Karam Chand

Substrate specificity of acetoxy derivatives of coumarins and quinolones towards Calreticulin mediated transacetylation: Investigations on antiplatelet function

Calreticulin transacetylase (CRTAase) is known to catalyze the transfer of acetyl group from polyphenolic acetates (PA) to certain receptor proteins (RP), thus modulating their activity. Herein, we studied for the first time the substrate specificity of CRTAase towards N-acetylamino derivatives of coumarins and quinolones. This study is endowed with antiplatelet action by virtue of causing CRTAase catalyzed activation of platelet Nitric Oxide Synthase (NOS) by way of acetylation leading to the inhibition of ADP/Arachidonic acid (AA)-dependent platelet aggregation. Among all the N-acetylamino/acetoxy coumarins and quinolones screened, 7-N-acetylamino-4-methylcoumarin (7-AAMC, 17) was found to be the superior substrate to platelet CRTAase and emerged as the most promising antiplatelet agent both in vitro and in vivo. Further it caused the inhibition of cyclooxygenase-1 (Cox-1) resulting in the down regulation of thromboxane A2 (TxA2), modulation of tissue factor and the inhibition of platelet aggregation. It was also found effective in the inhibition of LPS induced pro-thrombotic conditions.

Characterization of 4-methyl-2-oxo-1,2-dihydroquinolin-6-yl acetate as an effective antiplatelet agent

We have studied earlier a membrane bound novel enzyme Acetoxy Drug: protein transacetylase identified as Calreticulin Transacetylase (CRTAase) that catalyzes the transfer of acetyl groups from polyphenolic acetates (PAs) to the receptor proteins and thus modulating their biological activities. In this communication, we have reported for the first time that acetoxy quinolones are endowed with antiplatelet action by virtue of causing CRTAase catalyzed activation of platelet Nitric Oxide Synthase (NOS) by way of acetylation leading to the inhibition of ADP/Arachidonic acid (AA)-dependent platelet aggregation. The correlation of specificity of platelet CRTAase to various analogues of acetoxy quinolones with intracellular NO and consequent effect on inhibition of platelet aggregation was considered crucial. Among acetoxy quinolones screened, 6-AQ (4-methyl-2-oxo-1,2-dihydroquinolin-6-yl acetate/6-acetoxyquinolin-2-one, 22) was found to be the superior substrate to platelet CRTAase and emerged as the most active entity to produce antiplatelet action both in vitro and in vivo. 6-AQ caused the inhibition of cyclooxygenase-1 (Cox-1) resulting in the down regulation of thromboxane A2 (TxA2) and the inhibition of platelet aggregation. Structural modification of acetoxy quinolones positively correlated with enhancement of intracellular NO and antiplatelet action.

Synthesis and evaluation of c-Src kinase inhibitory activity of pyridin-2(1H)-one derivatives

Src kinase, a prototype member of the Src family of kinases (SFKs), is over-expressed in various human tumors, and has become a target for anticancer drug design. In this perspective, a series of eighteen 2-pyridone derivatives were synthesized and evaluated for their c-Src kinase inhibitory activity. Among them, eight compounds exhibited c-Src kinase inhibitory activity with IC50 value of less than 25μM. Compound 1-[2-(dimethylamino)ethyl]-5-(2-hydroxy-4-methoxybenzoyl)pyridin-2(1H)-one (36) exhibited the highest c-Src kinase inhibition with an IC50 value of 12.5μM. Furthermore, the kinase inhibitory activity of compound 36 was studied against EGFR, MAPK and PDK, however no significant activity was observed at the highest tested concentration (300μM). These results provide insights for further optimization of this scaffold for designing the next generation of 2-pyridone derivatives as candidate Src kinase inhibitors.

Specificity of calreticulin transacetylase to acetoxy derivatives of benzofurans: effect on the activation of platelet nitric oxide synthase.

Calreticulin Transacetylase (CRTAase) catalyzes the transfer of acetyl group(s) from polyphenolic acetates (PAs) to functional proteins, such as Glutathione S-transferase (GST), NADPH Cytochrome c reductase and Nitric Oxide Synthase (NOS) resulting in the modulation of biological activities. A comparison of the specificities of the acetoxy derivatives of coumarins, biscoumarins, chromones, flavones, isoflavones and xanthones has been carried out earlier by us with an aim to study the effect of nature and position of the acetoxy groups on the benzenoid ring and the position of the carbonyl group with respect to oxygen/nitrogen heteroatom for the catalytic activity of CRTAase. In this communication for the first time, we have studied the influence of differently substituted benzofurans on the CRTAase activity to study the effect of the replacement of pyran ring of coumarin with furan ring, presence of carbonyl at C-3, substitution of C-3 carbonyl group with acetoxy group and presence of various substituents (OAc/OH/Cl) on the benzenoid ring. It was observed that acetoxy derivatives of benzofurans lead to inhibition of ADP induced platelet aggregation by the activation of platelet Nitric Oxide Synthase catalyzed by CRTAase. Accordingly, the formation of NO in platelets by 3-oxo-2,3-dihydrobenzofuran-6,7-diyl diacetate (3a) was found to be comparable with that of model polyphenolic acetate (PA), 7,8-diacetoxy-4-methylcoumarin (DAMC).

Synthesis and Sensing Applications of Fluorescent 3-Cinnamoyl Coumarins.

