WO2020042729A1 - 6-双硫取代-2'-脱氧鸟苷类化合物及其制备方法和应用 - Google Patents

6-双硫取代-2'-脱氧鸟苷类化合物及其制备方法和应用 Download PDF

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WO2020042729A1
WO2020042729A1 PCT/CN2019/092094 CN2019092094W WO2020042729A1 WO 2020042729 A1 WO2020042729 A1 WO 2020042729A1 CN 2019092094 W CN2019092094 W CN 2019092094W WO 2020042729 A1 WO2020042729 A1 WO 2020042729A1
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compound
deoxyguanosine
mmol
substituted
formula
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French (fr)
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余露山
曾苏
张阳
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浙江大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/24—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 one nitrogen and one sulfur atom
    • 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
    • C07H19/16—Purine radicals
    • C07H19/173—Purine radicals with 2-deoxyribosyl as the saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives

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  • the invention relates to the technical field of medicinal chemistry, in particular to a 6-dithio-substituted-2'-deoxyguanosine compound, a preparation method thereof and application in an antitumor drug.
  • Cancer is the most important disease that endangers human health. According to the latest statistics of cancer in China in January 2019, the incidence of malignant tumors in China was 3.929 million and the deaths were 2.338 million in 2015 (statistical data is three years later). Malignant tumor deaths accounted for 23.9% of all deaths in China. In 2018, there were 18.1 million new cancer cases and 9.6 million cancer deaths worldwide. Due to the high degree of heterogeneity of tumors, some anti-tumor drugs can only be effective against certain types of tumors, and they are very resistant to drugs with repeated administration, which makes the tumor recurrence rate and mortality high. Therefore, the development of anticancer drugs has always been a hot spot in the development of new drugs.
  • the commonly used clinical treatment for colorectal cancer is 5-FU combined with oxaliplatin or irinotecan, but the effect is not good, especially for advanced patients.
  • the clinical incidence of malignant melanoma is low, metastatic malignant melanoma has no good clinical treatment at present, and the first-line treatment of dacarbazine has limited efficacy and large toxic and side effects. This shows that it is necessary to develop drugs for colorectal cancer and melanoma with good curative effect and low toxic and side effects, and it also has a very good market prospect.
  • Trx-1 Thioreductin-1
  • Trx-1 is a low molecular weight redox protein that promotes tumor growth, inhibits apoptosis, and up-regulates hypoxia-inducible factor-1 ⁇ and vascular endothelial growth factor. Trx-1 has been a popular target for discovering new anticancer drugs. Studies have shown that the disulfide compound PX-12 is a new thioredoxin inhibitor with a chemical name of 1-methylpropyl-2-imidazolyl disulfide, and its disulfide bond is the key activity of the drug molecule reaction Region, which triggers its molecularly targeted anticancer effect by reducing the activity of thioredoxin reductase and thioredoxin.
  • telomeres are located at the end of eukaryotic linear chromosomes, and their role is to maintain chromosome integrity and control the cell division cycle. Due to the "terminal replication problem", oxidative damage and other replication-related end processing events, telomeres gradually shorten each round of DNA replication in normal somatic cells. Telomerase reduces telomere shortening by adding hexameric telomere DNA (TTAGGG) repeats to the ends of linear chromosomes in the cell. Therefore, in telomerase-negative cells, because the telomeres are shortened, the cells undergo replicative senescence, but in telomerase-positive cells, the cells are immortalized and may develop cancer.
  • TTAGGG hexameric telomere DNA
  • telomere activity Most normal somatic human cells do not have telomerase activity, except for germ cells, stem cells, activated lymphocytes, but it is almost universally detected in about 85% to 90% of primary human cancers.
  • drugs targeting telomerase targets Compared with other methods, it has high tumor specificity and small side effects, mainly including four categories: 1 direct inhibition of telomerase enzyme activity; 2 Directly targeting telomere integrity, thereby promoting rapid telomere dysfunction and cancer growth inhibition; 3 vaccines targeting telomerase; 4 viruses targeting telomerase, of which the most studied are telomerase enzyme activity Direct suppression.
  • 2-Deoxyguanosine 6-thio-dG is a newly discovered small molecule telomerase inhibitor.
  • the chemical name is nucleoside 6-thio-2'-deoxyguanosine, which can be recognized by telomerase and Integration into de novo synthesized telomeres results in telomere dysfunction. Since most normal human cells do not have telomerase activity except for germ cells, stem cells, and activated lymphocytes, it is almost universally detected in about 85% to 90% of primary human cancers. Therefore, this The drugs have a broad spectrum of tumor suppressive effects.
  • 6-thio-dG is in the preclinical development stage, and Phase I studies are expected to be conducted in the second to third quarter of 2019.
  • Trx-1 and telomerase play a key role in the occurrence and development of tumors.
  • This research group designed a variety of Trx-1 and telomerase with both Trx-1 and telomerase.
  • Enzyme-inhibiting dual-target small-molecule compounds have yielded promising candidate drugs A, B, C, and D that have the effect of treating colon cancer and melanoma. It is planned to complete the comprehensive evaluation of the drugability of drug candidates A, B, C, and D through the implementation of this topic, and basically complete the preclinical evaluation of A, B, C, and D according to the requirements of the application, to promote innovative candidates with independent intellectual property rights
  • the drug stands out and lays the foundation for subsequent clinical research on new drugs.
  • the invention provides a 6-disulfide-substituted-2'-deoxyguanosine compound, a preparation method and application thereof, an enzyme activity test and an anti-tumor activity (such as a cell growth inhibitory activity test and a nude mouse tumor-bearing test) of such compounds. ) Have achieved better results.
  • R is C1-8 alkyl, C1-8 fluoroalkyl, C1-8 chloroalkyl, C1-8 hydroxyalkyl, C1-8 ketoalkyl,-(C1-4 alkyl) R1,- (C1-4alkyl) NH 2 ,-(C1-4alkyl) NH (C1-4alkyl), phenyl optionally substituted by R2, 5-membered aromatic ring optionally substituted by R2, any A C3-6 cycloalkyl optionally substituted with R2, a 5- to 6-membered heteroaromatic ring containing at least one nitrogen, optionally substituted with R2;
  • R1 is phenyl, phenyl optionally substituted with -F, -Cl, -OH, -NH 2 , -C1-4 alkyl, -OCH 3 , -CONH 2 or -CONHCH 3 , optionally substituted with R2 Substituted 5- to 6-membered heteroaromatic rings containing at least one nitrogen;
  • R2 is C1-4 alkyl, -CN, -OH, -OMe, -NH 2 , -NHMe, -N (CH 3 ) 2 , -F, -CF 3 , -CO (C1-4 alkyl),- COCH 2 CH 2 OH, -CONHCH 3 .
  • the 6-dithio-substituted-2'-deoxyguanosine compound is 6- (sec-butyldithio) -2'-deoxyguanosine having the structure of Formula A, and 6- (pair of the structure of Formula B Fluorobenzyldisulfide) -2'-deoxyguanosine, 6- (tert-butyldisulfide) -2'-structure of formula C or 6- (isopropyldisulfide) -2 of formula D '-Deoxyguanosine;
  • Another object of the present invention is to provide a method for preparing the above-mentioned 6-dithio-substituted-2'-deoxyguanosine compound, which can be achieved by the following steps:
  • R is as described in Formula I.
  • a method for preparing a 6-dithio-substituted-2'-deoxyguanosine compound includes the following steps:
  • R in the structures of Formula 5, 6, and 4 has the same meaning as R in the structure of Formula I.
  • step 1) the ratio of the amount of each of the compound 1, acetonitrile, acetic anhydride, triethylamine, and 4-dimethylaminopyridine is 50mmol to 70mmol: 130mL to 170mL: 150mmol to 210mmol: 250mmol to 350mmol: 3mmol to 9mmol . More preferably, the ratio of the respective amounts of the compound 1, acetonitrile, acetic anhydride, triethylamine, and 4-dimethylaminopyridine is 55 mmol to 65 mmol: 140 mL to 160 mL: 170 mmol to 190 mmol: 280 mmol to 320 mmol: 5 mmol to 7 mmol.
  • the ratio of the amounts of each of the compound 1, acetonitrile, acetic anhydride, triethylamine, and 4-dimethylaminopyridine is 60 mmol: 150 mL: 180 mmol: 300 mmol: 6 mmol.
  • step 2) the dichloromethane solution of the compound 2, dichloromethane, triethylamine, 2,4,6-trimethylbenzenesulfonyl chloride, 4-dimethylaminopyridine, and N-methylpyrrolidine
  • the ratio of the amount of thioacetic acid dissolved in methylene chloride is 15mmol to 25mmol: 150mL to 250mL: 5mL to 15mL: 30mmol to 50mmol: 2mmol to 6mmol: 10mL to 30mL: 20mL to 40mL.
