pyridine-3-sulfonamide Derivatives with Potential Antifungal

17 Pages·2017·5.48 MB·English

molecules
Article
Syntheses of Novel 4-Substituted N-(5-amino-1H-
1,2,4-triazol-3-yl)pyridine-3-sulfonamide Derivatives
with Potential Antifungal Activity
KrzysztofSzafran´ski1,* ID,JarosławSławin´ski1,* ID,AnnaKe˛dzia2andEwaKwapisz2
1 DepartmentofOrganicChemistry,MedicalUniversityofGdan´sk,Al.Gen.J.Hallera107,
80-416Gdan´sk,Poland
2 DepartmentofOralMicrobiology,MedicalUniversityofGdan´sk,ul.De˛bowa25.,80-204Gdan´sk,Poland;
[email protected](A.K.),[email protected](E.K.)
* Correspondence:[email protected](K.S.),[email protected](J.S.);
Tel.:[phone](K.S.),[phone](J.S.);Fax:[phone](K.S.&J.S.)
Received:10October2017;Accepted:3November2017;Published:7November2017
Abstract: Candidiasis represent a serious threat for patients with altered immune responses.
Therefore,wehaveundertakenthesynthesisofcompoundscomprisingapyridine-3-sulfonamide
scaffold and known antifungally active 1,2,4-triazole substituents. Thus a series of novel
4-substituted N-(5-amino-1H-1,2,4-triazol-3-yl)pyridine-3-sulfonamides have been synthesized
by multistep reactions starting from 4-chloropyridine-3-sulfonamide via N(cid:48)-cyano-N-[(4-
substitutedpyridin-3-yl)sulfonyl]carbamimidothioateswhichwerefurtherconvertedwithhydrazine
hydratetothecorresponding1,2,4-triazolederivatives26–36.Thefinalcompoundswereevaluatedfor
antifungalactivityagainststrainsofthegeneraCandida,Geotrichum,Rhodotorula,andSaccharomycess
isolatedfrompatientswithmycosis. Manyofthemshowgreaterefficacythanfluconazole,mostly
towards Candida albicans and Rhodotorula mucilaginosa species, with MIC values ≤ 25 µg/mL.
Adockingstudyofthemostactivecompounds26,34and35wasperformedshowingthepotential
modeofbindingtoCandidaalbicanslanosterol14α-demethylase. Alsoinvitrocytotoxicityofselected
compoundshavebeenevaluatedontheNCI-60celllinepanel.
Keywords: sulfonamides;pyridine-3-sulfonamides;1,2,4-triazole;antifungalagents;Candidaalbicans;
anticancerscreening
1. Introduction
Thearylsulfonamides,derivedfromtheantibacterialdrugsulfanilamide,constituteanimportant
class of biologically active compounds with a broad spectrum of pharmacological applications.
Versatilebiologicalpropertiesofsulfonamidesincludeantibacterial[1,2],antidiabetic[3],diuretic[4],
antiglaucoma[5],antiviral[6,7],anti-inflammatory[8,9]oranticanceractivity[10–12]. Ourlong-term
studies on pyridine-3-sulfonamides derivatives were focused on the synthesis of 4-substituded
pyridine-3-sulfonamideswithbothprimaryandsecondarysulfonamidemoieties,andexploration
of their multidirectional biological activity: inhibition of carbonic anhydrase isozymes [13–15],
invitro anticancer properties [15,16] and antibacterial activity [15]. Due to the fact that mycoses
arebecomingagrowingpublichealthproblem,mostlyforpatientswithalteredimmunefunctionasa
consequenceofprematurebirth,organtransplantation,primaryimmunedeficiency,HIV/AIDSor
cancerchemotherapy[17,18],weintendedtoextendourresearchtoantifungalactivity. Thereforewe
havesynthesizedaseriesofnovelN-(5-amino-1H-1,2,4-triazol-3-yl)pyridine-3-sulfonamidederivatives
andthenevaluatedthemagainstyeastandyeastlikespecies. Triazolederivatives(commonlynamed
“azoles”togetherwithimidazolederivatives)usuallyshowanantifungalmolecularmechanismof
Molecules2017,22,1926;doi:10.3390/molecules22111926 www.mdpi.com/journal/molecules
Molecules2017,22,1926 2of17
Molecules 2017, 22, 1926 2 of 17
amctoiolencbualasre dmoenchinahniibsimtio onf oafctthioenc ybtaoscehdro omne iPn-h4i5b0i-tdioenp eonfd tehnet clayntoocshterrooml1e4 αP--4d5e0m-detehpyelnadseen(Ct YlaPn5o1s)t[e1r9o]l
a1n4dα-adreemtheethmyolasstec o(CmYmPo51n)l y[1u9s]e adngdr oaurep tohfe amntoifsut ncogmalmdrounglys [u2s0e,2d1 g].rDouupe otof iatnstgifruenatgiaml dprourgtasn [c2e0,,t2h1e].
tDopuiec toof intse wgraeantt iifmunpgoarltadnecreiv, tahtiev teospiiscs otifl lnwewid ealnytiefxupnlgoarle dde[r2i2v–a2ti5v]e.sA icst ustaillll ywgirdeealtya etxtepnlotiroend i[s2b2e–i2n5g].
pAacidtutaolltyh egsryeantt haetsteisnotifonne iws bheyibnrgid pcaoimd ptoo uthned ss,yanmthoensgisw ohf incehwth heynberwidly cpomrespeonutenddsd,e armivoatnivge wschaincha ltshoe
bneewinlcylu pdreedse,nthteadt idnveroilvvaetitvweos cdaifnf earlesnot bpeh ianrcmluadcoepdh, othraest ,ionnveolovfew twhioc hdiifsfear1e,n2t, 4p-thraiarzmoalecorpinhgor[2e6s,– 2o8n]e.
Fouf rtwhhericmho ries ,aa lo1n,2g,4w-tirtihaztholeed reivnegl o[p2m6–e2n8t].o fFourrgtahneircmsoyrnet,h easloisntge cwhnitihq utehse, ndeewvemloepthmoednst aollfo woringgantoic
dsiyrnetchtemsiosd tieficchantiioqnueosf, snidewe cmhaeitnhoodfs1 ,a2l,l4o-wtriianzgo lteo sdciarfefcotl dmhoadvieficeamtieorng eodf [s2i9d,e3 0c]h,apirno voifd 1in,2g,4a-ttroiaozlotloe
mscoadffiofyldt hheavexe iestminegrgleeadd [2s9tr,3u0c]t,u prerso.viding a tool to modify the existing lead structures.
