Synthesis, docking simulation, biological evaluations and 3D-QSAR study of 1,4-dihydropyridines as calcium channel blockers

Article abstract of DOI:10.1248/cpb.c17-00186

Resurgence to target L-type voltage-dependent calcium channels has been applied by the synthesis of two series of nifedipine analogues where the ortho- or a meta-nitrophenyl ring is retained. A pre-synthetic molecular docking study with a receptor model followed by molecular alignment has been performed on 47 compounds to predict the most active member. The IC₅₀ values revealed that some of the compounds are similar to or more active than nifedipine. Substitution of groups at the 3- and 5-positions of the dihydropyridine (DHP) ring gave 3k, which is more active than nifedipine. Our valid three-dimensional quantitative structure–activity relationship (3D-QSAR) model prefigures the influence of lipophilicity, bulkiness and chelating effects of the C3 and C5 substituents. Bulky groups interfere with ring-to-ring hydrophobic interaction with tyrosine (Tyr)⁴³¹¹ and limit the efficiency of increasing the length of the hydrocarbon chain of esters at the 3- and 5-positions of the DHP ring as an approach to increasing the activity. The presence of a chelating substituent on the phenyl ring at the 4-position of the DHP ring ensures strong binding to the receptor and hence stabilization of the closed-channel conformation. The validation of 3D-QSAR model indicated its proficiency in predicting activity of newly compounds belonging to the same chemical class.

Full text of DOI:10.1248/cpb.c17-00186

Vol. 65, No. 10
Chem. Pharm. Bull. 65, 893–903 (2017)
893
Regular Article
Synthesis, Docking Simulation, Biological Evaluations and 3D-QSAR
Study of 1,4-Dihydropyridines as Calcium Channel Blockers
, a
Tarek Fathy El-Moselhy,a Peter Ayoub Sidhom,* Eman Ahmed Esmat,a and
Nageh Ahmed El-Mahdyb
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Tanta University; Tanta 31527, Egypt: and
b Department of Pharmacology, Faculty of Pharmacy, Tanta University: Tanta 31527, Egypt.
Received March 3, 2017; accepted July 18, 2017
Resurgence to target L-type voltage-dependent calcium channels has been applied by the synthesis of
two series of nifedipine analogues where the ortho- or a meta-nitrophenyl ring is retained. A pre-synthetic
molecular docking study with a receptor model followed by molecular alignment has been performed on 47
compounds to predict the most active member. The IC50 values revealed that some of the compounds are sim-
ilar to or more active than nifedipine. Substitution of groups at the 3- and 5-positions of the dihydropyridine
(DHP) ring gave 3k, which is more active than nifedipine. Our valid three-dimensional quantitative struc-
ture–activity relationship (3D-QSAR) model prefigures the influence of lipophilicity, bulkiness and chelating
effects of the C3 and C5 substituents. Bulky groups interfere with ring-to-ring hydrophobic interaction with
tyrosine (Tyr)4311 and limit the efficiency of increasing the length of the hydrocarbon chain of esters at the
3- and 5-positions of the DHP ring as an approach to increasing the activity. The presence of a chelating
substituent on the phenyl ring at the 4-position of the DHP ring ensures strong binding to the receptor and
hence stabilization of the closed-channel conformation. The validation of 3D-QSAR model indicated its profi-
ciency in predicting activity of newly compounds belonging to the same chemical class.
Key words calcium channel; dihydropyridine; docking; synthesis; three-dimensional quantitative structure–
activity relationship (3D-QSAR)
Precise regulation of calcium homeostasis is crucial for tool for subsequent researchers to test their compounds related
many physiological functions.1) Divergent types of calcium to ortho- or a meta-substituted phenyl ring.
channels and pumps can control the influx of calcium ions into
cells.2,3) Consequently, targeting calcium channels is advanta-
Experimental
geously beneficial to yield useful drugs. CaV1.2 blockers can
Chemicals, Reagents and Instruments Chemicals were
be roughly categorized into three different chemical classes: obtained from Sigma-Aldrich, U.S.A. and were used without
1,4-dihydropyridine (DHP) derivatives, phenylalkylamine de- further purification. Melting points were determined on a Stu-
rivatives and benzothiazepine derivatives. Among them, DHP art SMP10 capillary melting point apparatus and are uncor-
derivatives have the most significant pharmacological impor- rected. TLC was used to monitor the progress and/or comple-
tance. For example, amlodipine is among the top-five best tion of the reactions using a pre-coated sheet (Eastman Kodak
selling drugs in the treatment of cardiovascular diseases. Vari- Co., Silca 60 F254) and was visualized with UV light at
ous modulators can potentiate or block calcium entry through 254nm. Elemental analysis was performed by Micro Analyti-
L-type calcium channels.4–6) Calcium antagonists have a cal Center, Faculty of Science, Cairo University, Giza, Egypt
1
versatile pharmacological activity such as antihypertensive and were within 0.4% of the calculated values. H-NMR
and antianginal,7–9) antitumor,10,11) anti- inflammatory,12,13) anti- was obtained using a Bruker Advance-II 400 Spectrometer on
tubercular,14,15) anticonvulsant and antithrombotic.16,17)
400MHz using tetramethylsilane (TMS) as internal standard.
1,4-DHPs bind selectively to L-type calcium channel pro-
Chemistry. General Procedure for the Synthesis
tein, precisely at the transmembrane domain IIIS6 and IVS6 of Symmetrical Esters of Dialkyl 4-(Nitrophenyl)-2,6-
regions of the α1 subunit.18) The twelve approved DHP cal- dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (3a–l) To
cium antagonists developed are vasodilators.19) Vasodilatation a 50-mL round-bottomed flask, ammonium acetate (0.162g,
is due to the uncoupling of the contractile mechanism of vas- 2.10mmol) was added to a stirring solution of 2-nitrobenzal-
cular smooth muscle, which requires Ca2+.20)
dehyde or 3-nitrobenzaldehyde (0.254g, 1.65mmol) and the
Structure–activity relationship (SAR) of DHP21) and the corresponding alkyl acetoacetate (3.35mmol) in methanol or
clinical usage of nifedipine have promoted us to synthesize 2-propanol (10mL). The reaction mixture was protected from
two series of nifedipine analogues where the ortho- or a light and heated under reflux for 12–24h. After cooling, the
meta-nitrophenyl ring is retained. A pre-synthetic molecular precipitate was filtered and purified by crystallization from
docking study has guided the research scaffold to predict the methanol or 2-propanol to afford the corresponding product.
most active member of the two series. Hence, the building of
Dimethyl 2,6-Dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridi-
a reliable three dimensional quantitative (3D-Q)SAR model ne-3,5-dicarboxylate (3a)
(based on the biological IC50 values of not only ortho- or a
Recrystallization from methanol afforded (0.480g, 79%):
meta-nitrophenyl ring but also ortho- or a meta-chlorophenyl melting point (mp): 186–188°C.22)
ring) could optimize the outcome of our work and provide a
*To whom correspondence should be addressed. e-mail: Peter.ayoub@pharm.tanta.edu.eg
© 2017 The Pharmaceutical Society of Japan

894
Chem. Pharm. Bull.
