CN108640959B - 3' -deoxy-3 ',4' -didehydro nucleoside compounds and application thereof - Google Patents
3' -deoxy-3 ',4' -didehydro nucleoside compounds and application thereof Download PDFInfo
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- 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
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- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
- C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
Abstract
The invention relates to the field of antivirus, in particular to preparation of nucleoside compounds (I) and application thereof in resisting HIV virus, Hepatitis C Virus (HCV), Hepatitis B Virus (HBV), rabies virus and Zika virus.
Description
Technical Field
The invention relates to the use of 3' -deoxy-3 ',4' -didehydronucleosides for the treatment or prophylaxis of viral infections. More specifically, the invention describes the use of 3' -deoxy-3 ',4' -didehydro nucleosides for the treatment or prevention of infection by rabies virus, HIV virus, HCV virus, HBV virus, Zika virus.
Background
Nucleoside compounds, currently there are over 10 approved for the treatment of Human Immunodeficiency Virus (HIV), or Hepatitis C Virus (HCV), and other viruses. A challenge in developing antiviral therapies is to inhibit viral replication without damaging the host cells. In general, in order to exhibit antiviral activity, nucleoside analogs must be metabolically converted by host cell enzymes into their corresponding triphosphate forms. The triphosphate forms, nucleoside polymerase inhibitors, mimic natural nucleotides in that they compete with one (CTP, UTP, TTP, ATP, or GTP) of the five naturally occurring nucleoside 5' -triphosphates (NTPs) for RNA or DNA extension. Nucleoside analogs therefore inhibit viral replication by acting as a chain terminator or a delayed chain terminator.
Acquired Immune Deficiency Syndrome (AIDS) is an infectious disease caused by infection with the HIV virus that seriously compromises people's life and health. According to WHO statistics, the number of AIDS patients is more than 4000 ten thousand, 500 ten thousand patients are newly added every year, and 300 ten thousand deaths are caused every year. At present, the clinical treatment method of AIDS is mainly high-efficiency anti-retroviral therapy, which not only effectively controls HIV replication, but also can rebuild the immune function of AIDS patients, thus opening a hope for the treatment of AIDS. People hope to completely eliminate HIV in vivo by HAART, thereby achieving the aim of completely curing AIDS. However, subsequent practice has shown that, although HAART can maximally inhibit viral replication in patients and reduce plasma viral load (virus load) to levels that cannot be detected by conventional detection methods, the virus persists in infected patients and rebounds to pre-treatment levels once drug treatment is discontinued (Ho, D.D. heated HIV administration or diagnosis, 1998.280: 1866-1867.). One important reason for the difficulty of complete elimination of HIV in vivo is that HIV-1 can be latent in resting memory CD4+ T cells, which are generated by transformation of a small fraction of HIV-infected activated CD4+ T cells, whose integrated provirus lacks transcriptional activity and, therefore, is not attacked by the immune system and antiretroviral drugs. Although infected individuals carry a low number of latently infected cells, the rate of attenuation is so slow that complete elimination by HAART treatment alone is not possible during the individual's lifetime. Therefore, the latent HIV-infected resting CD4+ T cells constitute a major part of the viral reservoir (reservoir) in the body and are also a great obstacle to the complete elimination of HIV by current clinical treatments. The molecular mechanism of HIV-1 latent infection cell formation is thought to be related to chromatin state at the integration site, the presence of inhibitory nucleosome nuc-1, epigenetic modifications represented by acetylation, host transcription factors such as NF-. kappa.B and viral transcriptional activator Tat. Based on this mechanism, there have been proposed therapeutic strategies for eliminating the latent viral reservoir, i.e. attempts to accelerate the elimination of the viral reservoir by inducing pro-viral expression of HIV-latently infected cells by drugs, reactivating the latent virus, and killing the activated latently infected cells in combination with highly effective anti-retroviral therapy and under the action of the human immune system (Richman et al. the Challenge of finishing a Current for HIV Infection, Science, 2009, 1304, 323). Although several treatment schemes are provided clinically, the result is still not satisfactory, whether the activator is ineffective or effective but has great toxic and side effects, and no new anti-AIDS drug with independent intellectual property rights is yet on the market in China. Therefore, the research and development of a novel intervention drug which has independent intellectual property rights, safety, effectiveness and low price and can clear the HIV-1 virus repository is of great significance.
