Classifications

• • 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
  • C07D471/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • 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
  • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

• the invention relates to compounds, compositions, and methods for the treatment of human immunodeficiency virus (HIV) infection. More particularly, the invention provides novel inhibitors of HIV, pharmaceutical compositions containing such compounds, and methods for using these compounds in the treatment of HIV infection. The invention also relates to methods for making the compounds hereinafter described.
• HIV human immunodeficiency virus
• AIDS Acquired immunodeficiency syndrome
• HIV-infected individuals consists of a combination of approved anti-retroviral agents. Close to four dozen drugs are currently approved for HIV infection, either as single agents, fixed dose combinations or single tablet regimens; the latter two containing 2-4 approved agents. These agents belong to several different classes, targeting either a viral enzyme or the function of a viral protein during the virus replication cycle.
• agents are classified as either nucleotide reverse transcriptase inhibitors (NRTIs), non nucleotide reverse transcriptase inhibitors (NNRTIs), protease inhibitors (Pis), integrase strand transfer inhibitors (INSTIs), or entry inhibitors (one, maraviroc, targets the host CCR5 protein, while the other, enfiivirtide, is a peptide that targets the gp41 region of the viral gpl60 protein).
• a pharmacokinetic enhancer cobicistat or ritonavir
• ARVs antiretroviral agents
• the present invention discloses a compound of Formula I, or a pharmaceutically acceptable salt thereof:
• R°, R 1 , and R 2 are each independently hydrogen, Cl, F, -OMe, -CN, -Ci-C 3 alkyl, or -C3-C5 cycloalkyl, wherein -C1-C3 alkyl may be optionally substituted with from 1-3 fluorines;
• G 2 is hydrogen, 6 membered aryl, 5-6 membered heteroaryl, -CVCx alkyl. -C3-C7 cycloalkyl, or -C1-C3 alkyl wherein -C1-C3 alkyl is substituted with G 5 ;
• G 3 is hydrogen, methyl, fluoro, chloro, phenyl, OC1-C3 alkyl, or OPh;
• G 4 is hydrogen, methyl, fluoro, chloro, phenyl, OC1-C3 alkyl, or OPh;
• G 5 is -OG 6 , -C3-C7 cycloalkyl, 6 membered aryl, 5 membered heteroaryl, 4-7 membered heterocycle, -C(0)N(G 7 )(G 8 ), C(0)OH, -S0 2 (G 7 ), -N(G 7 )(G 8 ), -SOi-morpholine, C(O)- morpholine, or one of the following:
• G 6 is -C1-C6 alkyl, -C3-C7 cycloalkyl, 6 membered aryl, 5 membered heteroaryl, 4-7 membered heterocycle;
• G 7 is -C1-C6 alkyl or C3-C6 cycloalkyl
• G 8 is -C1-C6 alkyl or C3-C6 cycloalkyl
• R 3 is hydrogen, Cl, or F
• R 4 is hydrogen, C1-C3 alkyl, or cyclopropyl wherein cyclopropyl or C1-C3 cycloalkyl is optionally substituted with 1-3 fluorines;
• R 5 is Ci-Ce alkyl, C 3 -C 6 cycloalkyl, orN(G 7 )(G 8 );
• W is selected from:
• R 6 is methyl optionally substituted with 1 to 3 fluorines.
• the present invention discloses a pharmaceutical composition comprising a compound of Formula I or a pharmaceutically acceptable salt thereof.
• the present invention discloses a method of treating HIV infection comprising administering a composition comprising a compound of Formula I or a pharmaceutically acceptable salt thereof to a patient.
• the present invention discloses a compound of Formula (I) or pharmaceutically acceptable salt thereof for use in therapy.
• the present invention discloses a compound of Formula (I) or pharmaceutically acceptable salt thereof for use in treating HIV infection.
• the present invention discloses the use of a compound of Formula (I) or pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of HIV infection.
• R°, R 1 , and R 2 are positioned as shown below
• R°, R 1 , and R 2 are each independently selected from hydrogen, Cl, F, -OCH 3 , -CN, or - CH 3 with the proviso that substituents Cl, -OMe, and -CH 3 may not be used more than twice and substituent -CN may not be used more than once.