Vol. 65, No. 10 (2017)
Diethyl 2,6-Dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-
3,5-dicarboxylate (3b)
Diisopropyl 2,6-Dimethyl-4-(3-nitrophenyl)-1,4-dihydropy-
ridine-3,5-dicarboxylate (3i)
Recrystallization from methanol afforded 0.434g (66%):
Recrystallization from methanol afforded (0.465g, 70%):
mp: 128–130°C.22)
Rf=0.79 (CHCl3–MeOH, 95:5); mp: 116–118°C; 1H-NMR
Diisopropyl 2,6-Dimethyl-4-(2-nitrophenyl)-1,4-dihydropy- (400MHz, CDCl3) δ: 1.11 (d, J=6.0Hz, 6H, CH(CH3)2), 1.28
ridine-3,5-dicarboxylate (3c) (d, J=6.0Hz, 6H, CH(CH3)2), 2.36 (s, 6H, C2-CH3, C6-CH3),
Recrystallization from methanol afforded (0.358g, 54%): 4.92–5.00 (m, J=6.0, 6.4Hz, 2H, 2 of CH(CH3)2), 5.08 (s,
Rf=0.65 (CHCl3–MeOH, 95:5); mp: 132–134°C; 1H-NMR 1H, C4-H), 5.87 (s, 1H, NH), 7.38 (t, J=7.6, 8.0Hz, 1H, aro-
(400MHz, CDCl3) δ: 0.67 (d, J=5.9, 6.4Hz, 6H, CH(CH3)2), matic C(5′)H), 7.65 (d, J=7.6Hz, 1H, aromatic C(6′)H), 8.02
1.02 (d, J=6.4Hz, 6H, CH(CH3)2), 2.41 (s, 6H, C2-CH3, (d, J=8.0Hz, 1H, aromatic C(4′)H), 8.15 (s, 1H, aromatic
C6-CH3), 4.72–4.78 (m, J=6.0Hz, 2H, 2 of CH(CH3)2), C(2′)H)); Anal. (%): (C21H26N2O6), Calcd (Found): C 62.67
5.31 (s, 1H, C4-H), 5.51 (s, 1H, NH), 7.61 (t, J=7.5Hz, 1H, (62.68), H 6.51 (6.45), N 6.96 (7.10).
aromatic C(5′)H), 7.77(t, J=8.4Hz, 1H, aromatic C(4′)H), 7.86
Diisobutyl 2,6-Dimethyl-4-(3-nitrophenyl)-1,4-dihydropyri-
(d, J=7.5Hz, 1H, aromatic C(6′)H), 8.08 (d, J=8.4Hz, 1H, dine-3,5-dicarboxylate (3j)
aromatic C(3′)H); Anal. (%): (C21H26N2O6), Calcd (Found): C
62.67 (62.68), H 6.51 (6.45), N 6.96 (7.10).
Recrystallization from 2-propanol afforded (0.376g, 53%):
Rf=0.82 (CHCl3–MeOH, 95:5); mp: 95–97°C; 1H-NMR
Diisobutyl 2,6-Dimethyl-4-(2-nitrophenyl)-1,4-dihydropyri- (400MHz, CDCl3) δ: 0.88 (d, J=6.8Hz, 6H, CH(CH3)2), 0.92
dine-3,5-dicarboxylate (3d) (d, J=6.8Hz, 6H, CH(CH3)2), 1.87–1.97 (m, J=6.4, 6.8Hz,
Recrystallization from 2-propanol afforded (0.412g, 58%): 2H, 2 of CH(CH3)2), 2.39 (s, 6H, C2-CH3, C6-CH3), 3.85 (d,
Rf=0.69 (CHCl3–MeOH, 95:5); mp: 135–137°C; 1H-NMR J=6.4Hz, 4H, 2 of COOCH2), 5.16 (s, 1H, C4-H), 5.99 (s,
(400MHz, CDCl3) δ: 0.79 (d, J=6.8Hz, 6H, CH(CH3)2), 0.83 1H, NH), 7.39 (t, J=7.6, 8.0Hz, 1H, aromatic C(5′)H), 7.68
(d, J=6.8Hz, 6H, CH(CH3)2), 1.80–1.86 (m, J=6.4Hz, 6.8Hz, (d, J=7.6Hz, 1H, aromatic C(6′)H), 8.02 (d, J=8.0Hz, 1H,
2H, 2 of CH(CH3)2), 2.31 (s, 6H, C2-CH3, C6-CH3), 3.76 (d, aromatic C(4′)H), 8.15 (s, 1H, aromatic C(2′)H); Anal. (%):
J=6.4Hz, 4H, 2 of COOCH2), 5.08 (s, 1H, C4-H), 5.66 (s, 1H, (C23H30N2O6), Calcd (Found): C 64.17 (64.07), H 7.02 (6.74),
NH), 7.19 (t, J=8.0Hz, 1H, aromatic C(5′)H), 7.31 (t, J=7.6, N 6.51 (6.55).
8.0Hz, 1H, aromatic C(4′)H), 7.58 (d, J=8.0Hz, 1H, aromatic
Bis(2-methoxyethyl) 2,6-Dimethyl-4-(3-nitrophenyl)-1,4-di-
C(6′)H), 7.93 (d, J=7.6Hz, 1H, aromatic C(3′)H); Anal. (%): hydropyridine-3,5-dicarboxylate (3k)
(C23H30N2O6), Calcd (Found): C 64.17 (64.07), H 7.02 (6.74),
N 6.51 (6.55).
Recrystallization from methanol afforded (0.538g, 75%):
Rf=0.45 (CHCl3–MeOH, 95:5); mp: 118–120°C; 1H-NMR
Bis(2-methoxyethyl) 2,6-Dimethyl-4-(2-nitrophenyl)-1,4-di- (400MHz, CDCl3) δ: 2.35 (s, 6H, C2-CH3, C6-CH3), 3.34
hydropyridine-3,5-dicarboxylate (3e) (s, 6H, 2 of OCH3), 3.51–3.59 (m, J=4.00, 6.80Hz, 4H, 2
Recrystallization from methanol afforded (0.588g, 82%): of COOCH2CH2O), 4.12–4.22 (m, J=4.00, 7.4Hz, 4H, 2 of
Rf=0.38 (CHCl3–MeOH, 95:5); mp: 124–126°C; 1H-NMR COOCH2), 5.13 (s, 1H, C4-H), 6.23 (s, 1H, NH), 7.35–7.39 (t,
(400MHz, CDCl3) δ: 2.36 (s, 6H, C2-CH3, C6-CH3), 3.36 J=7.6, 8.00. Hz, 1H, aromatic C(5′)H), 7.69 (d, J=7.6Hz, 1H,
(s, 6H, 2 of OCH3), 3.54–3.57 (m, J=4.0, 4.8Hz, 4H, 2 aromatic C(6′)H), 8.00 (d, J=8.0Hz, 1H, aromatic C(4′)H),
of COOCH2CH2O), 4.13–4.24 (m, J=4.0, 4.8Hz, 4H, 2 8.13 (s, 1H, aromatic C(2′)H); Anal. (%): (C21H26N2O8), Calcd
of COOCH2), 5.14 (s,1H, C4-H), 6.07 (s, 1H, NH), 7.28 (t, (Found): C 58.06 (57.78), H 6.03 (5.92), N 6.45 (6.63).
J=8.0Hz, 1H, aromatic C(5′)H), 7.38 (t, J=7.6, 8.0Hz, 1H, ar-
Dibenzyl 2,6-Dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridi-
omatic C(4′)H), 7.70 (d, J=8.0Hz, 1H, aromatic C(6′)H), 8.02 ne-3,5-dicarboxylate (3l)
(d, J=7.6Hz, 1H, aromatic C(3′)H); Anal. (%): (C21H26N2O8),
Calcd (Found): C 58.06 (57.80), H 6.03 (6.11), N 6.45 (6.64).
