Synthesis and SAR study of new hydroxy and chloro-substituted 2,4-diphenyl 5H-chromeno[4,3-b]pyridines as selective topoisomerase IIα-targeting anticancer agents

As part of our effort to develop potential topoisomerase IIα (topo IIα) targeting anticancer agents, we systematically designed a new series of hydroxy and chloro-substituted 2,4-diphenyl 5H-chromeno[4,3-b]pyridines. Total eighteen compounds were synthesized and tested for their ability to inhibit the function of topo I and IIα, and proliferation of human breast (T47D), colorectal (HCT15), and cervix (HeLa) cancer cells. Except compound 11, all of the tested compounds displayed selective topo IIα inhibitory activity. Compounds 8–18, 22, 24, and 25 showed excellent topo IIα inhibitory activity than a positive control, etoposide. Most of the compounds appeared to be superior to reference compounds in their antiproliferative activity. Structure-activity relationship (SAR) study has shown that it is better to place the hydroxyphenyl group at the 4-position of the central pyridine for superior topo IIα inhibition and antiproliferative activity. Similarly, the 3′-, or 4′-hydroxyphenyl substitution at the 2- and 4-positon of pyridine ring is important for better activity than 2′-substitution.     

Development of 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines and their salts as potent cytotoxic agents and topoisomerase I/IIα inhibitors

A novel series of 35 angularly fused pentacyclic 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines and 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chlorides were designed and synthesized. Their cytotoxic activities were investigated against six human cancer cell lines (NCIH23, HCT15, NUGC-3, ACHN, PC-3, and MDA-MB-231). Among all screened compounds; 28, 30, 34, 35, 46, 48, 52, and 53 compounds exhibited potent cytotoxic activities against all tested human cancer cell lines. Further, these potent lead cytotoxic agents were evaluated against human Topoisomerase I and IIα inhibition. Among them, the compound 48 exhibited dual Topoisomerase I and IIα inhibition especially at 20?μM concentrations the compound 48 exhibited 1.25?times more potent Topoisomerase IIα inhibitory activity (38.3%) than the reference drug etoposide (30.6%). The compound 52 also exhibited excellent (88.4%) topoisomerase I inhibition than the reference drug camptothecin (66.7%) at 100?μM concentrations. Molecular docking studies of the compounds 48 and 52 with topo I discovered that they both intercalated into the DNA single-strand cleavage site where the compound 48 have van der Waals interactions with residues Arg364, Pro431, and Asn722 whilst the compound 52 have with Arg364, Thr718, and Asn722 residues. Both the compounds 48 and 52 have π–π stacking interactions with the stacked DNA bases. The docking studies of the compound 48 with topo IIα explored that it was bound to the topo IIα DNA cleavage site where etoposide was situated. The benzo[f]chromeno[4,3-b][1,7]naphthyridine ring of the compound 48 was stacked between the DNA bases of the cleavage site with π–π stacking interactions and there were no hydrogen bond interactions with topo IIα.     

Synthesis and biological evaluation of 2-phenol-4-chlorophenyl-6-aryl pyridines as topoisomerase II inhibitors and cytotoxic agents

A new series of 2-phenol-4-chlorophenyl-6-aryl pyridines were designed, synthesized, and evaluated for topoisomerase (topo) I and II inhibitory activities as well as cytotoxic activity against four different human cancer cell lines such as HCT15, T47D, DU145, and Hela. Most of the tested compounds exhibited stronger topo II inhibitory activity at 100 μM as compared to etoposide. All the compounds, except 39, did not show topo I inhibitory activity. Interestingly, compounds that showed better topo II inhibition than etoposide have ortho- or para-chlorophenyl at 4-position of central pyridine, and none of the compounds possess meta-chlorophenyl. SAR study revealed the importance of ortho- or para-chlorophenyl at 4-position of the central pyridine for selective topo II inhibitory activity. Similarly, all compounds possessing meta- or para-hydroxyphenyl moieties showed moderate to significant cytotoxic effects. Particularly, compounds 27–37, and 39 which showed excellent cytotoxicity (IC₅₀ = 0.68–1.25 μM) against T47D breast cancer cells suggest the importance of meta- or para-hydroxyphenyl moiety at 2-position of the central pyridine for the design of anticancer agents with related scaffolds.     

