Synthesis and structure-function analysis of Fe(II)-form-selective antibacterial inhibitors of Escherichia coli methionine aminopeptidase

Methionine aminopeptidase (MetAP) is a promising target for the development of novel antibacterial, antifungal and anticancer therapy. Based on our previous results, catechol derivatives coupled with a thiazole or thiophene moiety showed high potency and selectivity toward the Fe(II)-form of Escherichia coli MetAP, and some of them clearly showed antibacterial activity, indicating that Fe(II) is likely the physiologically relevant metal for MetAP in E. coli and other bacterial cells. To further understand the structure-function relationship of these Fe(II)-form selective MetAP inhibitors, a series of catechol derivatives was designed and synthesized by replacement of the thiazole or thiophene moiety with different five-membered and six-membered heterocycles. Inhibitory activities of these newly synthesized MetAP inhibitors indicate that many five- and six-membered rings can be accommodated by MetAP and potency on the Fe(II)-form can be improved by introducing substitutions on the heterocyles to explore additional interactions with the enzyme. The furan-containing catechols 11–13 showed the highest potency at 1 μM on the Fe(II)-form MetAP, and they were also among the best inhibitors for growth inhibition against E. coli AS19 strain. These findings provide useful information for the design and discovery of more effective MetAP inhibitors for therapeutic applications.     

Synthesis and biological evaluation of tricyclic anilinopyrimidines as IKKβ inhibitors

A series of tricyclic anilinopyrimidines were synthesized and evaluated as IKKβ inhibitors. Several analogues, including tricyclic phenyl (10, 18a, 18c, 18d, and 18j) and thienyl (26 and 28) derivatives were shown to have good in vitro enzyme potency and excellent cellular activity. Pharmaceutical profiling of a select group of tricyclic compounds compared to the non-tricyclic analogues suggested that in some cases, the improved cellular activity may be due to increased clog P and permeability.     

A single amino acid difference between archaeal and human type 2 methionine aminopeptidases differentiates their affinity towards ovalicin

In almost all living cells, methionine aminopeptidase (MetAP) co-translationally cleaves the initiator methionine in at least 70% of the newly synthesized polypeptides. MetAPs are typically classified into Type 1 and Type 2. While prokaryotes and archaea contain only either Type 1 or Type 2 MetAPs respectively, eukaryotes contain both types of enzymes. Almost all MetAPs published till date cleave only methionine from the amino terminus of the substrate peptides. Earlier experiments on crude Type 2a MetAP isolated from Pyrococcus furiosus (PfuMetAP2a) cosmid protein library was shown to cleave leucine in addition to methionine. Authors in that study have ruled out the PfuMetAP2a activity against leucine substrates and assumed it to be a background reaction contributed by other contaminating proteases. In the current paper, using the pure recombinant enzyme, we report that indeed activity against leucine is directly carried out by the PfuMetAP2a. In addition, the natural product ovalicin which is a specific covalent inhibitor of Type 2 MetAPs does not show efficient inhibition against the PfuMetAP2a. Bioinformatic analysis suggested that a glycine in eukaryotic MetAP2s (G222 in human MetAP2b) and asparagine (N53 in PfuMetAP2a) in archaeal MetAP2s positioned at the analogous position. N53 side chain forms a hydrogen bond with a conserved histidine (H62) at the entrance of the active site and alters its orientation to accommodate the ovalicin. This slight orientational difference of the H62, reduces affinity of the ovalicin by 300,000-fold when compared with the HsMetAP2b inhibition. This difference in the activity is partly reduced in the case of N53G mutation of the PfuMetAP2a.     

Multisubstituted quinoxalines and pyrido[2,3-d]pyrimidines: Synthesis and SAR study as tyrosine kinase c-Met inhibitors

Two series of new analogues were designed by replacing the quinoline scaffold of our earlier lead 2 (zgwatinib) with quinoxaline and pyrido[2,3-d]pyrimidine frameworks. Moderate c-Met inhibitory activity was observed in the quinoxaline series. Among the pyrido[2,3-d]pyrimidine series, compounds 13a–c possessing an O-linkage were inactive, whilst the N-linked analogues 15a–c retained c-Met inhibitory potency. Highest activity was observed in the 3-nitrobenzyl analog 15b that showed an IC₅₀ value of 6.5 nM. Further structural modifications based on this compound were undergoing.     

