Classifications

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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06086—Dipeptides with the first amino acid being basic
  • C07K5/06095—Arg-amino acid
• • 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
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C259/00—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups
  • C07C259/04—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids
  • C07C259/06—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids having carbon atoms of hydroxamic groups bound to hydrogen atoms or to acyclic carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06008—Dipeptides with the first amino acid being neutral
  • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
  • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06008—Dipeptides with the first amino acid being neutral
  • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
  • C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06008—Dipeptides with the first amino acid being neutral
  • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06086—Dipeptides with the first amino acid being basic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the invention pertains to compounds which are inhibitors of metalloproteases and, in particular, to compounds which inhibit the TNF- ⁇ converting enzyme.
• Tumor necrosis factor- ⁇ (TNF- ⁇ , also known as cachectin) is a mammalian protein capable of inducing a variety of effects on numerous cell types.
• TNF- ⁇ was initially characterized by its ability to cause lysis of tumor cells and is produced by activated cells such as mononuclear phagocytes, T-cells, B-cells, mast cells and NK cells.
• mononuclear phagocytes TNF- ⁇ is initially synthesized as a membrane-bound protein of approximately 26 kD. A 17 kD fragment of the 26 kD membrane-bound TNF- ⁇ is "secreted" and combines with two other secreted TNF- ⁇ molecules to form a circulating 51 kD homotrimer.
• TNF- ⁇ is a principal mediator of the host response to gram-negative bacteria.
• Lipopolysaccharide LPS, also called endotoxin
• endotoxin derived from the cell wall of gram- negative bacteria
• TNF- ⁇ secreted has only been recently elucidated. Kriegler et al. Cell, 53, 45-53, (1988) conjectured that TNF- ⁇ "secretion" is due to the cleaving of the 26 kD membrane-bound molecule by a proteolytic enzyme or protease. Scuderi et. al., J. Immunology, 143. 168-173 (1989), suggested that the release of TNF- ⁇ from human leukocyte cells is dependent on one or more serine proteases, e.g., a leukocyte elastase or trypsin.
• serine proteases e.g., a leukocyte elastase or trypsin.
• a serine protease inhibitor p-toluenesulfonyl-L-arginine methyl ester
• Scuderi et. al. suggested that the arginine methyl ester competes for the arginine-binding site in the enzyme's reactive center and thereby blocks hydrolysis.
• the lysine and phenylalanine analogs of the inhibitor reportedly failed to mimic the arginine methyl ester.
• protease which causes the cleavage of the TNF- ⁇ molecule into the 17 kD protein is, in fact, a metalloprotease which is believed to reside in the plasma membrane of cells producing TNF- ⁇ .
• the physicochemical characteristics of the enzyme have not been published.
• proteases recognize a specific amino acid sequence. Some proteases primarily recognize residues located N-terminal of the cleaved bond, some recognize residues located C-terminal of the cleaved bond, and some proteases recognize residues on both sides of the cleaved bond.
• Metalloprotease enzymes utilize a bound metal ion, generally Zn2 + , to catalyze the hydrolysis of the peptide bond. Metalloproteases are implicated in joint destruction (the matrix metalloproteases), blood pressure regulation (angiotensin converting enzyme), and regulation of peptide-hormone levels (neutral endopeptidase-24.11).
• hydroxamate moities and fused or conjugated bicycloaryl substituents possess hydroxamate moities and fused or conjugated bicycloaryl substituents.
• the myriad potential gelatinase inhibitors covered by the generic formula in EPA 489,577 are amino acid derivatives optionally possessing a hydroxamate group.
• Hydroxamate derivatives useful as angiotensin converting enzyme (ACE) inhibitors are reported in EPO 498,665.
• TACE TNF- ⁇ converting enzyme
• TACE inhibitors would therefore have clinical utility in treating conditions characterized by over-production or unregulated production of TNF- ⁇ .
• a particularly useful TACE inhibitor for certain pathological conditions would selectively inhibit TACE while not affecting TNF- ⁇ (also known as lymphotoxin) serum levels.
• the over-production or unregulated production of TNF- ⁇ has been implicated in certain conditions and diseases, for example: I. Systemic Inflammatory Response Syndrome, which includes:
• Cardiovascular Disease which includes:
• IV. Infectious Disease which includes:
• Mycobacterium tuberculosis Mycobacterium avium intracellulare
• Allergic/Atopic Diseases which includes:
• Dermatologic which includes:
• Neurologic which includes:
• XIV. Renal which includes: Nephrotic syndrome
• Inhibitors of TACE would prevent the cleavage of cell-bound TNF- ⁇ thereby reducing the level of TNF- ⁇ in serum and tissues. Such inhibitors would be of significant clinical utility and could be potential therapeutics for treating the above TNF- ⁇ -related disorders.
