Inhibition of Alzheimer's BACE-1 by 2,6-dialkyl-4-chromon-3-yl-1,4-dihydropyridine-3,5-dicarboxylates

Article abstract of DOI:10.1007/s00044-015-1367-z

Alzheimer's disease is the most common cause of dementia in the elderly, and no disease-modifying therapy is yet available for this devastating pathology. Deposition of different physicochemical forms of amyloid-β peptides is a critical phase in the patho

Full text of DOI:10.1007/s00044-015-1367-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1367-z
ORIGINAL RESEARCH
Inhibition of Alzheimer’s BACE-1 by 2,6-dialkyl-4-chromon-3-yl-
1,4-dihydropyridine-3,5-dicarboxylates
Nima Razzaghi-Asl1,2 Neha Aggarwal3 Smriti Srivastava3 Virinder S. Parmar3
•
•
•
•
Ashok K. Prasad3 Ramin Miri4 Luciano Saso5 Omidreza Firuzi4
•
•
•
Received: 14 September 2014 / Accepted: 9 April 2015
Ó Springer Science+Business Media New York 2015
Abstract Alzheimer’s disease is the most common cause of
dementia in the elderly, and no disease-modifying therapy is
yet available for this devastating pathology. Deposition of
different physicochemical forms of amyloid-b peptides is a
critical phase in the pathogenesis of Alzheimer’s disease.
b-Site amyloid precursor protein cleaving enzyme 1 (BACE-
1) is a major enzyme responsible for amyloid-b production;
therefore, inhibition of this enzyme represents a promising
approach for the discovery of amyloid-b-lowering agents. In
this study, a series of novel 2,6-dialkyl-4-chromon-3-yl-1,4-
dihydropyridine-3,5-dicarboxylates (14–23) were synthesized
6.84 6.62 (23) to 51.32 1.04 (14) percent enzyme inhi-
bitions at the concentration of 10 lM. The structure–activity
relationship study showed that the presence of 4-[7-
(ethanoyloxy)-4-oxo-4H-chromen-3-yl] moiety at C4 position
of dihydropyridine ring (14, 16 and 18) confers higher activity
compared with other substitutions at this position. Docking
simulation predicted a key H-bond interaction between Asp32
residue and dihydropyridine NH group. Moreover, all docked
dihydropyridines made good hydrophobic contacts with S1
and S2 subpockets of BACE-1. A good correlation between
estimated binding affinities (pKi) and experimental BACE-1
inhibitory activities at 10 lM was obtained (R2 = 0.639). The
findings of this study suggested that 2,6-dialkyl-4-chromon-3-
yl-1,4-dihydropyridine-3,5-dicarboxylates could be promising
scaffolds for the discovery of novel BACE-1 inhibitors for
management of Alzheimer’s disease.
¨
and assessed as BACE-1 inhibitors using the Forster reso-
nance energy transfer-based enzyme assay. Synthesized di-
hydropyridines exhibited weak-to-relatively-good BACE-1
inhibitory activities. Enzyme inhibitory activities ranged from
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-015-1367-z) contains supplementary
material, which is available to authorized users.
Keywords Alzheimer Á BACE-1 Á Dihydropyridine Á
Inhibitor
& Omidreza Firuzi
foomid@yahoo.com;
firuzio@sums.ac.ir
Introduction
1
Department of Medicinal Chemistry, School of Pharmacy,
Ardabil University of Medical Sciences, Ardabil, Iran
Alzheimer’s disease (AD) is a neurodegenerative disorder
characterized by a progressive decline in memory and other
cognitive functions and is the main cause of dementia in
the elderly (McKhann et al., 1984; Verdon et al., 2007;
Reitz et al., 2011). The main neuropathological hallmarks
of AD are the presence of extracellular amyloid-b (Ab)
plaques and intracellular neurofibrillary tangles in the
brain. Accumulation of different physicochemical forms of
Ab peptides in the extracellular space of the brain is be-
lieved to begin the neurodegeneration in patients afflicted
with AD (Selkoe, 2008). Proteolytic cleavage of the large
transmembrane amyloid-b precursor protein (APP) by the
2
Drug and Advanced Sciences Research Center, School of
Pharmacy, Ardabil University of Medical Sciences, Ardabil,
Iran
3
Bioorganic Laboratory, Department of Chemistry, University
of Delhi, Delhi, 110 007, India
4
Medicinal and Natural Products Chemistry Research Center,
Shiraz University of Medical Sciences, 71348-53734 Shiraz,
Iran
5
Department of Physiology and Pharmacology ‘‘Vittorio
Erspamer’’, Sapienza University of Rome, P. le Aldo Moro 5,
00185 Rome, Italy
123

Med Chem Res
consecutive actions of b-secretase and c-secretase enzymes
results in the production of Ab peptides (Vassar, 2002).
b-Secretase (b-site APP cleaving enzyme 1 or BACE-1) is
a type I membrane-associated aspartyl protease and is the rate-
limiting enzyme in Ab production. BACE-1 is an attractive
target for the discovery of disease-modifying therapies in AD
(Sinha et al., 1999). Final generation of Ab peptides is carried
out by subsequent cleavage of APP by c-secretase; however,
inhibition of this enzyme may be associated with some
mechanism-based adverse effects (John et al., 2003).
points were determined on Buchi M-560 instrument and
are uncorrected. The IR spectra are recorded on a Perk-
inElmer model 2000 FT-IR spectrometer by making KBr
disk for solid samples and thin film for oils. The 1H and 13
C
NMR spectra were recorded on JEOL ALPHA-400 spec-
trometer at 400 MHz and 100.5 MHz, respectively, using
TMS as internal standard. The chemical shift values were
on d scale, and the coupling constants (J) were in Hz.
