The discovery of sulfonylated dipeptides as potent VLA-4 antagonists.

Directed screening of a carboxylic acid-containing combinatorial library led to the discovery of potent inhibitors of the integrin VLA-4. Subsequent optimization by solid-phase synthesis afforded a series of sulfonylated dipeptide inhibitors with structural components that when combined in a single hybrid molecule gave a sub-nanomolar inhibitor as a lead for medicinal chemistry. Preliminary metabolic studies led to the discovery of substituted biphenyl derivatives with low picomolar activities. SAR and pharmacokinetic characterization of this series are presented.

Substituted 2-aminopyridines as inhibitors of nitric oxide synthases.

A series of substituted 2-aminopyridines was prepared and evaluated as inhibitors of human nitric oxide synthases (NOS). 4,6-Disubstitution enhanced both potency and specificity for the inducible NOS with the most potent compound having an IC50 of 28 nM.

Structural requirements for human inducible nitric oxide synthase substrates and substrate analogue inhibitors.

Inducible nitric oxide synthase (iNOS; EC 1.14.13.39) catalyzes the NADPH-dependent oxidation of one of the free guanidino nitrogens of L-Arg to form nitric oxide and L-citrulline. Analogues of L-Arg and the inhibitor, L-N6-(1-iminoethyl)lysine, were used to define structural elements required for the binding and catalysis of compounds. L-Arg analogues with sequentially shorter methylene spacing between the guanidino group and the amino acid portion of the molecule were not iNOS substrates but were reversible inhibitors. L-Arg analogues such as agmatine with a hydroxyl substitution at the 2-amino position were substrates. Desaminoarginine was not a substrate but a reversible inhibitor. Desaminoarginine, agmatine, and argininic acid bound to the enzyme to give type I difference spectra similar to that of L-Arg. The amidino compounds L-N6-(1-iminoethyl)lysine, L-N5-(1-iminoethyl)ornithine, and N5-(1-iminoethyl)cadaverdine, but not N6-(1-iminoethyl)-6-aminocaproic acid, were NADPH-dependent, irreversible inactivators of iNOS. For both the L-Arg and L-N6-(1-iminoethyl)lysine analogues, the 2-amino group appeared to play an important role in catalytic events leading to either substrate turnover or mechanism-based inactivation. Inactivation of iNOS by L-N6-(1-iminoethyl)lysine was NADPH- and dioxygen-dependent, but low incorporation of radiolabel with DL--4, 5-3H]-N6-(1-iminoethyl)lysine indicates that the mechanism of enzyme inactivation is not covalent modification of the protein.

Inhibition of stromelysin-1 (MMP-3) by P1'-biphenylylethyl carboxyalkyl dipeptides.

Carboxyalkyl peptides containing a biphenylylethyl group at the P1' position were found to be potent inhibitors of stromelysin-1 (MMP-3) and gelatinase A (MMP-2), in the range of 10-50 nM, but poor inhibitors of collagenase (MMP-1). Combination of a biphenylylethyl moiety at P1', a tert-butyl group at P2', and a methyl group at P3' produced orally bioavailable inhibitors as measured by an in vivo model of MMP-3 degradation of radiolabeled transferrin in the mouse pleural cavity. The X-ray structure of a complex of a P1-biphenyl inhibitor and the catalytic domain of MMP-3 is described. Inhibitors that contained halogenated biphenylylethyl residues at P1' proved to be superior in terms of enzyme potency and oral activity with 2(R)-[2-(4'-fluoro-4-biphenylyl)ethyl]-4(S)-n-butyl-1,5-pentane dioic acid 1-(alpha(S)-tert-butylglycine methylamide) amide (L-758,354, 26) having a Ki of 10 nM against MMP-3 and an ED50 of 11 mg/kg po in the mouse pleural cavity assay. This compound was evaluated in acute (MMP-3 and IL-1 beta injection in the rabbit) and chronic (rat adjuvant-induced arthritis and mouse collagen-induced arthritis) models of cartilage destruction but showed activity only in the MMP-3 injection model (ED50 = 6 mg/kg iv).

Inhibition of matrix metalloproteinases by N-carboxyalkyl peptides.