• the invention relates to compounds of formula I:
• X is hydroxamic acid, thiol, phosphoryl or carboxyl; m is 0, 1 or 2; R***, R 2 and R3 each independent of the other is hydrogen, alkylene(cycloalkyl),
• B is unsubstituted or substituted C2 to Cs alkylene; and the pharmaceutically acceptable salts thereof.
• the compounds of formula I are useful as metalloprotease inhibitors, and particularly useful as inhibitors of the TNF- ⁇ converting enzyme (TACE).
• the invention also relates to a method of treating a mammal having a disease characterized by an overproduction or an unregulated production of TNF- ⁇ .
• the method comprises the steps of administering to the mammal a composition comprising an effective amount of a biologically active compound of formula II:
• X is hydroxamic acid, thiol, phosphoryl or carboxyl; m is 0, 1 or 2;
• R-, R 2 and R-3 each independent of the other is hydrogen, alkylene(cycloalkyl), OR 4 , SR 4 , N(R )(R 5 ), halogen, substituted or unsubstituted Ci to C ⁇ alkyl, Ci to
• R 4 and R--> are each, independent of the other, hydrogen or substituted or unsubstituted Ci to C8 alkyl; n is 0, 1 or 2;
• Y is hydrogen, unsubstituted or substituted Ci to C8 alkyl, alkylene(cycloalkyl), the group -R 8 -COOR 9 or the group -R 1 ⁇ N(R 1 !(R 12 ); wherein R8 is Ci to C ⁇ alkylene; R ⁇ is hydrogen or Ci to C alkyl; R-® is unsubstituted or substituted Cl to C8 alkylene; and R- ⁇ - and R 2 are each, independent of the other, hydrogen or Cl to C8 alkyl; provided that when n is 1, A is a protected or an unprotected ⁇ -amino acid radical; and when n is 2, A is the same or different protected or unprotected ⁇ -amino acid radical; and the pharmaceutically acceptable salts thereof; wherein the compound is capable of reducing serum TNF- ⁇ levels by at least 80% when administered at 25mg kg in a murine model of LPS-induced sepsis syndrome; and a pharmaceutically acceptable carrier.
• compositions for treating the above-listed disorders comprising a compound according to formula II and protein having TNF- binding activity.
• the invention is directed to a compound of formula I:
• X is hydroxamic acid, thiol, phosphoryl or carboxyl; m is 0, 1 or 2;
• R ⁇ , R 2 and R 3 each independent of the other is hydrogen, alkylene(cycloalkyl), OR 4 , SR 4 , N(R 4 )(R 5 ), halogen, substituted or unsubstituted Ci to C8 alkyl, Ci to
• R6 is substituted or unsubstituted Ci to C ⁇ alkyl and R 7 is OR 4 , SR 4 , N(R 4 )(R 5 ) or halogen, wherein R 4 and R 5 are each, independent of the other, hydrogen or substituted or unsubstituted Cl to C8 alkyl; n is 0, 1 or 2; provided that when n is 1, A is a protected or an unprotected ⁇ -amino acid radical; provided that when n is 1 , A is a protected or an unprotected ⁇ -amino acid radical; when n is 2, A is the same or different protected or unprotected ⁇ -amino acid radical; and
• B is unsubstituted or substituted C2 to C8 alkylene; and the pharmaceutically acceptable salts thereof.
• the compounds of formula I are useful as inhibitors of TNF- ⁇ secretion, and particularly useful as inhibitors of the TNF- ⁇ converting enzyme (TACE).
• TACE TNF- ⁇ converting enzyme
• the invention also relates to a method for treating a mammal having a condition or a disease characterized by overproduction or unregulated production of TNF- ⁇ , comprising administering to the mammal a composition comprising an effective amount of a biologically active compound of formula II:
• X is hydroxamic acid, thiol, phosphoryl or carboxyl; m is 0, 1 or 2;
• R , R 2 and R 3 each independent of the other is hydrogen, alkylene(cycloalkyl),
• R 4 and R5 are each, independent of the other, hydrogen or substituted or unsubstituted Cl to C ⁇ alkyl; n is 0, 1 or 2; Y is hydrogen, unsubstituted or substituted Cl to C8 alkyl, alkylene(cycloalkyl), the group -R 8 -COOR 9 or the group -R 10 N(R 1 ⁇ (R 12 ); wherein R 8 is Ci to C ⁇ alkylene; R 9 is hydrogen or Cl to C8 alkyl; R 0 is unsubstituted or substituted Ci to
• R-*- - • ⁇ and R* 2 are each, independent of the other, hydrogen or Cl to
• A is a protected or an unprotected ⁇ -amino acid radical; and when n is 2, A is the same or different protected or unprotected ⁇ -amino acid radical; and the pharmaceutically acceptable salts thereof; wherein the compound is capable of reducing serum TNF levels by at least 80% when administered at 25mg/kg in a murine model of LPS-induced sepsis syndrome; and a pharmaceutically acceptable carrier.
