CN107698639B - N-formate hypoxia-activated prodrug of gemcitabine phosphate and application thereof - Google Patents
N-formate hypoxia-activated prodrug of gemcitabine phosphate and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
- C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
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- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
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Abstract
JixitaThe structure of the N-formate hypoxia activated prodrug of the ripelyl phosphate conforms to the general formula (I)
Wherein: r is:
a is
R' is isopropyl or benzyl, and Ar is phenyl or o-methylbenzyl. The medicine can delay the metabolism of the medicine by deoxycytidine deaminase, has stronger cytotoxicity under the hypoxic condition, and can be used for preparing medicines for treating tumors.
Description
Technical Field
The invention belongs to the field of pharmacy, and provides an N-formate hypoxia-activated prodrug of gemcitabine phosphate and application thereof.
Background
Gemcitabine is a nucleoside antitumor drug, the action mechanism of the drug is nucleotide metabolism antagonism, and after intracellular triphosphoration in vivo, the drug specifically interferes the metabolism of nucleic acid by inhibiting the synthesis of deoxynucleoside triphosphates (dNTPs), interfering cell replication by being doped into DNA or RNA molecules, competitively inhibiting the action of DNA polymerase and the like, prevents the division and the reproduction of cells and finally leads to the death of tumor cells. Gemcitabine, after entering the body, is rapidly and completely metabolized by deoxycytidine deaminase in the liver, kidney, blood and other tissues, and is converted to the inactive metabolite 2 ' -deoxy-2 ', 2 ' -difluorouridine.
Aminoacylation of nucleoside antitumor drugs can delay the metabolism of the drugs by deoxycytidine deaminase, for example, Enocitabine (Enocitabine) which is a derivative obtained by aminoacylation of Cytarabine (Cytarabine) has stronger and more durable antitumor effect than Cytarabine. However, aminoacylation does not reduce the toxic side effects of the drug on normal tissues.
Nucleoside antitumor drugs are easy to generate drug resistance, and the phosphate prodrug thereof can reduce the drug resistance and has good antitumor effect, wherein gemcitabine phosphate prodrug NUC-1031 has entered clinical research (Journal of Medicinal Chemistry 2014, 57, 1531-. However, gemcitabine phosphate prodrug cannot delay its metabolism by deoxycytidine deaminase and reduce the toxic side effects of the drug on non-tumor tissues. Aminoacylation of gemcitabine phosphate prodrug (WO2015/134334) can delay the metabolism of drug by deoxycytidine deaminase, and still cannot reduce the toxic side effects of drug on non-tumor tissues.
With rapid tumor growth, part of the tumor tissue is located further and further away from the nearest blood vessels and the oxygen supply is inadequate, resulting in tumor hypoxia (Nature review cancer 2002,2: 38-47). The traditional antitumor drugs have good lethality to tumors near blood vessels, but have limited effect on tumors in hypoxic regions. The tumor hypoxia activated prodrug can specifically release anti-tumor active ingredients in a tumor hypoxia area, thereby killing tumors in the hypoxia area (Chinese Journal of Cancer 2014,33: 80-86). The hypoxic activated prodrug has tumor targeting property, so that the hypoxic activated prodrug has better safety, and has more excellent anti-tumor effect when being used together with the traditional anti-tumor drug. Among them, TH302 has been studied clinically and has a good therapeutic effect on pancreatic cancer and the like (Journal of Clinical Oncology 2015, 33, 1475-1482).
The invention of China 201610649914.X discloses a gemcitabine ProTide hypoxic activated prodrug and an application thereof, wherein a hypoxic activating group is introduced on a side chain of gemcitabine phosphate to enable the prodrug to generate hypoxic activation selectivity and reduce toxic and side effects on normal tissues. But still does not delay the metabolism of the drug by deoxycytidine deaminase.
The N-formate hypoxia-activated prodrug of gemcitabine phosphate obtained by the invention aminoacylates gemcitabine phosphate, can delay the metabolism of a drug by deoxycytidine deaminase, increase the action time of the drug and reduce the dosage of the drug. And moreover, a hypoxic activating group is introduced in the amino acylation process, so that the derivative has low cytotoxicity under normal oxygen environment and high cytotoxicity under hypoxic condition, can specifically exert an anti-tumor effect on tumors in tumor hypoxic areas, reduces toxic and side effects on other tissues, has an excellent anti-cancer effect and good safety on the tumors, and can be used for preparing medicaments for treating the tumors.
Disclosure of Invention
The technical problem to be solved is as follows: the invention provides an N-formate hypoxia-activated prodrug of gemcitabine phosphate derivatives and application thereof. The medicine can delay the metabolism of the medicine by deoxycytidine deaminase, increase the action time of the medicine, reduce the dosage of the medicine, has smaller cytotoxicity under normal oxygen environment and stronger cytotoxicity under hypoxic condition, and can be used for preparing the medicine for treating tumors.