Isosteric analogs of lenalidomide and pomalidomide: Synthesis and biological activity

A series of analogs of the immunomodulary drugs lenalidomide (1) and pomalidomide (2), in which the amino group is replaced with various isosteres, was prepared and assayed for immunomodulatory activity and activity against cancer cell lines. The 4-methyl and 4-chloro analogs 4 and 15, respectively, displayed potent inhibition of tumor necrosis factor-α (TNF-α) in LPS-stimulated hPBMC, potent stimulation of IL-2 in a human T cell co-stimulation assay, and anti-proliferative activity against the Namalwa lymphoma cell line. Both of these analogs displayed oral bioavailability in rat.     

Nitric oxide inhibits ATPase activity and induces resistance to topoisomerase II-poisons in human MCF-7 breast tumor cells

BackgroundTopoisomerase poisons are important drugs for the management of human malignancies. Nitric oxide (^?NO), a physiological signaling molecule, induces nitrosylation (or nitrosation) of many cellular proteins containing cysteine thiol groups, altering their cellular functions. Topoisomerases contain several thiol groups which are important for their activity and are also targets for nitrosation by nitric oxide.MethodsHere, we have evaluated the roles of ^?NO/^?NO-derived species in the stability and activity of topo II (α and β) both in vitro and in human MCF-7 breast tumor cells. Furthermore, we have examined the effects of ^?NO on the ATPase activity of topo II.ResultsTreatment of purified topo IIα and β with propylamine propylamine nonoate (PPNO), an NO donor, resulted in inhibition of the catalytic activity of topo II. Furthermore, PPNO significantly inhibited topo II-dependent ATP hydrolysis. ^?NO-induced inhibition of these topo II (α and β) functions resulted in a decrease in cleavable complex formation in MCF-7 cells in the presence of m-AMSA and XK469 and induced significant resistance to both drugs in MCF-7 cells.ConclusionPPNO treatment resulted in the nitrosation of the topo II protein in MCF-7 cancer cells and inhibited both catalytic-, and ATPase activities of topo II. Furthermore, PPNO significantly affected the DNA damage and cytotoxicity of m-AMSA and XK469 in MCF-7 tumor cells.General significanceAs tumors express nitric oxide synthase and generate ^?NO, inhibition of topo II functions by ^?NO/^?NO-derived species could render tumors resistant to certain topo II-poisons in the clinic.     

Structure optimization and preliminary bioactivity evaluation of N-hydroxybenzamide-based HDAC inhibitors with Y-shaped cap

Histone deacetylase inhibitors (HDACIs) are effective small molecules in the treatment of human cancers. In our continuing efforts to develop novel N-hydroxyterephthalamide-based HDACIs, herein we report the design and development of a new class of N-hydroxybenzamide-based HDACIs. In this new class of analogs, we inserted an ethylene moiety in the linker and used indole as a part of the Y-shaped cap group. Biological characterization identified compounds 4o, 4p, 4q and 4t to show improved HDAC inhibition, while no isoform selectivity for HDACs was observed. These compounds also exhibited improved anti-proliferative activity against multiple cancer cell lines when compared to their parent compound and positive control SAHA.     

Design,synthesis and biological evaluation of novel perimidine o-quinone derivatives as non-intercalative topoisomerase II catalytic inhibitors

For the development of novel anticancer agents, we designed and synthesized a total of 37 perimidine o-quinone derivatives containing the o-quinone group at the A or B ring and different substituents (alkyl groups, aryl groups or heterocycles) at the C ring of the compounds. The structure-activity relationships (SARs) were established based on the cytotoxicity data of compounds from the HL-60, Huh7, Hct116, and Hela cell lines. The cytotoxicity results showed that most compounds exhibited potent cytotoxicity. In particular, compound b-12 showed the best anti-proliferative activity (IC₅₀ ≤ 1 μM) against four cancer cell lines and strong potency against the HL-60/MX2 (0.47 μM) cell line, which is resistant to Topo II poisons. Further studies showed that b-12 exhibited potent Topo IIα inhibitory activity (IC₅₀ = 7.54 μM) compared with Topo I, which acted as a class of non-intercalative Topo IIα catalytic inhibitor by inhibiting the ATP binding site of Topo II. Cell apoptosis and cell cycle assays confirmed that b-12 could induce the apoptosis of Huh7 cells in a dose-dependent manner.     

