NEW APPROACHES OF 4-ARYL-2-HYDRAZINOTHIAZOLE DERIVATIVES SYNTHESIS, MOLECULAR DOCKING, AND BIOLOGICAL EVALUATIONS
HTML Full TextNEW APPROACHES OF 4-ARYL-2-HYDRAZINOTHIAZOLE DERIVATIVES SYNTHESIS, MOLECULAR DOCKING, AND BIOLOGICAL EVALUATIONS
Magda H. Abdellattif * 1, Mostafa A. Hussien 2, 3 and Eman Alzahrani 4
Department of Pharmaceutical Chemistry, Deanship of Scientific Research 1, Department of Chemistry, Faculty of Science 4, Taif University, 21974, KSA.
Department of Chemistry 2, Faculty of Science, King Abdulaziz University, KSA.
Department of Chemistry 3, Faculty of Science, Port Said University, Egypt.
ABSTRACT: In the field of the preparation of chemotherapeutic agents with new mechanism distinct from currently approved drugs is important. However, the clinically effective approved small inhibitor molecule is still lacking. Previous reports indicated that 4-aryl-2-hydrazinothiazole derivatives provided a useful start point to develop HAT inhibitors (histone acetyltransferase). Consequently, preparation and biological evaluation of a focused library of 4-aryl-2-hydrazinothiazole based derivatives as useful as anti-cancer agents. Synthesis of 4-aryl-2-hydrazinothiazoles (1a-d), (2a-d), (3a-c), (4a-b), (5a-b), (6a-b) studied by either conventional method and free solvent microwave one-pot method, and the synthesized compounds were proved by spectroscopic methods (IR, 1HNMR, 13CNMR, GC-Ms, and elemental analysis). The activity against different cancer cell lines and the antimicrobial activities different organisms also studied, and all of the synthesized compounds were of high activity as an antibacterial for gram positive and gram negative; however, it showed also an antifungal activity were the most active compounds were 4a, 4b, 5b, 6a. It was found that all of the prepared thiazoles derivatives were active toward many cell lines (breast, liver, and prostate) where the more active compounds were 4a, 4b, and 6a. Molecular docking studies guided and proved the biological activities of these novel 4-aryl-2-hydrazinothiazoles.
Keywords: |
Antibacterial, Anticancer, Green synthesis, Microwave-assisted synthesis, Molecular modeling, 4-Aryl-2-hydrazinothiazol
INTRODUCTION: As the second leading cause of death after cardiovascular diseases, cancer is a cumbersome health problem. The ultimate cure for cancer is still lacking. Therefore the development of new anticancer agents is needed to satisfy clinical needs. This necessitates enhancing the currently available therapeutic toolbox for combating cancer.
This might be achieved by development of novel therapeutic agents based on the less exploited validated targets and thus providing novel anticancer agents with new mechanisms of actions. Apparently, this would also assist in overcoming the emerging resistance to anticancer.
Epigenetic modifications control the compactness of chromatin structure and hence accessibility of DNA genes. Accordingly, expression of genes would be affected by these epigenetic modifications which include alterations of histones via lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation and lysine ubiquitination and simulation.
The process of acetylation of histones is controlled by writers (histone acetyl transferases; HAT) and erasers (histone deacetylases; HDAC). While considerable efforts have been directed to HDAC inhibitors as anticancer, few reports are found for the development of HAT inhibitors 1. Based on homology, HATs are classified into several families. One of these families, four are the most studied and understood which are the GNAT family; the p300/CBP family; the MYST family and the Rtt109 family 2. The role of two members of the GNAT family, namely PCAF and GCN5, is well documented in cancer 3.
Also, it has been reported that GCN5 plays an important role in cell cycle progression and transcription of the gene of epidermal growth factor 4, 5. Accordingly, inhibition of GCN5 would lead to anti-proliferative effect. Furthermore, another two members of HAT, namely p300 and CBP were reported to have important roles in cells’ G1/S transition 6. Also, downregulation of p300 was found to result in anti-proliferative activity in human melanocytes 7. Consequently, development of histone acetyltransferase inhibitors could result in molecules with potential anticancer activity.
FIG. 1: 4-ARYL-2-HYDARZINOTHIAZOLE DERIVATIVES AS LEADS FOR DEVELOPING HAT INHIBITORS
HAT inhibitor commercially available research probe from Sigma Aldrich
The 4-Aryl-2-hydarzinothiazole nucleus is a promising scaffold for the development of HAT inhibitors. Recently, cyclopentylidene - [4- (4′-chlorophenyl) thiazol-2-yl) hydrazine was reported 8 as HAT inhibitor. It is commercially available from Sigma-Aldrich Company as a research probe modulating GCN5 member of HAT (CAS Number 357649-93-5). An opening of the cyclopentane ring to afford 1- (4- (4-chlorophenyl) thiazol-2-yl) -2- (propan-2-ylidene) hydrazine was reported to inhibit GCN5and p300 members of the HATs and possess anti-proliferative activity in neuroblastoma and glioblastoma cell lines 9. These reports indicate that these structures provide a useful starting point to develop anticancer agents based on HAT inhibition mechanism.
Green chemistry, sustainable synthetic efficiency is becoming important issues in contemporary synthetic practice 10. Abolishing or minimizing the burden of chemical processes on environmental, ecological systems and limited resources is crucial for the survival of forthcoming generations. Replacing organic solvents with water minimize harmful wastes of organic synthesis. In addition, reduction of the required reaction time would result in less consumption of energy. Minimizing energy input required to achieve chemical transformations is not only an economical issue, which is, of course, an important issue, but also is crucial to minimize the environmental burden of power generation plants.
Microwave assisted synthesis is a promising tool to achieve these goals of sustainable green synthesis. It enables dramatic reduction of reaction time and hence more efficient energy metrics of the chemical process. Moreover, the microwave assisted synthesis of 2-amino-4-arylthiazoles in aqueous medium has been recently developed 11. Therefore, it is appealing to develop the corresponding strategy for the green synthesis of the 4-Aryl-2-hydra-zinothiazole nucleus of the proposed research 12-15.
RESULTS AND DISCUSSION:
Chemistry: Synthesis of 4-aryl-2-substituted thiazoles derivatives:
SCHEME 1
SCHEME 2
SCHEME 3
SCHEME 4
Synthesis of 5-aryl-3-substitutedthiazolo [2, 3-c][1,2,4] triazoles derivatives:
SCHEME 5
TABLE 1: STRUCTURES AND IUPAC NAMES OF THE COMPOUNDS
Many attempts were carried out to prepare 1-(4-m-tolylthiazol-2-yl)hydrazine (B) where R = CH3 for derivatives prepared, the first attempt was carried out by the conventional method under reflux for 2hrs., by refluxing thiocarbohydrazide with, 2-bromo-1-p-tolylethanone in glacial acetic acid 16, 17, 18, 19. Where the second method was carried out using microwave free reaction for 1.5-2 min, using the two above compounds without any solvents or liquid media 20. The formed compound was confirmed by its IR, 1HNMR, 13CNMR, and elemental analysis data, the main observation was the presence two broad medium bands one at 600, and the other was at 1400 cm-1, these bands were proved before as a finger print band as evidence of the presence of thiazole ring 21. Not only the time factor or the solvent-free factor but also maximum yield product % obtained in comparison to the conventional method. The structure of the produced compounds was confirmed by IR, 1HNMR, 13CNMR, and elemental analysis, [methodology part].
The main goal of this work was reached by the preparation of different compounds of fused or open structure nitrogen system in a hope to reach different active HAT enzymes.
TABLE 2: COMPARISON YIELD / TIME REACTION USING TWO SYNTHETIC TECHNIQUES
Compound | Conventional method | Microwave method | ||
Yield % | Time (h) | Yield % | Time (min) | |
1a | 78 | 2-3 | 96 | 1.5-2 |
1b | 77 | 2-3 | 94 | 1.5-2 |
1c | 72 | 2-3 | 95 | 1.5-2 |
1d | 78 | 2-3 | 96 | 1.5-2 |
2a | 67 | 5 | 94 | 2-3 |
2b | 66 | 4-5 | 93 | 2-3 |
2c | 62 | 4-5 | 93 | 2-3 |
2d | 67 | 4-5 | 92 | 2-3 |
3a | 73 | 4-6 | 98 | 3-3.5 |
3b | 75 | 4-6 | 97 | 3-3.5 |
3c | 71 | 4-6 | 97 | 3-3.5 |
4a | 72 | 8 | 96 | 3.5 |
4b | 70 | 8 | 97 | 3.9 |
5a | 68 | 12 | 95 | 4 |
5b | 74 | 10 | 95 | 3.7 |
6a | 70 | 14 | 96 | 2.9 |
6b | 70 | 14 | 96 | 3.5 |
B** | 63 | 9 | 80 | 4.5 |
One could notice from the table above that microwave method was more efficient, although domestic microwave one pot multi-component was used, the advantages this method are less time to consume and high yield product.
Also, most of the compounds obtained were racemic as a result of using mechanical, thermal energy of domestic microwave. B** intermediate was separated and also characterized which gave indication and proof that the reactions moved in the right way either by a conventional method or by microwave irradiation method. The presence of a short-medium band between 600-700 cm-1 indicated the presence of aliphatic C-S bond of -S-CH3.
