One-Pot Multicomponent Synthesis of Thiourea...

Research ArticleOne-Pot Multicomponent Synthesis of Thiourea Derivatives inCyclotriphosphazenes Moieties

Zainab Ngaini1 Wan Sharifatun Handayani Wan Zulkiplee2

and Ainaa Nadiah Abd Halim1

1Faculty of Resource Science and Technology Universiti Malaysia Sarawak 94300 Kota Samarahan Sarawak Malaysia2Centre for Pre-University Studies Universiti Malaysia Sarawak 94300 Kota Samarahan Sarawak Malaysia

Correspondence should be addressed to Zainab Ngaini nzainabunimasmy

Received 15 March 2017 Revised 21 April 2017 Accepted 23 April 2017 Published 3 July 2017

Academic Editor Liviu Mitu

Copyright copy 2017 Zainab Ngaini et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In this study hexasubstituted thiourea was carried out via reaction of isothiocyanato cyclophosphazene intermediates with a seriesof aromatics amines and amino acids in a one-pot reaction system The reaction was not as straightforward as typical thioureasynthesis Six unexpected thiourea derivatives 3andashf were formed in the presence of cyclotriphosphazene moieties in good yields(53ndash82) The structures of 3andashf were characterized by elemental analysis and FTIR 1H 13C and 31P NMR spectroscopiesThe occurrence of reverse thioureas formation in a one-pot reaction system is discussed The possible binding interaction ofthe synthesised thiourea 3a-b in comparison to the predicted phenyl thiourea 5a-b and the targeted 4a with enzyme enoyl ACPreductase (FabI) is also discussed Molecular docking of the targeted hexasubstituted thiourea 4a is able to give higher bindingaffinity of minus75 kcalmol compared to 5a-b (minus59 kcalmol and minus63 kcalmol) and thiourea 3a-b (minus45 kcalmol and minus47 Kcalmol)

1 Introduction

Thiourea is widely studied and claimed to be used in manyapplications such as herbicides pharmaceutical agents pes-ticides rodenticides vulcanization accelerator and scaffoldsin organic synthesis [1] In the synthesis of thiourea isoth-iocyanate is formed as a reactive intermediate and easilyconverted to other side product during isolation [2] Manystudies reported on the direct reaction of isothiocyanateintermediate with amines after isolation of KCl to producethiourea in good purity [3]

Several studies reported on monosubstituted thioureawhich consists of one thiourea moiety either as a ligandbearing aromatic halogen or alkyl substituents [4] or asa complex compound coordinated with heavy metal center[5] Multisubstituted thioureas have gained more interestamong researchers due to the increase of their pharmaceu-tical properties Our recent studies on thiourea reportedthat compounds that consist of more than one thioureamoiety possess better antimicrobial activities [6ndash8] It was

due to the presence of more active sites of thiourea moietiescontaining C=S C=O and N-H groups which are easilyprotonated under acidic condition and interacted with thecarboxyl and phosphate groups of the bacterial surfacesthus enhancing the biological activities [7] Various meth-ods have been reported to make this versatile group ofthiourea derivatives easily accessible with excellent yields[2 9ndash12]

Hexakisphosphazenes bearing thioureas moieties havebeen reported from the stepwise reaction of the isolatedisothiocyanate intermediates with a series of aliphatic aminesvia P-Cl substitution of hexachlorocyclotriphosphazene [13]Hexachlorocyclotriphosphazene a cyclic inorganic com-pound with alternating phosphorus and nitrogen atoms hassparked great interest among researchers for an excellent can-didate in constructing hexasubstitutedmolecules [14 15]Thesubstitution of P-Cl bonds with various types of nucleophilesallowed the construction of phosphazenes-based ligandswithdifferent types of physical and chemical properties [16] Awide range of hexasubstituted phosphazene derivatives with

HindawiJournal of ChemistryVolume 2017 Article ID 1509129 7 pageshttpsdoiorg10115520171509129

2 Journal of Chemistry

various substituents such as hydroxyl amino andmany otherfunctional groups had been reported [13 15 16]

To the best of our knowledge no studies reported onthe synthesis of hexasubstituted thiourea onto cyclotriphos-phazene moieties bearing six units of amino acid or aromaticamines Our previous studies reported on thiourea bearingaromatic amine with excellent antibacterial properties [6 17]In continuation to our previouswork in this article we reporton the synthesis of thiourea compounds with hexachlorocy-clotriphosphazene as a hexasubstituted precursor in a typicalone-pot reaction system [11]The plausiblemechanismwhichleads to the unexpected final products is discussed Thebinding interaction of the synthesised thiourea via molecu-lar docking interaction in comparison to predicted phenylthiourea and the targeted compoundwith enzyme enoyl ACPreductase (FabI) is also thoroughly discussed

2 Materials and Methods

Hexachlorocyclotriphosphazene (99) was purchased fromAldrich Potassium thiocyanate aniline 120588-toluidine 120588ndashanisidine glycine L-alanine and L-phenyl alanine wereobtained fromMerck and used without purification Acetonewas distilled over magnesium sulphate anhydrous All otherreagents and solvent were used as received

