Ultrasonic synthesis of tyramine derivatives as novel inhibitors of α-glucosidase in vitro

Article abstract of DOI:10.3109/14756366.2016.1142983

Tyramine derivatives 3–27 were synthesized by using conventional and environmental friendly ultrasonic techniques. These derivatives were then evaluated for the first time for their α-glucosidase (Sources: Saccharomyces cerevisiae and mammalian rat-intest

Full text of DOI:10.3109/14756366.2016.1142983

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Ultrasonic synthesis of tyramine derivatives as
novel inhibitors of α-glucosidase in vitro
Hina Siddiqui, Muhammad Arslan Bashir, Kulsoom Javaid, Arsalan Nizamani,
Huma Bano, Sammer Yousuf, Atta-ur Rahman & M. Iqbal Choudhary
To cite this article: Hina Siddiqui, Muhammad Arslan Bashir, Kulsoom Javaid, Arsalan
Nizamani, Huma Bano, Sammer Yousuf, Atta-ur Rahman & M. Iqbal Choudhary (2016):
Ultrasonic synthesis of tyramine derivatives as novel inhibitors of α-glucosidase in vitro,
Journal of Enzyme Inhibition and Medicinal Chemistry, DOI: 10.3109/14756366.2016.1142983
To link to this article: http://dx.doi.org/10.3109/14756366.2016.1142983
Published online: 25 Feb 2016.
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–12
2016 Taylor & Francis. DOI: 10.3109/14756366.2016.1142983
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RESEARCH ARTICLE
Ultrasonic synthesis of tyramine derivatives as novel inhibitors of
a-glucosidase in vitro
Hina Siddiqui1*, Muhammad Arslan Bashir1, Kulsoom Javaid1, Arsalan Nizamani1, Huma Bano1, Sammer Yousuf1,
Atta-ur-Rahman1, and M. Iqbal Choudhary1,2*
1H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan and
2Department of Biochemistry, King Abdul Aziz University, Jeddah-21452, Saudi Arabia
Abstract
Keywords
Tyramine derivatives 3–27 were synthesized by using conventional and environmental friendly
ultrasonic techniques. These derivatives were then evaluated for the first time for their
a-glucosidase (Sources: Saccharomyces cerevisiae and mammalian rat-intestinal acetone
powder) inhibitory activity by using in vitro mechanism-based biochemical assays.
Compounds 7, 14, 20, 21 and 26 were found to be more active (IC50 ¼ 49.7 0.4, 318.8 3.7,
23.5 0.9, 302.0 7.3 and 230.7 4.0 mM, respectively) than the standard drug, acarbose
(IC50 ¼ 840.0 1.73 mM (observed) and 780 0.028 mM (reported)) against a-glucosidase
obtained from Saccharomyces cerevisiae. Kinetic studies were carried out on the most active
members of the series in order to determine their mode of inhibition and dissociation
constants. Compounds 7, 20 and 26 were found to be the competitive inhibitors of
a-glucosidase. These compounds were also screened for their protein antiglycation, and
dipeptidyl peptidase-IV (DPP-IV) inhibitory activities. Only compounds 20, 22 and 27 showed
weak antiglycation activity with IC50 values 505.27 5.95, 581.87 5.50 and 440.58 2.74 mM,
respectively. All the compounds were found to be inactive against DDP-IV enzyme. Inhibition of
a-glucosidase, DPP-IV enzymes and glycation of proteins are valid targets for the discovery of
antidiabetic drugs. Cytotoxicity of compounds 3–27 was also evaluated by using mouse
fibroblast 3T3 cell lines. All the compounds were found to be noncytotoxic. The current study
describes the synthesis a-glucosidase inhibitory activity of derivatives, based on a natural
product tyramine template. The compounds reported here may serve as the starting point for
the design and development of novel a-glucosidase inhibitors as antidiabetic agents.
Diabetes, a-glucosidase inhibition,
postprandial hyperglycemia, tyramine,
ultrasonic synthesis
History
Received 10 September 2015
Revised 11 December 2015
Accepted 21 December 2015
Published online 23 February 2016
Introduction
an important target for drug discovery against diabetes. In
addition to this, glycation of proteins in hyperglycemia leads to
late diabetic complications. The inhibition of protein glycation is
vigorously studied to slow down the on-set of diabetic
complications.
Tyramine (4-hydroxyphenethylamine) is a naturally occurring
monoamine, derived from the amino acid tyrosine. It is reported
to possess various biological properties, such as antioxidant5, a1-
adrenergic agonistic6 and monoamine oxidase7. It is reported that
administration of tyramine to diabetic rats reduces the hypergly-
cemia, even at doses that have low effects on cardiovascular
variables8.
The synthesis of imines or Schiff’s bases has special signifi-
cance in organic synthesis and medicinal chemistry9. For
example, they have been widely used in asymmetric synthesis
of a-aminonitriles, hydrogenation and cycloaddition reactions10.
They have been reported to possess various biological activities,
such as antimalarial, antibacterial, antifungal, antiviral, etc11.
These compounds also have various applications in coordination
chemistry11. The current study describes the synthesis of new
imine derivatives of tyramine (3–27) by using conventional and
ultrasonic-assisted chemistry and evaluation of their antidiabetic
activity by using a-glucosidase, DPP-IV inhibition and protein
antiglycation assays.
a-Glucosidase (EC 3.2.1.20) is an exo-type glycosidase enzyme
that catalyzes the release of monosaccharides from the nonredu-
cing end of the carbohydrates. a-Glucosidase inhibitors (AGIs)
reduce the absorption of glucose from the small intestine. As a
result, they can modulate the postprandial glucose levels in blood
and serve as antidiabetic drugs1,2,3. Acarbose, miglitol and
voglibose are a-glucosidase inhibitors used for the treatment of
type-2 diabetes4. However, these drugs cause multiple side
effects, such as diarrhea, abdominal discomfort, etc. A need of
new, safe and effective inhibitors of a-glucosidase is therefore
greatly felt. Similarly, dipeptidyl peptidase-IV (DPP-IV) is an
important enzyme involve in the cleavage of X-proline dipeptides
from the N-terminus of polypeptides. The inhibition of DPP-IV is
Address for correspondence: Hina Siddiqui, H.E.J. Research Institute of
Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi, 75270, Pakistan. E-mail:
hinahej@gmail.com
M. Iqbal Choudhary, H.E.J. Research Institute of Chemistry, International
Center for Chemical and Biological Sciences, University of Karachi,
Pakistan; Department of Biochemistry, Faculty of Science, King Abdul
Aziz University, Saudi Arabia. E-mail: hej@cyber.net.pk

