CN112940044B - Berberine coupled cisplatin compound, and preparation method and application thereof - Google Patents
Berberine coupled cisplatin compound, and preparation method and application thereof Download PDFInfo
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- 229940093265 berberine Drugs 0.000 title claims abstract description 173
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
- C07F15/0086—Platinum compounds
- C07F15/0093—Platinum compounds without a metal-carbon linkage
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Abstract
The invention relates to the technical field of pharmaceutical chemicals, in particular to a berberine coupled cisplatin compound, and a preparation method and application thereof. The invention provides a berberine-coupled cisplatin compound, which has a structure shown in the following formula I. The results of the examples show that the berberine-coupled cisplatin compound provided by the invention can effectively inhibit the proliferation activity of intestinal cancer cells and obviously reduce the killing property of cisplatin on normal intestinal epithelial cells, and the in-vivo experiments of mice prove that the berberine-coupled cisplatin compound has better tumor inhibiting effect than cisplatin.
Description
Technical Field
The invention relates to the technical field of pharmaceutical chemicals, in particular to a berberine coupled cisplatin compound, and a preparation method and application thereof.
Background
Colorectal cancer is the fourth most common malignant tumor and the fifth most lethal malignant tumor in China, and seriously endangers life and health of people. At present, surgery excision is mainly adopted in combination with radiotherapy and chemotherapy, but the postoperative recurrence rate of patients is high, and serious toxic and side effects limit the effective improvement of the survival time of intestinal cancer patients. Therefore, the development of new high-efficiency low-toxicity anti-intestinal cancer targeted drugs has become a very interesting and urgent task.
Platinum drugs are one of the most representative drugs in the field of chemotherapeutic drugs, and play an irreplaceable role in the clinical treatment of various cancers. Cisplatin is one of the three most widely used antitumor drugs worldwide as a first-generation platinum drug, but lacks selectivity to cancer cells, has serious toxic and side effects to normal cells, lacks sensitivity to digestive tract tumors, and limits further clinical application of cisplatin.
Disclosure of Invention
In view of the above, the invention provides a berberine-coupled cisplatin compound, a preparation method and application thereof, and the berberine-coupled cisplatin compound provided by the invention can effectively inhibit proliferation activity of intestinal cancer cells and obviously reduce the killing property of cisplatin on normal intestinal epithelial cells, and in vivo experiments of mice prove that the berberine-coupled cisplatin compound has better tumor inhibiting effect than cisplatin.
The invention provides a berberine coupling cisplatin compound, which has a structure shown in a formula I:
the invention provides a preparation method of berberine coupled cisplatin compound, which comprises the following steps:
carrying out hydroxylation reaction on berberine to obtain hydroxylated berberine;
carrying out nucleophilic substitution reaction on the hydroxylated berberine, butyl bromoacetate and an organic alkaline catalyst in a first polar organic solvent to obtain berberine with protected hydroxyl;
deprotection and carboxylation reaction are carried out on the berberine with protected hydroxyl and trifluoroacetic acid in a second polar organic solvent, so as to obtain carboxylated berberine;
carrying out oxidation reaction on cisplatin and hydrogen peroxide to obtain tetravalent cisplatin;
and (3) carrying out condensation reaction on the carboxylated berberine, tetravalent cisplatin and a condensation reaction catalyst in a polar organic solvent of the condensation reaction to obtain the berberine coupling cisplatin compound.
Preferably, the molar ratio of the hydroxylated berberine to the bromobutyl acetate to the organic alkaline catalyst is 2 (2.4-3) to 2-4.
Preferably, the organic basic catalyst comprises N, N-diisopropylethylamine and/or triethylamine; the nucleophilic substitution reaction is carried out at room temperature for 8-12 h.
Preferably, the volume ratio of the hydroxyl-protected berberine to the trifluoroacetic acid is (50-100) mg:1mL;
the volume ratio of the trifluoroacetic acid to the second polar organic solvent is 1 (2-3).
Preferably, the molar ratio of the carboxylated berberine to the tetravalent cisplatin is 6:5.
Preferably, the condensation reaction catalyst comprises triethylamine and O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroboric acid;
the mol ratio of the triethylamine to the O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroboric acid is (1-1.5): (1-1.5).
Preferably, the temperature of the condensation reaction is 60 ℃ and the time is 12 hours.
Preferably, the first polar organic solvent and the second polar organic solvent independently comprise dichloromethane, N-dimethylformamide or acetonitrile; the polar organic solvent for the condensation reaction includes DMSO or N, N-dimethylformamide.
The invention provides the berberine-coupled cisplatin compound disclosed by the technical scheme or the application of the berberine-coupled cisplatin compound obtained by the preparation method disclosed by the technical scheme in preparation of anti-intestinal cancer drugs.
