Synthesis Characterization and Antibacterial, Antifungal Activity of N-(Benzyl Carbamoyl or ...

21 Pages·2015·1.77 MB·English

HindawiPublishingCorporation
InternationalJournalofMedicinalChemistry
Volume2013,ArticleID436397,20pages
http://dx.doi.org/10.1155/2013/436397
Research Article
Synthesis Characterization and Antibacterial,
Antifungal Activity of N-(Benzyl Carbamoyl or
Carbamothioyl)-2-hydroxy Substituted Benzamide
and 2-Benzyl Amino-Substituted Benzoxazines
TysonBelz,SalehIhmaid,JasimAl-Rawi,andStevePetrovski
SchoolofPharmacyandAppliedScience,LaTrobeUniversity,P.O.Box199,Bendigo,UIC3552,Australia
CorrespondenceshouldbeaddressedtoJasimAl-Rawi;[email protected]
Received17June2013;Revised8August2013;Accepted11August2013
AcademicEditor:ArmandoRossello
Copyright©2013TysonBelzetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
NewN-(benzylcarbamothioyl)-2-hydroxysubstitutedbenzamides13,20,and21weresynthesizedusingsodiumbicarbonateand
benzylaminewith2-thioxo-substituted-1,3-benzoxazines6,10a,b,11c,and12a–n.The2-thioxo-substituted-1,3-oxazines6,10a-
b,11d12a–n,and26wereconvertedtothecorresponding2-methylthio-substituted-1,3-oxazines14a–land24whichwerethen
convertedto2-benzylamino-substituted-benzoxazines15a–ibyrefluxingwithbenzylamine.Products15a,b,e,f,andgwerealso
synthesizedbyboilingthecorrespondingN-(benzylcarbamothioyl)-2-hydroxysubstitutedbenzamides13a,b,f,l,andminacetic
acid.2-Oxo-substituted-1,3-benzoxazines22and25werepreparedbytreatingthecorresponding2-methylthio-substituted-1,3-
oxazines14and24withdiluteHCl.TheN-(benzylcarbamoyl)-2-hydroxysubstitutedbenzamide23wassynthesizedfromthe
1 13
reactionof2-oxo-substituted-1,3-benzoxazine22withbenzylamine.ThenewproductswerecharacterizedusingIR, H,and C
NMRinadditiontomicroanalysis.Selectedcompoundsweretestedinvitroforantibacterialandantifungiactivityandthemost
activecompoundswerefoundtobethe4-(substituted-benzylamino)-2-hydroxybenzoicacids9aandd(M.chlorophenolicum,MIC
50and25𝜇gmL−1,resp.),N1,N3-bis(benzylcarbamothioyl)-4,6-dihydroxy-substitutedphthalamides20aand20c(B.subtilisMIC
12.5,50𝜇gmL−1,resp.)and21(M.chlorophenolicum,MIC50𝜇gmL−1).
1.Introduction The Topliss method [14] was used in the optimization
ofsalicylicacidderivativesforpotentialuseasantibacterial
Thesearchfornewantibacterialcompoundsisachallenging
agents. The employment and analysis of physicochemical
task as bacteria are continuously developing resistance to
parameters and molecular electronic surfaces which high-
antimicrobial compounds; however, infections due to such
lighttheelectronic,lipophilic,andstericfeaturesmaybeuse-
bacterialstrainsareinfrequentalthoughpotentiallyfatal[1–
fulguidelinesinthecontinuoussearchfornew,moreeffective
3].Thisongoingproblemhasresultedinthesearchfornewer,
3-amino-salicylic acid analogs. The synthesis of the urea or
moreeffectiveantibacterialcompounds[1–3].
thioureaproduct(3,Scheme1)waspreviouslyachievedfrom
Urea, thiourea 3 (X=O or S), and benzo-1,3-oxazine
thereactionofbenzoylisocyanateorbenzoylisothiocyanate1
compounds5and6(Scheme1)havebeenshowntopossess
(X=OorS,resp.)withamines2[15–17].
antibacterial and antifungal properties [4–11]. The benzyl
thiourea analogue 3 has been reported to show activity The substitution R on the aromatic ring could be alkyl,
againstGram-positivebacteria[12]. alkoxy, –OC=OCH3 in positions 2, 3, 4, 5, and 6, and the
TheN-benzoyl-2-hydroxybenzamides[13]areimportant R1-N-R2inproduct3couldbealiphatic,aromatic,orcyclic
pharmacophores for antibacterial activity in which the 2- aminesubstitutions.Limitationassociatedwiththismethod
hydroxygroup(hydrogenbondingdonor)contributestothe is that R, the substitution on the aromatic ring, cannot be
activity, the imide linker (preferred) or urea linker retains –OH or RN–, which is desirable for antimicrobial activity,
activityandfreeNHisrequiredforhighactivity. particularlythehydroxygroup[18].
2 InternationalJournalofMedicinalChemistry
Furthermore, the synthesis of 2-amino-substituted-1,3- l R = 7-OH,-8-CH3, and m R = 8-OH), 18, and 19 were
benzoxazine 5 was achieved through the reaction of the synthesized from the reaction of the substituted 2-hydroxy
correspondingamine2withethyl2-cyanobenzoate4[19–21]. benzoicacidwithfreshlypreparedPh3P(SCN)2 (Schemes2
However,againthelimitationinthismethodisthattheRin and3)followingthereportedconditions[22,34]withsome
product5cannotbe–OHor–NH2. modifications(seeExperimental).Itisworthnotingthatthe
Thereactionofsubstituted2-thio-1,3-benzoxazine-4-one reaction of 4-benzylideneamino-2-hydroxybenzoic acid 8d
6 (Scheme1) with primary and secondary amines showed formedthecorresponding7-(benzylidene)amino-2-thio-1,3-
considerableinterestintheliterature. benzoxazineswhichwerehydrolysedduringtheisolationof
The secondary amine 2 (dimethyl, diethyl, and cyclic the product and gave 7-amino-2-thio-1,3-benzoxazines 10b.
amines) reaction with 2-thioxo-2,3-substituted 1,3-benzo- However, the reaction of 4-amino-2-hydroxybenzoic acid 7
xazin-4-one 6 gave only 2-amino-substituted-1,3-benzo- withthefreshlypreparedPh3P(SCN)2 failedtoproducethe
xazine5[19,20,22,23].Reactionswithprimaryamineslead expectedproduct10bhowevergaveacomplex,unidentifiable
to the opening of the oxazine ring and produce thiourea moleculecontainingatriphenylphosphenegroupattachedto
typeanalogues(compound3R=2-OHandR1NR2=–HN- theoxazineproduct.
