Two centuries of biodiversity discovery and loss in Singapore.

There is an urgent need for reliable data on the impacts of deforestation on tropical biodiversity. The city-state of Singapore has one of the most detailed biodiversity records in the tropics, dating back to the turn of the 19th century. In 1819, Singapore was almost entirely covered in primary forest, but this has since been largely cleared. We compiled more than 200 y of records for 10 major taxonomic groups in Singapore (>50,000 individual records; >3,000 species), and we estimated extinction rates using recently developed and novel statistical models that account for "dark extinctions," i.e., extinctions of undiscovered species. The estimated overall extinction rate was 37% (95% CI [31 to 42%]). Extrapolating our Singapore observations to a future business-as-usual deforestation scenario for Southeast Asia suggests that 18% (95% CI [16 to 22%]) of species will be lost regionally by 2100. Our extinction estimates for Singapore and Southeast Asia are a factor of two lower than previous estimates that also attempted to account for dark extinctions. However, we caution that particular groups such as large mammals, forest-dependent birds, orchids, and butterflies are disproportionately vulnerable.

Design and exploration of C-3 benzoic acid bioisosteres and alkyl replacements in the context of GSK3532795 (BMS-955176) that exhibit broad spectrum HIV-1 maturation inhibition.

GSK3532795 (formerly BMS-955176) is a second-generation HIV-1 maturation inhibitor that has shown broad spectrum antiviral activity and preclinical PK predictive of once-daily dosing in humans. Although efficacy was confirmed in clinical trials, the observation of gastrointestinal intolerability and the emergence of drug resistant virus in a Phase 2b clinical study led to the discontinuation of GSK3532795. As part of the effort to further map the maturation inhibitor pharmacophore and provide additional structural options, the evaluation of alternates to the C-3 phenyl substituent in this chemotype was pursued. A cyclohexene carboxylic acid provided exceptional inhibition of wild-type, V370A and ΔV370 mutant viruses in addition to a suitable PK profile following oral dosing to rats. In addition, a novel spiro[3.3]hept-5-ene was designed to extend the carboxylic acid further from the triterpenoid core while reducing side chain flexibility compared to the other alkyl substituents. This modification was shown to closely emulate the C-3 benzoic acid moiety of GSK3532795 from both a potency and PK perspective, providing a non-traditional, sp3-rich bioisostere of benzene. Herein, we detail additional modifications to the C-3 position of the triterpenoid core that offer effective replacements for the benzoic acid of GSK3532795 and capture the interplay between these new C-3 elements and C-17 modifications that contribute to enhanced polymorph coverage.

Discovery and Optimization of Novel Pyrazolopyrimidines as Potent and Orally Bioavailable Allosteric HIV-1 Integrase Inhibitors.

The standard of care for HIV-1 infection, highly active antiretroviral therapy (HAART), combines two or more drugs from at least two classes. Even with the success of HAART, new drugs with novel mechanisms are needed to combat viral resistance, improve adherence, and mitigate toxicities. Active site inhibitors of HIV-1 integrase are clinically validated for the treatment of HIV-1 infection. Here we describe allosteric inhibitors of HIV-1 integrase that bind to the LEDGF/p75 interaction site and disrupt the structure of the integrase multimer that is required for the HIV-1 maturation. A series of pyrazolopyrimidine-based inhibitors was developed with a vector in the 2-position that was optimized by structure-guided compound design. This resulted in the discovery of pyrazolopyrimidine 3, which was optimized at the 2- and 7-positions to afford 26 and 29 as potent allosteric inhibitors of HIV-1 integrase that exhibited low nanomolar antiviral potency in cell culture and encouraging PK properties.

Primary Localized Cutaneous Amyloidosis Affecting Female Individuals of a Pakistani Pedigree.

