Synthesis, evaluation of pharmacological activity, and molecular docking of 1,4-dihydropyridines as calcium antagonists

Article abstract of DOI:10.1248/cpb.c15-00737

1,4-Dihydropyridine (DHP) is an important class of calcium antagonist. It inhibits the influx of extracellular Ca²⁺ through L-type voltage-dependent calcium channels. Two series of nifedipine analogues were synthesized and evaluated as calcium

Full text of DOI:10.1248/cpb.c15-00737

Vol. 64, No. 4
Chem. Pharm. Bull. 64, 297–304 (2016)
297
Regular Article
Synthesis, Evaluation of Pharmacological Activity, and Molecular
Docking of 1,4-Dihydropyridines as Calcium Antagonists
,a
Moataz Ahmed Shaldam,* Mervat Hamed El-Hamamsy,b Dalia Osama Saleh,c and
Tarek Fathy El-Moselhyb
a Department of Medicinal Chemistry, Faculty of Pharmacy, Delta University for Science and Technology; Gamassa
b
35712, Egypt: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Tanta University; Tanta 31527,
c
Egypt: and Department of Pharmacology, National Research Center; Dokki 12622, Egypt.
Received September 28, 2015; accepted January 5, 2016; advance publication released online January 27, 2016
1,4-Dihydropyridine (DHP) is an important class of calcium antagonist. It inhibits the influx of extracel-
lular Ca2+ through L-type voltage-dependent calcium channels. Two series of nifedipine analogues were syn-
thesized and evaluated as calcium antagonists. The ortho-nitrophenyl ring of nifedipine was replaced with an
ortho- or a meta-chlorophenyl substituent. The IC50 values revealed that some of the compounds are similar
to or more active than nifedipine. Substitution with groups of suitable bulkiness, such as ethyl ester, at the
3- and 5-positions of the DHP ring gave 3h, which is approximately three-fold more active than nifedipine
as a calcium antagonist. A docking study with the DHP receptor model was performed to interpret the dif-
ferences in calcium antagonist activities. The molecular docking study demonstrated that the lipophilicity
of the substituted phenyl group at the 4-position of the DHP ring is an important factor that could increase
the activity of the calcium antagonist taking the steric factor into consideration. Bulky groups interfere with
ring-to-ring hydrophobic interaction with Tyr1460 and limit the efficiency of increasing the length of the hy-
drocarbon chain of esters at the 3- and 5-positions of the DHP ring as an approach to increase activity. The
presence of a chelating substituent on the phenyl ring at the 4-position of the DHP ring may ensure strong
binding to the receptor and hence stabilization of the closed-channel conformation.
Key words 1,4-dihydropyridine (DHP); calcium channel blocker; pharmacological evaluation; synthesis; mo-
lecular docking
Calcium is essential in all living organisms. Different types sized. A series of symmetric achiral DHPs (3a–k) has been
of calcium channels can control the influx of calcium ions into synthesized by using classical Hantzsch condensation25) as
cells.1) The influx of calcium ion into the cell takes place in shown in Chart 1. The asymmetric chiral DHPs series (6a–n)
both normal functioning and in pathologic processes.2,3) This has been synthesized by a modified Hantzsch reaction as
raises the importance of calcium channels as targets for many described by Meyer et al.,26) as shown in Chart 1. The syn-
drugs. Various drugs can modulate either agonist or antago- thesized compounds have either 2-chlorophenyl or 3-chloro-
nist, the calcium ion entry through L-type Ca2+ channels.4–7) phenyl ring at the 4-position of the DHP ring. All structural
Calcium antagonists have a various pharmacological activ- features necessary to reserve pharmacological activity were
ity including antihypertensive and antianginal agent,8–10) anti- considered. The preparation, collection and purification of the
tumor,11) anti-inflammatory,12,13) antitubercular,4) analgesic14) products were carried out in the absence of oxidizing agents
and antithrombotic.15,16)
and in darkness. The elemental analysis results for the synthe-
1,4-Dihydropyridines (DHPs) bind selectively to L-type sized compounds were within 0.4% of the calculated values
1
calcium channel protein.17) They can promote either channel and the H-NMR spectra for the synthesized compounds in
activation or inhibition.5,18) DHP calcium antagonists yet de- CDCl3 were compatible with the assigned structures. The
veloped are vasodilators.19) Vasodilatation is due to the uncou- structures of test compounds were shown in Fig. 1.
pling of the contractile mechanism of vascular smooth muscle,
Pharmacology All synthesized compounds were sub-
jected to evaluating their intrinsic calcium antagonist activity
which requires Ca2+.20)
Several studies that targeted structure–activity relationship using high K+ contraction guinea-pig ileal longitudinal smooth
(SAR) of DHP action at the L-type channel have focused on muscle (GPILSM) method.27–30) This method depends on the
the importance of the lipophilicity of DHPs in increasing cal- elevation of cytosolic Ca2+ concentration upon high potassi-
cium antagonist activity.21–24) Chlorophenyl ring is a lipophilic um-induced membrane depolarization through DHP-sensitive
group that can be introduced in 4-position of the DHP ring calcium channels.31,32)
and hence increases the lipophilicity of the target compounds.
The IC50 values were extracted from the graph using
In this research, ortho- and meta-chlorophenyl rings in 4-po- Graphpad Prism V6.01 software. A triplicate experiment was
sition of the DHP ring was used. Also side chain esters in performed for each compound. The calculated standard error
3- and 5-positions were designed to increase lipophilicity to of the mean (S.E.M.) reflected non-significant variation in the
investigate lipophilic effect on calcium antagonist activity.
results. The results were presented as IC50 S.E.M. in Table
1. These results were tested statistically for significance using
one-way ANOVA following post hoc test in SPSS 21 software.
