AU4830799A - Bifunctional antagonists of cytokine-sensitive protein kinase activation cascades and methods for use as anti-inflammatory agents - Google Patents

Description

WO 00/00491 PCTIUS99/14320 PATENT BIFUNCTIONAL ANTAGONISTS OF CYTOKINE-SENSITIVE PROTEIN KINASE ACTIVATION CASCADES 5 AND METHODS FOR USE AS ANTI-INFLAMMATORY AGENTS RELATED PATENT APPLICATIONS This application is a continuation-in-part of US Patent Application Serial Number 08/858,778 filed May 19, 1997 (now pending), which is in turn a continuation-in-part of US Patent Application Serial Number 08/367,102, filed 10 December 29, 1994 (now pending), which is in turn a continuation-in-part of US Patent Application Serial No. 08/482,55 1, filed June 7, 1995 (now allowed). STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under Grant No. GM 23200 awarded by the National Institutes of Health. The government may have 15 certain rights in the invention. BACKGROUND FOR THE INVENTION 1. Field of the Invention The invention relates to antagonists of cytokine-sensitive, mitogen activated protein kinase activation and their use as anti-inflammatory agents. - 1- WO 00/00491 PCTIUS99/14320 2. History of the Related Art Two of the MAPK (mitogen activated protein kinase) signalling cascades are initiated by proinflammatory cytokines (PICs) and other stress factors, such as ultraviolet light, ionizing radiation, tissue trauma, cytotoxic agents (e.g., anisomycin 5 and arsentine), heat shock and ceramide. These "PIC sensitive" signalling cascades lead to activation of the c-Jun N-terminal protein kinase (JNK; also known as SAPK, for "stress-activated protein kinase") and a newly discovered protein kinase, p38 (also known as CSBP, for "cytokine suppressive anti-inflammatory drug binding protein"). Activation of JNK and p38 is related to the onset and maintainence of 10 acute and chronic inflammatory conditions such as localized fibrosis, cystic fibrosis, ultraviolet light induced cutaneous immune suppression, cell senescence and apoptosis, gastrointestinal inflammation (e.g., inflammatory bowel disease) and pulmonary inflammation (e.g., chronic bronchitis and asthma). Inhibition of PIC synthesis and/or release by stressed cells can reduce MAPK activation-related 15 inflammation by avoiding initiation of the PIC sensitive MAPK signalling cascades. Different anti-inflammatory agents approach this goal in different ways. For example, pentoxifylline suppresses synthesis of PICs by inhibiting cAMP phosphodiesterase activity. Corticosteroids inhibit transcription of PICs. Cytokine suppressive anti-inflammatory drugs (CSAIDs) are believed to inhibit PIC synthesis 20 by binding a MAPK required for cytokine mRNA translation; these compounds selectively interfere with activation of the p38 signaling cascade. None of the drugs are completely effective in suppressing PIC synthesis or release and many have side effects that are use-limiting. SUMMARY OF THE INVENTION 25 The invention provides compounds which are bifunctional in that they block PIC initiation of both of the PIC sensitive MAPK activation cascades. In particular, the invention combines a novel antagonist of the JNK activation cascade with another PIC inhibitor, most preferably an antagonist of the p38 activation cascade. The components of the resulting "bifunctional activation cascade -2- WO 00/00491 PCT/US99/14320 antagonists" act synergistically to antagonize PIC-sensitive MAPK activation to a greater extent than could be achieved by either compound alone. In particular, the bifunctional activation cascade antagonists of the invention inhibit up to 100% of PIC release by stressed cells. In constrast, 70-80% 5 PIC inhibition is the maximum level achievable by known PIC-sensitive MAPK antagonists. Advantageously, bifunctional activation cascade antagonists do not pose the risk of side effects associated with certain anti-inflammatory agents, such as the sleeplessness and anxiety induced by methylxanthine-based cAMP phosphodiesterase inhibitors (e.g., pentoxifylline). Further, the potency of the inventive compounds 10 permits their use at lower dosages than are required by use of their constituents alone. The JNK activation cascade antagonist constituent of the inventive bifunctional activation cascade antagonists consists of heterocyclic molecules with biologically active side chains; specifically, purine, pteridine, thiadiazolopyrimidine, quinalozine and isoquinolone based compounds and water-soluble morpholinoethyl 15 esters thereof. Such compounds and methods for their synthesis are described in detail in commonly owned US Patent Application Serial Nos.08/858,778, 08/367,102 and 08/482,551, the disclosures of which are incorporated herein as though set forth in full. The p38 activation cascade antagonist constituents are any compound which interferes with the initiation or completion of the p38 activation cascade. In 20 one aspect of the invention, the p38 activation cascade antagonist is a pyridylimidazole. An especially useful class of pyridylimidazoles for use in the invention are the CSAIDs. The JNK and p38 activation cascade antagonist constituents of the bifunctional activation cascade antagonists are preferably conjugated together. Thus, 25 in one aspect of the invention, the constituents of the bifunctional activation cascade antagonists are conjugated to one another via a bond which is severable in vivo, such as ester, amide or azo linkages. In another aspect of the invention, unconjuated JNK and p38 activation cascade antagonist constituents are mixed in a pharmaceutically acceptable carrier. In yet another aspect of the invention, conjugated or unconjugated -3- WO 00/00491 PCTIUS99/14320 JNK and p38 constituents of the bifunctional activation cascade antagonists are combined with a delivery vehicle, such as a colloidal dispersion system. The invention further encompasses the following conjugated bifunctional activation cascade antagonists: CC~ CH 0 0 p N 0 N N

C;

3

H

7 N 5 wherein n is any number of carbon atoms from 1 to 7, 0 or N; RI, if present, is H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or aralkyl having less than 7 carbon atoms; R 6 and R 7 are H, OH or OR , in any combination; Z is N or C; 10 X, where Z is C, is H, halogen, N 3 , NO, NH 2 , NHR , N(R )2 or COR 1; and, A is H, halogen, N 3 , NO, NH 2 , NHR , N(R )2 or COR 1 . -4- WO 00/00491 PCT/US99/14320 BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 is a bar graph depicting inhibition of cell growth arrest in 3T3 fibroblasts according to the invention after growth arrest was induced through deprivation of the cells of serum. The cells were incubated and grown to 90% 5 confluence in serum. The medium was then removed and replaced with serum-free medium. To assess the effect of an inventive compound (no. 37, a pteridine) on cell senescence in the presence of ceramide, aliquots of the cells were incubated with different concentrations of each. Concentrations of compound no. 37 are indicated by the insert legend while concentrations of ceramide are indicated along the x axis. 10 Inhibitory effects were assessed as a measure of DNA synthesis; [ 3 H] thymidine incorporation detection is indicated along the y axis. FIGURE 2 is a bar graph depicting inhibition of cell apoptosis in human (Jurkat) T lymphocytes according to the invention. The inhibitory activity of two inventive compounds (nos. 37 and 6 (a purine)) was tested in comparison to like 15 activity of pentoxyfilline and a control compound, Ro 20-1724. Activation of the sphingomyelin signal transduction pathway was stimulated by incubation of the cells with an anti-FAS monoclonal antibody (which binds CD95, a cell surface receptor which triggers cell apoptosis). Percent inhibition was measured as a function of the number of cells which excluded vital dye erythrosin B. Percent inhibition is indicated 20 along the y axis while the concentration of compounds tested is indicated along the x axis. FIGURE 3 is a bar graph depicting inhibition of activity on the part of CaPK in Jurkat cells according to the invention. The inhibitory activity of a compound of the invention (no. 37) was tested in the presence of either ceramide or 25 anti-FAS. Inhibition of CaPK activity was measured as a function of phosphorylation and detected by autoradiography. The compounds the cells were incubated in are indicated along the y axis while the percent control (i.e., inhibition of CaPK) is indicated along the x axis. Shorter bars indicate greater relative inhibition. -5- WO 00/00491 PCT/US99/14320 FIGURES 4 (a) through (g) are copies of spectrographs indicative of absorbance of inventive compounds no. 37, no. 6, no. 37 in combination with no. 6, oxo variants of nos. 37 and 6, as well as, for comparison, PABA (p-amino benzoic acid, a common sunscreen additive) and isoquinolone. The inventive compounds 5 absorbed through most of the UVB wavelength, while a mixture of compound nos. 37 and 6 absorbed throughout the UVB wavelength. FIGURES 5(a) and (b) depict, respectively, the results of an enzyme linked immunosorbent assay (ELISA) for TNF-a production by bacterial lipopolysaccharide (endotoxin) stimulated human monocytes incubated with the 10 compounds of the invention and a control compound (Ro-1724, that is a known and specific inhibitor of phosphodiesterase type IV [the predominant isoform of phosphodiesterase found in monocytes and neutrophils]). Compounds tested are identified by the number assigned to them in Table 1. The horizontal axis of each graph shows the amount of each compound tested (in pM) while the vertical axis 15 shows the IC 50 values for TNF-a production as a percentage of the production in the presence of only the control compound. FIGURE 6 is a graph depicting the results of an assay for in vivo leukopenia in mouse blood in response to lipopolysaccharide (LPS). Leukopenia induced by LPS is mediated by TNF. Hence, this model assesses both TNF 20 production and action. Compounds tested for inhibition of leukopenia are identified by the number assigned to them in Table 1. Along the x axis of the graph, the numbers correspond to the number of white blood cells detected as cells/ml of fluid. -6- WO 00/00491 PCT/US99/14320 The results (shown by bars) are expressed in terms of a percentage of the leukopenia response (based on neutrophil content) to pure LPS, in absence of other compounds. FIGURE 7 depicts the results of an assay for inhibition by compounds of the invention (nos. 37 and 6) of the effects of a cell permeable ceramide analog (C 2 5 ceramide), dihydro ceramide and diacyl glycerol on TNF-a production by human monocytes. Inhibition of TNF-a production was measured by ELISA; the results are indicated in pg/ml of TNF-a along the x axis. FIGURE 8 depicts the results of an assay for inhibition by a compound of the invention (no. 37) to prevent the stimulatory effects of C 2 - ceramide or protein 10 kinase C activity in human lymphocyte extracts. Inhibitory effects were assessed as a measure of DNA synthesis; [3H] thymidine incorporation detection is indicated along the y axis. FIGURE 9 depicts the results of an assay for in vitro TNF-a production by human macrophages in response to lipopolysaccharide (LPS) and inhibition of that 15 production by pteridine and isoquinolone compounds of the invention (nos. 37 and 11-49). Along the x axis of the graph, the numbers correspond to the concentration of TNF-a detected in pg/ml. FIGURE 10 depicts inhibition of PDGF induced fibroblast proliferation among 3T3 fibroblasts in response to the inventive compounds. The compounds 20 tested are identified along the x axis by the numbers assigned to them in Table 1. Inhibitory effects were assessed as a measure of DNA synthesis; [ 3 H] thymidine incorporation detection is indicated along the y axis. -7- WO 00/00491 PCT/US99/14320 FIGURE 11 depicts inhibition of EGF induced fibroblast proliferation among 3T3 fibroblasts in response to the inventive compounds. The compounds tested are identified along the x axis by the numbers assigned to them in Table 1. Inhibitory effects were assessed as a measure of DNA synthesis; [ 3 H] thymidine 5 incorporation detection is indicated along the y axis. FIGURE 12 depicts data indicative of the apoptopic protective characteristics of the compounds of the invention as represented by compounds 1C 261 (Compound 37) and 11-49. Human lymphocytes were cultured in serum aliquots with the concentrations of the inventive compounds indicated along the x axis of the 10 FIGURE. Protective effects were measured over 4 days as a function of the length of survival of the cultured cells in the presence of the inventive compounds as compared to survival of the cells in the absence of the inventive compounds. 100% survival (y axis) means that a number of treated cells all survived throughout the test period while an equal number of untreated cells died. 15 FIGURES 13(a) through (c) show the structure of commercially available isoquinoline structures whose inhibitory effect with respect to production of TNF-a by human monocytes prior to modification to add side chain substituents according to the invention was tested. Except for Compound S52,626-6 (6,7-dimethoxy- 1(2H) isoquinoline, which possessed mild inhibitory activity as shown in FIGURE 10) none 20 of the tested compounds possessed any such inhibitory activity prior to their modification according to the invention, even at concentrations up to 500 gM. -8 - WO 00/00491 PCT/US99/14320 DETAILED DESCRIPTION OF THE INVENTION Unless otherwise noted, each of the references cited in the following disclosure is incorporated herein by reference for the purpose of illustrating the level of knowledge and skill in the art with respect to the subject matter discussed. In view 5 of this disclosure, those of ordinary skill in the art will be familiar with, or can readily ascertain, equivalents to the techniques, compounds and other material discussed in each reference suitable for use in the invention. I. Composition and Activity 10 of the Bifunctional Activation Cascade Antagonists The bifunctional activation cascade antagonists comprise a JNK activation cascade antagonist constituent as described in commonly owned US Patent Application Serial Nos.08/858,778, 08/367,102 and 08/482,551 (which describe the constituent compounds but do not suggest their selectivity for antagonizing the JNK 15 activation cascade) and another anti-inflammatory compound which antagonizes the p38 activation cascade. Each constituent inhibits PIC production, especially production of TNFa. Examples of JNK and p38 activation cascade antagonists are identified in Table I and compared to other anti-inflammatory agents. In the Table, the CSAID compounds which begin with "SK&F" or "SB" are manufactured by 20 Smith-Kline -9- WO 00/00491 PCTIUS99/14320 Beecham Pharmaceuticals, USA. The "FR" compound is manufactured by Fuji Laboratories, Japan. The "SR" compound is manufactured by Sanofi Recherche, France. Other compounds listed are commercially available from different sources. It should be noted that the term "CSAID" is used in this disclosure as a 5 generic term for p38 activation cascade antagonists, especially pyridylimidazole compounds. However, it is acknowledged that CSAID is also used as a trademark by SmithKlineBeecham Pharmaceuticals for specific pyridylimidazole compounds which antagonize p38 activation. Such compounds are included in, but do not exclusively comprise, the class of compounds referred to herein as "CSAIDs". -10- WO 00/00491 PCT/US99/14320 TABLE I COMPOUND CLASS ACTIVITY EXAMPLES JNK activation cascade antagonists PIC Described in US inhibitors, Patent Application affect JNK Serial activation Nos.08/858,778, cascade 08/367,102 and 08/482,551 CSAIDS PIC SK&F 86002 (also inhibitors, inhibits CO/5-LO), affects p38 SK&F 104351, activation SK&F 105561, cascade SK&F 105809, SK&F 202190, SB 203580, SB 206718, PD 098059 SR31747 FR133605 5 NSAID prosotoglandin endoperoxidase inhibit CO ibuprofen (PGHS) and 5-lipooxygenase inhibitors and/or 5-LO; naproxen (combination compounds also known as tend to cyclooxygenase(CO)/lipooxygenase (5- stimulate PIC LO) dual inhibitors) production 10 corticosteroids PIC inhibitor cortisone, fluticasone propionate, beclomethosone, prednisolone phosphodiesterase (PDE) inhibitors inhibit PDE in rolipram (inhibits signalling 4-PDE) transduction RO- 1724 (same pathways target activity) Of the classes of compounds listed in Table I, the NSAIDs and PDE inhibitors have, respectively, undesirable PIC stimulatory activity and side-effects. Similarly, while useful, corticosteroids pose the risk of certain undesirable side-effects -11- WO 00/00491 PCT/US99/14320 and are not as specific in their PIC inhibitory activity as CSAIDs. For this reason, pyridylimidazole compounds, especially CSAIDs, are the compounds of choice for use as partners to JNK activation cascade antagonists to form the bifunctional activation cascade antagonists of the invention, which may be administered alone or 5 together with other anti-inflammatory agents and medicaments as indicated. Administered alone, CSAIDs inhibit 70-80% or less of TNFa release in an art-accepted model of stress-induced inflammation (Example 2). CSAIDs predominantly antagonize activation of p38 kinase, but do not affect JNK activation to a signficant degree. Conversely, the activity of the JNK activation cascade antagonist 10 constituents of the bifunctional activation cascade antagonists of the invention is predominantly directed toward inhibition of PIC-stimulated JNK activation. These constituents of the inventive compounds have little impact on p38 activation. Surprisingly, combination of CSAIDs with JNK activation cascade antagonists produces a synergistic effect which gives the resulting bifunctional 15 activation cascade antagonists greater anti-inflammatory effect than is achieved by either compound alone. For example, as measured by suppression of TNFa release in stressed cells, up to 100% inhibition is achieved by bifunctional activation cascade antagonists (Example 1). The JNK activation cascade antagonist and CSAID constituents of the 20 bifunctional activation cascade antagonists of the invention antagonize activation of different MAPK pathways in a non-overlapping manner. While the invention is not to be limited by any particular theory as to its mechanism of action, it is likely that the increased potency of bifunctional activation cascade antagonists as compared to their -12- WO 00/00491 PCTIUS99/14320 constituent components is owing to a synergism produced by their non-overlapping effects on both of the PIC-sensitive MAPK signalling pathways. Thus, the bifunctional activation cascade antagonists of the invention exert synergistically enhanced, non-additive, pathway-specific effects on the JNK and p38 activation 5 cascade pathways. As such, the compounds can provide greater anti-inflammatory potency, with relatively low risk of additive toxicity, than would be expected from a combination of anti-inflammatory agents. Descriptions of representative CSAIDs and JNK activation cascade antagonists useful as constituents of the bifunctional activation cascade antagonists of 10 the invention are provided in Section II, A and B of this disclosure. For example, CSAID SB 203580 (inhibits inflammation in several animal models; Badger, et al.,J.Pharmacol.Exp.Ther., 3:1453-1461, 1996) and SB 210313 (Boehm, et al., J.Med.Chem., 39:3929-3927, 1996). Particularly potent JNK activation cascade antagonist constituents are compounds 54 and 54a (morpholinyl esters of Compound 15 52, described in Example XVII; isoquinilones), compound 6 (a purine) or compound 37 (a pteridine). Representative structures for each of these high potency constitutents are: N F (1)~ N N CSAID SB203580 -13- WO 00/00491 PCT/US99/14320 ( 2 ) 0 O N O

