WO1995029672A1 - HALOMETHYL AMIDES AS IL-1β PROTEASE INHIBITORS - Google Patents

HALOMETHYL AMIDES AS IL-1β PROTEASE INHIBITORS Download PDF

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WO1995029672A1
WO1995029672A1 PCT/US1995/005347 US9505347W WO9529672A1 WO 1995029672 A1 WO1995029672 A1 WO 1995029672A1 US 9505347 W US9505347 W US 9505347W WO 9529672 A1 WO9529672 A1 WO 9529672A1
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Prior art keywords
chloroacetamide
dichlorobenzyl
aralkyl
alkyl
compound
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PCT/US1995/005347
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French (fr)
Inventor
Roland E. Dolle
James M. Rinker
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Sanofi Winthrop, Inc.
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Priority to JP7528446A priority Critical patent/JPH09512556A/en
Priority to MX9605196A priority patent/MX9605196A/en
Priority to AU24634/95A priority patent/AU705321B2/en
Priority to EP95918877A priority patent/EP0758891A4/en
Publication of WO1995029672A1 publication Critical patent/WO1995029672A1/en
Priority to NO964472A priority patent/NO964472L/en
Priority to FI964342A priority patent/FI964342A0/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/12—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by halogen atoms or by nitro or nitroso groups
    • C07C233/13—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by halogen atoms or by nitro or nitroso groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C07C233/46—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/47—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
    • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
    • C07C255/24—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms containing cyano groups and singly-bound nitrogen atoms, not being further bound to other hetero atoms, bound to the same saturated acyclic carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
    • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
    • C07C255/24—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms containing cyano groups and singly-bound nitrogen atoms, not being further bound to other hetero atoms, bound to the same saturated acyclic carbon skeleton
    • C07C255/25—Aminoacetonitriles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/01—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms
    • C07C311/02—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C311/03—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton having the nitrogen atoms of the sulfonamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C311/06—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton having the nitrogen atoms of the sulfonamide groups bound to hydrogen atoms or to acyclic carbon atoms to acyclic carbon atoms of hydrocarbon radicals substituted by carboxyl groups

