CN102286047A

CN102286047A - 2'-deoxidized-2'-fluorin-4'-triazole substituted-beta-D cytidine analogue as well as preparation method and application thereof

Info

Publication number: CN102286047A
Application number: CN2011102713813A
Authority: CN
Inventors: 常俊标; 武杰; 宋传君
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2011-09-14
Filing date: 2011-09-14
Publication date: 2011-12-21

Abstract

The invention relates to a pyrimidine nucleoside derivative as well as a preparation method and application thereof, in particular to a 2'-deoxidized-2'-fluorin-4'-triazole substituted-beta-D cytidine analogue as well as a preparation method and application thereof to antitumor, antivirus and other aspects, belonging to the field of pharmacochemistry. The pyrimidine nucleoside derivative as a compound has the structural general formula as the specification, and the compound as well as salt and a prodrug thereof are used for preparing antitumor and antivirus medicaments or medicinal preparations for treating, preventing or relieving tumors and viruses, and have better activity.

Description

2 '-deoxidation-2 '-fluoro-4 '-triazole replacement-β-D cytidine analog, its preparation method and application thereof

Technical field

The present invention relates to miazines nucleoside derivates, preparation method and application thereof, relating in particular to 2 '-position fluorine replaces, the miazines nucleoside derivates that 4 '-position triazole replaces, its preparation method and in the application of aspect such as antitumor and antiviral belong to the pharmaceutical chemistry field.

Technical background

Cancer remains one of main disease of the most common and serious threat human life health, is the killer of second maximum of human health.The existing means of treatment cancer comprise: excision, and radiotherapy, chemical medicinal treatment, methods such as immunotherapy, interventional therapy, importing treatment, or the merging of these methods is used.In all these methods, chemotherapy is most widely used and has been used for multiple different cancer, especially those cancers that almost can't treat with additive method.Be used to clinically although much have the compound of chemotherapy effect, because curative effect is outstanding inadequately, therapeutic domain is narrow, and most of chemotherapy all are only limited to the deterioration that delays cancer and are not real healing.Simultaneously, because tumour cell produces resistance to some medicines, tumour and transfer thereof usually can't be treated with chemotherapy.Some tumour has the intrinsic resistance to the chemotherapeutics of some kinds.Tumour produces in therapeutic process gradually to the resistance of chemotherapeutics, and therefore, the effect of existing chemotherapeutics when treatment different sorts tumour is subjected to great restriction.In addition, the chemotherapeutics of a lot of different sorts treatment cancers all produces very serious toxic side effect, thereby causes chemotherapy not proceed down.So, seek chemical anticarcinogenic drug new, that better efficacy, toxic side effects are little clinically and press for safeguarding that human health remains.

Various viruses are as hepatitis B, third liver and acquired immune deficiency syndrome (AIDS) etc., very serious to the harm that the mankind bring.Bird flu is arrived period, does not almost have medicine and can treat.In 6,000,000,000 populations of the whole world, 1/2 people lives in the high popular district of hepatitis B virus (HBV), and about 2,000,000,000 people have been proved to be HBV and have infected, and 3～400,000,000 people are the HBV chronic infection, wherein 15%～25% finally will die from liver cirrhosis and liver cancer.In preceding 10 the disease causes of the death in the whole world, hepatitis B accounts for the 7th, and be about 100 ten thousand examples because of hepatitis B death person every year.Hepatitis B virus is the major disease that threatens human health.Be faced with many difficult problems in treatment HBV medicine, as resistance, drug target is unclear, produces resistance etc.Still need to research and develop anti-HBV new drug efficient, low toxicity.

It is global popular that hepatitis C is, and is the main reason of America and Europe and Japan and other countries hepatopathy in whole latter stage.Suffer from third liver according to about 1.8 hundred million people in the statistics whole world in 2009 of the World Health Organization, and about 3.5 ten thousand examples of annual New Development hepatitis C case.The whole world was doubled by the death toll that the relevant hepatopathy of third liver causes in per 10 years approximately, and the present third liver mortality ratio comes the tenth in all diseases of the whole world.Also there is not hepatitis C vaccine at present.The ribavirin (ribavirin) and the glycol interferon alpha-2b combination therapy chronic hepatitis C of U.S. Valeant invention, be considered to conventional treatment, HCV rate of virological response (SVR) only is 47%～54%, and also has the part patient to have to discontinue medication because of its severe side effect.Also have numerous patient's myrrhs to use.Therefore, research and development are efficient, the anti-third liver new drug of low toxicity is extremely urgent.

Virus of AIDS (HIV) is the major disease of serious threat human health.Though the misery that existing several nucleoside medicines are used to slow down aids patient, mostly curative effect is not high, and toxic side effect is big, and is very easy to produce resistance.Therefore, research ucleosides anti-AIDS new drug remains the key areas that the mankind defeat serious disease.

Natural product picodna DNA and the modified modification of nucleic acid RNA monomer nucleosides and the natural product class medicine that obtains, toxic side effect is lower.Up to the present, there be more than 70 nucleoside medicine to be widely used in treating diseases such as cancer, virus.Yet, still can not satisfy the needs of safeguarding human health, still need research and develop out better ucleosides new drug, optimal medicine should be to treat virus and tumour simultaneously, can play even more important effect to human health.

Summary of the invention

It is antitumor that the object of the invention is to provide a class to have, anticancer, antiviral, and 2 '-position fluorine of infection and anti-hyperplasia diffusion isoreactivity replaces the miazines nucleoside derivates that 4 '-position triazole replaces; Another purpose is to provide its preparation method; Purpose is to provide its application aspect the antiviral and antitumor drug in preparation again.

