Dihydropyrano [2,3-c] pyrazole: Novel in vitro inhibitors of yeast α-glucosidase

Article abstract of DOI:10.1016/j.bioorg.2016.01.008

Inhibition of α-glucosidase enzyme activity is a reliable approach towards controlling post-prandial hyperglycemia associated risk factors. During the current study, a series of dihydropyrano[2,3-C] pyrazoles (1-35) were synthesized and evaluated for thei

Full text of DOI:10.1016/j.bioorg.2016.01.008

Bioorganic Chemistry 65 (2016) 61–72
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Dihydropyrano [2,3-c] pyrazole: Novel in vitro inhibitors of yeast
a-glucosidase
Hamdy Kashtoh a, Munira Taj Muhammad b, Jalaluddin J.A. Khan a, Saima Rasheed b, Ajmal Khan b,
Shahnaz Perveen c, Kulsoom Javaid b, Atia-tul-Wahab d, Khalid Mohammed Khan b,
,
⇑
M. Iqbal Choudhary a,b,d,
⇑
a Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia
b H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
c PCSIR Laboratories Complex, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi 75280, Pakistan
d Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, 75270, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of
hyperglycemia associated risk factors. During the current study, a series of dihydropyrano[2,3-c] pyra-
zoles (1–35) were synthesized and evaluated for their -glucosidase inhibitory activity. Compounds 1,
4, 22, 30, and 33 were found to be the potent inhibitors of the yeast -glucosidase enzyme.
Mechanistic studies on most potent compounds reveled that 1, 4, and 30 were non-competitive inhibitors
(Ki = 9.75 0.07, 46 0.0001, and 69.16 0.01 M, respectively), compound 22 is a competitive inhibitor
(Ki = 190 0.016 M), while 33 was an uncompetitive inhibitor (Ki = 45 0.0014 M) of the enzyme.
a-glucosidase enzyme activity is a reliable approach towards controlling post-prandial
Received 11 November 2015
Revised 25 January 2016
Accepted 31 January 2016
Available online 1 February 2016
a
a
l
Keywords:
Dihydropyrano [2,3-c] pyrazole
l
l
Finally, the cytotoxicity of potent compounds (i.e. compounds 1, 4, 22, 30, and 33) was also evaluated
against mouse fibroblast 3T3 cell line assay, and no toxicity was observed. This study identifies non-
a
-Glucosidase inhibition
Post-prandial hyperglycemia
Diabetes
cytotoxic novel inhibitors of
a
-glucosidase enzyme for further investigation as anti-diabetic agents.
Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction
a-glucosidase inhibitors (AGIs) have been developed to reduce
post-prandial hyperglycemia in diabetic individuals [5,6]. Vogli-
bose, acarbose and miglitol are widely used AGIs (as oral antidia-
betic drugs) for the management of hyperglycemia. However,
these drugs are known for several gastrointestinal adverse effects,
such as abdominal discomfort, diarrhea, and flatulence [7]. In addi-
tion, these drugs have low efficacy with high IC50 values. Based on
the crucial role of a-glucosidase enzyme and the inefficiencies of
existing drugs, there is an urgent need to identify safe and effective
inhibitors.
Diabetes, a metabolic disorder, is characterized by chronic
hyperglycemia. Type 2 diabetes mellitus is mainly associated with
deficiency in insulin secretion, and insulin sensitivity [1]. Defects
in insulin functioning result in high blood glucose level in diabetic
patients [2]. The delayed insulin secretion, right after the meals,
results in sudden increase in blood glucose levels, known as
post-prandial hyperglycemia [3]. Prolonged hyperglycemia causes
macro- and micro-vascular complications, e.g. atherosclerosis,
and cardiovascular disorders [4]. Therefore, effective control of
hyperglycemia in type-2 diabetes includes the inhibition of carbo-
The current study describes the synthesis and
a-glucosidase
inhibitory activity in vitro of dihydropyrano [2,3-c] pyrazole
hydrate hydrolyzing enzymes (e.g.
mammals, -glucosidase enzyme is found in the small intestine
(i.e. in the brush borders of the enterocytes of the jejunum). It cat-
alyzes the hydrolysis of 1,4- bonds of the unabsorbed oligo- and
di-saccharides, and converts them into mono-saccharides which
are absorbed in the upper jejunum, resulting in hyperglycemia.
a-glucosidase, a-amylase). In
derivatives 1–35. As per literature survey, this is the first report
of a-glucosidase inhibitory activity of this class of compounds.
a
Fused heterocyclic compounds are focus of intense research,
because of their pharmacological importance. Among the fused
heterocycles, pyranopyrazoles have attracted major scientific
interest, when Junek and Aigner synthesized them for the first time
in mid-19th century. Since then pyranopyrazole nucleus is a versa-
tile source of biologically important molecules [8].
a
⇑
Corresponding authors at: Department of Biochemistry, Faculty of Science, King
There are 4 possible isomers of pyranopyrazole i.e. pyrano [2,3-
c] pyrazole, pyrano [4,3-c] pyrazole, pyrano [3,2-c] pyrazole and
Abdulaziz University, Jeddah 21589, Saudi Arabia (M.I. Choudhary).
E-mail addresses: khalid.khan@iccs.edu, hassaan2@super.net.pk (K.M. Khan),
iqbal.choudhary@iccs.edu (M.I. Choudhary).
http://dx.doi.org/10.1016/j.bioorg.2016.01.008
0045-2068/Ó 2016 Elsevier Inc. All rights reserved.

