WO2022258035A1 - 一种胆碱碳酸酯类前药及其制备方法和应用 - Google Patents

一种胆碱碳酸酯类前药及其制备方法和应用 Download PDF

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WO2022258035A1
WO2022258035A1 PCT/CN2022/098013 CN2022098013W WO2022258035A1 WO 2022258035 A1 WO2022258035 A1 WO 2022258035A1 CN 2022098013 W CN2022098013 W CN 2022098013W WO 2022258035 A1 WO2022258035 A1 WO 2022258035A1
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prodrug
triptolide
drug
choline
carbonate
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French (fr)
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刘敏
张芷依
王睿峰
陆伟跃
谢操
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复旦大学
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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • 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
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/41—Preparation of salts of carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/41—Preparation of salts of carboxylic acids
    • C07C51/412—Preparation of salts of carboxylic acids by conversion of the acids, their salts, esters or anhydrides with the same carboxylic acid part
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C53/00—Saturated compounds having only one carboxyl group bound to an acyclic carbon atom or hydrogen
    • C07C53/08—Acetic acid
    • C07C53/10—Salts thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/22—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms

Definitions

  • the invention belongs to the field of pharmacy, and relates to a class of choline carbonate prodrugs that can release the original drug through the conversion of plasma cholinesterase in vivo, in particular to a tripterygium wilfordii prodrug, its preparation method and its application in tumor treatment .
  • Pancreatic cancer is one of the common digestive system tumors, the incidence rate of which ranks ninth in the world, and the five-year survival rate of patients is less than 8%.
  • the reasons for the low survival rate of pancreatic cancer patients are the low early diagnosis rate, high degree of malignancy and poor treatment effect of pancreatic cancer.
  • chemotherapy is the first choice for clinical treatment of pancreatic cancer, which can be used for preoperative treatment to shrink tumors and postoperative treatment to eliminate residual tumor cells.
  • gemcitabine+nab-PTX and FOLFIRINOX have certain therapeutic effects on pancreatic cancer
  • other chemotherapy regimens have poor therapeutic effects on pancreatic cancer.
  • new drug treatments such as anti-angiogenic therapy, deinterstitial therapy, and immunotherapy, have all failed in the exploration of pancreatic cancer treatment. Therefore, the development of effective chemotherapeutic drugs for pancreatic cancer has important clinical significance.
  • Triptolide is an epoxidized diterpene lactone compound isolated from the plant Tripterygium wilfordii, which has multi-target mechanism of inducing apoptosis, interfering with cell cycle and inhibiting tumor angiogenesis. It is a broad-spectrum tumor suppressor. Although triptolide has good antitumor activity, its poor water solubility and serious side effects restrict its clinical application.
  • a prodrug refers to a compound that is inactive or low in activity in vitro after chemical structure modification, and releases the active original drug through enzymatic or non-enzymatic conversion in vivo to exert its medicinal effect.
  • prodrugs are mainly to increase the water solubility of the drug, improve the bioavailability of the drug, increase the stability of the drug, and reduce the toxicity and side effects.
  • prodrug design has attracted more and more attention in new drug research, and great progress has been made in nervous system drugs, antitumor drugs and antiviral drugs.
  • Plasma cholinesterase also known as pseudocholinesterase or butyrylcholinesterase, is a non-specific esterase synthesized in the liver and released into plasma. Plasma cholinesterase can hydrolyze various cholines such as acetylcholine, butyrylcholine and succinate choline, as well as many esters, peptides and amides.
  • the object of the present invention is to provide a kind of choline carbonate prodrug and its preparation method and application.
  • the present invention aims to provide a prodrug of triptolide, which can release triptolide in vivo through the conversion of plasma cholinesterase to play an anti-tumor effect, improve the water solubility of triptolide, and Reduced the toxic and side effects of triptolide.
  • the C14-hydroxyl of triptolide is esterified to synthesize triptolide choline carbonate, referred to as TD-1704.
  • TD-1704 exhibits good anti-tumor activity, low toxicity and side effects, and good water solubility, and has clinical application prospects.
  • the design of the choline carbonate prodrug can also provide new ideas for the preparation of prodrugs of other drugs.
  • TD-1704 refers to: triptolide choline carbonate acetate compound having the structure shown in formula III, or its solvate or polymorph:
  • AsPC-1 refers to: human metastatic pancreatic adenocarcinoma cells.
  • MIA PaCa-2 refers to: Human pancreatic cancer cells.
  • SGC-7901 refers to: human gastric adenocarcinoma cells.
  • PANC-1 refers to: human pancreatic cancer cells.
  • MTT method refers to the thiazolium blue colorimetric method.
  • PBS phosphate buffered saline
  • DIPEA Diisopropylethylamine
  • FBS means: fetal bovine serum
  • DMEM Dulbecco's Modified Eagle's Medium.
  • i.v. administration means: intravenous administration.
  • i.p. administration means: intraperitoneal administration.
  • TPL refers to: triptolide
  • the first aspect of the present invention provides a kind of choline carbonate prodrug, described choline carbonate prodrug is the compound with the structure shown in general formula I:
  • R-OH is an antineoplastic drug containing a hydroxyl group
  • the antineoplastic drug is selected from one or more of the following: triptolide, paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, podophyllotoxin, doxorubicin, epirubicin, daunorubicin, irinotecan, gemcitabine, vincristine, or vinblastine;
  • X- is pharmaceutically acceptable organic anion and inorganic anion, wherein, preferably:
  • the organic anion is selected from one or more of: formate, acetate, oxalate, succinate, fumarate, tartrate or citrate; and/or
  • the inorganic anion is selected from one or more of the following: hydrochloride, sulfate, bisulfate, sulfite, nitrate, carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, hydrogen bromate, hydroiodide, or borate.
  • the choline carbonate prodrug is a triptolide prodrug, preferably a compound having a structure shown in formula II, or a pharmaceutically acceptable Accepted salts, solvates or polymorphs:
  • Y - is a pharmaceutically acceptable organic anion and inorganic anion.
  • the organic anion is selected from one or more of the following: formate, acetate, oxalate, succinate, fumarate, citrate or tartrate; and/or
  • the inorganic anion is selected from one or more of the following: hydrochloride, sulfate, bisulfate, sulfite, nitrate, carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, hydrogen bromate, hydroiodide, or borate.
  • the choline carbonate prodrug of the first aspect of the present invention wherein, the choline carbonate prodrug is the acetate of triptolide prodrug, the compound having the structure shown in formula III, or its solvent Compounds or polymorphs:
  • a second aspect of the present invention provides a method for preparing the choline carbonate prodrug described in the first aspect, the method comprising:
  • the choline carbonate prodrug is obtained by modifying the hydroxyl group of the active site of the drug to obtain its choline carbonate;
  • the drug is an antineoplastic drug, an anti-infection drug, an anti-cardiovascular system disease drug, an anti-lymphatic system disease drug, an anti-immune system disease drug and/or an analgesic obtained by chemically obtaining its choline carbonate derivative drug.
  • the method according to the second aspect of the present invention comprises: reacting N,N-dimethylethanolamine with 4-(nitrophenyl) chloroformate to obtain 2-(dimethylamino)ethyl (4 -Nitrophenyl) carbonate, then carry out transesterification reaction with triptolide C14-position hydroxyl, obtain 2-(dimethylamino) ethyl triptolide carbonate, methyl iodide can prepare the obtained choline carbonate prodrugs.
  • a third aspect of the present invention provides a pharmaceutical composition comprising:
  • composition according to the third aspect of the present invention wherein, the pharmaceutical preparation form of the pharmaceutical composition is selected from one or more of the following: solution, tablet, capsule, granule, powder or drop pill.
  • the fourth aspect of the present invention provides the choline carbonate prodrug described in the first aspect, the alkali carbonate prodrug prepared according to the method described in the second aspect or the pharmaceutical composition described in the third aspect during preparation Application in antineoplastic drugs, anti-infective drugs, anti-cardiovascular system disease drugs, anti-lymphatic system disease drugs, anti-immune system disease drugs or analgesic drugs.
  • the choline carbonate prodrug has low activity in vitro and/or releases the original drug through conversion of plasma cholinesterase in vivo to exert antitumor effect. tumor effect.
  • the tumor is selected from one or more of the following: pancreatic cancer, gastric cancer, bile duct cancer, colon cancer, rectal cancer, liver cancer, breast cancer, ovarian cancer, non-small cell lung cancer or lymphoma.
  • a fifth aspect of the present invention provides a drug for treating cancer, infection, cardiovascular and cerebrovascular diseases, lymphatic system diseases and/or immune system diseases, said drug comprising: the choline carbonate pro-choline carbonate described in the first aspect Drug, alkali carbonate prodrug prepared according to the method described in the second aspect or the pharmaceutical composition described in the third aspect.
  • a sixth aspect of the present invention provides an analgesic drug, said drug comprising: the choline carbonate prodrug described in the first aspect, the alkali carbonate prodrug prepared according to the method described in the second aspect, or The pharmaceutical composition described in the third aspect.
