Hexafluoroisopropanol as a Bioconjugation Medium of Ultrafast, Tryptophan-Selective Catalysis.

The past decade has seen a remarkable growth in the number of bioconjugation techniques in chemistry, biology, material science, and biomedical fields. A core design element in bioconjugation technology is a chemical reaction that can form a covalent bond between the protein of interest and the labeling reagent. Achieving chemoselective protein bioconjugation in aqueous media is challenging, especially for generally less reactive amino acid residues, such as tryptophan. We present here the development of tryptophan-selective bioconjugation methods through ultrafast Lewis acid-catalyzed reactions in hexafluoroisopropanol (HFIP). Structure-reactivity relationship studies have revealed a combination of thiophene and ethanol moieties to give a suitable labeling reagent for this bioconjugation process, which enables modification of peptides and proteins in an extremely rapid reaction unencumbered by noticeable side reactions. The capability of the labeling method also facilitated radiofluorination application as well as antibody functionalization. Enhancement of an α-helix by HFIP leads to its compatibility with a certain protein, and this report also demonstrates a further stabilization strategy achieved by the addition of an ionic liquid to the HFIP medium. The nonaqueous bioconjugation approaches allow access to numerous chemical reactions that are unavailable in traditional aqueous processes and will further advance the chemistry of proteins.

Synthesis and biological evaluation of novel imidazo[1,2-a]pyridine-oxadiazole hybrids as anti-proliferative agents: Study of microtubule polymerization inhibition and DNA binding.

Efforts towards the development of potential anticancer agents, a new series of imidazo[1,2-a]pyridine-oxadiazole hybrids were synthesized and evaluated for their in vitro anticancer activity against lung cancer (A549) and prostate cancer (PC-3, DU-145) cell lines. Amongst the compounds tested, 6d showed the highest potency on A549 cells with an IC50 value of 2.8 ± 0.02 µM. Flow cytometric analysis of compound 6d treated A549 cells showed apoptosis induction by annexin-v/PI dual staining assay and the effect of 6d on different phases of cell cycle was also analyzed. Target based studies demonstrated the inhibition of tubulin polymerization by 6d at an IC50 value of 3.45 ± 0.51 µM and its effective binding with CT-DNA. Further, the molecular modelling studies revealed that 6d has a prominent binding affinity towards α/ß-tubulin receptor with admirable physico-chemical properties.

Cinnamide derived pyrimidine-benzimidazole hybrids as tubulin inhibitors: Synthesis, in silico and cell growth inhibition studies.

An approach in modern medicinal chemistry to discover novel bioactive compounds is by mimicking diverse complementary pharmacophores. In extension of this strategy, a new class of piperazine-linked cinnamide derivatives of benzimidazole-pyrimidine hybrids have been designed and synthesized. Their in vitro cytotoxicity profiles were explored on selected human cancer cell lines. Specifically, structural comparison of target hybrids with tubulin-DAMA-colchicine and tubulin-nocodazole complexes has exposed a deep position of benzimidazole ring into the αT5 loop. All the synthesized compounds were demonstrated modest to interesting cytotoxicity against different cancer cell lines. The utmost cytotoxicity has shown with an amine linker of benzimidazole-pyrimidine series, with specificity toward A549 (lung cancer) cell line. The most potent compound in this series was 18i, which inhibited cancer cell growth at micromolar concentrations ranging 2.21-7.29 µM. Flow cytometry studies disclosed that 18i inhibited the cells in G2/M phase of cell cycle. The potent antitumor activity of 18i resulted from enhanced microtubule disruption at a similar level as nocodazole on ß-tubulin antibody, explored using immunofluorescence staining. The most active compound 18i also inhibited tubulin polymerization with an IC50 of 5.72 ± 0.51 µM. In vitro biological analysis of 18i presented apoptosis induction on A549 cells with triggering of ROS generation and loss of mitochondrial membrane potential, resulting in DNA injury. In addition, 18i displayed impairment in cellular migration and inhibited the colony formation. Notably, the safety profile of most potent compound 18i was revealed by screening against normal human pulmonary epithelial cells (L132: IC50: 69.25 ± 5.95 µM). The detailed binding interactions of 18i with tubulin was investigated by employing molecular docking, superimposition and free energy analyses. Thus remarks made in this study established that pyrimidine-benzimidazole hybrids as a new class of tubulin polymerization inhibitors with significant anticancer activity.

Synthesis and Preclinical Evaluation of Indole Triazole Conjugates as Microtubule Targeting Agents that are Effective against MCF-7 Breast Cancer Cell Lines.