• the invention includes pharmaceutical compositions containing a compound according to formula I as the active component.
• pharmaceutical compositions comprising a compound according to formula II and a protein which binds TNF are described.
• An example of a protein which binds TNF is an anti-TNF antibody or a soluble TNF receptor which is described in EPA 0418014, assigned to the assignee of the instant application. The disclosure of EPA 0418014 is incorporated herein by reference.
• Alkyl means a straight or branched, univalent, saturated or unsaturated hydrocarbon group of 1 to 8 carbon atoms. Alkyl groups include the straight-chain groups methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, vinyl, allyl, butenyl, pentenyl, hexenyl, heptenyl and octenyl as well as the branched isomers thereof.
• Substituted alkyl means an alkyl group substituted with one or more of hydroxy, amino, halogen, or thiol.
• Alkylene means a bivalent alkyl group as defined above.
• Substituted alkylene means an alkylene group substituted with one or more of hydroxy, amino, halogen or thiol groups.
• Aryl means an aromatic or heteroaromatic group, including for example, phenyl, naphthyl, pyridyl, quinolyl, thienyl, furyl and the like, optionally substituted with one or more of Cl to C ⁇ alkyl, hydroxy, amino, halogen, thiol or alkyl groups.
• Alkylene(cycloalkyl) refers to groups of the structure -R1 3 -R-- 4 wherein R* 3 is an alkylene as defined above, and R-- 4 is a univalent cyclic alkane radical, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.
• Alkylenearyl means the group -R15-R 16, wherein Rl5 is a substituted or unsubstituted alkylene group as defined above, and R 16 is a substituted or unsubstituted aryl group as defined above.
• ⁇ -Amino acid refers to any of the 22 common amino acids, e.g., alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamine, glutamic acid, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.
• Protected side chain of an ⁇ -amino acid means the side chains of the amino acid are permanently or temporarily coupled to a chemical group which protects or prevents the side chain from undesired branching, structural modification or rearrangement which can occur during subsequent synthetic steps.
• Use of such protecting groups for these purposes is well known in the art, as are the protecting groups themselves. Examples of common protecting groups are N-tert-butyloxycarbonyl (Boc) and N-9- fluorenylmethyloxycarbonyl (Fmoc).
• Bioly active as used in defining certain compounds of formula II, designates a compound capable of (a) inhibiting secretion of TNF- ⁇ ; (b) preventing cleavage of membrane-bound TNF- ⁇ by TACE; or (c) reducing serum TNF levels by at least 80% when administered at 25 mg/kg in a standard murine model of LPS-induced sepsis syndrome.
• preferred radicals for X are hydroxamic acid, thiol and phosphoryl. More preferred X radicals are hydroxamic acid and thiol, while the most preferred radical is hydroxamic acid.
• the preferred value for m is 1.
• R* or R 2 radicals are hydrogen, Ci to C alkyl and Ci to C alkylenearyl. Where R* or R 2 is alkyl, preferred is Ci to C6 alkyl and most preferred is Ci to C4 alkyl. Where R* or R 2 is alkylenearyl, preferred alkylene groups are Cl to C6 alkylene, and more preferred is Ci to C4 alkylene; and preferred aryl groups are phenyl and substituted phenyl. The most preferred alkylenearyl group for Rl or R 2 is Ci to C4 alkylenephenyl. The most preferred group for R* is hydrogen and the most preferred group for R 2 is isobutyl.
• R 3 radicals are substituted and unsubstituted Ci to C ⁇ alkyl and Ci to C ⁇ alkylenearyl.
• R 3 is alkyl, preferred is Ci to C6 alkyl and more preferred is Ci to C4 alkyl, with t- butyl being most preferred.
• R 3 is Ci to C ⁇ alkylenearyl, preferred alkylene groups are Ci to C6 alkylene, and more preferred is Ci to C4 alkylene; and preferred aryl groups are phenyl, naphthyl, and thienyl, each optionally substituted with hydroxy, amino, halogen, thiol or alkyl groups.
• R 3 Preferred groups for R 3 are therefore Ci to C4 alkylenephenyl, Ci to C4 alkylenenaphthyl, and Cl to C4 alkylenethienyl. More preferred is Ci to C4 alkylenenaphthyl, with methylenenaphthyl being most preferred.
• R 3 is a protected or unprotected side chain of a naturally occurring ⁇ -amino acid
• R 3 preferably is an arginine, lysine, tryptophan or tyrosine side chain.
• the most preferred radicals for R 3 are t-buyl, methylene(cyclohexyl) and methylene-(2'naphthyl).
• the radical A is preferably an unprotected naturally-occurring amino acid residue. More preferred naturally-occurring residues are the alanyl radical or an unprotected seryl radical. The most preferred radical for A is an alanyl residue. Further preferred compounds are those where n is 0 or 1, while most preferably n is 1.