Definitions

  • This invention relates to a series of novel non-peptides which exhibit selective in vitro and in vivo inhibition of interleukin-l ⁇ converting enzyme, to compositions containing the novel non-peptides and to methods for therapeutic utility.
  • the interleukin l ⁇ converting enzyme inhibitors described in this invention comprise novel ⁇ -halomethyl amides and ⁇ -halomethyl suifonamides which possess particular utility in the treatment of inflammatory and immune-based diseases of lung, central nervous system, and connective tissues.
  • Interleukin-l ⁇ (IL-1 ⁇ ) protease (also known as interleukin-l ⁇ converting enzyme or ICE) is the enzyme responsible for processing of the biologically inactive 31 kD precursor IL— l ⁇ to the biologically active 17 kD form (Kostura, M.J.; Tocci, M.J.; Limjuco, G.; Chin, J.; Cameron, P.; Hillman, A.G.; Chartrain, N.A.; Schmidt, J.A., Prod Nat. Acad. Sc (1989), fi£, 5227-5231 and Black, R.A.; Kronheim, S.R.; Sleath, P.R., FEBS Let.. (1989), £47, 386-391 ).
  • IL— l ⁇ has also been proposed to act as a mediator of a wide variety of diseases, including rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, sepsis, acute and chronic myelogenous leukemia and osteoporosis (Dinarello, C.A.; Wolff, S.M., New En ⁇ l. J. Med.. (1993), 2k 106).
  • IL— I ⁇ receptor antagonist has been used to demonstrate the intermediacy of IL— l ⁇ in a number of human diseases and animal models (Hannum, C.H.; Wilcox, C.J.; Arend, W.P.; Joslin, G.G.; Dripps, D.J.; Heimdal, P.L; Armes, L.G.; Sommer, A.; Eisenberg, S.P.; Thompson, R.C., Nature. (1990), 343.
  • WO 9309135 published 11 May 1993, teaches that peptide-based aspartic acid arylacyloxy-and aryoxymethyl ketones are potent inhibitors of ICE in vitro. These compounds also specifically inhibited ICE in the whole cell (in vivo) by their ability to inhibit the formation of mature IL-1 ⁇ in whole cells. These ICE inhibitors also demonstrated utility in reducing fever and inflammation/swelling in rats.
  • IL-1 is present in affected tissues in ulcerative colitis in humans.
  • IL-1 ⁇ levels correlate with disease severity.
  • administration of 1 L-1 ra reduced tissue necrosis and the number of inflammatory cells in the colon.
  • IL-1 ra supresses joint swelling in the PG-APS model of arthritis in rats. See Schwab, J.H.; Anderle, S.K.; Brown, R.R.; Dalldorf, F.G. and Thompson, R.C., "Pro- and Anti-Inflammatory Roles of lnterelukin-1 in Recurrence of Bacterial Cell Wall- Induced Arthritis in Rats". Infect. Immun. (1991 ) 52; 4436-4442.
  • IL-1 ra shows efficacy in an small open-label human Rheumatoid Arthritis trial. See, Lebsack, M.E.; Paul, C.C.; Bloedow, C.C.; Burch, F.X.; Sack, M.A.; Chase, W., and Catalano, M.A. "Subcutaneous IL-1 Receptor Antagonist in Patients with Rheumatoid Arthritis", Arth. Rheum. (1991 ) 24; 545.
  • IL-1 appears to be an autocrine growth factor for the proliferation of chronic myelogenous leukemia cells. Both IL-1 ra and slL-1 R inhibit colony growth in cells removed from leukemia patients.
  • R1 independently selected from alkyl, haloalkyl and alkoxyalkyl
  • R2 H, alkyl, (CH2)-alkenyi, aralkyl, heteroaralkyl, carboxyalkyl, cyanoalkyl, aryl, heteroaryl;
  • R3 H, alkyl, (CH2)-alkenyl, aralkyl, heteroaralkyl, aryl, heteraryl;
  • Alkyl is defined as a saturated aliphatic hydrocarbon which may be either straight - or branched chain. Preferred groups have no more than 12 carbon atoms and may be methyl, ethyl, and structural isomers of propyl, butyl, up to dodecyl.
  • Haloalkyl is defined as an alkyl radical substituted by one or more halogen (F, Cl, Br, I). For example chloromethyl, dichloromethyl, fluoromethyl, difluoromethyl, fluorochloromethyl.
  • Alkoxyalkyl is defined as an alkyl radical substituted by an alkoxy group. For example methoxymethyl.
  • Aryl is defined as a phenyl on naphthyl ring which may be unsubstituted or substituted wherein one or more of the hydrogen atoms has been replaced by the same or different substituents including halo, alkyl, aryl, nitro, cyano, amino, alkylacylamino, hydroxyl, alkoxy, haloalkyl.
  • Halo means iodo, bromo, chloro, fluoro.
  • Carboxyalkyl means an alkyl radical substituted by a carboxyl group. For example, carboxymethyl.
  • Alkyl means an alkyl radical substituted with an aryl ring.
  • benzyl 4- chlorobenzyl.
  • Heteroaryl means pyridyl, thienyl or furanyl and structural isomers thereof.
  • Heteroaralkyl means an alkyl radical substituted by an heteroaryl ring. For example 2-thienyl ethyl.
  • Alkenyl is defined as an alkyl group containing one or more sites of unsaturation. For example, ethenyl, ethynl, 1-butenyl, 2-butynyl, 1 ,3-hexadienyl.
  • Cyanoalkyl means an alkyl radical substituted by a cyano group. For example, cyano ethyl.
  • the present invention also concerns the pharmaceutical composition and method of treatment of IL-l ⁇ protease mediated disease states or disorders in a mammal in need of such treatment comprising the administration of IL-l ⁇ protease inhibitors of formula (A) as the active agent.
  • disease states and disorders include: infectious diseases, such as meningitis and salpingitis; septic shock, respiratory diseases; inflammatory conditions, such as arthritis, cholangitis, colitis, encephalitis, endocerolitis, hepatitis, pancreatitis and reperfusion injury, immune-based diseases, such as hypersensitivity; auto-immune diseases, such as multiple sclerosis; bone diseases; and certain tumors and leukemias.
  • the present invention has particular utility in the modulation of processing of IL- l ⁇ for the treatment of rheumatoid arthritis.
  • Levels of IL-l ⁇ are known to be elevated in the synovial fluid of patients with the disease. Additionally, IL-l ⁇ stimulates the synthesis of enzymes believed to be involved in inflammation, such as collagenase and PLA2, and produces joint destruction which is very similar to rheumatoid arthritis following intra-articular injection in animals.
  • an effective amount of a compound of the invention or a pharmaceutical composition thereof is administered to the subject in need of, or desiring, such treatment.
  • These compounds or compositions may be administered by any of a variety of routes depending upon the specific end use, including orally, parenterally (including subcutaneous, intraarticular, intramuscular and intravenous administration), rectally, buccally (including sublingually), transdermally or intranasally. The most suitable route in any given case will depend upon the use, the particular active ingredient, and the subject involved.
  • the compound or composition may also be administered by means of controlled-release, depot implant or injectable formulations as described more fully herein.
  • the active ingredient in amounts between about 0.1 and 100 mg/kg body weight, most preferably from about 0.1 to 30 mg/kg body weight for human therapy, the active ingredient will be administered preferably in the range of from about 0.1 to about 20-50 mg/kg/day.
  • This administration may be accomplished by a single administration, by distribution over several applications or by slow release in order to achieve the most effective results.
  • administration When administered as a single dose, administration will most preferably be in the range of from about 0.1 to mg/kg to about 10 mg/kg.
  • compositions comprising as an active ingredient a compound of the present invention in admixture with a pharmaceutically acceptable, non-toxic carrier.
  • a pharmaceutically acceptable, non-toxic carrier such compositions may be prepared for use for parenteral (subcutaneous, intraarticular, intramuscular or intravenous) administration, particularly in the form of liquid solutions or suspensions; for oral or buccal administration, particularly in the form of tablets or capsules; or intranasally, particularly in the form of powders, nasal drops or aerosols.
