Picomolar inhibitor of reverse transcriptase featuring significantly improved metabolic stability

Considering the undesirable metabolic stability of our recently identified NNRTI 5(t1/2=96 min)in human liver microsomes,we directed our efforts to improve its metabolic stability by introducing a new favorable hydroxymethyl side chain to the C-5 position of pyrimidine.This strategy provided a series of novel methylol-biphenyl-diarylpyrimidines with excellent anti-HIV-1 activity.The best compound 9g was endowed with remarkably improved metabolic stability in human liver microsomes(t1/2=2754 min),which was about 29-fold longer than that of 5(t1/2=96 min).This compound conferred picomolar inhibition of WT HIV-1(EC50=0.9 nmol/L)and low nanomolar activity against five clinically drug-resistant mutant strains.It maintained particularly low cytotoxicity(CC50=264μmol/L)and good selectivity(SI=256,438).Molecular docking studies revealed that compound 9g exhibited a more stable conformation than 5 due to the newly constructed hydrogen bond of the hydroxymethyl group with E138.Also,compound 9g was characterized by good safety profiles.It displayed no apparent inhibition of CYP enzymes and h ERG.The acute toxicity assay did not cause death and pathological damage in mice at a single dose of 2 g/kg.These findings paved the way for the discovery and development of new-generation anti-HIV-1 drugs.

Development of fluorine-substituted NH_(2)-biphenyl-diarylpyrimidines as highly potent non-nucleoside reverse transcriptase inhibitors:Boosting the safety and metabolic stability

Our recent studies for nonnucleoside reverse transcriptase inhibitors identified a highly potent compound JK-4b against WT HIV-1(EC_(50)=1.0 nmol/L),but the poor metabolic stability in human liver microsomes(t_(1/2)=14.6 min)and insufficient selectivity(SI=2059)with high cytotoxicity(CC_(50)=2.08μmol/L)remained major issues associated with JK-4b.The present efforts were devoted to the introduction of fluorine into the biphenyl ring of JK-4b,leading to the discovery of a novel series of fluorine-substituted NH_(2)-biphenyl-diarylpyrimidines with noticeable inhibitory activity toward WT HIV-1 strain(EC_(50)=1.8–349 nmol/L).The best compound 5t in this collection(EC_(50)=1.8 nmol/L,CC_(50)=117μmol/L)was 32-fold in selectivity(SI=66,443)compared to JK-4b and showed remarkable potency toward clinically multiple mutant strains,such as L100I,K103N,E138K,and Y181C.The metabolic stability of 5t was also significantly improved(t_(1/2)=74.52 min),approximately 5-fold higher than JK-4b in human liver microsomes(t_(1/2)=14.6 min).Also,5t possessed good stability in both human and monkey plasma.No significant in vitro inhibition effect toward CYP enzyme and hERG was observed.The single-dose acute toxicity test did not induce mice death or obvious pathological damage.These findings pave the way for further development of 5t as a drug candidate.

Driving efficiency in a high-throughput metabolic stability assay through a generic high-resolution accurate mass method and automated data mining

Improving analytical throughput is the focus of many quantitative workflows being developed for early drug discovery.For drug candidate screening,it is common practice to use ultra-high performance liquid chromatography(U-HPLC)coupled with triple quadrupole mass spectrometry.This approach certainly results in short analytical run time;however,in assessing the true throughput,all aspects of the workflow needs to be considered,including instrument optimization and the necessity to re-run samples when information is missed.Here we describe a high-throughput metabolic stability assay with a simplified instrument set-up which significantly improves the overall assay efficiency.In addition,as the data is acquired in a non-biased manner,high information content of both the parent compound and metabolites is gathered at the same time to facilitate the decision of which compounds to proceed through the drug discovery pipeline.

Evaluation of the metabolism of PEP06,an endostatin-RGDRGD 30-amino-acid polypeptide and a promising novel drug for targeting tumor cells

PEP06 is a novel endostatin-Arg-Gly-Asp-Arg-Gly-Asp(RGDRGD)30-amino-acid polypeptide featuring a terminally fused RGDRGD hexapeptide at the N terminus.The active endostatin fragment of PEP06 directly targets tumor cells and exerts an antitumoral effect.However,little is known about the kinetics and degradation products of PEP06 in vitro or in vivo.In this study,we investigated the in vitro metabolic stability of PEP06 after it was incubated with living cells obtained from animals of different species;we further identified the degradation characteristics of its cleavage products.PEP06 underwent rapid enzymatic degradation in multiple types of living cells,and the liver,kidney,and blood play important roles in the metabolism and clearance of the peptides resulting from the molecular degradation of PEP06.We identified metabolites of PEP06 using full-scan mass spectrometry(MS)and tandem MS(MS2),wherein 43 metabolites were characterized and identified as the degradation metabolites from the parent peptide,formed by successive losses of amino acids.The metabolites were C and N terminal truncated products of PEP06.The structures of 11 metabolites(M6,M7,M16,M17,M21,M25,M33,M34,M39,M40,and M42)were further confirmed by comparing the retention times of similar full MS spectrum and MS2 spectrum information with reference standards for the synthesized metabolites.We have demonstrated the metabolic stability of PEP06 in vitro and identified a series of potentially bioactive downstream metabolites of PEP06,which can support further drug research.

Discovery of highly selective and orally available benzimidazole-based phosphodiesterase 10 inhibitors with improved solubility and pharmacokinetic properties for treatment of pulmonary arterial hypertension

Optimization efforts were devoted to discover novel PDE10 A inhibitors in order to improve solubility and pharmacokinetics properties for a long-term therapy against pulmonary arterial hypertension(PAH)starting from the previously synthesized inhibitor A.As a result,a potent and highly selective PDE10 A inhibitor,14·3 HC1(half maximal inhibitory concentration,IC50=2.8 nmol/L and>3500-fold selectivity)exhibiting desirable solubility and metabolic stability with a remarkable bioavailability of50%was identified with the aid of efficient methods of binding free energy predictions.Animal PAH studies showed that the improvement offered by 14·3 HCl[2.5 mg/kg,oral administration(p.o.)]was comparable to tadalafil(5.0 mg/kg,p.o.),verifying the feasibility of PDE10 A inhibitors for the antiPAH treatment.The crystal structure of the PDE10 A-14 complex illustrates their binding pattern,which provided a guideline for rational design of highly selective PDE10 A inhibitors.

Design,synthesis and biological evaluation of LpxC inhibitors with novel hydrophilic terminus

In order to develop novel LpxC inhibitors with good activities and metabolic stability, two series of compounds with hydrophilic terminus have been synthesized and their in vitro antibacterial activities against Escherichial coil and Pseudomonas aemginosa were evaluated. Especially, compounds 22b and c exhibited comparable antibacterial activities to CHIR-090 and better metabolic stability than CHIR-090 and LPC-011 in liver microsomes （rat and mouse）, which indicated the terminal methylsulfone may be a preferred structure in the design of LpxC inhibitors and worthy of further investigations.