Physical Measurement Melting points were determined bythe open tube capillary method and were uncorrectedInfrared spectra (]cmminus1) were recorded as KBr pellets ona Perkin Elmer 1605 FTIR spectrophotometer 1H and 13CNMR spectra were recorded on a JEOL ECA 500 spectrom-eter at 500MHz (1H) and 125MHz (13C) respectively withthe chemical shifts 120575 (ppm) being reported relative toDMSO-d6as standard The chemical shifts for 31P NMR are relative

to the internal standard of 85 phosphoric acid CHNSmicroanalyses were performed by use of a FLASHEA 1112CHNS analyser

21 General Procedure for the Synthesis of 3andashf A mixtureof hexachlorocyclotriphosphazene (035 g 10mmol) in dryacetone (150mL) was added dropwise into a solution ofpotassium thiocyanate (087 g 90mmol) in dry acetone(150mL)Themixturewas stirred for 1 h at room temperatureto form intermediate 2 The white potassium chloride (KCl)was filtered The filtrate was added to amine (60mmol)in dry acetone (150mL) and heated under reflux for 18 hThe mixture was cooled to room temperature and filteredThe filtrate was evaporated in vacuum to form a yellowishpowderThe crudewas recrystallized in EtOH CH

3CN (1 1)

The general procedure for the preparation of 3andashf utilised adifferent type of amines (g mmol) and yields as follows

Phenylthiourea (3a) [18] Aniline (5650 120583L 6mmol) (73yield) as a white crystal mp 1532ndash1535∘C (lit [18] 163∘C)

p-Tolylthiourea (3b) [19] p-Toluidine (0643 g 6mmol)(82 yield) as a white crystal mp 1678ndash1688∘C (lit [19]182ndash186∘C)

(4-Methoxyphenyl) Thiourea (3c) [18] p-Anisidine (0739 g6mmol) (68 yield) as grey powder mp 1724ndash1732∘C (lit[18] 193∘C)

2-(Carbamothioylamino) Acetic Acid (3d) [20] Glycine(0451 g 6mmol) (53 yield) as a yellowish powder mp1331ndash1345∘C (lit [20] 176ndash179∘C)

2-(Carbamothioylamino) Propanoic Acid (3e) L-alanine(0534 g 6mmol) (62 yield) as a yellow powder mp1388ndash1395∘C ]max (KBrcm

minus1) 3229 (OH) 3010 (NH) 2968(CH) 1698 (COOH) 1226 (C=S) 120575H (500MHz DMSO-d6) 159 (3H d J = 68 CH

3) 510 (1H q CH) 1011 (2H

s NH2) 1054 (1H s NH) 120575C (125MHz DMSO-d

6) 170

(CH3) 619 (CH) 1735 (COOH) 1806 (C=S) Calculated for

C4H8N2O2S C 3240 H 540 N 1890 S 2160 found C

3174 H 498 N 1879 S 2164

2-(Carbamothioylamino)-3-phenyl-propanoic Acid (3f ) L-phenyl alanine (0990 g 6mmol) (61 yield) as a yellowcrystal mp 1983ndash1989∘C ]max (KBrcm

minus1) 3172 (OH) 3100(NH) 2911 (CH) 1740 (COOH) 1452 (Ar-C) 1249 (C=S) 120575H(500MHz DMSO-d

6) 389 (2H d J = 138 CH

2) 542 (1H

q CH) 705 (2H d J = 63 Ar-H) 728 (3H m Ar-H) 1019(2H s NH

2) 1063 (1H s NH) 120575C (125MHz DMSO-d

6)

359 (CH2) 670 (CH) 1278 1289 1298 1345 (Ar-C) 1725

(COOH) 1799 (C=S) Calculated forC10H12N2O2S C 5360

H 540 N 1250 S 1430 found C 5335 H 531 N 1217S 1402

22 Antibacterial Screening Antibacterial activities of 3andashfwere analysed against E coli (ATCC 8739) using the tur-bidimetric kinetic method The Gram-negative E coli werecultured on a Luria-Bertani plate agar at 37∘CThen a colonyof the inoculums was transferred and allowed to grow inmedia containing nutrient broth at 37∘C with permanentstirring at 250 rpm for overnight 02mL of inoculums wasinoculatedwith 10mLof culturemedium that has been addedwith increasing concentration of synthesised compoundsdissolved in DMSO The mixture was shaken at 180 rpm at37∘C The negative control was medium broth of inoculumswith solvent The aliquots of each replicate were taken onevery 1 h interval for 6 hThe transmittance (119879) was recordedusing UV-Visible Spectrophotometer Optima SP-300 Theantibacterial activity was determined by plotting a graph ofln119873119905versus time The ln119873119905 value represents the number of

colony forming unitsmL which followed the expression ofln119873119905= 271 minus 856119879 [21]

23 Molecular Docking Molecular docking studies on theseries of 3a-b 4a and 5a-bwere carried out using AutoDockVina 112 program [22] The polar hydrogens of the synthe-sised compounds and protein were added with AutoDockTools 156 [23] before docking using Auto-Dock Vina pro-gram In Auto-Dock Vina program the cubic grid box of60∘A sizes (119909 119910 and 119911) with a spacing of 0375∘A wascentered to the active site of the protein The X-ray crystalstructure of the enzyme enoyl ACP reductase (FabI) of E

