Novel pyridine-2,4,6-tricarbohydrazide derivatives: Design, synthesis, characterization and in vitro biological evaluation as α- And β-glucosidase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2014.10.007

A range of novel pyridine 2,4,6-tricarbohydrazide derivatives (4a-4h) were synthesized and its biological inhibition towards α- and β-glucosidases was studied. Most of the compounds demonstrate to be active against α-glucosidase, and quite inactive/comple

Full text of DOI:10.1016/j.bioorg.2014.10.007

Bioorganic Chemistry 57 (2014) 148–154
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Novel pyridine-2,4,6-tricarbohydrazide derivatives: Design, synthesis,
characterization and in vitro biological evaluation as
a- and b-glucosidase inhibitors
a
a
b,
⇑
c
a
Sadaf Riaz , Islam Ullah Khan , Muhammad Yar , Muhammad Ashraf , Tanzeel Ur Rehman ,
c
d
e
e
Ayesha Shaukat , Syed Babar Jamal , Vera C.M. Duarte , Maria J. Alves
a
Department of Chemistry, GC University, Lahore 54000, Pakistan
b
Interdisciplinary Research Center in Biomedical Materials, COMSATS Institute of Information Technology, Lahore 54000, Pakistan
Department of Biochemistry and Biotechnology, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad 44000, Pakistan
Department of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
c
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 July 2014
Available online 6 November 2014
A range of novel pyridine 2,4,6-tricarbohydrazide derivatives (4a–4h) were synthesized and its biological
inhibition towards
against -glucosidase, and quite inactive/completely inactive against b-glucosidase. A number of com-
pounds were found to be more active against -glucosidase than the reference compound acarbose
IC50 38.25 ± 0.12 M); being compound 4d with the p-hydroxy phenyl motive the most active (IC50
0.24 ± 0.72 M). Molecular modeling studies show the interactions of compound 4d with the active site
of target -glucosidase kinase.
a- and b-glucosidases was studied. Most of the compounds demonstrate to be active
a
a
Keywords:
Diabetes
(
l
2
l
a-Glucosidase
a
b-Glucosidase
Pyridine-2,4,6-tricarbohydrazide
Acarbose
Ó 2014 Elsevier Inc. All rights reserved.
MMFF94X + Solvation
1
. Introduction
Diabetes is one of those chronic diseases, which are considered
to be potent inhibitors of both HIV replication and HIV mediated
syncytium development [9]. Acarbose [10], miglitol [11], voglibose
[12] and nojirimycin [13] are well known drugs, which are
prescribed to control blood glucose levels (Fig. 1). These drugs
are effective, but show a number of side effects, like abdominal
distension, flatulence, meteorism and diarrhea [14]. Potent and
among the main death causing factors in the world. WHO proposed
that diabetes will be the 7th major disease responsible for most of
the deaths by 2030. In fact, the number of diabetes patients has
become more than double since 1980–2008 [1]. To cure diabetes,
we need to control the postprandial glucose levels with the inhibi-
safer a-glucosidase inhibitors are highly desired. A major synthetic
focus has been put on non-glucosidic based inhibitors [15,16]. Our
tion of
border surface of the small intestine, which reversibly inhibit
digestive -glucosidase and decreases glucose levels produced
a-glucosidase [2,3]. a-Glucosidase is located on the brush
group has recently discovered pyrimidine derivatives as significant
a-glucosidase inhibitors, 4-methylphenyl bearing pyrimidine
a
(ethyl 6-methyl-4-(4-methylphenyl)-2-oxo-1,2,3,4-tetrahydropyr-
from dietary complex carbohydrate and starch. Hence, it decreases
absorption of glucose into the blood stream and reduces plasma
glucose levels.
imidine-5-carboxylate) was identified as most active of them all
(IC50 112.21 ± 0.97 lM) [17].
