CN110251685B - Synthesis method and application of taxol-berberine nano-drug - Google Patents
Synthesis method and application of taxol-berberine nano-drug Download PDFInfo
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- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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- A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention discloses a synthesis method of a paclitaxel-berberine nano-drug, which realizes the self-assembly of a coupled drug by designing a paclitaxel-berberine coupled drug molecule containing a disulfide bond to obtain the anticancer nano-drug. The paclitaxel-berberine nano-drug GSH provided by the invention has sensitive response, can realize the rapid release of the drug at the tumor part, improves the utilization efficiency of the drug, and shows obvious cancer cell inhibition effect on various cancer cells.
Description
Technical Field
The invention relates to a drug synthesis technology, in particular to a GSH (glutathione) responsive taxol-berberine nano-drug as well as a preparation method and application thereof.
Background
Cancer mortality is becoming more and more severe worldwide. Many scientists have made outstanding contributions in the work of combating cancer. Among the numerous treatments, chemotherapy remains the primary cancer treatment. However, most anticancer drugs face obstacles such as low solubility, large side effects, and multidrug resistance before reaching the focal region. In order to solve the above problems, nano drug delivery systems such as micelles, liposomes, dendrimers, and vesicles have received much attention. However, the preparation of nanocarriers is extremely complex, and degradation, metabolism and excretion of nanocarriers can cause significant toxicity problems. Therefore, it is very important to develop nano-drug formulations without toxic solvents or nano-carriers.
Paclitaxel is a complex natural product separated and extracted from the bark of taxus brevifolia, can inhibit the growth of cancer cells by blocking the normal division of the cells, and is a first-line medicine for the current clinical chemotherapeutics. Clinical studies have confirmed that paclitaxel is mainly suitable for ovarian cancer, breast cancer and non-small cell lung cancer, and has certain therapeutic effects on esophageal cancer, head and neck cancer and other malignant tumors. However, paclitaxel has poor water solubility, which makes administration difficult, and greatly limits the therapeutic application of this natural active product to tumors. The polyoxyethylene castor oil and ethanol are used as cosolvent clinically, however, the polyoxyethylene castor oil may cause anaphylactic reaction of patients, and the use of the polyoxyethylene castor oil reduces the anticancer activity of paclitaxel. Meanwhile, the paclitaxel has the defects of strong toxic and side effects, easy generation of drug resistance, incapability of penetrating through a blood brain barrier and the like due to poor selectivity.
Berberine is a water-soluble isoquinoline alkaloid which has been found to be effective against diarrhea, diabetes, metabolic syndrome, polycystic ovary syndrome, coronary artery disease, hyperlipidemia, obesity, and fatty liver, among other diseases, in addition to having a superior antimicrobial effect. In recent years, many studies have found that berberine has a good anti-tumor effect, and can selectively accumulate in mitochondria of tumor cells, and induce the mitochondrial permeability of cancer cells to change by reducing the mitochondrial membrane potential, so that the cancer cells die.
The reduction-responsive drug carrier is a drug delivery system with molecules connected by disulfide bonds with reduction responsiveness to form a nanostructure. Disulfide bonds are very stable at normal body temperature, pH, and oxidizing conditions of the human body, and are reduced to generate sulfhydryl groups in the presence of a certain amount of reducing agents such as glutathione reductase (GSH) or Dithiothreitol (DTT). An oxidation-reduction potential just exists inside and outside the cancer cell, the concentration (0.5-10 mmol/L) of glutathione in the cell is more than 200 times of that (2-20 mu mol/L) of glutathione outside the cell, and the concentration of glutathione outside the cell is not enough to reduce disulfide bonds. Therefore, the nano-drug containing disulfide bonds is reduced by GSH after entering target cells, and the connected disulfide bonds are broken to generate sulfydryl, so that the drug is effectively and quickly released.
The carrier-free nano-drug with tumor targeting property is formed by self-assembling the hydrophobic drug paclitaxel and the hydrophilic drug berberine through disulfide bonds in water, so that the anti-tumor effect is achieved, and more importantly, a plurality of clinical safety problems caused by the nano-carrier are solved.