Journal of Chemistry 3

Acetone

Acetonereux

4

12

KSCNAcetonereux

3andashf

Cl

Cl

ClCl

Cl

Cl

N

NN

N

N

N

N

NH

NN

P

P PP

S

P

P

P

P P

SCN

SCN

SCN

NCS

NCS

NCS

R㰀NH2

R㰀NH2

NH2

R㰀

NHCSNHR㰀 NHCSNHR㰀

NHCSNHR㰀

NHCSNHR㰀NHCSNHR㰀

NHCSNHR㰀

a = C6H5

b = C6H5CH3

c = C6H5OCH3

d = CH2(COOH)

e = (CH3)CH(COOH)

f = (C6H5)CH2CH(COOH)

R㰀

Scheme 1 The synthesis of 3andashf

coli (PDB entry 1C14) was obtained from Protein Data Bank(httpwwwrcsborgpdbhomehomedo) [7 24]

3 Results and Discussion

31 Chemistry Thesynthesis of the proposed hexasubstitutedthioureas 4andashf was prepared via reaction of hexachloro-cyclotriphosphazene with potassium thiocyanate to formisothiocyanates phosphazene intermediates followed by typ-ical thiourea reaction with a series of amines derivativesin a one-pot reaction system All compounds were sub-jected to IR spectroscopy and showed the disappearance ofV(NCS) at 2140ndash1990 cmminus1 and the formation of V(N-H) at3276ndash3010 cmminus1The formation of thiourea was evidenced bythe strong absorption peak at 1265ndash1227 cmminus1 correspondingto V(C=S) which shifted to the lower frequency due to theattachment of more electronegative nitrogen atoms [25] Theabsorbance peak attributed to the formation of V(P=N) asym-metric vibration at 1400ndash1200 cmminus1 [26 27] but however wasnot observed This phenomenon was also transpired in 31PNMR spectra where no phosphorus moieties were present

Further characterization of the synthesised compoundsvia 1H NMR showed the presence of thiourea (-NHCSNH-)represented by two NH peaks at 980ndash930 ppm and 333ndash330 ppm The higher resonance of NH peaks in 3dndashf at1084ndash1053 ppm and 1018ndash1001 ppm was due to downfieldeffect resulting from the formation of intramolecular hydro-gen bond between the hydrogen atom from thioureamoietieswith oxygen atom from the carboxylic acid group [5] 13CNMR spectra of compound 3andashf showed good agreementwith the corresponded structures with the presence of C=Speak at 1812ndash1809 ppm [28ndash30]

Elemental analysis of the synthesised compoundsafforded low carbon percentage in each compound whichindicated the formation of 3andashf Based on the IR 1H 13Cand 31P NMR spectra it was suggested that 3andashf were

synthesised in one-pot reaction system and not the targetedmolecule 4andashf (Scheme 1)

The presence of hexachlorocyclotriphosphazene in aone-pot reaction system is envisaged not only formingisothiocyanate intermediate 2 via P-Cl substitution but alsogenerating Clminus from the partially soluble KCl in acetone [31]The free chlorine ions deprotonate amines in the reactionsystem and form HCl and anionic amines which in turnreacted with hydrogen thiocyanate [18] and formed 3andashfTheplausible mechanism for the formation of 3andashf is shown inScheme 2

32 Antibacterial Activity Compounds 3andashf were furtherinvestigated for antibacterial activities by plotting the graphof ln119873

119905versus time Compounds 3andashf were examined at the

concentration of 50 ppm 80 ppm and 100 ppm against wild-type E coli at 37∘CThe result indicated that compounds 3andashfshowed poor inhibition against E coli The MIC graph forcompounds 3andashf as shown in Figure 1 was determined byextrapolating the concentration at the zero-growth rate of Ecoli (120583 = 0) [32]TheMIC values for all compounds 3andashf wereobserved to exceed 220 ppm Compounds withMIC value upto 400 ppm are considered to have inhibition activity againstgrowth of Gram-negative bacteria but only compounds withMIC value smaller than 220 ppm can be suggested for clinicalpurposes [33]

Like other typical Gram-negative bacteria the cell wallof E coli is made up from thin layer of peptidoglycanand an outer membrane constituted of lipopolysaccharidelipoprotein and phospholipids [34] In view of this thelarge molecular weight compound is required to coat the cellsurface and prevent the leakage of intercellular componentsof the bacteria [32]

33 Molecular Docking Design and Optimisation For a betterunderstanding of the interaction between thiourea deriva-tives and Gram-negative bacteria E coli molecular docking


Amines

1 2

KClN=C=S(excess)

H-N=C=S N+

++

+

++ +

+

PPN N

N N

H

H

H

HH

H

R㰀R㰀

R㰀 R㰀

3andashf

Cl

Cl

Cl

S

ClK+K+

Clminus

Clminus

S=C=NS=C=N

N=C=S

H+

minusminus

minusminus

Clminus

H2N

minus∙∙ ∙

∙N

minus∙∙ ∙

∙N

∙ ∙N

Scheme 2 Mechanism on the formation of 3andashf

000

050

100

150

200

250

300

350

0 50 100 150 200

3a3b3c

3d3e3f

(ppm)

