Bioisosterism in Drug Discovery and Development - An Overview.

Bioisosterism is a unique approach used by medicinal chemists for the reasonable modification of lead compounds into safer, more clinically effective, economical, and therapeutically attractive drugs. It is one of the most crucial lead modification tools, widely applied in the field of rational drug design to amplify the desired activity and eliminate undesirable properties, thus facilitating the optimization of pharmacokinetic profile and achievement of target selectivity. This review demonstrates the importance of bioisosterism in the process of drug discovery and development and highlights its relevance in the molecular evolution of many classes of drugs such as antibacterial sulfonamides, anticancer drugs, antivirals, antifungals, anthelmintics, local anesthetics, barbiturates, antidepressants, antihistamines, proton pump inhibitors and work carried out by our team of researchers. The role of bioisosterism as a strategy to achieve inhibition of enzymes such as thymidylate synthase, DNA polymerase, reverse transcriptase and several others has also been pointed out. There are no limits to the classes of drugs where bioisosterism has been successfully applied.

Epigenetic modulation of macrophage polarization- perspectives in diabetic wounds.

Diabetes is a chronic metabolic disorder that poses a global burden to healthcare. Increasing incidence of diabetes-related complications in the affected population includes a delay in wound healing that often results in non-traumatic limb amputations. Owing to the intricacies of the healing process and crosstalk between the multitude of participating cells, the identification of hyperglycaemia-induced changes at both cellular and molecular levels poses a challenge. Macrophages are one of the key participants in wound healing and continue to exert functional changes at the wound site since the time of injury. In the present review, we discuss the role of these cells and their aberrant functions in diabetic wounds. We have extensively studied the process of macrophage polarization (MP) and its modulation through epigenetic modifications. Data from both pre-clinical and clinical studies on diabetes have co-related hyperglycaemia induced changes in gene expression to an increased incidence of diabetic complications. Hyperglycaemia and oxidative stress, create an environment prone to changes in the epigenetic code, that is manifested as an altered inflammatory gene expression. Here, we have attempted to understand the different epigenetic modulations that possibly contribute towards dysregulated MP, resulting in delayed wound healing.

Evaluation of Novel 3-Hydroxyflavone Analogues as HDAC Inhibitors against Colorectal Cancer.

Alteration of epigenetic enzymes is associated with the pathophysiology of colon cancer with an overexpression of histone deacetylase 8 (HDAC8) enzyme in this tissue. Numerous reports suggest that targeting HDAC8 is a viable strategy for developing new anticancer drugs. Flavonols provide a rich source of molecules that are effective against cancer; however, their clinical use is limited. The present study investigated the potential of quercetin and synthetic 3-hydroxyflavone analogues to inhibit HDAC8 enzyme and evaluated their anticancer property. Synthesis of the analogues was carried out, and cytotoxicity was determined using MTT assay. Nonspecific and specific HDAC enzyme inhibition assays were performed followed by the expression studies of target proteins. Induction of apoptosis was studied through annexin V and caspase 3/7 activation assay. Furthermore, the analogues were assessed against in vivo colorectal cancer. Among the synthesized analogues, QMJ-2 and QMJ-5 were cytotoxic against HCT116 cells with an IC50 value of 68 ± 2.3 and 27.4 ± 1.8 µM, respectively. They inhibited HDAC enzyme in HCT116 cells at an IC50 value of 181.7 ± 22.04 and 70.2 ± 4.3 µM, respectively, and inhibited human HDAC8 and 1 enzyme at an IC50 value of <50 µM with QMJ-5 having greater specificity towards HDAC8. A reduction in the expression of HDAC8 and an increase in acetyl H3K9 expression were observed with the synthesized analogues. Both QMJ-2 and QMJ-5 treatment induced apoptosis through the activation of caspase 3/7 evident from 55.70% and 83.55% apoptotic cells, respectively. In vivo studies revealed a significant decrease in colon weight to length ratio in QMJ-2 and QMJ-5 treatment groups compared to DMH control. Furthermore, a reduction in aberrant crypt foci formation was observed in the treatment groups. The present study demonstrated the potential of novel 3-hydroxyflavone analogues as HDAC8 inhibitors with anticancer property against colorectal cancer.