Results and Discussion
Chemistry Two series of DHP derivatives were synthe- The results were statistically significant (p=0.000). This indi-
*To whom correspondence should be addressed. e-mail: dr_moutaz_986@yahoo.com
© 2016 The Pharmaceutical Society of Japan

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Vol. 64, No. 4 (2016)
Table 1. Pharmacological Activity of the Synthesized Compounds and
Nifedipine as a Reference Compound
Compound No.
IC50 (
S.E.M.)a)
3a
3b
3c
3d
3e
3f
4.27×10−10
2.00×10−10
4.02×10−10
5.44×10−10
2.07×10−10
8.57×10−10
4.88×10−10
1.59×10−10
4.10×10−10
7.00×10−10
4.32×10−10
2.39×10−10
3.55×10−10
4.83×10−10
7.76×10−10
5.87×10−10
2.68×10−10
7.21×10−10
6.98×10−10
6.01×10−10
2.81×10−10
2.71×10−10
5.97×10−10
7.46×10−10
2.60×10−10
6.01×10−10
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
0.114)
0.037)
0.002)
0.192)
0.006)
0.058)
0.020)
0.112)
0.123)
0.061)
0.131)
0.119)
0.253)
0.087)
0.151)
0.073)
0.133)
0.106)
0.012)
0.041)
0.116)
0.084)
0.056)
0.162)
0.114)
0.201)
3g
3h
3i
3j
3k
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
Nifed
Chart 1. Synthesis of Symmetrical Compounds (3a–k) and Asymmetri-
cal Compounds (6a–n)
a) IC50 S.E.M. is determined graphically from dose–response curve in molar con-
centration and each compound is tested in triplicate.
Fig. 1. Chemical Structures of the Synthesized 1,4-Dihydropyridines; Symmetrical Compounds (3a–k) and Asymmetrical Compounds (6a–n)
cates that the obtained results were due to the intrinsic activ- f–h along with the reference molecule, nifedipine, into the
ity of each compound. The synthesized compounds showed DHPs receptor model active site using Molegro Virtual Dock-
activity similar to, higher or lower than the reference com- er (MVD) V6.0. The pharmacological activity results of the
pound; nifedipine. The most active compound was 3h which is target compounds revealed that 3f and h were the least and the
three times more active than nifedipine.
highest active calcium antagonists among this series, respec-
Molecular Docking Molecular docking study was antici- tively. 3a and g have methyl ester group at 3- and 5-positions
pated to interpret the comparative differences in the binding similar to nifedipine. This gives the opportunity to explore the
interactions of the synthesized compounds with marked activ- effect of introducing the chlorophenyl ring at 4-position of the
ity at molecular level. This study was carried out on 3a and DHP ring.

Vol. 64, No. 4 (2016)
Chem. Pharm. Bull.
299
Fig. 2. Nifedipine Interaction with the DHP Receptor Model Showing H-Bond with Tyr1149, Hydrophobic Attraction Force with Tyr1460, Chelate with
Ca2+ Cofactor and Hydrophobic Interaction with the Lipophilic Bracelet (Leu397, Leu745, Met1158 and Ile1468) and with Ile1150
Nifedipine affinity to the receptor can be explained by in- ester groups in both nifedipine and the two compounds 3a and
teraction with seven residues (Fig. 2). NH group of the DHP g. The down methyl ester group stabilizes the lipophilic brace-
ring makes H-bond with Tyr1149. Hydrophobic attraction force let while the other methyl ester group is projected in the water
is observed between the phenyl ring of nifedipine and Tyr1460
.
lake core of the receptor and participates in chelating the Ca2+
The nitro group and oxygen of the carbonyl group in nife- cofactor (Fig. 3). Hydrogen bonding between NH group of the
dipine chelate the Ca2+ cofactor of the selectivity filter. The DHP ring and hydroxyl group of Tyr1149 was observed in 3a
down ester methyl group makes hydrophobic interaction with and g interaction with the receptor.
the lipophilic bracelet (Leu397, Leu745, Met1158 and Ile1468). The
Increasing lipophilicity is directly proportional to the
methyl group of DHP ring makes hydrophobic interaction calcium antagonist activity24) however; steric hindrance is a
with Ile1150
limitation to this rule. When observing the IC50 values of this
.
Comparing the IC50 values of 3a and g with nifedipine, series, it is notable that this series cannot tolerate side chain
both are more active than nifedipine. This can be explained by more than total four carbon atoms (ethyl ester at 3- and 5-po-
the effect of introducing chlorophenyl ring in the synthesized sitions). 3f is the least active among the series. This compound
compounds. Chlorine atom and nitro group are electron with- has benzyl ester substituent with total ten carbon atom side
drawing groups however chlorine atom is lipophilic in nature chain (five extended carbon atom at 3- and 5-positions). This
in contrast to the hydrophilic nature of the nitro group. Chlo- bulky side chain may interfere with the affinity axis (Tyr1149
rophenyl ring increases the lipophilicity of the synthesized and Tyr1460) binding33) which may result in abolishing ring to
compounds. Also ring to ring hydrophobic interaction be- ring hydrophobic attraction force between the chlorophenyl
tween the substituted phenyl ring in nifedipine and the phenyl ring and Tyr1460. The down benzyl ester group phenyl ring
ring in Tyr1460 was preserved in the synthesized compounds. makes the affinity axis instead of important stabilization of
There is a non-significant difference in activity regarding to the hydrophobic bracelet. This greatly decreases the activity of
the position of chlorine atom on the phenyl ring which was 3f regardless of increasing the lipophilicity of the compound
obvious from the IC50 values of 3a and g (4.27×10−10 M and (Fig. 4).
