Synthesis, antidepressant and antimicrobial activities of some novel stearic acid analogues

Article abstract of DOI:10.1016/j.ejmech.2012.06.025

Stearic acid, a saturated fatty acid was isolated from the microalga Spirulina platensis. Some novel stearic acid analogues having 1,3,4-oxadiazole, 1,2,4-triazole and 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole are synthesized and characterized by IR, NMR a

Full text of DOI:10.1016/j.ejmech.2012.06.025

European Journal of Medicinal Chemistry 54 (2012) 931e935
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, antidepressant and antimicrobial activities of some novel stearic
acid analogues
,
a
b
a
Selvaraj Jubie a , Patil Nilesh Ramesh , Palanichamy Dhanabal , Rajagopal Kalirajan ,
*
Nithyanantham Muruganantham b, Anthoniswamy Shanish Antony c
,
1
a Department of Pharmaceutical Chemistry, JSS College of Pharmacy, Rock lands, Ooty 643 001, Tamilnadu, India
b Department of Phytopharmacy and Phytomedicine, JSS College of Pharmacy, Ooty, India
c Department of Pharmacology, JSS College of Pharmacy, Ooty, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Stearic acid, a saturated fatty acid was isolated from the microalga Spirulina platensis. Some novel stearic
acid analogues having 1,3,4-oxadiazole, 1,2,4-triazole and 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole are
synthesized and characterized by IR, NMR and mass spectral analysis. All the synthesized compounds
were screened for antimicrobial activity by using cup plate method. The synthesized compounds have
been further screened for their antidepressant activity in swiss albino mice by forced swim test (FST),
midbrain dopamine has been estimated and quantified. All the compounds showed good antimicrobial
activity and compound 6 showed significant antidepressant activity.
Received 28 September 2011
Received in revised form
7 June 2012
Accepted 13 June 2012
Available online 23 June 2012
Keywords:
Spirulina platensis
Stearic acid
Ó 2012 Elsevier Masson SAS. All rights reserved.
Antidepressant
Estimation of dopamine antimicrobial
1. Introduction
During the last two decades, the chemistry of 1,2,4-triazole,
1,3,4-oxadiazoles, 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole and their
Spirulina platensis, a blue green microalga, has been used since
ancient times as a source of food because of its high protein and
nutritional value. Recently, this alga is being widely studied, not
only for nutritional reasons but also for its reported medicinal
properties. Sseveral studies have shown that Spirulina or its
extracts could prevent or inhibit cancer in humans and animals and
recent works have indicated that this species has immuno-
promoting effects. S. platensis was also reported to possess anti-
microbial activity [1e4]. Many seed oils, fatty acids (FA) and their
derivatives are known for their antimicrobial, antifungal and pes-
ticidal activities [5]. Saturated fatty acids are of interest since they
have lower melting points and exhibit good oxidative stability [6].
Fatty acids function as the key ingredients of antimicrobial food
additives which inhibit the growth of unwanted microorganisms
[7]. Also, fatty acids have been an emerging interest in treating
neuropsychological disorders such as depression and schizo-
phrenia [8,9].
derivatives have received considerable attention owing to their
synthetic and effective biological importance [10e13].
On the basis of this background the present work has
been aimed to synthesize some novel stearic acid analogues having
1,3,4-oxadiazole, 1,2,4-triazole and 1,2,4-triazolo-[3,4-b]-1,3,4-
thiadiazole substitution and to evaluate their antidepressant
activity and antimicrobial activity.
2. Results and discussion
2.1. Chemistry
We have synthesized three novel stearic acid analogues such as
2-(heptadecyl) e 5 - phenyl ꢀ1, 3,4-oxadiazole, 2-(heptadecyl)-5-
phenyl-1,2,4-triazole and 6 (-heptadecyl)-3-phenyl-[1,2,4] triazolo
[3,4-b]1,3,4-thiadiazole as illustrated in Scheme 1. Structures of all
the compounds were established on the basis of elemental analysis,
IR, 1H NMR and mass spectral data. Three novel heterocyclic
compounds such as 1,3,4- oxadiazole,1,2,4- triazole and
1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole substituted stearic acid
analogues were synthesized according to the Scheme. Benzoic acid
hydrazide 2 was obtained by the reaction of methyl benzoate with
* Corresponding author. Tel.: þ91 423 2443393; fax: þ91 423 2442947.