22.. RReessuullttss aanndd DDiissccuussssiioonn
2.1. Chemistry
2.1. Chemistry
ThegeneralsyntheticroutesforthepreparationofthedesiredN-(5-amino-1H-1,2,4-triazol-3-yl)
The general synthetic routes for the preparation of the desired N-(5-amino-1H-1,2,4-triazol-3-yl)
pyridine-3-sulfonamides26–36areshowninScheme1.
pyridine-3-sulfonamides 26–36 are shown in Scheme 1.

Scheme 1. Synthesis of 4-substituted N-(5-amino-1H-1,2,4-triazol-3-yl)-pyridine-3-sulfonamides 26–36.
Scheme1.Synthesisof4-substitutedN-(5-amino-1H-1,2,4-triazol-3-yl)-pyridine-3-sulfonamides26–36.
The necessary starting materials, i.e. 4-substituted pyridine-3-sulfonamides 2–6, 8–11 and 13–14
were obtained according to the previously described methods [13,15], (see Scheme S1 in
Supplementary Materials). Thus, target N-(5-amino-1H-1,2,4-triazol-3-yl)pyridine-3-sulfonamides
Molecules2017,22,1926 3of17
Thenecessarystartingmaterials,i.e. 4-substitutedpyridine-3-sulfonamides2–6,8–11and13–14
wereobtainedaccordingtothepreviouslydescribedmethods[13,15],(seeSchemeS1inSupplementary
Materials). Thus, target N-(5-amino-1H-1,2,4-triazol-3-yl)pyridine-3-sulfonamides 26–36 were
Molecules 2017, 22, 1926 3 of 17
synthesizedinconvenienttwo-stepreactions[31]bytreatmentofprimarypyridine-3-sulfonamides
2–6,82–6–1316a wndere1 3s–y1n4thwesiitzhedd iimn ceothnvyelnNie-ncty tawnoo-ismteipn oredaictthioioncs a[r3b1]o nbya ttereiantmboeinlti nogf parciemtoanrye pinyrtihdeinpe-r3e-s ence
of anshuylfdonroamusidpeos t2a–s6s, i8u–m11 caanrdb 1o3n–a1t4e w, aitnhd ditmheenthyinl Nth-ceyanneoxitmsintaogdeithoifocthareborenaactet iionn boseilqinuge ancceeto,nwe iitnh 99%
hydrtahzei nperehseyndcrea otef ainnhaycdertoounsi tproilteasosriuemth caanroblonaattree, flaundx t(hSecnh einm tehe1 )n.ext stage of the reaction sequence,
wFaitchi l9e9i%so hlaytdiroanzionfe Nhy(cid:48)-dcryaaten ion- Nac-e[t(o4n-iStruilbes ot.r- petyhraindoinl a-3t -ryefll)usxu l(fSocnhyeml]cea 1r)b. amimidothioatederivatives
15–19inwFahciicleh itsholeat4i-opny orfi dNin′-ecysaunbos-Nti-t[u(4e-nStuibssta.n-peylreidcitnro-3n-ydl)osunlaftoinnygl]4ca-arbryamlpiimpiedroatzhiinoeatme doeieritvya,tciovenss isted
15–19 in which the 4-pyridine substituent is an electron donating 4-arylpiperazine moiety, consisted
of acidification of the initially formed potassium salts with 4% hydrochloric acid to pH 7, giving
of acidification of the initially formed potassium salts with 4% hydrochloric acid to pH 7, giving the
thedesiredpureproduct(Scheme1). Ontheotherhand, inthecaseofcompoundscontainingan
desired pure product (Scheme 1). On the other hand, in the case of compounds containing an
electron withdrawing substituent, such as 1H-pyrazol-1-yl (compounds 8–11) or alkylthio groups
electron withdrawing substituent, such as 1H-pyrazol-1-yl (compounds 8–11) or alkylthio groups
(compounds13and14),wefoundthatthecorrespondingpotassiumsaltsoftype20A–25Awerestable
(compounds 13 and 14), we found that the corresponding potassium salts of type 20A–25A were
andastmabelne aabnlde taomdeinraebcltei stool adtiiroenct wishoelantioanci dwifiheend atocidpiHfie7d, two hpilHe i7s,o lwahtiiolen iosoflpatriootno noaf tperdotcoonmatpedo unds
20–25cormeqpouuirnedds s2t0r–o2n5 greeqruaicreidd isfitcroantigoenr atocidpifHica1ti–o2n wtoi tphH4 1%–2 hwyidthr o4c%h lhoyrdicroacchildor.icI nacitdh.e Inn ethxet nsetexpt , the
previsoteups,l tyheo pbrteavinioeudslmy oebthtayinleNd (cid:48)m-ceytahnylo N-N′-c-y[(apnyor-Nid-i[n(p-y3r-iydli)ns-u3l-fyol)nsyullf]ocnarybl]acamrbimamidimotidhoiothaitoeaste1s5 1–52–525i n the
formino tfhteh feorcmo rorfe tshpeo cnodrriensgpopnodtiansgs ipuomtasssiaulmts s1a5ltAs –1255AA–2,5iAn, rinea rcetaicotnionw witihth9 999%% hhyyddrraazzinine ehyhdyrdatrea te in
boilining baocileintogn aictertioleni(tcriolem (pcoomupnodusn2d6s –2360–)30o)r oer tehthaannooll ((ccoommppoouunndds s313–13–63) 6fu)rfnuirsnheisdh tehde Nth-(e5-Nam-(i5n-oa-m ino-
1H-1,2,4-triazol-3-yl)pyridine-3-sulfonamide derivatives 26–36 in moderate to good yields of 40–69%
1H-1,2,4-triazol-3-yl)pyridine-3-sulfonamidederivatives26–36inmoderatetogoodyieldsof40–69%
(Scheme 1).
(Scheme1).
Interestingly, it has been found, that treatment of protonated form of methyl N′-cyano-N-
Interestingly, it has been found, that treatment of protonated form of methyl N(cid:48)-cyano-N-
[(pyridin-3-yl)sulfonyl]carbamimidothioates 20–23 with 99% hydrazine hydrate led to the formation
(pyridin-3-yl)sulfonyl]carbamimidothioates20–23with99%hydrazinehydrateledtotheformationof
of only compound 37 having the hydrazine moiety in position 4 instead of a pyrazol-1-yl ring
only(cSocmhepmoeu 2n)d. 37havingthehydrazinemoietyinposition4insteadofapyrazol-1-ylring(Scheme2).

