Iraqi J Pharm Sci, Vol.21(1) 2012 Synthesis of thiadiazole derivatives 98 Synthesis and Preliminary Antimicrobial Study of 2-Amino-5- Mercapto-1,3,4-Thiadiazole Derivatives Husam A. Ameen* and Ahlam J. Qasir *,1 *Department of Pharmaceutical Chemistry, College of Pharmacy, University of Baghdad, Baghdad, Iraq ** Ibn-Zuhr Hospital ,Ministry of Health ,Baghdad, Iraq. Abstract Nitrogen heterocycles are of a special interest because they constitute an important class of natural and non natural products, many of which exhibit useful biological activities.Among these nitrogen heterocycles are 1, 3, 4-thiadiazole containing compounds. The therapeutic effects of these derivatives have been well studied for a number of pathological conditions including inflammation, pain, or hypertension. Moreover, synthesis of thiadiazoles has attracted wide-spread attention due to their diverse applications as antibacterial, anticancer, antifungal anti-inflammatory and antidepressant agents.According to this information’s new derivatives of 1, 3, 4-thiadiazole were designed and synthesized and in the hope of having some activities as antibacterial and antifungal. These are:  4-(((5-mercapto-1,3,4-thiadiazol-2-yl)imino)methyl)-2-methoxyphenol.  Tert-butyl (1- ((2- ((5- (methylsulfonyl) -1,3,4- thiadiazol-2-yl) amino) -2- oxoethyl)amino) -1- oxopropan - 2-yl) carbamate. Key words: 1, 3, 4-thiadiazole, biological activity, peptides, Schiff base. -4، 1،3-ميركابتى -5-امينى -2تخهيق ودراست تمهيذيت مضاداث انمايكروباث نمشتقاث ايادايازولث أحالو جميم قصير* و** أمين عامرحساو ،1 * فسع انكًٍٍبء انصٍدالٍَخ ،كهٍخ انصٍدنخ ، جبيعخ ثغداد ،ثغداد ، انعساق . .شْس ، ٔشازح انصحخ ، ثغداد ، انعساق اثٍ** يسزشفى الخالصــــة ، انطجٍعٍخ ٔغٍس نهًُزجبد انطجٍعٍخ فئخ ْبيخ رشكم ألَٓبرًثم أْزًبيب خبصب انٍُزسٔجٍٍ يٍ انًسكجبد انغٍس يزجبَسخ ًْ انًسكجبد انزً رحزٕي عهى حهقخ انٍُزسٔجٍٍ يٍ يٍ ثٍٍ ْرِ انًسكجبد انغٍس يزجبَسخ .ًفٍدحان األَشطخ انجٍٕنٕجٍخ ٌحًمٔكثٍس يُٓب ازرفبع أٔ، ٔاألنى ، االنزٓبة ثًب فً ذنك انحبالد انًسضٍخ نعدد يٍ يدزٔسخ انًشزقبد انعالجً نٓرِ انزأثٍس ثٍبدٌبشٔل. – 4، 3، 1 يضبد نهجساثٍى،، ثسجت رطجٍقبرّ انًزُٕعخ يثم اَزشبزأاسع االْزًبو صُبعخ ثٍبدٌبشٔل، فقد اجزرثذ ٔعالٔح عهى ذنك .ضغظ اندو رحضٍس يشزقبد رى رصًٍى ٔٔاعزًبدا عهى ْرِ انًعهٕيبد ، .، يضبد نالنزٓبثبد ٔيضبدح نالكزئبةبديضبد نهفطسٌ، يضبد نهسسطبٌ : ْٔرِ ًْ.يع رٕقع احزًبل اٌ ٌكٌٕ نٓب فعبنٍخ كًضبد ثكزٍسٌب ٔيضبد فطسي ثٍبدٌبشٔل – 4، 3، 1حهقخ جدٌدح يٍ  4-(((5-mercapto-1,3,4-thiadiazol-2-yl)imino)methyl)-2-methoxyphenol.  Tert-butyl (1-((2-((5-(methylsulfonyl)-1,3,4-thiadiazol-2-yl)amino)-2-oxoethyl)amino)-1- oxopropan-2-yl) carbamate. ، انفعانيت انبايىنىجيت ، بينيذاث ، قاعذة شيف . يازولاياداث – 4، 3، 1انكهماث انمفتاحيت : Introduction Over the past decade, drug resistance has become a growing problem in the treatment of infectious diseases caused by bacteria, fungi and viruses. In particular, resistance of bacterial pathogens to current antibiotics has emerged as a measure health problem. This is especially true in case of infectious diseases such as pneumonia, meningitis and tuberculosis, which would once have been easily treated with antibiotics, but is no longer so readily treated. At present, all widely used antibiotics, including some of the agents such as streptogramins and new generation flouroquinolones are subjected to bacterial resistance. The search for new antimicrobial agents is one of the most challenging tasks to the medicinal chemist (1) . Thiadiazole Thiadiazole is a five membered heterocyclic compounds that show various types of biological activity. It contains two nitrogen atoms and one sulphur atom as hetero atoms. There are several isomers of thiadiazole (Fig. 1), that is a- 1, 2, 3 Thiadiazole , b- 1,2,4 Thiadiazole, c- 1,3,4 Thiadiazole, d- 1, 2, 5 