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HK1210619B - Nucleoside phosphoramidate compounds for use in the treatment of hcv - Google Patents

Nucleoside phosphoramidate compounds for use in the treatment of hcv Download PDF

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Publication number
HK1210619B
HK1210619B HK15111394.2A HK15111394A HK1210619B HK 1210619 B HK1210619 B HK 1210619B HK 15111394 A HK15111394 A HK 15111394A HK 1210619 B HK1210619 B HK 1210619B
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HK
Hong Kong
Prior art keywords
alkyl
phenyl
alkenyl
alkynyl
halogenated
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HK15111394.2A
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German (de)
French (fr)
Chinese (zh)
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HK1210619A1 (en
Inventor
王勇
赵立文
张先
毕胜
高毅平
陈宏雁
王德忠
南阳
张仓
李玉秀
张迪
Original Assignee
南京圣和药业股份有限公司
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Priority claimed from PCT/CN2014/073004 external-priority patent/WO2014135107A1/en
Publication of HK1210619A1 publication Critical patent/HK1210619A1/en
Publication of HK1210619B publication Critical patent/HK1210619B/en

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Description

Technical field
The present invention belongs to the field of pharmaceutical chemistry, and in particular relates to a series of new nucleoside phosphoramidate compounds, compositions containing the nucleoside phosphoramidate compounds, and said compounds or compositions for use in the treatment of viral infection diseases, especially for use in the treatment of viral hepatitis.
Background
Hepatitis C virus (HCV) infection is a worldwide epidemic disease, and there are more than 200 million chronic infected individuals globally. Chronic infection rate of HCV is 15% in Egypt, 4.8% in Pakistan, and 3.2% in China, and these countries rank the world top three. Clinical manifestations of hepatitis C viral infection are diverse, ranging from mild symptoms such as inflammation to serious illnesses such as liver cirrhosis or liver cancer. Chronic hepatitis C can also be complicated by some extrahepatic manifestations, including rheumatoid arthritis, keratoconjunctivitis sicca, lichen planus, glomerulonephritis, mixed cryoglobulinemia, B-cell lymphoma and porphyria cutanea tarda, etc., which may be caused by the body's abnormal immune response. Furthermore, various complications can occur during the stage of HCV-induced cirrhosis decompensation, such as ascites, abdominal infections, upper gastrointestinal bleeding, hepatic encephalopathy, hepatorenal syndrome, liver failure and other symptoms.
HCV belongs to the Flaviviridae family hepatovirus genus, and its genetic structure is similar to the other two genera in Flaviviridae family, i.e. pestivirus genus and flavivirus genus. Currently, the standard methods of treatment of HCV infection include interferon alone, and combination therapy of interferon and ribavirin. However, only 50% patients respond to these methods, and interferon has significant side effects, such as flu-like symptoms, weight reduction, fatigue and weakness, and combination therapy of interferon and ribavirin has considerable side effects, including hemolysis, anemia and fatigue, etc.
In addition, drugs developed for the treatment of HCV infection include protease inhibitors, thiazolidine derivatives, thiazolidines and N-benzanilides, phenan-threnequinone, helicase inhibitors, nucleoside polymerase inhibitors and gliotoxin, antisense phosphorothioate oligodeoxy nucleotides, inhibitors of IRES-dependent translation, ribozymes and nucleoside analogs, etc.
Currently, using nucleoside phosphate compounds for the treatment of infection with the virus from the Flaviviridae family, particularly for the treatment of HCV infection is an important research direction in this field. WO 2006/065335 disclosed a fluorinated pyrrolo[2,3,d]pyrimidine nucleoside compound for inhibiting HCV virus. US 2006/0241064 disclosed a nucleoside compound for treating viral infection caused by the virus from the Flaviviridae family, such as HCV. WO 2008/121634 disclosed a nucleoside phosphoramidate compound for treating viral infections of mammals.
Notwithstanding the above disclosure, there is still a great need for compounds effective for use in the treatment and/or prevention of HCV infection.
Summary of the invention
An objective of the present invention is to provide a novel nucleoside phosphoramidate compound of general formula I for the treatment and/or prevention of HCV infection: or a stereoisomer, salt, hydrate, solvate or crystal thereof, wherein
  1. (1) R1 is selected from C1-6 alkyl;
  2. (2) R2 is selected from halogen;
  3. (3) R3 is selected from OH, H, and C1-4 alkoxy;
  4. (4) R4 is selected from H, C1-6 alkyl, and halogenated C1-6 alkyl;
  5. (5) R5 is selected from C1-6 alkyl, and halogenated C1-6 alkyl;
  6. (6) R6 is selected from the following moieties:
    1. a) phenyl-Y-, wherein Y is absent or selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-6 alkenyl-(CO)-, C2-6 alkynyl-(CO)-, O, S, amino and -N(C1-6 alkyl), and wherein the phenyl is optionally substituted by one or more groups selected from C1-6 alkyl, halogen, nitro, C1-6 alkoxy, cyano, C2-6 alkenyl, C2-6 alkynyl, C1-6 acylamino, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, amino, N(C1-6 alkyl)2 and C1-6 alkyl NHCO, or the phenyl and a five- or six-membered ring taken together form a benzo five-membered ring or benzo six-membered ring; and
  7. (7) R7 is selected from H, halogen, C1-6 alkyl, halogenated C1-6 alkyl, C1-6 alkoxy, halogenated C1-6 alkoxy, NO2, CN, C1-6 alkyl-NH-CO-, hydroxy, mono-C1-6 alkylamino, di-C1-6 alkylamino, C1-6 alkyl-S-, C2-6 alkenyl-S-, C2-6 alkynyl-S-, C1-6 alkyl-SO-, C2-6 alkenyl-SO-, C2-6 alkynyl-SO-, C1-6 alkyl-SO2- C2-6 alkenyl-SO2-, C2-6 alkynyl-SO2-, C1-6 alkyl-OSO2-, C2-6 alkenyl-OSO2-, C2-6 alkynyl-OSO2-.
Another objective of the present invention is to provide a preparation method of the nucleoside phosphoramidate compound of general formula I or a stereoisomer, salt, hydrate, solvate or crystal thereof.
Still another objective of the present invention is to provide a composition comprising a nucleoside phosphoramidate compound of general formula I of the present invention or a stereoisomer, salt, hydrate, solvate or crystal thereof and a pharmaceutically acceptable carrier, and a composition comprising a nucleoside phosphoramidate compound of general formula I of the present invention or a stereoisomer, salt, hydrate, solvate or crystal thereof and another antiviral drug.
Still another objective of the present invention is to provide a nucleoside phosphoramidate compound of general formula I of the present invention or a stereoisomer, salt, hydrate, solvate or crystal thereof, for use in the treatment and/or prevention of hepatitis C viral infection; as well as a nucleoside phosphoramidate compound of general formula I of the present invention or a stereoisomer, salt, hydrate, solvate or crystal thereof for use in the treatment and/or prevention of hepatitis C viral infection.
For the above objectives, the present invention provides the following technical solutions:
  • In a first aspect, the present invention provides a novel nucleoside phosphoramidate compound of general formula I: or a stereoisomer, salt, hydrate, solvate or crystal thereof, wherein (1) R1 is selected from C1-6 alkyl; preferably, R1 is selected from C1-3 alkyl; more preferably, R1 is CH3;(2) R2 is selected from halogen; preferably, R2 is F;(3) R3 is selected from OH, H, C1-4 alkoxy; preferably, R3 is selected from OH, H, methoxy; more preferably, R3 is OH;(4) R4 is selected from H, C1-6 alkyl, halogenated C1-6 alkyl; preferably, R4 is selected from H, C1-6 alkyl; more preferably, R4 is selected from H and C1-4 alkyl;(5) R5 is selected from C1-6 alkyl, halogenated C1-6 alkyl; preferably, R5 is selected from C1-6 alkyl; more preferably, R5 is selected from C1-4 alkyl;(6) R6 is selected from: a) phenyl-Y-, wherein Y is absent or selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-6 alkenyl-(CO)-, C2-6 alkynyl-(CO)-, O, S, amino and -N(C1-6 alkyl), and wherein the phenyl is optionally substituted by one or more groups selected from C1-6 alkyl, halogen, nitro, C1-6 alkoxy, cyano, C2-6 alkenyl, C2-6 alkynyl, C1-6 acylamino, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, amino, N(C1-6 alkyl)2 and C1-6 alkyl NHCO, or the phenyl and a five- or six-membered ring taken together form a benzo five-membered ring or benzo six-membered ring; preferably, R6 is selected from: a) phenyl-Y-, wherein Y is absent or selected from C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C2-4 alkenyl-(CO)-, C2-4 alkynyl-(CO)-, O, S, amino and -N(C1-4 alkyl), and wherein the phenyl is optionally substituted by one or more groups selected from C1-4 alkyl, halogen, nitro, C1-4 alkoxy, cyano, C2-4 alkenyl, C2-4 alkynyl, C1-4 acylamino, halogenated C1-4 alkyl, halogenated C1-4 alkoxy, amino, N(C1-4 alkyl)2 and C1-4 alkyl NHCO, or the phenyl and a five- or six-membered ring taken together form a benzo five-membered ring or benzo six-membered ring; more preferably, R6 is selected from: a) phenyl, phenyl-C1-3 alkyl-, phenyl-C2-3 alkenyl-, phenyl-C2-3 alkynyl-, phenyl-O-, phenyl-S-, phenyl-NH-, phenyl-N(C1-3 alkyl)-, phenyl-ethenyl-(CO)- and naphthyl-ethenyl-(CO)-, wherein the phenyl is optionally substituted by one or more groups selected from C1-4 alkyl, halogen, nitro, C1-4 alkoxy, cyano, C2-4 alkenyl, C2-4 alkynyl, C1-4 acylamino, halogenated C1-4 alkyl, halogenated C1-4 alkoxy, amino, N(C1-4 alkyl)2 and C1-4 alkyl NHCO, or the phenyl and a group selected from phenyl, oxazolyl, pyrazinyl and pyrrolyl taken together form a naphthyl, benzoxazolyl, benzo [b]pyrazinyl or benzo [b]pyrrolyl. Still more preferably, R6 is selected from: a) phenyl, , phenyl-(CH2)-, phenyl-=-, phenyl-=-C(O)- and phenyl-≡ -, wherein the phenyl is optionally substituted by one or more groups selected from C1-4 alkyl, halogen, nitro, C1-4 alkoxy, cyano, C2-4 alkenyl, C2-4 alkynyl, C1-4 acylamino, halogenated C1-4 alkyl, halogenated C1-4 alkoxy, amino, N(C1-4 alkyl)2 and C1-4 alkyl NHCO; and(7) R7 is selected from H, halogen, C1-6 alkyl, halogenated C1-6 alkyl, C1-6 alkoxy, halogenated C1-6 alkoxy, NO2, CN, C1-6 alkyl-NH-CO-, hydroxy, mono-C1-6 alkylamino, di-C1-6 alkylamino, C1-6 alkyl-S-, C2-6 alkenyl-S-, C2-6 alkynyl-S-, C1-6 alkyl-SO-, C2-6 alkenyl-SO-, C2-6 alkynyl-SO-, C1-6 alkyl-SO2-, C2-6 alkenyl-SO2-, C2-6 alkynyl-SO2-, C1-6 alkyl-OSO2-, C2-6 alkenyl-OSO2-, C2-6 alkynyl-OSO2-; preferably, R7 is selected from H, halogen, C1-6 alkyl, halogenated C1-6 alkyl, C1-6 alkoxy, halogenated C1-6 alkoxy, NO2, CN, C1-6 alkyl-NH-CO-; more preferably, R7 is selected from H, halogen, C1-4 alkyl, halogenated C1-4 alkyl, C1-4 alkoxy, halogenated C1-4 alkoxy, NO2, CN, C1-4 alkyl-NH-CO-.