  • the seventh aspect of the present invention provides a method for treating cancer, infection, cardiovascular and cerebrovascular diseases, lymphatic system diseases and/or immune system diseases, the method comprising: administering the first The choline carbonate prodrug according to one aspect, the alkali carbonate prodrug prepared according to the method described in the second aspect, or the pharmaceutical composition described in the third aspect.
  • the eighth aspect of the present invention provides a method for analgesia, the method comprising: administering the choline carbonate prodrug described in the first aspect to a subject in need, according to the method described in the second aspect
  • the base carbonate prodrug prepared by the method or the pharmaceutical composition described in the third aspect.
  • the present invention provides a design concept of choline carbonate prodrugs.
  • the invention provides a triptolide prodrug TD-1704.
  • the present invention also provides a preparation method of the triptolide biliary prodrug TD-1704 and its application in tumor treatment.
  • the prodrug synthesis method provided by the present invention is to obtain choline carbonate by modifying the hydroxyl group of the active site of the drug, thereby preparing prodrugs with different physicochemical properties.
  • Drugs can be anti-tumor drugs (such as triptolide, etc.), anti-infective drugs, anti-cardiovascular system disease drugs, anti-lymphatic system disease drugs, anti-immune system disease drugs obtained by chemical methods and analgesics, etc.
  • the triptolide prodrug TD-1704 is prepared by using the method provided by the invention.
  • TD-1704 can be administered intravenously for tumor treatment.
  • the experimental results of the present invention show that: TD-1704 can be quickly converted into the original drug triptolide under the action of plasma cholinesterase to play an anti-tumor effect. While improving the water solubility of triptolide, it retains a good It has excellent antitumor activity, reduces toxic and side effects, and has a good clinical application prospect in tumor treatment.
  • Triptolide prodrug TD-1704 was synthesized by chemical reaction, its purity was determined by HPLC, and its structure was characterized by MS and MRI.
  • TD-1704 after incubation with plasma cholinesterase and serum was investigated.
  • Nude mice bearing human pancreatic cancer cell PANC-1 orthotopic tumor model were injected with TD-1704 through tail vein, and the anti-tumor effect of TD-1704 was evaluated by survival time.
  • Nude mice bearing human gastric adenocarcinoma cell SGC-7901 subcutaneous tumor model were injected with TD-1704 through the tail vein, and the antitumor effect and safety of TD-1704 were evaluated by using tumor volume, tumor weight, serum liver function and serum renal function as indicators.
  • Nude mice bearing human-transferred pancreatic adenocarcinoma cell AsPC-1 orthotopic tumor model were injected with TD-1704 through the tail vein, and the anti-tumor effect of TD-1704 was evaluated by survival time.
  • Nude mice bearing human-transferred pancreatic adenocarcinoma cell AsPC-1 subcutaneous tumor model were injected with TD-1704 through the tail vein, and the antitumor effect of TD-1704 was evaluated by tumor volume and tumor weight.
  • TD-1704 Healthy mice were injected with TD-1704 through the tail vein, and the safety of TD-1704 was evaluated by blood routine, liver function, kidney function, pathological sections of major organs, and body weight.
  • the choline carbonate prodrugs of the present invention may have, but are not limited to, the following beneficial effects:
  • the choline carbonate prodrug of the present invention can be administered intravenously for tumor treatment.
  • the choline carbonate prodrug of the present invention can be rapidly converted into the original drug triptolide under the action of plasma cholinesterase so as to play an anti-tumor effect. While improving the water solubility of triptolide, it retains the It has good anti-tumor activity, reduces toxic and side effects, and has a good clinical application prospect in tumor treatment.
  • Figure 1 shows the chemical reaction diagram of TD-1704
  • Figure 1 shows the chemical reaction scheme used to prepare TD-1704.
  • FIG. 2 shows the HPLC and ESI-MS spectra of TD-1704
  • Chromatographic method Chromatographic column: Diamonsil@C18, 5 ⁇ m, 200 ⁇ 4.6mm; mobile phase: A: water (containing 0.1% acetic acid), B: acetonitrile (containing 0.1% acetic acid); elution program: 0 ⁇ 15min, 5% B ⁇ 50% B; flow rate 0.7mL/min; column temperature: 25°C; detection wavelength: 214nm, 254nm.
  • HPLC purity of the intermediate 2-(dimethylamino)ethyl triptolide carbonate is above 95%; ESI-MS: m/z[M+H] + is 476.2, consistent with the theoretical molecular weight.
  • Figure 3 shows the enzyme conversion reaction diagram of TD-1704 in vivo
  • Figure 3 shows the conversion process of TD-1704 into triptolide by plasma cholinesterase.
  • Figure 4 shows the degradation rate of TD-1704 in plasma cholinesterase and rat serum
  • Figure 4A shows the degradation rate of TD-1704 under the action of plasma cholinesterase
  • Figure 4B shows the degradation rate of TD-1704 in rat serum. The results showed that TD-1704 could be rapidly converted into triptolide in plasma cholinesterase and rat serum.
  • Figure 5 shows the growth inhibitory effect of TD-1704 on MIA PaCa-2, PANC-1, AsPC-1 and SGC-7901 cells before and after serum incubation
  • TD-1704 itself had low anti-tumor activity, but incubation with serum could significantly improve the anti-tumor activity of TD-1704.
  • Figure 6 shows the pharmacokinetic curves of triptolide and TD-1704 in rats
  • TD-1704 can be rapidly converted into triptolide in rats with a half-life of about 20 minutes.
  • Figure 7 shows the pharmacodynamic evaluation of human pancreatic cancer cell PANC-1 orthotopic tumor model nude mice
  • Figure 8 shows the pharmacodynamic evaluation of human gastric adenocarcinoma cell SGC-7901 subcutaneous tumor model in nude mice
  • Figure 8A shows the curve of tumor volume change over time of SGC-7901 subcutaneous tumor model nude mice
  • Figure 8B shows the comparison of tumor tissue weights of SGC-7901 subcutaneous tumor model nude mice on the 16th day after administration. The results showed that intravenous administration of TD-1704 could significantly inhibit the growth of tumor tissue in nude mice of SGC-7901 subcutaneous tumor model.
  • Figure 9 shows the TD-1704 safety evaluation
  • Figure 10 shows the pharmacodynamic evaluation of human metastatic pancreatic adenocarcinoma cell AsPC-1 orthotopic tumor model in nude mice
  • Figure 11 shows the pharmacodynamic evaluation of human metastatic pancreatic adenocarcinoma cell AsPC-1 subcutaneous tumor model in nude mice
  • Figure 11A shows the curve of the tumor volume of AsPC-1 subcutaneous tumor model nude mice over time;
  • Figure 11B shows the comparison of the tumor tissue weight of AsPC-1 subcutaneous tumor model nude mice on the 16th day after administration;
  • Figure 11C shows The in vivo and ex vivo photos of the tumor tissue of AsPC-1 subcutaneous tumor model nude mice are shown.
  • the tumor volume inhibition rate and tumor weight inhibition rate of triptolide group and TD-1704 low- and middle-dose groups were 65.0%/54.1%, 61.4%/57.7% and 99.9%/99.9%, respectively.
  • the results showed that intravenous administration of TD-1704 could significantly inhibit the growth of tumor tissue in AsPC-1 subcutaneous tumor model nude mice, and its antitumor activity was comparable to that of triptolide. The effect gradually increases.
  • Figure 12 and Figure 13 show the safety evaluation of TD-1704 in healthy mice.
  • Figure 12 shows the content of each index in the blood of healthy mice, specifically, Figure 12 shows red blood cell (RBC), white blood cell (WBC), hemoglobin (HGB), platelet (PLT), lymphocyte (Lymph ), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA) and blood urea nitrogen (UREA).
  • RBC red blood cell
  • WBC white blood cell
  • HGB hemoglobin
  • PHT platelet
  • Lymph lymphocyte
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • CREA creatinine
  • UAA blood urea nitrogen
  • Figure 13 shows the curves of each tissue section and body weight of healthy mice over time, specifically, Figure 13A shows heart, liver, spleen, lung and kidney tissue sections; Time change curve.
  • TD-1704 significantly reduced the effects of triptolide on liver function, kidney function, spleen and body weight. Compared with triptolide TPL, TD-1704 has good biological safety.
  • Fig. 2 shows the HPLC and ESI-MS collection of illustrative plates of TD-1704, chromatographic method: chromatographic column: Diamonsil@C18, 5 ⁇ m, 200 * 4.6mm; Mobile phase: A: water (containing 0.1% acetic acid), B: acetonitrile ( Contains 0.1% acetic acid); Elution program: 0 ⁇ 15min, 5%B ⁇ 50%B; Flow rate: 0.7mL/min; Column temperature: 25°C; Detection wavelength: 214nm, 254nm.