1
Signals from OH to NH groups in H NMR spectra were
confirmed by D2O exchange. HR-ESI-TOF-MS analyses
were carried out on a microTOF-Q instrument from Bruker
Daltonics, Bremen. Reactions were conducted under an
atmosphere of nitrogen when anhydrous solvents were
used. Analytical TLCs were performed on pre-coated
Merck silica gel 60F254 plates, and the spots were detected
under UV light. Silica gel (100–200 mesh) was used for
column chromatography.
Previous studies have demonstrated that BACE-1
knockout mice have negligible Ab production (Bassil and
Grossberg, 2009), and several research groups have gen-
erated various BACE-1 inhibitors (Silvestri, 2009; Ghosh
et al., 2012) that have lowered Ab production in vitro and
in vivo (Lahiri et al., 2014).
In the earlier attempts, peptidic BACE-1 inhibitors were
designed mimicking the conformation of substrates at
transition state (Ghosh et al., 2012), but the major draw-
back of peptidic BACE-1 inhibitors is related to their poor
pharmacokinetic profile, which prevents them from being
further developed to oral bioavailable CNS drugs. Thus,
recent attempts have been directed towards the discovery
of small molecule BACE-1 inhibitors (Silvestri, 2009).
1,4-Dihydropyridine is a privileged structure with a
wide range of pharmacological activities (Miri et al., 2008;
Kumar et al., 2011). Little attention has been directed to-
wards the BACE-1 blocking activity of these compounds
(Choi et al., 2010). Choi and coworkers have reported
novel N-methylsulfonamide-1,4-dihydropyridine deriva-
tives as BACE-1 inhibitors that exhibited IC50 values of
8–30 lM in a cell-based assay (Choi et al., 2010). We have
also previously observed that 3,5-bis-N-(aryl/heteroaryl)
carbamoyl-4-aryl-1,4-dihydropyridines show high potential
for inhibition of BACE-1 (Razzaghi-Asl et al., 2013).
In continuation of our interest in the discovery of non-
peptidic BACE-1 inhibitors (Razzaghi-Asl et al., 2013;
Edraki et al., 2013) and also to further expand the capacity
of 1,4-dihydropyridines as privileged small molecule
medicinal scaffolds (Chhillar et al., 2006, 2009; Miri et al.,
2010, 2012), we herein report the synthesis, assessment of
in vitro BACE-1 inhibitory activity and molecular docking
study of a series of 2,6-dialkyl-4-chromon-3-yl-1,4-dihy-
dropyridine-3,5-dicarboxylates.
General procedure for synthesis of compounds
14–23
The synthesis of compounds 14–23 has been achieved
starting from 2,4-dihydroxyacetophenone (1) and 2,5-di-
hydroxyacetophenone (2), which were mono-acetylated at
C4 or C5 hydroxyl group with acetic anhydride followed
by the Vilsmeier–Haack formylation reaction on resultant
acetylated compounds 3 and 4 to give 7-acetoxy-3-for-
mylchromone (5) and 6-acetoxy-3-formylchromone (6),
respectively. The chromones 5 and 6 were deacetylated
with NaOH to give the corresponding hydroxychromones 7
and 8. Hantzsch pyridine reaction on 7-hydroxy-3-for-
mylchromone (7) and 6-hydroxy-3-formylchromone (6)
with various b-ketoesters in the presence of catalytic
amount of Ba (NO3)2 yielded the desired hydroxy-
chromonyldihydropyridines 9–13 in 39–42 % overall
yields from dihydroxyacetophenones.
Further, the acylation of the chromonyldihydropyridines
9–13 with acetic and hexanoic anhydrides in THF afforded
a series of 7-O-acetoxychromonyldihydropyridines 14, 16
and 18, 7-O-hexanoylchromonyldihydropyridines 15, 17
and 19, 6-O-acetoxychromonyldihydropyridines 20 and 22
and 6-O-hexanoylchromonyldihydropyridines 21 and 23 in
70–85 % yields. Characteristic data of synthesized com-
pounds are as follows:
Dimethyl 40-(7-hydroxychromon-3-yl)-20,60-dimethyl-10,40-
dihydropyridine-30,50-dicarboxylate (9) Compound 9 was
obtained as yellow solid in 65 % yield; M.P. 283–285 °C;
IR (KBr) mmax 3314, 3244, 1663, 1626, 1599, 1506, 1340,
1223, 1124, 1019, 847 and 781 cm-1, 1H NMR (400 MHz,
DMSO): d 2.20 (6H, s), 3.56 (6H, s), 4.81 (1H, s), 6.76
(1H, d, J = 2.2 Hz), 6.87 (1H, dd, J = 8.8 and 2.2 Hz),
7.76 (1H, s), 7.84 (1H, d, J = 8.8 Hz), 8.32 (1H, s) and
Materials and methods
Chemistry
Chemicals were obtained from Sigma-Aldrich Co. (USA)
and SD Fine Ltd., Mumbai, India. AR-grade organic sol-
vents were used without any further purification. Melting
123

Med Chem Res
8.92 (1H, s); 13C NMR (100.5 MHz, DMSO): d 18.12,
32.97, 50.60, 98.00, 101.96, 114.78, 117.02, 125.43,
126.82, 146.56, 153.34, 157.16, 162.15, 167.48 and
174.80; HR-ESI-TOF-MS: m/z 408.1041 [M ? Na]?, cal-
culated for [C20H19NO7 ? Na]? 408.1054.
281–284 °C; IR (KBr) mmax 3308, 3241, 1703, 1657, 1636,
1
1622, 1471, 1333, 1218, 1116, 1094 and 748 cm-1, H
NMR (400 MHz, DMSO): d 1.10 (6H, t, J = 6.4 Hz), 2.17
(6H, s) 3.92–4.01 (4H, m), 4.78 (1H, s), 7.14 (1H, dd,
J = 8.7 and 2.8 Hz), 7.25 (1H, d, J = 2.8 Hz), 7.42–7.45
(1H, m), 7.86 (1H, s), 8.30 (1H, s) and 8.84 (1H, s); 13C
NMR (100.5 MHz, DMSO): d 14.07, 18.41, 33.18, 58.96,
98.23, 107.66, 119.40, 121.44, 122.46, 126.54, 146.32,
154.26, 154.53, 156.90, 167.09 and 174.91; HR-ESI-TOF-
MS: m/z 436.1343 [M ? Na]?, calculated for [C22H23-
NO7 ? Na]? 436.1367.