Recrystallization from 2-propanol afforded (0.420g, 52%):
Rf=0.91 (CHCl3–MeOH, 95:5); mp: 99–101°C; 1H-NMR
Dibenzyl 2,6-Dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridi- (400MHz, CDCl3) δ: 1.67 (s, 1H, NH), 2.38 (s, 6H, C2-
ne-3,5-dicarboxylate (3f) CH3, C6-CH3), 5.04 (d, J=12.4Hz, 2H, COOCH2), 5.12 (d,
Recrystallization from 2-propanol afforded (0.525g, 65%): J=12.4Hz, 2H, COOCH2), 5.88 (s, 1H, C4-H), 7.19–7.24 (m,
Rf=0.89 (CHCl3–MeOH, 95:5); mp: 102–104°C; 1H-NMR 11H, aromatic OCH2C6H5), 7.31 (t, J=7.6, 8.0Hz, 1H, aro-
(400MHz, CDCl3) δ: 1.65 (s, 1H, NH), 2.39 (s, 6H, C2- matic C(5′)H), 7.48 (d, J=7.6Hz, 1H, aromatic C(6′)H), 7.97
CH3, C6-CH3), 5.04 (d, J=5.50Hz, 2H, COOCH2), 5.26 (d, (d, J=8.0Hz, 1H, aromatic C(4′)H), 8.03 (s, 1H, aromatic
J=5.50Hz, 2H, COOCH2), 5.87 (s, 1H, C4-H), 7.20–7.26 (m, C(2′)H); Anal. (%): (C29H26N2O6), Calcd (Found): C 69.87
11H, aromatic OCH2C6H5), 7.31 (t, J=8.4Hz, 1H, aromatic (69.71), H 5.26 (5.11), N 5.62 (5.98).
C(5′)H), 7.38 (t, J=7.6, 8.4Hz, 1H, aromatic C(4′)H), 7.48
General Procedure for the Synthesis of Asymmetrical
(d, J=7.6Hz, 1H, aromatic C(6′)H), 8.97 (d, J=8.4Hz, 1H, Esters of Dialkyl 4-(Nitrophenyl)-2,6-dimethyl-1,4-dihy-
aromatic C(3′)H); Anal. (%): (C29H26N2O6), Calcd (Found): C dropyridine-3,5-dicarboxylate (6a–j) To a 50-mL round-
69.87 (69.62), H 5.26(4.98), N 5.62 (6.06).
Dimethyl 2,6-Dimethyl-4-(3-nitrophenyl)-1,4-dihydropyri- or 3-nitrobenzaldehyde (0.254g, 1.65mmol), alkyl acetoacetate
dine-3,5-dicarboxylate (3g) (1.65mmol) and alkyl 3-aminocrotonate (1.65mmol) in metha-
Recrystallization from methanol afforded (0.364g, 60%), nol or 2-propanol (10mL). The reaction mixture was protected
mp: 188–190°C.22)
from light and heated under reflux for 9–24h. After cooling,
Diethyl 2,6-Dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine- the precipitate was filtered and purified by crystallization from
bottomed flask were added a mixture of 2-nitrobenzaldehyde
3,5-dicarboxylate (3h)
methanol or 2-propanol to afford the corresponding product.
Recrystallization from methanol afforded 0.355g (54%); mp:
132–134°C.22)

Vol. 65, No. 10 (2017)
Chem. Pharm. Bull.
895
3-Ethyl 5-Methyl 2,6-Dimethyl-4-(2-nitrophenyl)-1,4-dihyd- (d, J=8.0Hz, 1H, aromatic C(3′)H); Anal. (%): (C23H22N2O6),
ropyridine-3,5-dicarboxylate (6a)
Calcd (Found): C 65.39 (65.06), H 5.52 (5.26), N 6.63 (6.54).
3-Ethyl 5-Methyl 2,6-Dimethyl-4-(3-nitrophenyl)-1,4-dihyd-
Recrystallization from methanol afforded (0.216g, 62%):
Rf=0.76 (CHCl3–MeOH, 95:5); mp: 142–144°C; 1H-NMR ropyridine-3,5-dicarboxylate (6f)
(400MHz, CDCl3) δ: 1.02 (t, J=7.4Hz, 3H, CH2CH3), 1.58
Recrystallization from methanol afforded (0.213g, 59%):
(s, 6H, C2-CH3, C6-CH3), 2.51 (s, 3H, COOCH3), 3.95–4.03 Rf=0.79 (CHCl3–CH3OH 95:5); mp: 162–164°C; 1H-NMR
(m, J=7.4Hz, 2H, COOCH2), 5.37 (s, 1H, C4-H), 5.72 (s, 1H, (400MHz, CDCl3) δ: 1.10 (t, J=7.4Hz, 3H, CH2CH3), 2.49 (s,
NH), 7.46 (t, J=8.0Hz, 1H, aromatic C(5′)H), 7.55 (t, J=7.6, 6H, C2-CH3, C6-CH3), 3.57 (s, 3H, COOCH3), 3.90–4.21 (m,
8.0Hz, 1H, aromatic C(4′)H), 7.75 (d, J=8.0Hz, 1H, aromatic J=7.4Hz, 2H, COOCH2), 5.29(s, 1H, C4-H), 5.70 (s, 1H, NH),
C(6′)H), 7.95 (d, J=7.6Hz, 1H, aromatic C(3′)H); Anal. (%): 7.39 (t, J=8.5Hz, 1H, aromatic C(5′)H), 7.69 (d, J=7.5Hz, 1H,
(C18H20N2O4), Calcd (Found): C 59.99 (59.74), H 5.59 (5.27), N aromatic C(6′)H), 8.00 (d, J=8.5Hz, 1H, aromatic C(4′)H),
7.77 (8.00).
3-Ethyl 5-Isopropyl 2,6-Dimethyl-4-(2-nitrophenyl)-1,4- (Found): C 58.99 (58.70), H 5.59 (5.23), N 7.77 (7.94).
dihydropyridine-3,5-dicarboxylate (6b) 3-Ethyl 5-Isopropyl 2,6-Dimethyl-4-(3-nitrophenyl)-1,4-di-
8.13 (s, 1H, aromatic C(2′)H); Anal. (%): (C18H20N2O4), Calcd
Recrystallization from methanol afforded (0.455g, 71%): hydropyridine-3,5-dicarboxylate (6g)
Rf=0.73 (CHCl3–MeOH, 95:5); mp: 115–117°C; 1H-NMR
Recrystallization from methanol afforded (0.449g, 70%):
(400MHz, CDCl3) δ: 1.11 (d, J=6.0Hz, 3H, CH(CH3)2), Rf=0.77 (CHCl3–MeOH, 95:5); mp: 132–134°C; 1H-NMR
1.21–1.27 (m, J=6.0, 7.2Hz, 6H, CH(CH3)2 and CH2CH3), 2.36 (400MHz, CDCl3) δ: 1.10 (d, J=6.0Hz, 3H, CH(CH3)2), 1.21
(s, 6H, C2-CH3, C6-CH3), 4.05–4.14 (m, J=3.6, 7.2Hz, 2H, (d, J=7.2Hz, 3H, CH(CH3)2), 1.26 (t, J=6.0Hz, 3H, CH2CH3),
COOCH2), 4.92–4.99 (m, J=6.0, 6.4Hz, 1H, CH(CH3)2), 5.09 2.36 (s, 6H, C2-CH3, C6-CH3), 4.07–4.15 (m, J=3.6, 7.2Hz,
(s, 1H, C4-H), 6.10 (s, 1H, NH), 7.44 (t, J=8.0, 8.4Hz, 1H, 2H, COOCH2), 4.93–5.00 (m, J=6.0, 6.4Hz, 1H, CH(CH3)2),
aromatic C(5′)H), 7.69(t, J=8.0Hz, 1H, aromatic C(4′)H), 8.05 5.11 (s, 1H, C4-H), 5.07 (s, 1H, NH), 7.38 (t, J=7.6, 8.0Hz,
(d, J=8.4Hz, 1H, aromatic C(6′)H), 8.18 (d, J=8.0Hz, 1H, 1H, aromatic C(5′)H), 7.65 (d, J=7.6Hz, 1H, aromatic C(6′)H),
aromatic C(3′)H); Anal. (%): (C20H24N2O6), Calcd (Found): C 8.02 (d, J=8.0Hz, 1H, aromatic C(4′)H), 8.14 (s, 1H, aromatic
61.84 (62.00), H 6.23 (6.32), N 7.21 (7.27).
