WO2016074596A1 - Nucleoside phosphoramidate derivative and application thereof - Google Patents

Abstract

The present invention provides a nucleoside phosphoramidate derivative and an application thereof, which is specifically a compound represented by formula I, or a pharmaceutically acceptable acidic salt, a solvate or a hydrate thereof. R₁ is C_1-4 alkyl; R₂ is any substituted phenyl or naphthyl, and a substituent group thereof is selected from C_1-4 alkyl and C_1-4 alkoxy; and R₃ is amino acid acyl or peptide acyl. The compound in the present invention can be used to prepare a drug for preventing or treating mammalian infectious diseases, and especially used to prepare drugs for preventing or treating hepatitis c, liver cirrhosis, and a liver cancer.

Description

Nucleoside phosphoramidate derivatives and their applications Technical field

The present invention belongs to the technical field of medicinal chemistry, and more particularly to a novel nucleoside phosphoramidate derivative, a pharmaceutically acceptable acid salt, solvate or hydrate, and their preparation for mammalian infectivity Use in diseases, prevention or treatment of drugs for hepatitis C, cirrhosis, and liver cancer.

Background technique

Hepatitis C virus (HCV) infection is a serious health problem that causes chronic liver disease in a large number of infected individuals, which in turn develops into cirrhosis and liver cancer. According to the World Health Organization, there are more than 200 million infected individuals worldwide, and at least 3 to 4 million people are infected each year. Once infected, approximately 20% of people can clear the virus, but the remaining people may carry HCV for the rest of their lives. 10% to 20% of chronically infected individuals eventually develop liver-destructive sclerosis or cancer. Current therapeutics for HCV infection include recombinant interferon, therapy alone or in combination with the nucleoside analog ribavirin, and Girard-listed sofosbuvir. While sofosbuvir is metabolized in the body by nearly 90% into inactive metabolites. Therefore, HCV infection treatment drugs with high bioavailability, good liver selectivity and high efficacy are still urgent clinical needs.

Summary of the invention

It is an object of the present invention to provide a novel nucleoside phosphoramidate derivative having antiviral viral infection activity.

A second object of the present invention is to provide a pharmaceutical composition comprising the above nucleoside phosphoramidate derivative.

A third object of the present invention is to provide a use of the above-described nucleoside phosphoramidate derivative or pharmaceutical composition for preventing or treating infectious diseases in a mammal, particularly for preventing or treating hepatitis C, liver cirrhosis, and liver cancer-related diseases. .

In particular, the invention provides a nucleoside phosphoramidate derivative, such as a compound of formula I, or a pharmaceutically acceptable acid salt, solvate or hydrate thereof:

among them:

R ₁ is a C _1-4 alkyl group;

R ₂ is an optionally substituted phenyl or naphthyl group, the substituent of which is selected from a C _1-4 alkyl group, a C _1-4 alkoxy group;

R ₃ is an amino acid acyl group or a polypeptide acyl group.

In a preferred embodiment of the invention, R ₁ in the compound of the formula I according to the invention is methyl, ethyl or isopropyl; further preferably isopropyl.

In a preferred embodiment of the invention, R ₂ in the compound of the formula I according to the invention is a phenyl or naphthyl group; further preferably a phenyl group.

In a preferred embodiment of the invention, R ₃ in the compounds of the formula I according to the invention is a natural amino acid acyl group, a non-natural amino acid acyl group or a dipeptidyl group.

In a preferred embodiment of the invention, R ₃ in the compounds of the formula I according to the invention is a natural amino acid acyl group.

In a preferred embodiment of the invention, R ₃ in the compound of the formula I according to the invention is glycyl, alanyl, prolyl, leucyl, isoleucyl, phenylalanyl, tryptophanyl , tyrosyl, aspartyl, asparaginyl, glutamyl, glutaminyl, lysyl, methionyl, seryl, threonyl, cysteinyl, prolyl, Histidine or arginyl.

In a preferred embodiment of the invention, R ₁ in the compound of formula I according to the invention is methyl, ethyl or isopropyl; R ₂ is phenyl or naphthyl; R ₃ is glycyl, alanyl , prolyl, leucyl, isoleucyl, phenylalanyl, tryptoyl, tyrosyl, aspartyl, asparaginyl, glutamyl, glutaminyl, lysyl, A methionyl group, a seryl group, a threonyl group, a cysteinyl group, a prolyl group, a histidine group, and an arginyl group.