4.88×10−10 M, respectively). This may indicate that substitution
The highest active compound among the synthesized series
at ortho- or meta-positions makes the phenyl ring non copla- was 3h which has the optimum side chain length. This com-
nar with the DHP ring. Although nifedipine contains nitro pound is three times more active than nifedipine. The ethyl
group that included in the bidentate chelation of Ca2+ cofac- ester group makes the proper stabilization of the hydrophobic
tor and absence of this property in the chlorine atom in the bracelet and minimal projection in the water lake. The phenyl
synthesized series, this series has higher calcium antagonist ring in 4-position of DHP ring makes ring to ring hydrophobic
activity than nifedipine. This again implies the importance attraction with Tyr1460. The chlorine atom in meta-position
of the ring lipophilicity in the calcium antagonist activity of dose not interfere with this interaction, in contrast to ortho-
DHPs.33) Ester groups at 3- and 5-positions are simply methyl position. The chlorine atom in ortho-position decreases to

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Vol. 64, No. 4 (2016)
Fig. 3. Molecular Docking of 3a (Top) and 3g (Bottom) into the Active Site of DHP Receptor Model Showing Interaction Similar to Nifedipine (H-
Bond with Tyr1149, Hydrophobic Attraction Force with Tyr1460, Chelate with Ca2+ Cofactor and Hydrophobic Interaction with the Lipophilic Bracelet
and with Ile1150
)
some extent ring to ring hydrophobic attraction force due to Esters of Dialkyl 4-(Chlorophenyl)-2,6-dimethyl-1,4-di-
clash with the Ca2+ cofactor of the selectivity filter (Fig. 5).
hydropyridine-3,5-dicarboxylate (3a–k) To 50mL round-
bottomed flask, ammonium acetate (0.162g, 2.10mmol)
was added to a stirring solution of 2-chlorobenzaldehyde or
Experimental
Chemistry Melting points were determined on a Stuart 3-chlorobenzaldehyde (0.254g, 1.81mmol) and the corre-
SMP10 capillary melting point apparatus and are uncor- sponding alkyl acetoacetate (3.62mmol) in 10mL methanol or
rected. Elemental analyses were performed by Micro Ana- 2-propanol. The reaction mixture was protected from light and
lytical Center, Faculty of Science, Cairo University, Giza, heated under reflux for 12–24h. After cooling, the precipitate
Egypt and were within
1H-NMR spectra were recorded on
(500MHz) spectrometer. Samples were dissolved in CDCl3.
Coupling constants are reported in Hz. All organic reagents 3,5-dicarboxylate (3a)
0.4% of the calculated values. was filtered and purified by crystallization from methanol or
JEOL-ECA500 2-propanol to afford the corresponding product.
Dimethyl 4-(2-Chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-
a
used were obtained from Sigma-Aldrich Company and were
Recrystallization from methanol afforded 0.480g (79%) of
used without further purification. Reactions were monitored the desired compound, mp: 186–188°C (lit. mp 186–188°C34)).
by TLC using pre-coated sheet (Fastman Kodak Co., Silca
Diethyl 4-(2-Chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-
60F254) and were visualized with UV light at 254nm. Oily 3,5-dicarboxylate (3b)
products were purified with preparative TLC (Silca gel) using
chloroform as eluent.
Recrystallization from methanol afforded 0.434g (66%) of
the desired compound, mp: 128–130°C (lit. mp 128–130°C34)).
General Procedure for the Synthesis of Symmetrical

Vol. 64, No. 4 (2016)
Chem. Pharm. Bull.
301
Fig. 5. Molecular Docking of 3h into the Active Site of DHP Recep-
tor Model Showing Interaction Like Nifidepine (H-Bond with Tyr1149
Hydrophobic Attraction Force with Tyr1460, Chelate with Ca2+ Cofactor
and Hydrophobic Interaction with the Lipophilic Bracelet and with Ile1150
and Proper Chlorine Atom Position Away from the Hydrophilic Core of
the Receptor
,
Fig. 4. Molecular Docking of 3f into the Active Site of DHP Receptor
Model Showing That the Bulky Benzyl Ester Group Makes the Affinity
Axis (Tyr1149 and Tyr1460) Binding Instead of Important Stabilization of
the Hydrophobic Bracelet
)
Diisopropyl 4-(2-Chlorophenyl)-2,6-dimethyl-1,4-dihydropyri- matic C(6′)H), 7.38 (d, J=7.5Hz, 1H, aromatic C(3′)H).