CDATA[Background: Microtubules are considered to be an important therapeutic target for most of the anticancer drugs. These are highly dynamic structures comprising of α-tubulin and ß-tubulin which are usually heterodimers and found to be involved in cell movement, intracellular trafficking, and mitosis inhibition of which might kill the tumour cells or inhibit the abnormal proliferation of cells. Most of the tubulin polymerization inhibitors, such as Vinca alkaloids, consist of Indole as the main scaffold. The literature also suggests using triazole moiety in the chemical entities, potentiating the inhibitory activity against cell proliferation. So, in our study, we used indole triazole scaffolds to synthesize the derivatives against tubulin polymerization. OBJECTIVE: The main objective of this study to synthesize indole triazole conjugates by using environmentally friendly solvents (green chemistry) and click chemistry. To carry out the MTT assay and tubulin polymerization assay for the synthesized indole triazole conjugates. METHODS: All the synthesized molecules were subjected to molecular docking studies using Schrodinger suite and the structural confirmation was performed by Mass, proton-NMR and carbon-NMR, documented in DMSO and CDCL3. Biological studies were performed using DU145 (prostate cancer), A-549 (lung cancer) and, MCF-7 (breast cancer), cell lines obtained from ATCC were maintained as a continuous culture. MTT assay was performed for the analogues using standard protocol. Cell cycle analysis was carried out using flow cytometry. RESULTS: The Indole triazole scaffolds were synthesized using the principles of Green chemistry. The triazole formation is mainly achieved by using the Click chemistry approach. Structural elucidation of synthesized compounds was performed using Mass spectroscopy (HR-MS), Proton-Nuclear Magnetic Spectroscopy (1H-NMR) and Carbon-Nuclear Magnetic Spectroscopy (13C-NMR). The XP-docked poses and free energy binding calculations revealed that 2c and 2g molecules exhibited the highest docking affinity against the tubulin-colchicine domain (PDB:1SA0). In vitro cytotoxic assessment revealed that 2c and 2g displayed promising cytotoxicity in MTT assay (with CTC50 values 3.52µM and 2.37µM) which are in good agreement with the computational results. 2c and 2g also arrested 63 and 66% of cells in the G2/M phase, respectively, in comparison to control cells (10%) and tubulin polymerization inhibition assay revealed that 2c and 2g exhibited significant inhibition of tubulin polymerization with IC50 values of 2.31µM, and 2.62µM, respectively in comparison to Nocodazole, a positive control, resulted in an IC50 value of 2.51µM. CONCLUSION: Indole triazole hybrids were synthesized using click chemistry, and docking studies were carried out using Schrodinger for the designed molecules. Process Optimization has been done for both the schemes. Twelve compounds (2a-2l) have been successfully synthesized and analytical evaluation was performed using NMR and HR-MS. In vitro evaluation was for the synthesized molecules to check tubulin polymerization inhibition for antiproliferative action. Among the synthesized compounds, 2c and 2g have potent anticancer activities by inhibiting tubulin polymerization.

Development of ß-carboline-benzothiazole hybrids via carboxamide formation as cytotoxic agents: DNA intercalative topoisomerase IIα inhibition and apoptosis induction.

In quest of promising anticancer agents, the pharmacophores of natural (ß-carboline) and synthetic origin (benzothiazole) were adjoined by a carboxamide bridge and three-point diversification was accomplished. The in vitro cytotoxic ability of the compounds was established on adherent and suspension human cancer cell lines and compounds 8u and 8f advanced as pre-eminent molecules with IC50 values of 1.46 and 1.81 µM respectively in A549 cell line. The cytospecificity was entrenched for potent compounds 8u and 8f by evaluating against normal human lung epithelial cells and selectivity index was calculated. Furthermore, EtBr displacement, relative viscosity and gel-based topoisomerase II target assays unveiled the intercalative topo-II inhibitory capability and DNA binding studies (absorbance) revealed the dissociation constant (Kd) for compounds 8u and 8f as 98 and 103 µM respectively. Additionally, cell-based flow cytometric assays like Annexin-V/PI dual staining aids in the quantification of apoptosis induced and JC-1 staining disclosed the depolarization of mitochondrial membrane potential by compound 8u in A549 cells in a dose-dependent manner. Moreover, wound healing assay established the inhibition of in vitro cell migration by compound 8u on A549 cells. In addition, molecular docking studies proved the binding of compounds 8u and 8f in the active site of DNA complexed with topo IIα and stabilized by interactions with DNA base pairs and amino acid residues. Remarkably, the compounds 8u and 8f follow Lipinski's rule of five and are in the recommended range for Jorgensen's rule of three with a minimal violation and other pharmacokinetic parameters revealing druggability of the synthesized hybrids.

Design and synthesis of thiadiazolo-carboxamide bridged ß-carboline-indole hybrids: DNA intercalative topo-IIα inhibition with promising antiproliferative activity.

