New oxadiazole derivatives of isonicotinohydrazide in the search for antimicrobial agents: Synthesis and in vitro evaluation

Article abstract of DOI:10.2298/JSC110123155M

Structural modifications of the front line antitubercular drug isoniazid provide lipophilic adaptations of the drug in which the hydrazide moiety of isoniazid is replaced by 1,3,4-oxadiazole heterocycles to eliminate in vivo acetylation by arylamine N-ace

Full text of DOI:10.2298/JSC110123155M

J. Serb. Chem. Soc. 77 (1) 9–16 (2012)
UDC 547.78+547.233+547.282.1:615.28–188
JSCS–4244
Original scientific paper
New oxadiazole derivatives of isonicotinohydrazide in the search
for antimicrobial agents: Synthesis and in vitro evaluation
1
2
3
4
*
MANAV MALHOTRA , MOHIT SANDUJA , ABDUL SAMAD and AAKASH DEEP
1Department of Pharmaceutical Chemistry, Meerut Institute of Engineering and Technology,
Bypass Road-Baghpat Crossing, Meerut-250005, Uttar Pradesh, India, 2Department of
Pharmaceutical Chemistry, ISF College of Pharmacy, Ferozepur Road, Moga-142001,
India, 3Department of Pharmaceutical Chemistry, College of Pharmacy in Al-Kharj,
King Saud University, Riyadh, Saudi Arabia and 4Department of Pharmaceutical
Sciences, Maharshi Dayanand University, Rohtak-124001, India
(Received 23 January, revised 24 June 2011)
Abstract: Structural modifications of the front line antitubercular drug isoniazid
provide lipophilic adaptations of the drug in which the hydrazide moiety of iso-
niazid is replaced by 1,3,4-oxadiazole heterocycles to eliminate in vivo acetyla-
tion by arylamine N-acetyltransferase, which results in the formation of inac-
tive acetylated drug. In the present study, a series of sixteen oxadiazole deriva-
tives were synthesized and characterized by IR, 1H-NMR, 13C-NMR and mass
spectral studies. All the synthesized compounds were evaluated for their anti-
microbial activity by broth dilution method against two Gram-positive bacterial
strains (Bacillus subtilis and Staphylococcus aureus), two Gram-negative bac-
terial strains (Pseudomonas aeruginosa and Escherichia coli) and two fungal
strains (Candida albicans and Aspergillus niger). The minimum inhibitory
concentrations of the compounds were in the range of 1.56–50 µg ml-1 against
the bacterial and fungal strains. The results revealed that all the synthesized
compounds have a significant biological activity against the tested microorga-
nisms. Among the synthesized derivatives 4g, 4h, 4m and 4p were found to be
the most effective antimicrobial compounds.
Keywords: 1,3,4-oxadiazoles; antimicrobial activity; isoniazid; Mannich bases;
lipophilicity.
INTRODUCTION
One of the key objectives of organic and medicinal chemists is to design and
1
synthesize molecules having potent therapeutic values. The rapid development
of resistance to existing antimicrobial drugs generates a serious challenge to the
scientific community. Consequently, there is a vital need for the development of
new antimicrobial agents having potent activity against the resistant microorga-
*Corresponding author. E-mail: aakashdeep82@gmail.com
doi: 10.2298/JSC110123155M
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MALHOTRA et al.
2–5
nism.
1,3,4-Oxadiazoles have played an important role in medicinal chemis-
try, pesticide chemistry, polymer science and they are the building blocks in the
6
construction of new molecular systems for biologically active molecules. Many
1,3,4-oxadiazoles display a remarkable biological activity, such as antimicro-
7,8
13
9
10
11
12
bial,
anti-HIV, antitubercular, antimalarial, analgesic, anti-inflamma-
tory. The oxadiazole pharmacophore has a key property that influences the abi-
lity of a drug to reach the target by transmembrane diffusion and show potent
14
antimicrobial activity. Inspired by the above facts and in continuation of an on-
going research program in the field of the synthesis and determination of the an-
15,16
timicrobial activity of medicinally important compounds,
the synthesis and
antimicrobial evaluation of new oxadiazole derivatives are reported herein.