3-(2-Methoxyethyl) 5-Methyl 2,6-Dimethyl-4-(2-nitrophen- (62.04), H 6.23 (5.94), N 7.21 (6.96).
C(2′)H); Anal. (%): (C20H24N2O6), Calcd (Found): C 61.84
yl)-1,4-dihydropyridine-3,5-dicarboxylate (6c)
3-(2-Methoxyethyl) 5-Methyl 2,6-Dimethyl-4-(3-nitrophenyl)-
Recrystallization from methanol afforded (0.329g, 51%): 1,4-dihydropyridine-3,5-dicarboxylate (6h)
Rf=0.50 (CHCl3–MeOH, 95:5); mp: 110–112°C; 1H-NMR
Recrystallization from methanol afforded (0.432g, 67%):
(400MHz, CDCl3) δ: 2.30 (s, 6H, C2-CH3, C6-CH3), 3.30 Rf=0.52 (CHCl3–MeOH, 95:5); mp: 126–128°C; 1H-NMR
(s, 6H, OCH3, COOCH3), 4.03–4.09 (m, J=3.6,4.0Hz, (400MHz, CDCl3) δ: 2.32 (s, 6H, C2-CH3, C6-CH3),
2H, COOCH2CH2O), 4.24–4.30 (m, 2H, J=4.0, 4.8Hz 3.30 (s, 6H, OCH3 and COOCH3), 4.02–4.08 (m,
COOCH2CH2O), 5.47 (s, 1H, C4-H), 5.90 (s, 1H, NH), 7.28 J=4.0Hz, 2H, COOCH2CH2O), 4.24–4.30 (m, J=4.0Hz, 2H,
(t, J=6.0, 8.0Hz, 1H, aromatic C(5′)H), 7.48 (t, J=7.2, COOCH2CH2O), 5.89 (s, 1H, C4-H), 5.98 (s, 1H, NH), 7.26
7.6Hz, 1H, aromatic C(4′)H), 7.54 (d, J=7.2Hz, 1H, aromatic (t, J=8.0Hz, 1H, aromatic C(5′)H), 7.48 (d, J=7.5Hz, 1H,
C(6′)H), 7.75 (d, J=8.0Hz, 1H, aromatic C(3′)H); Anal. (%): aromatic C(6′)H), 7.53 (d, J=8.0Hz, 1H, aromatic C(4′)H),
(C19H22N2O7), Calcd (Found): C 58.46 (58.32), H 5.68 (5.69), 7.75 (s, 1H, aromatic C(2′)H); Anal. (%): (C19H22N2O5), Calcd
N 7.18 (7.11).
(Found): C 58.46 (58.32), H 5.68 (5.69), N 7.18 (7.11).
3-Ethyl 5-(2-Methoxyethyl) 2,6-Dimethyl-4-(3-nitrophenyl)-
1,4-dihydropyridine-3,5-dicarboxylate (6i)
3-Ethyl 5-(2-Methoxyethyl) 2,6-Dimethyl-4-(2-nitrophenyl)-
1,4-dihydropyridine-3,5-dicarboxylate (6d)
Recrystallization from methanol afforded (0.440g, 66%):
Recrystallization from methanol afforded (0.487g, 73%):
Rf=0.53 (CHCl3–MeOH (95:5); mp: 105–107°C; 1H-NMR Rf=0.56 (CHCl3–MeOH, 95:5); mp: 120–122°C; 1H-NMR
(400MHz, CDCl3) δ: 1.17 (t, J=7.2Hz, 3H, CH2CH3), 2.33 (400MHz, CDCl3) δ: 1.17 (t, J=4.0Hz, 3H, CH2CH3), 2.31
(s, 6H, C2-CH3, C6-CH3), 3.31 (s, 3H, OCH3), 3.50–3.62 (m, (s, 6H, C2-CH3, C6-CH3), 3.33 (s, 3H,OCH3), 3.53–3.62 (m,
J=4.00,6.8Hz, 2H, COOCH2CH2O), 3.96–4.17 (m, J=4.00, J=4.0, 7.2Hz, 2H, COOCH2CH2O), 4.02–4.12 (m, J=4.0,
6.8Hz, 2H, COOCH2CH3), 4.24–4.29 (m, J=4.00, 4.8Hz, 6.4Hz, 2H, CH2CH3), 4.16–4.27 (m, J=4.0, 6.4Hz, 2H,
2H, COOCH2CH2O), 5.87 (s, 1H, C4-H), 5.89 (s, 1H, NH), COOCH2CH2O), 5.86 (s, 1H, C4-H), 7.26 (t, J=7.6, 8.0Hz, 1H,
7.26 (t, J=7.6, 8.00Hz, 1H, aromatic C(5′)H), 7.49 (t, J=7.2, aromatic C(5′)H), 7.48 (d, J=7.6Hz, 1H, aromatic C(6′)H),
7.6Hz, 1H, aromatic C(4′)H), 7.54 (d, J=8.00Hz, 1H, aromatic 7.53 (d, J=8.0Hz, 1H, aromatic C(4′)H), 7.73 (s, 1H, aromatic
C(6′)H), 7.74 (d, J=7.6Hz, 1H, aromatic C(3′)H); Anal. (%): C(2′)H); Anal. (%): (C20H24N2O7), Calcd (Found): C 59.40
(C20H24N2O7), Calcd (Found): C 59.4 (59.13), H 5.98 (5.81), N (59.09), H 5.98 (5.85), N 6.93 (6.99).
6.93 (6.92).
