Discovery of BMS-986144, a Third-Generation, Pan-Genotype NS3/4A Protease Inhibitor for the Treatment of Hepatitis C Virus Infection.

The discovery of a pan-genotypic hepatitis C virus (HCV) NS3/4A protease inhibitor based on a P1-P3 macrocyclic tripeptide motif is described. The all-carbon tether linking the P1-P3 subsites of 21 is functionalized with alkyl substituents, which are shown to effectively modulate both potency and absorption, distribution, metabolism, and excretion (ADME) properties. The CF3Boc-group that caps the P3 amino moiety was discovered to be an essential contributor to metabolic stability, while positioning a methyl group at the C1 position of the P1' cyclopropyl ring enhanced plasma trough values following oral administration to rats. The C7-fluoro, C6-CD3O substitution pattern of the P2* isoquinoline heterocycle of 21 was essential to securing the targeted potency, pharmacokinetic (PK), and toxicological profiles. The C6-CD3O redirected metabolism away from a problematic pathway, thereby circumventing the time-dependent cytochrome P (CYP) 450 inhibition observed with the C6-CH3O prototype.

P3-P4 ureas and reverse carbamates as potent HCV NS3 protease inhibitors: Effective transposition of the P4 hydrogen bond donor.

A series of tripeptidic acylsulfonamide inhibitors of HCV NS3 protease were prepared that explored structure-activity relationships (SARs) at the P4 position, and their in vitro and in vivo properties were evaluated. Enhanced potency was observed in a series of P4 ureas; however, the PK profiles of these analogues were less than optimal. In an effort to overcome the PK shortcomings, modifications to the P3-P4 junction were made. This included a strategy in which one of the two urea N-H groups was either N-methylated or replaced with an oxygen atom. The former approach provided a series of regioisomeric N-methylated ureas while the latter gave rise to P4 reverse carbamates, both of which retained potent NS3 inhibitory properties while relying upon an alternative H-bond donor topology. Details of the SARs and PK profiles of these analogues are provided.

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.

Functionalized triazines as potent HCV entry inhibitors.

A series of potent and novel acylsulfonamide-bearing triazines were synthesized and the structure-activity relationships (SARs) as HCV entry inhibitors were evaluated. This acylsulfonamide series was derived from an early lead, 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoic acid wherein the carboxylic acid was replaced with an acylsulfonamide moiety. This structural modification provided a class of compounds which projected an additional vector off the terminus of the acylsulfonamide functionality as a means to drive activity. This effort led to the discovery of potent analogues within this series that demonstrated sub-nanomolar EC50 values in the HCV pseudotype particle (HCVpp) assay.

Structure-activity relationships of 4-hydroxy-4-biaryl-proline acylsulfonamide tripeptides: A series of potent NS3 protease inhibitors for the treatment of hepatitis C virus.

The design and synthesis of a series of tripeptide acylsulfonamides as potent inhibitors of the HCV NS3/4A serine protease is described. These analogues house a C4 aryl, C4 hydroxy-proline at the S2 position of the tripeptide scaffold. Information relating to structure-activity relationships as well as the pharmacokinetic and cardiovascular profiles of these analogues is provided.

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).

FGF21 protects against ox-LDL induced apoptosis through suppressing CHOP expression in THP1 macrophage derived foam cells.

BACKGROUND: FGF21,as a member of the fibroblast growth factor superfamily, is an important endogenous regulator to systemic glucose and lipid metabolism. Elevated serum FGF21 levels have been reported in subjects with coronary heart disease and carotid artery plaques. The formation and apoptosis of foam cell, induced by ox-LDL and oxysterols, are key steps in the development of atherosclerosis. METHODS: In this study, THP1 derived macrophages were induced into foam cells by ox-LDL or sterols. The formation and apoptosis of foam cells treated with or without FGF21 were analyzed. RESULTS: We demonstrated that the accumulation of cholesterol was decreased after FGF21 treatment in THP1 macrophage derived foam cells. Consistently, the apoptosis of macrophage was alleviated dramatically with FGF21 treatment. ERK1/2 knockdown didn't abrogate the effect of FGF21 on THP1 macrophage derived foam cells. However, FGF21 suppressed the induced expression of CHOP and DR5 in THP1 macrophage derived foam cells. CONCLUSION: FGF21 protects against the formation and apoptosis of THP1 macrophages derived foam cells through suppressing the expression of CHOP.