The berberine coupling cisplatin compound provided by the invention is used for coupling berberine and tetravalent cisplatin, and can be reduced into berberine and cisplatin under the action of a reducing medium. The berberine coupled cisplatin compound provided by the invention is reduced in cytoplasm and releases cisplatin and berberine after entering cancer cells, and the cisplatin is combined with cell genome DNA to form adducts after entering cells, so that transcription and replication of cancer cell DNA are affected, and finally double-strand break and apoptosis of cancer cell DNA are induced; berberine inhibits intestinal cancer cell proliferation by inhibiting Wnt signal pathway protein to finally lead to intestinal cancer cell cycle arrest. In addition, the coupling of berberine and cisplatin changes the response of intestinal cancer cells to cisplatin, improves the selectivity of the intestinal cancer cells to cancer cells, and further improves the accumulation of platinum in the cancer cells so as to enhance the killing effect of the intestinal cancer cells on tumor cells and reduce the toxic and side effects of the intestinal cancer cells on normal intestinal epithelial cells. The berberine coupled cisplatin compound provided by the invention obviously improves the anti-intestinal cancer activity of cisplatin through the above way. The results of the examples show that the berberine-coupled cisplatin compound provided by the invention can effectively inhibit the proliferation activity of intestinal cancer cells and obviously reduce the killing property of cisplatin on normal intestinal epithelial cells, and the in-vivo experiments of mice prove that the berberine-coupled cisplatin compound has better tumor inhibiting effect and tumor selectivity than cisplatin.
Drawings
FIG. 1 is a mass spectrum of a berberine-coupled cisplatin compound prepared in example 1;
FIG. 2 is a nuclear magnetic resonance chart of the berberine-coupled cisplatin compound prepared in example 1;
FIG. 3 is a high performance liquid chromatography contrast chart of berberine coupled cisplatin compound, berberine, cisplatin and the product of test example 3 prepared in example 1;
FIG. 4 is a comparative diagram of immunofluorescence experiment of berberine-coupled cisplatin compound, berberine and cisplatin mixture, berberine and cisplatin treated HCT116 cells obtained in example 1 after 24 h;
FIG. 5 is a graph showing comparison of results of a berberine-coupled cisplatin compound, a berberine-cisplatin mixture, berberine and cisplatin Westen Blot experiment prepared in example 1;
FIG. 6 is a graph showing comparison of cell cycle test results of single dye detection of berberine coupled cisplatin compound, berberine and cisplatin mixture, berberine and cisplatin PI prepared in example 1;
FIG. 7 is a graph showing comparison of apoptosis detection results of berberine-coupled cisplatin compound, berberine-cisplatin mixture, berberine and cisplatin Annexin V/PI double-dyeing method prepared in example 1;
FIG. 8 is a graph showing the comparison of the intracellular platinum content of berberine-coupled cisplatin compound, berberine and cisplatin mixture and after treatment of SW620 and HCT116 cells prepared in example 1;
FIG. 9 is a graph showing the comparison of the intracellular platinum content of berberine-coupled cisplatin compound, berberine-cisplatin mixture and cisplatin treated HIEC-6 cells prepared in example 1;
FIG. 10 is a graph showing the effect of berberine-coupled cisplatin compound and cisplatin prepared in example 1 on nude mice transplantation tumor.
Detailed Description
The invention provides a berberine coupling cisplatin compound, which has a structure shown in a formula I:
the berberine coupling cisplatin compound provided by the invention couples berberine and tetravalent cisplatin, and can be reduced into berberine and cisplatin under the action of a reducing medium. The berberine can inhibit Wnt signal path to inhibit intestinal cancer growth, and has killing power to intestinal cancer cells, but no toxicity to normal intestinal epithelial cells; cisplatin enters cells and forms adducts by combining with cell genome DNA, so that the transcription and replication of the DNA are affected, and finally DNA double strand break and apoptosis are induced; the berberine coupled cisplatin compound changes the response of intestinal cancer cells to cisplatin, thereby improving the accumulation of intracellular platinum and further enhancing the killing effect of the intestinal cancer cells on tumor cells. The berberine coupled cisplatin compound obviously improves the anti-intestinal cancer activity of cisplatin through the way.
The invention provides a preparation method of berberine coupled cisplatin compound, which comprises the following steps:
carrying out hydroxylation reaction on berberine to obtain hydroxylated berberine;
carrying out nucleophilic substitution reaction on the hydroxylated berberine, butyl bromoacetate and an organic alkaline catalyst in a first polar organic solvent to obtain berberine with protected hydroxyl;
deprotection and carboxylation reaction are carried out on the berberine with protected hydroxyl and trifluoroacetic acid in a second polar organic solvent, so as to obtain carboxylated berberine;
carrying out oxidation reaction on cisplatin and hydrogen peroxide to obtain tetravalent cisplatin;
and (3) carrying out condensation reaction on the carboxylated berberine, tetravalent cisplatin and a condensation reaction catalyst in a polar organic solvent of the condensation reaction to obtain the berberine coupling cisplatin compound.
In the present invention, the raw materials are commercially available products well known to those skilled in the art unless specified otherwise.
The preparation method of the berberine coupled cisplatin compound provided by the invention comprises the following specific synthetic route:
the berberine is subjected to hydroxylation reaction to obtain hydroxylated berberine; in the present invention, the hydroxylation reaction is preferably described with reference to "Antibacterial activity and structure-activity relationships ofberberine diagnostics Eur J Med Chem (1996) 31,469-478"; in the invention, the specific steps of the hydroxylation reaction are as follows: at N 2 Under the protection, 1g berberine is reacted for 15min at 190 ℃, cooled to room temperature and added with 100mL with volume ratio of 1:1, washing the mixed solution of ethanol and methanol, and separating and purifying the washed precipitate by a high performance liquid chromatography to obtain the hydroxylated berberine, wherein the eluting agent used by the high performance liquid chromatography is the mixed solution of dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol is 20:1.
