A Facile one-pot synthesis of 2-Arylamino-5-Aryloxylalkyl-1,3,4-Oxadiazoles and their urease inhibition studies

Article abstract of DOI:10.1111/cbdd.12297

A one-pot method for the synthesis of structural type urease inhibitors, 2-amino-1,3,4-oxadiazoles, was developed. The structures of the compounds were established using spectroanalytical techniques and unambiguously confirmed by single-crystal X-ray analysis of compound 3o. The synthesized compounds were tested against jack beans urease, and most of the compounds (3c, 3g, 3j, 3k, 3n, 3r-3v) were found more active than the standard. The most potent compound (3u) had an IC₅₀ value of 6.03 ± 0.02 μm as compared to the IC₅₀ value of the standard (thiourea; 22.0 ± 1.2 μm). The prominent urease inhibition activity of these compounds may serve as an important finding in the development of less toxic and more potent antiulcer drugs. The compounds were also investigated against four bacterial strains, and some of the compounds (3g and 3r) were found more potent than the standard drug (ciprofloxacin) against all the tested strains. The MIC value for compound 3g was 0.156 μmol/mL against the tested bacterial strains. One-pot synthesis of submitted 1,3,4-oxadiazoles was successfully achieved. The synthesized compounds were found very potent inhibitors of urease, besides exhibiting antibacterial activities.

Full text of DOI:10.1111/cbdd.12297

Received Date : 07-Oct-2013
Revised Date : 16-Dec-2013
Accepted Date : 20-Jan-2014
Article type : Research Article
A facile one pot synthesis of 2-arylamino-5-aryloxylalkyl-1,3,4-
oxadiazoles and their urease inhibition studies
Tashfeen Akhtara,d, Muhammad Ashfaq Khanb, Jamshed Iqbalb*, Peter G. Jonesc and Shahid
Hameedd*
aDepartment of Chemistry, Mirpur University of Science and Technology (MUST), Mirpur-10250,
Azad Jammu and Kashmir, Pakistan
bDepartment of Pharmaceutical Sciences, COMSATS Institute of Information Technology,
Abbottabad-22060, Pakistan
cInstitut for Anorganische und Analytische Chemie, Technische Universität Braunschweig,
Hagenring 30, 38106 Braunschweig, Germany
dDepartment of Chemistry, Quaid-i-Azam University, Islamabad-45320, Pakistan.
Corresponding author: S. Hameed, shahidhameed@daad-alumni.de; shameed@qau.edu.pk;
Phone: +92 51 9064 2133; Fax: +92 51 9064 2241 or J. Iqbal, drjamshed@ciit.net.pk.
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as an
'Accepted Article', doi: 10.1111/cbdd.12297
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Key words: One pot synthesis, Acid hydrazides, O xadiazoles, Phenoxy acids, Urease inhibition
,
Crystal structure, Antibacterial
Abstract
A one pot method for the synthesis of structural type urease inhibitors, 2-amino-1,3,4-
oxadiazoles, was developed. The structures of the compounds were established using
spectroanalytical techniques and unambiguously confirmed by single crystal X-ray analysis of
compound 3o. The synthesized compounds were tested against jack beans urease and most of
the compounds (3c, 3g, 3j, 3k, 3n, 3r-3v) were found more active than the standard. The most
potent compound (3u) had an IC50 value of 6.03 ± 0.02 µM as compared to the IC50 value of the
standard (thiourea; 22.0 ± 1.2 µM). The prominent urease inhibition activity of these
compounds may serve as an important finding in the development of less toxic and more
potent antiulcer drugs. The compounds were also investigated against four bacterial strains and
some of the compounds (3g and 3r) were found more potent than the standard drug
(ciprofloxacin) against all the tested strains. The MIC value for compound 3g was 0.156
µmol/mL against the tested bacterial strains.
