Synthesis, Antiphospholipase A2, Antiprotease, Antibacterial Evaluation and Molecular Docking

17 Pages·2016·2.11 MB·English

molecules
Article
Synthesis, Antiphospholipase A , Antiprotease,
2
Antibacterial Evaluation and Molecular Docking
Analysis of Certain Novel Hydrazones
NahedN.E.El-Sayed1,2,*,AhmedM.Alafeefy3,MohammedA.Bakht4,VijayH.Masand5,
AliAldalbahi6,NanChen7,ChunhaiFan7andAbirBenBacha8
1 DepartmentofChemistry,CollegeofScience,“GirlsSection”,KingSaudUniversity,P.O.Box22452,
Riyadh11495,SaudiArabia
2 NationalOrganizationforDrugControlandResearch,Giza35521,Egypt
3 DepartmentofChemistry,KulliyyahofScience,InternationalIslamicUniversity,P.O.Box141,
25710Kuantan,Malyasia;[email protected]
4 DepartmentofPharmaceuticalChemistry,CollegeofPharmacy,PrinceSattamBinAbdulazizUniversity,
P.O.Box173,Al-kharj11942,SaudiArabia;[email protected]
5 DepartmentofChemistry,VidyaBharatiCollege,Camp,Amravati,Maharashtra444602,India;
[email protected]
6 ChemistryDepartment,KingSaudUniversity,P.O.Box2455,Riyadh11451,SaudiArabia;
[email protected]
7 DivisionofPhysicalBiology&BioimagingCenter,ShanghaiSynchrotronRadiationFacility,
ShanghaiInstituteofAppliedPhysics,ChineseAcademyofSciences,Shanghai201800,China;
[email protected](N.C.);[email protected](C.F.)
8 BiochemistryDepartment,ScienceCollege,KingSaudUniversity,P.O.Box22452,Riyadh11495,
SaudiArabia;[email protected]
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:DerekJ.McPhee
Received:21October2016;Accepted:28November2016;Published:2December2016
Abstract: Some novel hydrazone derivatives 6a–o were synthesized from the key intermediate
4-Chloro-N-(2-hydrazinocarbonyl-phenyl)-benzamide 5 and characterized using IR, 1H-NMR,
13C-NMR,massspectroscopyandelementalanalysis. Theinhibitorypotentialagainsttwosecretory
phospholipaseA (sPLA ),threeproteaseenzymesandelevenbacterialstrainswereevaluated. The
2 2
resultsrevealedthatallcompoundsshowedpreferentialinhibitiontowardshGIIAisoformofsPLA
2
ratherthanDrG-IBwithcompounds6land6ebeingthemostactive.Thetestedcompoundsexhibited
excellentantiproteaseactivityagainstproteinaseKandproteasefromBacillussp. withcompound
6l being the most active against both enzymes. Furthermore, the maximum zones of inhibition
againstbacterialgrowthwereexhibitedbycompounds;6a,6m,and6oagainstP.aeruginosa;6a,6b,
6d,6f,6l,6m,6n,and6oagainstSerratia;6kagainstS.mutans;andcompounds6a,6d,6e,6m,and
6nagainstE.feacalis. Thedockingsimulationsofhydrazones6a–owithGIIAsPLA ,proteinaseK
2
andhydrazones6a–ewithglutamine-fructose-6-phosphatetransaminasewereperformedtoobtain
informationregardingthemechanismofaction.
Keywords: benzo[d][1,3]oxazin-4-one;aroylhydrazides;N-acylhydrazones;hydrazones;benzylidene
hydrazides;phospholipases;proteases;antimicrobialevaluation;moleculardocking
1. Introduction
Hydrazonesbearingazomethinemoietycanbeformedbycondensationofhydrazidesoraroyl
hydrazides with aldehydes [1–4]. These compounds are most widely used as building blocks for
thesynthesisof3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazoles[5],differentmono-,di-and
Molecules2016,21,1664;doi:10.3390/molecules21121664 www.mdpi.com/journal/molecules
Molecules2016,21,1664 2of17
trisubstitutedhydrazines[6,7],azetidin-2-ones,thiazolidin-4-onesandmethylthiazolidines[8,9],and
formazanderivatives[10,11]. Hydrazonesarewellknowntoexhibitawidespectrumofbiological
activities [12] and have been utilized as useful candidates for development of antimalarial [13],
antitumor[14],antiviral[15,16],antimicrobial[17],antioxidant[18],analgesic,anti-inflammatoryand
anti-ulcerogenicagents[19,20].
SecretedphospholipasesA (sPLA s)areenzymesfoundinmammalsandanimalvenomsthat
2 2
catalyzehydrolysisofglycerophospholipidsatthesn-2esterpositiontoreleaseafreefattyacidand
alysophospholipid[21]. MammalianphospholipaseshavebeencategorizedintotengroupsIB,IIA,
IIC,IID,IIE,IIF,III,V,XandXIIA[22,23]. IthasbeenreportedthatsPLA splayakeyroleinagood
2
numberofbio-chemicalprocesses,thus,ononethehand,groupIBsPLA hasbeenproposedtobe
2
involvedinvariousphysiologicalandpathophysiologicalprocessessuchascellularproliferation,cell
migration,hormonerelease[24]andapoptosisinneuronalcells[25]. Inaddition,higherlevelsofthis
enzymeweredetectedintheserumofpatientwithchronicrenalfailure[26]andacutelunginjury[27]
comparedtohealthycontrols.
On the other hand, the GIIA sPLA has been correlated to inflammatory diseases, which
2
is due to elevated levels of this enzyme were detected in the fluids of patients suffering from
variousinflammatorydiseasessuchasrheumatoidarthritis[28],acutelunginjury-acuterespiratory
distresssyndrome(ALI-ARDS)[29]andpancreatitis-associatedadrenalinjuryinacutenecrotizing
pancreatitis[30],aswellasinvariouscancers[31]. Therefore,seekingpotentandselectiveinhibitors
to this sPLA may be considered an effective approach to develop novel anti-inflammatory and
2
antitumoragents.