• R°, R 1 , and R 2 are positioned as shown above and R°, R 1 , and R 2 are each independently selected from hydrogen, F, Cl or -CH 3 with the proviso that at least one of the groups R°, R 1 and R 2 is hydrogen.
• R°, R 1 , and R 2 are positioned as shown above and R° is fluorine, R 1 is fluorine, and R 2 is hydrogen.
• G 3 and G 4 are independently selected from hydrogen, methyl, fluoro, chloro, or OC1-C2 alkyl with the proviso that at least one of G 3 and G 4 must be hydrogen;
• R 3 is hydrogen, Cl, or F;
• R 4 is hydrogen, C 1 -C 3 alkyl, or cyclopropyl wherein C1-C 3 alkyl is optionally substituted with 1-3 fluorines and cyclopropyl is optionally substituted with 1-2 fluorines; and
• R 5 is C1-C 3 alkyl or C 3 -C4 cycloalkyl.
• R 3 is chloride, R 4 is methyl, 2,2-difluoroethyl, or 2,2,2-trifluoroethyl; and R 5 is methyl or cyclopropyl.
• W is
• R 6 is methyl optionally substituted with one fluorine and R 7 is methyl optionally substituted with 1 to 3 fluorines.
• G 2 is hydrogen, -CVCx alkyl, or -C1-C3 alkyl wherein -C1-C3 alkyl is substituted with G 5 ;
• G 5 is -OG 6 , -C3-C6 cycloalkyl, 6 membered aryl, 5 membered heteroaryl, 4-7 membered heterocycle, -C(0)N(G 7 )(G 8 ), -S02(G 7 ), -N(G 7 )(G 8 ), -SO2- morpholine, C(0)-morpholine, or one of the following:
• G 7 is -C1-C3 alkyl or C3-C6 cycloalkyl; and G 8 is -C1-C3 alkyl or C3-C6 cycloalkyl.
• G 2 is one of the following:
• the compounds and salts of this invention have the stereochemistry shown below
• the compounds and salts of this invention have the stereochemistry shown below
• Preferred compounds and salts of this invention include:
• Preferred compounds and salts of this invention include:
• salts of compounds of formula (I) are pharmaceutically acceptable. Such salts may be acid addition salts or base addition salts; for a review of suitable pharmaceutically acceptable salts see Berge et al, J. Pharm, Sci., 66, 1-19, 1977.
• acid addition salts are selected from the hydrochloride, hydrobromide, hydroiodide, sulphate, bisulfate, nitrate, phosphate, hydrogen phosphate, acetate, benzoate, succinate, saccharate, fumarate, maleate, lactate, citrate, tartrate, gluconate, camsylate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate.
• base addition salts include metal salts (such as sodium, potassium, aluminium, calcium, magnesium and zinc) and ammonium salts (such as isopropylamine, diethylamine, diethanolamine salts).
• metal salts such as sodium, potassium, aluminium, calcium, magnesium and zinc
• ammonium salts such as isopropylamine, diethylamine, diethanolamine salts.
• Other salts such as trifluoroacetates and oxalates
• All possible stoichiometric and non-stoichiometric forms of the salts of compounds of formula (I) are included within the scope of the invention.
• Acid and base addition salts may be prepared by the skilled chemist, by treating a compound of formula (I) with the appropriate acid or base in a suitable solvent, followed by
• the invention includes all stereoisomeric forms of the compounds including enantiomers and diastereomers including atropisomers.
• the term homochiral is used as a descriptor, per accepted convention, to describe a structure which is a single stereoisomer. Absolute stereochemistry was not assigned in all cases. Thus, the compound is drawn at the chiral center as unspecified but labelled as homochiral and in the procedures it is identified by its properties such as for example first eluting off a normal or chiral column per the conventions of chemists. It should be noted that the provided experimental procedures teach how to make the exact compound even if not drawn with absolute configuration. Methods of making and separating stereoisomers are known in the art.