As part of our ongoing medicinal chemistry research interests
[39–43] and vast medicinal potential of pyridine hydrazides have
convinced us to synthesize novel pyridine hydrazide derivatives
In addition,
ment of carbohydrate initiated diseases such as hepatitis, cancer
and viral infections [4–6]. -Glucosidase inhibitors are also known
for their antitumor, anti-viral and immunoregulatory activities
7–10]. From -glucosidase inhibitors, deoxynojirimycin (DNJ), N-
butyl-deoxynojirimycin (NB-DNJ) and castanospermine are known
a-glucosidase inhibitors are also used for the treat-
a
and evaluate their a-glucosidase inhibition potential.
Pyridine derivatives display a broad range of biological activities
such as antimicrobial, antioxidant, anti-inflammatory [18–20], anti-
cancer [21], analgesic, agonistic, hypotensive, hallucinogenic and
anti-metastatic [22–24]. In particular it has been fused as a core
skeleton in drug candidates involved in enzyme inhibitions [25].
Pyridine bearing hydrazide moieties such as isonicotinic acid
[
a
⇑
Corresponding author. Fax: +92 42 35321090.
E-mail address: drmyar@ciitlahore.edu.pk (M. Yar).
http://dx.doi.org/10.1016/j.bioorg.2014.10.007
045-2068/Ó 2014 Elsevier Inc. All rights reserved.
0

S. Riaz et al. / Bioorganic Chemistry 57 (2014) 148–154
149
OH 8
hydrazide (INH, isoniazid) [26] and pyridine-2,6-dicarbohydrazide
are pharmacologically useful targets as anticonvulsants [27],
antidepressants [28], anti-inflammatory [29], antimalarials [30],
antimycobacterials [31], anticancer [32], and antimicrobials
[33–36] (Fig. 2).
The docking method is used to estimate the conformation of the
compounds and its orientation within the binding site of the
enzyme. Docking studies aims: (1) specific structural modeling,
7
6
9
HO
5
4
OH
N
1
2
HO
OH
3
H C
HO
N
H
3
O
1
1
OH
10
HO
OH
O
OH O
OH
OH
HO
OH O
OH
O
Miglitol
OH
(
2) the accurate prediction of the activity [37]. MOE-Dock [38]
Acarbose
O
HO
was selected because allow ligands to be flexible during docking
so that they can adjust their conformations in the binding pocket
of the receptor.
In this paper, we report a facile synthesis and characterization
of novel pyridine derivatives, their in vitro a-glucosidase inhibition
OH
OH
H
N
OH
OH
HO
HO
HO
HO
HO OH N
H
OH
activity and molecular modeling to demonstrate the pattern of
binding interactions of our synthesized compounds into the
OH
Voglibose
Deoxynojirimycin
targeted
a-glucosidase.
Fig. 1. Acarbose, voglibose, deoxynojirimycin and miglitol structures.
2
. Results and discussion
2.1. Chemistry
H
O
N
Compounds 3 and 4a–4f were synthesized in good yields and
NH2
purity from commercially available pyridine tricarboxylic acid
(Scheme 1). Pyridine tricarboxylic acid was heated under reflux
in the presence of methanol and sulfuric acid which resulted in
trimethyl pyridine-2,4,6-tricarboxylate 2 in excellent yield (95%)
[44,45]. Treatment of ester 2 with hydrazine in the presence of
methanol (Scheme 1) afforded novel pyridine-2,4,6-tricarbohyd-
H
N
H
N
H N
N
NH2
2
N
O
O
Isoniazid
Pyridine-2,6-dicarbohydrazide
Fig. 2. The molecular structures of biologically well-known pyridine hydrazides.
.
razide 3 in 60% yield.
COOH
CONHNH2
COOCH3
CH OH
3
NH -NH (80%)
2 2
H SO
2
4
C H OH
2 5
HOOC
N
COOH
reflux
H COOC
N
COOCH3
H NHNOC
N
CONHNH2
3
4 h
2
6
h
1
2
60%
3
9
5%
Scheme 1. Synthesis of pyridine-2,4,6-tricarbohydrazide.
Table 1
Reaction conditions and yields of products 4a–h.