Proteasesorproteinasesenzymesareproteolyticenzymesthatbreakdownproteinsintopeptides
andaminoacids. Theyareclassifiedintodifferentclassesdependingontheirsourceandfunctions[32]
suchasproteinaseK,proteasefromBacillusandprotease-esperase. Biologically,proteasesareinvolved
in controlling many biological pathways in the life cycle of human, plants, animals, insects and
pathogens such as fungi, bacteria and viruses. Many studies reported their involvement in the
pathogenicprocessesofhumandiseasessuchasbleedingdisorders[33],inflammation[34],cancer[35],
hypertension[36]andAIDS[37]. Therefore,proteasesinhibitors’canbeconsideredastargetsindrug
designfordevelopingtherapeuticsandpreventionofdiseases[38].
In continuation to our previous efforts towards synthesis of novel biologically active
compounds[39–41],wereporthereinthesynthesisofnovelhydrazones6a–oandevaluationoftheir
phospholipases,proteasesandbacterialinhibitoryactivities. Thedockingsimulationsofhydrazones
6a–oagainstGIIAsPLA ,proteinaseKandhydrazones6a–eagainstglutamine-fructose-6-phosphate
2
transaminasewereperformedinordertoobtaininformationregardingthemechanismofaction.
2. ResultsandDiscussion
2.1. Chemistry
Benzoylationofanthranillicacid1with4-chlorobenzoylchloride2inmethylenechlorideinthe
presenceoftriethylamineaffordedthecorrespondingamidoacid3whichuponboilingwithexcess
of acetic anhydride underwent intramolecular cyclization and afforded benzo[d][1,3]oxazin-4-one
derivative 4. Nucleophilic attack by the amino group of hydrazine hydrate on the carbonyl
group of the latter benzoxazinone, and subsequent ring opening gives the key intermediate
4-Chloro-N-(2-hydrazinocarbonyl-phenyl)-benzamide 5. Condensation of hydrazino derivative 5
withavarietyofaromaticaldehydesyieldedthedesiredhydrazones6a–o(Scheme1). Thestructures
oftheseproductswereconfirmedbasedontheirspectraldataandelementalanalyses. Ingeneral,
theIRspectraofcompounds6a–odisplayedtwoabsorptionbandsatν 3421–3292cm−1,which
max
areattributedtothetwo(NH)groups,andtwoabsorptionbandsatν 1684–1657cm−1duetothe
max
twocarbonylgroups. Inaddition,the(C=N)groupsappearedaround1598–1579cm−1. Moreover,
the1H-NMRspectraofthesecompoundsindicatedthedisappearanceofthespectrallineduetoNH
2
Molecules2016,21,1664 3of17
groupofthestartinghydrazide5andappearanceoftwobroadsingletsignalsatδ12.37–11.08ppm
correspondingto2NHgroups,singletsignalsatδ8.84–8.30ppmduetothecharacteristicazomethines’
protons(N=CH)inadditiontosignalsattributedtothealiphaticandaromaticprotonsattheexpected
chemicaMloslehcuilfest 2v01a6l,u 21e, s1.66A4 nalogously,the13C-NMRspectraprovedthepresencethetwoC3 =ofO 17 groups
atδ 165.61–163.86ppm,azomethinecarbons’at151.46–145.10ppmandthealiphaticandaromatic
C chemical shift values. Analogously, the 13C-NMR spectra proved the presence the two C=O groups
carbonsaat tδCth 1e65e.6x1p–e1c6t3e.8d6 cphpemm, aiczaolmsehthifitnev acalurbeosn.sT’ ahte 1m51a.4s6s–1s4p5e.1c0t rpapomf aanlldo tfheth aelipshyanttich eansidz eardomcoamticp ounds
revealedcatrhboenes xaits ttheen ecxepeocftetdh echpemariecanlt sihoifnt vpaeluaekss. .ThFei nmaalslys ,spthecetrCa ,oHf a,llN of ethleem syennthtaeslizaenda clyomsepsouonfdasl lofthe
synthesrizeveedalceodm thpeo euxnisdtesncwe eorfe thine pagarreenetm ioenn ptewakitsh. Ftihneallpyr, othpeo Cse, dH,s tNru ecletumreenst.al analyses of all of the
synthesized compounds were in agreement with the proposed structures.
Cl
Cl
O
NH O
2
OH NH N
+ Cl TEA, CH2Cl2 OH Ac2O O
O Cl
O O
1 2 3 4
NH NH
2 2
Cl Cl
O ArCHO/ AcOH cat. O
NH Ar NH
HN N HN NH2
O O
6a-o 5
a, Ar = Ph b, Ar = 4-Cl-C H
6 4
c, Ar = 4-OH,3-OMe-C H d, Ar = 3,4,5-(OMe) -C H
6 3 3 6 2
e, Ar = 2,4-Cl-C H f, Ar = 4-OMe-C H
6 3 6 4
g, Ar = 4-N,N-di-Me-C H h, Ar = 2-OMe-C H
6 4 6 4
i, Ar = 4-Me-C H j, Ar = 4-COOH-C H
6 4 6 4
K, Ar = CH=CH-C H l, Ar = 2-COOH-C H
6 5 6 4
m, Ar = 3-OH,4-OMe-C H n, Ar = 4-cyclohexyl-C H
6 3 6 4
o, Ar = 3-OMe-C H
6 4
Scheme 1. The synthesis of Schiff bases 6a–o.
Scheme1.ThesynthesisofSchiffbases6a–o.
2.2. Biological Evaluation
2.2. BiologicalEvaluation
2.2.1. Phospholipases Inhibitory Activity
2.2.1. PhospholipasesInhibitoryActivity
The phospholipase inhibitory activity of hydrazones 6a–o have been detected against two
Thedipffheroesnpt hcolalispseass eofi nshPiLbAit2os,r ynaamcteilvyi thyuomfahny gdrroauzpo nIIeAs 6sPaL–Ao2h (ahvGe-IbIAee) nadnde tedcrotemdedagarayi ngsrtotuwp oIBd ifferent
classesosfPLsPAL2 A(DrsG,-nIBa)m. Tehlye ohbutaminaend grersouultps IrIeAveaslPeLd Athat( hcoGm-IpIoAu)nadns d6cd, r6odm, 6eed aanrdy 6gl reoxuhpibiItBeds sPeLleActiv(eD rG-IB).
inhibition2 against hGIIA rather than DrG-IB by 520.67% ± 4.04%, 55.67% ± 4.72%, 72.67% ± 2.51% a2nd
Theobtainedresultsrevealedthatcompounds6c,6d,6eand6lexhibitedselectiveinhibitionagainst
74.33% ± 3.21%, respectively, as shown in Figure 1 and Table S1.
hGIIAratheCrothnvaenrsDelryG, t-hIBe lboyw5es0t. 6in7h%ib±itor4y.0 a4c%tiv,i5ty5 .a6g7a%ins±t t4h.i7s 2e%nz,y7m2.e6 7(9%% ±± 22%.5)1 %waas nddisp7l4a.y3e3d% b±y 3.21%,
respecticvoemlyp,oausnsdh 6onw. TnhienseF riegsuulrtes a1rea nind agTraebelmeeSn1t .with the data obtained in our previous work [42].