Biology: Bacteria and fungi had developed resistance strains against currently available antimicrobial agents, and therefore it is essential for medicinal chemists to design and synthesize novel antimicrobial agents having less toxicity and more potent effects in much lesser time. In continuation to this, chemists have successfully synthesized effective agents based on heterocyclic compounds. The shining examples are Furamizole, Nasapadil, Tazobactam, and Cefatrizine. Thiazoles derivatives have various types of pharmacological effects, including antimicrobial 22. Numerous examples show that thiazoles and its derivatives play an important role in development of antimicrobial agents. Thiazoles derivatives have been synthesized by the condensation of phenylenediamine with acids, their nitriles, imidates and orthoesters 23. It had previously synthesized triazines derivatives and had received some exiting antimicrobial results 24, 25. Therefore, it focused on benzimidazole-based thiazoles derivatives which possess diverse chemical structures. These hybrid structures may be useful for the development of antimicrobial agents. The development of efficient preparation of thiazoles and triazines based compounds played a key role in modern organic synthesis 26.
The minimal inhibitory concentration for some of the newly synthesized compounds showed high significant activity against g-positive and g-negative and antifungal activities. Among the screened compounds, 1b, 1c, 2a, 2b, 3a, 4a, 4a, 5a, and 6a exhibited strong antimicrobial activity 6. A series of 4-(p-Aryl)-1, 3-thiazol-2-ylhydrazines derivatives are evaluated their antimicrobial activity against g-positive (S. aureus and S. pyogenes), g-negative bacteria (E. coli and P. aeruginosa) and strains of fungi (C. albicans, A. niger, and A. clavatus), they showed significant antimicrobial activity against tested micro-organisms 27.
Antimicrobial:
a) Antibacterial: Table 3 and 1, showed in-vitro experiment for the mean of antimicrobial effects on g-positive bacteria (SA), that revealed the effect of this dissolved substances as antimicrobial (antibacterial) and were done in the hrs of exposures included (1, 3, 5 and 7h). The mean antimicrobial effects were calculated during the exposure hrs, and the averages were in descending order by the percentage of the antibacterial as illustrated in Table 3 and Fig. 1.
Bacteria had developed resistance strains against currently available antimicrobial agents, synthesize novel antimicrobial less toxicity and more potent effects in much lesser time. The shining examples are Furamizole, Nasapadil, Tazobactam, and Cefatrizine. Benzimidazoles have various types of pharmacological effects, including antimicrobial 22, 23, 24, 25, 26. The minimal inhibitory concentration for some of the newly synthesized compounds showed high significant activity against g-positive.
Among the screened compounds, the thiazoles derivatives 1b, 1c, 2a, 2b, 3a, 4a, 4a, 5a, and 6a exhibited strong antimicrobial activity 25, 26, 27.
TABLE 3: IN-VITRO EXPERIMENT FOR THE MEAN OF ANTIMICROBIAL EFFECTS ON G-POSITIVE BACTERIA
Compound | 1h | 3h | 5h | 7h | Mean |
1b | 28 | 41 | 61 | 80 | 54 |
1c | 32 | 46 | 72 | 100 | 62.50 |
2a | 34 | 56 | 84 | 100 | 68.50 |
2b | 48 | 71 | 100 | 100 | 79.60 |
3a | 31 | 59 | 78 | 100 | 67.00 |
4a | 38 | 62 | 86 | 100 | 71.50 |
4a | 42 | 73 | 100 | 100 | 78.80 |
5a | 46 | 79 | 100 | 100% | 81.5 |
6a | 57 | 83 | 98 | 100 | 84.5 |
FIG. 1: IN-VITRO EXPERIMENT FOR THE MEAN OF ANTIMICROBIAL EFFECTS ON G-POSITIVE BACTERIA
Table 4 and Fig. 2 showed in-vitro experiment for the mean of antimicrobial effects on g-negative bacteria (EC), that revealed the effect of this dissolved substances as antimicrobial (anti-bacterial) and were done in the hrs of exposures included (1, 3, 5 and 7h). The mean antibacterial effects were calculated during the exposure hrs and the averages were in descending order by the percentage of the antibacterial as shown in Table 4 and Fig. 2. The minimal inhibitory concentration for some of the newly synthesized compounds showed high significant activity against g-negative bacteria 26, 27.
TABLE 4: IN-VITRO EXPERIMENT FOR THE MEAN OF ANTIMICROBIAL EFFECTS ON G-NEGATIVE BACTERIA % (*EC)
Compound | 1h | 3h | 5h | 7h | Mean |
1b | 27 | 38 | 61 | 86 | 53 |
1c | 29 | 40 | 76 | 100 | 61.3 |
2a | 30 | 51 | 80 | 100 | 65.3 |
2b | 41 | 68 | 82 | 100 | 72.8 |
3a | 28 | 51 | 79 | 100 | 64.5 |
4a | 32 | 53 | 83 | 100 | 67.0 |
4a | 38 | 67 | 89 | 100 | 73.5 |
5a | 40 | 70 | 93 | 100 | 78 |
6a | 46 | 78 | 95 | 100 | 81 |
FIG. 2: IN-VITRO EXPERIMENT FOR THE MEAN OF ANTIMICROBIAL EFFECTS ON G-NEGATIVE BACTERIA (*EC)
b) Antifungal: Table 5 and 3 showed in-vitro experiment for the mean of antimicrobial effects on (CA), that revealed the effect of this dissolved substances as antimicrobial antifungal and were done in the h of exposures included (1, 3, 5 and 7h). The mean antifungal effects were calculated during the exposure hrs and the averages were in descending order by the percentage of the antifungal as shown in Table 5 and Fig. 3. The minimal inhibitory concentration for some of the newly synthesized compounds showed high significant activity against fungi activities 26, their antimicrobial activity fungi (C. albicans) showed significant antimicrobial activity against tested microorganisms 28.
TABLE 5: IN-VITRO EXPERIMENT FOR THE MEAN OF ANTIMICROBIAL EFFECTS ON FUNGI (*CA) %
Compound | 1h | 3h | 5h | 7h | Mean |
1b | 23 | 33 | 54 | 100 | 52.5 |
1c | 24 | 37 | 60 | 89 | 52.5 |
2a | 22 | 41 | 69 | 95 | 56.8 |
2b | 31 | 48 | 79 | 100 | 64.5 |
3a | 23 | 51 | 71 | 96 | 60.3 |
4a | 25 | 40 | 59 | 83 | 51.8 |
4a | 29 | 58 | 83 | 98 | 67.5 |
5a | 39 | 64 | 89.5 | 100 | 75 |
6a | 43 | 70.5 | 93.5 | 100 | 84.5 |
FIG. 3: IN-VITRO EXPERIMENT FOR THE MEAN OF ANTIMICROBIAL EFFECTS ON FUNGI (*CA)
2. Anticancer: On the basis of computer modelling technique and the differences in structures of the compounds. As mentioned in literature, many attempts to get HAT inhibitor, New series of 4-Aryl-2-hydrazinothiazole containing nucleus were prepared and investigated toward different thin layers, selected series of the synthetic thiazoles 1b, 1c, 2a, 2b, 3a, 4a, 4a, 5a and 6a were tested in-vitro anti-cancer assays against three thin lines of breast cancer, liver cancer, and prostate cancer at constant concentration of 1×10-5 M for each compound and the cultured were incubated for 48 h. Then percentage of inhibition of cell growth was compared with untreated control cells. The percentage of inhibition for each cell line was illustrated in Table 6, it was found that all of them could inhibit the cell growth by varied extents, but in fact the most effective compounds were 5a, 6a.
TABLE 6: IN-VITRO ANTI-CANCER ACTIVITY INHIBITION %
Compound | MCF-7
(breast cancer) |
HEPG-2
(liver cancer) |
Prostate
Cancer (PC-3) |
1b | 44.62 | 62.02 | 43.2 |
1c | 33.43 | 59.21 | 65.32 |
2a | 24.42 | 53.06 | 61.9 |
2b | 48.56 | 33.6 | 67.35 |
3a | 49.63 | 57.64 | 81.42 |
4a | 51.09 | 61.04 | 88.1 |
4a | 56 | 63 | 87.09 |
5a | 48.5 | 66.8 | 90 |
6a | 47.55 | 47.81 | 84.3 |
FIG. 4: INHIBITION EFFECTS OF THE TESTED COMPOUND AT 10 µM CONCENTRATION AGAINST THREE THIN LINES
The novel synthesized compounds were tested for their antitumor activity against three cell lines, MCF-7 (breast cancer), PC-3 (prostate) and HEPG-2 (liver cancer). Compounds 4a, 5a showed the most potent activity, followed by compound 6a; 3b showed the lowest activity Table 7. These results are in agreement with those obtained by previous researchers 29, 30, 31, 32. These researchers reported that 4- aryl thiazole derivatives had anticancer activity both in-vivo and in-vitro.
From Table 6 and Fig. 4 it was found that the most effective compounds for prostate cell line was 5a, 6a, while for 4a was the most effective for hepatic cell line. All of the compounds are active and cytotoxicity of the target compounds were illustrated in Table 7 and Fig. 5, IC50.