  • TD-1704 Dissolve the intermediate in dichloromethane, add iodomethane dropwise, and stir at room temperature. After 24 hours, the organic solvent and excess methyl iodide were removed by evaporation, dissolved in H 2 O, and the supernatant was separated and purified by high performance liquid chromatography using an acetonitrile/water (containing 0.1% acetic acid) system to obtain TD-1704. The purity of TD-1704 was detected and identified by HPLC, ESI-MS and NMR.
  • TD-1704 Mix the rat serum with the PBS solution of TD-1704, incubate on a shaker at 37°C, and measure the concentrations of TD-1704 and triptolide in the system at 1min, 5min, 10min, 20min, 30min, 60min and 120min, respectively.
  • the degradation of TD-1704 in rat serum is shown in Figure 4B.
  • FIG. 4 shows the degradation rate of TD-1704 in plasma cholinesterase and rat serum.
  • FIG. 4A shows the degradation rate of TD-1704 under the action of plasma cholinesterase
  • FIG. 4B shows the degradation rate of TD-1704 in rat serum. The results showed that TD-1704 could be rapidly converted into triptolide in plasma cholinesterase and rat serum.
  • MTT assay was used to determine the in vitro growth inhibitory effect of TD-1704 on human gastric adenocarcinoma cells SGC-7901, human pancreatic cancer cells MIA PaCa-2 and PANC-1, and human metastatic pancreatic adenocarcinoma cells AsPC-1 before and after serum incubation.
  • the cells in logarithmic growth phase were digested with 0.25% trypsin and blown into single cells.
  • the cells were suspended in DMEM medium containing 10% FBS, and seeded in 96-well cell culture at a density of 3000 cells per well. In the plate, the volume of each well is 0.2mL, set aside three wells plus cell-free culture solution as blank wells, and culture in a carbon dioxide incubator for 24 hours.
  • the drugs in each group were diluted sequentially and equally with cell culture medium. Aspirate the cell culture medium in the 96-well plate, and add 200 ⁇ L of serial concentrations of the drug solution to each well. Three replicate wells were set up for each concentration, and three wells with only culture solution added were set aside as control wells.
  • Figure 5 shows the growth inhibitory effect of TD-1704 on MIA PaCa-2, PANC-1, AsPC-1 and SGC-7901 cells before and after serum incubation. It can be seen from Fig. 5A that after incubation of AsPC-1 cells with triptolide before/after serum incubation and TD-1704 before/after serum incubation for 48 hours, the IC 50 values were 23.0, 30.5,/and 25.1 nM. It can be seen from Figure 5B that after PANC-1 cells were incubated with triptolide before/after serum incubation and TD-1704 before/after serum incubation for 48 hours, the IC 50 values were 16.2, 13.4, 53.4 and 19.4nM.
  • Fig. 6 shows the pharmacokinetic curves of triptolide and TD-1704 in rats. It can be seen from Figure 6 and Table 1 that TD-1704 can be rapidly converted into triptolide in rats with a half-life of about 20 minutes.
  • PANC-1 pancreatic cancer cell
  • the nude mice Eighteen days after the tumor was planted, the nude mice were divided into groups, and saline, low-dose TD-1704 (0.31 mg/kg) and high-dose TD-1704 (0.61 mg/kg) were injected into the tail vein respectively. Administration once a day for 23 consecutive days. The survival time of the nude mice was recorded, and the survival curve of the nude mice is shown in FIG. 7 .
  • Fig. 7 shows the pharmacodynamic evaluation of human pancreatic cancer cell PANC-1 orthotopic tumor model nude mice. It can be seen from Figure 7 that the median survival times of the PBS group, TD-1704 intravenous low-dose group and TD-1704 intravenous high-dose group were 51, 67 and 84 days, respectively. The results showed that intravenous administration of TD-1704 could significantly prolong the survival time of PANC-1 orthotopic tumor model nude mice.
  • An animal model of subcutaneous tumor of human gastric adenocarcinoma cell SGC - 7901 was constructed, and the size of the tumor was observed regularly. Respectively tail vein injection of saline, low-dose TD-1704 (0.31mg/kg), middle-dose TD-1704 (0.46mg/kg) and high-dose TD-1704 (0.61mg/kg), intraperitoneal injection of triptolide ( 0.2mg/kg). The drug was administered once a day for 14 consecutive days, and the animals were sacrificed on the 16th day to obtain tumor tissues.
  • the long diameter (a) and short diameter (b) of the tumor were measured every other day, and the tumor volume of nude mice in each group was calculated according to the formula, and the curve of tumor volume over time was drawn (Fig. 8A), and the tumor tissue weight was weighed (Fig. 8B ).
  • V tumor volume 0.5(a ⁇ b 2 )
  • Fig. 8 shows the pharmacodynamic evaluation of human gastric adenocarcinoma cell SGC-7901 subcutaneous tumor model in nude mice.
  • Figure 8A shows the curve of tumor volume change over time of SGC-7901 subcutaneous tumor model nude mice;
  • Figure 8B shows the comparison of tumor tissue weights of SGC-7901 subcutaneous tumor model nude mice on the 16th day after administration. The results showed that intravenous administration of TD-1704 could significantly inhibit the growth of tumor tissue in nude mice of SGC-7901 subcutaneous tumor model.
  • An animal model of subcutaneous tumor of human gastric adenocarcinoma cell SGC - 7901 was constructed, and the size of the tumor was observed regularly. Respectively tail vein injection of PBS, low-dose TD-1704 (0.31mg/kg), medium-dose TD-1704 (0.46mg/kg) and high-dose TD-1704 (0.61mg/kg), intraperitoneal injection of triptolide (0.2 mg/kg). Dosing once a day for 14 consecutive days. On the 16th day, 500 ⁇ L of plasma from nude mice was collected, centrifuged, and serum was collected for liver and kidney function testing and analysis. The results are shown in FIG. 9 .
  • Figure 9 shows the safety evaluation of TD-1704. It can be seen from Figure 9A that there is no difference in blood aspartate aminotransferase concentration between the PBS group, triptolide intraperitoneal group, and TD-1704 intravenous low-, medium-, and high-dose groups, indicating that triptolide intraperitoneal administration and TD-1704 intravenous administration The medicine has no damage to the liver.
  • Example 5 An animal model of human metastatic pancreatic adenocarcinoma cell AsPC-1 tumor in situ was constructed in the same way. Seven days after seeding the tumor, the nude mice were randomly divided into 5 experimental groups, namely the PBS group, the triptolide group, the TD-1704 low-dose group, the TD-1704 middle-dose group and the TD-1704 high-dose group. Group 10. The low, medium and high doses of TD-1704 were 0.2mg/kg/d, 0.6mg/kg/d and 1.0mg/kg/d, respectively, administered i.v. The dose of triptolide group was 0.2 mg/kg/d, administered i.p.
  • the administration method is as follows: administer twice a day (with an interval of 12 hours), administer for 3 days, stop for 1 day, and repeat three cycles successively.
  • the survival time of the nude mice was recorded, and the survival curve of the nude mice is shown in FIG. 10 .
  • Fig. 10 shows the pharmacodynamic evaluation of human metastatic pancreatic adenocarcinoma cell AsPC-1 orthotopic tumor model in nude mice. It can be seen from Fig. 10 that the median survival times of the PBS group, triptolide group and TD-1704 intravenous low, medium and high dose groups were 30, 43.5, 39.5, 59.5 and 69 days, respectively. The results showed that intravenous administration of TD-1704 could significantly prolong the survival time of AsPC-1 orthotopic tumor model nude mice, and its antitumor activity was comparable to that of triptolide. The effect gradually increases.
  • Example 5 An animal model of human metastatic pancreatic adenocarcinoma AsPC-1 subcutaneous tumor was constructed in the same way.
  • the tumor size was 100 mm 3
  • the nude mice were randomly divided into 4 experimental groups, namely PBS group, triptolide group, TD-1704 low-dose group and TD-1704 middle-dose group, with 6 mice in each group.
  • the low and medium doses of TD-1704 were 0.2mg/kg/d and 0.6mg/kg/d, respectively, administered iv.
  • the dose of triptolide group was 0.2 mg/kg/d, administered ip.
  • the administration method is as follows: administer twice a day (with an interval of 12 hours), administer for 3 days, stop for 1 day, and repeat three cycles successively.
  • FIG. 11 shows the pharmacodynamic evaluation of human metastatic pancreatic adenocarcinoma cell AsPC-1 subcutaneous tumor model in nude mice.
  • Figure 11A shows the curve of the tumor volume of AsPC-1 subcutaneous tumor model nude mice over time
  • Figure 11B shows the comparison of tumor tissue weight in AsPC-1 subcutaneous tumor model nude mice on the 16th day after administration
  • 11C shows the in vivo and ex vivo photos of the tumor tissue of the AsPC-1 subcutaneous tumor model nude mice.
  • the tumor volume inhibition rate and tumor weight inhibition rate of triptolide group and TD-1704 low- and middle-dose groups were 65.0%/54.1%, 61.4%/57.7% and 99.9%/99.9%, respectively.
  • the results showed that intravenous administration of TD-1704 could significantly inhibit the growth of tumor tissue in AsPC-1 subcutaneous tumor model nude mice, and its antitumor activity was comparable to that of triptolide. The effect gradually increases.