Rabies is one of the oldest infectious diseases, and is a zoonosis caused by Rabies virus (RABV). Rabies is the most acute infectious disease of human patients to date, and once it occurs, the mortality rate is nearly 100%. As reported by the World Health Organization (WHO), about 55000 people die worldwide each year from rabies, most of which is distributed in africa and asia. In recent years, about 3000 people die of rabies per year in China, most of rabies is caused in vast rural areas or urban and rural junctions, and the rabies is mainly infected by being bitten by animals with poison. RABV enters the bite site with saliva, enters the nearest nerve fibers after a short incubation in the muscle, and gradually migrates to the brain, and the more close the wound site is to the brain, the faster its onset is. Once in the nervous system, ravv replication and migration will not interfere easily. Post-exposure treatment (PEP) is the most effective way to prevent and control rabies, and generally takes three measures, depending on the degree of bite: 1) flushing the wound with 0.1% soap water; 2) injecting anti-RABV immunoglobulin into the wound, and neutralizing the virus at the wound; 3) emergency immunization of 4-5 rabies vaccines. Currently, many people attach importance to the immunization of rabies vaccine only, neglecting wound management, and allow RABV to submerge into the brain before the vaccine immunization produces enough neutralizing antibodies (usually taking 2-4 weeks), resulting in immune failure. Because of the high price of anti-RABV immunoglobulins, they are difficult to apply widely to wound management, and require a certain scale of medical facilities to receive anti-RABV immunoglobulin treatments, it is difficult to treat wounds at the first time after a bite. Therefore, there is an urgent need to develop a drug which can effectively inhibit RABV at a wound site, is inexpensive, and can be widely used in wide medical underdeveloped regions.
Hepatitis C Virus (HCV) has infected more than 1.8 million people worldwide. It is estimated that four million people are newly infected each year, and 70% of them will develop chronic hepatitis. In developed countries, HCV is responsible for 50-76% of all liver cancer cases and two-thirds of all liver transplants. The standard therapy polyethylene glycol a in combination with ribavirin (a nucleoside drug) only works in 50-60% of patients and is associated with significant side effects. Therefore, new HCV drugs are urgently needed.
At present, 1.3 hundred million people in China are carriers of hepatitis B virus, namely, the proportion of carriers with positive hepatitis B surface antigen (Hbs Ag) accounts for 10.34 percent of the total number of people. Approximately one fourth of hepatitis B virus carriers will eventually develop chronic hepatitis, cirrhosis, and primary liver cancer. In addition, there are about 1200 or more ten thousand chronic hepatitis patients in the country, and about 30 million people die of liver disease every year among these patients, and about 15 million people die of liver cancer. Worse still, about 40% of pregnant women carrying hepatitis B surface antigen (Hbs Ag) can vertically transmit through mother and infant, so that 80-90 ten thousand of newborn infants (with the proportion of about 80-90%) become carriers of chronic hepatitis B virus, which not only seriously threatens human health, but also brings heavy economic burden to families and society. The treatment of chronic hepatitis B mainly adopts comprehensive treatments of antivirus, immunoregulation, liver function improvement, hepatic fibrosis resistance and the like, but the antivirus treatment is the most important and key treatment measure. At present, anti-Hepatitis B Virus (HBV) drugs are continuously generated, and especially nucleoside drugs (nucleosides) become hot spots of antiviral drug research in recent years, and rapid progress is achieved.
Zika virus (Zika virus) is a mosquito-borne virus that is transmitted primarily by Aedes mosquitoes. This virus was first discovered in 1947 by chance through the yellow fever monitoring network in rhesus monkeys of the bush-kanka forest of lindera indica, and subsequently in 1952 in the population of lindera and tanzania. On day 7 of 5 month 2015, the health organization in the united states and the world health organization announced that the brazilian health authority confirmed the prevalence of zika virus in northeastern brazilian, which was prevalent mainly in africa, southeast asia and pacific islands, however, since 5 month 2015, transmission of zika was reported in several countries in america, and it was predicted that other countries with zika vector mosquitoes will have cases in succession. In the next less than one year, as of day 10, 3 months, the number of countries and regions where the world health organization reported locally transmitted cases has increased to 52. As far as 2016, 11 months and 3, 13 input cases exist in China. At present, aspirin and nonsteroidal anti-inflammatory drugs are mostly used for treating Zika virus, but Zika and dengue fever have the possibility of simultaneous infection, and the drugs can increase the risk of dengue fever hemorrhage. It is now known that Zika virus of the Flaviviridae family, the genus flavivirus, is a spherical, single-stranded, positive-stranded RNA virus, 40-70nm in diameter, enveloped, approximately 10.8kb in length, encoding approximately 3400 amino acids. Zika virus is not particularly resistant, but flavivirus viruses are generally acid and heat intolerant. The NS3 protease is essential for proteolytic processing of its polyprotein to make the virus replicate, making NS3pro an attractive therapeutic target for inhibiting viral proliferation, and the NS2B-NS3 protease complex of zika virus plays a key regulatory role throughout the virus' life cycle, and only after the protease complex is activated and completes a series of hydrolysis reactions, the virus initiates the replication process. Therefore, the nonstructural protein NS2B-NS3 protease complex of Zika virus also becomes a key antiviral drug target, so that the inhibitor screening of the nonstructural protease of Zika virus has great significance for the development of drugs related to Zika virus.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: the 3' -deoxy-3 ',4' -didehydro nucleoside compounds are used for treating or preventing rabies virus, HIV virus, HCV virus, HBV virus and Zika virus infected hosts. The 3' -deoxy-3 ',4' -didehydronucleosides are converted enzymatically in the host cell to their corresponding triphosphate forms as prodrugs of monophosphates. The nucleoside polymerase inhibitors in triphosphate form mimic natural nucleotides, competing with their naturally occurring nucleoside triphosphates for RNA or DNA extension, thereby inhibiting viral replication by acting as a chain terminator or delayed chain terminator.