TABLE 7: IN-VITRO ANTICANCER ACTIVITY 4- AGAINST MCF-7 (BREAST CANCER) HEPG-2 (LIVER CANCER) AND PC-3 (PROSTATE CANCER)
Compound | MCF-7
(breast cancer) |
HEPG-2
(liver cancer) |
Prostate
Cancer (PC-3) |
IC50 (µg/mL) | |||
1b | 35.50 | 34.02 | 35.26 |
1c | 20.72 | 25.46 | 25.23 |
2a | 31.23 | 32.76 | 21.7 |
2b | 35.5 | 29.80 | 27.53 |
3a | 29.02 | 37.15 | 18.42 |
4a | 21.50 | 13.87 | 14.1 |
4a | 21.72 | 13.4 | 11.6 |
5a | 26.8 | 19.9 | 19.4 |
6a | 27.01 | 23.80 | 24.06 |
FIG. 5: CYTOTOXICITY OF COMPOUND SERIES AGAINST THREE THIN LINES
Molecular Docking Modeling: Molecular modelling studies were performed using Molecular Operating Environment (MOE, 2016) software on an Intel Core i3 processor 1.9 GHz, 4 GB memory with Windows 10, 64-bit operating system. Energy minimizations were performed with RMSD gradient of 0.05 kcal/ mol and MMFF94X force field using MOE and partial charges were automatically calculated. The X-ray crystallographic structures were obtained from Protein Data Bank (PDB), the PDB files are 3eqm, 3cqx, and 4ihz 33.
The target enzymes were prepared for docking by:
- Removing the ligand from the active site of the enzyme.
- Addition of hydrogen atoms to the structure with their standard geometry.
- Detecting the active site in the enzyme by MOE Alpha Site Finder.
- The obtained pocket was saved as Moe to be used in predicting the ligand enzyme interactions at the active site and docking of the compounds.
A-3EQM: Oxidoreductase / Breast Cancer:
FIG. 6: THE DIFFERENT BINDING MODES OF 1b, 1c, 2b, 6a, 3b, 4a, 4b, 6b AND X WITH 3EQM PROTEIN
TABLE 8: OXIDOREDUCTASE / 3EQM: OXIDOREDUCTASE INTERACTIONS
Mol | S | Msd | rmsd_refine | E_conf | E_place | E_score1 | E_refine | E_score2 |
1b | -5.81 | 8.11 | 1.08 | 41.41 | -79.14 | -8.56 | -28.01 | -5.81 |
1c | -6.29 | 7.94 | 3.50 | 27.81 | -64.83 | -9.58 | -31.89 | -6.29 |
2b | -7.22 | 7.49 | 2.37 | 30.78 | -80.88 | -10.72 | -38.42 | -7.22 |
6a | -7.64 | 5.98 | 0.61 | 41.69 | -93.99 | -9.64 | -38.69 | -7.64 |
3b | -8.15 | 8.50 | 1.49 | 67.49 | -61.74 | -10.70 | -25.87 | -8.15 |
4a | -8.53 | 9.35 | 2.25 | 89.88 | -87.33 | -10.88 | -47.18 | -8.53 |
4b | -9.06 | 7.53 | 1.56 | 58.18 | -78.72 | -11.04 | -43.00 | -9.06 |
6b | -7.05 | 8.53 | 0.85 | 45.19 | -78.51 | -9.97 | -28.45 | -7.05 |
X | -7.34 | 7.11 | 1.59 | 91.74 | -102.96 | -10.42 | -40.70 | -7.34 |
Where, S= Final score, which is the score of the last stage that was not set to none. Rmsd = The root mean square deviation of the pose, in Å, from the original ligand. This field is present if the site definition was identical to the ligand definition. rmsd_refin e= The root mean square deviation between the pose before refinement and the pose after refinement. E_conf = The energy of the conformer. If there is a refinement stage, this is the energy calculated at the end of the refinement. Note that for Forcefield refinement, by default, this energy is calculated with the solvation option set to Born. E_place = Score from the place-ment stage. E_score 1, E_score 2 = Score from rescoring stages 1 and 2. E_refine = Score from the refinement stage, calculated to be the sum of the van der Waals electrostatics and solvation energies, under the Generalized Born solvation model (GB/VI).
From analysis of docked structures it was concluded that the binding models of compound 1b, 1c, 2b, 6a, 3b, 4a, 4b, 6b and X to the crystal structure of 3EQM prostate protein cancer molecule and from the results showed in Table 8, it was found that all of the tested compounds were interacted with the protein through a hydrogen bond, ionic and metal interaction. The values of interaction energies revealed that compounds 4b, 4a and 3b have the most stable interaction than the other complexes and free ligands with a minimum binding energy of - 9.06 to -8.15 Kcal mol -1 see Fig. 7.
B-3CQX: Protein Binding Liver Cancer:
FIG. 7: THE DIFFERENT BINDING MODES OF 1b, 1c, 2b, 6a, 3b, 4a, 4b, 6b AND X WITH 3CQX PROTEIN
TABLE 9: 3CQX: INTERACTIONS
Mol | S | Rmsd | rmsd_refine | E_conf | E_place | E_score1 | E_refine | E_score2 |
1b | -5.32 | 13.24 | 2.02 | 42.47 | -31.86 | -9.04 | -26.49 | -5.32 |
1c | -5.56 | 15.00 | 2.74 | 71.69 | -64.64 | -9.56 | -32.40 | -5.56 |
2b | -6.35 | 12.61 | 1.62 | 32.36 | -65.18 | -10.97 | -32.02 | -6.35 |
6a | -6.42 | 12.69 | 1.36 | 43.38 | -50.94 | -9.20 | -27.96 | -6.42 |
3b | -6.93 | 13.02 | 1.10 | 52.79 | -61.24 | -10.18 | -37.45 | -6.93 |
4a | -7.04 | 12.06 | 1.33 | 87.86 | -62.76 | -10.65 | -40.07 | -7.04 |
4b | -7.20 | 13.59 | 1.37 | 48.15 | -53.90 | -10.12 | -37.70 | -7.20 |
6b | -5.79 | 12.06 | 1.28 | 44.95 | -51.82 | -8.91 | -27.88 | -5.79 |
X | -6.05 | 12.70 | 1.18 | 92.33 | -48.54 | -8.19 | -30.51 | -6.05 |
From analysis of docked structures it was concluded that the binding models of compound 1b, 1c, 2b, 6a, 3b, 4a, 4b, 6b and X to the crystal structure of 3CQX prostate protein cancer molecule and from the results showed in Table 9, it was found that all of the tested compounds were interacted with the protein through a hydrogen bond, ionic and metal interaction. The values of interaction energies revealed that compounds 4b, 4a, and 3b have the most stable interaction than the other complexes and free ligands with a minimum binding energy of – 7.20 to -6.93 Kcal mol-1 see Fig. 8.
C-4IHZ: Hydrolase Inhibitor:
FIG. 8: THE DIFFERENT BINDING MODES OF 1b, 1c, 2b, 6a, 3b, 4a, 4b, 6b AND X WITH 4ihz PROTEIN
TABLE 10: 4ihz/ INTERACTIONS
Mol | S | rmsd | rmsd_refine | E_conf | E_place | E_score1 | E_refine | E_score2 |
1b | -4.61 | 8.77 | 3.49 | 40.32 | -25.97 | -7.20 | -20.80 | -4.61 |
1c | -4.45 | 11.83 | 5.85 | 69.54 | -3.53 | -7.61 | -19.88 | -4.45 |
2b | -5.26 | 9.55 | 2.12 | 31.43 | -9.94 | -5.85 | -21.81 | -5.26 |
6a | -5.49 | 12.80 | 2.36 | 42.81 | 22.18 | -5.23 | -25.54 | -5.49 |
3b | -6.25 | 12.18 | 2.18 | 50.61 | -16.26 | -7.07 | -29.36 | -6.25 |
4a | -6.62 | 8.69 | 1.94 | 84.38 | 48.13 | -7.88 | -28.63 | -6.62 |
4b | -6.61 | 12.54 | 1.93 | 52.16 | 9.43 | -8.06 | -31.74 | -6.61 |
6b | -5.02 | 10.94 | 4.17 | 48.17 | 91.12 | 0.36 | -20.57 | -5.02 |
X | -5.45 | 12.04 | 5.44 | 94.75 | 142.28 | 4.24 | -24.82 | -5.45 |
FIG. 9: KEY OF INTERACTIONS OF PROTEINS AND MOLECULES
From analysis of docked structures it was concluded that the binding models of compound 1b, 1c, 2b, 6a, 3b, 4a, 4b, 6b and X to the crystal structure of 4ihz prostate protein cancer molecule and from the results showed in Table 10, it was found that all of the tested compounds were interacted with the protein through a hydrogen bond, ionic and metal interaction. The values of interaction energies revealed that compounds 4a, 4b, and 3b have the most stable interaction than the other complexes and free ligands with a minimum binding energy of – 6.62 to -6.25 Kcal mol -1 see Fig. 8. It concludes that all compounds have the good agreement of Molecular docking data with their Biological and anti-cancer Activity showing that Compounds 4a and 4b have the most Biological and anti-cancer activity as shown in Table 11.