  • the ratio of the amount of acetic acid in dichloromethane used is 18 mmol to 22 mmol: 180 mL to 220 mL: 8 mL to 12 mL: 35 mmol to 45 mmol: 3 mmol to 5 mmol: 15 mL to 25 mL: 25 mL to 35 mL.
  • the compound 2 dichloromethane, triethylamine, 2,4,6-trimethylbenzenesulfonyl chloride, 4-dimethylaminopyridine, N-methylpyrrolidine in a dichloromethane solution
  • the ratio of the amount of thioacetic acid dissolved in dichloromethane is 20 mmol: 200 mL: 10 mL: 40 mmol: 4 mmol: 20 mL: 30 mL.
  • the dichloromethane solution of N-methylpyrrolidine is composed of N-methylpyrrolidine and dichloromethane in a volume ratio of 1: 0.5-1.5, and the dichloromethane solution of thioacetic acid is 1 in volume ratio. : Composition of 1 to 3 thioacetic acid and dichloromethane.
  • the dichloromethane solution of N-methylpyrrolidine is composed of N-methylpyrrolidine and dichloromethane in a volume ratio of 1: 1
  • the dichloromethane solution of thioacetic acid is composed of a volume ratio. It is composed of 1: 2 thioacetic acid and dichloromethane.
  • step 3 the ratio of the amount of the thiol, thiourea, H 2 O, ethanol, concentrated hydrochloric acid, and H 2 O 2 of the formula 5 is 50 mmol to 80 mmol: 40 mmol to 60 mmol: 15 mL to 30 mL: 50 mL to 80mL: 4mL-7mL: 5mL-8mL.
  • the ratio of the amount of thiol, thiourea, H 2 O, ethanol, concentrated hydrochloric acid, and H 2 O 2 in the structure of Formula 5 is 60 mmol to 70 mmol: 45 mmol to 55 mmol: 20 mL to 25 mL: 60 mL to 70 mL: 5mL ⁇ 6mL: 6mL ⁇ 7mL.
  • the ratio of the amount of thiol, thiourea, H 2 O, ethanol, concentrated hydrochloric acid, and H 2 O 2 of the structure of Formula 5 is 65 mmol: 50 mmol: 22.5 mL: 65 mL: 5.5 mL: 6.5 mL.
  • the mass percentage of the concentrated hydrochloric acid is 35% to 37%.
  • step 4 the ratio of the amount of the compound 3, semicarbazide (dithiooxy) imide, methanol, and NaHCO 3 aqueous solution is 1 g to 2.5 g: 2 mmol to 8 mmol: 15 mL to 35 mL: 5 mL to 10 mL.
  • the NaHCO 3 aqueous solution is composed of NaHCO 3 and water in an amount ratio of 5 mmol to 10 mmol: 5 mL to 10 mL.
  • the ratio of the amount of the compound 3, semicarbazide (dithiooxy) imide, methanol, and NaHCO 3 aqueous solution is 1.3 g to 1.7 g: 4 mmol to 6 mmol: 22.5 mL to 27.5 mL: 6 mL to 9 mL.
  • the NaHCO 3 aqueous solution is composed of NaHCO 3 and water in an amount ratio of 6mmol to 9mmol: 6mL to 9mL.
  • the ratio of the amount of the compound 3, semicarbazide (dithiooxy) imide, methanol, and NaHCO 3 aqueous solution is 1.5 g: 5 mmol: 25 mL: 7.5 mL, and the amount of the NaHCO 3 aqueous solution is 7.5 mmol: 7.5 mL of NaHCO 3 and water.
  • the ratio of the amount of the compound 4, the amount of methanol, and the amount of ammonia water is 1 mmol to 3 mmol: 20 mL to 60 mL: 20 mL to 60 mL. More preferably, the ratio of the amount of the compound 4, the amount of methanol, and the amount of ammonia water is 1.5 mmol to 1.5 mmol: 35 mL to 45 mL: 35 mL to 45 mL. Most preferably, the ratio of the amount of the compound 4, the amount of methanol, and the amount of ammonia water is 2 mmol: 40 mL: 40 mL.
  • Another object of the present invention is to provide an application of the 6-bisthio-substituted-2'-deoxyguanosine derivative in the preparation of an antitumor drug.
  • Pharmacological studies have confirmed that the compounds provided by the present invention can be used to inhibit the activity of thioredoxin reductase and thioredoxin (TrxR / Trx-1), and can also be used to treat cancer.
  • the 6-bisthio-substituted-2'-deoxyguanosine derivative can be used for preparing antitumor drugs, and has an inhibitory effect on various cancer cells.
  • the antitumor drugs are used to prevent or / and treat colon cancer, lung cancer, and skin melanoma , Breast cancer, leukemia, liver cancer, gastric cancer, one or two or more (including two) drugs.
  • the antitumor drug is used to inhibit the activity of thioredoxin Trx.
  • the present invention has the following advantages:
  • the 6-dithio-substituted-2'-deoxyguanosine derivative of the present invention was discovered by the research group in the process of systematic structural modification of PX-12 and 6-thio-dG, and it is a new compound not reported in the literature.
  • FIG. 1 is a graph showing changes in tumor volume of nude mice in each group during administration of a tumor-bearing mouse model of HCT116 cells in the present invention
  • Figure 2 is the HE staining results of HCT116 cell tumor-bearing mouse model after administration (group D); (a) is heart, (b) is lung, (c) is kidney, (d) is liver, ( e) is the intestine and (f) is the tumor tissue;
  • Figure 3 is a graph showing the staining results of DAPI (living cells) and TUNEL (apoptotic cells) and MERGE (superimposed) staining of tumor tissues in each group after administration of a tumor-bearing mouse model of HCT116 cells in the present invention
  • A for compound A
  • B for compound B
  • C for compound C
  • D for compound D
  • E for compound 6-thio-dG
  • F for compound PX-12
  • G blank group, for solvent
  • Fig. 4 is a graph showing changes in body weight and tumor volume of nude mice in each group during the administration of A375 cell tumor-bearing mouse model, wherein Fig. 4 (a) is the change in body weight and Fig. 4 (b) is the change in tumor volume.
  • reaction solution was cooled to 0-5 ° C with an ice-water bath, and a DCM (10 ml) solution of N-methylpyrrolidine (10 ml) prepared in advance was added dropwise. Stir in a water bath for 0.5 h and at room temperature at 25 ° C for another 0.5 h. After the reaction solution was cooled to 0-5 ° C again with an ice water bath, a DCM (dichloromethane, 20ml) solution of thioacetic acid (10mL) prepared in advance was added dropwise, and the internal temperature was maintained at 5 ° C. After naturally warming to room temperature of 25 ° C, it was stirred overnight (16h).
  • the preparation method is the same as that in Preparation Examples 3-5.
  • P-fluorobenzyl mercaptan is used to replace sec-butyl mercaptan, and thiourea and p-fluorobenzyl mercaptan are reacted in concentrated hydrochloric acid and hydrogen peroxide to obtain p-fluorobenzyl semicarbazide (two Thioxy) imide was reacted with compound 3 under sodium bicarbonate, and finally hydrolyzed under the action of ammonia to obtain 0.6 g of compound B with a purity of 96% and a beige solid.
  • the preparation method is the same as that in Preparation Examples 3-5, tert-butyl mercaptan is used to replace sec-butyl mercaptan, and thiourea and tert-butyl mercaptan are reacted under concentrated hydrochloric acid and hydrogen peroxide to obtain tert-butylaminourea (dithiooxy )
  • tert-butylaminourea dithiooxy
  • the imide is then reacted with compound 3 under sodium bicarbonate and finally hydrolyzed under the action of ammonia water to obtain 0.59 g of compound C with a purity of about 95% and a pale yellow powder.
  • the preparation method is the same as in Preparation Examples 3-5.
  • Isopropyl mercaptan is used to replace sec-butyl mercaptan, and thiourea is reacted with isopropyl mercaptan under concentrated hydrochloric acid and hydrogen peroxide to obtain isopropylaminourea (dithiooxy )
  • the imide is then reacted with compound 3 under sodium bicarbonate, and finally hydrolyzed under the action of ammonia water to obtain 0.62 g of compound D with a purity of about 98% and a white powder.
  • Ellman reaction is the reaction of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) and thiol (-SH) to form a yellow substance, which has absorption at 412nm.
  • DTNB 5,5'-dithiobis (2-nitrobenzoic acid)
  • -SH thiol
  • All experiments were performed on 96-well plates. Before starting the enzyme reaction, prepare 5 groups of corresponding test compound stock solutions at different concentrations.
