A macrolactam inhibitor of T helper type 1 and T helper type 2 cytokine biosynthesis for topical treatment of inflammatory skin diseases.

T lymphocytes play a critical part in inflammatory skin diseases but are targeted by available therapies that have only partial efficacy, significant side-effects, or both. Because psoriasis, atopic dermatitis, and allergic contact hypersensitivity are associated with T helper type 1 (Th1), T helper type 2 (Th2), or mixed Th1-Th2 cell subsets and cytokine types, respectively, there is a need for a better broad-based inhibitor. The macrolactam ascomycin analog, ABT-281, was found to inhibit potently T cell function across species and to inhibit expression of multiple cytokines in human peripheral blood leukocytes which have been found in human skin disease cells and tissues. These included immunoregulatory Th1 (interleukin-2 and interferon-gamma) and Th2 (interleukin-4 and interleukin-5) cytokines. ABT-281 was shown to have potent topical activity (ED50 = 0.6% in acetone/olive oil) in a stringent swine model of allergic contact hypersensitivity, but its potency was markedly reduced compared with ascomycin when administered systemically due to more rapid clearance. Topical application of 3% ABT-281 in acetone/olive oil over 25% of the body surface in swine resulted in undetectable blood levels. Compared with a wide potency range of topical corticosteroids in clinical formulations, 0.3% and 1% ABT-281 ointments profoundly inhibited dinitrochlorobenzene-induced contact hypersensitivity in the pig by 78% and 90%, respectively, whereas super-potent steroids such as clobetasol propionate only inhibited in the 50% range and mild to moderate potency steroids such as fluocinolone acetonide were inactive. The potent topical activity of ABT-281 in swine, its superior efficacy, its rapid systemic clearance following uptake into the bloodstream, and its ability to inhibit cytokine biosynthesis of both Th1 and Th2 cell subsets, suggests that it will have a broad therapeutic value in inflammatory skin diseases, including psoriasis, atopic dermatitis, and allergic contact dermatitis.

High-level expression of a gene encoding the human complement factor C5a in Escherichia coli.

The synthetic C5a gene was initially found to be expressed poorly in Escherichia coli. We undertook studies to determine the reasons for poor expression and to increase expression. The work was focused on the role of the mRNA structure in C5a expression and stability of its product in E. coli. We present data on the effects of varying the sequence at the 5' end of mRNA as well as different ribosome-binding sites on expression. Evaluation of the stability of C5a showed rapid degradation of C5a in wild-type E. coli (half-life 3-5 min). Screening of several protease-deficient strains of E. coli showed that C5a was much more stable in an htpR strain carrying a mutation in the sigma subunit of RNA polymerase that is specific for heat shock promoters. The mutation is associated with a proteolytic deficiency. The half-life of C5a was increased to 20 min. By manipulating the expression vector, the regulatory region for the C5a gene, the host strain, growth conditions and methods for recovering the protein, C5a levels were increased 300-fold over previously reported amounts to about 3% of total cellular protein.

Structure-activity profiles of macrolactam immunosuppressant FK-506 analogues.

The immunosuppressive agent FK-506 has received much attention due to its efficacy and potency in the areas of transplant rejection and autoimmune disease. Calcineurin, a Ca(2+)-calmodulin activated phosphatase, was recently implicated in the immunosuppressive mechanism of FK-506. In our ongoing search for superior immunosuppressive agents, we have synthesized several analogues of FK-506 and tested their mechanistic and immunosuppressive actions. It was found that C-18 hydroxyl analogues of ascomycin, an analogue of FK-506 also called FR900520, bound tightly to immunophilin FKBP-12, but do not show any immunosuppressive activity in vitro or in vivo despite good bioavailability. Further, they reverse the inhibition of calcineurin caused by FK-506/FKBP-12 complex.

Glycerides as prodrugs. 2. 1,3-Dialkanoyl-2-(2-methyl-4-oxo-1,3-benzodioxan-2-yl)glycerides (cyclic aspirin triglycerides) as antiinflammatory agents.

A series of 1,3-dialkanoyl-2-(2-methyl-4-oxo-1,3-benzodioxan-2-yl)glycerides ("cyclic aspirin triglycerides") was synthesized. They demonstrated essentially all the systemic antiinflammatory activity associated with aspirin in the carrageenin-induced rat paw edema test. Examination of the rat stomachs showed that the 1,3-didecanoyl derivative did not cause gastric lesions.