According to one aspect of the invention, the invention provides the application of the 3' -deoxy-3 ',4' -didehydro nucleoside compound of the formula (I) in treating or preventing rabies virus, HIV virus, HCV virus, HBV virus and Zika virus and a preparation method of the formula (I).
Wherein:
b is selected from adenine and derivatives
Guanine and derivatives
Cytosine
Thymidine
Wherein the content of the first and second substances,
x is independently selected from:
R1and R2Independently selected from: c1-3Alkyl radical, C2-6Alkenyl radical, C2-6Alkynyl, C1-6Alkoxy, halogen, C1-6Haloalkyl, C1-6Amide, -NHSO3C1-6Alkyl, nitro, cyano;
z is selected from:
(1) is H, OH, halogen, C1-20Alkyl radical, C3-6Cycloalkyl radical, C1-6Haloalkyl, aryl or heteroaryl.
(2)
Wherein
Y is selected from O or S;
R1and R2Independently selected from:
(a)OR3wherein R is3Is H, C1-20Alkyl radical, C3-6Cycloalkyl radical, C1-6Haloalkyl, aryl or heteroaryl, including but not limited to phenyl optionally substituted with 1-3 substituents,the 1-3 substituents are independently selected from: c1-3Alkyl radical, C2-6Alkenyl radical, C2-6Alkynyl, C1-6Alkoxy, halogen, C1-6Haloalkyl, C1-6Amide, -NHSO3C1-6Alkyl, nitro, cyano, (CH)2)1- 6CO2R4、-N(R4)2、-SO2N(R4)2 and COR5；
R4Independently is H or C1-6An alkyl group;
R5is-OR4or-N (R)4)2。
(b)
Wherein n is 0 to 6,
R6is H, C1-3An alkyl group;
R7independently selected from H, C1-20Alkyl radical, C3-6Cycloalkyl radical, C1-6Haloalkyl, aryl or heteroaryl, including, but not limited to, phenyl optionally substituted with 1-3 substituents independently selected from: c1-3Alkyl radical, C2-6Alkenyl radical, C2-6Alkynyl, C1-6Alkoxy, halogen, C1-6A haloalkyl group.
The preparation route is as follows:
the method comprises the following basic steps:
catalyzing the compound 3 '-deoxy-3', 4 '-didehydro-1', 2 ', 5' -triacetyl ribose (II) and protected or free basic group with titanium tetrachloride to obtain 3 '-deoxy-3', 4 '-didehydro-2', 5 '-diacetyl ribose (III), hydrolyzing with ammonia methanol solution to obtain 3' -deoxy-3 ',4' -didehydro-ribose (IV), and mixing with PYCl3(wherein, Y is O or S) reacting and then alcoholyzing to obtain a compound of 3 '-deoxy-3'4 '-didehydro-5' -O-phosphoribonucleoside (I).
