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GSK3640254 is an HIV-1 maturation inhibitor (MI) that exhibits significantly improved antiviral activity toward a range of clinically relevant polymorphic variants with reduced sensitivity toward the second-generation MI GSK3532795 (BMS-955176). The key structural difference between GSK3640254 and its predecessor is the replacement of the

In the literature, C-N coupling methods for the reaction of iodo-oxazole with 2-pyridinone were found to be low yielding. C-N coupling using silver benzoate additives with CuI catalysts and 4,7-dimethoxy-1,10-phenanthroline ligands has been developed to afford synthetically useful yields of the desired heterobicycle product. The reaction conditions are applied to the coupling of a range of iodo-heterocycles with 2-pyridinone. The coupling of a variety of NH-containing heterocycles with 4-iodo-oxazole is also demonstrated. The use of 2-, 4-, or 5-iodo-oxazole allows for the coupling of pyridinone to each oxazole position.

BMS-955176 is a second-generation human immunodeficiency virus type 1 (HIV-1) maturation inhibitor (MI). A first-generation MI, bevirimat, showed clinical efficacy in early-phase studies, but ∼50% of subjects had viruses with reduced susceptibility associated with naturally occurring polymorphisms in Gag near the site of MI action. MI potency was optimized using a panel of engineered reporter viruses containing site-directed polymorphic changes in Gag that reduce susceptibility to bevirimat (including V362I, V370A/M/Δ, and T371A/Δ), leading incrementally to the identification of BMS-955176. BMS-955176 exhibits potent activity (50% effective concentration [EC 50 ], 3.9 ± 3.4 nM [mean ± standard deviation]) toward a library ( n = 87) of gag/pr recombinant viruses representing 96.5% of subtype B polymorphic Gag diversity near the CA/SP1 cleavage site. BMS-955176 exhibited a median EC 50 of 21 nM toward a library of subtype B clinical isolates assayed in peripheral blood mononuclear cells (PBMCs). Potent activity was maintained against a panel of reverse transcriptase, protease, and integrase inhibitor-resistant viruses, with EC 50 s similar to those for the wild-type virus. A 5.4-fold reduction in EC 50 occurred in the presence of 40% human serum plus 27 mg/ml of human serum albumin (HSA), which corresponded well to an in vitro measurement of 86% human serum binding. Time-of-addition and pseudotype reporter virus studies confirm a mechanism of action for the compound that occurs late in the virus replication cycle. BMS-955176 inhibits HIV-1 protease cleavage at the CA/SP1 junction within Gag in virus-like particles (VLPs) and in HIV-1-infected cells, and it binds reversibly and with high affinity to assembled Gag in purified HIV-1 VLPs. Finally, in vitro combination studies showed no antagonistic interactions with representative antiretrovirals (ARVs) of other mechanistic classes. In conclusion, BMS-955176 is a second-generation MI with potent in vitro anti-HIV-1 activity and a greatly improved preclinical profile compared to that of bevirimat.

HIV-1 maturation inhibition (MI) has been clinically validated as an approach to the control of HIV-1 infection. However, identifying an MI with both broad polymorphic spectrum coverage and good oral exposure has been challenging. Herein, we describe the design, synthesis, and preclinical characterization of a potent, orally active, second generation HIV-1 MI, BMS-955176 (2), which is currently in Phase IIb clinical trials as part of a combination antiretroviral regimen.

We have recently reported on the discovery of a C-3 benzoic acid (1) as a suitable replacement for the dimethyl succinate side chain of bevirimat (2), an HIV-1 maturation inhibitor that reached Phase II clinical trials before being discontinued. Recent SAR studies aimed at improving the antiviral properties of 2 have shown that the benzoic acid moiety conferred topographical constraint to the pharmacophore and was associated with a lower shift in potency in the presence of human serum albumin. In this manuscript, we describe efforts to improve the polymorphic coverage of the C-3 benzoic acid chemotype through modifications at the C-28 position of the triterpenoid core. The dimethylaminoethyl amides 17 and 23 delivered improved potency toward bevirimat-resistant viruses while increasing C24 in rat oral PK studies.

