Synthesis, In Silico Prediction, and In Vitro Evaluation of Anti-tumor Activities of Novel 4'-Hydroxybiphenyl-4-carboxylic Acid Derivatives as EGFR Allosteric Site Inhibitors.

INTRODUCTION: Allosteric inhibition of EGFR Tyrosine Kinase (TK) is currently among the most attractive approaches for designing and developing anti-cancer drugs to avoid chemoresistance exhibited by clinically approved ATP-competitive inhibitors. The current work aimed to synthesize new biphenyl-containing derivatives that were predicted to act as EGFR TK allosteric site inhibitors based on molecular docking studies. METHOD: A new series of 4'-hydroxybiphenyl-4-carboxylic acid derivatives, including hydrazine-1-carbothioamide (S3-S6) and 1,2,4-triazole (S7-S10) derivatives, were synthesized and characterized using IR, 1HNMR, 13CNMR, and HR-mass spectroscopy. Compound S4 had a relatively high pharmacophore-fit score, indicating that it may have biological activity similar to the EGFR allosteric inhibitor reference, and it scored a relatively low ΔG against EGFR TK allosteric site, indicating a high likelihood of drug-receptor complex formation. Compound S4 was cytotoxic to the three cancer cell lines tested, particularly HCT-116 colorectal cancer cells, with an IC50 value comparable to Erlotinib. Compound S4 induced the intrinsic apoptotic pathway in HCT-116 cells by arresting them in the G2/M phase. RESULT: All of the new derivatives, including S4, met the in silico requirements for EGFR allosteric inhibitory activity. CONCLUSION: Compound S4 is a promising EGFR tyrosine kinase allosteric inhibitor that warrants further research.

New Carbothioamide and Carboxamide Derivatives of 3-Phenoxybenzoic Acid as Potent VEGFR-2 Inhibitors: Synthesis, Molecular Docking, and Cytotoxicity Assessment.

INTRODUCTION/BACKGROUND: Because of the well-established link between angiogenesis and tumor development, the use of antiangiogenic therapeutics, such as those targeting VEGFR-2, presents a promising approach to cancer treatment. In the current study, a set of five hydrazine-1-- carbothioamide (compounds 3a-e) and three hydrazine-1-carboxamide derivatives (compounds 4a-c) were successfully synthesized from 3-phenoxybenzoic acid. These compounds were specially created as antiproliferative agents with the goal of targeting cancer cells by inhibiting VEGFR-2 tyrosine kinase. MATERIALS AND METHODS: The new derivatives were synthesized by conventional organic methods, and their structure was versified by IR, 1HNMR, 13CNMR, and mass spectroscopy. In silico investigation was carried out to identify the compounds' target, molecular similarity, ADMET, and toxicity profile. The cytotoxic activity of the prepared compounds was evaluated in vitro against three human cancer cell lines (DLD1 colorectal adenocarcinoma, HeLa cervical cancer, and HepG2 hepatocellular carcinoma). The effects of the leading compound on cell cycle progression and apoptosis induction were investigated by flow cytometry, and the specific apoptotic pathway triggered by the treatment was evaluated by RT-PCR and immunoblotting. Finally, the inhibitory activities of the new compounds against VEGFR-2 was measured. RESULTS: The designed derivatives exhibited comparable binding positions and interactions to the VEGFR-2 binding site to that of sorafenib (a standard VEGFR-2 tyrosine kinase inhibitor), as determined by molecular docking analysis. Compound 4b was the most cytotoxic compound, achieving the lowest IC50 against HeLa cells. Compound 4b, a strong representative of the synthesized series, induced cell cycle arrest at the G2/M phase, increased the proportion of necrotic and apoptotic HeLa cells, and activated caspase 3. The EC50 value of compound 4b against VEGFR-2 kinase activity was comparable to sorafenib's. CONCLUSION: Overall, the findings suggest that compound 4b has a promising future as a starting point for the development of new anticancer drugs.

Synthesis, docking study, and antitumor evaluation of benzamides and oxadiazole derivatives of 3-phenoxybenzoic acid as VEGFR-2 inhibitors.

