WO2009075960A1 - Ccr5 antagonists as therapeutic agents - Google Patents

Ccr5 antagonists as therapeutic agents Download PDF

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Publication number
WO2009075960A1
WO2009075960A1 PCT/US2008/081672 US2008081672W WO2009075960A1 WO 2009075960 A1 WO2009075960 A1 WO 2009075960A1 US 2008081672 W US2008081672 W US 2008081672W WO 2009075960 A1 WO2009075960 A1 WO 2009075960A1
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WIPO (PCT)
Prior art keywords
methyl
amιno
oxy
fluorophenyl
pyrιdιnyl
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PCT/US2008/081672
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French (fr)
Inventor
Wieslaw Mieczyslaw Kazmierski
Pek Yoke Chong
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Smithkline Beecham Corporation
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Publication of WO2009075960A1 publication Critical patent/WO2009075960A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

  • HIV human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • ARC AIDS-related complex
  • CCR5 In addition to CD4, HIV requires a co-receptor for entry into target cells
  • the chemokine receptors function together with CD4 as co-receptors for HIV
  • the chemokine receptors CXCR4 and CCR5 have been identified as the mam co-receptors for HIV-1 CCR5 acts as a major co-receptor for fusion and entry of macrophage-tropic HIV into host cells
  • CCR5 antagonists are thought to be useful as therapeutic agents active against HIV Nitrogen-containing heterocyclic compounds that are CCR5 antagonists are disclosed in
  • the present invention features compounds that are useful in the inhibition of HIV replication, the prevention of infection by HIV, the treatment of infection by HIV and in the treatment of AIDS and/or ARC, either as pharmaceutically acceptable salts or pharmaceutical composition ingredients
  • the present invention further features methods of treating AIDS, methods of preventing infection by HIV, and methods of treating infection by HIV as monotherapy or in combination with other antivirals, anti-infectives, immunomodulators, antibiotics or vaccines
  • the present invention also features pharmaceutical compositions, comprising the above-mentioned compounds that are suitable for the prevention or treatment of CCR5-related diseases and conditions
  • the present invention further features processes for making the above-mentioned compounds
  • the present invention features a compound selected from the group consisting of 5- ⁇ [((3-fluorophenyl) ⁇ 1-[(2-methyl-6- ⁇ [4-(4-morphol ⁇ nylmethyl)phenyl]oxy ⁇ -3- pyridinyljmethylj ⁇ -piperidinyljaminojcarbonyljaminoj ⁇ -pyridmecarboxamide b ⁇ s(tr ⁇ fluoroacetate),
  • pharmaceutically acceptable carrier refers to a carrier that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the therapeutic agent
  • Pharmaceutically acceptable salts of the compounds according to the invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases
  • suitable acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, glutamic, maleic, mandelic, phosphoric, glycollic, lactic, salicyclic, succinic, toluene- p-sulfonic, tartaric, acetic, citric, orotic, toluenesulfonic, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfon ⁇ c and benzenesulfonic acids
  • Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts
  • Salts derived from appropriate bases include alkali metal (e g sodium), alkaline earth metal (e g , magnesium), ammonium, NW 4 + (wherein W is C 1 ⁇ alkyl) and other amine salts
  • Physiologically acceptable salts of a hydrogen atom or an ammo group include salts of organic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic and succinic acids, organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids and inorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamic acids
  • Physiologically acceptable salts of a compound with a hydroxy group include the anion of said compound in combination with a suitable cation such as Na + , NH 4 + , and NW 4 + (wherein W is a C ⁇ alky
  • any reference to any of the above compounds also includes a reference to a pharmaceutically acceptable salt thereof
  • Salts of the compounds of the present invention may be made by methods known to a person skilled in the art
  • treatment of a compound of the present invention with an appropriate base or acid in an appropriate solvent will yield the corresponding salt
  • the present invention features compounds according to the invention for use in medical therapy, for example for the treatment or prophylaxis of viral infections such as an HIV infections and associated conditions
  • Reference herein to treatment extends to prophylaxis as well as the treatment of established infections, symptoms, and associated clinical conditions such as AIDS related complex (ARC), Kaposi's sarcoma, and AIDS dementia
  • the present invention features use of the compounds of the present invention in the manufacture of a medicament for the treatment or prophylaxis of a CCR5-related disease or condition, for example, a viral infection, for example, an HIV infection
  • a viral infection for example, an HIV infection
  • the present invention provides a method for the treatment or prevention of the symptoms or effects of a viral infection in an infected animal, for example, a mammal including a human, which comprises treating said animal with a pharmaceutically effective amount of a compound according to the invention
  • the viral infection is a retroviral infection, in particular an HIV infection
  • a further aspect of the invention includes a method for the treatment or prevention of the symptoms or effects of an HBV infection
  • the compounds according to the invention may also be used in adjuvant therapy in the treatment of HIV infections or HIV-associated symptoms or effects, for example Kaposi's sarcoma
  • the compounds of the present invention may also be used in the prevention or treatment of other CCR5-related diseases and conditions, including neuropathic pain, multiple sclerosis, rheumatoid arthritis, autoimmune diabetes, chronic implant rejection, asthma, rheumatoid arthritis, Crohns Disease, inflammatory bowel disease, chronic inflammatory disease, glomerular disease, nephrotoxic serum nephritis, kidney disease, Alzheimer's Disease , autoimmune encephalomyelitis, arterial thrombosis, allergic rhinitis, arteriosclerosis, Sjogren's syndrome (dermatomyositis), systemic lupus erythematosus, graft rejection, cancers with leukocyte infiltration of the skin or organs, human papilloma virus infection, prostate cancer, wound healing, amyotrophic lateral sclerosis, immune mediated disorders
  • CCR5-related diseases and conditions including neuropathic pain, multiple sclerosis, rheumatoid arthritis,
  • the present invention further provides a method for the treatment of a clinical condition in an animal, for example, a mammal including a human which clinical condition includes those which have been discussed hereinbefore, which comprises treating said animal with a pharmaceutically effective amount of a compound according to the invention
  • the present invention also includes a method for the treatment or prophylaxis of any of the aforementioned diseases or conditions
  • the present invention provides the use of a compound according to the invention in the manufacture of a medicament for the treatment or prophylaxis of any of the above mentioned viral infections or conditions
  • the above compounds according to the invention and their pharmaceutically acceptable derivatives may be employed in combination with other therapeutic agents for the treatment of the above infections or conditions
  • Combination therapies according to the present invention comprise the administration of a compound of the present invention or a pharmaceutically acceptable derivative thereof and another pharmaceutically active agent
  • the active ⁇ ngred ⁇ ent(s) and pharmaceutically active agents may be administered simultaneously in either the same or different pharmaceutical compositions or sequentially in any order
  • the amounts of the active ⁇ ngred ⁇ ent(s) and pharmaceutically active agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect
  • acyclic nucleosides for example acyclovir, valaciclovir, famciclovir, ganciclovir, and penciclovir
  • acyclic nucleoside phosphonates for example (S)-1-(3-hydroxy-2-phosphonyl- methoxypropyl)cytos ⁇ ne (HPMPC), [[[2-(6-am ⁇ no-9H-pur ⁇ n-9- yljethoxyjmethyljphosphinyhdenejbisfoxymethylenej ⁇ -dimethylpropanoic acid (bis-POM PMEA, adefovir dipivoxil), [[(1 R)-2-(6-am ⁇ no-9H-pur ⁇ n-9-yl)-1- methylethoxy]methyl]phosphon ⁇ c ac ⁇ d (tenofovir), and (R)-[[2-(6-Am ⁇ n
  • the present invention further includes the use of a compound according to the invention in the manufacture of a medicament for simultaneous or sequential administration with at least another therapeutic agent, such as those defined hereinbefore
  • Compounds of the present invention may be administered with an agent known to inhibit or reduce the metabolism of compounds, for example ritonavir Accordingly, the present invention features a method for the treatment or prophylaxis of a disease as hereinbefore described by administration of a compound of the present invention in combination with a metabolic inhibitor Such combination may be administered simultaneously or sequentially
  • a suitable dose for each of the above-mentioned conditions will be in the range of 0 01 to 250 mg per kilogram body weight of the recipient (e g a human) per day, preferably in the range of 0 1 to 100 mg per kilogram body weight per day and most preferably in the range 0 5 to 30 mg per kilogram body weight per day and particularly in the range 1 0 to 20 mg per kilogram body weight per day Unless otherwise indicated, all weights of active ingredient are calculated as the parent compound of formula (I), for salts or esters thereof, the weights would be increased proportionally
  • the desired dose may be presented as one, two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day In some cases the desired dose may be given on alternative days
  • These sub-doses may be administered in unit dosage forms, for example, containing 10 to 1000 mg or 50 to 500 mg, preferably 20 to 500 mg, and most preferably 50 to 400 mg of active ingredient per unit dosage form While it is possible for the active ingredient to be administered alone it is preferable to present it
  • phrases include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and intravitreal) administration
  • the compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy Such methods represent a further feature of the present invention and include the step of bringing into association the active ingredients with the carrier, which constitutes one or more accessory ingredients
  • the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product
  • the present invention further includes a pharmaceutical composition as hereinbefore defined wherein a compound of the present invention or a pharmaceutically acceptable derivative thereof and another therapeutic agent are presented separately from one another as a kit of parts
  • compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time
  • patches suitably contain the active compound 1) in an optionally buffered, aqueous solution or 2) dissolved and/or dispersed in an adhesive or 3) dispersed in a polymer
  • a suitable concentration of the active compound is about 1 % to 25%, preferably about 3% to 15%
  • the active compound may be delivered from the patch by electrotransport or iontophoresis as generally described in Pharmaceutical Research 3 (6), 318 (1986)
  • Pharmaceutical compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, caplets, cachets or tablets each containing a predetermined amount of the active ingredients, as a powder or granules, as a solution or a suspension in an aqueous or non-aqueous liquid, or as an o ⁇ l- ⁇ n-water liquid emulsion or a water- ⁇ n-o ⁇ l liquid
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as a powder or granules, optionally mixed with a binder (e g povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e g sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent Molded tablets may be made by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredients therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut
  • compositions suitable for topical administration in the mouth include lozenges comprising the active ingredients in a flavored base, usually sucrose and acacia or tragacanth, pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia, and mouthwashes comprising the active ingredient in a suitable liquid carrier
  • compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray Pharmaceutical compositions containing in addition to the active ingredient such carriers as are known in the art to be appropriate
  • compositions for rectal administration may be presented as a suppository with a suitable carrier comprising, for example, cocoa butter or a salicylate or other materials commonly used in the art
  • a suitable carrier comprising, for example, cocoa butter or a salicylate or other materials commonly used in the art
  • the suppositories may be conveniently formed by admixture of the active combination with the softened or melted carr ⁇ er(s) followed by chilling and shaping in molds
  • Pharmaceutical compositions suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the pharmaceutical composition isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs
  • the pharmaceutical compositions may be presented in unit-dose
  • Unit dosage pharmaceutical compositions include those containing a daily dose or daily subdose of the active ingredients, as hereinbefore recited, or an appropriate fraction thereof
  • compositions of this invention may include other agents conventional in the art having regard to the type of pharmaceutical composition in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents
  • Example I was synthesized from intermediates 5 and 9 as described for example III
  • Example III 4-[(5- ⁇ [4-((3-fluorophenyl) ⁇ [(4-fluorophenyl)am ⁇ no]carbonyl ⁇ am ⁇ no)-1- p ⁇ per ⁇ d ⁇ nyl]methyl ⁇ -6-methyl-2-pyr ⁇ d ⁇ nyl)oxy]- ⁇ /-[2-(methyloxy)ethyl]benzam ⁇ de was synthesized as follows
  • Example IV ⁇ /-(3-fluorophenyl)- ⁇ /'-(4-fluorophenyl)- ⁇ /- ⁇ 1-[(2-methyl-6- ⁇ [4-(4- morphol ⁇ nylcarbonyl)phenyl]oxy ⁇ -3-pyr ⁇ d ⁇ nyl)methyl]-4-p ⁇ per ⁇ d ⁇ nyl ⁇ urea
  • Example V was prepared from intermediate 245-( ⁇ [butyl(4-p ⁇ per ⁇ d ⁇ nyl)am ⁇ no]carbonyl ⁇ am ⁇ no)-2,4- difluorobenzamide and 4-[(5-formyl-6-methyl-2-pyr ⁇ d ⁇ nyl)oxy]- ⁇ /-(2-hydroxyethyl)benzam ⁇ de by the method described in example III
  • Lymphocytes were separated from whole blood by density gradient centrifugation with Lymphocyte Separation Medium (LSM) Whole blood was diluted 1 1 with sterile phosphate buffered saline (PBS) and overlaid onto LSM in 50 mL conical centrifuge tubes Samples were centrifuged at 500 x g at room temperature for 30 minutes PBLs were then gently removed from the interface between LSM and aqueous phases and washed twice in sterile PBS The cells were resuspended in 150 mL of RPMI containing 20% fetal bovine serum, 10% T-cell growth factor (IL-2), 50ug/mL gentamicin (RPMI 20/10/50) and 5ug/mL Phytohaemagluttinin and incubated at 37 0 C, 5% CO 2 for 2 to 3 days On the second or third day, the cells were washed once with PBS and counted for assay PHA stimulated PBLs were centrifuged at
  • HOS Assay HOS-Luc cells were created by introducing an HIV-1 LTR-I uciferase reporter into HOS-
  • CD4 CCR5 (NIH AIDS Research and Reference Reagents Program, cat #3318) The cells were propagated in DMEM complete medium Cells were split 1 20 by standard trypsinization when confluency reached 80% (roughly every 2 to 3 days) Black-walled 96-well tissue culture plates were seeded with HOS-Luc at 6x10 3 cells per well in 50 ⁇ L DMEM containing 2% FBS and placed in a humidified incubator at 37 0 C, 5% CO 2 overnight The following day, test compounds were serially diluted in 4-fold increments at 2X the final concentration in DMEM + 2% FBS + 0 2% DMSO Diluted compound (50 ⁇ L) was transferred to the HOS-Luc cells and the plates were placed in a humidified incubator at Zl 0 C, 5% CO 2 for 1 hr An additional 60 ⁇ L of 2X compound was transferred to a clear- walled 96-well tissue culture plate and 60 ⁇ L of virus (diluted 1 50 in tissue culture media) was added
  • RLU Relative Light Unit
  • Non-fasted male CD-1 mice (Charles River Laboratories, Raleigh NC), ranging in weight from 28 g to 30 g, were used for each study Test Article Administration and Sample Collection
  • a single dose (10 mL/kg of 0 1 mg free base/mL) was administered via tail vein
  • animals received a single dose (10 mL/kg of 0 1 or 0 3 mg free base/mL) by an oral gavage needle
  • Blood (approximately 0 8 mL) was taken via cardiac puncture from three mice/dose group at each timepoint of 0 083 (IV only), 0 167 (IV only), 0 25, 0 5, 1 , 2, 4, 6, 8, and 24 h and placed on ice Syringes used for sample collection contained approximately 10 ⁇ L EDTA Plasma was harvested by centrifugation for 3 mm at 4000 x g and 4°C and stored at -80 0 C until analysis
  • mice were orally administered a single dose (10 mL/kg of 0 1 or 0 3 mg free base/mL) by an oral gavage needle
  • Non-compartmental pharmacokinetic parameters [terminal plasma half-life (Ua), maximum plasma concentration (C max ), time of maximum plasma concentration (T max ) plasma clearance (CL), steady-state volume of distribution (V ss ), and area under the plasma concentration-time curve extrapolated to infinite time (AUC 0 - ⁇ )] were calculated based on the composite plasma concentration time data using WinNonlin Professional 4 1 (Pharsight, Mountain View CA)
  • Dose-normalized AUC 0 ⁇ (DNAUC 0 ⁇ oo) was obtained by dividing the AUC o ⁇ ⁇ with its respective dose and expressed as unit dose AUC 0 ⁇ (ng*h/mL/mg/kg)
  • test compound For intravenous administration, a single dose of test compound was administered into the jugular cannula followed by a 1 mL flush with saline
  • animals received a single dose by an oral gavage needle
  • Additional animals received capsule doses (shown in the following table) which were administered by a small capsule- dosing syringe (Harvard Apparatus)
  • Plasma samples were taken from the jugular cannula at 0 083 (IV only), 0 167 (IV only), 0 25, 0 5, 1 , 2, 4, 6, 8, and 24 h postdose and placed on ice Syringes used for sample collection contained approximately 10 ⁇ L EDTA Plasma was harvested by centrifugation for 3 mm at 4000 x g and 4°C and stored at -80 0 C until analysis
  • Plasma samples were thawed to room temperature and vortex-mixed Standards were prepared in pooled rat plasma containing EDTA Quality control (QC) samples were prepared from separately weighed sample stocks Ahquots (50 ⁇ L) of standards, QCs and study samples were pipetted into 96-well plates Acetonitrile (200 ⁇ L) containing internal standard was added to each well, samples vortex-mixed, and plates centrifuged for 20 mm at 2055 x g and 4°C The acetonitrile supernatant (150 ⁇ L) was transferred to clean 96 well plates containing 100 ⁇ L of 0 1% formic acid in water and assayed by LC/MS/MS The analytical system consisted of a CTC HTS PAL autosampler, Hewlett Packard (HP) 1100 binary pump and Applied Biosystems API 4000 mass spectrometer Samples (10 ⁇ L) were injected onto a 30 x 2 mm, 3m ⁇ cron, Atlantis C
  • Non-compartmental pharmacokinetic parameters [terminal plasma half-life (Ua), maximum plasma concentration (C max ), time of maximum plasma concentration (T max ) plasma clearance (CL), steady-state volume of distribution (V ss ), and area under the plasma concentration-time curve extrapolated to infinite time (AUC 0 - ⁇ )] were calculated based on the individual plasma concentration time data using WinNonlin Professional 4 1 (Pharsight, Mountain View CA)
  • Dose-normalized AUC 0 ⁇ (DNAUC 0 ⁇ ) was obtained by dividing the AUC o ⁇ ⁇ by its respective dose and expressed as unit dose AUC 0 ⁇ (ng*h/mL/mg/kg)
  • Oral bioavailability (F) was calculated using the following equation
  • Example XIII hERG dofetilide binding assay
  • Pepstatin A and PMSF were prepared as concentrated stocks in absolute ethanol (pepstatin 50Ox, PMSF 100x) and were added just before use
  • the blender was plunged into ice for 5 mins after the first burst and 10-40 mins after the final burst to allow foam to dissipate
  • the material was then spun at 50Og for 20 mins and the supernatant spun for 36 mins at 48,00Og
  • the pellet was resuspended in the same buffer as above but without PMSF and Pepstatin A
  • the material was then forced through a 0 6 mm needle, made up to the required volume, ahquoted and stored frozen at -80 °C
  • the membrane-bead suspension was mixed with tritiated dofetilide (final concentration of 7 nM/well) and dispensed into a white Greiner polypropylene 384- well plate (10 ml/well), containing 0 1 ml of test compound in DMSO
  • the assay plates were centrifuged at 10,000 rpm for 1 minute and left to equilibrate for 2-3 hrs at room temperature before reading on a ViewLuxTM luminescence imager Concentration response curve data were generated from an 11 -point inhibition curve (top assay concentration of 60 mM and a 1 4 step-dilution), a four parameter curve-fit being applied
  • the concentration-response curve was analyzed using a non linear equation, 4-parameter logistics, giving a determination of plC50

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Abstract

The present invention relates to compounds useful in the treatment of CCR5-related diseases and disorders, for example, useful in the inhibition of HIV replication, the prevention or treatment of an HIV infection, and in the treatment of the resulting acquired immune deficiency syndrome (AIDS).

