Rapid discovery and crystallography study of highly potent and selective butylcholinesterase inhibitors based on oxime-containing libraries and conformational restriction strategies.

Butyrylcholinesterase is regarded as a promising drug target in advanced Alzheimer's disease. In order to identify highly selective and potent BuChE inhibitors, a 53-membered compound library was constructed via the oxime-based tethering approach based on microscale synthesis. Although A2Q17 and A3Q12 exhibited higher BuChE selectivity versus acetylcholinesterase, the inhibitory activities were unsatisfactory and A3Q12 did not inhibit Aß1-42 peptide self-induced aggregation. With A2Q17 and A3Q12 as leads, a novel series of tacrine derivatives with nitrogen-containing heterocycles were designed based on conformation restriction strategy. The results demonstrated that 39 (IC50 = 3.49 nM) and 43 (IC50 = 7.44 nM) yielded much improved hBuChE inhibitory activity compared to the lead A3Q12 (IC50 = 63 nM). Besides, the selectivity indexes (SI = AChE IC50 / BChE IC50) of 39 (SI = 33) and 43 (SI = 20) were also higher than A3Q12 (SI = 14). The results of the kinetic study showed that 39 and 43 exhibited a mixed-type inhibition against eqBuChE with respective Ki values of 1.715 nM and 0.781 nM. And 39 and 43 could inhibit Aß1-42 peptide self-induced aggregation into fibril. X-ray crystallography structures of 39 or 43 complexes with BuChE revealed the molecular basis for their high potency. Thus, 39 and 43 are deserve for further study to develop potential drug candidates for the treatment of Alzheimer's disease.

Discovery of potent and selective Cdc25 phosphatase inhibitors via rapid assembly and in situ screening of Quinonoid-focused libraries.

Cell division cycle 25 (Cdc25) phosphatase is an attractive target for drug discovery. The rapid assembly and in situ screening of focused combinatorial fragment libraries using efficient modular reactions is a highly robust strategy for discovering bioactive molecules. In this study, we have utilized miniaturized synthesis to generate several quinonoid-focused libraries, by standard CuAAC reaction and HBTU-based amide coupling chemistry. Then the enzyme inhibition screening afforded some potent and selective Cdc25s inhibitors. Compound M5N36 (Cdc25A: IC50 = 0.15 ± 0.05 µM; Cdc25B: IC50 = 0.19 ± 0.06 µM; Cdc25C: IC50 = 0.06 ± 0.04 µM) exhibited higher inhibitory activity than the initial lead NSC663284 (Cdc25A: IC50 = 0.27 ± 0.02 µM; Cdc25B: IC50 = 0.42 ± 0.01 µM; Cdc25C: IC50 = 0.23 ± 0.01 µM). Moreover, M5N36 displayed about three-fold more potent against Cdc25C than Cdc25A and B, indicating that M5N36 could act as a relatively selective Cdc25C inhibitor. Cell viability evaluation, western blotting and molecular simulations provided a mechanistic understanding of the activity of M5N36. It showed promising anti-growth activity against the MDA-MB-231 cell line and desirable predicted physicochemical properties. Overall, M5N36 was proven to be a promising novel Cdc25C inhibitor.

In situ click chemistry-based rapid discovery of novel HIV-1 NNRTIs by exploiting the hydrophobic channel and tolerant regions of NNIBP.

HIV-1 RT has been considered as one of the most important targets for the development of anti-HIV-1 drugs for their well-solved three-dimensional structure and well-known mechanism of action. In this study, with HIV-1 RT as target, we used miniaturized parallel click chemistry synthesis via CuAAC reaction followed by in situ biological screening to discover novel potent HIV-1 NNRTIs. A 156 triazole-containing inhibitor library was assembled in microtiter plates and in millimolar scale. The enzyme inhibition screening results showed that 22 compounds exhibited improved inhibitory activity. Anti-HIV-1 activity results demonstrated that A3N19 effected the most potent activity against HIV-1 IIIB (EC50 = 3.28 nM) and mutant strain RES056 (EC50 = 481 nM). The molecular simulation analysis suggested that the hydrogen bonding interactions of A3N19 with the main chain of Lys101 and Lys104 was responsible for its potency. Overall, the results indicated the in situ click chemistry-based strategy was rational and might be amenable for the future discovery of more potent HIV-1 NNRTIs.

