Synthesis and structure-activity relationships of new antimicrobial active multisubstituted benzazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2003.11.014

A series of multisubstituted benzoxazoles, benzimidazoles, and benzothiazoles (5-7) as non-nucleoside fused isosteric heterocyclic compounds was synthesized and tested for their antibacterial activities against various Gram-positive and Gram-negative bacteria and antifungal activity against the fungus Candida albicans. Microbiological results indicated that the synthesized compounds possessed a broad spectrum of activity against the tested microorganisms at MIC values between 100 and 3.12 μg/ml. Structure-activity relationships (SAR) studies revealed that benzothiazole ring system enhanced the antimicrobial activity against Staphylococcus aureus. In these sets of non-nucleoside fused heterocyclic compounds electron withdrawing groups at position 5 of the benzazoles increased the activity against C. albicans.

Full text of DOI:10.1016/j.ejmech.2003.11.014

European Journal of Medicinal Chemistry 39 (2004) 291–298
www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis and structure–activity relationships of new antimicrobial active
multisubstituted benzazole derivatives
IlkayYildiz-Oren a, IsmailYalcin a, Esin Aki-Sener a,*, Nejat Ucarturk b
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Tandogan, 06100 Ankara, Turkey
b Department of Microbiology, Faculty of Pharmacy, Ankara University, Tandogan, 06100 Ankara, Turkey
Received 27 May 2003; received in revised form 24 November 2003; accepted 26 November 2003
Abstract
A series of multisubstituted benzoxazoles, benzimidazoles, and benzothiazoles (5–7) as non-nucleoside fused isosteric heterocyclic
compounds was synthesized and tested for their antibacterial activities against various Gram-positive and Gram-negative bacteria and
antifungal activity against the fungus Candida albicans. Microbiological results indicated that the synthesized compounds possessed a broad
spectrum of activity against the tested microorganisms at MIC values between 100 and 3.12 µg/ml. Structure–activity relationships (SAR)
studies revealed that benzothiazole ring system enhanced the antimicrobial activity against Staphylococcus aureus. In these sets of
non-nucleoside fused heterocyclic compounds electron withdrawing groups at position 5 of the benzazoles increased the activity against
C. albicans.
© 2003 Elsevier SAS. All rights reserved.
Keywords: Synthesis; Antimicrobial activity; Benzoxazole; Benzimidazole; Benzothiazole
1. Introduction
activity was inferior to the quinolone type antibacterial
agents such as norfloxacin or fleroxacin [2]. Moreover, a
The number of cases of multidrug resistant bacterial infec-
tions is increasing at an alarming rate. As well as the clini-
cians have become reliant on vancomycin as the antibiotic
for serious infections resistant to traditional agents [1] there
is still need for the new classes of antibacterial agents.
Substituted benzimidazoles, benzoxazoles, and ben-
zothiazoles are found to be associated with various chemo-
therapeutical activities such as antibacterial, antifungal, anti-
tumor, antiviral activities [2–9]. A series of 5-formyl-,
5-(aminocarbonyl)-, or 5- and 6-nitro derivatives of 2-(4-
methoxyphenyl)benzimidazoles, benzoxazoles and ben-
zothiazoles were synthesized and determined as topoi-
somerase I inhibitors [4]. In evaluating their cytotoxicity,
these new topoisomerase I poisons exhibited no significant
cross-resistance against cell lines and indicated minimum or
no DNA binding affinity. Substituted pyrimido[1,6-
a]benzimidazoles were also synthesized as a new class of
potent DNA gyrase inhibitors; however, their antibacterial
benzoxazole
derivative,
3-(4,7-dichlorobenzoxazol-2-
(L-
ylmethylamino)-5-ethyl-6-methylpyridin-2(1H)-one
697,661), was observed as a specific non-nucleoside reverse
transcriptase inhibitor for the human immunodeficiency vi-
rus HIV-1 type and combined therapy with zidovudine and
L-697,661 achieved a marked decrease of viraemia in some
primary HIV infected patients [3,10,11].
Additionally, a derivative of campotothecin having a ben-
zoxazole ring system within its structure was found to be
significantly more potent than campotothecin as inhibitors of
topoisomerase I [12].
Currently, a new series of benzothiazoles have been syn-
thesized as antitumor agents and showed potent inhibitory
activity against human breast cancer cell lines in vitro and in
vivo [6]. Among them, lysyl-amide of 2-(4-amino-3-
methylphenyl)-5- fluorobenzothiazole had been selected for
phase 1 clinical evaluation [13].
UK-1 is a unique natural anticancer active product having
bis(benzoxazole) structure, which is isolated from a strain of
Streptomyces sp. 517-02 [14]. Its semisynthetic derivatives
such as methyl UK-1 (MUK-1) and dimethyl UK-1 (DMUK-
* Corresponding author.
E-mail address: sener@pharmacy.ankara.edu.tr (N. Ucarturk).
© 2003 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2003.11.014

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I. Yildiz-Oren et al. / European Journal of Medicinal Chemistry 39 (2004) 291–298
1) both have activity against Gram-positive and Gram-
negative bacteria [15,16].
siella pneumoniae, Pseudomonas aeruginosa as Gram-
negative bacteria and the yeast Candida albicans by using
twofold serial dilution technique and compared to ampicillin,
amoxycillin, tetracycline, streptomycin, clotrimazole and
haloprogin as standard drugs. All the biological results of the
compounds were given in Table 2. The combined data re-
ported that the compounds were able to inhibit the in vitro
growth of screened microorganisms showing MIC values
between 100 and 3.12 µg/ml.