  • Healthy ICR mice were randomly divided into 5 groups, 6 mice in each group, half male and half male. They were PBS group, triptolide group, TD-1704 low-dose group, TD-1704 middle-dose group and TD-1704 high-dose group.
  • the low, medium and high doses of TD-1704 were 0.2mg/kg/d, 0.6mg/kg/d and 1.0mg/kg/d, respectively, administered i.v.
  • the dose of triptolide group was 0.1 mg/kg, administered i.p.
  • the administration method is as follows: administer twice a day (with an interval of 12 hours), administer for 3 days, stop for 1 day, and repeat four cycles in turn. The mice were weighed every two days.
  • FIG. 12 Organs such as mouse heart, liver, spleen, lung and kidney were embedded in paraffin, pathologically sectioned, and pathologically observed after HE staining ( FIG. 13A ). The body weight of nude mice was plotted over time ( FIG. 13B ).
  • FIG. 12 shows red blood cells (RBC), white blood cells (WBC), hemoglobin (HGB), platelets (PLT), lymphocytes (Lymph), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA) in blood And urea nitrogen (UREA) content.
  • Fig. 13 shows the curves of various tissue sections and body weight of healthy mice over time. Specifically, Fig. 13A shows tissue sections of heart, liver, spleen, lung and kidney; Fig. 13B shows the curve of body weight of healthy mice over time. The results showed that TD-1704 significantly reduced the effects of triptolide on liver function, kidney function, spleen and body weight. Compared with triptolide TPL, TD-1704 has good biological safety.

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Abstract

本发明提供了一种胆碱碳酸酯类前药，所述胆碱碳酸酯类前药在体内可经血浆胆碱酯酶的转化释放出原药的胆碱碳酸酯类前药及其制备方法和在肿瘤治疗中的应用。具体涉及一种雷公藤甲素前药及其制备方法和在肿瘤治疗中的应用。实验显示：雷公藤甲素前药改善了雷公藤甲素溶解性能，具有良好的水溶性；雷公藤甲素前药在体外活性低，在体内可经血浆胆碱酯酶迅速转化为雷公藤甲素，发挥抗肿瘤作用；雷公藤甲素前药显著延长了胰腺癌原位瘤模型裸鼠的生存期，有效抑制了胃癌皮下瘤模型裸鼠的肿瘤生长；雷公藤甲素前药具有较好的安全性。本发明的雷公藤甲素前药可用于胰腺癌和胃癌等多种原发肿瘤和转移肿瘤的治疗。

Description

一种胆碱碳酸酯类前药及其制备方法和应用
相关申请的交叉引用
本申请要求2021年06月11日提交的第CN202110653742.4号中国发明专利申请的优先权，所述申请以引用的方式整体并入本文。
技术领域
本发明属于药学领域，涉及一类在体内可经血浆胆碱酯酶转化释放出原药的胆碱碳酸酯类前药，具体涉及一种雷公藤前药及其制备方法和在肿瘤治疗中的应用。
背景技术
胰腺癌是常见的消化系统肿瘤之一，全球肿瘤发病率排在第九位，患者的五年生存率低于8％。导致胰腺癌患者生存率低的原因在于，胰腺癌早期诊断率低、恶性程度高和治疗效果差。目前，化疗是胰腺癌临床治疗的首选方法，可用于缩小肿瘤的术前治疗和消除残留肿瘤细胞的术后治疗。然而，除了吉西他滨+nab-PTX和FOLFIRINOX对胰腺癌有一定的治疗作用外，其它化疗方案对胰腺癌的治疗效果差。而且，新型药物治疗，如抗血管生成治疗、去间质化治疗和免疫治疗等，用于胰腺癌治疗探索均告失败。因此，开发胰腺癌的有效化疗药物研究具有重要的临床意义。
雷公藤甲素是从卫矛科植物雷公藤中分离提取出的一种环氧化二萜内酯化合物，具有诱导细胞凋亡、干预细胞周期和抑制肿瘤新生血管形成等多靶点作用机制，是一种广谱肿瘤抑制剂。尽管雷公藤甲素具有良好的抗肿瘤活性，但其水溶性差和毒副作用大制约了其临床应用。前药，是指药物经过化学结构修饰后得到的在体外无活性或活性低、在体内经酶或非酶的转化释放出活性原药从而发挥药效的化合物。前药的设计主要在于增加药物水溶性、提高药物生物利用度、增加药物稳定性，以及减小毒副作用等。目前，前药设计在新药研究中越来越受到人们的重视，已经在神经系统药物、抗肿瘤药物和抗病毒药物方面取得了很大的进展。血浆胆碱酯酶又称为假性胆碱酯酶或丁酰胆碱酯酶，是由肝脏中合成释放到血浆中的一种非特异性的酯酶。血浆胆碱酯酶能够水解乙酰胆碱、丁酰胆碱和琥珀酸胆碱等多种胆碱，以及水解许多酯类、肽类及酰胺类化合物。
发明内容
本发明的目的在于提供一种胆碱碳酸酯类前药及其制备方法和应用。
具体地，本发明旨在提供一种雷公藤甲素前药，在体内可经血浆胆碱酯酶的转化释 放出雷公藤甲素发挥抗肿瘤作用，提高了雷公藤甲素的水溶性，并降低了雷公藤甲素的毒副作用。本发明的优选实施方案对雷公藤甲素的C14-位羟基进行酯化反应,合成了雷公藤甲素胆碱碳酸酯，简称TD-1704。研究证明，TD-1704展现出良好的抗肿瘤活性、较低毒副作用和良好的水溶性，具有临床应用前景。同时，该胆碱碳酸酯盐类前药的设计，也可为其他药物的前药制备提供新的思路。
在阐述本发明内容之前，定义本文中所使用的术语如下：
术语“TD-1704”是指：具有式III所示结构的雷公藤甲素胆碱碳酸酯醋酸盐化合物、或其溶剂化物或多晶型物：
Figure PCTCN2022098013-appb-000001
术语“AsPC-1”是指：人转移胰腺腺癌细胞。
术语“MIA PaCa-2”是指：人胰腺癌细胞。
术语“SGC-7901”是指：人胃腺癌细胞。
术语“PANC-1”是指：人胰腺癌细胞。
术语“MTT法”是指：噻唑蓝比色法。
术语“PBS”是指：磷酸盐缓冲液。
术语“DIPEA”是指：二异丙基乙基胺。
术语“FBS”是指：胎牛血清。
术语“DMEM”是指：达尔伯克氏改良伊格尔氏培养基。
术语“i.v.给药”是指：静脉给药。
术语“i.p.给药”是指：腹腔给药。
术语“TPL”是指：雷公藤甲素。
本发明的第一方面提供了一种胆碱碳酸酯类前药，所述胆碱碳酸酯类前药为具有通式I所示结构的化合物：
Figure PCTCN2022098013-appb-000002
式I中：
R-OH为含有羟基的抗肿瘤药物，所述抗肿瘤药物选自以下一种或多种：雷公藤甲素、紫杉醇、多西他赛、卡巴他赛、喜树碱、羟基喜树碱、鬼臼毒素、阿霉素、表阿霉素、 柔红霉素、伊立替康、吉西他滨、长春新碱或长春花碱；
