WO2009058921A1 - Ccr5 antagonists as therapeutic agents - Google Patents

Ccr5 antagonists as therapeutic agents Download PDF

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Publication number
WO2009058921A1
WO2009058921A1 PCT/US2008/081665 US2008081665W WO2009058921A1 WO 2009058921 A1 WO2009058921 A1 WO 2009058921A1 US 2008081665 W US2008081665 W US 2008081665W WO 2009058921 A1 WO2009058921 A1 WO 2009058921A1
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Prior art keywords
amino
methyl
oxy
pyridinyl
phenyl
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PCT/US2008/081665
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French (fr)
Inventor
Wieslaw Mieczyslaw Kazmierski
Pek Yoke Chong
Maosheng Duan
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Smithkline Beecham Corporation
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Publication of WO2009058921A1 publication Critical patent/WO2009058921A1/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

Definitions

  • 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.
  • AIDS acquired immunodeficiency syndrome
  • ARC AIDS-related complex
  • 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 main 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.
  • 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 a compound selected from the group consisting of:
  • 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.
  • treatment 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.
  • prophylaxis refers to preventing a disease or condition or preventing the occurrence of symptoms of such a disease or condition, in a patient.
  • patient refers to a mammal, including a human.
  • 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
  • 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-sulfonic 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 1J( alkyl) and other amine salts.
  • Physiologically acceptable salts of a hydrogen atom or an amino 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 iJ( 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.
  • ARC AIDS related complex
  • Kaposi's sarcoma Kaposi's sarcoma
  • AIDS dementia AIDS related complex
  • 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 CCR5-related disease or condition for example, 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 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.
  • 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.
  • 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 ingredient(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 ingredient(s) and pharmaceutically active agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect.
  • Such therapeutic agents include agents that are effective for the treatment of viral infections or associated conditions.
  • agents include acyclic nucleosides, for example acyclovir, valaciclovir, famciclovir, ganciclovir, and penciclovir, acyclic nucleoside phosphonates, for example (S)-1-(3-hydroxy-2-
  • 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
  • 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.
  • 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.
  • 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.
  • 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
  • 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).
  • 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 oil-i ⁇ -water liquid emulsion or a water-in-oil 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 hydroxypropylmethyl cellulose
  • lubricant e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose
  • disi ⁇ tegra ⁇ t e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose
  • 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.
  • 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 carriers) followed by chilling and shaping in molds.
  • compositions suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the pharmaceutical composition isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents; and liposomes or other microparticulate systems which are designed to
  • compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use.
  • sterile liquid carrier for example water for injection
  • 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.
  • 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 XlII-XV, at Tables 1 , 2, and 3, the compounds of the present invention demonstrate good oral bioavailability and favorable cardiovascular profile.
  • reaction mixture was diluted with dichloromethane and washed with 5% w/v aqueous K 2 CO 3 . 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 6% methanol in dichloromethane to provide the product as foam.
  • e-Chloro-S-pyridinecarbonitrile (2.29g, 16.5 mmol) was dissolved in dichloromethane (60 mL). To this solution was added urea hydrogen peroxide (4.7 g, 49.6 mmol). After the resulting mixture was cooled down to 0 0 C on an ice bath, trifluoroacetic acid anhydride (6.3 mL, 45 mmol) was slowly introduced by syringe. The reaction was stirred for an additional 3 hours at room temperature before quenched with satd. aq. Na 2 S 2 ⁇ 3 and extracted with dichloromethane. The combined organic layers were then washed with satd. aq.
  • intermediate 33 (780 mg, 2.6 mmol) was treated with POCI 3 (10 mL) under reflux condition for 1 hour. After evaporation of the solvents, the residue was dissolved in dichloromethane and washed with satd. aq. NaHCO 3 . The organic phase was then dried over anhydrous sodium sulfate. Evaporation of the solvents gave a light gray solid, which was triturated with ether to afford intermediate 34 as an off white solid (720 mg, 86%).
  • PBLs Peripheral Blood Lymphocytes
  • LSM Lymphocyte Separation Medium
  • 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 LSWI and aqueous phases and washed twice in sterile PBS.
  • PBS sterile phosphate buffered saline
  • 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.
  • IL-2 T-cell growth factor
  • RPMI 20/10/50 50ug/mL gentamicin
  • PHA stimulated PBLs were centrifuged at 260 x g for 15 min, washed once with PBS, centrifuged as before, resuspended to 8x10 6 cells/mL in RPMI 20/10/50, and 50 ⁇ L 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 0 C, 5% CO 2 for 1 hr. An additional 60 ⁇ L of diluted
  • RT assay buffer 500 mM KCI, 50 mM DTT and 0.5% NP40 in distilled water
  • 40 ⁇ L of RT assay buffer (1.25 mM EGTA, 125 mM Tric/HCI, 12.5 mM MgCI 2 , 68 Ci/mmole methyl- 3 H deoxythymidine-5'-triphosphate, and 0.62 O.D.
  • the first wash consisted of approximately 300 ⁇ L of 5% Na 2 HPO 4 , followed by one wash of approximately 300 ⁇ L of distilled water, and a final wash consisting of 100 ⁇ L of 95% ethanol.
  • the plates were allowed to dry completely at room temperature, bottom-sealed with an adhesive backing, and 50 ⁇ L of scintillation cocktail was added. The plates were topsealed and read in a Topcount (Packard) luminometer at 108/well.
  • C log 10 (IC 50 )
  • Compounds of the invention have IC 50 values less than 1OnM.
  • HOS-Luc cells were created by introducing an HIV-1 LTR-luciferase 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 37 0 C, 5% CO 2 for Victoria.'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 to each well and thoroughly mixed. The virus input was previously determined by titrating the virus under assay
  • the HIV/compound mixture (100 ⁇ L) was transferred to the black-walled plates containing 100 ⁇ L of cells/compound. The plates were placed in a humidified incubator at 37 0 C, 5% CO 2 for four days.
  • RLU Relative Light Unit
  • Vm a x upper bound of response
  • Compounds of the invention have IC 50 values less than 1OnM.
  • 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.
  • a single dose (10 mLJkg 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 min 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.
  • Plasma samples were thawed to room temperature and vortex-mixed. Aliquots (50 ⁇ L) of standards and study samples were pipetted into 96-weil 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 ⁇ L) containing internal standard was added to each well, samples vortex-mixed, and plates ce ⁇ trifuged for 10 min at 2055 x g and 4 0 C.
  • the acetonitrile supernatant (100 ⁇ L) was transferred to clean 96 well plates containing 100 ⁇ L or 200 ⁇ 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 365 mass spectrometer. Samples (10 ⁇ L) were injected onto a 30 x 2 mm, ⁇ micron, Luna CN column
  • No ⁇ -compartmental pharmacokinetic parameters [terminal plasma half-life (t 1/2 ), maximum plasma concentration (C max ), time of maximum plasma concentration (T ma x) plasma clearance (CL), steady-state volume of distribution (V ss ), and area under the plasma concentration-time curve extrapolated to infinite time (AUCo- * ,)] were calculated based on the composite plasma concentration time data using WinNonlin Professional 4.1 (Pharsight, Mountain View CA) .
  • Dose-normalized AUC D - K o (DNAUC o- ⁇ ) was obtained by dividing the AUC 0 ⁇ owith its respective dose and expressed as unit dose AUC o ⁇ « (ng*h/mL/mg/kg).
  • Oral bioavailability (F) was calculated using the following equation.
  • F (%) [(DNAUCo-., oral)/(DNAUC 0 ⁇ , IV] * 100%
  • Example XIV Pharmacokinetics in rat
  • 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).
  • 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 ⁇ L
  • animals received a single dose by an oral gavage needle.
  • Plasma was harvested by centrifugation for 3 min at 4000 x g and 4°C and stored at -80 c C until analysis. Spleen and liver were also dissected, immediately frozen 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. Aliquots (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 min at 2055 x g and 4 0 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, 3micron, Atlantis C18 column (Waters) using a gradient eluting system at a flow rate of 0.75 mL/rnin. 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 min. The 5% A:95% B composition was held for 0.5 min followed by a linear gradient to 90% A:10% B at 2.0 min. 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. Aliquots (50 ⁇ L) 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,
  • 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 ⁇ L) were injected onto a 30 x 2 mm, 2.5micron, 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 hydroxideiacetonitrile (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 min. 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).
  • Non-compartmental pharmacokinetic parameters [terminal plasma half-life maximum plasma concentration (C max ), time of maximum plasma concentration (Tmax) 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 - ⁇ o (DNAUC o ⁇ ⁇ ) 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:
  • F (%) [(DNAUCo-,, oral)/(DNAUC 0 . ⁇ , IV] * 100% where DNAUC 0 -O, IV was the mean DNAUCo- ⁇ following IV dose administration.
  • Example XV hERG dofetilide binding assay
  • 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 0 C in a humidified CO 2 atmosphere.
  • Membranes were prepared from cells cultured in 1800 cm 2 roller bottles. All buffers and equipment were cooled before use and kept in ice throughout the process. The cells were harvested with HBSS containing NaHCO 3 (8.4 mM) and EDTA (0.6 mM) and spun down at 250 g for 5 mins at 4 0 C.
  • 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, aliquoted and stored frozen at -80 0 C.
  • Compound potencies were determined by a radioligand ( 3 H-dofetilide) competition 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), MgCI 2 (1.2 mM) and pH adjusted to 7.4 using KOH (5 M). The final potassium concentration in the assay was 13 mM.
  • pluronic acid was added to the buffer to get a 0.2% final concentration.
  • 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 reference compounds used were: astemizole, dofetilide, quinidine and cisapride.
  • % inhibition 100 x (1- (sample - NSB) / (TB - NSB), where sample was the signal observed in a particular unknown sample well.
  • 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.