EXPERIMENTAL
Material and methods
Melting points of the synthesized compounds were determined in open-glass capillaries
on Stuart SMP10 melting point apparatus and are uncorrected. The purity of the compounds
was checked by thin layer chromatography (TLC). Silica gel, 0.25 mm, 60 GF254, precoated
sheets obtained from Merck, (Germany) were used for the TLC and the spots were visualized
by iodine vapor/ultraviolet light. The IR spectra were obtained on a Perkin-Elmer 1600 FTIR
spectrometer in KBr pellets. The 1H-NMR spectra were recorded in DMSO-d6 solutions on a
Varian-Mercury 300 MHz spectrometer using tetramethylsilane as the internal reference. The
13C-NMR spectra were recorded in DMSO-d6 solutions on a Bruker Avance II 400 spec-
trometer at using tetramethylsilane as the internal reference. The mass spectra were recorded
on a Shimadzu GCMS-QP 1000 EX. The elemental analyses were performed on an ECS 4010
elemental combustion system. The necessary chemicals were purchased from Loba Chemie,
Fluka and Aldrich.
(E)-N’-(2-Methoxybenzylidene)isonicotinohydrazide (1a)
A mixture of 2-methoxybenzaldehyde (1.36 g, 0.01 mol) and isoniazid (1.37 g, 0.0100
mol) in 15 ml of absolute ethanol was refluxed for 7 h. The completion of reaction was
confirmed by TLC. The reaction mixture was then poured into ice-cold water and the obtained
precipitate was filtered and dried in an oven at a low temperature. The product was
recrystallized from absolute ethanol.17 Yield 68 %; m.p. 204–207 °C.
(E)-N’-3-((Dimethylamino)methyl)-2-methoxybenzylidene)isonicotinohydrazide (2a)
(E)-N’-(2-Methoxybenzylidene)isonicotinohydrazide (612 mg, 0.00240 mol) along with
(0.10 ml, 0.0036 mol) of formaldehyde and (0.0024 mol) of dimethylamine was placed in 100
ml round bottom flask to which 50 ml of absolute ethanol was added. The pH was adjusted to
4 with hydrochloric acid and the mixture refluxed for 35 h. Completion of the reaction was
confirmed by TLC. The reaction mixture was then poured into a beaker and concentrated on a
water bath. The reaction mixture was allowed to cool to room temperature and then diethyl
ether was added. The reaction mixture was kept for 3–5 h in a refrigerator, filtered and washed
with n-hexane. The product was recrystallized from absolute ethanol. Yield 78 %; m.p. 222–
–225 °C.
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ANTIMICROBAIL ACTIVITY OF OXADIAZOLE DERIVATIVES
11
1-(2-(3-((Dimethylamino)methyl)-2-methoxyphenyl)-5-(pyridin-4-yl)-1,3,4-oxadiazol-
-3(2H)-yl)ethanone (3a)
A mixture of (E)-N’-3-((dimethyamino)methyl)-2-methoxybenzylidene)isonicotinohyd-
razide (3.57 g, 0.0100 mol) with an excess of acetic anhydride was refluxed for 7 h until the
completion of the reaction, which was confirmed by TLC. The excess acetic anhydride was
distilled off and the residue was poured onto crushed ice. The solid thus obtained was filtered;
washed with water and then recrystallized with aqueous methanol. Yield 75 %; m.p. 168–170 °C.
General procedure for synthesis of the substituted oxadiazoles 4a–p
A mixture of 3a (0.010 mol) and an equimolar amount of an appropriate aromatic amine
(0.010 mol) was added to 25 ml absolute ethanol with a drop of glacial acetic acid and heated
under reflux for 7–9 h. The obtained precipitate was filtered off; washed with ethanol and
recrystallized from absolute ethanol to obtain compounds 4a–p.