X -为药学上可接受的有机阴离子和无机阴离子，其中，优选地：
所述有机阴离子选自以下一种或多种：甲酸根、醋酸根、草酸根、琥珀酸根、富马酸根、酒石酸根或柠檬酸根；和/或
所述无机阴离子选自以下一种或多种：盐酸根、硫酸根、硫酸氢根、亚硫酸根、硝酸根、碳酸根、碳酸氢根、磷酸根、磷酸氢根、磷酸二氢根、氢溴酸根、氢碘酸根或硼酸根。
根据本发明第一方面的胆碱碳酸酯类前药，其中，所述胆碱碳酸酯类前药为雷公藤甲素前药，优选为具有式II所示结构的化合物、或其药学上可接受的盐、溶剂化物或多晶型物：
Figure PCTCN2022098013-appb-000003
式II中：
Y -为药学上可接受的有机阴离子和无机阴离子。
根据本发明第一方面的胆碱碳酸酯类前药，其中，
所述有机阴离子选自以下一种或多种：甲酸根、醋酸根、草酸根、琥珀酸根、富马酸根、柠檬酸根或酒石酸根；和/或
所述无机阴离子选自以下一种或多种：盐酸根、硫酸根、硫酸氢根、亚硫酸根、硝酸根、碳酸根、碳酸氢根、磷酸根、磷酸氢根、磷酸二氢根、氢溴酸根、氢碘酸根或硼酸根。
根据本发明第一方面的胆碱碳酸酯类前药，其中，所述胆碱碳酸酯类前药为雷公藤甲素前药的醋酸盐，具有式III所示结构的化合物，或其溶剂化物或多晶型物：
Figure PCTCN2022098013-appb-000004
本发明的第二方面提供了制备第一方面所述的胆碱碳酸酯类前药的方法，所述方法包括：
通过对药物活性部位的羟基进行修饰得到其胆碱碳酸酯，从而得到所述胆碱碳酸酯类前药；
其中，所述药物为通过化学方法得到其胆碱碳酸酯衍生物的抗肿瘤药物、抗感染药物、抗心脑血管系统疾病药物、抗淋巴系统疾病药物、抗免疫系统疾病药物和/或镇痛药物。
根据本发明第二方面的方法，其中，所述方法包括：N,N-二甲基乙醇胺与4-(硝基苯基)氯甲酸酯反应得到2-(二甲氨基)乙基(4-硝基苯基)碳酸酯，再与雷公藤甲素C14-位羟基进行酯交换反应，得到2-(二甲氨基)乙基雷公藤甲素碳酸酯，碘甲烷甲基化可制备得到所述胆碱碳酸酯类前药。
本发明的第三方面提供了一种药物组合物，所述药物组合物包括：
第一方面所述的胆碱碳酸酯类前药；和
药学上可接受的载体。
根据本发明第三方面的药物组合物，其中，所述药物组合物的药物制剂形式选自以下一种或多种：溶液剂、片剂、胶囊剂、颗粒剂、散剂或滴丸。
本发明的第四方面提供了第一方面所述的胆碱碳酸酯类前药、按照第二方面所述的方法制备的碱碳酸酯类前药或第三方面所述的药物组合物在制备抗肿瘤药物、抗感染药物、抗心脑血管系统疾病药物、抗淋巴系统疾病药物、抗免疫系统疾病药物或镇痛药物中的应用。
根据本发明第四方面的应用，其中，所述抗肿瘤药物中，所述胆碱碳酸酯类前药在体外活性较低和/或在体内经血浆胆碱酯酶转化释放出原药发挥抗肿瘤作用。
根据本发明第四方面的应用，其中，所述肿瘤选自以下一种或多种：胰腺癌、胃癌、胆管癌、结肠癌、直肠癌、肝癌、乳腺癌、卵巢癌、非小细胞肺癌或淋巴肿瘤。
本发明的第五方面提供了一种治疗癌症、感染、心脑血管系统疾病、淋巴系统疾病和/或免疫系统疾病的药物，所述药物包括：第一方面所述的胆碱碳酸酯类前药、按照第二方面所述的方法制备的碱碳酸酯类前药或第三方面所述的药物组合物。
本发明的第六方面提供了一种镇痛药物，所述药物包括：第一方面所述的胆碱碳酸酯类前药、按照第二方面所述的方法制备的碱碳酸酯类前药或第三方面所述的药物组合物。
本发明的第七方面提供了一种用于治疗癌症、感染、心脑血管系统疾病、淋巴系统疾病和/或免疫系统疾病的方法，所述方法包括：对有需要的受试者给予第一方面所述的胆碱碳酸酯类前药、按照第二方面所述的方法制备的碱碳酸酯类前药或第三方面所述的药物组合物。
本发明的第八方面提供了一种用于镇痛的方法，所述方法包括：对有需要的受试者给予第一方面所述的胆碱碳酸酯类前药、按照第二方面所述的方法制备的碱碳酸酯类前药或第三方面所述的药物组合物。
由此，本发明提供了一种胆碱碳酸酯类前药的设计思路。本发明提供了一种雷公藤甲素前药TD-1704。本发明还提供了上述雷公藤甲素胆前药TD-1704的制备方法以及其在肿瘤治疗中的应用。
根据本发明一个具体的实施方式，本发明提供的前药合成方法，是通过对药物活性部位的羟基进行修饰得到其胆碱碳酸酯，从而制备得到具有不同物化性质的前药。药物可以是通过化学方法得到其胆碱碳酸酯衍生物的抗肿瘤药物(如雷公藤甲素等)、抗感染药物、抗心脑血管系统疾病药物、抗淋巴系统疾病药物、抗免疫系统疾病药物和镇痛药物等。
利用本发明提供的方法制备得到了雷公藤甲素前药TD-1704。TD-1704可进行静脉给药，用于肿瘤治疗。本发明的实验结果表明：TD-1704可在血浆胆碱酯酶的作用下快速转化为原药雷公藤甲素从而发挥抗肿瘤作用，在提高雷公藤甲素水溶性的同时，保留了较好的抗肿瘤活性，降低了毒副作用，在肿瘤治疗中具备良好的临床应用前景。
1.TD-1704的制备
通过化学反应合成了雷公藤甲素前药TD-1704，HPLC进行纯度测定，MS和MRI进行结构表征。
2.TD-1704体外降解和体外药效学评价
考察了TD-1704与血浆胆碱酯酶和血清孵育后的降解情况。MTT法考察了雷公藤甲素和TD-1704与血清孵育前后对人转移胰腺腺癌细胞AsPC-1、人胰腺癌细胞MIA PaCa-2和PANC-1，以及人胃腺癌细胞SGC-7901的生长抑制作用。
3.TD-1704体内药物动力学
考察了TD-1704在大鼠体内的药物动力学性质。
4.TD-1704体内药效学评价和安全性评价
荷人胰腺癌细胞PANC-1原位瘤模型裸鼠尾静脉注射TD-1704，以生存时间为指标评价TD-1704的抗肿瘤作用。
荷人胃腺癌细胞SGC-7901皮下瘤模型裸鼠尾静脉注射TD-1704，以瘤体积、瘤重、血清肝功能和血清肾功能为指标评价TD-1704的抗肿瘤作用和安全性。
荷人转移胰腺腺癌细胞AsPC-1原位瘤模型裸鼠尾静脉注射TD-1704，以生存时间为指标评价TD-1704的抗肿瘤作用。荷人转移胰腺腺癌细胞AsPC-1皮下瘤模型裸鼠尾静脉注射TD-1704，以瘤体积和瘤重为指标评价TD-1704的抗肿瘤作用。
健康小鼠尾静脉注射TD-1704，以血常规、肝功能、肾功能、主要脏器病理切片以及体重等指标评价了TD-1704的安全性。
本发明的胆碱碳酸酯类前药可以具有但不限于以下有益效果：
1.本发明的胆碱碳酸酯类前药可进行静脉给药，用于肿瘤治疗。
2.本发明的胆碱碳酸酯类前药可在血浆胆碱酯酶的作用下快速转化为原药雷公藤甲素从而发挥抗肿瘤作用，在提高雷公藤甲素水溶性的同时，保留了较好的抗肿瘤活性，降低了毒副作用，在肿瘤治疗中具备良好的临床应用前景。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1示出了TD-1704的化学反应图
图1示出了用于制备TD-1704的化学反应图。
图2示出了TD-1704的HPLC和ESI-MS图谱
色谱方法：色谱柱：Diamonsil@C18,5μm,200×4.6mm；流动相：A：水(含0.1％醋酸)，B：乙腈(含0.1％醋酸)；洗脱程序：0～15min，5％B～50％B；流速0.7mL/min；柱温：25℃；检测波长：214nm，254nm。由图2A和2B所示，中间体2-(二甲氨基)乙基雷公藤甲素碳酸酯的HPLC纯度均达到95％以上；ESI-MS：m/z[M+H] +为476.2，与理论分子量相符。由图2C和2D可见，TD-1704的HPLC纯度均达到95％以上；ESI-MS：TD-1704的阳离子部分m/z[B] +为490.2，与理论分子量相符。
图3示出了TD-1704在体内的酶转化反应图
图3示出了TD-1704经血浆胆碱酯酶转化为雷公藤甲素的过程。
图4示出了TD-1704在血浆胆碱酯酶和大鼠血清中的降解速率
图4A示出了TD-1704在血浆胆碱酯酶作用下的降解速率，图4B示出了TD-1704在大鼠血清中的降解速率。结果说明，TD-1704在血浆胆碱酯酶和大鼠血清中均能快速转化为雷公藤甲素。
图5示出了TD-1704在血清孵育前后对MIA PaCa-2、PANC-1、AsPC-1和SGC-7901细胞生长抑制作用
由图5A可见，AsPC-1细胞分别与血清孵育前/后的雷公藤甲素、血清孵育前/后的TD-1704孵育48小时后，IC 50为23.0、30.5、/和25.1nM。