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 main 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 a compound selected from the group consisting of:
Λ/-(4-{[5-({4-[(3-fluorophenyl)({[6-(methyloxy)-3-pyridinyl]amino}carbonyl)amino]-1- piperidiπyl}methyl)-6-methyl-2-pyridinyl]oxy}phenyl)methanesulfonamide;
5-({[(H[2-methyl-6-({4-[(methylsulfonyl)amino]phenyl}oxy)-3-pyridinyl]methyl}-4- piperidiπyl)(pheπyl)amino]carbonyl}amino)-2-pyridinecarboxamide;
5-({[(3-fluorophenyl)(H[2-methyl-6-({4-[(methylsulfonyl) amino]phenyl}oxy)-3- pyridinyl]methyl}-4-piperidiπyl)amino]carbonyl}amino)-2-pyridinecarboxamide;
5-({KH[6-({4-[(ethylsulfonyl)amino]phenyI}oxy)-2-methyl-3-pyridinyl]methylH- piperidinyl)(phenyl)amino]carbonyl}amino)-2-pyridiπecarboxamide;
5-({[(H[6-({4-[(ethylsulfonyl)amino]phenyl}oxy)-2-methyl-3-pyridinyl]methyl]-4- piperidinyl)(3-fluorophenyl)amino]carbonyl}amiπo)-2-pyridinecarboxamide;
5-({[(H[2-methyl-6-({2-methyl-4-I(methylsulfony[)amino]phenyl}oxy)-3- pyridinyl]methyl}-4-piperidinyl)(pheπyl)amino]carbonyI}amino)-2- pyridinecarboxamide;
5-({[(1-{[6-({2-chloro-4-[(methylsulfonyl)amino]phenyl}oxy)-2-methyl-3- pyridinyl]methyl}-4-piperidinyl)(phenyl)amino]carbonyl}amino)-2- pyridinecarboxamide;
5-({[(3-fIuoraphenyl)(1-{[4-methyI-2-({4-[(methylsulfonyl)amino]pheπyl}oxy)-5- pyrimidinyl]methyl}-4-piperidinyl)amino]carbonyl}amiπo)-2-pyridinecarboxamide;
5-({[(1-{[2-ethyl-6-({4-[(methylsulfonyl)amino]pheπyl}oxy)-3-pyridinyl]methyl}-4- piperidiny[)(phenyl)amino]carbonyl}amino)-2-pyridinecarboxamide;
5-({[(H[2-cyclopropyl-6-({4-[(methylsulfonyl)amino]phenyl}oxy)-3-pyridinyI]methyl}- 4-piperidinyl)(phenyl)amino]carboπyl}amino)-2-pyridinecarboxamide; 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-sulfonic 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 C1J( alkyl) and other amine salts. Physiologically acceptable salts of a hydrogen atom or an amino 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 CiJ(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 ingredient(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 ingredient(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)cytosine (HPWIPC), [[[2-(6-amino-9H-purin-9- y[)ethoxy]methyl]phosphinyliciene]bis(oxymethylene)-2,2-dimethylpropanoic acid (bis-POM PMEA, adefovir dipivoxil), [[(1 R)-2-(6-amino-9H-purin-9-yl)-1- methylethoxy]methyl]phosphonic acid (tenofovir), and (R)-[[2-(6-Amino-9H-purin-9- yl)-1 -methylethoxy]methyl]phosphoπic acid bis-(isopropoxycarbonyloxymethyl)ester (bis-POC-PMPA), nucleoside reverse transcriptase inhibitors, for example 3'-azido- 3'-deoxythymidine (AZT, zidovudine), 2',3'-dideoxycytidine (ddC, zalcitabine), 2', 3'- dideoxyadenosine, 2',3'-dideoxyinosine (ddl, didanosine), 2',3'-didehydrothymidine (d4T, stavudine), (-)-cjs-1-(2-hydroxymethyl)-1 ,3-oxathiolane 5-yl)-cytosine (iamivudine), cjs-1-(2-(hydroxymethyl)-1 ,3-oxathiolan-5-yl)-5-fluorocytosine (FTC), (-)-cis-4-[2-amino-6-(cyclopropylamino)-9JH-purin-9-yl]-2-cyclopentene-1-methanol (abacavir), and ribavirin, protease inhibitors, for example indinavir, ritonavir, nelfinavir, amprenavir, saquinavir, fosamprenavir, lopinavir, tipranavir, interferons such as α-iπterferon, immunomodulators such as interieukin 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~ methylpheπyl)amino)-2,6-dichloro-beπzeneacetarnide (loviride), 1-[3- (isopropylamino)-2-pyridyl]-4-[5-(methanesulfonamido)-1H-indol-2- ylcarbonyljpiperazine monomethanesulfonate (delavirdine), (S)-6-chloro-4- (cyclopropylethynyl)-i ,4-dihydro-4-(trifluoromethyl)-2H-3, 1 -benzoxazin-2-one (efavirenz, DMP 266), rilpivirine, 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 bs 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 oil-iπ-water liquid emulsion or a water-in-oil 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, disiπtegraπt (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 carriers) followed by chilling and shaping in molds.
Pharmaceutical compositions suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the pharmaceutical composition isotonic with the blood of the intended recipient; and aqueous and nonaqueous 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-dried (lyophilized) 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 XlII-XV, at Tables 1 , 2, and 3, the compounds of the present invention 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
^(^{[S-^-KS-fluoropheny^dβ-CmethyloxyJ-S-pyridinyllaminolcarbony^aminol-i- piperidiπyl}methyl)-6-methyl-2-pyridiπyl]oxy}phenyl)methaπesulfonamide
Figure imgf000013_0001
Intermediate 1
1 ,1-dimethylethyl 4-[(3-fluorophenyl)({[6-( methyloxy)-3- pyridinyl]ammo}carbonyl)amino]-1-piperidinecarboxylate
Prepared (in 31 % yield) as described for intermediate 10.
1H NMR (400 MHz, CHLOROFORM-d) 6 ppm 7.83 (d, J=2.93 Hz, 1 H), 7.63 (dd,
J=8.80, 2.93 Hz, 1 H), 7.41 - 7.49 (m, 1 H), 7.17 (td, J=8.25, 2.57 Hz, 1 H), 7.00 -
7.04 (m, 1 H), 6.92 - 6.98 (m, 1 H), 6.63 (d, J=8.80 Hz, 1 H), 5.71 (s, 1 H), 4.53 -
4.67 (m, 1 H), 4.04 - 4.19 (m, 2 H), 3.83 (s, 3 H), 2.70 - 2.85 (m, 2 H), 1.75 - 1.87
(m, 2 H), 1.37 (s, 9 H), 1.17 - 1.29 (m, 2 H). ES-LCMS: m/z 445.2 (M+H).
Figure imgf000013_0002
Intermediate 2
W-(3-fluorophenyl)-W-[6-( methyloxy)-3-pyridinyl]-N-4-piperidinylurea Prepared (in quantitative yield) as described for intermediate 14.
The synthesis of intermediate aldehyde 6
^^NHSOjIWs Nc^-W.. H0 ' Nr! JL _ ,NHSO2Me
Cl DMF X ^^s^^ ^n0'
3 80 'C
DIBAL1 CH2CI21 O -C ^,NHSO2Me
6
The synthesis of intermediate 3 was accomplished as described in the literature [Singh et al., (1991) Synthesis pp.894-896].
Preparation of phenol 4 was accomplished as in J. Med. Chew. 1999, 42, 1041. To a stirred 0 °C suspension of 4-aminophenol (30Og, 2.75 mol) in 3L WIeOH was slowly added methanesulfonyl chloride (106 mL, 1.37 mol). The reaction mixture was then stirred at room temperature for 2h and evaporated to give a brown solid residue. The residue was stirred vigorously in 3.5 L 1N HCI for 1h. The solution was filtered to obtain the phenol 4 as an off-white solid, which was washed with ice-cold water and dried in vacuo over NaOH pellets. Yield obtained was 65% (166.2g). 1H NMR of intermediate 4 MHz, DWISO-Cf6) δ 9.36 (s, 1 H), 9.17 (s, 1 H), 6.99 - 7.06 (m, 2 H), 6.70 - 6.75 (m, 2 H), 2.84 (s, 3 H). LRMS: (M-H)" calcd for C7H9NO3S - H, 186; found, 186
Solution of chloropyridine 3 (25g, 0.16 mol), phenol 4 (33.7g, 0.18 mol), and Cs2CO3 (106.8g, 0.33 mol) in dry DWIF (350 mL) was stirred at 80 °C overnight. The reaction mixture was then filtered and rinsed with CH2CI2. The filtrate was concentrated to a residue by rotary evaporation, and then diluted with water. The pH was adjusted to 7 by the addition of 2M aqueous HCI and then extracted with CH2CI2. The organic extracts were pooled, dried (Na2SO4), filtered and evaporated to provide a dark brown solid. To this was added 200 mL Et2O, and the solution was stirred overnight and then filtered. The light brown solid was washed with a small amount of Et2O, and dried to provide intermediate 5 as a light brown solid (46.3g, 93%). 1H NWIR of 5 (400 MHz, DMSO-Cf6) δ 9.77 (br. s., 1 H), 8.20 (d, J=8.43 Hz, 1 H), 7.20 - 7.26 (m, 2 H), 7.12 - 7.17 (m, 2 H), 6.95 (d, J=8.07 Hz, 1 H), 2.98 (s, 3 H), 2.48 (s, 3 H). LRMS: (M+H)* calcd for C14H13N3O3S + H, 304; found, 304.
