Synthesis, ADME, docking studies and in vivo anti-hyperglycaemic potential estimation of novel Schiff base derivatives from octadec-9-enoic acid

Article abstract of DOI:10.1016/j.bioorg.2018.12.004

A new series of octadec-9-enoic acid schiff base entities (S1-S30) were designed and synthesized targeting peroxisome proliferator activated receptor-gamma for agonist action. Molinspiration software (online) was used to estimate drug like molecular properties of the metabolites. Docking disquisition on co-crystallized protein of PPAR-γ (PDB ID 1FM9) was carried out which showed S21, S10 and S7 as best situated in the vital sites of receptor having docking scores ?9.19, ?8.68 and ?8.64 respectively. Free binding energy measured using model of Maestro 9.0 and was in range of from ?40.01 and ?80.54 kcal/mol, significant when compared with pioglitazone (?51.58 Kcal/mol). Seven best docked derivatives were assessed for in-vivo oral glucose tolerance on normal rats and anti-hyperglycaemic activity by streptozotocin induced diabetes model. S21 unveiled to be the best measured analogue among all the synthesized entities. Encouraging outcomes motivates fatty acids for further development of more effective and safer compounds.

Full text of DOI:10.1016/j.bioorg.2018.12.004

Accepted Manuscript
Synthesis, ADME, Docking Studies and in vivo Anti-Hyperglycaemic Potential
Estimation of Novel Schiff Base Derivatives from Octadec-9-enoic Acid
Garima Kapoor, Dharam Pal Pathak, Rubina Bhutani, Asif Husain, Sandeep
Jain, Md. Azhar Iqbal
PII:
S0045-2068(18)31051-4
DOI:
https://doi.org/10.1016/j.bioorg.2018.12.004
YBIOO 2668
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 September 2018
28 November 2018
3 December 2018
Please cite this article as: G. Kapoor, D. Pal Pathak, R. Bhutani, A. Husain, S. Jain, Md. Azhar Iqbal, Synthesis,
ADME, Docking Studies and in vivo Anti-Hyperglycaemic Potential Estimation of Novel Schiff Base Derivatives
from Octadec-9-enoic Acid, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.12.004
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Synthesis, ADME, Docking Studies and in vivo Anti-Hyperglycaemic Potential
Estimation of Novel Schiff Base Derivatives from Octadec-9-enoic Acid
Garima Kapoor a*, Dharam Pal Pathak a, Rubina Bhutani a, Asif Husain b, Sandeep Jain c, Md.
Azhar Iqbal b
a
Department of Pharmaceutical Chemistry, Delhi Institute of Pharmaceutical Sciences and
Research, New Delhi, India
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard
University), New Delhi, India
c
Department of Pharmaceutical Chemistry, Guru Jambeshwar University of Science and
Technology, Hisar, Haryana, India
*Authors for correspondence
Address: Department of Pharmaceutical Chemistry, Delhi Institute of Pharmaceutical
Sciences and Research, New Delhi-110017, India.
Phone: +91-9643503380
E-mail addresses: kapoor27garima@gmail.com (G. Kapoor), drasifhusain@yahoo.com (A.
Husain).

ABSTRACT
A new series of octadec-9-enoic acid schiff base entities (S1-S30) were designed and
synthesized targeting peroxisome proliferator activated receptor-gamma for agonist action.
Molinspiration software (online) was used to estimate drug like molecular properties of the
metabolites. Docking disquisition on co-crystallized protein of PPAR-γ (PDB ID 1FM9) was
carried out which showed S21, S10 and S7 as best situated in the vital sites of receptor having
docking scores -9.19, -8.68 and -8.64 respectively. Free binding energy measured using
model of Maestro 9.0 and was in range of from -40.01 and -80.54 kcal/mol, significant when
compared with pioglitazone (-51.58 Kcal/mol). Seven best docked derivatives were assessed
for in-vivo oral glucose tolerance on normal rats and anti-hyperglycaemic activity by
streptozotocin induced diabetes model. S21 unveiled to be the best measured analogue among
all the synthesized entities. Encouraging outcomes motivates fatty acids for further
development of more effective and safer compounds.
Keywords: Fatty acids; Hyperglycaemia; Molecular docking; PPAR; Schiff base; STZ

1. INTRODUCTION
Diabetes Mellitus type 2 or Non-Insulin dependent Diabetes Mellitus (NIDDM) is long-term
metabolic disease [1] which is 4th ruling cause of death in world; and execution of which
requires wide financial and public responsibility [2]. The International Diabetes Federation
(IDF) has assessed that the world over ubiquity of diabetes has forecasted to rise from 415
million today to 642 million in 2040 [3]. In diabetes, there is imbalance in carbohydrate and
lipid metabolism by insulin. Insulin is secreted from pancreatic beta cells in reaction to rising
plasma glucose, with various factors reorganizing its secretion [4].The increase in glucose
level characterized by T2DM assists spreading of complications and result in metabolic
abnormalities. Chronic hyperglycemia brings various disorders related to heart, kidney and
eyes [5].
Recently peroxisome proliferator activated receptors (PPARs) have appeared to be one of the
key governors of the nutrient and gene synergic reactions. PPARs exist in three subtypes α, β
and γ, in different species. They are a group of nuclear hormone receptor isoform that
manages dietary fat and are also a goal for the development of therapy for type 2 diabetes,
obesity and cardiovascular disease. They also control the expression of genes involved in
lipid balance and activates gene transcription to variety of drugs and natural fatty acids [5].
PPARs are target for structurally diverse fatty acids, eicosanoids and hypolipidemic drugs
[7,8].
Fatty acids are significant nutritive and healthy component implicated in heart and metabolic
diseases [9]. Diets rich in stearic acid and other saturated fatty acids are favourable for
diabetes mellitus patients [10]. Oleic and linoleic acids innervate insulin secretion in high-
glucose concentrations which specify that free fatty acids (FFAs) rise glucose-stimulated
insulin secretion (GSIS) [4].Newly designed fatty acids work on gene expression by binding
to and triggering PPAR subtypes to fall off blood glucose level [6]. Derivatives of stearic acid

like bromstearate induced marked hypoglycaemia in intact animals, normalized
hypeglycemia in rats with alloxan induced diabetes for 24h after administration as when
given by intra peritoneal injection, they inhibited the oxidation of fatty acids and
gluconeogenesis and enhanced the glucose oxidation in intact animals and in rats with
diabetes. Inhibitors of fatty acid oxidation are suggested for the pathogenesis therapy of
diabetes [11]. Substituted fatty acids and its derivatives having long-chain stimulate PPAR
gamma to a some extend and act as their endogenous ligand [12].Nitro fatty acids which
contain nitro groups have distinguished PPAR ligand property exhibiting particular PPAR
interactions of electrophilic fatty acids [13].
Schiff bases are also a remarkable class of ligands in pharmaceutical chemistry field having
synthetic flexibility, constructional resemblance with natural organic molecules and also
because of imine group (N=CH-) which explains the mechanism of transformation and
racemisation reaction in biological system [14].They exhibit many pharmacological functions
inclusive of antibacterial [15,16], antifungal [16], anti-diabetic [17,18], antitumor [19], anti-
cancer [20], herbicidal [21], and anti-inflammatory [22] activities. Schiff bases are also used
as raw material in the industries [23,24]. Schiff base ligands have shown PTP1B inhibition
potential pursuing to develop as novel anti-diabetic drugs [17]. The Schiff base derivative
synthesized from 3-hydroxyflavone and metformin resulted in good anti-diabetic activity,
establishing new complexes decreasing primary and secondary complications of type 2
diabetes mellitus [18].
In view of aforementioned points, increasing diabetic population and side effects associated
with them, we have looked into the importance and significance of natural component like
fatty acid, its derivatives and schiff base derivatives in treating type II diabetes in particular,
which inspired us to schedule the present organized study accommodating heterocyclic schiff