In a more preferred embodiment of the invention, R ₁ in the compound of formula I of the invention is isopropyl; R ₂ is phenyl; R ₃ is glycyl, alanyl, prolyl, leucyl , isoleucyl, phenylalanyl, tryptoyl, tyrosyl, aspartyl, asparaginyl, glutamyl, glutaminyl, lysyl, methionyl, seryl , threonyl, cysteinyl, prolyl, histidyl, arginyl.

In a particularly preferred embodiment of the invention, the compounds provided herein are selected from one of the following compounds, or a pharmaceutically acceptable acid salt, solvate or hydrate thereof:

In an embodiment of the invention, the derivative provided by the invention comprises an enantiomer and a racemate of a compound of formula I.

In an embodiment of the invention, the derivative of the invention comprises a compound of formula I or a pharmaceutically acceptable acid salt thereof, including but not limited to a salt of a compound with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, Nitric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, acetic acid, maleic acid, fumaric acid, malic acid, mandelic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid or succinic acid.

The "C _1-4 alkyl group" in the present invention means a linear or branched saturated hydrocarbon group having 1, 2, 3 or 4 carbon atoms, including but not limited to methyl, ethyl, n-propyl or isopropyl. Base, n-butyl and the like.

The "optionally substituted" in the present invention means having a substituent or having no substituent.

The "C _1-4 alkoxy group" in the present invention means a linear or branched alkyl-substituted oxy group having ₁ , 2, 3 or 4 carbon atoms, that is, "C _1-4 alkyl-O"-".

The "amino acid acyl group" in the present invention means a group formed by the absence of -OH in the carboxyl group of the amino acid (NH2-R-C(=O)-OH) (NH2-R-C(=O)-). The amino acids referred to herein include 20 kinds of natural amino acids, and also include various unnatural amino acids, such as: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), Proline (Pro), phenylalanine (Phe), tryptophan (Trp), methionine (Met), glycine (Gly), serine (Ser), threonine (Thr), cysteine (Cys ), tyrosine (Tyr), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg) ), histidine (His), and the like.

The "polypeptide acyl group" in the present invention refers to a group in which a carboxyl group (-C(=O)-OH) in a polypeptide lacks -OH, and the polypeptide herein includes a peptide which is passed through two, three or more amino acids. The small molecule compound formed by the bond is preferably a dipeptide including, but not limited to, a glycoglutapeptide, a glycyrrhizin, a propionol dipeptide, a propyl dipeptide, and the like.

In a second aspect, the present invention provides a process for the preparation of the above nucleoside phosphoramidate derivative of the compound of formula I or a pharmaceutically acceptable acid salt, solvate or hydrate thereof, comprising the steps of:

The compound of the formula II is reacted with a compound of the formula III under a condensing agent or a compound of the formula II with a compound of the formula IV, followed by removal of the amino protecting group to give a compound of the formula I

Wherein the compound of formula II, the compound of formula III and the compound of formula IV, R ₁ , R ₂ , R ₃ are as defined in the compound of formula I, the condensing agent may be carbonyl diimidazole (CDI), N, N'-diisopropyl Carbodiimide (DIC), N,N'-dicyclohexylcarbodiimide (DCC), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride ( EDC·HCl), O-(7-nitrobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), benzotriazole-N,N, N', N'-tetramethylurea hexafluorophosphate (HBTU), the Cbz group is a benzyloxycarbonyl protecting group that protects the amino group.

As a preferred embodiment, the present invention provides a process for the preparation of the above nucleoside phosphoramidate derivative of the compound of formula I or a pharmaceutically acceptable acid salt, solvate or hydrate thereof, which comprises formula II The compound or a salt thereof is dissolved in an organic solvent, and the base is added in a batchwise manner, and then reacted with the compound of the formula IV, and then the Cbz protecting group is removed by catalytic hydrogenation to obtain a compound of the formula I; or the compound of the formula III is reacted with a condensing agent, and added. The base is reacted with an organic solvent of the compound of the formula II or a salt thereof, and then Cbz is removed by catalytic hydrogenation. The protecting group provides a compound of formula I which can be further purified by conventional methods such as recrystallization, column chromatography and the like if necessary. Here, the base may be an inorganic base or an organic base, and may be selected from sodium carbonate, potassium carbonate, sodium hydrogencarbonate, potassium hydrogencarbonate, triethylamine, or N,N-diisopropylethylamine. The compound of formula I is salted with an aqueous solution or aqueous solution of an acid to give the acid salt of the compound of formula I.

