)JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/ijmc/2016/9346585.pdfthat Z is the most active against Pseudomonas aeruginosa ( mm) and least active against Candida

Research ArticleSyntheses and Antibiotic Evaluation of 2-[(2R4R)-4-Carboxy-2-hydroxypyrrolidin-1-yl]carbonylbenzene-15-dicarboxylic Acidsand 2-Carbamoylbenzene-15-dicarboxylic Acid Analogues

Abdulrazaq Tukur1 Isaac Asusheyi Bello1

Neil Anthony Koorbanally2 and James Dama Habila1

1Department of Chemistry Ahmadu Bello University Zaria 810001 Nigeria2School of Chemistry and Physics University of KwaZulu-Natal Private Bag X 54001 Durban 4000 South Africa

Correspondence should be addressed to James Dama Habila habilajames2005yahoocom

Received 27 October 2015 Accepted 16 December 2015

Academic Editor Gangadhar B Bagihalli

Copyright copy 2016 Abdulrazaq Tukur et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Our search for new antibiotics led to the syntheses and biological evaluation of new classes of dicarboxylic acid analoguesThe syntheses involve nucleophilic addition of different substituted benzylamine aniline alkylamine and 4-hydroxyl-L-prolinewith carbamoylbenzoic acid The results of the antimicrobial activity as indicated by the zone of inhibition (ZOI) showedthat Z10 is the most active against Pseudomonas aeruginosa (32mm) and least active against Candida stellatoidea (27mm) andVancomycin Resistant Enterococci (VRE) (27mm) whileZ7 shows the least zone of inhibition (22mm) againstMethicillin ResistantStaphylococcus aureus (MRSA)Theminimum inhibition concentration (MIC) determination reveals thatZ10 inhibits the growth oftested microbes at a low concentration of 625 120583gmL while Z9 and Z12 inhibits the growth of most microbes at a concentration of125 120583gmL recording the least MIC The Minimum BactericidalFungicidal Concentration (MBCMFC) results revealed that Z10has the highest bactericidalfungicidal effect on the test microbes at a concentration of 125 120583gmL with the exception of Candidastellatoidea and Vancomycin Resistant Enterococci (VRE) with MBCMFC of 25120583gmL The result of this investigation reveals thepotential of the target compounds (Z1minus357minus12) in the search for new antimicrobial agents

1 Introduction

Development of novel bioactive drugs in chemical warfareagainst bacteria fungi and other infectious diseases hasbecome an important and challenging task for the syntheticandmedicinal chemistsMany research programs are tailoredtowards the design and synthesis of new drugs for theirchemotherapeutic application The emergence of antimicro-bial resistance threatens the effective prevention and treat-ment of an ever increasing range of infections caused by bac-teria parasites virus and fungi New resistance mechanismsare emerging and spreading globally the appearance andwidespread use of fake and substandard drugs have furthercompounded the problem [1] The HIV epidemic around theworld has led to an increase in the number of immunocom-promised patients which in turn has led to an increase in

the number of systemic bacterial and fungal infections [2]Compounds containing carboxylic acid functional groups areplaying a major role in the field of medicine Generally theyplay an active and critical role in the biochemistry of humanor animal physiology They have been involved in studiesas antibacterial [3] anti-inflammatory [4] antiplatelet [5]antimicrobial [6] anticancer [7] antifungal [8] and analgesicand antiseptic [9]

Currently there are more than 450 clinically approveddrugs containing a free carboxylic acid group [10] To thebest of our knowledge no biological studies and syntheses ofthese compounds have been reported We present here thesyntheses of different substituted carboxylic and dicarboxylicacid analogues with electron withdrawing and donatinggroups (OH OCH

3

CH3

NO2

and F) and explored theirpotentials as antibacterial and antifungal drugs

Hindawi Publishing CorporationInternational Journal of Medicinal ChemistryVolume 2016 Article ID 9346585 8 pageshttpdxdoiorg10115520169346585

2 International Journal of Medicinal Chemistry

Cl HO

R = Z1ndash35ndash12

(ii) RNH2 (15 eq) DCM reflux 2h

O

O

O

O O

OH

R

O

O

(i) KOHaq (5) 30min

Scheme 1 Preparation of carbamoylbenzoic and carbamoyldibenzoic acid

2 Materials and Methods

21 General Experimental Details All chemicals and sol-vents were purchased from Sigma-Aldrich (Germany) andused as purchased without further purification Thin-layerchromatography was performed using precoated silica gel 60(F254

) from MERCK (Germany) Spots on the TLC plateswere visualized under UV light (254 nm and 366 nm) and byheating with 10 sulphuric acid inMeOHThemelting pointwas recorded using a Gallenkamp melting point apparatusThe UV-VIS analysis was carried out on a Perkin ElmerLambda 35 UVVIS Spectrometer scanning from 200 to400 nm Absorbance was measured with a 1 cm quartz cellThe infrared spectra of the solid products were recordedon a Perkin Elmer Spectrum 100 FTIR spectrometer usingATR sampling accessory 1H and 13C-NMR spectra wererecorded using Bruker AvanceIII 400MHz Spectrometer atroom temperature (400MHz for 1H and 100MHz for 13C)using TMS as reference Chemical shift values (120575) werereported in parts per million (ppm) relative to TMS andcoupling constants are given in Hz The solvent used forthesemeasurements was deuteratedDMSOMultiplicities aregiven as follows singlet (s) doublet (d) doublet of doublets(dd) triplet (t) and multiplet (m)

