WO1996029088A1 - Novel piperazine derivatives as type il-1 receptor antagonists - Google Patents

Abstract

Piperazine derivatives which act as potent and highly specific interleukin-1 receptor antagonists. Piperazine derivatives have now been discovered which will bind to the interleukin-1 receptor but will not activate it. As a result, these compounds are useful in the treatment of diseases which are mediated by IL-1. Included among the IL-1 receptor antagonists of the present invention are substituted piperazines which contain amino acid derivatives. The IL-1 receptor antagonists of the present invention are also useful in assays for the determination of IL-1 activity.

Description

NOVEL PIPERAZINE DERIVATIVES AS TYPE IL-1 RECEPTOR ANTAGONISTS

BACKGROUND OF THE INVENTION

Cytokines are extracellular proteins that modify the behavior of cells and control major physiological processes. A number of cytokines are produced by activated macrophages and are considered proinflammatory. For example, interleukin-l (IL-1) has been shown to mediate such diseases as rheumatoid arthritis, inflammatory bowel disease, sepsis, sepsis syndrome, osteoporosis, ischemic injury, reperfusion injury, asthma, psoriasis and cachexia.

IL-1 exists in various forms and the genes which encode two of these forms, IL-lα and IL-1/3, have been cloned. Unless otherwise noted, "IL-l" refers to either or both IL-lα and IL-ljS. These two genes are both located in chromosome 2; each gene contains 7 exons, and the two genes are homologous in a region of the sixth exon. Both IL-l and IL-1/3 initially exist as 31 kD precursors but are processed by proteases to produce the amino terminus of the 17.5 kD mature proteins. Receptors for IL-l recognize the α and β forms, and both forms have similar biological properties. See Dinarello, Blood, 77(8): 1627-1652 (1991), incorporated herein by reference. As noted above, the biological properties of IL-l include mediating many immunological and inflammatory responses to infection and tissue injury. Because of the role of IL-l in these important processes, the therapeutic benefits of IL-l and derivatives of IL-l have been extensively studied. See U.S. Patent Nos. 5,075,288 and 5,077,219, incorporated herein by reference. Inappropriate production or response to IL-l has been shown to influence many chronic inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, encephalitis, glomerulonephritis, and respiratory distress syndrome. See Bender and Lee, Ann. Rep. Med. Chem. , 25:185-193 (1989); and U.S. Patent No. 5,075,222, particularly columns 1 to 3, each of which is incorporated herein by reference. One feature of the regulation of IL-l is the existence of a powerful natural inhibitor, the interleukin-l receptor antagonist. See United States Patent No. 5,075,222 (which describes some IL-l inhibitors, or receptor antagonists, which are useful in the treatment of IL-l mediated diseases). Other IL-l inhibitors have been extensively studied, as reviewed in Larrick, Immunol. Today, 10(2):61-66 (1989), incorporated herein by reference. IL-l inhibitors include the naturally occurring IL-lra protein and soluble IL-l receptor, as well as derivatives of IL-lα and IL-13 produced by recombinant DNA technology, as discussed in Dinarello, supra. See also PCT patent publication Nos. 91/08285, published 13 June 1991, and 91/02127, published 14 Nov. 1991, incorporated herein by reference.

Traditional therapies for the treatment of diseases mediated by IL-l {e.g. rheumatoid arthritis and inflammatory bowel disease) include the use of indomethacin and other non-steroidal anti-inflammatory drugs and salicylates. More recently, the IL-l receptor antagonist has been shown to be effective in the treatment of rheumatoid arthritis. Other therapies include the use of vasoactive drugs, antibiotics, jS-receptor stimulants including isopretenol and dopamine, and α-receptor blocking agents (e.g., phenoxybenzamine and phentolamine) for the treatment of sepsis and septic shock. Osteoporosis has been treated with estrogen, vitamin D and fluoride. Asthma has been treated with a variety of therapies including α or β adrenergic stimulants to dilate airways, methyxanthine to improve movement of airway mucus, gluccocorticoids to reduce airway inflammation and anticholinergics for broncodilation.

In addition to IL-l inhibitors and therapeutic agents which inhibit IL-l mediated diseases, others have focused on understanding the IL-IR. For a review, see Dower and Urdal, Immunol. Today, 8(2):46-51 (1987), incorporated herein by reference. Two distinct naturally occurring types of the IL-IR are known to exist, and the corresponding genes have been cloned and expressed, as reported in Dower et al. , J. Clin. Immunol. , 10 (6):289-299 (1990); PCT patent publication No. 91/00742; U.S. Patent No. 4,968,607, and McMahon et al , EMBO J., 10(10):2821-2832 (1991), each of which is incorporated herein by reference. The type I receptor (IL-lRtI) is 80 kD in size, while the type II receptor (IL-lRtϋ) is 60 kD in size. A number of studies regarding whether IL-lRtI and IL-lRtll have different affinities for ligands have been conducted; see Slack et al. , J. Biol. Chem. , 268:2513-2524 (1993) and Hannum et al , Nature, 343:336-340 (1990).

The availability of cloned genes for IL-lRtI and ILlRtll, including a soluble IL-lRtI derivative, has facilitated the search for agonists and antagonists of these important receptors. The availability of the recombinant receptor protein allows the study of receptor-ligand interaction in a variety of random and semi-random peptide diversity generation systems. These systems include the "peptides on plasmids" system described in U.S. Patent No. 5,270, 170, the "peptides on phage" system described in U.S. patent application Serial No. 718,577, filed June 20, 1991, and in Cwirla et al , Proc. Natl. Acad. Sci. USA, 87:6378-6382 (1990), and the "very large scale immobilized polymer synthesis" system described in U.S. Patent No. 5,143,854; PCT patent publication No. 90/15070, published December 13, 1990; U.S. patent application Serial No. 624,120, filed December 6, 1990; Fodor et al , Science, 251:767-773 (15 Feb. 1991); Dower and Fodor (1991) Ann. Rep. Med. Chem. , 26:271-180 (1991); and U.S. patent application Serial No. 805,727, filed December 6, 1991; each of the foregoing patent applications and publications is incorporated herein by reference.

Despite the availability of IL-l receptors and assays for antagonist discovery, many current therapeutic agents suffer from side effects or simply do not produce the desired result. Accordingly, what is needed in the art are new compounds which can effectively treat IL-l mediated diseases and which can be used as standards or competitive inhibitors in assays for the determination of IL-l activity. The present invention provides such compounds and related advantages as well.

SUMMARY OF THE INVENTION

Piperazine derivatives which act as potent and highly specific interleukin-l receptor antagonists have now been discovered. These piperazine derivatives will bind to the interleukin-l receptor but will not activate it. As a result, these compounds are useful in the treatment of diseases which are mediated by IL-l. Included among the IL-l receptor antagonists of the present invention are substituted piperazines which contain amino acid derivatives. These IL-l receptor antagonists of the present invention are also useful in assays for the determination of IL-l activity.

The invention also provides pharmaceutical compositions containing the piperazine derivatives described above. These compositions are useful in clinical applications including the treatment of rheumatoid arthritis, inflammatory bowel disease, sepsis, sepsis syndrome, osteoporosis, ischemic injury, reperfusion injury, asthma, psoriasis and cachexia. The compounds of the invention may be used alone or in combination with other therapeutic agents.

In other aspects, the present invention provides libraries of piperazine derivatives which are attached to a solid support, or attached to multiple solid supports. The present invention further provides methods of preparing the libraries as well as methods of screening the libraries to determine relative binding efficiencies of the attached piperazine derivatives to the IL-l receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows one approach to the solid phase synthesis of compounds of the present invention, whether individually or as a member of a library. Figure 2 shows the structures of a number of commercially available carboxylic acids (general formula R' — CO₂H) which are useful in synthesizing compounds and libraries of the present invention. These carboxylic acids can be used to cap the amino terminus of the peptide portion, or they can provide the — A — aryl portion which is attached to the piperazine group (as — CO — aryl).

Figure 3 shows the structures of a number of commercially available sulfonyl chlorides (general formula R" — SO₂CI) which are useful in synthesizing compounds and libraries of the present invention. These sulfonyl chlorides can be used in the same manner as the carboxylic acids described above.

Figure 4 shows the structures of a representative number of commercially available isocyanates (general formula R'" — NCO) which are useful in synthesizing compounds and libraries of the present invention. These isocyanates can be used in the same manner as the carboxylic acids and sulfonyl chlorides described above.

Figure 5 shows a synthetic scheme for the preparation of differentially protected piperazinyl glutamic acid.

Figure 6 illustrates a method used for the solid phase preparation of compound A.

Figure 7 shows a synthetic scheme used for the preparation of the library, LI.

Figures 8 and 9 show the building blocks used for X_] and X₂, respectively, in the preparation of library LI. The letters C and N denote the carbonyl portion of an amide linkage and the amine portion of a second linkage, respectively.

Figures 10, 11 and 12 show the building blocks used as X_] in the construction of libraries L2-L4.

Figure 13 shows the ten carboxylic acids used for X₂ in the preparation of L2

Figure 14 shows the ten isocyanates used for X₂ in the preparation of L3.

Figure 15 shows the ten sulfonyl chlorides used for X₂ in the preparation of L4.

Figures 16 and 17 show the synthetic schemes for the preparation of libraries L5-L8. Figures 18-22 show the building blocks used in the construction of libraries L5-L8.

Figure 23 shows six active pools from the libraries L1-L3.

Figure 24 shows the structures and activity of four compounds prepared based on the activity associated with the pools shown in Figure 23.

Figure 25 shows the structures of three active pools from L5.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations and Definitions

The following abbreviations are used herein: AcOH, acetic acid; Boc, t- butoxycarbonyl; BOP, benzotriazol-l-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate; CDI, carbonyl dϋmidizole; DCM, dichloromethane; DIEA, diisopropylethylamine; DME, dimethoxyethane; DMF, dimethylformamide; EDC, l-(3- dimethylaminopropyl)-3-ethylca_bodiimide hydrochloride; EGF-R, Epidermal Growth Factor-Receptor; EtOAc, ethyl acetate; Fmoc, fluorenylmethoxycarbonyl; HATU, O-(7- azabenzotriazol-l-yl)-l,l,3,3-tetramethyl-uronium hexafluorophosphate; HBTU, O- (benzotriazol- 1 -yl)- 1 , 1 ,3 ,3-tetramethyluronium hexafluorophosphate; HOPAC , 4- hydroxyphenylacetic acid; OBt, hydroxybenzotriazole radical; NHDF, Normal Human Dermal Fibroblast; NMP, N-methylpyrrolidone; Pfp, pentafluorophenyl ester; TFA, trifluoroacetic acid.

