Synthesis, in vitro biological activities and in silico study of dihydropyrimidines derivatives

Article abstract of DOI:10.1016/j.bmc.2015.09.001

We describe here the synthesis of dihydropyrimidines derivatives 3a-p, and evaluation of their α-glucosidase enzyme inhibition activities. Compounds 3b (IC₅₀ = 62.4 ± 1.5 μM), 3c (IC₅₀ = 25.3 ± 1.26 μM), 3d (IC₅₀ = 12.4 ±

Full text of DOI:10.1016/j.bmc.2015.09.001

Accepted Manuscript
Synthesis, In vitro Biological Activities and In Silico Study of Dihydropyrimi-
dines Derivatives
Assem Barakat, Mohammad Shahidul Islam, Abdullah Mohammed Al-Majid,
Hazem A. Ghabbour, Hoong-Kun Fun, Kulsoom Javed, Rehan Imad, Sammer
Yousuf, M. Iqbal Choudhary, Abdul Wadood
PII:
S0968-0896(15)30026-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.09.001
BMC 12548
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 May 2015
29 August 2015
1 September 2015
Please cite this article as: Barakat, A., Islam, M.S., Al-Majid, A.M., Ghabbour, H.A., Fun, H-K., Javed, K., Imad,
R., Yousuf, S., Iqbal Choudhary, M., Wadood, A., Synthesis, In vitro Biological Activities and In Silico Study of
Dihydropyrimidines Derivatives, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.
2015.09.001
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Synthesis, In vitro Biological Activities and In Silico Study of
Dihydropyrimidines Derivatives
Assem Barakat 1,2,*,†, Mohammad Shahidul Islam1,*†, Abdullah Mohammed
Al-Majid1,†, Hazem A. Ghabbour3,†, Hoong-Kun Fun3,4,†, Kulsoom Javed5, Rehan
Imad6, Sammer Yousuf5, M. Iqbal Choudhary1,5 and Abdul Wadood7
1
2
Department of Chemistry, College of Science, King Saud University, P. O. Box
2455, Riyadh-11451, Saudi Arabia.
Department of Chemistry, Faculty of Science, Alexandria University, P. O. Box 426,
Ibrahimia, 21321 Alexandria, Egypt.
3
4
5
6
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud
University, P. O. Box 2457, Riyadh 11451, Saudi Arabia.
X-Ray Crystallography Unit, School of Physics, Universiti Sains Malaysia, Penang,
11800 Malaysia.
H.E.J. Research Institute of Chemistry, International Center for Chemical and
Biological Sciences, University of Karachi, Karachi-75270, Pakistan.
Dr. Panjwani Center for Molecular Medicine and Drug Research, International
Center for Chemical and Biological Sciences, University of Karachi,
Karachi-75270, Pakistan.
7
Department of Biochemistry, Abdul Wali Khan University, Mardan-23200, Pakistan
† These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: ambarakat@ksu.edu.sa
(A.B.); shahid.10amui@gmail.com (M.S.I); Tel.: +966-11467-5884 (A.B.); Fax:
+966-11467-5992 (A.B.).
Received: / Accepted: / Published:
1

Abstract
We describe here the synthesis of dihydropyrimidines derivatives 3a-p, and evaluation
of their -glucosidase enzyme inhibition activities. Compounds 3b (IC50 = 62.4±1.5
M), 3c (IC50 = 25.3±1.26 M), 3d (IC50 = 12.4±0.15 M), 3e (IC50 = 22.9±0.25 M),
3g (IC50 = 23.8±0.17 M), 3h (IC50 = 163.3±5.1 M), 3i (IC50 = 30.6±0.6 M), 3m
(IC50 = 26.4±0.34 M), and 3o (IC50 = 136.1±6.63 M) were found to be potent
α-glucosidase inhibitors in comparison to the standard drug acarbose (IC50 = 840±1.73
M). The compounds were also evaluated for their in vitro cytotoxic activity against
PC-3, HeLa, and MCF-3 cancer cell lines, and 3T3 mouse fibroblast cell line. All
compounds were found to be non cytotoxic, except compounds 3f and 3m (IC50
=
17.79±0.66 M - 20.44±0.30 M), which showed a weak cytotoxic activity against the
HeLa, and 3T3 cell lines. In molecular docking simulation study, all the compounds
were docked into the active site of the predicted homology model of α-glucosidase
enzyme. From the docking result, it was observed that most of the synthesized
compounds showed interaction through carbonyl oxygen atom and polar phenyl ring
with active site residues of the enzyme.
Keywords: Spiro heterocycles; pyrimidines; α-glucosidase inhibitors; cytotoxicity;
cancer cell lines; molecular docking
1.
Introduction
α-Glucosidase (EC 3.2.1.20) enzyme catalyzes the hydrolysis of complex carbohydrates
into α-glucose. Type 2 diabetes mellitus (DM), known as non insulin-dependent DM, is
a common disorder of glucose and fat metabolism which causes immense sufferings to
patients [1]. Diabetes is characterized by high levels of blood sugar, which ultimately
can cause serious complications in the kidneys, eyes and cardiovascular system.
2

Therefore, the treatment of diabetes primarily focuses on reducing fluctuations of sugar
levels in blood and controlling subsequent complications [2]. α-Glucosidase is an
important mediator of glucose release in the small intestine [3]. Therefore, inhibition of
α-glucosidase can significantly decrease the postprandial hyperglycemia as a key
strategy in the control of type 2 DM. Recently, α-glucosidase inhibitors have been
reported to retard the absorption of glucose and improve postprandial hyperglycemia
[4]. Consequently, it was found useful to develop new glucosidase inhibitors [5].
Several types of α-glucosidase inhibitors have been reported, including natural products,
such as anthocyanins, curcuminoids, triterpenoids, flavonoids, and xanthones, and
synthetic compounds [6–11]. However, the role of these inhibitors in the treatment of
diabetes, through delaying the absorption of sugar, has not been validated through
clinical studies. Since 1980, the prevalence of diabetes has increased from 153 million
to 347 million in 2008 [12]. Based on WHO projection, diabetes will become the
seventh leading cause of death globally by 2030. One therapeutic approach for diabetes
is to suppress the postprandial hyperglycemia by inhibition of carbohydrate-hydrolyzing
enzymes, such as α-glucosidase [13,14]. α-Glucosidase inhibitors reversibly inhibit
digestive -glucosidase, which retards the liberation of glucose from dietary complex
carbohydrate and starch, delaying absorption of glucose into the bloodstream and, thus
reducing the plasma glucose levels. Inhibition of α-glucosidase has drawn a special
interest by the pharmaceutical research community as a remedy for the treatment of
carbohydrate mediated diseases, such as cancer, viral infections, diabetics and hepatitis
[15–17]. The inhibitors of α-glucosidase are known to possess antiviral, antitumor,
antidiabetic, and immunoregulatory activities [18–20]. In addition, α-glucosidase
inhibitors, such as N-butyl-deoxynojirimycin (NB-DNJ), deoxynojirimycin (DNJ), and
castanospermine are potent inhibitors of the HIV replication and HIV mediated
3