3-Benzyl 5-Methyl 2,6-Dimethyl-4-(3-nitrophenyl)-1,4-di-
3-Benzyl 5-Methyl 2,6-Dimethyl-4-(2-nitrophenyl)-1,4-di- hydropyridine-3,5-dicarboxylate (6j)
hydropyridine-3,5-dicarboxylate (6e)
Recrystallization from methanol afforded (0.446g, 64%):
Recrystallization from methanol afforded (0.481g, 69%): Rf=0.73 (CHCl3–MeOH, 95:5); mp: 114–116°C; 1H-NMR
Rf=0.70 (CHCl3–MeOH, 95:5); mp: 108–110°C; 1H-NMR (400MHz, CDCl3) δ: 2.26 (s, 3H, COOCH3), 3.55 (s, 6H, C2-
(400MHz, CDCl3) δ: 2.25 (s, 3H, COOCH3), 3.54 (s, 6H, C2- CH3, C6-CH3), 3.59 (s, 2H, COOCH2), 5.09 (s, 1H, C4-H), 6.10
CH3, C6-CH3), 3.56 (s, 2H, COOCH2), 5.10 (s, 1H, C4-H), 6.11 (s, 1H, NH), 7.07–7.27 (m, 11H, aromatic OCH2C6H5), 7.22 (t,
(s, 1H, NH), 7.09–7.18 (m, 11H, aromatic OCH2C6H5), 7.22 (t, J=7.6, 8.0Hz, 1H, aromatic C(4′)H), 7.48 (d, J=7.6Hz, 1H,
J=7.6Hz, 1H, aromatic C(5′)H), 7.47 (t, J=7.6, 8.0Hz, 1H, ar- aromatic C(6′)H), 7.89 (d, J=8.0Hz, 1H, aromatic C(3′)H)
omatic C(4′)H), 7.88 (d, J=7.6Hz, 1H, aromatic C(6′)H), 7.97 7.97 (s, 1H, aromatic C(2′)H); Anal. (%): (C23H22N2O6), Calcd

896
Chem. Pharm. Bull.
Vol. 65, No. 10 (2017)
(Found): C 65.39 (65.75), H 5.52 (5.10), N 6.63 (6.50).
Discovery Studio 3.1 visualizer (Accelrys Software Inc., San
Pharmacological Evaluation A written permission was Diego, CA, U.S.A.). The docking parameters are saved in a
obtained from the Research Ethics Committee (REC) of configuration file which is provided as an example in the sup-
Faculty of Pharmacy, Tanta University and the National Re- porting data.
search Center (NRC) where pharmacological evaluation was
3D-QSAR Studies Using Comparative Molecular Field
performed in accordance with Guide for the Care and Use of Analysis (CoMFA) Method CoMFA is an alignment-
Laboratory Animals 8th Edition 2011 since Egypt has not yet dependent descriptor method. Molecular field interaction ener-
compiled its own guide.
gies are calculated and correlated with biological activities by
The intestine of male guinea-pigs weighing 300–450g was using multivariate statistical analyses. In this research, we aim
removed above the ileocecal junction and longitudinal smooth to generate a QSAR model based on 47 ligands with different
muscle segments of 2cm length were mounted under a resting core structures to generate a useful model for the prediction
tension of 0.5g. The segments were maintained at 37°C in a of novel ligands after. All ligands were drawn into Marvin
10mL jacketed organ bath (ML870B6/C, PanLab). The organ Sketch 5.11.4. The most energetically favored conformer was
bath was filled with a physiological saline solution of the saved as (*. mol2) file format by VAMPS module in BIOVIA
following composition (mmol): NaCl (137), CaCl2 (1.8), KCl Materials Studio 8.29) CoMFA has been carried out using
(2.7), MgSO4 (1.1), sodium hydride (NaH)2PO4 (0.4), NaHCO3 SYBYL CERTARA X2.1.1.30)
(12) and glucose (5), and was continuously aerated with car-
The SYBYL engine consists of 2D and 3D studies: we
bogen (oxygen–carbon dioxide; 95:5) at 37°C. The muscles selected the 3D studies to explain QSAR for the newly synthe-
were equilibrated for 1h with a solution changed every 15min. sized compounds. The molecules were aligned by the template
The contractions were recorded with a force displacement based techniques using the basic nucleus. 3D descriptors such
transducer (MLT0201, PanLab) connected to amplifier. Stocks as Tripos force field, electrostatic, steric and hydrophobic field
of the tested compounds (0.01mM in ethanol) were stored pro- types, with cut-off values of 30.0 and 20.0 kcal/mol, respec-
tected from light. Dilutions were made with double distilled tively with Gasteiger charge type were selected for building
water.23)
In order to study the effects of synthesized DHP on KCl-
the model.
The set of molecules was divided into training and test set
induced contraction of ileum, at the first step, several contrac- based on the OptiSim Diversity Algorithm leading to a train-
tions with KCl (40mM) were made. No significant differences ing set of 32 molecules which had been aligned was placed in
between KCl-induced contractions were observed as stability a rectangular grid. The minus Log the half maximal inhibi-
of tissue and thereafter the main experiments started. At this tory concentration (pIC50) values of DHPs were used as the
step, KCl (40mM) elicited contraction was recorded again and dependent variables, CoMFA values as independent variable.
the peak of the first phase (phasic contraction) was considered A better value of q2 was obtained when hydrophobicity de-
as a control. The contractile response was taken as the 100% scriptors were used as an additional independent variable.
value for the tonic (slow) component of the response. Then, Regression analyses of these variables were performed using
tested compounds were cumulatively added and compound the Partial least square (PLS) algorithms with default param-
induced relaxation of contracted muscle was expressed as per- eters. To acquire the optimal principal components (PCs) and
cent of control.
q2 value for PLS procedure, the cross-validation analyses were
The IC50 of each compound (molar concentration needed to performed by using leave-one-out (LOO) with 3 principal
produce 50% relaxation on contracted ileal smooth muscle) components. Both of the predicted pIC50 in the training set
was graphically determined by the concentration–response and the test set were obtained from the CoMFA model.
curves. Each segment was treated with only one compound.
Nifedipine was used as a reference compound. A triplicate ficient for a predictive model. To determine the best CoMFA
experiment was performed for each compound. model, linear regressions were analyzed for the observed and
Though a high value of q2 is important, it alone is not suf-
Data was statistically analyzed using SPSS 21.0 program predicted values of the test set compounds with intercept
(SPSS, Chicago, IL, U.S.A.).24) The results were recorded as (squared correlation coefficient r2, slope k) and without in-
the mean standard error of the mean (S.E.M.) and they were tercept (squared correlation coefficient r20, slope k′). Models
evaluated statistically using one-way ANOVA following post are considered acceptable if they meet all of the following
hoc test.
conditions: 0.85≤k≤1.15 or 0.85≤k′≤1.15; q2>0.5, r2>0.6 and
Molecular Docking Herewith, we correspond to the situ- r2Pred>0.6.31)
ation where only the sequence of the target protein (and not
the 3D structure) is available, and then its similarity with
protein sequences in database(s) is analyzed. Consequently,
Results and Discussion
Chemistry The synthetic routes of the two DHP series
suitable coordinates of the DHP receptor model were used.25) which are based on Hantzsch condensation32) or a modi-
The Zhorov model was selected as the target receptor for a fied Hantzsch reaction are summarized in Chart 1. A series
molecular docking study. All ligands were drawn into Marvin of symmetric achiral DHPs (3a–l) has been synthesized by
Sketch 5.11.4.26) The most energetically favored conformer using classical Hantzsch condensation.33) The asymmetric
was saved as (*. mol2) file format for docking. The optimal chiral DHPs series (6a–j) has been synthesized by a modified
geometry of the ligands was determined during the docking Hantzsch reaction as described by Meyer et al.,33) In spite the
process. The Molecular docking study was performed on se- commonness of Hantzsch condensation, we still use due to its
lected molecules along with the reference molecule (nifedip- simplicity as well as the high yield of the product as a one-pot
ine) into the DHPs receptor model active site using AutoDock reaction. The synthesized compounds have either 2-nitro-
Vina 1.1.2 protocol.27,28) Docking results were visualized using phenyl or 3-nitrophenyl ring at the 4-position of the DHP ring.

Vol. 65, No. 10 (2017)
Chem. Pharm. Bull.
897
Table 1. Chemical Structures and the Results of in Vitro Calcium Chan-
nel Antagonist Activity Presented as IC50 S.E.M. of the Synthesized
1,4-Dihydropyridines; Symmetrical Compounds (3a–l) and Asymmetrical
Compounds (6a–j)
Compd.
