Studies towards the Design and Synthesis of Novel 1,5-Diaryl-1H-imidazole-4-carboxylic Acids and 1,5-Diaryl-1H-imidazole-4-carbohydrazides as Host LEDGF/p75 and HIV-1 Integrase Interaction Inhibitors.

Two targeted sets of novel 1,5-diaryl-1H-imidazole-4-carboxylic acids 10 and carbohydrazides 11 were designed and synthesized from their corresponding ester intermediates 17, which were prepared via cycloaddition of ethyl isocyanoacetate 16 and diarylimidoyl chlorides 15. Evaluation of these new target scaffolds in the AlphaScreenTM HIV-1 IN-LEDGF/p75 inhibition assay identified seventeen compounds exceeding the pre-defined 50% inhibitory threshold at 100 µM concentration. Further evaluation of these compounds in the HIV-1 IN strand transfer assay at 100 µM showed that none of the compounds (with the exception of 10a, 10l, and 11k, with marginal inhibitory percentages) were actively bound to the active site, indicating that they are selectively binding to the LEDGF/p75-binding pocket. In a cell-based HIV-1 antiviral assay, compounds 11a, 11b, 11g, and 11h exhibited moderate antiviral percentage inhibition of 33-45% with cytotoxicity (CC50) values of >200 µM, 158.4 µM, >200 µM, and 50.4 µM, respectively. The antiviral inhibitory activity displayed by 11h was attributed to its toxicity. Upon further validation of their ability to induce multimerization in a Western blot gel assay, compounds 11a, 11b, and 11h appeared to increase higher-order forms of IN.

Advances in the development of HIV integrase strand transfer inhibitors.

HIV-1 integrase (IN) is a key enzyme in viral replication that catalyzes the covalent integration of viral cDNA into the host genome. Currently, five HIV-1 IN strand transfer inhibitors (INSTIs) are approved for clinical use. These drugs represent an important addition to the armamentarium for antiretroviral therapy. This review briefly illustrates the development history of INSTIs. The characteristics of the currently approved INSTIs, as well as their future perspectives, are critically discussed.

Petra/Osiris/Molinspiration and Molecular Docking Analyses of 3-Hydroxy-Indolin-2-one Derivatives as Potential Antiviral Agents.

BACKGROUND: Studies on the interaction between bioactive molecules and HIV-1 virus have been the focus of recent research in the scope of medicinal chemistry and pharmacology. OBJECTIVE: Investigating the structural parameters and physico-chemical properties of elucidating and identifying the antiviral pharmacophore sites. METHODS: A mixed computational Petra/Osiris/Molinspiration/DFT (POM/DFT) based model has been developed for the identification of physico-chemical parameters governing the bioactivity of 22 3-hydroxy-indolin-2-one derivatives of diacetyl-L-tartaric acid and aromatic amines containing combined antiviral/antitumor/antibacterial pharmacophore sites. Molecular docking study was carried out with HIV-1 integrase (pdb ID: 5KGX) in order to provide information about interactions in the binding site of the enzyme. RESULTS: The POM analyses of physico-chemical properties and geometrical parameters of compounds 3a-5j, show that they are bearing a two combined (O,O)-pockets leading to a special platform which is able to coordinate two transition metals. The increased activity of series 3a-5j, as compared to standard drugs, contains (Osp2,O sp3,O sp2)-pharmacophore site. The increase in bioactivity from 4b (R1, R2 = H, H) to 3d (R1, R2 = 4-Br, 2-OCH3) could be attributed to the existence of π-charge transfer from para-bromo-phenyl to its amid group (COδ---NHδ+). Similar to the indole-based reference ligand (pdb: 7SK), compound 3d forms hydrogen bonding interactions between the residues Glu170, Thr174 and His171 of HIV-1 integrase in the catalytic core domain of the enzyme. CONCLUSION: Study confirmed the importance of oxygen atoms, especially from the methoxy group of the phenyl ring, and electrophilic amide nitrogen atom for the formation of interactions.

