Synthesis, characterisation and biological activity of novel 4-thiazolidinones, 1,3,4-oxadiazoles and some related compounds

Article abstract of DOI:10.1016/S0223-5234(01)01326-5

Two novel series of 4-thiazolidinone derivatives namely 2-substituted-3-{[4-(4-methoxybenzoylamino)benzoyl]amino}-4-thiazolidinones (7a-e) and 2-[4-(4-methoxybenzoylamino)benzoylhydrazono]-3-alkyl-4-thiazolidinones (5a-c) together with 2-[4-(4-methoxybenzoylamino)phenyl]-5-(substituted enyl)amino-1,3,4-oxadiazoles (6a-c) have been synthesised as title compounds. N¹-[4-(4-methoxybenzoylamino)benzoyl]-N²-substituted methylene hydrazines (3a-e) and 1-[4-(4-methoxybenzoylamino)benzoyl]-4-substituted phenyl thiosemicarbazides (4a-f) were also prepared and used as intermediate to give the title compounds. All synthesised compounds were screened for their antimycobacterial activity against Mycobacterium tuberculosis H37Rv and antimicrobial activities against various bacteria and fungi. Compounds 7a and 7b were found as the most active derivatives demonstrating 90 and 98 percent inhibition of mycobacterial growth of M. tuberculosis H37Rv in the primary screen at 6.25 μg mL^-1, respectively. However, level II assay revealed that the MIC values were not less than 6.25 μg mL^-1. None of the compounds showed significant antimicrobial activity against the microorganisms used whereas 3a and 7a inhibited the growth of several bacteria and fungi.

Full text of DOI:10.1016/S0223-5234(01)01326-5

European Journal of Medicinal Chemistry 37 (2002) 197–206
www.elsevier.com/locate/ejmech
Original article
Synthesis, characterisation and biological activity of novel
4-thiazolidinones, 1,3,4-oxadiazoles and some related compounds
a
S¸. Gu¨niz Ku¨c¸u¨kgu¨zel a,*, E. Elc¸in Oruc¸ , Sevim Rollas a, Fikrettin S¸ahin b,
c
8
Ahmet Ozbek
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara Uni6ersity, 81010 Haydarpasa, Istanbul, Turkey
b Atatu¨rk Uni6ersity, Biotechnology Application and Research Centre, Erzurum, Turkey
c Department of Microbiology and Clinical Microbiology, Atatu¨rk Uni6ersity, Medical School, Erzurum, Turkey
Received 12 September 2001; received in revised form 23 November 2001; accepted 23 November 2001
Abstract
Two novel series of 4-thiazolidinone derivatives namely 2-substituted-3-{[4-(4-methoxybenzoylamino)benzoyl]amino}-4-thiazo-
lidinones (7a–e) and 2-[4-(4-methoxybenzoylamino)benzoylhydrazono]-3-alkyl-4-thiazolidinones (5a–c) together with 2-[4-(4-
methoxybenzoylamino)phenyl]-5-(substituted phenyl)amino-1,3,4-oxadiazoles (6a–c) have been synthesised as title compounds.
N1-[4-(4-methoxybenzoylamino)benzoyl]-N2-substituted methylene hydrazines (3a–e) and 1-[4-(4-methoxybenzoylamino)benzoyl]-
4-substituted phenyl thiosemicarbazides (4a–f) were also prepared and used as intermediate to give the title compounds. All
synthesised compounds were screened for their antimycobacterial activity against Mycobacterium tuberculosis H37R6 and
antimicrobial activities against various bacteria and fungi. Compounds 7a and 7b were found as the most active derivatives
demonstrating 90 and 98% inhibition of mycobacterial growth of M. tuberculosis H37R6 in the primary screen at 6.25 mg mL−1
,
respectively. However, level II assay revealed that the MIC values were not less than 6.25 mg mL−1. None of the compounds
showed significant antimicrobial activity against the microorganisms used whereas 3a and 7a inhibited the growth of several
´
bacteria and fungi. © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: 4-thiazolidinone; 1,3,4-oxadiazole; hydrazide–hydrazone; thiosemicarbazide; antimycobacterial activity; BACTEC 460 radiometric
system
ene hydrazines and 1-[4-(4-methoxybenzoylamino)-
benzoyl]-4-alkyl–aryl thiosemicarbazides were de-
scribed.