Thiadiazole (2) . Figure 1: Structures of Thiadiazole isomers 1 Corresponding author E- mail : ahlamqasir@yahoo.com Received : 24/12/2011 Accepted : 21/4/2012 mailto:ahlamqasir@yahoo.com Iraqi J Pharm Sci, Vol.21(1) 2012 Synthesis of thiadiazole derivatives 99 It acts as a “hydrogen binding domain” and “two-electron donor system”. Thiadiazole acts as a bioisosteric replacement of thiazole moiety. So, it is act as a one component of third and fourth generation cephalosporin (3) .1, 3, 4-Thiadiazole is the isomer of thiadiazole series. A glance at the standard reference works shows that more studies have been carried out on the 1,3,4 Thiadiazole than all the other isomers combined. Members of this ring system have found their way in to such diverse applications as pharmaceuticals, oxidation inhibitors and metal complexing agents (4) . During last few years there has been intense investigation of different classes of thiadiazole compounds, many of which known to possess interesting biological properties such as antimicrobial (5) ,antituberculosis (6) , anti- inflammatory (7) , analgesic (8) , anticonvulsants (9) , antihypertensive (10) , antioxidant (11) , anticancer (12) and antifungal (13) ,antiviral (4) ,antidepressant (14) activity.Among them 2,5- disubstituted 1,3,4-thiadiazole derivatives posses interesting biological activity probably conferred to them due to strong aromaticity of the ring system which leads to great in vivo stability and generally, a lack of toxicity for higher vertebrates, including humans when diverse functional group that interact with biological receptor are attached to aromatic ring (3,15) .Schiff bases are known to have biological activities such as antibacterial (16) , antifungal (17) , antitumor (18) and antioxidant activities (19) . Schiff bases appear to be important intermediates in a number of enzymatic reactions involving interaction of an. enzyme with an amino or a carbonyl group of the substrate. One of the most prevalent types of catalytic mechanisms in biochemical processes involves condensation of a primary amine in an enzyme, usually that of a lysine residue, with a carbonyl group of the substrate to form an imine, or Schiff base (20) . The biological properties of thiadiazole derivatives aroused our interest in to design and synthesize new derivatives of thiadiazole then subjected them to investigate their possible biological activity (as antibacterial & antifungal activities). Experimental work Chemicals and Equipments Boc-L-alanine, Carbon disulfide, Dicyclohexylcarbodiimide (DCC ), Hydrogen peroxide, 1-Hydroxybenzotriazole (HOBt), Dimethylsulfoxide (DMSO), Glycine, Methyl iodide, N-Methyl morpholine (NMM), Thionyl chloride, Vanillin.All the solvents and materials used were of Analar type and used without further purification. Infrared spectral determination was performed for all compounds in KBr disk, using FTIR at the collage of science (Baghdad University).Elemental analysis has been done using Carlo Erba elemental analyzer. The analysis was done in Al-al Bayt University in Al-Mafraq (Jordan). Chemical synthesis Synthesis of 2-amino -5-mercapto -1, 3, 4- thiadiazole compound (I) (21) Thiosemicarbazide (4g , 0.043 mole) was suspended in absolute ethanol (30ml) in round bottom flask (250ml), anhydrous sodium carbonate (2.23g, 0.021mole) and CS2 (9.5g , 0.125mole) were then added respectively with continues stirring.The reactant mixture was refluxed for 5hours; the reaction mixture was then allowed to cool to room temperature and filtered. The filtrate was evaporated under vacuum then cold distilled water (90 ml) was added, acidification with concentrated HCl drop by drop was carried out, white –yellowish precipitate was