Next, to a stirred suspension of intermediate 5 (46.3g, 152.6 mmol) in 1 L CH2CI2 at 0 0C was added DIBAL (300 mL of a 1M solution in CH2CI2, 300 mmol) dropwise via an addition funnel, over 2h. After addition was complete, the reaction mixture was stirred for 2h at 0 "C. When TLC showed that the reaction was complete, 700 mL sat'd aqueous solution of Rochelle's salt was added carefully and the biphasic mixture was stirred overnight, then filtered. The aqueous layer was extracted with
CH2CI2 and the combined organic layers were dried (Na2SO4), filtered, and evaporated to give a brown oil, which was purified by chromatography (0-50% EtOAc/hexanes) to provide intermediate 6 as an off-white solid (29.2g, 62%). 1H NMR of intermediate 6 (400 MHz, DMSO-Cf6) δ 10.18 (s, 1 H), 9.78 (s, 1 H), 8.19 (d, J=8.43 Hz, 1 H), 7.24 - 7.29 (m, 2 H), 7.16 - 7.21 (m, 2 H), 6.95 (d, J=8.43 Hz, 1 H), 3.02 (s, 3 H), 2.63 (s, 3 H). LRMS: (M+H)+ calcd for C14H14N2O4S + H, 307; found, 307.
The title compound, N-(4-{[5-({4-[(3-fluorophenyl)({[6-(methyloxy)-3- pyridinyl]amino}carbonyl)amino]-1-piperidinyI}rnethyl)-6-methyl-2- pyridinyl]oxy}phenyl)methanesulfonamide (example I), was synthesized from intermediates 2 and 6 using reductive amination procedure described in example Vl 1H NMR (CDCI3, 400 MHz) δ 7.85 (d, J=2.93 Hz, 1 H), 7.79 (br. s., 1 H), 7.61 (dd, J=8.80, 2.57 Hz, 1 H), 7.42 - 7.48 (m, 2 H), 7.19 - 7.24 (m, 2 H), 7.15 (td, J=8.25, 1.83 Hz, 1 H), 7.00 - 7.06 (m, 3 H), 6.96 (dt, J=8.98, 2.10 Hz, 1 H), 6.61 (d, J=9.17 Hz, 1 H), 6.50 (d, J=8.07 Hz, 1 H), 5.75 (s, 1 H), 4.42 - 4.57 (m, 1 H), 3.81 (s, 3 H), 3.33 (s, 2 H), 2.93 (s, 3 H), 2.82 (d, J=11.36 Hz, 2 H), 2.36 (s, 3 H), 2.11 (t, J=11.36 Hz, 2 H), 1.81 (d, J=11.36 Hz, 2 H), 1.27 - 1.42 (m, 2 H). HRMS: (M+H)+ calcd for C32H36FN6O5S + H, 635.2446; found, 635.2446.
Example Il
5-({[(1-{[2-methyl-6-({4-[(methylsulfonyI)amino]phenyl}oxy)-3-pyridinyl]methyI}-4- piperidinyl)(phenyl)amino]carbonyl}amino)-2-pyridinecarboxamide
Figure imgf000015_0001
Example III
5-({[(3-fluorophenyl)(1-{[2-methyl-6-({4-[(methylsulfonyl) amino]phenyl}oxy)-3- pyridinyl]methyl}-4-piperidinyl)amino]carbonyl}amino)-2-pyridinecarboxamide
Figure imgf000016_0001
Synthesis of intermediates 13 and 14
Figure imgf000016_0002
Intermediate 7 4-(phenylamιno)-1 -piperidinecarboxylate
Figure imgf000016_0003
A mixture of 4-Boc-pιpeπdone (50 0 g, 0 25 mol, 1 equiv) and aniline (22 8 mL, 025 mol, 1 equiv) in dichloroethane (400 mL) was treated with tιtanιum(IV) isopropoxide (928 mL, 0 31 mol, 1 25 equiv) and heated to 800C for 4 h The reaction was concentrated down and the residue taken up in ethanol (600 mL) and cooled to 0 0C Using a mechanical stirrer and an internal temperature probe sodium borohydride (14 2 g, 0 37 mol, 1 5 equiv) was added in small amounts over 1 5 h, while maintaining the intermal temperature ≤32 0C The reaction was allowed to warm to RT over 18 h and then quenched with H2O The titanium salts were removed by filtration and washed with excess ethyl acetate The entire filtered mixture was concentrated down and the residue taken up in ethyl acetate and filtered thru a pad of celite The celite was washed with additional ethyl acetate and the organics were concentrated down to give a brown solid The solid was suspended in hexanes, stirred at RT for 18 h and filtered to give 7 (56 g 81% yield) as a white solid
1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.14 - 7.22 (m, 2 H), 6.72 (t, J=LZZ Hz, 1 H), 6.62 (d, J=7.87 Hz, 2 H), 4.05 (s, 2 H), 3.37 - 3.49 (m, 1 H), 2.93 (s, 2 H), 2.03 (s, 2 H), 1.53 (s, 1 H), 1.42 - 1.52 (m, 9 H), 1.38 (d, J=19.41 Hz, 2 H). ES- LCMS: m/z 299.1 (M+Na).
Intermediate 8
Figure imgf000017_0001
Sodium triacetoxyborohydride (79.7 g, 0.37 mol, 1.5 equiv) was added portionwise over 1 h to a solution of 4-BOC-piperidone (50.0 g, 0.25 mol, 1 equiv), 3- fluoroaniline (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 00C. 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 hexaπes:ethyl acetate, stirred overnight and filtered to give 8 (67 g, 91% yield) as a white solid.
1H NMR (400 MHz, DMSO-Cf6) δ ppm 6.96 - 7.05 (m, 1 H), 6.29 - 6.40 (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 - 3.42 (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 9 i .i-dimethylethy^-Ktfθ-cyano-S-pyridiπylJaminoJcarbonylXphenylJaminol-i- piperidinecarboxylate txjjy
A solution of the 5-amino-2-pyridinecarbonitrile (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-dimethylethyl 4-(phenylamino)-1-piperidinecarboxylate 7 (774 mg, 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-dimethylethyl 4-[{[(6-cyano-3-pyridinyl)amino]carbonyl}(phenyl)amino]-1- piperidinecarboxylate, 9 (822 mg, 70 % yield) as a white solid. 1H NMR (400 MHz, CHLOROFORM-cO δ 8.13 - 8.20 (m, 2 H), 7.50 - 7.59 (m, 4 H), 7.19 - 7.23 (m, 2 H), 6.11 (s, 1 H), 4.57 - 4.68 (m, 1 H), 4.09 - 4.19 (m, 2 H), 2.73 - 2.86 (m, 2 H), 1.80 - 1.89 (m, 2 H), 1.38 (s, 9 H), 1.20 - 1.33 (m, 2 H). ES-LCMS: mil 422.1 (M+H).
Intermediate 10
1 ,1-dimethylethyl 4-[{[(6-cyano-3-pyridiπyl)amino]carbonyl}(3-fluorophenyl)amino]-1- piperidinecarboxylate
Figure imgf000018_0001
Prepared (in 77 % yield) as described above for intermediate 9, except that 8 was used as substrate
1H NMR (400 MHz, CHLOROFORM-cf) 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, 6.41 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 11
1 , 1 -dimethylethyl 4-[({[6-(aminocarbonyl)-3-pyridinyl]amino}carbonyl)(phenyl)amino]-
1 -piperidinecarboxylate
H'NΛIXINOB
A solution of 1 ,1-dimethylethyl 4-[{[(6-cyano-3 pyridinyl)amino]carbonyl}(phenyl)arnino]-1-piperidinecarboxylate, 9 (822.1 mg, 1.95
mmol), urea hydrogen peroxide (2.2 g, 23.4 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-dimethylethyl 4-[({[6-(aminocarbonyl)-3- pyridinyl]amiπo}cart>onyl)(ρhenyl)amino]-1-piperidiπecarboxylate, 11 (848.4 mg, 99 % yield) as a white solid. 1H NMR (400 MHz, CHLOROFORM-tf) δ 8.34 (d, J=2.56 Hz, 1 H), 8.07 (d, J=8.61 Hz, 1 H), 7.88 (dd, J=8.61 , 2.56 Hz, 1 H), 7.69 (br. s., 1 H), 7.49 - 7.57 (m, 3 H), 7.21 - 7.25 (m, 2 H), 6.04 (s, 1 H), 5.42 (br. s., 1 H), 4.61 - 4.71 (m, 1 H), 4.08 - 4.22 (m, 2 H), 2.74 - 2.89 (m, 2 H), 1.80 - 1.90 (m, 2 H), 1.39 (s, 9 H), 1.21 - 1.32 (m, 2 H). ES-LCMS: m/z 440.2(M+H).
Intermediate 12
1,1-dimethylethyl 4-[({[6-(aminocarbonyl)-3-pyridiπyl]amino}carbonyl)(3- fluoropheny!)amino]-1-piperidinecarboxylate
Figure imgf000019_0001
Prepared (in quantitative yield) as described above for intermediate 11
1H NMR (400 MHz, CHLOROFORM-of) 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), 5.47 (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 13 amino]carbonyl}amino)-2-pyridinecarboxamide
Figure imgf000019_0002
A solution of 1,1-dimethylethyl 4-[({[6-(aminocarbonyl)-3- pyridiπyl]amino}carbonyl)(phenyl)amino]-1-piperidinecarboxylate 11 (848 mg, 1.9
mrnol) 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% iPrOH/CH2CI2. The organic extracts were dried (Na2SO4) and concentrated to provide 5-({[phenyl(4- piperidinyl)amino]carbonyl}amino)-2-pyridinecarboxamide, 13 in quantitative yield as a white solid. ES-LCMS: m/z 340.1 (M+H).
Intermediate 14
Figure imgf000020_0001
Prepared (in quantitative yield) as described above for intermediate 13 ES-LCMS: m/z 358.1 (M+H).
