Ravi Naik

Bioactive benzofuran derivatives: moracins A-Z in medicinal chemistry.

Benzofuran heterocycles are fundamental structural units in a variety of biologically active natural products as well as synthetic materials. Over the time, benzofuran derivatives have attracted many researchers due to the broad scope of their biological activity, which include anticancer, antimicrobial, immunomodulatory, antioxidant and anti-inflammatory properties. Egonol, homoegonol and moracin families are biologically active natural products containing benzofuran heterocycle as basic structural units. This paper focuses on the moracin family (moracin A to Z). Morus, a genus of flowering plants in the family Moraceae, comprises 10-16 species of deciduous trees commonly known as mulberries. The root bark, stem bark and leaves of Morus alba, M. lhou, Morus macroura are the main sources for arylbenzofuran derivatives including the moracins. A large volume of research has been carried out on moracins and their derivatives, which has shown the pharmacological importance of this benzofuran heterocyclic nucleus. In this mini-review, we attempt to highlight the importance of moracins, as they have been a major source for drug development. Herein, we also summarize the current state of the art concerning the synthesis and medicinal use of moracins A-Z.

Identification of malate dehydrogenase 2 as a target protein of the HIF-1 inhibitor LW6 using chemical probes

Hypoxia-inducible factor (HIF) regulates tumor angiogenesis and metastasis in response to low oxygen tension. In the presence of oxygen, HIF-1a is rapidly degraded through the ubiquitin–proteasome pathway. In hypoxic conditions, stabilized HIF-1a dimerizes with HIF-1b. The HIF-1a/b heterodimer binds to hypoxia response elements (HRE) in gene promoters and induces the expression of target genes involved in angiogenesis, metastasis, glycolysis, cell proliferation, and resistance to apoptosis. Increased expression of HIF-1a in many solid tumors correlates with aggressive tumor growth, therapeutic resistance, and a poor clinical outcome. HIF-1a shifts the metabolism from oxidative phosphorylation to anaerobic glycolysis. Therefore, HIF-1a is an important therapeutic target for cancer. We previously synthesized and evaluated aryloxyacetylamino benzoic acid analogues. LW6 (1 in Figure 1A) potently inhibited HIF-1a accumulation by degrading HIF1a without affecting the HIF-1a mRNA levels during hypoxia. LW6, which is commercially available, has been used in various studies as an HIF-1a inhibitor. However, the molecular target of LW6 remains unknown. To identify a drug target, chemical biological methods such as activity-based probes (ABPs), photoaffinity labeling, biotinylation, and click conjugation have been used. Herein, we identify the molecular target of 1 using chemical probes. Cellular images and direct protein interactions of 1 were examined in living cells with a series of chemical probes (2–6), which were designed using the structure– activity relationship (SAR) of 1. Synthesis and characterization data for these probes are available in the Supporting Information. The distribution of drug molecules within subcellular compartments can provide information about the mechanism of drug action. The intracellular localization of LW6 was visualized through click chemistry with probe 2, containing an acetylene group, in colon cancer HCT116 cells (Figure 1A). Both 1 and 2 suppressed HIF-1a accumulation (Figure 1B) and HRE-luciferase activity (Figure 1A; Supporting Information, Figure S12). Subsequently, the cellular localization of probe 2 was determined by a click reaction with an azidelinked Alexa Fluor 488 molecule. Notably, copper-catalyzed azide–alkyne cycloadditions (click reactions) are highly specific and efficient bio-orthogonal reactions to visualize intracellular probe distribution. We found that compound 2 was localized primarily in the cytoplasm (Figure 1C). The colocalization of compound 2 (3 mm) with the mitochondriaselective probe, MitoTracker (500 nm), indicated that 2 is specifically localized in the mitochondria, whereas the localization of an adamantyl-free probe 4 was not observed (Figure S13). The mitochondrial localization of probe 2 was Figure 1. Biological activities and cellular localization of a chemical probe for LW6. A) Formula of 1 and its clickable probe 2. B) Inhibitory effects of 1 and probe 2 on HIF-1a accumulation were determined by immunoblot analysis. C) Localization of probe 2 (3 mm, green) was detected through a click reaction using azide-linked Alexa Fluor 488 in HCT116 cells. Mitochondria were selectively stained with the MitoTracker probe (red). Nuclei (blue) were stained with 4,6-diamidino-2phenylindole (DAPI). D) Competitive binding of probe 2 (3 mm) to its target molecules in the presence or absence of 1 (10 mm). Scale bars = 20 mm.