由图5B可见，PANC-1细胞分别与血清孵育前/后的雷公藤甲素、血清孵育前/后的TD-1704孵育48小时后，IC 50为16.2、13.4、53.4和19.4nM。
由图5C可见，MIA PaCa-2细胞分别与血清孵育前/后的雷公藤甲素、血清孵育前/后的TD-1704孵育48小时后，IC 50为19.8、23.3、172和48.3nM。
由图5D可见，SGC-7901细胞分别与血清孵育前/后的雷公藤甲素、血清孵育前/后的TD-1704孵育48小时后，IC 50为8.26、20.0、2055.9和28.6nM。
结果说明，TD-1704本身抗肿瘤活性低，经血清孵育可显著提高TD-1704的抗肿瘤活性。
图6示出了雷公藤甲素及TD-1704在大鼠体内的药动学曲线
由图6可见，TD-1704可在大鼠体内快速转化为雷公藤甲素，半衰期在20min左右。
图7示出了人胰腺癌细胞PANC-1原位瘤模型裸鼠的药效学评价
由图7可见，PBS组、TD-1704静脉低剂量组和TD-1704静脉高剂量组的中位生存时间分别为51、67和84天。结果表明，TD-1704静脉给药可显著延长PANC-1原位瘤模型裸鼠的生存时间。
图8示出了人胃腺癌细胞SGC-7901皮下瘤模型裸鼠的药效学评价
图8A示出了SGC-7901皮下瘤模型裸鼠的瘤体积随时间的变化曲线；图8B示出了给药后第16天SGC-7901皮下瘤模型裸鼠的肿瘤组织重量比较。结果表明，TD-1704静脉给药可显著抑制SGC-7901皮下瘤模型裸鼠的肿瘤组织生长。
图9示出了TD-1704安全性评价
由图9A可见，PBS组、雷公藤甲素组腹腔组和TD-1704静脉低、中、高剂量组的血中谷草转氨酶浓度没有差异，说明雷公藤甲素腹腔给药和TD-1704静脉给药对肝脏无损伤。由图9B可见，与PBS组比较，TD-1704静脉低、中、高剂量组的血尿素氮水平无差异，雷公藤甲素腹腔给药组的血尿素氮水平偏高，说明TD-1704对肾脏无损伤，雷公藤甲素对肾脏有一定的损伤。结果表明，TD-1704体内的安全性较好，优于雷公藤甲素。
图10示出了人转移胰腺腺癌细胞AsPC-1原位瘤模型裸鼠的药效学评价
由图10可见，PBS组、雷公藤甲素组和TD-1704静脉低、中和高剂量组的中位生存时间分别为30、43.5、39.5、59.5和69天。结果表明，TD-1704静脉给药可显著延长AsPC-1原位瘤模型裸鼠的生存时间，其抗肿瘤活性与与雷公藤甲素相当，且随着剂量的增加，TD-1704的抗肿瘤作用逐渐增强。
图11示出了人转移胰腺腺癌细胞AsPC-1皮下瘤模型裸鼠的药效学评价
图11A示出了AsPC-1皮下瘤模型裸鼠的瘤体积随时间的变化曲线；图11B示出了给药后第16天AsPC-1皮下瘤模型裸鼠的肿瘤组织重量比较；图11C示出了AsPC-1皮 下瘤模型裸鼠的肿瘤组织在体和离体照片。雷公藤甲素组和TD-1704低、中剂量组的肿瘤体积抑瘤率和肿瘤重量抑瘤率分别为65.0％/54.1％、61.4％/57.7％和99.9％/99.9％。结果表明，TD-1704静脉给药可显著抑制AsPC-1皮下瘤模型裸鼠的肿瘤组织生长，其抗肿瘤活性与与雷公藤甲素相当，且随着剂量的增加，TD-1704的抗肿瘤作用逐渐增强。
图12和图13示出了健康小鼠的TD-1704安全性评价。
图12示出了健康小鼠血液中各项指标的含量，具体地，图12示出了血液中红细胞(RBC)、白细胞(WBC)、血红蛋白(HGB)、血小板(PLT)、淋巴细胞(Lymph)、谷草转氨酶(AST)、谷丙转氨酶(ALT)、肌酐(CREA)和尿素氮(UREA)的含量。
图13示出了健康小鼠的各组织切片和体重随时间的变化曲线，具体地，图13A示出了心、肝、脾、肺和肾组织切片；图13B示出了健康小鼠体重随时间的变化曲线。
结果表明，TD-1704显著降低了雷公藤甲素对肝功能、肾功能、脾脏和体重的影响。与雷公藤甲素TPL比较，TD-1704具有良好的生物安全性。
实施发明的最佳方式
下面通过具体的实施例进一步说明本发明，但是，应当理解为，这些实施例仅仅是用于更详细具体地说明之用，而不应理解为用于以任何形式限制本发明。
本部分对本发明试验中所使用到的材料以及试验方法进行一般性的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。本领域技术人员清楚，在上下文中，如果未特别说明，本发明所用材料和操作方法是本领域公知的。
通过下述实施例将有助于进一步理解本发明，但并不限制本发明的内容。
实施例1
TD-1704的制备与表征
将N,N-二甲基乙醇胺和DIPEA加入二氯甲烷中，氮气保护下冰浴冷却至0±5℃，对硝基苯基氯甲酸酯缓慢加入上述体系中。撤去冰浴，待反应液温度恢复至室温后，将雷公藤甲素加入该体系，氮气保护下室温搅拌。24h后蒸发除去有机溶剂，冰乙醚洗涤沉淀，残余沉淀以乙腈溶解，采用乙腈/水(含0.1％醋酸)体系经高效液相色谱法分离纯化得中间体N,N-二甲基乙基雷公藤甲素碳酸酯。HPLC、ESI-MS和NMR进行中间体的纯度检测和鉴定。
图2示出了TD-1704的HPLC和ESI-MS图谱，色谱方法：色谱柱：Diamonsil@C18,5μm,200×4.6mm；流动相：A：水(含0.1％醋酸)，B：乙腈(含0.1％醋酸)；洗脱程序：0～15min，5％B～50％B；流速0.7mL/min；柱温：25℃；检测波长：214nm，254nm。如图2A和2B所示，中间体2-(二甲氨基)乙基雷公藤甲素碳酸酯的HPLC纯度均达到95％以上；质谱检测结果显示中间体的分子离子峰m/z[M+H] +为476.2，与理论 分子量相符。中间体经1H-NMR表征，结构正确，解析如下： 1H-NMR(400MHz,Chloroform-d,δ)：4.83(s,1H),4.69(s,2H),4.39(q,2H,C30-CH 2-),3.83(d,1H),3.58–3.41(m,3H),2.95-2.85(m,2H,C32-CH 2-),2.70(d,1H),2.49(s,6H,C34,35-CH 3),2.32(d,1H),2.06-1.86(m,3H),1.58(dd,1H),1.26-1.18(m,1H),1.06(s,3H),0.99(d,3H),0.85(d,3H)。
将中间体溶于二氯甲烷，滴加碘甲烷，室温下搅拌。24h后蒸发除去有机溶剂和过量碘甲烷，用H 2O溶解，上清液采用乙腈/水(含0.1％醋酸)体系经高效液相色谱法分离纯化得TD-1704。HPLC、ESI-MS和NMR对TD-1704进行纯度检测和鉴定。如图2C和2D所示，TD-1704的HPLC纯度均达到95％以上；质谱检测结果显示，TD-1704的阳离子部分m/z[B] +为490.2，与理论分子量相符。TD-1704经 1H-NMR表征，结构正确，解析如下： 1H NMR(400MHz,DMSO-d 6,δ)):4.85(d,2H,C17-CH 2-),4.80(d,1H,),4.60(s,2H,C30-CH 2-),3.97(d,2H),3.74(s,2H),3.68(d,1H),3.13(s,9H,C34,35,36-CH 3),2.63(s,1H),2.23(s,1H),2.10(d,1H),1.81(d,1H),1.54(d,1H),1.30(s,1H),1.50(s,1H),1.05(d,1H),0.90(m,6H,C20-CH 3)。
实施例2
TD-1704酶降解和血清降解试验
称取一定量的血浆胆碱酯酶溶于0.5mL PBS。将0.5mL TD-1704的PBS溶液加入上述酶溶液中，37℃摇床孵育。分别于1min、5min、15min、30min、60min、120min和240min取样50μL(补加50μL PBS)，加入150μL乙腈溶液终止反应，过0.22μm滤膜，进样HPLC。考察TD-1704与血浆胆碱酯酶共孵育时的降解情况，结果见图4A。
将大鼠血清与TD-1704的PBS溶液混合，37℃摇床孵育，分别于1min、5min、10min、20min、30min、60min和120min时测定体系中TD-1704以及雷公藤甲素的浓度，考察TD-1704在大鼠血清中的降解情况，见过见图4B。
