WO2009058923A1 - Ccr5 antagonists as therapeutic agents - Google Patents

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 Al DS-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 VVO2001087839. 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:

5-({[butyl(1-([2-methyl-6-({4-[(methylsulfonyl)amino]phenyl}thio)-3-pyridinyl]methyl}-4- piperidinyl)amino]carbonyl}amino)-2,4-difluorobenzamide;

2,4-difluoro-5-({[(3-fluorophenyl)(1-{[2-methyl-6-({4-[(methylsulfonyl)amino]ρhenyl}thio)- 3-pyridinyl]methyl}-4-piperidinyl)amino]carbonyl}amino)benzamide; 2,4-difluoro-5-({[(1-{[2-methyl-6-({4-[(methylsulfonyl)amino]phenyl}thio)-3- pyridinyl]methyl}-4-piperidinyl){phenyl)amino]carbonyl}amino)benzamide; 2,4-difluoro-5-{[((3-fluorophenyl){1-[(2-methyl-6-{[4-(methylsulfonyl)phenyl]amino}-3- pyridiπyl)methyl]-4-piperidinyl}amino)carbonyl]amino}benzamide; 5-({[{1-[(6-{[4-({[2-(ethyloxy)ethyl]amino}carbonyl)-2-methylphenyl]amino}-2-methyl-3- pyridinyl)methyl]-4-piperidinyl}(3-fluorophenyl)amino]carbonyl}amino)-2,4- difluorobenzamide;

Λ/-(4-[[5-({4-[{[(4-chlorophenyl)amino]carbonyl}(phenyl)amino]-1-piperidinyl}methyl)-2- pyridinyl]thio}phenyl)methanesulfonamide; 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, naphthaIene-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 C1-4 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 C1-4alkyl 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 (HPMPC), [[[2-(6-amino-ΘH-purin-9- yl)ethoxy]methyl]phosphinylidene]bis(oxymethylene)-2,2-dimethylpropanoic acid (bis- POWI PWlEA, 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]phosphonic 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), (-)-cis-1-(2-hydroxymethyl)-1 ,3-oxathiolane 5-yl)-cytosine (lamivudine), cis-1-(2-(hydroxymethyi)-1 ,3-oxathiolan-5-yl)-5-fluorocytosine (FTC), (- )-cis-4-[2-amino-6-(cyclopropylamino)-9H-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- interferon, immunomodulators such as interleukin Il or thymosin, granulocyte macrophage colony stimulating factors, erythropoetin, soluble CD4 and genetically engineered derivatives thereof, non-nucleoside reverse transcriptase inhibitors (NNRTIs), for example nevirapine (BI-RG-587), alpha-((2-acetyl-5- methylphenyl)amino)-2,6-dichloro-benzeneacetamide (loviride), 1 -[3- (isopropylamino)-2-pyridyl]-4-[5-(methanesulfonamido)-1 H-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, iπtegrase inhibitors, or fusion inhibitors, for example T-20 and T-1249.

The present invention further includes the use of a compound according to the invention in the manufacture of a medicament for simultaneous or sequential administration with at least another therapeutic agent, such as those defined hereinbefore.

Compounds of the present invention may be administered with an agent known to inhibit or reduce the metabolism of compounds, for example ritonavir. Accordingly, the present invention features a method for the treatment or prophylaxis of a disease as hereinbefore described by administration of a compound of the present invention in combination with a metabolic inhibitor. Such combination may be administered simultaneously or sequentially.

In general a suitable dose for each of the above-mentioned conditions will be in the range of 0.01 to 250 mg per kilogram body weight of the recipient (e.g. a human) per day, preferably in the range of 0.1 to 100 mg per kilogram body weight per day and most preferably in the range 0.5 to 30 mg per kilogram body weight per day and particularly in the range 1.0 to 20 mg per kilogram body weight per day. Unless otherwise indicated, all weights of active ingredient are calculated as the parent compound of formula (I); for salts or esters thereof, the weights would be increased proportionally. The desired dose may be presented as one, two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. In some cases the desired dose may be given on alternative days. These sub-doses may be administered in unit dosage forms, for example, containing 10 to 1000 mg or 50 to 500 mg, preferably 20 to 500 mg, and most preferably 50 to 400 mg of active ingredient per unit dosage form.

While it is possible for the active ingredient to be administered alone it is preferable to present it as a pharmaceutical composition. The compositions of the present invention comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof and optionally other therapeutic agents. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the composition and not injurious to the patient.

Phamaceutical compositions include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and intravitreal) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods represent a further feature of the present invention and include the step of bringing into association the active ingredients with the carrier, which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

The present invention further includes a pharmaceutical composition as hereinbefore defined wherein a compound of the present invention or a pharmaceutically acceptable derivative thereof and another therapeutic agent are presented separately from one another as a kit of parts.

Compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Such patches suitably contain the active compound 1) in an optionally buffered, aqueous solution or 2) dissolved and/or dispersed in an adhesive or 3) dispersed in a polymer. A suitable concentration of the active compound is about 1% to 25%, preferably about 3% to 15%. As one particular possibility, the active compound may be delivered from the patch by electrotransport or iontophoresis as generally described in Pharmaceutical Research '3 (6), 318 (1986).

Pharmaceutical compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, caplets, cachets or tablets each containing a predetermined amount of the active ingredients; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-iπ-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, disintegrant (e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Molded tablets may be made by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredients therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

Pharmaceutical compositions suitable for topical administration in the mouth include lozenges comprising the active ingredients in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Pharmaceutical compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray Pharmaceutical compositions containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Pharmaceutical compositions for rectal administration may be presented as a suppository with a suitable carrier comprising, for example, cocoa butter or a salicylate or other materials commonly used in the art. The suppositories may be conveniently formed by admixture of the active combination with the softened or melted carrier(s) followed by chilling and shaping in molds.

Pharmaceutical compositions suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain 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 9 and 10 at Tables 1 and 2, 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 1 δ-Cflbutylfi-lp-methyl-e-KΦKmethylsulfonyOaminolphenylMhioJ-a-pyridinyOmethylM- piperidinyl)amino]carbonyl}amino)-2,4-difluorobenzamide

The synthesis of intermediate 8

Intermediate 1 2,4-difluoro-5-nitrobeπzoic acid

Nitric Acid (120 mL) was added slowly to a cold solution of sulfuric acid (200 mL). 2,4- difluorobenzoic acid (65 g, 0.41 mol, 1 equiv) was added portionwise over 1.5 h and the reaction allowed to warm to RT. The reaction mixture was heated to 50 ⁰C for 2h and then poured slowly onto ice. The product was extracted into ethyl acetate, dried over

Na₂SO₄, filtered and concentrated to give intermediate 1 (82 g, 98% yield) as a yellow solid.

¹H NMR (400 MHz, DMSO-d_β) δ ppm 8.56 (t, J=7.87 Hz, 1 H), 7.82 (t, J=10.71 Hz, 1 H).

Intermediate 2 methyl 2,4-difluoro-5-nitrobenzoate

Sulfuric acid (3 mL) was added to a solution of 2,4-difluoro-5-nitrobenzoic acid intermediate 1 (100 g, 0.49 mol, 1 equiv) in methanol (700 mL) and the reaction heated to reflux for 18 h. The reaction mixture was concentrated and the residue taken up in water and brought to pH 8 by addition of 1N NaOH. The product was extracted into ethyl acetate, dried over Na₂SO₄, filtered and concentrated. The residue was purified by silica gel flash column chromatography (0 → 25% EtOAc: Hexanes) to give intermediate 2 (90 g, 85% yield) as a tan solid.

¹H NMR (400 MHz, DMSO-Cf₆) δ ppm 8.56 - 8.64 (m, 1 H), 7.83 - 7.94 (m, 1 H), 3.85 - 3.92 (m, 3 H).

Intermediate 3 methyl 5-amino-2,4-difluorobenzoate

Iron powder (12.3 g, 0.22 mol, 4 equiv) was added to a solution of methyl 2,4-difluoro-5- nitrobenzoate intermediate 2 (12.0 g, 0.06 mol, 1 equiv) in acetic acid (150 mL) and water (150 mL) and the reaction was heated to 40⁰C for 1.5 h. The reaction mixture was filtered thru a pad of celite and the filtered solution extracted with ethyl acetate. The organics were washed with satd. aq. NaCI, dried over Na₂SO₄, filtered and concentrated to give intermediate 3 (10.Og, 96% yield) as a brown solid.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.27 (dd, J=9.98, 7.42 Hz, 1 H), 7.14 (t, J=10.99 Hz, 1 H), 5.28 (s, 2 H), 3.77 (s, 3 H). ES-LCMS: m/z 189.0 (M+H).

