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The synthesis and biological activities of rapamycin (I) analogs modified at the C-40 position are reported. Emphasis placed on compounds that potentially have an improved safety profile on account of their shorter in vivo half-life when compared with rapamycin.

Interleukin-8 modulation is implicated in many inflammatory and cancer diseases. Starting from a mass-screening hit, the synthesis and structure-activity relationship of 2-amino-3-heteroarylquinoxalines as non-peptide, small molecule interleukine-8 receptor antagonists have been developed. The optimized derivatives, PD 0210293 (13y) and PD 0220245 (13r), show inhibition of both IL-8 receptor binding and IL-8-mediated neutrophil chemotaxis.

Several novel 4-substituted 4-arylpiperidines were synthesized. The chemistry leading to 4-(4-chlorophenyl)-4-fluoropiperidine ( 6 ), 4-azido-4-(4-chlorophenyl)piperidine ( 7 ) and 4-(4-chlorophenyl)-4-methylpiperidine ( 8 ) is described.

C24-Deoxyascomycin was prepared in a two-step process from ascomycin and evaluated for its immunosuppressant activity relative to ascomycin and FK506. An intermediate in the synthetic pathway, Delta(23,24)-dehydroascomycin, was likewise evaluated. Despite lacking the hydrogen-bonding interactions associated with the C24-hydroxyl moiety of ascomycin, C24-deoxyascomycin was found to be equipotent to the parent compound both in its immunosuppressive potency and in its interaction with the immunophilin, FKBP12. Conversely, Delta(23,24)-dehydroascomycin which also lacks the same hydrogen-bonding interactions did not exhibit this potency. NMR studies were conducted on the FKBP12/C24-deoxyascomycin complex in an attempt to understand this phenomenon at the molecular level. The NMR structures of the complexes formed between FKBP12 and ascomcyin or C24-deoxyascomcyin were very similar, suggesting that hydrogen-bonding interactions with the C24 hydroxyl moiety are not important for complex formation.

A series of novel pyrazole carboxamides is disclosed that demonstrate strong immunosuppressant activity in rodent and human mixed leukocyte response (MLR) assays (IC501 microM). The synthesis, biological activity, mode of action, and pharmacokinetic properties of this new lead series are discussed.

Abstract: Drug therapy for the major inflammatory skin diseases, which include atopic dermatitis, psoriasis and allergic contact dermatitis, is often inadequate due to poor efficacy, toxicity, or both. Much research has focused on the macrolactam T cell inhibitors as a promising new class of agents for immunotherapy, and medicinal chemistry efforts to design novel ascomycin analogs have produced clinically promising agents. A synthetic program to modify the ascomycin nucleus to alter its physicochemical properties and promote systemic clearance is described. A biologic screening strategy to identify analogs with reduced systemic activity and rapid phannacokinetic elimination led to identification of the clinical candidate, ABT-281. A swine contact hypersensitivity model was used as a stringent indicator of skin penetration as human doses of topical corticosteroids produced inhibition only in the 50% range and ED50 values were 100-fold less potent than in rat. Also, cyclosporine was confirmed to be topically inactive in swine, as seen in human. ABT-281 had topical potency equal to tacrolimus (FKS06) despit_e a severalfold lower potency for inhibiting swine T cells in vitro, consistent-with superior skin penetration. ABT-281 was found to have a shorter duration of action after i.v. dosing in monkeys using an ex vivo whole blood IL-2 production assay. Systemic potency was reduced by 30- fold or more in rat popliteal lymph node hyperplasia and contact hypersensitivity assays. Following i.v. or i.p. administration in the swine contact hypersensitivity model, ABT-281 was 19- and 61-fold less potent, respectively, than FK506. Pharmacokinetic studies showed that ABT-281 had a shorter half life and higher rate of clearance than FKS06 in all three species. The potent topical activity and reduced sytemic exposure of ABT- 281 may thus provide both efficacy and a greater margin of safety for topical therapy of skin diseases.

Ascomycin ( 1a ), a macrolactam antifungal antibiotic disclosed by Arai in 1962, 1 was found to display immunosuppressive activity more than 2 decades later by Okuhara and coworkers at Fujisawa. 2 Ascomycin ( 1a ) and FK506 ( 1b ) bind to a peptidyl-prolyl-isomerase, FKBP, a necessary but insufficient condition for drug activity. Both FK506 and ascomycin exist as a mixture of slowly interconverting cis and trans amide rotamers. It has also been shown that only the trans amide rotamer binds to FKBP. 24-epi-Ascomycin ( 3 ), 24-oxo-22-norascomyin ( 9a ), and 22-norascomycin ( 9b ), obtained by semisynthesis from ascomycin, exist as single rotamers on the NMR time scale. Their synthesis and the biological consequences of this observation are discussed.

Small acyclic model compounds were synthesized to examine the importance of the C(9) carbonyl group of KF506 for FKBP binding. 4-(4′-Methoxyphenyl)-1-butyl (3″, 4″, 5″-trimethoxyphenylglyoxyl) pipecolate (compound 2d), containing the N-(glyoxyl)pipecolate motif of FK506, was found to bind to FKBP with IC50 of 16 μM. Replacement of the carbonyl group at the position corresponding to C(9) of FK506 with small nonpolar groups (hydrogen, methylene or methyl group, compounds 2a–2c) significantly reduced the binding affinity.