R1
CONHN CH
CONHNH2
Glacial acetic acid
GAA)
HC NHNOC
N
CONHN CH
(
CH OH
3
H NHNOC
N
CONHNH2
2
Aldehydes
RT-Reflux
4a-4h
3
R1
R1
Entry
Sample code
Time (h)
R1
Yield (%)
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
4f
3
4
3
4
5
6
5
3
H
82
75
95
85
86
70
60
69
o-OH
m-OH
p-OH
p-N-(CH
m-NH
-cinnamyl
p-OCH
3 2
)
2
4g
4h
3

1
50
S. Riaz et al. / Bioorganic Chemistry 57 (2014) 148–154
Table 2
- and b-Glucosidase inhibition activity, and IC50 values (mean ± SEM, n = 3) of pyridine-2,4,6-tricarbohydrazide 3 and its derivatives 4a–4h.
a
Entry
Sample code
a
-Glucosidase from baker’s yeast
b-Glucosidase from almonds
%Inhibition (0.5 mM)
2.83 ± 0.95
%Inhibition (0.5 mM)
IC50
–
28.1 ± 0.4
26.8 ± 0.3
22.5 ± 0.7
20.4 ± 0.3
>500
l
M ± SEMa
IC50 l
M ± SEMa
1
2
3
4
5
6
7
8
9
3
14.3 ± 0.9
98.1 ± 0.5
98.6 ± 0.4
95.7 ± 0.8
93.8 ± 0.7
44.9 ± 0.9
40.1 ± 1.2
99.1 ± 0.6
98.1 ± 0.8
–
–
–
–
–
–
–
–
–
b
4a
4b
4c
4d
4e
4f
NI
4.33 ± 1.92
6.39 ± 1.64
NI
NI
NI
NI
NI
>500
23.3 ± 0.4
28.1 ± 0.4
4g
4h
a
All reactions were performed in triplicates and averaged, and SEM is standard mean error of the experiments.
No inhibition detected at 0.5 mM.
b
Fig. 3. 2D Interaction analysis of compounds in the active site of the enzyme: (A) 3 and (B) 4c.
Pyridine-2,4,6-tricarbohydrazide 3 was treated with a wide
a reference compound. The IC50 values of these compounds are
listed in Table 2.
range of selected aldehydes in the presence of methanol and few
drops of glacial acetic acid. Good to excellent yields of the corre-
sponding Schiff bases 4a–4h (60–95%) were obtained as shown
in Table 1.
Pyridine 2,4,6-tricarbohydrazide 3 (Table 2, Entry 1) was found
to be very weak inhibitor (% inhibition of 14.29 ± 1.21). In the Schiff
bases series 4a–4h, the un-substituted compounds (4a and 4g) or
and those containing a hydroxyl group (4b–d) were the most
active, and with comparable inhibition. Compounds bearing amino
substituents (4e and 4f) showed to be inactive. In the phenol deriv-
atives cases (4b–4d) the higher activity observed could be due to
their proton donating ability, possibly related to reversible proton-
ation [46]. Among these phenol compounds, 4d with p-hydroxyl
2
2
.2. Biological
.2.1.
The synthesized compounds 3 and 4a–4h were screened for
their -glucosidase inhibitory activity and acarbose was used as
a-Glucosidase activity from baker’s yeast
a
Fig. 4. 2D Interaction analysis of compounds in the active site of the enzyme: (A) 4b and (B) 4h.

S. Riaz et al. / Bioorganic Chemistry 57 (2014) 148–154
151
Fig. 5. 2D interaction analysis of compound 4d in the active site of the enzyme.
Fig. 6. 2D interaction analysis of compound 4g in the active site of the enzyme.
group is the most active. This is may be due to the higher acidity of
2.2.3. Molecular modeling studies for a-glucosidase
compound 4d compared to the other phenol derivatives. On the
other hand the lack of inhibition activity of compounds bearing
amino groups (4e and 4f), may be connected to its protonation
inability. The presence of p-methoxy in the phenyl group seems
2.2.3.1. Interaction analysis of compound 3 and 4c. In case of com-
pound 3, a total of five interactions with the residues of the binding
site were observed. Asn89, Gln108 and Glu110 interacts with
formohydrazide moieties attached to pyridine ring. Whereas,
Lys78 make arene-hydrogen bonding with pyridine ring as shown
in Fig. 3A. The interaction analysis of compound 4c (Fig. 3B)
revealed hydroxyl benzylidene formohydrazide as an important
interacting moiety, as three amino acid residues Glu110, His172
and Arg287 interact with the very same chemical moiety.