22 General Procedure for the Synthesis of Z1ndash357ndash12 Asolution of 13-dioxo-2-benzofuran-5-carbonyl chloride (5 g238mmol) was weighed into a round bottom flask con-taining 100mL of KOHaq (5) and then stirred for 30minthe solid product (13-dioxo-2-benzofuran-5-carboxylic acid)formed was then filtered under suction The products werepure enough for further reactionwithout further purificationthis acid (03 g 156mmol) was further transferred to a 10mLround bottom flask containing dichloromethane (7mL) anda magnetic stirrer and 15 equiv of different classes of aminewere each added individually to the respective flasks and themixture refluxed for at least two hours The heat was thenremoved and the reaction stirred for a further 30min Thereactionmixtures were allowed to stand at room temperaturefor a further 30min the solid precipitates formed werethen filtered under suction and washed thoroughly withdichloromethane to remove any excess amines (Scheme 1)After drying they were recrystallized from dichloromethaneto give the purified products (Table 1)

4-[(4-Fluorobenzyl)carbamoyl]benzene-15-dicarboxylic Acid(Z1) Bone white solid powder (99 yield) was prepared

according to the general procedure from 13-dioxo-2-benzofuran-5-carboxylic acid 2 (03 g 156mmol) 4-fluoro-benzylamine (026 g 21mmol) and DCM (7mL) as solventand purified by recrystallization with DCM Melting point173ndash175∘C UV analysis 120582max(log 120576) 245 (439) 300 (392) IR(cmminus1) 2872 (COOH) 1603 and 1536 (CONH) 896 (=CH)and 828ndash808 (C=C) 1H-NMR (400MHz DMSO-d6) 120575H872 (d 1H J = 132Hz H-3) 818 (d 1H J = 808Hz H-6)796 (d 1H J = 812Hz H-4) 754 (q 2H J = 260 568HzH-21015840 61015840) 726 (d 2H J = 884Hz H-31015840 51015840) 403 (s 2H H-10)13C-NMR (100MHz DMSO-d6) 16833 (C-7) 16788 (C-9)16781 (C-8) 16194 (d 119869CF = 24273Hz C-41015840) 13721 (C-2)13682 (C-1) 13443 (C-5) 13357 (C-6) 13239 (C-4) 13125 (d119869CF = 308Hz C-11015840) 13102 (d 119869CF = 828Hz C-21015840 61015840) 13046(C-3) 11532 (d 119869CF = 2146Hz C-31015840 51015840) 4164 (C-10) GC-MS (mz rel int) 3171 [M]+ (74) 2991 (100) 2441 (24) 1221(23) LRMS 3133 [MminusNa]+ for C

16

H12

FNO5

calculatedmass3173 (see Appendixes 1ndash7 available online as SupplementaryMaterial at httpdxdoiorg10115520169346585)

4-[(3-Fluorobenzyl)carbamoyl]benzene-15-dicarboxylic Acid(Z2) Bone white solid powder (55 yield) was pre-pared according to the general procedure from 13-dioxo-2-benzofuran-5-carboxylic acid 2 (03 g 156mmol) 3-fluorobenzylamine (026 g 21mmol) and DCM (7mL) assolvent and purified by recrystallization with DCM Meltingpoint 200-201∘C UV analysis (120582max(log 120576)ethanol) 250 (426)300 (348) IR (cmminus1) 2868 (COOH) 1271 and 1246 (O-C)1691 and 1501 (CONH) 1691 and 1574 (C=C) and 913 (=CH)1H-NMR (400MHz DMSO-d6) 120575H 872 (s 1H H-6) 818(d 1H J = 804Hz H-3) 796 (dd 1H J = 180 804Hz H-4) 746ndash740 (m 1H H-51015840) 733ndash727 (m 3H H-21015840 H-61015840 N-H) 733ndash727 (m 3H H-21015840 H-61015840 N-H) 718ndash714 (m 1H J =246 866Hz H-41015840) 400 (s 2H H-10) 13C-NMR (100MHzDMSO-d6) 16821 (C-7) 16774 (C-9) 16769 (C-8) 16204 (d119869CF = 24196Hz C-31015840) 13936 (d 119869CF = 853Hz C-11015840) 13699(C-1) 13451 (C-2) 13358 (C-3) 13358 (C-5) 13242 (C-6)13043 (d 119869CF = 836Hz C-51015840) 13042 (C-4) 12444 (d 119869CF =271Hz C-61015840) 11512 (d 119869CF = 2167Hz C-21015840) 11459 (d 119869CF =2073Hz C-41015840) 4235 (C-10) GC-MS (mz rel int) 3171[M]+ (72) 2991 (100) 2261 (24) 1221 (21) LRMS 3171 [M minusNa]+ for C

16

H12

FNO5

calculated 3173 (Appendixes 8ndash15)

2-[(2-Fluorobenzyl)carbamoyl]benzene-15-dicarboxylic Acid(Z3) Bone white solid powder (35 yield) was preparedaccording to the general procedure from 13-dioxo-2-benzofuran-5-carboxylic acid 2 (03 g 156mmol)

International Journal of Medicinal Chemistry 3

Table 1 The synthesised compounds and their percentage yield ()

Sample R Product Yield ()

