CN115260263A - Alkyl sulfur aryl clavudine phosphamide compound and application thereof - Google Patents

Alkyl sulfur aryl clavudine phosphamide compound and application thereof Download PDF

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CN115260263A
CN115260263A CN202210563714.8A CN202210563714A CN115260263A CN 115260263 A CN115260263 A CN 115260263A CN 202210563714 A CN202210563714 A CN 202210563714A CN 115260263 A CN115260263 A CN 115260263A
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沙薇
刘洋
吴霖霖
张豪龙
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Abstract

The invention provides an alkyl sulfur aryl clavudine phosphoramide compound which is shown as a formula (I). Compared with the prior art, the alkyl sulfur aryl cladribine phosphoramide compound provided by the invention maintains unique anti-HBV activity, reduces the whole body exposure of cladribine, eliminates possible toxic and side effects, provides a new choice for combined medication, and possibly provides a new functional cure scheme for HBV patients in the future.

Description

Alkyl sulfur aryl clavudine phosphamide compound and application thereof
Technical Field
The invention belongs to the technical field of medicine synthesis, and particularly relates to an alkyl sulfur aryl clevudine phosphoramide compound and an application thereof.
Background
Chronic hepatitis b is an infectious disease caused by Hepatitis B Virus (HBV) infection, and clinically manifested as pain, weakness, enlargement of liver and spleen, etc., with continuous replication of HBV, liver fibrosis and cirrhosis can be gradually formed, even liver cancer is induced, endangering the life of patients. Currently, hepatitis B cure is a common target in China and even all over the world. The current clinical anti-HBV therapeutic drugs are Nucleotide Analogs (NAs) and polyethylene glycol interferon alpha (PEG-IFN alpha). The long-term application of the nucleotide antiviral drugs can generate certain drug resistance, promote viral gene mutation or deletion, and the drug is easy to relapse once the drugs are stopped, while the polyethylene glycol interferon can obviously improve the liver function of a patient, the combination of the two drugs can enhance the treatment effect, inhibit HBV replication to the maximum extent, relieve liver function injury and delay the progress of chronic hepatitis B, but cannot directly clear covalent closed circular DNA (cccDNA) in a hepatocyte nucleus, and HBV infection is difficult to completely cure. Nevertheless, nucleoside (acid) antiviral drugs are still the most promising therapeutic drugs for chronic hepatitis B at present.
The cladribine is a non-natural L-nucleoside thymidine analogue, is a first-generation nucleotide Active Site Polymerase Inhibitor (ASPIN), is a liver tissue targeting drug, has a remarkable virus inhibition effect and a liver biochemical improvement effect, still has a remarkable persistent effect (the persistent effect is a vital advantage, and most hepatitis B patients need long-term treatment) after treatment for 6 months, and has a remarkable virus control effect. The action mechanism of the clevudine is different from other nucleoside drugs, is not a DNA chain terminator, but is only a nucleotide which inhibits each stage of DNA synthesis by non-competitive twisting of the active site of polymerase to ensure that the synthesis of virus DNA chain cannot smoothly proceed by generating triphosphate L-FMAU-TP through a series of intracellular phosphorylation to be combined at the active site and induce the conformational change of the polymerase, and is possibly complementary with other schemes which seek to functionally cure HBV.
However, since cladribine does not have the advantage of liver targeting and has been found in previously published clinical studies, cladribine causes reversible proximal skeletal myopathy in subjects, such that it is no longer being developed for the treatment of chronic hepatitis b, which may be the result of mitochondrial dysfunction in patients. WO2017/223421 by alberta delayata et al reports a novel liver targeting molecule ATI2173, which aims to eliminate adverse reactions such as skeletal myopathy by improving pharmacokinetic characteristics of cladribine and reducing systemic exposure level, but the ability of active metabolites thereof to enter liver tissues is not significantly improved.
Therefore, the development of a new generation of ASPIN with the same mechanism of action, higher activity and less toxic side effects is an urgent clinical need to be met.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide alkylthio aryl cladribine phosphoramides and their use, which reduce the total exposure of cladribine while retaining the unique anti-HVB activity, and eliminate possible toxic side effects; compared with ATI-2173, the compound involved in the invention has higher biological transformation efficiency which is unpredictable, so that the compound has the advantages of low dosage and small toxic and side effect in clinic.