The effects of epidural/spinal opioids in labour analgesia on neonatal outcomes: a meta-analysis of randomized controlled trials.

PURPOSE: Epidural/spinal opioids are increasingly used to relieve parturients' pain in labour. Some studies indicate that opioids can induce side effects in neonates, such as respiratory depression and neurobehavioural changes. This meta-analysis aimed to clarify the effects of opioids in labour analgesia on neonates. SOURCE: PubMed, Cochrane Central Register of Controlled Trials (CENTRAL), and EMBASE™ were searched for relevant randomized controlled trials (RCTs). The neonatal data of Apgar scores, Neurological and Adaptive Capacity Scores (NACS), and umbilical cord pH values were extracted. Statistical analyses were carried out using Review Manager 5.2 and Stata(®) 10. PRINCIPAL FINDINGS: Twenty-one trials with 2,859 participants were included in our meta-analysis. No difference in the incidence of Apgar scores < 7 was shown between the opioid and control groups at one minute (risk difference [RD] 0.0%, 95% confidence interval [CI]: -3.0 to 2.0, P = 0.78; I (2) = 0%, 95% CI: 0 to 50) and at five minutes (RD -1.0%, 95% CI: -2.0 to 1.0, P = 0.31; I(2) = 0%, 95% CI: 0 to 50). No significant differences were found in the NACS at two hours (mean difference [MD] -0.35, 95% CI: -1.70 to 1.01, P = 0.62; I(2) = 0%, 95% CI: 0 to 79) and at 24 hr (MD -0.45, 95% CI: -1.36 to 0.46, P = 0.33; I(2) = 3%, 95% CI: 0 to 26). Also, no significant differences were found in umbilical cord artery pH (MD -0.02, 95% CI: -0.06 to 0.03, P = 0.48; I(2) = 80%, 95% CI: 46 to 92) and vein pH (MD -0.03, 95% CI: -0.07 to 0.00, P = 0.08; I(2) = 77%, 95% CI: 36 to 91). No significant publication bias was found. CONCLUSION: The common doses of fentanyl and sufentanil used with an epidural/spinal technique in labour analgesia are safe for neonates up to 24 hr after delivery. In future studies, more attention should be paid to the long-term side effects in neonates.

The discovery of asunaprevir (BMS-650032), an orally efficacious NS3 protease inhibitor for the treatment of hepatitis C virus infection.

The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhibitor of the NS3/4A enzyme is currently in phase III clinical trials for the treatment of hepatitis C virus infection. The discovery of 24 was enabled by employing an isolated rabbit heart model to screen for the cardiovascular (CV) liabilities (changes to HR and SNRT) that were responsible for the discontinuation of an earlier lead from this chemical series, BMS-605339 (1), from clinical trials. The structure-activity relationships (SARs) developed with respect to CV effects established that small structural changes to the P2* subsite of the molecule had a significant impact on the CV profile of a given compound. The antiviral activity, preclincial PK profile, and toxicology studies in rat and dog supported clinical development of BMS-650032 (24).

Discovery and early clinical evaluation of BMS-605339, a potent and orally efficacious tripeptidic acylsulfonamide NS3 protease inhibitor for the treatment of hepatitis C virus infection.

The discovery of BMS-605339 (35), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropylacylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1' site of the protease. The identification of 35 was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK). This was achieved through modulation of the P2* subsite of the inhibitor which identified the isoquinoline ring system as a key template for improving PK properties with further optimization achieved through functionalization. A methoxy moiety at the C6 position of this isoquinoline ring system proved to be optimal with respect to potency and PK, thus providing the clinical compound 35 which demonstrated antiviral activity in HCV-infected patients.

