New C2- and N3-Modified Thieno[2,3-d]Pyrimidine Conjugates with Cytotoxicity in the Nanomolar Range.

AIMS: The aim of the current study was to develop and explore a series of new cytotoxic agents based on the conjugation between the thieno[2,3-d]pyrimidine moiety and a second pharmacophore at the C2 or N3 position. BACKGROUND: As the thieno[2,3-d]pyrimidine core is a bioisostere of the 4-anilinoquinazoline, various new thienopyrimidine derivatives were synthesized by modifying the structure of the clinically used anticancer quinazoline EGFR inhibitors of the first generation - gefitinib, and second-generation - dacomitinib and canertinib. It was reported that some thieno[2,3-d]pyrimidine derivatives showed improved EGFR inhibitory activity. On the other hand, the benzimidazole heterocycle is present as a pharmacophore unit in the structure of many clinically used chemotherapeutic agents. Some 2-aminobenzimidazole derivatives, possessing anticancer activity, demonstrated EGFR inhibition and the benzimidazole derivative EGF816 is currently in the second phase of clinical trials. OBJECTIVE: The objectives of the study were the design of a novel series thieno[2,3-d]pyrimidines, synthesis of the compounds and investigation of their effects towards human cancer HT-29, MDA-MB-231, HeLa, HepG2 and to normal human Lep3 cell lines. (American Type Culture Collection, ATCC, Rockville, MD, USA). METHODS: The synthetic protocol implemented cyclocondensation of 2-amino-thiophenes and nitriles in an inert medium, aza- Michael addition to benzimidazole derivatives and nucleophylic substitution at the N3 place. MTS test was used in order to establish the cytotoxicity of the tested compounds. SAR analysis and in silico assessment of the inhibitory potential towards human oncogenic V599EB-Raf were performed using Molinspiration tool and Molecular Operating environment software. RESULTS: The MTS test data showed that almost all studied thieno[2,3-d]pyirimidines (9-13, 21-22 and 25) manifest high inhibitory effect on cell proliferation at nanomolar concentrations, whereas compounds 9 (IC50 = 130 nM) and 10 (IC50 = 261 nM) containing amino acid moiety, and 21 (IC50 = 108 nM) possessing two thienopyrimidine moieties attached to a 1,3-disubstituted benzimidazole linker, revealed many times lower toxicity against Lep3 cells compared to the cancer cells. Thienopyrimidines 11-13 possessed high selectivity against HeLa cells. Compound 13 showed high inhibitory activity against MDA-MB-231 and HepG2, with IC50 1.44 nM and 1.11 nM respectively. To outline the possible biological target of the studied coumpounds, their potential to interact with human oncogenic V599EB-Raf was explored by a docking study. As a result, it was suggested that the benzimidazolyl and glycyl fragments could enhance the binding ability of the new compounds by increasing the number of hydrogen bond acceptors and by stabilizing the inactive form of the enzyme. CONCLUSION: The thienopyrimidines tested in vitro for human cancer HT-29, MDA-MB-231, HeLa, HepG2 and normal human Lep3 cell lines demonstrated cytotoxicity in the nanomolar range. It was established that compounds 9, 10 and 21 showed many times lower toxicity against normal Lep3 cells that can provide a high selectivity towards all four cancer cell lines at small concentrations. Based on the analysis of the structure-activity relationship, the observed trends in the cytotoxicity could be related to the lipophilicity and the topological polar surface area of the tested compounds. The docking study on the potential of the new thieno[2,3-d]pyrimidine-4-ones to interact with mutant V599EB-Raf showed that the compounds might be able to stabilize the enzyme in its inactive form.

Synthesis, in vitro biological activity and docking of new analogs of BIM-23052 containing unnatural amino acids.

