Design, synthesis, and biological evaluation of N-(pyridin-3-yl)pyrimidin-4-amine analogues as novel cyclin-dependent kinase 2 inhibitors for cancer therapy.

The discovery and development of CDK2 inhibitors has currently been validated as a hot topic in cancer therapy. Herein, a series of novel N-(pyridin-3-yl)pyrimidin-4-amine derivatives were designed and synthesized as potent CDK2 inhibitors. Among them, the most promising compound 7l presented a broad antiproliferative efficacy toward diverse cancer cells MV4-11, HT-29, MCF-7, and HeLa with IC50 values of 0.83, 2.12, 3.12, and 8.61 µM, respectively, which were comparable to that of Palbociclib and AZD5438. Interestingly, these compounds were less toxic on normal embryonic kidney cells HEK293 with high selectivity index. Further mechanistic studies indicated 7l caused cell cycle arrest and apoptosis on HeLa cells in a concentration-dependent manner. Moreover, 7l manifested potent and similar CDK2/cyclin A2 nhibitory activity to AZD5438 with an IC50 of 64.42 nM. These findings revealed that 7l could serve as ahighly promisingscaffoldfor CDK2 inhibitors as potential anticancer agents and functional probes.

Synthesis and biological evaluation of novel 5-substituted/unsubstituted triazolothiadiazines as tubulin depolymerizing and vascular disrupting agents with promising antitumor activity.

A novel series of 5-substituted/unsubstituted [1,2,4]triazolo[3,4-b][1,3,4] thiadiazine compounds has been achieved successfully through chemoselective reduction of the C = N bond, based on our prior work. Initial biological evaluation illustrated that the most active derivative 7j exhibited significant cell growth inhibitory activity toward MCF-7, A549, HCT116, and A2780 with the IC50 values of 0.75, 0.94, 2.90, and 4.15 µM, respectively. Most importantly, all the representative analogs did not demonstrate obvious cytotoxic activity against the non-tumoural cell line HEK-293 (IC50 > 100 µM). The mechanism study revealed that 7j caused the G2 /M phase arrest, induced cell apoptosis in HeLa cells in a concentration-dependent manner, and also showed potent tubulin polymerization inhibitory effect. Meanwhile, 7j exerted significant antivascular activity in the wound-healing and tube formation assays. These observations indicate that 5-unsubstituted 6,7-dihydro-5H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine scaffold might be considered as a potential lead for antitubulin inhibitors to develop highly efficient anticancer agents with potent selectivity over normal human cells.

Rational design, synthesis and biological evaluation of novel 2-(substituted amino)-[1,2,4]triazolo[1,5-a]pyrimidines as novel tubulin polymerization inhibitors.

Following our previously reported compound 3, we designed and synthesized a series of new 2-(substituted amino)- [1,2,4]triazolo[1,5-a]pyrimidines as potential tubulin polymerization inhibitors. Among them, analogue 4k, having a 3-hydroxy-4-methoxyphenylamino group, was observed to display excellent antiproliferative activity toward HeLa, HCT116, A549, and T47D with the IC50 values of 0.31, 1.28, 3.99 and 10.32 µM, respectively, which were approximately 32, 48, 4, and 5-fold improvement compared with 3. Importantly, 4k possessed significant selectivity in inhibiting cancer cell lines over the normal HEK293 cells. Moreover, futher mechanism analysis demonstrated that 4k caused G2/M arrest, induced cells apoptosis in HeLa cells, and manifested significant tubulin polymerization inhibitory activity with the IC50 value of 4.9 µM, which is comparable to CA-4 (IC50 = 4.2 µM). The observations performed in this study reveal that 2-arylamino- [1,2,4]triazolo[1,5-a]pyrimidines represent a novel class of tubulin polymerization inhibitors with potent antiproliferative efficacy.

Efficient synthesis and evaluation of novel 6-arylamino-[1,2,4]triazolo[4,3-a]pyridine derivatives as antiproliferative agents.

Based on our previous work, a series of novel 6-arylamino-[1,2,4]triazolo[4,3-a]pyridine derivatives were synthesized, and evaluated for antiproliferative activities. SAR studies revealed that inserting an amino linkage between 6­aryl group and [1,2,4]triazolo[4,3-a]pyridine core led to amuch broaderantitumorspectrum, and the most promising compound 8 l exerted potent andbroad-spectrum antiproliferative activity toward HeLa, HCT116, MCF-7, and A549 cell lines, with IC50 values in the micromolar range of 5.98-12.58 µM, which were more active than the positive control 5-FU. The mechanism investigation illustrated that 8 l dose-dependently caused cell cycle arrest at the G2/M phase, and induced cell apoptosis in HeLa cells. Consequently, these findings suggest the 6-arylamino-[1,2,4]triazolo[4,3-a]pyridines afford significant potential for the discovery of a new highly efficient anticancer agents.