2
H. Siddiqui et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Over the years, sonochemistry (ultrasonic-assisted chemistry) Chromatograms, developed on TLC plates, were visualized under
has emerged as an important tool in organic synthesis. It is a ultraviolet light at 254 nm for fluorescence quenching spots and
type of green chemistry with several advantages, including 365 nm for florescent spots.
cost-effectiveness, environmental friendly nature and time and
yield efficiency12.
Ultrasonic equipment: Ultrasonic-mediated reactions were
conducted in Elma Ultrasonic Cleaner LC30H (Elma
During the current study, the Schiff’s bases or benzylidinea- Schmidbauer GmbH, Germany). All experiments were performed
mine derivatives of tyramine (3–27) were synthesized because with output power of 240 W.
these classes have been reported to have a wide range of
biological activities, including enzyme inhibition activities13. General procedure for the synthesis of compounds 3–27
Moreover, compounds containing benzoyl moiety are also
reported to have enzyme-inhibiting activities14. All the com-
pounds were found to be new, except 9, 17, 18, 19, 21, 22 and 25.
These derivatives have not been reported yet for their a-gluco-
sidase inhibitory activity. An in vitro mechanism-based biochem-
ical assay was employed to assess their a-glucosidase (source:
Saccharomyces cerevisiae) inhibitory activity. Compounds 7, 14,
20, 21 and 26 were found to be more active (IC50 ¼ 49.7 0.4,
318.8 3.7, 23.5 0.9, 302.0 7.3 and 230.7 4.0 mM, respect-
ively) than the standard drug, acarbose (IC50 ¼ 840.0 1.73 mM
(obtained) and 780 0.028 mM (reported15)). The cytotoxicity of
these compounds was also evaluated. In addition, this series of
compounds was also screened for antiglycation and dipeptidyl
peptidase-IV (DPP-IV) inhibitory activities.
Both yeast and human a-glucosidase share about 40%
sequence homology16. As a result, inhibitors of yeast a-glucosi-
dase may also inhibit a-glucosidase enzyme obtained from human
and other sources. Based on this assumption, we checked
compounds 3–27 for the inhibition of a-glucosidase obtained
from mammalian source (rat-intestinal acetone powder). We
observed that compounds 20 and 22 possess good inhibitory activ-
ity with the IC50 ¼ 175.4 3.17 and 173.9 3.8 mM, respect-
ively, if compared with standard acarbose (IC50 ¼ 67.1 4.96 mM
(observed) and IC50 ¼ 52.8 mM or 34.11 mg/mL (reported17)).
However, the inhibitory activity of these compounds against
human a-glucosidase remained to be studied.
NH
1- C
H OH, CH COOH
Reflux, 3-4 hrs
2
5 3
O
N
R
2
+
H
R
2- C
H
OH, CH COOH
HO
2
5
3
HO
Sonication (0.5- 1 hr)
1
2a-y
3-27
Conventional method: About 1.0 mmol of aldehyde and
ethanol (15 mL) were mixed together, along with the 0.8 mmol
of acetic acid (0.05 mL). The reaction mixture was heated at
reflux for 10–15 min, followed by the addition of tyramine
(1 mmol or 0.137 g). The reaction mixture was allowed to reflux,
and progress of the reaction was monitored by TLC. Upon
completion (3–4 h), the solvent was evaporated under vacuo. The
reaction mixture was washed with a mixture of hexanes-ethyl
acetate (8:2), followed by washing with hexane. The solid product
obtained was pure enough to be analyzed by spectroscopic
techniques, such as 1H- and 13C-NMR, IR and mass spectroscopy.
In some cases, the compounds were purified by re-crystallization
or column chromatography.
Ultrasonication method: A reaction flask containing aldehyde
(1.0 mmol), 15 mL ethanol and 0.05 mL acetic acid (0.8 mmol),
was immersed in an ultrasonic bath containing water at room
temperature. The mixture was exposed to ultrasound irradiation
for varying time durations (Table 1). The reaction progress was
monitored by TLC. After completion, the solvent was evaporated
and washed with a mixture of hexanes and ethyl acetate to afford
the desired pure products, similar reaction was performed with
heating (80 ꢁC) as well.
Experimental
(3). 1H-NMR
Chemistry
4-(2-(2-Nitrobenzylideneamino)ethyl)phenol
(300 MHz, DMSO-d6): d 9.11 (s, 1H, OH), 8.47 (s, 1H, CH),
8.01 (d, J ¼ 7.8 Hz, 1H), 7.94 (d, J ¼ 6.9 Hz, 1H), 7.80 (t,
J ¼ 7.2 Hz, 1H), 7.78 (q, J ¼ 1.2 Hz, 1H), 7.04 (d, J ¼ 8.4 Hz, 2H),
6.66 (d, J ¼ 8.1 Hz, 2H), 3.81 (t, J ¼ 6.9 Hz, 2H), 2.82 (t,
J ¼ 7.2 Hz, 2H); 13C-NMR (75 MHz, DMSO-d6): d 156.9 (C-2),
155.5 (C-40), 148.8 (C-200), 133.4 (C-500), 131.1 (C-100), 130.0 (C-
10), 129.7 (C-20, C-60), 129.6 (C-600), 129.3 (C-400), 124. 1 (C-300),
115.0 (C-30, 50), 62.3 (C-80), 35.7 (C-70); MS (direct probe,
positive EI) m/z 270.1 (M+), 163.1, 107.1; HREI-MS: Calculated
for C15H14N2O3; 270.1004, Observed; 270.1003; ꢁmax; 3421.5,
3080.1, 2927.7, 1608, 1517.9, 1338.5, 1253.6, 1105.1, 825.5.
General experimental conditions
Electron impact mass spectra (EI-MS) were recorded on a JEOL
JMS-600H mass spectrometer with a MASPEC data system. The
1H-NMR spectra were recorded in deuterated solvents on Bruker
Avance 300, 400, 500 and 600 MHz NMR spectrometers. The
chemical shifts (ꢀ) and coupling constants (J) were measured in
ppm and Hz, respectively, relative to SiMe4 as internal standard.
Thin-layer chromatography (TLC) analyses were performed on
precoated ALUGRAM, SIL G/UV254 aluminum plates (Kieselgel
60, 20 ꢀ 20, 0.5 mm thick, E. Merk, Germany). The reagents and
solvents were obtained from Aldrich (St. Louis, Missouri (MO)), E.
Merck Darmstadt, (Germany), and Fluka (Buchs, Switzerland).
Reagents and solvents were used without purification.
4-(2-(4-Nitrobenzylideneamino)ethyl)phenol
(300 MHz, CDCl3): d 8.25 (d, J ¼ 8.7 Hz, 2H), 8.16 (s, 1H),
(4). 1H-NMR
Table 1. Comparison between sonication and conventional synthesis of selected tyramine derivatives.
Conventional Ultrasonic with heating Ultrasonic without heating
Compound
Time (hours)
Yield %
Time (hours)
Yield %
Time (hours)
Yield %
3
4
5
6
7
8
9
4
4
4
4
4
4
4
87
85
91
81
84
88
90
1.0
0.30
1.0
1.0
0.30
1.0
88
84
90
90
85
91
87
2
1
82
82
90
85
84
89
85
3.5
1.0
1.0
1.0
1.0
0.58