The invention provides an alkyl sulfur aryl clavudine phosphoramide compound, which is shown as a formula (I):
Figure BDA0003657469660000021
wherein R is1～R3Each independently selected from hydrogen, deuterium, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl;
R4selected from substituted or unsubstituted alkyl;
a is selected from substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl.
Preferably, the substituents in the substituted alkyl, substituted aryl, substituted cycloalkyl and substituted heterocyclic group are independently selected from one or more of deuterium, halogen, C1-C10 alkoxy, C1-C10 deuterated alkoxy, C6-C20 aryl, C6-C20 deuterated aryl, cyano, carboxyl, ester group, amide group, amino and hydroxyl.
Preferably, said R1～R3Each independently selected from hydrogen, deuterium, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C6-C20 aryl, substituted or unsubstituted C3-C20 cycloalkyl, and substituted or unsubstituted C2-C20 heterocyclyl;
R4selected from substituted or unsubstituted C1-C10 alkyl;
a is selected from substituted or unsubstituted C6-C20 aryl, substituted or unsubstituted C3-C20 cycloalkyl and substituted or unsubstituted C2-C20 heterocyclic radical.
Preferably, the alkyl sulfur aryl clavudine phosphoramide compound has a structure shown in a formula (II) and/or a formula (III):
Figure BDA0003657469660000031
preferably, the alkyl sulfur aryl clavudine phosphoramide compound has a structure shown in a formula (IV):
Figure BDA0003657469660000032
wherein, the carbon atom with the X is chiral carbon atom, and the configuration is R-configuration or S configuration.
Preferably, the alkyl sulfur aryl clavudine phosphoramide compound has a structure shown in a formula (V):
Figure BDA0003657469660000041
preferably, the alkyl sulfur aryl clavudine phosphoramide compound has a structure shown in formula (VI) or formula (VII):
Figure BDA0003657469660000042
preferably, the alkylthio aryl claftalidamide compounds have a structure represented by any one of formulae (VI-1) to (VI-4) and formulae (VII-1) to (VII-4):
Figure BDA0003657469660000043
the invention also provides a pharmaceutical composition which comprises one or more of the alkyl sulfur aryl cladribine phosphoramide compound, the pharmaceutically acceptable salt, the hydrate, the solvate and the crystal thereof.
The invention also provides application of one or more of the alkyl sulfur aryl clavudine phosphoramide compound, the pharmaceutically acceptable salt, the hydrate, the solvate and the crystal thereof in preparing a medicament for preventing and/or treating diseases caused by hepatitis viruses.
The invention provides an alkyl sulfur aryl clavudine phosphoramide compound, which is shown as a formula (I). Compared with the prior art, the alkyl sulfur aryl cladribine phosphoramide compound provided by the invention maintains unique anti-HBV activity, reduces the whole body exposure of cladribine, eliminates possible toxic and side effects, provides a new choice for drug combination, and possibly provides a new functional cure scheme for HBV patients in the future.
Drawings
FIG. 1 is a graph showing the change of Clevidine-TP content in Huh7 cells with time after the alkyl thio aryl clavudine phosphoramides obtained in examples 1 and 2 of the present invention were treated.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The invention provides an alkyl sulfur aryl clavudine phosphoramide compound, which is shown as a formula (I):
Figure BDA0003657469660000051
wherein R is1～R3Each independently is hydrogen, deuterium, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl; preferably hydrogen, deuterium, a substituted or unsubstituted C1-C10 alkyl group, a substituted or unsubstituted C6-C20 aryl group, a substituted or unsubstituted C3-C20 cycloalkyl group, a substituted or unsubstituted C2-C20 heterocyclic group; more preferably hydrogen, deuterium, a substituted or unsubstituted C1 to C8 alkyl group, a substituted or unsubstituted C6 to C15 aryl group, a substituted or unsubstituted C3 to C15 cycloalkyl group, a substituted or unsubstituted C2 to C15 heterocyclic group; further preferably hydrogen, deuterium, a substituted or unsubstituted C1-C5 alkyl group, a substituted or unsubstituted C6-C10 aryl group, a substituted or unsubstituted C3-C10 cycloalkyl group, or a substituted or unsubstituted C2-C10 heterocyclic group; most preferred is hydrogen, deuterium, substituted or unsubstituted C1 to C3 alkyl group, substituted or unsubstituted C6 to C8 aryl group, substituted or unsubstituted C3 to C8 cycloalkyl group, and substituted or unsubstituted C2 to C8 heterocyclic group.
