Azole antimicrobial pharmacophore-based tetrazoles: Synthesis and biological evaluation as potential antimicrobial and anticonvulsant agents

Article abstract of DOI:10.1016/j.bmc.2009.02.004

The azole pharmacophore is still considered a viable lead structure for the synthesis of more efficacious and broad spectrum antimicrobial agents. Potential antibacterial and antifungal activities are encountered with some tetrazoles. Therefore, this stud

Full text of DOI:10.1016/j.bmc.2009.02.004

Bioorganic & Medicinal Chemistry 17 (2009) 2410–2422
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Azole antimicrobial pharmacophore-based tetrazoles: Synthesis and biological
evaluation as potential antimicrobial and anticonvulsant agents q
b
b
Sherif A. F. Rostom a, , Hayam M. A. Ashour , Heba A. Abd El Razik ,
*
Abd El Fattah H. Abd El Fattah c, Nagwa N. El-Din d
a Department of Chemistry, Faculty of Science, King Abdulaziz University, PO Box 80203, Jeddah 21589, Saudi Arabia
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt
c Department of Microbiology, High Institute of Public Health, Alexandria University, Alexandria, Egypt
d Department of Pharmacology, Faculty of Medicine, Alexandria University, Alexandria 21521, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The azole pharmacophore is still considered a viable lead structure for the synthesis of more efficacious
and broad spectrum antimicrobial agents. Potential antibacterial and antifungal activities are encoun-
tered with some tetrazoles. Therefore, this study presents the synthesis and antimicrobial evaluation
of a new series of substituted tetrazoles that are structurally related to the famous antimicrobial azole
pharmacophore. A detailed discussion of the structural elucidation of some of the newly synthesized
compounds is also described. Antimicrobial evaluation revealed that twenty compounds were able to dis-
play variable growth inhibitory effects on the tested Gram positive and Gram negative bacteria with spe-
cial efficacy against the Gram positive strains. Meanwhile, six compounds exhibited moderate antifungal
activity against Candida albicans and Aspergillus fumigatus. Structurally, the antibacterial activity was
encountered with tetrazoles containing a phenyl substituent, while the obtained antifungal activity
was confined to the benzyl variants. Compounds 16, 18, 24 and 27 were proved to be the most active
antibacterial members within this study with a considerable broad spectrum against all the Gram posi-
tive and negative strains tested. A distinctive anti-Gram positive activity was displayed by compound 18
Received 5 November 2008
Accepted 5 February 2009
Available online 8 February 2009
Keywords:
1H-Tetrazoles
Azoles
Pyrazoles
1,3,4-Oxadiazoles
1,3,4-Triazoles
Antibacterial activity
Antifungal activity
Anticonvulsant activity
against Staphylococcus aureus that was equipotent to ampicillin (MIC 6.25
lg/mL).
On the other hand, twelve compounds were selected to be screened for their preliminary anticonvulsant
activity against subcutaneous metrazole (ScMet) and maximal electroshock (MES) induced seizures in
mice. The results revealed that five compounds namely; 3, 5, 13, 21, and 24 were able to display notice-
able anticonvulsant activity in both tests at 100 mg/kg dose level. Compounds 5 and 21 were proved to be
the most active anticonvulsant members in this study with special high activity in the ScMet assay (%
protection: 100% and 80%, respectively).
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
ever, in recent years, much attention has been focused on address-
ing the problem of multi-drug resistant (MDR) bacteria and fungi
Research and development of potent and effective antimicrobial
agents represents one of the most important advances in therapeu-
tics, not only in the control of serious infections, but also in the pre-
vention and treatment of some infectious complications of other
therapeutic modalities such as cancer chemotherapy and surgery.
Over the past decade, fungal infection became an important com-
plication and a major cause of morbidity and mortality in immuno-
compromised individuals such as those suffering from
tuberculosis, cancer or AIDS and in organ transplant cases.1 How-
resulting from the widespread use and misuse of classical antimi-
crobial agents.2 Such serious global health problem demands a re-
newed effort seeking the development of new antimicrobial agents
effective against pathogenic microorganisms resistant to currently
available treatments.
Among the important pharmacophores responsible for antimi-
crobial activity, the azole scaffold (A; Fig. 1) is still considered a
viable lead structure for the synthesis of more efficacious and
broad spectrum antimicrobial agents. Azoles (imidazole and tria-
zole derivatives) inhibit the synthesis of sterols in fungi by inhibit-
ing cytochrome P450-dependent 14a-lanosterol demethylase (P-
q
This work was presented in the 6th AFMC International Medicinal Chemistry
45014DM), which removes the methyl group on C-14 of lanosterol,
a key intermediate step in the formation of ergosterol in the fungal
cell membrane.3 Moreover, it was recently found that generation of
Symposium, Istanbul, Turkey, July 8–11/2007, as a poster PC-21.
* Corresponding author. Tel.: +966 507654566; fax: +9662 6400000 22327.
E-mail address: sherifrostom@yahoo.com (S.A.F. Rostom).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.004

S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 2410–2422
2411
R1
O
R
O
Cl
Cl
N
N
N
N
N
N
Cl
R
A
B
General Structure of the target compounds
R = benzyl or phenyl group
Azole Pharmacophore
R = halogenated aryl or heterocyclic ring
R1 = heterocylic ring
(pyrazoles, 1,3,4-oxadiazoles, 1,3,4-triazoles)
HO
O
N
N
N
N
D
C
Denzimol
Nafimidone
Figure 1. (A) The Azole pharmacophore, (B) general structure of the target compounds, (C) nafimidone, (D) denzimol.
reactive oxygen species (ROS) is important for the antifungal activ-
ity of miconazole, pointing to an additional mode of action for this
azole.4 Additionally, it has been reported that remarkable antibac-
terial activity against methicillin-resistant strains of Staphylococcus
aureus (MRSA) was ascribed to some azoles, particularly miconaz-
ole.5 Furthermore, some azoles were proved to be effective inhibi-
denzimole (D) (Fig. 1) are two independently discovered represen-
tatives of this group and possess a profile of activity similar to that
of phenytoin or carbamazepine but more distinct than barbiturates
and valproic acid.27,28 Structure–activity relationship studies re-
vealed that anticonvulsant properties of this group are associated
with the presence of an imidazole ring, a small oxygen functional
group in addition to a lipophilic aryl portion facilitating penetra-
tion of the blood–brain barrier.29 Consequently, it was considered
worthwhile to investigate the anticonvulsant activity of some of
the newly synthesized compounds comprising both the above-
mentioned pharmacophore and the tetrazole ring which is re-
ported to contribute to some anticonvulsant activity.17,30
tors of enoyl acyl carrier protein reductase (FabI),
a novel
antibacterial target.6 However, the frequent use of azoles has re-
sulted in clinically resistant strains of fungi and bacteria.7 The pri-
mary structural requirement for the antimicrobial azole class is a
weakly basic imidazole or triazole ring bonded by a nitrogen–car-
bon linkage to the rest of the structure. At the molecular level, the
amidine nitrogen (N-3 in the imidazole or N-4 in the triazole) is be-
lieved to bind to the heme iron of enzyme-bound cytochrome P450
to prevent oxidation of steroidal substrates by the enzyme. Most of
them possess aromatic rings that are believed to mimic the corre-
sponding non-polar steroidal portion of the substrate for the en-
2. Results and discussion
2.1. Chemistry
zyme. The non-polar functionality confers
a high degree of
lipophilicity to the antifungal azoles.8 Various attempts have been
undertaken to modify the structures of the so far effective azole
drugs in order to improve their antimicrobial potency and selectiv-
ity,9–11 however, few reports have discussed the contribution of
the tetrazole moiety in such pharmacophore in spite of the poten-
tial antibacterial and antifungal activities encountered with some
tetrazoles.12–17
Motivated by these findings, and in continuation of our ongoing
efforts endowed with the discovery of nitrogenated heterocycles
with potential chemotherapeutic activities,18–26 it was planned to
synthesize and investigate the antimicrobial activity of a new ser-
ies of substituted tetrazoles having the general formula (B) that are
structurally related to the famous azole pharmacophore (A)
(Fig. 1). The target compounds were designed so that the imidazole
ring was isosterically replaced with a tetrazole one, keeping almost
the same dimensions and substitution pattern of such type of com-
pounds. Moreover, the aralkyl moiety attached to the secondary
alcoholic group was varied to include other functionalities that
are known to contribute to a variety of antimicrobial activities
including the ester, acid, acid hydrazide and thiosemicarbazide
groups. Additionally, it was considered of interest to incorporate
other chemotherapeutically-active heterocyclic rings (pyrazoles,
oxadiazoles and triazoles) within the structure, hoping to impart
some synergism to the target compounds.
Synthesis of the intermediate and target compounds was per-
formed according to the reactions outlined in Schemes 1–3. In
Scheme 1, the starting compounds 5-(benzyl or phenyl)-1H-tetra-
zole 1, 2 were prepared following a previously reported literature
procedure.31 Alkylation of these tetrazoles with 4-chlorophenacyl
bromide in refluxing acetone containing anhydrous potassium car-
bonate afforded the ethanone derivatives 3, 4 in good yields. A lit-
erature survey revealed that alkylation of 5-substituted tetrazoles
affords generally mixtures of 1,5- and 2,5-regioisomers,32,33,13
which could be separated by silica gel chromatography.34,35
However in the present study, compounds 3 and 4 were purified
by fractional crystallization from diethyl ether followed by crystal-
lization from the proper solvent. Furthermore, 1H NMR studies of
disubstituted tetrazoles have shown that protons of CH2 group at-
tached to N1 of 1,5-disubstituted tetrazoles are more shielded than
the corresponding protons of 2,5-disubstituted tetrazoles.36–38
Moreover, their 13C NMR spectra have shown that the tetrazole-
C5 carbon atom of 1,5-disubstituted tetrazoles is more shielded
than the corresponding carbon of the 2,5-disubstituted tetra-
zoles.36–38 Based on these reported observations, compound 3
(R = CH2–C6H5) was designated as 1,5-disubstituted tetrazole since
its 1H NMR spectrum displayed a signal for N–CH2 protons at d
6.32 ppm, while its 13C NMR spectrum showed a C5 resonance at
d 156.17 ppm. Meanwhile, compound 4 (R = C6H5) was designated
as 2,5-disubstituted tetrazole as evidenced from its 1H NMR that
exhibited a signal for N–CH2 protons at d 6.72 ppm, whereas its
13C NMR spectrum showed a signal for tetrazole-C5 atom at d
On the other hand, literature survey revealed that the newly
synthesized compounds are structurally related to a distinct class
of antiepileptic drugs; the aralkylimidazoles. Nafimidone (C) and

