Development of an efficient semisynthetic modification of FR901459 via a novel regioselective N,O-acyl migration

HCV utilizes cellular protein cyclophilins in the virus replication cycle and cyclophilin inhibitors have become a new class of anti-HCV agents. In our screening of natural products, we identified a unique cyclosporin analogue, FR901459, as a cyclophilin inhibitor with potent anti-HCV activity. In this work, we developed an efficient synthetic methodology to prepare FR901459 derivatives via an N, O-acyl migration reaction. This method allows us to efficiently manipulate the amino acid residues at the 3 position while avoiding lengthy total synthesis for each compound. By using this methodology, we discovered 4, which has superior anti-HCV activity and decreased immunosuppressive activity compared to FR901459.     

Inhibition of Mycobacterium tuberculosis InhA: Design,synthesis and evaluation of new di-triclosan derivatives

Multi-drug resistant tuberculosis (MDR-TB) represents a growing problem for global healthcare systems. In addition to 1.3 million deaths in 2018, the World Health Organisation reported 484,000 new cases of MDR-TB. Isoniazid is a key anti-TB drug that inhibits InhA, a crucial enzyme in the cell wall biosynthesis pathway and identical in Mycobacterium tuberculosis and M. bovis. Isoniazid is a pro-drug which requires activation by the enzyme KatG, mutations in KatG prevent activation and confer INH-resistance. ‘Direct inhibitors’ of InhA are attractive as they would circumvent the main clinically observed resistance mechanisms. A library of new 1,5-triazoles, designed to mimic the structures of both triclosan molecules uniquely bound to InhA have been synthesised. The inhibitory activity of these compounds was evaluated using isolated enzyme assays with 2 (5-chloro-2-(4-(5-(((4-(4-chloro-2-hydroxyphenoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) exhibiting an IC₅₀ of 5.6 µM. Whole-cell evaluation was also performed, with 11 (5-chloro-2-(4-(5-(((4-(cyclopropylmethoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) showing the greatest potency, with an MIC₉₉ of 12.9 µM against M. bovis.     

In vitro cytotoxic potential of extracts from Aristolochia foetida Kunth against MCF-7 and bMECs cell lines

The aim of this study was to evaluate the cytotoxic potential of Aristolochia foetida Kunth. Stems and leaves of A. foetida Kunth (Aristolochiaceae) have never been investigated pharmacologically. Recent studies of species of the Aristolochiaceae family found significant cytotoxic activities. Hexane, dichloromethane, ethyl acetate and methanol extracts were analyzed by ¹H NMR and GC–MS to know the metabolites in each extract. In GC–MS analysis, the main compounds were methyl hexadecanoate (3); hexadecanoic acid (4); 2-butoxyethyl dodecanoate (9); ethyl hexadecanoate (20); methyl octadeca-9,12,15-trienoate (28) and (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid (40). The results showed a significant reduction in cell viability of the MCF-7 (breast cancer) cell line caused by organic extracts in a dose-dependent manner. The cytotoxicity activity of the dichloromethane extract from the stems (DSE) showed IC₅₀ values of 45.9 μg/mL and the dichloromethane extract of the leaves (DLE) showed IC₅₀ values of 47.3 μg/mL. DSE and DLE had the highest cytotoxic potential in an in vitro study against the MCF-7 cell line and non-tumor cells obtained from the bovine mammary epithelial (bMECs). DSE and DLE induced a loss in mitochondrial membrane potential (ΔΨm) and can cause cell death by apoptosis through the intrinsic pathway in the MCF-7 cell line. DSE and DLE are cytotoxic in cancer cells and cause late apoptosis. Higher concentrations of DSE and DLE are required to induce a cytotoxic effect in healthy mammary epithelial cells. This is the first report of the dichloromethane extract of A. foetida Kunth that induces late apoptosis in MCF-7 cancer cells and may be a candidate for pharmacological study against breast cancer.     