After obtaining the hydroxyl berberine, butyl bromoacetate and the organic alkaline catalyst are subjected to nucleophilic substitution reaction in a first polar organic solvent to obtain the berberine with protected hydroxyl.
In the present invention, the organic basic catalyst preferably comprises N, N-diisopropylethylamine and/or triethylamine, more preferably comprises N, N-diisopropylethylamine or triethylamine, most preferably comprises N, N-diisopropylethylamine, and in the present invention, the molar ratio of the hydroxylated berberine, butyl bromoacetate and organic basic catalyst is preferably 2 (2.4-3): 2-4, more preferably 2 (2.5-2.6): 2.2-3.8, most preferably 2:2.4:1; in the present invention, the first polar organic solvent preferably includes dichloromethane, N-dimethylformamide or acetonitrile, more preferably includes dichloromethane, and the amount of the first polar organic solvent is not specifically limited, so long as the dissolution of the reaction raw materials can be achieved, and in the specific embodiment of the present invention, the mass-to-volume ratio of the hydroxymethylcyanidine to the first polar organic solvent is preferably 1g:50mL.
In the present invention, the temperature of the nucleophilic substitution reaction is preferably room temperature, and the time is preferably 8 to 12 hours. In the present invention, the affinity substitution reaction is preferably carried out under stirring, and the present invention is not particularly limited to the specific embodiment of stirring, and may be carried out in a manner well known to those skilled in the art.
The reaction solution after the nucleophilic substitution reaction is preferably subjected to a post-treatment comprising solvent removal and separation and purification, wherein the solvent removal means is preferably reduced pressure distillation, the temperature of the reduced pressure distillation is preferably room temperature, the time of the reduced pressure distillation is not particularly required, the first polar organic solvent is completely evaporated, the equipment of the reduced pressure distillation is not particularly required, and the reduced pressure distillation is performed in a rotary evaporator in a specific embodiment of the present invention. The method is characterized in that the method is used for separating and purifying the solid crude product after the solvent is removed, the method is preferably high performance liquid chromatography, no special requirements are imposed on the specific implementation mode of the high performance liquid chromatography, in the specific embodiment of the method, the high performance liquid chromatography is stage elution, the chromatographic column is a silica gel column, the eluent is a mixed solution of dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol is 100:1 and 50:1.
After berberine with protected hydroxyl is obtained, the berberine with protected hydroxyl and trifluoroacetic acid are subjected to deprotection and carboxylation reaction in a second polar organic solvent to obtain carboxylated berberine; in the present invention, the second polar organic solvent preferably comprises dichloromethane, N-dimethylformamide or acetonitrile, more preferably comprises dichloromethane, and the volume ratio of the hydroxyl group-protected berberine to trifluoroacetic acid is preferably (50 to 100) mg:1mL, the volume ratio of trifluoroacetic acid to the second organic solvent is preferably 1:3.
In the present invention, the temperature of the deprotection and carboxylation reaction is preferably room temperature and the time is preferably 3 hours. The hydroxyl-protected berberine is preferably dissolved in trifluoroethylene and a second polar organic solvent for deprotection reaction, and in the present invention, the deprotection and carboxylation reaction are preferably carried out under stirring conditions, and the stirring embodiment is not particularly required, and the deprotection and carboxylation reaction can be carried out in a manner well known to a person skilled in the art.
The reaction solution after the deprotection and carboxylation reaction is preferably subjected to post-treatment, wherein the post-treatment preferably comprises solvent removal, precipitation and separation and purification, the solvent removal mode is preferably reduced pressure distillation, the temperature of the reduced pressure distillation is preferably room temperature, the time of the reduced pressure distillation is not particularly required, the second polar organic solvent is completely evaporated, the equipment of the reduced pressure distillation is not particularly required, and in a specific embodiment of the invention, the reduced pressure distillation is performed in a rotary evaporator. The reaction solution after removing the solvent is preferably subjected to precipitation and precipitation, wherein the precipitation solvent for precipitation and precipitation is preferably anhydrous diethyl ether, after precipitation and precipitation, the solid crude product obtained after precipitation and precipitation is preferably subjected to separation and purification, the separation and purification mode is preferably high performance liquid chromatography, the specific implementation mode of the reversed phase high performance liquid chromatography is not particularly required, the high performance liquid chromatography is stage elution in the specific embodiment of the invention, the chromatographic column is a silica gel column, the eluent is a mixed solution of dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol is 100:1 and 50:1.
The invention carries out oxidation reaction on cisplatin and hydrogen peroxide to obtain tetravalent cisplatin; in the invention, the mass concentration of the hydrogen peroxide is preferably 15%, and in the invention, the hydrogen peroxide with the mass concentration of 15% is preferably obtained by diluting commercially available hydrogen peroxide with the mass concentration of 30% by water. The mass ratio volume of cisplatin to hydrogen peroxide is preferably 30mg:1mL. In the present invention, the oxidation reaction is preferably carried out under stirring, and the present invention is not particularly limited to the specific embodiment of stirring, and may be carried out in a manner well known to those skilled in the art.