Among the synthesized compounds, 2-(phenoxy-
methyl)benzothiazole 6a was found the most active deriva-
tive at an MIC value of 3.12 µg/ml against S. aureus. The
derivatives 5c, 5o, 6a-c, 7b, 7g, 7h, and 7p exhibited
significant antibacterial activity with MIC values of 25 µg/ml
for the Gram-negative enterobacter P. aeruginosa, which is
effective in nosocomial infections and often resistant to anti-
biotic therapy, providing higher potencies than the compared
standard drugs.
In the last few years, we reported the synthesis of several
2,5-disubstitutedbenzoxazole and benzimidazole derivatives
as the antimicrobial agents [17–25]. The structure–activity
relationships (SAR) of previously synthesized compounds
indicated that the fused heterocyclic nucleus was important
for the antimicrobial activity.
The goal at the outset of this research was to develop more
effective antimicrobial analogues of 2,5,6-trisubstituted ben-
zoxazole, benzimidazole, and benzothiazole. The strategy
employed was to examine the effect of the isosteric hetero-
cyclic nucleus and the positions of 2, 5 and 6 against some
Gram-positive and Gram-negative bacteria and the fungus
C. albicans. Therefore, in this study, antimicrobial activity
and the SAR of a series of newly (5–7) and previously
(10–12) [26,27] synthesized multisubstituted benzoxazoles,
benzimidazoles, benzothiazoles, and oxazolo(4,5-b)pyri-
dines were described in order to reveal the lead optimization
against the tested various bacteria and the fungus C. albi-
cans.
Table 2 indicated that thiazolo ring instead of oxazolo and
imidazolo moieties in the fused heterocyclic system im-
proved the potency as threefold against S. aureus (com-
pounds 6a–c). Substitution of Z with -NH-group reduced the
antibacterial activity against S. feacalis (compounds 7n and
7o).
2. Chemistry
When a nitro group was attached at position 5 of the fused
heterocyclic system caused twofold better potency against
E. coli. However, holding a carboxylmethyl group on posi-
tion R1 or possessing a pyridine ring as a six membered
moiety at the fused ring system slightly reduced the activity
against K. pneumoniae. It should be pointed out that besides
benzothiazole derivatives and the compounds having a chlo-
rine atom on position R1 of benzoxazole and benzimidazole
rings were also performed better activity against the enteric
Gram-negative rod P. aeruginosa.
On the other hand, the SAR results against C. albicans
revealed that the electron withdrawing groups such as chlo-
rine or nitro attached at position 5 of the heterocyclic nucleus
increased the potency. Besides, the bridge groups such as
thiomethylene or ethyl between benzazole and phenyl en-
hanced the activity against the same fungus.
As a result, we could point out while benzothiazole as a
nucleus in these series increased the activity against S. au-
reus, amino methyl group as a bridge element between fused
heterocyclic ring and phenyl decreased the activity against
S. faecalis. In these sets of non-nucleoside fused heterocyclic
compounds electron withdrawing groups at position 5 of the
fused ring system increased the activity against C. albicans.
These observations provide some predictions in order to
design further antimicrobial active compounds prior to their
synthesis following with molecular modeling studies.
The general synthetic procedure was employed to prepare
compounds 5a–o (except 5e,5j), 6a-c, and 7a-p (except 7e,
7i, 7m) (Table 1) involving the reaction of the appropriate
carboxylic acids 1a/b/c/d or e with suitable 4,5,6-trisubsti-
tuted anilines 2, 3, 4 by refluxing in the presence of dehy-
drating agents in one step procedure as shown in Scheme 1.
For the preparation of the compounds 5 and 6, trimethylsilyl
polyphosphate ester (PPSE) was used as the cyclodehydra-
tion reagent in the ring closure reactions [28]. During the
synthesis of benzimidazole derivatives 7a-p (except 7e, 7i,
7m) aqueous hydrochloric acid was used as the condensation
reagent according to well-known Philips’ method [29].
Preparation of the compounds 5e, 5j, 5p, 7e, 7i, and 7m,
which are holding 5- carboxylmethyl group on benzoxazole
and benzimidazole ring systems, was carried out as outlined
in Scheme 2. The primarily synthetic steps involved the
methyl esterification of 3-amino-4-hydroxybenzoic acid 8a
and 3,4-diaminobenzoic acid 8b to obtain the compounds 9a,
b. After the treatment of 9a or b with 1a/b or c in PPSE, 5-
carboxylmethyl-2-substitutedbenzoxazoles 5e, 5j, 5p and
benzimidazoles 7e,7i, 7m were obtained.
The chemical, physical and spectral data of the newly
synthesized compounds 5, 6, 7 are reported in Table 1.
3. Results and discussion
4. Experimental protocols
The minimum inhibitory concentrations (MIC) of the
newly and previously synthesized compounds determined
against Staphylococcus aureus, Streptococcus faecalis, Ba-
cillus subtilis as Gram-positive and Escherichia coli, Kleb-
4.1. Chemistry
Unless otherwise noted, materials were obtained from
commercially suppliers and used without further purification.