R1
R2
X
IC50
(
S.E.M.)a)
Series 1 (3a–l)
3a (Nifed.)
–CH3
–C2H5
—
—
—
—
—
—
—
—
—
—
—
—
2-NO2 6.32×10−10
(
0.035)
0.035)
0.041)
0.173)
0.091)
0.058)
0.032)
0.153)
0.161)
0.066)
0.002)
0.058)
3b
3c
2-NO2
2-NO2
2-NO2
2-NO2
2-NO2
3-NO2
3-NO2
3-NO2
3-NO2
3-NO2
3-NO2
1.45×10−9
1.60×10−9
1.44×10−9
4.40×10−10
3.07×10−9
(
–CH(CH3)2
(
(
(
(
3d
3e
3f
–CH2CH(CH3)2
–CH2CH2OCH3
–CH2C6H5
3g
3h
3i
–CH3
2.60×10−9
2.10×10−9
2.88×10−9
2.99×10−9
(
–C2H5
(
–CH(CH3)2
–CH2CH(CH3)2
–CH2CH2OCH3
–CH2C6H5
(
(
3j
3k
3l
3.81×10−10
(
(
2.83×10−9
Series 2 (6a–j)
6a
6b
–C2H5
–CH3 2-NO2
–C2H5 2-NO2
2.83×10−9
1.21×10−9
(
(
0.058)
0.121)
0.261)
0.133)
0.113)
0.012)
0.321)
0.118)
0.104)
0.089)
–CH(CH3)2
6c
6d
6e
6f
–CH2CH2OCH3 –CH3 2-NO2
–CH2CH2OCH3 –C2H5 2-NO2
9.6×10−10
1.61×10−9
2.35×10−9
3.34×10−9
1.32×10−9
2.40×10−9
1.78×10−9
2.48×10−9
(
(
(
(
(
(
(
(
–CH2C6H5
–C2H5
–CH3 2-NO2
–CH3 3-NO2
–C2H5 3-NO2
Chart 1. Synthesis of Symmetrical Compounds (3a–l) and Asymmetri-
cal Compounds (6a–j)
6g
6h
6i
–CH(CH3)2
–CH2CH2OCH3 –CH3 3-NO2
–CH2CH2OCH3 –C2H5 3-NO2
All structural features necessary to reserve pharmacological
activity were considered. Divergent acetoacetate esters were
selected to give a variety of side chains which could help in
building the 3D-QSAR model and the biological evaluation.
Some acetoacetate is common and other is new such as the
methoxy ethyl derivative. This type of selection provides a
6j
–CH2C6H5
–CH3 3-NO2
a) The molar concentration of antagonist test compound causing a 50% in the
tonic contractile response (IC50 S.E.M.) in guinea-pig ileum smooth muscle by KCl
(80mmol/L) was determined graphically by dose–response curve. Each compound is
tested in triplicate.
suitable range of bulkiness and electrostatic effects which is IC50 share a similar tendency suggesting that the hyperten-
complied with SAR of DHPs.
sive effect was produced by inhibitory action against L-type
The preparation, collection and purification of the products calcium channels. In the series of symmetric DHPs deriva-
were carried out in the absence of oxidizing agents and in tives (3a–l), increasing lipophilic nature of R1 enhances the
darkness for enhanced photo-stability.34) The elemental analy- pharmacological activity to a certain limit. Therefore, 3f and
sis results for the synthesized compounds were within 0.4% l express high IC50 values proving our concept. Accordingly,
of the calculated values and the Proton-NMR spectra for the the chloro-derivatives should express a higher pharmacologi-
synthesized compounds in CDCl3 were compatible with the cal activity than the nitro-derivatives which is represented in
assigned structures. The structures of test compounds were Table 2.
shown in Table 1.
Molecular Docking Computational molecular docking is
Pharmacological Evaluation The tested DHPs displayed widely used for the study of protein–ligand interactions and
varying levels of intrinsic calcium antagonist activity using for drug discovery and development. Typically, the process
high K+ contraction guinea-pig ileal longitudinal smooth starts with a target of known structure, such as a crystallo-
muscle (GPILSM) method.35–39) The IC50 values ranged from graphic structure of an enzyme of medicinal interest or a ho-
0.381 nM for 3k to 3.34nM for 6f. The IC50 values were ex- mology model. Docking is then used to predict the bound con-
tracted from the graph using Graphpad Prism V6.01 software. formation and the binding free energy of small molecules to
A triplicate experiment was performed for each compound. the target. Single docking experiments are useful for exploring
The calculated S.E.M. reflected non-significant variation in the function of the target, and virtual screening- in which a
the results. The results were presented as IC50 S.E.M. in large library of compounds is docked and ranked-may be used
Table 1. These results were tested statistically for significance to identify new inhibitors for drug development.
using one-way ANOVA following post hoc test in SPSS 21
To our knowledge, several theoretical models of CaV1.2-
software. The results were statistically significant (p=0.000). DHPs have been reported. In 2001, Zhorov et al. built two
This indicates that the obtained results were due to the intrin- models of the pore region of CaV1.2 from rabbit cardiac mus-
sic activity of each compound. The synthesized compounds cle, and docked nifedipine into the active site to explore the
showed activity similar to, higher or lower than the reference interactions of agonists and antagonists.40) In 2003, Lipkind
compound; nifedipine. The most active compound was 3k and Fozzard constructed the inner pore structure of CaV1.2
which is approximately two times more active than nifedipine. and predicted the binding conformations of nifedipine, phe-
A comprehensive study of the activity data shows that nylalkylamine and agonist Bay K8644 by using the molecular

898
Chem. Pharm. Bull.
Vol. 65, No. 10 (2017)
Table 2. Actual and Predicted Biological Activities
IC50 (nM)
(Obs.)
PIC50
(Obs.)
PIC50
(Pred.)
Code
Residues
cLogP
Training Set from Nitro Analogues (17 ligands)
3c
3d
3e
3g
3h
3i
1.60
1.45
0.44
2.60
2.10
2.88
2.99
1.21
0.96
1.61
2.35
3.34
1.32
2.40
2.87
1.72
2.49
8.7959
8.386
8.902
−0.1061
−0.468
0.2075
4.8008
6.0388
3.0448
3.2048
4.2628
4.8808
6.1188
3.6538
4.4918
3.0848
3.6138
1.8884
3.7338
4.5718
3.1648
3.6938
4.9234
8.854
9.3565
8.585
9.149
8.6975
8.7505
8.7257
8.551
−0.1125
−0.0727
−0.1851
−0.0104
−0.0047
0.2231
8.6778
8.5406
8.5406
8.9172
9.0177
8.7932
8.6289
8.4763
8.8794
8.6198
8.5421
8.7645
8.6038
Fig. 1. General Structure of DHP Drugs, with the Adopted Nomencla-
ture Highlighted42)
3j
6a
6b
6c
6d
6e
6f
8.9219
8.7946
8.8299
8.6141
8.3474
8.7561
8.6829
8.7278
8.6644
8.5177
These computational results provided some valuable infor-
mation on L-type Ca2+ channels (LTCC)-DHP recognition,
but the reliability of the predictions may be questionable be-
cause the template used in the homology modeling of CaV1.2
is the K+ channel, where the structures of VGCC and the K+
channel are quite different, especially in the intervening P-
loop.2)
Recently, the crystal structure of the voltage-gated calcium
channel from bacterium Arcobacter butzleri (CaVAb) was
reported (PDB entry: 4MS2), with a pore-motif that is struc-
turally related to vertebrate VGCCs. The residues forming
the inner low-affinity site of CaVAb are still present in verte-
brate channels. Therefore, construction of the 3D structure of
human CaV1.2 based on the crystal structure of CaVAb is more
reasonable than that of the K+ channel.44)
In the current study, we performed pre-synthetic docking of
21 DHPs derivatives into the active site of Zhorov model using
AutoDock Vina 1.1.2 protocol. The crystal structure of Zhorov
model was obtained from the scientist himself with a permis-
sion of a scientific use. Besides, we are working now on a ho-
mology model based on bacterium Arcobacter butzleri (CaAb)
and the docking results will be compared and published in
the recent future. In the meantime, the overall architecture of
the mammalian skeletal muscle CaV1.1 channel was recently
elucidated at a resolution of ca. 4–6Å by cryo-electron mi-
croscopy.44) However, a higher-resolution structural analysis of
mammalian CaV channels has not yet been achieved.