Substituted 2H-isoquinolin-1-ones as potent Rho-kinase inhibitors: Part 3, aryl substituted pyrrolidines

The discovery and SAR of a series of β-aryl substituted pyrrolidine 2H-isoquinolin-1-one inhibitors of Rho-kinase (ROCK) derived from 2 is herein described. SAR studies have shown that aryl groups in the β-position are optimal for potency. Our efforts focused on improving the ROCK potency of this isoquinolone class of inhibitors which led to the identification of pyrrolidine 32 which demonstrated a 10-fold improvement in aortic ring (AR) potency over 2.     

Expression and characterization of Mycobacterium tuberculosis methionine aminopeptidase type 1a

Methionine aminopeptidase (MetAP) carries out the cotranslational N-terminal methionine excision and is essential for bacterial survival. Mycobacterium tuberculosis expresses two MetAPs, MtMetAP1a and MtMetAP1c, at different levels in growing and stationary phases, and both are potential targets to develop novel antitubercular therapeutics. Recombinant MtMetAP1a was purified as an apoenzyme, and metal binding and activation were characterized with an activity assay using a fluorogenic substrate. Ni(II), Co(II) and Fe(II) bound tightly at micromolar concentrations, and Ni(II) was the most efficient activator for the MetAP-catalyzed substrate hydrolysis. Although the characteristics of metal binding and activation are similar to MtMetAP1c we characterized before, MtMetAP1a was significantly more active, and more importantly, a set of inhibitors displayed completely different inhibitory profiles on the two mycobacterial MetAPs in both potency and metalloform selectivity. The differences in catalysis and inhibition predicted the significant differences in active site structure.     

Design and synthesis of a novel 1H-pyrrolo[3,2-b]pyridine-3-carboxamide derivative as an orally available ACC1 inhibitor

We initiated our structure-activity relationship (SAR) studies for novel ACC1 inhibitors from 1a as a lead compound. Our initial SAR studies of 1H-Pyrrolo[3,2-b]pyridine-3-carboxamide scaffold revealed the participation of HBD and HBA for ACC1 inhibitory potency and identified 1-methyl-1H-pyrrolo[3,2-b]pyridine-3-carboxamide derivative 1c as a potent ACC1 inhibitor. Although compound 1c had physicochemical and pharmacokinetic (PK) issues, we investigated the 1H-pyrrolo[3,2-b]pyridine core scaffold to address these issues. Accordingly, this led us to discover a novel 1-isopropyl-1H-pyrrolo[3,2-b]pyridine-3-carboxamide derivative 1k as a promising ACC1 inhibitor, which showed potent ACC1 inhibition as well as sufficient cellular potency. Since compound 1k displayed favorable bioavailability in mouse cassette dosing PK study, we conducted in vivo Pharmacodynamics (PD) studies of this compound. Oral administration of 1k significantly reduced the concentration of malonyl-CoA in HCT-116 xenograft tumors at a dose of 100 mg/kg. Accordingly, our novel series of potent ACC1 inhibitors represent useful orally-available research tools, as well as potential therapeutic agents for cancer and fatty acid related diseases.     

Novel peptidomimetics as BACE-1 inhibitors: Synthesis,molecular modeling,and biological studies

Aiming at identifying new scaffolds for BACE-1 inhibition devoid of the pharmacokinetic drawbacks of peptide-like structures, we investigated a series of novel peptidomimetics based on a 1,4-benzodiazepine (BDZ) core 1a–h and their seco-analogues 2a–d. We herein discuss synthesis, molecular modeling and in vitro studies which, starting from 1a, led to the seco-analogues (R)-2c and (S)-2d endowed with BACE-1 inhibition properties in the micromolar range both on the isolated enzyme and in cellular studies. These data can encourage to pursue these analogues as hits for the development of a new series of BACE-1 inhibitors active on whole-cells.     