Z1F

NHminus

HO

O O

OHNH

O

F99

Z2

FNHminus

HO

O O

OHNH

OF

55

Z3

F

NHminus HO

O O

OHNH

O F

35

Z5

OH

COOH

minusN N

OO

OHOH

O

HO

OHO

78

Z7

F F

NHminus

NH

O

O

OHHO

OF

F

90

Z8 CH3minusHNNH

O

O

OHHO

O

CH3 54

Z9 OCH3

minusNHNH

O

O

OHHO

O

OCH3

24

Z10NHminus

OHHO

O O

NH

O

65

Z11

N

CH3

CH3Nminus OHHO

O O

NH

O

N

CH3

CH3 79


Table 1 Continued


Z12 Nminus

NO2

OHHO

O O

NH

O

NO236

2-fluorobenzylamine (026 g 21mmol) and DCM (7mL) assolvent and purified by recrystallization with DCM Meltingpoint 198ndash200∘C UV analysis 120582max(log 120576) 245 (424) 300(343) IR (cmminus1) 2839 (COOH) 1272 and 1248 (O-C) 1684and 1574 (CONH) 1613 and 1500 (C=C) and 907 (=CH)t61H-NMR (400MHz DMSO-d6) 120575H 871 (s 1H H-6) 815(d 1H J = 807Hz H-3) 794 (dd 1H J = 176 807HzH-4) 756ndash753 (m 1H Ar-H H-41015840 N-H) 739ndash734 (m 1HAr-H H-61015840) 724ndash719 (m 2H Ar-H H-31015840 H-51015840) 398 (s 1HH-10) 13C-NMR (100MHz DMSO-d6) 16885 (C-7) 16803(C-9) 16795 (C-8) 16015 (d 119869CF = 24357Hz C-21015840) 13853(C-2) 13636 (C-1) 13431 (C-5) 13353 (C-6) 13222 (C-3)13042 (C-4) 13042 (d 119869CF = 399Hz C-41015840) 12976 (d 119869CF= 813Hz C-61015840) 12478 (d 119869CF = 1461Hz C-11015840) 12444 (d119869CF = 344Hz C-51015840) 11515 (d 119869CF = 2114Hz C-31015840) 3677 (d119869CF = 424Hz C-10) GC-MS (mz rel int) 3171 [M]+ (91)2991 (100) 2441 (32) 1221 (24) LRMS 3131 [M minus Na]+ forC16

H12

FNO5

calculated mass 3173 (Appendixes 16ndash23)

2-[(2R4R)-4-Carboxy-2-hydroxypyrrolidin-1-yl]carbonylbenzene-15-dicarboxylic Acid (Z5) White solid powder (78yield) was prepared according to the general procedurefrom 13-dioxo-2-benzofuran-5-carboxylic acid 2 (03 g156mmol) 4-hydroxy-L-proline (028 g 21mmol) andDCM (7mL) as solvent and purified by recrystallization withDCM Melting point 187ndash180∘C UV analysis 120582max(log 120576) 245(402) 300 (318) IR (cmminus1) 2855 (COOH) 1285 and 1250(O-C) 1689 and 1574 (CONH) 1639 and 1502 (C=C) and916 (=CH) 1H-NMR (400MHz DMSO-d6) 120575H 843 (s 1HH-6) 805 (dd 1H J = 14 804Hz H-4) 796 (dd 1H J =292 772Hz H-3) 441 (t H J = 432Hz H-21015840) 431 (t 1H J= 852Hz H-41015840) 332 [(dd 1H J = 404 1208Hz Hc-51015840) 306(d 1H J = 1208Hz Hd-51015840)] 221 [(dd 1H J = 772 1340HzHa-31015840) 206 (m 1H J = 432 108Hz Hb-31015840)] 13C-NMR(100MHz DMSO-d6) 17041 (C-7) 16792 (C-10) 16734 (C-9) 16621 (C-8) 13787 (C-2) 13322 (C-1) 13229 (C-5) 13125(C-4) 13103 (C-6) 13028 (C-3) 6875 (C-21015840) 5798 (C-41015840)5323 (C-51015840) 3731 (C-31015840) GC-MS (mz rel int) 3231 [Mminus4]+(100) 2261 (29) 2821 (18) 1221 (13) LRMS 3243 [M minusNa]+for C14

H13

NO8


2-[(24-Difluorobenzyl)carbamoyl]benzene-15-dicarboxylicAcid (Z7) Pale yellow solid powder (90 yield) wasprepared according to the general procedure from 13-dioxo-2-benzofuran-5-carboxylic acid 2 (03 g 156mmol)24-difluorobenzylamine (03 g 21mmol) and toluene(7mL) as solvent and purified by recrystallization withDCM Melting point 152ndash154∘C UV analysis 120582max(log 120576) 245

(386) 300 (295) IR (cmminus1) 2878 2631 (COOH) 1273 and1297 (O-Cacid) 1687 and 1561 (CONH) 1603 and 1507 (C=C)and 847 (=CH) 1H-NMR (400MHz DMSO-d6) 120575H 874 (dH J = 180Hz H-6) 824 (d H J = 812Hz H-4) 801 (ddH J = 188 808Hz H-3) 764 (q H J = 852 1516Hz H-61015840)735 (td H J = 248 196 252 100Hz H-51015840) 720 (td H J =232 212 248 836Hz H-31015840) 407 (s 2H H-10) 13C-NMR(100MHz DMSO-d6) 16746 (C-7) 16746 (C-9) 16716(C-8) 16134 (q 119869CF = 1222 24621Hz C-41015840) 16055 (q 119869CF =1243 24981Hz C-21015840) 13805 (C-2) 13473 (C-5) 13370 (C-1) 13363 (C-6) 13277 (C-4) 13269 (q 119869CF = 565 1006HzC-61015840) 11789 (d 119869CF = 349Hz C-11015840) 11177 (q 119869CF = 3651761Hz C-51015840) 10404 (t 119869CF = 2563Hz C-31015840) 3541 (d 119869CF= 358Hz C-10) GC-MS (mz rel int) 3311 [M + 4]+ (100)2441 (32) 3001 (16) 1031 (10) LRMS 3331 [M minus Na]+ forC16

H11

F2

NO5


2-(Butylcarbamoyl)benzene-15-dicarboxylic Acid (Z8) Bonewhite solid powder (54 yield) was prepared accordingto the general procedure from 13-dioxo-2-benzofuran-5-carboxylic acid 2 (03 g 156mmol) butylamine (015 g21mmol) and DCM (7mL) as solvent and purified byrecrystallization with DCM Melting point 118ndash120∘C UVanalysis 120582max(log 120576) 245 (408) 300 (326) IR (cmminus1) 30612959 (COOH) 1239 and 1267 (O-Cacid) 1654 and 1562(CONH) 1622 and 1516 (C=C) and 780 (=CH) 1H-NMR(400MHz DMSO-d6) 120575H 871 (s 1H H-6) 819 (d 1H J =796Hz H-3) 797 (d 1H J = 796Hz H-4) 281 (t 2H J= 716Hz H-11015840) 153 (m 2H J = 692 716Hz H-21015840) 134(q 2H J = 716Hz H-31015840) 088 (t 3H J = 716Hz H-41015840)13C-NMR (100MHz DMSO-d6) 16799 (C-7) 16784 (C-9)16779 (C-8) 13703 (C-2) 13652 (C-1) 13445 (C-5) 13355 (C-6) 13247 (C-3) 13048 (C-4) 3844 (C-11015840) 2912 (C-21015840) 1907(C-31015840) 1343 (C-41015840) GC-MS (mz rel int) 2611 [M+ 4]+ (41)2181 (100) 1851 (74) 2611 (41) LRMS 2611 [M minus Na]+ forC13