E-mail address: jubiejawahar@gmail.com (S. Jubie).
1
Off campus e JSS University, Mysore.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.025

932
S. Jubie et al. / European Journal of Medicinal Chemistry 54 (2012) 931e935
NH2NH2
C6H5COOC2H5
1
a
d
C6H5CONHNH2
C6H5CONHNH-C-S K
2
S
2a
b
e
N
N
N
N
C17H35
O
C
O
C6H5
3
C6H5
SH
N
c
OH
C17H35
NH2
5
f
N
N
C17H35
N
N
N
C6H5
S
NH2
C6H5
4
N
N
C17H35
6
Scheme 1. Synthesis of the compounds. Reagents and conditions. a) NH2NH2, reflux 8 h. b) POCl3,C17H35COOH, reflux, 8 h, neutralization with NaOH, c) NH2NH2, reflux, 8 h,
acidification with HCl upto pH3 d) CS2/Aq.KOH/C2H5OH,stirring 10 h e) NH2NH2 reflux, 3 h, acidification with HCl. f) POCl3,C17H35COOH, reflux, 8 h, neutralization with NaOH.
hydrazine hydrate in the presence of absolute ethanol. Acid
hydrazides can be considered as useful intermediates leading to the
formation of some heterocyclic rings such as 1,2,4-triazole, 1,3,4-
thiadiazole, 1,3,4-oxadiazole. Condensation of acid hydrazides
with various aromatic carboxylic acids in the presence of boiling
phosphorous oxychloride yields 1,3,4-oxadiazole. In the present
study, in place of aromatic acids a saturated carboxylic acid such as
stearic acid was used for the synthesis of our aimed heterocyclic
compounds. Compound 2 on treatment with stearic acid in the
presence of phosphorous oxychloride afforded 2- (heptadecyl) e 5 -
phenyl ꢀ1, 3, 4 e oxadiazole 3. (Scheme 1). The reaction of 3 with
compounds. The preliminary structural activity relationship
showed that introduction of fused ring system such as [1,2,4] tri-
azolo[3,4-b]1,3,4-thiadiazole substitution exhibited significant
antidepressant activity than oxadiazole and triazole substitution.
Also the lipophilicity data suggested that compounds 3, 4 and 6
were most lipophilic in nature (Clog P 9.28, 9.62 and 10.37) and
showed highest antidepressant activity (Table 2).
2.3. Effect of stearic acid analogues on dopamine levels
Further to confirm the antidepressant activity we have esti-
mated the dopamine levels of midbrain region. The data showed
either increase or maintenance of dopamine levels after treatment
with test or standard drugs (Table 3). The retention of dopamine at
midbrain region might be the cause to exhibit significant antide-
pressant activity for the synthesized compounds.
Dopamine concentration in striatal region was measured by
HPLC equipped with UV spectrophotometer detector. All animals of
the test groups showed significant difference in the dopamine
excess hydrazine produced
2 - (heptadecyl)e5ephenyl-1,2,4-
triazole 4 (Scheme 1). On the other hand potassium dithiocarba-
zinate [2a] was prepared from compound 2, by using potassium
hydroxide, carbon disulphide and ethanol as a solvent. Compound
2a had been undergone ring closure reaction with excess hydrazine
resulted in the formation of 4-amino-5-phenyl-4H-1,2,4 triazole-3-
thiol 5 Compound 5 was cyclized with stearic acid to get 6(-hep-
tadecyl)-3-phenyl-[1,2,4] triazolo[3,4-b]1,3,4-thiadiazole 6 using
POCl3 as a solvent.