The technical scheme is as follows: gemcitabine phosphate N-formate hypoxia-activated prodrug, the structure of which conforms to the general formula (I)
The N-formate hypoxia-activated prodrug of gemcitabine phosphate has a specific chemical structure shown as the following formula 1-6:
the application of the compound or the pharmaceutically acceptable salt thereof in preparing the medicine for treating the tumor.
The medicine for treating tumor contains the compound or its pharmaceutically acceptable salt as effective component.
Cell growth inhibition tests show that the compounds shown in the invention have lower cytotoxicity compared with gemcitabine phosphate prodrug NUC-1031. Liver homogenate stability studies show that the compounds of the invention produce lower concentrations of the active ingredient gemcitabine monophosphate under normal oxygen conditions than gemcitabine phosphate prodrug NUC-1031, suggesting that the compounds of the invention are metabolized at a lower rate under normal oxygen conditions. Under the hypoxic condition, the compound disclosed by the invention generates gemcitabine monophosphate with higher concentration, which indicates that the compound can generate larger cytotoxicity under the hypoxic condition.
It should be noted that the attachment of the hypoxic group to the gemcitabine phosphate ester has a great effect on maintaining the hypoxic selectivity of the drug, and if the hypoxic group is attached to the amino group of the gemcitabine phosphate ester in other ways, the hypoxic selectivity of the drug cannot be maintained. In the case of compound 7, a study of the stability of liver homogenates showed that gemcitabine monophosphate, the active ingredient, could not be produced under normal oxygen conditions or under hypoxic conditions. If the methyl group on the side chain of the hypoxic group is deleted, the hypoxic selectivity of the drug is obviously reduced. In the case of compound 8, the study of the stability of liver homogenates showed that the difference in the concentration of gemcitabine monophosphate, the active ingredient, was significantly reduced under normoxic or hypoxic conditions.
Has the advantages that: the medicine can delay the metabolism of the medicine by deoxycytidine deaminase, increase the action time of the medicine, reduce the dosage of the medicine, has smaller cytotoxicity in a normal oxygen environment and stronger cytotoxicity in a hypoxic condition, has excellent anti-tumor effect and good safety, and can be used for preparing the medicine for treating tumor.
Drawings
FIG. 1 is a graph showing the growth inhibitory effect of Compound 6 of interest on human BxPC-3 nude mouse subcutaneous transplantable tumors.
Detailed Description
The following examples are given to enable a person skilled in the art to fully understand the invention, but do not limit it in any way.
Example 1: synthesis of target compound 1-target compound 6:
synthesis of Compound 1
The synthetic route is as follows:
triphosgene (0.178g, 0.6mmol) was dissolved in 4mL of toluene at-78 deg.C, pyridine (0.047g, 0.6mmol) in 1mL of toluene was added, followed by 1- (4-nitrophenyl) ethanol (0.72g, 0.4mmol) dissolved in 40mL of toluene slowly and stirred at room temperature for 24 h. After completion of the reaction, toluene was distilled off under reduced pressure, the resulting residue was dissolved in N, N-dimethylformamide (2.5mL), gemcitabine phosphate prodrug 1a (0.14g,0.27mmol), pyridine (61. mu.L, 0.75mmol) were added at 4 ℃, stirred at room temperature for 24 hours, the solvent was distilled off under reduced pressure, dissolved in ethyl acetate (30mL), washed with water, the organic layer was dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography to obtain a white solid (1, 97 mg).
Referring to the procedure for compound 1, compounds 2-6 were synthesized.
Table 1 main raw materials of the compounds of the examples and1H NMR
example 2: research on in vitro inhibition effect of target compound on tumor cell proliferation
Collecting tumor cells in logarithmic growth phase, adding 0.25% pancreatin, digesting for 3min, suspending the cells with 10% calf serum RPMI-1640, counting, and adjusting cell concentration to 1 × 105One cell per mL, 100. mu.L/well inoculated in a Top-count special 96-well cell culture plate at 37 ℃ in 5% CO2And (5) incubating for 24 h. The cells were then divided into experimental and control groups, and the experimental groups were added with target compound solutions (0.1nM, 1nM, 10nM, 100nM), each in four duplicate wells, and the volume of each well was made up to 200 μ L. Continuously culturing for 72 hr after each group is added with sample, and adding into each well before culturing3H-TdR 3×105Bq, determining CPM (count per minute) value of each well by using Top-count. Calculating the half Inhibition Concentration (IC) of each experimental group drug to cell proliferation50)。
TABLE 1 half maximal Inhibitory Concentration (IC) of the target compounds on tumor cell proliferation (72 hours)50，nM)
The above experimental results show that: the in vitro inhibition effect of the example compounds (1-6) on tumor cell proliferation is significantly lower than that of gemcitabine and NUC-1031, which indicates that the example compounds of the invention have less cytotoxicity.