Somatostatin (SST) is an endogenous cyclic tetradecapeptide hormone that exerts multiple biological activities via a family of five receptors. BIM-23052 (DC-23-99) D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH2 is a linear SST analog with established in vitro GH-inhibitory activity and high affinity to sstr5, sstr3 and sstr2. The different SSTR subtypes are expressed in different tissues and in some tumor cells. Based on this finding, a series of new analogs of BIM-23052 with expected antitumor activity have been synthesized. The Thr at position 6 in BIM-23052 was replaced by the conformationally hindered Tle, Aib, Ac5c and Ac6c of the new analogs. The peptides were synthesized by standard solid-phase peptide chemistry methods, Fmoc strategy. The cytotoxic effects of the compounds were tested in vitro against a panel of tumor cell lines: HT-29, MDA-MB-23, Hep-G2, HeLa and the normal human diploid cell line Lep-3. All five somatostatin receptor subtypes were modeled and docking was performed to determine the binding affinity of the analogs. The new peptides exhibited different concentration-dependent antiproliferative effect on the tumor cell lines after 24 h of treatment. The compound 3B (Aib6) demonstrated the most pronounced antiproliferative effects on HepG-2 cells with the IC50 = 0.01349 nM. Docking confirmed that all compounds bind well to SST receptors with preference to sstr3 and sstr5, which is most probably the reason for the observed biological effects.

Synthesis, Cytotoxicity and Antioxidant Activity of New Analogs of RC-121 Synthetic Derivatives of Somatostatin.

BACKGROUND: Based on the structure of RC-121 (D-Phe-c (Cys-Tyr-D-Trp-Lys-Val-Cys)-Thr-NH2, - synthetic derivatives of somatostatin), some analogs were synthesized and tested for in vitro cytotoxic and antioxidant activity. OBJECTIVES: The new analogs were modifyed at position 5 with Dap (diaminopropanoic acid), Dab (diaminobutanoic acid) and Orn and at position 6 with the unnatural amino acids Tle (t-leucine). METHODS: The in vitro cytotoxic effects of the substances were investigated against a panel of human tumor cell lines HT-29 (Human Colorectal Cancer Cell Line), MDA-MB-23 (Human Breast Cancer Cell Line), Hep G-2 (Human Hepatocellular Carcinoma Cell Line) and HeLa (cervical cancer cell line). The antioxidant capacities were tested by ORAC (Oxygen Radical Antioxidant Capacity) and HORAC (Hydroxyl Radical Averting Capacity) methods. RESULTS: All substances expressed significantly higher antioxidant capacity by comparison with galic acid and Trolox. All substances showed considerable antioxidant capacity as well. Compound 2T (D-Phe-c(Cys-Tyr-DTrp- Dap-Tle-Cys)-Thr-NH2)had the highest antioxidant effect. The compound 4T (D-Phe-c(Cys-Tyr-D-Trp- Orn-Tle-Cys)-Thr-NH2) displayed antiproliferative effect on HeLa cells with IC50 30 µM. The peptide analog 3T (D-Phe-c(Cys-Tyr-D-Trp-Lys-Tle-Cys)-Thr-NH2) exerted the most pronounced inhibition on the cell vitality up to 53%, 56% and 65% resp. against MDA-MB-23, Hep G-2, HeLa in the higher tested concentration. CONCLUSION: The somatostatin analogs showed moderate influence on the vitality of different tumor cells and could be used in changing their pathology.

Synthesis, anticancer activity and photostability of novel 3-ethyl-2-mercapto-thieno[2,3-d]pyrimidin-4(3H)-ones.