H15

NO5


2-[(3-Methoxypropyl)carbamoyl]benzene-15-dicarboxylicAcid (Z9) White solid powder (24 yield) was preparedaccording to the general procedure from 13-dioxo-2-benzofuran-5-carboxylic acid 2 (03 g 156mmol) 3-methoxypropylamine (019 g 21mmol) and DCM (7mL) assolvent and purified by recrystallization with DCM Meltingpoint 135-136∘C UV analysis 120582max(log 120576) 245 (368) 300(248) IR (cmminus1) 3136 2932 (COOH) 1356 and 1297 (O-Cacid) 1622 and 1529 (CONH) 1477 (C=C) and 752 (=CH)1H-NMR (400MHz DMSO-d6) 120575H 867 (d H J = 124Hz)812 (d H J = 800Hz H-4) 791 (dd H J = 120 800Hz


H-3) 339 (t 2H J = 600Hz H-31015840) 322 (s H H-41015840) 286(t 2H J = 728Hz H-11015840) 182 (qt 2H J = 648 700 1348HzH-21015840) 13C-NMR (100MHz DMSO-d6) 16904 (C-7) 16819(C-9) 16808 (C-8) 13991 (C-2) 13583 (C-1) 13415 (C-5)13345 (C-6) 13205 (C-4) 13034 (C-3) 6892 (C-31015840) 5787(C-41015840) 3652 (C-11015840) 2744 (C-21015840) GC-MS (mz rel int) 2811[M]+ (28) 2071 (100) 321 (93) 45 (77) LRMS 2771 [MminusNa]+for C13

H15

NO6


2-(Benzylcarbamoyl)benzene-15-dicarboxylic Acid (Z10)Cream coarse powder (65 yield) was prepared accordingto the general procedure from 13-dioxo-2-benzofuran-5-carboxylic acid 2 (03 g 156 mmol) benzylamine (023 g21mmol) and DCM (7mL) as solvent and purified byrecrystallization with DCM Melting point 163ndash165∘C UVanalysis 120582max(log 120576) 245 (378) 300 (278) IR (cmminus1) 33963034 (COOH) 1361 and 1288 (O-Cacid) 1622 and 1551(CONH) 1499 (C=C) and 693 (=CH) 1H-NMR (400MHzDMSO-d6) 120575H 872 (d 1H J = 160Hz H-6) 815 (d 1H J =804Hz H-3) 794 (dd 1H J = 160 804Hz H-4) 746ndash731(d 5H Ar-H H-2101584061015840310158405101584041015840) 400 (s 2H H-10) 13C-NMR(100MHz DMSO-d6) 16903 (C-7) 16815 (C-9) 16804 (C-8) 13938 (C-11015840) 13605 (C-2 C-5) 13423 (C-1) 13352 (C-6)13214 (C-3) 13040 (C-4) 12846 (C-21015840 C-61015840) 12845 (C-31015840C-51015840) 12791 (C-41015840) GC-MS (mz rel int) 2951 [M + 4]+(100) 278 (34) 2081 (25) 1041 (24) LRMS 2951 [M minus Na]+for C16

H13

NO5


2-[(46-Dimethylpyridin-2-yl)carbamoyl]benzene-15-dicarb-oxylic Acid (Z11) Bone white powder (79 yield) wasprepared according to the general procedure from 13-dioxo-2-benzofuran-5-carboxylic acid 2 (03 g 156mmol)2-amino-46-dimethylpyridine (026 g 21mmol) and DCM(7mL) as solvent and purified by recrystallizationwithDCMMelting point 189-190∘C UV analysis 120582max(log 120576) 245 (431)300 (388) IR (cmminus1) 3331 3097 (COOH) 1343 and 1296 (O-Cacid) 1675 and 1534 (CONH) 1604 and 1477 (C=C) and 752(=CH) 1H-NMR (400MHz DMSO-d6) 120575H 858 (d 1H J =148Hz H-6) 809 (d 1H J = 804Hz H-4) 804 (dd 1H J =168 804Hz H-3) 647 (s 2H H-31015840H-51015840) 233 (s 3H 41015840 61015840-CH3

) 13C-NMR (100MHz DMSO-d6) 16793 (C-7) 16763(C-9) 16679 (C-8) 15486 (C-21015840) 15481 (C-61015840) 14741 (C-41015840)13832 (C-2) 13424 (C-1) 13257 (C-5) 13228 (C-6) 13149(C-4) 13080 (C-3) 11324 (C-51015840) 10820 (C-31015840) 2112 (61015840-CH3

) 1914 (41015840-CH3

) GC-MS (mz rel int) 3142 [M]+ (5)321 (100) 43 (65) 2071 (20) 771 (18) LRMS 3041 [M+Na]+for C16

H14

N2

O5


2-[(2-Nitrophenyl)carbamoyl]benzene-15-dicarboxylic Acid(Z12) Pale brownpowder (36yield)was prepared accordingto the general procedure from 13-dioxo-2-benzofuran-5-carboxylic acid 2 (03 g 156mmol) 2-nitrobenzylamine(029 g 21mmol) and DCM (7mL) as solvent and purifiedby recrystallization with DCM Melting point 204-205∘CUV analysis 120582max(log 120576) 245 (426) 300 (348) IR (cmminus1)3317 3062 (COOH) 1290 and 1249 (O-Cacid) 1679 and 1574(CONH) 1500 (C=C) and 752 (=CH) 1H-NMR (400MHzDMSO-d6) 120575H 821 (d 3H J = 148Hz H-4510158406) 812 (dd2H J = 156 796Hz H-3 H-31015840) 775 (d 2H J = 796Hz