The title compound, Example II, 5-({[(1-{[2-methyl-6-({4-
[(rnethylsuIfonyI)arnino]phenyl}oxy)-3-pyridinyl]rnethyl}-4- piperidinyl)(phenyl)amino]carbonyl}amino)-2-pyridinecarboxamid, was prepared from intermediates 13 and 6 using reductive amination procedure described in example Vl
1H NMR (CDCI3, 400 MHz) δ 8.29 (d, J=2.56 Hz, 1 H), 8.07 (br. s., 1 H), 8.02 (d,
J=8.61 Hz, 1 H), 7.87 (dd, 7=8.61 , 2.56 Hz, 1 H), 7.66 (d, J=4.21 Hz, 1 H), 7.41 -
7.53 (m, 4 H), 7.23 - 7.28 (m, 2 H), 7.19 - 7.23 (m, 2 H), 7.01 - 7.06 (m, 2 H), 6.50
(d, J=8.24 Hz, 1 H), 6.08 (s, 1 H), 6.01 (d, J=4.21 Hz, 1 H), 4.46 - 4.57 (m, 1 H),
3.33 (s, 2 H), 2.95 (s, 3 H), 2.82 (d, J=11.35 Hz, 2 H), 2.36 (s, 3 H), 2.07 - 2.17 (m, 2
H), 1.82 (d, J=10.62 Hz, 2 H), 1.29 - 1.43 (m, 2 H). HRMS: (M+H)* calcd for
C32H35N7O6S + H, 630.2499; found, 630.2487.
The title compound, Example III, 5-({[(3-fluorophenyI)(1-{[2-methyI-6-({4- [(methylsulfonyl)arnino]phenyl}oxy)-3-pyridinyI]rnethyl}-4- piperidinyl)amino]carboπyl}amino)-2-pyridinecarboxamide was prepared from
intermediates 14 and 6 using reductive animation procedure descπbed in example Vl
1H NMR (CDCI3, 400 MHz) δ 8 33 (d, J=I 38 Hz, 1 H), 8 04 (d, J=8 61 Hz, 1 H), 7 89 (dd, J=8 61 , 2 56 Hz, 1 H), 7 74 (br s , 1 H), 7 66 (d, J=A 21 Hz, 1 H), 743 - 7 52 (m, 2 H), 723 - 7 28 (m, 2 H), 7 17 - 7 23 (m, 1 H), 7 02 - 7 08 (m, 3 H), 6 97 (dt, J=B 97, 2 20 Hz, 1 H), 6 52 (d, J=8 06 Hz, 1 H), 6 09 (s, 1 H), 5 90 (d, J=4 21 Hz, 1 H), 445 - 4 57 (m, 1 H), 3 35 (s, 2 H), 2 97 (s, 3 H), 2 84 (d, J=11 17 Hz, 2 H), 2 37 (s, 3 H), 207 - 2 18 (m, 2 H), 1 82 (d, J=10 99 Hz, 2 H), 1 30 - 1 44 (m, 2 H) HRMS (M+H)+ calcd for C32H34FN7O5S + H, 6482404, found, 648 2404
Example IV
5-({[(H[6-({4-[(ethylsulfoπyl)amino]phenyl}oxy)-2-methyl-3-pyπdιnyl]methylH- pιperιdιnyl)(3-fluorophenyl)amιno]carbonyl}amιno)-2-pyrιdιnecarboxamιde
Figure imgf000021_0001
Intermediate 15 Λ/-{4-[(5-cyano-6-methyl-2-pyπdιnyl)oxy]phenyl}ethanesulfonamιde
Figure imgf000021_0002
Prepared (in 92 % yield) as described for intermediate 5
1H NMR (400 MHz, CHLOROFORM-cQ δ 7 84 (d, J=8 80 Hz, 1 H), 7 25 - 7 30 (m, 2 H), 7 11 - 7 16 (m, 2 H), 677 (d, J=843 Hz, 1 H), 644 (s, 1 H), 3 16 (q, J=I 33 Hz, 2 H), 261 (s, 3 H), 1 42 (t, J=I 33 Hz, 3 H) ES-LCMS m/z 318 3 (M+H)
Intermediate 16 Λ/-{4-[(5-formyl-6-methyl-2-pyπdιnyl)oxy]phenyl}ethanesulfonamιde i U
Prepared (in 69 % yield) as descπbed for intermediate 6
1H NMR (400 MHz, CHLOROFORM-d) δ 10.23 (s, 1 H), 8.10 (d, J=8.43 Hz, 1 H), 7.27 - 7.34 (m, 2 H), 7.12 - 7.17 (m, 3 H), 6.78 (d, J=8.43 Hz, 1 H), 3.17 (q, J=7.33 Hz, 2 H), 2.72 (s, 3 H), 1.41 (t, J=I 52 Hz, 3 H). ES-LCMS: m/z 321.0 (M+H).
Figure imgf000022_0001
BO -C lLΛoΛsJ ° ° CH * CI * 'UΛoΛiJ' ° °
3
The title compound, 5-({[(1-{[6-({4-[(ethylsulfonyI)amino]phenyl}oxy)-2-rrιethyl-3- pyridinyl]methyl}-4-piperidinyl)(3-fluorophenyl)amino]carbonyl}amino)-2- pyridinecarboxamide, was prepared from intermediates 16 and 14 using reductive aminatioπ described in example Vl
1H NMR (CDCI3, 400 MHz) δ 8.33 (d, J=2.56 Hz, 1 H), 8.05 (d, J=B.61 Hz, 1 H), 7.89 (dd, J=8.61 , 2.56 Hz, 1 H), 7.65 (d, J=4.21 Hz, 1 H), 7.42 - 7.54 (m, 3 H), 7.17 - 7.28 (m, 3 H), 7.02 - 7.08 (m, 3 H), 6.97 (dt, J=8.97, 2.20 Hz, 1 H), 6.52 (d, J=8.24 Hz, 1 H), 6.06 (s, 1 H), 5.83 (d, J=4.03 Hz, 1 H), 4.45 - 4.57 (m, 1 H), 3.35 (s, 2 H), 3.09 (q, J=7.33 Hz, 2 H), 2.84 (d, J=11.35 Hz, 2 H), 2.38 (s, 3 H), 2.14 (t, J=11.08 Hz, 2 H), 1.82 (d, ,/=11.17 Hz, 2 H), 1.38 - 1.43 (m, 2 H), 1.35 (t, J=7.33 Hz, 3 H). HRWIS: (M+H)+ calcd for C33H36FN7O6S + H, 662.2561 ; found, 662.2566
Example V
5-({[(H[6-({4-[(ethylsulfonyI)amino]phenyl}oxy)-2-methyl-3-pyridinyl]methyl}-4- piperidinyl)(phenyl)amino]carbonyl}amino)-2-pyridinecarboxamide
AinJyCoJx0
Prepared from intermediates 16 and 13 using reductive amination described in example VI
1H NMR (CDCI3, 400 MHz) δ 8.29 (d, J=2.56 Hz, 1 H), 8.04 (d, J=8.61 Hz, 1 H), 7.88 (dd, ./=8.61, 2.56 Hz, 1 H), 7.75 (br. s., 1 H), 7.65 (d, J=4.39 Hz, 1 H), 7.41 - 7.54
(m, 4 H), 7.18 - 7.28 (m, 4 H), 7.01 - 7.06 (m, 2 H), 6.50 (d, J=8.24 Hz, 1 H), 6.06 (s, 1 H), 5.91 (d, J=4.03 Hz, 1 H), 4.46 - 4.57 (m, 1 H), 3.33 (s, 2 H), 3.09 (q, J=7.33 Hz, 2 H), 2.83 (d, J=11.35 Hz, 2 H), 2.37 (s, 3 H), 2.13 (t, J=11.17 Hz, 2 H), 1.82 (d, J=11.17 Hz, 2 H), 1.37 - 1.43 (m, 2 H), 1.34 (t, J=7.33 Hz, 3 H). HRMS: (M+H)+ calcd for C33H37N7O5S + H, 644.2655; found, 644.2655.
Example Vl
5-({[(H[2-methyl-6-({2-methyl-4-[(methylsulfoπyl)amino]phenyl}oxy)-3- pyridiπyl]methyl}-4-piperidinyl)(pheπyl)amino]carbonyl}amino)-2- pyridinecarboxamide
Figure imgf000023_0001
Example VII
5-({[(1-{[6-({2-chloro-4-[(methylsulfonyI)amino]phenyl}oxy)-2-methyl-3- pyridiπyl]methyl}-4-piperidinyl)(phenyl)amino]carboπyl}amino)-2- pyridinecarboxamide
Figure imgf000023_0002
DCE
Figure imgf000024_0001
Intermediate 17 N-(4-hydroxy-3-rnethylphenyl)methanesulfoπamide
Figure imgf000024_0002
Synthesized by the method described for intermediate 4
1H NMR (CDCI3, 400 MHz) δ 2.2 (s, 3 H) 2.9 <s, 3 H) 4.7 (s, 1 H) 6.1 (s, 1 H) 6.7 (d, J=8.4 Hz, 1 H) 7.0 (d, J=8.2 Hz, 1 H) 7.0 (s, 1 H). LCMS: (M-H)* calcd for C8H11NO3S-H, 200; found, 200. intermediate 18 Λ/-(3-chloro-4-hydroxyphenyl)methanesulfonamide
Figure imgf000024_0003
Synthesized by the method described for intermediate 4
1H NMR (CDCI3, 400 MHz) δ 3.0 (s, 3 H) 5.5 (s, 1 H) 6.2 (s, 1 H) 7.0 (d, J=8.8 Hz, 1 H) 7.1 (m, 1 H) 7.3 (m, 1 H). LCMS: (M-H)+ calcd for C7HBCINO3S-H, 220; found, 220.
Intermediate 19 Λ/-{4-[(5-cyano-6-methyl-2-pyridinyl)oxy]-3-methylphenyl}methanesulfonamide
Figure imgf000025_0001
Synthesized by the method described for intermediate 5
1H NWlR (CDCI3, 400 MHz) S 2.1 (s, 3 H) 2.6 (s, 3 H) 3.1 (s, 3 H) 6.4 (s, 1 H) 6.7 (d, J=BA Hz, 1 H) 7.0 (d, J=8.4 Hz, 1 H) 7.1 (m, 1 H) 7.2 (d, J=2.6 Hz, 1 H) 7.8 (d, J=8.4 Hz, 1 H). LCMS: (M+H)* calcd for C15H15N3O3Si-H, 318; found, 318.