2412
S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 2410–2422
Cl
N
N
N
1, 3, 5, 7, 9, 11: R = CH2C6H5
+
NH
Br
R
O
2, 4, 6, 8, 10, 12: R = C6H5
1,2
i
N
R
N
N
R
N
N
N
N
N
ii
OH
O
Cl
Cl
3,4
5,6
v
iii
N
N
N
R
N
R
R
N
N
N
O
O
O
N
N
N
N
N
NH2
O
O
O
N
H
OH
O
vi
iv
Cl
11,12
Cl
9,10
Cl
7,8
Scheme 1. Reagents and conditions: (i) dry acetone, K2CO3, reflux; (ii) NaBH4, ethanol, room temperature; (iii) ethyl bromoacetate, sodium hydride, room temperature; (iv)
sodium hydroxide, ethanol, reflux; (v) 2-chloroacetic acid, sodiumhydride, room temperature; (vi) hydrazine hydrate, ethanol, reflux.
164.93 ppm. In addition, more spectroscopic evidence for the reg-
iochemical assignment of the two ethanone derivatives 3, 4 was
obtained by the aid of nuclear overhauser effect (NOE) and heter-
onuclear multiple bond correlation (HMBC) experiments. NOE
hydrazine hydrate gave the corresponding acetohydrazides 11,
12, respectively, which were employed as key intermediates for
synthesis of the target compounds presented in Scheme 2.
Thus, condensing the acetohydrazides 11, 12 with 4-chloro-
benzaldehyde in boiling ethanol furnished the corresponding azo-
methines 13, 14, respectively. It should be noted down here that,
compounds having the arylidene–hydrazide structure may exist
as E/Z geometrical isomers about C@N double bond and as cis/trans
amide conformers at the CO–NH moiety (Fig. 3).40,41 In this respect,
the DMSO-d6 1H NMR spectra of these compounds confirmed their
existence as E geometrical isomers, which coincides with the liter-
ature findings for analogous compounds containing the imine
(C@N) functionality.41 On the other hand, further interpretation
of their DMSO-d6 1H NMR spectra revealed presence of three sets
of signals at d 3.78–3.97, 4.17–4.45, 7.84–8.13, 8.17–8.68 ppm
and d 11.12–11.20, 11.39–11.44 ppm attributed to OCH2, N@CH
and CO–NH groups of the cis and trans conformers, respectively.
According to the literature, the upfield lines of the N@CH and
CONH protons were assigned to the cis conformer of the amide
structure, whereas, the downfield lines were due to the trans con-
former.42 Cyclocondensation of acetohydrazides 11, 12 with acetyl
acetone resulted in formation of the pyrazole derivatives 15, 16.
Furthermore, synthesis of 1,3,4-oxadiazol-2-thiol 17, 18 was
achieved by treatment of 11, 12 with carbon disulfide in boiling
ethanol containing potassium hydroxide. Treatment of 11 (R = ben-
zyl) and 12 (R = phenyl) with aryl isothiocyanates in ethanol at
room temperature afforded the corresponding thiosemicarbazides
19–21. Unexpectedly, when the phenyl analog 12 was refluxed
with ary lisothiocyanates in ethanol, the 1,3,4-triazole derivatives
experiment for compound
3 showed a correlation between
N–CH2 protons at d 6.32 ppm and benzyl CH2 protons at d
4.25 ppm (Fig. 2). Moreover, HMBC spectrum for compound 3
revealed a strong correlation between the protons of the N–CH2
group at 6.32 ppm and the tetrazole-C5 carbon at 156.17 ppm, a
feature that was not observed in compound 4 (R = C6H5). Reduction
of the ethanone derivatives 3, 4 using sodium borohydride at room
temperature resulted in the formation of the corresponding etha-
nol derivatives 5, 6 in excellent yields. Being chiral molecules,
the geminal hydrogens of the (N–CH2) moiety adjacent to the chi-
ral centre (CH) experience different magnetic environments.39
Hence, their 1H NMR spectra showed both the N–CH2 and CH
methine protons as double doublets at about d 4.43–4.94 and
4.92–5.30 ppm, respectively (see Section 4), in addition to D2O-
exchangeable signals at d 6.04 and 5.99 ppm due to OH protons.
Alkylation of 5, 6 with ethyl bromoacetate at room temperature
using sodium hydride yielded the requisite acetate derivatives 7,
8 in moderate yields. Here, it is worth mentioning OCH2 protons
of 7 were found to be relatively more shielded than those of com-
pound 8, providing a substantial proof for their assigned 1,5- and
2,5-disubstituted structures. Synthesis of acetic acid derivatives 9
and 10 was accomplished either through alkylation of the ethanol
derivatives 5, 6 with chloroacetic acid using sodium hydride
(method A), or by hydrolysis of the esters 7 and 8 using 5% sodium
hydroxide (method B). Refluxing the esters 7, 8 in ethanolic

S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 2410–2422
2413
N
N
R
R
N
N
Cl
O
O
N
N
N
N
N
O
O
N
H
N
N
13, 15, 17: R = CH2C6H5
14, 16, 18: R = C6H5
Cl
Cl
13,14
15,16
ii
i
N
R
N
N
N
R
O
N
N
N
N
N
N
NH2
O
SH
O
N
O
H
iii
Cl
Cl
12: R = C6H5
v
11,12
17,18
iv
N
N
N
N
SH
N
N
N
N
R
N
N
SH
N
N
N
N
N
O
O
N
N
H
N
H
N
R1
O
N
O
R1
R1
N
H
vi
S
19: R = CH2C6H5, R1 = C6H5
20: R = CH2C6H5, R1 = 4-Cl-C6H4
Cl
Cl
Cl
22: R1 = C6H5
23: R1 = 4-Cl-C6H4
24: R1 = C6H5
25: R1 = 4-Cl-C6H4
19: R = CH2C6H5, R1 = C6H5
20: R = CH2C6H5, R1 = 4-Cl-C6H4
21: R =C6H5, R1 = C6H5
Scheme 2. Reagents and conditions: (i) 4-chlorobenzaldehyde, ethanol, reflux; (ii) acetyl acetone, ethanol, reflux; (iii) carbon disulfide, potassium hydroxide; (iv) aryl
isothiocyanate, ethanol, room temperature; (v) aryl isothiocyanate, ethanol, reflux; (vi) 5% aq sodiumcarbonate, reflux.
24, 25 were directly obtained. On the other hand, synthesis of the
1,3,4-triazole derivatives 22, 23 was achieved through refluxing
the thiosemicarbazides 19, 20 in 5% sodium carbonate solution.
Finally, cyclodesulfurisation of the intermediate thiosemicar-
bazides 19 and 21 to the 1,3,4-oxadiazole derivatives 26, 27 was
performed using freshly prepared yellow mercuric oxide in boiling
dioxane (Scheme 3). At this stage, it was attempted to prepare the
analogous 1,3,4-thiadiazole derivatives 28 and 29 through treat-
ment of the thiosemicarbazides 19, 21 with concd sulfuric acid fol-
lowing different reported procedures, however, all these trials
were abortive. Surprisingly, two unexpected products namely; a
tetrazolobenzazepine 30 and a styryl tetrazole derivative 31 were
isolated instead as evidenced from their elementary analyses, 1H
NMR, 13C NMR, HMBC, and mass spectral data. The 1H NMR spec-
trum of compound 30 showed complete absence of signals rele-
vant to the 1,3,4-thiadiazole structure, and appearance of signals
corresponding to the N–CH2, benzyl CH2 and CH protons in addi-
tion to a reduction in the expected number of aromatic protons
(8 instead of 9 protons). Moreover, an obvious change in the chem-
ical shifts and splitting pattern of the previously mentioned pro-
tons was observed (see Section 4). 13C NMR spectrum of the
same compound displayed signals attributed to five quaternary
carbons instead of the expected eight carbons and eight aromatic
carbons instead of the expected thirteen. Its mass spectrum
showed a molecular ion peek at m/z 296 instead of the expected
1,3,4-thiadiazole structure m/z 483. The found values of the ele-
mentary analysis of this compound were in favor of the cyclized
tetrazolobenzazepine structure. Moreover, HMQC (Heteronuclear
Multiple Quantum Coherence) data were in accordance with the
assigned structure. More evidence to assess the new structure of
30 was obtained from HMBC experiment that showed a strong cor-
relation between the CH carbon (d 42.73 ppm) and the benzyl C3–H

2414
S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 2410–2422
N
R
N
N
R
N
N
N
N
N
N
N
N
N
N
O
N
O
O
H
S
H
Cl
Cl
26: R = CH2C6H5
27: R =C6H5
28: R = CH2C6H5
29: R =C6H5
N
N
N
i
N
19: R = CH2C6H5
ii
N
R
N
O
H
N
N
H
N
N
O
N
H
S
Cl
30
Cl
19: R = CH2C6H5
21: R =C6H5
N
N
N
N
21: R =C6H5
31
Cl
Scheme 3. Reagents and conditions: (i) HgO, dioxane, reflux; (ii) sulfuric acid, 0 °C, 15 min.
N
N
4.2
N
Cl
Cl
N
6.32
H
N
H
O
H
N
Tet
N
Figure 2. NOE correlation of compound 3.
Tet
N
H
O
O
Cl
trans, E
trans, Z
(d 6.8 ppm) which confirms the process of internal cyclization. The
reaction mechanism for such cyclization was suggested to take
place through protonation of the ether oxygen followed by cleav-
age of the side chain leaving a secondary carbocation. The latter
was then subjected to a nucleophilic attack by the benzyl ortho
carbon leading to cyclization to the seven-membered azepine ring.
Concerning the styryl tetrazole derivative 31, investigation of its 1H
NMR spectrum revealed absence of signals relevant to the 1,3,4-
thiadiazole structure as well as disappearance of signals due N–
Cl
H
N
H
H
O
N
O
N
N
H
CH2 and CH protons. Instead, two doublets at
d 7.66 and
Tet
Cl
8.50 ppm with a coupling constant of 14.6 Hz corresponding to
two vinylic protons in the E configuration were displayed. Other
aromatic protons of the phenyl and 4-chlorophenyl moieties were
located at their expected chemical shifts. In addition, its 13C NMR,
HMBC spectral data and the found values of the elementary analy-
sis of this compound agreed with the styryl rather than the 1,3,4-
thiadiazolyl structure. The formation of such derivative was sug-
gested to proceed via protonation of the ether oxygen followed
by cleavage of the side chain leaving a secondary carbocation
which loses a proton from the neighboring carbon to form the vinyl
CH@CH double bond.
Tet
cis, Z
cis, E
N
N
N
O
N
Tet =
R
Cl
Figure 3. E/Z geometrical isomers and cis/trans conformers of compounds 13 and
14.

