Gemcitabine ProTide hypoxia-activated prodrug and application thereof

A gemcitabine ProTide hypoxic-activated prodrug and a use thereof in the preparation of a medicament for treating tumors. The general structural formula thereof is formula (A), wherein: one of R1 and R2 is a hypoxic-activated group of —C(R3R4)ArNO2, and the other is an alkyl group of 1 to 6 carbon atoms, a phenyl group or —CH2Ar, wherein R3 and R4 are —H or a methyl group, and —Ar is an aromatic ring compound. The gemcitabine ProTide hypoxic-activated prodrug described in the present invention has a stronger cytotoxicity under a hypoxic condition, has excellent anti-tumor effects and is very safe; the present invention can be used along with other anti-tumor drugs to exert a better anti-tumor activity, and can be used in the preparation of a medicament for treating tumors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the national phase entry of International Application No. PCT/CN2017/095794, filed on Aug. 3, 2017, which is based upon and claims priority to Chinese Patent Application No. 201610649914.X, filed on Aug. 9, 2016, and Chinese Patent Application No. 201710610509.1, filed on Jul. 25, 2017, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention belongs to the field of pharmacy, and provides a gemcitabine ProTide hypoxic-activated prodrug and a use thereof.

BACKGROUND

Gemcitabine is a nucleoside anti-tumor drug. The mechanism of action of this drug is to antagonize nucleotide metabolism. After intracellular triphosphorylation in vivo, gemcitabine specifically interferes with nucleic acid metabolism and prevents cell division and reproduction by inhibiting the synthesis of deoxynucleoside triphosphate (dNTPs), interfering with cell replication by being incorporated into DNA or RNA molecules, competitively inhibiting DNA polymerase, and the like, thus eventually causing the death of tumor cells. Nucleoside anti-tumor drugs are prone to drug resistance, but ProTide prodrugs thereof can reduce the occurrence of drug resistance and have a good anti-tumor effect, among which a gemcitabine ProTide prodrug NUC-1031 has been clinically studied (Journal of Medical Chemistry 2014, 57, 1531-1542). However, ProTide prodrugs cannot reduce the toxic and side effects of drugs on non-tumor tissues.

With the rapid growth of tumors, some tumor tissues are farther and farther away from the nearest blood vessel, and oxygen supply is insufficient, resulting in tumor hypoxia (Nature review cancer 2002, 2: 38-47). Traditional anti-tumor drugs have good lethality to tumors near blood vessels, but have limited effects on tumors in hypoxic regions. Tumor hypoxic-activated prodrugs can specifically release anti-tumor active constituents in tumor hypoxic regions, thus killing tumors in the hypoxic regions (Chinese Journal of Cancer 2014, 33: 80-86). Hypoxic-activated prodrugs have a tumor targeting property, thus having a better safety performance, and a better anti-tumor effect when used in combination with traditional anti-tumor drugs, among which TH302 has been clinically studied and has a good therapeutic effect on pancreatic cancer (Journal of Clinical Oncology 2015, 33, 1475-1482).

SUMMARY

Technical Problem

The present invention provides a gemcitabine ProTide hypoxic-activated prodrug and a use thereof. The prodrug has a stronger cytotoxicity under a hypoxic condition, has excellent anti-tumor effects and is very safe, can be used along with traditional anti-tumor drugs to exert a good anti-tumor activity at a small dose, and can be used in the preparation of a medicament for treating tumors.

Technical Solution

The chemical structural formula of the gemcitabine ProTide hypoxic-activated prodrug is:

wherein one of R1and R2is a hypoxic-activated group of —C(R3R4)ArNO2, the other is an alkyl group of 1 to 6 carbon atoms, a phenyl group or —CH2Ar, R3and R4are —H or a methyl group, and —Ar is an aromatic ring compound.

As a preferred scheme, for the gemcitabine ProTide hypoxic-activated prodrug, the structure of R1is:

R2is an alkyl or benzyl group of 1 to 6 carbon atoms, R3is —H or a methyl group, and R4is a methyl group.

As a preferred scheme, for the gemcitabine ProTide hypoxic-activated prodrug, R1is a phenyl group, and the structure of R2is:

R3is —H or a methyl group, and R4is a methyl group.