Disclosure of Invention
The purpose of the invention is as follows: in view of the above problems of the existing cancer treatments and drug carriers, an object of the present application is to provide a paclitaxel-berberine conjugate nano-drug; the second purpose of the invention is to provide a preparation method of the paclitaxel-berberine coupling nano-drug; the invention also aims to provide the application of the paclitaxel-berberine conjugate nano-medicament.
The technical scheme is as follows: the synthesis method of the taxol-berberine nano-medicament comprises the following steps:
(1) taking berberine hydrochloride, and obtaining demethylberberine under the conditions of high temperature and vacuum;
(2) taking demethyl berberine to react with bromoethanol to obtain hydroxyethyl substituted demethyl berberine;
(3) dissolving hydroxyethyl-substituted demethylberberine in methanol, adding methanol solution of sodium borohydride, and reacting to obtain reduced hydroxyethyl berberine;
(4) reacting reduced hydroxyethyl berberine with dithiodipropionic acid under the conditions that 4-Dimethylaminopyridine (DMAP) is taken as a catalyst and Dicyclohexylcarbodiimide (DCC) is taken as a condensing agent to obtain the dithiodipropionic acid berberine;
(5) reacting the berberine dithiodipropionate with the taxol to obtain a taxol-berberine coupling prodrug;
(6) reacting the paclitaxel-berberine conjugate prodrug with N-bromosuccinimide (NBS) to obtain a paclitaxel-berberine conjugate drug;
(7) dissolving the paclitaxel-berberine conjugate drug in an organic solvent, dripping into poor solvent water, performing ultrasonic treatment, and blow-drying the organic solvent to obtain the paclitaxel-berberine nano drug.
In the step (3), hydroxyethyl-substituted demethyl berberine is dissolved in methanol, then a methanol solution of sodium borohydride is slowly added, and the reaction is carried out for 12-24 hours in ice bath to obtain the reduced hydroxyethyl berberine. Wherein the mass ratio of the hydroxyethyl-substituted demethylberberine to the sodium borohydride is 1: 1-2.
In the step (4), firstly, dissolving dithiodipropionic acid, N' -dicyclohexylcarbodiimide and 4-dimethylaminopyridine in pyridine in sequence, stirring for 15-30 minutes under the ice bath inert gas condition to obtain a first mixed solution, then slowly adding the reduced hydroxyethyl berberine dichloromethane solution into the first mixed solution, and reacting for 24-48 hours under the ice bath condition to obtain dithiodipropionic acid berberine.
In the step (4), the amount of dithiodipropionic acid is 1-2 times of that of reduced hydroxyethyl berberine.
In the step (4), the amount of DCC is 1-2 times of the amount of the reduced hydroxyethyl berberine.
In the step (5), the mass ratio of the dithiodipropionic acid berberine to the paclitaxel is 1: 1.
Further, in the step (5), 4-dimethylaminopyridine is used as a catalyst, N' -dicyclohexylcarbodiimide is used as a condensing agent, and the amount of DCC is 1-2 times that of berberine dithiodipropionate.
In the step (6), the paclitaxel-berberine conjugate prodrug is dissolved in a chloroform solution, stirred for 30min, then slowly added into the chloroform solution of NBS, and continuously reacted for 24 h.
In the step (6), the ratio of the paclitaxel-berberine conjugate prodrug to the NBS substance is 1: 1-1.2.
In the step (7), the reaction temperature is between room temperature and 50 ℃.
In the step (7), the organic solvent is selected from acetone, isopropanol, methanol, ethanol, pyridine, dimethyl sulfoxide or N, N-dimethylformamide.
In the step (7), the temperature of the ultrasound is room temperature, the power of the ultrasound is 300W, and the time of the ultrasound is 10-60 min.
The nano-drug prepared by the method and the application thereof in preparing anti-cancer drugs are also within the protection scope of the invention. Wherein the tumor comprises lung cancer and liver cancer.