Some derivatives of 3-ethyl-2-mercapto-thieno[2,3-d]pyrimidin-4(3H)-ones were synthesized using ethyl 2-aminothiophene-3-carboxylates as precursors in order to estimate their cytotoxicity, respectively proliferative activity. Thienopyrimidinones containing thiosemicarbazide as well as 1,3,4-thiadiazole moieties were evaluated for their cytotoxical effect on four cancer cell lines: HT-29, breast cancer cells MDA-MB-231, HeLa, HepG2 as well as human diploid cell line Lep-3. Compounds 5b, 6a and 6b revealed cytotoxicity to the four studied cancer cell lines. The highst cytotoxicity against MDA-MB-31 exhibited the thiosemicarbazide 5b with IC50 2.31.10(-4) µM, but most active towards HT-29 cell lines was thienopyrimidine 6c with IC50 0.001 µM. Compound 6a showed the highest inhibitory activity with IC50 - 0.99 µM to human liver carcinoma HepG2 cells and low cytotoxicity towards Lep3 (IC50 = 191 µM). The thienopyrimidine derivative linked to thiadiazole 6b was toxic to the four studied cancer cell lines, especially to HeLa (IC50-0.83 µM), and besides that the compound demonstrated toxicity to Lep 3 cells at very high concentration 89 × 10(3) µM. The solid-state photostability of the derivatives 5a-c and 6a-c was tested by irradiation with UV light. All of the studied compounds show solid-state photostability in 240 min of irradiation. Using MOE software molecular docking of the three ligands 5b, 6b and 7 was accomplished into an internal pocket formed by the activation segment and the P-loop of (V599E)B-Raf. It was established that the binding of the ligands to (V599E)B-Raf promotes an inactive conformation of the enzyme.

Synthesis and in vitro antitumor activity of new octapeptide analogs of somatostatin containing unnatural amino acids.

Some modified octapeptide analogs of somatostatin with the following structure D-Phe-c(Cys-Phe-D-Trp-Xxx-Yyy-Cys)-Thr-NH2, where Xxx is Lys or Orn and Yyy is Aib (α-aminoisobutyric acid), Ac5c (1-aminocyclopentanecarboxylic acid) or Ac6c (1-aminocyclohexane carboxylic acid) have been synthesized. The peptides were prepared by standard Fmoc-solid phase peptide chemistry method. The direct disulphide bond formation has been employed on the solid phase by Tl(CF3CO2)3. The cytotoxic effects of the compounds were tested in vitro against a panel of tumor cell lines: HT-29 (human colorectal cancer cell line), MDA-MB-23 (human breast cancer cell line), Hep-G2 (human hepatocellular carcinoma cell line), HeLa (cervical cancer cell line) and normal human diploid cell line Lep-3. The new peptides exhibited different concentration-dependent antiproliferative effect against the tumor cell lines after 24 h treatment. The compounds were most effective to the HT-29 tumor cells. The compound 4C (Orn(5), Aib(6)) demonstrated the most pronounced antiproliferative effects on HT-29 cells with the IC50 = 0.0199 µM.

Synthesis and antiproliferative activity of some new thieno[2,3-d]pyrimidin-4(3H)-ones containing 1,2,4-triazole and 1,3,4-thiadiazole moiety.

Some new thieno[2,3-d]pyrimidin-4(3H)-ones containing 1,2,4-triazole and 1,3,4-thiadiazole moiety were synthesized using thieno[2,3-d]pyrimidin-3(4H)-yl)acetohydrazides as precursors in order to determine their cytotoxicity. Compounds 5, 7-8 and 10-18 were evaluated for their cytotoxical effect on four cancer cell lines: human colorectal cancer cell line HT-29, breast cancer cells MDA-MB-231, cervical cancer cells HeLa, human liver carcinoma HepG2 and human normal diploid cell line Lep3. Exclusively high cytotoxic activity of compounds 8, 16 and 17 against MDA-MB-231 cells was ascertained and the calculated IC50 values were 3.91·10(-2), 1.2·10(-3) and 3.74·10(-2) µM respectively. Thienopyrimidinones 10, 15 and 17 exhibited high cytotoxicity against HT-29 cell and the IC50 values were in the range 1.56·10(-3) µM- 0.13 µM. To HeLa cell lines cytotoxicity demonstrated compounds 8, 10, 11, 13 and 15-18 but the substance 13 was the most toxic with IC50 - 9.5·10(-4) µM. Distinctly high antiproliferative activity of derivatives 10, 14-15 and 17-18 was estimated against Hep G2, compound 15 showed IC50 - 0.21 µM. Proliferative effects to Lep 3 demonstrated compounds 5, 7-8, 11-14, 16, 18 whose EC50 values were from 0.12 to 2.21 µM. The biological data highlighted that the nature and the position of the substituents influence both the cytotoxicity to the cancer cells and the proliferation properties to Lep3 of the tested compounds.

