Discovery of N-arylalkyl-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxamide derivatives as HCV NS5B polymerase inhibitors

The metal ion chelating β-N-hydroxy-γ-ketocarboxamide pharmacophore was integrated into a quinazolinone scaffold, leading to N-arylalkyl-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxamide derivatives as hepatitis C virus (HCV) NS5B polymerase inhibitors. Lead optimization led to the identification of N-phenylpropyl carboxamide 9 k (IC(50) =8.8 μM). Compound 9 k possesses selectivity toward HCV1b replicon Ava.5 cells (EC(50) =17.5 μM) over parent Huh-7 cells (CC(50) =187.5 μM). Compound 9 k effects a mixed mode of NS5B inhibition, with NTP-competitive displacement properties. The interaction between 9 k and NS5B is stabilized by the presence of magnesium ions. Docking studies showed that the binding orientation of 9 k occupies the central portions of both magnesium-mediated and NTP-ribose-response binding sites within the active site region of NS5B. As a result, 3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxamide derivatives are disclosed herein as novel, mainly active site inhibitors of HCV NS5B polymerase.     

Discovery and structure-activity relationship studies of a unique class of HIV-1 integrase inhibitors

HIV-1 integrase (IN) is an essential enzyme for viral replication and a validated target for the development of drugs against AIDS. Currently there are no approved drugs that target IN. However, new IN inhibitors are under clinical investigation. As more IN inhibitors enter human drug trials, there is a growing need for the design of novel lead compounds with diverse structural scaffolds and promising pharmacokinetic properties to counteract the difficulties observed with first-generation IN inhibitors. We have identified a novel class of IN inhibitors through the systematic exploration of structure-activity relationships in a series of linomide analogues. The predicted bound conformation of the most active analogues inside the IN active site also supports the observed structure-activity correlation in this new compound class.     

Synthesis and structure-activity relationship studies of HIV-1 virion infectivity factor (Vif) inhibitors that block viral replication

The human immunodeficiency virus 1 (HIV-1) virion infectivity factor (Vif) protein, essential for in vivo viral replication, protects the virus from innate antiviral cellular factor apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (APOBEC3G; A3G) and is an attractive target for the development of novel antiviral therapeutics. We have evaluated the structure-activity relationships of N-(2-methoxyphenyl)-2-((4-nitrophenyl)thio)benzamide (RN-18), a small molecule recently identified as an inhibitor of Vif function that blocks viral replication only in nonpermissive cells expressing A3G, by inhibiting Vif-A3G interactions. Microwave-assisted cross-coupling reactions were developed to prepare a series of RN18 analogues with diverse linkages and substitutions on the phenyl rings. A dual cell-based assay system was used to assess antiviral activity against wild-type HIV-1 in both nonpermissive (H9) and permissive (MT4) cells that also allowed evaluation of specificity. In general, variations of phenyl substitutions were detrimental to antiviral potency and specificity, but isosteric replacements of amide and ether linkages were relatively well tolerated. These structure-activity relationship data define structural requirements for Vif-specific activity, identify new compounds with improved antiviral potency and specificity, and provide leads for further exploration to develop new antiviral therapeutics.     

Design, synthesis, biological evaluation, and molecular modeling studies of TIBO-like cyclic sulfones as non-nucleoside HIV-1 reverse transcriptase inhibitors

TIBO- and TBO-like sulfone derivatives 1 and 2 were designed, synthesized, and tested for their ability to block the replication cycle of HIV-1 in infected cells. The anti-HIV-1 activities of sulfones 3, which were intermediates in the syntheses of 1 and 2, were also evaluated. Surprisingly, the sulfone analogues of TIBO R82913 (compounds 1) were inactive, whereas interesting results were obtained for truncated derivatives 2. Compound 2 w was the most potent among this series in cell-based assays (EC50=0.07 microM, CC50>200 microM, SI>2857). It was twofold less potent than R82913, but more selective. An X-ray crystallographic analysis was carried out to establish the absolute configuration of 2 w and its enantiomer 2 x, which were obtained by semipreparative HPLC of 2 v, one of the most potent racemates. Compounds 1-3 were proven to target HIV-1 RT. In fact, representative derivatives inhibited recombinant HIV-1 RT in vitro at concentrations similar to those active in cell-based assays. 3D QSAR studies and docking simulations were developed on TIBO- and TBO-like sulfone derivatives to rationalize their anti-HIV-1 potencies and to predict the activity of novel untested sulfone derivatives. Predictive 3D QSAR models were obtained with a receptor-based alignment by docking of TIBO- and TBO-like derivatives into the NNBS of RT.     

