Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/08—Bridged systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/08—Bridged systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
  • C07D498/08—Bridged systems

Definitions

• the disclosure generally relates to the novel compounds of formula I, including their salts, which have activity against hepatitis C virus (HCV) and are useful in treating those infected with HCV.
• HCV hepatitis C virus
• the disclosure also relates to compositions and methods of using these compounds.
• HCV Hepatitis C virus
• HCV Hepatitis C virus
• a substantial fraction of these HCV infected individuals develop serious progressive liver disease, including cirrhosis and hepatocellular carcinoma (Lauer, G.M. et al., N. Engl. J. Med., 345:41-52 (2001)).
• HCV is a positive-stranded RNA virus. Based on a comparison of the deduced amino acid sequence and the extensive similarity in the 5 '-untranslated region, HCV has been classified as a separate genus in the Flaviviridae family. All members of the Flaviviridae family have enveloped virions that contain a positive stranded RNA genome encoding all known virus-specific proteins via translation of a single, uninterrupted, open reading frame.
• this polyprotein In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce the structural and non- structural (NS) proteins.
• NS structural and non- structural
• HCV the generation of mature non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases.
• the first one is believed to be a metalloprotease and cleaves at the NS2- NS3 junction; the second one is a serine protease contained within the N-terminal region of NS3 (also referred to as NS3 protease) and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4A cleavage site, and in trans, for the remaining NS4A-NS4B, NS4B-NS5A, NS5A-NS5B sites.
• the NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components.
• NS5B (also referred to as HCV polymerase) is a RNA-dependent RNA polymerase that is involved in the replication of HCV.
• the HCV NS5B protein is described in "Structural Analysis of the Hepatitis C Virus RNA Polymerase in Complex with Ribonucleotides" (Bressanelli, S. et al., J.
• the invention provides technical advantages, for example, the compounds are novel and are effective against hepatitis C. Additionally, the compounds provide advantages for pharmaceutical uses, for example, with regard to one or more of their mechanism of action, binding, inhibition efficacy, target selectivity, solubility, safety profiles, or bioavailability. DESCRIPTION OF THE INVENTION
• the invention encompasses compounds of formula I, including pharmaceutically acceptable salts, and compositions and methods of treatment using these compounds.
• One aspect of the invention is a compound of formula I
• R 1 is CO 2 R 5 or CONR 6 R 7 ;
• R 2 is furanyl, pyrrolyl, thienyl, pyrazolyl, isoxazolyl, isothiazolyl, imidazolyl, oxazolyl, thiazolyl, oxadiazolyl, triazolyl, or tetrazolyl; and R 2 is substituted with 1 substituent selected from the group consisting of cycloalkyl, tetrahydrofuranyl, tetrahydropyranyl, phenyl, pyrimidinyl, pyrazinyl, pyridinonyl, benzimidazolyl, piperidinyl substituted with 0-1 alkyl substituents, and pyridinyl substituted with 0-1 alkyl substituents; and R 2 is substituted with 1 substituent selected from CO 2 R 5 ,
• R 2 is substituted with 0-1 substituents selected from oxo, amino, alkyl, and haloalkyl;
• R 3 is hydrogen, halo, alkyl, alkenyl, hydroxy, benzyloxy, or alkoxy;
• R 4 is cycloalkyl
• R 5 is hydrogen or alkyl
• R 6 is hydrogen, alkyl, alkylSO 2 , cycloalkylSO 2 , haloalkylSO 2 , (R 9 )(R 10 )NSO 2 , or (R 1 ⁇ SO 2 ;
• R 7 is hydrogen or alkyl
• R 8 is hydrogen, alkyl, cycloalkyl, (cycloalkyl)alkyl, alkylcarbonyl, cycloalkylcarbonyl, haloalkylcarbonyl, alkoxycarbonyl, alkylSO 2 , cycloalkylSO 2 , haloalkylSO 2 , aminocarbonyl, (alkylamino)carbonyl, (dialkylamino)carbonyl, benzyl, benzyloxycarbonyl, or pyridinyl;
• R 9 is hydrogen or alkyl
• R 10 is hydrogen or alkyl
• R 11 is azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperidinyl, homopiperazinyl, or homomo ⁇ holinyl, and is substituted with 0-1 alkyl substituents;
• R 12 is hydrogen, alkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl,
• R 13 is azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperidinyl, homopiperazinyl, or homomo ⁇ holinyl, and is substituted with 0-3 substituents selected from halo, alkyl, cycloalkyl, alkoxyalkyl, amino, alkylamino, dialkylamino, R 11 , aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, (R u )alkyl, or CO 2 R 5 ;
• R , 13 is a [4.3.0] or [3.3.0] bicyclic diamine attached to the carbonyl through one nitrogen, and is substituted with 0-2 R 8 substituents;
• R 14 is hydrogen, alkyl, cycloalkyl, (cycloalkyl)alkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, or benzyl;
• R 15 is hydrogen, alkyl, cycloalkyl, (cycloalkyl)alkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, or benzyl; or NR 14 R 13 taken together is azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, N-(alkyl)piperazinyl, morpholinyl, thiomorpholinyl, homopiperidinyl, or homomorpholinyl;
• R 16 is hydrogen or alkyl
• R 17 is hydrogen, alkyl, or cycloalkyl
• X is methylene, a bond, or absent; or a pharmaceutically acceptable salt thereof.
• R 1 is CO 2 R 5 or CONR 6 R 7 ;
• R 2 is pyrazolyl, isoxazolyl, or imidazolyl, and is substituted with 1 substituent selected from the group consisting of cycloalkyl, tetrahydropyranyl, phenyl, pyrimidinyl, pyrazinyl, pyridinonyl, benzimidazolyl, piperidinyl substituted with 1 alkyl substituent, and pyridinyl substituted with 0-1 alkyl substituents; and R 2 is substituted with 1 substituent selected from CO 2 R 5 , CON(R 12 ) 2 , and COR 13 ; and R 2 is substituted with 0-1 alkyl substituents;
• R is alkoxy
• R 4 is cycloalkyl
• R 5 is hydrogen or alkyl
• R 6 is alkylSO 2 , cycloalkylSO 2 , or (R 9 )(R 10 )NSO 2 ;
• R 7 is hydrogen
• R 8 is hydrogen, alkyl, or (cycloalkyl)alkyl
• R 12 is alkyl or alkoxyalkyl
• R 13 is azetidinyl, piperidinyl, piperazinyl, morpholinyl, homopiperazinyl, or homomorpholinyl, and is substituted with 0-3 substituents selected from halo, alkyl, cycloalkyl, or alkoxyalkyl;
• R 16 is hydrogen; R 17 is alkyl; and X is a bond; or a pharmaceutically acceptable salt thereof.
• Another aspect of the invention is a compound of formula I where:
• R 1 is CO 2 R 5 or CONR 6 R 7 ;
• R 2 is pyrazolyl, isoxazolyl, or imidazolyl, and is substituted with 1 substituent selected from cyclopropyl, cyclobutyl, tetrahydropyranyl, phenyl, pyrimidinyl, pyrazinyl, pyridinonyl, benzimidazolyl, N-methylpiperidinyl, pyridinyl or methylpyridinyl; and R 2 is substituted with 1 substituent selected from CO 2 R 5 , CON(R 12 ) 2 , and COR 13 ; and R 2 is substituted with 0-1 methyl substituent; R 3 is methoxy;
• R 4 is cyclohexyl
• R 5 is hydrogen or alkyl
• R 6 is isopropylSO2, isobutylSO2, cyclopropylSO2, or Me 2 NSO 2 ;
• R 7 is hydrogen
• R 8 is hydrogen, methyl, ethyl, or (cyclopropyl)methyl
• R 12 is isopropyl or methoxyethyl
• R 13 is difluoroazetidinyl, difluoropiperidinyl, methylpiperazinyl, cyclopentylpiperazinyl, trimethylpiperazinyl, morpholinyl, dimethylmorpholinyl, (methoxymethyl)mo ⁇ holinyl, N-methylhomopiperazinyl, or homomo ⁇ holinyl;
• R 1 is CO 2 R 5 or CONR 6 R 7 ;
• R is furanyl, pyrrolyl, thienyl, pyrazolyl, isoxazolyl, isothiazolyl, imidazolyl, oxazolyl, thiazolyl, oxadiazolyl, triazolyl, or tetrazolyl; and R 2 is substituted with 1 substituent selected from cycloalkyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl substituted with 0-1 alkyl substituents, and pyridinyl substituted with 0-1 alkyl substituents; and R is substituted with 1 substituent selected from CO 2 R , CON(R 12 )2, and COR 13 ; and R 2 is substituted with 0-1 substituents selected from oxo, amino, alkyl, and haloalkyl;
• R 3 is hydrogen, halo, alkyl, alkenyl, hydroxy, benzyloxy, or alkoxy;
• R 4 is cycloalkyl
• R 5 is hydrogen or alkyl
• R 6 is hydrogen, alkyl, alkylSO 2 , cycloalkylSO 2 , haloalkylSO 2 , (R 9 )(R 10 )NSO 2 , or (R 1 ⁇ SO 2 ;
• R 7 is hydrogen or alkyl
• R 8 is hydrogen, alkyl, cycloalkyl, (cycloalkyl)alkyl, alkylcarbonyl, cycloalkylcarbonyl, haloalkylcarbonyl, alkoxycarbonyl, alkylSO 2 , cycloalkylSO 2 , haloalkylSO 2 , aminocarbonyl, (alkylamino)carbonyl, (dialkylamino)carbonyl, benzyl, benzyloxycarbonyl, or pyridinyl;
• R 9 is hydrogen or alkyl
• R 10 is hydrogen or alkyl
• R 11 is azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperidinyl, homopiperazinyl, or homomorpholinyl, and is substituted with 0-1 alkyl substituents;
• R 12 is hydrogen, alkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, or (R ⁇ )alkyl;
• R 13 is azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperidinyl, homopiperazinyl, or homomorpholinyl, and is substituted with 0-3 substituents selected from alkyl, alkoxyalkyl, amino, alkylamino, dialkylamino, R 11 , aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, (R ⁇ )alkyl, or
• R .13 is a [4.3.0] or [3.3.0] bicyclic diamine attached to the carbonyl through one nitrogen, and is substituted with 0-2 R 8 substituents;
• R 14 is hydrogen, alkyl, cycloalkyl, (cycloalkyl)alkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, or benzyl;
• R 15 is hydrogen, alkyl, cycloalkyl, (cycloalkyl)alkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, or benzyl; or NR 14 R 13 taken together is azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, N-(alkyl)piperazinyl, morpholinyl, thiomo ⁇ holinyl, homopiperidinyl, or homomorpholinyl;
• R 16 is hydrogen or alkyl
• R 17 is hydrogen, alkyl, or cycloalkyl
• X is methylene, a bond, or absent; or a pharmaceutically acceptable salt thereof.