Glycerides as prodrugs. 3. Synthesis and antiinflammatory activity of [1-(p-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetyl]glycerides (indomethacin glycerides).

Mono-, bis-, and tris[1-(p-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetyl]glycerides and 1,3-dialkanoyl-2-[1-(p-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetyl]glycerides were synthesized and evaluated for antiinflammatory activity in the rat paw carrageenin edema assay. Three of the most active compounds (4, 18a, and 18e) were tested in the rat adjuvant arthritis model and found to be essentially equivalent in activity to indomethacin. On a molar basis, the acute gastric irritating properties of 18a and 18e were seven to eight times less than indomethacin, resulting in a 2.5- to 3-fold improvement in the ratio of antiedema activity to ulcerogenicity.

Glycerides as prodrugs. 1. Synthesis and antiinflammatory activity of 1,3-bis(alkanoyl)-2-(O-acetylsalicyloyl)glycerides (aspirin triglycerides).

A series of 1,3-bis(alkanoyl)-2-(O-acetylsalicyloyl)glycerides (aspirin triglycerides) having aspirin at the 2 position of glycerol and fatty acids at the 1 and 3 positions was prepared. The compounds were administered orally and tested for efficacy in the rat paw edema test, and the stomachs were examined for the presence of lesions. The results showed that the members of this series in which the fatty acids are of intermediate chain length (C4-C12) do not cause gastric lesions and have essentially all the systemic activity associated with aspirin.

Identification and synthesis of a receptor binding site of human 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.

Hydroxamic acid inhibitors of 5-lipoxygenase.

The hydroxamic acid functionality can be incorporated in a variety of simple molecules to produce potent inhibitors of 5-lipoxygenase. As an example of this, the structure-activity relationships in a series of omega-phenylalkyl and omega-naphthylalkyl hydroxamic acids are presented. Among the features described are the influence of hydrophobicity, aryl substitution, and modifications of the hydroxamate group on enzyme inhibitory potency. To assist in the selection of more potent hydroxamic acid inhibitors, a simple hypothesis about the nature of enzyme-inhibitor binding was devised. In this hypothesis, the structures of compounds were matched to a proposed geometry of arachidonic acid when bound to the enzyme. Compounds that match best without extending into disfavored regions were predicted to be the best inhibitors. Three series of hydroxamates selected according to this approach are described. Within these series are some of the most potent inhibitors of 5-lipoxygenase reported to date.

Synthesis and 5-lipoxygenase inhibitory activity of 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid analogues.

A series of eicosatetraenes (2-24) were designed, synthesized, and evaluated in vitro for inhibitory activity against 5-lipoxygenase (20000g supernatant from homogenized rat basophilic leukemia cells). All compounds were found to be active with the potencies (IC50's) ranging from 0.19 to 97 microM. Compounds containing the hydroxamic acid functionality (10-12) exhibited the best activity (IC50 = 0.19-2.8 microM). The most potent inhibitor was 5-[(hydroxyamino)carbonyl]methyl]-6,8,11,14-eicosatetraenoic acid (11), which was 10 times more active than the C-1 hydroxamates of arachidonic acid or 5-HETE. Cyclization of the linear eicosanoids 2 and 14 in the C-1 to C-5 region produced compounds (21 and 24, respectively) with several-fold greater potency.

Structure-function studies in a series of carboxyl-terminal octapeptide analogues of anaphylatoxin C5a.

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.

Structural requirements for the inhibition of 5-lipoxygenase by 15-hydroxyeicosa-5,8,11,13-tetraenoic acid analogues.