For realizing the object of the invention, the present invention has carried out structural modification to ribonucleoside, and synthesized 2 '-position fluorine and replaced, miazines nucleosides and epimer thereof that 4 '-position triazole replaces, and comprise its spendable various salt and derivative in pharmacy.It has following general structure, and comprises operable salt in its pharmacy, ester, prodrug or its metabolite.

Described R is one of following substituting group: C ₄-C ₈Straight chained alkyl, pyridyl or thienyl, ethanoyl, carboxyl, the formicester base, the cyclopropane base, the tertiary butyl, the cyclopentanol base, (trimethylammonium) silylation, the 1-methylcyclohexyl, phenyl is by C ₁-C ₅The phenyl that alkyl, hydroxyl or methoxyl group replace,

n=?2-9,

，-CH ₂-O-ph ,

，

Deng.

It can pass through active compound (I) or its prodrug or its 5 '-phosphoric acid ester and organic acid or inorganic acid reaction and form salt, exists with the form of salt.

It can be one of following compound and be not limited only to these compounds:

Reaction scheme, method are as follows:

Compound ISynthetic: known compound 2 '-deoxidation-2 '-fluoro-4 '-azido-beta-D-cytidine is joined in the mixed solution of the trimethyl carbinol and water, after the stirring and dissolving, add corresponding alkynes, and adding N, it is alkalescence that N '-diisopropylethylamine (DIPEA) adjusts solution, adds the acetonitrile solution of CuI again under lucifuge.Add sodium ascorbate under the nitrogen atmosphere, normal temperature stirs down.Evaporated under reduced pressure solution obtained compound with column chromatography separating purification after reaction finished I

That compound of being contained in the general formula of the present invention and salt thereof and prodrug are used for preparation treatment, prevent or alleviate is antitumor, antiviral or pharmaceutical preparation.Be particularly useful for treating or alleviate the cancer that tissue or organ tumor cell cause.The preferred colorectal carcinoma of indication cancer, liver cancer, lymphoma, lung cancer, the esophageal carcinoma, mammary cancer, central nerve neuroma, melanoma, ovarian cancer, cervical cancer, kidney, leukemia, prostate cancer, carcinoma of the pancreas, cancer of the stomach etc.Compound in the general formula of the present invention also can be used for treating or prevents or alleviate the disease that virus causes.The preferred hepatitis B of indication virus, hepatitis C, virus of AIDS, yellow fever virus (YFV), respiratory syncytial virus (RSV), hsv (HSV), bovine viral diarrhea virus (BVDV), hepatitis G virus (HGV), GB virus-B(GBV-B), dengue virus (Dengue), ERC group virus (HRV), poliovirus (Poliovirus), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV) etc.

The compound salt form can be by negative ion formation suitable in group that has positive charge in the molecule (as amido) and the pharmacy in the general formula of the present invention.Suitable negative ion includes, but are not limited to fluorochemical, muriate, bromide, iodide, vitriol, nitrate, phosphoric acid salt, tartrate, metilsulfate, trifluoroacetate, maleate, acetate etc.Spendable salt can also be formed by electronegative group (carboxyl) in the compound and positive ion in the pharmacy.Can be used for positively charged ion of this purpose such as sodium salt, sylvite, magnesium salts, calcium salt and organic amine salt ion such as tetramethyl-ammonium salt ion, TBuA salt ion and other organic ion.

Innovative point of the present invention and advantage are: the nucleoside pyrimidine glycosyl is modified, synthesized series 2 '-position fluorine and replaced the miazines nucleoside derivates that 4 '-position triazole replaces, these compounds have antitumor, anticancer, antiviral, infection and anti-hyperplasia diffusion isoreactivity; The preparation method is feasible; Be applied to treat virus and tumour medicine, have good application prospect.

Embodiment

By following specific embodiment the present invention is better illustrated.

The general formula compound according to the present invention ( I) synthetic route and in conjunction with the embodiments invention is further specified, but be not to limit the scope of the invention.

Embodiment 1:

2 '-deoxidation-2 '-fluoro-4 '-(4-cyclopropane base-1,2,3-triazol radical)-β-D-cytidine ( 1) preparation

In the single port bottle of 50 mL, add 0.2 mmol 2 '-deoxidation-2 '-fluoro-4 '-azido-beta-D-cytidine, the trimethyl carbinol (1 mL), water (1 mL), stirring at room dissolving.Add ring third acetylene (0.24 mmol) and N, N '-diisopropylethylamine (DIPEA) (0.3 mmol).Adding CuI(0.01 mmol under the lucifuge) 1 mL acetonitrile solution.Add NaAsc(0.01 mmol under the nitrogen atmosphere), normal temperature stirs after 24 hours down, adds NaAsc (0.01 mmol), continues stirring at normal temperature 24 hours, and the TLC monitoring reaction is complete to example reaction.The evaporated under reduced pressure solvent, column chromatography (methylene dichloride: methyl alcohol=10:1) separate obtain white solid ( 1), fusing point: 156-158 oC, productive rate 85%; HRMS [M+Na]: 375.1174;

¹H-NMR?(CD ₃OD,?300?MHz)?δ:?7.94?(1H,?d,? J?=?7.4?Hz),?7.85?(1H,?s),?6.80?(1H,?dd,? J?=?11.4,?5.0?Hz),?5.97 (1H,?br),?5.36?(1H,?dt,? J?=?54.0,?4.8?Hz),?4.29?(1H,?d,? J?=?12.5?Hz),?4.24?(1H,?d,? J?=?12.5?Hz),?1.00-0.78?(5H,?M).