Wherein in the course of the said route,
base B is selected from adenine and derivatives
Guanine and derivatives
Cytosine
Thymidine
Wherein, X is independently selected from: c1-3Alkyl radical, C2-6Alkenyl radical, C2-6Alkynyl, C1-6Alkoxy, halogen, C1-6A halogenated alkyl group,
C1-6Amide, -NHSO3C1-6Alkyl, nitro, cyano;
R1and R2Independently selected from:
(a)OR3wherein R is3Is H, C1-20Alkyl radical, C3-6Cycloalkyl radical, C1-6Haloalkyl, aryl or heteroaryl, including but not limited to
Not limited to phenyl optionally substituted with 1 to 3 substituents independently selected from: c1-3Alkyl, aryl, heteroaryl, and heteroaryl,
C2-6Alkenyl radical, C2-6Alkynyl, C1-6Alkoxy, halogen, C1-6Haloalkyl, C1-6Amide, -NHSO3C1-6Alkyl, aryl, heteroaryl, and heteroaryl,
Nitro, cyano, (CH)2)1-6CO2R4、-N(R4)2、-SO2N(R4)2 and COR5；
R4Independently is H or C1-6An alkyl group;
R5is-OR4or-N (R)4)2。
In accordance with another aspect of the present invention, there are provided in vitro activity assays and data for the inhibition of rabies, HIV, HCV, HBV, Zika virus by compounds of formula (I).
Detailed Description
Example 1
3 '-deoxy-3', 4 '-didehydro-5' -O-phosphate ribocytidine (Compounds 4a-e)
The preparation route is as follows:
preparation of intermediate 3' -deoxy-3 ',4' -didehydro-2 ', 5 ' -diacetyl ribocytidine (Compound 2)
Cytosine (11.0g, 0.1mol) is suspended in 180mL of anhydrous toluene, 15mL of anhydrous DMF and 20mL of hexamethyldisilazane are added, the mixture is heated and refluxed for 4h, the system is completely clarified, cooled to 70 ℃, and evaporated to dryness under reduced pressure. 3 '-deoxy-3', 4 '-didehydro-1', 2 ', 5' -triacetyl ribose (25.8g, 0.1mol), anhydrous dichloromethane (200 mL), titanium tetrachloride (1.0 mL) were added at room temperature, the reaction was carried out at room temperature for 5 hours, filtration was carried out, the filtrate was washed with water, the organic phase was dried over anhydrous sodium sulfate, and filtration and concentration were carried out to obtain 2, 25.2g of an intermediate compound (yield: 85.2%).
Preparation of intermediate 3' -deoxy-3 ',4' -didehydro-ribocytidine (Compound 3)
Adding compound 2(15.0g, 0.05mol) into 100mL of 10% ammonia methanol solution, stirring at room temperature for 2h, concentrating under reduced pressure after the reaction is finished, adding ethanol into the residue, heating to completely dissolve, filtering, and cooling while stirring for crystallization. Filtered and dried, and white solid, i.e., intermediate 3' -deoxy-3 ',4' -didehydro-cytidine (compound 3), 7.52g, yield 75.3%.
Preparation of 3 '-deoxy-3', 4 '-didehydro-5' -phosphodiethyl ribocytidine (Compound 4a)
Dissolving phosphorus oxychloride (5.0g, 0.033mol) in 50mL triethyl phosphate, adding the compound 3(7.4g, 0.033mol) at 0 ℃, reacting for 2h, filtering after the reaction is completed to obtain a white solid, adding the white solid into 50mL absolute ethyl alcohol, reacting for 4h at 60 ℃, reacting for complete, and recovering ethyl alcohol under reduced pressure. After purification by silica gel column chromatography, a white-like solid, i.e., 5.0g of 3 '-deoxy-3', 4 '-didehydro-5' -O-diethyl phosphate ribocytidine was obtained in a yield of 50.5%.
Compound 4 a: ESI M/z 362[ M +1 ]]。1H NMR(600MHz，DMSO)，δ：9.01 (1H，d)，8.10(2H，s，NH2)，5.82(1H，d)，5.21(1H，d),4.82(1H， dd)，4.75(1H，m)，4.66(2H，s)，4.07(4H，q)，1.26(6H，t)。
4b, 4c, 4d, 4e can be prepared by the method of preparation 4 a.
Example 2
3 '-deoxy-3', 4 '-didehydro-5' -O-phosphoribosylthymidine (Compounds 7a-e)
The preparation route is as follows:
preparation of intermediate 3' -deoxy-3 ',4' -didehydro-2 ', 5 ' -diacetylribothymidine (Compound 5)
Thymine (12.60g, 0.1mol) was added to 200mL of anhydrous dichloromethane, and 3 '-deoxy-3', 4 '-didehydro-1', 2 ', 5' -triacetyl ribose (25.6g, 0.1mol) and titanium tetrachloride (1.0 mL) were added at room temperature to react for 5 hours at room temperature, followed by filtration, washing of the filtrate with water, drying of the organic phase with anhydrous sodium sulfate, filtration and concentration to obtain 5, 24.1g of an intermediate compound with a yield of 81.5%.