We have synthesized two novel fluorescent 3-(4-diethylaminocinnamoyl) coumarins that exhibit fluorescence quenching upon exposure to a nerve agent simulant, diethylchlorophosphate (DCP), providing a basis for rapid and sensitive DCP chemosensing. Furthermore, these coumarin derivatives display two-photon fluorescence upon illumination with near-infrared laser pulses and their two-photon (TP) absorption cross-section was evaluated. The potential for TP bio-imaging of these compounds was investigated by their cellular uptake in HeLa cells by TP confocal microscopy.

Chromenone and quinolinone derivatives as potent antioxidant agents

The antioxidant activity (AOA) of three different classes of phenolic compounds viz chromen-2-ones, chromen-4-ones, and quinolin-2-ones was systematically studied using DPPH, ABTS, FRAP, and in vitro lipid peroxidation inhibition assays. The effect of incorporation of hydrophobic group on AOA was also studied. In DPPH, ABTS, and FRAP assays, the highest AOA was registered for the dihydroxy chromenones among all the phenolic derivatives. Presence of alkyl group led to reduction in AOA in the above three assays. However, in lipid peroxidation inhibition assay for selected compounds, incorporation of alkyl group led to enhancement in AOA. The AOA of few compounds was observed to be more than three times higher in comparison to standard “Trolox” in lipid peroxidation inhibitory assay.

Calreticulin transacetylase: A novel enzyme-mediated protein acetylation by acetoxy derivatives of 3-alkyl-4-methylcoumarins

Our earlier investigations culminated in the discovery of a unique membrane-bound enzyme Calreticulin transacetylase (CRTAase) in mammalian cells catalyzing the transfer of acetyl group from polyphenolic acetates (PAs) to certain functional proteins viz. Glutathione S-transferase (GST), NADPH Cytochrome c reductase and Nitric oxide synthase (NOS) resulting in the modulation of their biological activities. In order to develop SAR study, herein, we studied the influence of alkyl group at C-3 position of acetoxy coumarins on the CRTAase activity. The alkylated acetoxy coumarins lead to inhibition of catalytic activity of GST, and ADP induced platelet aggregation by the way of activation of platelet Nitric oxide synthase (NOS). Furthermore, the increase in size of the coumarin C-3 alkyl group was found to decrease the CRTAase activity.

Synthesis, (1)H and (13)C NMR assignment of novel 2-pyridone derivatives.

The synthesis of the 2-pyridone core structure is an attractive target for the synthetic organic chemist owing to its significance inmedicinal chemistry. In addition, 2-pyridone constitutes a structural component of several naturally occurring lead compounds, such as Huperzine A, Fredericamycin A, Camptothecin (CTP), Ilicicolin H, and Pyridoxatin. Many drugs containing pyridone skeleton have been released into the clinical world and a few more are under clinical trials, e.g. Amrinone, Milrinone or Primacor are used as cardiotonic agents for the treatment of heart failure, Perampanel is used for the treatment of Parkinson’s disease, Pirfenidone is used for the treatment of idiopathic pulmonary fibrosis, and Ciclopiroxolamine is used for the topical treatment of dermal infections. The potential clinical applicability and comparatively low toxicity of 2-pyridones have led the interest of many researchers to explore the utility of this moiety for better and varied pharmacological activities. Apart from the medicinal properties, 2-pyridone derivatives serve as viable synthons to pyridine, piperidine, quinolizidine, and indolizidine alkaloids and pyridone-tethered systems for dyes and pigments. Because the substitution of various functional groups on pyridin-2(1H)-one nucleus greatly affects the chemical shift values in the magnetic resonance of the proton and carbon nucleus, herein, we have synthesized a series of N-substituted derivatives of benzoylpyridin2-(1H)-ones and studied the impact of the different substituents on the chemical shifts of the protons and carbons of pyridone ring using one-dimensional (1D) and two-dimensional (2D) NMR spectroscopic techniques. This study may be helpful for the data exploration and structural elucidation of newer naturally occurring and synthetic 2-pyridone derivatives.

Acetamide Derivatives of Chromen‐2‐ones as Potent Cholinesterase Inhibitors

Alzheimers disease (AD), a neurodegenerative disorder, is a serious medical issue worldwide with drastic social consequences. Inhibition of cholinesterase is one of the rational and effective approaches to retard the symptoms of AD and, hence, consistent efforts are being made to develop efficient anti‐cholinesterase agents. In pursuit of this, a series of 19 acetamide derivatives of chromen‐2‐ones were synthesized and evaluated for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory potential. All the synthesized compounds exhibited significant anti‐AChE and anti‐BChE activity, with IC50 values in the range of 0.24–10.19 μM and 0.64–30.08 μM, respectively, using donepezil hydrochloride as the standard. Out of 19 compounds screened, 3 compounds, viz. 22, 40, and 43, caused 50% inhibition of AChE at 0.24, 0.25, and 0.25 μM, respectively. A kinetic study revealed them to be mixed‐type inhibitors, binding with both the CAS and PAS sites of AChE. The above‐selected compounds were found to be effective inhibitors of AChE‐induced and self‐mediated Aβ1–42 aggregation. ADMET predictions demonstrated that these compounds may possess suitable blood–brain barrier (BBB) permeability. Hemolytic assay results revealed that these compounds did not lyse human RBCs up to a thousand times of their IC50 value. MTT assays performed for the shortlisted compounds showed them to be negligibly toxic after 24 h of treatment with the SH‐SY5Y neuroblastoma cells. These results provide insights for further optimization of the scaffolds for designing the next generation of compounds as lead cholinesterase inhibitors.