alkyl)whilewithNH2 CH2Phgave3R=2-OHandR1NR2 7-(Substituted benzylamino)-2-thioxo-2,3-dihydro-4H-
=NHCH2Phmixedwith2-(benzylamino)-4H-benz[e]-1,3- 1,3-benzoxazin-4-one 11a–c were synthesized from the
oxazin-4-one(5R=HandR1NR2=–NHCH2Ph[19,20].The reaction of 4-(substituted-benzylamino)-2-hydroxybenzoic
abovereactionproducedlowyieldsoramixtureofproducts acids 9a–c with the freshly prepared Ph3P(SCN)2 [22, 32]
13aand15a(Scheme2)whichwasdifficulttoseparate. (Scheme2). The structures of the new products 11a–c were
1 13
In this work, we developed simple general procedures confirmedusing H,and CNMRandmicroanalysis.
for the synthesize of N-(benzyl carbamothioyl)-2-hydroxy- Similarly,thesubstituted-2-thioxo-1,3-benzoxazin-4-one
benzamides13,20,and21,N-(benzylcarbamoyl)-2-hydroxy- 6 and 12b–n prepared using a previously reported [22,
substituted-benzamides 23, and 2-(benzyl amino)-sub- 23, 33] method gave products with identical physical and
stituted-1,3-benzoxazin-4-one 15 and 26. The antibacterial spectroscopicdata.
andantifungalactivitywasevaluatedforanumberofthese The benz-bis-(1,3-oxazine) 18a–c and 19 were prepared
products with the intension of producing novel products from the reaction of dihydroxy-dicarboxybenzoic acids
that can be used to eliminate problematic bacteria in the 16a–cand17withthefreshlypreparedPh3P(SCN)2 [22,32]
environmental and medical settings. These compounds according to the previously reported method [34] for the
couldpotentiallyresultinnovelantibiotic. synthesisof18cwithsomemodificationtoimprovetheyield.
The structures of the new dibenzoxines products 18a.b
1 13
and 19 were confirmed using H and CNMR and micro-
2.ResultsandDiscussion
analysis(Scheme4).
2.1.Chemistry
2.1.3. Synthesis of the 2-Methylthio-substituted-1,3-oxazines.
2.1.1.SynthesisofSubstituted2-HydroxyAromaticCarboxylic 2-Methylthio-substituted-1,3-oxazines14a–i(aR=H,bR=
Acids. 5-Substituted-4,6-dihydroxybenzene-1,3-dicarboxylic 8-CH3,cR=8-Ph,dR=7-OCH3,eR=7-OCH2CH3,f R
acids 16a–c, and 2,3-dihydroxybenzene-1,4-dicarboxylic =7-OH,gR=7-OH,-8-CH3,hR=AcNH,andIR=NH2)
acid17werepreparedbycarboxylationof2-substituted-1,3- and24werepreparedbythereactionofCH3Iinthepresence
hydroxy-benzene and 1,2-dihydroxy-benzene, respectively, of NaHCO3 with substituted-1,3-oxazines 6, 10a,b, 11c, and
according to the previously reported method [24]. Comp- 12 (a R = 8-CH3, b R = 8-Ph, e R = 7-OCH2CH3, h R = 7-
ound 7 was selectively acetylated in the presence of NaOH OCH3,kR=7-OH,andlR=7-OH,-8-CH3)accordingtothe
andaceticanhydridewiththereactionmaintainedatpH6-7 previously reported procedure [35]. Physical properties, IR,
in accordance with the reported procedure [25–27] to give 1HNMR,and13CNMR,collectedforproducts14a–Iwere
theacid8aanexcellentyield(82%)(Scheme2). foundtobeidenticaltothereporteddata[35].Thestructures
Allowingcompound7toreactwithsubstitutedbenzalde- ofnewproduct,14h,i,and24wereconfirmedwithanalysis
hyde gave the corresponding Schiff base 8b-c (Scheme2) oftheIR,1H,and13CNMRdata.
[28–33].ReductionofSchiffbase8b-cwithsodiumborohy- Compounds14a–iand24were used inthesynthesisof
dride gave 4-(substituted-benzylamino)-2-hydroxybenzoic compounds15a–Iand26withnofurtherpurification.
acids 9a–c with high yield (73–85%). The structures of
1
the prepared acids were confirmed using mp, IR, H, and
13 2.1.4. Synthesis of Benzyl Thiourea 13a–q, 20a, c, and 21.
CNMRwhichwasingoodagreementwiththepreviously
Initially, the equimolar reaction of compound 6 with ben-
reportedphysicalandspectroscopydata[26–28].
zylamine in dioxane was repeated according to the earlier
reported method [19, 20], in which the mixture that was
2.1.2.SynthesisofSubstituted2-Thio-substituted-benzoxazines. refluxedfor4hoursgave13aandwasa46%yield(Scheme3).
The 2-thio-substituted-1,3-benzoxazines 6, 10, 11, 12 (a R However,whenthereactionwascarriedoutusingsolvent
= 8-CH3, b R = 8-Ph, c R = 6-Br, d R = 6-OCH2CH3, e free conditions, excess benzylamine was added directly to
R = 7-OCH2CH3, f R = 8-OCH2CH3, g R = 6-OCH3, h powered2-thio-1,3-benzoxazine6andthemixturewasleftat
R = 7-OCH3, i R = 8-OCH3, j R = 6,8-I, k R = 7-OH, roomtemperaturefor2days,thebenzylthioureaproduct13a
InternationalJournalofMedicinalChemistry 3
R 3 R 3
R
4 2 1 4 2
R
NH 1
5 1 N=C + R2 5 1 HN N R
6 =X 6 2
1 O 2 O X
3
R
R
OCN 1
R
O N
OEt
4 + 2 R2
O N
O S
O
N + 2
H 5
O
6
Scheme1:Previoussynthesisofureaorthiourea3(X=OorS)and2-aminobenzo1,3-oxazine5.
H2N OH R OH R1 O S
i ii
O O 8a, d NH
OH OH O
7 8 10
aR=AcNH aR1=AcNH
bR=phenyl-C=N bR1=NH2
cR=2-Hydroxy-phenyl-C=N
dR=3-Ethoxy-2-hydroxy-phenyl-C=N
R2 8b–ieii R2
R3 R1 R3 R1
iv
HN OH HN O S
O NH
9 11
OH O
aR1=R2=R3=H aR1=R2=R3=H
bR2=R3=H, R1=OH bR2=R3=H, R1=OH
cR1=OH,R2=OEt,R3=H cR1=OH,R2=OEt,R3=H
Scheme2:Synthesisof7-N-substituted-1,3-oxazines10-11startingfrom2-hydroxy-substitutedbenzoicacids8and9.Reactionconditions:
(i) compound a (Ac)2O, b PhCH=O, c 2-hydroxy-C6H4-CH=O, and d 3-ethoxy-2-hydroxy-C6H3-CH=O, (ii) Ph3P(SCN)2 in CH2Cl2,
(iii)NaBH4,and(iv)Ph3P(SCN)2inCH2Cl2.