Hereafter, to distinguish between the two sides of the DHP
ring, as suggested by Goldmann and Stoltefuss,45) the pre-
ferred conformation of the DHP ring will be regarded as a
flattened boat with C4 as the bow, the axial aryl ring as the
bowsprit, and the N1 atom as the stern (Fig. 1a). Accordingly,
the two sides of the DHP ring will be referred to the port side
(left) and the starboard side (right; Fig. 1b).
−0.0367
0.0148
0.1289
0.1233
6g
6h
6i
−0.0631
−0.1857
0.1001
6j
0.0861
Training Set from Chloro Analogues (15 ligands)
5a*
5b
5c
5d
5e
5f
0.43
0.23
0.35
0.48
0.77
0.59
0.27
0.72
0.70
0.60
0.28
0.27
0.6
9.3665
9.4559
9.4559
9.3118
9.1135
9.2291
9.5686
9.1427
9.1549
9.2218
9.5528
9.5686
9.2218
9.1249
9.5850
9.5697
9.4517
9.4037
9.4483
9.0374
9.1732
9.5054
9.4504
9.0852
9.3811
9.4067
9.4302
9.2821
9.4068
9.5182
−0.2032
0.0042
4.1442
4.7038
4.7082
5.5418
5.2372
6.1608
4.1348
4.6638
5.9686
4.3992
5.2286
5.2372
5.8476
4.2674
4.3592
0.0522
−0.1365
0.0761
0.0559
5g
5h
5i
0.0632
−0.3077
0.0697
5j
−0.1593
0.1461
5k
5l
0.1384
5m
5n
5o
−0.0603
−0.2819
0.0668
0.75
0.26
Test Set from Nitro Analogues (5 ligands)
Nifed
3b
0.63
1.45
3.07
0.38
2.83
9.2007
8.8386
8.5129
9.4202
8.5482
9.1709
8.7666
8.4758
9.3607
8.5806
0.0298
0.072
3.1248
4.1828
6.274
3f
0.0371
0.0595
−0.0324
3k
3.1248
6.642
3l
Test Set from Chloro Analogues (10 ligands)
4a*
4b
4c
4d
4e
4f
0.42
0.20
0.40
0.54
0.20
0.86
0.48
0.16
0.41
0.70
9.3768
9.699
9.412
−0.0352
0.2505
3.8702
5.2328
5.8508
7.0546
4.0948
7.612
9.4485
9.5076
9.2622
9.5725
8.9548
9.3672
9.3986
9.3185
9.4923
9.3979
9.2676
9.699
−0.1097
0.0054
Based on our previous studies,46) the best binding poses of
nifedipine showed that NH group of the DHP ring makes H-
bond with Tyr4311 in the stern-to-Tyr4311 orientation. This was
proved by the docking scores, and the surrounding residues of
ligands, especially the residues that have essential impact on
the binding affinity between LTCC and DHPs (Table 3).
It can be observed in Fig. 2, that the dihydropyridine ring
of DHPs fits into the cleft formed by the IIIS6, IIIS5 and
IVS6 segments, and the plane of the DHP ring is parallel to
0.1265
9.0655
9.3188
9.7959
9.3872
9.1549
0.1107
4g
4h
4i
−0.0484
0.3973
4.3906
4.9282
7.3046
6.0666
0.0687
4j
−0.3374
*The chemical structures of the chloro-derivatives are provided in the supporting
data.
docking approach.41) In 2007, Cosconati et al. constructed the the pore axis, while the 4-aryl substituent is perpendicular to
central pore of CaV1.2 and explored the binding modes of nine the pore axis.45) The starboard side of the heterocyclic ring
different DHPs.42) In 2009, Tikhonov and Zhorov constructed faces upward, and the portside side at the ortho position to
two structural models of CaV1.2 in open and closed states, and the NH group faces towards the selectivity filter. Most of the
explored the possible binding structures of (S)-nimodipine.43)
ligands fit into a hydrophobic pocket surrounded by Phe2322
,

Vol. 65, No. 10 (2017)
Chem. Pharm. Bull.
899
Fig. 2. (a) The 2D and (b) 3D Amino Acid Residues Interaction of Nifedipine and CaV1.2 Model
Table 3. The Surrounding Residues of the Best Docking Poses of DHPs Binding in the Active Site of LTCC Are Shown Below, and Experimentally
Found DHP-Sensing Residues Are in Bold Type
Ligand
Nifed.
Surrounding residues
Docking score (kcal/mol)
Ile4318, Val1322, Thr3245, Thr3248, Phe3247, Ser3247, Thr4248, Ile3311, Met3319, Ile3314, Ile4315,
Met3318, Ala3315, Phe3322, Tyr4311.
−7.0
3k
3f
Ile4318, Val1322, Thr3245, Thr3248, Phe3247, Ser3247, Thr4248, Ile3311, Met3319, Ile3314, Ile4315,
Met3318, Ala3315, Phe3322, Tyr4311.
−8.4
−6.6
Ile4318, Val1322, Thr3245, Thr3248, Phe3247, Ser3247, Thr4248, Ile3311, Met3319, Ile3314, Ile4315,
Met3318, Ala3315, Phe3322, Tyr4311.
Ile1150, Phe1176, Met3318 and Met3319, and form aryl–aryl interac- explore more potent inhibitors, 3D-QSAR models were built
tion with Phe3322 or Tyr4311. The nitro group and oxygen of the using the 3D-QSAR protocol of SYBYL CERTARA® X2.1.1.
carbonyl group in nifedipine chelate the Ca2+ cofactor of the In this study, 47 compounds bearing variable cores either nitro
selectivity filter.
derivatives or chloro derivatives46) with definite IC50 values
In Fig. 3, the bowsprit group of 3k points upward and then were selected as the model dataset. By convention, the pIC50
approaches the Ca2+ ion chelated by the acid residues of the scale (−log IC50), in which higher values indicate exponential-
selectivity filter, while the bowsprit group of nifedipine can- ly greater potency, is used as a method to measure inhibitory
not due to the side chain length privilege in case of 3k. In activity. The training and test set was chosen by the OptiSim
addition, the portside group of 3k is closer to the selectivity Diversity Algorithm in SYBYL. The diversity algorithm re-
filter in comparison with that of nifedipine. These results are sulted in a perfect selection for both training and test sets. The
consistent with previous studies that the antagonistic action training set contains 32 ligands with varieties of ortho and
of DHP derivatives is directly associated with the Ca2+ occu- meta substituents on the phenyl ring and diverse side chains
pancy of the selectivity filter.
which could give a reliable data for QSAR model away from
The steric effect of the 2 phenyl rings pushes the crucial over fit of data. The test set contains 15 ligands. 3k (the high-
Tyr4311 away from NH of DHP ring also the nitro and carbonyl est biologically active compound in the nitro series) is selected
groups that chelate Ca2+ ion not directed to the lumen of the in concordance with 3e selection in the training set to reflect
selectivity filter. This could show the inability of 3f to un- the reliability of the basis of diversity algorithms.
dergo well allosteric inhibition (Fig. 4).