1. Introduction
4-Thiazolidinone derivatives have been demonstrated
to possess antibacterial [1–6], antifungal [2,7–10], anti-
convulsant [11–14], anticancer [15] and antituberculosis
[16,17] activities. Compounds MKT 077 [18] and HP-
236 [19] have been reported as a registered antitumour
and antipsychotic agents, respectively (Fig. 1).
4-Thiazolidinones have been reported as novel inhi-
bitors of the bacterial enzyme Mur B which was a
precursor acting during the biosynthesis of peptidogly-
can [20]. Moreover, anticonvulsant [21,22], antibacterial
[23] and antifungal [24] properties of several N1-[4-(4-
methoxybenzoylamino)benzoyl]-N2-substituted methyl-
* Correspondence and reprints.
E-mail address: gunizkguzel@hotmail.com (S¸.G. Ku¨c¸u¨kgu¨zel).
Fig. 1.
´
0223-5234/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0223-5234(01)01326-5

198
S¸.G. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 37 (2002) 197–206
In view of the above observations, the synthesis of
NMR and EI-mass spectral data. Physical and spectral
data are given Tables 1 and 2.
novel 4-thiazolidinone derivatives starting from either
hydrazide–hydrazones or aroylthiosemicarbazides were
aimed at investigating biological activities of these
compounds.
In the UV spectra of arylthiosemicarbazide deriva-
tives 4e and 4f, which were obtained from the reaction
of the hydrazide 2 with 4-methyl–methoxyphenyl iso-
thiocyanates, two absorption maxima were shown at
205–206 and 292 nm [25]. The 1H-NMR spectra of
compounds 4e and 4f displayed the ꢀCONHNHꢀ,
ꢀCONHNHCSꢀ and ꢀCSNHC6H4ꢀ resonances of
thiosemicarbazides at 10.42–10.41, 9.74–9.68 and
9.62–9.57 ppm as singlets, respectively [26]. The struc-
tures of 4a–d were described previously [24]. The UV
spectra of hydrazide–hydrazone derivatives 3a–e,
which were obtained by the action of appropriate alde-
hydes on the hydrazide 2, exhibited characteristic K
bands arising from chromophoric ꢀCꢁNꢀ group at
312–365 nm [27]. Of these series, only compound 3a
containing a nitro function, showed batochromic shift.
2. Chemistry
Synthetic route to compounds 3a–e, 4a–f, 5a–c,
6a–c, 7a–e are shown in Fig. 2. Ethyl 4-(4-methoxy-
benzoylamino)benzoate (1) and 4-(4-methoxybenzoy-
lamino)benzoyl hydrazine (2) were prepared according
to the method described by Rollas et al. [24]. Conden-
sation of 2 with appropriate aldehydes yielded the
corresponding hydrazide–hydrazones 3a–e, which on
condensation with mercaptoacetic acid afforded 4-thia-
zolidinones 7a–e. Acylthiosemicarbazides 4a–f were
prepared by the addition of 4-(4-methoxybenzoy-
lamino)benzoyl hydrazine (2) to various isothio-
cyanates. Of these compounds, 4a (R=ꢀCH3), 4b
(R=ꢀC2H5), 4c (R=ꢀCH2ꢀCHꢁCH2) and 4d (R=
ꢀC6H5) have been reported elsewhere [24] whereas com-
pounds 4e and 4f were synthesised as novel compounds
in the present study. Compounds 4a–f were reacted
with ethyl bromoacetate in the presence of anhydrous
sodium acetate to give the desired 4-thiazolidinones
(5a–c). However, formation of desired 4-thiazolidi-
nones from (4d–f) failed and instead 1,3,4-oxadiazoles
(6a–c) were obtained (Fig. 2). Structures of these com-
1
In the H-NMR spectra of compounds 3a–e, the sig-
nals representing the azomethine protons appeared at
8.33–8.46 ppm whereas hydrazide–hydrazone NꢀH
protons (ꢀCONHNꢁCHꢀ) resonated at 11.59–12.18
ppm [27,28].
1
The H-NMR spectra of 5a–c, which were obtained
from reaction of 4a–c with ethyl bromoacetate and
anhydrous sodium acetate in absolute ethanol, showed
single N1ꢀH (amide, CONH) and N2ꢀH (4-thiazolidi-
none, CONHNꢁ) resonances at 10.19–10.29 and
10.59–10.71 ppm, respectively. The SꢀCH2 resonances
of 5a–c were observed in the 3.95–4.09 ppm range. In
1
1
pounds were characterised using UV, H-NMR, 13C-
the H-NMR spectra of 6a–c, SꢀCH2 resonances were
Fig. 2. Key: (a) H2NꢀNH2·H2O–EtOH; (b) RꢀCHꢁO–EtOH; (c) RꢀNCS–EtOH; (d) HSꢀCH2ꢀCOOH–benzene; (e) BrꢀCH2ꢀCOOC2H5–
NaOAc–EtOH.