4 InternationalJournalofMedicinalChemistry
R 8 1 3 8󳰀
O S R OH
7 4 7󳰀 9󳰀
8a 2 NaHCO3:PhCH2NH2 2 2󳰀 4󳰀
6 4a 4 NH 1:1 MeOH:H2O 5 1 1󳰀 HN 3󳰀 HN 6󳰀
5 6 5󳰀
O
O S
6,10a–b,11c, and12a–m 13
a:R=H (75%) j:R=3-OCH3 (82%)
b:R=3-CH3 (79%) k:R=3,5-I (75%)
c:R=3-Ph (80%) l:R=4-OH (87%)
d:R=5-Br (82%) m:R=4-OH-3-CH3 (78%)
NaHCO3: CH3I Acetic acid: reflux2hrs e:R=5-OCH2CH3 (87%) n:R=3-OH (72%)
f:R=4-OCH2CH3 (73%) o:R=NH2 (47%)
g:R=3-OCH2CH3 (69%) p:R=AcNH (78%)
h:R=5-OCH3 (69%) q:R=4[3-OEt,2-OH(C6H3-)]NH- (70%)
i:R=4-OCH3 (69%) 6󳰀
5󳰀 7󳰀
2󳰀
R O S R O HN 4󳰀
CH
3
PhCH NH 3󳰀
2 2
Dioxane
N N
O O
14a–i 15
From13to15 From14to15
15a:R=H (75%) 15a:R=H (75%) 15f:R=7-OH (77%)
15b:R=8-CH3 (67%) 15b:R=8-CH3 (72%) 15g:R=7-OH,-8-CH3 (74%)
15e:R=7-OCH2CH3 (61%) 15c:R=8-Ph (65%) 15h:R=AcNH (47%)
15f:R=7-OH (67%) 15d:R=7-OH3 (83%) 15i:R=NH2 (47%)
15g:R=7-OH-8-CH3 (70%) 15e:R=7-OCH2 CH3 (80%)
Scheme3:SynthesisofN-(benzylcarbamothioyl)-2-hydroxy-substitutedbenzamide13,substituted2-benzylamino-1,3-benzoxazines15.
had decreased yield (19%), and none of the cyclic analogue WithslightmodificationtotheprocedureB,byaltering
15a could be isolated. When 2-thio-1,3-benzoxazine 6 was the ratio of benzylamine to starting material (3:1) and the
allowed to react with excess benzylamine (4-fold) and the reaction time to 16 hours, the bis-oxazines 18a,c and 19
mixturewasthenheatedtorefluxindioxanefor2hours,the were found to react in a similar fashion to give substituted
openproduct13awasagainisolatedin18%yield. bis(benzyl carbamothioyl) analogues 20a,c (yield 63, 49%)
To overcome this low yield and possible mixture for- and21(yield63%)(Scheme4).
mation, the reaction procedure was modified in which the
The previously prepared N-(benzyl carbamothioyl)-2-
benzoxazines 6, 10a-b, 11d, and 12b–m were mixed with
hydroxybenzamide13awascharacterizedbycomparisonof
NaHCO3 andsuspendedin1:1mixtureofmethanol,water 1 13
∘ its physical data (mp, IR, H and C NMR spectra) with
andthemixturewasthenheatedto40 Cforafewminutes.
values found in the literature [12, 19, 20]. The structures of
Excess(1.5-fold)benzylaminewasaddeddropwiseatroom
newbenzylthioureacompounds13b–q,20a,c,and21were
temperature and left stirring for 4 hours. Products 13a–
1 13
q was isolated according to the general procedure B (see confirmed using IR, H NMR and C NMR spectroscopy
1
Experimental)andtheyieldweremoderatetohigh(69–87%) andmicroanalysis.The HNMRandIRspectraalsoshoweda
(Scheme3). highcorrelationwiththepreviouslypreparedbenzylthiourea
InternationalJournalofMedicinalChemistry 5
R R R
HO OH S O O S HO OH
H H H H
HO OH i HN NH ii N N N N
O O O O Ph S O O S Ph
16a–c 18a–c aR=H 20a, c
bR=OH
OH OH S cR=CH3 OH H H
N N
OH HO S
O HN O
i ii
H H
OH O S N N O HN
O
O NH Ph S O Ph
17 19 O 21
Scheme4:Synthesisofsubstituted-N,N-bis(benzylcarbamothioyl)-dihydroxy-isoandtetraphthalamides20a,c,and21.Reactionconditions:
(i)Ph3P(SCN)2inCH2Cl2and(ii)NaHCO3,PhCH2NH2.
1 󸀠
13a [19, 20]. In the H NMR spectra, the CH2, H-5 of the literature[19,20].Thestructuresofnew2-benzylamino-1,3-
benzyl amine in compounds 13a–q, 20a,c, and 21 appeared benzoxazinecompounds15b–gwereconfirmedusingIR,1H
as a doublet at ∼𝛿 4.9ppm and the 4󸀠-NH, appeared as a NMR,and13CNMRspectroscopyandmicroanalysis.Inthe
tripletat∼𝛿11.0ppminallcases.Assignmentofthecarbon- 1
HspectratheCH2ofthebenzylamineappearsasadoublet
13 chemical shifts was made using the previous reported at ∼𝛿 4.5ppm. The previously analysed 1H and 13C NMR
1 13
chemicalshiftsof 13a[19,20].The Hand CNMRspectra spectra of the parent 2-methylthio-1,3-benzoxazines 14 [35]
of the parent 2-thioxo-2H-benz[e]-1,3-oxazin-4(3H)-one 12 wereusedtoaidintheanalysisofthenewproducts15b–gs.
were also used to aid with structural identification. The
1 13
simulated H and C NMR spectra using ChemDraw V12
2.1.6.Synthesisof2-Oxo-substituted-benzoxazines22a–hand
ultra were also used as references to aid the analysis of the
1 13 25. 2-Methylthio-substituted-benzoxazines 14a, b, and d–g
observed Hand CNMRspectraofthenewproducts.
wereallowedtoreactwith10%HClaccordingtothegeneral
procedureEandgave2-dione-1,3-benzoxazines22a–hwith
good to excellent yield (Scheme5); however, product 22h
was produce if 40% HCl was used in the hydrolysis of
2.1.5.Synthesisof2-Benzylamino-(substituted)-benz-1,3-ox-
14h. Similarly, the 2,8-dioxo product 25 was prepared from
azines15. Asmentionedearlierthereactionofthe2-thio-1,3-
the hydrochloric acid hydrolysis of the corresponding 2,8-
benzoxazine6withaprimaryaminedidnotconsistentlygive
dimethylthio-analogue24(Scheme5).
thecyclicproduct13a[19,20].