GSK3532795, formerly known as BMS-955176 (1), is a potent, orally active, second-generation HIV-1 maturation inhibitor (MI) that advanced through phase IIb clinical trials. The careful design, selection, and evaluation of substituents appended to the C-3 and C-17 positions of the natural product betulinic acid (3) was critical in attaining a molecule with the desired virological and pharmacokinetic profile. Herein, we highlight the key insights made in the discovery program and detail the evolution of the structure-activity relationships (SARs) that led to the design of the specific C-17 amine moiety in 1. These modifications ultimately enabled the discovery of 1 as a second-generation MI that combines broad coverage of polymorphic viruses (EC

The design and synthesis of a series of C28 amine-based betulinic acid derivatives as HIV-1 maturation inhibitors is described. This series represents a continuation of efforts following on from previous studies of C-3 benzoic acid-substituted betulinic acid derivatives as HIV-1 maturation inhibitors (MIs) that were explored in the context of C-28 amide substituents. Compared to the C-28 amide series, the C-28 amine derivatives exhibited further improvements in HIV-1 inhibitory activity toward polymorphisms in the Gag polyprotein as well as improved activity in the presence of human serum. However, plasma exposure of basic amines following oral administration to rats was generally low, leading to a focus on moderating the basicity of the amine moiety distal from the triterpene core. The thiomorpholine dioxide (TMD) 20 emerged from this study as a compound with the optimal antiviral activity and an acceptable pharmacokinetic profile in the C-28 amine series. Compared to the C-28 amide 3, 20 offers a 2- to 4-fold improvement in potency towards the screening viruses, exhibits low shifts in the EC50 values toward the V370A and ΔV370 viruses in the presence of human serum or human serum albumin, and demonstrates improved potency towards the polymorphic T371A and V362I virus variants.

The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhibitor of the NS3/4A enzyme is currently in phase III clinical trials for the treatment of hepatitis C virus infection. The discovery of 24 was enabled by employing an isolated rabbit heart model to screen for the cardiovascular (CV) liabilities (changes to HR and SNRT) that were responsible for the discontinuation of an earlier lead from this chemical series, BMS-605339 (1), from clinical trials. The structure-activity relationships (SARs) developed with respect to CV effects established that small structural changes to the P2* subsite of the molecule had a significant impact on the CV profile of a given compound. The antiviral activity, preclincial PK profile, and toxicology studies in rat and dog supported clinical development of BMS-650032 (24).

The discovery of BMS-605339 (35), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropylacylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1' site of the protease. The identification of 35 was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK). This was achieved through modulation of the P2* subsite of the inhibitor which identified the isoquinoline ring system as a key template for improving PK properties with further optimization achieved through functionalization. A methoxy moiety at the C6 position of this isoquinoline ring system proved to be optimal with respect to potency and PK, thus providing the clinical compound 35 which demonstrated antiviral activity in HCV-infected patients.

The discovery of a back-up to the hepatitis C virus NS3 protease inhibitor asunaprevir (2) is described. The objective of this work was the identification of a drug with antiviral properties and toxicology parameters similar to 2, but with a preclinical pharmacokinetic (PK) profile that was predictive of once-daily dosing. Critical to this discovery process was the employment of an ex vivo cardiovascular (CV) model which served to identify compounds that, like 2, were free of the CV liabilities that resulted in the discontinuation of BMS-605339 (1) from clinical trials. Structure–activity relationships (SARs) at each of the structural subsites in 2 were explored with substantial improvement in PK through modifications at the P1 site, while potency gains were found with small, but rationally designed structural changes to P4. Additional modifications at P3 were required to optimize the CV profile, and these combined SARs led to the discovery of BMS-890068 (29).

In this article, we describe the characteristic (15)N chemical shifts of isatin oxime ethers and their isomer nitrone. These oxime ethers and nitrones are the alkylation reaction products of isatin oximes. In our study, the (15)N chemical shifts observed in these oxime ethers were in the 402-408 (or 22-28) ppm range, although those for their corresponding nitrone series were in the 280-320 (or -100 to -60) ppm range. This remarkable difference in (15)N NMR chemical shift values could potentially be used to determine the O- versus N-alkylation of oximes, even when only one isomer is available. In this paper, the differences in (15)N NMR chemical shifts serve as the basis for a discussion about how to distinguish both regioisomers derived from the oximes alkylation.