In a preferred embodiment, the present invention provides a compound of general formula I or a stereoisomer, salt, hydrate, solvate or crystal thereof, wherein R1 is methyl, ethyl, propyl or isopropyl;R2 is F;R3 is OH, H or methoxy;R4 is H, methyl, ethyl, n-propyl or isopropyl;R5 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl;R6 is phenyl-Y- or heterocyclyl-Y-, wherein Y is absent or selected from C1-3 alkyl, C2-3 alkenyl, C2-3 alkynyl, C2-3 alkenyl-(CO)-, C2-3 alkynyl-(CO)-, O, S, amino and -N(C1-3 alkyl), and wherein the phenyl or the heterocyclyl is optionally substituted by one or more groups selected from C1-3 alkyl, halogen, nitro, C1-3 alkoxy, cyano, C2-3 alkenyl, C2-3 alkynyl, C1-3 acylamino, halogenated C1-3 alkyl, halogenated C1-3 alkoxy, amino, N(C1-3 alkyl)2 and C1-3 alkyl NHCO, or the phenyl and a five- or six-membered ring taken together form a benzo five-membered ring or benzo six-membered ring.
Surprisingly, the inventors of the present inventionhavefound that, when R6 group and the oxygen group both of which are attached to the benzene ring in general formual I are at the para- or meta- position to each other, especiallywhere R6is selected from phenyl-Y- and heterocyclyl-Y-,the compound according to the present invention has a very excellent anti-HCV activity. Without wishing to be bound by any existing theory, the inventors of the present invention believe that, the compound of general formual I wherein R6 group particularly selected from phenyl-Y-, and the phenyl-Y- or heterocyclyl-Y- is positioned either para or meta to the -O- group attached to the benzene ring in the structure of phenyl-Y-phenyl-O- or heterocyclyl-Y-phenyl-O-, exhibits excellent anti-HCV activity.
In a further preferred embodiment,the present invention provides a compound of general formula Ior a stereoisomer, salt, hydrate, solvate or crystal thereof, wherein R1 is methyl or ethyl;R2 is F;R3 is OH or H;R4 is H, methyl or ethyl;R5 is methyl, ethyl, n-propyl or isopropyl;R6 is phenyl-Y-, wherein Y is absent or selected from C1-3 alkyl, C2-3 alkenyl, C2-3 alkynyl, C2-3 alkenyl-(CO)- and C2-3 alkynyl-(CO)-, and wherein the phenyl is optionally substituted by one or more groups selected from C1-3 alkyl, halogen, nitro, C1-3 alkoxy, cyano, C2-3 alkenyl, C2-3 alkynyl, C1-3 acylamino, halogenated C1-3 alkyl, halogenated C1-3 alkoxy, amino, N(C1-3 alkyl)2 and C1-3 alkyl NHCO, or the phenyl and a group selected from oxazolyl, pyrazinyland pyrrolyl taken together form a benzoxazolyl, benzo[b]pyrazinyl or benzo[b]pyrrolyl, wherein R6 group"phenyl-Y and the oxygen group both of which are attached to the benzene ring areatthe para- or meta-position to each other.
In a further preferred embodiment, the present invention provides a compound of general formula I or a stereoisomer, salt, hydrate, solvate or crystal thereof, wherein R1 is methyl;R2 is F;R3 is OH;R4 is H, methyl or ethyl;R5 is methyl, ethyl, n-propyl or isopropyl;R6 is phenyl-Y-, wherein Y is absent or selected from C1-3 alkyl, C2-3 alkenyl, C2-3 alkynyl, C2-3 alkenyl-(CO)-and C2-3 alkynyl-(CO)-, and wherein the phenyl is optionally substituted by one or more groups selected from C1-3 alkyl, halogen, nitro, C1-3 alkoxy, halogenated C1-3 alkyl, halogenated C1-3 alkoxy, cyano, C2-3 alkenyl, C2-3 alkynyl, C1-3 acylamino, amino and C1-3 alkyl NHCO, or the phenyl and a group selected from oxazolyl, pyrazinyland pyrrolyl taken together form a benzoxazolyl, benzo[b]pyrazinyl or benzo[b]pyrrolyl, wherein R6 group"phenyl-Y-"and the oxygen groupboth of which are attached to the benzene ring are at the para- or meta- positionto each other.
In a further preferred embodiment, the present invention provides a compound of general formula I or a stereoisomer, salt, hydrate, solvate or crystal thereof, whereinR1 is methyl; R2 is F;R3 is OH; R4 is H or methyl; R5 is isopropyl; R6 is phenyl-Y-, wherein Y is absent or selected from C1-3 alkyl, C2-3 alkenyl, C2-3 alkynyl, C2-3 alkenyl-(CO)-and C2-3 alkynyl-(CO)-, and wherein the phenyl is optionally substituted by one or more groups selected from C1-3 alkyl, halogen, nitro, C1-3 alkoxy, halogenated C1-3 alkyl, halogenated C1-3 alkoxy, cyano and C1-3 alkyl NHCO, or the phenyl and a group selected from oxazolyl, pyrazinyland pyrrolyl taken together form a benzoxazolyl, benzo[b]pyrazinyl or benzo[b]pyrrolyl, wherein R6 group"phenyl-Y-" and the oxygen group both of which are attached to the benzene ring are at the para- or meta- position to each other.
In a further preferred embodiment, the present invention provides a compound of general formula I or a stereoisomer, salt, hydrate, solvate or crystal thereof, wherein R6 is phenyl or benzyl, preferably, the phenyl or benzyl and the oxygen group both of which are attached to the benzene ring are at the para- or meta- position to each other.
In other embodiments, the present invention provides acompound of general formula I or a stereoisomer, salt, hydrate, solvate or crystal thereof.
In some embodiments, more preferably, the present invention provides a compound of general formula Ia: or a stereoisomer, salt, hydrate, solvate or crystal thereof, wherein
  1. (1) R6 is selected from:
    1. a) phenyl-Y-, wherein Y is absent or selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-6 alkynyl-(CO)-, C2-6 alkynyl-(CO)-, O, S, amino and -N(C1-6 alkyl), and wherein the phenyl is optionally substituted by one or more groups selected from C1-6 alkyl, halogen, nitro, C1-6 alkoxy, cyano, C2-6 alkenyl, C2-6 alkynyl, C1-6 acylamino, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, amino, N(C1-6 alkyl)2 and C1-6 alkyl NHCO, or the phenyl and a five- or six-membered ring taken together form a benzo five-membered ring or benzo six-membered ring;
    preferably, R6 is selected from:
    1. a) phenyl-Y-, wherein Y is absent or selected from C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C2-4 alkenyl-(CO)-, C2-4 alkynyl-(CO)-, O, S, amino and -N(C1-4 alkyl), and wherein the phenyl is optionally substituted by one or more groups selected from C1-4 alkyl, halogen, nitro, C1-4 alkoxy, cyano, C2-4 alkenyl, C2-4 alkynyl, C1-4 acylamino, halogenated C1-4 alkyl, halogenated C1-4 alkoxy, amino, N(C1-4 alkyl)2 and C1-4 alkyl NHCO, or the phenyl and a five- or six-membered ring taken together form a benzo five-membered ring or benzo six-membered ring;
    more preferably, R6 is selected from:
    1. a) phenyl, phenyl-C1-3 alkyl-, phenyl-C2-3 alkenyl-, phenyl-C2-3 alkynyl-, phenyl-O-, phenyl-S-, phenyl-NH-, phenyl-N(C1-3 alkyl)-, phenyl-ethenyl-(CO)- and naphthyl-ethenyl-(CO)-, wherein the phenyl is optionally substituted by one or more groups selected from C1-4 alkyl, halogen, nitro, C1-4 alkoxy, cyano, C2-4 alkenyl, C2-4 alkynyl, C1-4 acylamino, halogenated C1-4 alkyl, halogenated C1-4 alkoxy, amino, N(C1-4 alkyl)2 and C1-4 alkyl NHCO, or the phenyl and a group selected from phenyl, oxazolyl, pyrazinyl and pyrrolyl taken together form a naphthyl, benzoxazolyl, benzo[b]pyrazinyl or benzo[b]pyrrolyl.