1
to have no biological effect, since compound 4a (R = H) and 4h
1
(
R = p-OMe) gave the same IC50 value (28.12 ± 0.44
l
M).
The overall order of synthesized pyridine derivatives based on
IC50 value was as follows:
4
d > 4c > 4g > 4b > 4a ¼ 4h > 4e ¼ 4f
2.2.3.2. Interaction analysis of compound 4b and 4h. The molecular
2
.2.2. Inhibition towards b-glucosidase from almonds
All the synthesized pyridine derivatives (3, 4a–4h) were
structure of compound 4b is very much similar to compound 4c.
As observed in compound 4c, the hydroxyl benzylidene formohyd-
razide moieties were found as the interacting feature of the
compound. The active site residues Tyr93, Gln108, Glu110 and
Thr295 interact with compound as shown in Fig. 4 A. In case of
compound 4h, five interactions were observed. The active site
investigated for their b-glucosidase inhibitory potential. The three
compounds 3, 4b and 4c were found to be very weak inhibitors,
whereas remaining compounds were completely inactive against
b-glucosidase (Table 2).

1
52
S. Riaz et al. / Bioorganic Chemistry 57 (2014) 148–154
and 13C NMR spectra were recorded at 300 MHz on JEOL-Lambda
NMR instrument. Chemical shifts are quoted as d ppm and the
coupling constants J in Hz. Signals are described as s (singlet), d
Table 3
Molecular docking results showing binding energies and docking score.
Entry
Sample code
Docking score
Binding energy (GBVI/WSA)
(
doublet), m (multiplet) and br (broad). Melting points were
1
2
3
4
5
6
7
8
9
3
4a
4b
4c
4d
4e
4f
4g
ꢀ6.3050
ꢀ12.0689
ꢀ11.9923
ꢀ12.6625
12.7878
ꢀ12.6011
ꢀ23.9313
ꢀ23.4106
ꢀ24.1839
ꢀ26.2320
ꢀ18.1921
ꢀ17.2923
ꢀ21.8861
ꢀ20.7101
ꢀ22.3214
measured on a Buchi 434 melting point apparatus and are
uncorrected. Combustion analysis was performed on a Elementar,
variomicrocube, Germany.
ꢀ7.8998
4
.1. Synthesis of trimethyl pyridine-2,4,6-tricarboxylate 2
ꢀ7.6350
ꢀ11.8713
ꢀ10.9936
ꢀ11.5348
4h
Acarbose
Trimethyl pyridine-2,4,6-tricarboxylate 2 was prepared using
1
0
reported method [44]. Pyridine tricarboxylic acid 1 (10 g, 47 mmol)
in methanol (70 mL) with concentrated sulfuric acid, 98% (5 mL)
was heated under reflux for six hours until complete disappear-
ance of compound 1. The solution was neutralized with CaCO3,
filtered and solvent was evaporated under reduced pressure. The
crude product was recrystallised from toluene to give a white
crystalline product; Yield: 95%; m.p.: 161 °C IR (
cm ): 3462, 3304 (NH stretch), 2976, 1737 (C@O), 1644 (C@N),
6 H
1488 (CAN); H NMR (DMSO-d ) d : 8.54 (2H, apparent s), 3.95
(9H, apparent s).
residues Gln108, Glu110, Phe170 and Asp267 interact with
methoxy benzylidene formohydrazide moieties of the compound.
Phe170 make arene-arene bonding with anisole ring of methoxy
benzylidene formohydrazide moiety (Fig. 4B).
m
max, KBr,
ꢀ1
1
2
.2.3.3. Interaction analysis of compound 4d. The molecular struc-
ture of compound 4d is closely related to compound 4b. A total
of seven interactions were observed in case of compound 4d:
Glu69, Lys78, and Gly268, Gln108, and Glu110 as shown in Fig. 5.