DOI: 10.3109/14756366.2016.1142983
Ultrasonic synthesis of tyramine derivatives
3
7.84 (d, J ¼ 8.7 Hz, 2H), 7.06 (d, J ¼ 8.1 Hz, 2H), 6.74 (d, 225.1154, Observed; 225.1136; ꢁmax; 3417.6, 2927.7, 1645.2,
J ¼ 8.4 Hz, 2H), 3.88 (t, J ¼ 6.3 Hz, 2H), 2.98 (t, J ¼ 7.2 Hz, 2H); 1512.1, 1450.4, 1380.9, 1243.1, 823.5.
13C-NMR (100 MHz, DMSO-d6): d 159.5 (C-2), 155.5 (C-40),
148.4 (C-400), 141.6 (C-100), 129.6 (C-200,600), 129.6 (C-10), 128.7
(C-20, 60), 123.8 (C-300, 500), 115.0 (C-30, 50), 62.4 (C-80), 35.8
(C-70); MS (direct probe, positive EI) m/z 270.1 (M+), 163.0,
133.1, 107.0; HREI-MS: Calculated for C15H14N2O3; 270.1004,
Observed; 270.1011; ꢁmax; 3222.8, 3047.3, 1508.2, 1244.0, 981.7,
827.4.
4-(2-(3-Chlorobenzylideneamino)ethyl)phenol (10). 1H-NMR
(300 MHz, DMSO-d6): d 9.13 (s, 1H), 8.23 (s, 1H), 7.73 (s,
1H), 7.66 (d, J ¼ 7.2 Hz, 1H), 7.52 (t, J ¼ 8.1 Hz, 2H), 7.46 (t,
J ¼ 7.8 Hz, 1H), 7.02 (t, J ¼ 8.1 Hz, 2H), 6.66 (d, J ¼ 8.4 Hz, 2H),
3.76 (t, J ¼ 7.2 Hz, 2H), 2.82 (t, J ¼ 7.2 Hz, 2H); 13C-NMR
(100 MHz, DMSO-d6): d 160.0 (C-2), 155.5 (C-40), 138.2 (C-100),
133.5 (C-300), 130.6 (C-10), 130.2 (C-400), 129.7 (C-50), 129.6
(C-20, 60), 127.0 (C-600), 126.5 (C-50), 115.0 (C-30, 50), 62.2 (C-80),
35.9 (C-70); MS (direct probe, positive EI) m/z 261.2 ([M + 2]+),
259.2 (M+), 152.1, 125.1, 107.0; HREI-MS: Calculated for
C15H14ClNO; 259.0764, Observed; 259.0764; ꢁmax; 3334.7,
2923.9, 1602.7, 1514.0, 1382.9, 1261.4, 821.6.
4-(2-(3-Nitrobenzylideneamino)ethyl)phenol
(5). 1H-NMR
(600 MHz, DMSO-d6): d 9.14 (s, 1H), 8.55 (t, J ¼ 1.2 Hz, 1H),
8.39 (s, 1H), 8.29 (dd, J ¼ 1.2 Hz, 1H), 8.14 (d, J ¼ 7.8 Hz, 1H),
7.74 (t, J ¼ 7.8 Hz, 1H), 7.02 (d, J ¼ 8.4 Hz, 2H), 6.65 (d,
J ¼ 8.4 Hz, 2H), 3.80 (t, J ¼ 7.2 Hz, 2H), 2.84 (t, J ¼ 7.8 Hz, 2H);
13C-NMR (100 MHz, DMSO-d6): d 159.2 (C-2), 155.5 (C-40),
148.1 (C-300), 137.6 (C-10), 133.9 (C-600), 130.8 (C-10), 129.7 (C-
20, 60), 124.9 (C-500), 121.8 (C-400), 115.0 (C-30, 50), 62.2 (C-80),
35.8 (C-70); MS (direct probe, positive EI) m/z 270.3 (M+), 163.1,
135.1, 107.1; HREI-MS: Calculated for C15H14N2O3; 270.1004,
Observed; 270.0979; ꢁmax; 3085.9, 2931.6, 1641.3, 1517.9,
1348.1, 1240.1, 1099.3, 821.6.
4-(2-(3-Methylbenzylideneamino)ethyl)phenol (11). 1H-NMR
(300 MHz, DMSO-d6): d 9.12 (s, 1H, OH), 8.18 (s, 1H), 7.52
(s, 1H), 7.48 (d, J ’¼ 7.5 Hz, 1H), 7.33 (t, J ¼ 7.5 Hz, 1H), 7.26
(d, J ¼ 7.5 Hz, 1H), 7.02 (t, J ¼ 8.4 Hz, 2H), 6.67 (t, J ¼ 4.5 Hz,
2H), 3.72 (t, J ¼ 7.2 Hz, 2H), 2.81 (t, J ¼ 7.2 Hz, 2H), 2.32 (s, 3H);
13C-NMR (75 MHz, DMSO-d6): d 160.8 (C-2), 155.4 (C-40),
137.8 (C-100), 136.1 (C-300), 131.1 (C-10), 129.8 (C-400), 129.6
(C-20, 60), 128.5 (C-200), 128.0 (C-500), 125.1 (C-600), 114.9 (C-30,
50), 62.4 (C-80), 36.0 (C-70), 20.8 (CH3); MS (direct probe,
positive EI) m/z 239.2 (M+), 132.1, 105.1; HREI-MS: Calculated
for C16H17NO; 239.1310, Observed; 239.1294; ꢁmax; 2916.2,
1639.4, 1514.0, 1450.4, 1244.0, 1043.4, 815.8.
4-(2-(2-Bromo-4-methylbenzylideneamino)ethyl)phenol (6). 1H-
NMR (300 MHz, DMSO-d6): d 9.12 (s, 1H), 8.4 (s, 1H), 7.82 (d,
J ¼ 7.8 Hz, 1H), 7.48 (s, 1H), 7.25 (d, J ¼ 8.1 Hz, 1H), 7.02 (d,
J ¼ 8.4 Hz, 2H), 6.65 (d, J ¼ 8.4 Hz, 2H), 3.79 (t, J ¼ 6.9 Hz, 2H),
2.82 (t, J ¼ 7.2 Hz, 2H), 2.31 (s, 3H) MS (direct probe, positive
EI) m/z 319.0 ([M + 2]+), 317.0 (M+), 209.9, 184.9, 107.0; HREI-
MS: Calculated for C16H16BrNO; 317.0415, Observed 317.0406;
4-(2-(2-Methylbenzylideneamino)ethyl)phenol (12). 1H-NMR
(300 MHz, DMSO-d6): d 9.12 (s, 1H), 8.44 (s, 1H), 7.75 (d,
J ¼ 7.5 Hz, 1H), 7.32 (t, J ¼ 6.6 Hz, 1H), 7.24 (t, J ¼ 9.9 Hz, 2H),
7.03 (d, J ¼ 8.1 Hz, 2H), 6.66 (d, 2H, J ¼ 8.1 Hz, 2H), 3.78 (t, J’ ¼
7.2 Hz, 2H), 2.82 (t, J ¼ 7.2 Hz, 2H), 2.38 (s, 3H); 13C-NMR
(75 MHz, DMSO-d6): d 159.6 (C-2), 155.4 (C-40), 137.2 (C-200),
133.9 (C-100), 130.7 (C-10), 129.9 (C-400), 129.9 (C-600), 129.7 (C-
20, 60), 127.1 (C-300), 125.9 (C-500), 114.9 (C-30, 50), 62.8 (C-80),
36.1 (C-70), 18.8 (C-CH3); MS (direct probe, positive EI) m/z
239.2 (M+), 132.1, 105.1; HREI-MS: Calculated for C16H17NO;
239.1310, Observed; 239.1327; ꢁmax; 2916.2, 2873.7, 1602.7,
1510.2, 1456.2, 1247.9, 817.8.
ꢁ
max; 3334.7, 2927.7, 1596.9, 1508.2, 1261.4, 823.5.
4-(2-(4-Chlorobenzylideneamino)ethyl)phenol
(7). 1H-NMR
(600 MHz, DMSO-d6): d 9.13 (s, 1H), 8.23 (s, 1H), 7.72 (d,
J ¼ 9.0 Hz, 2H), 7.01 (d, J ¼ 8.4 Hz, 2H), 6.64 (d, J ¼ 8.4 Hz, 2H),
3.74 (t, J ¼ 7.2 Hz, 2H), 2.79 (t, J ¼ 7.8 Hz, 2H); 13C-NMR
(75 MHz, DMSO-d6): d 159.7 (C-2), 155.5 (C-40), 135.1 (C-400),
135.0 (C-100), 129.9 (C-10), 129.7 (C-20, 60), 129.4 (C-20, 60), 128.8
(C-300, 500), 115.0 (C-30, 50), 62.3 (C-80), 36.0 (C-70); MS (direct
probe, positive EI) m/z 261.1 ([M + 2]+), 259.1 (M+), 152.1,
125.0, 107.0; HREI-MS: Calculated for C15H14ClNO; 259.0764,
Observed; 259.0763; ꢁmax; 3417.6, 1643.2, 1506.3, 1238.2,
1091.6, 819.7.
4-(2-(4-Methoxybenzylideneamino)ethyl)phenol (13). 1H-NMR
(300 MHz, DMSO-d6): d 9.11 (s, 1H), 8.15 (s, 1H), 7.65 (d,
J ¼ 8.7 Hz, 2H), 7.02 (q, J ¼ 8.4 Hz, 4H), 6.65 (d, J ¼ 8.4 Hz, 2H),
3.78 (s, 3H), 3.70 (t, J ¼ 7.2 Hz, 2H), 2.79 (t, J ¼ 7.2 Hz, 2H); 13C-
NMR (75 MHz, DMSO-d6): d 161.0 (C-400), 160.0 (C-2), 155.4
(C-40), 130.0 (C-10), 129.6 (C-200, 600), 129.3 (C-20, 60), 129.0 (C-
100), 114.9 (C-30, 50), 114.0 (C-300, 500), 62.3 (C-80), 55.2
(C-OCH3), 36.2 (C-70); MS (direct probe, positive EI) m/z 255.0
(M+), 148.1, 121.1; HREI-MS: Calculated for C16H17NO2;
255.1259, Observed; 255.1241; ꢁmax; 2923.9, 1606.6, 1512.1,
1259.4, 1031.8, 821.6.
4-(2-(2-Chlorobenzylideneamino)ethyl)phenol
(8). 1H-NMR
(600 MHz, DMSO-d6): d 9.13 (s, 1H), 8.53 (s, 1H), 7.94 (dd,
J ¼ 1.8 Hz, 1H), 7.49 (m, J ¼ 1.2 Hz, 2H), 7.40 (m, 1H), 7.02 (d,
J ¼ 7.8 Hz, 2H), 6.65 (t, J ¼ 3.0 Hz, 2H), 3.81 (q, J ¼ 6.6 Hz, 2H),
2.82 (t, J ¼ 7.2 Hz, 2H); 13C-NMR (100 MHz, DMSO-d6): d 157.0
(C-80), 155.5 (C-40), 133.8 (C-10), 132.7 (C-200), 132.0 (C-400),
129.8 (C-10), 129.7 (C-20, 60), 128.0 (C-600), 127.4 (C-500), 115.0
(C-30, 50), 62.5 (C-80), 35.8 (C-70); MS (direct probe, positive EI)
m/z 261.1 ([M + 2]+), 259.1 (M+), 156.0, 139.0, 108.0; HREI-MS:
Calculated for C15H14ClNO; 259.0764, Observed; 259.0744;
ꢁ
max; 2929.7, 1616.2, 1510.2, 1448.4, 1247.9, 1029.9, 812.0.
4-(2-(4-(Benzyloxy)benzylideneamino)ethyl)phenol
(14). 1H-
NMR (300 MHz, DMSO-d6): d 9.13 (s, 1H), 8.14 (s, 1H), 7.64
(d, J ¼ 8.7 Hz, 2H), 7.46 (t, J ¼ 6.9 Hz, 2H), 7.41 (t, J ¼ 6.6 Hz,
2H), 7.36 (t, J ¼ 5.4 Hz, 1H), 7.06 (q, 4H), 6.64 (d, J ¼ 8.1 Hz,
2H), 5.14 (s, 2H), 3.70 (t, J ¼ 7.2 Hz, 2H), 2.78 (t, J ¼ 7.2 Hz, 2H);
13C-NMR (75 MHz, DMSO-d6): d 160.1 (C-400), 159.9 (C-2),
155.4 (C-40), 136.7 (C-100), 129.9 (C-10), 129.6 (C-20, 60), 129.3
(C-200, 600), 129.1 (C-100), 128.4 (C-300, 500), 127.8 (C-400), 127.7 (C-
200, 600), 114.9 (C-30, 50), 114.8 (C-300, 500), 69.3 (C-2a), 62.4 (C-80),
36.2 (C-70); MS (direct probe, positive EI) m/z 331.1 (M+), 224.1,
107.0, 91.1; HREI-MS: Calculated for C22H21NO2; 331.1572,
4-(2-(Benzylideneamino)ethyl)phenol (9). 1H-NMR (400 MHz,
00
00
DMSO-d6): d 8.13 (s, 1H), 7.68 (q, J6 , 5 ¼ 1.5 Hz, J ¼ 6.0 Hz,
2H), 7.39 (dd, J ¼ 1.5 Hz, 3H), 7.08 (d, J ¼ 8.0 Hz, 2H), 6.73
(d, J ¼ 8.0 Hz, 2H), 3.82 (t, J ¼ 7.5 Hz, 2H), 2.94 (t, J ¼ 7.5 Hz,
2H); 13C-NMR (75 MHz, DMSO-d6): d 160.8 (C-2), 155.5 (C-
40), 136.1 (C-100), 130.5 (C-10), 130.0 (C-400), 129.7 (C-20, 60),
128.6 (C-200, 600), 127.7 (C-300, 500), 115.0 (C-30, 50), 62.4 (C-80),
36.1 (C-70); MS (direct probe, positive EI) m/z 225.1 (M+),
118.0, 107.0, 91.0; HREI-MS: Calculated for C15H15NO;