S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 2410–2422
2415
Table 2
2.2. Biological evaluation
Minimal inhibitory concentrations (MIC,
compounds against some fungal strains
l
g/mL) of the active newly synthesized
2.2.1. In vitro antibacterial and antifungal activities
Compound no.
Candida albicans
Aspergillus fumigatus
All the newly synthesized compounds were evaluated for their
in vitro antibacterial activity against S. aureus (ATCC 6538), Bacillus
subtilis (NRRL B-14819) and Micrococcus luteus (ATCC 21881) as
examples of Gram positive bacteria and Escherichia coli (ATCC
25922), Pseudomonas aeruginosa (ATCC 27853) and Klebsiella pneu-
monia (clinical isolate) as examples of Gram negative bacteria.
They were also evaluated for their in vitro antifungal potential
against Candida albicans, Candida tropicalis and Candida krusei as
representatives of fungi and Aspergillus niger, Aspergillus fumigatus
and Tricophyton rubrum as examples of moulds. All the fungal and
mould strains were clinical isolates, identified with conventional
morphological and biochemical methods. Agar-diffusion method
was used for determination of the preliminary antibacterial and
antifungal activity. Ampicillin trihydrate (antibiotic), clotrimazole
and miconazole (antifungals) were used as reference drugs. The re-
sults were recorded for each tested compound as the average
diameter of inhibition zones (IZ) of bacterial or fungal growth
around the discs in mm. The minimum inhibitory concentration
(MIC) measurement was determined for compounds that showed
significant growth inhibition zones (P14 mm) using the two-fold
a
15
17
22
23
26
30
Cb
100
100
50
25
25
—
6.25
3.12
—
—
—
—
—
25
12.5
3.12
Mc
a
(À): Totally inactive (MIC P 200
lg/mL).
b
c
C: Clotrimazole (standard broad spectrum antifungal agent).
M: Miconazole (standard broad spectrum antifungal agent).
12.5
50
12.5
l
g/mL). The analogs 14, 15, 23, 24, 25, 26 and 27 (MIC
g/mL) exhibited 25% of the potency of ampicillin (MIC
g/mL) against the same species. M. luteus was proved to be
l
l
the least sensitive Gram positive microorganism to most of the
tested compounds. Only four compounds namely; 15, 16, 18 and
24 (MIC 50
wards M. luteus which was 25% of the activity of ampicillin (MIC
12.5 g/mL) (Table 1). On the other hand, investigation of antibac-
lg/mL) exhibited moderate growth inhibitory effect to-
serial dilution method.43 The MIC (
lg/mL) values of the active
l
terial activity of the active compounds against the three tested
Gram negative strains revealed that two analogs namely 18 and
27 were able to produce moderate growth inhibitory activity
compounds against the tested bacterial and fungal strains are re-
corded in Tables 1 and 2.
Regarding the antibacterial activity, the results revealed that 20
out of the tested 27 compounds displayed variable inhibitory ef-
fects on the growth of the tested Gram positive and Gram negative
bacterial strains. In general, most of the tested compounds re-
vealed better activity against the Gram positive rather than the
Gram negative bacteria. Among the Gram positive bacteria tested,
two strains namely; S. aureus and B. subtilis showed relative high
sensitivity towards the tested compounds. In this view, compound
against E. coli (MIC 25
ampicillin (MIC 6.25 g/mL). Whereas, compounds 16, 23, 24, 26
and 30, (MIC 50 g/mL), exhibited mild activity against the same
lg/mL) which was 25% of the activity of
l
l
organism. Meanwhile, the tested P. aeruginosa and K. pneumonia
strains were proved to be weakly sensitive to most of the tested
compounds. Among these, compounds 18 and 24 showed moder-
ate growth inhibitory profiles against these organisms (MIC
50
l
g/mL), which were about 25% of the activity of ampicillin.
Concerning the antifungal activity of the tested compounds,
18 was equipotent to ampicillin (MIC 6.25
whereas the analogs 16, 24 and 27 (MIC 12.5
active than ampicillin. Moreover, compounds 15, 23, 25 and 26
(MIC 25 g/mL) showed 25% of the activity of ampicillin against
lg/mL) against S. aureus,
lg/mL) were 50% less
only two organisms namely; C. albicans and A. fumigatus showed
certain sensitivity against some of the tested compounds, whereas
the rest of the fungal strains were totally insensitive to the same
compounds. Five compounds namely; 15, 17, 22, 23 and 26 exhib-
ited moderate growth inhibitory action on C. albicans with MIC
l
the same organism. With regard to the activity against B. subtilis,
the best activity was displayed by compounds 16 and 18 (MIC
25 lg/mL), which represented half the potency of ampicillin (MIC
Table 1
Minimal inhibitory concentrations (MIC,
l
g/mL) of the active newly synthesized compounds against some Gram positive and Gram negative bacterial strains
Compound no.
S. aureus ATCC 6538
B. subtilis NRRL B-14819
M. luteus ATCC 21881
E. coli ATCC 25922
P. aeruginosa ATCC
27853
K. pneumonia (clinical
isolate)
a
8
10
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
30
31
Ab
100
100
100
100
50
25
12.5
50
6.25
100
100
50
50
25
12.5
25
25
12.5
50
100
6.25
—
—
—
—
—
—
—
100
100
100
50
—
25
100
—
—
100
50
50
100
50
25
50
100
6.25
—
—
—
—
—
—
100
—
50
—
—
—
—
—
100
—
—
100
—
—
—
—
—
—
—
—
100
—
100
—
—
—
—
—
50
100
—
100
—
100
—
100
50
50
25
100
25
—
100
100
50
50
100
50
100
—
—
100
100
50
50
50
50
50
—
—
—
100
50
100
—
100
—
—
12.5
—
12.5
—
12.5
12.5
a
(À): Totally inactive (MIC P 200
l
g/mL).
b
A: Ampicillin trihydrate (standard broad spectrum antibiotic).

2416
S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 2410–2422
range of 25–100
miconazole (MICs 6.25 and 3.12
antifungal agents utilized in this assay. Only one compound
namely; tetrazolobenzazepine 30, was able to inhibit the growth
l
g/mL, when compared with clotrimazole and
g/mL, respectively), the standard
group with phenyl one (compounds 24 and 25) enhanced both
l
the antimicrobial spectrum and potential of these compounds.
Consequently, a broad spectrum antibacterial activity was dis-
played by the 1,3,4-triazole derivative 24 (R1 = C6H5), with a spe-
of A. fumigatus at MIC of 25
lg/mL (Table 2).
cial growth inhibitory effect on S. aureus (MIC 12.5 lg/mL).
A close examination of the structures of the active compounds
presented in Tables 1 and 2 revealed that, their antimicrobial
activity is strongly bound to the nature of the substituent at the
tetrazole-C5, together with the substituent linked to the alkoxy
part of the structure. In general, it could be clearly recognized that
potential antibacterial activity was encountered with tetrazoles
containing a phenyl substituent, while the obtained antifungal
activity was confined to those comprising the benzyl group. More-
over, the unsubstituted ethanone and ethanol derivatives 3–6 to-
gether with the analogs substituted with open chain
counterparts like the esters, acids and acid hydrazides 7–12
Introduction of a chlorine atom to the structure as in compound
25 (R1 = 4-Cl–C6H4) resulted in about 50% reduction in the antibac-
terial activity. Finally, the unexpected tetrazolobenzazepine 30 and
styryltetrazole 31 derivatives revealed mild antibacterial activity
against some Gram positive and Gram negative bacteria with
MIC range of 50–100
appreciable antifungal activity against A. fumigatus at MIC of
25 g/mL (Table 2).
lg/mL. Meanwhile, the analog 30 showed
l
2.2.2. Preliminary anticonvulsant screening
Twelve compounds namely; 3–6, 13, 15, 16, 18, 21, 22, 24 and
30, were selected to be screened for their preliminary anticonvul-
sant properties against subcutaneous metrazole (ScMet)44 and
maximal electroshock (MES)45 induced seizures in mice. Wister al-
bino mice of either sex, weighing 25–30 g and 3 months old were
showed weak or no antimicrobial activity (MIC P100 lg/mL). An
exception is the azomethines 13, 14 which showed relative better
antibacterial profile when compared with the parent acid hydra-
zides 11, 12. Derivatization of the latter compounds into thiose-
micarbazides 19–21 offered very limited improvement in the
antibacterial spectrum. On the other hand, cyclization of the pre-
mentioned functionalities provided a variety of heterocycles that
possess remarkable antibacterial and antifungal spectra. In this re-
spect, cyclocondensation of the acid hydrazides 11, 12 to the pyr-
azoles 15, 16 resulted in an obvious improvement in the
antimicrobial spectrum. Among these, compound 16 (R = C6H5)
was able to exert about 50% of the activity of ampicillin against
the Gram positive S. aureus and B. subtilis, whereas it showed mild
Table 3
Preliminary anticonvulsant screening of some representative compounds
Compound no.
Dose (mg/kg)
Anticonvulsant activitya
ScMetb
% Protection
Mesc
% Protection
d
3
30
100
300
0/6
3/6
0/6
0
50
0
—
—
50
0
3/6
—
4
30
100
300
—
0/6
—
—
0
—
—
—
0
—
0/6e
—
activity against the Gram negative E. coli (MIC 50 lg/mL). On the
other hand, cyclization of the thiosemicarbazides 19–21 resulted
in a series of 1,3,4-oxadiazole and 1,3,4-triazole derivatives with
significantly improved antimicrobial potentials. Regarding the 2-
substituted-1,3,4-oxadiazoles structure variants 17, 18, 26 and
27; the antimicrobial activity of these compounds appears to be
closely related to the nature of the substituent at position-2 of
the 1,3,4-oxadiazole counterpart. The 2-thiol substituent (com-
pounds 17 and 18) proved to be the favorite functionality and re-
sulted in a remarkable improvement in antibacterial spectrum. In
this respect, compound 18 (R = C6H5) showed a broad antibacterial
spectrum against all the tested Gram positive and negative strains,
with a unique potency against S. aureus that was equipotent to
5
30
100
300
0/6
6/6
3/6
0
100
50
—
4/6
—
—
67
—
6
30
100
300
—
0/6
—
—
0
—
—
0/6
—
—
0
—
13
15
16
18
21
22
24
30
a
30
100
300
—
3/6
—
—
50
—
—
3/6
—
—
50
—
30
100
300
—
0/6
—
—
0
—
—
0/6
—
—
0
—
ampicillin (MIC 6.25 lg/mL). However, it was devoid of any anti-
fungal activity. Referring to its benzyl analog 17, it was noticeably
less active than 18 as revealed from its activity against the three
30
100
300
—
0/6
—
—
0
—
—
0/6
—
—
0
—
30
100
300
—
2/6
—
—
33
—
—
0/6
—
—
0
—
tested Gram positive strains (MIC range 50–100
was totally inactive against Gram negative bacteria. However, it
showed weak antifungal activity against C. albicans (MIC 100 g/
lg/mL), while it
l
30
100
300
0/6
5/6
0/6
0
80
0
—
4/6
—
—
67
—
mL). On the other hand, introduction of an aminophenyl moiety
at position-2 of the 1,3,4-oxadiazole counterpart (compounds 26,
27) resulted in a noticeable change in both the antimicrobial po-
tential and spectrum of these compounds. Thus, compound 26
(R = CH2–C6H5) showed a remarkable antifungal activity against
30
100
300
—
0/6
—
—
0
—
—
2/6
—
—
33
—
C. albicans (MIC 25 lg/mL) together with a moderate antibacterial
30
100
300
—
2/6
—
—
33
—
—
2/6
—
—
33
—
profile. Replacing the benzyl group with a phenyl one as in 27
(R = C6H5) resulted in an obvious improvement in the antibacterial
spectrum against both Gram positive and negative bacteria with
30
100
300
—
0/6
—
—
0
—
—
—
33
—
2/6f
—
special high activity against S. aureus (MIC 12.5
lg/mL) and
E. coli (MIC 25 g/mL), however, it lacked any antifungal activity.
l
With regard to the substituted 1,3,4-triazoles 22–25, the nature
of the substituents seems to manipulate the antimicrobial activity.
Among the benzyltetrazoles 22, 23, the analog 23 (R1 = 4-Cl–C6H4)
revealed an acceptable broad antibacterial spectrum particularly
against Gram positive bacteria, with special high activity against
Convulsions were detected within 30 min of seizure induction.
b
Subcutaneous metrazole test (number of animals protected/number of animals
tested).
Maximal electroshock test (number of animals protected/number of animals
tested).
c
d
Not determined.
Dead following tonic extension.
One animal died following tonic extension.
e
f
S. aureus (MIC 25
lg/mL), and a remarkable antifungal potential
against C. albicans (MIC 25
l
g/mL). Replacement of the benzyl

