CN115466269A

CN115466269A - Choline carbonate prodrug and preparation method and application thereof

Info

Publication number: CN115466269A
Application number: CN202110653742.4A
Authority: CN
Inventors: 刘敏; 张芷依; 王睿峰; 陆伟跃; 谢操
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2021-06-11
Filing date: 2021-06-11
Publication date: 2022-12-13
Also published as: WO2022258035A1

Abstract

The invention belongs to the field of pharmacy, and relates to a choline carbonate prodrug capable of releasing original medicine through the conversion of plasma cholinesterase in vivo, a preparation method thereof and application thereof in tumor treatment. In particular to a triptolide prodrug TD-1704, a preparation method and application thereof in tumor treatment. The experiment shows that: TD-1704 improves triptolide solubility, and has good water solubility; TD-1704 is low in vitro activity, can be quickly converted into triptolide by plasma cholinesterase in vivo, and plays an anti-tumor role; TD-1704 obviously prolongs the survival time of the pancreatic cancer orthotopic tumor model nude mouse, and effectively inhibits the tumor growth of the gastric cancer subcutaneous tumor model nude mouse; TD-1704 has better security. The TD-1704 provided by the invention can be used for treating various primary tumors and metastatic tumors such as pancreatic cancer, gastric cancer and the like.

Description

Choline carbonate prodrug and preparation method and application thereof

Technical Field

The invention belongs to the field of pharmacy, relates to a choline carbonate prodrug which can be converted by plasma cholinesterase in vivo to release a raw drug, and particularly relates to a tripterygium prodrug TD-1704, a preparation method thereof and application thereof in tumor treatment.

Background

Pancreatic cancer is one of the common digestive system tumors, the global tumor incidence ranks ninth, and the five-year survival rate of patients is less than 8%. The low survival rate of pancreatic cancer patients is caused by low early diagnosis rate, high malignancy and poor treatment effect of pancreatic cancer. Chemotherapy is currently the first choice for the clinical treatment of pancreatic cancer, and can be used for both preoperative treatment to shrink tumors and postoperative treatment to eliminate residual tumor cells. However, in addition to some therapeutic effects of gemcitabine + nab-PTX and FOLFIRINOX on pancreatic cancer, other chemotherapeutic regimens have poor therapeutic effects on pancreatic cancer. Moreover, new drug therapies, such as anti-angiogenic therapy, disintermesenteric therapy, immunotherapy, etc., have failed to be explored for pancreatic cancer treatment. Therefore, the research of developing effective chemotherapeutic drugs for pancreatic cancer has important clinical significance.

Triptolide is an epoxidized diterpene lactone compound separated and extracted from tripterygium wilfordii which is a Celastraceae plant, has multi-target action mechanisms of inducing apoptosis, intervening cell cycle, inhibiting tumor neovascularization and the like, and is a broad-spectrum tumor inhibitor. Although triptolide has good antitumor activity, the poor water solubility and the toxic and side effects greatly restrict the clinical application of triptolide. Prodrug is a compound obtained by modifying the chemical structure of a drug, which has no or low activity in vitro and releases active drug substances through enzymatic or non-enzymatic conversion in vivo to exert the drug effect. The prodrug is designed to increase the water solubility of the drug, improve the bioavailability of the drug, increase the stability of the drug, reduce toxic and side effects and the like. At present, the design of the prodrug is more and more emphasized by people in the research of new drugs, and great progress is made in the aspects of nervous system drugs, anti-tumor drugs and antiviral drugs. Plasma cholinesterase, also known as pseudocholinesterase or butyrylcholinesterase, is a nonspecific esterase that is synthesized in the liver and released into the plasma. The plasma cholinesterase can hydrolyze multiple cholines such as acetylcholine, butyrylcholine and succinic acid choline, and many esters, peptides and amides.

In conclusion, the invention aims to provide the triptolide prodrug which can release triptolide through the transformation of plasma cholinesterase in vivo to play an anti-tumor role, improve the water solubility of the triptolide and reduce the toxic and side effects of the triptolide. The invention carries out esterification reaction on C14-hydroxyl of triptolide, and synthesizes triptolide choline carbonate, TD-1704 for short. Researches prove that the TD-1704 shows good antitumor activity, low toxic and side effects and good water solubility, and has a clinical application prospect. Meanwhile, the design of the choline carbonate salt prodrug can also provide a new idea for preparing prodrugs of other medicines.

Disclosure of Invention

The invention provides a design idea of a choline carbonate prodrug.

The invention provides a triptolide prodrug TD-1704.