Identification of highly potent and selective Cdc25 protein phosphatases inhibitors from miniaturization click-chemistry-based combinatorial libraries.

Cell division cycle 25 (Cdc25) protein phosphatases play key roles in the transition between the cell cycle phases and their association with various cancers has been widely proven, which makes them ideal targets for anti-cancer treatment. Though several Cdc25 inhibitors have been developed, most of them displayed low activity and poor subtype selectivity. Therefore, it is extremely important to discover novel small molecule inhibitors with potent activities and significant selectivity for Cdc25 subtypes, not only served as drugs to treat cancer but also to probe its mechanism in transitions. In this study, miniaturized parallel click chemistry synthesis via CuAAC reaction followed by in situ biological screening were used to discover selective Cdc25 inhibitors. The bioassay results showed that compound M2N12 proved to be the most potent Cdc25 inhibitor, which also act as a highly selective Cdc25C inhibitor and was about 9-fold potent than that of NSC 663284. Moreover, M2N12 showed remarkable anti-growth activity against the KB-VIN cell line, equivalent to that of PXL and NSC 663284. An all-atom molecular dynamics (MD) simulation approach was further employed to probe the significant selectivity of M2N12 for Cdc25C relative to its structural homologs Cdc25A and Cdc25B. Overall, above results make M2N12 a promising lead compound for further investigation and structural modification.

Recent applications of click chemistry in drug discovery.

Introduction: Click chemistry has been exploited widely in the past to expedite lead discovery and optimization. Indeed, Copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry is a bioorthogonal reaction of widespread utility throughout medicinal chemistry and chemical biology. Areas covered: The authors review recent applications of CuAAC click chemistry to drug discovery based on the literature published since 2013. Furthermore, the authors provide the reader with their expert perspectives on the area including their outlook on future developments. Expert opinion: Click chemistry reactions are an important part of the medicinal chemistry toolbox and offer substantial advantages to medicinal chemists in terms of overcoming the limitations of useful chemical synthesis, increasing throughput, and improving the quality of compound libraries. To explore new chemical spaces for drug-like molecules containing a high degree of structural diversity, it may be useful to merge the diversity-oriented synthesis and 'privileged' substructure-based strategy with bioorthogonal reactions using sophisticated automation and flow systems to improve productivity. Large compound libraries obtained in this way should be of great value for the discovery of bioactive compounds and therapeutic agents.

Overview of Recent Strategic Advances in Medicinal Chemistry.

Introducing novel strategies, concepts, and technologies that speed up drug discovery and the drug development cycle is of great importance both in the highly competitive pharmaceutical industry as well as in academia. This Perspective aims to present a "big-picture" overview of recent strategic innovations in medicinal chemistry and drug discovery.

Targeting the hydrophobic channel of NNIBP: discovery of novel 1,2,3-triazole-derived diarylpyrimidines as novel HIV-1 NNRTIs with high potency against wild-type and K103N mutant virus.