  • the compounds When administered orally (or rectally) the compounds will usually be formulated into a unit dosage form such as a tablet , capsule, suppository or cachet.
  • a unit dosage form such as a tablet , capsule, suppository or cachet.
  • Such formulations typically include a solid, semi-solid or liquid carrier or diluent.
  • Exemplary diluents and vehicles are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, aginates, tragacanth, gelatin, syrup, methylcellulose, polyoxyethylene sorbitar monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, and magnesium stearate.
  • compositions may be prepared by any of the methods well-known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences. 17th edition, Mack Publishing Company, Easton, PA, 1985.
  • Formulations for parenteral administration may contain as common excipients sterile water or saline, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like.
  • Examples of vehicles for parenteral administration include water, aqueous vehicles such as saline, Ringer's solution, dextrose solution, and Hank's solution and nonaqueous vehicles such as fixed oils (such as corn, cottonseed, peanut, and sesame), ethyl oleate, and isopropyl myristate.
  • aqueous vehicles such as saline, Ringer's solution, dextrose solution, and Hank's solution
  • nonaqueous vehicles such as fixed oils (such as corn, cottonseed, peanut, and sesame), ethyl oleate, and isopropyl myristate.
  • Sterile saline is a preferred vehicle and the compounds are sufficiently water soluble to be made up as a solution for all foreseeable needs.
  • the vehicle may contain minor amounts of additives such as substances that enhance solubility, isotonicity, and chemical stability, e.g., antioxidants, buffers, and preservatives.
  • Formulations for nasal administration may be solid and contain as excipients, for example, lactose or dextran, or may be aqueous or oily solutions for administration in the form of nasal drops or metered spray.
  • excipients include sugars, calcium stearate, magnesium stearate, pregelatinated starch, and the like.
  • surfactant acids such as for example, glycocholic acid, cholic acid, taurocholic acid, ethocholic acid, desoxychoiic acid, chenodesoxycholic acid, dehydrocholic acid, glycodeoxy-cholic acid, and the like (See, B.H. Vickery, "LHRH and its Analogs-Contraception and Therapeutic Applications", Pt. 2, B.H. Vickery and J.S. Nester, Eds., MTP Press, Lancaster, UK, 1987).
  • Part A To 1 g of 2,4-dichlorobenzaldehyde in 6 mL of 1 ,2-dichloroethane was added 428 ⁇ L of allyl amine, 280 ⁇ L of acetic acid, and 1.8 g of NaBH(OAc)3 in the given order. After 30 minutes, the reaction mixture was diluted with chloroform and saturated aqueous NaHC ⁇ 3. The layers were separated and the organic layer was dried (MgS ⁇ 4) and concentrated in vacuo affording a colorless oil. Flash chromatography (15% EtOAc-hexane) afforded 476 mg (38%) of l as a colorless oil.
  • Part B 476 mg of 1 was dissolved in 5 mL of methylene chloride and 306 ⁇ L of Et3N was added. The reaction mixture was cooled to 0°C and 175 ⁇ L of chloroacetyl chloride was added and the mixture was stirred for 2 h. The reaction was then diluted with chloroform and washed twice with water. The combined organic layers were dried (MgS ⁇ 4) and concentrated in vacuo provided a white solid. Flash chromatography
  • Part A 100 g of 2,4-dichlorobenzylamine was dissolved into 600 mL of CH2CI2 and the reaction mixture was cooled to 0°C. Next, 89 mL of Et3N was added followed by the dropwise addition of 50 mL of chloroacetyl chloride. The reaction mixture was stirred for 24 h. The reaction was then washed twice with H2O, dried (MgS ⁇ 4) and concentrated in vacuo affording a solid which was triturated with 10% hexane-EtOAc affording 131 g (97%) of 3_ as a pure white solid.
  • Part B 500 mg of 3_ was dissolved in 2 mL of THF and 2 mL (10 equiv) of benzyl bromide was added. Next, 276 mg of potassium t-butoxide in 7 mL of THF was added dropwise to the reaction mixture which was then stirred for 30 minutes and finally was concentrated in vacuo. The residue was dissolved in chloroform and washed twice with H2O, dried (MgS ⁇ 4) and concentrated in vacuo affording a yellow oil. Flash chromotography (15% EtOAc-hexane) afforded 500 mg (73%) of 4 as a white solid. Low Resolution Mass Spec, m/z (relative intensity): 356 (M+H; 11 ), 196 (4), 91 (100)
  • Partially purified IL-1 ⁇ protease is stored at -80°C, thawed on ice, and preincubated for 10 minutes at 37°C with 2.5 mM dithiothreitol in a buffer solution containing 10 mM Tris-HCI (pH 8.0) and 25% (v/w) glycerol.
  • Inhibitors are prepared as stock solutions in dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • the protease is preincubated with inhibitor in a volume of 20 ⁇ L in a 1.5 mL polypropylene microcentrifuge tube for 15 minutes at 37°C.
  • the volume of compound added to the assay is adjusted to yield a DMSO concentration in the preincubation of ⁇ 15% (v/v).
  • the enzyme assay is then initiated by the addition of substrate (TRITC-AYVHDAPVRS-NH2) (SEQ I.D. No. 1 ) to yield a final concentration of 67 ⁇ M in a final volume of 30 ⁇ L.
  • substrate TRITC-AYVHDAPVRS-NH2
  • SEQ I.D. No. 1 substrate
  • the reaction are carried out for 60 minutes at 37°C in the dark and are terminated by the addition of 10 mL of 10% trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • the samples are analyzed by high pressure liquid chromatography using a reverse phase (C18) column and elution with an acetonitrile/water/TFA gradient. Substrate and product are monitored by their absorbance at 550 nm and elute at 4.2 and 5.2 minutes, respectively.
  • IC50 In vivo inhibition
  • Human monocytes were isolated from heparinized leukopheresis units obtained through Biological Specialty Corporation (Lansdale, PA). Monocytes were purified by Ficoll-Hupaque (Pharmacia Fine Chemicals, Piscataway, NJ) gradient centrifugation and more than 95% pure monocyte populations obtained by centrifugal elutriation. The assay was performed on duplicate samples of freshly isolated human monocytes, cultured in suspension at 37°C and rotated gently in conical bottom polypropylene tubes (Sardstedt Inc., Princeton, NJ). Human monocytes at a concentration of 5 x 10 6 cells/mL were resuspended in 1 mL of RPMI 1640 (a common tissue buffer from M.A.
  • RPMI 1640 a common tissue buffer from M.A.
  • FCS fetal calf serum
  • the cells were treated either with a compound of the invention (i.e. test compound) or with a non-inhibitor (control compound, typically 0.03% DMSO) for 15 minutes and then activated with 0.01 % fixed Staphylococcus aureus (The Enzyme Center, Maiden, MA) for 1 hour.
  • the cells were then centrifuged and resuspended in 1 mL of cysteine, methionine-free RPMI media containing 1% dialyzed FCS (Hyclone).
  • the cells were pretreated with a test compound or control compound for 15 minutes after which 0.01 % fixed S. aureus plus 100 ⁇ Ci Tran 35-S label (ICN, Irvine, CA) was added and the cells incubated at 37°C for 1 hour. After incubation, cells were centrifuged, washed once in phosphate buffer saline and resuspended in 1 mL RPMI containing 1% fetal calf serum. The cells were again pretreated with a test or control compound for 15 minutes and then 0.01 % S. aureus for 2 hours. At the end of the incubation, cells were centrifuged and supernates saved for immunoprecipitation.
  • Cells were washed once in phosphate buffer saline and then lysed in RIPA, a continuous cell media buffer containing 2 mM phenylmethylsulfonyl fluoride, 10 mM iodoacetate, 1 ⁇ g/mL pepstatin A, 1 ⁇ g/mL leupeptin and 0.5 TI ) aprotinin.
  • the IL-l ⁇ proteins were then precipitated with 70 ⁇ L protein A sepharose, resuspended in 60 ⁇ L SDS sample buffer and run on 15% SGD-PAGE gels. Autoradiography was performed on dried gels and the amount of radioactivity (counts per minute, cpm) quantitated using a Betascope 603 analyzer. Data Analysis
  • Example 1 The compound in Example 1 had in vivo IC50 of ⁇ 10 ⁇ M.