As used herein, the term "alkyl" refers to a saturated hydrocarbon radical which may be straight-chain or branched-chain (for example, ethyl, isopropyl, t-amyl, or 2,5- dimethylhexyl) or cyclic (for example cyclobutyl, cyclopropyl or cyclopentyl). This definition applies both when the term is used alone and when it is used as part of a compound term, such as "aralkyl" and similar terms. Preferred alkyl groups are those containing 1 to 6 carbon atoms. All numerical ranges in this specification and claims are intended to be inclusive of their upper and lower limits.

The term "alkenyl" as used herein refers to an alkyl group as described above which contains one or more sites of unsaturation.

The term "alkoxy" refers to an alkyl radical as described above which also bears an oxygen substituent which is capable of covalent attachment to another hydrocarbon radical (such as, for example, methoxy, ethoxy and t-butoxy). The term "aryl" refers to an aromatic substituent which may be a single ring or multiple rings which are fused together, linked covalently or linked to a common group such as an ethylene or methylene moiety. The aromatic rings may each contain heteroatoms, for example, phenyl, naphthyl, biphenyl, diphenylmethyl, 2,2-diphenyl-l-ethyl, thienyl, pyridyl and quinoxalyl. The aryl moieties may also be optionally substituted with halogen atoms, or other groups such as nitro, carboxyl, alkoxy, phenoxy and the like. Additionally, the aryl radicals may be attached to other moieties at any position on the aryl radical which would otherwise be occupied by a hydrogen atom (such as, for example, 2-pyridyl, 3-pyridyl and 4-pyridyl). The terms "arylalkyl", "arylalkenyl" and "aryloxyalkyl" refer to an aryl radical attached directly to an alkyl group, an alkenyl group, or an oxygen which is attached to an alkyl group, respectively.

The term "acyl" refers to a radical produced from an organic acid by removal of the hydroxyl group. Examples of acyl radicals include acetyl, pentanoyl, benzoyl, 4- hydroxybenzoyl, pivaloyl and 4-hydroxyphenylacetyl.

The term "protecting group" as used herein, refers to any of the groups which are designed to block one reactive site in a molecule while a chemical reaction is carried out at another reactive site. More particularly, the protecting groups used herein can be any of those groups described in Greene, et al , Protective Groups In Organic Chemistry, 2nd Ed., John Wiley & Sons, New York, NY, 1991, incorporated herein by reference. The proper selection of protecting groups for a particular synthesis will be governed by the overall methods employed in the synthesis. For example, in "light-directed" synthesis, discussed below, the protecting groups will be photolabile protecting groups such as Nvoc, MeNPOC, and those disclosed in co-pending Application PCT/US93/ 10162 (filed October 22, 1993), incorporated herein by reference. In other methods, protecting groups may be removed by chemical methods and include groups such as Fmoc, Dmt and others known to those of skill in the art.

The term "protected amino acid" refers to an amino acid, typically an α-amino acid having either or both the amine functionality and the carboxylic acid functionality suitably protected by one of the groups described above. Additionally, for those amino acids having reactive sites or functional groups on a side chain (i.e. , serine, tyrosine, glutamic acid), the term "protected amino acid" is meant to refer to those compounds which optionally have the side chain functionality protected as well.

The term "activating agent" refers to those groups which, when attached to a particular functional group or reactive site, render that site more reactive toward covalent bond formation with a second functional group or reactive site. For example, the group of activating groups which are useful for a carboxylic acid include simple ester groups and anhydrides. The ester groups include alkyl, aryl and alkenyl esters and in particular such groups as 4-nitrophenyl, pentafiuorophenyl and N-succinimidyl. Other activating agents are known to those of skill in the art.

The term "pharmaceutically or therapeutically acceptable carrier" refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the host or patient.

The term "therapeutically- or pharmaceutically-effective amount" as applied to the compositions of the instant invention refers to the amount of composition sufficient to induce a desired biological result. That result can be alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In the present invention, the result will typically involve a decrease in the immunological and/or inflammatory responses to infection or tissue injury.

For the compounds of the invention which contain amino acid or peptide fragments, the amino acid residues are denoted by single-letter or three-letter designations following conventional practices. The designations for gene-encoded amino acids are as follows:

o

Commonly encountered amino acids which are not gene-encoded may also be used in the present invention. These amino acids and their abbreviations include omithine (Orn); aminoisobutyric acid (Aib); benzothiophenylalanine (BtPhe); albizziin (Abz); t- butylglycine (Tie); phenylglycine (PhG); cyclohexylalanine (Cha); norleucine (Nle); 2- naphthylalanine (2-Nal) or Nal(2); 1-naphthylalanine (1-Nal); 2-thienylalanine (2-Thi); l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); N-methylisoleucine (N-Melle); homoarginine (Har); Nα-methylarginine (N-MeArg); phosphotyrosine (pTyr or pY); pipecolinic acid (Pip); 4-chlorophenylalanine (4-ClPhe); 4-fluorophenylalanine (4-FPhe); and sarcosine (Sar). All of the amino acids used in the present invention may be either the D- or L- isomer. The L-isomers are preferred when not otherwise specified.

In addition to compounds consisting of naturally-occurring amino acid and other non-gene-encoded amino acids, other peptidomimetics are also useful in the compounds of the present invention. In particular, compounds having one or more amide linkages optionally replaced by an isostere can be used. In the context of the present invention, for example, —CONH— may be replaced by — CH₂NH— , — NHCO— , — SO₂NH— , — CH₂O— , — CH₂CH₂— , — CH₂S— , — CH₂SO— , — CH=CH— (cis or trans), — COCH₂— , — CH(OH)CH₂— and 1,5-disubstituted tetrazole such that the radicals linked by these isosteres would be held in similar orientations to radicals linked by CONH. For a general review of these and other isosteres see, Spatola, A.F., in Chemistry and

Biochemistry of Amino Acids, Peptides and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983). See also, Spatola, A.F., Peptide Backbone Modifications (general review), Vega Data, Vol. 1, Issue 3, (March 1983); Morley, Trends Pharm Sci (general review), pp. 463-468 (1980); Hudson, D. et al, Int J Pept Prot Res, 14: 177-185 (1979) (-CH₂NH-, CH₂CH₂-); Spatola et al, Life Sci, 38: 1243-1249 (1986) (-CH₂-S);

Hann J. Chem. Soc. Perkin Trans. 1, 307-314 (1982) (-CH-CH-, as and trans); Almquist et al. , J Med Chem, 23:1392-1398 (1980) (-COCH₂-); Jennings-White et al, Tetrahedron Lett, 23:2533 (1982) (-COCH₂-); Szelke et al , European Appln. EP 45665 CA: 27:39405 (1982) (-CH(OH)CH₂-); Holladay et al, Tetrahedron Lett, 24:4401-4404 (1983) (-C(OH)CH₂-); and Hruby, Life Sci, 31:189-199 (1982) (-CH₂-S-); each of which is incorporated herein by reference. Compounds and fragments having isosteric replacements may have significant advantages over those compounds and fragments with the natural amide linkages. Some of the advantages include, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others. The compounds of the present invention may also be labeled via covalent attachment of one or more labels, directly or through a spacer (e.g., an amide group), to position(s) on the compound that are predicted by quantitative structure-activity data and/or molecular modeling to be non-interfering. Such non-interfering positions generally are positions that do not form direct contacts with the L-1R protein or other macromolecules(s) (e.g., immunoglobulin superfamily molecules) to which the compound binds to produce the therapeutic effect. Additionally, derivatization (e.g., labeling) of compounds should not substantially interfere with the desired biological or pharmacological activity of the compound.

Piperazine Derivatives

The compounds of the present invention bind to the interleukin-l receptor but will not activate it. These antagonists are represented by the formula:

The group A in this formula represents a linking group which can be a single bond, -, —CONH—, — COZ¹— , — CONHZ¹— , — CON(Z²)— , — CON^Z¹— or — SO₂ — , in which the letter Z¹ represents a alkyl, alkenyl or alkynyl linking group and Z² represents a hydrogen, alkyl or aryl substituent. In preferred embodiments, A is a single bond, —CO—, —COZ¹—, or —CONH—, more preferably —COZ¹—.

The symbol R represents a single amino acid or a fragment comprising from about 2 to about 20 amino acids. The fragment has the formula (AA')_n in which AA is an amino acid and i is an integer denoting its position upstream from the glutamine residue. An AA¹ at any position may be the same or different from an AA¹ at any other position. AA¹ is preferably Tyr, Trp, Glu, Pro, Gly, pTyr, 1-Nal, Phe, or Thr, more preferably Trp, Tyr, or pTyr; AA², when present, is preferably Tyr, Trp, BtPhe, Abz, Glu, Pro, Gly, pTyr, 1- Nal, Phe, or Thr, more preferably Trp, Tyr, Phe or 1-Nal; AA³ when present is pTyr, Glu, D or L-Asp, Gly, D or L-Ala, Aib, Val, D-Lys or D-Phe, preferably pTyr, Ala, D- Ala or Gly and n is 3 to 20; AA⁴, when n is 4 to 20, is preferably Pro, L-Pip, N-MeAla or Sar; AA⁵, when n is 5 to 20 or 5 to 10 is preferably Thr, alloThr, t-Leucine, Val, Ser, Nle, He or Ala; AA⁶ , when n is 6 to 20, or 6 to 10 is preferably Tip, 4-NO₂Phe, Phe or 1-Nal; AA⁷, when n is 7 to 20 or 7 to 10 is preferably Glu, pTyr, Asp, Gin, Met, Glu-5- ethyl ester, or Ala; and AA⁸, when n is 8 to 20 or 8 to 10 is preferably Phe, 4-ClPhe, 4- FPhe, Tyr, 4-CH₃OPhe, 4-NO₂Phe, 4-NH₂Phe, Tip, His, 3-pyridylalanine, thienylalanine, benzothienylalanine, phenylglycine, 1-Nal, or 2-Nal. The most preferred embodiments are those in which R is a peptide of formula (AA¹^ where n is from 2 to 10 and AA¹ is Tyr, Tip or pTyr, and AA² is Tip or Tyr.