I. Yildiz-Oren et al. / European Journal of Medicinal Chemistry 39 (2004) 291–298
293
Table 1
Physical, preparation, and spectral data of the synthesized compounds 5–7
Compound Y
number
Z
R
R1
R2
Mp (°C)
Yield Reaction Reaction
IR (cm–1
)
1H-NMR d ppm(J = Hz)
(recrystallization (%)
time (h) temperature
(°C)
solvent) a
5a
5b
O
O
O
O
H
H
H
H
H
146–147 (154–
156) [30] (A)
49
22
1.5
140
3100, 2940, 1605–
1500, 1455–1390,
7.70–7.20 (m, 9H, C-4 H, C-5 H, C-6 H,
C-7 H and phenyl protons), 5.20 (s, 2H,
1300–1045, 950–700 CH2)
NO2
134 (B)
3
120
3120, 2950, 1630–
1595, 1533, 1458,
1345, 1230–1060,
950–695
8.58 (d, IH, C-4 H, J4,6 = 3.5 Hz),
8.20 and 8.45 (dd, 1H, C-6 H,
J6,7 = 8.8 Hz, J6,4 = 3.5 Hz), 7.60 (d, IH,
C-7 H, J7,6 = 8.8 Hz), 7.40–6.80 (m, 5H,
phenyl protons
5c
O
O
O
O
H
H
Cl
H
H
86 (85–88) [31] 17
(C)
3
2
140
160
3110, 2975, 1610–
1500, 1460–1390,
1295–1050, 945–695
3110, 2890, 1630–
1585, 1525, 1458,
1348, 1270–1060,
955–700
d 7.50–7.00 (m, 8H, C-4 H, C-6 H, C-7
H and phenyl protons), 5.20 (s, 2H, CH2)
5d
NO2 138–139 (D)
42
8.50–7.50 (m, 3H, C-4 H, C-5 H, and C-7
H), 7.40–6.80 (m, 5H, phenyl protons),
5.37 (s, 2H, CH2)
5e
O
O
H
COOCH3 H
121–122 (B)
57
3
110
3100, 2995–2900,
1735, 1630–1500,
1440, 1250, 1230–
1050, 985–690
8.46–8.43 (d, 1H, C-4 H, J4,6 = 1.60 Hz),
8.19–8.06 (dd, 1H, C 6 H, J6,7 = 8.80 Hz,
J6,4 = 1.60 Hz), 7.64–7.52 (dd, 1H, C-7
H, J7,6 = 8.80 Hz), 7.40–6.90 (m, 5H,
phenyl protons), 5.34 (s, 2H, CH2), 3.95
(s, 3H, CH3)
5f
O
O
O
O
Cl H
H
H
81–82 (85–86)
[31] (B)
56
74
2.5
2.5
110
110
3120, 2960–2900,
1630–1585, 1460–
1500, 1245–1042,
949–660
d 7.80–7.21 (m, 6H, C-4 H, C-5 H, C-6
H, C-7 H, C-3′ H and C-5′ H), 7.04–6.95
(dd, 2H, C-2′ H and C-6′ H and C-6′ H,
J2′,3′ and J6′,5′ = 9.2 Hz), 5.29 (s, 2H,
CH2)
8.60 (d, 1H, C-4 H, J4,6 = 2.40 Hz), 8.40–
8.20 (dd, 1H, C-6 H, J6,7 = 8.96 Hz,
J6,4 = 2.40 Hz), 7.65 (d, 1H, C-7 H,
J7,6 = 8.96 Hz), 7.35–7.23 (dd, 2H, C-3′
H and C-5′ J3′,2′ and J5′,6′ = 9.12 Hz),
7.00–6.90 (dd, 2H, C-2′ H and C-6′ H,
J2′,.3′ and J5′,6′ = 9.12 Hz), 5.34 (2H, s)
5g
Cl NO2
140–141 (D)
3110, 2960–2890,
1630–1590, 1538,
1440, 1350, 1240–
1050, 940–660
5h
O
O
Cl H
NO2 147–148 (A)
23
5
130
3148, 2950–2880,
1635–1585, 1525,
1435, 1340, 1235–
1065, 960–665
8.49–8.46 (d, 1H, C-7 H, J7,5 = 2.24 Hz),
8.40–8.20 (dd, 1H, C-5 H,
J5,4 = 8.76 Hz, J5,7 = 2.24 Hz), 7.90–7.80
(d, 1H, C-4 H, J4,5 = 8.76 Hz), 7.35–7.23
(dd, 2H, C-3′ H and C-5′, J3′,2′ and
J5′,6′ = 9.26 Hz), 7.03–6.90 (dd, 2H, c-2′
H and C-6′ H, J2′,6′ and J6′,5′ = 9.26 Hz),
5.35 (2H, s)
5i
5j
O
O
O
O
Cl Cl
NO2 88–89 (B)
39
48
3
4
120
150
3130, 2940–2900,
1600–1580, 1540,
1448, 1350, 1285–
1080, 965–660
8.10 (s, 1H, C-7 H), 7.90 (s, 1H, C-4 H),
7.34–7.23 (dd, 2H, C-3′ H and C-5′ H,
J3′,2′ and J5′,6′ = 9.26 Hz), 7.00–6.90 (dd,
2H, C-2′ H and C-6′ H, J2′,3′ and J5′,6′
9.26 Hz), 5.34 (s, 2H, CH2)
8.46–8.43 (d, 1H, C-4 H, J4,6 = 1.60 Hz),
8.19–8.07 (dd, 1H, C-6 H,
J6,7 = 8.60 Hz, J6,4 = 1.60 Hz), 7.63–7.52
(dd, 1H, C-7 H, J7,6 = 8.60 Hz) 7.33–
7.21 (dd, 2H, C-3′ H and C-5′ H, J3′,2′
and J5′,6′ = 9.26 Hz), 7.03–6.90 (dd, 2H,
C-2′ H and C-6′ H, J2′,3′, and
J6′,5′ = 9.26 Hz), 5.29 (s, 2H, CH2) 3.95
(s, 3H, CH3)
Cl COOCH3 H