base derivatives of fatty acid, expecting superior antihyperglycemic products with fewer
unwanted secondary effects than the prevailing ones.
2. MATERIAL AND METHODS
2.1. General
The reagents and solvents were purchased from commercial merchandisers like Merck,
Sigma-Aldrich etc. which were taken for reactions without refining them further. The
advancement of the reactions was verified by thin layer chromatography (TLC) on precoated
aluminium sheet plates (Merck, Germany) at 254 nm using UV-visualizer and iodine
chamber as detectors of spots on it. Melting points of the synthesized derivatives were
calculated by open capillary method working on Icon- Instruments electric melting point
apparatus and were not corrected. Infrared (IR) spectra were analysed by Bruker ATR
1
spectrophotometer. Nuclear magnetic resonance (NMR), H (300 MHz) and 13C NMR (75
MHz) spectra of all the prepared analogues were recorded on a Bruker Advance II
spectrometer DPX 300 in CDCl3 or DMSO-d6 as solvent. Tetramethyl silane (TMS) was used
as internal standard and chemical shifts were indicated as δ values which were reported in
parts per million (ppm). The mass spectrometric data of the series (at room temperature)
were resoluted on LCMS/LCQ of Agilent, Advantage-Max mass spectrophotometer.
Elemental analysis was executed by Perkin-Elmer 240 elemental analyzer for C, H and N
elements of each derived congener and results were in range of ± 0.4% when compared with
theoretical values. The protocol for synthetic procedure and different substitutions are
summarized in Scheme 1.

Methanol in
benzene solvent
O
O
H2SO4
(1)
Oleic acid
[Octadec-9-enoic acid]
OH
OCH3
Hydrazine hydrate (80%)
Ethanol
O
(2)
HN NH2
Ethanol /
Different
Aldehydes
Few Drops of
Glacial acetic acid
S1-S30
O
R
HN N
HO
OCH3
OCH3
R=
OH
OCH3
OH
,
,
,
,
OCH3
OCH3
OCH3
O
Cl
N
,
,
,
,
OH
,
,
Cl
O2N
Br
OCH3
,
,
,
F
F
Br
F
Cl
,
,
,
,
F
F
Cl
Cl
H3CO
F
Br
OCH3
,
,
,
,
H3CO
H3CO
NO2
Cl
HO
N
OCH3
,
,
Cl
,
,
HO
Scheme 1:- Protocol for the synthetic scheme of novel fatty acid derivatives.
2.2. Synthesis
2.2.1. Synthesis of methyl oleate (1) [6]
Oleic acid or (E)-octadec-9-enoic acid (0.25M) and methanol (1.24M) were taken in and
poured in a three neck 250mL round bottom flask. This round bottom flask was assembled on

ice box and magnetic stirrer. 2mL of H2SO4 was added drop by drop by separating funnel
into it while stirring. After H2SO4 is finished, the mixture was refluxed for 7h on water bath
in which condenser was fixed with a glass tube containing Na2SO4 anhydrate and cotton at
the end. The product obtained was vaporized for elimination of solvent and methanol using
rotary evaporator. 100mL of n-hexane was added to the residue and then washed 3 times with
water. Na2SO4 anhydrous was added to the resulting solution and kept for 24h before
filtering. Then the residue was again purified using rotary evaporator to evaporate n-hexane
and gave methyl oleate as a product in liquid form.
The yield of methyloleate was 91 %, m.p. 15oC and b.p. 358oC, IR: (KBr, cm-1) 2925, 2854
1
(C-H), 1742 (C=O), 1658 (C=C), 1169 (C-OC), 722 [(CH2)n]. H NMR: (CDCl3, δ, ppm):
5.31 (2H, m, CH=CH), 3.7 (3H, s, OCH3), 2.4 (2H, t, CH2), 1.89 (4H, m, 2×CH2), 1.6 (2H, m,
CH2), 1.29 (20H, m, 10×CH2), 0.89 (3H, s, CH3). ESI-MS: m/z 297 (M+H). Anal. calcd. for
C19H36O2: C, 76.97; H, 12.24%. Found: C, 76.53; H, 12.56%.
2.2.2. Synthesis of stearohydrazide (2)
Methyl oleate (0.1M) was poured in the 100mL RBF having 80% hydrazine hydrate (0.1M)
which was refluxed with stirring for 9h using ethanol as solvent. The resultant mixture was
taken in a beaker and precipitate was obtained by keeping the beaker on ice box for few
minutes and then was filtered. The recrystallization was done from ethanol to get
stearohydrazide [6, 25]. The yield was 84%, m.p. 107oC, IR: (KBr, cm-1) 3316-3100 (NH-
1
NH2), 2921, 2853 (C-H), 1629 (O=C-NH), 1160 (C-O-C), 720 [(CH2)n]. H NMR: (CDCl3,
δ, ppm): 8.38 (1H, s, NH), 3.75 (2H, s, NH2), 2.31 (2H, t, CH2–CONH), 1.62 (2H, m,
CH2CH2–CO), 1.27 (28H, s, 14×CH2), 0.91 (3H, t, CH3). ESI-MS: m/z 299 (M+H). Anal.
calcd. for C18H38N2O: C, 72.42; H, 12.83; N, 9.38%. Found: C, 72.71; H, 12.29; N, 9.49%.
2.2.3. General Procedure for the Preparation of Schiff bases (S1-S30)

To the solution of (2) (0.1M) in 20 mL ethanol, an appropriate aldehyde (0.1M) was added.
3mL of glacial acetic acid was added to this mixture and then refluxed with continuous
stirring for about 8h on Parallel synthesizer. The solvent was evaporated and the mixture was
poured onto crushed ice. The precipitate thus obtained was then filtered and washed with ice
cold water. The resultant residue was recrystallized from benzene to give final schiff bases
derivatives (S1-S30) [26-27].
2.2.4.1. N'-(4-hydroxybenzylidene)stearohydrazide (S1)
Yield: 59%, m.p.: 274–276°C, IR: (KBr, cm-1) 2856 (RCH3), 2925 (C-H phenyl), 1621
1
(C=O amide), 1482 (C=C phenyl) 1667 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.86 (3H, t,
CH3), 1.23 (28H, s, 14xCH2), 1.51-1.55 (2H, m, CH2), 2.15 (2H, t, CH2), 5.32 (1H, s, -OH),
6.83 [2H, d, 2xCH-(Ar)], 7.47 [2H, d, 2xCH-(Ar)], 7.86 (1H, s, -NH), 8.04 (1H, s, N=CH).
13C NMR: (DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 116.0, 126.4,
130.6, 143.0, 160.8, 167.5. ESI-MS: m/z 403 (M+H); Anal. calcd. for C25H42N2O2: C, 74.29;
H, 10.80; N,6.96%. Found: C, 74.29; H, 10.80; N, 6.67%.
2.2.4.2. N'-(3-hydroxybenzylidene)stearohydrazide (S2)
Yield: 51%, m.p.: 285–288°C, IR: (KBr, cm-1) 2854 (R-CH3), 2923 (C-H phenyl), 1620
1
(C=O amide), 1479 (C=C phenyl) 1668 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.90 (3H, t,
CH3), 1.31 (28H, m, 14xCH2), 1.44-1.53 (2H, m, CH2), 2.11 (2H, t, CH2), 5.01 (1H, s, -OH),
6.67 [1H, d, CH-(Ar)], 7.01-7.3 [3H, m, 3x-CH-(Ar)], 7.99 (1H, s, -NH), 8.1 (H, s, N=CH).
13C NMR: (DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 115.0, 118.2,
121.8, 130.3, 135.8, 143.0, 158.6, 167.5. ESI-MS: m/z 403 (M+H). Anal. calcd. for
C25H42N2O2: C, 74.54; H, 10.51; N,6.96%. Found: C, 74.28; H, 10.90; N, 6.72%.
2.2.4.3. N'-(2-hydroxybenzylidene)stearohydrazide (S3)
Yield: 66%, m.p.: 262–264°C, IR: (KBr, cm-1) 2866 (R-CH3), 2919 (C-H phenyl), 1620
1
(C=O amide), 1480 (C=C phenyl) 1666 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.85 (3H, t,