The reaction of 2-methylthio-(substituted)-1,3-benzoxa-
zines14(aR=H,bR=8-CH3,cR=8-Ph,dR=7-OCH3,e 2.1.7.SynthesisofSubstituted-N-(benzylcarbamoyl)-2-hydro-
R=7-OCH2CH3,fR=7-OH,gR=7-OH,-8-CH3,andhR= xybenzamides 23a–g. The synthesis of the substituted-
AcNH-)and24withexcess(5-fold)benzylamineaccording N-(benzyl carbamoyl)-2-hydroxybenzamides 23a–g was
tothegeneralprocedureCgave2-benzylamino-substituted- achieved using the reaction of the relevant benzoxazine-2,
1,3-benzoxazines15a–hand26withmoderatetohighyields 4-di-one 22a–h with excess benzylamine in dioxane with
62–83%(Schemes3and5). refluxedaccordingtothegeneralprocedureF.
Thereactionofbenzylaminewith2-methylthio-benzoxa-
zinetookplacewithnotraceofthethioureaanalogue13. Structure Elucidation of Substituted-N-(benzyl carbamoyl)-
Followingtheirsuccessfulsynthesis,manyoftheN-(ben- 2-hydroxybenzamides 23a–g. The structures of the newly
zyl carbamothioyl)-2-hydroxybenzamides 13a, b, f, and g preparedsubstitutedureacompounds23a–gwereconfirmed
1 13
were then cyclised by refluxing in acetic acid for 2 hours usingIRand Hand CNMRspectroscopyandmicroanal-
1
according to the general procedure D (Scheme3) and gave ysis. The H NMR and IR spectra supported the proposed
thecorresponding2-benzylamino-1,3-benzoxazines15a,b,f, structuresandshowedsomecorrelationwiththepreviously
andgfairtogoodyields. prepared benzyl thiourea 13a–q, 20a, c, and 21. In the 1H
Previouslyprepared2-benzylamino-1,3-benzoxazine15a spectra, the methylene CH2 (H-11 of compounds 23a–g) of
was characterized by comparison of the physical data (mp, the benzyl amine appears to shift up field as a doublet at
IR, and 1H and 13C NMR spectra) with that found in the ∼𝛿4.3ppm.TheNH(H-10ofcompounds23a–g)appearsas
6 InternationalJournalofMedicinalChemistry
R O R OH
O
14a, b, and d-g10%HCL PhCH2NH2 H H
NH N N
O O O Ph
22 23
aR=H eR=7-OH aR=H eR=4-OH,3-CH3
bR=8-CH3 fR=7-OH,8-CH3 bR=3-CH3 fR=AcNH-
cR=7-OCH3 gR=AcNH cR=4-OCH2CH3 gR=NH2
dR=7-OCH2CH3 hR=NH2 dR=4-OH
CH3 CH3
S O O S O O O O
18c 10%HCL
N N HN NH
O O
O O
24 25
PhCH NH
2 2
CH
H 3 H
N O O N
N
N
O O
26
Scheme5:Synthesisof2-dione-1,3-benzoxazines22a–Iand25a,bfromthe2-methylthio-1,3-benzoxazines14a,b,d–g,and24a-b.
abroadtripletat∼𝛿8.5ppm.The1Hand13CNMRspectra potentiallypathogenictohumansandanimalsandothersare
oftheparent2-oxo-1,3-benzoxazines22and25. problematic in an environmental setting. Furthermore, the
bacterial species selected have different cell wall composi-
tions,thatis,someareGram-negativeandsomeareGram-
2.2.BiologicalTesting
positivestrains.Someantimicrobialagentsinhibitbacteriaby
2.2.1.BrothDilutionSusceptibilityTesting. Inthisstudy,some interactingwithcomponentsofthecellwallthatareabsent
of the newly prepared compounds were tested and showed in Gram-negative bacteria [37]; therefore, the selection of
antimicrobial activity against 8 different bacterial strains strains was carefully selected with the possibility that an
and 4 cultures of fungi. The bacterial species investigated inhibitory compound would also hint to its mechanism of
were P. aeruginosa, B. subtilis, S. aureus, A. baumannii, E. action. Based on the results obtained it is clear that the
coli, S. agalactiae M. smegmatis, and M. chlorophenolicum. Gram-negative strains, that is, P. aeruginosa, E. coli, and A.
The antifungal evaluation was determined against A. niger, baumanniiwere least affectedbythecompoundsandwhen
A. corymbifera, R. oryzae, and A. alternata. The minimal inhibitionwasobserveditwasathighlevels200𝜇gmL−1 or
inhibitory concentrations (MICs) and minimal fungicidal higher. Interestingly, compound 9d seemed to have a more
concentrations(MFCs),definedasthelowestconcentration dramatic effect Gram-positive strains with the exception of
of drug that inhibits the growth of bacteria or fungi in the M.smegmatis.Despitetheeffectsthatsomeofthecompounds
inoculums’,weredeterminedusingthebrothdilutionmeth- hadonthebacterialstrains,itappearsthatthesecompounds
ods. The compounds which demonstrated MIC and MFC are not so effective when tested on the four fungal cultures
valueslessthan300and200𝜇gmL−1,respectively,arelisted chosenwiththeexceptionof 9donA.corymbifera.
inTables1and2.Accordingto[36],antimicrobialagentsare Based on the results obtained for each of the newly
effectiveonarangeofbacterialspeciesatlowconcentrations, synthesisedcompounds,itisevidentthatcompound9dhas
that is, <128𝜇gmL−1. Therefore, we conducted our MIC morepotenteffectwhencomparedtotheothers.Thisreveals
experimentsusingconcentrationsashighas300𝜇gmL−1.We thatdespitethefactthatmostofthecompoundsdonotseem
also selected a range of bacterial and fungal species to test tohaveanoteworthyeffectonthestrains,compound9disof
our newly synthesised compounds. Some of the species are interestandfurtherinvestigationisrequired.
InternationalJournalofMedicinalChemistry 7
Table1:BrothdilutionsusceptibilityMICvaluesforinhibitiongrowthofbacteria(𝜇gmL−1).