C

3

H

7 0 Me O N N OMe COMPOUNDS 54 and 54a -14- WO 00/00491 PCTIUS99/14320 (3) O R1 , NyJ N, I /X A ''Y R2 Where R 2 is (CH 2

)

3 COOEt COMPOUND 6 (4) F N 0N N HO"

CH

3 CHO O N N N N N13H 5 COMPOUND 37 The constituents of the bifunctional activation cascade antagonists may be co-administered as separate compounds. However, to ameliorate their systemic activity, the constituents may be conjugated to one another by a bond which is severable in vivo and delivered as a prodrug or combined as separate constituents 10 within a single unit delivery vehicle, such as a liposome. -15- WO 00/00491 PCTIUS99/14320 Suitable conjugation means include amide, ester and aso linkages, which are separable in vivo by, for example, the action of proteinases or esterases. A particularly useful conjugation bond that is cleavable in vivo is a tertiary N acyloxymethyl amide bond (Moreira, et al., Tetrahedron Lett., 35:7107-7110 (1994), 5 the disclosure of which is incorporated herein to illustrate the steps for formation of such bonds). Briefly, tertiary N acyloxymethyl amide bonds are formed to stabilize carboxylic acid or secondary amide moities on prodrugs (i.e., drugs bound to inert carriers by a bond which is separable in vivo). The bonds involve direct coupling of a 10 RCoNR 2

CH

2 (secondary amide) moiety to an R 3

CO

2 H drug. -16- WO 00/00491 PCT/US99/14320 Examples of bifunctional activation cascade antagonists bound by ester linkages are: (5) N IH N NN N R 0 N 0 N N F

C

3

H

7 N F 0 0 OMe N Oz N N N a OMe SB203313/COMPOUND 54 or 54a -17- WO 00/00491 PCT/US99/14320 (6) C CH 3 N N N C) N N N

C

3

H

7 SB203580/COMPOUND 37 (7) F \ 0 0 N O., N N NN 0 N N

C

3

H

7 SB203313/COMPOUND 37 5 wherein, for all compounds, n is any number of carbon atoms from 1 to 7, O or N; RI, if present, is H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or aralkyl having less than 7 carbon atoms;

R

6 and R 7 are H, OH or OR,, in any combination; 10 Z is N or C; X, where Z is C, is H, halogen, N 3 , NO, NH 2 , NHR,, N(R,)2 or CORI; and, A is H, halogen, N 3 , NO, NH 2 , NHR,, N(R,)2 or COR,. -18- WO 00/00491 PCT/US99/14320 The bifunctional activation cascade antagonists of the invention may be prepared in a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers preferred for use with the bifunctional activation cascade antagonists of the invention may include sterile aqueous of non-aqueous solutions, suspensions, and 5 emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/ aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's 10 or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxi dants, chelating agents, and inert gases and the like. A composition of bifunctional activation cascade antagonists may also be lyophilized using means well known in the 15 art, for subsequent reconstitation and use according to the invention. Absorption promoters, detergents and chemical irritants (e.g., keritinolytic agents) can enhance transmission of a bifunctional activation cascade antagonist to a target tissue. For reference concerning general principles regarding absorption promoters and detergents which have been used with success in mucosal delivery of 20 organic and peptide-based drugs, see Chien, Novel Drug Delivery Systems, Ch. 4 (Marcel Dekker, 1992). Examples of suitable nasal absorption promoters in particular are set forth at Chien, supra at Ch. 5, Tables 2 and 3; milder agents are preferred. Suitable agents -19- WO 00/00491 PCT/US99/14320 for use in the method of this invention for mucosal/nasal delivery are also described in Chang, et al., Nasal Drug Delivery, "Treatise on Controlled Drug Delivery", Ch. 9 and Table 3-4B thereof, (Marcel Dekker, 1992). Suitable agents which are known to enhance absorption of drugs through skin are described in Sloan, Use of Solubility 5 Parameters from Regular Solution Theory to Describe Partitioning-Driven Processes, Ch. 5, "Prodrugs: Topical and Ocular Drug Delivery" (Marcel Dekker, 1992), and at places elsewhere in the text. All of these references are incorporated herein for the sole purpose of illustrating the level of knowledge and skill in the art concerning drug delivery techniques. 10 A colloidal dispersion system may be used for targeted delivery of the bifunctional activation cascade antagonist to an inflamed tissue. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A particularly convenient and effective colloidal system is a liposome. 15 Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 pm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. Compounds can be encapsulated within the aqueous interior and be delivered to cells in an active form. In addition to 20 mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. The composition of a liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination -20- WO 00/00491 PCTIUS99/14320 with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, 5 phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. 10 The targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo 15 endothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of 20 localization. The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be -21- WO 00/00491 PCT/US99/14320 incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various well known linking groups can be used for joining the lipid chains to the targeting ligand (see, e.g., Yanagawa, et al., Nuc.Acids Symp.Ser., 19:189 (1988); Grabarek, et al., 5 Anal.Biochem., 185:131 (1990); Staros, et al., Anal.Biochem., 156:220 (1986) and Boujrad, et al., Proc.Natl.AcadSci. USA, 90:5728 (1993)). Examples of targeting ligands are receptor ligands, antigens, enzymes and monoclonal antibodies. Such targeting ligands can be prepared according to conventional techniques (e.g., peptide synthesis) and many are commercially available. -22- WO 00/00491 PCT/US99/14320 II. Bifunctional Activation Cascade Antagonist Constituents .JNK Activation Cascade Antagonist Constituents JNK activation cascade antagonist constituents useful in the invention 5 include all of the constituents described in commonly owned US Patent Application Serial Nos.08/858,778, 08/367,102 and 08/482,551, whose disclosures are incorporated herein. For ease of reference, details of the structures of each of these compounds, as well as additional compounds of similar activity, as well as methods for their synthesis, are provided below. 10 JNK activation cascade antagonists of the invention generally comprise purines, pteridines, thiadiazolopyrimidines and quinazolines prepared according to the schemes described below. For reference, the techniques used in synthesizing the compounds are adaptations of the well-known Traube Synthesis protocol (Lister, "Purines" (Wiley-Interscience, 1971), at p. 220), beginning with 4,5 15 diaminopyrimidines; to wit: (1) for the purines in general, see Brown, "The Chemistry ofHeterocyclic Compounds: Fused Pyrimidines ", Part II, The Purines, 1971), at pp. 31-90; (2) for the 9-dieazapurines in particular, see Fox, et al., J. Org. Chem., 43:2536, 1978; (3) for the pteridines, see, for a description of the standard Timmis reaction, Nishigaki, et al., Heterocycles, L5:757-759, 1981; Timmis, Nature, 164:13 20 9, 1949, (or other standard Traube-like protocols for preparing pteridines by ring closure of diaminopyfimidines using a two carbon reagent); and, (4) for the pyrimidines, see, Schrage and Hitchings, J. Org. Chem., 16:207, 1951. -23- WO 00/00491 PCTIUS99/14320 JNK activation cascade antagonists, intermediates and compounds tested for comparison of activity to the compounds of the invention are identified in the discussion below by the numbers assigned to each compound in Table 2. -24- WO 00/00491 PCT/US99/14320 TABLE 2 LA C 0 0 A ( (d 4-) -4 0 0 -4 z It 1 2 m 04 e 0 C1 '-4 oN co- (N, ... . . ... '-4 -4 rd- - ZZC 'U 04 Loo I w -1 04 2 0. H (N LA -25- WO 00/00491 PCTIUS99/1 4320 .. . .. . CN 0 HA %0 Cu 0 0 z -4 CCu H 0 P4 .4 ... .. .. z z 0 z 0 0 0n C-aH '-4 o- s -0C, Cu I C .- .... 0 z .... .... 0. a-26 WO 00/00491 PCTIUS99/1 4320 z4 z 0 0 00 0 . .... ..... 10 0 C' .00 0 0 0 ' C.) CN o 0 o co Ca4 0) o ~C4 C)) 0 m c.. $4 'zC4 C14 (Dz CLC. on o o- - 0 z 'U~C c" oc)0 C) __ -27-* WO 00/00491 PCT/US99/14320 z 0 0N LOA fLn >1z .C:) -4 '-4 04. 0 00 A 0 tH M zCI 0 z -- 0 0 ..... .... z Z-() 0)z A z s-4 r a NN (NN)r o 028- WO 00/0049 1 PCTIUS99/14320 .. .. .. . 0 z H A ........ . ..... .... .. . . . 4-i -~: j:' ji:S0j'*j'j -4 X. H4 10 P4 C) N C ai C) N a, ,a 1-4 0 - 0 00 H Z Mia MiI E M0C. -29 WO 00/00491 PCTIUS99/14320 4-4 zz 00 44l 64 0' 0 It I 0 co I >1N

Z-

Z -4 0 0 - 0 0 04 Q4 (4 00 o- co a%' . ...... ....... LI I - 1 -30 WO 00/00491 PCT/US99/14320 4J4 ij .. ... . . C~C EAz z z 0 U oN o H -4 Z-0 Z-(. 01 2l 0 .... ) C) .. .... . 031 WO 00/00491 PCTIUS99/1 4320 E-4 P'4 EE z $4. *0 14 .p $4 C 02 0U 0 0u w o 9co -32 WO 00/00491 PCTIUS99/1 4320 4J4 2 ON C4 r4 41 .00 -CM HH HH Nn m -33- WO 00/00491 PCTIUS99/14320 .. . .. . ... .. .. . ... . .. . ........ 0 ...... ...... ... .. .. 4 4 ..... .... )., .) ............. E- z co CC) oc 04 C%4 .. . ..... tItp '-4 o zZ~z 14 0 0 C) C .03 -34 WO 00/00491 PCT/US99/1 4320 r44 Q) E4 011 w In w twE 4J o: 0 4. 0) -4 1 -H" 0 4 ............ 4 H4 a 0) 0 0 o- 0 0 04 *0 4.4 0 -. q Ed -,4 C -4n -4 0 0 H 0 x~ -4Z4 -44 .4....Z.......... 0 ' 4 0 %D0 > LO -- a n ,_ 0 0-4 to4~ 02 w 4 M 0 cd1 -- 4,- to (d - 0 O ~4J -4 H G) 04V 0 0 .0. (d 0 4 o . % ..... 0. ... 0 ty z s~,~4 r- E m M' ('4 . ... ... ... ... -35-J - WO 00/00491 PCT/US99/14320 Purines Purines JNK activation cascade antagonists have the general formula (8): 0 I X A I Z Y2 R2 where Z is N or CH and R, is (CH 2 )nA, where: A is NH 2 , acyloxy, SO 3 H, P0 4

H

2 , NNO(OH), SO 2

NH

2 , PO(OH)NH 2 , 5 SO 2 R, or COOR where R is H, an alkyl having from 1 to 4 carbon atoms, an alkenyl having from 1 to 4 carbon atoms, tetrazolyl or benzyl; n is any number of atoms from 1 to 7 having saturated and/or unsaturated carbon to carbon bonds, which atoms may include an oxygen or nitrogen atom in place of a carbon atom to form, respectively, ether or amino 10 linkages; and, preferably, R, is a o-carboxyalkyl, o-carboxyalkenyl, or o carboxyaryl having from 1 to 8 carbon atoms, wherein the aromatic group further has as a substituent A (as defined above); -36- WO 00/00491 PCT/US99/14320

R

2 is H, an alkyl (including aliphatic and alicyclic, and heteroalicyclic forms), alkenyl, aralkyl having 1 to 7 carbon atoms or a o-hydroxyalkyl having from 1 to 7 carbon atoms;

R

3 is the same as R 2 ; and 5 X is H, any halogen, OH, SH, OR', or SR', where R' is an alkyl, alkenyl, phenyl or benzyl having from 1 to 4 carbon atoms. These compounds are synthesized per the below synthesis scheme (as described in further detail in the Examples). -37- WO 00/00491 PCT/US99/14320 SCHEME 1 (PURINES) Three basic synthesis protocols were utilized in Scheme I; to wit: Method A 0 CH 3 0 HN N> a N 0~ N 0--)'N N I I