Intermediate 4

1 ,1-dimethylethyl 4-(butylamino)-1-piperidinecarboxylate

Sodium triacetoxyborohydride (88.5 g, 0.42 mol, 1.6 equiv) was added portioπwise over 1 h to a solution of 4-BOC-piperidone (52.0 g, 0.26 mol, 1 equiv), π-butyl amine (30.9 ml_, 0.31 mol, 1.2 equiv) and acetic acid (44.7 ml_, 0.78 mol, 3 equiv) in dichloromethane (700 ml_) at 0⁰C. The reaction mixture was allowed to warm to RT over 2 h and then slowly poured into cold 3.3N NaOH (1 L) with stirring. The reaction mixture was then separated and the organics dried over Na₂SO₄, filtered and concentrated to give intermediate 4 (66 g, 98% yield) as an oil.

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 4.03 (s, 2 H), 2.77 (t, J=11.99 Hz, 2 H), 2.52 - 2.66 (m, 3 H), 1.82 (d, J=11.90 Hz, 2 H), 1.39 - 1.52 (m, 11 H), 1.29 - 1.39 (m, 2 H), 1.16 - 1.29 (m, 2 H), 1.06 (s, 1 H), 0.84 - 0.96 (m, 3 H).

Intermediate 5

1 ,1-dimethylethyl 4-{butyl[({2,4-difluoro-5-

[(methyloxyjcarbonyllphenyljaminojcarbonyllaminoj-i-piperidinecarboxylate

A solution of methyl 5-amino-2,4-difluorobenzoate intermediate 3 (5.5 g, 29.5 mmol, 1 equiv) in dichloromethane (100 mL) at -40 ⁰C was treated with pyridine (7.16 mL, 88.6 mmol, 3.0 equiv) and 1.9 M phosgene in toluene (23.3 mL, 44.3 mmol, 1.5 equiv). After 1.5 h, a solution of 1,1-dimethylethyl 4-(butylamino)-1-piperidiπecarboxylate intermediate 4 (9.1 g, 35.4 mmol, 1.2 equiv) in dichloromethane (50 mL) was added and the reaction was allowed to warm to RT for 18 h. The reaction was diluted with dichloromethane, washed with satd. aq. NaHCO₃, dried over Na₂SO₄, filtered and concentrated to give intermediate 5 (13.1 g, 95% yield) a white solid.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.09 (s, 1 H), 7.89 - 7.99 (m, 1 H), 7.42 (t, J=10.62 Hz, 1 H), 3.88 - 3.99 (m, 1 H), 3.85 (s, 3 H), 3.11 - 3.22 (m, 2 H), 2.96 (d, J=12.27 Hz, 2 H), 2.41 - 2.53 (m, 3 H), 1.43 - 1.59 (m, 5 H), 1.21 - 1.35 (m, 2 H), 1.20 (s, 9 H), 0.90 (t, J=7.33 Hz, 3 H). ES-LCMS: m/z 468.1 (M-H).

Intermediate 6

5-({[butyl(Ht(1 ,1-dimethylethyl)oxy]carbonyl}-4-piperidinyl)amino]carbonyl}amino)-2,4- difluorobenzoic acid

A solution of 1 ,1-dimethylethyl 4-{butyl[({2,4-difluoro-5- [(methyloxy)carbonyl]phenyl}amino)carbonyl]amino}-1-piperidinecarboxylate intermediate 5 (13.0g, 27.7 mmol, 1 equiv) and 1N lithium hydroxide (15 mL) in tetrahydrofuran (30 mL) was stirred at RT for 2 h. The reaction was concentrated down and then acidified to pH 2 by addition of 1 N hydrochloric acid. The aqueous layer was extracted with ethyl acetate and the organics dried over Na₂SC^, filtered and concentrated to give intermediate 6 (12.0 g, 95% yield) as a white solid. 1H NMR (400 MHz, DMSO-d_B) δ ppm 8.09 (s, 1 H), 7.81 - 7.91 (m, 1 H), 7.29 - 7.38 (m, 1 H), 3.93 - 4.07 (m, 1 H), 3.24 - 3.38 (m, 2 H), 3.09 - 3.20 (m, 2 H), 2.72 (d, J=6.97 Hz, 2 H), 1.52 - 1.65 (m, 4 H), 1.42 - 1.51 (m, 2 H), 1.34 - 1.41 (m, 9 H), 1.19 - 1.33 (m, 2 H), 0.87 (t, J=7.33 Hz, 3 H). ES-LCMS: m/z 454.43 (M-H).

Intermediate 7

1 , 1 -dimethylethyl 4-[({[5-(aminocarbonyl)-2,4- difluorophenyl]amino}carbonyl)(butyl)amino]-1-piperidinecarboxylate

A solution of 5-({[butyl(1-{[(1 ,1-dimethylethyl)oxy]carbonyl}-4- piperidiπyl)amino]carbonyl}amino)-2,4-difluorobenzoic acid intermediate 6 (12.0, 26.3 mmol, 1 equiv) in Λ/,Λ/-dimethylformamide (100 mL) was treated with HATU (10.0 g, 26.3 mmol, 1 equiv) followed by Λ/,Λ/-diisopropylethylamine (9.2mL, 52.6 mmol,

2 equiv). The reaction was stirred at RT for 15 min before the addition of ammonia hydroxide (2.6 mL, 39.4 mmol, 1.5 equiv). The reaction was stirred at RT for 2 h and then partitioned between ethyl acetate and water. The organics were washed with satd. aq. NaHCO₃, dried over Na₂SO₄, filtered and concentrated. The residue was purified by silica gel flash column chromatography (0 → 5% 2N NH₃ in MeOH : CH₂CI₂) to give intermediate 7 (10.1 g; 80% yield) as a white solid.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.04 (s, 1 H), 7.55 - 7.70 (m, 3 H), 7.29 (t, J=10.35 Hz, 1 H), 3.98 (s, 3 H), 3.24 - 3.29 (m, 1 H), 3.03 - 3.19 (m, 2 H), 2.57 - 2.79 (m, 2 H), 1.50 - 1.65 (m, 3 H), 1.45 (s, 2 H), 1.37 (s, 9 H), 1.16 - 1.30 (m, 2 H), 0.86 (t, J=7.23 Hz,

3 H). ES-LCMS: m/z 453.4 (M-H).

Intermediate 8 5-({[butyl(4-piperidinyl)amino]carbonyl}amino)-2,4-difluorobenzarnide

A solution of 1,1-dimethylethyl 4-[({[5-(aminocarbony|)-2,4- difluorophenyl]amino}carboπyl)(butyl)amino]-1-piperidinecarboxylate intermediate 7 (3.0 g, 6.60 mmol, 1 equiv) in 4N HCI in dioxaπe (16.5 mL, 66.0 mol, 10 equiv) was stirred at RT for 2 h and concentrated to a yellow solid. The solid was dissolved in water and washed with ethyl acetate. The aqueous layer was made basic (pH 8) by addition of 1N NaOH and then extracted with dichloromethane. The organics were dried over Na₂SO₄, filtered and concentrated to give intermediate 8 (1.9g, 81% yield) as a yellow solid.

¹H NMR (400 MHz, DMSO-Cf₆) δ ppm 8.00 (s, 1 H), 7.58 - 7.72 (m, 3 H), 7.30 (t, J=10.45 Hz, 1 H), 3.86 - 3.97 (m, 1 H), 3.57 - 3.62 (m, 1 H), 3.30 (s, 2 H), 3.10 - 3.19 (m, 2 H), 2.90 - 3.00 (m, 2 H), 2.39 - 2.47 (m, 1 H), 1.69 - 1.79 (m, 1 H), 1.41 - 1.58 (m, 4 H), 1.20 - 1.34 (m, 2 H), 0.88 (t, J=7.33 Hz, 3 H). ES-LCMS: m/z 355.1 (M+H).

Intermediate 10 θ-chloro^-methyl-S-pyridinecarbaldehyde

Dibal-H

10

At 0 ⁰C, to a stirred solution of 6-chloro-2-methyl-3-pyridinecarbonitrile ([Singh et al., (1991) Synthesis pp. 894-896) (1.53g, lOmmol) in dichloroππethane (3OmL) was added dibal-H (12mL, 1Wl in CH2C12). The resulting mixture was stirred for one hour before it was quenched with saturated citric acid solution. Stirring was continued for additional one hour. The reaction mixture was then diluted with dichloromethane (10OmL) and washed with saturated sodium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate. After evaporation of solvents, the crude product was purified by column chromatography (silical gel, 0 to 30% ethyl acetate in hexane) to afford 950mg (61%) of 6-chloro-2-methyl-3-pyridiπecarbaldehyde. 1 H NMR (400 MHz, CHLOROFORM-d) δ ppm 10.28 (s, 1 H), 8.04 (d, J=8.07 Hz, 1 H), 7.33 (d, J=8.07 Hz, 1 H), 2.84 (s, 3 H).