Embodiment 2:

2 '-deoxidation-2 '-fluoro-4 '-(4-phenyl-1,2,3-triazol radical)-β-D-cytidine ( 2) preparation

In the single port bottle of 50 mL, add 0.2 mmol 2 '-deoxidation-2 '-fluoro-4 '-azido-beta-D-cytidine, the trimethyl carbinol (1 mL), water (1 mL), stirring at room dissolving.Add phenylacetylene (0.24 mmol) and N, N '-diisopropylethylamine (DIPEA) (0.3 mmol).Adding CuI(0.01 mmol under the lucifuge) 1 mL acetonitrile solution.Add NaAsc(0.01 mmol under the nitrogen atmosphere), normal temperature stirs down, and the TLC monitoring reaction is complete to example reaction, reacts completely in about 20 hours.The evaporated under reduced pressure solvent, column chromatography (methylene dichloride: methyl alcohol=10:1) separate obtain faint yellow solid ( 2), fusing point: 163-164 oC, productive rate 91%; HRMS [M+Na]: 411.1163;

¹H-NMR?(CD ₃OD,?300?MHz)?δ:?8.51?(1H,?s),?7.97?(1H,?d,? J?=?7.6?Hz),?7.85?(2H,?d,? J?=?7.2?Hz),?7.44-7.30?(3H,?m),?6.91?(1H,?dd,? J?=?11.9,?4.8?Hz),?6.00?(1H,?br),?5.43?(1H,?dt,? J?=?53.8,?4.5?Hz),?4.41?(1H,?d,? J?=?12.2?Hz),?4.34?(1H,?d,? J?=?12.2?Hz).

Embodiment 3:

2 '-deoxidation-2 '-fluoro-4 '-(4-p-methylphenyl-1,2,3-triazol radical)-β-D-cytidine ( 3) preparation

In the single port bottle of 50 mL, add 0.2 mmol 2 '-deoxidation-2 '-fluoro-4 '-azido-beta-D-cytidine, the trimethyl carbinol (1 mL), water (1 mL), stirring at room dissolving.Adding is to methylbenzene acetylene (0.24 mmol) and N, N '-diisopropylethylamine (DIPEA) (0.3 mmol).Adding CuI(0.01 mmol under the lucifuge) 1 mL acetonitrile solution.Add NaAsc(0.01 mmol under the nitrogen atmosphere), normal temperature stirs down, and the TLC monitoring reaction is complete to example reaction, reacts completely in about 20 hours.The evaporated under reduced pressure solvent, column chromatography (methylene dichloride: methyl alcohol=10:1) separate obtain faint yellow solid ( 3), fusing point: 171-172 oC, productive rate 90%; HRMS [M+Na]: 425.1317;

¹H-NMR?(CD ₃OD,?300?MHz)?δ:?8.46?(1H,?s),?7.98?(1H,?d,? J?=?7.5?Hz),?7.73?(2H,?d,? J?=?8.1?Hz),?7.23?(2H,?d,? J?=?8.1?Hz),?6.91?(1H,?dd,? J?=?11.3,?5.0?Hz),?6.02?(1H,?br),?5.43?(1H,?dt,? J?=?54.1,?4.7?Hz),?4.37?(2H,?m),?2.34?(1H,?s).

Embodiment 4:

2 '-deoxidation-2 '-fluoro-4 '-(4-to ethylphenyl-1,2,3-triazol radical)-β-D-cytidine ( 4) preparation

In the single port bottle of 50 mL, add 0.2 mmol 2 '-deoxidation-2 '-fluoro-4 '-azido-beta-D-cytidine, the trimethyl carbinol (1 mL), water (1 mL), stirring at room dissolving.Adding is to ethylbenzene acetylene (0.24 mmol) and N, N '-diisopropylethylamine (DIPEA) (0.3 mmol).Adding CuI(0.01 mmol under the lucifuge) 1 mL acetonitrile solution.Add NaAsc(0.01 mmol under the nitrogen atmosphere), normal temperature stirs down, and the TLC monitoring reaction is complete to example reaction, reacts completely in about 20 hours.The evaporated under reduced pressure solvent, column chromatography (methylene dichloride: methyl alcohol=10:1) separate obtain faint yellow solid ( 4), fusing point: 153-154 oC, productive rate 93%; HRMS [M+Na]: 439.1496;

¹H-NMR?(CD ₃OD,?300?MHz)?δ:?8.45(1H,?s),?7.98?(1H,?d,? J?=?7.4?Hz),?7.76?(2H,?d,? J?=?8.1?Hz),?7.26?(2H,?d,? J?=?8.1?Hz),?6.90?(1H,?d,? J?=?7.4?Hz),?5.99?(1H,?br),?5.42?(1H,?d,? J?=?54.0?Hz),?4.40?(1H,?d,? J?=?12.4?Hz),?4.33?(1H,?d,? J?=?12.4?Hz),?2.66?(2H,?q,? J?=?7.6?Hz),?1.26?(3H,?t,? J?=?7.6?Hz).