145–146 (B)
3130, 2990–2880,
1743, 1630–1500,
1440, 1250, 1210–
1045, 940–660
5k
O
O
S
S
H
H
H
H
H
37–38 (37–38)
[32] (D)
33
30
3
3
140
110
3100, 2995–2880,
1620–1575, 1460,
7.60–7.20 (m, 9H, C-4 H, C-5 H, C-6 H,
C-7 H and phenyl protons) 4.27 (s, 2H,
1250–1020, 950–690 CH2)
5m
NO2
53–54 (B)
3120, 2925, 1630–
1575, 1531, 1490–
1445, 1345, 1260–
1070, 970–700
8.50 (d, 1H, C-4 H, J4,6 = 2.08 Hz), 8.30–
8.20 (dd, 1H, C-6 H, J6,7 = 8.90 Hz,
J6,4 = 2.08 Hz), 7.60–7.50 (d, 1H, C-7 H,
J7,6 = 8.90 Hz), 7.46–7.22 (m, 5H, phe-
nyl protons), 4.33 (s, 2H, CH2)
5n
O
S
H
H
NO2 57–58 (D)
32
2.5
115
3140, 2940, 1630–
1579, 1535, 1425,
1345, 1275–1060,
960–695
8.30–7.80 (m, 2H, C-5 H and C-7 H), 7.60
(d, 1H, C-4 H, J4,5 = 9.10 Hz), 7.39–7.20
(m, 5H, phenyl protons), 4.33 (s, 2H,
CH2)
(continued on next page)

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I. Yildiz-Oren et al. / European Journal of Medicinal Chemistry 39 (2004) 291–298
Table 1
(continued)
Compound Y
number
Z
S
R
H
R1
Cl
R2
Mp (°C)
Yield Reaction Reaction
IR (cm–1
)
1H-NMR d ppm(J = Hz)
(recrystallization (%)
time (h) temperature
(°C)
solvent) a
5o
O
NO2 78–79 (B)
36
50
2.5
130
3125, 2970, 1630–
1570, 1540, 1445,
1345, 1260–1000,
960–695
3120, 2995–2880,
1735, 1625–1585,
1430, 1260, 1220–
1090, 975–690
8.05 (s, 1H, C-7 H), 7.80 (s, 1H, C-4 H),
7.40–7.20 (s, 5H, phenyl protons), 4.30 (s,
2H, CH2)
5p
O
S
H
H
COOCH3 H
59–60 (B)
3
110
8.37–8.34 (dd, 1H, C-4 H,
J4,6 = 1.60 Hz), 8.14–8.01 (dd, 1H, C-6
H, J6,7 = 8.48 Hz, J6,4 = 1.60 Hz), 7.50–
7.10 (m, 6H, C-7 H and phenyl protons),
4.31 (s, 2H, CH2), 3.94 (s, 3H, CH3)
6a
6b
S
S
O
O
H
H
H
77–78 (82–83)
[33] (B)
72
47
3
4
140
140
3100, 2950, 1605–
1500, 1445, 1260–
1058, 940–670
3100, 2950, 1605–
1500, 1445, 1260–
1050, 820–658
8.00–7.80 (m, 2H, C-4 and (C-7 H), 7.40–
6.90 (m, 7H, C-5 H, C-6 H and phenyl
protons), 5.47 (s, 2H, CH2)
8.09–7.39 (m, 4H, C-4 H, C-5 H, C-6 H
and C-7 H), 7.30–7.20 (dd, 2H, C-3′ H
and C-5′ H, J3′,2′ and J5′,6′ = 9.11 Hz)
6.90 (dd, 2H, C-2′ H and C-6′ H, J2′,3′
and J6′,5′ = 9.11 Hz) 5.45 (s, 2H, CH2)
Cl H
95–96 (83–86)
[34] (118) [35]
(B)
6c
7a
7b
7c
S
S
H
H
H
H
H
H
H
H
H
43–44 (41–42)
[33] (B)
43
94
50
37
3.5
2.5
5
140
100
100
100
3100, 2940, 1583–
1480, 1435, 1250–
1030, 935–695
3095–2700, 1605–
1500, 1449, 1340–
1020, 922–695
3100–2700, 1600–
1500, 1450, 1350–
1035, 925–695
3340, 3100, 2950,
1630–1555, 1510,
1470, 1345, 1250–
1050, 1000–655
7.90–7.10 (m, 9H, C-4 H, C-5 H, C-5 H,
C-6 H, C-7 H and phenyl protons), 4.46
(s, 2H, CH2)
7.67–6.99 (m, 9H, C-4 H, C-5 H, C-6 H,
C-7 H and phenyl protons), 5.35 (s, 2H,
CH2)
NHO
NHO
NHO
H
159–161 (160–
161) [35] (A)
Cl
128–129 (129)
[36] (A)
7.60–6.90 (m, 8H, C-4 H, C-6 H, C-7 H
and phenyl protons), 5.29 (s, 2H, CH2)
NO2
185–186 (186–
187) [36] (A)
2
8.20–7.60 (m, 2H, C-4 H and C-6 H)
7.30–6.80 (m, 6H, C-7 H and phenyl pro-
tons), 5.20 (s, 2H, CH2)
7d
7e
NHO
NHO
H
H
CH3
H
167–168 (170)
[36] (A)
19
32
5
3
100
120
3080–2680, 1600–
1500, 1460, 1310–
1030, 945–695
3600, 3080, 2980–
2880, 1729, 1630–
1500, 1440, 1240,
1180–1040, 1000–695
7.60–6.90 (m, 8H, C-4 H, C-6 H, C-7 H
and phenyl protons), 5.30 (s, 2H, CH2),
2.44 (s, 3H, CH3).