-0

Intermediate 9 ^DMF intermediate 11

Preparation of intermediate 9, N-(4-mercaptopheπyl)methaπesulfonamide

At O⁰C, to a stirred solution of {4-[(4-aminophenyl)dithio]phenyl}amiπe(2.5g, lOmmol) in dichloromethane (10OmL) and pyridine (1.74g, 22 mmol) were slowly added methanesulfonyl chloride (2.3g, 20mmol) and 4-N,N-dimethylaminopyridine (catalyst). The resulting mixture was stirred overnight at room temperature. The products were precipitated and collected by filtration as pink solid. LC-MS indicated a mixture of N, N'-dimesylated product (53%) and mono-mesylated product (47%). The newly generated solid above was dissolved in 10OmL of methanol. To this solution under nitrogen atmosphere was added sodium borohydride (2.3g, ΘOmmol) at O⁰C portionwise. After addition, the reaction was warmed up to the room temperature and allowed for an additional 4-hour stirring before quenched with saturated sodium bicarbonate solution. The mixture was then concentrated down to half of its volume and the solid was filtered off through celite. The filtrate was extracted with dichloromethane (3X 5OmL)). The combined organic phases were dried over anhydrous sodium sulfate. After evaporation of solvents, N-(4- mercaptophenyl)methanesulfonamide intermediate 9 was obtained as solid (1.Og , 25% over two steps). 1 H NMR (400 MHz, DMSO-d6) δ ppm 9.62 (s, 1 H), 7.24 (m, 2 H), 7.08 (m, 2 H), 2.93 (s, 3 H).

Preparation of intermediate 11, N-{4-[(5-formyl-6-methyl-2-pyridinyl)thio]phenyl} methaπesulfonamide

To a stirred solution of N-(4-mercaptophenyl)methanesulfonamide intermediate 9 (1.0g,4.9mmol) in N, N-dimethylformamide (3OmL) were added 6-chloro-2-methyl-3- pyridinecarbaldehyde intermediate 10 (0.763g, 4.9mmol) and cesium carbonate (1.6g, 4.9mmol)). The resulting mixture was heated to 60⁰C for two hours. After completion of the reaction, the mixture was diluted with ethyl acetate (10OmL) and washed with sodium bicarbonate solution. The organic phase was then dried over anhydrous sodium sulfate. After evaporation of solvents, the crude product was purified by column chromatography (silica gel, 0 to 50% ethyl acetate in hexane) to give 844 mg (53%) of N-{4-[(5-formyl-6-methyl-2- pyridinyl)thio]phenyl}methanesulfonamide intermediate 11 as solid. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 10.21 (s, 1 H), 7.83 (d, J=8.43 Hz, 1 H), 7.60 (m, 2 H), 7.31 (m, 2 H), 7.02 (s, 1 H), 6.75 (d, J=8.43 Hz, 1 H), 3.12 (s, 3 H), 2.80 (s, 3 H).

5-({[butyi(1-{[2-methyl-6-({4-[(methylsulfonyl)amino]pheπyl}thio)-3-pyridinyl]methyl}-4- piperidinyl)amino)carbonyl}amino)-2,4-difluorobenzamide, example 1

At 0 ⁰C, to a stirred solution of 5-({[butyl(4-piperidinyl)amino]carbonyl}amino)-2,4- difluorobenzamide intermediate B (117mg, 0.33mmol) in N, N-dimethylformamide were added intermediate 11 N-{4-{(5-formyl-6-methyl-2- pyridinyl)thio]phenyl}methanesulfonamide (100mg, 0.31mmol), sodium triacetoxylborohydride (95mg, 0.45mmol) and acetic acid. The resulting mixture was stirred while naturally warming up to the room temperature overnight. After the completion of the reaction, the mixture was diluted with ethyl acetate (10OmL) and the washed with saturated sodium bicarbonate solution. The organic phase was dried over sodium sulfate. After evaporation of the solvents, the crude product was purified by column chromatography (silical gel, 0 to 60% CH2CI2 mixed withi 0%MeOH/2 M NH3 in CH2CI2) to give 70 mg (32%) of ( 5-({[butyl(H[2-methyl-6-({4-

[(methylsulfonyl)amino]phenyl}thio)-3-pyridinyl]methyl}-4- piperidinyl)amino]carbonyl}amino)-2,4-difluorobenzamide (70 mg, 32%) as solid. 1H NMR (400 MHz, METHANOL-d4) δ ppm 7.86 (m, 1 H), 7.54 (m, 2 H), 7.48 (d, J=8.06 Hz, 1 H), 7.33 (m, 2 H), 7.12 (m, 1 H), 6.68 (d, J=7.70 Hz, 1 H), 4.02 (m, 1 H), 3.45 (s, 2 H), 3.24 (m, 2 H), 3.03 (s, 3 H), 2.52 (s, 3 H), 2.15 (m, 2 H), 1.80 (m, 2 H), 1.72 (m, 2 H), 1.59-(m, 2 H), 1.36 (m, 2 H), 0.96 (m, 3 H). HRMS m/z (M+H)+ calcd: 661.2442, obsd: 661.2466.

Example 2

2,4-difluoro-5-({[(3-fluorophenyl)(1-{[2-methyl-6-({4-[(methylsuifonyl)amino] pheπyl}thio)-3-pyridiny!]methyl}-4-piperidinyl)amino]carbonyl}amino)benzamide

Synthesis of Intermediate 16

Intermediate 12

1 , 1 -dimethylethyl 4-[(3-fluorophenyl)amino]-1 -piperidinecarboxylate

Sodium triacetoxyborohydride (79.7 g, 0.37 mol, 1.5 equiv) was added portioπwise over 1 h to a solution of 4-BOC-piperidoπe (50.0 g, 0.25 mol, 1 equiv), 3-fluoroaniliπe (28.9 mL, 0.30 mol, 1.2 equiv) and acetic acid (86.2 mL, 1.51 mol, 6 equiv) in dichloroethane ( 700 mL) at 0 ⁰C. The reaction mixture was allowed to warm to RT over 2 h and then slowly poured into cold 3.3N NaOH (1 L) with stirring. The reaction mixture was then separated and the organics dried over Na_∑SO^ filtered and concentrated to give a tan solid. The solid was suspended in 3:1 hexanes:ethyl acetate, stirred overnight and filtered to give 12 (67 g, 91% yield) as a white solid.

1 H NMR (400 MHz, DMSO-c/₆) 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 13

1 , 1 -dimethylethyl 4-[[({2,4-difluoro-5-[(methyloxy)carbonyl]phenyl}amino)carbonyl](3- fluoropheny!)amino]-1-piperidinecarboxylate

A solution of methyl 5-amiπo-2,4-difluorobenzoate intermediate 3 (5.0 g, 26.5 mmol, 1 equiv) in dichloromethane (150 mL) at -40⁰C was treated with pyridine (6.4 ml_, 79.7 mmol, 3.0 equiv) and 1.9 M phosgene in toluene (20.1 mL, 39.8 mmol, 1.5 equiv). After 1.5 h, the reaction was concentrated down and dried under vac. The residue was taken up in dichloromethane (150 mL) and 1 ,1-dimethylethyl 4-[(3-fluorophenyl)amino]-1- piperidinecarboxylate intermediate 12 (8.6 g, 29.2 mmol, 1.1 equiv) was added and the reaction was heated to 40 ⁰C 18 h. The reaction was diluted with dichloromethane, washed with satd. aq. NaHCO₃, dried over Na₂SO₄, filtered and concentrated. The residue was purified by silica gel flash column chromatography (0 → 30% ethyl acetate : hexanes) to give intermediate 13 (12.6 g, 94% yield) a white solid. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.65 (dd, J=8.88, 7.78 Hz, 1 H), 7.44 - 7.55 (m, 1 H), 7.17 - 7.24 (m, 1 H), 7.04 (d, J=7.87 Hz, 1 H), 6.92 - 6.99 (m, 1 H), 6.80 (t, J=10.26 Hz, 1 H), 5.96 (d, J=3.11 Hz, 1 H), 4.56 - 4.70 (m, 1 H), 4.03 - 4.26 (m, 2 H), 3.80 - 3.92 (m, 3 H), 2.79 (s, 2 H), 1.85 (d, J=13.37 Hz, 2 H), 1.39 (s, 9 H), 1.19 - 1.35 (m, 2 H). ES-LCMS: rn/z 530.1 (M+Na).