S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 2410–2422
2417
utilized with accommodation conditions maintained at 25 °C. Di-
methyl sulfoxide (DMSO) was used for dissolving pentylenetetraz-
ole (metrazole) and the test compounds, whereas the control
experiments were performed with solvent alone. The compounds
were administered intraperitoneally (ip) at doses of 30, 100 and
300 mg/kg body weight, 30 min before the induction of seizures.
A minimal time of 30 min subsequent to subcutaneous administra-
tion of metrazole was used for seizure detection.
The results presented in Table 3 reveal that, out of the com-
pounds tested, five compounds namely; 3, 5, 13, 21, and 24 were
able to display noticeable anticonvulsant activity in both the ScMet
and MES tests with percentage protection range of 33–100%. It
could be obviously recognized that the prominently active com-
pounds 3, 5 and 21 exerted their anticonvulsant activity at
100 mg/kg dose level, however, no activity was observed at doses
of 30 or 300 mg/kg. Among these, compounds 5 (% protection:
ScMet: 100 and MES: 67%) and 21 (% protection: ScMet: 80 and
MES: 67%) were proved to be the most active members in this
study. Furthermore, at a dose level of 100 mg/kg, compound 18
showed mild activity only in the ScMet test (33% protection),
whereas, compounds 22 and 30 revealed the same activity only
in the MES test (33% protection). The rest of the tested compounds
namely; 4, 6, 15 and 16 lacked any anticonvulsant activity in both
tests at the same dose levels used.
Further interpretation of the results revealed that, in spite of the
observable activity of the benzyl derivatives 3 and 5 in both tests,
their phenyl analogs 4 and 6 were devoid of any anticonvulsant
activity. Moreover, it is clear that good anticonvulsant activity
was confined to compounds including a free oxygen functionality
such as carbonyl or hydroxyl groups (compounds 3 and 5, respec-
tively), and to those containing an alkoxy group comprising an
open-chain substituted hydrazinocarbonyl group attached to the
methoxy substituent at the ethylene bridge as in the azomethine
13 and the substituted thiosemicarbazide 21. This observation is
concordant with previous literature findings for the structural
requirements of anticonvulsant activity for arylalkyl imidazoles
represented by Nafimidone and Denzimole.46–48 Cyclization of the
alkoxy side chain to different heterocyclic ring systems as in com-
pounds 15, 16, 18, 22 and 24, resulted in an obvious reduction in
the anticonvulsant potential. In this view, while compounds com-
prising the 1,3,4-oxadiazolyl (18) and the 1,3,4-triazolyl (22 and
24) moieties exhibited mild anticonvulsant activity, those contain-
ing a pyrazolyl counterpart (15 and 16) were devoid of any activity.
open chain counterparts like the esters, acids, acid hydrazides and
thiosemicarbazides showed weak or no antimicrobial activity (MIC
P100 lg/mL). However, cyclization of the pre-mentioned func-
tionalities provided a variety of heterocycles with improved anti-
bacterial and antifungal spectra. Among these, the 1,3,4-
oxadiazole and 1,3,4-triazole derivatives revealed significant broad
spectrum antimicrobial activities. Compounds 16, 18, 24 and 27
proved to be the most active antibacterial members within this
study with a considerable broad spectrum against all the Gram po-
sitive and negative strains tested. Among these, particular anti-
Gram positive activity was displayed by compound 18 against S.
aureus that was equipotent to ampicillin (MIC 6.25 lg/mL).
On the other hand, twelve compounds were selected to be
screened for their preliminary anticonvulsant activity against sub-
cutaneous metrazole (ScMet) and maximal electroshock (MES) in-
duced seizures in mice. The results revealed that five compounds
were able to display noticeable anticonvulsant activity in both tests
at 100 mg/kg dose level. Good anticonvulsant activity was confined
to compounds including a free oxygen functionality such as carbonyl
or hydroxyl groups, and to those containing an alkoxy group with an
open chain substituent. Cyclization of the alkoxy side chain to differ-
ent heterocyclic ring systems resulted in an obvious reduction in the
anticonvulsant potential. Compounds 5 and 21 were proved to be
the most active anticonvulsant members in this study with special
high activity in the ScMet assay (% protection: 100% and 80%, respec-
tively). Finally, these compounds represent new structure scaffolds
that could be further optimized for future development of more po-
tent and selective antimicrobial and/or anticonvulsant agents.
4. Experimental
4.1. Chemistry
Melting points were determined in open glass capillaries on a
Stuart melting point apparatus and were uncorrected. The infrared
(IR) spectra were recorded on Perkin–Elmer 1430 infrared spectro-
photometer using the KBr plate technique. 1H NMR, 13C NMR, NOE,
HMQC and HMBC spectra were determined on Jeol spectrometer
(500 MHz) at the Microanalytical unit, Faculty of Science, Alexan-
dria University using tetramethylsilane (TMS) as internal standard
and DMSO-d6 as solvent (chemical shifts in d, ppm). Splitting pat-
terns were designated as follows: s: singlet; d: doublet; dd: doublet
of doublet; t: triplet; m: multiplet. Mass spectra were carried out
using a Schimadzu GCMS-QP-1000EX mass spectrometer at 70ev,
Faculty of Science, Cairo University. Microanalyses were performed
at the Microanalytical Unit, Faculty of Science, Cairo University and
at the Central lab, Faculty of Pharmacy, Alexandria University,
Egypt. The found values were within 0.4% of the theoretical values.
Silica gel 60GF 254 was used for column chromatography. Follow
up of the reactions and checking the purity of the compounds were
made by TLC on silica gel-protected glass plates and the spots were
detected by exposure to UV-lamp at k 254.
3. Conclusion
The main aim of the present investigation is to synthesize and
investigate the antimicrobial activity of new tetrazole-containing
compounds that are structurally related to the famous antimicro-
bial azole pharmacophore, with the hope of discovering new struc-
ture leads serving as potential broad spectrum antimicrobial
agents. The obtained results revealed that twenty compounds were
able to display variable growth inhibitory effects on the tested
Gram positive and Gram negative bacterial strains. Meanwhile, five
compounds exhibited moderate antifungal activity against C. albi-
cans, whereas, only one analog was able to inhibit the growth of
A. fumigatus. In general, most of the tested compounds showed bet-
ter activity against the Gram positive rather than the Gram nega-
tive bacteria, particularly S. aureus and B. subtilis. Structurally, the
antimicrobial potential of the active compounds is dependent on
the nature of the substituents: remarkable antibacterial activity
was encountered with tetrazoles containing a phenyl substituent,
while the obtained antifungal activity was confined to those com-
prising the benzyl group. Moreover, the unsubstituted ethanone
and ethanol derivatives, together with the analogs substituted with
4.1.1. 1-(4-Chlorophenyl)-2-(5-substituted-1H-tetrazol-1-yl or
2H-tetrazol-2-yl)ethanones (3,4)
A mixture of the appropriate tetrazole 1, 2 (75 mmol), 4-chlor-
ophenacyl bromide (17.6 g, 75 mmol) and anhydrous potassium
carbonate (10.4 g, 75 mmol) in dry acetone (120 mL) was heated
under reflux with stirring for 8 h. The reaction mixture was left
to cool and filtered. The filtrate was evaporated under reduced
pressure and the residual solid mass was triturated with diethyl
ether and the separated solid was filtered, dried and crystallized.
4.1.1.1. 2-(5-Benzyl-1H-tetrazol-1-yl)-1-(4-chlorophenyl)etha-
none (3). White crystals, (ethyl acetate/n-hexane). Yield: 75%;