Antimicrobial activity
The synthesized compounds were evaluated for their in vitro antimicrobial activity
against the Gram-positive bacteria Staphylococcus aureus (MTCC 96) and Bacillus subtilis
(MTCC 121), the Gram-negative bacteria Escherichia coli (MTCC 40) and Pseudomonas
aeruginosa (MTCC 2453) and the fungal strains Candida albicans (MTCC 227) and Asper-
gillus niger (MTCC 8189). Antimicrobial activity was assessed by the serial two-fold dilution
technique. Amoxicillin was used as the standard drug for the antibacterial activity and nystatin
was used as the standard drug for the antifungal activity. All the compounds were dissolved in
dimethyl sulfoxide to give a concentration of 10 µg ml-1. The two-fold dilutions of the test and
standard compounds were prepared in double strength nutrient broth I.P. (bacteria) or Sabou-
raud dextrose broth I.P. (fungi).18 The stock solution was serially diluted to give concen-
trations of 50–0.78 µg ml-1 in the nutrient broth. The inoculum size was approximately 106
colony forming units (CFU ml-1). The tubes were incubated at 37±1°C for 24 h (bacteria), 25
°C for C. albicans and 35°C for A. niger for 7 days. Subsequently, the inoculated culture tubes
were macroscopically examined for turbidity. The culture tube showing turbidity (lower con-
centration) and the culture tube showing no turbidity (higher concentration) gave the mi-
nimum inhibitory concentration (MIC) for the compound.
RESULTS AND DISCUSSION
The syntheses of the target compounds were performed according to the out-
line given in Scheme 1. Compounds 4a–p were readily prepared in good yield
and purity. Firstly, an equimolar mixture of 2-methoxybenzaldehyde and isonico-
tinohydrazide was refluxed to form (E)-N’-(2-methoxybenzylidene)isonicotino-
hydrazide (1a), then its reaction with formaldehyde and dimethylamine formed
(E)-N’-(3-((dimethyamino)methyl)-2-methoxybenzylidene)isonicotinohydrazide
(2a), which on treatment with acetic anhydride yielded 1-(2-(3-((dimethylami-
no)methyl)-2-methoxyphenyl)-5-(pyridin-4-yl)-1,3,4-oxadiazol-3(2H)-yl)ethanone
(3a) and in the last reaction with substituted aromatic amines, it afforded a series
of (Z)-N-(1-(2-(3-((dimethylamino)methyl)-2-methoxyphenyl)-5-(pyridin-4-yl)-
-1,3,4-oxadiazol-3(2H)-yl)ethylidene)benzenamine derivatives (4a–p). The struc-
ture, melting points and yields the synthesized compounds 4a–p are given in
Table I. The purity of the compounds was checked by TLC and elemental analyses.
Available online at www.shd.org.rs/JSCS/
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MALHOTRA et al.
Scheme 1. Synthetic route for the formation of the title compounds.
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ANTIMICROBAIL ACTIVITY OF OXADIAZOLE DERIVATIVES
13
TABLE I. Physical data of the synthesized compounds 4a–4p
Compd.