2.2. Biological activity
Table 1
Antidepressant screening by forced swim test.
The stearic acid analogues 3,4 and 6 were evaluated for anti-
depressant activity by FST in rats at a dose of 30 mg/kg and
compared with the standard drug (Diazepam, 30 mg/kg). Antide-
pressant activity was assessed as mean immobility time in seconds
and data are presented as Mean ꢁ S.E.M in Table 1. Results of FST
revealed that compounds 3, 4 and 6 exhibited significant (P < 0.01)
reduction in immobility time compared to control while compound
6 showed the most potent antidepressant activity among the titled
Compound
Duration of immobility in sec. (mean ꢁ S.E.M)
Control
3
4
6
80.67 ꢁ 0.882
70.00 ꢁ 1.065**
68.83 ꢁ 1.167**
62.67 ꢁ 1.520***
52.97 ꢁ 0.77***
Clomipramine
Data was analysed by Dunnet’s test. n ¼ 6; (***) equals p < 0.001, (**) equals p < 0.01
when compared with control.

S. Jubie et al. / European Journal of Medicinal Chemistry 54 (2012) 931e935
933
Table 2
4. Experimental protocol
Physicochemical data of compounds 3, 4 and 6.
Yielda (%)
M.P.(◦C)
R.f.b
Clog Pc
4.1. Materials and methods
Compound
3
4
6
65
62
68
72e78
76e79
71e74
0.20
0.23
0.30
9.28
9.62
10.37
The used chemicals were purchased from Fluka chemicals. Their
purity was checked by GC. All solvents were purified by distillation
and if necessary residual water was removed. The composition of
solvents and eluents are given in volume ratios of the components.
Plant powder of S. platensis was collected from Antenna Research
Foundation Pvt. Limited, Madurai, Tamilnadu, India. Stearic acid
methyl ester was isolated according to the previously reported
method [14]. It was hydrolysed to give free stearic acid. Products
were purified by column chromatography and identified using
different spectral techniques.
Melting points were taken in glass capillary tubes on a Veego
VMP-1 apparatus and are uncorrected. The 1H NMR were recorded
on Bruker DRX-300 (300 MHz FT-NMR) using deuterated chloro-
form as solvent and TMS as internal standard. The IR spectra of
compounds were recorded on Shimadzu FT-IR spectrometer by KBr
pellet technique and are expressed in cmꢀ1. The mass spectra of
compounds were recorded on JEOL GCMATE II GCeMS.
a
After recrystallization from ethanol.
Ethyl acetate: Chloroform (9:1).
Calculated using Cambridge software 8.0.
b
c
levels when compared with control. Compound 6 increased the
dopamine concentration while the compounds 3& 4 decreased the
dopamine levels.
2.4. Antimicrobial screening
The results of preliminary antimicrobial testing of compounds
are shown in Tables 4 and 5. The investigation of antibacterial
screening data revealed that all the three tested compounds were
found to have moderate to good bacterial inhibition. The antibac-
terial potency has been calculated as per the procedure given in
Indian Pharmacopoeia. The results revealed that compound 3
showed pronounced antibacterial activities against Escherichia coli
with 93.30% potency and Staphylococcus aureus with 94.60%
potency. Also it showed moderate antibacterial activities against
Bacillus subtilis and P. aeruginosa with 75.78%, 60.95% respectively.
Compound 6 showed pronounced antibacterial activities against B.
subtilis and P. aeruginosa with 93.88%. It showed moderate activities
against E. coli with 84.08% potency and S. aureus with 79.35%
4.2. Synthesis of benzoic acid hydrazide 2
Hydrazine hydrate (10 mmol, 1.25 ml) was placed in a round
bottom flask fitted with a reflux condenser. To this solution ethyl
benzoate (10 mmol, 1.5 ml) was added dropwise and heated gently
for 15 min. Then absolute alcohol (5 ml) was added through the
condenser to produce clear solution and refluxed for 4e5 h. The
ethanol was distilled off and cooled. The crystals were filtered off by
the addition of ice water and recrystallised with ethanol.
