The mechanism of anticancer effects of some pyrrolopyrimidine derivatives on HT-29 human colon cancer cells.

In the present work, the mechanism of anticancer activity of some pyrrolopyrimidine derivatives was evaluated. Compounds 5 and 8 exhibiting significant antiproliferative activity against HT-29 cells with IC50 values of 4.17 µM and 2.96, arrested the cells at the G2/M phase and significantly induced apoptosis. The apoptotic potential of the compounds has been verified via ELISA assay, which resulted in increased BAX, PUMA, BIM, and cleaved caspase 3 expression and decreased BCL-XL and MCL-1 protein levels in HT-29 cells. Moreover, the immunofluorescence technique showing that compounds 5 and 8-treatment reduced Ki67 immunolocalization and increased the caspase 3 and p53 immunolocalization confirmed the apoptotic activity. While treatment of HT-29 cells to compounds 5 and 8 inhibited Akt and ERK1/2, there are no alterations in JNK and p38 signaling pathways. According to molecular docking results, compounds 5 and 8 occupied the active site of Akt kinase and showed important hydrogen bonding interactions with key amino acids. Also, siRNA-mediated depletion of BIM, PUMA, and BAX/BAK expression decreased apoptotic response in HT-29 cells upon exposure to compound 5 and compound 8. Compounds 5 and 8 trigger the activation of mitochondrial apoptosis in HT-29 cells. Additionally, we found that proapoptotic BH3-only proteins BIM and PUMA are required for the full engagement of mitochondrial apoptosis signaling. However, p53 was dispensable for compound 5- or compound 8-induced apoptosis in HT-29 cells.

Effects of pyrrolopyrimidine derivatives on cancer cells cultured in vitro and potential mechanism.

In this study, the anticancer activities of some pyrrolopyrimidine derivatives were evaluated. Compound 3 is the most cytotoxic compound on MCF-7 cancer cells with an IC50 value of 23.42 µM. Also, compound 3 induced apoptosis and the ROS(+) cell population in MCF-7 cells. Moreover, it significantly reduced MMP-9 activity, having 42.16 ± 5.10% and 58.28 ± 1.96% inhibitory activities at 10 µM and 50 µM concentrations, respectively. Molecular docking results supported the activity, showing key hydrogen bonds with the binding site of MMP-9. Therefore, compound 3 might be a lead compound for the development of potent MMP-9 inhibitors.

New aldo-keto reductase 1C3 (AKR1C3) inhibitors based on the hydroxytriazole scaffold.

The aldo-keto reductase 1C3 (AKR1C3) enzyme is considered an attractive target in Castration Resistant Prostate Cancer (CRPC) because of its role in the biosynthesis of androgens. Flufenamic acid, a non-selective AKR1C3 inhibitor, has previously been subjected to bioisosteric modulation to give rise to a series of compounds with the hydroxytriazole core. In this work, the hit compound of the previous series has been modulated further, and new, more potent, and selective derivatives have been obtained. The poor solubility of the most active compound (cpd 5) has been improved by substituting the triazole core with an isoxazole heteronucleous, with similar enzymatic activity being retained. Potent AKR1C3 inhibition is translated into antiproliferative effects against the 22RV1 CRPC cellular model, and the in-silico design, synthesis and biological activity of new compounds are described herein. Compounds have also been assayed in combination with two approved antitumor drugs, abiraterone and enzalutamide.

Novel pyrrolopyrimidine derivatives induce p53-independent apoptosis via the mitochondrial pathway in colon cancer cells.

