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NOVEL UREA AND THIOUREA DERIVATIVES OF THIAZOLE-GLUTAMIC ACID CONJUGATE AS POTENTIAL INHIBITORS OF MICROBES AND FUNGI
© 2013 n A. Sharma, R. Suhas, S. Chandan, D. C. Gowda#
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore, Karnataka, 570006 India
Received March 21, 2013; in final form, April 17, 2013
Since discovery and development of effective as well as safe drugs has brought a progressive era in human healthcare that is accompanied by the appearance of drug resistant bacterial strains, there is constant need of new antibacterial agent having novel mechanisms of action to act against the harmful pathogens. In the present study, several N-terminal substituted urea/thiourea derivatives were synthesized by the reaction of glutamic acid and 3-(1-piperazinyl)-1,2-benzisothiazole with various substituted phenyl isocyan-ates/isothiocyanates. Elemental analysis, IR, XH NMR, 13C NMR and mass spectral data confirmed the structure of the newly synthesized compounds. The derivatives were investigated for their antibacterial and antifungal activities against various pathogens of human origin by agar well diffusion method and microdilution method. The preliminary antimicrobial bioassay reveals that the compounds containing fluoro and bromo as substituents showed promising antimicrobial activity.
Keywords: glutamic acid, thiazole, conjugation, urea/thiourea, antimicrobial
DOI: 10.7868/S0132342313050138
INTRODUCTION
Development of new antimicrobial agents with novel structure and mode of action remains the primary goal of scientists for the solution of increasing bacterial resistance gained by microorganism to classical antimicrobial agents [1]. Nitrogen containing heterocycles with sulfur atom are an important class of compounds in medicinal chemistry. Thiazoles and their derivatives being an integral part of many potent biologically active molecules such as sulfathiazole (antimicrobial drug), ritonavir (antiretroviral drug), abafungin (antifungal drug) with trade name Abasol cream and bleomycin and tiazofurin (antineoplastic drugs) have been explored previously [2]. The benzothiazole ring is present in various marine or terrestrial natural compounds which have useful biological activities [3]. Benzothiazole derivatives have attracted a great deal of interest due to their anticancer [4], antitumor [5], antibacterial, antimicrobial and fungicidal activities [6]. Urea/thiourea derivatives display a wide range ofbiological activities including antibacterial, antifungal, antitubercular, antithyroid, an-tihelmintic, rodenticidal, insecticidal, herbicidal, and plant growth regulator properties [7—9].
Abbreviations: Boc — tert-Butyloxycarbonyl; EDCI — 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; Het — 3-(1-Piperazi-nyl)-1,2-benzisothiazole; HOBt — N-Hydroxybenzotriazole; NMM — N-Methyl morpholine; TFA — Trifluoroacetic acid. # Corresponding author (phone: +91 821 2419664; e-mail: dchannegowda@yahoo.co.in).
The therapeutical applications of amino acids have received a considerable attention in respiratory physiology, cardiology, renal failure, neurological disorders, and congenital defects. In recent years, various drug— amino acid conjugates were reported [10] and it is well known that amino acids are essential starting materials for cell growth and building blocks for protein synthesis. Therefore, a possible strategy for increasing the transport of lipophilic compounds of biological interest across cell membrane is the conjugation with ami-no acids [11]. Glutamic acid is a flexible molecule and various conformations corresponding to different groups present may define different bioactive conformations that activate each single type of glutamate receptors [12].
These apart, previous investigations from our group [13—20] have shown that compounds derived from amino acid/peptide conjugates act as biologically active agents. In continuation of these efforts, it has been considered of interest to incorporate all these biologically active components together to give a confined structure for evaluating its antimicrobial activity.
RESULTS AND DISCUSSION
Boc-Glu(OBzl)-OH was conjugated to Het using EDCI/HOBt as coupling agent and NMM as base to get compound (I). The side chain benzyl group of (I) was deprotected with 1N NaOH/MeOH to get (II) which was conjugated with Het to get conjugate (III).
Boc group of (III) was deblocked using TFA and further reacted with isocyanates and isothiocyanates to get urea and thiourea derivatives respectively (Scheme). All the derivatives (IVa—u) were obtained in high yields. The evidence for the formation of derivatives was obtained by IR, XH NMR, 13C NMR, mass and elemental analysis. The presence of strong peak at 3304—3399 cm-1, 2029-2048 cm-1 and 1629-1642 cm-1 in IR spectra
confirmed absorption bands for -NH-, -C=S, and -C=O groups, respectively. 1H NMR spectra for all the synthesized compounds showed multiplets for the aromatic protons at 6 7.21-7.93 and urea/thiourea NH protons at 6 8.03-8.25 and 8.23-8.77, respectively. 13C NMR spectral analysis of the urea and thiourea showed typical absorptions in the range 6 154.49155.36 and 6 180.62-180.86, respectively.