dine-3,5-dicarboxylate (3c) Dibenzyl 4-(2-Chlorophenyl)-2,6-dimethyl-1,4-dihydropyri-
Recrystallization from methanol afforded 0.355g (50%), dine-3,5-dicarboxylate (3f)
mp: 132–134°C, Anal. (%): (C21H26ClNO4), Calcd (Found),
TLC purification afforded 0.706g (80%), pure oil. (%):
C: 64.36 (64.22), H: 6.69 (6.46), N: 3.57 (3.53). 1H-NMR (C29H26ClNO4), Calcd (Found), C: 71.38 (71.22), H: 5.37 (5.35),
(500MHz, CDCl3) δ: 1.05 (d, J=5.9Hz, 6H, CH(CH3)2), 1.22 N: 2.87 (2.79). 1H-NMR (500MHz, CDCl3) δ: 2.25 (s, 6H,
(d, J=5.9Hz, 6H, CH(CH3)2), 2.28 (s, 6H, C2-CH3 and C6- C2-CH3 and C6-CH3), 5.03 (d, J=20.1Hz, 2H, COOCH2), 5.06
CH3), 4.85–5.01 (m, 2H, 2 of CH(CH3)2), 5.36 (s, 1H, C4-H), (d, J=21.3Hz, 2H, COOCH2), 5.42 (s, 1H, C4-H), 5.80 (s, 1H,
5.67 (s, 1H, NH), 7.02 (t, J=8.5Hz, 1H, aromatic C(5′)H), 7.11 NH), 7.01–7.07 (m, 2H, aromatic C(5′)H and C(4′)H), 7.13–7.31
(t, J=7.5Hz, 1H, aromatic C(4′)H), 7.21 (d, J=8.5Hz, 1H, aro- (m, 12H, aromatic OCH2C6H5, C(6′)H and C(3′)H).
matic C(6′)H), 7.37 (d, J=7.5Hz, 1H, aromatic C(3′)H).
Dimethyl 4-(3-Chlorophenyl)-2,6-dimethyl-1,4-dihydropyri-
Diisobutyl 4-(2-Chlorophenyl)-2,6-dimethyl-1,4-dihydropyri- dine-3,5-dicarboxylate (3g)
dine-3,5-dicarboxylate (3d)
Recrystallization from methanol afforded 0.201g (60%
Recrystallization from methanol afforded 0.160g (21%), yield) of the desired compound, mp: 188–190°C (lit. mp
mp: 135–137°C, Anal. (%): (C23H30ClNO4), Calcd (Found), 188–190°C34)).
C: 65.78 (65.90), H: 7.20 (6.95), N: 3.34 (3.26). 1H-NMR
Diethyl 4-(3-Chlorophenyl)-2,6-dimethyl-1,4-dihydropyri-
(500MHz, CDCl3) δ: 0.80 (d, J=5.8Hz, 6H, CH(CH3)2), dine-3,5-dicarboxylate (3h)
0.86 (d, J=5.8Hz, 6H, CH(CH3)2), 1.88–2.01 (m, 2H, 2 of
Recrystallization from methanol afforded 0.196g (54%
CH(CH3)2), 2.31 (s, 6H, C2-CH3 and C6-CH3), 3.70–3.90 (m, yield) of the desired compound, mp: 132–134°C (lit. mp
4H, 2 of COOCH2), 4.50 (s, 1H, C4-H), 5.62 (s, 1H, NH), 7.02 132–134°C34)).
(t, J=8.5Hz, 1H, aromatic C(5′)H), 7.12 (t, J=7.5Hz, 1H, aro-
Diisoproyl 4-(3-Chlorophenyl)-2,6-dimethyl-1,4-dihydropyri-
matic C(4′)H), 7.22 (d, J=8.5Hz, 1H, aromatic C(6′)H), 7.40 dine-3,5-dicarboxylate (3i)
(d, J=7.5Hz, 1H, aromatic C(3′)H).
Recrystallization from methanol afforded 0.567g (80%),
Bis(2-methoxyethyl) 4-(2-Chlorophenyl)-2,6-dimethyl-1,4- mp: 116–118°C, Anal. (%): (C21H26ClNO4), Calcd (Found),
dihydropyridine-3,5-dicarboxylate (3e)
C: 64.36 (64.09), H: 6.69 (6.39), N: 3.57 (3.58). 1H-NMR
Recrystallization from methanol afforded 0.445g (58%), (500MHz, CDCl3) δ: 1.08 (d, J=5.9Hz, 6H, CH(CH3)2), 1.20
mp: 124–126°C, Anal. (%): (C21H26ClNO6), Calcd (Found), (d, J=5.9Hz, 6H, CH(CH3)2), 2.23 (s, 6H, C2-CH3 and C6-
C: 59.50 (59.68), H: 6.18 (6.38), N: 3.30 (3.34). 1H-NMR CH3), 4.87–4.95 (m, 2H, 2 of CH(CH3)2), 5.58 (s, 1H, C4-H),
(500MHz, CDCl3) δ: 2.27 (s, 6H, C2-CH3 and C6-CH3), 3.27 7.06–7.23 (m, 3H, aromatic C(6′)H, C(5′)H and C(4′)H), 7.27
(s, 6H, 2 of OCH3), 3.46–3.63 (m, 4H, 2 of COOCH2CH2O), (s, 1H, aromatic C(2′)H).
4.04–4.24 (m, 4H, 2 of COOCH2), 5.37 (s, 1H, C4-H), 5.65
(s, 1H, NH), 7.03 (t, J=8.5Hz, 1H, aromatic C(5′)H), 7.11 (t, dine-3,5-dicarboxylate (3j)
J=7.5Hz, 1H, aromatic C(4′)H), 7.23 (d, J=8.5Hz, 1H, aro- Recrystallization from methanol afforded 0.175g (23%), mp:
Diisobutyl 4-(3-Chlorophenyl)-2,6-dimethyl-1,4-dihydropyri-

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Chem. Pharm. Bull.