Intermediate 14

5-({[(1 -{[(1 , 1 -dimethylethyOoxylcarbonylM-pipeiϊdiriylxs- fluorophenylJaminolcarbony^amiπoJ-Σ^-difluorobenzoic acid

A solution of 1,1-dimethylethyl 4-[[({2,4-difluoro-5- [(methyloxy)carbonyl]phenyl}amino)carbonyl](3-fluorophenyl)amino]-1- piperidinecarboxylate intermediate 13 (12.6 g, 24.9 mmol, 1 equiv) and 1N lithium hydroxide (50 mL) in tetrahydrofuran (200 mL) was stirred at RT for 72 h. The reaction was concentrated down and then acidified to pH 3 by addition of 1N hydrochloric acid. The aqueous layer was extracted with ethyl acetate and the orgaπics dried over Na₂SO₄, filtered and concentrated to give intermediate 14 (10.6 g, 86% yield) as a white solid. ¹H NMR (400 MHz, DMSO-cf_e) δ ppm 7.89 (t, J=8.42 Hz, 1 H), 7.43 - 7.52 (m, 1 H), 7.22 - 7.33 (m, 3 H), 7.13 - 7.20 (m, 1 H), 7.05 - 7.12 (m, 1 H), 4.29 - 4.40 (m, 1 H), 3.86 - 3.97 (m, 2 H), 2.72 (s, 2 H), 1.76 (s, 2 H), 1.24 - 1.32 (m, 9 H), 1.00 - 1.16 (m, 2 H). ES- LCMS: m/z 492.1 (M-H).

Intermediate 15

1,1-dimethylethyl 4-[({[5-(aminocarbonyl)-2,4-difluorophenyl]amino}carbonyl)(3- fluorophenyl)amino]-1-piperidinecarboxylate

A solution of 5-({[(1-{[(1 ,1-dimethylethyl)oxy]carbonyl}-4-piperidinyl)(3- fluorophenyl)amino]carbonyl}amino)-2,4-difluorobenzoic acid intermediate 14 (6.6, 13.3 mmol, 1 equiv) in Λ/,Λ/-dimethylformamide (100 ml_) was treated with HATU (5.0 g, 13.3 mmol, 1 equiv) followed by Λ/,/V-diisopropylethylamine (4.6 mL, 36.7 mmol, 2 equiv). The reaction was stirred at RT for 15 min before the addition of ammonia hydroxide (1 3 mL, 20.0 mmol, 1.5 equiv). The reaction was stirred at RT for 4 h and then partitioned between ethyl acetate and water. The organics were washed with satd. aq. NaHCθ₃, dried over Na₂SO₄, filtered and concentrated. The residue was purified by silica gel flash column chromatography (0 → 10% 2N NH₃ in MeOH : CH₂CI₂) to give intermediate 15 (5.8 g; 88% yield) as a tan solid.

¹H NMR (400 MHz, DMSO-Cf₆) δ ppm 7.68 (t, J=8.42 Hz, 1 H), 7.60 (s, 2 H), 7.44 - 7.54 (m, 1 H), 7.23 - 7.32 (m, 3 H), 7.13 - 7.19 (m, 1 H), 7.09 (dd, J=7.87, 0.92 Hz, 1 H), 4.29 - 4.42 (m, 1 H), 3.86 - 3.99 (m, 2 H), 2.79 (s, 2 H), 1.78 (d, J=11.35 Hz, 2 H), 1.30 (s, 9 H), 1.02 - 1.16 (m, 2 H). ES-LCMS: m/z 515.3 (M+Na).

Intermediate 16 2,4-difluoro-5-({[(3-fluorophenyl)(4-piperidinyl)amino]carbonyl}amino)benzamide

A solution of 1 ,1-dimethylethyl 4-[({[5-(aminocarbonyl)-2,4- difluorophenyl]amino}carbonyl)(3-fluoropheπyl)amiπo]-1-piperidinecarboxylate intermediate 15 (5.8 g, 11.7 mmol, 1 equiv) in 4N HCI in dioxane (29.0 ml_, 117.7 mmoi, 10 equiv) was stirred at RT for 2 h and concentrated to a yellow solid. The solid was dissolved in water and washed with ethyl acetate. The aqueous layer was made basic (pH 8) by addition of 1N NaOH and then extracted with dichloromethane. The organics were dried over Na₂SO₄, filtered and concentrated to give intermediate 16 (4.1g, 89 % yield) as a yellow solid

¹H NMR (400 MHz, DMSO-αtø δ ppm 7.67 - 7.74 (m, 1 H), 7.60 (s, 2 H), 7.45 - 7.54 (m, 1 H), 7.24 - 7.31 (m, 2 H), 7.21 (s, 1 H), 7.05 - 7.16 (m, 2 H), 4.20 - 4.33 (m, 1 H), 3.29 (s, 1 H), 2.82 - 2.92 (m, 2 H), 2.38 - 2.50 (m, 2 H), 1.69 (dd, J=11.54, 1.65 Hz, 2 H), 1.00 - 1.14 (m, 2 H). ES-LCMS: m/z 393.3 (M+H).

2,4-Difluoro-5-({[(3-fluorophenyl)(1-{[2-methyl-6-({4-[(methylsulfonyl)amino] phenyl}thio)-3-pyridinyl]methyl}-4-piperidinyl)amino]carbonyl}amino)benzamide (103mg, 41%) was obtained as solid from intermediate 11 N-{4-[(5-formyl-6-methyl- 2-pyridinyl)thio]pheπyl}methanesulfonamide (100mg, 0.31 mmol) and intermediate 16 2,4-difluoro-5-({[(3-fluorophenyl)(4-piperidinyl)amino]carbonyl}amino)benzamide (129mg, 0.33mmol) and sodium triacetoxyborohydride (95mg, 0.45mmol) in N, N- dimethylformamide following the procedure outlined in example 1. 1 H NMR (400 MHz, METHANOL-d4) δ ppm 7.97 (m, 1 H), 7.52 (m, 4 H), 7.39 (d, J=8.07 Hz, 1 H), 7.28 (m, 4 H), 7.10 (m, 4 H), 6.63 (d, J=8.07 Hz, 1 H), 4.38 (m, 1 H), 3.39 (s, 2 H), 3.02 (s, 3 H), 2.85 (d, J=12.10 Hz, 2 H), 2.45 (s, 3 H), 2.14 (m_r2 H), 1.84 (m, 2 H), 1.41 (m, 2 H). HRMS m/z (M+H)+ calcd: 699.2035, obsd: 699.2045.

Example 3 2,4-difluoro-5-({[(1-{[2-methyl-6-({4-[(methylsulfonyl)amino]phenyi}thio)-3- pyridinyl]methyl}-4-piperidinyl)(phenyl)amiπo]carbonyl}amino)benzamide

The synthesis of intermediate 21

Intermediate 17

1 ,1-dimethylethyl 4-(phenylamino)-1-piperidiπecarboxylate

A mixture of 4-Boo-piperidone (50.0 g, 0.25 mol, 1 equiv) and aniline (22.8 mL, 0.25 mol, 1 equiv) in dichloroethane (400 mL) was treated with titanium (IV) isopropoxide (92.8 mL, 0.31 mol, 1.25 equiv) and heated to 80 OC for 4 h. The reaction was concentrated down and the residue taken up in ethanol (600 mL) and cooled to 0 OC. 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 OC. 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 intermediate 17 (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=7.33 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 18 terf-butyl 4-[({[2,4-difluoro-5-

(methoxycarbonylJphenyπaminoJcarbonylXphenyOaminolpiperidine-i-carboxylate

Intermediate 18 was prepared from intermediates 3 and 17 as described for intermediate

5.

¹H NMR (400 MHz, DMSOd₆) δ ppm 8.06 (d, J=8.06 Hz, 1 H) 7.40 - 7.53 (m, 3 H) 7.35

(t, J=10.71 Hz, 1 H) 7.26 (dd, J=8.15, 1.37 Hz, 2 H) 7.02 (s, 1 H) 4.34 - 4.50 (m, 1 H)

3.98 (d, J=6.04 Hz, 2 H) 3.81 (s, 3 H) 2.74 (br. s., 2 H) 1.76 (d, J=1.47 Hz, 2 H) 1.29 (s,

9 H) 1.07 (qd, 2 H). ES-LCMS: m/z 490.4 (M+H).

Intermediate 19

5-({[[1-(tert-butoxycarboπyl)piperidin-4-yl](phenyl)amino]carbonyl}amino)-2,4- difluorobenzoic acid

Intermediate 19 was prepared using chemistry identical to that described for intermediate 6.

¹H NMR (400 MHz, DMSOd₆) δ ppm 8.03 (t, J=8.52 Hz, 1 H) 7.39 - 7.52 (m, 3 H) 7.23 ■ 7.34 (m, 3 H) 6.99 (s, 1 H) 4.39 (tt, J=12.00, 3.66 Hz, 1 H) 3.80 - 4.00 (m, 2 H) 2.64 (d, J=2.01 Hz, 2 H) 1.78 (d, J=10.62 Hz, 2 H) 1.29 (s, 9 H) 0.98 - 1.14 (m, J=12.50, 12.38, 12.38, 4.49 Hz, 2 H). ES-LCMS: m/z 476.6 (M+H).