An extensive study of the requirements for effective binding of N-carboxyalkyl peptides to human stromelysin, collagenase, and to a lesser extent, gelatinase A has been investigated. These efforts afforded inhibitors generally in the 100-400 nM range for these matrix metalloproteinases. The most significant increase in potency was obtained with the introduction of a beta-phenylethyl group at the P1' position, suggesting a small hydrophobic channel into the S1' subsite of stromelysin. One particular compound, N-[1(R)-carboxyethyl]-alpha(S)-(2-phenylethyl)glycyl-L-leucine,N- phenylamide (79a), is relatively selective for rabbit stromelysin with a K(i) = 6.5 nM and may prove useful for elucidating the role of endogenously-produced stromelysin in lapine models of tissue degradation.

Substituted 4,6-diaminoquinolines as inhibitors of C5a receptor binding.

The anaphylatoxin C5a is implicated in a number of inflammatory diseases. It is a highly cationic protein with 13 of 74 amino acids being either arginine or lysine. A search focusing on positively charged molecules, particularly amine-containing functionalities, led to the discovery of substituted 4,6-diaminoquinolines 1 [N,N'-bis(4-amino-2-methyl-6-quinolyl)urea] and 7 [6-N-(2-chlorocinnamoyl)-4,6-diamino-2-methylquinoline] as inhibitors of C5a receptor binding. These two compounds inhibited the binding of radiolabeled C5a to its receptor isolated from human neutrophils with IC50's = 3.3 and 12 micrograms/mL, respectively. Our efforts to enhance their potencies by chemical modification revealed a narrow profile of potency for effective C5a receptor binding inhibition.

Conformation of MeAla6-cyclosporin A by NMR. Relationship of sidechain orientation of the MeBmt-1, MeLeu-9, and MeLeu-10 residues to immunosuppressive activity.

MeAla6-cyclosporin A (MeAla6-CsA) is a unique CsA analog that shows weak immunosuppressive activity and yet binds strongly to the proposed cytosolic protein receptor, cyclophilin (CyP). Preliminary 1H NMR data showed significant chemical shift differences between spectra of MeAla6-CsA and CsA, suggesting different preferred conformations. A more detailed study, however, revealed that the backbone conformations of the two molecules are essentially identical, and that the differences can be accounted for, principally, by the sidechain motions of the MeBmt-1, MeLeu-9, and -10 residues. ROE and coupling constant data show that in MeAla6-CsA, the preferred chi 1 rotamers for MeLeu-9 and -10 are + 180 degrees (T), whereas in CsA there is a more even distribution of rotamer populations for MeLeu-10, and a preferred -60 degrees (G-) chi 1 rotamer for MeLeu-9. Similar data argue that the sidechain of MeBmt-1 is more restricted in its motion in MeAla-CsA than in CsA. Temperature studies suggest that these preferred rotamers for MeAla6-CsA may increase the stability of the hydrogen bond between NH(7) and CO(11), but prevent particular residues, especially the essential MeBmt-1 sidechain, from adopting orientations required to elicit immunosuppressive activity. The significant changes observed in the preferred orientations for the sidechains of the MeBmt-1, MeLeu-9, and MeLeu-10 residues in MeAla6-CsA argue that the particular orientations which they assume in CsA are not essential for cyclophilin binding.

Unexpected up-regulation of gene expression by cyclosporin A and FK-506 in a T-cell lymphoma: both immunosuppressants augment Ly-6E antigen induction by interferon-gamma in the presence of ionomycin.

Cyclosporin A (CsA) and FK-506 inhibit lymphokine gene activation in T-cells. In the present study, we investigated the effects of these immunosuppressants on the regulation of a non-lymphokine molecule, the Ly-6E surface antigen, in the YAC-1 T-cell lymphoma. These cells do not normally express Ly-6E mRNA or Ly-6E surface molecules but are induced to do so upon treatment with IFN-gamma. At submicromolar concentrations, CsA or FK-506 did not alter this induction. However, at higher concentrations (1-12 microM), they both increased the induction of Ly-6E mRNA expression. Cyclosporin A or FK-506 also markedly affected Ly-6E induction when the cultures were co-treated with the calcium ionophore, ionomycin. In the absence of CsA or FK-506, ionomycin suppressed Ly-6E induction by IFN-gamma. Both immunosuppressants reversed this inhibitory effect and increased Ly-6E mRNA and Ly-6E surface expression to levels that were 2- to 3-fold higher than in cells induced with IFN-gamma alone. In this system, the two immunosuppressants were active at pharmacologically relevant concentrations, similar to those inhibiting normal T-cell activation, with FK-506 being 30- to 50-fold more potent than CsA. The ability of CsA analogs to enhance Ly-6E induction in the presence of ionomycin also correlated with their immunosuppressive activity. Therefore, through mechanisms apparently related to those involved in their immunosuppressive action, both CsA and FK-506 convert the negative effect of ionomycin on IFN-gamma-mediated Ly-6E induction into an overall positive effect. The YAC-1 cell model, described here, provides a unique example of upregulation of gene expression by these two immunosuppressants.