62
H. Kashtoh et al. / Bioorganic Chemistry 65 (2016) 61–72
pyrano [3,4-c] pyrazole. Among them, pyrano [2,3-c] pyrazoles
play an important role in pharmacological and medicinal chem-
istry [9]. They have several activities, such as analgesic, anti-
inflammatory, anti-bacterial, anti-microbial, anti-platelet, Chk1
kinase inhibitory, and vasodilatory [10–15]. Pyrano [2,3-d] pyrim-
of the compound 1 (DMSO-d6, 300 MHz) showed a singlet at d
12.18 due to NH of the pyrazolone ring. Phenyl ring showed two
2H doublets at d 8.21 for H-30/H-50 ðJ3 ;2 =5 ;6 ¼ 8:7 Hz), and 7.46
0
0
0
0
for H-20/H-60 ðJ2 ;3 =6 ;5 ¼ 8:7 Hz). A 2H singlet at d 7.03 was due
to NH2 group. Another singlet at d 5.08 was assigned to H-4 of
the pyran ring, while a 3H singlet at d 1.75 was due to the methyl
attached to the pyrazolone ring.
0
0
0
0
idine derivatives have been reported for
a-glucosidase inhibitory
activity [16]. We, therefore, decided to evaluate pyrano [2,3-c]
pyrazole derivatives for their a-glucosidase inhibitory activity.
The 13C NMR spectra showed a total of 12 carbons (two of them
are overlapped), including 5 methine, 1 methyl, and 8 quaternary
carbons. The downfield signal at d 120.4 was assigned to the nitrile
carbon. Carbons of the pyrazolone ring appeared at d 154.6 (C-8),
135.8 (C-3), and 96.5 (C-9), while the methyl carbon appeared at
d 9.7. The pyran carbons appeared at d 161.4 (C-6), 55.9 (C-5),
and 35.8 (C-4). The downfield signals appeared at d 123.8, 128.8,
146.3, and 152.0 were assigned to the aromatic carbons C-20/C-60,
C-30/C-50, C-10, and C-40, respectively. The C-20/C-60 phenyl protons
resonating at d 7.46 showed 3J correlation with C-4 (d 35.8) of
2. Results and discussion
2.1. Chemistry
Multi-component (one-pot) synthesis of pyrano [2,3-c] pyra-
zoles is widely reported in the presence of various catalysts, such
as acetylene dicarboxylates [17], CsCO3 supported on hydroxy apa-
tite coated Ni0.5 Zn0.5 Fe2O4 magnetic nanoparticles [18], and with
4
pyran ring, and J correlation of H-4 (d 4.81) with C-30 (d 128.8)
L-proline [19]. In present study we synthesized pyrano [2,3-c]
indicated the attachment of pyran moiety with phenyl ring. The
methyl protons appeared at d 1.78 showed 4J coupling with C-8
(d 154.6) and 3J coupling with C-9 (d 96.5) indicated the attach-
ment of pyrazolone moiety with pyran ring. The key HMBC inter-
actions are mentioned in Fig. 1.
pyrazoles by the reaction of pyrazolones with corresponding ben-
zylidenes in the presence of triethylamine at room temperature in
only 25–30 min.
In a typical reaction, equimolar amount of pyrazolone and tri-
ethylamine were stirred for 2 min at ambient temperature, and
corresponding benzylidenes (1.0 equivalent generated from
malononitrile) were added. The reaction mixture was stirred for
25–30 min, and progress of reaction was monitored by TLC. The
desired products appeared as precipitates and filtered. The wash-
ing of resulting precipitates was carried out with dichloromethane
to afford pure products (Scheme 1). The structures of the pure
products were elucidated by spectroscopic analyses.
The HREI-MS of compound 1 showed the M+ at m/z 297.0644,
corresponding to C14H11N5O3 (Calcd 297.0682). The base peak at
m/z 231 was due to the loss of a malononitrile moiety (C3H2N2)
from the M+, while a peak at m/z 175 was due to the fragment
C8H7N4O, resulted from the loss of NO2 substituted phenyl ring
from the M+. Another fragment m/z 271 was due to the loss of a
CN group from the M+. The EI-MS fragmentation pattern of
compound-1 is represented in Fig. 2.
Structures of other compounds i.e. 2–35 were also deduced
based on spectral studies and comparison with 1, as presented in
the experimental section. Out of 35 synthesized compounds, 13
were found to be known (i.e. 1, 3–6, 15–17, 19, 20, 24, 30, 31)
[20–22], while rest were identified as new compounds.
2.2. General structure elucidation
The structures of dihydropyrano [2,3-c] pyrazole derivatives
were deduced by spectroscopic analyses. The 1H NMR spectrum
N
H2N
NC
O
N
H3C
OH
NH
DCM , TEA
R.T.
+
R
N N
CH3
N
H
R
1-35
H
O
N
N
H
N
C
H
R
N
N
H
N
H
H
R
H
O
N
C
N
+
O
H
H
R
N
N
N N
H
O
H
N N
O
R
O
H
H2N
NC
CN
NH
N
NH
N
N
H
CH3
R
Scheme 1. Synthesis of dihydropyrano [2,3-c] pyrazoles.

H. Kashtoh et al. / Bioorganic Chemistry 65 (2016) 61–72
63
Compounds 1, 4, 22, 30, and 33 were found to be potent mem-
bers, while the rest of the compounds showed less than 50% inhi-
bition, and thus considered as inactive. Compound 1 was the most
1
N
H2N
O
161.1
6
154.6
8
4.81, s
potent analog of this series (IC50 = 54.2 2.02
pared with standard drug, i.e. acarbose (IC50 = 937.0 1.6
Compounds 4, 22, 30, and 33 were also found to be significantly
active (IC50 values between 60.8 and 180.2 M).
lM), when com-
H
H
N
2
l
M).
55.9
12.18, s
9
5
96.5
4
120.4
l
135.8
NC
A preliminary structure–activity relationship (SAR) for the com-
pounds 1–35 suggested that the activity depends mainly on the
substitutions on aromatic ring (R). Apparently, dihydropyrano
[2,3-c] pyrazole moiety interact with the enzyme through hydro-
gen bonding. However, different substitutions on aromatic ring
(R) may also influence the inhibitory potential.
35.8
9.7
7.46, d
CH3
146.3
H
H
(J= 8.7 Hz.)
1.78, s
123.8
2'
6'
123.8
In this series, compound 1 was found to be the most active
analog. This may be due to the presence of p-nitro substituted aro-
matic ring (R) which may facilitate its interaction with the enzyme.
Mechanistic studies revealed that it is a non-competitive inhibitor
(Fig. 3). Interestingly, the activity was completely lost with an
ortho and meta substituted nitro group, as in compounds 2 and 3.
Additionally compounds with an OH, along with ortho substituted
nitro group, were also unable to inhibit the activity of enzyme, as is
the case with compound 35.
128.8
128.8
3'
4'
152.0
5'
H
H
8.21, d
8.21, d
(J= 8.7 Hz)
NO2
Fig. 1. Key HMBC correlations of compound 1.
Compound 30 (IC50 = 60.8 0.51 lM), the second most active
member of the series, have benzyloxy at para, and methoxy at meta
positions of the aromatic ring. The potent activity of this com-
pound may be due to the combined effects of these moieties. The
3. Bioactivity
kinetic studies on compound 30 showed that it is
a non-
3.1. In-vitro a-glucosidase inhibitory activity
competitive inhibitor (Fig. 6). Both, benzyloxy and methoxy moi-
eties facilitate the interaction of compound 30 with the allosteric
site of the enzyme. To further understand the contribution of ben-
zyloxy moiety, we compared the activities of compound 30 with 33
and 32. Results revealed that the inhibitory activity of 33 is less as
compared to 30, as the benzyloxy moiety is substituted at ortho
position and no OCH3 group was present (i.e. compound 33;
In search of new
several classes of lead compounds previously [23–25]. However,
this is the first report of the -glucosidase inhibitory activity of
dihydropyrano [2,3-c] pyrazole class of compounds.
-Glucosidase enzyme from (Saccharomyces cerevisiae, EC
3.2.1.20) was used to evaluate the -glucosidase inhibitory activity
of compounds 1–35 (Table 1). Based on literature report, in
different species of -glucosidase, four highly conserved regions
are present, having Asp and Glu, as the catalytic residues. Thus
-glucosidase from yeast is extensively used for the preliminary
identification of -glucosidase inhibitors [26]. However, the inhibi-
a-glucosidase inhibitors, we have identified
a
a
a
IC50 = 163.6 1.51 lM). The activity is completely lost when only
benzyloxy is substituted at para position, as observed in compound
32. These results suggest that the presence of benzyloxy moiety at
para position is only beneficial when OCH3 group (i.e. an electron
donating group) is also present on the benzene ring (i.e. compound
30). Compound 22, the third most active compound of the series
a
a
a
tory activity of these compounds against human
remained to be determined.
a-glucosidase
(IC50 = 159.7 4.21
lM), has a benzyloxy moiety at meta, and a
hydroxyl group (electron donating group) at para positions. Again
H2N
NC
O
N
NH
H2N
NC
O
N
-NO2 Ph
NH
CH3
CH3
O2N
NO2
C8H7N4O•
m/z 175
1
-CN
O
N
H2N
O
NH
N
NH
CH3
CH3
NO2
NO2
•
C11H9N3O3
C13H12N4O3
m/z 271
m/z 231
Fig. 2. EI-MS Fragmentation pattern of compound 1.