A series of isatin oximes was alkylated with alkyl halides and under Mitsunobu conditions to generate O-alkylated oxime ether and N-alkylated nitrone products. Alkylation of the sodium salts of oximes and with alkyl iodides produced predominantly the N-alkylated nitrones while alkylation of and with the harder electrophiles alkyl bromides and alkyl chlorides, gave mostly the O-alkylated products, oxime ethers . Interestingly, alkylation of oxime under Mitsunobu conditions with isopropyl alcohol produced the N-alkylated nitrone (E)-7bd as the major product. However, alkylation of oxime , which incorporates a sterically bulky bromine substituent at the C-4 position of the isatin heterocycle, with 2-bromo- and 2-iodo-propane or with isopropyl alcohol under Mitsunobu conditions gave exclusively the O-alkylated product, oxime ether (Z)-6cd. The oxime ether and nitrone products and , respectively, were characterized by LC/MS, 1H NMR, and 13C NMR. In addition, the structures of oxime ether (E)-6bd and nitrone were determined by X-ray crystallography. J. Heterocyclic Chem., 46, 432 (2009).

A series of benzimidazole-based inhibitors of respiratory syncytial virus (RSV) fusion were optimized for antiviral potency, membrane permeability and metabolic stability in human liver microsomes. 1-Cyclopropyl-1,3-dihydro-3-[[1-(4-hydroxybutyl)-1H-benzimidazol-2-yl]methyl]-2H-imidazo[4,5-c]pyridin-2-one (6m, BMS-433771) was identified as a potent RSV inhibitor demonstrating good bioavailability in the mouse, rat, dog and cynomolgus monkey that demonstrated antiviral activity in the BALB/c and cotton rat models of infection following oral administration.

BMS-433771 is a potent inhibitor of respiratory syncytial virus (RSV) replication in vitro. Mechanism of action studies have demonstrated that BMS-433771 halts virus entry through inhibition of F protein-mediated membrane fusion. BMS-433771 also exhibited in vivo efficacy following oral administration in a mouse model of RSV infection (C. Cianci, K. Y. Yu, K. Combrink, N. Sin, B. Pearce, A. Wang, R. Civiello, S. Voss, G. Luo, K. Kadow, E. Genovesi, B. Venables, H. Gulgeze, A. Trehan, J. James, L. Lamb, I. Medina, J. Roach, Z. Yang, L. Zadjura, R. Colonno, J. Clark, N. Meanwell, and M. Krystal, Antimicrob. Agents Chemother.48:413-422, 2004). In this report, the in vivo efficacy of BMS-433771 against RSV was further examined in the BALB/c mouse and cotton rat host models of infection. By using the Long strain of RSV, prophylactic efficacy via oral dosing was observed in both animal models. A single oral dose, administered 1 h prior to intranasal RSV inoculation, was as effective against infection as a 4-day b.i.d. dosing regimen in which the first oral dose was given 1 h prior to virus inoculation. Results of dose titration experiments suggested that RSV infection was more sensitive to inhibition by BMS-433771 treatment in the BALB/c mouse host than in the cotton rat. This was reflected by the pharmacokinetic and pharmacodynamic analysis of the efficacy data, where the area under the concentration-time curve required to achieve 50% of the maximum response was ∼7.5-fold less for mice than for cotton rats. Inhibition of RSV by BMS-433771 in the mouse is the result of F1-mediated inhibition, as shown by the fact that a virus selected for resistance to BMS-433771 in vitro and containing a single amino acid change in the F1 region was also refractory to treatment in the mouse host. BMS-433771 efficacy against RSV infection was also demonstrated for mice that were chemically immunosuppressed by cyclophosphamide treatment, indicating that compound inhibition of the virus did not require an active host immune response.

BMS-433771 was found to be a potent inhibitor of respiratory syncytial virus (RSV) replication in vitro. It exhibited excellent potency against multiple laboratory and clinical isolates of both group A and B viruses, with an average 50% effective concentration of 20 nM. Mechanism-of-action studies demonstrated that BMS-433771 inhibits the fusion of lipid membranes during both the early virus entry stage and late-stage syncytium formation. After isolation of resistant viruses, resistance was mapped to a series of single amino acid mutations in the F1 subunit of the fusion protein. Upon oral administration, BMS-433771 was able to reduce viral titers in the lungs of mice infected with RSV. This new class of orally active RSV fusion inhibitors offers potential for clinical development.