    Still more preferably, R6 is selected from:
    1. a) phenyl, phenyl-(CH2)-, phenyl-=-, phenyl-=-C(O)- and phenyl-≡ -, wherein the phenyl is optionally substituted by one or more groups selected from C1-4 alkyl, halogen, nitro, C1-4 alkoxy, cyano, C2-4 alkenyl, C2-4 alkynyl, C1-4 acylamino, halogenated C1-4 alkyl, amino, N(C1-4 alkyl)2 and C1-4 alkyl NHCO, or the phenyl and a group selected from oxazolyl, pyrazinyl and pyrrolyl taken together form a benzoxazolyl, benzo[b]pyrazinyl or benzo[b]pyrrolyl; and
  2. (2) R7 is selected from H, halogen, C1-6 alkyl, halogenated C1-6 alkyl, C1-6 alkoxy, halogenated C1-6 alkoxy, NO2, CN and C1-6 alkyl-NH-CO-; preferably, R7 is selected from H, halogen, C1-4 alkyl, halogenated C1-4 alkyl, C1-4 alkoxy, halogenated C1-4 alkoxy, NO2, CN and C1-4 alkyl-NH-CO-; more preferably, R7 is selected from H, fluoro, chloro, methyl, ethyl, n-propyl, isopropyl, fluoromethyl, difluoromethyl, trifluoromethyl, fluoroethyl, difluoroethyl, trifluoroethyl, methoxy, ethoxy, n-propoxy, isopropoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, fluoroethoxy, difluoroethoxy, trifluoroethoxy, NO2, CNand C1-4 alkyl-NH-CO-.
In a preferred embodiment, the present invention provides a compound of general formula Ia or a stereoisomer, salt, hydrate, solvate or crystal thereof, wherein R6 is phenyl-Y-, wherein Y is absent or selected from C1-3 alkyl, C2-3 alkenyl, C2-3 alkynyl, C2-3 alkenyl-(CO)-, C2-3 alkynyl-(CO)-, O, S, amino and - N(C1-3 alkyl), and wherein the phenyl or heterocyclyl is optionally substituted by one or more groups selected from C1-3 alkyl, halogen, nitro, C1-3 alkoxy, cyano, C2-3 alkenyl, C2-3 alkynyl, C1-3 acylamino, halogenated C1-3 alkyl, halogenated C1-3 alkoxy, amino, NH(C1-3 alkyl), N(C1-3 alkyl)2 and C1-3 alkyl NHCO, or the phenyl and a five- or six-membered ring taken together form a benzo five-membered ring or benzo six-membered ring.Preferably, the present invention provides the compound of general formula Ia or a stereoisomer, salt, hydrate, solvate or crystal thereof, wherein R6 group "phenyl-Y-" and the oxygen group both of which are attached to the benzene ring are at the para- or meta- position to each other.
In a further preferred embodiment, the present invention provides a compound of general formula Ia or a stereoisomer, salt, hydrate, solvate or crystal thereof, wherein R6 is phenyl-Y-, wherein Y is absent or selected from C1-3 alkyl, C2-3 alkenyl, C2-3 alkynyl, C2-3 alkenyl-(CO)- and C2-3 alkynyl-(CO)-, and wherein the phenyl is optionally substituted by one or more groups selected from C1-3 alkyl, halogen, nitro, C1-3 alkoxy, cyano, C2-3 alkenyl, C2-3 alkynyl, C1-3 acylamino, halogenated C1-3 alkyl, halogenated C1-3 alkoxy, amino, NH(C1-3 alkyl), N(C1-3 alkyl)2 and C1-3 alkyl NHCO, or the phenyl and a group selected from oxazolyl, pyrazinyl and pyrrolyl taken together form a benzoxazolyl, benzo[b]pyrazinyl or benzo[b]pyrrolyl, wherein R6 group and the oxygen group both of which are attached to the benzene ring are at the para- or meta- position to each other.
In a further preferred embodiment, the present invention provides a compound of general formula Ia or a stereoisomer, salt, hydrate, solvate or crystal thereof, wherein R6 is phenyl-Y-, wherein Y is absent or selected from C1-3 alkyl, C2-3 alkenyl, C2-3 alkynyl, C2-3 alkenyl-(CO)- and C2-3 alkynyl-(CO)-, and wherein the phenyl is optionally substituted by one or more groups selected from C1-3 alkyl, halogen, nitro, C1-3 alkoxy, cyano, C2-3 alkenyl, C2-3 alkynyl, halogenated C1-3 alkyl, halogenated C1-3 alkoxy, C1-3 acylamino, amino and C1-3 alkyl NHCO, or the phenyl and a group selected from oxazolyl, pyrazinyl and pyrrolyl taken together form a benzoxazolyl, benzo[b]pyrazinyl or benzo[b]pyrrolyl, wherein R6 group and the oxygen group both of which are attached to the benzene ring are at the para- or meta- position to each other.
In a further preferred embodiment, the present invention provides a compound of general formula Ia or a stereoisomer, salt, hydrate, solvate or crystal thereof, wherein R6 is phenyl-Y-, wherein Y is absent or selected from C1-3 alkyl, C2-3 alkenyl, C2-3 alkynyl, C2-3 alkenyl-(CO)- and C2-3 alkynyl-(CO)-, and wherein the phenyl is optionally substituted by one or more groups selected from C1-3 alkyl, halogen, nitro, C1-3 alkoxy, cyano, halogenated C1-3 alkyl, halogenated C1-3 alkoxy and C1-3 alkyl NHCO, or the phenyl and a group selected from oxazolyl, pyrazinyl and pyrrolyl taken together form a benzoxazolyl, benzo[b]pyrazinyl or benzo[b]pyrrolyl, wherein R6 group and the oxygen group both of which are attached to the benzene ring are at the para- or meta- position to each other.
In a further preferred embodiment, the present invention provides a compound of general formula Ia or a stereoisomer, salt, hydrate, solvate or crystal thereof, wherein R6 is phenyl- or benzyl-(i.e., phenyl-CH2-), preferably, the phenyl or benzyl and the oxygen group both of which are attached to the benzene ring are at the para- or meta- position to each other.
The present invention provides the following specific compounds: and
In another aspect, the present invention provides a preparation method of a compound of general formula I or Ia according to the present invention, comprising the following steps:
  1. a)reacting compound (1) with phosphorus oxychloride under analkine condition, followed by adding compound (2), and then adding pentafluorophenol, to obtain compound (3);
  2. b) reacting compound (3) with compound (4) at low temperature, to give the subject compound (5).
wherein R1, R2, R3, R4, R5, R6 and R7 are as defined in general formula I above.
In a third aspect, the present invention provides a pharmaceutical composition comprising a compound of general formula I or Ia according to the present invention, or a stereoisomer, salt, hydrate, solvate or crystal thereof.
In some embodiments, the present invention provides a pharmaceutical composition comprising a compound of general formula I or Ia, or a stereoisomer, salt, hydrate, solvate or crystal thereof, and additional anti-HCV therapeutic agents selected from one or more of the followings: HCV NS3 protease inhibitors, HCV NS5B RNA-dependent RNA polymerase inhibitors, nucleoside analogs, interferon α, pegylated interferon, ribavirin, levovirin, viramidine, TLR7 agonists, TLR9 agonists, cyclophilin inhibitors, α-glucosidase inhibitors, NS5A inhibitors and NS3 helicase inhibitors.
A pharmaceutical formulation may be formulated by mixing the compound of formula I or Ia of the present invention, or a stereoisomer, salt, hydrate, solvate or crystal thereof with a pharmaceutically acceptable carrier, diluent or excipient for suitable for oral or parenteral administration. Methods of administration include, but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal and oral routes. The formulations may be administered by any routes, for example by infusion or bolus injection, or via epithelial or mucocutaneous (such as oral or rectal mucosa, etc.) absorption. The administration may be systematic or topical. Examples of formulations for oral administration include solid or liquid dosage forms, specifically, comprising tablets, pills, granules, powder, capsules, syrups, emulsions, suspensions and the like. The formulations may be prepared by methods well known in the art, and may comprise conventional carriers, diluents or excipients in the field of pharmaceutical formulation.
"Flaviviridae viruses" means any virus of the Flaviviridae family, including those viruses that infect human or non-human animals, such as flavivirus, pestivirus and hepatitis C virus. The compounds and compositions of the present invention may be used particularly for the treatment or preventive treatment of HCV infection.
In another aspect, the present invention provides a compound of general formula I or Ia of the present invention, or a stereoisomer, salt, hydrate, solvate or crystal thereof for use in the prevention or treatment of viral infection diseases, especially Flaviviridae family viral infection diseases, as well as a compound of general formula I or Ia of the present invention, or a stereoisomer, salt, hydrate, solvate or crystal thereof for use in the prevention and/or treatment of viral infection diseases, especially for use in the prevention and/or treatment of HCV infection diseases, such as HCV viral hepatitis. Examples of such diseases include acute hepatitis C, chronic hepatitis C, and mixed infection of hepatitis C and hepatitis B or hepatitis D.
Definition
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as that commonly understood by a person skilled in the art.
The term "stereoisomer" refers to isomers created by a different spatial arrangement of atoms in molecules, including the cis and trans isomers, and enantiomers and conformational isomers. All stereoisomers are within the scope of the present invention. A single stereoisomer of the compound of the present invention may be substantially free of other isomers, or mixed into, for example racemates, or mixed with all other stereoisomers.
The term "salt" refers to a pharmaceutically acceptable salt formed by the compounds of the present invention with an acid. Said acid may be selected from: phosphoric acid, sulfuric acid, hydrochloric acid, hydrobromic acid, citric acid, maleic acid, malonic acid, mandelic acid, succinic acid, fumaric acid, acetic acid, lactic acid, nitric acid, sulfonic acid, p-toluenesulfonic acid, malic acid, methanesulfonic acid or the like.