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95–97°C, Anal. (%): (C23H30ClNO4), Calcd (Found), C: 65.78 (t, J=8.5Hz, 1H, aromatic C(5′)H), 7.11 (t, J=7.5Hz, 1H, aro-
(65.55), H: 7.20 (6.94), N: 3.34 (3.33). 1H-NMR (500MHz, matic C(4′)H), 7.21 (d, J=8.5Hz, 1H, aromatic C(6′)H), 7.38
CDCl3) δ: 0.87 (d, J=5.8Hz, 6H, CH(CH3)2), 0.90 (d, (d, J=7.5Hz, aromatic 1H, C(3′)H).
J=5.8Hz, 6H, CH(CH3)2), 1.80–1.96 (m, 2H, 2 of CH(CH3)2),
5-Isobutyl 3-Methyl 4-(2-Chlorophenyl)-2,6-dimethyl-1,4-
2.32 (s, 6H, C2-CH3 and C6-CH3), 3.83 (d, J=6.0Hz, 4H, 2 of dihydropyridine-3,5-dicarboxylate (6d)
COOCH2), 4.89 (s, 1H, C4-H), 5.79 (s, 1H, NH), 6.99–7.21(m,
Recrystallization from methanol afforded 0.215g (32%), mp:
3H, aromatic C(6′)H, C(5′)H and C(4′)H), 7.28 (s, 1H, aro- 158–160°C, Anal. (%): (C20H24ClNO4), Calcd (Found), C: 63.57
matic C(2′)H).
Bis(2-methoxyethyl) 4-(3-Chlorophenyl)-2,6-dimethyl-1,4- CDCl3) δ: 0.78 (d, J=6.4Hz, 6H, CH(CH3)2), 1.88–1.99 (m,
dihydropyridine-3,5-dicarboxylate (3k) 1H, CH(CH3)2), 2.28 (s, 6H, C2-CH3 and C6-CH3), 3.58 (s,
(63.40), H: 6.40 (6.15), N: 3.71 (3.57). 1H-NMR (500MHz,
Recrystallization from methanol afforded 0.476g (62%), mp: 3H, COOCH3), 3.84 (d, J=7.3Hz, 2H, COOCH2CH), 5.30
118–120°C, Anal. (%): (C21H26ClNO6), Calcd (Found), C: 59.50 (s, 1H, C4-H), 5.68 (s, 1H, NH), 6.97 (t, J=8.5Hz, 1H, aro-
(59.19), H: 6.18 (5.92), N: 3.30 (3.28). 1H-NMR (500MHz, matic C(5′)H), 7.11 (t, J=7.5Hz, 1H, aromatic C(4′)H), 7.23
CDCl3) δ: 2.31 (s, 6H, C2-CH3 and C6-CH3), 3.33 (s, 6H, 2 (d, J=8.5Hz, 1H, aromatic C(6′)H), 7.38 (d, J=7.5Hz, 1H,
of OCH3), 3.49–3.62 (m, 4H, 2 of COOCH2CH2O), 4.06–4.24 aromatic C(3′)H).
(m, 4H, 2 of COOCH2), 4.96 (s, 1H, C4-H), 6.99–7.15 (m, 2H,
3-Ethyl 5-Isobutyl 4-(2-Chlorophenyl)-2,6-dimethyl-1,4-
aromatic C(6′)H and C(5′)H); 7.19 (d, J=8.8Hz, 1H, aromatic dihydropyridine-3,5-dicarboxylate (6e)
C(4′)H); 7.26 (s, 1H, aromatic C(2′)H).
Recrystallization from methanol afforded 0.196g (28%), mp:
General Procedure for the Synthesis of Asymmetrical 114–116°C, Anal. (%): (C21H26ClNO4), Calcd (Found), C: 64.36
1
Esters of Dialkyl 4-(Chlorophenyl)-2,6-dimethyl-1,4-dihy- (64. 09), H: 6.69 (6.39), N: 3.57 (3.59). H-NMR (500MHz,
dropyridine-3,5-dicarboxylate (6a–n) To a 50mL round- CDCl3) δ: 0.74 (d, J=6.5Hz, 6H, CH(CH3)2), 1.15 (t, J=6.1Hz,
bottomed flask were added a mixture of 2-chlorobenzaldehyde 3H, CH2CH3), 1.84–1.99 (m, 1H, CH(CH3)2), 2.27 (s, 6H,
or 3-chlorobenzaldehyde (0.250g, 1.78mmol), alkyl aceto- C2-CH3 and C6-CH3), 3.75 (d, J=7.1Hz, 2H, COOCH2CH),
acetate (1.78mmol) and alkyl 3-aminocrotonate (1.78mmol) 3.96–4.21 (m, 2H, COOCH2CH3), 5.34 (s, 1H, C4-H), 5.77
in 10mL methanol or 2-propanol. The reaction mixture was (s, 1H, NH), 6.98 (t, J=8.5Hz, 1H, aromatic C(5′)H), 7.11 (t,
protected from light and heated under reflux for 9–24h. After J=7.5Hz, 1H, aromatic C(4′)H), 7.23 (d, J=8.5Hz, 1H, aro-
cooling, the precipitate was filtered and purified by crystalli- matic C(6′)H), 7.37 (d, J=7.5Hz, 1H, aromatic C(3′)H).
zation from methanol or 2-propanol to afford the correspond-
ing product.