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Abstract

Disclosed are compounds, compositions and methods for inhibiting interleukin-1β protease activity, the compounds having formula (A) described herein.

Description

HALOMETHYL AMIDES AS IL- 7β PROTEASE INHIBITORS
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to a series of novel non-peptides which exhibit selective in vitro and in vivo inhibition of interleukin-lβ converting enzyme, to compositions containing the novel non-peptides and to methods for therapeutic utility. More particularly, the interleukin lβ converting enzyme inhibitors described in this invention comprise novel α-halomethyl amides and α-halomethyl suifonamides which possess particular utility in the treatment of inflammatory and immune-based diseases of lung, central nervous system, and connective tissues.
Reported Developments
Interleukin-lβ (IL-1 β) protease (also known as interleukin-lβ converting enzyme or ICE) is the enzyme responsible for processing of the biologically inactive 31 kD precursor IL— lβ to the biologically active 17 kD form (Kostura, M.J.; Tocci, M.J.; Limjuco, G.; Chin, J.; Cameron, P.; Hillman, A.G.; Chartrain, N.A.; Schmidt, J.A., Prod Nat. Acad. Sc (1989), fi£, 5227-5231 and Black, R.A.; Kronheim, S.R.; Sleath, P.R., FEBS Let.. (1989), £47, 386-391 ). In addition to acting as one of the body's early responses to injury and infection, IL— lβ has also been proposed to act as a mediator of a wide variety of diseases, including rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, sepsis, acute and chronic myelogenous leukemia and osteoporosis (Dinarello, C.A.; Wolff, S.M., New Enαl. J. Med.. (1993), 2k 106). A naturally occurring IL— Iβ receptor antagonist has been used to demonstrate the intermediacy of IL— lβ in a number of human diseases and animal models (Hannum, C.H.; Wilcox, C.J.; Arend, W.P.; Joslin, G.G.; Dripps, D.J.; Heimdal, P.L; Armes, L.G.; Sommer, A.; Eisenberg, S.P.; Thompson, R.C., Nature. (1990), 343. 336-340; Eisenberg, S.P.; Evans, R.J.; Arend, W.P.; Verderber, E.; Brewer, M.T.; Hannum, C.H.; Thompson, R.C., Nature (1990), 2 2, 341 -346; Ohlsson, K.; Bjork, P.; Bergenfeldt, M.; Hageman, R.; Thompson, R.C., Nature. (1990), 42, 550-552; Wakabayashi, G., FASEB. (1991 ),
-ι- 338-343; Pacifici, R.; et al. Proc. Natl. Acad. Sci. (1989), fig, 2398-2402 and Yamamoto, I.; et al. Cancer Rsh (1989), 49_, 4242-4246). The specific role of IL— lβ in inflammation and immunomodulation is supported by the recent observation that the cowpox virus employs an inhibitor of ICE to suppress the inflammatory response of its host (Ray, CA. et al, C , (1992), 22, 597-604).
In summary, the utility of ICE inhibitors in modifying certain IL— 1 mediated disease states has been suggested and demonstrated in vivo by several workers in the field. The following review of the current state of the art in ICE research further supports such utility of ICE inhibitors:
1 ) WO 9309135, published 11 May 1993, teaches that peptide-based aspartic acid arylacyloxy-and aryoxymethyl ketones are potent inhibitors of ICE in vitro. These compounds also specifically inhibited ICE in the whole cell (in vivo) by their ability to inhibit the formation of mature IL-1 β in whole cells. These ICE inhibitors also demonstrated utility in reducing fever and inflammation/swelling in rats.
2) Patients with Lyme disease sometimes develop Lyme arthritis. B. burgdorferi, the causative agent of Lyme disease, is a potent inducer of IL-1 synthesis by mononuclear cells. Miller et al. (Miller, L.C.; Lynch, E.A. Isa, S.; Logan, J.W.; Dinarello, C.A.; and Steere, A.C., "Balance of synovial fluid IL-1 β and IL-1 Receptor Antagonist and Recovery from Lyme arthritis", Lancet (1993) 341 : 146-148) showed that in patients who recovered quickly from Lyme Arthritis, the balance in synovial fluid of IL-1 -beta and IL-1 ra was in favor of IL-ra. When the balance was shifted in favor of IL-1 β, it took significantly longer for the disease to resolve. The conclusion was that the excess IL-1 ra blocked the effects of the IL-1 β in the patients studied.
3) IL-1 is present in affected tissues in ulcerative colitis in humans. In animal models of the disease, IL-1 β levels correlate with disease severity. In the model, administration of 1 L-1 ra reduced tissue necrosis and the number of inflammatory cells in the colon.
See, Cominelli, F.; Nast, C.C.; Clark, B.D.; Schindler, R., Llerena, R.; Eysselein, V.E.; Thompson, R.C.; and Dinarello, C.A.; "lnterleukin-1 Gene Expression, Synthesis, and Effect of Specific IL-1 Receptor Blockade in Rabbit Immune Complex Colitis" J. Clin. Investigations (1990) Vol. 22, pp, 972-980.
4) IL-1 ra supresses joint swelling in the PG-APS model of arthritis in rats. See Schwab, J.H.; Anderle, S.K.; Brown, R.R.; Dalldorf, F.G. and Thompson, R.C., "Pro- and Anti-Inflammatory Roles of lnterelukin-1 in Recurrence of Bacterial Cell Wall- Induced Arthritis in Rats". Infect. Immun. (1991 ) 52; 4436-4442.
5) IL-1 ra shows efficacy in an small open-label human Rheumatoid Arthritis trial. See, Lebsack, M.E.; Paul, C.C.; Bloedow, C.C.; Burch, F.X.; Sack, M.A.; Chase, W., and Catalano, M.A. "Subcutaneous IL-1 Receptor Antagonist in Patients with Rheumatoid Arthritis", Arth. Rheum. (1991 ) 24; 545.
6) IL-1 appears to be an autocrine growth factor for the proliferation of chronic myelogenous leukemia cells. Both IL-1 ra and slL-1 R inhibit colony growth in cells removed from leukemia patients.
See, Estrov, Z.; Kurzrock, R.; Wetzler, M.; Kantarjian, H.; Blake, M.; Harris, D.; Gutterman, J.U.; and Talpaz, M., "Supression of Chronic Myelogenous Leukemia Colony Growth by lnterleukin-1 (IL-1 ) Receptor Antagonist and Soluble IL-1 Receptors: a Novel Application for Inhibitors of IL-1 Activity". Blood (1991 ) Z2; 1476-1484.
7) As in 6) above, but for acute myelogenous leukemia rather than chronic myelogenous leukemia.
See, Estrov, 2.; Kurzrock, R.; Estey, E.; Wetzler, M.; Ferrajoli, A.; Harris, D.; Blake, M.; Guttermann, J.U.; and Talpaz, M. "Inhibition of Acute Myelogenous Leukemia Blast Proliferation by lnterleukin-1 (IL-1 ) Receptor Antagonist and Soluble IL-1 Receptors". (1992, Blood 79: 1938-1945.
An effective therapy has yet to be fully developed commercially for the treatment of IL-lβ mediated inflammatory diseases. Consequently, there is a need for therapeutic agents effective in the treatment and prevention of these diseases. SUMMARY OF THE INVENTION
According to the present invention, there is provided a compound of the formula (A) or a pharmaceutically acceptable salt thereof:
I 2
R3-N-Y-Rl (A) wherein:
Y = CO or Sθ2;
R1 = independently selected from alkyl, haloalkyl and alkoxyalkyl;
R2 = H, alkyl, (CH2)-alkenyi, aralkyl, heteroaralkyl, carboxyalkyl, cyanoalkyl, aryl, heteroaryl; and
R3 = H, alkyl, (CH2)-alkenyl, aralkyl, heteroaralkyl, aryl, heteraryl;
"Alkyl" is defined as a saturated aliphatic hydrocarbon which may be either straight - or branched chain. Preferred groups have no more than 12 carbon atoms and may be methyl, ethyl, and structural isomers of propyl, butyl, up to dodecyl.
"Haloalkyl" is defined as an alkyl radical substituted by one or more halogen (F, Cl, Br, I). For example chloromethyl, dichloromethyl, fluoromethyl, difluoromethyl, fluorochloromethyl.
"Alkoxyalkyl" is defined as an alkyl radical substituted by an alkoxy group. For example methoxymethyl.
"Aryl" is defined as a phenyl on naphthyl ring which may be unsubstituted or substituted wherein one or more of the hydrogen atoms has been replaced by the same or different substituents including halo, alkyl, aryl, nitro, cyano, amino, alkylacylamino, hydroxyl, alkoxy, haloalkyl. "Halo" means iodo, bromo, chloro, fluoro.
"Carboxyalkyl" means an alkyl radical substituted by a carboxyl group. For example, carboxymethyl.
"Aralkyl" means an alkyl radical substituted with an aryl ring. For example benzyl, 4- chlorobenzyl.
"Heteroaryl" means pyridyl, thienyl or furanyl and structural isomers thereof.
"Heteroaralkyl" means an alkyl radical substituted by an heteroaryl ring. For example 2-thienyl ethyl.
"Alkenyl" is defined as an alkyl group containing one or more sites of unsaturation. For example, ethenyl, ethynl, 1-butenyl, 2-butynyl, 1 ,3-hexadienyl.
"Cyanoalkyl" means an alkyl radical substituted by a cyano group. For example, cyano ethyl.
The present invention also concerns the pharmaceutical composition and method of treatment of IL-lβ protease mediated disease states or disorders in a mammal in need of such treatment comprising the administration of IL-lβ protease inhibitors of formula (A) as the active agent. These disease states and disorders include: infectious diseases, such as meningitis and salpingitis; septic shock, respiratory diseases; inflammatory conditions, such as arthritis, cholangitis, colitis, encephalitis, endocerolitis, hepatitis, pancreatitis and reperfusion injury, immune-based diseases, such as hypersensitivity; auto-immune diseases, such as multiple sclerosis; bone diseases; and certain tumors and leukemias.