Introduction
1,3,4-Oxadiazoles constitute an important class of five membered heterocyclic
pharmacophores. They have numerous biological applications such as anti-tumour (1),
antibacterial (2), anti-inflammatory (3), anti-HIV (4), anti-convulsant (5), enzyme inhibition (6),
NR2B receptors (7) and hypoglycemic (8,9). The insecticidal (10,11), fungicidal (12), herbicidal
(13) and antimicrobial (4,14,15,16) activities have also been reported for this class of
compounds. Besides, being used as core nucleus in liquid crystalline compounds (17),
oxadiazoles are also used as fluorescent materials in organic light emitting diodes (18,19).
Different methods for the synthesis of 1,3,4-oxadiazoles have recently been reviewed along
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with other five membered heterocycles (20). 2-Amino-1,3,4-oxadiazles are commonly
synthesized by cyclization of corresponding semicarbazide or thiosemicarbazide derivatives (21-
23). The use of semicarbazides requires harsh reaction conditions such as the use of SOCl2,
POCl3 or P2O5. The use of phosphonium salts and other reagents result in significant by-
products and are thus limited to solid phase synthesis. For the cyclization of
thiosemicarbazides, different reagent like DCC or EDC or highly reactive alkylating agents have
been used (24). H2S scavengers such as mercuric or lead oxide are another important approach
for the synthesis of these compounds (25). Thiosemicarbazides may easily be purified by
precipitation from the reaction mixture or recrystallization, but it is not necessary (26). The use
of H2S scavengers in the cyclization of thiosemicarbazides (25) provoked us to investigate their
use in one pot procedure. In the past, we have reported the synthesis of different chiral azoles
from thiosemicarbazide intermediates and their biological applications (27,28) and faced the
problem of lower yields. To overcome the problem of low yields in step-wise procedure, we
investigated a one pot approach for the synthesis of 2-arylamino-1,3,4-oxadiazoles (Scheme 1)
and explored the pharmacological applications of the products.
Methods and Materials
General
Melting points of the compounds were measured in open capillaries using Gallenkamp melting
point apparatus (MP-D) and are uncorrected. Infrared (IR) spectra were recorded on a Thermo
Scientific Nicolet 6700FT-IR spectrophotometer (USA) using the ATR (attenuated total
reflectance) facility. Mass spectra were measured on a MAT-112-S spectrometer at 70 eV. The
1H and 13C NMR spectra were run on a Bruker Avance 300 MHz NMR spectrophotometer and
signals calibrated to the residual solvent signal (chemical shifts in δ ppm). Elemetal analysis was
determined using Leco CHNS-932 Elemental Analyzer, Leco Corporation (USA) and were within
± 0.4 % of the theoretical values. The X-ray crystallography was performed on an Oxford
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Diffraction Xcalibur E diffractometer. The isothiocyanates (2a-e) were commercial products of
Sigma-Aldrich. Compounds 1a-g were prepared according to a reported procedure (28).
Synthesis of compounds 3a-3u
All the 2-arylamino-5-aryloxyalkyl-1,3,4-oxadiazoles were synthesized by the same general
procedure: Aryloxyalkanoic acid hydrazide (1a-g) was dissolved in methanol (30 mL) and
methanolic solution (10 mL) of arylisothiocyanate (2a-e, 1.0 eq) added slowly with stirring. The
resulting mixture was refluxed and consumption of arylisothiocyanate, i.e., formation of
thiosemicarbazide (3-4 hr) monitored by tlc (petroleum ether-ethyl acetate; 6 : 4). After
complete consumption of arylisothiocyanate required quantity of mercuric acetate (1.0 eq) was
added and the reflux continued. After another 2-3 hours, tlc indicated the formation of one
major product, i.e., the product of the cyclization of thiosemicarbazide to 2-aminooxadiazole.
The reaction mixture was cooled down to room temperature, filtered and concentrated in
vacuo. The resulting solid was either recrystallized from aqueous ethanol or purified by flash
column chromatography. The structures of the synthesized compounds (3a-3u) were
established using spectroscopic techniques.