8.20–7.00 (m, 8H, C-4 H, C-6 H, C-7 H
and phenyl protons), 4.60 (s, 2H, CH2),
3.90 (s, 3H, CH3)
COOCH3 H
82–83
7f
NHO Cl H
NHO Cl Cl
NHO Cl CH3
H
H
H
178 (178–180)
[37] (D)
29
35
14
56
20
19
14
3.5
100
100
100
130
3100–2700, 1600–
1495, 1440, 1355–
1055, 825–645
3120–2650, 1620–
1495, 1440, 1350–
1010, 925–660
3100–2700, 1630–
1500, 1460, 1300–
1020, 970–650
3370, 3100, 2980–
2870, 1712, 1630–
1500, 1440, 1365,
1320–1040, 975–670
7.60–6.80 (m, 8H, C-4 H, C-5 H, C-6 H,
C-7 H and phenyl protons), 5.36 (s, 2H,
CH2)
7.70 6.90 (m, 7H, C-4 H, C-7 H and phe-
nyl protons), 5.29 (s, 2H, CH2)
7g
7h
7i
182–183 (186)
[36] (A)
150–151(148)
[36] (E)
7.42–6.54 (m, 7H, C-4 H, C-6 H, C-7 H
and phenyl protons), 5.31 (s, 2H, CH2),
2.45 (s, 3H, CH3)
7.88–7.00 (m, 7H, C-4 H, C-6 H, C-6 H,
C-7 H and phenyl protons), 5.30 (s, 2H,
CH2), 3.90 (s, 3H, CH3)
NHO Cl COOCH3 H
97–98
7j
NHS
NHS
H
H
H
H
H
132–133 (135–
136) [32] (A)
40
28
4
5
115
100
3100, 3000–2500,
1600–1520, 1440,
7.50–7.07 (m, 9H, C-4 H, C-5 H, C-6 H,
C-7 H and phenyl protons), 4.36 (s, 2H,
1300–1020, 1020–670 CH2)
7k
NO2
58–60 (A)
3400–2800, 1630–
1590, 1475, 1345,
1200–1020, 950–695 (m, 6H, C-7 H and phenyl protons), 4.38
(s, 2H, CH2)
8.40 (d, 1H, C-4 H), 7.60 (dd, 1H, C-6 H,
J6,7 = 7.2 Hz, J6,4 = 2.32 Hz), 7.40–7.10
7m
NHS
H
COOCH3 H
142–143
24
2
120
3100–2600, 1730,
1630–1510, 1440,
1320, 1250–1025,
970–700
8.19–7.30 (m, 8H, C-4 H, C-6 H, C-7 H
and phenyl protons), 4.70 (s, 2H, CH2),
3.95 (s, 3H, CH3)
7n
7o
7p
NHNHH
H
H
H
H
159–160 (163)
[38] (D)
47
41
17
40
60
4
100
100
100
3490, 3100–2650,
d 7.50–6.30 (m, 9H, C-4 H, C-5 H, C-6
1600–10 500, 1420,
H, C-7 H and phenyl protons), 4.54 (s,
1340–1060, 1000–695 2H, CH2)
NHNHH CH3
NHCH2H Cl
76–77 (F)
3340, 3100–2800,
1600–1510, 1450,
1315–1015, 940–695 2.40 (s, 3H, CH3)
3150–2700, 1635–
1500, 1440, 1335–
1020, 930–700
7.60–6.50 (m, 8H, C-4 H, C-6 H, C-7 H
and phenyl protons), 4.51 (s, 2H, CH2),
123–124 (D)
7.50–6.90 (m, 8H, C-4 H, C-6 H, C-7 H
and phenyl protons), 3.19 (s, 4H, CH2)
a (A), EtOH/H2O; (B), CH2Cl2/hexane; (C), diethyl ether/petroleum ether; (D), benzene/petroleum ether; (E), MeOH/H2O; (F), CHCl3/hexane.

I. Yildiz-Oren et al. / European Journal of Medicinal Chemistry 39 (2004) 291–298
295
Scheme 1
The reaction mixtures were protected from moist air by means
of a calcium chloride drying tube and stirred magnetically.
The compounds 5, 6, and 7 were synthesized as new products
except 5a, 5c, 5f, 5k, 6a–c, 7a–d, 7f–7h, and 7j [30–38].
The structures of known compounds were supported only by
1H-NMR spectral and elemental analyses data that are in
agreement with the proposed structures. All TLC was run on
Kieselgel HF254 chromatoplates (0.3 mm) with a fluorescent
indicator, employing variety of solvents for routine monitor-
ing of reaction mixtures and confirming the homogeneity of
analytical samples. Merck silica gel (230–400 mesh) and
CHCl3 as a solvent were used for flash column chromato-
graphic separations. Melting points were obtained on a Mel-
Temp (Buchi SMP 20) capillary melting point apparatus and
are uncorrected. IR spectra were recorded on Pye Unicam
1
SP-1025 with KBr pellets. H-NMR spectra were obtained
with a Bruker AC 80 MHz spectrometer and TMS was used
as an internal standard. Elemental analyses were carried out
with a Perkin-Elmer model 240-C apparatus. The results of
the elemental analyses (C, H, N) were within 0.4% of the
calculated amounts.