Has the advantages that: in the co-assembled GSH response type taxol-berberine nano-medicament prepared by the invention, the hydrophobic medicament is taxol, the hydrophilic medicament is berberine, and by utilizing the special structure of taxol-berberine molecules, the water solubility of the hydrophobic medicament is improved, and the newly obtained molecules can form nano-particles in aqueous solution, so that the passive targeting property of the medicament is improved, the medicament effect is enhanced, and possible side effects are reduced. The new drug molecule designed by the invention has determined result, is easy to repeat and characterize, and has better treatment effect.
Drawings
FIG. 1 is a hydrogen spectrum of hydroxyethyl substituted berberine;
FIG. 2 is a high resolution mass spectrum of hydroxyethyl substituted berberine;
FIG. 3 is a hydrogen spectrum of reduced hydroxyethylberberine;
FIG. 4 is a high resolution mass spectrum of reduced hydroxyethylberberine;
FIG. 5 is a hydrogen spectrum of berberine dithiodipropionate;
FIG. 6 is a high resolution mass spectrum of berberine dithiodipropionate;
FIG. 7 is a hydrogen spectrum of a paclitaxel-berberine conjugate prodrug;
FIG. 8 is a high resolution mass spectrum of a paclitaxel-berberine conjugate prodrug;
FIG. 9 shows the hydrogen spectrum of the paclitaxel-berberine conjugate drug;
FIG. 10 is a high resolution mass spectrum of paclitaxel-berberine conjugate drugs;
FIG. 11 is a transmission electron microscope image of paclitaxel-berberine conjugate nano-drug;
FIG. 12 is a graph showing the distribution of the particle size of paclitaxel-berberine conjugate nano-drugs;
FIG. 13 is a graph of in vitro cytotoxicity (MTT) against A549 of paclitaxel-berberine conjugate nano-drug and paclitaxel with drug concentration on the abscissa and cancer cell survival rate on the ordinate;
FIG. 14 is a graph of cumulative release of paclitaxel-berberine conjugate nano-drug in PBS at 37 deg.C in the presence/absence of 10mM Glutathione (GSH), with time on the abscissa and cumulative drug release on the ordinate.
Detailed Description
For the clear understanding of the technical solutions of the present invention, the technical solutions of the present invention are further described in detail below with reference to the accompanying drawings and examples. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
EXAMPLE 1 Synthesis of demethylberberine
Adding berberine hydrochloride 10g into 250mL round bottom flask, heating at 190 deg.C under 30mmHg for 60min to change yellow solid powder into dark red powder gradually to obtain demethylberberine crude product. Column chromatography (eluent: chloroform/methanol 20/1) to obtain dark red demethylberberine powder 6.92g with yield 80%.
EXAMPLE 2 Synthesis of hydroxyethyl-substituted Berberine
Into a 250mL round-bottom flask were added 10g (31.14mmol) of desmethylberberine prepared in example 1, and acetonitrile (CH) 3 CN)20mL, potassium carbonate 5.16g (37.37mmol), stirring at 50 ℃ for 30min, then adding bromoethanol 4.67g (37.37mmol), heating under reflux for 24h, column chromatography (elution machine: chloroform/methanol 20/1) to yield 12.2g yellow hydroxyethyl-substituted berberine powder in 88% yield. 1 H NMR(600MHz，MeOD)δ9.95(s，1H)，8.62(s，1H)，8.05(d， J＝9.1Hz，1H)，8.00(d，J＝9.0Hz，1H)，7.59(s，1H)，6.93(s，1H)，6.13(s，2H)，4.99-4.93(m， 2H)，4.54-4.50(m，2H)，4.12(s，3H)，4.00-3.96(m，2H)，3.31-3.26(m，2H)；ESI-MS m/z [M+H] + 366.13315. Fig. 1 and 2 are a hydrogen spectrum and a high-resolution mass spectrum of hydroxyethyl-substituted berberine, respectively, which demonstrate the success of preparing the compound.