Description

CCR5 ANTAGONISTS AS THERAPEUTIC AGENTS
BACKGROUND OF THE INVENTION
The human immunodeficiency virus ("HIV") is the causative agent for acquired immunodeficiency syndrome ("AIDS"), a disease characterized by the destruction of the immune system, particularly of CD4+ T-cells, with attendant susceptibility to opportunistic infections, and its precursor AIDS-related complex ("ARC"), a syndrome characterized by symptoms such as persistent generalized lymphadenopathy, fever and weight loss
In addition to CD4, HIV requires a co-receptor for entry into target cells The chemokine receptors function together with CD4 as co-receptors for HIV The chemokine receptors CXCR4 and CCR5 have been identified as the mam co-receptors for HIV-1 CCR5 acts as a major co-receptor for fusion and entry of macrophage-tropic HIV into host cells These chemokine receptors are thought to play an essential role in the establishment and dissemination of an HIV infection Therefore, CCR5 antagonists are thought to be useful as therapeutic agents active against HIV Nitrogen-containing heterocyclic compounds that are CCR5 antagonists are disclosed in
WO2006030925, WO2004080966, and WO2001087839 However, such compounds can demonstrate limited oral bioavailability or unfavorable cardiovascular profile
We have now discovered a series of small molecule nonpeptide compounds that are useful as inhibitors of HIV replication
BRIEF DESCRIPTION OF THE INVENTION
The present invention features compounds that are useful in the inhibition of HIV replication, the prevention of infection by HIV, the treatment of infection by HIV and in the treatment of AIDS and/or ARC, either as pharmaceutically acceptable salts or pharmaceutical composition ingredients The present invention further features methods of treating AIDS, methods of preventing infection by HIV, and methods of treating infection by HIV as monotherapy or in combination with other antivirals, anti-infectives, immunomodulators, antibiotics or vaccines The present invention also features pharmaceutical compositions, comprising the above-mentioned compounds that are suitable for the prevention or treatment of CCR5-related diseases and conditions The present invention further features processes for making the above-mentioned compounds
DETAILED DESCRIPTION OF THE INVENTION
The present invention features a compound selected from the group consisting of 5-{[((3-fluorophenyl){1-[(2-methyl-6-{[4-(4-morpholιnylmethyl)phenyl]oxy}-3- pyridinyljmethylj^-piperidinyljaminojcarbonyljaminoj^-pyridmecarboxamide bιs(trιfluoroacetate),
5-{[((3-fluorophenyl){1-[(2-methyl-6-{[4-(4-morpholιnylcarbonyl)phenyl]oxy}-3- pyridinyljmethylj^-piperidinyljaminojcarbonyljaminoj^-pyridmecarboxamide, 4-[(5-{[4-((3-fluorophenyl){[(4-fluorophenyl)amιno]carbonyl}amιno)-1-pιperιdιnyl]methyl}-6- methyl-2-pyrιdιnyl)oxy]-Λ/-[2-(methyloxy)ethyl]benzamιde, Λ/-(3-fluorophenyl)-ΛΛ(4-fluorophenyl)-Λ/-{1-[(2-methyl-6-{[4-(4- morpholιnylcarbonyl)phenyl]oxy}-3-pyrιdιnyl)methyl]-4-pιperιdιnyl}urea, S-IKbutylli-KΘ-^-fP-hydroxyethylJaminolcarbonylJphenylJoxyl^-methyl-S- pyrιdιnyl}methyl)-4-pιperιdιnyl]amιno}carbonyl)amιno]-2,4-dιfluorobenzamιde,
2,4-dιfluoro-5-{[((3-fluorophenyl){1-[(2-methyl-6-{[4-(1 ,3-oxazol-5-yl)phenyl]oxy}-3- pyrιdιnyl)methyl]-4-pιperιdιnyl}amιno)carbonyl]amιno}benzamιde,
4-{[5-({4-[({[5-(amιnocarbonyl)-2,4-dιfluorophenyl]amιno}carbonyl)(3-fluorophenyl)amιno]-1- pιperιdιnyl}methyl)-6-methyl-2-pyrιdιnyl]oxy}phenyl (i-methylethyl)carbamate, 2,4-dιfluoro-5-[({(3-fluorophenyl)[1-({2-methyl-6-[(4-{2-[(1-methylethyl)amιno]-2- oxoethyl}phenyl)oxy]-3-pyrιdιnyl}methyl)-4-pιperιdιnyl]amιno}carbonyl)amιno]benzamιde, and pharmaceutically acceptable salts thereof The term "pharmaceutically effective amount" refers to an amount of a compound of the invention that is effective in treating a CCR5-related disease, for example a virus infection, for example an HIV infection, in a patient either as monotherapy or in combination with other agents The term "treatment" as used herein refers to the alleviation of symptoms of a particular disorder in a patient, or the improvement of an ascertainable measurement associated with a particular disorder, and may include the suppression of symptom recurrence in an asymptomatic patient such as a patient in whom a viral infection has become latent The term "prophylaxis" refers to preventing a disease or condition or preventing the occurrence of symptoms of such a disease or condition, in a patient As used herein, the term "patient" refers to a mammal, including a human
The term "pharmaceutically acceptable carrier" refers to a carrier that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the therapeutic agent
Pharmaceutically acceptable salts of the compounds according to the invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases Examples of suitable acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, glutamic, maleic, mandelic, phosphoric, glycollic, lactic, salicyclic, succinic, toluene- p-sulfonic, tartaric, acetic, citric, orotic, toluenesulfonic, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonιc and benzenesulfonic acids Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts
Salts derived from appropriate bases include alkali metal (e g sodium), alkaline earth metal (e g , magnesium), ammonium, NW4 + (wherein W is C1^ alkyl) and other amine salts Physiologically acceptable salts of a hydrogen atom or an ammo group include salts of organic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic and succinic acids, organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids and inorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamic acids Physiologically acceptable salts of a compound with a hydroxy group include the anion of said compound in combination with a suitable cation such as Na+, NH4 +, and NW4 + (wherein W is a C^alkyl group)
Any reference to any of the above compounds also includes a reference to a pharmaceutically acceptable salt thereof Salts of the compounds of the present invention may be made by methods known to a person skilled in the art For example, treatment of a compound of the present invention with an appropriate base or acid in an appropriate solvent will yield the corresponding salt The present invention features compounds according to the invention for use in medical therapy, for example for the treatment or prophylaxis of viral infections such as an HIV infections and associated conditions Reference herein to treatment extends to prophylaxis as well as the treatment of established infections, symptoms, and associated clinical conditions such as AIDS related complex (ARC), Kaposi's sarcoma, and AIDS dementia
The present invention features use of the compounds of the present invention in the manufacture of a medicament for the treatment or prophylaxis of a CCR5-related disease or condition, for example, a viral infection, for example, an HIV infection According to another aspect, the present invention provides a method for the treatment or prevention of the symptoms or effects of a viral infection in an infected animal, for example, a mammal including a human, which comprises treating said animal with a pharmaceutically effective amount of a compound according to the invention According to one aspect of the invention, the viral infection is a retroviral infection, in particular an HIV infection A further aspect of the invention includes a method for the treatment or prevention of the symptoms or effects of an HBV infection
The compounds according to the invention may also be used in adjuvant therapy in the treatment of HIV infections or HIV-associated symptoms or effects, for example Kaposi's sarcoma
The compounds of the present invention may also be used in the prevention or treatment of other CCR5-related diseases and conditions, including neuropathic pain, multiple sclerosis, rheumatoid arthritis, autoimmune diabetes, chronic implant rejection, asthma, rheumatoid arthritis, Crohns Disease, inflammatory bowel disease, chronic inflammatory disease, glomerular disease, nephrotoxic serum nephritis, kidney disease, Alzheimer's Disease , autoimmune encephalomyelitis, arterial thrombosis, allergic rhinitis, arteriosclerosis, Sjogren's syndrome (dermatomyositis), systemic lupus erythematosus, graft rejection, cancers with leukocyte infiltration of the skin or organs, human papilloma virus infection, prostate cancer, wound healing, amyotrophic lateral sclerosis, immune mediated disorders
The present invention further provides a method for the treatment of a clinical condition in an animal, for example, a mammal including a human which clinical condition includes those which have been discussed hereinbefore, which comprises treating said animal with a pharmaceutically effective amount of a compound according to the invention The present invention also includes a method for the treatment or prophylaxis of any of the aforementioned diseases or conditions
In yet a further aspect, the present invention provides the use of a compound according to the invention in the manufacture of a medicament for the treatment or prophylaxis of any of the above mentioned viral infections or conditions The above compounds according to the invention and their pharmaceutically acceptable derivatives may be employed in combination with other therapeutic agents for the treatment of the above infections or conditions Combination therapies according to the present invention comprise the administration of a compound of the present invention or a pharmaceutically acceptable derivative thereof and another pharmaceutically active agent The active ιngredιent(s) and pharmaceutically active agents may be administered simultaneously in either the same or different pharmaceutical compositions or sequentially in any order The amounts of the active ιngredιent(s) and pharmaceutically active agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect
Examples of such therapeutic agents include agents that are effective for the treatment of viral infections or associated conditions Among these agents are acyclic nucleosides, for example acyclovir, valaciclovir, famciclovir, ganciclovir, and penciclovir, acyclic nucleoside phosphonates, for example (S)-1-(3-hydroxy-2-phosphonyl- methoxypropyl)cytosιne (HPMPC), [[[2-(6-amιno-9H-purιn-9- yljethoxyjmethyljphosphinyhdenejbisfoxymethylenej^^-dimethylpropanoic acid (bis-POM PMEA, adefovir dipivoxil), [[(1 R)-2-(6-amιno-9H-purιn-9-yl)-1- methylethoxy]methyl]phosphonιc acιd (tenofovir), and (R)-[[2-(6-Amιno-9H-purιn-9-yl)-1- methylethoxy]methyl]phosphonιc acid bιs-(ιsopropoxycarbonyloxymethyl)ester (bis-POC- PMPA), nucleoside reverse transcriptase inhibitors, for example 3'-azιdo-3'-deoxythymιdιne (AZT, zidovudine), 2',3'-dιdeoxycytιdιne (ddC, zalcitabine), 2',3'-dιdeoxyadenosιne, 2', 3'- dideoxyinosme (ddl, didanosine), 2',3'-dιdehydrothymιdιne (d4T, stavudme), (-)-αs-1-(2- hydroxymethyl)-1 ,3-oxathιolane 5-yl)-cytosιne (lamivudine), cιs-1-(2-(hydroxymethyl)-1 ,3- oxathιolan-5-yl)-5-fluorocytosιne (FTC), (-J-cis^-β-amino-θ-fcyclopropylaminoJ-ΘH-puπn-θ- yl]-2-cyclopentene-1 -methanol (abacavir), and ribavirin, protease inhibitors, for example indinavir, ritonavir, nelfinavir, amprenavir, saquinavir, fosamprenavir, lopinavir, tipranavir, interferons such as α-ιnterferon, immunomodulators such as interleukin Il or thymosin, granulocyte macrophage colony stimulating factors, erythropoetin, soluble CD4 and genetically engineered derivatives thereof, non-nucleoside reverse transcriptase inhibitors (NNRTIs), for example nevirapine (BI-RG-587), alpha-((2-acetyl-5-methylphenyl)amιno)- 2,6-dιchloro-benzeneacetamιde (loviride), 1-[3-(ιsopropylamιno)-2-pyrιdyl]-4-[5- (methanesulfonamιdo)-1 H-ιndol-2-ylcarbonyl]pιperazιne monomethanesulfonate (delavirdine), (SJ-θ-chloro^-fcyclopropylethynylJ-i ^-dihydro^-ftnfluoromethylJ^H-S.I- benzoxazιn-2-one (efavirenz, DMP 266), nlpivirine, integrase inhibitors, or fusion inhibitors, for example T-20 and T-1249
The present invention further includes the use of a compound according to the invention in the manufacture of a medicament for simultaneous or sequential administration with at least another therapeutic agent, such as those defined hereinbefore
Compounds of the present invention may be administered with an agent known to inhibit or reduce the metabolism of compounds, for example ritonavir Accordingly, the present invention features a method for the treatment or prophylaxis of a disease as hereinbefore described by administration of a compound of the present invention in combination with a metabolic inhibitor Such combination may be administered simultaneously or sequentially