Preparation of intermediate 3' -deoxy-3 ',4' -didehydroribothymidine (Compound 6)
Adding compound 5(16.10g, 0.05mol) into 100mL of 10% ammonia methanol solution, stirring at room temperature for 2h, concentrating under reduced pressure after the reaction is finished, adding ethanol into the residue, heating to dissolve completely, filtering, and cooling while stirring for crystallization. Filtration and drying were carried out to obtain a white solid, i.e., intermediate 3' -deoxy-3 ',4' -didehydroribothymidine (compound 6), 8.42g, with a yield of 70.1%.
Preparation of 3 '-deoxy-3', 4 '-didehydro-5' -O-diethylphosphoribosylthymidine (Compound 7a)
Dissolving phosphorus oxychloride (5.00g, 0.033mol) in 50mL of triethyl phosphate, adding the compound 6(8.00g, 0.033mol) at 0 ℃, reacting for 3h, filtering after the reaction is completed to obtain a white solid, adding the white solid into 50mL of absolute ethyl alcohol, reacting for 4h at 60 ℃, reacting completely, and recovering the ethyl alcohol under reduced pressure. After silica gel column chromatography purification, a white solid, i.e., 4.87g of 3 '-deoxy-3', 4 '-didehydro-5' -O-diethyl phosphate ribothymidine, was obtained with a yield of 40.1%.
Compound 7 a: ESI M/z 377[ M +1 ]]。1H NMR(600MHz，DMSO)，δ：9.81 (1H，s，NH)，7.43(1H，d)，6.12(1H，d)，4.86(1H，m)，4.78(1H， dd)，4.60(2H，d)，4.01(4H，q)，2.38(3H，s)，1.24(6H，t)。
7b, 7c, 7d, 7e can be prepared by the method of preparation 7 a.
Example 3
3 '-deoxy-3', 4 '-didehydro-5' -O-phosphoribosyladenosine (Compounds 10a-e)
Preparation of intermediate 3' -deoxy-3 ',4' -didehydro-2 ', 5 ' -diacetyl riboadenosine (Compound 8)
Adenine (13.7g, 0.1mol) was suspended in 250mL of anhydrous toluene, 20mL of anhydrous DMF and 20mL of hexamethyldisilazane were added, and the mixture was refluxed for 7 hours, and the system was completely clarified, cooled to 70 ℃ and evaporated to dryness under reduced pressure. 3 '-deoxy-3', 4 '-didehydro-1', 2 ', 5' -triacetyl ribose (25.8g, 0.1mol), anhydrous dichloromethane 200mL, titanium tetrachloride 1.0mL were added at room temperature, the reaction was carried out for 5h at room temperature, the filtrate was washed with water, the organic phase was dried over anhydrous sodium sulfate, and the filtrate was concentrated to obtain 8, 25.5g of an intermediate compound with a yield of 76.8%.
Preparation of intermediate 3' -deoxy-3 ',4' -didehydroriboadenosine (Compound 9)
Adding compound 8(16.67g, 0.05mol) into 100mL of 10% ammonia methanol solution, stirring at room temperature for 2h, concentrating under reduced pressure after the reaction is finished, adding ethanol into the residue, heating to dissolve completely, filtering, and cooling while stirring for crystallization. Filtration and drying were carried out to obtain a white solid, i.e., intermediate 3' -deoxy-3 ',4' -didehydroriboadenosine (Compound 9), 7.47g, yield 60.1%.
Preparation of 3 '-deoxy-3', 4 '-didehydro-5' -O-diethyl phosphate riboadenosine (Compound 10a)
Dissolving phosphorus oxychloride (3.77g, 0.025mol) in 50mL triethyl phosphate, adding the compound 9(6.27g, 0.025mol) at 0 ℃, reacting for 4h, filtering after the reaction is completed to obtain a white solid, adding the white solid into 50mL absolute ethyl alcohol, reacting for 6h at 60 ℃, reacting for complete reaction, and recovering the ethyl alcohol under reduced pressure. After purification by silica gel column chromatography, a white-like solid, i.e., 4.93g of compound 3 '-deoxy-3', 4 '-didehydro-5' -O-diethyl phosphate riboadenosine was obtained in a yield of 51.3%.
