Patent Grant
10683321
Provided herein are compounds, methods and pharmaceutical compositions for use in treatment of viral infections, including hepatitis C virus infections in hosts in need thereof. In certain embodiments, 4′-OR nucleosides are provided which can display remarkable efficacy and bioavailability for the treatment of, for example, HCV infection in a human.
The hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. (Boyer, N. et al., J. Hepatol. 32:98-112, 2000). HCV causes a slow growing viral infection and is the major cause of cirrhosis and hepatocellular carcinoma (Di Besceglie, A. M. and Bacon, B. R., Scientific American, October: 80-85, 1999; Boyer, N. et al., J. Hepatol. 32:98-112, 2000). It is estimated there are about 130-150 million people with chronic hepatitis C virus infection. Hepatitis C-related liver diseases cause approximately 350,000 to 500,000 deaths each year.
HCV infection becomes chronic in about 55-85% of cases, with many patients initially being asymptomatic. About 15 to 30% of patients with chronic hepatitis due to HCV develop cirrhosis within about 20 years. (Hepatitis C Fact Sheet, World Health Organization Fact Sheet No., 164, April 2014). Development of cirrhosis due to HCV also increases the risk of hepatocellular cancer (The Merck Manual Online, Chronic Hepatitis, available at www.merckmanuals.com/professional/hepatic_and_biliary_disorders/hepatitis/chronic_hepatitis.html, last revision February 2014).
In light of the fact that HCV infection has reached epidemic levels worldwide, and has tragic effects on the infected patient, there remains a strong need to provide new effective pharmaceutical agents to treat hepatitis C that have low toxicity to the host. Further, given the rising threat of other flaviviridae infections, there remains a strong need to provide new effective pharmaceutical agents that have low toxicity to the host. Therefore, there is a continuing need for effective treatments of flavivirus infections and HCV infections.
Provided herein are compounds useful, for example, for the treatment of flavivirus infections such as HCV infections. The compounds are 4′-OR nucleosides. In certain embodiments the 4′-OR nucleosides can display remarkable efficacy or bioavailability, or both, for the treatment of, for example, HCV infection in a human.
In certain embodiments, the compounds provided herein are useful in the prevention and treatment of Flaviviridae infections and other related conditions such as anti-Flaviviridae antibody positive and Flaviviridae-positive conditions, chronic liver inflammation caused by HCV, cirrhosis, fibrosis, acute hepatitis, fulminant hepatitis, chronic persistent hepatitis and fatigue. These compounds or formulations can also be used prophylactically to prevent or retard the progression of clinical illness in individuals who are anti-Flaviviridae antibody or Flaviviridae-antigen positive or who have been exposed to a Flaviviridae. In particular embodiments, the Flaviviridae is hepatitis C. In certain embodiments, the compounds are used to treat any virus that replicates through an RNA-dependent RNA polymerase.
A method for the treatment of a Flaviviridae infection in a host, including a human, is also provided that includes administering an effective amount of a compound provided herein, administered either alone or in combination or alternation with another anti-Flaviviridae agent, optionally in a pharmaceutically acceptable carrier.
In certain embodiments, provided herein are compounds according to Formula 1001:
or a pharmaceutically acceptable salt thereof, wherein:
Base is a natural or unnatural nucleobase;
PD is hydrogen,
W is S or O; one of X and Y is hydrogen, —OR1, —SR1, —NR1R2, an O-linked amino acid residue or derivative thereof, an N-linked amino acid residue or derivative thereof, or an N-linked amino alcohol residue or derivative thereof; and the other of X and Y is OR1 or —NR1R2;
each of Z1, Z2, Z3, and Z4 is independently —H, —OH, —OCH3, —N3, —NH2, —CH3, —Cl, or —F; or, in the alternative, Z4 and Y, together with the atoms to which they are attached, combine to form a six-membered heterocyclic ring wherein Z4 and Y together represent a single divalent —O—, and X is —OR1, —SR1, —NR1R2, an O-linked amino acid residue or derivative thereof, an N-linked amino acid residue or derivative thereof, or an N-linked amino alcohol residue or derivative thereof; and each of Z1, Z2, and Z3 is independently —H, —OH, —OCH3, —N3, —NH2, —CH3, —Cl, or —F;
each R1 is independently alkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkylcarbonylthioalkyl, alkoxycarbonylalkyl, arylalkoxycarbonylalkyl, alkylcarbonylalkoxy(arylalkyl), cycloalkylcarbonylalkoxyl, alkoxycarbonylaminoalkylcarbonylthioalkyl, hydroxylalkylcarbonylthioalkyl, or aminoalkylcarbonylalkoxycarbonylthioalkyl; and each R2 is independently hydrogen or alkyl;
R is alkyl or alkynyl;
with the proviso that the compound of formula 1001 is not 4′-methoxyuridine, 4′-methoxycytidine, 4′-ethoxycytidine, 4′-methoxyadenosine, 3′-deoxy-3′-fluoro-4′-methoxythymidine, 2′, 3′-dideoxy-3′-fluoro-4′-methoxyadenosine, 2′, 3′-dideoxy-3′-fluoro-4′-methoxyuridine, 2′, 3′-dideoxy-3′-fluoro-4′-methoxycytidine, 2′, 3′-dideoxy-3′-fluoro-4′-methoxyguanosine, or 5′-mono, di, or triphosphate thereof.
In certain embodiments, provided herein are compounds according to Formula I:
or a pharmaceutically acceptable salt thereof, wherein:
Base is a natural or unnatural nucleobase;
PD is hydrogen,
W is S or O; one of X and Y is hydrogen, —OR1, —SR1, —NR1R2, an O-linked amino acid residue or derivative thereof, an N-linked amino acid residue or derivative thereof, or an N-linked amino alcohol residue or derivative thereof; and the other of X and Y is —OR1 or —NR1R2;
each of Z1, Z2, Z3, and Z4 is independently —H, —OH, —N3, —NH2, —CH3, —Cl, or —F; or, in the alternative, Z4 and Y, together with the atoms to which they are attached, combine to form a six-membered heterocyclic ring wherein Z4 and Y together represent a single divalent—O—, and X is —OR1, —SR1, —NR1R2, an O-linked amino acid residue or derivative thereof, an N-linked amino acid residue or derivative thereof, or an N-linked amino alcohol residue or derivative thereof; and each of Z1, Z2, and Z3 is independently —H, —OH, —N3, —NH2, —CH3, —Cl, or —F;
each R1 is independently alkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkylcarbonylthioalkyl, alkoxycarbonylalkyl, arylalkoxycarbonylalkyl, alkylcarbonylalkoxy(arylalkyl), cycloalkylcarbonylalkoxyl, alkoxycarbonylaminoalkylcarbonylthioalkyl, hydroxylalkylcarbonylthioalkyl, or aminoalkylcarbonylalkoxycarbonylthioalkyl; and each R2 is independently hydrogen or alkyl;
R is alkyl or alkynyl;
with the proviso that the compound of formula I is not 4′-methoxyuridine, 4′-methoxycytidine, 4′-ethoxycytidine, 4′-methoxyadenosine, 3′-deoxy-3′-fluoro-4′-methoxythymidine, 2′, 3′-dideoxy-3′-fluoro-4′-methoxyadenosine, 2′, 3′-dideoxy-3′-fluoro-4′-methoxyuridine, 2′, 3′-dideoxy-3′-fluoro-4′-methoxycytidine, 2′, 3′-dideoxy-3′-fluoro-4′-methoxyguanosine, or 5′-mono, di, or triphosphate thereof.
In one aspect, the compounds provided herein are provided or administered in combination with a second therapeutic agent, such as one useful for the treatment or prevention of HCV infections. Exemplary second therapeutic agents are provided in detail elsewhere herein.
In another aspect, provided herein are pharmaceutical compositions, single unit dosage forms, and kits suitable for use in treating or preventing disorders such as HCV infections which comprise a therapeutically or prophylactically effective amount of a compound provided herein, e.g., of any of Formulae 1001, I-XX, 2a-2e, 5a2, 5c, 6a-8a, 6b, 7b, 113, 114, 20, 21, 23, 24, 13a, 13ai, 13aii, 14a, 14ai, 14aii, 16a, 122, 122, 123, 123i, 123ii, 131-136, 9a-9g, 208-214, 220, 222, 223, 224, 230, 233, 301-314, 17a, 402-414, 18a, 502-514, 520, 530 and 601-614 or a pharmaceutically acceptable salt, optionally with a therapeutically or prophylactically effective amount of a second therapeutic agent such as one useful for the treatment or prevention of HCV infections.
In certain embodiments, a method of treatment of a liver disorder is provided comprising administering to an individual in need thereof a treatment effective amount of a 4′-OR nucleoside compound.
Flaviviridae which can be treated are, e.g., discussed generally in Fields Virology, Sixth Ed., Editors: Knipe, D. M., and Howley, P. M., Lippincott Williams & Wilkins Publishers, Philadelphia, Pa., Chapters 25-27, 2013. In a particular embodiment of the invention, the Flaviviridae is HCV. In an alternate embodiment, the Flaviviridae is a flavivirus or pestivirus. In certain embodiments, the Flaviviridae can be from any class of Flaviviridae. In certain embodiments, the Flaviviridae is a mammalian tick-borne virus. In certain embodiments, the Flaviviridae is a seabird tick-borne virus. In certain embodiments, the Flaviviridae is a mosquito-borne virus. In certain embodiments, the Flaviviridae is an Aroa virus. In certain embodiments, the Flaviviridae is a Dengue virus. In certain embodiments, the Flaviviridae is a Japanese encephalitis virus. In certain embodiments, the Flaviviridae is a Kokobera virus. In certain embodiments, the Flaviviridae is a Ntaya virus. In certain embodiments, the Flaviviridae is a Spondweni virus. In certain embodiments, the Flaviviridae is a Yellow fever virus. In certain embodiments, the Flaviviridae is an Entebbe virus. In certain embodiments, the Flaviviridae is a Modoc virus. In certain embodiments, the Flaviviridae is a Rio Bravo virus.
Specific flaviviruses which can be treated include, without limitation: Absettarov, Aedes, Alfuy, Alkhurma, Apoi, Aroa, Bagaza, Banzi, Bukalasa bat, Bouboui, Bussuquara, Cacipacore, Calbertado, Carey Island, Cell fusing agent, Cowbone Ridge, Culex, Dakar bat, Dengue 1, Dengue 2, Dengue 3, Dengue 4, Edge Hill, Entebbe bat, Gadgets Gully, Hanzalova, Hypr, Ilheus, Israel turkey meningoencephalitis, Japanese encephalitis, Jugra, Jutiapa, Kadam, Kamiti River, Karshi, Kedougou, Kokobera, Koutango, Kumlinge, Kunjin, Kyasanur Forest disease, Langat, Louping ill, Meaban, Modoc, Montana myotis leukoencephalitis, Murray valley encephalitis, Nakiwogo, Naranjal, Negishi, Ntaya, Omsk hemorrhagic fever, Phnom-Penh bat, Powassan, Quang Binh, Rio Bravo, Rocio, Royal Farm, Russian spring-summer encephalitis, Saboya, St. Louis encephalitis, Sal Vieja, San Perlita, Saumarcz Reef, Sepik, Sokuluk, Spondweni, Stratford, Tembusu, Tick-borne encephalitis, Turkish sheep encephalitis, Tyuleniy, Uganda S, Usutu, Wesselsbron, West Nile, Yaounde, Yellow fever, Yokose, and Zika.
Pestiviruses which can be treated are discussed generally in Fields Virology, Sixth Ed., Editors: Knipe, D. M., and Howley, P. M., Lippincott Williams & Wilkins Publishers, Philadelphia, Pa., Chapter 25, 2013. Specific pestiviruses which can be treated include, without limitation: bovine viral diarrhea virus (“BVDV”), classical swine fever virus (“CSFV,” also called hog cholera virus), and border disease virus (“BDV”).
Provided herein are compounds, compositions and methods useful for treating liver disorders such as HCV infection in a subject. Further provided are dosage forms useful for such methods.
When referring to the compounds provided herein, the following terms have the following meanings unless indicated otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.
The term “alkyl,” as used herein, unless otherwise specified, refers to a saturated straight or branched hydrocarbon. In certain embodiments, the alkyl group is a primary, secondary, or tertiary hydrocarbon. In certain embodiments, the alkyl group includes one to ten carbon atoms, i.e., C1 to C10 alkyl. In certain embodiments, the alkyl group is methyl, CF3, CCl3, CFCl2, CF2Cl, ethyl, CH2CF3, CF2CF3, propyl, isopropyl, butyl, isobutyl, secbutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, or 2,3-dimethylbutyl. The term includes both substituted and unsubstituted alkyl groups, preferably unsubstituted or halogenated alkyl groups. In certain embodiments, the alkyl group is a fluorinated alkyl group. Non-limiting examples of moieties with which the alkyl group can be substituted include halogen (fluoro, chloro, bromo or iodo), hydroxyl, carbonyl, cycloalkyl, aralkyl, sulfanyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991, hereby incorporated by reference.
The term “lower alkyl,” as used herein, and unless otherwise specified, refers to a saturated straight or branched hydrocarbon having one to six carbon atoms, i.e., C1 to C6 alkyl. In certain embodiments, the lower alkyl group is a primary, secondary, or tertiary hydrocarbon. The term includes both substituted and unsubstituted moieties, preferably unsubstituted.
The term “upper alkyl,” as used herein, and unless otherwise specified, refers to a saturated straight or branched hydrocarbon having seven to thirty carbon atoms, i.e., C7 to C30 alkyl. In certain embodiments, the upper alkyl group is a primary, secondary, or tertiary hydrocarbon. The term includes both substituted and unsubstituted moieties, preferably unsubstituted.
The term “cycloalkyl,” as used herein, unless otherwise specified, refers to a saturated cyclic hydrocarbon. In certain embodiments, the cycloalkyl group may be a saturated, and/or bridged, and/or non-bridged, and/or a fused bicyclic group. In certain embodiments, the cycloalkyl group includes three to ten carbon atoms, i.e., C3 to C10 cycloalkyl. In some embodiments, the cycloalkyl has from 3 to 15 (C3-15), from 3 to 10 (C3-10), or from 3 to 7 (C3-7) carbon atoms. In certain embodiments, the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexylmethyl, cycloheptyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, decalinyl or adamantyl. The term includes both substituted and unsubstituted cycloalkyl groups, including halogenated cycloalkyl groups. In certain embodiments, the cycloalkyl group is a fluorinated cycloalkyl group. Preferably the cycloalkyl is unsubstituted or fluorinated. Non-limiting examples of moieties with which the cycloalkyl group can be substituted include halogen (fluoro, chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary.
“Alkylene” refers to divalent saturated aliphatic hydrocarbon groups including those having from one to eleven carbon atoms which can be straight-chained or branched. In certain embodiments, the alkylene group contains 1 to 10 carbon atoms. The term includes both substituted and unsubstituted moieties, preferably unsubstituted. This term is exemplified by groups such as methylene (—CH2—), ethylene (—CH2CH2—), the propylene isomers (e.g., —CH2CH2CH2— and —CH(CH3)CH2—) and the like. The term includes halogenated alkylene groups. In certain embodiments, the alkylene group is a fluorinated alkylene group. Non-limiting examples of moieties with which the alkylene group include halogen (fluoro, chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl, amino, alkylamino, alkylaryl, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, and phosphonate, either unprotected, or protected as necessary.
“Alkenyl” refers to monovalent olefinically unsaturated hydrocarbon groups, in certain embodiments, having up to about 11 carbon atoms, including from 2 to 8 carbon atoms, or from 2 to 6 carbon atoms, which can be straight-chained or branched and having at least 1 including from 1 to 2, site of olefinic unsaturation. The term includes both substituted and unsubstituted moieties, preferably unsubstituted. Exemplary alkenyl groups include ethenyl (i.e., vinyl, or —CH═CH2), n-propenyl (—CH2CHCH2), isopropenyl (—C(CH3)═CH2), and the like. The term includes halogenated alkenyl groups. In certain embodiments, the alkenyl group is a fluorinated alkenyl group. Non-limiting examples of moieties with which the alkenyl group can be substituted include halogen (fluoro, chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary.
The term “cycloalkenyl,” as used herein, unless otherwise specified, refers to an unsaturated cyclic hydrocarbon. In certain embodiments, cycloalkenyl refers to mono- or multicyclic ring systems that include at least one double bond. In certain embodiments, the cycloalkenyl group may be a bridged, non-bridged, and/or a fused bicyclic group. In certain embodiments, the cycloalkyl group includes three to ten carbon atoms, i.e., C3 to C10 cycloalkyl. In some embodiments, the cycloalkenyl has from 3 to 10 (C3-10), or from 4 to 7 (C4-7) carbon atoms. The term includes both substituted and unsubstituted cycloalkenyl groups, including halogenated cycloalkenyl groups. In certain embodiments, the cycloalkenyl group is a fluorinated cycloalkenyl group or unsubstituted. Non-limiting examples of moieties with which the cycloalkenyl group can be substituted include halogen (fluoro, chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary.
“Alkenylene” refers to divalent olefinically unsaturated hydrocarbon groups, in certain embodiments, having up to about 11 carbon atoms or from 2 to 6 carbon atoms which can be straight-chained or branched and having at least 1 or from 1 to 2 sites of olefinic unsaturation. This term is exemplified by groups such as ethenylene (—CH═CH—), the propenylene isomers (e.g., —CH═CHCH2— and —C(CH3)═CH— and —CH═C(CH3)—) and the like. The term includes both substituted and unsubstituted alkenylene groups, including halogenated alkenylene groups. In certain embodiments, the alkenylene group is a fluorinated alkenylene group or unsubstituted. Non-limiting examples of moieties with which the alkenylene group can be substituted include halogen (fluoro, chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary.
“Alkynyl” refers to acetylenically unsaturated hydrocarbon groups, in certain embodiments, having up to about 11 carbon atoms or from 2 to 6 carbon atoms which can be straight-chained or branched and having at least 1 or from 1 to 2 sites of alkynyl unsaturation. Non-limiting examples of alkynyl groups include acetylenic, ethynyl (—C≡CH), propargyl (—CH2C≡CH), and the like. The term includes both substituted and unsubstituted alkynyl groups, including halogenated alkynyl groups. In certain embodiments, the alkynyl group is a fluorinated alkynyl group or unsubstituted. Non-limiting examples of moieties with which the alkynyl group can be substituted include halogen (fluoro, chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary.
The term “aryl,” as used herein, and unless otherwise specified, refers to a substituent derived from an aromatic ring. In an embodiment, an aryl group is a C6-C12 aryl group. In an embodiment, an aryl group is phenyl, biphenyl or naphthyl. The term includes both substituted and unsubstituted moieties. An aryl group can be substituted with any described moiety, including, but not limited to, one or more moieties include halogen (fluoro, chloro, bromo or iodo), alkyl, haloalkyl, hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991.
“Alkoxy” and “alkoxyl” refer to the group —OR′ where R′ is alkyl or cycloalkyl as defined herein. In certain embodiments, the alkoxyl or alkoxy group is —OR′, wherein R′ is alkyl or cycloalkyl, and wherein alkyl is C1 to C10 alkyl and cycloakyl is C3 to C15 cycloalkyl. Alkoxy and alkoxyl groups include, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.
“Alkylcarbonyloxy” refers to a radical —O—C(O)-alkyl, wherein alkyl is a defined herein.
“Alkoxylcarbonyl” refers to a radical —C(O)-alkoxyl where alkoxyl is as defined herein.
“Alkoxylalkylcarbonyl” refers to a radical —C(O)-alkyl-alkoxyl where alkoxyl and alkyl are as defined herein.
“Alkoxylcarbonylalkyl” refers to a radical -alkyl-C(O)-alkoxyl where alkoxyl and alkyl are as defined herein.
“Alkoxylcarbonylamino” refers to a radical -amino-C(O)-alkoxyl where alkoxyl and amino are as defined herein.
As used herein, “(alkoxylcarbonyl)(alkoxylcarbonylamino)alkyl” refers to an alkyl radical substituted with both an alkoxylcarbonyl and an alkoxylcarbonylamino group, where “alkoxylcarbonyl” and “alkoxylcarbonylamino” are as described herein. In an embodiment, the term refers to a radical of formula
wherein n is an integer selected over the range of 1-10, AA is —C(O)—O—R100, AN is —NH—C(O)—O—R101, and each of R100 and R101 is independently lower alkyl. In an embodiment, each of R100 and R101 is independently C1-C5 alkyl.
“Amino” refers to the group —NHR′ or —NH—, wherein R′ is hydrogen, alkyl, or cycloalkyl.
“Amino alcohol” refers to the radical —NHLOH, wherein L is alkylene.
“Carboxyl” or “carboxy” refers to the radical —C(O)OH.
The term “alkylamino” or “arylamino” refers to an amino group that has one or two alkyl or aryl substituents, respectively. In certain embodiments, the alkyl substituent is upper alkyl. In certain embodiments, the alkyl substituent is lower alkyl. In another embodiment, the alkyl, upper alkyl, or lower alkyl is unsubstituted.
“Halogen” or “halo” refers to chloro, bromo, fluoro or iodo.
“Monoalkylamino” refers to the group alkyl-NR′—, wherein R′ is hydrogen, alkyl, or cycloalkyl.
“Thioalkoxyl” refers to the group —SR′ where R′ is alkyl or cycloalkyl.
The term “heterocyclo” or “heterocyclic” refers to a monovalent monocyclic non-aromatic ring system and/or multicyclic ring system that contains at least one non-aromatic ring, wherein one or more of the non-aromatic ring atoms are heteroatoms independently O, S, or N; and the remaining ring atoms are carbon atoms. In certain embodiments, the heterocyclo or heterocyclic group has from 3 to 20, from 3 to 15, from 3 to 10, from 3 to 8, from 4 to 7, or from 5 to 6 ring atoms. Heterocyclo groups are bonded to the rest of the molecule through the non-aromatic ring. In certain embodiments, the heterocyclo is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include a fused or bridged ring system, and in which the nitrogen or sulfur atoms may be optionally oxidized, the nitrogen atoms may be optionally quaternized, and some rings may be partially or fully saturated, or aromatic. The heterocyclo may be attached to the main structure at any heteroatom or carbon atom which results in the creation of a stable compound. Examples of such heterocyclic radicals include, but are not limited to, azepinyl, benzodioxanyl, benzodioxolyl, benzofuranonyl, benzopyranonyl, benzopyranyl, benzotetrahydrofuranyl, benzotetrahydrothienyl, benzothiopyranyl, benzoxazinyl, β-carbolinyl, chromanyl, chromonyl, cinnolinyl, coumarinyl, decahydroisoquinolinyl, dihydrobenzisothiazinyl, dihydrobenzisoxazinyl, dihydrofuryl, dihydroisoindolyl, dihydropyranyl, dihydropyrazolyl, dihydropyrazinyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dioxolanyl, 1,4-dithianyl, furanonyl, imidazolidinyl, imidazolinyl, indolinyl, isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isochromanyl, isocoumarinyl, isoindolinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazolidinonyl, oxazolidinyl, oxiranyl, piperazinyl, piperidinyl, 4-piperidonyl, pyrazolidinyl, pyrazolinyl, pyrrolidinyl, pyrrolinyl, quinuclidinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydrothienyl, thiamorpholinyl, thiazolidinyl, tetrahydroquinolinyl, and 1,3,5-trithianyl. In certain embodiments, heterocyclic may also be optionally substituted as described herein. Non-limiting examples of moieties with which the heterocyclic group can be substituted include halogen (fluoro, chloro, bromo or iodo), hydroxyl, carbonyl, alkoxycarbonyl, alkoxycarbonylalkyl, sulfanyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, and phosphonate, either unprotected, or protected as necessary.