• R 1 is CONR 6 R 7 ;
• R 6 is alkylSO 2 , cycloalkylSO 2 , haloalkylSO 2 , (R 9 ) 2 NSO 2 , or (R 10 )SO 2 ;
• R 7 is hydrogen
• Another aspect of the invention is a compound of formula I where R " is pyrazolyl substituted with 1 substituent selected from cyclopropyl, cyclobutyl,
• Another aspect of the invention is a compound of formula I where R 3 is hydrogen.
• Another aspect of the invention is a compound of formula I where R is methoxy.
• Another aspect of the invention is a compound of formula I where R 4 is cyclohexyl.
• Another aspect of the invention is a compound of formula I where R 6 is
• Another aspect of the invention is a compound of formula I where X is methylene
• Another aspect of the invention is a compound of formula I where X is a bond
• Another aspect of the invention is a compound of formula I where X is absent
• Another aspect of the invention is a compound of formula I according to the following stereochemistry
• Another aspect of the invention is a compound of formula I according to the following stereochemistry
• Another aspect of the invention is a compound of formula I according to the following stereochemistry
• Another aspect of the invention is a compound of formula I according to the following stereochemistry
• any instance of a variable substituent including R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , and X, can be used independently with the scope of any other instance of a variable substituent.
• the invention includes combinations of the different aspects.
• Alkyl means a straight or branched alkyl group composed of 1 to 6 carbons.
• Alkenyl means a straight or branched alkyl group composed of 2 to 6 carbons with at least one double bond.
• Alkynyl means a straight or branched alkyl group composed of 2 to 6 carbons with at least one triple bond.
• Cycloalkyl means a monocyclic ring system composed of 3 to 7 carbons.
• Haloalkyl and haloalkoxy include all halogenated isomers from monohalo to perhalo.
• Terms with a hydrocarbon moiety include straight and branched isomers for the hydrocarbon portion. Parenthetic and multiparenthetic terms are intended to clarify bonding relationships to those skilled in the art. For example, a term such as ((R)alkyl) means an alkyl substituent further substituted with the substituent R.
• the invention includes all pharmaceutically acceptable salt forms of the compounds. Pharmaceutically acceptable salts are those in which the counter ions do not contribute significantly to the physiological activity or toxicity of the compounds and as such function as pharmacological equivalents. These salts can be made according to common organic techniques employing commercially available reagents.
• anionic salt forms include acetate, acistrate, besylate, bromide, chloride, citrate, fumarate, glucouronate, hydrobromide, hydrochloride, hydroiodide, iodide, lactate, maleate, mesylate, nitrate, pamoate, phosphate, succinate, sulfate, tartrate, tosylate, and xinofoate.
• Some cationic salt forms include ammonium, aluminum, benzathine, bismuth, calcium, choline, diethylamine, diethanolamine, lithium, magnesium, meglumine, 4-phenylcyclohexylamine, piperazine, potassium, sodium, tromethamine, and zinc.
• Some of the compounds of the invention possess asymmetric carbon atoms (see, for example, the compound below).
• the invention includes all stereoisomeric forms, including enantiomers and diastereomers as well as mixtures of stereoisomers such as racemates. Some stereoisomers can be made using methods known in the art. Stereoisomeric mixtures of the compounds and related intermediates can be separated into individual isomers according to methods commonly known in the art
• the compounds may be made by methods known in the art including those described below and including variations within the skill of the art. Some reagents and intermediates are known in the art. Other reagents and intermediates can be made by methods known in the art using readily available materials.
• the variables (e.g., numbered "R" substituents) used to describe the synthesis of the compounds are intended only to illustrate how to make the compounds and are not to be confused with variables used in the claims or in other sections of the specification. The following methods are for illustrative purposes and are not intended to limit the scope of the invention. [0032] Abbreviations used in the schemes generally follow conventions used in the art.
• LC-MS retention time 2.81 min; 613 m/z (M ⁇ +).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 2 min, a hold time of 1 min, and an analysis time of 3 min where solvent A was 10% MeOH / 90% H 2 O / 0.1% trifluoroacetic acid and solvent B was 10% H 2 O / 90% MeOH / 0.1% trifluoroacetic acid.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the microwave vessel was capped under an inert atmosphere of nitrogen and the reaction heated at 16O 0 C for 40 minutes.
• the reaction was combined with previous experiments run under identical experimental conditions.
• the mixture was diluted with ethyl acetate and washed with 1.0N aqueous hydrochloric acid.
• the acidic aqueous phases were combined and back extracted one time using ethyl acetate.
• the ethyl acetate fractions were combined and washed sequentially with saturated aqueous sodium bicarbonate and brine.
• the organic phase was dried over MgSCU, filtered and solvent removed in vacuo to obtain an orange-amber foam.
• the crude product was adsorbed onto 3.
• the elution conditions employed a flow rate of 5 ml/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the resulting product as an amber foam was dissolved in dichloromethane and benzene added. The volatiles were removed in vacuo using a rotary evaporator. The process of dissolution in dichloromethane/benzene and removal was repeated to aid in removal of free TFA in the reaction. The product as an amber foam was dried in vacuo at room temperature overnight of yield 920mg of the title compound as an amorphous yellow solid.
• LC-MS retention time 1.57min; 621m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a Waters XTERRA® MS 7u Cl 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• LC-MS retention time 1.68 min;726 m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the sample was dissolved in acetonitrile / DMF (1: 1) (4ml) purified using a PHENOMENEX® Luna C18 30 x 100mm 1Ou column and monitored using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the title compound was collected between 10.4 and 11.4 minutes.
• the product fraction were combined and volatiles removed in vacuo.
• the trifluoroacetic acid salt was formed by dissolving the product in dichloromethane( ⁇ 12ml) and filtering using Whatman autovial 0.45uM filter, then adding TFA (35 ⁇ L, 0.454 mmol) then remove volatiles in vacuo using a rotary evaporator.
• the final product was dried in vacuo at room temperature.
• the 1 H NMR exhibited characteristics of restricted rotation and/or salt formation with broadening and splitting of spectra peaks.
• LC-MS retention time 1.71 min; 726m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou Cl 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H2O / 10 niM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 niM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction was capped under a nitrogen atmosphere and stirred at room temperature for 19hrs.
• the reaction was diluted with ethyl acetate (400ml) and washed with l.ON aqueous hydrochloric acid (2x60ml).
• the aqueous layers were combined and back extracted with ethyl acetate (2x75ml).
• the organic phases were combined and washed with brine and dried over magnesium sulfate and filtered.
• To the yellow filtrate was added 10ml of 2.0M hydrogen chloride in diethyl ether, volatiles were then removed in vacuo using a rotary evaporator.