Diethyl 40-(7-hydroxychromon-3-yl)-20,60-dimethyl-10,40-
dihydropyridine-30,50-dicarboxylate (10) Compound 10
was obtained as yellow solid in 63 % yield; M.P.
241–243 °C; IR (KBr) mmax 3619, 3310, 1702, 1678, 1625,
1500, 1306, 1252, 1201, 1095, 848 and 748 cm-1 1H
,
NMR (400 MHz, DMSO): d 1.14 (6H, t, J = 7.3 Hz), 2.20
(6H, s), 3.93–4.08 (4H, m), 4.79 (1H, s), 6.78 (1H, d,
J = 2.2 Hz), 6.87 (1H, dd, J = 8.8 and 2.2 Hz), 7.78 (1H,
s), 7.85 (1H, d, J = 8.8 Hz), 8.32 (1H, s) and 8.85 (1H, s);
13C NMR (100.5 MHz, DMSO) d 14.20, 18.27, 33.24,
58.83, 98.22, 101.97, 114.73, 117.11, 125.90, 126.81,
146.43, 153.65, 157.11, 162.08, 166.99 and 174.52; HR-
ESI-TOF-MS: m/z 436.1356 [M ? Na]?, calculated for
[C22H23NO7 ? Na]? 436.1367.
Diethyl 40-(7-acetoxychromon-3-yl)-20,60-dimethyl-10,40-di-
hydropyridine-30,50-dicarboxylate (14) Compound 14
was obtained as yellow solid in 78 % yield; M.P.
197–199 °C; IR (KBr) mmax 3340, 1767, 1702, 1666, 1634,
1493, 1367, 1204, 1183, 1020, 912 and 745 cm-1 1H
,
NMR (400 MHz, CDCl3): d 1.25 (6H, t, J = 7.3 Hz), 2.28
(6H, s), 2.34 (3H, s), 4.06–4.15 (4H, m), 4.86 (1H, s), 7.10
(1H, dd, J = 8.8 and 2.2 Hz), 7.24 (1H, d, J = 2.2 Hz),
7.89 (1H, s), 8.00 (1H, s) and 8.13 (1H, d, J = 8.8 Hz); 13
C
Diethyl 40-(7-hydroxychromon-3-yl)-20,60-dipropyl-10,40-di-
hydropyridine-30,50-dicarboxylate (11) Compound 11
was obtained as yellow solid in 62 % yield; M.P.
177–179 °C; IR (KBr) mmax 3619, 3306, 2968, 1702, 1677,
NMR (100.5 MHz, CDCl3): d 14.34, 19.49, 21.10, 35.49,
59.49, 97.94, 110.94, 119.13, 122.94, 125.35, 126.73,
147.37, 153.95, 155.05, 156.36, 167.72, 168.60 and
176.34; HR-ESI-TOF-MS: m/z 478.1465 [M ? Na]?, cal-
culated for [C24H25NO8 ? Na]? 478.1472.
1624, 1499, 1300, 1245, 1198, 1099, 848 and 768 cm-1
,
1H NMR (400 MHz, DMSO) d 0.87 (6H, t, J = 7.4 Hz),
1.12 (6H, t, J = 7.3 Hz), 1.43–1.54 (4H, m), 2.39–2.46
(2H, m), 2.66–2.73 (2H, m), 3.92–4.05 (4H, m), 4.81 (1H,
s), 6.74 (1H, d, J = 2.2 Hz), 6.84 (1H, dd, J = 8.8 and
2.2 Hz), 7.66 (1H, s), 7.82 (1H, d, J = 8.8 Hz), 8.74 (1H,
s) and 10.64 (1H, s); 13C NMR (100.5 MHz, DMSO): d
13.73, 14.17, 21.88, 32.73, 32.91, 58.90, 98.08, 101.96,
114.72, 117.03, 126.09, 126.87, 150.48, 153.27, 157.11,
162.08, 166.74 and 174.42; HR-ESI-TOF-MS: m/z
492.1969 [M ? Na]?, calculated for [C26H31NO7 ? Na]?
492.1993.
Diethyl
40-(7-hexanoyloxychromon-3-yl)-20,60-dimethyl-
10,40-dihydropyridine-30,50-dicarboxylate (15) Compound
15 was obtained as yellow solid in 70 % yield; M.P.
193–195 °C; IR (KBr) mmax 3311, 2961, 1764, 1698, 1663,
1632, 1495, 1309, 1213, 1179, 1123, 1093, 846 and
1
777 cm-1, H NMR (400 MHz, CDCl3): d 0.94 (3H, t,
J = 7.0 Hz), 1.25 (6H, t, J = 7.3 Hz), 1.37–1.42 (4H, m),
1.75–1.79 (2H, m), 2.29 (6H, s), 2.59 (2H, t, J = 7.2 Hz),
4.07–4.13 (4H, m), 4.86 (1H, s), 7.08 (1H, dd, J = 8.8 and
2.2 Hz), 7.22 (1H, d, J = 2.2 Hz), 7.44 (1H, s), 7.99 (1H,
s) and 8.14 (1H, d, J = 8.8 Hz); 13C NMR (100.5 MHz,
CDCl3): d 13.87, 14.35, 19.61, 22.26, 24.44, 31.18, 34.29,
35.39, 59.44, 98.22, 110.89, 119.14, 122.88, 125.38 and
126.79, 147.05, 154.11, 154.98 and 156.38, 167.69, 171.51
and 176.32; HR-ESI-TOF-MS: m/z 534.2079 [M ? Na]?,
calculated for [C28H33NO8 ? Na]? 534.2098.