R4The alkyl group is preferably a substituted or unsubstituted C1-C10 alkyl group, more preferably a substituted or unsubstituted C1-C8 alkyl group, still more preferably a substituted or unsubstituted C1-C5 alkyl group, still more preferably a substituted or unsubstituted C1-C3 alkyl group, and most preferably a substituted or unsubstituted C1-C2 alkyl group.
A is substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl; preferably a substituted or unsubstituted C6 to C20 aryl group, a substituted or unsubstituted C3 to C20 cycloalkyl group, a substituted or unsubstituted C2 to C20 heterocyclic group; more preferably a substituted or unsubstituted C6 to C15 aryl group, a substituted or unsubstituted C3 to C15 cycloalkyl group, or a substituted or unsubstituted C2 to C15 heterocyclic group; further preferably a substituted or unsubstituted C6-C10 aryl group, a substituted or unsubstituted C3-C10 cycloalkyl group, a substituted or unsubstituted C2-C10 heterocyclic group; most preferred is a substituted or unsubstituted C6 to C8 aryl group, a substituted or unsubstituted C3 to C8 cycloalkyl group, or a substituted or unsubstituted C2 to C8 heterocyclic group.
In the present invention, the heteroatom in the heterocyclic group is not particularly limited as long as it is a heteroatom known to those skilled in the art, and in the present invention, one or more of an oxygen atom, a sulfur atom and a nitrogen atom is preferable.
In the invention, the substituent groups in the substituted alkyl, substituted aryl, substituted cycloalkyl and substituted heterocyclic group are respectively and independently one or more of deuterium, halogen, C1-C10 alkoxy, C1-C10 deuterated alkoxy, C6-C20 aryl, C6-C20 deuterated aryl, cyano, carboxyl, ester group, amide group, amino and hydroxyl; more preferably one or more of deuterium, halogen, alkoxy of C1-C8, deuterated alkoxy of C1-C8, aryl of C6-C15, deuterated aryl of C6-C15, cyano, carboxyl, ester group, amide group, amino and hydroxyl; more preferably one or more of deuterium, halogen, C1-C6 alkoxy, C1-C6 deuterated alkoxy, C6-C10 aryl, C6-C10 deuterated aryl, cyano-group, carboxyl, ester group, amide group, amino group and hydroxyl group; more preferably one or more of deuterium, halogen, alkoxy of C1-C4, deuterated alkoxy of C1-C4, aryl of C6-C10, deuterated aryl of C6-C10, cyano, carboxyl, ester group, amide group, amino and hydroxyl; most preferably one or more of deuterium, halogen, alkoxy of C1-C2, deuterated alkoxy of C1-C2, aryl of C6-C10, deuterated aryl of C6-C10, cyano, carboxyl, ester group, amide group, amino and hydroxyl.
The alkyl sulfur aryl clavudine phosphoramide compound provided by the invention has chirality, wherein chiral atoms can be phosphorus atoms or carbon atoms.
When the chiral atom is a phosphorus atom, the alkyl sulfur aryl clavudine phosphoramide compound has a structure shown in a formula (II) and/or a formula (III); i.e. the phosphorus atom is a chiral atom, the configuration of which can be R-configuration, S-configuration or a mixture of the two.
Figure BDA0003657469660000071
Wherein, R is1～R3、R4The above is the same as A, and the description is omitted here.
When the chiral atom is a carbon atom, the alkyl sulfur aryl clavudine phosphoramide compound has a structure shown in a formula (IV):
Figure BDA0003657469660000072
wherein, the carbon atom with the X is chiral carbon atom, and the configuration is R-configuration or S configuration, or the mixture of the two.
According to the present invention, it is further preferred that the alkylthio aryl cladribine phosphoramide compound has a structure represented by formula (V):
Figure BDA0003657469660000073
according to the present invention, it is further preferred that the alkylthio aryl cladribine phosphoramide compound has a structure represented by formula (VI) or formula (VII):
Figure BDA0003657469660000081
according to the present invention, it is further preferred that the alkylthio arylklufback phosphoramides have a structure represented by any one of the formulae (VI-1) to (VI-4) and the formulae (VII-1) to (VII-4):
Figure BDA0003657469660000082
the invention provides an alkyl sulfur aryl cladribine phosphoramide compound, which belongs to a typical ProTide (McGuigan) prodrug, and the design principle is that nucleoside phosphonic acid/phosphoric acid drugs are respectively connected with polar groups through phosphoester bonds/phosphoramide bonds (aryl modules/amino acid ester groups) to form a phosphoester/phosphoramide prodrug, the polarity of molecules is reduced by masking the polar groups to increase the membrane permeability, and the proto-type drug is released by specific enzyme hydrolysis after the current drug is absorbed into the body.