• the invention includes all tautomeric forms of the compounds.
• the invention includes atropisomers and rotational isomers.
• the scope of any instance of a variable substituent can be used independently with the scope of any other instance of a variable substituent.
• the invention includes combinations of the different aspects.
• the stereochemistry of all the centers were not unambiguously assigned so they can be referred to as diastereomer 1 and diastereomer 2 or enantiomer 1 or enantiomer 2 etc. and these are understood by chemists skilled in the art.
• atropisomers can be observed and these are understood to convert at slow or fast rates or even not at all depending on the conditions for handling the compound.
• Atropisomers are referred to as mixtures of atropisomers where they interconvert at ambient temperatures or as atropisomer 1 and atropisomer 2 where they were isolated. Since the compounds are identified by their properties rather than exact structural assignment from a crystal structure, it is understood in the art that where not specified, atropisomers are covered and inferred to be covered by the chemical structure.
• preferred routes of administration are oral and by injection to deliver subcutaneously. Therefore, preferred pharmaceutical compositions are those compositions suitable for these routes of administration, for example tablets or injectable compositions.
• the compounds of this invention are believed to act as Capsid Inhibitors.
• the compounds of the present invention and their salts, solvates, or other pharmaceutically acceptable derivatives thereof may be employed alone or in combination with other therapeutic agents.
• the compounds of the present invention and any other pharmaceutically active agent(s) may be administered together or separately and, when administered separately, administration may occur simultaneously or sequentially, in any order.
• the amounts of the compounds of the present invention and the other pharmaceutically active agent(s) and the relative timings of administration will be selected to achieve the desired combined therapeutic effect.
• the administration in combination of a compound of the present invention and salts, solvates, or other pharmaceutically acceptable derivatives thereof with other treatment agents may be in combination by administration concomitantly in: (1) a unitary pharmaceutical composition including multiple compounds; or (2) separate pharmaceutical compositions each including one of the compounds.
• the combination may be administered separately in a sequential manner wherein one treatment agent is administered first and the other second or vice versa, and the different agents could be administered on different schedules if appropriate.
• Such sequential administration may be close in time or remote in time.
• the amounts of the compound(s) of Formulas I, II, or III or salts thereof and the other pharmaceutically active agent(s) and the relative timings of administration will be selected to achieve the desired combined therapeutic effect.
• the compounds of the present invention may be used in combination with one or more agents useful in the prevention or treatment of HIV.
• the compounds of the invention according to the various embodiments can be made by various methods available in the art, including those of the following schemes in the specific examples which follow.
• the structure numbering and variable numbering shown in the synthetic schemes may be distinct from, and should not be confused with, the structure or variable numbering in the claims or the rest of the specification.
• the variables in the schemes are meant only to illustrate how to make some of the compounds of the invention. Abbreviations used in the schemes generally follow conventions used in the art.
• reaction mixture (became a clear solution after T3P addition) was stirred at -25 °C to 10 °C over 4.5 h, then N-(7-amino-4-chloro-l- methyl-lH-indazol-3-yl)-N-(4-methoxybenzyl)methanesulfonamide (6 g, 15.19 mmol) was added and the mixture was stirred for 18 h while warming to rt.
• the reaction mixture was diluted with ethyl acetate, washed with 1N NaOH, then water, then 0.5 M citric acid, then water, then dried over NaiSOr and concentrated in vacuo.
• reaction mixture was stirred for 2 h after which the reaction mixture was diluted with water and extracted with ethyl acetate. The combined EtOAc extractions were washed with brine, dried over Na2SC>4, and concentrated in vacuo.
• the crude product was purified via silica gel flash
• H2SO4 (0.425 L, 0.34 V) and 70% HNO3 (0.85 kg, 13.49 mol, 1.30 equiv.) at 0 °C] was added to the above reaction mixture at below 10 °C [Note: Reaction is slightly exothermic (3-6 °C); so that addition is preferred at lower temperature].