Yield: 58.82%. MP: 112e116 ꢂC, Rf value: 0.20, TLC:
eluent ¼ Ethyl acetate: Chloroform (4:1),
potency. The Compound
4 showed pronounced antibacterial
activities against E. coli and B. subtils with 93.30% potency. It
showed moderate activities against S. aureus with 75.78% potency
and P. aeruginosa with 59.45%. All the compounds showed good
antifungal activity as compared to standard. Compound 6 showed
good antifungal activity against Candida albicans with 92.57%
potency.
IR (KBr cmꢀ1): 1662 (C]O), 3020 (AreCH str), 3300 (NeH str),
1487 (CeN str).
4.3. Synthesis of 2-(heptadecyl-5-phenyl-1, 3, 4-oxadiazole) 3
A mixture of benzoic acid hydrazide (10 mmol), stearic acid
(10 mmol) and phosphorus oxychloride (10 ml) was refluxed for
8 h, cooled and poured on crushed ice. The solid separated was
filtered, and treated with dil. NaOH, washed with water and
recrystallised from ethanol.
3. Conclusion
Owing to the biological importance of fatty acids, three novel
fatty acid analogues have been synthesized and confirmed by
physical parameters and consistent with their IR, 1H NMR and mass
spectra. The synthesized compounds show significant antidepres-
sant activity by forced swim test when compared to the control and
moderate to good antimicrobial activities when compared to the
standards tetracycline and amphotericin B. In particular compound
6 showed significant antidepressant activity. From the present
study, it can be concluded that our synthesized stearic acid
analogues have significant antidepressant and antimicrobial
activities.
IR (KBr) cmꢀ1; 3057.27 (Ar CeH), 1602.90 (C]N), 1591 (C]C),
1290.42
(CeOeC),
769.62,
690.54
(AreCH).1H
NMR
( 300 MHZ,CDCl3)
d; 7.4e8.2 (m, 5H, AreH), 2.55 (t, 2H, CH2), 1.2 (s,
28H, CH2), 1.9 (m, 2H, CH2), 0.96 (t, 3H, CH3) ppm. FAB-MS: 384
(Mþ, 100%): 232 (C17H35COþ),267(C17H35CNþ)m/zþ2103(C6H5CNþ)
m/z-2.Anal.Calcd.for.C25H40N2O :C,78.07; H,10.48; N,7.28. Found
:C,78.17; H,10.61; N,7.37%.
4.4. Synthesis of 2-(heptadecyl)-5-phenyl-1,2,4-triazole 4
A
mixture of oxadiazole (10 mmol), hydrazine hydrate
(10 mmol) and absolute alcohol (10 ml) was refluxed for 5 h. Then,
potassium hydroxide was added to the reaction mixture and the
precipitate formed was filtered. The solid obtained was acidified
with conc. HCl up to pH-3 and washed with water. The resultant
solid was recrystallised from ethanol.
Table 3
Effect of control, stearic acid analogous on concentrations of dopamine level in brain.
Groups
Concentration of dopamine (ng/mg of protein)
Control
Clomipramine
Comp. 3
Comp. 4
Comp. 6
0.399 ꢁ 0.151
0.420 ꢁ 0.181###
0.250 ꢁ 0.035##
0.367 ꢁ 0.031##
1.455 ꢁ 0.030###
IR (KBr) cmꢀ1; 3057.27 (NeH), 2955.04(AreCH), 1602.90 (C]N),
1572.04 (C]C), 767.69, 688.61 (AreCH). 1H NMR (300 MHZ,CDCl3)
d; 7.45e7.55 (m, 5H, AreH), 7.1 (s, 1H, NH2), 2.8 (m, 2H, CH2), 1.2
Data was analysed by Dunnet’s test. n ¼ 6; (###) equals p < 0.001, (##) equals
p < 0.01 when compared with control.