4.2. Synthesis of pyridine 2,4,6-tricarbohydrazide 3
Hydrazine hydrate (80%, 5 eq.) was added to a stirred solution
of trimethyl pyridine-2,4,6-tricarboxylate 2 (5 g, 24 mmol) in
ethanol (50 mL) [47]. The reaction mixture was heated at reflux for
2
4
.2.3.4. Interaction analysis of compound 4g. In case of compound
g, a total of four interactions were observed: Thr91, Gln108 and
Glu110 interact with formo hydrazide group of phenylallylidene
formohydrazide moiety, whereas, Phe170 interacts through
arene-arene bonding with terminal benzene ring (Fig. 6).
4
h and allowed to cool to RT, filtered, dried and recrystallized from
ethanol to obtain pure product 3 (3 g, 70%); m.p.: 295 °C; IR (
m
max
,
ꢀ1
KBr pellets, cm ): 3317 (NH stretch), 1649 (C@O), 1623 (C@N),
In order to rationalize the obtained IC50 values for the synthe-
1
1
235 (CAN), 1063(NAN); H NMR (DMSO-d
6 H
) d : 10.66 (2H,
sized compounds against a-glucosidase, docking score and binding
apparent s), 10.42 (1H, s), 8.478 (2H, s), 4.66 (4H, s), 3.94 (2H, s).
energy for all compounds have been calculated (Table 3). The
Generalized-Born Volume Integral/Weighted Surface Area (GBVI/
WSA) is a scoring function which estimates the free energy of bind-
ing of the ligand from a given pose. For all scoring functions, lower
scores indicate more favorable poses [50]. In molecular docking
studies, binding energies and docking score are the primary bases
for the selection of active and non-active compounds.
4
4
.3. General procedure for the synthesis of compounds (4a–4h)
.3.1. General method A
Compounds 4a–4h were prepared by following the method
reported by Hearn et al. [48] with modifications. Pyridine 2,4,6-tri-
carbohydrazide 3 (1 equiv.) was dissolved into absolute ethanol
(
50 mL) in the presence of glacial acetic acid (2–3 mL). The reaction
mixture was stirred at RT and then treated with selected aldehyde
3.3 equiv.). After being refluxed for 3–5 h, the reaction mixture was
3
. Conclusion
(
In summary, a facile synthesis of a range of pyridine 2,4,6-tri-
cooled, and placed in refrigerator overnight. The resulting solid was
filtered, washed with water (200 mL), and n-hexane (30 mL).
carbohydrazide derivatives bearing different level of substituents
was achieved in good to excellent yields. The -glucosidase inhib-
a
itory activity of these pyridine derivatives was evaluated; acarbose
was used as the reference compound. Structure activity relation-
ship demonstrated that the inhibition potential of the synthesized
4
.3.2. N’-2,N’-4-dibenzylidene-6-((2benzylidenehydrazinyloxy)methyl)
pyridine-2,4-dicarbohydrazide (4a)
Compound 4a was prepared by following general method A:
pyridine 2,4,6-tricarbohydrazide 3 (500 mg, 2.6 mmol, 1 equiv.),
benzaldehyde (0.29 mL, 2.9 mmol), 3 h reflux. White amorphous
solid; Yield: 82%; m.p. 310–312 °C; Rf (n-hexane: EtOAc 1:1) 0.5;
IR (
1
compounds is greatly dependent upon the nature of R ; com-
pounds bearing hydroxyl-phenyl groups were comparatively more
active, while amino compounds were inactive. Compound 4d with
p-hydroxyl-phenyl motif was the most active among this series
ꢀ1
m
max, KBr pellets, cm ): 1660 (C@O), 1551 (C@N), 1199
(
IC50 20.24 ± 0.72
lM). Molecular modeling was performed on
1
(
(
CAN), 3412 (NH); H NMR (DMSO-d
2H, s), 8.25 (2H, s), 8.12(1H, s), 7.84 (6H, s), 7.50 (6H, apparent
: C, 67.30; H, 4.48; N,
6 H
) d : 12.38 (3H, s), 8.84
the inactive parent trihydrazide compound 3 together with the
most active compounds (4b–d, 4g, 4h) for comparison purposes
and understanding of the interaction patterns responsible for the
inhibition activity.
