Synthesis of new imine-/amine-bearing imidazo[1,2-a]pyrimidine derivatives and screening of their cytotoxic activity.

Imidazo[1,2-a]pyrimidine derivatives bearing imine groups (3a-e) were successfully synthesized in moderate to good yields using microwave-assisted heating. Corresponding amine derivatives (4a-e) were also obtained by the reduction reaction of the imine derivatives (3a-e). All synthesized products were characterized by FT-IR, 1H NMR, 13C NMR, and LC-MS spectroscopic techniques. In silico ADMET, Lipinski, and drug-likeness studies of the compounds were conducted and all were found to be suitable drug candidates. The cytotoxicity of the potential drug molecules was screened against the breast cancer cell lines MCF-7 and MDA-MB-231 and the healthy model HUVEC by the sulforhodamine B method. According to the antiproliferative studies, compounds 3d and 4d showed remarkable inhibition of MCF-7 cells with IC50 values of 43.4 and 39.0 µM and of MDA-MB-231 cells with IC50 values of 35.9 and 35.1 µM, respectively. In particular, compound 3d selectively inhibited the proliferation of MCF-7 1.6-fold and MDA-MB-231 2.0-fold relative to healthy cells. Moreover, the apoptotic mechanism studies indicated that compound 4d induced apoptosis by moderately increasing the ratio of Bax/Bcl-2 genes. Imidazo[1,2-a]pyrimidine derivative 3d, a promising cytotoxic agent, may be helpful in the discovery of new and more efficient anticancer agents for breast cancer treatment.

Analysis of two non-pharmacological pain management methods for vaccine injection pain in infants: A randomized controlled trial.

OBJECTIVES: This study was performed to investigate the efficiency of local heat and cold application to decrease vaccine-associated pain among infants 2-6 months of age. METHODS: This was a randomized controlled trial. The study universe comprised infants aged 2-6 months who were brought to 4 family health centers in the Safranbolu district of Karabuk Province, Turkey, for a pneumococcal vaccination June 1-November 30, 2016. A total of 96 infants (heat application: 31, cold application: 32, and control group: 33) were enrolled in the the study. The data were collected using an infant information form and the Face, Legs, Activity, Cry, Consolability (FLACC) pain scale. RESULTS: The mean FLACC score of the infants was 5.531±1.934 in the cold application group, 8.710±1.346 in the heat application group, and 9.152±1.661 in the control group. The difference between the mean scores of the groups was statistically significant (KW=49.043; p=0.000). CONCLUSION: Local cold and heat application methods applied to the vaccination area before a pneumoccal vaccine reduced vaccine-associated pain in the infants, and the application of cold was more effective than heat.

New nimesulide derivatives with amide/sulfonamide moieties: Selective COX-2 inhibition and antitumor effects.

Seventeen new amide/sulfonamide containing nimesulide derivatives were synthesized and characterized by several spectroscopic techniques and primarily investigated for their inhibitory potential on COX enzymes and other pro-inflammatory factors. Experimental analyses showed that among seventeen compounds, N8 and N10 have remarkable potency and selectivity for the COX-2 enzyme over COX-1 at very low doses as compared to nimesulide. Moreover, both N8 and N10 selectively reduced the Lipopolysaccharide (LPS)-stimulated COX-2 mRNA expression level while the COX-1 level remained stable. Both PGE2 release and nitric oxide production in macrophage cells were significantly suppressed by the N8 and N10 treatment groups. In silico ADME/Tox, molecular docking and molecular dynamics (MD) simulations were also conducted. Additionally, all compounds were also screened in a panel of cancer cell lines for their antiproliferative properties by MTT and SRB assays. Compound N17 exhibited a considerable antiproliferative effect on the colon (IC50: 9.24 µM) and breast (IC50: 11.35 µM) cancer cell lines. N17 exposure for 48 h decreased expression of anti-apoptotic protein BCL-2 and increased the expression of apoptogenic BAX. Besides, the BAX/BCL-2 ratio was increased with visible ultrastructural changes and apoptotic bodies under scanning electron microscopy. In order to investigate the structural and dynamical properties of selected hits on the target structures, multiscale molecular modeling studies are also conducted. Our combined in silico and in vitro results suggest that N8 and N10 could be further developed as potential nonsteroidal anti-inflammatory drugs (NSAIDs), while cytotoxic N17 might be studied as a potential lead compound that could be developed as an anticancer agent.

Microwave assisted, sequential two-step, one-pot synthesis of novel imidazo[1,2-a]pyrimidine containing tri/tetrasubstituted imidazole derivatives.