Scheme 2. Proposed mechanism of the formation of undesirable product: N-(5-amino-1H-1,2,4-
Scheme 2. Proposed mechanism of the formation of undesirable product: N-(5-amino-1H-1,2,4-
triazol-3-yl)-4-hydrazinopyridine-3-sulfonamide (37).
triazol-3-yl)-4-hydrazinopyridine-3-sulfonamide(37).
The proposed explanation of the observed reaction pattern (Scheme 2) can be justified by the
kTnhoewpnr foapcto tsheadt coemxpploaunnadtsi ownitoh fthteh e1-aolbksyel-r2v-ceydanroe-a3c-(tpioyrnidpinaet-t3e-rsnulf(oSncyhle)gmuaen2id)incea nstrbuectjuurset aifidoepdt ba ythe
knowzwnitftaecritonthica ftorcmo m[32p]o; tuhnerdesforwe,i tshimtihlaer m1-eathlkyly Nl-′2-c-ycaynaon-No--3[(-p(ypryidriind-i3n-yel-)3su-slfuolnfyoln]-ycal)rgbaumanimididinotehisotartue cture
adopdteraivaztwivietst e2r0i–o2n3i tchafto arlmso s[h3o2w]; sttrhoenrge afocirdei,c psriompielratriesm sheothuyldl bNe p(cid:48)-rceysaennto i-nN th-[e( pfoyrrmid oinf i-n3n-yerl )ssaultlsf.o nyl]-
carbaImntrimamidooletchuiloaar teprdoetroinvaattiiovne sof2 0t–h2e3 ptyhraitdianleso nsihtroowgenst raotnomg afcaicdiilictaptreos ptehret ieasttaschko uolfd tbhee pnrietrsoegnetni nthe
nucleophile at position 4, which leads to the substitution of the pyrazole ring by hydrazine. In the
formofinnersalts. Intramolecularprotonationofthepyridinenitrogenatomfacilitatestheattackof
final step the strong acidic N-cyanoisothiourea moiety is cyclized to the 1,2,4-triazole ring.
thenitrogennucleophileatposition4,whichleadstothesubstitutionofthepyrazoleringbyhydrazine.
All the synthesized compounds were characterized by IR and 1H-NMR spectroscopy and
InthefinalstepthestrongacidicN-cyanoisothioureamoietyiscyclizedtothe1,2,4-triazolering.
elemental analyses (C, H, N). In the IR spectra of the methyl N′-cyano-N-[(pyridin-3-yl)sulfonyl]
All the synthesized compounds were characterized by IR and 1H-NMR spectroscopy and
carbamimidothioates 15–25 the characteristic absorption bands corresponding to the stretching vibration
elemental analyses (C, H, N). In the IR spectra of the methyl N(cid:48)-cyano-N-[(pyridin-3-yl)sulfonyl]
of cyanide (C≡N) group appeared in the range of 2161–2178 cm−1 and disappeared in the final
carbaNm-aimmiindoo-1thHi-o1a,2t,e4s-tr1ia5z–o2l5 dtehreivacthivaersa c2t6e–r3is7t icaftaebr scoyrcplitzioatnionb.a nSdimsilcaorlryr etshpeo n1Hd-iNngMRto stpheectrsatr eotfc hing
compounds 15–25 show singlets at 2.15–2.38 ppm corresponding to the SCH3 group which do not
Molecules2017,22,1926 4of17
vibration of cyanide (C≡N) group appeared in the range of 2161–2178 cm−1 and disappeared in
thefinalN-amino-1H-1,2,4-triazolderivatives26–37aftercyclization. Similarlythe1H-NMRspectra
ofcompounds15–25showsingletsat2.15–2.38ppmcorrespondingtotheSCH groupwhichdonot
3
occurinthe1H-NMRspectraofthetriazolederivatives. ThestronglyacidicNHprotonofcompounds
15–19occursasverybroadsignalwithachemicalshiftover13.6ppm. Surprisinglythecorrresponding
NH proton signal of compounds 20–25 is not observed at 14 ppm, however a substantial water
signal usually occurring in DMSO-d at 3.35 ppm is found downfield as a broad signal at about
6
6ppm,probablyduetofastexchangewiththeacidicNHproton. Compounds26–37howeverare
characterizedbybroadaminoNH groupsignalsintherangeof5.82–6.05ppm.
2
2.2. AntifungalActivity
Compounds 26–36 were evaluated for their invitro antifungal activity on 31 yeast strains
belonging to the genera of Candida, Geotrichum, Rhodotorula, and Saccharomycess isolated from
patients with candidiasis of the oral cavity and respiratory tract (Candida albicans—eight strains,
Candida glabrata—four strains, Candida guilliermondii—two strains, Candida krusei—three strains,
Candidalusitaniae—twostrains, Candidaparapsilosis—threestrains, Candidatropicalis—threestrains,
Candidautilis—onestrain,Geotrichumcandidum—twostrains,Rhodotorulamucilaginosa—twostrains,
Saccharomycescerevisiae—onestrain).Alsosixstandardizedstrainswereusedinthetest:Candidaalbicans
ATCC10231,CandidaglabrataATCC66032,CandidakruseiATCC14243,CandidalusitaniaeATCC34499,
CandidaparapsilosisATCC22019andCandidatropicalisATCC750. Theminimalinhibitoryconcentration
(MIC),definedasconcentrationoftestedcompoundwhichcausescompletegrowthinhibition,was
determinedbymeansofthedilutiontechniqueintheSabouraud’sagar(Table1). Fluconazolewas
chosenasreferencedrug.
Among tested yeast-like species, Candida albicans turned out to be the most sensitive to the
testedderivatives, showingsusceptibilitytoeightcompounds(26, 28, 30–32and34–36)withMIC
valueslowerorcomparablethanthereferencedrug(MIC=25–100µg/mL).Agoodactivityprofile
against Rhodotorula mucilaginosa was also observed for compounds 26, 28, 32, 35, and 36, which
exhibitedgreaterefficacythanfluconazole(MIC≥100µg/mL).Furthermore,severalcompounds
showedoutstandingactivitytowardssomeyeaststrains,i.e.,compound36againstCandidaglabrata
(MIC ≥ 25 µg/mL), 26 against Candida guilliermondii (MIC ≥ 12.5 µg/mL), 32 and 35 against
Candida krusei (MIC ≥ 25 µg/mL and MIC ≥ 12.5 µg/mL) and 31 against Candida tropicalis (MIC
≥ 25 µg/mL). Lack of sensitivity to the test compounds was however observed for strains of
Candidaparapsilosis,GeotrichumcandidumandSaccharomycescerevisiae.