The invention provides a preparation method of the triptolide gallbladder prodrug TD-1704 and application thereof in tumor treatment.

Specifically, the method for synthesizing the prodrug provided by the invention is to modify the hydroxyl of the active site of the drug to obtain choline carbonate, so as to prepare the prodrug with different physicochemical properties. The medicine can be antitumor medicine (such as triptolide) prepared from choline carbonate derivatives by chemical method, anti-infective medicine, cardiovascular and cerebrovascular system disease resisting medicine, lymphatic system disease resisting medicine, immune system disease resisting medicine, analgesic medicine, etc.

The triptolide prodrug TD-1704 is prepared by the method provided by the invention. TD-1704 can be administered intravenously for tumor treatment. The experimental results of the invention show that: TD-1704 can be quickly converted into original triptolide under the action of plasma cholinesterase so as to play an anti-tumor role, and the triptolide has the advantages of improving the water solubility of triptolide, retaining better anti-tumor activity, reducing toxic and side effects, and having good clinical application prospect in tumor treatment.

Preparation of TD-1704

The triptolide prodrug TD-1704 is synthesized through chemical reaction, HPLC is used for purity determination, and MS and MRI are used for structure characterization.

2 TD-1704 in vitro degradation and in vitro pharmacodynamic evaluation

The degradation of TD-1704 after incubation with plasma cholinesterase and serum was examined. The MTT method investigates the growth inhibition effect of triptolide and TD-1704 and serum on human metastatic pancreatic adenocarcinoma cells AspC-1, human pancreatic cancer cells MIA PaCa-2 and PANC-1 and human gastric adenocarcinoma cells SGC-7901 before and after incubation.

TD-1704 pharmacokinetics in vivo

The pharmacokinetic properties of TD-1704 in rats were examined.

In vivo pharmacodynamic evaluation and safety evaluation of TD-1704

The nude mouse tail vein injection of the PANC-1 in-situ tumor model of the human pancreatic cancer cells is TD-1704, and the survival time is used as an index to evaluate the anti-tumor effect of the TD-1704.

The human gastric adenocarcinoma cell SGC-7901 subcutaneous tumor model nude mouse is injected with TD-1704 in tail vein, and the antitumor effect and safety of TD-1704 are evaluated by taking tumor volume, tumor weight, serum liver function and serum kidney function as indexes.

Drawings

FIG. 1, chemical reaction diagram of TD-1704, FIG. 1 shows the chemical reaction diagram for preparing TD-1704.

FIG. 2 HPLC and ESI-MS spectra of TD-1704

The chromatographic method comprises the following steps: a chromatographic column: diamonsil @ C18,5 μm, 200X 4.6mm; mobile phase: a: water (containing 0.1% acetic acid), B: acetonitrile (containing 0.1% acetic acid); elution procedure: 0 to 15min,5% to B to 50% by weight; the flow rate is 0.7mL/min; column temperature: 25 ℃; detection wavelength: 214nm,254nm. As shown in the diagrams A and B, the HPLC purity of the intermediate 2- (dimethylamino) ethyl triptolide carbonate is over 95 percent; ESI-MS: m/z [ M + H] ⁺ Is 476.2, corresponding to the theoretical molecular weight. As can be seen from the graphs C and D, the HPLC purity of TD-1704 is more than 95%; ESI-MS: cationic moiety m/z [ B ] of TD-1704] ⁺ 490.2, consistent with the theoretical molecular weight.

FIG. 3 shows the in vivo enzyme conversion reaction of TD-1704, and FIG. 3 shows the process of converting TD-1704 into triptolide by plasma cholinesterase.

FIG. 4, degradation rates of TD-1704 in plasma cholinesterase and rat serum

FIG. 4A shows the degradation rate of TD-1704 by plasma cholinesterase, and FIG. 4B shows the degradation rate of TD-1704 in rat serum. The results show that TD-1704 can be quickly converted into triptolide in plasma cholinesterase and rat serum.

FIG. 5, TD-1704 inhibition of MIA PaCa-2, PANC-1, asPC-1 and SGC-7901 cell growth before and after serum incubation

As can be seen in FIG. A, the IC50 of the AspC-1 cells after 48 hours of incubation with triptolide before and after serum incubation and TD-1704 before and after serum incubation were 23.0, 30.5,/and 25.1nM.

As shown in FIG. B, the IC was determined after incubation of PANC-1 cells with triptolide before and after serum incubation and TD-1704 before and after serum incubation for 48 hours ₅₀ 16.2, 13.4, 53.4 and 19.4nM.