The present invention has particular utility in the modulation of processing of IL- lβ for the treatment of rheumatoid arthritis. Levels of IL-lβ are known to be elevated in the synovial fluid of patients with the disease. Additionally, IL-lβ stimulates the synthesis of enzymes believed to be involved in inflammation, such as collagenase and PLA2, and produces joint destruction which is very similar to rheumatoid arthritis following intra-articular injection in animals.
In the practice of this invention an effective amount of a compound of the invention or a pharmaceutical composition thereof is administered to the subject in need of, or desiring, such treatment. These compounds or compositions may be administered by any of a variety of routes depending upon the specific end use, including orally, parenterally (including subcutaneous, intraarticular, intramuscular and intravenous administration), rectally, buccally (including sublingually), transdermally or intranasally. The most suitable route in any given case will depend upon the use, the particular active ingredient, and the subject involved. The compound or composition may also be administered by means of controlled-release, depot implant or injectable formulations as described more fully herein.
In general, for the uses as described in the instant invention, it is expedient to administer the active ingredient in amounts between about 0.1 and 100 mg/kg body weight, most preferably from about 0.1 to 30 mg/kg body weight for human therapy, the active ingredient will be administered preferably in the range of from about 0.1 to about 20-50 mg/kg/day. This administration may be accomplished by a single administration, by distribution over several applications or by slow release in order to achieve the most effective results. When administered as a single dose, administration will most preferably be in the range of from about 0.1 to mg/kg to about 10 mg/kg.
The exact dose and regimen for administration of these compounds and compositions will necessarily be dependent upon the needs of the individual subject being treated, the type of treatment, and the degree of affliction or need. In general, parenteral administration requires lower dosage than other methods of administration which are more dependent upon absorption.
A further aspect of the present invention relates to pharmaceutical compositions comprising as an active ingredient a compound of the present invention in admixture with a pharmaceutically acceptable, non-toxic carrier. As mentioned above, such compositions may be prepared for use for parenteral (subcutaneous, intraarticular, intramuscular or intravenous) administration, particularly in the form of liquid solutions or suspensions; for oral or buccal administration, particularly in the form of tablets or capsules; or intranasally, particularly in the form of powders, nasal drops or aerosols.
When administered orally (or rectally) the compounds will usually be formulated into a unit dosage form such as a tablet , capsule, suppository or cachet. Such formulations typically include a solid, semi-solid or liquid carrier or diluent. Exemplary diluents and vehicles are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, aginates, tragacanth, gelatin, syrup, methylcellulose, polyoxyethylene sorbitar monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, and magnesium stearate.
The compositions may be prepared by any of the methods well-known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences. 17th edition, Mack Publishing Company, Easton, PA, 1985. Formulations for parenteral administration may contain as common excipients sterile water or saline, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. Examples of vehicles for parenteral administration include water, aqueous vehicles such as saline, Ringer's solution, dextrose solution, and Hank's solution and nonaqueous vehicles such as fixed oils (such as corn, cottonseed, peanut, and sesame), ethyl oleate, and isopropyl myristate. Sterile saline is a preferred vehicle and the compounds are sufficiently water soluble to be made up as a solution for all foreseeable needs. The vehicle may contain minor amounts of additives such as substances that enhance solubility, isotonicity, and chemical stability, e.g., antioxidants, buffers, and preservatives. For oral administration, the formula can be enhanced by the addition of bile salts and also by the addition of acylcamitines (Am. J. Physiol. 251 :332 (1986)). Formulations for nasal administration may be solid and contain as excipients, for example, lactose or dextran, or may be aqueous or oily solutions for administration in the form of nasal drops or metered spray. For buccal administration typical excipients include sugars, calcium stearate, magnesium stearate, pregelatinated starch, and the like.
When formulated for nasal administration the absorption across the nasal mucous membrane is enhanced by surfactant acids, such as for example, glycocholic acid, cholic acid, taurocholic acid, ethocholic acid, desoxychoiic acid, chenodesoxycholic acid, dehydrocholic acid, glycodeoxy-cholic acid, and the like (See, B.H. Vickery, "LHRH and its Analogs-Contraception and Therapeutic Applications", Pt. 2, B.H. Vickery and J.S. Nester, Eds., MTP Press, Lancaster, UK, 1987).
DETAILED DESCRIPTION OF THE INVENTION
The compounds of this invention were prepared by using the general synthetic methods described in the Schemes below.
In Scheme I, the desired amine (Formula 1 ) was either purchased commercially or prepared by reductive amination of an aldehyde (Formula 2) and an amine (Formula 3), and then acylated or sulfonylated with an appropriate acid or sulfonyl chloride. This afforded compounds of the type in Formula 4.
Alternatively (Scheme II), direct alkylation of an acylated amine (Formula 5) was performed to give differentially N,N-disubstituted amides of the type in Formula 6. The alkylation reaction proceeds nicely using potassium t-butoxide as a base and tetrahydrofuran as a solvent.
Methods for the preparation of acid chlorides, sulfonyl chlorides, reductive amination and alkylation of amines are well known in the art. See "Advanced Organic Chemistry", J. March, eds. McGraw-Hill Book Co., Second Edition, 1977.
Scheme I
R3-NH2 + R2 - CHO (formula 2) (formula 3)
NaBH(OAc)3
R3-NH + Cl-Y-R! — ► R3-N-Y-R1
I CH2C12 I
R2 R2 (formula 1) (formula 4)
Scheme II
Figure imgf000011_0002
(formula 5)
Figure imgf000011_0001
Example 1
Preparation of N-AIM-N-(2.4-dichlorobenzvn chloroacetamide (2^.
Figure imgf000012_0001
_
Figure imgf000012_0002
Part A: To 1 g of 2,4-dichlorobenzaldehyde in 6 mL of 1 ,2-dichloroethane was added 428 μL of allyl amine, 280 μL of acetic acid, and 1.8 g of NaBH(OAc)3 in the given order. After 30 minutes, the reaction mixture was diluted with chloroform and saturated aqueous NaHCθ3. The layers were separated and the organic layer was dried (MgSθ4) and concentrated in vacuo affording a colorless oil. Flash chromatography (15% EtOAc-hexane) afforded 476 mg (38%) of l as a colorless oil.
Part B: 476 mg of 1 was dissolved in 5 mL of methylene chloride and 306 μL of Et3N was added. The reaction mixture was cooled to 0°C and 175 μL of chloroacetyl chloride was added and the mixture was stirred for 2 h. The reaction was then diluted with chloroform and washed twice with water. The combined organic layers were dried (MgSθ4) and concentrated in vacuo provided a white solid. Flash chromatography
(15% EtOAc-hexane) afforded 500 mg of £ as a white solid: Low Resolution Mass Spec, m/z (relative intensity): 292 (M+H; 100), 256 (76), 174 (12), 159 (34), 146 (5) Example 2
Preparation of N-Benzyl-N-i"2.4-dichlorobenzyl) chloroacetamide (4).
Figure imgf000013_0001
Figure imgf000013_0002
Part A: 100 g of 2,4-dichlorobenzylamine was dissolved into 600 mL of CH2CI2 and the reaction mixture was cooled to 0°C. Next, 89 mL of Et3N was added followed by the dropwise addition of 50 mL of chloroacetyl chloride. The reaction mixture was stirred for 24 h. The reaction was then washed twice with H2O, dried (MgSθ4) and concentrated in vacuo affording a solid which was triturated with 10% hexane-EtOAc affording 131 g (97%) of 3_ as a pure white solid.
Part B: 500 mg of 3_ was dissolved in 2 mL of THF and 2 mL (10 equiv) of benzyl bromide was added. Next, 276 mg of potassium t-butoxide in 7 mL of THF was added dropwise to the reaction mixture which was then stirred for 30 minutes and finally was concentrated in vacuo. The residue was dissolved in chloroform and washed twice with H2O, dried (MgSθ4) and concentrated in vacuo affording a yellow oil. Flash chromotography (15% EtOAc-hexane) afforded 500 mg (73%) of 4 as a white solid. Low Resolution Mass Spec, m/z (relative intensity): 356 (M+H; 11 ), 196 (4), 91 (100)
Using the methods described in Examples 1 and 2, the following were also prepared: Example 3 N-(2.4-Dichlorobenzvfl-N-methvi chloroacetamide
Low Resolution Mass Spec, m/z (relative intensity): 266 (M+H; 16), 232 (53), 214 (19), 188 (100), 173 (16).
Example 4 N-Benzyl-N-(3-chlorobenzv0 chloroacetamide
Low Resolution Mass Spec, m/z (relative intensity): 308 (M+H; 36), 216 (5), 182 (17), 106 (15), 91 (100).
Example s
N-Benzyl-N-(2.5-dichlorobenzyl) chloroacetamide
Low Resolution Mass Spec, m/z (relative intensity): 342 (M+H; 58), 306 (15), 182 (32), 106 (23), 91 (100).
Example 6 N-(4-Chiorobenzyl) chloroacetamide