Molecular Alignment One of the most crucial problems
As a conclusion, we can understand the best binding mode in most of the alignment-based 3D-QSAR methods is that
of DHPs in the active site by molecular alignment of highly their results are highly sensitive to the manner in which the
active ligands (Fig. 5) to describe the amino acid interactions bioactive conformations of all the molecules are superimposed
and be correlated with docking scores shown in Table 3.
over each other. In cases, where all the molecules in a data set
3D-QSAR Studies Using CoMFA 3D-QSAR Method In have a common rigid core structure, molecules can be aligned
order to give a systematic evaluation on DHPs as CCB and to easily using least-square fitting procedure. However, in case

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Chem. Pharm. Bull.
Vol. 65, No. 10 (2017)
Fig. 3. (a) The 2D, (b) Cartoon Presentation of (c) Amino Acid Residues Interaction of 3k and CaV1.2 Model
Fig. 4. (a) The 2D, (b) Cartoon Presentation of (c) Amino Acid Residues Interaction of 3f and CaV1.2 Model
of structural heterogeneity in the dataset, alignment of highly SYBYL align database methods (Fig. 6).
flexible molecules becomes quite difficult and time consum-
Model Validation To seek the most robust QSAR model,
ing.
different sets of molecular descriptors either 2D or 3D have
In CoMFA studies, 3D structures of the molecules are been chosen. The results of each model have been presented
required to be aligned based on a suitable conformational in Table 4. According to the previous data, model 3 has
template and its substructure, which is assumed to be a “bio- been chosen due to the relatively high (Q2) and (R2) values.
active” conformation. A compound with the highest pIC50 Better values of R2 and Q2 were obtained when the calculated
value was used as the template for molecular alignment. Three logarithm of the 1-octanol–water partition coefficient of the
alignment figures are shown to describe the nitro-analogues non-ionized molecules (cLogP) was used as an additional
series, chloro-analogues series and all 47 ligands using the independent variable that signifies the need of hydrophobicity

Vol. 65, No. 10 (2017)
Chem. Pharm. Bull.
901
Fig. 5. Molecular Alignment of Ligands in the Activesite of CaV1.2 Model Showing the Best Binding Mode
Fig. 6. Molecular Alignment of (a) Nitro-Analogues Series, (b) Chloro-Analogues Series and (c) 47 Ligands in Concordance with (d) the Optimal
Binding Mode of DHPs
Table 4. Different QSAR Models with the Cross-Validated Coefficient
Table 5. Validation Criteria for Our QSAR Model
(Q2) and Correlation Coefficient (R2)
R2
Q2
R2pred
r0
r0′
Standard
error
Model
Molecular descriptors
(Q2)
(R2)
0.939
K
0.686
K′
0.968
0.893
S.E.
0.9111
RMSE
0.902
Model 1
Model 2
CoMFA
0.641
0.660
0.840
0.847
0.939
0.161366
0.159536
0.117327
0.897
0.1173277
0.025284
cLogP*, CoMFA
Model 3 cLogP, CoMFA, Hydrophobe 0.686
Rp2red > 0.6,
r2 − r 02/r2 <0.1, r2 − r 0,2/ r2 < 0.1 and
0.85 ≤ k ≤1.15 or 0.85 ≤ k′≤1.15
for high biological activity.
In Table 5, we present the data of our QSAR model and
In the CoMFA contour maps, the steric field is illustrated in
PLS results to prove its reliability and predictability according green (bulky favorable) and yellow (bulky unfavorable) con-
to the validation criteria of Golbraikh and Tropsha47) (Fig. 7).
tours (Fig. 8-left). The steric favorable green contours near the
phenyl ring, C-3 and C-5 substituents of the DHP ring indi-

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Chem. Pharm. Bull.
Vol. 65, No. 10 (2017)
Fig. 7. Regression Line and QSAR Equation of Our QSAR Model Which Show the Most Active 3k as a Test Compound Exactly on the Regression
Line
Fig. 8. CoMFA Contour Maps Based on 3k, (Left) Steric Field
(Right) electrostatic field.
cated the importance of bulky groups in these areas for inhibi- of DHPs. With an electronegative substituent, such as nitro
tory activity. The presence of bulky hydrophilic groups on the groups and extra oxygen atom in the side chain of 3k rational-
phenyl ring increased the inhibitory activity in comparing 4e ize the large percent of electrostatic negative charge desirable
and 5e which is proved by large % Steric/Hydrophobic desir- (62.65%) than nifedipine (65.59%). In case of 4e and 5e, the
able contribution 27.17 and 34.12%, respectively. 3k shows a lower % of contribution favors the nitro substitutions.
less percent (6.35%) compared to chloro derivatives but higher
than nifedipine (5.13%).
Conclusion
One of steric unfavorable yellow contours was near C-2 and
Despite the ever-increasing load of patients with hyperten-
C-4 dimethyl groups of the DHP ring (Fig. 8-right), suggest- sion and related cardiovascular diseases, our attention toward
ing that a bulky group in this region decreased inhibitory ac- the ailment is requires reconsideration and efforts toward drug
tivity as illustrated by the fact that pIC50 of 3k is higher than discovery in this field are little. Recently, hypertension has
3l and f which have benzyl group in the yellow region causing become a condition rather than a disease in our present-day
a penalty and rationalize the high % Steric/Hydrophobic un- lifestyle, but it is the root cause for various other cardiovas-
desirable contribution of them (7.89 and 9.86%, respectively cular, neural and renal conditions. So, the calendar years 2015
(Graphical abstract contains the colored image).
and 2016 carry efforts to reach the crystal structure of CaV1.2
In the CoMFA electrostatic contour map (Fig. 8), red con- to maximize the production of new antihypertensive agents.
tours near the substituent on the phenyl ring and the carbonyl We attempted to develop new molecules as lead compounds
groups of the ester linkages, indicated that electronegative for their development in new antihypertensive agents by
groups around these areas increased the blocking activity modulating the L-type calcium channels. Our endeavor fo-

Vol. 65, No. 10 (2017)
Chem. Pharm. Bull.
Suresh B., Nanjan M., Lett. Drug Des. Discov., 5, 7–14 (2008).
903
cuses on developing a 3D-QSAR model based on synthesized
compounds guided by pre-synthetic molecular docking. Dock-
ing scores yield a high activity of 3k and e compared to refer-
ence nifedipine. After synthesis, the biological activity proves
our docking results which are 0.44 and 0.38nM respectively.
To develop a reliable 3D-QSAR model, we have used another
core structure (chloro-derivatives) for diversity. The results of
validation either internally or externally show the reliability
and predictability of the model. All methods of drug design
used in this research indicate that the need of lipophilicity
outweighs the importance of electronegativity of substituents
reflected by the higher pIC50 and predicted pIC50 of chloro de-
rivatives. Although our results are built on a homology model
of Ca2+ channels relied on K+ channels, we are working on a
model based on bacterial calcium channel to give more benefi-
cial data in the future.
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