Therapeutic Strategies for Metabolic Diseases: Small‐Molecule Diacylglycerol Acyltransferase (DGAT) Inhibitors

Metabolic diseases such as atherogenic dyslipidemia, hepatic steatosis, obesity, and type II diabetes are emerging as major global health problems. Acyl‐CoA:diacylglycerol acyltransferase (DGAT) is responsible for catalyzing the final reaction in the glycerol phosphate pathway of triglycerol synthesis. It has two isoforms, DGAT‐1 and DGAT‐2, which are widely expressed and present in white adipose tissue. DGAT‐1 is most highly expressed in the small intestine, whereas DGAT‐2 is primarily expressed in the liver. Therefore, the selective inhibition of DGAT‐1 has become an attractive target with growing potential for the treatment of obesity and type II diabetes. Furthermore, DGAT‐2 has been suggested as a new target for the treatment of DGAT‐2‐related liver diseases including hepatic steatosis, hepatic injury, and fibrosis. In view the discovery of drugs that target DGAT, herein we attempt to provide insight into the scope and further reasons for optimization of DGAT inhibitors.

Synthesis and structure-activity relationship study of chemical probes as hypoxia induced factor-1α/malate dehydrogenase 2 inhibitors.

A structure-activity relationship study of hypoxia inducible factor-1α inhibitor 3-aminobenzoic acid-based chemical probes, which were previously identified to bind to mitochondrial malate dehydrogenase 2, was performed to provide a better understanding of the pharmacological effects of LW6 and its relation to hypoxia inducible factor-1α (HIF-1α) and malate dehydrogenase 2 (MDH2). A variety of multifunctional probes including the benzophenone or the trifluoromethyl diazirine for photoaffinity labeling and click reaction were prepared and evaluated for their biological activity using a cell-based HRE-luciferase assay as well as a MDH2 assay in human colorectal cancer HCT116 cells. Among them, the diazirine probe 4a showed strong inhibitory activity against both HIF-1α and MDH2. Significantly, the inhibitory effect of the probes on HIF-1α activity was consistent with that of the MDH2 enzyme assay, which was further confirmed by the effect on in vitro binding activity to recombinant human MDH2, oxygen consumption, ATP production, and AMP activated protein kinase (AMPK) activation. Competitive binding modes of LW6 and probe 4a to MDH2 were also demonstrated.

Synthesis and structure-activity relationship of (E)-phenoxyacrylic amide derivatives as hypoxia-inducible factor (HIF) 1α inhibitors.

A series of (E)-phenoxyacrylic amide derivatives were synthesized and evaluated as hypoxia inducible factor (HIF) 1α inhibitors. The present structure-activity relationship study on this series identified the morpholinoethyl containing ester 4p as a potent inhibitor of HIF-1α under hypoxic conditions (IC50=0.12 μM in a cell-based HRE reporter assay) in HCT116 cells. The representative compound 4p suppressed hypoxia-induced HIF-1α accumulation and targeted gene expression in a dose-dependent manner. The effect of HIF-1α inhibition by 4p was further demonstrated by its inhibitory activity on in vitro tube formation and migration of cells, which may be valuable for development of novel therapeutics for cancer and tumor angiogenesis.

Identification of Targets of the HIF-1 Inhibitor IDF-11774 Using Alkyne-Conjugated Photoaffinity Probes.