A series of novel pyrrolopyrimidine urea derivatives were synthesized and evaluated for their anticancer activity against colon cancer cell lines. Compounds showed the remarkable cytotoxic activity on HCT-116 wt cell line. The most potent compound 4c (IC50 = 0.14 µM) induced apoptosis in HCT-116 wt and HCT-116 p53-/- cell lines. Otherwise, treatment of HCT-116 BAX-/-BAK-/- cells with compound 4c didn't lead to activation of apoptosis, suggesting that compound 4c induces apoptotic cell death by activating BAX/BAK-dependent pathway. Moreover, while the compound 4c increase the activation of caspase-3 and caspase-9 levels in HCT-116 wt and HCT-116 p53-/- cells, caspase-3 or caspase-9 activation was not observed in HCT-116 BAX-/-BAK-/- cells. In addition, compound 4c induced mitochondrial apoptosis in cells grown as oncospheroids, which better mimic the in vivo milieu of tumors. 4c treatment also activated JNK along with inhibition of prosurvival kinases such as Akt and ERK 1/2 in HCT-116 wt and HCT-116 p53 -/- cells as well as in HCT-116 BAX-/-BAK-/- cells. Notably, our results indicated that compound 4c induced mitochondrial apoptosis through activation p53-independent apoptotic signaling pathways.

Synthesis and anticancer activity of some pyrimidine derivatives with aryl urea moieties as apoptosis-inducing agents.

A new series of pyrimidine derivatives containing aryl urea moieties was designed and synthesized. The anticancer activities of all compounds were evaluated in vitro against colon and prostat cancer cell lines by MTT assay. Among these compounds, 4b exhibited the highest cytotoxic activity against SW480 cancer cell line with IC50 value of 11.08 µM. Mechanistic studies showed that compound 4b arrested cell cycle at G2/M phase and induced apoptosis through upregulating Bax, Ikb-α and cleaved PARP and downregulating Bcl-2 expression levels. Moreover, compound 4b induced loss of mitochondrial membrane potential in SW480 cells. These results suggest that pyrimidine with urea moieties could be a template for designing new anticancer agents.

Novel indole hydrazide derivatives: Synthesis and their antiproliferative activities through inducing apoptosis and DNA damage.

A series of novel indole hydrazide derivatives was synthesized and evaluated for their anticancer activities. Compound 12 exhibited the highest antiproliferative activity against the MCF-7 cell line, with an IC50 value of 3.01 µM. Treatment of MCF-7 cells with compound 12 led to cell cycle arrest at the G0/G1 phase and also displayed a significant annexin V binding pattern, indicating that compound 12 is effective in apoptotic cell death. The Western blot analysis showed that compound 12 increased the expression of proapoptotic Bax and decreased the levels of the antiapoptotic Bcl-2 protein. It was also observed that MCF-7 cells treated with compound 12 showed reduced levels of procaspase-3 and -9 proteins. Moreover, compound 12 treatment induced a significant DNA damage in MCF-7 cells by increasing H2AX and ATM phosphorylation.

Design, synthesis and in vitro apoptotic mechanism of novel pyrrolopyrimidine derivatives.

In this work we described the synthesis and evaluation of cytotoxic and apoptotic activity of novel pyrrolopyrimidine derivatives against A549, PC3 and MCF-7 cells. Among the synthesized compounds, 6b, 8a, 9a and 7a, 8b displayed the significant cytotoxic activities against A549 and PC3 cells with IC50 value of 0.35, 1.48, 1.56 and 1.04, 1.89 µM, respectively. It was found that A549 cells were more sensitive to synthesized compounds than PC3 and MCF-7 cells. In order to evaluate the mechanism of cytotoxic activity in A549, compounds 6b, 8a and 9a were selected for further studies. Annexin V binding assay and western blot analysis results revealed that 6b, 8a and 9a induced apoptosis in A549 cells by intrinsic apoptotic pathway through the activation pro-apoptotic proteins such as Bim, Bax, Bak, Puma and deactivation of anti-apoptotic proteins including Bcl-2, Mcl-1 and Bcl-XL accompanied by the activation of caspase-3, caspase-9 and cleavage of PARP. Also, compounds 6b, 8a and 9a triggered apoptosis in HCT116 wt cells via activation of caspase-3 and caspase-9, but not in HCT116 Bax/Bak KO cells, indicating resistance to 6b, 8a and 9a treatment.

Cytotoxic and Apoptotic Effects of Novel Pyrrolo[2,3-d]Pyrimidine Derivatives Containing Urea Moieties on Cancer Cell Lines.