After carboxylated berberine and tetravalent cisplatin are obtained, the carboxylated berberine, tetravalent cisplatin and a condensation reaction catalyst are mixed in an organic solvent for condensation reaction to carry out condensation reaction, and the berberine coupling cisplatin compound is obtained.
In the invention, the mol ratio of the carboxylated berberine to the tetravalent cisplatin is preferably 6:5; the condensation reaction catalyst preferably comprises triethylamine and O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroboric acid, and the molar ratio of the triethylamine to the O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroboric acid is preferably (1-1.5): (1-1.5). In the present invention, the polar organic solvent of the condensation reaction preferably includes DMSO or N, N-dimethylformamide, more preferably includes DMSO. In the invention, the mass-volume ratio of the total mass of the carboxyl berberine and the tetravalent cisplatin to the polar organic solvent of the condensation reaction is preferably 15mg:1mL.
In the present invention, the condensation reaction preferably includes the steps of: first mixing carboxylated berberine and a polar organic solvent of partial condensation reaction to obtain carboxylated berberine solution;
performing second mixing on tetravalent cisplatin, a condensation reaction catalyst and the residual polar organic solvent of the condensation reaction to obtain tetravalent cisplatin mixed solution;
and thirdly mixing the tetravalent cisplatin mixed solution and the carboxylated berberine solution.
In the present invention, the volume ratio of the polar organic solvent of the partial condensation reaction to the polar organic solvent of the remaining condensation reaction is preferably 1:1, in the present invention, the third mixing mode is preferably dropwise adding, and in the specific embodiment of the present invention, the dropwise adding is to drop the tetravalent cisplatin mixed solution into the carboxylated berberine solution. The invention has no special requirement on the dropping speed.
In the present invention, the temperature of the condensation reaction is preferably 60℃and the time is preferably 12 hours, and in the present invention, the condensation reaction is preferably carried out under stirring, and the stirring is not particularly limited to the specific embodiment of the present invention, and may be carried out in a manner well known to those skilled in the art.
After the condensation reaction is finished, the condensation reaction liquid cooled to room temperature is preferably subjected to post-treatment, the post-treatment preferably comprises solvent removal, separation and purification and drying, the solvent removal mode is preferably reduced pressure distillation, the temperature of the reduced pressure distillation is preferably room temperature, the time of the reduced pressure distillation is not particularly required, the polar organic solvent of the condensation reaction is completely evaporated, the equipment of the reduced pressure distillation is not particularly required, and in the specific embodiment of the invention, the reduced pressure distillation is performed in a rotary evaporator. The method is characterized in that the method is used for separating and purifying the solid crude product obtained after the solvent is removed, the separation and purification mode is preferably reverse-phase high-performance liquid chromatography, the specific implementation mode of the reverse-phase high-performance liquid chromatography is not particularly required, in the specific embodiment of the method, the reverse-phase high-performance liquid chromatography is stage elution, the chromatographic column is C-18 column, and the eluent is a mixed solution of water and acetonitrile. After separation and purification, the berberine coupled cisplatin pure product is preferably dried, wherein the drying mode is preferably freeze drying, the temperature of the freeze drying is preferably 0 ℃, and the time is preferably 12 hours.
The preparation method provided by the invention can ensure that berberine-coupled cisplatin is synthesized with higher efficiency and better yield, and the pure berberine-coupled cisplatin is light yellow solid powder, and in the invention, the ratio of the mass of the berberine-coupled cisplatin compound to the mass of berberine is 1:20.
The invention provides the berberine-coupled cisplatin compound according to the technical scheme or the application of the berberine-coupled cisplatin compound obtained by the preparation method according to the technical scheme in preparing the anti-colorectal cancer medicine.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention.
Example 1
Reference "Antibacterial activity and structure-activity relationships ofberberine analogs. Eur J Med Chem (1996) 31,469-478", in N 2 Under the protection, 1000mg berberine is reacted for 15min at 190 ℃, cooled to room temperature and added with 100ml with the volume ratio of 1:1, separating and purifying by high performance liquid chromatography after washing the mixed solution of ethanol and methanol, wherein the eluting agent used by the high performance liquid chromatography is the mixed solution of dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol is 20:1, so as to obtain the hydroxylated berberine;
carrying out affinity substitution reaction on 320mg of hydroxylated berberine and 234mg of tert-butyl bromoacetate in 10mL of dichloromethane solution containing 260mg of N, N-diisopropylethylamine at room temperature under stirring for 8 hours, removing dichloroethane from the reaction liquid after nucleophilic substitution reaction at room temperature by adopting a rotary evaporator to obtain a solid crude product, adopting a high performance liquid chromatography, using a silica gel column, a mixed solution of dichloromethane and methanol (the volume ratio of dichloromethane to methanol is 100:1 and 50:1) as an eluent, and adopting stage elution to separate and purify the solid crude product to obtain berberine with protected hydroxyl;
dissolving 100mg of berberine with protected carboxyl and 3mL of trifluoroacetic acid in 1mL of dichloromethane, carrying out deprotection reaction for 3h at room temperature under stirring, removing dichloroethane from the reaction liquid after deprotection reaction at room temperature by adopting a rotary evaporator, adding anhydrous diethyl ether to obtain a solid crude product, adopting high performance liquid chromatography, adopting a silica gel column, a mixed solution of dichloromethane and methanol (the volume ratio of dichloromethane to methanol is 100:1 and 50:1) as an eluent, and adopting stage elution to separate and purify the solid crude product to obtain carboxylated berberine;
carrying out oxidation reaction on 300mg of cisplatin and 10mL of hydrogen peroxide with mass concentration of 15% under the condition of stirring to obtain tetravalent cisplatin;
carrying out first mixing on 300mg of carboxylated berberine and 10ml of LDMSO under the condition of stirring to obtain a carboxylated berberine solution, carrying out second mixing on 330mg of cisplatin, 120mg of triethylamine, 456mg of O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroboric acid and 10ml of LDMSO under the condition of stirring to obtain a tetravalent cisplatin mixed solution, dripping the tetravalent cisplatin mixed solution into the carboxylated berberine solution, carrying out condensation reaction under the condition of stirring, removing DMSO from the condensation reaction solution cooled to room temperature by adopting a rotary evaporator after the reaction temperature is 60 ℃ for 12 hours to obtain a solid crude product, adopting a reversed phase high performance liquid chromatography, using a C-18 chromatographic column, taking a mixed solution of water and acetonitrile as an eluent, adopting a stage elution mode to separate and purify the solid crude product, and carrying out freeze drying on the obtained berberine coupled cisplatin pure product at 0 ℃ for 12 hours after separation and purification to obtain 90mg of berberine coupled cisplatin compound.