CH3), 1.24 (28H, s, 14xCH2), 1.53-1.64 (2H, m, CH2), 2.18 (2H, t, CH2), 5.32 (1H, s, -OH),
6.78-6.85 [2H, m, 2xCH-(Ar)], 7.05-7.13 [H, m, CH-(Ar)], 7.21-7.33 [H, m, CH-(Ar)], 7.88
(1H, s, -NH), 8.07 (H, s, N=CH). 13C NMR: (DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4,
29.6, 29.7, 31.9, 38.5, 116.0, 118.5, 121.5, 130.6, 143.0, 161.1, 167.5. ESI-MS: m/z 403
(M+H). Anal. calcd. for C25H42N2O2: C, 74.54; H, 10.51; N,6.96%. Found: C, 74.18; H,
10.80; N, 6.73%.
2.2.4. N'-(3,4,5-trimethoxybenzylidene)stearohydrazide (S4)
Yield: 58%, m.p.: 275–277°C, IR: (KBr, cm-1) 2865 (R-CH3), 3001 (C-H phenyl), 1626
1
(C=O amide), 1484 (C=C phenyl) 1659 (-N=CH), 1265 (-OCH3). H NMR: (CDCl3, δ,
ppm): 0.85 (3H, t, CH3), 1.23 (28H, s, 14xCH2), 1.49-1.58 (2H, m, CH2), 2.6 (2H, t, CH2),
3.49 (9H, s, 3-OCH3), 6.94-6.97 [2H, m, 2xCH-(Ar)], 7.88 (1H, s, -NH), 8.09 (H, s, N=CH).
13C NMR: (DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 56.2, 56.5,
106.7, 128.1, 141.5, 143.0, 150.9, 167.5. ESI-MS: m/z 477 (M+H). Anal. calcd. for
C28H48N2O4: C, 70.55; H, 10.15; N,5.88%. Found: C, 70.42; H, 10.52; N, 5.31%.
2.2.4.5. N'-(3,4-dimethoxybenzylidene)stearohydrazide (S5)
Yield: 48%, m.p.: 254–258°C, IR: (KBr, cm-1) 2779 (R-CH3), 2991 (C-H phenyl), 1622
1
(C=O amide), 1481 (C=C phenyl) 1665 (-N=CH), 1261 (-OCH3). H NMR: (CDCl3, δ,
ppm): 0.80 (3H, t, CH3), 1.16 (28H, s, 14xCH2), 1.49-1.51 (2H, m, CH2), 2.16 (2H, t, CH2),
3.71 (6H, s, 2-OCH3), 6.9-7.2 [2H, m, 2xCH-(Ar)], 7.82 (1H, s, -NH), 8.01 (H, s, N=CH).
13C NMR: (DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 56.2, 114.4,
115.4, 122.5, 127.1, 143.0, 149.9, 152.1, 167.5. ESI-MS: m/z 447 (M+H). Anal. calcd. for
C27H46N2O3: C, 72.60; H, 10.38; N,6.27%. Found: C, 72.21; H, 10.49; N, 6.13%.
2.2.4.6. N'-(4-hydroxy-3-methoxybenzylidene)stearohydrazide (S6)
Yield: 51%, m.p.: 271–273°C, IR: (KBr, cm-1) 2876 (R-CH3), 2988 (C-H phenyl), 1626
1
(C=O amide), 1471 (C=C phenyl) 1665 (-N=CH), 1262 (-OCH3). H NMR: (CDCl3, δ,

ppm): 0.85 (3H, t, CH3), 1.23 (28H, s, 14xCH2), 1.47-1.57 (2H, m, CH2), 2.17 (2H, t, CH2),
3.34 (3H, s, -OCH3), 5.3 (1H, s, -OH), 6.71-6.83 [(1H, m, CH-(Ar)], 7.0-7.2 [(2H, m, 2xCH-
13
(Ar)], 7.85 (1H, s, -NH), 8.03 (1H, s, N=CH). C NMR: (DMSO-d6, δ, ppm): 14.1, 22.8,
26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 56.2, 114.8, 117.0, 122.9, 127.4, 143.0, 148.0, 151.5, 167.5.
ESI-MS: m/z 432 (M+H). Anal. calcd. for C26H44N2O3: C, 72.18; H, 10.25; N,6.48%. Found:
C, 72.55; H, 10.61; N, 6.12%.
2.2.4.7. N'-(4-(dimethylamino)benzylidene)stearohydrazide (S7)
Yield: 46%, m.p.: 279–281°C, IR: (KBr, cm-1) 2888 (R-CH3), 2976 (C-H phenyl), 1621
1
(C=O amide), 1470 (C=C phenyl) 1665 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.95 (3H, t,
CH3), 1.24 (28H, s, 14xCH2), 1.51-1.55 (2H, m, CH2), 2.18 (2H, t, CH2), 2.85 (6H, s, 2x-
NCH3), 6.61-6.79 [(2H, m, CH-(Ar)], 7.34-7.41 [(2H, m, 2xCH-(Ar)], 7.99 (1H, s, -NH), 8.1
(1H, s, N=CH). 13C NMR: (DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5,
40.3, 114.4, 123.3, 130.1, 143.0, 151.9, 167.5. ESI-MS: m/z 430 (M+H). Anal. calcd. for
C27H47N3O: C, 75.47; H, 11.03; N,9.79%. Found: C, 75.09; H, 11.39; N, 9.48%.
2.2.4.8. N'-(furan-2-ylmethylene)stearohydrazide (S8)
Yield: 58%, m.p.: 287–289°C, IR: (KBr, cm-1) 2878 (R-CH3), 2989 (C-H phenyl), 1625
1
(C=O amide), 1470 (C=C phenyl) 1665 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.91 (3H, t,
CH3), 1.27 (28H, s, 14xCH2), 1.51-1.54 (2H, m, CH2), 2.13 (2H, t, CH2), 6.31-6.49 [(2H, m,
CH-(Ar)], 7.09 (1H, s, -NH), 7.31-7.42 [(H, m,-OCH-(furan)], 7.5 (1H, s, N=CH). 13C NMR:
(DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 109.4, 109.9, 134.7, 143.9,
149.1, 167.5. ESI-MS: m/z 377 (M+H). Anal. calcd. for C23H40N2O2 C, 73.36; H, 10.71;
N,7.44%. Found: C, 73.33; H, 10.41; N, 7.04%.
2.2.4.9. N'-(2,4-dimethylbenzylidene)stearohydrazide (S9)
Yield: 57%, m.p.: 255–257°C, IR: (KBr, cm-1) 2678 (R-CH3), 2990 (C-H phenyl), 1598
1
(C=O amide), 1476 (C=C phenyl) 1666 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.89 (3H, t,