• the elution conditions employed a flow rate of 5 ml/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the sample was dissolved in acetonitrile / DMF (1: 1) (2mL) purified using a PHENOMENEX® Luna C18 30 x 100mm 1Ou column and monitored using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the product exhibited rotomeric peaks with retention time of 8.5minutes and a shoulder peak at 8.9minutes.
• the product fractions were combined and the volatiles removed in vacuo using a rotary evaporator. Place the off white pale yellow product on vacuum pump and dry in vacuo for approximately lhr, then dissolve in dichloromethane and transfer to a 25ml pear shaped flask, add TFA (30 ⁇ L, 0.389 mmol), then remove volatiles in vacuo using a rotary evaporator. Transfer product to a CMDD vial using dichloromethane then concentrate using a nitrogen sweep and finally remove remainder of solvent in vacuo using a rotary evaporator.
• LC-MS retention time 1.46 min; 809 m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the sample was dissolved in acetonitrile / DMF (1 :1) (2ml) purified using a PHENOMENEX® Luna Cl 8 30 x 100mm 1Ou column and monitored using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• LC-MS retention time 1.49 min; 767m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou Cl 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction was purified on a Shimadzu high pressure liquid chromatography system employing DISCOVERY VP® software interfaced with a SCL-IOA controller, SIL-IOA autosampler and FRC-IOA fraction collector.
• the sample was dissolved in acetonitrile / DMF (3:1) (4ml) purified using a PHENOMENEX® Luna C18 30 x 100mm 1Ou column and monitored using a SPD- 10AV UV-Vis detector at a detector wave length of 22OnM.
• the retention time of product is 9.3minutes. Combine product fractions and remove volatiles in vacuo using a rotary evaporator using a vacuum pump.
• the product was briefly dried in vacuo at room temperature and the TFA salt made by dissolving in dichloromethane and adding TFA (60 ⁇ L, 0.779 mmol). The volatiles were removed in vacuo and the sample re-dissolved in dichloromethane, filtered through a 0.45uM syringe filter and the solution concentrated. An additional amount of TFA (15.0 ⁇ L, 0.195 mmol) was added to the product solution and the volatiles were removed in vacuo using a rotary evaporator to obtain an orange solid. The title compound was dried in vacuo at room temperature overnight to yield 35.8mg (34%) as an amorphous orange solid.
• LC-MS retention time 1.59 min; 806 m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H2O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction was diluted in acetonitrile / DMF (1 : 1) (2ml) purified using a PHENOMENEX® Luna C18 30 x 100mm 1Ou column and monitored using a SPD- IOAV UV-Vis detector at a detector wave length of 22OnM.
• the product fractions were combined and the volatiles were removed in vacuo using a rotary evaporator.
• the product was briefly dried in vacuo and then dissolved in dichloromethane and TFA (30 ⁇ L, 0.389 mmol) was added.
• the volatiles were removed in vacuo using a rotary evaporator.
• the sample was re-dissolved in approximately 2ml of dichloromethane and filtered through a 0.45uM syringe filter into a vial.
• the product solution was concentrated under a nitrogen sweep then TFA (15 ⁇ L, 0.195 mmol) was added and the volatiles removed in vacuo using a rotary evaporator resulting in an orange foam.
• LC-MS retention time 1.65 min;795 m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction mixture was diluted with acetonitrile and a small amount of water to effect solubilization of the sample to a total volume of 2ml and purified using a Waters XTERRA® Prep MS Cl 8 OBD, 5uM, 30mm x 100mm column and monitored using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the product exhibited rotomeric like peak splitting with retention time s of 9.2 and 9.4minutes.
• the product fractions were combined and volatiles removed in vacuo using a rotary evaporator.
• the product was dried in vacuo for ⁇ lhr then dissolved in dichloromethane ( ⁇ 3ml) and filtered through an ACRODISC® 0.45uM syringe filter using a norm jet syringe which was pre-rinsed using dichloromethane in to a 35ml flask.
• TFA (30 ⁇ L, 0.389 mmol) was added to the solution and the volatiles were removed in vacuo using a rotary evaporator.
• the sample was redissolved in dichloromethane and transferred into a vial. The solution was concentrated using a nitrogen sweep and final volatiles were removed in vacuo using a rotary evaporator.
• LC-MS retention time 1.59 min; 821 m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the product was purified on a Shimadzu high pressure liquid chromatography system employing DISCOVERY VP® software interfaced with a SCL-IOA controller, SIL-IOA autosampler and FRC-IOA fraction collector.
• the reaction mixture was diluted with acetonitrile to 2ml and purified using a Waters XTERRA ( R ) Prep MS Cl 8 OBD, 5uM, 30mm x 100mm column and monitored using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the product exhibited rotomeric like splitting with retention time s of 10.5 and 10.8minutes.
• the product fractions were combined and volatiles removed in vacuo using a rotary evaporator.
• the sample was further purified on a Shimadzu high pressure liquid chromatography system employing DISCOVERY VP® software interfaced with a SCL-IOA controller, SIL-IOA autosampler and FRC-IOA fraction collector.
• the sample was dissolved in acetonitrile (ImL) and purified using a Waters Sunfire Prep C 18 OBD, 5uM 19mm x 100mm column and monitored using a SPD- 1 OAV UV-Vis detector at a detector wave length of 22OnM.
• LC-MS retention time 1.71 min; 807m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou Cl 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H2O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction mixture was diluted with acetonitrile to 2ml and purified using a Waters XTERRA® Prep MS Cl 8 OBD, 5uM, 30mm x 100mm column and monitored using a SPD-IOAV UV-Vis detector at a detector wave length of 22OnM.
• the product exhibited rotomeric like splitting with retention time s of 9.3 and 9.6 minutes.
• the product fractions were combined and volatiles removed in vacuo using a rotary evaporator.
• the product was dried in vacuo for approximately lhr then dissolved in dichloromethane ( ⁇ 4ml) and filtered through an ACRODISC® 0.45uM syringe filter using a norm jet syringe which was pre-rinsed using dichloromethane.
• TFA (30 ⁇ L, 0.389 mmol) was added to the solution and the volatiles were removed in vacuo using a rotary evaporator.
• the product was dried in vacuo at room temperature yielding 40.9mg (45.2%) as an amorphous orange solid.
• LC-MS retention time 1.60 min; 81 InVz (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou Cl 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction mixture was diluted with acetonitrile to 2ml and purified using a Waters XTERRA ( R ) Prep MS Cl 8 OBD, 5uM, 30mm x 100mm column and monitored using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the product exhibited an asymmetric peak with retention time of 9.0 minutes.
• the product fractions were combined and volatiles removed in vacuo using a rotary evaporator.
• the product was dried in vacuo for ⁇ lhr then dissolved in dichloromethane ( ⁇ 3ml) and filtered through an ACRODISC® 0.45uM syringe filter using a norm jet syringe which was pre-rinsed using dichloromethane in to a 35ml flask.
• TFA 60 ⁇ L, 0.779 mmol
• LC-MS retention time 1.74 min; 8O8m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou Cl 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction was capped under a nitrogen atmosphere and heated in a microwave to 14O 0 C for 40 minutes.
• the reaction was diluted with ethyl acetate and washed with LON aqueous hydrochloric acid.
• the aqueous phase was back extracted one time using ethyl acetate.
• the organic phases were combined and sequentially washed with saturated sodium bicarbonate and brine.
• the organic phase was dried over sodium sulfate, filtered and solvent removed in vacuo.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the product was dissolved in approximately 125ml of ethyl acetate and washed two times with 75ml of aqueous 1.0N hydrochloric acid. The aqueous layers were combined and backed extracted with ethyl acetate. The organic extracts were combined and washed with brine and dried over magnesium sulfate, filtered and the solvent removed in vacuo to give an amorphous orange solid.
• the product was dissolved in dichloromethane and benzene added to the solution. The volatiles were removed in vacuo using a rotary evaporator. The product was re-dissolved in dichloromethane and solvent removed in vacuo to give a amorphous orange foam (1.28g).
• Analytical grade sample was purified by reverse phase HPLC: The sample was purified on a Shimadzu high pressure liquid chromatography system employing DISCOVERY VP® software interfaced with a SCL-IOA controller, SIL- 1OA autosampler and FRC-IOA fraction collector. The sample (40mg) was dissolved in acetonitrile / DMF (1: 1) (2ml) purified using a Waters Sunfire Prep Cl 8 OBD, 5uM 19mm x 100mm column and monitored using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• Retention time of product is 11.5minutes. Remove volatiles from product in vacuo using a rotary evaporator then transfer to a vial in dichloromethane.
• LC-MS retention time 1.90 min; 615m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 niM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the material was purified by trituration.
• the solid reaction residue was heated to reflux in 20ml of methanol then 7ml of de-ionized water added.
• the material was allowed to cool and stand for 1.5 hours then filtered.
• the mustard colored precipitate was rinsed with a small amount of 15% water in methanol (%v/v).
