Synthesis, Characterization and Antimicrobial Evaluation with DFT Study of New Two-Amino-4-(4-Chlorophenyl) Thiazole Derivatives

79 Synthesis, Characterization and Antimicrobial Evaluation with DFT Study of New Two-Amino-4-(4-Chlorophenyl) Thiazole Derivatives Nedaa A. A. Rahim and Ammar A Mahmood Kubba* Department of Pharmaceutical Chemistry, College of Pharmacy ,University of Baghdad, Baghdad, Iraq. Abstract 2-amino-4-(4-chloro phenyl)-1,3-thiazole (1) was synthesized by refluxing thiourea with parachloro phenacyl bromide in absolute methanol. The condensation of amine compound (1) with phenylisothiocyanate in the presence of pyridine will produce 1-(4-(4-chlorophenyl)thiazol-2-yl)-3phenylthiourea(2), which is upon treatment with 2,4 dinitrophenyl hydrazine by conventional method, afforded 1( 4 ( 4 – chlorophenyl ) thiazol – 2 – yl ) – 3 phenylhydrazonamide,N' ( 2 , 4 dinitrophenyl) ,(3).The characterization of the titled compounds were performed utilizing FTIR spectroscopy, HNMR and CHNS elemental analysis, and by measurements of their physical properties. The synthesized compounds had been screened for their, in vitro preliminary antimicrobial activity against three Gram-positive bacteria: (Staph. aureus, Micrococcus luteus and Bacillus subtilis) and three Gram-negative bacteria : (Pseud.aeruginosa, E.coli and Proteus mirabilis) ,and two fungal strains(Candida albicans and Candida glabrata ), using a minimum inhibitory concentration (MIC) of 100 μg\ml of test compound, by well diffusion method. The derivatives showed moderate antibacterial activity against Gram-positive Staphylococcus aureus and Bacillus subtilis & high antifungal activity against Candida glabrata and Candida albicans. Computational study was performed to calculate some of thermodynamic parameters by using density functional theory (DFT)


Introduction
Thiazoles are significantly important heterocyclic that represent interesting properties depending on the linked groups to the thiazole (1) . 2-aminothiazoles are a subclass of thiazole family that are of high interest due to their broad biological activity., fungicidal (2) , antioxidative (3) , analgesic (4) , bactericidal (5) and anti-inflammatory (5) ,anti-HIV (6) , antitubercular (7) , Thiazole containing N=C=S moiety have been used as antiphychotics (8) and antimalarial (9) , and as herbicidal (10) .Thiazoles have broadly been engaged as effective γsecretase inhibitors to hinder Alzheimer's disease (11) and have a lot of derivations and because of its derivations type have a lot of application in various industries, medical science, and pharmacy.In 1887, Hantzsch et al identified and established molecular structure of thiazoles. In 1889, the first derivation of thiazoles identified by Prop et al and was named 2-amino thiazole (12) . After that until now, many derivations identified and reported (13,14). Thiazole and related compounds are called 1 , 3 -azoles ( nitrogen and one other heteroatom in a fivemembered ring ). They are isomeric with the 1 , 2bazoles , the nitrogen , and sulfur compound being called isothiazole (15) compounds from readily available reagents is one of the major challenges in organic chemistry. Therefore to meet the facile results of these tough challenges thiazole nucleus was being considered. Among the wide variety of heterocycles that have been explored for developing pharmaceutically molecules (16) .Our study focuses on derivatization of new molecules starting from the parent nucleus 2amino-4-(4-chloro phenyl) 1,3-thiazole (1), by treatment with phenyl isothiocyanate, to yield (2), then refluxing it with to-2,4-dinitrophenyl hydrazine to produce (3), subsequently evaluation of the produced compounds in vitro for their preliminary antimicrobial activity ,also using DFT study to identify the changes in electronic structure responsible for farmacological action (17) .

Material and Methods
Chemicals used during the synthesis, supplied by hyper-chem/china. Completion of reactions and the purity of compounds were ascertained by thin-layer chromatography (TLC) , using Silica gel GF254 (type 60) precoated Aluminium sheets, Merck (Germany) exposed to UV-254nm light and the eluent used is n-hexane: ethyl acetate (6.0:4.0) for compound (1) , n-hexane: ethyl acetate (8.0:2.0) for compound (2) and n-hexane: ethyl acetate (4.0:6.0) for compound (3).Melting points were measured by using Stuart SMP3 melting point apparatus in open capillary tubes, and are uncorrected. The infrared spectra were performed in KBr disc, (υ,cm -1 ), using FTIR Spectrophotometer (Shimadzu, WQF-520, Japan) at the University of Baghdad College of Education for Pure Sciences Ibn Al-Haitham. The CHNS elemental microanalysis of the final synthesized products was done using (Elemental Vario MICRO cube instrument, Germany) in the University of Mustansiriya-College of Pharmacy.

General method for the Synthesis of 2-Amino-4-( 4 -chlorophenyl ) thiazole (1) Method 1 ( 18) :
A mixture of p-chloro phenacyl bromide (0.01 mol,2.3g) in 100 ml of absolute alcohol and thiourea (0.02 mol, 1.5g) were taken in a round bottom flask and refluxed for 3 h. Then the reaction mixture was cooled and precipitated with cold water. The solid separated was collected by filtration and the solvent was removed under vacuum. The residue obtained was dried, recrystallized from ethanol.  ( 19) A mixture of p-chloro phenacyl bromide (0.0064 mol, 1.5 g), and thiourea (0.0064 mol, 0.489 g) in 10 ml of DMSO was stirred at room temperature until completion of the reaction. The time of the reaction was monitored by a stopwatch. The progress of the reaction was monitored by thin-layer chromatography. On completion of the reaction, the reaction mixture was poured into crushed ice. The precipitated product was filtered and dried and recrystallized from ethanol. The product was pure enough (single spot on TLC) for all practical purposes.