Design, synthesis and antiproliferative properties of some new 5-substituted-2-iminobenzimidazole derivatives.

Some new 1,3,5-substituted-2,3-dihydro-2-imino-benzimidazoles were synthesized under solid-liquid phase transfer catalysis conditions using 5-substituted-2-aminobenzimidazoles as precursors in order to assess their cytotoxicity respectively proliferative activity. The structures of the compounds were confirmed by IR, (1)H NMR, (13)C NMR and elemental analysis. Compounds 9-10, 12 and 16-17 were evaluated for their cytotoxical effect on four cancer cell lines: HT-29, breast cancer cells MDA-MB-231, HeLa, HepG2 and as well as human diploid cell line Lep-3. Significant cytotoxicity of hydrazone 16 against MDA-MB-231 was established by biologically study, the IC50 was 6.2 nM while the EC50 value to Lep 3 is 0.21 nM. Relative high antiproliferative effects of the acetate 12 and compound 16 against HT-29 were ascertained and the calculated IC50 values were IC50 - 0.85 nM and IC50 - 2.83 nM respectively. Cytotoxic activity against HeLa and HepG2 cells was demonstrated by hydrazone 17, IC50 was 7.2 nM and 117 nM respectively. All tested compounds revealed proliferative activities to human diploid cell line Lep-3. The EC50 values were in the range from 0.05 to 16.91 nM. The obtained results prove the selective cytotoxicity of the tested compounds and are promising for further evaluation of the investigated compounds in vivo experiments using experimentally induced tumors in laboratory animals.

Syntheses, DNA binding and anticancer profiles of L-glutamic acid ligand and its copper(II) and ruthenium(III) complexes.

A new multidentate ligand (L) has been synthesized by the controlled condensation of L-glutamic acid with formaldehyde and ethylenediamine. Cu(II) and Ru(III) metal ion complexes of the synthesized ligand have also been prepared. The ligand and the metal complexes were purified by chromatography and characterized by spectroscopy and other techniques. Molar conductance measurements suggested ionic nature of the complexes. The ligand and the complexes are soluble in water with quite good stabilities; essential requirements for effective anticancer drugs. DNA binding constants (Kbs) for copper and ruthenium complexes were 1.8 x 103 and 2.6 x 103 M-1 while their Ksv values were 7.9 x 103, and 7.3 x 103; revealing strong binding of these complexes with DNA. Hemolytic assays of the reported compounds indicated their significantly less toxicity to RBCs than the standard anticancer drug letrazole. Anticancer profiles of all the compounds were determined on HepG2, HT-29, MDA-MB-231 and HeLa human cancer cell lines. All the compounds have quite good activities on HeLa cell lines but the best results were of CuL on HepG2, HT-29 and MDA-MB-231 cell lines.

Cytotoxic activity of new racemic and optically active N-phosphonoalkyl bicyclic ß-amino acids against human malignant cell lines.

The cytotoxic effects of novel racemic and optically active constrained N-phosphonoalkyl bicyclic ß-amino acids were tested against a panel of human tumor cell lines. All of the compounds investigated exhibited different concentration-dependent antiproliferative effects against the HT-29, MDA-MB-231, HepG2 and HeLa cell lines after 24 h treatment. The most sensitive cells were the HeLa cells at various concentrations of the four compounds tested. The aminophosphonate 3 exerted the most pronounced antiproliferative effect against the HeLa cells (inhibition of the cell vitality up to 70% at 0.5 mg/ml) and was not toxic to the normal Lep3 cells at lower concentration. Furthermore, the N-phosphonophenyl derivatives 1 and 2 displayed antiproliferative effect against mainly the MDA-MB-231 tumour cells at higher concentration.

Synthesis, characterization and cytotoxicity of some novel 1,3-disubstituted-2,3-dihydro-2-iminobenzimidazoles.