CH3), 1.23 (28H, s, 14xCH2), 1.51-1.56 (2H, m, CH2), 2.1 (2H, t, CH2), 2.33 (6H, s, 2xAr-
CH3), 6.9-6.98 [(2H, m, CH-(Ar)], 7.39-7.44 [(1H, m, CH-(Ar)], 8.02 (1H, s, -NH), 8.3 (1H,
s, N=CH). 13C NMR: (DMSO-d6, δ, ppm): 14.1, 18.2, 22.8, 24.6, 26.0, 29.4, 29.6, 29.7,
31.9, 38.5, 123.6, 126.2, 129.0, 131.0, 138.7, 140.6, 143.0, 167.5. ESI-MS: m/z 414 (M+H).
Anal. calcd. for C27H46N2O C, 78.20; H, 11.18; N,6.76%. Found: C, 78.60; H, 11.39; N,
6.39%.
2.2.4.10. N'-(3-chlorobenzylidene)stearohydrazide (S10)
Yield: 63%, m.p.: 270–272°C, IR: (KBr, cm-1) 2658 (R-CH3), 2996 (C-H phenyl), 1628
1
(C=O amide), 1477 (C=C phenyl) 1668 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.91 (3H, t,
CH3), 1.29 (28H, s, 14xCH2), 1.47-1.51 (2H, m, CH2), 2.03 (2H, t, CH2), 6.9-7.2 [(2H, m,
13
CH-(Ar)], 7.6-7.66 [(2H, m, 2xCH-(Ar)], 8.0 (1H, s, -NH), 8.24 (1H, s, N=CH). C NMR:
(DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 127.3, 129.3, 130.3, 131.2,
134.4, 135.2, 143.0, 167.5. ESI-MS: m/z 422 (M+H). Anal. calcd. for C25H41ClN2O C,
71.31; H, 9.81; N,6.65%. Found: C, 71.20; H, 9.47; N, 6.91%.
2.2.4.11. N'-(2-chlorobenzylidene)stearohydrazide (S11)
Yield: 69%, m.p.: 210–212°C, IR: (KBr, cm-1) 2875 (R-CH3), 2991 (C-H phenyl), 1616
1
(C=O amide), 1464 (C=C phenyl) 1669 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.94 (3H, t,
CH3), 1.21 (28H, s, 14xCH2), 1.41-1.59 (2H, m, CH2), 2.18 (2H, t, CH2), 6.9-7.2 [(2H, m,
CH-(Ar)], 7.2 [1H, d, CH-(Ar)],7.68 [1H, d, CH-(Ar)], 7.99 (1H, s, -NH), 8.14 (1H, s,
N=CH). 13C NMR: (DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 127.0,
129.3, 130.6, 132.5, 133.3, 143.0, 144.0, 167.5. ESI-MS: m/z 421 (M+H). Anal. calcd. for
C25H41ClN2O: C, 71.31; H, 9.49; N, 6.65%. Found: C, 71.19; H, 9.41; N, 6.48%.
2.2.4.12. N'-(2-bromobenzylidene)stearohydrazide (S12)
Yield: 61%, m.p.: 267–269°C, IR: (KBr, cm-1) 2881 (R-CH3), 2998 (C-H phenyl), 1626
1
(C=O amide), 1459 (C=C phenyl) 1664 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.96 (3H, t,

CH3), 1.37 (28H, s, 14xCH2), 1.49-1.51 (2H, m, CH2), 2.08 (2H, t, CH2), 7.2-7.28 [(2H, m,
13
CH-(Ar)], 7.5-7.59 [2H, m, 2xCH-(Ar)], 7.88 (1H, s, -NH), 8.3 (1H, s, N=CH). C NMR:
(DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 121.7, 127.9, 131.4, 131.8,
133.3, 135.3, 143.0, 167.5. ESI-MS: m/z 466 (M+H). Anal. calcd. for C25H41BrN2O: C,
64.50; H, 8.88; N, 6.02%. Found: C, 64.17; H, 8.96; N, 6.35%.
2.2.4.13. N'-(3-methoxybenzylidene)stearohydrazide (S13)
Yield: 51%, m.p.: 224–226°C, IR: (KBr, cm-1) 2875 (R-CH3), 2998 (C-H phenyl), 1636
1
(C=O amide), 1461 (C=C phenyl) 1663 (-N=CH), 1265 (-OCH3). H NMR: (CDCl3, δ,
ppm): 0.86 (3H, t, CH3), 1.34 (28H, s, 14xCH2), 1.58-1.77 (2H, m, CH2), 2.18 (2H, t, CH2),
3.64 (3H, s, -OCH3), 6.56-6.66 [(1H, m, CH-(Ar)], 7.1-7.3 [(2H, m, 2xCH-(Ar)], 7.95 (1H, s,
-NH), 8.13 (1H, s, N=CH). 13C NMR: (DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7,
31.9, 38.5, 55.9, 113.4, 116.6, 121.5, 129.9, 134.8, 143.0, 148.0, 160.8, 167.5. ESI-MS: m/z
417 (M+H). Anal. calcd. for C26H44N2O2: C, 74.95; H, 10.64; N,6.72%. Found: C, 74.57; H,
10.29; N, 6.94%.
2.2.4.14. N'-(2-nitrobenzylidene)stearohydrazide (S14)
Yield: 67%, m.p.: 241–243°C, IR: (KBr, cm-1) 2768 (R-CH3), 3006 (C-H phenyl), 1668
1
(C=O amide), 1471 (C=C phenyl) 1667 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.95 (3H, t,
CH3), 1.23 (28H, s, 14xCH2), 1.59-1.66 (2H, m, CH2), 2.5 (2H, t, CH2), 7.89-7.92 [(3H, m,
3xCH-(Ar)], 7.95-8.01 [(1H, m, CH-(Ar)], 8.28 (1H, s, -NH), 8.39 (1H, s, N=CH). 13C
NMR: (DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 121.2, 126.3,
130.1, 132.0, 135.0, 143.0,148.9, 167.5. ESI-MS: m/z 432 (M+H). Anal. calcd. for
C25H41N3O3: C, 69.57; H, 9.57; N, 9.74%. Found: C, 69.22; H, 9.81; N, 9.45%.
2.2.4.15. N'-(2-methylbenzylidene)stearohydrazide (S15)
Yield: 52%, m.p.: 248–250°C, IR: (KBr, cm-1) 2778 (R-CH3), 2999 (C-H phenyl), 1638
1
(C=O amide), 1466 (C=C phenyl) 1669 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.92 (3H, t,

CH3), 1.13 (28H, s, 14xCH2), 1.56-1.64 (2H, m, CH2), 2.1 (2H, t, CH2), 2.45 (3H, s, Ar-CH3),
6.9-7.01 [(2H, m, CH-(Ar)], 7.37-7.41 [(1H, m, CH-(Ar)], 7.92 (1H, s, -NH), 8.1 (1H, s,
13
N=CH). C NMR: (DMSO-d6, δ, ppm): 14.1, 17.9, 22.8, 24.6, 26.0, 29.4, 29.6, 29.7, 31.9,
38.5, 125.9, 126.6, 129.1, 129.2, 131.0, 138.8, 143.0, 167.5. ESI-MS: m/z 401 (M+H). Anal.
calcd. for C26H44N2O C, 77.94; H, 11.07; N,6.99%. Found: C, 77.63; H, 11.32; N, 6.68%.
2.2.4.16. N'-(4-chlorobenzylidene)stearohydrazide (S16)
Yield: 65%, m.p.: 291–293°C, IR: (KBr, cm-1) 2618 (R-CH3), 3016 (C-H phenyl), 1636
(C=O amide), 1444 (C=C phenyl) 1665 (-N=CH). 1H NMR: (CDCl3, δ, ppm): 0.87 (3H, t,
CH3), 1.26 (28H, s, 14xCH2), 1.47-1.58 (2H, m, CH2), 2.19 (2H, t, CH2), 7.16-7.29 [(2H, m,
13
2xCH-(Ar)], 7.59-7.61 [2H, m, CH-(Ar)], 7.86 (1H, s, -NH), 8.1 (1H, s, N=CH). C NMR:
(DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 129.0, 130.6, 131.9, 136.6,
143.0, 167.5. ESI-MS: m/z 422 (M+H). Anal. calcd. for C25H41ClN2O: C, 71.31; H, 9.81; N,
6.65%. Found: C, 71.70; H, 9.48; N, 6.39%.
2.2.4.17. N'-(4-fluorobenzylidene)stearohydrazide (S17)
Yield: 61%, m.p.: 288–290°C, IR: (KBr, cm-1) 2818 (R-CH3), 2966 (C-H phenyl), 1625
1
(C=O amide), 1464 (C=C phenyl) 1668 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.90 (3H, t,
CH3), 1.25 (28H, s, 14xCH2), 1.67-1.75 (2H, m, CH2), 2.34 (2H, t, CH2), 7.06-7.17 [(2H, m,
13
2xCH-(Ar)], 7.62-7.7 [2H, m, CH-(Ar)], 7.86 (1H, s, -NH), 8.6 (1H, s, N=CH). C NMR:
(DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 115.6, 129.4, 130.8,
143.0,165.2, 167.5. ESI-MS: m/z 405 (M+H). Anal. calcd. for C25H41FN2O: C, 74.21; H,
10.21; N, 6.92%. Found: C, 74.66; H, 10.13; N, 6.59%.
2.2.4.18. N'-(3-fluorobenzylidene)stearohydrazide (S18)
Yield: 57%, m.p.: 218–220°C, IR: (KBr, cm-1) 2724 (R-CH3), 2969 (C-H phenyl), 1626
(C=O amide), 1444 (C=C phenyl) 1666 (-N=CH). 1H NMR: (CDCl3, δ, ppm): 0.87 (3H, t,
CH3), 1.25 (28H, s, 14xCH2), 1.53-1.59 (2H, m, CH2), 2.14 (2H, t, CH2), 7.06-7.3 [(3H, m,