Tricyclic 4,4-dimethyl-3,4-dihydrochromeno[3,4-d]imidazole derivatives as microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors: SAR and in vivo efficacy in hyperalgesia pain model

A series of substituted tricyclic 4,4-dimethyl-3,4-dihydrochromeno[3,4-d]imidazole derivatives have been synthesized and their mPGES-1 biological activity has been disclosed in detail. Structure-activity relationship (SAR) optimization provided inhibitors with excellent mPGES-1 potency and low to moderate PGE₂ release A549 cell potency. Among the mPGES-1 inhibitors studied, 7, 9 and 11l provided excellent selectivity over COX-2 (>200-fold) and >70-fold selectivity for COX-1 except 11l, which exhibited dual mPGES-1/COX-1 activity. Furthermore, the above tested mPGES-1 inhibitors demonstrated good metabolic stability in liver microsomes, high plasma protein binding (PPB) and no significant inhibition observed in clinically relevant CYP isoforms. Besides, selected mPGES-1 tool compounds 9 and 11l provided good in vivo pharmacokinetic profile and oral bioavailability (%F = 33 and 85). Additionally, the representative mPGES-1 tool compounds 9 and 11l revealed moderate in vivo efficacy in the LPS-induced thermal hyperalgesia guinea pig pain model.     

2,4-Diamino-quinazolines as inhibitors of β-catenin/Tcf-4 pathway: Potential treatment for colorectal cancer

The synthesis and SAR of a series of 2,4-diamino-quinazoline derivatives as β-catenin/Tcf-4 inhibitors are described. This series was developed by modifying the initial lead 1, which was identified by screening of our compound library and found to inhibit the β-catenin/Tcf-4 pathway. Replacement of the biphenyl moiety in compound 1 with the N-phenylpiperidine-4-carboxamide chain as in 2, resulted in a number of new analogues, which are potent inhibitors of the β-catenin/Tcf-4 pathway. Compound such as 16k exhibited good cellular potency, solubility, metabolic stability and oral bioavailability.     

Constrained (l-)-S-adenosyl-l-homocysteine (SAH) analogues as DNA methyltransferase inhibitors

Potent SAH analogues with constrained homocysteine units have been designed and synthesized as inhibitors of human DNMT enzymes. The five membered (2S,4S)-4-mercaptopyrrolidine-2-carboxylic acid, in 1a, was a good replacement for homocysteine, while the corresponding six-member counterpart was less active. Further optimization of 1a, changed the selectivity profile of these inhibitors. A Chloro substituent at the 2-position of 1a, compound 1d, retained potency against DNMT1, while N⁶ alkylation, compound 7a, conserved DNMT3b2 activity. The concomitant substitutions of 1a at both 2- and N⁶ positions reduced activity against both enzymes.     

Synthesis and bioactivities evaluation of l-pyroglutamic acid analogues from natural product lead

A series of l-pyroglutamic acid analogues from natural product lead were designed and synthesized, as well as their antifungal activities against Phytophthora infestans, neuritogenic activities, antibacterial activities and anti-inflammatory activities are described. The bioassays and SAR study showed that the majority of l-pyroglutamic acid esters have a significant antifungal activity against P. infestans, especially 2d and 2j demonstrated the best activities with EC₅₀ values of 1.44 and 1.21?μg?mL^?1, which were about seven times that of commercial azoxystrobin (7.85?μg?mL^?1). Moreover, compounds 2e, 2g and 4d displayed anti-inflammatory activity against LPS-induced NO production in BV-2 microglial cells; neuritogenic activity in NGF-induced PC-12 cells is the same activity. This study demonstrates that compounds 2d and 2j are potential drugs to control P. infestans.     

2-Aminobenzimidazoles as potent Aurora kinase inhibitors

This Letter describes the discovery and key structure–activity relationship (SAR) of a series of 2-aminobenzimidazoles as potent Aurora kinase inhibitors. 2-Aminobenzimidazole serves as a bioisostere of the biaryl urea residue of SNS-314 (1c), which is a potent Aurora kinase inhibitor and entered clinical testing in patients with solid tumors. Compared to SNS-314, this series of compounds offers better aqueous solubility while retaining comparable in vitro potency in biochemical and cell-based assays; in particular, 6m has also demonstrated a comparable mouse iv PK profile to SNS-314.     