Urease inhibition assay
In vitro inhibitory studies on urease were determined by indophenols method which measures
the liberation of ammonia from the reaction (29). Changes in the absorbance at 625 nm of
indophenols blue produced in the Berthelot reaction after incubation for 30 min at 30°C was
used to assess the amount of ammonia.
In short, 96-well plates were used for the assay mixture and incubated for 30 minutes at 30°C.
The ingredients of the assay mixture were: 40 μL buffer (100 mM urea, 0.01 M K2HPO4, 1 mM
EDTA and 0.01 M LiCl2, pH 8.2) along with 10 µL of enzyme (5 U/mL) and 10 μL of test
compound. In addition to this, equal volumes (40μL each) of phenol reagents (1% w/v phenol
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and 0.005% w/v sodium nitroprusside) and alkali reagent (0.5% w/v NaOH and 0.1% active
chloride NaOCl) were added to each well. After 30 min, the absorbance was measured with the
help of a microplate reader (Bio-TekELx 800TM, Instruments, Inc. USA). The reactions were
exercised in triplicate. The standard inhibitor in the current assay was thiourea. The IC50 values
of the compounds were determined.
Antibacterial activity
The serial dilution method was used to analyze in vitro antibacterial activities of the compounds
by minimum inhibitory concentration (MIC) (30). Further, two Gram negative bacteria namely,
Escherichia coli and Shigella flexneri, and two Gram positive bacteria namely, Staphylococcus
aureus and Bacillus subtilis were deployed in the present antibacterial assay. These bacterial
strains were stored and sub-cultured for compound testing using Muller-Hinton broth (Merck)
at 37 °C. Moreover, for the production of cell suspension of about 10−5 CFU mL−1 (colony-
forming units per mL), the cells were suspended in saline solution. Serial dilutions to final
concentrations of 2.5, 1.25, 0.625, 0.313 and 0.156 µg/mL were prepared in the test tubes of
the compounds under study that were previously dissolved in N,N-dimethylformamide (DMF).
Twenty four hour (24 h) old inoculum having 100 μL was added to each test tube. The lowest
inhibitory concentration of the test compounds were determined by MIC method that shows
the zone of inhibition after 18 h, for incubating 24 h at 37 °C. Ciprofloxacin was used as the
standard drug in the assay.
X-Ray Structure Determination of Compound 3o
Crystal data: Triclinic, P , a = 5.5706(4), b = 9.7318(6), c = 15.8489(8) Å, α = 95.550(5), β =
1
93.081(5), γ = 95.573(5)°, V = 849.40 Å3, Z = 2, μ = 2.6 mm–1, Dx = 1.598 Mg/m3. Data collection
and reduction: A colourless prism 0.4 × 0.15 × 0.10 mm was mounted on a glass fibre in inert oil
and transferred to the cold gas stream of the diffractometer (Oxford Diffraction Xcalibur E). A
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total of 23036 intensities were recorded to 2θmax 56.6° using monochromated Mo Kα radiation
(λ = 0.71073 Å); 3983 of these were independent (Rint = 0.027). Absorption corrections were
performed on the basis of multi-scans. Structure refinement: The structure was refined
anisotropically on F2 using the program SHELXL-97 (31). The NH hydrogen was refined freely but
using a distance restraint; the methyl was refined as an idealised rigid group allowed to rotate
but not tip; other hydrogen atoms were included using a riding model starting from calculated
positions. The final wR2 for all reflections was 0.066 for 222 parameters, with R1 = 0.026 for
reflections with I > 2σ(I); S = 1.09, max. Δρ = 0.69 e Å–3.
Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre as
supplementary publication no. CCDC-864708. Copies of the data can be obtained free of charge
from www.ccdc.cam.ac.uk/data_request/cif.