  • the total volume of each well of the TrxR / Trx-1 reaction is 60 ⁇ L, including 100 mM HEPES, 5 mM EDTA (HE buffer, pH 7.5), 1 mM NADPH, 1.0 ⁇ MTrxR, 0.8 ⁇ MTrx-1, 2.5 mg / mL bovine insulin and the required concentration of Test compound. No test compound was added to the control group. The blank group did not add test compounds, TrxR and Trx-1. The assay plate was shaken on a shaker for 30 seconds to thoroughly mix the reactants.
  • TrxR / Trx-1 activity% (A test- A kb ) / (A control -A kb ), that is, the relative percentage of TrxR / Trx-1 activity was inhibited by adding 2'-deoxyguanosine derivatives
  • the activity is plotted against the activity without 2'-deoxyguanosine derivatives, and the data is fitted to a concentration response curve with a variable slope.
  • the IC 50 value is calculated from the curve.
  • Table 1 The experimental results are shown in Table 1.
  • Trx-1 inhibitor PX-12 As can be seen from Table 1, the clinical phase II Trx-1 inhibitor PX-12 was used as a positive control, and the sulfur-containing anti-tumor drugs Cladribine and anti-tumor with a 2'-deoxyguanosine-like structure already on the market Candidate drug 6-thio-2'-deoxyguanosine (6-thio-dG) was used as a negative control. Some of the compounds with this new structure showed good TrxR / Trx-1 inhibitory activity. The TrxR / Trx-1IC 50 of B and Compound D reached 9.78 and 4.26 ⁇ M, respectively, which were close to the positive control PX-12, which provided a new basis for the research on the preparation of Trx targeted cancer treatment drugs.
  • MTT test 6-disulfide substituted-2′-deoxyguanosine derivatives inhibit tumor cell growth
  • the MTT method was used to determine the tumor cell growth inhibitory activity of candidate compounds. Take human tumor cells in logarithmic growth phase, digest with 0.25% trypsin for a certain time, centrifuge, resuspend and count with a blood cell counting plate to prepare a cell suspension, and adjust the cell suspension to 1.0 ⁇ 10 4 ⁇ 1 ⁇ 10 5 / mL. The cell suspension was inoculated into a 96-well culture plate, 100 ⁇ L per well, and cultured in a saturated humidity, 37 ° C. and 5% CO 2 incubator for 24 hours, and the cells were completely adhered. Dilute the test compound to the required concentration with complete medium. Aspirate the complete medium from the 96-well culture plate inoculated with human tumor cells.
  • the antitumor compound 6-thio-2'-deoxyguanosine (6-thio-dG), the Trx inhibitor PX-12, and antitumor compounds having a similar structure of 2'-deoxyguanosine that have been marketed The drug Cladribine is used as a positive control. Most of the test compounds A, B, C, D and positive controls have a significant inhibitory effect on the growth of human colon cancer cells HCT116, SW620, HT29, and human melanoma cells A375. The growth of human gastric cancer cells BGC, human leukemia cells K562, and human liver cancer cells HepG2 has a secondary inhibitory effect.
  • test compound B and D have more obvious inhibitory effects on tumor cell proliferation, and provide research for the preparation of cancer treatment drugs, especially for melanoma and colorectal cancer. Out of direction.
  • Nude mouse tumor-bearing test 6-disulfide-substituted-2'-deoxyguanosine derivatives inhibit tissue growth of subcutaneously transplanted tumors in nude mice
  • human colon cancer cell line HCT116 as the research object, we established a human colon cancer subcutaneous transplantation tumor model in 32 immunodeficiency nude mice. Take the 24th-generation HCT116 cells in logarithmic growth phase, digest with trypsin to prepare a single-cell suspension, and inoculate 1 ⁇ 10 7 cells subcutaneously in the right armpit of male nude mice about 5 weeks later. After inoculation, intraperitoneal injection of immunosuppressants Cyclophosphamide (100 mg / kg) for two consecutive days. Tumor growth was seen from 9 to 10 days.
  • nude mice were divided into 7 groups by random number table, including test drug group, positive control group, and blank control group, respectively: Group A: Compound A; Group B: Compound B; Group C: Compound C; Group D: Compound D; Group E: Compound 6-thio-dG; Group F: Compound PX-12; Group G : Blank group without giving any compound. Except 3 in group C, 4 in group G, and 5 in other 5 groups, marked, tail vein injection, administration once every two days, administration for 24 days, 12 times, dose based on compound A, the first 8 times 2mg / kg, 10 mg / kg for the last 4 times, and other molar amounts.
  • FIG. 1 Changes in tumor volume of nude mice in each group during the administration are shown in FIG. 1.
  • the slope of tumor volume change in nude mice in each group is: D ⁇ B ⁇ A ⁇ 6-thio-dG ⁇ PX-12 ⁇ C ⁇ KB, so the inhibitory effect on the growth of subcutaneously transplanted tumor tissue in nude mice is strong: D> B ⁇ A ⁇ 6-thio-dG> PX-12> C> KB.
  • Test compound D inhibited the growth of transplanted tumor tissues better than the positive control 6-thio-dG and PX-12
  • test compound B and A inhibited the growth of transplanted tumor tissues closer to the positive control 6-thio-dG than the positive control PX-12
  • D, B, and A showed the expected dual-target synergy.
  • the results of TUNEL staining are shown in Fig. 3.
  • the apoptosis rate can be calculated by the proportion of tumor tissue apoptotic dye staining, so as to speculate the strength of apoptosis in each group: group D> group A ⁇ B group ⁇ E group (6-thio -dG group)> C group> F group (PX-12 group)> G group (KB group). It is basically consistent with the tumor volume growth inhibition in nude mice.
  • Table 3 shows the results of blood biochemical indicators of nude mice in each group during the administration.
  • DTIC dacarbazine
  • Designed administration groups were: Compound D 10mg / kg group; 6-thio-dG + PX12 combined drug group (dose equivalent to 2 times the molar amount of compound D); DTIC 20.4mg / kg group (dose equivalent to 4 times) Moles of compound D) and the blank solvent group.
  • the experimental results are shown in Figure 4.
  • the weight was significantly higher than that of the DITC group, the 6-thio-dG and PX-12 combination group, indicating that the body can quickly reverse the toxicity after continuous administration.
  • the compound D had significantly more tumor suppressive effect and lower toxicity than the combination inhibitor group and body weight change.