Some new 1,3-disubstituted-2,3-dihydro-2-iminobenzimidazoles were synthesized using 1-(un)substituted-2-aminobenzimidazoles as precursors in order to determine their cytotoxicity. The structures of the compounds were confirmed by IR, (1)H NMR, (13)C NMR and elemental analysis. Compounds 4, 7-11 and 13-14 were evaluated for their cytotoxical effect on two cancer cell lines: human colorectal cancer cell line HT-29, breast cancer cells MDA-MB-231 and as well as normal spleen cells. The distinctly marked antiproliferative activity of 1,3-bis(3-phenylpropyl-1)-1,3-dihydro-2H-benzimidazol-2-imine hydro bromide 7, N-(aminopropyl)-2-(3-{2-[(aminopropyl)-amino]-2-oxoethyl}-2-imino-2,3-dihydro-1H-benzimidazol-1-yl)acetamide 9 and 1,3-bis[2-(4-methylpiperazin-1-yl)-2-oxoethyl]-2,3-dihydro-2H-benzimidazol-2-imine 11 against human colorectal cancer cell line HT-29 was ascertained and the calculated IC(50) were 9.26, 0.56 and 0.013 nM respectively. Compounds 4, 9, 10 and 13 exhibited relative high cytotoxic activity against MDA-MB-231 cells. The calculated IC(50) values were in the range 0.123-1.65 nM. All tested compounds excluding compound 1,3-bis[2-(4-methylpiperazin-1-yl)-2-oxoethyl]-2,3-dihydro-2H-benzimidazol-2-imine (11) revealed proliferative activities to normal spleen cells. The computed EC(50) values varied from 0.05 to 16.91 nM.

Self-assembly of novel molecular complexes of 1,10-phenanthroline and 5-amino-1,10-phenanthroline and evaluation of their in vitro antitumour activity.

Novel molecular complexes of 1,10-phenanthroline (phen) and 5-amino-1,10-phenanthroline (5-NH2-phen) [(5-NH2-phen)2(phen) (H2O)3 (1), (phen)2(imidazole) (H+) (BF4-) (2), (phen)2(benzimidazole) (H+) (BF4-) (3), (5-NH2-phen)4(H2O)3 (4), and (phen)3 (indole) (H+) (BF4-) (5)] were synthesized via self-assembly processes and their in vitro anticancer activity was investigated. The structures of the compounds were confirmed by UV, FTIR, CIMS(CH4) and elemental analysis. The crystal structure of 2 was determined by X-ray diffraction. Cytotoxicity of the substances was measured using the cultivated human tumour cell lines HepG2, HEp-2, and 8-MB-GA. The tested substances showed different activity depending on the cell line and amount used. Substances 2 and 3 were not toxic to the non-tumour cells (Lep-3), but significantly toxic to all tumour ones. This is not the case with compounds 4 and 5, which are non-toxic towards carcinogenic cell lines, but even stimulate both HepG2 and HEp-2.

Current major cancer targets for nanoparticle systems.

This review presents some common features of nanoparticles - activity, toxicity and biological activity. Humans are exposed to tiny particles via dust storms, volcanic ash, and other natural processes and the body systems are well adapted to protect from these potentially harmful intruders. Technological advancement has also changed the character of particulate pollution, increasing the proportion of nanometer-sized particles - "nanoparticles" and expanding the variety of chemical compositions. Studies have shown a strong correlation between particulate air pollution levels, respiratory and cardiovascular diseases, various cancers, and mortality. Adverse effects of nanoparticles on human health depend on individual factors such as genetics and existing disease, as well as exposure, and nanoparticle chemistry, size, shape, agglomeration state, and electromagnetic properties. The key to understand the toxicity of nanoparticles is their size, smaller than cells and cellular organelles, which allows them to penetrate these basic biological structures, disrupting their normal function. Examples of toxic effects include tissue inflammation, and altered cellular redox balance toward oxidation, causing abnormal function or cell death. Some of these materials have desirable characteristics for industrial applications, as nanostructured materials often exhibit beneficial properties, from UV absorbance in sunscreen to oil-less lubrication of motors. In the sense of the huge surrounding positive and negative influence of known and unknown NP-impacts it seems very important to understand and forecast the processes in the body, due to the interaction between these two sides - organism. How nanoparticles can be used as drug delivery systems and imaging devices to increase the efficacy per dose of therapeutic or imaging contrast agents; how nanoparticles will be further developed to improve their functionality in cancer treatment and imaging? How reacts the immune system of the organism after introducing nanoparticles with the aim to defeat tumors? Here the aim was to discuss the right and wrong applications of NP and to answer to some of these questions. In the mean time there will appear much more investigations because of the important application of the NP not only as drug delivery systems, but as diagnostics as well.