A series of novel imidazo[1,2- a ]pyrimidine containing tri/tetrasubstituted imidazole derivatives (1-10) has been synthesized via sequential two-step, one-pot, multicomponent reaction using imidazo[1,2- a ]pyrimidine-2-carbaldehyde, benzil, primary amines, and ammonium acetate catalyzed by p -toluenesulfonic acid under microwave-assisted conditions. The results showed that target compounds can be obtained from a wide range of primary amines bearing different functional groups with moderate to good yields (46%-80%) under optimum reaction conditions. This method provides a green protocol for imidazo[1,2- a ]pyrimidine containing tri/tetrasubstituted imidazole derivatives due to ethyl alcohol as a green solvent, microwave irradiation as a greener heating method and one-pot multicomponent reaction as a green technique. The synthesized compounds have been elucidated using various spectroscopic tools such as FT-IR, 1H NMR, 13 C NMR, and MS.

Prodrugs for nitroreductase based cancer therapy-4: Towards prostate cancer targeting: Synthesis of N-heterocyclic nitro prodrugs, Ssap-NtrB enzymatic activation and anticancer evaluation.

In this study, various N-heterocyclic nitro prodrugs (NHN1-16) containing pyrimidine, triazine and piperazine rings were designed and synthesized. The final compounds were identified using FT-IR, 1H NMR, 13C NMR as well as elemental analyses. Enzymatic activities of compounds were conducted by using HPLC analysis to investigate the interaction of substrates with Ssap-NtrB nitroreductase enzyme. MTT assay was performed to evaluate the toxic effect of compounds against Hep3B and PC3 cancer cell lines and healthy HUVEC cell. It was observed that synthesized compounds NHN1-16 exhibited different cytotoxic profiles. Pyrimidine derivative NHN3 and triazine derivative NHN5 can be good drug candidates for prostate cancer with IC50 values of 54.75 µM and 48.9 µM, respectively. Compounds NHN6, NHN10, NHN12, NHN14 and NHN16 were selected as prodrug candidates because of non-toxic properties against three different cell models. The NHN prodrugs and Ssap-NtrB combinations were applied to SRB assay to reveal the prodrug capabilities of these selected compounds. SRB screening results showed that the metabolites of all selected non-toxic compounds showed remarkable cytotoxicity with IC50 values in the range of 1.71-4.72 nM on prostate cancer. Among the tested compounds, especially piperazine derivatives NHN12 and NHN14 showed significant toxic effect with IC50 values of 1.75 nM and 1.79 nM against PC3 cell compared with standart prodrug CB1954 (IC50: 1.71 nM). Novel compounds NHN12 and NHN14 can be considered as promising prodrug candidates for nitroreductase-prodrug based prostate cancer therapy.

Prodrugs for nitroreductase-based cancer therapy-3: Antitumor activity of the novel dinitroaniline prodrugs/Ssap-NtrB enzyme suicide gene system: Synthesis, in vitro and in silico evaluation in prostate cancer.

Prodrugs for targeted tumor therapies have been extensively studied in recent years due to not only maximising therapeutic effects on tumor cells but also reducing or eliminating serious side effects on healthy cells. This strategy uses prodrugs which are safe for normal cells and form toxic metabolites (drugs) after selective reduction by enzymes in tumor tissues. In this study, prodrug candidates (1-36) containing nitro were designed, synthesized and characterized within the scope of chemical experiments. Drug-likeness properties of prodrug candidates were analyzed using DS 2018 to investigate undesired toxicity effects. In vitro cytotoxic effects of prodrug canditates were performed with MTT assay for human hepatoma cells (Hep3B) and prostate cancer cells (PC3) and human umbilical vein endothelial cells (HUVEC) as healthy control. Non-toxic compounds (3, 5, 7, 10, 12, 15, 17, 19 and 21-23), and also compounds (1, 2, 5, 6, 9, 11, 14, 16, 20 and 24) which had low toxic effects, were selected to examine their suitability as prodrug canditates. The reduction profiles and kinetic studies of prodrug/Ssap-NtrB combinations were performed with biochemical analyses. Then, selected prodrug/Ssap-NtrB combinations were applied to prostate cancer cells to determine toxicity. The results of theoretical, in vitro cytotoxic and biochemical studies suggest 14/Ssap-NtrB, 22/Ssap-NtrB and 24/Ssap-NtrB may be potential prodrug/enzyme combinations for nitroreductase (Ntr)-based prostate cancer therapy.

PRODRUGS FOR NITROREDUCTASE BASED CANCER THERAPY- 2: Novel amide/Ntr combinations targeting PC3 cancer cells.