Current chemotherapeutic agents have several limitations, including lack of selectivity, the development of undesirable side effects, and chemoresistance. As a result, there is an unmet need for the development of novel small molecules with minimal side effects and the ability to specifically target tumor cells. A new series of 3-phenoxybenzoic acid derivatives, including 1,3,4-oxadiazole derivatives (4a-d) and benzamides derivatives (5a-e) were synthesized; their chemical structures were confirmed by Fourier-transform infrared spectroscopy, 1H nuclear magnetic resonance (NMR), 13C NMR, and mass spectra; and various physicochemical properties were determined. The antiproliferative activities of the new derivatives were evaluated by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Three compounds (4b, 4c, and 4d) exhibited cytotoxicity against two of the three cell lines tested, five compounds (3, 4a, 5a, 5b, and 5e) were toxic to one cell line, while two compounds (5c and 5d) were not cytotoxic to any of the three cell lines tested in the current study. Based on docking scores, MTT assay findings, and vascular endothelial growth factor receptor 2 (VEGFR-2) kinase activity data, Compound 4d was selected for further biological investigation. Flow cytometry was used to determine the mode of cell death (apoptosis vs. necrosis) and the effect on cell cycle progression. Compound 4d arrested HepG2 hepatocellular carcinoma cells in the G2/M phase and activated both the intrinsic and extrinsic apoptosis pathways. In conclusion, Compound 4d has shown promising results for future research as a potent VEGFR-2 tyrosine kinase inhibitor.

New Indole-6-Carboxylic Acid Derivatives as Multi-Target Antiproliferative Agents: Synthesis, in Silico Studies, and Cytotoxicity Evaluation.

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) are commonly overexpressed in cancers making them appealing targets for cancer therapeutics. Two groups of indole-6-carboxylic acid derivatives, hydrazone derivatives targeting EGFR and oxadiazole derivatives targeting VEGFR-2, were synthesized and characterized using FT-IR, 1 H-NMR, 13 CNMR, and HR-MS techniques. Binding patterns to potential molecular targets were studied using molecular docking and compared to standard EGFR and VEGFR-2 inhibitors. The newly synthesized compounds were cytotoxic to the three cancer cell lines tested (HCT-116, HeLa, and HT-29 cell lines) as evaluated by the MTT assay. Compound 3 b (EGFR-targeting) and compound 6 e (VEGFR-2-targeting) possessed the highest antiproliferation activity, were cancer-selective, arrested cancer cells in the G2/M phase, induced the extrinsic apoptosis pathway, and had the highest EGFR/VEGFR-2 enzyme inhibitory activity, respectively. The structure-activity relationships of the new compounds showed that the presence of an aryl or heteroaryl fragment attached to a linker is required for the anti-tumor activity. In conclusion, the findings of the current study suggest that compounds 3 b and 6 e are promising cytotoxic agents that act by inhibiting EGFR and VEGFR-2 tyrosine kinases, respectively.

Design, Synthesis, in silico and in vitro Evaluation of New Combretastatin A-4 Analogs as Antimitotic Antitumor Agents.

BACKGROUND: Combretastatin A-4 (CA-4) binds ß-tubulin at the colchicine-binding site preventing tubulin from polymerizing into microtubules. CA-4 and cis combretastatin analogs isomerize to the trans form resulting in decreased cytotoxicity and anti-tubulin activity. However, the excellent anti-cancer potential and relatively simple molecular structure of CA-4 provide an encouraging starting point for the development of new, more stable and more potent anti-tubulin compounds. OBJECTIVE: This study aimed to synthesize a new series of compounds derived from 4-(3,4,5- trimethoxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione derivatives (compounds 10-12) with substituted phenyl group at C5 of the triazole ring (B-ring) as analogs of CA-4, with different alkyl and aryl side chain substituents at the triazole moiety, resulting in the permanent cis configuration of the two phenyl rings. Moreover, the anti-cancer activities of the new compounds were assessed. METHODS: Chemical synthesis was carried out by conventional organic methods. The newly synthesized CA-4 analogs were characterized by FT-IR, 1HNMR, 13CNMR, and HR-MS(ESI) techniques. Molecular docking studies, including docking score (ΔG), ADMET, DFT, and molecular similarities, were performed. The anti-proliferative activity of the new compounds against three human cancer cell lines (A549, Hep G2, and HCT-116) and the normal cell line WI-38 was evaluated using the MTT assay, and their ability to inhibit tubulin polymerization, and consequently, their effects on cell cycle progression and induction of apoptosis were assessed. RESULTS: Molecular docking studies showed that compounds 11b and 11d exhibited the highest docking scores (-13.30 and -14.01 Kcal/mol, respectively) into the colchicine-binding site, scores very close to the reference drug colchicine (-13.50 Kcal/mol), and that hydrogen bonding and hydrophobic interaction are essential for binding. The most active cytotoxic compound, 11b, had potent IC50 values against the three human cancer cell lines (3.83, 10.20, and 10.67 µM against Hep G2, HCT- 116, and A549, respectively) while exhibiting low cytotoxicity against non-cancer-human WI-38, suggesting that compound 11b targets rapidly growing cancer cells. Moreover, compound 11b exhibited potent anti-tubulin activity which was comparable to CA-4. Targeting microtubules caused cell cycle arrest at the G2/M phase resulting in the induction of apoptosis. CONCLUSION: These findings indicate that compound 11b is a promising ß-tubulin-binding compound with antimitotic action that has the potential to treat cancer.