2418
S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 2410–2422
mp: 130–132 °C; IR (KBr, cmÀ1): 1693 (C@O), 1589 (C@N). 1H
NMR (d ppm): 4.25 (s, 2H, benzyl CH2), 6.32 (s, 2H, N-CH2),
7.15–7.26 (m, 5H, benzyl-H), 7.66, 8.0 (2d, J = 8.4 Hz, 4H, chloro-
phenyl C3,5–H & C2,6–H). 13C NMR (d ppm): 28.36 (benzyl CH2),
53.63 (N–CH2), 127.52 (benzyl C4), 129.07 (benzyl C3,5), 129.47
(chlorophenyl C3,5), 129.60 (benzyl C2,6), 130.83 (chlorophenyl
C2,6), 132.96 (chlorophenyl C1), 135.50 (benzyl C1), 139.91 (chlo-
rophenyl C4), 156.17 (tetrazole-C5), 190.79 (C@O). Anal. Calcd for
C16H13ClN4O (312.75): C, 61.44; H, 4.19; N, 17.91. Found: C,
61.32; H, 4.53; N, 17.97.
(20:1) then with mixtures containing decreasing amounts of petro-
leum ether and finally with methylene chloride alone. The sepa-
rated products were then crystallized.
4.1.3.1. Ethyl2-[2-(5-benzyl-1H-tetrazol-1-yl)-1-(4-chloro-
phenyl)ethoxy]acetate (7). White crystals, (methylene chloride/
petroleum ether 60/80). Yield: 40%; mp: 80–81 °C; IR (KBr,
cmÀ1): 1745 (C@O), 1600 (C@N); 1247, 1133, 1090, 1046 (C–O–
C); 1H NMR (d ppm): 1.10 (t, J = 6.9 Hz, 3H, CH2CH3), 3.76, 3.90
(2d, J = 16.4 Hz, 2H, OCH2), 3.96 (q, J = 6.9 Hz, 2H, CH2CH3), 4.31,
4.34 (2d, J = 16.1 Hz, 2H, benzyl CH2), 4.60 (dd, J = 14.5, 4.2 Hz,
1H, N–CH2), 4.65 (dd, J = 14.5, 8 Hz, 1H, N–CH2), 4.84 (dd, J = 8,
4.2 Hz, 1H, CH), 7.21–7.32 (m, 5H, benzyl-H), 7.35, 7.43 (2d,
J = 8.1 Hz, 4H, chlorophenyl C2,6–H & C3,5–H). Anal. Calcd for
C20H21ClN4O3 (400.86): C, 59.92; H, 5.28; N, 13.98. Found: C,
59.67; H, 5.03; N, 13.72.
4.1.1.2. 1-(4-Chlorophenyl)-2-(5-phenyl-2H-tetrazol-2-yl)etha-
none (4). White crystals, (ethyl acetate/n-hexane). Yield: 85%;
mp: 159–160 °C; IR (KBr, cmÀ1): 1691 (C@O), 1589 (C@N). 1H
NMR (d ppm): 6.72 (s, 2H, N-CH2), 7.50–7.57 (m, 3H, phenyl
C3,4,5–H), 7.66 (d, J = 8.4 Hz, 2H, chlorophenyl C3,5–H), 8.07 (d,
J = 8.4 Hz, 4H, phenyl C2,6–H & chlorophenyl C2,6–H). 13C NMR (d
ppm): 59.57 (N–CH2), 126.91 (phenyl C2,6), 127.32 (phenyl C4),
129.7 (chlorophenyl C3,5), 129.84 (phenyl C3,5), 130.84 (chloro-
phenyl C2,6), 131.18 (phenyl C1), 132.95 (chlorophenyl C1),
140.11 (chlorophenyl C4), 164.93 (tetrazole-C5), 190.97 (C@O).
Anal. Calcd for C15H11ClN4O (298.73): C, 60.31; H, 3.71; N, 18.76.
Found: C, 60.92; H, 3.61; N, 19.16.
4.1.3.2. Ethyl2-[1-(4-chlorophenyl)-2-(5-phenyl-2H-tetrazol-2-
yl)ethoxy]acetate (8). White crystals, (methylene chloride/petro-
leum ether 60/80). Yield: 35%; mp: 68–70 °C; IR (KBr, cmÀ1):
1745 (C@O), 1596 (C@N); 1207, 1125, 1090, 1045 (C–O–C); 1H
NMR (d ppm): 1.0 (t, J = 6.9 Hz, 3H, CH2CH3), 3.86 (d, J = 16.4 Hz,
1H, OCH2), 3.91 (q, J = 6.9 Hz, 2H, CH2CH3), 4.04 (d, J = 16.4 Hz,
1H, OCH2), 4.96 (dd, J = 13.6, 3.2 Hz, 1H, N–CH2), 5.11 (dd,
J = 13.6, 8.4 Hz, 1H, N–CH2), 5.16 (dd, J = 8.4, 3.2 Hz, 1H, CH),
7.42, 7.45 (2d, J = 8.4 Hz, 4H, chlorophenyl C2,6–H & C3,5–H),
7.49–7.55 (m, 3H, phenyl C3,4,5–H), 8.03 (d, J = 7.65 Hz, 2H, phenyl
C2,6–H). Anal. Calcd for C19H19ClN4O3 (386.83): C, 58.99; H, 4.95; N,
14.48. Found: C, 58.30; H, 4.87; N, 14.25.
4.1.2. 1-(4-Chlorophenyl)-2-(5-substituted 1H-tetrazol-1-yl or
2H-tetrazol-2-yl)ethanols (5, 6)
To a well stirred suspension of compound 3 or 4 (30 mmol) in
ethanol (80 mL), sodium borohydride (4.5 g, 120 mmol) was added
portion wise over a period of 1 h. Stirring at room temperature was
maintained for further 24 h and the reaction mixture was then
poured onto crushed ice. The separated solid was filtered, washed
with water, dried and crystallized.
4.1.4. 2-[1-(4-Chlorophenyl)-2-(5-substituted 1H-tetrazol-1-yl
or 2H-tetrazol-2-yl)ethoxy]acetic acids (9, 10)
Method A: To a well stirred solution of compounds 5 or 6
(1.5 mmol) in dry tetrahydrofuran (5 mL), sodium hydride
(0.054 g, 2.25 mmol) was added and the mixture was stirred at
room temperature for 1 h. Chloroacetic acid (0.14 g, 1.5 mmol)
was then added and the mixture was left stirred at room temper-
ature overnight. The reaction mixture was evaporated to dryness
under reduced pressure and the remaining residue was triturated
with diethyl ether. The separated solid was filtered, dissolved in
water, neutralized with dil HCl and the obtained product was fil-
tered, dried and crystallized.
4.1.2.1. 2-(5-Benzyl-1H-tetrazol-1-yl)-1-(4-chlorophenyl) etha-
nol (5). White crystals, (ethanol/water). Yield: 87%; mp: 114–
115 °C; IR (KBr, cmÀ1): 3355 (OH), 1598 (C@N); 1H NMR (d
ppm): 4.23, 4.28 (2d, J = 16.4 Hz, 2H, benzyl CH2), 4.43 (dd,
J = 14.2, 7.8 Hz, 1H, N–CH2), 4.52 (dd, J = 14.2, 4 Hz, 1H, N–CH2),
4.92 (dd, J = 7.8, 4 Hz, 1H, CH), 6.04 (s, 1H, OH, D2O exchangeable)
7.19–7.32 (m, 5H, benzyl-H), 7.35, 7.38 (2d, J = 8.4 Hz, 4H, chloro-
phenyl C2,6–H & C3,5–H). Anal. Calcd for C16H15ClN4O (314.77): C,
61.05; H, 4.80; N, 17.80. Found: C, 61.45; H, 4.76; N, 18.27.
MethodB: A mixture of the selected ester 7 or 8 (2.7 mmol) and 5%
sodium hydroxide (2.6 ml) in ethanol (15 mL) was heated under re-
flux for 2 h. After evaporation of the reaction mixture to dryness, the
residue was dissolved in water and acidified with dil HCl. The sepa-
rated solid was filtered, washed with water, dried and crystallized.
4.1.2.2. 1-(4-Chlorophenyl)-2-(5-phenyl-2H-tetrazol-2-yl)etha-
nol (6). White crystals, (ethanol). Yield: 89%; mp: 120–121 °C; IR
(KBr, cmÀ1): 3320 (OH), 1592 (C@N); 1H NMR (d ppm): 4.85–4.94
(m, 2H, N–CH2), 5.20–5.30 (m, 1H, CH), 5.99 (d, J = 5 Hz, 1H, OH,
D2O exchangeable), 7.39, 7.45 (2d, J = 8.4 Hz, 4H, chlorophenyl
C2,6–H & C3,5–H), 7.50–7.56 (m, 3H, phenyl C3,4,5–H), 8.01–8.06
(m, 2H, phenyl C2,6–H). Anal. Calcd for C15H13ClN4O (300.74): C,
59.91; H, 4.36; N, 18.63. Found: C, 60.37; H, 4.31; N, 18.88.
4.1.4.1. 2-[2-(5-Benzyl-1H-tetrazol-1-yl)-1-(4-chlorophenyl)eth-
oxy]acetic acid (9). White crystals (ethanol). Yield: 35% (method
A), 85% (method B); mp: 132–133 °C; IR (KBr, cmÀ1): 3166–2625
(OH), 1730 (C@O), 1596 (C@N); 1245, 1135, 1088, 1049 (C–O–C);
1H NMR (d ppm): 3.70, 3.86 (2d, J = 16.4 Hz, 2H, OCH2), 4.30, 4.36
(2d, J = 16.4 Hz, 2H, benzyl CH2), 4.61 (dd, J = 14.89, 4.78 Hz, 1H,
N–CH2), 4.65 (dd, J = 14.89, 8.22 Hz, 1H, N–CH2), 4.86 (dd, J = 8.22,
4.78 Hz, 1H, CH), 7.22–7.31 (m, 5H, benzyl-H), 7.33, 7.42 (2d,
J = 8.4 Hz, 4H, chlorophenyl-H), 12.67 (br s, 1H, OH, D2O exchange-
able). Anal. Calcd for C18H17ClN4O3 (372.81): C, 57.99; H, 4.60; N,
15.03. Found: C, 58.33; H, 4.75; N, 15.04.
4.1.3. Ethyl2-[1-(4-chlorophenyl)-2-(5-substituted 1H-tetrazol-
1-yl or 2H-tetrazol-2-yl)ethoxy]acetates (7, 8)
To a well stirred solution of compounds 5 or 6 (10 mmol) in dry
tetrahydrofuran (15 mL), sodium hydride (0.36 g, 15 mmol) was
added portion wise and the mixture was stirred at room tempera-
ture for 1 h. Ethyl bromoacetate (1.67 g, 1.1 mL, 10 mmol) was
added dropwise and the reaction mixture was left stirred at room
temperature overnight. The reaction mixture was concentrated un-
der reduced pressure, poured onto cold water (100 mL) and ex-
tracted with methylene chloride (2 Â 50 mL). The combined
organic extracts were washed with water (2 Â 30 mL), dried over
anhydrous sodium sulfate, concentrated under vacuum to a small
volume and chromatographed on a silica gel column eluting first
with a mixture of methylene chloride/petroleum ether (60/80)
4.1.4.2. 2-[1-(4-Chlorophenyl)-2-(5-phenyl-2H-tetrazol-2-
yl)ethoxy]acetic acid (10). White crystals (ethanol/water). Yield:
34% (method A), 82% (method B); mp: 126–128 °C; IR (KBr,
cmÀ1): 3166–2625 (OH), 1701 (C@O), 1593 (C@N); 1235, 1115,
1088, 1045 (C–O–C); 1H NMR (d ppm): 3.78, 3.96 (2d, J = 16.8 Hz,
2H, OCH2), 4.96 (dd, J = 13.74, 4.59 Hz, 1H, N–CH2), 5.09 (dd,