The symbol m represents an integer of from 1 to 3 and the symbol R¹ represents a hydrogen, alkyl, aryl, acyl or sulfonyl group. The R¹ groups which are attached to R are covalently bound to the N-terminal nitrogen of the amino acid or peptide. Additionally, the R¹ groups may be the same or different. Suitable alkyl groups include methyl, ethyl, t-butyl and the like. Thus, R¹ can represent multiple hydrogen or alkyl groups to form an ammonium ion at the N-terminus of the compound. For example, R^l-R can represent Mβ₃N⁺ — Nal(2) in which the (2) designates the ring position through which naphthalene is attached to alanine. Aryl groups which are useful include phenyl, naphthyl, pyridyl and thienyl as well as versions substituted with groups such as hydroxyl, hydroxyalkyl, alkoxy, halides (fluoro, chloro, bromo and iodo), nitro and carboxylic acids, esters and amides. Suitable acyl groups include acetyl, propionyl, butanoyl, benzoyl, phenylacetyl, succinoyl, glutaroyl, 4-hydroxyphenylacetyl, 3-(4-hydroxyphenyl)propionyl, 3-phenylpropionyl, 4- hydroxybenzoyl, 3,4-dihydroxyphenylacetyl, 3-nitro-4-hydroxyphenylacetyl, 4- hydroxymethylphenylacetyl and 4-nitrophenylacetyl. Suitable sulfonyl groups include phenylsulfonyl, naphthylsulfonyl, pentafluorophenylsulfonyl, 3- trifluoromethylphenylsulfonyl, 4-t-butylphenylsulfonyl and mesitylsulfonyl. Preferred groups for R¹ will depend on the number and nature of the amino acid residues which are present in R. For example, when n is 2, preferred acyl groups for R¹ are acetyl, propionyl, 4-hydroxybenzoyl, 4-hydroxyphenylacetyl, 4-hydroxyphenylpropionyl, 3,4- dihydroxyphenylacetyl, 3-nitro-4-hydroxyphenylacetyl, 4-hydroxymethylphenylacetyl and 4-nitrophenylacetyl. When n is 1, a preferred acyl group for R¹ is 3-pyridylacryloyl.

Of the remaining groups, X¹, X² and X³ are the same or different, each being H, —OH, — Z¹— OH, — OZ², — F, — Cl, — Br, —I, — NO₂, — SO₂NH₂, — CO₂H,

— CONH₂, — CONHZ², — NH₂, — NHZ², — NZ² ₂ or — CO₂Z², in which the letters Z¹ and Z² have the same meanings provided above. Optionally, two of the groups (i.e. , X¹ and X²) can be combined to form a ring fused to the phenyl ring. In one group of preferred embodiments, X¹ is 4-OH, 4-Z¹-OH, or 4-OZ² and X² is H, —OH, — Z¹— OH, — OZ², — CONH₂, —CONHZ², or — CO₂Z².

In one particularly preferred embodiment, R¹ is acetyl, R is — YW — , A is — COCH₂ — , X¹ is 4-hydroxy, and X² and X³ are both hydrogen. In another particularly preferred embodiment, R¹ is acetyl, R is — WY — , A is — COCH₂ — , X¹ is 4-hydroxy, and X² and X³ are both hydrogen. In yet another particularly preferred embodiment, R¹ is acetyl, R is — WY— , A is — COCH₂CH₂— , X¹ is 4-hydroxy, and X² and X³ are both hydrogen. In still another particularly preferred embodiment, R¹ is acetyl, dimethyl- or trimethylammonium, R is — FEWTPGWY— , A is — COCH₂— , X¹ is 4-hydroxy, and X² and X³ are both hydrogen. In another particularly preferred embodiment, R¹ is acetyl, R is — WpY— , A is — COCH₂— , X¹ is 4-hydroxy, and X² and X³ are both hydrogen.

The interleukin-l receptor antagonists of the present invention may be prepared by standard synthetic methods which are known to those of skill in the art. Portions of the compounds can be prepared using solid phase techniques which are extensively described and used in the art to prepare peptides. The compounds may also be prepared using liquid phase amino acid coupling methods which are also well known in the art. See, M. Bodanszky and A. Bodanszky, The Practice of Peptide Synthesis (1984) and M. Bodanszky, Principles of Peptide Synthesis (1984).

In one synthetic methodology directed toward the compounds of the present invention, a protected glutamic acid is first tethered to a solid support. A variety of solid supports can be used, for example, a Knorr linker is suitable for formation of an amide linkage to the side-chain carboxylic acid of the glutamic acid residue. Other suitable resins include, for example, PAL or XAL. Subsequent steps involve deprotection of the amino group and sequential coupling of amino acids to the amine terminus. Alternatively, an appropriate peptide can be synthesized by other conventional means and coupled to the resin-bound glutamic acid in a more convergent synthesis. Following formation of the peptide portion of the compound, the remaining carboxylic acid moiety of the resin-bound glutamic acid is deprotected. To the unmasked carboxylic acid is coupled a piperazine derivative, using conventional techniques such as BOP/HOBt, DCC/HOBt or, HATU. The piperazine derivatives are similarly synthesized by a variety of methods, typically involving protection and deprotection steps to selectively alkylate or acylate the ring nitrogens. In one approach, commercially available t-Boc-piperazine is N-acylated with, for example, (4-hydroxyphenyl)acetic acid using standard methods (i.e., BOP/HOBt, DCC/HOBt, HATU, CDI or thionyl chloride). Following acylation, the t-Boc protecting group is removed and the acylated piperazine is then coupled to the resin-bound glutamic acid. Cleavage of the compound from the resin is accomplished using standard methods. One of skill in the art will recognize that a number of alternative procedures exist for the preparation of the present compounds, including reversing the order of synthesis on the resin-bound glutamic acid, and using other reagents for the acylations and couplings which are described in, for example, March, Advanced Organic Chemistry, Fourth Edition, Wiley-Interscience, NY, (1992), incorporated herein by reference.

In Vitro Uses

The compounds of the invention are useful in vitro as unique tools for understanding the biological role of IL-l, including the evaluation of the many factors thought to influence, and be influenced by, the production of IL-l and the receptor binding process. The present compounds are also useful in the development of other compounds that bind to the IL-l Rtl, because the present compounds provide important information on the relationship between structure and activity that should facilitate such development.

The compounds are also useful as competitive inhibitors in assays to screen for new IL-l receptor Mockers. In such assay embodiments, the compounds of the invention can be used without modification or can be modified in a variety of ways; for example, by labeling, such as covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal. In any of these assays, the materials thereto can be labeled either directly or indirectly. Possibilities for direct labeling include label groups such as: radiolabels such as ¹²⁵I, enzymes (U.S. Patent No. 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels (U.S. Patent No. 3,940,475) capable of monitoring the change in fluorescence intensity, wavelength shift, or fluorescence polarization. Possibilities for indirect labeling include biotinylation of one constituent followed by binding to avidin coupled to one of the above label groups. The compounds may also include spacers or linkers in cases where the compounds are to be attached to a solid support.

The compounds of the invention can also be used in assays as probes for determining the expression of the IL-l Rtl on the surface of cells. Such an assay is useful for determining the degree of cellular immunological and inflammatory response, for example to infection and tissue injury. Typically, the cells under study will be exposed to the compounds for a period sufficient for the compounds to bind to the receptor(s) exposed on the cell surface. The cells are then separated from the non-bound compounds and unreacted cells, e.g., by affinity chromatography or the use of a cell sorter, to identify whether binding of the compounds to the receptor has occurred.

Thus, the compositions and methods of the present invention also can be used in vitro for testing a patient's susceptibility to varying treatment regimens for disorders associated with the overproduction of IL-l or an improper response to IL-l using an in vitro diagnostic method whereby a specimen is taken from the patient and is treated with a IL-lRtI binding, IL-l blocking compound of the present invention to determine the effectiveness and amount of the compound necessary to produce the desired effect. The blocking compound and dosage can be varied. After the blocking compounds are screened, then the appropriate treatment and dosage can be selected by the physician and administered to the patient based upon the results. Therefore, this invention also contemplates use of a blocking compound of this invention in a variety of diagnostic kits and assay methods.

The compounds of the invention can also be administered to warm blooded animals, including humans, to block the binding of IL-lα or IL-ljS to the IL-lRtI in vivo. Thus, the present invention encompasses methods for therapeutic treatment of IL-l related disorders that comprise administering a compound of the invention in amounts sufficient to block or inhibit the binding of IL-l to the IL-IR in vivo. For example, the peptides and compounds of the invention can be administered to treat symptoms related to the overproduction of IL-l or an improper response to IL-l. Since the biological effects of IL-l include immunologic properties, such as T-cell activation, increased IL-2R expression, B-cell activation via induction of IL-6, natural killer cell activity, and lympholάne gene expression; pro-inflammatory properties such as fever, sleep, anorexia, neuropeptide release, gene expression for complement, suppression of P450 synthesis, endothelial cell activation, neutrohilia, increased adhesion molecule expression, neutrophil priming, eosinophil degranulation, hypotension, myocardial suppression, neutrophil tissue infiltration, beta islet cell cytotoxicity, hyperlipidemia, cyclooxygenase and lipoxygenase gene expression, synthesis of collagenases and collagens, and osteoblast activation, the compositions and methods described herein will find use for the treatment and/or prevention of a variety of IL-l related disorders. See, e.g., Dinarello (1991) Blood £: 1627-1652, which is incorporated herein by reference. Examples of specific disorders having such symptoms include but are not limited to, atherlerosclerosis, rheumatoid arthritis, osteoporosis, HIV infection and AIDS, bacterial infection, respiratory distress syndrome, coal miner pneumonoconiosis, alcoholic cirrhosis, cuprophane hemodialysis, cardiopulmonary bypass, chronic hepatitis B, thermal injury, reticulohistiocytosis, sarcoidosis, tuberculosis, obstructive jaundice, Paget's disease and osteomalacia, IDDM, Kawasaki's disease, inflammatory bowel disease, sepsis, toxic shock, and luteal phase. Accordingly, additional aspects of the invention are directed to pharmaceutical compositions containing the compounds of the invention. The antagonists of the invention may be administered in conventional formulations. One common formulation might include a saline solution buffered to pH 7.4, and suitable for administration by injection. Formulations for bolus administration are also useful, and comprise the selected antagonist with pharmaceutically acceptable excipients such as starch or gum arabic as binding agents. Other typical formulations may be found in Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, latest edition.

In some embodiments the compounds of the present invention are present as salts, typically pharmaceutically acceptable salts. As used herein the term "pharmaceutically acceptable salts" refers to salts which are obtained in the usual manner. Examples include treatment of compounds of general formula I with non-toxic inorganic or organic acids or bases. Suitable bases include ammonia, sodium hydroxide, potassium hydroxide, lithium hydroxide, morpholine, trimethylamine, and piperidine. Suitable acids include HCI,

HOAc, citric acid, lactic acid, glutamic acid and others known to those of skill in the art.

The preparation of pharmaceutically acceptable salts is described in Berge, et al , J. Pharm. Sci. 66:1-19 (1977), incorporated herein by reference. Additionally, salts may be exchanged using techniques such as ion-exchange chromatography.

Systemic administration of the compounds is typically carried out by injection, preferably by intravenous injection. Alternatively, intramuscular, intraperitoneal or subcutaneous injection may be used. Other forms of systemic administration of the compounds such as transdermal or transmucosal administration are also possible. Oral administration may also be used with properly formulated enteric or encapsulated formulations.

The dosage used will depend on such factors as the choice of antagonist, the route of administration, the nature of the formulation, the nature of the patient's illness and the judgment of the attending physician. Typically, the dosage will be in the range of 0.1-100 μg/kg of subject. More preferably, the dosage will be in the range of 1-50 μg/kg of subject.