• Preferred radicals for B are C2 to C alkylene. More preferred radicals are C2 to C4 alkylene, with dimethylene being most preferred.
• Y is preferably hydrogen, unsubstituted or substituted Ci to C8 alkyl or the group -R10N(R1 1)(R1 2 ).
• Rl I and R ⁇ 2 preferably are each independently hydrogen or Ci to C6 alkyl.
• More preferred R*0 radicals are unsubstituted or substituted Ci to C4 alkylene, with dimethylene being most preferred.
• More preferred radicals for RlO and Rl * • are hydrogen or Ci to C4 alkyl, with hydrogen being most preferred.
• the inhibitor compounds may be prepared by converting the carboxylic acid or ester compound (Io), wherein R is H or Ci to C alkyl, and P is CBZ, BOC, FMOC or other suitable protective group (Greene T., Wuts P., "Protective Groups in Organic Synthesis", 2nd Ed.; Wiley: New York, 1991; Chapter 7), to the corresponding hydroxamic acid or hydroxamic ester compound (Ip).
• R is H or Ci to C alkyl
• P is CBZ, BOC, FMOC or other suitable protective group
• R' is H, TMS, t-Bu, Bzl or other group made by treating these compounds, or an activated form of the carboxylic acid, (Bodanszky, M., Bodanszky, A., "The Practice of Peptide Synthesis”; Springer- Verlag: Berlin, 1984; Chapter II) with a hydroxylamine reagent under conditions which effect the conversion. This is followed by the subsequent removal of the protective group P and R' to generate compound (Iq).
• a hydroxylamine reagent described above can be hydroxylamine or alternatively, it can be an O-protected hydroxylamine such as commercially available O-trimethylsilyl hydroxylamine, O-tert-butylhydroxylamine, or O-benzylhydroxylamine.
• precursor compound (Io) may be carried out by condensing the dicarboxylate compound (Ie), with the amine (In), wherein R" is an activating group
• compound (Ie) may be typically carried out as follows: the sodium salt of the 2-oxocarboxylate compound (la), is esterified with benzyl bromide to produce the benzyl ester (lb).
• benzyl bromide to produce the benzyl ester (lb).
• Several examples of compound (la) are commercially available as various salts or carboxylic acids. Others can be made synthetically (see, for example, Nimitz, J. et al., /. Org. Chem. 46:211, 1981; and Weinstock, L.et al., Synth. Commun. 1J:943, 1981).
• the benzyl ester compound (lb) is treated with a Wittig reagent, typically methyl or tert-butyl triphenylphosphoranylidene acetate, to form the alkene (Ic), as a mixture of E- and Z- isomers.
• a Wittig reagent typically methyl or tert-butyl triphenylphosphoranylidene acetate
• Reduction of the alkene compound (Ic) is carried out with hydrogen, in the presence of an appropriate catalyst (typically palladium on activated charcoal), to both hydrogenate the double bond and to remove the benzyl ester, giving the mono-ester compound (Id) as a enantiomeric mixture.
• Compound (Ie) is obtained by treating the mono-ester compound (Id) using any of a variety of conventional carboxylate activation procedures.
• the preparation of the amine compound (In) is achieved by condensing the compound (II) with the amine compound (Ik), wherein P' is an amine protective group other than P, and R" is an activating group such as an active ester, anhydride or other group that causes condensation with the amine terminus of (Ik) to occur with formation of a peptide bond, to give compound (Im). Removal of P is accomplished under appropriate conditions (Bodanszky, M.; Bodanszky, A., "The Practice of Peptide Synthesis”; Springer-Verlag: Berlin, 1984; Chapter III) to produce compound (In), either as corresponding amine or the amine salt.
• Compound (II) is prepared from the commercially available N-protected carboxylic acid, or which can be synthesized by standard methods. Preparation of (Ik) is carried out by condensing the compound (Ii) with mono- protected diamine (Ih) wherein P is an amine protective group such as CBZ, BOC, FMOC or other suitable protective group; and P' is an amine protective group other than P, and R" is an activating group such as an active ester, anhydride or other group that causes condensation with the unprotected amine terminus of compound (Ih) to occur with formation of a amide bond to give compound (Ij). Removal of P' under appropriate conditions is accomplished to produce compound (Ik), either as the corresponding amine or the amine salt.
• P is an amine protective group such as CBZ, BOC, FMOC or other suitable protective group
• R" is an activating group such as an active ester, anhydride or other group that causes condensation with the unprotected amine terminus of compound (
• Precursor compound (Ih) is prepared in two steps from the amine-nitrile (If).
• compound (If) is available commercially and others can be easily synthesized by classical methods.
• the amine-nitrile (If) is protected with an appropriate protective group reagent to produce the protected amine-nitrile (Ig).