4.1.1. Synthesis of methyl 3-amino-4-hydroxybenzoate (9a)
and methyl 3,4-diaminobenzoate (9b)
For the esterification, dry HCl treated over 30 ml MeOH
until the weight increased 10%, and the compounds 8a or b
(3 g, 19.6 or 19.7 mmol) were added to the mixture and
heated under reflux with stirring for 4 h [39]. The mixture
was dissolved by adding water and the crude product 9a or b
was precipitated after the reaction mixture was neutralized
with K2CO3. The precipitate was filtered, washed with water
Scheme 2

296
I. Yildiz-Oren et al. / European Journal of Medicinal Chemistry 39 (2004) 291–298
Table 2
Antimicrobial activity results (MIC, µg/ml) of the newly (5–7) and previously (10–12) [26,27] synthesized compounds with the standard drugs
Microorganisms a
Gram-positive
Gram-negative
Fungus
C.a.
25
25
25
25
25
25
25
25
25
25
12.5
12.5
12.5
12.5
12.5
50
50
25
25
12.5
12.5
25
25
25
12.5
25
25
12.5
12.5
12.5
25
25
25
25
25
Compound number
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5m
5n
5o
5p
6a
6b
6c
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7m
7n
7o
7p
10a
10b
10c
10d
10e
10f
10g
10h
10i
11a
11b
12a
12b
12c
X
Y
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
Z
R
R1
H
NO2
Cl
R2
H
H
H
NO2
H
H
H
NO2
NO2
H
H
H
NO2
NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
S.a.
25
25
25
25
25
50
50
50
50
50
50
50
50
50
50
3.12
6.25
6.25
25
25
25
50
25
50
50
50
50
50
50
50
50
50
25
25
25
25
50
50
50
50
25
50
50
100
50
50
25
1.56
1.56
1.56
3.12
–
S.f.
50
50
50
100
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
100
100
50
25
50
50
50
50
50
50
25
50
50
50
50
50
50
1.56
1.56
1.56
100
–
B.s.
12.5
25
6.25
12.5
25
50
25
12.5
12.5
25
E.c.
50
25
50
50
50
50
25
50
50
50
50
25
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
25
50
50
50
25
25
50
50
50
50
50
50
25
50
50
50
50
50
50
12.5
3.12
3.12
1.56
–
K.p.
25
25
25
25
50
25
25
25
25
50
25
25
25
25
50
25
25
25
25
25
25
25
50
25
25
25
50
25
25
50
25
25
25
25
25
25
25
12.5
25
25
25
25
50
50
25
25
25
25
12.5
3.12
1.56
–
P.a.
50
50
25
50
50
50
50
50
50
50
50
50
50
25
50
25
25
25
50
25
50
50
50
25
25
50
50
50
50
50
50
50
25
25
50
25
25
50
50
25
25
50
50
50
25
50
50
>200
>200
50
100
–
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
O
O
O
O
O
O
O
O
O
O
S
S
S
S
S
O
O
S
O
O
O
O
O
O
O
O
O
S
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
Cl
H
H
H
H
H
H
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
H
H
H
H
Cl
H
H
H
H
COOCH3
H
NO2
H
Cl
COOCH3
H
NO2
H
25
12.5
6.25
25
50
25
25
12.5
25
25
50
50
50
25
25
50
50
12.5
25
25
Cl
COOCH3
H
H
H
H
Cl
NO2
CH3
COOCH3
H
S
S
S
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
O
O
O
O
O
O
O
O
Cl
CH3
COOCH3
H
NO2
COOCH3
H
CH3
Cl
CH3
H
Cl
Cl
CH3
H
Cl
S
S
NH
NH
CH2
O
O
O
O
O
O
S
S
S
O
O
S
6.25
12.5
12.5
25
CH3
NO2
H
H
CH3
H
H
CH3
H
H
H
25
12.5
12.5
25
25
6.25
25
12.5
25
25
12.5
25
12.5
1.56
1.56
1.56
50
25
25
50
50
12.5
25
25
12.5
12.5
12.5
25
12.5
–
CH3
H
H
H
Cl
O
O
NH
NH
NH
N
CH
CH
CH
S
CH2
CH3
CH3
H
H
Ampicillin
Amoxycillin
Tetracycline
Streptomycin
Clotrimazol
Haloprogin
–
–
–
6.25
3.12
–
–
–
–
–
–
–
a Abbreviations: E.c., Escherichia coli; K.p., Klepsiella pneumoniae; P.a., Pseudomonas aeruginosa; S.a., Staphylococcus aureus; S.f., Streptococcus
faecalis; B.s., Bacillus subtilis; C.a., Candida albicans.

I. Yildiz-Oren et al. / European Journal of Medicinal Chemistry 39 (2004) 291–298
297
and recrystallized from aqueous EtOH and dried under
vacuum over a night. 9a: 76.5% yield; mp 109 °C (111 °C)
[40], 9b: 76% yield; mp 104 °C (108 °C) [41].
maintained at the Microbiology Department of Faculty Phar-
macy, Ankara University.