HCV NS5A replication complex inhibitors. Part 5: Discovery of potent and pan-genotypic glycinamide cap derivatives

The isoquinolinamide series of HCV NS5A inhibitors exemplified by compounds 2b and 2c provided the first dual genotype-1a/1b (GT-1a/1b) inhibitor class that demonstrated a significant improvement in potency toward GT-1a replicons compared to that of the initial program lead, stilbene 2a. Structure–activity relationship (SAR) studies that uncovered an alternate phenylglycine-based cap series that exhibit further improvements in virology profile, along with some insights into the pharmacophoric elements associated with the GT-1a potency, are described.     

Identification of new pyrrolo[2,3-d]pyrimidines as potent VEGFR-2 tyrosine kinase inhibitors: Design,synthesis, biological evaluation and molecular modeling

Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in cancer angiogenesis. In the current study, a series of novel pyrrolo[2,3-d]pyrimidine based-compounds was designed and synthesized as VEGFR-2 inhibitors, in accordance to the structure activity relationship (SAR) studies of known type II VEGFR-2 inhibitors. The newly synthesized compounds were evaluated for their ability to inhibit VEGFR-2 kinase enzyme in vitro. All the tested compounds demonstrated highly potent dose-related VEGFR-2 inhibition with IC₅₀ values in nanomolar range. Among these compounds, pyrrolo[2,3-d]pyrimidine derivatives carrying biaryl urea moieties (12d and 15c) exhibited IC₅₀ values of 11.9 and 13.6 nM respectively. Additionally, most of the newly synthesized final compounds were tested on 60 human cancer cell lines. Docking of these compounds into the inactive conformation of VEGFR-2 was performed which showed comparable binding modes to that of the FDA approved VEGFR-2 kinase inhibitors. These newly discovered potent kinase inhibitors could be considered as potential candidates for the development of new targeted anticancer agent.     

Optimization of a binding fragment targeting the “enlarged methionine pocket” leads to potent Trypanosoma brucei methionyl-tRNA synthetase inhibitors

Potent inhibitors of Trypanosoma brucei methionyl-tRNA synthetase were previously designed using a structure-guided approach. Compounds 1 and 2 were the most active compounds in the cyclic and linear linker series, respectively. To further improve cellular potency, SAR investigation of a binding fragment targeting the “enlarged methionine pocket” (EMP) was performed. The optimization led to the identification of a 6,8-dichloro-tetrahydroquinoline ring as a favorable fragment to bind the EMP. Replacement of 3,5-dichloro-benzyl group (the EMP binding fragment) of inhibitor 2 using this tetrahydroquinoline fragment resulted in compound 13, that exhibited an EC₅₀ of 4 nM.     

New structure–activity relationship studies in a series of N,N-bis(cyclohexanol)amine aryl esters as potent reversers of P-glycoprotein-mediated multidrug resistance (MDR)

As a continuation of previous research on a new series of potent and efficacious P-gp-dependent multidrug resistant (MDR) reversers with a N,N-bis(cyclohexanol)amine scaffold, we have designed and synthesized several analogs by modulation of the two aromatic moieties linked through ester functions to the N,N-bis(cyclohexanol)amine, aiming to optimize activity and to extend structure–activity relationships (SAR) within the series. This scaffold, when esterified with two different aromatic carboxylic acids, gives origin to four geometric isomers (cis/trans, trans/trans, cis/cis and trans/cis).The new compounds were tested on doxorubicin-resistant erythroleukemia K562 cells (K562/DOX) in the pirarubicin uptake assay. Most of them resulted in being potent modulators of the extrusion pump P-gp, showing potency values ([I]_0.5) in the submicromolar and nanomolar range. Of these, compounds 2b, 2c, 3d, 5a–d and 6d, showed excellent efficacy with a α_max close to 1. Selected compounds (2d, 3a, 3b, 5a–d) were further studied to evaluate their doxorubicin cytotoxicity potentiation (RF) on doxorubicin-resistant erythroleukemia K562 cells and were found able to enhance significantly doxorubicin cytotoxicity on K562/DOX cells.The results of both pirarubicin uptake and the cytotoxicity assay, indicate that the new compounds of the series are potent P-gp-mediated MDR reversers. They present a structure with a mix of flexible and rigid moieties, a property that seems critical to allow the molecules to choose the most productive of the several binding modes possible in the transporter recognition site.In particular, compounds 5c and 5d, similar to the already reported analogous isomers 1c and 1d,²⁹ are potent and efficacious modulators of P-gp-dependent MDR and may be promising leads for the development of MDR-reversal drugs.