1,4-Dihydropyridinebutyrolactone-derived ring-opened ester and amide analogs targeting BET bromodomains.

Based on a previously reported 1,4-dihydropyridinebutyrolactone virtual screening hit, nine lactone ring-opened ester and seven amide analogs were prepared. The analogs were designed to provide interactions with residues at the entrance of the ZA loop of the testis-specific bromodomain (ZA) channel to enhance the affinity and selectivity for the bromodomain and extra-terminal (BET) subfamily of bromodomains. Compound testing by AlphaScreen showed that neither the affinity nor the selectivity of the ester and lactam analogs was improved for BRD4-1 and the first bromodomain of the testis-specific bromodomain (BRDT-1). The esters retained affinity comparable to the parent compound, whereas the affinity for the amide analogs was reduced 10-fold. A representative benzyl ester analog was found to retain high selectivity for BET bromodomains as shown by a BROMOscan. X-ray analysis of the allyl ester analog in complex with BRD4-1 and BRDT-1 revealed that the ester side chain is located next to the ZA loop and solvent exposed.

Concise synthesis and preliminary biological evaluation of new triazolylthioacetone derivatives bearing pyridine, pyrazine, and 3,4,5-trimethoxybenzyl fragment.

Based on our previous work, a series of novel triazolylthioacetones incorporating pyridine, pyrazine, and 3,4,5-trimethoxybenzyl fragment were synthesized, and evaluated for antiproliferative activities and interactions with tubulin. Some analogues exhibited moderate to excellent potency, with the most promising compound IIc possessing IC50 values of 0.62, 1.46, and 3.65 µM against HT-29, HCT116, and HepG2 tumor cells, respectively, which were comparable with the positive control CA-4. Mechanistical studies revealed that IIc concentration-dependently caused cell cycle arrest at the G2/M phase in HCT116 tumor cells, and displayed a significant inhibition of tubulin polymerization with an IC50 value of 12.7 µM. Moreover, molecular docking analysis suggested that IIc could occupy the colchicine-binding site in a similar way with typical tubulinpolymerizationinhibitors. These results highlighted the 4-amino-triazolylthioacetone scaffold as potential tubulin polymerization inhibitors for development of highly efficient anticancer agents.

Development of pteridin-7(8H)-one analogues as highly potent cyclin-dependent kinase 4/6 inhibitors: Synthesis, structure-activity relationship, and biological activity.

CDK4/6 have been validated as the cancer therapeutic targets. Here, we describe a series of pteridin-7(8H)-one analogues as potent CDK4/6 inhibitors. Among them, the most promising compound 7s demonstrated remarkable and broad-spectrum antiproliferative activities toward HCT116, HeLa, MDA-MB-231, and HT-29 cells with IC50 values of 0.65, 0.70, 0.39, and 2.53 µM, respectively, which were more potent than that of the anticancer drug Palbociclib. Interestingly, 7s also manifested the greatest inhibitory activities toward both CDK4/cyclin D3 and CDK6/cyclin D3 (IC50 = 34.0 and 65.1 nM, respectively), which was comparable with Palbociclib. Additionally, molecular simulation indicated that 7s bound efficiently at the ATPbindingsitesofCDK4 and CDK6. Further mechanistic studies revealed that compound 7s could concentration-dependently induce cell cycle arrest and apoptosis in HeLa cells. Takentogether, 7s represents a promising novel CDK4/6 inhibitor for the potential treatment of cancer.

Design, synthesis and antiproliferative activity of novel 2,4-diamino-5-methyleneaminopyrimidine derivatives as potential anticancer agents.

In order to discover new anticancer agents, 25 novel 2,4-diamino-5-methyleneaminopyrimidine derivatives were designed and synthesized based on our previous work via a ring-opening strategy. Among them, compared with 5-FU, compound 7i exhibited 4.9-, 2.9-, 2.1-, and 3.0-fold improvement in inhibiting HCT116, HT-29, MCF-7, and HeLa cells proliferation with IC50 values of 4.93, 5.57, 8.84, and 14.16 µM, respectively. Moreover, further mechanistic studies indicated that compound 7i could concentration-dependently induce cell cycle arrest and apoptosis in HCT116 cells. These findings revealed that 2,4-diamino-5-methyleneaminopyrimidine scaffold has potential for further investigation to explore novel anticancer agents.