The conjoining of salient pharmacophoric properties directing the development of prominent cytotoxic agents was executed by constructing thiadiazolo-carboxamide bridged ß-carboline-indole hybrids. On the evaluation of in vitro cytotoxic potential, 12c exhibited prodigious cytotoxicity among the synthesized new molecules 12a-k, with an IC50 < 5 µM in all the tested cancer cell lines (A549, MDA-MB-231, BT-474, HCT-116, THP-1) and the best cytotoxic potential was expressed in lung cancer cell line (A549) with an IC50 value of 2.82 ± 0.10 µM. Besides, another compound 12a also displayed impressive cytotoxicity against A549 cell line (IC50: 3.00 ± 1.40 µM). Further target-based assay of these two compounds 12c and 12a revealed their potential as DNA intercalative topoisomerase-IIα inhibitors. Additionally, the antiproliferative activity of compound 12c was measured in A549 cells by traditional apoptosis assays revealing the nuclear, morphological alterations, and depolarization of membrane potential in mitochondria and externalization of phosphatidylserine in a concentration-dependent manner. Cell cycle analysis unveiled the G0/G1 phase inhibition and wound healing assay inferred the inhibition of in vitro cell migration by compound 12c in lung cancer cells. Remarkably, the safety profile of compound 12c was disclosed by screening against normal human lung epithelial cell line (BEAS-2B: IC50: 71.2 ± 7.95 µM) with a selectivity index range of 14.9-25.26. Moreover, Molecular modeling studies affirm the intercalative binding of compound 12c and 12a in the active pocket of topo-IIα. Furthermore, in silico prediction of physico-chemical parameters divulged the propitious drug-like properties of the synthesized derivatives.

Exploration of carbamide derived pyrimidine-thioindole conjugates as potential VEGFR-2 inhibitors with anti-angiogenesis effect.

The development of new small molecules from known structural motifs through molecular hybridization is one of the trends in drug discovery. In this connection, we have combined the two pharmacophoric units (pyrimidine and thioindole) in a single entity via molecular hybridization strategy along with introduction of urea functionality at C2 position of pyrimidine to increase the efficiency of H-bonding interactions. Among the synthesized conjugates 12a-aa, compound 12k was found to exhibit significant IC50 values 5.85, 7.87, 6.41 and 10.43 µM against MDA-MB-231 (breast), HepG2 (liver), A549 (lung) and PC-3 (prostate) cancer cell lines, respectively. All these compounds were further evaluated for their inhibitory activities against VEGFR-2 protein. The results specified that among the tested compounds, 12d, 12e, 12k, 12l, 12p, 12q, 12t and 12u prominently suppressed VEGFR-2, with IC50 values of 310-920 nM in association to the positive control (210 nM). Angiogenesis inhibition was evident by tube formation assay in HUVECs and cell-invasion by transwell assay. The mechanism of cellular toxicity on MDA-MB-231 was found through depolarisation of mitochondrial membrane potential, increased ROS production and subsequent DNA damage resulting in apoptosis induction. Moreover, clonogenic and wound healing assays designated the inhibition of colony formation and cell migration by 12k in a dose-dependent manner. Molecular docking studies also shown that compound 12k capably intermingled with catalytically active residues GLU-885, ASP-1046 of the VEGFR-2 through hydrogen-bonding interactions.

Ring-opening cyclization of activated spiro-aziridine oxindoles with heteroarenes: a facile synthetic approach to spiro-oxindole-fused pyrroloindolines.

Herein, we report a facile tandem approach for the synthesis of both spiro-oxindole-fused pyrroloindolines and benzofurano-pyrrolidines via a Lewis acid-catalyzed domino ring-opening with concomitant ring annulation using activated spiro-aziridines and heteroarenes. This method offers a new class of novel spiro-fused polycyclic pyrrolidines in a one-pot and sustainable manner with good yields and high diastereoselectivity. In addition, the structure of 3d was confirmed by single X-ray crystallography analysis.

Design and synthesis of substituted dihydropyrimidinone derivatives as cytotoxic and tubulin polymerization inhibitors.