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199

200
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S¸.G. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 37 (2002) 197–206
201
3. Results and discussion
not observed. Moreover, elemental analyses revealed
that 6a–c had no sulfur atom leading to the conclusion
that the structures of these compounds must be diffe-
rent from 4-thiazolidinone.
3.1. In 6itro e6aluation of antimycobacterial acti6ity
against Mycobacterium tuberculosis H37R6
These observations demonstrated that 6a–c are 1,3,4-
oxadiazole. Cesur et al. [29] have been reported pre-
viously a similar case. N1ꢀH (amide, CONH) and N3ꢀH
(1,3,4-oxadiazole, NH) protons of 6a–c were shown at
10.35 and 10.60 (for 6a), 10.04–10.74, 10.11–10.57
ppm (for 6b and 6c), respectively.
The synthesised compounds 3a–e, 4e, 4f, 5a–c, 6a–c
and 7a–e were tested in vitro for their antimycobacte-
rial activity. Primary screen was conducted at 6.25 mg
mL−1 against M. tuberculosis H37R6 in BACTEC 12B
medium using the BACTEC 460 radiometric system
[30]. Rifampin was used as the standard in the tests.
Compounds effecting B90% inhibition in the primary
screen (MIC \6.25 mg mL−1) were not evaluated
further. Compounds demonstrating at least 90% inhibi-
tion in the primary screen were re-tested at lower
concentration in a broth microdilution assay alamar
Blue. The results of antimycobacterial activity studies
are presented in Table 3. The antimycobacterial activity
was observed only for compounds 7a and 7b with 90
and 98% inhibitions at 6.25 mg mL−1, respectively.
Level II assay revealed that the MIC values were not
less than 6.25 mg mL−1. These results failed to provide
evidence for any reliable structure–activity relation-
ships. However, 7a and 7b which had promising an-
timycobacterial properties could be regarded as lead
compounds for further development. Although, all test
compounds were dissolved in DMSO, it was observed
that the solvent showed no activity in these assays at
the level that were used for screening. This was most
probably because compounds were initially dissolved in
DMSO in the minimal volume, then serially diluted
with media to the testing concentration, so there was
1
The H-NMR spectra of 7a–e which were obtained
from reaction of 3a–e with mercaptoacetic acid in dry
benzene showed single N1ꢀH (amide, CONH) and
N2ꢀH (4-thiazolidinone, CONHNꢂ) resonances at
10.21–10.33 and 10.48–10.72 ppm. In the 1H-NMR
spectra, methylene protons of the 4-thiazolidinone ring
displayed two signals appearing as doublets at 3.83–
3.96 and 3.87–4.00 ppm due to the non-equivalent,
geminal methylene protons [10] interacting with the
chiral centre at position 2. This phenomenon was not
observed with 5a–c lacking the asymmetric carbon. The
methin protons of 4-thiazolidinones 7a–e showed reso-
nances at 5.84–6.08 ppm. Methyl protons of the
methoxy function gave a singlet at 3.75–3.85 ppm in
each case.
EIMS of the selected compounds 5b, 6b displayed
molecular ions at m/z 412, 400 which confirmed their
molecular weights, respectively. Molecular ion was not
detected in the mass spectrum of compound 7b. The
fragment ion at m/z 270 peak which formed by the loss
of 195 fragment was observed. The major fragmenta-
tion pathway appeared by cleavage of CH3OꢀC6H4ꢀ
CONHꢀ bonds of amide moiety.