Design,synthesis, and structure-activity relationships of new benzofuro[3,2-b]pyridin-7-ols as DNA topoisomerase II inhibitors

Human DNA topoisomerases have become attractive targets for developing more effective anticancer drugs. In this study, a series of new benzofuro[3,2-b]pyridin-7-ols were designed and synthesized for the first time and screened for their topoisomerase I and II inhibitory and antiproliferative activity. Structure-activity relationships revealed the position of ortho- and para-hydroxyl group at 2-phenyl ring, and meta-hydroxyl group at 4-phenyl ring of benzofuro[3,2-b]pyridin-7-ol are important for potent and selective topo II inhibitory activity. Compound 11 showed the most selective and potent topo II inhibition (100% inhibition at 100?µM) and strongest antiproliferative activity (IC₅₀?=?0.86?µM) than all the positive controls in HeLa cell line.     

Cytotoxic activity of hirsutanone,a diarylheptanoid isolated from Alnus glutinosa leaves

BackgroundThe low efficacy of cancer therapy for the treatment of patients with advanced disease makes the development of new anticancer agents necessary. Because natural products are a significant source of anticancer drugs, it is important to explore cytotoxic activity of novel compounds from natural origin.PurposeThe aim of this work is to evaluate the cytotoxic capacity of hirsutanone, a diarylheptanoid isolated from Alnus glutinosa leaves. Hirsutanone cytotoxic way of action was also studied.Material and methodsThe cytotoxic ability of Alnus glutinosa leaves ethyl acetate extract was studied over HeLa and PC-3 cell lines, with the MTT colorimetric assay. Hirsutanone was isolated from this extract using chromatographic methods, and its structure elucidated by spectroscopic analysis. HT-29 cell viability after hirsutanone treatment was determined using SRB assay. In order to understand hirsutanone way of action, cytotoxicity was evaluated adding the diarylheptanoid and antioxidants. DNA topoisomerase II (topo II) poison activity, was also evaluated using purified topo II and a supercoiled form of DNA that bears specific topo II recognition and binding region; topo II poisons stabilize normally transient DNA-topo II cleavage complexes, and lead an increased yield of linear form as a consequence of a lack of double-strand breaks rejoining.ResultsThe diarylheptanoid hirsutanone was isolated from Alnus glutinosa (L.) Gaertn. (Betulaceae) leaves extract that showed cytotoxic activity against PC-3 and HeLa cell lines. Hirsutanone showed cytotoxic activity against HT-29 human colon carcinoma cells. Pre-treatment with the antioxidants NAC (N-acetylcysteine) and MnTMPyP (Mn(III)tetrakis-(1-methyl-4-pyridyl)porthyrin) reduced this activity, suggesting that reactive oxygen species (ROS) participate in hirsutanone-induced cancer cell death. Using human topo II and a DNA supercoiled form, hirsutanone was found to stabilize topo II-DNA cleavage complexes, acting as a topo II poison.ConclusionOur data suggest that, like curcumin, an induction of oxidative stress and topo II-mediated DNA damage may play a role in hirsutanone-induced cancer cell death. Since both compounds share similar structure and cytotoxic profile, and curcumin is in clinical trials for the treatment of cancer, our results warrant further studies to evaluate the anticancer potential of hirsutanone.     

Design,synthesis, biological evaluation and molecular docking studies of novel 3-aryl-4-anilino-2H-chromen-2-one derivatives targeting ERα as anti-breast cancer agents

The estrogen receptor (ER) has played an important role in breast cancer development and progression and is a central target for anticancer drug discovery. In order to develop novel selective ERα modulators (SERMs), we designed and synthesized 18 novel 3-aryl-4-anilino-2H-chromen-2-one derivatives based on previously reported lead compounds. The biological results indicated that most of the compounds presented potent ERα binding affinity and possessed better anti-proliferative activities against MCF-7 and Ishikawa cell lines than the positive control tamoxifen. The piperidyl substituted compounds such as 16d and 18d demonstrated strong ERα binding affinities and excellent anti-proliferative activities respectively. Compound 18d displayed the most potent ERα binding affinity with RBA value of 2.83%, while 16d exhibited the best anti-proliferative activity against MCF-7 cells with IC₅₀ value of 4.52 ± 2.47 μM. Further molecular docking studies were also carried out to investigate binding pattern of the newly synthesized compounds with ERα. All these results together with the structure–activity relationships (SARs) indicated that these 3-aryl-4-anilino-2H-chromen-2-one derivatives with basic side chain could serve as promising leads for further optimization as novel SERMs.     