The use of nitroreductases (NTR) that catalyze the reduction of nitro compounds by using NAD(P)H in GDEPT (Gene-directed enzyme prodrug therapy) studies which minimize toxicity at healthy cells and increases concentration of drugs at cancer cells is remarkable. Discovery of new prodrug/NTR combinations is necessary to be an alternative to known prodrug candidates such as CB1954, SN23862, PR-104A. For this aim, nitro containing aromatic amides (A1-A23)2 were designed, synthesized, performed in silico ADMET and molecular docking techniques in this study. Prodrug candidates were studied on reduction potentials with Ssap-NtrB by HPLC system. Also, cyototoxic properties and prodrug ability of these amides were investigated using different cancer cell lines such as Hep3B and PC3. As a result of theoretical and biological studies, combinations of A5, A6 and A20 with Ssap-NtrB can be suggested as potential prodrugs/enzyme combinations at NTR based cancer therapy compared with CB1954/NfsB.

Prodrugs for Nitroreductase Based Cancer Therapy- 1: Metabolite Profile, Cell Cytotoxicity and Molecular Modeling Interactions of Nitro Benzamides with Ssap-NtrB.

BACKGROUND: Directed Enzyme Prodrugs Therapy (DEPT) as an alternative method against conventional cancer treatments, in which the non-toxic prodrugs is converted to highly cytotoxic derivative, has attracted an ample attention in recent years for cancer therapy studies. OBJECTIVE: The metabolite profile, cell cytotoxicity and molecular modeling interactions of a series of nitro benzamides with Ssap-NtrB were investigated in this study. METHOD: A series of nitro-substituted benzamide prodrugs (1-4) were synthesized and firstly investigated their enzymatic reduction by Ssap-NtrB (S. saprophyticus Nitroreductase B) using HPLC analysis. Resulting metabolites were analyzed by LC-MS/MS. Molecular docking studies were performed with the aim of investigating the relationship between nitro benzamide structures (prodrugs 1-4) and Ssap-NtrB at the molecular level. Cell viability assay was conducted on two cancer cell lines, hepatoma (Hep3B) and colon (HT-29) cancer models and healthy cell model HUVEC. Upon reduction of benzamide prodrugs by Ssap-NtrB, the corresponding amine effectors were tested in a cell line panel comprising PC-3, Hep3B and HUVEC cells and were compared with the established NTR substrates, CB1954 (an aziridinyl dinitrobenzamide). RESULTS: Cell viability assay resulted in while prodrugs 1, 2 and 3 had no remarkable cytotoxic effects, prodrug 4 showed the differential effect, showing moderate cytotoxicity with Hep3B and HUVEC. The metabolites that obtained from the reduction of nitro benzamide prodrugs (1-4) by Ssap-NtrB, showed differential cytotoxic effects, with none toxic for HUVEC cells, moderate toxic for Hep3B cells, but highly toxic for PC3 cells. CONCLUSION: Amongst all metabolites of prodrugs after Ssap-NtrB reduction, N-(2,4- dinitrophenyl)-4-nitrobenzamide (3) was efficient and toxic in PC3 cells as comparable as CB1954. Kinetic parameters, molecular docking and HPLC results also confirm that prodrug 3 is better for Ssap-NtrB than 1, 2 and 4 or known cancer prodrugs of CB1954 and SN23862, demonstrating that prodrug 3 is an efficient candidate for NTR based cancer therapy.

Biological evaluation and molecular docking studies of nitro benzamide derivatives with respect to in vitro anti-inflammatory activity.

A series of nitro substituted benzamide derivatives were synthesized and evaluated for their potential anti-inflammatory activities in vitro. Firstly, all compounds (1-6) were screened for their inhibitory capacity on LPS induced nitric oxide (NO) production in RAW264.7 macrophages. Compounds 5 and 6 demonstrated significantly high inhibition capacities in a dose-dependent manner with IC50 values of 3.7 and 5.3µM, respectively. These two compounds were also accompanied by no cytotoxicity at the studied concentrations (max 50µM) in macrophages. Molecular docking analysis on iNOS revealed that compounds 5 and 6 bind to the enzyme more efficiently compared to other compounds due to having optimum number of nitro groups, orientations and polarizabilities. In addition, 5 and 6 demonstrated distinct regulatory mechanisms for the expression of the iNOS enzyme at the mRNA and protein levels. Specifically, both suppressed expressions of COX-2, IL-1ß and TNF-α significantly, at 10 and 20µM. However, only compound 6 significantly and considerably decreased LPS-induced secretion of IL-1ß and TNF-α. These results suggest that compound 6 may be a multi-potent promising lead compound for further optimization in structure and as well as for in vivo validation studies.

Modification of existing antibiotics in the form of precursor prodrugs that can be subsequently activated by nitroreductases of the target pathogen.

The use of existing antibiotics in the form of prodrug followed by activation using enzymes of pathogenic origin could be a useful approach for antimicrobial therapy. To investigate this idea, a common antibiotic, sulfamethoxazole has been redesigned in the form of a prodrug by simple functional group replacement. Upon reductive activation by a type I nitroreductase from a pathogen, the drug displayed enhanced antimicrobial capacity. This strategy could improve the efficacy and selectively of antibiotics and reduce the incidence of resistance.