Example 3: examination of the production of active metabolite Gemcitabine monophosphate concentration of the Compound of interest in liver homogenates
NADPH start system preparation: accurately weighing NADPNa2G-6-P-Na, G-6-PDH and MgCl2Adding water to dissolve the mixture in a proper amount and fixing the volume, wherein the system contains 2 mmol.L-1NADPNa2，40mmol·L-1G-6-P-Na，4U·L-1G-6-PDH，40mmol·L- 1MgCl2And storing at-20 ℃.
Sample preparation: firstly, adding a proper amount of sample methanol solution into an EP tube, volatilizing the solvent in a water bath, adding a Tris buffer solution, homogenizing rat liver, and carrying out vortex mixing. Carrying out pre-incubation for 5min at 37 ℃ in a constant-temperature oscillation water tank. Add NADPH to start the system 200. mu.L, vortex well to start the reaction. The final reaction volume was 400. mu.L, containing 1.0 mmol. multidot.L-1 1NADPNa2，20mmol·L-1G-6-P-Na，2U·L-1G-6-PDH，20mmol·L-1MgCl2The mass concentration of the liver homogenate protein is 2.0 mg-mL-1The final substrate concentration was 0.5. mu. moL. L-1. Incubating in a water bath at 37 ℃. After incubation for 60min, 0.4mL of acetonitrile was added to stop the reaction. In parallel, 5 parts.
Sample treatment: after the reaction is terminated by acetonitrile, the mixture is mixed evenly by vortex and ultrasonic treatment for 5min, and the mixture is centrifuged at high speed (13000 r.min)-1At 4 ℃ for 20min, taking supernatant, and volatilizing the supernatant in a water bath at 37 ℃ under nitrogen flow. Redissolving the residue with 400. mu.L methanol, sonicating to complete dissolution, and centrifuging at high speed (13000r min)-120min, 4 ℃), the supernatant was subjected to HPLC analysis, and the concentration of gemcitabine monophosphate was determined and compared with the concentration of gemcitabine monophosphate produced by NUC-1031.
TABLE 2 comparison of Gemcitabine monophosphate concentration of the target Compound in liver homogenates
| Compound numbering | Gemcitabine monophosphate concentration (% relative) |
| 1 | 6 |
| 2 | 7 |
| 4 | 5 |
| 5 | 6 |
| 6 | 5 |
| 7 | 0 |
| 8 | 82 |
| NUC-1031 | 100 |
The above experimental results show that: the target compounds 1-6 produced gemcitabine monophosphate in liver homogenates at significantly lower concentrations than NUC-1031, with target compound 7 being undetectable and target compound 8 producing gemcitabine slightly less than NUC-1031.
Example 4: stability study of target compound in hypoxic state in liver homogenate
Reference example 3 was run, where the samples were incubated, the solution was treated with nitrogen for 20 minutes before adding the samples and incubated under nitrogen for 60 minutes after adding the samples. The centrifugate was subjected to HPLC to determine the concentrations of the objective compound and O-desmethyllenvatinib.
TABLE 3 Gemcitabine monophosphate concentration of the target Compound in liver homogenates under hypoxic conditions
The above experimental results show that: gemcitabine monophosphate is produced in liver homogenates at significantly higher concentrations than in normoxic state for the target compounds 1-6 in hypoxic state. Gemcitabine monophosphate could not be detected in target compound 7, suggesting that gemcitabine monophosphate could not be effectively released by compound 7 under hypoxic conditions. While target compound 8 produced gemcitabine monophosphate at a concentration slightly higher than the normoxic state, suggesting that compound 8 is not hypoxia selective.
Example 5: growth inhibition of target compound 6 on human BxPC-3 nude mouse subcutaneous transplantation tumor
Taking BxPC-3 human pancreatic cancer cells in logarithmic growth phase at 5 × 106Individual cell 0.2mL-1Only a-1The concentration of (2) is inoculated to the subcutaneous back of a nude mouse, and after the length and diameter of the transplanted tumor of the nude mouse are all more than or equal to 5mm, the similar volume of the tumor body is calculated according to the length and diameter of the transplanted tumor. The mice were divided into 5 groups by randomized block design assignment.
Administration protocol 40 model animals were randomly divided into a negative control group, a low dose group (compound 6, 0.15mmol/kg), a high dose group (compound 6, 0.6mmol/kg), and gemcitabine hydrochloride group (0.2mmol/kg), administered by intraperitoneal injection (2 times per week) for 3 weeks, and the nude mice were sacrificed after one week of drug withdrawal. The animal body weight was also measured.
The inhibitory effect and body weight change are shown in fig. 1: after administration, each group showed significant tumor growth inhibition, and all the nude mice in the test groups showed no significant difference in body weight, but all were smaller than the control group. The high dose group showed better therapeutic effect and safety than the gemcitabine group.
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1.
Application in preparing medicine for treating pancreatic cancer.
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| CN106146583A (en) * | 2014-11-17 | 2016-11-23 | 常州方圆制药有限公司 | Novel cytidine derivatives and application thereof |
| CN106279321A (en) * | 2016-08-09 | 2017-01-04 | 南京医科大学 | Gemcitabine ProTide weary oxygen activation prodrug and application thereof |
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