Bacteria
# P.aeruginosa B.subtilis S.aureus A.baumannii E.coli S.agalactiae M.smegmatis M.chloro
9c 200 200 >200 200 200 n/a >200 50
9d 200 100 25 200 200 n/a >200 25
13c >300 >300 >300 >300 >300 >300 n/a n/a
13d 200 >300 >300 200 >300 300 n/a n/a
13e >300 >300 300 >300 >300 >300 n/a n/a
13g >300 >300 >300 >300 >300 >300 n/a n/a
13j >300 >300 >300 >300 >300 200 n/a n/a
13l >300 >300 300 >300 >300 >300 n/a n/a
13m 200 200 300 >300 >300 >300 n/a n/a
13n 300 >300 300 300 >300 300 n/a n/a
20a >200 12.5 >200 >200 >200 n/a >200 >200
20c >200 25 >200 >200 >200 n/a >200 >200
21 >200 25 200 >200 >200 n/a 100 50
22g 200 >200 >200 200 >200 n/a >200 200
Table 2: Broth dilution susceptibility MFC values for inhibition listedinTables3and4andweretestedinduplicatewiththe
growthoffungi(𝜇gmL−1). averagegiven.Anyzoneofinhibitionthatwasnotedaround
thediscwasconsideredsensitiveandthezoneofclearlywas
Fungi
noted.Theseresultsaremoreusefulforcompoundsthatwere
# R.oryzae A.niger A.corymbifera A.alternata
difficult to dissolve, but equally, these results can indicate
9c 200 >200 >200 >200 resistanceifthecompounddoesnotdiffusethroughtheagar
9d 200 >200 100 >200 properly.
22g >200 >200 >200 >200 Based on the results obtained in Section2.2.1, it is clear
20a >200 >200 >200 >200 thatcompound9disofinterest.Basedontheresultsobtained
20c >200 >200 >200 >200 in Table3, compound 9d has an inhibitor effect on M.
21 >200 >200 200 200 smegmatis but in the MIC study had no effect. This could
indicate solubility problems with the compound when in
solution; however, this is only speculative; further studies
are required to reveal the cause. In addition, compound
2.2.2. Disc Diffusion Susceptibility Testing. Disc diffusion
9d seems to have no inhibitory effect on S. aureus but in
susceptibility testing was performed on compounds with
the MIC studies had a dramatic affect. This difference in
poor solubility in broth dilution susceptibility testing. The
resultisunusualbutclearlyindicatesthatdifferentmethods
preliminary antimicrobial testing was achieved using the
couldrevealdifferentresultsandthereforeitisimportantto
standard agar disk diffusion methods. Compounds that
performbothmethodspriortofurtherinvestigationontheir
inhibitedcertainbacteriaorfungiaresummarizedin(Tables
inhibitoryeffects.
3and4).
IntheMICstudies,weused300𝜇gmL−1 asthehighest
The concentrations of the prepared compounds were
10−4𝜇gmL−1 (seeExperimental).Thecontroldataisusedto cut-off level. If a compound has an inhibitory effect on
any strain that is greater than this level, then this should
determineifthebacterialstrainsareresistant(R)orsensitive
be revealed in the disc diffusion assay. However, further
(S) to the prepared compounds tested. The disc diffusion
investigation is required as some of these compounds are
assaywasusedasapreliminaryguideforallcompoundsand
dissolved in DMSO and when applied to bacterial cultures
usedincorrespondencewiththebrothdilutionmethodfor
determining MIC/MFC values. This method is particularly cancomeoutofsolution.Thediscdiffusionassaysseem to
useful when MIC/MFC values are unable to be determined indicate some sensitivity to fungal cultures despite the fact
using the broth dilution method due to the compounds thattheywereundetectableintheMICstudies.
insolubility. The insoluble compounds zones of inhibition
therefore can be determined in millimeters relative to the 2.3. The Structure Activity Relationships of the Tested Com-
controlandusedasaroughguide.Sincethezoneofinhibition pounds(BrothDilution). TheresultsinTables1and2show
ofclearancemaybeaffectedbyotherparameters,suchas,the that the 4-(benzylamino)-2-hydroxybenzoic acid derivative
nutrientagardepthoftheplateandsolventused,theresults 9dshowedthebroadestrangeofactivityofthecompounds
shownusingthismethodthereforeshouldbeusedasaguide. tested, exhibiting activity against the Gram-positive and
MIC/MFC values are determined using the broth dilution Gram-negativebacteriaandalsoM. chlorophenolicum.Fur-
method. All compounds that showed clearance zones are thermore, compound 9d showed to be more active than
8 InternationalJournalofMedicinalChemistry
Table3:ResultsshowingDiscdiffusionsusceptibilityforthesynthesisedcompounds.
Bacteria
# P.aeruginosa B.subtilis S.aureus A.baumannii E.coli S.agalactiae M.smegmatis M.chloro
8c S(2mm) R R R R n/a S(2mm) R
8d S(2mm) S(3mm) R R R n/a S(2mm) S(2mm)
9c S(2mm) S(2mm) R R R n/a S(2mm) S(2mm)
9d S(2mm) S(4mm) R R R n/a S(2mm) S(2mm)
13b R S(6mm) S(5mm) R R S(5mm) n/a n/a
13k R S(2mm) S(5mm) S(2mm) R S(5mm) n/a n/a
13f R S(2mm) S(5mm) S(2mm) R S(5mm) n/a n/a
15d R S(2mm) S(5mm) S(2mm) R S(5mm) n/a n/a
15e R R R R R R n/a n/a
13o R R R R R — R R
13p R R R R R n/a S(2mm) R
13q R R R R R n/a R R
20a R S(2mm) R R R n/a R R
20c R S(2mm) R R R n/a S(3mm) S(4mm)
21 R S(4mm) R R R n/a R R
22h R R R R R n/a S(3mm) S(3mm)
22i R R R R R n/a S(2mm) R
23g R R R R R n/a R R
Table 4: Disc diffusion susceptibility for the synthesised com- clearing was greater than the control which was indicative
pounds. that the strain was sensitive to the compound, whereas a
zoneofclearingequaltothecontrolindicatedresistance.The
Fungi
resultsrevealthatnoneofthecompoundshadaninhibitory
# R.oryzae A.niger A.corymbifera A.alternata effect on E. coli at concentration 104𝜇gmL−1 (Table3). The
8c R R R R
B. subtilis bacterial species tested showed inhibitory effects
8d S(2mm) S(3mm) S(5mm) R
tomostofthecompoundstested,forexample,13dinhibited
9c R R R R
S. aureus most strongly and compounds 13k, 13f, and 13d
9d R R R R
inhibitedgrowthofA.baumannii,B.subtilis,andS.agalactiae
13p S(2mm) R R R
(Table3). Some bacterial species that were sensitive to a
13q S(2mm) S(2mm) S(3mm) R
21c S(2mm) S(2mm) S(3mm) R compound showed similar sized zones of inhibition. One
20a R R R R example was 13k which exhibited activity of 2mm for A.
