Unsymmetric dihydropyridines bearing 2-pyridyl methyl carboxylate as modulators of P-glycoprotein; Synthesis and biological evaluation in resistant and non-resistant cancer cells

Article abstract of DOI:10.1139/cjc-2018-0351

Multi-drug resistance (MDR) in cancer cells is often associated with overexpression of P-glycoprotein (P-gp or ABCB1 or MDR1); therefore, modulators of this transporter might be helpful in overcoming MDR. In this study, 16 novel unsymmetrical dihydropyridine (DHP) derivatives bearing 2-pyridyl methyl carboxylate at C₃ and a nitroimidazole or nitrophenyl ring at C₄ positions of the DHP ring were synthesized. Their cytotoxicity was tested against four human cancer cells by MTT assay. The reversal capacity of MDR was examined in P-gp overexpressing cells (MES-SA/DX5) by measuring the alteration of doxorubicin's IC₅₀ and performing flow cytometric determination of intracellular rhodamine 123 accumulation. The calcium channel blocking (CCB) activity, as a side effect of DHPs, was tested on the ileum of a guinea pig. Molecular docking was performed to explain the binding mode of compounds. Two derivatives, 4a and 4c, containing 4-nitrophenyl at C₄ and possessing methyl (4a) and iso-propyl (4c) carboxylates at the C₅ position of DHP core demonstrated superior cytotoxic and MDR reversal activities and lower CCB effect. Docking analysis confirmed the importance of the 4-nitrophenyl ring for P-gp inhibitory activity. Some of the synthesized DHP derivatives with considerable MDR reversal capacity could be promising compounds for further discovery of useful agents for management of drug resistant cancer.

Full text of DOI:10.1139/cjc-2018-0351

Page 1 of 43
Unsymmetric dihydropyridines bearing 2-pyridyl methyl carboxylate as
modulators of P-glycoprotein; synthesis and biological evaluation in
resistant and non-resistant cancer cells
Maryam Nejatia,b, Hossein Sadeghpourb, Sara Ranjbarc, Katayoun Javidniaa, Najmeh
Edrakia, Luciano Sasod, Omidreza Firuzia*and Ramin Miria*
aMedicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences,
Shiraz, Iran
bDepartment of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences,
Shiraz, Iran
cPharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
dDepartment of Physiology and Pharmacology ‘‘Vittorio Erspamer’’, Sapienza University of Rome,
Rome, Italy
Ramin Miri, PhD, Medicinal and Natural Products Chemistry Research Center, Shiraz
University of Medical Sciences, Shiraz, Iran, Tel: +98-71-3230-3872, Fax: +98-71-3233-
2225, E-mail: mirir@sums.ac.ir
Omidreza Firuzi, MD PhD, Medicinal and Natural Products Chemistry Research
Center, Shiraz University of Medical Sciences, Shiraz, Iran, Tel: +98-71-3230-3872, Fax:
+98-71-3233-2225, E-mail: firuzio@sums.ac.ir
1

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Abstract
Multidrug resistance (MDR) in cancer cells is often associated with overexpression of P-
glycoprotein (P-gp or ABCB1 or MDR1), therefore, modulators of this transporter might be
helpful in overcoming MDR. In this study, 16 novel unsymmetrical dihydropyridine (DHP)
derivatives bearing 2-pyridyl methyl carboxylate at C3 and nitroimidazole or nitrophenyl ring
at C4 positions of the DHP ring were synthesized. Their cytotoxicity was tested against 4
human cancer cells by MTT assay. MDR reversal capacity was examined in P-gp
overexpressing cells (MES-SA/DX5) by measuring alteration of doxorubicin’s IC50 and
performing flow cytometric determination of intracellular rhodamine 123 accumulation.
Calcium channel blocking (CCB) activity, as a side effect of DHPs, was tested on guinea pig
ileum. Molecular docking was performed to explain the binding mode of compounds. Two
derivatives, 4a and 4c, containing 4-nitrophenyl at C4 while possessing methyl (4a) and iso-
propyl carboxylates (4c) at C5 position of DHP core demonstrated superior cytotoxic and
MDR reversal activities and lower CCB effect. Docking analysis confirmed the importance
of 4-nitrophenyl ring for P-gp inhibitory activity. Some of the synthesized DHP derivatives
with considerable MDR reversal capacity, could be promising compounds for further
discovery of useful agents for management of drug resistant cancer.
Keywords: Cancer, ABC transporters, Antineoplastic Agents, Molecular docking, Structure-
activity relationship
2