FIG. 1 is a mass spectrum of a berberine-coupled cisplatin compound prepared in example 1;
FIG. 2 is a nuclear magnetic resonance chart of the berberine-coupled cisplatin compound prepared in example 1;
as can be seen from the nuclear magnetic spectrum of FIG. 1, the berberine-coupled cisplatin compound prepared in example 1 of the present invention has a molecular weight of 696.0, which is actually measured and is consistent with a molecular weight 690.06 obtained by theoretical calculation; as can be seen from the nuclear magnetic resonance chart of FIG. 2, the H atom chemical shift of the berberine-coupled cisplatin compound prepared in example 1 of the present invention belongs to a definite one-to-one correspondence.
Example 2
Reference "Antibacterial activity and structure-activity relationships ofberberine analogs. Eur J Med Chem (1996) 31,469-478", in N 2 Under the protection, 1g berberine is reacted for 15min at 190 ℃, cooled to room temperature and added with 100ml with the volume ratio of 1:1, and separating and purifying by high performance liquid chromatography, wherein the eluent used in the high performance liquid chromatography is the mixed solution of dichloromethane and methanolThe volume ratio of the dichloromethane to the methanol is 20:1, and the hydroxylated berberine is obtained;
carrying out affinity substitution reaction on 320mg of hydroxylated berberine and 234mg of tert-butyl bromoacetate in 10mL of dichloromethane solution containing 260mg of N, N-diisopropylethylamine at room temperature under stirring for 8 hours, removing dichloroethane from the reaction liquid after nucleophilic substitution reaction at room temperature by adopting a rotary evaporator to obtain a solid crude product, adopting a high performance liquid chromatography, using a silica gel column, a mixed solution of dichloromethane and methanol (the volume ratio of dichloromethane to methanol is 100:1 and 50:1) as an eluent, and adopting stage elution to separate and purify the solid crude product to obtain berberine with protected hydroxyl;
dissolving 100mg of berberine with protected carboxyl and 3mL of trifluoroacetic acid in 1mL of dichloromethane, carrying out deprotection reaction at room temperature for 3h, removing dichloroethane from the reaction solution after deprotection reaction at room temperature by adopting a rotary evaporator, adding anhydrous diethyl ether to obtain a solid crude product, adopting a high performance liquid chromatography, using a silica gel column, a mixed solution of dichloromethane and methanol (the volume ratio of dichloromethane to methanol is 100:1 and 50:1) as an eluent, and adopting stage elution to separate and purify the solid crude product to obtain carboxylated berberine;
carrying out oxidation reaction on 300mg of cisplatin and 10mL of hydrogen peroxide with mass concentration of 15% under the condition of stirring to obtain tetravalent cisplatin;
carrying out first mixing on 300mg of carboxylated berberine and 10mL of DMSO under the condition of stirring to obtain carboxylated berberine solution, carrying out second mixing on 330mg of cisplatin, 120mg of triethylamine, 456mg of O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroboric acid and 10mL of MSO under the condition of stirring to obtain tetravalent cisplatin mixed solution, dripping the tetravalent cisplatin mixed solution into the carboxylated berberine solution, carrying out condensation reaction under the condition of stirring, removing DMSO from the condensation reaction solution cooled to room temperature by adopting a rotary evaporator after the reaction temperature is 60 ℃ for 12 hours to obtain a solid crude product, adopting a reversed phase high performance liquid chromatography, using a C-18 chromatographic column, adopting a mixed solution of water and acetonitrile as eluent, adopting a mode of stage elution to separate and purify the solid crude product, and carrying out freeze drying on the obtained berberine coupled cisplatin pure product at 0 ℃ after separation and purification for 12 hours to obtain 90mg of berberine coupled cisplatin compound.
The mass spectrum and nuclear magnetic resonance chart of the berberine-coupled cisplatin compound prepared in example 2 are similar to those of the product in example 1.