Dimethyl 40-(6-hydroxychromon-3-yl)-20,60-dimethyl-10,40-
dihydropyridine-30,50-dicarboxylate (12) Compound 12
was obtained as yellow solid in 66 % yield; M.P.[300 °C;
IR (KBr) mmax: 3363, 3234, 1705, 1655, 1621, 1503, 1472,
1342, 1216, 1115, 1022 and 773 cm-1
;
1H NMR
(400 MHz, DMSO): d 2.17 (6H, s), 3.52 (6H, s), 4.80 (1H,
s), 7.14 (1H, dd, J = 8.8 and 2.9 Hz), 7.24 (1H, d,
J = 2.9 Hz), 7.40 (1H, d, J = 8.8 Hz) and 7.84 (1H, s),
8.91 (1H, s), 9.93 (1H, s); 13C NMR (100.5 MHz, DMSO):
d 18.15, 33.12, 50.61, 98.03, 107.59, 119.47, 122.61,
125.02, 125.04, 146.61, 149.11, 153.96, 154.55, 167.48 and
175.22; HRMS (ESI positive mode): m/z 408.1048
[M ? Na]?, calculated for [C20H19NO7 ? Na]? 408.1054.
Dimethyl 40-(7-acetoxychromon-3-yl)-20,60-dimethyl-10,40-
dihydropyridine-30,50-dicarboxylate (16) Compound 16
was obtained as yellow solid in 80 % yield; M.P.
236–238 °C; IR (KBr) mmax 3333, 1776, 1679, 1636, 1614,
1442, 1337, 1222, 1182, 1017, 849 and 778 cm-1 1H
,
NMR (400 MHz, CDCl3): d 2.29 (6H, s), 2.34 (3H, s), 3.66
(6H, s), 4.87 (1H, s), 7.09 (1H, dd, J = 8.8 and 2.2 Hz),
7.22 (1H, d, J = 2.2 Hz), 7.40 (1H, s), 7.98 (1H, s) and
8.14 (1H, dd, J = 8.8 and 2.2 Hz); 13C NMR (100.5 MHz,
CDCl3): d 19.37, 21.08, 35.31, 50.81, 97.91, 110.94,
Diethyl 40-(6-hydroxychromon-3-yl)-20,60-dimethyl-10,40-
dihydropyridine-30,50-dicarboxylate (13) Compound 13
was obtained as yellow solid in 64 % yield; M.P.
123

Med Chem Res
119.16, 122.86, 125.44 and 126.72, 147.53, 154.00, 154.87
156.36, 168.14, 168.57 and 176.40; HR-ESI-TOF-MS: m/z
450.1142 [M ? Na]?, calculated for [C22H21NO8 ? Na]?
450.1159.
175.91; HR-ESI-TOF-MS: m/z 590.2713 [M ? Na]?, cal-
culated for [C32H41NO8 ? Na]? 590.2724.
Diethyl 40-(6-acetoxychromon-3-yl)-20,60-dimethyl-10,40-di-
hydropyridine-30,50-dicarboxylate (20) Compound 20
was obtained as yellow solid in 83 % yield; M.P.
145–148 °C; IR (KBr) mmax 3340, 1764, 1676, 1632, 1572,
Dimethyl 40-(7-hexanoyloxychromon-3-yl)-20,60-dimethyl-
10,40-dihydropyridine-30,50-dicarboxylate (17) Compound
17 was obtained as yellow solid in 77 % yield; M.P.
181–182 °C; IR (KBr) mmax 3318, 2953, 1763, 1706, 1670,
1
1476, 1327, 1217, 1126, 1024 and 776 cm-1, H NMR
(400 MHz, CDCl3): d 1.24 (6H, t, J = 7.3 Hz), 2.30 (6H,
s), 2.31 (3H, s), 4.05–4.14 (4H, m), 4.86 (1H, s), 6.52 (1H,
s), 7.33 (1H, dd, J = 8.7 and 2.8 Hz), 7.43 (1H, d,
J = 8.7 Hz), 7.82 (1H, d, J = 2.8 Hz), 7.97 (1H, s); 13C
NMR (100.5 MHz, CDCl3): d 14.47, 19.84, 21.10, 35.38,
59.87, 98.38, 117.26, 119.80, 125.03, 125.86, 127.20,
146.32, 153.47, 154.74, 167.74, 169.30 and 175.90; HR-
ESI-TOF-MS: m/z 478.1469 [M ? Na]?, calculated for
[C24H25NO8 ? Na]? 478.1472.
1636, 1438, 1315, 1217, 1131, 1092, 846 and 778 cm-1
,
1H NMR (400 MHz, CDCl3): d 0.94 (3H, t, J = 7.3 Hz),
1.36–1.42 (4H, m), 1.77 (2H, pentet, J = 7.3 Hz), 2.29
(6H, s), 2.58 (2H, t, J = 7.7 Hz), 3.66 (6H, s), 4.86 (1H, s),
7.08 (1H, dd, J = 8.8 and 2.2 Hz), 7.22 (1H, d,
J = 2.2 Hz), 7.39 (1H, s), 7.98 (1H, s) and 8.13 (1H, d,
J = 8.8 Hz); 13C NMR (100.5 MHz, CDCl3): d 13.84,
19.36, 22.23, 24.39, 31.15, 34.27, 35.32, 50.86, 97.91,
110.90, 119.21, 122.79,125.42, 126.80, 147.55, 154.17,
154.86, 156.39, 168.16, 171.47 and 176.44; HR-ESI-TOF-
MS: m/z 506.1763 [M ? Na]?, calculated for [C26H29
NO8 ? Na]? 506.1785.
Diethyl
40-(6-hexanoyloxychromon-3-yl)-20,60-dimethyl-
10,40-dihydropyridine-30,50-dicarboxylate (21) Compound
21 was obtained as yellow solid in 82 % yield; M.P.