As a preferred scheme, for the gemcitabine ProTide hypoxic-activated prodrug, the structure of R1is:

As a preferred scheme, for the gemcitabine ProTide hypoxic-activated prodrug, R1is —CH2Ar, —Ar is a benzene ring with an electron donating group, and the structure of R2is:

R3is —H or a methyl group, and R4is a methyl group.

As a preferred scheme, the structure of the gemcitabine ProTide hypoxic-activated prodrug is as follows:

As a preferred scheme, the structure of the gemcitabine ProTide hypoxic-activated prodrug is as follows:

A use of the above compound or pharmaceutically acceptable salt thereof in the preparation of a medicament for treating tumors.

A use of a composition of the above compound or pharmaceutically acceptable salt thereof and gemcitabine hydrochloride in the preparation of a medicament for treating tumors.

A medicament for treating tumors, of which the effective component being the above gemcitabine ProTide hypoxic-activated prodrug or pharmaceutically acceptable salt thereof.

A medicament for treating tumors, of which the effective component being the composition of the above gemcitabine ProTide hypoxic-activated prodrug or pharmaceutically acceptable salt thereof and gemcitabine hydrochloride.

It should be pointed out that our research found that the cytotoxicity of compounds 001-021 under a hypoxic condition was significantly higher than that under a normal oxygen condition. The cytotoxicity of a target compound (e.g., compound 022) obtained by introducing a hypoxic-activated group into an amino position of gemcitabine ProTide prodrug NUC-1031 under a hypoxic condition was not significantly different from that under a normal oxygen condition (see Table 1). It indicated that the introduction position of the hypoxic-activated group was specific for maintaining the hypoxic-activated function of drugs.

It should also be pointed out that our research found that when R2was a hypoxic-activated group, if R3and R4were both H (such as compound 023), the cytotoxicity of the target compound under a hypoxic condition was not significantly different from that under a normal oxygen condition (see Table 1), while when R1was a hypoxic-activated group, if R3and R4were both H (such as compounds 013-017), the cytotoxicity of the target compound under a hypoxic condition was significantly higher than that under a normal oxygen condition (see Table 1).

When R1was a hypoxic-activated group, R2was —CH2Ar, and Ar was a phenyl group with an electron donating group, the target compound showed a stronger cytotoxicity under a hypoxic condition (such as compounds 018-021); when Ar was a phenyl group having no substituent group or having an electron with-drawing group (such as compound 024), the cytotoxicity of the target compound under a hypoxic condition differed slightly from that under a normal oxygen condition.

Taking compound 001 as an example, the anti-tumor effect and safety performance of the target compound were investigated. The target compound showed a significant anti-tumor growth effect (seeFIG. 1andFIG. 2). At 4 times of treatment dose, compared with a control group, there was no significant difference in animal weight, indicating that the target compound had good safety performance (seeFIG. 3). Combined use with traditional anti-tumor drugs such as gemcitabine can generate better anti-tumor effect (seeFIG. 2).

Advantageous Effect

The gemcitabine ProTide hypoxic-activated prodrug of the present invention has a small cytotoxicity in a normal oxygen environment and strong cytotoxicity under a hypoxic condition, therefore, the gemcitabine ProTide hypoxic-activated prodrug can specifically play an anti-tumor effect on tumors in tumor hypoxic regions, reduce toxic and side effects on other tissues, has an excellent anti-cancer effect and good safety performance, can be used together with traditional anti-tumor drugs such as gemcitabine to generate a good anti-tumor effect at a small dose, and can be used for preparing medicaments for treating tumors. Further research finds that at 4 times of effective dose, the gemcitabine ProTide hypoxic-activated prodrug provided by the present invention has no obvious toxic effect increase compared with low dose.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The following embodiments enable those skilled in the art to fully understand the present invention, but do not limit the present invention in any way.

Embodiment 1: Synthesis of Target Compounds 001-021

Synthesis of 3′-O-(t-butyloxycarboryl) gemcitabine

Compounds 002-021 were synthesized with the same method.