S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 2410–2422
2419
J = 13.74, 8.02 Hz, 1H, N–CH2), 5.15 (dd, J = 8.02, 4.59 Hz, 1H, CH),
7.42 (s, 4H, chlorophenyl-H), 7.49–7.55 (m, 3H, phenyl C3,4,5–H),
8.01 (d, J = 7.1 Hz, 2H, phenyl C2,6–H), 12.68 (s, 1H, OH, D2O
exchangeable). Anal. Calcd for C17H15ClN4O3 (358.78): C, 56.91;
H, 4.21; N, 15.62. Found: C, 55.60; H, 4.31; N, 15.42.
J = 15.8 Hz, 2H, OCH2, cis conformer), 4.28, 4.45 (2d,
J = 16.1 Hz, 2H, OCH2; trans conformer), 4.8–5.4 (m, 3H, N–
CH2 & CH), 7.40–7.70 (m, 11H, phenyl C3,4,5–H & two chloro-
phenyl-H), 7.84 (s, 1H, N@CH, cis conformer), 8.03 (m, 2H, phe-
nyl C2,6–H), 8.17 (s, 1H, N@CH, trans conformer), 11.12, 11.39
(2s, 2H, 2CONH, cis and trans conformers). Anal. Calcd for
C24H20Cl2N6O2 (495.36): C, 58.19; H, 4.07; N, 16.97. Found: C,
57.71; H, 4.09; N, 16.66.
4.1.5. 2-[1-(4-Chlorophenyl)-2-(5-substituted 1H-tetrazol-1-yl
or 2H-tetrazol-2-yl)ethoxy]acetohydrazides (11, 12)
To a solution of the acetate ester 7 or 8 (20 mmol) in absolute
ethanol (50 mL), hydrazine hydrate 98% (8 g, 160 mmol) was
added and the reaction mixture was heated under reflux for 4 h.
For compound 11, the reaction mixture was evaporated to dryness
under reduced pressure and the residual mass was crystallized. For
compound 12, the reaction mixture was left overnight and the sep-
arated solid product was filtered, dried and crystallized.
4.1.7. 2-[1-(4-Chlorophenyl)-2-(5-substituted1H-tetrazol-1-yl or
2H-tetrazol-2-yl)ethoxy]-1-(3,5-dimethyl-1H-pyrazol-1-
yl)ethanones (15, 16)
A mixture of equimolar amounts of 11 or 12 (1 mmol) and acet-
yl acetone (1 mmol) in ethanol (10 mL) was heated under reflux for
6 h. The reaction mixture was concentrated to approximately half
of its volume and allowed to cool to room temperature. The sepa-
rated solid was filtered, washed with ether, dried and crystallized.
4.1.5.1. 2-[2-(5-Benzyl-1H-tetrazol-1-yl)-1-(4-chlorophenyl)eth-
oxy]acetohydrazide (11). White crystals (ethanol/water). Yield:
73%; mp: 30–31 °C; IR (KBr, cmÀ1): 3330, 3274 (NH2, NH), 1682
(C@O), 1615 (C@N); 1200, 1104, 1040 (C–O–C); 1H NMR (d ppm):
3.76 (s, 2H, NH2, D2O exchangeable), 3.80, 3.86 (2d, J = 16.4 Hz, 2H,
OCH2), 4.33, 4.37 (2d, J = 16.3 Hz, 2H, benzyl CH2), 4.60 (dd, J = 14.6,
4.5 Hz, 1H, N–CH2), 4.64 (dd, J = 14.6, 7.8 Hz, 1H, N–CH2), 4.86 (dd,
J = 7.8, 4.5 Hz, 1H, CH), 7.20–7.33 (m, 5H, benzyl-H), 7.37, 7.46 (2d,
J = 8.2 Hz, 4H, chlorophenyl C2,6–H & C3,5–H), 8.84 (s, 1H, NH, D2O
exchangeable). Anal. Calcd for C18H19ClN6O2Á1H2O (404.86): C,
53.40; H, 5.23; N, 20.75. Found: C, 53.75; H, 4.84; N, 20.60.
4.1.7.1. 2-[2-(5-Benzyl-1H-tetrazol-1-yl)-1-(4-chlorophenyl)eth-
oxy]-1-(3,5-dimethyl-1H-pyrazol-1-yl)ethanone
(15). White
crystals (ethanol). Yield: 53%; mp: 152–153 °C; IR (KBr, cmÀ1):
1740 (C@O), 1655 (C@N); 1210, 1131, 1090 (C–O–C); 1H NMR (d
ppm): 2.05, 2.39 (2s, 6H, 2CH3), 4.34, 4.37 (2d, J = 16.8 Hz, 2H, ben-
zyl CH2), 4.49–4.70 (m, 4H, OCH2 & N–CH2), 4.97 (dd, J = 6.85, 4.6
Hz, 1H, CH), 6.12 (s, 1H, pyrazole C4–H), 7.20–7.44 (m, 9H, ben-
zyl-H & chlorophenyl-H). Anal. Calcd for C23H23ClN6O2 (450.92):
C, 61.26; H, 5.14; N, 18.64. Found: C, 61.66; H, 4.99; N, 18.39.
4.1.5.2. 2-[1-(4-Chlorophenyl)-2-(5-phenyl-2H-tetrazol-2-yl)eth-
oxy]acetohydrazide (12). White crystals (ethanol). Yield: 75%; mp:
130–131 °C; IR (KBr, cmÀ1): 3334–3274 (NH2, NH), 1685 (C@O),
1620 (C@N); 1202, 1104, 1045 (C–O–C); 1H NMR (d ppm): 3.79 (s,
2H, NH2, D2O exchangeable), 4.22 (s, 2H, OCH2), 4.94 (dd, J = 13.7,
4.6 Hz, 1H, N–CH2), 5.12 (dd, J = 13.7, 8 Hz, 1H, N–CH2), 5.20 (dd,
J = 8, 4.6 Hz, 1H, CH), 7.45 (s, 4H, chlorophenyl-H), 7.52–7.61 (m,
3H, phenyl C3,4,5–H), 8.0–8.1 (m, 2H, phenyl C2,6–H), 8.92 (s, 1H,
NH, D2O exchangeable). Anal. Calcd for C17H17ClN6O2 (372.81): C,
54.77; H, 4.60. N, 22.54. Found: C, 54.59; H, 4.70; N, 22.43.
4.1.7.2. 2-[1-(4-Chlorophenyl)-2-(5-phenyl-2H-tetrazol-2-yl)eth-
oxy]-1-(3,5-dimethyl-1H-pyrazol-1-yl)ethanone
(16). White
crystals (ethanol). Yield: 65%; mp: 160–161 °C; IR (KBr, cmÀ1):
1742 (C@O), 1655 (C@N); 1201, 1137, 1090 (C–O–C); 1H NMR (d
ppm): 2.03, 2.33 (2s, 6H, 2CH3), 4.59, 4.73 (2d, J = 17.5 Hz, 2H,
OCH2), 4.99 (dd, J = 13.76, 3.0 Hz, 1H, N–CH2), 5.14 (dd, J = 13.76,
7.47 Hz, 1H, N–CH2), 5.26 (dd, J = 7.47, 3.0 Hz, 1H, CH), 6.08 (s,
1H, pyrazole C4–H), 7.40–7.60 (m, 7H, phenyl C3,4,5–H & chloro-
phenyl-H), 8.0 (d, J = 7.1 Hz, 2H, phenyl C2,6–H). Anal. Calcd for
C22H21ClN6O2 (436.89): C, 60.48; H, 4.84; N, 19.24. Found: C,
60.17 H, 4.89; N, 18.92.
4.1.6. (E)-2-[1-(4-Chlorophenyl)-2-(5-substituted-1H-tetrazol-1-
yl or 2H-tetrazol-2-yl)ethoxy]-N1-(4-chlorobenzylidene)aceto-
hydrazides (13,14)
A solution of the selected acetohyrazide 11 or 12 (1 mmol) in
ethanol (10 mL) was heated under reflux with an equimolar
amount of 4-chlorobenzaldehyde for 2 h. The reaction mixture
was left for an overnight and the precipitated solid was filtered,
washed with ethanol, dried and crystallized.
4.1.8. 5-{[1-(4-Chlorophenyl)-2-(5-substituted1H-tetrazol-1-yl
or 2H-tetrazol-2-yl)ethoxy]methyl}-1,3,4-oxadiazole-2-thiols
(17, 18)
A mixture of 11 or 12 (5 mmol), potassium hydroxide (5 mmol)
and carbon disulfide (5 mL) in ethanol (50 ml) was heated under
reflux for 12 h. The reaction mixture was concentrated, cooled, di-
luted with water and acidified with dil HCl. The separated solid
was filtered, washed with ethanol, dried and crystallized.
4.1.6.1. (E)-2-[2-(5-Benzyl1H-tetrazol-1-yl)-1-(4-chlorophenyl)eth-
oxy]-N1-(4-chlorobenzylidene)acetohydrazide (13). White crystals
(ethanol). Yield: 82%; mp: 191–192 °C; IR (KBr, cmÀ1): 3312 (NH),
1697 (C@O), 1607 (C@N); 1232, 1124, 1087, 1041 (C–O–C); 1H NMR
(d ppm): 3.78, 3.87 (2d, J = 14.55 Hz, OCH2, cis conformer), 4.17–4.38
(m, 4H, benzyl CH2 & OCH2; trans conformer), 4.64 (dd, J = 15.27,
3.82 Hz, 1H, N–CH2), 4.72 (dd, J = 15.27, 7.85 Hz, 1H, N–CH2), 4.89
(dd, J = 7.85, 3.82 Hz, 1H, CH), 7.20–7.28 (m, 13H, benzyl-H & two chlo-
rophenyl-H), 8.13, 8.68 (2s, 2H, two N@CH, cis and trans conformers),
11.20, 11.44 (2s, 2H, 2CONH, cis and trans conformers), Anal. Calcd
for C25H22Cl2N6O2 (509.39): C, 58.95; H, 4.35; N, 16.50. Found: C,
58.58; H, 3.84; N, 15.74.
4.1.8.1. 5-{[2-(5-Benzyl-1H-tetrazol-1-yl)-1-(4-chlorophenyl)eth-
oxy]methyl}-1,3,4-oxadiazole-2-thiol (17). White crystals (ethanol).
Yield: 73%; mp: 230–231 °C; IR (KBr, cmÀ1): 3125 (NH), 2788 (SH),
1635, 1598 (C@N), 1513, 1270, 1095, 940 (NCS), 1199, 1129, 1061
(C–O–C); 1H NMR (d ppm): 4.24 (s, 2H, OCH2), 4.28, 4.32 (2d,
J = 14.5 Hz, 2H, benzyl CH2), 4.62 (dd, J = 14.5, 4 Hz, 1H, N–CH2), 4.72
(dd, J = 14.5, 8.4 Hz, 1H, N–CH2), 4.91 (dd, J = 8.4, 4 Hz, 1H, CH), 7.17–
7.32 (m, 5H, benzyl-H), 7.34, 7.43 (2d, J = 7.25 Hz, 4H, chlorophenyl-
H). 14.5 (s, 1H, SH, D2O exchangeable). Anal. Calcd for C19H17ClN6O2S
(428.90): C, 53.21; H, 4.00. Found: C, 53.01; H, 4.55; N. 19.64.
4.1.8.2. 5-{[1-(4-Chlorophenyl)-2-(5-phenyl-2H-tetrazol-2-yl)-
ethoxy]methyl}-1,3,4-oxadiazole-2-thiol (18). White crystals
(ethanol). Yield: 72%; mp: 172–173 °C; IR (KBr, cmÀ1): 3136
(NH), 2774 (SH), 1619, 1595 (C@N), 1511, 1288, 1083, 940 (NCS),
1249, 1153, 1054 (C–O–C); 1H NMR (d ppm): 4.43, 4.51 (2d,
J = 14.2 Hz, 2H, OCH2), 5.01 (dd, J = 17, 4.15 Hz, 1H, N–CH2), 5.20
4.1.6.2. (E)-2-[1-(4-Chlorophenyl)-2-(5-phenyl-2H-tetrazol-
2yl)ethoxy]-N1-(4-chlorobenzylidene)acetohyrazide
(14). White crystals (ethanol). Yield: 85%; mp: 198–199 °C; IR
(KBr, cmÀ1): 3188 (NH), 1685 (C@O), 1594 (C@N), 1232, 1121,
1086, 1011 (C–O–C); 1H NMR (d ppm): 3.93, 3.97 (2d,