Example 3
Reference "Antibacterial activity and structure-activity relationships ofberberine analogs. Eur J Med Chem (1996) 31,469-478", in N 2 Under the protection, 1g berberine is reacted for 15min at 190 ℃, cooled to room temperature and added with 100ml with the volume ratio of 1:1, separating and purifying by high performance liquid chromatography after washing the mixed solution of ethanol and methanol, wherein the eluting agent used by the high performance liquid chromatography is the mixed solution of dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol is 20:1, so as to obtain the hydroxylated berberine;
carrying out affinity substitution reaction on 320mg of hydroxylated berberine and 234mg of tert-butyl bromoacetate in 10mL of dichloromethane solution containing 260mg of N, N-diisopropylethylamine at room temperature under stirring for 8 hours, removing dichloroethane from the reaction liquid after nucleophilic substitution reaction at room temperature by adopting a rotary evaporator to obtain a solid crude product, adopting a high performance liquid chromatography method, using a silica gel column, a mixed solution of dichloromethane and methanol (the volume ratio of dichloromethane to methanol is 100:1 and 50:1) as an eluent, and adopting stage elution to separate and purify the solid crude product to obtain hydroxyl-protected berberine;
dissolving 100mg of berberine with protected carboxyl and 3mL of trifluoroacetic acid in 1mL of dichloromethane, carrying out deprotection reaction for 3h at room temperature under stirring, removing dichloroethane from the reaction liquid after deprotection reaction at room temperature by adopting a rotary evaporator, adding anhydrous diethyl ether to obtain a solid crude product, adopting high performance liquid chromatography, adopting a silica gel column, a mixed solution of dichloromethane and methanol (the volume ratio of dichloromethane to methanol is 100:1 and 50:1) as an eluent, and adopting stage elution to separate and purify the solid crude product to obtain carboxylated berberine;
carrying out oxidation reaction on 300mg of cisplatin and 10mL of hydrogen peroxide with mass concentration of 15% under the condition of stirring to obtain tetravalent cisplatin;
carrying out first mixing on 300mg of carboxylated berberine and 10ml of LDMSO under the condition of stirring to obtain a carboxylated berberine solution, carrying out second mixing on 330mg of cisplatin, 120mg of triethylamine, 456mg of O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroboric acid and 10ml of LDMSO under the condition of stirring to obtain a tetravalent cisplatin mixed solution, dripping the tetravalent cisplatin mixed solution into the carboxylated berberine solution, carrying out condensation reaction under the condition of stirring, removing DMSO from the condensation reaction solution cooled to room temperature by adopting a rotary evaporator after the reaction temperature is 60 ℃ for 12 hours to obtain a solid crude product, adopting a reversed phase high performance liquid chromatography, using a C-18 chromatographic column, taking a mixed solution of water and acetonitrile as an eluent, adopting a stage elution mode to separate and purify the solid crude product, and carrying out freeze drying on the obtained berberine coupled cisplatin pure product at 0 ℃ for 12 hours after separation and purification to obtain 90mg of berberine coupled cisplatin compound.
The mass spectrum and nuclear magnetic resonance chart of the berberine-coupled cisplatin compound prepared in example 3 are similar to those of the product in example 1.
Test example 1
Carrying out reduction reaction on the berberine-coupled cisplatin compound prepared in the embodiment 1 in a reduction medium, wherein the reduction medium is sodium ascorbate, the reaction time is 12h, the reaction temperature is 37 ℃, and products after the reduction reaction are detected by high performance liquid chromatography, and comparing the berberine-coupled cisplatin compound with berberine and cisplatin, wherein the berberine-coupled cisplatin compound is recorded as: B-D, carboxylated berberine is marked as: b-1, cisplatin was labeled as DDP, and in order to make cisplatin detectable in high performance liquid chromatography, cisplatin was chelated with the chelating agent DDTC, labeled as Pt (DDTC) 2 The high performance liquid chromatogram is shown in fig. 3, and it can be obtained from fig. 3 that the berberine-coupled cisplatin compound obtained in example 1 of the present application can be successfully cut off in a reducing medium, and carboxylated berberine and cisplatin can be released with higher efficiency.
Test example 2
The berberine prepared in example 1 of the present invention was detected by MTT methodCoupled cisplatin compounds have cytotoxic effects on human colon carcinoma cells HCT116/SW620 and human normal intestinal epithelial cells HIEC-6. The specific experiments are divided into five groups, namely, berberine independent administration group (Ber group), berberine coupled cisplatin compound administration group (B-D group), cisplatin independent administration group (DDP group) and berberine and cisplatin combined administration group (B+D group), after the cells are treated for 24 hours by the different administration groups, the MTT experiment detects the inhibition effect of the drugs on cell proliferation and calculates corresponding IC 50 Specific experimental data are shown in table 1, and the IC50 results of MTT show that berberine-coupled cisplatin compound has better effect on tumor cell proliferation inhibition compared with berberine or cisplatin alone or in combination with berberine and cisplatin, and that berberine-coupled cisplatin compound has much lower cytotoxicity on intestinal normal epithelial cells (HIEC-6) than cisplatin. Compared with normal cells, the berberine-coupled cisplatin compound prepared in the embodiment 1 of the invention shows selectivity on tumor cells.