• the reaction mixture was stirred at 5-10 °C for 2-3 h. After completion of the reaction (monitored by TLC), it was quenched with ice cold water (18.75 L, 15 V) at below 25 °C. Then the reaction mass was allowed warm to room temperature and stirred for 2 h. The solids were isolated by filtration and then were washed with water (2.5 L, 2.0 V).
• Step-2a To a solution of DMSO (5.9 L, 5.0 V)) in a round-bottom flask was added 2,6- dichloro-3-nitrobenzaldehyde (1.17 kg, 5.31 mol, 1.0 equiv.) at room temperature. After being stirred for 30 min at room temperature, hydroxylamine hydrochloride (0.63 kg, 9.04 mol, 1.70 equiv.) was added and the reaction mass was stirred at room temperature for 3 h. After completion of the reaction (monitored by TLC), the reaction mass was quenched by the addition of ice-cold water (18.0 L, 15.0 V) added at a rate sufficient to maintain the temperature below 30 °C (Observation: Solids formed upon water addition).
• the reaction mass was stirred at room temperature for 60-90 min.
• the solids were isolated by filtration; washed with water (2.5 L, 2.0 V); followed by washing with a mixture of acetone and hexanes (6.0 L, 1 : 1 ratio). Bulk residual water was removed from the solids by maintaining vacuum filtration for 60-90 min.
• the wet solid was initially air dried and then finally dried in a hot air oven at 50-55 °C for 10-12 h (until moisture content was not more than 1.0 %) to get the dried target product, 2,6-dichloro-3-nitrobenzaldehyde oxime (1.22 kg, 92% yield) as an off-white solid.
• the crude product (which contains 10-20% of 2,6-dichloro-3- nitrobenzonitrile) was used directly in the next step without further purification.
• Step-2b To a stirred solution of the crude oxime (preparation described above, 1.13 kg, 4.80 mol, 1.0 equiv.) in DCM (9.04 L, 8.0 V) at 0-5 °C was added triethylamine (“TEA”,
• the solids were isolated via filtration and then were washed with water (2.25 L, 3.0 V).
• the wet solid was washed with a 1 : 1 ratio mixture of acetone (1.875 L, 2.5 V) and hexanes (1.875 L, 2.5 V).
• Bulk residual water was removed from the solids by maintaining vacuum filtration for 60-90 min.
• the wet solid was finally dried in a hot air oven for 7-8 h at 50 °C (until moisture content reaches below 1.5%) to get the dried product, 4-chloro-7-nitro-li/-indazol-3 -amine (549.0 g, 75% yield) as a brick red- colored solid.
• reaction temperature was slowly raised to room temperature and stirring was continued an additional 2 h at the same temperature.
• reaction mass was quenched by the addition of ice-cold water (15.0 L, 30.0 V) and the resulting mixture was then stirred for 6-8 h at room temperature.
• the solids were isolated via filtration and were then washed with water (1.5 L, 3.0 V).
• the wet solid was washed with IPA (1.5 L, 3.0 V) followed by hexanes (1.0 L, 2.0 V). Bulk residual water was removed from the solids by maintaining vacuum filtration for 60-90 min.
• Step 5 Preparation of '-(4-chloro- 1 -methyl-7-nitro- l//-indazol-3-yl)methanesulfonamide
• Step 5a To a solution of 4-chloro- l-methyl-7-nitro-li/-indazol-3 -amine (625.0 g, 2.76 mol, 1.0 equiv.) in DCM (6.25 L, 10.0 V) at 0-5 °C. was added triethylamine (TEA) (837.0 g, 8.27 mol, 3.0 equiv.); followed by the addition of 4-dimethylaminopyridine (DMAP) (20.60 g, 0.165 mol, 0.06 equiv.).
• TEA triethylamine
• DMAP 4-dimethylaminopyridine
• Step 6 Preparation of '-(4-chloro- 1 -methyl-7-nitro- l /-indazol-3-yl)-/V-(4- methoxybenzyl)methanesulfonamide
• the mixture was poured into ice cold water (19.05 L, 30.0 V) [Note: Slow quenching with vigorous stirring is preferred to avoid clumping as the product precipitates] .