4
H. Siddiqui et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Observed; 331.1563; ꢁmax; 3429.2, 2860.2, 1647.1, 1606.6, 116.0, 106.9; HREI-MS: Calculated for C16H14N2O; 250.1106,
1255.6, 1037.6, 825.5.
Observed; 250.1100; ꢁmax; 3435.0, 2925.8, 1641.3, 1508.2,
1448.4, 1377.1, 1238.2, 1109.0, 829.3.
4-(2-(2-Methoxybenzylideneamino)ethyl)phenol (15). 1H-NMR
(300 MHz, DMSO-d6): d 9.11 (s, 1H), 8.53 (s, 1H), 7.81 (dd, 4-(2-(Naphthalen-1-ylmethyleneamino)ethyl)phenol (19). 1H-
J ¼ 1.2 Hz, 1H), 7.44 (m, J ¼ 1.5 Hz, 1H), 7.07 (m, 4H), 6.69 (t, NMR (300 MHz, DMSO-d6): d 9.11 (s, 1H), 8.99 (t, J ¼ 2.4 Hz,
J ¼ 13.2 Hz, 2H), 3.74 (t, J ¼ 7.2 Hz, 2H), 2.79 (t, J ¼ 7.2 Hz, 1H), 8.84 (s, 1H), 8.02 (q, J ¼ 8.1 Hz, 2H), 7.87 (d,
13
2H); C-NMR (100 MHz, DMSO-d6): d 158.3 (C-200), 155.9 J ¼ 6.9 Hz, 1H), 7.59 (m, 3H), 7.09 (d, J ¼ 8.4 Hz, 2H), 6.67 (d,
(C-2), 155.5 (C-40), 132.0 (C-400), 130.0 (C-10), 130.0 (C-20, 60), J ¼ 6.9 Hz, 1H), 3.89 (t, J ¼ 6.9 Hz, 2H), 2.91 (t, J ¼ 7.2 Hz, 2H);
126.5 (C-600), 124.0 (C-10), 120.5 (C-500), 115.0 (30, 50), 111.8 13C-NMR (75 MHz, DMSO-d6): d 161.1 (C-2), 155.5 (C-40),
(C-300), 63.0 (C-80), 55.6 (C-CH3), 36.5 (C-70); MS (direct 133.4 (C-100), 131.3 (C-1000), 130.7 (C-10), 130.6 (C-400), 129.9 (C-
probe, positive EI) m/z 255.0 (M+), 148.0, 121.0; HREI-MS: 900), 129.8 (C-20, 60), 129.0 (C-500), 128.5 (C-300), 127.1 (C-600),
Calculated for C16H17NO2; 255.1259, Observed; 255.1243; 126.1 (C-700), 125.4 (C-200), 124.6 (C-800), 115.0 (C-30, 50), 63.3
ꢁ
825.5.
max; 3446.6, 2902.7, 1602.7, 1456.2, 1380.9, 1249.8, 1026.1, (C-80), 36.2 (C-70); MS (direct probe, positive EI) m/z 275.2 (M+),
168.0, 141.1; HREI-MS: Calculated for C19H17NO; 275.1310,
Observed; 275.1331; ꢁmax: 3431.1, 2918.1, 1639.4, 1508.2,
1444.6, 1338.5, 1232.4, 1103.2, 813.9.
4-(2-(3-Methoxybenzylideneamino)ethyl)phenol (16). 1H-NMR
(300 MHz, DMSO-d6): d 9.14 (s, 1H, OH), 8.20 (s, 1H, CH),
7.36 (t, J ¼ 7.5 Hz, 1H), 7.26 (d, J ¼ 8.4 Hz, 2H), 7.03 (d,
J ¼ 8.4 Hz, 4H), 6.66 (d, J ¼ 8.4 Hz, 3H), 3.77 (s, 3H), 3.74 (t,
J80,70 ¼ 7.5 Hz, 2H), 2.81 (t, J70,80 ¼ 7.2 Hz, 2H); 13C-NMR
(75 MHz, DMSO-d6): d 160.7 (C-2), 159.4 (C-300), 155.4 (C-40),
137.6 (C-10), 129.8 (C-10), 129.7 (C-50), 129.6 (C-20, 60),
120.6 (C-600), 116.6 (C-400), 114.9 (C-30, 50), 111.9 (C-200),
62.3 (C-80), 55.1 (C-OCH3), 36.0 (C-70); MS (direct probe,
positive EI) m/z 255.1 (M+), 148.0, 121.0, 107.0; HREI-MS:
4-(2-(4-(Dimethylamino)benzylideneamino)ethyl)phenol
(20). 1H-NMR (300 MHz, DMSO-d6): d 9.09 (s, 1H), 8.05 (s,
1H), 7.50 (d, J ¼ 8.7 Hz, 2H), 7.02 (d, J ¼ 8.4 Hz, 2H), 6.71 (q,
J ¼ 8.7 Hz, 4H), 3.66 (t, J ¼ 7.5 Hz, 2H), 2.77 (t, J ¼ 7.2 Hz, 2H);
13C-NMR (100 MHz, DMSO-d6): d 160.2 (C-2), 155.4 (C-40),
151.7 (C-200, 400), 130.1 (C-10), 129.6 (C-20, 60), 129.0 (C-200, 600),
124.0 (C-100), 114.9 (C-30, 50), 111.5 (C-300, 500), 62.5 (C-80), 40.2
(N(CH3)2), 36.4 (C-70); MS (direct probe, positive EI) m/z 268.2
(M+), 161.0, 146.1, 134.1, 117.0; HREI-MS: Calculated for
Calculated for C16H17NO2; 255.1259, Observed; 255.1247; ꢁmax
;
C17H20N2O; 268.1576, Observed; 268.1596; ꢁmax; 2898.8,
2852.5, 1610.5, 1514.0, 1452.3, 1373.2, 1242.1, 1178.4, 812.0.
3421.5, 2935.5, 1639.4, 1585.4, 1510.2, 1271.0,1242.1, 1039.6,
819.7.
2-((4-Hydroxyphenethylimino)methyl)phenol
(21). 1H-NMR
4-(2-(4-Methylbenzylideneamino)ethyl)phenol (17). 1H-NMR
(300 MHz, DMSO-d6): d 9.13 (s, 1H, OH), 8.18 (s, 1H, CH),
7.59 (d, J ¼ 7.8 Hz, 2H), 7.24 (d, J ¼ 7.8 Hz, 2H), 7.02 (d,
J ¼ 8.4 Hz, 2H), 6.65 (d, J ¼ 8.4 Hz, 2H), 3.73 (t, J ¼ 7.2 Hz, 2H),
2.80 (t, J ¼ 7.2 Hz, 2H), 2.32 (s, 3H, CH3); 13C-NMR (75 MHz,
DMSO-d6): d 160.6 (C-2), 155.5 (C-40), 140.2 (C-400), 133.6
(C-100), 129.9 (C-10), 1129.7 (20, 60), 129.2 (200, 600), 127.7 (C-300,
500), 114.9 (C-30, 50), 62.4 (C-80), 36.1 (C-70), 21.0 (CH3); MS
(direct probe, positive EI) m/z 239.1 (M+), 132.0, 105.0; HREI-
MS: Calculated for C16H17NO; 239.1310, Observed; 239.1305;
(300 MHz, DMSO-d6): d 13.52 (s, 1H), 9.15 (s, 1H), 8.44 (s,
1H), 7.37 (d, J ¼ 6.6 Hz, 1H), 7.32 (m, 1H), 7.03 (d, J ¼ 8.4 Hz,
1H), 6.87 (t, J ¼ 6.9 Hz, 2H), 6.67 (d, J ¼ 8.4 Hz, 2H), 3.79 (t,
J ¼ 6.9 Hz, 2H), 2.84 (t, J ¼ 7.2 Hz, 2H); 13C-NMR (75 MHz,
DMSO-d6): d 165.8 (C-200), 160.8 (C-2), 155.6 (C-40), 132.2 (C-
600), 131.5 (C-10), 129.6 (C20, 60), 129.3 (C-400), 118.5 (C-10),
118.3 (C-500), 116.4 (C-300), 115.1 (C-30, 50), 60.0 (C-80), 35.8 (C-
70); MS (direct probe, positive EI) m/z 241.2 (M+), 134.1, 107.1;
HREI-MS: Calculated for C15H15NO2; 241.1103, Observed;
241.1093; ꢁmax; 3377.1, 2925.8, 1635.5, 1448.4, 1380.9, 1230.5,
1159.1, 1004.8, 839.0.
ꢁmax; 3421.6, 2937.3, 2852.5, 1645.2, 1510.2, 1452.3, 1380.9,
1238.2, 815.8.
X-ray crystallographic analysis of compound 17: C16H17N1O1,
Mr ¼ 239.31,
Orthorhomibic,
space
group
˚
P212121,
5-((4-Hydroxyphenethylimino)methyl)-2-methoxyphenol
(22). 1H-NMR (300 MHz, DMSO-d6): d 9.10 (s, 1H), 8.09 (s,
1H), 7.29 (s, 1H), 7.08 (m, 3H), 6.80 (d, J ¼ 8.1 Hz, 1H), 6.66 (d,
J ¼ 8.4 Hz, 2H), 3.78 (s, 3H), 3.68 (t, J ¼ 7.2 Hz, 2H), 2.78 (t,
J ¼ 7.5 Hz, 2H); 271.0, 164.0; 13C-NMR (100 MHz, DMSO-d6): d
160.3 (C-2), 155.4 (C-40), 149.1 (C-400), 147.8 (C-500), 130.0 (C-
100), 129.6 (C-20, 60), 127.9 (C-10), 122.4 (C-200), 115.1 (C-300),
114.9 (C-30, 50), 109.8 (C-600), 62.3 (C-80), 55.4 (OCH3), 36.3
(C-70); MS (direct probe, positive EI) m/z 271.0 (M+), 164.0, 132,
107, 82.9; HREI-MS: Calculated for C16H17NO3; 271.1208,
Observed; 271.1214; ꢁmax; 3440.8, 2956.7, 1662.5, 1585.4,
1515.9, 1431.1, 1323.1, 1232.4, 1128.3, 1020.3, 821.6.
˚
˚
a ¼ 5.6936(10) A, b ¼ 14.614(3) A, c ¼ 15.597(3) A, a3 ¼ b ¼
ꢁ
˚
g ¼ 90 , V ¼ 1297.8(4) A3, Z ¼ 4, ꢂcalc ¼ 1.225 mg/m3, F(000)
˚
¼ 512, Mo Kao ¼ 0.71073 A, max/min transmission 0.9909/
0.9768, crystal dimensions 0.31 ꢀ 0.23 ꢀ 0.12, 1.91ꢁ 5y525.48ꢁ,
7105 reflections were collected, of which 2360 reflections were
observed (Rint ¼ 0.0258). The R values were: R1 ¼ 0.0431,
wR2 ¼ 0.1224 for I 4 2s(I) and R1 ¼ 0.0499, wR2 ¼ 0.1649
for all data; max/min residual electron density: 0.670/ꢂ0.449e
Aꢁꢂ3. Crystallographic data for compound 17 has been deposited
in the Cambridge Crystallographic Data Center. The crystallo-
graphic information can be directly from CCDC Data Center
(CCDC 1422584).
4-(2-(2-Chloro-4-fluorobenzylideneamino)ethyl)phenol
4-(2-(4-Cyanobenzylideneamino)ethyl)phenol
(18). 1H-NMR
(23). 1H-NMR (300 MHz, CD3OD): d 8.44 (s, 1H), 7.97 (d,
J600,500 ¼ 6.3 Hz, 1H), 7.26 (dd, J ¼ 2.7 Hz, J ¼ 2.4 Hz, 1H), 7.17
(m, 1H), 7.02 (d, J ¼ 8.4 Hz, 2H), 6.69 (d, J ¼ 8.7 Hz, 2H), 3.86 (t,
J ¼ 6.9 Hz, 2H), 2.91 (t, J ¼ 6.9 Hz, 2H); 13C-NMR (75 MHz,
DMSO-d6): d 164.7 (C-400), 161.4 (C-80), 155.9 (C-40), 155.5 (C-
200), 134.8, 134.6 (C-10), 129.9 (C-600), 129.8 (C-10), 129.7 (C-20,
60), 117.1, 116.7 (C-300), 115.3 (C-500), 114.9 (C-30, 50), 62.4 (C-
80), 35.8 (C-70); MS (direct probe, positive EI) m/z 279.1
(300 MHz, DMSO-d6): d 9.12 (s, 1H), 8.32 (s, 1H), 7.92 (q,
J ¼ 8.7 Hz, J ¼ 11.1 Hz, 4H), 7.02 (d, J ¼ 8.4 Hz, 2H), 6.66 (d,
J ¼ 8.4 Hz, 2H), 3.81 (t, J ¼ 6.9 Hz, 2H), 2.84 (t, J ¼ 7.2 Hz, 2H);
13C-NMR (100 MHz, DMSO-d6): d 159.8 (C-2), 155.5 (C-40),
140.0 (C-10), 132.6 (C-10, 300, 500), 129.6 (C-20, 60), 128.3 (C-200,
600), 118.5 (C-CN), 114.9 (C-30, 50), 112.6 (C-400), 62.4 (C-80),
35.8 (C-70); MS (direct probe, positive EI) m/z 251.0 (M+), 143.0,