Embodiment 5:

2 '-deoxidation-2 '-fluoro-4 '-[4-(1-methylcyclohexyl)-1,2,3-triazol radical]-β-D-cytidine ( 5) preparation

In the single port bottle of 50 mL, add 0.2 mmol 2 '-deoxidation-2 '-fluoro-4 '-azido-beta-D-cytidine, the trimethyl carbinol (1 mL), water (1 mL), stirring at room dissolving.Add 3-cyclohexyl propine (0.24 mmol) and N, N '-diisopropylethylamine (DIPEA) (0.3 mmol).Adding CuI(0.01 mmol under the lucifuge) 1 mL acetonitrile solution.Add NaAsc(0.01 mmol under the nitrogen atmosphere), normal temperature stirs after 48 hours down, adds NaAsc(0.01 mmol), continuing stirring at normal temperature 24 hours, the TLC monitoring reaction is complete to example reaction.The evaporated under reduced pressure solvent, column chromatography (methylene dichloride: methyl alcohol=10:1) separate obtain white solid ( 5), fusing point: 148-150 oC, productive rate 80%; HRMS [M+Na]: 431.1779;

¹H-NMR?(CD ₃OD,?300?MHz)?δ:?7.94?(1H,?d,? J?=?7.5?Hz),?7.88?(1H,?s),?6.82?(1H,?dd,? J?=?11.5,?4.6?Hz),?5.98?(1H,?br),?5.37?(1H,?d,? J?=?54?Hz),?4.32?(1H,?d,? J?=?12.4?Hz),?4.26?(1H,?d,? J?=?12.4?Hz),?2.59?(2H,?d,? J?=?6.8?Hz),?1.73-1.15?(11H,?m).

Embodiment 6:

2 '-deoxidation-2 '-fluoro-4 '-(the 4-tertiary butyl-1,2,3-triazol radical)-β-D-cytidine ( 6) preparation

In the single port bottle of 50 mL, add 0.2 mmol 2 '-deoxidation-2 '-fluoro-4 '-azido-beta-D-cytidine, the trimethyl carbinol (1 mL), water (1 mL), stirring at room dissolving.Add 3,3-dimethyl-ethyl acetylene (0.24 mmol) and N, N '-diisopropylethylamine (DIPEA) (0.3 mmol).Adding CuI(0.01 mmol under the lucifuge) 1 mL acetonitrile solution.Add NaAsc(0.01 mmol under the nitrogen atmosphere), normal temperature stirs after 48 hours down, adds NaAsc(0.01 mmol), continuing stirring at normal temperature 48 hours, the TLC monitoring reaction is complete to example reaction.The evaporated under reduced pressure solvent, column chromatography (methylene dichloride: methyl alcohol=10:1) separate obtain white solid ( 6), fusing point: 159-161 oC, productive rate 75%; HRMS [M+Na]: 391.1472;

¹H-NMR?(CD ₃OD,?300?MHz)?δ:?7.95?(1H,?d,? J?=?7.5?Hz),?7.87?(1H,?s),?6.82?(1H,?dd,? J?=?11.3,?5.1?Hz),?5.99?(1H,?br),?5.37?(1H,?dt,? J?=?54.3,?4.9?Hz),?4.31(1H,?d,? J?=?12.2?Hz),?4.26?(1H,?d,? J?=?12.2?Hz),?1.35?(9H,?s).

Embodiment 7:

4 '-(4-normal-butyl-1,2,3-triazol radical)-2 '-deoxidation-2 '-fluoro-beta-D-cytidine ( 7) preparation

In the single port bottle of 50 mL, add 0.2 mmol 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine, the trimethyl carbinol (1 mL), water (1 mL), stirring at room dissolving.Add 1-hexin (0.24 mmol) and N, N '-diisopropylethylamine (DIPEA) (0.3 mmol).Adding CuI(0.01 mmol under the lucifuge) 1 mL acetonitrile solution.Add NaAsc(0.01 mmol under the nitrogen atmosphere), normal temperature stirs after 48 hours down, adds NaAsc (0.01 mmol), continues stirring at normal temperature 48 hours, and the TLC monitoring reaction is complete to example reaction.The evaporated under reduced pressure solvent, column chromatography (methylene dichloride: methyl alcohol=10:1) separate obtain white solid ( 7), fusing point: 133-135 oC, productive rate 70%;

¹H-NMR?(CD ₃OD,?300?MHz)?δ:?7.95?(1H,?d,? J?=?7.6?Hz),?7.90?(1H,?s),?6.83?(1H,?dd,? J?=?11.4,?5.1?Hz),?5.98?(1H,?d,? J?=?6.2?Hz),?5.38?(1H,?dt,? J?=?54.1,?4.8?Hz),?4.32(1H,?d,? J?=?13.0?Hz),?4.27?(1H,?d,? J?=?12.3?Hz),?2.71?(2H,? J?=?7.5?Hz),?1.71-1.61?(2H,?m),?1.44-1.28?(2H,?m),?0.94?(3H,?t, ?J?=?7.3?Hz).

Embodiment 8:

4 '-(4-cyclopentanol base-1,2,3-triazol radical)-2 '-deoxidation-2 '-fluoro-beta-D-cytidine ( 8) preparation

In the single port bottle of 50 mL, add 0.2 mmol 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine, the trimethyl carbinol (1 mL), water (1 mL), stirring at room dissolving.Add cyclopentanol base hexin (0.24 mmol) and N, N '-diisopropylethylamine (DIPEA) (0.3 mmol).Adding CuI(0.01 mmol under the lucifuge) 1 mL acetonitrile solution.Add NaAsc(0.01 mmol under the nitrogen atmosphere), normal temperature stirred 24 hours down, and the TLC monitoring reaction is complete to example reaction.The evaporated under reduced pressure solvent, column chromatography (methylene dichloride: methyl alcohol=10:1) separate obtain white solid ( 8), fusing point: 165-167 oC, productive rate 86%;

¹H-NMR?(CD ₃OD,?300?MHz)?δ:?7.98?(1H,?s),?7.93?(1H,?d,? J?=?7.5?Hz),?6.81?(1H,?dd,? J?=?12.0,?5.1?Hz),?5.97?(1H,?d,? J?=?7.5?Hz),?5.38?(1H,?dt,? J?=?54.3,?4.8?Hz),?4.32(1H,?d,? J?=?12.6?Hz),?4.26?(1H,?d,? J?=?12.6?Hz),?2.15-1.81?（8H,?m）.