TABLE 11: COMPARISON BETWEEN MOLECULAR DOCKING DATA AND BIOLOGICAL AND ANTICANCER ACTIVITY
Comp. | (+ve Gram) | (-ve Gram) | Fungi | MCF-7 (breast cancer) | Liver cancer | Prostate | Colon cancer | Leukemia | Docking | ||
MCF-7 | HEPG-2 | Cancer(PC-3) | (HT29) | MOLT-4 | 3eqm | 3cqx | 4ihz | ||||
1b | 28% | 27% | 23% | 44.62 | 62.02* | 43.20 | 33.60 | 53.20 | -5.81 | -5.32 | -4.61 |
1c | 32% | 29% | 24% | 33.43 | 59.21 | 65.32 | 45.20 | 55.60 | -6.29 | -5.56 | -4.45 |
2a | 34% | 30% | 22% | 24.42 | 53.06 | 61.90 | 36.10 | 52.30 | |||
6a | 48% | 41% | 31% | 48.56 | 33.60 | 67.35 | 54.10 | 43.20 | -7.64 | -6.42 | -5.26 |
3a | 31% | 28% | 23% | 49.63 | 57.64 | 81.42 | 77.30 | 51.90 | |||
4a | 38% | 32% | 25% | 51.09 | 61.04 | 88.10 | 65.20 | 58.40 | -8.15 | -6.93 | -5.49 |
4b | 42% | 38% | 29% | 56 | 63.00 | 87.09 | 76.70 | 63.40 | -8.53 | -7.04 | -6.25 |
5a | 46% | 40% | 39% | 48.50 | 66.80 | 90.00 | 66.70 | 46.50 | |||
6b | 57% | 46% | 43% | -6.62 | -6.62 | -6.62 | -6.62 | -6.62 | -6.62 | -6.62 | -6.62 |
X | -7.34 | -6.05 | -6.61 | ||||||||
2b | -7.22 | -6.35 | -5.02 | ||||||||
3b | -9.06 | -7.2 | -5.45 |
* The activity with high impact related to the average have underline format
Methodology:
1. Chemistry: All chemicals and reagents used were from sigma Aldrich analytical grade, and they were used without further purifying. Where Microwave synthesis was carried out using domestic Microwave system. Melting points measurements were determined on (Pyrex capillary) Gallenkamp apparatus. Infrared Spectra were recorded with a Thermo Nicolet Nexus 470 FT-IR spectrometer in the range 4000 - 400 cm−1 using potassium bromide disks. 1H-NMR spectra, 13C-NMR spectra were recorded on Burker AM250 NMR spectrometer using as DMSO-d6 and CDCl3 solvents for the samples; chemical shifts will be recorded in δ (ppm) units, relative to Me4Si as an internal standard. The mass spectra will be recorded on Shimadzu LCMS-QP 800 LC-MS. Thin-Layer Chromatography (TLC) was carried out on pre-coated Merck silica gel F254 plates. Elemental analysis was obtained using Perkin Elmer 2400 II series CHN Analyser.
Each compound series was synthesized by two methods as mentioned before method A (Conventional method), and method B (Microwave method).
1a: [4-(p-Tolyl)-1, 3-thiazol-2-yl]hydrazine:
Method A: A mixture of 4'-methylbromo acetophenone (0.01 mol), with thiosemicarbazide (0.01mol) in glacial acetic acid (2 mmol) was refluxed under stirring for 2 - 3 h, after that the mixture cooled at room temperature, the precipitate was collected by filtration and give yellowish crystals.
Method B: A mixture of 4'-methylbromo acetophenone (0.01 mol), with thiosemicarbazide (0.01mol), was dissolved in methylene chloride/ methanol (4:1, 20 ml), then added silica gel of (1.0 g, 200-400 mesh), the solvent was removed by vaporization, the dried crude was transferred into a glass beaker and irradiated for 1.5-2 min in a domestic microwave oven, the product was tested and separated on silica gel column using methylene chloride as eluent.
1a: m.p 301 ºC, IR, (KBr, ʋ Cm-1), 810 (Ar), 2900 (-CH3), 3450 (NH2), 3300 (NH), 1HNMR, δ 1.8 (s, 3H, CH3), 3.36(3H, NH, NH2), 7.29 (dd, 2H, Ar), 7.5 (s, H, thiophene ring), 7.7 (dd, 2H, Ar), 13C NMR δ: 19.28, 77.2, 121.1, 127.5, 132.8, 139.6, 151.3, 156.4, MS (m/z): 205.3 (M+), Anal. calcd: (205.3), C-58.51; H-5.40; N-20.47, found: C-58.0; H-5.2; N-20.5.
1b: 4-(p-Bromophenyl)-1,3-thiazol-2-yl]hydrazine:
Method A: A mixture of 4'-nitro-bromo acetophenone (0.01 mol), with thiosemicarbazide (0.01mol) in glacial acetic acid (2 mmol) was refluxed under stirring for 2 - 3 h, after that the mixture cooled at room temperature, the precipitate was collected by filtration and give yellowish to orange crystals.
Method B: A mixture of 4'-nitro-bromo acetophenone (0.01 mol), with thiosemicarbazide (0.01mol), was dissolved in methylene chloride/ methanol (4:1, 20 ml), then added silica gel of (1.0 g, 200-400 mesh), the solvent was removed by vaporization, the dried crude was transferred into a glass beaker and irradiated for 1.5-2 min in a domestic microwave oven, the product was tested and separated on silica gel column using methylene chloride as eluent.
1b: m.p 340 ºC, IR (KBr, ʋ Cm-1), 520 (-Br), 3450 (NH2), 3150 (NH), 780 (Ar), 1HNMR δ 3.8 (3H, NH, NH2), 7.2-7.7 (m, 4H, Ar), 7.51 (s, H, thiophene ring), 13CNMR δ: 77.2, 120.6, 122.1, 129.8, 135.6, 138.7, 151.5, 159.4, MS (m/z): 269.5 (M+), Anal. calcd: (269), C-58.51; H-5.40; N-20.47, found: C-58.51; H-5.40; N-20.47.
1c: [4-(p-Nitrophenyl)-1, 3-thiazol-2-yl] hydrazine:
Method A: A mixture of 4'-bromobromoaceto phenone (0.01 mol), with thiosemicarbazide (0.01 mol) in glacial acetic acid (2 mmol) was refluxed under stirring for 2 - 3 h, after that the mixture cooled at room temperature, the precipitate was collected by filtration and give dark orange crystals.
Method B: A mixture of 4'-bromo-bromo
acetophenone (0.01 mol), with thiosemicarbazide (0.01mol), was dissolved in methylene chloride/ methanol (4:1, 20 ml), then added silica gel of (1.0 g, 200-400mesh), the solvent was removed by vaporization, the dried crude was transferred into a glass beaker and irradiated for 1.5 - 2 min in a domestic microwave oven, the product was tested and separated on silica gel column using methylene chloride as eluent.
1c: M.P 334 ºC, IR, (KBr, ʋ Cm-1), 810 (Ar), 1530 (NO2), 3450 (NH2), 3300 (NH), 1HNMR δ 3.8(s, 3H, NH, NH2), 6.8 (dd, 2H, Ar), 7.5 (s H, CHS), 7.6 (dd, 2H, Ar, J = 9.2), 13 NMR δ: 77.8, 116.35, 134.26, 151.3, 154.5, MS (m/z): 206.9 (M+), Anal. calcd: (207), C-52.16; H-4.38; N-20.27, found: C-52.1; H-4.4; N-20.3.
1d: p-(2-Hydrazino-1, 3-thiazol-4-yl) phenol:
Method A: A mixture of 4'-Hyroxy-bromo acetophenone (0.01 mol), with thiosemicarbazide (0.01mol) in glacial acetic acid (2 mmol) was refluxed under stirring for 2-3h, after that the mixture cooled at room temperature, the precipitate was collected by filtration and give orange crystals.
Method B: A mixture of 4'-Hyroxy-bromo-acetophenone (0.01 mol), with thiosemicarbazide (0.01 mol), was dissolved in methylene chloride/ methanol (4:1, 20 ml), then added silica gel of (1.0 g, 200 - 400 mesh), the solvent was removed by vaporization, the dried crude was transferred into a glass beaker and irradiated for 1.5-2 min in a domestic microwave oven, the product was tested and separated on a silica gel column using methylene chloride as eluent
1d: m.p above 350 ºC, IR, (KBr, ʋ Cm-1), 3500 (OH), 3400 (NH2), 3310 (NH), 800 (Ar), 1HNMR δ 3.4 (s, 4H: OH, NH, NH2), 6.8 (dd, 2H, Ar, J = 8.5), 7.2 (s, H, CHS), 7.5 (dd, 2H, Ar), 13 NMR δ 77.8, 113.4, 121, 128.3, 129.9, 149, 151.5, 156.3, MS (m/z): 236.5 (M+), Anal. Calcd: (236), C-45.75; H-3.41; N-23.71, Found: C-44.8; H-3.3; N-23.71.
2a: 1-{2-[4-(p-Tolyl)-1,3-thiazol-2-yl]hydrazino}-1-ethanone:
Method A: A mixture of 1a (0.01 mol), with acetyl chloride (0.01mol) in glacial acetic acid (30 ml) was refluxed under stirring for 5 h, after that the mixture cooled at room temperature, then poured on crushed ice, the precipitate was collected by filtration and crystallization from ethanol and give orange to brown crystals
Method B: A mixture of 1a (0.01 mol), with acetyl chloride (0.01mol) was mixed with methylene chloride/methanol (4:1, 20 ml), then added silica gel of (1.0 g, 200 - 400 mesh), the solvent was removed by vaporization, the dried crude was transferred into a glass beaker and irradiated for 2 - 3 min in a domestic microwave oven, the product was tested and separated on silica gel column using methylene chloride as eluent.