In vitro and in vivo anticancer studies of 2'-hydroxy chalcone derivatives exhibit apoptosis in colon cancer cells by HDAC inhibition and cell cycle arrest.

Considering the therapeutic values of bioflavonoids in colon cancer treatment, six 2'-hydroxy chalcones (C1-C6) were synthesized, characterized and screened for in vitro cytotoxicity on human colon carcinoma (HCT116) and African green monkey kidney epithelial cells (Vero). Only C5 showed selective cytotoxicity against HCT116 cells. Other potent cytotoxic compounds were C1, C2 and C3. Further screening included enzyme inhibition studies on histone deacetylase (HDAC) enzyme where C1 showed lowest IC50 value (105.03 µM). Based on cytotoxicity data C1, C2 and C3 were selected for further in vitro mechanistic studies, namely apoptotic studies (Acridine orange/Ethidium bromide (AO/EB) and Annexin V), cell cycle analysis using propidium iodide (PI) stain and in vivo anticancer efficacy in 1,2-dimethyl hydrazine (DMH) induced colorectal carcinoma in Wistar rats. The compounds induced apoptosis in more than 30 % cells in AO/EB and Annexin V staining. They also showed cell cycle arrest in G2/M phase with PI staining. They showed a significant reduction in aberrant crypt foci formation and adenocarcinoma count along with a significant (p<0.05) reduction in TNF-α levels as compared to DMH control at 100 mg/kg dose. Thus, it can be concluded that the synthesized 2'-hydroxychalcones were effective against colon adenocarcinoma in in vitro and in vivo studies.

Novel flavonol analogues as potential inhibitors of JMJD3 histone demethylase-A study based on molecular modelling.

Epigenetic modulation of gene expression has drawn enormous attention among researchers globally in the present scenario. Since their discovery, Jmj-C histone demethylases were identified as useful markers in understanding the role of epigenetics in inflammatory conditions and in cancer as well. This has created arousal of interest in search of suitable candidates. Potential inhibitors from various other scaffolds such as hydroxyquinolines, hydroxamic acids and triazolopyridines have already been identified and reported. In this direction, our present study attempts to target one of the important members of the family- namely JMJD3 (also known as KDM6B), that plays a pivotal role in inflammatory and immune reactions. Using molecular modeling approaches, myricetin analogues were identified as promising inhibitors of JMJD3. Extensive literature review showed myricetin as the most promising flavonol inhibitor for this enzyme. It served as a prototype for our study and modification of it's scaffold led to generation of analogues. The ZINC database was used as a repository for natural compounds and their analogues. Using similarity search options, 65 analogues of myricetin were identified and screened against JMJD3 (PDB ID: 4ASK), using the high throughput virtual screening and ligand docking tools in Maestro Molecular Modeling platform (version 10.5) from Schrödinger, LLC. 8 analogues out of 65 were identified as the most appropriate candidates which gave the best pose in ligand docking. Their binding mode and energy calculations were analysed using induced fit docking (IFD) and prime-MMGBSA tool, respectively. Thus, our findings highlight the most promising analogues of myricetin with comparable binding affinity as well as binding energy than their counterparts that could be taken for further optimisation as inhibitors of JMJD3 in both in vitro and in vivo screening studies.

Isolation of isoflavones from Iris kashmiriana Baker as potential anti proliferative agents targeting NF-kappaB.