• P is typically CBZ, BOC or FMOC groups, but can be any other suitable group.
• the protected amine-nitrile (Ig) undergoes reduction with a reagent such as borane-methyl sulfide complex or sodium borohydride/cobalt (II) chloride, to give the mono-protected diamine (Ih) which can be isolated as its amine salt.
• Compound (Ii) is prepared from the carboxyl form of the corresponding P'-protected dipeptide or P'-protected amino acid by conventional methods, or can be purchased commercially.
• the compounds of formula II may be administered orally, parenterally, via inhalation, transdermally, intra-nasally, intra-ocularlly, mucosally, rectally and topically. Such administration may be in dosage unit formulations containing conventional adjuvants and carrier materials.
• parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intracisternal injection or infusion techniques.
• the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
• carrier materials are well known, and are described, for example, in European Patent Application No. 0 519 748, incorporated herein by reference. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
• the following examples are illustrative of the invention. Thin layer chromotagraphy was performed using silica gel 60 F254 plates. Reaction schemes for Examples 1 through 9 are appended and follow Example 14.
• Compound A refers to the compound N- ⁇ D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl ⁇ L-3-(2'naphthyl)- alanyl-L-alanine amide described by Spatola et. al., Peptides: Chemistry and Biology, Proceedings of the 12th American Peptide Symposium, eds. Smith, J.A., Rivier, J.E., ESCOM, Leiden, Netherlands. Compound A was prepared using the following procedure, and a reaction scheme therefor is appended as reaction scheme A.
• the diastereomers of (A) were separated and purified by reverse phase HPLC using a Ci column, eluting with water containing 0.1% trifluoracetic acid with a gradient of acetonitrile (0-60% in 30 minutes) and also containing 0.1% trifluoroacetic acid, ("Method A"), to give a purified early eluting diastereomer and a purified late eluting diastereomer, which had retention times of 21 and 23 minutes respectively.
• TLC Rf 0J3 (chloroform-methanol 9:l)
• the reaction was stirred at -15 °C for 2 hours, then at room temperature for 18 hours.
• the N,N- dimethylformamide was removed in vacuo and the resulting solid was dissolved in 1 liter of hot ethyl acetate.
• the hot solution was washed with IM HC1 (3x150 ml), water (150 ml), saturated sodium bicarbonate (3x150 ml) and finally with brine (150 ml). After drying over anhydrous magnesium sulfate, the hot solution was concentrated in vacuo.
• the non- homogeneous solution was transferred to a flask containing 100 ml of absolute ethanol, and heated until it became homogeneous.
• the hot solution was dried over a small amount of anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain a solid.
• the solid was triturated with cold 1 :3 ethyl acetate- hexane and collected by filtration to give 1.46g (71% yield) of L-3-(2'-naphthyl)alanyl-L- alanine, 2-(benzyloxy-carbonyl-amino)-ethyl amide (lj) as a white solid.
• the diastereomers of (1) were separated by reverse phase HPLC using a Ci ⁇ column and eluting with water containing 0.1% trifluoroacetic acid with a gradient of acetonitrile (0-60% in 30 minutes) also containing 0.1% trifluoroacetic acid (hereinafter "Method A”).
• the purified diastereomers (In) and (lo) had retention times of 20 and 22 minutes, respectively.
• Diastereomer (In) showed the following NMR data.
• 4-methylpentanoyl chloride 12(b) was prepared by adding dropwise with stirring, 38ml (0.52 mol) of thionyl chloride to 50g (0.43 mol) of 4-methylvaleric acid over 30 minutes. The mixture was heated during the addition, leading to vigorous HC1 gas evolution. When the thionyl chloride addition was completed, the reaction mixture was refluxed for 1 hour. The reaction mixture was distilled, with collection of the distillate between 135 and 148 °C.
• reaction mixture was cooled to -78 °C and 34.6ml (0.25 mol) of 12(b) was added over 10 minutes. Stirring was continued at -78 °C for one hour, then the reaction mixture was allowed to stir at room temperature overnight. The tetrahydrofuran was removed in vacuo by rotary evaporation to produce an orange residue.
• reaction mixture was stirred at -5 °C for 20 minutes, then a solution of 25.0g (0.0934 mol) of 12(d) dissolved in 380 ml of anhydrous tetrahydrofuran (pre-cooled to -5°C) was added.
• the reaction was stirred at -5 °C for 2 hours, then water (50ml) was added.
• the reaction was allowed to warm to room temperature.
• the tetrahydrofuran was removed by rotary evaporation to produce a residue.
• the residue was dissolved in ethyl acetate (250ml) and washed with water (125ml) and brine (125ml).
• the reaction was filtered to remove the solid, using 500ml of dichloromethane and 250ml of water to rinse the solid collected.
• the filtrate was tranferred to a separatory funnel and the layers were separated.