BACKGROUND: Pyrrolo[2,3-d]pyrimidines have been recently reported to have anticancer activities through inhibition of different targets such as, Epidermal Growth Factor Receptor (EGFR) tyrosine kinase, Janus Kinase (JAK), mitotic checkpoint protein kinase (Mps1), carbonic anhydrase, MDM-2. On the other hand, aryl urea moieties which are found in some tyrosine kinase inhibitors such as Sorafenib and Linifanib have aroused recent attention as responsible for anticancer activities. The aims of this paper are to synthesize pyrrolo[ 2,3-d]pyrimidine derivatives containing urea moiety and evaluate their anti-cancer activity against human lung cancer cell line (A549), prostate cancer cell line (PC3), human colon cancer cell line (SW480) and human breast cancer cell line (MCF-7). METHODS: A series of new pyrrolo[2,3-d]pyrimidines containing urea moieties have been synthesized as Scheme 1. In vitro cytotoxicity of target compounds were evaluated against, SW480, PC3, A549 and MCF-7 human cancer cell lines using a MTT assay. In order to evaluate the mechanism of cytotoxic activity of compounds 9e, 10a and 10b, having the best cytotoxic activity, Annexin V binding assay, cell cycle analysis and western blot analysis were performed. RESULTS: Among the target compounds, 10a (IC50 = 0.19 µM) was found to be the most potent derivative against PC3 cells. Compound 10b and 9e showed the strong cytotoxic activity against MCF-7 and A549 cells with IC50 value of 1.66 µM and 4.55 µM, respectively. Flow cytometry data suggest that the cytotoxic activity of the compounds on cancer cells might be mediated by apoptosis revealing a significant increase in the percentage of late apoptotic cells and causing a cell cycle arrest at different stages. Western blot analysis of apoptosis marker demonstrated that these compounds induce apoptosis through the intrinsic pathway. CONCLUSION: Compound 9e displayed the strongest cytotoxicity against A549 cancer cell line, and induced late apoptosis in A549, as confirmed by cell cycle arrest in G0/G1 phase. In addition, compound 9e reduced expression of the anti-apoptotic protein Bcl-2 and enhanced expression of the pro-apoptotic protein Bax, besides increased caspase-9 and caspase-3, as well as cleavage of PARP levels. These results suggest that compound 9e showed a cytotoxic effect in A549 cells through activation of the mitochondrial apoptotic pathway. Further studies will be undertaken in our laboratory to improve cytotoxic activity of compound 9e and to identify the biological targets of 9e which are responsible for anticancer activity.

The Effects of 1,3,5-trisubstituted Indole Derivatives on Cell Growth, Apoptosis and MMP-2/9 mRNA Expression of MCF-7 Human Breast Cancer Cells.

BACKGROUND: Matrix metalloproteinases are known as extracellular matrix degrading enzymes and have important role on tumor progression. OBJECTIVE: This study reports the effects of 1,3,5-trisubstituted indole derivatives on cytotoxicity, apoptosis and MMP- 2/MMP-9 mRNA expression of MCF-7 human breast carcinoma cells. METHOD: The cytotoxic effects of the compounds on MCF-7 cells were performed by MTT test, and cell proliferation was determined via BrdU incorporation. The apoptotic effects were observed by cell death detection elisa. The effects of the compounds on MMP-2/-9 enzyme activity and mRNA expression were also performed. RESULTS: The compounds inhibited the proliferation of MCF-7 breast carcinoma cells significantly in a dose dependent manner. All compounds were able to induce DNA fragmentation, especially compound 1. The IC50 values of compound 2 and 4 for MMP-2 were 0.42 µM and 1.88 µM, respectively. MMP-2 mRNA expression results were correlated with the inhibition of enzyme activity, such compound 4 inhibited MMP-2 mRNA expression at all treated concentrations. Docking simulation has also been performed to analyze the binding mode of compounds and the results showed that compound 2, the most active compound, formed a hydrogen bond with Glu202 for binding to the MMP-2 active site. In addition, the hydrophobic parts of compound 2 are in contact with nonpolar surface areas of MMP-2, such as His201, His211, Tyr223 and Tyr193. CONCLUSION: According to the molecular docking results along with the biological assay data, it is suggested that compound 2 might be used for further design and development of MMP-2 inhibitors.