23 7 3
s) 7.48 (3H, s); Anal. Calcd. for C29H N O
1
8.94. Found: C, 66.30; H, 4.34; N, 18.17.
4.3.3. N’-2, N’-4, N’-6-tris(2-hydroxybenzylidene)pyridine-2,4,6-
4
. Experimental
tricarbohydrazide (4b)
The compound 4b was prepared according to general method A:
pyridine 2,4,6-tricarbohydrazide 3 (500 mg, 2.6 mmol), salicylalde-
hyde (0.27 mL, 2.9 mmol), 4 h reflux. White amorphous solid
All chemicals and solvents used are of analytical grade and were
purchased from Sigma Aldrich and Merck Chemical Company and
used without further purification. TLC was run on the silica-coated
aluminum sheets (silica gel 60 F254, E Merck, Germany) and visu-
alized in low UV light. IR spectra in KBr pellets were recorded on
(735 mg); Yield: 75%; m.p.: 324–325 °C; R
f
(n-hexane: EtOAc,
ꢀ1
1:1) 0.42; IR (
1254 (CAN), 3375 (NH); H NMR (DMSO-d
11.28 (3H, s), 9.70 (2H, s), 8.69 (3H, d, J = 9.9 Hz), 8.64 (3H, d,
mmax, KBr pellets, cm ): 1665 (C@O), 1609 (C@N),
1
6 H
) d : 12.22 (3H, s),
1
the FT-IR Perkin Elmer spectrum BX spectrophotometer. H NMR

S. Riaz et al. / Bioorganic Chemistry 57 (2014) 148–154
153
J = 9.3 Hz), 8.52 (3H, s), 7.33(3H, d, J = 9.2 Hz), 6.88 (3H, t,
J = 6.3 Hz); Anal. Calcd for C29 : C, 61.59; H, 4.10; N,
7.34. Found: C, 60.30; H, 4.34; N, 16.17.
4.3.9. N’-2,N’-4,N’-6-tris(4-methoxybenzylidene)pyridine-2,4,6-
tricarbohydrazide (4h)
The Compound 4h was prepared according to general method
A: pyridine 2,4,6-tricarbohydrazide 3 (500 mg, 2 mmol, 1 equiv.),
4-methoxybenzaldehyde (0.88 g, 6.5 mmol), 3 h reflux. White
23 7 6
H N O
1
4.3.4. N’-2, N’-4, N’-6-tris(3-hydroxybenzylidene)pyridine-2,4,6-
tricarbohydrazide (4c)
amorphous solid; Yield: 69%; m.p.: 248–250 °C; R
EtOAc, 1:1) 0.4; IR (m
f
(n-hexane:
max, KBr, cm ): 1724 (C@O), 1557 (C@N),
1277 (CAN), 3430 (NH); 1H NMR (DMSO-d
) d : 12.31 (3H, s),
ꢀ1
Compound 4c was prepared by following general A: pyridine
2
,4,6-tricarbohydrazide 3 (500 mg, 2 mmol, 1 equiv.), 3-hydroxy
benzaldehyde (0.8 g, 6.5 mmol), 3 h reflux. White amorphous
solid; Yield: 95%, m.p.: 280°C; R (n-hexane: EtOAc, 1:1) 0.4; IR
max, KBr pellets, cm ): 1660 (C@O), 1607 (C@N), 1254 (CAN),
6
H
9.85(2H, s), 8.70 (2H, s), 8.68 (1H, s), 7.75 (4H, apparent d,
J = 8.1Hz), 7.70 (2H, d, J = 8.4 Hz), 7.06–7.01 (6H, s), 3.81(9H, s);
f
ꢀ1
(m
Anal. Calcd for C32
29 7 6
H N O : C, 63.25; H, 4.81; N, 16.14. Found: C,
1
3
355(NH). H NMR (DMSO-d
2H, s), 8.66 (3H, d, J = 9.9 Hz), 8.60 (3H, d, J = 9.3 Hz), 8.53 (3H,
s), 7.23 (3H, t, J = 9.2 Hz), 6.83 (3H, t, J = 6.2 Hz); Anal. Calcd for
: C, 61.59; H, 4.10; N, 17.34. Found: C, 60.20; H, 4.32;
6 H
) d : 12.32 (3H, s), 9.69 (3H, s), 9.44
61.25; H, 4.21; N, 16.04.