In particular, for the compound having an additional reactive functional group in the R ₃ group such as MJ10707, MJ10708, MJ10710, MJ10711, MJ10713, MJ10714, MJ10715, MJ10716, MJ10717, MJ10718, MJ10719, MJ10720, etc., the corresponding side chain is added in the present invention. Protection, adding a deprotection step after the condensation reaction.

In a third aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned nucleoside phosphoramidate derivative of Formula I, or a pharmaceutically acceptable acid salt, solvate or hydrate thereof, the pharmaceutical composition A pharmaceutically acceptable carrier or diluent may also be included, and the pharmaceutical composition may also include other active ingredients to form a combination or a synergistic composition. The pharmaceutical composition can be administered by intravenous injection, by injection into tissue, intraperitoneally, orally or intranasally. The pharmaceutical composition may be in the form of a solution, a dispersion, a suspension, a powder, a capsule, a tablet, a pill, a time release capsule, a time release tablet, and a time release pill. The pharmaceutical composition is administered at a dose of 5 to 5000 mg/day.

In a fourth aspect, the present invention provides the above nucleoside phosphoramidate derivative, such as a compound of formula I, or a pharmaceutically acceptable acid salt, solvate or hydrate thereof, for use in the preparation of a medicament for preventing or treating infectious diseases in a mammal, in particular It is used in the preparation of drugs for preventing or treating hepatitis C, liver cirrhosis and liver cancer.

Compared with the prior art, the novel nucleoside phosphoramidate derivatives of the present invention have significant anti-HCV activity, and the concentration of liver active metabolites, which is a key indicator of anti-HCV activity, is even higher than that of sofosbuvir.

DRAWINGS

Figure 1 shows the concentration (ng/g) of the liver active metabolite GS-461203 after administration of 80 mg/kg of the compound of the present invention and sofosbuvir to the SD rats.

detailed description

The embodiments of the present invention are exemplified below by way of examples, and those skilled in the art, under the teachings of the present invention, according to the prior art, the improvements of the embodiments of the present invention still belong to the protection of the present invention. Within the scope.

The source of the compound starting material used in the examples was: all reagents were purchased from a reagent company, and the compound of formula II was synthesized by the method of sofosbuvir with reference to J. Org. Chem. 2011, 76, 8311-8319.

Nuclear magnetic resonance spectroscopy data was collected and processed by a Bruker AV-300 NMR spectrometer.

Example 1 Synthesis of MJ10701

(1) Synthesis of intermediate M1

Cbz-Gly-OH (209 mg, 1 mmol) was dissolved in 2 ml of N,N-dimethylformamide (DMF), cooled to -5 ° C, DIC (63.1 mg, 0.5 mmol) was added with stirring, and reacted at room temperature for 30 min. Continue to cool to -5 ° C, add S1 (265mg, 0.5mmol) in 2ml DMF solution, triethylamine (60.7mg, 0.6mmol), catalytic amount of 4-dimethylaminopyridine (DMAP), react at room temperature for 4h, the reaction is over The mixture was poured into 10 ml of water and extracted with 10 ml of ethyl acetate. The organic layer was evaporated. 60.1%. MS (m/z): 721.2 [M + 1] ⁺ .

(2) Synthesis of MJ10701

M1 (360 mg, 0.5 mmol) was dissolved in 3 ml of methanol, 5% Pd/C 360 mg was added, and the reaction was carried out for 20 min under hydrogen. After the reaction was completed, Pd/C was filtered off, and the filtrate was evaporated to dryness. Powder (MJ10701) 118 mg, yield 40.2%. MS (m/z): 587.2 [M+1] ⁺ ; ¹ H-NMR (DMSO-d6) δ: 0.89-1.36 (m, 12H), 3.31-3.34 (m, 2H), 3.77-3.83 (m, 2H), 4.00-4.08 (m, 1H), 4.22-4.30 (m, 2H), 4.84-4.89 (m, 1H), 5.33-5.48 (m, 1H), 5.64-5.67 (m, 2H), 6.01- 6.11 (m, 2H), 7.16-7.22 (m, 3H), 7.35-7.40 (m, 2H), 7.70-7.75 (m, 1H), 7.96 (s, 1H).