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Abstract

本发明公开了一种6-双硫取代-2'-脱氧鸟苷类化合物及其制备方法和在抗肿瘤药物中的应用，本发明以2'-脱氧鸟苷作为结构母核，设计并合成了系列全新的硫氧还原蛋白-1(Trx-1)抑制剂，本发明化合物为式Ⅰ结构，并对该类化合物进行了Trx-1活性抑制测试，显示出明显的抑制活性。体内外研究表明，这些化合物对多种类型肿瘤表现出显著的抑瘤作用，可应用于人体细胞增殖相关的肿瘤的药物中，为抗癌药物的研究提供了新的途径。

Description

6-双硫取代-2’-脱氧鸟苷类化合物及其制备方法和应用 技术领域
本发明涉及药物化学技术领域，具体涉及一种6-双硫取代-2’-脱氧鸟苷类化合物及其制备方法和在抗肿瘤药物中的应用。
背景技术
癌症是危害人类健康的最主要的疾病。据2019年1月我国最新的癌症统计数据显示，2015(统计数据晚3年)年我国恶性肿瘤发病约392.9万人，死亡233.8万人，恶性肿瘤死亡占居民全部死因的23.9％。而2018年全球范围内有1810万癌症新发病例和960万癌症死亡病例。由于肿瘤的高度异质性，一些抗肿瘤药物只能对某种或是某类肿瘤有效，而且随着重复给药的进行又非常容易耐药，这使得肿瘤的复发率和死亡率居高不下，因此，抗癌药物的研发始终是新药研发的热点。
目前，临床上结直肠癌的常用治疗方案是5-FU联合奥沙利铂或伊立替康，但效果都不佳，特别是对中晚期病人。恶性黑色素瘤虽然临床上发病率较低，但转移性恶性黑色素瘤目前临床上没有好的治疗手段，一线用药达卡巴嗪疗效有限且毒副作用大。由此可见，开发疗效好、毒副作用低的结直肠癌和黑色素瘤治疗药物非常必要，同时也具有非常好的市场前景。
硫氧还原蛋白-1(Trx-1)是一种促进肿瘤生长，抑制细胞凋亡，上调低氧诱导因子-1α和血管内皮生长因子的低分子量的细胞氧化还原蛋白。Trx-1一直是发现新型抗癌药物的热门靶点。研究表明，二硫类化合物PX-12作为新型硫氧还原蛋白抑制剂，化学名称为1-甲基丙基-2-咪唑基二硫醚，其二硫键是该药物分子发生反应的关键活性区域，通过降低硫氧还原蛋白还原酶和硫氧还原蛋白的活性而触发了其分子靶向性的抗癌作用。目前，国内外对Trx-1促进肿瘤生长的机制，PX-12的抑瘤作用机制进行了充分的研究，并对PX-12的构效关系和结构优化等进行了研究。虽然，在临床II期试验中PX-12对进展性胰腺癌患者(一线化疗方案失败患者)未表现出显著的抑瘤作用，但是其对结直肠癌的治疗作用还是被认为值得期待。
端粒位于真核线性染色体末端，作用是保持染色体的完整性和控制细胞分裂周期。由于“末端复制问题”，氧化损伤和其他复制相关的末端处理事件，端粒在正常体细胞中每轮DNA复制逐渐缩短。端粒酶通过在细胞中的线性染色体末端添加六聚体端粒DNA(TTAGGG)重复来减少端粒缩短。因而，在端粒酶阴性的细胞中，因为端粒缩短，细胞会进行复制性衰老，但在端粒酶阳性的细胞中，细胞会永生化，有可能发展为癌症。大多数正常的体细胞人体细胞不具有端粒酶活性，除了生殖细胞，干细胞，激活的淋巴细胞，但在约85％至90％的原发性人类癌症中几乎普遍检测到它。近年来，针对端粒酶靶点的药物设计和研究越来越受到人们的关注，相对其他方法其肿瘤特异性高，副作用较小，主要包括四类：①端粒酶酶活性的直接抑制；②直接靶向端粒完整性，从而促进快速端粒功能障碍和癌症生长抑制；③靶向端粒酶的疫苗；④靶向端粒酶的病毒，其中研究最多的是端粒酶酶活性的直接抑制。目前已有多个针对该靶点的候选新药处于临床试验研究阶段，如imetelstat，GV1001，GRNVAC1等。2-脱氧鸟苷类化合物6-thio-dG是最新发现的小分子端粒酶抑制剂，化学名称是核苷6-硫代-2'-脱氧鸟苷，可被端粒酶识别，并被整合到从头合成的端粒中，导致端粒功能障碍。由于除了生殖细胞，干细胞，激活的淋巴细胞外，大多数正常的人体细胞不具有端粒酶活性，但在约85％至90％的原发性人类癌症中几乎普遍检测到它，因此，该类药物具有较为广谱的肿瘤抑制作用。目前，6-thio-dG正处于临床前开发阶段，预计在2019年2-3季度会进行临床I期研究。
综上所述，Trx-1和端粒酶在肿瘤的发生和发展过程中起着关键性的作用，本课题组针对Trx-1和端粒酶设计了多种同时具有Trx-1和端粒酶抑制作用的双靶点小分子化合物，获得了颇具苗头的具有治疗结肠癌和黑色素瘤作用的候选药物A、B、C、D。拟通过本课题的实施，完成候选药物A、B、C、D的成药性综合评价，并按申报要求基本完成A、B、C、D的临床前评价，促使具有自主知识产权的创新性候选药物脱颖而出，为后续的新药临床研究奠定基础。
本项目的开展实施，将加强和提升我国在抗肿瘤创新药物方面的研发能力，促进具有我国自主知识产权的抗肿瘤创新药物的研制，为我国医药产业提供科技支撑和保障。同时对我国的人口健康、社会和经济的可持续发展有着重要的战略意义。
发明内容
本发明提供了一种6-双硫取代-2’-脱氧鸟苷类化合物及其制备方法和应用，这类化合物的酶活性试验和抗肿瘤活性(细胞生长抑制活性试验和裸鼠荷瘤试验)都达到了较好的效果。
一种6-双硫取代-2’-脱氧鸟苷类化合物，为式Ⅰ结构：
Figure PCTCN2019092094-appb-000001
其中，R为C1-8烷基、C1-8氟烷基、C1-8氯烷基、C1-8羟基烷基、C1-8酮基烷基、-(C1-4烷基)R1、-(C1-4烷基)NH 2、-(C1-4烷基)NH(C1-4烷基)、任选地被R2取代的苯基、任选地被R2取代的5元芳环、任选地被R2取代的C3-6环烷基、任选地被R2取代的至少含1个氮的5至6元杂芳环；
R1为苯基、任选地被-F、-Cl、-OH、-NH 2、-C1-4烷基、-OCH 3、-CONH 2或-CONHCH 3取代的苯基、任选地被R2取代的至少含1个氮的5至6元杂芳环；
R2为C1-4烷基、-CN、-OH、-OMe、-NH 2、-NHMe、-N(CH 3) 2、-F、-CF 3、-CO(C1-4烷基)、-COCH 2CH 2OH、-CONHCH 3。
进一步优选，所述的6-双硫取代-2’-脱氧鸟苷类化合物为式A结构的6-(仲丁基双硫)-2’-脱氧鸟苷、式B结构的6-(对氟苄基双硫)-2’-脱氧鸟苷、式C结构的6-(叔丁基双硫)-2’-脱氧鸟苷或式D结构的6-(异丙基双硫)-2’-脱氧鸟苷；
A：6-(仲丁基双硫)-2’-脱氧鸟苷
Figure PCTCN2019092094-appb-000002
B：6-(对氟苄基双硫)-2’-脱氧鸟苷
Figure PCTCN2019092094-appb-000003
C：6-(叔丁基双硫)-2’-脱氧鸟苷
Figure PCTCN2019092094-appb-000004
D：6-(异丙基双硫)-2’-脱氧鸟苷
Figure PCTCN2019092094-appb-000005
本发明的另一个目的是提供上述6-双硫取代-2’-脱氧鸟苷类化合物的制 备方法，可以通过以下步骤实现：
合成反应式如下：
Figure PCTCN2019092094-appb-000006
R如通式I所述。
一种6-双硫取代-2’-脱氧鸟苷类化合物的制备方法，包括以下步骤：
1)将化合物1、乙腈、乙酸酐和三乙胺加入至反应器中，再加入4-二 甲氨基吡啶，加完后氮气保护下搅拌反应10～26h(进一步优选为16～20h，最优选18h)，析出白色固体，经过过滤洗涤得到化合物2；
Figure PCTCN2019092094-appb-000007
2)将化合物2和二氯甲烷加入至反应器中，再依次加入三乙胺、2,4,6-三甲基苯磺酰氯和4-二甲氨基吡啶，加完后在氮气保护下升温至回流，搅拌反应10～24h(进一步优选为14～18h，最优选16h)，之后用水淬灭，萃取后反应液再用冰水浴冷却至0-5℃后，将N-甲基吡咯烷的二氯甲烷溶液滴入，滴完后继续冰水浴下搅拌0.3h～1.5h(进一步优选0.5h)，环境温度15℃～35℃再搅拌0.3h～1.5h(进一步优选为0.5h)，反应液再次用冰水浴冷却至0-5℃后，将硫代乙酸的二氯甲烷溶液逐滴滴入，反应器内温度保持在0-6℃(进一步优选为5℃)，滴完后自然升温至环境温度15℃～35℃后搅拌反应10～24h(进一步优选为14～18h，最优选16h)，待反应完成后，加水淬灭，再用二氯甲烷萃取，经洗涤、干燥得到化合物3；