CH

3 OH 3 theobromine 4, R 2 =CH,COEt 5,

R

2

=(CH

2

)

2 COOEt 6, R2=(CH 2

)

3 COOEt 2, R 2

=(CH

2

)

3 COOH b 7, R 2

=(CH

2

)

4 COOEt 8, R 2

=CH

2 PhCOOMc (para) 0

OH

3 0

OH

3 O N Br 0 N 0<N N

CH

3

OH

3 9 10, R2=(CH2) 3 C00Et 11, R 2

'=CH

2 PhCOOMe (para) (a) RIX, DMF,

K

2 C0 3 (b) Br 2 , Ac 2 O (c) NaSH, EtOH 0 OH 3

N

1 N 0~<N >==s I H

H

3 12, R 2

=(CH

2

)

3 COOEt 13, R 2

=CH

2 PhCOOEt 5 Generally, theobromine was used as the starting material under conditions to ensure N- I alkylation took place in lieu of 0-6 alkylation. Compounds 4 through -38- WO 00/00491 PCT/US99/14320 8, 10 and 11 (Table I) were prepared by this method. Compounds 10 and 11 in particular were prepared by a variation of the alkylation method in which theobromine was first brominated to give 8-bromotheobromine (compound 9), then alkylated. The 8-bromo substituent was also displaced by NaSH to yield the corresponding 8-thioxo 5 derivatives, compounds 12 and 13. O 0 Method B

R

1 N HNa N X o N NH 2 O N NH 2

CH

3 CH 3 14 15, H n-propyl b, c 16, H n-hexyl 17, H n-heptyl 18, N=O n-propyl 19, N=O n-hexyl 20, N=O n-heptyl 22 21, NH 2 n-propyl 22, NH 2 n-hexyl d or a 23, NH2 n-heptyl 0 H N N I H CH3 (a) RIX, DMF,

K

2 C0 3 24, RI=n-hexyl Y=O (b) NaNO 2 , HCI 25, RI=H Y=S (c) Pd/H 2 (d) urea, 140C (e) potassium ethyl xanthate This method is essentially based on the Traube purine synthesis protocol referred to supra. The method was used to prepare 1,3,8-trisubstituted xanthines bearing no alkyl group at the N-7 position. In this procedure, the N-1 substituted 10 pyrimidine was alkylated at position N-3. Formation of the purine ring was complete -39- WO 00/00491 PCT/US99/14320 by nitrosation, reduction of the nitroso to the amine by catalytic hydrogenation, then ring closure using urea or potassium ethyl xanthate to provide compounds 24 and 25 (respectively, 8-oxo and 8-thioxo derivatives). A detailed description of this protocol is provided in the Examples. 5 Method C 0 0 HN H

NH

2 a HN N> 0 NN

C

3

H

7

C

3

H

7 26, X = H 27,X=N=0 29 28, X = NH2 b R CH 3 O

CH

3 N NI , c HN 0 <N N - 0' N N'

C

3

H

7 C 3

H

7 31, RI=(CH 2 )3COOEt 30 (a) diethoxymethyl acetate (b) CH 2 1, NaOH (c) RIX, DMF,

K

2 CO3 -40- WO 00/00491 PCT/US99/14320 The above method was utilized to prepare the N-3 propylpurines. The starting material used was n-propyl urea condensed with ethyl cyanoacetate in the presence of sodium ethoxide to yield the 6-amino-1 -propylpyrimidinedione in moderate yield. Commercially available 3-n-propylxanthine could also be used as the 5 starting material. Ring closure was accomplished as described in method A, except that diethoxymethyl acetate was used as the source of carbon in the ring closure step. Sequential alkylations were then performed using alkyl halides to yield the final compound 31 (ethyl 4-(2,3,6,7-tetrahydro-2,6-dioxo-7-methyl-3-n-propyl-1H-purin-1 10 yl)butonoic acid). A detailed description of this protocol is provided in the Examples. Pteridines Pteridine JNK activation cascade antagonists have the general formula (9): 0 R1 N R4 N O N N' R2 -41- WO 00/00491 PCTIUS99/14320 R, is (CH 2 )nA, where: A is NH 2 , acyloxy, SO 3 H, P0 4

H

2 , NNO(OH), S0 2

NH

2 , PO(OH)NH 2 ,

SO

2 R, or COOR where R is H, an alkyl having from 1 to 4 carbon atoms, an alkenyl having from 1 to 4 carbon atoms, tetrazolyl or benzyl; 5 n is any number of atoms from 1 to 7 having saturated and/or unsaturated carbon to carbon bonds, which atoms may include an oxygen or nitrogen atom in place of a carbon atom to form, respectively, ether or amino linkages; and, preferably, R, is a w-carboxyalkyl, o-carboxyalkenyl, or w 10 carboxyaryl having from 1 to 8 carbon atoms, wherein the aromatic group further has as a substituent A (as defined above);

R

2 is H, an alkyl (including aliphatic and alicyclic, and heteroalicyclic forms), alkenyl, aralkyl having 1 to 7 carbon atoms or a o-hydroxyalkyl having from 1 to 7 carbon atoms; 15 R 4 is the same as R 2 , OH or an O-alkyl having from 1 to 5 carbon atoms;

R

5 is the same as R 2 , OH or an O-alkyl having from 1 to 5 carbon atoms; and, Z is N or CH. -42- WO 00/00491 PCT/US99/14320 These compounds are synthesized per the below synthesis scheme (which is described in further detail in the Examples.) -43- WO 00/00491 PCT/US99/14320 SCHEME 11 (PTERIDINES) 00 32, R 2 Me a HN NH 2 b,c Rj 1 N N Et 27, R 2 -n-G 3

H

7 - 0

-

1 22, R 2 =Me 0 N NH 2 x~ ~~E Ut1 3 28, R 2 Me 3 H 40, R 1 =H a~c 2, R2 C3H741, Rl=(CH 2

)

3 dOOEt or 41a', R 1

=(CH

2

)

3 COOH d,c 0 R , NNX R 4R N 0N z 0 N N R5) I ON N

CH

3 I 39, R 1

=C

5

H

1 3

R

4 =COOMe R 5 =COOMe 42, R 1 =H R 4 =phenyl R 5 =H 34, R 1 =H R 2 =Me 43, Rl=(CH2) 3 COOH R 4 =phenyl , 5 =H 35, R 1 =H R 2

,C

3

H

7 36, R 1 = (CH 2

)

3 COOEt R 2 =Me 36a, R 1 = (CH 2

)

3 COOH R 2 =Mc 37, R 1 = (CH 2

)

3 COOEt R 2

=C

3

H

7 (a) Na 2

S

2

O

4 , H 2 0 38, R 1 = CH 2 CH=CHCOOMe R 2 =Me (b) 3,4-hexandione (c) RIX, DMF, K 2 C0 3 (d) glYoxal:sodium bisulfite (e) phenethylamine (0) dimethylacetylene dicarboxylate -44- WO 00/00491 PCTIUS99/14320 Method C (above) was chosen as a convenient method to produce N-alkyls in preference to O-alkyls in this group. Method C was modified to this end as follows: 0 0 32, R 2 = Me a HN NH2 b,c RlsN N Et 27, R 2 =n-C 3

H

7 22, R 2 = Me O NH 2 O N 1~' 0 N N Et 2 CH 3 33, R 2 = Me 40 RI=H a,c 28, R2=C3H7 41, R=(CH 2

)

3 COOEt or 41a, R 1

=(CH

2

)

3 COOH f d,c 0 Rj-,N N R 4 R 0N 0 N N R5

CH

3 0 N N R2 39, R 1

=C

5

H

13

R

4 =COOMe R 5 =COOMe 42, R 1 =H R 4 =phenyl RH 34, RI=H R2=Me 43, R 1

=(CH

2

)

3 COOH R 4 =phenyl R5=H 35, RI=H R 2

=C

3

H

7 36, RI= (CH 2

)

3 COOEt R 2 =Me 36a, RI= (CH 2

)

3 COOH R 2 =Me 37, RI= (CH 2

)

3 COOEt R 2

=C

3

H

7 (a) Na 2

S

2 0 4 , H 2 0 38, RI= CH 2 CH=CHCOOMe R 2 =Me (b) 3,4-hexandione (c) RIX, DMF, K 2 C0 3 (d) glyoxal:sodium bisulfite (e) phenethylamine (f) dimethylacetylene dicarboxylate -45- WO 00/00491 PCT/US99/14320 Synthesis of the pteridines was based on orthodiaminopyrimidines as precursors. Ring closure of the orthodiamines (compounds 33 and 28) was accomplished with a two carbon source (e.g., glyoxal) to produce compounds 34 and 35 (N-I substituted pteridines). Alkylation at N-3 as described with respect to 5 Method A produced the desired pteridines (compounds 36-38). Further, use of 3,4 hexanedione in the ring closure step produced a more lipophilic derivative (compound 41; 6,7-diethyl pteridine). Condensation of compound 22 with dimethylacetylene dicarboxylate formed compound 39 (1,3-dialkylpteridine), while treatment of compound 27 phenethyl amine followed by alkylation provided compound 43 (6 10 phenyl dialkyl pteridine). Both of the latter protocols utilized a Timmis reaction to produce the desired products. A detailed description of these protocols is provided in the Examples. Thiadiazolopyrimidines Thiadiazolopyrimidine JNK activation cascade antagonists have the 15 general formula (10): 0

R

1 N N 0 N N R2 - 4 6- WO 00/00491 PCT/US99/14320 R, is (CH 2 )nA, where: A is NH 2 , acyloxy, SO 3 H, PO 4

H

2 , NNO(OH), SO 2

NH

2 , PO(OH)NH 2 ,

SO

2 R, or COOR where R is H, an alkyl having from 1 to 4 carbon atoms, an alkenyl having from 1 to 4 carbon atoms, tetrazolyl or benzyl; 5 n is any number of atoms from 1 to 7 having saturated and/or unsaturated carbon to carbon bonds, which atoms may include an oxygen or nitrogen atom in place of a carbon atom to form, respectively, ether or amino linkages; and and, preferably, R, is a o-carboxyalkyl, o-carboxyalkenyl, or o 10 carboxyaryl having from 1 to 8 carbon atoms, wherein the aromatic group further has as a substituent A (as defined above); and

R

2 is H, an alkyl (including aliphatic and alicyclic, and heteroalicyclic forms), alkenyl, aralkyl having 1 to 7 carbon atoms or a o-hydroxyalkyl having from 1 to 7 carbon atoms. 15 These compounds are synthesized per the below synthesis scheme (as described in further detail in the Examples). -47- WO 00/00491 PCT/US99/14320 SCHEME III (THIADIAZOLOPYRIMIDINES) Method C (above) was chosen as a convenient method to produce N-alkyls in preference to 0-alkyls in this group. Method C was modified to this end as 5 follows: 0 0 HN

NH

2 abN N 1a,b N R N2 I R2 33, R 2 = Me 28, R 2

=C

3

H

7 45, Ri=H R2=Me 46, R 1 =H R 2

=C

3

H

7 47, Ri=(CH 2

)

3 COOEt R 2 =Me 47a, Ri=(CH 2

)

3 COOH R 2 =Me 48, Ri=(CH 2

)

3 COOEt R 2

=C

3

H

7 0 23 a,b R N S 0 ~N ~N

CH

3 44, Ri=n-hexyl -48- WO 00/00491 PCT/US99/14320 Synthesis of the pyrimidines was based on orthodiaminopyrimidines as precursors. Ring closure of the orthodiamines was accomplished by treatment with thionyl chloride in the presence of pyridine. Alkylation of these intermediates produced compounds 47 and 48 (disubstituted pyrimidines). A detailed description of 5 this protocol is provided in the Examples. -49- WO 00/00491 PCTIUS99/14320 Isoquinolones Isoquinolone JNK activation cascade antagonists have the general formula (11): 0 R2 N R3 R7 R4 5 R 2 is (CH 2 )A, where: A is NH 2 , acyloxy, SO 3 H, P0 4

H

2 , NNO(OH), SO 2

NH

2 , PO(OH)NH 2 ,

SO

2 R, or COOR where R is H, an alkyl having from 1 to 4 carbon atoms, an alkenyl having from 1 to 4 carbon atoms, tetrazolyl or benzyl; n is any number of atoms from 1 to 7 having saturated and/or unsaturated 10 carbon to carbon bonds, which atoms may include an oxygen or nitrogen atom in place of a carbon atom to form, respectively, ether or amino linkages; -50- WO 00/00491 PCT/US99/14320 and, preferably, R 2 is a o-carboxyalkyl, o-carboxyalkenyl, or o carboxyaryl having from 1 to 8 carbon atoms, wherein the aromatic group further has as a substituent A (as defined above); and

R

3 is H, an alkyl (including aliphatic and alicyclic, and heteroalicyclic 5 forms), alkenyl, aralkyl having 1 to 7 carbon atoms or a o-hydroxyalkyl having from 1 to 7 carbon atoms;

R

4 is H, OH, NH 2 or O-alkyl having from 1-7 carbon atoms; R, is H, OH, NO, NO 2 , NH 2 an O-alkyl having from 1-4 carbon atoms, or X where; 10 X is H, any halogen, OH, SH, OR', or SR', where R' is an alkyl, alkenyl, phenyl or benzyl having from 1 to 4 carbon atoms; and,

R

7 is H, OH, NO, NO 2 , NH 2 an O-alkyl having from 1-7 carbon atoms, or X where: X is H, any halogen, OH, SH, OR, or SR', where R' is an alkyl, alkenyl, 15 phenyl or benzyl having from 1 to 7 carbon atoms. These compounds were synthesized by purchasing isoquinolines from Aldrich Chemical and adding side chains to the ring structure with respect to the compounds of the invention. Only the 6,7-dimethoxy-1(2H)-isoquinoline compound -51- WO 00/00491 PCT/US99/14320 (Aldrich # S52,626-6) had any inhibitory effect on TNF-a production prior to addition of the side chains described above. -52- WO 00/00491 PCTIUS99/14320 Quinazolines Quinazoline JNK activation cascade antagonists are synthesized according to the following scheme and are represented in structure by compound 52: Scheme IV 5 (DIDEAZAPTERIDINES OR QUINAZOLINES) Compound 52 (a quinazoline derivative; ethyl 4-(1-methyl-2,4 dioxoquinazol-3-yl)butanoic acid) was produced as follows: o 0 0 a,b N N a0 , R 1 N c,d , N1 OIN HN0N

CH

3 CH3 CH 3 49 50, Rj=(CH 2

)

3 COOH 52, R 1

=(CH

2

)

3 COOEt 51, R 1

=(CH

2

)

3 C00Et 52a, R 1

=(CH

2

)

3 COOH -53- WO 00/00491 PCTIUS99/14320 The starting material for this protocol was N-methyl isatoic anhydride. A detailed description of this protocol is provided in the examples. -54- WO 00/00491 PCTIUS99/14320 Increasing water solubility The carboxylic ester and acid derivatives of the compounds described above can be converted to substituted carboxylic esters, where the substituent of the ester increases the water solubility of the compounds. o 0 0 a,b N c,d N HN