(
4.4. a-Glucosidase assay procedure
29 23 7 6
C H N O
N, 16.06.
The
modifications as given by Pierre et al. [49]. Total volume of 100
reaction mixture contained, 70
10 L (0.5 mM) test compound, followed by the addition of 10
(0.0234 units, Sigma Inc.)
were mixed, preincubated for 10 min at 37 °C and pre-read at
400 nm. The reaction was initiated by the addition of 10 L of
0.5 mM substrate (p-nitrophenyl glucopyranoside, Sigma Inc.).
After 30 min of incubation at 37 °C, absorbance of the yellow color
produced due to the formation of p-nitrophenol was measured at
400 nm using Synergy HT (BioTek, USA) 96-well microplate reader.
Acarbose was used as positive control. All experiments were car-
ried out in triplicates. The percent inhibition was calculated by
the following equation;
a-glucosidase inhibition activity was performed with slight
lL
4.3.5. N’-2,N’-4,N’-6-tris(4-hydroxybenzylidene)pyridine-2,4,6-
lL 50 mM phosphate buffer, pH 6.8,
tricarbohydrazide (4d)
The compound 4d was prepared by following general method
A: pyridine 2,4,6-tricarbohydrazide 3 (500 mg, 2 mmol, 1 equiv.),
l
lL
a-glucosidase enzyme. The contents
4
-hydroxy benzaldehyde (0.8 g, 6.5 mmol), 4 h reflux. White
amorphous solid. Yield: 85%, m.p.: 324 °C; R (n-hexane: EtOAc,
max, KBr, cm ): 1660 (C@O), 1607 (C@N), 1254
l
f
ꢀ
1
1
:1) 0.4; IR (
CAN), 3355(NH). H NMR (DMSO-d
3H, s), 8.88 (2H, s), 8.66 (3H, d, J = 9.9 Hz), 7.65 (6H, s), 6.86 (6H,
: C, 61.59; H, 4.10; N, 17.34. Found:
m
1
(
(
6 H
) d : 12.19 (3H, s), 10.00
s); Anal. Calcd for C29
23 7 6
H N O
C, 60.10; H, 4.22; N, 16.04.
4
2
.3.6. N’-2,N’-4,N’-6-tris(4-(dimethylamino)benzylidene)pyridine-
,4,6-tricarbohydrazide (4e)
Inhibitionð%Þ ¼ ðabs of controlꢀabs of test=abs of controlÞꢁ100
.
IC50 values (concentration at which there is 50% in enzyme
catalyzed reaction) were calculated using EZ-Fit Enzyme Kinetics
Software (Perrella Scientific Inc. Amherst, USA).
Compound 4d was prepared by general method A: pyridine
2
,4,6-tricarbohydrazide 3 (500 mg, 2 mmol, 1 equiv.), 4-(dimethyl-
amino)benzaldehyde (0.97 g, 6.5 mmol), 5 h reflux. White amor-
phous solid; Yield: 86%; m.p.: 240–242 °C; R (n-hexane: EtOAc,
max, KBr, cm ): 1660 (C@O), 742 (CACl), 1557
f
ꢀ1
1
(
(
:1) 0.6; IR (
C@N), 1200 (CAN), 3400 (NH); H NMR (DMSO-d
3H, s), 8.60 (2H, s), 8.51(2H, s), 7.65–7.68 (6H, m), 7.57 (1H, s),
m
1
6 H
) d : 12.12
4.5. b-Glucosidase assay procedure
6
6
38 10 3
.7 (6H, d, J = 9 Hz), 3.00 (18H, s); Anal. Calcd for C35H N O : C,
5.00; H, 5.92; N, 21.66. Found: C, 64.10; H, 4.92; N, 20.01.