Table 3
Antimycobacterial activity of 5a–c, 6a–c, 7a–e, 3a–e and 4e, 4f
Compound
R
MIC (mg mL−1
)
% Inhibition
Level II
5a
5b
5c
6a
6b
6c
7a
7b
7c
7d
7e
3a
3b
3c
3d
3e
ꢀCH3
ꢀC2H5
\6.25
\6.25
\6.25
\6.25
\6.25
\6.25
B6.25
B6.25
\6.25
\6.25
\6.25
\6.25
\6.25
\6.25
\6.25
\6.25
\6.25
\6.25
0.25
0
0
0
7
0
–
–
–
–
–
–
ꢀCH2ꢀCHꢁCH2
ꢀC6H5
ꢀC6H4ꢀCH3(4)
ꢀC6H4ꢀOCH3(4)
5-nitro-2-furyl
ꢀC6H4ꢀF(4)
ꢀC6H4ꢀCl(4)
ꢀC6H4ꢀBr(4)
ꢀC6H3ꢀOH,OC2H5(4,3)
5-nitro-2-furyl
ꢀC6H4ꢀF(4)
ꢀC6H4ꢀCl(4)
ꢀC6H4ꢀBr(4)
ꢀC6H3ꢀOH,OC2H5(4,3)
ꢀC6H4ꢀCH3(4)
ꢀC6H4ꢀOCH3(4)
–
0
90
98
40
11
0
0
2
3
0
0
0
0
98
\6.25
\6.25
–
–
–
–
–
–
–
–
–
–
–
4e
4f
Rifampin

202
S¸.G. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 37 (2002) 197–206
4. Experimental
very minimal residual DMSO in the assayed medium.
Controls were run in each assay to verify this fact.
4.1. Chemistry
3.2. In 6itro e6aluation of antimicrobial acti6ity
Benzocaine, Et2O, BzCl, ethyl and allyl isothio-
cyanates were purchased from Merck Company. All the
other chemicals used in the experiments were purchased
from Fluka. All m.p. were recorded on a Bu¨chi 530
m.p. apparatus and uncorrected. UV spectra were
recorded on a Shimadzu UV 2100S spectrophotometer
Compounds 3a–e, 4e, 4f, 5a–c, 6a–c and 7a–e were
screened in vitro for their antimicrobial activity against
Acinobacter baumanii (18 strains), A. calcoaceticus (12
strains), A. lwoffii (five strains), A. johnsonii (three
strains), Agrobacterium tumefaciens (10 strains), A. ra-
diobacter (eight strains), A. rubi (four strains), Alcalige-
nes xylosoxydans (2 starins), A. faecalis (two strains),
Bacillus anthracis (two strains), B. amyloeiquefaciens
(six strains), Bacillus anthracis (two strains), B. bre6is
(13 strains), B. cereus (15 strains), B. licheriformis (eight
strains), B. magaterium (three strains), B. popiliae (one
strain), B. subtilis (38 strains), Bre6ibacillus agrii (four
strains), Burkholdria cepecia (15 strains), B. gladioli
(three strains), Cla6ibacter michiganese (two strains),
Curtobacterium flaccumfaciens (one strain), Enterobac-
ter aerogenes (eight strains), E. cloacae (two strains),
Erwinia caroto6ora (three strains), Escherichia coli (21
strains), Edwardsiella spp (four strains), Fla6obacterium
blastinum (four strains), Klebsiella pneumoniae (three
strains), K. oxytoca (14 strains), Neisseria spp (seven
strains), Pantonea agglomerans (18 strains), Pseu-
domonas aeruginosa (18 strains), P. syringae p6s. (52
strains), Proteus mirabilis (seven strains), Serratia lique-
faciens (five strains), Staphylococcus aureus (80 strains),
S. epidermis (43 strains), Stenotrophomonas maltophilia
(46 strains), Streptococcus pneumoniae (10 strains), S.
pyogenes (41 strains), Xanthomonas campestris p6s. (41
strains) for bactericidal and bacteriostatic effects, Al-
ternaria alternata (10 strains), Aspergillus niger (10
strains), Fusarium graminearum (10 strains), Microspo-
rum canis (two strains), Penicillum spp. (10 strains),
Trichoderma spp. (10 strains), Rhizoctonia solani (10
strains), Trichophyton spp. (two strains) at 1 and 0.5 mg
mL−1 using disc-diffusion method [31]. Penicillin, Ce-
faclor, Ceftizoxime, Isepamisin, Clindamycin, Ery-
thromycin, Vancomycin, Teicoplanin, Imipenem,
Meropenem, Ampicillin-sulbactam, Levofloksasin for
gram (+) bacteria and Isepamisin, Amikacin,
Netilmicin, Ceftriaxone, Cefoperazone, Cefepime, Cef-
tizoxime, Trimethoprim-Sulphametoxazol, Imipenem,
Meropenem, Aztreonam, Ampicillin, Ofloxacin for
gram (−) bacteria were used as standards in the tests.
All compounds were found inactive against tested bac-
teria and fungi, whereas 3a and 7a showed varying
degrees of inhibition against several bacteria and fungi.