CH

3 OH 3 OH 3 49 50, Ri=(CH 2

)

3 COOH 52, R 1

=(CH

2

)

3 COOEt 51, Ri=(CH 2

)

3 COOEt 52a, Ri=(CH 2

)

3 COOH 5 Particular substituents that increase the water solubility of the compounds effectively are aminoalkyl esters. The amino group of the aminoalkyl ester preferably is a secondary or tertiary amino group. The amino substituents have between 1 and 6 carbon atoms, one of which can be replaced by an oxygen atom or nitrogen atom. The preferred aminoalkyl is a tertiary amine and the amino substituents are preferably are 10 ether groups. The most preferred aminoalkyl is N-morpholinoethyl. The carboxylic ester and carboxylic acid derivatives can be converted to the morpholinoethyl ester form by the method shown in Scheme V. -55- WO 00/00491 PCT/US99/14320 Compound 53 (an isoquinolone derivative; ethyl (6,7-dimethoxy-1(2H) isoquinolonyl)butanoic acid was converted to compound 54, a morpholinoethyl ester (isolated as a hydrochloride salt) as follows: Scheme V 5 (MORPHOLINOETHYL DERIVATIVES: COMPOUND 54) MeO 0 MeO N 0 53 1) NaOH or HCI, heat 2) SOC1 2 3) morpholinoethanol 4) HCI MeO 0 MeO N O N 0 O .HCI 54 -56- WO 00/00491 PCT/US99/14320 CSAID Constituents CSAIDs are available from, for example, the sources listed in the legend to Table 1. Chemical descriptions and data demonstrating activity for many of these compounds have been published in the art in, for example, US Patent No. 5,658,903; 5 US Patent No. 5,593,992; US Patent No. 5,317,019; US Patent No. 4,794,114; US Patent No. 4,778,806; Published PCT Application No. W09725045; Published PCT Application W09725047; Published PCT Application W09725048; Published PCT Application No. W09640143; European Patent No. 565582; Austrian Patent No. 586907; Griswold, et al., Drugs Exp.Clin.Res., 19:243-248 (1993)(SK&F 86002, 10 105809 and 104351; in vivo=32, 48 and 34 mg/kg, respectively); Marshall, et al., Biochem.Pharmacol., 42:813-824 (1991) (SK&F 105809; in vitro data for inhibition of CO, 5-LO, IL-I and PGHS); Griswold, et al., J Immunol.Methods, 195:1-5 (1996) (SB 203580); and Badger,et al.,JPharmacol.Exp.Ther., 279:1453-1461 (1996) (in vivo data for SB 203580); Boehm, et al., J.Med Chem., 39:3929-3937 (1997) (SB 15 210313)). Each of these references are incorporated herein for ease of reference in identifying CSAIDs for use in the invention. For further ease of reference, the chemical names of several known CSAIDs are set forth below: SB 210313: 1-[3-(4-morpholinyl)propyl]-4-(4-fluorophenyl-5-(4 20 pyridyl)imidazole -57- WO 00/00491 PCT/US99/1 4320 SK&F 105809: 2-(4-methylsulfinylphenyl)-3 -(4-pyridyl)-6,7 dihydro- [5HI-pyrrolo[ 1,2-a]imidazole SK&F 105561: 2-(4-methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro [5H]-pyrrolo[ 1,2-a]imidazole 5 SB 203580: 4-(4-fluorophenyl)-2-(4-methylsulfinyl)-5-(4 pyridyl)imidazole SK&F 86002: 5-(4-pyridyl)-6(4-flurophenyl)-2,3 dihydroimidazole(2, 1 -b)thia zol -58- WO 00/00491 PCT/US99/14320 A representative CSAID synthesis scheme (shown for SB 210313) is: N H N NH R F N / CH 2 OH 3 N CH 2 OTBDMS 4 -pyridylcarbinol LDA / THF CIOC F H N S CH 3 0 - H 3 CO