Low Resolution Mass Spec, m/z (relative intensity): 308 (M+H; 22), 274 (13), 230 (11 ), 125 (90), 91 (100).
Example 7 N-Benzyl-N-(3.4-dichlorobenzyl) chloroacetamide
Low Resolution Mass Spec, m/z (relative intensity): 342 (M+H; 20), 106 (20), 91 (100). Example 8 N-Benzyl-N-(2-chlorobenzvπ chioroacetamide
Low Resolution Mass Spec, m/z (relative intensity): 308 (M+H; 50), 272 (14), 182 (15), 125 (10), 106 (22), 91 (100).
Example 9 N-Benzyl-N-(2.3-dichlorobenzyl) chloroacetamide
Low Resolution Mass Spec, m/z (relative intensity): 342 (M+H; 36), 306 (15), 182 (13), 106 (17), 91 (100).
Example 10
N-Cyanoethyl-N-(2.4-dichlorobenzyl) methoxyacetamide
H NMR (CDCI3) δ 7.44-7.06 (m, 3H, Ar), 4.70 (s, 2H, (OCH2-0) 4.23 and 4.11 (two singlets, 2H (rotamers), ArCH2-N) 3.63 and 3.55 (two triplets, 2H (rotamers) J = 6.53 Hz each, N-CH2-CH2) 2.68 and 2.63 (two triplets, 2H, J = 6.53Hz each (rotamers) CH2-CN)
Example 11 N-Cyanomethyl-N-(2.4-dichlorobenzyl) chloromethylsulfonamide
1H NMR (CDCI3) δ 7.55-7.28 (m, 3H, Ar), 4.72 (s, 2H, S02CH2-CI) 4.58 (s, 2H, ArCH2N) 3.70 (t, 2H, J = 7.02Hz, N-CH2CH2) 2.61 (t, 2H, J = 7.21 Hz, CH2-CN).
Example 12
N-Cyanoethyl-N-(2.4-dichlorobenzyl) propionamide
Low Resolution Mass Spec, m/z (relative intensity): 285 (M+H; 72), 249 (100), 188 (7), 159 (9), 109 (6). Example 13 N-Cvanoethyl-N-(2.4-dichlorobenzv0 fluoroacetamide
Low Resolution Mass Spec, m/z (relative intensity): 289 (M+H; 100), 253 (40), 159 (20).
Example 14 N-(2.4-Dichlorobenzv0-N-r(3-phenyπpropyπ chloroacetamide
Low Resolution Mass Spec, m/z (relative intensity):
370 (M+H; 62), 336 (53), 302 (23), 185 (40), 159 (69), 125 (31 ), 93 (100).
Example 15 f(N-ChloroacetyO-N-f2.4-dichlorobenzylϊ| αlycine
Low Resolution Mass Spec, m/z (relative intensity): 311 (M+H;100), 274 (46), 232 (16), 159 (11 ), 115 (5).
Example 16 N-(2.4-Dichlorobenzv0-N-[(2-thienyl)ethvπ chloroacetamide
Low Resolution Mass Spec, m/z (relative intensity): 364 (M+H; 100), 326 (7), 266 (12), 159 (29), 110 (56).
Example 17 N-(2.4-Pichlorobenzyl)-N-f(2-thienyl)methyπ chloroacetamide
1H NMR (CDCI3) δ 7.49-6.98 (m, 6H, Ar), 4.78 and 4.66 (two singlets, 2H (rotamers) Ar-CH2-N), 4.75 (s, 2H, COCH2-CI), 4.28 and 4.12 (two singlets, 2H, N-CH2-thiophene) Example 18 N-(3-ChlorobenzvD chloroacetamide
Resolution Mass Spec, m/z (relative intensity): (M+H; 80), 182 (77), 153 (1 1 ), 141 (16), 125 (100), 106 (42).
Example 19 N-(2.3-Dichlorobenzyl) chloroacetamide
Resolution Mass Spec, m/z (relative intensity): (M+H; 59), 216 (100), 159 (74), 106 (42).
Example 20
N-(2,5-Dichlorobenzy0 chloroacetamide
Resolution Mass Spec, m/z (relative intensity): (M+H; 95), 216 (100), 159 (95), 141 (13), 106 (89).
Example 21 N-(2,4-DichlorobenzyP chloroacetamide
Resolution Mass Spec, m/z (relative intensity): (M+H; 38), 217 (20), 185 (43.6), 159 (25), 132 (19), 110 (20), 93 (100), 75 (32).
Example 22 N-|T2.4-Dichlorophenv0ethvπ chloroacetamide
Resolution Mass Spec, m/z (relative intensity): (M+H; 23), 232 (11 ), 185 (56), 139 (9), 170 (13), 93 (100), 75 (24). Compounds of the present invention were tested for IL-lβ protease inhibition activity according to the following protocols:
In Vitro
Partially purified IL-1 β protease is stored at -80°C, thawed on ice, and preincubated for 10 minutes at 37°C with 2.5 mM dithiothreitol in a buffer solution containing 10 mM Tris-HCI (pH 8.0) and 25% (v/w) glycerol. Inhibitors are prepared as stock solutions in dimethyl sulfoxide (DMSO). The protease is preincubated with inhibitor in a volume of 20 μL in a 1.5 mL polypropylene microcentrifuge tube for 15 minutes at 37°C. The volume of compound added to the assay is adjusted to yield a DMSO concentration in the preincubation of <15% (v/v). The enzyme assay is then initiated by the addition of substrate (TRITC-AYVHDAPVRS-NH2) (SEQ I.D. No. 1 ) to yield a final concentration of 67 μM in a final volume of 30 μL. The reaction are carried out for 60 minutes at 37°C in the dark and are terminated by the addition of 10 mL of 10% trifluoroacetic acid (TFA). Following the addition of 115 μL of 0.1% TFA, the samples are analyzed by high pressure liquid chromatography using a reverse phase (C18) column and elution with an acetonitrile/water/TFA gradient. Substrate and product are monitored by their absorbance at 550 nm and elute at 4.2 and 5.2 minutes, respectively.
The compound in example 1- possesses IL-1 β protease inhibition (IC50 = <1.0 μM).
In Vivo
In vivo inhibition (IC50) was determined as follows:
Human monocytes were isolated from heparinized leukopheresis units obtained through Biological Specialty Corporation (Lansdale, PA). Monocytes were purified by Ficoll-Hupaque (Pharmacia Fine Chemicals, Piscataway, NJ) gradient centrifugation and more than 95% pure monocyte populations obtained by centrifugal elutriation. The assay was performed on duplicate samples of freshly isolated human monocytes, cultured in suspension at 37°C and rotated gently in conical bottom polypropylene tubes (Sardstedt Inc., Princeton, NJ). Human monocytes at a concentration of 5 x 106 cells/mL were resuspended in 1 mL of RPMI 1640 (a common tissue buffer from M.A. Bioproducts, Walkersville, MD) containing 1% fetal calf serum (FCS) (HyClone, Logan, UT) and 50 μg/mL gentamycin (Gibco, Grand Island, NY). The cells were treated either with a compound of the invention (i.e. test compound) or with a non-inhibitor (control compound, typically 0.03% DMSO) for 15 minutes and then activated with 0.01 % fixed Staphylococcus aureus (The Enzyme Center, Maiden, MA) for 1 hour. The cells were then centrifuged and resuspended in 1 mL of cysteine, methionine-free RPMI media containing 1% dialyzed FCS (Hyclone). The cells were pretreated with a test compound or control compound for 15 minutes after which 0.01 % fixed S. aureus plus 100 μCi Tran 35-S label (ICN, Irvine, CA) was added and the cells incubated at 37°C for 1 hour. After incubation, cells were centrifuged, washed once in phosphate buffer saline and resuspended in 1 mL RPMI containing 1% fetal calf serum. The cells were again pretreated with a test or control compound for 15 minutes and then 0.01 % S. aureus for 2 hours. At the end of the incubation, cells were centrifuged and supernates saved for immunoprecipitation. Cells were washed once in phosphate buffer saline and then lysed in RIPA, a continuous cell media buffer containing 2 mM phenylmethylsulfonyl fluoride, 10 mM iodoacetate, 1 μg/mL pepstatin A, 1 μg/mL leupeptin and 0.5 TI ) aprotinin.
For the immunoprecipitations, an equal volume of 1% dry milk in RIPA buffer plus 50 μL of resuspended protein A sepharose CL-4B (Pharmacia, Piscataway, New York) was added to supernates and 1 mL of 4% dry milk containing protein A sepharose CL-4B to cell lysates and samples rotated for 30 minutes at 4°C. Beads were then centrifuged down, samples transferred to fresh tubes and incubated overnight with 40 μg rabbit anti-human IL-lβ polyclonal antibody (Genzyme, Cambridge, MA). The IL-lβ proteins were then precipitated with 70 μL protein A sepharose, resuspended in 60 μL SDS sample buffer and run on 15% SGD-PAGE gels. Autoradiography was performed on dried gels and the amount of radioactivity (counts per minute, cpm) quantitated using a Betascope 603 analyzer. Data Analysis
In the monocyte pulse chase assay, each test parameter was run in duplicate.
Data was collected from the Beta Scope using a personal computer, then transferred to the VAX system for calculation of mean cpm and standard deviation of the mean.
When test compounds were evaluated, the percent inhibition of release of mature IL-lβ was calculated as follows:
100 x [1 - (cells treated with stimuli + test compound - unstimulated cells)/(cells treated with stimuli + control compound- unstimulated cells)]
These % inhibition values were then used to calculate IC50 value for each compound. Since the human monocyte pulse chase assay uses primary cells from different donors, each test compound was run in 2-3 separate experiments, using monocytes from 2-3 different donors.
The compound in Example 1 had in vivo IC50 of <10 μM.
SEQUENCE LISTING (1) GENERAL INFORMATION:
(i) APPLICANT: Dolle, Roland E.
Rinker, James M.
(ii) TITLE OF INVENTION: Halomethyl Amides As IL-lβ Protease Inhibitors
(iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Sterling Winthrop Inc.
(B) STREET: 9 Great Valley Parkway
(C) STREET: P.O. Box 3026
(D) CITY: Malvern (E) STATE: PA
(F) COUNTRY: USA
(G) ZIP: 19355
(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette, 3.5 inch, 2.0 MB storage
(B) COMPUTER: Apple Macintosh
(C) OPERATING SYSTEM: Macintosh 7.1
(D) SOFTWARE: Microsoft Word 5.1a (vi) CURRENT APPLICATION DATA;
(A) APPLICATION NUMBER: To be Assigned
(B) FILING DATE: 4/29/94
(C) CLASSIFICATION: 1811 (viii) ATTORNE /AGENT INFORMATION:
(A) NAME: Doreen M. Wells
(B) REGISTRATION NUMBER: 34,278
(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (610) 889-8684
(B) TELEFAX: (610) 889-6364
(2) INFORMATION FOR SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: -1
(D) OTHER INFORMATION: /label= TRITC /note= "TRITC is tetramehylrhodamine isothiocyanate".
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 11 (D) OTHER INFORMATION: /label= Xaa /note= "Xaa is NH2".
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1
Ala Tyr Val His Asp Ala Pro Val Arg Ser 1 5 10