The alkyl sulfur aryl clavudine phosphoramide compound provided by the invention maintains unique anti-HBV activity, reduces the whole body exposure of the clavudine, eliminates possible toxic and side effects, provides a new choice for combined medication, and is possible to provide a new functional cure scheme for HBV patients in the future.
The invention also provides a preparation method of the alkyl sulfur aryl clavudine phosphoramide compound, which comprises the following steps: reacting the compound shown in the formula (A) with clevudine to obtain the alkyl sulfur aryl clevudine phosphoramide compound.
Figure BDA0003657469660000091
Wherein R is1～R3Each independently selected from hydrogen, deuterium, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl;
R4selected from substituted or unsubstituted alkyl;
a is selected from substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl.
The reaction is preferably carried out in the presence of a catalyst; the catalyst is preferably tert-butyl magnesium chloride; the reaction is preferably carried out in an organic solvent; the organic solvent is preferably a mixed solvent of tetrahydrofuran and N-methylpyrrolidone; the volume ratio of tetrahydrofuran to N-methylpyrrolidone is preferably 10: (1 to 5), more preferably 10: (2 to 4), more preferably 10:3; in the present invention, it is preferable that after mixing the clarithrone, the organic solvent and the catalyst, heating and stirring are carried out, the compound represented by the formula (A) is added to carry out a reaction to obtain the alkylthio arylclarithrone phosphoramide compound; the compound represented by the formula (A) is added in the form of a solution thereof; the solvent of the solution is preferably tetrahydrofuran; the heating and stirring temperature is preferably 40-60 ℃, more preferably 45-55 ℃, and further preferably 50 ℃; the heating and stirring time is preferably 20-50 min, and more preferably 30-40 min; the reaction temperature is preferably 20 ℃ to 30 ℃, and more preferably 25 ℃; the reaction time is preferably 10 to 15 hours, more preferably 10 to 14 hours, and still more preferably 12 to 13 hours.
After the reaction is finished, preferably pouring the reaction solution into a saturated ammonium chloride solution, extracting with ethyl acetate, washing an organic phase with a saturated sodium chloride solution, drying, and concentrating to obtain the alkyl sulfur aryl clavudine phosphoramide compound; or separating the concentrated substance by high performance liquid chromatography to obtain alkyl sulfur aryl clavudine phosphoramide compounds; the stationary phase of the high performance liquid chromatography is preferably a C18 chromatographic column, more preferably Welch Xitinate C18; the mobile phase of the high performance liquid chromatography comprises a mobile phase A and a mobile phase B; the mobile phase A is preferably ammonia water; the mobile phase B is preferably acetonitrile; the elution procedure of the high performance liquid chromatography is preferably that the mobile phase B rises from 20-30% to 50-60% within 15-25 min, more preferably that the mobile phase B rises from 20-30% to 50-60% within 20min, and even more preferably that the mobile phase B rises from 25-30% to 55-60% within 20 min; further carrying out high performance liquid chromatography separation and preferably carrying out chiral resolution; the stationary phase preferably adopted for chiral resolution is DAICEL CHIRALPAK AD; the mobile phase for chiral resolution comprises a mobile phase A and a mobile phase B; the mobile phase A during chiral separation is preferably 0.1% ammonia water; the mobile phase B is preferably ethanol and/or isopropylamine; the elution procedure of the chiral resolution is preferably equal gradient elution; the concentration of the mobile phase B is preferably 40% to 60%, more preferably 45% to 55%, and still more preferably 50% by volume percentage.
In the present invention, the compound represented by the formula (a) is preferably prepared according to the following steps: reacting a compound represented by the formula (B) with a compound represented by the formula (C) and/or a salt compound thereof in the presence of an organic base to obtain a compound represented by the formula (A).
