Design, synthesis, and SAR of heterocycle-containing antagonists of the human CCR5 receptor for the treatment of HIV-1 infection.

Replacement of the large hydantoin-indole moiety from our previous work with a variety of smaller heterocyclic analogues gave rise to potent CCR5 antagonists having binding affinity comparable to the hydantoin analogues. The synthesis, SAR, and biological profiles of this class of antagonists are described.

Discovery of human CCR5 antagonists containing hydantoins for the treatment of HIV-1 infection.

A series of hydantoin derivatives has been discovered as highly potent nonpeptide antagonists for the human CCR5 receptor. The synthesis, SAR, and biological profiles of this class of antagonists are described.

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.

Oleanane triterpenes from Junellia tridens.

Three novel triterpenes, 3,4-seco-olean-12-ene-3,28-dioic acid (4), 3alpha-hydroxyolean-11-en-28,13beta-olide (5), and 3alpha-hydroxyoleane-11:13(18)-dien-28-oic acid (6), were isolated from the aerial parts of the Argentinean shrub, Junellia tridens. Another five compounds-oleanolic (1), oleanonic (2), and epioleanolic acids (3), all biosynthetically related to the three new oleananes, and epibetulinic acid (7) and sitosterol (8)-were also isolated. Structures were elucidated primarily by 1D and 2D NMR and mass spectrometry, and all protons and carbons of the three novel compounds were fully assigned by NMR. We report the minimum inhibitory concentrations of these compounds against Mycobacterium tuberculosis and conclude that they are responsible for antitubercular activity originally observed in the crude plant extract. LC-MS data is provided on the occurrence of triterpenes 1-6 in six other species of Junellia.

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).

Comparison of the structure of human recombinant short form stromelysin by multidimensional heteronuclear NMR and X-ray crystallography.

Stromelysin-1 is a matrix metalloprotease that has been implicated in a number of degenerative diseases. Here we present the refined NMR solution structure of the catalytic domain of stromelysin-1 complexed with a small inhibitor and compare it to the X-ray crystal structure of the same complex. The structures are similar in global fold and show an unusual bottomless S1' subsite. There are differences, however, in the least well defined regions, Phe83-Ile89, His224-Phe232 and Pro249- Pro250, reflecting the lack of NOE data and large B-factors. The region His224-Phe232 contains residues of the S1' subsite and, consequently, small differences are observed in this subsite. Hydrogen-bond data show that, in contrast to the crystal structure, the solution structure lacks a hydrogen bond between the amide of Tyr223 and the carbonyl of the P3' residue. Analysis of bound water shows two tightly bound water molecules both in the solution and the crystal structure; neither of these waters are in the inhibitor binding site.

p-Nitrobenzyl side-chain protection for solid-phase synthesis.

A solid-phase supported peptide synthesis (SPPS) strategy using p-nitrobenzyl (pNB) esters, thioethers and carbamates for side-chain protection is described. The synthesis of Fmoc p-nitrobenzyl side-chain protected amino acids of lysine, cysteine, glutamic acid and aspartic acid are synthesized and incorporated into the synthesis of tetramers by standard Fmoc methodology using Wang polystyrene resins. Deprotection is carried out on resin in mildly acidic reducing conditions, using a solution of DMF, SnCl2, phenol and HOAc. The yellow by-products associated with the deprotection of the pNB protecting group are then removed by treatment with a solution of benzene sulfinic acid in DMF. The methodology is successfully extended in the synthesis of a peptide with multiple pNB protecting groups.

Glutathione-S-transferase activates novel alkylating agents.

Alkylating agents which are activated by glutathion-S-transferases (GSTs) have been designed and synthesized. The model compound gamma-glutamyl-alpha-amino-beta-[(2-ethyl N,N,N',N'-tetraethylphosphorodiamidate) sulfonyl]propionylglycine (1) and the nitrogen mustards gamma-glutamyl-alpha- amino-beta-[[2-ethyl N,N,N',N'-tetrakis (2-chloroethyl)phosphorodiamidate] sulfonyl]propionylglycine (2) and gamma-glutamyl-alpha-amino-beta-[[2-ethyl-N,N,N',N'-tetrakis(2- chloroethyl)phosphorodiamidate]sulfonyl]-propionyl-(R)-(-)-phenylg lycine (3) were prepared via multistep chemical synthesis. The compounds were tested with recombinant human A1-1, M1a-1a and P1-1 GSTs. HPLC studies showed that the compounds were differentially and catalytically cleaved by biologically relevant concentrations of the GSTs. Mass spectral studies of the cleavage mixture of 2 showed that M1a-1a GST liberated the cytotoxic phosphate moiety needed for efficacy as an alkylating agent. Cell culture studies with MCF-7 breast cancer cells showed that 1 was not toxic at 200 microM, while 2 and 3 showed IC50S of 40.6 and 37.5 microM, respectively, for the same cell line. MCF-7 cells transfected to overexpress P1-1 GST showed enhanced sensitivity with 2 and 3, with IC50S of 20.9 and 9.5 microM, respectively. This result correlates well with the rates of cleavage of 2 and 3 by P1-1 GST observed in vitro and demonstrates that higher levels of cellular P1-1 GST will give increased sensitivity to these drugs.