Primary localized cutaneous amyloidosis is a group of rare conditions where amyloid deposition is limited to the skin without systemic manifestations. Most cases are sporadic; however, mutations in the oncostatin M receptor (OSMR) and interleukin-31 receptor A (IL31RA) genes can cause a familial form of the condition in up to 10% of cases. Here, we describe a family in which 8 female individuals are affected by either macular amyloidosis or amyloidosis cutis dyschromica. To the best of our knowledge, a sex-specific expression or the coexistence of 2 different phenotypes of primary localized cutaneous amyloidosis in 1 pedigree has not yet been reported.

Enantioselective Synthesis of a γ-Secretase Modulator via Vinylogous Dynamic Kinetic Resolution.

Two efficient asymmetric routes to γ-secretase modulator BMS-932481, under investigation for Alzheimer's disease, have been developed. The key step for the first route involves a challenging enantioselective hydrogenation of an unfunctionalized trisubstituted alkene to establish the benzylic stereocenter, representing a very rare case of achieving high selectivity on a complex substrate. The second route demonstrates the first example of a vinylogous dynamic kinetic resolution (VDKR) ketone reduction, where the carbonyl and the racemizable stereocenter are not contiguous, but are conjugated through a pyrimidine ring. Not only did this transformation require both catalyst and substrate control to correctly establish the two stereocenters, but it also necessitated that the nonadjacent benzylic center of the ketone substrate be more acidic than that of the alcohol product to make the process dynamic. DFT computations aided the design of this novel VDKR pathway by reliably predicting the relative acidities of the intermediates involved.

BMS-986163, a Negative Allosteric Modulator of GluN2B with Potential Utility in Major Depressive Disorder.

There is a significant unmet medical need for more efficacious and rapidly acting antidepressants. Toward this end, negative allosteric modulators of the N-methyl-d-aspartate receptor subtype GluN2B have demonstrated encouraging therapeutic potential. We report herein the discovery and preclinical profile of a water-soluble intravenous prodrug BMS-986163 (6) and its active parent molecule BMS-986169 (5), which demonstrated high binding affinity for the GluN2B allosteric site (Ki = 4.0 nM) and selective inhibition of GluN2B receptor function (IC50 = 24 nM) in cells. The conversion of prodrug 6 to parent 5 was rapid in vitro and in vivo across preclinical species. After intravenous administration, compounds 5 and 6 have exhibited robust levels of ex vivo GluN2B target engagement in rodents and antidepressant-like activity in mice. No significant off-target activity was observed for 5, 6, or the major circulating metabolites met-1 and met-2. The prodrug BMS-986163 (6) has demonstrated an acceptable safety and toxicology profile and was selected as a preclinical candidate for further evaluation in major depressive disorder.

Potent Inhibitors of Hepatitis C Virus NS3 Protease: Employment of a Difluoromethyl Group as a Hydrogen-Bond Donor.

The design and synthesis of potent, tripeptidic acylsulfonamide inhibitors of HCV NS3 protease that contain a difluoromethyl cyclopropyl amino acid at P1 are described. A cocrystal structure of 18 with a NS3/4A protease complex suggests the presence of a H-bond between the polarized C-H of the CHF2 moiety and the backbone carbonyl of Leu135 of the enzyme. Structure-activity relationship studies indicate that this H-bond enhances enzyme inhibitory potency by 13- and 17-fold compared to the CH3 and CF3 analogues, respectively, providing insight into the deployment of this unique amino acid.

Discovery of non-zwitterionic aryl sulfonamides as Nav1.7 inhibitors with efficacy in preclinical behavioral models and translational measures of nociceptive neuron activation.

Since zwitterionic benzenesulfonamide Nav1.7 inhibitors suffer from poor membrane permeability, we sought to eliminate this characteristic by replacing the basic moiety with non-basic bicyclic acetals and monocyclic ethers. These efforts led to the discovery of the non-zwitterionic aryl sulfonamide 49 as a selective Nav1.7 inhibitor with improved membrane permeability. Despite its moderate cellular activity, 49 exhibited robust efficacy in mouse models of neuropathic and inflammatory pain and modulated translational electromyogram measures associated with activation of nociceptive neurons.