Epoxomicin (1), a peptide alpha',beta'-epoxyketone isolated from the actinomycete strain No.Q996-17, possesses potent in vivo anti-tumor and anti-inflammatory activities. In this paper, we report the first syntheses of epoxomicin, [3H]-epoxomicin, and a biotinylated epoxomicin analog as well as the absolute configuration of the epoxide stereocenter. The natural product and derivatives have permitted the first identification of the proteasome as the specific cellular target of epoxomicin.

The inhibition of new blood vessel formation (angiogenesis) is an effective means of limiting both the size and metastasis of solid tumors. The leading anti-angiogenic compound, TNP-470, has proven to be effective in in vitro and in animal model studies, and is currently being tested in phase III antitumor clinical trials. Despite many detailed pharmacological studies, little is known of the molecular mode of action of TNP-470. Using a derivative of the TNP-470 parent compound, the fungal metabolite, fumagillin, we have purified a mammalian protein that is selectively and covalently bound by this natural product. This fumagillin binding protein was found to be a metalloprotease, methionine aminopeptidase (MetAP-2), that is highly conserved between human and Saccharomyces cerevisiae . In the absence of MetAP-1, a distantly related methionine aminopeptidase, MetAP-2 function is essential for vegetative growth in yeast. We demonstrate that fumagillin selectively inhibits the S. cerevisiae MetAP-2 protein in vivo . The binding is highly specific as judged by the failure of fumagillin to inhibit MetAP-1 in vivo . Hence, these results identify MetAP-2 as an important target of study in the analysis of the potent biological activities of fumagillin.

The N-trifluoroacetyl derivative of the novel β-amino acid (2S,3S,8S,9S)-Adda methyl ester 4 has been prepared from the D-glucose derived oxazoline 7. A key step in the construction of 4 was the generation of the E , E decadienoate system via dissolving metal reduction of vicinal bis -mesylate 15.

The advanced rapamycin intermediate 26 has been prepared by an iterative sigmatropic sequence incorporating the diastereoselective [2,3] Wittig rearrangement of a D -glucofuranose-derived tertiary allylic ether.

The N-trifluoroacetyl derivative of the novel β-amino acid (2S,3S,8S,9S)-Adda methyl ester 4 has been prepared from the D-glucose derived oxazoline 7. A key step in the construction of 4 was the generation of the E , E decadienoate system via dissolving metal reduction of vicinal bis -mesylate 15.

Epoxomicin (1), a peptide alpha',beta'-epoxyketone isolated from the actinomycete strain No.Q996-17, possesses potent in vivo anti-tumor and anti-inflammatory activities. In this paper, we report the first syntheses of epoxomicin, [3H]-epoxomicin, and a biotinylated epoxomicin analog as well as the absolute configuration of the epoxide stereocenter. The natural product and derivatives have permitted the first identification of the proteasome as the specific cellular target of epoxomicin.

A series of bezimidazole-isatin oximes were prepared and profiled as inhibitors of respiratory syncytial virus (RSV) replication in cell culture. Structure-activity relationship studies were directed toward optimization of antiviral activity, cell permeability and metabolic stability in human liver micorosomes (HLM). Parallel combinatorial synthetic chemistry was employed to functionalize isatin oximes via O-alkylation which quickly identified a subset of small, lipophilic substituents that established good potency for the series. Further optimization of the isatin oxime derivatives focused on introduction of nitrogen atoms to the isatin phenyl ring to provide a series of aza-isatin oximes with significantly improved PK properties. Several aza-isatin oximes analogs displayed targeted metabolic stability in HLM and permeability across a confluent monolayer of CaCo-2 cells. These studies identified several compounds, including 18i, 18j and 18n that demonstrated antiviral activity in the BALB/c mouse model of RSV infection following oral dosing.

Exchange of amide protons with deuterium results in changes of13C chemical shifts for carbon atoms near the site of substitution (CH—N and N—C=O). There is a measurable decrease in the isotopic shifts of CH—N for cycloalkylacetamides as ring size increases from 3 to 8 and for lactams as ring size increases from 5 to 9. The isotopic shifts of N—C=O for lactams also decrease as ring size increases from 5 to 9 but remains fairly constant for cycloalkylacetamides (three- to eight-membered rings).

