br Results and discussion br Conclusion A
Results and discussion
Conclusion A new series of 2-mercapto-quinazolin-4-one analogues is designed and synthesized to possess electron withdrawing or donating functional groups (Cl or CH3O) at position 6- or 7-, 4-methoxyphenyl function at position 3. The 2-mercapto function is used to connect to either 1,2,4-triazole, or 1,3,4-thiadiazole via a methylene bridge. The obtained compounds were evaluated as inhibitors of bovine liver DHFR. Compound 17 proved to be the most active inhibitor in this study with IC50 of 0.01μM (eight fold more active than MTX). As an application of the use of DHFR inhibitors, the synthesized compounds were tested for their in vitro anticancer and antimicrobial activities. The docking study indicated that compound 17 the most active DHFR inhibitor in this study bind into active binding region and interact with the hinge domain through the amino induced pluripotent stem cells residues Ala9 and Phe34 via hydrogen bond and arene-arene interactions. Due to the lengthened structure of this inhibitor, it targets the hydrophobic back pocket. Moreover, due to its bulkiness, this compound may fit into the hydrophobic pocket, causing favorable contacts in DHFR enzyme active site. Such hypothesis has been proposed as explanation of the unique activity of 17 in particular among the synthesized compounds. Therefore, the obtained mark points could be used as template model for further development and future optimization of new DHFR inhibitors.
Experimental The compounds were synthesized at Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences & Pharmaceutical Industries, Future University, Cairo. Melting points (°C) were determined on Mettler FP80 melting point apparatus and are uncorrected. Microanalyses were performed on a Perkin-Elmer 240 elemental analyzer. All of the new compounds were analyzed for C, H and N and agreed with the proposed structures within ±0.4% of the theoretical values. 1H, 13C NMR spectra were recorded on a on Bruker 400MHz FT spectrometer (NMR facility, Faculty of Pharmacy, Cairo University); chemical shifts are expressed in δ ppm with reference to TMS. Mass spectral (MS) data were obtained on a Perkin Elmer, Clarus 600 GC/MS and Joel JMS-AX 500 mass spectrometers. Thin layer chromatography was performed on pre-coated (0.25mm) silica gel GF254 plates (E. Merck, Germany), compounds were detected with 254nm UV lamp. Silica gel (60–230mesh) was employed for routine column chromatography separations. DHFR inhibition activity experiments were performed at Pharmacology Department, Faculty of Pharmaceutical Sciences & Pharmaceutical Industries, Future University, Cairo. Bovine liver DHFR enzyme, methotrexate (MTX) was used in the assay (Sigma Chemical Co, USA). Anticancer activity screening was performed utilizing HepG2 liver, MCF-7 breast, A549 lung and Caco2 colon human tumor cell lines obtained from the American Type Culture Collection (Rockville, MD, USA). Antimicrobial screening was performed at Microbiology Department, Faculty of Pharmaceutical Sciences & Pharmaceutical Industries, Future University, Cairo, using two Gram-positive bacteria obtained from ATCC (American Type Culture Collection) S. aureus ATCC 25923, B. subtilis ATCC 19659 and two Gram-negative bacteria: (E. coli ATCC 25922 P. aeruginosa ATCC 27853). Tested fungi were one yeast-like fungus (Candida albicans ATCC 10231) and one molds (Aspergillus flavus ATCC 10124).
Acknowledgements Thanks are due to Science and Technology Development Fund – Egypt, for the Research Support Technology Development Grant (RSTDG) no. 12626.
Sulphadoxine-pyrimethamine (SP) is an anti-malarial combination drug that was once commonly used, but that has been abandoned as first-line treatment due to increasing resistance in malaria parasites. At present, SP is used for intermittent preventive treatment with SP during pregnancy (IPTp-SP) in most Sub-Saharan African countries. IPTp-SP helps in preventing malaria related morbidity during pregnancy, like maternal anemia and low birth weight of the offspring . However, because of a continuous increase in SP resistant , there are now also concerns about the efficacy of IPTp-SP. Mutations in two genes, dihydropterate synthase ( and dihydrofolate reductase (, encoding the target proteins of sulphadoxine and pyrimethamine respectively, are associated with resistance against SP. Each of these (S436, A436, K540, A581, K613) and (N51, C59, S108, I164) mutations contribute to the level of clinical resistance and especially the double (A437, K540) with triple (N51, C59, S108) combination (quintuple mutant) and the more recently upcoming sextuple mutant (additional A581 mutation) are associated with SP resistance , . In East Africa, prevalence of the quintuple mutation is high and has reached near 100% saturation in some areas, not surprisingly there are reports of IPTp-SP having lost effectiveness or even having detrimental effects in these countries , . In contrast, in West Africa S436 and A437 and the triple mutations are fairly common, but the K540 and consequently the quintuple mutation is hardly described . Efficacy of IPTp-SP is therefore still sufficient, but surveillance of possible emergent SP resistance mutations is extremely important . In an intervention trial situated around Nanoro, Burkina Faso, pregnant women were followed up from first antenatal care visit (ANC) to delivery as described by Scott Informed consent was obtained for all participants. All women received standard care at the ANC, including IPTp-SP, and additionally women from intervention villages received monthly home screening for malaria with rapid diagnostic tests. Women with positive RDTs were treated with artemether-lumefantrine. At each ANC visit, blood spots on filter papers were collected. These filter papers were used for DNA extraction. eal-time PCR positive samples from first ANC (random selection) and from delivery collected from May 2014 till September 2015 were included for mutation analyses. An additional sample set was collected from the general population (GP). For this sample set a cross-sectional survey was done in April and May 2015 in the same area as the longitudinal study in pregnant women. From randomly selected households two members (non-pregnant and aged≥6months old) were enrolled after informed consent was obtained. For underaged children informed consent was given by parents or guardians. Blood spots on filter papers were collected and processed similarly to the longitudinal study. A random selection of real-time PCR positive samples were included in mutation analyses performed in the Netherlands. One blood spot was punched out from each filter paper using skin biopsy punchers (acuderm inc, USA). DNA was extracted by first lysing the filter papers in polypropylene tubes on a roller bank for 30min using EasyMAG lysis buffer (bioMérieux). Subsequently the lysed fluid was transferred to EasyMAG vessels and incubated with magnetic bead silica (bioMérieux) according to manufacturer’s instructions. The DBS 1.0 protocol was used to extract the DNA in the NucliSENS EasyMAG. Extraction controls were included (positive filter papers of blood spots spiked with diluted FCR3 or 3D7 culture, and negative filter papers with blood spots of uninfected erythrocytes).