Figure BDA0003657469660000101
The salt compound of the compound shown in the formula (C) is preferably hydrochloride thereof; the organic base is preferably triethylamine; the reaction is preferably carried out in an organic solvent; the organic solvent is preferably dichloromethane; the reaction temperature is preferably-5 ℃ to 5 ℃, and more preferably 0 ℃; the reaction time is preferably 1 to 2 hours; after the reaction is finished, concentrating, adding an ethyl acetate solvent, filtering, concentrating, and purifying by column chromatography to obtain a compound shown in a formula (A); the eluent for column chromatography purification is preferably petroleum ether and ethyl acetate; the volume ratio of the petroleum ether to the ethyl acetate is preferably 50:1 to 1:1, more preferably 20:1 to 1:1, more preferably 10:1 to 1:1, more preferably 6:1 to 2:1, most preferably 4:1; the stationary phase of column chromatography purification is preferably silica gel; the particle size of the stationary phase is preferably 200-300 meshes.
The compound represented by the formula (B) is preferably prepared according to the following steps: reacting 4-nitrobenzene phosphorus dichloride with a compound shown in a formula (D) in the presence of organic base to obtain a compound shown in a formula (B).
Figure BDA0003657469660000102
Wherein the organic base is preferably triethylamine; the reaction is preferably carried out in an organic solvent; the organic solvent is preferably dichloromethane; in the invention, preferably, 4-nitrobenzene phosphorus dichloride is dissolved in an organic solvent, the temperature is reduced to minus 30 ℃ to minus 25 ℃, then an organic solution containing the compound shown in the formula (D) and organic alkali is dripped, the temperature is increased to 20 ℃ to 30 ℃ after the dripping is finished, more preferably 25 ℃, and the reaction is carried out to obtain the compound shown in the formula (B); the reaction time is preferably 30 to 90min, more preferably 50 to 80min, and still more preferably 60min.
The invention also provides a pharmaceutical composition, which comprises one or more of the alkyl sulfur aryl clevudine phosphoramide compounds, the pharmaceutically acceptable salts, the hydrates, the solvates and the crystals thereof.
The invention also provides the application of one or more of the alkyl sulfur aryl clavudine phosphoramide compounds, the medicinal salts thereof, the hydrates thereof, the solvates thereof and the crystals thereof in the preparation of medicaments for preventing and/or treating diseases caused by hepatitis viruses.
In the present invention, the hepatitis virus is preferably hepatitis B virus.
In order to further illustrate the present invention, the following examples are provided to describe the alkyl thio aryl clavudine phosphoramide compounds and their applications in detail.
The reagents used in the following examples are all commercially available.
Example 1
Figure BDA0003657469660000111
Synthesis of intermediate 3
4-Nitrophenyldichlorophosphate (5.00 g) was dissolved in dichloromethane (10.0 mL), the temperature was lowered to-30 ℃ to-25 ℃, a solution of Compound 1A (3.01 g) and triethylamine (2.17g, 2.99mL) in dichloromethane (10.0 mL) was added dropwise while controlling the temperature, after the addition was raised to 25 ℃, stirring was carried out for 1 hour, a solution of L-isopropyl alanine hydrochloride (3.93 g) in dichloromethane (10.0 mL) and triethylamine (4.15g, 5.71 mL) was added dropwise while lowering the temperature to 0 ℃, and the mixture was allowed to react at 0 ℃ for 1 hour. LCMS (RT =1.06 min) showed complete consumption of starting material. The solution was concentrated, dissolved in ethyl acetate (20.0 mL), filtered under suction, and the filter cake was washed with ethyl acetate (20.0 mL) and concentrated. The residue was purified by column chromatography (200 to 300 mesh silica gel, petroleum ether/ethyl acetate = 4/1) to obtain compound 3 (4.80 g, yield 54.0%) as a white solid.