141–143 °C; IR (KBr) mmax 3328, 2980, 1724, 1692, 1674,
1625, 1477, 1330, 1210, 1164, 1087, 826 and 776 cm-1
,
Diethyl 40-(7-acetoxychromon-3-yl)-20,60-dipropyl-10,40-di-
hydropyridine-30,50-dicarboxylate (18) Compound 18
was obtained as yellow solid in 85 % yield; M.P.
189–191 °C; IR (KBr) mmax 3330, 2968, 1764, 1700, 1668,
1H NMR (400 MHz, CDCl3): d 0.93 (3H, t, J = 7.2 Hz),
1.24 (6H, t, J = 7.1 Hz), 1.37–1.39 (4H, m), 1.71–1.77
(2H, m), 2.30 (6H, s), 2.56 (2H, t, J = 7.2 Hz), 4.05–4.10
(4H, m), 4.86 (1H, s), 6.38 (1H, s), 7.32 (1H, dd, J = 9.2
and 2.7 Hz), 7.43 (1H, d, J = 9.2 Hz), 7.81 (1H, d,
J = 2.7 Hz) and 7.97 (1H, s); 13C NMR (100.5 MHz,
CDCl3): d 13.86, 14.42, 19.94, 22.26, 24.69, 31.23, 34.22,
35.34, 59.62, 98.78, 117.54, 119.41, 125.02, 125.86 and
127.26, 146.31, 147.34, 153.50 and 154.90, 167.59, 172.17
and 176.15; HR-ESI-TOF-MS: m/z 534.2082 [M ? Na]?,
calculated for [C28H33NO8 ? Na]? 534.2098.
1637, 1500, 1441, 1201, 1177, 1091, 907 and 770 cm-1
,
1H NMR (400 MHz, CDCl3): d 0.95 (6H, t, J = 7.2 Hz),
1.22 (6H, t, J = 7.3 Hz), 1.59 (4H, brs), 2.31 (3H, s),
2.40–2.48 (2H, m), 2.78–2.85 (2H, m), 4.02–4.14 (4H, m),
4.89 (1H, s), 6.18 (1H, s), 7.04 (1H, dd, J = 8.7 and
2.3 Hz), 7.17 (1H, d, J = 2.3 Hz), 7.87 (1H, s) and 8.14
(1H, d, J = 8.7 Hz); 13C NMR (100.5 MHz, CDCl3): d
13.99, 14.27, 21.10, 22.06, 34.75, 35.29, 59.54, 98.25,
110.86, 118.86, 122.99, 125.77, 126.90, 150.85, 153.81,
154.89, 156.32, 167.31, 168.62 and 175.83; HR-ESI-TOF-
MS: m/z 534.2076 [M ? Na]?, calculated for [C28H33
NO8 ? Na]? 534.2098.
Dimethyl 40-(6-acetoxychromon-3-yl)-20,60-dimethyl-10,40-
dihydropyridine-30,50-dicarboxylate (22) Compound 22
was obtained as yellow solid in 85 % yield; M.P.
276–277 °C; IR (KBr) mmax 3331, 1766, 1683, 1634, 1619,
1
1476, 1330, 1220, 1126, 1024 and 775 cm-1, H NMR
Diethyl
40-(7-hexanoyloxychromon-3-yl)-20,60-dipropyl-
(400 MHz, CDCl3): d 2.28 (6H, s), 2.32 (3H, s), 3.65 (6H,
s), 4.88 (1H, s), 7.35 (1H, dd, J = 9.2 and 2.8 Hz),
7.44–7.47 (2H, m), 7.78 (1H, d, J = 2.8 Hz) and 8.02 (1H,
s); 13C NMR (100.5 MHz, CDCl3): d 19.52, 20.99, 35.22,
50.87, 98.14, 117.33, 119.44, 125.03, 125.71 and 127.24,
147.22, 153.47, 154.85, 168.11, 169.31 and 176.39; HR-
ESI-TOF-MS: m/z 450.1144 [M ? Na]?, calculated for
[C22H21NO8 ? Na]? 450.1159.
10,40-dihydropyridine-30,50-dicarboxylate (19) Compound
19 was obtained as yellow solid in 83 % yield; M.P.
139–141 °C; IR (KBr) mmax 3331, 2966, 1766, 1697, 1667,
1634, 1500, 1442, 1201, 1136, 1052, 846 and 769 cm-1
,
1H NMR (400 MHz, CDCl3): d 0.89–0.97 (9H, m), 1.22
(6H, t, J = 7.4 Hz), 1.35–1.39 (4H, m), 1.59–1.65 (4H, m),
1.70–1.78 (2H, m) 2.39–2.48 (2H, m), 2.56 (2H, t,
J = 7.3 Hz), 2.78–2.85 (2H, m), 4.02–4.15 (4H, m), 4.89
(1H, s), 6.20 (1H, s), 7.03 (1H, dd, J = 8.7 and 2.3 Hz),
7.16 (1H, d, J = 2.3 Hz), 7.87 (1H, s) and 8.14 (1H, d,
J = 8.7 Hz); 13C NMR (100.5 MHz, CDCl3): d 13.85,
13.99, 14.26, 22.12, 22.24, 24.42, 31.15, 34.26, 34.70,
35.42, 59.49, 98.07, 110.84, 118.91, 122.88, 125.66,
126.73, 151.13, 153.97, 154.96, 156.35, 167.34, 171.51 and
Dimethyl 40-(6-hexanoyloxychromon-3-yl)-20,60-dimethyl-
10,40-dihydropyridine-30,50-dicarboxylate (23) Compound
23 was obtained as yellow solid in 85 % yield; M.P.