The proteasome regulates cellular processes as diverse as cell cycle progression and NF-κB activation. In this study, we show that the potent antitumor natural product epoxomicin specifically targets the proteasome. Utilizing biotinylated-epoxomicin as a molecular probe, we demonstrate that epoxomicin covalently binds to the LMP7, X, MECL1, and Z catalytic subunits of the proteasome. Enzymatic analyses with purified bovine erythrocyte proteasome reveal that epoxomicin potently inhibits primarily the chymotrypsin-like activity. The trypsin-like and peptidyl-glutamyl peptide hydrolyzing catalytic activities also are inhibited at 100- and 1,000-fold slower rates, respectively. In contrast to peptide aldehyde proteasome inhibitors, epoxomicin does not inhibit nonproteasomal proteases such trypsin, chymotrypsin, papain, calpain, and cathepsin B at concentrations of up to 50 μM. In addition, epoxomicin is a more potent inhibitor of the chymotrypsin-like activity than lactacystin and the peptide vinyl sulfone NLVS. Epoxomicin also effectively inhibits NF-κB activation in vitro and potently blocksin vivoinflammation in the murine ear edema assay. These results thus define epoxomicin as a novel proteasome inhibitor that likely will prove useful in exploring the role of the proteasome in variousin vivoandin vitrosystems.

Derivatives of the epoxy-β-aminoketone containing natural product eponemycin have been prepared in order to study the molecular mode of action of this anti-angiogenic compound. Synthesis and use of a biotinylated dihydroeponemycin analogue demonstrated that dihydroeponemycin forms a covalent adduct with at least two intracellular proteins in human endothelial cells. Pretreatment of cells with a five equivalent excess of dihydroeponemycin precluded biotin-dihydroeponemycin binding indicating a specific interaction between natural product and the target proteins. This biotin-dihydroeponemycin derivative will prove useful in the purification and identification of eponemycin receptors.

Dynamic protein palmitoylation has been proposed to regulate GTP-binding proteins by controlling their membrane association and thus their access to key signaling proteins. While the palmitoyl protein thioesterase(s) responsible for depalmitoylation of plasma membrane-associated signaling proteins has (have) not been identified, the lysosomal palmitoyl protein thioesterase 1 (PPT1) has proven useful in in vitro studies of membrane localization requirements of GTP-binding proteins. We have previously reported the binding of the antiproliferative cyclic depsipeptide didemnin B to PPT1. To investigate the nature of this binding and its possible effects on PPT1 enzymatic activity, human PPT1 was expressed in an insect cell baculoviral system, and inhibition assays were performed using both [3H]palmitoyl Ha-Ras and myristoyl-CoA as PPT1 substrates. Didemnin B was shown to inhibit recombinant human PPT1 with a Ki of 92 nM. Kinetic analysis of this inhibition revealed that didemnin B inhibits PPT1 uncompetitively. Providing biochemical support for an uncompetitive mode of inhibition, in vitro binding studies of PPT1 and didemnin indicate that the natural product binds preferentially to the enzyme-substrate complex PPT1-palmitoyl-CoA. As the first described inhibitor of PPT1, didemnin B may prove to be a useful tool in the investigation of protein palmitoylation regulation.

Molecular mechanics calculations can be used in conjunction with hand-held models for a more complete approach to the instruction of stereochemistry.

IR spectroscopy is presented as a convenient means to monitor the formation of acyl (and alkyl) cations from corresponding acid chlorides, with Lewis acids at low temperature in the solid state. Phenylacetyl chloride is co-deposited (under vacuum at −173°C with antimony pentafluoride on a KBr window), as in the ‘molecular beam’ method of cation generation. The initial IR spectrum of the deposition shows (in addition to starting materials) that a small amount of phenylacetyl cation (2274 cm−1) has been formed. Warming the solid matrix to −123°C Promotes the smooth conversion of acid chloride to acyl cation. Ultraviolet irradiation (through quartz windows) at −123°C to −73°C facilitates this conversion but does not result in the loss of carbon monoxide from the acyl cation to form benzyl cation. When benzyl chloride is used in place of phenylacetyl chloride, there is no noticeable difference between IR spectra taken before and after warming and/or photolysis of the solid deposition.