휇times10

minus2

Figure 1 MIC graph for 3andashf

studies were carried out and optimised by comparing 3a-bwith the predicted phenyl thiourea 5a-b and the targeted 4aThe studies were carried out via molecular docking to theactive site of the enzyme enoyl ACP reductase (FabI) of E coli(PDB entry 1C14) usingAutoDockVina 112 program [7 22ndash24] The compounds and binding interactions are shown inTable 1 The binding affinity of the compounds was evaluatedbased on binding free energies (Δ119866119887 kcalmol) [35]

The binding model of thiourea and the predicted phenylthiourea 5a-b is depicted in Table 1 Compounds 3a-b showedbinding free energy of minus45 kcalmol and minus47 kcalmolrespectively Based on the importance properties of the aro-matic group in earlier studies [6ndash8] the optimisation studyviamolecular docking was carried out to evaluate the bindingfree energy of 3a-b in comparison to the predicted phenylthiourea 5a-b The presence of another aromatic group in5a-b demonstrated for a higher binding affinity with the

free energy of minus59 kcalmol and minus63 kcalmol respectivelyThe additional aromatic group in 5a-b is strongly bound toenzyme enoyl ACP reductase (FabI) of E coli through 120587-120587 bond interactions (yellow colour cylindrical wireframe)with hydrophobic pockets of Phe 1251 The hydrophobicinteraction between phenyl rings has increased the lipophilic-ity of the compound [7 33] The binding affinity of 5b isslightly higher than 5a due to the electron donating inductiveeffect of the substituted methyl group which provides betterinteractions network with the active site residues [36] Theabsence of aromatic ring was accountable for lesser bindingaffinity resulting in less activity in 3a-b [37]

Due to the importance of phenyl groups for a betterbinding affinity it is noteworthy to analyse the significance ofhexasubstituted thiourea moieties onto cyclotriphosphazene4a Based on Table 1 the presence of six thiourea moietiesin 4a showed the highest binding affinity with a free energyof minus75 kcalmol Apart from the 120587-120587 bond interactions withPhe 1251 4awas observed to interact with the enzyme via twohydrogen bonds (green colour sphere) The NH groups in 4aare forming hydrogen bondingwith C=O andNHofAla 1152The bonding provides specificity and stabilisation of bindingbetween 4a and enzyme active site which consequentlyenhanced the binding affinity [38 39] Other basic residuessuch as Pro 1154 Ile 1153 Val 1213 Ala 1254 Hoh 2087 Hoh2067 Arg 171 and Gly 242 were observed in the vicinity ofcompound 4a which suggested that a strong electrostaticinteraction was also involved in the binding process [40]

4 Conclusions

In summary the thiourea derivatives 3andashf were unexpectedlysynthesised from the reaction of amines with excess thio-cyanates groups in a one-pot reaction systemThe isolation ofisothiocyanato cyclophosphazene intermediates could be thebestmethod to give hexasubstituted thioureasThe formationof HCl in the reaction condition was envisaged to be respon-sible for the deprotonation of amines thus reducing the


Table 1 Molecular docking images of thiourea derivatives

Compound Docking image

3aHN

SNH2

3bHN

S

NH2

5aHN

HN

S

5bHN

HN

S

4

S

SPPP

S

S

SS

NH

NHHN

HNHN

HNN

N

H

H NH

H

NNNNN

NHH

possible formation of hexasubstituted thioureas Biologicalactivities of thiourea 3andashf showed poor inhibitions towardsE coli Molecular docking interaction study thoroughlyexplained the binding interactions of the selected thiourea3a-b compared to the binding affinity with the predicted 5a-band the targeted 4a Based on the molecular docking study itcan be concluded that the targeted hexasubstituted thioureaas in 4a is envisaged to give better binding affinity comparedto monothiourea 3andashf

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors would like to acknowledge Universiti MalaysiaSarawak and the Ministry of Higher Education forfinancial support through C09SpSTG1359161 andFRGSST0l(0l)12982015(15) They acknowledge UniversitiMalaysia Terengganu Malaysia for providing CHNSelemental analysis services

References

[1] P K Mohanta S Dhar S K Samal H Ila and H Junjappa ldquo1-(Methyldithiocarbonyl)imidazole A useful thiocarbonyl trans-fer reagent for synthesis of substituted thioureasrdquo Tetrahedronvol 56 no 4 pp 629ndash637 2000


[2] N Sun B Li J Shao et al ldquoA general and facile one-pot processof isothiocyanates from amines under aqueous conditionsrdquoBeilstein Journal of Organic Chemistry vol 8 pp 61ndash70 2012

[3] A Saeed N Abbas H Rafique S Rashid and A HameedldquoSynthesis characterization and antibacterial activity of some1-aroyl-3-aryl thioureardquo Chemistry vol 18 no 5 pp 152ndash1582009