Intermediate 20 tert-butyl 4-[({[5-(aminocarbonyl)-2,4- difluorophenyπaminoJcarbonylHphenylJaminolpiperidine-i-carboxylate

Intermediate 20 was prepared in a manner identical to that described for intermediate 7. ¹H NMR (400 MHz, DMSOd₆) δ ppm 7.75 - 7.86 (m, 1 H) 7.55 - 7.64 (m, 2 H) 7.47 (d, J=7.51 Hz, 3 H) 7.25 (dd, J=6.04, 2.01 Hz, 3 H) 6.94 - 7.01 (m, 1 H) 4.30 - 4.47 (m, 1 H) 3.83 - 4.01 (m, 2 H) 2.64 - 2.85 (m, 2 H) 1.69 - 1.87 (m, 2 H) 1.29 (s, 9 H) 1.07 (m, 2 H). ES-LCMS: m/z 497.3 (M+Na).

Intermediate 21 2,4-difluoro-5-({[phenyl(piperidin-4-yl)amino]carbonyl}amino)benzamide

Intermediate 21 was prepared identically to chemistry described towards the intermediate 16.

¹H NMR (400 MHz, DMSOd₆) δ ppm 7.84 (td, J=5.68, 2.75 Hz, 1 H) 7.60 (br. s., 2 H) 7.38 - 7.54 (m, 3 H) 7.17 - 7.32 (m, J=7.65, 5.06, 5.06, 2.56 Hz, 3 H) 6.86 (br. s, 1 H) 4.30 (d, J=3.11 Hz, 1 H) 3.54 (spt, J=2.59 Hz, 2 H) 2.86 (d, J=11.72 Hz, 2 H) 1.86 (br. s., 1 H) 1.67 (br. s., 2 H) 1.07 (dt, J=6.00, 2.95 Hz, 2 H)ES-LCMS: m/z 375.4 (M+H).

Example 3: 2,4-Difluoro-5-({[(1 -{[2-methyl-6-({4-[(methylsulfonyl)amino]phenyl}thio)- 3-pyridinyl]methyl}-4-piρeridinyl)(phenyl)amino]carbonyl}amino)benzarnide (35mg, 33%) was obtained as solid from intermediate 11 N-{4-[(5-formyI-6-methyl-2- pyridinyl)thio]phenyl}methanesulfonamide (50mg, 0.155mmo!) and intermediate 21 2,4-difluoro-5-({[phenyl(4-piperidinyl)amiπo]carbonyl}amino)benzamide hydrochloride (63mg, 0.155mrnol) and sodium triacetoxylborohydride (51mg, 0.23mmo!) in N, N- dimethylformamide following the procedure outlined in example 1. 1 H NMR (400 MHz, METHANOL-d4) δ ppm 8.08 (m, 1 H), 7.51 (m, 5 H), 7.37 (d, J=8.06 Hz, 1 H), 7.31 (m, 4 H), 7.04 (t, J=10.26 Hz, 1 H), 6.61 (d, J=8.07 Hz, 1 H), 4.40 (m, 1 H), 3.36 (m, 2 H), 3.02 (s, 3 H), 2.84 (m, 2 H), 2.42 (s, 3 H), 2.13 (m, 2 H), 1.83 (d, J=11.00 Hz, 2 H), 1.38 (dd, J=12.46, 3.30 Hz, 2 H). HRMS m/z (M+H)+ calcd: 681.2129, obsd: 681.2132.

Example 4

2,4-difluoro-5-{[((3-fluorophenyl){1-[(2-methyl-6-{[4-(methylsulfonyl)phenyl]amino}-3- pyridinyl)methyl]-4-piperidinyl}amino)carbonyl]amino}benzamide

Example 4 was prepared according to the scheme below.

Preparation of 2-methyl-6-{[4-(rnethylsulfonyl)phenyl]amino}-3- pyridinecarbonitrile, intermediate 24

6-Chloro-2-methyl-3-pyridinecarbonitrile (153mg, 1mmol) and 4- (methylsulfonyl)aniliπe hydrochloride (207 mg, 1mmol) were mixed in a sealed tube followed by additions of cesium carbonate (975mg, 3mmol), Pd2(dba)3 (52 mg, 0.05mmol), 2-dicyclohexylphosphino-2'-(N, N'-dimethylamino)biphenyl (40mg, O.immol) and dimethoxyethane (5mL). The resulting mixture was purged with N2 (3X) before heated to 80 ⁰C overnight. After cooled downed to room temperature, the reaction mixture was diluted with ethyl acetate (10OmL) and washed with saturated sodium bicarbonate solution. The organic phase was then dried over anhydrous sodium sulfate. Evaporation of solvents gave the crude product which was purified by column chromatography (silical gel, 0 to 70% ethyl acetate in hexaπe) to afford 40 mg of 2-methyl-6-[[4-(methylsulfonyl) phenyl]amino}-3-pyridinecarbonitrile intermediate 22 as solid. 1 H NMR (400 MHz, DMSO-d6) δ ppm 7.94 (m, 3 H), 7.83 (m, 2 H), 6.83 (d, J=8.80 Hz, 1 H), 3.14 (s, 3 H), 2.58 (s, 3 H). LC-MS m/z (M+H)+ calcd: 288, obsd: 288.1 Preparation of 2-methyl-6-{[4-(methylsulfoπyl)phenyl]amino}-3-pyridinecarbaldehyde, intermediate 23

At 0 °C, to a stirred solution of 2-methyl-6-{[4-(methylsulfoπyl)phenyl]amino}-3- pyridinecarboπitrile intermediate 22 (40mg, 0.13mmol) in dichloromethane (15mL) was added dibal-H (0.4mL, 1M in CH2CI2). The resulting mixture was stirred for one hour before it was quenched with saturated citric acid solution. Stirring was continued for additional one hour. The reaction mixture was diluted with dichloromethane (5OmL) and washed with saturated sodium bicarbonate solution. The organic phase was then dried over anhydrous sodium sulfate. After evaporation of solvents, 2-methyl-6-{[4-(rnethylsulfonyl)phenyl] amino}-3-pyridinecarbaldehyde intermediate 23 was obtained and used for the next step directly. LC-MS m/z (M+H)+ calcd: 291 , obsd: 291.1

2,4-difluoro-5-{[((3-fluorophenyl){1-[(2-methyl-6-{[4-(methylsulfonyl)phenyl]amino}-3- pyridinyl)methyl]-4-piperidinyl}amino)carbonyl]arnino}beπzamide, example 4.

2,4-Difluoro-5-{[((3-fiuorophenyl){1-[(2-methyl-6-{[4-(methylsulfonyl)pheπyl]amino}-3- pyridinyl)methyI]-4-piperidinyl}amino)carbonyl]amino}benzamide (5.7mg, 13.5%) was obtained as solid from 2-rnethyl-6-{[4-(methylsulfonyl)phenyl]amino}-3- pyridinecarbaldehyde intermediate 23 (17.4mg, 0.06mmol) and intermediate 16 2,4-difluoro-5-({[(3-fluorophenyl)(4-piperidinyl)amino]carbonyl}amino)benzamide (26mg, O.Oδmmol) and sodium triacetoxyborohydride (26mg, 0.12mmol) following the procedure outlined in example 1. 1 H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.65 (t, J=8.80 Hz, 1 H), 8.02 (s, 1 H), 7.82 (d, J =8.80 Hz, 2 H), 7.51 (m, 4 H), 7.21 (m, 1 H), 7.06 (d, J =7.70 Hz, 1 H), 6.Θ8 (m, 1 H), 6.89 (m, 1 H), 6.83 (m, 1 H), 6.71 (d, J =8.07 Hz, 1 H), 6.50 (s, 1 H), 5.90 (d, J =1.47 Hz, 2 H), 4.54 (s, 1 H), 3.37 (m, 2 H), 3.04 (s, 2 H), 2.84 (m, 5 H), 2.46 (s, 3 H), 2.13 (m, 2 H), 1.85 (m, 2 H), 1.42 (s, 2 H). LCMS m/z (M+H)+ calcd: 667, obsd: 667.5

Example 5

5-({[{1-[(6-{[4-({[2-(ethyloxy)ethyl]amino}carbonyl)-2-methylphenyl]amino}-2-methyl-3- pyridinyl)methyl]-4-piperidinyl}(3-fluorophenyl)amino]oarbonyl}amino)-2,4- difluorobenzamide

Example 5 was prepared as in the scheme below.

Preparation of 4-amino-N-[2-(ethyloxy)ethyl]-3-methylbenzamide intermediate 24

To a stirred solution of 4-amino-3-methylbenzoic acid (1.52g, lOmmol) in N₁N- dimethylformamide (3OmL) were added 2-ethoxyethylamine (1 ,8g, 20mmol), triethyl amine (1.01g, lOmmol) and HATU (3.8g, 10mmol), sequentially. The resulting mixture was stirred for 4 hours at room temperature before it was diluted with ethyl acetate (20OmL) and washed with saturated sodium bicarbonate solution and brine. The organic phase was then dried over anhydrous sodium sulfate. After evaporation of solvents, the crude product was purified by column chromatography (silical gel, 0 to 90% ethyl acetate in hexane) to afford 1.9g (86%)of 4-amino-N-f2-(ethyloxy)ethyl]-3- methylbenzamide intermediate 24 as oil. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.52 (s, 1 H), 7.46 (dd, J =8.07, 2.20 Hz, 1 H), 6.66 (d, J =8.43 Hz, 1 H), 6.41 (s, 1 H), 3.58 (m, 6 H), 2.19 (s, 3 H), 1.20 (m, 3 H). LCMS m/z (M+H)+ calcd: 223, obsd: 223.3.