192–193 °C; IR (KBr) mmax 3333, 2953, 1759, 1697, 1672,
1621, 1497, 1311, 1225, 1143, 1053, 827 and 771 cm-1
,
1H NMR (400 MHz, CDCl3): d 0.93 (3H, t, J = 7.8 Hz),
123

Med Chem Res
Scheme 1 Synthesis of 2,6-
dialkyl-4-chromon-3-yl-1,4-
dihydropyridine-3,5-
dicarboxylates (14–23)
1.37–1.40 (4H, m), 1.75 (2H, pentet, J = 7.8 Hz), 2.28
(6H, s), 2.56 (2H, t, J = 7.8 Hz), 3.65 (6H, s), 4.88 (1H, s),
7.33–7.35 (2H, m), 7.44 (1H, d, J = 8.7 Hz), 7.78 (1H, d,
J = 2.7 Hz) and 8.01 (1H, s); 13C NMR (100.5 MHz,
CDCl3): d 13.86, 19.54, 22.26, 24.54, 31.18, 34.19, 35.19,
50.86, 98.20, 117.34, 119.41, 125.02, 125.72, 127.31,
147.14, 147.32, 153.42, 154.82, 168.09, 172.19 and
176.42; HR-ESI-TOF-MS: m/z 506.1767 [M ? Na]?, cal-
culated for [C26H29NO8 ? Na]? 506.1785.
energy transfer (FRET) assay with recombinant human
BACE-1 and quenched fluorescent peptide substrate on the
basis of Swedish mutant APP sequence (SEVNLDAEFK).
FRET assay kit was purchased from Life Technologies,
and the assay was performed on the basis of the
manufacturer instructions using a multimode microplate
reader (BMG LABTECH, Polar star, Germany).
For carrying out the assay, the provided BACE-1 (pu-
rified baculovirus-expressed) and the peptide substrate (Rh-
EVNLDAEFK-Quencher) as well as the stock solutions of
inhibitors dissolved in DMSO were diluted in the assay
buffer (50 mM sodium acetate, pH 4.5). The final con-
centration of DMSO was kept below 4 %. BACE-1 and test
samples (10 ll of each) were placed in 96-well plates.
Then, BACE-1 substrate (10 ll) was added to the mixture
BACE-1 enzyme inhibition assay
All the synthesized compounds 14–23 were assessed for
their BACE-1 enzyme inhibitory activity. Enzyme inhibi-
¨
tion activities were determined using a Forster resonance
123

Med Chem Res
and the plates were incubated at 25 °C for 90 min in the
dark. The reaction was stopped with 2.5 M sodium acetate,
and fluorescence signals were recorded at 544 nm (exci-
tation) and 590 nm (emission) wavelengths to monitor the
hydrolysis of the substrate. OM99-2 (Glu-Val-Asn-Leu-W-
Ala-Ala-Glu-Phe, Calbiochem) was used as a reference
inhibitor compound.
analysis was performed on the docked results using a root
˚
mean square (RMS) tolerance of 2 A. Schematic 2D rep-
resentations of the ligand–receptor interactions were all
generated using LIGPLOT (Wallace et al., 1995).
Results and discussion
Chemistry
The percentage of enzyme inhibition for each concen-
tration of test compounds was calculated using the Eq. (1):
.
ðEÀDÞ
% Enzyme inhibition ¼
ðEÀSÞ Â 100
ð1Þ
Synthesis of desired 2,6-dialkyl-4-chromon-3-yl-1,4-dihy-
dropyridine-3,5-dicarboxylate derivatives was conducted
following the routes depicted in Scheme 1. The structures
of all the synthesized compounds 3–23 were unambigu-
ously established on the basis of their spectral (IR, 1H
NMR, 13C NMR and HRMS) data analysis. The structures
of known compounds 3–8 were further confirmed by the
comparison of their physical and spectral data with those
reported in the literature (Nohra et al., 1974; Kumar et al.,
2007; Chand et al., 2014).
where E is the fluorescence emission of maximum enzyme
activity (wells containing substrate plus enzyme), D indi-
cates fluorescence emission for wells containing the test
compounds (dihydropyridine plus substrate and enzyme),
and S represents the baseline fluorescence emission. Each
experiment was repeated three times, and BACE-1 inhi-
bition values were presented as mean S.E.
Molecular docking study
Determination of BACE-1 inhibition
Flexible-ligand docking studies were carried out using
AutoDock version 4.2 (Morris et al., 2009).
BACE-1 enzyme inhibitory activities of synthesized 1,4-
dihydropyridines were determined using a FRET-based kit.
Activities were measured as the percentages of enzyme
inhibition at the concentrations of 10 and 50 lM of test
compounds (Table 1).
X-ray crystallographic holo structures of BACE-1 were
all retrieved from the Brookhaven Protein Data Bank
(2B8L, 2B8V, 2IS0, 2IRZ, 2QMF, 2VJ9, 2VKM; http://
www.rcsb.org/).
The protein structure was subjected to optimization step
in order to minimize the crystallographically induced bond
clashes using steepest descent method by GROMACS
package (Van Der Spoel et al., 2005). For preparation of a
target protein as a template, cognate ligand and all crys-
tallographic water molecules were removed from the ori-
ginal receptor structure. All the preprocessing steps for
receptor crystallographic file (PDB code 2IRZ) were per-
formed within WHAT IF server (http://swift.cmbi.ru.nl/
servers/html/) and AutoDock Tools 1.5.4 program (ADT)
(Sanner, 1999; Morris et al., 2009). ADT program was
used to merge nonpolar hydrogens into related carbon
atoms of the receptor, and Kollman charges were also as-
signed. For docked ligands, Gasteiger charges were as-
signed and torsions degrees of freedom were also allocated
by ADT program.
The main focus of this study was to find out novel di-
hydropyridine-based BACE-1 inhibitors with the aim of
finding important structural prerequisites for an optimal
BACE-1 inhibitory activity. Enzyme inhibitions at 10 lM
were used for comparing the potencies of test compounds.