In general a suitable dose for each of the above-mentioned conditions will be in the range of 0 01 to 250 mg per kilogram body weight of the recipient (e g a human) per day, preferably in the range of 0 1 to 100 mg per kilogram body weight per day and most preferably in the range 0 5 to 30 mg per kilogram body weight per day and particularly in the range 1 0 to 20 mg per kilogram body weight per day Unless otherwise indicated, all weights of active ingredient are calculated as the parent compound of formula (I), for salts or esters thereof, the weights would be increased proportionally The desired dose may be presented as one, two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day In some cases the desired dose may be given on alternative days These sub-doses may be administered in unit dosage forms, for example, containing 10 to 1000 mg or 50 to 500 mg, preferably 20 to 500 mg, and most preferably 50 to 400 mg of active ingredient per unit dosage form While it is possible for the active ingredient to be administered alone it is preferable to present it as a pharmaceutical composition The compositions of the present invention comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof and optionally other therapeutic agents Each carrier must be acceptable in the sense of being compatible with the other ingredients of the composition and not injurious to the patient
Phamaceutical compositions include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and intravitreal) administration The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy Such methods represent a further feature of the present invention and include the step of bringing into association the active ingredients with the carrier, which constitutes one or more accessory ingredients In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product
The present invention further includes a pharmaceutical composition as hereinbefore defined wherein a compound of the present invention or a pharmaceutically acceptable derivative thereof and another therapeutic agent are presented separately from one another as a kit of parts
Compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time Such patches suitably contain the active compound 1) in an optionally buffered, aqueous solution or 2) dissolved and/or dispersed in an adhesive or 3) dispersed in a polymer A suitable concentration of the active compound is about 1 % to 25%, preferably about 3% to 15% As one particular possibility, the active compound may be delivered from the patch by electrotransport or iontophoresis as generally described in Pharmaceutical Research 3 (6), 318 (1986) Pharmaceutical compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, caplets, cachets or tablets each containing a predetermined amount of the active ingredients, as a powder or granules, as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oιl-ιn-water liquid emulsion or a water-ιn-oιl liquid emulsion The active ingredient may also be presented as a bolus, electuary or paste
A tablet may be made by compression or molding, optionally with one or more accessory ingredients Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as a powder or granules, optionally mixed with a binder (e g povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e g sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent Molded tablets may be made by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredients therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach
Pharmaceutical compositions suitable for topical administration in the mouth include lozenges comprising the active ingredients in a flavored base, usually sucrose and acacia or tragacanth, pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia, and mouthwashes comprising the active ingredient in a suitable liquid carrier
Pharmaceutical compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray Pharmaceutical compositions containing in addition to the active ingredient such carriers as are known in the art to be appropriate
Pharmaceutical compositions for rectal administration may be presented as a suppository with a suitable carrier comprising, for example, cocoa butter or a salicylate or other materials commonly used in the art The suppositories may be conveniently formed by admixture of the active combination with the softened or melted carrιer(s) followed by chilling and shaping in molds Pharmaceutical compositions suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the pharmaceutical composition isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs The pharmaceutical compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dned (lyophihzed) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described
Unit dosage pharmaceutical compositions include those containing a daily dose or daily subdose of the active ingredients, as hereinbefore recited, or an appropriate fraction thereof
It should be understood that in addition to the ingredients particularly mentioned above the pharmaceutical compositions of this invention may include other agents conventional in the art having regard to the type of pharmaceutical composition in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents
Compounds of the present invention demonstrate advantageous properties compared with CCR5 antagonists disclosed in WO2006030925, WO2004080966, and
WO2001087839 As shown in Examples IX, X, Xl, XII, XIII, Tables 1 , 2, and 3, representative compounds of the present invention may demonstrate good oral bioavailability and favorable cardiovascular profile
The following examples are intended for illustration only and are not intended to limit the scope of the invention in any way
Example I
5-{[((3-fluorophenyl){1-[(2-methyl-6-{[4-(4-morpholιnylmethyl)phenyl]oxy}-3- pyridinylJmethylH-piperidinylJaminoJcarbonylJamino^-pyridinecarboxamide bιs(trιfluoroacetate)
Figure imgf000011_0001
The synthesis of intermediate 5
Figure imgf000011_0002
Figure imgf000011_0003
Intermediate 2 4-(4-morpholιnylmethyl)phenol
HOXJ 1^o
A solution of 4-hydroxybenzaldehyde (1 g, 8 2 mmol), morpholine (1 mL, 11 5 mmol) and NaBH(OAc)3 (3 5 g, 16 4 mmol) in CH2CI2 (20 mL) was stirred at RT for 24 h The reaction mixture was washed with sat'd aq NaHCO3, dried (Na2SO4), concentrated, and the residue was purified by silica gel flash column chromatography (0→10% MeOH (2M NH3) CH2CI2) to provide 4-(4-morpholιnylmethyl)phenol 2 (1 6 g, quant ) as an off white solid 1H NMR (400 MHz, CHLOROFORM-d) δ 7 09 - 7 15 (m, 2 H), 6 67 - 6 72 (m, 2 H), 3 68 - 3 74 (m, 4 H), 3 43 (s, 2 H), 2 42 - 2 52 (m, 4 H), 2 10 (s, 1 H) ES-LCMS m/z 194 2 (M+H)
Intermediate 4 2-methyl-6-{[4-(4-morpholιnylmethyl)phenyl]oxy}-3-pyrιdιnecarbonιtrιle
Figure imgf000011_0004
Prepared from 3 and 2 in 88 % yield as described for intermediate 33. 1H NMR (400 MHz, CHLOROFORM-d) δ 7 78 (d, J=8 61 Hz, 1 H), 7 31 - 7 37 (m, 2 H), 7 02 - 7 08 (m, 2 H), 6 68 (d, J=842 Hz, 1 H), 3 66 - 3 71 (m, 4 H), 348 (s, 2 H), 2 58 (s, 3 H), 2 41 - 246 (m, 4 H) ES-LCMS m/z 310 2 (M+H) Intermediate 5
2-methyl-6-{[4-(4-morpholιnylmethyl)phenyl]oxy}-3-pyrιdιnecarbaldehyde
Figure imgf000012_0001
A solution of 2-methyl-6-{[4-(4-morpholιnylmethyl)phenyl]oxy}-3-pyrιdιnecarbonιtrιle, 4 (8204 mg, 2 7 mmol), sodium hypophosphite hydrate (327 mg, 3 7 mmol), and catalytic Raney nickel in pyridine (17 mL), acetic acid (8 mL) and water (6 mL) was heated at 40 °C for 6 h, then filtered through celite, washing with CH2CI2 The filtrate was evaporated and the residue was purified by silica gel flash column chromatography (10→100% EtOAc hexanes) to provide 2-methyl-6-{[4-(4-morpholιnylmethyl)phenyl]oxy}-3- pyndinecarbaldehyde 5 (221 4 mg, 27 % yield) as a clear oil 1H NMR (400 MHz, CHLOROFORM-d) δ 10 22 (s, 1 H), 8 07 (d, J=843 Hz, 1 H), 7 35 - 7 40 (m, 2 H), 7 08 -
7 14 (m, 2 H), 6 73 (d, J=8 80 Hz, 1 H), 3 70 - 3 75 (m, 4 H), 3 52 (s, 2 H), 2 74 (s, 3 H), 2 45 - 2 51 (m, 4 H) ES-LCMS m/z 313 2 (M+H)
Synthesis of intermediate 9
Figure imgf000012_0002
Intermediate 6 4-[(3-fluorophenyl)amιno]-1-pιperιdιnecarboxylate
Figure imgf000012_0003
Sodium triacetoxyborohydride (79 7 g, 0 37 mol, 1 5 equiv) was added portionwise over 1 h to a solution of 4-BOC-pιperιdone (50 0 g, 0 25 mol, 1 equiv), 3-fluoroanιlιne (28 9 mL, 0 30 mol, 1 2 equiv) and acetic acid (86 2 mL, 1 51 mol, 6 equiv) in dichloroethane ( 700 mL) at 0 0C The reaction mixture was allowed to warm to RT over 2 h and then slowly poured into cold 3 3N NaOH (1L) with stirring The reaction mixture was then separated and the organics dried over Na2SO4, filtered and concentrated to give a tan solid The solid was suspended in 3 1 hexanes ethyl acetate, stirred overnight and filtered to give 6 (67 g, 91 % yield) as a white solid
1H NMR (400 MHz, DMSO-cfe) δ ppm 6 96 - 7 05 (m, 1 H), 6 29 - 640 (m, 2 H), 6 18 - 6 25 (m, 1 H), 5 80 (d, J=8 24 Hz, 1 H), 3 81 (s, 2 H), 3 30 - 342 (m, 1 H), 2 87 (s, 2 H), 1 81 (s, 2 H), 1 32 - 1 42 (m, 9 H), 1 09 - 1 24 (m, 2 H) Intermediate 7
1 ,1-dιmethylethyl 4-[{[(6-cyano-3-pyrιdιnyl)amιno]carbonyl}(3-fluorophenyl)amιno]-1- piperidmecarboxylate
Figure imgf000013_0001
A solution of the 5-amιno-2-pyrιdιnecarbonιtrιle (333 mg, 2 8 mmol) in CH2CI2 (13 mL) at 0 0C was treated with pyridine (670 μL, 8 4 mmol) and 1 9 M phosgene in toluene (2 2 mL, 4 2 mmol) The reaction was allowed to warm to RT, stirred for 24 h, and then evaporated, dried in vacuo and redissolved in CH2CI2 (20 mL) To this was added 1 ,1-dιmethylethyl 4- [(3-fluorophenyl)amιno]-1-pιperιdιnecarboxylate 6 (2 8 mmol) and the solution was stirred at RT for 24h The mixture was concentrated and purified by silica gel flash column chromatography (0→40% EtOAc CH2CI2) to give 1 ,1-dιmethylethyl 4-[{[(6-cyano-3- pyridinyl)amino]carbonyl}(3-fluorophenyl)amino]-1-piperidinecarboxylate 7 (77 % yield) 1H NMR (400 MHz, CHLOROFORM-d) δ 8 24 (d, J=2 56 Hz, 1 H), 8 15 (dd, J=8 61 , 2 38 Hz, 1 H), 7 58 (d, J=8 61 Hz, 1 H), 7 54 (td, J=8 15, 641 Hz, 1 H), 7 24 - 7 30 (m, 1 H), 7 02 - 7 06 (m, 1 H), 6 97 (dt, J=8 88, 2 15 Hz, 1 H), 6 09 (s, 1 H), 4 55 - 4 69 (m, 1 H),
4 05 - 4 23 (m, 2 H), 2 73 - 2 86 (m, 2 H), 1 79 - 1 88 (m, 2 H), 1 39 (s, 9 H), 1 19 - 1 33 (m, 2 H) ES-LCMS m/z 440 1 (M+H)
Intermediate 8 1 ,1-dιmethylethyl 4-[({[6-(amιnocarbonyl)-3-pyrιdιnyl]amιno}carbonyl)(3- fluorophenyl)amιno]-1-pιperιdιnecarboxylate
Figure imgf000014_0001
A solution of 1 ,1-dιmethylethyl 4-[{[(6-cyano-3-pyrιdιnyl)amιno]carbonyl}(3- fluorophenyl)amιno]-1-pιperιdιnecarboxylate 7 (1 95 mmol), urea hydrogen peroxide (2 2 g, 234 mmol), and solid potassium carbonate (80 7 mg, 0 59 mmol) in acetone (15 mL) and water (7 5 mL) was stirred at RT for 24 h The reaction mixture was then evaporated and extracted with CHCI3, dried (Na2SO4), and concentrated to provide 1 ,1-dιmethylethyl 4- [({[θ-faminocarbonylJ-S-pyπdinylJaminoJcarbonylXS-fluorophenylJamino]-'!- piperidmecarboxylate 8 (99 % yield) as a white solid 1H NMR (400 MHz, CHLOROFORM-d) δ 8 38 (d, J=2 38 Hz, 1 H), 8 10 (d, J=8 61 Hz, 1 H), 7 91 (dd, J=8 70, 2 47 Hz, 1 H), 7 72 (br s , 1 H), 7 52 (td, J=8 10, 6 32 Hz, 1 H), 7 22 -
7 28 (m, 1 H), 7 03 - 7 07 (m, 1 H), 6 98 (dt, J=8 84, 2 17 Hz, 1 H), 6 05 (s, 1 H), 547 (br s , 1 H), 4 59 - 4 70 (m, 1 H), 4 09 - 4 23 (m, 2 H), 2 74 - 2 86 (m, 2 H), 1 80 - 1 89 (m, 2 H), 1 39 (s, 9 H), 1 21 - 1 33 (m, 2 H) ES-LCMS m/z 458 2(M+H) Intermediate 9
Figure imgf000014_0002
A solution of 1 ,1-dιmethylethyl 4-[({[6-(amιnocarbonyl)-3-pyrιdιnyl]amιno}carbonyl)(3- fluorophenyl)amιno]-1-pιperιdιnecarboxylate 8 (1 9 mmol) in 4N HCI/dioxane (4 8 mL) and CH2CI2 (15 mL) was stirred at RT for 2 h The reaction mixture was then evaporated, sat'd aq NaCO3 was added and the solution was extracted with 15% ιPrOH/CH2CI2 The organic extracts were dried (Na2SO4) and concentrated to provide 5-({[(3-fluorophenyl)(4- pιperιdιnyl)amιno]carbonyl}amιno)-2-pyrιdιnecarboxamιde 9 in quantitative yield as a white solid ES-LCMS m/z 358 1 (M+H)
Example I was synthesized from intermediates 5 and 9 as described for example III
Figure imgf000015_0001
1H NMR (CD3OD, 400 MHz) δ 8 59 - 8 63 (m, J=1 83 Hz, 1 H), 7 98 (s, 2 H), 7 84 (d, J=8 43 Hz, 1 H), 7 51 - 7 59 (m, 3 H), 7 22 - 7 31 (m, 3 H), 7 13 - 7 20 (m, 2 H), 6 88 (d, J=8 07 Hz, 1 H), 4 64 - 4 76 (m, J=3 30 Hz, 1 H), 4 38 (s, 2 H), 4 34 (s, 2 H), 3 98 - 4 10 (m, 2 H), 3 67 - 3 82 (m, 2 H), 3 52 - 3 62 (m, 2 H), 3 35 - 3 47 (m, 2 H), 3 15 - 3 33 (m, 4
H), 2 45 (s, 3 H), 2 18 (d, J=13 56 Hz, 2 H), 1 70 - 1 86 (m, J=11 73 Hz, 2 H) HRMS (M+H)+ calcd for C36H40FN7O4 + H, 654 3204, found, 654 3210
Example Il 5-{[((3-fluorophenyl){1-[(2-methyl-6-{[4-(4-morpholιnylcarbonyl)phenyl]oxy}-3- pyridinylJmethylH-piperidinylJaminoJcarbonyriamino^-pyridinecarboxamide
Figure imgf000015_0002
Intermediate 10 yl-2-pyrιdιnyl)oxy]benzoate A mixture of θ-chloro^-methyl-S-pyπdinecarbonitπle 3 (2 5O g, 164 mmol, 1eq ), phenylmethyl 4-hydroxybenzoate (3 70 g, 16 4 mmol, 1 eq ) and cesium carbonate (10 68 g, 32 8 mmol, 2 eq ) in anhydrous dimethylformamide (50 mL) was heated at 50 C under a nitrogen atmosphere for 45 mm The reaction mixture was evaporated in vacuo and triturated with water The product slurry was filtered, washed and dried under high vacuum to provide phenylmethyl 4-[(5-cyano-6-methyl-2-pyrιdιnyl)oxy]benzoate 10 as a solid (4 77 g, 84%) 1H NMR (400 MHz, CDCI3) δ 8 14 (d, J = 8 7 Hz, 2 H), 7 86 (d, J = 84 Hz, 1 H),
7 46-7 34 (m, 5 H), 7 19 (d, J = 8 6 Hz, 2 H), 6 82 (d, J = 8 5 Hz, 1 H), 5 37 (s, 2 H), 2 60 (s, 3 H), MS m/z 345 (M+H)+
Intermediate 11 4-[(5-cyano-6-methyl-2-pyrιdιnyl)oxy]benzoιc acid
Figure imgf000016_0001