Preparation of N-[2-(ethyloxy)ethyl]-4-[(5-formyl-6-methyl-2-pyridinyl)amino]-3- methylbenzamide, intermediate 25

β-Chloro^-methyl-S-pyridinecarbaldehyde intermediate 10 (156mg, 1mmol) and 4- amino-N-[2-(ethyloxy)ethyl]-3-methylbenzamide intermediate 24 (266mg, 1.2mmol) were mixed in a sealed tube followed by additions of cesium carbonate (975mg, 3mmol), Pd2(dba)3 (46mg, O.Oδmmol), 2-dicyclohexylphosphino-2'-(N, N'- dimethylaminojbiphenyl (40mg, O.immol) and dimethoxyethane (5mL). The resulting mixture was purged with N2 (3X) before being heated to 90 ⁰C overnight. After cooled down to room temperature, the reaction mixture was diluted with ethyl acetate (10OmL) and washed with saturated sodium bicarbonate solution. The organic phase was then dried over anhydrous sodium sulfate. After evaporation of solvents, the crude product was purified by column chromatography (silical gel, 0 to 80% ethyl acetate in hexane) to afford 145mg (43%) of N-[2-(ethyloxy)ethyl]-4-[(5-formyl-6- methyl-2-pyridinyl)amino]-3-methylbenzamide intermediate 25 as solid. 1H NMR (400 WIHz, CHLOROFORM-d) δ ppm 10.12 (s, 1 H), 7.92 (d, J=8.80 Hz, 1 H), 7.73 (s, 1 H), 7.62 (m, 1 H), 6.83 (s, 1 H), 6.63 (m, 2 H), 6.53 (s, 1 H), 3.60 (m, 6 H), 2.75 (s, 3 H), 2.34 (S, 3 H), 1.23 (m, 3 H). LCMS m/z (M+H)+ calcd: 342, obsd: 342.1. 5-({[{1-[{6-{[4-({[2-(ethyloxy)ethyl]amiπo}carbonyl)-2-tτiethylphenyl]amino}-2-nπethyl-3- pyridiπyl)methyl]-4-piperidinyl}(3-fluorophenyl)amino]carbonyl}arnino)-2,4- difluorobenzamide, example S.

To a stirred solution of N-[2-(ethyloxy)ethyl]-4-[(5-formyl-6-methyl-2-pyridinyl)amino]- 3-methylbenzamide intermediate 25 (34mg, O.immol) and intermediate 16 2,4- difluoro-5-({[(3-fluorophenyl)(4-piperidinyl)amino]carbonyl}amino)benzamide (40mg, O.immol) in dichloroethane(IOmL) was added titanium (V) isopropoxide (36μL, 0.12mmol). The resulting mixture was heated to 80°C for one hour. After the reaction was cooled down to room temperature, sodium triacetoxyborohydride (33mg, 0.15mmol) was added. Stirring was continued overnight before the reaction was quenched with saturated sodium bicarbonate solution. The layers were separated and the aqueous phase was further extracted with dichloromethane (2X 2OmL). The combined organic layers were then dried over anhydrous sodium sulfate. After evaporation of solvents, the crude product was purified by column chromatography (silica gel, 0 to 60% CH2CI2 mixed with 10 % of methaπol/2M ammonia in CH2CI2) to give 50mg (69%) of 5-({[{1-[(6-{[4-({[2-(ethyloxy)ethyl]amino}carbonyl)-2- methylphenyl]amiπo}-2-methyI-3-pyridinyl)rnethyl]-4-piperidinyl}(3- fluorophenyl)amino]carbonyl}amino)-2,4-difluorobenzamide, example 5, as solid. 1 H NMR (400 MHz, METHANOL-d4) δ ppm 7.95 (m, 1 H), 7.66 (m, 2 H), 7.54 (m, 2 H), 7.40 (d, J=8.42 Hz, 1 H), 7.22 (m, 1 H), 7.09 (m, 3 H), 6.67 (d, J =8.24 Hz, 1 H), 4.38 (m, 1 H), 3.55 (m, 6 H), 3.37 (s, 2 H), 2.88 (m, 2 H), 2.37 (s, 3 H), 2.28 (m, 3 H), 1.83 (m, 2 H), 1.42 (m, 2 H), 1.20 (m, 3 H). HRMS m/z (M+H)+ calcd: 718.3329, obsd: 718.3329.

Example 6

N-(4-[[5-({4-[{[(4-chlorophenyl)amino]carbonyl}(phenyl)amino]-1-piperidinyl}methyl)-2- pyridinyl]thio}phenyl)methanesulfonamide gunχjθ>s

Intermediate 26 i .i-dimethylethyl ^KK^chlorophenyOaminolcarbonylXphenylJaminol-i- piperidinecarboxylate

A solution of 1,1-dimethylethyl 4-(phenylamino)-1-piperidinecarboxylate intermediate 17 (10.Og, 36.18 mmol, 1 equiv) and p-chloroisocyanate (6.6 g, 43.42 mmol, 1.2 equiv) in dichloroethane (100 mL) was stirred at RT for 18h. The suspension was diluted with excess dichloromethane, washed with 1N hydrochloric acid, dried over Na2SO4, filtered and concentrated to give a white solid. The solid was suspended in ether, stirred for 5 h, and filtered to give intermediate 26 (14.6 g, 94% yield) as a white solid.

1H NWlR (400 MHz, DMSO-d6) δ ppm 7.30 - 7.49 (m, 6 H), 7.14 - 7.24 (m, 4 H), 4.40 (t, J=11.99 Hz, 1 H), 3.82 - 4.00 (m, 2 H), 2.74 (s, 2 H), 1.75 (d, J=11.54 Hz, 2 H), 1.28 (s, 9 H), 0.94 - 1.14 (m, 2 H). ES-LCMS: m/z 452.2 (M+Na). Intermediate 27

N'-(4-chlorophenyl)-N-phenyl~N-4-piperidinylurea hydrochloride

A solution of 1 ,1-dimethylethyl 4-[{[(4-chlorophenyl)amino]carbonyl}(phenyl)amino]-1- piperidinecarboxylate intermediate 26 (14.6 g, 34.12 mmol, 1 equiv) in 4N HCI in dioxane (42.6 mL, 0.17 mol, 5 equiv) was stirred at RT for 2h and concentrated to a white solid. The solid was suspended in ether, stirred for 4 h and filtered to give intermediate 27 (11.8 g, 94% yield) as a white solid.

1H NMR (400 MHz, DMSO-d6) δ ppm 8.81 (s, 1 H)₁ 8.15 (s, 1 H), 7.39 - 7.53 (m, 4 H), 7.32 - 7.39 (m, 3 H), 7.15 - 7.28 (m, 3 H), 4.44 - 4.61 (m, 1 H), 3.20 (s, 2 H), 2.97 (s, 2 H), 1.90 (s, 2 H), 1.45 (d, J=12.82 Hz, 2 H). ES-LCMS: m/z 330.0 (M+H).

interm

ediate 10 DMF

Intermediate 28

6-[(4-nitrophenyl)thio]-3-pyridinecarbaldehyde

To a stirred solution of β-Chloro-S-pyridinecarbaldehyde Intermediate 10 (141 mg, 1 mmoi) in 1OmL of N, N-dimethylformamide were added 4-nitrobenzenethiol (199mg, 1mmol) and cesium carbonate (325mg, 1mmol). The resulting mixture was then heated to 80⁰C overnight. After it was cooled down to the room temperature, the reaction mixture was diluted with ethyl acetate (10OmL) and washed with saturated sodium bicarbonate solution. The organic layer was then dried over anhydrous sodium sulfate before evaporation of solvents. The crude product was purified by column chromatography (siiical gel, 0 to70%, CH2CI2 mixed with10% MeOH/ 2M NH₃ in CH2CI2) to afford 200mg (77%) of 6-[(4-nitropheπyl)thio]-3-pyridinecarbaldehyde Intermediate 28 as solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.01 (s, 1 H), 8.90 (s, 1 H), 8.29 (d, J=8.78 Hz, 2 H), 8.12 (dd, J=8.42, 2.20 Hz, 1 H), 7.86 (d, J=8.97 Hz, 2 H), 7.46 (s, 1 H). LC-MS m/z (M+H)+ calcd: 261, obsd: 261.