Novel 4-Oxo-4,10-dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine-3-carboxylic Acid Derivatives as HIV-1 Integrase Inhibitors: Synthesis, Docking Studies, Molecular Dynamics Simulation and Biological Activities.

BACKGROUND: HIV-1 integrase (IN) has been considered as an important target for the development of novel anti-HIV-1 drugs. OBJECTIVE: The aim of this study was to design novel groups of HIV IN inhibitors. METHODS: In this study, we presented a novel series of 4-oxo-4,10-dihydrobenzo[4,5]imidazo[1,2- a]pyrimidine-3-carboxylic acid derivatives by structural modification of N-arylindole ß-diketoacids as a well-known group of IN inhibitors. RESULTS: Based on in-vitro anti-HIV-1 activity in a cell-based assay, compounds 5, 6a and 6k displayed moderate to good inhibitory activity with EC50 values of 4.14, 1.68 and 0.8 µM, respectively. However, integrase inhibition assay showed that most of the analogues did not have significant effects against integrase enzyme except compound 5 with an IC50 value of 45 µM. Our results indicated that compound 6k was the best one among synthesized compounds with an EC50 of 0.8 µM and SI of 175. Docking and molecular dynamics simulation studies were also performed to provide some insights into the probable mechanism of tested compounds. CONCLUSION: These findings suggest that 4-oxo-4,10-dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine-3- carboxylic acid derivatives may consider as promising lead compounds for the development of new anti-HIV-1 drugs.

Discovery of novel HIV-1 integrase-LEDGF/p75 allosteric inhibitors based on a pyridine scaffold forming an intramolecular hydrogen bond.

We have discovered HIV-1 novel integrase-LEDGF/p75 allosteric inhibitors (INLAIs) based on a pyridine scaffold forming an intramolecular hydrogen bond. Scaffolds containing a pyridine moiety have been studied extensively and we have already reported that substituents extending from the C1 position contributed to the antiviral potency. In this study, we designed a new pyridine scaffold 2 with a substituent at the C1 position. Interestingly, during attempts at optimization, we found that the direction of the C1 substituents with an intramolecular hydrogen bond contributed to the antiviral potency. Compound 34f exhibited better antiviral potency against WT and the T174I mutant (EC50 (WT) = 6.6 nM, EC50 (T174I) = 270 nM) than BI 224436 (EC50 (WT) = 22 nM, EC50 (T174I) > 5000 nM).

Structure-based amelioration of PXR transactivation in a novel series of macrocyclic allosteric inhibitors of HIV-1 integrase.

Previous studies have identified a series of imidazo[1,2-a]pyridine (IZP) derivatives as potent allosteric inhibitors of HIV-1 integrase (ALLINIs) and virus infection in cell culture. However, IZPs were also found to be relatively potent activators of the pregnane-X receptor (PXR), raising the specter of induction of CYP-mediated drug disposition pathways. In an attempt to modify PXR activity without affecting anti-HIV-1 activity, rational structure-based design and modeling approaches were used. An X-ray cocrystal structure of (S,S)-1 in the PXR ligand binding domain (LBD) allowed an examination of the potential of rational structural modifications designed to abrogate PXR. The introduction of bulky basic amines at the C-8 position provided macrocyclic IZP derivatives that displayed potent HIV-1 inhibitory activity in cell culture with no detectable PXR transactivation at the highest concentration tested.

Design, synthesis and SAR study of novel C2-pyrazolopyrimidine amides and amide isosteres as allosteric integrase inhibitors.

The design, synthesis and structure-activity relationships associated with a series of C2-substituted pyrazolopyrimidines as potent allosteric inhibitors of HIV-1 integrase (ALLINIs) are described. Structural modifications to these molecules were made in order to examine the effect on potency and, for select compounds, pharmacokinetic properties. We examined a variety of C2-substituted pyrazolopyrimidines and found that the C2-amide derivatives demonstrated the most potent antiviral activity of this class against HIV-1 infection in cell culture.