Discovery of (S,E)-3-(2-fluorophenyl)-N-(1-(3-(pyridin-3-yloxy)phenyl)ethyl)-acrylamide as a potent and efficacious KCNQ2 (Kv7.2) opener for the treatment of neuropathic pain.

Acrylamide (S)-6, a potent and efficacious KCNQ2 (Kv7.2) opener, demonstrated significant activity in two models of neuropathic pain and in the formalin test, suggesting that KCNQ2 openers may be useful in the treatment of neuropathic pain including diabetic neuropathy.

Preclinical Profile and Characterization of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir (BMS-650032).

Asunaprevir (ASV; BMS-650032) is a hepatitis C virus (HCV) NS3 protease inhibitor that has demonstrated efficacy in patients chronically infected with HCV genotype 1 when combined with alfa interferon and/or the NS5A replication complex inhibitor daclatasvir. ASV competitively binds to the NS3/4A protease complex, with K(i) values of 0.4 and 0.24 nM against recombinant enzymes representing genotypes 1a (H77) and 1b (J4L6S), respectively. Selectivity was demonstrated by the absence of any significant activity against the closely related GB virus-B NS3 protease and a panel of human serine or cysteine proteases. In cell culture, ASV inhibited replication of HCV replicons representing genotypes 1 and 4, with 50% effective concentrations (EC(50)s) ranging from 1 to 4 nM, and had weaker activity against genotypes 2 and 3 (EC(50), 67 to 1,162 nM). Selectivity was again demonstrated by the absence of activity (EC(50), >12 µM) against a panel of other RNA viruses. ASV exhibited additive or synergistic activity in combination studies with alfa interferon, ribavirin, and/or inhibitors specifically targeting NS5A or NS5B. Plasma and tissue exposures in vivo in several animal species indicated that ASV displayed a hepatotropic disposition (liver-to-plasma ratios ranging from 40- to 359-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥110-fold above the inhibitor EC(50)s observed with HCV genotype-1 replicons. Based on these virologic and exposure properties, ASV holds promise for future utility in a combination with other anti-HCV agents in the treatment of HCV-infected patients.

(R)-2-(4-Phenylbutyl)dihydrobenzofuran derivatives as melatoninergic agents.

(R)-2-(4-Phenylbutyl)dihydrobenzofuran derivatives (e.g., 3 and 4) were synthesized as novel melatoninergic ligands with significantly lower vasoconstrictive activity in vitro in the rat tail artery. Binding affinity assays were performed on cloned human MT1 and MT2 receptors stably expressed in NIH3T3 cells.

(S,E)-N-[1-(3-heteroarylphenyl)ethyl]-3-(2-fluorophenyl)acrylamides: synthesis and KCNQ2 potassium channel opener activity.

Replacement of the morpholinyl moiety in (S,E)-N-[1-(3-morpholinophenyl)ethyl]-3-phenylacrylamide (1) with heteroaryl groups led to the identification of (S,E)-N-1-[3-(6-fluoropyridin-3-yl)phenyl]ethyl-3-(2-fluorophenyl)acrylamide (5) as a potent KCNQ2 potassium channel opener. Among this series of heteroaryl substituted acrylamides, (S,E)-N-1-[3-(1H-pyrazol-1-yl)phenyl]ethyl-3-(2-fluorophenyl)acrylamide (9) exhibits balanced potency and efficacy. The syntheses and the KCNQ2 opener activity of this series of acrylamides are described.

Synthesis and KCNQ2 opener activity of N-(1-benzo[1,3]dioxol-5-yl-ethyl, N-[1-(2,3-dihydro-benzofuran-5-yl)-ethyl, and N-[1-(2,3-dihydro-1H-indol-5-yl)-ethyl acrylamides.