2420
S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 2410–2422
(dd, J = 17, 7.65 Hz, 1H, N–CH2), 5.30 (dd, J = 7.65, 4.15 Hz, 1H, CH),
7.40–7.70 (m, 7H, phenyl C3,4,5–H & chlorophenyl-H), 8.02–8.09
(m, 2H, phenyl C2,6–H), 14.44 (s, 1H, SH, D2O exchangeable). Anal.
Calcd for C18H15ClN6O2S (414.87): C, 52.11; H, 3.64; N, 20.26.
Found: C, 51.88; H, 4.05; N, 19.93.
CH2), 4.56 (dd, J = 14.5, 4.4 Hz, 1H, N–CH2), 4.58 (dd, J = 14.5,
6.8 Hz, 1H, N–CH2), 4.75 (dd, J = 6.8, 4.4 Hz, 1H, CH), 7.02–7.57
(m, 14H, Ar–H), 13.98 (s, 1H, SH, D2O exchangeable). Anal. Calcd
for C25H22ClN7OS (504.01): C, 59.58; H, 4.40; N, 19.45. Found: C,
59.81; H, 4.80; N, 19.10.
4.1.9. N4-Aryl-N1-{[1-(4-chlorophenyl)-2-(5-substituted1H-tetrazol-
1-yl or 2H-tetrazol-2-yl)ethoxy]acetyl} thiosemicarbazides (19–21)
A mixture of equimolar amounts of 11 or 12 (5 mmol) and the
selected aryl isothiocyanate (5 mmol) in absolute ethanol (20 mL)
was stirred at room temperature for 3 h. The reaction mixture
cleared then a heavy white precipitate separated out. It was fil-
tered, washed with ether, dried and crystallized.
4.1.10.2. 5-{[2-(5-Benzyl-1H-tetrazol-1-yl)-1-(4-chlorophenyl)-
ethoxy]methyl}-4-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol
(23). White crystals (ethanol/water). Yield: 84%; mp: 108–110 °C;
IR (KBr, cmÀ1): 3422 (NH), 2761 (SH), 1597 (C@N); 1220, 1092,
1013 (C–O–C); 1H NMR (d ppm): 3.98, 4.02 (2d, J = 12.25 Hz, 2H,
OCH2), 4.12, 4.16 (2d, J = 16.05 Hz, 2H, benzyl CH2), 4.49 (dd,
J = 14.53, 3.8 Hz, 1H, N–CH2), 4.58 (dd, J = 14.53, 7.25 Hz, 1H, N–
CH2), 4.73 (dd, J = 7.25, 3.8 Hz, 1H, CH), 7.04–7.52 (m, 13H, Ar–
H), 14.0 (s, 1H, SH, D2O exchangeable). Anal. Calcd for
C25H21Cl2N7OS (538.45): C, 55.76; H, 3.93; N, 18.21. Found: C,
55.51; H, 3.62; N, 17.96.
4.1.9.1. N1-{[2-(5-Benzyl-1H-tetrazol-1-yl)-1-(4-chlorophenyl)eth-
oxy]acetyl}-N4-phenylthiosemicarbazide
(19). White
crystals
(dioxane/water). Yield: 80%; mp: 186–187 °C; IR (KBr, cmÀ1): 3250,
3182 (NH), 1697 (C@O), 1597 (C@N), 1552, 1278, 1089, 973 (NCS),
1237, 1124, 1058, 1032 (C–O–C); 1H NMR (d ppm): 3.77, 3.86 (2d,
J = 14.5 Hz, 2H, OCH2), 4.30 (s, 2H, benzyl CH2), 4.66 (dd, J = 15.28,
4.6 Hz, 1H, N–CH2), 4.71 (dd, J = 15.28, 6.87 Hz, 1H, N–CH2), 4.92
(dd, J = 6.87, 4.6 Hz, 1H, CH), 7.12–7.32 (m, 10H, benzyl-H & phe-
nyl-H), 7.34, 7.43 (2d, J = 8.4 Hz, 4H, chlorophenyl-H), 9.58 (s, 2H,
NHC@S & NH-phenyl, D2O exchangeable), 9.93 (s, 1H, NH–C@O).
Anal. Calcd for C25H24ClN7O2S (522.02): C, 57.52; H, 4.63; N, 18.78.
Found: C, 57.84; H, 5.00; N, 18.77.
4.1.11. 5-{[1-(4-Chlorophenyl)-2-(5-phenyl-2H-tetrazol-2-yl)
ethoxy]methyl}-4-substituted4H-1,2,4-triazole-3-thiols (24, 25)
A mixture of equimolar amounts of 12 (0.75 g, 2 mmol) and the
proper aryl isothiocyanate (2 mmol) in ethanol (20 mL) was heated
under reflux for 3 h. The reaction mixture was left to attain room
temperature and the precipitated product was filtered, washed
with ethanol, dried and crystallized.
4.1.11.1. 5-{[1-(4-Chlorophenyl)-2-(5-phenyl-2H-tetrazol-2-
yl)ethoxy]methyl}-4-phenyl-4H-1,2,4-triazole-3-thiol
4.1.9.2. N1-{[2-(5-Benzyl-1H-tetrazol-1-yl)-1-(4-chlorophenyl)eth-
oxy]acetyl}-N4-(4-chlorophenyl)thiosemicarbazide
(20). White
(24). White crystals (ethanol). Yield: 82%; mp: 218–219 °C; IR
(KBr, cmÀ1): 3421 (NH), 2771 (SH), 1595 (C@N); 1219, 1092,
1045 (C–O–C); 1H NMR (d ppm): 4.09 (s, 2H, OCH2), 4.83 (dd,
J = 13.75, 4 Hz, 1H, N–CH2), 4.96 (dd, J = 13.75, 8.21 Hz, 1H, N–
CH2), 5.04 (dd, J = 8.21, 4 Hz, 1H, CH), 7.14–7.41 (m, 9H, N-phe-
nyl-H & chlorophenyl-H), 7.51–7.57 (m, 3H, phenyl C3,4,5–H), 8.01
(d, J = 7.63 Hz, 2H, phenyl C2,6–H), 13.94 (s, 1H, SH, D2O exchange-
able). Anal. Calcd for. C24H20ClN7OS (489.98): C, 58.83 H, 4.11; N,
20.01. Found: C, 58.81; H, 4.80; N, 19.91
crystals (dioxane/water). Yield: 84%; mp: 179–180 °C; IR (KBr,
cmÀ1): 3255, 3182 (NH), 1700 (C@O), 1599 (C@N), 1558, 1279,
1
1089, 975 (NCS), 1196, 1126, 1034 (C–O–C); H NMR (d ppm): 3.77,
3.86 (2d, J = 14.5 Hz, 2H, OCH2), 4.3 (s, 2H, benzyl CH2), 4.63 (dd,
J = 12.23, 6.5 Hz, 1H, N–CH2), 4.67 (dd, J = 12.23, 5.35 Hz, 1H, N–
CH2), 4.93 (dd, J = 6.5, 5.35 Hz, 1H, CH), 7.12–7.33 (m, 9H, benzyl-H
& N4-chlorophenyl-H), 7.35, 7.43 (2d, J = 8.4 Hz, 4H, chlorophenyl-
H), 9.57 (s, 2H, NHC@S & NHC6H4–Cl, D2O exchangeable), 9.92 (s,
1H, NHC@O, D2O exchangeable). Anal. Calcd for C25H23Cl2N7O2S
(556.47): C, 53.96; H, 4.17; N, 17.62. Found: C, 53.70; H, 4.01; N, 17.50.
4.1.11.2. 5-{[1-(4-Chlorophenyl)-2-(5-phenyl-2H-tetrazol-2-
yl)ethoxy]methyl}-4-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol
(25). White crystals (ethanol). Yield: 84%; mp: 204–205 °C; IR
(KBr, cmÀ1): 3415 (NH), 2761 (SH), 1580 (C@N),1246, 1087, 1046
(C–O–C); 1H NMR (d ppm): 4.13 (s, 2H, OCH2), 4.85 (dd, J = 13.75,
3.8 Hz, 1H, N–CH2), 4.99 (dd, J = 13.75, 8.4 Hz, 1H, N–CH2), 5.06
(dd, J = 8.4, 3.8 Hz, 1H, CH), 7.18, 7.21, 7.35, 7.39 (4d, J = 8.4 Hz,
8H, N-chlorophenyl-H & chlorophenyl-H), 7.50–7.57 (m, 3H, phe-
nyl C3,4,5–H). 8.02 (d, J = 7 Hz, 2H, phenyl C2,6–H), 13.99 (s, 1H,
SH, D2O exchangeable). Anal. Calcd for C24H19Cl2N7OS (524.43):
C, 54.97; H, 3.65; N, 18.70. Found: C,54.52; H, 3.48; N, 18.01.
4.1.9.3. N4-Phenyl-N1-{[2-(5-phenyl-2H-tetrazol-2-yl)-1-(4-
chlorophenyl)ethoxy]acetyl}thiosemicarbazide
(21). White
crystals (dioxane/water). Yield: 69%; mp: 175–176 °C; IR (KBr,
cmÀ1): 3347, 3322, 3221 (NH), 1727 (C@O), 1596 (C@N); 1513,
1265, 1088, 932 (NCS), 1210, 1108, 1041 (C–O–C); 1H NMR (d
ppm): 3.86, 3.93 (2d, J = 14.55 Hz, 2H, OCH2), 5.0 (dd, J = 6.85,
5.35 Hz, 1H, CH), 5.13 (dd, J = 17.5, 6.85 Hz, 1H, N–CH2), 5.17 (dd,
J = 17.5, 5.35 Hz, 1H, N–CH2), 7.1–7.54 (m, 12H, phenyl C3,4,5–H,
N4-phenyl-H & chlorophenyl-H), 7.95–8.02 (m, 2H, phenyl C2,6
–
H), 9.51 (s, 2H, NHC@S & –NH phenyl, D2O exchangeable). Anal.
Calcd for C24H22ClN7O2S (508.0): C, 56.74; H, 4.37; N, 19.30. Found:
C, 56.46; H, 4.41; N, 19.06.
4.1.12. 5-Phenylamino-2-{[2-(5-substituted1H-tetrazol-1-yl or
2H-tetrazol-2-yl)ethoxy]methyl}-1,3,4-oxadiazoles (26, 27)
A mixture of the selected thiosemicarbazide 19 or 21 (1 mmol),
freshly prepared yellow mercuric oxide (0.22 g, 1 mmol) in dioxane
(20 mL) was heated under reflux for 4 h then filtered while hot. The
filtrate was evaporated to dryness under reduced pressure and the
remaining residue was triturated with diethyl ether, then filtered
and crystallized.
4.1.10. 5-{[2-(5-Benzyl-1H-tetrazol-1-yl)-1-(4-chlorophenyl)
ethoxy]methyl}-4-substituted 4H-1,2,4-triazole-3-thiols (22, 23)
A suspension of the selected thiosemicarbazide 19 or 20
(1.1 mmol) in 10 mL 5% Na2CO3 solution was heated under reflux
for 2 h. The reaction mixture was cooled, filtered and the filtrate
was acidified using 2 N HCl. The obtained precipitate was filtered,
washed with water, dried and crystallized.
4.1.12.1. 2-{[2-(5-Benzyl-1H-tetrazol-1-yl) 1-(4-chlorophenyl)
ethoxy]methyl}-5-phenylamino-1,3,4-oxadiazole (26). Whitecrys-
tals (CH2Cl2/ethanol). Yield: 52%; mp: 114–116 °C; IR (KBr, cmÀ1):
3259 (NH), 1617 (C@N), 1247, 1208, 1110, 1034 (C–O–C); 1H NMR (d
ppm): 4.0–4.09 (m, 4H, OCH2 & benzyl CH2), 4.44 (dd, J = 14.73,
4.78 Hz, 1H, N–CH2), 4.50 (dd, J = 14.73, 7.66 Hz, 1H, N–CH2), 4.67
(dd, J = 7.66, 4.78 Hz, 1H, CH), 7.0–7.54 (m, 14 H, benzyl-H, phenyl-H
4.1.10.1. 5-{[2-(5-Benzyl-1H-tetrazol-1-yl)-1-(4-chlorophenyl)-
ethoxy]methyl}-4-phenyl-4H-1,2,4-triazole-3-thiol (22). White
crystals (ethanol/water). Yield: 85%; mp: 191–192 °C; IR (KBr,
cmÀ1): 3433 (NH), 2766 (SH), 1593 (C@N), 1249, 1108, 1054 (C–
O–C); 1H NMR (d ppm): 4.06 (s, 2H, OCH2), 4.17 (s, 2H, benzyl