Discovery of highly potent tubulin polymerization inhibitors: Design, synthesis, and structure-activity relationships of novel 2,7-diaryl-[1,2,4]triazolo[1,5-a]pyrimidines.

By removing 5-methyl and 6-acetyl groups in our previously reported compound 3, we designed a series of novel 2,7-diaryl-[1,2,4]triazolo[1,5-a]pyrimidine derivatives as potential tubulin polymerization inhibitors. Among them, compound 5e displayed low nanomolar antiproliferative efficacy on HeLa cells which was 166-fold higher than the lead analogue 3. Interestingly, 5e displayed significant selectivity in inhibiting cancer cells over HEK-293 (normal human embryonic kidney cells). In addition, 5e dose-dependently arrested HeLa in G2/M phase through the alterations of the expression levels of p-cdc2 and cyclin B1, and caused HeLa cells apoptosis by regulation of expressions of cleaved PARP. Further evidence demonstrated that 5e effectively inhibited tubulin polymerization and was 3-fold more powerful than positive control CA-4. Moreover, molecular docking analysis indicated that 5e overlapped well with CA-4 in the colchicine-binding site. These studies demonstrated that 2,7-diaryl-[1,2,4]triazolo[1,5-a]pyrimidine skeleton might be used as the leading unit to develop novel tubulin polymerization inhibitors as potential anticancer agents.

Synthesis, biological evaluation, and structure-activity relationships of new tubulin polymerization inhibitors based on 5-amino-1,2,4-triazole scaffold.

Based on our previous research, thirty new 5-amino-1H-1,2,4-triazoles possessing 3,4,5-trimethoxyphenyl moiety were synthesized, and evaluated for antiproliferative activities. Among them, compounds IIa, IIIh, and IIIm demonstrated significant antiproliferative activities against a panel of tumor cell lines, and the promising compound IIIm dose-dependently caused G2/M phase arrest in HeLa cells. Furthermore, analogue IIa exhibited the most potent tubulinpolymerization inhibitory activity with an IC50 value of 9.4 µM, and molecular modeling studies revealed that IIa formed stable interactions in the colchicine-binding site of tubulin, suggesting that 5-amino-1H-1,2,4-triazole scaffold has potential for further investigation to develop novel tubulin polymerization inhibitors with anticancer activity.

Synthesis and antiproliferative evaluation of novel 8-cyclopentyl-7,8-dihydropteridin-6(5H)-one derivatives as potential anticancer agents.

Based on our previous work, a novel class of 8-cyclopentyl-7,8-dihydropteridin-6(5H)-one derivatives were synthesized and evaluated as antiproliferative agents. Structure-activity relationship analysis revealed that the greatest activities were achieved with a 4-(4-methylpiperazin-1-yl)aniline group at C-2 position of dihydropteridin-6(5H)-one core, and the most promising compound 6k demonstrated comparable antiproliferative activity with Palbociclib and more potent than our parent derivative 4 toward four cell lines including HCT-116, HeLa, HT-29, and MDA-MB-231 with IC50 values of 3.29, 6.75, 7.56, and 10.30 µM, respectively. Moreover, the mechanism studies revealed that compound 6k could induce cell cycle arrest at G2/M phase via a concentration-dependent manner. In general, these preliminary observations suggested that these compounds could serve as promising scaffolds for further modification to develop novel and highly potent cancer therapy agents.

Synthesis, and biological evaluation of 3,6-diaryl-[1,2,4]triazolo[4,3-a]pyridine analogues as new potent tubulin polymerization inhibitors.

On the basis of our previous work, twenty-nine novel [1,2,4]triazolo[4,3-a]pyridines possessing 3,4,5-trimethoxylphenyl groups were designed, synthesized, and evaluated as tubulin polymerization inhibitors. The bioassay results revealed that some of the compounds displayed excellent antiproliferative efficacies in the nanomolar range against HeLa cells, and the most promising derivative 7i demonstrated almost comparable activity to that of the reference CA-4 and sixty-two fold more potent than the parent compound 6 with an IC50 value of 12 nM. Importantly, 7i exhibited high selectivity over the normal human embryonic kidney HEK-293 cells (IC50 > 100 µM). Further mechanism studies revealed that 7i significantly arrested cell cycle at G2/M phase, induced apoptosis with a dose-dependent manner, and disrupted microtubule networks. Additionally, the most active compound 7i effectively inhibited tubulin polymerization with a value similar to that of CA-4 (3.4 and 4.2 µM, respectively). Furthermore, molecular docking analysis suggested that 7i well occupied the colchicine binding pocket of tubulin. The present study highlights that compound 7i is a novel potential tubulin polymerization inhibitor and deserves further investigation for the treatment of cancers.