Bioisosteric replacement studies led to the identification of N-(1-benzo[1,3]dioxol-5-yl-ethyl)-3-(2-chloro-phenyl)-acrylamide ((S)-3) as a highly potent KCNQ2 opener, and 3-(2,6-difluoro-phenyl)-N-[1-(2,3-dihydro-benzofuran-5-yl)-ethyl]-acrylamide ((S)-4), and N-[1-(2,3-dihydro-1H-indol-5-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide ((S)-5) as highly efficacious KCNQ2 openers. In contrast, their respective R enantiomers showed significantly less or no appreciable KCNQ2 opener activity even at the highest concentration tested (10 microM). Because of its high potency and moderate efficacy as well as its convenient synthesis, (+/-)-3 was selected as a reference compound for analyzing efficacies of KCNQ openers in electrophysiology studies. Compounds (S)-4 and (S)-5 demonstrated significant activity in reducing neuronal hyperexcitability in rat hippocampal slices. The synthesis and the KCNQ2 opener activity of these acrylamides are described.

N-[2-[2-(4-Phenylbutyl)benzofuran-4-yl]cyclopropylmethyl]acetamide: an orally bioavailable melatonin receptor agonist.

N-[2-[2-(4-Phenylbutyl)benzofuran-4-yl]cyclopropylmethyl]acetamide 3a was synthesized as an orally bioavailable agonist at MT1 and MT2 melatonin receptors with significantly low vasoconstrictive activity.

Synthesis and structure-activity relationship of novel benzoxazole derivatives as melatonin receptor agonists.

A series of benzoxazole derivatives was synthesized and evaluated as melatoninergic ligands. The binding affinity of these compounds for human MT(1) and MT(2) receptors was determined using 2-[(125)I]-iodomelatonin as the radioligand. From this series of benzoxazole derivatives, compounds 14 and 17 were identified as melatonin receptor agonists.

Synthesis and structure-activity relationship of acrylamides as KCNQ2 potassium channel openers.

A new class of acrylamides was synthesized, and the effects of these analogues on outward potassium current were evaluated by using two electrode voltage clamp recordings from Xenopus laevis oocytes expressing cloned mKCNQ2 channels. SAR studies indicated that the pharmacophore of the acrylamide series includes the (S) absolute configuration at the (1-phenyl)ethyl moiety and the alpha,beta-unsaturated acrylamide functionality with a free NH. This study identified (S)-N-[1-(3-morpholin-4-yl-phenyl)-ethyl]-3-phenyl-acrylamide ((S)-1) and (S)-N-[1-(4-fluoro-3-morpholin-4-yl-phenyl)-ethyl]-3-(4-fluoro-phenyl)-acrylamide ((S)-2) as KCNQ2 openers for further electrophysiological evaluations. These two acrylamides demonstrated significant activity in the cortical spreading depression model of migraine as we reported previously.

(S)-N-[1-(4-cyclopropylmethyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide is a potent and efficacious KCNQ2 opener which inhibits induced hyperexcitability of rat hippocampal neurons.

(S)-N-[1-(4-Cyclopropylmethyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide ((S)-2) was identified as a potent and efficacious KCNQ2 opener. This compound demonstrated significant activity in reducing neuronal hyperexcitability in rat hippocampal slices, and the inhibition mediated by (S)-2 was reversed by the KCNQ blocker linopirdine.

Design and synthesis of benzoxazole derivatives as novel melatoninergic ligands.

A novel series of benzoxazole derivatives was synthesized and evaluated as melatoninergic ligands. The binding affinity of these compounds for human MT(1) and MT(2) receptors was determined using 2-[(125)I]-iodomelatonin as the radioligand. The results of the SAR studies in this series led to the identification of compound 28, which exhibited better MT(1) and MT(2) receptor affinities than melatonin itself. This work also established the benzoxazole nucleus as a melatoninergic pharmacophore, which served as an isosteric replacement to the previously established alkoxyaryl core.