Enlightened by our previous efforts to modify diarylpyrimidines as HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) and the reported crystallographic studies, we designed and synthesized novel 1,2,3-triazole-derived diarylpyrimidine derivatives via the CuAAC "click reaction", to make additional interactions with the hydrophobic channel in the NNRTI binding pocket. The newly synthesized compounds were evaluated for anti-HIV potency in MT-4 cells. All the compounds showed favorable activity against the wild-type HIV-1 strain with an EC50 of 0.013-5.62 µM. Interestingly, some compounds displayed remarkable potency in inhibiting K103N mutant virus, a key drug-resistant mutant to NNRTIs. Among them, meta-methylbenzoate (ZL2, EC50(IIIB) = 0.020 µM, EC50(K103N) = 0.043 µM, CC50 > 241.52 µM), para-methylbenzoate (ZL3, EC50(IIIB) = 0.013 µM, EC50 (K103N) = 0.022 µM, CC50 > 241.52 µM) and para-phenol (ZL7, EC50(IIIB) = 0.014 µM, EC50 (K103N) = 0.054 µM, CC50 = 2.1 µM) derivatives are the three most promising compounds which are superior to the first-line antiretroviral drug efavirenz (EC50(IIIB) = 0.003 µM, EC50 (K103N) = 0.11 µM, CC50 > 6.34 µM) against the K103N mutant strain. More encouragingly, ZL2 and ZL3 exhibited much lower cytotoxicity and a high selection index of >10 000 compared with all the control drugs (AZT, 3TC, NVP, EFV, and ETV). The detailed structure-activity relationship (SAR), enzymatic inhibitory activity and docking study of the representative compounds are also discussed. Furthermore, the preliminary physicochemical properties and the early metabolic stability of representative compounds were examined to evaluate their drug-like properties.

Identification of Dihydrofuro[3,4- d]pyrimidine Derivatives as Novel HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors with Promising Antiviral Activities and Desirable Physicochemical Properties.

To address drug resistance to HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs), a series of novel diarylpyrimidine (DAPY) derivatives targeting "tolerant region I" and "tolerant region II" of the NNRTIs binding pocket (NNIBP) were designed utilizing a structure-guided scaffold-hopping strategy. The dihydrofuro[3,4- d]pyrimidine derivatives 13c2 and 13c4 proved to be exceptionally potent against a wide range of HIV-1 strains carrying single NNRTI-resistant mutations (EC50 = 0.9-8.4 nM), which were remarkably superior to that of etravirine (ETV). Meanwhile, both compounds exhibited comparable activities with ETV toward the virus with double mutations F227L+V106A and K103N+Y181C. Furthermore, the most active compound 13c2 showed favorable pharmacokinetic properties with an oral bioavailability of 30.96% and a half-life of 11.1 h, which suggested that 13c2 is worth further investigation as a novel NNRTI to circumvent drug resistance.

Discovery of novel 1,4-disubstituted 1,2,3-triazole phenylalanine derivatives as HIV-1 capsid inhibitors.

The HIV-1 capsid (CA) protein plays crucial roles in both early and late stages of the viral life cycle, which has intrigued researchers to target it to develop anti-HIV drugs. Accordingly, in this research, we report the design, synthesis and biological evaluation of a series of novel phenylalanine derivatives as HIV-1 CA protein inhibitors using the Cu(I)-catalyzed azide and alkyne 1,3-dipolar cycloaddition (CuAAC) reaction. Among this series of inhibitors, compound II-10c displayed a remarkable anti-HIV activity (EC50 = 2.13 µM, CC50 > 35.49 µM). Furthermore, surface plasmon resonance (SPR) binding assays showed that compounds II-10c and PF-74 (lead compound) have similar affinities to HIV-1 CA monomer. Further investigation showed that the weak permeability and water solubility of representative compounds were probably the important factors that restricted their cell-based activity. Preliminary structure-activity relationships (SARs) were inferred based on the activities of these compounds, and their known structure. The most promising new compound was studied with molecular dynamics simulation (MD) to determine the preferred interactions with the drug target. Finally, the activities of members of this series of inhibitors were deeply inspected to find the potential reasons for their anti-HIV-1 activity from various perspectives. This highlights the important factors required to design compounds with improved potency.

Contemporary medicinal-chemistry strategies for the discovery of selective butyrylcholinesterase inhibitors.

Butyrylcholinesterase (BChE) is considered a promising drug target for the treatment of moderate to severe Alzheimer's disease (AD). Here, we review medicinal-chemistry strategies that are currently available for the discovery of selective BChE inhibitors.

Discovery of potent HIV-1 non-nucleoside reverse transcriptase inhibitors by exploring the structure-activity relationship of solvent-exposed regions I.