The structure-activity analysis revealed that among the 4-(4-phenylpiperazin-1-yl)pyridine-
3-sulfonamidederivatives26–30compound26,withanunsubstitutedbenzenering26orcompound28
substitutedwithfluorineatomaresomeofthemostactivecompounds.Ontheotherhand,compounds
containing one or two chlorine atoms in the phenylpiperazine substituent like 27 and 29 didn’t
demonstrateanyantifungalactivity. Similarlyintheseriesof1H-pyrazolederivatives31–34,lackofa
substituent(compounds31,34)orthepresenceofamethylgroup(compound32)attheposition4of
thepyrazoleringexertedapositiveeffectonantifungalproperties,whilethelongaliphaticn-butyl
chainincompound33resultedinlossofactivity.
2.3. InSilicoDockingStudies
To assess the possible ability of the new compounds to bind to lanosterol 14α-demethylase
(CYP51),whichismoleculartargetforclinicallyusedazole-antifungals,moleculardockingstudies
wereperformed. Forthesestudiescompounds26,34and35werechosen,duetotheirhighestoverall
activityandvariedtypesofsubstituentsinposition4ofthepyridinering(N-phenylpiperazinein26,
pyrazolein34andanalkylthiomoietyin35). Alsofluconazole,andposaconazole,drugsknownto
inhibitCYP51,weretakenasthepositivecontrol.
Molecules2017,22,1926 5of17
Table1.TheMICvalues(µg/mL)obtainedforcompounds26–36andfluconazole(Fl)againstyeaststrainsusedinassay.
Candida Candida Candida Candida Candida Candida Candida Geotrichum Rhodotorula Saccharomyces
Candidaalbicans
Compd. M.W: glabrata guilliermondii krusei lusitaniae parapsilosis tropicalis utilis candidum mucilaginosa cerevisiae
(9strains)
(5strains) (2strains) (4strains) (3strains) (4strains) (4strains) (1strain) (2strains) (2strains) (1strain)
26 400.46 ≤61.020;;5*0;;*1;0*;0*;1A00; *;*;*;*;100B 12.5;25 100;*;*;100C 100;*;12.5D *;*;*;*,E 100;*;*;100F * *;* ≤6.2;50 *
27 434.90 *;*;*;*;*;*;*;*;*A *;*;*;*;*B *;* *;*;*;*C *;*;*,D *;*;*;*,E *;*;*;*,F * *;* *;* *
28 418.45 12.5;1*2;.*5;;*1;0*0A;100;*; *;*;*;*;*B 50;100 100;*;*;*C 50;100;50D *;*;*;*E *;*;*;*F 100 *;* 12.5;25 *
29 469.35 *;*;*;*;*;*;*;*;*A *;*;*;*;*B *;* *;*;*;*C *;*;*D *;*;*;*E *;*;*;*F * *;* *;* *
30 430.48 12.5;10*;0*;;1*0A0;*;*;*; *;*;*;*;*B *;* *;*;*;*C *;*;*D *;*;*;*E *;*;*;*F * *;* 100;* *
31 334.36 25;50;*;*;*;*;*;*;*A *;*;*;*;*B *;* *;*;*;*C *;*;*D *;*;*;*E 25;*;*;50F * *;* 100;* *
32 348.38 25;50;100A;*;*;*;*;*;* 50;*;*;*B *;* 25;*;*;50C *;*;*D *;*;*;*E 100*;*;100F 100 *;* 25;25 *
33 390.46 *;*;*;*;*;*;*;*;*A *;*;*;*;*B *;* *;*;*;*C *;*;*D *;*;*;*E *;*;*;*F * *;* *;* *
34 362.41 ≤6.2;≤*6;*.2;;*1A00;*;*;*; *;*;*;*;*B *;* 100;*;*;*C 50;100;100D *;*;*;*E *;*;*;*F * *;* 100;200 *
35 362.43 ≤160.20;;1*;2*.5;;*;5500;1A00; 100;100B;*;*;100 100;* 12.5;*;*;50C *;*;*D *;*;*;*E *;*;*;*F * *;* 6.2;100 *
36 329.36 12.5;5*0;;*;110000;1A00;*;*; 25;100;*;*;100B 50;100 100;*;*;100C 100;*;100D *;*;*;*E *;*;*;*F * *;* 25;100 *
Fl 306.28 12.5;50;##;A#;#;#;#;#; 25;50;#;#;25B 12.5;50 25;5500C;50; 121.25.;51D2.5; 6.2;31.21.5E;25; 25;50;#;6.2F 25 12.5;25 #;# #
M.W.—molecularweight;*≥200(µg/mL);#>100(µg/mL);A—standardstrainCandidaalbicansATCC10231;B—standardstrainCandidaglabrataATCC66032;C—standardstrainCandida
kruseiATCC14243;D—standardstrainCandidalusitaniaeATCC34499;E—standardstrainCandidaparapsilosisATCC2201;F—standardstrainCandidatropicalisATCC750.
Molecules2017,22,1926 6of17
BMeolcecauuless e201C7,a 2n2d, 1i9d2a6 albicans turned out to be the most sensitive from among the teste6d ofs 1p7 ecies
dockiMnoglecoulfest 2h0e17c, 2o2m, 19p2o6 undswasperformedinthecrystallographicstructureofC.albicansl6a onf o17s terol
14α-demeBtehcyaulassee C[a3n3d]id(Pa DalBbicIaDns: 5tFuSrnAe)d coou-ct rtyos tbael litzhee dmwositt hsepnossitaivceo nfraozmo lea,moobntga itnheed tefsrtoemd stpheeciPerso tein
dockiBnegc aouf steh eC caonmdidpao ualnbdicsa nws atsu prneerdfo romuet dt oi nb teh eth cer ymstoasllto gsernapsihtiivce s tfrruocmtu raem oofn Cg. tahlbei ctaensst eldan sopsteecrieosl
DataBankwebsite. Thedockingsimulationsintotheactivesiteoftheenzymewasperformedusing
1d4oαck-dinegm oeft htyhlea sceo m[3p3o] u(PnDdsB wIDas:5 pFeSrAfo) rcmo-ecdr yisnt athlleiz cerdy swtaitlhlo gproaspahcoicn astzroulcet,u orbet oaifn Ce.d a flbroicman sth lae nPorsoteterionl
theAutodockVinasoftware[34].