Formulations

Using a method of the invention, the receptor antagonists are formulated into any of a number of preparations for topical, oral, or injectable administration. Additionally, the compounds can be formulated into topical preparations either for local therapy or for transdermal delivery systemically.

Formulations include a therapeutically effective concentration of the compound of interest in a dermatological vehicle. The amount of the compound to be administered, and its concentration in the topical formulations, depends upon the vehicle selected, the clinical condition of the patient, the side effects and the stability of the compound in the formulation. Thus, the physician will employ the appropriate preparation containing the appropriate concentration in the formulation, as well as the amount of formulation administered, depending upon clinical experience with the patient in question or with similar patients.

The concentration of the compounds of the present invention for topical formulations is in the range of about 0.1 mg/mL to about 30 mg/mL. Typically, the concentration of the compound for topical formulations is in the range of about 0.3 mg/mL to about 10 mg/mL. Solid dispersions of the compounds as well as solubilized preparations can be used. Thus, the precise concentration to be used in the vehicle will be subject to modest experimental manipulation in order to optimize the therapeutic response. Suitable vehicles include gels such as hydrogels, oil-in-water or water-in-oil emulsions using mineral oils, petrolatum and the like. Topical preparations typically include vehicles suitable for use on the skin (including the corneal/coηjunctival epithelium), including emollients, emulsifiers, wax, fats, alcohols, and/or oils.

One example of a topical formulation includes about 1 % of the appropriate agent by weight; about 35-40% alcohol, predominantly ethanol and isopropyl alcohol; about 30% propylene glycol; about 15% polyethylene glycol 400 (PEG 400); about 10% water; and small amounts, such as about 1 % or less, of each of glycerin, sodium laurel sulfate, stabilizers, preservatives, humectants, thickeners, and chemicals selected for the addition of color or scent. The therapeutic compound is optionally formulated into a transdermal therapeutic system for topical application. See Barry, Dermatological Formulations, (1983), especially at page 181 and literature cited therein. While such topical delivery systems were originally designed primarily for transdermal administration of low molecular weight drugs, they are capable of percutaneous delivery of other drugs. They may be readily adapted to administration of the therapeutic compounds of the invention by appropriate selection of the rate-controlling microporous membrane.

The therapeutic compound can also be administered by aerosol to achieve localized delivery to the lungs, skin, or mucous membranes, for example. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A nonaqueous (e.g., fluorocarbon propellent) suspension could be used. Sonic nebulizers preferably are used in preparing aerosols. Sonic nebulizers minimize exposing the therapeutic compound to shear, which can result in degradation of the drug.

Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the compound together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. The formulations are sterile. Aerosols generally are prepared from isotonic solutions. Preparations of the therapeutic compounds either for systemic or local delivery may be employed and may contain excipients as described above for parenteral administration and other excipients used in a topical preparation such as cosol vents, surfactants, oils, humectants, emollients, preservatives, stabilizers and antioxidants. Any pharmacologically acceptable buffer may be used, e.g. , tris or phosphate buffers. The compounds of the present invention can also be formulated into pills, tablets, powders, caplets and the like for oral administration. Additionally, the compounds can be prepared for injectable use, preferably in an aqueous medium such as a sodium chloride solution of about 0.1 % . Other examples of suitable aqueous vehicles are water, various 10 saline solutions, Ringer's solution, dextrose solution, and Hank's solution.

Thus, a composition of the invention includes an effective amount of the therapeutic compound which may be formulated with conventional, pharmaceutically acceptable, vehicles for parenteral administration by injection. Formulations may also include small amounts of adjuvants such as buffers and preservatives to maintain isotonicity, physiological and pH stability.

Preferably the compositions are presented for administration in unit dosage forms. The term "unit dosage form" refers to physically discrete units suitable as unitary doses for human subjects and animals, each unit containing a predetermined quantity of active material calculated to produce a desired pharmaceutical effect in association with the required pharmaceutical diluent, carrier or vehicle. Examples of unit dosage forms include vials, ampules, tablets, caplets, pills, powders, granules, eyedrops, oral or ocular solutions or suspensions, ocular ointments, and oil-in-water emulsions. Means of preparation, formulation and administration are known to those of skill, see generally Remington's Pharmaceutical Science 15th ed., Mack Publishing Co., Easton, PA (1980). A number of references teach preparation of patches for transdermal delivery. See, for instance, U.S. Patent No. 5,286,491 to Amkraut, assigned to ALZA Corporation. See Havener, W.H., Ocular Pharmacology, C.V. Mosby Co., St. Louis (1983) for ophthalmic preparations.

Slow Release Delivery Slow or extended-release delivery systems, including any of a number of biopolymers (biological-based systems), systems employing liposomes, and polymeric delivery systems, can be utilized with the compositions described herein to provide a continuous or long-term source of the therapeutic compound. Such slow release systems are applicable to formulations, for example, for topical, ophthalmic, oral, and parenteral use.

Conditions for Therapy

Any number of inflammatory conditions are susceptible to treatment with compounds of the present invention. Preferred examples include inflammation of the skin such as dermatitis including contact dermatitis, allergic dermatitis, seborrheic dermatitis, eczema, psoriasis, scleroderma, and dermal manifestations of lupus. Other examples include inflammation of the joints such as rheumatoid arthritis, lupus arthritis, degenerative joint disease, and autoimmune joint inflammation of various etiologies. Additional inflammatory conditions, such as delayed hypersensitivity reactions; inflammatory bowel disease including ulcerative colitis and Crohn's disease; autoimmune conditions; eye conditions such as conjunctivitis, scleritis and uveitis; and mucositis are candidates for treatment with the invention. Moreover, the invention includes the use of the present compounds as second active agents in a transdermal delivery device to provide anti-inflammatory effects. Examples of transdermal delivery devices include transdermal delivery patches and iontophoretic delivery apparatuses. In one embodiment, the condition for therapy is dictated by the first active ingredient. The present compounds are used as a second active agent for their anti- inflammatory effects. For instance, transdermal patches and iontophoretic apparatuses are known to cause some local skin irritation or inflammation due to the mechanics or their use or to the compounds used to adhere the device to the skin. Accordingly, one or more of the present compounds are added to the device, either a patch or an iontophoretic apparatus, so that the compound's anti-inflammatory effects can prevent or minimize the local irritation to the skin.

Alternatively, the present compounds can be the primary therapeutic agent in a transdermal delivery system. For example, one or more of the compounds can be applied topically for either local or systemic absorption. In cases in which local therapy is desired, for instance, the use of a transdermal patch or iontophoretic delivery can insure deep or full thickness dermal penetration when indicated.

Dosages and Schedules

In therapeutic applications, the dosages of the present compounds used in accordance with the invention vary depending on a number of factors including, for example, the condition being treated; the age, weight, and clinical condition of the recipient patient; and the experience and judgment of the clinician or practitioner administering the therapy.

The dosage of the specific compound for treatment depends upon many factors that are well known to those skilled in the art. An effective amount of the compound is that which provides either subjective relief of symptoms or an objectively identifiable improvement as noted by the clinician or other qualified observer. The dosing range varies with the compound used, the route of administration and the potency of the particular compound. The dosages of the present compounds used to practice the invention include dosages effective to result in the desired anti-inflammatory effect. Estimation of appropriate dosage for the individual patient is well within the skill of the ordinary prescribing physician or other appropriate health care practitioner. As a guide, the practitioner can use conventionally available advice from a source such as the Physician 's Desk Reference, 48th Edition, Medical Economics Data Production Co., Montvale, New Jersey 07645-1742 (1994), ("PDR" hereinafter). An initial oral dose of about 10 to about 180 mg per day of the present compounds is recommended. The dosage may be increased, usually in increments of about 10 to 100 mg, to a maximum of about 480 mg per day. When the dosage is in the high range, such as at 480 mg per day, it is preferable to provide a divided dosage of 240 mg twice a day or every 12 hours.

When the compound is injected, such as intravenously, the usual initial dose is about 0.1 to about 10 mg as a bolus over at least 2 minutes. This dosage may be repeated in about 30 minutes after the initial dose. For pediatric patients, the intravenous dosage is about 0.01 to about 0.2 mg per kg body weight, usually given in a single dose of about 0.15 to about 2 mg over at least 2 minutes. For children, a maximum of about 0.3 mg per kg body weight is typically given. Usually, it is not advisable to exceed 10 mg as a single intravenous dose.

Where necessary, the dosage can be repeated daily, or sometimes twice a day, until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy. Once a therapeutic effect is achieved, the dosage can be tapered or discontinued.

Administration

In general, the route of administration is topical (including administration to the eye, scalp, and mucous membranes), oral, or parenteral (injectable).

Topical delivery of the compounds of the present invention includes delivery by direct application to the skin or mucous membranes, by transdermal patches, and by iontophoretic delivery apparatuses. The transdermal patch or iontophoretic apparatus can include the present compounds either as a first active agent for treating inflammation as the primary condition, or as a second active agent to treat inflammation associated with the local irritant effects of the transdermal patch or iontophoretic device. A preferred way to practice the invention is to apply the compound, in a cream or oil based carrier, directly to the dermal lesions. Typically, the concentration of therapeutic compound in a cream or oil is 1-2%. Alternatively, an aerosol can be used topically whenever appropriate, such as to the skin, oral mucosa, and upper and lower respiratory tracts. The compounds can also be orally administered. Topical administration is preferred in treatment of skin lesions, including lesions of the scalp, lesions of the cornea (keratitis), and lesions of mucous membranes where such direct application is practical. Shampoo formulations are sometimes advantageous for treating scalp lesions such as seborrheic dermatitis and psoriasis of the scalp. Mouthwash and oral paste formulations can be advantageous for mucous membrane lesions. Oral administration is a preferred alternative for treatment of skin lesions and other lesions where direct topical application is not as practical, and it is a preferred route for other applications. Other preferred routes of administration include oral; intra-articular (direct injection into a joint); subcutaneous (SQ); intra-dermal; directly to the upper or lower respiratory tracts, usually in aerosol form; intravascular, such as intravenous (IV) or intraarterial; intramuscular (IM); intra-lesional (directly into or around a lesion); intrathecal; ocular and intra-ocular, such as topical application to the cornea, sclera or conjunctiva, choroidal injection, transscleral injection or placing a scleral patch, and selective arterial catheterization; and intraperitoneal (IP). When injected, the drug can be delivered as a bolus, a short term infusion or a continuous, longer term infusion.

Intra-articular injection is a preferred alternative in the case of treating one or only a few (such as 2-6) joints. Usually, the compound is delivered in an aqueous solution of about 10-20 mg/mL. Additionally, the compound is injected directly into lesions (intra- lesion administration) in appropriate cases. Intra-dermal administration is an alternative for dermal lesions such as those of psoriasis.