TABLE 1 inhibition of cell proliferation by different drug treatment groups
| Cellline | Ber(μM) | DDP(μM) | B+D(μM) | B-D(μM) |
| HCT116 | 57.76 | 32.29 | 30.22 | 3.99 |
| SW620 | 41.74 | 31.26 | 30.75 | 5.22 |
| HIEC-6 | ＞200 | 25 | 36.54 | ＞200 |
Test example 3
Immunofluorescence experiments are used for detecting the action mechanism of the berberine coupled cisplatin compound prepared in the embodiment 1 of the invention on HCT116 of human colon cancer cells. The results of immunofluorescence detection after 24H treatment of HCT116 cells with a blank control group (control), a berberine-alone group (Ber group), a berberine-coupled cisplatin compound group (B-D group), a cisplatin-alone group (DDP group) and a berberine-cisplatin combined group (B+D group) are shown in FIG. 4, and it can be seen from FIG. 4 that the expression level of the B-D group gamma H2A protein (DNA damage marker protein) prepared in example 1 is obviously increased, which indicates that the berberine-coupled cisplatin compound prepared in example 1 of the invention can interfere with DNA replication after treatment of cells, resulting in DNA damage breakage and further affecting tumor cell proliferation.
Test example 4
The action mechanism of the berberine-coupled cisplatin compound prepared in the embodiment 1 of the invention on HCT116 of human colon cancer cells is detected by using a Westen Blot experiment. The HCT116 cells are treated by a blank control group (control), a berberine independent drug group (Ber group), a berberine coupled cisplatin compound drug group (B-D group), a cisplatin independent drug group (DDP group) and a berberine and cisplatin combined drug group (B+D group) respectively for 24 hours, the result of Westen Blot experiments shows that the expression level of gamma H2A protein (DNA damage marker protein) in the B-D group is obviously increased, as shown in figure 5, in addition, the Westen Blot experiments also show that related protein Caspase-3 and PARP which can obviously induce apoptosis of the HCT116 cells after being treated by the B-D group can be subjected to shearing activation, and the expression of P53 is induced to be up-regulated, so that the berberine coupled cisplatin compound prepared by the invention example 1 can induce DNA fracture and apoptosis of cells after being treated by the cells, and cell cycle arrest is caused, thereby influencing proliferation of tumor cells.
Test example 5
The action mechanism of the berberine coupled cisplatin compound prepared in the embodiment 1 of the invention on HCT116 of human colon cancer cells is detected by a PI single-dyeing method. HCT116 cells were treated with a control group (control), a berberine-alone group (Ber group), a berberine-coupled cisplatin compound group (B-D group), a cisplatin-alone group (DDP group) and a berberine-cisplatin combination group (B+D group), respectively, and the results of the PI single-dye detection cell cycle blocking experiments showed that the B-D group treated cells were blocked in the S phase, as shown in Table 2 and FIG. 6, indicating that the berberine-coupled cisplatin compound prepared in example 1 of the present invention could cause cell cycle blocking after treating cells, thereby affecting tumor cell proliferation.
TABLE 2 results of experiments for detecting cell cycle arrest by PI single-dye method for different drug-treated groups
Test example 6
Annexin V/PI double-staining method is used for detecting the action mechanism of berberine coupled cisplatin compound prepared in the embodiment 1 of the invention on HCT116 of human colon cancer cells. The treatment of HCT116 cells for 24h by a blank control group (control), a berberine independent drug group (Ber group), a berberine coupled cisplatin compound drug group (B-D group), a cisplatin independent drug group (DDP group) and a berberine and cisplatin combined drug group (B+D group) shows that the apoptosis rate of HCT116 cells induced by the B-D group is 52.4% and is far higher than that of cisplatin at the same concentration, and the effect of inducing apoptosis of cells and further influencing proliferation of tumor cells is shown by the berberine coupled cisplatin compound prepared by the embodiment 1 of the invention after the treatment of cells as shown in the table 3 and the figure 7.
TABLE 3 Annexin V/PI double-staining method for detecting apoptosis of different drug-treated groups
| HCT116 cells | Control | Ber(μM) | DDP(μM) | B+D(μM) | B-D(μM) |
| Apoptosis rate% | 5.33 | 4.93 | 7.82 | 61.3 | 4.92 |
Test example 7
The uptake of berberine coupled cisplatin compounds prepared in example 1 of the present invention on B-D by human colon cancer HCT116 and SW620 cells and human normal intestinal epithelial cell HIEC-6 was determined by inductively coupled plasma mass spectrometry (ICP-MS) experimental method. Treating HCT116 cells, SW620 cells with a control group (control), a berberine-coupled cisplatin compound administration group (B-D group), a cisplatin single administration group (DDP group) and a berberine-cisplatin combined administration group (B+D group) for 6 hours; treating HIEC-6 cells with a control group (control), a berberine-coupled cisplatin compound administration group (B-D group), and a cisplatin alone administration group (DDP group) for 6 hr; the intracellular platinum accumulation amount of the cells is detected by ICP-MS experiments, the experimental results are shown in table 4, fig. 8 and fig. 9, and the intake of the tested tumor cells to B-D is obviously higher than that of cisplatin, which is 13.27 times and 6.85 times of that of the cells respectively; and normal cells also have a large difference in platinum uptake for B-D and cisplatin. The test result shows that the large increase of the accumulation amount of the berberine coupled cisplatin compound prepared in the embodiment 1 of the invention in HCT116 and SW620 cells is one of the important reasons for the enhanced cytotoxicity of B-D; and the selectivity in tumor cells is related to the difference between the uptake and intracellular accumulation of tumor cells and normal cells.