Compound 3a is more active against gram (+) than
gram (−) bacteria and has a better antibacterial acti-
vity than compound 7a. The results of antimicrobial
activity are given in Tables 4 and 5.
1
(1 mg/100 mL in EtOH). H-NMR spectra were ob-
tained on a Bruker AVANC-DPX 400 instrument. Ele-
mental analyses were performed on a Carlo Erba 1106
instrument and the results were in acceptable range.
4.1.1. Preparation of ethyl
4-(4-methoxybenzoylamino)benzoate (1) and
4-(4-methoxybenzoylamino)benzoyl hydrazine (2)
These compounds were prepared according to the
method described by Rollas et al. [24].
4.1.2. Synthesis of N1-[4-(4-methoxybenzamido)-
benzoyl]-N2-substituted alkylidene hydrazines (3a–e)
and 1-[4-(4-methoxybenzamido)-benzoyl]-4-substituted
phenylthiosemicarbazides (4a–f)
A solution of 0.01 mol of 4-(4-methoxybenzoy-
lamino)benzoyl hydrazine (2) and equimolar amount of
appropriate aldehyde (for 3a–e)/appropriate isothio-
cyanate (for 4a–f) in 100 mL of EtOH was heated
under reflux for 2 h. The precipitate obtained was
filtered-off, washed with water and cleaned twice with
boiling EtOH. Physical and spectral data of 3a–e and
4e, 4f are given in Table 1. Compounds 4a–d were
previously described [24] [4a (R=ꢀCH3) m.p. 234–
235 °C, lit. [24] 227–230 °C; 4b (R=ꢀC2H5) m.p. 230,
lit. [24] 231 °C; 4c (R=ꢀCH2ꢀCHꢁCH2) m.p. 218, lit.
[24] 216–218 °C and 4d (R=ꢀC6H5) m.p. 249–
250 °C, lit. [24] 250 °C].
4.1.3. Synthesis of 2-[4-(4-methoxybenzoylamino)-
benzoylhydrazono]-3-alkyl-4-thiazolidinones (5a–c)
0.01 mol of appropriate thiosemicarbazide 4a–c and
0.011 mol of ethyl bromoacetate were refluxed in 30 mL
of absolute EtOH in the presence of 0.04 mol of
anhydrous NaOAc for 9 h. The reaction mixture was
cooled, diluted with water and allowed to stand
overnight. The solid precipitated was washed with wa-
ter, dried and washed with hot EtOH to give 5a–c.
Analysis for 5b: R: ꢀC2H5. C20H20N4O4S. Molecular
weight: 412.40. m.p. 247–250 °C. Yield: 67%. UV
1
(EtOH) umax (nm) (log m): 296 (4.70), 223 (4.24). H-
NMR (400 MHz, DMSO-d6), ppm: 1.11 (t, 3H,
CH3ꢀCH2ꢀ); 3.67 (q, 2H, CH3ꢀCH2ꢀ); 3.76 (s, 3H,
ꢀOꢀCH3); 3.95 (s, 2H, ꢀSꢀCH2); 6.99–7.89 (m, 8H,
ArꢀH); 10.19 (s, 1H, N1ꢀH); 10.63 (s, 1H, N2ꢀH).
13C-NMR (100.6 MHz, DMSO-d6), ppm: 13.02

S¸.G. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 37 (2002) 197–206
203
Table 4
Antimicrobial activity of 3a–e and 4e, 4f
Number of strains
tested
Bactericidal
effect
Bactericidal
effect
Bacteriostatic
effect
Bacteriostatic
effect
(1 mg mL−1
)
(0.5 mg mL−1
)
(1 mg mL−1
)
(0.5 mg mL−1
)
3a
3a
3a
3a
Acinetobacter baumanii
Acinetobacter calcoaceticus
Acinetobacter lwoffii
Acinetobacter johnsonii
Agrobacterium tumefaciens
Agrobacterium radiobacter
Agrobacterium rubi
Bacillus anthracis
Bacillus amyloeiquefaciens
Bacillus bre6is
18
12
5
3
10
8
4
2
6
13
15
8
3
1
38
4
15
3
2
1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
7–9 mm
7–11 mm
6–8 mm
7–9 mm
7–11 mm
4–7 mm
7 mm
6–13 mm
7–10 mm
–
3–5 mm
5–7 mm
3–5 mm
4–5 mm
–
3–4 mm
4 mm
4–8 mm
4–7 mm
–
Bacillus cereus
Bacillus licheriformis
Bacillus magaterium
Bacillus popilliae
Bacillus substilis
Bre6ibacillus agrii
Burkholdria cepacia
Burkholdria gladioli
Cla6ibacter michiganense
Curtobacterium flaccumfaciens
Enterobacter aerogenes
–
–
3–4 mm
2 mm
–
1–2 mm
–
–
8
2–3 mm/3 strain;
–/5 strain
Enterobacter cloacae
Erwinia caroto6ora
Escherichia coli
2
3
21
–
–
–
–
–
–
–
–
–
–
–
1–2 mm/7 strains;
–/14 strains
Fla6obacterium blastinum
Klebsiella pneumoniae
Neisseria spp
Pantoea agglomerans
Pseudomonas aeruginosa
Pseudomonas syringae p6s.