[4] N A Nordin T W Chai B L Tan et al ldquoNovel syntheticmonothiourea aspirin derivatives bearing alkylated amines aspotential antimicrobial agentsrdquo Journal of Chemistry vol 2017no 1 pp 1ndash7 2017

[5] M K Rauf Imtiaz-ud-Din A Badshah et al ldquoSynthesisstructural characterization and in vitro cytotoxicity and anti-bacterial activity of some copper (I) complexes with N N1015840-disubstituted thioureasrdquo Journal of Inorganic Biochemistry vol103 no 8 pp 1135ndash1144 2009

[6] W S H Wan Zullkiplee A N Abd Halim Z Ngaini MA Mohd Ariff and H Hussain ldquoBis-Thiourea bearing aryland amino acids side chains and their antibacterial activitiesrdquoPhosphorus Sulfur and Silicon and the Related Elements vol 189no 6 pp 832ndash838 2014

[7] A N Abd Halim and Z Ngaini ldquoSynthesis and bacteriostaticactivities of bis (thiourea) derivatives with variable chainlengthrdquo Journal of Chemistry vol 2016 no 1 pp 1ndash7 2016

[8] W S H Wan Zullkiplee M A Mohd Ariff H HussainW M Khairul and Z Ngaini ldquoBacteriostatic activities ofN-substituted tris-thioureas bearing amino acid and anilinesubstituentsrdquo Phosphorus Sulfur and Silicon and the RelatedElements pp 1ndash5 2016

[9] W Fathalla M Cajan J Marek and P Pazdera ldquoOne-potquinazolin-4-yl-thiourea synthesis via N-(2-cyanophenyl)ben-zimidoyl isothiocyanaterdquo Molecules vol 6 no 7 pp 588ndash6022001

[10] N Azizi A Khajeh-Amiri H Ghafuri and M BolourtchianldquoToward a practical and waste-free synthesis of thioureas inwaterrdquoMolecular Diversity vol 15 no 1 pp 157ndash161 2011

[11] K Appalanaidu T Dadmal N Jagadeesh Babu and R MKumbhare ldquoAn improved one-potmulticomponent strategy forthe preparation of thiazoline thiazolidinone and thiazolidinolscaffoldsrdquo RSC Advances vol 5 no 107 pp 88063ndash88069 2015

[12] V Strukil M D Igrc L Fabian et al ldquoAmodel for a solvent-freesynthetic organic research laboratory Click-mechanosynthesisand structural characterization of thioureas without bulk sol-ventsrdquo Green Chemistry vol 14 no 9 pp 2462ndash2473 2012

[13] H R Allcock J S Rutt and M Parvez ldquoSynthesis of cyclicphosphazenes with isothiocyanato thiourethane and thioureaside groups X-ray crystal structure of N3P3(NMe2)3(NCS)3rdquoInorganic Chemistry vol 30 no 1 pp 1776ndash1782 1991

[14] Z Ngaini and N I A Rahman ldquoSynthesis and characterizationof chalconesubstituted phosphazenesrdquo Canadian Journal ofChemistry vol 88 no 7 pp 654ndash658 2010

[15] Z Ngaini and N I A Rahman ldquoSynthesis and characterizationof cyclotriphosphazenes bearing chalcones derivativesrdquo Phos-phorus Sulfur and Silicon and the Related Elements vol 185 no3 pp 628ndash633 2010

[16] R K Voznicova J Taraba J Prıhoda andM Alberti ldquoThe syn-thesis and characterization of new aminoadamantane deriva-tives of hexachloro-cyclo-triphosphazenerdquo Polyhedron vol 27no 9-10 pp 2077ndash2082 2008

[17] Z Ngaini M A Mohd Arif H Hussain E S Mei DTang and D H A Kamaluddin ldquoSynthesis and antibacterial

activity of acetoxybenzoyl thioureas with aryl and amino acidside Chainsrdquo Phosphorus Sulfur and Silicon and the RelatedElements vol 187 no 1 pp 1ndash7 2012

[18] P Venkatesh and S N Pandeya ldquoSynthesis characterisationand anti-inflammatory activity of some 2-amino benzothiazolederivativesrdquo International Journal of ChemTech Research vol 1no 4 pp 1354ndash1358 2009

[19] R L Smith and R T Williams ldquoThe metabolism of arylth-ioureas - IV p-chorophenyl- and p-tolyl-thioureardquo Journal ofMedicinal and Pharmaceutical Chemistry vol 4 no 1 pp 147ndash162 1961

[20] National Center for Biotechnology Information PubChemCom-pound Database Apr 2017 CID = 3040094 httpspubchemncbinlmnihgovcompound3040094

[21] N B PappanoO PCentorbi andFH Ferretti ldquoDeterminationof minimum concentration inhibitory chalcone derivativesrdquoRevise Microbiology vol 2 no 1 pp 183ndash188 1990

[22] O Trott and A J Olson ldquoAutoDock Vina improving the speedand accuracy of docking with a new scoring function efficientoptimization and multithreadingrdquo Journal of ComputationalChemistry vol 31 no 2 pp 455ndash461 2010