ConverAsse lmy,antyh eclinloicwale sstutdiinesh riebvietoalreyd tahcatti tvhiet ylevaeglsa oinf ssPtLtAh2iss aeren iznycrmeaese(d9 i%n d±iffe2re%nt) inwflaasmmdiastporlya yed by
conditions such as rheumatoid arthritis [28], these active hydrazones can be proposed as potential
compound6n. Theseresultsareinagreementwiththedataobtainedinourpreviouswork[42].
candidates to explore their utility for such ailment.
AsmanyclinicalstudiesrevealedthatthelevelsofsPLA sareincreasedindifferentinflammatory
2
conditionssuchasrheumatoidarthritis[28],theseactivehydrazonescanbeproposedaspotential
candidatestoexploretheirutilityforsuchailment.
Molecules2016,21,1664 4of17
Molecules 2016, 21, 1664 4 of 17
Molecules 2016, 21, 1664 4 of 17

Figure 1. Inhibitory activity (%) of compounds 6a–o against sPLA2 (hG-IIA and DrG-IB).
Figure1.Inhibitoryactivity(%)ofcompounds6a–oagainstsPLA (hG-IIAandDrG-IB).
2
2.2.2. Proteases Inhibitory Activity
Figure 1. Inhibitory activity (%) of compounds 6a–o against sPLA2 (hG-IIA and DrG-IB).
2.2.2. ProteasesInhibitoryActivity
The synthesized compounds were also screened for in vitro anti-proteases activity against the
2.2.2. Proteases Inhibitory Activity
Tcohmemsyenrctihalelsyi zavedailcaobmle pporoutenadsse-wesepreeraasles, oprsoctreeiennaseed Kf oarndin pvroittreoasaen otbi-tpairnoetde afrsoems aBcatciivlliutys sapg. aTihnes tthe
commscerreceiTnaihlnlegy s yraenvstuahlietlssa ibzsehledo wcponrm oitnpe oaFusinegd-uesrse wp 2ee rraean sadel s,Top asbrcolreet eeSin2ne adres vfeoeraK lienda v ntidhtraotp artnhoteti e-ptaerssoetetedoa bscetosam ianpcetoiduvintfdyr soa mgdaisinpBslaatc ytiehlldeu s sp.
Thesvccoarmreieemndi enrdcgeigarlreleyse usa lvtoasfi lsaphbroloewt epnarsoietnse aFisniegh-ueibsripetieo2rnaa.s neT,d hpeTr oamtbelianexaiSms2eu rKme va neindahl peibdriottotehrayas teat ochtbeivtatiteiinseetsed da fgrcoaoimnms Btp aopcirulolnutesdi snspad.s ieTs hpKel a yed
(s8c6re ±e n2i.n6g4) ,r epsruolttesa sseh ofrwonm iBn acFiilgluusr e( 724 .6a6n d± 2T.a8b8l)e a nS2d rpervoetaelaesde -ethspate rtahsee t(e3s9t.e3d3 ±co 3m.2p1o) uwnedrse deixshpilbaiyteedd
varied degrees of proteases inhibition. The maximum inhibitory activities against proteinase K
bvyar cieodm pdoeugrnede s6 lo. f proteases inhibition. The maximum inhibitory activities against proteinase K
(86±2.64),proteasefromBacillus(74.66±2.88)andprotease-esperase(39.33±3.21)wereexhibited
(86 ± 2.64), protease from Bacillus (74.66 ± 2.88) and protease-esperase (39.33 ± 3.21) were exhibited
bycobmyp coomunpdou6nl.d 160l0. Proteinase K
)
(% 90 Protease from Bacillus sp
Activity y (%) 178090000 EPPsrrooptteeeriaanssaees efr oKm Bacillus sp
ory tivit 6800 Esperase
tc 70
biA 50
nhiory 4600
se Ibit 3500
Protease Inhi 234000
a 10
e
t 20
o 0
r
P
10 6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 6o
0 Compound
6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 6o
Figure 2. Antiprotease activity (%) of compounds 6a–o against different protease enzymes.
Compound

Furthermore, the next highest inhibitory activities against proteinase K were exhibited by
Figure 2. Antiprotease activity (%) of compounds 6a–o against different protease enzymes.
compFoiugunrdes 26.eA (n74ti.p6r6o%te ±a s2e.5a1c%tiv) iatynd(% 6)mo f(6co9m% p±o 4u.n5d8%s6),a –wohailgea itnhset ndeifxfte rheingthpesrto tienahsiebietnozryy macetsi.vities
againFsut rpthroetremasoer efr, otmhe B naceixlltu sh sigph. ewste rien ehxibhiitboirteyd a bcyti vcoitmiesp oaugnadinss 6t bp (r7o2t.e3i3n%as ±e 2K.5 1w%e)r ae nedx 6hgib (i7te2d.3 3b%y
F±co u6mr.4tp2ho%eur)nm. dos r6ee, (t7h4e.66n%ex ±t 2h.5i1g%he) satndin 6hmib (i6to9%ry ±a 4c.t5i8v%it)i,e wshaiglea itnhes tnepxrto hteiginhaesste inKhibwiteorrey aecxthiviibtiietes d by
compaoguainnIdst tsi sp6 wreoot(er7ta4hs. e6m 6fer%onmti±o Bn2ainc.5igl1l ut%hsa )stp am.n wodset6r oem fe tx(hh6e9i bc%iotme±dp bo4yu.5 nc8do%ms s)p,howouwhnedidlse g6tobho e(d7n 2a.en3x3tit%phr ±oig t2eh.a5es1es% tin)i nhahinbidibt i6iotgno ( ra7yg2a.a3ic3nt%sitv ities
againp±s rt6o.t4pe2ri%noat)se. ea Kse anfrdo mproBteaacsiel lfurosms pB.aciwlluesr sep.e, xash dibeipteicdtedb yin cFoigmurpeo 2u. nItd wsas6 bals(o7 2n.o3t3ic%ed ±tha2t .t5h1e%re )waans d 6g
It is worth mentioning that most of the compounds showed good antiprotease inhibition against
(72.33%±6.42%).