Table 1 In vitro BACE-1 enzyme inhibitory activities of synthesized
1,4-dihydropyridine compounds
Compound
Inhibition at 50 lM (%)a
Inhibition at 10 lM (%)
14
15
16
17
18
19
20
21
22
23
a
37.27 2.64
9.05 3.56
39.30 2.63
3.10 6.39
23.78 3.95
12.65 3.36
32.54 2.78
3.03 0.93
44.10 1.80
37.58 3.14
51.32 0.60
15.15 4.51
36.74 2.99
19.09 1.31
32.15 5.26
41.40 4.09
22.91 2.00
12.24 3.27
34.79 8.29
6.84 3.82
Lamarckian genetic algorithm (LGA) was applied to
simulate the binding between dihydropyridine and BACE-1
active site. One hundred independent genetic algorithm
(GA) runs were considered for each ligand under study. For
Lamarckian GA method, 5.0 9 107 maximum number of
evaluations, 27,000 maximum generations, a gene mutation
rate of 0.02 and a crossover rate of 0.8 were used. A grid of
60 9 60 9 60 points in x, y and z direction was built on the
Values represent mean standard error (SE) of three independent
experiments. IC50 value for OM99-2 determined as the reference
BACE-1 inhibitor was 0.003 0.001 lM
˚
center of BACE-1 catalytic site (spacing 0.375 A). Cluster
123

Med Chem Res
Table 2 Docking validation results for different holo PDB structures of BACE-1 using AutoDock 4.2
PDB code Structure of cognate ligand
Resolution GGA Maximum number Population in RMSD from
runs
˚
(A)
of energy
evaluations
the optimum
cluster (%)
reference
structure (A)
˚
2B8L
1.70
100
5.0 9 106
47
0.515
2B8V
1.80
100
5.0 9 106
80
0.533
2IRZ
1.80
100
5.0 9 106
88
0.478
2IS0
2.20
100
5.0 9 106
27
0.414
2QMF
1.75
100
5.0 9 106
18
1.157
123

Med Chem Res
Table 2 continued
PDB code Structure of cognate ligand
Resolution GGA Maximum number Population in RMSD from
˚
(A)
runs
of energy
evaluations
the optimum
cluster (%)
reference
structure (A)
˚
2VJ9
1.60
100
5.0 9 106
34
0.479
2VKM
2.05
100
5.0 9 106
25
1.616
in spite of bearing the 4-[7-(hexanoyloxy)-4-oxo-4H-
chromen-3-yl] group at the C4 formation of the
dihydropyridine ring, exhibited the second top
BACE-1 inhibitory activity.
Assessment of the structure activity relationship of syn-
thesized derivatives resulted in the following observations:
1. Some of the dihydropyridine derivatives exhibited
higher BACE-1 inhibitory activity at 10 lM compared
with the activity exhibited at 50 lM. The wells
containing the 50 lM concentration of these com-
pounds had generally very high fluorescence emissions
from the beginning of incubation time (data not
shown), and it was assumed that these compounds
had an interference with the FRET assay at higher
concentrations. However, low aqueous solubility at
higher amounts due to the lipophilic structure of these
dihydropyridine compounds could have also contribut-
ed to this issue.
5. Compound 19 was the only agent bearing n-propyl
substituents at the C2 and C6 positions of dihydropy-
ridine ring. This could provide additional lipophilic
contacts with the active site and probably explain its
higher activity.
6. Among the compounds bearing 4-[7-(ethanoyloxy)-4-
oxo-4H-chromen-3-yl] moiety at C4 of the dihydropy-
ridine ring (14, 16 and 18), compound 14 with
ethoxycarbonyl at C3/C5 and methyl moieties at C2/
C6 was the most active compound. This rule seemed to
be quite different among compounds bearing 4-[6-
(ethanoyloxy)-4-oxo-4H-chromen-3-yl] at C4, in which
the presence of methoxy carbonyl at C3/C5 rendered the
compound 22 more active than the compound 20.
2. Compounds 14, 16, 18, 19 and 22 were the most active
agents, and all of them exhibited [30 % enzyme
inhibition at 10 lM.
3. The presence of the 4-[7-(ethanoyloxy)-4-oxo-4H-
chromen-3-yl] moiety at C4 of the dihydropyridine
ring (14, 16 and 18) seemed to confer higher activity in
comparison with 4-[6-(ethanoyloxy)-4-oxo-4H-chro-
men-3-yl] (20 and 22), 4-[7-(hexanoyloxy)-4-oxo-4H-
chromen-3-yl] (15, 17 and 19) and 4-[6-(hexanoy-
loxy)-4-oxo-4H-chromen-3-yl] (21 and 23) sub-
stituents at this position.
Molecular docking
The 3D structures of protein targets are valuable sources of
information in modern drug discovery. In this regard,
available X-ray crystallographic data from the protein data
bank have facilitated the performance of docking simula-
tion studies. In docking, stereoelectronic fit of ligand and
its receptor is modeled (Putta and Beroza, 2007). Au-
toDock is a qualified docking program that has facilitated
the process of drug design (The AutoDock Web site. http://
autodock.scripps.edu) (Sellers et al., 2010).
4. Inappropriate orientation of bulkier 4-[7-(hexanoy-
loxy)-4-oxo-4H-chromen-3-yl] and 4-[6-(hexanoy-
loxy)-4-oxo-4H-chromen-3-yl] groups in the active
site of BACE-1 may partly explain the observed order.
An exception to this notion was compound 19, which
123

Med Chem Res
With the aim of achieving possible ligand–receptor
binding features, we focused on molecular docking of
synthesized dihydropyridine derivatives on the active site
of BACE-1 as a protein target for treatment of AC. For this
purpose, docking validation step was performed by re-
docking of the co-crystallized conformation of cognate li-
gands into 3D structures of BACE-1. PDB structures were
chosen on the basis of crystallographic resolutions and also
relative similarity of co-crystallized ligands to the dihy-
dropyridine structures (http://www.rcsb.org/).