We developed a hypoxia-inducible factor-1 (HIF-1) inhibitor, IDF-11774, as a clinical candidate for cancer therapy. To understand the mechanism of action of IDF-11774, we attempted to isolate target proteins of IDF-11774 using bioconjugated probes. Multifunctional chemical probes containing sites for click conjugation and photoaffinity labeling were designed and synthesized. After fluorescence and photoaffinity labeling of proteins, two-dimensional electrophoresis (2DE) was performed to isolate specific molecular targets of IDF-11774. Heat shock protein (HSP) 70 was identified as a target protein of IDF-11774. We revealed that IDF-11774 inhibited HSP70 chaperone activity by binding to its allosteric pocket, rather than the ATP-binding site in its nucleotide-binding domain (NBD). Moreover, IDF-11774 reduced the oxygen consumption rate (OCR) and ATP production, thereby increasing intracellular oxygen tension. This result suggests that the inhibition of HSP70 chaperone activity by IDF-11774 suppresses HIF-1α refolding and stimulates HIF-1α degradation. Taken together, these findings indicate that IDF-11774-derived chemical probes successfully identified IDF-11774s target molecule, HSP70, and elucidated the mode of action of IDF-11774 in inhibiting HSP70 chaperone activity and stimulating HIF-1α degradation in cancer cells.

Discovery of indolyl acrylamide derivatives as human diacylglycerol acyltransferase-2 selective inhibitors

A series of indolyl acrylamide derivatives was synthesized as potential diacylglycerol acyltransferase (DGAT) inhibitors. Furfurylamine containing indolyl acrylamide derivative 5h exhibited the most potent DGAT inhibitory activity using microsomes prepared from rat liver. Further evaluation against human DGAT-1 and DGAT-2 identified indolyl acrylamide analogues as selective inhibitors against human DGAT-2. In addition, the most potent compound 5h inhibited triglyceride synthesis dose-dependently in HepG2 cell lines.

Efficient and Convenient Method for Synthesis of Benzofuran-3-acetic Acids and Naphthafuran-acetic Acids

Abstract Herein, we report an efficient and convenient method for synthesis of benzofuran-3-acetic acids and naphthafuran-acetic acids 5a–p by the reaction of substituted-4-bromomethylcoumarins with aqueous sodium hydroxide at refluxing temperature. The obtained products are characterized by infrared, 1H NMR, 13C NMR, and mass spectral data. Structures 5a and 5e are confirmed by their single x-ray diffraction studies. The advantages of this method are good yields, easy workup, and no chromatographic purifications. GRAPHICAL ABSTRACT

Chemical biology approach for the development of hypoxia inducible factor (HIF) inhibitor LW6 as a potential anticancer agent.

Intratumoral hypoxia has long been considered to be a driving force in tumor progression as well as a negative prognostic factor in human cancers. The discovery of hypoxia inducible factors (HIFs), which mediate transcriptional responses to changes in oxygen levels, has renewed enthusiasm for drug discovery and the development of targeted therapies in this field. LW6 represents an important new class of small molecules that inhibit HIF-1; it has been major source for diverse lead compounds including HIF-1α inhibitors. Through a chemical biology approach, LW6-derived chemical probes were successfully utilized for the identification of the direct targeting of a protein in cancer. LW6 provides a valuable platform for the discovery and development of small molecule inhibitors of HIF-1α-dependent tumor progression, metabolic reprogramming, and angiogenesis.Graphical Abstract

Methyl 3-(3-(4-(2,4,4-Trimethylpentan-2-yl)phenoxy)-propanamido)benzoate as a Novel and Dual Malate Dehydrogenase (MDH) 1/2 Inhibitor Targeting Cancer Metabolism

Previously, we reported a hypoxia-inducible factor (HIF)-1 inhibitor LW6 containing an (aryloxyacetylamino)benzoic acid moiety inhibits malate dehydrogenase 2 (MDH2) using a chemical biology approach. Structure-activity relationship studies on a series of (aryloxyacetylamino)benzoic acids identified selective MDH1, MDH2, and dual inhibitors, which were used to study the relationship between MDH enzyme activity and HIF-1 inhibition. We hypothesized that dual inhibition of MDH1 and MDH2 might be a powerful approach to target cancer metabolism and selected methyl-3-(3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanamido)-benzoate (16c) as the most potent dual inhibitor. Kinetic studies revealed that compound 16c competitively inhibited MDH1 and MDH2. Compound 16c inhibited mitochondrial respiration and hypoxia-induced HIF-1α accumulation. In xenograft assays using HCT116 cells, compound 16c demonstrated significant in vivo antitumor efficacy. This finding provides concrete evidence that inhibition of both MDH1 and MDH2 may provide a valuable platform for developing novel therapeutics that target cancer metabolism and tumor growth.