3-Methyl
5-(2-Methoxyethyl)
4-(2-Chlorophenyl)-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (6f)
5-Ethyl 3-Methyl 4-(2-Chlorophenyl)-2,6-dimethyl-1,4-
dihydropyridine-3,5-dicarboxylate (6a)
Recrystallization from methanol afforded 0.347g (51%),
mp: 103–105°C, Anal. (%): (C19H22ClNO5), Calcd (Found),
Recrystallization from methanol afforded 0.189g (30%), C: 60.08 (59.76), H: 5.84 (5.61), N: 3.69 (3.44). 1H-NMR
mp: 113–115°C, Anal. (%): (C18H20ClNO4), Calcd (Found), (500MHz, CDCl3) δ: 2.26 (s, 6H, C2-CH3 and C6-CH3), 3.26
C: 61.80 (62.09), H: 5.76 (5.50), N: 4.00 (4.18). 1H-NMR (s, 3H, OCH3), 3.52–3.58 (m, 2H, COOCH2CH2O), 3.66 (s,
(500MHz, CDCl3) δ: 1.17 (t, J=6.1Hz, 3H, CH2CH3), 2.25 (s, 3H, COOCH3), 4.05–4.26 (m, 2H, COOCH2CH2O), 5.42 (s,
6H, C2-CH3 and C6-CH3), 3.59 (s, 3H, COOCH3), 3.98–4.14 1H, C4-H), 5.77 (s, 1H, NH), 7.02 (t, J=8.5Hz, 1H, aromatic
(m, 2H, COOCH2), 5.40 (s, 1H, C4-H), 5.69 (s, 1H, NH), 7.01 C(5′)H), 7.11 (t, J=7.5Hz, 1H, aromatic C(4′)H), 7.22 (d,
(t, J=8.5Hz, 1H, aromatic C(5′)H), 7.13 (t, J=7.5Hz, 1H, aro- J=8.5Hz, 1H, aromatic C(6′)H), 7.36 (d, J=7.5Hz, 1H, aro-
matic C(4′)H), 7.22 (d, J=8.5Hz, 1H, aromatic C(6′)H), 7.37 matic C(3′)H).
(d, J=7.5Hz, 1H, aromatic C(3′)H).
5-Isopropyl 3-Methyl 4-(2-Chlorophenyl)-2,6-dimethyl-1,4- methyl-1,4-dihydropyridine-3,5-dicarboxylate (6g)
dihydropyridine-3,5-dicarboxylate (6b) Recrystallization from methanol afforded 0.394g (56%), mp:
3-Ethyl 5-(2-Methoxyethyl) 4-(2-Chlorophenyl)-2,6-di-
Recrystallization from methanol afforded 0.166g (25%), mp: 70–72°C, Anal. (%): (C20H24ClNO5), Calcd (Found), C: 60.99
151–153°C, Anal. (%): (C19H22ClNO4), Calcd (Found), C: 62.72 (61.31), H: 6.14 (5.80), N: 3.56 (3.53). 1H-NMR (500MHz,
(62.42), H: 6.09 (5.84), N: 3.85 (3.89). 1H-NMR (500MHz, CDCl3) δ: 1.17 (t, J=6.1Hz, 3H, CH2CH3), 2.30 (s, 6H, C2-
CDCl3) δ: 1.00 (d, J=5.9Hz, 3H, CH(CH3)2), 1.19 (d, J=5.9Hz, CH3 and C6-CH3), 3.29 (s, 3H, OCH3), 3.44 (t, J=5.3Hz, 2H,
3H, CH(CH3)2), 2.22 (s, 3H, C2-CH3), 2.32 (s, 3H, C6-CH3), COOCH2CH2O), 3.51–3.61 (m, 2H, COOCH2CH3), 4.00–4.10
3.55 (s, 3H, COOCH3), 4.85–5.05 (m, 1H, CH(CH3)2), 5.31 (m, 2H, COOCH2CH2O), 4.41 (s, 1H, C4-H), 4.85 (s, 1H, NH),
(s, 1H, C4-H), 5.71 (s, 1H, NH), 7.00 (t, J=8.5Hz, 1H, aro- 7.02 (t, J=8.5Hz, 1H, aromatic C(5′)H), 7.11 (t, J=7.5Hz, 1H,
matic C(5′)H), 7.11 (t, J=7.5Hz, 1H, aromatic C(4′)H), 7.21 aromatic C(4′)H), 7.21 (d, J=8.5Hz, 1H, aromatic C(6′)H),
(d, J=8.5Hz, 1H, aromatic C(6′)H), 7.35 (d, J=7.5Hz, 1H, 7.37 (d, J=7.5Hz, 1H, aromatic C(3′)H).
aromatic C(3′)H).
5-Benzyl 3-Methyl 4-(2-Chlorophenyl)-2,6-dimethyl-1,4-
3-Ethyl 5-Isopropyl 4-(2-Chlorophenyl)-2,6-dimethyl-1,4- dihydropyridine-3,5-dicarboxylate (6h)
dihydropyridine-3,5-dicarboxylate (6c)
TLC purification afforded 0.601g (81% yield), pure oil,
Recrystallization from methanol afforded 0.483g (71%), mp: Anal. (%): (C23H22ClNO4), Calcd (Found), C: 67.07 (66.89),
1
115–117°C, Anal. (%): (C20H24ClNO4), Calcd (Found), C: 63.57 H: 5.38 (5.47), N: 3.40 (3.36). H-NMR (500MHz, CDCl3) δ:
(63.38), H: 6.40 (6.09), N: 3.71 (3.57). 1H-NMR (500MHz, 2.27 (s, 3H, COOCH3), 3.47 (s, 6H, C2-CH3 and C6-CH3), 3.60
CDCl3) δ: 1.44 (t, J=6.1Hz, 3H, CH2CH3), 1.18 (d, J=6.0Hz, (s, 2H, COOCH2), 4.98 (s, 1H, C4-H), 5.44 (s, 1H, NH), 7.01
3H, CH(CH3)2), 1.24 (d, J=6.0Hz, 3H, CH(CH3)2), 2.21 (s, 6H, (t, J=8.5Hz, 1H, C(5′)H), 7.08–7.15 (m, 8H, aromatic C(4′)H,
C2-CH3 and C6-CH3), 3.82–4.20 (m, 2H, COOCH2), 4.70–5.01 C(6′)H, C(3′)H and OCH2C6H5).