13
3xCH-(Ar)], 7.42-7.6 [1H, m, CH-(Ar)], 7.91 (1H, s, -NH), 8.09 (1H, s, N=CH). C NMR:
(DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 114.1, 117.8, 124.8, 130.5,
135.4, 143.0, 163.0, 167.5 ESI-MS: m/z 405 (M+H). Anal. calcd. for C25H41FN2O: C,
74.21; H, 10.21; N, 6.92%. Found: C, 74.71; H, 10.59; N, 6.56%.
2.2.4.19. 3 N'-(2-fluorobenzylidene)stearohydrazide (S19)
Yield: 63%, m.p.: 280–282°C, IR: (KBr, cm-1) 2674 (R-CH3), 2989 (C-H phenyl), 1656
1
(C=O amide), 1464 (C=C phenyl) 1676 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.93 (3H, t,
CH3), 1.39 (28H, s, 14xCH2), 1.47-1.56 (2H, m, CH2), 2.21 (2H, t, CH2), 6.9-7.3 [(3H, m,
13
3xCH-(Ar)], 7.6 [1H, t, CH-(Ar)], 8.1 (1H, s, -NH), 8.36(1H, s, N=CH). C NMR: (DMSO-
d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 115.6, 118.2, 124.5, 130.8, 132.7,
143.0, 159.7, 167.5 ESI-MS: m/z 405 (M+H). Anal. calcd. for C25H41FN2O: C, 74.21; H,
10.21; N, 6.92%. Found: C, 74.33; H, 10.60; N, 6.54%.
2.2.4.20. N'-(4-bromobenzylidene)stearohydrazide (S20)
Yield: 61%, m.p.: 225–227°C, IR: (KBr, cm-1) 2866 (RCH3), 2955 (C-H phenyl), 1629
1
(C=O amide), 1472 (C=C phenyl) 1661 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.96 (3H, t,
CH3), 1.29 (28H, s, 14xCH2), 1.43-1.57 (2H, m, CH2), 2.12 (2H, t, CH2), 7.5-7.67 [(4H, m,
13
4xCH-(Ar)], 7.86 (1H, s, -NH), 8.16(1H, s, N=CH). C NMR: (DMSO-d6, δ, ppm): 14.1,
22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 125.4, 131.4, 131.8, 132.8, 143.0, 167.5. ESI-MS:
m/z 466 (M+H). Anal. calcd. for C25H41BrN2O: C,64.50; H, 8.88; N, 6.02%. Found: C,
64.15; H, 8.49; N, 6.40%.
2.2.4.21. N'-(3-bromobenzylidene)stearohydrazide (S21)
Yield: 54%, m.p.: 216–218°C, IR: (KBr, cm-1) 2861 (RCH3), 2945 (C-H phenyl), 1619
1
(C=O amide), 1442 (C=C phenyl) 1651 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.89 (3H, t,
CH3), 1.31(28H, s, 14xCH2), 1.51-1.58 (2H, m, CH2), 2.17 (2H, t, CH2), 7.2-7.7 [(4H, m,
13
4xCH-(Ar)], 8.16 (1H, s, -NH), 8.21(1H, s, N=CH). C NMR: (DMSO-d6, δ, ppm): 14.1,

22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 123.2, 128.2, 131.1, 132.7, 134.0, 136.0, 143.0, 167.5.
ESI-MS: m/z 466 (M+H). Anal. calcd. for C25H41BrN2O: C,64.50; H, 8.88; N, 6.02%.
Found: C, 64.81; H, 8.66; N, 6.31%.
2.2.4.22. N'-(4-methoxybenzylidene)stearohydrazide (S22)
Yield: 64%, m.p.: 256–258°C, IR: (KBr, cm-1) 2769 (R-CH3), 2996 (C-H phenyl), 1612
(C=O amide), 1461 (C=C phenyl) 1655 (-N=CH), 1266 (-OCH3) 1H NMR: (CDCl3, δ, ppm):
0.91 (3H, t, CH3), 1.29 (28H, s, 14xCH2), 1.39-1.53 (2H, m, CH2), 1.98-2.18 (2H, t, CH2),
3.77 (3H, s, Ar-OCH3), 6.8-7.1 [2H, d, 2xCH-(Ar)], 7.5 [2H, d, 2xCH-(Ar)], 7.92 (1H, s, -
NH), 8.19 (1H, s, N=CH). 13C NMR: (DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7,
31.9, 38.5, 55.9, 114.4, 126.1, 130.2, 143.0, 163.0, 167.5. ESI-MS: m/z 417 (M+H). Anal.
calcd. for C26H44N2O2: C, 74.95; H, 10.64; N,6.72%. Found: C, 74.59; H, 10.28; N, 6.56%.
2.2.4. N'-(2-methoxybenzylidene)stearohydrazide (S23)
Yield: 68%, m.p.: 245–247°C, IR: (KBr, cm-1) 2714 (R-CH3), 2919 (C-H phenyl), 1616
1
(C=O amide), 1414 (C=C phenyl) 1676 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.82 (3H, t,
CH3), 1.33 (28H, s, 14xCH2), 1.41.56 (2H, m, CH2), 2.14-2.33 (2H, t, CH2), 3.73 (3H, s, Ar-
OCH3), 6.8-7.01 [2H, m, 2xCH-(Ar)], 7.2-7.66 [2H, m, 2xCH-(Ar)], 7.86 (1H, s, -NH), 8.23
(1H, s, N=CH). 13C NMR: (DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5,
55.9, 114.4, 116.9, 121.2, 130.2, 132.1, 143.0, 160.5, 167.5. ESI-MS: m/z 417 (M+H). Anal.
calcd. for C26H44N2O2: C, 74.95; H, 10.64; N,6.72%. Found: C, 74.56; H, 10.31; N, 6.64%.
2.2.4.5. N'-(2,6-dichlorobenzylidene)stearohydrazide (S24)
Yield: 58%, m.p.: 214–218°C, IR: (KBr, cm-1) 2761 (RCH3), 2985 (C-H phenyl), 1639
1
(C=O amide), 1449 (C=C phenyl) 1655 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.89 (3H, t,
CH3), 1.27 (28H, s, 14xCH2), 1.51-1.61 (2H, m, CH2), 2.18-2.21 (2H, t, CH2), 7.26-7.39
[(3H, m, 3xCH-(Ar)], 7.96 (1H, s, -NH), 8.19 (1H, s, N=CH). 13C NMR: (DMSO-d6, δ,
ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 127.1, 131.9, 133.9, 135.4, 143.0, 167.5.