2a: M.P above 350 ºC, IR, (KBr, ʋ Cm-1), 3000 (CH3), 3300 (NH), 3320 (NH amide), 1650 (C=O amide), 1HNMR δ 1.5 (s, 3H, CH3), 1.77 (s, 3H, CH3), 6.4 (s, 2H, NH), 7.3 (dd, 2H, Ar), 7.7 (dd, 2H, Ar, J = 9.3), 7.6 (s, H, HCS), 13 NMR δ: 14.5, 19.8, 77.6, 121, 127.5, 135.9, 138.7, 151.5, 156.4, 172, MS (m/z): 247.7 (M+), Anal. calcd (248), C-58.28; H-5.30; N-16.99, found: C-57.60; H-5.00; N-16.8.
2b: Phenyl {2- [4-(p- tolyl) -1, 3-thiazol-2 -yl] hydrazine} formaldehyde:
Method A: A mixture of 1a (0.01 mol), with benzoyl chloride (0.01 mol) in glacial acetic acid (30 ml ) was refluxed under stirring for 4 - 5 h, after that the mixture cooled at room temperature, then poured on crushed ice, the precipitate was collected by filtration and crystallization from ethanol and give orange crystals.
Method B: A mixture of 1a (0.01 mol), with benzoyl chloride (0.01mol) was mixed with methylene chloride/methanol (4:1, 20 ml), then added silica gel of (1.0 g, 200 - 400 mesh), the solvent was removed by vaporization, the dried crude was transferred into a glass beaker and irradiated for 2 - 3 min in a domestic microwave oven, the product was tested and separated on silica gel column using methylene chloride as eluent.
2b: M.P above 350 ºC, IR, (KBr, ʋ Cm-1), 2950 (CH3), 3400 (NH), 3350 (NH amide), 1650 (C=O amide) 1HNMR, δ 1.5 (s, 3H, CH3), 6.1 (s, 2NH), 7.2-7.8 (m, 4HAr, 5Hph, H, HCS), 13 NMR δ: 19.8, 77.1, 121, 127.5, 128.7, 130, 133.6, 136, 139, 154.5, 156, 170, MS (m/z): 309.5 (M+), Anal. calcd: (310), C-66.0; H-5.0; N-13.0, found: C-65.99; H-4.89; N-13.58.
2c: {2-[4-(p- Hydroxyphenyl)-1, 3-thiazol -2-yl] hydrazino}phenylformaldehyde:
Method A: A mixture of 1d (0.01 mol), with acetyl chloride (0.01 mol) in glacial acetic acid (30 ml) was refluxed under stirring for 4 - 5 h, after that the mixture cooled at room temperature, then poured on crushed ice, the precipitate was collected by filtration and crystallization from ethanol and give orange to brown crystals.
Method B: A mixture of 1d with acetyl chloride (0.01mol) was mixed with methylene chloride/ methanol (4:1, 20 ml), then added silica gel of (1.0 g, 200 - 400 mesh), the solvent was removed by vaporization, the dried crude was transferred into a glass beaker and irradiated for 2 - 3 min in a domestic microwave oven, the product was tested and separated on silica gel column using methylene chloride as eluent.
2c: M.P above ºC, IR, (KBr, ʋ Cm-1), 2890 (2 CH3), 3300 (NH), 3320 (NH amide), 1650 (C=O amide) , 3540 (OH, phenolic), 1HNMR δ 5.8 (s, H, OH, 2NH), 7.1-7.6 (m, 4HAr, 5Hph, 1HCS), 13 NMR δ: 77.8, 116.35, 121, 128.8, 133.5, 134.5, 151, 153.6, 156.7, 170, MS (m/z): 311 (M+), Anal. calcd: (311), C-61.72; H-4.21; N-13.50, found: C-60.9; H-4.4; N-13.50.
2d: 1-{2-[4-(p-Hydroxyphenyl)-1, 3-thiazol-2-yl] hydrazino}-1-ethanone:
Method A: A mixture of 1d (0.01 mol), with benzoyl chloride (0.01 mol) in glacial acetic acid (30 ml) was refluxed under stirring for 4 - 5 h, after that the mixture cooled at room temperature, then poured on crushed ice, the precipitate was collected by filtration and crystallization from ethanol and give orange crystals.
Method B: A mixture of 1d with benzoyl chloride (0.01mol) was mixed with methylene chloride/ methanol (4:1, 20 ml), then added silica gel of (1.0 g, 200-400 mesh), the solvent was removed by vaporization, the dried crude was transferred into a glass beaker and irradiated for 2-3 min in a domestic microwave oven, the product was tested and separated on silica gel column using methylene chloride as eluent.
2d: M.P above 350 ºC, IR, (KBr, ʋ Cm-1), 2950 (2 CH3), 3300 (NH), 3320 (NH amide), 1650 (C=O amide) , 3600 (OH, phenolic), 650 (Ar) 1HNMR, δ 1.46 (s, 3H, CH3), 6.09 (s, OH, 2NH), 7.1-7.5 (m, 4H, Ar, H, HCS), 13 NMR δ: 14.8, 77.8, 116.4, 121, 128.9, 134.4, 151.1, 153.5, 156.2, 172.1, MS (m/z): 247 (M+), Anal. calcd: (247), C-53.00; H-4.45; N-16.86, found: C-52.8; H-4.7; N-17.00.
3a: 2- [(o-Nitrophenyl) methylidene]-1 -[4-(p-tolyl)-1, 3-thiazol-2-yl]hydrazine:
Method A: A mixture of 1a (0.01 mol), with 2-nitrobenzaldehyde (0.01 mol) in chloro acetyl chloride (10 ml ) in DMF (15 ml) was reflxed under stirring for 6 h, after that the mixture cooled at room temperature, then poured on crushed ice, the precipitate was collected by filtration and crystallization from suitable, proper solvent.
Method B: A mixture of 1a (0.01 mol), with 2-nitrobenzaldehyde (0.01 mol) in chloro acetyl chloride (10 ml) was mixed with methylene chloride/methanol (4:1, 20 ml), then added silica gel of (1.0 g, 200 - 400 mesh), the solvent was removed by vaporization, the dried crude was transferred into a glass beaker and irradiated for 2 - 3 min in a domestic microwave oven, the product was tested and separated on a silica gel column using methylene chloride as eluent.
3a: M.P above 350 ºC, IR, (KBr, ʋ Cm-1), 2850 (CH3), 1000 (C=N free), 3100 (NH), 1340 (NO2), 780 (2Ar), 1HNMR δ 1.46 (s, 3H, CH3), 5.09 (s, NH), 6.9-7.7 (m, 4H, Ar, 4HAr`, H, HCS), 13 NMR δ: 19.8, 77.8, 112.4, 116.1, 121, 127.3, 129.9, 135, 139.5, 151, 156, MS (m/z): 338.4 (M+), Anal. calcd: (339), C-60.34; H-4.17; N-16.56, Found: C-60.6; H-4.3; N-16.45.
3b: 4-Bromo-2- nitro-6- ({2- [4- (p-tolyl)-1, 3-thiazol-2-yl]hydrazono}methyl)phenol:
Method A: A mixture of 1a (0.01 mol), with 5-bromo-3-nitrosalicylaldehyde (0.01 mol) in chloro acetyl chloride (10 ml) in DMF (15 ml) was refluxed under stirring for 4 h, after that the mixture cooled at room temperature, then poured on crushed ice, the precipitate was collected by filtration and crystallization from suitable proper solvent.
Method B: A mixture of 1a (0.01 mol), with 5-bromo-3-nitrosalicylaldehyde (0.01mol) in chloro acetyl chloride (10 ml ) was mixed with methylene chloride/methanol (4:1, 20 ml), then added silica gel of (1.0 g, 200 - 400 mesh), the solvent was removed by vaporization, the dried crude was transferred into a glass beaker and irradiated for 2-2.5 min in a domestic microwave oven, the product was tested and separated on silica gel column using methylene chloride as eluent.
3b: M.P above 350 ºC IR, (KBr, ʋ Cm-1 ), 2900 (CH3), 3330 (NH), 980 (C=N), 3600 (OH), 1525 (NO2), 570 (Br), 800 (2Ar), 1HNMR δ 1.46 (s, 3H, CH3), 5.09 (s, OH, NH), 7.1-7.7 (m, 4H, Ar, 1HAr`), 7.5(s, H, HCS), 13NMR δ: 19.8, 77.8, 112.4, 116.1, 118.3, 121, 127.5, 129.9, 135.9, 137.3, 138.2, 139.5, 151.5, 156.4, MS (m/z): 433.8 (M+), Anal. calcd: ( 434), C-47.12; H-3.02; N-12.93, found: C-46.8; H-3.5; N-12.7.
3c: 1- [(p-Chlorophenyl) methylidene]-2- [4- (p-tolyl)-1, 3-thiazol-2-yl]hydrazine:
Method A: A mixture of 1a (0.01 mol), with 4-chlorobenzaldehydealdehyde (0.01 mol) in benzoyl chloride (10 ml), in DMF (15 ml) was refluxed under stirring for 4 h, after that the mixture cooled at room temperature, then poured on crushed ice, the precipitate was collected by filtration and crystallization from suitable, proper solvent.
Method B: A mixture of 1a (0.01 mol), with 4-chlorobenzaldehydealdehyde (0.01 mol) in benzoyl chloride chloride (10 ml), was mixed with methylene chloride / methanol (4:1, 20 ml), then added silica gel of (1.0 g, 200 - 400 mesh), the solvent was removed by vaporization, the dried crude was transferred into a glass beaker and irradiated for 2 - 3 min in a domestic microwave oven, the product was tested and separated on silica gel column using methylene chloride as eluent.