Results and Discussion
Chemistry
The selected α-aryloxypropanoic/butanoic acid hydrazides 1a-g were prepared starting from
different substituted phenols (32) and α-bromopropanoic acid, followed by esterification and
hydrazinolysis (scheme 1). The hydrazides were reacted with arylisothiocyanates in refluxing
methanol and consumption of the reactants was monitored by thin layer chromatography (tlc).
After the consumption of reactants (3-4 hr), required quantity (1.0 eq) of mercuric acetate
(Hg(OAc)2) was added and the reflux continued. The reaction mixture gradually turned black
indicating the formation of mercuric sulfide (HgS). After the addition of Hg(OAc)2, it took
another 2-3 hr for completion of the reaction (tlc). The product was isolated by suction
filtration. Recently, a one pot procedure for the synthesis of 1,3,4-oxadiazoles involving resin
bound reagents has also been reported (23) but the procedure involves very long reaction
times and the resin bound reagents are expensive too. In contrast, the procedure being
reported here has the clear advantage of reaction completion in a much shorter time and the
reagents are relatively inexpensive.
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Seven different aryloxyalkanoic acid hydrazides and five arylisothiocyanates were utilized in the
current procedure. The investigated one pot procedure is clearly advantageous over the step
wise procedure (22) in that: no isolation of the intermediate thiosemicarbazides is required, it is
less time consuming and gives better purified yields.
The IR spectra of compounds 3a-u indicated the formation of 1,3,4-oxadiazole ring by the
presence of weak CN absorptions in the region of 1620-1580 cm-1. A single absorption for NH
was observed in the range 3270-3250 cm-1. In 1H NMR spectra, the cyclization to 1,3,4-
oxadiazoles was confirmed by the appearance of only one downfield singlet in the range of δ
11.67-8.47 with one proton integral, assigned to the only NH proton. The integration of
aromatic signals also confirmed the cyclization by the presence of two benzene rings with
varied substitution pattern. The signals for aromatic protons were observed as two multiplets
for p-substituted aminophenyl moiety and two multiplets for p-substituted phenoxy moiety.
The 1H NMR spectra of compounds 3e, 3i, 3k, and 3o contained three signals for the 3,4-
dichlorophenoxy moiety in the aromatic region; a doublet (4J = 3.0 Hz) assigned to proton H-2
residing between the chlorine and oxygen substituents, the second doublet (3J = 9.0 Hz) for
4
proton H-5 ortho to the oxygen atom and a doublet of doublet (3J = 9.0 Hz, J = 3.0 Hz) for
proton H-6 ortho to the chlorine atom. In the aliphatic region, the signal for CH was observed
downfield from δ 5.23 to 5.86 either as a triplet or a quartet depending on the nature of R
group. In 13C NMR spectra, the signals for imine (CN) carbon were observed in the region of
158-161 ppm. The carbon signals for the compounds (3c-f) bearing 4-fluorophenoxy moiety
were observed as doublets with different JC,F values depending on the number of intervening
bonds. The structures of the compounds 3a-u were further confirmed by the mass spectral
analysis. The mass fragmentation patterns were in accordance with the structural features of
the molecules (figure 1). The most important fragments for the cleavage of oxadiazole nucleus
were observed at m/z = 119 + X and m/z = 132/133 + R + R´, corresponding to the C–O–C-N and
N–N–C–O cleavage. Other fragments containing aminoaryl and aryloxy moieties were also
observed. The observed fragments led to the confirmation of the suggested structures for 2,5-
disubstituted 1,3,4-oxadiazoles. The purity of the synthesized compounds was ascertained by
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elemental analysis and the calculated values for CHN were found in a very good agreement
with the observed data.
The structures of the synthesized compounds were also confirmed by single crystal X-Rays
analysis of compound 3o. Except for the ethyl group, the molecule has an "L"-shape (Figure 2).