H

3 CO' /

CH

3 OTBOMS O Cu(OAc) -- 0 - 0 F F -59- WO 00/00491 PCT/US99/14320 N N H N OHC CN CN N F - NH4 0 Ac F T.F. Gallagher, et al. Bioorg. Med. Chen. Lett. 5, 1171-1176, 1995 and US Patent 5656644. KOH / t-BuOH RX NH N - 0 N 'NH \ R F -60- WO 00/00491 PCT/US99/14320 Although not preferred as partners for JNK activation cascade antagonists in the bifunctional activation cascade antagonists of the invention, corticosteroids and other medicaments, such as antibiotics or different anti-inflammatory agents, may also be administered with the inventive compounds if clinically indicated. 5 III. Kits for Use in Practicing the Methods of the Invention For use in the methods described below, kits are also provided by the invention. Such kits may include any or all of the following: a bifunctional activation cascade antagonist; a pharmaceutically acceptable carrier (may be pre-mixed with the 10 bifunctional activation cascade antagonist) or suspension base for reconstituting lyophilized bifunctional activation cascade antagonists; additional medicaments; a sterile vial for each bifunctional activation cascade antagonist and additional medicament (if any), or a single vial for mixtures thereof; device(s) for use in delivering the composition to a host; assay reagents for detecting indicia that the anti 15 inflammatory sought have been achieved in treated animals and a suitable assay device. IV. Methods for Use of the Inventive Compounds. The bifunctional activation cascade antagonists of the invention may be 20 administered to a mammalian host to retard cellular responses associated with TNF-a and IL-1 production, release and MPAK system activation. As exemplified herein, the methods of the invention are expected to be of particular use in providing -61- WO 00/00491 PCT/US99/14320 protection against inflammation and associated excess formation of fibrotic tissue. In particular, with reference to demonstrated efficacy for the JNK activation cascade antagonist and CSAID constituents of bifunctional activation cascade antagonists, these compounds can be expected to have therapeutic efficacy in the treatment of 5 arthritis, spondylitis, bone resorption (osteoarthritis), sepsis, septic shock, endotoxic shock, inflammatory bowel disease, asthma, bronchitis, chronic pulmonary inflammatory disease, silicosis, repurfusion injury, graft versus host reachtions, allograft rejection, fever, viral and bacterial infection, inflammation of the joints, psoriasis and eczema, radiation dermatitis, cell senescence and apoptosis, multiple 10 sclerosis and other inflammatory conditions. The bifunctional activation cascade antagonists of the invention are administered to a host using any available method and route suitable for drug delivery, including ex vivo methods, as well as systemic or localized routes. Those of ordinary skill in the clinical arts will be familiar with, or can readily ascertain, means for drug 15 delivery, which are briefly discussed below. The entrance point for many inflammatory stimulants into a host is through the skin or mucosa. Thus, delivery methods and routes which target the skin (e.g., for cutaneous and subcutaneous conditions) or mucosa (e.g., for respiratory, ocular, lingual or genital conditions) will be especially useful. 20 Intranasal administration means are particularly useful in addressing respiratory inflammation, particularly inflammation mediated by antigens transmitted -62- WO 00/00491 PCT/US99/14320 from the nasal passages into the trachea or broncheoli. Such means include inhalation of aerosol suspensions or insufflation. Nebulizer devices suitable for delivery of drug compositions to the nasal mucosa, trachea and bronchioli are well-known in the art and will therefore not be described in detail here. For general review in regard to 5 intranasal drug delivery, those of ordinary skill in the art may wish to consult Chien, Novel Drug Delivery Systems, Ch. 5 (Marcel Dekker, 1992). Dermal routes of administration, as well as subcutaneous injections, are useful in addressing allergic reactions and inflammation in the skin. Examples of means for delivering drugs to the skin are topical application of a suitable 10 pharmaceutical preparation, transdermal transmission, injection and epidermal administration. For transdermal transmission, absorption promoters or iontophoresis are suitable methods. For review regarding such methods, those of ordinary skill in the art may wish to consult Chien, supra at Ch. 7. lontophoretic transmission may be 15 accomplished using commercially available "patches" which deliver their product continuously via electric pulses through unbroken skin for periods of several days or more. Use of this method allows for controlled transmission of pharmaceutical compositions in relatively great concentrations, permits infusion of combination drugs and allows for contemporaneous use of an absorption promoter. 20 An exemplary patch product for use in this method is the LECTRO PATCH trademarked product of General Medical Company of Los Angeles, CA. This product electronically maintains reservoir electrodes at neutral pH and can be adapted to provide dosages of differing concentrations, to dose continuously and/or to dose periodically. Preparation and use of the patch should be performed according to -63- WO 00/00491 PCT/US99/14320 the manufacturer's printed instructions which accompany the LECTRO PATCH product; those instructions are incorporated herein by this reference. Opthalmic administration (e.g., for treatment of allergic conjunctivitis) involves invasive or topical application of a pharmaceutical preparation to the eye. 5 Eye drops, topical cremes and injectable liquids are all examples of suitable mileaus for delivering drugs to the eye. Systemic administration involves invasive or systemically absorbed topical administration of pharamaceutical preparations. Topical applications as well as intravenous and intramuscular injections are examples of common means for systemic 10 administration of drugs. The compounds of the invention vary in potency. A summary of the potency of each JNK activation cascade antagonist (expressed as a percentage of inhibition of intracellular responses to LPS, namely, the production of TNF-a, where responses to pure LPS=100% and are measured as the concentration of the inventive 15 compound needed to inhibit TNF-a production by 50%) is provided in Table 2, elsewhere above. The relative potencies of other antiinflammatory constituents, including CSAIDs, are known in the art. For example, the IC 50 (level at which 50% of TNFa release is inhibited) for CSAIDs are published (see, e.g., Griswold, et al., Drugs 20 Exp.Clin.Res., 19:243-248 (1993)(SK&F 86002, 105809 and 104351; in vivo=32, 48 and 34 mg/kg, respectively); Marshall, et al., Biochem.Pharmacol., 42:813-824 (1991) (SK&F 105809; in vitro data for inhibition of CO, 5-LO, IL-1 and PGHS); Griswold, et al., J.Immunol.Methods, 195:1-5 (1996) (SB 203580); and Badger, et al., -64- WO 00/00491 PCT/US99/14320 J'PharmacoLExp. Ther., 279:1453-1461 (1996) (in vivo data for SB 203580); Boehm, et al., J.Med Chem., 39:3929-3937 (1997) (SB 210313)). The maximum potency of any of these compounds administered alone is about 70-80% TNFa inhibition (Example I). Those of ordinary skill in the art will 5 recognize that lesser or greater dosages of the compounds of the invention may be required depending on the potency of the particular compound being administered. Dosages of the compounds of the invention will vary depending on the age, weight and presenting condition of the host to be treated, as well as the potency of the particular compound administered. Such variables will readily be accounted for 10 by those of ordinary skill in the clinical art. In particular, dosages will be adjusted upward or downward for each recipient based on the severity of the condition to be treated and accessibility of the target cells to the pharmaceutical formulations of the invention. Where possible, it will be preferable to administer the pharmaceutical formulations of the 15 invention locally at the site of the target cells; e.g., into inflamed skin or by infusion to another organ of the host. Thus, dosages will also vary depending on the route of administration and the extent to which the formulations of the invention are expected to reach target cells before dilution or clearance of the formulation. Taking these factors into account, acceptable CSAID and JNK activation 20 cascade antagonist dosage ranges for each compound administered separately are about 0.1 to 80 mg/kg/day for oral and parenteral routes; about 0.1-150 mg/kg/day for topical routes and about 0.01-1 mg/kg/day for respiratory routes. Due to their -65- WO 00/00491 PCT/US99/14320 enhanced potency, bifunctional activation cascade antagonists are administered in lower dosages (e.g., at about a 10-40% lower dosage). Those of ordinary skill in the clinical arts will be able to determine medically sound dosing schedules for patients with particular presenting conditions, taking into account the severity of the condition, 5 the patient's overall health, patient age and weight, and other clinically relevant factors. These dosages may be combined with other conventional pharmaceutical therapies for inflammation and fibrosis; e.g., corticosteroids. Those of ordinary skill in the art will be familiar with, or can readily ascertain, the identity and clinical signs of specific inflammatory conditions, and can 10 identify clinical signs of improvement therein (such as reductions in serum levels of TNF-a and improvement in clinical health) in addition to those exemplified herein. -66- WO 00/00491 PCT/US99/14320 V. Methods for Identification of Therapeutically Effective Analogues of Bifunctional activation cascade antagonists Those of ordinary skill in the art will be familiar with means to develop 5 analogues to the bifunctional activation cascade antagonists and constituents specifically described herein which, although not structurally identical thereto, may possess the same biological activity. Such compounds are within the scope of the invention and may be identified according to the protocols described below and in the Examples. 10 Though exposure of cells to the compounds of the invention under controlled conditions, the responsiveness of cells to inflammatory agents and intracellular mechanisms therefor can be investigated. This information will not only better elucidate the intracellular pathways responsible for cellular responses to particular stimuli, but will also aid in the identification of anti-inflammatory and anti 15 fibrosis therapeutic compounds. To identify and select therapeutic compounds for use in treating conditions such as inflammation and fibrosis, lymphocytes, monocytes, neutrophils, intracellular components such as microsomes or immunologically naive animals are exposed to a PIC and the candidate therapeutic compound. Specifically, a control is incubated with 20 a known amount of the inflammatory or fibroblast proliferation inducing agent. Treatment groups are exposed to the same amount of inflammatory or fibroblast proliferation inducing agent as well as aliquots of the candidate therapeutic compound. Inflammatory responses or fibroblast proliferation in each group are -67- WO 00/00491 PCT/US99/14320 detected by conventional means known to those of skill in the art (such as the assay steps described in the examples) and compared. The invention having been fully described, examples illustrating its practice are set forth below. These examples should not, however, be considered to 5 limit the scope of the invention, which is defined by the appended claims. In the examples, the abbreviation "min." refers to minutes, "hrs" and "h" refer to hours, and measurement units (such as "ml") are referred to by standard abbreviations. "mp" refers to melting point. -68- WO 00/00491 PCT/US99/14320 EXAMPLE I INHIBITION OF PIC ACTIVATION OF THE MAPK SYSTEM BY BIFUNCTIONAL ACTIVATION CASCADE INHIBITORS As an in vitro model of MAPK system activation, peripheral blood 5 monocyte cells (PBMC) were obtained from healthy human donors and stimulated with a PIC-sensitive-MAPK activator, namely lipopolysaccharide (LPS, Sigma). TNFa production was measured as the hallmark of MAPK cascade activation antagonism. The PBMC were isolated from heparinized normal human blood by ficoll 10 histopaque density centrifugation. The cells were plated in 96 well plates with a JNK activation cascade antagonist (compound 54 or compound 37), a CSAID (SB 210313) or a bifunctional activation cascade antagonist composed of compound 54 or 37 and SB 210313. The latter was synthesized as described in Boehm, et al., , J.Med.Chem., 39:3929-3937 (1997). SB210313 is 1-[3-(4-morpholinyl)propyl]-4-(4-flurophenyl)-5 15 (4-pyridyl) and is crystallized from acetone/hexane and stored as a dry powder at room temperature. The synthesized SB210313 compound tested is referred to below as 1-105. Each compound tested was added to the wells in equal volumes at concentrations of 10tm or 5 0pm. The plated cells were suspended in RPMI 1640 20 supplemented with 20% autologous plasma and glutamine and plated at a density of 5X10' cells/ml. Cells were incubated in the plates for 60 minutes then each well received LPS at a final concentration of 10 tg/ml. 18 hours later, 1 OOpI of each well -69- WO 00/00491 PCT/US99/14320 was collected and assayed for TNFa content using a comercially available mouse anti human TNFa antibody (Upstate Biotechnology, Inc.). In the TNFa inhibition assay, compound 54 achieved 50% inhibition (IC 50 ) at 10p.im. The IC 50 for compound 37 was achieved at 20-25pm.. The IC 50 for 5 SB210313, one of the most potent CSAIDs, was achieved at 1.6 to 3.2p1m. However, the maximal level of inhibition achieved by any of the compounds was only 70-80%. In startingly contrast, the bifunctional activation cascade antagonist composed of compound 37 (the JNK activation cascade antagonist of lesser potency as compared to compound 54) and SB 210313 achieved IC 5 0 in as little as 0.78 rim, a 10 level more than merely additive of the activities of each constituent alone. Thus, the bifunctional activation cascade antagonists of the invention achieve synergy between the JNK activation cascade antagonist and CSAID constituents. EXAMPLE II EFFECT OF JNK ACTIVATION CASCADE ANTAGONISTS AND CSAIDs 15 ON THE JNK ACTIVATION CASCADE To demonstrate the probable efficacy of bifunctional activation cascade antagonists in antagonizing activation of JNK, the data below demonstrating the efficacy of JNK activation cascade antagonists in this context are provided. MOLT-4 human lymphoblastoid cells were used as models of JNK 20 activation in the presence of anisomycin. -70- WO 00/00491 PCT/US99/14320 MOLT-4 cells were preincubated with compound 54 to demonstrate the effect of compound 54 on JNK activation. The compound was dissolved in phosphate buffered saline and provided to the cells in concentrations of 50Pm or 10ptm. Incubations were made for intervals of 15, 30 or 60 minutes. Each incubated cell 5 sample was activated with 50nM anisomycin for 30 minutes. Cells exposed only to PBS served as a control and, for comparison, one group of cell samples were exposed to compound 54 alone (without anisomycin) or anisomycin alone (without compound 54). After the activation period, the MOLT-4 cell samples were washed three 10 times and lysed. 100ptg protein samples were used as reagents to measure enzyme activity in the MOLT-4 cell samples by a standard binding kinase assay, using GST c Jun as the ligand and substrate. For comparison, RAW mouse macrophages were stressed with a different stressor and the inhibitory potency of compound 54 vis-a-vis JNK activation was 15 tested. The cells were pre-incubated with compound 54 for 60 minutes at increasing concentrations and activated with 1 ig/ml LPS for 30 minutes. The cells were washed three times and lysed before testing. The IC 50 s for compound 54 varied from 10-50ptm, depending on the stressor, the time of preincubation and the cell line studied. Specifically, the IC 50 for 20 compound 54 with respect to anisomycin-induced JNK activation was achieved in MOLT-4 cells at 1 Opm after 60 minutes of incubation. In contrast, virtually no -71- WO 00/00491 PCT/US99/14320 change in JNK activation is achieved by CSAIDs in similar models (see, e.g., Kumar, et al., Biochem.Biophys.Res. Commun., 235:533-538 (1997)). As demonstrated in Example I, the bifunctional activation cascade antagonists of the invention provide the activity of each antagonistic constituent in a 5 synergistic manner. Thus, the bifunctional activation cascade antagonists provide the same activity of each constituent alone at greater potency. EXAMPLE III EFFECT OF CSAIDs AND JNK ACTIVATION CASCADE ANTAGONISTS ON THE p38 ACTIVATION CASCADE 10 To demonstrate the probable efficacy of bifunctional activation cascade antagonists in antagonizing activation of p38, the data below demonstrating the activity of JNK activation cascade antagonists and of CSAIDs in this context are provided. Lymphocyte and monocyte extracts were prepared under similar conditions 15 and were immunoprecipitated with affinity purified goat polyclonal antibody against the 20 amino acid N terminus of p38 (Santa Cruz Biotechnology), together with protein A SEPHAROSETM. The immunoprecipitates were washed, mixed with glutathione S-transferase-ATF2 SEPHAROSETM beads as substrate, and resuspended in kinase buffer containing [y- 32 P]ATP. Samples were processed as described in the 20 preceding Example, separated by SDS-PAGE and analyzed by autoradiography with Coomassie blue staining. Densitometry tracings of the 49 kDa GST-ATF2 band -72- WO 00/00491 PCT/US99/14320 showed that compound 54 does not block p38 kinase activation to an appreciable extent (only about 20% inhibition was achieved at 100 pm). In contrast, similar assays have demonstrated that CSAIDs inhibit activation of p38, but not of other kinases (see, e.g., Wilson, et aL, Chem.Biol., 4:423 5 431 (1997)). These data, and those set forth in Example II, demonstrate that the JNK activation cascade antagonist and CSAID constituents of the bifunctional activation cascade antagonists of the invention antagonize activation of different MAPK pathways in a non-overlapping manner. 10 As demonstrated in Example I, the bifunctional activation cascade antagonists of the invention provide the activity of each antagonistic constituent in a synergistic manner. Thus, the bifunctional activation cascade antagonists provide the same activity of each constituent alone at greater potency. -73- WO 00/00491 PCT/US99/14320 EXAMPLE IV INHIBITION OF CELL SENESCENCE AFTER SERUM DEPRIVATION IN SERUM-DEPENDENT CELLS BY JNK ACTIVATION CASCADE ANTAGONISTS 5 To demonstrate the probable efficacy of bifunctional activation cascade antagonists in inhibiting cell senescence, the data below demonstrating the efficacy of JNK activation cascade antagonists in this context are provided. Many cell types are dependent upon serum factors for growth. Thus, deprivation of such cells of serum provides a model for assessment of compounds to 10 modulate cell responses to intracellular ceramide-mediated signal transduction. In particular, withdrawal of serum from serum-dependent cell cultures produces increased intracellular levels of endogenous ceramide and may also increase intracellular levels of endogenous diacyl glycerol (see, e.g., Jayadev, et al., J.Biol.Chem., 270:2047-2052, 1995). 15 To evaluate the inhibitory effect of the compounds of the invention on ceramide-associated conditions in vitro, the serum withdrawal model was used. Specifically, 3T3 fibroblast cells were seeded in 96 well microtiter plates in DMEM in the presence of 10% fetal bovine serum. The cells were incubated to 90% confluence. The medium was removed, the cells washed and reincubated in serum-free 20 DMEM. Compound no. 37 and cell permeable ceramide were added to the wells at concentrations of, respectively, 0, 4, 40 or 400 piM compound no. 37 and 0, 5 or 10 tM of ceramide. After 24 hrs. incubation, 0.5 tCi of [ 3 H] thymidine was added to -74- WO 00/00491 PCT/US99/14320 each well for 2 hrs. DNA synthesis in the tested cell population was assessed by conventional techniques for detection of [ 3 H] thymidine incorporation. The results of this assay are indicated in FIGURE 1 and establish the cell senescence inhibitory efficacy of the inventive compounds (as represented by compound no. 37). 5 As demonstrated in Example I, the bifunctional activation cascade antagonists of the invention provide the activity of each antagonistic constituent in a synergistic manner. Thus, the bifunctional activation cascade antagonists provide the same activity of each constituent alone at greater potency. -75- WO 00/00491 PCT/US99/14320 EXAMPLE V INHIBITION OF CELL APOPTOSIS AFTER CD95 STIMULATION BY JNK ACTIVATION CASCADE ANTAGONISTS To demonstrate the probable efficacy of bifunctional activation cascade 5 antagonists in inhibiting cell apoptosis, the data below demonstrating the efficacy of JNK activation cascade antagonists in this context are provided. Engagement of cell surface receptor CD95 (also known as Fas/Apo-1 antigen) triggers cell apoptosis. DX2 is a functional anti-FAS (CD95) antibody which will, on binding of CD95, activate the Smase catalysis of sphingomyelin hydrolysis 10 and production of ceramide (see, re DX2, Cifone, et al., J.Exp.Med, 177:1547-1552, 1993, the disclosure of which is incorporated herein by reference for use in accessing the DX2 antibody). Thus, binding of CD95 is a model for induction of apoptosis via the sphingomyelin signal transduction pathway. To assess the inhibitory effect of the compounds of the invention on 15 ceramide-mediated cell apoptosis, human T lymphoblasts (Jurkat) were suspended at 2x10 6 cells per ml in RPMI-1640 supplemented with insulin, transferrin, selenium and glutamine. After incubation for 2 hrs. at room temperature with either compound no. 37, compound no. 6, pentoxifylline or a control compound (Ro-1724), 25 ng/ml of anti-FAS antibody was added to each suspension. After another 2 hrs., cell apoptosis 20 was measured as a function of the number of cells (counted by hemocytometer) that excluded the vital dye erythrosin B. The results of the experiment are shown in FIGURE 2 and establish the apoptosis inhibitory efficacy of the compounds of the invention (as represented by compounds nos. 6 and 37, particularly the latter). -76- WO 00/00491 PCT/US99/14320 As demonstrated in Example I, the bifunctional activation cascade antagonists of the invention provide the activity of each antagonistic constituent in a synergistic manner. Thus, the bifunctional activation cascade antagonists provide the same activity of each constituent alone at greater potency. 5 EXAMPLE VI INHIBITION OF THE ACTIVITY OF CERAMIDE ACTIVATED PROTEIN KINASE BY JNK ACTIVATION CASCADE ANTAGONISTS To demonstrate the probable efficacy of bifunctional activation cascade antagonists in inhibiting activation of ceramide activated protein-kinase activation (at 10 a site recognized by MAPK kinases), the data below demonstrating the efficacy of JNK activation cascade antagonists in this context are provided. Ceramide-activated protein kinase (CaPK) is a 97 kDa protein which is exclusively membrane-bound and is believed to serve a role in the sphingomyelin signal transduction pathway. In particular, CaPK is believed to mediate 15 phosphorylation of a peptide derived from the amino acid sequence surrounding Thr 69 of the epidermal growth factor receptor (i.e., amino acids 663-681). This site is also recognized by MAPK kinases. Thus, the effect of the JNK activation cascade antagonists on CaPK -77- WO 00/00491 PCT/US99/14320 activity in cells is indicative of the effect that the compounds exert on signal transduction in the sphingomyelin pathway. To that end, Jurkat cells were suspended at 2x 106 cells per ml in RPMI 1640 medium. After incubation for 2 hrs., either compound 37, 20 pLM of ceramide or 5 25 ng/ml of anti-FAS antibody DX2 were added to each suspension and incubated for 15 mins. After centrifugation and washing, the cells were separately homogenized in a dounce homogenizer. Ceramide kinase levels in each test sample were assayed as described by Liu, et al., J.Biol.Chem., 269:3047-3052, 1994 (the disclosure of which is 10 incorporated herein for reference and use in assaying ceramide kinase). Briefly, the membrane fraction was isolated from each test sample of treated cell homogenate by ultracentrifugation and run on a 10% PAGE gel. The gel was washed with guanidine HCL, and renatured in HEPES buffer. Then [ 32 P]-ATP was added to the gel and left there for 10 mins. Thereafter, the gel was extensively washed with 5% TCA. 15 Autophos-phorylated kinase was detected by autoradiography. The results of this assay are indicated in FIGURE 3, and establish the CaPK inhibitory efficacy of the compounds of the invention (as represented by compound 37). As demonstrated in Example I, the bifunctional activation cascade 20 antagonists of the invention provide the activity of each antagonistic constituent in a -78- WO 00/00491 PCT/US99/14320 synergistic manner. Thus, the bifunctional activation cascade antagonists provide the same activity of each constituent alone at greater potency. -79- WO 00/00491 PCT/US99/14320 EXAMPLE VII ABSORBANCE OF UVB RADIATION BY JNK ACTIVATION CASCADE ANTAGONISTS To demonstrate the probable efficacy of bifunctional activation cascade 5 antagonists in inhibiting absorbance of UVB radiation, the data below demonstrating the efficacy of JNK activation cascade antagonists in this context are provided. Radiation (particularly in the UVB wavelength) is a major cause of skin damage (including apoptosis) in humans. As indicated elsewhere above, the sphingomyelin signal transduction pathway is believed to be involved in at least the 10 early stages of development of radiation induced dermatoses (including radiation dermatitis, sunburn and UVB induced immune suppression from radiation damage to Langerhans cells in the skin- see, e.g., Haimovitz-Friedman, et al., J.Exp.Med., 180:525-535, 1994 (cellular responses to ionizing radiation); and, Kurimoto and Streilein, J.Immunol., 145:3072-3078, 1992 (cutnaceous immune suppression from 15 UVB exposure)). Thus, a compound which will inhibit cell responses to stimulus of the sphingomyelin signal transduction pathway by radiation and can be administered topically at the site of exposure would be of great benefit in retarding the damage associated with radiation exposure (e.g., through exposure to sunlight or radiation). To assess the radiation absorbing abilities of the compounds of the 20 invention, the ultraviolet spectra of compounds of the invention (nos. 6 and 37, alone, in combination and as 8-oxo derivatives) were evaluated and compared to those of a commercially available sunscreen additive (PABA) and isoquinoline. The spectra were identified using a KONTRON analytical instrument. As indicated in FIGURE 4, -80- WO 00/00491 PCTIUS99/14320 the compounds of the invention (as represented by compounds nos. 6 and 37) absorbed through most of the UVB region, indicating efficacy in absorbing radiation. Surprisingly, a mixture of compound nos. 6 and 37 proved to absorb throughout the UVB region. Thus, given the somewhat greater absorbance characteristics of 5 compound 37 vis-a-vis compound 6, it can be reasonably expected that mixtures of the two in ratios of 1:1 or greater (favoring compound 37) will have substantial synergistic efficacy in absorbing radiation and retarding its effects on cells. EXAMPLE VIII INHIBITION OF TNF-a PRODUCTION BY JNK ACTIVATION CASCADE 10 ANTAGONISTS(OTHER THAN COMPOUND 54) To demonstrate the probable efficacy of bifunctional activation cascade antagonists in inhibiting TNFa production, the data below demonstrating the efficacy of JNK activation cascade antagonists in this context are provided. As shown in FIGURE 5(a), compounds of the invention having N-I chain 15 lengths from 2-5 carbons are especially useful in inhibiting TNF-a production in vitro, while N-I chain lengths of about 4 carbons (with a terminal ester) appear to be optimal in this respect (as compared to a control compound; FIGURE 5(b)). Further, the esterified compounds were significantly more effective inhibitors of TNF-a production than their carboxylic counterparts. These data were obtained as follows: 20 Peripheral blood mononuclear cells were isolated from normal human blood on Hypaque-Ficoll density gradients. A portion of the isolated cells were further purified by adherence to gelatin coated flasks. -81- WO 00/00491 PCTIUS99/14320 100 pl aliquots of monocytes were placed onto 96 well microtiter plates at a density of 5x10 5 cells/ml in RPMI-1640 medium containing 10% fetal bovine serum. After incubation for 24 hrs., various concentrations of the test compounds (FIGURE 10) were added to the plated cells in a volume of 100 Pl and incubated for 1 5 hr. After incubation, 1 jig/ml of LPS was added to each well. 18 hrs. after exposure of the plated cells to LPS, 100 il of medium was collected from each well and assayed (by ELISA) for release of TNF-a, using recombinant human TNF as a standard. The sensitivity of the assay ranged from 10 100 pg/ml. 10 As demonstrated in Example I, the bifunctional activation cascade antagonists of the invention provide the activity of each antagonistic constituent in a synergistic manner. Thus, the bifunctional activation cascade antagonists provide the same activity of each constituent alone at greater potency. EXAMPLE IX 15 IN VIVO AND IN VITRO LEUKOPENIA IN RESPONSE TO LPS AND INHIBITION OF SAME BY JNK ACTIVATION CASCADE ANTAGONISTS To demonstrate the probable efficacy of bifunctional activation cascade antagonists in inhibiting in vivo leukopenia, the data below demonstrating the efficacy of JNK activation cascade antagonists in this context are provided. 20 As shown in FIGURES 6 through 10 and Table 1, the compounds of the invention effectively reduce cellular response to LPS, a known inducer of TNF-a -82- WO 00/00491 PCT/US99/14320 production. In the presence of ceramide, the inhibitory activity of the compounds of the invention on LPS induced leukopenia (a phenomenon dependent on TNF-a induced surface expression of the P-selection class of adhesion molecules) was enhanced (FIGURE 6). However, the inhibitory activity of the compounds of the 5 invention was essentially unaffected by diacylglycerol (FIGURE 7), indicating that the mode of action of the compounds of the invention are not dependent on hydrolysis of phosphatidic acid. These data were obtained as follows: The leukopenia inhibitory capacity of the test compounds was determined by intraperitoneal administration of 0.5 pig of LPS in saline to ICR female mice (age 10 6-8 weeks; weight 19-23 g). One hour before receiving the LPS, the mice received the test compound by intraperitoneal injection at a dose of 50 mg/kg (in isotonic saline). Two hours after injection of LPS, 200 il of blood was collected from each mouse into a heparinized tube and the total count of nucleated cells determined in a 15 hemocytometer. Calcium independent protein kinase activity was measured, using a 1% triton X-100 extract of Jurkat cells (5X10 8 /ml). The reaction mixture consisted of 20 mM Tris HC pH 7.5, 20 mM MgCl 2 , 20 pM ATP containing 200,000 cpm [y32P] ATP, and 50 pM Myelin Basic Protein. The extract was pre-incubated with A) 20 compound 37 B) compound 37 with or without 10 pM ceramide C) ceramide or D) dihydro ceramide for 15 minutes, followed by addition of substrate and ATP, and incubation at 300 C for 5 minutes. The total count of nucleated cells was measured in -83- WO 00/00491 PCT/US99/14320 a hemocytometer (FIGURE 8). The same protocol was followed to obtain the results shown in FIGURE 7 (with the addition of diacyl glycerol to some of the test mixtures). An isoquinoline compound of the invention (compound 52) was also tested 5 in vitro for its inhibitory efficacy with respect to LPS induced TNF-a production in human cells. Human macrophages were cultured in 96 well microtiter plates and incubated with LPS. Aliquots of the stimulated cells were then incubated with, respectively, 0.1, 1, 10, 100 or 1000 pM of compound 52, compound 37 and a 10 commercially available isoquinoline (6,7-dimethoxy-1(2H)-isoquinoline from Aldrich Chemical; labelled S52-626-6 in FIGURE 9) which, like the compounds of the invention, has an oxygen ortho to a ring nitrogen but, unlike the compounds of the invention, lacks a side chain substituent as described above. The inhibitory efficacy of each compound was 15 measured as a function of TNF-a reduction in pg/ml. The results of the experiment are indicated in FIGURE 11 and establish that the compounds of the invention (represented by compound 52 and compound 37) have inhibitory efficacy with respect to reduction in LPS induced TNF-a production by human cells. Other isoquinolines tested did not exert inhibitory activity in the absence of the side chain substituents 20 added according to the invention. -84- WO 00/00491 PCT/US99/14320 These experiments were repeated in human lymphocytes. The protective abilities of the compounds tested is demonstrated by the data in FIGURES 12 and 13. As demonstrated in Example I, the bifunctional activation cascade antagonists of the invention provide the activity of each antagonistic constituent in a 5 synergistic manner. Thus, the bifunctional activation cascade antagonists provide the same activity of each constituent alone at greater potency. EXAMPLE X IN VIVO ANTI-INFLAMMATORY EFFICACY OF CSAIDs The effects of CSAIDs on inflammation in an in vivo model of arthritis 10 have been previously demonstrated. The following references are relevant in this regard and are incorporated herein for ease of reference by those in the art: Boehm, et al., JMed.Chem., 39:3929-3937 (1996) (SB 210313) Badger, et al., J.Pharmacol.Exp. Ther., 279:1453-1461 (1996) (SB 203580) As demonstrated in Example I, the bifunctional activation cascade 15 antagonists of the invention provide the activity of each antagonistic constituent in a synergistic manner. Thus, the bifunctional activation cascade antagonists provide the same activity of each constituent alone at greater potency. EXAMPLE XI FIBROBLAST PROLIFERATION IN RESPONSE TO LPS AND INHIBITION 20 OF SAME BY JNK ACTIVATION CASCADE ANTAGONISTS -85- WO 00/00491 PCT/US99/14320 To demonstrate the probable efficacy of bifunctional activation cascade antagonists in inhibiting fibrosis, the data below demonstrating the efficacy of JNK activation cascade antagonists in this context are provided. As shown in FIGURE 10, PDGF induced fibroblast proliferation was 5 selectively inhibited by the compounds of the invention. In addition, the compounds were shown not to be cytostatic or cytotoxic, insofar as they did not alter EGF triggered mitogenesis in the cells tested (FIGURE 11). These data were obtained as follows: Mouse fibroblast line 3T3 cells (American Type Culture Collection #CCL 10 92) were seeded into 96 well plates in complete medium and allowed to grow to confluence. The medium was then replaced with medium-free serum and the cells incubated for 24 hrs. The test compounds were then incubated with the cells for 1 hr before addition of 5 ng/ml human PDGF or EGF was added to each well. After another 24 15 hrs, 1 pCi of [ 3 H]-thymidine was added to each well. 4 hrs later the cells were harvested onto glass fiber filters and the cellular incorporation of [ 3 H]-thymidine was measured by liquid scintillation counting (FIGURES 10 and 11). As demonstrated in Example I, the bifunctional activation cascade antagonists of the invention provide the activity of each antagonistic constituent in a 20 synergistic manner. Thus, the bifunctional activation cascade antagonists provide the same activity of each constituent alone at greater potency. -86- WO 00/00491 PCTIUS99/14320 EXAMPLE XII SYNTHESIS OF COMPOUNDS 2,4-8 AND 10-13 General Alkylation Procedure for Compounds 4-8, 10, 11 (Method A): Theobromine or 8-bromotheobromine (2 mmol) was combined with 5 anhydrous K 2 C0 3 (2.5 mmol) and dry DMF (15 mL) and the mixture was brought to 75 C. The appropriate alkyl halide (2.5 mmol) was added and the mixture was stirred at 75 0 C for 2-18 h. The reaction mixture was cooled, poured into water (125 mL) and extracted with ethyl acetate (2 x 75 mL). The organic layer was dried over magnesium sulfate and evaporated to yield a colorless oil or white solid which was 10 triturated with ethyl ether. The resulting solid, often analytically pure, may be purified further if desired by crystallization from a small amount of ethanol. Yields 58-89%. Compounds 15-17, 31, 36-38, 41, 43, 47, and 48 (described below) were prepared by this same procedure only using the appropriate precursors in place of theobromine. 15 General Thiation Procedure for Compounds 12 and 13: The 8-bromoxanthine 10 or 11 (0.25 mmol) was suspended in anhydrous ethanol (10 mL) and heated to reflux. NaSH.(H 2 0)x (2.5 mmol) was added and the mixture became clear, green almost immediately. The mixture was stirred under reflux for 30 min, cooled and evaporated onto silica gel. Flash column 20 chromatography using 5-7% MeOH in CH 2 Cl 2 provided a 63% and 75% yield of 12 and 13, respectively as white solids. Note: Compound 13 was found by 'H NMR to be the ethyl ester due to transesterification under the reaction conditions. -87- WO 00/00491 PCTIUS99/14320 'H NMR spectra and elemental analyses or exact mass data were consistent with the assigned structures. EXAMPLE XIII SYNTHESIS OF COMPOUNDS 24, 25,31 AND INTERMEDIATES 5 General Procedure for C-Nitrosation of Pyrimidines (Compounds 18-20, 27, and 32): The pyrimidine (15 mmol) was suspended in N HCl (30 mL) and an aqueous solution of sodium nitrite (20 mmol in 10 mL) was dripped in with stirring over 10 min. The suspension went from off-white to purple almost immediately. 10 Stirring was continued for lh, pH adjusted to 5 with ammonia water and the purple solid product collected to provide 75-90% yield after drying. The characteristic lack of the C-5 proton in the 'H NMR was evident for each compound (Table 2). General Procedure for the Reduction of 5-Nitroso to 5-Amino Pyrimidines (Compounds 21-23, 28, and 33): 15 The 5-nitrosopyrimidine (15 mmol) was suspended in water (50 mL) and heated to 80-90'C. With stirring, sodium hydrosulfite (45 mmol) was added in portions over 5 min. The color quickly changed from purple to light green and stirring was continued an additional 10 min. The mixture was cooled in ice and filtered. The filtered solid was washed with cold water, EtOH and Et 2 0 to provide the 20 orthodiamine in 70-88% yield as a tan to pale green solid. Synthesis of 1 -n-Hexyl-3 -methyluric acid intermediate (24): -88- WO 00/00491 PCT/US99/14320 The nitrosopyrimidine 19 (270 mg, 1.06 mmol) was dissolved in ethanol (20 mL) with warming and palladium on carbon (75 mg, 10%) was added under argon. Hydrogenation was performed at room temperature and 15 psi for 2 h, filtered to remove catalyst and evaporated to dryness. The residue was combined with urea 5 (600 mg, 10 mmol) and heated neat on the hot plate with stirring. The temperature reached 140'C which produced a clear melt and was maintained for about 10 min. with additional urea added (1 g). Upon cooling the melt solidified and was dissolved in n NaOH (25 mL) and boiled with decolorizing carbon for 10 min., filtered and acidified to pH 3-4 while hot. The resulting precipitate was collected after cooling 10 and washed with water and dried to yield 160 mg (57%) of 24 as an off-white solid with the following characteristics: mp >290'C dec. 'H NMR (500 MHZ, DMSO-d 6 ) 5 11.80 and 10.73 (2s, 2H, N-7 H, N-9 H), 3.78 (t, 2H, N-CH 2 ), 3.30 (s, 3H, N-CH 3 , under H20 signal), 1.48 (in, 2H, 2'CH 2 ), 1.24 (in, 6H, 3', 4', 5' CH 2 ), 0.85 (t, 3H, CH 3 ). Analysis: C, 2