• the lower(dichloromethane) layer was filtered and concentrated in vacuo by rotary evaporation to produce a dark oil.
• the oil was purified with two successive flash chromatography columns [each column: 500 grams of silica gel 60, eluted with 1900ml of 1:4 ethyl acetate: hexane, and 1000 ml of ethyl acetate] to produce 26.6 (65% yield) of 12(f) as a viscous oil.
• the diastereomers (2) and (3) can be made from L-3-(2'-naphthyl)alanine amide hydrochloride (8b) and compound (2d), using the sequence of reactions used to prepare Compound (1) from Compounds (lj) and (Id). Compounds (2) and (3) were separated by reverse phase HPLC as described above.
• Compound (5b) was prepared from Compound (5a) in 87% yield, by the method used to prepare Compound (lj).
• TLC Rf 0J 1 (chloroform-isopropanol 9:1); -1H NMR (CDCl3) ⁇ 1.28(d,3H), 1.43(m,lH), 1.70(m,4H), 3.30(m,6H), 3.91(m,2H) 4.34(m,lH), 5.03(s,2H), 5J l(s,2H), 5.22(s,2H), 5.50(m,lH), 7.01(m,lH), 7.33(bmJ5H), 7.76(dJH), 9.25(m,lH), 9.41(m,lH); 13 C NMR (CDCI3) ⁇ 17.7, 24.5, 31J, 40.3, 40.6, 44.1, 48.6, 54.1, 66.7, 66.9, 68.9, 127.9, 128.0, 128.1, 128.2, 128.
• Compound (5c) was prepared from Compounds (5b) and (Id) in 88% yield, as a mixture of diastereomers, with the method used to prepare Compound (Ik).
• !H NMR (d6-DMSO; mixture of diastereomers) ⁇ 0.79(bm,6H), 1.06(m,lH), 1.13 & 1.20(d, 3H), 1.52(bm,6H), 2.40(m,lH), 2.71(m,lH), 3.03(bm,5H), 3.47 & 3.54(s,3H), 3.88(m, 2H), 4J8(m,2H), 5.00(s,2H), 5.04(s,2H), 5.24(s,2H), 7.35(bm,18H), 7.59 & 7.71(dJH), 7.66 & 7.94(t,lH), 8J3 & 8.45(d,lH); 13 C NMR(d6-DMSO); mixture of diastereomers) ⁇ 17.
• Hydroxamate (5d) was prepared from Compound (5c) in 78% yield as a mixture of diastereomers.
• Compound (6b) was prepared from Compounds (6a) and (Id) in 69% yield using the method previously described to prepare Compound (A2).
• TLC Rf 0.21 and 0.29 (chloroform-isopropanol 9: 1);
• H NMR (d6-DMSO; mixture of diastereomers) ⁇ 0.81(m,3H), 0.88(m,3H), 1.17 & 1.23(d,3H), 1.40(bm,8H), 2.46(m,3H), 2.78(m,lH), 2.98(m,2H), 3.54 & 3.56(s, 3H), 4.08(m,lH), 4J6(mJH), 5.00(s,2H), 7.04(m,lH), 7.23(tJH), 7.34(m,6H), 7.58 & 7.68(d,lH), 8J0 & 8.42(d,lH).
• Compound (6c) was prepared from Compound (6b) in 48% yield, using the method previously described to prepare (A3).
• the diastereomers (6A) and (6B) were prepared from Compound (6c) by the method used to prepare Compound (1) from Compound (lm).
• HPLC purification (method A) produced an early-eluting iso er (6A) and a late-eluting isomer (6B).
• Compound (7b) was prepared from Compound (7a) as a mixture of diastereomers in 64% yield with the method used to synthesize Compound (6b).
• TLC Rf 0.53 and 0.57 (chloroform-isopropanol 9:1); IH NMR (d6-DMSO; mixture of diastereomers) ⁇ 0.60 & 0.68(d,3H), 0.76 & 0.82(d,3H), 1.04(m,lH), 1J9 & 1.26(d,3H), 1.40(m,2H), 2.31(bm, 2H), 2.68(m,2H), 3.05(mJH), 3.48 & 3.55(s,3H), 4.20(mJH),4.44(mJH), 5.03 & 5.04(s,2H), 6.87(m,2H), 7.06(bsJH), 7J5(m,3H), 7.38(bm,5H), 7.69 & 7.78(dJH), 8J5 & 8.
• Compound (7c) was prepared from Compound (7b) in 48% yield with the method used to prepare Compound (6c). A single diastereomer of Compound (7c) was isolated by
• the dicyclohexylurea by-product was removed by filtration, and the filtrate was transferred to a flask containing 1.5 ml (0.022 mol) of concentrated NH4OH. After the mixture had stirred at room temperature for 1 hour, the solvent was removed in vacuo to give a residue. The residue was dissolved in ethyl acetate (350 ml) and washed with water (100 ml), IM HCL (100 ml), water (100 ml), saturated sodium bicarbonate solution (100 ml) and finally with brine (100 ml). After drying over anhydrous magnesium sulfate, the solution was filtered and concentrated in vacuo to produce a solid.