Ascomycin (1) is a close analogue of the immunosuppressant FK-506 (2) which differs slightly in the side chain at position 21 (ethyl vs allyl). Structurally unique ascomycin and FK-506 are the subjects of intense chemical research because of their application in organ transplantation and autoimmune disease. We have been interested in studying the effect of structural modification and conformational change on biological activity. This paper reports the fermentation and structural determination of ascomycin as well as the synthesis and structure confirmation of a series of pyrazole analogues (3 and 4) derived from ascomycin.

The synthesis and structure-activity relationships of C-terminal octapeptide analogues of anaphylatoxin C5a have been studied. The introduction of hydrophobic amino acids into the N-acetylated native octapeptide (N-Ac-His-Lys-Asp-Met-Gln-Leu-Gly-Arg-OH) (1) has led to an analogue with 100 times more activity than the native octapeptide in inhibiting the binding of 125I-labeled anaphylatoxin C5a to human neutrophil membrane receptors. The observed apparent binding Ki's for the compounds (8-10) are in the range of 1-3 microM, and they possess nearly full agonist activity, despite the fact that these analogues are one-eighth or -ninth the size of the natural ligand anaphylatoxin C5a.

C5a is a 74 amino acid polypeptide that likely plays an important role in the pathogenesis of a number of inflammatory diseases. Therefore, the discovery of a C5a antagonist is of considerable therapeutic interest. A series of peptides designed to survey various regions of the molecule was synthesized by solid-phase peptide synthesis and evaluated for receptor-binding activity with polymorphonuclear leukocyte membranes. The C-terminal octapeptide (Ac-His-Lys-Asp-Met-Gln-Leu-Gly-Arg-OH) was identified as the smallest fragment which possessed reasonable C5a receptor binding activity.

A novel series of barbituric acid derivatives were identified as selective inhibitors of alpha4beta7 MAdCAM (mucosal addressin cell adhesion molecule-1) interactions via a high throughput screening exercise. These inhibitors were optimized to submicromolar potencies in whole cell adhesion assays, retaining their selectivity over alpha4beta1 VCAM.

A novel class of functional ligands for the human glucocorticoid receptor is described. Substituents in the C-10 position of the tetracyclic core are essential for glucocorticoid receptor (GR) selectivity versus other steroid receptors. The C-5 position is derivatized with meta-substituted aromatic groups, resulting in analogues with a high affinity for GR (K(i) = 2.4-9.3 nM) and functional activity comparable to prednisolone in reporter gene assays of glucocorticoid-mediated gene transcription. The biological activity of these novel quinolines was also prednisolone-equivalent in whole cell assays of glucocorticoid function, and compound 13 was similar to prednisolone (po ED(50) = 2.8 mpk for 13 vs ED(50) = 1.2 mpk for prednisolone) in a rodent model of asthma (sephadex-induced eosinophil influx).

Ascomycin 2, a close analogue of the immunosuppressant FK506 1, was modified to incorporate a hydroxyl group at the C-33 position. This increased the aqueous solubility of ascomycin by a hundred-fold at pH 7.4 and by approximately 300-fold at pH 6.5. Ascomycin 3 also exhibited an excellent immunosuppressive activity in vitro, as tested in a human mixed lymphocyte proliferation (HuMLR) assay, and in vivo using a rat popliteal lymph node (rPLN) hyperplasia assay.

Stereoselective syntheses of several nonpeptide fragments that function as Leu10-Val11 scissile bond replacements in human angiotensinogen are presented. The opening of N-protected aminoalkyl epoxide 3 with a variety of sulfur, oxygen, nitrogen, and carbon nucleophiles is a key reaction in the preparation of these novel fragments 4-8. The coupling of these fragments to protected dipeptides that mimic positions 8 and 9 in angiotensinogen produces inhibitors of human renin even though the molecules contain no functionality beyond what is formally the Val11 side chain of angiotensinogen. R groups that closely resemble that of the Val side chain are preferable; thus, isopropyl greater than or equal to higher alkyl greater than phenyl greater than substituted phenyl. Sulfur is the best X group; oxidation leads to slight (X = SO2) and significant (X = SO) decreases in inhibitory potency. One such inhibitor, 60, has an IC50 of 13 nM when tested with purified human renin at pH 6.0. The significant activity of these small inhibitors is thought to be due in part to the hydroxyl group of the fragment functioning as a transition-state analogue. Of these, the inhibitors that contain histidine show marked selectivity toward renin over a related aspartic proteinase, pepsin.

Stereoselective syntheses of several nonpeptide sulfidoethanol fragments that function as Leu10-Val11 (P1-P1') scissile bond replacements in human angiotensinogen are presented. These fragments are prepared from a variety of amino acids with formal P1 side chains varying in size and lipophilicity by converting them to their corresponding N-protected aminoalkyl epoxide 5 followed by ring opening with isopropyl mercaptan. The coupling of these fragments to either Boc-Phe-Ala-OH or Boc-Phe-His-OH produces inhibitors of human renin, 6 and 7, respectively, which are compared to a series of dipeptide-aldehyde inhibitors, 4, by molecular modeling and biochemical methods. Qualitatively, histidine-containing (P2) inhibitors 7 possess greater inhibitory potency than their corresponding alanine (P2) analogues 6, which are more potent than the corresponding aldehydic inhibitors from series 4. Within a given series, inhibitors with the cyclohexylmethyl P1 side chain are more potent than the benzyl analogues, which in turn are more potent than cyclohexyl or isobutyl derivatives. Inhibitors with parger P1 side chains (e.g. adamantylmethyl and benzhydryl) are much less active. The inhibitory potency of these compounds against human renin is discussed in terms of specific interactions with the enzyme.