The term “heteroaryl” refers to a monovalent monocyclic aromatic group and/or multicyclic aromatic group that contain at least one aromatic ring, wherein at least one aromatic ring contains one or more heteroatoms which are independently O, S, or N in the ring. Heteroaryl groups are bonded to the rest of the molecule through the aromatic ring. Each ring of a heteroaryl group can contain one or two O atoms, one or two S atoms, and/or one to four N atoms, provided that the total number of heteroatoms in each ring is four or less and each ring contains at least one carbon atom. In certain embodiments, the heteroaryl has from 5 to 20, from 5 to 15, or from 5 to 10 ring atoms. Examples of monocyclic heteroaryl groups include, but are not limited to, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl, triazinyl and triazolyl. Examples of bicyclic heteroaryl groups include, but are not limited to, benzofuranyl, benzimidazolyl, benzoisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl, benzotriazolyl, benzoxazolyl, furopyridyl, imidazopyridinyl, imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofuranyl, isobenzothienyl, isoindolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl, pyridopyridyl, pyrrolopyridyl, quinolinyl, quinoxalinyl, quinazolinyl, thiadiazolopyrimidyl, and thienopyridyl. Examples of tricyclic heteroaryl groups include, but are not limited to, acridinyl, benzindolyl, carbazolyl, dibenzofuranyl, perimidinyl, phenanthrolinyl, phenanthridinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxazinyl and xanthenyl. In certain embodiments, heteroaryl may also be optionally substituted as described herein.
The term “alkylaryl” refers to an aryl group with an alkyl substituent, wherein aryl and alkyl are as defined herein. The term “aralkyl” or “arylalkyl” refers to an alkyl group with an aryl substituent, wherein aryl and alkyl are as defined herein.
The term “alkylheterocyclo” refers to a heterocyclo group with an alkyl substituent. The term “heterocycloalkyl” refers to an alkyl group with a heterocyclo substituent.
The term “alkylheteroaryl” refers to a heteroaryl group with an alkyl substituent. The term “heteroarylalkyl” refers to an alkyl group with a heteroaryl substituent.
As used herein, the term “hydantoinyl” refers to the group
where RXX and RYY are each independently hydrogen or lower alkyl.
As used herein, the term “hydantoinylalkyl” refers to the group -alkyl-hydantoinyl, where alkyl and hydantoinyl are as described herein.
As used herein, the term “aminoalkyl” refers to an alkyl group with an amino substituent, where alkyl and amino are as described herein.
As used herein, the term “carboxylalkyl” refers to the group -alkyl-C(O)OH, where alkyl is as described herein.
As used herein, the term “aminoiminoaminoalkyl” refers to the group -alkyl-amino-C(═NH)-amino, where alkyl and amino are as described herein.
As used herein, the term “aminocarbonylalkyl” refers to the group -alkyl-C(O)-amino, where alkyl and amino are as described herein.
As used herein, the term “sulfanylalkyl” refers to the group -alkyl-SH, where alkyl is as described herein.
As used herein, the term “carbamoylalkyl” refers to the group -alkyl-C(O)-amino, where alkyl and amino are as described herein.
As used herein, the term “alkylsulfanylalkyl” refers to the group -alkyl-S-alkyl, where alkyl is as described herein.
As used herein, the term “hydroxylarylalkyl” refers to the group -alkyl-aryl-OH, where alkyl and aryl are as described herein.
As used herein, the term “hydroxylalkyl” refers to the group -alkyl-OH, where alkyl is as described herein.
Unless expressly stated to the contrary, substitution by a named substituent is permitted on any atom in a chain or ring provided such substitution is chemically allowed and results in a stable compound. A “stable” compound is a compound which can be prepared and isolated and whose structure and properties remain or can be caused to remain essentially unchanged for a period of time sufficient to allow use of the compound for the purposes described herein (e.g., therapeutic or prophylactic administration to a subject).
To the extent substituents and substituent patterns provide for the existence of tautomers (e.g., keto-enol tautomers) in the compounds described herein, all tautomeric forms of these compounds, whether present individually or in mixtures, are within the scope of the present disclosure. Compounds of the present disclosure having a hydroxy substituent on a carbon atom of a heteroaromatic ring are understood to include compounds in which only the hydroxy is present, compounds in which only the tautomeric keto form (i.e., an oxo substituent) is present, and compounds in which the keto and enol forms are both present.
The term “protecting group” as used herein and unless otherwise defined refers to a group that is added to an oxygen, nitrogen or phosphorus atom to prevent its further reaction or for other purposes. A wide variety of oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis.
“Pharmaceutically acceptable salt” refers to any salt of a compound provided herein which retains its biological properties and which is not toxic or otherwise undesirable for pharmaceutical use. Such salts may be derived from a variety of organic and inorganic counter-ions well known in the art. Such salts include, but are not limited to: (1) acid addition salts formed with organic or inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic, trifluoroacetic, trichloroacetic, propionic, hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic, phthalic, lauric, methanesulfonic, ethanesulfonic, 1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic, camphoric, camphorsulfonic, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic, 3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric, gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic, cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2) base addition salts formed when an acidic proton present in the parent compound either (a) is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion or an aluminum ion, or alkali metal or alkaline earth metal hydroxides, such as sodium, potassium, calcium, magnesium, aluminum, lithium, zinc, and barium hydroxide, ammonia or (b) coordinates with an organic base, such as aliphatic, alicyclic, or aromatic organic amines, such as ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N′-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, N-methylglucamine piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and the like.
Pharmaceutically acceptable salts further include, by way of example only and without limitation, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium and the like, and when the compound contains a basic functionality, salts of non-toxic organic or inorganic acids, such as hydrohalides, e.g. hydrochloride and hydrobromide, sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate, malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate, tartarate, citrate, benzoate, 3-(4-hydroxybenzoyl)benzoate, picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate), ethanesulfonate, 1,2-ethane-disulfonate, 2-hydroxyethanesulfonate, benzenesulfonate (besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4-toluenesulfonate, camphorate, camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate, glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl sulfate, gluconate, benzoate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclohexylsulfamate, quinate, muconate and the like.
As used herein, the term “nucleobase” refers to the base portion of a nucleoside or nucleotide. In certain embodiments, a nucleobase is a purine or pyrimidine base, as defined herein.
The term “purine” or “pyrimidine” base refers to, but is not limited to, adenine, N6-alkylpurines, N6-acylpurines (wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl), N6-benzylpurine, N6-halopurine, N6-vinylpurine, N6-acetylenic purine, N6-acyl purine, N6-hydroxyalkyl purine, N6-alkylaminopurine, N6-thioalkyl purine, N2-alkylpurines, N2-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine, 5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or 4-mercaptopyrmidine, uracil, 5-halouracil, including 5-fluorouracil, C5-alkylpyrimidines, C5-benzylpyrimidines, C5-halopyrimidines, C5-vinylpyrimidine, C5-acetylenic pyrimidine, C5-acyl pyrimidine, C5-hydroxyalkyl purine, C5-amidopyrimidine, C5-cyanopyrimidine, C5-iodopyrimidine, C6-iodo-pyrimidine, C5—Br-vinyl pyrimidine, C6—Br-vinyl pyrimidine, C5-nitropyrimidine, C5-amino-pyrimidine, N2-alkylpurines, N2-alkyl-6-thiopurines, 5-azacytidine, 5-azauracil, triazolopyridine, imidazolopyridine, pyrrolopyrimidine, and pyrazolopyrimidine. Purine bases include, but are not limited to, guanine, adenine, hypoxanthine, 7-deazaguanine, 7-deazaadenine, 2,6-diaminopurine, 6-chloropurine, 7-fluoro-7-deazaguanine, 7-fluoro-7-deazaadenine, 2-amino-6-chloropurine, 6-methoxypurine, 6-ethoxypurine, 2-amino-6-methoxypurine and pyrrolotriazine. Functional oxygen and nitrogen groups on the base can be protected as necessary or desired. Suitable protecting groups are well known to those skilled in the art, and include trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups, and acyl groups such as acetyl and propionyl, methanesulfonyl, and p-toluenesulfonyl.
The term “acyl” or “O-linked ester” refers to a group of the formula C(O)R′, wherein R′ is alkyl or cycloalkyl (including lower alkyl), carboxylate residue of amino acid, aryl including phenyl, alkaryl, arylalkyl including benzyl, alkoxyalkyl including methoxymethyl, aryloxyalkyl such as phenoxymethyl; or substituted alkyl (including lower alkyl), aryl including phenyl optionally substituted with chloro, bromo, fluoro, iodo, C1 to C4 alkyl or C1 to C4 alkoxy, sulfonate esters such as alkyl or arylalkyl sulphonyl including methanesulfonyl, the mono, di or triphosphate ester, trityl or monomethoxy-trityl, substituted benzyl, alkaryl, arylalkyl including benzyl, alkoxyalkyl including methoxymethyl, aryloxyalkyl such as phenoxymethyl. Aryl groups in the esters optimally comprise a phenyl group. In particular, acyl groups include acetyl, trifluoroacetyl, methylacetyl, cyclpropylacetyl, propionyl, butyryl, hexanoyl, heptanoyl, octanoyl, neo-heptanoyl, phenylacetyl, 2-acetoxy-2-phenylacetyl, diphenylacetyl, α-methoxy-α-trifluoromethyl-phenylacetyl, bromoacetyl, 2-nitro-benzeneacetyl, 4-chloro-benzeneacetyl, 2-chloro-2,2-diphenylacetyl, 2-chloro-2-phenylacetyl, trimethylacetyl, chlorodifluoroacetyl, perfluoroacetyl, fluoroacetyl, bromodifluoroacetyl, methoxyacetyl, 2-thiophencacetyl, chlorosulfonylacetyl, 3-methoxyphenylacetyl, phenoxyacetyl, tert-butylacetyl, trichloroacetyl, monochloro-acetyl, dichloroacetyl, 7H-dodecafluoro-heptanoyl, perfluoro-heptanoyl, 7H-dodeca-fluoroheptanoyl, 7-chlorododecafluoro-heptanoyl, 7-chloro-dodecafluoro-heptanoyl, 7H-dodecafluoroheptanoyl, 7H-dodeca-fluoroheptanoyl, nona-fluoro-3,6-dioxa-heptanoyl, nonafluoro-3,6-dioxaheptanoyl, perfluoroheptanoyl, methoxybenzoyl, methyl 3-amino-5-phenylthiophene-2-carboxyl, 3,6-dichloro-2-methoxy-benzoyl, 4-(1,1,2,2-tetrafluoro-ethoxy)-benzoyl, 2-bromo-propionyl, omega-aminocapryl, decanoyl, n-pentadecanoyl, stearyl, 3-cyclopentyl-propionyl, 1-benzene-carboxyl, O-acetylmandelyl, pivaloyl acetyl, 1-adamantane-carboxyl, cyclohexane-carboxyl, 2,6-pyridinedicarboxyl, cyclopropane-carboxyl, cyclobutane-carboxyl, perfluorocyclohexyl carboxyl, 4-methylbenzoyl, chloromethyl isoxazolyl carbonyl, perfluorocyclohexyl carboxyl, crotonyl, 1-methyl-1H-indazole-3-carbonyl, 2-propenyl, isovaleryl, 1-pyrrolidinecarbonyl, 4-phenylbenzoyl.
The term “amino acid” refers to naturally occurring and synthetic α, β, γ, or δ amino acids, and includes but is not limited to, amino acids found in proteins, i.e. glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine. In certain embodiments, the amino acid is in the L-configuration. In certain embodiments, the amino acid is in the D-configuration. In certain embodiments, the amino acid is provided as a substituent of a compound described herein, wherein the amino acid is a residue including alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleuccinyl, β-prolinyl, β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl, β-serinyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl, β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl, and β-histidinyl.
The term “amino acid derivative” refers to a group derivable from a naturally or non-naturally occurring amino acid, as described and exemplified herein. Amino acid derivatives are apparent to those of skill in the art and include, but are not limited to, ester, amino alcohol, amino aldehyde, amino lactone, and N-methyl derivatives of naturally and non-naturally occurring amino acids. In an embodiment, an amino acid derivative is provided as a substituent of a compound described herein, wherein the substituent is —NRX-G1(SC1)—C(O)-Q1, wherein Q1 is —SRY, —NRYRY or alkoxyl, RR is hydrogen or alkyl, SC is a side chain of a naturally occurring or non-naturally occurring amino acid, G1 is C1-C2 alkylene, and Rx is hydrogen or RX and SC1, together with the atoms to which they are attached, combine to form a five-membered heterocyclic ring. In an embodiment, an amino acid derivative is provided as a substituent of a compound described herein, wherein the substituent is —O—C(O)-G2(SC2)—NH-Q2, wherein Q2 is hydrogen or alkoxyl, SC2 is a side chain of a naturally occurring or non-naturally occurring amino acid and G2 is C1-C2 alkylene. In certain embodiments, Q2 and SC2, together with the atoms to which they are attached, combine to form a five-membered heterocyclic ring. In certain embodiments, each of G1 and G2 is independently C1 alkylene and each of SC1 and SC2 is independently hydrogen, alkyl, arylalkyl, heterocycloalkyl, carboxylalkyl, heteroarylalkyl, aminoalkyl, hydroxylalkyl, aminoiminoaminoalkyl, aminocarbonylalkyl, sulfanylalkyl, carbamoylalkyl, alkylsulfanylalkyl or hydroxylarylalkyl. In an embodiment, an amino acid derivative is provided as a substituent of a compound described herein, wherein the amino acid derivative is in the D-configuration. In an embodiment, an amino acid derivative is provided as a substituent of a compound described herein, wherein the amino acid derivative is in the L-configuration. In another embodiment, the amino derivative is —NRX-G1(SC1)—C(O)-Q1, where RX is H, G1(Sc1) is CH(CH3) and Q1 is O—C3-C6 unsubstituted alkyl, where CH(CH3) is a D-configuration and in further embodiment a L-configuration.
The term “substantially free of” or “substantially in the absence of” with respect to a nucleoside composition refers to a nucleoside composition that includes at least 85% or 90% by weight, in certain embodiments 95%, 98%, 99% or 100% by weight, of the designated diastereomer of that nucleoside. In certain embodiments, in the methods and compounds provided herein, the compounds are substantially free of other diastereomers.
Similarly, the term “isolated” with respect to a nucleoside composition refers to a nucleoside composition that includes at least 85%, 90%, 95%, 98%, 99% to 100% by weight, of the nucleoside, the remainder comprising other chemical species or disastereomer.
“Solvate” refers to a compound provided herein or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate.
“Isotopic composition” refers to the amount of each isotope present for a given atom, and “natural isotopic composition” refers to the naturally occurring isotopic composition or abundance for a given atom. Atoms containing their natural isotopic composition may also be referred to herein as “non-enriched” atoms. Unless otherwise designated, the atoms of the compounds recited herein are meant to represent any stable isotope of that atom. For example, unless otherwise stated, when a position is designated specifically as “H” or “hydrogen,” the position is understood to have hydrogen at its natural isotopic composition.
“Isotopic enrichment” refers to the percentage of incorporation of an amount of a specific isotope at a given atom in a molecule in the place of that atom's natural isotopic abundance. For example, deuterium enrichment of 1% at a given position means that 1% of the molecules in a given sample contain deuterium at the specified position. Because the naturally occurring distribution of deuterium is about 0.0156%, deuterium enrichment at any position in a compound synthesized using non-enriched starting materials is about 0.0156%. The isotopic enrichment of the compounds provided herein can be determined using conventional analytical methods known to one of ordinary skill in the art, including mass spectrometry and nuclear magnetic resonance spectroscopy.
“Isotopically enriched” refers to an atom having an isotopic composition other than the natural isotopic composition of that atom. “Isotopically enriched” may also refer to a compound containing at least one atom having an isotopic composition other than the natural isotopic composition of that atom.
As used herein, “alkyl,” “cycloalkyl,” “alkenyl,” “cycloalkenyl,” “alkynyl,” “aryl,” “alkoxy,” “alkoxycarbonyl,” “alkoxycarbonylalkyl,” “amino,” “carboxyl,” “alkylamino,” “arylamino,” “thioalkyoxy,” “heterocyclo,” “heteroaryl,” “alkylheterocyclo,” “alkylheteroaryl,” “acyl,” “aralkyl,” “alkaryl,” “purine,” “pyrimidine,” “carboxyl” and “amino acid” groups optionally comprise deuterium at one or more positions where hydrogen atoms are present, and wherein the deuterium composition of the atom or atoms is other than the natural isotopic composition.
Also as used herein, “alkyl,” “cycloalkyl,” “alkenyl,” “cycloalkenyl,” “alkynyl,” “aryl,” “alkoxy,” “alkoxycarbonyl,” “alkoxycarbonylalkyl,” “carboxyl,” “alkylamino,” “arylamino,” “thioalkyoxy,” “heterocyclo,” “heteroaryl,” “alkylheterocyclo,” “alkylheteroaryl,” “acyl,” “aralkyl,” “alkaryl,” “purine,” “pyrimidine,” “carboxyl” and “amino acid” groups optionally comprise carbon-13 at an amount other than the natural isotopic composition.
Also as used herein, “alkyl,” “cycloalkyl,” “alkenyl,” “cycloalkenyl,” “alkynyl,” “aryl,” “alkoxy,” “alkoxycarbonyl,” “alkoxycarbonylalkyl,” “carboxyl,” “alkylamino,” “arylamino,” “thioalkyoxy,” “heterocyclo,” “heteroaryl,” “alkylheterocyclo,” “alkylheteroaryl,” “acyl,” “aralkyl,” “alkaryl,” “purine,” “pyrimidine,” “carboxyl” for the different formulae described herein (including different referred-to embodiments) are each unsubstituted.
As used herein, EC50 refers to a dosage, concentration or amount of a particular test compound that elicits a dose-dependent response at 50% of maximal expression of a particular response that is induced, provoked or potentiated by the particular test compound.
As used herein, the IC50 refers to an amount, concentration or dosage of a particular test compound that achieves a 50% inhibition of a maximal response in an assay that measures such response.
The term “host,” as used herein, refers to any unicellular or multicellular organism in which the virus can replicate, including cell lines and animals, and in certain embodiments, a human. Alternatively, the host can be carrying a part of the Flaviviridae viral genome, whose replication or function can be altered by the compounds of the present invention. The term host specifically includes infected cells, cells transfected with all or part of the Flaviviridae genome and animals, in particular, primates (including chimpanzees) and humans. In most animal applications of the present invention, the host is a human patient. Veterinary applications, in certain indications, however, are clearly anticipated by the present invention (such as chimpanzees).
As used herein, the terms “subject” and “patient” are used interchangeably herein. The terms “subject” and “subjects” refer to an animal, such as a mammal including a non-primate (e.g., a cow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey such as a cynomolgus monkey, a chimpanzee and a human), and for example, a human. In certain embodiments, the subject is refractory or non-responsive to current treatments for hepatitis C infection. In another embodiment, the subject is a farm animal (e.g., a horse, a cow, a pig, etc.) or a pet (e.g., a dog or a cat). In certain embodiments, the subject is a human.
As used herein, the terms “therapeutic agent” and “therapeutic agents” refer to any agent(s) which can be used in the treatment or prevention of a disorder or one or more symptoms thereof. In certain embodiments, the term “therapeutic agent” includes a compound provided herein. In certain embodiments, a therapeutic agent is an agent which is known to be useful for, or has been or is currently being used for the treatment or prevention of a disorder or one or more symptoms thereof.
“Therapeutically effective amount” refers to an amount of a compound or composition that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease. A “therapeutically effective amount” can vary depending on, inter alia, the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated.
“Treating” or “treatment” of any disease or disorder refers, in certain embodiments, to ameliorating a disease or disorder that exists in a subject. In another embodiment, “treating” or “treatment” includes ameliorating at least one physical parameter, which may be indiscernible by the subject. In yet another embodiment, “treating” or “treatment” includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. In yet another embodiment, “treating” or “treatment” includes delaying the onset of the disease or disorder.
As used herein, the terms “prophylactic agent” and “prophylactic agents” as used refer to any agent(s) which can be used in the prevention of a disorder or one or more symptoms thereof. In certain embodiments, the term “prophylactic agent” includes a compound provided herein. In certain other embodiments, the term “prophylactic agent” does not refer a compound provided herein. For example, a prophylactic agent is an agent which is known to be useful for, or has been or is currently being used to prevent or impede the onset, development, progression and/or severity of a disorder.
As used herein, the phrase “prophylactically effective amount” refers to the amount of a therapy (e.g., prophylactic agent) which is sufficient to result in the prevention or reduction of the development, recurrence or onset of one or more symptoms associated with a disorder, or to enhance or improve the prophylactic effect(s) of another therapy (e.g., another prophylactic agent).
Compounds
Provided herein are 4′-OR nucleoside compounds useful for the treatment of Flaviviridae infections such as HCV infection. The 4′-OR nucleoside compounds can be formed as described herein and used for the treatment of Flaviviridae infections such as HCV infection.
The compounds described herein may optionally be used in the form of a pharmaceutically acceptable salt. It is understood that references to compounds or pharmaceutically salts thereof would include compounds in present form as well as in different forms, such as polymorphs and solvates (including hydrates), as applicable.
In certain embodiments, provided herein are compounds according to Formula 1001:
or a pharmaceutically acceptable salt thereof, wherein:
Base is a natural or unnatural nucleobase;
PD is hydrogen,
W is S or O; one of X and Y is hydrogen, —OR1, —SR1, —NR1R2, an O-linked amino acid residue or derivative thereof, an N-linked amino acid residue or derivative thereof, or an N-linked amino alcohol residue or derivative thereof; and the other of X and Y is OR1 or NR1R2;
each of Z1, Z2, Z3, and Z4 is independently —H, —OH, —OCH3, —N3, —NH2, —CH3, —Cl, or —F; or, in the alternative, Z4 and Y, together with the atoms to which they are attached, combine to form a six-membered heterocyclic ring wherein Z4 and Y together represent a single divalent —O—, and X is —OR1, —SR1, —NR1R2, an O-linked amino acid residue or derivative thereof, an N-linked amino acid residue or derivative thereof, or an N-linked amino alcohol residue or derivative thereof; and each of Z1, Z2, and Z3 is independently —H, —OH, —OCH3, —N3, —NH2, —CH3, —Cl, or —F;
each R1 is independently alkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkylcarbonylthioalkyl, alkoxycarbonylalkyl, arylalkoxycarbonylalkyl, alkylcarbonylalkoxy(arylalkyl), cycloalkylcarbonylalkoxyl, alkoxycarbonylaminoalkylcarbonylthioalkyl, hydroxylalkylcarbonylthioalkyl, or aminoalkylcarbonylalkoxycarbonylthioalkyl; and each R2 is independently hydrogen or alkyl;
R is alkyl or alkynyl;
with the proviso that the compound of formula 1001 is not 4′-methoxyuridine, 4′-methoxycytidine, 4′-ethoxycytidine, 4′-methoxyadenosine, 3′-deoxy-3′-fluoro-4′-methoxythymidine, 2′, 3′-dideoxy-3′-fluoro-4′-methoxyadenosine, 2′, 3′-dideoxy-3′-fluoro-4′-methoxyuridine, 2′, 3′-dideoxy-3′-fluoro-4′-methoxycytidine, 2′, 3′-dideoxy-3′-fluoro-4′-methoxyguanosine, or 5′-mono, di, or triphosphate thereof.