Synthesis and biological evaluation of benzofuran-based 3,4,5-trimethoxybenzamide derivatives as novel tubulin polymerization inhibitors.

A new series of derivatives characterized by the presence of the 3,4,5-trimethoxylbenzamide substituted benzofurans were synthesized and evaluated for antiproliferative activity against four cancer cell lines and one normal human cell line. Among them, derivative 6g with greatest cytotoxicity significantly inhibited the growth of MDA-MB-231, HCT-116, HT-29 and HeLa cell lines with IC50 values of 3.01, 5.20, 9.13, and 11.09 µM, respectively. Importantly, 6g possessed excellent selectivity over non-tumoral cell lines HEK-293 (IC50 > 30 µM). Moreover, mechanistic studies revealed that 6g induced HeLa cells arrested in G2/M phase in a concentration-dependent manner, and inhibited polymerization of tubulin via a consistent way with CA-4. In general, these observations suggest that 6g is a promising anti-cancer lead and is worth further investigation to generate potential antitumor agents.

Synthesis and biological evaluation of novel pyrazolo[3,4-b]pyridines as cis-restricted combretastatin A-4 analogues.

Twenty-six novel pyrazolo[3,4-b]pyridine-bridged analogues of combretastatin A-4 possessing 3,4,5-trimethoxylphenyl groups, were synthesized and evaluated for their antiproliferative and tubulin polymerization inhibitory activities. Preliminary biological evaluation demonstrated that some of the target compounds displayed significant antiproliferative effectagainst four different cell lines including MCF-7, MDA-MB-231, HeLa and Kyse150. The most active analogue 6n was found to induce HeLa cells arrest in the G2/M phase in a dose-dependent manner. Molecular modeling studies indicated that derivative 6n most likely occupies the colchicine site of tubulin. The initial results suggest that the 3,4,5-trimethoxyphenyl substituted pyrazolo[3,4-b]pyridine could serve as a promising scaffold for development of potent tubulin inhibitors as anticancer agents.

Design, synthesis and biological evaluation of novel indole-based oxalamide and aminoacetamide derivatives as tubulin polymerization inhibitors.

A series of novel indole-based oxalamide and aminoacetamide derivatives were designed, synthesized, and evaluated for antiproliferative activities. Preliminary results revealed that compound 8g exhibited significant antiproliferative effect against PC-3, HeLa and HCT-116 cell lines. Flow cytometric analysis of the cell cycle demonstrated the compound 8g induced the cell cycle arrest at G2/M phase in HeLa cell lines. Immunocytochemistry revealed loss of intact microtubule structure in cells treated with 8g andinhibition of tubulinpolymerization. Additionally, molecular docking analysis suggested that 8g formed stable interactions in the colchicine-binding site of tubulin. These preliminary results demonstrated that a new class of novel indole-based oxalamide and aminoacetamide derivatives described in the investigation could be developed as potential scaffolds to new anticancer agents.

Novel [1,2,4]triazolo[1,5-a]pyrimidine derivatives as potent antitubulin agents: Design, multicomponent synthesis and antiproliferative activities.

As restricted CA-4 analogues, a novel series of [1,2,4]triazolo[1,5-a]pyrimidines possessing 3,4,5-trimethoxylphenyl groups has been achieved successfully via an efficient one-pot three-component reaction of 3-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazol-5-amine, 1,3-dicarbonyl compounds and aldehydes. Initial biological evaluation demonstrated some of target compounds displayed potent antitumor activity in vitro against three cancer cell lines. Among them, the most highly active analogue 26 inhibited the growth of HeLa, and A549 cell lines with IC50 values at 0.75, and 1.02 µM, respectively, indicating excellent selectivity over non-tumoural cell line HEK-293 (IC50 = 29.94 µM) which suggested that the target compounds might possess a high safety index. Moreover, cell cycle analysis illustrated that the analogue 26 significantly induced HeLa cells arrest in G2/M phase, meanwhile the compound could dramatically affect cell morphology and microtubule networks. In addition, compound 28 exhibited potent anti-tubulin activity with IC50 values of 9.90 µM, and molecular docking studies revealed the analogue occupied the colchicine-binding site of tubulin. These observations suggest that [1,2,4]triazolo[1,5-a]pyrimidines represent a new class of tubulin polymerization inhibitors and well worth further investigation aiming to generate potential anticancer agents.

Discovery of novel pyrimidine-based benzothiazole derivatives as potent cyclin-dependent kinase 2 inhibitors with anticancer activity.