H-41015840 61015840) 13C-NMR (100MHz DMSO-d6) 16841 (C-7)16747 (C-9) 16595 (C-8) 13734 (C-2) 13242 (C-1) 13176(C-4 510158406) 12929 (C-3 C-31015840) 12858 (C-41015840 61015840) GC-MS(mz rel int) 3311 [M minus H]+ (94) 321 (100) 44 (71) 2071(67) 2441 (39) LRMS 3332 [M minus 3H]+ for C

15

H10

N2

O7


3 Biological Assay

31 Clinical Isolate The test compounds (Z1minus357minus12) wereevaluated on the following isolates obtained from theDepartment of Medical Microbiology Ahmadu Bello Uni-versity Teaching Hospital Zaria Nigeria (ABUTH) Can-dida stellatoidea Candida tropicalis Candida krusei Can-dida albicans Shigella dysenteriae Salmonella typhi Kleb-siella pneumonia Pseudomonas aeruginosa Proteus vulgarisEscherichia coli Corynebacteriumulcerans Streptococcus pyo-genes Staphylococcus aureus Vancomycin Resistant Entero-cocci (VRE) and Methicillin Resistant Staphylococcus aureus(MRSA)

32 Antimicrobial Susceptibility Test The cork and borediffusion method as reported by Karou et al [11] was used todetermine the antimicrobial activity of the test compoundsPure cultures of the organism were inoculated on to MuellerHinton Agar (MERCK) and incubated for 24 h at 38∘C forbacteria and 48 h at 34∘C for fungi About 5 discrete colonieswere aseptically transferred using sterile wire loops intotubes containing sterile normal saline (085 NaCl) andwere adjusted to a turbidity of 05 McFarland Standard Thesuspensions were then inoculated on the surface of sterileMueller-Hinton Agar plates using sterile cotton swabs Asterile 6mm diameter Cork borer was used to make holes(wells) into the set of inoculated Mueller-Hinton Agar Thewells were filled with different concentration of the testcompounds The plates were then incubated all the testswere performed in triplicate and the antimicrobial activitieswere determined as mean diameter of inhibition zone (mm)produced by the test compounds

33 Minimum Inhibition Concentration (MIC) TheMIC wasdetermined for the compounds using microbroth dilutionmethod in accordance with National Committee for ClinicalLaboratory Standard [12] Serial dilution of the least concen-tration of the compounds that showed activity was preparedusing test tubes containing 9mL of double strength nutrientbroth (OXOID) The test tubes were inoculated with the sus-pension of the standardized inoculum and incubated at 38∘Cfor 24 h MICs were recorded as the lowest concentration ofthe compounds showing no visible growth (turbidity) in thebroth

34 Minimum Bactericidal and Minimum Fungicidal Con-centration (MBCMFC) TheMBCMFC was determined byaseptically inoculating aliquots of culture from theminimuminhibition concentration (MIC) tubes that showedno growthon sterile nutrient Agar (OXOID) plates incubated at 38∘Cfor bacteria and 34∘C for fungi for 48 h The MBCMFC was


Table 2 Zone of inhibition (mm)

Test organism Z1 Z2 Z3 Z5 Z7 Z8 Z9 Z10 Z11 Z12 Sp FlMRSA 26 28 mdash mdash 22 28 mdash 29 28 mdash 35 mdashVRE mdash 24 mdash 29 mdash 24 20 27 mdash 24 mdash mdashS aureus 27 29 24 mdash 24 27 mdash 29 29 26 37 mdashS pyogenes 25 mdash 27 27 mdash mdash 24 mdash mdash 24 34 mdashC ulcerans mdash 24 23 mdash 26 24 28 30 mdash mdash 32 mdashE coli 29 27 26 29 26 mdash mdash mdash 27 25 37 mdashP vulgaris mdash mdash 26 28 mdash mdash 25 mdash 25 27 mdash mdashP aeruginosa mdash 24 mdash 24 30 31 28 32 mdash 24 35 mdashK pneumonia 27 29 28 mdash mdash mdash 25 29 30 27 39 mdashS typhi mdash 27 26 mdash 29 26 26 30 mdash 24 42 mdashS dysenteriae 29 mdash mdash 29 25 mdash 27 mdash 24 mdash 40 mdashC albicans 24 mdash 27 24 mdash 28 24 29 29 27 mdash 35C krusei 27 28 mdash 26 25 24 22 28 24 27 mdash 35C tropicalis mdash 26 mdash 24 mdash 26 26 29 23 24 mdash 30C stellatoidea 26 27 28 29 27 28 24 27 27 23 mdash 34mdash not determined

Table 3 Minimum inhibitory concentration (MIC) (120583gmL)

Test organism Z1 Z2 Z3 Z5 Z7 Z8 Z9 Z10 Z11 Z12MRS 1250 62 mdash mdash 62 62 mdash 62 62 mdashVRE mdash 62 mdash 62 mdash 125 125 62 mdash 125S aureus 62 125 125 mdash 62 62 mdash 62 62 125S pyogenes 125 62 62 62 mdash mdash 125 mdash mdash 125C ulcerans mdash 125 125 mdash 125 mdash 62 62 mdash mdashE coli 62 62 125 62 125 125 62 mdash 62 125P vulgaris mdash mdash 125 62 125 mdash mdash mdash 125 62P aeruginosa mdash 125 mdash 125 mdash mdash 125 62 mdash 125K pneumonia 62 62 62 mdash 62 62 62 62 62 62S typhi mdash mdash 125 mdash mdash mdash 125 62 mdash 125S dysenteriae 62 62 mdash 62 62 125 125 mdash 125 mdashC albicans 125 mdash 62 125 125 62 125 62 62 62C krusei 62 62 mdash 125 mdash 125 125 62 125 62C tropicalis mdash 125 mdash 125 62 125 125 62 125 125C stellatoidea 125 62 62 62 mdash 62 125 62 62 125mdash no MIC

recorded as the lowest concentration of compounds showingno bacterialfungal growth at all