Embodiment 2: Research on In-Vitro Inhibitory Effect of Target Compound on Tumor Cell Proliferation Under Normal Oxygen State and Hypoxic State

Take tumor cells in the logarithmic growth phase, add 0.25% pancreatin for digestion for 3 min, use RPMI-1640 containing 10% calf serum for suspension culture of the cells, count the number, adjust cell concentration to 1×105cells/mL, inoculate to a Top-count dedicated 96-well cell culture plate at 100 μL/well, and incubate at 37° C. and 5% CO2for 24 h; divide the cells into experimental groups and control groups, and add a target compound solution (0.001 μg/mL, 0.01 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL) to the experimental groups, wherein each concentration corresponded to four wells, and the volume of each well was made up to 200 μL; after adding samples, continue to culture for 72 h for each group (for hypoxic groups, continue to culture for 72 h at 5% CO2, 95% N2), add3H-TdR 3×105Bq to each group before culture ended, and measure the CPM (count per minute) value of each well with Top-count; and calculate the median inhibition concentration (IC50) of drugs in each experimental group on cell proliferation.

The above experimental results show that gemcitabine, NUC-1031 and compounds 022-024 have no significant difference in in-vitro inhibition on tumor cell proliferation under normal oxygen and hypoxic conditions, and compounds (1-021) of the embodiments of the present invention have significant difference (10-50 times) in in-vitro inhibition on tumor cell proliferation under normal oxygen and hypoxic conditions, indicating that the compounds of the embodiments of the present invention have a stronger cytotoxicity for tumors in hypoxic regions.

Embodiment 3: Growth Inhibition Effect of Target Compound on Orthotopic Transplantation Tumor of Human BxPC-3 Nude Mice

Take BxPC-3 human pancreatic cancer cells in the logarithmic growth phase, inoculate subcutaneously on the back of nude mice at a concentration of 5×106cells·0.2 mL−1·mouse−1, establish a human BxPC-3 nude mice subcutaneous transplantation tumor model, take out after growing into a 1 cm subcutaneous transplantation tumor, remove the central necrotic tissue under an aseptic condition, and select and cut the surrounding healthy tumor tissue into 1 mm3tissue blocks.

Preparation of surgical orthotopic transplantation model: intraperitoneally anesthetize nude mice with pentobarbital sodium (50 mg/Kg), make a cut beside the left upper rectus abdominis muscle to expose spleen and tail of pancreas, cut open capsula pancreatis, implant a tumor block into the tail of pancreas near splenic artery, and suture the capsula pancreatis.

Administration scheme: model animals were randomly divided into an experimental group (compound 001), a control group, a gemcitabine group and an NUC-1031 group 3 weeks after operation, from the third week after operation, the nude mice were injected intraperitoneally (0.2 mmol/kg, twice per week) for 4 weeks, the nude mice were killed one week after drug withdrawal, and pancreatic tumor tissues were taken and weighed. SeeFIG. 1for inhibition effect: growth inhibition effect of target compound on orthotopic transplantation tumor of human BxPC-3 nude mice. After administration, the pancreatic tumor tissue quality of nude mice in the experimental group (compound 001) was significantly lower than those of the gemcitabine group and the NUC-1031 group, indicating a better tumor growth inhibition effect.

Embodiment 4: Growth Inhibition Effect of Target Compound on Subcutaneous Transplantation Tumor of Human BxPC-3 Nude Mice

Take BxPC-3 human pancreatic cancer cells in the logarithmic growth phase, inoculate subcutaneously on the back of nude mice at a concentration of 5×106cells·0.2 mL−1·mouse−1, three weeks later, after the long diameters of the transplanted tumors in nude mice were all ≥5 mm, calculate the similar volume of tumor bodies based on the long diameter and short diameter of the transplanted tumors. Nude mice were divided into 5 groups by a random block design and allocation method according to the tumor volume.

Administration scheme: 50 model animals were randomly divided into a negative control group, a low-dose group (compound 001, 0.1 mmol/kg), a high-dose group (compound 001, 0.4 mmol/kg), a gemcitabine hydrochloride group (0.2 mmol/kg), and a combined administration group (compound 001, 0.1 mmol/kg+ gemcitabine hydrochloride, 0.1 mmol/kg), intraperitoneally injected (twice per week) for 3 weeks, and killed one week after drug withdrawal. At the same time, animal weight was measured and the eye condition of the animal was observed.

SeeFIG. 2for inhibition effect andFIG. 3for weight change: after administration, each group showed a significant tumor growth inhibition effect, and the high-dose group and the combined administration group showed a better therapeutic effect. The weights of nude mice in all experimental groups had no significant difference, but were smaller than those of the control groups.

The above examples are only to illustrate the technical concept and features of the present invention, with the purpose of enabling those familiar with the technology to understand the content of the present invention and implement it accordingly, but do not limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be included within the scope of protection of the present invention.