Two novel series of human immunodeficiency virus-1 (HIV-1) non-nucleoside reverse transcriptase inhibitors (NNRTIs) bearing a thiophene[3,2-d]pyrimidine scaffold and sulfonamide linker in the right wing have been identified, which demonstrated activity against the wild-type (WT) HIV-1 strain in MT-4 cells with inhibitory concentrations ranging from micromolar to submicromolar. Especially, against the mutant strains K103N and E138K, most compounds exhibited more potent activity than against WT HIV-1. Compound 7 (EC50  = 0.014, 0.031 µM) achieved the most potent activity against the two mutants, being more effective than that of nevirapine (NVP, EC50  = 7.572, 0.190 µM) and comparable to that of etravirine (ETV, EC50  = 0.004, 0.014 µM). Molecular docking experiments on the novel analogs have also suggested that the extensive network of main chain hydrogen bonds are important in the binding mode, which may provide valuable insights for further optimization.

Discovery of phenylalanine derivatives as potent HIV-1 capsid inhibitors from click chemistry-based compound library.

The HIV-1 capsid (CA) protein plays essential roles in both early and late stages of HIV-1 replication and is considered an important, clinically unexploited therapeutic target. As such, small drug-like molecules that inhibit this critical HIV-1 protein have become a priority for several groups. Therefore, in this study we explore small molecule targeting of the CA protein, and in particular a very attractive inter-protomer pocket. We report the design, parallel synthesis, and anti-HIV-1 activity evaluation of a series of novel phenylalanine derivatives as HIV-1 CA protein inhibitors synthesized via Cu(I)-catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction. We demonstrate robust inhibitory activity over a range of potencies against the HIV-1 NL4-3 reference strain. In particular, compound 13m exhibited the greatest potency and lowest toxicity within this new series with an EC50 value of 4.33 µM and CC50 value of >57.74 µM (SI > 13.33). These values are very similar to the lead compound PF-74 (EC50 = 5.95 µM, CC50 > 70.50 µM, SI > 11.85) in our assay, despite significant structural difference. Furthermore, we demonstrate via surface plasmon resonance (SPR) binding assays that 13m interacts robustly with recombinant HIV-1 CA and exhibits antiviral activity in both the early and late stages of HIV-1 replication. Overall, the novel parallel synthesis and structure-activity relationships (SARs) identified within this study set the foundation for further rational optimization and discovery of CA-targeting compounds with improved potency.

Design, synthesis and biological evaluation of tacrine-1,2,3-triazole derivatives as potent cholinesterase inhibitors.

We report herein the design and synthesis of a series of 11 novel tacrine-1,2,3-triazole derivatives via a Cu(i)-catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction. The newly synthesized compounds were evaluated for their inhibition activity against Electrophorus electricus acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) as potential drug targets for Alzheimer's disease (AD). Among the designed compounds, compound 8a2 exhibited potent inhibition against AChE and BChE with IC50 values of 4.89 µM and 3.61 µM, respectively. Further structure-activity relationship (SAR) and molecular modeling studies may provide valuable insights into the design of better tacrine-triazole analogues with potential therapeutic applications for AD.

Design, synthesis, and antiviral evaluation of novel hydrazone-substituted thiophene[3,2-d]pyrimidine derivatives as potent human immunodeficiency virus-1 inhibitors.

In the previous studies of our laboratory, the thiophene[3,2-d]pyrimidine was identified as a promising scaffold for seeking highly potent HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). In this study, we designed, synthesized, and biologically evaluated a series of thiophene[3,2-d]pyrimidine derivatives with changed linker between the thiophenepyrimidine core and the right wing. Some of the synthesized compounds exhibited excellent HIV-1 inhibitory potency with low (double-digit) nanomolar 50% effective concentration (EC50 ) values. Among them, compound 13a exhibited the most potent anti-HIV-1 activity (EC50  = 21.2 nM), which was 10-fold greater than that of NVP (EC50  = 281 nM). Moreover, 13a showed much lower cytotoxicity (CC50  = 183 µM) and higher selection index (SI = 8,632) than NVP, ETV, and AZT. Besides, some physicochemical properties and water solubility were calculated or measured. The preliminary structure-activity relationships and molecular simulation studies of these compounds were also discussed comprehensively to provide valuable direction for further design and optimization.