In certain embodiments, provided herein are compounds according to Formula I:
or a pharmaceutically acceptable salt thereof, wherein:
Base is a natural or unnatural nucleobase;
PD is hydrogen,
W is S or O; one of X and Y is hydrogen, —OR1, —SR1, —NR1R2, an O-linked amino acid residue or derivative thereof, an N-linked amino acid residue or derivative thereof, or an N-linked amino alcohol residue or derivative thereof; and the other of X and Y is —OR1 or —NR1R2;
each of Z1, Z2, Z3, and Z4 is independently —H, —OH, —N3, —NH2, —CH3, —Cl, or —F; or, in the alternative, Z4 and Y, together with the atoms to which they are attached, combine to form a six-membered heterocyclic ring wherein Z4 and Y together represent a single divalent —O—, and X is —OR1, —SR1, —NR1R2, an O-linked amino acid residue or derivative thereof, an N-linked amino acid residue or derivative thereof, or an N-linked amino alcohol residue or derivative thereof; and each of Z1, Z2, and Z3 is independently —H, —OH, —N3, —NH2, —CH3, —Cl, or —F;
each R1 is independently alkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkylcarbonylthioalkyl, alkoxycarbonylalkyl, arylalkoxycarbonylalkyl, alkylcarbonylalkoxy(arylalkyl), cycloalkylcarbonylalkoxyl, alkoxycarbonylaminoalkylcarbonylthioalkyl, hydroxylalkylcarbonylthioalkyl, or aminoalkylcarbonylalkoxycarbonylthioalkyl; and each R2 is independently hydrogen or alkyl;
R is alkyl or alkynyl.
In certain embodiments according to Formula 1001 or I, the nucleobase is purine, pyrimidine, adenine, N6-alkylpurines, N6-acylpurines, N6-benzylpurine, N6-halopurine, N6-vinylpurine, N6-acetylenic purine, N6-acyl purine, N6-hydroxyalkyl purine, N6-alkylaminopurine, N6-thioalkyl purine, N2-alkylpurines, N2-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine, 5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or 4-mercaptopyrmidine, uracil, 5-halouracil, 5-fluorouracil, C5-alkylpyrimidines, C5-benzylpyrimidines, C5-halopyrimidines, C5-vinylpyrimidine, C5-acetylenic pyrimidine, C5-acyl pyrimidine, C5-hydroxyalkyl purine, C5-amidopyrimidine, C5-cyanopyrimidine, C5-iodopyrimidine, C6-iodo-pyrimidine, C5—Br-vinyl pyrimidine, C6—Br-vinyl pyrimidine, C5-nitropyrimidine, C5-amino-pyrimidine, N2-alkylpurines, N2-alkyl-6-thiopurines, 5-azacytidine, 5-azauracil, triazolopyridine, imidazolopyridine, pyrrolopyrimidine, pyrazolopyrimidine, guanine, hypoxanthine, 7-deazaguanine, 7-deazaadenine, 2,6-diaminopurine, 6-chloropurine, 7-fluoro-7-deazaguanine, 7-fluoro-7-deazaadenine, 2-amino-6-chloropurine, 6-methoxypurine, 6-ethoxypurine, 2-amino-6-methoxypurine, or pyrrolotriazine;
each R1 is independently alkyl, cycloalkyl, aryl, heteroaryl, aryl-alkyl, heteroaryl-alkyl, alkyl-carbonyl-thioalkyl, alkoxy-carbonyl-alkyl, aryl-alkoxy-carbonyl-alkyl, alkyl-carbonyl-alkoxy-(arylalkyl), cycloalkyl-carbonyl-alkoxyl, alkoxycarbonyl-amino-alkyl-carbonyl-thio-alkyl, hydroxyl-alkyl-carbonyl-thioalkyl, or amino-alkyl-carbonyl-alkoxy-carbonyl-thio-alkyl; and each R2 is independently hydrogen or alkyl;
each alkyl is independently straight or branched C1-10 alkyl, unsubstituted or substituted with one or more halogen, fluoro, chloro, bromo, iodo, hydroxyl, carbonyl, C3-15 cycloalkyl, phenyl C1-10 alkyl, biphenyl C1-10 alkyl, naphthyl C1-10 alkyl, sulfanyl, amino, C1-10 alkyl-amino, phenyl-amino, biphenyl-amino, naphthyl-amino, C1-10 alkoxy, phenyloxy, biphenyloxy, naphthyloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate; each cycloalkyl is independently a C3-15 cycloalkyl, unsubstituted or substituted with one or more halogen, fluoro, chloro, bromo, iodo, hydroxyl, carbonyl, sulfanyl, amino, C1-10 alkylamino, phenyl-amino, biphenyl-amino, naphthylamino, C1-10 alkoxy, phenyloxy, biphenyloxy, naphthyloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, and phosphonate;
each aryl is independently phenyl, biphenyl, or naphthyl, unsubstituted or substituted by one or more halogen, fluoro, chloro, bromo, iodo, C1-10 alkyl, halo C1-10 alkyl, hydroxyl, amino, C1-10 alkyl-amino, phenyl-amino, biphenyl-amino, naphthyl-amino, C1-10 alkoxy, phenyloxy, biphenyloxy, naphthyloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate;
each heteroaryl is independently furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl, triazinyl, triazolyl, benzofuranyl, benzimidazolyl, benzoisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl, benzotriazolyl, benzoxazolyl, furopyridyl, imidazopyridinyl, imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofuranyl, isobenzothienyl, isoindolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl, pyridopyridyl, pyrrolopyridyl, quinolinyl, quinoxalinyl, quinazolinyl, thiadiazolopyrimidyl, thienopyridyl, acridinyl, benzindolyl, carbazolyl, dibenzofuranyl, perimidinyl, phenanthrolinyl, phenanthridinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxazinyl, or xanthenyl;
each arylalkyl is independently aryl C1-10 alkyl; wherein aryl is defined above; each heteroarylalkyl is independently heteroaryl C1-10 alkyl; wherein heteroaryl is defined above;
each alkoxy is independently —OR′ wherein R′ is alkyl or cycloalkyl and wherein alkyl is C1-10 alkyl and cycloalkyl is C3-15 cycloalkyl;
each alkynyl is independently straight or branched C2-11 alkynyl, unsubstituted or substituted by one or more halogen, fluoro, chloro, bromo, iodo, hydroxyl, carbonyl, sulfanyl, amino, C1-10 alkyl-amino, phenyl-amino, biphenyl-amino, naphthyl-amino, C1-10 alkoxy, phenyloxy, biphenyloxy, naphthyloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate.
In certain embodiments, the compound of Formula 1001 or I is not 4′-methoxyuridine, 4′-methoxycytidine, 4′-ethoxycytidine, 4′-methoxyadenosine, 3′-deoxy-3′-fluoro-4′-methoxythymidine, 2′, 3′-dideoxy-3′-fluoro-4′-methoxyadenosine, 2′, 3′-dideoxy-3′-fluoro-4′-methoxyuridine, 2′, 3′-dideoxy-3′-fluoro-4′-methoxycytidine, 2′, 3′-dideoxy-3′-fluoro-4′-methoxyguanosine, or 5′-mono, di, or triphosphate thereof.
In certain embodiments, Z3 is —H, and each of Z1, Z2, and Z4 is independently —H, —OH, —OCH3, —N3, —NH2, —CH3, —Cl, or —F.
In certain embodiments, when Z1 is —H, Z2 is —OH or —H, Z3 is —H, Z4 is —OH or —F, then PD is
W is S or O; one of X and Y is hydrogen, —OR1, —SR1, —NR1R2, an O-linked amino acid residue or derivative thereof, an N-linked amino acid residue or derivative thereof, or an N-linked amino alcohol residue or derivative thereof; and the other of X and Y is —OR1 or —NR1R2; wherein R1 and R2 are defined as above.
In certain embodiments, when Z is —H, Z2 is —OH, Z3 is —H, Z4 is —OH, then PD is
W is S or O; one of X and Y is hydrogen, —OR1, —SR1, —NR1R2, an O-linked amino acid residue or derivative thereof, an N-linked amino acid residue or derivative thereof, or an N-linked amino alcohol residue or derivative thereof; and the other of X and Y is —OR1 or —NR1R2; wherein R1 and R2 are defined as above.
In certain embodiments, when Z1 is —H, Z2 is —H, Z3 is —H, Z4 is —F, then PD is
W is S or O; one of X and Y is hydrogen, —OR1, —SR1, —NR1R2, an O-linked amino acid residue or derivative thereof, an N-linked amino acid residue or derivative thereof, or an N-linked amino alcohol residue or derivative thereof; and the other of X and Y is —OR1 or —NR1R2; wherein R1 and R2 are defined as above.
In certain embodiments, when Z1 is —H, Z2 is —OH, Z3 is —H, Z4 is —N3, then PD is
W is S or O; one of X and Y is hydrogen, —OR1, —SR1, —NR1R2, an O-linked amino acid residue or derivative thereof, an N-linked amino acid residue or derivative thereof, or an N-linked amino alcohol residue or derivative thereof; and the other of X and Y is —OR1 or —NR1R2; wherein R1 and R2 are defined as above.
In certain embodiments, R is unsubstituted or substituted alkyl; PD, Base, Z1, Z2, Z3, and Z4 are defined as above. In certain embodiments, R is methyl, ethyl, or isopropyl.
In certain embodiments, R is unsubstituted or substituted alkynyl; PD, Base, Z1, Z2, Z3, and Z4 are defined as above. In certain embodiments, R is ethynyl or propargyl.
In certain embodiments, Z1 is —H, Z2 is —OH and Z3 is —H, Z4 is —OH; and PD, Base, and R are defined as above.
In certain embodiments, Z1 is —H, Z2 is —F, Z3 is —H, and Z4 is —OH; and PD, Base, and R are defined as above.
In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —F; and PD, Base, and R are defined as above.
In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —NH2; and PD, Base, and R are defined as above.
In certain embodiments, Z1 is —H, Z2 is —OH, and Z3 and Z4 are each —H; and PD, Base, and R are defined as above.
In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —N3; and PD, Base, and R are defined as above.
In certain embodiments, Z1 is —H, Z2 is —OCH3 and Z3 is —H, Z4 is —OH; and PD, Base, and R are defined as above.
In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —F; and PD, Base, and R are defined as above.
In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —NH2; and PD, Base, and R are defined as above.
In certain embodiments, Z1 is —H, Z2 is —OCH3, and Z3 and Z4 are each —H; and PD, Base, and R are defined as above.
In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —N3; and PD, Base, and R are defined as above.
In certain embodiments, PD is
W is S or O; one of X and Y is hydrogen, —OR1, —SR1, —NR1R2, an O-linked amino acid residue or derivative thereof, an N-linked amino acid residue or derivative thereof, or an N-linked amino alcohol residue or derivative thereof; and the other of X and Y is —OR1 or —NR1R2;
each of Z1, Z2, Z3, and Z4 is independently —H, —OH, —OCH3, —N3, —NH2, —CH3, —Cl, or —F;
R is alkyl or alkynyl;
each R1 is independently alkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkylcarbonylthioalkyl, alkoxycarbonylalkyl, arylalkoxycarbonylalkyl, alkylcarbonylalkoxy(arylalkyl), cycloalkylcarbonylalkoxyl, alkoxycarbonylaminoalkylcarbonylthioalkyl, hydroxylalkylcarbonylthioalkyl, or aminoalkylcarbonylalkoxycarbonylthioalkyl; and each R2 is independently hydrogen or alkyl. In certain embodiments described in this paragraph, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —OH. In certain embodiments described in this paragraph, Z1 is —H, Z2 is —F, Z3 is —H, and Z4 is —OH. In certain embodiments described in this paragraph, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —F. In certain embodiments described in this paragraph, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —NH2. In certain embodiments described in this paragraph, Z1 is —H, Z is —OH, and Z3 and Z4 are each —H. In certain embodiments described in this paragraph, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —N3. In certain embodiments described in this paragraph, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —OH. In certain embodiments described in this paragraph, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —F. In certain embodiments described in this paragraph, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —NH2. In certain embodiments described in this paragraph, Z1 is —H, Z2 is —OCH3, and Z3 and Z4 are each —H. In certain embodiments described in this paragraph, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —N3.
In certain embodiments, PD is
W is S or O; Y and Z4, together with the atoms to which they are attached, combine to form a six-membered heterocyclic ring wherein Y and Z4 together represent a single divalent —O—, and X is OR, —SR1, —NR1R2, an O-linked amino acid residue or derivative thereof, an N-linked amino acid residue or derivative thereof, or an N-linked amino alcohol residue or derivative thereof; and each of Z1, Z2, and Z3 is independently —H, —OH, —OCH3, —N3, —NH2, —CH3, —Cl, or —F;
R is alkyl or alkynyl;
each R1 is independently alkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkylcarbonylthioalkyl, alkoxycarbonylalkyl, arylalkoxycarbonylalkyl, alkylcarbonylalkoxy(arylalkyl), cycloalkylcarbonylalkoxyl, alkoxycarbonylaminoalkylcarbonylthioalkyl, hydroxylalkylcarbonylthioalkyl, or aminoalkylcarbonylalkoxycarbonylthioalkyl; and each R2 is independently hydrogen or alkyl. In certain embodiments described in this paragraph, Z1 is —H, Z2 is —OH, and Z3 is —H. In certain embodiments described in this paragraph, Z1 is —H, Z2 is —F, and Z3 is —H. In certain embodiments described in this paragraph, Z1 is —H, Z2 is —OCH3, and Z3 is —H.
In certain embodiments, provided herein are compounds according to any of Formulae II-IIb:
or a pharmaceutically acceptable salt thereof, wherein: each X is independently —OR1, —SR1, —NR1R2, an O-linked amino acid residue or derivative thereof, an N-linked amino acid residue or derivative thereof, or an N-linked amino alcohol residue or derivative thereof; and Base, W, R, Z1, Z2, and Z3 are as described in the context of Formula 1001 or I. In certain embodiments, Z1 is —H, Z2 is —OH, and Z3 is —H. In certain embodiments, Z1 is —H, Z2 is —F, and Z3 is —H. In certain embodiments, Z1 is —H, Z2 is —OCH3, and Z3 is —H.
In certain embodiments, provided herein are compounds according to any of Formulae III-IIIb:
or a pharmaceutically acceptable salt thereof, wherein: R3 is alkyl, alkoxyl, hydroxylalkyl, acylalkyl, or alkoxylcarbonylalkyl; and Base, Y, R, Z1, Z2, Z3, and Z4 are as described in the context of Formula 1001 or I. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —OH. In certain embodiments, Z1 is —H, Z2 is —F, Z3 is —H, and Z4 is —OH. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —F. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —NH2. In certain embodiments, Z1 is —H, Z2 is —OH, and Z3 and Z4 are each —H. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —N3. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —OH. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —F. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —NH2. In certain embodiments, Z1 is —H, Z2 is —OCH3, and Z3 and Z4 are each —H. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —N3.
In certain embodiments, provided herein are compounds according to any of Formulae IV-IVbii:
or a pharmaceutically acceptable salt thereof, wherein: R9 is methyl or hydroxymethyl; and Base, R, Z1, Z2, Z3, and Z4 are as described in the context of Formula 1001 or I. In certain embodiments, R9 is hydroxymethyl. In certain embodiments, R is methyl. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —OH. In certain embodiments, Z1 is —H, Z2 is —F, Z3 is —H, and Z4 is —OH. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —F. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —NH2. In certain embodiments, Z1 is —H, Z2 is —OH, and Z3 and Z4 are each —H. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —N3. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —OH. In certain embodiments, Z is —H, Z2 is —OCH3, Z3 is —H, and Z4 is F. In certain embodiments, Z1 is H, Z2 is OCH3, Z3 is H, and Z4 is NH2. In certain embodiments, Z1 is —H, Z2 is —OCH3, and Z3 and Z4 are each —H. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —N3.
In certain embodiments, provided herein are compounds according to any of Formulae V-Vbii:
or a pharmaceutically acceptable salt thereof, wherein Base, R, Z1, Z2, Z3, and Z4 are as described in the context of Formula 1001 or I. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z is —OH. In certain embodiments, Z1 is —H, Z2 is —F, Z3 is —H, and Z4 is —OH. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —F. In certain embodiments, Z1 is —H, Z is —OH, Z is —H, and Z4 is —NH2. In certain embodiments, Z1 is —H, Z2 is —OH, and Z3 and Z4 are each —H. In certain embodiments, Z1 is —H, Z is —OH, Z3 is —H, and Z4 is —N3. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —OH. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —F. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —NH2. In certain embodiments, Z is —H, Z2 is —OCH3, and Z3 and Z4 are each —H. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —N3.
In certain embodiments, provided herein are compounds according to any of Formulae VI-VIbii:
or a pharmaceutically acceptable salt thereof, wherein R10 is alkyl, hydroxylalkyl, acylalkyl, or alkoxylcarbonylalkyl and Ar is an aryl group; and Base, R, Z1, Z2, Z3, and Z4 are as described in the context of Formula 1001 or I. In certain embodiments, R10 is isopropyl group and Ar is phenyl group. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —OH. In certain embodiments, Z1 is —H, Z2 is —F, Z3 is —H, and Z4 is —OH. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —F. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —NH2. In certain embodiments, Z1 is —H, Z2 is —OH, and Z3 and Z4 are each —H. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —N3. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —OH. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —F. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —NH2. In certain embodiments, Z1 is —H, Z2 is —OCH3, and Z3 and Z4 are each —H. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —N3.
In certain embodiments, provided herein are compounds according to any of Formulae I-VIbii, wherein each Base is independently:
or tautomeric form thereof, wherein:
R4 is independently hydrogen, hydroxyl, alkoxyl, amino, or aminoalkyl;
R5 is independently hydrogen, hydroxyl, amino, or alkoxyl;
R6 is independently hydrogen, halogen, or alkyl; and
R7 is independently hydrogen, hydroxyl, or amino.
In certain embodiments, provided herein are compounds according to any of Formulae VII-X:
or a pharmaceutically acceptable salt thereof, wherein: PD, R, Z1, Z2, Z3, and Z4 are as described in the context of Formula 1001 or I; each R6 is independently hydrogen, halogen, or alkyl; each R7 is independently hydrogen, hydroxyl, or amino. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —OH. In certain embodiments, Z1 is —H, Z2 is —F, Z3 is —H, and Z4 is —OH. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —F. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —NH2. In certain embodiments, Z1 is —H, Z2 is —OH, and Z3 and Z4 are each —H. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —N3. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —OH. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —F. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —NH2. In certain embodiments, Z1 is —H, Z2 is —OCH3, and Z3 and Z4 are each —H. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —N3.
In certain embodiments, provided herein are compounds according to any of Formulae XI-XX:
or a pharmaceutically acceptable salt thereof, wherein PD, R, Z1, Z2, Z3, and Z4 are as described in the context of Formula 1001 or I. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —OH. In certain embodiments, Z1 is —H, Z2 is —F, Z3 is —H, and Z4 is —OH. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —F. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —NH2. In certain embodiments, Z1 is —H, Z2 is —OH, and Z3 and Z4 are each —H. In certain embodiments, Z1 is —H, Z2 is —OH, Z3 is —H, and Z4 is —N3. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —OH. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —F. In certain embodiments, Z1 is —H, Z2 is —OCH3, Z3 is —H, and Z4 is —NH2. In certain embodiments, Z1 is —H, Z2 is —OCH3, and Z3 and Z4 are each H. In certain embodiments, Z1 is H, Z2 is OCH3, Z3 is H, and Z4 is —N3.
In certain embodiments provided herein is a compound according to any of Formulae 2a-2e, 5a2, 5c, 6a-8a, 6b, 7b, 113, 114, 20, 21, 23, and 24:
or a pharmaceutically acceptable salt thereof.
In certain embodiments provided herein is a compound according to any of Formulae 13a, 13ai, 13aii, 14a, 14ai, 14aii, 16a, 122, 122i, 123, 123i, and 123ii:
or a pharmaceutically acceptable salt thereof.
In certain embodiments provided herein is a compound according to any of Formulae 131-136:
or a pharmaceutically acceptable salt thereof.
In certain embodiments provided herein is a compound according to any of Formulae 9a-9g:
or a pharmaceutically acceptable salt thereof.
In certain embodiments provided herein is a compound according to any of Formulae 208-214, 220, 222, 223, 224, 230, and 233:
or a pharmaceutically acceptable salt thereof.
In certain embodiments provided herein is a compound according to any of Formulae 17a and 402-414:
or a pharmaceutically acceptable salt thereof.
In certain embodiments provided herein is a compound according to any of Formulae 18a, 502-514, 520 and 530:
or a pharmaceutically acceptable salt thereof.
In certain embodiments provided herein is a compound according to any of Formulae 601-614:
or a pharmaceutically acceptable salt thereof.
In certain embodiments provided herein is a compound according to any of Formulae 301-314:
or a pharmaceutically acceptable salt thereof.
In some embodiments, provided herein are:
Optically Active Compounds
It is appreciated that compounds provided herein have several chiral centers and may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that any racemic, optically-active, diastereomeric, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound provided herein, which possess the useful properties described herein is within the scope of the invention. It being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).
In particular, since the 1′ and 4′ carbons of a nucleoside are chiral, their non-hydrogen substituents (the base and the CHOR groups, respectively) can be either cis (on the same side) or trans (on opposite sides) with respect to the sugar ring system. The four optical isomers therefore are represented by the following configurations (when orienting the sugar moiety in a horizontal plane such that the oxygen atom is in the back): cis (with both groups “up”, which corresponds to the configuration of naturally occurring β-D nucleosides), cis (with both groups “down”, which is a non-naturally occurring β-L configuration), trans (with the C2′ substituent “up” and the C4′ substituent “down”), and trans (with the C2′ substituent “down” and the C4′ substituent “up”). The “D-nuclcosides” are cis nuclcosides in a natural configuration and the “L-nucleosides” are cis nucleosides in the non-naturally occurring configuration.
Likewise, most amino acids are chiral (designated as L or D, wherein the L enantiomer is the naturally occurring configuration) and can exist as separate stereoisomers.
The compounds of and formula described herein may have one or more chiral (asymmetric) centers. Unless specified, the present invention encompasses all stereoisomeric forms of the compounds. Unspecified centers of asymmetry that are present in the compounds can all independently of one another have (R) or (S) configuration. When bonds to a chiral atom are depicted as straight lines in a provide structure, or when a compound name is recited without an (R) or (S) chiral designation for a chiral atom, it is understood that both the (R) and (S) configurations of each such chiral atom, and hence each enantiomer or diastereomer and mixtures thereof, are embraced.
Examples of methods to obtain stereoisomerically pure materials are known in the art, and include at least the following.