(s, 28H, CH2), 1.8e1.9 (m, 2H, CH2), 0.96 (t, 3H, CH3) ppm. FAB-MS;
398 (Mþ,100%)0.103(C6H5CNþ)m/z ꢀ2103. Anal.Calcd. for.

934
S. Jubie et al. / European Journal of Medicinal Chemistry 54 (2012) 931e935
Table 4
Antibacterial activity of 3, 4 & 6.
Compd.
Ec
Pa
Sa
Bs
Conc. (
500
m
g/ml)
Conc. (
m
g/ml)
1000
Conc. (
500
m
g/ml)
1000
Conc. (
m
g/ml)
1000
1000
% pot
500
% pot
% pot
500
% pot
3
4
6
Std
1.4
1.6
1.6
1.8
1.8
1.7
1.9
2.3
93.30
93.30
84.08
e
1.6
1.3
1.5
1.6
1.7
1.6
1.9
2.2
60.95
59.45
93.88
e
1.4
1.3
1.6
1.8
2.1
1.8
1.7
1.9
94.60
75.78
79.35
e
1.3
1.2
1.3
1.3
1.6
1.5
1.6
2.0
75.78
93.30
93.88
e
Sa eStaphylococcus aureus Pa e Pseudomonas aeruginosa.
Ec e Escherichia coli Bs e Bacillus subtilis.
C25H42N4:C,75.33; H,10.62; N,14.05. Found :C,75.37; H,10.74;
N,14.37%.
were conducted between 9 and 14 h. Each mouse was used only
once.
4.6.2. Anti depressant activity
4.5. Synthesis of 4[amino]-5-phenyl-4H-1,2,4-triazole-3-thiol 5
The activity was screened by forced swim test (FST) [15]. Forced
swimming test is a behavioural test used to assess the efficacy of an
antidepressant drug. The synthesized compounds were used for
the study. They were housed under standard laboratory conditions
for a week before the experiments. The housing conditions were
maintained at controlled temperature (23 ꢂC) (30 mg/kg) and
standard drug clomipramine (30 mg/kg) were suspended in 0.5%
carboxymethylcellulose (CMC) and were injected intraperitoneally
(i.p), 30 min prior to the test. Control animals received the vehicle
only. The mice were forced to swim individually in a glass jar (tank)
(25 ꢃ 12 ꢃ 25 cm3) containing fresh water of 15 cm height and
maintained at 25 ꢁ 3 ꢂC. After an initial 2 min period of vigorous
activity, each animal assumed a typical immobile posture. A mouse
was considered to be immobile when it remains floating in the
water without struggling, making only minimum movements of its
limbs necessary to keep its head above water. The total duration of
immobility was recorded during the next 4 min of a total 6 min test.
The change in immobility duration was studied after administering
drugs in separate groups of 6 animals. The percentage change from
control [(Test ꢃ 100)-100 was calculated for each mouse.
A mixture of carbon disulphide (15 mmol, 11.2 g), potassium
hydroxide (15 mmol, 8.5 g) in absolute ethanol (125 ml), benzoic
acid hydrazide (10 mmol,1.36 g) was stirred for 16 h. To the resulted
solution dry ether (250 ml) was added and the precipitate potas-
sium dithiocarbazinate was collected by filtration. The potassium
salt was obtained by the above procedure was near to the quanti-
tative yield and used directly without purification. A suspension of
the potassium salt (10 mmol, 1.25 g), hydrazine hydrate (3 ml) and
water (2 ml) was refluxed for 3e4 h. The colour changes to green,
with the evolution of hydrogen sulphide gas. After cooling the
solution white precipitate was obtained. It was acidified with conc.
hydrochloric acid. The product was collected by filtration and
recrystallised with ethanol.
Yield: 88%; m.p. 211e216 ꢂC, Rf value: 0.22, IR (KBr) cmꢀ1;943.3
(NeCeS), 1278 (NeNeC), 3365(NeH), 696.33 (CeS), 3082 (AreCH).