Staphylococcus aureus
4
3
7
18
14
52
80
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
5–9 mm
3–4 mm
–
–
–
–
–
–
5–9 mm/74 strain;
–/6 strains
4–10 mm
2–4 mm/74 strain;
–/6 strains
2–3 mm
Staphylococcus epidermis
Stenotrophomonas maltophilia
Streptococcus pneumoniae
43
46
10
–
–
–
–
–
–
–
–
3–7 mm/8 strains;
–/2 strains
6–9 mm/35 strains;
–/6 strains
–
1–2 mm/7 strains;
–/3 strains
2–4 mm/31 strains;
–/10 strains
–
Streptococcus pyogenes
41
–
–
–
Xanthomonas campestris p6s.
140
2–4 mm/
22 strains;
–/118 strains
Total
655
–
–
*, For 3b, 3c, 3d and 3e effect bacteriostatic against Escherichia coli: 2–3 mm (1 mg mL−1 concentration); *, for 4e and 4f effect bacteriostatic
against Enterobacter aerogenes, E. coli Streptococcus pyogenes and Xanthomonas campestris: 2–4 mm (1 mg mL−1 concentration).
4.1.4. Synthesis of 2-[4-(4-methoxybenzoylamino)-
phenyl]-5-(substituted phenyl) amino-1,3,4-oxadiazoles
(6a–c)
(NꢀCH2ꢀCH3); 33.1 (SꢀCH2); 38.4 (NꢀCH2ꢀCH3);
56.32 (OCH3);114.53; 120.32; 127.52; 129.06; 130.61;
143.17 (Aromatic C); 156.87 (4-thiazolidinone CꢁN);
163.12 (hydrazone CꢁO); 165.16 (amide CꢁO); 172.5
(4-thiazolidinone CꢁO). EIMS (70 eV, m/z) (%): 412
[M+] (14.74); 338 (3.78); 271 (2.95); 270 (7.95); 254
(14.10); 135 (100); 120 (2.12); 107 (6.38); 92 (7.40).
Compounds 6a–c were obtained from 4d–f as de-
scribed for 4a–c. Analysis for 6b: R: ꢀC6H4ꢀCH3 (4).
C23H20N4O3. Molecular weight: 400.44. m.p. 299–

204
S¸.G. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 37 (2002) 197–206
Table 5
Antimicrobial activity of 5a–c, 6a–c and 7a–e
Number of strains
tested
Bactericidal
effect
Bactericidal
effect
Bacteriostatic
effect
Bacteriostatic
effect
(1 mg mL−1
)
(0.5 mg mL−1
)
(1 mg mL−1
)
(0.5 mg mL−1
)
7a
7a
7a
7a
Acinetobacter baumanii
Acinetobacter calcoaceticus
Acinetobacter lwoffii
Acinetobacter johnsonii
Agrobacterium tumefaciens
Agrobacterium radiobacter
Agrobacterium rubi
Bacillus anthracis
Bacillus amyloliquefaciens
Bacillus bre6is
Bacillus cereus
Bacillus licheriformis
Bacillus magaterium
Bacillus popilliae
18
12
5
3
10
8
4
2
6
13
15
8
3
1
38
4
15
3
2
1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
3–5 mm
5–7 mm
4–7 mm
6–9 mm
5–9 mm
5–7 mm
5 mm
4–8 mm
3–7 mm
–
–
–
–
–
–
–
2–3 mm
3–4 mm
2–3 mm
4–5 mm
–
3–4 mm
3 mm
3–5 mm
2–3mm
–
–
–
–
–
–
–
–
Bacillus substilis
Bre6ibacillus agrii
Burkholdria cepacia
Burkholdria gladioli
Cla6ibacter michiganense
Curtobacterium flaccumfaciens
Enterobacter aerogenes
Enterobacter cloacae
Erwinia caroto6ora
8
2
3
21
Escherichia coli
2–3 mm/
15 strains;
−/6 strains
Fla6obacterium blastinum
Klebsiella pneumoniae
Neisseria spp
4
3
7
18
14
52
80
43
46
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Pantoea agglomerans
Pseudomonas aeruginosa
Pseudomonas syringae p6s.