A solution of phenylmethyl 4-[(5-cyano-6-methyl-2-pyrιdιnyl)oxy]benzoate 10 (1 70 g, 4 94 mmol) in a one-to-one mixture of ethyl acetate and ethanol (100 mL) was combined with palladium on barium sulfate (0 35 g) and stirred under a balloon of hydrogen gas for 3 days The mixture was filtered, evaporated in vacuo and dried under high vacuum to provide 4-[(5-cyano-6-methyl-2-pyrιdιnyl)oxy]benzoιc acid 11 as a solid (1 45 g) 1H NMR (400 MHz, CDCI3) δ 8 17 (d, J = 8 8 Hz, 2 H), 7 88 (d, J = 84 Hz, 1 H), 7 24 (d, J = 8 8 Hz, 2 H), 6 86 (d, J = 8 5 Hz, 1 H), 2 62 (s, 3 H), MS m/z 255 (M+H)+ Intermediate 12
4-[(5-formyl-6-methyl-2-pyrιdιnyl)oxy]benzoιc acιd
Figure imgf000016_0002
A solution of 4-[(5-cyano-6-methyl-2-pyrιdιnyl)oxy]benzoιc acid 11 (1 113 g, 4 38 mmol, 1 eq ) and triethylamine (1 25 mL, 8 76 mmol, 2 eq ) in dichloromethane (65 mL) under nitrogen was cooled and maintained at -30 C The mixture was treated with diisobutylaluminum hydride (1 0 M in CH2CI2, 15 3 mL, 15 3 mmol, 3 5 eqs ) and stirred for approximately 15 mm Additional diisobutylaluminum hydride (5 0 mL, 5 0 mmol, 1 1 eqs ) was added and the reaction was quenched with slow addition of methanol after 10 mm The mixture was evaporated in vacuo and partitioned between ethyl acetate and 1N NaHSO4 The layers were separated and the aqueous phase was back-extracted twice with ethyl acetate The combined organic layers were washed with brine, dried over anhydrous magnesium sulfate, filtered and evaporated in vacuo to a solid residue of 4-[(5- formyl-6-methyl-2-pyrιdιnyl)oxy]benzoιc acid 12 (874 mg, 78%) 1H NMR (400 MHz, DMSO-Cf6) δ 12 97 (br, 1 H), 10 18 (s, 1 H), 8 23 (d, J = 8 3 Hz, 1 H), 7 99 (d, J = 8 6 Hz, 2 H), 7 28 (d, J = 8 6 Hz, 2 H), 7 06 (d, J = 8 5 Hz, 1 H), 2 61 (s, 3 H), MS m/z 258 (M+H)+
Intermediate 13
2-methyl-6-{[4-(4-morpholιnylcarbonyl)phenyl]oxy}-3-pyrιdιnecarbaldehyde
Figure imgf000017_0001
A solution of 4-[(5-formyl-6-methyl-2-pyrιdιnyl)oxy]benzoιc acid 12 (300 mg, 1 17 mmol, 1 eq ) and carbonyldiimidazole (378 mg, 2 33 mmol, 2 eq ) in acetonitrile (7 5 mL) was heated at 77 °C under nitrogen for 2 hr After cooling to ambient temperature, morpholine (510 μL, 5 83 mmol, 5 eq ) was added and the reaction was stirred for 15 mm and then evaporated in vacuo The residue was partitioned between ethyl acetate and 1N NaHSO4 After separating the layers, the aqueous phase was back-extracted twice with ethyl acetate The combined organic layers were washed with brine, dried over anhydrous magnesium sulfate, filtered and evaporated in vacuo to a residue Purification on flash grade silica gel eluting with 1 1 ethyl acetate hexanes provided 2-methyl-6-{[4-(4- morpholιnylcarbonyl)phenyl]oxy}-3-pyrιdιnecarbaldehyde 13 (215 mg, 57%) as a solid 1H NMR (400 MHz, DMSO-Cf6) δ 10 18 (s, 1 H), 8 20 (d, J = 8 6 Hz, 1 H), 7 48 (d, J = 8 3 Hz, 2 H), 7 25 (d, J = 8 3 Hz, 2 H), 7 02 (d, J = 8 3 Hz, 1 H), 3 62-3 38 (m, 8 H), 2 61 (s, 3 H), MS /T)/z 327 (M+H)+
Example II: 5-{[((3-fluorophenyl){1 -[(2-methyl-6-{[4-(4-morpholιnylcarbonyl)phenyl]oxy}-3- pyridinylJmethylH-piperidinylJaminoJcarbonylJamino^-pyridinecarboxamide
Figure imgf000017_0002
was prepared from 9 and 13 as described in Example III
1H NMR (400 MHz, CD3OD) δ 8 57 (d, J = 2 2 Hz, 1 H), 7 96-7 89 (m, 2 H), 7 66 (d, J = 8 2 Hz, 1 H), 7 52 (m, 1 H), 745 (d, J = 8 5 Hz, 2 H), 7 24 (m, 1 H), 7 15-7 09 (m, 3 H), 6 72 (d, J = 8 2 Hz, 2 H), 445 (m, 1 H), 3 68 (br, 8 H), 3 51 (s, 2 H), 2 95 (d, J = 11 8 Hz, 2 H), 2 38 (s, 3 H), 2 28 (app t, J = 11 8 Hz, 2 H), 1 88 (d, J = 12 0 Hz, 2 H), 1 45 (app qd, J = 12 2, 4 1 Hz, 2 H) HRMS (M+H)+ calcd for C36H38FN7O5 + H, 668 2997, found, 668 3009 LR/MS M+H = 668
Example III
4-[(5-{[4-((3-fluorophenyl){[(4-fluorophenyl)amιno]carbonyl}amιno)-1-pιperιdιnyl] methyl }-6- methyl-2-pyrιdιnyl)oxy]-Λ/-[2-(methyloxy)ethyl]benzamιde was synthesized as described below
The synthesis of intermediate 15
Figure imgf000018_0002
Intermediate 14
1 ,1-dιmethylethyl 4-((3-fluorophenyl){[(4-fluorophenyl)amιno]carbonyl}amιno)-1- piperidmecarboxylate o
A solution of 1 ,1-dιmethylethyl 4-[(3-fluorophenyl)amιno]-1-pιperιdιnecarboxylate 6 (40 0 g, 0 13 mol, 1 equiv) in dichloroethane (400 mL) was treated with N, N- diisopropylethylamme (56 8 mL, 0 32 mol, 2 4 equiv) and 1 9 M phosgene in toluene (85 8 mL, 0 16 mol, 1 2 equiv) and then heated to 40 0C for 45 mm 4-fluoroanιlιne (15 4 mL, 0 16 mol, 1 2 equiv) was added and then reaction was allowed to continue to stir at 40 0C for another 18 h The reaction was diluted with dichloromethane, washed with satd aq NaHCO3, dried over Na2SO4, filtered and concentrated to give a white solid The solid was suspended in ether, stirred for 18 h and filtered to give 14 (38 0 g, 65% yield) as a white solid
1H NMR (400 MHz, DMSO-cfe) δ ppm 742 - 7 54 (m, 2 H), 7 28 - 7 36 (m, 2 H), 7 20 - 7 28 (m, 1 H), 7 09 - 7 15 (m, 1 H), 6 95 - 7 07 (m, 3 H), 4 32 - 4 45 (m, 1 H), 3 88 - 3 99 (m, 2 H), 2 73 (s, 2 H), 1 76 (d, J=11 54 Hz, 2 H), 1 23 - 1 34 (m, 9 H), 1 01 - 1 16 (m, 2 H) ES- LCMS m/z 432 1 (M+H)
Intermediate 15
Figure imgf000019_0001
A solution of 1 ,1-dιmethylethyl 4-((3-fluorophenyl){[(4-fluorophenyl)amιno]carbonyl}amιno)- 1-pιperιdιnecarboxylate 14
(50 5 g, 0 12 mol, 1 equiv) in 4N HCI in dioxane (117 1 mL, 047 mol, 4 equiv) was stirred at RT for 4h and concentrated to a white solid The solid was suspended in ethyl acetate, stirred for 2 h and filtered to give 15 (43 0 g, 97% yield) as a white solid 1H NMR (400 MHz, DMSO-cfe) δ ppm 8 93 (d, J=10 62 Hz, 1 H), 8 24 (s, 1 H), 7 62 (s, 1 H), 7 44 - 7 55 (m, 1 H), 7 31 - 7 40 (m, 2 H), 7 24 - 7 31 (m, 1 H), 7 18 (dd, J=9 98, 1 92 Hz, 1 H), 7 08 (d, J=8 06 Hz, 1 H), 7 00 (t, J=8 88 Hz, 2 H), 4 42 - 4 60 (m, 1 H), 3 13 - 3 27 (m, 2 H), 2 96 (s, 2 H), 1 91 (s, 2 H), 1 49 (d, J=12 82 Hz, 2 H) ES-LCMS m/z 332 2 (M+H)
Figure imgf000019_0002
Synthesis of intermediate 16 4-[(5-formyl-6-methyl-2-pyrιdιnyl)oxy]-Λ/-[2-(methyloxy)ethyl]benzamιde
Figure imgf000019_0003
A solution of 4-[(5-formyl-6-methyl-2-pyrιdιnyl)oxy]benzoιc acid 12 (350 mg, 1 36 mmol, 1 eq ) and carbonyldiimidazole (441 mg, 2 72 mmol, 2 eq ) in acetonitrile (10 mL) was heated at 77 C under nitrogen for 2 hr After cooling to ambient temperature, 2- (methyloxy)ethanamιne (590 μL, 6 80 mmol, 5 eq ) was added and the reaction was stirred for 15 mm and then evaporated in vacuo The residue was partitioned between ethyl acetate and 1N NaHSO4 After separating the layers, the aqueous phase was back- extracted three times with ethyl acetate The combined organic layers were washed with brine, dried over anhydrous magnesium sulfate, filtered and evaporated in vacuo to a solid residue of 4-[(5-formyl-6-methyl-2-pyrιdιnyl)oxy]-Λ/-[2-(methyloxy)ethyl]benzamιde 16 (262 mg, 61 %) 1H NMR (400 MHz, CDCI3) δ 10 26 (s, 1 H), 8 13 (d, J = 8 4 Hz, 1 H), 7 85 (d, J = 8 6 Hz, 2 H), 7 22 (d, J = 8 5 Hz, 2 H), 6 84 (d, J = 8 4 Hz, 1 H), 649 (br, 1 H), 3 67 (m, 2 H), 3 57 (m, 2 H), 340 (s, 3 H), 2 72 (s, 3 H), MS m/z 315 (M+H)+
Example III , 4-[(5-{[4-((3-fluorophenyl){[(4-fluorophenyl)amιno]carbonyl}amιno)-1- pιperιdιnyl]methyl}-6-methyl-2-pyrιdιnyl)oxy]-Λ/-[2-(methyloxy)ethyl]benzamιde was synthesized as follows
Figure imgf000020_0001
A mixture of intermediate 164-[(5-formyl-6-methyl-2-pyrιdιnyl)oxy]-Λ/-[2- (methyloxy)ethyl]benzamιde (37 mg, 0 096 mmol, 1 eq ), intermediate 15 Λ/-(3- fluorophenyl)-Λ/'-(4-fluorophenyl)-Λ/-4-pιperιdιnylurea hydrochloride (30 mg, 0 096 mmol, 1 eq ), diisopropylethylamine (20 μL, 0 115 mmol, 1 2 eq ) and sodium triacetoxyborohydride (41 mg, 0 193 mmol, 2 eq ) in dichloromethane (2 5 mL) was stirred under nitrogen for 16 hrs The reaction mixture was diluted with dichloromethane and washed with 5% w/v aqueous K2CO3 After separation of the layers, the aqueous phase was back-extracted with dichloromethane The combined organic layers were dried over anhydrous sodium sulfate, filtered, evaporated in vacuo and purified by preparative TLC eluting with 13% 2M methanols NH3 in dichloromethane to provide 4-[(5-{[4-((3-fluorophenyl){[(4- fluorophenyl)amιno]carbonyl}amιno)-1-pιperιdιnyl]methyl}-6-methyl-2-pyrιdιnyl)oxy]-Λ/-[2- (methyloxy)ethyl]benzamιde as a foam (39 mg, 64%) 1H NMR (400 MHz, CD3OD) δ 7 85 (d, J = 8 7 Hz, 2 H), 7 66 (d, J = 8 3 Hz, 1 H), 7 52 (app q, J = 7 9 Hz, 1 H), 7 25-7 20 (m, 3 H), 7 13-7 07 (m, 4 H), 6 95 (app t, J = 8 8 Hz, 2 H), 6 71 (d, J = 8 3 Hz, 1 H), 442 (m, 1 H), 3 55 (s, 4 H), 3 46 (s, 2 H), 3 37 (s, 3 H), 2 91 (d, J = 11 6 Hz, 2 H), 2 39 (s, 3 H), 2 20
(app t, J = 11 6 Hz, 2 H), 1 85 (d, J = 11 8 Hz, 2 H), 1 42 (app qd, J = 12 2, 3 7 Hz, 2 H) HRMS (M+H)+ calcd for C35H37F2N5O4 + H, 630 2892, found, 630 2887 LR/MS M+H : 630
Example IV Λ/-(3-fluorophenyl)-Λ/'-(4-fluorophenyl)-Λ/-{1-[(2-methyl-6-{[4-(4- morpholιnylcarbonyl)phenyl]oxy}-3-pyrιdιnyl)methyl]-4-pιperιdιnyl}urea
Figure imgf000021_0001
A mixture of 2-methyl-6-{[4-(4-morpholιnylcarbonyl)phenyl]oxy}-3-pyrιdιnecarbaldehyde (25 mg, 0 077 mmol, 1 eq ), intermediate 15; Λ/-(3-fluorophenyl)-Λ/'-(4-fluorophenyl)-Λ/-4- piperidinylurea hydrochloride (27 mg, 0 077 mmol, 1 eq ), diisopropylethylamine (16 μL, 0 092 mmol, 1 2 eq ) and sodium triacetoxyborohydride (32 mg, 0 153 mmol, 2 eq ) in dichloromethane (2 5 mL) was stirred under nitrogen for 16 hrs The reaction mixture was diluted with dichloromethane and washed with 5% w/v aqueous K2CO3 After separation of the layers, the aqueous phase was back-extracted with dichloromethane The combined organic layers were dried over anhydrous sodium sulfate, filtered, evaporated in vacuo and purified by preparative TLC eluting with 5% 2M methanolic NH3 in dichloromethane to provide W-(3-fluorophenyl)-W-(4-fluorophenyl)-W-{1-[(2-methyl-6-{[4-(4- morpholιnylcarbonyl)phenyl]oxy}-3-pyrιdιnyl)methyl]-4-pιperιdιnyl}urea as a foam (29 mg, 59%) 1H NMR (400 MHz, CD3OD) δ 7 64 (d, J = 8 2 Hz, 1 H), 7 53-744 (m, 3 H), 7 23- 7 19 (m, 3 H), 7 14-7 06 (m, 4 H), 6 93 (t, J = 8 8 Hz, 2 H), 6 70 (d, J = 8 2 Hz, 1 H), 4 40
(m, 1 H), 3 67 (br, 8 H), 344 (s, 2 H), 2 89 (d, J = 11 2 Hz, 2 H), 2 37 (s, 3 H), 2 18 (app t, J = 11 5 Hz, 2 H), 1 84 (d, J = 12 2 Hz, 2 H), 1 41 (app qd, J = 12 2, 3 6 Hz, 2 H) HRMS (M+H)+ calcd for C36H37F2N5O4 + H, 642 2892, found, 642 2899 LR/MS M+H = 642 Example V
S-IKbutylli-Ke-p-fP-hydroxyethyOaminolcarbonylJphenyOoxyl^-methyl-S- pyrιdιnyl}methyl)-4-pιperιdιnyl]amιno}carbonyl)amιno]-2,4-dιfluorobenzamιde
Figure imgf000022_0001
The synthesis of intermediate 24
Figure imgf000022_0002
Intermediate 17
2,4-dιfluoro-5-nιtrobenzoιc acid
Figure imgf000022_0003
Nitric Acid (120 mL) was added slowly to a cold solution of sulfuric acid (200 mL) 2,4- difluorobenzoic acid (65 g, 0 41 mol, 1 equiv) was added portionwise over 1 5 h and the reaction allowed to warm to RT The reaction mixture was heated to 50 0C for 2h and then poured slowly onto ice The product was extracted into ethyl acetate, dried over Na2SO4, filtered and concentrated to give intermediate 17 (82 g, 98% yield) as a yellow solid 1H NMR (400 MHz, DMSO-cfe) δ ppm 8 56 (t, J=I 87 Hz, 1 H), 7 82 (t, J=10 71 Hz, 1 H) Intermediate 18
Figure imgf000023_0001
Sulfuric acid (3 mL) was added to a solution of 2,4-dιfluoro-5-nιtrobenzoιc acid intermediate 17(100 g, 049 mol, 1 equiv) in methanol (700 mL) and the reaction heated to reflux for 18 h The reaction mixture was concentrated and the residue taken up in water and brought to pH 8 by addition of 1N NaOH The product was extracted into ethyl acetate, dried over Na2SO4, filtered and concentrated The residue was purified by silica gel flash column chromatography (0 → 25% EtOAc Hexanes) to give intermediate 18 (90 g, 85% yield) as a tan solid
1H NMR (400 MHz, DMSO-cfe) δ ppm 8 56 - 8 64 (m, 1 H), 7 83 - 7 94 (m, 1 H), 3 85 - 3 92 (m, 3 H) Intermediate 19 methyl 5-amιno-2,4-dιfluorobenzoate
Figure imgf000023_0002
Iron powder (12 3 g, 0 22 mol, 4 equiv) was added to a solution of methyl 2,4-dιfluoro-5- nitrobenzoate intermediate 18 (12 0 g, 0 06 mol, 1 equiv) in acetic acid (150 mL) and water (150 mL) and the reaction was heated to 40 0C for 1 5 h The reaction mixture was filtered thru a pad of celite and the filtered solution extracted with ethyl acetate The organics were washed with satd aq NaCI, dried over Na2SO4, filtered and concentrated to give intermediate 19 (10 Og, 96% yield) as a brown solid 1H NMR (400 MHz, DMSO-cfe) δ ppm 7 27 (dd, J=9 98, 742 Hz, 1 H), 7 14 (t, J=10 99 Hz, 1 H), 5 28 (s, 2 H), 3 77 (s, 3 H) ES-LCMS m/z 189 0 (M+H)
Intermediate 20
1 ,1-dιmethylethyl 4-(butylamιno)-1-pιperιdιnecarboxylate
Figure imgf000024_0001
Sodium triacetoxyborohydride (88 5 g, 0 42 mol, 1 6 equiv) was added portionwise over 1 h to a solution of 4-BOC-pιperιdone (52 0 g, 0 26 mol, 1 equiv), π-butyl amine (30 9 mL, 0 31 mol, 1 2 equiv) and acetic acid (44 7 mL, 0 78 mol, 3 equiv) in dichloromethane (700 mL) at
0 0C The reaction mixture was allowed to warm to RT over 2 h and then slowly poured into cold 3 3N NaOH (1L) with stirring The reaction mixture was then separated and the organics dried over Na2SO4, filtered and concentrated to give intermediate 20 (66 g, 98% yield) as an oil
1H NMR (400 MHz, CHLOROFORM-d) δ ppm 4 03 (s, 2 H), 2 77 (t, J=11 99 Hz, 2 H), 2 52 - 2 66 (m, 3 H), 1 82 (d, J=11 90 Hz, 2 H), 1 39 - 1 52 (m, 11 H), 1 29 - 1 39 (m, 2 H), 1 16 -
1 29 (m, 2 H), 1 06 (s, 1 H), 0 84 - 0 96 (m, 3 H)
Intermediate 21
1 ,1-dιmethylethyl 4-{butyl[({2,4-dιfluoro-5-
[(methyloxyjcarbonyljphenyljaminojcarbonyljaminoj-i-piperidinecarboxylate
Figure imgf000024_0002