proteinase K and protease from Bacillus sp., as depicted in Figure 2. It was also noticed that there was
Molecules2016,21,1664 5of17
Itisworthmentioningthatmostofthecompoundsshowedgoodantiproteaseinhibitionagainst
proteinaseKandproteasefromBacillussp.,asdepictedinFigure2. Itwasalsonoticedthattherewas
alignmentininhibitoryactivityagainstthesetwoenzymes,asanysinglecompoundthatshowedhigh
inhibitorypotentialagainstoneofthemalsoreactedinasimilarfashiontowardstheotherone.
Astheanti-inflammatoryactivitiesofchemicalcompoundscanbeexpressedbyphospholipase
A (hGIIA) and/or through protease inhibitor potentials [42], compound 6l, which was found to
2
bethemostactivecandidateagainstbothphospholipasesA (sPLA )andproteaseenzymesunder
2 2
investigation, may be proposed as promising potential anti-inflammatory agent for treatment of
ulcerativecolitis.
2.2.3. AntibacterialScreening
Finally,compounds6a–owerefurtherexaminedforinvitroantibacterialactivity. Preliminary
screening against eleven strains of Gram-positive and Gram-negative bacteria was performed by
adoptingstandardprotocol[43].Theantibacterialpotencywasdeterminedbymeasuringtheinhibition
zones. Alltestswereperformedinduplicatesandmeansofinhibitionzoneswererecordedinmmas
presentedinTable1.
Analysis of the screening data revealed that the inhibition activity produced by some of the
testedcompoundswasfoundtobegoodtoexcellentcomparedtotheusedreferencedrugtetracycline.
Amongtheelevenstrainsthatwouldbeconsideredmoresusceptibletoinhibitionbyoneormoreofthe
syntheticcompoundswere;P.aeruginosa,Serratia,S.aureus,S.mutansandE.feacaliswithgenusSerratia
beingthemostsensitiveonewhichwasinhibitedbyninehydrazones. Thehighestinhibitiontowards
thispathogenwasdisplayedbycompounds6a,6b,6d,6e,6f,6l,6m,6n,and6owithinhibitionzones
of15.5±0.0,12.5±0.0,12±0.0,14±1.0,14±1.0,13±0.01,14.5±1.5,14.5±0.5and12.5±0.5,
respectively. ThehighestinhibitoryactivityagainstP.aeruginosawasexertedbycompounds6a,6m,
and6owithinhibitionzonesof17±1.0,18.5±0.5and18.5±0.5,respectively. Althoughcompounds
6j, 6l and 6n produced the highest inhibition zones against S. aureus strain, they were considered
to be less effective compared to the reference drug tetracycline. Moreover, the maximum zone of
inhibition(24.5±0.5)wasexhibitedbycompound6kagainstS.mutans. Finally,thereferencedrug
tetracyclinewasfoundtobecompletelyinactiveagainstE.feacalis,whilecompounds6a,6d,6e,6m,
and6nexhibitedthemaximuminhibitionzonesof18.5±0.5,18.0. ±1.0,18.5±0.5,13.5±1.5and
18.5 ± 0.5, respectively. The rest of the compounds showed moderate inhibition against all other
bacterialstrains.
2.3. MolecularDockingAnalysis
Basedonthedataobtainedfromdifferentbiologicalevaluations,compounds6a–oweredocked
against proteinase K, GIIA sPLA , and compounds 6a–e against glutamine-fructose-6-phosphate
2
transaminase(GlcN6P)synthase(GlmS,L-glutamine: D-fructose-6Pamidotransferase,EC2.6.1.16)in
ordertoprovideaconceivablerationalefortheobservedactivities.
2.3.1. DockingSimulationsforCompounds6a–oinActivesiteofGIIAsPLA
2
GIIAsPLA isalowmolecularweightenzyme(14kDa)withsevendisulfidebondswithahighly
2
conserved Ca2+-binding loop and a catalytic dyad consisting of His47/Asp91 along with active a
hydrophobicregionlinedneartheN-terminalhelix[44].
Molecules2016,21,1664 6of17
Table1.Antibacterialactivitiesofthesynthesizedhydrazones6a–o.
Gram-NegativeBacteria Gram-PositiveBacteria
Comp#
E.coli p.aeruginosa K.Pneumoniae Salmonella Serratia S.aureus S.aureusALA1 MRSAATCC3 S.mutans E.feacalis B.subtilis
6a 0±0 17±1 0±0 8±1 15.5±0 0±0 10±0 12±0 12±0.5 18.5±0.5 0±0
6b 14.5±1.5 12±0 0±0 12.5±0.5 12.5±0.5 8±0.5 0±0 6±0 0±0 9.5±0.5 0±0
6c 1±0 1.5±0 0±0 10.25±1 1.4±1 0±0 0±0 8±0.5 0.6±1.2 1.3±1 0±0
6d 14.5±0.5 10±0 12.2±1 6.5±0 12±0 8.5±0.5 0±0 0±0 10±0.0 18±1 0±0
6e 10±0 12.5±0.1 12.5±0.5 4±0 14±1 8.5±0.5 0±0 14±1 8±1 18.5±0.5 0±0
6f 0±0 10.5±0.1 4.5±0.1 10±0 14±0.1 0±0 0±0 0±0 8±1 0±0 0±0
6g 0±0 10.5±0.5 8±1 0±0 10.5±0.5 8.5±0 0±0 0±0 0±0 0±0 0±0
6h 8±0 12.5±0.7 0±0 0±0 10±0 8.5±0.7 0±0 0±0 0±0 0±0 0±0
6i 8.5±0.5 10.5±0.5 0±0 0±0 8.5±0.5 10.5±1.5 0±0 0±0 0±0 0±0 0±0
6j 8±1 10±0 0±0 0±0 8.5±0.5 19.5±1.5 0±0 0±0 0±0 0±0 0±0
6k 10±0 13.5±1.5 14.5±1.5 0±0 10±0 10.5±1.5 0±0 0±0 24.5±0.5 0±0 8.5±0.5
6l 14±1 11±0 11±0 8±1 13±0.1 18.5±0.5 0±0 0±0 14±2 0±0 0±0
6m 10±1 18.5±0.5 0±0 4±0 14.5±1.5 8.5±0.5 0±0 8.5±0.5 0±0 13.5±1.5 0±0
6n 10±0 0±0 13±1 0±0 12.5±0.5 14±1 0±0 10±0 15±1 18.5±0.5 0±0
6o 10±0 18.5±0.5 10±0 0±0 12.5±0.5 0±0 0±0 0±0 9±0.6 0±0 0±0
TCN 19±0.1 17±0.2 17±0.5 16±0.1 12±0.3 31±0.6 29±0.4 18±0.3 27±1.2 0±0 15±0.5
Comp#=Compoundnumber;TCN=Tetracycline.