3c: M.P above 350 ºC, IR, (KBr, ʋ cm-1), 850 (Cl), 2950 (CH3), 980 (C=N), 690 (Ar), 3350 (NH), 1HNMR, δ 1.5(s, 3H, CH3), 5.1(s, H, NH, 7.39 (m, 2H, Ar), 7.5 (m, 2H, Ar`), 7.58(s, H, HSN), 7.6 (m, 2H, Ar ), 7.7 (m, 2H, Ar)13 NMR, 19.8, 77.7, 77.72, 77.8, 121.07, 126.66, 127.5, 128.95, 129.85, 134.75, 135.86, 138.66, MS (m/z), 327.1,(M+), Anal. calcd: (327.83), C-63.00; H-4.01; N-12.86, found: C-62.28; H-4.3; N-12.82.
4a: (p-Chlorophenyl) (dimethylamino) {2- [4-(p-tolyl)-1, 3-thiazol-2 yl]hydrazino}methane:
Method A: A mixture of 1a (0.01mol) with dimethylamine (0.01 mol), 4-chlorobenzaldehyde (0.01 mol) in glacial acetic acid (20 ml) was refluxed for 6 - 8 h, after that the mixture cooled at room temperature, then poured on crushed ice, the precipitate was collected by filtration and crystallization from suitable, proper solvent.
Method B: A mixture of 1a (0.01mol) with dimethylamine (0.01 mol), 4-chlorobenzaldehyde (0.01 mol) was mixed with methylene chloride/ methanol (4:1, 20 ml), then added silica gel of (1.0 g, 200-400 mesh), the solvent was removed by vaporization, the dried crude was transferred into a glass beaker and irradiated for 2 - 3 min in a domestic microwave oven, the product was tested and separated on silica gel column using methylene chloride as eluent.
4a: M.P above 350 ºC, IR, (KBr, ʋ cm-1), 2900 (CH3), 740 (Cl), 800 (Ar), 3400 (2NH), 1250 (C-N), 1HNMR δ 1.49 (s, 3H, CH3), 1.9 (s, 6H, 2CH3), 3.6 (s, NH), 5.4 (s, H, CH), 7.1-7.7 (m, 4H, Ar, 4HAr`) , 7.52 (s, H, HCS), 13 NMR δ : 19.8, 36.2, 77.8, 87.1, 121, 126.5, 127.7, 129.9, 134.8, 138.2, 151.5, 156.4, MS (m/z): 372.3 (M+), Anal. calcd: (372), C-61.2; H-5.7; N-15.00, found: C-61.19; H-5.68; N-15.02.
4b: (p-Chlorophenyl) (dimethylamino) {2- [4- (p-nitrophenyl)-1, 3-thiazol -2 -yl] hydrazino} methane:
Method A: A mixture of 1c (0.01 mol) with dimethylamine (0.01 mol), 4-chlorobenzaldehyde (0.01 mol) in glacial acetic acid (20 ml) was refluxed for 6-8 h, after that the mixture cooled at room temperature, then poured on crushed ice, the precipitate was collected by filtration and crystallization from suitable proper solvent.
Method B: A mixture of 1c (0.01mol) with dimethylamine (0.01 mol), 4-chlorobenzaldehyde (0.01 mol) was mixed with methylene chloride/ methanol (4:1, 20 ml), then added silica gel of (1.0 g, 200-400 mesh), the solvent was removed by vaporization, the dried crude was transferred into a glass beaker and irradiated for 2-3 min in a domestic microwave oven, the product was tested and separated on silica gel column using methylene chloride as eluent.
4b (C18H18ClN5O2S): m.p above 350 ºC, IR, (KBr, ʋ cm-1), 1525(NO2), 770 (Cl), 800 (Ar), 3400 (2NH), 1250 (C-N), 1HNMR δ 1.8 (s, 6H, 2CH3), 3.6 (s, NH), 5.14 (s, H, CH), 7.1 (dd, 2H, Ar, J = 8.5), 7.4 (dd, 2H, Ar, J = 8.9), 7.56 (dd, 2H, Ar`, J = 9.5), 7.8 (dd, 2H, Ar`, J = 9.3), 7.5 (s, H, HCS), 13 NMR δ : 19.8, 36.2, 77.8, 87.7, 113.3, 126.6, 128, 129.7, 134.8, 148.2, 151, 156.6, MS (m/z): 403.1, (M+), Anal. calcd: (403), C-53.6; H-4.5; N-17.5, found: C-53.53; H-4.49; N-17.34
5a: 8- (p-Bromophenyl)-2- methyl-6 -thia-1.3.4-triaza bicyclo[3.3.0]octa-2,4,7-triene:
Method A: A mixture of 1c (0.01mol) with acetic anhydride (0.01 mol), in glacial acetic acid (20 ml) and sod. acetate was refluxed for 12 h, after that the mixture cooled at room temperature, then poured on crushed ice, the precipitate was collected by filtration and crystallization from suitable proper solvent.
Method B: A mixture of 1c (0.01 mol) with acetic anhydride (0.01 mol), acetic acid (20 ml) and sod. acetate was mixed with methylene chloride/ methanol (4:1, 20 ml), then added silica gel of (1.0 g, 200-400 mesh), the solvent was removed by vaporization, the dried crude was transferred into a glass beaker and irradiated for 3 - 4 min in a domestic microwave oven, the product was tested and separated on a silica gel column using methylene chloride as eluent.
5a: M.P 293.4 ºC, IR, (KBr, ʋ cm-1), 2150 (triazene), 2850 (CH3), 650 (Br), 1HNMR δ 1.6 (s, 3H, CH3), 7.6-7.8 (m, 4H, Ar, 4HAr`), 8 (s, H, HCS), 13 NMR δ: 19.8, 78, 120.7, 123.9, 129.1, 132.3, 139.6, 157.2, 168, MS (m/z): 294, (M+), Anal. calcd: (294), C-44.91; H-2.74; N-14.28, found: C-44.86; H-2.64; N-14.00.
5b:2-(p-Chlorophenyl)-8-(p-nitrophenyl)-6-thia-1.3.4-triazabicyclo[3.3.0]octa-2,4,7-triene:
Method A: A mixture of 3c (0.01 mol) with phI(AcO)2 (0.01 mol), in glacial acetic acid (20 ml) and sod. acetate was refluxed for 10-12 h, after that the mixture cooled at room temperature, then poured on crushed ice, the precipitate was collected by filtration and crystallization from the suitable proper solvent.
Method B: A mixture of 3c (0.01 mol) with phI(AcO)2 (0.01 mol) and sod. acetate was mixed with methylene chloride/methanol (4:1, 20 ml), then added silica gel of (1.0 g, 200-400 mesh), the solvent was removed by vaporization, the dried crude was transferred into a glass beaker and irradiated for 3 - 4 min in a domestic microwave oven, the product was tested and separated on silica gel column using methylene chloride as eluent.
5b: M.P above 350 ºC IR, (KBr, ʋ cm-1), 1525(NO2), 770 (Cl), 800 (2Ar), 1250 (C-N), 1HNMR 7.1 (dd, 2H, Ar, J = 9), 7.73 (dd, 2H, Ar, J = 8.7), 7.67 (dd, 2H, Ar`, J = 9), 7.93 (dd, 2H, Ar`, J = 9), 8.2 (s, H, HCS), δ: 77.9, 113.4, 126.3, 128.1, 129.5, 129.8, 132.7, 140.7, 141.7, 148.9, 157.6, 167.8, MS (m/z): 356.8 (M+), Anal. calcd: (357), C-53.9; H-2.6; N-15.66, found: C-53.86; H-2.54; N-15.70.
6a: p- (2- Phenoxy -6 -thia-1.3.4- triazabicyclo [3.3.0] octa-2,4,7-trien-8-yl)phenol:
Method A: A mixture of 1d (0.01 mol) with carbondisulfide (0.01 mol), methyl iodide (0.01 mol), in ethanol (15 ml) with KOH (5%, 15 ml) / THF (10 ml) was refluxed for 14 h, which gave in-situ B** as intermediate which could be separated, where B** refluxed in the reaction medium with phenol, then cooled at room temperature, then poured on crushed ice, the precipitate was collected by filtration and crystallization from suitable, proper solvent.
6b: p-(2-Anilino-6-thia-1.3.4-triazabicyclo [3.3.0] octa-2, 4, 7-trien-8-yl)phenol:
Method A: A mixture of 1d (0.01 mol) with carbondisulfide (0.01 mol), methyl iodide (0.01 mol), in ethanol (15 ml) with KOH (5%, 15 ml)/ THF (10 ml) was refluxed for 4 h, which gave in-situ B** as intermediate which could be separated, where B** refluxed in the reaction medium with aniline, then cooled at room temperature, then poured on crushed ice, the precipitate was collected by filtration and crystallization from suitable, proper solvent.
Method B: A mixture of 1d (0.01 mol) with carbondisulfide (0.01 mol), methyl iodide (0.01 mol), in ethanol (15 ml) with KOH (alcoholic) (5%, 15 ml) then added silica gel of (1.0 g, 200-400 mesh), the solvent was removed by vaporization, the dried crude was transferred into a glass beaker and irradiated for 3 - 4.5 min in a domestic microwave oven, the product was tested and separated on silica gel column using methylene chloride as eluent. Which gave in-situ B** as intermediate which could be separated, and investigated, then B** was subjected to direct irradiation with phenol to give 6a or with aniline to give 6b.