4 Results and Discussion

41 Chemistry Our synthetic approach involved two steps(Scheme 1) In the first step the acid chloride is hydrolysed bythe aqueous KOH to form the corresponding carboxylic acidThe reaction takes place at room temperature under stirringcondition for 30min The second step involves nucleophilicaddition of the substituted amine which attack the anhydridering breaking it open to form the second carboxylic acidgroup and an amide This reaction was carried out in DCMunder reflux condition for at least 2 h The overall yields aregiven in the experimental section and they ranges between 35and 99

The structures of the compounds were confirmed by theuse of 1H and 13C NMR with application of 2D NMR wherenecessary

42 Biological Results The synthesised compounds weretested against eleven bacteria including two resistance bac-teria Methicillin Resistant Staphylococcus aureus (MRSA)Vancomycin Resistant Enterococci (VRE) and four fungiThe test compounds had significant zones of inhibitionsagainst all tested organism as compared to the standard drugCompound Z10 had the best activity among the compoundstested with zone of inhibition ranging from 32 to 27mm onthe test microbes but was not able to inhibit five bacteria (Sdysenteriae P aeruginosa P vulgaris E coli and S pyogenes)(Table 2)Thiswas the compoundwithout any substitution onthe benzyl ring Compounds Z7 and Z11 had zones rangingfrom 30 to 22mm while Z1 Z2 and Z5 were in the rangebetween 29 and 24mmThe zone of inhibition of Z3 Z9 andZ12 was in the range between 28 and 20mm as compared tothe zone of the standard drugs (32 to 40mm)

Minimum inhibitory concentration (MIC) results(Table 3) reveal that a low concentration of 625120583gmL ofthe test compounds (Z1minus3 Z57minus12) inhibited the growth of


Table 4 Minimum bactericidalfungicidal concentration (MBCMFC) (120583gmL)

Test organism Z1 Z2 Z3 Z5 Z7 Z8 Z9 Z10 Z11 Z12

MRS 25 125 mdash mdash 50 125 mdash 125 125 mdashVRE mdash 50 mdash 125 mdash 50 50 25 mdash 50S aureus 25 125 50 mdash 50 25 mdash 125 125 25S pyogenes 25 mdash 25 25 mdash mdash 25 mdash mdash 25C ulcerans mdash 25 50 mdash 25 mdash 25 mdash mdash mdashE coli 125 25 25 125 25 50 125 125 25 25P vulgaris mdash mdash 25 125 25 mdash mdash mdash 25 25P aeruginosa mdash 25 mdash 50 mdash mdash 25 mdash mdash 25K pneumonia 25 125 125 mdash 125 125 125 125 125 25S typhi mdash mdash 25 mdash mdash mdash 25 125 mdash 50S dysenteriae 125 25 mdash 125 125 25 25 125 25 mdashC albicans 50 mdash 25 25 mdash 125 50 125 125 25C krusei 25 125 mdash 25 25 25 50 125 50 25C tropicalis mdash 25 mdash 50 mdash 25 25 125 50 50C stellatoidea 25 25 125 125 25 125 50 25 25 50mdash no MBCMFC

the resistance bacteria (MRSA) the only exception was Z1which inhibited at 125 120583gmL Three of the compounds(Z2510) inhibited the resistance VRE at a concentration of625 120583gmL while Z8 Z9 and Z12 inhibited VRE at a higherconcentration of 125120583gmL Generally other test microbeshave shown MIC ranging between 625 and 50 120583gmL Thesetest compounds were also found to be both bactericidaland fungicidal at a concentration of 125120583gmL as recordedby the minimum bactericidalfungicidal concentration(MBCMFC) analysis (Table 4)

5 Conclusion

The different substituted test compounds were successfullysynthesised and their structures were confirmed by NMRanalysis Generally 2-(benzylcarbamoyl)benzene-15-dicarb-oxylic acid (Z10) showed a better activity against resistancebacteria MRSA and VRE and other microbes Althoughthe test compounds were not as active as the standarddrugs sparfloxacin and fluconazole the compounds may beemployed in situations where there is resistance to antimicro-bial drugs CompoundZ10 is therefore a lead candidate in thesearch for an antimicrobial agent

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors wish to thank the School of Chemistry andPhysics University of KwaZulu-Natal Durban South Africafor providing facilities to carry out this research

References

[1] Y Smith and K Kajimura ldquoBactericidal activities of disin-fectants against vancomycin-resistant enterococcirdquo Journal ofHospital Infection vol 50 no 2 pp 140ndash144 2002

[2] J D Habila H R Kandappa P Govender and N A Koor-banally ldquoSynthesis and antifungal studies of (2E)-N-benzyl-N1015840-phenylbut-2-enediamide and (2E)-N N1015840-dibenzylbut-2-enediamide analoguesrdquo Journal of Chemistry vol 2013 ArticleID 281341 7 pages 2013

[3] M E Falagas A P Grammatikos and A MichalopoulosldquoPotential of old-generation antibiotics to address current needfor new antibioticsrdquoExpert Review ofAnti-InfectiveTherapy vol6 no 5 pp 593ndash600 2008

[4] M Sharma and S M Ray ldquoSynthesis and biological evalua-tion of amide derivatives of (56-dimethoxy-23-dihydro-1H-inden-1-yl)acetic acid as anti-inflammatory agents with reducedgastrointestinal ulcerogenecityrdquo European Journal of MedicinalChemistry vol 43 no 10 pp 2092ndash2102 2008