Staphylococcus aureus
Staphylococcus epidermis
Stenotrophomonas maltophilia
4–7 mm
6–9 mm
–
2–3 mm
2–4 mm
–
2–3 mm/
7 strains;
−/39 strains
–
Streptococcus pneumoniae
10
3–5 mm/
5 strains;
−/5 strains
5–7 mm
–
1–2 mm/
5 strains;
−/5 strains
2–3 mm
–
–
Streptococcus pyogenes
Xanthomonas campestris p6s.
41
140
–
–
–
3–4 mm/
39 strains;
−/101 strains
Total
655
–
–
–
*, Bacteriostatic effect of 5a, 5b, 5c, 6a, 6b and 6c against E. coli and Enterobacter aerogenes: 2–5 mm (1 mg mL−1 concentration); *, bacteriostatic
effect of 7b, 7c, 7d and 7e against E. coli and Xanthomonas campestris: 2–3 mm (1 mg mL−1 concentration); standards used for Gram(+) bacteria:
Penicillin, Cefaclor, Ceftizoxime, Isepamisin, Clindamycin, Erythromycin, Vancomycin, Teicoplanin, Imipenem, Meropenem, Ampicillin-sulbac-
tam, Levofloksasin (5–15 mm zone); standards used for Gram(−) bacteria: Isepamisin, Amikacin, Netilmicin, Ceftriaxon, Cefoperazon, Cefepim,
Ceftizoxime, Trimethoprim-Sulphametoxazol, Imipenem, Meropenem, aztreonam, Ampicillin, Ofloxacin (5–15 mm zone).
303 °C. Yield: 76%. UV (EtOH) umax (nm) (log m): 321
(4.57), 254 (4.23). 1H-NMR (400 MHz, DMSO-d6),
ppm: 2.27 (s, 3H, ꢀC6H4ꢀCH3); 3.85 (s, 3H, ꢀOꢀCH3);
7.07–8.01 (m, 12H, ArꢀH); 10.04–10.74 (d, 2H, N1ꢀH
and N3ꢀH). 13C-NMR (100.6 MHz, DMSO-d6), ppm:
21.18 (C6H4ꢀCH3); 56.31 (OCH3);114.53; 117.92;
119.43; 121.24; 127.03; 127.47; 130.33; 130.62; 131.52;
137.08 (Aromatic C); 142.62; 158.43 (oxadiazole CꢁN);
166.07 (amide CꢁO). EIMS (70 eV, m/z) (%): 400 [M+]
(10.00); 135(100); 107 (9.50).

S¸.G. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 37 (2002) 197–206
205
4.1.5. Synthesis of 2-substituted-3-{[4-(4-methoxy-
benzoylamino)benzoyl]amino}-4-thiazolidinones (7a–e)
A mixture of 3a–e (0.01 mol) and mercaptoacetic
acid (0.01 mol) was refluxed in dry C6H6 (50 mL) using
a Dean–Stark water separator. Excess C6H6 was evapo-
rated in vacuo. The resulting residue was triturated
with saturated NaHCO3 solution until CO2 evolution
ceased. The solid was washed with water, dried and
recrystallised from ethanol–water. Anal. for 7b: R:
ꢀC6H4F(4). C24H20FN3O4S. Molecular weight: 465.50.