(m, 1H, CH(CH3)2), 5.28 (s, 1H, C4-H), 5.72 (s, 1H, NH), 6.98

Vol. 64, No. 4 (2016)
Chem. Pharm. Bull.
303
5-Ethyl 3-Methyl 4-(3-Chlorophenyl)-2,6-dimethyl-1,4- 5.71 (s, 1H, NH), 7.04–7.15 (m, 2H, aromatic C(6′)H and
dihydropyridine-3,5-dicarboxylate (6i) C(5′)H), 7.19 (d, J=8.8Hz, 1H, aromatic C(4′)H), 7.27 (s, 1H,
Recrystallization from methanol afforded 0.302g (48%), mp: aromatic C(2′)H)
118–120°C, Anal. (%): (C18H20ClNO4), Calcd (Found), C: 61.80
Pharmacology Male guinea-pigs weighing 300–450g
(61.78), H: 5.76 (5.85), N: 4.00 (3.69). 1H-NMR (500MHz, were sacrificed and the intestine was removed above the il-
CDCl3) δ: 1.09 (t, J=6.1Hz, 3H, CH2CH3), 2.25 (s, 6H, C2- eocecal junction and longitudinal smooth muscle segments
CH3 and C6-CH3), 3.61 (s, 3H, COOCH3), 3.90–4.30 (m, 2H, of 2cm length were mounted under a resting tension of 0.5g.
COOCH2), 4.89 (s, 1H, C4-H), 5.66 (s, 1H, NH), 6.85–7.15 (m, The segments were maintained at 37°C in a 10mL jacketed
1H, aromatic C(6′)H and C(5′)H), 7.19 (d, J=8.8Hz, 1H, aro- organ bath (ML870B6/C, PanLab). The organ bath was filled
matic C(4′)H), 7.31 (s, 1H, C(2′)H).
5-Isopropyl 3-Methyl 4-(3-Chlorophenyl)-2,6-dimethyl-1,4- tion (mmol): NaCl (137), CaCl2 (1.8), KCl (2.7), MgSO4 (1.1),
dihydropyridine-3,5-dicarboxylate (6j) NaH2PO4 (0.4), NaHCO3 (12) and glucose (5), and was contin-
with a physiological saline solution of the following composi-
Recrystallization from methanol afforded 0.257g (39%), uously aerated with carbogen (oxygen–carbon dioxide; 95:5)
mp: 133–135°C, Anal. (%): (C19H22ClNO4), Calcd (Found), at 37°C. The muscles were equilibrated for 1h with a solution
C: 62.72 (62.63), H: 6.09 (5.83), N: 3.85 (4.16). 1H-NMR changed every 15min. The contractions were recorded with a
(500MHz, CDCl3) δ: 1.07 (d, J=5.9Hz, 3H, CH(CH3)2), 1.23 force displacement transducer (MLT0201, PanLab) connected
(d, J=5.9Hz, 3H, CH(CH3)2), 2.24 (s, 6H, C2-CH3 and C6- to amplifier. Stocks of the tested compounds (10−6 M in etha-
CH3), 3.62 (s, 3H, COOCH3), 4.78–5.13 (m, 2H, CH(CH3)2, nol) were stored protected from light. Dilutions were made
C4-H), 5.69 (s, 1H, NH), 7.04–7.22 (m, 3H, aromatic C(6′)H, with double distilled water.
C(5′)H and C(4′)H), 7.28 (s, 1H, aromatic C(2′)H).
3-Ethyl 5-Isopropyl 4-(3-Chlorophenyl)-2,6-dimethyl-1,4- induced contraction of ileum, at the first step, several contrac-
dihydropyridine-3,5-dicarboxylate (6k) tions with KCl (40mM) were made. No significant differences
In order to study the effects of synthesized DHP on KCl-
Recrystallization from methanol afforded 0.252g (37%), between KCl-induced contractions were considered as stability
mp: 117–119°C, Anal. (%): (C20H24ClNO4), Calcd (Found), of tissue and thereafter the main experiments were started. At
C: 63.57 (63.49), H: 6.40 (6.13), N: 3.71 (3.80). 1H-NMR this step, KCl (40mM)-elicited contraction was recorded again
(500MHz, CDCl3) δ: 0.87 (d, J=5.9Hz, 3H, CH(CH3)2), 0.90 and the peak of the first phase (phasic contraction) was con-
(d, J=5.9Hz, 3H, CH(CH3)2), 1.88 (t, J=6.1Hz, 3H, CH2CH3), sidered as a control. The contractile response was taken as the
2.34 (s, 6H, C2-CH3 and C6-CH3), 3.72–3.88 (m, 3H, COOCH2, 100% value for the tonic (slow) component of the response.
COOCH), 5.01 (s, 1H, C4-H), 5.71 (s, 1H, NH), 6.92–7.16 (m, Then, tested compounds were cumulatively added and com-
2H, aromatic C(6′)H and C(5′)H), 7.19 (d, J=8.8Hz, 1H, aro- pound induced relaxation of contracted muscle was expressed
matic C(4′)H), 7.28 (s, 1H, aromatic C(2′)H).