Compound 10 a: ESI M/z 386[ M +1 ]]。1H NMR(600MHz，DMSO)，δ：8.37 (1H，s)，8.21(1H，s)，7.12(2H，s，NH2)，5.76(1H，d)，4.80(1H， d)，4.45(2H，s)，4.22(1H,dd)，3.88(4H，q)，1.23(6H，t)。
10b, 10c, 10d, 10e can be prepared by the method of preparation 10 a.
Example 4
3 '-deoxy-3', 4 '-didehydro-5' -O-phosphate riboguanosine (Compounds 13a-e)
The preparation route is as follows:
preparation of intermediate 3' -deoxy-3 ',4' -didehydro-2 ', 5 ' -diacetyl riboguanosine (Compound 11)
Adenine (15.3g, 0.1mol) was suspended in 250mL of anhydrous toluene, 20mL of anhydrous DMF and 20mL of hexamethyldisilazane were added, and the mixture was refluxed for 7 hours under heating to completely clarify the system, cooled to 70 ℃ and evaporated to dryness under reduced pressure. 3 '-deoxy-3', 4 '-didehydro-1', 2 ', 5' -triacetyl ribose (25.8g, 0.1mol), anhydrous dichloromethane (200 mL), titanium tetrachloride (1.0 mL) were added at room temperature, the reaction was carried out at room temperature for 5 hours, filtration was carried out, the filtrate was washed with water, the organic phase was dried over anhydrous sodium sulfate, and filtration and concentration were carried out to obtain intermediate compound 11, 24.5g, yield 70.2%.
Preparation of intermediate 3' -deoxy-3 ',4' -didehydroriboguanosine (Compound 12)
Adding compound 11(17.55g, 0.05mol) into 100mL of 10% ammonia methanol solution, stirring at room temperature for 2h, concentrating under reduced pressure after the reaction is finished, adding ethanol into the residue, heating to dissolve completely, filtering, and cooling while stirring for crystallization. Filtration and drying were carried out to obtain a white solid, i.e., intermediate 3' -deoxy-3 ',4' -didehydroriboguanosine (compound 9), 10.69g, yield 80.5%.
Preparation of 3 '-deoxy-3', 4 '-didehydro-5' -O-diethyl phosphate riboguanosine (Compound 13a)
Dissolving phosphorus oxychloride (3.77g, 0.025mol) in 50mL triethyl phosphate, adding the compound 12(6.67g, 0.025mol) at 0 ℃, reacting for 5h, filtering after the reaction is completed to obtain a white solid, adding the white solid into 60mL absolute ethyl alcohol, reacting for 5h at 60 ℃, reacting for complete reaction, and recovering the ethyl alcohol under reduced pressure. After purification by silica gel column chromatography, a white-like solid, i.e., 4.27g of 3 '-deoxy-3', 4 '-didehydro-5' -O-diethyl phosphate riboguanosine was obtained in a yield of 42.7%.
Compound 13 a: ESI M/z 402[ M +1 ]]。1H NMR(600MHz，DMSO)，δ：8.20 (2H,s,NH2)，8.0(1H,s,NH)，7.51(1H，s)，6.22(1H，d)，4.80(1H,d)， 4.50(2H，s)，4.04(1H，dd)，3.98(4H，q)，1.23(6H，t)。
13b, 13c, 13d, 13e can be prepared by the method of preparation 13 a.
Example 5
In vitro anti-HIV-1 Activity assay (MTT method) of 3 '-deoxy-3', 4 '-didehydro-5' -O-phospho nucleosides
The test steps are as follows:
(1) the different concentrations of 3 '-deoxy-3', 4 'to be tested'Didehydro-5' -O-phosphonucleoside drug samples were diluted in 96-well cell plates in a 1640 medium dilution format to maintain 100. mu.L of each well, 50. mu.L (about 10000) of MT-4 cells were added to each well, and 50. mu.L of 100TCID was added50The HIV-1 virus liquid of (1). And setting a plurality of normal cell control and virus infection control holes at the same time, and setting a drug AZT positive control group.
(2)37℃，5％CO2The cell culture box is used for culturing for 48 hours. On the third day, 20. mu.L of 2-fold concentrated 1640 solution was added to each well, and the culture was continued for 48 hours.
(3) Fixing and dyeing: discarding the culture medium, adding fixing solution 200 μ L, and fixing at room temperature for 5 min; gently washing cells with PBS for 2 times, adding 100 μ L staining solution (MTT), and incubating for 50 min; the blue spots were counted by microscope. The virus inhibition rate was calculated according to the following formula, and the half Inhibitory Concentration (IC) was obtained by fitting the test data using software50). The results of the experiments are shown in the following table.