64
H. Kashtoh et al. / Bioorganic Chemistry 65 (2016) 61–72
Table 1
a
-Glucosidase inhibition activity of compounds 1–35.
.
Compounds
R
IC50 SEM (lM)
1
54.2 2.02
2
NA
3
4
NA
180.2 2.5
5
6
7
8
NA
NA
NA
NA
9
NA
NA
10
11
NA
12
13
NA
NA

H. Kashtoh et al. / Bioorganic Chemistry 65 (2016) 61–72
65
Table 1 (continued)
Compounds
R
IC50 SEM (lM)
14
NA
15
16
17
NA
NA
NA
18
19
NA
NA
20
NA
NA
21
22
159.7 4.21
23
24
NA
NA
25
26
27
NA
NA
NA
28
NA
(continued on next page)

66
H. Kashtoh et al. / Bioorganic Chemistry 65 (2016) 61–72
Table 1 (continued)
Compounds
R
IC50 SEM (lM)
29
NA
30
60.8 0.51
31
32
NA
NA
33
163.6 1.51
34
NA
35
NA
Acarbose (standard drug)
937 1.6
its activity may be due the combined effect of benzyloxy and elec-
tron donating hydroxyl moieties. When we compared its activity
plots. Ki was calculated by secondary re-plot of Lineweaver–Burk
plots, and finally the Dixon plot helped to deduce the Ki. Compound
with compound 30 (IC50 = 159.7 4.21 vs 60.8 0.51
l
M),
a
1 was found to be a non-competitive inhibitor (Ki = 9.75 0.07
(Fig. 3). Fig. 3 shows that in the presence of compound 1, the Km of
-glucosidase enzyme was not affected, while the Vmax of the
lM)
decreased activity was observed, which again indicated that ben-
zyloxy moiety at para position, along with OCH3 group at meta, is
essential for activity.
a
enzyme decreased, this indicated
a non-competitive type of
Compound 4, the 4th most potent compound of the series, pos-
sesses dichloro substituents at ortho and para positions of the aro-
inhibition.
The kinetics studies on compound 4 indicated that it is a
non-competitive inhibitor (Ki = 46.0 0.0001 M) (Fig. 4). The
Lineweaver–Burk plots showed that in the presence of compound
4, the Km of -glucosidase enzyme was not affected, while the Vmax
of enzyme decreased.
The kinetic studies on compound 22 showed that it is a compet-
itive inhibitor (Ki = 190 0.016 lM) (Fig. 5). The Lineweaver–Burk
plots showed that in the presence of compound 22, the Vmax was
not affected, while the Km increased.
The kinetic studies on compound 30 showed that it is a
non-competitive inhibitor (Ki = 60 0.002
Lineweaver–Burk plots indicated the Km of
matic ring (IC50 = 180.2 1.5
lM). Its relatively lower activity, as
l
compared to compound 1, may be due to the chloride substitu-
tions, incapable of forming hydrogen bond with the amino acid
residues of the enzyme, as compared to nitro group. The kinetics
studies on compound 4 indicated that it is a non-competitive inhi-
bitor (Fig. 4), interacting with the active site or with the allosteric
site of the enzyme. Interestingly, the activity was completely lost
when chloro substitutions were present at ortho positions of the
aromatic ring, as in compounds 5 (having o-dichloro benzyl group),
and 6 (having o-chloro benzyl group). The bromo and methoxy
substituted compounds 7–14, 23–29, and 34 were found to be
inactive.
a
l
a
M) (Fig. 6). The
-glucosidase enzyme
was not affected, while the Vmax of enzyme decreased.
These results indicate that the
a-glucosidase inhibitory activi-
The kinetic studies on compound 33 showed that it is
ties of the dihydropyrano [2,3-c] pyrazole derivatives mainly
depend on the benzyloxy moiety at para, along with OCH3 group
at meta positions.
an uncompetitive inhibitor (Ki = 45 0.0014 lM) (Fig. 7). The
Lineweaver–Burk plots indicated that both Vmax and Km values
were decreased, which shows an uncompetitive type of inhibition.
3.2. Kinetic studies
3.3. Cytotoxicity studies
To deduce the inhibition mechanism of this series, the kinetics
studies were performed on all potent analogs (i.e. 1, 4, 22, 30, and
33), with different concentrations of test compounds and sub-
strates. The type of inhibition was determined by Lineweaver–Burk
Cytotoxicity of all potent compounds (i.e. 1, 4, 22, 30, and 33)
was evaluated by using the rat fibroblast 3T3 cell line [18]. Results
revealed that at 30
lM concentration, all compounds were non-
cytotoxic to the cells (Table 2).

H. Kashtoh et al. / Bioorganic Chemistry 65 (2016) 61–72
67
(A)
(A)
0.8
0.6
0.4
0.2
0
0.1
0
-0.2
-0.4
-0.6
-0.8
-0.1
-8
-6
-4
-2
0
2
4
6
8
1/Substrate (4-Nitrophenyl-α-D-glucopyranoside)
-4
-2
0
2
4
1/Substrate (4-Nitrophenyl-α-D-glucopyranoside)
(B)
(B) 0.08
0.01
0.06
0.04
0.02
0
0
-0.02
-0.04
-0.06
-0.08
-0.01
-200
0
200
Compound 4
-20
-10
0
10
20
Fig. 4. The inhibition of
plot of reciprocal of rate of reaction (velocities) versus reciprocal of substrate in the
absence (N), and in presence of 15 M (h), 250 (d), and 500 M (s) of
compound 4. The figure (B) is the secondary replot of Lineweaver–Burk plot
between the slopes of each line on Lineweaver–Burk plot versus different
concentrations of compound 4.
a-glucosidase by compound 4 (A) is the Lineweaver–Burk
(Compound
)
1
l
lM
l
Fig. 3. The inhibition of
plot of reciprocal of rate of reaction (velocities) versus reciprocal of substrate in the
M (j), and 20 (h) of compound 1.
a-glucosidase by compound 1 (A) is the Lineweaver–Burk
absence (4), and in presence of 15
l
lM
Figure (B) is the secondary replot of Lineweaver–Burk plot between the slopes of
each line on Lineweaver–Burk plot versus different concentrations of compound 1.
precipitates were washed with water to remove the unreacted
pyrazolone to obtain pure products. Melting points were recorded
for crystalline substances.
Table 2
Cytotoxicity evaluation of active and potent compounds.
Compounds
Cytotoxicity (3T3 cell line)
IC50 (lM) SEM
4.1.1. 6-Amino-3-methyl-4-(4-nitrophenyl)-2,4-dihydropyrano[2,3-c]
pyrazole-5-carbonitrile (1)
1
4
22
30
33
>30
>30
>30
>30
>30
Yield: 72%; m.p. 230 °C, 1H NMR (DMSO-d6) d 12.18 (s, 1H, H-2),
8.21 (d, 2H, J3 ;2 =5 ;6 ¼ 8:4 Hz, H-30, H-50), 7.46 (d, 2H, J2 ;3 =6 ;5
¼
0
0
0
0
0
0
0
0
8:4 Hz, H-20, H-60), 7.03 (s, 2H, NH2), 4.81 (s, 1H, H-4), 1.79 (s,
3H, CH3); 13C NMR; (DMSO-d6) d 161.0 (C-6), 154.6 (C-8), 152.0
(C-40), 146.3 (C-10), 135.8 (C-3), 128.8 (C-30, C-50), 123.8 (C-20, C-
60), 120.4 (CN), 96.5 (C-9), 55.9 (C-5), 35.8 (C-4), 9.7 (C-10). HREI-
MS: M+ m/z 297.0644, Calcd 297.0682 (C14H11N5O3), 231 (100%),
175 (90%).
NA: not active; SEM: standard error mean.
4. Experimental
4.1. Synthesis of compounds 1–35
4.1.2. 6-Amino-3-methyl-4-(3-nitrophenyl)-2,4-dihydropyrano[2,3-c]
pyrazole-5-carbonitrile (2)
Dichloromethane (10 mL) was taken in a round-bottomed flask,
into which 1.0 equivalent (1 mmol) of triethylamine and pyra-
zolone were poured. The mixture was stirred for 2 min without
heating. To this mixture, 1.0 equivalent of corresponding pre-
synthesized benzylidene from malononitrile was added. Then the
mixture was agitated for 25–30 min. The reaction was observed
by TLC. The desired products appeared as precipitates. The
Yield: 73%; m.p. 216 °C, 1H NMR (DMSO-d6) d 12.18 (s, 1H, H-2),
8.11 (dt, 1H, J4 ;5 ¼ 6:9 Hz, J4 ;6 =4 ;2 ¼ 2:4 Hz, H-40), 8.01 (t, 1H,
0
0
0
0
0
0
J5 ;4 =5 ;6 ¼ 2:4 Hz, H-50), 7.65 (m, 1H, H-60), 7.02 (s, 2H, –NH2),
4.86 (s, 1H, H-4), 1.79 (s, 3H, CH3); HREI-MS: M+ m/z 297.0850,
Calcd 297.0862 (C14H11N5O3), 231 (95%), 109 (100%), 66 (50%).
0
0
0
0