S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 2410–2422
2421
& chlorophenyl-H), 10.45 (s, 1H, NH, D2O exchangeable). Anal. Calcd
for C25 H22ClN7O2 (487.94): C, 61.54; H, 4.54; N, 20.09. Found: C,
61.24; H, 4.63; N, 20.34.
of the test compound in DMSO (1 mg/mL) were placed on an agar
plate seeded with the appropriate test organism in triplicates. The
utilized test organisms were: S. aureus (ATCC 6538), B. subtilis
(NRRL B-14819) and M. luteus (ATCC 21881) as examples of Gram
positive bacteria and E. coli (ATCC 25922), P. aeruginosa (ATCC
27853) and K. pneumonia (clinical isolate) as examples of Gram
negative bacteria. They were also evaluated for their in vitro anti-
fungal potential against C. albicans, C. tropicalis and C. krusei as rep-
resentatives of fungi and A. niger, A. fumigatus and T. rubrum as
examples of moulds. All of these fungal strains were clinical iso-
lates, identified with conventional morphological and biochemical
methods. Ampicillin trihydrate (antibiotic), clotrimazole and mico-
nazole (antifungals) were used as reference standards. DMSO alone
was used as control at the same above-mentioned concentration.
The plates were incubated at 37 °C for 24 h for bacteria and for
7 days for fungi. Compounds that showed significant growth inhi-
bition zones (P14 mm) using the twofold serial dilution tech-
nique, were further evaluated for their minimal inhibitory
concentrations (MICs).
4.1.12.2. 2-{[1-(4-chlorophenyl)-2-(5-Phenyl-2H-tetrazol-2-
yl)ethoxy]methyl}-5-phenylamino-1,3,4-oxadiazole (27). White
crystals (CH2Cl2/ethanol). Yield: 49%; mp: 127–128 °C; IR (KBr,
cmÀ1): 3314 (NH), 1607 (C@N), 1260, 1228, 1180, 1031 (C–O–C);
1H NMR (d ppm): 4.14, 4.17 (2d, J = 12.58 Hz, 2H, OCH2), 4.82
(dd, J = 13.75, 4.4 Hz, 1H, N–CH2), 4.96 (dd, J = 13.75, 8.23 Hz, 1H,
N–CH2), 5.05(dd, J = 8.23, 4.4 Hz, 1H, CH), 7.12–7.43 (m, 9H, phe-
nylamino-H & chlorophenyl-H), 7.48–7.58 (m, 3H, phenyl C3,4,5
–
H), 8.01 (d, J = 7 Hz, 2H, phenyl C2,6–H), 10.36 (s, 1H, NH, D2O
exchangeable). Anal. Calcd for C24H20ClN7O2 (473.91): C, 60.82;
H, 4.25; N, 20.69. Found: C, 60.57; H, 4.22; N, 20.78.
4.1.13. General procedure for the synthesis of 6-(4-chlorophenyl)-
5,6-dihydro-11H-tetrazolo[1,5-a]benz[d]azepine (30) and 2-(4-
chlorostyryl)-5-phenyl-2H-tetrazole (31)
The appropriate thiosemicarbazide 19 or 21 (1 mmol) was dis-
solved in ice-cold concd H2SO4 (4 mL) and stirred for 15 min at
0 °C followed by further stirring for 15 min at room temperature.
The reaction mixture was poured onto crushed ice and the ob-
tained precipitate was filtered, washed with water and crystallized.
4.2.1.2. In vitro antibacterial activity (minimal inhibitory con-
centration (MIC) measurement). The microdilution susceptibility
test in Müller–Hinton Broth (Oxoid) was used for the determina-
tion of antibacterial activity.43 Stock solutions of the tested com-
pounds and ampicillin trihydrate were prepared in DMSO at
4.1.13.1. 6-(4-Chlorophenyl)-5,6-dihydro-11H-tetrazolo[1,5-
a]benz[d]azepine (30). White crystals (ethanol). Yield: 52%;
mp: 169–170 °C; IR (KBr, cmÀ1): 1617 (C@N); 1H NMR (d
ppm): 4.26, 4.72 (2d, J = 16.8 Hz, 2H, tetrazolobenzazepine
C11–H), 4.91 (dd, J = 17.96, 9.16 Hz, 1H, tetrazolobenzazepine
C6–H), 5.11 (dd, J = 17.96, 9.16 Hz, 2H, tetrazolobenzazepine
C5–H), 6.83–6.92 (m, 1H, tetrazolobenzazepine C7–H), 7.20–
7.30 (m, 2H, tetrazolobenzazepine C8–H & C9–H), 7.31 (d,
J = 7.6 Hz, 2H, chlorophenyl C2,6–H), 7.40–7.49 (m, 3H, tetra-
zolobenzazepine C10–H & chlorophenyl C3,5–H). 13C NMR (d
ppm): 28.43 (tetrazolobenzazepine C11), 42.73 (tetra-
zolobenzazepine C6), 51.07 (tetrazolobenzazepine C5), 127.48,
127.84 (tetrazolobenzazepine C8 & C9), 128.58 (chlorophenyl
concentration of 800
centrations of (400, 200,. . .6.25
l
g/mL followed by two-fold dilution at con-
lg/ml). The microorganism sus-
pensions at 106 CFU/ml (Colony Forming Unit/mL) concentration
were inoculated to the corresponding wells. Plates were incubated
at 36 °C for 24–48 h and the minimal inhibitory concentrations
(MIC) were determined. Control experiments were also done. All
assays were performed in duplicate and results are represented
in Table 1.
4.2.1.3. In vitro antifungal susceptibility assay. MIC of the six ac-
tive compounds 15, 17, 22, 23, 26 and 30 against C. albicans and A.
fumigatus was determined by broth microdilution testing in accor-
dance with the guidelines in NCCLS document M27-A and M38-
P.49,50 Briefly stock solutions were prepared in DMSO for clotrima-
zole, miconazole and the compounds. Serial twofold dilution of the
compounds was made in RPMI1640 medium buffered to pH 7.0
with 0.165 M 4-morpholinepropanesulfonic acid (MOPS) buffer
as outlined in NCCLS M27-A document. Aliquots of 0.1 mL of each
compound were dispensed into the wells of plastic microdilution
microtiter plates so that the final concentration of solvent did
not exceed 1% in any well. An inoculum of the organisms at
106 CFU/mL (Colony Forming Unit/mL) concentration was pre-
C3
& C5), 128.97 (tetrazolobenzazepine C7), 129.5 (tetra-
zolobenzazepine C10), 129.79 (chlorophenyl C2 & C6), 131.88
(chlorophenyl C4), 133.76 (tetrazolobenzazepine C10a), 137.99
(chlorophenyl C1), 139.89 (tetrazolobenzazepine C6a), 151.63
(tetrazolobenzazepine-C11a); MS, m/z (relative abundance%):
297 [M++1, (17.3)], 296 [M+, (26.9)], 240 (40.4), 179 (73.1),
178 (92.3), 131 (42.3), 91 (50), 89 (86.5), 77 (50), 63 (100), 51
(100). Anal. Calcd for C16H13ClN4 (296.75): C, 64.76; H, 4.42;
N, 18.88. Found: C, 64.65; H, 4.35; N, 18.51.
pared and 100 lL of the individual fungal inoculum was added to
4.1.13.2. 2-(4-Chlorostyryl)-5-phenyl-2H-tetrazole (31). White
crystals (ethanol). Yield: 48%; mp: 130–131 °C; IR (KBr, cmÀ1):
1616 (C@N); 1H NMR (d ppm): 7.47 (d, J = 8 Hz, 2H, chlorophenyl
C2,6–H), 7.53–7.60 (m, 3H, phenyl C3,4,5–H), 7.66 (d, J = 14.6 Hz,
1H, NCH@CH–C6H4–Cl), 7.78 (d, J = 8 Hz, chlorophenyl C3,5–H),
8.06–8.17 (m, 2H, phenyl C2,6–H), 8.50 (d, J = 14.6 Hz, 1H, N–
CH@CH). 13C NMR (d ppm): 123.41 (NCH@CH), 123.65 (–NCH@CH),
126.26 (phenyl C4), 126.51 (phenyl C2 & C6), 128.77 (chlorophenyl
C3 & C5), 129.15 (phenyl C3 & C5), 129.19 (chlorophenyl C2 & C6),
130.77 (phenyl C1), 131.85 (chlorophenyl C1), 133.84 (chlorophenyl
C4), 163.87 (tetrazole-C5). Anal. Calcd for C15H11ClN4 (282.73): C,
63.72; H, 3.92; N, 19.82. Found: C, 63.58; H, 3.31; N, 19.86.
each well of the microtiter plate containing the reference drug or
the compound. The plates were incubated at 25 °C for 72 h. After
the completion of incubation, the broth microdilution wells were
examined and the growth in each well was compared with that
of the control. The MIC of each compound was defined as the low-
est concentration that produced 80% inhibition in the growth of the
organism compared with that of the control. All assays were per-
formed in duplicate and the results are presented in Table 2.
4.2.2. Preliminary anticonvulsant screening
4.2.2.1. Subcutaneous metrazole seizure pattern test. This test
was carried out following the procedure described by Chaturvedi
et al.44 Wister albino mice of either sex, weighing 25–30 g and
3 months old were utilized throughout the assay. They were kept
in the animal house under standard conditions of light and temper-
ature (25 °C) with free access to food and water. The animals were
randomly divided into groups each of six rats. Dimethyl sulfoxide
(DMSO) was used for dissolving metrazole and the test com-
4.2. Biological evaluation
4.2.1. In vitro antibacterial and antifungal activities
4.2.1.1. Inhibition zone (IZ) measurement. Standard sterilized
filter paper discs (5 mm diameter) impregnated with a solution

2422
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