• the product was dried in vacuo at room temperature to give 445mg of a mustard yellow solid.
• a second crop of product was obtained from the mother liquor by dissolving with the addition of methanol and the subsequent concentration in vacuo using a rotary evaporator.
• LC-MS retention time 1.95 min; 72OnVz (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction was capped under a nitrogen atmosphere and stirred at room temperature for 19hrs.
• the reaction was diluted with ethyl acetate (125ml) and washed with l.ON aqueous hydrochloric acid.
• the aqueous layers were combined and back extracted Ix with ethyl acetate.
• the organic layers were combined and washed sequentially with l.ON aqueous hydrochloric acid and brine.
• the organic phase was dried over MgSCU, filtered and solvent removed in vacuo.
• the sample was dried in vacuo at room temperature to give 232mg of product as a amorphous orange/amber solid.
• a small analytical grade sample was purified by reverse phase HPLC.
• the 89mg of product was purified on a Shimadzu high pressure liquid chromatography system employing DISCOVERY VP® software interfaced with a SCL-IOA controller, SIL-IOA autosampler and FRC-IOA fraction collector.
• the sample was dissolved in acetonitrile / DMF (1 :1) (2mL) and purified using a Waters Sunfire Prep Cl 8 OBD, 5uM 19mm x 100mm column and monitored using a SPD- 10AV UV-Vis detector at a detector wave length of 22OnM.
• Product peak eluted as broad peak collected from 9.97min to 11.18min. Remove volatiles for product fractions in vacuo using a SPEED VAC® with heat on low setting. The purified title compound was isolated (49.8mg) as a amorphous yellow solid. The remainder of the reaction product was carried forward into amide coupling reactions without further purification.
• LC-MS retention time 1.23 min; 692m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 3.0x50mm column using a SPD-IOAV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• DMAP (8.5 mg, 0.397 mmol) was added to the reaction followed by the amine reagent, morpholine (25.0 ⁇ L, 0.287 mmol).
• the reaction was capped under a nitrogen atmosphere and stirred at room temperature for 36hrs.
• the product was purified on a Shimadzu high pressure liquid chromatography system employing DISCOVERY VP® software interfaced with a SCL-IOA controller, SIL-IOA autosampler and FRC- 1OA fraction collector.
• the reaction was diluted to 2ml with acetonitrile and purified using a Waters Sunfire Prep Cl 8 OBD, 5uM 19mm x 100mm column and monitored using a SPD-IOAV UV-Vis detector at a detector wave length of 22OnM.
• LC-MS retention time 1.19 min;761 m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction was diluted to 2ml with acetonitrile and purified using a Waters Sunfire Prep Cl 8 OBD, 5uM 19mm x 100mm column and monitored using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the product peak appears to be rotomeric with splitting and broadening, retention time is 6.18 to 7.33 minutes.
• the title compound (61.9mg) was isolated as a amorphous yellow solid trifluoroacetic acid salt.
• LC-MS retention time 1.26 min;800m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou Cl 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction was diluted to 2ml with acetonitrile and purified using a Waters Sunfire Prep Cl 8 OBD, 5uM 19mm x 100mm column and monitored using a SPD-IOAV UV-Vis detector at a detector wave length of 22OnM.
• LC-MS retention time 1.61min; 805m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou Cl 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction solution was filtered through a 0.45uM syringe filter and filtrate solution was purified using a Waters Sunfire Prep Cl 8 OBD, 5uM 19mm x 100mm column and monitored using a SPD-IOAV UV-Vis detector at a detector wave length of 22OnM.
• the title compound (51.6
• LC-MS retention time 1.55 min; 803 m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction was diluted to 2ml with acetonitrile and purified using a Waters Sunfire Prep Cl 8 OBD, 5uM 19mm x 100mm column and monitored using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H2O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction was diluted to 2ml with acetonitrile and purified using a Waters Sunfire Prep Cl 8 OBD, 5uM 19mm x 100mm column and monitored using a SPD-IOAV UV-Vis detector at a detector wave length of 22OnM.
• LC-MS retention time 1.75min; 789m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou Cl 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H2O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction solution was filtered through a 0.45uM syringe filter and purified using a Waters Sunfire Prep Cl 8 OBD, 5uM 19mm x 100mm column and monitored using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• Two 2 ml injection were performed for purification.
• Retention time of product was 6.0min. Remove volatiles from the product fractions in vacuo using a SPEED VAC® set on low heat. The title compound (60.4mg) was isolated as a amorphous yellow solid trifluoroacetic acid salt. 1 H NMR spectrum exhibits characteristics of restricted rotation ⁇ salt formation with broadening of peaks and splitting of peaks.
• LC-MS retention time 1.61 min; 802m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the product was purified on a Shimadzu high pressure liquid chromatography system employing DISCOVERY VP® software interfaced with a SCL-IOA controller, SIL-IOA autosampler and FRC-IOA fraction collector.
• Product purification was accomplished using a Waters Sunfire Prep Cl 8 OBD, 5uM 19mm x 100mm column monitored using a SPD-IOAV UV-Vis detector at a detector wave length of 22OnM.
• LC-MS retention time 1.68 min; 815 m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 3.0x50mm column using a SPD-IOAV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• tert-butyl 13-cyclohexyl-6-((2E,Z)-3- (dimethylamino)-2-(ethoxycarbonyl)-2-propenoyl)-3-methoxy-7H-indolo[2,l- a][2]benzazepine-10-carboxylate (1900 mg, 3.10 mmol) was dissolved in ethanol (20 mL) . Cyclobutylhydrazine (267 mg, 3.10 mmol) and triethylamine (1.426 mL, 10.23 mmol) were added. The reaction mixture was then heated at 160 0 C under microwave condition for 2 hr.
• the sample was dissolved in acetonitrile / DMF (1 :1) (4ml) purified using a Waters Sunfire Prep Cl 8 OBD, 5uM 19mm x 100mm column and monitored using a SPD- 10AV UV-Vis detector at a detector wave length of 22OnM.
• First eluting product is 7H-indolo[2,l-a][2]benzazepine-10-carboxylic acid, 13-cyclohexyl-6-[l-cyclopropyl-4-(ethoxycarbonyl)-lH-pyrazol-5-yl]-3- methoxy-, 1,1-dimethylethyl ester with retention time of 12.5 minutes with the minor component product being 7H-indolo[2,l-a][2]benzazepine-10-carboxylic acid, 13- cyclohexyl-6-[ 1 -cyclopropyl-4-(ethoxycarbonyl)- lH-pyrazol-3-yl]-3-methoxy-, 1,1- dimethylethyl ester with a retention of 15.6 minutes.
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 70% solvent A / 30% solvent B to 0% solvent A / 100% solvent B, a gradient time of 5min, a hold time of 1 min, and an analysis time of 6 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H2O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 70% solvent A / 30% solvent B to 0% solvent A / 100% solvent B, a gradient time of 5min, a hold time of 1 min, and an analysis time of 6 min where solvent A was 5% acetonitrile / 95% H2O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction was cooled under a nitrogen atmosphere and propane-2-sulfonamide (105 mg, 0.636 mmol) was added to the reaction followed by DBU (0.096 mL, 0.636 mmol).
• the reaction was immerse in oil bath and heated at reflux under nitrogen atmosphere overnight.
• the reaction was diluted with ethyl acetate (50 mL) and the organic layer washed sequentially with 1.0N aqueous hydrochloric acid (50 mL) and 0. IM aqueous Na ⁇ PCU (50 mL). The organic layer was dried in vacuo to yield 124 mg of the title product as a yellow foam.
• LC-MS retention time 1.55 min; 643 m/z (MH).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou Cl 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• reaction was capped under a nitrogen atmosphere and stir at room temperature for 1 hour then morpholine (38 ⁇ L, 0.44 mmol) was added. The reaction was capped under a nitrogen atmosphere and stirred at room temperature overnight. [00186] The reaction was diluted with ethyl acetate (50 mL) and washed sequentially with 1.0N aqueous hydrochloric acid (25 mL), 0. IM aqueous NaH 2 PCU (25 mL). The organic phase was concentrated overnight in vacuo at room temperature to yield 78 mg of a yellow amorphous solid.
• the title compound was further purified on a Shimadzu high pressure liquid chromatography system employing DISCOVERY VP® software interfaced with a SCL-IOA controller, SIL- 1OA autosampler and FRC-IOA fraction collector.
• the sample was dissolved in acetonitrile / DMF (1 :1) (total volume 2ml) purified using a Waters Sunfire Prep Cl 8 OBD, 5uM 19mm x 100mm column and monitored using a SPD- IOAV UV-Vis detector at a detector wave length of 22OnM.
• the sample was run as two ImI injections.
• the run time of the second Prep HPLC run was truncated to 15 minutes base on data from the first run.