4.5.1. Synthesis of 6(-heptadecyl-3-phenyl-[1,2,4]triazolo[3,4-b])
1,3,4-thiadiazole 6
A mixture of 1,2,4 triazole (10 mmol), stearic acid (10 mmol) and
phosphorus oxychloride (10 ml) was refluxed for 8 h, cooled and
poured on crushed ice. The solid separated was filtered, and treated
with dil. NaOH, washed with water and recrystallised from ethanol.
IR (KBr) cmꢀ1;2955.04 (AreCH), 1602.90 (C]N), 1573.97 (C]C),
4.6.3. Estimation of dopamine
Dissected striata were immediately frozen on dry ice and stored
at ꢀ80 ꢂC. Striatal tissue was sonicated in 0.1 M perchloric acid
771.55 (AreCH). 1H NMR (300 MHZ,CDCl3)
d; 7 (m, 5H, AreH), 2.8(t,
(100
mg/mg tissue). The supernatant fluids were taken for
measurement of dopamine levels by HPLC. Briefly, 20 ml superna-
2H, CH2),1.2 (s, 28H, CH2),1.7 (m, 2H, CH2), 0.9 (t, 3H, CH3) ppm. FAB-
MS; 440 (Mþ,100%). 253 (CH2-C17H35),124 (C3N4S) 103 (C6H5CN
m/zþ4), 79(C6H5 m/zþ2).Anal.Calcd.for.C26H40N4S:C,70.86; H,9.15;
N,12.71.Found:C,70.37; H,9.15; N,12.37%.
tant fluid was isocratically eluted through an 4.6 mm C18 column
with a mobile phase containing 50 mM ammonium phosphate pH
4.6, 25 mM hexane sulfonic acid pH 4.04, 5% acetonitrile and
detected by UV detector. The flow rate used was 0.5 ml/min. Par-
acetamol (100 mg/ml) was used as the internal standard. Concen-
4.6. Biology
tration of dopamine was expressed as nanogrammes per
milligramme of protein [16].
4.6.1. Animals required
Inbred male albino mice (Swiss strain) weighing between 20
and 30 g and the relative humidity of 50 ꢁ 5%. They received
a standard diet and water ad libitum. The animals were transferred
to the laboratory 1 h before the start of the experiment. The insti-
tutional animal ethics committee approved the study. Experiments
4.7. Antimicrobial activity
Compounds 3, 4 and 6 were tested for in vitro antimicrobial
activity against the Gram- positive bacteria S. aureus and B. subtilis,
the Gram negative bacteria Salmonella typhimuriu, E. coli and fungi
C. albicans. The primary screen was carried out by agar disc diffu-
sion method [17,18]. Standard inoculum (1e2 ꢃ 107 c.f.u./Ml 0.5
McFarland standards) was introduced on to the surface of sterile
agar plates, using a sterile glass spreader for even distribution. Disc
measuring 6 mm in diameter, sterilized by dry heat at 140 ꢂC for 1 h,
were prepared from whatman No.1 filter paper. The sterile discs
previously soaked in a known concentration of the test compounds
were placed in nutrient agar medium. Solvent and growth controls
were kept. Tetracycline and amphotericin-B were used as negative
Table 5
Antifungal activity of 3, 4 & 6.
Compd.
Ca conc (mg/ml)
500
1000
% Pot
3
4
6
1.5
1.5
1.5
2.0
2.2
1.9
56.71
65.97
92.57
Ca e Candida albicans.

S. Jubie et al. / European Journal of Medicinal Chemistry 54 (2012) 931e935
935
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control. The plates were inverted and incubated for 24 h at 37 ꢂC.
The susceptibility was assessed on the basis of diameter of zone of
inhibition against Gram positive and Gram negative strains of
bacteria and fungal strain. Inhibition zones were measured and the
percentage potency was calculated.
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Acknowledgements
This study was supported by financial grant under Department
of Science and Technology (DST) (SR/SO/HS-0143-2009) India.
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