As seen in FIG. C, IC was determined after 48 hours of incubation of MIA PaCa-2 cells with triptolide before and after serum incubation and TD-1704 before and after serum incubation ₅₀ 19.8, 23.3, 172 and 48.3nM.

As seen in FIG. D, the IC50 of SGC-7901 cells after 48 hours incubation with triptolide before/after serum incubation and TD-1704 before/after serum incubation were 8.26, 20.0, 2055.9 and 28.6nM.

The result shows that the anti-tumor activity of the TD-1704 is low, and the anti-tumor activity of the TD-1704 can be obviously improved by serum incubation.

FIG. 6 shows the pharmacokinetic profiles of triptolide and TD-1704 in rats

TABLE 1 pharmacokinetic parameters of triptolide and TD-1704 in rats

As can be seen from the figure and the table, TD-1704 can be rapidly converted into triptolide in a rat body, and the half-life period is about 20 min.

FIG. 7 shows pharmacodynamic evaluation of human pancreatic cancer cell PANC-1 orthotopic tumor model nude mice

As can be seen, median survival times were 51, 67 and 84 days for the saline, TD-1704 intravenous low dose, and TD-1704 intravenous high dose groups, respectively. The result shows that the survival time of PANC-1 orthotopic tumor model nude mice can be remarkably prolonged by TD-1704 intravenous administration.

FIG. 8 shows pharmacodynamic and safety evaluation of human gastric adenocarcinoma SGC-7901 subcutaneous tumor model nude mice

FIG. A is a graph showing the tumor volume of nude mice model SGC-7901 subcutaneous tumor as a function of time; panel B is a comparison of tumor tissue weights in nude mice model SGC-7901 subcutaneous tumors 16 days after dosing. The result shows that the tumor tissue growth of nude mice of SGC-7901 subcutaneous tumor model can be remarkably inhibited by TD-1704 intravenous administration.

FIG. 9, TD-1704 evaluation of safety

As can be seen from the graph A, there was no difference in blood glutamic oxaloacetic transaminase concentrations in the normal saline group, the celiac group of triptolide group and the TD-1704 intravenous low, medium and high dose groups, indicating that celiac administration of triptolide and intravenous administration of TD-1704 had no damage to the liver. As can be seen from the graph B, compared with the normal saline group, the blood urea nitrogen levels of the TD-1704 venous low, medium and high dose groups are not different, and the blood urea nitrogen level of the triptolide intraperitoneal administration group is higher, which indicates that the TD-1704 has no damage to the kidney and the triptolide has certain damage to the kidney. The result shows that the TD-1704 has better safety in vivo and is superior to triptolide.

Detailed Description

The invention will be further understood by reference to the following examples, but is not limited to the scope of the following description.

Example 1

Preparation and characterization of TD-1704

Adding N, N-dimethylethanolamine and DIPEA into dichloromethane, cooling to 0 +/-5 ℃ in an ice bath under the protection of nitrogen, and slowly adding p-nitrophenyl chloroformate into the system. Removing ice bath, adding triptolide into the system after the temperature of the reaction solution is returned to room temperature, and protecting with nitrogenStirring at room temperature. Evaporating to remove organic solvent after 24h, washing the precipitate with glacial ethyl ether, dissolving the residual precipitate with acetonitrile, and separating and purifying by high performance liquid chromatography with acetonitrile/water (containing 0.1% acetic acid) system to obtain intermediate N, N-dimethyl ethyl triptolide carbonate. Purity detection and identification of the intermediates were performed by HPLC, ESI-MS and NMR. As shown in fig. 2A and 2B, the HPLC purity of the intermediate was above 95%; the mass spectrum detection result shows that the molecular ion peak M/z [ M + H ] of the intermediate] ⁺ Is 476.2, corresponding to the theoretical molecular weight. The intermediate is characterized by 1H-NMR, has a correct structure and is resolved as follows: ¹ H-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)。

the intermediate is dissolved in dichloromethane, methyl iodide is added dropwise, and stirring is carried out at room temperature. After 24H the organic solvent and excess methyl iodide are removed by evaporation, using H ₂ Dissolving O, separating and purifying the supernatant by adopting an acetonitrile/water (containing 0.1% acetic acid) system through high performance liquid chromatography to obtain TD-1704.TD-1704 was checked and characterized for purity by HPLC, ESI-MS and NMR. As shown in FIGS. 2C and 2D, the HPLC purity of TD-1704 was above 95%; mass spectrometry results show that the cation portion of TD-1704 is m/z [ B ]] ⁺ 490.2, consistent with the theoretical molecular weight. TD-1704 Jing ¹ H-NMR characterization shows that the structure is correct, and the analysis is as follows: ¹ H 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 )。

example 2

TD-1704 enzyme degradation and serum degradation assay

An amount of plasma cholinesterase was weighed out and dissolved in 0.5mL PBS. 0.5mL of TD-1704 in PBS was added to the enzyme solution and incubated at 37 ℃ with a shaker. Sampling 50 μ L (supplemented with 50 μ L PBS) at 1min, 5min, 15min, 30min, 60min, 120min and 240min, adding 150 μ L acetonitrile solution to terminate the reaction, filtering with 0.22 μm filter membrane, and introducing sample HPLC. The degradation of TD-1704 with plasma cholinesterase was examined and the results are shown in FIG. 4A.