A solution of methyl 5-amιno-2,4-dιfluorobenzoate intermediate 19 (5 5 g, 29 5 mmol, 1 equiv) in dichloromethane (100 mL) at -40 0C was treated with pyridine (7 16 mL, 88 6 mmol, 3 0 equiv) and 1 9 M phosgene in toluene (23 3 mL, 44 3 mmol, 1 5 equiv) After 1 5 h, a solution of 1 ,1-dιmethylethyl 4-(butylamιno)-1-pιperιdιnecarboxylate intermediate 20 (9 1 g, 35 4 mmol, 1 2 equiv) in dichloromethane (50 mL) was added and the reaction was allowed to warm to RT for 18 h The reaction was diluted with dichloromethane, washed with satd aq NaHCO3, dried over Na2SO4, filtered and concentrated to give intermediate 21 (13 1 g, 95% yield) a white solid
1H NMR (400 MHz, DMSO-cfe) δ ppm 8 09 (s, 1 H), 7 89 - 7 99 (m, 1 H), 7 42 (t, J=10 62 Hz, 1 H), 3 88 - 3 99 (m, 1 H), 3 85 (s, 3 H), 3 11 - 3 22 (m, 2 H), 2 96 (d, J=12 27 Hz, 2 H), 2 41 - 2 53 (m, 3 H), 1 43 - 1 59 (m, 5 H), 1 21 - 1 35 (m, 2 H), 1 20 (s, 9 H), 0 90 (t, J=I 33 Hz, 3 H) ES-LCMS m/z 468 1 (M-H)
Intermediate 22 S-røbutyHI-fKI .I-dimethylethylJoxyJcarbonylJ^-pipendinylJaminoJcarbonylJamino)^^- difluorobenzoic acid
Figure imgf000025_0001
A solution of 1 ,1-dιmethylethyl 4-{butyl[({2,4-dιfluoro-5- [(methyloxyjcarbonyljphenyljaminojcarbonyljaminoj-i-piperidinecarboxylate intermediate 21 (13 Og, 27 7 mmol, 1 equiv) and 1 N lithium hydroxide (15 mL) in tetrahydrofuran (30 mL) was stirred at RT for 2 h The reaction was concentrated down and then acidified to pH 2 by addition of 1N hydrochloric acid The aqueous layer was extracted with ethyl acetate and the organics dried over Na2SO4, filtered and concentrated to give intermediate 22 (12 0 g, 95% yield) as a white solid
1H NMR (400 MHz, DMSO-cfe) δ ppm 8 09 (s, 1 H), 7 81 - 7 91 (m, 1 H), 7 29 - 7 38 (m, 1 H), 3 93 - 4 07 (m, 1 H), 3 24 - 3 38 (m, 2 H), 3 09 - 3 20 (m, 2 H), 2 72 (d, J=Q 97 Hz, 2 H), 1 52 - 1 65 (m, 4 H), 1 42 - 1 51 (m, 2 H), 1 34 - 1 41 (m, 9 H), 1 19 - 1 33 (m, 2 H), 0 87 (t, J=I 33 Hz, 3 H) ES-LCMS m/z 45443 (M-H)
Intermediate 23
1 ,1-dιmethylethyl 4-[({[5-(amιnocarbonyl)-2,4 dιfluorophenyl]amιno}carbonyl)(butyl)amιno]-
1 -piperidinecarboxylate
Figure imgf000025_0002
A solution of 5-({[butyl(1-{[(1 ,1-dιmethylethyl)oxy]carbonyl}-4- pιperιdιnyl)amιno]carbonyl}amιno)-2,4-dιfluorobenzoιc acid intermediate 22 (12 0, 26 3 mmol, 1 equiv) in Λ/,Λ/-dιmethylformamιde (100 mL) was treated with HATU (10 0 g, 26 3 mmol, 1 equiv) followed by Λ/,Λ/-dιιsopropylethylamιne (9 2mL, 52 6 mmol, 2 equiv) The reaction was stirred at RT for 15 min before the addition of ammonia hydroxide (2 6 mL, 394 mmol, 1 5 equiv) The reaction was stirred at RT for 2 h and then partitioned between ethyl acetate and water The organics were washed with satd aq NaHCO3, dried over Na2SO4, filtered and concentrated The residue was purified by silica gel flash column chromatography (0 → 5% 2N NH3 in MeOH CH2CI2) to give intermediate 23 (10 1 g, 80% yield) as a white solid
1H NMR (400 MHz, DMSO-cfe) δ ppm 8 04 (s, 1 H), 7 55 - 7 70 (m, 3 H), 7 29 (t, J=10 35 Hz, 1 H), 3 98 (s, 3 H), 3 24 - 3 29 (m, 1 H), 3 03 - 3 19 (m, 2 H), 2 57 - 2 79 (m, 2 H), 1 50 - 1 65 (m, 3 H), 1 45 (s, 2 H), 1 37 (s, 9 H), 1 16 - 1 30 (m, 2 H), 0 86 (t, J=I 23 Hz, 3 H) ES-LCMS m/z 453 4 (M-H)
Intermediate 24 5-({[butyl(4-pιperιdιnyl)amιno]carbonyl}amιno)-2,4-dιfluorobenzamιde
Figure imgf000026_0001
A solution of 1 ,1-dιmethylethyl 4-[({[5-(amιnocarbonyl)-2,4- dιfluorophenyl]amιno}carbonyl)(butyl)amιno]-1-pιperιdιnecarboxylate intermediate 23 (3 0 g, 6 60 mmol, 1 equiv) in 4N HCI in dioxane (16 5 mL, 66 0 mol, 10 equiv) was stirred at RT for 2 h and concentrated to a yellow solid The solid was dissolved in water and washed with ethyl acetate The aqueous layer was made basic (pH 8) by addition of 1 N NaOH and then extracted with dichloromethane The organics were dried over Na2SO4, filtered and concentrated to give intermediate 24 (1 9g, 81% yield) as a yellow solid 1H NMR (400 MHz, DMSO-cfe) δ ppm 8 00 (s, 1 H), 7 58 - 7 72 (m, 3 H), 7 30 (t, J=10 45 Hz, 1 H), 3 86 - 3 97 (m, 1 H), 3 57 - 3 62 (m, 1 H), 3 30 (s, 2 H), 3 10 - 3 19 (m, 2 H), 2 90
- 3 00 (m, 2 H), 2 39 - 247 (m, 1 H), 1 69 - 1 79 (m, 1 H), 1 41 - 1 58 (m, 4 H), 1 20 - 1 34 (m, 2 H), 0 88 (t, J=I 33 Hz, 3 H) ES-LCMS m/z 355 1 (M+H)
Example V
Figure imgf000027_0001
was prepared from intermediate 245-({[butyl(4-pιperιdιnyl)amιno]carbonyl}amιno)-2,4- difluorobenzamide and 4-[(5-formyl-6-methyl-2-pyrιdιnyl)oxy]-Λ/-(2-hydroxyethyl)benzamιde by the method described in example III
1H NMR (400 MHz, CD3OD) δ 7 88-7 81 (m, 3 H), 7 72 (d, J = 8 2 Hz, 1 H), 7 14-7 07 (m, 3 H), 6 75 (d, J = 8 2 Hz, 1 H), 4 02 (m, 1 H), 3 69 (t, J = 5 9 Hz, 2 H), 3 50-3 45 (m, 4 H), 3 24 (m, 2 H), 2 97 (d, J = 11 6 Hz, 2 H), 2 46 (s, 3 H), 2 18 (app t, J = 11 5 Hz, 2 H), 1 82 (app qd, J = 12 0, 3 7 Hz, 2H), 1 71 (m, 2 H), 1 60 (m, 2 H), 1 36 (m, 2 H), 0 95 (t, J = 7 4 Hz, 3 H)
HRMS (M+H)+ CaICd TOr C33H40F2N6O5 + H, 639 3107, found, 639 3107 LR/MS M+H = 639
Example Vl
2,4-dιfluoro-5-{[((3-fluorophenyl){1-[(2-methyl-6-{[4-(1 ,3-oxazol-5-yl)phenyl]oxy}-3- pyrιdιnyl)methyl]-4-pιperιdιnyl}amιno)carbonyl]amιno}benzamιde
Figure imgf000027_0002
Intermediate 25
Figure imgf000028_0001
4-methyl-5-[4-(methyloxy)phenyl]-1 ,3-oxazole
A solution of p-anisaldehyde (2 mL, 16 4 mmol), solid K2CO3 (3 8 g, 29 6 mmol) and tosylmethylisocyanide (3 8 g, 19 5 mmol) in MeOH (20 mL) was heated under refluc for 6 h Sat'd aq NaHCO3 was then added to the suspension and the mixture was extracted with CH2CI2 The organic extracts were dried (Na2SO4), concentrated, and the residue was purified by silica gel flash column chromatography (0→33% EtOAc hexanes) to give 4- methyl-5-[4-(methyloxy)phenyl]-1 ,3-oxazole, 25 (879 8 mg, 31 % yield) as an off white solid 1 H NMR (400 MHz, CHLOROFORM-d) δ 7 80 (s, 1 H), 7 48 - 7 53 (m, 2 H), 7 17 (s, 1 H),
6 85 - 6 90 (m, 2 H), 3 76 (s, 3 H) ES-LCMS m/z 176 1 (M+H)
Intermediate 26
Figure imgf000028_0002
4-(4-methyl-1 ,3-oxazol-5-yl)phenol
To a -78 °C solution of 4-methyl-5-[4-(methyloxy)phenyl]-1 ,3-oxazole, 25 (879 8 mg, 5 0 mmol) in CH2CI2 (10 mL) was slowly added BBr3 (10 mL of a 1M solution in CH2CI2) The reaction mixture was allowed to warm to RT and stirring was continued for 8 h The solution was cooled to 0 °C and Et2O (20 mL) and MeOH (1 mL) were slowly added followed by sat'd aq NaHCO3 The organic layer was isolated and the aqueous layer was extracted with CH2CI2 The organic layers were combined, dried (Na2SO4), concentrated and the residue was redissolved in MeOH (10 mL) and stirred for 2 h The solution was then concentrated and passed through a short plug of silica gel, eluting with 5% MeOH in CH2CI2 to provide 4-(4-methyl-1 ,3-oxazol-5-yl)phenol, 26 in quantitative yield as a light brown solid 1 H NMR (400 MHz, METHANOL-ώ,) δ 8 14 (s, 1 H), 7 51 - 7 55 (m, 2 H), 7 28
(s, 1 H), 6 82 - 6 87 (m, 2 H) ES-LCMS m/z 162 1 (M+H)
Intermediate 27
Figure imgf000028_0003
,3-oxazol-5-yl)phenyl]oxy}-3-pyrιdιnecarbonιtrιle
Prepared from 3 and 26 (in 75 % yield) as described for intermediate 33 1 H NMR (400 MHz, CHLOROFORM-d) δ 7 91 (s, 1 H), 7 82 (d, J=8 42 Hz, 1 H), 7 66 - 7 70 (m, 2 H), 7 33 (s, 1 H), 7 16 - 7 21 (m, 2 H), 6 78 (d, J=8 61 Hz, 1 H), 2 58 (s, 3 H) ES-LCMS m/z 278 2 (M+H)
Intermediate 28
Figure imgf000029_0001
2-methyl-6-{[4-(1 ,3-oxazol-5-yl)phenyl]oxy}-3-pyrιdιnecarbaldehyde Prepared (in 12 % yield) as described for intermediate 5
1 H NMR (400 MHz, CHLOROFORM-d) δ 10 24 (s, 1 H), 8 12 (d, J=843 Hz, 1 H), 7 93 (s, 1 H), 7 68 - 7 74 (m, 2 H), 7 35 (s, 1 H), 7 21 - 7 26 (m, 2 H), 6 83 (d, J=843 Hz, 1 H), 2 73
(s, 3 H) ES-LCMS m/z 281 3 (M+H)
Synthesis of Intermediate 32
Figure imgf000029_0002
Intermediate 29
1 ,1-dιmethylethyl 4-[[({2,4-dιfluoro-5-[(methyloxy)carbonyl]phenyl}amιno)carbonyl](3- fluorophenyl)amιno]-1-pιperιdιnecarboxylate
Figure imgf000029_0003
A solution of methyl 5-amιno-2,4-dιfluorobenzoate intermediate 19 (5 O g, 26 5 mmol, 1 equiv) in dichloromethane (150 mL) at -40 0C was treated with pyridine (6 4 mL, 79 7 mmol, 3 0 equiv) and 1 9 M phosgene in toluene (20 1 mL, 39 8 mmol, 1 5 equiv) After 1 5 h, the reaction was concentrated down and dried under vac The residue was taken up in dichloromethane (150 mL) and 1 ,1-dιmethylethyl 4-[(3-fluorophenyl)amιno]-1- piperidmecarboxylate intermediate 6 (8 6 g, 29 2 mmol, 1 1 equiv) was added and the reaction was heated to 40 0C 18 h The reaction was diluted with dichloromethane, washed with satd aq NaHCO3, dried over Na2SO4, filtered and concentrated The residue was purified by silica gel flash column chromatography (0 → 30% ethyl acetate hexanes) to give intermediate 29 (12 6 g, 94% yield) a white solid
1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8 65 (dd, J=8 88, 7 78 Hz, 1 H), 744 - 7 55 (m, 1 H), 7 17 - 7 24 (m, 1 H), 7 04 (d, J=I 87 Hz, 1 H), 6 92 - 6 99 (m, 1 H), 6 80 (t, J=10 26 Hz, 1 H), 5 96 (d, J=Z 11 Hz, 1 H), 4 56 - 4 70 (m, 1 H), 4 03 - 4 26 (m, 2 H), 3 80 - 3 92 (m, 3 H), 2 79 (s, 2 H), 1 85 (d, J=13 37 Hz, 2 H), 1 39 (s, 9 H), 1 19 - 1 35 (m, 2 H) ES-LCMS m/z 530 1 (M+Na)
Intermediate 30 5-({[(1-{[(1 ,1-dιmethylethyl)oxy]carbonyl}-4-pιperιdιnyl)(3- fluorophenyl)amιno]carbonyl}amιno)-2,4-dιfluorobenzoιc acid
Figure imgf000030_0001
A solution of 1 ,1-dιmethylethyl 4-[[({2,4-dιfluoro-5- [(methyloxyJcarbonylJphenylJaminoJcarbonylKS-fluorophenylJaminoJ-i- piperidmecarboxylate intermediate 29 (12 6 g, 24 9 mmol, 1 equiv) and 1N lithium hydroxide (50 mL) in tetrahydrofuran (200 mL) was stirred at RT for 72 h The reaction was concentrated down and then acidified to pH 3 by addition of 1N hydrochloric acid The aqueous layer was extracted with ethyl acetate and the organics dried over Na2SO4, filtered and concentrated to give intermediate 30 (10 6 g, 86% yield) as a white solid 1H NMR (400 MHz, DMSO-cfe) δ ppm 7 89 (t, J=842 Hz, 1 H), 7 43 - 7 52 (m, 1 H), 7 22 - 7 33 (m, 3 H), 7 13 - 7 20 (m, 1 H), 7 05 - 7 12 (m, 1 H), 4 29 - 440 (m, 1 H), 3 86 - 3 97
(m, 2 H), 2 72 (s, 2 H), 1 76 (s, 2 H), 1 24 - 1 32 (m, 9 H), 1 00 - 1 16 (m, 2 H) ES-LCMS /D/Z 492 1 (M-H)
Intermediate 31 1 ,1-dιmethylethyl 4-[({[5-(amιnocarbonyl)-2,4-dιfluorophenyl]amιno}carbonyl)(3- fluorophenyl)amιno]-1-pιperιdιnecarboxylate
Figure imgf000031_0001
A solution of 5-({[(1-{[(1 ,1-dιmethylethyl)oxy]carbonyl}-4-pιperιdιnyl)(3- fluorophenyl)amιno]carbonyl}amιno)-2,4-dιfluorobenzoιc acid intermediate 30 (6 6, 13 3 mmol, 1 equiv) in Λ/,Λ/-dιmethylformamιde (100 mL) was treated with HATU (5 0 g, 13 3 mmol, 1 equiv) followed by Λ/,Λ/-dιιsopropylethylamιne (4 6 mL, 36 7 mmol, 2 equiv) The reaction was stirred at RT for 15 mm before the addition of ammonia hydroxide (1 3 mL, 20 0 mmol, 1 5 equiv) The reaction was stirred at RT for 4 h and then partitioned between ethyl acetate and water The organics were washed with satd aq NaHCO3, dried over Na- 2SO4, filtered and concentrated The residue was purified by silica gel flash column chromatography (0 → 10% 2N NH3 in MeOH CH2CI2) to give intermediate 31 (5 8 g, 88% yield) as a tan solid
1H NMR (400 MHz, DMSO-cfe) δ ppm 7 68 (t, J=842 Hz, 1 H), 7 60 (s, 2 H), 7 44 - 7 54 (m, 1 H), 7 23 - 7 32 (m, 3 H), 7 13 - 7 19 (m, 1 H), 7 09 (dd, J=I 87, 0 92 Hz, 1 H), 4 29 - 442 (m, 1 H), 3 86 - 3 99 (m, 2 H), 2 79 (s, 2 H), 1 78 (d, J=11 35 Hz, 2 H), 1 30 (s, 9 H), 1 02 - 1 16 (m, 2 H) ES-LCMS m/z 515 3 (M+Na)
Intermediate 32 2,4-dιfluoro-5-({[(3-fluorophenyl)(4-pιperιdιnyl)amιno]carbonyl}amιno)benzamιde
Figure imgf000031_0002
A solution of 1 ,1-dιmethylethyl 4-[({[5-(amιnocarbonyl)-2,4- difluorophenyl]amino}carbonyl)(3-fluorophenyl)amino]-1-piperidinecarboxylate intermediate 31 (5 8 g, 11 7 mmol, 1 equiv) in 4N HCI in dioxane (29 0 mL, 117 7 mmol, 10 equiv) was stirred at RT for 2 h and concentrated to a yellow solid The solid was dissolved in water and washed with ethyl acetate The aqueous layer was made basic (pH 8) by addition of 1 N NaOH and then extracted with dichloromethane The organics were dried over Na2SO4, filtered and concentrated to give intermediate 32 (4 1g, 89 % yield) as a yellow solid
1H NMR (400 MHz, DMSO-cfe) δ ppm 7 67 - 7 74 (m, 1 H), 7 60 (s, 2 H), 7 45 - 7 54 (m, 1
H), 7 24 - 7 31 (m, 2 H), 7 21 (s, 1 H), 7 05 - 7 16 (m, 2 H), 4 20 - 4 33 (m, 1 H), 3 29 (s, 1
H), 2 82 - 2 92 (m, 2 H), 2 38 - 2 50 (m, 2 H), 1 69 (dd, J=11 54, 1 65 Hz, 2 H), 1 00 - 1 14
(m, 2 H) ES-LCMS m/z 393 3 (M+H)
Example Vl
2,4-dιfluoro-5-{[((3-fluorophenyl){1-[(2-methyl-6-{[4-(1 ,3-oxazol-5-yl)phenyl]oxy}-3- pyrιdιnyl)methyl]-4-pιperιdιnyl}amιno)carbonyl]amιno}benzamιde
Figure imgf000032_0001
was obtained from intermediates 28 and 32 as descrived in example III 1H NMR (CDCI3, 400 MHz) δ 8 62 (t, J=8 70 Hz, 1 H), 7 88 (s, 1 H), 7 58 - 7 65 (m, 2 H),
7 42 - 7 52 (m, 2 H), 7 27 (s, 1 H), 7 16 - 7 22 (m, 1 H), 7 12 - 7 16 (m, 2 H), 7 04 (dd, J=I 78, 1 01 Hz, 1 H), 6 97 (dt, J=9 02, 2 17 Hz, 1 H), 6 80 (t, J=10 53 Hz, 1 H), 6 59 (d, J=8 24 Hz, 1 H), 6 51 (d, J=9 71 Hz, 1 H), 6 02 (s, 1 H), 5 89 (d, J=2 75 Hz, 1 H), 446 - 4 58 (m, 1 H), 3 36 (s, 2 H), 2 85 (d, J=11 17 Hz, 2 H), 2 39 (s, 3 H), 2 14 (t, J=11 26 Hz, 2 H), 1 78 - 1 88 (m, 2 H), 1 31 - 1 47 (m, 2 H) HRMS (M+H)+ calcd for C35H3IF3N6O4 + H,
657 2437, found, 657 2433
Example VII
4-{[5-({4-[({[5-(am|nocarbonyO-2>4-d|f|uoro^^ pιperιdιnyl}methyl)-6-methyl-2-pyrιdιnyl]oxy}phenyl (i-methylethyl)carbamate
Figure imgf000033_0001
Intermediate 33: 2-methyl-6-({4-[(phenylmethyl)oxy]phenyl}oxy)-3-pyrιdιnecarbonιtrιle
Figure imgf000033_0002