[5] P X Zhang H Lin C Qu et al ldquoDesign synthesis and in vitroantiplatelet aggregation activities of ferulic acid derivativesrdquoJournal of Chemistry vol 2015 Article ID 376527 7 pages 2015

[6] A A Kadi E S Al-Abdullah I A Shehata E E Habib TM Ibrahim and A A El-Emam ldquoSynthesis antimicrobialand anti-inflammatory activities of novel 5-(1-adamantyl)-134-thiadiazole derivativesrdquo European Journal of Medicinal Chem-istry vol 45 no 11 pp 5006ndash5011 2010

[7] X-H Yang L Xiang X Li et al ldquoSynthesis biological eval-uation and molecular docking studies of 134-thiadiazol-2-amide derivatives as novel anticancer agentsrdquo Bioorganic andMedicinal Chemistry vol 20 no 9 pp 2789ndash2795 2012

[8] S H Lee I-O Kim C S Cheong and B Y ChungldquoSynthesis and antifungal activities of N-aryl-4-phenyl-3-(4-phenoxyphenyl)butanamidesrdquo Archiv der Pharmazie vol 332no 10 pp 333ndash336 1999

[9] R K Pal H Yasmin L Nahar et al ldquoSynthesis of 5 6-dichloroindan-1-acids and their tetrazolyl derivatives as anal-gesic and anti-inflammatory agentsrdquo Medicinal Chemistry vol8 no 5 pp 874ndash882 2012


[10] K A M Abouzid N A Khalil E M Ahmed and SA-B Zaitone ldquoSynthesis and biological evaluation of newheteroaryl carboxylic acid derivatives as anti-inflammatory-analgesic agentsrdquoChemical and Pharmaceutical Bulletin vol 61no 2 pp 222ndash228 2013

[11] D Karou A Savadogo A Canini et al ldquoAntibacterial activityof alkaloids from Sida acutardquo African Journal of Biotechnologyvol 5 no 2 pp 195ndash200 2006

[12] National Committee for Clinical Laboratory Standards Per-formance Standards for Antimicrobial Disk Susceptibility TestsNCCLS Document M2-A9 Wayne Pa USA 9th edition 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

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International Journal ofPhotoenergy


Carbohydrate Chemistry

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Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


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Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


Cl HO

R = Z1ndash35ndash12

(ii) RNH2 (15 eq) DCM reflux 2h

O

O

O

O O

OH

R

O

O

(i) KOHaq (5) 30min

Scheme 1 Preparation of carbamoylbenzoic and carbamoyldibenzoic acid

2 Materials and Methods

21 General Experimental Details All chemicals and sol-vents were purchased from Sigma-Aldrich (Germany) andused as purchased without further purification Thin-layerchromatography was performed using precoated silica gel 60(F254

) from MERCK (Germany) Spots on the TLC plateswere visualized under UV light (254 nm and 366 nm) and byheating with 10 sulphuric acid inMeOHThemelting pointwas recorded using a Gallenkamp melting point apparatusThe UV-VIS analysis was carried out on a Perkin ElmerLambda 35 UVVIS Spectrometer scanning from 200 to400 nm Absorbance was measured with a 1 cm quartz cellThe infrared spectra of the solid products were recordedon a Perkin Elmer Spectrum 100 FTIR spectrometer usingATR sampling accessory 1H and 13C-NMR spectra wererecorded using Bruker AvanceIII 400MHz Spectrometer atroom temperature (400MHz for 1H and 100MHz for 13C)using TMS as reference Chemical shift values (120575) werereported in parts per million (ppm) relative to TMS andcoupling constants are given in Hz The solvent used forthesemeasurements was deuteratedDMSOMultiplicities aregiven as follows singlet (s) doublet (d) doublet of doublets(dd) triplet (t) and multiplet (m)

22 General Procedure for the Synthesis of Z1ndash357ndash12 Asolution of 13-dioxo-2-benzofuran-5-carbonyl chloride (5 g238mmol) was weighed into a round bottom flask con-taining 100mL of KOHaq (5) and then stirred for 30minthe solid product (13-dioxo-2-benzofuran-5-carboxylic acid)formed was then filtered under suction The products werepure enough for further reactionwithout further purificationthis acid (03 g 156mmol) was further transferred to a 10mLround bottom flask containing dichloromethane (7mL) anda magnetic stirrer and 15 equiv of different classes of aminewere each added individually to the respective flasks and themixture refluxed for at least two hours The heat was thenremoved and the reaction stirred for a further 30min Thereactionmixtures were allowed to stand at room temperaturefor a further 30min the solid precipitates formed werethen filtered under suction and washed thoroughly withdichloromethane to remove any excess amines (Scheme 1)After drying they were recrystallized from dichloromethaneto give the purified products (Table 1)

4-[(4-Fluorobenzyl)carbamoyl]benzene-15-dicarboxylic Acid(Z1) Bone white solid powder (99 yield) was prepared

according to the general procedure from 13-dioxo-2-benzofuran-5-carboxylic acid 2 (03 g 156mmol) 4-fluoro-benzylamine (026 g 21mmol) and DCM (7mL) as solventand purified by recrystallization with DCM Melting point173ndash175∘C UV analysis 120582max(log 120576) 245 (439) 300 (392) IR(cmminus1) 2872 (COOH) 1603 and 1536 (CONH) 896 (=CH)and 828ndash808 (C=C) 1H-NMR (400MHz DMSO-d6) 120575H872 (d 1H J = 132Hz H-3) 818 (d 1H J = 808Hz H-6)796 (d 1H J = 812Hz H-4) 754 (q 2H J = 260 568HzH-21015840 61015840) 726 (d 2H J = 884Hz H-31015840 51015840) 403 (s 2H H-10)13C-NMR (100MHz DMSO-d6) 16833 (C-7) 16788 (C-9)16781 (C-8) 16194 (d 119869CF = 24273Hz C-41015840) 13721 (C-2)13682 (C-1) 13443 (C-5) 13357 (C-6) 13239 (C-4) 13125 (d119869CF = 308Hz C-11015840) 13102 (d 119869CF = 828Hz C-21015840 61015840) 13046(C-3) 11532 (d 119869CF = 2146Hz C-31015840 51015840) 4164 (C-10) GC-MS (mz rel int) 3171 [M]+ (74) 2991 (100) 2441 (24) 1221(23) LRMS 3133 [MminusNa]+ for C