Claims

WHAT IS CLAIMED IS:
1. A compound of the formula (A) or a pharmaceutically acceptable salt thereof:
R3-N-Y-R, (A) wherein:
Y = CO or S0 ;
R1 = independently selected from alkyl, haloalkyl and alkoxyalkyl;
R2 = H, alkyl, (CH2)-aikenyl, aralkyl, heteroaralkyl, carboxyalkyl, cyanoaikyl, aryl, heteroaryl; and
R3 = H, alkyl, (CH2)-alkenyl, aralkyl, heteroaralkyl, aryl, heteraryl.
2. The compound according to claim 1 wherein:
Y = CO;
R1 = haloalkyl; R2 = (CH2)-alkenyl; aralkyl; and
R3 = aralkyl.
3. The compound according to claim 1 selected from the group consisting of: N- Allyl-N-(2,4-dichlorobenzy!) chloroacetamide, N-Benzyl-N-(2,4-dichlorobenzyl) chloro- acetamide, N-Benzyl-N-(3-chlorobenzyl) chloroacetamide, N-Benzyl-N-(2,5-dichloro- benzyl) chloroacetamide, N-Benzyl-N-(3,4-dichlorobenzyl) chloroacetamide, N-Benzyl- N-(2-chlorobenzyl) chloroacetamide, N-Benzyl-N-(2,3-dichlorobenzyl) chloro¬ acetamide.
4. The compound according to claim 1 selected from the group consisting of: N- Cyanoethyl-N-(2,4-dichlorobenzyl) methoxyacetamide, N-Cyanomethyl-N-(2,4-di- chlorobenzyl) chloromethylsulfonamide, N-Cyanoethyl-N-(2,4-dichlorobenzyl) propion- amide, N-Cyanoethyl-N-(2,4-dichlorobenzyl) fluoroacetamide. 5. The compound according to claim 1 selected from the group consisting of: N- (2,4-Dichlorobenzyl)-N-methyl chloroacetamide, N-(4-Chlorobenzyl) chloroacetamide, N-(3-chlorobenzyl) chloroacetamide, N-(2,3-dichlorobenzyl) chloroacetamide, N-(2,
5- dichlorobenzyl) chloroacetamide, N-(2,4-dichlorobenzyl) chloroacetamide.
6. The compound according to claim 1 selected from the group consisting of: N- (2,4-Dichlorobenzyl)-N-[(3-phenyl) propyl] chloroacetamide, [(N-Chloroacetyl)-N-(2,4- dichlorobenzyl)] glycine, N-(2,4-dichlorobenzyl)-N-[(2-thienyl)ethyl] chloroacetamide, N- (2,4-dichlorobenzyl)-N-[(2-thienyl)methyl] chloroacetamide, N-[(2,4-dichlorophenyl)- ethyl] chloroacetamide.
7. A pharmaceutical composition for inhibiting interleukin-1 β protease comprising a compound of the formula (A) or a pharmaceutically acceptable salt thereof:
Figure imgf000023_0001
wherein:
Y = CO or S02;
R1 = independently selected from alkyl, haloalkyl and alkoxyalkyl;
R2 = H, alkyl, (CH2)-alkenyl, aralkyl, heteroaralkyl, carboxyalkyl, cyanoalkyl, aryl, heteroaryl; and
R3 = H, alkyl, (CH2)-alkenyl, aralkyl, heteroaralkyl, aryl, heteraryl.
8. The pharamceutical composition of claim 7 comprising a compound of formula (A) or a pharmaceutically acceptable salt thereof wherein:
Y = CO;
R1 = haloalkyl;
R2 = (CH2)-alkenyl; aralkyl; and R3 = aralkyl.
9. The pharmaceutical composition of claim 7 wherein said compound is selected from one of the groups described in claims 3 to 6.
10. A method of inhibiting interleukin-1 β protease activity in a mammal in need of such treatment comprising administering to said mammal an effective inhibitory amount of a pharmaceutical composition comprising a compound of the formula(A) or a pharmaceutically acceptable salt thereof:
R3— N -Y— R, (A) wherein: Y = CO or Sθ2;
Ri = independently selected from alkyl, haloalkyl and alkoxyalkyl;
R2 = H, alkyl, (CH2)-alkenyl, aralkyl, heteroaralkyl, carboxyalkyl, cyanoalkyl, aryl, heteroaryl; and
R3 = H, alkyl, (CH2)-alkenyl, aralkyl, heteroaralkyl, aryl, heteraryl.
11. The method of claim 10 comprising said compound of formula (A) or a pharmaceutically acceptable salt thereof wherein:
Y = CO;
Ri = haloalkyl;
R2 = (CH2)-alkenyl; aralkyl; and R3 = aralkyl.
12. The method of claim 10 wherein said compound is selected from one of the groups described in claims 3 to 6.
PCT/US1995/005347 1994-04-29 1995-04-28 HALOMETHYL AMIDES AS IL-1β PROTEASE INHIBITORS WO1995029672A1 (en)

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JP7528446A JPH09512556A (en) 1994-04-29 1995-04-28 Halomethyl amides as IL-1β protease inhibitors
MX9605196A MX9605196A (en) 1994-04-29 1995-04-28 HALOMETHYL AMIDES AS IL-1'beta' PROTEASE INHIBITORS.
AU24634/95A AU705321B2 (en) 1994-04-29 1995-04-28 Halomethyl amides as IL-1beta protease inhibitors
EP95918877A EP0758891A4 (en) 1994-04-29 1995-04-28 HALOMETHYL AMIDES AS IL-1-g(b) PROTEASE INHIBITORS
NO964472A NO964472L (en) 1994-04-29 1996-10-21 Halo methylamides as IL-1 prosthesis inhibitors
FI964342A FI964342A0 (en) 1994-04-29 1996-10-28 Halomethylamides as IL-1beta protease inhibitors