ESI-MS: m/z 422 (M+H). Anal. calcd. for C25H40Cl2N2O: C, 65.92; H, 8.85; N, 6.15%.
Found: C, 65.59; H, 8.47; N, 6.33%.
2.2.4.6. N'-(3-(trifluoromethyl)benzylidene)stearohydrazide (S25)
Yield: 51%, m.p.: 221–223°C, IR: (KBr, cm-1) 2801 (RCH3), 2995 (C-H phenyl), 1632
(C=O amide), 1452 (C=C phenyl) 1652 (-N=CH). 1H NMR: (CDCl3, δ, ppm): 0.86 (3H, t,
CH3), 1.34 (28H, s, 14xCH2), 1.49-1.56 (2H, m, CH2), 2.15 (2H, t, CH2), 7.2 [1H, t, CH-
(Ar)], 7.49 [(1H, d, CH-(Ar)], 7.61 [(1H, d, CH-(Ar)], 7.82 [(1H, s, CH-(Ar)], 8.03 (1H, s, -
NH), 8.11(1H, s, N=CH). 13C NMR: (DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7,
31.9, 38.5, 124.2, 125.6, 127.5, 129.2, 131.1, 132.5, 134.1, 143.0, 167.5. ESI-MS: m/z 455
(M+H). Anal. calcd. for C26H41F3N2O: C,68.69; H, 9.09; N, 6.16%. Found: C, 68.33; H, 9.40;
N, 6.51%.
2.2.4.7. N'-(2,4,6-trimethoxybenzylidene)stearohydrazide (S26)
Yield: 54%, m.p.: 249–252°C, IR: (KBr, cm-1) 2799 (R-CH3), 2992 (C-H phenyl), 1627
1
(C=O amide), 1441 (C=C phenyl) 1654 (-N=CH), 1251 (-OCH3). H NMR: (CDCl3, δ,
ppm): 0.80 (3H, t, CH3), 1.16 (28H, s, 14xCH2), 1.49-1.51 (2H, m, CH2), 2.16 (2H, t, CH2),
3.76 (9H, s, 3xAr-OCH3), 5.93 [2H, s, 2xCH-(Ar)], 7.89 (1H, s, -NH), 8.17 (1H, s, N=CH).
13C NMR: (DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 55.9, 92.8,
102.7, 143.0, 162.5, 165.0, 167.5. ESI-MS: m/z 477 (M+H). Anal. calcd. for C28H48N2O4: C,
70.55; H, 10.15; N,5.88%. Found: C, 70.91; H, 10.44; N, 5.59%.
2.2.4.8. N'-benzylidenestearohydrazide (S27)
Yield: 61%, m.p.: 257–259°C, IR: (KBr, cm-1) 2758 (R-CH3), 3001 (C-H phenyl), 1641
1
(C=O amide), 1476 (C=C phenyl) 1658 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.81 (3H, t,
CH3), 1.29 (28H, s, 14xCH2), 1.47-1.59 (2H, m, CH2), 2.13 (2H, t, CH2), 7.2-7.3 [(3H, m,
3xCH-(Ar)], 7.51-7.59 [(2H, m, 2xCH-(Ar)], 8.11 (1H, s, -NH), 8.2 (1H, s, N=CH). 13C
NMR: (DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 127.3, 129.3,

130.3, 131.2, 134.4, 135.2, 143.0, 167.5. ESI-MS: m/z 387 (M+H). Anal. calcd. for
C25H42N2O C, 77.67; H, 10.95; N,7.25%. Found: C, 77.41; H, 10.76; N, 7.39%.
2.2.4.9. N'-(5-chloro-2-hydroxybenzylidene)stearohydrazide (S28)
Yield: 67%, m.p.: 235–237°C, IR: (KBr, cm-1) 2796 (R-CH3), 2914 (C-H phenyl), 1620
1
(C=O amide), 1475 (C=C phenyl) 1655 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.87 (3H, t,
CH3), 1.24 (28H, s, 14xCH2), 1.41-1.55 (2H, m, CH2), 2.03 (2H, t, CH2), 5.03 (1H, s, -OH),
6.7 [2H, d, 2xCH-(Ar)], 7.13 [1H, m, CH-(Ar)], 7.5 [H, d, CH-(Ar)], 7.98 (1H, s, -NH), 8.17
13
(H, s, N=CH). C NMR: (DMSO-d6, δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5,
117.4 119.9, 127.0, 130.7, 132.6, 143.0, 159.2, 167.5. ESI-MS: m/z 438 (M+H). Anal. calcd.
for C25H41ClN2O2: C, 68.70; H, 9.46; N,6.41%. Found: C, 68.39; H, 9.07; N, 6.80%.
2.2.4.10. N'-(4-(diethylamino)-2-hydroxybenzylidene)stearohydrazide (S29)
Yield: 53%, m.p.: 217–220°C, IR: (KBr, cm-1) 2758 (R-CH3), 2896 (C-H phenyl), 1618
1
(C=O amide), 1471 (C=C phenyl) 1658 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.85 (3H, t,
CH3), 1.13-1.21 [6H, m, 2xCH3 of (diethylamino)] 1.19 (28H, s, 14xCH2), 1.47-1.53 (2H, m,
CH2), 2.03 (2H, t, CH2), 3.39 [4H, q, 2xCH2 (diethylamino)], 5.03 (1H, s, -OH), 6.1-6.2 [(2H,
m, 2xCH-(Ar)], 7.3 [(1H, m, CH-(Ar)], 7.9 (1H, s, -NH), 8.14 (1H, s, N=CH). 13C NMR:
(DMSO-d6, δ, ppm):, 13.0, 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 44.7, 99.2, 107.0,
108.0, 131.5, 143.0, 153.5, 162.0, 167.5. ESI-MS: m/z 474 (M+H). Anal. calcd. for
C29H51N3O2 C, 73.52; H, 10.85; N,8.87%. Found: C, 73.12; H, 10.47; N, 8.49%.
2.2.4.10. N'-(4-chloro-3-nitrobenzylidene)stearohydrazide (S30)
Yield: 63%, m.p.: 270–272°C, IR: (KBr, cm-1) 2758 (R-CH3), 2992 (C-H phenyl), 1628
1
(C=O amide), 1473 (C=C phenyl) 1658 (-N=CH). H NMR: (CDCl3, δ, ppm): 0.96 (3H, t,
CH3), 1.17 (28H, s, 14xCH2), 1.49-1.59 (2H, m, CH2), 2.19 (2H, t, CH2), 7.69 [(2H, d, 2xCH-
13
(Ar)], 8.41 [(1H, s, CH-(Ar)], 8.0 (1H, s, -NH), 8.24 (1H, s, N=CH). C NMR: (DMSO-d6,
δ, ppm): 14.1, 22.8, 26.0, 29.4, 29.6, 29.7, 31.9, 38.5, 125.5, 129.9, 131.2, 132.8, 136.7,

143.0, 148.9, 167.5. ESI-MS: m/z 467 (M+H). Anal. calcd. for C25H40ClN3O3 C, 64.43; H,
8.65; N,9.02%. Found: C, 64.09; H, 8.28; N, 9.41%.
2.3. In silico computational studies
2.3.1. Physicochemical Property Prediction: Lipinski Rule
Physicochemical property prediction is very crucial for the drafting and synthesis of novel
PPAR gamma agonists with enhanced biological profile. The drug-resemblance characters of
new identities were tested by applying Lipinski’s rule of five by using online Molinspiration
property calculation toolkit [28]. The requirement is that the derivatives should not have more
than 5 and 10 hydrogen bond donors or acceptors respectively, MW less than 500 amu, and
partition coefficients value between octanol and water (log P(oct/wat)) less than 5. For the
compound to be active orally, it should not violate more than one of these rules. Estimation of
mi log P, H-bond donors, H-bond acceptors; number of rotatable bonds, molecular volume
and molecular weight of the novel series designed were predicted by this toolkit. Additional
basic specifications like Topological Polar Surface Area (TPSA) were also calculated to
identify membrane penetration of new compounds having low CNS bioavailability to get best
oral drug identities. ynthetic analogues with values are selected for orally
active drug candidates and are chosen for developing new molecules. Aforementioned online
prediction software assisted in perceiving novel collection of molecules with acceptable
biological activity [29-30]. The %ABS (% Ab) value for synthesized derivatives was
evaluated by putting TPSA value in the following formula: %Ab = 109 – [0.345 ×TPSA]
[31].
2.3.2. Molecular docking and Prime MMGBSA [29]
Docking estimations for getting binding patterns of all novel compounds was completed by
using Maestro 9.0 Schrodinger program, New York, USA on PDB ID-1FM9 which is a
PPAR-γ crystal captured by RCSB. The elucidations of divergent process of binding of these