Discovery of a Hepatitis C Virus NS5B Replicase Palm Site Allosteric Inhibitor (BMS-929075) Advanced to Phase 1 Clinical Studies.

The hepatitis C virus (HCV) NS5B replicase is a prime target for the development of direct-acting antiviral drugs for the treatment of chronic HCV infection. Inspired by the overlay of bound structures of three structurally distinct NS5B palm site allosteric inhibitors, the high-throughput screening hit anthranilic acid 4, the known benzofuran analogue 5, and the benzothiadiazine derivative 6, an optimization process utilizing the simple benzofuran template 7 as a starting point for a fragment growing approach was pursued. A delicate balance of molecular properties achieved via disciplined lipophilicity changes was essential to achieve both high affinity binding and a stringent targeted absorption, distribution, metabolism, and excretion profile. These efforts led to the discovery of BMS-929075 (37), which maintained ligand efficiency relative to early leads, demonstrated efficacy in a triple combination regimen in HCV replicon cells, and exhibited consistently high oral bioavailability and pharmacokinetic parameters across preclinical animal species. The human PK properties from the Phase I clinical studies of 37 were better than anticipated and suggest promising potential for QD administration.

Asymmetric Synthesis of the Major Metabolite of a Calcitonin Gene-Related Peptide Receptor Antagonist and Mechanism of Epoxide Hydrogenolysis.

An asymmetric synthesis of the major metabolite of the calcitonin gene-related peptide recepotor antagonist BMS-846372 is presented. The variously substituted cyclohepta[b]pyridine ring system represents an underexplored ring system and showed some unexpected chemistry. Reactivities of epoxide and ketone functional groups on the cycloheptane ring were extensively controlled by a remote bulky TIPS group. The rate difference of the hydrogenolysis between two diastereomeric epoxide intermediates shed some light on the mechanism of epoxide hydrogenolysis, and further, deuterium labeling studies revealed more mechanistic details on this well-known chemical transformation for the first time.

Discovery of a Potent Acyclic, Tripeptidic, Acyl Sulfonamide Inhibitor of Hepatitis C Virus NS3 Protease as a Back-up to Asunaprevir with the Potential for Once-Daily Dosing.

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).

Targeting the BACE1 Active Site Flap Leads to a Potent Inhibitor That Elicits Robust Brain Aß Reduction in Rodents.

By targeting the flap backbone of the BACE1 active site, we discovered 6-dimethylisoxazole-substituted biaryl aminothiazine 18 with 34-fold improved BACE1 inhibitory activity over the lead compound 1. The cocrystal structure of 18 bound to the active site indicated two hydrogen-bond interactions between the dimethylisoxazole and threonine 72 and glutamine 73 of the flap. Incorporation of the dimethylisoxazole substitution onto the related aminothiazine carboxamide series led to pyrazine-carboxamide 26 as a very potent BACE1 inhibitor (IC50 < 1 nM). This compound demonstrated robust brain Aß reduction in rat dose-response studies. Thus, compound 26 may be useful in testing the amyloid hypothesis of Alzheimer's disease.

Insight to the thermodynamic stability of molecular crystals through crystallographic studies of a multipolymorph system.

Five solvent-free polymorphs of a pharmaceutical compound were discovered during polymorph screening. Out of the five polymorphs, only one has strong intermolecular N-H···N hydrogen bonding, whereas the others exhibit only weak C-H···N and π-π stacking interactions in addition to all the other weak C-H···X and van der Waals interactions. The relative thermodynamic stability relationships among the polymorphs are not intuitive and quite complex due to enantiotropic phase behavior. For instance, the polymorph with the most efficient packing (i.e., highest density) is not always the most thermodynamically stable form, and the polymorph with strong intermolecular interactions is not thermodynamically more stable than the polymorph with weak intermolecular interactions at all temperatures. Nevertheless, systematic examination and comparison of the molecular packing and intermolecular interactions of these polymorphs provide insight into the importance of H-bonding and packing efficiency to the thermodynamic stability of a crystalline form, and how these effects are dependent on temperature. This study seeks to correlate single-crystal structure features with experimentally established thermodynamic stability, and provides an example where a polymorph with only van der Waals forces and weak intermolecular interactions can be more stable than a polymorph that displays strong H-bonding in its structural make-up.