New Combretastatin Analogs as Anticancer Agents: Design, Synthesis, Microtubules Polymerization Inhibition, and Molecular Docking Studies.

A new series of 4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole-3-thiol derivatives were synthesized as analogs for the anticancer drug combretastatin A-4 (CA-4) and characterized using FT-IR, 1 H-NMR, 13 CNMR, and HR-MS techniques. The new CA-4 analogs were designed to meet the structural requirements of the highest expected anticancer activity of CA-4 analogs by maintaining ring A 3,4,5-trimethoxyphenyl moiety, and at the same time varying the substituents effect of the triazole moiety (ring B). In silico analysis indicated that compound 3 has higher total energy and dipole moment than colchicine and the other analogs, and it has excellent distribution of electron density and is more stable, resulting in an increased binding affinity during tubulin inhibition. Additionally, compound 3 was found to interact with three apoptotic markers, namely p53, Bcl-2, and caspase 3. Compound 3 showed strong similarity to colchicine, and it has excellent pharmacokinetics properties and a good dynamic profile. The in vitro anti-proliferation studies showed that compound 3 is the most cytotoxic CA-4 analog against cancer cells (IC50 of 6.35 µM against Hep G2 hepatocarcinoma cells), and based on its selectivity index (4.7), compound 3 is a cancer cytotoxic-selective agent. As expected and similar to colchicine, compound 3-treated Hep G2 hepatocarcinoma cells were arrested at the G2/M phase resulting in induction of apoptosis. Compound 3 tubulin polymerization IC50 (9.50 µM) and effect on Vmax of tubulin polymerization was comparable to that of colchicine (5.49 µM). Taken together, the findings of the current study suggest that compound 3, through its binding to the colchicine-binding site at ß-tubulin, is a promising microtubule-disrupting agent with excellent potential to be used as cancer therapeutic agent.

Novel 5-bromoindole-2-carboxylic Acid Derivatives as EGFR Inhibitors: Synthesis, Docking Study, and Structure Activity Relationship.