syncytium formation in vitro [20]. Currently, glucosidic based -glucosidase inhibitors,
such as nojirimycin [21], miglitol [22], acrabose [23], and voglibose [24] are clinically
used to control blood glucose levels in diabetic patients (Fig. 1). Although effective,
these inhibitors exhibit adverse side effects such as abdominal distension, flatulence,
meteorism and possibly diarrhea [25]. In order to search for effective and safer
-glucosidase inhibitors, efforts are now focused on the development of non-glucosidic
based inhibitors [26, 27].
Dihydropyrimidines (DHPMs) are pharmacologically important molecules due to their
diverse biological activities, such as antibacterial, antihypertensive, antiviral, and
antagonistic properties [28–32]. Pyrimidines also occupy a unique position in the
medicinal chemistry, as they are part of nucleic acids [29]. Recently, pyrimidine
derivatives have been reported as potent inhibitors of the enzymes responsible for
diabetes [33], and particularly, pyrimidine-fused heterocycles, which are identified as
specific α-glucosidase inhibitors (Fig. 1) [34].
4

Figure. 1. Known α-glucosidase inhibitors
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of the target compounds 3a-p
The desired diazaspiro[5.5]undecane-1,3,5,9-tetraone derivatives 3a-p [35], bearing
different substituents, were prepared in excellent yields (up to 99%), as shown in
Scheme-1. The preparation of 3a-p was carried out via double Michael addition of
N,N-dimethyl barbituric acid 1 into diaryledine 2a-p, mediated by NHEt2. Notably, a
variety of functional groups, such as hydroxyl, methoxy and chloro were tolerated under
our new reaction protocol. The chemical structures of all the synthesized compounds
were deduced with the help of physical and spectroscopic methods.
Scheme 1. Double Michael addition reaction as key step in the synthesis of spirocyclic
products 3a-p.
5

2.1.2. Single-Crystal X-Ray Crystal Diffraction Studies
The chemical structures of 3a, 3h, 3i, 3n, and 3o were unambiguously deduced with the
aid of single-crystal X-ray diffraction techniques, and have been reported before [35e,f].
6

Compounds 3g and 3p were obtained as single-crystals by slow diffusion with diethyl
ether, in dichloromethane at room temperature for 2 days. Data was collected on a
Bruker APEX-II D8 Venture area diffractometer, equipped with graphite
monochromatic Mo Kα radiation at 100 (2) K was used. Cell refinement and data
reduction was carried out by Bruker SAINT. SHELXS-97 [36, 37] was used to solve the
structures. The final refinement was carried out by full-matrix least-squares techniques
with anisotropic thermal data for non-hydrogen atoms on F2. All the hydrogen atoms
were placed in calculated positions (Table S1-S6, supplementary material). Similarity
and simulation restraints were applied for the disordered atoms. The crystal structures of
3g, and 3p are shown in Fig. 2. The structures of 3g, and 3p were confirmed by
single-crystal X-ray diffraction analysis (Bruker AXS GmbH). CCDC- 1042003; and
CCDC-1042005 contain the supplementary crystallographic data for these compounds.
3g
3p
Figure. 2. The ORTEP diagram of the final X-ray model of compounds 3g and 3p with
displacement ellipsoids drawn at 30% probability level.
7

2.2. Biological Activity
2.2.1. α-Glucosidase inhibition assay
Compounds 3a-3p were evaluated for their in vitro α-glucosidase inhibitory activity, in
comparison to acarbose as a standard inhibitor (IC50 = 840±1.73 M). Compounds 3b
(IC50 = 62.4±1.5 M), 3c (IC50 = 25.3±1.26 M), 3d (IC50 = 12.4±0.15 M), 3e (IC50 =
22.9±0.25 M), 3g (IC50 = 23.8±0.17 M), 3h (IC50 = 163.3±5.1 M), 3i (IC50
=
30.6±0.6 M), 3m (IC50 = 26.4±0.34 M), and 3o (IC50 = 136.1±6.63 M) were found
to be more potent α-glucosidase inhibitors than the standard α-glucosidase inhibitors
drug acarbose (IC50 = 840±1.73 M). Among all tested compounds, o,o-dichloro
substituted phenyl ring containing compound 3d (IC50 = 12.4±0.15 M) was found to
be the most active member of the series. A decrease in activity due to positional effects
of chloro groups on phenyl ring was observed for compound 3e (IC50 = 22.9±0.25 M),
having o,p dichloro substituted phenyl ring instead of o,o-dichloro substituted phenyl
ring. Further decrease in activity was observed for compound 3c (IC50 = 25.3±1.26 M)
containing a mono chloro susbtituant, instead of dichloro group, attached to the benzene
ring
of
the
basic
2,4-dimethyl-7,11-diphenyl-2,4-diazaspiro[5.5]-
undecane-1,3,5,9-tetraone moiety. The aforementioned observation of the change in
α-glucosidase inhibition activity due to substitution pattern of halogen on phenyl ring
was further supported by comparing the inhibition activity of meta bromo substituted
phenyl ring containing 3m (IC50 = 26.4±0.34 M) with that of para bromo substituted
phenyl ring containing 3f, which found to be inactive. However potent activity was
observed for 3g (IC50 = 23.8±0.17 M) which has electron withdrawing nitro group at
meta position instead of electronegative bromine, as observed in compound 3m. Several
fold decrease in activity was observed for compound 3h (IC50 = 163.3±5.1 M), as
compared to 3b (IC50 = 62.4±1.5 M). The decrease in activity may be due to the
8