4.2.1. Antibacterial assay
The cultures were obtained in Mueller-Hinton broth
(Difco) for all the bacteria after 24 h of incubation at
37 1 °C. Testing was carried out ion Mueller-Hinton broth
at pH 7.4 and the twofold serial dilution technique was
applied. The final inoculum size was 105 CFU/ml. A set of
tubes containing only inoculated broth was kept as control.
After incubation for 24 h at 37 1 °C, the last tube with no
growth of microorganism was recorded to represent MIC
expressed in µg/ml. Every experiment in the antibacterial
assay was replicated twice in order to define the MIC values.
4.1.2. General method for the synthesis of substituted
benzoxazoles (5a–p), benzothiazoles (6a–c),
2-substituted-5-carboxymethyl-benzimidazoles (7e, 7i, 7m),
method A
A mixture of corresponding carboxylic acids 1a/b/ or c
(5 mmol) and appropriate 4- and/or 5-disubstituted
2-aminophenols 2, or methyl 3-amino-4-hydroxybenzoate
9a or 2-aminothiophenol 3 or methyl 3,4-diaminobenzoate
9b (6.9 mmol) was heated under reflux with stirring at vari-
ous temperatures and time in 15 ml PPSE. At the end of the
reaction period, the mixture was taken to 30 ml dichlo-
romethane and neutralized with 50 ml 1 N NaOH solution.
The organic layer was separated and the aqueous solution
extracted with 3 × 25 ml portions of CH2Cl2. The combined
extracts were dried on Na2SO4, filtered and the solvent was
removed with rotary evaporator. The residue was purified by
flash chromatography, eluting with CHCl3 and the obtained
product was recrystallized.
4.2.2. Antifungal assay
The yeast C. albicans was maintained in Sabouraud dex-
trose broth (Difco) after incubation for 24 h at 25 1 °C.
Testing was performed in Sabouraud dextrose broth at pH
7.4 and the twofold serial dilution technique was applied.
The final inoculum size was 104 CFU/ml. A set of tubes
containing only inoculated broth was kept as control. After
incubation for 48 h at 25 1 °C, the last tube with no growth
of yeast was recorded to represent MIC expressed in µg/ml.
Every experiment in the antifungal assay was replicated
twice in order to define the MIC values.
4.1.3. General method for the synthesis of substituted
benzimidazoles (7a–p), method B
A mixture of corresponding carboxylic acids 1a/b/c/d or e
(10 mmol) and appropriate 5-substituted-2-phenylen-
diamines 4 (10 mmol) was boiled under reflux with stirring
for various time in 15 ml 6 N HCl. At the end of the reaction
period, the mixture was neutralized with excess of NaHCO3.
The collected precipitate washed with water, dried in
vacuum, purified by flash chromatography, eluting with
CHCl3 and recrystallized.
Acknowledgements
We would like to thank the Research Fund of Ankara
University (Grant No.2001 08 03 030) and SBAG-COST-
B16-1 (102S291) for the financial support in this research.
4.2. Microbiology
References
For determining both the antibacterial and the antifungal
activity, the synthesized compounds and the control drugs
were dissolved in absolute ethanol (0.8 mg/ml) [42]. Further
dilutions were prepared at the required quantities of 400,
200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78 µg/ml
concentrations. The MIC were determined by using the
method of twofold serial dilution technique [9,26,42,43]. In
order to ensure that the solvent per se had no effect on
bacterial growth, a control test was also performed contain-
ing inoculated broth supplemented with only ethanol at the
same dilutions used in our experiments and found inactive in
culture medium. All the compounds were tested for their in
vitro growth inhibitory activity against different bacteria and
a fungus C. albicans RSKK 628. The origin of bacterial
strains was S. aureus ATCC 6538, S. faecalis ATCC 10541,
B. subtilis ATCC 6033 as Gram-positive and E. coli ATCC
10536, K. pneumoniae ATCC 52211, P. aeruginosa RSKK
355 as Gram-negative bacteria. The RSKK strains used in
this study were obtained from the culture collection of Refik
Saydam Health Institution of Health Ministry, Ankara and
[1] S.K. Fridkin, R.P. Gaynes, Clin. Chest Med. 20 (1999) 303–316.
[2] C. Hubschwerlen, P. Pflieger, J.L. Specklin, K. Gubernator,
H. Gmunder, P. Angehrn, I. Kompis, J. Med. Chem. 35 (1992) 1385–
1388.
[3] L. Perrin, A. Rakik, S. Yearly, C. Baumberger, S. Kinloch-de Loies,
M. Pechiere, B. Hirschel, AIDS 10 (1996) 1233–1237.
[4] J.S. Kim, Q. Sun, B. Gatto, C. Yu, A. Liu, L.F. Liu, E.J. LaVoie,
Bioorg. Med. Chem. 4 (1996) 621–630.
[5] J.S. Kim, C. Yu, A. Liu, L.F. Liu, E.J. LaVoie, J. Med. Chem. 40
(1997) 2818–2824.
[6] D.F. Shi, T.D. Bradshaw, S. Wrigley, C.J. McCall, P. Lelieveld, I. Fich-
tner, M.F.G. Stevens, J. Med. Chem. 39 (1996) 3375–3384.
[7] R. Zhou, E.B. Skibo, J. Med. Chem. 39 (1996) 4321–4331.
[8] O.T. Arpaci, E.A. Sener, I. Yalcin, N. Altanlar, Arch. Pharm. Phar.
Med. Chem. 6 (2002) 283–288.