16

H12

FNO5

calculatedmass3173 (see Appendixes 1ndash7 available online as SupplementaryMaterial at httpdxdoiorg10115520169346585)

4-[(3-Fluorobenzyl)carbamoyl]benzene-15-dicarboxylic Acid(Z2) Bone white solid powder (55 yield) was pre-pared according to the general procedure from 13-dioxo-2-benzofuran-5-carboxylic acid 2 (03 g 156mmol) 3-fluorobenzylamine (026 g 21mmol) and DCM (7mL) assolvent and purified by recrystallization with DCM Meltingpoint 200-201∘C UV analysis (120582max(log 120576)ethanol) 250 (426)300 (348) IR (cmminus1) 2868 (COOH) 1271 and 1246 (O-C)1691 and 1501 (CONH) 1691 and 1574 (C=C) and 913 (=CH)1H-NMR (400MHz DMSO-d6) 120575H 872 (s 1H H-6) 818(d 1H J = 804Hz H-3) 796 (dd 1H J = 180 804Hz H-4) 746ndash740 (m 1H H-51015840) 733ndash727 (m 3H H-21015840 H-61015840 N-H) 733ndash727 (m 3H H-21015840 H-61015840 N-H) 718ndash714 (m 1H J =246 866Hz H-41015840) 400 (s 2H H-10) 13C-NMR (100MHzDMSO-d6) 16821 (C-7) 16774 (C-9) 16769 (C-8) 16204 (d119869CF = 24196Hz C-31015840) 13936 (d 119869CF = 853Hz C-11015840) 13699(C-1) 13451 (C-2) 13358 (C-3) 13358 (C-5) 13242 (C-6)13043 (d 119869CF = 836Hz C-51015840) 13042 (C-4) 12444 (d 119869CF =271Hz C-61015840) 11512 (d 119869CF = 2167Hz C-21015840) 11459 (d 119869CF =2073Hz C-41015840) 4235 (C-10) GC-MS (mz rel int) 3171[M]+ (72) 2991 (100) 2261 (24) 1221 (21) LRMS 3171 [M minusNa]+ for C

16

H12

FNO5

calculated 3173 (Appendixes 8ndash15)

2-[(2-Fluorobenzyl)carbamoyl]benzene-15-dicarboxylic Acid(Z3) Bone white solid powder (35 yield) was preparedaccording to the general procedure from 13-dioxo-2-benzofuran-5-carboxylic acid 2 (03 g 156mmol)




Z1F

NHminus

HO

O O

OHNH

O

F99

Z2

FNHminus

HO

O O

OHNH

OF

55

Z3

F

NHminus HO

O O

OHNH

O F

35

Z5

OH

COOH

minusN N

OO

OHOH

O

HO

OHO

78

Z7

F F

NHminus

NH

O

O

OHHO

OF

F

90

Z8 CH3minusHNNH

O

O

OHHO

O

CH3 54

Z9 OCH3

minusNHNH

O

O

OHHO

O

OCH3

24

Z10NHminus

OHHO

O O

NH

O

65

Z11

N

CH3

CH3Nminus OHHO

O O

NH

O

N

CH3

CH3 79


Table 1 Continued


Z12 Nminus

NO2

OHHO

O O

NH

O

NO236


H12

FNO5



H13

NO8




H11

F2

NO5



H15

NO5





H15

NO6



H13

NO5




) 1914 (41015840-CH3


H14

N2

O5




15

H10

N2

O7


3 Biological Assay





















5 Conclusion




Acknowledgment


References























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




Z1F

NHminus

HO

O O

OHNH

O

F99

Z2

FNHminus

HO

O O

OHNH

OF

55

Z3

F

NHminus HO

O O

OHNH

O F

35

Z5

OH

COOH

minusN N

OO

OHOH

O

HO

OHO

78

Z7

F F

NHminus

NH

O

O

OHHO

OF

F

90

Z8 CH3minusHNNH

O

O

OHHO

O

CH3 54

Z9 OCH3

minusNHNH

O

O

OHHO

O

OCH3

24

Z10NHminus

OHHO

O O

NH

O

65

Z11

N

CH3

CH3Nminus OHHO

O O

NH

O

N

CH3

CH3 79


Table 1 Continued


Z12 Nminus

NO2

OHHO

O O

NH

O

NO236


H12

FNO5



H13

NO8




H11

F2

NO5



H15

NO5





H15

NO6



H13

NO5




) 1914 (41015840-CH3


H14

N2

O5




15

H10

N2

O7


3 Biological Assay





















5 Conclusion




Acknowledgment


References























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Table 1 Continued


Z12 Nminus

NO2

OHHO

O O

NH

O

NO236


H12

FNO5



H13

NO8




H11

F2

NO5



H15

NO5





H15

NO6



H13

NO5




) 1914 (41015840-CH3


H14

N2

O5




15

H10

N2

O7


3 Biological Assay





















5 Conclusion




Acknowledgment


References























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



H15

NO6



H13

NO5




) 1914 (41015840-CH3


H14

N2

O5




15

H10

N2

O7


3 Biological Assay





















5 Conclusion




Acknowledgment


References























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

















5 Conclusion




Acknowledgment


References























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






5 Conclusion




Acknowledgment


References























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of














Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Documents

)JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/ijmc/2016/9346585.pdfthat Z is the most active against Pseudomonas aeruginosa ( mm) and least active against Candida