DOI: 10.3109/14756366.2016.1142983
Ultrasonic synthesis of tyramine derivatives
5
([M + 2]+), 277.1 (M+), 170.0, 143.0, 107.0; HREI-MS: phosphate buffer (pH 6.8) at 37 ꢁC. The enzyme (0.2 U/mL) in
Calculated for C15H13ClFNO; 277.0670, Observed; 277.0673; phosphate buffer saline was incubated with various concentrations
ꢁ
1238.2, 1029.9, 908.4, 8, 821.6.
max; 3078.2, 2916.2, 1639.4, 1596.9, 1512.1, 1450.4, 1380.9, of test compounds at 37 ꢁC for 15 min. About 0.7 mM of the
substrate, p-nitrophenyl-a-D-glucopyranoside, was added absorb-
ance change at 400 nm was recorded up to 30 min by multiplate
reader. DMSO (7.0% final) was used as control. Acarbose was
used as positive control. Following formula was used to calculate
the % inhibition:20
4-(2-(2,4-Dichlorobenzylideneamino)ethyl)phenol
(24). 1H-
NMR (400 MHz, DMSO-d6): d 9.14 (s, 1H, OH), 8.48 (s, 1H),
7.94 (d, J ¼ 8.8 Hz, 1H), 7.68 (d, J ¼ 1.6 Hz, 1H), 7.49 (dd,
J ¼ 1.6 Hz, J ¼ 1.2 Hz, 1H), 7.02 (d, J ¼ 8.4 Hz, 2H), 6.65 (d,
J ¼ 8.0 Hz, 2H), 3.82 (t, J ¼ 7.2 Hz, 2H), 2.82 (t, J ¼ 7.2 Hz, 2H);
13C-NMR (100 MHz, DMSO-d6): d 156.1 (C-2), 155.5 (C-40),
135.8 (C-100), 134.6 (C-10), 131.7 (C-200), 129.7 (C-20, 60), 129.6
(C-300), 129.3 (C-600), 127.9 (C-40), 115.0 (C-30, 50), 62.5 (C-80),
35.8 (C-70); MS (direct probe, positive EI) m/z 295.0 ([M + 2]+),
293.0 (M+), 186.0, 159.0, 107.0; HREI-MS: Calculated for
C15H13Cl2NO; 293.0374, Observed; 293.0368, ꢁmax; 2922.0,
1637.5, 1591.2, 1510.2, 1450.4, 1377.1, 1236.3, 1101.3, 1035.7,
817.8.
% Inhibition ¼ 100 ꢂ ðOD test well/OD controlÞ ꢀ 100
DPP-IV inhibitory assay
Dipeptidyl peptidase (DPP-IV) (Human), (E.C. 3.4.1.4.5) inhibi-
tory activity was evaluated by using glycine-proline-p-nitroaniline
(Gly-Pro-PNA) as substrate, and sitagliptin as standard inhibitor.
Test samples (10 mL) were pipetted onto a 96-well microplate
along with DPP-IV (0.025 /mL) and Tris-HCl buffer pH 8.0 were
preincubated for 20 min at 37 ꢁC. Reaction was initiated with the
addition of substrate (0.2 mM). The microplate was then
incubated for 50 min at 37 ꢁC. (Spectramax 384, Molecular
Devices, Sunnyvale, CA,USA).
4-Chloro-2-(((4-hydroxyphenethyl)imino)methyl)phenol
(25). 1H-NMR (300 MHz, DMSO-d6): d 13.55 (s, 1H, OH), 9.18
(s, 1H, OH), 8.42 (s, 1H), 7.47 (d, J ¼ 2.4 Hz, 1H), 7.32 (d,
J ¼ 2.4 Hz, 1H), 7.02 (d, J ¼ 8.4 Hz, 2H), 6.87 (d, J ¼ 8.8 Hz, 1H),
6.66 (d, J ¼ 8.0 Hz, 2H), 3.78 (t, J ¼ 7.2 Hz, 2H), 2.83 (t,
J ¼ 6.8 Hz, 2H); 13C-NMR (100 MHz, DMSO-d6): d 164.7 (C-
200), 160.0 (C-2), 155.7 (C-40), 131.9 (C-400), 130.5 (C-10), 129.7
(C-20, 60), 129.2 (C-600), 121.5 (C-100), 119.5 (C-500), 118.7 (C-300),
115.1 (C-30, 50), 59.8 (C-80), 35.7 (C-70); MS (direct probe,
positive EI) m/z 277.0 ([M + 2]+), 275.0 (M+), 168.0, 141.0,
107.1, 91.1, 77.1; HRESI-MS: Calculated for C15H14ClNO2;
275.0713, Observed; 275.0791; ꢁmax; 2806.2, 2607.6, 1643.2,
1515.9, 1460.0, 1259.4, 1170.7, 1016.4, 829.3.
Absorbance was measured at 405 nm. The following formula
was used to calculate the % inhibition:21
Inhibition ð%Þ ¼ ½ðA negative control ꢂ A ControlÞ
ꢂ ðA sample ꢂ A sample controlÞꢃ=
ꢀ ðA negative control ꢂ AcontrolÞ ꢀ 100%
Protein glycation inhibition assay
Bovine serum albumin (10 mg/mL), methyl glyoxal (14 mM),
various concentrations of the test compounds (prepared in DMSO,
10% final concentration) and 0.1 M phosphate buffer (pH 7.4)
containing sodium azide (30 mM) was incubated under aseptic
conditions at 37 ꢁC for 9 days. After 9 days, each sample was
examined for the development of specific fluorescence (excita-
tion, 330 nm; emission, 420 nm) against sample blank. Rutin was
used as a positive control. All of the experiments were carried out
in a 96-well microplate reader (SpectraMax M5, Molecular
Devices, USA). The percent inhibition of AGEs formation in the
test sample versus control was calculated for each inhibitor
compound by using the following formula:22
4-Fluoro-2-(((4-hydroxyphenethyl)imino)methyl)phenol
(26). 1H-NMR (300 MHz, DMSO-d6): d 13.25 (bs, 1H, OH),
9.17 (bs, 1H, OH), 8.42 (s, 1H), 7.28 (d, J ¼ 3.0 Hz, 1H), 7.19 (m,
1H), 7.03 (d, J ¼ 9.0 Hz, 2H), 6.88 (q, J ¼ 3.0 Hz, 1H), 6.67 (d,
J ¼ 9.0 Hz, 2H), 3.79 (t, J ¼ 6.0 Hz, 2H), 2.84 (t, J ¼ 9.0 Hz, 2H);
13C-NMR (75 MHz, DMSO-d6): d 164.7, 164.6 (C-2, J ¼ 3.0 Hz),
156.9, 156.9 (C-200, J ¼ 0.7 Hz), 156.0, 155.6 (C-500,
J ¼ 32.25 Hz), 152.9 (C-40), 129.6 (C-20, 60), 129.2 (C-10),
119.2, 118.9 (C-100, J ¼ 23.2 Hz), 118.6, 118.5 (C-400,
J ¼ 6.7 Hz), 117.7, 117.6 (C-300, J ¼ 7.5 Hz), 116.6, 116.3 (C-600,
J ¼ 23.2 Hz), 115.1 (C-30, 50), 60.1 (C-80), 35.7 (C-70); MS (direct
probe, positive EI) m/z 259.2 (M+), 152.1, 125.0, 106.9, 94.9,
76.9; HRESI-MS: Calculated for C15H14NO2F: 259.1009,
Observed: 259.1086; ꢁmax; 2925.8, 1614.3, 1496.7, 1454.2,
1245.9, 1151.4, 1010.6, 835.1, 767.6.
%Inhibition ¼ ð1 ꢂ fluorescence of test sample=
ꢀFluorescence of the control groupÞ ꢀ 100
2-Bromo-4-chloro-6-(((4-hydroxyphenethyl)imino)methyl)phenol
(27). 1H-NMR (300 MHz, DMSO-d6): d 14.55 (bs, 1H, OH),
9.23 (s, 1H, OH), 8.40 (s, 1H), 7.69 (d, J ¼ 3.0 Hz, 1H), 7.39 (d,
J ¼ 3.0 Hz, 1H), 7.03 (d, J ¼ 6.0 Hz, 2H), 6.68 (d, J ¼ 6.0 Hz, 2H),
3.83 (t, J ¼ 6.0 Hz, 2H), 2.88 (t, J ¼ 9.0 Hz, 2H); 13C-NMR
(100 MHz, DMSO-d6): d 165.2 (C-200), 163.8 (C-2), 155.8 (C-40),
135.6 (C-400), 130.8 (C-10), 129.6 (C-20, 60), 128.3 (C-600), 117.5
(C-500), 116.5 (C-100), 115.2 (C-30, 50), 114.9 (C-300), 55.4 (C-80),
34.9 (C-70); MS (direct probe, positive EI) m/z 357.2 ([M + 2]+),
355.2 (M+), 248.1, 221.1, 167.1, 107.1, 77.1, 44.0; HRESI-MS:
Calculated for C15H13BrClNO2; 354.6262, Observed; 355.0052;
Cytotoxicity evaluation on 3T3 cells
3-[4, 5-Dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide
(MTT) assay was used to determine the cytotoxicity of the
compounds. Mouse fibroblast 3T3 cells were grown in Dulbecco’s
modified Eagle medium (DMEM), along with 5% fetal bovine
serum (FBS), 100 mg/mL of streptomycin and 100 IU/mL of
penicillin in 75-cm2 flasks. The flasks were then incubated at
37 ꢁC in 5% CO2. Cells were harvested, followed by counting with
hemocytometer and dilution with a medium. Cell culture was
prepared with the concentration of 5 ꢀ 104 cells/mL, followed by
introduction (100 mL/well) into 96-well plates. Medium was
removed after overnight incubation. Freshly prepared medium
(200 mL) was added with various concentrations of test com-
pounds (1–30 mM). After 48 h, 200 mL MTT dye (0.5 mg/mL)
was added into all wells and again incubated for 4 h. DMSO
(100 mL) was added to each well. The formation of formazan dye
ꢁmax; 3008.7, 1637.5, 1492.8, 1369.4, 1228.6, 1139.9, 1043.4,
831.3.
ꢃ-Glucosidase inhibitory activity
a-Glucosidase (Saccharomyces cerevisiae, EC 3.2.1.20), (Sigma
catalog # G0660) inhibitory activity was evaluated by using 0.1 M