Example 3 Synthesis of reduced hydroxyethyl Berberine
Into a 250mL round bottom flask were added 10g (22.47mmol) of the hydroxyethylberberine prepared in example 2, and methanol (CH) 3 OH)20mL, stirring in an ice bath, and then adding sodium borohydride (NaBH) 4 ) (1.28g, 33.71mmol) in methanol for 12h to give a crude reduced hydroxyethylberberine product which is purified by column chromatography (eluent: chloroform/methanol 20/1) to obtain white reduced hydroxyethyl berberine powder 8.29g with yield 95%. 1 H NMR(600MHz，CDCl3)δ6.88(d，J＝8.4Hz， 1H)，6.79(d，J＝8.4Hz，1H)，6.72(s，1H)，6.59(s，1H)，5.91(s，2H)，4.26(d，J＝15.5Hz，1H)，4.16 -4.12(m，1H)，4.04(dd，J＝10.3，5.8Hz，1H)，3.88-3.81(m，5H)，3.57-3.51(m，2H)，3.22(dd， J＝15.9，3.3Hz，1H)，3.17(dd，J＝10.4，4.7Hz，1H)，3.10(d，J＝11.2Hz，1H)，2.82(dd，J＝15.5， 11.6Hz，1H)，2.63(dd，J＝21.8，10.6Hz，2H)；ESI-MS m/z[M+H]And 370.16589. FIGS. 3 and 4 are the hydrogen and high resolution mass spectra of reduced hydroxyethylberberine, respectively, demonstrating the success of the preparation of the compound.
EXAMPLE 4 Synthesis of Berberine Dithiodipropionate
2.84g (13.54mmol) of dithiodipropionic acid, a catalytic amount of 4-Dimethylaminopyridine (DMAP), Dicyclohexylcarbodiimide (DCC) (2.80g, 13.54mmol) and 20mL of pyridine (pydine) are added into a 100mL round-bottom flask, stirred under the protection of nitrogen, stirred for 30min under ice bath, a dichloromethane solution of reduced hydroxyethyl berberine (5g, 13.54mmol) is added into the reaction system, and the reaction is continued for 48h under the ice bath to obtain a crude product of the dithiodipropionic acid berberine, the solvent pyridine is removed by reduced pressure distillation, and then column chromatography (eluent: chloroform/methanol ═ 20/1) is carried out for separation to obtain 4.18g of reduced dithiodipropionic acid berberine powder with the yield of 55%. 1 H NMR(600MHz，MeOD)δ7.03(s，2H)，6.91(s，1H)，6.71(s，1H)，5.96(s，2H)，4.47(s， 1H)，4.42-4.37(m，2H)，4.35-4.30(m，2H)，3.86(d，J＝3.7Hz，3H)，3.72(s，1H)，3.64(dd，J＝ 17.0，4.3Hz，1H)，3.37(s，1H)，3.22(s，1H)，3.11-3.09(m，1H)，3.07-2.99(m，2H)，2.96(dd，J＝ 13.5，6.2Hz，4H)，2.82(t，J＝6.9Hz，2H)，2.66(t，J＝7.0Hz，2H)，2.59(t，J＝6.1Hz，1H)； ESI-MS m/z[M+H]And 562.15688. Fig. 5 and fig. 6 are the hydrogen spectrogram and the high-resolution mass spectrogram of the berberine dithiodipropionate, respectively, which prove the success of the preparation of the compound.
EXAMPLE 5 Synthesis of Berberine Dithiodipropionate
5.69g (27.08mmol) of dithiodipropionic acid, a catalytic amount of 4-Dimethylaminopyridine (DMAP), Dicyclohexylcarbodiimide (DCC) (2.80g, 13.54mmol) and 20mL of pyridine (pydine) are added into a 100mL round-bottomed flask, the mixture is stirred for 30min under ice bath with protection of nitrogen, a dichloromethane solution of reduced hydroxyethyl berberine (5g, 13.54mmol) is added into the reaction system, the reaction is continued for 48h under ice bath to obtain a crude product of the dithiodipropionic acid berberine, the solvent pyridine is removed by reduced pressure distillation, and then column chromatography (eluent: chloroform/methanol ═ 20/1) is carried out for separation to obtain 4.79g of reduced dithiodipropionic acid berberine powder with the yield of 63%.