Molecular dimensions (e.g. of the five-membered ring, see figure caption) may be regarded as
normal. The molecular packing involves layers parallel to the bc plane (Figure 3) in which
molecules are linked by short contacts ClLCl 3.43, ClLO1 3.34 and N3LBr 3.53 Å. The N3-H03
group, a classical hydrogen bond donor, links the layers via the interaction to N2 of a molecule
related by a axis translation.
Urease Inhibition Studies
All the synthesized 1,3,4-oxidiazole derivatives were investigated for their urease inhibitory
activities against Jack beans urease. Among investigated compounds, 3c, 3g, 3j, 3k, 3n, 3r, 3s, 3t
and 3u were found more potent with higher inhibition as compared to the IC50 value of the
standard inhibitor (thiourea) as shown in table 1. The presence of ethyl group rather than
methyl at the alkoxycarbon, as well as strong electron withdrawing groups (fluoro or nitro) at
position 4 of the arylmino group was found more effective in imparting inhibitory characters to
these compounds. Compound 3u has the least IC50 value among the most potent compounds in
the current study. This may be attributed to the presence of highly electron withdrawing nitro
group. Since the fluorine atom has the highest electro-negativity and electron withdrawing
capacity, therefore, the second most active compound 3g possesses a fluorine substituent at
position 4 of the arylamino group. The compound 3t ranked the second least IC50 value after 3u
and 3g. Compounds 3g, 3t and 3u are three times more potent than the standard as can be observed
from their IC50 values. It appears that greater the electron withdrawing ability of the 4-arylamino
part of the molecule, least IC50 value is the outcome.
Table 1. Urease inhibition activities of the synthesized compounds (3a-u).
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Compd.
Ar
R
X
IC50+SEM (µM)
3a
4-ClC6H4
Me Me 26.2 ± 2.2
3b
3c
3d
3e
3f
4-BrC6H4
Me Me 33.5 ± 1.4
4-MeC6H4 Me Me 9.82 ± 0.08
3,4-Cl2C6H3 Me Me 41.0 ± 1.3
4-BrC6H4
Et Me 47.4 ± 3.4
3,4-Cl2C6H3 Et Me 47.4 ± 2.2
3g
4-BrC6H4
4-ClC6H4
Me F
Me F
6.21 ± 0.04
3h
3i
45.9 ± 5.2
45.3 ± 8.1
12.4 ± 0.06
3,4-Cl2C6H3 Me F
4-MeC6H4 Me F
3j
3k
3l
4-BrC6H4
4-ClC6H4
Me Cl
Me Cl
11.6 ± 0.05
33.2 ± 2.7
42.8 ± 6.7
11.5 ± 0.07
57.2 ± 6.3
45.5 ± 4.2
35.8 ± 1.2
3m
3n
3o
3p
3q
3,4-Cl2C6H3 Me Cl
4-MeC6H4 Me Cl
4-BrC6H4
4-BrC6H4
Et Cl
Et Br
4-MeC6H4 Et Br
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3r
4-BrC6H4
Et
F
F
F
7.65 ± 0.06
8.02 ± 0.07
7.42 ± 0.03
3s
3,4-Cl2C6H3 Et
4-MeC6H4 Et
3t
3u
4-BrC6H4
Et NO2 6.03 ± 0.02
22.0 ± 1.2
Thiourea
Antibacterial activity
The antibacterial activity of the synthesized 1,3,4-oxidiazole derivatives was evaluated using
two strains in each category; Gram-positive bacteria namely Staphylococcus aureus and Bacillus
subtilis, and Gram-negative bacteria namely, Escherichia coli and Shigella flexneri. The
reference drug used in the assay was ciprofloxacin. The minimum inhibitory concentrations of
the compounds were determined by using Muller-Hinton broth (Merck). The compounds
demonstrated excellent antibacterial activity against the tested strains.