5-Isobutyl 3-Methyl 4-(3-Chlorophenyl)-2,6-dimethyl-1,4-
dihydropyridine-3,5-dicarboxylate (6l)
as percent of control.
The IC50 of each compound (molar concentration needed to
produce 50% relaxation on contracted ileal smooth muscle)
Recrystallization from methanol afforded 0.166g (24%), mp: were graphically determined from the concentration–response
96–98°C, Anal. (%): (C20H24ClNO4), Calcd (Found), C: 63.57 curves. Each segment was treated with only one compound.
(63.75), H: 6.40 (6.23), N: 3.71 (3.91). 1H-NMR (500MHz, Nifedipine was used as reference compound. A triplicate ex-
CDCl3) δ: 0.80 (d, J=6.4Hz, 6H, CH(CH3)2), 1.83–1.93 (m, periment was performed for each compound. This protocol
1H, CH(CH3)2), 2.28 (s, 6H, C2-CH3 and C6-CH3), 3.61 (s, was approved by Tanta University ethics committee.
3H, COOCH3), 3.79 (d, J=6.5Hz, 1H, COOCH2), 3.85 (d,
Statistic Data Analysis Data were statistically analyzed
J=6.5Hz, 1H, COOCH2) 4.91 (s, 1H, C4-H), 5.74 (s, 1H, NH), using SPSS 21.0 program (SPSS, Chicago, IL, U.S.A.). The re-
7.01–7.22 (m, 3H, aromatic C(6′)H, C(5′)H and C(4′)H), 7.28 sults are recorded as the mean S.E.M. and they were evaluat-
(s, 1H, aromatic C(2′)H).
3-Methyl 5-(2-Methoxyethyl) 4-(3-Chlorophenyl)-2,6-dimeth-
yl-1,4-dihydropyridine-3,5-dicarboxylate (6m)
ed statistically using one-way ANOVA following post hoc test.
Molecular Docking Docking process requires the 3D
structure of both protein and ligands. Coordinates of the DHP
Recrystallization from methanol afforded 0.198g (29%), receptor model developed by Shaldam et al.33) were used. All
mp: 106–108°C, Anal. (%): (C19H22ClNO5), Calcd (Found), ligands were drawn into Marvin Sketch V5.1.3.35) The most
C: 60.08 (59.98), H: 5.84 (5.55), N: 3.69 (3.39). 1H-NMR energetically favored conformer was saved as (*.mol2) file for-
(500MHz, CDCl3) δ: 2.28 (s, 6H, C2-CH3 and C6-CH3), 3.33 mat for docking. The optimal geometry of the ligands was de-
(s, 3H, OCH3), 3.45–3.55 (m, 2H, COOCH2CH2O), 3.63 (s, 3H, termined during the docking process. Docking was performed
COOCH3), 4.03–4.35 (m, 2H, COOCH2CH2O), 5.01 (s, 1H, in a multistep procedure using Molegro Virtual Docker V6.0
C4-H), 5.79 (s, 1H, NH), 7.01–7.22 (m, 3H, aromatic C(6′)H, program. Firstly, nifedipine was placed in the proposed active
C(5′)H and C(4′)H), 7.26 (s, 1H, aromatic C(2′)H).
3-Ethyl 5-(2-Methoxyethyl) 4-(3-Chlorophenyl)-2,6-dimeth- as the most crucial part of the ligand that forms H-bond with
yl-1,4-dihydropyridine-3,5-dicarboxylate (6n) the receptor. Consequently, docking template was generated in
site defined by SAR.33) The NH-group of DHP is considered
Recrystallization from methanol afforded 0.203g (29%), attempts to align all ligands to the orientation of nifedipine.
mp: 116–118°C, Anal. (%): (C20H24ClNO5), Calcd (Found), This is a demanding to bias the formation of H-bond with
C: 60.99 (61.00), H: 6.14 (6.36), N: 3.56 (3.88). 1H-NMR Tyr1149—an important DHP sensing residue—while takes the
(500MHz, CDCl3) δ: 1.16 (t, J=6.1Hz, 3H, CH2CH3), 2.29 orientation described by SAR.33) Docking template consisted
(s, 6H, C2-CH3 and C6-CH3), 3.34 (s, 3H, OCH3), 3.52 (t, of one hydrogen bond donor, two rings, three hydrogen bond
J=4.5Hz, 2H, COOCH2CH2O), 4.07–4.15 (m, 2H, CH2CH3), acceptors and five steric moieties. Iterative simplex algorithm
4.20 (t, J=4.5Hz, 2H, COOCH2CH2O), 4.98 (s, 1H, C4-H), was chosen to perform docking process with 15 runs per

304
Chem. Pharm. Bull.
Vol. 64, No. 4 (2016)
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Conclusion
The lipophilicity of the substituent at 4-position of DHP
ring is an important factor that may increase the activity of
the calcium antagonist. On the other hand, this substituent
should not be too bulky to interfere with ring to ring hydro-
phobic interaction with Tyr1460. Consequently, this interference
makes increasing the length of ester side chain is not an ef-
fective approach to increase the calcium antagonist activity.
The presence of chelating group substituent at 4-position of
DHP ring may guarantee the ring to ring hydrophobic interac-
tion and chelates the Ca2+ cofactor making more binding and
blocking effect to the receptor. The presence of substituent at
ortho- or meta-position of 4-phenyl ring is essential to accom-
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Conflict of Interest The authors declare no conflict of
interest.
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