Discovery of MK‐8742: An HCV NS5A Inhibitor with Broad Genotype Activity

The NS5A protein plays a critical role in the replication of HCV and has been the focus of numerous research efforts over the past few years. NS5A inhibitors have shown impressive in vitro potency profiles in HCV replicon assays, making them attractive components for inclusion in all oral combination regimens. Early work in the NS5A arena led to the discovery of our first clinical candidate, MK‐4882 [2‐((S)‐pyrrolidin‐2‐yl)‐5‐(2‐(4‐(5‐((S)‐pyrrolidin‐2‐yl)‐1H‐imidazol‐2‐yl)phenyl)benzofuran‐5‐yl)‐1H‐imidazole]. While preclinical proof‐of‐concept studies in HCV‐infected chimpanzees harboring chronic genotype 1 infections resulted in significant decreases in viral load after both single‐ and multiple‐dose treatments, viral breakthrough proved to be a concern, thus necessitating the development of compounds with increased potency against a number of genotypes and NS5A resistance mutations. Modification of the MK‐4882 core scaffold by introduction of a cyclic constraint afforded a series of tetracyclic inhibitors, which showed improved virologic profiles. Herein we describe the research efforts that led to the discovery of MK‐8742, a tetracyclic indole‐based NS5A inhibitor, which is currently in phase 2b clinical trials as part of an all‐oral, interferon‐free regimen for the treatment of HCV infection.     

Synthesis and anti-HIV activity of aryl-2-[(4-cyanophenyl)amino]-4-pyrimidinone hydrazones as potent non-nucleoside reverse transcriptase inhibitors

A series of novel diarylpyrimidines (DAPYs) with a ketone hydrazone substituent on the methylene linker between the pyrimidine nucleus and the aryl moiety at the C‐4 position were synthesized, and their antiviral activity against human immunodeficiency virus (HIV)‐1 in MT‐4 cells was evaluated. Most compounds of this class exhibited excellent activity against wild‐type HIV‐1, with EC₅₀ values in the range of 1.7–13.2 nM . Of these compounds, 2‐bromophenyl‐2‐[(4‐cyanophenyl)amino]‐4‐pyrimidinone hydrazone ( 9 k ) displayed the most potent anti‐HIV‐1 activity (EC₅₀=1.7±0.6 nM ), with excellent selectivity for infected over uninfected cells (SI=5762). In addition, the 4‐methyl phenyl analogue 9 d (EC₅₀=2.4±0.2 nM , SI=18461) showed broad spectrum HIV inhibitory activity, with EC₅₀ values of 2.4±0.2 nM against wild‐type HIV‐1, 5.3±0.4 μM against HIV‐1 double‐mutated strain RES056 (K103N+Y181C), and 5.5 μM against HIV‐2 ROD strain. Furthermore, structure–activity relationship (SAR) data and molecular modeling results for these compounds are also discussed.     

Structure-activity studies on suramin analogues as inhibitors of NAD+-dependent histone deacetylases (sirtuins)

Suramin is a symmetric polyanionic naphthylurea originally used for the treatment of trypanosomiasis and onchocerciasis. Suramin and diverse analogues exhibit a broad range of biological actions in vitro and in vivo, including, among others, antiproliferative and antiviral activity. Suramin derivatives usually target purinergic binding sites. Class III histone deacetylases (sirtuins) are amidohydrolases that require nicotinamide adenine dinucleotide (NAD(+)) as a cofactor for their catalytic mechanism(.) Deacetylation of the target proteins leads to a change in conformation and alters the activity of the proteins in question. Suramin was reported to inhibit human sirtuin 1 (SIRT1). We tested a diverse set of suramin analogues to elucidate the inhibition of the NAD(+)-dependent histone deacetylases SIRT1 and SIRT2 and discovered selective inhibitors of human sirtuins with potency in the two-digit nanomolar range. In addition, the structural requirements for the binding of suramin derivatives to sirtuins were investigated by molecular docking. The recently published X-ray crystal structure of human SIRT5 in complex with suramin and the human SIRT2 structure were used to analyze the interaction mode of the novel suramin derivatives.     