Design, synthesis and SAR study of bridged tricyclic pyrimidinone carboxamides as HIV-1 integrase inhibitors.

The design, synthesis and structure-activity relationships associated with a series of bridged tricyclic pyrimidinone carboxamides as potent inhibitors of HIV-1 integrase strand transfer are described. Structural modifications to these molecules were made in order to examine the effect on potency towards wild-type and clinically-relevant resistant viruses. The [3.2.2]-bridged tricyclic system was identified as an advantageous chemotype, with representatives exhibiting excellent antiviral activity against both wild-type viruses and the G140S/Q148H resistant virus that arises in response to therapy with raltegravir and elvitegravir.

Synthesis and biological evaluation of bis-N2,N2'-(4-hydroxycoumarin-3-yl)ethylidene]-2,3-dihydroxysuccinodihydrazides.

A series of N2,N2'-bis[4-hydroxycoumarin-3-yl)ethylidene]-2,3-dihydroxysuccino-hydrazides, containing 4-hydroxycoumarin, hydrazine and tartaric acid moieties, have been prepared and examined for possible biological activity. Several of these compounds exhibit promising HIV-1 integrase inhibition (IC50 = 3.5 µM), and anti-T. brucei (32% viability) and anti-mycobacterial (Visual MIC90 = 15.63 µM) activity.

Synthesis, biological evaluation and in silico modeling of novel integrase strand transfer inhibitors (INSTIs).

Although a relatively wide range of therapeutic options is currently available for the treatment of HIV/AIDS, it is still among the most serious and virulent diseases and is associated with a high mortality rate. Integrase strand transfer inhibitors (INSTIs), e.g., FDA-approved dolutegravir (DTG), bictegravir (BIC) and cabotegravir (CAB), have recently been included in standard highly active antiretroviral therapy (HAART) schemes as one of the five major components responsible for the most beneficial clinical outcome. In this paper, we describe a combinatorial amide synthesis, biological evaluation and in silico modeling of new INSTIs containing heteroaromatic bioisosteric substitution instead of the well-studied halogen-substituted benzyl fragment. With the focus on the mentioned diversity point, a medium-sized library of compounds was selected for synthesis. A biological study revealed that many molecules were highly active INSTIs (EC50 < 10 nM). Two compounds 1{4} and 1{26} demonstrated picomolar antiviral activity that was comparable with CAB and were more active than DTG and BIC. Molecular docking study was performed to evaluate the binding mode of compounds in the active site of HIV-1 IN. In rats, lead compound 1{26} showed two-fold greater bioavailability than CAB and had a similar half-life. Compound 1{26} and its sodium salt were considerably more soluble in water than the parent drugs. Both molecules were very stable in human liver microsomes and plasma, demonstrated high affinity towards plasma proteins and did not show cytochrome (CYP) inhibition. This benefit profile indicates the great potential of these molecules as attractive candidates for subsequent evaluation as oral long-acting drugs and long-acting nanosuspension formulations for intramuscular injection.

Novel Benzoxazin-3-one Derivatives: Design, Synthesis, Molecular Modeling, Anti-HIV-1 and Integrase Inhibitory Assay.

INTRODUCTION: Integrase is a validated drug target for anti-HIV-1 therapy. The second generation integrase inhibitors display π-stacking interaction ability with 3'-end nucleotide as a streamlined metal chelating pharmacophore. METHODS: In this study, we introduced benzoxazin-3-one scaffold for integrase inhibitory potential as bioisostere replacement strategy of 2-benzoxazolinone. RESULTS: Molecular modeling studies revealed that amide functionality alongside oxadiazole heteroatoms and sulfur in the second position of oxadiazole ring could mimic the metal chelating pharmacophore. The halobenzyl ring occupies hydrophobic site created by the cytidylate nucleotide (DC-16). CONCLUSION: The most potent and selective compound displayed 110 µM IC50 with a selectivity index of more than 2.