EXAMPLE 6 Synthesis of Berberine Dithiodipropionate
5.69g (27.08mmol) of dithiodipropionic acid, a catalytic amount of 4-Dimethylaminopyridine (DMAP), Dicyclohexylcarbodiimide (DCC) (2.80g, 27.08mmol) and 20mL of pyridine (pydine) are added into a 100mL round-bottom flask, stirred under nitrogen protection for 30min under ice bath, a dichloromethane solution of reduced hydroxyethyl berberine (5g, 13.54mmol) is added into the reaction system, and the reaction is continued for 48h under ice bath to obtain a crude product of the dithiodipropionic acid berberine, the solvent pyridine is removed by reduced pressure distillation, and then column chromatography (eluent: chloroform/methanol ═ 20/1) is carried out for separation to obtain 6.08g of reduced dithiodipropionic acid berberine powder with the yield of 80%.
EXAMPLE 7 Synthesis of paclitaxel-Berberine conjugate prodrug
1.7g (2.97mmol) of the berberine dithiodipropionate synthesized in example 6, a catalytic amount of 4-Dimethylaminopyridine (DMAP), Dicyclohexylcarbodiimide (DCC) (0.61g, 2.97mmol) and 10mL of pyridine (pydine) are added into a 100mL round-bottom flask, the mixture is stirred for 30min under ice bath under the protection of nitrogen, a dichloromethane solution of Paclitaxel (PTX) (2.54g, 2.97mmol) is added into the reaction system, the reaction is continued for 48h under ice bath to obtain a crude product of the paclitaxel-berberine coupling drug precursor, the solvent pyridine is removed by reduced pressure distillation, and then column chromatography (eluent: chloroform/methanol: 50/1) is carried out for separation to obtain 2.94g of the paclitaxel-berberine coupling drug precursor with the yield of 71%. 1 H NMR(600MHz，CDCl3)δ8.14(d，J＝7.6Hz，2H)，7.75(d，J＝7.2Hz， 2H)，7.61(t，J＝7.4Hz，1H)，7.55-7.48(m，3H)，7.44-7.37(m，6H)，7.34(d，J＝7.5Hz，1H)， 7.06(s，1H)，6.87(d，J＝8.4Hz，1H)，6.78(d，J＝8.3Hz，1H)，6.72(s，1H)，6.58(d，J＝6.5Hz，1H)， 6.29(s，1H)，6.24(t，J＝8.7Hz，1H)，5.98(d，J＝9.0Hz，1H)，5.91(s，2H)，5.68(d，J＝7.1Hz，1H)， 5.53(s，1H)，4.97(d，J＝9.0Hz，1H)，4.44(t，J＝8.5Hz，1H)，4.38(t，J＝9.4Hz，2H)，4.32(d，J＝ 8.5Hz，1H)，4.26(dd，J＝17.7，14.3Hz，2H)，4.22-4.16(m，2H)，3.81(d，J＝10.5Hz，4H)，3.51(s， 2H)，3.22(d，J＝15.0Hz，1H)，3.12(d，J＝24.4Hz，2H)，2.91(t，J＝7.0Hz，2H)，2.87(d，J＝5.1 Hz，2H)，2.85-2.75(m，4H)，2.65(d，J＝14.5Hz，2H)，2.54(d，J＝14.7Hz，2H)，2.45(s，3H)， 2.40-2.33(m，1H)，2.22(s，3H)，2.19-2.13(m，1H)，1.94(s，3H)，1.91-1.81(m，2H)，1.68(s， 3H)，1.23(s，3H)，1.13(s，3H)；ESI-MS m/z[M+H]And 1397.47749. Fig. 7 and fig. 8 are the hydrogen spectrum and high resolution mass spectrum of paclitaxel-berberine conjugate prodrug, respectively, which demonstrate the success of the preparation of the compound.