formed, the precipitate was collected by filtration, and washed with distilled water, re-crystallized using hot distilled water. The physical appearance, percentage of yield, melting point and Rf values were listed in tablet 1, the elemental analysis results are presented in table 2 while the IR data are shown in table 3. . Table 1: Physical appearance, percentage of yield, melting points, Rf values of intermediates and final compounds Compound No. Physical appearance Yield % Melting point ( o C) Rf Values Observed Reported A B I Faint yellow crystal 65% 232-234 230-232 0.40 0.33 II Yellow powder 80% 177-178 178-181 0.45 0.38 III Yellow powder 75% 205-208 - 0.30 0.48 IV Orange powder 77% 245-246 - 0.55 0.28 V Dark yellow powder 62% 185-188 - 0.22 0.29 A) Chloroform: ethanol (9:1), B) Acetone: Ethyl acetate (1:2) (25) . Iraqi J Pharm Sci, Vol.21(1) 2012 Synthesis of thiadiazole derivatives 100 Table 2: Elemental microanalysis of the final compounds Compound Molecular weight Chemical formula Elemental microanalysis % Element Calculated Observed IV 267.01 C10H9N3O2S2 C 44.93 45.027 H 3.39 3.386 N 15.72 15.951 O 11.97 11.473 S 23.99 24.163 V 573.17 C26H31N5O6S2 C 54.43 55.242 H 5.45 5.493 N 12.21 12.577 O 16.73 16.086 S 11.18 10.624 Table 3: Characteristic IR absorptions of compounds I- V Compound Band (cm -1 ) Interpretation I 3330,3317 2614 1608 1554 1172 1058,1122 N-H stretching vibration of the Primary amines. S-H stretching of thiol. C=N stretching of thiadiazole ring moiety. N-H bending. C-N stretching of the thiadiazole ring moiety. C=S stretching vibration gives evidence that compound (I) can exist in two tautomeric form, thiol form and thion form. II 3332,3282 2945,2873 1629 1521 1373 1139 N-H stretching vibration of the Primary amines. C-H stretching vibration of CH3 (symmetrical & asymmetrical). C=N stretching vibration of the thiadiazole ring moiety. N-H bending. C-H bending vibration of CH3. C-N stretching of thiadiazole ring moiety. III 3398,3423 2939,2864 1624 1498 1423 1319, 1168 N-H stretching vibration of the Primary amines. C-H stretching vibration of CH3. C=N stretching vibration of the thiadiazole ring moiety. N-H bending. C-H bending vibration of CH3. S=O stretching vibration of sulfone (symmetrical & asymmetrical). IV 3200-3500 3097-3141 2925 2554 1614 1554 1477,1363 1363 1174 1058 756 O-H stretching of phenol. C-H stretching vibration of aromatic ring. C-H stretching vibration of CH3. S-H stretching of thiol. C=N stretching. C=C stretching of aromatic ring. C-H bending of saturated carbon. O-H bending of phenol. C-O stretching of ether overlap with C=S stretching.. Aromatic C-H in plane bending. Aromatic C-H out of plane bending. V 3328 2930 2850 1671 1625 1574 1338,1149 1226 1180 N-H stretching of amide. C-H asymmetrical stretching vibration of CH3&CH2. C-H symmetrical stretching vibration of CH3&CH2. C=O stretching of amide and carbamate. C=N stretching. N-H bending of amide overlap with C=C stretching of aromatic ring. S=O symmetrical & asymmetrical stretching of sulfone. C-O stretching of ester. C-N stretching. Iraqi J Pharm Sci, Vol.21(1) 2012 Synthesis of thiadiazole derivatives 101 Synthesis of 5-(methylthio)-1, 3, 4- thiadiazole-2-amine compound (II) (22) Compound (I) (1.33 g, 0.01mole) was dissolved in the minimum volume of distilled water and sufficient volume of KOH (85%) solution was added under stirring at room temperature, after (5-10 minute), the solution was brought to 0°C in ice bath and then methyl Iodide (0.625ml, 0.01mole) was added with vigorous stirring at a rate of 1drop every 2min, continuous stirring was maintained for 3hours. the solution then was filtered, the precipitate was washed with water, dried to give compound (II), which was used without further purification. The physical appearance, percentage of yield, melting point and Rf values were listed in tablet 1, the elemental analysis results are presented in table 2 while the IR data are shown in table 3. Synthesis of 5-(methylsulfonyl)-1, 3, 4- thiadiazole-2-amine, compound (III) (23) Compound (II) (0.147 g, 1mmol) was dissolved in ethanol (95%) (30ml), H2O2 (0.068 g, 2mmol) was added with continues stirring at room temperature for 2 hrs. Then the excess of solvent was evaporated to give compound (III), which was used without further purification. The physical appearance, percentage yield, melting point and Rf values were listed in tablet 1, the elemental analysis results are presented in table 2 while the IR data are shown in table 3. Synthesis of 4-(((5-mercapto-1, 3, 4- thiadiazol-2-yl) imino) methyl)- 2methoxyphenol, (compound IV) (24) Compound I (0.266 g, 2 mmole) was suspended in 25 ml of absolute ethanol then vanillin (0.304 g, 2 mmole)dissolved in 25 ml of absolute ethanol solution was added. The mixture was refluxed for 8 hrs, and then left overnight. The solvent was evaporated in vacuum and the residue was re-crystallized from methanol. The physical appearance, percentage of yield, melting point and Rf values were listed in tablet 1, the elemental analysis results are presented in table 2 while the IR data are shown in table 3. Synthesis of tert-butyl (1-((2-((5- (methylsulfonyl)-1, 3, 4-thiadiazol-2-yl) amino)-2-oxoethyl) amino)-1-oxopropan-2-yl) carbamate, (compound V) (25) To a stirred solution of the dipeptide (Boc-Ala-Gly-OH) (0.246 g, 1 mmole) in DMF (3 ml), NMM (0.11 ml, 1 mmole) was added followed by stirring for 10 minutes. Solution of compound III (0.179 g, 1 mmole) in DMF (3 ml) was added to the reaction mixture. The mixture was then cooled to (- 15°C), then HOBt (0.3 g, 2 mmole) was added followed by DCC (0.23 g, 1 mmole) with stirring which was continued for 72 hrs., at (0°C) and for 48 hrs., at ambient temperature. Ethyl acetate (10 ml) was added to the reaction mixture which was then filtered to get rid of N, N-dicyclohexylurea (DCU). The filtrate was evaporated to dryness under vacuum, and the residue was re-dissolved in ethyl acetate (10 ml), the excess DCU which was still adhesive on the peptide residue was precipitated out and filtered. The clear filtrate was washed twice with (5 ml) HCl (0.1 N) solution, once with (10 ml) D.W. , and with (10 ml) saturated NaCl solution using the sepertaory funnel. The ethyl acetate layer was dried using anhydrous magnesium sulfate then the solvent was evaporated to get compound V which was re- crystallized from (Ethyl acetate: Petroleum ether 40-60) mixture. Physical appearance, percentage of yield, melting point and Rf value are listed in table 1, the elemental analysis results are presented in table 2 while the IR data are shown in table 3. Antimicrobial activity (26) The synthesized compounds were screened for the presence of antibacterial constituents against four strains of bacteria i.e. Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, beta-hemolytic- Streptococcus pyogenes and one species of fungi i.e. against Candida albicans by disc diffusion method. Nutrient agar was used as culture medium for bacterial growth; blood agar was used as culture medium for beta- hemolytic-Streptococcus pyogenes growth, while fungi were subcultured in Sabouraud dextrose agar medium. All compounds were dissolved in DMSO at concentration 0.625 mg/disc * . Gentamycin [CN] (30mcg/disc for bacteria), Amoxicillin/clavulanic acid [AMC] (30mcg/disc for bacteria), and ketoconozole (100 units/disc for fungi) was used as reference antibiotic and DMSO as control. The zones of