Intermediate 20 Λ/-{3-chloro-4-[(5-cyano-6-methyl-2-pyridinyl)oxy]phenyl}methanesulfonamide
a
Synthesized by the method described for intermediate 5
1H NMR (CDCI3, 400 MHz) δ 2.6 (s, 3 H) 3.1 (s, 3 H) 6.6 (s, 1 H) 6.9 (d, J=8.4 Hz, 1 H) 7.2 (d, J=1.5 Hz, 2 H) 7.4 (s, 1 H) 7.9 (d, J=8.8 Hz, 1 H). LCMS: (M+H)* calcd for C14H12CIN3O3S+H, 338; found, 338.
Intermediate 21
Λ/-{4-[(5-formyl-6-methyl-2-pyridinyl)oxy]-3-methylphenyl}methanesulfoπamide
Figure imgf000025_0002
Synthesized from intermediate 19 by the method described for intermediate 6 1H NMR (CDCI3, 400 MHz) δ 2.2 (s, 3 H) 2.7 (s, 3 H) 3.1 (s, 3 H) 6.7 (m, 2 H) 7.1 (d, J=8.8 Hz, 1 H) 7.1 (m, 1 H) 7.2 (s, 1 H) 8.1 (d, J=8.4 Hz, 1 H) 10.2 (s, 1 H). LCMS: (M+H)+ calcd for C15Hi6N2O4StH, 321 ; found, 321.
Intermediate 22 W-{3-chloro-4-[(5-formyl-6-methyl-2-pyridinyl)oxy]phenyl}methanesulfonamide
Synthesized from intermediate 20 by the method described for intermediate 6 1H NMR (CDCI3, 400 MHz) δ 2.7 (s, 3 H) 3.1 (s, 3 H) 6.8 (s, 1 H) 6.9 (d, J=8.6 Hz, 1 H) 7.2 (m, 2 H) 7.4 (d, J=2.2 Hz, 1 H) 8.1 (d, J=8.1 Hz, 1 H) 10.2 (s, 1 H).LCMS: (M+H)+ calcd for C14H13CIN2O4StH, 341; found, 341.
5-({t(H[2-methyl-6-({2-methyl-4-[(methylsulfonyl)amino]phenyl}oxy)-3- pyridinyl]methyl}-4-piperidinyl)(phenyl)amino]carbonyl}amino)-2- pyridinecarboxamide (example
Figure imgf000026_0001
A mixture of Λ/-{4-[(5-formyl-6-methyl-2-pyridinyI)oxy]-3- methylphenyljmethanesulfonamide 21 (47.2 mg, 0.15 mmol, 1.0 eq) and 5- ({[phenyl(4-piperidinyI)amino]carbonyl}amino)-2-pyridinecarboxamide 13 (50 mg; 0.15 mmol, 1.0 eq) and NaBH(OAc)3 (62.5 mg; 0.30 mmol, 2.0 eq) in 1,2- dichloroethane (8.0 mL) was stirred overnight at RT. The mixture was diluted with excess DCM and washed with saturated aq. NaHCO3. Organics separated and aqueous layer back extracted with DCM. Organics combined, dried over Na2SO4, filtered and concentrated down. Residue was purified by ISCO FC in 0-4 % in MeOH(W/ 2M NH3):CH2CI2 and then by preparative TLC to give 5-({[(1-{[2-methyl-6- ({2-methyl-4-[(methylsulfonyl)amino]ρhenyl}oxy)-3-pyridinyl]methyl}-4- piperidiπyl)(phenyl)amino]carbonyl}amino)-2-pyridinecarboxarnide as a white solid (55 mg, 58 % yield). 1H NMR (CDCI3, 400 MHz) δ 8.30 (d, J=2.38 Hz, 1 H), 8.05 (d, J=8A2 Hz, 1 H), 7.87 (dd, J=8.61, 2.56 Hz, 1 H), 7.63 (d, J=3.30 Hz, 1 H), 7.48 - 7.55 (m, 3 H), 7.43 (d, J=8.42 Hz, 1 H), 7.22 (s, 2 H), 7.11 (d, J=2.56 Hz, 1 H), 7.01 - 7.06 (m, 1 H), 6.95 - 6.99 (m, 1 H), 6.43 (br. s., 1 H), 6.41 (d, J=8.24 Hz, 1 H), 6.02
(s, 1 H), 5.45 (br. s., 1 H), 4.47 - 4.58 (m, 1 H), 3.34 (s, 2 H), 3.00 (s, 3 H), 2.84 (d, J=11.72 Hz, 2 H), 2.37 (s, 3 H), 2.17 (s, 3 H), 2.09 - 2.17 (m, 2 H), 1.83 (d, J=11.35 Hz, 2 H), 1.31 - 1.44 (m, 2 H). HRMS: (M+H)* calcd for C33H37N7O5S + H, 644.2655; found, 644.2644.
The reaction procedure and purification process to afford the title compound, 5-({[(1- {[6-({2-chloroJl~[(methyIsulfonyl)amino]phenyl}oxy)-2-methyl-3-pyridinyl]methyl}-4- piperidiπyl)(phenyl)amino]carboπyl}amino)-2-pyridinecarboxamide (example VII), are identical to that described for example Vl, except that in addition to 13, intermediate 22 W-{3-chloro^4~[(5-formyl-6-rnethyl-2- pyridinyl)oxy]phenyl}methanesulfonamide was used as aldehyde. 5-({[(1-(J6-({2- chloro-4-[(methylsulfonyl)amino]phenyI}oxy)-2-methyl-3-pyridinyl]methylH- piperidinyl)(ρhenyI)amino]carbonyl}amino)-2-pyridinecarboxamide was obtained as a white solid (56 mg, 57 % yield). 1H NMR (CDCI3, 400 MHz) 8 8.30 (d, J=2.56 Hz, 1 H), 8.06 (d, J=8.42 Hz, 1 H), 7.87 (dd, J=8.61, 2.38 Hz, 1 H), 7.63 (d, J=4.21 Hz, 1 H), 7.45 - 7.54 (m, 4 H), 7.35 (d, J=2.01 Hz, 1 H), 7.22 (d, J=1.65 Hz, 2 H), 7.09 - 7.17 (m, 2 H), 6.61 (br. s., 1 H), 6.57 (d, J=8.24 Hz, 1 H), 6.01 (br. s., 1 H), 5.46 (d, J=2.56 Hz, 1 H), 4.48 - 4.58 (m, 1 H), 3.34 (s, 2 H), 3.03 (s, 3 H), 2.84 (d, J=11.17 Hz, 2 H), 2.33 (s, 3 H), 2.15 (t, J=11.26 Hz, 2 H), 1.83 (d, J=12.64 Hz, 2 H), 1.31 - 1.45 (m, 2 H). HRMS: (M+H)+ calcd for C32H34CIN7O5S + H, 664.2109; found, 664.2117.
Example VIII
5-({[(3-fluorophenyl)(1-{[4-methyl-2-({4-[(methylsulfonyl)amino]phenyl}oxy)-5- pyrimidinyI]methyl}-4-piperidinyl)amino]carbonyl}amino)-2-pyridinecarboxamide
ΛXxjcrώjorYe
Figure imgf000028_0001
23 65 % 24 42 %
Figure imgf000028_0002
25 42 % 26
Figure imgf000028_0003
Intermediate 23
Ethyl-Σ-Chloro-Φfnethyl-δ-pyrirnidinecarboxylate
Figure imgf000028_0004
The synthesis of ethyl 2-ch|oro-4-methyl-5-pyrimidiπecarboxylate 23 was accomplished as described in the literature [Palanki et al., (2000) J. Med.Chem. pp3995-4004]. 1H NMR (CDCI3, 400 MHz) δ 1.4 (t, ,7=7.1 Hz, 3 H) 2.8 (s, 3 H) 4.4 (q, J=7.1 Hz1 2 H) 9.0 (s, 1 H). LCMS: (M+H)+ calcd for CBH9CIN2O2 + H, 201; found, 201.
Intermediate 24
Ethyl 4-methyl-2-({4-[(methylsulfonyl)amiπo]pheπyl}oxy)-5-pyrimidinecarboxylate
Figure imgf000028_0005
A mixture of Ethyl^-Chloro^-methyl-S-pyrimidinecarboxylate 23 (930 mg, 4.65 mmol), phenol 4 (870 mg, 4.65 mmol), and Cs2CO3 (2.27 g, 7.0 mmol) in dry DMF (20 mL) was stirred at 80 0C under N2 overnight. The reaction mixture was then
filtered and rinsed with CH2CI2. The filtrate was concentrated to a residue by rotary evaporation, and then diluted with water. It was extracted with 10 % MeOH in CH2CI2. The organic extracts were dried over Na2SO4, filtered and evaporated. The residue was purified by ISCO FC with 0-50 % EtOAC in hexane to provide ethyl 4- methyl-2-({4 [(methylsulfonyl)amino]phenyl}oxy)-5-pyrimidinecarboxylate 24 as a white solid (1.07 g, 66 %).
1H NMR (CDCI3, 400 MHz) δ 1.4 (t, J=7.1 Hz, 3 H) 2.8 (s, 3 H) 3.0 (s, 3 H) 4.4 (q, J=7.1 Hz, 2 H) 6.5 (s, 1 H) 7.2 (m, 2 H) 7.3 (m, 2 H) 9.0 (s, 1 H). LCMS: (M+H)+ calcd for C15H17N3O5S + H, 352; found, 352.