HN

4 0 3 (C, H, N; Table 2). 15 Synthesis of 3-Methyl-8-thiouric acid (25) intermediate: The pyrimidinediamine 33 (100 mg, 0.63 mmol) was combined with potassium ethyl xanthate (810 mg, 5 mmol) and DMF (10 mL) and heated at 100 0 C. The suspension became green almost immediately and reaction was complete after 30 min. by TLC. After a total reaction time of 1 h, the mixture was cooled, filtered and 20 washed with Et 2 0, dried to yield an off-white solid (310 mg) which presumably contained the unreacted potassium ethyl xanthate and the potassium salt of the desired -89- WO 00/00491 PCT/US99/14320 product. The solid was suspended in water (5 mL) and heated to dissolve. Glacial acetic acid was added to pH 5 and a vigorous effervescence was noted. A white solid formed which was filtered warm and washed with water, then ethanol and dried to yield 99 mg (79%) of the title compound. 'H NMR (DMSO-d 6 ) 6 13.40, 12.92 and 5 11.80 (3br s, 3H, NHs), 3.28 (s, 3H, CH 3 ). Analysis: C 6

H

6

N

4 0 2 S (C, H, N; Table 2). Synthesis of 3-n-Propylxanthine (29) intermediate: The pyrimidinediamine 28 (750 mg) was combined with diethoxymethyl acetate (7 mL) and heated at 80'C for 2 h. The mixture was evaporated to dryness and water (5 mL) was added and the mixture heated for 20 min. to near boiling. The 10 resulting solution was then allowed to evaporate slowly to yield off-white crystals. Yield 680 mg (86%); mp 282-284'C, Lit." 291-292 0 C. EXAMPLE XIV SYNTHESIS OF COMPOUNDS 36-39,41 AND 43 AND INTERMEDIATES General Procedure for Ring Closure of Pyrimidine-diamines to Pteridines: 15 The orthodiamine 28 or 33 (2 mmol) was suspended in water (20 mL) and heated to above 70 C before a solution of glyoxal-sodium bisulfite addition product (10 mmol in 25 mL water) was added with stirring. The pale green suspension slowly became light amber and clear. After heating 5 min TLC indicated reaction was complete. The mixture was cooled and extracted with ethyl acetate (5 x 40 mL), dried 20 over MgSO 4 and evaporated to yield the 1-methyl (34) or 1 -n-propylpteridine (35) in -90- WO 00/00491 PCT/US99/14320 71 and 78 %, respectively. 'H NMR showed the appearance of two aromatic signals at about 8.74 and 8.55 as doublets (J = 2.5 Hz) for both compounds. Synthesis of 6,7-Diethyl-1-methylpteridine-2,4-dione (40) intermediate: Compound 33 (200 mg, 1.27 mmol) was suspended in acetonitrile (5 mL) 5 and 3,4-hexanedione (185 ptL, 1.52 mmol) was added. The mixture was heated at 70'C for 15 min with minimal product formation due to insolubility of 33. Therefore DMF (3 mL) and water (3 mL) were added and the temperature was raised to 100 0 C. After 90 min total reaction time the mixture was cooled and poured into water (100 mL) and extracted with ethyl acetate (3 x 75 mL). The organic layer was dried over 10 MgSO 4 and evaporated to provide the colorless crystalline product. Yield 240 mg (81%); mp 218-222'C; 'H NMR (DMSO-d 6 ) 6 11.78 (br s, 1H, NH), 3.46 (s, 3H,

NCH

3 ), 2.95 and 2.93 (2q, 4H, 2CH 2 of ethyls), 1.28 and 1.23 (2t, 6H, 2CH 3 of ethyls). Analysis: C 1