Synthesis of DNA interactive C3-trans-cinnamide linked β-carboline conjugates as potential cytotoxic and DNA topoisomerase I inhibitors

A series of new C3-trans-cinnamide linked β-carboline conjugates has been synthesized by coupling between various β-carboline amines and substituted cinnamic acids. Evaluation of their anti-proliferative activity against a panel of selected human cancer cell lines such as A549 (lung cancer), MCF-7 (breast cancer), B16 (melanoma), HeLa (cervical cancer) and a normal cell line NIH3T3 (mouse embryonic fibroblast cell line), suggested that the newly designed conjugates are considerably active against all the tested cancer cell lines with IC₅₀ values 13–45?nM. Moreover, the conjugates 8v and 8x were the most active against MCF-7 cells (14.05?nM and 13.84?nM respectively) and also even potent on other cell lines tested. Further, detailed investigations such as cell cycle analysis, apoptosis induction study, topoisomerase I inhibition assay, DNA binding affinity and docking studies revealed that these new conjugates are DNA interactive topoisomerase I inhibitors.     

4β-[4′-(1-(Aryl)ureido)benzamide]podophyllotoxins as DNA topoisomerase I and IIα inhibitors and apoptosis inducing agents

A series of 4β-[4′-(1-(aryl)ureido)benzamide]podophyllotoxin congeners (11a–l) were synthesized and evaluated for their cytotoxic activity against six human cancer cell lines. Some of the compounds like 11a, 11h, 11k and 11l showed significant anti-proliferative activity in Colo-205 cells and were superior to etoposide. The flow-cytometric analysis studies indicated that these compounds show strong G1 cell cycle arrest, as well exhibited improved inhibitory activities on DNA topoisomerase I and IIα enzymes. These compounds induce apoptosis by up regulating caspase-3 protein as observed by ELISA and Western blotting analysis. In addition, a brief structure–activity relationship studies within the series along with docking results of representative compounds 11a, 11h, 11k, 11l were presented.     

Synthesis of novel benzothiazole amides: Evaluation of PPAR activity and anti-proliferative effects in paraganglioma,pancreatic and colorectal cancer cell lines

The reduced activation of PPARs has a positive impact on cancer cell growth and viability in multiple preclinical tumor models, suggesting a new therapeutic potential for PPAR antagonists. In the present study, the benzothiazole amides 2a-g were synthesized and their activities on PPARs were investigated. Transactivation assay showed a moderate activity of the novel compounds as PPARα antagonists. Notably, in cellular assays they exhibited cytotoxicity in pancreatic, colorectal and paraganglioma cancer cells overexpressing PPARα. In particular, compound 2b showed the most remarkable inhibition of viability (greater than 90%) in two paraganglioma cell lines, with IC₅₀ values in the low micromolar range. In addition, 2b markedly impaired colony formation capacity in the same cells. Taken together, these results show a relevant anti-proliferative potential of compound 2b, which appears particularly effective in paraganglioma, a rare tumor poorly responsive to chemotherapy.     

Anti-proliferative effect of chalcone derivatives through inactivation of NF-κB in human cancer cells

To investigate the anti-proliferative effect of NF-κB inhibitor, a series of analogs of (E)-1-(2-hydroxy-6-(isopentyloxy)phenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one (5a) were prepared and evaluated for their NF-κB inhibition and anti-proliferative activity against various human cancer cell lines. Compounds (E)-1-(2-(3,3-dimethylbutoxy)-6-hydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one (5e) and (E)-4-(3-(2-(3,3-dimethylbutoxy)-6-hydroxyphenyl)-3-oxoprop-1-enyl)benzenesulfonamide (5p) showed good NF-κB inhibition as well as potent anti-proliferative activity. SAR studies showed that all the compounds with potent or moderate NF-κB inhibition displayed good anti-proliferative activity. All the analogs (5b–r) maintained a good correlation between their NF-κB inhibition and anti-proliferative activity though the extent is not directly proportional to each other.     