Discovery of 3H-imidazo[4,5-b]pyridines as potent c-Met kinase inhibitors: design, synthesis, and biological evaluation

To identify novel c-Met inhibitors, sequences and crystal structures of the human kinome were analyzed to find interesting hinge binders that have been underexplored within the tyrosine kinase subfamily. Through this study, the imidazolopyridine ring was selected as a novel c-Met hinge-binding inhibitor scaffold. A series of derivatives was prepared, and the structure-activity relationships were studied. Among these, one compound in particular showed excellent activities in enzymatic and cellular assays, good in vitro metabolic stability, and favorable pharmacokinetic parameters. When administered orally, the compound inhibited tumor growth in an NIH-3T3/TPR-Met xenograft model and did not show adverse effects on body weight. The present work not only conceptually demonstrates a new route for designing novel kinase inhibitors by using known structural information of ligand-hinge interactions but also provides a series of imidazolopyridine derivatives as potent c-Met inhibitors.     

Structure-activity relationship studies of 3-aroylindoles as potent antimitotic agents

The concise synthesis and structure-activity relationship (SAR) studies of 3-aroylindoles were carried out in an effort to improve the potency and solubility of anticancer drug candidate BPR0L075 (8) by exploring structure modifications through three regimens: substitution of the B ring, at the N1 position, and of the 3-carbonyl linker. The SAR information revealed that the methoxy group of the B ring could be replaced with an electron-donating group such as methyl (in compound 9) or N,N-dimethylamino (in compound 13) while retaining both strong cytotoxic and antitubulin activities. The introduction of amide (compounds 30-33) and carbamate (compounds 34-37) functionalities at the N1 position of 8 gave analogues with potent antiproliferative activities. The cytotoxic potency of 8 was improved by replacing the carbonyl group with sulfide (compound 41) or oxygen (compound 43), indicating that the carbonyl moiety is important but not essential. The N,N-dimethylamino derivative 13 not only displayed potent cytotoxicity and antitubulin activity, but also showed a markedly improved physicochemical profile relative to the parent compound.     

Synthesis and structure-activity relationships of FAAH inhibitors: cyclohexylcarbamic acid biphenyl esters with chemical modulation at the proximal phenyl ring

Fatty acid amide hydrolase (FAAH) is a serine hydrolase that catalyzes the intracellular hydrolysis of fatty acid ethanolamides such as anandamide and oleoylethanolamide. Targeting this enzyme may have important therapeutic potentials owing to the multiple physiological roles of these amides. Cyclohexylcarbamic acid biphenyl-3-yl ester (URB524) was one of the most promising FAAH inhibitors so far described. We report the modulation of the electronic and steric features of the proximal phenyl ring of this compound by introducing a series of substituents at the ortho and para positions. pIC50 values were found to correlate with molecular features thought to be involved in the recognition step such as steric hindrance and hydrogen-bonding ability. Derivatives with small polar groups at the para position of the proximal phenyl ring were slightly better FAAH inhibitors than the parent compound URB524.     

Design, synthesis, and biological activity of urea derivatives as anaplastic lymphoma kinase inhibitors

In anaplastic large-cell lymphomas, chromosomal translocations involving the kinase domain of anaplastic lymphoma kinase (ALK), generally fused to the 5' part of the nucleophosmin gene, produce highly oncogenic ALK fusion proteins that deregulate cell cycle, apoptosis, and differentiation in these cells. Other fusion oncoproteins involving ALK, such as echinoderm microtubule-associated protein-like 4-ALK, were recently found in patients with non-small-cell lung, breast, and colorectal cancers. Recent research has focused on the development of inhibitors for targeted therapy of these ALK-positive tumors. Because kinase inhibitors that target the inactive conformation are thought to be more specific than ATP-targeted inhibitors, we investigated the possibility of using two known inhibitors, doramapimod and sorafenib, which target inactive kinases, to design new urea derivatives as ALK inhibitors. We generated a homology model of ALK in its inactive conformation complexed with doramapimod or sorafenib in its active site. The results elucidated why doramapimod is a weak inhibitor and why sorafenib does not inhibit ALK. Virtual screening of commercially available compounds using the homology model of ALK yielded candidate inhibitors, which were tested using biochemical assays. Herein we present the design, synthesis, biological activity, and structure-activity relationships of a novel series of urea compounds as potent ALK inhibitors. Some compounds showed inhibition of purified ALK in the high nanomolar range and selective antiproliferative activity on ALK-positive cells.     