Synthesis,biological evaluation and anti-proliferative mechanism of fluorine-containing proguanil derivatives

Proguanil, a member of biguanide family, has excellent anti-proliferative activities. Fluorine-containing compounds have been demonstrated to have super biological activities including enhanced binding interactions, metabolic stability, and reduced toxicity. In this study, based on the intermediate derivatization methods, we synthesized 13 new fluorine-containing proguanil derivatives, and found that 7a,7d and 8e had much lower IC₅₀ than proguanil in 5 human cancerous cell lines. The results of clonogenic and scratch wound healing assays revealed that the inhibitory effects of derivatives 7a,7d and 8e on proliferation and migration of human cancer cell lines were much better than proguanil as well. Mechanistic study based on representative derivative 7a indicated that this compound up-regulates AMPK signal pathway and downregulates mTOR/4EBP1/p70S6K. In conclusion, these new fluorine-containing derivatives show potential for the development of cancer chemotherapeutic drugs.     

Discovery of isoquinolinone and naphthyridinone-based inhibitors of poly(ADP-ribose) polymerase-1 (PARP1) as anticancer agents: Structure activity relationship and preclinical characterization

The exploitation of GLU988 and LYS903 residues in PARP1 as targets to design isoquinolinone (I & II) and naphthyridinone (III) analogues is described. Compounds of structure I have good biochemical and cellular potency but suffered from inferior PK. Constraining the linear propylene linker of structure I into a cyclopentene ring (II) offered improved PK parameters, while maintaining potency for PARP1. Finally, to avoid potential issues that may arise from the presence of an anilinic moiety, the nitrogen substituent on the isoquinolinone ring was incorporated as part of the bicyclic ring. This afforded a naphthyridinone scaffold, as shown in structure III. Further optimization of naphthyridinone series led to identification of a novel and highly potent PARP1 inhibitor 34, which was further characterized as preclinical candidate molecule. Compound 34 is orally bioavailable and displayed favorable pharmacokinetic (PK) properties. Compound 34 demonstrated remarkable antitumor efficacy both as a single-agent as well as in combination with chemotherapeutic agents in the BRCA1 mutant MDA-MB-436 breast cancer xenograft model. Additionally, compound 34 also potentiated the effect of agents such as temozolomide in breast cancer, pancreatic cancer and Ewing’s sarcoma models.     

Synthesis of novel 2-pyrrolidinone and pyrrolidine derivatives and study of their inhibitory activity against autotaxin enzyme

Autotaxin (ATX), a glycoprotein (~125 kDa) isolated as an autocrine motility factor from melanoma cells, belongs to a seven-membered family of ectonucleotide pyrophosphatase/phosphodiesterase (ENPP), and exhibits lysophospholipase D activity. ATX is responsible for the hydrolysis of lysophosphatidylcholine (LPC) to produce the bioactive lipid lysophosphatidic acid (LPA), which is upregulated in a variety of pathological inflammatory conditions, including fibrosis, cancer, liver toxicity and thrombosis. Given its role in human disease, the ATX-LPA axis is an interesting target for therapy, and the development of novel potent ATX inhibitors is of great importance. In the present work a novel class of ATX inhibitors, optically active derivatives of 2-pyrrolidinone and pyrrolidine heterocycles were synthesized. Some of them exhibited interesting in vitro activity, namely the hydroxamic acid 16 (IC₅₀ 700 nM) and the carboxylic acid 40b (IC₅₀ 800 nM), while the boronic acid derivatives 3k (IC₅₀ 50 nM), 3l (IC₅₀ 120 nM), 3 m (IC₅₀ 180 nM) and 21 (IC₅₀ 35 nM) were found to be potent inhibitors of ATX.     

A novel potent metal-binding NDM-1 inhibitor was identified by fragment virtual,SPR and NMR screening

NDM-1 can hydrolyze nearly all available β-lactam antibiotics, including carbapenems. NDM-1 producing bacterial strains are worldwide threats. It is still very challenging to find a potent NDM-1 inhibitor for clinical use. In our study, we used a metal-binding pharmacophore (MBP) enriched virtual fragment library to screen NDM-1 hits. SPR screening helped to verify the MBP virtual hits and identified a new NDM-1 binder and weak inhibitor A1. A solution NMR study of ¹⁵N-labeled NDM-1 showed that A1 disturbed all three residues coordinating the second zinc ion (Zn2) in the active pocket of NDM-1. The perturbation only happened in the presence of zinc ion, indicating that A1 bound to Zn2. Based on the scaffold of A1, we designed and synthesized a series of NDM-1 inhibitors. Several compounds showed synergistic antibacterial activity with meropenem against NDM-1 producing K. pneumoniae.     