• LC-MS retention time 1.94 min; 712 m/z (MH).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou Cl 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction was cooled under a nitrogen atmosphere and propane-2-sulfonamide (28 mg, 0.23 mmol) was added to the reaction followed by DBU (0.26 mL, 0.17 mmol).
• the reaction was immerse in oil bath and heated at reflux under nitrogen atmosphere overnight.
• the reaction was diluted with ethyl acetate (50 mL) and the organic layer washed sequentially with 1.0N aqueous hydrochloric acid (50 mL) and 0.1M aqueous NaH 2 PO 4 (50 mL). The organic layer was dried in vacuo to yield 31 mg of the title product as a yellow foam.
• LC-MS retention time 1.78 min; 643 m/z (MH).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou Cl 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction was capped under a nitrogen atmosphere and stir at room temperature for 1 hour then morpholine (19 ⁇ L, 0.22 mmol) was added. The reaction was capped under a nitrogen atmosphere and stirred at room temperature overnight. [00197] The reaction was diluted with ethyl acetate (50 mL) and washed sequentially with 1.0N aqueous hydrochloric acid (25 mL), 0. IM aqueous NaH ⁇ PO 4 (25 mL). The organic phase was concentrated overnight in vacuo at room temperature to yield 33mg of a yellow amorphous solid.
• the title compound was further purified on a Shimadzu high pressure liquid chromatography system employing DISCOVERY VP® software interfaced with a SCL-IOA controller, SIL- 1OA autosampler and FRC-IOA fraction collector.
• the sample was dissolved in acetonitrile / DMF (1 :1) (total volume 2ml) purified using a Waters Sunfire Prep Cl 8 OBD, 5uM 19mm x 100mm column and monitored using a SPD- IOAV UV-Vis detector at a detector wave length of 22OnM.
• the sample was run as two ImI injections.
• the run time of the second Prep HPLC run was truncated to 15 minutes base on data from the first run.
• LC-MS retention time 1.91 min; 712 m/z (MH).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou Cl 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the sample 76816-035-a (63mg) was dissolved in acetonitrile / DMF mixture (2: 1, 2ml) purified using a PHENOMENEX® Luna Cl 8 30 x 100mm 1Ou column and monitored using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• LC-MS retention time 4.21min (88%); 625m/z (MH-) and 5.23min (12%); 625m/z (MH-).
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 4.6x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min , a gradient of 70% solvent A / 30% solvent B to 0% solvent A / 100% solvent B, a gradient time of 5min, a hold time of 2 min, and an analysis time of 7 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray.
• the elution conditions employed a flow rate of 4 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 5min, a hold time of 2 min, and an analysis time of 7 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMAS S® Platform for LC in electrospray mode. The intermediate was used without further purification.
• the elution conditions employed a flow rate of 4 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 5min, a hold time of 2 min, and an analysis time of 7 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 niM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 niM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction was placed under a nitrogen atmosphere and stirred at room temperature for 18.5hrs.
• the reaction was diluted with ethyl acetate and washed with LON aqueous hydrochloric acid.
• the aqueous phases were combined and back extracted with ethyl acetate.
• the organic layers were combined and sequentially washed with 1.0N aqueous hydrochloric acid and brine.
• the organic solution was dried over magnesium sulfate, filtered and the solvent removed in vacuo using a rotary evaporator to give an amorphous orange solid/foam which was dried in vacuo to give 415mg of crude product.
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou Cl 8 4.6x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 5min, a hold time of 2 min, and an analysis time of 7 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode. [00210] The crude product was used without any further purification in subsequent pyrazole synthesis.
• Temp. 8O 0 C
• a Waters LCT mass spectrometer with 4 way MUX source The sample was analyzed using an Ascentis 4.6x50mm 5uM C18 column.
• the elution conditions employed a flow rate of 2 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 8min, a hold time of 1 min, and an analysis time of 9 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H2O / 95% acetonitrile / 10 mM ammonium acetate.
• the elution conditions employed a flow rate of 2 ml/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 8min, a hold time of 1 min, and an analysis time of 9 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• Triethyl amine (65.9 ⁇ L, 0.473 mmol) was added to the reaction followed by 4-hydrazinyl-3-methylpyridine hydrochloride, 0.4 H 2 O (26.7 mg, 0.160 mmol).
• the reaction was capped under a nitrogen atmosphere and heated in a microwave to 14O 0 C for 40 minutes.
• HPLC analysis indicates reaction incomplete with starting material present. Recap reaction under a nitrogen atmosphere and heat reaction at 14O 0 C for an additional 40 minutes.
• the reaction was diluted with ethyl acetate and washed sequentially with 1.0N aqueous hydrochloric acid. The aqueous layer was back extracted with ethyl acetate and the organic phases combined.
• the elution conditions employed a flow rate of 4 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 5min, a hold time of 2 min, and an analysis time of 7 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode
• the elution conditions employed a flow rate of 4 ml/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 5min, a hold time of 2 min, and an analysis time of 7 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the carboxylic acid intermediate was used without further purification: [00220] In a 2 dram vial, the above carboxylate acid as the TFA salt (85.4mg, 0.107 mmol) was dissolved in THF (ImL). Carbonyldiimidazole (43.5 mg, 0.268 mmol) was added to the reaction then the reaction was caped under a nitrogen atmosphere and stir at room temperature for 1 hour then heated to 7O 0 C for 2 hours followed by cooling to room temperature.
• the reaction was diluted to 2ml with acetonitrile with the addition of a few drops of TFA and purified using a Waters Sunfire Prep Cl 8 OBD, 5uM 19mm x 150mm column and monitored using a SPD-IOAV UV-Vis detector at a detector wave length of 22OnM.
• LC data was recorded on a Shimadzu LC- 1 OAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• HPLC data was recorded on a LC-IOAS liquid chromatograph equipped with a PHENOMENEX® 1Ou C18 3.0xl60mm column using a SPD-10AV UV-Vis detector at a wave length of 22OnM.
• the elution conditions employed a flow rate of 1 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 30 min, where solvent A was 10% acetonitrile / 90% H 2 O / 10 mM TFA and solvent B was 10% H 2 O / 90% acetonitrile / 10 mM TFA.
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 1Ou Cl 8 3.0x50mm column using an SPD-10AV UV-Vis detector at a wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile / 95% H 2 O / 10 mM ammonium acetate and solvent B was 5% H 2 O / 95% acetonitrile / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the rxn was stirred for 1 hour. LCMS indicates the rxn was done, 754.19 at 4.05 minutes. It was diluted with ether, washed with saturated ammonium chloride then brine, dried (MgSO 4 ) and evaporated giving a yellow foam. The foam was dissolved in DCM, the solution was added to a Thompson silica gel cartridge and it was eluted with ethyl acetate/methanol (0% to 5%). The appropriate fractions (TLC) were combined and evaporated giving a light yellow foam. The foam was dissolved in ether and crystals formed upon setting. The mixture was filtered giving the product (341 mg, 0.452 mmol, 74.2 % yield) as slightly yellow powder.
• the rxn was stirred for 1 hour. It was diluted with ether, washed with saturated ammonium chloride then brine, dried (MgSO 4 ) and evaporated giving a yellow foam. The foam was dissolved in DCM, the solution was added to a Thompson silica gel cartridge and it was eluted with ethyl acetate/methanol (5% to
• the rxn was stirred for 1 hour. LCMS indicates the rxn was complete after 10 minutes, 740.30 at 3.96 minutes. It was diluted with ether, washed with saturated ammonium chloride then brine, dried (MgSO 4 ) and evaporated giving a yellow foam. The foam was dissolved in DCM, the solution was added to a Thompson silica gel cartridge and it was eluted with ethyl acetate/methanol (0% to 10%). The appropriate fractions (TLC) were combined and evaporated giving a light yellow film.
• the rxn was stirred over night. LCMS indicates the rxn was done, 753.2/755.2 at 3.08 minutes.
• the rxn was diluted with DCM, washed with 0.1 N HCl (aqueous) then brine, dried (MgSO 4 ) and evaporated giving a yellow syrup.
• the syrup was dissolved in DCM, the solution was added to a Thompson silica gel cartridge and it was eluted with hexane/ethyl acetate (30% to 100%). The appropriate fractions (TLC) were combined and evaporated giving a creamy white solid. The solid was triturated in ether/hexane and filtered giving the product (39 mg, 0.051 mmol, 48% yield) as a white powder.
• the rxn was stirred over night. LCMS indicates the rxn was done, 767.3/767.2 at 3.10 minutes.
• the rxn was diluted with DCM, washed with 0.1 N HCl (aqueous) then brine, dried (MgSO 4 ) and evaporated giving a yellow syrup.
• the syrup was dissolved in DCM, the solution was added to a Thompson silica gel cartridge and it was eluted with hexane/ethyl acetate (30% to 100%). The appropriate fractions (TLC) were combined and evaporated giving a creamy white solid.