68
H. Kashtoh et al. / Bioorganic Chemistry 65 (2016) 61–72
0.4
(A)
(A)
0.6
0.4
0.2
0
0.2
0
-0.2
-0.4
-0.6
-0.2
-0.4
-20
0
20
-4
-2
0
2
4
1/Substrate (4-Nitrophenyl-α-D-glucopyranoside)
1/Substrate (4-Nitrophenyl-α-D-glucopyranoside)
0.06
0.04
0.02
0
(B) 0.04
0.02
(B)
0
-0.02
-0.02
-0.04
-0.04
-0.1 -0.08 -0.06 -0.04 -0.02
0
0.02 0.04 0.06 0.08 0.1
Compound 22
-100 -80 -60 -40 -20
0
20 40 60 80 100
Fig. 5. The inhibition of
Lineweaver–Burk plot of reciprocal of rate of reaction (velocities) versus reciprocal
a
-glucosidase enzyme by compound 22 (A) is the
Compound 30
of substrate (p-nitrophenyl-
and in the presence of 62.5
secondary replot of Lineweaver–Burk plot between the slopes of each line on
Lineweaver–Burk plot versus different concentrations of compound 22.
a
l
-D-glucopyranoside) in the absence of inhibitor (N),
M (j), and 250 M (h) of compound 22. (B) is the
Fig. 6. The inhibition of
a
-glucosidase enzyme by compound 30 (A) is the
l
Lineweaver–Burk plot of reciprocal of rate of reaction (velocities) versus reciprocal
of substrate (p-nitrophenyl- -D-glucopyranoside) in the absence of inhibitor (.)
and in the presence of 50 M ( ), 70 M (h) of compound 30. (B) is
a
l
r
lM (4), and 90 l
the secondary replot of Lineweaver–Burk plot between the slopes of each line on
Lineweaver–Burk plot versus different concentrations of compound 30.
4.1.3. 6-Amino-3-methyl-4-(2-nitrophenyl)-2,4-dihydropyrano[2,3-c]
pyrazole-5-carbonitrile (3)
4.1.5. 6-Amino-4-(2,6-dichlorophenyl)-3-methyl-2,4-dihydropyrano
[2,3-c]pyrazole-5-carbonitrile (5)
Yield: 56%; m.p. 220 °C, 1H NMR (DMSO-d6) d 12.18 (s, 1H, H-2),
Yield: 64%; m.p. 245 °C, 1H NMR (DMSO-d6) d 12.08 (s, 1H, H-2),
7. 86 (dd, 1H, J3 ;5 ¼ 7:8 Hz, J3 ;4 ¼ 0:6 Hz, H-30), 7.68 (dt, 1H,
0
0
0
0
7.53 (dd, 1H, J3 ;4 ¼ 8:1 Hz, J3 ;5 ¼ 1:5 Hz, H-30), 7.39 (m, 2H, H-40,
H-50), 6.98 (s, 2H, NH2), 5.56 (s, 1H, H-4), 1.76 (s, 3H, Me); 13C
NMR (DMSO-d6) d 162.0 (C-6), 155.4 (C-8), 135.8 (C-20), 135.1 (C-
60), 134.8 (C-10), 134.4 (C-3), 130.8 (C-30), 129.5 (C-40), 128.4 (C-
50), 120.0 (CN), 94.7 (C-9), 52.8 (C-5), 32.4 (C-4), 9.2 (C-10). HREI-
MS: M+ m/z 320.0229, Calcd 320.0232 (C14H10Cl2N4O), [M+2]+
322 (50%), 219 (90%), 175 (100%), 126 (15%), 99 (10%), 66 (15%).
0
0
0
0
J4 ;5 ¼ 7:8 Hz, J4 ;3 ¼ 0:6 Hz, H-40), 7.50 (dt, 1H, J5 ;6 ¼ 7:2 Hz,
0
0
0
0
0
0
J5 ;4 = ¼ 0:9 Hz, H-50), 7.32 (dd, 1H, J6 ;4 ¼ 7:8 Hz, J6 ;5 ¼ 0:9 Hz, H-
60), 7.00 (s, 2H, NH2), 5.08 (s, 1H, H-4), 1.76 (s, 3H, CH3); 13C
NMR (DMSO-d6) d 161.1 (C-6), 154.9 (C-8), 149.1 (C-20), 137.5 (C-
10), 134.5 (C-3), 133.6 (C-40), 131.2 (C-60), 128.3 (C-50), 123.5 (C-
30), 120.2 (CN), 96.3 (C-9), 55.9 (C-5), 31.3 (C-4), 9.4 (C-10). HREI-
MS: M+ m/z 297.0857, Calcd 297.0862 (C14H11N5O3), 280 (100%),
249 (75%), 185 (50%).
0
0
0
0
0
0
4.1.6. 6-Amino-4-(2-chlorophenyl)-3-methyl-2,4-dihydropyrano[2,3-
c]pyrazole-5-carbonitrile (6)
Yield: 56%; m.p. 235 °C, 1H NMR (DMSO-d6) d 12.10 (s, 1H, H-2),
4.1.4. 6-Amino-4-(2,4-dichlorophenyl)-3-methyl-2,4-dihydropyrano
[2,3-c]pyrazole-5-carbonitrile (4)
7.42 (dd, 1H, J3 ;5 ¼ 7:5 Hz, J3 ;4 ¼ 1:5 Hz, H-30), 7.28 (dq, 2H,
0
0
0
0
Yield: 55%; m.p. 195 °C, 1H NMR (DMSO-d6) d 12.14 (s, 1H, H-2),
J5 ;4 ¼ 7:5 Hz, J4 ;5 ¼ 1:5 Hz, H-40, H-50), 7.18 (dd, 1H,
0
0
0
0
7.57 (d, 1H, J3 ;5 ¼ 2:1 Hz, H-30), 7.41 (dd, 1H, J5 ;6 ¼ 8:4 Hz,
J6 ;5 ¼ 7:2 Hz, J6 ;4 ¼ 1:5 Hz, H-60), 6.91 (s, 2H, NH2), 5.05 (s, 1H,
H-4), 1.75 (s, 3H, Me); 13C NMR (DMSO-d6) d 161.2 (C-6), 154.9
(C-8), 140.9 (C-20), 135.3 (C-3), 131.9 (C-10), 130.7 (C-30), 129.4
(C-40), 128.6 (C-50), 127.7 (C-60), 120.4 (CN), 96.8 (C-9), 55.6 (C-
5), 33.4 (C-4), 9.5 (C-10). HREI-MS: M+ m/z 286.0622, Calcd
0
0
0
0
0
0
0
0
J5 ;3 ¼ 2:1 Hz, H-50), 7.22 (d, 1H, J6 ;5 ¼ 8:4 Hz, H-60), 6.97 (s, 2H,
NH2), 5.05 (s, 1H, H-4), 1.77 (s, 3H, Me); HREI-MS: M+ m/z
320.0222, Calcd 320.0232 (C14H10Cl2N4O) [M+2]+ 322 (35%), 280
(100%), 249 (75%), 185 (50%).
0
0
0
0