Example 2 Synthesis of MJ10702

(1) Synthesis of intermediate M2

Cbz-Ala-OH (223 mg, 1 mmol) was dissolved in 2 ml of N,N-dimethylformamide (DMF), cooled to -5 ° C, DIC (63.1 mg, 0.5 mmol) was added with stirring, and reacted at room temperature for 30 min. Continue to cool to -5 ° C, add S1 (265mg, 0.5mmol) in 2ml DMF solution, triethylamine (60.7mg, 0.6mmol), catalytic amount of 4-dimethylaminopyridine (DMAP), react at room temperature for 4h, the reaction is over The mixture was poured into 10 ml of water and extracted with 10 ml of ethyl acetate. The organic layer was evaporated. 58.3%. MS (m/z): 735.2 [M + 1] ⁺ .

(2) Synthesis of MJ10702

M2 (368 mg, 0.5 mmol) was dissolved in 3 ml of methanol, 5% Pd/C 360 mg was added, and the reaction was carried out for 20 min under hydrogen. After the reaction was completed, Pd/C was filtered off, and the filtrate was evaporated to dryness. Powder (MJ10702) 137 mg, yield 45.8%. MS (m/z): 601.2 [M+1] ⁺ ; ¹ H-NMR (DMSO-d6) δ: 0.88-1.37 (m, 15H), 3.22-3.24 (m, 1H), 3.76-3.82 (m, 2H), 3.99-4.08 (m, 1H), 4.21-4.29 (m, 2H), 4.83-4.90 (m, 1H), 5.32-5.49 (m, 1H), 5.62-5.66 (m, 2H), 6.01- 6.09 (m, 2H), 7.15-7.22 (m, 3H), 7.35-7.41 (m, 2H), 7.71-7.73 (m, 1H), 7.99 (s, 1H).

Example 3 Synthesis of MJ10703

(1) Synthesis of intermediate M3

Cbz-Val-OH (251 mg, 1 mmol) was dissolved in 2 ml of N,N-dimethylformamide (DMF), cooled to -5 ° C, DIC (63.1 mg, 0.5 mmol) was added with stirring, and reacted at room temperature for 30 min. Continue to cool to -5 ° C, add S1 (265mg, 0.5mmol) in 2ml DMF solution, triethylamine (60.7mg, 0.6mmol), catalytic amount of 4-dimethylaminopyridine (DMAP), react at room temperature for 4h, the reaction is over The mixture was poured into 10 ml of water and extracted with 10 ml of EtOAc. EtOAc (EtOAc m. 50.6%. MS (m/z): 763.3 [M + 1] ⁺ .

(2) Synthesis of MJ10703

M3 (381 mg, 0.5 mmol) was dissolved in 3 ml of methanol, 5% Pd/C 360 mg was added, and the reaction was carried out under hydrogen for 20 min. After the reaction was completed, Pd/C was filtered off, and the filtrate was evaporated to dryness. Powder (MJ10703) 159 mg, yield 50.6%. MS (m/z): 629.2 [M+1] ⁺ ; ¹ H-NMR (DMSO-d6) δ: 0.89-1.36 (m, 18H), 1.85-1.91 (m, 1H), 3.23 - 3.25 (m, 1H), 3.77-3.83 (m, 2H), 3.99-4.07 (m, 1H), 4.22-4.29 (m, 2H), 4.82-4.90 (m, 1H), 5.33-5.50 (m, 1H), 5.64 5.67 (m, 2H), 6.02-6.10 (m, 2H), 7.16-7.22 (m, 3H), 7.35-7.40 (m, 2H), 7.70-7.73 (m, 1H), 7.95 (s, 1H).

Example 4 Synthesis of MJ10704

The target compound was obtained in the same manner as in Example 3, using benzyloxycarbonyl-protected amino group as the starting material. The yield was 51.3%. MS (m/z): 643.2 [M + 1] ⁺ .

Example 5 Synthesis of MJ10705

The target compound was obtained in the same manner as in Example 3, using the benzyloxycarbonyl-protected amino-isoleucine as a starting material, yield 41.1%. MS (m/z): 643.2 [M + 1] ⁺ .

Example 6 Synthesis of MJ10706

The phenylalanine which protected the amino group with a benzyloxycarbonyl group was used as a starting material, and the title compound was obtained in the same manner as in Example 3 in a yield of 40.7%. MS (m/z): 677.2 [M + 1] ⁺ .