• the resulting solids were isolated via filtration and washed with water (1.90 L, 3.0 V); then the solids were washed with hexanes (1.27 L, 2.0 V). Bulk residual water was removed from the solids by maintaining vacuum filtration for 60-90 min.
• the isolated solid was dissolved in Ethyl acetate (12.7 L, 20.0 V) and charcoal was added (63.5 g). The mixture was heated to 60-70 °C and then stirred for 30-45 min. at that temperature.
• Step 7 Preparation of '-(7-Amino-4-chloro- 1 -methyl- l /-indazol-3-yl)-/V-(4- methoxybenzyl)methanesulfonamide
• Step 1 Preparation of 4-chloro- 1 -(2, 2-difluoroethyl)-7-nitro-li7-indazol-3 -amine
• the solids were isolated via filtration and were then washed with water (540 mL, 3.0 V). The wet solid was washed with hexanes (0.9 L, 5.0 V). Bulk residual water was removed from the solids by maintaining vacuum filtration for 60-90 min. The wet solid was dried in a hot air oven for 7-8 h at 50 °C (until the moisture content was below 1.0%).
• the isolated material, 4-chloro- 1 -(2.2-difluorocthyl)-7-nitro- l//-indazol-3-aminc (160 g, 71% yield), was used in the next step without further purification.
• Step 2 Preparation of A'-(4-chloro- 1 -(2.2-difluoroethyl)-7-nitro- l//-indazol-3-yl)methane sulfonamide
• Step 2a To a solution of 4-chloro- l-(2,2-difluoroethyl)-7-nitro-li/-indazol-3 -amine
• Step 2b To a stirred solution of A'-(4-chloro- 1 -(2.2-difluoroethyl)-7-nitro- l//-indazol-
• the mixture was poured into ice cold water (4.8 L, 60.0 V) [Note: Slow quenching with vigorous stirring is preferred to avoid clumping as the product precipitates].
• the resulting solids were isolated via filtration and washed with water (480 mL, 3.0 V); then the solids were washed with hexanes (320 mL, 2.0 V). Bulk residual water was removed from the solids by maintaining vacuum filtration for 1-2 h.
• the isolated solid was dissolved in ethyl acetate (1.6 L, 10.0 V) and charcoal was added (16.0 g). The mixture was heated to 60-70 °C and then stirred for 30-45 min. at that temperature.
• the mixture was filtered while hot (40-50 °C) through a pad of Celite and the Celite pad was then extracted with ethyl acetate (800 mL, 5.0 V).
• the combined filtrates were concentrated to dryness under reduced pressure at below 50 °C.
• ethyl acetate 160 mL, 1.0 V.
• the suspension was stirred for 30 min.
• the solids were isolated via filtration and then were washed with hexanes (320 mL, 2.0 V). Residual water was removed from the solids by maintaining vacuum filtration for 45-60 min.
• Step 1 Preparation of A'-(4-chloro- 1 -(2.2-difluorocthyl)-7-nitro- l /-indazol-3- yl)cyclopropanesulfonamide
• Step 2 Preparation of A'-(4-chloro- 1 -(2.2-difluoroethyl)-7-nitro- l//-indazol-3-yl)-A'-(4- methoxybenzyl)cyclopropanesulfonamide
• the mixture was poured into ice cold water (3.0 L, 30.0 V) [Note: Slow quenching with vigorous stirring is preferred to avoid clumping as the product precipitates] .
• the resulting solids were isolated via filtration and washed with water (300 mL, 3.0 V); then the solids were washed with hexanes (300 mL, 3.0 V). Bulk residual water was removed from the solids by maintaining vacuum filtration for 1-2 h.
• the wet solid was dissolved in ethyl acetate (500 mL, 5.0 V) and charcoal was added (10.0 g). The mixture was heated to 60-70 °C and then stirred for 30-45 minutes at that temperature.