[23] G M Morris H Ruth W Lindstrom et al ldquoSoftware news andupdates AutoDock4 and AutoDockTools4 automated dockingwith selective receptor flexibilityrdquo Journal of ComputationalChemistry vol 30 no 16 pp 2785ndash2791 2009

[24] S George M B Ramzeena S V Ram S K Selvaraj S Rajanand T K Ravi ldquoDesign docking synthesis and anti E coliscreening of novel thiadiazolo thiourea derivatives as possibleinhibitors of enoyl ACP reductase (FabI) enzymerdquo BangladeshJournal of Pharmacology vol 9 no 1 pp 49ndash53 2014

[25] Y K Shao and J X Si ldquoSynthesis and herbicidal activity ofN-(o-flourophenoxyacetyl) thiourea activities and related fusedheterocyclic compoundrdquo Arkivoc vol 10 pp 63ndash68 2006

[26] K Moriya T Masuda T Suzuki S Yano and M Kaji-wara ldquoLiquid crystalline phase transition in hexakis (4-(n-(41-alkoxyphenyl) iminomethyl) phenoxy) cyclotriphosphazenerdquoMolecular Crystals and Liquid Crystals vol 318 no 1 pp 267ndash278 1998

[27] E Cil M Arslan and A O Gorgulu ldquoSynthesis and character-isationof benzyl andbenzoyl substituted oxime-phosphazeesrdquoPolyhedron vol 25 no 18 pp 3526ndash3532 2006

[28] G G Muccioli J Wouters G K E Scriba W Poppitz JH Poupaert and D M Lambert ldquo1-Benzhydryl-3-phenylureaand 1-benzhydryl-3-phenylthiourea derivatives New templatesamong the CB1 cannabinoid receptor inverse agonistsrdquo Journalof Medicinal Chemistry vol 48 no 23 pp 7486ndash7490 2005

[29] Y-H Shen and D-J Xu ldquoPhenylthioureardquoActa Crystallograph-ica Section E Structure Reports Online vol 60 no 7 pp o1193ndasho1194 2004

[30] R S Correa O Estevez-Hernandez J Ellena and J Duque ldquo1-(2-Furoyl)-3-(o-tolyl)thioureardquo Acta Crystallographica SectionE Structure Reports Online vol 64 no 8 p o1414 2008

[31] H R Allcock Phosphorus-Nitrogen Compounds Cyclic Linearand High Polymeric Systems Academic Press Elsevier NewYork NY USA 1972

[32] M De Los Angeles Alvarez V E P Zarelli N B Pappano andN B Debattista ldquoBacteriostatic action of synthetic polyhydrox-ylated chalcones against Escherichia colirdquo Biocell vol 28 no 1pp 31ndash34 2004

[33] H Arslan N Duran G Borekci C K Ozer and C AkbayldquoAntimicrobial activity of some thiourea derivatives and their


nickel and copper complexesrdquoMolecules vol 14 no 1 pp 519ndash527 2009

[34] J L Ramos S Marques and K N Timmis ldquoTranscriptionalcontrol of the Pseudomonas TOL plasmid catabolic operonsis achieved through an interplay of host factors and plasmid-encoded regulatorsrdquo Annual Review of Microbiology vol 51 pp341ndash373 1997

[35] P-C Lv H-Q Li J Sun Y Zhou and H-L Zhu ldquoSynthesisand biological evaluation of pyrazole derivatives containingthiourea skeleton as anticancer agentsrdquo Bioorganic and Medici-nal Chemistry vol 18 no 13 pp 4606ndash4614 2010

[36] N K N A Zawawi M Taha N Ahmat et al ldquoSynthesis invitro evaluation and molecular docking studies of biscoumarinthiourea as a new inhibitor of 120572-glucosidasesrdquo BioorganicChemistry vol 63 pp 36ndash44 2015

[37] E Tatar S Karakus S G Kucukguzel et al ldquoDesignsynthesis and molecular docking studies of a conjugatedthiadiazolendashthiourea scaffold as antituberculosis agentsrdquo Bio-logical and Pharmaceutical Bulletin vol 39 no 4 pp 502ndash5152016

[38] M Meyer P Wilson and D Schomburg ldquoHydrogen bondingand molecular surface shape complementarity as a basis forprotein dockingrdquo Journal of Molecular Biology vol 264 no 1pp 199ndash210 1996

[39] S Purser P RMoore S Swallow and V Gouverneur ldquoFluorineinmedicinal chemistryrdquoChemical Society Reviews vol 37 no 2pp 320ndash330 2008

[40] Y He Y Wang L Tang et al ldquoBinding of puerarin tohuman serum albumin a spectroscopic analysis and moleculardockingrdquo Journal of Fluorescence vol 18 no 2 pp 433ndash4422008

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Analytical ChemistryInternational Journal of


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various substituents such as hydroxyl amino andmany otherfunctional groups had been reported [13 15 16]