• the rxn was stirred over night. LCMS indicates the rxn was done, 766.2/768.2 at 2.83 minutes.
• the rxn was diluted with DCM, washed with 0.1 N HCl (aqueous) then brine, dried (MgSO 4 ) and evaporated giving a yellow syrup.
• the syrup was dissolved in DCM, the solution was added to a Thompson silica gel cartridge and it was eluted with hexane/ethyl acetate (30% to 100%). The appropriate fractions (TLC) were combined and evaporated giving a creamy white solid.
• the rxn was stirred over night. LCMS indicates the rxn was done, 764.2/766.2 at 2.67 minutes.
• the rxn was diluted with DCM, washed with 0.1 N HCl (aqueous) then brine, dried (MgSO 4 ) and evaporated giving a yellow syrup.
• the syrup was dissolved in DCM, the solution was added to a Thompson silica gel cartridge and it was eluted with DCM/methanol (0% to 5%). The appropriate fractions (TLC) were combined and evaporated giving a creamy white solid. The solid was triturated in ether/hexane and filtered giving the product (52 mg, 0.067 mmol, 63.2 % yield) as a yellow powder.
• the rxn was stirred over night. LCMS indicates the rxn was done, 778.3/780.2 at 2.67 minutes.
• the rxn was diluted with DCM, washed with 0.1 N HCl (aqueous) then brine, dried (MgSO 4 ) and evaporated giving a yellow syrup.
• the syrup was dissolved in DCM, the solution was added to a Thompson silica gel cartridge and it was eluted with DCM/methanol (0% to 5%). The appropriate fractions (TLC) were combined and evaporated giving a creamy white solid. The solid was triturated in hexane/ether and filtered giving the product (53 mg, 0.067 mmol, 64.6 % yield) as a yellow powder.
• the rxn was stirred over night. LCMS indicates the rxn was done, 764.2/766.2 at 3.03 minutes.
• the rxn was diluted with DCM, washed with 0.1 N HCl (aqueous) then brine, dried (MgSO 4 ) and evaporated giving a yellow syrup.
• the syrup was dissolved in DCM, the solution was added to a Thompson silica gel cartridge and it was eluted with DCM/methanol (0% to 5%). The appropriate fractions (TLC) were combined and evaporated giving a creamy white solid. The solid was triturated in ether/hexane and filtered giving the product (51 mg, 0.066 mmol, 61.9 % yield) as a light yellow powder.
• the rxn was stirred over night. LCMS indicates the rxn was done, 778.2/780.2 at 3.15 minutes.
• the rxn was diluted with DCM, washed with 0.1 N HCl (aqueous) then brine, dried (MgSO 4 ) and evaporated giving a yellow syrup.
• the syrup was dissolved in DCM, the solution was added to a Thompson silica gel cartridge and it was eluted with DCM/methanol (0% to 5%). The appropriate fractions (TLC) were combined and evaporated giving a creamy white solid. The solid was triturated in ether/hexane and filtered giving the product (50 mg, 0.063 mmol, 60.9 % yield) as a creamy white powder.
• LCMS 679.29 at 4.00 minutes.
• LCMS pos/neg 768.2/770.2 at 2.48 minutes.
• the rxn was diluted with DCM, washed with saturated NaHCO 3 (aqueous) then brine, dried (MgSCU) and evaporated giving a yellow film.
• the film was dissolved in DCM, the solution was added to a Thompson silica gel cartridge and it was eluted with DCM/methanol (0% to 10%). The appropriate fractions (TLC) were combined and evaporated giving a light yellow solid.
• LC-MS retention time 2.74 min; m/z (MH+): 612.
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a Waters SunFire 5u Cl 8 4.6x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 2 min, a hold time of 2 min, and an analysis time of 4 min where solvent A was 10% MeOH / 90% H 2 O / 0.1% trifluoroacetic acid and solvent B was 10% H 2 O / 90% MeOH / 0.1% trifluoroacetic acid.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• LC-MS retention time 3.14 min; m/z (MH+): 664.
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a Waters SunFire 5u Cl 8 4.6x50mm column using a SPD-IOAV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 2 min, a hold time of 2 min, and an analysis time of 4 min where solvent A was 10% MeOH / 90% H 2 O / 0.1% trifluoroacetic acid and solvent B was 10% H 2 O / 90% MeOH / 0.1% trifluoroacetic acid.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• Step 1 Preparation of 13-cyclohexyl-6-(l-cyclopropyl-4-(ethoxycarbonyl)-3- isopropyl-l/f-pyrazol-5-yl)-3-methoxy-7H-indolo[2,l-a][2]benzazepine-10- carboxylic acid
• LC-MS retention time 2.44 min; m/z (M ⁇ +): 608.
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a Waters SunFire 5u Cl 8 4.6x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 2 min, a hold time of 2 min, and an analysis time of 4 min where solvent A was 10% MeOH / 90% H 2 O / 0.1% trifluoroacetic acid and solvent B was 10% H 2 O / 90% MeOH / 0.1% trifluoroacetic acid.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• Step 2 Preparation of ethyl 5-(13-cyclohexyl-10-((isopropylsulfonyl)carbamoyl)-3- methoxy-7H-indolo[2,l-a][2]benzazepin-6-yl)-l-cyclopropyl-3-isopropyl-lH- pyrazole-4-carboxylate
• EDC 151 mg, 0.790 mmol
• 13- cyclohexyl-6-(l-cyclopropyl-4-(ethoxycarbonyl)-3-isopropyl-lH-pyrazol-5-yl)-3- methoxy-7H-indolo[2,l-a][2]benzazepine-10-carboxylic acid 320 mg, 0.527 mmol
• propane-2-sulfonamide (227 mg, 1.843 mmol
• DMAP (193 mg, 1.58 mmol) in dichloroethane (5 mL) at room temperature. The reaction was allowed to stir overnight.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 2 min, a hold time of 2 min, and an analysis time of 4 min where solvent A was 5% MeOH / 95% H 2 O / 10 rnM ammonium acetate and solvent B was 5% H 2 O / 95% MeOH / 10 mM ammonium acetate.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• the reaction was allowed to stir for 6 hrs and then was concentrated and diluted with THF (6 mL), EtOH (6 mL), and NaOH (6 mL, 6.00 mmol, IM aq.). The reaction was allowed to stir overnight. An additional amount of NaOH (6 mL, 6.00 mmol, IM aq.) was added and the reaction was allowed to stir for 3 days at rt. An additional amount of NaOH (6 mL, 6.00 mmol, IM aq) was added and the reaction was allowed to stir overnight. The reaction was then heated to 6O 0 C for 7 hrs then allowed to cool and stir at room temperature overnight.
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 3u Cl 8 2.0x30mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 2 min, a hold time of 2 min, and an analysis time of 4 min where solvent A was 10% acetonitrile / 90% H 2 O / 0.1% trifluoroacetic acid and solvent B was 10% H 2 O / 90% acetonitrile / 0.1% trifluoroacetic acid.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• Example KPl Preparation of 13-cyclohexyl-6-(l-cyclopropyl-4-(((2R,6S)-2,6- dimethyl-4-morpholinyl)carbonyl)-3-isopropyl-lH-pyrazol-5-y I)-N- (isopropylsulfonyl)-3-methoxy-7H-indolo[2,l-a][2]benzazepine-10-carboxamide
• LC-MS retention time 2.36 min; m/z (MH+): 782.
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 3u C 18 2.0x30mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 2 min, a hold time of 1 min, and an analysis time of 3 min where solvent A was 10% acetonitrile / 90% H 2 O / 0.1% trifluoroacetic acid and solvent B was 10% H 2 O / 90% acetonitrile / 0.1% trifluoroacetic acid.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• Example KP2 Preparation of 13-cyclohexyl-6-(l-cyclopropyl-3-isopropyl-4- (((3R,5S)-3,4,5-trimethyl-l-piperazinyl)carbonyl)-lH-pyrazol-5-yl)-N-
• HATU (65.0 mg, 0.171 mmol) was added to a stirring solution of 5-(13- cyclohexyl-10-((isopropylsulfonyl)carbamoyl)-3-methoxy-7H-indolo[2,l- a][2]benzazepin-6-yl)-l-cyclopropyl-3-isopropyl-lH-pyrazole-4-carboxylic acid (78 mg, 0.114 mmol), (2S,6R)-l,2,6-trimethylpiperazine dihydrochloride (46 mg, 0.23 mmol), DIEA (80 ⁇ L, 0.456 mmol) in DMF (1 mL) at room temperature.
• the reaction was allowed to stir for 2 hours.
• the reaction was purified by preparative reverse phase ⁇ PLC on a Cl 8 column using a TFA buffered ⁇ 2 O/C ⁇ 3CN gradient, and concentrated to give the titled compound (31 mg, 0.033 mmol, 29 % yield) consistent by LCMS and NMR, with sufficient purity by HPLC.