Cancer is possibly one of the most devastating and complex disease and therefore involves chemotherapy as one of the frontline strategies in its therapy. However, expected toxicity and resistance with chemotherapeutic agents encourage us to use the plant derived natural chemotherapeutic sources at the clinical stage of cancer therapy. In view of this strategy, herein new glycosides and isoflavonoids were isolated from Iris kashmiriana Baker and subjected to structure elucidation followed by their biological evaluation. Isolated compounds and their derivatives were purified by the column chromatography and structural identification was made by a combination of various spectroscopic technique vis. UV, IR, 1H NMR, 13C NMR, DEPT, 2-D NMR and mass spectrometry coupled with chemical analysis. Furthermore, an in silico library of isolated isoflavones and its analogues were designed. NF-kappaB (transcription factor that facilitates angiogenesis, inflammation, invasion and metastasis) was selected as a target to evaluate the anticancer and antioxidant activity of isoflavones and its analogues. Designed library of isoflavones and analogues were docked into the active site of NF-kappa B and the most active 15 analogues were selected for synthesis. Finally, all compounds were evaluated for their cytotoxicity against various cell lines and antioxidant activity with different methods that demonstrate their anti-cancer and anti-oxidant potential. The cell cycle specificity of the cytotoxicity was further analyzed by corresponding analysis, using flow cytometer. Most of the compounds exhibit moderate activity, whereas the 5,7,8-trihydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one, 5,7,8-trihydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one, 5,7,8-triacetoxyoxy-3-(4-methoxyphenyl)-4H-chromen-4-one and 6,7-diacetoxyoxy-3-(4-methoxyphenyl)-4H-chromen-4-one showed distinct broad-spectrum anticancer activity with IC50 values ranges between 3.8 and 5.6 µg/mL. Cell cycle analysis indicates that these compounds induced cell cycle arrest at G2/M phase.

Targets in anticancer research--A review.

Cancer is a complex disease characterized by a loss in the normal cell regulatory mechanisms that govern cell survival, proliferation, and differentiation. Current chemotherapeutics, as anticancer agents, are developing resistance to single drug and also to treatment therapies involving multiple drugs. Cross resistance associated with the specificity and selectivity of existing drugs has restricted the application of chemotherapy. Alternatively, these limitations have given better insight in understanding the underlying molecular mechanisms responsible for the development of various stages in cancer. In the light of this, continuous efforts are being made in order to identify and validate newer anticancer targets. This review presents some of the important targets that have been already reported, such as aromatase, farnesyl transferase, histone deacetylase, tyrosine kinase and cyclin-dependent kinase. A few molecules designed against these targets have successfully reached clinical trials. However, only limited marketed drugs are available from these classes. Besides, the review also highlights some of the other important targets and strategies that have also drawn considerable attention in the area of anticancer drug development such as, cancer stem cells and monoclonal antibodies. Further, the integration of the tools in molecular biology with the results from preclinical and clinical trials would strengthen the effectiveness of treatment regimens in cancer patients. There lies a much scope for designing promising lead compounds and treatment therapies against these established targets.

In vitro and in vivo evaluation of novel cinnamyl sulfonamide hydroxamate derivative against colon adenocarcinoma.

The potential of cinnamic acid as an anti-inflammatory and anti-cancer agent has been studied previously. In our investigation, novel bio-isosters of cinnamyl sulfonamide hydroxamate were synthesized, characterized and confirmed for their structure and evaluated for cytotoxicity. Three NCEs namely, NMJ-1, -2 and -3 showed cell-growth inhibition in 6 human cancer cell lines with IC50 at the range of 3.3±0.15-44.9±2.6 µM. The hydroxamate derivatives of cinnamyl sulfonamide are reported inhibitors of HDAC enzyme. Thus, the effectiveness of these molecules was determined by whole cell HDAC assay in HCT 116 cell line. NMJ-2 (0.41±0.01 µM) exhibited better enzyme inhibition (IC50) compared to SAHA (2.63±0.07). In order to evaluate induction of apoptosis by treatment, Hoechst 33342 and AO/EB nuclear staining methods were used. Further, cell cycle analysis, Annexin V binding and caspase 3/7 activation assays were performed by flow cytometry where NMJ-2 significantly arrested the cell cycle at G2/M phase, increased Annexin V binding to the cell surface and activation of caspase-3/7. Bax/Bcl-2 ratio was observed by Western blot and showed an increase with NMJ-2 treatment. This was comparable to standard SAHA. The acute toxicity study (OECD-425) showed that NMJ-2 was safe up to 2000 mg/kg in rats. 1,2-Dimethyl hydrazine (DMH) was used to produce experimental colon adenocarcinoma in Wistar rats. 5-FU and NMJ-2 (100 mg/kg p.o. and 10 mg/kg i.p. once daily for 21 days, respectively) were administered to the respective groups. Both treatments significantly reduced ACFs, adenocarcinoma count, TNF-α, IL-6, nitrite and nitrate levels in colonic tissue. Our findings indicate that NMJ-2 has potent anti-cancer activity against colon cancer, by acting through HDAC enzyme inhibition and activation of intrinsic mitochondrial apoptotic pathway, with additional anti-inflammatory activity.