BACKGROUND: The indole backbone is encountered in a class of N-heterocyclic compounds with physiological and pharmacological effects such as anti-cancer, anti-diabetic, and anti-HIV. These compounds are becoming increasingly popular in organic, medicinal, and pharmaceutical research. Nitrogen compounds' hydrogen bonding, dipole- dipole interactions, hydrophobic effects, Van der Waals forces, and stacking interactions have increased their relevance in pharmaceutical chemistry due to their improved solubility. Indole derivatives, such as carbothioamide, oxadiazole, and triazole, have been reported to act as anti-cancer drugs due to their ability to disrupt the mitotic spindle and prevent human cancer cell proliferation, expansion, and invasion. OBJECTIVES: To synthesize new 5-bromoindole-2-carboxylic acid derivatives that function as EGFR tyrosine kinase inhibitors as deduced through molecular docking studies. METHODS: Different derivatives of indole (carbothioamide, oxadiazole, tetrahydro pyridazine-3,6-dione, and triazole) were synthesized and evaluated through different chemical, spectroscopic methods (IR, 1HNMR, 13CNMR, and MS) and assessed in silico and in vitro for their antiproliferative activities against A549, HepG2, and MCF-7 cancer cell lines. RESULTS: According to molecular docking analyses, compounds 3a, 3b, 3f, and 7 exhibited the strongest EGFR tyrosine kinase domain binding energies. In comparison to erlotinib, which displayed some hepatotoxicity, all of the evaluated ligands displayed good in silico absorption levels, did not appear to be cytochrome P450 inhibitors, and were not hepatotoxic. The new indole derivatives were found to decrease cell growth of three different types of human cancer cell lines (HepG2, A549, and MCF-7), with compound 3a being the most powerful while still being cancer-specific. Cell cycle arrest and the activation of apoptosis were the results of compound 3a's inhibition of EGFR tyrosine kinase activity. CONCLUSION: The novel indole derivatives, compound 3a in particular, are promising anti-cancer agents which inhibit cell proliferation by inhibiting EGFR tyrosine kinase activity.

New Niflumic Acid Derivatives as EGFR Inhibitors: Design, Synthesis, In silico Studies, and Anti-proliferative Assessment.

BACKGROUND: 1,3,4-oxadizole and pyrazole derivatives are very important scaffolds for medicinal chemistry. A literature survey revealed that they possess a wide spectrum of biological activities including anti-inflammatory and antitumor effects. OBJECTIVES: To describe the synthesis and evaluation of two classes of new niflumic acid (NF) derivatives, the 1,3,4-oxadizole derivatives (compounds 3 and (4A-E) and pyrazole derivatives (compounds 5 and 6), as EGFR tyrosine kinase inhibitors in silico and in vitro. METHODS: The designed compounds were synthesized using conventional organic synthesis methods. The antitumor activities of the new NF derivatives against HepG2 hepatocellular carcinoma and A549 non-small cell lung cancer cell lines were assessed in vitro via MTT assay, flow cytometry, RT-PCR, as well as via molecular docking studies. RESULTS: The cytotoxicity results indicated that the newly synthesized NF derivatives were cytotoxic against the two cancer cell lines, with compound 6 being the most cytotoxic, achieving the lowest IC50 concentration. Furthermore, compound 6 targeted EGFR tyrosine kinase leading to cell cycle arrest at the G2/M cell cycle phase and induction of apoptosis. The in vitro biological investigation results matched those of the molecular docking analysis. In conclusion, the new NF derivatives, specifically compound 6, exhibited favorable pharmacokinetic features and are promising EGFR tyrosine kinase inhibitors. CONCLUSION: A series of niflumic acid derivatives (3, 4A-E, 5, and 6) were successfully created, and FT-IR, ^{1H, ₁₃CNMR, and HRMS were used to confirm their chemical structures. According to molecular docking studies, compounds 3, 5, and 6 have the highest docking scores (ΔG), and most tested compounds have a good pharmacokinetic profile. Results of compound 6 in vitro antitumor activities showed that it is a promising EGFR tyrosine kinase inhibitor.}

Vitamin B12 enhances the antitumor activity of 1,25-dihydroxyvitamin D3 via activation of caspases and targeting actin cytoskeleton.

BACKGROUND: 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is an effective anticancer agent, and when combined with other agents it shows superior activities. Vitamin B12 has been shown to contribute to increasing the effectiveness of anticancer drugs when used in combination. Thus, the current study aimed at investigating the anticancer potential of the combination of 1,25(OH)2D3 and vitamin B12. METHODS: MTT assay was used to determine the cytotoxic activity of combining 1,25(OH)2D3 and vitamin B12 against six different cancer cell lines and one normal cell line. The surviving fraction after clonogenic assay was measured, and the effects of 1,25(OH)2D3/B12 combination on the activity of different caspases, cell adhesion, actin cytoskeleton, cell morphology, and percentage of polarized cells were evaluated. RESULTS: Vitamin B12 did not cause cytotoxicity, however, it enhanced the cytotoxicity of 1,25(OH)2D3 against cancer cells. The cytotoxic effects of 1,25(OH)2D3 and its combination with vitamin B12 was not evident in the normal mammary MCF10A cell line indicating cancer cell-specificity. The cytotoxic effects of 1,25(OH)2D3/B12 combination occurred in a dose-dependent manner and was attributed to apoptosis induction which was mediated by caspase 4 and 8. Moreover, 1,25(OH)2D3/B12-treated cells showed enhanced inhibition of clonogenic tumor growth, reduced cell adhesion, reduced cell area, reduced percentage of cell polarization, and disorganized actin cytoskeleton resulting in reduced migratory phenotype when compared to cells treated with 1,25(OH)2D3 alone. CONCLUSION: 1,25(OH)2D3 and vitamin B12 exhibited synergistic anticancer effects against different cancer cell lines. The combination therapy of 1,25(OH)2D3 and vitamin B12 may provide a potential adjunctive treatment option for some cancer types.