V
(III)
TFA, 45 min, rt
O
NMM, DMF
N— S x-c6h4-n=c=z
0°C-rt
O
(IVa—u)
(IVa) X = H, Z = O (IVb) X = H, Z = S (IVc) X = 2OCH3, Z = O (IVd) X = 2OCH3, Z = S (IVe) X = 3OCH3, Z = O (IVf) X = 3OCH3, Z = S (IVg) X = 4OCH3, Z = O (IVh) X = 4OCH3, Z = S
Scheme
(IVi) X = 2Br, Z = S (IVq) X = 2F, Z = O (IVj) X = 3Br, Z = O (IVr) X = 2F, Z = S (IVk) X = 3Br, Z = S (IVs) X = 3F, Z = O (IVl) X = 4Br, Z = O (IVt) X = 4F, Z = O (IVm) X = 3Cl, Z = O (IVu) X = 4F, Z = S (IVn) X = 3Cl, Z = S (IVo) X = 4Cl, Z = O (IVp) X = 4Cl, Z = S
In the present study, synthesized compounds (I, II, rial and antifungal activities against microbial strains III, IVa—u) were evaluated for their in vitro antibacte- such as Gram negative bacteria (Escherichia coli, Ral-
stonia solanacearum and Xanthomonas oryzae), Gram positive bacteria (Klebsiella pneumoniae) and fungi (Aspergillus niger, Aspergillusflavus and Fusarium oxy-sporum) by agar well diffusion method and microdilution method. Known antibiotics like Streptomycin (the reference antibacterial drug) and Bavistin (the reference antifungal drug) were used for comparison. The results of antimicrobial testing against tested strains were obtained as zone of inhibition (mm) and minimum inhibitory concentration (^g/mL) as tabulated in Table 1 and 2 respectively.
It is evident from the results that the heterocycle conjugated glutamic acid (I) has exhibited enhanced activity compared to heterocycle tested alone which is inactive. When side chain benzyl ester was removed, the resultant COOH derivative (II) showed increase in activity. This may be due to the increase in the polarity of the compound [18] which would help the molecule to interact or penetrate more through the cell membrane of microbes and there by inactivating them. This indicates that increase in polarity has an impact on the activity. Further, this was conjugated to Het to get double heterocyclic conjugate (III) which showed slight enhancement in the activity than (I). This explained that double conjugation play a role in improving the activity.
Among the derivatives synthesized, compounds containing thiourea group (IVb, d, f, h, i, k, n, p, r, u) have exhibited enhanced activity compared to their urea analogues (IVi, c, e, g, j, l, m, o, q, s, t). This would be due to electronegativity and larger size of sulfur atom which increases its ability to interact both with electron poor and with electron rich functional groups which help the molecule bind to receptor more efficiently [21]. Thioureas containing thiocarbonyl group act as versatile ambidendate donor ligand which would help these molecules to bind the receptor [22].
The result of the derivatives largely depends on the nature of the substituent group on the phenyl ring of urea/thiourea. The effectiveness detected for the derivatives seems to be governed in parts by the presence of the substituents on the phenyl ring of urea/thiourea. Compounds without substituents (halogen or OMe) have shown less activity than their counterparts. This shows that presence of substituent on the phenyl ring is critical to enhance the activity. In general, compounds with electron withdrawing groups were more active when compared with electron donating groups. It seems also interesting to point out that among the isomeric substituted compounds the position of the substituent exerts in general, a certain effect on the activity against all the microorganisms. The preferential order of the substituent for the activity was found to be p > o > m. In the series, fluoro containing derivatives (IVq—u) showed effective activity when compared with chloro (IVm—p) and bromo (IVi—l) derivatives, while the methoxy (IVc—h) is comparable. This may be due to electronic, lipophilic and steric parameters [23] in
halogens due to which increase in activity was witnessed. The presence of fluorine in the molecules may increase the lipophilicity and thus enhance the rate of cell penetration and transport of the inhibitor to an active site. The higher polarizability due to the C—F bond may give new possibilities for binding to the receptor [24]. Also the results indicate that increase in the size and decrease in the electronegativity of the halogens renders the compounds less active. Further, activity towards gram negative bacteria was more compared to gram positive microorganisms. This feature may be attributed to the presence of more electron withdrawing elements in the compounds which would help in crossing the microbial membrane and thereby rupturing the cells.
To summarize, a series of urea/thiourea derivatives of glutamic acid conjugated to Het was synthesized and characterized. These compounds were screened for their antibacterial and antifungal activities and it was found that som
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