In some embodiments, provided is a composition of a 4′-OR nucleoside compound that comprises a substantially pure designated stereoisomer of the 4′-OR nucleoside compound. In certain embodiments, in the methods and compounds of this invention, the compounds are substantially free of other stereoisomers. In some embodiments, a composition includes a compound that is at least 85%, 90%, 95%, 98%, 99% or 100% by weight, of the compound, the remainder comprising other chemical species or stereoisomers.
Isotopically Enriched Compounds
Also provided herein are isotopically enriched compounds, including but not limited to isotopically enriched 4′-OR nucleoside compounds.
Isotopic enrichment (for example, deuteration) of pharmaceuticals to improve pharmacokinetics (“PK”), pharmacodynamics (“PD”), and toxicity profiles, has been demonstrated previously with some classes of drugs. See, for example, Lijinsky et. al., Food Cosmet. Toxicol., 20: 393 (1982); Lijinsky et. al., J. Nat. Cancer Inst., 69: 1127 (1982); Mangold et. al., Mutation Res. 308: 33 (1994); Gordon et. al., Drug Metab. Dispos., 15: 589 (1987); Zello et. al., Metabolism, 43: 487 (1994); Gately et. al., J. Nucl. Med., 27: 388 (1986); Wade D, Chem. Biol. Interact. 117: 191 (1999).
Isotopic enrichment of a drug can be used, for example, to (1) reduce or eliminate unwanted metabolites, (2) increase the half-life of the parent drug, (3) decrease the number of doses needed to achieve a desired effect, (4) decrease the amount of a dose necessary to achieve a desired effect, (5) increase the formation of active metabolites, if any are formed, and/or (6) decrees the production of deleterious metabolites in specific tissues and/or create a more effective drug and/or a safer drug for combination therapy, whether the combination therapy is intentional or not.
Replacement of an atom for one of its isotopes often will result in a change in the reaction rate of a chemical reaction. This phenomenon is known as the Kinetic Isotope Effect (“KIE”). For example, if a C—H bond is broken during a rate-determining step in a chemical reaction (i.e. the step with the highest transition state energy), substitution of a deuterium for that hydrogen will cause a decrease in the reaction rate and the process will slow down. This phenomenon is known as the Deuterium Kinetic Isotope Effect (“DKIE”). (See, e.g., Foster et al., Adv. Drug Res., vol. 14, pp. 1-36 (1985); Kushner et al., Can. J. Physiol. Pharmacol., vol. 77, pp. 79-88 (1999)).
The magnitude of the DKIE can be expressed as the ratio between the rates of a given reaction in which a C—H bond is broken, and the same reaction where deuterium is substituted for hydrogen. The DKIE can range from about 1 (no isotope effect) to very large numbers, such as 50 or more, meaning that the reaction can be fifty, or more, times slower when deuterium is substituted for hydrogen. High DKIE values may be due in part to a phenomenon known as tunneling, which is a consequence of the uncertainty principle. Tunneling is ascribed to the small mass of a hydrogen atom, and occurs because transition states involving a proton can sometimes form in the absence of the required activation energy. Because deuterium has more mass than hydrogen, it statistically has a much lower probability of undergoing this phenomenon.
Tritium (“T”) is a radioactive isotope of hydrogen, used in research, fusion reactors, neutron generators and radiopharmaceuticals. Tritium is a hydrogen atom that has 2 neutrons in the nucleus and has an atomic weight close to 3. It occurs naturally in the environment in very low concentrations, most commonly found as T2O. Tritium decays slowly (half-life=12.3 years) and emits a low energy beta particle that cannot penetrate the outer layer of human skin. Internal exposure is the main hazard associated with this isotope, yet it must be ingested in large amounts to pose a significant health risk. As compared with deuterium, a lesser amount of tritium must be consumed before it reaches a hazardous level. Substitution of tritium (“T”) for hydrogen results in yet a stronger bond than deuterium and gives numerically larger isotope effects. Similarly, substitution of isotopes for other elements, including, but not limited to, 13C or 14C for carbon, 33S, 34S, or 36S for sulfur, 15N for nitrogen, and 17O or 18O for oxygen, may lead to a similar kinetic isotope effect.
For example, the DKIE was used to decrease the hepatotoxicity of halothane by presumably limiting the production of reactive species such as trifluoroacetyl chloride. However, this method may not be applicable to all drug classes. For example, deuterium incorporation can lead to metabolic switching. The concept of metabolic switching asserts that xenogens, when sequestered by Phase I enzymes, may bind transiently and re-bind in a variety of conformations prior to the chemical reaction (e.g., oxidation). This hypothesis is supported by the relatively vast size of binding pockets in many Phase I enzymes and the promiscuous nature of many metabolic reactions. Metabolic switching can potentially lead to different proportions of known metabolites as well as altogether new metabolites. This new metabolic profile may impart more or less toxicity.
The animal body expresses a variety of enzymes for the purpose of eliminating foreign substances, such as therapeutic agents, from its circulation system. Examples of such enzymes include the cytochrome P450 enzymes (“CYPs”), esterases, proteases, reductases, dehydrogenases, and monoamine oxidases, to react with and convert these foreign substances to more polar intermediates or metabolites for renal excretion. Some of the most common metabolic reactions of pharmaceutical compounds involve the oxidation of a carbon-hydrogen (C—H) bond to either a carbon-oxygen (C—O) or carbon-carbon (C—C) pi-bond. The resultant metabolites may be stable or unstable under physiological conditions, and can have substantially different pharmacokinctic, pharmacodynamic, and acute and long-term toxicity profiles relative to the parent compounds. For many drugs, such oxidations are rapid. These drugs therefore often require the administration of multiple or high daily doses.
Therefore, isotopic enrichment at certain positions of a compound provided herein will produce a detectable KIE that will affect the pharmacokinetic, pharmacologic, and/or toxicological profiles of a compound provided herein in comparison with a similar compound having a natural isotopic composition.
Preparation of Compounds
The compounds provided herein can be prepared, isolated or obtained by any method apparent to those of skill in the art. Compounds provided herein can be prepared according to the Exemplary Preparation Scheme provided below. Reaction conditions, steps and reactants not provided in the Exemplary Preparation Scheme would be apparent to, and known by, those skilled in the art.
In the Exemplary Preparation Scheme, Base is as described in the context of Formula 1001 or I. Additional steps and reagents not provided in the Exemplary Preparation Scheme would be known to those of skill in the art. Exemplary methods of preparation are described in detail in the Examples herein.
Pharmaceutical Compositions and Methods of Administration
4′-OR nucleoside compounds can be formulated into pharmaceutical compositions using methods available in the art and those disclosed herein. Any of the compounds disclosed herein can be provided in the appropriate pharmaceutical composition and be administered by a suitable route of administration.
The methods provided herein encompass administering pharmaceutical compositions containing at least one compound as described herein, including a compound of any of general Formulae 1001, I-XX, 2a-2e, 5a2, 5c, 6a-8a, 6b, 7b, 113, 114, 20, 21, 23, 24, 13a, 13ai, 13aii, 14a, 14ai, 14aii, 16a, 122, 122i, 123, 123i, 123ii, 131-136, 9a-9g, 208-214, 220, 222, 223, 224, 230, 233, 301-314, 17a, 402-414, 18a, 502-514, 520, 530 and 601-614, if appropriate in the salt form, either used alone or in the form of a combination with one or more compatible and pharmaceutically acceptable carriers, such as diluents or adjuvants, or with another anti-HCV agent.
In certain embodiments, the second agent can be formulated or packaged with the compound provided herein. Of course, the second agent will only be formulated with the compound provided herein when, according to the judgment of those of skill in the art, such co-formulation should not interfere with the activity of either agent or the method of administration. In certain embodiments, the compound provided herein and the second agent are formulated separately. They can be packaged together, or packaged separately, for the convenience of the practitioner of skill in the art.
In clinical practice the active agents provided herein may be administered by any conventional route, in particular orally, parenterally, rectally or by inhalation (e.g. in the form of aerosols). In certain embodiments, the compound provided herein is administered orally.
Use may be made, as solid compositions for oral administration, of tablets, pills, hard gelatin capsules, powders or granules. In these compositions, the active product is mixed with one or more inert diluents or adjuvants, such as sucrose, lactose or starch.
These compositions can comprise substances other than diluents, for example a lubricant, such as magnesium stearate, or a coating intended for controlled release.
Use may be made, as liquid compositions for oral administration, of solutions which are pharmaceutically acceptable, suspensions, emulsions, syrups and elixirs containing inert diluents, such as water or liquid paraffin. These compositions can also comprise substances other than diluents, for example wetting, sweetening or flavoring products.
The compositions for parenteral administration can be emulsions or sterile solutions. Use may be made, as solvent or vehicle, of propylene glycol, a polyethylene glycol, vegetable oils, in particular olive oil, or injectable organic esters, for example ethyl oleate. These compositions can also contain adjuvants, in particular wetting, isotonizing, emulsifying, dispersing and stabilizing agents. Sterilization can be carried out in several ways, for example using a bacteriological filter, by radiation or by heating. They can also be prepared in the form of sterile solid compositions which can be dissolved at the time of use in sterile water or any other injectable sterile medium.
The compositions for rectal administration are suppositories or rectal capsules which contain, in addition to the active principle, excipients such as cocoa butter, semi-synthetic glycerides or polyethylene glycols.
The compositions can also be aerosols. For use in the form of liquid aerosols, the compositions can be stable sterile solutions or solid compositions dissolved at the time of use in apyrogenic sterile water, in saline or any other pharmaceutically acceptable vehicle. For use in the form of dry aerosols intended to be directly inhaled, the active principle is finely divided and combined with a water-soluble solid diluent or vehicle, for example dextran, mannitol or lactose.
In certain embodiments, a composition provided herein is a pharmaceutical composition or a single unit dosage form. Pharmaceutical compositions and single unit dosage forms provided herein comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (e.g., a compound provided herein, or other prophylactic or therapeutic agent), and a typically one or more pharmaceutically acceptable carriers or excipients. In a specific embodiment and in this context, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” includes a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water can be used as a carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
Typical pharmaceutical compositions and dosage forms comprise one or more excipients. Suitable excipients are well-known to those skilled in the art of pharmacy, and non-limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a subject and the specific active ingredients in the dosage form. The composition or single unit dosage form, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
Lactose free compositions provided herein can comprise excipients that are well known in the art and are listed, for example, in the U.S. Pharmocopia (USP) SP (XXI)/NF (XVI). In general, lactose free compositions comprise an active ingredient, a binder/filler, and a lubricant in pharmaceutically compatible and pharmaceutically acceptable amounts. Exemplary lactose free dosage forms comprise an active ingredient, microcrystalline cellulose, pre gelatinized starch, and magnesium stearate.
Further encompassed herein are anhydrous pharmaceutical compositions and dosage forms comprising active ingredients, since water can facilitate the degradation of some compounds. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical arts as a means of simulating long term storage in order to determine characteristics such as shelf life or the stability of formulations over time. See, e.g., Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed., Marcel Dekker, New York, 1995, pp. 379 80. In effect, water and heat accelerate the decomposition of some compounds. Thus, the effect of water on a formulation can be of great significance since moisture and/or humidity are commonly encountered during manufacture, handling, packaging, storage, shipment, and use of formulations.
Anhydrous pharmaceutical compositions and dosage forms provided herein can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient that comprises a primary or secondary amine can be anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected.
An anhydrous pharmaceutical composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions can be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.
Further provided are pharmaceutical compositions and dosage forms that comprise one or more compounds that reduce the rate by which an active ingredient will decompose. Such compounds, which are referred to herein as “stabilizers,” include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.
The pharmaceutical compositions and single unit dosage forms can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such compositions and dosage forms will contain a prophylactically or therapeutically effective amount of a prophylactic or therapeutic agent, in certain embodiments, in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject. The formulation should suit the mode of administration. In a certain embodiment, the pharmaceutical compositions or single unit dosage forms are sterile and in suitable form for administration to a subject, for example, an animal subject, such as a mammalian subject, for example, a human subject.
A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, intramuscular, subcutaneous, oral, buccal, sublingual, inhalation, intranasal, transdermal, topical, transmucosal, intra-tumoral, intra-synovial and rectal administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to human beings. In an embodiment, a pharmaceutical composition is formulated in accordance with routine procedures for subcutaneous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocamne to ease pain at the site of the injection.
Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a subject, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil in water emulsions, or a water in oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a subject; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a subject.
The composition, shape, and type of dosage forms provided herein will typically vary depending on their use. For example, a dosage form used in the initial treatment of viral infection may contain larger amounts of one or more of the active ingredients it comprises than a dosage form used in the maintenance treatment of the same infection. Similarly, a parenteral dosage form may contain smaller amounts of one or more of the active ingredients it comprises than an oral dosage form used to treat the same disease or disorder. These and other ways in which specific dosage forms encompassed herein will vary from one another will be readily apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa. (2000).
Generally, the ingredients of compositions are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
Typical dosage forms comprise a compound provided herein, or a pharmaceutically acceptable salt, solvate or hydrate thereof lie within the range of from about 0.1 mg to about 1000 mg per day, given as a single once-a-day dose in the morning or as divided doses throughout the day taken with food. Particular dosage forms can have about 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 100, 200, 250, 500 or 1000 mg of the active compound.
Oral Dosage Forms
Pharmaceutical compositions that are suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa. (2000).
In certain embodiments, the oral dosage forms are solid and prepared under anhydrous conditions with anhydrous ingredients, as described in detail herein. However, the scope of the compositions provided herein extends beyond anhydrous, solid oral dosage forms. As such, further forms are described herein.
Typical oral dosage forms are prepared by combining the active ingredient(s) in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, micro crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.
Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid excipients are employed. If desired, tablets can be coated by standard aqueous or non-aqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.
For example, a tablet can be prepared by compression or molding. Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
Examples of excipients that can be used in oral dosage forms include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.
Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre gelatinized starch, and mixtures thereof. The binder or filler in pharmaceutical compositions is typically present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form.
Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL PH 101, AVICEL PH 103 AVICEL RC 581, AVICEL PH 105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. A specific binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC 581. Suitable anhydrous or low moisture excipients or additives include AVICEL PH 103™ and Starch 1500 LM.
Disintegrants are used in the compositions to provide tablets that disintegrate when exposed to an aqueous environment. Tablets that contain too much disintegrant may disintegrate in storage, while those that contain too little may not disintegrate at a desired rate or under the desired conditions. Thus, a sufficient amount of disintegrant that is neither too much nor too little to detrimentally alter the release of the active ingredients should be used to form solid oral dosage forms. The amount of disintegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art. Typical pharmaceutical compositions comprise from about 0.5 to about 15 weight percent of disintegrant, specifically from about 1 to about 5 weight percent of disintcgrant.
Disintegrants that can be used in pharmaceutical compositions and dosage forms include, but arc not limited to, agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, pre gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.
Lubricants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional lubricants include, for example, a syloid silica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore, Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co. of Plano, Tex.), CAB O SIL (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, Mass.), and mixtures thereof. If used at all, lubricants are typically used in an amount of less than about 1 weight percent of the pharmaceutical compositions or dosage forms into which they are incorporated.
Delayed Release Dosage Forms
Active ingredients such as the compounds provided herein can be administered by controlled release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; and 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480; 5,733,566; 5,739,108; 5,891,474; 5,922,356; 5,972,891; 5,980,945; 5,993,855; 6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363; 6,264,970; 6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358; and 6,699,500; each of which is incorporated herein by reference in its entirety. Such dosage forms can be used to provide slow or controlled release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the active ingredients provided herein. Thus encompassed herein are single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled release.
All controlled release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of an optimally designed controlled release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled release formulations include extended activity of the drug, reduced dosage frequency, and increased subject compliance. In addition, controlled release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and can thus affect the occurrence of side (e.g., adverse) effects.
Most controlled release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, temperature, enzymes, water, or other physiological conditions or compounds.
In certain embodiments, the drug may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In certain embodiments, a pump may be used (see, Sefton, CRC Crit. Ref Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in a subject at an appropriate site determined by a practitioner of skill, i.e., thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)). The active ingredient can be dispersed in a solid inner matrix, e.g., polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethyleneterephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinylalcohol and cross-linked partially hydrolyzed polyvinyl acetate, that is surrounded by an outer polymeric membrane, e.g., polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetate copolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride, vinylchloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble in body fluids. The active ingredient then diffuses through the outer polymeric membrane in a release rate controlling step. The percentage of active ingredient in such parenteral compositions is highly dependent on the specific nature thereof, as well as the needs of the subject.
Parenteral Dosage Forms
In certain embodiments, provided are parenteral dosage forms. Parenteral dosage forms can be administered to subjects by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Because their administration typically bypasses subjects' natural defenses against contaminants, parenteral dosage forms are typically, sterile or capable of being sterilized prior to administration to a subject. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.
Suitable vehicles that can be used to provide parenteral dosage forms are well known to those skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
Compounds that increase the solubility of one or more of the active ingredients disclosed herein can also be incorporated into the parenteral dosage forms.
Transdermal, Topical & Mucosal Dosage Forms
Also provided are transdermal, topical, and mucosal dosage forms. Transdermal, topical, and mucosal dosage forms include, but are not limited to, ophthalmic solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions, or other forms known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences, 16th, 18th and 20th eds., Mack Publishing, Easton Pa. (1980, 1990 & 2000); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia (1985). Dosage forms suitable for treating mucosal tissues within the oral cavity can be formulated as mouthwashes or as oral gels. Further, transdermal dosage forms include “reservoir type” or “matrix type” patches, which can be applied to the skin and worn for a specific period of time to permit the penetration of a desired amount of active ingredients.
Suitable excipients (e.g., carriers and diluents) and other materials that can be used to provide transdermal, topical, and mucosal dosage forms encompassed herein are well known to those skilled in the pharmaceutical arts, and depend on the particular tissue to which a given pharmaceutical composition or dosage form will be applied. With that fact in mind, typical excipients include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3 diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof to form lotions, tinctures, creams, emulsions, gels or ointments, which are nontoxic and pharmaceutically acceptable. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art. See, e.g., Remington's Pharmaceutical Sciences, 16th, 18th and 20th eds., Mack Publishing, Easton Pa. (1980, 1990 & 2000).
Depending on the specific tissue to be treated, additional components may be used prior to, in conjunction with, or subsequent to treatment with active ingredients provided. For example, penetration enhancers can be used to assist in delivering the active ingredients to the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols such as ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; and various water soluble or insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate).
The pH of a pharmaceutical composition or dosage form, or of the tissue to which the pharmaceutical composition or dosage form is applied, may also be adjusted to improve delivery of one or more active ingredients. Similarly, the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery. Compounds such as stearates can also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of one or more active ingredients so as to improve delivery. In this regard, stearates can serve as a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery enhancing or penetration enhancing agent. Different salts, hydrates or solvates of the active ingredients can be used to further adjust the properties of the resulting composition.
Dosage and Unit Dosage Forms
In human therapeutics, the doctor will determine the posology which he considers most appropriate according to a preventive or curative treatment and according to the age, weight, stage of the infection and other factors specific to the subject to be treated. In certain embodiments, doses are from about 1 to about 1000 mg per day for an adult, or from about 5 to about 250 mg per day or from about 10 to 50 mg per day for an adult. In certain embodiments, doses are from about 5 to about 400 mg per day or 25 to 200 mg per day per adult. In certain embodiments, dose rates of from about 50 to about 500 mg per day are also contemplated.
In further aspects, provided are methods of treating or preventing an HCV infection in a subject by administering, to a subject in need thereof, an effective amount of a compound provided herein, or a pharmaceutically acceptable salt thereof. The amount of the compound or composition which will be effective in the prevention or treatment of a disorder or one or more symptoms thereof will vary with the nature and severity of the disease or condition, and the route by which the active ingredient is administered. The frequency and dosage will also vary according to factors specific for each subject depending on the specific therapy (e.g., therapeutic or prophylactic agents) administered, the severity of the disorder, disease, or condition, the route of administration, as well as age, body, weight, response, and the past medical history of the subject. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
In certain embodiments, exemplary doses of a composition include milligram or microgram amounts of the active compound per kilogram of subject or sample weight (e.g., about 10 micrograms per kilogram to about 50 milligrams per kilogram, about 100 micrograms per kilogram to about 25 milligrams per kilogram, or about 100 microgram per kilogram to about 10 milligrams per kilogram). For compositions provided herein, in certain embodiments, the dosage administered to a subject is 0.140 mg/kg to 3 mg/kg of the subject's body weight, based on weight of the active compound. In certain embodiments, the dosage administered to a subject is between 0.20 mg/kg and 2.00 mg/kg, or between 0.30 mg/kg and 1.50 mg/kg of the subject's body weight.
In certain embodiments, the recommended daily dose range of a composition provided herein for the conditions described herein lie within the range of from about 0.1 mg to about 1000 mg per day, given as a single once-a-day dose or as divided doses throughout a day. In certain embodiments, the daily dose is administered twice daily in equally divided doses. In certain embodiments, a daily dose range should be from about 10 mg to about 200 mg per day, in other embodiments, between about 10 mg and about 150 mg per day, in further embodiments, between about 25 and about 100 mg per day. It may be necessary to use dosages of the active ingredient outside the ranges disclosed herein in some cases, as will be apparent to those of ordinary skill in the art. Furthermore, it is noted that the clinician or treating physician will know how and when to interrupt, adjust, or terminate therapy in conjunction with subject response.
Different therapeutically effective amounts may be applicable for different diseases and conditions, as will be readily known by those of ordinary skill in the art. Similarly, amounts sufficient to prevent, manage, treat or ameliorate such disorders, but insufficient to cause, or sufficient to reduce, adverse effects associated with the composition provided herein are also encompassed by the herein described dosage amounts and dose frequency schedules. Further, when a subject is administered multiple dosages of a composition provided herein, not all of the dosages need be the same. For example, the dosage administered to the subject may be increased to improve the prophylactic or therapeutic effect of the composition or it may be decreased to reduce one or more side effects that a particular subject is experiencing.
In certain embodiment, the dosage of the composition provided herein, based on weight of the active compound, administered to prevent, treat, manage, or ameliorate a disorder, or one or more symptoms thereof in a subject is 0.1 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 10 mg/kg, or 15 mg/kg or more of a subject's body weight. In another embodiment, the dosage of the composition or a composition provided herein administered to prevent, treat, manage, or ameliorate a disorder, or one or more symptoms thereof in a subject is a unit dose of 0.1 mg to 200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.
In certain embodiments, treatment or prevention can be initiated with one or more loading doses of a compound or composition provided herein followed by one or more maintenance doses. In such embodiments, the loading dose can be, for instance, about 60 to about 400 mg per day, or about 100 to about 200 mg per day for one day to five weeks. The loading dose can be followed by one or more maintenance doses. In certain embodiments, each maintenance does is, independently, about from about 10 mg to about 200 mg per day, between about 25 mg and about 150 mg per day, or between about 25 and about 80 mg per day. Maintenance doses can be administered daily and can be administered as single doses, or as divided doses.