Modified 2,4-diaryl-5H-indeno[1,2-b]pyridines with hydroxyl and chlorine moiety: Synthesis,anticancer activity,and structure–activity relationship study

As a part of ongoing studies in developing novel anticancer agents, a series of modified 2,4-diaryl-5H-indeno[1,2-b]pyridines were designed, and synthesized by introducing hydroxyl and chlorine moieties. They were evaluated for topoisomerase inhibitory activity and cytotoxicity against HCT15, T47D, and HeLa cancer cell lines. This modification allowed us to demonstrate structure–activity relationship (SAR) study with respect to the non-substituted 2,4-diaryl-5H-indeno[1,2-b]pyridines. Compounds (2, 3, 4, 5, 8, and 9) with meta or para hydroxyl group on 2 or 4-phenyl ring have enhanced topo I and II inhibitory activity and cytotoxicity. However, additional substitution of chlorine group on furyl or thienyl ring (11, 12, 14, 16–18) generally reduced topo I and II inhibitory activity but improved cytotoxicity. The observation of cytotoxic properties and SAR study according to the position of hydroxyl and chlorine group will provide valuable insight for further study of development of novel anticancer agents with related scaffolds.     

Design,synthesis and molecular modeling study of certain VEGFR-2 inhibitors based on thienopyrimidne scaffold as cancer targeting agents

Different series of novel thieno [2,3-d]pyrimidine derivative (9a-d,10a-f,l,m and 15a-m) were designed, synthesized and evaluated for their ability to in vitro inhibit VEGFR-2 enzyme. Also, the cytotoxicity of the final compounds was tested against a panel of 60 different human cancer cell lines by NCI. The VEGFR-2 enzyme inhibitory results revealed that compounds 10d, 15d and 15 g are among the most active inhibitors with IC₅₀ values of 2.5, 5.48 and 2.27 µM respectively, while compound 10a remarkably showed the highest cell growth inhibition with mean growth inhibition (GI) percent of 31.57%. It exhibited broad spectrum anti-proliferative activity against several NCI cell lines specifically on human breast cancer (T7-47D) and renal cancer (A498) cell lines of 85.5% and 77.65% inhibition respectively. To investigate the mechanistic aspects underlying the activity, further biological studies like flow cytometry cell cycle together with caspase-3 colorimetric assays were carried on compound 10a. Flow cytometric analysis on both MCV-7 and PC-3 cancer cells revealed that it induced cell-cycle arrest in the G0-G1phase and reinforced apoptosis via activation of caspase-3. Furthermore, molecular modeling studies have been carried out to gain further understanding of the binding mode in the active site of VEGFR-2 enzyme and predict pharmacokinetic properties of all the synthesized inhibitors.     

Monocyclic 4-amino-6-(phenylamino)-1,3,5-triazines as inhibitors of human DNA topoisomerase IIα

Human DNA topoisomerase IIα (htIIα) is a validated target for the development of anticancer agents. Starting from the available information about the binding of the purine-based htIIα inhibitors in the ATP binding site we designed a virtual screening campaign combining structure-based and ligand-based pharmacophores with a molecular docking calculation searching for compounds that would contain a monocycle mimetic of the purine moiety. We discovered novel 4-amino-6-(phenylamino)-1,3,5-triazines 6, 7 and 11 as monocyclic htIIα inhibitors targeting the ATP binding site. Compound 6 from the 1,3,5-triazine series also displayed cytotoxicity properties in hepatocellular carcinoma (HepG2) cell lines and selectivity against human umbilical vein endothelial (HUVEC) cell lines.     

Substrate specificity plays an important role in uncoupling the catalytic and scaffolding activities of rat testis DNA topoisomerase IIα

Abstract

Topoisomerase II (topo II) is a dyadic enzyme found in all eukaryotic cells. Topo II is involved in a number of cellular processes related to DNA metabolism, including DNA replication, recombination and the maintenance of genomic stability. We discovered a correlation between the development of postnatal testis and increased binding of topo IIα to the chromatin fraction. We used this observation to characterize DNA-binding specificity and catalytic properties of purified testis topo IIα. The results indicate that topo IIα binds a substrate containing the preferred site with greater affinity and, consequently, catalyzes the conversion of form I to form IV DNA more efficiently in contrast to substrates lacking such a site. Interestingly, topo IIα displayed high-affinity and cooperativity in binding to the scaffold associated region. In contrast to the preferred site, however, high-affinity binding of topo IIα to the scaffold-associated region failed to result in enhanced catalytic activity. Intriguingly, competition assays involving scaffold-associated region revealed an additional DNA-binding site within the dyadic topo IIα. These results implicate a dual role for topo IIα in vivo consistent with the notion that its sequestration to the chromatin might play a role in chromosome condensation and decondensation during spermatogenesis.