Intermediate 25 Λ/-(4-{[5-(hydroxymethyl)-4-methyl-2-pyrimidinyl]oxy}phenyl)methanesulfonamide
Figure imgf000029_0001
DIBAL (1.2 mL 1 M in CH2CI2, 1.2 mmol) was added dropwise to a solution of ethyl 4-methyl-2-({4 [(methylsulfonyl)amino]phenyl}oxy)-5-pyrimidinecarboxylate 24 (175 mg, 0.5 mmol) in CH2CI2 at -78 °C under N2. The reaction mixture was stirred for 1h at
-78 "C and then allowed to warm up to 0 °C and stirred for 0.5 h. Sat'd aqueous solution of Rochelle's salt was added carefully and the biphasic mixture was stirred for 1h. The organic layer was separated and the aqueous layer was extracted withiO % MeOH in CH2CI2 and the combined organic layers were dried (Na2SO4), filtered, and evaporated. The residue was purified by ISCO FC with 0-5 % MeOH (w/ 2M NH3) in CH2CI2 to give Λ/-(4-{[5-(hydroxymethyl)-4-rnethyl-2- pyrirnidinyl]oxy}phenyl)methanesulfonarnide 25 as a white solid (64 mg, 42%). 1H NMR (CDCI3, 400 MHz) δ 2.5 (s, 3 H) 3.0 (s, 3 H) 4.7 (d, J=5.3 Hz, 2 H) 6.4 (s, 1 H) 7.2 (m, 2 H) 7.3 (m, 2 H) 8.4 (s, 1 H). LCMS: (M+Hf calcd for C13H15N3O4S + H, 310; found, 310.
Intermediate 26 Λ/-{4-[(5-formyl-4-methyl-2-pyrimidiπyl)oxy]phenyl}methanesulfonamide
Figure imgf000030_0001
MnO2 (340 mg, 3.9 mmol) was added to a solution of Λ/-(4-fJ5-(hydroxymethyl)-4- methyl-2-pyrimidinyl]oxy}phenyl)methanesulfonarnide 25 (62 mg, 0.2 mmol) in CH2CI2 at -RT . The reaction mixture was stirred for 48 h. Filtered off the inorganics and filtrate was evaporated. The residue was purified by ISCO FC with 0-25 % EtOAC in CH2CI2 to give Λ/-{4-[(5-formyl-4-methyl-2- pyrimidinyl)oxy]phenyl}methanesulfonamide 26 as a white solid (26 mg, 42%), 1H NMR (CDCI3, 400 MHz) δ ppm 2.8 (s, 3 H) 3.0 (s, 3 H) 6.5 (s, 1 H) 7.2 (m, 2 H) 7.3 (m, 2 H) 8.8 (s, 1 H) 10.2 (s, 1 H). LCMS: (M+Hf calcd for C13H13N3O4S + H, 308; found, 308.
The title compound, 5-({[(3-fluorophenyl)(1-[[4-methyl-2-({4-
[(methylsulfonyl)amino]phenyl}oxy)-5-pyrirnidinyl]methyl}-4- piperidinyl)amino]carbonyl}amino)-2-ρyridinecarboxamide, was synthesized from intermediates 26 and 14 using reductive amination procedure described in example
Vl
White solid (21 mg, 30 % yield).
1H NMR (CDCI3, 400 MHz) δ 8.35 (d, J=2.57 Hz, 1 H), 8.19 (s, 1 H), 8.09 (d, J=8.80
Hz, 1 H), 7.89 (dd, J=8.43, 2.57 Hz, 1 H), 7.65 (s, 1 H), 7.48 - 7.56 (m, 1 H), 7.23 -
7.29 (m, 4 H), 7.17 - 7.20 (m, 2 H), 7.06 (d, J=8.43 Hz, 1 H), 6.95 - 7.01 (m, 1 H),
6.01 (s, 1 H), 5.48 (br. s., 1 H), 4.46 - 4.60 (m, 1 H), 3.38 (s, 2 H), 3.03 (s, 3 H), 2.85
(d, J=11.73 Hz, 2 H), 2.46 (s, 3 H), 2.19 (t, J=12.46 Hz, 2 H), 1.81 - 1.91 (m, 2 H),
1.33 - 1.38 (m, 2 H). HRMS: (M+H)+ calcd for C31H33FN8OsS + H, 649.2357; found,
649.2354.
Example IX
5-({[(1-{[2-ethyl-6-({4-[(methylsulfonyl)amino]phenyl}oxy)-3-pyridinyl]methyl}-4- piperidiπyl)(phenyl)amino]carbonyl}amino)-2-pyridinecarboxamide
Figure imgf000031_0001
Intermediate 27 6-{[(1 ,1-dιmethylethyI)(dιmethyl)sιlyI]oxy}-2-methyl-3-pyrιdιnecarbonitπle
Figure imgf000031_0002
A slurry of 2-methyl-6-oxo-1 ,6-dιhydro-3-pyrιdιnecarbonιtπle (2 0O g, 149 mmol, 1eq ) in dimethylformamide (20 rtiL) was cooled under nitrogen to 0-5 °C Tπethylamine (2 5 mL, 17 9 mmol, 1 2 eq ) and f-butyldimethylsilyl chloride (247 g, 16 4 mmol, 1 1 eq ) was added After stirring approximately 1hrs, additional tπethylamine (2 δ mL, 17 9 mmol, 1 2 eq ) and t-butyldιmethylsιlyl chloride (247 g, 164 mmol, 1 1 eq ) was added After stirring another hour, the reaction was evaporated in vacuo and the residue was purified on flash grade silica gel eluting with 0-20% ethyl acetate in hexanes to provide -{[(1,1- dιmethylethyl)(dιmethyl)sιlyl]oxy}-2-methyl-3-pyπdιnecarbonιtrιle 27 (1 659 g, 45%) as an oil 1H NMR (400 MHz, CDCI3) δ 769 (d, J = 84 Hz, 1 H), 6 56 (d, J = 8 5 Hz, 1 H)1 260 (s, 3 H), 097 (s, 9 H), 033 (s, 6 H)
Intermediate 28 2-ethyl-6-oxo-1 ,6-dihydro-3-pyrιdιnecarbonιtπle
Figure imgf000032_0001
A solution of 6-{[(1 ,1-dimethylethyI)(dimethyI)silyl]oxy}-2-rnethyI-3- pyridinecarbonitrile 27 (1.123 g, 4.52 mmol, 1 eq.) in tetrahydrofuran (10 mL) was cooled under nitrogen to -78 "C. Lithium diisopropylamide (2.0 M, 2.7 mL, 5.4 mmol, 1.2 eq.) was added dropwise via syringe. After stirring 10 min., methyl iodide (0.34 mL, 5.43 mmol, 1.2 eq.) was added via syringe. After stirring an additional 15 min., the reaction was quenched with glacial acetic and warmed to ambient temperature. The resulting slurry was filtered and the mother liquor was combined with glacial acetic acid (1 mL) and heated to 50 °C for 45 min. The reaction mixture was evaporated in vacuo and crystallized from ethyl acetate. The resulting slurry was cooled to 0-5 °C and filtered. The solid, contaminated with diisopropylamine acetate salt, was dried under high vacuum to provide 2-ethyl-6-oxo-1 ,6-dihydro-3- pyridinecarboπitrile 28 (653 mg). 1H NMR (400 MHz, DMSOd6) δ 7.58 (d, J = 9.4 Hz, 1 H), 6.25 (d, J = 9.3 Hz, 1 H), 2.62 (q, J = 7.6 Hz, 2 H), 1.18 (t, J = 7.6 Hz, 3 H); MS nVz 149 (M+H)*.
Intermediate 29 β-chloro^-ethyl-S-pyridinecarbonitrile
Figure imgf000032_0002
A mixture of 2-ethyl-6-oxo-1 ,6-dihydro-3-pyridinecarbonitrile 28 (610 mg, 4.12 mmol, 1 eq.) and phosphorus oxychloride (5 mL) was heated at reflux for 1.5 hrs. The reaction was evaporated in vacuo to a residue and partitioned between dichloromethane and water. The mixture was brought to basic pH with concentrated aqueous ammonium hydroxide and the layers were separated. The aqueous phase was back-extracted with dichloromethane and the combined organic layers were dried over anhydrous magnesium sulfate, filtered and evaporated in vacuo. The crude product was purified on flash silica gel eluting with 20% ethyl acetate in hexanes to provide 6-chloro-2-ethyl-3-pyridinecarbonitrile 29 as a white solid (242
mg, 38% from 6-{[(1 ,1-dimethylethy!)(dimethyl)silyl]oxy}-2-methyl-3- pyridinecarboπitrile). 1H NMR (400 MHz, CDCI3) δ 7.83 (d, J = 8.2 Hz, 1 H), 7.29 (d, J = 8.2 Hz, 1 H), 3.04 (q, J = 7.6 Hz, 2 H), 1.36 (t, J = 7.6 Hz, 3 H); MS m/z 167 (M+H)+.
Intermediate 30 N-{4-[(5-cyano-6-ethyl-2-pyridinyl)oxy]phenyl}methanesulfonamide
Figure imgf000033_0001
A mixture of e-chloro^-ethyl-S-pyridinecarbonϊtrile 29 (223 mg, 1.34 mmol, 1.0 eq.), Λ/-(4-hydroxyphenyl)methanesulfonamide 4 (250 mg, 1.34 mmol, 1.0 eq.), and cesium carbonate (545 mg, 1.67 mmol, 1.25 eq.) in dimethyl formamide (3 ml.) was heated at 80 °C for 1.5 hrs. under nitrogen. The reaction mixture was evaporated in vacuo and partitioned between ethyl acetate and water. The layers were separated and the aqueous layer 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. The crude product was purified on flash grade silica gel eluting with 40 % ethyl acetate in hexanes to provide N -{4-[(5-cyano-6- ethyl-2-pyridinyl)oxy]phenyl}methanesulfonamide 30 as a foam (200 mg, 47%). 1H NMR (400 MHz, CDCI3) δ 7.84 (d, J = 8.5 Hz, 1 H), 7.27 (d, J = 9.0 Hz, 2 H), 7.16 (d, J = 8.9 Hz, 2 H), 6.78 (d, J = 8.5 Hz, 1 H), 6.32 (br, 1 H), 3.04 (s, 3 H)1 2.91 (q, J = 7.6 Hz, 2 H), 1.22 (t, J = 7.4 Hz, 3 H); MS m/z 318 (M+H)+.