The term "solvate" refers to a solid or liquid complex formed by coordination of the compound of the present invention with a solvent molecule. Hydrate is a special form of solvate, wherein the compound coordinates with water. In the context of the present invention, hydrate is a preferred solvate.
The term "crystal" refers to all solid forms formed by the compound of the present invention, comprising crystalline and amorphous forms.
The term "alkyl" refers to a straight-chain, branched chain or cyclic saturated hydrocarbon group, preferably with 6 or less carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, t-butyl, cyclobutyl, n-pentyl, isopentyl, neopentyl, cyclohexyl, n-hexyl, isohexyl, 2, 2-dimethylbutyl and 2,3-dimethylbutyl. The term "C1-6 alkyl" means a straight-chain, branched chain or cyclic saturated hydrocarbon group with 1-6 carbon atoms. The term "C1-4 alkyl" means a straight-chain, branched chain or cyclic saturated hydrocarbon group with 1-4 carbon atoms.
The term "alkenyl" means a straight-chain or branched chain unsaturated hydrocarbon group containing one or more carbon-carbon double bonds (C=C), preferably with 2 to 6 carbon atoms, more preferably with 2 to 4 carbon atoms, and most preferably with 2 carbon atoms. The term "C2-6 alkenyl" refers to an unsaturated hydrocarbon group of 2 to 6 carbon atoms containing 1 or 2 carbon-carbon double bonds. The term "C2-4 alkenyl" refers to an unsaturated hydrocarbon group of 2 to 4 carbon atoms containing 1 or 2 carbon-carbon double bonds.
The term "alkynyl" means a straight-chain or branched chain unsaturated hydrocarbon group containing one or more carbon-carbon triple bond (C=C), preferably with 2 to 6 carbon atoms, more preferably with 2 to 4 carbon atoms, most preferably with 2 carbon atoms. The term "C2-6 alkynyl" refers to an unsaturated hydrocarbon group of 2 to 6 carbon atoms containing 1 or 2 carbon-carbon triple bonds. The term "C2-4 alkynyl" refers to an unsaturated hydrocarbon group of 2 to 4 carbon atoms containing 1 or 2 carbon-carbon triple bonds.
The term "alkoxy" refers to -O- alkyl.
The term "halogen" refers to fluorine, chlorine, bromine and iodine.
The term "halogenated alkyl" means an alkyl substituted by at least one halogen atom.
The term "heterocyclyl" refers to a cyclic group containing at least one heteroatom, wherein the heteroatom may be N, O or S, including single heterocyclyl and fused heterocyclyl. The single heterocyclyl group includes, but is not limited to furan, thiophene, pyrrole, thiazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-thiadiazole, oxazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole, pyridine, pyrimidine, pyridazine, pyrazine, tetrahydrofuran, pyrrolidine, piperidine, piperazine, morpholine, isoxazoline,etc. The fused heterocyclyl group includes, but is not limited to quinoline, isoquinoline, indole, benzofuran, benzothiophene, purine, acridine, carbazole, fluorene, chromene ketone, fluorenone, quinoxaline, 3,4-dihydronaphthalenone, dibenzofuran, dibenzofuran hydride, benzoxazolyl and the like.
The term "benzo five-membered ring" and "benzo six-membered ring" mean a fused ring group.
Detailed description of the invention
The following examples are provided to illustrate the present invention without limiting the invention to the particulars of these examples. The reagents and starting materials used in the present invention are commercially obtained.
Example1:(2S)-2-((([1,1'-biphenyl]-4-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
Step 1: preparation of (2S)-2-((((S)-pentafluorophenoxy)([1,1'-biphenyl]-4-yloxy)phosphoryl)amino)propanoic acid isopropyl ester
To a reaction flask were added 0.9g phosphorus oxychloride (5.87mmol) and 30mL dichloromethane. The mixture was cooled to -60°C and a solution of 1g p-phenylphenol (5.87mmol) and 0.6g triethylamine (5.87mol) in dichloromethane were added dropwise slowly. After the addition was complete, the mixture was reacted overnight at room temperature, cooled to 0°C, and 0.9g L-alanine isopropyl ester hydrochloride (5.3mmol) was added. The mixture was cooled to - 60°C, and a solution of 1.34g triethylamine (13mmol) in dichloromethane (20mL) was added dropwise. After the addition was complete, the temperature was raised to -5°C, then a solution of 1g pentafluorophenol (5.3mmol) and 0.8g triethylamine (8mmol) in dichloromethane (15mL) was added dropwise to the above mixture solution. The resultant mixture was stirred for 1 hour at -5°C. After completion of the reaction, water was added for extraction, then the mixture was dried, concentrated and separated by silica gel column chromatography to give the title compound.
Step 2: preparation of (2S)-2-((([1,1'-biphenyl]-4-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
To a reaction flask were added (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine (26mg,0.1mmol) and 1mL tetrahydrofuran. A solution of 1M tert-butyl magnesium chloride (0.25mmol) in THF (0.25mL) was added dropwise under nitrogen protection in an ice bath. After the addition was complete, the mixture was reacted for 4 hours at room temperature, and a solution of the above-obtained phosphate intermediate of p-phenylpheno (70mg, 0.13mmol) in tetrahydrofuran (1.5mL) was added dropwise in an ice bath. After the addition, the mixture was reacted overnight at room temperature. After the reaction was complete, the reaction was quenched by adding 6mL of 2N HCl in an ice bath. The resultant mixture was extracted with ethyl acetate, washed with saturated sodium bicarbonate solution, dried, concentrated, and separated and purified by silica gel column chromatography to give the title compound. 1HNMR(300MHz,DMSO)δ:11.51(s,1H,pyrimidineN-H),7.68-7.62(m,4H,Ar-H,pyrimidine-H),7.60-7.56(d,1H,Ar-H),7.48-7.43(t,2H,Ar-H),7.38-7.30(m,3H,Ar-H),6.12-6.00(m,2H,tetrahydrofuran-H),5.84(d,1H,pyrimidine-H),5.56(d,1H,P-NH),4.90-4.82(m,1H,-(CH3)2C-H),4.62-4.34(m,1H,tetrahydrofuran-OH),4.30-4.22(m,1H,(CH3)C(NH)-H),4.06-4.00(m,1H,tetrahydrofuran-H),3.88-3.77(m,2H,P-O-CH2-H),1.30-1.20(m,9H,3×CH3),1.58(d,3H,CH3). ESI-MS m/z: [M+H]+ =606.2.
Example 2: (2S)-2-((([1,1'-biphenyl]-3-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using m-phenylphenol, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1HNMR(300MHz,DMSO)δ:11.50(s,1H,pyrimidineN-H),7.68-7.55(m,3H,Ar-H,pyrimidine-H),7.50-7.36(m,6H,Ar-H),7.21(d,1H,Ar-H),6.13-6.00(m,2H,tetrahydrofuran-H),5.88-5.84(d,1H,pyrimidine-H),5.57-5.52(d,1H,P-NH),4.86-4.82(m,1H,-(CH3)2C-H),4.40-4.38(m,1H,tetrahydrofuran-OH),4.28-4.26(m,1H,(CH3)C(NH)-H),4.04-4.02(m,1H,tetrahydrofuran-H),3.88-3.80(m,2H,P-O-CH2-H),1.28-1.20(m,6H,2×CH3),1.14-1.11(d,6H, 2×CH3). ESI-MS m/z: [M+H]+ =606.2.
Example 3: (2S)-2-((([1,1'-biphenyl]-2-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using o-phenylphenol, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1HNMR(300MHz,DMSO)δ:11.49(s,1H,pyrimidine-N-H),7.56-7.05(m,10H,Ar-H,pyrimidine-H),6.38-5.89(m,2H,tetrahydrofuran-H),5.80(d,1H,pyrimidine-H),5.42(d,1H,P-NH),4.86-4.75(m,1H,-(CH3)2C-H),4.24-4.18(m,1H,tetrahydrofuran-OH),4.14-4.08(m,1H,(CH3)C(NH)-H),3.99-3.89(m,1H,tetrahydrofuran-H),3.85-3.72(m,2H,P-O-CH2-H), 1.27-1.23(m,3 H,CH3),1.19-1.07(d,9H, 3×CH3). ESI-MS m/z: [M+H]+ =606.2.
Example 4: (2S)-2-(((2-benzylphen-1-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using o-benzylphenol, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1HNMR(300MHz,DMSO)δ:11.48(s,1H,pyrimidine-N-H),7.53-7.05(m,10H,Ar-H,pyrimidine-H),6.36-5.88(m,2H,tetrahydrofuran-H),5.81(d,1H,pyrimidine-H),5.43(d,1H,P-NH),4.85-4.71(m,1H,-(CH3)2C-H),4.21-4.15(m,1H,tetrahydrofuran-OH),4.12-4.01(m,1H,(CH3)C(NH)-H),3.92-3.89(m, 1H,tetrahydrofuran-H),3.81-3.73(m,2H,P-O-CH2-H),1.32-1.31(m,2H, CH2)1.23-1.21(m,3H,CH3),1.11-1.01(d,9H, 3×CH3). ESI-MS m/z: [M+H]+ =620.2.
Example 5: (2S)-2-((([4'-fluoro-1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using 4-fluoro-4'-hydroxybiphenyl, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1HNMR(300MHz,CDCl3)δ:8.09(s,1H,pyrimidineN-H),7.55-7.49(m,6H,Ar-H,),7.32(d,1H,Ar-H),7.17-7.11(d,1H,pyrimidine-H),6.23-6.18(m,1H,tetrahydrofuran-H),5.76(d,1H,pyrimidine-H),5.09-5.00(m,1H,P-NH),4.62-4.49(m,2H,-(CH3)2C-H),4.16-4.12(m,1H,tetrahydrofuran-OH),4.04-3.97(m,2H,(CH3)C(NH)-H),3.78-3.71 (m,1H,tetrahydrofuran-H),1.49(m,2H,P-O-CH2-H),1.42-1.39(m,6H,2×CH3),1.30-1.24(d,6H, 2×CH3). ESI-MS m/z: [M+H]+ =624.3.