It should, of course, be understood that the compositions and methods of this invention can be used in combination with other agents exhibiting the ability to modulate IL-l synthesis, release, and/or binding. Examples of such agents include, but are not limited to disease modifying antirheumatic drugs chloroquine, auranofin, sodium aurothiomalate, and dexamethasone (see, e.g., Lee et al , Proc. Natl Acad. Sci. , 85:1204 (1988)); tenidap (see, e.g., Otterness, 3rd Interscience World Conference on Inflammation, Monte-Carlo, Abstr. p. 371 (March, 1989); antioxidants, such as nordihydroguaiaretic acid (see, e.g., Lee et al , Int. J. Immunopharmacol , 10:835 (1988)), probucol (see, e.g. , Ku et al , Am. J. Cardiol , 62:778 (1988)), and disulfiram (see, e.g., Marx, Science, 239:257 (1988)); pentoxifylline (see, e.g., Sullivan et al , Infect. Immun. , 56: 1722 (1988)); denbufylline (see, e.g., Mandell et al PCT publication WO 89/015145 (1989); romazarit (see, e.g., Machin et al (1988) U.S. Patent No. 4,774,253); tiaprofenic acid; dexamethasone; and natural macromolecular IL-l inhibitors (see, e.g. , Rosenstreich et al. in "Lymphokines", E. Pick, Ed., 14:6 Academic Press (1987) and Larrick, Immunol. Today, 10:6 (1989)); as well as the other agents described in Bender and Lee, Annual Reports in Medicinal Chemistry Chapter 20: Pharmacological Modulation of Interleukin-l, pp. 185-193 (1989), which is incorporated herein by reference.

Libraries of IL-l Receptor Ligands

In another aspect, the present invention provides libraries of IL-l receptor ligands, each member of the library having the formula:

in which S¹ represents a solid support, L represents a bond, spacer or a linking group, and A, R, R¹, m, X¹, X² and X³ represent the groups described above for the compounds of the invention. The solid support may be biological, nonbiological, organic, inorganic, or a combination of any of these, existing as particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates, slides, etc. The solid support is preferably flat but may take on alternative surface configurations. For example, the solid support may contain raised or depressed regions on which synthesis takes place. In some embodiments, the solid support will be chosen to provide appropriate light- absorbing characteristics. For example, the support may be a polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon, or any one of a variety of gels or polymers such as (poly)tetrafluoroethylene, (poly)vinylidendifluoride, polystyrene, polycarbonate, or combinations thereof. Other suitable solid support materials will be readily apparent to those of skill in the art.

Preferably, the surface of the solid support will contain reactive groups, which could be carboxyl, amino, hydroxyl, thiol, or the like. More preferably, the surface will be optically transparent and will have surface Si — OH functionalities, such as are found on silica surfaces. Attached to the solid support is an optional spacer or linking group, L. The spacer molecules are preferably of sufficient length to permit the IL-l receptor ligands in the completed member of the library to interact freely with receptors exposed to the library. The spacers, when present, are typically 6-50 atoms long to provide sufficient exposure for the attached IL-l receptor ligand. The spacer or linking group, L, is comprised of a surface attaching portion and a ligand attaching site. The surface attaching portion is that part of L which is directly attached to the solid support. This portion can be attached to the solid support via carbon-carbon bonds using, for example, supports having (poly)trifluorochloroethylene surfaces, or preferably, by siloxane bonds (using, for example, glass or silicon oxide as the solid support). Siloxane bonds with the surface of the support are formed in one embodiment via reactions of surface attaching portions bearing trichlorosilyl or trialkoxysilyl groups. The spacer or linking group will also have a ligand attaching site. Functional groups which are suitable for attachment to an IL-l receptor ligand include amines, hydroxyl, thiol, and carboxyl. The surface attaching portion and the ligand attaching site can be separated by a variety of groups including alkylene groups (e.g., ethylene, propylene, butylene, etc.), aryl acetylene, ethylene glycol oligomers containing 2-14 monomer units, diamines, diacids, amino acids, peptides, or combinations thereof. Additionally, this portion of the spacer can be selected based upon its hydrophilic/hydrophobic properties to improve presentation of the ligands to the receptors. Preferably, this portion of L is constructed of polyethyleneglycols, alkylene, poly alcohol, polyester, polyamine, polyphosphodiester and combinations thereof.

In one group of preferred embodiments, the spacer or linking group is formed from bis(2-hydroxyethyl)-aminopropyltriethoxysilane, 2-hydroxyethylaminopropyltriethoxysilane, aminopropyltriethoxysilane or hydroxypropyltriethoxysilane.

In other embodiments, a portion of L can be a linking group which is cleavable, preferably a photocleavable linking group. Photocleavable linking groups have been described in co-pending application USSN 08/374,492, filed January 17, 1995, the contents of which are incorporated herein by reference.

Attached to the distal end of L is an IL-l receptor ligand. The ligands which are attached to preselected regions of a solid support, or alternatively to individual solid supports are each independently compounds of formula (I) which have been described above. These compounds are each attached to a linking group or spacer through the carbamoyl functionality ( — CONH₂) of the glutamine residue

The library can have virtually any number of different members, and will be limited only by the number or variety of compounds desired to be screened in a given application and by the synthetic capabilities of the practitioner. In one group of embodiments, the library will have from 2 up to 100 members. In other groups of embodiments, the library will have between 100 and 10000 members, and between 10000 and 1000000 members, preferably on a solid support. In preferred embodiments, the library will have a density of more than 100 members at known locations per cm², preferably more than 1000 per cm², more preferably more than 10,000 per cm².

Preparation of the Libraries The libraries of the present invention can be prepared using a variety of solid phase techniques which are known to those of skill in the art. A general description of the preparation is provided with reference to Figure 1.

As shown in Figure 1 (illustrating only a single compound synthesis), a linking group is attached to a solid support to provide a derivatized solid support having a plurality of available ligand attaching sites. To the sites in derivatized support is attached a piperazinylglutamine scaffold which is selectively protected on each of the available amine functional groups (shown as P¹ and P²). Selective deprotection of either amino group allows independently, the controlled site modification of each member of the library. The building blocks used in the synthesis can be selected from, for example, amino acids and peptides (described in detail above with reference to the compounds of the invention), carboxylic acids, sulfonic acids and isocyanates. In one group of embodiments, each chemically distinct member of the library will be synthesized on a separate solid support. A more thorough discussion of the various solid phase techniques can be found in the patents and publications provided below.

Libraries on a Single Substrate Light-Directed Methods For those embodiments using a single solid support, the novel piperazine derivatives and libraries of IL-l receptor ligands of the present invention can be formed using, for example, "light directed" methods (which are one technique in a family of methods known as VLSIPS™ methods). These methods are described in U.S. Patent No. 5,143,854, previously incorporated by reference.

Flow Channel or Spotting Methods

Additional methods applicable to library synthesis on a single substrate are described in co-pending Applications Ser. No. 07/980,523, filed November 20, 1992, and 07/796,243, filed November 22, 1991, incorporated herein by reference for all purposes. In the methods disclosed in these applications, reagents are delivered to the substrate by either (1) flowing within a channel defined on predefined regions or (2) "spotting" on predefined regions. However, other approaches, as well as combinations of spotting and flowing, may be employed. In each instance, certain activated regions of the substrate are mechanically separated from other regions when the monomer solutions are delivered to the various reaction sites.

Pin-Based Methods

Another method which is useful for the preparation of compounds and libraries of the present invention involves "pin based synthesis. " This method is described in detail in U.S. Patent No. 5,288,514, previously incorporated herein by reference. The method utilizes a substrate having a plurality of pins or other extensions. The pins are each inserted simultaneously into individual reagent containers in a tray. In a common embodiment, an array of 96 pins/containers is utilized. O 96/29088 ,.„ PCT/TJS96/03955

23

Libraries on Multiple Substrates Bead Based Methods

Yet another method which is useful for synthesis of compounds and libraries of the present invention involves "bead based synthesis." A general approach for bead based synthesis is described in co-pending Application Ser. Nos. 07/762,522 (filed September 18, 1991); 07/946,239 (filed September 16, 1992); 08/146,886 (filed November 2, 1993); 07/876,792 (filed April 29, 1992) and PCT/US93/04145 (filed April 28, 1993), the disclosures of which are incorporated herein by reference.

In one group of preferred embodiments, a library of IL-l receptor ligands is prepared using bead-based synthesis. In brief, beads are suitably modified with a spacer or linking group, for example, aminoalkyltriethoxysilane, to provide beads having amino groups as synthesis initiation sites. Photocleavable linking groups are attached to the amino groups to provide derivatized beads having a general formula:

-L-S¹ in which S¹ is the solid support and L is a combination of spacer (aminoalkylsilane) and photocleavable linking group. Attached to the photocleavable linking group is a basic piperazinyl-glutamine scaffold, for example, N^α-Fmoc-N-Boc-piperazinyl-α-glutamine. One of skill in the art will understand that a number of protecting groups are available for use in preparing the libraries of the present invention. Selection of the protecting groups will depend on conditions for their selective removal as well as their compatibility with other features of the library members which are being synthesized. For example, when a photocleavable linker is used in preparation of the libraries, the remaining protecting groups should be selectively removable by chemical means (i.e. , the use of dilute acid or base, or hydrogenolysis). Thus, Fmoc and Boc are only two examples of selectively removable protecting groups. Others are known to one of skill in the art and can be found in, for example, Greene, et al., Protective Groups In Organic Chemistry, 2nd Ed., John Wiley & Sons, New York, NY, 1991, previously incorporated herein by reference. Following attachment of a suitably protected piperazinyl-glutamine scaffold to the linker on the solid support, the scaffold can be selectively derivatized. This derivatization is achieved by first removing one protecting group and attaching the desired building blocks to the free amine. Next, the second protecting group is removed and the free amine is appropriate building blocks are covalently attached to that site. The diversity of piperazine derivatives which can be synthesized by this route is a result of the combinatorial array of building blocks which can be used. For example, the building blocks can be selected from compounds having a variety of functional groups, including amino acids and peptides, carboxylic acids, sulfonyl chlorides and isocyanates. A number of commercially available carboxylic acids, sulfonyl chlorides and isocyanates can be attached to the piperazine nitrogen, and/or to the amine terminus of an amino acid or peptide which is attached to the N^α position of the glutamine residue. Figures 2-4 provide the structures of representative commercially available building blocks. Additionally, one of skill in the art will understand that for those ligands having peptides attached at the N^α position, the peptide can be attached in one synthetic reaction, or it can be constructed at that position via sequential addition of amino acid monomers.

The libraries of the present invention can be used for development of new therapeutic agents as well as for the evaluation of existing therapeutic candidates. For example, libraries comprising a plurality of IL-l receptor ligands can be synthesized having a variety of capping groups attached at either the terminal peptide amine position or on the piperazine moiety. The ligands can be cleaved from the solid support using, for example, photochemical means and evaluated for efficacy against a variety of diseases states associated with unwanted IL-l receptor activity. Alternatively, a library of ligands can be prepared in which each ligand has a known affinity for the IL-l receptor. Following preparation of the library, a solution of labeled receptor is incubated with the library to attach the receptor to the various ligands. A displacement assay, using drug candidates can then be carried out by incubating the library with the drug candidate and determining the extent to which the drug candidate displaces the receptor from the ligands.