6
H. Siddiqui et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
within the cells was calculated by measuring the absorbance at
The stereochemistry of the products was deduced by NOESY-
540 nm, using a multiplate reader (SpectraMax plus, Molecular NMR and single-crystal X-ray diffraction analyses. Figure 1
Devices). The cytotoxicity was estimated as the concentration shows the key NOESY interactions in the compounds 21 and 17.
causing 50% inhibition of 3T3 cells (IC50). Following formula In compound 21, strong NOE correlations were observed between
was used to calculate the % inhibition:
the C-2 olefinic proton and the C-600 aromatic proton. NOE
between C-2 olefinic proton and methylenic protons at C-80 was
also observed. These correlations indicated an E configuration of
the C¼N double bond. The NOE correlation between the two
hydroxyls at C-40 and C-200 may well because of the intramo-
lecular chemical exchange rather than NOE18.
To further study the stereochemisty of the double bond,
NOESY experiment was performed on another member of the
series (compound 17). A similar NOESY correlation was
observed between C-2 olefinic proton and C-600/C-200 aromatic
proton and C-80 methylene protons. Single-crystal X-ray diffrac-
tion analysis of compound 17 unambiguously established the E
configuration of the C¼N double bond in compounds 3–27
(Figure 2).
% Inhibition ¼ 100 ꢂ ððmean of O:D: of test compound
ꢂ mean of O:D: of negative control=
ðmean of O:D: of positive control
ꢂ mean of O:D: of negative controlÞ ꢄ 100Þ
The data were processed by using a software called Soft-Max
Pro (Molecular Devices).23
Statistical analysis
The EZ-Fit Enzyme Kinetics Program (Perrella Scientific Inc.,
Amherst, USA) was employed to calculate the IC50 values. All the
experiments were performed in triplicate, and results are reported
as the Standard Error of Mean values.
Bioactivity
a-Glucosidase obtained from yeast has been widely used for the
identification of in vitro inhibitors of a-glucosidase enzyme.
However, mammalian a-glucosidase (rat intestinal acetone
powder) activity may serve as better test model for the identi-
fication, design and development of antidiabetic agents,
because of its close similarities with the mammalian sys-
tems. a-Glucosidase enzymes from bacterial, yeast and
mammalian sources are known to have fairly conserved active
sites19. Therefore, compounds 3–27 have been studied on both
assays.
Compounds 3–27 were evaluated for their a-glucosidase
(Saccharomyces cerevisiae) inhibitory activity in vitro
(Table 2). Compounds 7, 14, 20, 21 and 26 were found to
be more active (IC50 ¼49.7 0.4, 318.8 3.7, 23.5 0.9,
302.0 7.3 and 230.7 4.0 mM, respectively) than the standard
drug, acarbose (IC50 ¼ 840.0 1.73 mM), while other members of
the series exhibited less than 50% inhibition. Compounds 3–27
were also screened against a-glucosidase obtained from mamma-
lian source (rat-intestinal acetone powder) also. We observed that
Results and discussion
Chemistry
Schiff base derivatives of tyramine 3–27 were synthesized in a
single step by using various aldehydes. Both conventional and
ultrasonic methods were used for the synthesis. In the conven-
tional method, resulting compounds were purified by solvent–
solvent extraction and obtained in good to excellent yields. In a
few cases, the compounds were purified by using normal phase
column chromatography. It was observed that ultrasonic proced-
ure (with or without heating) not only provides excellent yields,
but no major purification of resultant compounds was required.
After completion of the reaction, the solvent was evaporated, and
the washing yielded the resultant products. Table 1 shows
comparison of reaction time and yields between the conventional
and sonication (with or without heating) techniques for selected
compounds.
Figure 1. Key NOESY correlations of
compounds 17 and 21.
5'
5'
HO 4'
HO 4'
6'
6'
H
H
H
H
8'
8'
6''
1'
3'
6''
1'
1''
2''
3'
1''
2''
5''
4''
CH3
2
5''
4''
N
2
N
2'
7'
2'
7'
1
1
HO
3''
3''
17
21
Figure 2. ORTEP drawing of the
single-crystal X-ray structure of
compound 17.