TABLE 4 platinum content in cells after treatment of HCT116, SW620 and HIEC-6 cells with different drug treatment groups
Test example 8
Nude mice with transplants were continuously dosed for 16 days with a blank (control), cisplatin alone (DDP) and berberine coupled cisplatin compound (B-D), and body weight statistics (D in fig. 9) and nude mice transplant tumor growth, including volume statistics (a in fig. 10) and weight statistics (c in fig. 10) of the transplants were recorded, and nude mice were sacrificed 16 days after dosing and photographed by dissecting the transplants (B in fig. 10); analysis a in fig. 10 shows that the control tumor volume grew rapidly, while the DDP and B-D tumor volumes were also increasing, the tumor growth was significantly inhibited; analysis from c in fig. 10 shows that DDP group treated mice inhibited tumor growth to some extent, while B-D group significantly inhibited tumor growth, with statistical significance being analyzed by two-way variance using GraphPad Prism software. Statistical significance is expressed as p <0.05, p <0.01 and p <0.001. Statistical results show that B-D has obvious effect of inhibiting the growth of tumor cells in vivo; and the weight statistical graph of the mice (shown as D in fig. 10) shows that the weight of the mice in the DDP group has a slight decrease trend, but the mice in the B-D group have no obvious change, and the safety of the B-D in the mice is reflected to a certain degree; analysis from B in fig. 10 shows that the tumor volumes of DDP and B-D groups were significantly smaller than the control group and the B-D group tumor volumes were significantly smaller than the DDP group 16 days after dosing, and the differences were statistically significant.
In conclusion, the berberine-coupled cisplatin compound provided by the invention has three aspects of enhancing the inhibition activity of intestinal cancer cells, reducing the toxicity to normal cells and inhibiting tumors in nude mice.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A berberine coupled cisplatin compound having a structure as shown in formula I:
2. the method for preparing berberine-coupled cisplatin compound as claimed in claim 1, comprising the steps of:
carrying out hydroxylation reaction on berberine to obtain hydroxylated berberine;
carrying out nucleophilic substitution reaction on the hydroxylated berberine, butyl bromoacetate and an organic alkaline catalyst in a first polar organic solvent to obtain berberine with protected hydroxyl;
deprotection and carboxylation reaction are carried out on the berberine with protected hydroxyl and trifluoroacetic acid in a second polar organic solvent, so as to obtain carboxylated berberine;
carrying out oxidation reaction on cisplatin and hydrogen peroxide to obtain tetravalent cisplatin;
and (3) carrying out condensation reaction on the carboxylated berberine, tetravalent cisplatin and a condensation reaction catalyst in a polar organic solvent of the condensation reaction to obtain the berberine coupling cisplatin compound.
3. The preparation method according to claim 2, wherein the molar ratio of the hydroxylated berberine to the bromobutyl acetate to the organic alkaline catalyst is 2 (2.4-3): 2-4.
4. A process according to claim 2 or 3, wherein the organic basic catalyst is N, N-diisopropylethylamine and/or triethylamine; the nucleophilic substitution reaction is carried out at room temperature for 8-12 h.
5. The preparation method according to claim 2, wherein the mass ratio of the hydroxyl-protected berberine to the trifluoroacetic acid is (50-100) mg:1mL;
the volume ratio of the trifluoroacetic acid to the second polar organic solvent is 1 (2-3).
6. The method of claim 2, wherein the molar ratio of carboxylated berberine to tetravalent cisplatin is 6:5.
7. The preparation method according to claim 2, wherein the condensation reaction catalyst is triethylamine and O-benzotriazol-N, N' -tetramethylurea tetrafluoroboric acid;
the mol ratio of the triethylamine to the O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroboric acid is (1-1.5): (1-1.5).
8. The method according to any one of claims 2, 6 and 7, wherein the condensation reaction is carried out at a temperature of 60 ℃ for a period of 12 hours.
9. The method of claim 2, wherein the first polar organic solvent and the second polar organic solvent are independently dichloromethane, N-dimethylformamide or acetonitrile; the polar organic solvent of the condensation reaction is DMSO or N, N-dimethylformamide.
10. The application of the berberine-coupled cisplatin compound as claimed in claim 1 or the berberine-coupled cisplatin compound obtained by the preparation method as claimed in claims 2-9 in preparing anti-intestinal cancer drugs.
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| AU2018316175B2 (en) * | 2017-08-11 | 2023-02-23 | The Regents Of The University Of Michigan | Inhibitors of MEK/PI3K, JAK/MEK, JAK/PI3K/mTOR and MEK/PI3K3k/mTOR biological pathways and methods for improving lymphatic uptake, bioavailability, and solubility of therapeutic compounds |
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| CN105622674A (en) * | 2016-02-29 | 2016-06-01 | 东南大学 | Tetravalent platinum complex with bioactive group and preparation method of tetravalent platinum complex |
| WO2020042996A1 (en) * | 2018-08-29 | 2020-03-05 | 中国科学院上海药物研究所 | Oxaliplatin coupled prodrug, and preparation method and use thereof |
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