20c R R R R baumannii and B. subtilis and 5mm for S. aureus and S.
22h R R R R agalactiae. The same applies to compounds 8d, 9c, and 9d
22i R R R R whichhadshowna2mmclearancezoneagainstP.aeruginosa
25b R R R R M. smegmatis M. chlorophenolicum. Similarly to the broth
dilution results, compounds 21 and 9d were found to be
activeagainstthreefungispecies,R.oryzae,A.niger,andA.
others against S. aureus with an MIC value of 25𝜇g/mL. corymbifera,withclearancezones2–5mm,respectively.
Compounds20a,cand21(bis-thioureaproducts)werefound
All the compounds tested showed a 2–4mm zone of
to be particularly active towards Gram-positive B. subtilis
clearingformostofthesusceptiblespecieswiththeexception
at MIC values of 12.5, 25, and 25𝜇gmL−1. In addition, ofcompound13bwhichhadalarger6mmzoneofinhibition.
compound 21 also showed activity towards M. smegmatis
This larger zone indicates a hypersensitive effect on the
(MIC 50𝜇gmL−1). Other synthesized compounds which bacterialspecies;however,itisspecificforthecompoundand
showedaninhibitoryeffectwere13nwhichhadaninhibitory
species.Becausethemechanismofactionofthecompound
effect on four bacterial species at 300𝜇gmL−1 and 13d and is unknown, it is difficult to explain the reason for the
13m which had an inhibitory effect on two of the bacterial
hypersensitive effect. One possible explanation is that B.
species at concentration 200𝜇gmL−1. Interestingly, E. coli subtilisencodesaproteinthatcantransportcompound13b
wasnotinhibitedbyanyofthecompounds. into the cell and this has a more toxic effect than those
working from outside the cell. A similar phenomenon has
2.4. The Chemical Compounds Activity and Structural Rela- been shown with bacterial mercury resistance where the
tionships of the Antimicrobial Assay Results (From Disk Dif- presenceofamercuryimportproteindisplaysalargerzone
fusion Assay). In the presence of a compound, a zone of ofclearinginadiscdiffusionassay[38].
InternationalJournalofMedicinalChemistry 9
The data obtained revealed patterns of inhibition, espe- 0.01mol) was allowed to react with 4-amino-2-hydroxy-
cially those conducted with the disc diffusion assay. This benzoic acid 7 (1.53g, 0.01mol) for 1 hour according to
suggeststhatasimilarmechanismofactioncouldbeinvolved the reported procedure [29, 30] and gave solid which
intheinhibitionofgrowth. recrystallised from methanol to give 8d 2.95g, 98% as
red crystals, mp 185–188∘C decomp. ]max (KBr)/cm−1 1655
1
3.Conclusion (C=O), 1622, 1600 (C=N): HNMR (200MHz, 300K, d6-
acetone)𝛿8.93(s,1H,H-8),8.02(d,1H,JH6,H5=8.8Hz,H-6),
In conclusion, we have prepared seven new compounds of 7.33 (dd, 1H, JH15,H14 = 7.8Hz, JH15,H13 = 1.6Hz, H-15), 7.24
2-benzylamino-substituted-1,3-benzoxazines, nineteen new (dd,1H,JH13,H14 =8.0Hz,JH13,H15 =1.6Hz,H-13),6.91–7.09
N-(benzyl carbamothioyl)-substituted-benzamide and have (m, 3H, H-3,H-5, and H-14), 4.24 (q, 2H, JH16,H17 = 7.0Hz,
evaluated some for their activity against bacteria and H-16), 1.41 (t, 3H, JH17,H16 = 7.0Hz, H-17). Product 8d was
fungi.ItappearsthatN-(benzylcarbamothioyl)-substituted- usedimmediatelyinthesynthesisof 9d.
benzamidehasshownantibacterialactivitysuchas20a,20c,
21,13d,13m,and13n.
We are in the process of synthesising new substituted 4.1.4.Synthesisof4-Substituted-(benzylamino)-2-hydroxyben-
products by replacing the benzyl group of N-(benzyl car- zoicAcids9a–d
bamothioyl)by6-aminopenicillanicacidandtesttheirbac-
General Procedure A. In slight modification to a previous
teriaactivity.
reported method, [28] the appropriate 4-substituted ((ben-
zylidene) amino)-hydroxybenzoic acids 8b-c reduced using
4.Experimental sodiumborohydride(2equiv).
4.1.Chemistry. InfraredspectrawereobtainedusingaPerkin
1 13 4-(Benzylamino)-2-hydroxybenzoic Acid 9a. 2-Hydroxy-4-
Elmer FT-IR 1720x spectrometer. H NMR and C NMR
{[(𝐸)-benzylidene] amino}benzoicacid8b(2.41g, 10mmol)
spectra were obtained using a Bruker AC 200 NMR spec-
1 was allowed to react with sodium borohydride (0.76g,
trometerat200and50MHz,respectively.All HNMRand
13 20mmol) according to general procedure A. The resulting
C NMR spectral results are recorded as chemical shifts
solid was recrystallised from methanol/water to give 9a
(in𝛿)CrDelaCtli3vesotolvetnhteainntder3n9a.l4TppMmS ifnorDpMroStoOn-da6ndsol7v7e.0ntppfomr (1.76g,73%),mp122–125∘C.]max (KBr)1/cm−1 3500–3200br
13 (OH), 3024, 2569 (NH), 1632(C=O); HNMR (200MHz,
C NMR. Microanalysis was performed by Chemical and
Micro analytical Services (CMAS), Australia. Melting point 340Kd6-DMSO)𝛿11.34(bs,1H,OHofCOOHexchangeable
determinations were carried out using a Stuart Scientific withD2O),7.5(d,1H,JH6,H5=8.6Hz,H-6),7.21–7.47(m,6H,
(SMP3) melting point apparatus and all melting points are 5xCH,8-NHexchangeablewithD2O),6.22(dd,1H,JH5,H6=
uncorrected. 8.6Hz,JH5,H8=2.0Hz,H-5),6.03(d,1H,JH3,H5=2.0Hz,H-
3),4.38(s,2H,H-9).3.32(2-OHunderthewaterenvelope);
13CNMR(50MHz,340K,d6-DMSO)𝛿171.7(C-7),163.2(C-
4.1.1. Starting Materials. The stating reagents benzyl amine, 2),154.8(C-4),139.1(C-10),130.1(C-6),128.1,126.9,126.6(C-
sodiumhydrogencarbonate,methyliodide-amino-2-hydro- 12, C-11 and C-13), 105.3 (C-5), 100.4 (C-1), 96.8 (C-3), 45.8
xybenzoic acid, and dry 1,4-dioxane were purchased from (C-9).Theresultingproduct9awasnotstableandwasused
AldrichChemicalCompanyandwereusedasreceived. immediatelyinthegenralprocedureB.