Discovery of Novel Diarylpyrimidine Derivatives as Potent HIV-1 NNRTIs Targeting the "NNRTI Adjacent" Binding Site.

A novel series of diarylpyrimidine derivatives, which could simultaneously occupy the classical NNRTIs binding pocket (NNIBP) and the newly reported "NNRTI Adjacent" binding site, were designed, synthesized, and evaluated for their antiviral activities in MT-4 cell cultures. The results demonstrated that six compounds (20, 27 and 31-34) showed excellent activities against wild-type (WT) HIV-1 strain (EC50 = 2.4-3.8 nM), which were more potent than that of ETV (EC50 = 4.0 nM). Furthermore, 20, 27, 33, and 34 showed more potent or equipotent activity against single mutant HIV-1 strains compared to that of ETV. Especially, 20 showed marked antiviral activity, which was 1.5-fold greater against WT and 1.5- to 3-fold greater against L100I, K103N, Y181C, Y188L, and E138K when compared with ETV. In addition, all compounds showed lower toxicity (CC50 = 5.1-149.2 µM) than ETV (CC50 = 2.2 µM). The HIV-1 RT inhibitory assay was further conducted to confirm their binding target. Preliminary structure-activity relationships (SARs), molecular modeling, and calculated physicochemical properties of selected compounds were also discussed comprehensively.

Further Exploring Solvent-Exposed Tolerant Regions of Allosteric Binding Pocket for Novel HIV-1 NNRTIs Discovery.

Based on the detailed analysis of the binding mode of diarylpyrimidines (DAPYs) with HIV-1 RT, we designed several subseries of novel NNRTIs, with the aim to probe biologically relevant chemical space of solvent-exposed tolerant regions in NNRTIs binding pocket (NNIBP). The most potent compound 21a exhibited significant activity against the whole viral panel, being about 1.5-2.6-fold (WT, EC50 = 2.44 nM; L100I, EC50 = 4.24 nM; Y181C, EC50 = 4.80 nM; F227L + V106A, EC50 = 17.8 nM) and 4-5-fold (K103N, EC50 = 1.03 nM; Y188L, EC50 = 7.16 nM; E138K, EC50 = 3.95 nM) more potent than the reference drug ETV. Furthermore, molecular simulation was conducted to understand the binding mode of interactions of these novel NNRTIs and to provide insights for the next optimization studies.

Current insights into anti-HIV drug discovery and development: a review of recent patent literature (2014-2017).

INTRODUCTION: To deal with the rapid emergence of drug resistance challenges, together with the difficulty to eradicate the virus, off-target effects and significant cumulative drug toxicities, it is still imperative to develop next-generation anti-HIV agents with novel chemical classes or new mechanisms of action. AREAS COVERED: We primarily focused on current strategies to discover novel anti-HIV agents. Moreover, examples of anti-HIV lead compounds were mainly selected from recently patented publications (reported between 2014 and 2017). In particular, 'privileged structure'-focused substituents decorating approach, scaffold hopping, natural-product diversification and prodrug are focused on. Furthermore, exploitation of new compounds with unexplored mechanisms of action and medicinal chemistry strategies to deplete the HIV reservoir were also described. Perspectives that could inspire future anti-HIV drug discovery are delineated. EXPERT OPINION: Even if a large number of patents have been disclosed recently, additional HIV inhibitors are still required, especially novel chemical skeletons displaying a unexploited mechanism of action. Current medicinal chemistry strategies are inadequate, and appropriate and new methodologies and technologies should be exploited to identify novel anti-HIV drug candidates in a time- and cost- effective manner.

Discovery of novel diarylpyrimidines as potent HIV-1 NNRTIs by investigating the chemical space of a less explored "hydrophobic channel".