To the best of our knowledge no studies reported onthe synthesis of hexasubstituted thiourea onto cyclotriphos-phazene moieties bearing six units of amino acid or aromaticamines Our previous studies reported on thiourea bearingaromatic amine with excellent antibacterial properties [6 17]In continuation to our previouswork in this article we reporton the synthesis of thiourea compounds with hexachlorocy-clotriphosphazene as a hexasubstituted precursor in a typicalone-pot reaction system [11]The plausiblemechanismwhichleads to the unexpected final products is discussed Thebinding interaction of the synthesised thiourea via molecu-lar docking interaction in comparison to predicted phenylthiourea and the targeted compoundwith enzyme enoyl ACPreductase (FabI) is also thoroughly discussed

2 Materials and Methods

Hexachlorocyclotriphosphazene (99) was purchased fromAldrich Potassium thiocyanate aniline 120588-toluidine 120588ndashanisidine glycine L-alanine and L-phenyl alanine wereobtained fromMerck and used without purification Acetonewas distilled over magnesium sulphate anhydrous All otherreagents and solvent were used as received

Physical Measurement Melting points were determined bythe open tube capillary method and were uncorrectedInfrared spectra (]cmminus1) were recorded as KBr pellets ona Perkin Elmer 1605 FTIR spectrophotometer 1H and 13CNMR spectra were recorded on a JEOL ECA 500 spectrom-eter at 500MHz (1H) and 125MHz (13C) respectively withthe chemical shifts 120575 (ppm) being reported relative toDMSO-d6as standard The chemical shifts for 31P NMR are relative

to the internal standard of 85 phosphoric acid CHNSmicroanalyses were performed by use of a FLASHEA 1112CHNS analyser

21 General Procedure for the Synthesis of 3andashf A mixtureof hexachlorocyclotriphosphazene (035 g 10mmol) in dryacetone (150mL) was added dropwise into a solution ofpotassium thiocyanate (087 g 90mmol) in dry acetone(150mL)Themixturewas stirred for 1 h at room temperatureto form intermediate 2 The white potassium chloride (KCl)was filtered The filtrate was added to amine (60mmol)in dry acetone (150mL) and heated under reflux for 18 hThe mixture was cooled to room temperature and filteredThe filtrate was evaporated in vacuum to form a yellowishpowderThe crudewas recrystallized in EtOH CH

3CN (1 1)

The general procedure for the preparation of 3andashf utilised adifferent type of amines (g mmol) and yields as follows

Phenylthiourea (3a) [18] Aniline (5650 120583L 6mmol) (73yield) as a white crystal mp 1532ndash1535∘C (lit [18] 163∘C)

p-Tolylthiourea (3b) [19] p-Toluidine (0643 g 6mmol)(82 yield) as a white crystal mp 1678ndash1688∘C (lit [19]182ndash186∘C)

(4-Methoxyphenyl) Thiourea (3c) [18] p-Anisidine (0739 g6mmol) (68 yield) as grey powder mp 1724ndash1732∘C (lit[18] 193∘C)

2-(Carbamothioylamino) Acetic Acid (3d) [20] Glycine(0451 g 6mmol) (53 yield) as a yellowish powder mp1331ndash1345∘C (lit [20] 176ndash179∘C)

2-(Carbamothioylamino) Propanoic Acid (3e) L-alanine(0534 g 6mmol) (62 yield) as a yellow powder mp1388ndash1395∘C ]max (KBrcm

minus1) 3229 (OH) 3010 (NH) 2968(CH) 1698 (COOH) 1226 (C=S) 120575H (500MHz DMSO-d6) 159 (3H d J = 68 CH

3) 510 (1H q CH) 1011 (2H

s NH2) 1054 (1H s NH) 120575C (125MHz DMSO-d

6) 170

(CH3) 619 (CH) 1735 (COOH) 1806 (C=S) Calculated for

C4H8N2O2S C 3240 H 540 N 1890 S 2160 found C

3174 H 498 N 1879 S 2164

2-(Carbamothioylamino)-3-phenyl-propanoic Acid (3f ) L-phenyl alanine (0990 g 6mmol) (61 yield) as a yellowcrystal mp 1983ndash1989∘C ]max (KBrcm

minus1) 3172 (OH) 3100(NH) 2911 (CH) 1740 (COOH) 1452 (Ar-C) 1249 (C=S) 120575H(500MHz DMSO-d

6) 389 (2H d J = 138 CH

2) 542 (1H

q CH) 705 (2H d J = 63 Ar-H) 728 (3H m Ar-H) 1019(2H s NH

2) 1063 (1H s NH) 120575C (125MHz DMSO-d

6)

359 (CH2) 670 (CH) 1278 1289 1298 1345 (Ar-C) 1725

(COOH) 1799 (C=S) Calculated forC10H12N2O2S C 5360

H 540 N 1250 S 1430 found C 5335 H 531 N 1217S 1402

22 Antibacterial Screening Antibacterial activities of 3andashfwere analysed against E coli (ATCC 8739) using the tur-bidimetric kinetic method The Gram-negative E coli werecultured on a Luria-Bertani plate agar at 37∘CThen a colonyof the inoculums was transferred and allowed to grow inmedia containing nutrient broth at 37∘C with permanentstirring at 250 rpm for overnight 02mL of inoculums wasinoculatedwith 10mLof culturemedium that has been addedwith increasing concentration of synthesised compoundsdissolved in DMSO The mixture was shaken at 180 rpm at37∘C The negative control was medium broth of inoculumswith solvent The aliquots of each replicate were taken onevery 1 h interval for 6 hThe transmittance (119879) was recordedusing UV-Visible Spectrophotometer Optima SP-300 Theantibacterial activity was determined by plotting a graph ofln119873119905versus time The ln119873119905 value represents the number of

colony forming unitsmL which followed the expression ofln119873119905= 271 minus 856119879 [21]