Intermediate 29

N'-(4-chlorophenyl)-N-[1-({6-[(4-nitrophenyI)thio]-3-pyridiπyl}methyl)-4-piperidinyl]-N- pheπylurea

To a stirred solution of 6-[(4-nitrophenyl)thio]-3-pyridinecarbaldehyde intermediate 28 (200mg, 0.77mmol) in dichloromethane (1OmL) were added N'-(4-chlorophenyI)- N-pheπyl-N-4-piperidinylurea intermediate 27 (260mg, 0.79mmol), sodium triacetoxylborohydride (487mg, 2.31 mmol). The resulting mixture was stirred at the room temperature overnight before it was quenched by saturated sodium bicarbonate solution. The layers were separated and the aqueous layer was extracted with dichloromethane (3X 3OmL). The combined organic layers were dried over sodium sulfate. After evaporation of solvents, the crude product was purified by column chromatography (silical gel, 0 to70%, CH2CI2 mixed with10%MeOH/2 M NH3 in CH2CI2) to afford 330mg (75%) of N'-(4-chlorophenyl)-N-[1-({6-[(4-nitrophenyl)thio]-3- pyridinyl}methyl)-4-piperidiπyl]-N-phenylurea intermediate 29 as solid 1H NMR (400 MHz, DMSO-d6) δ ppm 8.34 (m, 1 H), 8.18 (m, 2 H), 7.64 (m, 2 H), 7.45 (m, 4 H), 7.35 (m, 4 H), 7.18 (m, 4 H), 4.27 (m, 1 H), 3.42 (m, 2 H), 2.77 (m, 2 H), 2.03 (m, 2 H), 1.72 (m, 2 H), 1.21 (m, 3 H). LC-MS m/z (M+H)+ calcd: 574.1 , obsd: 574.1.

Example 6

To a stirred solution of N'-(4-chlorophenyl)-N-[1-({6-[(4-nitrophenyl)thio]-3- pyridinyl}methyl)-4-piperidinyl]-N-phenylurea intermediate 29 (330mg, 0.58mmol) in a mixture of acetic acid (5mL) and water (5mL was added iron powder (128mg, 2.3mmol). The reaction mixture was heated to 40 ⁰C for 40 minutes before it was concentrated down. The residual was partitioned between ethyl acetate (10OmL) and saturated sodium bicarbonate solution (5OmL). The layers were separated and the organic layer was dried over anhydrous sodium sulfate. Evaporation of the solvents gave 250mg (81%) of the desired aniline which was used for the next step. At -78⁰C, to a solution of the aniline generated above in pyridine (3 mL) was added methanesulfonyl chloride (43μL, 0.55mmol). The mixture was stirred while naturally warmed up to room temperature over 2 hours. After the reaction was diluted with ethyl acetate (10OmL), the excess pyridine was washed out with water and brine. The organic phase was then dried over anhydrous sodium sulfate. Evaporation of the solvents and column chromatography (silica gel, 0 to70%, CH2CI2 mixed with10%MeOH/2 M NH3 in CH2CI2) provided 180mg (63%) of N-(4-{[5-{{4-[{[(4- chlorophenyl)amino]carbonyl}(phenyl)amino]-1-piperidinyl}methyl)-2- pyridinyl]thio}phenyl)methanesulfoπamide example 6 as solid. 1H NMR (400 MHz, METHANOL-d4) δ ppm 8.21 (s, 1 H), 7.48 (m, 6 H), 7.28 (m, 4 H), 7.19 (m, 4 H), 6.86 (d, J=8.06 Hz, 1 H), 4.39 (m, 1 H), 3.41 (s, 2 H), 3.01 (s, 3 H), 2.83 (m, 2 H)₁ 2.12 (s, 2 H), 1.81 (s, 2 H), 1.38 (d, J=17.40 Hz, 2 H), 1.27 (s, 2 H), 0.88 (m, 2 H). HRMS m/z (M+H)+ calcd: 622.1708, obsd: 622.1702.

Example 7: Biological Activity PBL Assay

Whole blood samples were obtained from normal human donors. Peripheral Blood Lymphocytes (PBLs) were separated from whole blood by density gradient centrifugation with Lymphocyte Separation Medium (LSM). Whole blood was diluted 1:1 with sterile phosphate buffered saline (PBS) and overlaid onto LSM in 50 mL conical centrifuge tubes. Samples were centrifuged at 500 x g at room temperature for 30 minutes. PBLs were then gently removed from the interface between LSM and aqueous phases and washed twice in sterile PBS. The cells were resuspended in 150 mL of RPMI containing 20% fetal bovine serum, 10% T-cell growth factor (IL-2), 50ug/mL gentamicin (RPMI 20/10/50) and 5ug/mL Phytohaemagluttinin and incubated at 37oC, 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% DIWSO. Fifty (50) uL of diluted compound was transferred to the PBLs and placed in a humidified incubator at 37oC, 5% CO2 for 1hr. An additional 60 μL of diluted compound was transferred to a separate 96-well plate containing 60 μL of HIV-1 Ba-L (diluted 1 :50 into RPWII 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 37oC, 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 -80oC 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/HCl, 12.5 mM MgCI2, 68 Ci/mmole methyl-3H deoxythymidine-5'-triphosphate, and 0.62 O. D. units/mL of poly(rA) p(dT)12-18 in distilled water). The plates were thoroughly mixed on a platform shaker and placed in a humidified incubator at 37oC, 5% CO2 for 2h. Whatman Unifilter DE-81 96-well plates were placed on a vacuum manifold and 100 μL of the RT reaction was added. Full vacuum from an in-house line was applied until all of the reaction volume had seeped through the plate. Each plate was then washed a total of three times with all washes performed under full, in-house vacuum. All wash buffers were delivered with a 12 channel Nunc plate washer connected to a carboy containing at least 2 liters of buffer. The first wash consisted of approximately 300 μL of 5% Na2HPO4, followed by one wash of approximately 300 μL of distilled water, and a final wash consisting of 100 μL of 95% ethanol. The plates were allowed to dry completely at room temperature, bottom-sealed with an adhesive backing, and 50 μL of scintillation cocktail was added. The plates were topsealed and read in a Topcount (Packard) luminometer at 10s/well.

Raw data from the PBL assay were expressed as Counts Per Minute (CPM) and normalized according to the following formula:

Log10(CPM) - Log10 (Geometric mean PC) * Log10 (Geometric mean NC) - Log10

(Geometric mean PC)

Where:

PC = Positive Control = mock infected wells in column 12 on each assay plate

NC = Negative Control = untreated but infected wells in column 11 on each assay plate

The potency of test compounds are reported as IC50 values derived from the unconstrained four parameter logistic equation, defined as: y = A + ((B-C))/(1 +(10^Λx)/10^ΛC)^ΛD)

Where:

A = minimum y

D = slope factor

B = maximum y x = Iog10(molar compound concentration)

C = log10(IC50)

Compounds of the invention have IC50 values less than 1OnM.

Example 8: 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 37oC, 5% CO2 for 1hr. 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 37oC, 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) Iuminometer 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 1C50 values derived from the four parameter Hill equation, defined as: y=Vmax*(1 -(x^Λn/(K^Λn+x^Λn)))+Y2 Where: x = Log10[compound] y = normalized response data

Vmax = upper bound of response

K = IC50

Y2 = lower bound or baseline of response π = hill coefficient Compounds of the invention have IC50 values less than 1OnM.

Example 9: 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).

Biood 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 - 80⁰C until analysis.

For oral solution administration, animals received a single dose by an oral gavage needle. Animals (n=2) were euthanized at 1 , 2, 4, and 24 h postdose, and blood samples (approximately 1 mL with EDTA anticoagulant) taken by cardiac puncture and placed on ice. Plasma was harvested by centrifugation for 3 min at 4000 x g and 4°C and stored at -80⁰C until analysis. Spleen and liver were also dissected, immediately frozen and stored at -80⁰C until analysis. Sample Analysis

Plasma samples were thawed to room temperature and vortex-mixed. Standards were prepared in pooled rat plasma containing EDTA. Quality control (QC) samples were prepared from separately weighed sample stocks. 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 4°C. The acetonitrile supernatant (150 μL) was transferred to clean 96 well plates containing 100 μL of 0.1% formic acid in water and assayed by LC/MS/MS.

The analytical system consisted of a CTC HTS PAL autosampler, Hewlett Packard (HP) 1100 binary pump and Applied Biosystems API 4000 mass spectrometer. Samples (10 μL) were injected onto a 30 x 2 mm, 3micron, Atlantis C18 column (Waters) using a gradient eluting system at a flow rate of 0.75 mL/min. Mobile phases consisted of A) water containing 0.5% formic acid and B) acetonitrile containing 0.5% formic acid. The initial mobile phase composition of 90% A: 10% B was followed by a linear gradient to 5% A:95% B at 1 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 liver 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 4⁰C. 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.βmicron, Atlantis C18 column (Waters) using an isocratic eluting system at a flow rate of 0.8 mL/min. Mobile phases consisted of A) water containing 0.1% acetic acid, pH adjusted to 6.2 with ammonium hydroxide:acetonitrile (95:5) and B) acetonitrile containing 0.1% acetic acid. The mobile phase composition was 50% A:50% B and total run time was 1.1 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 (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 individual plasma concentration time data using

WinNonlin Professional 4.1 (Pharsight, Mountain View CA) . Dose-normalized AUC

0->∞ (DNAUC 0-»∞) was obtained by dividing the AUC 0-s>-∞ by its respective dose and expressed as unit dose AUCO→∞ (ng*h/mL/mg/kg). Oral bioavailability (F) was calculated using the following equation;

F (%) = [(DNAUCO-∞, oral)/(DNAUCO-∞, IV] * 100% where DNAUC0-∞, IV was the mean DNAUCO-∞ following IV dose administration.