Antidiabetic activity of benzopyrone analogues in nicotinamide-streptozotocin induced type 2 diabetes in rats.

Benzopyrones are proven antidiabetic drug candidate in diabetic drug discovery. In this view novel synthetic benzopyrone analogues were selected for testing in experimental diabetes. Type 2 diabetes (T2D) was induced in Wistar rats by streptozotocin (60 mg/kg, i.p.) followed by nicotinamide (120 mg/kg i.p.). Rats having fasting blood glucose (FBG)>200 mg/dL, 7 days after T2D-induction, are selected for the study. Test compounds and standard treatment were continued for 15 days. FBG, oral glucose tolerance test (OGTT), and insulin tolerance test (ITT) were determined on 21st day after induction of T2D. Plasma lipids and serum insulin were estimated. Homeostatic model assessment (HOMA-IR) was then calculated from serum insulin. Rats were sacrificed and pancreas was isolated for histopathological observations. Oxidative stress markers were estimated in liver homogenate. Quercetin, a natural product with benzopyrone ring, showed significant hypoglycemic activity comparable to glibenclamide. Treatment with test compounds lowered the FBG and insulin resistance was significant alleviated as determined by OGTT, HOMA-IR, and ITT. There was significant normalisation of liver antioxidant enzymes compared to diabetic rats indicating that all the synthesised benzopyrone analogues are beneficial in reducing oxidative stress and are on par with the standard quercetin and glibenclamide in experimental T2D.

Iminoflavones combat 1,2-dimethyl hydrazine-induced aberrant crypt foci development in colon cancer.

The aim of the present study was to evaluate the antitumor potential of iminoflavones in in vitro and in vivo anticancer models. Preliminary screening in various cancer cell lines revealed four potential iminoflavones out of which IMF-8 was taken based on its activity against colon cancer cells. This was further confirmed by observing the nuclear changes in the cells by AO/EB and Hoechst 33342 staining studies. In vivo activity was assessed by dimethyl hydrazine-(DMH-) induced colon cancer model in rats. Animals were administered DMH (20 mg/kg, b.w. for 10 weeks and 30 mg/kg b.w., i.p. for 10 weeks) and were supplemented with (IMF-8) iminoflavone-8 (200 mg/kg, p.o. for 14 days). Results showed that DMH induced 100% aberrant crypt foci (ACF) and polyps which were significantly reduced in the IMF-8 treated group. IMF-8 significantly increased the catalase and GSH levels whereas it reduced the TNF- α and IL-6 levels markedly which suggests the antioxidative and anti-inflammatory actions of flavonoids present in IMF-8. The histopathological images of the IMF-8 treated colon showed no signs of mucosal crypt abscess. These findings suggest that the semi-synthetic iminoflavones, IMF-8, effectively inhibit DMH-induced ACFs and colonic crypts by alleviating the oxidative stress and suppressing the inflammation.

Antidiabetic activity of 3-hydroxyflavone analogues in high fructose fed insulin resistant rats.