presence of para methoxy (–OCH3) substituted phenyl ring of 3h, instead of toluene
moiety of 3b. A decrease in activity was also observed when methyl on the phenyl ring
of 3b (IC50 = 62.4±1.5 M) was replaced with a bulky trifloro methyl group in 3o (IC50
= 136.1±6.63 M). All other compounds were found to be inactive. The results are
summarized in Table 1.
2.2.2. Cytotoxic activity
Compounds 3a-3p were also tested for their cytotoxic activity against PC-3, Hela,
MCF-7 and 3T3 cell lines. Compound 3m showed a weak cytotoxic effect against HeLa
(IC50 = 17.79±0.66 M) and 3T3 (IC50 = 19.81±0.18 M)) cell lines, whereas,
compound 3f showed a weak cytotxicity against 3T3 (IC50 = 20.44±0.30 M) cell line
only. Doxorubicine was used as the standard drug to compare the activities against Hela
(IC50 = 0.51±0.15 M), and MCF-3 (IC50 = 0.924±0.01 M) cell lines, while
cycloheximide (IC50 = 0.26±0.12 M) was used as standard drug against 3T3 cell line.
The cytotoxic activities of these compounds may be due to the presence of bromo
substituted phenyl ring of the basic 2,4-dimethyl-7,11-diphenyl-2,4- diazaspiro[5.5]
undecane- 1,3,5,9-tetraone moiety . All tested compounds were found to be non
cytotoxic against PC-3 cell line. The results are summarized in Table 1.
Table 1. Results of various biological assays on compounds 3a-3p.
α-Glucosidase
Cytotoxicity Assay
Compounds
Inhibition
(IC50 = M)
PC3 (IC50 = M)
Hela (IC50 = M)
MCF-7 (IC50 = M)
3T3 (IC50 = M)
>30
>30
>30
>30
NA
>30
>30
>30
NA
3a
3b
62.4±1.5
9

>30
>30
NA
>30
NA
>30
NA
NA
NA
>30
NA
NA
>30
NA
NA
NA
>30
25.3±1.26
12.4±0.15
22.9±0.25
NA
3c
3d
3e
3f
>30
>30
>30
>30
>30
>30
>30
>30
20.44±0.30
>30
>30
>30
23.8±0.17
163.3±5.1
30.6±0.6
NA
3g
3h
3i
>30
>30
>30
>30
>30
>30
>30
>30
>30
3j
>30
>30
>30
NA
3k
3l
>30
>30
>30
NA
>30
17.79±0.66
>30
19.81±0.18
>30
26.4±0.34
NA
3m
3n
3o
3p
>30
>30
>30
>30
136.1±6.63
NA
>30
>30
>30
Doxorubicin
1.69±0.29
Doxorubicin
0.51±0.15 M
Doxorubicin
0.92±0.01
Cycloheximide
0.26±0.12
Acarboses
840±1.73
STD.
2.3. Computational studies
2.3.1. Homology Model and Molecular Docking study of α-glucosidase enzyme
The crystallographic structure of α-glucosidase enzyme has not been solved yet.
However, since a few homology models have been reported [37-40], we attempted to
build the 3D structure for α-glucosidase by comparative homology modeling technique
using the same propriety as described by Burke et al [41]. The sequence in fasta format
of α-glucosidase was retrieved from UniProt (access code P53341). Template selection
10

search was performed by means of MOE-Search tools against the PDB-database
implemented in MOE 2010.11. The 1.30 Å resolving crystallographic structure of
Saccharomyces cerevisiae isomaltase (SCI) (PDB code: 3AJ7) [42] with 72.4% was
selected as the template for modeling. The 3D structure was built by means of MOE
homology modeling tools. The predicted 3D model was subjected to energy
minimization up to 0.05 gradients. Before docking simulation, ligands and protein were
prepared by means of MOE 2010.11 software. 3D structure of all synthesized
compounds was built in MOE of Molecular Builder program. Finally, a database was
created in which all the ligands were converted into their particular 3D structures and
this database was used as input file MOE-docking. Subsequently, the energy of
compounds was minimized up to 0.05 gradient using MMFF94x force field. Energy
minimization of the database was followed by the preparation of protein for docking
purposes. Most macromolecular crystal structures contain little or no hydrogen
coordinate data due to limited resolution, and thus protonation was accomplished prior
to docking using Protonate 3D tools implemented in MOE. Protonation was followed by
energy minimization up to 0.05 gradient, using Amber99 force field. The database was
docked into the active site of protein using the Triangular Matching docking method,
and 30 conformations of each ligand protein complex were generated with docking
score (S). Each complex was analyzed for interactions and their 3D pose was taken.
2.3.2. Molecular docking simulation
From the molecular docking simulation study, it was observed that the top ranked
conformation of all the compounds were well accommodated inside the active site of
the predicted homology model of α-glucosidase enzyme. From the docking result, it was
observed that most of the synthesized compounds showed interaction through carbonyl
oxygen atom and polar phenyl ring with active site residues of the enzyme.
11

In case of the most active compound, 3d, in the series of sixteen synthesized
compounds, two hydrogen bonds, one arene-arene and one arene-cation interactions
were found with the catalytically active site residues Arg439, Arg212, Asp214, and
Asp349, respectively, as shown in Figure 3a. It was observed that both Arg439 and
Arg212 established hydrogen bonds (donor) with the carbonyl oxygen atoms of
pyrimidine and hexanone ring of compound 3d. Asp214 and Asp349 were found in
making phi-bonds with the phenyl ring of the compound. The highest activity (IC50
value 2.46 ± 0.008) of compound 3d in the series might be attributed to the
delocalization effect of electrons, which is possible up to some extent, or might be due
to the position of polar group (Cl) over the benzene ring, i.e., ortho with respect to the
hexanone moiety of the compound. As a result of this effect compound 3d showed good
interactions with the active site residues of the enzyme (Fig. 3a). The activity of
compound 3d is more as compared to compounds 3c and 3e, although there is little
difference in their structures. In case of compound 3c the chlorine atoms are attached at
para position of the phenyl ring, whereas in case of compound 3e the chlorine moiety is
present at ortho and para positions of the phenyl ring of the compound. The position of
chlorine atoms attached to the phenyl ring might play a very important role in the
activities of these compounds. For example, when chlorine atoms present at meta or
para positions of the compounds (compound 3c and 3d) the activity decreases. From
the docking results it was observed that these compounds show less interaction (Fig. 3b)
as compared to compound 3d.
The docking conformation of compound 3m showed that the carbonyl oxygen of
pyrimidine ring established hydrogen bond with the active site residue Arg312, and
12

phenyl ring of the compound formed arene-cation interaction with Arg212. Furthermore,
the compound showed hydrophobic interaction (Fig. 3c) with the active site residues,
Phe157 and Phe300, respectively. In case of compound 3f, poor interactions (Fig. 3d)
with active site residues were observed. These poor interactions might be one of reasons
for the lack of activity for this compound. Overall, the docking results showed that
when halogen moiety is present at ortho and meta positions (3d, 3e and 3m) showed
good interactions (Fig. 3a) and good biological activity (Table 1) whereas poor
interactions (Fig. 3c) and poor biological activity (Table 1) were observed when present
at para positions of phenyl ring (compounds 3c, 3f and 3n).
Over all the docking results showed that halogens moiety present at para position or
hydrophobic groups attached to the phenyl ring of the compounds (3c, 3f, 3i and 3j)
showed less interaction. These poor interactions might be one of the reasons for poor
biological activities of these compounds.
13