EXAMPLE 8 Synthesis of paclitaxel-Berberine conjugate prodrug
1.7g (2.97mmol) of the berberine dithiodipropionate synthesized in example 6, a catalytic amount of 4-Dimethylaminopyridine (DMAP), Dicyclohexylcarbodiimide (DCC) (1.22g, 5.94mmol) and 10mL of pyridine (pydine) are added into a 100mL round-bottomed flask, the mixture is stirred for 30min under ice bath under the protection of nitrogen, a dichloromethane solution of Paclitaxel (PTX) (2.54g, 2.97mmol) is added into the reaction system, the reaction is continued for 48h under ice bath to obtain a crude paclitaxel-berberine coupling drug precursor, the solvent pyridine is removed by reduced pressure distillation, and then column chromatography (eluent: chloroform/methanol ═ 50/1) is carried out for separation to obtain 3.52g of the paclitaxel-berberine coupling drug precursor with the yield of 85%.
EXAMPLE 9 Synthesis of paclitaxel-Berberine conjugate drugs
2g (1.43 mmol) of the paclitaxel-berberine conjugate drug precursor synthesized in example 8 and 20mL of chloroform (CHCl3) are added into a 100mL round-bottom flask, stirred at room temperature for 30min, then a chloroform solution of N-bromosuccinimide (NBS) (254.52mg, 1.43mmol) is added into the reaction system, and the reaction is continued at room temperature for 24h to obtain a crude product of the paclitaxel-berberine conjugate drug, and the crude product is separated by column chromatography (eluent: chloroform/methanol ═ 10/1) to obtain 1.09g of the paclitaxel-berberine conjugate drug with the yield of 52%. 1 H NMR(600MHz，MeOD)δ9.75(s，1H)，8.70(s，1H)，8.10(d，J＝8.4Hz，3H)，8.00(d， J＝9.1Hz，1H)，7.79(d，J＝8.5Hz，2H)，7.67(d，J＝8.6Hz，1H)，7.65(s，1H)，7.60(d，J＝7.9Hz， 2H)，7.52(d，J＝7.5Hz，1H)，7.47-7.43(m，6H)，7.26(t，J＝7.2Hz，1H)，6.94(s，1H)，6.40(s， 1H)，6.10(s，2H)，6.01(d，J＝10.1Hz，1H)，5.78(d，J＝6.8Hz，1H)，5.61(d，J＝7.2Hz，1H)，5.46 (d，J＝6.7Hz，1H)，4.97(d，J＝7.9Hz，1H)，4.94-4.91(m，2H)，4.67(t，J＝4.0Hz，2H)，4.56(d，J ＝8.2Hz，2H)，4.29(d，J＝11.0Hz，1H)，4.16(s，2H)，4.10(s，3H)，3.76(d，J＝7.2Hz，1H)，3.27- 3.24(m，2H)，2.86(d，J＝6.3Hz，2H)，2.81(dd，J＝14.5，8.1Hz，4H)，2.67(t，J＝6.8Hz，2H)，2.37 (s，3H)，2.13(s，3H)，1.87(s，2H)，1.76(dd，J＝14.6，4.6Hz，2H)，1.63(s，3H)，1.28(s，3H)，1.12(s， 3H)，1.09(s，3H)，0.90(t，J＝7.0Hz，1H)；ESI-MS m/z[M+H]And 1393.44253. FIGS. 9 and 10 are the hydrogen spectrum and high-resolution mass spectrum of paclitaxel-berberine conjugate drug, respectively, which demonstrate the success of the preparation of the compound.
EXAMPLE 10 Synthesis of paclitaxel-Berberine conjugate drugs
Into a 100mL round-bottomed flask were added 2g (1.43 mmol) of the paclitaxel-berberine conjugate prodrug synthesized in example 8, chloroform (CHCl) 3 )20mL, stirring at room temperature for 30min, adding a chloroform solution of N-bromosuccinimide (NBS) (306.14mg, 1.72mmol) into the reaction system, reacting at room temperature for 24h to obtain crude product of paclitaxel-berberine conjugate, performing column chromatography (eluent: chloroform/methanol 10/1) to obtain 0.94g of paclitaxel-berberine conjugate drug with 45% yield.