Additionally, the libraries prepared according to the methods described above can be used to screen for IL-l receptor activity. In one group of embodiments, a solution containing a marked (labeled) receptor is introduced to the library and incubated for a suitable period of time. The library is then washed free of unbound receptor and the ligands having high affinity for the receptor are identified by identifying those regions on the surface of the library where markers are located. Suitable markers include, but are not limited to, radiolabels, chromophores, fluorophores, chemiluminescent moieties, and transition metals. Alternatively, the presence of receptors may be detected using a variety of other techniques, such as an assay with a labelled enzyme, antibody, and the like. Other techniques using various marker systems for detecting bound receptor will be readily apparent to those skilled in the art.

In a preferred embodiment, a library prepared on a single solid support (using, for example, the VLSIPS™ technique) can be exposed to a solution containing marked receptor. The receptor can be marked in any of a variety of ways, but in one embodiment marking is effected with a radioactive label. The marked receptor binds with high affinity to an immobilized ligand on the surface. After washing the surface free of unbound receptor, the surface is placed proximate to x-ray film or phosphorimagers to identify the ligands that are recognized by the receptor. Alternatively, a fluorescent marker may be provided and detection may be by way of a charge-coupled device (CCD), fluorescence microscopy or laser scanning. When autoradiography is the detection method used, the marker is a radioactive label, such as ³²P. The marker on the surface is exposed to X-ray film or a phosphorimager, which is developed and read out on a scanner. An exposure time of about 1 hour is typical in one embodiment. Fluorescence detection using a fluorophore label, such as fluorescein, attached to the receptor will usually require shorter exposure times.

Quantitative assays for receptor concentrations can also be performed according to the present invention. In a direct assay method, the surface containing localized probes prepared as described above, is incubated with a solution containing a marked receptor for a suitable period of time. The surface is then washed free of unbound receptor. The amount of marker present at predefined regions of the surface is then measured and can be related to the amount of receptor in solution. Methods and conditions for performing such assays are well-known and are presented in, for example, L. Hood et al , Immunology, Benjamin/Cummings (1978), and E. Harlow et al , Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). See, also U.S. Pat. No. 4,376,110 for methods of performing sandwich assays. The precise conditions for performing these steps will be apparent to one skilled in the art.

In instances where the libraries are synthesized on beads in a number of containers, the beads are exposed to a receptor of interest. In a preferred embodiment the receptor is fluorescently or radioactively labelled. Thereafter, one or more beads are identified that exhibit significant levels of, for example, fluorescence using one of a variety of techniques. For example, in one embodiment, mechanical separation under a microscope is utilized. The identity of the molecule on the surface of such separated beads is then identified using, for example, NMR, mass spectrometry, and sequencing of the associated peptide, or the like. In another embodiment, automated sorting (i.e. , fluorescence activated cell sorting) can be used to separate beads (bearing probes) which bind to receptors from those which do not bind. Typically the beads will be labeled and identified by methods disclosed in Needels, et al , Proc. Natl Acad. Sci. , USA 90:10700-10704 (1993), incorporated herein by reference. The foregoing description and the following examples are offered primarily for illustration and not as limitations. It will be readily apparent to those of ordinary skill in the art that the operating conditions, materials, procedural steps and other parameters of the system described herein may be further modified or substituted in various ways without departing from the spirit and scope of the invention. EXAMPLES

EXAMPLE 1

This example describes the preparation of the resin-bound peptide, -Resin

N-Fmoc-L-Glutamic acid γ-t-butyl ester (4.43 g, 10 mmol, from Sigma Chemical

Co., St. Louis, Missouri, USA) was combined with allyl bromide (22 mL) and diisopropyl ethyl amine (3.5 mL, 20 mmol) and heated to reflux under an atmosphere of N₂ for 1 hour. The reaction mixture was cooled and diluted with EtOAc then extracted with 0.1 N HCI. The aqueous layer was extracted with EtOAc and the combined organic portions were washed with 0.05 N NaHCO₃, saturated NaCl, dried over MgSO₄ and filtered. The organic solvent was removed under reduced pressure and the resulting oil was dried under vacuum. A mixture of TFA and CH₂C1₂ (15 mL each) was added and the reaction mixture was stirred at room temperature for two hours then concentrated under reduced pressure. The residual oil was diluted with EtOAc, washed with saturated NaCl, dried over MgSO₄, and filtered. The solvent was removed under reduced pressure and the solid was triturated with EtOAc/hexane to provide Fmoc-L-glutamic acid α-allyl ester.

Fmoc-protected Knorr resin (5.0 g, from Novabiochem) was shaken with a 50:50 mixture of piperidine and NMP for 30 min to remove the Fmoc protecting groups from the resin. The resin was then washed with DMF (5X) and treated with a mixture of Fmoc-L- glutamic acid α-allyl ester (4.44 g), DIEA (3.84 mL), and 38 mL of a 1: 1: 1 0.3 M mixture of HOBt/HBTU/NMP. After 1 hour, the resin was washed with DMF (5X), MeOH, then dried under vacuum.

The resulting resin-bound Fmoc-L-glutamic acid α-allyl ester was subsequently reacted under standard solid phase synthesis conditions first with Fmoc-Y-(O-t-butyl), then with Fmoc-W-(Boc). Removal of the Fmoc group from the tryptophan residue followed by acylation of the terminal amine with acetic anhydride provided the resin-bound triamino acid fragment,

The allyl ester was removed by shaking the resin with Pd PPh_{j j} in a mixture of DMSO, THF, 0.1 N HCI, and morpholine (10:10:5:0.5). The resin was then washed with methylene chloride and DMF and used without further purification.

In a similar manner, the following resin-bound (via the 7-caιboxy group of glutamic acid) fragments were constructed and used in preparation of the compounds of the invention: Ac-Y-W-Q, Ac-W-pY-Q, and Ac-F-E-W-T-P-G-W-Y-Q.

EXAMPLE 2

This example describes a general procedure for the preparation of N-acylated piperazine derivatives which are useful in the preparation of the peptide piperazine derivatives.

General Procedure:

To a solution of carboxylic acid (20 mmol, 2.0 equivalents) in DME (10 mL) is added N-t-butoxycarbonylpiperazine (10 mmol), followed by HOBt (20 mmol, 2.0 equiv.) and EDC (20 mmol, 2.0 equiv.). The mixture is stirred at room temperature overnight then filtered. The filtrate is concentrated, then suspended in EtOAc and 10% citric acid solution. The precipitate is filtered off and the aqueous layer is extracted with EtOAc. The combined organic extracts are washed with saturated NaHCO₃, 10% citric acid, saturated NaCl, dried over MgSO₄, filtered and evaporated to dryness. The product can be purified by recrystallization, column chromatography or preparative HPLC.

The resulting Nl-t-Boc-N4-acylated piperazine is dissolved in methylene chloride and treated with TFA (4 equivalents). After stirring at room temperature overnight, the reaction mixture is concentrated under reduced pressure to provide N-acylated piperazines. The following acylated piperazines were prepared using the general procedures outlined above: N-(4-hydroxybenzoyl)piperazine, N-(4-hydroxyphenylacetyl)piperazine, N- (3-(4-hydroxyphenyl)propionyl)piperazine, N-(4-hydroxymethylbenzoyl)piperazine, and N- (4'-hydroxy-4-biphenylcarbonyl)piperazine. Zo

EXAMPLE 3

This example describes the general procedure for coupling a substituted piperazine to a resin-bound tripeptide.

To the resin-bound tripeptide from Example 1 above (250 mg, 0.125 mmol) was added 4-hydroxyphenylacetylpiperazine (0.50 mmol, 4.0 equiv.), 1.5 mL of 0.3 M HOBt in HBTU/NMP (0.50 mmol, 4.0 equiv.) and 174 μh of diisopropylethylamine (1 mmol, 8 equiv.). The mixture was shaken overnight, filtered, then washed with DMF (5X) and MeOH (3X). The peptide was cleaved from the resin by shaking the resin with using 95% TFA/H₂O (5 mL) and triisopropylsilane (75 μL) for two hours. The resin was filtered off and washed with TFA and methylene chloride. The combined filtrate and washes were concentrated and the product was precipitated with EtjO. The product was further purified by HPLC to provide,

The compounds in Table 1 below were prepared using the general methods or by modifications of the general methods which would be apparent to one of skill in the art. Phenylpiperazine (used in the preparation of 6 and 7 is available from commercial sources. Alkyl (or acyl) piperazines are prepared either by alkylation (or acylation) of N-t-Boc- piperazine followed by removal of the protecting group, or by alkylation of N4-t-Boc- piperazin-2-one, followed by reduction of the amide and removal of the protecting group.

Table 1 ELrl Receptor Antagonists

Compound (R¹)™* — A— — X 1*

AcYW— — CO— 4— OH

AcYW— — COCH, 4— OH

AcWY— —COCH,— 4— OH

AcWY— — COCH₂CH₂— 4— OH

AcFEWTPGWY- — COCH,— 4— OH

AcYW- bond H

AcWY— bond H

8 AcWY- 4— OH

AcWY- — COCHo 4— OH

10 AcWY- 4— CH₂OH

11 AcWY— 4— (4'-C₆H₄' OH)

12 AcWpY— — COCHn 4— OH

13 3-Pyridylacryloyl-W- — COCH, 4— OH

X² and X are hydrogen EXAMPLE 4

This example illustrates the preparation of (Me₃N)Nal(2)-Glu-Trp-Thr-Pro-D-Ala- Trp-Tyr-(NMe)Gln-X where X = piperazine acylated with 4-hydroxyphenylacetic acid, according to the scheme provided below.

N-Boc-N-methyl-L-Glutamic acid 7-benzyl ester (from Bachem Bioscience, Inc.) was combined with 9-fluorenylmethanol (1 eq), 4-dimethylaminopyridine (0.1 eq) and water soluble carbodiimide (EDC) (1 eq) in DMF at 0°C, modifying a procedure described by Kessler, et al , Tetrahedron Lett. 24:281 (1983)). After 1 h the ice bath was removed and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo, diluted with ethyl acetate and then extracted with saturated NaHCO₃ and saturated NaCl. The organic layer was dried over MgSO₄ and filtered. The organic solvent was removed in vacuo and the resulting gum was dried under vacuum to provide the Fmoc ester which was used without further purification. The 7-benzyl ester was removed using a procedure described by Anantharamaiah, et al., J. Chem. Soc. Perkin Trans. I, p. 490 (1977). The protected glutamate derivative was dissolved in a 1:1 mixture of cyclohexene and ethanol containing a suspension of 5% Pd/C. The resulting mixture was heated at reflux until starting material was consumed. The reaction mixture was cooled and filtered to remove the Pd/C catalyst and then concentrated in vacuo.