Synthesis and evaluation of novel radioiodinated PSMA targeting ligands for potential radiotherapy of prostate cancer

Radioligand therapy (RLT) using prostate-specific membrane antigen (PSMA) targeting ligands is an attractive option for the treatment of Prostate cancer (PCa) and its metastases. We report herein a series of radioiodinated glutamate-urea-lysine-phenylalanine derivatives as new PSMA ligands in which l-tyrosine and l-glutamic acid moieties were added to increase hydrophilicity concomitant with improvement of in vivo targeting properties. Compounds 8, 15, 19a/19b and 23a/23b were synthesized and radiolabeled with ¹²⁵I by iododestannylation. All iodinated compounds displayed high binding affinities toward PSMA (IC₅₀ = 1–13 nM). In vitro cell uptake studies demonstrated that compounds containing an l-tyrosine linker moiety (8, 15 and 19a/19b) showed higher internalization than MIP-1095 and 23a/23b, both without the l-tyrosine linker moiety. Biodistribution studies in mice bearing PC3-PIP and PC3 xenografts showed that [¹²⁵I]8 and [¹²⁵I]15 with higher lipophilicity exhibited higher nonspecific accumulations in the liver and intestinal tract, whereas [¹²⁵I]19a/19b and [¹²⁵I]23a/23b containing additional glutamic acid moieties showed higher accumulations in the kidney and implanted PC3-PIP (PSMA+) tumors. [¹²⁵I]23b displayed a promising biodistribution profile with favorable tumor retention, fast clearance from the kidney, and 2–3-fold lower uptake in the liver and blood than that observed for [¹²⁵I]MIP-1095. [^125/131I]23b may serve as an optimal PSMA ligand for radiotherapy treatment of prostate cancer over-expressing PSMA.     

Design,synthesis, and biological activity of new endomorphin analogs with multi-site modifications

Endomorphin (EM)-1 and EM-2 are the most effective endogenous analgesics with efficient separation of analgesia from the risk of adverse effects. Poor metabolic stability and ineffective analgesia after peripheral administration were detrimental for the use of EMs as novel clinical analgesics. Therefore, here, we aimed to establish new EM analogs via introducing different bifunctional d-amino acids at position 2 of [(2-furyl)Map⁴]EMs. The combination of [(2-furyl)Map⁴]EMs with D-Arg² or D-Cit² yielded analogs with enhanced binding affinity to the μ-opioid receptor (MOR) and increased stability against enzymatic degradation (t_1/2 > 300 min). However, the agonistic activities of these analogs toward MOR were slightly reduced. Similar to morphine, peripheral administration of the analog [D-Cit², (2-furyl)Map⁴]EM-1 (10) significantly inhibited the pain behavior of mice in multiple pain models. In addition, this EM-1 analog was associated with reduced tolerance, less effect on gastrointestinal mobility, and no significant motor impairment. Compared to natural EMs, the EM analogs synthesized herein had enhanced metabolic stability, bioavailability, and analgesic properties.     

Analogs of nitrofuran antibiotics are potent GroEL/ES inhibitor pro-drugs

In two previous studies, we identified compound 1 as a moderate GroEL/ES inhibitor with weak to moderate antibacterial activity against Gram-positive and Gram-negative bacteria including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, and SM101 Escherichia coli (which has a compromised lipopolysaccharide biosynthetic pathway making bacteria more permeable to drugs). Extending from those studies, we developed two series of analogs with key substructures resembling those of known antibacterials, nitroxoline (hydroxyquinoline moiety) and nifuroxazide/nitrofurantoin (bis-cyclic-N-acylhydrazone scaffolds). Through biochemical and cell-based assays, we identified potent GroEL/ES inhibitors that selectively blocked E. faecium, S. aureus, and E. coli proliferation with low cytotoxicity to human colon and intestine cells in vitro. Initially, only the hydroxyquinoline-bearing analogs were found to be potent inhibitors in our GroEL/ES-mediated substrate refolding assays; however, subsequent testing in the presence of an E. coli nitroreductase (NfsB) in situ indicated that metabolites of the nitrofuran-bearing analogs were potent GroEL/ES inhibitor pro-drugs. Consequently, this study has identified a new target of nitrofuran-containing drugs, and is the first reported instance of such a unique class of GroEL/ES chaperonin inhibitors. The intriguing results presented herein provide impetus for expanded studies to validate inhibitor mechanisms and optimize this antibacterial class using the respective GroEL/ES chaperonin systems and nitroreductases from E. coli and the ESKAPE bacteria.     