Design, synthesis and biological evaluation of imidazole and oxazole fragments as HIV-1 integrase-LEDGF/p75 disruptors and inhibitors of microbial pathogens.

We describe here the synthesis of libraries of novel 1-subtituted-5-aryl-1H-imidazole, 5-aryl-4-tosyl-4,5-dihydro-1,3-oxazole and 5-aryl-1,3-oxazole fragments via microwave (MW)-assisted cycloaddition of para-toluenesulfonylmethyl isocyanide (TosMIC) to imines and aldehydes. The compounds obtained were biologically evaluated in an AlphaScreen HIV-1 IN-LEDGF/p75 inhibition assay with six imidazole-based compounds (16c, 16f, 17c, 17f, 20a and 20d) displaying more than 50% inhibition at 10 µM, with IC50 values ranging from 7.0 to 30.4 µM. Additionally the hypothesis model developed predicts all active scaffolds except 20d to occupy similar areas as the N-heterocyclic (A) moiety and two aromatic rings (B and C) of previously identified inhibitor 5. These results indicate that the identified compounds represent a viable starting point for their use as templates in the design of next generation inhibitors targeting the HIV-1 IN and LEDGF/p75 protein-protein interaction. In addition, the in vitro antimicrobial properties of these fragments were tested by minimum inhibitory concentration (MIC) assays showing that compound 16f exhibited a MIC value of 15.6 µg/ml against S. aureus, while 17f displayed a similar MIC value against B. cereus, suggesting that these compounds could be further developed to specifically target those microbial pathogens.

Chromenone derivatives as a versatile scaffold with dual mode of inhibition of HIV-1 reverse transcriptase-associated Ribonuclease H function and integrase activity.

A number of compounds targeting different processes of the Human Immunodeficiency Virus type 1 (HIV-1) life cycle have been developed in the continuing fight against AIDS. Coumarin-based molecules already proved to act as HIV-1 Protease (PR) or Integrase (IN) inhibitors and also to target HIV-1 reverse transcriptase (RT), blocking the DNA-dependent DNA-polymerase activity or the RNA-dependent DNA-polymerase activity working as common NNRTIs. In the present study, with the aim to exploit a coumarin-based scaffold to achieve the inhibition of multiple viral coded enzymatic functions, novel 4-hydroxy-2H, 5H-pyrano (3, 2-c) chromene-2, 5-dione derivatives were synthesized. The modeling studies calculated the theoretical binding affinity of the synthesized compounds on both HIV-1 IN and RT-associated Ribonuclease H (RNase H) active sites, which was confirmed by biological assays. Our results provide a basis for the identification of dual HIV-1 IN and RT RNase H inhibitors compounds.

Carboxylate-functionalized foldamer inhibitors of HIV-1 integrase and Topoisomerase 1: artificial analogues of DNA mimic proteins.

Inspired by DNA mimic proteins, we have introduced aromatic foldamers bearing phosphonate groups as synthetic mimics of the charge surface of B-DNA and competitive inhibitors of some therapeutically relevant DNA-binding enzymes: the human DNA Topoisomerase 1 (Top1) and the human HIV-1 integrase (HIV-1 IN). We now report on variants of these anionic foldamers bearing carboxylates instead of phosphonates. Several new monomers have been synthesized with protecting groups suitable for solid phase synthesis (SPS). Six hexadecaamides have been prepared using SPS. Proof of their resemblance to B-DNA was brought by the first crystal structure of one of these DNA-mimic foldamers in its polyanionic form. While some of the foldamers were found to be as active as, or even more active than, the original phosphonate oligomers, others had no activity at all or could even stimulate enzyme activity in vitro. Some foldamers were found to have differential inhibitory effects on the two enzymes. These results demonstrate a strong dependence of inhibitory activity on foldamer structure and charge distribution. They open broad avenues for the development of new classes of derivatives that could inhibit the interaction of specific proteins with their DNA target thereby influencing the cellular pathways in which they are involved.