Example 7 Synthesis of MJ10709

The target compound was obtained in the same manner as in Example 3, using benzyloxycarbonyl-protected amino-aspartic acid as the starting material. The yield was 52.7%. MS (m/z): 644.2 [M + 1] ⁺ .

Example 8 Synthesis of MJ10712

The target compound was obtained in the same manner as in Example 3, using benzyloxycarbonyl-protected amino-amino glutamine as a starting material in a yield of 45.2%. MS (m/z): 658.2 [M+1] ⁺ .

Example 9 Synthesis of MJ10718

The title compound was obtained in the same manner as in Example 3, using benzyloxycarbonyl-protected amino-proline as the starting material. The yield was 41.1%. MS (m/z): 627.2 [M + 1] ⁺ .

Example 10 Synthesis of MJ10707

(1) Synthesis of intermediate M7

Cbz-Trp(Boc)-OH (438 mg, 1 mmol) was dissolved in 2 ml of N,N-dimethylformamide (DMF), cooled to -5 ° C, and DIC (63.1 mg, 0.5 mmol) was added with stirring at room temperature The reaction was continued for 30 min, and the temperature was further lowered to -5 ° C. S1 (265 mg, 0.5 mmol) in 2 ml of DMF solution, triethylamine (60.7 mg, 0.6 mmol), catalytic amount of 4-dimethylaminopyridine (DMAP), and reaction at room temperature for 4 h were added. The reaction mixture was poured into 10 ml of water, and the mixture was extracted with EtOAc (3 mL). The yield was 63.2%. MS (m/z): 950.3 [M + 1] ⁺ .

(2) Synthesis of MJ10707

M7 (475 mg, 0.5 mmol) was dissolved in 10 ml of methanol, and hydrogen chloride gas was introduced. After the reaction was completed, 5% Pd/C 300 mg was added, and the reaction was carried out for 20 min under hydrogen. After the reaction was completed, Pd/C was filtered off, and saturated sodium hydrogencarbonate was used. The solution was quenched, the solvent was evaporated, and then evaporated to silica gel column chromatography to afford white powder (MJ10707) 109 mg. MS (m/z): 716.2 [M + 1] ⁺ .

Example 11 Synthesis of MJ10708

The tyrosine which protected the amino group with benzyloxycarbonyl group and benzyl group to protect the phenolic hydroxyl group was used as a starting material, and the target compound was obtained in the same manner as in Example 3 in a yield of 38.1%. MS (m/z): 693.2 [M+1] ⁺ .

Example 12 Synthesis of MJ10710

The target compound was obtained in the same manner as in Example 3, using a benzyloxycarbonyl-protected amino group aspartic acid side chain carboxybenzyl ester as a starting material. The yield was 39.9%. MS (m/z): 645.2 [M + 1] ⁺ .

Example 13 Synthesis of MJ10711

The target compound was obtained in the same manner as in Example 3, using the benzyloxycarbonyl-protected amino group as the starting material of the glutamic acid side chain carboxybenzyl ester. The yield was 41.5%. MS (m/z): 659.2 [M + 1] ⁺ .

Example 14 Synthesis of MJ10713

The target compound was obtained in the same manner as in Example 3 using benzyloxycarbonyl bis-protected amino lysine as a starting material in a yield of 53.5%. MS (m/z): 658.2 [M+1] ⁺ .

Example 15 Synthesis of MJ10714

The methionine protecting the amino group with a benzyloxycarbonyl group as a starting material was similar to that of Example 3 except that the catalytic hydrogenation was carried out using Raney Ni as a catalyst to obtain a target compound in a yield of 31.3%. MS (m/z): 661.2 [M + 1] ⁺ .

Example 16 Synthesis of MJ10715

The target compound was obtained in the same manner as in Example 3, using a benzyloxycarbonyl group to protect the aminobenzyl group to protect the hydroxyl group as a starting material, and the yield was 43.6%. MS (m/z): 617.2 [M + 1] ⁺ .

Example 17 Synthesis of MJ10716

The target compound was obtained in the same manner as in Example 3, using benzyloxycarbonyl as a starting material to protect the aminobenzyl group to protect the hydroxy group. The yield was 39.1%. MS (m/z): 631.2 [M + 1] ⁺ .

Example 18 Synthesis of MJ10717

A benzyloxycarbonyl group-protected aminotrityl-protected thiol-containing cysteine was used as a starting material, and a method similar to that of Example 10 was carried out except that catalytic hydrogenation was carried out using Raney Ni as a catalyst to obtain a target compound in a yield of 23.1%. MS (m/z): 633.2 [M + 1] ⁺ .

