There was a good alignment profile between compound

There was a good alignment profile between compound 26 (IC50 0.06μM) and MTX (IC50 0.08μM) explaining its activity pattern (Fig. 5a). Fig. 5b clearly indicates a different alignment profiles where carbonyl groups are not on the same side between 25 (IC50>100.0μM) and MTX which is in consistency with the obtained experimental data. The surface map for the DHFR binding pocket was calculated (Fig. 6). MTX showed to occupy the whole space lying into the groove pocket (Fig. 6a). Compound 26 active site alignment showed a pattern which resemble that of MTX, filling all the area of the active site with its bulkiness and fit into the hydrophobic pocket, forming favorable binding contact (Fig. 6b). Results for 26 showed larger blue hydrophobic areas which are responsible for the interaction with amino Phleomycin residues inside the enzyme active pocket. On the other hand, compound 25 structure was pointed out toward the surface wall of the active site and deprived of any receptor exposure clashes explaining its poor DHFR inhibitory activity (Fig. 6c). The hydrophobic distributions of the least active compound 25, showed less lipophilic moieties and hence the required lipophilicity for effective binding to DHFR is absent.
Conclusion A new series of 2,4-substituted-1,3-thiazoles and thiazolo[4,5-d]pyridazine both bearing the 2-thioureido function with anticipated antitumor and DHFR inhibitory activity were synthesized. A new method to prepare the thiazolo[4,5-d]pyridazine heterocycle was adopted. Compound 26 proved to be the most active DHFR inhibitor with IC50 of 0.06μM (merely comparable to MTX, IC50 0.08μM); while compounds 22 and 23 were active with IC50 of 0.1 and 2.5μM, respectively. Compound 4 showed antitumor activity against NCI-H522 non-small cell lung, HT29 colon, and T-47D breast cancers with GI values of 40.4, 30.7 and 27.7%, respectively; while compound 20 showed 31.4, 25.2, 37.7, 25.1, 41.0GI% against NCI-H522 non-small cell lung, HT29 colon, SK-OV-3 ovarian, MCF7 breast and T-47D breast cancers respectively. In addition compound 21 showed antitumor activity against NCI-H522 non-small cell lung, HT29 colon and TK-10 renal with GI values of 31.7, 29.4, and 34.7%, respectively. Compound 26 proved lethal to HS 578T breast cancer cell line with IC50 value of 0.8μM, inducing cell cycle arrest and apoptosis through increasing the percentage of cells in Pre-G1 and enhancing G2/M phase. Structure activity correlations of the investigated compounds revealed that the 3-[4-chlorophenyl)thioureido]- series is more active than the 3-[(4-methoxyphenyl)-thioureido]- counterparts; also the type of substituent at positions 7- of the thiazolo[4,5-d]pyridazine affected the DHFR inhibition potency. The order of activity in the 3-[(4-chloro-phenyl)thioureido]- series, was 4-BrPh>4-CH3OPh>pH>4-CH3Ph; while in the 3-[(4-methoxy-phenyl)-thioureido]-series was 4-CH3OPh>pH>4-CH3Ph=4-BrPh. Meanwhile, compound 26 showed high affinity binding energy value of 46.65Kcal/mol toward Phe 31 residue which is linked to the thiazolo[4,5-d]pyridazine ring while Arg 22 residue is linked to 7-Phenyl moiety in addition to a network of π-π interaction and hydrogen bonding. The obtained data could be used as template for further development of new DHFR inhibitors.
Experimental Melting points (°C) were determined by open capillary tube method using (Bio Cote SMP 10) apparatus and they are uncorrected. Microanalyses were performed using FLASH 2000 CHNS/O analyzer, Thermo Scientific at the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Nasr City, Cairo. 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 Bruker Avance III HD FT‐high resolution‐ NMR 400MHz Center for Drug Discovery Research and Development Faculty of Pharmacy‐Ain Shams University, chemical shifts are expressed in δ ppm with reference to TMS. Mass spectrum was carried out on Direct Inlet part to mass analyzer in Thermo Scientific GCMS model ISQ at the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Nasr City, Cairo. Thin layer chromatography was performed on precoated (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 and Biochemistry Department, Faculty of Pharmacy; Future University in Egypt. Bovine liver DHFR enzyme and methotrexate (MTX) were used in the assay (Sigma Chemical Co, USA). Elisa reader SN208125 Bio Tek MQX200 and Software program GEN5 at wave length 340nm were used to measure the changes in absorbance. In vitro antitumor testing was conducted at the NCI’s disease-oriented human cell lines assay facility, Bethesda, MD, USA. Cell Cycle analysis of the effect of compound 26 on HS 578T breast cancer cell line was performed at the confirmatory diagnostic unit, VACSERA, Egypt. Molecular modeling study was conducted using Discovery Studio Client version 2.5 software. Enzyme structure, starting coordinate of hDHFR enzyme in tertiary complex with reduced-nicotinamide adenine dinucleotide phosphate (NADPH) and MTX, code ID 1U70, was obtained from the Protein Data Bank of Brookhaven National Laboratory [45].