Molecules2016,21,1664 7of17
ThMeodleocucleks 2in01g6, s21i,m 16u64l ationsfor6a–oinactivesiteofGIIAsPLA2 arepresentedinSupp7l oef m18 entary
Materials,FigureS1. Resultsofdockingsimulationsforcompound6l,themostactiveanti-GIIAsPLA
2
enzyme, deTphice tdedockhienrge saimsualartieopnrse fsoern 6taa–toiv ien ianctiFvieg usitree o3f, GreIIvAesaPlLeAd2 tahrea tpirtesienntteerda cint sSwupipthlemheyndtarroyp hobic
Materials, Figures S1. Results of docking simulations for compound 6l, the most active anti-GIIAsPLA2
andpolarresiduesofactivesiteofGIIAsPLA . Compound6lhasadopted“U”shapewithitscentral
enzyme, depicted here as a representative 2in Figure 3, revealed that it interacts with hydrophobic
benzeneringorientedtowardsthepolarresiduesviz. His6,Gly22,Asp48,andVal30. The–CONH–N–
and polar residues of active site of GIIAsPLA2. Compound 6l has adopted “U” shape with its central
moietyibsenrezsepneo nrisnigb olerifeonrteidn ttoewraacrtdiso nthse wpoitlahr Prehseid5uaens dvizH. Hisi4s76,. GTlhye22–, CAOsp–4N8, Han–dN V=alm30o. Tiehtey –bCrOidNgHin–gN–th etwo
benzenemroiinetgys isa rsesapponosliabrlel ifnork ienrtehraacstioinncs owriptho Prahtee5d angdo Hodis4fl7e. Txhibe i–liCtOy–tNoHth–eNl=i gmaoniedty, hbreindgciengsu thceh twaofl exible
moietybisenbzeennee firicnigasl faos raf puorltahre lrinakmer ehnads minceonrtpso.rTathede –gCoolda fnledxi–bCiliOtyO toH thger oliguapnsd,a hreenocrei esuncthe da tfolewxibalred Leu2
moiety is beneficial for further amendments. The –Cl and –COOH groups are oriented toward Leu2
and Val3 due to polar interactions. The –CO– group of –CONH– group is close to Gly22. Hence,
and Val3 due to polar interactions. The –CO– group of –CONH– group is close to Gly22. Hence,
–CONH–groupisimportantgroupforretentioninfutureoptimizations.
–CONH– group is important group for retention in future optimizations.

Figure 3. Docking pose for compound 6l as a representative, in the active site of GIIAsPLA2.
Figure3.Dockingposeforcompound6lasarepresentative,intheactivesiteofGIIAsPLA .
2
2.3.2. Docking Simulations for Compounds 6a–o in Active Site of Proteinase K
2.3.2. DockingSimulationsforCompounds6a–oinActiveSiteofProteinaseK
Proteinase K (EC 3.4.21.64, protease K, endopeptidase K, Tritirachium alkaline proteinase,
ProTtreitiinraacsheiumK a(lbEuCm s3e.r4i.n2e1 p.6ro4t,einparsoet, eaansde TrKiti,raecnhiduomp aelbputimd apsroeteKin,asTe rKit)i risa ac hbiruoamd-saplekcatrluinme seprrinoet einase,
protease belonging to Peptidase family S8 with ability to digest proteins. It is used for the destruction of
Tritirachiumalbumserineproteinase,andTritirachiumalbumproteinaseK)isabroad-spectrumserine
proteins in cell lysates (tissue, and cell culture cells) and for the release of nucleic acids [45].
proteasebelongingtoPeptidasefamilyS8withabilitytodigestproteins. Itisusedforthedestruction
The docking simulations for hydrazones 6a–o in active site of proteinase K are presented in
ofproteSinupspinlemceelnltlayrysa Mteaste(triisaslsu, eF,igaunrdesc eS2ll. cRuelstuulrtse ocfe ldlso)ckainndg sfoimruthlaetiorenlse afosre coofmnpuoculnedic 6alc, itdhse [m45os].t
Thaectdivoec kanintigprostieminualsaet Kio, ndsepfiocrtedh yhderrea azso an eresp6reas–eontaintivaec itni vFeigsuirtee 4o, frepveraolteedi nthaaste itK inaterreacptsr ewsiethn ted in
SupplemhyednrtoaprhyobMic aatnedr ipaolsla,r FreigsiudrueesS o2f. acRtievseu slittes oof fpdrooteciknianseg Ks.i mCoumlaptoiuonnds 6flo irs ucnoamblpe otou oncdcu6ply, tthhee most
activeacnotmipprloettee isnpaascee oKf ,adcteipveic stieted ohf etrhee aesnzayrmeep. rIets ienntetraatcitvse winithF pigoularre r4es,irdeuvees avliezd. Athsna1t6i1t, iAntsenr1a6c2,t swith
hydrophSeorb22ic4 aanndd Hpoisl6a9r. rIne saiddduietisono,f iat chtaisv eHs-bitoendo ffoprrmoatetiionna wseitKh .HC2Oom (apt oduisntadnc6el oisf 2u.8n6a bAl°e), tporoescecnut pythe
inside the active site of Proteinase K, due to the –COOH group present on benzene ring. Hence, the
completespaceofactivesiteoftheenzyme.Itinteractswithpolarresiduesviz.Asn161,Asn162,Ser224
–COOH group is beneficial. Another important interaction is arene-cation interaction between the
andHis69. Inaddition,ithasH-bondformationwithH O(atdistanceof2.86A◦),presentinsidethe
benzene ring possessing –Cl atom with H2O (at distance2 of 4.08 A°), present inside the active site of
activesiteofProteinaseK,duetothe–COOHgrouppresentonbenzenering. Hence,the–COOH
groupis beneficial. Anotherimportantinteractionisarene-cationinteractionbetweenthebenzenering
possessing–ClatomwithH O(atdistanceof4.08A◦),presentinsidetheactivesiteofproteinaseK.