R
Molecular formula Molecular weight
Melting point, °C Yield, %
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
H
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
2-Br
3-Br
C25H27N5O2
C25H26FN5O2
C25H26FN5O2
C25H26FN5O2
C25H26ClN5O2
C25H26ClN5O2
C25H26ClN5O2
C25H26BrN5O2
C25H26BrN5O2
C25H26BrN5O2
C25H26N6O4
C25H26N6O4
C25H26N6O4
C26H29N5O3
C26H29N5O3
C26H29N5O3
429.5
447.5
447.5
447.5
463.9
463.9
463.9
508.4
508.4
508.4
474.5
474.5
474.5
459.2
459.2
459.2
205–207
195–197
187–189
212–214
178–180
185–187
218–220
228–230
235–237
215–217
223–225
210–212
217–219
213–215
207–209
210–212
72
69
72
64
65
59
69
77
64
65
79
65
73
56
73
65
4-Br
2-NO2
3-NO2
4-NO2
2-OCH3
3-OCH3
4-OCH3
Nevertheless, the structures of all new compounds synthesized were confirmed
1
13
by IR, H-NMR and C-NMR spectroscopy. The analytic and spectral data for
all the compounds synthesized in this study are given in the Supplementary
material to this paper. The IR spectra of all the compounds 4a–p showed
absorption bands at around 2993–2955, 2868–2839, 1679–1664, 1589–1557, 1189–
–1
–1174, 1097–1018 cm regions, confirming the presence of CH, CH , C=N, C=C,
2
C–N, C–O groups, respectively. The, the chemical shifts, multiplicities, and
1
coupling constants of the signals in the H-NMR spectra of the respective deri-
vatives 4a–p verified their structures. The spectra of most compounds showed
the characteristic 4H protons of pyridine at around δ 8.98–8.15 ppm, the charac-
teristic protons of phenyl at δ 7.95–6.45 ppm, the 1H proton of oxadiazole at around
δ 5.73–5.37 ppm, the 3H protons of O–CH at around δ 3.88–3.64 ppm, the 2H
3
protons of Ar–CH –N at around δ 3.69–3.52 ppm, the 6H protons of N–2CH at
2
3
around δ 2.49–2.26 ppm and the 3H protons of CH at δ 1.21–1.05 ppm. The
3
13
C-NMR spectra of compounds 4a–p exhibited characteristic signals of
–N=C–CH at around δ 164.77–164.18 ppm, phenyl at δ 155.87–119.15 ppm,
3
pyridine at δ 149.78–124.11 ppm, oxadiazole at δ 155.17–154.13, O–CH at δ
3
57.63–54.29, Ar–CH –N at δ 54.73–53.92 ppm, N-2CH at δ 47.38–45.49 ppm
2
3
and N–C–CH at δ 22.77–15.13 ppm.
3
Antimicrobial activity
The compounds were evaluated for their antimicrobial properties in compa-
rison to the control antibacterial drug amoxicillin and antifungal drug nystatin.
The determined MIC values of compounds 4a–p are listed in Table II. The inves-
tigation of antibacterial screening data revealed that all the tested compounds
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MALHOTRA et al.
showed moderate to good bacterial inhibition. The compounds 4g, 4h, 4m and 4p
displayed excellent activity against the Gram-positive bacteria B. subtilis and S.
aureus and good activity against the Gram-negative bacteria P. aeruginosa and
E. coli. Compounds 4c, 4d and 4f showed moderate antibacterial activity, while
compounds 4a, 4b, 4e and 4o were less active against the tested bacterial strains.
Of all the synthesized derivatives, compound 4a was found to be the least active
compound against most of the bacterial strain. From, these results, it could be ge-
neralized that the p-chloro-, o-bromo-, p-nitro- and p-methoxy-substituted oxadi-
azole derivatives showed higher antibacterial activity compared to the other ana-
logues.