[9] O.T. Arpaci, I. Oren, N. Altanlar, Il Farmaco 57 (2002) 175–181.
[10] S. Staszewski, F.E. Massari, A. Kober, R. Göhler, S. Durr,
K.W. Anderson, C.L. Schneider, J.A. Waterburry, K.K. Bakshi,
V.I. Taylor, et al., J. Infect Dis. 171 (1995) 1159–1165.
[11] D.B. Olsen, S.S. Carroll, J.C. Culberson, J.A. Shafer, L.C. Kuo,
Nucleic Acids Res. 22 (1994) 1437–1443.
[12] M.R. Peel, M.W. Milstead, D.D. Sternbach, M. Besterman, P. Leitner,
B. Morton, Bioorg. Med. Chem. Lett. 5 (1995) 2129–2132.

298
I. Yildiz-Oren et al. / European Journal of Medicinal Chemistry 39 (2004) 291–298
[13] I. Hutchinson, S.A. Jennings, B.R. Vishnuvajjala, A.D. Westwell,
M.F.G. Stevens, J. Med. Chem. 45 (2002) 744–747.
[14] M. Ueki, K. Ueno, S. Miyadoh, K. Abe, K. Shibata, M. Tanguchi,
S. Oi, J. Antibiot. 46 (1993) 1089–1094.
[28] J.M. Aizpurua, C. Palomo, Soc. Chim. France Bull. (1984) 142–144.
[29] M.A. Phillips, J. Chem. Soc. (1928) 2393–2399.
[30] T.O. Olagbemiro, M.O. Agho, O.J. Abayeh, J.O. Amupitan, Recl.
Trav. Chim. Pays-Bas. 115 (1996) 337–338.
[15] M. Ueki, K. Shibata, M. Taniguchi, J. Antibiot. 51 (1998) 883–885.
[16] M. Ueki, M. Taniguchi, J. Antibiot. 50 (1997) 788–790.
[17] I.Yalcin, E. Sener, T. Ozden, S. Ozden,A.Akin, Eur. J. Med. Chem. 25
(1990) 705–708.
[18] I. Yalcin, I. Oren, E. Sener, A. Akin, N. Uçartürk, Eur. J. Med. Chem.
27 (1992) 401–406.
[19] I. Yalcin, E. Sener, Int. J. Pharm. 98 (1993) 1–8.
[20] E. Sener, I. Yalcin, E. Sungur, Quant. Struc. Act. Rel. 10 (1991)
223–228.
[21] E. Sener, I.Yalcin, O. Temiz, I. Oren,A.Akin, N. Uçartürk, Il Farmaco
52 (1997) 99–103.
[31] L.G.S. Brooker, F.L. White, US 2,478,366, 1949.
[32] J.E. Cranham, W.A.W. Cummings, A.M. Johnston, H.A. Stevenson, J.
Sci. Food Agr. 9 (1958) 143–147; Chem. Abstr. 52 (1958) 11773f.
[33] A. Somat, R. Guglielmetti, J. Metzger, Helv. Chim. Acta 55 (1972)
1783–1801.
[34] L.G.S. Brooker, F.L. White, US 2,494,031 (1950); Chem. Abstr. 44
(1950) 7686g.
[35] F.E. King, R.M. Acheson, J. Chem. Soc. (1949) 1396–1400.
[36] F. Gümüs, T.G.Altuntas, T. Saygun, T. Özden, S. Özden, Pharm. Belg.
44 (1989) 398–402.
[37] Chugai Pharmaceutical Co. Japan 7538 (67); Chem. Abstr. 68 (1964)
29698y.
[22] O.T. Arpaci, I. Oren, E. Sener, I. Yalcin, N. Ucarturk, Il Farmaco 53
(1998) 337–341.
[38] G. Kaupp, K. Sailer, Angew. Chem. 102 (1990) 917–919.
[39] H. Gilman, R. Adams, H.T. Clarke, J.B. Conant, C.S. Marvel,
C.R. Noller, F.C. Whitmore, Organic Synthesis, second ed, Collective,
Volume I, John Wiley and Sons, Inc, New York, 1941, pp. 237–238.
[40] A. Einhorn, E. Rubbert, Annalen der Chem. 325 (1902) 305–339.
[41] P.R. Thomas, G.J. Tyler, J. Chem. Soc. (1957) 2197–2202.
[42] S. Shadomy, A. Espinel, A Manual of Clinical Microbiology, Am.
Soc. Microbiol (1980) 647 Washington, DC.
[23] I. Oren, O. Temiz, I. Yalcin, E. Sener, A. Akin, N. Altanlar, Eur. J.
Pharm. Sci. 7 (1998) 153–160.
[24] E.A. Sener, O.T. Arpacý, I. Yalcin, N. Altanlar, Il Farmaco 55 (2000)
397–405.
[25] E. Sener, H. Turgut, I. Yalcin, I. Oren, L. Turker, N. Celebi, Inter. J.
Pharm. 110 (1994) 109–115.
[26] I. Oren, O. Temiz, I. Yalcin, E. Sener, A. Akin, N. Uçartürk, Arzneim.
Forsch. 47 (1997) 1393–1397.
[43] E.S. Charles, V.K. Agrawal, S. Sharma, R.N. Iyer, Eur. J. Med. Chem.
Chim. Ther. 14 (1979) 435–438.
[27] A.Akbay, I. Oren, O.T.Arpaci, E.A. Sener, I.Yalcin,Arzneim. Frosch.
53 (2003) 266–271.