EXAMPLE 11 Synthesis of paclitaxel-Berberine conjugate drugs
Into a 100mL round-bottomed flask were added 2g (1.43 mmol) of the paclitaxel-berberine conjugate prodrug synthesized in example 8, chloroform (CHCl) 3 )20mL, stirring at room temperature for 30min, adding chloroform solution of N-bromosuccinimide (NBS) (254.52mg, 1.43mmol) into the reaction system, reacting at 60 deg.C for 24h to obtain crude product of paclitaxel-berberine conjugate, performing column chromatography (eluent: chloroform/methanol 10/1) to obtain 1.46g of paclitaxel-berberine conjugate drug with 70% yield.
EXAMPLE 12 preparation of Taxol-Berberine Nanomedicine
2mg of the paclitaxel-berberine conjugate drug synthesized in example 11 is dissolved in 1mL of acetone, and then the solution is dropped into 10mL of deionized water and stirred, and ultrasonic treatment is carried out at the power of 300W for 30min at room temperature, and the organic solvent in the solution is blown off, so as to obtain the paclitaxel-berberine nano drug.
EXAMPLE 13 preparation of Taxol-Berberine Nanomedicine
2mg of the paclitaxel-berberine conjugate drug synthesized in example 11 is dissolved in 1mL of acetone, and then the solution is dropped into 10mL of deionized water and stirred, and the organic solvent in the solution is blown off by ultrasonic waves with 300W power for 45min at room temperature, so that the paclitaxel-berberine nano drug is obtained.
EXAMPLE 14 preparation of Taxol-Berberine Nanomedicine
2mg of the paclitaxel-berberine conjugate drug synthesized in example 11 is dissolved in 1mL of acetone, and then the solution is dropped into 10mL of deionized water and stirred, and the organic solvent in the solution is blown off by ultrasonic treatment at the power of 300W for 60min at room temperature, so that the paclitaxel-berberine nano drug is obtained.
Example 15 preparation of Taxol-Berberine Nanomedicine
2mg of the paclitaxel-berberine conjugate drug synthesized in example 11 is dissolved in 1mL of isopropanol, then dropped into 10mL of deionized water and stirred, and the organic solvent in the solution is blown off by ultrasonic treatment at 300W power for 60min at room temperature, so as to obtain the paclitaxel-berberine nano drug.
EXAMPLE 16 preparation of Taxol-Berberine Nanomedicine
2mg of the paclitaxel-berberine conjugate drug synthesized in example 11 is dissolved in 1mL of methanol, then the solution is dropped into 10mL of deionized water and stirred, and the organic solvent in the solution is blown off by ultrasonic treatment at 300W power for 60min at room temperature, so that the paclitaxel-berberine nano drug is obtained.
Example 17 related characterization of paclitaxel-berberine Nanoparticulates
The morphology of the nanoparticles of example 16 was measured by transmission electron microscopy and showed that all nanoparticles showed a rod-like structure. The Dynamic Light Scattering (DLS) method detects the particle size of the nanoparticles in example 16, and the result shows that the average particle size of all the nanoparticles is 100-250 nm. FIG. 11 is the nanoparticle morphology, with regular rod-like structure, and FIG. 12 is the average particle size of paclitaxel-berberine nanoparticles measured by dynamic light scattering method, which is 165 nm.
Example 18 MTT method cancer cell viability assay
Inoculating A549 human lung cancer cell in 96-well culture plate with 5% CO 2 After culturing for 24h in an incubator at 37 ℃, adding different concentrations into each holeThe paclitaxel-berberine nano-drug solution and the paclitaxel solution of example 16 are 100 μ L each, so that the final drug concentrations are 0.625, 1.25, 2.5, 5, 10, 20 and 40 μ M, respectively, and the culture is continued for 24 h; separately 50. mu.L of MTT was added, followed by 5% CO 2 Continuously culturing for 4h in an incubator at 37 ℃, removing the culture medium, adding 150 mu L of DMSO, shaking uniformly on a plate shaker, reading a plate at 495nm by an enzyme-labeling instrument, and calculating the cell inhibition rate according to the measured absorbance value. Data are presented as mean ± SD (n-3).