A new series of diarylpyrimidines (DAPYs) were designed, synthesized and evaluated as novel HIV-1 NNRTIs to further explore the chemical space surrounding the "hydrophobic channel" of the NNRTI binding pocket (NNIBP), guided by the comprehensive analysis of X-ray structural biology data of HIV-1 RT/NNRTI complexes and molecular modeling. Encouragingly, most of the synthesized DAPYs were found to be active against the HIV-1 wild-type (WT) strain with EC50 values ranging from 3 nM to 63 nM, and displayed significantly reduced cytotoxicity compared with etravirine (ETV) and rilpivirine (RPV). Among them, two most promising compounds Z10 (EC50 = 3 nM) and Z13 (EC50 = 3 nM) showed equivalent potency against the HIV-1 WT strain to the reference drugs efavirenz (EFV, EC50 = 3 nM) and ETV (EC50 = 3 nM). Notably, Z13 also showed the most potent activity against HIV-1 mutant strains including K103N (EC50 = 10 nM), E138K (EC50 = 22 nM) and RES056 (EC50 = 0.935 µM). Against mutant strains Y181C, Y188L and F227L + V106A, Z17 showed double-digit nanomolar inhibitory activity with EC50 values 27 nM, 98 nM and 30 nM, respectively. The structure-activity relationships (SARs) and molecular docking studies provided important clues for further molecular elaboration. Collectively, this study provides useful information to guide lead optimization and drug discovery via the exploration of this seldom investigated region.

Discovery of Thiophene[3,2-d]pyrimidine Derivatives as Potent HIV-1 NNRTIs Targeting the Tolerant Region I of NNIBP.

Our previous studies led us to conclude that thiophene[3,2-d]pyrimidine is a promising scaffold for diarylpyrimidine (DAPY)-type anti-HIV agents with potent activity against resistance-associated human immunodeficiency virus (HIV) variants (J. Med. Chem. 2016, 59, 7991-8007; J. Med. Chem. 2017, 60, 4424-4443). In the present study, we designed and synthesized a series of thiophenepyrimidine derivatives with various substituents in the right wing region of the structure with the aim of developing new interactions with the tolerant region I of the binding pocket of the HIV-1 non-nucleoside reverse transcriptase (NNRTI), and we evaluated their activity against a panel of mutant HIV-1 strains. All the derivatives exhibited moderate to excellent potency against wild-type (WT) HIV-1 in MT-4 cells. Among them, sulfonamide compounds 9b and 9d were single-figure-nanomolar inhibitors with EC50 values of 9.2 and 7.1 nM, respectively. Indeed, 9a and 9d were effective against the whole viral panel except RES056. Notably, both compounds showed potent antiviral activity against K103N (EC50 = 0.032 and 0.070 µM) and E138K (EC50 = 0.035 and 0.045 µM, respectively). Furthermore, 9a and 9d exhibited high affinity for WT HIV-1 RT (IC50 = 1.041 and 1.138 µM, respectively) and acted as classical NNRT inhibitors (NNRTIs). These results are expected to be helpful in the design of thiophenepyrimidine-based NNRTIs with more potent activity against HIV strains with RT mutations.

Structure-Based Optimization of Thiophene[3,2-d]pyrimidine Derivatives as Potent HIV-1 Non-nucleoside Reverse Transcriptase Inhibitors with Improved Potency against Resistance-Associated Variants.

This work follows on from our initial discovery of a series of piperidine-substituted thiophene[3,2-d]pyrimidine HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTI) ( J. Med. Chem. 2016 , 59 , 7991 - 8007 ). In the present study, we designed, synthesized, and biologically tested several series of new derivatives in order to investigate previously unexplored chemical space. Some of the synthesized compounds displayed single-digit nanomolar anti-HIV potencies against wild-type (WT) virus and a panel of NNRTI-resistant mutant viruses in MT-4 cells. Compound 25a was exceptionally potent against the whole viral panel, affording 3-4-fold enhancement of in vitro antiviral potency against WT, L100I, K103N, Y181C, Y188L, E138K, and K103N+Y181C and 10-fold enhancement against F227L+V106A relative to the reference drug etravirine (ETV) in the same cellular assay. The structure-activity relationships, pharmacokinetics, acute toxicity, and cardiotoxicity were also examined. Overall, the results indicate that 25a is a promising new drug candidate for treatment of HIV-1 infection.