Example 19 Synthesis of MJ10719

The target compound was obtained in the same manner as in Example 10 by the benzyloxycarbonyl-protected aminotrityl group-protected side chain heterocyclic nitrogen histidine as a starting material in a yield of 27.3%. MS (m/z): 667.2 [M + 1] ⁺ .

Example 20 Synthesis of MJ10720

The target compound was obtained in the same manner as in Example 10 by using benzyloxycarbonyl-protected aminotrityl-protected arginine as a starting material, and the yield was 29.6%. MS (m/z): 686.2 [M + 1] ⁺ .

Example 21 Determination of GS-461203 in the liver after intragastric administration of test compounds in rats

GS-461203 is an active metabolite formed by the anti-hepatitis drug sofosbuvir in vivo. It passes NS5B polymerase. It can be incorporated into the RNA of hepatitis C virus and exerts an antiviral effect like a chain terminator. The content of GS-461203 in the liver after administration can reflect the strength of the compound against hepatitis C virus.

54 SD rats were divided into 3 groups, and the test solution of sofosbuvir and the patented compound was administered by gavage at a dose of 80 mg/kg, respectively, at 0.5, 1, 2, 4, 8, and 24 hours after administration. The animals were sacrificed by inhalation of carbon dioxide (3/time/group), and the liver was perfused with ice bath saline, and the liver samples were taken and placed in an -80 °C environment.

Rat liver samples were added with ice bath 70% aqueous methanol solution (including EDTA, etc.), homogenized, centrifuged, and the supernatant was taken in an appropriate amount, evaporated, and dissolved in an aqueous solution of acetonitrile containing 0.1% formic acid, which was used as a test solution.

The content of GS-461203 was determined by LC/MS/MS method, and the liver exposure (AUC) was calculated. The test compound liver GS-461203AUC was higher than sofosbuvir, MJ10702 group and MJ10703 group were significantly higher than sofosbuvir group. The results are shown in Table 1.

Table 1 Exposure of GS-461203 in the liver (ALC) after intragastric administration in rats

Compound	Dose (mg/kg)	AUC 0-t (ug.h/g)
Sofosbuvir	80	54.2
MJ10701	80	65.2
MJ10702	80	79.1
MJ10703	80	90.9
MJ10704	80	67.7
MJ10705	80	60.4
MJ10706	80	61.1
MJ10707	80	70.3
MJ10708	80	69.9
MJ10709	80	70.7
MJ10710	80	59.7
MJ10711	80	77.3
MJ10712	80	59.1
MJ10713	80	75.6
MJ10714	80	58.1
MJ10715	80	63.5
MJ10716	80	70.2
MJ10717	80	62.1

MJ10718	80	68.5
MJ10719	80	71.5
MJ10720	80	63.1

Example 22 Pharmacokinetics of GS-331007 in plasma after intragastric administration of test compounds in rats

GS-331007 is an inactive metabolite formed by the anti-hepatitis drug sofosbuvir in vivo. It is also the main metabolite of sofosbuvir. Its presence indirectly reflects the bioavailability of sofosbuvir in vivo, and the patented compounds undergo metabolism in vivo. As a process of nucleoside monophosphate and then dephosphorylation to GS-331007, the amount of GS-331007 in the test plasma can reflect the overall absorption of the inventive compound in vivo.

SD rats were divided into several groups, with 27 rats in each group. The test solutions of sofosbuvir and patented compounds were administered orally at a dose of 50 mg/kg, respectively, and 0.5, 1, 2, 3, 4 after administration. At 6, 8, 10, and 12 hours, animal plasma was collected (3/time/group).

The concentration of metabolite GS-331007 in rat plasma was quantitatively determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The pharmacokinetic parameters were calculated by WinNonlin 6.2 software according to the non-compartmental model. The results are shown in Table 2.