• LC-MS retention time 1.75 min; m/z (MH+): 795.
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 3u C18 2.0x30mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 2 min, a hold time of 1 min, and an analysis time of 3 min where solvent A was 10% acetonitrile / 90% H 2 O / 0.1% trifluoroacetic acid and solvent B was 10% H 2 O / 90% acetonitrile / 0.1% trifluoroacetic acid.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• Example KP3 Preparation of 13-cyclohexyl-6-(l-cyclopropyl-3-isopropyl-4-((3- methyl-3,8-diazabicyclo[3.2.1]oct-8-yl)carbonyl)-l/f-pyrazol-5-yl)-N-
• HATU (65.0 mg, 0.171 mmol) was added to a stirring solution of 5-(13- cyclohexyl-10-((isopropylsulfonyl)carbamoyl)-3-methoxy-7H-indolo[2,l- a][2]benzazepin-6-yl)-l-cyclopropyl-3-isopropyl-lH-pyrazole-4-carboxylic acid (78 mg, 0.114 mmol), 3-methyl-3,8-diazabicyclo[3.2.1]octane dihydrochloride (45 mg, 0.23 mmol), DIEA (80 ⁇ L, 0.456 mmol) in DMF (1 mL) at room temperature.
• LC-MS retention time 1.78 min; m/z (MH+): 793.
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 3u Cl 8 2.0x30mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 2 min, a hold time of 1 min, and an analysis time of 3 min where solvent A was 10% acetonitrile / 90% H 2 O / 0.1% trifluoroacetic acid and solvent B was 10% H 2 O / 90% acetonitrile / 0.1% trifluoroacetic acid.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• Example KP4 Preparation of 13-cyclohexyl-6-(l-cyclopropyl-3-isopropyl-4-(3-oxa- 8-azabicyclo[3.2.1]oct-8-ylcarbonyl)-lH-pyrazol-5-yl)-N-(isopropylsulfonyl)-3- methoxy-7H-indolo[2, 1 -a] [2]benzazepine- 10-carboxamide
• HATU (65.0 mg, 0.171 mmol) was added to a stirring solution of 5-(13- cyclohexyl-10-((isopropylsulfonyl)carbamoyl)-3-methoxy-7H-indolo[2,l- a][2]benzazepin-6-yl)-l-cyclopropyl-3-isopropyl-lH-pyrazole-4-carboxylic acid (78 mg, 0.114 mmol), 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride (34 mg, 0.23 mmol), DIEA (80 ⁇ L, 0.456 mmol) in DMF (1 mL) at room temperature.
• the reaction was allowed to stir for 2 hours.
• the reaction was purified by preparative reverse phase HPLC on a Cl 8 column using a TFA buffered H 2 OZMeOH gradient, and concentrated to give the titled compound (30 mg, 0.038 mmol, 34 % yield) consistent by LCMS and NMR, with sufficient purity by HPLC.
• LC-MS retention time 2.36 min; m/z (MH+): 780.
• LC data was recorded on a Shimadzu LC-IOAS liquid chromatograph equipped with a PHENOMENEX® Luna 3u C18 2.0x30mm column using a SPD-10AV UV-Vis detector at a detector wave length of 22OnM.
• the elution conditions employed a flow rate of 5 ml/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 2 min, a hold time of 1 min, and an analysis time of 3 min where solvent A was 10% acetonitrile / 90% H 2 O / 0.1% trifluoroacetic acid and solvent B was 10% H 2 O / 90% acetonitrile / 0.1% trifluoroacetic acid.
• MS data was determined using a MICROMASS® Platform for LC in electrospray mode.
• Solvent A 10% MeOH-90% H 2 O-0.1 % TFA;
• Solvent B 90% MeOH-10% H 2 O-0.1% TFA;
• Solvent B 10% H 2 O / 90% MeOH / 0.1% Trifluoroacetic Acid
• Step 1 Preparation of compound 9-1 [00291] To a solution of cyclopropanamine (0.8 g, 14.01 mmol) and TEA (1.673 mL, 12.00 mmol) in ether (20 mL) was added ethyl 2-bromoacetate (1.109 mL, 10 mmol). The reaction mixture was stirred at rt overnight. The solvent was concentrated and the residue was diluted with hexane. The solid was filtered off and the filtrate was concentrated to afford an oil as compound 9-1 (1.432 g, 100%). The crude product was used in next step.
• Step 4 Preparation of compound 9-4 [00294] In a 5 mL microwave vessel was added compound 1-3 (0.694 g, 0.947 mmol), compound 9-3 (0.290 g, 0.947 mmol) and copper(I) iodide (0.018 g, 0.095 mmol). The mixture was degassed with N2, then Tetrakis (0.109 g, 0.095 mmol) was added. The reaction mixture was heated in oil bath at 120 0 C for 5 h. The reaction mixture was cooled to rt, concentrated to remove most solvent.
• Step 6 Preparation of compound 9-6 [00296] To a mixture of compound 9-5 (0.20 g, 0.354 mmol) in THF (3 mL) was added CDI (0.126 g, 0.778 mmol). The mixture was heated at 50 0 C for 0.5 h, then cooled down. Propane-2-sulfonamide (0.13 g, 1.055 mmol) and DBU (0.176 mL, 1.167 mmol) were added. The reaction mixture was stirred at rt overnight.
• the protein was expressed with an 18 amino acid C-terminal truncation to enhance the solubility.
• the E. coli competent cell line BL21(DE3) was used for expression of the protein. Cultures were grown at 37 0 C for ⁇ 4 hours until the cultures reached an optical density of 2.0 at 600 nm. The cultures were cooled to 20 0 C and induced with 1 mM IPTG. Fresh ampicillin was added to a final concentration of 50 ⁇ g/ml and the cells were grown overnight at 20 0 C.
• Cell pellets (3L) were lysed for purification to yield 15-24 mgs of purified NS5B.
• the lysis buffer consisted of 20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 0.5% triton X-IOO, 1 mM DTT, ImM EDTA, 20% glycerol, 0.5 mg/ml lysozyme, 10 mM MgCl 2 , 15 ug/ml deoxyribonuclease I, and Complete TM protease inhibitor tablets (Roche). After addition of the lysis buffer, frozen cell pellets were resuspended using a tissue homogenizer.
• aliquots of the lysate were sonicated on ice using a microtip attached to a Branson sonicator.
• the sonicated lysate was centrifuged at 100,000 x g for lhr at 4 0 C and filtered through a 0.2 ⁇ m filter unit (Corning).
• the protein was purified using three sequential chromatography steps: Heparin sepharose CL-6B, polyU sepharose 4B, and Dual-Glo SP sepharose (Pharmacia).
• the chromatography buffers were identical to the lysis buffer but contained no lysozyme, deoxyribonuclease I, MgCl 2 or protease inhibitor and the NaCl concentration of the buffer was adjusted according to the requirements for charging the protein onto the column.
• Each column was eluted with a NaCl gradient which varied in length from 5-50 column volumes depending on the column type.
• the resulting purity of the enzyme is >90% based on SDS-PAGE analysis.
• the enzyme was aliquoted and stored at -80 0 C.
• HCV RdRp genotype Ib assays were run in a final volume of 60 ⁇ l in 96 well plates (Costar 3912).
• the assay buffer is composed of 20 mM Hepes, pH 7.5, 2.5 mM KCl, 2.5 mM MgCl 2 , 1 mM DTT, 1.6 U RNAse inhibitor (Promega N2515), 0.1 mg/ml BSA (Promega R3961), and 2 % glycerol. All compounds were serially diluted (3 -fold) in DMSO and diluted further in water such that the final concentration of DMSO in the assay was 2%.
• HCV RdRp genotype Ib enzyme was used at a final concentration of 28 nM.
• a polyA template was used at 6 nM, and a biotinylated oligo-dT12 primer was used at 180 nM final concentration. Template was obtained commercially (Amersham 27-4110). Biotinylated primer was prepared by Sigma Genosys. 3H-UTP was used at 0.6 ⁇ Ci (0.29 ⁇ M total UTP). Reactions were initiated by the addition of enzyme, incubated at 30 0 C for 60 min, and stopped by adding 25 ⁇ l of 50 mM EDTA containing SPA beads (4 ⁇ g/ ⁇ l, Amersham RPNQ 0007). Plates were read on a Packard TOPCOUNT® NXT after >lhr incubation at room temperature. Modified HCV NS5B RdRp Enzyme Assay
• a modified enzyme assay was performed essentially as described for the standard enzyme assay except for the following:
• the biotinylated oligo dT12 primer was precaptured on streptavidin-coated SPA beads by mixing primer and beads in assay buffer and incubating at room temperature for one hour. Unbound primer was removed after centrifugation.