图4示出了TD-1704在血浆胆碱酯酶和大鼠血清中的降解速率。其中，图4A示出了TD-1704在血浆胆碱酯酶作用下的降解速率，图4B示出了TD-1704在大鼠血清中的降解速率。结果说明，TD-1704在血浆胆碱酯酶和大鼠血清中均能快速转化为雷公藤甲素。
实施例3
TD-1704体外药效试验
采用MTT法测定TD-1704在血清孵育前后对人胃腺癌细胞SGC-7901、人胰腺癌细胞MIA PaCa-2和PANC-1、人转移胰腺腺癌细胞AsPC-1的体外生长抑制作用。取对数生长期的各株细胞，用0.25％胰蛋白酶消化并吹打成单个细胞，细胞悬浮于含10％FBS的DMEM培养液中，以每孔3000个细胞的密度接种于96孔细胞培养板中，每孔体积0.2mL，留出三孔加不含细胞的培养液作为空白孔，二氧化碳培养箱内培养24小时。用 细胞培养液将各组药物依次等倍稀释。吸去96孔板内细胞培液，各孔加入200μL系列浓度的药液。每个浓度均设三复孔，留出三个仅加入培养液的孔作为对照孔。培养48小时后在实验孔、对照孔和空白孔中加入MTT试剂(5mg/mL)20μL孵育4小时，弃去孔内培养液，每孔加入二甲亚砜150μL，振荡使生成的蓝紫色结晶充分溶解后，用酶标仪测定各孔在490nm处的吸光度(A)，按照以下公式计算细胞存活率，结果见图5：
存活率＝(A490实验孔-A490空白孔)/(A490对照孔-A490空白孔)×100％
图5示出了TD-1704在血清孵育前后对MIA PaCa-2、PANC-1、AsPC-1和SGC-7901细胞生长抑制作用。由图5A可见，AsPC-1细胞分别与血清孵育前/后的雷公藤甲素、血清孵育前/后的TD-1704孵育48小时后，IC 50为23.0、30.5、/和25.1nM。由图5B可见，PANC-1细胞分别与血清孵育前/后的雷公藤甲素、血清孵育前/后的TD-1704孵育48小时后，IC 50为16.2、13.4、53.4和19.4nM。由图5C可见，MIA PaCa-2细胞分别与血清孵育前/后的雷公藤甲素、血清孵育前/后的TD-1704孵育48小时后，IC 50为19.8、23.3、172和48.3nM。由图5D可见，SGC-7901细胞分别与血清孵育前/后的雷公藤甲素、血清孵育前/后的TD-1704孵育48小时后，IC 50为8.26、20.0、2055.9和28.6nM。结果说明，TD-1704本身抗肿瘤活性低，经血清孵育可显著提高TD-1704的抗肿瘤活性。
实施例4
TD-1704药物动力学试验
SD大鼠共6只，体重在200g左右，按随机原则分成2组，每组3只。分别给予等摩尔剂量的雷公藤甲素与TD-1704溶液。其中雷公藤甲素溶于含10％丙二醇生理盐水溶液，按200μg/kg的剂量尾静脉注射给药。TD-1704组尾静脉注射306μg/kg的TD-1704生理盐水溶液。给药后在1min、5min、10min、15min、30min、45min、1h、1.5h、2h、3h、4h和6h分别采集大鼠全血约0.5mL。将采集的全血4000rpm/min离心10min后取上层血浆100μL，加入1mol/L的HCL溶液20μL，以及500ng/mL的内标卡马西平溶液100μL，充分混匀，再加入0.7mL乙酸乙酯。涡旋混匀2min，于4000rpm/min离心10min，取上层有机相移到EP管中，重复萃取一次，两次有机相合并后用氮气吹干，最后以100μL流动相溶液充分溶解残渣，过孔径0.22um微孔滤膜，取上清1μL进样。LC-MS分析求得各时间点大鼠血浆中的TD-1704和雷公藤甲素的浓度，得到TD-1704在大鼠体内药代动力学曲线，结果见图6和表1。
表1、雷公藤甲素及TD-1704在大鼠体内的药动学参数
Figure PCTCN2022098013-appb-000005
Figure PCTCN2022098013-appb-000006
图6示出了雷公藤甲素及TD-1704在大鼠体内的药动学曲线。由图6和表1可见，TD-1704可在大鼠体内快速转化为雷公藤甲素，半衰期在20min左右。
实施例5
TD-1704体内药效学评价
1.人胰腺癌细胞PANC-1细胞原位瘤药效试验
构建人胰腺癌细胞PANC-1原位瘤动物模型：取对数生长期的PANC-1细胞，每只裸鼠接种2×10 6个细胞(分散于40μL PBS缓冲液中)。裸鼠麻醉后，于右侧腹部(可见皮下有一深色阴影，为脾脏)纵向剪开皮肤，约1cm切口，接着剪开肌肉层，细胞接种于胰腺尾端。种瘤18天后，裸鼠分组，尾静脉分别注射生理盐水、低剂量TD-1704(0.31mg/kg)和高剂量TD-1704(0.61mg/kg)。每日给药一次，连续给药23天。记录裸鼠的生存时间，裸鼠生存曲线见图7。
图7示出了人胰腺癌细胞PANC-1原位瘤模型裸鼠的药效学评价。由图7可见，PBS组、TD-1704静脉低剂量组和TD-1704静脉高剂量组的中位生存时间分别为51、67和84天。结果表明，TD-1704静脉给药可显著延长PANC-1原位瘤模型裸鼠的生存时间。
2.人胃腺癌细胞SGC-7901皮下瘤药效试验
构建人胃腺癌细胞SGC-7901皮下瘤动物模型，定期观察肿瘤大小，待肿瘤大小为150mm 3时，分组进行试验。分别尾静脉注射生理盐水、低剂量TD-1704(0.31mg/kg)、中剂量TD-1704(0.46mg/kg)和高剂量TD-1704(0.61mg/kg)、腹腔注射雷公藤甲素(0.2mg/kg)。每日给药一次，连续给药14天，第16天处死动物，取肿瘤组织。隔天测量肿瘤的长径(a)及短径(b)，根据公式计算各组裸鼠肿瘤体积，绘制肿瘤体积随时间的变化曲线(图8A)，并在称量肿瘤组织重量(图8B)。肿瘤体积计算公式：
V 瘤体积＝0.5(a×b 2)
图8示出了人胃腺癌细胞SGC-7901皮下瘤模型裸鼠的药效学评价。图8A示出了SGC-7901皮下瘤模型裸鼠的瘤体积随时间的变化曲线；图8B示出了给药后第16天SGC-7901皮下瘤模型裸鼠的肿瘤组织重量比较。结果表明，TD-1704静脉给药可显著抑制SGC-7901皮下瘤模型裸鼠的肿瘤组织生长。
实施例6
TD-1704安全性评价
构建人胃腺癌细胞SGC-7901皮下瘤动物模型，定期观察肿瘤大小，待肿瘤大小为150mm 3时，分组进行试验。分别尾静脉注射PBS、低剂量TD-1704(0.31mg/kg)、中剂量TD-1704(0.46mg/kg)和高剂量TD-1704(0.61mg/kg)、腹腔注射雷公藤甲素(0.2mg/kg)。每日给药一次，连续给药14天。第16天，取裸鼠血浆500μL、离心，取血清进行肝和肾功能检测分析，结果见图9。
图9示出了TD-1704安全性评价。由图9A可见，PBS组、雷公藤甲素组腹腔组和TD-1704静脉低、中、高剂量组的血中谷草转氨酶浓度没有差异，说明雷公藤甲素腹腔给药和TD-1704静脉给药对肝脏无损伤。由图9B可见，与PBS组比较，TD-1704静脉低、中、高剂量组的血尿素氮水平无差异，雷公藤甲素腹腔给药组的血尿素氮水平偏高，说明TD-1704对肾脏无损伤，雷公藤甲素对肾脏有一定的损伤。结果表明，TD-1704体内的安全性较好，优于雷公藤甲素。
实施例7
TD-1704体内药效学评价
1.人转移胰腺腺癌细胞AsPC-1原位瘤药效试验
实施例5同法构建人转移胰腺腺癌细胞AsPC-1原位瘤动物模型。种瘤7天后，将裸小鼠随机分为5个实验组，分别为PBS组、雷公藤甲素组、TD-1704低剂量组、TD-1704中剂量组和TD-1704高剂量组，每组10只。TD-1704低、中和高剂量分别为0.2mg/kg/d、0.6mg/kg/d和1.0mg/kg/d，i.v.给药。雷公藤甲素组剂量为0.2mg/kg/d，i.p.给药。给药方法如下：每天给药两次(间隔12h)，给药3天，停药1天，依次重复三个循环。记录裸鼠的生存时间，裸鼠生存曲线见图10。
图10示出了人转移胰腺腺癌细胞AsPC-1原位瘤模型裸鼠的药效学评价。由图10可见，PBS组、雷公藤甲素组和TD-1704静脉低、中和高剂量组的中位生存时间分别为30、43.5、39.5、59.5和69天。结果表明，TD-1704静脉给药可显著延长AsPC-1原位瘤模型裸鼠的生存时间，其抗肿瘤活性与与雷公藤甲素相当，且随着剂量的增加，TD-1704的抗肿瘤作用逐渐增强。