The carboxylic acid produced was coupled to p-methylbenzhydryl amine resin (from Peptides International) via a procedure described in Stewart and Young, "Solid Phase Peptide Synthesis" (1984). The resin was neutralized with 10% DIEA/DCM for 10 min, washed with DCM (3x) and DMF (3x) and treated with a mixture of Boc-N-methyl- L-glutamic acid α-fluorenyl methyl ester, DCC (1 eq), HOBt (1 eq) in NMP. After 2 h, the resin was washed with DMF (3x), 1:1 DCM/MeOH (3x), MeOH (3x), and DCM (3x).

The resulting resin-bound Boc protected glutamic acid derivative was treated with 50% TFA/DCM for 30 min, washed with DCM (3x), neutralized with 5% DIEA/DCM for 5 min, washed with DCM (3x) and DMF (3x). Boc-Tyr(BrZ) was coupled to the resulting secondary amine via a procedure described by Angell, et al, Tetrahedron Lett. 33:5981 (1994). Boc-Tyr(BrZ) (2.5 equivalents), DIC (2.5 eq.) and HOAc (2.5 eq.) were dissolved in DMF and added to the resin. The pH was monitored by holding moist pH paper over the reaction mixture and the pH was kept above pH 9 with the addition of DIEA. After 2 h, the resin was filtered, washed with DMF (3x), 1: 1 DCM/MeOH (3x), and DCM (3x). Subsequent couplings of Boc amino acids were performed under standard solid phase synthesis conditions.

The resulting resin-bound fragment (with the fragment coupled to the resin via the carboxy goup of glutamic acid), Boc-Nal(2)-Glu(OBn)-T_p-Thr(OBn)-Pro-D-Ala-Trp- Tyr(BrZ)-(NMe)Gln-OFmoc, was treated with 20% piperidine/DMF for 20 min to remove the fluorenylmethyl ester. This was coupled to the 4-hydroxyphenylacerylpiperazine as described in Example 3.

The permethylation of the N-terminal amine was performed by a modification of the procedure described by Macielag, et al U.S. Patent No. 5,422,341. The terminal Boc group was removed by TFA/DCM and the resulting ammonium salt was neutralized as previously described. This peptide-resin was suspended in 20 mL of DMF. To this suspension, CH₃I (400 eq) and K₂CO₃ (20 eq) were added. The reaction mixture was vortexed for 18 h. The resin was filtered and washed with DMF (3x), 1: 1 water/MeOH (3x), water (2x), DMF (3x) and MeOH (3x). Deblocking and cleavage of the peptide from the resin was performed by standard

HF procedures as described in Stewart and Young. The crude peptide was extracted from the resin with 50% aqueous HOAc, diluted with water and lyophilized. EXAMPLE 5

This example illustrates the preparation of a variety of bead-based libraries which incorporate a piperazinylglutamate motif. These libraries have a broad spectrum of activity and are useful for screening IL-l receptors for activity. The libraries are based on the structure activity relationships discovered for compound A, below.

ICgr l uM

Compound A

To prepare the desired libraries it was necessary to develop a suitable synthesis of the differentially protected piperazine glutamic acid scaffold as shown in Figure 5. Thus, Fmoc-glutamic acid benzyl ester was coupled to Boc-piperazine. Hydrogenolysis of the benzyl protecting group was carried out under standard conditions (H₂ atmosphere, Pd/C catalyst in ethanol) to provide a 91 % yield of the desired carboxylic acid. This compound was of sufficient purity to be used in library synthesis without further purification.

To demonstrate that libraries could be prepared and to validate the chemical synthesis on a solid support, compound A was prepared on a Tentagel S — NH₂ resin using a photolabile linking group (PL) as shown in Figure 6. The use of the photolabile linking group results in the release of an amide upon photolysis following completion of the modified peptide.

Libraries were prepared as outlined in Figure 7. An initial library, LI , was prepared having the general formula:

Generic Structure for Ll

Thirty-six unnatural amino acid building blocks were selected for each of the two positions (X_i and X₂, see Figures 8 and 9), resulting in a library of 1296 compounds.

Other libraries, L2-L4, have also been prepared based on compound A, in which the phosphotyrosine and naphthylalanine groups were kept fixed and replacements for the two 4-hydroxyphenylacetic acid (HOPAC) capping groups were investigated. In these libraries, carboxylic acids were used to generate amides, isocyanates were used to generate ureas and sulfonyl chlorides were used to generate sulfonamides. The generic structures of these libraries are depicted below.

Libraries L2-L4

X,= amide, urea and suitonamide

X ₂= suitonamide

In each of these libraries, thirty-six building blocks were used for X_l consisting of twelve each of carboxylic acids (see Figure 10), isocyanates (see Figure 11) and sulfonyl chlorides (see Figure 12). For library L2, the X₂ group formed an amide from each of the ten carboxylic acids in Figure 13. For library L3, the X₂ group formed a urea from each of the ten isocyanates in Figure 14. For library L4, the X₂ group formed a sulfonamide from each of the ten sulfonyl chlorides in Figure 15. As a result, each library, L2, L3 and L4 consisted of 360 compounds.

Still other libraries, L5-L8, were prepared having the general structures provided in Figures 16 and 17. The four libraries were prepared using either commercially available alkyl piperazines (2) or acylated piperazines which were formed during synthesis (2). Additionally, this group of libraries used tyrosine in place of phosphotyrosine. Two synthetic strategies were used for the preparation of the four libraries and are outlined in Figures 16 and 17. As shown in Figure 16, construction of the peptide beginning with the N-terminus results in the scrambling of stereochemistry at the glutamine residue. Alternatively, optically pure libraries can be prepared using the methodology of Figure 17, in which the piperazine is derivatized at the peptide C-terminus followed by subsequent construction of the peptide at the N-terminus. The four libraries were completed using the groups X_j and X₂ as shown in Figures 18-22.

Libraries L1-L3 and L5-L8 were screened in competitive binding assays according to procedures described below. In each case, compound A was used as a positive control and a photolysis buffer (1 % hydrazine in DMSO) was used as a negative control. The results indicated that in each of the libraries LI, L2 and L3, there were several "pools" which were comparable in affinity to the positive control (IC₅₀ = 1 μM). Figure 23 shows the structures of compounds represented in the active pools. The six pools were deconvoluded and the structures of the most active compounds were identified. Four of these compounds were synthesized (compounds B, C, D, and E) and their IC₅₀ values were determined. Structures and activity are shown in Figure 24.

Library L5 did not exhibit any active pools, however L6 provided the three active pools shown in Figure 25.

EXAMPLE 6

Assay for Interleukin-l Receptor Antagonist Activity

The compounds of the present invention may be tested for interleukin-l receptor antagonist activity by using a competitive assay and labeled IL-lα. In this assay, stock solutions of each compound were prepared. The appropriate amount of the compound was dissolved in DMSO, and then nineteen volumes of binding buffer (RPMI 1640, 1 % BSA, 20 mM HEPES, pH 7.2-7.3, and 0.1 % sodium azide) were added to yield a 1 mM compound, 5 % DMSO stock solution.

One assay utilized a truncated IL-l Rtl which had been immobilized on 96-well plates with an appropriate antibody, typically a non-blocking high affinity 35 antibody. In other assays, cells expressing "full-length" IL-lRtI were used with results that yielded IC₅₀ values about 5 to 10 fold lower than those determined using the truncated receptor. The cells were seeded onto Falcon 3072 96- well plates at about 10⁵ cells per well, and the plates were incubated overnight at 37^* C in media containing serum. The following morning, the cells were checked to ensure that the cells were confluent and adhered to the bottom of the wells.

According to either assay protocol, the plates were then washed three times with binding buffer, and then 50 mL of binding buffer and 25 mL of a compound solution (either the stock solution or a dilution thereof; each stock was subjected to five three fold dilutions) were added to each well. Then 25 mL of binding buffer containing ¹²⁵I-IL-lα (final concentration of 90 pM) were added to each well to begin the assay. Each assay was carried out in duplicate. The plates were then incubated for two hours at 4^*C.

After the two hour incubation, the wells were rinsed three times with ice cold PBS (a semi-automated cell harvesting device was used to conduct the rinse). The receptors or cells were then detached from the plates by adding 100 mL of 0.1 N NaOH to each well and incubating the plates at room temperature for 20 minutes. After the 20 minute incubation, about 75 mL of the suspension was counted on a gamma counter, and the IC₅₀ for each compound was determined using computer assistance and the results of the gamma counting. Table 2 lists IC₅₀ values for representative compounds of the invention.

36 PC17US96/03955

Table 2 Interleukin-l Receptor Antagonist Activity

* v Xl¹ is 4-OH, X² and X³ are both H

EXAMPLE 7

This example illustrate the inhibition of IL-l induced EGF-R down-regulation in NHDF cells by Compound 5.

This assay for EGF-R down regulation in NHDF cells is a functional assay which is designed to illustrate cellular responses to compounds which inhibit the action of IL-l. NHDF cells are plated into a 24-well plate at about 10⁵ cells per well, and the plates were incubated overnight at 37 °C in media containing serum. The following morning, the cells were checked to ensure that the cells were confluent and adhered to the bottom of the wells.

The plates were then washed three times with binding buffer, and a compound solution (either the stock solution or a dilution thereof; each stock was subjected to five three fold dilutions) was added to each well. The cells were preincubated with the compounds for 2 minutes at 37°C, then incubated with IL-l plus the compound for 20 minutes at 37°C. The concentration of IL-l was held constant at 5 pM (IC₅₀ for Ll-lα is 1.8 pM). ¹²⁵I EGF was added and the cells were incubated for 3 hours at 4°C. The cells were then washed and the amount of EGF which remained bound to the cells was determined. Following this protocol, the EC₅₀ for Compound 5 was found to be 3.5 μM.

EXAMPLE 8

This example illustrates the use of the compounds of the present invention in a topical formulation to treat psoriasis, a skin inflammatory condition. An adult patient having severe cutaneous manifestations of psoriasis is selected for therapy. The patient is evaluated with screening tests including vital signs with special attention to blood pressure and any tendency to orthostasis, a complete blood count, a urine analysis, and peripheral blood tests for blood urea nitrogen, sodium, potassium, calcium, and creatinine. A chest x-ray (CXR) and electrocardiogram (EKG) are also performed. The practitioner makes notations of the severity and extent of the dermal psoriatic plaques and performs biopsies if indicated.

The patient is treated with a topical piperazine agent in a cream vehicle of concentration 2% (weight/volume) applied directly to the psoriatic skin areas three to four times a day until a therapeutic benefit is achieved. Thereafter, the cream is applied less frequently, as needed, to maintain the benefit. The patient is evaluated in follow up visits with the practitioner with special attention to blood pressure, including any orthostatic changes, and cardiac status.