• Step 3 Preparation of A'-(7-amino-4-chloro- 1 -(2.2-difluorocthyl)- l//-indazol-3-yl)-A'-(4- methoxybenzyl)cyclopropanesulfonamide
• Step 1 Preparation of 4-chloro-7-nitro- 1 -(2.2.2-trifluorocthyl)- l//-indazol-3-aminc
• the mixture was stirred for 5-10 min, then to the stirred mixture at 10-15 °C was added 2,2,2- trifluoroethyl trifluoromethanesulfonate (60.18 g, 0.26 mol, 1.1 equiv.) at a rate sufficient to maintain the reaction mass below 20 °C (Note: slow addition is preferred for obtaining more favorable regio-selectivity).
• the reaction mass was allowed to slowly warm to room temperature and was then stirred at the same temperature for 2 h. After completion of the reaction (monitored by TLC), the reaction mass was quenched via the addition of ice-cold water (1.5 L, 30.0 V) and the resulting mixture was allowed to warm to room temperature with stirring for 6-8 h.
• the solids were isolated via filtration and were then washed with water (150 mL, 3.0 V). The wet solid was washed with hexanes (250 mL, 5.0 V) and then bulk residual water was removed from the solids by maintaining vacuum filtration for 60-90 min. The wet solid was dried in a hot air oven for 7-8 h at 50 °C (until the moisture content was below 1.0%).
• the isolated material 4-chloro-7-nitro- 1 -(2.2.2-trifluoroethyl)- l//-indazol- 3-amine (45.0 g, 60% yield), was used directly in the next step without further purification.
• Step 2 Preparation of v'-(4-chloro-7-nitro- l -(2.2.2-trifluoroethyl)- l//-indazol-3- yl)methanesulfonamide
• Step 2a To a solution of 4-chloro-7-nitro-l -(2,2,2-trifluoroethyl)- li/-indazol-3 -amine
• the mixture was diluted with water (200 mL, 10.0 V) and then stirred at room temperature for 15 min.
• the organic layer was separated, and the aqueous layer was extracted with DCM (200 mL, 10.0 V).
• the combined organic layers were washed with 10% brine solution (60 mL, 3.0 V), dried over NaiSCri, filtered, and concentrated to afford the crude solids.
• Step 2b To a stirred solution of/V-(4-chloro-7-nitro-l -(2,2,2-trifluoroethyl)- IH- indazol-3-yl)-/V-(methylsulfonyl)methanesulfonamide (entirety of the material prepared above) in ethanol (200 mL, 10.0 V) at room temperature was added slowly aq. 5% NaOH solution (140 mL, 7.0 V) [Note: Slow addition is preferred via dropping funnel]. The reaction mass was stirred at the same temperature for 2 h.
• Example preparation for TLC analysis An aliquot of the reaction solution (-1.0 ml) was acidified by the addition of aq. 2.0 N HC1 to reach pH 2-3; then the mixture was extracted with ethyl acetate and the organic phase was analyzed by TLC], the reaction mass was cooled to 0-5 °C and the pH was adjusted to 2-3 by the addition of aq. 2.0 N HC1 (100 mL, 5.0 V) while maintain the temperature below 10 °C [Note: Precipitation occurred upon addition of HC1 and increased with stirring] . The reaction mixture was warmed to room temperature and then stirred for 1.5-2.0 h.
• Step 3 Preparation of '-(4-chloro-7-nitro- l -(2.2.2-trifluorocthyl)- l//-indazol-3-yl)- '-(4- methoxybenzyl)methanesulfonamide
• the mixture was poured into ice cold water (2.0 L, 40.0 V) [Note: Slow quenching with vigorous stirring is preferred to avoid clumping as the product precipitates] .
• the resulting solids were isolated via filtration and washed with water (150 mL, 3.0 V); then the solids were washed with hexanes (150 mL, 3.0 V). Bulk residual water was removed from the solids by maintaining vacuum filtration for 1-2 h.
• the solids were dissolved in ethyl acetate (500 mL, 10.0 V) and to the solution was added charcoal (5.0 g). The mixture was heated to 60-70 °C and then stirred at that temperature for 30-45 min.