Figure. 3. Molecular docking conformations of compounds 3d (a), 3g (b), 3m (c) and 3f
(d) in the active site of the α-glucosidase enzyme.
3. Conclusion
In conclusion, we have synthesized spiro heterocyclic molecules by double Michael
addition reaction in excellent yield. The synthesized compounds were evaluated in vitro
against their cytotoxities including anti-cancer, and α-glucosiade enzyme inhibition
assay. Moreover, the molecular docking study was performed and the docking results
showed that halogen moiety present at the ortho and meta positions showed good
interactions and good biological activity as compared to para position. In addition
hydrophobic groups attached to the phenyl ring of the compounds (3c, 3f, 3i and 3j)
showed less interaction. The results showed promising activities and can be considered
for further studies.
14

4. Experimental
4.1. General remarks
All glassware was oven–dried before use and the reactions were conducted under inert
atmosphere. Progress of the reaction was monitored by TLC (Merck Silica Gel 60
F–254 thin layer plates; Merck, Schwalbach; Hessen, Germany). The chemicals were
purchased from Aldrich (Gillingham, Dorset, UK), and Fluka Chemie GmbH (Buchs,
Switzerland), etc, and were used without further purification, unless otherwise stated.
Petroleum ether (PE), hexane and ethyl acetate were distilled prior to use, especially for
column chromatography. All the major solvents were dried by using slandered drying
techniques mentioned in the literature. Melting points were measured on a Gallen-kamp
melting point apparatus in open glass capillaries, and are uncorrected. IR Spectra were
measured as KBr pellets on a Nicolet 6700 FT-IR spectrophotometer (Thermo Fisher
Scientific, Madison,WI, USA). The NMR spectra were recorded on a Jeol-400 NMR
1
13
spectrometer (Peabody, MA, USA). H-NMR (400 MHz), and C-NMR (100 MHz)
were run in deuterated chloroform (CDCl3). Chemical shifts (δ) are referred in ppm and J
-coupling constants are given in Hz. Mass spectrometric analysis was conducted by
using ESI mode on AGILENT Technologies 6410–triple quad LC/MS instrument (Santa
Clara, CA, USA). Elemental analysis was carried out on Elmer 2400 Elemental Analyzer,
CHN mode (Waltham, MA, USA).
General procedure of double Michael addition reaction for the synthesis of
spiro-compounds 3a-m (GP1).
A solution of N,N-dimethyl barbituric acid (1) (2 mmol) and diarylidene acetone
derivatives (2a-p) (2 mmol) in 10 mL of dry CH2Cl2 were charged into a 50 mL round
bottom flask under inert atmsopher. Et2NH (2.5 mmol) was then added to the reaction
mixture and stirred at room temperature for up to 1.5 – 2 hours, until TLC showed
15

complete consumption of both the reactants. After completion of the reaction, the crude
product was directly subjected to column chromatography, using 100 – 200 mesh silica
gel and ethyl acetate/n-hexane (2:8, v/v) as an eluent to afford the pure products 3a-p.
The solid products were further crystallized from a mixture of CHCl3/n-heptane.
4.2.1. 2,4-Dimethyl-7,11-diphenyl-2,4-diazaspiro[5.5]undecane-1,3,5,9-tetraone (3a)
[35e, 42]
Diarylidene acetone 2a (468.2 mg, 2 mmol) reacted with compound 1 (312.1 mg, 2
mmol) according to GP1 yielded white solid spiro-product 3a (765 mg, 1.96 mmol,
98%); m.p. 125–127 oC; 1H NMR (400 MHz, CDCl3) δ: 2.59 & 2.63 (dd, 2H, J =15.36
Hz, 4.40 Hz, CH2(e)), 2.85 (s, 3H, –NCH3), 3.01 (s, 3H, –NCH3), 3.72 (t, 2H, J =14.7
Hz, CH2(a)), 3.99 & 4.03 (dd, 2H, J = 14.7 Hz, 4.40 Hz, CH), 7.06–7.08 (m, 4H, Ar-H),
13
7.21–7.26 (m, 6H, Ar-H); C NMR (100 MHz, CDCl3) δ: 27.98, 28.39, 42.99, 50.55,
60.95, 127.56, 128.69, 128.94, 137.17, 149.70, 169.04, 170.71, 208.29; IR (KBr, cm–1)
νmax = 2959, 2925, 1716, 1675, 1484, 1422, 1381, 1125, 755, 706; [Anal. Calcd. for
C23H22N2O4: C, 70.75; H, 5.68; N, 7.17; Found: C, 70.69; H, 5.65; N, 7.01]; LC/MS
(ESI, m/z): [M+], calculated 390.21, C23H22N2O4 found 390.16. CCDC-1007513
4.2.2. 2,4-Dimethyl-7,11-di-p-tolyl-2,4-diazaspiro[5.5]undecane-1,3,5,9-tetraone (3b)
Diarylidene acetone 2b (524.3 mg, 2 mmol) reacted with compound 1 (312.1 mg, 2
mmol) according to GP1 yielded white solid spiro-product 3b (802 mg, 1.92 mmol,
o
1
96%); m.p. 122 – 124 C; H-NMR (400 MHz, CDCl3) δ: 2.25 (s, 3H, CH3), 2.55 &
2.58 (dd, 2H, J = 14.7 Hz, 4.40 Hz, CH2(e)), 2.87 (s, 3H, –NCH3), 3.01 (s, 3H, –NCH3),
3.68 (t, 2H, J = 14.7 Hz, CH2(a)), 3.94 & 3.98 (dd, 2H, J = 13.9 Hz, 4.4 Hz, CH), 6.93 (d,
4H, J = 8.0 Hz, Ar-H), 7.01 (d, 4H, J = 8.0 Hz, Ar-H); 13C-NMR (100 MHz, CDCl3) δ:
21.13 27.94, 28.41, 43.15, 50.19, 61.08, 127.39, 129.58, 134.19, 138.39, 149.65, 169.29,
170.88, 208.63; IR (KBr, cm–1) νmax = 3019, 2970, 1740, 1678, 1441, 1370, 1221, 902,
16