Total volume of 100
0 mM phosphate buffer, pH 6.8, 10
L (1.2 units/mL; G0395; Sigma)
of b-glucosidase from almonds. The contents were mixed,
pre-incubated for 10 min at 37 °C and pre-read at 400 nm. The
l
L reaction mixture contained, 70
lL
5
lL (0.5 mM) test compound,
followed by the addition of 10
l
4.3.7. N’2, N’4, N’6-tris(3-aminobenzylidene)pyridine-2,4,6-
tricarbohydrazide (4f)
The compound 4f was prepared by general method A: Pyridine
,4,6-tricarbohydrazide 3 (500 mg, 2 mmol, 1 equiv.), 3-amino-
reaction was initiated by the addition of 10
lL of 0.5 mM substrate
2
(
p-nitrophenyl b-glucopyranoside, Sigma Inc.). After 30 min of
incubation at 37 °C, absorbance of the yellow color produced due
to the formation of p-nitrophenol was measured at 400 nm using
6-well microplate reader. All experiments were carried out in
triplicates. The percent inhibition was calculated by the following
equation;
benzaldehyde (0.78 g, 6.5 mmol), 6 h reflux. Yellow amorphous
solid. Yield: 70%; m.p.: 320–321 °C; R (n-hexane: EtOAc, 1:1)
max, KBr, cm ): 1724 (C@O), 1557 (C@N), 1277 (CAN),
f
ꢀ1
0.5; IR (
3430 (NH); H NMR (DMSO-d
9.97 (1H, s), 8.63 (3H, d, J = 6.9 Hz), 8.59 (2H, s), 8.53 (3H, d,
m
9
1
6
H
) d : 12.20(3H, s), 10.02 (2H, s),
J = 5.1 Hz), 7.66 (2H, d, J = 8.1 Hz), 7.58 (1H, d, J = 8.1 Hz), 6.88
3H, d, J = 6.3 Hz), 3.97 (6H, s); Anal. Calcd for C29 : C,
(
26 10 3
H N O
6
1.91; H, 4.66; N, 24.90. Found: C, 60.10; H, 3.92; N, 23.60.
Inhibitionð%Þ ¼ ðabs of control ꢀ abs of test=abs of controlÞ
ꢁ
100:
4.3.8. N’-2, N’-4, N’-6-tris(3-phenylallylidene)pyridine-2,4,6-
tricarbohydrazide (4g)
The compound 4g was prepared by general method A: pyridine
,4,6-tricarbohydrazide 3 (500 mg, 2 mmol, 1 equiv.), cinnamalde-
4.6. Protein preparation
2
hyde (0.86 g, 6.5 mmol), 5 h reflux. Yellow amorphous solid; Yield:
The protein molecule included in our study, a-glucosidase
6
0%; m.p.: 276 °C; R
f
(n-hexane: EtOAc, 1:1) 0.6; IR (
m
max, KBr,
kinase was obtained from Protein Data Bank. Water molecules
were removed and the 3D protonation of the protein molecule
was carried out. The energy of the protein molecule was minimized
using the Energy minimization algorithm of MOE tool. The
following parameters were used for energy minimization; gradi-
ent: 0.05, Force Field: MMFF94X + Solvation, Chiral Constraint:
Current Geometry. Energy minimization was terminated when
ꢀ1
1
cm ): 1724 (C@O), 1557 (C@N), 1277 (CAN), 3430 (NH);
H
NMR (DMSO-d
6
) d
.50(6H, d, J = 6.0 Hz), 7.67 (3H, d, J = 7.5 Hz), 7.46–7.35 (6H, s),
29 7 3
.18 (3H, d, J = 8.7 Hz), 3.97 (3H, s); Anal. Calcd for C35H N O
H
: 12.24 (3H, s), 8.79 (3H, s), 8.62 (2H, s),
8
7
(
1
595): C, 70.57; H, 4.91; N, 16.46. Found: C, 69.10; H, 3.91; N,
5.82.

1
54
S. Riaz et al. / Bioorganic Chemistry 57 (2014) 148–154
the root mean square gradient falls below the 0.05. The minimized
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