A study of the correlation between cyclophilin binding and in vitro immunosuppressive activity of cyclosporine A and analogues.

In order to establish whether CyP is the pharmacologically relevant CsA receptor, the CyP binding v immunosuppressive activity was measured for an extensive, structurally varied group of CsA analogues. Overall, CyP binding was found to parallel immunosuppressive activity. Other than MeAla6-CsA, the few exceptions to the correlation could be ascribed to cellular metabolism. These results strongly implicate CyP or a related protein in the mechanism of action of cyclosporine.

Pharmacology of the dual inhibitor of cyclooxygenase and 5-lipoxygenase 3-hydroxy-5-trifluoromethyl-N-(2-(2-thienyl)-2-phenyl-ethenyl)-benzo (b)thiophene-2-carboxamide.

3-Hydroxy-5-trifluoromethyl-N-(2-(2-thienyl)-2-phenyl-ethenyl)- benzo (b) thiophene-2-carboxamide (L-652,343) is an inhibitor of cyclooxygenase and 5-lipoxygenase in vitro and inhibits the synthesis of the products of both these pathways in whole cells. L-652,343 is an inhibitor of the acute edema induced by carrageenan in vivo and is active topically in suppressing arachidonic acid induced inflammation in the skin. The compound is an effective inhibitor of the chronic inflammation of adjuvant and type II collagen induced polyarthritis. L-652,343 is an extremely potent analgesic in models of yeast and platelet activating factor induced hyperalgesia in rats and phenylbenzoquinone-induced writhing in mice. The fever induced by Brewer's yeast is lowered by L-652,343. The ulcerogenicity and gastric bleeding induced by L-652,343 is extremely low, providing a favorable therapeutic index which is superior to that of indomethacin, piroxicam and phenylbutazone.

Synthesis and immunoadjuvant activities of 2-acetamido-5-O-acetyl-6-O-acyl-2-deoxy-3-O-[(R)-2-propionyl-L-alanyl-D- isoglutamine]-D-glucofuranoses as potential prodrug forms of 6-O-Acyl Derivatives of N-acetylmuramyl dipeptide.

2-Acetamido-5-O-acetyl-6-O-acyl-2-deoxy-3-O-[(R)-2-propionyl-L-alanyl-D- isoglutamine]-D-glucofuranoses, designed as prodrug forms of the corresponding immunoadjuvant-active 6-O-acyl derivatives of N-acetylmuramyl dipeptide (MDP), were synthesized from benzyl 2-acetamido-2-deoxy-5, 6-O-isopropylidene-beta-D-glucofuranoside and found, when administered to mice in an aqueous medium, to elevate antibody production against bovine serum albumin. The 5,6-di-O-acetyl derivative 8 exhibited activity similar to that of MDP at 50 micrograms/dose. The antibody titer measured for the 5-O-acetyl-6-O-stearoyl compound 9 was comparable to that obtained with 6-O-stearoyl-MDP at 50 micrograms, and both compounds were more active than MDP at 5 micrograms. The more lipophilic 5-O-acetyl-6-O-[2-(behenoyloxy)isobutyryl] compound 10 was considerably more active than MDP at both 50 and 5 micrograms; moreover, its potent adjuvant activity was not diminished at the lower dose. The three 5-O-acetylated 6-O-acylated dipeptidyl furanose derivatives also significantly stimulated production of circulating antibodies against hepatitis B vaccine in mice; titers were comparable to those observed with the alum-adsorbed vaccine. The range of immunoadjuvant activities obtained with 8-10 and control compounds supports a prodrug mechanism for this class of furanoid MDP analogues.

Synthesis of 2-methyl-[2-acetamido-4-O-acetyl-6-O-benzyl-3-O-(2-butenyl)-1,2-dideoxy-alpha-D -glucopyrano]-[2,1-d]-2-oxazoline, a versatile intermediate for the synthesis of complex oligosaccharides of bacterial cell-wall, human milk, and blood-group substances.