Biological activity and apoptotic signaling pathway of C11-functionalized cephalostatin 1 analogues.

Cephalostatin 1, a potent anti-cancer agent, is a natural bis-steroidal alkaloid that causes cell death in the subnanomolar to picomolar ranges via an atypical apoptosis pathway. Although cephalostatin 1 is a highly effective anticancer drug, its availability limits its utilization. We previously reported the synthesis of two 12'α-hydroxy derivatives of cephalostatin 1 that induce cell death by activating the ER stress apoptosis signaling pathway. For the current work, we synthesized six C11-functionalized cephalostatin 1 analogues (CAs) to evaluate their biological activity. For the cytotoxic compounds, the induced apoptotic pathway was investigated. The C11-functionalized cephalostatin 1 analogues 5 and 6 (CA5 and CA6) were found to exhibit cytotoxic activity against K-562 leukemia cells, MCF-7 breast cancer cells and DU-145 prostate cancer cells, while the remaining four analogues did not show anti-tumor activities against any of the cell lines. Our results indicated that CA5 and CA6 induced cell death via the atypical ER-dependent apoptosis pathway; they increased the expression of Smac/DIABLO, an inhibitor of inhibitors of apoptosis (IAPs), which in turn facilitated the activation of different caspases including the ER-caspase 4 without cytochrome c release from mitochondria. CA5 and CA6 are promising anticancer agents due to their low GI50, the remarkable apoptosis pathway they induce which can overcome chemoresistance, and their very low toxicity to normal cells making them cephalostatin 1 utilizable alternatives.

Cephalostatin 1 analogues activate apoptosis via the endoplasmic reticulum stress signaling pathway.

The current study was conducted to compare the cytotoxicity of two stereospecific cephalostatin 1 analogues (CAs) against several human normal cell types and cancer cell lines and to determine their cytotoxic mechanism. Both CA analogues induced apoptosis and were cytotoxic with 50% growth inhibition (GI50) at ~1µM or less in six human cancer cell lines but neither analogue at 10µM killed more than 14% of any of three types of normal human cells suggesting their cytotoxicity is cancer-specific. CA treatment inhibited clonogenic tumor growth and activated caspase 3 and 9 but not caspase 8. CA-induced apoptosis was inhibited by the pan caspase inhibitor indicating the importance of caspase activation. CA treatment released smac/DIABLO but not cytochrome c from mitochondria and induced phosphorylation of eIF-2 and the activation of procaspase 4 in cancer cells, similar to cell treatment with thapsigargin, a known endoplasmic reticulum (ER) stress inducer. Finally, cells pretreated with a caspase 4 inhibitor were resistant to CA-induced apoptosis. In conclusion, both CAs induced apoptosis by triggering ER stress. Because of their ease of synthesis and low GI50, these cephalostatin analogues represent promising anticancer drugs.

2-deoxy-D-Glucose Synergizes with Doxorubicin or L-Buthionine Sulfoximine to Reduce Adhesion and Migration of Breast Cancer Cells.