Figure PCTCN2019092094-appb-000008
3)在反应器中将式5结构的硫醇和硫脲溶于H 2O和乙醇中，冰水浴冷却至0-5℃后，将浓盐酸滴加，滴完后再将H 2O 2滴加，反应器内温度保持在0-6℃(进一步优选为5℃)，滴完后氮气保护下自然升温至环境温度15℃～35℃反应10～24h(进一步优选为14～18h，最优选16h)，之后过滤洗涤得式6结构的氨基脲(二硫氧基)酰亚胺；
Figure PCTCN2019092094-appb-000009
4)将化合物3、式6结构的氨基脲(二硫氧基)酰亚胺和甲醇加入到反应器中，再将NaHCO 3水溶液逐滴滴加，滴完后环境温度15℃～35℃反应10～24h(进一步优选为14～18h，最优选16h)，反应完成后，经后处理得到化合物4；
Figure PCTCN2019092094-appb-000010
5)将化合物4和甲醇加入到反应器中，再向反应器滴加氨水，滴完后环境温度15℃～35℃反应10～24h(进一步优选为14～18h，最优选16h)，经后处理得到式Ⅰ结构的6-双硫取代-2’-脱氧鸟苷类化合物；
Figure PCTCN2019092094-appb-000011
其中，式5、式6、式4结构中的R与式Ⅰ结构中的R具有相同意义。
步骤1)中，所述的化合物1、乙腈、乙酸酐、三乙胺、4-二甲氨基吡啶各用量之比为50mmol～70mmol：130mL～170mL：150mmol～210mmol：250mmol～350mmol：3mmol～9mmol。进一步优选，所述的化合物1、乙腈、乙酸酐、三乙胺、4-二甲氨基吡啶各用量之比为55mmol～65mmol：140mL～160mL：170mmol～190mmol：280mmol～320mmol：5mmol～7mmol。最优选的，所述的化合物1、乙腈、乙酸酐、三乙胺、4-二甲氨基吡啶各用量之比为60mmol：150mL：180mmol：300mmol：6mmol。
步骤2)中，所述的化合物2、二氯甲烷、三乙胺、2,4,6-三甲基苯磺酰氯、4-二甲氨基吡啶、N-甲基吡咯烷的二氯甲烷溶液、硫代乙酸的二氯甲烷 溶的用量之比为15mmol～25mmol：150mL～250mL：5mL～15mL：30mmol～50mmol：2mmol～6mmol：10mL～30mL：20mL～40mL。进一步优选，所述的化合物2、二氯甲烷、三乙胺、2,4,6-三甲基苯磺酰氯、4-二甲氨基吡啶、N-甲基吡咯烷的二氯甲烷溶液、硫代乙酸的二氯甲烷溶的用量之比为18mmol～22mmol：180mL～220mL：8mL～12mL：35mmol～45mmol：3mmol～5mmol：15mL～25mL：25mL～35mL。最优选地，所述的化合物2、二氯甲烷、三乙胺、2,4,6-三甲基苯磺酰氯、4-二甲氨基吡啶、N-甲基吡咯烷的二氯甲烷溶液、硫代乙酸的二氯甲烷溶的用量之比为20mmol：200mL：10mL：40mmol：4mmol：20mL：30mL。
所述的N-甲基吡咯烷的二氯甲烷溶液由体积比1：0.5～1.5的N-甲基吡咯烷和二氯甲烷组成，所述的硫代乙酸的二氯甲烷溶液由体积比1：1～3的硫代乙酸和二氯甲烷组成。最优选，所述的N-甲基吡咯烷的二氯甲烷溶液由体积比1：1的N-甲基吡咯烷和二氯甲烷组成，所述的硫代乙酸的二氯甲烷溶液由体积比1：2的硫代乙酸和二氯甲烷组成。
步骤3)中，所述的式5结构的硫醇、硫脲、H 2O、乙醇、浓盐酸、H 2O 2的用量之比为50mmol～80mmol：40mmol～60mmol：15mL～30mL：50mL～80mL：4mL～7mL：5mL～8mL。进一步优选，所述的式5结构的硫醇、硫脲、H 2O、乙醇、浓盐酸、H 2O 2的用量之比为60mmol～70mmol：45mmol～55mmol：20mL～25mL：60mL～70mL：5mL～6mL：6mL～7mL。最优选的，所述的式5结构的硫醇、硫脲、H 2O、乙醇、浓盐酸、H 2O 2的用量之比为65mmol：50mmol：22.5mL：65mL：5.5mL：6.5mL。
所述的浓盐酸的质量百分数为35％～37％。
步骤4)中，所述的化合物3、氨基脲(二硫氧基)酰亚胺、甲醇、NaHCO 3水溶液的用量之比为1g～2.5g：2mmol～8mmol：15mL～35mL：5mL～10mL，所述的NaHCO 3水溶液由用量比5mmol～10mmol：5mL～10mL的NaHCO 3和水组成。进一步优选，所述的化合物3、氨基脲(二硫氧基)酰亚胺、甲醇、NaHCO 3水溶液的用量之比为1.3g～1.7g：4mmol～6mmol：22.5mL～27.5mL：6mL～9mL，所述的NaHCO 3水溶液由用量比6mmol～9mmol：6mL～9mL的NaHCO 3和水组成。最优选，所述的化合物3、氨基脲(二硫氧基)酰亚胺、甲醇、NaHCO 3水溶液的用量之比为1.5g：5mmol：25mL：7.5mL，所述的NaHCO 3水溶液由用量比7.5mmol：7.5mL的NaHCO 3和水组成。
步骤5)中，所述的化合物4、甲醇、氨水的用量之比为1mmol～3mmol：20mL～60mL：20mL～60mL。进一步优选，所述的化合物4、甲醇、氨水的用量之比为1.5mmol～1.5mmol：35mL～45mL：35mL～45mL。最优选，所述的化合物4、甲醇、氨水的用量之比为2mmol：40mL：40mL。
本发明的另一个目的是提供所述6-双硫取代-2’-脱氧鸟苷类衍生物在制备抗肿瘤药物中的应用。药理研究证实，本发明提供的这类化合物可用来抑制硫氧还原蛋白还原酶和硫氧还原蛋白(TrxR/Trx-1)的活性，也可以用来治疗癌症。
6-双硫取代-2’-脱氧鸟苷衍生物可用于制备抗肿瘤药物，对各种癌细胞具有抑制作用，所述的抗肿瘤药物为预防或/和治疗结肠癌、肺癌、皮肤黑色素瘤、乳腺癌、白血病、肝癌、胃癌药物中的一种或者两种以上(包括两种)。
所述的抗肿瘤药物用于抑制硫氧还原蛋白Trx的活性。
与现有技术相比，本发明具有如下优点：
本发明6-双硫取代-2’-脱氧鸟苷衍生物是本课题组在对PX-12和6-thio-dG进行系统结构改造的过程中发现的，是未见文献报道的新化合物，具有完全自主知识产权；体内外抗肿瘤活性研究表明，其对不同肿瘤细胞的生长抑制活性普遍高于其类似物PX-12和6-thio-dG，高效广谱，且毒性相对较小；作用机制研究表明，该化合物除了抑制硫氧还原蛋白还原酶和硫氧还原蛋白(TrxR/Trx-1)的活性，还可阻断端粒酶，具有双靶点作用。
附图说明
图1为本发明中HCT116细胞荷瘤鼠模型给药过程中各组裸鼠肿瘤体积的变化图；
图2为本发明中HCT116细胞荷瘤鼠模型给药后HE染色结果(D组)；其中，(a)为心，(b)为肺，(c)为肾，(d)为肝，(e)为肠，(f)为肿瘤组织；
图3为本发明中HCT116细胞荷瘤鼠模型给药后各组肿瘤组织DAPI(活细胞)和TUNEL(凋亡细胞)以及MERGE(叠加)染色结果图；A：给化合物A；B：给化合物B；C：给化合物C；D：给化合物D；E：给化合物6-thio-dG；F：给化合物PX-12；G：空白组，给溶剂；
图4为A375细胞荷瘤鼠模型给药过程中各组裸鼠体重和肿瘤体积的变 化图，其中，图4(a)为体重变化，图4(b)为肿瘤体积变化。
具体实施方式
本发明结合实施例作进一步的说明。以下的实施例是说明本发明，而不是以任何方式限制本发明。
实施例1(制备二乙酰基-2'-脱氧鸟苷2)
Figure PCTCN2019092094-appb-000012
将化合物1(17.11g，60mmol)，MeCN(乙腈，150mL)，Ac 2O(乙酸酐，18.38g，180mmol)和TEA(三乙胺，30.36g，300mmol)依次加入至500ml单口瓶中，再缓慢加入DMAP(4-二甲氨基吡啶，0.73g，6mmol)，加完后氮气保护下室温25℃搅拌过夜(18h)。析出大量白色固体，TLC检测反应完全。停止反应，抽滤，滤饼再用MeCN(10mL×3)洗，抽干，再用油泵拉干。得到20.1g化合物2(经相关图谱数据确认为式2结构的化合物)，白色固体，收率95.2％。