6a: M.P above 350 ºC, IR, (KBr, ʋ cm-1), 800 (2Ar), 1250 (C-N), 2150 (triazene) 1100 (C-O), 3550 (OH), 1HNMR 4.8 (s, H, OH), 7.2-7.8 (m, 4H, Ar, 4HAr`), 7.9 (s, H, HCS), 13 NMR δ: 77.8, 114.1, 116.4, 119.8, 128, 129, 134.3, 153.5, 157.1, 158.1, 160, 168.1, MS (m/z): 309 (M+), Anal. calcd : (309) C-62.12; H-3.58; N-13.58, found: C-62.00; H-3.68; N-13.6.
6b: M.P above 350 ºC, IR, (KBr, ʋ cm-1), 800 (2Ar), 1250 (C-N), 2150 (triazene) 1100 (C-O), 3600 (NH), 3500 (OH), 1HNMR 4.6 (s, H, OH, NH), 6.8-7.7 (m, 4H, Ar, 4HAr`), 7.6 (s, H, HCS), 13 NMR δ: 77.8, 114.1, 116.4, 117.8, 128, 129, 134.3, 148.5, 158.1, 160, 169, MS (m/z): 307.6 (M+), Anal. calcd: (308), C-62.32; H-4.0; N-18.17, found: C-62.2; H-3.92; N-18.2.
B**: M.P 255.4 ºC, IR, (KBr, ʋ cm-1), 800 (Ar), 630 (C-S), 3000 (CH3), 1HNMR 1.8 (s, 3H, CH3), 7.56-7.8 (m, 6H, 2HAr, 4HAr`), 8.05 (s, H, HCS), 13 NMR δ: 77.9, 126.33, 126.55, 127.45, 129, 131.3, 141.6, 157.22, 167,8, MS (m/z): 307.6 (M+), Anal. calcd: (308), C-62.32; H-4.0; N-18.17, found: C-62.2; H-3.92; N-18.2.
Biology
Antimicrobial: Collected the chemical subject samples under study in sterile and dry Eppendorf tubes with the specific information for each sample. Weight the understudy quantity of each sample and were dissolved in acetone, mixed for (2-5) min. Prepared 2 mL of each acetone dissolved subject in Wizerman tube for each microbe under test and added the data on the tube 34.
Collected the microbial identified isolates as pure and clear colonies, then made a microbial distilled water suspension of each as McFarland conc. with special information setting 35.
Applied a mixture of (acetone dissolved subject + microbial suspension), then mix the ingredients and calculated the time. Cultured got from each mixture every time recorded (1, 3, 5 and 7 h), that did on suitable dish media with data recorded on each plate, incubated at (35 - 37) ºC for (24 - 48) h. Followed the results of microbial cultures growth and recorded the number of colonies in each dish 36. Determined the percentage of microbial death by applying the Equation:
[(Colony no. / 300 × 100) -100] 37
Statistical analysis of the results, calculation of the arithmetic mean for each chemical materials subject samples under study that were showed in the form of tables and graphs 38.
Anticancer:
Cell Culture: Many human Cell lines were obtained from Egyptian Holding Company for Biological Products & Vaccines (VACSERA), Giza, Egypt, and maintained in the tissue culture unit. The cells were routinely grown in RBMI-1640 medium, supplemented with 10% heat-inactivated FBS, 50 units/mL of Penicillin and 50 mg/mL of Streptomycin and maintained in a humidified atmosphere containing 5% CO2. They were acting as a monolayer culture by serial sub-culturing. The reagents of the cell cultured were obtained from Lonza (Basel, Switzerland). The activity of the compounds under investigation as anticancer was determined against MCF-7 cells (breast cancer), PC-3 (prostate cancer), and HEPG-2 cells (liver cancer) 39.
Cytotoxicity: The sulforhodamine B (SRB) was used to determine Cytotoxicity using assay method as previously described by 40. Collections of multi times growing cells were done by using 0.25% Trypsin EDTA and seeded in 96 well plates at 1000-2000 cells/well in RBMI-1640-supplemented medium. After 24 h, cells were incubated for 72 h with multiple different concentrations of the synthesized under investigation compounds. After 72 h treatments, the cells were fixed with 10% trichloroacetic acid for one h at 4 °C. Wells were stained for 10 min at room temperature with 0.4% SRBC (sulforhodamine B) dissolved in 1% acetic acid. The plates were subjected to air dry for 24 h, and the dye was solubilized with Tris HCl for 5 min on a shaker at 1600 rpm. OD (the optical density) of each well was measured spectrophotometrically at 564 nm with an ELISA microplate reader (ChroMate, 4300, FL, USA). Calculations of IC50 values were according to the Boltzmann sigmoidal equation for the response of concentration curve by nonlinear regression fitting models (Graph Pad, Prism Version 5).
C. Modeling: Molecular docking studies were carried out using the Molecular Operating Environment (MOE) docking software 41, 42. The Three Dimensional (3D) crystal structure of GPb was downloaded from the Protein Data Bank 43, 44. The allosteric site as characterized in the crystal structure of the selected proteins was used as the target site. Before the docking experiment was performed, the compounds prepared for docking by minimizing its energy at the B3LYP/631G (d) level of theory. Partial charges were calculated by Gasteiger’s method. Since most macromolecular crystal structures contain little or no hydrogen coordinate data due to limited resolution, protonation was done before docking using protonate 3D tools implemented in MOE. Protonation was followed by energy minimization up to a 0.05 Gradient using an Amber 99 force field. The compounds were docked into the protein’s active site using the Triangular Matching docking method, and thirty conformations of the title compound and protein complex were generated with a docking score (S). The complexes were analyzed for interactions, and their 3D images were taken by using visualizing tool PyMol. The docking protocol predicted the same conformation as was present in the crystal structure with an RMSD value within the allowed range of 2.0 Å 45, 46. The superposition of the docked conformation and co-crystalized ligand is shown. Amongst the docked conformations, the top-ranked conformation was visualized for ligand-protein interactions in PyMol software.
CONCLUSION: In brief, synthesis of thiazoles derivatives by domestic microwave method was more efficient than conventional for many reasons like (time, yield, easy ability, solvents, etc.) and also gives us a guide to prepare many compounds by this way. Many of 4-Aryl-2-hydrazinothiazole derivatives were obtained, but actually the openly structured derivatives were more efficient as antibacterial and as anticancer agents, this may be due to the free active N of (NH2, NH-) in some compounds or free (O) especially for six series compounds.
Molecular docking modeling for the synthesized compounds directed us to the effective power of some compounds which is agreed with anticancer and antimicrobial experiments. Some of the compounds showed two active sites at docking results which may be latter lead us to another investigation for them.
ACKNOWLEDGEMENT: The authors are very grateful to Taif University, Taif, KSA, because this work was financially supported by Taif University, Taif, KSA, under project number 1/438/4938.
CONFLICT OF INTEREST: The author(s), declare that there is no conflict of interest.
REFERENCES:
- Gajer JM, Furdas SD, Gründer A, Gothwal M, Heinicke U, Keller K, Colland F, Fulda S, Pahl HL, Fichtner I, Sippl W and Jung M: Histone acetyltransferase inhibitors block neuroblastoma cell growth in-vivo. Oncogenesis 2015; 4: 137.
- Wapenaar H and Dekker FJ: Histone acetyltransferases: challenges in targeting bi-substrate enzymes. Clinical Epigenetics 2016; 8: 59.
- Alhamwe BA, Khalaila R, Wolf J, von Bülow V, Harb H, Alhamdan F, Hii CS, Prescott SL, Ferrante A, Renz H, Garn H, and Potaczek DP: Histone modifications and their role in epigenetics of atopy and allergic diseases. Allergy, Asthma & Clinical Immunology 2018; 1-16.
- Stefanowicz D, Ullah J, Lee K, Shaheen F, Olumese E, Fishbane N, Koo HK, Hallstrand TS, Knight DA and Hackett TL: Epigenetic modifying enzyme expression in asthmatic airway epithelial cells and fibroblasts. BMC Pulm Med 2017; 17(1): 24.
- Cao Z, Wu R, Gao D, Xu T, Luo L, Li Y, Han J, and Zhang Y: Maternal histone acetyltransferase KAT8 is required for porcine preimplantation embryo development. Oncotarget 2017; 8(52): 90250-90261
- Blais LMJA: Transcriptional control of stem cell fate by E2Fs and pocket proteins. Front Genet 2015; 6: 161.
- Bennett DC: Genetics of melanoma progression: the rise and fall of cell senescence. Pigment Cell Melanoma Res. 2016; 29(2):122-40.
- Chimenti F, Bizzarri B, Maccioni E, Secci D, Bolasco A, Chimenti P, Fioravanti R, Granese A, Carradori S, Tosi F, Ballario P, Vernarecci S and Filetici P: A novel histone acetyltransferase inhibitor modulating Gcn5 network: cyclopentylidene- [4- (4' -chlorophenyl) thiazol -2 -yl) hydrazone. Med. Chem. 2009; 52: 530.