Adopt compound 1Synthetic method and route, can get compound by (TMS) acetylene and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 9

Adopt compound 2Synthetic method and route, by getting compound to propylbenzene acetylene and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 10

Adopt compound 2Synthetic method and route, by getting compound to butylbenzene acetylene and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 11

Adopt compound 2Synthetic method and route, by getting compound to amylbenzene acetylene and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 12

Adopt compound 2Synthetic method and route, can get compound by o-methyl-benzene acetylene and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 13

Adopt compound 2Synthetic method and route, can get compound by a methylbenzene acetylene and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 14

Adopt compound 7Synthetic method and route, can get compound by 1-octyne and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 15

Adopt compound 7Synthetic method and route, can get compound by 1-n-heptylacetylene and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 16

Adopt compound 7Synthetic method and route, can get compound by 1-decine and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 17

Adopt compound 2Synthetic method and route, can get compound by propargylamine and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 18

Adopt compound 2Synthetic method and route, can get compound by propine methylbenzylamine and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 19

Adopt compound 8Synthetic method and route, can get compound by 10-undecyne-1-1 alcohol and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 20

Adopt compound 8Synthetic method and route, can get compound by 3-butyne-2-alcohol and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 21

Adopt compound 8Synthetic method and route, can get compound by 3-butine-1-alcohol and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 22

Adopt compound 8Synthetic method and route, can get compound by 4-pentyne-1-alcohol and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 23

Adopt compound 8Synthetic method and route, can get compound by 1-hexin-3-alcohol and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 24

Adopt compound 8Synthetic method and route, can get compound by methylpentynol and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 25

Adopt compound 8Synthetic method and route, can get compound by 1-heptyne-3-alcohol and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 26

Adopt compound 8Synthetic method and route, can get compound by 1-octyne-3-alcohol and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 27

Adopt compound 2Synthetic method and route, can get compound by 1-phenyl-2-propine-1-alcohol and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 28

Adopt compound 2Synthetic method and route, can get compound by 2-phenyl-3-butyne-2-alcohol and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 29

Adopt compound 2Synthetic method and route, can get compound by 3-hydroxyl-phenylacetylene and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 30

Adopt compound 2Synthetic method and route, can get compound by 2-acetylene thiophene and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 31

Adopt compound 2Synthetic method and route, can get compound by 3-acetylene thiophene and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 32

Adopt compound 2Synthetic method and route, can get compound by 2-ethynyl pyridine and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 33

Adopt compound 2Synthetic method and route, can get compound by 3-ethynyl pyridine and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 34

Adopt compound 2Synthetic method and route, can get compound by 4-ethynyl pyridine and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 35

Adopt compound 1Synthetic method and route, can get compound by 3-crotonylene-ketone and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 36

Adopt compound 1Synthetic method and route, can get compound by propynoic acid and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 37

Adopt compound 1Synthetic method and route, can get compound by propynoic acid methyl esters and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 38

Adopt compound 1Synthetic method and route, can get compound by phenyl propargyl ether and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 39

Adopt compound 2Synthetic method and route, can get compound by 4-anisole acetylene and compound 4 '-azido--2 '-deoxidation-2 '-fluoro-beta-D-cytidine reaction 40

The above, only being part embodiment of the present invention, is not that the present invention is done any pro forma restriction, any simple modification that every foundation technical spirit of the present invention is done the foregoing description, equivalent variations and modification all belong in the technical solution of the present invention scope.

Synthetic compound of the present invention can be used for antitumor and activeconstituents virus drugs, can use separately, also can with other anti-tumor virus drugs drug combinations.In the drug combination therapeutic process of indication of the present invention, comprise that at least a The compounds of this invention of utilization or its reactive derivative use to increase general curative effect with other one or more anti-tumor virus drugs.Dose during drug combination should be decided according to rational therapy effect obtained under the different situations with administration time.

The medicament compatibility of being contained comprises the effective dose of the compound in the general formula.This medicament compatibility can contain a kind of effective antitumour, antiviral Chemical Composition and medicinal formula." effective dose " herein refers to the consumption that can produce required this compound of result of treatment for institute's treatment target.This effective dose or dosage can be by experience person being arranged according to the suggestion of different situations and difference.Such as, the tumour of being treated is different with viral species, the usage difference of medicine; Whether shared etc. with other methods of treatment such as other antitumor drugs or antiviral, dosage all can change.Can make any spendable preparation formulation.If some has alkalescence or acidic cpd and can form avirulent acid or salt, can use the form of the salt of this compound.Spendable organic acid salt comprises spendable anion salt on the physiology in the pharmacy, as p-methyl benzenesulfonic acid salt, metilsulfate, acetate, benzoate, Citrate trianion, tartrate, maleate, succinate, ascorbate salt and glycerophosphate etc.Spendable inorganic salt comprise muriate, bromide, fluorochemical, iodide, vitriol, nitrate, supercarbonate, carbonate, phosphoric acid salt etc.Can make the form of described salt if any the compound of alkalescence and suitable acid as amine.The compound of carboxylic-acid can form spendable salt with basic metal or alkaline-earth metal.