H

14

N

4 0 2 (C, H, N; Table 2). Synthesis of 1-Methyl-6-phenylpteridine-2,4-dione (42) intermediate: 15 The nitrosopyrimidine 32 (220 mg, 1.28 mmol) was mixed thoroughly with phenethyl amine hydrochloride (1.5 g, 9.5 mmol) and heated in an open beaker on the hot plate. After a few minutes at about 160'C the purple reaction mixture fused to a brown paste. TLC indicated many products so sulfolane (1 mL) was added and heat was continued for 15 min. The reaction mixture was heated in water (10 20 mL) and then diluted 50 mL in water and extracted with ethyl acetate (2 x 50 mL), the organic layer dried over MgSO 4 and then concentrated. The residue was flash -91- WO 00/00491 PCT/US99/14320 chromatographed on silica gel using 4% MeOH in CH 2 Cl 2 . Yield 75 mg (23%) of 42 as a pale yellow-orange solid. mp >307'C dec.; 'H NMR (500 MHZ, DMSO-d 6 ) 11.95 (br s, 1H, NH), 9.37 (s, 1H, C-7 H), 8.17 (m, 2H, 2',6' phenyl), 7.55 (m, 3H, 3',4',5' phenyl), 3.51 (s, 3H, NCH 3 ). Anal. C 13

HION

4 0 2 (C, H, N). -92- WO 00/00491 PCT/US99/14320 EXAMPLE XV SYNTHESIS OF COMPOUNDS 44,47 AND 48 General Method for Ring Closure of Pyrimidines to Thiadiazolo pyrimidines (Compounds 44-46): 5 The orthodiamine 23, 27, or 32 (2.3 mmol) was suspended in dry acetonitrile (5 mL) and dry pyridine (1.5 mL) was added. Thionyl chloride (1 mL, 13.7 mmol) was added quickly and the mixture, which became clear and darkened, was heated at 60'C for 10 min. The mixture was then cooled and poured into n HCl (40 mL) with stirring. The resulting yellow solution was extracted with ethyl acetate 10 (3 x 40 mL), dried over MgSO 4 and evaporated to yield a pale yellow solid which was triturated with ether. Yield 65-74%. Alkylation of these intermediates yielded the disubstituted products 47 and 48. EXAMPLE XVI 15 SYNTHESIS OF COMPOUNDS 50 AND 52 Ethyl 4 -[(2-methylamino)benzoyl]aminobutanoate (51): A mixture of N-methylisatoic anhydride (3.5 g, 19.8 mmol) was combined with 4-aminobutyric acid (2.5 g, 24.3 mmol) in dry DMF (50 mL) and heated at 100 C for 2 h. TLC indicated reaction to be complete and the DMF was removed in 20 vacuo. The residue was used directly for esterification which was accomplished by dissolving the residue in 100% ethanol (50 mL) and adding chlorotrimethyl silane (2.5 mL, 20 mmol). The mixture was heated at 65 C for 6 h and then evaporated to yield -93.- WO 00/00491 PCT/US99/14320 a brown syrup. Crude yield 87% from isatoic anhydride. A small sample was purified for characterization and biological testing by preparative TLC using 7% MeOH in CH 2 Cl 2 . The remainder of the material was used directly for preparation of compound 52. Analysis: C1 4

H

20

N

2 0 3 (C, H, N; Table 2). 5 Ethyl 1-Methyl-1, 4 -dihydro-2,4-dioxo-3(2H)-quinazolinebutanoate (52): The residue from 51 was combined with ethyl chloroformate (10 mL) and heated at 90'C for 1 h. The mixture was cooled and poured into saturated aqueous sodium bicarbonate (50 mL) with stirring and after 10 min extracted with ethyl acetate 10 (2 x 75 mL). The organic layer was dried over MgSO 4 and evaporated to yield a brown syrup. The crude product was flash chromatographed on silica using 3% MeOH in CH 2 Cl 2 to yield g (%) of 52 as a thick oil. 'H NMR (500 MHZ, DMSO-d 6 ) 8 7.27-7.42 (2m, 4H, C-5,6,7,8), 4.04 (t, 2H, CH 2 of ethyl), 3.88 (in, 2H, NCH 2 ), 3.11 (s, 3H, NCH 3 ), 2.33 (t, 2H, 2'CH 2 ), 1.71 (in, 2H, 3'CH 2 ). Analysis: C, 5 Hi 8

N

2 0 4 (C, 15 H, N). EXAMPLE XVII SYNTHESIS OF HIGHLY WATER SOLUBLE DERIVATIVES Acid Hydrolysis of Compound 53. Compound 53 (1.63 g, 5.1 mmol) was combined with n HCl (30 mL) and 20 was heated at boiling for 75 min. The mixture was cooled and the resulting solid which formed was filtered, washed with cold water, and dried to yield 1.5 g -94- WO 00/00491 PCT/US99/14320 (quantitative conversion) of the free carboxylic acid of suitable purity for esterification (as judged by TLC). Esterification to form morpholinoethyl ester Compound 54. The carboxylic acid obtained above in this Example (1.0 g, 3.4 mmol) was 5 dissolved in dichloromethane (25 mL) with warming and then thionyl chloride (1 mL, 13.7 mmol) was added followed by 3 drops of DMF. After a few minutes a white solid precipitated (acid chloride intermediate), but the reaction mixture was allowed to stir at room temperature overnight. The mixture was evaporated to remove excess thionyl chloride and the residue was suspended in dry acetonitrile (25 mL). To this 10 mixture was added morpholinoethanol (1.24 mL, 10.2 mmol) and heated at 80'C for 5 minutes. The solid suspension became clear almost immediately and after cooling, the reaction mixture was evaporated onto silica gel and loaded on a flash silica gel column and eluted with methanol - dichloromethane 5/95. The fractions of pure product were pooled and evaporated to yield an off-white residue which was dissolved 15 in isopropyl alcohol (5 mL) and concentrated HCl (1 mL) was added. Upon concentration in vacuo, an off-white solid formed. Yield after drying 0.93 g (62%), mp 90'C. Proton NMR confirms structure assignment as the morpholino ethyl ester of Compound 53: 'H NMR (DMSO-d 6 , 500 MHZ) 8 7.56 and 7.15 (2s, 2H, C-5 and C-8 aromatics), 7.35 and 6.55 (2d, 2H, C-3 and C-4 aromatics), 4.37 (m, 2H, C-l' of 20 ester), 3.92 (m, 2H, C-4 of acyl), 3.86 and 3.84 (2s, 6H, OMe's), 3.95, 3.45, 3.10 (3m, 8H, morpholino and C-2'), 2.38 (t, 2H, C-2 of acyl), 1.92 (m, 2H, C-3 of acyl). -95- WO 00/00491 PCTIUS99/14320 The invention having been fully described, modifications thereof may be apparent to those of ordinary skill in the art. Such modifications are within the scope of the invention as defined by the appended claims. -96-

Claims (66)

1. A compound for antagonizing activation of c-Jun kinase and p38 comprised of a first constituent having the formula: 0 R1 N Y, I X A/ Z Y2 R2 wherein: 5 R, is a terminally substituted normal alkyl having from 1 to 7 carbon atoms, a terminally substituted alkenyl having from 2 to 7 carbon atoms, a terminally substituted ether having from 2 to 6 carbon atoms, a terminally substituted secondary amine having from 2 to 6 carbon atoms, or substituted aryl having less than 8 carbons, where said terminal group is 10 NH 2 , substituted amino, acyloxy, SO 3 H, P04H 2 , NNO(OH), SO 2 NH 2 , PO(OH)NH 2 , SO 2 R or COOR, where R is H, an alkyl having from 1 to 4 carbon atoms, an alkenyl having from 1 to 4 carbon atoms, tetrazolyl, benzyl, or an alkylamino, where the alkyl group has from 1 to 4 carbon atoms and the amino is NH 2 or a substituted amino where the substituents -97- WO 00/00491 PCTIUS99/14320 on the amino have 1 to 6 carbon atoms, one of which can be replaced by an oxygen atom or nitrogen atom; Z is C, CH, or N; R 2 is an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or aralkyl having 5 less than 7 carbon atoms when Z is C, R 2 is a halogen, NO, amino, or substituted amino when Z is CH, or R 2 is H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or aralkyl having less than 7 carbon atoms when Z is N; A is CO when Z is N, or CR when Z is C or CH, where R 5 is H, an alkyl, 10 a cyclic alkyl, a heterocyclic alkyl, alkenyl, aryl or aralkyl having less than 7 carbon atoms, OH, or an O-alkyl having from 1 to 5 carbon atoms and there is a double bond between Z and A when Z is C and a single bond between Z and A when Z is CH; Yj is N, NR 6 or CR 6 , where R 6 is H, NO, an amino, a substituted amino, an 15 alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or aralkyl having less than 7 carbon atoms; Y 2 is N or CH; and X is S when Yj and Y 2 are N, CR 7 when Y, is NR 6 where R 7 is H, OH, SH, Br, Cl, or I, or =C(R 3 )-C(R 4 )= when Yi is N, CH or CR 6 and Y 2 is N or 20 CH, where each of R 3 and R 4 , independently, is H, an alkyl, a cyclic alkyl, -98- WO 00/00491 PCTIUS99/14320 a heterocyclic alkyl, alkenyl, aryl, alkylcarboxyl, or aralkyl having less than 7 carbon atoms, SH, OH or an O-alkyl having from 1 to 5 carbon atoms, or a salt thereof; and, 5 a second constituent consisting of a pyridinylimidazole conjugated to the first constituent; wherein the first constituent selectively antagonizes c-Jun kinase activation and the second constituent selectively antagonizes p38 activation. -99- WO 00/00491 PCT/US99/14320

2. The compound according to Claim 1, wherein the first constituent has the formula: 0 0 R NR R1 NY1 R3 N N. R5 R or O N Y2 R4 2 R 2 wherein 5 R, is a terminally substituted normal alkyl having from 1 to 7 carbon atoms, a terminally substituted alkenyl having from 2 to 7 carbon atoms, a terminally substituted ether having from 2 to 6 carbon atoms, a terminally substituted secondary amine having from 2 to 6 carbon atoms, or substituted aryl having less than 8 carbons, where said terminal group is 10 NH 2 , substituted amino, acyloxy, SO 3 H, P04H2, NNO(OH), SO 2 NH 2 , PO(OH)NH 2 , SO 2 R or COOR where R is H, an alkyl having from 1 to 4 carbon atoms, an alkenyl having from 1 to 4 carbon atoms, tetrazolyl, benzyl, or an alkylamino, where the alkyl group has from 1 to 4 carbon atoms and the amino is NH 2 or a substituted amino where the substituents 15 on the amino have 1 to 6 carbon atoms, one of which can be replaced by an oxygen atom or nitrogen atom; -100- WO 00/00491 PCTIUS99/14320 R 2 is an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or aralkyl having less than 7 carbon atoms; each of Y, and Y 2 , independently, is N or CH; each of R 3 and R4, independently, is H, SH, OH, an alkyl, a cyclic alkyl, a 5 heterocyclic alkyl, alkenyl, aryl, alkylcarboxyl, or aralkyl having less than 7 carbon atoms, or OH or an O-alkyl having from 1 to 5 carbon atoms; R 5 , when present, is H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, aryl or aralkyl having less than 7 carbon atoms, OH, or an O-alkyl having from 1 to 5 carbon atoms; and 10 R 6 is NO, an amino, or a substituted amino; or a salt thereof.

3. The compound of Claim 2 wherein the first constituent has the formula: o 0 R1 R: or R1 N N R 3 R 0 N N R4 R2 -101- WO 00/00491 PCT/US99/14320 wherein R, is a terminally substituted normal alkyl having from 1 to 7 carbon atoms, a terminally substituted alkenyl having from 2 to 7 carbon atoms, a terminally substituted ether having from 2 to 6 carbon atoms, a terminally 5 substituted secondary amine having from 2 to 6 carbon atoms, or substituted aryl having less than 8 carbons, where said terminal group is NH 2 , substituted amino, acyloxy, SO 3 H, PO 4 2 , NNO(OH), SO 2 NH 2 , PO(OH)NH 2 , SO 2 R or COOR where R is H, an alkyl having from 1 to 4 carbon atoms, an alkenyl having from 1 to 4 carbon atoms, tetrazolyl, 10 benzyl, or an alkylamino, where the alkyl group has from 1 to 4 carbon atoms and the amino is NH 2 or a substituted amino where the substituents on the amino have 1 to 6 carbon atoms, one of which can be replaced by an oxygen atom or nitrogen atom; R 2 , when present, is an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or is aralkyl having less than 7 carbon atoms; and each of R 3 and R 4 , independently, is H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, aryl, alkylcarboxyl, or aralkyl having less than 7 carbon atoms, or OH or an O-alkyl having from 1 to 5 carbon atoms; or a salt thereof. -102- WO 00/00491 PCT/US99/14320

4. The compound of Claim 3, wherein the first constituent has the following substituents: R, is a terminally substituted normal alkyl having from 1 to 7 carbon atoms, where said terminal group and is NH 2 , substituted amino, or 5 COOR, where R is H, an alkyl having from 1 to 4 carbon atoms, or an alkylamino, where the alkyl group has from 1 to 4 carbon atoms and the amino is a substituted amino where the substituents on the amino have 1 to 6 carbon atoms, one of which can be replaced by an oxygen atom or nitrogen atom; 10 R 2 , when present, is an alkyl having less than 7 carbon atoms; and each of R 3 and R 4 , independently, is H, an alkyl, aryl, alkylcarboxyl, or aralkyl having less than 7 carbon atoms, or an O-alkyl having from 1 to 5 carbon atoms; or a salt thereof. -103- WO 00/00491 PCTIUS99/14320

5. The compound of Claim 4 wherein the first constituent has the formula: 0 R1 NR 3 6aR4 wherein R, is a terminally substituted normal alkyl having from 1 to 7 carbon atoms 5 where said terminal group is COOR, where R is H, an alkyl having from 1 to 4 carbon atoms, or an alkylamino, where the alkyl group has from 1 to 4 carbon atoms and the amino is a morpholino group; and each of R 3 and R 4 , independently, is H, an alkyl having less than 7 carbon atoms, or an O-alkyl having from 1 to 5 carbon atoms; 10 or a salt thereof. -104- WO 00/00491 PCT/US99/14320

6. The compound of Claim 5, wherein the first constituent has the following substituents: R, is a terminally substituted normal alkyl having from 1 to 4 carbon atoms where said terminal group is COOR where R is an N-morpholinoalkyl 5 group; and, each of R 3 and R 4 , independently, is an O-alkyl having from 1 to 5 carbon atoms; or a salt thereof.