• the diastereomers (8) and (9) can be made from L-3-(2'-naphthyl)alanine amide hydrochloride (8b) and (Id), using the sequence of reactions used to prepare Compound (1) from Compounds (lj) and (Id).
• N-BOC-L-3-(2'-naphthyl)alanyl-L-(O-benzyl)serine amide (10a) was prepared from N-BOC-L-3-(2'-naphthyl)alanine and L-(O-benzyl)serine amide in 80% yield with the method used to prepare (7a).
• Compound (10c) was prepared from Compounds (10b) and (Id) as a mixture of diastereomers in 97% yield following the method used to prepare Compound (Ik).
• TLC Rf 0.69 and 0.73 (chloroform-isopropanol 9: 1); l U NMR (d6-DMSO; mixture of diastereomers) ⁇ 0.25 & 0.40(d,3H), 0.68 & 0.79(d,3H), l.OO(mJH), 1.32(m,2H), 2.31(bm,3H), 2.64(mJH), 2.98(mJH), 3.37 & 3.50(s,3H), 3.68(m,2H), 4.48(mJH), 4.49 & 4.53(s,2H), 4.72(mJH), 7.35(bm,6H), 7.44(m,4H), 7.78(m,4H), 7.93 & 7.99(dJH), 8.30 & 8.49(dJH); 13 C N
• Compound (10) was prepared from Compound (lOd) in 84% yield with the method used to prepare Compound (In). HPLC retention times: 25.2 and 27J minutes (method A). MS: mle All (M+).
• Compound (lib) was prepared from Compounds (11a) and (Id), in 86% yield using the method previously described to prepare Compound (A2).
• TLC Rf 0.57 and 0.62 (chloroform-isopropanol 9:1); iH NMR (d6-DMSO; mixture of diastereomers) ⁇ 0.23 & 0.40(d,3H), 0.70 & 0.79(d,3H), 1.01(m,2H), 1J8 & 1.26(d,3H), 1.32(m,2H), 2.22(m,2H), 2.53(d,3H), 2.92(mJH), 3.22(mJH), 3.38 & 3.39(s,3H), 4.22(mJH), 4.63(mJH), 7.44(m,4H), 7.73(sJH), 7.81(m,4H), 8.22 & 8.46(dJH).
• Compound (11) was prepared from Compound (lib) in 23% yield using the method previously described to prepare Compound (A3).
• N-Boc-L-tert-leucine 13(b) was prepared by treating L-tert-leucine (Aldrich Chemical) with di-tert-butyl dicarbonate and diisopropylethyl amine in dimethylfluoride (DMF). Then (13b) was treated with NHS and dicyclohexylcarbodiimide (DCC) in anhydrous tertrahydrofuran to produce N-Boc-L-tert- leucine N-hydroxysuccinimidyl ester, which then is coupled with (lh) from Reaction
• L-tert-leucine Aldrich Chemical
• DCC dicyclohexylcarbodiimide
• the following example demonstrates the selective in vitro inhibition of T-cell TNF- ⁇ secretion, as compared to TNF- ⁇ and IFN- ⁇ secretion, by Compound 1.
• Human peripheral blood T-cells were purified from peripheral blood mononuclear cells by rosetting with 2-aminoethylisothiouronium bromide hydrobromide-treated sheep erythrocytes. After hypotonic lysis of sheep erythrocytes, monocytes were depleted by plastic adherence for one hour at 37 °C.
• the peripheral blood T-cells were stimulated with anti-CD3 antibody (OKT3) which was immobilized on the culture wells at 10 ⁇ g/ml in PBS plus 10 ng/ml of the phorbol ester, PMA.
• Culture medium comprised RPMI 1640 medium containing 10% fetal bovine serum, 50 U/ml penicillin, and 50 ⁇ g/ml streptomycin. The stimulation was performed in the presence or absence of the inhibitor Compound 1 (200 ⁇ M), and TNF- ⁇ in the medium was assayed by ELISA. Results are shown in Table I.
• Compound 1 inhibited TNF- ⁇ release by 72% and 63%, respectively, while there was no inhibitory effect on the release of TNF- ⁇ or interferon- ⁇ .
• Compound 1 clearly demonstrates selective inhibition of TNF- ⁇ secretion and has no effect on either
• TNF- ⁇ or interferon- ⁇ secretion are TNF- ⁇ or interferon- ⁇ secretion.
• This example describes the effects of Compound 1 on cell surface TNF- ⁇ for human
• T-cells which have been stimulated by PMA and ionomycin.
• the alloreactive human T-cell clone, PL-1 does not express cell surface TNF- ⁇ in the absence of stimulation.