H. Kashtoh et al. / Bioorganic Chemistry 65 (2016) 61–72
69
7.00 (d, 1H, J5 ;4 ¼ 8:7 Hz, H-50), 6. 88 (s, 2H, NH2), 4.94 (s, 1H, H-
4), 3.77 (s, 3H, OMe), 1.80 (s, 3H, Me); HREI-MS: M+ m/z
360.0218, Calcd 360.0222 (C15H13BrN4O2), [M+2]+ 362 (34%), 363
281 (55%), 262 (100%), 175 (98%). 109 (17%).
0
0
0.08
0.06
0.04
0.02
0
(A)
4.1.9. 6-Amino-4-(5-bromo-2-methoxyphenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (9)
Yield: 68%; m.p. 235 °C, 1H NMR (DMSO-d6) d 11.34 (s, 1H, H-2),
7.42 (m, 2H, H-20, H-60), 7.30 (m, 2H, H-40, H-50), 6.16 (s, 2H, NH2),
4.68 (s, 1H, H-4), 1.92 (s, 3H, Me); HREI-MS: M+ m/z 330.0121,
Calcd 330.0116 (C14H11BrN4O), [M+2]+ 330 (35%) 264 (37%), 175
(100%), 128 (29%), 109 (60%), 66 (25%).
-0.02
-0.04
-0.06
-0.08
4.1.10. 6-Amino-4-(4-bromo-2,6-dimethoxyphenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (10)
Yield: 79%; m.p. 220 °C, 1H NMR (DMSO-d6) d 12.02 (s, 1H, H-2),
7.22 (s, 1H, H-30), 6.83 (s, 2H, NH2), 6.74 (s, 1H, H-50), 4.89 (s, 1H, H-
4), 3.71 (s, 3H, OMe), 3.68 (s, 3H, OMe), 1.80 (s, 3H, Me); HREI-MS:
M+ m/z 390.0316, Calcd 390.0328 (C16H15BrN4O3), [M+2]+ 392
(35%), 359 (32%), 327 (41%), 214 (100%).
-4
-2
0
2
4
1/Substra te (4-Nitrophenyl- α-D-glucopyranoside)
4.1.11. 6-Amino-4-(3-bromo-4-methoxyphenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (11)
0.06
0.04
0.02
0
(B)
Yield: 79%; m.p. 215 °C, 1H NMR (DMSO-d6) d 12.12 (s, 1H, H-2),
7.31 (d, 1H, J6 ;5 ¼2.1 Hz, H-60), 7.17 (dd, 1H, J2 ;6 ¼8.4 Hz,
0
0
0
0
J2 ;5 ¼2.1 Hz, H-20), 7.07 (d, 1H, J5 ;6 ¼8.4 Hz, H-50), 6.89 (s, 2H,
NH2), 4.58 (s, 1H, H-4), 3.82 (s, 3H, OMe), 1.80 (s, 3H, Me); 13C
NMR (DMSO-d6) d 162.7 (C-6), 154.6 (C-30), 154.0 (C-8), 138.2 (C-
10), 135.6 (C-3), 131.5 (C-20), 127.9 (C-60), 120.6 (CN), 112.5 (C-
50), 110.3 (C-40), 97.3 (C-9), 57.0 (C-5), 56.1 (OMe), 34.9 (C-4), 9.7
(C-10). HREI-MS: M+ m/z 360.0234, Calcd 360.0222 (C15H13BrN4-
O2), [M+2]+ 362 (14.5%), 294 (85%), 175 (100%), 115 (18%), 66
(67%).
0
0
0
0
-0.02
-0.04
4.1.12. 6-Amino-4-(2,6-dimethoxyphenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (12)
Yield: 77%; m.p. 222 °C, 1H NMR (DMSO-d6) d 11.99 (s, 1H, H-2),
-200
0
200
7.17 (t, 1H, J3 ;4 ¼8.1 Hz, H-30), 6.5 (m, 2H, H-40, H-50) 6.78 (s, 2H,
NH2), 4.89 (s, 1H, H-4), 3.71 (s, 3H, OMe), 3.63 (s, 3H, OMe), 1.79
(s, 3H, Me); HREI-MS: M+ m/z 312.1231, Calcd 312.1222
(C16H16N4O3), 297 (29%), 281 (47%), 214 (100%). 175 (62%).
0
0
Compound 33
Fig. 7. The inhibition of
Lineweaver–Burk plot of reciprocal of rate of reaction (velocities) versus reciprocal
of substrate (p-nitrophenyl- -D-glucopyranoside) in the absence of inhibitor (
and in the presence of 31.25 M (j), and 125 M (h) of compound 33.
(B) is the secondary replot of Lineweaver–Burk plot between the slopes of each line
on Lineweaver–Burk plot versus different concentrations of compound 33.
a-glucosidase enzyme by compound 33 (A) is the
a
r)
lM (4), 62.5
l
l
4.1.13. 6-Amino-4-(4-ethoxy-3-methoxyphenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (13)
Yield: 74%; m.p: 237 °C, 1H NMR (DMSO-d6) d 12.04 (s, 1H, H-2),
6.85 (d, 1H, J6 ;5 ¼8.1 Hz, H-50), 6.80 (s, 2H, NH2), 6.74 (br. s, 1H, H-
0
0
286.0621 (C14H11ClN4O), [M+2]+ 288 (46%), 175 (100%), 128 (60%),
66 (25%).
20), 6. 65 (br. d, 1H, J6 ;5 =6 ;2 ¼ 8:1 Hz, H-60), 4.53 (s, 1H, H-4), 3.97
0
0
0
0
00;200
(q, 2H, J1
1.32 (t, 3H, J2
¼ 6:6 Hz, CH2), 3.69 (s, 3H, OMe), 1.81 (s, 3H, Me),
¼ 6:6 Hz, Me); HREI-MS: M+ m/z 326.1382, Calcd
00;100
4.1.7. 6-Amino-4-(2-bromophenyl)-3-methyl-2,4-dihydropyrano[2,3-
c]pyrazole-5-carbonitrile (7)
326.1379 (C17H18N4O3), 281 (90%), 250 (80%), 162 (15%).
Yield: 77%; m.p. 240 °C, 1H NMR (DMSO-d6) d 12.12 (s, 1H, H-2),
4.1.14. 6-Amino-4-(3,4-dimethoxyphenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (14)
7.59 (d, 1H, J3 ;4 ¼ 7:8 Hz, H-30), 7.36 (t, 1H, J4 ;3 =4 ;5 ¼ 7:5 Hz, H-40),
7.19 (m, 2H, H-50, H-60), 6.94 (s, 2H, NH2), 5.05 (s, 1H, H-4), 1.75 (s,
3H, Me); 13C NMR (DMSO-d6) d 161.1 (C-6), 154.8 (C-8), 142.5 (C-
20), 135.3 (C-3), 132.5 (C-30), 130.8 (C-40), 128.8 (C-50), 128.3 (C-60),
122.3 (C-10), 120.2 (CN), 96.9 (C-9), 55.9 (C-5), 35.8 (C-4), 9.6 (C-
10). HREI-MS: M+ m/z 330.0124, Calcd 330.0116 (C14H11BrN4O),
[M+2]+ 334 (25%), 185 (100%), 175 (80%), 128 (80%), 109 (15%),
66 (20%).
0
0
0
0
0
0
Yield: 65%; m.p. 231 °C, 1H NMR (DMSO-d6) d 12.05 (s, 1H, H-2),
6.89 (br. d, 1H, J6 ;5 ¼8.1 Hz, H-60), 6.80 (s, 2H, NH2), 6.74 (s, 1H, H-
0
0
20), 6.69 (d, 1H, J5 ;6 ¼8.1 Hz, H-50), 4. 53 (s, 1H, H-4), 3.71 (s, 3H,
OMe), 3.68 (s, 3H, OMe), 1.81 (s, 3H, Me); HREI-MS: M+ m/z
312.1220, Calcd 312.1222 (C16H16N4O3), 246 (42%), 215 (10%),
189 (8.6%), 175 (16%).
0
0
4.1.15. 6-Amino-3-methyl-4-phenyl-2,4-dihydropyrano[2,3-c]
pyrazole-5-carbonitrile (15)
4.1.8. 6-Amino-4-(2-bromo-6-methoxyphenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (8)
Yield: 57%; m.p. 229 °C, 1H NMR (DMSO-d6) d 12.07 (s, 1H, H-2),
7.33 (m, 2H, H-30, H-40), 7.23 (m, 3H, H-20, H-50, H-60), 6.84 (s, 2H,
NH2), 4. 58 (s, 1H, H-4), 1.77 (s, 3H, Me); 13C NMR (DMSO-d6) d
Yield: 71%; m.p. 245 °C, 1H NMR (DMSO-d6) d 12.06 (s, 1H, H-2),
7.38 (dd, 1H, J4 ;2 ¼ 8:7 Hz, J4 ;6 ¼ 2:4 Hz, H-40), 7.06 (s, 1H, H-50),
0
0
0
0