The inhibition rate is [1- (number of blue spots of experimental group-number of blue spots of cell control group)/(number of blue spots of virus control group-number of blue spots of cell control group) ]. 100%.
TABLE 1 in vitro Activity test results against HIV-1 Virus
The experimental results are as follows:
the results of the experiments show that 3 '-deoxy-3', 4 '-didehydro-5' -O-phosphate nucleosides, in which the IC of a part of the active compound is50Compared with the first-line anti-HIV drug AZT, other 3 '-deoxy-3', 4 '-didehydro-5' -O-phosphate nucleosides also show stronger in-vitro HIV inhibition effect.
Example 6
In vitro anti-HCV Activity assay for 3 '-deoxy-3', 4 '-didehydro-5' -O-Phosphotidylsenosides
The method comprises the following steps:
(1) huh7.5.1 cells in good growth state were added to a 96-well plate at about 100. mu.L/well (about 10000 cells), DMEM medium was added thereto, and the mixture was incubated at 37 ℃ with 5% CO2Culture boxAnd culturing for 24 h.
(2) The test 3 '-deoxy-3', 4 '-didehydro-5' -O-phosphate nucleoside compound and the control drug RBV (ribavirin) were added diluted (10-fold) with DMSO gradients, 6 gradients were set for each compound, three wells were repeated for each gradient, blank and negative control wells were set simultaneously, and medium was added to a final volume of 200 μ L per well. Placing at 37 deg.C and 5% CO2Culturing in an incubator.
(3) Culturing for 72h, adding 20 μ L of WST-1 solution into each well, and placing 96-well culture plate in 5% CO2Incubate at 37 ℃ for 2 h. Shaking the 96-well plate on a shaking table for one minute to thoroughly mix the system to be detected, and measuring OD on a microplate reader450The value of (c).
(4) The growth inhibition rate of the test compound on Huh7.5.1 cells is calculated by software processing, and the IC of the test compound is obtained50The value is obtained.
Growth inhibition rate ═ 100% 1- (experimental well OD value/negative control OD value) ].
TABLE 2 in vitro Activity test results against HCV Virus
The experimental results are as follows:
test results show that compared with ribavirin, 3 '-deoxy-3', 4 '-didehydro-5' -O-phosphate nucleoside shows stronger in-vitro inhibition of HCV virus activity.
Example 7
In vitro anti-HBV Activity test of 3 '-deoxy-3', 4 '-didehydro-5' -O-phosphate nucleosides
The method comprises the following steps:
(1) taking a bottle of Hep G2.2.15 cells full of culture flask, digesting with 0.25% pancreatin to obtain single cell suspension, counting, and adjusting cell concentration to 1 × 104M L-1, connected toSeeded in 96-well cell culture plates at 100. mu.L per well. Place the plates in CO2Medium, 37 deg.C, 5.0% CO2Culturing for 24h under the condition.
(2) After the cells grow into a monolayer, 1640 culture media containing 3 '-deoxy-3', 4 '-didehydro-5' -O-nucleoside phosphate at different concentrations are respectively added, and a lamivudine positive control group and a blank control group are set at the same time, and 3 duplicate wells of each concentration are cultured for 5 days.
(3) To each well was added MTT 20. mu.L containing crystal violet, 37 ℃, 5.0% CO2Incubating for 4h under the condition, discarding the supernatant, adding 200 mu L of DMSO into each well, placing in a micro-oscillator, slightly oscillating until the crystal violet is almost completely dissolved, and detecting the OD value of each well at 490nm (reference wavelength 630nm) by using a 168-plus-1000 XC type enzyme standard instrument.
Inhibition rate [ (blank control well OD value-administration well OD value)/blank control well OD value ]. 100%
TABLE 3 in vitro Activity test results against HBV virus
The experimental results are as follows:
test results show that compared with ribavirin, the 3' -deoxy-3 ',4' -didehydronucleoside has stronger in-vitro HBV virus inhibition activity.
Example 8
In vitro rabies virus activity assay for 3' -deoxy-3 ',4' -didehydro-nucleosides
The method comprises the following steps:
1, diluting the drug samples with different concentrations to be tested in a 96-well cell plate by using 1640 culture solution at a multiple ratio to keep 100 mu L of drug solution in each well, adding 50 mu L (about 10000) of cells which are generated into a single-layer SK-N-SH cell and digested by pancreatin into each well, and adding 50 mu L of 100TCID50The rabies virus Strain (SAD) virus liquid. Several wells were also set for normal cell control and virus infection control.