实施例2(制备二乙酰基-6-硫代-2’-脱氧鸟苷3)
Figure PCTCN2019092094-appb-000013
将化合物2(7.03g，20mmol)和DCM(二氯甲烷，200mL)加入至250mL三口瓶中，再依次加入TEA(三乙胺，10mL)，2,4,6-三甲基苯磺酰氯(8.75g，40mmol)和DMAP(4-二甲氨基吡啶，0.49g，4mmol)，加完后在氮气保护下升温至回流，搅拌过夜(16h)。取样后用水淬灭，萃取后反应液再用冰水浴冷却至0-5℃后，将预先配好的N-甲基吡咯烷(10ml)的DCM(10ml)溶液滴入，滴完后继续冰水浴下搅拌0.5h，室温25℃再搅拌0.5h。反应液再 次用冰水浴冷却至0-5℃后，将预先配好的硫代乙酸(10mL)的DCM(二氯甲烷，20ml)溶液逐滴滴入，内温保持在5℃，滴完后自然升温至室温25℃后搅拌过夜(16h)。待反应完成后，加水(100mL)淬灭，再用DCM(二氯甲烷，100mL×4)萃取，饱和NaCl水(100mL)洗一次，无水硫酸钠干燥，硅胶拌样旋干，过柱(DCM→DCM:甲醇MeOH＝100:1→50:1→20:1→10:1)。得到5g化合物3(经相关图谱数据确认为式3结构的化合物)，浅黄色固体，收率71％。
实施例3(制备仲丁基氨基脲(二硫氧基)酰亚胺)
Figure PCTCN2019092094-appb-000014
将仲丁硫醇(5.86g，65mmol)和硫脲(3.81g，50mmol)溶于H 2O(22.5mL)和EtOH(乙醇，65mL)中，冰水浴冷却至0-5℃后，将浓盐酸(质量百分数36.5％，5.5ml)缓慢滴加，滴完后再将H 2O 2(6.5mL)逐滴滴加，内温保持在5℃，滴完后氮气保护下自然升温至室温25℃反应过夜(16h)。直接旋干，油泵拉干，得仲丁基氨基脲(二硫氧基)酰亚胺粗品。
实施例4(制备二乙酰基-6-二硫叔丁基-2’-脱氧鸟苷4a)
Figure PCTCN2019092094-appb-000015
将化合物3(粗品，1.5g)，仲丁基氨基脲(二硫氧基)酰亚胺(1.84g，5mmol)和MeOH(25mL)加入到100mL单口瓶中，加完后再将预先配好的NaHCO 3(0.63g，7.5mmol)的H 2O(7.5mL)溶液逐滴滴加，滴完后室温25℃搅拌过夜(16h)。反应完成后，加硅胶拌样旋干，过柱(DCM→DCM:MeOH＝100:1→50:1→20:1)。得到1.2g化合物4a(经相关图谱数据确认为式4a结构的化合物)，浅黄色油状固体。
实施例5(制备6-(仲丁基双硫)-2’-脱氧鸟苷A)
Figure PCTCN2019092094-appb-000016
将化合物4a(0.91g，2mmol)和MeOH(40mL)加入到100mL单口瓶中，再缓慢滴加氨水(40mL)，滴完后室温25℃搅拌过夜(16h)。TLC(薄层色谱)检测反应完全，停止反应，旋干，油泵拉干。得到0.7g化合物A，纯度在96％，米黄色固体。
化合物A表征数据如下：
1H NMR(DMSO-D 6,600MHz)δ8.17(s,1H),6.59(s,2H),6.14(t,J＝6.0Hz,1H),6.25(d,J＝4.2Hz,1H),4.93-4.90(m,1H),4.29(s,1H),3.75-3.41(m,1H),3.51-3.48(m,1H),3.44-3.41(m,1H),3.03-3.01(m,1H),2.56-2.51(m,1H),2.17-2.14(m,1H),1.59-1.54(m,1H),1.45-1.40(m,1H),1.18(d,J＝6.0Hz,3H),0.88(t,J＝6.0Hz,3H)； 13C NMR(DMSO-D 6,151MHz)δ159.8,158.2,151.4,139.8,124.4,87.7,82.9,70.8,61.7,47.5,40.1,28.3,19.6,11.3；MS(ESI)(m/z):calcd for C 14H 21N 5O 3S 2(M+H +):372.11；Found:372.27.
实施例6(制备6-(对氟苄基双硫)-2’-脱氧鸟苷B)
Figure PCTCN2019092094-appb-000017
制备方法同制备实施例3-5，用对氟苄基硫醇替换仲丁硫醇，由硫脲与对氟苄基硫醇在浓盐酸、双氧水下反应，所得对氟苄基氨基脲(二硫氧基)酰亚胺再与化合物3在碳酸氢钠下反应，最后在氨水作用下水解，得到0.6g化合物B，纯度在96％，米黄色固体。
化合物B表征数据如下：
1H NMR(DMSO-D 6,600MHz)δ8.25(d,J＝3.0Hz,1H),7.40(t,J＝7.2Hz,2H),7.12(t,J＝8.4Hz,2H),6.77(s,2H),6.25-6.23(m,1H),5.34(t,J＝3.6Hz,1H),5.01(d,J＝5.4Hz,1H),4.38(s,1H),4.23(s,2H),3.84(m,1H),3.59-3.57(m,1H),3.52-3.50(m,1H),2.63-2.61(m,1H),2.26-2.23(m,1H)； 13C NMR(DMSO-D 6,151MHz)δ161.6(d,J＝243.6Hz),159.9,157.9,151.5,139.8,132.8,131.8(d,J＝8.3Hz),124.4,115.2(d,J＝21.3Hz),87.8,82.9,70.7,61.7,41.0,40.1；MS(ESI)(m/z):calcd for C 17H 18FN 5O 3S 2(M+H +):423.08；Found:424.39.
实施例7(制备6-(叔丁基双硫)-2’-脱氧鸟苷C)
Figure PCTCN2019092094-appb-000018
制备方法同制备实施例3-5，用叔丁基硫醇替换仲丁硫醇，由硫脲与叔丁基硫醇在浓盐酸、双氧水下反应，所得叔丁基氨基脲(二硫氧基)酰亚胺再与化合物3在碳酸氢钠下反应，最后在氨水作用下水解，得到0.59g化合物C，纯度在95％左右，淡黄色粉末。
化合物C表征数据如下：
1H NMR(CDCl 3,600MHz)δ7.75(s,1H),6.26-6.23(m,1H),5.64(br,2H),5.34(s,2H),4.74(d,J＝5.4Hz,1H),4.20(s,1H),3.96(d,J＝12.6Hz,1H),3.77(d,J＝12.0Hz,1H),2.99-2.95(m,1H),2.30-2.27(m,1H),1.34(s,9H)； 13C NMR(CDCl 3,151MHz)δ162.1,158.5,149.6,140.5,126.9,89.4,87.6,73.4,63.6,49.7,40.5,30.0；MS(ESI)(m/z):calcd for C 14H 21N 5O 3S 2(M+H +):371.11；Found:372.23.
实施例8(制备6-(异丙基双硫)-2’-脱氧鸟苷D)
Figure PCTCN2019092094-appb-000019
制备方法同制备实施例3-5，用异丙基硫醇替换仲丁硫醇，由硫脲与异丙基硫醇在浓盐酸、双氧水下反应，所得异丙基氨基脲(二硫氧基)酰亚胺再与化合物3在碳酸氢钠下反应，最后在氨水作用下水解，得到0.62g化合物D，纯度在98％左右，白色粉末。
化合物D表征数据如下：
1H NMR(CDCl 3,600MHz)δ7.76(s,1H),6.34(d,J＝12.0Hz,1H),6.26-6.23(m,1H),5.39(s,2H),4.72(d,J＝4.8Hz,1H),4.19(s,1H),3.93(d,J＝12.6Hz,1H),3.75(d,J＝12.0Hz,1H),3.23-3.19(m,1H),2.96-2.91(m,1H),2.67-2.51(m,1H),2.30-2.27(m,1H),1.31(s,3H),1.30(s,3H)； 13C NMR(CDCl 3,151MHz)δ162.0,158.6,149.7,140.5,126.8,89.2,87.4,73.1,63.5,41.8,40.4,22.51,22.47；MS(ESI)(m/z):calcd for C 13H 19N 5O 3S 2(M+H +):357.09；Found:358.04.