Mixing rat serum with PBS solution of TD-1704, incubating at 37 deg.C with shaking table, measuring the concentrations of TD-1704 and triptolide in the system at 1min, 5min, 10min, 20min, 30min, 60min and 120min, respectively, and examining the degradation of TD-1704 in rat serum, as shown in FIG. 4B.

Example 3

TD-1704 in vitro potency assay

The in vitro growth inhibition 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 is determined by adopting an MTT method. Each cell line in the logarithmic growth phase was digested with 0.25% trypsin and blown into single cells, the cells were suspended in DMEM medium containing 10% FBS, seeded into 96-well cell culture plates at a density of 3000 cells per well, and the wells were 0.2mL per well, three wells were left, and the cells were cultured in a carbon dioxide incubator for 24 hours while adding a medium containing no cells as blank wells. Each group of drugs was sequentially diluted in equal times with cell culture medium. The cell culture medium in the 96-well plate was aspirated, and 200. Mu.L of each well was added. Three more wells were set for each concentration, leaving three wells to which culture medium alone was added as control wells. After 48 hours of culture, 20 μ L of MTT reagent (5 mg/mL) was added to the experimental, control and blank wells and incubated for 4 hours, the culture medium in the wells was discarded, 150 μ L of dimethyl sulfoxide was added to each well, the resulting bluish violet crystals were sufficiently dissolved by shaking, the absorbance (a) at 490nm was measured in each well using a microplate reader, and the cell viability was calculated according to the following formula, as shown in fig. 5:

survival = (a 490 experimental wells-a 490 blank wells)/(a 490 control wells-a 490 blank wells) × 100%.

Example 4

TD-1704 pharmacokinetic testing

The SD rats are 6 in total, the body weight is about 200g, and the SD rats are divided into 2 groups of 3 rats according to a random principle. The triptolide and TD-1704 solution are administered at equimolar doses, respectively. Wherein the triptolide is dissolved in a normal saline solution containing 10% propylene glycol and is administered by caudal vein injection according to the dosage of 200 mug/kg. TD-1704 group tail vein was injected with 306. Mu.g/kg of TD-1704 physiological saline solution. About 0.5mL of whole blood was collected from rats at 1min, 5min, 10min, 15min, 30min, 45min, 1h, 1.5h, 2h, 3h, 4h and 6h after administration. Collecting whole blood, centrifuging at 4000rpm/min for 10min, collecting upper layer plasma 100 μ L, adding 1mol/L HCL solution 20 μ L and 500ng/mL internal standard carbamazepine solution 100 μ L, mixing well, and adding 0.7mL ethyl acetate. Mixing by vortex for 2min, centrifuging at 4000rpm/min for 10min, transferring the upper organic phase into an EP tube, extracting repeatedly once, mixing the two organic phases, blowing with nitrogen, dissolving the residue with 100 μ L of mobile phase solution, passing through a microporous membrane with the diameter of 0.22 μm, and sampling the supernatant with 1 μ L. The LC-MS analysis determines the concentration of TD-1704 and triptolide in the plasma of rats at each time point, and the in vivo pharmacokinetic curve of TD-1704 in rats is obtained, and the result is shown in FIG. 6 and Table 1.

TABLE 1

Example 5

TD-1704 in vivo pharmacodynamic evaluation

1. Drug effect test of human pancreatic cancer cell PANC-1 cell in situ tumor

Constructing a human pancreatic cancer cell PANC-1 in-situ tumor animal model: taking PANC-1 cells in logarithmic growth phase, inoculating 2 × 10 cells to each nude mouse ⁶ Individual cells (dispersed in 40 μ L PBS buffer). After anesthesia, the skin was cut longitudinally at the right abdomen (dark shade under the skin, spleen) for about 1cm incision followed by cutting the muscle layer and cells were seeded at the tail end of the pancreas. 18 days after the breeding, the nude mice were divided into groups and injected with normal saline, low dose TD-1704 (0.31 mg/kg) and high dose TD-1704 (0.61 mg/kg) into the tail vein, respectively. The administration was once daily for 23 days. The survival time of the nude mice was recorded and the survival curve of the nude mice is shown in fig. 7.