Cytotoxicity and effects of 1,10-phenanthroline and 5-amino-1,10-phenanthroline derivatives on some immunocompetent cells.

The biological activity of previously synthesized compounds [(phen)(3)(H(+))(2)(NO(3)(-))(2) (1), Pd(5-NH(2)-phen)(2)(NO(3))(2) (2) and Pd(phen)(2)(NO(3))(2)(H(2)O) (3)] was investigated in vivo. The three compounds did not show any histological alterations in the observed lung, liver, spleen and lymph nodes of White Wistar rats. The propidium iodine staining did not discover any cytotoxic effect of the tested derivatives. The tests for immunological response predominantly showed stimulation of the antibody-producing B-cells and lower or no stimulation of the T-cells. The LIF-test showed better stimulation of all lymphocytes with 1, followed by 2 and 3. Substance 3 showed highest stimulating effect on B-cell blood lymphocytes in all doses (maximum in the lowest dose), whereas the impact of 2 is weaker and that of 1 is the weakest. The T-cell immune response after treatment with substance 1 is best influenced by dose of 1mg in the spleen cell-fraction, followed by 3 (5 mg).

Antitumour activity of novel 1,10-phenanthroline and 5-amino-1,10-phenanthroline derivatives.

Synthesis and impact on the tumour growth of palladium(II) complex of 5-amino-1,10-phenanthroline Pd(5-NH(2)-phen)(2)(NO(3))(2) and the protonated dimer (phen)(2)(H(+))(BF(4)(-)) have been described. In the reported experiments a cancerous (100% lethality) myeloid subcutaneous tumour (with Graffi-tumour origin) in hamsters was used. The animals were injected i.p. with different doses of the substances. The longest mean survival time (1.65 times longer than the controls) was achieved when the substance Pd(5-NH(2)-phen)(2)(NO(3))(2) was injected into the animals. One of the animals even survived until the 71st day, which is 2.2 times longer than the controls. The compound (phen)(2)(H(+))(BF(4)(-)) prolonged the mean life-time of the animals 1.4 times in comparison to the controls. On the other hand, the Pd(II) complex of 1,10-phenanthroline, Pd(phen)(2)(H(2)O)(NO(3))(2), did not reveal any antitumour activity. Our experience concerning the effect of other drugs on this tumour has shown a survival time no longer than 4-5d after the death of the controls.

Synthesis, cytotoxicity and effects of some 1,2,4-triazole and 1,3,4-thiadiazole derivatives on immunocompetent cells.

Novel derivatives of 4,5-substituted-1,2,4-triazole-thiones and 2,5-substituted-1,3,4-thiadiazoles were synthesized and evaluated for their cytotoxicity. The biological study indicated that compounds 4-ethyl-5-(4,5,6,7-tetrahydro-1-benzothien-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 13, N-ethyl-5-(4,5,6,7-tetrahydro-1-benzothien-2-yl)-1,3,4-thiadiazol-2-amine 16, 4-amino-5-(4,5,6,7-tetrahydro-1-benzothien-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 20 and 4-amino-5-(5-phenylthien-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 21 possessed high cytotoxicity in vitro against thymocytes. The corresponding IC(50) values were 0.46 microM, 5.2 x 10(-6)microM, 0.012 microM and 1.0 x 10(-6)microM. Most toxic against lymphocytes was compound 21, IC(50) - 0.012 microM. The tested compounds showed a general stimulation effect on B-cells' response.