LCMS(ESI)m/z:455.1[M+H]+
Synthesis of 4A and 4B
Clavudine (570 mg) was weighed out and dissolved in a mixed solvent of tetrahydrofuran (10.0 mL) and N-methylpyrrolidone (3.00 mL), tert-butylmagnesium chloride (1.7M, 2.58mL) was added dropwise to the above system, the mixture was stirred at 50 ℃ for 30 minutes, compound 3A (1.99 g) was added dropwise to tetrahydrofuran (10.0 mL), and the mixture was stirred at 25 ℃ for 12 hours. LCMS (RT =1.656 min) showed complete consumption of starting material. The reaction mixture was poured into a saturated ammonium chloride solution (15.0 mL), and extracted with ethyl acetate (15.0 mL,10.0 mL). The organic phase was washed with saturated sodium chloride solution (10.0 mL), dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (column: welch Xtimate C18 250 × 70mm #10um; mobile phase: 0.1% ammonia-acetonitrile; B%:25% -55%,20 min) and chiral resolution (column: DAICEL CHIRALPAK AD (250 mm × 30mm,10 μm; mobile phase: 0.1% ammonia ethanol; B%:50% -50%,11 min) to give white solids, 4A (59.4 mg, 100% purity) and 4B (127 mg, 100% purity).
LCMS(ESI)m/z:576.2[M+H]+；
The obtained compound 4A is analyzed by nuclear magnetic resonance, and the result of nuclear magnetic resonance hydrogen spectrum is obtained1HNMR(400MHz,MeOD)δppm 1.23-1.27(m,6H),1.36-1.42(m,3H),1.84 (d,J＝1.20Hz,3H),2.47(s,3H),3.88-3.98(m,1H),4.10-4.15(m,1H),4.28 -4.42(m,3H),4.95(br,dd,J＝3.60,2.12Hz,1H),4.98-5.02(m,1H),6.18- 6.29(m,1H),7.18-7.31(m,4H),7.49-7.54(m,1H)。
The obtained compound 4B is analyzed by nuclear magnetic resonance, and the result of nuclear magnetic resonance hydrogen spectrum is obtained1H NMR(400MHz,MeOD)δppm 1.22-1.27(m,6H)1.32-1.38(m,3H)1.82 -1.87(m,3H)2.46-2.51(m,3H)3.88-3.98(m,1H)4.09-4.16(m,1H)4.34- 4.47(m,3H)4.94-4.97(m,1H)4.97-5.02(m,1H)6.19-6.29(m,1H)7.16- 7.23(m,2H)7.26-7.34(m,2H)7.54-7.61(m,1H)。
Example 2
Figure BDA0003657469660000131
Synthesis of intermediate 6
4-mercaptophenol (12.6 g) and iodoethaneAlkane (15.6 g.) was added to acetone (100 ml), and potassium carbonate (11.7 g.) was added. After stirring at room temperature for 17 hours, the mixture was filtered through celite, and the filtrate was concentrated under reduced pressure. The resulting residue was dissolved in Et2O (100 ml) and washed with 2m naoh (2 × 60ml). The combined aqueous layers were acidified with 1M HCl and Et2O (2x 100ml). The combined organic layers were washed with anhydrous Na2SO4Drying, concentration, column chromatography (CyH/EtOAc 5.
Synthesis of intermediate 8
4-Nitrobenzene phosphorus dichloride (5.00 g) was dissolved in dichloromethane (10.0 mL), the temperature was reduced to-30 ℃ to-25 ℃, a solution of Compound 6 (3.31 g) and triethylamine (2.96g, 4.08mL) in dichloromethane (10.0 mL) was added dropwise while controlling the temperature, the mixture was stirred for 1 hour while increasing the temperature to 25 ℃, a solution of L-isopropyl alanine hydrochloride (3.93 g) in dichloromethane (10.0 mL) and triethylamine (4.15g, 5.71 mL) were added dropwise while reducing the temperature to 0 ℃, and the mixture was allowed to react at 0 ℃ for 1 hour. LCMS (RT =1.103 min) showed complete consumption of starting material. The solution was concentrated, dissolved in ethyl acetate (20.0 mL), filtered, and the filter cake was washed with ethyl acetate (20.0 mL) and concentrated. The residue was purified by column chromatography (silica, petroleum ether/ethyl acetate = 50/1-1/1) to give compound 8 (4.80 g, yield 52.4%) as a white solid.