inhibition were determined at the end of an incubation period of 24 hr at 35 ◦ C for bacteria growth and 5 days at 28 ◦ C for Fungi growth. The inhibition zone values are summarized in table 4. This study is done in collage of science (Baghdad University). * Three different concentrations (0.25 mg/disc, 0.5 mg/disc, and 0.625 mg/disc) were tried and the above concentration 0.625 mg/disc gave the best results. Iraqi J Pharm Sci, Vol.21(1) 2012 Synthesis of thiadiazole derivatives 102 Table 4: Antimicrobial screening data (zone of inhibition in mm) for final compounds (IV-V) Compound Staphylococcus aureus Streptococcus pyogenes Escherichia coli Klebsiella pneumonia Candida albicans IV 27 30 22 15 32 V 28 35 18 16 25 Gentamycine - - 22 18 - Augumentin 24 20 - - - ketoconozole - - - - 30 DMSO - - - - - The zone of inhibition of tested compounds shows, the 2-amino-5-mercapto-1, 3, 4 thiadiazole derivatives encompass potent bioactivities against bacterial and fungal strains, due to the strong bioactivity of our synthesized cycle and to the substituent groups that we added to the cycle. Results and Discussion The 2-amino-5-mercapto-1,3,4- thiadiazole was synthesized through steps of reactions starting from thiosemicarbazide with carbon disulphide in basic medium.Alkylation of compound (I) this step includes the synthesis of thioether or alkyl sulfide, it was done by treatment of a thiol with a base, such as KOH, giving the corresponding thiolate ion (RS - ). This undergoes reaction with a primary or secondary alkyl halide to give a sulfide. The reaction occurs by an SN2 mechanism, analogous to the Williamson synthesis of ethers. Thiolate anions are among the best nucleophiles known, and product yields are usually high in these SN2 reactions. Thioethers can be oxidized to sulfoxides by one equivalent of 30% H2O2 or by many other oxidizing agents , including H2O2 –flavin catalyst; Sulfoxides can be further oxidized to sulfones by another equivalent of H2O2.Primary amines(compound I), add to aldehydes (vanillin) to yield Imines R2C=NR,Conventional solution method was used as a coupling method between the dipeptide (Boc-Ala-Gly-OH) and compound III for synthesizing compound V. Dicyclohexylcarbodiimide (DCC) was used as a coupling reagent in amide bond formation; while 1-hydroxybenzotriazole (HOBt) or N- hydroxysuccinamide (HOSu) were used to increase the yields of the product and suppress racemization. Scheme 1: General scheme of synthesis compounds Iraqi J Pharm Sci, Vol.21(1) 2012 Synthesis of thiadiazole derivatives 103 Conclusion The synthesis of these proposed compounds was successfully achieved by following the stated procedures as previously described. The results obtained from this investigation indicated that the strategy adapted for the synthesis of the designed derivatives was successful, since the conformity of synthesized compounds was achieved according to the data shown by the physical and chemical analysis including (TLC, melting point, IR and Elemental microanalysis (CHNSO). compound IV & V show good antimicrobial activity comparable with marketable compounds. The antimicrobial evaluation indicated that the newly synthesized compound IV, showed highest antimicrobial activity in comparing to Augumentin for gram positive bacteria, gentamycin for gram negative bacteria and ketoconazole for fungi.Compound V showed a good antimicrobial activity, highest activity against Streptococcus pyogenes compared to Augumentin. All the synthesized compounds have excellent antifungal activity. Acknowledgment The authors gratefully thank the College of Pharmacy, University of Baghdad for supporting this research. . References 1. 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