As a result, it was found (as shown in FIG. 13) that paclitaxel-berberine nano-drug (IC) was present after 24 hours of culture 50 The inhibition effect of 1.29 mu M on A549 is better than that of taxol (IC) 50 ＝1.55μM)。
Example 19 paclitaxel-berberine Nanoparticulate Release test
The release of paclitaxel and berberine from the nano-drug was measured by dialysis. The paclitaxel-berberine nano-drug of example 16 was diluted with Phosphate Buffered Saline (PBS) to give a 60 μ M solution. 1mL of the solution was taken into a dialysis bag (MwCO: 2000), and dialyzed for 55 hours in 30mL of PBS or a PBS buffer solution containing 10mM glutathione. A2 mL sample was taken from the dialysis bag for fluorescence detection (excitation wavelength: 380nm) at a predetermined time, and 2mL of PBS was supplemented.
As a result, it was found (as shown in FIG. 14) that the paclitaxel-berberine nano-drug is very stable in a solution without glutathione. In contrast, in the presence of glutathione, around 50% of the drug is rapidly released within 10 hours.
Claims (10)
1. A synthesis method of paclitaxel-berberine nano-drugs is characterized by comprising the following steps:
(1) taking berberine hydrochloride, and obtaining demethylberberine under the conditions of high temperature and vacuum;
(2) taking demethyl berberine to react with bromoethanol to obtain hydroxyethyl substituted demethyl berberine;
(3) dissolving hydroxyethyl-substituted demethyl berberine in methanol, adding a methanol solution of sodium borohydride, and reacting to obtain reduced hydroxyethyl berberine;
(4) taking reduced hydroxyethyl berberine and dithiodipropionic acid to react under the conditions that 4-dimethylaminopyridine is taken as a catalyst and N, N' -dicyclohexylcarbodiimide is taken as a condensing agent to obtain the dithiodipropionic acid berberine;
(5) reacting the berberine dithiodipropionate with the taxol to obtain a taxol-berberine coupling prodrug;
(6) reacting the paclitaxel-berberine conjugate prodrug with N-bromosuccinimide to obtain a paclitaxel-berberine conjugate drug;
(7) dissolving the paclitaxel-berberine conjugate drug in an organic solvent, dripping into poor solvent water, performing ultrasonic treatment, and blow-drying the organic solvent to obtain the paclitaxel-berberine nano drug.
2. The synthesis method according to claim 1, wherein in the step (3), the reaction is carried out in an ice bath, and the mass ratio of the hydroxyethyl-substituted demethylberberine to sodium borohydride is 1: 1-2.
3. The synthesis method according to claim 1, wherein in the step (4), dithiodipropionic acid, N' -dicyclohexylcarbodiimide and 4-dimethylaminopyridine are dissolved in pyridine in sequence, the mixture is stirred for 15-30 minutes under an ice bath inert gas condition to obtain a first mixed solution, then a reduced hydroxyethyl berberine dichloromethane solution is slowly added into the first mixed solution, and the mixture reacts for 24-48 hours under an ice bath condition to obtain dithiodipropionic acid berberine.
4. The method according to claim 1, wherein in the step (4), the amount of dithiodipropionic acid is 1-2 times that of reduced hydroxyethyl berberine.
5. The method according to claim 1, wherein in the step (4), the amount of N, N' -dicyclohexylcarbodiimide substance is 1 to 2 times that of the reduced hydroxyethylberberine.
6. The method according to claim 1, wherein in step (5), the ratio of the amounts of the substances of berberine dithiodipropionate and paclitaxel is 1: 1.
7. The synthesis method according to claim 1, wherein in the step (6), the paclitaxel-berberine conjugate prodrug is dissolved in a chloroform solution, stirred for 30-60 min, and then slowly added into the chloroform solution of N-bromosuccinimide to continue the reaction for 24-48 h.
8. The method of claim 1, wherein in step (6), the ratio of the amount of paclitaxel-berberine conjugate prodrug to N-bromosuccinimide is 1: 1-1.2.
9. The method according to claim 1, wherein in the step (7), the organic solvent is selected from acetone, isopropanol, methanol, ethanol, pyridine, dimethyl sulfoxide and N, N-dimethylformamide.
10. Use of the nano-drug prepared by the method of any one of claims 1 to 9 in the preparation of an anti-lung cancer drug.
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