BACKGROUND: Cancer metastasis depends on cell motility which is driven by cycles of actin polymerization and depolymerization. Reactive oxygen species (ROS) and metabolic oxidative stress have long been associated with cancer. ROS play a vital role in regulating actin dynamics that are sensitive to oxidative modification. The current work aimed at studying the effects of sub-lethal metabolic oxidative stress on actin cytoskeleton, focal adhesion and cell migration. MATERIALS AND METHODS: T47D human breast cancer cells were treated with 2-deoxy- D-glucose (2DG), L-buthionine sulfoximine (BSO), or doxorubicin (DOX), individually or in combination, and changes in intracellular total glutathione and malondialdehyde (MDA) levels were measured. The expression of three major antioxidant enzymes was studied by immunoblotting, and cells were stained with fluorescent- phalloidin to evaluate changes in F-actin organization. In addition, cell adhesion and degradation ability were measured. Cell migration was studied using wound healing and transwell migration assays. RESULTS: Our results show that treating T47D human breast cancer cells with drug combinations (2DG/BSO, 2DG/DOX, or BSO/DOX) decreased intracellular total glutathione and increased oxidized glutathione, lipid peroxidation, and cytotoxicity. In addition, the drug combinations caused a reduction in cell area and mitotic index, prophase arrest and a decreased ability to form invadopodia. The formation of F-actin aggregates was increased in treated T47D cells. Moreover, combination therapy reduced cell adhesion and the rate of cell migration. CONCLUSIONS: Our results suggest that exposure of T47D breast cancer cells to combination therapy reduces cell migration via effects on metabolic oxidative stress.

Feasibility of collecting umbilical cord blood in jordan and the effect of maternal and neonatal factors on hematopoietic stem cell content.

BACKGROUND: Cord blood transplant is an accepted treatment for many malignant and non-malignant diseases. We sought to determine the feasibility of collecting cord blood in Jordan and the effect of maternal and fetal factors on the quality of the cord blood units. METHODS: A total of 124 cord blood units were collected, and 75 (60%) cord blood units were included in this analysis. Cord blood volume, total nucleated cell (TNC) count, cell viability and CD34(+) content were measured, and clonogenic assay was performed. RESULTS: The mean volume of the collected units was 68.9 ml (range 40-115) with mean nucleated cell count of 6.5 x 10(8) (range 1-23.0). Our results showed a positive correlation between the volume of cord blood and TNC count (p=0.008), cell viability (p=0.001), CD34(+) content (p=0.034) and the length of the umbilical cord (p=0.011). In addition, our results showed an inverse relation between the Colony Forming Unit-Granulocyte Macrophage (CFU-GM) concentration and the gestation duration (p=0.038). CONCLUSION: We conclude that it is feasible to collect cord blood units in Jordan with excellent TNC and CD34(+) cell content. The volume of cord blood collected was associated with higher TNC count and CD34(+) count. Efforts toward establishing public cord blood banks in our area are warranted.

Non-overlapping activities of ADF and cofilin-1 during the migration of metastatic breast tumor cells.

BACKGROUND: ADF/cofilin proteins are key modulators of actin dynamics in metastasis and invasion of cancer cells. Here we focused on the roles of ADF and cofilin-1 individually in the development of polarized migration of rat mammary adenocarcinoma (MTLn3) cells, which express nearly equal amounts of each protein. Small interference RNA (siRNA) technology was used to knockdown (KD) the expression of ADF and cofilin-1 independently. RESULTS: Either ADF KD or cofilin KD caused cell elongation, a reduction in cell area, a decreased ability to form invadopodia, and a decreased percentage of polarized cells after 180 s of epidermal growth factor stimulation. Moreover, ADF KD or cofilin KD increased the rate of cell migration and the time of lamellipodia protrusion but through different mechanisms: lamellipodia protrude more frequently in ADF KD cells and are more persistent in cofilin KD cells. ADF KD cells showed a significant increase in F-actin aggregates, whereas cofilin KD cells showed a significant increase in prominent F-actin bundles and increased cell adhesion. Focal adhesion area and cell adhesion in cofilin KD cells were returned to control levels by expressing exogenous cofilin but not ADF. Return to control rates of cell migration in ADF KD cells was achieved by expression of exogenous ADF but not cofilin, whereas in cofilin KD cells, expression of cofilin efficiently rescued control migration rates. CONCLUSION: Although ADF and cofilin have many redundant functions, each of these isoforms has functional differences that affect F-actin structures, cell adhesion and lamellipodial dynamics, all of which are important determinants of cell migration.