m.p. 257–258 °C. Yield: 74%. UV (EtOH) umax (nm)
(log m): 293 (4.53), 222 (4.06). 1H-NMR (400 MHz,
DMSO-d6), ppm: 3.76 (s, 3H, ꢀOꢀCH3); 3.83, 3.87 (2d
and each 1H, ꢀSꢀCH2, J=15.9 Hz); 5.86 (s, 1H,
ꢀNꢀCHꢀSꢀ); 6.98–7.87 (m, 12H, ArꢀH); 10.21 (s, 1H,
N1ꢀH); 10.48 (s, 1H, N2ꢀH). 13C-NMR (100.6 MHz,
DMSO-d6), ppm: 30.19 (SꢀCH2); 56.32 (OCH3); 61.94
(SꢀCHꢀN); 114.52; 116.20; 116.41; 120.24; 126.70;
127.36; 129.26; 130.03; 130.89; 130.98; 135.45; 143.90
(Aromatic C); 162.99 (hydrazone CꢁO); 166.06 (amide
CꢁO); 170.04 (4-thiazolidinone CꢁO). EIMS (70 eV,
m/z) (%): 270 [M+−195] (2.53); 254 (2.33); 197 (3.92);
150 (3.20); 135 (100); 123 (2.93); 122 (12.26); 120 (3.73);
107 (5.86); 92 (7.07).
at least 30, the increase in GI (DGI) from the previous
day in the control was compared with that in the drug
vial. The following formula was used to interpret
results:
DGI control\DGI drug=susceptible
DGI controlBDGI drug=resistant
If a clear susceptibility pattern (the difference of DGI
of control and the drug bottle) was not seen at the time
the control GI is 30 the vials were read for 1 or 2
additional days to establish a definite pattern of DGI
differences.
4.2.2. Antimicrobial acti6ity
In the present study, disc-diffusion method was
utilised for the determination of antibacterial and anti-
fungal activities of the synthesised compounds. What-
man filter paper discs were impregnated with test
compounds, dissolved in DMSO at 0.5 and 1.0 mg
mL−1. Tryptic Soy Agar (TSA) and Potato Dextrose
Agar (PDA) were used as growth media for bacterial
and fungal strains, respectively. The bacteria to be
tested were initially grown as 24–48 h fresh cultures in
TSA. Each bacterial strain was then suspended in 0.1
M buffer; diluted to an inoculum size to have an optical
density of 0.1 at 600 nm (ca. 108 CFU mL−1) and 0.1
mL of these suspensions were disseminated onto plates
containing TSA. After drying the suspensions keeping
the plates in a sterilizer, paper discs impregnated with
test compounds were placed onto plates maintaining
equal distance between each disc (eight discs/plate).
Following incubation of the plates at 26 and 30 °C for
24–48 h, inhibition zones surrounding the test discs
were measured. Test plates were then followed up for
further 7 days in order to determine whether any
compound was bactericidal or bacteriostatic.
For the determination of antifungal activity, fungal
isolates were cultivated in PDA for 4–10 days. A
micelle disc, obtained from pure fungal culture, at the
centre of test plates and discs containing the test com-
pounds at the margins in equal distance from micelle
disc were placed. These plates were then incubated at
25 °C for 4–10 days. Antifungal activity of the test
compounds was evaluated measuring inhibition zones
when fungal micelles covered up the plate surface.
Paper discs with only DMSO were used as negative
controls. In all determinations, tests were performed
duplicate and the results were reported as mean of these
values.
4.2. Microbiology
4.2.1. In 6itro e6aluation of antimycobacterial acti6ity
against M. tuberculosis H37R6
Primary screen was conducted at 6.25 mg mL−1
against M. tuberculosis H37R6 in BACTEC 12B
medium using the BACTEC 460 radiometric system
[30]. Compounds effecting B90% inhibition in the
primary screen (MIC \6.25 mg mL−1) were not evalua-
ted further. Compounds demonstrating at least 90%
inhibition in the primary screen were re-tested at lower
concentration (MIC) in a broth microdilution assay
alamar Blue. The MIC was defined as the lowest con-
centration inhibiting 99% of the inoculum.
4.2.1.1. BACTEC radiometric method of susceptibility
testing. Inocula for susceptibility testing were either
from a positive BACTEC isolation vial with a growth
index (GI) of 500 or more, or a suspension of orga-
nisms isolated earlier on a conventional medium.
The culture was well mixed with a syringe and 0.1
mL of a positive BACTEC culture was added to each
of the vials containing the test drugs. The drug vials
contained rifampin (0.25 mg mL−1). A control vial was
inoculated with a 1:100 dilution of the culture. A
suspension equivalent to a Mc Farland No. 1 standard
was prepared in the same manner as a BACTEC posi-
tive vial, when growth from a solid medium was used.
Each vial was tested immediately on a BACTEC
instrument to provide CO2 in the headspace. The vials
were incubated at 37 °C and tested daily with a
BACTEC instrument. When the GI in the control reads
Acknowledgements
This work was supported by the Research Fund of
Marmara University, project number: 1998/62. We

206
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