Synthesis, Biological, and Computational Evaluation of Novel 1,3,5-Substituted Indolin-2-one Derivatives as Inhibitors of Src Tyrosine Kinase.

Several substituted indolin-2-one derivatives were synthesized and evaluated for their activities against Src kinase. Several compounds showed activity against Src, with IC50 values in the low micromolar range. Among them, compound 2f showed the most significant activity with an IC50 value of 1.02 µM. Molecular docking studies have been performed for evaluation of the binding modes of compound 2f into the Src active site. The docking structure of compound 2f disclosed that the indole NH forms a hydrogen bond with the carbonyl of Met341. These results suggest that our novel compound 2f is a promising compound for the further development of indole-based drugs targeting Src kinase.

Evaluation of novel aminomethyl indole derivatives as Src kinase inhibitors and antioxidant agents.

BACKGROUND: Oxidative stress has been implicated in aging and in a variety of diseases affecting the nervous, respiratory, cardiovascular and gastrointestinal system in humans. Reactive oxygen species (ROS) have been associated with mechanisms to activate kinases, such as protein tyrosine kinases, which may initiate malignant transformation. Significant evidences of the activation of protein kinases by oxidative stress brought increased attention to the role of antioxidants in these mechanisms. Therefore, recent efforts have focused on revealing the relationship between protein kinase inhibition and the levels of ROS production. METHODS: Antioxidant properties of aminomethyl indole derivatives were investigated by employing various in vitro systems, microsomal NADPH-dependent inhibition of lipid peroxidation (LP), interaction of 2,2-diphenyl-1-picrylhydrazyl and scavenging of superoxide anion radical by virtue of superoxide dismutase inhibitory activity. In vitro tyrosine kinase assays of the aminomethyl indole derivatives were evaluated by changes in the enzymatic activity of pp60(c-Src) tyrosine kinase through alterations in the phosphorylation level of immobilized kinase substrate. RESULTS: Analysis of the antioxidant effects of indole 1a-c, bromo indole 2a-c and phenyl indole 3a-c derivatives revealed almost equal inhibition against LP for 5-bromo indole 2a-c and phenyl indole 3a-c derivatives and slight inhibition against superoxide dismutase only for 1a and 1c. Nonsubstituted compounds at position 5 showed half-inhibition of LP. Compound 1a has tyrosine kinase inhibition with an IC(50) of 102.6 ± 1.16 µM. CONCLUSION: The substitution feature at position 5 of the indole ring certainly plays an important role in both tyrosine kinase inhibition and antioxidant capacity. While certain lipophilicity of this substitution is important for antioxidant activity, it may, on the other hand, have a negative impact on the inhibition of Src kinase.

Investigation of aminomethyl indole derivatives as hyaluronidase inhibitors.

Hyaluronidase inhibitors are of potential therapeutic value for the treatment of a variety of diseases, such as cancer, arthrosis, or bacterial infections. Potent and selective hyaluronidase inhibitors are not known so far, and current approaches to the development of hyaluronidase inhibitors are limited. Elevated levels of hyaluronan (HA) are connected with most malignant tumours. Therefore, the search for drugs modulating the hyaluronidase activity became very important. In the present study, a new series of aminomethyl indole derivatives (AMIDs) were tested for inhibition of bovine testes hyaluronidase (BTH). In vitro assays were performed using stains-all at pH 7 and Morgan-Elson reaction at pH 3.5. Among the AMIDs, 3-[(4-methylpiperazin-1-yl)methyl]-5-phenyl-1H-indole (9) was found to be active with 23% inhibition at 50 microM and pH 7. All the other inhibitors showed less activity at pH 3.5 and pH 7. These activity results demonstrated that compounds with phenyl substitution at position 5 have higher activity. The results confirmed that more lipophilic compounds have better inhibition against the hyaluronidase enzyme.