LCMS(ESI)m/z:469.2[M+H]+。
Synthesis of 9A and 9B
Clavudine (555 mg) was weighed out and dissolved in a mixed solvent of tetrahydrofuran (10.0 mL) and N-methylpyrrolidone (3.00 mL), tert-butylmagnesium chloride (1.7M, 2.51mL) was added dropwise to the above system, and the mixture was stirred at 50 ℃ for 30 minutes, then tetrahydrofuran (10.0 mL) of Compound 8 (2.00 g) was added dropwise, and the mixture was stirred at 25 ℃ for 12 hours. LCMS (RT =1.77 min) showed complete consumption of starting material. The reaction mixture was poured into a saturated ammonium chloride solution (15.0 mL), and extracted with ethyl acetate (15.0 mL,10.0 mL). The organic phase was washed with saturated sodium chloride solution (10.0 mL), dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (column: welch Xtimate C18. About. 70mmmm #10 μm; mobile phase: 0.1% ammonia-acetonitrile; B%:30% -60%,20 min) and chiral resolution (column: DAICEL CHIRALPAK AD (250mm. About. 30mm,10 μm); mobile phase: 0.1% ammonia isopropylamine; B%:50% -50%,11 min) to give compounds 9A (60.2 mg, 98.9%) and 9B (70.6 mg, 100% purity) as white solids.
LCMS(ESI)m/z:590.2[M+H]+；
The obtained compound 9A is analyzed by nuclear magnetic resonance, and the result of nuclear magnetic resonance hydrogen spectrum is obtained1H NMR(400MHz,MeOD)δppm 1.16-1.25(m,6H),1.25-1.29(m,3H),1.36 -1.42(m,3H),1.79-1.87(m,3H),2.89-2.97(m,2H),3.90-3.96(m,1H), 4.10-4.15(m,1H),4.28-4.43(m,3H),4.94-4.97(m,1H),4.98-5.03(m,1 H),6.19-6.27(m,1H),7.20-7.25(m,2H),7.31-7.36(m,2H),7.50-7.54(m, 1H)。
The obtained compound 9B is analyzed by nuclear magnetic resonance, and the result of nuclear magnetic resonance hydrogen spectrum is obtained1H NMR(400MHz,MeOD)δppm 1.18-1.25(m,6H),1.27(t,J＝7.20Hz,3H), 1.33-1.37(m,3H),1.79-1.89(m,3H),2.86-2.97(m,2H),3.86-3.98(m,1 H),4.11-4.20(m,1H),4.34-4.51(m,3H),4.95-4.96(m,1H),4.97-5.02(m, 1H),5.08-5.10(m,1H),6.17-6.28(m,1H),7.17-7.24(m,2H),7.33-7.38 (m,2H),7.54-7.62(m,1H)。
Figure BDA0003657469660000141
As can be seen from the above reaction process, the active molecule of Clavudine 5' -triphosphate produced by a series of intracellular phosphorylation events in clevudine is effective, and the prodrug design bypasses the first phosphorylation step, delivers the Clavudine monophosphate to the liver where it is further converted to the active Clavudine triphosphate. The concentration of the compound converted to the active product, clevudine triphosphate, in cells after incubation in Huh7 cells was quantified and indirectly reflected in the biological activity of the compound.
Day 1, huh7 cells were plated at a density of 0.5 x 10^ 6/well in 12-well plates with 5% CO2Culturing overnight in a cell culture box at 37 ℃; on day 2, each compound was prepared in 20mM stock solution in terms of reference molecular weight, diluted to a working concentration of 20. Mu.M, and added to Huh7 cellsSetting 3 wells, and plating a total of 8 cell plates with 7 compounds; cells were collected for intracellular concentration determination of clevudine triphosphate at 4 different time points (1 h, 3h, 6h, 24 h) respectively, specifically using DPBS to rinse the cells and then blot the residual liquid in the wells, adding 800 μ L of internal standard in MeOH/Water (v/v, 70) to each tube of cell sample, calibration standard and quality control sample, after thorough vortexing of the mixture, 12000xg, centrifuged for 15 minutes at 4 ℃,70 μ L of supernatant was placed in a 96 well plate and centrifuged for 5 minutes at 4000rpm at 4 ℃,10 μ L was taken and sci was processed by MultiQuant 3.0.2 Software (ex, MA, USA) using an acquire UPLC System with BEH C18 column to analyze the sample by LC-MS/MS.
The method comprises the steps of adding a test compound with a specified concentration into Huh7 cells to treat the cells, collecting the cells at different time points, and quantitatively analyzing the Clevadine-TP content in the Huh7 cells after the compound treatment by adopting an ultra performance liquid chromatography triple quadrupole mass spectrometry, wherein the obtained result graph is shown in figure 1. From the results in FIG. 1, it is shown that the test compounds were all converted to cladribine triphosphate and that over time the content of cladribine triphosphate in Huh7 cells gradually increased and the levels were significantly better than ATI-2173.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. An alkyl sulfur aryl clavudine phosphoramide compound is shown in a formula (I):
Figure FDA0003657469650000011
wherein R is1～R3Each independently selected from hydrogen, deuterium, substituted or unsubstituted alkyl, substituted or unsubstitutedSubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl;
R4selected from substituted or unsubstituted alkyl;
a is selected from substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl.