TABLE II. Antimicrobial screening results of the tested compounds (minimum inhibitory
concentration, µg ml-1)
Gram-positive bacteria
B. subtilis S. aureus P. aeruginosa E. coli
(MTCC121) (MTCC96) (MTCC2453) (MTCC40) (MTCC227) (MTCC8189)
Gram-negative bacteria
Fungal strain
C. albicans A. nigar
Compound
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
6.25
12.5
6.25
6.25
12.5
12.5
3.12
12.5
3.12
3.12
6.25
3.12
1.56
6.25
25
12.5
50
50
25
3.12
12.5
3.12
12.5
12.5
3.12
3.12
6.25
25
25
6.25
25
1.56
12.5
6.25
3.12
12.5
3.12
6.25
3.12
6.25
6.25
6.25
3.12
50
25
12.5
3.12
12.5
12.5
3.12
3.12
6.25
12.5
12.5
25
12.5
12.5
12.5
6.25
1.56
6.25
6.25
6.25
6.25
1.56
6.25
12.5
12.5
25
12.5
3.12
12.5
12.5
6.25
12.5
6.25
3.12
6.25
1.56
3.12
12.5
6.25
12.5
12.5
3.12
25
25
12.5
6.25
6.25
6.25
12.5
6.25
12.5
1.56
3.12
Amoxicillin
Nystatin
0.12
–
0.25
–
0.15
–
0.20
–
–
0.30
–
0.78
Concerning the antifungal activity of the tested compounds, only two fungal
strains were selected C. albicans and A. niger and the result of antifungal screen-
ing data revealed that all the synthesized compounds showed variable degrees of
inhibition against the tested fungi. The investigation of antibacterial screening
data revealed that all the tested compounds showed moderate to good fungal inhi-
bition as compare to standard drug nystatin. Of the screened compounds, 4g, 4h,
4m and 4p exhibited the highest antifungal activity against both fungal strains,
while compounds 4e, 4l and 4n showed moderate antifungal activity. Among all
the synthesized derivatives, compound 4i was found to be the least active com-
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ANTIMICROBAIL ACTIVITY OF OXADIAZOLE DERIVATIVES
15
pound against the fungal strains. From these result, it could be generalized that
the p-fluoro-, o-bromo-, p-nitro- and m-methoxy-substituted oxadiazole deriva-
tives 4g, 4h, 4m and 4p showed higher antifungal activity than the other analo-
gues. It was also observed that the derivatives having a chloro-, bromo-, nitro-
and methoxy- substituent at the ortho and meta positions were not as potent as
the derivatives having same substituent at the para position. Thus, from the ob-
tained results it was found that the nature and position of the substituent had
marked effects on antibacterial and antifungal activity.
SUPPLEMENTARY MATERIAL
Аnalytical and spectral data of synthesized compounds are available electronically from
http://www.shd.org.rs/JSCS/, or from the corresponding author on request.
И З В О Д
НОВИ ОКСАДИАЗОЛНИ ДЕРИВАТИ ИЗОНИКОТИНОХИДРАЗИДА У ПОТРАЗИ ЗА
АНТИМИКРОБНИМ АГЕНСИМА: СИНТЕЗА И IN VITRO ЕВАЛУАЦИЈА
MANAV MALHOTRA1, MOHIT SANDUJA2, ABDUL SAMAD3 и AAKASH DEEP4
1Department of Pharmaceutical Chemistry, Meerut Institute of Engineering and Technology, Bypass Road-
-Baghpat Crossing, Meerut-250005, Uttar Pradesh, India, 2Department of Pharmaceutical Chemistry, ISF
College of Pharmacy, Ferozepur Road, Moga-142001, India, 3Department of Pharmaceutical Chemistry,
College of Pharmacy in Al-Kharj, King Saud University, Riyadh, Saudi Arabia и 4Department of
Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak-124001, India
Структурним модификацијама најважнијег лека против туберкулозе, изониjазида, доби-
јени су деривати који имају израженије липофилне особине услед замене хидразидне функ-
ције 1,3,4-оксадиазолским хетероцикличним делом структуре. На тај начин се спречава in
vivo ацетиловање ензимом ариламин-N-ацетилтрансфераза чиме су добијани неактивни
ацетиловани деривати. У овом раду приказано је шеснаест нових оксадиазолских деривата
1
који су окарактерисани спектралним анализама (IR, H-NMR, 13C-NMR и MS). Испитана је
антимикробна активност синтетисаних деривата према Грам-позитивним (Bacillus subtilis и
Staphylococcus aureus), Грам-негативним сојевима (Pseudomonas aeruginosa и Escherichia
coli) и сојевима гљива (Candida albicans и Aspergillus niger). Минималне инхибиторне
концентрација једињења налазе се у опсегу 1,56–50 µg ml-1 према сојевима бактерија и
гљива. Резултати показују да једињења показују изражену активност, од којих су нај-
активнији деривати 4g, 4h, 4m и 4p.
(Примљено 23. јануара , ревидирано 24. јуна 2011)
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