Definitions

  • 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
  • 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.
  • 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.
  • 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.
  • the compound has the structure shown below (6-AP A-sodium-cotton):
  • the tumor includes, but is not limited to, colon cancer, breast cancer, melanoma, lung cancer or prostate cancer.
  • the tumor is colon cancer.
  • a pharmaceutical composition comprising: an effective amount of said compound or a combination thereof; and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is for use in the prevention and treatment of tumors.
  • the pharmaceutical composition further comprises: an effective amount of 5-fluorouracil.
  • a method of preparing a pharmaceutical composition comprising: mixing an effective amount of the compound or a combination thereof with a pharmaceutically acceptable carrier.
  • the method further comprises: mixing an effective amount of 5-fluorouracil with the compound or a combination thereof, and a pharmaceutically acceptable carrier.
  • a kit comprising the compound; or a pharmaceutical composition is provided.
  • the pharmaceutical composition is for use in the prevention and treatment of tumors.
  • 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.
  • Figure 2 Compound 7 inhibits cell growth and induces apoptosis with a time and dose dependent effect.
  • CT26 cells were tested for cell proliferation after treatment with different concentrations of compound 7 for different periods of time.
  • 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).
  • 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 ⁇ and 10 ⁇ 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, ⁇ -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 ⁇ /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 ⁇ 0.52. ( ⁇ ⁇ 0.01 is indicated by *).
  • CT26 tumor-bearing mice were grouped according to (A).
  • the tumor-bearing mice were intragastrically administered with compound 7 at a dose of 10 ⁇ /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 ( ⁇ ⁇ 0.01 with *; ⁇ ⁇ 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 ⁇ / kg body weight for 12 consecutive days, compound 7 or 1 was administered by gavage, and the control group was given 10% ethanol (NC; ).
  • Gossypol has not been clinically used for anti-tumor because of its low water solubility and side effects in the body. Treatment.
  • 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.
  • R1-R4 are independently selected from OH, H or 0.
  • the compound has the structure shown below:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a drug or composition for preventing and treating tumors.
  • 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.
  • 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
  • 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.
  • 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”.
  • 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.
  • 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.
  • the preferred pharmaceutical composition is an injection or oral preparation.
  • 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.
  • 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.
  • 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.
  • 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.
  • the compounds of the invention may be used in combination with other active ingredients or therapeutic agents (e.g., other anti-tumor drugs, etc.).
  • the other active ingredient or therapeutic agent is 5-fluorouracil.
  • 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 compound of the present invention has low toxicity and is safe to use.
  • 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.
  • the reagents used were purchased from GL Biochem (Shanghai) Co., Ltd. unless otherwise stated.
  • Mouse colon cancer cell line CT26 obtained from ATCC Bioresource Center, USA
  • breast cancer cell line 4T1 obtained from ATCC Bioresource Center, USA
  • 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
  • RMPI-1640 medium HyClone, Logan, UT
  • Human colon cancer cell line HCT116 obtained from ATCC Bioresource Center of the United States
  • 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
  • 10% fetal bovine serum with DMEM HexClone
  • 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 ⁇ l 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.
  • MTT 3-(4,5-methylazo)-2,5-diphenyltetrazolium salt
  • CT26 cells were seeded in 96-well plates at 2 x 10 3 cells/ ⁇ M 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.
  • 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.
  • the inventors used the software AutoDock Vina (http://vina.scripps.edu/) to anti-apoptotic protein
  • Bcl-2 Protein Data Bank code 1YSW
  • Bcl-xL PDB code 1BXL
  • the cell lysate was electrophoresed on a 10% SDS-PAGE gel and then transferred to nitric acid.
  • 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 ⁇ -actin (Santa Cruz Biotechnology, Santa Cruz) , CA) antibodies were incubated overnight at 4 °C.
  • HRP horseradish peroxidase
  • mice 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 ⁇ ⁇ /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 ⁇ 0.52.
  • Compound 7 and natural gossypol 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 ⁇ /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 ⁇ , and stained with hematoxylin-eosin, and observed under the microscope.
  • 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.
  • Compound 3-9 had a certain killing effect on tumor cells, especially Compound 7.
  • 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 ⁇ .
  • Compound 7 also had a significant killing effect on mouse melanoma cell line B 16-F 10 , human lung cancer cell line ⁇ 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.
  • 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.
  • Example 3 Compound 7 downregulates its protein expression level by binding to the BH3 domain of Bcl-2 and Bcl-xL.
  • 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.
  • Compound 7 has an antitumor activity in vitro.
  • the inventors gave CT26 tumor-bearing mice a compound dose of 10 ⁇ /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 ( ⁇ ⁇ 0.01) (Fig. 4 ⁇ ).
  • 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 present inventors administered Compound 7 to a CT26 tumor-bearing mouse at a dose of 10 ⁇ m ⁇ /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 ( ⁇ ⁇ 0.01) (Fig. 4C;).
  • mice 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).

Abstract

L'invention divulgue des dérivés de gossypol, des procédés de préparation et des utilisations de ceux-ci dans la préparation de médicaments antitumoraux. Les compositions pharmaceutiques contenant lesdits dérivés de gossypol et les procédés de préparation de celles-ci sont également divulgués.
PCT/CN2011/076482 2010-07-02 2011-06-28 Utilisation de dérivés de gossypol lors de la préparation de médicaments antitumoraux WO2012000421A1 (fr)

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CN103265560B (zh) * 2013-05-17 2016-05-04 中国科学院上海高等研究院 棉酚/棉酮衍生物及其制备方法和抗肿瘤药物中的应用
CN104817472B (zh) * 2015-03-25 2016-08-24 武汉大学 一种新型棉酚衍生物及其制备方法和抗肿瘤应用
CN105503627B (zh) * 2015-12-10 2017-10-03 武汉大学 一种新型棉酚希夫碱衍生物及其制备方法与应用
CN107417580A (zh) * 2017-09-12 2017-12-01 陕西科技大学 一类具有抗肿瘤活性的棉酚‑l‑精氨酸席夫碱类化合物及其合成方法
US9758486B1 (en) * 2017-03-27 2017-09-12 Shaanxi University Of Science And Technology Edaravone-gossypol derivatives with antitumor activities and a method of preparing the same
US9758503B1 (en) * 2017-03-27 2017-09-12 Shaanxi University Of Science And Technology Coumarin-gossypol derivatives with antitumor activities and a method of preparing the same
CN107573318A (zh) * 2017-09-12 2018-01-12 陕西科技大学 一种具抗肿瘤活性的新型棉酚席夫碱类衍生物及其合成方法

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