D14aαta-d Beamnekt hwyelbasseit e[.3 T3]h (eP dDoBc kIiDn:g5 FsiSmAu) lcaoti-ocnryss itnatloli zthede awctitivhe p soitsea coof nthazeo elne,z yombtea iwneads pfreormfo rtmhee dP ruostienign
First,inordertovalidatethedockingmethodology,weperformedaredockingofposaconazole,
tDhaet Aa Buatondko wcke bVsiintea. sTohftew daorcek [i3n4g] .s imulations into the active site of the enzyme was performed using
theformerco-crystallizedligandwithCYP51,initsactivesite. Therootmeansquaredeviation(RMSD)
the AFuirtsotd, ionc kor Vdeinr ato s ovfatlwidaartee t[h34e ]d. ocking methodology, we performed a redocking of posaconazole, the
valuefobremtwFerir ecseot,n -icnrt yhosretdaetlorli ptzoe rdvaa lnliigdkaaentded t phwreie tdhdo iCccktYienPdg5 1mc,o einnth fiootsdr moalcoatigtvyieo, nwsiteae pn. eTdrhftoehr rmeooeodtb mas ereeradvnoe scdqkuiXnag-rr eoa fdy peocvrsiaaytcsiootnanal z(sRotlMreu,S tcDhtue) reof
liganvdfoa,rlwmueae rsb ec1ot.w1-c9er8eynsÅt athl(laeiz vteodap ll uirgeaanbnkedel dow wpitrh2e dCÅiYctpPe5od1i n,c oitnni nfitodsr miaccaatittvieoesn st ihatena.dt T tthhheee rdooobocst ekmrivneeagdnp Xsrq-orutaaoyrc eoc rdlyewsvtaaials tisvotranul i(cdRtuaMrteeS dDo)f).
Tlvigahlaeunedd b,o wectkawisne 1eg.n1 9pt8ha eÅr a t(moap ev traealurnsek eobdeb ltpoawrien d2ei cdÅte fpdoo rcinottnh ifenodrbmiecaasttteiossn ct ohaarnet ddth tech oedn oofcboksrienmrgva eptdrioo Xtno-srcaosylh wcorawyss stvaatlhl isdatratutiecndtvu).re es toigf ated
complioguannTddh,s ewd daisso pc1kl.1ai9ny8g m Åpo a(arrae vmfaaeluvteeor rbsa eobloblweta ei2ns eÅtidm p foaoitrne ttd hineb dibnicedastti enssgc tohfrraeetd et hceeon ndeforogcrkmyi,na−gti o9pn.r9os tksochcaoolwl/ wsm atosh lavtfa oliirndvcaeotesmtdig)p.a oteudn d26,
−9.3ckocmalp/Tohmueno ddlsof ocdkriisncpoglam pypa mroaoumrneed tfea3rvs5o oraabnbtdalein− ees9dt.i 1mfokarct etahdle/ b mbineodslti nfsogc rofrrceeodem ecnpoeonrfugonyrm,d −a39t.4i9o, nkresc asslph/meocwotlis vf oethrly ac,to timhnavpneosufltniugdac to2e6nd,a zole
−co9.m3 pkocualn/mdso dl ifsoprl acoym mpoorue nfdav 3o5r aabnlde e−s9t.i1m kactaeld/ mbionl dfionrg c formeep eonuenrdg y3,4 −, 9r.e9s pkceaclt/imveolyl ,f othr acno mflupcoounnadz o2l6e,
(−8.1kcal/mol),butlessbeneficialthenposaconazole(−13.2kcal/mol,Table2).
(−−98.3.1 kkccaall//mmooll )f,o br ucto lmesps obuennde f3ic5i aal nthde −n9 p.1o ksaccaol/nmaozol lfeo r(− c1o3m.2p kocuanl/dm 3o4l,, Traesbplee c2t)i.v ely, than fluconazole
(−8.1 kcal/mol), but less beneficial then posaconazole (−13.2 kcal/mol, Table 2).

Figure 1. Superimposition of computed binding geometry of compound 26 (green) and conformation
Figure1.Superimpositionofcomputedbindinggeometryofcompound26(green)andconformation
oFfi gcuor-cer 1y.s Staulpliezreidm lpigoasnitdio pno osfa ccoomnapzuotleed (r beidn)d iinnsgid gee othme estorlyv oenf tc oamccpesosuibnlde 2s6u r(fgarceee no)f aCnYdP c5o1n afcotrimvea stiioten.
ofco-crystallizedligandposaconazole(red)insidethesolventaccessiblesurfaceofCYP51activesite.
of co-crystallized ligand posaconazole (red) inside the solvent accessible surface of CYP51 active site.

Figure 2. Superimposition of the computed binding geometry of compounds 26 (green), 34 (purple)
aFnigdu 3r5e (2b.l uSeu)p. Aertiommpso ostihtieorn t hoafn t hcaer cboomn:p suutlfeudr —biynedllionwg ,g neiotrmogeetrny— ovfi ocolemt, poxoyugnedns— 2r6e d(g, rheyednr)o, g3e4n (—pugrrepyle. )
Figuraen2d. 3S5u (bpleurei)m. Aptoosmitsi oonthoerf tthhaenc coamrbponu:t seudlfbuirn—dyienlglogwe, onmitreotgreyno—fvcioomletp, ooxuyngdens—26re(dg,r heyednr)o,g3e4n(—pgurrepyl.e )and
35(blue).Atomsotherthancarbon:sulfur—yellow,nitrogen—violet,oxygen—red,hydrogen—grey.
Molecules2017,22,1926 7of17
Molecules 2017, 22, 1926 7 of 17

FFiigguurree 33.. TThhee ttwwoo--ddiimmeennssioionnaallp preresesenntatatitoionno fofe neznyzmyme-eli-gliagnadndin itnertearcaticotnioonf ocfo mcopmopuonudnsd2s6 ,2364, 3a4n dan3d5
a3n5 danthde tahcet iavcetisviete siotfe CoYf CP5Y1P(5P1D (PBDIDB: 5IDFS:5AF)S.A).