Table 2 Pharmacokinetic parameters of GS-331007 in plasma after intragastric administration of sofosbuvir and test compounds in rats

Compound	Dose (mg/kg)	C max (ng/g)	t 1/2 (hr)	AUC last (hr*ng/g)
Sofosbuvir	50	1653	2.56	6987
MJ10702	50	1865	3.12	8567
MJ10703	50	1743	2.37	7359
MJ10704	50	1712	3.21	7132
MJ10705	50	1663	2.43	7764
MJ10706	50	1581	3.11	7931
MJ10715	50	1547	3.47	7513
MJ10716	50	1639	2.38	7079
MJ10718	50	1987	2.33	8832

Example 23 Solubility determination

Sofosbuvir does not form salts under acidic and basic conditions, and is almost insoluble in neutral aqueous solutions. The patented compounds can form salts with acids, increase their solubility in water, and provide convenience in formulation.

Preparation of the patented compound hydrochloride: The free base of the patented compound was dissolved in ethyl acetate, and a solution of ethyl chloride in ethyl acetate was added dropwise, stirred for 10 min, petroleum ether was added to precipitate a precipitate, and the precipitate was washed with petroleum ether for 3 times, and removed under reduced pressure. Solvent, the hydrochloride of the product.

Preparation of reference standard solution: Sofosbuvir, the hydrochloride salt of the patented compound, was dissolved in methanol to prepare a solution of 0.5 mg/ml.

The sample to be tested is dissolved in water, precipitated at the bottom, filtered, and the absorbance coefficient is determined by ultraviolet spectrophotometry. The sample concentration is calculated. The test shows that the solubility of the patented compound is greater than 6 mg/ml, and the results of MJ10702 and MJ10703 are shown in Table 4.

Table 4 Solubility of the inventive compound

Compound	Sofosbuvir	MJ10702 hydrochloride	MJ10703 hydrochloride
Solubility	<2mg/ml	>11mg/ml	>10mg/ml

Claims (10)

1. a compound of formula I, or a pharmaceutically acceptable acid salt, solvate or hydrate thereof:

   

   among them:

   R ₁ is a C _1-4 alkyl group;

   R ₂ is an optionally substituted phenyl or naphthyl group, the substituent of which is selected from a C _1-4 alkyl group, a C _1-4 alkoxy group;

   R ₃ is an amino acid acyl group or a polypeptide acyl group.
2. The acid salt, solvate or hydrate thereof of claim 1 or a pharmaceutically acceptable compound of formula I as claimed in claim wherein, R ₁ is methyl, ethyl or isopropyl.
3. A compound of formula I, or a pharmaceutically acceptable acid salt, solvate or hydrate thereof, according to claim 1, wherein R ₂ is phenyl or naphthyl.
4. A compound of formula I, or a pharmaceutically acceptable acid salt, solvate or hydrate thereof, according to claim 1, wherein R ₃ is a natural amino acid acyl group, a non-natural amino acid acyl group or a dipeptidyl group.
5. A compound of formula I, or a pharmaceutically acceptable acid salt, solvate or hydrate thereof, according to claim 4, wherein R ₃ is glycyl, alanyl, prolyl, leucyl, isoluminescence Aminoacyl, phenylalanyl, tryptoyl, tyrosyl, aspartyl, asparaginyl, glutamyl, glutaminyl, lysyl, methionyl, seryl, threonine An acyl group, a cysteinyl group, a prolyl group, a histidine group, and an arginyl group.
6. A compound of formula I, or a pharmaceutically acceptable acid salt, solvate or hydrate thereof, according to claim 1, wherein R ₁ is methyl, ethyl or isopropyl; R ₂ is phenyl or naphthyl ; R ₃ is glycyl, alanyl, prolyl, leucyl, isoleucyl, phenylalanyl, tryptoyl, tyrosyl, aspartyl, asparaginyl, glutamine Acyl, glutaminyl, lysyl, methionyl, seryl, threonyl, cysteinyl, prolyl, histidyl, arginyl.
7. A compound of formula I according to claim 1 or a pharmaceutically acceptable acid salt, solvate or hydrate thereof, wherein the compound is selected from the group consisting of:

   

   

   
8. A pharmaceutical composition comprising a compound of formula I as claimed in any one of claims 1 to 7, or a pharmaceutically acceptable acid salt, solvate or hydrate thereof.
9. Use of a compound of formula I according to any one of claims 1 to 7, or a pharmaceutically acceptable acid salt, solvate or hydrate thereof, for the manufacture of a medicament for the prophylaxis or treatment of a viral infection in a mammal.
10. Use of a compound of formula I according to any one of claims 1 to 7, or a pharmaceutically acceptable acid salt, solvate or hydrate thereof, for the manufacture of a medicament for the prophylaxis or treatment of hepatitis C, cirrhosis or liver cancer.

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