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US23642594A 1994-04-29 1994-04-29
US08/236,425 1994-04-29

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WO1995029672A1 true WO1995029672A1 (en) 1995-11-09

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JP (1) JPH09512556A (en)
CN (1) CN1147201A (en)
AU (1) AU705321B2 (en)
CA (1) CA2189036A1 (en)
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US5843904A (en) * 1995-12-20 1998-12-01 Vertex Pharmaceuticals, Inc. Inhibitors of interleukin-1βconverting enzyme
US5874424A (en) * 1995-12-20 1999-02-23 Vertex Pharmaceuticals Incorporated Inhibitors of interleukin-1β converting enzyme
US6008217A (en) * 1995-12-20 1999-12-28 Vertex Pharmaceuticals Incorporated Inhibitors of interleukin-1β converting enzyme
US6162828A (en) * 1995-03-31 2000-12-19 Takeda Chemical Industries, Ltd. Cysteine protease inhibitor
US6204261B1 (en) 1995-12-20 2001-03-20 Vertex Pharmaceuticals Incorporated Inhibitors of interleukin-1β Converting enzyme inhibitors
US6426413B1 (en) 1998-03-09 2002-07-30 Vertex Pharmaceuticals Incorporated Inhibitors of caspases
EP1248612A1 (en) * 2000-01-06 2002-10-16 Merck Frosst Canada Inc. Novel compounds and compositions as protease inhibitors
US6531474B1 (en) 1998-03-19 2003-03-11 Vertex Pharmaceuticals Incorporated Inhibitors of caspases
US7332514B2 (en) 2002-08-30 2008-02-19 Japan Tobacco Inc. Dibenzylamine compound and medicinal use thereof
US7417029B2 (en) 2000-05-19 2008-08-26 Vertex Pharmaceuticals Incorporated Prodrug of an ice inhibitor
US7531570B2 (en) 2004-05-27 2009-05-12 Vertex Pharmaceuticals Incorporated Treatment of diseases using ICE inhibitors
US7772366B2 (en) 1994-06-17 2010-08-10 Vertex Pharmaceuticals Incorporated Inhibitors of interleukin-1β converting enzyme
US7906677B2 (en) 1999-09-27 2011-03-15 Novartis Ag Process for phenylacetic acid derivatives
US9352010B2 (en) 2011-07-22 2016-05-31 The J. David Gladstone Institutes Treatment of HIV-1 infection and AIDS

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CN100441186C (en) * 2003-12-03 2008-12-10 和记黄埔医药企业有限公司 Medical use of artificial lywrine

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Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7772366B2 (en) 1994-06-17 2010-08-10 Vertex Pharmaceuticals Incorporated Inhibitors of interleukin-1β converting enzyme
US6162828A (en) * 1995-03-31 2000-12-19 Takeda Chemical Industries, Ltd. Cysteine protease inhibitor
US8119631B2 (en) 1995-12-20 2012-02-21 Vertex Pharmaceuticals Incorporated Inhibitors of interleukin-1β converting enzyme
US6008217A (en) * 1995-12-20 1999-12-28 Vertex Pharmaceuticals Incorporated Inhibitors of interleukin-1β converting enzyme
US5874424A (en) * 1995-12-20 1999-02-23 Vertex Pharmaceuticals Incorporated Inhibitors of interleukin-1β converting enzyme
US6204261B1 (en) 1995-12-20 2001-03-20 Vertex Pharmaceuticals Incorporated Inhibitors of interleukin-1β Converting enzyme inhibitors
US6258948B1 (en) 1995-12-20 2001-07-10 Vertex Pharmaceuticals, Incorporated Inhibitors of Interleukin-1β converting enzyme
US6423840B1 (en) 1995-12-20 2002-07-23 Vertex Pharmaceuticals Incorporated Inhibitors of interleukin-1β converting enzyme
US7790713B2 (en) 1995-12-20 2010-09-07 Vertex Pharmaceuticals Incorporated Inhibitors of interleukin-1β converting enzyme
US6162790A (en) * 1995-12-20 2000-12-19 Vertex Pharmaceuticals Incorporated Inhibitors of interleukin-1β converting enzyme
US5843904A (en) * 1995-12-20 1998-12-01 Vertex Pharmaceuticals, Inc. Inhibitors of interleukin-1βconverting enzyme
US6426413B1 (en) 1998-03-09 2002-07-30 Vertex Pharmaceuticals Incorporated Inhibitors of caspases
US6531474B1 (en) 1998-03-19 2003-03-11 Vertex Pharmaceuticals Incorporated Inhibitors of caspases
US7358273B2 (en) 1998-03-19 2008-04-15 Vertex Pharmaceuticals Incorporated Inhibitors of caspases
US8691848B2 (en) 1998-03-19 2014-04-08 Vertex Pharmaceuticals Incorporated Inhibitors of caspases
US7906677B2 (en) 1999-09-27 2011-03-15 Novartis Ag Process for phenylacetic acid derivatives
EP1248612A1 (en) * 2000-01-06 2002-10-16 Merck Frosst Canada Inc. Novel compounds and compositions as protease inhibitors
EP1248612A4 (en) * 2000-01-06 2005-09-07 Merck Frosst Canada Inc Novel compounds and compositions as protease inhibitors
US9994613B2 (en) 2000-05-19 2018-06-12 Vertex Pharmaceuticals Incorporated Prodrug of an ICE inhibitor
US8022041B2 (en) 2000-05-19 2011-09-20 Vertex Pharmaceuticals Incorporated Prodrug of an ICE inhibitor
US8329662B2 (en) 2000-05-19 2012-12-11 Vertexd Pharmaceuticals Incorporated Prodrug of an ICE inhibitor
US7417029B2 (en) 2000-05-19 2008-08-26 Vertex Pharmaceuticals Incorporated Prodrug of an ice inhibitor
US9156880B2 (en) 2000-05-19 2015-10-13 Vertex Pharmaceuticals Incorporated Prodrug of an ice inhibitor
US9487555B2 (en) 2000-05-19 2016-11-08 Vertex Pharmaceuticals Incorporated Prodrug of an ice inhibitor
US7807701B2 (en) 2002-08-30 2010-10-05 Japan Tobacco Inc. Dibenzylamine compounds and pharmaceutical use thereof
US7332514B2 (en) 2002-08-30 2008-02-19 Japan Tobacco Inc. Dibenzylamine compound and medicinal use thereof
US7531570B2 (en) 2004-05-27 2009-05-12 Vertex Pharmaceuticals Incorporated Treatment of diseases using ICE inhibitors
US9352010B2 (en) 2011-07-22 2016-05-31 The J. David Gladstone Institutes Treatment of HIV-1 infection and AIDS
US9956260B1 (en) 2011-07-22 2018-05-01 The J. David Gladstone Institutes Treatment of HIV-1 infection and AIDS

Also Published As

Publication number Publication date
CA2189036A1 (en) 1995-11-09
EP0758891A1 (en) 1997-02-26
NO964472D0 (en) 1996-10-21
AU705321B2 (en) 1999-05-20
AU2463495A (en) 1995-11-29
NO964472L (en) 1996-10-21
CN1147201A (en) 1997-04-09
FI964342A (en) 1996-10-28
EP0758891A4 (en) 1997-12-03
MX9605196A (en) 1997-12-31
JPH09512556A (en) 1997-12-16
FI964342A0 (en) 1996-10-28

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