2. The alkylthio aryl krafft family compound of claim 1, wherein the substituents for the substituted alkyl, aryl, cycloalkyl and heterocyclyl groups are independently selected from deuterium, halogen, C1-C10 alkoxy, C1-C10 deuterated alkoxy, C6-C20 aryl, C6-C20 deuterated aryl, cyano, carboxyl, ester, amide, amino and hydroxyl.
3. The alkylthio aryl cladribine phosphoramide as claimed in claim 1 wherein R is1～R3Each independently selected from hydrogen, deuterium, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C6-C20 aryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C2-C20 heterocyclyl;
R4selected from substituted or unsubstituted C1-C10 alkyl;
a is selected from substituted or unsubstituted C6-C20 aryl, substituted or unsubstituted C3-C20 cycloalkyl and substituted or unsubstituted C2-C20 heterocyclic radical.
4. The alkylthio arylcladophosphamide compounds as claimed in claim 1, wherein the alkylthio arylcladophomide compounds have the structure represented by formula (II) and/or formula (III):
Figure FDA0003657469650000021
5. the alkylthio arylcladribine phosphoramide compound of claim 1, wherein the alkylthio arylcladribine phosphoramide compound has a structure represented by formula (IV):
Figure FDA0003657469650000022
wherein, the carbon atom with the star is a chiral carbon atom, and the configuration is R-configuration or S configuration.
6. The alkylthio arylcladribine phosphoramide compound of claim 1, wherein the alkylthio arylcladribine phosphoramide compound has a structure represented by formula (V):
Figure FDA0003657469650000023
7. the alkylthio arylcladribine phosphoramide compound of claim 1 wherein the alkylthio arylcladribine phosphoramide compound has a structure represented by formula (VI) or formula (VII):
Figure FDA0003657469650000031
8. the alkylthio arylcladophosphamides compound according to claim 1, wherein the alkylthio arylcladophosphamides compound has a structure represented by any one of formula (VI-1) to formula (VI-4) formula (VII-1) to formula (VII-4):
Figure FDA0003657469650000032
9. a pharmaceutical composition comprising one or more of the alkylsulfuryl clarfluoride compounds, pharmaceutically acceptable salts thereof, hydrates thereof, solvates thereof and crystals thereof as claimed in any one of claims 1 to 8.
10. Use of one or more alkylthioaryl claftdegumphane amides, pharmaceutically acceptable salts, hydrates, solvates and crystals thereof according to any one of claims 1 to 8 for the preparation of a medicament for the prophylaxis and/or treatment of diseases caused by hepatitis virus.
CN202210563714.8A 2022-05-23 2022-05-23 Alkyl sulfur aryl clavudine phosphamide compound and application thereof Pending CN115260263A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104761604A (en) * 2014-01-02 2015-07-08 江苏豪森药业股份有限公司 Uridine monophosphate analogue, and preparation method and applications thereof
CN107286190A (en) * 2016-04-13 2017-10-24 刘沛 The preparation of oxyl benzylamino phosphoric acid/phosphate derivatives of nucleosides and its medical usage
CN107428792A (en) * 2014-12-15 2017-12-01 埃默里大学 For treating the phosphamide of hepatitis type B virus
CN109689065A (en) * 2016-06-24 2019-04-26 埃默里大学 For treating the phosphinylidyne aminate of hepatitis type B virus

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104761604A (en) * 2014-01-02 2015-07-08 江苏豪森药业股份有限公司 Uridine monophosphate analogue, and preparation method and applications thereof
CN107428792A (en) * 2014-12-15 2017-12-01 埃默里大学 For treating the phosphamide of hepatitis type B virus
CN107286190A (en) * 2016-04-13 2017-10-24 刘沛 The preparation of oxyl benzylamino phosphoric acid/phosphate derivatives of nucleosides and its medical usage
CN109689065A (en) * 2016-06-24 2019-04-26 埃默里大学 For treating the phosphinylidyne aminate of hepatitis type B virus

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