The structural requirements for inhibition of RBL-1 (rat basophilic leukemia) 5-lipoxygenase by 15-hydroxyeicosa-5,8,11,13-tetraenoic acid (15-HETE, 1) were studied by systematic chemical modifications of the molecule at the hydroxyl and carboxyl groups, the double bonds, and the carboxylate and omega side chains. The most potent inhibitors were analogues that contained a 5,8-cis,cis-diene system and acted as alternate substrates for the enzyme. However, several analogues in which the 5,8-diene had been reduced were also found to inhibit the enzyme. Inhibition of 5-lipoxygenase by 15-hydroxyeicosa-11,13-dienoic acid (15-HEDE) analogues was optimal in compounds that generally contained a free carboxyl group, a carboxylate side chain of nine carbons, an omega side chain of five or six carbons, a cis,trans- or trans,cis-11,13-diene or 11,13-diyne system, and a 15-hydroxyl group. Conversion of 15-HEDE to its 16-membered lactone reduced but did not eliminate 5-lipoxygenase inhibitory activity. In contrast, a 3- to 10-fold enhancement of activity occurred when 5,15-diHETE (58) or 5-HETE (56) were cyclized to their respective delta-lactones. Molecular modeling of 15-HEDE analogues, modified in the C11-C15 region, showed that inactive analogues protrude into regions in space not occupied by active analogues. These structural studies indicate that multiple regions are important for 5-lipoxygenase inhibition by both 15-HETE and 15-HEDE analogues and that no single region plays a predominant role in inhibition.

Nonsteroidal anti-inflammatory drugs as scaffolds for the design of 5-lipoxygenase inhibitors.

Representative nonsteroidal anti-inflammatory drug (NSAID) cyclooxygenase inhibitors such as ibuprofen, naproxen, and indomethacin were used as orally bioavailable scaffolds to design selective 5-lipoxygenase (5-LO) inhibitors. Replacement of the NSAID carboxylic acid group with a N-hydroxyurea group provided congeners with selective 5-LO inhibitory activity.

Hydroxamic acid inhibitors of 5-lipoxygenase: quantitative structure-activity relationships.

An evaluation of the quantitative structure-activity relationships (QSAR) for more than 100 hydroxamic acids revealed that the primary physicochemical feature influencing the in vitro 5-lipoxygenase inhibitory potencies of these compounds is the hydrophobicity of the molecule. A significant correlation was observed between the octanol-water partition coefficient of the substituent attached to the carbonyl of the hydroxamate and in vitro inhibitory activity. This correlation held for hydroxamic acids of diverse structure and with potencies spanning 4 orders of magnitude. Although the hydrophobicity may be packaged in a variety of structural ways and still correlate with potency, the QSAR study revealed two major exceptions. Specifically, the hydrophobicity of portions of compounds in the immediate vicinity of the hydroxamic acid functionality does not appear to contribute to increased inhibition and the hydrophobicity of fragments beyond approximately 12 A from the hydroxamate do not influence potency. The QSAR study also demonstrated that inhibitory activity was enhanced when there was an alkyl group on the hydroxamate nitrogen, when electron-withdrawing substituents were present and when the hydroxamate was conjugated to an aromatic system. These observations provide a simple description of the lipoxygenase-hydroxamic acid binding site.

4-hydroxythiazole inhibitors of 5-lipoxygenase.

4-Hydroxythiazoles have been identified as potent inhibitors of 5-lipoxygenase in vitro exhibiting IC50's of less than 1 microM. An investigation of structure-activity relationships showed that the most potent inhibitors of this series are the 5-phenyl derivatives. The corresponding thiazolidin-4-one analogues were found to be relatively inactive. The 4-hydroxythiazoles were active inhibitors against 5-lipoxygenase in both intact rat polymorphonuclear leukocytes and human whole blood. The compounds were also selective inhibitors of 5-lipoxygenase, displaying only weak activity against other related enzymes, cyclooxygenase and 12- and 15-lipoxygenase.

Synthesis and structure-activity relationships of a series of novel benzopyran-containing platelet activating factor antagonists.

A class of N-substituted tetrahydrobenzopyrano[3,4-c]pyridines, I, have been identified as antagonists of platelet activating factor (PAF). The structural features essential for PAF binding were determined by systematic modification of three sites in the molecule. While O-alkyl analogues had little effect on binding potency, N-alkyl analogues exhibited a wide range of activity. Structural changes in the core ring system generally resulted in a loss of binding activity. Optimization of the N- and O-substituents resulted in the analogues 25-27 which exhibited Ki values ranging between 131 and 167 nM in a [3H]PAF binding assay. Compound 23 was also active in a model of PAF-induced shock in the mouse following intravenous administration.

2-[(Phenylthio)methyl]pyridine derivatives: new antiinflammatory agents.