The computed geometries of compounds 26, 34 and 35 suggest however different binding
The computed geometries of compounds 26, 34 and 35 suggest however different binding
patterns of the tested compound than that of well-known azole antifungal drugs like fluconazole,
patterns of the tested compound than that of well-known azole antifungal drugs like fluconazole,
and posaconazole [35]. Generally, the 1,2,4-triazole moiety in the investigated compounds doesn’t
andposaconazole[35]. Generally,the1,2,4-triazolemoietyintheinvestigatedcompoundsdoesn’t
interact covalently with the CYP51 heme, instead is directed towards hydrophobic tunnel
interactcovalentlywiththeCYP51heme,insteadisdirectedtowardshydrophobictunnelconnecting
connecting the chamber adjacent to the heme with the protein surface (Figure 1 and 2). Furthermore,
thechamberadjacenttothehemewiththeproteinsurface(Figures1and2). Furthermore,theNH
othreN NHH ogrr oNuHp2o gfrtohuepa omf itnhoet raimazinoloetrriianzgoloef rcinogm opfo cuonmdpso2u6n,d34s 2a6n,d 343 5anfodr m35s fhorymdrso hgyednrboognend sbownidths
2
with Ser378. The space over the Fe ion, which supposed to be occupied by the triazole, is filled with
Ser378. The space over the Fe ion, which supposed to be occupied by the triazole, is filled with
the pyridine ring stabilized by a π-π interaction with Phe228 (Table 2, Figure 3).On the other hand
thepyridineringstabilizedbyaπ-πinteractionwithPhe228(Table2,Figure3).Ontheotherhand
the substituent from position 4 of the pyridine scaffold is directed deeply inside the active pocket
the substituent from position 4 ofthe pyridine scaffold is directed deeply inside theactive pocket
(previously occupied by the difluorophenyl ring of posaconazole) interacting with the porphyrin
(previouslyoccupiedbythedifluorophenylringofposaconazole)interactingwiththeporphyrinring
ring and active site residues (Figure 1 and 2). Location of substituents from the position 4 in the
andactivesiteresidues(Figures1and2). Locationofsubstituentsfromtheposition4inthebinding
binding cleft may be responsible for large differences in activity of synthesized compounds
cleftmayberesponsibleforlargedifferencesinactivityofsynthesizedcompoundscorrelatedwith
correlated with relatively minor changes in the structure of this substituent (e.g., compounds 26 and
relativelyminorchangesinthestructureofthissubstituent(e.g.,compounds26and29or33and34).
29 or 33 and 34). It is possible to suppose that this low molecular compounds (MW < 470) with
Itispossibletosupposethatthislowmolecularcompounds(MW<470)withsimpleandmodular
simple and modular structure, and easily susceptible to structural diversification with substituents
structure,andeasilysusceptibletostructuraldiversificationwithsubstituentsinposition4ofpyridine
in position 4 of pyridine ring makes the N-triazolopyridine sulfonamide scaffold a suitable molecule
ringmakestheN-triazolopyridinesulfonamidescaffoldasuitablemoleculeforfurtherhittolead
for further hit to lead optimization in designing anti-Candida drugs.
optimizationindesigninganti-Candidadrugs.
Molecules2017,22,1926 8of17
Table2. Predictedbindingaffinityandnon-covalentinteractionbetweencompounds26,34and35
andtheactivesiteofCYP51.
Compound MoleculeFragmentInvolvedin Interacting Typeof Interaction
BindingAffinity Interaction Residue Interaction Distance[Å]
LEU376 π-alkyl 4.93
SER378 hydrogenbond 2.67
5-amino-1H-1,2,4-triazole
PHE380 π-donorhydrogen 4.93
MET508 π-sulfur 2.67
PHE228 π-π 5.13
26 pyridine-3-sulfonamidescaffold GLY307 π-donorhydrogen 3.56
−9.9(kcal/mol)
TYR132 π-alkyl 5.24
HEME π-alkyl 5.22
HEME π-σ 3.23
4-phenylpiperazinesubstituent
ILE304 π-alkyl 5.42
ILE131 π-σ 3.70
ILE131 π-σ 3.51
LEU376 5.06
5-amino-1H-1,2,4-triazole 2.75(OH)
SER378 hydrogenbond
2.77(C=O)
PHE233 π-π 2.29
34 PHE228 π-π 5.05
−9.3(kcal/mol) pyridine-3-sulfonamidescaffold LEU376 π-alkyl 5.41
MET508 π-alkyl 5.38
LEU376 π-alkyl 4.03
ILE131 π-alkyl 4.66
3,5-diethyl-1H-pyrazolesubstituent HEME π-σ 3.69
HEME π-π 4.97
HEME π-alkyl 3.71
LEU376 π-alkyl 4.39
5-amino-1H-1,2,4-triazole SER378 hydrogenbond 1.83
TYR118 hydrogenbond 2.19
35 LEU376 π-alkyl 5.27
−9.1(kcal/mol) pyridine-3-sulfonamidescaffold PHE228 π-π 5.66
ILE131 π-alkyl 5.16
benzylthiosubstituent HEME π-σ 4.18
HEME π-σ 3.74
2.4. AnticancerActivity
Recently, we have shown that 4-(4-arylpiperazin-1-yl)pyridine-3-sulfonamides 2–6 exhibited
moderateantitumoractivity[15],whilesomeoftheirsulfonylureaderivatives[13]showedantitumor
activity either beneficial or comparable to the clinically tested diarylsulfonylurea sulofenur [16].
Thispromptedustoevaluateobtainednewcompounds20,26,28–31and34–36intheNCI-60Human
TumorCellLinesScreenintheNationalCancerInstitute(Bethesda,MD,USA).Thepreliminaryassay
wasperformedatasinglehighconcentrationof10µMagainsttheNCIpanelof60celllinesderived
fromninedifferenthumancancertypes: leukemia,non-small-celllungcancer(NSCLC),colon,central
nervoussystem(CNS),melanoma, ovarian, renal, prostateandbreastcancer. Inresultsinhibition
growthpercent(IGP)comparedtono-drugcontrol,havebeencalculated,(dataobtainedforthemost
sensitivecelllinesareshownTableS1inSupplementaryMaterials). Inthisseries,moderatecytostatic
activity (IGP ≥ 10%) of particular compounds, was observed only for 1 to 5 cell lines from whole
NCI-60panel. ThehighestIGPvalueof23%wasfoundforcompound34(R=3,5-diethyl-1H-pyrazole)
towardstheMCF7breastcancercelllineandforcompound36(IGP=21%;R=-SCH CONH )towards
2 2
theSNB-75braincancerline. Nosignificantrelationshipwasfoundbetweencytostaticactivityand
thestructureoftestedcompounds,withtheexceptionofincreasedsensitivityofrenaltumorUO-31
(IGP=10–17%)tocompounds26,28–30with4-phenylpiperazinesubstituentatthe4-positionofthe
pyridinering.