2
Interestingly,thecompoundpossessesintramolecularH-bondformation(distance2.09A◦)between
Molecules2016,21,1664 8of17
Molecules 2016, 21, 1664 8 of 17
the–NHpr–otpeianratseo fK–. CInOteNreHsti–ngglyro, uthpe wcoimthpo–uCnOd –pposasretssoefs –iCntOraNmoHle–cNul–arg Hro-buopn.d TfhorimsaHti-obno (nddistiasnpcer obably
respons2i.b09le Afo°)r bthetewsepeenc tihfiec –oNriHen– taptairot nofa n–dCOshNaHp–e gorfotuhpe wmitohl e–cCuOle– ipnasritd eoft h–CeOacNtHiv–eNs–it eg.roTuhpe. cTohmis pound
6lhasaHc-qbuonirde dis pwroebiradbly“ Jr”esopron“sLib”les fhoar pthee isnpseicdifeic tohreienatcattiivone asnitde s.hTahpee obf ethnez menoelercuinleg inpsoidses ethses iancgtiv–eC OOH
groupissitcel.o Tsheer tcoomthpeouancdti v6el hsaitse arceqsuiidreude sw.eTirhdi s“Ji”n dori c“aLt”e sshthapaet tihnseid–eC tOheN aHcti–vea nsidte–. CThOe NbeHn–zeNn–e grirnogu psare
possessing –COOH group is closer to the active site residues. This indicates that the –CONH– and –
importantforfuturemodifications.
CONH–N– groups are important for future modifications.

Figure 4. Docking pose for compound 6l as a representative, in the active site of proteinase K.
Figure4.Dockingposeforcompound6lasarepresentative,intheactivesiteofproteinaseK.
2.3.3. Docking Simulations for Compounds 6a–e in Active Site of
2.3.3. DGolcuktainmginSei-mFruuclatotsioe-n6s-PfhoorsCphoamtep Toraunnsdamsi6naa–see inActiveSiteofGlutamine-Fructose-6-Phosphate
Transaminase
The molecular mechanism of multifarious reaction catalyzed by glucosamine-6-phosphate
Th(eGlcmNo6Ple) csuynlathrasme e(GchlmaSn, iLs-mgluotafmmineu: lDti-ffraurcitoouses-6Pre aamctidioontracnasftearlaysez,e EdC b2.y6.1g.1l6u) ceonszaymmei ncoem-6p-prisheos sphate
both ammonia transfer (L-glutamine to Fru-6-P) and sugar isomerization (fructosamine-6-phosphate
(GlcN6P)synthase(GlmS,L-glutamine: D-fructose-6Pamidotransferase,EC2.6.1.16)enzymecomprises
to glucosamine-6-phosphate). This reaction is the initiation of the pathway leading to the eventual
bothammoniatransfer(L-glutaminetoFru-6-P)andsugarisomerization(fructosamine-6-phosphate
production of uridine 5′-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), a product that is present in
toglucoalsl aclmasisn oef -o6r-gpahniosmsps,h bautte i)n. fTuhngisi arneda cbtaicotenriais itt ihs eutiinliziteida ttoio cnonosftruthcte mpaactrhomwoaleyculeleasd eisnsegnttioal tfhore theev entual
productcieolln woafll uasrsideminbely5, (cid:48)s-udcihp ahso as npuhmob-Ner- oafc aemtyinl-oD s-ugglaurc-coosnatmaininineg (mUaDcrPom-GollceNcuAlesc,) c,oampprroisdinugc cthtihtiant ainsdp resent
inallclmasasnonofporrogteainnsis imn sf,ubnguit, ianndfu pnegpitiadnogdlybcaacnt earnida liitpiospuoltiislaizccehdartiodecso inns tbrauctcetrima. aIcnr othme oplreockualreysoteics sential
cell, the inhibition of GlcN6P synthase even for a small time is fatal. Fortunately, because of the longer
forthecellwallassembly,suchasanumberofaminosugar-containingmacromolecules,comprising
lifespan of human, the running down of amino sugar pool for a short time is not deadly. Consequently,
chitin and mannoproteins in fungi, and peptidoglycan and lipopolisaccharides in bacteria. In the
it has been proposed as a possible target for developing antibacterial and antifungal agents [46–49].
prokaryoticcell,theinhibitionofGlcN6Psynthaseevenforasmalltimeisfatal. Fortunately,because
It is evident that recognition of a molecule by a particular enzyme is dependent on the structural
ofthelopnrogpeerrtliiefse sopf atnhaotf mhuolmecaunle, tahnedr uitsn ndiinstgribduotwionn ooff athme inmoosleucgualarr peoleocltrfoosrtaatics hpoorttentitmiale (iMsnEoP)t. deadly.
ConseqFuuertnutrlmy,oirte,h aansalbyseies nofp lirgoapndo–sreedcepatsora ipntoersascitbiolenst aforrg GeltcNfo-r6-dP esvynetlhoapsein rgevaeanlteidb tahcatte lrigiaanldasn hdavainntgi fungal
agents[p4r6im–4ar9y] .amido groups can form stable hydrogen bonds with the amino acids residues present in
Ititshee vbiinddeinntgt shiatet orfe cthoeg fnuintigoanl eonfzaymmeo tlheucsu tlheebyy maapy asretrivceu alas r“aennczhyomrs”e tios dloecpk ethned iennhtibointorth ine tshtreu ctural
properties of that molecule and its distribution of the molecular electrostatic potential (MEP).