672, 520; [Anal. Calcd. for C25H26N2O4: C, 71.75; H, 6.26; N, 6.69; Found: C, 71.58; H,
6.37; N, 6.81]; LC/MS (ESI, m/z): [M+], calculated 418.3, C25H26N2O4 found 418.19.
4.2.3.7,11-Bis(4-chlorophenyl)-2,4-dimethyl-2,4-diazaspiro[5.5]undecane-1,3,5,9-tetrao
ne (3c)
Diarylidene acetone 2c (604.1 mg, 2 mmol) reacted with compound 1 (312.1 mg, 2
mmol) according to GP1 yielded white solid spiro-product 3c (889 mg, 1.92 mmol,
97%); m.p. 211 – 213 oC; 1H-NMR (400 MHz, CDCl3) δ: 2.55 & 2.58 (dd, 2H, J = 14.7
Hz, 4.40 Hz, CH2(e)), 2.89 (s, 3H, –NCH3), 3.04 (s, 3H, –NCH3), 3.64 (t, 2H, J = 14.7
Hz, CH2(a)), 3.95 & 3.98 (dd, 2H, J = 14.7 Hz, 4.40 Hz, CH), 6.99 (d, 4H, J = 8.80 Hz,
13
Ar-H), 7.22 (d, 4H, J = 8.80 Hz, Ar-H); C-NMR (100 MHz, CDCl3) δ: 28.49, 28.68,
42.94, 49.87, 60.58, 128.86, 129.01, 135.25, 135.52, 149.41, 168.75, 170.44, 207.20; IR
(KBr, cm–1) νmax = 3015, 2970, 1740, 1678, 1437, 1369, 1218, 904, 672, 521; [Anal.
Calcd. for C23H20Cl2N2O4: C, 60.14; H, 4.39; N, 6.10; Found: C, 59.97; H, 4.46; N,
6.19]; LC/MS (ESI, m/z): [M+], calculated 458.1, C23H20Cl2N2O4 found 458.08.
4.2.4.7,11-Bis(2,6-dichlorophenyl)-2,4-dimethyl-2,4-diazaspiro[5.5]undecane-1,3,5,9-te
traone (3d)
Diarylidene acetone 2d (734 mg, 2 mmol) reacted with compound 1 (312.1 mg, 2
mmol) according to GP1 yielded white solid spiro-product 3d (936 mg, 1.78 mmol,
o
1
89%); m.p. 149 – 151 C; H-NMR (400 MHz, CDCl3) δ: 2.58 & 2.62 (dd, 2H, J =
16.1 Hz, 4.40 Hz, CH2(e)), 2.93 (s, 3H, –NCH3), 3.26 (s, 3H, –NCH3), 3.42 (t, 2H, J =
15.4 Hz, CH2(a)), 4.67 & 4.71 (dd, 2H, J = 13.9 Hz, 4.40 Hz, CH), 7.04 (d, 2H, J = 8.8
Hz, Ar-H), 7.15 & 7.17 (dd, 2H, J = 8.8 Hz, 2.20 Hz, Ar-H), 7.38 (d, 2H, J = 2.2 Hz,
Ar-H); 13C-NMR (100 MHz, CDCl3) δ: 28.51, 29.10, 43.55, 45.20, 57.36, 128.28,
130.55, 133.99, 134.98, 149.55, 167.90, 169.91, 205.63; IR (KBr, cm–1) νmax = 3015,
2970, 2030, 1977, 1722, 1776, 1585, 1470, 1446, 1376, 1223, 1106, 1050, 528, 751,
17

521, 469; [Anal. Calcd. for C23H18Cl4N2O4: C, 52.30; H, 3.43; N, 5.30; Found: C,
52.41; H, 3.45; N, 5.37]; LC/MS (ESI, m/z): [M+], calculated 526.10.1, C23H18Cl4N2O4
found 526.00.
4.2.5.7,11-Bis(2,4-dichlorophenyl)-2,4-dimethyl-2,4-diazaspiro[5.5]undecane-1,3,5,9-te
traone (3e)
Diarylidene acetone 2e (734 mg, 2 mmol) reacted with compound 1 (312.1 mg, 2 mmol)
according to GP1 yielded white solid spiro-product 3e (957 mg, 1.82 mmol, 91%); m.p.
o
1
185 – 187 C; H-NMR (400 MHz, CDCl3) δ: 2.57 & 2.61 (dd, 2H, J = 16.1 Hz, 4.40
Hz, CH2(e)), 2.96 (s, 3H, –NCH3), 3.26 (s, 3H, –NCH3), 3.42 (t, 2H, J = 15.4 Hz, CH2(a)),
4.67 & 4.71 (dd, 2H, J = 15.4 Hz, 4.4 Hz, CH), 7.11 – 7.17 (m, 2H, Ar-H), 7.21 (s, 2H,
Ar-H), 7.11 – 7.38 (m, 2H, Ar-H); 13C-NMR (100 MHz, CDCl3) δ: 28.70, 29.14, 43.57,
45.24, 57.36, 127.75, 129.86, 130.50, 131.91, 133.02, 134.00, 149.55, 169.43, 169.91,
205.60; IR (KBr, cm–1) νmax = 2920, 1718, 1673, 1444, 1375, 1108, 1045, 828, 747, 465;
[Anal. Calcd. for C23H18Cl4N2O4: C, 52.30; H, 3.43; N, 5.30; Found: C, 52.41; H,
3.45; N, 5.37]; LC/MS (ESI, m/z): [M+], calculated 526.10.1, C23H18Cl4N2O4 found
526.00.
4.2.6.7,11-Bis(4-bromophenyl)-2,4-dimethyl-2,4-diazaspiro[5.5]undecane-1,3,5,9-tetrao
ne (3f)
Diarylidene acetone 2f (780 mg, 2 mmol) reacted with compound 1 (312.1 mg, 2 mmol)
according to GP1 yielded white solid spiro-product 3f (1.0 g, 1.86 mmol, 93%); m.p.
o
1
205 – 207 C; H-NMR (400 MHz, CDCl3) δ: 2.55 & 2.58 (dd, 2H, J = 14.7 Hz, 4.4
Hz, CH2(e)), 2.90 (s, 3H, –NCH3), 3.02 (s, 3H, –NCH3), 3.59 (t, 2H, J = 14.7 Hz, CH2(a)),
3.94 & 3.97 (dd, 2H, J = 14.7 Hz, 4.40 Hz, CH), 6.92 (d, 4H, J = 8.80 Hz, Ar-H),
13
7.37(d, 4H, J = 8.8 Hz, Ar-H); C-NMR (100 MHz, CDCl3) δ: 28.16, 28.59, 42.82,
49.95, 60.40, 122.90, 129.25, 129.86, 132.22, 132.42, 149.90, 169.53, 170.48, 207.15;
18