Molecules2017,22,1926 9of17
3. MaterialsandMethods
3.1. GeneralInformation
Thefollowinginstrumentsandparameterswereused: meltingpoints: BoethiusHMKapparatus
(VebAnalytic,Dresden,Germany);IRspectra: KBrpellets,400–4000cm−1ThermoMattsonSatellite
FTIRspectrophotometer(ThermoMattson,Madison,WI,USA);1H-and13C-NMRspectra: Gemini
200 at 200 and 50 MHz (13C), respectively, or Unity 500 Plus apparatus at 500 MHz and 125 MHz
(13C) (Varian, Palo Alto, CA, USA); chemical shifts are expressed at δ in ppm values relative to
TMSasstandard. LC-MSanalysesofcompound37wasperformedona: LCMS-ESI-IT-TOFLC-20A
mass spectrometer (Shimadzu Scientific Instruments, Columbia, MD, USA) in positive ion mode.
Elementalanalyses(C,H,N)wereperformedon2400SeriesIICHNElementalAnalyzer(PerkinElmer,
Shelton,CT,USA).Thin-layerchromatographywasperformedonsilicagel60F254TLCplates(Merck,
Darmstadt,Germany)usingbenzene/ethanol(4:1)asmobilephases,andvisualizedwithUVlight254
or366nm.
Marvin ver. 17.9, was used for drawing, displaying and characterizing chemical structures,
substructuresandreactions(ChemAxon,http://www.chemaxon.com,2017).
Thestarting4-chloro-3-pyridinesulfonamide(1)wascommerciallyavailable(AlfaAesar,Karlsruhe,
Germany), and primary 4-substituted pyridine-3-sulfonamides 2–14 were obtained according to
previouslydescribedmethods(2–11[15]and12–14[13]).
3.2. Synthesis
3.2.1. ProcedureforthePreparationofMethylN(cid:48)-Cyano-N-{[4-(piperazin-1-yl)pyridin-3-yl]sulfonyl}
carbamimidothioates15–19
Amixtureofappropriate4-(piperazin-1-yl)pyridine-3-sulfonamide(2–6)(2.5mmol),dimethyl
N-cyanodithioiminocarbonate (0.40 g, 2.75 mmol) and anhydrous potassium carbonate (0.33 g,
2.5 mmol) in dry acetone (7.5 mL) was stirred under reflux for 18 h. The reaction mixture was
evaporated under reduced pressure, residue was dissolved in water 5 mL, and extracted with
dichloromethane(3×5mL).Organiclayerwasremovedandwaterphasewasacidifiedwithdiluted
hydrochloricacidtopH=7. Theprecipitateobtainedwascollectedbyfiltrationanddried. Inthis
manner,thefollowingproductswereobtained.
MethylN(cid:48)-cyano-N-{[4-(4-phenylpiperazin-1-yl)pyridin-3-yl]sulfonyl}carbamimidothioate(15). Startingfrom
4-(4-phenylpiperazin-1-yl)pyridine-3-sulfonamide2(0.76g)thetitlecompoundwasobtained. Yield
0.87g,(84%);m.p. 241–244◦C;IR(KBr): 3177(NH),3092(C -H),2922(C-H),2161(C≡N),1645,1599,
Ar
1528(C=C,C=N)cm−1;1H-NMR(500MHz,DMSO-d )δ: 2.36(s,3H,S-CH ),3.33(m,4H,2×CH ),
6 3 2
4.07(m,4H,2×CH ),6.80(t. 1H,H-4Ph),6.98(d. 2H,H-2,6Ph),7.23(t. 2H,H-3,5Ph),7.36(d. 1H,
2
H-5pyrid.),8.31(d. 1H,H-6pyrid.),8.82(s. 1H,H-2pyrid.),13.65(brs,1H,NH)ppm. Anal. calcd. for
C H N O S (416.52);C,51.90;H,4.84;N,20.18. Found: C,51.42;H,4.70;N,20.17.
18 20 6 2 2
Methyl N-[4-[4-(4-chlorophenyl)piperazin-1-yl]pyridin-3-yl}sulfonyl-N(cid:48)-cyanocarbamimidothioate (16).
Starting from 4-[4-(4-chlorophenyl)piperazin-1-yl]pyridine-3-sulfonamide 3 (0.88 g) the title
compound was obtained. Yield 1.01 g, (90%); m.p. 254–257 ◦C dec.; IR (KBr): 3172 (NH), 3090
(C -H),2987,2924(C-H),2162(C≡N),1647,1572,1486(C=C,C=N),1326,1127(SO )cm−1;1H-NMR
Ar 2
(500MHz,DMSO-d )δ: 2.36(s,3H,S-CH ),3.34(m,4H,2×CH ),4.06(m,4H,2×CH ),7.00(d,
6 3 2 2
2H,H-2,6Ph),7.25(d,2H,H-3,5Ph),7.36(d,1H,H-5pyrid.),8.30(d,1H,H-6pyrid.),8.81(s,1H,
H-2pyrid.), 13.74(brs, 1H,NH)ppm; 13C-NMR(DMSO-d , 50MHz)δ: 15.60; 47.69; 50.55; 113.53;
6
116.50; 116.99; 122.89; 126.19; 128.97; 139.97; 145.53; 149.30; 156.03; 173.86 ppm. Anal. calcd. for
C H ClN O S (450.97);C,47.94;H,4.25;N,18.64. Found: C,47.76;H,4.14;N,18.30.
18 19 6 2 2
Molecules2017,22,1926 10of17
Methyl N(cid:48)-cyano-N-{[4-[4-(4-fluorophenyl)piperazin-1-yl]pyridin-3-yl]sulfonyl}carbamimidothioate (17).