2.人转移胰腺腺癌细胞AsPC-1皮下瘤药效试验
实施例5同法构建人转移胰腺腺癌细胞AsPC-1皮下瘤动物模型。待肿瘤大小为100mm 3时，将裸小鼠随机分为4个实验组，分别为PBS组、雷公藤甲素组、TD-1704低剂量组和TD-1704中剂量组，每组6只。TD-1704低和中剂量分别为0.2mg/kg/d和0.6mg/kg/d，i.v.给药。雷公藤甲素组剂量为0.2mg/kg/d，i.p.给药。给药方法如下：每天给药两次(间隔12h)，给药3天，停药1天，依次重复三个循环。隔天测量肿瘤的长径(a)、短径(b)和裸小鼠体重，按照瘤体积计算公式V＝0.5×a×b 2计算各组裸鼠肿瘤体积，绘制肿瘤体积随时间的变化曲线(图11A)，给药第16天处死裸小鼠，取肿瘤组织称重(图11B)。拍摄裸小鼠肿瘤组织的在体和离体照片(图11C)。
图11示出了人转移胰腺腺癌细胞AsPC-1皮下瘤模型裸鼠的药效学评价。其中，图11A示出了AsPC-1皮下瘤模型裸鼠的瘤体积随时间的变化曲线；图11B示出了给药后第16天AsPC-1皮下瘤模型裸鼠的肿瘤组织重量比较；图11C示出了AsPC-1皮下瘤模型裸鼠的肿瘤组织在体和离体照片。雷公藤甲素组和TD-1704低、中剂量组的肿瘤体积抑瘤率和肿瘤重量抑瘤率分别为65.0％/54.1％、61.4％/57.7％和99.9％/99.9％。结果表明，TD-1704静脉给药可显著抑制AsPC-1皮下瘤模型裸鼠的肿瘤组织生长，其抗肿瘤活性与与雷公藤甲素相当，且随着剂量的增加，TD-1704的抗肿瘤作用逐渐增强。
实施例8
健康小鼠的TD-1704安全性评价
将健康ICR小鼠随机分为5组，每组6只，雌雄各半。分别为PBS组、雷公藤甲素组、TD-1704低剂量组、TD-1704中剂量组和TD-1704高剂量组。TD-1704低、中和高剂量分别为0.2mg/kg/d、0.6mg/kg/d和1.0mg/kg/d，i.v.给药。雷公藤甲素组剂量为0.1mg/kg，i.p.给药。给药方法如下：每天给药两次(间隔12h)，给药3天，停药1天，依次重复四个循环。每隔两天称量小鼠体重。给药结束后，小鼠眼眶取血，等量分成和/或样3500rpm离心5min，取血浆检测肝功能生化指标(谷丙转氨酶和谷草转氨酶)、肾功能生化指标(肌酐和尿素氮)(图12)。取小鼠心、肝、脾、肺和肾等脏器石蜡包埋，病理切片，HE染色后进行病理学观察(图13A)。绘制裸小鼠体重随时间的变化曲线(图13B)。
图12示出了血液中红细胞(RBC)、白细胞(WBC)、血红蛋白(HGB)、血小板(PLT)、淋巴细胞(Lymph)、谷草转氨酶(AST)、谷丙转氨酶(ALT)、肌酐(CREA)和尿素氮(UREA)的含量。图13示出了健康小鼠的各组织切片和体重随时间的变化曲线。具体地，图13A示出了心、肝、脾、肺和肾组织切片；图13B示出了健康小鼠体重随时间的变化曲线。结果表明，TD-1704显著降低了雷公藤甲素对肝功能、肾功能、脾脏和体重的影响。与雷公藤甲素TPL比较，TD-1704具有良好的生物安全性。
尽管本发明已进行了一定程度的描述，明显地，在不脱离本发明的精神和范围的条件下，可进行各个条件的适当变化。可以理解，本发明不限于所述实施方案，而归于权利要求的范围，其包括所述每个因素的等同替换。

Claims (15)

  1. 一种胆碱碳酸酯类前药，其特征在于，所述胆碱碳酸酯类前药为具有通式I所示结构的化合物：
    Figure PCTCN2022098013-appb-100001
    式I中：
    R-OH为含有羟基的抗肿瘤药物，所述抗肿瘤药物选自以下一种或多种：雷公藤甲素、紫杉醇、多西他赛、卡巴他赛、喜树碱、羟基喜树碱、鬼臼毒素、阿霉素、表阿霉素、柔红霉素、伊立替康、吉西他滨、长春新碱或长春花碱；
    X -为药学上可接受的有机阴离子和无机阴离子，其中，优选地：
    所述有机阴离子选自以下一种或多种：甲酸根、醋酸根、草酸根、琥珀酸根、富马酸根、酒石酸根或柠檬酸根；和/或
    所述无机阴离子选自以下一种或多种：盐酸根、硫酸根、硫酸氢根、亚硫酸根、硝酸根、碳酸根、碳酸氢根、磷酸根、磷酸氢根、磷酸二氢根、氢溴酸根、氢碘酸根或硼酸根。
  2. 根据权利要求1所述的胆碱碳酸酯类前药，其特征在于，所述胆碱碳酸酯类前药为雷公藤甲素前药，优选为具有式II所示结构的化合物、或其药学上可接受的盐、溶剂化物或多晶型物：
    Figure PCTCN2022098013-appb-100002
    式II中：
    Y -为药学上可接受的有机阴离子和无机阴离子。
  3. 根据权利要求2所述的胆碱碳酸酯类前药，其特征在于：
    所述有机阴离子选自以下一种或多种：甲酸根、醋酸根、草酸根、琥珀酸根、富马酸根、柠檬酸根或酒石酸根；和/或
    所述无机阴离子选自以下一种或多种：盐酸根、硫酸根、硫酸氢根、亚硫酸根、硝酸根、碳酸根、碳酸氢根、磷酸根、磷酸氢根、磷酸二氢根、氢溴酸根、氢碘酸根或硼酸根。
  4. 根据权利要求1至3中任一项所述的胆碱碳酸酯类前药，其特征在于，所述胆碱碳酸酯类前药为雷公藤甲素前药的醋酸盐，具有式III所示结构的化合物，或其溶剂化物或多晶型物：
    Figure PCTCN2022098013-appb-100003
  5. 制备权利要求1至4中任一项所述的胆碱碳酸酯类前药的方法，其特征在于，所述方法包括：
    通过对药物活性部位的羟基进行修饰得到其胆碱碳酸酯，从而得到所述胆碱碳酸酯类前药；
    其中，所述药物为通过化学方法得到其胆碱碳酸酯衍生物的抗肿瘤药物、抗感染药物、抗心脑血管系统疾病药物、抗淋巴系统疾病药物、抗免疫系统疾病药物和/或镇痛药物。
  6. 根据权利要求5所述的方法，其特征在于，所述方法包括：N,N-二甲基乙醇胺与4-(硝基苯基)氯甲酸酯反应得到2-(二甲氨基)乙基(4-硝基苯基)碳酸酯，再与雷公藤甲素C14-位羟基进行酯交换反应，得到2-(二甲氨基)乙基雷公藤甲素碳酸酯，碘甲烷甲基化可制备得到所述胆碱碳酸酯类前药。
  7. 一种药物组合物，其特征在于，所述药物组合物包括：
    权利要求1至4中任一项所述的胆碱碳酸酯类前药；和
    药学上可接受的载体。
  8. 根据权利要求7所述的药物组合物，其特征在于，所述药物组合物的药物制剂形式选自以下一种或多种：溶液剂、片剂、胶囊剂、颗粒剂、散剂或滴丸。
  9. 权利要求1至4中任一项所述的胆碱碳酸酯类前药、按照权利要求5或6所述的方法制备的碱碳酸酯类前药或权利要求7或8所述的药物组合物在制备抗肿瘤药物、抗感染药物、抗心脑血管系统疾病药物、抗淋巴系统疾病药物、抗免疫系统疾病药物或镇痛药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述抗肿瘤药物中，所述胆碱碳酸酯 类前药在体外活性较低和/或在体内经血浆胆碱酯酶转化释放出原药发挥抗肿瘤作用。
  11. 根据权利要求9或10所述的应用，其特征在于，所述肿瘤选自以下一种或多种：胰腺癌、胃癌、胆管癌、结肠癌、直肠癌、肝癌、乳腺癌、卵巢癌、非小细胞肺癌或淋巴肿瘤。
  12. 一种治疗癌症、感染、心脑血管系统疾病、淋巴系统疾病和/或免疫系统疾病的药物，其特征在于，所述药物包括：权利要求1至4中任一项所述的胆碱碳酸酯类前药、按照权利要求5或6所述的方法制备的碱碳酸酯类前药或权利要求7或8所述的药物组合物。
  13. 一种镇痛药物，其特征在于，所述药物包括：权利要求1至4中任一项所述的胆碱碳酸酯类前药、按照权利要求5或6所述的方法制备的碱碳酸酯类前药或权利要求7或8所述的药物组合物。
  14. 一种用于治疗癌症、感染、心脑血管系统疾病、淋巴系统疾病和/或免疫系统疾病的方法，其特征在于，所述方法包括：对有需要的受试者给予权利要求1至4中任一项所述的胆碱碳酸酯类前药、按照权利要求5或6所述的方法制备的碱碳酸酯类前药或权利要求7或8所述的药物组合物。
  15. 一种用于镇痛的方法，其特征在于，所述方法包括：对有需要的受试者给予权利要求1至4中任一项所述的胆碱碳酸酯类前药、按照权利要求5或6所述的方法制备的碱碳酸酯类前药或权利要求7或8所述的药物组合物。
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