DOI: 10.3109/14756366.2016.1142983
Ultrasonic synthesis of tyramine derivatives
7
Table 2. a-glucosidase inhibitory activity, and cytotoxicity of compounds 3–27.
a-Glucosidase
Mammalian Rat-intestinal
acetone powder
Saccharomyces cerevisiae
S. #.
3
-R Group
Yield %
87
IC50 SEM [mM]
IC50 SEM [mM]
Cytotoxicity [mM]
430
–
–
NO
2
4
5
85
91
–
–
–
–
430
430
NO
2
NO
2
6
81
–
–
430
CH
3
Br
7
8
84
88
49.7 0.4
–
–
430
430
Cl
–
Cl
9
90
83
–
–
–
–
430
430
10
Cl
11
12
81
83
–
–
–
–
430
430
CH
3
CH
3
13
14
86
75
–
–
–
430
430
OCH
3
318.8 3.69
O
(continued )

8
H. Siddiqui et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Table 2. Continued
a-Glucosidase
Mammalian Rat-intestinal
Saccharomyces cerevisiae
acetone powder
IC50 SEM [mM]
–
S. #.
15
-R Group
Yield %
78
IC50 SEM [mM]
Cytotoxicity [mM]
430
–
OCH
3
16
81
–
–
430
OCH
CH
3
17
18
19
85
87
90
–
–
–
–
–
–
430
430
430
3
CN
20
21
79
89
23.5 0.9
175.4 3.17
430
430
N(CH )
3 2
302.0 7.3
–
OH
22
23
78
81
–
–
173.9 3.8
430
430
OH
OCH
F
3
–
Cl
24
25
76
84
–
–
–
–
430
Cl
Cl
–
Cl
OH
(continued )