Startingfrom4-[4-(4-fluorophenyl)piperazin-1-yl]pyridine-3-sulfonamide4(0.84g)thetitlecompound
wasobtained.Yield0.76g,(70%);m.p.248–251◦C;IR(KBr):3175(NH),3088(C -H),2925,2850(C-H),
Ar
2161(C≡N),1646,1570,1509(C=C,C=N),1301,1128(SO )cm−1;1H-NMR(500MHz,DMSO-d )δ:
2 6
2.35(s,3H,S-CH ),3.26(brs,4H,2×CH ),4.04(brs,4H,2×CH ),6.99(m,2H,H-2,6Ph),7.06(t,2H,
3 2 2
H-3,5Ph),7.35(d,1H,H-5pyrid.),8.29(d. 1H,H-6pyrid.),8.80(s,1H,H-2pyrid.),13.75(brs,1H,NH)
ppm. Anal. calcd. forC H FN O S (434,51);C,49.76;H,4.41;N,19.34. Found: C,49.72H,4.36;
18 19 6 2 2
N,19.40.
MethylN(cid:48)-cyano-N-{[4-[4-(3,4-dichlorophenyl)piperazin-1-yl]pyridin-3-yl]sulfonyl}carbamimidothioate(18).
Starting from 4-[4-(3,4-dichlorophenyl)piperazin-1-yl]pyridine-3-sulfonamide 5 (0.97 g) the title
compoundwasobtained. Yield0.82g,(68%);m.p. 251–254◦C;IR(KBr): 3167(NH),3093(C -H),
Ar
2925,2843(C-H),2161(C≡N),1648,1594,1570,1480(C=C,C=N),1329,1125(SO )cm−1;1H-NMR
2
(500MHz,DMSO-d )δ: 2.35(s,3H,S-CH ),3.40(brs,4H,2×CH ),4.04(brs,4H,2×CH ),6.95(dd,
6 3 2 2
1H,H-6Ph)7.17(d,1H,H-2Ph),7.33(d,1H,H-5pyrid.),7.40(d,1H,H-5Ph),8.29(d. 1H,H-6pyrid.),
8.81(s,1H,H-2pyrid.),13.80(brs,1H,NH)ppm. Anal. calcd. forC H Cl N O S (485.41);C,44.54;
18 18 2 6 2 2
H,3.74;N,17.31. Found: C,44.69;H,3.50;N,17.31.
MethylN(cid:48)-cyano-N-{[4-[4-(2-methoxyphenyl)piperazin-1-yl]pyridin-3-yl]sulfonyl}carbamimidothioate(19).
Starting from 4-[4-(2-methoxyphenyl)piperazin-1-yl]pyridine-3-sulfonamide 6 (0.87 g) the title
compoundwasobtained. Yield0.81g,(73%);m.p. 238–240◦C;IR(KBr):3198(NH),3032(C -H),2964,
Ar
2918,2850(C-H),2168(C≡N),1639,1596,1501,1483(C=C,C=N),1341,1146(SO )cm−1;1H-NMR
2
(500 MHz, DMSO-d ) δ: 2.35 (s, 3H, S-CH ), 3.13 (s, 4H, 2 × CH ), 3.80 (s, 3H, CH ), 4.03 (s, 4H,
6 3 2 3
2×CH ),6.89–6.97(m,4H,Ph.),7.35(d,1H,H-5pyrid.),8.29(d. 1H,H-6pyrid.),8.80(s,1H,H-2
2
pyrid.),13.60(brs,1H,NH)ppm. Anal. calcd. forC H N O S (446.55);C,51.10;H,4.97;N,18.82.
19 22 6 3 2
Found: C,50.73;H,4.88N,18.41.
3.2.2. ProcedureforthePreparationofMethylN-(Pyridin-3-yl)sulfonyl-N(cid:48)-
cyanocarbamimidothioate20–25
A mixture of the appropriate 4-substituted pyridine-3-sulfonamide 8–11,13,14 (2.5 mmol),
dimethyl N-cyanodithioiminocarbonate (0.40 g, 2.75 mmol) and anhydrous potassium carbonate
(0.33 g, 2.5 mmol) in dry acetone (7.5 mL) was stirred under reflux for 12 h. The precipitate was
collectedbyfiltration,anddried,thensuspendedinwater(2.5mL)andslowlyadjustedtopH2with
4%solutionofHCl(analogously—adjustingsuspensiontopH7with4%solutionofHClresultsin
potassiumsalts20A–25A).Afterstirringatroomtemperaturefor2h,theprecipitatewasfilteredoff,
washedwithcoldwater(2×1mL),anddried. Inthismanner,thefollowingproductswereobtained.
Methyl N(cid:48)-cyano-N-{[4-(3,5-dimethyl-1H-pyrazol-1-yl)pyridin-3-yl]sulfonyl}carbamimidothioate (20).
Startingfrom4-(3,5-dimethyl-1H-pyrazol-1-yl)pyridine-3-sulfonamide8(0.63g)thetitlecompound
wasobtained. Yield0.63g,(72%);m.p. 172–175◦C;IR(KBr): 3501(NH),3090(C -H),2930(C-H),
Ar
2162(C≡N),1628,1477(C=C,C=N),1289,1147(SO )cm−1;1H-NMR(500MHz,DMSO-d )δ: 2.04(s,
2 6
3H,CH ),2.13(s,3H,CH ),2.16(s,3H,CH ),6.06(s,1H,H-4pyrazole),7.47(d,1H,H-5pyrid.),8.84
3 3 3
(d,1H,H-6pyrid.),9.12(s,1H,H-2pyrid.) ppm. Anal. calcd. forC H N O S (350.42);C,44.56;H,
13 14 6 2 2
4.03;N,23.98. Found: C,44.26;H,3.87;N.23.65.
Methyl N(cid:48)-cyano-N-{[4-(3,4,5-trimethyl-1H-pyrazol-1-yl)pyridin-3-yl]sulfonyl}carbamimidothioate (21).
Startingfrom4-(3,4,5-trimethyl-1H-pyrazol-1-yl)pyridine-3-sulfonamide9(0.66g)thetitlecompound
wasobtained. Yield0.57g,(63%);m.p. 181–183◦C;IR(KBr): 3087(C -H),2925,2854(C-H),2167
Ar
(C≡N),1573,1470(C=N),1302,1152,1147(SO )cm−1;1H-NMR(500MHz,DMSO-d )δ: 1.93(s,3H,
2 6
CH ),1.99(s,3H,CH ),2.15(s,3H,CH ),2.16(s,3H,CH ),7.55(d,1H,H-5pyrid.),8.88(d,1H,H-6
3 3 3 3
pyrid.),9.14(s. 1H,H-2pyrid.) ppm. Anal. calcd. forC H N O S (364.45);C,46.14;H,4.43;N,
14 16 6 2 2
23.06. Found: C,46.30;H,4.15;N,22.91.