70
H. Kashtoh et al. / Bioorganic Chemistry 65 (2016) 61–72
160.8 (C-6), 154.7 (C-8), 144.4 (C-10), 135.5 (C-3), 128.4 (C-20, C-60),
127.4 (C-30, C-50), 126.7 (C-40), 120.7 (CN), 97.6 (C-9), 57.1 (C-5),
36.2 (C-4), 9.7 (C-10). HREI-MS: M+ m/z 252.1010, Calcd
252.1011 (C14H12N4O), 185 (80%), 175 (100%), 109 (40%), 66 (20%).
4.1.22. 6-Amino-4-(3-(benzyloxy)-4-hydroxyphenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (22)
Yield: 68%; m.p. 113 °C, 1H NMR (DMSO-d6) d 11. 98 (s, 1H, H-2),
7.33 (m, 4H, H-100, H-200, H-500, H-50), 7.17 (t, 1H, J4
¼6.0 Hz,
00;300=400;500
H-400), 7.07 (d, 1H, J2 ;3 ¼ 9:0 Hz, H-20), 7.01 (dd, 1H, J3 ;2 ¼ 6:3 Hz,
0
0
0
0
J3 ;4 ¼1.2 Hz, H-30), 6.91 (t, 1H, J3
¼ 7:5 Hz, H-300), 6.75 (s,
0
0
00;200=300;400
4.1.16. 6-Amino-3-methyl-4-(naphthalen-1-yl)-2,4-dihydropyrano
[2,3-c]pyrazole-5-carbonitrile (16)
2H, NH2), 5.12 (d, 2H, JCH2,CH3 = 2.7 Hz, OCH2), 5.03 (s, 1H, H-4),
1.73 (s, 3H, Me); HREI-MS: M+ m/z 374.1371, Calcd 374.1379
(C21H18N4O3), 292 (10%), 267 (62%), 201 (5%), 175 (4%), 91 (100%).
Yield: 64%; m.p. 218 °C, 1H NMR (DMSO-d6) d 12.04 (s, 1H, H-2),
8.18 (s, 1H, H-20), 7.954 (m, 1H, H-60), 7.84 (d, 1H, J7 ;8 ¼ 8:1 Hz, H-
70), 7.49 (m, 2H, H-40, H-50), 7.36 (s, 1H, H-80), 6.87 (s, 2H, NH2), 5.41
(s, 1H, H-4), 1.53 (s, 3H, Me); HREI-MS: M+ m/z 302.1165, Calcd
302.1168 (C18H14N4O), 206 (37%), 162 (40%), 115 (29%), 109
(50%), 66 (35%).
0
0
4.1.23. 6-Amino-4-(3-bromo-4-methoxyphenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (23)
Yield: 53%; m.p. 101 °C, 1H NMR (DMSO-d6) d 12.10 (s, 1H, H-2),
7.29 (d, 1H, J2 ;6 ¼2.1 Hz, H-20), 7.16 (dd, 1H, J5 ;6 ¼8.7 Hz,
0
0
0
0
J5 ;2 ¼2.1 Hz, H-50), 7.06 (d, 1H, J6 ;5 ¼8.7 Hz, H-60), 6.86 (s, 2H,
NH2), 4.57 (s, 1H, H-4), 3.81 (s, 3H, OMe), 1.79 (s, 3H, Me); HREI-
MS: M+ m/z 360.0245, Calcd 360.0222 (C15H13BrN4O2), [M+2]+
362 (34%), 296 (32%), 237 (9%), 175 (100%), 109 (34%).
0
0
0
0
4.1.17. 6-Amino-3-methyl-4-(4-(methylthio)phenyl)-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (17)
Yield: 72%; m.p. 228 °C, 1H NMR (DMSO-d6) d 12.08 (s, 1H, H-2),
7.21 (d, 2H, J2 ;3 =6 ;5 ¼8.1 Hz, H-20, H-60), 7.11 (d, 2H, J3 ;4 =5 ;6
¼
0
0
0
0
0
0
0
0
8.1 Hz, H-30, H-50), 6.84 (s, 2H, NH2), 4.55 (s, 1H, H-4), 2.44 (s,
3H, SCH3), 1.78 (s, 3H, Me); HREI-MS: M+ m/z 298.0894, Calcd
298.0888 (C15H14N4OS), 232 (100%), 185 (27%), 175 (66%). 109
(34%).
4.1.24. 6-Amino-4-(3-chloro-4,5-dimethoxyphenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (24)
Yield: 50%; m.p. 98 °C, 1H NMR (DMSO-d6) d 12.07 (s, 1H, H-2),
7.03 (d, 1H, J6 ;2 ¼8.7 Hz, H-60), 6.87 (br. s, 3H, H-50, NH2), 4.97 (s,
1H, H-4), 3.80 (s, 3H, OMe), 3.72 (s, 3H, OMe), 1.76 (s, 3H, Me);
HREI-MS: M+ m/z 346.0811, Calcd 346.0833 (C16H15ClN4O3), [M
+2]+ 348 (24%), 223 (13%), 175 (100%), 66 (16%).
0
0
4.1.18. 6-Amino-3-methyl-4-(5-methylfuran-2-yl)-2,4-dihydropyrano
[2,3-c]pyrazole-5-carbonitrile (18)
Yield: 76%; m.p. 197 °C, 1H NMR (DMSO-d6) d 12.11 (s, 1H, H-2),
6.89 (s, 2H, NH2), 6.02 (d, 1H, J5 ;4 ¼3.0 Hz, H-50), 5.95 (d, 1H,
0
0
4.1.25. 6-Amino-4-(4-chloro-2,3-dimethoxyphenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (25)
J4 ;5 ¼ 3:0 Hz, H-40), 4.68 (s, 1H, H-3), 2.17 (s, 3H, CH3-30), 1.98 (s,
0
0
13
3H, Me); C NMR (DMSO-d6) d 161.4 (C-6), 154.7 (C-10), 153.8
Yield: 52%; m.p. 118 °C, 1H NMR (DMSO-d6) d 12.06 (s, 1H, H-2),
(C-40), 150.6 (C-8), 135.7 (C-20), 120.5 (CN), 106.1 (C-20), 106.1 (C-
10), 95.2 (C-9), 54.18 (C-5), 29.8 (C-4), 13.3 (C-60), 9.5 (C-10).