To develop novel CDK2 inhibitors as anticancer agents, a series of novel pyrimidine-based benzothiazole derivatives were designed and synthesized. Initial biological evaluation demonstrated some of target compounds displayed potent antitumor activity in vitro against five cancer cell lines. Especially, the analogue 10s exhibited approximately potency with AZD5438 toward four cells including HeLa, HCT116, PC-3, and MDA-MB-231 with IC50 values of 0.45, 0.70, 0.92, 1.80 µM, respectively. More interestingly, the most highly active compound 10s in this study also possessed promising CDK2/cyclin A2 inhibitory activities with IC50 values of 15.4 nM, which was almost 3-fold potent than positive control AZD5438, and molecular docking studies revealed that the analogue bound efficiently with the CDK2 binding site. Further studies indicated that compound 10s could induce cell cycle arrest and apoptosis in a concentration-dependent manner. These observations suggest that pyrimidine-benzothiazole hybrids represent a new class of CDK2 inhibitors and well worth further investigation aiming to generate potential anticancer agents.

Facile one-pot synthesis, antiproliferative evaluation and structure-activity relationships of 3-amino-1H-indoles and 3-amino-1H-7-azaindoles.

A highly efficient method has been developed for the one-pot synthesis of substituted 3-amino-1H-indole and 3-amino-1H-7-azaindole derivatives starting from ethyl 2-cyanophenylcarbamate/ethyl 3-cyanopyridin-2-ylcarbamate, and α-bromoketones in good to excellent yields. All newly synthesized analogues were screened for their antiproliferative activities against four cancer cell lines. The most promising compound 8v demonstrated 13-, 5-, and 1.4-fold improvement compared to fluorouracil in inhibiting HeLa, HepG2, and MCF-7 cell proliferation with IC50 values of 3.7, 8.0, and 19.9 µM, respectively. Furthermore, 8v induced significant cell cycle arrest at the G2/M phase in HeLa cell lines via a concentration-dependent manner. These encouraging findings indicate that the common 3-amino-1H-7-azaindole is a very favorable scaffold for the design of novel anticancer small-molecule drugs.

Synthesis, biological evaluation, and molecular docking investigation of 3-amidoindoles as potent tubulin polymerization inhibitors.

A series of novel 3-amidoindole derivatives possessing 3,4,5-trimethoxylphenyl groups were synthesized and evaluated for their antiproliferative and tubulin polymerization inhibitory activities. Some of them demonstrated moderate to potent activities in vitro against six cancer cell lines including MCF-7, MDA-MB-231, BT549, T47D, MDA-MB-468, and HS578T. The most active compound 27 inhibited the growth of T47D, BT549, and MDA-MB-231 cell lines with IC50 values at 0.04, 3.17, and 6.43 µM, respectively. Moreover, the flow cytometric analysis clearly revealed that compound 27 significantly inhibited growth of breast cancer cells through arresting cell cycle in G2/M phase via a concentration-dependent manner. In addition, the compound also exhibited the most potent anti-tubulin activity with IC50 values of 9.5 µM, which was remarkable, compared to CA-4. Furthermore, molecular docking analysis demonstrated the interaction of the compound 27 at the colchicine-binding site of tubulin. These preliminary results suggest that compound 27 is a very promising tubulin-binding agent and is worthy of further investigation aiming to the development of new potential anticancer agents.

Discovery and optimization of 3,4,5-trimethoxyphenyl substituted triazolylthioacetamides as potent tubulin polymerization inhibitors.

Based on our previous research, three series of new triazolylthioacetamides possessing 3,4,5-trimethoxyphenyl moiety were synthesized, and evaluated for antiproliferative activities and inhibition of tubulin polymerization. The most promising compounds 8b and 8j demonstrated more significant antiproliferative activities against MCF-7, HeLa, and HT-29 cell lines than our lead compound 6. Moreover, analogues 8f, 8j, and 8o manifested more potent antiproliferative activities against HeLa cell line with IC50 values of 0.04, 0.05 and 0.16 µM, respectively, representing 100-, 82-, and 25-fold improvements of the activity compared to compound 6. Furthermore, the representative compound, 8j, was found to induce significant cell cycle arrest at the G2/M phase in HeLa cell lines via a concentration-dependent manner. Meanwhile, compound 8b exhibited the most potent tubulin polymerization inhibitory activity with an IC50 value of 5.9 µM, which was almost as active as that of CA-4 (IC50 = 4.2 µM). Additionally, molecular docking analysis suggested that 8b formed stable interactions in the colchicine-binding site of tubulin.