In certain embodiments, a dose of a compound or composition provided herein can be administered to achieve a steady-state concentration of the active ingredient in blood or serum of the subject. The steady-state concentration can be determined by measurement according to techniques available to those of skill or can be based on the physical characteristics of the subject such as height, weight and age. In certain embodiments, a sufficient amount of a compound or composition provided herein is administered to achieve a steady-state concentration in blood or serum of the subject of from about 300 to about 4000 ng/mL, from about 400 to about 1600 ng/mL, or from about 600 to about 1200 ng/mL. In some embodiments, loading doses can be administered to achieve steady-state blood or serum concentrations of about 1200 to about 8000 ng/mL, or about 2000 to about 4000 ng/mL for one to five days. In certain embodiments, maintenance doses can be administered to achieve a steady-state concentration in blood or serum of the subject of from about 300 to about 4000 ng/mL, from about 400 to about 1600 ng/mL, or from about 600 to about 1200 ng/mL.
In certain embodiments, administration of the same composition may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months. In other embodiments, administration of the same prophylactic or therapeutic agent may be repeated and the administration may be separated by at least at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.
In certain aspects, provided herein are unit dosages comprising a compound, or a pharmaceutically acceptable salt thereof, in a form suitable for administration. Such forms are described in detail herein. In certain embodiments, the unit dosage comprises 1 to 1000 mg, 5 to 250 mg or 10 to 50 mg active ingredient. In particular embodiments, the unit dosages comprise about 1, 5, 10, 25, 50, 100, 125, 250, 500 or 1000 mg active ingredient. Such unit dosages can be prepared according to techniques familiar to those of skill in the art.
The dosages of the second agents are to be used in the combination therapies provided herein. In certain embodiments, dosages lower than those which have been or are currently being used to prevent or treat HCV infection are used in the combination therapies provided herein. The recommended dosages of second agents can be obtained from the knowledge of those of skill. For those second agents that are approved for clinical use, recommended dosages are described in, for example, Hardman et al., eds., 1996, Goodman & Gilman's The Pharmacological Basis Of Basis Of Therapeutics 9th Ed, Mc-Graw-Hill, New York; Physician's Desk Reference (PDR) 57th Ed., 2003, Medical Economics Co., Inc., Montvale, N.J., which are incorporated herein by reference in its entirety.
In various embodiments, the therapies (e.g., a compound provided herein and the second agent) are administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart. In various embodiments, the therapies are administered no more than 24 hours apart or no more than 48 hours apart. In certain embodiments, two or more therapies are administered within the same patient visit. In other embodiments, the compound provided herein and the second agent are administered concurrently.
In other embodiments, the compound provided herein and the second agent are administered at about 2 to 4 days apart, at about 4 to 6 days apart, at about 1 week part, at about 1 to 2 weeks apart, or more than 2 weeks apart.
In certain embodiments, administration of the same agent may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months. In other embodiments, administration of the same agent may be repeated and the administration may be separated by at least at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.
In certain embodiments, a compound provided herein and a second agent are administered to a patient, for example, a mammal, such as a human, in a sequence and within a time interval such that the compound provided herein can act together with the other agent to provide an increased benefit than if they were administered otherwise. For example, the second active agent can be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect. In certain embodiments, the compound provided herein and the second active agent exert their effect at times which overlap. Each second active agent can be administered separately, in any appropriate form and by any suitable route. In other embodiments, the compound provided herein is administered before, concurrently or after administration of the second active agent.
In certain embodiments, the compound provided herein and the second agent are cyclically administered to a patient. Cycling therapy involves the administration of a first agent (e.g., a first prophylactic or therapeutic agents) for a period of time, followed by the administration of a second agent and/or third agent (e.g., a second and/or third prophylactic or therapeutic agents) for a period of time and repeating this sequential administration. Cycling therapy can reduce the development of resistance to one or more of the therapies, avoid or reduce the side effects of one of the therapies, and/or improve the efficacy of the treatment.
In certain embodiments, the compound provided herein and the second active agent are administered in a cycle of less than about 3 weeks, about once every two weeks, about once every 10 days or about once every week. One cycle can comprise the administration of a compound provided herein and the second agent by infusion over about 90 minutes every cycle, about 1 hour every cycle, about 45 minutes every cycle. Each cycle can comprise at least 1 week of rest, at least 2 weeks of rest, at least 3 weeks of rest. The number of cycles administered is from about 1 to about 12 cycles, more typically from about 2 to about 10 cycles, and more typically from about 2 to about 8 cycles.
In other embodiments, courses of treatment are administered concurrently to a patient, i.e., individual doses of the second agent are administered separately yet within a time interval such that the compound provided herein can work together with the second active agent. For example, one component can be administered once per week in combination with the other components that can be administered once every two weeks or once every three weeks. In other words, the dosing regimens are carried out concurrently even if the therapeutics are not administered simultaneously or during the same day.
The second agent can act additively or synergistically with the compound provided herein. In certain embodiments, the compound provided herein is administered concurrently with one or more second agents in the same pharmaceutical composition. In another embodiment, a compound provided herein is administered concurrently with one or more second agents in separate pharmaceutical compositions. In still another embodiment, a compound provided herein is administered prior to or subsequent to administration of a second agent. Also contemplated are administration of a compound provided herein and a second agent by the same or different routes of administration, e.g., oral and parenteral. In certain embodiments, when the compound provided herein is administered concurrently with a second agent that potentially produces adverse side effects including, but not limited to, toxicity, the second active agent can advantageously be administered at a dose that falls below the threshold that the adverse side effect is elicited.
Kits
Also provided are kits for use in methods of treatment of a liver disorder such as HCV infections. The kits can include a compound or composition provided herein, a second agent or composition, and instructions providing information to a health care provider regarding usage for treating the disorder. Instructions may be provided in printed form or in the form of an electronic medium such as a floppy disc, CD, or DVD, or in the form of a website address where such instructions may be obtained. A unit dose of a compound or composition provided herein, or a second agent or composition, can include a dosage such that when administered to a subject, a therapeutically or prophylactically effective plasma level of the compound or composition can be maintained in the subject for at least 1 days. In some embodiments, a compound or composition can be included as a sterile aqueous pharmaceutical composition or dry powder (e.g., lyophilized) composition.
In some embodiments, suitable packaging is provided. As used herein, “packaging” includes a solid matrix or material customarily used in a system and capable of holding within fixed limits a compound provided herein and/or a second agent suitable for administration to a subject. Such materials include glass and plastic (e.g., polyethylene, polypropylene, and polycarbonate) bottles, vials, paper, plastic, and plastic-foil laminated envelopes and the like. If e-beam sterilization techniques are employed, the packaging should have sufficiently low density to permit sterilization of the contents.
Methods of Use
Provided herein is a method for inhibiting replication of a virus in a host, which comprises contacting the host with a therapeutically effective amount of a 4′-OR nucleoside disclosed herein, e.g., a 4′-OR nucleoside compound of any of Formulae 1001, I-XX, 2a-2e, 5a2, 5c, 6a-8a, 6b, 7b, 113, 114, 20, 21, 23, 24, 13a, 13ai, 13aii, 14a, 14ai, 14aii, 16a, 122, 122i, 123, 123i, 123ii, 131-136, 9a-9g, 208-214, 220, 222, 223, 224, 230, 233, 301-314, 17a, 402-414, 18a, 502-514, 520, 530 and 601-614, or a pharmaceutically acceptable salt, or active metabolite thereof.
Provided herein is a method for inhibiting replication of a virus in a cell, which comprises contacting the cell with a therapeutically effective amount of a 4′-OR nuclcoside compound disclosed herein, e.g., a 4′-OR nucleoside compound of any of Formulae 1001, I-XX, 2a-2e, 5a2, 5c, 6a-8a, 6b, 7b, 113, 114, 20, 21, 23, 24, 13a, 13ai, 13aii, 14a, 14ai, 14aii, 16a, 122, 122i, 123, 123i, 123ii, 131-136, 9a-9g, 208-214, 220, 222, 223, 224, 230, 233, 301-314, 17a, 402-414, 18a, 502-514, 520, 530 and 601-614, or a pharmaceutically acceptable salt, or active metabolite thereof.
Provided herein is a method for inhibiting replication of a virus, which comprises contacting the virus with a therapeutically effective amount of a 4′-OR nucleoside compound disclosed herein, e.g., a 4′-OR nucleoside compound of any of Formulae 1001, I-XX, 2a-2e, 5a2, 5c, 6a-8a, 6b, 7b, 113, 114, 20, 21, 23, 24, 13a, 13ai, 13aii, 14a, 14ai, 14aii, 16a, 122, 122i, 123, 123i, 123ii, 131-136, 9a-9g, 208-214, 220, 222, 223, 224, 230, 233, 301-314, 17a, 402-414, 18a, 502-514, 520, 530 and 601-614, or a pharmaceutically acceptable salt, or active metabolite thereof.
Provided herein is a method for inhibiting the activity of a polymerase, which comprises contacting the polymerase with a 4′-OR nucleoside triphosphate compound disclosed herein, e.g., a 4′-OR nucleoside triphosphate compound of any of Formulae 1001, I-XX, 9a-9g, 208-214, 220, 222, 223, 224, 230, 233, 520, 530 or a pharmaceutically acceptable salt, or active metabolite thereof.
In certain embodiments, provided herein are methods for the treatment and/or prophylaxis of a host infected with Flaviviridae that includes the administration of an effective amount of a 4′-OR nucleoside compound disclosed herein, e.g., a 4′-OR nucleoside compound of any of Formulae 1001, I-XX, 2a-2e, 5a2, 5c, 6a-8a, 6b, 7b, 113, 114, 20, 21, 23, 24, 13a, 13ai, 13aii, 14a, 14ai, 14aii, 16a, 122, 122i, 123, 123i, 123ii, 131-136, 9a-9g, 208-214, 220, 222, 223, 224, 230, 233, 301-314, 17a, 402-414, 18a, 502-514, 520, 530 and 601-614, or a pharmaceutically acceptable salt, or active metabolite thereof.
In certain embodiments, provided herein are methods for treating an HCV infection in a subject. In certain embodiments, the methods encompass the step of administering to the subject in need thereof an amount of a compound effective for the treatment or prevention of an HCV infection in combination with a second agent effective for the treatment or prevention of the infection. The compound can be any compound as described herein, and the second agent can be any second agent described in the art or herein. In certain embodiments, the compound is in the form of a pharmaceutical composition or dosage form, as described elsewhere herein.
In certain embodiments, provided herein are methods for the treatment and/or prophylaxis of a host infected with Flaviviridae that includes the administration of an effective amount of a compounds provided herein, or a pharmaceutically acceptable salt thereof. In certain embodiments, provided herein are methods for treating an HCV infection in a subject. In certain embodiments, the methods encompass the step of administering to the subject in need thereof an amount of a compound effective for the treatment or prevention of an HCV infection in combination with a second agent effective for the treatment or prevention of the infection. The compound can be any compound as described herein, and the second agent can be any second agent described in the art or herein. In certain embodiments, the compound is in the form of a pharmaceutical composition or dosage form, as described elsewhere herein.
Flaviviridae which can be treated are, e.g., discussed generally in Fields Virology, Fifth Ed., Editors: Knipe, D. M., and Howley, P. M., Lippincott Williams & Wilkins Publishers, Philadelphia, Pa., Chapters 33-35, 2006. In a particular embodiment of the invention, the Flaviviridae is HCV. In an alternate embodiment, the Flaviviridae is a flavivirus or pestivirus. In certain embodiments, the Flaviviridae can be from any class of Flaviviridae. In certain embodiments, the Flaviviridae is a mammalian tick-borne virus. In certain embodiments, the Flaviviridae is a seabird tick-borne virus. In certain embodiments, the Flaviviridae is a mosquito-borne virus. In certain embodiments, the Flaviviridae is an Aroa virus. In certain embodiments, the Flaviviridae is a Dengue virus. In certain embodiments, the Flaviviridae is a Japanese encephalitis virus. In certain embodiments, the Flaviviridae is a Kokobera virus. In certain embodiments, the Flaviviridae is a Ntaya virus. In certain embodiments, the Flaviviridae is a Spondweni virus. In certain embodiments, the Flaviviridae is a Yellow fever virus. In certain embodiments, the Flaviviridae is a Entebbe virus. In certain embodiments, the Flaviviridae is a Modoc virus. In certain embodiments, the Flaviviridae is a Rio Bravo virus.
Specific flaviviruses which can be treated include, without limitation: Absettarov, Aedes, Alfuy, Alkhurma, Apoi, Aroa, Bagaza, Banzi, Bukalasa bat, Bouboui, Bussuquara, Cacipacore, Calbertado, Carey Island, Cell fusing agent, Cowbone Ridge, Culex, Dakar bat, Dengue 1, Dengue 2, Dengue 3, Dengue 4, Edge Hill, Entebbe bat, Gadgets Gully, Hanzalova, Hypr, Ilheus, Israel turkey meningoencephalitis, Japanese encephalitis, Jugra, Jutiapa, Kadam, Kamiti River, Karshi, Kedougou, Kokobera, Koutango, Kumlinge, Kunjin, Kyasanur Forest disease, Langat, Louping ill, Meaban, Modoc, Montana myotis leukoencephalitis, Murray valley encephalitis, Nakiwogo, Naranjal, Negishi, Ntaya, Omsk hemorrhagic fever, Phnom-Penh bat, Powassan, Quang Binh, Rio Bravo, Rocio, Royal Farm, Russian spring-summer encephalitis, Saboya, St. Louis encephalitis, Sal Vieja, San Perlita, Saumarez Reef, Sepik, Sokuluk, Spondweni, Stratford, Tembusu, Tick-borne encephalitis, Turkish sheep encephalitis, Tyuleniy, Uganda S, Usutu, Wesselsbron, West Nile, Yaounde, Yellow fever, Yokose, and Zika.
Pestiviruses which can be treated are discussed generally in Fields Virology, Fifth Ed., Editors: Knipe, D. M., and Howley, P. M., Lippincott Williams & Wilkins Publishers, Philadelphia, Pa., Chapters 33-35, 2006. Specific pestiviruses which can be treated include, without limitation: bovine viral diarrhea virus (“BVDV”), classical swine fever virus (“CSFV,” also called hog cholera virus), and border disease virus (“BDV”).
In certain embodiments, the subject can be any subject infected with, or at risk for infection with, HCV. Infection or risk for infection can be determined according to any technique deemed suitable by the practitioner of skill in the art. In certain embodiments, subjects are humans infected with HCV.
In certain embodiments, the subject has never received therapy or prophylaxis for an HCV infection. In further embodiments, the subject has previously received therapy or prophylaxis for an HCV infection. For instance, in certain embodiments, the subject has not responded to an HCV therapy. For example, under current interferon therapy, up to 50% or more HCV subjects do not respond to therapy. In certain embodiments, the subject can be a subject that received therapy but continued to suffer from viral infection or one or more symptoms thereof. In certain embodiments, the subject can be a subject that received therapy but failed to achieve a sustained virologic response. In certain embodiments, the subject has received therapy for an HCV infection but has failed to show, for example, a 2 log10 decline in HCV RNA levels after 12 weeks of therapy. It is believed that subjects who have not shown more than 2 log10 reduction in serum HCV RNA after 12 weeks of therapy have a 97-100% chance of not responding.
In certain embodiments, the subject is a subject that discontinued an HCV therapy because of one or more adverse events associated with the therapy. In certain embodiments, the subject is a subject where current therapy is not indicated. For instance, certain therapies for HCV are associated with neuropsychiatric events. Interferon (IFN)-alfa plus ribavirin is associated with a high rate of depression. Depressive symptoms have been linked to a worse outcome in a number of medical disorders. Life-threatening or fatal neuropsychiatric events, including suicide, suicidal and homicidal ideation, depression, relapse of drug addiction/overdose, and aggressive behavior have occurred in subjects with and without a previous psychiatric disorder during HCV therapy. Interferon-induced depression is a limitation for the treatment of chronic hepatitis C, especially for subjects with psychiatric disorders. Psychiatric side effects are common with interferon therapy and responsible for about 10% to 20% of discontinuations of current therapy for HCV infection.
Accordingly, provided are methods of treating or preventing an HCV infection in subjects where the risk of neuropsychiatric events, such as depression, contraindicates treatment with current HCV therapy. In certain embodiments, provided are methods of treating or preventing HCV infection in subjects where a neuropsychiatric event, such as depression, or risk of such indicates discontinuation of treatment with current HCV therapy. Further provided are methods of treating or preventing HCV infection in subjects where a neuropsychiatric event, such as depression, or risk of such indicates dose reduction of current HCV therapy.
Current therapy is also contraindicated in subjects that are hypersensitive to interferon or ribavirin, or both, or any other component of a pharmaceutical product for administration of interferon or ribavirin. Current therapy is not indicated in subjects with hemoglobinopathies (e.g., thalassemia major, sickle-cell anemia) and other subjects at risk from the hematologic side effects of current therapy. Common hematologic side effects include bone marrow suppression, neutropenia and thrombocytopenia. Furthermore, ribavirin is toxic to red blood cells and is associated with hemolysis. Accordingly, in certain embodiments, provided are methods of treating or preventing HCV infection in subjects hypersensitive to interferon or ribavirin, or both, subjects with a hemoglobinopathy, for instance thalassemia major subjects and sickle-cell anemia subjects, and other subjects at risk from the hematologic side effects of current therapy.
In certain embodiments, the subject has received an HCV therapy and discontinued that therapy prior to administration of a method provided herein. In further embodiments, the subject has received therapy and continues to receive that therapy along with administration of a method provided herein. The methods can be co-administered with other therapy for HBC and/or HCV according to the judgment of one of skill in the art. In certain embodiments, the methods or compositions provided herein can be co-administered with a reduced dose of the other therapy for HBC and/or HCV.
In certain embodiments, provided are methods of treating a subject that is refractory to treatment with interferon. For instance, in some embodiments, the subject can be a subject that has failed to respond to treatment with one or more agents including interferon, interferon α, pegylated interferon α, interferon plus ribavirin, interferon α plus ribavirin, and pegylated interferon α plus ribavirin. In some embodiments, the subject can be a subject that has responded poorly to treatment with one or more agents including interferon, interferon α, pegylated interferon α, interferon plus ribavirin, interferon α plus ribavirin, and pegylated interferon α plus ribavirin. A pro-drug form of ribavirin, such as taribavirin, may also be used.
In certain embodiments, the subject has, or is at risk for, co-infection of HCV with HIV. For instance, in the United States, 30% of HIV subjects are co-infected with HCV and evidence indicates that people infected with HIV have a much more rapid course of their hepatitis C infection. Maier and Wu, 2002, World J Gastroenterol 8:577-57. The methods provided herein can be used to treat or prevent HCV infection in such subjects. It is believed that elimination of HCV in these subjects will lower mortality due to end-stage liver disease. Indeed, the risk of progressive liver disease is higher in subjects with severe AIDS-defining immunodeficiency than in those without. See, e.g., Lesens et al., 1999, J Infect Dis 179:1254-1258. In certain embodiments, compounds provided herein have been shown to suppress HIV in HIV subjects. Thus, in certain embodiments, provided are methods of treating or preventing HIV infection and HCV infection in subjects in need thereof.
In certain embodiments, the compounds or compositions are administered to a subject following liver transplant. Hepatitis C is a leading cause of liver transplantation in the U.S., and many subjects that undergo liver transplantation remain HCV positive following transplantation. In certain embodiments, provided are methods of treating such recurrent HCV subjects with a compound or composition provided herein. In certain embodiments, provided are methods of treating a subject before, during or following liver transplant to prevent recurrent HCV infection.
Assay Methods
Compounds can be assayed for HCV activity according to any assay known to those of skill in the art.
Further, compounds can be assayed for accumulation in liver cells of a subject according to any assay known to those of skill in the art. In certain embodiments, a compound can be administered to the subject, and a liver cell of the subject can be assayed for the compound or a derivative thereof, e.g. a nucleoside, nucleoside phosphate or nucleoside triphosphate derivative thereof.
In certain embodiments, a 4′-OR nucleoside compound is administered to cells, such as liver cells, in vivo or in vitro, and the nucleoside triphosphate levels delivered intracellularly are measured, to indicate delivery of the compound and triphosphorylation in the cell. The levels of intracellular nucleoside triphosphate can be measured using analytical techniques known in the art. Methods of detecting ddATP are described herein below by way of example, but other nucleoside triphosphates can be readily detected using the appropriate controls, calibration samples and assay techniques.
In certain embodiments, ddATP concentrations are measured in a sample by comparison to calibration standards made from control samples. The ddATP concentrations in a sample can be measured using an analytical method such as HPLC LC MS. In certain embodiments, a test sample is compared to a calibration curve created with known concentrations of ddATP to thereby obtain the concentration of that sample.
In certain embodiments, the samples are manipulated to remove impurities such as salts (Na+, K+, etc.) before analysis. In certain embodiments, the lower limit of quantitation is about ˜0.2 pmol/mL for hepatocyte cellular extracts particularly where reduced salt is present.
In certain embodiments, the method allows successfully measuring triphosphate nucleotides formed at levels of 1-10,000 pmol per million cells in e.g. cultured hepatocytes and HcpG2 cells.
Second Therapeutic Agents
In certain embodiments, the compounds and compositions provided herein are useful in methods of treatment of a liver disorder, that comprise further administration of a second agent effective for the treatment of the disorder, such as HCV infection in a subject in need thereof. The second agent can be any agent known to those of skill in the art to be effective for the treatment of the disorder, including those currently approved by the FDA.
In certain embodiments, a compound provided herein is administered in combination with one second agent. In further embodiments, a compound provided herein is administered in combination with two second agents. In still further embodiments, a compound provided herein is administered in combination with two or more second agents.
As used herein, the term “in combination” includes the use of more than one therapy (e.g., one or more prophylactic and/or therapeutic agents). The use of the term “in combination” does not restrict the order in which therapies (e.g., prophylactic and/or therapeutic agents) are administered to a subject with a disorder. A first therapy (e.g., a prophylactic or therapeutic agent such as a compound provided herein) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy (e.g., a prophylactic or therapeutic agent) to a subject with a disorder.
As used herein, the term “synergistic” includes a combination of a compound provided herein and another therapy (e.g., a prophylactic or therapeutic agent) which has been or is currently being used to prevent, manage or treat a disorder, which is more effective than the additive effects of the therapies. A synergistic effect of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) permits the use of lower dosages of one or more of the therapies and/or less frequent administration of said therapies to a subject with a disorder. The ability to utilize lower dosages of a therapy (e.g., a prophylactic or therapeutic agent) and/or to administer said therapy less frequently reduces the toxicity associated with the administration of said therapy to a subject without reducing the efficacy of said therapy in the prevention or treatment of a disorder). In addition, a synergistic effect can result in improved efficacy of agents in the prevention or treatment of a disorder. Finally, a synergistic effect of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) may avoid or reduce adverse or unwanted side effects associated with the use of either therapy alone.
The active compounds provided herein can be administered in combination or alternation with another therapeutic agent, in particular an anti-HCV agent. In combination therapy, effective dosages of two or more agents are administered together, whereas in alternation or sequential-step therapy, an effective dosage of each agent is administered serially or sequentially. The dosages given will depend on absorption, inactivation and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. In certain embodiments, an anti-HCV (or anti-pestivirus or anti-flavivirus) compound that exhibits an EC50 of 10-15 μM. In certain embodiments, less than 1-5 μM, is desirable.