EXAMPLE 9

This example illustrates the use of the present compounds to treat rheumatoid arthritis, a systemic inflammatory condition.

An adult patient having rheumatoid arthritis involving multiple small joints, several large joints, and intermittent debilitating systemic symptoms is selected for treatment. Screening tests as in Example 7 are performed. At the practitioner's discretion, the patient's current medications are continued, and oral piperazine agent is added to the current regimen. The piperazine agent is started at 20 mg twice a day. If tolerated, the dosage is increased in increments of 20 mg daily until a therapeutic benefit is achieved. If needed, the piperazine agent is injected directly into joints which are especially problematic. An aqueous concentration of 10 mg/mL is used, and each joint treated is injected with 20-40 mg of the piperazine agent. The injection is repeated weekly if needed.

EXAMPLE 10

This example illustrates the use of a piperazine agent as a second active agent in a transdermal patch.

A patient is selected for treatment with a transdermal patch including a first active agent which is not verapamil. Because the patient has, in the past, experienced unacceptable local irritation from a transdermal patch, the piperazine agent is incorporated into the patch as a second active agent for its anti-inflammatory effects. The concentration of piperazine agent in the patch is about 0.5% by weight. The patch is applied according to the requirements of the first active agent. Because of the addition of the piperazine agent to the patch, the patient does not experience unacceptable local skin irritation at the site of application of the patch.

The above description is illustrative and not restrictive. Many variations of the invention will become apparent to those of skill in the art upon review of this disclosure. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents.

Claims

WHAT IS CLAIMED IS:

1. An interleukin-l receptor antagonist of formula I:

wherein:

A is a member selected from the group consisting of a single bond,

—CO—, —CONH—, —COZ¹—, — CON(Z²)— , — CON(Z²)Z¹— and — SO₂— ; R is a member selected from the group consisting of single amino acids and fragments comprising from 2 to 20 amino acids; R¹ is a member selected from the group consisting of H, alkyl, aryl, acyl and sulfonyl; m is an integer of from 1 to 3; X¹, X² and X³ are each members independently selected from the group consisting of H, —OH, — Z¹— OH, — OZ², — F, — Cl, — Br, —I,

— NO₂, — SO₂NH₂, — CO₂H, — CONH₂, —CONHZ², — NHZ²,

— NZ² ₂, — CO₂Z², and optionally where X¹ and X² taken together form a fused ring; wherein:

Z¹ is a linking group selected from the group consisting of alkyl, alkenyl and alkynyl; and Z² is a member selected from the group consisting of H, alkyl and aryl, and its pharmaceutically acceptable salts.

2. An interleukin-l receptor antagonist of claim 1 wherein R¹ is a member selected from the group consisting of acetyl, propionyl, 4-hydroxybenzoyl, 4- hydroxyphenylacetyl, 4-hydroxyphenylpropionyl, 3,4-dihydroxyphenylacetyl, 3-nitro-4- hydroxyphenylacetyl, 4-hydroxymethylphenylacetyl, 4-nitrophenylacetyl and 3- pyridylacryloyl. 3. An interleukin-l receptor antagonist of claim 1 wherein A is a member selected from the group consisting of — CO — , — COZ¹ — and — CONHZ¹ — , and R¹ is a member selected from the group consisting of acetyl, propionyl, 4-hydroxybenzoyl, 4- hydroxyphenylacetyl, 4-hydroxyphenylpropionyl, 3,4-dihydroxyphenylacetyl,

3-nitro-4- hydroxyphenylacetyl, 4-hydroxymethylphenylacetyl, 4-nitrophenylacetyl and 3- pyridylacryloyl.

4. An interleukin-l receptor antagonist of claim 1 wherein R is a peptide of the formula (AA')_n wherein AA represents an amino acid residue; i is an integer denoting the position downstream from the glutamine residue; and n is an integer of from 2 to 20; such that amino acid residues at any said position may be the same as or different from amino acid residues at any other said position.

5. An interleukin-l receptor antagonist of claim 1 wherein:

AA¹ is an amino acid selected from the group consisting of Tyr, Trp, Glu,

Pro, Gly, pTyr, Nal, Phe, BtPhe and Thr; and n is 2 to 10.

6. An interleukin-l receptor antagonist of claim 1 wherein:

AA¹ and AA² are each amino acids independently selected from the group consisting of Tyr, Trp, Glu, Pro, Gly, pTyr, Nal, Phe, BtPhe and Thr; and n is 2 to 10.

7. An interleukin-l receptor antagonist of claim 1 wherein:

AA¹ is an amino acid selected from the group consisting of Tyr and Trp; AA² and AA³ are each amino acids independently selected from the group consisting of Tyr, Trp, Glu, Pro, Gly, pTyr, Nal, Phe, BtPhe and

Thr; and n is 3 to 10.

8. An interleukin-l receptor antagonist of claim 1 wherein:

AA¹ and AA² are each amino acids independently selected from the group consisting of Tyr, Trp and pTyr; AA³ and AA⁴ are each amino acids independently selected from the group consisting of Tyr, Trp, Glu, Pro, Gly, pTyr, Nal, Phe, BtPhe and

Thr; and n is 4 to 10.

9. An interleukin-l receptor antagonist of claim 1 wherein: AA¹ is an amino acid selected from the group consisting of Tyr, Trp and pTyr; A A² is an amino acid selected from the group consisting of Tyr and Tip; AA³, AA⁴ and AA⁵ are each amino acids independently selected from the group consisting of Tyr, Trp, Glu, Pro, Gly, pTyr, Nal, Phe, BtPhe and Thr; R¹ is a member selected from the group consisting of acetyl, propionyl,

4-hydroxybenzoyl, 4-hydroxyphenylacetyl, 4- hydroxyphenylpropionyl, 3,4-dihydroxyphenylacetyl, 3-nitro-4- hydroxyphenylacetyl, 4-hydroxymethylphenylacetyl, 4- nitrophenylacetyl and 3-pyridylacryloyl; A is a member selected from the group consisting of — CO — , — COZ¹ — and —CONHZ¹—; X¹ is a member selected from the group consisting of H, — OH and

— Z¹— OH; X² is H; and n is 5 to 10.

10. An interleukin-l receptor antagonist of claim 1 wherein R¹ is acetyl, R is — YW— , A is — COCH₂— , X¹ is 4-hydroxy, and X² and X³ are both hydrogen.

11. An interleukin-l receptor antagonist of claim 1 wherein R¹ is acetyl, R is — WY— , A is — COCH₂— , X¹ is 4-hydroxy, and X² and X³ are both hydrogen.

12. An interleukin-l receptor antagonist of claim 1 wherein R¹ is acetyl, R is — WY— , A is — COCH₂CH₂— , X¹ is 4-hydroxy, and X² and X³ are both hydrogen.

13. An interleukin-l receptor antagonist of claim 1 wherein R¹ is acetyl, R is — FEWTPGWY— , A is — COCH₂— , X¹ is 4-hydroxy, and X² and X³ are both hydrogen.

14. An interleukin-l receptor antagonist of claim 1 wherein R¹ is acetyl, R is —WpY—, A is — COCH₂— , X¹ is 4-hydroxy, and X² and X³ are both hydrogen.

15. A pharmaceutical composition for the treatment of conditions mediated by unwanted interleukin-l activity which comprises a compound of the formula I:

wherein:

A is a member selected from the group consisting of a single bond,

—CO—, —CONH—, —COZ¹—, — CON(Z²)— , — CON^Z¹— and — SO₂ — ; R is a member selected from the group consisting of single amino acids and peptides comprising from 2 to 20 amino acids; R¹ is a member selected from the group consisting of alkyl, aryl, acyl and sulfonyl; m is an integer of from 1 to 3; X¹, X² and X³ are each members independently selected from the group consisting of H, —OH, — Z¹— OH, — OZ², — F, — Cl, — Br, —I,

— NO₂, — SO₂NH₂, — CO₂H, — CONH₂, —CONHZ², — NH₂,

— NHZ², — NZ² ₂, — CO₂Z², and optionally where X¹ and X² taken together form a fused ring; wherein:

Z¹ is a linking group selected from the group consisting of alkyl, alkenyl and alkynyl; and Z² is a member selected from the group consisting of H, alkyl and aryl; and pharmaceutically acceptable salts thereof in a pharmaceutically acceptable carrier.

16. A method of treating conditions mediated by unwanted interleukin-l receptor activity which method comprises administering to a subject in need of such treatment an effective amount of a compound of formula I:

wherein:

A is a member selected from the group consisting of a single bond,

—CO—, —CONH—, —COZ¹—, — CON(Z²)— , — CONfZ^Z¹— and — SO₂— ; R is a member selected from the group consisting of single amino acids and peptides comprising from 2 to 20 amino acids; R¹ is a member selected from the group consisting of alkyl, aryl, acyl and sulfonyl; m is an integer of from 1 to 3; X¹, X² and X³ are each members independently selected from the group consisting of H, —OH, — Z¹— OH, — OZ², — F, — Cl, — Br, —I,

— NO₂, — SO₂NH₂, — CO_jH, — CONH₂, —CONHZ², — NH₂,

— NHZ², — NZ² ₂, — CO₂Z², and optionally where X¹ and X² taken together form a fused ring; wherein:

Z¹ is a linking group selected from the group consisting of alkyl, alkenyl and alkynyl; and Z² is a member selected from the group consisting of H, alkyl and aryl; or its pharmaceutically acceptable salt.

17. A synthetic IL-l receptor ligand library comprising a plurality of different members, each member having the formula:

wherein,

S¹ is a solid support;

L is a spacer or linking group;

A is a member selected from the group consisting of a single bond,

—CO—, —CONH—, —COZ¹—, — CON(Z²)— , — CON(Z²)Z¹— and — SO₂— ; R is a member selected from the group consisting of single amino acids and fragments comprising from 2 to 20 amino acids; R¹ is a member selected from the group consisting of H, alkyl, aryl, acyl and sulfonyl; m is an integer of from 1 to 3; X¹, X² and X³ are each members independently selected from the group consisting of H, —OH, — Z¹— OH, — OZ², — F, — Cl, — Br, —I,

— NO₂, — SO₂NH₂, — CC^H, — CONH₂, —CONHZ², — NHZ²,

— NZ² ₂, — CO₂Z², and optionally where X¹ and X² taken together form a fused ring; wherein:

Z¹ is a linking group selected from the group consisting of alkyl, alkenyl and alkynyl; and Z² is a member selected from the group consisting of H, alkyl and aryl.

18. A synthetic IL-l receptor ligand library in accordance with claim 17, wherein said solid support is a bead.

19. A synthetic IL-l receptor ligand library in accordance with claim 17, wherein said linking group is photocleavable.

20. A synthetic IL-l receptor ligand library in accordance with claim 17, having at least 100 members.