HREI-MS: M+ m/z 256.0949, Calcd 256.0960 (C13H12N4O2), 214
(70%), 190 (100%), 133 (80%).
7.03 (d, 1H, J6 ;2 ¼2.0 Hz, H-20), 6.89 (br. s, 1H, H-60), 6.86 (s, 2H,
0
0
NH2), 4.97 (s, 1H, H-4), 3.80 (s, 3H, OMe), 3.72 (s, 3H, OMe), 1.76
(s, 3H, Me); HREI-MS: M+ m/z 346.0844, Calcd 346.0833 (C16H15
-
ClN4O3), [M+2]+ 348 (35%), 245 (67%), 201 (11%), 175 (100%), 83
(61%).
4.1.19. 6-Amino-4-(4-hydroxyphenyl)-3-methyl-2,4-dihydropyrano
[2,3-c]pyrazole-5-carbonitrile (19)
4.1.26. 4-(6-Amino-5-cyano-3-methyl-2,4-dihydropyrano[2,3-c]
pyrazol-4-yl)-2-methoxyphenyl acetate (26)
Yield: 71%; m.p. 234 °C, 1H NMR (DMSO-d6) d 12.02 (s, 1H, H-2),
0
0
0
0
Yield: 57%; m.p. 84 °C, 1H NMR (DMSO-d6) d 12.10 (s, 1H, H-2),
9.25 (s, 1H, H-40-OH), 6.95 (d, 2H, J2 ;3 =6 ;5 ¼8.4 Hz, H-20, H-60), 6.76
7.07 (d, 1H, J6 ;5 ¼7.8 Hz, H-60), 6.94 (d, 1H, J2 ;6 ¼1.2 Hz, H-20), 6.88
(s, 2H, NH2), 6.68 (d, 2H, J3 ;5 =3 ;4 ¼8.4 Hz, H-30, 50), 4.45 (s, 1H, H-4),
0
0
0
0
0
0
0
0
(s, 2H, NH2), 6.71 (dd, 1H, J5 ;3 ¼1.2 Hz, J5 ;6 ¼7.8 Hz, H-50), 4.62 (s,
1H, H-4), 3.71 (s, 3H, OMe), 2.22 (s, 3H, COCH3), 1.84 (s, 3H, Me);
HREI-MS: M+ m/z 340.1179, Calcd 340.1172 (C17H16N4O4), 298
(77%), 232 (78%), 175 (100%), 83 (39%).
13
1.77 (s, 3H, Me); C NMR (DMSO-d6) d 160.5 (C-6), 155.9 (C-40),
0
0
0
0
154.7 (C-8), 135.4 (C-10), 134.7 (C-3), 128.3 (C-20, C-60), 120.8
(CN), 115.0 (C-30, C-50), 98.0 (C-3), 57.7 (C-5), 35.4 (C-4), 9.7 (C-
10). HREI-MS: M+ m/z 268.086, Calcd 268.0960 (C14H12N4O2), 202
(85%), 109 (65%), 66 (100%).
4.1.27. 6-Amino-4-(4-bromo-2,5-dimethoxyphenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (27)
4.1.20. 6-Amino-4-(2,5-dimethoxyphenyl)-3-methyl-2,4-dihydropyrano
[2,3-c]pyrazole-5-carbonitrile (20)
Yield: 59%; m.p. 106 °C, 1H NMR (DMSO-d6) d 12.08 (s, 1H, H-2),
7.07 (s, 1H, H-30), 6.87 (s, 2H, NH2), 6.61 (s, 1H, H-60), 4.95 (s, 1H, H-
4), 3.76 (s, 3H, OMe), 3.62 (s, 3H, OMe), 1.77 (s, 3H, Me); HREI-MS:
M+ m/z 390.0328, Calcd 390.0328 (C16H15BrN4O3), [M+2]+ 392
(27%), 364 (7%), 245 (95%), 216 (27%), 175 (100%).
Yield: 63%; m.p. 104 °C, 1H NMR (DMSO-d6) d 11.99 (s, 1H, H-2),
6.94 (d, 1H, J3 ;4 ¼9.0 Hz, H-30), 6.79 (s, 2H, NH2), 6.75 (dd, 1H,
0
0
J4 ;3 ¼ 9:0 Hz, J4 ;6 ¼ 2:7 Hz, H-40), 6.49 (d, 1H, J6 ;4 ¼ 2:7 Hz, H-
60), 4.89 (s, 1H, H-4), 3.70 (s, 3H, OMe), 3.63 (s, 3H, OMe), 1.79 (s,
3H, Me); HREI-MS: M+ m/z 312.1222, Calcd 312.1222
(C16H16N4O3), 297 (44%), 281 (70%), 215 (100%).
0
0
0
0
0
0
4.1.28. 6-Amino-4-(4-bromo-3,5-dimethoxyphenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (28)
Yield: 57%; m.p. 102 °C, 1H NMR (DMSO-d6) d 12.10 (s, 1H, H-2),
6.90 (s, 2H, H-20, H-50), 6.54 (s, 2H, NH2), 4.65 (s, 1H, H-3), 3.76 (s,
6H, 2 OMe), 1.85 (s, 3H, Me); 13C NMR; (DMSO-d6) d 161.0 (C-6),
156.4 (C-10), 154.6 (C-8), 145.6 (C-10), 135.8 (C-3), 120.6 (CN),
104.1 (C-20, C-60), 97.9 (C-30, C-50), 96.7 (C-9), 56.4 (C-5), 56.2
(2 ꢀ OMe), 36.5 (C-4), 9.9 (C-10). HREI-MS: M+ m/z 390.0319, Calcd
390.0328 (C16H15BrN4O3), [M+2]+ 392 (32%), 324 (9%), 175 (100%),
109 (6%).
4.1.21. 6-Amino-4-(2,6-dimethoxyphenyl)-3-methyl-2,4-dihydropyrano
[2,3-c]pyrazole-5-carbonitrile (21)
Yield: 58%; m.p. 97 °C, 1H NMR (DMSO-d6) d 11.74 (s, 1H, H-2),
7.17 (t, 1H, J4 ;5 ;3 ¼ 8:1 Hz, H-40), 6.57 (m, 4H, NH2, H-30, H-50), 5.11
(s, 1H, H-4), 3.81 (s, 3H, OMe), 3.50 (s, 3H, OMe), 1.76 (s, 3H, Me);
HREI-MS: M+ m/z 312.1227, Calcd 312.1222 (C16H16N4O3), 297
(32%), 281 (100%), 175 (52.8%).
0
0
0