IR (KBr, cm–1) νmax = 3020, 1712, 1675, 1640, 1415, 1376, 1284, 1177, 1067, 979, 806,
547, 443; [Anal. Calcd. for C23H20Br2N2O4: C, 50.39; H, 3.68; N, 5.11; Found: C,
50.51; H, 3.73; N, 5.17]; LC/MS (ESI, m/z): [M+], calculated 546.11, C23H20Br2N2O4
found 545.98.
4.2.7.2,4-Dimethyl-7,11-bis(3-nitrophenyl)-2,4-diazaspiro[5.5]undecane-1,3,5,9-tetraon
e (3g)
Diarylidene acetone 2g (648 mg, 2 mmol) reacted with compound 1 (312.1 mg, 2
mmol) according to GP1 yielded white solid spiro-product 3g (912 mg, 1.9 mmol,
95%); m.p. 232 - 234 oC; 1H-NMR (400 MHz, CDCl3) δ: 2.66 & 2.69 (dd, 2H, J = 15.4
Hz, 4.4 Hz, CH2(e)), 2.87 (s, 3H, –NCH3), 3.06 (s, 3H, –NCH3), 3.75 (t, 2H, J = 14.7 Hz,
CH2(a)), 4.14 & 4.18 (dd, 2H, J = 13.9 Hz, 4.4 Hz, CH), 7.42 – 7.51 (m, 4H, Ar-H),
13
7.91(s, 2H, Ar-H), 8.13 (d, 2H, J = 8.0 Hz, Ar-H); C-NMR (100 MHz, CDCl3) δ:
28.36, 28.71, 42.63, 49.98, 60.09, 122.40, 124.01, 130.28, 133.95, 138.95, 148.64,
168.26, 169.84, 205.46; IR (KBr, cm–1) νmax = 2953, 1715, 1673, 1527, 1420, 1381, 901,
808, 731, 681, 451; [Anal. Calcd. for C23H20N4O8: C, 57.50; H, 4.20; N, 11.66: Found:
C, 57.56; H, 4.32; N, 11.43]; LC/MS (ESI, m/z): [M+], calculated 480.03, C23H20N4O8
found 480.13; CCDC-1042003.
4.2.8.7,11-Bis(4-methoxyphenyl)-2,4-dimethyl-2,4-diazaspiro[5.5]undecane-1,3,5,9-tetr
aone (3h)
Diarylidene acetone 2h (588 mg, 2 mmol) reacted with compound 1 (312.1 mg, 2
mmol) according to GP1 yielded white solid spiro-product 3h (873 mg, 1.94 mmol,
o
1
97%); m.p. 131 - 133 C; H-NMR (400 MHz, CDCl3) δ: 2.53 & 2.56 (dd, 2H, J =
14.7Hz, 4.4 Hz, CH2(e)), 2.88 (s, 3H, –NCH3), 3.01 (s, 3H, –NCH3), 3.68 (t, 2H, J =
14.7 Hz, CH2(a)), 3.72 (s, 3H, OCH3), 3.91 & 3.94 (dd, 2H, J = 13.9 Hz, 4.4 Hz, CH),
13
6.73 (d, 4H, J = 8.8 Hz, Ar-H), 6.96 (d, 4H, J = 8.8 Hz, Ar-H); C-NMR (100 MHz,
19

CDCl3) δ: 28.01, 28.45, 43.29, 49.73, 55.26, 61.39, 114.17, 128.64, 129.21, 149.98,
159.51, 169.25, 171.79, 208.54; IR (KBr, cm–1) νmax = 2957, 2838, 1713, 1670, 1609,
1510, 1449, 1420, 1248, 1031, 831, 729, 530, 452; [Anal. Calcd. for C25H26N2O6: C,
66.65; H, 5.82; N, 6.22; Found: C, 66.81; H, 5.71; N, 6.34]; LC/MS (ESI, m/z): [M+],
calculated 450.15, C25H26N2O6 found 450.18; CCDC-1004326.
4.2.9.2,4-Dimethyl-7,11-di(naphthalen-1-yl)-2,4-diazaspiro[5.5]undecane-1,3,5,9-tetrao
ne (3i)
Diarylidene acetone 2i (668 mg, 2 mmol) reacted with compound 1 (312.1 mg, 2 mmol)
according to GP1 yielded white solid spiro-product 3i (941 mg, 1.92 mmol, 96%); m.p.
218 - 220 oC; 1H-NMR (400 MHz, CDCl3) δ: 1.98 (s, 3H, –NCH3), 2.75 & 2.79 (dd, 2H,
J = 15.4 Hz, 4.4 Hz, CH2(e)), 3.22 (s, 3H, –NCH3), 3.89 (t, 2H, J = 15.0 Hz, CH2(a)),
5.11 & 5.14 (dd, 2H, J = 13.9 Hz, 4.4 Hz, CH), 7.33 – 7.39 (m, 4H, Ar-H), 7.47 (t, 2H,
J = 8.0 Hz, Ar-H), 7.57 (t, 2H, J = 8.0 Hz, Ar-H), 7.73 & 7.74 (dd, 2H, J = 7.6 Hz, 2.40
13
Hz, Ar-H), 7.80 (d, 2H, J = 8.1 Hz, Ar-H), 8.22 (d, 2H, J = 8.8 Hz, Ar-H); C-NMR
(100 MHz, CDCl3) δ: 28.03, 28.32, 44.37, 44.86, 59.34, 123.16, 124.20, 124.95, 126.19,
126.74, 128.94, 129.23, 130.91, 134.05, 134.22, 149.61, 169.47, 170.38, 208.33; IR
(KBr, cm–1) νmax = 3049, 2919, 1711, 1666, 1421, 1374, 1266, 1241, 1018, 773, 467;
[Anal. Calcd. for C31H26N2O4: C, 75.90; H, 5.34; N, 5.71; Found: C, 76.13; H, 5.41; N,
5.83]; LC/MS (ESI, m/z): [M+], calculated 490.23, C31H26N2O4 found 490.19;
CCDC-1004327.
4.2.10.2,4-Dimethyl-7-phenyl-11-(p-tolyl)-2,4-diazaspiro[5.5]undecane-1,3,5,9-tetraone
(3j)
Diarylidene acetone 2j (496 mg, 2 mmol) reacted with compound 1 (312 mg, 2 mmol)
according to GP1 yielded white solid spiro-product 3j (776 mg, 1.92 mmol, 96%); m p.
110 - 112 oC; 1H-NMR (400 MHz, CDCl3) δ: 2.25 (s, 3H, CH3), 2.55 & 2.59 (dd, 2H, J
20