Rational design,synthesis and biological evaluation of novel multitargeting anti-AD iron chelators with potent MAO-B inhibitory and antioxidant activity

A series of (3-hydroxypyridin-4-one)-coumarin hybrids were developed and investigated as potential multitargeting candidates for the treatment of Alzheimer's disease (AD) through the incorporation of iron-chelating and monoamine oxidase B (MAO-B) inhibition. This combination endowed the hybrids with good capacity to inhibit MAO-B as well as excellent iron-chelating effects. The pFe³⁺ values of the compounds were ranging from 16.91 to 20.16, comparable to more potent than the reference drug deferiprone (DFP). Among them, compound 18d exhibited the most promising activity against MAO-B, with an IC₅₀ value of 87.9 nM. Moreover, compound 18d exerted favorable antioxidant activity, significantly reversed the amyloid-β_1-42 (Aβ_1-42) induced PC12 cell damage. More importantly, 18d remarkably ameliorated the cognitive dysfunction in a scopolamine-induced mice AD model. In brief, a series of hybrids with potential anti-AD effect were successfully obtained, indicating that the design of iron chelators with MAO-B inhibitory and antioxidant activities is an attractive strategy against AD progression.     

Scaffold-hopping identifies furano[2,3-d]pyrimidine amides as potent Notum inhibitors

The carboxylesterase Notum is a key negative regulator of the Wnt signaling pathway by mediating the depalmitoleoylation of Wnt proteins. Our objective was to discover potent small molecule inhibitors of Notum suitable for exploring the regulation of Wnt signaling in the central nervous system. Scaffold-hopping from thienopyrimidine acids 1 and 2, supported by X-ray structure determination, identified 3-methylimidazolin-4-one amides 20–24 as potent inhibitors of Notum with activity across three orthogonal assay formats (biochemical, extra-cellular, occupancy). A preferred example 24 demonstrated good stability in mouse microsomes and plasma, and cell permeability in the MDCK-MDR1 assay albeit with modest P-gp mediated efflux. Pharmacokinetic studies with 24 were performed in vivo in mouse with single oral administration of 24 showing good plasma exposure and reasonable CNS penetration. We propose that 24 is a new chemical tool suitable for cellular studies to explore the fundamental biology of Notum.     

Identification of potent pyrazole based APELIN receptor (APJ) agonists

The apelinergic system comprises the apelin receptor and its cognate apelin and elabela peptide ligands of various lengths. This system has become an increasingly attractive target for pulmonary and cardiometabolic diseases. Small molecule regulators of this receptor with good drug-like properties are needed. Recently, we discovered a novel pyrazole based small molecule agonist 8 of the apelin receptor (EC₅₀ = 21.5 µM, K_i = 5.2 µM) through focused screening which was further optimized to initial lead 9 (EC₅₀ = 0.800 µM, K_i = 1.3 µM). In our efforts to synthesize more potent agonists and to explore the structural features important for apelin receptor agonism, we carried out structural modifications at N1 of the pyrazole core as well as the amino acid side-chain of 9. Systematic modifications at these two positions provided potent small molecule agonists exhibiting EC₅₀ values of <100 nM. Recruitment of β-arrestin as a measure of desensitization potential of select compounds was also investigated. Functional selectivity was a feature of several compounds with a bias towards calcium mobilization over β-arrestin recruitment. These compounds may be suitable as tools for in vivo studies of apelin receptor function.     