Table 1 Plasma Pharmacokinetic Parameter Estimates for Compounds in Non-Fasted Male Sprague-Dawley CD Rat Following Intravenous, Oral Solution and Oral Capsule

* WO2006/030925 Example 10: 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 DWIEM / 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 4°C. This spin was repeated after resuspending the pellets in 200 mis of the same solution. All subsequent steps were performed at 4⁰C. The cells were homogenized within a glass Waring blender for 2 x 15 sees in 200 mis of a solution containing HEPES (50 mM), leupeptin (0.1 mM), bacitracin (25mg/ml), EDTA (1mM), PMSF (1mM) and Pepstatin A (2mM). Pepstatin A and PMSF were prepared as concentrated stocks in absolute ethanol (pepstatin 500x, 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 -80⁰C.

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 ceπtrifuged at 10,000 rpm for 1 minute and left to equilibrate for 2-3 hrs at room temperature before reading on a ViewLux™ luminescence imager. Concentration response curve data were generated from an 11 -point inhibition curve (top assay concentration of 60 mM and a 1 :4 step-dilution), a four parameter curve-fit being applied.

3. Drugs and Materials

Compounds were dissolved in DMSO at a concentration of 10 mM. For concentration-response assays, dilutions were prepared in 100% DMSO using a 1 in 4 dilution step. The top assay concentration was 60 mM and the final DMSO assay concentration was 1%. On each test compound plate 0.1 ml of DMSO was included in one column to assess total binding (TB) and dofetilide or astemizole (10 mM final concentration) added to one column to determine non specific binding (NSB). Four reference compounds were included in each assay for quality control (QC) purposes. The reference compounds used were: astemizole, dofetilide, quinidine and cisapride.

4. Data Analysis

For a single concentration assay, the results of each well are expressed as % inhibition:

% inhibition = 100 x (1- (sample - NSB) / (TB - NSB), where sample was the signal observed in a particular unknown sample well.

For concentration-response assays, the results of each test well were expressed as % of controls. The normalization equation used was:

% control: 100 x (sample - NSB) / (TB - NSB)

The concentration-response curve was analyzed using a non linear equation, 4- parameter logistics, giving a determination of plC50.

Curve QC, plate QC (z' > 0.2) and assay QC (plC50 for reference compounds ± 0.5 log units of the rolling mean) were applied when analyzing all experiments.

Table 2

' WO2006/030925

Claims

Claims

1. A compound selected from the group consisting of

5-({[butyl(1-{[2-methyl-6-({4-[(methyIsulfonyl)amino]phenyl}thio)-3-pyridinyl]methyl}-4- piperidinyl)amino]carbonyl}amino)-2,4-difluorobenzamide;

2,4-difluoro-5-({[(3-fluorophenyl)(1-{[2-methyl-6-({4-[(methylsulfonyl)amino]phenyl}thio)-

3-pyridinyl]methyl}-4-piperidinyl)amino]carbonyl}amino)bβnzarnide;

2,4-difluoro-5-({[(1-{[2-methyl-6-({4-[(methylsulfonyl)amino]phenyl}thio)-3- pyridiny!]methyl}-4-piperidinyl)(pheπyl)amino]carbonyl}amino)ben2amide;

2,4-difluoro-5-{[((3-fluorophenyl){1-[(2-methyl-6-{[4-(metriylsulfonyl)pheπyl]amino}-3- pyridinyl)methyl]-4-piperidinyl}amino)carbonyl]amino}benzamide;

5-({[{1-[(6-{[4-({[2-(ethyloxy)ethyl]amino}carbony[)-2-methylphenyl]amino}-2-methyl-3- pyridinyl)methyI]-4-piperidinyl}(3-fluorophenyI)amino]carbonyl}amino)-2,4- difluorobeπzamide;

Λ/-(4-([5-({4-[{[(4-chlorophenyl)amino]carbonyl}(phenyl)amino]-1-piperidinyl}methyl)-2- pyridiπyl]thio}phenyl)methanesulfonamide; and pharmaceutically acceptable salts thereof.

2. A method of treatment or prevention of a HIV infection in a human comprising administering to said human an effective amount of a compound according to claim 1.

3. A method of treatment or prevention of neuropathic pain, multiple sclerosis, rheumatoid arthritis, autoimmune diabetes, chronic implant rejection, asthma, rheumatoid arthritis, Crohns Disease, inflammatory bowel disease, chronic inflammatory disease, glomerular disease, nephrotoxic serum nephritis, kidney disease, Alzheimer's Disease , autoimmune encephalomyelitis, arterial thrombosis, allergic rhinitis, arteriosclerosis, Sjogren's syndrome (dermatomyositis), systemic lupus erythematosus, graft rejection, cancers with leukocyte infiltration of the skin or organs, human papilloma virus infection, prostate cancer, wound healing, amyotrophic lateral sclerosis and immune mediated disorders in a human comprising administering to said human a pharmceutically effective amount of a compound according to claim 1.

4. A compound according to claim 1 for use in medical therapy.

5. Use of a compound according to claim 1 in the manufacture of a medicament for the treatment or prophylaxis of an HIV infection.

6. The use of a compound according to claim 1 in the manufacture of a medicament for the treatment or prophylaxis of neuropathic pain, multiple sclerosis, rheumatoid arthritis, autoimmune diabetes, chronic implant rejection, asthma, rheumatoid arthritis, Crohns Disease, inflammatory bowel disease, chronic inflammatory disease, glomerular disease, nephrotoxic serum nephritis, kidney disease, Alzheimer's Disease , autoimmune encephalomyelitis, arterial thrombosis, allergic rhinitis, arteriosclerosis, Sjogren's syndrome (dermatomyositis), systemic lupus erythematosus, graft rejection, cancers with leukocyte infiltration of the skin or organs, human papilloma virus infection, prostate cancer, wound healing, amyotrophic lateral sclerosis and immune mediated disorders.

7. A pharmaceutical composition comprising a pharmaceutically effective amount of a compound according to claim 1 together with a pharmaceutically acceptable carrier.

8. A pharmaceutical composition according to claim 7 in the form of a tablet or capsule.

9. A pharmaceutical composition according to claim 7 in the form of a liquid.

10. A method of treatment or prevention of an HIV infection in a human comprising administering to said human a composition comprising a compound according to claim 1 and another therapeutic agent.

11. A method according to claim 10, wherein said composition comprises another therapeutic agent selected from the group consisting of acyclic nucleosides, for example acyclovir, valaciclovir, famciclovir, ganciclovir, and penciclovir, acyclic nucleoside phosphoπates, for example (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 PMEA, adefovir dipivoxil), [[(1R)-2-(6-amino-9H-purin-9-yl)-1- methylethoxy]methyl]phosphonio acid (tenofovir), and (R)-[[2-(6-Amino-9H-purin-9- yl)-1 -methylethoxy]methyl]phosphonic 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'- dideoxyadenosiπe, 2',3'-dideoxyinosine (ddl, didanosine), 2',3'-didehydrothymidine (d4T, stavudine), (-)-cjs-1-(2-hydroxymethyl)-1 ,3-oxathiolane 5-yl)-cytosine (lamivudine), ds-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine (FTC), (- )-cis-4-[2-amino-6-(cyclopropylamino)-9H.-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-interferon, immuπomodulators such as interleukin Il or thymosin, granulocyte macrophage colony stimulating factors, erythropoetin, soluble CD₄ and genetically engineered derivatives thereof, non-nucleoside reverse transcriptase inhibitors (NNRTIs), for example nevirapine (BI-RG-587), alpha-((2-acetyl-5- rnethylphenyl)arnino)-2,6-dichloro-benzeπeacetamide (loviride), 1-[3- (isopropylamino)-2-pyridyl]-4-[5-(methanesulfonamido)-1H-indol-2- ylcarbonyl]piperazine monomethanesulfonate (delavirdine), (S)-6-chloro-4- (cyclopropylethyπyl)-i ,4-dihydro-4-(trifluoromethyl)-2H-3, 1 -benzoxazin-2-one (efavirenz, DMP 266),rilpiviriπe, integrase inhibitors, or fusion inhibitors, for example T-20 and T-1249.