Intermediate 31 : Λ/-{4-[(6-ethyl-5-formyl-2-pyridinyl)oxy]phenyl}methaπesulfonamide
Figure imgf000033_0002
A solution of N -{4-[(5-cyano-6-ethyl-2-pyridinyl)oxy]phenyl}methanesulfonamide 30 (197 mg, 0.621 mmol, 1.0 eq.) in dichloromethane (3 mL) was cooled under nitrogen to -50 0C. Diisobutylaluminum hydride (1.0 M in CH2CI2, 1.5 mL, 1.5 mmol, 2.4 eq.) was added in two equal portions separated by 10 min. After stirring an additional 15 min., the reaction was quenched by addition of 1N NaHSO4. After stirring for 1.5
hrs., the triphasic mixture was separated and the aqueous phase was back- extracted twice with dichloromethaπe. The combined organic layers were dried over anhydrous magnesium sulfate, filtered and evaporated in vacuo. The crude product was purified on flash silica gel eluting with 50% ethyl acetate in hexanes to provide Λ/-{4-[(6-ethyl-5-formyl-2-pyridinyl)oxy]phenyl}methanesulfonamide 31 as a solid (154 mg, 77%). 1H NMR (400 MHz, CDCI3) δ 10.27 (s, 1 H), 8.13 (d, J = 8.5 Hz, 1 H), 7.28 (d, J = 8.7 Hz, 2 H), 7.19 (d, J = 8.9 Hz, 2 H), 6.80 (d, J = 8.5 Hz, 1 H), 6.36 (br, 1 H), 3.08 (q, J = 7.6 Hz, 2 H), 3.05 (s, 3 H), 1.23 (t, J = 7.4 Hz, 3 H); MS rn/z 321 (M+H)+.
The title compound, 5-({[(1-{[2-ethyl-6-({4-[(methylsulfonyl)amino]phenyI}oxy)-3- pyridinyl]methyl}-4-piperidinyl)(pheπyl)arnino]carbonyl}amino)-2- pyridinecarboxamide, was prepared by reacting Λ/-{4-[(6-ethyl-5-formyl-2- pyridinyl)oxy]phenyl}methanesulfonamide 31 (25 mg, 0.078 mmol, 1.0 eq.), 5-({[(1- methyl-4-piperidinyl)(phenyl)amino]carbonyl}amino)-2-pyridinecarboxamide 13 (27 mg, 0.078 mmol, 1.0 eq.), and sodium triacetoxyborohydride (33 mg, 0.156 mmol, 2 eq.) in dichloromethane (2.5 mL), stirred under nitrogen for approximately 64 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 6% methanol in dichloromethane to provide the product as foam. Trituration with diethyl ether and evaporation of the solvent provided 5-({[(1-{[2- ethyl-6-({4-[(methylsulfonyl)amino]phenyl}oxy)-3-pyridinyl]methyl}-4- piperidinyl)(pheπyl)amino]carbonyl}amino)-2-pyridinecarboxamide (example IX) (23 mg, 46%) as a solid. 1H NMR (400 MHz, CD3OD) δ 8.53 (d, J = 2.5 Hz, 1 H), 7.93 (d, J = 8.5 Hz, 1 H), 7.88 (dd, J = 8.5, 2.4 Hz, 1 H), 7.60 (d, J = 8.3 Hz, 1 H), 7.54- 7.46 (m, 3 H), 7.29-7.24 (m, 4 H), 7.05 (d, J = 8.8 Hz, 2 H), 6.58 (d, J = 8.2 Hz, 1 H), 4.43 (m, 1 H), 3.43 (s, 2 H), 2.93 (s, 3 H), 2.87 (d, J = 11.2 Hz, 2 H), 2.69 (q, J = 7.6 Hz, 2 H), 2.16 (app t, J = 11.6 Hz, 2 H), 1.84 (d, J = 12.6 Hz, 2 H), 1.40 (app qd, J = 12.0, 3.6 Hz, 2 H), 1.09 (t, J = 7.5 Hz, 3 H). HRMS: (M+Hf calcd for C33H37N7O5S + H, 644.2655; found, 644.2682 LR/MS: M+H = 644.
Synthesis of intermediate 36
Figure imgf000035_0001
e-Chloro-S-pyridinecarbonitrile (2.29g, 16.5 mmol) was dissolved in dichloromethane (60 mL). To this solution was added urea hydrogen peroxide (4.7 g, 49.6 mmol). After the resulting mixture was cooled down to 00C on an ice bath, trifluoroacetic acid anhydride (6.3 mL, 45 mmol) was slowly introduced by syringe. The reaction was stirred for an additional 3 hours at room temperature before quenched with satd. aq. Na2S2θ3 and extracted with dichloromethane. The combined organic layers were then washed with satd. aq. NaHCO3, dried over anhydrous sodium sulfate and concentrated. The residue was purified by flash column chromatography (silical gel, 0-100% EtOAc/ hexane ) to provide intermediate 32 as a white solid (2.1 g, 82%). 1H NMR (400 MHz, CHLOROFORM- d) δ ppm 8.5 (d, J=1.6 Hz, 1 H), 7.6 (d, J=8.4 Hz, 1 H), 7.4 (dd, J=8.4, 1.6 Hz, 1 H). MS nVz 155 (M+H)+.
To a solution of intermediate 32 (616 mg, 4 mmol) in DMF (25mL) were added intermediate 4 (748 mg, 4 mmol) and Cs2CO3 (1 ,3 g, 4 mmol). The mixture was stirred for 4 hours at 45 0C before cooled down to room temperature and diluted with water. Intermediate 33 was precipitated and collected as white solid by filtration (1.1 g, 90%). 1H NMR (400 MHz, DMSO-d6) δ ppm 9.8 (s, 1 H), 9.0 (s, 1 H), 7.7 (s, 1 H), 7.2 (m, 2 H), 7.1 (d, J=8.8 Hz, 2 H), 3.0 (s, 3 H). MS m/z 306 (M+Hf.
At N2 atmosphere, intermediate 33 (780 mg, 2.6 mmol) was treated with POCI3 (10 mL) under reflux condition for 1 hour. After evaporation of the solvents, the residue was dissolved in dichloromethane and washed with satd. aq. NaHCO3. The organic phase was then dried over anhydrous sodium sulfate. Evaporation of the solvents gave a light gray solid, which was triturated with ether to afford intermediate 34 as an off white solid (720 mg, 86%). 1H NMR (400 MHz, CHLOROFORM-c/) δ ppm 7.9 (d, J=8.4 Hz, 1 H), 7.3 (d, J=4.4 Hz, 2 H), 7.1 (m, 2 H), 6.9 (d, J=8.6 Hz, 1 H), 3.0 (s, 3 H).
Intermediate 34 (533 mg, 1.6 mmol), cyclopropyl boronic acid (424 mg, 4.9 mmol), K3PO4 (1040 mg, 4.9 mmol) and PEPPSI™-IPr (87 mg) were mixed in schlenk flask charging with 20 mL of toluene. The mixture was purged with N2 (3X) before heated to 1000C for 20 hours. After cooled down to room temperature, the solid was filtered off and washed with EtOAc. The filtrate was evaporated and the residue was purified by chromatography (silical gel, 0-50% EtOAc/hexane) to provide intermediate 35 as a sloid (373 mg, 71%). 1H NMR (400 WlHz, CHLOROFORM-d) δ ppm 7.8 (d, J=8.4 Hz, 1 H), 7.3 (s, 2 H), 7.1 (d, J=8.8 Hz, 2 H), 6.7 (d, J=8.4 Hz, 1 H), 6.5 (s, 1 H), 3.0 (s, 3 H), 2.4 (dt, J=S.0, 3.9 Hz, 1 H), 1.0 (dd, J=7.7, 2.9 Hz, 2 H), 0.9 (dd, J=4.4, 2.9 Hz, 2 H). MS m/z 330 (M+H)+.
Intermediate 36 (440 mg, 94%) was synthesized from intermediate 35 (450 mg, 1.5 mmol) in a similar procedure as described in the preparation of intermediate 31. 1 H NMR (400 MHz, DMSO-Cf6) δ ppm 10.3 (s, 1 H), 8.1 (d, J=8.6 Hz, 1 H), 7.2 (d, J=8.8 Hz, 2 H), 7.1 (m, 2 H), 6.8 (d, J=8.4 Hz, 1 H), 2.9 (m, 4 H), 0.9 (d, J=7.7 Hz, 2 H), 0.7 (br. s., 2 H).
Example X
5-({[(1-{t2-cyclopropyl-6-({4-t(methylsulfonyl)amino]phenyl}oxy)-3-pyridinyl]methyl}- 4-piperidiπyl)(phenyl)amino]carbonyl}amino)-2-pyridinecarboxamide
Figure imgf000037_0001
^({[(HP-Cyclopropyl-β-^-KmethylsulfonyOaminolpheny^oxyJ-S-pyridinyllmethyl}- 4-piperidinyl)(phenyl)amino]carbonyl}amino)-2-pyridinecarboxarnide was prepared from intermediate 36 and 13 as decribed in Example IX. 1H NMR (400 MHz, CHLOROFORM-d) B ppm 8.3 (d, J=2.4 Hz, 1 H), 8.0 (d, J=8.6 Hz, 2 H), 7.6 (d, J=3.7 Hz, 1 H)1 7.5 (m, 5 H), 7.2 (d, J=8.6 Hz, 6 H), 7.0 (d, J=S.6 Hz, 2 H), 6.5 (d, J=8.2 Hz, 1 H), 6.0 (s, 1 H), 5.7 (s, 1 H), 4.5 (m, 1 H), 3.0 (s, 3 H), 2.9 (d, J=10.6 Hz, 2 H), 2.1 (m, 3 H), 1.8 (s, 2 H), 1.4 (s, 2 H), 0.8 (m, 4 H). HRMS: (M+Hf calcd for C34H37N7O5S + H, 656.2650; found, 656.2651.