23 Molecular Docking Molecular docking studies on theseries of 3a-b 4a and 5a-bwere carried out using AutoDockVina 112 program [22] The polar hydrogens of the synthe-sised compounds and protein were added with AutoDockTools 156 [23] before docking using Auto-Dock Vina pro-gram In Auto-Dock Vina program the cubic grid box of60∘A sizes (119909 119910 and 119911) with a spacing of 0375∘A wascentered to the active site of the protein The X-ray crystalstructure of the enzyme enoyl ACP reductase (FabI) of E


Acetone

Acetonereux

4

12

KSCNAcetonereux

3andashf

Cl

Cl

ClCl

Cl

Cl

N

NN

N

N

N

N

NH

NN

P

P PP

S

P

P

P

P P

SCN

SCN

SCN

NCS

NCS

NCS

R㰀NH2

R㰀NH2

NH2

R㰀


NHCSNHR㰀


NHCSNHR㰀

a = C6H5

b = C6H5CH3

c = C6H5OCH3

d = CH2(COOH)

e = (CH3)CH(COOH)


R㰀















Amines

1 2

KClN=C=S(excess)

H-N=C=S N+

++

+

++ +

+

PPN N

N N

H

H

H

HH

H

R㰀R㰀

R㰀 R㰀

3andashf

Cl

Cl

Cl

S

ClK+K+

Clminus

Clminus

S=C=NS=C=N

N=C=S

H+

minusminus

minusminus

Clminus

H2N

minus∙∙ ∙

∙N

minus∙∙ ∙

∙N

∙ ∙N


000

050

100

150

200

250

300

350

0 50 100 150 200

3a3b3c

3d3e3f

(ppm)

휇times10

minus2






4 Conclusions





3aHN

SNH2

3bHN

S

NH2

5aHN

HN

S

5bHN

HN

S

4

S

SPPP

S

S

SS

NH

NHHN

HNHN

HNN

N

H

H NH

H

NNNNN

NHH




Acknowledgments


References






















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Acetone

Acetonereux

4

12

KSCNAcetonereux

3andashf

Cl

Cl

ClCl

Cl

Cl

N

NN

N

N

N

N

NH

NN

P

P PP

S

P

P

P

P P

SCN

SCN

SCN

NCS

NCS

NCS

R㰀NH2

R㰀NH2

NH2

R㰀


NHCSNHR㰀


NHCSNHR㰀

a = C6H5

b = C6H5CH3

c = C6H5OCH3

d = CH2(COOH)

e = (CH3)CH(COOH)


R㰀















Amines

1 2

KClN=C=S(excess)

H-N=C=S N+

++

+

++ +

+

PPN N

N N

H

H

H

HH

H

R㰀R㰀

R㰀 R㰀

3andashf

Cl

Cl

Cl

S

ClK+K+

Clminus

Clminus

S=C=NS=C=N

N=C=S

H+

minusminus

minusminus

Clminus

H2N

minus∙∙ ∙

∙N

minus∙∙ ∙

∙N

∙ ∙N


000

050

100

150

200

250

300

350

0 50 100 150 200

3a3b3c

3d3e3f

(ppm)

휇times10

minus2






4 Conclusions





3aHN

SNH2

3bHN

S

NH2

5aHN

HN

S

5bHN

HN

S

4

S

SPPP

S

S

SS

NH

NHHN

HNHN

HNN

N

H

H NH

H

NNNNN

NHH




Acknowledgments


References






















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Amines

1 2

KClN=C=S(excess)

H-N=C=S N+

++

+

++ +

+

PPN N

N N

H

H

H

HH

H

R㰀R㰀

R㰀 R㰀

3andashf

Cl

Cl

Cl

S

ClK+K+

Clminus

Clminus

S=C=NS=C=N

N=C=S

H+

minusminus

minusminus

Clminus

H2N

minus∙∙ ∙

∙N

minus∙∙ ∙

∙N

∙ ∙N


000

050

100

150

200

250

300

350

0 50 100 150 200

3a3b3c

3d3e3f

(ppm)

휇times10

minus2






4 Conclusions





3aHN

SNH2

3bHN

S

NH2

5aHN

HN

S

5bHN

HN

S

4

S

SPPP

S

S

SS

NH

NHHN

HNHN

HNN

N

H

H NH

H

NNNNN

NHH




Acknowledgments


References






















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




3aHN

SNH2

3bHN

S

NH2

5aHN

HN

S

5bHN

HN

S

4

S

SPPP

S

S

SS

NH

NHHN

HNHN

HNN

N

H

H NH

H

NNNNN

NHH




Acknowledgments


References






















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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