Compounds 3g and 3r proved to be the most potent antibacterials against all the tested strains
in the current study. Compounds 3c, 3g, 3k, 3r, 3t and 3u were equipotent (MIC: 0.156μg/mL)
as the standard ciproloxicin against E. coli. Some of the compounds (3c, 3g, 3j, 3k, 3n, 3r, 3t and
3u) were four times more active than the standard (ciprofloxacin) against B. subtilis as shown in
table 2. In addition, the potency of compounds 3c, 3f, 3g, 3j, 3q, 3r and 3s against S. aureus is
comparable to that of ciprofloxacin. Compounds 3b and 3g were twice as potent as the
standard against S. flexneri. The activity of the rest of the compounds, except 3c, 3e, 3l and 3o,
was comparable to the standard against S. flexneri. The two most potent antibacterial
compounds (3g and 3r) possess a fluorine atom at position 4 of the arylamino group while a
bromine atom at position 4 of the arylkoxy group. Since halogens are electron withdrawing, the
electron withdrawing effect of these groups might be the possible reason for higher
antibacterial activity of the compounds.
Table 2. The MIC (µmol/mL) valuesa of 1,3,4-oxidiazole derivatives (3a-u) against bacterial
strains.
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Compd.
Bacterial strains and MIC values (µmol/mL)
E. coli B. subtilis S. aureus S. flexneri
3a
3b
3c
3d
3e
3f
0.625 0.625
0.625 0.625
0.156 0.156
0.313 0.313
1.250 1.250
0.625 0.625
0.156 0.156
0.313 1.250
0.625 0.625
0.313 0.156
0.156 0.156
0.313 0.625
1.250 0.625
0.625 0.156
0.625 0.625
0.313 0.625
1.250 0.625
0.156 0.156
0.625
0.313
0.156
0.313
0.625
0.156
0.156
1.250
1.250
0.156
0.313
0.625
0.625
0.313
0.313
0.156
0.313
0.156
0.313
0.156
0.625
0.313
0.625
0.313
0.156
0.313
0.313
0.313
0.313
0.625
0.313
0.313
0.625
0.313
0.313
0.313
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
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3s
3t
3u
0.313 0.156
0.156 0.156
0.156 0.156
0.156
0.313
0.625
0.156
0.313
0.313
0.313
0.313
Ciprofloxacin 0.156 0.625
avalues are the average of three experiments
Conclusions
The current study was directed to the design and synthesis of urease inhibitors. The designed
inhibitors were synthesized by a one pot procedure using variety of arloxyalkanoic acids and
arylisothiocyanates. The synthesized compounds, in general, were active as urease inhibitors
with compounds 3r, 3t and 3u exhibiting IC50 values almost three times lower than the standard
(table 1). Besides urease inhibition the synthesized compounds were investigated against four
different bacterial strains and compounds 3g and 3r were found active against all the tested
strains. Among others, compounds 3c, 3g, 3j, 3k, 3n, 3r, 3t and 3u exhibited four times low MIC
values than the standard (ciprofloxacin) against B. Subtilis (table 2). Overall, as the preliminary
investigations suggest, the designed molecules are not only strong urease inhibitors but may
also be utilized in the development of new more potent azole antibiotics.
Acknowledgment
The authors (S. Hameed and T. Akhtar) are thankful to Higher Education Commission of
Pakistan for the financial support through project No. 20-674/R&D/06/1764 under National
Research Program for Universities.
Conflict of Interest
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The authors have declared no conflict of interest.
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Supporting Information
Additional Supporting Information may be found in the online version of this article:
Appendix S1: Physical properties and spectral data of the target compounds is provided.
Figure Legends
Figure 1: Suggested mass fragmentation pattern.
Figure 2: Molecular structure of compound 3o in the crystal. Ellipsoids represent 50%
probability levels. Selected bond lengths (Å): O1-C1 1.3640(18), O1-C2 1.3709(18), N1-C1
1.299(2), N1-N2 1.4140(18), N2-C2 1.279(2).
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Figure 3: Packing diagram of compound 3o in the crystal, showing the layer structure. Dashed
lines indicate short intermolecular contacts (see text). One contact to the next layer is shown at
the top of the diagram.
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.