Design and synthesis of potent and selective azaindole-based Rho kinase (ROCK) inhibitors

Rho kinase plays a pivotal role in several cellular processes such as vasoregulation, making it a suitable target for the treatment of hypertension and related disorders. We discovered a new compound class of Rho kinase (ROCK) inhibitors containing a 7-azaindole hinge-binding scaffold tethered to an aminopyrimidine core. Herein we describe the structure-activity relationships elucidated through biochemical and functional assays. The introduction of suitable substituents at the 3-position of the bicyclic moiety led to an increase in activity, which was required to design compounds with favorable pharmacokinetic profile. Azaindole 32 was identified as a highly selective and orally available ROCK inhibitor able to cause a sustained blood pressure reduction in vivo.     

Unexpected novel binding mode of pyrrolidine-based aspartyl protease inhibitors: design, synthesis and crystal structure in complex with HIV protease

At present nine FDA-approved HIV protease inhibitors have been launched to market, however rapid drug resistance arising under antiviral therapy calls upon novel concepts. Possible strategies are the development of ligands with less peptide-like character or the stabilization of a new and unexpected binding-competent conformation of the protein through a novel ligand-binding mode. Our rational design of pyrrolidinedimethylene diamines was inspired by the idea to incorporate key structural elements from classical peptidomimetics with a non-peptidic heterocyclic core comprising an endocyclic amino function to address the catalytic aspartic acid side chains of Asp 25 and 25'. The basic scaffolds were decorated by side chains already optimized for the recognition pockets of HIV protease or cathepsin D. A multistep synthesis has been established to produce the central heterocycle and to give flexible access to side chain decorations. Depending on the substitution pattern of the pyrrolidine moiety, single-digit micromolar inhibition of HIV-1 protease and cathepsin D has been achieved. Successful design is suggested in agreement with our modelling concepts. The subsequently determined crystal structure with HIV protease shows that the pyrrolidine moiety binds as expected to the pivotal position between both aspartic acid side chains. However, even though the inhibitors have been equipped symmetrically by polar acceptor groups to address the flap water molecule, it is repelled from the complex, and only one direct hydrogen bond is formed to the flap. A strong distortion of the flap region is detected, leading to a novel hydrogen bond which cross-links the flap loops. Furthermore, the inhibitor addresses only three of the four available recognition pockets. It achieves only an incomplete desolvation compared with the similarly decorated amprenavir. Taking these considerations into account it is surprising that the produced pyrrolidine derivatives achieve micromolar inhibition and it suggests extraordinary potency of the new compound class. Most likely, the protonated pyrrolidine moiety experiences strong enthalpic interactions with the enzyme through the formation of two salt bridges to the aspartic acid side chains. This might provide challenging opportunities to combat resistance of the rapidly mutating virus.     

Design, synthesis, and biological evaluation of 1-[(biarylmethyl)methylamino]-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ols as potent antifungal agents: new insights into structure-activity relationships

We recently reported the design and synthesis of azole antifungal agents with a focus on modifications to the side chain appended to the propanol group. Herein we have identified a series of new 1-[(biarylmethyl)methylamino] derivatives with broad-spectrum antifungal activities against the most prevalent human pathogenic fungi (Candida spp. and Aspergillus fumigatus). Compounds containing a flexible benzylamine moiety were clearly shown to yield the best antifungal activities, without the need for a hydrogen-bond acceptor substituent directly attached to the para position. We were also able to determine that selected compounds are able to overcome gene overexpression and point mutations that lead to reduced susceptibility or resistance against current treatments, such as fluconazole. As the minor differences observed with small structural modifications cannot be explain with only a three-dimensional model of CYP51, adequate physicochemical parameters must be evaluated in terms of antifungal potency, bioavailability, and toxicity. Therefore, structure-activity relationship studies such as these reveal new insights for the development of future antifungal therapies.     

Design, synthesis, and in vitro antifungal activity of 1-[(4-substituted-benzyl)methylamino]-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ols

As part of our studies focused on the design of 1‐[((hetero)aryl‐ and piperidinylmethyl)amino]‐2‐phenyl‐3‐(1H‐1,2,4‐triazol‐1‐yl)propan‐2‐ols as antifungal agents, we report the development of new extended benzylamine derivatives substituted at the para position by sulfonamide or retrosulfonamide groups linked to alkyl or aryl chains. These molecules have broad‐spectrum antifungal activities not only against Candida spp., including fluconazole‐resistant strains, but also against a filamentous species (A. fumigatus). Concerning fluconazole resistance, selected compounds exhibit the capacity to overcome CDR and ERG11 gene upregulation and to maintain antifungal activity despite a recognized critical CYP51 substitution in C. albicans isolates. Synthesis, investigation of the mechanism of action by sterol analysis in a C. albicans strain, and structure–activity relationships (SARs) are reported.