H. Kashtoh et al. / Bioorganic Chemistry 65 (2016) 61–72
71
4.1.29. 6-Amino-4-(2-chloro-3-methoxyphenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (29)
4.2. In-vitro yeast
a-glucosidase inhibition assay
Yield: 58%; m.p. 119 °C, 1H NMR (DMSO-d6) d 12.09 (s, 1H, H-2),
a-Glucosidase inhibitory activity was evaluated at 37 °C, by using
7.27 (t, 1H, J5 ;4 =5 ;6 ¼ 7:8 Hz, H-50), 7.00 (d, 1H, J6 ;5 ¼7.8 Hz, H-60),
0.1 M phosphate buffer (pH6.8) [28]. The enzyme (EC 3.2.1.20, Saccha-
romyces cerevisiae purchased from sigma; catalog # G5003) (0.2 U/
mL) was incubated with test compounds and phosphate
buffer at 37 °C for 15 min. After 15 min of incubation, 0.7 mM of
0
0
0
0
0
0
6.90 (s, 2H, NH2), 6.72 (d, 1H, J4 ;5 ¼ 7:8 Hz, H-40), 3.84 (s, 3H,
OMe), 1.75 (s, 3H, Me); HREI-MS: M+ m/z 316.0720, Calcd
316.0727 (C15H13ClN4O2), [M+2]+ 318 (34%), 290 (7.5%), 215
(58.2%), 175 (100%).
0
0
substrate
i.e.
p-nitrophenyl-
a
-D-glucopyranoside
(Sigma;
catalog # N1377) was added and the change in absorbance was
recorded at 400 nm for 30 min by using spectrophotometer
(SpectraMax M2, Molecular Devices, CA, USA). Test compound was
replaced with DMSO in control experiments (7.5% final concentra-
tion). Acarbose was used as the standard inhibitor. The percent inhibi-
tion of each compound was calculated by using the following formula:
4.1.30. 6-Amino-4-(4-(benzyloxy)-3-methoxyphenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (30)
Yield: 53%; m.p. 135 °C, 1H NMR (DMSO-d6) d 12.05 (s, 1H, H-2),
7.42 (m, 5H, 200, 300, 400, 500, 600), 6.99 (d, 1H, J5 ;6 ¼8.4 Hz, H-50), 6.80
0
0
(s, 2H, NH2), 6.77 (d, 1H, J2 ;6 ¼1.5 Hz, H-20), 6.66 (dd, 1H,
0
0
% Inhibition ¼ 100 ꢁ ðOD test well=OD controlÞ ꢀ 100
J6 ;5 ¼8.4 Hz, J6 ;2 ¼1.5, H-60), 5.16 (s, 2H, OCH2), 3.84 (s, 3H,
OMe), 1.75 (s, 3H, Me); HREI-MS: M+ m/z 388.1591, Calcd
388.1535 (C22H20N4O3), 290 (7.5%), 215 (58.2%), 175 (100%).
0
0
0
0
4.2.1. Mechanistic studies
Mechanistic studies were carried out to determine the mecha-
nism of inhibition and ligand binding to the enzyme. The enzyme
solution (0.2 U/mL) was incubated with different concentrations
of inhibitor for 15 min at 30 °C. The reaction was then initiated
by adding different concentrations of substrate (0.1–0.8 mM),
and change in absorbance was measured for 30 min at 400 nm
on microtitre plate reader (Spectra Max M2, Molecular Devices,
CA, USA).
4.1.31. 6-Amino-3-methyl-4-(4-(trifluoromethyl)phenyl)-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (31)
Yield: 51%; m.p. 83 °C, 1H NMR, (DMSO-d6) d 12.15 (s, 1H, H-2),
7.70 (d, 2H, J3 ;2 =5 ;6 ¼ 7:8 Hz, H-30, H-50), 7.40 (d, 2H,
0
0
0
0
J2 ;3 =6 ;5 ¼7.8 Hz, H-20, H-60), 6.96 (s, 2H, NH2), 4.73 (s, 1H, H-2),
0
0
0
0
1.78 (s, 3H, Me); HREI-MS: M+ m/z 320.0885, Calcd 320.0885 (C15
-
H11F3N4O), 253 (11%), 185 (9%), 175 (100%).
4.3. Cytotoxicity evaluation of compounds on rat fibroblast 3T3 cell
lines
4.1.32. 6-Amino-4-(4-(benzyloxy)phenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (32
)
Yield: 62%; m.p. 94 °C, 1H NMR (DMSO-d6) d 12.06 (s, 1H, H-2),
The experiment was performed according to the method
described by Dimas et al. by using rat fibroblast 3T3 cells [27].
Briefly the 3T3-adherent cells (2 ꢀ 105 cells/mL) were cultured
overnight in a CO2 environment at 37 °C in a 96-well plate. After
7.43 (m, 5H, H-200, H-300, H-400, H-500, H-20), 7.25 (t, 1H,
J4
¼7.8 Hz, H-400), 6.88 (m, 1H, H-60), 6.84 (s, 2H, NH2),
00;300=400;500
6.77 (br. s, 2H, H-50, H-60), 5.04 (s, 2H, OCH2), 4.55 (s, 1H, H-4),
1.75 (s, 3H, Me); HREI-MS: M+ m/z 358.1437, Calcd 358.1430
(C21H18N4O2), (1.4%), 292 (3%), 201 (5%), 175 (6%).
24 h, supernatant was removed, and 50
(100–12.5 g/mL) along with 150 L of DMEM medium (supple-
mented with 5% v/v) fetal bovine serum, penicillin (100 units/mL)
and streptomycin (100 g/mL) were added to each well and fur-
ther incubated for 24 h. After completion of incubation, the med-
ium was aspirated carefully, and 50 L of MTT solution (3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) (2 mg/
mL in PBS) was added to each well and the plate was further incu-
bated for 4 h. After 4 h of incubation, MTT solution was aspirated,
lL of the test compounds
l
l
l
4.1.33. 6-Amino-4-(2-(benzyloxy)phenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (33)
l
Yield: 53%; m.p. 118 °C, 1H NMR (DMSO-d6) d 11.99 (s, 1H, H-2),
7.40 (m, 5H, H-200, H-300, H-400, H-500, H-600), 7.16 (t, 1H,
J3 ;4 =3 ;2 ¼7.2 Hz, H-30), 7.07 (br. d, 1H, J5 ;4 ¼8.1 Hz, H-50), 7.01
0
0
0
0
0
0
(br. d, 1H, J6 ;5 ¼7.2 Hz, H-60), 6.91 (t, 1H, J4 ;3 =4 ;5 ¼7.2 Hz, H-40),
6.77 (s, 2H, NH2), 5.12(s, 2H, OCH2), 5.02 (s, 1H, H-4), 1.72 (s, 3H,
CH3); HREI-MS: M+ m/z 358.1407 Calcd 358.1430 (C21H18N4O2),
292 (3%), 201 (5%), 175 (6%).
0
0
0
0
0
0
and the washing of the cells was carried out with PBS. 100 lL
DMSO was added to each well in order to dissolve the blue insol-
uble MTT formazan. 96-Well plate was then agitated for 15 min
at room temperature, and finally the absorbance was recorded at
540 nm by using microplate readers (SpectraMax Plus-384). The
percent viability was calculated as the relative ratio of absor-
bance/optical densities.
4.1.34. 6-Amino-4-(2-chloro-3,4-dimethoxyphenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (34)
Yield: 53%; m.p. 114 °C, 1H NMR (DMSO-d6) d 12.06 (s, 1H, H-2),
4.4. Statistical analysis
7.03 (d, 1H, J6 ;5 ¼8.7 Hz, H-60), 6.89 (d, 1H, J5 ;4 ¼8.7 Hz, H-50), 6.87
0
0
0
0
(s, 2H, NH2), 4. 97 (s, 1H, H-4), 3.80 (s, 3H, OMe), 3.72 (s, 3H, OMe),
The EZ-Fit Enzyme Kinetics program (Perrella Scientific Inc.,
Amherst, New Hampshire, USA) was employed to calculate the IC50
values. All graphs were plotted by using GraFit program (Version
7.0), (2010). Values of the correlation coefficients, intercepts, slopes,
and their standard errors were calculated by the linear regression
analysis by using the same program. Each point in the constructed
graphs represents the mean of the three experiments [29].
1.76 (s, 3H, Me); HREI-MS: M+ m/z 346.0851, Calcd 346.0833, (C16
-
H
15ClN4O3), [M+2]+ 348 (3%), 245 (91%), 230 (10%), 201 (23%), 175
(69%), 115 (13%).
4.1.35. 6-Amino-4-(2-hydroxy-6-nitrophenyl)-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (35)
Yield: 64%; m.p. 107 °C, 1H NMR (DMSO-d6) d 11.16 (s, 1H, H-2),
9.29 (s, 1H, OH), 8.68 (s, 1H, H-60), 8.15 (d, 1H, J4 ;2 ¼2.1 Hz, H-40),
0
0
5. Conclusion
8.02 (d, 1H, J2 ;4 ¼2.1 Hz, H-20), 7.38 (s, 2H, NH2), 5.19 (s, 1H, H-3),
2.07 (s, 3H, Me); HREI-MS: M+ m/z 313.0821, Calcd 313.0811
(C14H11N5O4), 213 (11%), 185 (9%), 175 (100%).
0
0
Compounds 1, 2, 4, 19, 22, 30, and 33 were identified as potent
inhibitors of the yeast a-glucosidase enzyme in vitro. The kinetics

72
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Acknowledgments
This work was supported by the Higher Education Commission
(HEC), under the National Research Program for Universities
(Pakistan, Project No. 20-1910). The authors are grateful to the
King Abdulaziz University, Jeddah, Saudi Arabia, for supporting
the study visit of one of the authors (Hamdy Kashtoh) to the Inter-
national Center for Chemical and Biological Sciences, University of
Karachi, Karachi-75270, Pakistan.
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