生物实验1：
Ellman试验——6-双硫取代-2’-脱氧鸟苷类衍生物类似物化合物对TrxR/Trx-1活性抑制作用
我们使用基于Ellman反应的TrxR/TR实验来测定候选化合物的抑制活性。Ellman反应是5，5’-二硫代双(2-硝基苯甲酸)(DTNB)与巯基(-SH)反应生成黄色物质，在412nm下有吸收。吸收的增加可等量对应TrxR介导的NADPH的减少，从而进一步判断TrxR/Trx-1的活性。所有实验都在96孔板上进行。在酶反应开始之前先制备5组不同浓度的相应待测化合物储备液。TrxR/Trx-1反应每孔总体积为60μL，包括100mM HEPES，5mM EDTA(HE buffer，pH 7.5)，1mM NADPH，1.0μMTrxR，0.8μMTrx-1，2.5mg/mL牛胰岛素和所需浓度的待测化合物。对照组不加待测化合物。空白组不加待测化合物、TrxR和Trx-1。将测定板在振荡器上摇动30秒以充分混合反应物。在37℃孵育30min后，然后加入100μL现配的包含6M盐酸胍、50mMTris和10mM DTNB的混合溶液终止反应。再孵育15min后，在412nm波长下 测定吸光度。最后，TrxR/Trx-1活性％＝(A 测-A kb)/(A control-A kb)，即TrxR/Trx-1活性相对百分率为加了2’-脱氧鸟苷类衍生物抑制了的活性相对于不加2’-脱氧鸟苷类衍生物的活性，作图，并将数据拟合为具有可变斜率的浓度响应曲线，从曲线中计算IC 50值，实验结果见表1。
表1 6-双硫取代-2’-脱氧鸟苷类衍生物的TrxR/Trx-1活性抑制
化合物 A B C D 6-thio-dG PX-12 Cladribine
IC 50(μM) 31.71 9.78 47.73 4.26 >100 2.55 >100
由表1可知，以临床Ⅱ期的Trx-1抑制剂PX-12作为阳性对照，以已经上市的具有2’-脱氧鸟苷类似结构的含硫抗肿瘤药物克拉屈滨(Cladribine)和抗肿瘤候选药物6-硫代-2’-脱氧鸟苷(6-thio-dG)作为阴性对照，该类全新结构的化合物中有部分化合物表现出了较好的TrxR/Trx-1抑制活性，其中化合物B和化合物D的TrxR/Trx-1IC 50分别达到了9.78和4.26μM，接近阳性对照PX-12，为制备靶向Trx的癌症治疗药物的研究提供了新的依据。
生物实验2：
MTT试验——6-双硫取代-2’-脱氧鸟苷类衍生物对肿瘤细胞生长抑制作用
我们使用MTT法对候选化合物的肿瘤细胞生长抑制活性进行测定。分别取对数生长期的人肿瘤细胞，用0.25％的胰酶消化一定时间，离心，重悬后用血细胞计数板计数，制备细胞悬液，调整细胞悬液至1.0×10 4～1×10 5个/mL。取细胞悬液接种于96孔培养板中，每孔100μL，置饱和湿度、37℃和5％CO 2培养箱中培养24h，待细胞完全贴壁。用完全培养基稀释受试化合物至所需浓度，将已接种人肿瘤细胞的96孔培养板的完全培养基吸出，加入含药培养基，每孔100μL，DMSO终浓度小于0.1％，置于培养箱中培养48h。96孔板中每孔加入MTT 10μL，继续培养4h。终止培养，小心吸去孔内培养液。每孔加入150μL DMSO，置摇床上低速振荡10min，使结晶物充分溶解。在酶联免疫检测仪OD 570nm处测量各孔的吸光值。同时设置调零孔(培养基、MTT、二甲基亚砜)，对照孔(细胞、相同浓度的药物溶解介质、培养液、MTT、二甲基亚砜)，以相应溶剂为对照，计算细胞生长抑制活性情 况。每组设定3-6个复孔，重复若干次。细胞活力(cell viability of control)＝(A 药物组-A 调零孔)/(A 对照孔-A 调零孔)*100％。作图，并将数据拟合为具有可变斜率的浓度响应曲线，从曲线中计算IC 50值，实验结果见表2。
表2 6-双硫取代-2’-脱氧鸟苷类衍生物的肿瘤细胞生长抑制(IC 50，μM)
Figure PCTCN2019092094-appb-000020
由表2可知，以抗肿瘤化合物6-硫代-2’-脱氧鸟苷(6-thio-dG)、Trx抑制剂PX-12和已经上市的具有2’-脱氧鸟苷类似结构的抗肿瘤药物克拉屈滨(Cladribine)作为阳性对照，受试化合物A、B、C、D及阳性对照大多对人结肠癌细胞HCT116、SW620、HT29、人黑色素瘤细胞A375的增值有明显的抑制作用，对人胃癌细胞BGC、人白血病细胞K562、人肝癌细胞HepG2的增值有次之的抑制作用、对人非小细胞肺癌A549、人乳腺癌细胞MCF7、Bcap37的增值抑制作用相对较弱，对人正常结肠上皮细胞NCM460的增殖几乎无抑制作用。其中，对人结直肠肿瘤细胞HCT116，受试化合物作用明显，且优于阳性对照。综合各类细胞，相较于受试化合物A、C，受试化合物B、D对肿瘤细胞增殖抑制作用效果更明显，为制备癌症治疗药物，特别是黑色素瘤和结直肠癌治疗药物的研究提供了方向。
生物实验3：
裸鼠荷瘤试验——6-双硫取代-2’-脱氧鸟苷类衍生物对裸鼠皮下移植瘤 组织生长抑制作用
1)人结肠癌细胞株HCT116荷瘤鼠模型
我们以人结肠癌细胞株HCT116为研究对象，对32只免疫缺陷裸鼠建立人结肠癌皮下移植瘤模型。取对数生长期的第24代HCT116细胞，用胰酶消化后制备单细胞悬液，于5周左右的雄性裸鼠右侧腋窝皮下接种1×10 7个细胞，接种后腹腔注射免疫抑制剂环磷酰胺(100mg/kg)，连续两天。9～10天见肿瘤生长，结肠癌细胞HCT116植入皮下后第20天，用随机数字表法将裸鼠分为7组，包括受试药组、阳性对照组和空白对照组，分别为：A组：给化合物A；B组：给化合物B；C组：给化合物C；D组：给化合物D；E组：给化合物6-thio-dG；F组：给化合物PX-12；G组：空白组，不给任何化合物。除C组3只，G组4只，其它5组均5只，标记，尾静脉注射，每两天给药一次，给药24天，12次，剂量以化合物A计，前8次2mg/kg，后4次10mg/kg，其他同摩尔量。肿瘤体积的计算方法是将肿瘤的长度作为肿瘤最长的直径，宽度作为相应的垂直直径，肿瘤体积V＝(长×宽 2)mm 3/2。最后一剂治疗后48小时，眼眶取血，然后处死动物。血液离心取上清，然后血生化分析。固定组织石蜡包埋切片，然后HE染色。冰冻组织中瘤块进行冰冻切片，然后DAPI和TUNEL染色，判断裸鼠肿瘤组织中细胞凋亡情况。实验结果如下。
给药过程中各组裸鼠肿瘤体积的变化如图1所示。各组裸鼠肿瘤体积变化斜率大小为：D<B≈A≈6-thio-dG<PX-12<C<KB，因而对裸鼠皮下移植瘤组织生长抑制作用强弱：D>B≈A≈6-thio-dG>PX-12>C>KB。受试化合物D抑制移植瘤组织生长能力强于阳性对照6-thio-dG和PX-12，受试化合物B和A抑制移植瘤组织生长能力与阳性对照6-thio-dG接近，强于阳性对照PX-12，D、B和A表现了预期的双靶点协同作用。
HE染色结果如图2所示，可通过观察各组织的形态判断化合物的毒副作用，结果表明各组之间无明显差异，化合物无明显毒副作用。
TUNEL染色结果如图3所示，可通过瘤组织凋亡染料染色所占比例计算凋亡率，从而推测各组细胞凋亡强弱：D组>A组≈B组≈E组(6-thio-dG组)>C组>F组(PX-12组)>G组(KB组)。基本与裸鼠肿瘤体积生长抑制情况相符。
给药过程中各组裸鼠血生化指标检测结果如表3所示。
表3 HCT116细胞荷瘤鼠模型给药过程中各组裸鼠血生化指标检测结果
Figure PCTCN2019092094-appb-000021
由表3可知，裸鼠各组血生化指标基本无显著性差异。
2)人黑色素瘤细胞A375荷瘤鼠模型
我们采用人恶性黑色素瘤细胞A375荷瘤鼠模型进一步研究了化合物D对人恶性黑色素瘤的抑制作用，方法基本同上。达卡巴嗪(Dacarbazine，DTIC)为黑色素瘤一线用药。设计给药组分别为：化合物D 10mg/kg组；6-thio-dG+PX12联合用药组(剂量分别相当于2倍的化合物D摩尔数量)；DTIC 20.4mg/kg组(剂量相当于4倍的化合物D摩尔数量)以及空白溶剂组。实验结果见图4。
结果发现，按摩尔数计算，化合物D给药剂量为阳性对照药DITC给药剂量的四分之一时，其抑瘤作用都显著高于DITC，而且其抑瘤作用也显著高于2倍摩尔给药剂量下6-thio-dG和PX-12联合用药组(见图4)。该结果表明，化合物D的抑制人恶性黑色素瘤活性要显著高于临床一线用药DITC，而且从大鼠体重来看，虽然给药初期化合物D组体重下降较快，但是给药6 天后体重开始逐渐升高，到给药13天后，体重明显超过DITC组、6-thio-dG和PX-12联合用药组，表明连续给药后机体能快速逆转该毒性。此外，从与6-thio-dG和PX-12联合用药组的抑瘤作用和体重变化相比，化合物D比联合用药组抑瘤作用显著增加，而且毒性更低。
此外，各组血生化指标基本无显著性差异，都未显示明显的毒性。

Claims (10)

  1. 一种6-双硫取代-2’-脱氧鸟苷类化合物，其特征在于，为式Ⅰ结构：
    Figure PCTCN2019092094-appb-100001
    其中，R为C1-8烷基、C1-8氟烷基、C1-8氯烷基、C1-8羟基烷基、C1-8酮基烷基、-(C1-4烷基)R1、-(C1-4烷基)NH 2、-(C1-4烷基)NH(C1-4烷基)、任选地被R2取代的苯基、任选地被R2取代的5元芳环、任选地被R2取代的C3-6环烷基、任选地被R2取代的至少含1个氮的5至6元杂芳环；