7. The compound of Claim 6, wherein R, is -(CH 2 ) 3 COOR where R is an N 10 morpholinoethyl group, and R 3 and R 4 each are methoxy groups, or a salt thereof. -105- WO 00/00491 PCTIUS99/14320

8. The compound of Claim 4, wherein the first constituent has the 0 formula: R1 N R 3 0 N N R4 R2 wherein R, is a terminally substituted normal alkyl having from 1 to 7 carbon atoms 5 where said terminal group is COOR, where R is H, an alkyl having from 1 to 4 carbon atoms, or an alkylamino, where the alkyl group has from 1 to 4 carbon atoms and the amino is a morpholino group; R 2 is an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or aralkyl having less than 7 carbon atoms; and 10 each of R 3 and R 4 , independently, is H, OH, SH, an alkyl, aryl, or aralkyl having less than 7 carbon atoms, or an 0-alkyl having from 1 to 5 carbon atoms; or a salt thereof.

9. The compound of Claim 8, wherein the first constituent has the following 15 substituents: -106- WO 00/00491 PCT/US99/14320 R, is a terminally substituted normal alkyl having from 1 to 4 carbon atoms where said terminal group is COOR where R is an N-morpholinoalkyl group; R 2 is an alkyl having less than 7 carbon atoms; and 5 each of R3 and R 4 , independently, is H, an alkyl, aryl, or alkylcarboxyl having less than 7 carbon atoms, or an O-alkyl having from 1 to 5 carbon atoms; or a salt thereof.

10. The compound of Claim 9, wherein the first constituent has the following 10 substituents: R, is -(CH 2 ) 3 COOR where R is an N-morpholinoethyl group; R2 is an alkyl having less than 4 carbon atoms; and each of R 3 and R 4 , independently, is H, an alkyl having 1 to 3 carbon atoms, phenyl, or methylcarboxyl; 15 or a salt thereof. -107- WO 00/00491 PCTIUS99/14320

11. The compound of Claim 10, wherein the first constituent has the following substituents: R2 is -(CH 2 ) 2 CH 3 ; and, R 3 and R 4 each are H; 5 or a salt thereof.

12. The compound according to Claim 1 wherein the first constituent has the formula: R1 R 1 N O N R N NN 0 N 02 NN R2 R2 wherein R, is a terminally substituted normal alkyl having from 1 to 7 carbon 10 atoms, a terminally substituted alkenyl having from 2 to 7 carbon atoms, a terminally substituted ether having from 2 to 6 carbon atoms, a terminally substituted secondary amine having from 2 to 6 carbon atoms, or substituted aryl having less than 8 carbons, where said terminal group is NH 2 , substituted amino, acyloxy, SO 3 H, PO 4 H 2 , NNO(OH), SO 2 NH 2 , -108- WO 00/00491 PCT/US99/14320 PO(OH)NH 2 ,SO 2 R or COOR, where R is H, an alkyl having from 1 to 4 carbon atoms, an alkenyl having from 1 to 4 carbon atoms, tetrazolyl, benzyl, or an alkylamino, where the alkyl group has from 1 to 4 carbon atoms and the amino is NH 2 or a substituted amino where the substituents 5 on the amino have 1 to 6 carbon atoms, one of which can be replaced by an oxygen atom or nitrogen atom; R 2 is H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or aralkyl having less than 7 carbon atoms; Y2is N or CH; 10 R 6 is H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or aralkyl having less than 7 carbon atoms; and R 7 is H, OH, SH, Br, Cl, or I; or a salt thereof; and, a second constituent consisting of a pyridinylimidazole; 15 wherein the first constituent selectively antagonizes c-Jun kinase activation and the second constituent selectively antagonizes p38 activation.

13. The compound of Claim 12, wherein the first constituent has the following substituents: -109- WO 00/00491 PCTIUS99/14320 R, is a terminally substituted normal alkyl having from 1 to 7 carbon atoms, where said terminal group is NH 2 , substituted amino, or COOR, where R is H, an alkyl having from 1 to 4 carbon atoms, or an alkylamino, where the alkyl group has from 1 to 4 carbon atoms and the amino is NH 2 5 or a substituted amino where the substituents on the amino have 1 to 6 carbon atoms, one of which can be replaced by an oxygen atom or nitrogen atom; R 2 is H, or an alkyl having less than 7 carbon atoms; Y 2 is N or CH; 10 R 6 is H, or an alkyl having less than 7 carbon atoms; and R 7 is H, OH, SH, or Br; or a salt thereof.

14. The compound of Claim 13, wherein R, is (CH 2 ) 3 COOR and R is an N morpholinoethyl group, or a salt thereof.

15 15. The compound of Claim 1, wherein R, is (CH 2 ) 3 COOR and R is an N morpholinoethyl group, or a salt thereof. -110- WO 00/00491 PCTIUS99/14320

16. The compound according to Claim 1, wherein the second constituent is a cytokine suppressive anti-inflammatory drug.

17. The compound according to Claim 16, wherein the cytokine suppressive anti-inflammatory drug is selected from the group of such drugs comprising 1-[3-(4 5 morpholinyl)propyl]-4-(4-fluorophenyl-5-(4-pyridyl)imidazole, 2-(4 methylsulfinylphenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a]imidazole, 2-(4 methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a]imidazole, 4-(4 fluorophenyl)-2-(4-methylsulfinyl)-5-(4-pyridyl)imidazole and 5-(4-pyridyl)-6(4 flurophenyl)-2,3-dihydroimidazole(2, 1 -b)thia zol. 10

18. The compound according to Claim 1, wherein the first and second constituents are conjugated to one another by a bond which is severable in vivo.

19. The compound according to Claim 18, wherein the bond is an amide, ester or azo bond.

20. The compound according to Claim 19, wherein the bond is a tertiary N 15 acyloxymethyl amide bond. -111- WO 00/00491 PCT/US99/14320

21. The compound according to Claim 1 mixed in a colloid.

22. The compound according to Claim 21, wherein the colloid is a liposome.

23. A pharmaceutical composition comprising the compound of Claim 1 and a pharmaceutically acceptable carrier. -112- WO 00/00491 PCT/US99/14320

24. A compound for antagonizing activation of c-Jun kinase and p38 having the formula: N F A R 6 NiO (cH2) N N\N N Z R7 x or N F 0 0 N R 6 f N O (CH2)nfN N N ,NdO N N R 7 R1 wherein 5 n is any number of carbon atoms from 1 to 7, 0 or N; RI, if present, is H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or aralkyl having less than 7 carbon atoms; R 6 and R, are H, OH or OR,, in any combination; Z is N or C; 10 X, where Z is C, is H, halogen, N 3 , NO, NH 2 , NHR,, N(R,)2 or COR,; and, -113- WO 00/00491 PCT/US99/14320 A is H, halogen, N 3 , NO, NH 2 , NHR,, N(R,)2 or COR,.

25. A compound according to Claim 24 having the formula: CH 3 0 C+ N ./ FNN N N N

26. A compound according to Claim 24 having the formula: N I H N 0 01- / \ / 0 N N 5 R 00 N F 0 N N C 3 H 7

27. A pharmaceutically acceptable composition comprising the compound according to Claim 24 mixed in a colloid. -114- WO 00/00491 PCT/US99/14320

28. The compound according to Claim 27, wherein the colloid is a liposome.

29. A pharmaceutical composition comprising the compound of Claim 24 and a pharmaceutically acceptable carrier.

30. A compound comprised of a first constituent having the formula: 0 R3 R 1 N 0 N 5 wherein R2 R, is (CH 2 )COO-Et; R 2 is CH 3 ; R3 is CH 3 ; and, Z is N; -115- WO 00/00491 PCT/US99/14320 further comprised of a second constituent conjugated to the first constituent and consisting of a pyridinylimidazole which selectively antagonizes p38 activation.

31. The compound according to Claim 30, wherein the pyridinylimidazole is a 5 cytokine suppressive anti-inflammatory drug.

32. The compound according to Claim 31, wherein the cytokine suppressive anti-inflammatory drug is selected from the group of such drugs comprising 1-[3-(4 morpholinyl)propyl]-4-(4-fluorophenyl-5-(4-pyridyl)imidazole, 2-(4 methylsulfinylphenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a]imidazole, 2-(4 10 methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a]imidazole, 4-(4 fluorophenyl)-2-(4-methylsulfinyl)-5-(4-pyridyl)imidazole and 5-(4-pyridyl)-6(4 flurophenyl)-2,3-dihydroimidazole(2,1 -b)thia zol.

33. The compound according to Claim 30, wherein the first and second constituents are conjugated to one another by a bond which is severable in vivo. -116- WO 00/00491 PCT/US99/14320

34. The compound according to Claim 33, wherein the bond is an amide, ester or azo bond.

35. The compound according to Claim 34, wherein the bond is a tertiary N acyloxymethyl amide bond. 5

36. The compound according to Claim 30, wherein the first and second constituents are mixed in a colloid.

37. The compound according to Claim 36, wherein the colloid is a liposome.

38. A pharmaceutical composition comprising the compound of Claim 30 and a pharmaceutically acceptable carrier. 10

39. A compound comprised of a first constituent having the formula: -117- WO 00/00491 PCT/US99/14320 0 RN N R4 O N N R5 R2 wherein R, is (CH 2 ) 3 COO-Et; R 2 is n-propyl; R4 is H; and 5 R 5 is H; further comprised of a second constituent conjugated to the first constituent and consisting of a pyridinylimidazole which selectively antagonizes p38 activation.

40. The compound according to Claim 39, wherein the pyridinylimidazole is a 10 cytokine suppressive anti-inflammatory drug.

41. The compound according to Claim 40, wherein the cytokine suppressive anti-inflammatory drug is selected from the group of such drugs comprising 1-[3-(4 -118- WO 00/00491 PCT/US99/14320 morpholinyl)propyl]-4-(4-fluorophenyl-5-(4-pyridyl)imidazole, 2-(4 methylsulfinylphenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a]imidazole, 2-(4 methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a]imidazole, 4-(4 fluorophenyl)-2-(4-methylsulfinyl)-5-(4-pyridyl)imidazole and 5-(4-pyridyl)-6(4 5 flurophenyl)-2,3-dihydroimidazole( 2 , 1 -b)thia zol.

42. The compound according to Claim 39, wherein the first and second constituents are conjugated to one another by a bond which is severable in vivo.

43. The compound according to Claim 42, wherein the bond is an amide, ester or azo bond. 10

44. The compound according to Claim 43, wherein the bond is a tertiary N acyloxymethyl amide bond.

45. The compound according to Claim 39 mixed in a colloid.

46. The compound according to Claim 45, wherein the colloid is a liposome. -119- WO 00/00491 PCT/US99/14320

47. A pharmaceutical composition comprising the compound of Claim 39 and a pharmaceutically acceptable carrier. -120- WO 00/00491 PCT/US99/14320

48. A compound comprised of a first constituent having the formula: MeO O MeO N 0 0 53 1) NaOH or HCI, heat 2) SOC1 2 3) morpholinoethanol 4) HCI MeO MeO N N 0 0 -HCI 54 further comprised of a second constituent conjugated to the first constitunt and consisting of a pyridinylimidazole which selectively antagonizes p38 activation.

49. The compound according to Claim 48, wherein the pyridinylimidazole is a 5 cytokine suppressive anti-inflammatory drug. -121- WO 00/00491 PCT/US99/14320

50. The compound according to Claim 49, wherein the cytokine suppressive anti-inflammatory drug is selected from the group of such drugs comprising 1-[3-(4 morpholinyl)propyl]-4-(4-fluorophenyl-5-(4-pyridyl)imidazole, 2-(4 methylsulfinylphenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a]imidazole, 2-(4 5 methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a]imidazole, 4-(4 fluorophenyl)-2-(4-methylsulfinyl)-5-(4-pyridyl)imidazole and 5-(4-pyridyl)-6(4 flurophenyl)-2,3 -dihydroimidazole(2, 1 -b)thia zol.

51. The compound according to Claim 48, wherein the first and second constituents are conjugated to one another by a bond which is severable in vivo. 10

52. The compound according to Claim 51, wherein the bond is an amide, ester or azo bond.

53. The compound according to Claim 52, wherein the bond is a tertiary N acyloxymethyl amide bond.

54. The compound according to Claim 48 mixed in a colloid. -122- WO 00/00491 PCT/US99/14320

55. The compound according to Claim 54, wherein the colloid is a liposome.

56. A pharmaceutical composition comprising the compound of Claim 48 and a pharmaceutically acceptable carrier.

57. A method for treating a vertebrate host for an inflammatory condition 5 comprising administering the compound of Claim 1 to the host.

58. The method according to Claim 57 wherein the inflammatory condition is arthritis, spondylitis, bone resorption, sepsis, septic shock, endotoxic shock, inflammatory bowel disease, asthma, bronchitis, chronic pulmonary inflammatory disease, silicosis, repurfusion injury, graft versus host reactions, allograft rejection, 10 fever, viral and bacterial infection, inflammation of the joints, psoriasis and eczema, radiation dermatitis, multiple sclerosis, cell senescence and apoptosis.

59. A method for treating a vertebrate host for an inflammatory condition comprising administering the compound of Claim 24 to the host. -123- WO 00/00491 PCTIUS99/14320

60. The method according to Claim 59 wherein the inflammatory condition is arthritis, spondylitis, bone resorption, sepsis, septic shock, endotoxic shock, inflammatory bowel disease, asthma, bronchitis, chronic pulmonary inflammatory disease, silicosis, repurfusion injury, graft versus host reactions, allograft rejection, 5 fever, viral and bacterial infection, inflammation of the joints, psoriasis and eczema, radiation dermatitis, multiple sclerosis, cell senescence and apoptosis.

61. A method for treating a vertebrate host for an inflammatory condition comprising administering the compound of Claim 30 to the host.

62. The method according to Claim 61 wherein the inflammatory condition is 10 arthritis, spondylitis, bone resorption, sepsis, septic shock, endotoxic shock, inflammatory bowel disease, asthma, bronchitis, chronic pulmonary inflammatory disease, silicosis, repurfusion injury, graft versus host reactions, allograft rejection, fever, viral and bacterial infection, inflammation of the joints, psoriasis and eczema, radiation dermatitis, multiple sclerosis, cell senescence and apoptosis. 15

63. A method for treating a vertebrate host for an inflammatory condition comprising administering the compound of Claim 39 to the host. -124- WO 00/00491 PCT/US99/14320

64. The method according to Claim 63 wherein the inflammatory condition is arthritis, spondylitis, bone resorption, sepsis, septic shock, endotoxic shock, inflammatory bowel disease, asthma, bronchitis, chronic pulmonary inflammatory disease, silicosis, repurfusion injury, graft versus host reactions, allograft rejection, 5 fever, viral and bacterial infection, inflammation of the joints, psoriasis and eczema, radiation dermatitis, multiple sclerosis, cell senescence and apoptosis.

65. A method for treating a vertebrate host for an inflammatory condition comprising administering the compound of Claim 48 to the host.

66. The method according to Claim 65 wherein the inflammatory condition is 10 arthritis, spondylitis, bone resorption, sepsis, septic shock, endotoxic shock, inflammatory bowel disease, asthma, bronchitis, chronic pulmonary inflammatory disease, silicosis, repurfusion injury, graft versus host reactions, allograft rejection, fever, viral and bacterial infection, inflammation of the joints, psoriasis and eczema, radiation dermatitis, multiple sclerosis, cell senescence and apoptosis. -125-