Synthesis, Molecular Modelling and Biological Studies of 3-hydroxypyrane- 4-one and 3-hydroxy-pyridine-4-one Derivatives as HIV-1 Integrase Inhibitors.

BACKGROUND: Despite the progress in the discovery of antiretroviral compounds for treating HIV-1 infection by targeting HIV integrase (IN), a promising and well-known drug target against HIV-1, there is a growing need to increase the armamentarium against HIV, for avoiding the drug resistance issue. OBJECTIVE: To develop novel HIV-1 IN inhibitors, a series of 3-hydroxy-pyrane-4-one (HP) and 3- hydroxy-pyridine-4-one (HPO) derivatives have been rationally designed and synthesized. METHODS: To provide a significant characterization of the novel compounds, in-depth computational analysis was performed using a novel HIV-1 IN/DNA binary 3D-model for investigating the binding mode of the newly conceived molecules in complex with IN. The 3D-model was generated using the proto-type foamy virus (PFV) DNA as a structural template, positioning the viral polydesoxyribonucleic chain into the HIV-1 IN homology model. Moreover, a series of in vitro tests were performed including HIV-1 activity inhibition, HIV-1 IN activity inhibition, HIV-1 IN strand transfer activity inhibition and cellular toxicity. RESULTS: Bioassay results indicated that most of HP analogues including HPa, HPb, HPc, HPd, HPe and HPg, showed favorable inhibitory activities against HIV-1-IN in the low micromolar range. Particularly halogenated derivatives (HPb and HPd) offered the best biological activities in terms of reduced toxicity and optimum inhibitory activities against HIV-1 IN and HIV-1 in cell culture. CONCLUSION: Halogenated derivatives, HPb and HPd, displayed the most promising anti-HIV profile, paving the way to the optimization of the presented scaffolds for developing new effective antiviral agents.

The Design of New HIV-IN Tethered Bifunctional Inhibitors Using Multiple Microdomain Targeted Docking.

Currently, used antiretroviral HIV therapy drugs exclusively target critical groups in the enzymes essential for the viral life cycle. Increased mutagenesis of their genes changes these viral enzymes, which once mutated can evade therapeutic targeting, effects which confer drug resistance. To circumvent this, our review addresses a strategy to design and derive HIV-Integrase (HIV-IN) inhibitors which simultaneously target two IN functional domains, rendering it inactive even if the enzyme accumulates many mutations. First we review the enzymatic role of IN to insert the copied viral DNA into a chromosome of the host T lymphocyte, highlighting its main functional and structural features to be subjected to inhibitory action. From a functional and structural perspective we present all classes of HIV-IN inhibitors with their most representative candidates. For each chosen compound we also explain its mechanism of IN inhibition. We use the recently resolved cryo EM IN tetramer intasome DNA complex onto which we dock various reference IN inhibitory chemical scaffolds such as to target adjacent functional IN domains. Pairing compounds with complementary activity, which dock in the vicinity of a IN structural microdomain, we design bifunctional new drugs which may not only be more resilient to IN mutations but also may be more potent inhibitors than their original counterparts. In the end of our review we propose synthesis pathways to link such paired compounds with enhanced synergistic IN inhibitory effects.

HIV-1 Integrase-Targeted Short Peptides Derived from a Viral Protein R Sequence.