Synthetic 3-hydroxyflavone analogues (JY-1, JY-2, JY-3, JY-4), were tested for antidiabetic activity in high-fructose-diet-fed (66 %, for 6 weeks) insulin-resistant Wistar rats (FD-fed rats). The fasting blood glucose, insulin, creatinine and AGEs were decreased to near normal upon treatment with test compounds. Insulin resistance markers such as HOMA-IR, K-ITT, plasma triglycerides, lipids, endogenous antioxidant defense and glycogen were restored in FD-fed rats after treatment with 3-hydroxyflavones. It is known that insulin resistance is partly because of oxidative stress and hence antioxidant activity was determined. They exhibited significant in vitro DPPH and ABTS radical scavenging activity (IC50: 10.66-66.63 µM). Test compounds inhibited ROS and NO production in RAW 264.7 cells (IC50: 10.39-42.63 µM) and they were found as potent as quercetin. Further, the test compounds inhibited lipid peroxidation at low concentrations (IC50: 99.61-217.47 µM). All test compounds at concentrations 100-200 µM protected calf thymus DNA-damage by Fenton reaction. In addition, test compounds inhibited protein glycation in different in vitro antiglycation assays. JY-2 showed maximum potency in all the stages of glycation which was comparable to the standard quercetin and aminoguanidine. Test compounds also enhanced the glucose uptake by L6 myotubes at an EC50 much lower than that of quercetin. Thus the synthetic 3-hydroxyflavones were found to have good antidiabetic activity by pleotropic and multimodal suppression of insulin resistance and enhancement of glucose uptake by skeletal muscles. These compounds are non-toxic at the doses tested. Further, the combined antioxidant and antiglycation activities of these molecules have complementary benefits in management of diabetes.

1-(2-Hy-droxy-5-methyl-phen-yl)-3-(2-methyl-phen-yl)prop-2-en-1-one.

In the title compound, C(17)H(16)O(2), the dihedral angle between the aromatic rings is 5.12 (13)° and an intra-molecular O-H⋯O hydrogen bond generates an S(6) ring.

1-(2-Hy-droxy-4-meth-oxy-phen-yl)-3-(4-methyl-phen-yl)prop-2-en-1-one.

The mol-ecule of the title compound, C(17)H(16)O(3), exists in the E conformation with respect to the central C=C bond, is almost planar(r.m.s. deviation = 0.003 Å) and has an intra-molecular O-H⋯O hydrogen bond, which generates an S(6) ring. In the crystal, mol-ecules are linked by C-H⋯O inter-actions.

1-(2-Hydr-oxy-5-methyl-phen-yl)-3-(3-methylthiophen-2-yl)prop-2-en-1-one.

In the structure of the title compound, C(15)H(14)O(2)S, the benzene ring is nearly coplanar with the thio-phene ring. The hydroxy group substituted at C2 position is in an antiperi-planar conformation with respect to the phenyl ring. The crystal structure exhibits weak intramolecular O-H⋯O hydrogen bonding.

Synthesis, Antiinflammatory and Antibacterial Activity of Novel Indolyl-isoxazoles.

Chalcones were synthesized by reacting indole-3-aldehyde, prepared by Vilsemeir Haack reaction with 4-substituted acetophenone in ethanolic KOH solution. These chalcones were immediately reacted with hydroxylamine hydrochloride in presence of glacial acetic acid as reagent to obtain the corresponding isoxazole derivatives. The synthesized heterocycles were characterized on the basis of physical, chemical tests and spectroscopic data. These compounds were tested for the acute antiinflammatory activity and antibacterial activity using carrageenan-induced rat paw edema method and cup-plate method, respectively.

Synthesis of Some New 3-Coumarinoyl Pyridinium and Quinolinium Bromides for their Antimicrobial Activity.

3-Bromo acetyl coumarins (4a-c) on treatment with methyl and ethyl esters of nicotinic acid, isonicotinic acid (5a-d) gave 3-coumrinoyl pyridinium bromides (7a-l) and with quinoline (6) gave 3-coumarinoyl quinolinium bromides (8a-c). The structures of the final compounds were established by their spectral data. The antimicrobial activitiy of all the newly synthesized test compounds were evaluated and some of them, showed good antibacterial activity.

Microwave-induced synthesis of schiff bases of aminothiazolyl bromocoumarins as antibacterials.

A fast and highly efficient method for the synthesis of some of the schiff bases of aminothiazolylbromocoumarin (4a-m) has been performed by microwave irradiation of 2'-amino-4'-(6-bromo-3-coumarinyl) thiazole (3) and substituted aromatic aldehydes (a-m). Microwave assisted reactions have resulted in better yields of the desired products than when prepared under conventional conditions. The resulting products were evaluated for their qualitative and quantitative antibacterial activity.