Antimicrobial screening
The antimicrobial activities of the synthesized derivatives were measured using well diffusion technique with a comparison to cefotaxime sodium(cefot.) and sulfamethoxazole (sulf.) as standard antibacterial agents ,and miconazole as standard antifungal agent, using dimethylsulfoxide (DMSO) as solvent and as a control, and it was run in the Ministry of Health/ National Center for Drug Control (2) and (3) in DMSO as shown in table 1.

Results and Discussion
Chemistry The 2-Amino-4-(4-chlorophenyl) thiazole (1) synthesis, was carried out according to Hantzsch method 22 , by reaction of phenacyl bromide with thiourea in the presence of absolute methanol for 3 hours. They are characterized by FTIR, due to the appearance primary amine (NH2) stretching at 3438 and 3284 cm -1 and then reaction of (1) with phenylisothiocyanate in the presence of pyridine to produce (2) which was characterized by FTIR, a band of( C=S) stretching displayed at 1254cm -1 , and NH stretching band at 3159 cm -1 ,and finally condensation of (2) with, 2,4 dinitrophenyl hydrazine using absolute ethanol as a solvent. to afford (3) which showed the characteristic NO2 ( asymmetrical and symmetrical)stretching at 1512 and 1331 cm -1 , respectively, also peak attributed to(C=N) band recorded at 1614 cm -1 .

Synthesis of compound (1)
The method of parent nucleus synthesis was described by Hantzsch, in 1887 the cyclization of α-halo carbonyl compounds by a great variety of reactants containing the N-C-S fragment of the ring is still the widely used method of synthesis of thiazoles ,as shown in the following scheme (2) (22) .

Synthesis of compound (3)
The reactivity of phenyl isothiocyanate is, determined by two active centers the nucleophilic nitrogen atom, and thiocarbonyl groups, which make them capable of participating in diverse type of addition and cyclization reactions. The unshared pair of electron of NH2 group of 2,4-dinitro phenyl hydrazine would attack thiocarbonyl carbon(C=S) of (2) and in the presence of acidic hydrogen of ethanol, H2S gas would be evolved, and the tatumeric structure of (3), would also formed (24) , as shown in the scheme 4.

Scheme4. Mechanism of Synthesis of (3) Anti microbial Activity
From the results illustrated in table1, showed that the new synthesized derivative (2 and 3) have moderate activity against Grampositive bacteria Staph. aureus and Bacillus subtilis also , both compounds have no antibacterial activity against Gram-negative bacteria.It is evident that the tested compounds demonstrated the most potent antifungal activity against Candida albicans and Candida glabrata.

Computational Studies Density function theory (DFT) study
In order to explore the theoreticalexperimental consistency, quantum chemical calculations were performed with complete geometry optimizations using standard Spartan 10 software. Geometry optimization was carried out by B3LYP/6-31G* level of theory. The chemical reactivity descriptors calculated using DFT are: total energy (E), log p and electronegativity (μ).
On the basis of frontier molecular orbitals, chemical hardness corresponds to the gap between the HOMO and LUMO , where ELUMO and EHOMO are the LUMO and HOMO energies. electronegativity (25) is defined as the negative of electronegativity of a molecule, Physically,( μ) describes the escaping tendency of electrons from an equilibrium system (26) . It is a measure of the stabilization in energy after a system accepts additional amount of electronic charge from the environment

Structural and electronic properties
DFT calculations were performed for compound 1-3. Optimized molecular structures of the most stable form are shown in Figure 1.

Comp.
No.  It can be seen that the energy gaps between HOMO and LUMO for compound 1 is 6.915, compound 2 is -5.85, compound 3 is 1.425. The lower value in the HOMO and LUMO energy gap explains the eventual charge transfer interaction taking place within the molecules. The larger the HOMO-LUMO energy gap, more stable/less reactive the molecule, for example, compound 1. The values of (μ ) for compounds 1, 2 and 3 are presented in (Table 1 row 5). The trend in electronegativity for compounds 1-3 are: 2 > 3 >1 . The greater the electronic chemical potential, the less stable or more reactive is the compound. Therefore, compound 2 is more reactive than compounds 3 and 1, respectively. Therefore, compound 2 is the most reactive, and 3 and 1 are the least reactive.

Electrostatic potential charges and related quantum chemical properties
The distribution of the electronic density (electrostatic potential charges), related quantum chemical parameters; HOMO/LUMO gap (Table 1, row 4), the trends in energy gap 1> 3> 2 .On the other hand log p provides a measure of a molecule's lipophilicity, where high log p-value indicates high lipophilicity, while low value suggests low lipophilicity. the partition coefficients of the compounds (log p), (Table 1, row6) were calculated for observed compounds 1-3. , The results indicated that compounds having the highest lipophilicity were 3 > 2 > 1 with corresponding values 6.66, 6.04 and 3.61,respectively. These values and properties are very useful and can be used in order to evaluate chemical properties and possibilities for interaction of compounds with biological macromolecules (receptors, enzymes) (27) .

Conclusion
A new derivatives of 2-amino thiazole were successfully synthesized using conventional method and tested for their preliminary antimicrobial activity,using well diffusion method. The synthesized compounds displayed slight antibacterial activities against Gram-positive types, while demonstrated no antibacterial activities against Gram-negative bacteria, and high antifungal activity against tested fungal species. The quantum chemistry calculations using the Density Function Theory (DFT) method was performed to study stability of compounds 1-3. The results showed that the compound 1 is more stable than compound 3, as was indicated via calculations of the larger the HOMO-LUMO energy gap parameter (28) .