4.1.2. Synthesis of 4-(Acetyl amino)-2-hydroxybenzoic Acid
2-Hydroxy-4-((2-hydroxybenzyl)amino)benzoicAcid9b.(E)-
8a. According to the previously reported method [29, 30,
2-Hydroxy-4-((2-hydroxybenzylidene)amino)benzoic acid
37], product 8a was prepared from the reaction of 4-
8c(2.6g,10mmol)wasallowedtoreactwithsodiumboro-
amino-2-hydroxybenzoic acid 7 and acetic anhydride and
hydride(0.76g,20mmol)accordingtogeneralprocedureA.
∘
recrystallisedfrom1,4-dioxane,82%yield,mp221–224 C(lit
The resulting solid was recrystallised from methanol/water
∘
[37] and mp 235 C). The physical and spectroscopic data is ∘
to give 9b as white crystals (2.20g, 85%), mp 184–186 C
consistentwiththeliteraturevalues[27,35]. decomp. ]max (KBr)/cm−1 3500–3200br (OH), 1614 (C=O);
1HNMR(200MHz,300Kd6-DMSO)𝛿11.45(bs,1H,8-NH
4.1.3.Synthesisof4-Substituted((Benzylidene)amino)-2-hyd- or 11-OH exchangeable with D2O), 9.61 (bs, 1H, 11-OH or
roxybenzoicAcidIntermediates8b–e. Accordingtothepre- 8-NH exchangeable with D2O) 7.44 (d, 1H, JH6,H5 = 8.8Hz,
viously reported method [29, 30], intermediates 8b–e were H-6),7.1–6.7(m,6H,H-13,H-15,H-14,H-12and11-OH),6.28
preparedfromtheappropriatesubstitutedbenzaldehydeand (dd, 1H, JH5,H6 = 8.8Hz, JH5,H3 = 1.1Hz, H-5), 5.92 (d, 1H,
4-amino-2-hydroxy-benzoicacid7. JH3,H5 =1.1Hz,H-3);13CNMR(50MHz,350K,d6-DMSO)
Products 8b, c, and e were not identified and used 𝛿171.9(C-7),163.5(C-2),155.2/155.0(C-11/C-4),131.2(C-6),
immediatelyinthesynthesisofcompound9a,b,andd. 128.5 (C-13), 127.9 (C-14), 125.0 (C-10), 119.1 (C-15), 115.3
(C-12), 105.6 (C-5), 100.6 (C-1), 97.0 (C-3), 41.1 (C-8). The
(Z)-4-((3-Ethoxy-2-hydroxybenzylidene)amino)-2-hydroxy- resulting product 9b was used immediately in the general
benzoic Acid 8d. 3-Ethoxy-2-hydroxybenzaldehyde (1.66g, procedureB.
10 InternationalJournalofMedicinalChemistry
4-((3-Ethoxy-2-hydroxybenzyl)amino)-2-hydroxybenzoic dryDCM(20mL)wasaddedtoamixtureoffreshlyprepared
Acid 9c.(E)-4-((3-Ethoxy-2-hydroxybenzylidene)amino)-2- Ph3P(SCN)2 (10mmol) according to the general procedure
hydroxybenzoic acid 8d (10mmol, 3.0g) was allowed to B. The resulting solid was isolated upon evaporation of the
reactwithsodiumborohydride(20mmol,0.76g)according DCM filtrate 10b (0.74g, 91% crude yield). The solid was
togeneralprocedureA.Theresultingsolidwascollectedand recrystallised from methanol, mp 250–253∘C decomp. ]max
recrystallised from methanol/water to give 9d (2.66g, 81%) (KBr)cm−13443,3328,(NH2),3059,2916(NH),1749(C=O),
as white crystals, mp 159–161∘C decomp. ]max (KBr)/cm−1 1679,1616(C=C),1205(C=S);1HNMR(200MHz,370K,d6-
3500–3200br(OH,NHabsorptionundertheOHenvelope), DMSO) 𝛿 13.04 (s, 1H, 3-NH), 7.58 (d, 1H, JH5,H6 = 8.6Hz,
1625(C=O);1HNMR(200MHz,300Kd6-DMSO)𝛿7.38(d, H-5), 6.65 (s, 2H, 7-NH2), 6.60 (dd, 1H, JH6,H5 = 8.6Hz,
1H,JH6,H5 =8.6Hz,H-6),6.85–6.65(m,3H,H-13,H-14and JH6,H8 =1.8Hz,H-6),6.37(d,1H,JH8,H6 =1.8Hz);13CNMR
H-15), 6.35 (bs, 4H, 3 x OH and NH), 6.17 (dd, 1H, JH5,H6 (50MHz,330K,d6-DMSO)𝛿182.4(C-2),157.3,156.9,156.5
= 8.6Hz, JH5,H3 = 1.8Hz, H-5), 5.87 (d, 1H, JH3,H5 = 1.8Hz, (C-4, C-8a, C-7), 128.1 (C-5), 112.7 (C-6), 102.3 (C-4a), 96.8
H-3), 4.23 (s, 2H, CH2NH, H-9), 4.05 (q, 2H, JH16,H17 = (C-8); Anal. Calcd. For C8H6N2O2S: C, 49.47; H, 3.11; N,
13
6.8Hz,H-16),1.35(t,3H,JH17,H16=6.8Hz,H-17); CNMR 14.42.Found:C,49.51;H,3.20;N,14.36.