Discovery of ORN0829, a potent dual orexin 1/2 receptor antagonist for the treatment of insomnia

Here, we present the design, synthesis, and SAR of dual orexin 1 and 2 receptor antagonists, which were optimized by balancing the antagonistic activity for orexin receptors and lipophilicity. Based on the prototype compound 1, ring construction and the insertion of an additional heteroatom into the resulting ring led to the discovery of orexin 1 and 2 receptor antagonists, which were 3-benzoyl-1,3-oxazinane derivatives. Within these derivatives, (−)-3h enabled a high dual orexin receptor antagonistic activity and a low lipophilicity. Compound (−)-3h exhibited potent sleep-promoting effects at a po dose of 1 mg/kg in a rat polysomnogram study, and optimal PK properties with a rapid T_max and short half-lives in rats and dogs were observed, indicating a predicted human half-life of 0.9–2.0 h. Thus, (−)-3h (ORN0829; investigation code name, TS-142) was selected as a viable candidate and is currently in clinical development for the treatment of insomnia.     

Studies on fragment-based design of allosteric inhibitors of human factor XIa

Human factor XIa (hFXIa) has emerged as an attractive target for development of new anticoagulants that promise higher level of safety. Different strategies have been adopted so far for the design of anti-hFXIa molecules including competitive and non-competitive inhibition. Of these, allosteric dysfunction of hFXIa’s active site is especially promising because of the possibility of controlled reduction in activity that may offer a route to safer anticoagulants. In this work, we assess fragment-based design approach to realize a group of novel allosteric hFXIa inhibitors. Starting with our earlier discovery that sulfated quinazolinone (QAO) bind in the heparin-binding site of hFXIa, we developed a group of two dozen dimeric sulfated QAOs with intervening linkers that displayed a progressive variation in inhibition potency. In direct opposition to the traditional wisdom, increasing linker flexibility led to higher potency, which could be explained by computational studies. Sulfated QAO 19S was identified as the most potent and selective inhibitor of hFXIa. Enzyme inhibition studies revealed that 19S utilizes a non-competitive mechanism of action, which was supported by fluorescence studies showing a classic sigmoidal binding profile. Studies with selected mutants of hFXIa indicated that sulfated QAOs bind in heparin-binding site of the catalytic domain of hFXIa. Overall, the approach of fragment-based design offers considerable promise for designing heparin-binding site-directed allosteric inhibitors of hFXIa.     

In-vivo hyperglycemic,antioxidant, histopathological changes,and simultaneous measurement of kaempferol verified by high-performance thin layer chromatography of Setaria italica in streptozotocin -induced diabetic rats

BackgroundSetaria italica (common name- foxtail, kangni) is one of the major food crops which is prominently cultivated in southern regions of India and in certain regions of Uttar Pradesh. Besides the crop’s consumption as a general source of carbohydrate rich cereal, the seeds of the crop are comprised of more fiber. So, it is recommended to add in the dietary supplementation of the diabetic people across the country.ObjectiveIn this paper, it intends to investigate the antidiabetic activity and antioxidant activity of S. italica (foxtail millet) seeds in diabetic rats.MethodsThe six genotypes of foxtail millets (S. italica) namely Kangni-1, Kangni-4, Kangni-5, Kangni-6, Kangni-7 & Kangni-10 respectively were subjected to in vitro investigations via. comprehensive metabolic panel (CMP) involving blood glucose study, Kidney & Liver function test, and antioxidant study (Catalase test; Glutathione S-transferase (GST); Superoxide Dismutase (SOD); glutathione (GSH); hiobarbituric acid reactive substances (TBARS) & Glutathione peroxidase (GPx) and were performed in vivo animal investigations in Wistar rats. The STZ induced diabetic rats were fed with doses of different S. italica seed aqueous extract to evaluate its anti-hyperglycemic activity by oral administration of SISAE. Further, it was compared with Glibenclamide which acts as one of the standard oral hypoglycemic agents.ResultsFrom achieved outcomes, a significant fall of blood glucose level (70%) produced 300 mg SISAE/kg b.w. after 6 h of extract administration. However, no change could be produced by these doses of the SISAE in normal rats’ blood glucose levels. A significant fall in glucose level along with significant glycemic control by lower HbA1c levels was observed in diabetic treated rats after 3 weeks of treatment with 300 mg of SISAE/kg b.w./day when comparing to untreated diabetic rats. Among these five genotypes of S. italica, the differences in the glycemic index were found. a significant fall could be found in blood glucose levels of Wistar rats, when every experimental rat was incorporating with the extract of different genotypes of Setaria italica L. Beauv than the rats treated with Glibenclamide in every 7 days of interval. The level of catalase, SOD, GST, GPx, GSH and TBARS showed variation while the rats were fed with the extract of S. italica in the liver test of rats. In kidney function test, the result shows that there is significant relationship between foxtail extract and kidney function of STZ induced diabetes rats. They show the change in their serum creatinine level, serum urea and serum uric acid.ConclusionThe result obtained from the study shows that the extract of S. italica seeds is capable for the hypolipidemic and antihyperglycemic activities, thereby, they serve as one of the good sources for herbal medicinal items.     