DOI: 10.3109/14756366.2016.1142983
Ultrasonic synthesis of tyramine derivatives
9
a-Glucosidase
Mammalian Rat-intestinal
acetone powder
Saccharomyces cerevisiae
IC50 SEM [mM]
230.7 4.0
S. #.
26
-R Group
Yield %
91
IC50 SEM [mM]
Cytotoxicity [mM]
–
–
F
OH
27
88
–
–
–
Cl
Br
OH
Tyramine (Substrate for the synthesis)
Acarbose (Standard)
Acarbose (Standard) Reported Value
Cycloheximide (Standard)
–
–
–
–
–
–
840.0 1.73
780 0.028
–
67.1 4.96
52.8 mM or 34.11 mg/mL
–
0.26 0.1
SEM: Standard Error of Mean.
Table 3. Antiglycation activityof compounds
3–27.
Table 4. The mode of inhibition of compounds 7, 20 and 26.
Compound
Ki (mM)
Km
Type of inhibition
Compound
IC50 SEM [mM]
7
20
60
9
89
736
0.89
0.9
0.9
0.9
Competitive
Competitive
Competitive
Competitive
3
4
5
–
–
–
26
Standard-Acarbose
6
–
7
–
8
–
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
–
–
–
–
–
–
–
–
found to be active against mammalian a-glucosidase only.
Similarly, compound 7, which was just about 2-fold less active
than compound 20, did not inhibit the mammalian a-glucosidase.
The limited data presented here suggested that the structure–
activity–relationship (SAR) for the yeast and mammalian (rat-
intestinal acetone powder) a-glucosidases is different; however,
certain correlations were also observed, for example, compound
20, the most active member of the series (IC50 ¼ 23.5 0.9 mM)
against a-glucosidase from Saccharomyces cerevisiae was
also found to be active against a-glucosidase obtained
from mammalian rat-intestinal acetone powder as well
(IC50 ¼ 175.4 3.17 mM).
–
–
–
505.27 6.0
–
581.87 5.50
–
–
–
–
Compound 20 was found to be the most active member of the
series (IC50 ¼ 23.5 0.9 mM), which possess electron-donating
dimethylamino group at the para or C-400 position. The activity
decreased to about half (IC50 ¼ 49.7 0.4 mM) when a chloro
group was present at C-400 position (as in compound 7). This may
be due to negative inductive effect of the halogens. Change in the
position of the chloro substituent from para to the ortho and meta
positions, resulted in a complete loss of activity, as in com-
pounds 8 and 10. The presence of the chloro groups at the C-400
25
26
27
Standard (Rutin)
440.58 2.74
294 1.15
SEM: Standard Error of Mean.
compounds 20 and 22 possess good inhibitory activity with the and C-200 also resulted in a complete loss of activity (as in
IC50 ¼175.4 3.17 and 173.9 3.8 mM, respectively, compared compound 24).
with standard drug, acarbose (IC50 ¼ 67.1 4.96 mM (Observed)
Compound 26 was found to be another active member of the
and IC50 ¼ 52.8 mM or 34.11 mg/mL (reported)). It is interesting to series (IC50 ¼ 230.7 4.0 mM), with hydroxyl and fluoro groups
note that compound 20 was found to be active against both at C-200 and C-500 positions, respectively. The activity decreased
mammalian and yeast a-glucosidase, while compound 22 was many folds when there was a substitution of a benzyloxy group at

10 H. Siddiqui et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
(A)
0.4
(B)
0.02
0
0.2
0
−0.2
−0.02
−0.4
−20
0
20
−140−120−100−80 −60 −40 −20
0
20 40 60 80 100 120 140
1/Substrate (4-Nitrophenyl -D-
glucopyranoside)
Compound 7
0.4
(C)
0.2
0
−0.2
−0.4
−140 −120 −100 −80 −60 −40 −20
0
20 40 60 80 100 120 140
Compound 7
Figure 3. The inhibition of a-glucosidase enzyme by compound 7, (A) is the Lineweaver–Burk plot of reciprocal of rate of reaction (velocities) versus
reciprocal of substrate (p-nitrophenyl a-D-glucopyranoside) in the absence of inhibitor and in the presence of 125.00 mM (*), 62.5 mM (ꢅ), 31.25 mM
(œ), 7.8 mM (D) and 3.9 mM (m) of compound 7. (B) Is the secondary re-plot of Lineweaver–Burk plot between the slopes of each line on Lineweaver–
Burk plot versus different concentrations of compound 7, (C) is the Dixon plot of reciprocal of rate of reaction (velocities) versus different
concentrations of compound 7.
the C-400 position (as in compound 14, IC50 ¼ 318.8 3.7 mM).
The cytotoxicity of compound 3–27 was evaluated against the
Substitution of a methoxy group at the C-400 and C-200 positions mouse fibroblast 3T3 cell lines. All compounds were turned out to
(ortho and meta) resulted in a complete loss of activity, as in be noncytotoxic (Table 2).
compounds 15 and 16. Although the methoxy group is also
Kinetic studies
electron donating but the high electro-negativity of oxygen may
be a reason for the lower activities of these compounds, in
comparison to compounds 20 and 26, which also possess electron-
donating groups.
In order to study the mechanism of inhibition, kinetics studies
were performed on the most active compounds 7, 20 and 26. The
study showed them to be competitive inhibitors with Ki values
60, 9 and 89 mM, respectively (Table 4). Lineweaver–Burk
plots were used to determine the type of inhibition. The
reciprocals of the rate of the reaction were plotted against the
reciprocal of substrate concentrations to monitor the effect of
the inhibitor on both Km and Vmax values. Figures 3, 4 and 5
showed no effect on Vmax of a-glucosidase enzyme in the
presence of different concentrations of compounds 7, 20 and
26, while the Km values increased. This showed a competitive
behavior. The secondary replots of Lineweaver–Burk plots
were plotted to determine the Ki values (Figures 3B, 4B and
Interestingly, among all the compounds with substitution at
C-200, only compound 21 showed enzyme inhibition activity
(IC50 ¼ 302.0 7.3 mM).
In order to further study the antidiabetic potential of the
compounds 3–27, they were evaluated for their protein glycation
and DPP IV inhibition activities. Only compounds 20, 22 and 27
showed weak antiglycation activity with IC50 values
505.27 5.95, 581.87 5.50 and 440.58 2.74 mM, respectively
(Table 3). All other compounds were found to be inactive in both
the assays.

(B)
(A)
1
0.8
0.04
0.02
0
0.6
0.4
0.2
0
−0.2
−0.4
−0.6
−0.8
−0.02
−0.04
−40
−20
0
20
40
−20
0
20
1/Substrate (4-Nitrophenyl -D-
glucopyranoside)
Compound 20
(C) 0.4
0.2
0
−0.2
−0.4
−40 −20
0
20
40
Compound 20
Figure 4. The inhibition of a-glucosidase enzyme by compound 20 (A) is the Lineweaver–Burk plot of reciprocal of rate of reaction (velocities) versus
reciprocal of substrate (p-nitrophenyl a-D-glucopyranoside) in the absence of inhibitor and in the presence of 31.25 mM (*), 25 mM (ꢅ), 15 mM (œ),
10 mM (g) and 7.8 mM (D) of compound 20. (B) Is the secondary re-plot of Lineweaver–Burk plot between the slopes of each line on Lineweaver–Burk
plot versus different concentrations of compound 20, (C) is the Dixon plot of reciprocal of rate of reaction (velocities) versus different concentrations of
compound 20.
(A)
(B)
0.4
0.2
0.04
0.02
0
0
−0.02
−0.04
−0.2
−0.4
−10
0
10
−0.2
0
0.2
1/S
Compound 26
(C)
0.4
0.2
0
−0.2
−0.4
−0.2
0
0.2
Compound 26
Figure 5. The inhibition of a-glucosidase enzyme by compound 26 (A) is the Lineweaver–Burk plot of reciprocal of rate of reaction (velocities) versus
reciprocal of substrate (p-nitrophenyl a-D-glucopyranoside) in the absence of inhibitor and in the presence of 200.00 mM (*), 125 mM (ꢅ), 62.5 mM
(œ), 31.25 mM (D) and 15.625 mM (m) of compound 26 (B) is the secondary replot of Lineweaver–Burk plot between the slopes of each line on
Lineweaver–Burk plot versus different concentrations of compound 26, (C) is the Dixon replot of Ki.

12 H. Siddiqui et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
in alloxan-induced diabetic rats. Am J Pharmacol Toxicol 2007;2:
178–88.
5B). By plotting the slope of each line in the Lineweaver–
Burk plots against different concentrations of compounds 7, 20
and 26, the Ki values were calculated. The Ki values were
deduced from Dixon plots, by plotting the reciprocal of the
rate of reaction against different concentrations of compounds
7, 20 and 26 (Figures 3C, 4C and 5C).
9. Patel MS, Patel DD, Patel VS, et al. Synthesis of Schiff bases of
N-based methylene derivatives. Int J Sci Res 2014;2:580–5.
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a short review of their antimicrobial activities. J Adv Res 2011;2:
1–8.
Conclusion
In conclusion, we have synthesized a class of novel a-glucosidase
inhibitors, based on a natural product template, tyramine. The
compounds reported here may serve as the starting points for the
design and development of novel a-glucosidase inhibitors as anti-
diabetic agents.
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Declaration of interest
The authors have declared that there are no conflicts of interest.
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