It has been recognized that drug-resistant variants of flaviviruses, pestiviruses or HCV can emerge after prolonged treatment with an antiviral agent. Drug resistance most typically occurs by mutation of a gene that encodes for an enzyme used in viral replication. The efficacy of a drug against the viral infection can be prolonged, augmented, or restored by administering the compound in combination or alternation with a second, and perhaps third, antiviral compound that induces a different mutation from that caused by the principle drug. Alternatively, the pharmacokinetics, biodistribution or other parameter of the drug can be altered by such combination or alternation therapy. In general, combination therapy is typically preferred over alternation therapy because it induces multiple simultaneous stresses on the virus.
Any of the viral treatments described in the Background of the Invention can be used in combination or alternation with the compounds described in this specification. Non-limiting examples of second agents include:
HCV Protease inhibitors: Examples include Medivir HCV Protease Inhibitor TMC 435 (simeprevir, Medivir, Tibotec, Johnson & Johnson); MK-5172 (Merck), MK-7009 (Merck), RG7227 (ITMN-191) (Roche/Pharmasset/InterMune), boceprevir (SCH 503034) (Schering), SCH 446211 (Schering), narlaprevir SCH900518 (Schering/Merck), ABT-450 (Abbott/Enanta), ACH-1625 (Achillion), BI 201335 (faldaprevir, Boehringer Ingelheim), PHX1766 (Phenomix), VX-500 (Vertex) and telaprevir (VX-950) (Vertex). Further examples of protease inhibitors include substrate-based NS3 protease inhibitors (Attwood et al., Antiviral peptide derivatives, PCT WO 98/22496, 1998; Attwood et al., Antiviral Chemistry and Chemotherapy 1999, 10, 259-273; Attwood et al., Preparation and use of amino acid derivatives as anti-viral agents, German Patent Pub. DE 19914474; Tung et al., Inhibitors of serine proteases, particularly hepatitis C virus NS3 protease, PCT WO 98/17679), including alphaketoamides and hydrazinourcas, and inhibitors that terminate in an electrophile such as a boronic acid or phosphonate (Llinas-Brunet et al, Hepatitis C inhibitor peptide analogues, PCT WO 99/07734); Non-substrate-based NS3 protease inhibitors such as 2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al., Biochemical and Biophysical Research Communications, 1997, 238, 643-647; Sudo K. et al., Antiviral Chemistry and Chemotherapy, 1998, 9, 186), including RD3-4082 and RD3-4078, the former substituted on the amide with a 14 carbon chain and the latter processing a para-phenoxyphenyl group; and Sch 68631, a phenanthrenequinone, an HCV protease inhibitor (Chu M. et al., Tetrahedron Letters 37:7229-7232, 1996);
SCH 351633, isolated from the fungus Penicillium griseofulvum, was identified as a protease inhibitor (Chu M. et al., Bioorganic and Medicinal Chemistry Letters 9:1949-1952). Eglin c, isolated from leech, is a potent inhibitor of several serine proteases such as S. griseus proteases A and B, α-chymotrypsin, chymase and subtilisin. Qasim M. A. et al., Biochemistry 36:1598-1607, 1997;
U.S. patents disclosing protease inhibitors for the treatment of HCV include, for example, U.S. Pat. No. 6,004,933 to Spruce et al., which discloses a class of cysteine protease inhibitors for inhibiting HCV endopeptidase 2; U.S. Pat. No. 5,990,276 to Zhang et al., which discloses synthetic inhibitors of hepatitis C virus NS3 protease; U.S. Pat. No. 5,538,865 to Reyes et a; WO 02/008251 to Corvas International, Inc., and U.S. Pat. No. 7,169,760, US2005/176648, WO 02/08187 and WO 02/008256 to Schering Corporation. HCV inhibitor tripeptides are disclosed in U.S. Pat. Nos. 6,534,523, 6,410,531, and 6,420,380 to Boehringer Ingelheim and WO 02/060926 to Bristol Myers Squibb. Diaryl peptides as NS3 serine protease inhibitors of HCV are disclosed in WO 02/48172 and U.S. Pat. No. 6,911,428 to Schering Corporation. Imidazoleidinones as NS3 serine protease inhibitors of HCV are disclosed in WO 02/08198 and U.S. Pat. No. 6,838,475 to Schering Corporation and WO 02/48157 and U.S. Pat. No. 6,727,366 to Bristol Myers Squibb. WO 98/17679 and U.S. Pat. No. 6,265,380 to Vertex Pharmaceuticals and WO 02/48116 and U.S. Pat. No. 6,653,295 to Bristol Myers Squibb also disclose HCV protease inhibitors. Further examples of HCV serine protease inhibitors are provided in U.S. Pat. No. 6,872,805 (Bristol-Myers Squibb); WO 2006000085 (Boehringer Ingelheim); U.S. Pat. No. 7,208,600 (Vertex); US 2006/0046956 (Schering-Plough); WO 2007/001406 (Chiron); US 2005/0153877; WO 2006/119061 (Merck); WO 00/09543 (Boehringer Ingelheim), U.S. Pat. No. 6,323,180 (Boehringer Ingelheim) WO 03/064456 (Boehringer Ingelheim), U.S. Pat. No. 6,642,204 (Boehringer Ingelheim), WO 03/064416 (Boehringer Ingelheim), U.S. Pat. No. 7,091,184 (Boehringer Ingelheim), WO 03/053349 (Bristol-Myers Squibb), U.S. Pat. No. 6,867,185, WO 03/099316 (Bristol-Myers Squibb), U.S. Pat. No. 6,869,964, WO 03/099274 (Bristol-Myers Squibb), U.S. Pat. No. 6,995,174, WO 2004/032827 (Bristol-Myers Squibb), U.S. Pat. No. 7,041,698, WO 2004/043339 and U.S. Pat. No. 6,878,722 (Bristol-Myers Squibb);
Thiazolidine derivatives which show relevant inhibition in a reverse-phase HPLC assay with an NS3/4A fusion protein and NS5A/5B substrate (Sudo K. et al., Antiviral Research, 1996, 32, 9-18), especially compound RD-1-6250, possessing a fused cinnamoyl moiety substituted with a long alkyl chain, RD4 6205 and RD4 6193;
Thiazolidines and benzanilides identified in Kakiuchi N. et al., J. EBS Letters 421, 217-220; Takeshita N. et al., Analytical Biochemistry, 1997, 247, 242-246;
A phenanthrenequinone possessing activity against protease in a SDS-PAGE and autoradiography assay isolated from the fermentation culture broth of Streptomyces sp., SCH 68631 (Chu M. et al., Tetrahedron Letters, 1996, 37, 7229-7232), and SCH 351633, isolated from the fungus Penicillium griseofulvum, which demonstrates activity in a scintillation proximity assay (Chu M. et al., Bioorganic and Medicinal Chemistry Letters 9, 1949-1952);
Helicase inhibitors (Diana G. D. et al., Compounds, compositions and methods for treatment of hepatitis C, U.S. Pat. No. 5,633,358; Diana G. D. et al., Piperidine derivatives, pharmaceutical compositions thereof and their use in the treatment of hepatitis C, PCT WO 97/36554);
HCV polymerase inhibitors, including nucleoside and non-nucleoside polymerase inhibitors, such as ribavirin, viramidine, clemizole, filibuvir (PF-00868554), HCV POL, NM 283 (valopicitabine), MK-0608, 7-Fluoro-MK-0608, MK-3281, IDX-375, ABT-072, ABT-333, ANA598, BI 207127, GS 9190, PSI-6130, R1626, PSI-6206, PSI-938, PSI-7851, GS-7977 (sofosbuvir, Pharmasset, Gilead), RG1479, RG7128, HCV-796 VCH-759 or VCH-916;
Gliotoxin (Ferrari R. et al., Journal of Virology, 1999, 73, 1649-1654), and the natural product cerulenin (Lohmann V. et al., Virology, 1998, 249, 108-118);
Interfering RNA (iRNA) based antivirals, including short interfering RNA (siRNA) based antivirals, such as Sirna-034 and others described in International Patent Publication Nos. WO/03/070750 and WO 2005/012525, and US Patent Publication No. US 2004/0209831;
HCV NS5A inhibitors, such as BMS-790052 (daclatasvir, Bristol-Myers Squibb), PPI-461 (Presidio Pharmaceuticals), PPI-1301 (Presidio Pharmaceuticals), IDX-719 (samatasvir, Idenix Pharmaceuticals), AZD7295 (Arrow Therapeutics, AstraZeneca), EDP-239 (Enanta), ACH-2928 (Achillion), ACH-3102 (Achillion), ABT-267 (Abbott), or GS-5885 (Gilead);
Antisense phosphorothioate oligodeoxynucleotides (S-ODN) complementary to sequence stretches in the 5′ non-coding region (NCR) of the virus (Alt M. et al., Hepatology, 1995, 22, 707-717), or nucleotides 326-348 comprising the 3′ end of the NCR and nucleotides 371-388 located in the core coding region of the HCV RNA (Alt M. et al., Archives of Virology, 1997, 142, 589-599; Galderisi U. et al., Journal of Cellular Physiology, 1999, 181, 251-257);
Inhibitors of IRES-dependent translation (Ikeda N et al., Agent for the prevention and treatment of hepatitis C, Japanese Patent Pub. JP-08268890; Kai Y. et al., Prevention and treatment of viral diseases, Japanese Patent Pub. JP-10101591);
HCV entry inhibitors, such as celgosivir (MK-3253) (MIGENIX Inc.), SP-30 (Samaritan Pharmaceuticals), ITX4520 (iTherX), ITX5061 (iTherX), PRO-206 (Progenics Pharmaceuticals) and other entry inhibitors by Progenies Pharmaceuticals, e.g., as disclosed in U.S. Patent Publication No. 2006/0198855;
Ribozymes, such as nuclease-resistant ribozymes (Maccjak, D. J. et al., Hepatology 1999, 30, abstract 995) and those disclosed in U.S. Pat. No. 6,043,077 to Barber et al., and U.S. Pat. Nos. 5,869,253 and 5,610,054 to Draper et al.; and
Nucleoside analogs developed for the treatment of Flaviviridae infections.
In certain embodiments, the compounds provided herein can be administered in combination with any of the compounds described by Idenix Pharmaceuticals in International Publication Nos. WO 01/90121, WO 01/92282, WO 2004/003000, 2004/002422 and WO 2004/002999.
Other patent applications disclosing the use of certain nucleoside analogs that can be used as second agents to treat hepatitis C virus include: PCT/CA00/01316 (WO 01/32153; filed Nov. 3, 2000) and PCT/CA01/00197 (WO 01/60315; filed Feb. 19, 2001) filed by BioChem Pharma, Inc. (now Shire Biochem, Inc.); PCT/US02/01531 (WO 02/057425; filed Jan. 18, 2002); PCT/US02/03086 (WO 02/057287; filed Jan. 18, 2002); U.S. Pat. Nos. 7,202,224; 7,125,855; 7,105,499 and 6,777,395 by Merck & Co., Inc.; PCT/EP01/09633 (WO 02/18404; published Aug. 21, 2001); US 2006/0040890; 2005/0038240; 2004/0121980; U.S. Pat. Nos. 6,846,810; 6,784,166 and 6,660,721 by Roche; PCT Publication Nos. WO 01/79246 (filed Apr. 13, 2001), WO 02/32920 (filed Oct. 18, 2001) and WO 02/48165; US 2005/0009737; US 2005/0009737; U.S. Pat. Nos. 7,094,770 and 6,927,291 by Pharmasset, Ltd.
Further compounds that can be used as second agents to treat hepatitis C virus are disclosed in PCT Publication No. WO 99/43691 to Emory University, entitled “2′-Fluoronucleosides”. The use of certain 2′-fluoronucleosides to treat HCV is disclosed.
Other compounds that can be used as second agents include 1-amino-alkylcyclohexanes (U.S. Pat. No. 6,034,134 to Gold et al.), alkyl lipids (U.S. Pat. No. 5,922,757 to Chojkier et al.), vitamin E and other antioxidants (U.S. Pat. No. 5,922,757 to Chojkier et al.), squalene, amantadine, bile acids (U.S. Pat. No. 5,846,964 to Ozeki et al.), N-(phosphonoacetyl)-L-aspartic acid, (U.S. Pat. No. 5,830,905 to Diana et al.), benzenedicarboxamides (U.S. Pat. No. 5,633,388 to Diana et al.), polyadenylic acid derivatives (U.S. Pat. No. 5,496,546 to Wang et al.), 2′,3′-dideoxyinosine (U.S. Pat. No. 5,026,687 to Yarchoan et al.), benzimidazoles (U.S. Pat. No. 5,891,874 to Colacino et al.), plant extracts (U.S. Pat. No. 5,837,257 to Tsai et al., U.S. Pat. No. 5,725,859 to Omer et al., and U.S. Pat. No. 6,056,961), and piperidines (U.S. Pat. No. 5,830,905 to Diana et al.).
In certain embodiments, a compound of any of Formulae 1001, I-XX, 2a-2e, 5a2, 5c, 6a-8a, 6b, 7b, 113, 114, 20, 21, 23, 24, 13a, 13ai, 13aii, 14a, 14ai, 14aii, 16a, 122, 122i, 123, 123i, 123ii, 131-136, 9a-9g, 208-214, 220, 222, 223, 224, 230, 233, 301-314, 17a, 402-414, 18a, 502-514, 520, 530 and 601-614, or pharmaceutically acceptable salt thereof or a composition comprising a compound of any of Formulae 1001, I-XX, 2a-2c, 5a2, 5c, 6a-8a, 6b, 7b, 113, 114, 20, 21, 23, 24, 13a, 13ai, 13aii, 14a, 14ai, 14aii, 16a, 122, 122i, 123, 123i, 123ii, 131-136, 9a-9g, 208-214, 220, 222, 223, 224, 230, 233, 301-314, 17a, 402-414, 18a, 502-514, 520, 530 and 601-614 or a pharmaceutically acceptable salt thereof is administered in combination or alternation with a second anti-viral agent including an interferon, a nucleotide analogue, a polymerase inhibitor, an NS3 protease inhibitor, an NS5A inhibitor, an entry inhibitor, a non-nucleoside polymerase inhibitor, a cyclosporine immune inhibitor, an NS4A antagonist, an NS4B-RNA binding inhibitor, a locked nucleic acid mRNA inhibitor, a cyclophilin inhibitor, and combinations thereof.
Exemplary Second Therapeutic Agents for Treatment of HCV
In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with an anti-hepatitis C virus interferon, such as Intron A® (interferon alfa-2b) and; Roferon A® (Recombinant interferon alfa-2a), Infergen® (consensus interferon; interferon alfacon-1), PEG-Intron® (pegylated interferon alfa-2b), and Pegasys® (pegylated interferon alfa-2a). In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with ribavirin and in combination or alternation with an anti-hepatitis C virus interferon. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with ribavirin, in combination or alternation with an anti-hepatitis C virus interferon, and in combination or alternation with an anti-hepatitis C virus protease inhibitor. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with an anti-hepatitis C virus interferon and without ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with an anti-hepatitis C virus interferon, in combination or alternation with an anti-hepatitis C virus protease inhibitor, and without ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with ribavirin, in combination or alternation with an anti-hepatitis C virus protease inhibitor, and in combination or alternation with an anti-hepatitis C virus NS5A inhibitor. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with an anti-hepatitis C virus protease inhibitor, and in combination or alternation with an anti-hepatitis C virus NS5A inhibitor, and without ribavirin, and without an interferon.
In certain embodiments, the anti-hepatitis C virus interferon is infergen, IL-29 (PEG-Interferon lambda), R7025 (Maxy-alpha), Belerofon, Oral Interferon alpha, BLX-883 (Locteron), omega interferon, multiferon, medusa interferon, Albuferon or REBIF®.
In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with an anti-hepatitis C virus polymerase inhibitor, such as ribavirin, viramidine, HCV POL, NM 283 (valopicitabine), MK-0608, 7-Fluoro-MK-0608, PSI-6130, R1626, PSI-6206, PSI-938, R1479, HCV-796, VX-950 (Telaprevir, Vertex), GS 9190 NN (Gilead), GS 9256 (Gilead), PSI-7792 (Pharmasset), BI 207127 (BI), R7128 (Roche), GS-7977 (sofosbuvir, Pharmasset, Gilead), PSI-938 (Pharmasset), VX-222 (Vertex), ALS-2200 (Vertex), ALS-2158 (Vertex), MK-0608 (Merck), TMC649128 (Medivir), PF-868554 (Pfizer), PF-4878691 (Pfizer), ANA598 (Roche), VCH-759 (Vertex), IDX184 (Idenix), IDX375 (Idenix), A-837093 (Abbott), GS 9190 (Gilead), GSK625433 (GlaxoSmithKline), ABT-072 (Abbott), ABT-333 (Abbott), INX-189 (Inhibitex), or EDP-239 (Enanta).
In certain embodiments, the one or more compounds provided herein can be administered in combination with ribavarin and an anti-hepatitis C virus interferon, such as Intron A® (interferon alfa-2b) and Pegasys® (Peginterferon alfa-2a); Roferon A® (Recombinant interferon alfa-2a), Infergen® (consensus interferon; interferon alfacon-1), PEG-Intron® (pegylated interferon alfa-2b), Zalbin (albinterferon alfa-2b), omega interferon, pegylated interferon lambda, and Pegasys® (pegylated interferon alfa-2a).
In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with an HCV NS5A inhibitor, such as BMS-790052 (daclatasvir, Bristol-Myers Squibb), PPI-461 (Presidio Pharmaceuticals), PPI-1301 (Presidio Pharmaceuticals), IDX-719 (samatasvir, Idenix Pharmaceuticals), AZD7295 (Arrow Therapeutics, AstraZeneca), EDP-239 (Enanta), ACH-2928 (Achillion), ACH-3102 (Achillion), ABT-267 (Abbott), or GS-5885 (Gilead).
In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with an anti-hepatitis C virus protease inhibitor such as ITMN-191, SCH 503034 (boceprevir), VX950 (telaprevir), VX985, VX500, VX813, PHX1766, BMS-650032 (asunaprevir, Bristol-Myers Squibb), GS 9256, BI 201335 (faldaprevir, Boehringer Ingelheim), IDX320, R7227, MK-7009 (vaniprevir), TMC 435 (simeprevir, Medivir, Tibotcc, Johnson & Johnson), BMS-791325, ACH-1625, ACH-2684, ABT-450, AVL-181, or Medivir HCV Protease Inhibitor.
In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with an anti-hepatitis C virus vaccine, such as TG4040, PeviPRO™, CGI-5005, HCV/MF59, GV1001, IC41, GNI-103, GenPhar HCV vaccine, C-Vaxin, CSL123, Hepavaxx C, ChronVac-C® or INNO0101 (E1).
In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with an anti-hepatitis C virus monoclonal antibody, such as MBL-HCV1, AB68 or XTL-6865 (formerly HepX-C); or an anti-hepatitis C virus polyclonal antibody, such as cicavir.
In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with an anti-hepatitis C virus immunomodulator, such as Zadaxin® (thymalfasin), SCV-07, NOV-205 or Oglufanide.
In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with cyclophilin inhibitor, such as Enanta cyclophilin binder, SCY-635, or Debio-025.
In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with Nexavar, doxorubicin, PI-88, amantadine, JBK-122, VGX-410C, MX-3253 (Ceglosivir), Suvus (BIVN-401 or virostat), PF-03491390 (formerly IDN-6556), G126270, UT-231B, DEBIO-025, EMZ702, ACH-0137171, MitoQ, ANA975, AVI-4065, Bavituxinab (Tarvacin), Alinia (nitrazoxanide) or PYN17.
In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with telaprevir, boceprevir, simeprevir, interferon alphacon-1, interferon alpha-2a, interferon alpha-2b, pegylated interferon alpha 2a, pegylated interferon alpha 2b, ribavirin, or combinations thereof.
In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with a protease inhibitor. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with telaprevir. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with boceprevir. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with simeprevir.
In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with a protease inhibitor and in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with telaprevir and in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with boceprevir and in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with simeprevir and in combination or alternation with ribavirin.
In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with a protease inhibitor and not in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with telaprevir and not in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with boceprevir and not in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with simeprevir and not in combination or alternation with ribavirin.
In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with an interferon. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with interferon alphacon-1. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with interferon alpha-2a. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with interferon alpha-2b. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with pegylated interferon alpha 2a. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with pegylated interferon alpha 2b.
In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with an interferon and in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with interferon alfacon-land in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with interferon alpha-2a and in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with interferon alpha-2b and in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with pegylated interferon alpha 2a and in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with pegylated interferon alpha 2b and in combination or alternation with ribavirin.
In certain embodiments, one or more compounds can be administered in combination or alternation with one or more of the second agents provided herein and not in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with an interferon and not in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with interferon alfacon-1 and not in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with interferon alpha-2a and not in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with interferon alpha-2b and not in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with pegylated interferon alpha 2a and not in combination or alternation with ribavirin. In certain embodiments, one or more compounds provided herein can be administered in combination or alternation with pegylated interferon alpha 2b and not in combination or alternation with ribavirin.
In an embodiment, provided herein is a method for the treatment of a host infected with a hepatitis C virus, comprising the administration of an effective treatment amount of a compound or composition described herein. In certain embodiments, the host is a human. In some embodiments, the administration directs a substantial amount of the compound, or pharmaceutically acceptable salt or stereoisomer thereof, to a liver of the host. In an embodiment, the compound or composition is administered in combination or alternation with a second anti-viral agent including an interferon, a nucleotide analogue, a polymerase inhibitor, an NS3 protease inhibitor, an NS5A inhibitor, an entry inhibitor, a non-nucleoside polymerase inhibitor, a cyclosporine immune inhibitor, an NS4A antagonist, an NS4B-RNA binding inhibitor, a locked nucleic acid mRNA inhibitor, a cyclophilin inhibitor, and combinations thereof. In an embodiment, the second anti-viral agent is telaprevir, boceprevir, simeprevir, interferon alphacon-1, interferon alpha-2a, interferon alpha-2b, pegylated interferon alpha 2a, pegylated interferon alpha 2b, ribavirin, or combinations thereof. In an embodiment, the second anti-viral agent is telaprevir, boceprevir, simeprevir, interferon alphacon-1, interferon alpha-2a, interferon alpha-2b, pegylated interferon alpha 2a, pegylated interferon alpha 2b, or combinations thereof, and further wherein the administration is not in combination or alternation with ribavirin.
As used herein, the symbols and conventions used in these processes, schemes and examples, regardless of whether a particular abbreviation is specifically defined, are consistent with those used in the contemporary scientific literature, for example, the Journal of the American Chemical Society or the Journal of Biological Chemistry. Specifically, but without limitation, the following abbreviations may be used in the examples and throughout the specification: g (grams); mg (milligrams); mL (milliliters); μL (microliters); mM (millimolar); M (micromolar); Hz (Hertz); MHz (megahertz); mmol (millimoles); hr or hrs (hours); min (minutes); MS (mass spectrometry); ESI (electrospray ionization); TLC (thin layer chromatography); HPLC (high pressure liquid chromatography); THF (tetrahydrofuran); CDCl3 (deuterated chloroform); AcOH (acetic acid); DCM (dichloromethane); DMSO (dimethylsulfoxide); DMSO-d6 (deuterated dimethylsulfoxide); EtOAc (ethyl acetate); MeOH (methanol); and BOC (t-butyloxycarbonyl).