2. Human gastric adenocarcinoma cell SGC-7901 subcutaneous tumor efficacy test

Constructing a human gastric adenocarcinoma SGC-7901 subcutaneous tumor animal model, periodically observing the tumor size until the tumor size is 150mm ³ At that time, the test is performed in groups. The tail vein is injected with normal saline, low dose TD-1704 (0.31 mg/kg), medium dose TD-1704 (0.46 mg/kg), high dose TD-1704 (0.61 mg/kg), and intraperitoneal injection of triptolide (0.2 mg/kg). Once daily for 14 consecutive days, animals were sacrificed on day 16 and tumor tissue was removed. The tumor length (a) and the tumor length (B) were measured every other day, the tumor volume of each group of nude mice was calculated according to the formula, the change of the tumor volume with time was plotted (fig. 8A), and the tumor tissue weight was weighed (fig. 8B). Tumor volume calculation formula:

V _{tumor volume} ＝0.5(a×b ² )。

Example 6

TD-1704 safety evaluation

Constructing a human gastric adenocarcinoma cell SGC-7901 subcutaneous tumor animal model, and regularly observing the tumor size until the tumor size is 150mm ³ At that time, the test is performed in groups. The tail vein is injected with normal saline, low dose TD-1704 (0.31 mg/kg), medium dose TD-1704 (0.46 mg/kg), high dose TD-1704 (0.61 mg/kg), and intraperitoneal injection of triptolide (0.2 mg/kg). The administration is once daily for 14 days. On

day

16, 500. Mu.L of nude mouse plasma was collected, centrifuged, and serum was collected for analysis of liver and kidney function tests, and the results are shown in FIG. 9.

Claims

1. A choline carbonate prodrug, wherein the prodrug is a compound having a structure according to formula I:

in formula I:

R-OH is antitumor drug containing hydroxyl, including triptolide, paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, podophyllotoxin, adriamycin, epirubicin, daunorubicin, irinotecan, gemcitabine, vincristine or vinblastine;

X ^- is pharmaceutically acceptable organic anion and inorganic anion, the organic anion comprises formate, acetate, oxalate, succinate, fumarate, tartrate or citrate; inorganic anions include hydrochloride, sulfate, bisulfate, sulfite, nitrate, carbonate, bicarbonate, phosphate, hydrogenphosphate, dihydrogenphosphate, hydrobromate, hydroiodide, or borate.

2. The choline carbonate prodrug according to claim 1, which is a triptolide prodrug, a compound having the structure of formula II, or a pharmaceutically acceptable salt, solvate, or polymorph thereof:

in formula II:

Y ^- is pharmaceutically acceptable organic anion and inorganic anion, wherein the organic anion comprises formate, acetate, oxalate, succinate, fumarate, citrate or tartrate; inorganic anions include hydrochloride, sulfate, bisulfate, sulfite, nitrate, carbonate, bicarbonate, phosphate, hydrogenphosphate, dihydrogenphosphate, hydrobromate, hydroiodide, or borate.

3. The choline carbonate prodrug according to claim 2, wherein the acetate salt of triptolide prodrug, the compound having the structure of formula III, or a solvate or polymorph thereof is designated TD-1704:

4. the choline carbonate prodrug according to claim 3, wherein TD-1704 is prepared by the following method: the N, N-dimethylethanolamine reacts with 4- (nitrophenyl) chloroformate to obtain 2- (dimethylamino) ethyl (4-nitrophenyl) carbonate, then the 2- (dimethylamino) ethyl triptolide carbonate is subjected to ester exchange reaction with C14-hydroxyl of triptolide to obtain the 2- (dimethylamino) ethyl triptolide carbonate, and methyl iodide is methylated to obtain TD-1704.

5. The choline carbonate prodrug according to any one of claims 1, 2 or 3, which has low activity in vitro and releases the drug to exert an antitumor effect in vivo by conversion with plasma cholinesterase.

6. The choline carbonate prodrug according to claim 3, for use in the treatment of tumors, including pancreatic cancer, gastric cancer, cholangiocarcinoma, colon cancer, rectal cancer, liver cancer, breast cancer, ovarian cancer, non-small cell lung cancer or lymphoid tumors.

7. The choline carbonate prodrug according to claim 3, wherein the pharmaceutical formulation comprises a solution, a tablet, a capsule, a granule, a powder or a drop pill.