molecules with cavities of the receptor were reported. The effective revealed focus in anti-
hyperglycaemic potential is PPAR gamma receptor. Protein data bank (PDB 1FM9) was
opened and the basic crystal structure was downloaded. Tool named Lig prep was used in
preparing ligands ie. novel hybrids and protein preparation wizard in drafting structure of
protein. Molecules of water which were not required were removed by software physically
from the framework. The structure of protein was intensified and then its energy was
minimized by root-mean-square deviation of 0.3 Å. Ligands energy was also minimized with
the OPLS (optimized potentials for liquid simulations) 2005 force field. Glide scores and free
binding energy of docked compounds was used in estimating binding affinities.
Software Maestro 9.0 is used to estimate binding energy of the ligands i.e. synthesized
compounds and pioglitazone against receptor having PDB ID 1FM9 which is crystal structure
of R γ through method called as rime molecular mechanics-generalized born surface
area (MMGBSA). Conclusions can be obtained by regulating MM-GBSA program instantly
from the file created by docking procedure. The perfect organization and binding
resemblance of the novel entities on the vital positions of the receptor with the nearby amino
acids are anticipated by docking glide score, free binding energy, H bond and pie-pie
bonding. The protein-ligand energy in coulomb-vdW is elucidated by the Emodel function.
2.4. In vitro study
2.4.1. PPAR-gamma transactivation assay [32]
Entities having good glide score (S4, S5, S7, S10, S14, S15 and S21) were checked for their
PPAR-gamma transactivation activity. Human embryonic kidney (HEK) 293 cells were
cultured in Dulbecco's Modified Eagle Medium (DMEM) with foetal bovine serum (FBS-
10% heat inactivated). Cells were incubated in 5% CO2 incubator at 37 °C till it reaches 70–
80% convergence. Cells were inoculated in 96-well plates containing 60000 cells/well. After
that, they were transfected with 2.5 μL of RE-Luciferase, . 7 μL of Rγ, 1. μl of

Renilla, and 2 μl of Lipofectamine. Five hours after transfection, cells were treated with the
synthesized derivatives (10μM) for 24h which were then collected by cell culture lysis buffer.
Luciferase activity was observed after 42h on luminometer using the luciferase assay kit
(Promega) as reported in the manufacturer’s instructions. Pioglitazone was taken as standard
drug.
2.4.2. Cytotoxicity study by MTT Assay [33]
In vitro cytoxicity study was performed on the selected compounds having good docking
glide scores and which were found to be PPAR gamma active, in order to carry out in vivo
study on the same. Breast cancer cell line (MCF-7) cells were plated at a density of 5×104
cells/mL were incubated for 48 h with different concentrations of derivatives (S4, S5, S7,
S10, S14, S15 and S21) dissolved in DMSO (the final volume of DMSO/medium was less
than 1% in experiments). Etoposide and DMSO were used as positive and negative controls,
respectively. After this, the medium was removed and 2 μL of a phenol red-free medium
containing MTT (1 mg/mL, final concentration) was added to all wells. After 4 h of
incubation, the culture medium was replaced with 1 μL of DM O to each well. The
absorbance was then measured on a spectrophotometer (SpectraMax; Molecular Devices) at
570 nm. Then the cells were exposed to different concentrations of derivatives for 48 h and
measurement of cell viability was taken by MTT method. The assay is based on the cleavage
of the yellow tetrazolium salt MTT [3-(4,5-dimethythiazol- 2-yl) -2,5-diphenyl tetrazolium
bromide] into purple formazan by metabolically active cells after reduction by mitochondrial
enzymes, which can be spectrophotometrically quantified at 570 nm. The amount of
formazan product generated is proportional to the number of living cells and leads to a
stronger color formation. The experiment was performed for three times and the values for
each were calculated from triplicate wells. IC50 values were determined applying nonlinear
regression analysis to the data for three dilutions.

2.5.In Vivo Study
Albino wistar rats (200–250 g) of either sex were taken from Animal House Center of Delhi
Institute of Pharmaceutical Sciences and Research, New Delhi for oral glucose tolerance and
anti-hyperglycaemic study. All the procured animals were divided and housed in different
polypropylene cages at standard laboratory conditions (temperature 22 ± 2oC and relative
humidity 45 ± 5% with 12h day: 12h night cycle) maintaining disinfectant state. All animals
were nourished with normal pellet diet and water ad libitum. The study on experimental
animals was performed after the approval of Institutional Animal Ethical Committee (IAEC)
through protocol IAEC ⁄ 2016-I/ Prot. No.05 corresponding to CPCSEA guidelines. Blood
glucose level was measured using automatic analysis (Accu-Chek Active Glucose, Roche
Diagnostics, Mannheim, Germany).
2.5.1. Oral glucose tolerance test in normal rats
S4, S5, S7, S10, S14, S15 and S21 entities were selected from all the synthesized derivatives
for oral glucose tolerance test according to their docking scores. The test was performed on
overnight fasted normal rats, divided into nine groups, six in each. Group 1 was taken as
normal and was given 0.1% (w/v) aqueous CMC solution (5 mL/kg) orally. Group 2 was
marked as standard group which was treated with suspension of 36 mg/kg b.w. of
pioglitazone in 0.1% (w/v) aqueous CMC. Groups 3-9 were served 100 mg/kg of the test
analogs S4, S5, S7, S10, S14, S15 and S21 respectively orally using oral gavage. Glucose
(5g/kg b.w) was given 30 min post-administration of CMC, standard drug and tested
compounds. Blood glucose level was measured by retro orbital plexus of rat eye at 0h (just
before), 30min and 90min after the oral administration of the selected test derivatives by
using Accu-chek Active TM Test strips in Accu-chek Active TM Test meter of Roche [34-
37].
2.5.2. Hypoglycaemic activity on streptozotocin-induced diabetic rats [38-42]

2.5.2.1. Drugs Used:
Pioglitazone was specified to rats in quantity of 36 mg/kg body weight in 0.1% (w/v) aqueous
CMC which is stated as a reference standard.
2.5.2.2. Induction of non-insulin dependent diabetes mellitus:
The non-insulin-dependent diabetes mellitus was created in overnight fasted rats, by
intraperitonial injection of freshly made streptozotocin solution in 0.1M citrate buffer (4.5pH)
at a dose of 60 mg/kg body weight. After this regiment, blood glucose level was observed
after 48 h to check hyperglycaemia. Diabetes was evolved and stabilized for 2 days and then
anti-diabetic study was done. The rats having permanent blood glucose level > 250 mg/dL
were accounted to be diabetic and were used in further study [35].
2.5.2.3. Group design
S4, S5, S7, S10, S14, S15 and S21 compounds were selected for the study by evaluating their
docking results. In this, the rats were divided into nine groups (n=6) after NIDDM induction.
Group 1 taken as diabetic control (0.1% (w/v) aqueous CMC solution,5 mL/kg b.w.), Group 2
served as reference standard received pioglitazone, 36mg/kg b.w. by oral route, suspended in
0.1% (w/v) aqueous CMC vehicle. Group 3-9 taken as test groups, received oral dose of 100
mg/kg b.w. of selected test derivatives S4, S5, S7, S10, S14, S15 and S21 respectively
through oral gavage.
2.5.2.4. Blood Glucose Measurement
Blood sample was collected from retro orbital plexus of the eye under light ether anaesthesia
using capillary tube and recorded at an interval of 0, 2, 4, and 6 h gap by Accu-chek Active
TM Test strips in Accu-chek Active TM Test meter of Roche.
2.5.3. Statistical analysis
Results of both the studies are expressed as means ± SEM for groups. Difference between
data of derivatives and control were tested by one-way analysis of variance ANOVA