In the general easy mixed solvent that is dissolved into organic solvent, water-soluble solvent and organic solvent or water-soluble solvent and water of the compound of containing in the formula of of the present invention.Water-soluble solvent preferred alcohols, poly ethylene glycol, N-methyl-2-pyrrolinone, N,N-dimethylacetamide, N, dinethylformamide, methyl-sulphoxide, second cyanogen with and share.Described pure particular methanol, ethanol, Virahol, glycerol or ethylene glycol.The compounds of this invention can mix with preparations carrier commonly used and make preparation.Compound dissolution is in water miscible organic solvent, non-protonic solvent, water-soluble lipid, cyclodextrin, lipid acid, phosphatide or in the mixed solvent of these solvents and make drug solution; Add physiological saline again and obtain the carbohydrate of 1-20%, as the aqueous solution of glucose.The preparation stabilization that makes therefrom also is used for animal and clinical.

With compound in the above-mentioned general formula is the product medicine that active ingredient is prepared into, can be by oral or parenteral route administration, also can be by transplant medicine pump in the body and additive method administration, the parenteral route administration of indication herein is meant subcutaneous intracutaneous, intramuscular, intravenously, intra-arterial, atrium in, in the synovial membrane, in the breastbone, in the sheath, interior, the intracranial injection of wound site or drip infusion technique etc.Use conventional method proportioning by the technician, mixing finally becomes needed pharmaceutical dosage form.Can be the outstanding solution of tablet, capsule, emulsion, pulvis, the little pin of intravenous administration, infusion solutions, lyophilized powder, dripping pill, emulsion suspension liquid, water, the aqueous solution, colloid, colloidal solution, sustained release preparation, nanometer formulation or be used for animal or clinical with other forms of formulation.

Compound in the general formula of the present invention is used for the treatment of or alleviates the preparation of the cancer drug of a certain tissue or organ.The indication cancer comprises but is not only limited to colorectal carcinoma, liver cancer, lymphoma, lung cancer, the esophageal carcinoma, mammary cancer, central nerve neuroma, melanoma, ovarian cancer, kidney, leukemia, prostate cancer, carcinoma of the pancreas etc.

Compound in the general formula of the present invention is used for the treatment of or prevents or alleviate the preparation that certain virus causes disease medicament.Indication virus includes but are not limited to hepatitis B (HBV), hepatitis C (HVC), virus of AIDS (HIV), yellow fever virus (YFV), respiratory syncytial virus (RSV), hsv (HSV), bovine viral diarrhea virus (BVDV), hepatitis G virus (HGV), GB virus-B(GBV-B), dengue virus (Dengue), ERC group virus (HRV), poliovirus (Poliovirus), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV) etc.

Antiviral and anti-tumor activity

The compound of being contained among the present invention has antiviral activity, comprises anti-AIDS HIV-1, HBV and HCV activity, and anti-tumor activity.Only enumerate the part example below and do an explanation.

One, the anti-HIV-1 replication activity is measured:

The compound of being contained among mensuration the present invention is to the active influence of HIV-1 virus replication.By with embodiment of the invention gained compound and 293T (source: after ATCC) cell is hatched 15 minutes, add VSVG/HIV-luc(NL4-3) virus strain, cultivated 48 hours for 37 ℃, with the gradient of infection of luciferase as reporter-gene assays HIV-1.(Zidovudine, AZT) (Xiamen steps and restrains medicine company limited) is as positive control with zidovudine.

Substratum: DMEM substratum (U.S. Gibco company), RPMI-1640, FBS; Compound method: RPMI-1640/DMEM+10%FBS.Experiment medium: dimethyl sulfoxide (DMSO) (DMSO, U.S. Sigma).

Experimental technique:

1, trial-product, reference substance preparation: given the test agent is weighed and is dissolved among the DMSO, and storage liquid concentration is 10mmol/L; Reference substance: the zidovudine and be dissolved in DMSO of weighing, storage liquid concentration is 10 mmol/L.

2, experimental procedure

2.1 the preparation of wild-type HIV-1 reorganization pseudovirus: transfection the day before yesterday, by 2.2 * 10 ⁶The density inoculation 293ET cell of individual cell is in 100 mm culture dish, calcium phosphate precipitation method cotransfection 3 μ g VSV-G plasmids and 8 μ g wild-type HIV-1 core genes with improvement, after the transfection 16 hours, wash cell and renew bright substratum continuation cultivation 32 hours with PBS, collect supernatant and, generate wild-type HIV-1 recombinant virus particle VSVG/HIV-WT through the membrane filtration of 0.45 μ m.

2.2 the p24 antigen measuring of HIV-1 reorganization pseudovirus: respectively get 450 μ l after the doubling dilution virus stock solution used wild-type, lysate with 50 μ l carries out cracking, according to p24 antigen ELISA test kit specification sheets (ZeptoMetrix, Cat:0801111), measure and calculate the p24 antigen concentration of wild-type recombinant virus stoste.

2.3 medicine detects the HIV-1 inhibition: infect the day before yesterday, the 293T cell is pressed every hole 6 * 10 ⁴Density be inoculated on 24 orifice plates, dissolve testing compound with DMSO, added in the cell culture fluid in preceding 15 minutes in infecting, the DMSO solvent is made blank, adds 0.5 ml virus liquid (virus stock solution used being diluted to 0.1 –, 0.5 ng p24/ml according to p24 concentration) again.Infected back 48 hours, remove supernatant, add 50 μ l cell pyrolysis liquid (Promega) lysing cell in every hole, 20 μ l product of cell lysis are added in the 30 μ l luciferase substrates (Promega) again, relative reactivity with the plain enzyme of FB15 fluorimetric detector (Sirius) Instrument measuring cell fluorescence, compare the half-inhibition concentration that the computerized compound duplicates wild-type HIV-1 with DMSO.