Example Xl: 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 LSWI 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 min, 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 370C, 5% CO2 for 1 hr. 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 -800C and then thawed to room temperature for 30 min. 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 deoxythymidine-5'-triphosphate, 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 370C, 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 108/well.
Raw data from the PBL assay were expressed as Counts Per Minute (CPM) and normalized according to the following formula:
Log10(CPM) - Log™ (Geometric mean PC) + Log10 (Geometric mean NC) - Log™ (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
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 = logio(molar compound concentration) C = log10(IC50)
Compounds of the invention have IC50 values less than 1OnM.
Example XII: Biological Activity
HOS Assay
HOS-Luc cells were created by introducing an HIV-1 LTR-luciferase 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 370C, 5% CO2 for ihr.'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 370C, 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 = Logio[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 XlII: 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 mLJkg 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 min 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. Aliquots (50 μL) of standards and study samples were pipetted into 96-weil 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 ceπtrifuged for 10 min at 2055 x g and 40C. 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, βmicron, 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 min. 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
Noπ-compartmental pharmacokinetic parameters [terminal plasma half-life (t1/2), 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 (AUCo-*,)] were calculated based on the composite plasma concentration time data using WinNonlin Professional 4.1 (Pharsight, Mountain View CA) . Dose-normalized AUC D-Ko (DNAUC o-∞) was obtained by dividing the AUC 0→owith its respective dose and expressed as unit dose AUC o→« (ng*h/mL/mg/kg). Oral bioavailability (F) was calculated using the following equation. F (%) = [(DNAUCo-., oral)/(DNAUC0Λ, 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 imgf000043_0001
* WO2006/030925
Example XIV: 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 min at 4000 x g and 4°C and stored at -80°C 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 min at 4000 x g and 4°C and stored at -80cC 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. Aliquots (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 min at 2055 x g and 40C. 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, 3micron, Atlantis C18 column (Waters) using a gradient eluting system at a flow rate of 0.75 mL/rnin. 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 min. The 5% A:95% B composition was held for 0.5 min followed by a linear gradient to 90% A:10% B at 2.0 min. 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. Aliquots (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 iiver or spleen homogenate and aliquots (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 min 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, 2.5micron, 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 hydroxideiacetonitrile (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 min. 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
Figure imgf000045_0001
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-κo (DNAUC o→∞) 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 (%) = [(DNAUCo-,, oral)/(DNAUC0.∞, IV] * 100% where DNAUC0-O, 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
Figure imgf000046_0001
* WO2006/030925
Example XV: 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 310C 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 40C. This spin was repeated after resuspending the pellets in 200 mis of the same solution. All subsequent steps were performed at 4αC. 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 (1 mM), PMSF (1mM) 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, aliquoted and stored frozen at -80 0C.
2. Experimental Protocol
Compound potencies were determined by a radioligand (3H-dofetilide) 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 (1.2 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 DWISO 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.
Figure imgf000048_0001
* WO2006/030925

Claims

Claims
1. A compound selected from the group consisting of
Λ/-(4-{[5-({4-[(3-fiuorophenyl)({[6-(methyloxy)-3-pyridinyI]amino}carbonyl)amino]-1- piperidinyl}methyl)-6-methyl-2-pyridinyl]oxy}phenyl)methanesulfonamide;
5-({[(H[2-methyl-6-({4-[(methyisulfonyl)amino]phenyI}oxy)-3-pyridinyl]methyl}-4- piperidinyl)(phenyl)amino]carbony!}amino)-2-pyridinecarboxamide;
^-({[(S-fluorophenyOCI-flZ-methyl-e-^KmetriylsulfonylJ aminolphenylJoxyJ-S- pyridinyl]methyl}-4-piPeridiπyl)amino]carbonyi}amino)-2-pyridinecarboxamide;
5-({[(1-{[6-({4-[(ethylsulfonyl)amino]pheπyl}oxy)-2-methyl-3-pyridinyl]methyl}-4- piperidinyl)(phenyl)amino]carbonyl}amino)-2-pyridinecarboxamide;
5-({t(H[6-({4-t(ethylsulfonyl)amino]phenyl}oxy)-2-methyl-3-pyridinyl]methyl}-4- piperidinyl)(3-fluorophenyl)amino]carbonyl}amino)-2-pyridinecarboxamide;
5-({[(H[2-methyl-6-({2-methyl-4-[(methylsulfonyl)amino]phenyl}oxy)-3- pyridinyl]methyl)~4-piperidinyl)(phenyi)amino]carbonyl}amino)-2- pyridinecarboxamide;
S-UKHtδ-KZ-chloro^-tCmethylsulfoπylJaminolphenylloxyJ^-methyl-S- pyridinyl]methyl}-4-piperidiπyl)(phenyl)amino]carbonyi}amino)-2- pyridinecarboxamide;
5-({[(3-fluorophenyl){1-{[4-methyl-2-({4-[(methylsulfonyl)amino]phenyI}oxy)-5- pyrimidinyl]methyl}-4-piperidinyl)amino]carbonyl}amino)-2-pyridinecarboxamide;
5-({[(1-{[2-ethyl-6-({4-[(methylsulfonyl)amino]pheπyI}oxy)-3-pyridinyl]methyl}-4- piperidinyl)(phenyi)amino]carbonyl}amiπo)-2-pyridinecarboxamide;
5^{[(1φ-cyclopropyl-δ-({4-[(niΘthylsulfonyl)aminolphenyl}oxy)-3-pyridinyl]mβlhyl}- 4-piperidinyl)(pheπyI)amino]carbonyl}amino)-2-pyridinecarboxamide; 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
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)cytosine (HPMPC), [[[2-(6-amino-9H-purin-9- yl)ethoxy]methyl]phosphinylidene]bis(oxymethylene)-2,2- dimethylpropanoic acid (bis-POM PMEEA, adefovir dipivoxil), [[(1 R)-2-(6-amino-9H- purin-9-yl)-1-methylethoxy]methyl]phosphonic acid (tenofovir), (R)-[[2-(6-Amino-9H- purin-9-yl)-1-methylethoxy]methy!]phosphonic acid bis- (isopropoxycarbonyloxymethyl)ester (bis-POC-PMPA), nucleoside reverse transcriptase inhibitors, 3'-azido-3'-deoxythymidine (AZT, zidovudine), 2', 3'- dideoxycytidine (ddC, zalcitabine), 2',3'-dideoxyadenosine, 2',3'-dideoxyinosine (ddl, didanosine), 2',3'-didehydrothymidine (d4T, stavudine), (-)-cis-1 -(2-hydroxymethyl)- 1 ,3-oxathiolane 5-yl)-cytosine (lamivudine), cis-1 -f2-(hydroxymethyl)-1.3- oxathiolan-5-yl)-5-fluorocytosine (FTC), (-J-cis-^-p-amino-β^cyclopropylaminoJ-ΘH- purin-9-yl]-2-cyclopentene-1 -methanol (abacavir), ribavirin, protease inhibitors, .
indinavir, ritonavir, nelfinavir, amprenavir, saquinavir, fosamprenavir, iopiπavir, 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), nevirapine (BI-RG-587), alpha-((2-acetyl-5- methylphenyl)amino)-2,6-dichloro-beπzeneacetamide (loviride), 1 -[3- (isopropylamino)-2-pyridyl]-4-[5-(methanesulfonamido)-1H-indol-2- ylcarbonyl]piperazine monomethanesulfonate (delavirdine), (S)-6-chloro-4- (cyclopropylethynyl)-i ,4-dihydro-4-(trifluoromethyl)-2H-3, 1 -benzoxazin-2-one (efavirenz, DMP 266), rilpivirine, integrase inhibitors, fusion inhibitors, T-20 and T- 1249.
PCT/US2008/081665 2007-10-31 2008-10-30 Ccr5 antagonists as therapeutic agents WO2009058921A1 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020025574A1 (en) 2018-08-03 2020-02-06 Bayer Aktiengesellschaft Process for the preparation of 6-(haloalkyl)-2-halo-5-acylpyridines and intermediates for this process
WO2020179859A1 (en) 2019-03-06 2020-09-10 第一三共株式会社 Pyrrolopyrazole derivative
WO2023014908A1 (en) * 2021-08-05 2023-02-09 Pipeline Therapeutics, Inc. Lpar1 antagonists and uses thereof

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WO2002079186A2 (en) * 2001-03-30 2002-10-10 F. Hoffmann-La Roche Ag Aminopiperidine derivatives as modulators of chemokine receptor activity
WO2006030925A1 (en) * 2004-09-13 2006-03-23 Ono Pharmaceutical Co., Ltd. Nitrogenous heterocyclic derivative and medicine containing the same as an active ingredient

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002079186A2 (en) * 2001-03-30 2002-10-10 F. Hoffmann-La Roche Ag Aminopiperidine derivatives as modulators of chemokine receptor activity
WO2006030925A1 (en) * 2004-09-13 2006-03-23 Ono Pharmaceutical Co., Ltd. Nitrogenous heterocyclic derivative and medicine containing the same as an active ingredient
CA2579501A1 (en) * 2004-09-13 2006-03-23 Ono Pharmaceutical Co., Ltd. Nitrogenous heterocyclic derivative and medicine containing the same as an active ingredient

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020025574A1 (en) 2018-08-03 2020-02-06 Bayer Aktiengesellschaft Process for the preparation of 6-(haloalkyl)-2-halo-5-acylpyridines and intermediates for this process
WO2020179859A1 (en) 2019-03-06 2020-09-10 第一三共株式会社 Pyrrolopyrazole derivative
WO2023014908A1 (en) * 2021-08-05 2023-02-09 Pipeline Therapeutics, Inc. Lpar1 antagonists and uses thereof

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