• cell surface TNF- ⁇ as well as the ligands for CD40 and 41 BB, are rapidly induced on the cell surface.
• Detection of cell surface TNF- ⁇ was performed by staining with an Fc fusion protein consisting of an Fc portion of a human IgGl molecule (IgGFc) coupled with an extracellular domain of TNF receptor (p80).
• IgGFc human IgGl molecule
• Detection of cell surface ligands for 41BB and CD40 was performed by staining with analogous Fc fusion proteins consisting of IgGFc and extracellular domains of 41BB and CD40, respectively.
• a fusion molecule consisting of IgGFc and the extracellular portion of the IL-4 receptor (EL-4R:Fc) was utilized as a negative control for staining, since PL-1 cells do not express cell-surface IL-4 in response to PMA stimulation.
• TNFR:Fc and IL-4R:Fc fusion proteins are described in EP 0 464 533, incorporated herein by reference.
• Compound 1 for increasing cell surface TNF- ⁇ is apparent.
• the effect of Compound 1 was specific for TNFR:Fc binding as no increase on 41BB:Fc or CD40:Fc binding was detected.
• a substantial increase in cell-surface TNF- ⁇ resulted in a 100-fold increase in TNFR:Fc binding in the presence of Compound 1 (MFI was 616) as compared to an MFI of 7 in absence of Compound 1, after 18 hours of stimulation. Under the same conditions, 41BB:Fc and CD40:Fc binding were enhanced only approximately 2-fold.
• mice Female Balb/c mice (18-20g) were injected i.v. with 400 ⁇ g of LPS. Simultaneously, the mice were injected subcutaneously with 500 ⁇ g of Compound A or Compound 1 in 0.5 ml of saline containing 0.02% DMSO. Control mice received LPS intravenously and saline/DMSO subcutaneously. Two hours following the LPS injection, serum was obtained and pooled from two mice in each treatment group. TNF- ⁇ levels were determined by ELISA and are shown in the following Table III.
• Compound 1 inhibits the secretion of TNF- ⁇ at least by 80%, and essentially by 100%, as the TNF- ⁇ levels were undetectable. Comparatively, Compound A reduced serum TNF- ⁇ levels by approximately 60% as compared to the saline/DMSO control.
• mice were injected i.v. with
• mice were injected subcutaneously with 500 ⁇ g
• Example 1 Compound 1 reduced serum TNF- ⁇ levels by 82% as compared to TNF- ⁇ levels in mice that received LPS only. As compared to mice that received LPS + saline, Compound 1 reduced serum TNF- ⁇ levels by 76%. In experiment 2, Compound 1 reduced serum TNF- ⁇ levels by 89% as compared to TNF- ⁇ levels in mice that received LPS only. As compared to mice that received LPS + saline, Compound 1 reduced serum TNF- ⁇ levels by 85%. In experiment 3, Compound 1 reduced serum TNF- ⁇ levels by 85% as compared to TNF- ⁇ levels in mice that received LPS only. As compared to mice that received LPS + saline, Compound 1 reduced serum TNF- ⁇ levels by 84%.
• Compound 1 reduced serum TNF- ⁇ levels by 85.4 ⁇ 2.98% as compared to TNF- ⁇ levels in mice that received LPS only. From Tables III and IV, Compound 1 effectively reduces serum TNF- ⁇ levels by at least 80% when administered at 25 mg/kg in a murine model of LPS-induced sepsis syndrome.
• mice Female Balb/c mice (18-20g) were injected i.v. with 450 ⁇ g of LPS. Simultaneously, the mice were injected subcutaneously with 250 ⁇ g of Compound A or Compound 1 in 0.25 ml of saline containing 0.02% DMSO. Control mice received LPS intravenously and saline/DMSO subcutaneously. Two hours following the LPS injection, serum was obtained from three mice in each treatment group. TNF- ⁇ levels were determined by ELISA. The results are expressed as the mean optical density (OD) obtained in the ELISA from each treatment group, and are shown in Table V. The background OD of the control sample was 0J62 ⁇ 0.003.
• Table V illustrates the effect of Compound 1 and Compound A on inhibiting serum
• TNF- ⁇ release in LPS -stimulated mice Compound 1 reduced serum TNF- ⁇ levels to those of the control, thereby indicating a complete inhibition of TNF- ⁇ secretion at 250 ⁇ g/ml.
• Each of Compound 1 and Compound A was diluted to 50 ⁇ M in normal mouse serum and incubated at 37°C. At various times, aliquots were withdrawn, diluted 100-fold into ice-cold PBS, and tested for inhibitory efficacy against purified TACE. After 40 minutes, Compound A showed a decrease in inhibitory effect corresponding to a 3-4 fold loss in concentration of the compound, and Compound 1 showed no decrease in inhibitory effect.
• N C-(CH 2 ) n -NH-P (Ig)

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