2.4 use the influence of MTS method detection compound pair cell survival: the 293T cell of logarithmic phase is seeded in 96 orifice plates every hole 100ul, 37 ℃, 5%CO by the cell density in 8000 ~ 10000/hole ₂After cultivating 24 h in the incubator, add testing compound and be blank (final concentration is 0.1%) with DMSO, 37 ℃, 5%CO ₂Continue in the incubator to cultivate 44 hours.In every hole, add the mixed solution that 20 μ l MTS/PMS now join, 37 ℃, 5%CO ₂Develop the color after continuing in the incubator to cultivate 4h.On enzyme connection detector, wavelength 490 nm and 650 nm(backgrounds) locate to detect the absorbance value (OD) in each hole, and calculate the survival rate of cell.

Record anti-HIV-1 virus activity (EC ₅₀) and cytotoxicity (CC ₅₀) see the following form:

Two, resistance of hepatitis B HBV external biological activity test:

2.1, the recovery and the cultivation of cell

From liquid nitrogen container, take out a frozen HepG2.2.15 cell, place 42 ℃ of water-baths to shake rapidly to thawing.In DMEM perfect medium (foetal calf serum 10%, G418 380ug/ml, penicillin 100U/ml, Streptomycin sulphate 100U/ml).Culture condition is 5% CO ₂, 37 ℃ of cultivations.Went down to posterity once in 2～3 days behind the cell normal growth.

2.2, the mtt assay detection compound is to the toxicity of HepG2.2.15 cell.

The MTT colorimetry detection of drugs of setting up according to Mosmann is to the toxicity of HepG2.2.15 cell.Cultured continuously 72 hours, every hole adds MTT 10ul, after 4 hours, abandoning supernatant, every hole adds DMSO 200ul, and microplate reader (detecting wavelength 490nm, reference wavelength 630nm) reads each hole OD value, the record result.The computerized compound is to HepG2.2.15 cell inhibiting percentage:

The inhibition percentage=

According to the inhibition percentage of medicine pair cell, calculate the half toxic concentration (TC of medicine pair cell ₅₀).

2.3, compound is to the HBV body outer suppressioning experiment

Selection as screening concentration, detects HBV surface antigen s and the antigenic influence of e in its pair cell nutrient solution less than compound half toxic concentration.Select the HepG2.2.15 cell of logarithmic phase for use, trysinization becomes single cell suspension, according to 2 * 10 ⁴The concentration of/ml is inoculated in 24 orifice plates, every hole 1ml, 3 holes of each concentration.Add and to contain the substratum of different pharmaceutical concentration, and to establish the cell that adds complete substratum be normal control.Collect the 3rd day, the 6th day, the 9th day cell conditioned medium liquid and cell, in-20 ℃ of preservations.Measure the expression of e antigen and surface antigen inside and outside the 3rd day, the 6th day, the 9th day the cell with the ELISA method.

2.4, the ELISA detection compound is to the influence of HBeAg and HBsAg in the HepG2.2.15 cell conditioned medium liquid

Carry out according to the test kit working method;

Measure: use the microplate reader reading, select dual wavelength 450/630nm, read each hole OD value.

The result judges: at first calculate the Cutoff value according to the average OD value of COV=negative control * 2.1, if sample OD value 〉=COV is positive, sample OD value≤COV is negative.

Compound number	Structure	IC ₅₀（μM）　（HBsAg）	IC ₅₀（μM）（HBeAg）	CC ₅₀（μM）（HepG2.2.15）
					1	0.12	1.0	833.88
2		0.1	0.25	566.30
					3	0.01	>1.0	497.63
4		0.02	>1.0	501.74
					5	0.06	0.46	534.79
6		0.07	0.55	764.68
					7	0.11	>1.0	701.85
8		0.03	>1.0	699.77
					9	0.14	>1.0	795.68
10		0.03	>1.0	503.44
					11	0.02	>1.0	488.97
12		0.03	>1.0	588.79

Three, anti-hepatitis C HCV activity:

The compound that the present invention is contained has the effect of anti-hepatitis C HCV.External anti-HCV activity experiment is as follows:

3.1, cell cultures

HCV replicating cell (Avva.5) is cultivated in the Eagle medium of the improved G418 that comprises 10% fetal bovine serum and 1mg/mL of Dulbecco ' s.The 293-Sip-L cell is cultivated in the Eagle medium of the hygromycin B of improved G418 that contains 10% fetal bovine serum and 250 μ g/mL of Dulbecco ' s and 150 μ g/mL.

3.2, HCV infects measuring method (RT-PCR method)

Cultivated 12 hours in comprising the HCV male substratum of 100 μ L with the cell in the Petri culture dish of 60mm diameter.Cell is cultivated in the fresh medium that does not comprise HCV and is changed every day once then.When cell detected HCV-RNA in infection after 7 days, cell needed trypsinized and cleans twice with the improved Eagle medium of centrifuging Dulbecco ' s.The top section (in contrast) of the washing second time and the cell of washing collect to carry out the RNA extraction in pairs and RT-PCR detects.Beta-actin mRNA measures simultaneously to do contrast.

3.4, the detection by quantitative of HCV-RNA

Adopt full-automatic PCR euzymelinked immunosorbent assay (ELISA) to come quantitative assay HCV-RNA(2.0 version, Roche Diagnostics, Branchburg, NJ).

The compounds of this invention 1Inhibition effect (seeing the following form) is arranged when 6,0.6 and 0.06 μ g/mL.The HCV-RNA level is measured with full-automatic PCR euzymelinked immunosorbent assay (ELISA).