Table 3 AutoDock-based binding affinities and lipophilicity indices
for 2,6-dialkyl-4-aryl-1,4-dihydropyridine-3,5-dicarboxylates in the
active site of BACE-1
Compound code
Estimated Ki (nM)
Estimated pKi
ClogP
14
15
16
17
18
19
20
21
22
23
46.33
83.93
4.334
4.076
3.995
3.816
4.310
4.512
4.079
3.885
4.122
3.724
3.21
4.80
2.58
4.10
4.85
6.10
3.19
4.77
2.57
4.02
101.20
152.92
48.93
30.76
In this way, applied AutoDock 4 program could be
tested for predictability of known binding poses
˚
(RMSD B 2 A) (Cosconati et al., 2010). Following this
83.33
130.45
75.57
rationale, BACE-1 structure with the PDB code of 2IRZ
188.83
(Rajapakse et al., 2006) was selected for our further
Fig. 1 2D schematic representations of the interactions of the most potent synthetic compounds, 14 (a) and 22 (b) with the active site of
BACE-1
123

Med Chem Res
Fig. 2 2D schematic representations of the interactions of compounds 18 (a) and 19 (b) with the active site of BACE-1
group and Thr72. Another important H-bond might be
expected between carbonyl moiety of chromenon and
Gln73. As can be seen in Fig. 1, major lipophilic
interactions with the active site of BACE-1 might be
observed within S1 and S2 subpockets.
docking simulations. Results of docking validation step are
summarized in Table 2.
Our docking procedure provided some insight into the
ligand–enzyme binding poses. Estimated free binding en-
ergies for dihydropyridine molecules are summarized in
Table 3. All the lipid/aqueous partition coefficients were
also estimated using online ALOGPS 2.1 program (Tetko
et al., 2001; Tetko and Tanchuk, 2002).
4. The exceptions to the above observation were com-
pounds 18 and 19 in which the presence of n-propyl
groups at C2 and C6 positions of dihydropyridine ring
might distort the molecule and hence avoid additional
lipophilic contacts with BACE-1 active site.
Following structure-binding relationships could be de-
veloped regarding the obtained in silico results:
5. Different H-bond patterns were observed for com-
pounds 18 and 19. Compound 18 made H-bond with
Ser325 side chain via its 4-[7-(ethanoyloxy)-4-oxo-
4H-chromen-3-yl] group, while compound 19 par-
ticipated in hydrogen bonding to the side chain of
Gln73 through carbonyl oxygen of the ester group
(Fig. 2).
1. In almost all docked dihydropyridine structures, a key
hydrogen bond between dihydropyridine’s NH and
Asp32 carboxylate oxygen supported a possible inter-
action with one of the catalytic dyad residues.
2. LIGPLOT diagrams showed that another participant of
BACE-1 catalytic dyad (Asp228) interacted with
dihydropyridine structures through hydrophobic
contacts.
6. All of the compounds bearing hexanoyloxy-4-oxo-4H-
chromen-3-yl group at C4 of the dihydropyridine ring
might participate in H-bond interaction with the NH
3. H-bond interaction with S1 subpocket of BACE-1 was
detected between the carbonyl oxygen of the ester
123

Med Chem Res
Fig. 3 Correlation of estimated BACE-1 binding affinities (pKi) with in vitro BACE-1 inhibition percentages at 10 lM (a) and 50 lM
(b) concentrations of synthesized 2,6-dialkyl-4-chromon-3-yl-1,4-dihydropyridine-3,5-dicarboxylates
moiety of Asn233 backbone through their hexanoy-
loxy carbonyl oxygen. The inappropriate orientation of
4-[7-(hexanoyloxy)-4-oxo-4H-chromen-3-yl] and 4-[6-
(hexanoyloxy)-4-oxo-4H-chromen-3-yl] groups in the
active site of BACE-1 may partly explain the lower
inhibitory activities of 15, 17, 21 and 23.
compound 19 with 4-[7-(hexanoyloxy)-4-oxo-4H-chro-
men-3-yl] group at C4 of the dihydropyridine ring was also
a potent agent. The presence of n-propyl substituents at C2
and C6 of dihydropyridine ring in compound 19, which is
expected to provide additional lipophilic contacts with the
BACE-1 active site, could probably explain its higher ac-
tivity. On the basis of docking simulation studies, a binding
pose indicating a key H-bond interaction between Asp32
residue and dihydropyridine NH might be expected in the
active site of BACE-1. Moreover, all docked dihydropy-
ridine structures made possibly good hydrophobic contacts
with some of the residues of S1 and S2 subpockets. The
results of the present study could guide toward rational
design of more potent BACE-1 blocking agents on the
basis of 2,6-dialkyl-4-(7-(alkyloyloxy)-4-oxo-4H-chro-
men-3-yl)-1,4-dihydropyridine-3,5-dicarboxylate scaffold.
7. Further inspection of the binding maps showed that
these four compounds may not form H-bonds with
Gln73 and Ser325 unlike the compounds bearing 4-[7-
(ethanoyloxy)-4-oxo-4H-chromen-3-yl] (14 and 16)
or 4-[6-(ethanoyloxy)-4-oxo-4H-chromen-3-yl] sub-
stituents (20 and 22).
Finally, we found that docking affinities (pKi) of 2,6-
dialkyl-4-aryl-1,4-dihydropyridine-3,5-dicarboxylates
could be used to predict the experimental BACE-1 inhi-
bition data at 10 lM (R2 = 0.639; Fig. 3a). Compounds 14
and 19 which were the most potent compounds in enzyme
inhibition assay showed also the highest pKi in the docking
study. However, a decent correlation was not observed
between pKi and the inhibition data at 50 lM (Fig. 3b),
possibly due to the reasons mentioned earlier in enzyme
inhibition section.
Acknowledgments The authors wish to thank the Vice-Provost for
Research of the Shiraz University of Medical Sciences for the fi-
nancial support to this project (Grant Number: 93-01-12-8102).
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