followed by dunnett’s comparison test. The values were taken to be significant at p<0.05 and
p<0.01.
3. Results and discussion
3.1. Chemistry
New Schiff base analogs of fatty acid showing agonism to PPAR were drafted and prepared
by Scheme 1. Stearohydrazide (2) required as starting material for derivatives was obtained
from methyl oleate (1) which was acquired from the reaction of hydrazine hydrate. To the
solution of stearohydrazide in ethanol, glacial acetic acid was added and refluxed with
vigorous stirring with appropriate substituted aldehydes to give respective novel chemical
schiff base entities (S1-S30). The structures of synthesized derivatives S1-S30 were
characterized by proton and carbon nuclear magnetic resonance and MASS spectroscopy.
The IR spectra developed bands in range of 1598-1656 (C=O amide) and 1651-1676 (-
N=CH). The 1H NMR spectrum showed singlet at 7.5-8.39 parts per million for protons of -
N=CH. δ value for the same was around 143.0 parts per million in 13C NMR spectra. Almost
all derivatives displayed weak molecular ion peaks reporting instability of ions. Elemental
analysis data were found in the range of ±0.4% for the theoretical values of the analyzed
elements (C, H, N).
The X-ray analysis of the compound S21 was done by Rigaku Ultima IV X-ray
Diffractometer (Fig.1).
Fig.1. X-ray analysis of S21

3.2. Computational study
3.2.1. In silico pharmacokinetic prediction
Prediction of Absorption, Distribution, Metabolism and Excretion by Molinspiration of novel
synthesized schiff bases (S1-S30) are represented in Table 1. For a drug to be active orally, it
should not violate more than one of Lipinski rules. In this study all the tested synthetic
entities pass the Lipinski screening test. The topological polar surface area values of our
synthesized compounds are from 41.46 to 87.29 Å, which engross additional structural
elaboration for the evolution of new analogues. Absorption percentage (% ABS) is found to
be in range of 78.88 to 94.69 which shows that all the derivatives have moderate to good
permeability into membrane.
Table 1: ADME property estimation

Compd. %ABS TPSAa n-
Code
Molecular Molecular Log nOHNHd nONe Lipinski’s
rotbb
Weight
402.62
402.62
402.62
476.70
446.68
432.65
429.69
376.58
414.68
421.07
421.07
465.52
416.65
431.62
400.65
421.07
404.61
404.61
404.61
465.52
465.52
416.65
416.65
455.51
454.62
476.70
Volume
432.10
432.10
432.10
500.72
47517
Pc
violation
(</= 1)
1
S1
87.71
87.71
87.71
85.13
88.32
84.53
93.57
90.16
94.69
94.69
94.69
94.69
91.50
78.88
94.69
94.69
94.69
94.69
94.69
94.69
94.69
91.50
91.50
94.69
94.69
85.13
61.69
61.69
61.69
69.16
59.93
70.92
44.70
54.60
41.46
41.46
41.46
41.46
50.70
87.29
41.46
41.46
41.46
41.46
41.46
41.46
41.46
50.70
50.70
41.46
41.46
69.16
18
18
18
21
20
19
19
18
18
18
18
18
19
19
18
18
18
18
18
18
18
19
19
18
19
21
8.88
8.87
9.04
8.93
8.93
8.80
9.10
8.77
9.31
9.27
9.27
9.30
9.07
9.03
9.19
9.27
9.12
9.11
9.11
9.31
9.30
9.08
9.07
9.43
9.33
9.07
2
2
2
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
4
4
4
6
5
5
4
4
3
3
3
3
4
6
3
3
3
3
3
3
3
4
4
3
3
6
S2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
S3
S4
S5
S6
457.64
469.99
405.65
457.20
437.62
437.62
441.96
449.62
447.41
440.64
437.62
429.01
429.01
429.01
441.96
441.96
449.62
449.62
451.15
455.38
500.72
S7
S8
S9
S10
S11
S12
S13
S14
S15
S16
S17
S18
S19
S20
S21
S22
S23
S24
S25
S26

S27
S28
S29
S30
94.69
87.71
86.59
78.88
41.46
69.16
64.93
87.29
18
18
21
19
386.62
437.07
473.75
424.08
495.63
511.61
460.95
9.06
9.25
9.30
9.24
1
2
2
1
3
4
5
6
1
1
1
1
466.07
b
c
aTopological polar surface area (TPSA). Number rotatable bonds (n-rotb). Log of partition
d
e
coefficient n-octanol/water (LogP). Hydrogen bond donors (nOHNH). Hydrogen bond
acceptor (nON).
3.2.2. Molecular docking
Molecular Docking has been reported for getting the binding patterns of novel compounds
with the vital positions of 3D PPAR-γ crystal form which was taken from protein data bank
(PDB ID-1FM9) having similarity with enzymes of humans. The work was completed by
using by Maestro 9.0 Schrodinger program. Glide scores and free binding energies of all
entities are shown in Table 2. S21, S10 and S7 were showing best scores as -9.19, -8.68 and -
8.64 respectively, thus are well positioned on the sites as compared to the standard
pioglitazone having docking score of -8.46. In this study we have explored binding approach
of three most promising derivatives of the series (S21, S10 and S7). Examination by
molecular docking of S21, S10 and S7 into the vital locations of enzyme specified different
molecular interrelationships and interactions (hydrogen bond, π-interaction, and hydrophobic
interactions) for good activity and affinity of the compounds. Illustrations of binding patterns
in 3D and 2D view of these best docked compounds (S21, S10 and S7) within 3.5Å are
shown in Fig.2, 3 and 4 respectively.
Compound S21 having highest docking score is the most effective derivative of the series.
S21 shows three hydrogen bond interactions between oxygen of -C=O group of compound
and amino acid HIE 449, HIP 323 and TYR 473, in which two hydrogen bonds, HIP 323 and
TYR 473 are similar to the standard drug, pioglitazone. It forms hydrophobic interaction with

Ile262, Pro269, Leu270, Cys285, Leu340, Ile341, Leu330, Ile326, Phe282, Leu453, Phet363,
Leu356 Ala278, Phe360, Ile281 and Leu353. The chain and aromatic ring also forms
hydrophobic interaction via weak van der Waals interactions with side chains non-polar
group of Phe287, Met348, Val339, Leu465 Ile456 and Met364. The polar interaction also
takes place with Gln283, Gln271, Ser342 and Gln286. The carboxy group of chain also form
hydrogen bond with Ser289, Hie449 and Tyr473 (Fig. 2).
A.

B.
Fig.2. A. 3D interrelationship picture of S21 (PDB ID: 1FM9) presenting hydrogen bonds
with yellow dot lines in relation to amino acid HIE 449, HIP 323 and TYR 473. B. 2D
docked picture of compound S21 showing hydrophobic interactions by green amino acid
residues.

In the derivative S10, second most active entity, showed one hydrogen bond interaction with
TYR 473 amino acid which is similar to the standard drug, pioglitazone and another
hydrogen bond is formed between hydrogen of -NH with MET 364 amino acid. There are
formation of hydrophobic interaction with Phe360, Cys285, Phe287, Pro269, Leu270, Ile262,
Ile341, Leu340, Ile326, Leu330, Tyr327, Phe282, Ile456, Leu453, Met364, Ala278, Leu353,
Phe363 Ile281 and Leu356. The chain of fatty acid and aromatic ring also forms hydrophobic
interaction via weak van der Waals interactions with side chains non-polar group of Val339,
Leu369and Leu465. The polar interaction also takes place with Gln283, Gln271, Ser342 and
Gln286. The carboxy group of chain also form hydrogen bond with Hip323, Ser289, Hie449
and Tyr473 (Fig. 3).
A.