2，37℃，5％CO2The cell culture box is used for culturing for 48 hours. On the third day, each well is supplemented with 2 times of concentrated 1640 solution 20. mu.LThe culture was continued for 48 h.
3, fixing and dyeing: discarding the culture medium, adding fixing solution 200 μ L, and fixing at room temperature for 5 min; gently washing cells with PBS for 2 times, adding 100 μ L staining solution (MTT), and incubating for 50 min; the blue spots were counted by microscope. The virus inhibition rate was calculated according to the following formula, and the half Inhibitory Concentration (IC) was obtained by fitting the test data using software50). The results of the experiments are shown in the following table.
The inhibition rate is [1- (number of blue spots of experimental group-number of blue spots of cell control group)/(number of blue spots of virus control group-number of blue spots of cell control group) ]. 100%.
TABLE 4 in vitro Activity test results against rabies Virus
The experimental results are as follows:
test results show that the 3' -deoxy-3 ',4' -didehydronucleoside has stronger activity of inhibiting rabies virus (SAD) in vitro.
Example 9
In vitro inhibition of 3' -deoxy-3 ',4' -didehydro-nucleosides against Zika Virus Activity
1, diluting the drug samples to be tested with different concentrations in 96-well cell plates by 1640 culture medium at a multiple ratio, keeping 100 μ L of the drug solution in each well, adding 50 μ L (about 10000) of grown monolayer Vero cells to each well, digesting with pancreatin, and adding 50 μ L100 TCID50Zika virus liquid of (1). Several wells were also set for normal cell control and virus infection control.
2，37℃，5％CO2The cell culture box is used for culturing for 48 hours. On the third day, 20. mu.L of 2-fold concentrated 1640 solution was added to each well, and the culture was continued for 48 hours.
3, fixing and dyeing: discarding the culture medium, adding fixing solution 200 μ L, and fixing at room temperature for 5 min; gently washing cells with PBS for 2 times, adding 100 μ L staining solution (MTT), and incubating for 50 min; the blue spots were counted by microscope. Calculating the virus inhibition rate according to the following formula, fitting the test data by using software to obtain the median inhibitory concentration(IC50). The results of the experiments are shown in the following table.
The inhibition rate is [1- (number of blue spots of experimental group-number of blue spots of cell control group)/(number of blue spots of virus control group-number of blue spots of cell control group) ]. 100%.
TABLE 5 in vitro Activity test results against Zika Virus
The experimental results are as follows:
test results show that the 3' -deoxy-3 ',4' -didehydro nucleoside compounds show stronger activity of inhibiting Zika virus in vitro.
The invention relates to the technical field of nucleoside compound antivirus, in particular to a 3 '-deoxy-3', 4 '-didehydro nucleoside compound which shows higher activity in inhibiting HIV-1, rabies virus, HCV, HBV and Zika disease, and shows that the 3' -deoxy-3 ',4' -didehydro nucleoside compound has great application potential in the aspect of antivirus.
Claims (6)
1. A3 ' -deoxy-3 ',4' -didehydronucleoside compound is characterized in that the chemical general formula is (I):
wherein:
b is selected from
Z is
Wherein
Y is selected from O or S;
R1and R2Independently selected from:
(a)OR3wherein R is3Selected from ethyl, isopropyl, n-butyl, n-hexyl, phenyl.
2. Use of the 3' -deoxy-3 ',4' -didehydronucleosides of claim 1 in the preparation of antiviral medicaments.
3. Use of the 3' -deoxy-3 ',4' -didehydro-nucleosides of claim 1 for the preparation of a medicament for the treatment of a host infected with HIV-1 or HIV-2.
4. Use of the 3' -deoxy-3 ',4' -didehydro-nucleosides of claim 1 in the preparation of a medicament for treating a rabies virus infected host.
5. Use of the 3' -deoxy-3 ',4' -didehydro-nucleosides of claim 1 in the preparation of a medicament for treating a host infected with hepatitis c, hepatitis b virus.
6. Use of the 3' -deoxy-3 ',4' -didehydro-nucleoside compound of claim 1 for the preparation of a medicament for treating a host infected with Zika virus.
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| Straightforward synthesis of 3"-deoxy-3",4"-didehydronucleoside-5"-aldehydes via 2",3"-O-orthoester group elimination: a simple route to 3",4"-didehydronucleosides;Magdalena Petrová，等;《Tetrahedron Letters》;20101030;第51卷;第6874-6876页 * |
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