Example 6: (2S)-2-((([4'-nitro -1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using 4-hydroxy-4'-nitro biphenyl, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1HNMR(300MHz,DMSO)δ:11.48(s,1H,pyrimidineN-H),8.29(d,2H,Ar-H,),7.94(d,2H,Ar-H),7.82(d,2H,Ar-H),7.56(d,1H,pyrimidine-H),7.38-7.34(m,2H,Ar-H),6.13-6.04(m,2H,tetrahydrofuran-H),5.83(d,1H,pyrimidine-H),5.56(d,1H,P-NH),4.88-4.85(m,1H,-(CH3)2C-H),4.40(m,1H,tetrahydrofuran-OH),4.28(m,1H,(CH3)C(NH)-H),4.04-4.02(m,1H,tetrahydrofuran-H)3.86-3.84(m,2H,P-O-CH2-H),1.28-1.25(m,6H,2×CH3),1.18-1.15(m,6H,2×CH3). ESI-MS m/z: [M+H]+ =651.4.
Example 7: (2S)-2-((((E)-4-(3,5-dimethoxyphenylethenyl)phen-1-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using (E)-4-(3,5-dimethoxyphenylethenyl)phenol, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1HNMR(300MHz,DMSO)δ:11.51(s,1H,N-H),8.19(d,1H,Ar-H),7.59(t,2H,Ar-H),7.20(m,4H,Ar-H),6.76(s,2H,Ar-H),6.41 (s,1H,Ar-H),6.10(s,1H,Ar-H),6.06(m,1H,-CH-),5.85(d,1H,N-H),5.57(d,1H,-CH-),4.85(q,1H,-CH-),4.37(m,2H,-CH-),4.27(m,1H,-OH),4.01(m,1H,-CH2-),3.78(s,6H,-CH3),1.29(s,3H,-CH3),1.25(s,3H,-CH3),1.15(d,6H,-CH3). ESI-MS m/z: [M+H]+ =692.2.
Example 8:(2S)-2-(((3-cinnamoylphen-1-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using 3-cinnamoylphenol, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1HNMR(300MHz,DMSO)δ:11.52(s,1H,pyrimidineN-H),7.88-7.82(m,1H,pyrimidine-H),7.78-7.72(m,3H,Ar-H),7.60-7.56(d,1H,Ar-H),7.46(s,1H,CH=CH),7.31-7.22(m,4H,Ar-H),6.63(d,1H,CH=CH),6.12-6.00(m,2H,tetrahydrofuran-H),5.82(d,1H,pyrimidine-H),5.53(d,1H,P-NH),4.91-4.82(m,1H,-(CH3)2C-H),4.63-4.34(m,1H,tetrahydrofuran-OH),4.30-4.22(m,1H,(CH3)C(NH)-H),4.06-4.00(m,1H,tetrahydrofuran-H),3.88-3.77(m,2H,P-O-CH2-H),1.30-1.20(m,9H,3×CH3),1.58(d,3H,CH3). ESI-MS m/z: [M+Na]+ =682.1.
Example 9:(2S)-2-(((4-(phenylethynyl)phen-1-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicacid isopropyl ester
The title compound was prepared according to the method described in Example 1 using 1-(4-hydroxyphenyl)-2-phenyl acetylene, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1HNMR(300MHz,DMSO)δ:11.49(s, 1H,pyrimidineN-H),7.71(d,2H,Ar-H,),7.56(m,4H,Ar-H,pyrimidine-H),7.42(s,2H,Ar-H,),7.27(m,1H),7.04(m,1H),6.16-6.03(m,2H,tetrahydrofuran-H),5.84(d,1H,pyrimidine-H),5.66-5.52(m,1H,P-NH),4.87-4.83(m,1H,-(CH3)2C-H),4.38(m,1H,tetrahydrofuran-OH),4.25(m,1H,(CH3)C(NH)-H),4.02(m,1H,tetrahydrofuran-H),3.79(m,2H,P-O-CH2-H),1.27-1.23(m,6H,2×CH3),1.16-1.14(m,6H,2×CH3). ESI-MS m/z: [M+H]+ =630.3.
Example 10: (2S)-2-(((2-(quinoxalin-5-yl)phen-1-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using 2-(quinoxalin-5-yl)phenol, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1HNMR(300MHz,DMSO)δ:11.48(s,1H,pyrimidineN-H),7.69-7.61(m,1H,pyrimidine-H),7.69-7.61(m,3H,Ar-H),7.61-7.58(d,1H,Ar-H),7.49-7.45(t,2H,quinoxaline-H),7.37-7.32(m,3H,Ar-H),6.11-6.03(m,2H,tetrahydrofuran-H),5.82(d,1H,pyrimidine-H),5.53(d,1H,P-NH),4.90-4.83(m,1H,-(CH3)2C-H),4.62-4.32(m,1H,tetrahydrofuran-OH),4.30-4.21(m,1H,(CH3)C(NH)-H),4.04-4.01(m,1H,tetrahydrofuran-H),3.83-3.74(m,2H,P-O-CH2-H),1.34-1.21(m,9H,3×CH3),1.53(d,3H,CH3). ESI-MS m/z: [M+H]+ =658.2.
Example 11: (2S)-2-((([4'- cyano-1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using 4'-cyano-4-hydroxybiphenyl, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1HNMR(300MHz,DMSO)δ:11.50(s,1H,pyrimidineN-H),7.98-7.82(m,8H,Ar-H),7.57(m,1H,pyrimidine-H),6.18-6.15(m,2H,tetrahydrofuran-H),5.91(d,1H,pyrimidine-H),5.59(d,1H,P-NH),4.92-4.87(m,1H,-(CH3)2C-H),4.68-4.36(m,2H,tetrahydrofuran-OH,(CH3)C(NH)-H),4.03-4.01 (m,1H,tetrahydrofuran-H),3.85-3.82(m,2H,P-O-CH2-H),1.40-1.29(m,9H,3×CH3),1.15-1.13(d,3H,CH3). ESI-MS m/z: [M+H]+=631.2.
Example 12: (2S)-2-(((4-((E)-4-fluorophenylethenylphenyl)-1-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
Step 1: preparation of (E)-4-(4-fluorophenylethenyl)phenol
To a 50mLthree-necked flask were added p-iodophenol(1.32g, 6mmol), p-fluorostyrene (610mg, 5mmol), tetrakis(triphenylphosphine)palladium (622mg, 0.5mmol) and cesium carbonate (4.89g, 15mmol), then added 20mL of 1,4-dioxane.Argon gas was passed through the solution, and the mixture was reacted at 80°C overnight (about 10h) until the solution turned black. After 24h, the reaction was complete by TLC monitoring. Dilute hydrochloric acid was slowly added dropwise at 0°C, and the mixture was extracted with ethyl acetate and separated by silica gel column chromatography to give a white solid. ESI-MS m/z: [M+H]+ =215.2.
Step 2: preparation of(2S)-2-((((E)-4-(4-fluorophenylethenylphenyl)-1-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using (E)-4-(4-fluorophenylethenyl)phenol, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1HNMR(300MHz,DMSO)δ:11.46(s,1H,pyrimidineN-H),7.64-7.58(m,5H,Ar-H,pyrimidine-H),7.23-7.15(m,6H,Ar-H,-CH=CH-),6.10-6.15(m,2H,tetrahydrofuran-H),5.91(d,1H,pyrimidine-H),5.79(d,1H,P-NH),4.91-4.88(m,1H,-(CH3)2C-H),4.66-4.32(m,2H,tetrahydrofuran-OH,(CH3)C(NH)-H),4.03-4.01(m,1H,tetrahydrofuran-H),3.85-3.82(m,2H,P-O-CH2-H),1.40-1.29(m,9H,3×CH3),1.15-1.13(d,3H,CH3). ESI-MS m/z: [M+H]+ =650.4.
Example 13: (2S)-2-((((4'-chloro-[1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using 4'-chloro-4-hydroxy[1,1'-biphenyl], phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1H NMR (300 MHz, DMSO-d6 ) δ ppm: 11.54 (s,1H),7.66-7.69(d,3H),7.55-7.59(d, 1H),7.49-7.52(d,2H),7.29-7.33(m,3H),6.12-6.16(m,2H),5.96-5.99(m,1H),5.34(m,1H),4.81-4.89 (m,1H),4.38-4.42(m,2H),3.84(m,1H),3.82-3.84(m,2H),1.05-1.14(m,12H). LC-MS m/z [M+H]+=640.
Example 14: (2S)-2-(((2-fluoro-[1,1'-biphenyl]-4-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
Step 1: preparation of 2-fluoro-4-hydroxy-[1,1'-biphenyl]
In a 250mL eggplant-shaped flask were added 1.06g phenylboronic acid, 2.38g 3-fluoro-4-iodophenol, 0.4g Pd(dppf)Cl2 and 9g cesium carbonate, then added 50mL 1,4-dioxane and 5mL water. The mixture was stirred at 90°C for 1.5h under nitrogen protection, then the reaction was stopped. The reaction solution was extracted with 100mL ethyl acetate and 50mL saturated sodium chloride solution, and washed with water(3X50mL).The organic phases were collected, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give the title compound. LC-MS m/z: [M+H]+=189.
Step 2: preparation of(2S)-2-(((2-fluoro-[1,1'-biphenyl]-4-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using 2-fluoro-4-hydroxy-[1,1'-biphenyl] prepared from Step 1, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1H NMR (400 MHz, DMSO-d6 ) δ ppm: 11.49(s,1H),7.51-7.82(m,6H),7.15-7.29(m,3H),6.12-6.15(m,1H),6.03(m,1H),5.97-5.99(m,1H),5.58(m,1H),4.80-4.89(m,1H),4.38-4.41(m,2H),4.01(m,1H),3.82-3.84(m,2H),1.05-1.14 (m, 12H). LC-MS m/z: [M+H]+= 624.