Furturmore, analysis of ligand–receptor interactions for GlcN-6-P synthase revealed that ligands
havingprimaryamidogroupscanformstablehydrogenbondswiththeaminoacidsresiduespresent
inthebindingsiteofthefungalenzymethustheymayserveas“anchors”tolocktheinhibitorin
thebindingpocketoftheenzyme. Inaddition,previousstructure–activityrelationshipexperiments
Molecules2016,21,1664 9of17
reveaMleoldecutlhesa 2t01p6,r 2e1s, e16n6c4 eofactiveelectrophiliccenterataproperpositionisrequiredforina9 cotf i1v7 ation
oftheenzymetotakeplace[49]. Byanalogy,hydrazones6a–ohavingthesamestructuralfeatures
binding pocket of the enzyme. In addition, previous structure–activity relationship experiments
representedbytwoprimaryamidogroupsandelectrophilicdoublebondmayserveasinhibitorsfor
revealed that presence of active electrophilic center at a proper position is required for inactivation of
bacterialGlcN6Psynthase. Thishypothesisisexaminedbydockinghydrazones6a–eintheactivesite
the enzyme to take place [49]. By analogy, hydrazones 6a–o having the same structural features
ofthebacterialenzyme(theseresultsarepresentedinSupplementaryMaterials,FigureS3).
represented by two primary amido groups and electrophilic double bond may serve as inhibitors for
Dockingsimulationsforthemostactivecandidate6awhichinhibitscollectivelythreebacterial
bacterial GlcN6P synthase. This hypothesis is examined by docking hydrazones 6a–e in the active site
strainosf tahned baecxtheribiailt ienngzyimnhei (btihteiosen rzeosunletss alraer gpererstehnatendt hino Ssueppprloedmuecnetadryb yMtahteeriraelfse, rFeingucereds rSu3g). tetracycline
against twDoockbiancgt esrimiaullsattrioanins sf,orS tehrera mtiaosat nacdtivEe. cfeaancdaildisa,ted 6eap wictheicdh hinehreibiatss caolrleepctriveseelyn ttharteive ebaincteFriiaglu re 5,
reveaslteradintsh aantdit eixnhtiebriaticntgs iwnhitihbitaiogno zoodnensu lmargbeerr tohfanr etshiodsue epsroodfuaccetdiv beys tihtee roeffegrleunccoe sdarmugin teet-r6a-cpychloinsep hate
(GlcNag6aPin)ssty tnwtho absaec.teIrtiaml asitnraliynsi,n Steerrraactitas wanidth Ep. ofelaacralaisn, ddehpyicdterdo phheoreb iacs rea sridepureesseonftatthivee aicnt iFviegusritee .5,I thas
adoprteevdeawledei rthdat“ Ji”t ionrte“raLc”ts swhiathp ea ignotohde naucmtibveer soift er.esTidhueeso xofy gacetnivea tsoitme sofo gfluthcoestawmoinea-m6-ipdheosgprhoautep sare
respo(GnslciNbl6eP)f osryntthheasfeo. rImt mataiionnly oinfteHra-cbtos nwditihn gpowlairt hantdh heypdorolaprhorbesici dreuseidsuSees rofA t4h0e 1ac(tdivies tsaintec. eIt 2h.a6s1 A◦),
GlnAad3o4p8t(eddi swtaeinrcde “1J”.9 o4rA “L◦)” asnhdapSee irnA th3e4 9ac(tdiviset asintec.e T1h.e7 1oxAy◦g)e.nH aetonmces, othf ethaem twidoe agmroiduep gsraoruepbs eanree ficial
responsible for the formation of H-bonding with the polar residues Ser A401 (distance 2.61 A°), Gln
forfuturedevelopment. Thearene-cationinteractionofbenzeneringofligandwiththepolarresidue
A348 (distance 1.94 A°) and Ser A349 (distance 1.71 A°). Hence, the amide groups are beneficial for
ArgA26hasstrengthenedthebindingofligandwiththereceptor. Thechlorineatomonthebenzene
future development. The arene-cation interaction of benzene ring of ligand with the polar residue Arg
ringoAf2t6h healsi gstarnendghthaesnhedy dthroe pbhinodbiincgi noft elirgaacntido wnsitwh tihthe trheceephtyodr.r oTpheh ochbliocrirnees iadtoumes oTnr pthAe 7b4en,zAelnae Ari6n0g2 and
ValAo3f 9th9et hliagtancdo nhsatsi thuytdertohpeholibpico pinhteilriacctrieogniso wniothf tthhee hcyadvriotyp.hTobhiec r–eCsiOdu-NesH T-rNp –A7m4,o Aieltay Ap6r0e2s aenndt bVeatlw een
the twA3o99b ethnazt ecnoensrtiitnugtes tahse laippooplhailricl irnekgieorn hoaf sthfeu cranviisthy.e Tdhhe i–gChOfl-NexHib-Nil–it myotoiettyh perelisgeannt dbe,twheenenc ethseu ch a
flexibtwleom boeinezteynies raindgvsa nast aag peooluasr flionrkfeur rhthase rfumrnoidshifiedc ahtiigohn sf.leTxihbeilibtye ntzo etnhee rliignagn,dw, hheicnhcei ssuacchti na gflaexsiablbe ridge
moiety is advantageous for further modifications. The benzene ring, which is acting as a bridge
betweenthetwo–CONH–groups,isorientedtowardpolarregionoftheactivesite,whichcomprises
between the two –CONH– groups, is oriented toward polar region of the active site, which comprises
polarresiduessuchasSerA401,CysA300,SerA303,SerA347,ThrA352,GlnA348andSerA349.
polar residues such as Ser A401, Cys A300, Ser A303, Ser A347, Thr A352, Gln A348 and Ser A349.

Figure 5. Docking pose for compound 6a, as a representative, in the active site of glucosamine-
Figure5. Dockingposeforcompound6a, asarepresentative, intheactivesiteofglucosamine-6-
6-phosphate (GlcN6P) synthase.
phosphate(GlcN6P)synthase.
3. Experimental Section
3. ExperimentalSection
3.1. Chemistry
3.1. Chemistry
3.1.1. General
3.1.1. General
All the chemicals were purchased from various suppliers, and were used without further
pAulrliftihcaetiochn,e umnilceassls otwheerrweispeu srtactheads. eMdelftrinogm povianrtsio wuesres umpepasluierresd, oann ad Gwalelernekaumsepd mwelittinhgo uptoifnut rther
purification,unlessotherwisestated. MeltingpointsweremeasuredonaGallenkampmeltingpoint
Molecules2016,21,1664 10of17
apparatus (Sanyo Gallenkamp, South borough, UK) in open glass capillaries and are uncorrected.