To a solution of θ-chloro^-methyl-S-pyridinecarbonitrile (1 00 g, 6 56 mmol) in N, N- dimethylformamide (10 mL) was added 4-[(phenylmethyl)oxy]phenol (1 44 g, 7 21 mmol) and cesium carbonate (4 27 g, 13 1 mmol) The reaction mixture was stirred at room temperature for 5 hours, after which the reaction mixture was concentrated in vacuo The resulting residue was diluted with ethyl acetate and filtered to remove any solid material The organic layer was washed with water and brine, then dried over sodium sulfate
Filtration and concentration, followed by flash chromatography (5% to 60%, ethyl acetate in hexanes) provided 2-methyl-6-({4-[(phenylmethyl)oxy]phenyl}oxy)-3-pyrιdιnecarbonιtrιle 33 (2 07 g, 99%) as a white solid 1H NMR (400 MHz, CDCI3) δ 7 78 (d, J = 8 5 Hz, 1 H), 7 46-7 32 (m, 5 H), 7 06 (d, J = 9 1 Hz, 2 H), 7 00 (d, J = 10 4 Hz, 2 H), 6 68 (d, J = 8 4 Hz, 1 H), 5 07 (s, 2 H), 2 62 (s, 3 H), MS m/z 317 (M+H)+
Intermediate 34: 6-[(4-hydroxyphenyl)oxy]-2-methyl-3-pyrιdιnecarbonιtrιle
Figure imgf000033_0003
To a suspension of 2-methyl-6-({4-[(phenylmethyl)oxy]phenyl}oxy)-3-pyrιdιnecarbonιtrιle 33 (2 07 g, 6 54 mmol) in ethanol (10 mL) was added 5 % palladium on barium sulfate (100 mg) The reaction mixture was stirred under a hydrogen balloon for 16 hours After LC/MS indicated ~ 25 % conversion to product, an additional 100 mg palladium catalyst was added and the reaction mixture was stirred under hydrogen (30 psi) at room temperature for an additional 24 hours Filtration and concentration provided 6-[(4-hydroxyphenyl)oxy]- 2-methyl-3-pyrιdιnecarbonιtrιle 34 (1 40 g, 95%) as a white solid 1H NMR (400 MHz,
CD3OD) δ 7 93 (d, J = 8 6 Hz, 1 H), 6 92 (d, J = 8 8 Hz, 2 H), 6 79 (d, J = 8 9 Hz, 2 H), 6 72 (d, J = 8 6 Hz, 1 H), 6 58 (s, 1 H), 2 53 (s, 3 H), MS m/z 227 (M+H)+
Intermediate 35: 6-[(4-hydroxyphenyl)oxy]-2-methyl-3-pyrιdιnecarbaldehyde
Figure imgf000034_0001
To a cold (-35 "C) solution of 6-[(4-hydroxyphenyl)oxy]-2-methyl-3-pyrιdιnecarbonιtrιle 34 (620 mg, 2 74 mmol) in dichloromethane (25 mL) was added diisobutylaluminum hydride (8 22 mL, 1 0 M solution in dichloromethane, 8 22 mmol) dropwise, maintaining cooling bath temperature between -40 °C and -35 °C The reaction mixture was stirred at -35 °C for an additonal 30 minutes The reaction mixture was quenched with methanol (3 mL), added dropwise, followed by saturated aqueous citric acid solution The reaction mixture was stirred at room temperature for 2 hours, then diluted with dichloromethane The organic layer was separated, washed with water, washed with saturated aqueous sodium bicarbonate solution, then dried over sodium sulfate Filtration and concentration provided 6-[(4-hydroxyphenyl)oxy]-2-methyl-3-pyrιdιnecarbaldehyde 35 (474 mg, 75%) as a white solid 1H NMR (400 MHz, CDCI3) δ 10 20 (s, 1 H), 8 07 (d, J = 8 6 Hz, 1 H), 6 99-6 94 (m, 3 H), 6 88 (d, J = 8 8 Hz, 2 H), 6 67 (d, J = 8 6 Hz, 1 H), 2 76 (s, 3 H), MS m/z 230 (M+H)+ Intermediate 36: 4-[(5-formyl-6-methyl-2-pyrιdιnyl)oxy]phenyl (i-methylethyl)carbamate
Figure imgf000034_0002
To a solution of 6-[(4-hydroxyphenyl)oxy]-2-methyl-3-pyrιdιnecarbaldehyde 35 (96 mg, 042 mmol) in tetrahydrofuran was added isopropyl isocyanate (62 μL, 0 63 mmol) The reaction mixture was stirred at room temperature for 10 minutes, then stirred at 60 °C for 2 hours The reaction mixture was cooled and quenched with ice water The excess tetrahydrofuran was removed in vacuo The aqueous mixture was extracted with ethyl acetate The organic layer was washed with water and brine, then dried over sodium sulfate Filtration and concentration provided 4-[(5-formyl-6-methyl-2-pyrιdιnyl)oxy]phenyl (i-methylethyl)carbamate 36 (122 mg, 92%) as a white solid 1H NMR (400 MHz, CDCI3) δ 10 21 (s, 1 H), 8 06 (d, J = 8 5 Hz, 1 H), 7 17-7 10 (m, 5 H), 6 73 (d, J = 8 4 Hz, 1 H), 4 97 (br d, J = 7 2 Hz, 1 H), 3 88 (m, 1 H), 2 72 (s, 3 H), 1 23 (d, J = 6 5 Hz, 6 H), MS m/z 315 (M+H)+
Example VII
Figure imgf000035_0001
4-{[5-({4-[({[5-(am|nocarbonyO-2>4-d|f|uoroPn pιperιdιnyl}methyl)-6-methyl-2-pyrιdιnyl]oxy}phenyl (i-methylethyl)carbamate To a mixture of 2,4-dιfluoro-5-({[(3-fluorophenyl)(4- pιperιdιnyl)amιno]carbonyl}amιno)benzamιde hydrochloride 32 (60 mg, 0 14 mmol) in dichloromethane was added Λ/,Λ/-dιιsopropylethylamιne (29 μL, 0 17 mmol) The resulting mixture was stirred at room temperature for 3 hours 4-[(5-formyl-6-methyl-2- pyrιdιnyl)oxy]phenyl (i-methylethyl)carbamate 36 (44 mg, 0 14 mmol) was added and the reaction mixture was stirred at room temperature for 6 hours Sodium triacetoxyborohydride (59 mg, 0 28 mmol) was added and stirred 16 hours at room temperature The reaction mixture was diluted with dichloromethane and quenched with saturated aqueous sodium bicarbonate solution The organic layer was dried over sodium sulfate Filtration and concentration, follwed by preparative TLC provided 4-{[5-({4-[({[5- (aminocarbonylJ^^-difluorophenylJaminoJcarbonylXS-fluorophenylJaminoJ-i- pιperιdιnyl}methyl)-6-methyl-2-pyrιdιnyl]oxy}phenyl (i-methylethyl)carbamate (33 mg, 34%) as a white solid
1H NMR (400 MHz, CD3OD) δ 7 97 (app t, J = 8 3 Hz, 1 H), 7 60-7 50 (m, 2 H), 7 24 (app td, J = 8 4, 24 Hz, 1 H), 7 16-7 03 (m, 7 H), 6 58 (d, J = 8 3 Hz, 1 H), 440 (m, 1 H), 3 76 (m, 1 H), 342 (s, 2 H), 2 89 (br d, J = 11 2 Hz, 2 H), 2 39 (s, 3 H), 2 16 (app t, J = 11 2 Hz, 2 H), 1 85 (d, J = 11 1 Hz, 2 H), 1 42 (app qd, J = 12 3, 3 7 Hz, 2 H), 1 20 (d, J = 6 5 Hz, 6 H), MS m/z 691 (M+H), HRMS C36H37F3N6O5 (M+H)+ calcd 691 2856, found 691 2856
Example VIII 2,4-dιfluoro-5-[({(3-fluorophenyl)[1-({2-methyl-6-[(4-{2-[(1-methylethyl)amιno]-2- oxoethyl}phenyl)oxy]-3-pyridinyl}methyl)-4-piperidinyl]amino}carbonyl)amino]benzamide
Figure imgf000036_0001
Intermediate 37 phenylmethyl (4-hyclroxyphenyl)acetate
Figure imgf000036_0002
A mixture of (4-hydroxyphenyl)acetιc acid (3 50 g, 23 0 mmol, 1 0 eq ), benzyl bromide (2 74 mL, 34 5 mmol, 1 5 eq ) and potassium bicarbonate (2 76 g, 27 6 mmol, 1 20 eq ) in dimethylformamide (30 mL) was heated under nitrogen at 40 °C for 4 hrs The reaction mixture was evaporated in vacuo and the residue was partitioned between ethyl acetate and 1 N NaHSO4 After separating the layers, the aqueous phase was back-extracted with ethyl acetate The combined organic layers were washed with saturated aqueous NaHCO3, 1N NaHSO4, saturated aqueous brine and dried over magnesium sulfate Evaporation in vacuo and drying under high vacuum provided phenylmethyl (4- hydroxyphenyl)acetate (5 196 g, 93%) as a solid 1H NMR (400 MHz, CDCI3) δ 7 36-7 28 (m, 5 H), 7 14 (d, J = 8 3 Hz, 2 H), 6 76 (d, J = 8 7 Hz, 2 H), 5 10 (s, 2 H), 4 64 (br, 1 H), 3 58 (s, 2 H), MS m/z 243 (M+H)+ Intermediate 38 phenylmethyl {4-[(5-cyano-6-methyl-2-pyridinyl)oxy]phenyl}acetate
Figure imgf000037_0001
A mixture of phenylmethyl (4-hydroxyphenyl)acetate 37 (794 mg, 3 28 mmol, 1 0 eq ), 6- chloro-2-methyl-3-pyrιdιnecarbonιtrιle (500 mg, 3 28 mmol, 1 0 eq ) and cesium carbonate (1 281 g, 3 93 mmol, 1 2 eq ) in anhydrous dimethylformamide (7 5 mL) was heated under nitrogen at 70 °C for 1 hr The reaction mixture was filtered through Celite and evaporated in vacuo The residue was triturated with water, filtered and dried under vacuum at 50 °C to provide phenylmethyl {4-[(5-cyano-6-methyl-2-pyrιdιnyl)oxy]phenyl}acetate 38 (1 05 g,
89%) as a solid 1H NMR (400 MHz, CDCI3) δ 7 81 (d, J = 8 6 Hz, 1 H), 7 38-7 30 (m, 7 H),
7 09 (d, J = 8 5 Hz, 2 H), 6 71 (d, J = 8 5 Hz, 1 H), 5 15 (s, 2 H), 3 69 (s, 2 H), 2 62 (s, 3 H), MS m/z 359 (M+H)+
Intermediate 39 {4-[(5-cyano-6-methyl-2-pyrιdιnyl)oxy]phenyl}acetιc acid
Figure imgf000037_0002
A solution of phenylmethyl {4-[(5-cyano-6-methyl-2-pyrιdιnyl)oxy]phenyl}acetate 38 (855 mg, 2 39 mmol, 1 0 eq ) in 1 1 ethyl acetate ethanol (20 mL) and palladium on barium sulfate (175 mg) was hydrogenated under a balloon of hydrogen over -64 hours The reaction mixture was filtered, evaporated in vacuo, and dried under high vacuum to provide {4-[(5-cyano-6-methyl-2-pyrιdιnyl)oxy]phenyl}acetιc acid as a solid 39 (630 mg, 98%) 1H NMR (400 MHz, CDCI3) δ 7 81 (d, J = 8 6 Hz, 1 H), 7 34 (d, J = 8 5 Hz, 2 H), 7 11 (d, J =
8 3 Hz, 2 H), 6 73 (d, J = 8 5 Hz, 1 H), 3 69 (s, 3 H), 2 62 (s, 2 H), MS m/z 269 (M+H)+
Intermediate 40 {4-[(5-formyl-6-methyl-2-pyrιdιnyl)oxy]phenyl}acetιc acid
Figure imgf000037_0003
A solution of {4-[(5-cyano-6-methyl-2-pyrιdιnyl)oxy]phenyl}acetιc acid 39 (400 mg, 1 49 mmol, 1 0 eq ) and triethylamine (416 μL, 2 98 mmol, 2 0 eq ) in dichloromethane (10 mL) was cooled under nitrogen to -30-40 0C and maintained Diisobutylalummum hydride (1 0 M in DCM, 5 22 mL, 5 22 mmol, 3 5 eq ) was added via syringe and the reaction was maintained After ~30 mm , additional diisobutylalummum hydride (2 0 mL, 2 0 mmol, 1 3 eq ) was added After stirring an additional 30 mm , the reaction was quenched with 1 N NaHSO4 and stirred for ~1 5 hours The layers were separated and the aqueous phase was back-extracted with dichloromethane The combined organic layers were dried over anhydrous magnesium sulfate, filtered, evaporated in vacuo and dried under high vacuum to provide 4-[(5-formyl-6-methyl-2-pyrιdιnyl)oxy]phenyl}acetιc acid 40 (253 mg, 63%) as a solid 1H NMR (400 MHz, CDCI3) δ 10 25 (s, 1 H), 8 09 (d, J = 8 5 Hz, 1 H), 7 35 (d, J = 8 4 Hz, 2 H), 7 14 (d, J = 8 5 Hz, 2 H), 6 76 (d, J = 8 5 Hz, 1 H), 3 70 (s, 2 H), 2 74 (s, 3 H), MS m/z 272 (M+H)+
Intermediate 41 2-{4-[(5-formyl-6-methyl-2-pyrιdιnyl)oxy]phenyl}-Λ/-(1- methylethyl)acetamιde
Figure imgf000038_0001
A mixture of 4-[(5-formyl-6-methyl-2-pyrιdιnyl)oxy]phenyl}acetιc acid 40 (176 mg, 0 649 mmol, 1 0 eq ) and carbonyldiimidazole (211 mg, 1 30 mmol, 2 0 eq ) in acetonitrile (5 mL) was heated at reflux under nitrogen for 2 hours lsopropylamine (275 μL, 3 20 mmol, 5 eq ) was added and the reaction was cooled to ambient temperature The reaction was evaporated in vacuo and the residue was partitioned between dichloromethane and 5% w/v K2CO3 After separating the layers, the aqueous phase was back-extracted four times with dichloromethane The combined organic phases were dried over anhydrous magnesium sulfate, filtered and evaporated in vacuo The residue was purified on flash grade silica gel eluting with 60% ethyl acetate in hexanes to provide 2-{4-[(5-formyl-6-methyl-2- pyrιdιnyl)oxy]phenyl}-Λ/-(1-methylethyl)acetamιde 41 (66 mg, 33%) 1H NMR (400 MHz, CDCI3) δ 10 25 (s, 1 H), 8 10 (d, J = 8 5 Hz, 1 H), 7 30 (d, J = 8 4 Hz, 2 H), 7 15 (d, J = 8 3 Hz, 2 H), 6 78 (d, J = 8 5 Hz, 1 H), 5 20 (br, 1 H), 4 09 (m, 1 H), 3 55 (s, 2 H), 2 73 (s, 3 H), 1 11 (d, J = 6 5 Hz, 6 H), MS m/z 313 (M+H)+
Example VIII
Figure imgf000039_0001
2,4-dιfluoro-5-[({(3-fluorophenyl)[1-({2-methyl-6-[(4-{2-[(1-methylethyl)amιno]-2- oxoethyl}phenyl)oxy]-3-pyrιdιnyl}methyl)-4 pιperιdιnyl]amιno}carbonyl)amιno]benzamιde A mixture of 2-{4-[(5-formyl-6-methyl-2-pyrιdιnyl)oxy]phenyl}-Λ/-(1-methylethyl)acetamιde 41 (25 mg, 0 080 mmol, 1 0 eq ), 2,4-dιfluoro-5-({[(3-fluorophenyl)(4- pιperιdιnyl)amιno]carbonyl}amιno)benzamιde hydrochloride 32 (34 mg, 0 080 mmol, 1 0 eq ), diisopropylethylamine (14 2 μL, 0 080 mmol, 1 0 eq ) and sodium triacetoxyborohydride (34 mg, 0 16 mmol, 2 0 eq ) in dichloromethane (3 mL) under nitrogen was stirred at ambient temperature for 16 hours The reaction mixture was diluted with dichloromethane and washed with 5% w/v aqueous K2CO3 After separation of the layers, the aqueous phase was back-extracted with dichloromethane The combined organic layers were dried over anhydrous sodium sulfate, filtered, evaporated in vacuo and purified by preparative TLC eluting with 7% 2M methanols NH3 in dichloromethane Further purification by preparative TLC eluting with 2% acetic acid in ethyl acetate and by reverse-phase semi-preparative HPLC eluting with 5-95% acetonitrile in 0 1% aqueous TFA followed by lyophilization provided 2,4-dιfluoro-5-[({(3-fluorophenyl)[1-({2-methyl-6-[(4- {2-[(1-methylethyl)amιno]-2-oxoethyl}phenyl)oxy]-3-pyrιdιnyl}methyl)-4- pιperιdιnyl]amιno}carbonyl)amιno]benzamιde (9 mg, 16%) 1H NMR (400 MHz, CD3OD) δ 7 92 (app t, J = 8 0 Hz, 1 H), 7 74 (d, J = 8 3 Hz, 1 H), 7 55 (app q, J = 74 Hz, 1 H), 7 32- 7 24 (m, 3 H), 7 19 (d, J = 7 7 Hz, 2 H), 7 10-7 01 (m, 3 H), 6 75 (d, J = 8 2 Hz, 1 H), 4 65 (m, 1 H), 4 30 (s, 2 H), 3 93 (m, 1 H), 3 53 (d, J = 11 9 Hz, 2 H), 346 (s, 2 H), 3 24 (m, 2 H), 2 45 (s, 3 H), 2 18 (d, J = 13 6 Hz, 2 H), 1 72 (app q, J = 12 8 Hz, 2 H), 1 13 (d, J = 6 5 Hz, 6 H) HRMS (M+H)+ calcd for C37H39F3N6O4 + H, 689 3063, found, 689 3058 LR/MS M+H =
689
Example IX: Biological Activity
PBL Assay Whole blood samples were obtained from normal human donors Peripheral Blood