Example 15: (2S)-2-((((3'-(N-methylcarbamoyl)-[1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
Step 1: 4'-hydroxy-N-methyl-[1,1'-biphenyl]-3-formamide
The title compound was prepared according to the method described in Step 1 of Example 14 using 4-iodophenol, 3-(N-methylcarbamoyl)phenylboronic acid, Pd(dppf)Cl2 and cesium carbonate as starting materials. LC-MS m/z: [M+H]+ =228.
Step 2:preparation of (2S)-2-((((3'-(N-methylcarbamoyl)-[1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using 4'-hydroxy-N-methyl-[1,1'-biphenyl]-3-formamide prepared from Step 1, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1H NMR(400 MHz,DMSO-d6 ) δ ppm:11.46-11.50(s,1H),7.95-7.96(m,1H),7.70-7.73 (m,1H),7.57-7.59(m,1H),7.35-7.37(m,2H),7.30-7.32(m,1H),7.28-7.31(m,1H),7.22-7.26(m,1H),7.18-7.20(m,1H),7.15-7.17(m,1H),6.08-6.10(m,1H),6.01(m,1H),5.97-6.02(m,1H),5.58(m,1H),4.81-4.88(m,1H),4.38-4.42(m,2H),4.05(m,1H),3.89-3.92(m,5H),1.16-1.28(m,12H). LC-MS m/z: [M-H]+ = 661.
Example 16: (2S)-2-((((3'-fluoro-[1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
Step 1: preparation of 4-(3-fluorophenyl) phenol
The title compound was prepared according to the method described in Step 1 of Example 14 using 4-hydroxyphenylboronic acid, 3-fluoroiodobenzene, Pd(dppf)Cl2 and cesium carbonate as starting materials. LC-MS m/z: [M+H]+ =189.
Step 2: preparation of(2S)-2-((((3'-fluoro-[1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using 4-(3-fluorophenyl)phenol prepared from Step 1, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1H NMR (400 MHz, DMSO-d6 ) δ ppm: 11.49(s,1H),7.95-7.97(m,1H),7.70-7.72(m, 1H),7.56-7.58(m,1H),7.36-7.38(m,2H),7.31-7.32(m,1H),7.28-7.30(m,1H),7.19-7.20(m,1H),7.16-7.17(m,1H),6.07-6.10(m,1H),6.03(m,1H),5.97-6.01(m,1H),5.56(m,1H),4.80-4.88(m,1H),4.38-4.41(m,2H),4.06(m,1H),3.82-3.86(m,2H),1.16-1.27(m,12H). LC-MS m/z: [M+H]+=624.
Example 17: (2S)-2-((((3'-chloro-[1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
Step 1: preparation of 4-(3-chlorophenyl) phenol
The title compound was prepared according to the method described in Step 1 of Example 14 using 4-hydroxyphenylboronic acid, 3-chloroiodobenzene, Pd(dppf)Cl2 and cesium carbonate as starting materials. LC-MS m/z: [M+H]+ =205.
Step 2: preparation of(2S)-2-((((3'-chloro-[1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using 4-(3-chlorophenyl)phenol prepared from Step 1, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1H NMR (400 MHz, DMSO-d6 ) δ ppm: 11.49(s,1H),7.51-7.82(m,6H),7.26-7.35(m, 3H),6.12-6.14(m,1H),6.11(m,1H),5.96-5.98(m,1H),5.88(m,1H),4.80-4.88(m,1H),4.38-4.42 (m,2H),4.01(m,1H),3.82-3.87 (m,2H),1.11-1.29(m,12H). LC-MS m/z: [M+H]+=640.
Example 18: (2S)-2-(((((4'-methoxy-[1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using 4-hydroxy-4'-methoxy-1,1'-biphenyl, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1HNMR(500MHz,DMSO)δ:11.49(s,1H),7.61-7.55(m,5H),7.28-7.23(m,2H),7.01(d,2H),6.05-6.01(m,2H),5.84-5.82(m,1H),5.57-5.55(m,1H),4.88-4.84(m,1H),4.41-4.37(m,1H),4.27-4.25(m,1H),4.05-4.01(m,1H)3.84-3.82(m,2H),2.333.79(s,3H)1.28-1.22(m,6H),1.18-1.15(m,6H). ESI-MS m/z: [M+H]+ =636.1.
Example 19: preparation of (2S)-2-(((((4'-trifluoromethyl-[1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
Step 1: preparation of 4-hydroxy-4'-trifluoromethyl-[1,1'-biphenyl]
2g 4-iodobenzotrifluoride and 1.2g p-hydroxyphenylboronic acid were added to a 100mL single-neck flask, and 2g potassium carbonate, 0.26g PdCl2(PPh3)2, 30mL 1,4-dioxane and 10mL water were added. The mixture was reacted for 4h at 70°C under nitrogen protection before the reaction was stopped. The resultant mixture was concentrated, and 10mL water was added. The mixture was extracted with ethyl acetate (2X10mL), dried, filtered, concentrated, and purified by column chromatography to give the title compound.
Step 2:preparation of (2S)-2-(((((4'-trifluoromethyl-[1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using 4-hydroxy-4'-trifluoromethyl-[1,1'-biphenyl] prepared from Step 1, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1HNMR(500MHz,DMSO)δ:11.49(s,1H),7.87(d,2H),7.80(d,2H),7.76(d,2H),7.57(d,1 H),7.36(d,2H),6.11-6.01(m,2H),5.88-5.83(m,1H),5.60-5.55(m,1H),4.89-4.84(m,1H),4.42-4.39(m,1H),4.28-4.26(m,1H),4.04-4.02(m,1H),3.85-3.84(m,2H),1.28-1.24(m,6H),1.16-1.14(m,6H). ESI-MS m/z: [M+H]+ =674.
Example 20: (2S)-2-(((((4'-chloro-2'-fluoro-[1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
Step 1: preparation of 4-hydroxy-4'-chloro-2'-fluoro-[1,1'-biphenyl]
The title compound was prepared according to the method described in Step 1 of Example 37 using 2-fluoro-4-chloroiodobenzene, p-hydroxyphenyl boronic acid, potassium carbonate and PdCl2(PPh3)2 as starting materials. 1HNMR(300MHz,CDCL3-d6 )δ ppm: 7.43-6.83 (m,7H),4.94(s,1H).
Step 2: preparation of (2S)-2-(((((4'-chloro-2'-fluoro-[1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using 4-hydroxy-4'-chloro-2'-fluoro-[1,1'-biphenyl] prepared from Step 1, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1HNMR(500MHz,DMSO)δ:11.48(s,1H),7.56-7.52(m,5H),7.39-7.30(m,3H),6.11-6.01(m,2H),5.86-5.82(m,1H),5.56-5.54(m,1H),4.87-4.85(m,1H),4.42-4.39(m,1H),4.28-4.26(m,1H,),4.04-4.02(m,1H)3.85-3.84(m,2H),1.28-1.24(m,6H),1.16-1.14(m,6H). ESI-MS m/z: [M+Na]+=680.
Example 21: (2S)-2-(((((3'-fluoro-4'-methyl-[1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
Step 1: preparation of 4-hydroxy-3'-fluoro-4'-methyl-[1,1'-biphenyl]
The title compound was prepared according to the method described in Step 1 of Example 19 using 3-fluoro-4-methyl-iodobenzene,p-hydroxyphenyl boronic acid, potassium carbonate and PdCl2(PPh3)2as starting materials. 1HNMR(300MHz,CDCL3-d6 )δ ppm: 7.45-6.87(m,7H),4.77(s,1H),2.31(s,3H).
Step 2: preparation of(2S)-2-(((((3'-fluoro-4'-methyl-[1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using 4-hydroxy-3'-fluoro-4'-methyl-[1,1'-biphenyl] prepared from Step 1, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1HNMR(500MHz,DMSO)δ:11.48(s,1H),7.68(d,2H),7.57(d,1H),7.44-7.29(m,5H)6.08-6.03(m,2H),5.85-5.82(m,1H),5.56-5.54(m,1H),4.88-4.83(m,1H),4.42-4.38(m,1H,),4.28-4.26(m,1H),4.04-4.02(m,1H),3.84-3.82(m,2H),2.26(d,3H),1.28-1.23(m,6H),1.19-1.14(m,6H). ESI-MS m/z: [M+Na]+=660.
Example 22: (2S)-2-((((4'-fluoro-3'-methyl-[1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
Step 1: preparation of 4-hydroxy-4'-fluoro-3'-methyl-[1,1'-biphenyl]
The title compound was prepared according to the method described in Step 1 of Example 19 using 4-fluoro-3-methyl-iodobenzene,p-hydroxyphenylboronic acid, potassium carbonate and PdCl2(PPh3)2as starting materials. 1H NMR (300 MHz, CDCL3-d6 ) δ ppm:7.52-7.42(m,2H),7.34-7.26(m,2H),7.06-6.87(m,3H),4.67(s,1H),2.32(s,3H).
Step 2: preparation of (2S)-2-((((4'-fluoro-3'-methyl-[1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using 4-hydroxy-4'-fluoro-3'-methyl-[1,1'-biphenyl] prepared from Step 1, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1HNMR(500MHz,DMSO)δ:11.50(s,1H),7.65-7.55(m,4H),7.47-7.45(m,1H),7.30-7.17(m,3H),6.11-5.99(m,2H,),5.85-5.83(m,1H),5.57-5.54(m,1H),4.90-4.84(m,1H,),4.38-4.36(m,1H),4.27-4.25(m,1H,),4.02-3.99(m,1H,),3.87-3.80(m,2H),2.30(s,3H),1.29-1.21 (m,6H),1.16-1.14(m,6H). ESI-MS m/z: [M+Na]+ =660.