2-Methyl-[2-acetamido-4-O-acetyl-6-O-benzyl-3-O-(2-butenyl)-1,2-dideoxy-alpha-D-glucopyrano]-[2,1-d]-2-oxazoline (2), a glycosylating agent in which the three hydroxyl groups are blocked with protecting groups of differing "persistence", is of utility in the synthesis of oligosaccharides containing highly branched 2-acetamido-2-deoxy-D-glucosyl residues, and it was synthesized in a ten-step sequence from 2-acetamido-2-deoxy-D-glucose via allyl 2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranoside (3). Alkylation of 3 with 2-butenyl (crotyl) bromide, hydrolysis of the benzylidene acetal group, benzylation of the 6-hydroxyl group, and acetylation of the 4-hydroxyl group afforded allyl 2-acetamido-4-O-acetyl-6-O-benzyl-3-O-(2-butenyl)-2-deoxy-beta-D-glucopyranoside(10). Treatment of 10 with chlorotris(tri-phenylphosphine)rhodium(I) gave mainly the corresponding 1-propenyl beta-glycoside, which was converted into oxazoline 2 by the action of mercuric chloride-mercuric oxide in acetonitrile. Glycosylation of benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-alpha-D-glucopyranoside with 2, and subsequent O-deacetylation at O-4' gave a glycosyl acceptor, benzyl 2-acetamido-4-O-[2-acetamido-6-O-benzyl-3-O-(2-butenyl) -2-deoxy-beta-D-glucopyranosyl]-3,6-di-O-benzyl-2-deoxy-alpha-D-glucopyranoside .

Insulin-like, and insulin-antagonistic, carbohydrate derivatives. The synthesis of aryl and aralkyl D-mannopyranosides and 1-thio-D-mannopyranosides.

A number of novel, aryl and aralkyl D-mannopyranosides and 1-thio-D-mannopyranosides were synthesized for evaluation of insulin-like and insulin-antagonistic properties. The substituted-phenyl alpha-D-mannopyranosides were prepared by the general procedure of Helferich and Schmitz-Hillebrecht, the substituted-phenyl 1-thio-alpha-D-mannopyranosides by a method corresponding to the Michael synthesis of aromatic glycosides, and the aralkyl 1-thio-alpha-D-mannopyranosides by aralkylation of 2,3,4,6-tetra-O-acetyl-1-thio-alpha-D-mannopyranose (15) and subsequent O-deacetylation. Compound 15 was obtained by basic cleavage of the amidino group in 2-S-(tetra-O-acetyl-alpha-D-mannopyranosyl)-2-thiopseudourea hydrobromide, the product of the reaction of tetra-O-acetyl-alpha-D-mannosyl bromide with thiourea. Benzyl 1-thio-beta-D-mannopyranoside, obtained by reaction of the sodium salt of 1-thio-beta-D-mannopyranose with alpha-bromotoluene, and benzyl 1-thio-alpha-L-mannopyranoside were also synthesized, in order to assess the stereospecificity of the biological activities measured.

Structure-activity relationships of aminoalkyl and -aryl glycosides having insulin-like activity.

A number of alkyl, aryl, and aralkyl glycosides (mono- and disaccharides) substituted in the aglycon with a primary amino group have been found to exert insulin-like activity on rat adipocytes in vitro. Systematic variations in the saccharide configuration, glycosidic linkage, aglycon moiety, and sugar substitution pattern were investigated to delineate structure-activity relationships. A high degree of structural specificity was observed. Maximal insulin mimicking activity was obtained with the 6-aminohexyl 1-thio-D-mannopyranosides; the beta anomer was more active than the alpha anomer. Modification of the sugar hydroxyl groups resulted, in most cases, in partial or complete loss of biological activity at the levels tested; however, in a few instances, sugar-modified derivatives did show enhanced insulin-like effects. Specific structural types evaluated are discussed in greater detail. 6-Aminohexyl 1-thio-beta-D-mannopyramoside also exhibited in vivo insulin-like effects on both diaphragm muscle and omental adipose tissues. The specificities for the sugar as well as the aglycon portions of these carbohydrate derivatives suggest that both parts of the molecule are involved in the expression of the full biological activity observed; their respective roles in the mechanism of the insulin-like activity are discussed.