= 14.7 Hz, 4.4Hz, CH2(e)), 3.87 (s, 3H, –NCH3), 3.01 (s, 3H, –NCH3), 3.72 (t, 2H, J =
14.7 Hz, CH2(a)), 3.94 & 3.98 (dd, 2H, J = 14.7 Hz, 4.40 Hz, CH), 6.92 (d, 4H, J = 8.1
13
Hz, Ar-H), 7.01 (d, 4H, J = 8.1 Hz, Ar-H), 7.21 – 7.25 (m, 1H, Ar-H); C-NMR (100
MHz, CDCl3) δ: 21.12, 27.97, 28.40, 44.17, 43.15, 50.19, 61.8, 127.39, 128.92, 129.57,
134.18, 138.39, 149.98, 168.18, 170.89, 208.66; IR (KBr, cm–1) νmax = 29.57, 2924,
1717, 1672, 1446, 1419, 1377, 1285, 814, 730, 560, 509; [Anal. Calcd. for C24H24N2O4:
C, 71.27; H, 5.98; N, 6.93; Found: C, 71.36; H, 6.07; N, 7.01]; LC/MS (ESI, m/z):
[M+], calculated 404.11, C24H24N2O4 found 404.17.
4.2.11.2,4-Dimethyl-7,11-di(thiophen-2-yl)-2,4-diazaspiro[5.5]undecane-1,3,5,9-tetraon
e (3k)
Diarylidene acetone 2k (520 mg, 2 mmol) reacted with compound 1 (312.1 mg, 2
mmol) according to GP1 yielded white solid spiro-product 3k (788 mg, 1.96 mmol,
o
1
98%); m.p. 136 – 138 C; H-NMR (400 MHz, CDCl3) δ: 2.80 & 2.83 (dd, 2H, J =
15.4 Hz, 4.4 Hz, CH2(e)), 3.11 (s, 3H, –NCH3), 3.14 (s, 3H, –NCH3), 3.66 (t, 2H, J =
14.6 Hz, CH2(a)), 4.34 & 4.37 (dd, 2H, J = 13.9 Hz, 4.4 Hz, CH), 6.84 (d, 2H, J = 3.7
13
Hz, Ar-H), 6.93 – 6.95 (m, 2H, Ar-H), 7.23 (d, 2H, J = 5.1 Hz, Ar-H); C-NMR (100
MHz, CDCl3) δ: 28.19, 28.61, 44.16, 45.44, 61.50, 125.48, 125.87, 126.96, 139.84,
149.83, 168.60, 170.90, 205.77; IR (KBr, cm–1) νmax = 2959, 2921, 1715, 1668, 1420,
1373, 1260, 1036, 799, 702, 501, 444; [Anal. Calcd. for C19H18N2O4S2: C, 56.70; H,
4.51; N, 6.96; Found: C, 56.76; H, 4.43; N, 7.03]; LC/MS (ESI, m/z): [M+], calculated
402.11, C19H18N2O4S2 found 402.07
4.2.12. 7,11-Di(furan-2-yl)-2,4-dimethyl-2,4-diazaspiro[5.5]undecane-1,3,5,9-tetraone
(3l)
Diarylidene acetone 2l (456 mg, 2 mmol) reacted with compound 1 (312.1 mg, 2 mmol)
according to GP1 yielded white solid spiro-product 3l (725 mg, 1.96 mmol, 98%); m.p.
21

114 - 116 oC; 1H-NMR (400 MHz, CDCl3) δ: 2.67 & 2.71 (dd, 2H, J = 15.4 Hz, 4.4 Hz,
CH2(e)), 3.08 (s, 3H, –NCH3), 3.10 (s, 3H, –NCH3), 3.49 (t, 2H, J = 14.7 Hz, CH2(a)),
4.08 & 4.11 (dd, 2H, J = 14.0 Hz, 4.4 Hz, CH), 6.05 (d, 2H, J = 3.6 Hz, Ar-H), 6.25 –
13
6.26 (m, 2H, Ar-H), 7.23 (d, 2H, J = 1.40 Hz, Ar-H); C-NMR (100 MHz, CDCl3) δ:
28.18, 28.80, 40.90, 43.32, 57.09, 107.39, 110.56, 142.56, 151.20, 151.32, 168.01,
170.75, 206.10; IR (KBr, cm–1) νmax = 3115, 1959, 1721, 1670, 1446, 1420, 1377, 1011,
922, 738, 465; [Anal. Calcd. for C19H18N2O6: C, 61.62; H, 4.90; N, 7.56; Found: C,
61.49; H, 5.11; N, 7.43]; LC/MS (ESI, m/z): [M+], calculated 370.18, C19H18N2O6 found
370.12.
4.2.13.7,11-Bis(3-bromophenyl)-2,4-dimethyl-2,4-diazaspiro[5.5]undecane-1,3,5,9-tetra
one (3m)
Diarylidene acetone 2m (780 mg, 2 mmol) reacted with compound 1 (312.1 mg, 2
mmol) according to GP1 yielded white solid spiro-product 3m (1037 mg, 1.90 mmol,
o
1
95%); m.p. 118 - 120 C; H-NMR (400 MHz, CDCl3) δ: 2.57 & 2.61 (dd, 2H, J =
14.7 Hz, 4.4 Hz, CH2(e)), 2.91 (s, 3H, –NCH3), 3.06 (s, 3H, –NCH3), 3.64 (t, 2H, J =
14.7 Hz, CH2(a)), 3.92 & 3.96 (dd, 2H, J = 14.7 Hz, 4.4 Hz, CH), 6.98 (d, 2H, J = 8.1Hz,
Ar-H), 7.11 (t, 2H, J = 8.1Hz, Ar-H), 7.22, (s, 2H, Ar-H), 7.37 (d, 2H, J = 7.4 Hz,
Ar-H); 13C-NMR (100 MHz, CDCl3) δ: 28.09, 28.57, 42.67, 50.01, 60.52, 123.11,
126.25, 130.53, 130.69, 131.97, 139.22, 149.44, 168.66, 170.29, 206.89; IR (KBr, cm–1)
νmax = 2959, 2921, 1710, 1667, 1423, 1349, 1285, 1256, 1070, 787, 749, 695, 443;
[Anal. Calcd. for C23H20Br2N2O4: C, 50.39; H, 3.68; N, 5.11; Found: C, 50.51; H,
3.73; N, 5.17]; LC/MS (ESI, m/z): [M+], calculated 546.11, C23H20Br2N2O4 found
545.98.
4.2.14.7,11-Bis(4-fluorophenyl)-2,4-dimethyl-2,4-diazaspiro[5.5]undecane-1,3,5,9-tetra
one (3n)
22