Example 23: (2S)-2-((((4'-methyl-[1,1'-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoic acid isopropyl ester
The title compound was prepared according to the method described in Example 1 using 4-hydroxy-4'-methyl-[1,1'-biphenyl], phosphorus oxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenol and (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine as starting materials. 1HNMR(500MHz,DMSO)δ:11.49(s,1H),7.58(d,1H,),7.53-7.51(m,4H),7.29-7.25(m,4H),6.10-6.02(m,2H,),5.88-5.82(m,1H,),5.60-5.55(m,1H),4.88-4.84(m,1H,),4.41-4.38(m,1H),4.27(m,1H,),4.04-4.02(m,1H)3.84-3.82(m,2H),2.33(s,3H),1.28-1.23(m,6H),1.18-1.14(m,6H). ESI-MS m/z: [M+H]+ =620.
Pharmacological and pharmacokinetic activity: Experimental example 1: antiviral activity assay of the compounds of the present invention in HCV replicon system 1. Experimental materials 1.1 Reagents:
Table 1: List of reagents
Name of reagents Supplier
DMEM medium Invitrogen
Fetal bovine serum(FBS) Gibco
L-Glutamine Invitrogen
penicillin-streptomycin solution Invitrogen
DPBS/Modified Hyclone
Trypsin/EDTA Invitrogen
Dimethyl sulfoxide (DMSO) Sigma
Bright-Glo™ Promega
CellTiter-Fluor™ Reagent Promega
1.2 Huh7 1b cell lines:
The cell line Huh7 1b, which was provided by WuXi AppTec Co., Ltd. in Shanghai, contained an HCV 1b replicon with a stable Luciferase (Luc) reporter. It was constructed by cloning HCV nonstructural protein gene, neo(G418 resistance) and luciferase reporter gene into the pBR vector through gene recombination technology. Then the vector carrying the HCV replicon was transfected into Huh7 cells, and the transfected Huh7 cells were screened by G418 resistance. The HCV replicon was stably replicated, and the related protein and luciferase were stably expressed in Huh7 cells. The cells model was used for in-vitro screen of anti-HCV compounds. The anti-HCV activity of the compounds was measured by detecting the level of luciferase expression. See Lohmann V, et al.1999. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science. 285(5424):110-113.
1.3 positive control:
The control drug used in this experimental example was the compund of Example 25 in WO 2008/121634 ( PCT/US2008/058183 ), i.e. (S)-2-{[(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyl-tetrahydro-furan-2-ylmethoxy]-phenoxy-phosphorylamino}-propanoic acid isopropyl ester. This compound was prepared according to the method described in J. Org. chem, 2011, 76, 8311-8319, and identified by H-NMR and mass spectrometry.
2. Experimental procedures:
  • 2.1 compound preparation: adding each of the compounds listed in Table 2 to 96-well pates using a POD™ 810 Automation Platform (LabCyte Corporation, USA), with 10µM of each of the compounds as a starting concentration and in duplicate; serially diluting each of the compounds in 3-fold fashion and formulating 10 concentrations, with the final concentration of DMSO being0.5%;
  • 2.2 cells preparation: seeding the Huh71b cells into 96-well plates respectively with a volume of 125µland 8X103 cells/well, and incubating in a37°C, 5% CO2 incubator for 72h;
  • 2.3 detection of cell activity: adding 30µl per cell of the Cell Titer-Fluor™ Reagent, incubating in a 37°C, 5% CO2incubator for 1h, measuring the fluorescence signal values with a fluorometer, and using the obtained data for calculation of cytotoxicities of the compounds;
  • 2.4 Bright-Glo detection: adding 100µl per well of the Bright-Glo™ Luciferase Substrate, using a chemiluminescence detection system EnVision™ (PerkinElmer, USA) to detect the fluorescence signal values within 5 minutes, and using the obtained data for calculation of potencies of the compounds.
  • 2.5 data processing: converting the obtained data to a percentage of cell viability (Viability%) using the following formula: CPD: fluorescence signal value of the well added with the compoundZPE (Zero percent effect): fluorescence signal value of the control well with zero percent effect.
Processing the raw data to calculate a percentage of inhibition (Inhibition%) using the following formula:
  • CPD: fluorescence signal value of the well added with the compound;
  • HPE (Hundred percent effect): fluorescence signal value of the control well with hundred percent effect;
  • ZPE (Zero percent effect): fluorescence signal value of the control well with zero percent effect.
The inhibition percentage was processed by the GraphPad Prism software to obtain the corresponding curves and EC50 values. The data was listed in Table 2. Table 2
Postive control 0.1168
Example 1 0.056
Example 2 0.1051
Example 3 0.1232
Example 4 undetected
Example 5 0.061
Example 6 undetected
Example 7 0.411
Example 8 undetected
Example 9 0.189
Example 10 undetected
Example 11 0.3141
Example 13 0.098
Example 14 0.123
Example 16 0.102
Example 17 0.068
Example 18 0.073
Example 20 0.061
Example 21 0.056
Example 22 0.050
Example 23 0.126
The above results have shown that the compounds of the present invention have the ability to inhibit HCV virus very efficiently, and compared to the positive control, have a superior or equal activity based on EC50 values.
Experimental example 2: Antiviral activity assay of the compounds of the present invention in the Cell-culture-derived infectious HCV (HCVcc) system 1. Experimental materials 1.1 Compounds
The compound according to the above example 1 was used in this experiment. The compound was formulated into a 10mM mother liquid with DMSO, diluted to 500nM by DMEM complete culture medium containing 0.5% of DMSO, followed by 4-fold dilution successively, and six concentrations were obtained. The positive control was diluted to 10µM by DMEM complete culture medium containing 0.5% of DMSO, followed by 4-fold successively, and six concentrations were obtained.
1.2 Cells
Huh 7.5.1 cells, provided by Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
1.3 Viruses
J399EM (HCV genotype2a) virus, i.e. a full length HCV mutant strain transfected with EGFP (enhanced green fluorescent protein) was used, which had the same infection capacity as the wild type JFH-1. The NS5A-EGFP fusion protein fluorescence was observed directly in infected cells by inserting EGFP coding sequence in the NS5Aregion. J399EM was provided by Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
1.4 Reagents
  • DMEM medium, purchased from Invitrogen Corporation, USA;
  • Fetal bovine serum (FBS), purchased from Sigma Corporation, USA;
  • L(+)-Glutamine, purchased from Invitrogen Corporation, USA;
  • Penicillin-streptomycin (Pen-Strep), purchased from Invitrogen Corporation, USA;
  • Phosphate buffered saline (PBS), purchased from Hyclone Corporation, USA;
  • Trypsin, purchased from Invitrogen Corporation, USA;
  • Dimethyl sulfoxide (DMSO), purchased from Sigma Corporation, USA;
  • Lysis buffer, purchased from Promega Corporation, USA;
  • MTT, purchased from Sigma Corporation, USA.
1.4 Instruments
EnVision®Multilabel Plate readers, purchased from Perkin-Elmer Corporation, USA.
2 Experimental procedures
1) Huh 7.5.1 cells preparation: collecting the Huh 7.5.1 cells in logarithmic phase, resuspending in DMEM complete culture medium, seeding into 96-well plates (7x103 cells/well), and incubating in a 37°C, 5% CO2 incubator for 24h; 2) virus infection: resuspending J399EM viruses in DMEM complete culture medium, adding the virus supernatant (MOI≈0.1) to the above 96-well plates, and washing with PBS after 8 hours of infection; 3) drug treatment: adding various concentrations of the compounds to the Huh 7.5.1 cells infected by J399EM viruses, with each concentration in duplicate; setting the control group with zero percent effect (ZPE) and the control group with hundred percent effect (HPE); replacing the compound with the DMEM complete culture medium containing 0.5% DMSO in ZPE group, and using the cells not infected with viruses in HPE group. 4) cells incubation: incubating the 96-well plates in a 37°C, 5% CO2 incubator for 72 hr; 5) anti-HCV activity detection: reading the relative fluorescence units (RFU) of each well by EnVision® Multilabel Plate readers after incubation, and calculating the anti-HCV activity of the compounds using the obtained data according to the following formula: wherein RFUZPE represents the relative fluorescence units of the control group with zero percent effect and RFUCPD represents the relative fluorescence units of the corresponding compound group, and RFUHPE represents the relative fluorescence units of the control group with hundred percent effect. 6) Cell viability detection: adding MTT solution to each well, incubating in a 37°C, 5% CO2 incubator for 4hr, followed by addition of MTT dissolving solution, reading optical density (OD) of each well at 570nm by the Multilabel Plate readers after 6hr, calculating the cytotoxicity of the compounds using the obtained data according to the following formula: wherein ODZPE represents the optical density of the control group with zero percent effect, and ODCPD represents the optical density of the corresponding compound; 7) Data processing: processing the Inhibition%, Viability% by GraphPad Prism Software respectively, and obtaining the half maximal effective concentration (EC50) values and half maximal cytotoxic concentration (CC50) values of the compounds against the HCVcc GT2a viruses. The results were listed in Table 3. Table 3
Positive control 0.173 >10
Example 1 0.039 >10
It was seen from Table 3 that the compound of Example 1 of the present invention had excellent antiviral activity and less cell toxicity compared with the positive control in the HCVcc system.
In addition, the experiments of the present invention also showed that using in vitro HCVcc system, the compounds prepared by the present invention, such as the compounds prepared in Examples 2, 5, 12, 16, 18, 28 and 38, had a low half maximal effective concentration (EC50) and a high half maximal cytotoxic concentration (CC50) against the HCVcc GT2a viruses, which demonstrated good inhibitory activity and small cytotoxicity.
Experimental example 3: Pharmacokinetic studies of the compounds of the present invention in SD rats 1. Experimental materials 1.1 Compounds
In this experiment, the compound of Example 1 was used, and the positive control drug was the same as above. Each of the compounds was added to 0.5% sodium carboxymethyl cellulose (CMC), and the mixture was vortexed to prepare a 10 mg/mL suspension for intragastric administration. The standard substance GS-461203 was the metabolite of the tested compounds, and the chemical name was (2'R)-2'-deoxy-2'-fluoro-2'-methyluridinetriphosphate, which was purchased from TriLink BioTechnologies Corporation, USA.