- Seccic D, Carradoric S, Bizzarric B, Bolascoc A, Ballariob P, Patramania Z, Fragapanea P, Vernareccib S, Canzonettab C and Fileticia P: Synthesis of a novel series of thiazole-based histone acetyltransferase inhibitors. Med. Chem. 2014; 22: 1680.
- Verma A and Kumar S: Selective Oxidative Decarbonylative Cleavage of Unstrained C(sp3)–C(sp2) Bond: Synthesis of Substituted Benzoxazinones. Lett. 2016; 18(17): 4388-4391.
- Jain KS, Bariwal JB, Kathiravan MK, Raskar VK, Wankhede GS, Londhe NA, and Dighe SN: An efficient and rapid synthesis of 2-amino-4-arylthiazoles employing microwave irradiation in water. Green and Sustainable Chemistry. 2011; 1: 36.
- Díaz JE, Mollo MC, and Orelli LR: Microwave-assisted cyclizations promoted by polyphosphoric acid esters: a general method for 1-aryl -2- iminoazacycloalkanes. Beilstein J. Org Chem. 2016; 12: 2026-2031.
- Suslick KS: Mechanochemistry and sonochemistry: concluding remarks. Faraday Discuss. 2014; 170: 411-422.
- Díaz JE, Mollo MC, and Orelli LR: Microwave-assisted cyclizations promoted by polyphosphoric acid esters: a general method for 1-aryl- 2- iminoazacycloalkanes. Beilstein J Org Chem. 2016; 12: 2026-2031.
- Dewan A, Sarmah M, Thakur AJ, Bharali P, and Bora U: Greener biogenic approach for the synthesis of palladium nanoparticles using papaya peel: An eco-friendly catalyst for C-C Coupling Reaction. ACS Omega 2018; 3(5): 5327 -5335.
- Kaur N: Role of microwaves in the synthesis of fused five-membered heterocycles with three N-heteroatoms. Synthetic Communications Reviews 2015; 45: 430-431.
- Varshney V, Mishra NN, Shukla PK, and Sahu DP: Novel 4-N-substituted aryl but-3-ene-1,2-dione derivatives of piperazinyl oxazolidinones as antibacterial agents. Med. Chem. Lett. 2009; 19: 6810-6812.
- Jambol AA, Hamid MHSA, Mirza AH, Islam MS, and Karim MR: Some novel Schiff bases from pyruvic acid with amines containing N & S donor atoms: Synthesis, Spectral Studies and X-Ray Crystal Structures. International Journal of Organic Chemistry 2017; 7: 42-56.
- El-Sayed HA, Moustafa AH, Haikal AZ, and El-Ashry ESH: Synthesis and antibacterial activity of some glucosyl- and ribosyl-pyridazine-3-ones. Nucleosides Nucleotides Nucleic Acids 2009; 28: 184-192.
- Saad HA, Youssef MM, and Mosselhi MA: Microwave assisted synthesis of some newly fused 1,2,4-Triazines bearing thiophene moieties with expected pharmacological activity. Molecules 2011; 16: 4937-4957.
- Notley SM: Langmuir 2012; 28: 14110-14113.
- Holla BS, Malini KV, Rao BS, Sarojini BK and Kumari NS: Synthesis of some new 2,4-disubstituted thiazoles as possible antibacterial and anti-inflammatory agents. Eur J Med Chem. 2003; 38(3): 313-18.
- Sekar U, Padalkar B, Gupta V, Pharangare K, Patil V and Umape P: Synthesis and antimicrobial activity of novel 2-substituted benzimidazole, benzoxazole and benzothiazole derivatives. J. Chem., 2016, 9: S1125-S1130.
- Alaqeel S: Synthetic approaches to benzimidazoles from ophenylenediamine: A literature review. J. Saudi Chem. Soc., 2017; 21: 229-237.
- Desai N, Joshi V, Rajpara K, Vaghani H, Satodiya H, and Bhatt K: Synthesis and evaluation of N-substituted thiazolidine-2,4-dione containing pyrazole as a potent antimicrobial agent. Anti-infective agent 2014; 12: 85-94.
- Desai N, Shihory N, and Kotadiya GB: Facile synthesis of benzimidazole bearing 2-pyridone derivatives as potential antimicrobial agents. Chem. Lett., 2014; 25: 305-307.
- Padmaja A, Rajasekhar C, Muralikrishna A, and Padmavathi V: Synthesis and antioxidant activity of oxazolyl/ thiazolyl sulfonyl methyl pyrazoles and isoxazoles. J. Med. Chem. 2011; 6: 5034-5038.
- Haggam R, El-Sayed H, Said S, Ahmed M, Moustafa A, and Abd-El-Noora R: O-glycosylation/alkylation and antimicrobial activity of 4, 6-diaryl-2-oxonicotinonitrile J. Heterocyclic. Chem. 2017; 54: 375-383.
- Desai N, Darshan P, and Darshita V: Synthesis and antimicrobial activity of some heterocyclic compounds bearing benzimidazole and pyrazoline motifs. Med. Chem. Res. 2017.
- EsmaeiliN, Ebrahimzadeh H, Abdi K, and Safarian S: Determination of some phenolic compounds in Crocus sativus corms and its antioxidant activities study. Pharmacogn. Mag., 2011; 7(25):74-80.
- Steven S, Richard LH, Stephen AY, and Weidbrauk DL: Clinical virology manual, ASM press, Edition 4th, 2009: 1-692.
- Karegoudar P, Karthikeyan MS, Prasad DJ, Mahalinga M, Holla BS, Kumari NS: Synthesis of some novel 2,4-disubstituted thiazoles as possible antimicrobial agents. European Journal of Medicinal” Chemistry 2008; 43(2): 261-267.
- Chen J, Wang T, Xu S, Lin A, Yao H, Xie W, Zhu Z, and Xu J: Design, synthesis and biological evaluation of novel nitric oxide-donating protoberberine derivatives as anti-tumor agents. European Journal of Medicinal Chemistry 2017; 132: 173-183.
- Walaa M, Nessma M and Gehad M: Mixed ligand complexes of the novel nano ferrocene based Schiff base ligand (HL): Synthesis, spectroscopic characterization, MOE studies, and antimicrobial/anticancer activities. Journal of Organometallic Chemistry 2017; 848: 288-301.
- Abdellattif MH, Helmy MM, and Eldeab HA: New methodology for the synthesis of coumarin derivatives as a potenet IJAPBC 2014; 3(4): 984-990.
- CLSI (Clinical and Laboratory Standards Institute) Performance for antimicrobial disk susceptibility tests; approved standard. 11. Wayne (PA), USA: Clinical and Laboratory Standards Institute; 2012.
- https://archive.org/stream/manualofmicrobio00amer/manualofmicrobio00amer_djvu.txt
- https://arxiv.org/pdf/1701.05870.pdf
- https://www.iarc.fr/en/publications/pdfs-online/epi/cancerepi/CancerEpi-3.pdf
- Macchia ML, Barontini S, Bertini S, Di Bussolo V, Fogli S, Giovannetti E, Grossi E, Minutolo F and Danesi R: Design, synthesis and characterization of the antitumor activity of novel ceramide analogues. Med. Chem., 2001; 44(23): 3994-4000.
- Subudhi BB, Chattopadhyay S, Mishra P and Kumar A: Current strategies for inhibition of chikungunya infection. Viruses 2018; 10(5): 235.
- El-Hashash MA, Rizk SA and El-Sayed AA: “Ultrasonic and solvent-free synthesis of regioselective diastereomeric adducts and heterocyclic products as an antibacterial Journal Advances in Chemistry 2017; 13(12): 6106-6117.
- Hegelund F, Larsen RW, and palmer MH: The vibration spectrum of thiazole between (600 - 1400 cm -1) revisited, a combined high resolution infrared and theoretical study. Journal of Molecular Spectroscopy 2007; 244(1): 63-78.
- Barakat A, Al-Majid AM, Soliman SM, Lotfy G, Ghabbour HA, Fun HK, Wadood A, Warad I and Sloop JC: new diethyl ammonium salt of the thiobarbituric acid derivative: synthesis, molecular structure investigations and docking studies. Molecules2015; 20 (11): 20642-658.
- Abdellattif MH, Maghrabi IA, Areef MMH, ElDeab HA, Mouneir SM, and Belal A: Efficient Microwave-assisted solvent-free synthesis and molecular docking studies of 2-pyridone derivatives as anticancer agents and evaluation of cytotoxic effects. Journal Advances of Chemistry 2016; 1 2: 4, 4351-4364.
- Hojjat-Farsangi M: Targeting non-receptor tyrosine kinases using small molecule inhibitors: an overview of recent advances. J Drug Target 2016; 24(3):192-211.
How to cite this article:
Abdellattif MH, Hussien MA and Alzahrani E: New approaches of 4-aryl-2-hydrazinothiazole derivatives synthesis, molecular docking, and biological evaluations. Int J Pharm Sci & Res 2018; 9(12): 5060-78. doi: 10.13040/IJPSR.0975-8232.9(12).5060-78.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
Article Information
4
5060-5078
1,102
1439
English
IJPSR
M. H. Abdellattif *, M. A. Hussien and E. Alzahrani
Department of Pharmaceutical Chemistry, Deanship of Scientific Research, Taif University, 21974, KSA.
m.hasan@tu.edu.sa
25 April, 2018
15 June, 2018
20 June, 2018
10.13040/IJPSR.0975-8232.9(12).5060-78
01 December, 2018