    R1为苯基、任选地被-F、-Cl、-OH、-NH 2、-C1-4烷基、-OCH 3、-CONH 2或-CONHCH 3取代的苯基、任选地被R2取代的至少含1个氮的5至6元杂芳环；
    R2为C1-4烷基、-CN、-OH、-OMe、-NH 2、-NHMe、-N(CH 3) 2、-F、-CF 3、-CO(C1-4烷基)、-COCH 2CH 2OH、-CONHCH 3。
  2. 根据权利要求1所述的6-双硫取代-2’-脱氧鸟苷类化合物，其特征在于，所述的6-双硫取代-2’-脱氧鸟苷类化合物为式A结构的6-(仲丁基双硫)-2’-脱氧鸟苷、式B结构的6-(对氟苄基双硫)-2’-脱氧鸟苷、式C结构的6-(叔丁基双硫)-2’-脱氧鸟苷或式D结构的6-(异丙基双硫)-2’-脱氧鸟苷；
    Figure PCTCN2019092094-appb-100002
  3. 根据权利要求1所述的6-双硫取代-2’-脱氧鸟苷类化合物的制备方法，其特征在于，包括以下步骤：
    1)将化合物1、乙腈、乙酸酐和三乙胺加入至反应器中，再加入4-二甲氨基吡啶，加完后氮气保护下搅拌反应10～26h，析出白色固体，经过过滤洗涤得到化合物2；
    Figure PCTCN2019092094-appb-100003
    2)将化合物2和二氯甲烷加入至反应器中，再依次加入三乙胺、2,4,6-三甲基苯磺酰氯和4-二甲氨基吡啶，加完后在氮气保护下升温至回流，搅拌反应10～24h，之后用水淬灭，萃取后反应液再用冰水浴冷却至0-5℃后，将N-甲基吡咯烷的二氯甲烷溶液滴入，滴完后继续冰水浴下搅拌0.3h～1.5h， 环境温度15℃～35℃再搅拌0.3h～1.5h，反应液再次用冰水浴冷却至0-5℃后，将硫代乙酸的二氯甲烷溶液逐滴滴入，反应器内温度保持在0-6℃，滴完后自然升温至环境温度15℃～35℃后搅拌反应10～24h，待反应完成后，加水淬灭，再用二氯甲烷萃取，经洗涤、干燥得到化合物3；
    Figure PCTCN2019092094-appb-100004
    3)在反应器中将式5结构的硫醇和硫脲溶于H 2O和乙醇中，冰水浴冷却至0-5℃后，将浓盐酸滴加，滴完后再将H 2O 2滴加，反应器内温度保持在0-6℃，滴完后氮气保护下自然升温至环境温度15℃～35℃反应10～24h，之后过滤洗涤得式6结构的氨基脲(二硫氧基)酰亚胺；
    Figure PCTCN2019092094-appb-100005
    4)将化合物3、式6结构的氨基脲(二硫氧基)酰亚胺和甲醇加入到反应器中，再将NaHCO 3水溶液逐滴滴加，滴完后环境温度15℃～35℃反应10～24h，反应完成后，经后处理得到化合物4；
    Figure PCTCN2019092094-appb-100006
    5)将化合物4和甲醇加入到反应器中，再向反应器滴加氨水，滴完后环境温度15℃～35℃反应10～24h(进一步优选为14～18h，最优选16h)，经后处理得到式Ⅰ结构的6-双硫取代-2’-脱氧鸟苷类化合物；
    Figure PCTCN2019092094-appb-100007
    其中，式5、式6、式4结构中的R与式Ⅰ结构中的R具有相同意义。
  4. 根据权利要求3所述的6-双硫取代-2’-脱氧鸟苷类化合物的制备方法，其特征在于，步骤1)中，所述的化合物1、乙腈、乙酸酐、三乙胺、4-二甲氨基吡啶各用量之比为50mmol～70mmol：130mL～170mL：150mmol～210mmol：250mmol～350mmol：3mmol～9mmol。
  5. 根据权利要求3所述的6-双硫取代-2’-脱氧鸟苷类化合物的制备方法，其特征在于，步骤2)中，所述的化合物2、二氯甲烷、三乙胺、2,4,6-三甲基苯磺酰氯、4-二甲氨基吡啶、N-甲基吡咯烷的二氯甲烷溶液、硫代乙酸的二氯甲烷溶的用量之比为15mmol～25mmol：150mL～250mL：5mL～15mL：30mmol～50mmol：2mmol～6mmol：10mL～30mL：20mL～40mL；
    所述的N-甲基吡咯烷的二氯甲烷溶液由体积比1：0.5～1.5的N-甲基吡咯烷和二氯甲烷组成，所述的硫代乙酸的二氯甲烷溶液由体积比1：1～3的硫代乙酸和二氯甲烷组成。
  6. 根据权利要求3所述的6-双硫取代-2’-脱氧鸟苷类化合物的制备方法，其特征在于，步骤3)中，所述的式5结构的硫醇、硫脲、H 2O、乙醇、浓盐酸、H 2O 2的用量之比为50mmol～80mmol：40mmol～60mmol：15mL～30mL：50mL～80mL：4mL～7mL：5mL～8mL；
    所述的浓盐酸的质量百分数为35％～37％。
  7. 根据权利要求3所述的6-双硫取代-2’-脱氧鸟苷类化合物的制备方法，其特征在于，步骤4)中，所述的化合物3、氨基脲(二硫氧基)酰亚胺、甲醇、NaHCO 3水溶液的用量之比为1g～2.5g：2mmol～8mmol：15mL～35mL：5mL～10mL，所述的NaHCO 3水溶液由用量比5mmol～10mmol：5mL～10mL的NaHCO 3和水组成。
  8. 根据权利要求3所述的6-双硫取代-2’-脱氧鸟苷类化合物的制备方法，其特征在于，步骤5)中，所述的化合物4、甲醇、氨水的用量之比为 1mmol～3mmol：20mL～60mL：20mL～60mL。
  9. 根据权利要求1或2所述的6-双硫取代-2’-脱氧鸟苷类化合物在制备抗肿瘤药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述的抗肿瘤药物为预防或/和治疗结肠癌、肺癌、皮肤黑色素瘤、乳腺癌、白血病、肝癌、胃癌药物中的一种或者两种以上。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002033128A2 (en) * 2000-10-18 2002-04-25 Pharmasset Limited Multiplex quantification of nucleic acids in diseased cells
WO2004094447A1 (ja) * 2003-04-22 2004-11-04 Kyushu Tlo Company, Limited チオヌクレオシド－ｓ－ニトロシル誘導体
CN109053841A (zh) * 2018-08-30 2018-12-21 浙江大学 6-双硫取代-2’-脱氧鸟苷类化合物及其制备方法和应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2679700A1 (en) * 2007-03-02 2008-09-12 Talia Miron Mercaptopurine derivatives as anticancer agents
CN102643247A (zh) * 2011-02-21 2012-08-22 中国科学院上海药物研究所 一类二硫醚类化合物及其制备方法和用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002033128A2 (en) * 2000-10-18 2002-04-25 Pharmasset Limited Multiplex quantification of nucleic acids in diseased cells
WO2004094447A1 (ja) * 2003-04-22 2004-11-04 Kyushu Tlo Company, Limited チオヌクレオシド－ｓ－ニトロシル誘導体
CN109053841A (zh) * 2018-08-30 2018-12-21 浙江大学 6-双硫取代-2’-脱氧鸟苷类化合物及其制备方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SANEYOSHI, MINEO ET AL.: "Synthetic Nucleosides and Nucleotides. X. Synthesis and Biological Activities of Several Purin-6-yl Benzyl Disulfides and Their Ribonucleosides", JOURNAL OF PHARMACOBIO-DYNAMICS, vol. 1, no. 3, 31 December 1978 (1978-12-31), XP002480550, ISSN: 0386-846X, DOI: 20190917140257X *
SANEYOSHI, MINEO ET AL.: "Synthetic Nucleosides and Nucleotides. X. Synthesis and Biological Activities of Several Purin-6-yl Benzyl Disulfides and Their Ribonucleosides", JOURNAL OF PHARMACOBIO-DYNAMICS, vol. 1, no. 3, 31 December 1978 (1978-12-31), XP002480550, ISSN: 0386-846X, DOI: 20190917140308A *

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