1.2 Animals
Male SD rats, 6-8 weeks old, weighing 237.0-268.4 g, provided by Shanghai Super-B&K laboratory animal Co. Limited.
1.3 Reagents
  • methanol (chromatographic pure), purchased from Spectrum Corporation, USA;
  • acetonitrile (chromatographic pure): purchased from Spectrum Corporation, USA.
1.4 Instruments
  • API 5500 LC-MS, purchased fromAB Corporation, USA.
2. Experimental method
1) administration: dividing SD rats into 4 groups, with 18 rats in each group, intragastrically administering with the compound in an amount of 50mg/kg. Fasting the rats for 10-15 hours prior to intragastric administration, and refeeding four hours after administration; 2) sampling: at 0.5h, 1h, 2h, 4h, 6h and 12h after administration, sacrificing the rats (n=3 at each time point for each group), collecting about 1g of each rat liver respectively, adding 3 volumes of pre-cooled methanol, homogenizing for 30s and storing the prepared liver homogenate in -80°C before analysis. 3) standard curve preparation: taking appropriate amount of GS-461203 stock solution, serially diluting with methanol to prepare standard solutions with concentrations of 30, 27, 10, 5, 2, 1, 0.5, 0.2 and 0.1 µg/mL, parallelly adding 10µL of each standard solution to 90 µL blank rat liver homogenate to prepare sample standard curves of concentrations of 3000, 2700, 1000, 500, 200, 100, 50, 20 and 10 ng/mL. 4) sample processing: adding 30µl liver homogenate sample or standard curve sample to 150µl solution of acetonitrile containing the internal standard (100 ng/mL), vortexing for 2min, centrifuging for 10min (6000 revolutions/min), and transferring the supernatant to injection vials; 5) sample data analysis: taking 5µL supernatant, injecting, and then using UPLC-MS/MS to detect the concentration of GS-461203 in the samples; according to the obtained drug concentration-time data of the liver homogenates, using the non-compartmental model of pharmacokinetic calculation software WinNonlin 6.2.1 to calculate pharmacokinetic parameters of metabolite GS-461203 of each of the tested compounds. The results were listed in Table 4. Table 4
GS-461203
Positive control 753 2.00 5627
Example 1 818 1.00 7106
It was seen from the data in Table 4 that the compound of Example 1 according to the present invention was better than that of the positive control in the peak concentration in liver tissue and the area under the plasma drug concentration-time curve of the metabolite, and had greater in vivo exposure level.
The above results show that the compounds of the present invention have the ability to efficiently inhibit HCV virus, and compared to the positive control, have equal or superior effects in EC50, less toxicity to the host cells, higher CC50, and good security. The compounds of the present invention have good prospects for use in the treatment of HCV infection.
Although the present invention has been described in detail above, it should be understood by those skilled in the art that various modifications and alterations can be made without departing from the scope of the present invention. The scope of the present invention is defined by the claims.

Claims (12)

  1. A nucleoside phosphoramidate compound of the following general formula I, or a stereoisomer, salt, hydrate, solvate or crystal thereof, wherein
    (1) R1 is selected from C1-6 alkyl;
    (2) R2 is selected from halogen;
    (3) R3 is selected from OH, H and C1-4 alkoxy;
    (4) R4 is selected from H, C1-6 alkyl and halogenated C1-6 alkyl;
    (5) R5 is selected from C1-6 alkyl and halogenated C1-6 alkyl;
    (6) R6 is selected from the following moieties:
    a) phenyl-Y-, wherein Y is absent or selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-6 alkenyl-(CO)-, C2-6 alkynyl-(CO)-, O, S, amino and -N(C1-6 alkyl), and wherein the phenyl is optionally substituted by one or more groups selected from C1-6 alkyl, halogen, nitro, C1-6 alkoxy, cyano, C2-6 alkenyl, C2-6 alkynyl, C1-6 acylamino, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, amino, N(C1-6 alkyl)2 and C1-6 alkyl NHCO, or the phenyl and a five- or six-membered ring taken together form a benzo five-membered ring or benzo six-membered ring; and
    (7) R7 is selected from H, halogen, C1-6 alkyl, halogenated C1-6 alkyl, C1-6 alkoxy, halogenated C1-6 alkoxy, NO2, CN, C1-6 alkyl-NH-CO-, hydroxy, mono-C1-6 alkylamino, di-C1-6 alkylamino, C1-6 alkyl-S-, C2-6 alkenyl-S-, C2-6 alkynyl-S-, C1-6 alkyl-SO-, C2-6 alkenyl-SO-, C2-6 alkynyl-SO-, C1-6 alkyl-SO2-, C2-6 alkenyl-SO2-, C2-6 alkynyl-SO2-, C1-6 alkyl-OSO2-, C2-6 alkenyl-OSO2-, C2-6 alkenyl-OSO2-.
  2. The compound according to claim 1, or the stereoisomer, salt, hydrate, solvate or crystal thereof, wherein R1 is selected from C1-3 alkyl, R2 is F, R3 is selected from OH, H and methoxy, R4 is selected from H and C1-6 alkyl, and R5 is selected from C1-6 alkyl.
  3. The compound according to claim 1 or 2, or the stereoisomer, salt, hydrate, solvate or crystal thereof, wherein R6 is selected from
    a) phenyl-Y-, wherein Y is absent or selected from C1-4 alkyl, C2-4alkenyl, C2-4 alkynyl, C2-4 alkenyl-(CO)-, C2-4 alkynyl-(CO)-, O, S, amino and -N(C1-4 alkyl), and wherein the phenyl is optionally substituted by one or more groups selected from C1-4 alkyl, halogen, nitro, C1-4 alkoxy, cyano, C2-4 alkenyl, C2-4 alkynyl, C1-4 acylamino, halogenated C1-4 alkyl, halogenated C1-4 alkoxy, amino, N(C1-4 alkyl)2 and C1-4 alkyl NHCO, or the phenyl and a five- or six-membered ring taken together form a benzo five-membered ring or benzo six-membered ring.
  4. The compound according to claim 3, or the stereoisomer, salt, hydrate, solvate or crystal thereof, wherein R6 is selected from
    a) phenyl, phenyl-C1-3 alkyl-, phenyl-C2-3 alkenyl-, phenyl-C2-3 alkynyl-, phenyl-O-, phenyl-S-, phenyl-NH-, phenyl-N(C1-3 alkyl)-, phenyl-ethenyl-(CO)- and naphthyl-ethenyl-(CO)-, wherein the phenyl is optionally substituted by one or more groups selected from C1-4 alkyl, halogen, nitro, C1-4 alkoxy, cyano, C2-4 alkenyl, C2-4 alkynyl, C1-4 acylamino, halogenated C1-4 alkyl, halogenated C1-4 alkoxy, amino, N(C1-4 alkyl)2 and C1-4 alkyl NHCO, or the phenyl and a group selected from phenyl, oxazolyl, pyrazinyl and pyrrolyl taken together form a naphthyl, benzoxazolyl, benzo[b]pyrazinyl or benzo[b]pyrrolyl.
  5. The compound according to claim 4, or the stereoisomer, salt, hydrate, solvate or crystal thereof, wherein R6 is selected from
    a) phenyl, phenyl-(CH2)-, phenyl-=-, phenyl-=-C(O)- and phenyl-≡-, wherein the phenyl is optionally substituted by one or more groups selected from C1-4 alkyl, halogen, nitro, C1-4 alkoxy, cyano, C2-4 alkenyl, C2-4 alkynyl, C1-4 acylamino, halogenated C1-4 alkyl, halogenated C1-4 alkoxy, amino, N(C1-4 alkyl)2 and C1-4 alkyl NHCO, or the phenyl and a group selected from oxazolyl, pyrazinyl and pyrrolyl taken together form a benzoxazolyl, benzo[b]pyrazinyl or benzo[b]pyrrolyl.
  6. The compound according to any one of claims 1 to 5, or the stereoisomer, salt, hydrate, solvate or crystal thereof, wherein R6 group " phenyl-Y-" and the oxygen group both of which are attached to the benzene ring are at the para- or meta- position to each other.
  7. The compound according to any one of claims 1 to 6, or the stereoisomer, salt, hydrate, solvate or crystal thereof, wherein R1 is CH3, R2 is F, R3 is OH, R4 is CH3, and R5 is isopropyl.
  8. The compound according to claim 7, or the stereoisomer, salt, hydrate, solvate or crystal thereof, wherein R6 is phenyl or benzyl.
  9. The compound according to claim 1, or the stereoisomer, salt, hydrate, solvate or crystal thereof, wherein the compound is selected from
  10. A pharmaceutical composition, comprising the compound according to any one of claims 1 to 9, or the stereoisomer, salt, hydrate, solvate or crystal thereof and a pharmaceutically acceptable carrier.
  11. A compound according to any one of claims 1 to 9, or the stereoisomer, salt, hydrate, solvate or crystal thereof or the composition according to claim 10, for use in the treatment of Flaviviridae family viral infection, especially hepatitis C viral infection.
  12. A compound according to any one of claims 1 to 9, or the stereoisomer, salt, hydrate, solvate or crystal thereof or the composition according to claim 10, for use as a medicament in the prevention of Flaviviridae family viral infection, especially hepatitis C viral infection.
HK15111394.2A 2013-03-08 2014-03-06 Nucleoside phosphoramidate compounds for use in the treatment of hcv HK1210619B (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CN201310075423 2013-03-08
CN201310075423 2013-03-08
PCT/CN2014/073004 WO2014135107A1 (en) 2013-03-08 2014-03-06 Novel nucleoside phosphoramidate compound and use thereof

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Publication Number Publication Date
HK1210619A1 HK1210619A1 (en) 2016-04-29
HK1210619B true HK1210619B (en) 2018-04-27

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