For all of the following examples, standard work-up and purification methods known to those skilled in the art can be utilized. Unless otherwise indicated, all temperatures are expressed in ° C. (degrees Centigrade). All reactions are conducted at room temperature unless otherwise noted. Synthetic methodologies illustrated herein are intended to exemplify the applicable chemistry through the use of specific examples and are not indicative of the scope of the disclosure.
Compounds 2a-2e, 5a2 and 5c were prepared according to the following scheme, Scheme 1.
Compound (1a):
Compound 1a was synthesized according to procedure described in J. Med. Chem., 2009, 52, 219-223 and in WO2005000864A1 Patent.
Compounds (1b) and (1c):
Compounds 1b and 1c were synthesized according to procedure described in J. Med. Chem., 2009, 52, 2971-2978.
Compound (1d):
Compound 1d was synthesized according to procedure described in J. Med. Chem., 2009, 52, 219-223.
Compound (1e)
Compound 1e was prepared according to the following scheme, Scheme 1′:
To a solution of Compound A1 (33.4 mmol) in anhydrous DMF (27 mL/mmol) was added methyltriphenoxyphosphonium iodide (33.4 mmol) under nitrogen at room temperature. The reaction mixture was stirred 1 h30 at room temperature. The reaction was monitored by LC/MS. Addition of methyltriphenoxyphosphonium iodide (33.4 mmol) and additional hour of stirring at room temperature were required for reaction completion. The reaction mixture was poured onto iced water (2 L) and extracted with ethyl acetate (5×1 L). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (DCM/MeOH: 1%, then 10%) to afford the expected intermediate A2 as a beige solid in 65% yield.
To a solution of intermediate A2 (21.3 mmol) in methanol (9.5 mL/mmol) was added dropwise at room temperature a solution of sodium methoxide 30% in methanol (21.3 mmol). The reaction mixture was stirred 1 h 30 at 60° C. The reaction was monitored by LC/MS. Dropwise addition at 30° C. of a solution of sodium methoxide 30% in methanol (85.2 mmol) and additional 1 h30 of stirring at 60° C. were required for reaction completion. The reaction mixture was cooled down to room temperature and Dowex-H+ (24 g) was added, followed by acetic acid (2 mL). The reaction mixture was filtered and washed with methanol. The filtrate was concentrated under reduced pressure. The crude residue was purified by 3 successive flash chromatographies on silica gel (DCM/MeOH: 0%, then 10%; petroleum ether/THF: 0 to 60%; DCM/EtOH: 1% to 3%) to afford the expected compound as a white solid with 90% purity in 41% yield. 1H NMR (DMSO-d6, 300 MHz) δ (ppm) 4.319-4.323 (m, 1H), 4.55-4.66 (m, 3H), 5.67 (d, J=8.10 Hz, 1H), 5.93 (d, J=4.50 Hz, 1H), 6.34-6.35 (m, 1H), 7.63 (d, J=8.10 Hz, 1H), 11.47 (s, 1H); MS (ESI) m/z=274.0 (MNa+).
General Method A:
To a suspension of the appropriate nucleoside (1a to 1e) (2.20 mmol) in methanol (2.27 mL/mmol) was added mCPBA (2.65 mmol) at room temperature under nitrogen. The reaction mixture was stirred for 2 hours or overnight. The reaction was monitored by LC/MS. The reaction mixture was quenched with a saturated solution of sodium sulfite (0.2 mL), and then concentrated under reduced pressure. The crude residue was partitioned with a mixture H2O/CH2Cl2. The aqueous layer was directly purified by preparative HPLC (H2O/CH3CN: 2 to 10%) to afford 2 separated compounds: the expected 13-D-ribo compound (2a to 2e) and the respective 4′ isomer α-L-lyxo.
Compound (2a)
Compound 2a was synthesized from compound 1a according to general method A. The expected compound was obtained as a white solid in 29% yield. 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 3.28 (s, 3H), 3.43 (dd, J=11.67 Hz and 5.56 Hz, 1H), 3.54 (dd, J=11.67 Hz and 5.33 Hz, 1H), 4.03-4.078 (m, 1H), 4.10-4.13 (m, 1H), 4.71 (d, J=7.34 Hz, 1H), 5.20 (t, J=5.60 Hz, 1H), 5.25 (d, J=6.60 Hz, 1H), 5.65 (d, J=7.96 Hz, 1H), 5.90 (d, J=4.85 Hz, 1H), 7.74 (d, J=8.14 Hz, 1H), 11.37 (s, 1H); MS (ESI) m/z=297.0 (MNa+).
Compound (2b)
Compound 2b was synthesized from compound 1b according to general method A. The expected compound was obtained as a white solid in 32% yield. 1H NMR (MeOD, 400 MHz) δ (ppm) 3.42 (s, 3H), 3.66 (d, J=11.82 Hz, 1H), 3.90 (d, J=11.82 Hz, 1H), 4.55 (dd, J=22.70 Hz and 6.00 Hz, 1H), 5.03 (dd, J=53.84 Hz and 5.82 Hz, 1H), 5.69 (d, J=8.07 Hz, 1H), 6.05 (d, J=18.95 Hz, 1H), 7.84 (d, J=8.07 Hz, 1H); MS (ESI) m/z=277.0 (MH+).
Compound (2c)
Compound 2c was synthesized from compound 1c according to general method A. The expected compound was obtained as a white solid in 22% yield. 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 3.25-3.27 (m, 3H), 4.51-4.58 (m, 1H), 4.65-4.80 (m, 1H), 5.09-5.11 (m, 1H), 5.77 (t, J=7.93 Hz, 1H), 5.94-5.97 (m, 1H), 6.05 (t, J=7.58 Hz, 1H), 7.44 (t, J=7.93 Hz, 1H), 11.43 (s, 1H); 19F NMR (DMSO-d6, 400 MHz) δ (ppm) −209.5; MS (ESI) m/z=277 (MH+).
Compound (2d)
Compound 2d was synthesized from compound 1d according to general method A. The expected compound was obtained as a white solid in 33% yield. 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 1.91 (dd, J=13.46 Hz and 7.67 Hz, 1H), 2.43-2.47 (m, 1H), 3.23 (s, 3H), 3.42 (d, J=5.55 Hz, 2H), 4.19-4.25 (m, 1H), 5.22 (t, J=5.67 Hz, 1H), 5.50 (d, J=5.55 Hz, 1H), 5.67 (d, J=8.15 Hz, 1H), 5.88 (d, J=6.37 Hz, 1H), 7.78 (d, J=8.15 Hz, 1H), 11.34 (s, 1H); MS (ESI) m/z=281.0 (MNa+).
Compound (2e)
Compound 2e was synthesized from compound 1e according to general method A. The expected compound was obtained as a white solid in 18% yield. 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 3.30 (s, 3H), 3.51 (dd, J=11.98 Hz and 5.92 Hz, 1H), 3.61 (dd, J=11.98 Hz and 5.66 Hz, 1H), 3.80 (d, J=7.31 Hz, 1H), 4.38-4.43 (m, 1H), 5.27 (t, J=5.79 Hz, 1H), 5.65 (d, J=8.10 Hz, 1H), 5.89 (d, J=4.15 Hz, 1H), 6.12 (d, J=5.92 Hz, 1H), 7.59 (d, J=8.10 Hz, 1H), 11.40 (s, 1H); MS (ESI) m/z=300.0 (MH+).
Compound (3a)
To a solution of compound 2a (0.51 mmol) in anhydrous pyridine (2.0 mL/mmol) was added acetic anhydride (2.0 mL/mmol) and DMAP (0.05 mmol). The reaction mixture was stirred 12 hours at room temperature and then concentrated under reduced pressure. The residue was dissolved in dichloromethane, washed with saturated NaHCO3, dried over Na2SO4 and concentrated under reduced pressure to afford the expected compound in 87% yield. 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 2.00 (s, 3H), 2.04 (s, 3H), 2.05 (s, 3H), 3.32 (s, 3H), 4.20 (d, J=11.96 Hz, 1H), 4.33 (d, J=11.96 Hz, 1H), 5.44 (dd, J=7.73 Hz and 3.57 Hz, 1H), 5.57 (d, J=7.73 Hz, 1H), 5.68 (d, J=8.05 Hz, 1H), 5.86 (d, J=3.50 Hz, 1H), 7.79 (d, J=8.05 Hz, 1H), 11.49 (s, 1H); MS (ESI) m/z=423.2 (MNa+).
Compound (3c)
To a solution of compound 2c (2.10 mmol) and DMAP (0.21 mmol) in anhydrous pyridine (10.0 mL/mmol) was added acetic anhydride (4.62 mmol). The reaction mixture was stirred 2 hours at 0° C. and then quenched with ice, diluted with ethyl acetate and washed with water. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (DCM/MeOH: 0 to 10%) to afford the expected compound in 65% yield; MS (ESI) m/z=359 (MH−).
Compound (4a)
To a solution of compound 3a (0.75 mmol) and 1,2,4-triazole (8.46 mmol) in dichloromethane (2.7 mL/mmol) and triethylamine (8.46 mmol) was added under nitrogen at 0° C. phosphorus oxychloride (1.95 mmol). The reaction mixture was stirred 2.5 hours at room temperature and then, quenched with ice and diluted with dichloromethane. The organic layer was washed with water, dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (DCM/MeOH: 0 to 10%) to afford the expected intermediate compound; MS (ESI) m/z=452.2 (MH+).
Compound (4c)
To a solution of compound 3c (1.36 mmol) and 1,2,4-triazole (15.37 mmol) in dichloromethane (10 mL/mmol) and triethylamine (15.37 mmol) was added under nitrogen at 0° C. phosphorus oxychloride (3.54 mmol). The reaction mixture was stirred 4 hours at room temperature and then, quenched with ice and diluted with dichloromethane. The organic layer was washed with water, dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (DCM/MeOH: 0 to 10%) to afford the expected compound as an oil in 35% yield; MS (ESI) m/z=412.2 (MH+).
Compound (5ai)
To a suspension of compound 4a (4.43 mmol) in THF (9.5 mL/mmol) was added ammonium hydroxide solution (23% mass; 0.75 mL/mmol). The reaction mixture was stirred 2 hours at room temperature. The reaction was monitored by LC/MS. The reaction mixture was quenched with a saturated NH4Cl solution, and extracted with DCM. The aqueous layer was extracted with a mixture of DCM/THF (1:1) and the combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to afford the expected compound as a white foam in 78% yield; MS (ESI) m/z=400.0 (MH+).
Compound (5a2)
To a solution of compound 4a (0.75 mmol) in THF (12 mL/mmol) was added ammonium hydroxide solution (0.9 mL/mmol). The reaction mixture was stirred 3.5 hours at room temperature and then, concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (DCM/MeOH: 0 to 10%) to afford the expected compound. This product was dissolved in ethyl acetate, and the mixture was washed with water and brine/The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to afford the expected compound as a white solid; 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 3.39-3.43 (m, 1H), 3.53-3.57 (m, 1H), 3.94-3.96 (m, 1H), 4.11-4.13 (m, 1H), 4.51 (brs, 1H), 5.10 (brs, 2H), 5.69-5.72 (m, 1H), 5.91-5.94 (m, 1H), 7.15-7.19 (m, 2H), 7.66-7.69 (m, 1H); MS (ESI) m/z=274.0 (MH+).
Compound (5c)
To a solution of compound 4c (0.47 mmol) in THF (12 mL/mmol) was added ammonium hydroxide solution (0.9 mL/mmol). The reaction mixture was stirred 1 hour at room temperature and then, concentrated under reduced pressure. The crude residue was dissolved in 7N ammonia solution in methanol (20 mL/mmol). The reaction mixture was stirred at room temperature overnight and concentrated under reduced pressure. The residue was purified by C18 chromatography (H2O/CH3CN: 0 to 10%) to afford the expected compound as a white solid in 80% yield; 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 3.44 (dd, J=11.74 Hz and 1.70 Hz, 1H), 3.52 (dd, J=11.74 Hz and 1.70 Hz, 1H), 4.26-4.34 (m, 1H), 4.88 (dd, J=53.87 Hz and 5.07 Hz, 1H), 5.76 (d, J=7.39 Hz, 1H), 6.06 (d, J=7.63 Hz, 1H), 7.26 (brs, 2H), 7.67 (d, J=7.51 Hz, 1H); 19F NMR (DMSO-d6, 376.5 MHz) δ (ppm) −212.61; MS (ESI) m/z=276 (MH+).
Compounds 6a-8a, 6b, and 7b were prepared according to the following scheme, Scheme 2:
Compound (6a)
Compound 6a was synthesized from compound 1a in ethanol according to general method A. The expected compounds were obtained as white solid as a 60/40 4′ isomer mixture. MS (ESI) m/z=289.0 (MH+).
Compound (6b)
Compound 6b was synthesized from compound 1b in ethanol according to general method A. The expected compounds were obtained as a white solid as a 60/40 4′ isomer mixture; 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 1.10 (t, J=7.03 Hz, 2.6H), 1.14 (t, J=7.03 Hz, 3.4H), 3.42-3.58 (m, 4H), 3.61-3.71 (m, 4H), 4.08 (t, J=4.61 Hz, 1.2H), 4.24-4.35 (m, 0.8H), 4.81-4.83 (m, 1.2H), 4.94-4.96 (m, 0.5H), 5.08-5.13 (m, 1.2H), 5.29-5.31 (m, 0.8H), 5.45 (dd, J=6.43 Hz and 4.48 Hz, 0.6H), 5.58 (dd, J=6.43 Hz and 4.48 Hz, 0.6H), 5.62 (d, J=8.06 Hz, 0.8H), 5.82 (d, J=8.14 Hz, 1.2H), 5.98-6.02 (m, 0.8H), 6.05 (d, J=5.40 Hz, 1.2H), 6.26 (dd, J=14.45 Hz and 6.54 Hz, 1.2H), 7.45 (d, J=8.14 Hz, 1.2H), 7.77 (d, J=8.06 Hz, 0.8H), 11.46 (brs, 2H); 19F NMR (DMSO-d6, 376.5 MHz) δ (ppm) −214.89, −196.16; MS (ESI) m/z=291.0 (MH+).
Compound (7a)
Compound 7a was synthesized from compound 1a in a solution of isopropanol in water (6.19 mol/L) according to general method A. The expected compound was obtained as a white solid in 9% yield; 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 1.07 (d, J=6.14 Hz, 3H), 1.12 (d, J=6.14 Hz, 3H), 3.40 (dd, J=11.62 Hz and 5.63 Hz, 1H), 3.54 (dd, J=11.62 Hz and 5.13 Hz, 1H), 4.00-4.04 (m, 1H), 4.06-4.15 (m, 2H), 4.49 (d, J=7.44 Hz, 1H), 5.12 (d, J=6.64 Hz, 1H), 5.24 (t, J=5.41 Hz, 1H), 5.65 (d, J=8.09 Hz, 1H), 5.93 (d, J=4.55 Hz, 1H), 7.77 (d, J=8.09 Hz, 1H), 11.34 (brs, 1H); MS (ESI) m/z=301.2 (MH−).
Compound (7b)
Compound 7b was synthesized from compound 1b in a solution of isopropanol in water (6.19 mol/L) according to general method A. The expected compound was obtained as a white solid; 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 1.07 (d, J=6.13 Hz, 3H), 1.11 (d, J=6.13 Hz, 3H), 3.42 (dd, J=11.74 Hz and 5.78 Hz, 1H), 3.72 (dd, J=11.65 Hz and 4.99 Hz, 1H), 4.12 (heptuplet, J=6.13 Hz, 1H), 4.26 (ddd, J=25.27 Hz, 9.34 Hz and 5.49 Hz, 1H), 4.98 (dd, J=53.61 Hz and 5.49 Hz, 1H), 4.99 (d, J=9.39 Hz, 1H), 5.36 (t, J=5.44 Hz, 1H), 5.62 (d, J=8.16 Hz, 1H), 6.02 (d, J=18.33 Hz, 1H), 7.80 (d, J=8.16 Hz, 1H), 11.41 (brs, 1H); 19F NMR (DMSO-d6, 376.5 MHz) δ (ppm) −196.68; MS (ESI) m/z=305.0 (MH+).
Compound (8a)
Compound 8a was synthesized from compound 1a in propargyl alcohol according to general method A. The expected compound was obtained as a white solid in 20% yield; 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 3.34 (t, J=2.42 Hz, 1H), 3.44 (dd, J=11.62 Hz and 4.68 Hz, 1H), 3.56 (dd, J=11.62 Hz and 4.50 Hz, 1H), 4.05-4.10 (m, 1H), 4.13-4.16 (m, 1H), 4.27 (dd, J=15.76 Hz and 2.43 Hz, 1H), 4.34 (dd, J=15.76 Hz and 2.43 Hz, 1H), 4.93 (d, J=7.14 Hz, 1H), 5.30-5.32 (m, 2H), 5.66 (d, J=8.11 Hz, 1H), 5.96 (d, J=4.80 Hz, 1H), 7.75 (d, J=8.11 Hz, 1H), 11.36 (brs, 1H); MS (ESI) m/z=297.0 (MH−).
Compounds 9a-9g, 17a, 18a, 220, 222, 223, 224, 230, 233, 520, 530 were prepared according to the following scheme, Scheme 3:
General Method B.
The following procedure was used to obtain compounds 9a to 9g, 220, 222, 223, 224, 230, 233 in tris triethyl ammonium salt form, 17a, 18a, 520, 530.
The appropriate nucleoside (100 mg) was dried in a flask under vacuum overnight. Trimethylphosphate (1.9 ml) and proton sponge (100 mg) were added to the flask and the reaction mixture was stirred under nitrogen cooled by an ice/water bath. Distilled phosphorus oxychloride (45 μl) was added and the reaction mixture was stirred during 4 hours with cooling. Tributylamine (0.32 ml) and tributylamine pyrophosphate (4.0 ml of a 0.5 M solution in DMF) were added and the reaction was allowed to stir for an additional 45 min with cooling. The reaction was quenched with triethylammonium bicarbonate (0.5 M, 20 ml) and the solvents were concentrated under reduced pressure. The crude was dissolved in 10 ml of water and purified using a Sephadex DEAE A-25 column with a linear gradient of 0-1 M NaCl buffered with 20 mM Tris-HCl (pH 7.0) (triphosphates eluted at ˜0.4 M NaCl; diphosphates eluted at ˜0.2 M NaCl; monophosphates eluted at ˜0.1 M NaCl) and desalted on a C18 column to afford the expected compound.
Compound (9a) (in Tris Triethyl Ammonium Salt Form)
White solid; MS (ESI) m/z=513.0 (MH−).
Compound (9b) (in Tris Triethyl Ammonium Salt Form)
White solid; MS (ESI) m/z=515.0 (MH−).
Compound (9c) (in Tris Triethyl Ammonium Salt Form)
White solid; MS (ESI) m/z=515.0 (MH−).
Compound (9d) (in Tris Triethyl Ammonium Salt Form)
White solid; MS (ESI) m/z=497.0 (MH−).
Compound (9e) (in Tris Triethyl Ammonium Salt Form)
White solid; MS (ESI) m/z=512.0 (MH−).
Compound (9f) (in Tris Triethyl Ammonium Salt Form)
White solid; MS (ESI) m/z=512.0 (MH−).
Compound (9g) (in Tris Triethyl Ammonium Salt Form)
White solid; MS (ESI) m/z=514.0 (MH−).
Compound (17a) (in Bis Triethyl Ammonium Salt Form)
Aqueous solution; MS (ESI) m/z=433.0 (MH−).
Compound (18a) (in Mono Triethyl Ammonium Salt Form)
Aqueous solution; MS (ESI) m/z=353.0 (MH−).
Compound (220): (in Tris Triethyl Ammonium Salt Form)
Aqueous solution; MS (ESI) m/z=577.0 (MH−).
Compound (222): (in Tris Triethyl Ammonium Salt Form)
Aqueous solution; MS (ESI) m/z=554.0 (MH−).
Compound (223): (in Tris Triethyl Ammonium Salt Form)
Aqueous solution; MS (ESI) m/z=538.0 (MH−).
Compound (224): (in Tris Triethyl Ammonium Salt Form)
Aqueous solution; MS (ESI) m/z=527.0 (MH−).
General Method C.
The following procedure was used to obtain compounds 230, 233 and 530 in triethyl ammonium salt form from compounds 220, 223 and 520.
The pure triphosphate or monophosphate (10-100 mg) was dissolved in water (10 ml). Pd/C (5%, 10 mg) was added and the reaction mixture was stirred over H2 with a hydrogen balloon for 30 min. The product was purified using a Sephadex DEAE A-25 column with a linear gradient of 0-1 M triethylammonium bicarbonate. The product containing fractions were pooled and concentrated and freeze-dried to give the desired compound.
Compound (230): (in Tris Triethyl Ammonium Salt Form)
Aqueous solution; MS (ESI) m/z=551.0 (MH−).
Compound (233): (in Tris Triethyl Ammonium Salt Form)
Aqueous solution; MS (ESI) m/z=512.0 (MH−).
Compound (530): (in Triethyl Ammonium Salt Form)
Aqueous solution; MS (ESI) m/z=391.1 (MH−).
Compounds 13a, 13a Dia 1, 13a Dia 2, 14a, 14a Dia 1, 14a Dia 2, and 16a were prepared according to the following synthetic scheme, Scheme 4:
Compound (10a)
To a solution of compound 5a1 (5.53 mmol) and 4-dimethylaminopyridine (11.1 mmol) in DCM (15 mL/mmol) was added at 0° C. benzyl chloroformate (8.30 mmol). The reaction mixture was stirred overnight at room temperature. The reaction was monitored by LC/MS. The reaction mixture was diluted with DCM (26.6 mL/mmol) and washed successively with a 1N HCl aqueous solution (6.7 mL/mmol), water (13.3 mL/mmol) and brine. The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (DCM/MeOH: 0 to 8%) to afford the expected compound as a white foam in 75% yield; 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 2.00 (s, 3H), 2.047 (s, 3H), 2.051 (s, 3H), 3.33 (s, 3H), 4.20 (d, J=11.97 Hz, 1H), 4.35 (d, J=11.97 Hz, 1H), 5.19 (s, 2H), 5.50 (dd, J=7.63 Hz and 2.89 Hz, 1H), 5.65 (d, J=7.63 Hz, 1H), 5.88 (d, J=2.89 Hz, 1H), 7.06 (d, J=7.39 Hz, 1H), 7.31-7.42 (m, 5H), 8.15 (d, J=7.55 Hz, 1H), 11.00 (brs, 1H); MS (ESI) m/z=534.2 (MH+).
Compound (11a)
A solution of compound 10a (1.21 mmol) in 7N ammonia solution in methanol (10 mL/mmol) was stirred at room temperature for 1 hour. The reaction was monitored by LC/MS. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (DCM/MeOH: 0 to 20%) to afford the expected compound as a white foam in 93% yield. 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 3.29 (s, 3H), 3.45 (dd, J=11.75 Hz and 5.58 Hz, 1H), 3.64 (dd, J=11.75 Hz and 5.22 Hz, 1H), 3.99-4.03 (m, 1H), 4.15 (dd, J=8.13 Hz and 6.53 Hz, 1H), 4.58 (d, J=8.31 Hz, 1H), 5.23-5.26 (m, 2H), 5.95 (d, J=3.47 Hz, 1H), 6.66 (brs, 1H), 7.02 (d, J=7.40 Hz, 1H), 7.26 (brs, 1H), 7.31-7.42 (m, 5H), 8.24 (d, J=7.58 Hz, 1H), 10.85 (brs, 1H); MS (ESI) m/z=408.0 (MH+).