Lymphocytes (PBLs) were separated from whole blood by density gradient centrifugation with Lymphocyte Separation Medium (LSM) Whole blood was diluted 1 1 with sterile phosphate buffered saline (PBS) and overlaid onto LSM in 50 mL conical centrifuge tubes Samples were centrifuged at 500 x g at room temperature for 30 minutes PBLs were then gently removed from the interface between LSM and aqueous phases and washed twice in sterile PBS The cells were resuspended in 150 mL of RPMI containing 20% fetal bovine serum, 10% T-cell growth factor (IL-2), 50ug/mL gentamicin (RPMI 20/10/50) and 5ug/mL Phytohaemagluttinin and incubated at 370C, 5% CO2 for 2 to 3 days On the second or third day, the cells were washed once with PBS and counted for assay PHA stimulated PBLs were centrifuged at 260 x g for 15 mm, washed once with PBS, centrifuged as before, resuspended to 8x106 cells/mL in RPMI 20/10/50, and 50 μL was distributed to 96-well tissue culture plates Test compounds were serially diluted in 4-fold increments at 2X the final concentration in RPMI 20/10/50 + 0 2% DMSO Fifty (50) uL of diluted compound was transferred to the PBLs and placed in a humidified incubator at 37°C, 5% CO2 for 1hr An additional 60 μL of diluted compound was transferred to a separate 96-well plate containing 60 μL of HIV-1 Ba-L (diluted 1 50 into RPMI 20/10/50) and thoroughly mixed The input of Ba-L was previously determined by titrating the virus under assay conditions without compound and selecting a dilution that gives a response in the linear range of the curve and a signal to background of at least 30 One hundred (100) μL of this mixture was transferred onto the PBL/compound mixture and placed in a humidified incubator at 37°C, 5% CO2 for seven days On day seven of the assay, 50 μL of culture supernatant was transferred to a new 96-well plate The plates were frozen at -80°C and then thawed to room temperature for 30 mm Ten (10) μL of reverse transcriptase (RT) extraction buffer (500 mM KCI, 50 mM DTT and 0 5% NP40 in distilled water) was added to the supernatant followed by the addition of 40 μL of RT assay buffer (1 25 mM EGTA, 125 mM Tric/HCI, 12 5 mM MgCI2, 68 Ci/mmole methyl-3H deoxythymιdιne-5'-trιphosphate, and 0 62 O D units/mL of poly(rA) p(dT)i2_i8 in distilled water) The plates were thoroughly mixed on a platform shaker and placed in a humidified incubator at 37°C, 5% CO2 for 2h Whatman Unifilter DE-81 96-well plates were placed on a vacuum manifold and 100 μL of the RT reaction was added Full vacuum from an in-house line was applied until all of the reaction volume had seeped through the plate Each plate was then washed a total of three times with all washes performed under full, in- house vacuum All wash buffers were delivered with a 12 channel Nunc plate washer connected to a carboy containing at least 2 liters of buffer The first wash consisted of approximately 300 μL of 5% Na2HPO4, followed by one wash of approximately 300 μL of distilled water, and a final wash consisting of 100 μL of 95% ethanol The plates were allowed to dry completely at room temperature, bottom-sealed with an adhesive backing, and 50 μL of scintillation cocktail was added The plates were topsealed and read in a Topcount (Packard) luminometer at 10s/well
Raw data from the PBL assay were expressed as Counts Per Minute (CPM) and normalized according to the following formula
Logio(CPM) - Log-io (Geometric mean PC) ÷ Log™ (Geometric mean NC) - Log10 (Geometric mean PC)
Where
PC = Positive Control = mock infected wells in column 12 on each assay plate NC = Negative Control = untreated but infected wells in column 11 on each assay plate
The potency of test compounds are reported as IC50 values derived from the unconstrained four parameter logistic equation, defined as y = A + ((B-C))/(1+(10Λx)/10ΛC)ΛD) Where
A = minimum y
D = slope factor
B = maximum y x = Iog10(molar compound concentration) C = 1Og10(IC50)
Compounds of the invention have IC50 values less than 1OnM
Example X: Biological Activity
HOS Assay HOS-Luc cells were created by introducing an HIV-1 LTR-I uciferase reporter into HOS-
CD4 CCR5 (NIH AIDS Research and Reference Reagents Program, cat #3318) The cells were propagated in DMEM complete medium Cells were split 1 20 by standard trypsinization when confluency reached 80% (roughly every 2 to 3 days) Black-walled 96-well tissue culture plates were seeded with HOS-Luc at 6x103 cells per well in 50 μL DMEM containing 2% FBS and placed in a humidified incubator at 370C, 5% CO2 overnight The following day, test compounds were serially diluted in 4-fold increments at 2X the final concentration in DMEM + 2% FBS + 0 2% DMSO Diluted compound (50 μL) was transferred to the HOS-Luc cells and the plates were placed in a humidified incubator at Zl0C, 5% CO2 for 1 hr An additional 60 μL of 2X compound was transferred to a clear- walled 96-well tissue culture plate and 60 μL of virus (diluted 1 50 in tissue culture media) was added to each well and thoroughly mixed The virus input was previously determined by titrating the virus under assay conditions without compound and selecting a dilution that gives a response in the linear range of the curve and a signal to background of at least 30 The HIV/compound mixture (100 μL) was transferred to the black-walled plates containing 100 μL of cells/compound The plates were placed in a humidified incubator at Zl0C, 5% CO2 for four days
Following the four-day incubation, 150 μL of supernatant was removed and 50 μL of reconstituted Luclite (Promega) was added to each well For cytotoxicity assessment, 50 μL of reconstituted CellTiter-GLo (Promega) was added to each well Each plate was topsealed and read in a Topcount (Packard) luminometer at 1s/well
Raw data from the HOS-Luc assay were expressed as Relative Light Units (RLU) and normalized according to the following formula
(RLU at drug [ ] / RLU no drug)*100 = % control
The potency of test compounds are reported as IC50 values derived from the four parameter Hill equation, defined as y=Vmax*(1-(xΛn/(KΛn+xΛn)))+Y2
Where x = Log10[compound] y = normalized response data
Vmax = upper bound of response
K = IC50
Y2 = lower bound or baseline of response n = hill coefficient
Compounds of the invention have IC50 values less than 1OnM
Example Xl: Pharmacokinetics in mouse
Non-fasted male CD-1 mice (Charles River Laboratories, Raleigh NC), ranging in weight from 28 g to 30 g, were used for each study Test Article Administration and Sample Collection
For intravenous administration, a single dose (10 mL/kg of 0 1 mg free base/mL) was administered via tail vein For oral solution administration, animals received a single dose (10 mL/kg of 0 1 or 0 3 mg free base/mL) by an oral gavage needle Blood (approximately 0 8 mL) was taken via cardiac puncture from three mice/dose group at each timepoint of 0 083 (IV only), 0 167 (IV only), 0 25, 0 5, 1 , 2, 4, 6, 8, and 24 h and placed on ice Syringes used for sample collection contained approximately 10 μL EDTA Plasma was harvested by centrifugation for 3 mm at 4000 x g and 4°C and stored at -800C until analysis
Mice were orally administered a single dose (10 mL/kg of 0 1 or 0 3 mg free base/mL) by an oral gavage needle
Blood (approximately 0 8 mL) was taken via cardiac puncture from three mice per dose group at each timepoint (0 5, 1 , 2, 4, 8, 12, and 24 h) and placed on ice Syringes used for sample collection contained approximately 10 μL EDTA Livers and spleens were also harvested immediately after blood collection, combined (n = 3 at each timepoint/dose group), and placed on ice Blood and tissue samples were stored at -800C until analysis
Sample Analysis
Plasma samples were thawed to room temperature and vortex-mixed Ahquots (50 μL) of standards and study samples were pipetted into 96-well plates Liver and spleen tissues were thawed to room temperature and weighed Each pooled liver and spleen sample was diluted with 4 and 9 volumes of water (mL/g), respectively Following homogenization (Tomtec Autogizer), homogenates were diluted 5-fold in pooled mouse plasma and analyzed with plasma samples Liver and spleen QC samples were prepared in homogenized blank liver or spleen homogenate Acetonitrile (200 μL) containing internal standard was added to each well, samples vortex-mixed, and plates centrifuged for 10 mm at 2055 x g and 4°C The acetonitrile supernatant (100 μL) was transferred to clean 96 well plates containing 100 μL or 200 μL of 0 1 % formic acid in water and assayed by LC/MS/MS The analytical system consisted of a CTC HTS PAL autosampler, Hewlett Packard (HP) 1100 binary pump and Applied Biosystems API 365 mass spectrometer Samples (10 μL) were injected onto a 30 x 2 mm, 3mιcron, Luna CN column (Phenomenex) using an isocratic eluting system at a flow rate of 0 8 mL/min Mobile phases consisted of A) water containing 0 1 % acetic acid, pH adjusted to 6 2 with ammonium hydroxide acetonitrile (95 5) and B) acetonitrile containing 0 1% acetic acid The mobile phase composition was 50% A 50% B and total run time was 1 1 mm Test compounds and internal standard were detected in the positive turboionspray mode by multiple reaction monitoring (MRM) of the transitions m/z 645 to m/z 291 , and m/z 665 to m/z 467, respectively Data were acquired and analyzed using Applied Biosystems Analyst version 1 4 1 software Liver and spleen samples were converted to ng/g following quantitation against the plasma calibration curves accounting for the appropriate dilution factor (25- and 50-fold, respectively)
Data Analysis
Non-compartmental pharmacokinetic parameters [terminal plasma half-life (Ua), maximum plasma concentration (Cmax), time of maximum plasma concentration (Tmax) plasma clearance (CL), steady-state volume of distribution (Vss), and area under the plasma concentration-time curve extrapolated to infinite time (AUC0-∞)] were calculated based on the composite plasma concentration time data using WinNonlin Professional 4 1 (Pharsight, Mountain View CA) Dose-normalized AUC 0→∞ (DNAUC 0→oo) was obtained by dividing the AUC o→∞with its respective dose and expressed as unit dose AUC 0→∞ (ng*h/mL/mg/kg) Oral bioavailability (F) was calculated using the following equation F (%) = [(DNAUCo-oo, oral)/(DNAUC0.∞, IV] * 100%
Table 1 Plasma Pharmacokinetic Parameter Estimates for Compounds in Non- fasted Male CD-1 Mice Following Intravenous (1 mg free base/kg) and Oral (1 or 2 or 3 mg free base/kg) Administration
Figure imgf000045_0001
* WO2006/030925
Example XII: Pharmacokinetics in rat
Male Sprague-Dawley CD rats (Charles River Laboratories, Raleigh NC), were either surgically implanted with a jugular cannula and housed in individual cages or no surgery was required Food and water were offered freely
Test Article Administration and Sample Collection
For intravenous administration, a single dose of test compound was administered into the jugular cannula followed by a 1 mL flush with saline For oral solution administration, animals received a single dose by an oral gavage needle Additional animals received capsule doses (shown in the following table) which were administered by a small capsule- dosing syringe (Harvard Apparatus)
Blood samples (approximately 0 5 mL each) were taken from the jugular cannula at 0 083 (IV only), 0 167 (IV only), 0 25, 0 5, 1 , 2, 4, 6, 8, and 24 h postdose and placed on ice Syringes used for sample collection contained approximately 10 μL EDTA Plasma was harvested by centrifugation for 3 mm at 4000 x g and 4°C and stored at -800C until analysis
For oral solution administration, animals received a single dose by an oral gavage needle Animals (n=2) were euthanized at 1 , 2, 4, and 24 h postdose, and blood samples (approximately 1 mL with EDTA anticoagulant) taken by cardiac puncture and placed on ice Plasma was harvested by centrifugation for 3 mm at 4000 x g and 4°C and stored at - 800C until analysis Spleen and liver were also dissected, immediately frozen and stored at -800C until analysis
Sample Analysis Plasma samples were thawed to room temperature and vortex-mixed Standards were prepared in pooled rat plasma containing EDTA Quality control (QC) samples were prepared from separately weighed sample stocks Ahquots (50 μL) of standards, QCs and study samples were pipetted into 96-well plates Acetonitrile (200 μL) containing internal standard was added to each well, samples vortex-mixed, and plates centrifuged for 20 mm at 2055 x g and 4°C The acetonitrile supernatant (150 μL) was transferred to clean 96 well plates containing 100 μL of 0 1% formic acid in water and assayed by LC/MS/MS The analytical system consisted of a CTC HTS PAL autosampler, Hewlett Packard (HP) 1100 binary pump and Applied Biosystems API 4000 mass spectrometer Samples (10 μL) were injected onto a 30 x 2 mm, 3mιcron, Atlantis C18 column (Waters) using a gradient eluting system at a flow rate of 0 75 mL/min Mobile phases consisted of A) water containing 0 5% formic acid and B) acetonitrile containing 0 5% formic acid The initial mobile phase composition of 90% A 10% B was followed by a linear gradient to 5% A 95% B at 1 mm The 5% A 95% B composition was held for 0 5 mm followed by a linear gradient to 90% A 10% B at 2 0 mm Data were acquired and analyzed using Applied BioSystems Analyst version 1 4 1 software