Infrared spectra (IR) were recorded using the KBr disc technique using a Perkin Elmer FT-IR
spectrophotometer1000(PerkinElmer,Waltham,MA,USA).1H-and13C-NMRspectrawererecorded
on a BRUCKER-PLUS NMR (Billerica, MA, USA) operating at 500 MHz in deuterated dimethyl
sulfoxide(DMSO-d ). ChemicalshiftsarereferredtointermsofppmandJ-couplingconstantsare
6
giveninHz.MassSpectrawererecordedonaShimadzuGCMS-QP5000instrument(Shimadzu,Tokyo,
Japan). ElementalanalysiswasdonetoevaluatethepresenceofC,H,andNbyPerkinElmer-series-II
andthefoundresultswerewithin±0.4%ofthetheoreticalvalues. Thebiologicalevaluationsofthe
productswerecarriedoutatKingSaudUniversity,Riyadh,KSA.
3.1.2. SyntheticProcedures
Compounds3and4werepreparedaccordingtoreportedprocedures[20].
4-Chloro-N-(2-(hydrazinecarbonyl)phenyl)benzamide5
Amixtureof2-(4-chlorophenyl)-4H-benzoyl[d][1,3]oxazin-4-one4(2.57g,10mmol)and10mL
ofhydrazinehydrate(80%)wasrefluxedfor1h. Evaporationoftheexcesshydrazineunderreduced
pressureandwashingoftheremainingsolidproductwithplentyofwateraffordthetitlecompound
inpureform.
Yield(95%);shinywhitepowder;m.p. 175–177◦C.IR(KBr)ν : 3316–3128(NH and2NH),
max 2
1673, 1635 (2 C=O), 1592 (C=N) cm−1; 1H-NMR (DMSO-d ) δ: 12.04 (s, 1H, NHCO), 11.92 (s, 1H,
6
NHCO),8.51(d,1H,Ar-H,J=8.3Hz),7.95(d,2H,2×Ar-H,J=8.4Hz),7.90(d,1H,Ar-H,J=7.6Hz),
7.76–7.62(m,3H,3×Ar-H),7.28(t,1H,Ar-H,J=7.5Hz),4.52(s,2H,NH );13C-NMR(DMSO-d )
2 6
δ: 165.27 (CO), 164.16 (CO), 138.2, 137.10, 132.43, 129.11, 128.72, 127.80, 125.445, 124.32, 120.93
(12 × Ar-C); MS (ESI): 290 [M+ + 1], Anal. Calcd. For C H ClN O : C (58.04%); H (4.17%);
14 12 3 2
N(14.50%);Found: C(58.08%);H(4.21%);N(14.53%).
SynthesisofCompounds6a–o
Generalprocedures: Amixtureof4-chloro-N-(2-(hydrazinecarbonyl)phenyl)benzamide5(2.89g,
10 mmol) and the appropriate aldehyde (10 mmol) was refluxed in absolute ethanol (15 mL) in
presenceoffewdropsofglacialaceticacidasacatalystforabout4hourandmonitoredwithTLC
(n-hexane:EtOAc,80:20)untilthereactionwascompletedasobservedbyappearanceofasinglenew
spot. Theresultantreactionmixturewascooledtoroomtemperatureandthesolidproductobtained
ineachexperimentwascollectedbyfiltrationandrecrystallizedfromethanoltoaffordthedesired
products6a–o
N-(2-(2-benzylidenehydrazine-1-carbonyl)phenyl)-4-chlorobenzamide(6a)Yield(89%);whitecrystals;m.p.
244–246 ◦C; IR (KBr) ν : 3336–3197 (2 NH), 1673, 1635 (2 C=O), 1592 (C=N) cm−1; 1H-NMR
max
(DMSO-d )δ: 12.14(s,1H,NHCO),11.94(s,1H,NHCO),8.51(d,1H,Ar-H,J=8.3Hz),8.47(s,1H,
6
N=CH),7.96(d,2H,2×Ar-H,J=8.4Hz),7.92(d,1H,Ar-H,J=7.6Hz),7.76–7.47(m,8H,8×Ar-H),
7.30 (t, 1H, Ar-H, J = 7.5 Hz); 13C-NMR (DMSO-d ) δ: 165.39 (CO), 164.00 (CO), 149.53 (N=CH),
6
139.53,137.42,134.49,133.63,133.06,130.86,129.48,129.36,129.10,127.75,123.85,121.85,121.75,121.31
(18 × Ar-C); MS(ESI):379(C H ClN O , [M+ +1]; Anal. Calcd. forC H ClN O : C(66.76%);
21 16 3 2 21 16 3 2
H(4.27%);N(11.12%);Found: C(66.72%);H(4.24%);N(11.14%).
4-Chloro-N-(2-(2-(4-chlorobenzylidene)hydrazine-1-carbonyl)phenyl)benzamide (6b) Yield (95%); white
powder;m.p. 252–256◦C;IR(KBr)ν : 3333–3201(2NH),1675,1639(2C=O),1598(C=N)cm−1;
max
1H-NMR (DMSO-d ) δ: 12.19 (s, 1H, NHCO), 11.95 (s, 1H, NHCO), 8.52 (d, 1H, Ar-H, J = 8.3 Hz),
6
8.45 (s, 1H, N=CH), 7.96 (d, 2H, 2 × Ar-H, J = 8.4 Hz), 7.90 (d, 1H, Ar-H, J = 7.8 Hz), 7.77 (d, 2H,
2×Ar-H,J=8.4Hz),7.65–7.61(m,3H,3×Ar-H),7.52(d,2H,2×Ar-H,J=8.4Hz),7.28(t,1H,Ar-H,
J=7.6Hz);13C-NMR(DMSO-d )δ: 165.46(CO),163.96(CO),148.13(N=CH),139.62,137.43,135.32,
6
133.58,133.42,133.10,129.67,129.44,129.33,129.07,123.76,121.71,121.08(18×Ar-C);MS(ESI):413