• the primer-bound beads were resuspended in 20 mM Hepes buffer, pH 7.5 and used in the assay at final concentrations of 20 nM primer and 0.67 ⁇ g/ ⁇ l beads.
• the FRET peptide (Anaspec, Inc.) (Taliani et al, Anal. Biochem., 240:60- 67 (1996)) contains a fluorescence donor, EDANS, near one end of the peptide and an acceptor, DABCYL, near the other end.
• the fluorescence of the peptide is quenched by intermolecular resonance energy transfer (RET) between the donor and the acceptor, but as the NS3 protease cleaves the peptide the products are released from RET quenching and the fluorescence of the donor becomes apparent.
• RET intermolecular resonance energy transfer
• the assay reagent was made as follows: 5X cell Luciferase cell culture lysis reagent from Promega (#E153A) diluted to IX with dH 2 O, NaCl added to 150 mM final, the FRET peptide diluted to 20 ⁇ M final from a 2 mM stock. [00312] To prepare plates, HCV replicon cells, with or without a Renilla luciferase reporter gene, were trypsinized and placed into each well of a 96-well plate with titrated test compounds added in columns 3 through 12; columns 1 and 2 contained a control compound (HCV protease inhibitor), and the bottom row contained cells without compound. The plates were then placed in a CO 2 incubator at 37 0 C.
• the signal to noise using an endpoint analysis after the reads was at least three-fold.
• plates were rinsed with PBS, 50 ul of DMEM (high glucose) without phenol red was added and plates were then used for luciferase assay using the Promega DUAL-GLO ( I ) Luciferase Assay System.
• Compound analysis was determined by quantification of the relative HCV replicon inhibition and the relative cytotoxicity values.
• cytoxicity values the average Alamar Blue fluorescence signals from the control wells were set as 100% non-toxic. The individual signals in each of the compound test wells were then divided by the average control signal and multiplied by 100% to determine percent cytotoxicity.
• HCV replicon inhibition values an average background value was obtained from the two wells containing the highest amount of HCV protease inhibitor at the end of the assay period. These numbers were similar to those obtained from na ⁇ ve Huh-7 cells.
• HCV Replicon Luciferase Reporter Assay [00316] The HCV replicon luciferase assay was developed to monitor the inhibitory effects of compounds described in the disclosure on HCV viral replication. Utilization of a replicon luciferase reporter assay was first described by Krieger et al (Krieger, N. et al., J. Virology, 75(10):4614-4624 (2001)).
• HUH-7 cells constitutively expressing the HCV replicon, were grown in Dulbecco's Modified Eagle Media (DMEM) (Gibco-BRL) containing 10% Fetal calf serum (FCS) (Sigma) and 1 mg/ml G418 (Gibco-BRL).
• DMEM Dulbecco's Modified Eagle Media
• FCS Fetal calf serum
• G418 G418
• EnduRen as substrate After 3 days incubation at 37 0 C, cells were analyzed for Renilla Luciferase activity using the EnduRen as substrate (Promega cat #E6485).
• the EnduRen substrate was diluted in DMEM and then added to the plates to a final concentration of 7.5 ⁇ M.
• the plates were incubated for 2 hrs at 37°C and then read immediately for 30 seconds with VIEWLUX® Imager (PerkinElmer) using a luminescence program.
• CC50 values were generated by multiplexing the EnduRen-containing plates with CELLTITER-BLUE® (Promega cat # G8082).
• compositions and Methods of Treatment The compounds demonstrate activity against HCV NS5B and can be useful in treating HCV and HCV infection. Therefore, another aspect of the invention is a composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. [00319] Another aspect of the invention is the use of a compound of formula I in the manufacture of a medicament for the treatment of hepatitis C. [00320] Another aspect of the invention is a composition further comprising a compound having anti-HCV activity.
• Another aspect of the invention is a composition where the compound having anti-HCV activity is an interferon. Another aspect of the invention is where the interferon is selected from interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, and lymphoblastoid interferon tau. [00322] Another aspect of the invention is a composition where the compound having anti-HCV activity is a cyclosporin. Another aspect of the invention is where the cyclosporin is cyclosporin A.
• Another aspect of the invention is a composition where the compound having anti-HCV activity is selected from the group consisting of interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, interfering RNA, anti-sense RNA, Imiqimod, ribavirin, an inosine 5'-monophospate dehydrogenase inhibitor, amantadine, and rimantadine.
• interleukin 2 interleukin 6, interleukin 12
• a compound that enhances the development of a type 1 helper T cell response interfering RNA, anti-sense RNA, Imiqimod, ribavirin, an inosine 5'-monophospate dehydrogenase inhibitor, amantadine, and rimantadine.
• Another aspect of the invention is a composition where the compound having anti-HCV activity is effective to inhibit the function of a target selected from HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH, and a nucleoside analog for the treatment of an HCV infection.
• a target selected from HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH, and a nucleoside analog for the treatment of an HCV infection.
• a target selected from HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV
• Another aspect of the invention is a method of inhibiting the function of the HCV replicon comprising contacting the HCV replicon with a compound of formula I or a pharmaceutically acceptable salt thereof.
• Another aspect of the invention is a method of inhibiting the function of the HCV NS5B protein comprising contacting the HCV NS5B protein with a compound of formula I or a pharmaceutically acceptable salt thereof.
• Another aspect of the invention is a method of treating an HCV infection in a patient comprising administering to the patient a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.
• the compound is effective to inhibit the function of the HCV replicon.
• the compound is effective to inhibit the function of the HCV
• Another aspect of the invention is a method of treating an HCV infection in a patient comprising administering to the patient a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, in conjunction with (prior to, after, or concurrently) another compound having anti-HCV activity.
• Another aspect of the invention is the method where the other compound having anti-HCV activity is an interferon.
• Another aspect of the invention is the method where the interferon is selected from interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, and lymphoblastoid interferon tau.
• Another aspect of the invention is the method where the other compound having anti-HCV activity is a cyclosporin.
• Another aspect of the invention is the method where the cyclosporin is cyclosporin A.
• Another aspect of the invention is the method where the other compound having anti-HCV activity is selected from interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, interfering RNA, anti-sense RNA, Imiqimod, ribavirin, an inosine 5'-monophospate dehydrogenase inhibitor, amantadine, and rimantadine.
• Another aspect of the invention is the method where the other compound having anti-HCV activity is effective to inhibit the function of a target selected from the group consisting of HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH, and a nucleoside analog for the treatment of an
• a target selected from the group consisting of HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH, and a nucleoside analog for the treatment of an
• Another aspect of the invention is the method where the other compound having anti-HCV activity is effective to inhibit the function of target in the HCV life cycle other than the HCV NS5B protein.
• “Therapeutically effective” means the amount of agent required to provide a meaningful patient benefit as understood by practitioners in the field of hepatitis and HCV infection. A therapeutically effective amount is that which is needed to provide a meaningful patient benefit.
• compositions comprised of a therapeutically effective amount of a compound or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier and may contain conventional excipients.
• Pharmaceutically acceptable carriers are those conventionally known carriers having acceptable safety profiles.
• compositions encompass all common solid and liquid forms including for example capsules, tablets, lozenges, and powders as well as liquid suspensions, syrups, elixirs, and solutions. Compositions are made using common formulation techniques, and conventional excipients (such as binding and wetting agents) and vehicles (such as water and alcohols) are generally used for compositions. See, for example, Remington 's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, PA (1985). [00341] Solid compositions are normally formulated in dosage units and compositions providing from about 1 to 1000 mg of the active ingredient per dose are preferred. Some examples of dosages are 1 mg, 10 mg, 100 mg, 250 mg, 500 mg, and 1000 mg.
• Liquid compositions are usually in dosage unit ranges. Generally, the liquid composition will be in a unit dosage range of 1-100 mg/rnL. Some examples of dosages are 1 mg/mL, 10 mg/mL, 25 mg/mL, 50 mg/mL, and 100 mg/mL. Generally, other agents will be present in a unit range similar to agents of that class used clinically. Typically, this is 1-100 mg/mL.
• the invention encompasses all conventional modes of administration; oral and parenteral methods are preferred. Generally, the dosing regimen will be similar to other agents used clinically. Typically, the daily dose will be 1-100 mg/kg body weight daily. Generally, more compound is required orally and less parenterally. The specific dosing regime, however, will be determined by a physician using sound medical judgment.
• the invention also encompasses methods where the compound is given in combination therapy. That is, the compound can be used in conjunction with, but separately from, other agents useful in treating hepatitis and HCV infection. In these combination methods, the compound will generally be given in a daily dose of 1-100 mg/kg body weight daily in conjunction with other agents. The other agents generally will be given in the amounts used therapeutically. The specific dosing regime, however, will be determined by a physician using sound medical judgment. [00345] Some examples of compounds suitable for compositions and methods are listed in Table 2.

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