HIV-1 integrase (IN) inhibitors represent a new class of highly effective anti-AIDS therapeutics. Current FDA-approved IN strand transfer inhibitors (INSTIs) share a common mechanism of action that involves chelation of catalytic divalent metal ions. However, the emergence of IN mutants having reduced sensitivity to these inhibitors underlies efforts to derive agents that antagonize IN function by alternate mechanisms. Integrase along with the 96-residue multifunctional accessory protein, viral protein R (Vpr), are both components of the HIV-1 pre-integration complex (PIC). Coordinated interactions within the PIC are important for viral replication. Herein, we report a 7-mer peptide based on the shortened Vpr (69⁻75) sequence containing a biotin group and a photo-reactive benzoylphenylalanyl residue, and which exhibits low micromolar IN inhibitory potency. Photo-crosslinking experiments have indicated that the peptide directly binds IN. The peptide does not interfere with IN-DNA interactions or induce higher-order, aberrant IN multimerization, suggesting a mode of action for the peptide that is distinct from clinically used INSTIs and developmental allosteric IN inhibitors. This compact Vpr-derived peptide may serve as a valuable pharmacological tool to identify a potential new pharmacologic site.

5-Hydroxypyrido[2,3-b]pyrazin-6(5H)-one derivatives as novel dual inhibitors of HIV-1 reverse transcriptase-associated ribonuclease H and integrase.

We reported herein the design, synthesis and biological evaluation of a series of 5-hydroxypyrido[2,3-b]pyrazin-6(5H)-one derivatives as HIV-1 reverse transcriptase (RT) ribonuclease H (RNase H) inhibitors using a privileged structure-guided scaffold refining strategy. In view of the similarities between the pharmacophore model of RNase H and integrase (IN) inhibitors as well as their catalytic sites, we also performed IN inhibition assays. Notably, the majority of these derivatives inhibited RNase H and IN at micromolar concentrations. Among them, compound 7a exhibited similar inhibitory activity against RNase H and IN (IC50RNase H = 1.77 µM, IC50IN = 1.18 µM, ratio = 1.50). To the best of our knowledge, this is the first reported dual HIV-1 RNase H-IN inhibitor based on a 5-hydroxypyrido[2,3-b]pyrazin-6(5H)-one structure. Molecular modeling has been used to predict the binding mode of 7a in complex with the catalytic cores of HIV-1 RNase H and IN. Taken together these results strongly support the feasibility of developing HIV-1 dual inhibitors from analog-based optimization of divalent metal ion chelators. Recently, the identification of dual inhibitors proved to be a highly effective strategy for novel antivirals discovery. Therefore, these compounds appear to be useful leads that can be further modified to develop more valuable anti-HIV-1 molecules with suitable drug profiles.

7-Step Flow Synthesis of the HIV Integrase Inhibitor Dolutegravir.

Dolutegravir (DTG), an important active pharmaceutical ingredient (API) used in combination therapy for the treatment of HIV, has been synthesized in continuous flow. By adapting the reported GlaxoSmithKline process chemistry batch route for Cabotegravir, DTG was produced in 4.5 h in sequential flow operations from commercially available materials. Key features of the synthesis include rapid manufacturing time for pyridone formation, one-step direct amidation of a functionalized pyridone, and telescoping of multiple steps to avoid isolation of intermediates and enable for greater throughput.

The discovery and preclinical evaluation of BMS-707035, a potent HIV-1 integrase strand transfer inhibitor.

BMS-707035 is an HIV-1 integrase strand transfer inhibitor (INSTI) discovered by systematic optimization of N-methylpyrimidinone carboxamides guided by structure-activity relationships (SARs) and the single crystal X-ray structure of compound 10. It was rationalized that the unexpectedly advantageous profiles of N-methylpyrimidinone carboxamides with a saturated C2-substitutent may be due, in part, to the geometric relationship between the C2-substituent and the pyrimidinone core. The single crystal X-ray structure of 10 provided support for this reasoning and guided the design of a spirocyclic series 12 which led to discovery of the morpholino-fused pyrimidinone series 13. Several carboxamides derived from this bicyclic scaffold displayed improved antiviral activity and pharmacokinetic profiles when compared with corresponding spirocyclic analogs. Based on the excellent antiviral activity, preclinical profiles and acceptable in vitro and in vivo toxicity profiles, 13a (BMS-707035) was selected for advancement into phase I clinical trials.