• the mixture was filtered while hot (40-50 °C) through a pad of Celite and the Celite pad was extracted with ethyl acetate (250 mL, 5.0 V). The combined filtrate was concentrated to dryness under reduced pressure at below 50 °C. The solids were combined with ethyl acetate
• Step 4 Preparation of '-(7-amino-4-chloro- 1 -(2,2,2-trifluoroethyl)- l /-indazol-3-yl)-/V-(4- methoxybenzyl)methanesulfonamide
• the reaction mixture was stirred at room temperature for 3 h. After completion of the reaction (monitored by in-process TLC/HPLC), the mixture was diluted with ethyl acetate (1.0 L, 20.0 V) and water (250 mL, 5.0 V). The mixture was stirred for 15 min. The mixture was filtered through a pad of Celite and the Celite pad was extracted with ethyl acetate (250 mL, 5.0 V). The bi -phasic filtrate was partition and the organic layer was reserved while the aqueous layer was extracted with ethyl acetate (500 mL, 10.0 V).
• Step 1
• reaction mixture was diluted with EtOAc and then washed with aq. 0.5 M citric acid, dried over NaiSCri and concentrated in vacuo.
• the resulting residue was purified on silica gel (330 g RediSep column) using 0-45% ethyl acetate in hexanes over 15 CV, then at 45 % EtOAc for 10 CV.
• reaction mixture was stirred for 1 h at rt, then the mixture was directly subjected to silica gel column chromatography (40 g RediSep column) using 10-100 % ethyl acetate in hexanes over 20 CV.
• Wavelength 215 and 254 nm.
• ESI+ Range 150 to 1500 Dalton.
• System Agilent 1290 Infinity II.
• Wavelength 215 and 254 nm.
• ESI+ Range 150 to 1500 Dalton.
• System Agilent 1290 Infinity II.
• IUPAC Chemical Names The IUPAC chemical names for each example are listed below. At this time these names are not recognized by common software such tools such as ChemDraw or JChem. Therefore, the chemical names used throughout the Examples section above were generated with ChemDraw and then appended with the correct P/M designation. The chemical names above can be converted to chemical structures using ChemDraw after the P/M nomenclature— e.g.,“(3P)-“— is removed.
• HIV cell culture assay - MT-2 cells, 293T cells and the proviral DNA clone of NL4-3 virus were obtained from the NIH AIDS Research and Reference Reagent Program.
• MT-2 cells were propagated in RPMI 1640 media supplemented with 10% heat inactivated fetal bovine serum (FBS), 100 pg/ml penicillin G and up to 100 units/mL streptomycin.
• FBS heat inactivated fetal bovine serum
• the 293T cells were propagated in DMEM media supplemented with 10% heat inactivated FBS, 100 pg/mL penicillin G and 100 pg/mL streptomycin.
• the recombinant virus was prepared through transfection of the recombinant NL4-3 proviral clone into 293T cells using Transit-293 Transfection Reagent from Mirus Bio LLC (Madison, WI). Supematent was harvested after 2-3 days and the amount of virus present was titered in MT-2 cells using luciferase enzyme activity as a marker by measuring luciferase enzyme activity.
• Luciferase was quantitated using the EnduRen Live Cell Substrate from Promega (Madison, WI). Antiviral activities of compounds toward the recombinant virus were quantified by measuring luciferase activity in MT-2 cells infected for 4-5 days with the recombinant virus in the presence of serial dilutions of the compound.
• cytotoxicity and the corresponding CC50 values were determined using the same protocol as described in the antiviral assay except that uninfected cells were used. Cytotoxicity was assessed on day 4 in uninfected MT2 cells by using an XTT (2,3-bis[2- Methoxy-4-nitro-5 -sulfophenyl] -2H-tetrazolium-5 -carboxy anilide inner salt)-based colorimetric assay (Sigma- Aldrich, St Louis, Mo).

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• 2019
  • 2019-09-17 JP JP2021515492A patent/JP7433303B2/ja active Active
  • 2019-09-17 US US17/273,748 patent/US11919897B2/en active Active
  • 2019-09-17 WO PCT/IB2019/057814 patent/WO2020058844A1/en not_active Ceased
  • 2019-09-17 EP EP19773190.4A patent/EP3853228A1/en not_active Withdrawn

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