Characterization of 2-chloro-N10-substituted phenoxazines for reversing multidrug resistance in cancer cells.

Twenty-one 2-chloro-N10-substituted phenoxazines have been synthesized and characterized as potential modulators of multidrug resistance (MDR). Many of the compounds, at a concentration of 100 microM, enhanced accumulation of vinblastine (VLB) in drug-resistant KB8-5 cells to a greater extent than the same concentration of verapamil (VRP). However, the effects on VLB accumulation were specific, because these derivatives had little activity in the parental drug-sensitive line KB3-1. The compounds slowed the efflux of VLB from KB8-5 cells, suggesting that the chlorophenoxazines, like VRP, can inhibit P-glycoprotein (P-gp)-mediated efflux of VLB from this cell line. Two of the chlorophenoxazine derivatives, and also VRP, were able to stimulate the vanadate-sensitive ATPase activity attributable to P-gp in membranes isolated from MDR1 baculovirus-infected Sf9 cells. This result suggests that these modulators exert their effect by directly interacting with P-gp. Apart from the parent unsubstituted molecule, 2-chlorophenoxazine, there was a good correlation between log10P and the ability of the compounds to enhance VLB accumulation in KB8-5. This suggests that lipophilicity of a modulator is important, but is not the sole determinant of potency. Within this series of compounds, the optimal structural features for MDR modulation include a hydrophobic phenoxazine ring with a -Cl atom in the C-2 position and a tertiary amine group four carbons from the tricyclic ring. Many of the agents at the IC10 concentration completely reversed the 37-fold VLB resistance in KB8-5 cells. The most active agents in KB8-5 were able to partially reverse VLB resistance in an MDR colon carcinoma cell line GC3/c1 and completely reversed the 86-fold VLB resistance in the MDR1-overexpressing breast carcinoma cell line BC19/3. These same agents could only partially sensitize BC19/3 cells to taxol and doxorubicin, suggesting that the chlorophenoxazine derivatives show some specificity for modulating VLB resistance.

Liquid secondary ionization mass spectrometry and collision-induced dissociation study of 2-chloro-N10-substituted phenoxazines.

Positive-ion liquid secondary ionization mass spectrometry in combination with 3-nitrobenzyl alcohol as the liquid matrix was used to investigate the mass spectral features of a set of 21 N10-substituted derivatives of 2-chlorophenoxazine. The N-10 substitution included propyl, butyl and acetyl groups containing various secondary amines (N,N-diethylamine, N,N-diethanolamine, morpholine, piperidine, pyrrolidone or beta-hydroxyethylpiperazine) or a chloro group. These compounds are potent multi-drug resistance modulators. The molecular ions are observed as M+ and [M + H]+ ions. In general, the fragmentation pathways of these molecules are similar and very straightforward. The phenoxazine ring system remains stable under the Cs+ ion beam bombardment conditions, while fragmentations are observed along the length of the alkyl and acetyl side-chains. The fragmentation reactions were corroborated by acquiring product ion and constant neutral loss tandem mass spectrometric scans of the pertinent ions.

Analysis of phenoxazine chemosensitizers: an electron ionization and keV-ion beam bombardment mass spectrometry study.

The mass spectral behavior of a set of eight 2- and 10-disubstituted phenoxazines putatively possessing anticancer drug enhancer properties was investigated. Both electron ionization (EI) and keV-ion beam bombardment (liquid secondary ion mass spectrometry, LSIMS) were used. As expected, EI led to extensive fragmentation to produce structurally characteristics ions. Except in one example, the molecular ions were reasonably abundant. Two different liquid matrices--sulfolane and 3-nitrobenzyl alcohol--were used to obtain LSIMS data. The use of the latter produced more stable molecular ions. Ion beam bombardment also produced several structure-specific fragments. A unique feature of the LSI spectra obtained using either of the above matrices is production of both M+. and [M + H]+ ions, with the former being more abundant in most cases. Adduct formation with the liquid matrices was also observed for many compounds.