Diarylideneacetone 2n (540 mg, 2 mmol) was reacted with compound 1 (312.1 mg, 2
mmol) according to GP1 to yield a white solid of spiro-product 3n (835 mg, 1.96 mmol,
98%); m.p. 175–177 °C; UV-Vis: 204 nm, 218 nm (sh) and 233 nm (sh) (in
Dichloromethane); 1H-NMR (CDCl3) δ: 2.55 and 2.59 (dd, 2H, J = 15.4 Hz, J = 4.4 Hz,
CH2(e)), 2.88 (s, 3H, -NCH3), 3.02 (s, 3H, –NCH3), 3.64 (t, 2H, J = 14.7 Hz, CH2(a)), 3.95
& 3.99 (dd, 2H, J = 14.0 Hz, J = 4.4Hz, CH), 6.89–6.94 (m, 4H, Ar-H), 7.01–7.04 (m,
4H, Ar-H): 13C-NMR (CDCl3) δ: 27.92, 28.34, 42.93, 49.56, 60.82, 115.73 and 115.95 (d,
J2 = 21.4 Hz), 115.10 and 129.18 (d, J3 = 7.65 Hz), 132.78 and 132.81 (d, J4 = 3.06 Hz),
149.33, 161.20 & 163.67 (d, J1 = 147 Hz), [ArC1, C2, C3& C4 are split into doublets due
19
to F], 168.74, 170.48, 207.27; IR (KBr, cm−1) νmax = 2920, 1718, 1618, 1507, 1418,
1378, 1223, 1159, 828, 753, 510, 466; [Anal. Calcd. for C23H20F2N2O4: C, 50.39; H,
3.68; N, 5.11; Found: C, 50.51; H, 3.73; N, 5.17]; LC/MS (ESI, m/z): [M+], calculated
426.21, C23H20F2N2O4 found 426.14.
4.2.15.(7R,11S)-2,4-Dimethyl-7,11-bis(4-(trifluoromethyl)phenyl)-2,4-diazaspiro[5.5]u
ndecane-1,3,5,9-tetraone (3o)
Diarylideneacetone 2o (684 mg, 2 mmol) was reacted with compound 1 (312.1 mg, 2
mmol) according to GP1 to yield the white solid spiro-product 3o (936 mg, 1.88 mmol,
94%); m p. 180–182 °C; UV-Vis: (in ethanol): 204nm and 232nm; 1H-NMR (CDCl3) δ:
2.70 and 2.74 (dd, 2H, J = 15.4 Hz, J = 4.4 Hz, CH2(e)), 3.02 (s, 3H, -NCH3), 3.04 (s, 3H,
–NCH3), 3.56 (t, 2H, J = 14.7 Hz, CH2(e)), 4.25 and 4.28 (dd, 2H, J = 14.0 Hz, Jae = 4.4
13
Hz, CH), 6.75–7.14 (m, 4H, Ar-H); C-NMR (CDCl3) δ: 28.21, 28.63, 44.17, 45.44,
61.51, 125.48, 125.87, 126.96, 139.84, 149.93, 168.60, 170.91, 205.78; IR (KBr, cm−1) νmax
23

= 2956, 2919, 1715, 1669, 1419, 1373, 1280, 702, 500, 444; [Anal. Calcd. for
C23H16F6N2O4: C, 55.43; H, 3.24; N, 5.62; Found: C, 55.29; H, 3.17; N, 5.75]; LC/MS
(ESI, m/z): [M+], calculated 498.19, C23H16F6N2O4 found 498.10.
Biological activities:
In vitro anti-cancer and a-glucosidase inhibition assays were performed to assess the
biological activity of synthesized compounds. Results are presented here as means ±
standard error from triplicate (n=3) observation. IC50 values were calculated by using
EZ-FIT, Enzyme kinetics software by Perrella Scientific.
In Vitro α-Glucosidase Inhibition Assay:
α-Glucosidase inhibition assay was performed spectrophotometrically. α-Glucosidase
from Saccharomyces cerevisiae (G0660-750UN, Sigma Aldrich), was dissolved in
phosphate buffer (pH 6.8., 50 mM). Test compounds were dissolved in 70% DMSO. In
96-well plates, 20μL of test sample, 20 μL of enzyme and 135 μL of buffer were added
and incubated for 15 minutes at 37°C. After incubation, 25 μL of p-nitrophenyl- α
-D-glucopyranoside (0.7 mM, Sigma Aldrich) was added and change in absorbance was
monitored for 30 minutes at 400 nm. Test compound was replaced by DMSO (7.5%
final) as control. Acarbose (Acarbose, Sigma Aldrich) was used as a standard inhibitor
[43].
Cytotoxic activity:
Cytotoxic activity of compounds was evaluated in 96-well flat-bottomed microplates by
using the standard MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium
bromide, MP) colorimetric assay [39]. For this purpose, PC3 cells (Prostrate Cancer)
were cultured in Dulbecco’s Modified Eagle Medium, supplemented with 10% of fetal
bovine serum (FBS, PAA), 100 IU/mL of penicillin and 100 µg/mL of streptomycin in
75 cm2 flasks, and kept in 5% CO2 incubator at 37o C. Exponentially growing cells were
24

harvested, counted with haemocytometer and diluted with a particular medium with 5%
FBS. Cell culture with the concentration of 1x105 cells/mL was prepared and introduced
(100 µL/well) into 96-well plates. After overnight incubation, medium was removed and
200 µL of fresh medium was added with different concentrations of compounds (1-30
µM). Stock solution, 20 mM of compounds were prepared in 100% DMSO and final
concentration of DMSO at 30 µM is 0.15%. After 48 hrs, 200 µL MTT (0.5 mg/mL)
was added to each well and incubated further for 4 hrs. Subsequently, 100 µL of DMSO
was added to each well. The extent of MTT reduction to formazan within cells was
calculated by measuring the absorbance at 570 nm, using a micro plate reader (Spectra
Max plus, Molecular Devices, CA, USA). The cytotoxicity was recorded as
concentration causing 50% growth inhibition (IC50) for PC3 cells. The percent
inhibition was calculated by using the following formula:
% inhibition = 100-((mean of O.D of test compound – mean of O.D of negative
control)/ (mean of O.D of positive control – mean of O.D of negative control)*100).
The results (% inhibition) were processed by using Soft- Max Pro software (Molecular
Device, USA).
Acknowledgments
The authors would like to extend their sincere appreciation to the Deanship of Scientific
Research at king Saud University for its funding this Research group NO (RG
-257-1436-1437).
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