Compound (12a)
To a solution of compound 11a (0.91 mmol) in anhydrous tetrahydrofuran (7 mL/mmol) was added dropwise under nitrogen tert-butylmagnesium chloride 1.0 M in THF (2.82 mmol). The reaction mixture was stirred at room temperature for 10 minutes and then, were added isopropyl (2R)-2-[[(4-nitrophenoxy)-phenoxy-phosphoryl]amino]propanoate (1.18 mmol) in anhydrous tetrahydrofuran (3 mL/mmol), followed by DMSO (3 mL/mmol). The reaction mixture was stirred at room temperature overnight. The reaction was monitored by LC/MS. The reaction mixture was diluted with DCM and quenched by addition of 0.5M phosphate buffer (pH=7). The organic layer was washed 3 times with brine and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (eluent: DCM/MeOH: 0 to 8%) to afford a mixture of diastereoisomers as a white foam in 34% yield. 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 1.12-1.14 (m, 6H), 1.19-1.22 (m, 3H), 3.31 (s, 3H), 3.75-3.82 (m, 1H), 4.01-4.22 (m, 4H), 4.77 (dd, J=8.42 Hz and 6.05 Hz, 1H), 4.81-4.88 (m, 1H), 5.18 (d, J=2.70 Hz, 2H), 5.42 (d J=6.43 Hz and 4.18 Hz, 1H), 5.95 (t, J=4.05 Hz, 1H), 6.01-6.11 (m, 1H), 7.02 (dd, J=19.74 Hz and 7.59 Hz, 1H), 7.14-7.23 (m, 3H), 7.33-7.40 (7H), 7.98 (dd, J=19.74 Hz and 7.59 Hz, 1H), 10.89 (brs, 1H); 31P NMR (DMSO-d6, 162 MHz) δ (ppm) 3.28 (s, 1P), 3.70 (s, 1P); MS (ESI) m/z=677.2 (MH+).
Compounds (13a, 13a Dia 1, and 13a Dia 2)
To a solution of compound 12a (0.78 mmol) in methanol (24.5 mL/mmol) was added Palladium on Carbon 10% (0.055 g/g). The reaction mixture was purged 3 times vacuo/nitrogen, then 3 times vacuo/hydrogen and stirred under hydrogen atmosphere for 1 h15 at room temperature. The reaction was monitored by LC/MS. The reaction mixture was filtered through a pad of celite, and the filtrate was concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (eluent: DCM/methanol: 0 to 16%) to afford the expected compound as a mixture of diastereoisomers in 77% yield. The 2 diastereoisomers were separated by chiral separation (Chiralpak ID 5; Hexane/EtOH: 1/1) to afford pure diastereoisomers.
Compound 13a Diastereomer 1; White solid; 31% yield; 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 1.14 (dd, J=6.22 Hz and 2.48 Hz, 6H), 1.20 (d, J=7.08 Hz, 3H), 3.73-3.83 (m, 1H), 4.01-4.12 (m, 3H), 4.17 (dd, J=7.98 Hz and 6.82 Hz, 1H), 4.68 (d, J=8.24 Hz, 1H), 4.85 (heptuplet, J=6.22 Hz, 1H), 5.29 (d, J=6.55 Hz, 1H), 5.67 (d, J=7.43 Hz, 1H), 5.94 (d, J=4.56 Hz, 1H), 6.00 (dd, J=12.78 Hz and 9.94 Hz, 1H), 7.15-7.23 (m, 5H), 7.34-7.38 (m, 2H), 7.51 (d, J=7.43 Hz, 1H); 31P NMR (DMSO-d6, 162 MHz) δ (ppm) 3.67 (s, 1P); MS (ESI) m/z=543.2 (MH+).
Compound 13a Diastereomer 2; White solid; 31% yield; 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 1.14 (d, J=6.22 Hz, 6H), 1.22 (d, J=7.04 Hz, 3H), 3.28 (s, 3H), 3.72-3.83 (m, 1H), 3.94-4.01 (m, 2H), 4.07-4.13 (m, 2H), 4.69 (d, J=8.41 Hz, 1H), 4.85 (heptuplet, J=6.22 Hz, 1H), 5.28 (d, J=6.54 Hz, 1H), 5.71 (d, J=7.45 Hz, 1H), 5.94 (d, J=4.36 Hz, 1H), 6.05 (dd, J=13.24 Hz and 10.13 Hz, 1H), 7.15-7.24 (m, 5H), 7.34-7.38 (m, 2H), 7.45 (d, J=7.45 Hz, 1H); 31P NMR (DMSO-d6, 162 MHz) δ (ppm) 3.28 (s, 1P); MS (ESI) m/z=543.2 (MH+).
Compounds (14a, 14a Dia 1, and 14a Dia2)
To a solution of compound 2a (3.50 mmol) and 2-(2,2-dimethylpropanoylsulfanyl)ethoxy-phosphinic acid (5.25 mmol) in pyridine (12 mL/mmol) was slowly added pivaloyl chloride (7.00 mmol) at 0° C. The reaction mixture was stirred for 1 hour at 0° C. and 2 hours at room temperature. The reaction was quenched with a 1M solution of NH4Cl (150 mL) and extracted with ethyl acetate. The organic layer was dried over Na2SO4) and concentrated under reduced pressure to afford the expected intermediate compound S-[2-[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxy-2-methoxy-tetrahydrofuran-2-yl]methoxyphosphonoyloxy]ethyl]-2,2-dimethyl-propanethioate. MS (ESI) m/z=505.0 (MNa+).
To a solution of the intermediate compound in dichloromethane (10 mL/mmol) and carbon tetrachloride (5 mL/mmol) were added triethylamine (21.00 mmol) and (L)-alanine isopropyl ester hydrochloride (10.50 mmol) at room temperature. The reaction mixture was stirred overnight at room temperature. The reaction mixture was directly purified by flash chromatography on silica gel (DCM/MeOH: 0 to 10%), followed by preparative MS/HPLC to afford the expected compound as a white solid as a mixture of diastereoisomers. The 2 diastereoisomers were separated by preparative MS/HPLC to afford pure diastereoisomers.
Compound 14a Diastereoisomer 1; White solid; 2.3% yield; 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 1.16-1.19 (m, 15H), 1.25 (d, J=7.14 Hz, 3H), 3.08-3.11 (m, 2H), 3.30 (s, 3H), 3.66-3.76 (m, 1H), 3.88 (dd, J=11.17 Hz and 5.48 Hz, 1H), 3.93-3.99 (m, 3H), 4.08-4.15 (m, 2H), 4.80 (d, J=7.24 Hz, 1H), 4.88 (heptuplet, J=6.21 Hz, 1H), 5.38 (d, J=6.10 Hz, 1H), 5.58-5.65 (m, 2H), 5.88 (d, J=4.65 Hz, 1H), 7.62 (d, J=8.07 Hz, 1H), 11.41 (brs, 1H); 31P NMR (DMSO-d6, 162 MHz) δ (ppm) 7.86 (s, 1P); MS (ESI) m/z=612.2 (MH+).
Compound 14a Diastereoisomer 2; White solid; 1.5% yield; 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 1.17-1.20 (m, 15H), 1.24 (d, J=7.08 Hz, 3H), 3.07 (t, J=6.44 Hz, 2H), 3.31 (s, 3H), 3.62-3.72 (m, 1H), 3.88-3.99 (m, 4H), 4.10-4.17 (m, 2H), 4.83 (d, J=7.56 Hz, 1H), 4.88 (heptuplet, J=6.20 Hz, 1H), 5.39 (d, J=6.15 Hz, 1H), 5.60-5.66 (m, 2H), 5.89 (d, J=4.44 Hz, 1H), 7.61 (d, J=8.15 Hz, 1H), 11.41 (brs, 1H); 31P NMR (DMSO-d6, 162 MHz) δ (ppm) 7.59 (s, 1P); MS (ESI) m/z=612.2 (MH+).
Compound (15a)
Compound 15a was synthesized from compound 11a as described for compound 14a.
Step 1: intermediate compound: MS (ESI) m/z=616.2 (MH+).
Step 2: The reaction mixture was directly purified by flash chromatography on silica gel (DCM/MeOH: 0 to 10%) to afford the expected compound as a colourless oil as a mixture of diastereoisomers in 6.6% yield; MS (ESI) m/z=745.4 (MH+).
Compound (16a)
To a solution of compound 15a (0.08 mmol) in ethanol (62.5 mL/mmol) was added Palladium black (1/1: w/w). The reaction mixture was purged 3 times vacuo/nitrogen, then 3 times vacuo/hydrogen and stirred under hydrogen atmosphere for 3 hours at room temperature. The reaction was monitored by LC/MS. The reaction mixture was filtered through a pad of celite, and the filtrate was concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (eluent: DCM/methanol: 0 to 20%), followed by preparative MS/HPLC to afford the expected compound as a mixture of diastereoisomers (Dia 1/Dia 2: 53/47) in 23% yield; 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 1.17-1.20 (m, 15H), 1.23-1.26 (m, 3H), 3.05-3.11 (m, 2H), 3.28 (s, 3H), 3.64-3.76 (m, 1H), 3.86-3.99 (m, 4H), 4.00-4.05 (m, 1H), 4.11-4.17 (m, 1H), 4.64 (t, J=8.53 Hz, 1H), 4.88 (heptuplet, J=6.21 Hz, 1H), 5.26-5.28 (m, 1H), 5.57-5.64 (m, 1H), 5.72 (dd, J=7.43 Hz and 2.61 Hz, 1H), 5.92 (dd, J=4.18 Hz and 2.48 Hz, 1H), 7.20-7.25 (m, 2H), 7.53 (d, J=7.30 Hz, 1H); 31P NMR (DMSO-d6, 162 MHz) δ (ppm) 7.58 (s, 0.47P), 7.78 (0.53P); MS (ESI) m/z=611.2 (MH+).
Compound (20)
Compound (20) was synthesized according to Scheme 5; White solid; 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 10.65 (brs, 1H), 7.81 (s, 1H), 6.50 (s, 2H), 6.18 (d, J=7.25 Hz, 1H), 5.75 (d, J=7.25 Hz, 1H), 5.33 (t, J=5.30 Hz, 1H), 4.64 (dd, J=9.51 Hz and 7.36 Hz, 1H), 4.47-4.43 (m, 1H), 3.71 (dd, J=11.71 Hz and 5.35 Hz, 1H), 3.50 (dd, J=11.93 Hz and 5.69 Hz, 1H), 3.31 (s, 3H); MS (ESI) m/z=339.0 (MH+).
Compound (21)
Compound (21) was synthesized according to Scheme 5; White solid; 1H NMR (MeOD, 400 MHz) δ (ppm) 7.96 (s, 1H), 5.97 (d, J=4.23 Hz, 1H), 4.57 (dd, J=6.99 Hz and 4.28 Hz, 1H), 3.85 (d, J=12.03 Hz, 1H), 3.69 (d, J=12.03 Hz, 1H), 3.46 (s, 3H); MS (ESI) m/z=339.2 (MH+).
Compound (22)
Compound (22) was synthesized according to procedure described in US 2013/0165400.
Compound (23)
Compound (23) was synthesized according to General Method A.
White solid; 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 11.38 (brs, 1H), 7.73 (d, J=7.96 Hz, 1H), 5.89 (d, J=4.51 Hz, 1H), 5.68 (d, J=7.96 Hz, 1H), 5.57 (d, J=7.16 Hz, 1H), 5.26 (t, J=5.57 Hz, 1H), 4.46 (t, J=7.16 Hz, 1H), 4.18 (dd, J=7.01 Hz and 4.43 Hz, 1H), 3.66 (dd, J=11.83 Hz and 5.48 Hz, 1H), 3.47 (dd, J=11.83 Hz and 5.77 Hz, 1H), 3.29 (s, 3H); MS (ESI) m/z=300.0 (MH+).
Compound (24)
Compound (24) was synthesized according to General Method A.
White solid; 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 11.40 (brs, 1H), 7.79 (d, J=8.08 Hz, 1H), 5.94 (d, J=4.20 Hz, 1H), 5.67 (d, J=8.08 Hz, 1H), 5.29 (t, J=5.54 Hz, 1H), 4.71 (d, J=8.39 Hz, 1H), 4.28 (dd, J=8.42 Hz and 6.47 Hz, 1H), 3.82 (dd, J=6.38 Hz and 4.17 Hz, 1H), 3.61 (dd, J=11.63 Hz and 5.49 Hz, 1H), 3.45 (dd, J=11.63 Hz and 5.71 Hz, 1H), 3.35 (s, 3H), 3.28 (s, 3H); MS (ESI) m/z=311.0 (MNa+).
Compound (122)
Compound (122) was synthesized according to Scheme 7.
Compound (122) Dia 1
White solid; 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 10.69 (brs, 1H), 7.84 (s, 1H), 7.35-7.31 (m, 2H), 7.18-7.14 (m, 3H), 6.54 (brs, 2H), 6.23-6.17 (m, 1H), 5.99 (dd, J=12.84 Hz and 10.02 Hz, 1H), 5.36 (d, J=9.08 Hz, 1H), 5.38-5.23 (m, 1H), 4.85-4.80 (m, 1H), 4.83 (heptuplet, J=6.27 Hz, 1H), 4.30 (dd, J=10.96 Hz and 5.95 Hz, 1H), 4.16 (dd, J=10.96 Hz and 5.95 Hz, 1H), 3.82-3.72 (m, 1H), 3.37 (s, 3H), 1.18 (d, J=6.85 Hz, 3H), 1.11 (d, J=6.27 Hz, 3H), 1.105 (d, J=6.27 Hz, 3H); 19F NMR (DMSO-d6, 376 MHz) δ (ppm) −196.49 (s, 1F); 31P NMR (DMSO-d6, 162 MHz) δ (ppm) 3.98 (s, 1P); MS (ESI) m/z=585.2 (MH+).
Compound (122) Dia 2
White solid; 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 7.78 (s, 1H), 7.37-7.33 (m, 2H), 7.23-7.15 (m, 3H), 6.57 (brs, 2H), 6.22-6.16 (m, 1H), 6.015 (dd, J=13.27 Hz and 10.32 Hz, 1H), 5.43-5.40 (m, 1H), 5.28 (dd, J=53.44 Hz and 5.78 Hz, 1H), 4.84 (heptuplet, J=6.15 Hz, 1H), 4.79-4.70 (m, 1H), 4.25 (dd, J=10.85 Hz and 5.81 Hz, 1H), 4.11 (dd, J=10.85 Hz and 5.30 Hz, 1H), 3.78-3.68 (m, 1H), 3.35 (s, 3H), 1.18 (d, J=6.82 Hz, 3H), 1.125 (d, J=6.15 Hz, 6H); 19F NMR (DMSO-d6, 376 MHz) δ (ppm) −196.31 (s, 1F); 31P NMR (DMSO-d6, 162 MHz) δ (ppm) 3.54 (s, 1P); MS (ESI) m/z=585.2 (MH+).
Compound (123)
Compound (123) was synthesized according to Scheme 7.
Compound (123) Mixture of Diastereoisomers
White solid; 1H NMR (MeOD, 400 MHz) δ (ppm) 7.65 (d, J=8.01 Hz, 0.6H), 7.58 (d, J=8.01 Hz, 0.4H), 7.42-7.38 (m, 2H), 7.31-7.21 (m, 3H), 5.93 (d, J=4.09 Hz, 0.6H), 5.86 (d, J=3.93 Hz, 0.4H), 5.73 (d, J=8.01 Hz, 0.4H), 5.67 (d, J=8.01 Hz, 0.6H), 5.00 (heptuplet, J=6.20 Hz, 1H), 4.65 (d, J=7.26 Hz, 0.6H), 4.59 (d, J=7.41 Hz, 0.4H), 4.43-4.35 (m, 1H), 4.29-4.15 (m, 2H), 3.98-3.89 (m, 1H), 3.47 (s, 1.8H), 3.43 (s, 1.2H), 1.37-1.34 (m, 3H), 1.26-1.23 (m, 6H); 31P NMR (MeOD, 162 MHz) δ (ppm) 3.97 (s, 0.6P), 3.35 (s, 0.4P); MS (ESI) m/z=569.2 (MH+).
Test compounds in the form of nucleoside triphosphates were examined for inhibitory activity against purified HCV polymerase in a standard assay. Bacterial expression constructs encoding the approximately 65 kDa HCV genotype 1b NS5B protein were used to generate recombinant HCV polymerases (with a deletion of the 21 carboxy terminal amino acids). Both the wild-type genotype 1b protein and protein containing the S282T mutation were expressed and purified for use in the enzymatic activity assay.
The enzymatic activity assay measured the inhibitory effect of increasing concentrations of test compound on the incorporation of α-[33P]-labeled nucleotide into trichloroacetic acid-precipitable material. Recombinant polymerase and synthetic RNA template were combined in reaction buffer containing ribonucleoside triphosphates, α-[33P]-labeled nucleotide and eight concentrations of test compound in three-fold dilutions. Reactions were incubated for two hours at 30° C.
Reactions were terminated by the addition of ice-cold trichloroacetic acid and sodium pyrophosphate to promote precipitation of newly-synthesized ribonucleic acid. Precipitable material from the reactions was collected by filtration onto 96-well filter plates, washed extensively with water, and quantified by liquid scintillation.
The inhibitory activity of test compounds was determined by fitting results to dose-response curves using XLfit software.
Results are provided in Table 1.
Huh-7-derived cell line (Zluc) that harbors an HCV genotype 1b replicon and a luciferase reporter gene was grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM GlutaMAX, 1% MEM nonessential amino acids, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 0.5 mg/mL Geneticin® (G418). For dose response testing the cells were seeded in 96-well plates at 7.5×103 cells per well in a volume of 50 μL, and incubated at 37° C./5% CO2. Drug solutions were made up freshly in Huh-7 media as 2× stocks. Ten additional 5-fold dilutions were prepared from these stocks in DMEM without G418. At least three hours after Zluc cells were seeded, drug treatment was initiated by adding 50 μL of drug dilutions to the plates in duplicate. Final concentrations of drug ranged from 100 μM to 0.0000512 μM. Cells were then incubated at 37° C./5% CO2. Alternatively, compounds were tested at two concentrations (1 μM and 10 μM). In all cases, Huh-7 (which do not harbors the HCV replicon) served as negative control. After 72 hours of incubation, the inhibition of HCV replication was measured by quantification of photons emitted after mono-oxygenation of 5′-fluoroluciferin to oxyfluoroluciferin by firefly luciferase. For this, media was removed from the plates via gentle tapping. Fifty microliters of ONE-glo luciferase assay reagent was added to each well. The plates were shaken gently for 3 min at room temperature and luminescence was measured on a Victor3 V 1420 multilabel counter (Perkin Elmer) with a 1 second read time using a 700 nm cut-off filter. The EC50 values were calculated from dose response curves from the resulting best-fit equations determined by Microsoft Excel and XLfit 4.1 software. When screening at two fixed concentrations, the results were expressed as % inhibition at 1 μM and 10 μM.
For cytotoxicity evaluation, Zluc cells were treated with compound as described herein, and cell viability was monitored using the CellTiter-Blue Cell Viability Assay (Promega) by adding 20 μL of the assay solution to each well. The plates were then incubated at 37° C./5% CO2 for at least 3 hours. Fluorescence was detected in plates using excitation and emission wavelengths of 560 and 590 nm, respectively, in a Victor3 V 1420 multilabel counter (Perkin Elmer) and CC50 values were determined using Microsoft Excel and XLfit 4.1 software.
Compounds presented in Table 2 below were assayed according to the replicon assay described herein.
Ms=Mouse; TP=triphosphate.
A single oral dose of Compound 1 at 10 mg/kg in PEG 200 (dose volume 5 mL/kg) was administered to nine CD-1 male mice. Five untreated animals were used for the collection of control plasma and liver. Terminal plasma and liver samples were collected from three animals per time point at 4, 12 and 24 hours post dose. Liver specimens were collected from all animals immediately after the incision. Freezing forceps stored in liquid nitrogen were used to freeze the liver before excision.
Plasma samples were analyzed for nucleoside by LC-MS/MS. The internal standard (IS) was either 2′-MeG-D3 or tiapride. For protein precipitation and extraction, each plasma sample (50 μL) was treated with 500 μL of 0.2% formic acid in acetonitrile and 20 μL of the internal standard working solution. After vortexing and centrifugation, 500 μL of the sample extracts were transferred to a new plate, dried under N2 at ˜28° C. and reconstituted with 75 μL of 0.2% FA in water. The extracts were chromatographed on an Aquasil C18 column using a gradient system of 0.2% formic acid in water and acetonitrile. The analytes were detected and quantified by tandem mass spectrometry in positive ion mode on an MDS Sciex API5000 equipped with a Turbo Ionspray® interface. The calibration range was 0.500 (LLOQ) to 200 ng/mL in mouse plasma.
Liver samples were analyzed for the active species nucleoside triphosphate by LC-MS/MS. The triphosphate levels were assayed by homogenizing (on ice) a known weight of mouse liver with 4× volume of 0.95 M trichloroacetic acid (TCA). Internal standard solution was added to the homogenate followed by neutralization with 20% ammonium hydroxide solution and addition of 500 μL 1% formic acid. The tissue samples were extracted by weak anion exchange solid phase extraction (SPE). Post extraction, the eluates were evaporated under nitrogen, followed by reconstitution before injection onto the LC-MS/MS system. The samples were chromatographed on a Luna NH2 column using a gradient system of ammonium acetate (1 mM to 20 mM and pH 8.0 to pH 10.0) in water and acetonitrile (70:30). The analyte was detected and quantified by tandem mass spectrometry in positive ion mode on an API4000 equipped with a Turbo Ionspray® interface.
Results are provided in Table 3 below.
1Single point concentration provided as follows: + ≤ 15 < ++ ≤ 50 < +++ ≤ 100 < ++++
2Integrated concentration provided as follows: + ≤ 150 < ++ ≤ 500 < +++ ≤ 1500 < ++++
While the claimed subject matter has been described in terms of various embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof. Accordingly, it is intended that the scope of the claimed subject matter is limited solely by the scope of the following claims, including equivalents thereof.
This application is the national stage application under 35 U.S.C. 371 of International Patent Application No. PCT/US14/070993, filed Dec. 17, 2014, which claims priority to U.S. Provisional Patent Application No. 61/917,687, filed Dec. 18, 2013. Each of the aforementioned PCT and provisional applications is incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2014/070993 | 12/17/2014 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2015/095419 | 6/25/2015 | WO | A |
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| 9073960 | Beigelman | Jul 2015 | B2 |
| 9126971 | Cheng | Sep 2015 | B2 |
| 20030236216 | Devos et al. | Dec 2003 | A1 |
| 20130165400 | Beigelman et al. | Jun 2013 | A1 |
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| 1999043691 | Sep 1999 | WO |
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| Number | Date | Country | |
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| 20180162898 A1 | Jun 2018 | US |
| Number | Date | Country | |
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| 61917687 | Dec 2013 | US |