Heteroarylamide smoothened inhibitors: Discovery of N-[2,4-dimethyl-5-(1-methylimidazol-4-yl)phenyl]-4-(2-pyridylmethoxy)benzamide (AZD8542) and N-[5-(1H-imidazol-2-yl)-2,4-dimethyl-phenyl]-4-(2- pyridylmethoxy)benzamide (AZD7254)

Aberrant hedgehog (Hh) pathway signaling is implicated in multiple cancer types and targeting the Smoothened (SMO) receptor, a key protein of the Hh pathway, has proven effective in treating metastasized basal cell carcinoma. Our lead optimization effort focused on a series of heteroarylamides. We observed that a methyl substitution ortho to the heteroaryl groups on an aniline core significantly improved the potency of this series of compounds. These findings predated the availability of SMO crystal structure in 2013. Here we retrospectively applied quantum mechanics calculations to demonstrate the o-Me substitution favors the bioactive conformation by inducing a dihedral twist between the heteroaryl rings and the core aniline. The o-Me also makes favorable hydrophobic interactions with key residue side chains in the binding pocket. From this effort, two compounds (AZD8542 and AZD7254) showed excellent pharmacokinetics across multiple preclinical species and demonstrated in vivo activity in abrogating the Hh paracrine pathway as well as anti- tumor effects.     

Physiological,biochemical and molecular evaluation of mungbean genotypes for agronomical yield under drought and salinity stresses in the presence of humic acid

Drought and salinity are potential threats in arid and semi arid regions. The current study was conducted with objective to optimize the production of different exotic genotypes of mungbean (NM-121-25, Chakwal M-6, DM-3 and PRI-Mung-2018) under drought and salinity stresses using humic acid in field experiments. One year tri-replicate field experiment was performed in RCBD using three factorial arrangement and effects of humic acid (60 kg ha^?1) were evaluated at physiological, biochemical, molecular and agronomical level under individual and integrated applications of drought (no irrigation till 15 days) and salinity (EC 6.4 dSM^?1). Data for physiological parameters (total chlorophyll, photosynthesis rate, stomatal conductance, transpiration rate and membrane damage), antioxidant enzymes (superoxide dismutase, catalase, peroxidase) and proline were collected on weekly basis since after the initiation of drought and salinity stresses. However data for agronomic characteristics (plant height, branches plant^?1, LAI, pods plant^?1, pod length and hundred seed weight) and grain carbohydrate content were collected after harvesting, while sampling for drought (VrDREB2A, VrbZIP17 and VrHsfA6a) and salinity (VrWRKY73, VrUBC1 and VrNHX1) related genes expression study was done after plants attained seedling stage. Under both individual and integrated applications of drought and salinity, all genotypes showed significant (p ≤ 0.05) increase in all traits excluding Cell membrane damage and proline during humic acid application. Likewise, genes expression revealed statistically distinct (p ≤ 0.05) up-regulation under humic acid treatment as compared to no humic acid treatment during both individual and integrated applications of drought and salinity. The genotype PRI-Mung-2018 recorded noteworthy performance during study. Moreover correlation and PCA analysis revealed that ultimate agronomical yield due to humic acid is an outcome of interconnection of physiological and biochemical parameters.