An operationally simple Biginelli protocol was employed for the synthesis of new C6-carbon based aryl α-haloacrylamide-linked dihydropyrimidinone derivatives. The synthesized compounds were appraised for their in vitro antiproliferative potential against a selected panel of human cancer cell lines especially MCF-7 (human breast cancer), MDA-MB-231 (human breast cancer), HCT-116 (human colon cancer), HCT-15 (human colorectal adenocarcinoma), HT-29 (human colon adenocarcinoma) and DU145 (human prostate cancer) along with normal lung fibroblasts (HFL-1). Preferably, compounds containing α-haloacrylamide (10a-g) functionality were found to exhibit most significant cytotoxicity (IC50 value 0.54 ±â€¯0.12 to 8.35 ±â€¯0.82 µM) against the listed cancer cell lines, particularly towards breast cancer cell lines MCF-7 and MDA-MB-231 (IC50 value 0.54 ±â€¯0.12 to 3.70 ±â€¯0.24 µM). In the seam of synthesized compounds, compound 10f exhibited potent antiproliferative activity against breast cancer cell lines namely MCF-7 (IC50 value 0.54 ±â€¯0.12 µM) and MDA-MB-231 (IC50 value 1.18 ±â€¯0.32 µM). Further to understand the underlying apoptosis mechanisms, different staining techniques such as AO/EB, DCFDA, and DAPI staining were performed. To know the extent of apoptosis and loss of mitochondrial membrane potential in MCF-7 cell lines, annexin V-FITC/PI and JC-1 were performed. Cell cycle analysis revealed that compound 10f arrested the cells at G2/M phase in a dose-dependent manner. The compound 10f also found to exhibit significant inhibition of tubulin polymerization (IC50 of 6.91 ±â€¯0.43 µM) with microtubule destabilizing properties. Molecular docking studies also revealed that compound 10f efficiently interacted with critical catalytically active residues Ser178, Val238, and Val318 of the α/ß-tubulin by a hydrogen bond.

Ru(II)-Catalyzed Regioselective Hydroxymethylation of ß-Carbolines and Isoquinolines via C-H Functionalization: Probing the Mechanism by Online ESI-MS/MS Screening.

A Ru(II)-catalyzed regioselective C-H activation toward hydroxymethylation of ß-carbolines and isoquinolines as effective directing groups has been developed, and the mechanism was probed by using online electrospray ionization-tandem mass spectrometry. The introduction of the hydroxymethyl group in the biologically relevant molecules routed via C-H functionalization remains an important task. Gratifyingly, this protocol draws attention to the regioselective formation of monohydroxymethylated ß-carboline/isoquinoline products exclusively.

Design and Synthesis of DNA-Interactive ß-Carboline-Oxindole Hybrids as Cytotoxic and Apoptosis-Inducing Agents.

A new series of (E)-3-[(1-aryl-9H-pyrido[3,4-b]indol-3-yl)methylene]indolin-2-one hybrids were synthesized and evaluated for their in vitro cytotoxic activity against a panel of selected human cancer cell lines, namely, HCT-15, HCT-116, A549, NCI-H460, and MCF-7, including HFL. Among the tested compounds, (E)-1-benzyl-5-bromo-3-{[1-(2,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl]methylene}indolin-2-one (10 s) showed potent cytotoxicity against HCT-15 cancer cells with an IC50 value of 1.43±0.26 µm and a GI50 value of 0.89±0.06 µm. Notably, induction of apoptosis by 10 s on the HCT-15 cell line was characterized by using different staining techniques, such as acridine orange/ethidium bromide (AO/EB) and DAPI. Further, to understand the mechanism of anticancer effects, various assays such as annexin V-FITC/PI, DCFDA, and JC-1were performed. The flow cytometric analysis revealed that compound 10 s arrests the HCT-15 cancer cells at the G0/G1 phase of the cell cycle. Additionally, western blot analysis indicated that treatment of 10 s on HCT-15 cancer cells led to decreased expression of anti-apoptotic Bcl-2 and increased protein expression of both pro-apoptotic Bax and caspase-3, -8, and -9, and cleaved PARP with reference to actin. Next, a clonogenic assay revealed the inhibition of colony formation in HCT-15 cancer cells by 10 s in a dose-dependent manner. Moreover, upon testing on normal human lung cells (HFL), the compounds were observed to be safer with a low toxicity profile. In addition, viscosity and molecular-docking studies showed that compound 10 s has typical intercalation with DNA.

Vanadium-Catalyzed Oxidative C(CO)-C(CO) Bond Cleavage for C-N Bond Formation: One-Pot Domino Transformation of 1,2-Diketones and Amidines into Imides and Amides.

A novel vanadium-catalyzed one-pot domino reaction of 1,2-diketones with amidines has been identified that enables their transformation into imides and amides. The reaction proceeds by dual acylation of amidines via oxidative C(CO)-C(CO) bond cleavage of 1,2-diketones to afford N,N'-diaroyl-N-arylbenzamidine intermediates. In the reaction, these intermediates are easily hydrolyzed into imides and amides through vanadium catalysis. This method provides a practical, simple, and mild synthetic approach to access a variety of imides as well as amides in high yields. Moreover, one-step construction of imide and amide bonds with a long-chain alkyl group is an attractive feature of this protocol.