(50MHz,300K,d6-DMSO)𝛿172.2(C-7),163.6(C-2),155.3
(C-4), 146.6 (C-12), 144.1 (C-11), 131.4 (C-6), 125.7 (C-10), 7-(Benzylamino)-2-thioxo-2H-benz[e][1,3]oxazin-4(3H)-one
120.3(C-14),119.2(C-15),111.8(C-13),105.8(C-1),100.2(C-5), 11a. In slight modification to previously reported general
96.7(C-3),64.4(C-16),40.8(C-9),15.0(C-17);Anal.Calcd. procedure B, 4-(benzylamino)-2-hydroxybenzoic acid 9a
ForC16H17NO5:C,63.36;H,4.62;N,5.65.Found:C,63.51; (0.94g, 4mmol) was allowed to react with the freshly
H,4.48;N,5.66. prepared Ph3P(SCN)2 (10mmol) at room temperature for
2 hours then under reflux for 16 hours. The resulting crude
4.1.5. Synthesis of Substituted-dihydroxy-di-carboxylic Acids
solids(0.94g,82%)werefiltered,collected,andrecrystallised
16 and 17. According to the previously reported general
fromtoluenetogive11a(0.88g,77%)asyellowcrystals,mp
procedures[23–26], theappropriatesubstitutedphenol was 210–212∘Cdecomp.]max(KBr)/cm−13301(9-NH)3068,2926
used in the synthesis substituted-dihydroxy-di-carboxylic 1
(3-NH),1689(C=O),1197(C=S); HNMR(200MHz,340K,
acids16and17. d6-DMSO) 𝛿 12.90 (bs, 1H, 3-NH exchangeable with D2O),
4.1.6.Synthesisof7-N-Substituted-amino-1,3-oxazines10a,b, 7.61 (d, 1H, JH5,H6 = 8.8Hz, H-5), 7.37–7.26 (m, 6H, Ar and
9-NHexchangeablewithD2O),6.73(dd,1H,JH6,H5=8.8Hz,
and11a,b,andd
JH6,H8=2.0Hz,H-6),6.42(d,1H,JH8,H6=2.0Hz,H-8),4.41
13
GeneralProcedureB.Thesubstituted-2-hydroxybenzoicacid (d, 2H, JH10,H9 = 5.9Hz, H-10); C NMR (50MHz, 340K,
was allowed to react with the freshly prepared Ph3P(SCN)2 d6-DMSO)𝛿182.1(C-2),157.2,156.5(C-4,C-8a),155.2(C-7),
accordingtopreviouslyreportedgeneralprocedure[22,34]. 138.2(C-11),(128.2,127.3,127.0,126.8),(C-13,C-12,C-14and
C-5),111.9(C-6),102.5(C-4a),95.1(C-8),45.8(C-10);Anal.
N-(4-Oxo-2-thioxo-3,4-dihydro-2H-benz[e][1,3]oxazin-7-yl)
Calcd.ForC15H12N2O2S:C,59.99;H,4.03;N,9.33.Found:
acetamide 10a. Slightly modified to the previously reported
C,59.83;H,4.14;N,9.45.
general procedure B [22, 34], 4-(acetyl amino)-2-
hydroxybenzoic acid 8a (1.56g, 8mmol) was allowed to 7-((2-Hydroxybenzyl)amino)-2-thioxo-2H-benz[e][1,3]oxaz-
reactwithfreshlypreparedPh3P(SCN)2 (10mmol)atroom in-4(3H)-one 11b. In slight modification to previously
temperature for 2 hours then under reflux for 16 hours. reportedgeneralprocedureB,2-hydroxy-4-((2-hydroxyben-
At the completion of the reaction, the PbBr2 filter cake zyl)amino)benzoic acid 9b (1.07g, 4mmol) was allowed to
was washed by acetic acid (150mL) to extract the desired react with the freshly prepared Ph3P(SCN)2 (10mmol) at
product.Theaceticacidfiltratewasevaporatedandminimal roomtemperaturefor2hoursthenunderrefluxfor16hours.
toluenewasaddedtodissolveanyoilwiththeproduct.The The resulting crude solid recrystallised from acetonitrile
∘
crude solid was filtered and recrystallised from ethanol to to give 11b (0.55g, 46%) as yellow crystals, mp 173–175 C
give 10a (1.22g, 65%) as light red crystals, mp 285–287∘C decomp.]max(KBr)/cm−13500–3000br(OH),3310(9-NH),
decomp. ]max (KBr)/cm−1 3290, 3183 (9-NH), 3072, 2923 2926 (3-NH), 1684 (C=O), 1192 (C=S); 1HNMR (200MHz,
(3-NH),1704(C=O),1188(C=S);1HNMR(200MHz,390K, 340K, d6-DMSO) 𝛿 12.86 (bs, 1H, 3-NH), 9.50 (bs, 1H,
d6-DMSO)𝛿13.38(bs,1H,9-NH),10.56(s,1H,3-NH),7.86 12-OH), 7.59 (d, 1H, JH5,H6 = 8.6Hz, H-5), 7.41 (bs, 1H,
(d, 1H, JH5,H6 = 8.6Hz, H-5), 7.77 (d, 1H, JH8,H6 = 1.8Hz, 9-NH), 7.05–7.20 (m, 2H, H-14, H-16), 6.69–6.88 (m, 3H,
H-8), 7.44 (dd, 1H, JH6,H5 = 8.6Hz, JH6,H8 = 1.8Hz, H-6), H-13,H-15andH-6),6.42(d,1H,JH8,H6=2.0Hz,H-8),4.32
2.11 (s, 3H, 11-CH3); 13C NMR (50MHz, 330K, d6-DMSO) (s,2H,H-10);13CNMR(50MHz,340K,d6-DMSO)𝛿182.2
𝛿 181.9 (C-2), 169.2 (C-10), 156.7 (C-8a), 146.0 (C-7), 127.3 (C-2), 157.3, 156.7, 155.4, 154.9 (C-4, C-8a, C-12 and C-7),
(C-5),116.3(C-6),109.6(C-4a),104.2(C-8),24.0(C-11)155.9 128.5, 127.9, 127.3 (C-14, C-16 and C-5) 123.9 (C-11), 118.8,
(C-4); Anal. Calcd. For C10H8N2O3S: C, 50.84; H, 3.41; N, 115.1 C-15, C-13), 111.9 (C-6), 102.2 (C-4a), 94.9 (C-8), 40.9
11.86C.Found:C,50.69;H,3.53;N,11.86. (C-10); Anal. Calcd. For C15H12N2O3: C, 59.99; H, 4.03; N,
9.33Found:C,59.83;H,4.14;N,9.45.
Synthesisof7-Amino-2-thioxo-2H-benz[e][1,3]oxazin-4(3H)-
one10b.Asuspensionof(E)-4-((3-ethoxy-2-hydroxybenzy- 7-((3-Ethoxy-2-hydroxybenzyl)amino)-2-thioxo-2H-benz[e]
lidene)amino)-2-hydroxybenzoic acid 8d (1.2g, 4mmol) in [1,3]oxazin-4(3H)-one 11c. In slight modification to