Plasma samples were thawed to room temperature and vortex-mixed Standards were prepared in pooled rat plasma containing EDTA Quality control (QC) samples in plasma were prepared from separately weighed sample stocks Ahquots (50 μL) of standards, QCs and plasma samples were pipetted into 96-well plates Liver and spleen tissues were weighed, thawed to room temperature and water (9 volumes, mL/g) added to each sample Following homogenization (Tomtec Autogizer), homogenate (10 μL) was added to pooled rat plasma (40 μL) containing EDTA in 96-well plates Liver and spleen QC samples were prepared in homogenized blank liver or spleen homogenate and ahquots (10 μL) added to pooled rat plasma (40 μL) containing EDTA in 96-well plates Acetonitrile (200 μL) containing internal standard was added to each well, samples vortex-mixed, and plates centrifuged for 20 mm at 2055 x g and 4°C The acetonitrile supernatant (100 μL) was transferred to clean 96 well plates containing 400 μL of 0 1 % formic acid in water and assayed by LC/MS/MS The analytical system consisted of a CTC HTS PAL autosampler, Hewlett Packard (HP) 1100 binary pump and Applied Biosystems API 365 mass spectrometer Samples (10 μL) were injected onto a 30 x 2 mm, 2 5mιcron, Atlantis C18 column (Waters) using an isocratic eluting system at a flow rate of 0 8 mL/min Mobile phases consisted of A) water containing 0 1 % acetic acid, pH adjusted to 6 2 with ammonium hydroxide acetonitrile (95 5) and B) acetonitrile containing 0 1% acetic acid The mobile phase composition was 50% A 50% B and total run time was 1 1 mm Data were acquired and analyzed using Applied BioSystems Analyst version 1 4 1 software Liver and spleen samples were converted to ng/g following quantitation against the plasma calibration curves accounting for the appropriate dilution factor (50-fold)
Data Analysis
Non-compartmental pharmacokinetic parameters [terminal plasma half-life (Ua), maximum plasma concentration (Cmax), time of maximum plasma concentration (Tmax) plasma clearance (CL), steady-state volume of distribution (Vss), and area under the plasma concentration-time curve extrapolated to infinite time (AUC0-∞)] were calculated based on the individual plasma concentration time data using WinNonlin Professional 4 1 (Pharsight, Mountain View CA) Dose-normalized AUC 0→∞ (DNAUC 0→∞) was obtained by dividing the AUC o→∞ by its respective dose and expressed as unit dose AUC0→∞ (ng*h/mL/mg/kg) Oral bioavailability (F) was calculated using the following equation
F (%) = [(DNAUC0,,, oral)/(DNAUC0-«, IV] * 100% where DNAUC0.∞, IV was the mean DNAUCo-∞ following IV dose administration
Table 2 Plasma Pharmacokinetic Parameter Estimates for Compounds in Non- Fasted Male Sprague-Dawley CD Rat Following Intravenous, Oral Solution and Oral Capsule
Example PO DNAUC0→« No (ng h/mL/mg/kg)
7(16)* 25
III 1848
IV 324
V 38
Vl 326
VII 305
VIII 550
1 WO2006/030925
Example XIII: hERG dofetilide binding assay
1 hERG Membrane Preparation The cloned hERG channel was heterologously expressed in Chinese hamster ovary
(CHO)-KI cells Cells were grown in medium comprising DMEM / Ham's F12 (with 15 mM HEPES), 10% FBS, 2 mM Glutamax and 500 mg/ml G418 (no selection pressure in final passage) at 31°C in a humidified CO2 atmosphere Membranes were prepared from cells cultured in 1800 cm2 roller bottles All buffers and equipment were cooled before use and kept in ice throughout the process The cells were harvested with HBSS containing
NaHCO3 (8 4 mM) and EDTA (0 6 mM) and spun down at 250 g for 5 mins at 4°C This spin was repeated after resuspending the pellets in 200 mis of the same solution All subsequent steps were performed at 40C The cells were homogenized within a glass Waring blender for 2 x 15 sees in 200 mis of a solution containing HEPES (50 mM), leupeptin (0 1 mM), bacitracin (25mg/ml), EDTA (1mM), PMSF (1 mM) and Pepstatin A
(2mM) Pepstatin A and PMSF were prepared as concentrated stocks in absolute ethanol (pepstatin 50Ox, PMSF 100x) and were added just before use The blender was plunged into ice for 5 mins after the first burst and 10-40 mins after the final burst to allow foam to dissipate The material was then spun at 50Og for 20 mins and the supernatant spun for 36 mins at 48,00Og The pellet was resuspended in the same buffer as above but without PMSF and Pepstatin A The material was then forced through a 0 6 mm needle, made up to the required volume, ahquoted and stored frozen at -80 °C
2 Experimental Protocol Compound potencies were determined by a radioligand (3H-dofetιlιde) competition assay In this assay, hERG-expressing CHO-K1 membranes (1 mg membranes/well ) were adhered to wheat germ agglutinin-coated SPA PEI imaging beads from Amersham (60 mg/well) in assay buffer, containing HEPES (25 mM), MgCI2 (I 2 mM) and pH adjusted to 7 4 using KOH (5 M) The final potassium concentration in the assay was 13 mM On the day of the assay, pluronic acid was added to the buffer to get a 0 2% final concentration
After one hour on ice, the membrane-bead suspension was mixed with tritiated dofetilide (final concentration of 7 nM/well) and dispensed into a white Greiner polypropylene 384- well plate (10 ml/well), containing 0 1 ml of test compound in DMSO The assay plates were centrifuged at 10,000 rpm for 1 minute and left to equilibrate for 2-3 hrs at room temperature before reading on a ViewLux™ luminescence imager Concentration response curve data were generated from an 11 -point inhibition curve (top assay concentration of 60 mM and a 1 4 step-dilution), a four parameter curve-fit being applied
3 Drugs and Materials Compounds were dissolved in DMSO at a concentration of 10 mM For concentration- response assays, dilutions were prepared in 100% DMSO using a 1 in 4 dilution step The top assay concentration was 60 mM and the final DMSO assay concentration was 1% On each test compound plate 0 1 ml of DMSO was included in one column to assess total binding (TB) and dofetilide or astemizole (10 mM final concentration) added to one column to determine non specific binding (NSB)
Four reference compounds were included in each assay for quality control (QC) purposes The reference compounds used were astemizole, dofetilide, quinidine and cisapride
4 Data Analysis For a single concentration assay, the results of each well are expressed as % inhibition % inhibition = 100 x (1- (sample - NSB) / (TB - NSB), where sample was the signal observed in a particular unknown sample well
For concentration-response assays, the results of each test well were expressed as % of controls The normalization equation used was
% control 100 x (sample - NSB) / (TB - NSB)
The concentration-response curve was analyzed using a non linear equation, 4-parameter logistics, giving a determination of plC50
Curve QC, plate QC (z' > 0 2) and assay QC (plC50 for reference compounds ± 0 5 log units of the rolling mean) were applied when analyzing all experiments
Table 3
Figure imgf000050_0001
1 WO2006/030925

Claims

Claims
1 A compound selected from the group consisting of 5-{[((3-fluorophenyl){1-[(2-methyl-6-{[4-(4-morpholιnylmethyl)phenyr]oxy}-3- pyridinylJmethylH-piperidinylJaminoJcarbonylJamino^-pyridinecarboxamide bιs(trιfluoroacetate),
S-røS-fluorophenylXI-p-methyl-θ-^-^-morpholmylcarbonylJphenyrioxyJ-S- pyridinylJmethylH-piperidinylJaminoJcarbonyriamino^-pyridinecarboxamide, 4-[(5-{[4-((3-fluorophenyl){[(4-fluorophenyl)amιno]carbonyl}amιno)-1-pιperιdιnyl]methyl}-6- methyl-2-pyrιdιnyl)oxy]-Λ/-[2-(methyloxy)ethyl]benzamιde, Λ/-(3-fluorophenyl)-Λ/'-(4-fluorophenyl)-Λ/-{1-[(2-methyl-6-{[4-(4- morpholιnylcarbonyl)phenyl]oxy}-3-pyrιdιnyl)methyl]-4-pιperιdιnyl}urea, 5-[({butyl[1-({6-[(4-{[(2-hydroxyethyl)amino]carbonyl}phenyl)oxy]-2-methyl-3- pyrιdιnyl}methyl)-4-pιperιdιnyl]amιno}carbonyl)amιno]-2,4-dιfluorobenzamιde,
2,4-dιfluoro-5-{[((3-fluorophenyl){1-[(2-methyl-6-{[4-(1 ,3-oxazol-5-yl)phenyl]oxy}-3- pyrιdιnyl)methyl]-4-pιperιdιnyl}amιno)carbonyl]amιno}benzamιde, 4-{[5-({4-[({[5-(amιnocarbonyl)-2,4<lιfluorophenyl]amιno}carbonyl)(3-fluoropheny^ pιperιdιnyl}methyl)-6-methyl-2-pyrιdιnyl]oxy}phenyl (i-methylethyl)carbamate, 2,4-dιfluoro-5-[({(3-fluorophenyl)[1-({2-methyl-6-[(4-{2-[(1-methylethyl)amιno]-2- oxoethyl}phenyl)oxy]-3-pyrιdιnyl}methyl)-4-pιperιdιnyl]amιno}carbonyl)amιno]benzamιde, and pharmaceutically acceptable salts thereof 2 A method of treatment or prevention of a HIV infection in a human comprising administering to said human an effective amount of a compound according to claim 1
3 A method of treatment or prevention of neuropathic pain, multiple sclerosis, rheumatoid arthritis, autoimmune diabetes, chronic implant rejection, asthma, rheumatoid arthritis, Crohns Disease, inflammatory bowel disease, chronic inflammatory disease, glomerular disease, nephrotoxic serum nephritis, kidney disease, Alzheimer's Disease , autoimmune encephalomyelitis, arterial thrombosis, allergic rhinitis, arteriosclerosis, Sjogren's syndrome (dermatomyositis), systemic lupus erythematosus, graft rejection, cancers with leukocyte infiltration of the skin or organs, human papilloma virus infection, prostate cancer, wound healing, amyotrophic lateral sclerosis and immune mediated disorders in a human comprising administering to said human a pharmceutically effective amount of a compound according to claim 1
4 A compound according to claim 1 for use in medical therapy
5 Use of a compound according to claim 1 in the manufacture of a medicament for the treatment or prophylaxis of an HIV infection
6 The use of a compound according to claim 1 in the manufacture of a medicament for the treatment or prophylaxis of neuropathic pain, multiple sclerosis, rheumatoid arthritis, autoimmune diabetes, chronic implant rejection, asthma, rheumatoid arthritis, Crohns Disease, inflammatory bowel disease, chronic inflammatory disease, glomerular disease, nephrotoxic serum nephritis, kidney disease, Alzheimer's Disease , autoimmune encephalomyelitis, arterial thrombosis, allergic rhinitis, arteriosclerosis, Sjogren's syndrome (dermatomyositis), systemic lupus erythematosus, graft rejection, cancers with leukocyte infiltration of the skin or organs, human papilloma virus infection, prostate cancer, wound healing, amyotrophic lateral sclerosis and immune mediated disorders
7 A pharmaceutical composition comprising a pharmaceutically effective amount of a compound according to claim 1 together with a pharmaceutically acceptable carrier
8 A pharmaceutical composition according to claim 7 in the form of a tablet or capsule
9 A pharmaceutical composition according to claim 7 in the form of a liquid
10 A method of treatment or prevention of an HIV infection in a human comprising administering to said human a composition comprising a compound according to claim 1 and another therapeutic agent 11 A method according to claim 10, wherein said composition comprises another therapeutic agent selected from the group consisting of acyclic nucleosides, acyclovir, valaciclovir, famciclovir, ganciclovir, penciclovir, acyclic nucleoside phosphonates, (S)-1-(3- hydroxy-2-phosphonyl-methoxypropyl)cytosιne (HPMPC), [[[2-(6-amιno-9H-purιn-9- yljethoxyjmethyljphosphinyhdenejbisfoxymethylenej^^-dimethylpropanoic acid (bis-POM PMEA, adefovir dipivoxil), [[(1 R)-2-(6-amιno-9H-purιn-9-yl)-1- methylethoxy]methyl]phosphonιc acid (tenofovir), (R)-[[2-(6-Amιno-9H-purιn-9-yl)-1- methylethoxy]methyl]phosphonιc acid bιs-(ιsopropoxycarbonyloxymethyl)ester (bis-POC- PMPA), nucleoside reverse transcriptase inhibitors, 3'-azιdo-3'-deoxythymιdιne (AZT, zidovudine), 2',3'-dιdeoxycytιdιne (ddC, zalcitabine), 2',3'-dιdeoxyadenosιne, 2', 3'- dideoxyinosme (ddl, didanosine), 2',3'-dιdehydrothymιdιne (d4T, stavudme), (-)-cιs-1-(2- hydroxymethyl)-1 ,3-oxathιolane 5-yl)-cytosιne (lamivudine), cιs-1-(2-(hydroxymethyl)-1 ,3- oxathιolan-5-yl)-5-fluorocytosιne (FTC), (-J-cis^-β-amino-θ-fcyclopropylaminoJ-ΘH-puπn-θ- yl]-2-cyclopentene-1 -methanol (abacavir), ribavirin, protease inhibitors, indinavir, ritonavir, nelfinavir, amprenavir, saquinavir, fosamprenavir, lopinavir, tipranavir, interferons, α- interferon, immunomodulators, interleukin Il or thymosin, granulocyte macrophage colony stimulating factors, erythropoetin, soluble CD4 and genetically engineered derivatives thereof, non-nucleoside reverse transcriptase inhibitors (NNRTIs), nevirapme (BI-RG-587), alpha-((2-acetyl-5-methylphenyl)amιno)-2,6-dιchloro-benzeneacetamιde (loviride), 1-[3- (isopropylamino)-2-pyridyl]-4-[5-(methanesulfonamido)-1 H-indol-2-ylcarbonyl]piperazme monomethanesulfonate (delavirdine), (SJ-θ-chloro^-fcyclopropylethynylJ-i ^-dihydro^- (trιfluoromethyl)-2H-3,1-benzoxazιn-2-one (efavirenz, DMP 266), nlpivirine, integrase inhibitors, fusion inhibitors, T-20 and T-1249
52
PCT/US2008/081672 2007-12-12 2008-10-30 Ccr5 antagonists as therapeutic agents WO2009075960A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104119316A (en) * 2014-06-25 2014-10-29 南通大学 Pyridine phenyl ether compound as well as preparation and application of pyridine phenyl ether compound

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001087839A1 (en) * 2000-05-17 2001-11-22 Astrazeneca Ab Pharmaceutically active piperidine derivatives, in particular as modulators of chemokine receptor activity
US20060229336A1 (en) * 2002-12-13 2006-10-12 Kazmierski Wieslaw M Ccr5 antagonists as therapeutic agents

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001087839A1 (en) * 2000-05-17 2001-11-22 Astrazeneca Ab Pharmaceutically active piperidine derivatives, in particular as modulators of chemokine receptor activity
US20060229336A1 (en) * 2002-12-13 2006-10-12 Kazmierski Wieslaw M Ccr5 antagonists as therapeutic agents

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104119316A (en) * 2014-06-25 2014-10-29 南通大学 Pyridine phenyl ether compound as well as preparation and application of pyridine phenyl ether compound

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