Four, anti-tumor activity:

4.1, cell and cell cultures

Cell cultures; The Raji cell is put 37 ° of C, and 5%CO2 cultivates in the cell culture incubator, and nutrient solution is the RPMI1640 nutrient solution that contains 10% calf serum, cell cultures 2～3d, take the logarithm vegetative period cell be used for the experiment.

4.2, tetrazolium bromide (MTT) reduction method detects inhibitory rate of cell growth

The cell of taking the logarithm vegetative period, adjusting cell density is 5 * 10 ⁴Individual/ml, be inoculated in respectively in three 96 well culture plates, nutrient solution is changed in 100 μ L/ holes behind the cell cultures 24h, and experimental group adds 0.3,1.5,3,6,12,24 μ mol/L N-Cu respectively, 200 μ L/ holes, every kind of drug level is one group.Blank group (being used for zeroing) only adds the RPMI1640 nutrient solution that contains 10% foetal calf serum and does not add cell, and control group adds the RPMI1640 nutrient solution that equal-volume contains 10% foetal calf serum.More than each group all establish 5 multiple holes.After cell continued respectively to cultivate 24h, 48h and 72h, every hole added MTT(5mg/ml) solution 20 μ L, continue to cultivate 4h.Supernatant liquor is abandoned in suction, adds DMSO 150 μ L/ holes, and about concussion mixing 5min, after the dissolving fully to be crystallized, detecting wavelength on the inherent enzyme-linked immunosorbent assay instrument of 20min is absorbancy (A) value in each hole, 570nm place.Experiment repeats 3 times, averages.Calculate the growth inhibition ratio of cell as follows.Inhibitory rate of cell growth=(1-experimental group average A value/control group average A value) * 100%.

The compound that the present invention is contained has antitumor action.Effect sees the following form part typical compound of the present invention to human lymphoma Raji cell inhibiting.

。

Claims

1.2 '-deoxidation-2 '-fluoro-4 '-triazole replacement-β-D cytidine analog or its salt is characterized in that, it has following general structure:

In the formula, R is one of following substituting group: C ₄-C ₈Straight chained alkyl, pyridyl or thienyl, ethanoyl, carboxyl, the formicester base, the cyclopropane base, the tertiary butyl, the cyclopentanol base, (trimethylammonium) silylation, the 1-methylcyclohexyl, phenyl is by C ₁-C ₅The phenyl that alkyl, hydroxyl or methoxyl group replace,

n?=?2-9，

，-CH ₂-O-ph?，

，

。

2. 2 '-deoxidation-2 ' as claimed in claim 1-fluoro-4 '-triazole replacement-β-D cytidine analog or its salt is characterized in that, are one of following compound:

。

3. the synthetic method of 2 '-deoxidation-2 ' as claimed in claim 1-fluoro-4 '-triazole replacement-β-D cytidine analog or its salt is characterized in that, and is synthetic as follows:

Compound 2 '-deoxidation-2 '-fluoro-4 '-azido-beta-D-cytidine is joined in the mixed solution of the trimethyl carbinol and water, after the stirring and dissolving, add corresponding alkynes, and add N, it is alkalescence that N '-diisopropylethylamine is adjusted solution, adds the acetonitrile solution of CuI again under lucifuge; Add sodium ascorbate under the nitrogen atmosphere, normal temperature stirs down, and evaporated under reduced pressure solution after reaction finishes obtains compound with column chromatography separating purification I

4. the application in antiviral, the antitumor drug of preparation of 2 '-deoxidation-2 ' as claimed in claim 1-fluoro-4 '-triazole replacement-β-D cytidine analog or its salt, it is characterized in that, it is applied to the preparation treatment, prevents or alleviates antitumor or antiviral and pharmaceutical preparation as activeconstituents.

5. the application in antiviral, the antitumor drug of preparation of 2 '-deoxidation-2 ' as claimed in claim 4-fluoro-4 '-triazole replacement-β-D cytidine analog or its salt, it is characterized in that, with it is that active ingredient is prepared into oral or the parenteral route form of medication, or passes through the form of medication of transplant medicine pump in the body.

6. the application in, the antitumor drug antiviral in preparation as claim 4 or 5 described 2 '-deoxidation-2 '-fluoro-4 '-triazole replacement-β-D cytidine analog or its salt is characterized in that active ingredient is one or more The compounds of this invention.

7. the application in, the antitumor drug antiviral in preparation as claim 4 or 5 described 2 '-deoxidation-2 '-fluoro-4 '-triazole replacement-β-D cytidine analog or its salt, it is characterized in that, be applied to treat the preparation of colorectal carcinoma, liver cancer, lymphoma, lung cancer, the esophageal carcinoma, mammary cancer, central nerve neuroma, melanoma, ovarian cancer, kidney, leukemia, prostate cancer or carcinoma of the pancreas medicine or pharmaceutical preparation.

8. antiviral in preparation as claim 4 or 5 described 2 '-deoxidation-2 '-fluoro-4 '-triazole replacement-β-D cytidine analog or its salt, application in the antitumor drug, it is characterized in that, be applied to treat hepatitis B (HBV), hepatitis C (HVC), virus of AIDS (HIV), yellow fever virus (YFV), respiratory syncytial virus (RSV), hsv (HSV), bovine viral diarrhea virus (BVDV), hepatitis G virus (HGV), GB virus-B(GBV-B), dengue virus (Dengue), ERC group virus (HRV), poliovirus (Poliovirus), varicella zoster virus (VZV), Epstein-Barr virus (EBV) or cytomegalovirus (CMV) medicine or preparation of pharmaceutical formulations.