Design and synthesis of 4-heteroaryl 1,2,3,4-tetrahydroisoquinolines as triple reuptake inhibitors.

A series of 4-bicyclic heteroaryl 1,2,3,4-tetrahydroisoquinoline inhibitors of the serotonin transporter (SERT), norepinephrine transporter (NET), and dopamine transporter (DAT) was discovered. The synthesis and structure-activity relationship (SAR) of these triple reuptake inhibitors (TRIs) will be discussed. Compound 10i (AMR-2), a very potent inhibitor of SERT, NET, and DAT, showed efficacy in the rat forced-swim and mouse tail suspension models with minimum effective doses of 0.3 and 1 mg/kg (po), respectively. At efficacious doses in these assays, 10i exhibited substantial occupancy levels at the three transporters in both rat and mouse brain. The study of the metabolism of 10i revealed the formation of a significant active metabolite, compound 13.

Discovery and preclinical characterization of the cyclopropylindolobenzazepine BMS-791325, a potent allosteric inhibitor of the hepatitis C virus NS5B polymerase.

Described herein are structure-activity relationship studies that resulted in the optimization of the activity of members of a class of cyclopropyl-fused indolobenzazepine HCV NS5B polymerase inhibitors. Subsequent iterations of analogue design and syntheses successfully addressed off-target activities, most notably human pregnane X receptor (hPXR) transactivation, and led to significant improvements in the physicochemical properties of lead compounds. Those analogues exhibiting improved solubility and membrane permeability were shown to have notably enhanced pharmacokinetic profiles. Additionally, a series of alkyl bridged piperazine carboxamides was identified as being of particular interest, and from which the compound BMS-791325 (2) was found to have distinguishing antiviral, safety, and pharmacokinetic properties that resulted in its selection for clinical evaluation.

Recognition of mitochondrial targeting sequences by the import receptors Tom20 and Tom22.

The Tom20 and Tom22 receptor subunits of the TOM (translocase of the outer mitochondrial membrane) complex recognize N-terminal presequences of proteins that are to be imported into the mitochondrion. In plants, Tom20 is C-terminally anchored in the mitochondrial membrane, whereas Tom20 is N-terminally anchored in animals and fungi. Furthermore, the cytosolic domain of Tom22 in plants is smaller than its animal/fungal counterpart and contains fewer acidic residues. Here, NMR spectroscopy was used to explore presequence interactions with the cytosolic regions of receptors from the plant Arabidopsis thaliana and the fungus Saccharomyces cerevisiae (i.e., AtTom20, AtTom22, and ScTom22). It was found that AtTom20 possesses a discontinuous bidentate hydrophobic binding site for presequences. The presequences on plant mitochondrial proteins comprise two or more hydrophobic binding regions to match this bidentate site. NMR data suggested that while these presequences bind to ScTom22, they do not bind to AtTom22. AtTom22, however, binds to AtTom20 at the same binding site as presequences, suggesting that this domain competes with the presequences of imported proteins, thereby enabling their progression along the import pathway.

A synthesis of N-bridged 5,6-bicylic pyridines via a mild cyclodehydration using the burgess reagent and discovery of a novel carbamylsulfonylation reaction.

Cyclodehydration using the Burgess reagent provided a novel approach toward the synthesis of N-bridged 5,6-bicylic pyridines including pyrolo-, imidazo-, and triazolopyridines under mild and neutral conditions. The methodology tolerates acid-sensitive functional groups. A novel addition product was observed between the resulting pyrrolo- or imidazopyridine and an additional equivalent of the Burgess reagent, producing the corresponding sulfonylcarbamate adduct.