Isozyme-specific glutathione-S-transferase inhibitors: design and synthesis.

Glutathione-S-transferase (GST) isozyme-selective inhibitors were designed by an empirically guided strategy. In the first phase, literature data were used to select C-terminal modifications which generated maximum variation in the catalytic efficiency (Vmax/Km) for glutathione (GSH) analogs used as substrates with different rat GSTs. Also, on the basis of literature data, the sulfhydryl group was functionalized with a selection of alkyl and aryl groups to maximize potential isozyme specificity. Affinity chromatography sorbents were prepared from these which showed isozyme selectivity for both rat tissue and recombinant human GST isozymes. Some of these compounds also showed selective inhibition of GST activity in catalysis of the reaction of 1-chloro-2,4-dinitrobenzene with GSH. In the second phase, electronic effects were explored through synthesis of an isostructural series of S-benzyl GSH ligands with different substituents on the aromatic ring. GST isozyme specificity for these ligands, measured by binding to derivatized sorbents, varied substantially, with hydrophobic substituents favoring the human GST M1a isozyme and electronegative moieties favoring GST P1. In the third phase, information obtained from testing both series of compounds was combined and used to prepare GSH analogs with chemical features responsible for isozyme specificity at both the C-terminus and the sulfur. This approach gave two new compounds which showed improved potency while still maintaining selectivity in the inhibition of GSTs. A detailed discussion of the logic used in the selection of functional groups for maximum potency and selectivity is included.

Photoisomerization mechanism of the rhodopsin chromophore: picosecond photolysis of pigment containing 11-cis-locked eight-membered ring retinal.

The primary photochemical event in rhodopsin is an 11-cis to 11-trans photoisomerization of its retinylidene chromophore to form the primary intermediate photorhodopsin. Earlier picosecond studies have shown that no intermediate is formed when the retinal 11-ene is fixed through a bridging five-membered ring, whereas a photorhodopsin-like intermediate is formed when it is fixed through a flexible seven-membered ring. Results from a rhodopsin analog formed from a retinal with locked 11-ene structure through the more flexible eight-membered ring (Ret8) are described. Incubation of bovine opsin with Ret8 formed two pigments absorbing at 425 nm (P425) and 500 nm (P500). P425, however, is an artifact because it formed from thermally denatured opsin or other proteins and Ret8. Excitation of P500 with a picosecond green pulse led to formation of two intermediates corresponding to photo- and bathorhodopsins. These results demonstrate that an appearance of early intermediates is dependent on the flexibility of the 11-ene and that the photoisomerization of P500 proceeds by stepwise changes of chromophore-protein interaction, which in turn leads to a relaxation of the highly twisted all-trans-retinylidene chromophore in photorhodopsin.

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.

2,3-Dihydro-5-benzofuranols as antioxidant-based inhibitors of leukotriene biosynthesis.

The enzymes that catalyze the oxidative metabolism of arachidonic acid have provided fertile ground for the development of useful therapeutic agents for nearly a quarter century. Inhibitors of the enzyme cyclooxygenase prevent the formation of the prostaglandins and thromboxanes and are clinically useful antiinflammatories and peripheral analgesics. More recently it has been discovered that the enzyme 5-lipoxygenase is the first step in the formation of a series of biologically important metabolites of arachidonic acid, the leukotrienes. Evidence suggests that an inhibitor of 5-lipoxygenase may be a useful therapeutic agent in the treatment of asthma, immediate hypersensitivity, and inflammation. Various antioxidants have been examined as inhibitors of 5-lipoxygenase in vitro. We were intrigued by recent reports that the 2,3-dihydro-5-benzofuranol ring system maximizes the stereoelectronic effects necessary for efficient hydrogen atom abstraction by peroxyl radicals. In this study we describe the synthesis of over 50 new 2,3-dihydro-5-benzofuranols and their biological evaluation as inhibitors of leukotriene biosynthesis in isolated human polymorphonuclear leukocytes. We show that the 2,3-dihydro-5-benzofuranol ring system, although not a potent inhibitor of leukotriene biosynthesis in itself, can provide a useful template for the design of antioxidant-based inhibitors of leukotriene biosynthesis. Furthermore, within a structural class the potency of a given analogue can be predicted on the basis of its overall calculated lipophilicity (log P). The data are interpreted in terms of a model in which the observed inhibition by this class of inhibitors is dependent on the intrinsic ability of the antioxidant to reduce the enzyme and on the fraction of the inhibitor that is partitioned into the membrane.