2-[(Phenylthio)methyl]pyridine derivatives inhibited the dermal reverse passive Arthus reaction (RPAR) in the rat. In the same model, indomethacin was inactive, and hydrocortisone was active. Compounds Ia-d also significantly reduced exudate volume and white blood cell accumulation in the pleural RPAR. This pattern of activity was similar to that of hydrocortisone and different from that of indomethacin.

3-[1-(2-Benzoxazolyl)hydrazino]propanenitrile derivatives: inhibitors of immune complex induced inflammation.

3-[1-(2-Benzoxazolyl)hydrazino]propanenitrile derivatives were evaluated in the dermal and pleural reverse passive Arthus reactions in the rat. In the pleural test these compounds were effective in reducing exudate volume and accumulation of white blood cells. This pattern of activity was similar to that of hydrocortisone and different from that of indomethacin. The structural requirements for inhibiting the Arthus reactions were studied by systematic chemical modification of 1. These structure-activity relationship studies revealed that nitrogen 1' of the hydrazino group is essential for activity and must be electron rich, whereas chemical modifications of other sites of 1 had only a modest effect on activity.

In vivo characterization of hydroxamic acid inhibitors of 5-lipoxygenase.

The hydroxamic acid functionally can be incorporated into simple molecules to produce potent inhibitors of 5-lipoxygenase. The ability of many of these hydroxamates to inhibit leukotriene synthesis in vivo has been measured directly with a rat peritoneal anaphylaxis model. Despite their potent enzyme inhibitory activity in vitro, many orally dosed hydroxamic acids only weakly inhibited leukotriene synthesis in vivo. This discrepancy is attributable at least in part to the rapid metabolism of hydroxamates to the corresponding carboxylic acids, which are inactive against the enzyme. A study of the structural features that affect this metabolism revealed that 2-arylpropionohydroxamic acids are relatively resistant to metabolic hydrolysis. Several members of this class of hydroxamates are described that are orally active inhibitors of leukotriene synthesis.

5-Lipoxygenase inhibitors: synthesis and structure-activity relationships of a series of 1-aryl-2H,4H-tetrahydro-1,2,4-triazin-3-ones.

Synthetic routes were developed to access a variety of novel 1-aryl-2H,4H-tetrahydro-1,2,4-triazin-3-one analogs which were evaluated as 5-lipoxygenase (5-LO) inhibitors. The parent structure, 1-phenylperhydro-1,2,4-triazin-3-one (4), was found to be a selective inhibitor of 5-LO in broken cell, intact cell, and human blood assays with IC50 values of 5-21 microM. In a rat anaphylaxis model, 4 blocked leukotriene formation with an ED50 = 7 mg/kg when administered orally. Compound 4 exhibited selectivity for inhibition of 5-LO with little activity against related enzymes: 12-LO from human platelets, 15-LO from soybean, and cyclooxygenase (COX) from sheep seminal vesicle. In pilot subacute toxicity testing, 4 did not produce methemoglobinemia in rats (400 mg/kg po daily for 9 days) or in dogs (200 mg/kg po daily for 28 days). These results indicated that the triazinone structure provided a 5-LO inhibitor template devoid of the toxicity problems observed in the related phenidone (1) and pyridazinone (3) classes of 5-LO inhibitors. The parent compound 4 is a selective, orally bioavailable 5-LO inhibitor which can serve as a useful reference standard for in vivo pharmacological studies involving leukotriene-mediated phenonmena.

N-(acyloxyalkyl)pyridinium salts as soluble prodrugs of a potent platelet activating factor antagonist.

Pyrrolothiazole 4 is a potent antagonist of platelet activating factor-mediated effects in a variety of in vitro and in vivo assays. Despite its positive activity in models of inflammation and septic shock, 4 lacks the aqueous solubility necessary for intravenous administration. This deficit was overcome by conversion of 4 to water-soluble pyridinium prodrugs. A two-step procedure was used to prepare a series of N-(acyloxyalkyl)pyridinium salts, all of which exhibited aqueous solubility of greater than 20 mg/mL. The rate of conversion of these prodrugs to 4 was faster in human plasma than in pH 7 aqueous buffer. This rate difference was shown to be due to serum enzymes since the conversion in plasma was significantly slower in the presence of a serine esterase inhibitor. A strong correlation between prodrug structure and buffer/plasma half-life was established. The N-(acetyloxymethyl)pyridinium prodrug 11 (ABT-299) is currently undergoing clinical evaluation for the treatment of sepsis.