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1. Introduction
Cancer is the main cause of death in the world and the annual number of new cancer cases is
estimated to rise from 14 million in 2012 to 22 million within the next 2 decades 1. Multidrug
resistance (MDR) in cancer, defined as the resistance of cancer cells to several different
chemotherapeutic agents, is an important cause of treatment failure 2,3. MDR appears to have
diverse and complex mechanisms. One of the most accepted classifications is the division to
classical or ATP-binding cassette (ABC) transporters-mediated MDR and atypical MDR that
involves transporter–independent mechanisms 4.
ABC efflux transporter proteins pump the anticancer agent out of the cancer cell and
do not allow its intracellular accumulation 2,5-8. One of these efflux pumps, P-glycoprotein (P-
gp, ABCB1 or MDR1), belongs to the family of ATP-binding cassette (ABC) transporters
and constitutes a major cause of MDR 9,10. This membrane protein is responsible for the
11
efflux of several compounds across the plasma membrane, including anticancer agents
.
There has been much effort focused towards the development of efficient MDR reversal
agents in order to restore the sensitivity of cancer cells to chemotherapeutic agents 6.
1,4-Dihydropyridine (1,4-DHP) derivatives have several pharmacological activities
and a number of them are being used for management of cardiovascular diseases due to their
12
calcium channel blocking (CCB) capacity . Aside from their CCB effect, these compounds
13-16
have been also studied for their MDR reversal activity
and their interactions with P-gp
have been assessed in previous reports 17-20. These compounds have also shown cytotoxic
18,21,22
effects against different cancer cells
and therefore, represent high potential for
discovery of anticancer and MDR reversal agents. Since the CCB capacity of DHPs may
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cause undesired cardiovascular effects, investigators have attempted to synthesize DHPs with
better cytotoxic and MDR reversal profile, while possessing low CCB effect 23,24
.
According to a previous study, DHPs with pyridine ring at C3 and C5 positions had
better MDR reversal activities 15. The previous studies of our group have also shown that
symmetric and fused DHPs containing pyridyl alkyl carboxylate moieties are potent P-
glycoprotein modulators 25-27
.
In continuation of our ongoing efforts on the structure-activity relationship (SAR) of
DHPs as cytotoxic and MDR reversal agents, in the present study, novel unsymmetric DHP
derivatives with 2-pyridyl methyl carboxylate at C3 while containing nitrophenyl or
nitroimidazole moieties at C4 and different alkyl carboxylates at C5 were synthesized and
their CCB, cytotoxic and MDR reversal activities were examined. Finally, in order to get
perception about the binding mode of the derivatives in the active site of P-gp, molecular
docking analysis was carried out.
2. Experimental section
2.1. General
Nifedipine, rhodamine 123, MTT (thiazolyl blue tetrazolium bromide) and 2,2,6-trimethyl-
4H-1,3-dioxin-4-one were purchased from Sigma-Aldrich. Alkyl acetoacetates (methyl, ethyl,
iso-propyl and tert-butyl acetoacetate), dimethyl sulfoxide and 2-pyridyl methyl alcohol were
obtained from Merck, Darmstadt, Germany. Penicillin-G/streptomycin and RPMI 1640 were
from Biosera, Ringmer, UK. FBS and doxorubicin were from Invitrogen, San Diego, CA and
Ebewe Pharma, Unterach, Austria, respectively. Melting points were determined by using an
electrothermal 9100 digital melting point apparatus and were uncorrected (Electrothermal,
Essex, UK). The reaction progress and purity of the synthesized compounds were checked on
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Merck aluminum plates precoated with silica gel 60 F-254Silica gel column chromatography
1
was performed with Silica gel 60G. H NMR spectra were recorded on a Bruker 500
spectrometer with TMS (tetramethylsilane) as an internal standard. IR spectra were measured
in KBr with a Perkin-Elmer FT-IR in the range of 600–4000 cm-1. Mass spectra were
recorded on an Agilent GC-MS.
2. 2. Synthesis
2.2.1. Synthesis of 2-pyridyl methyl-3-oxobutanoate (III)
2-pyridyl methyl alcohol I (16. 67mmol, 1.82 g) was added to the stirred solution of 2,2,6-
trimethyl-4H-1,3-dioxin-4-one II (16. 67mmol, 2.37 g) in xylene. The resulting mixture was
refluxed for 1 hr. After TLC (chloroform: methanol, 9:1) showed complete conversion the
solvent was removed under the reduced pressure. The final product III was purified using
column chromatography (petroleum ether:ethylacetate, 4:1).
IR (KCl) 3020 (C-H aromatic), 1747 (CO-ester), 1720 (CO-ketone) cm-1
2.2.2. General procedure for the synthesis of unsymmetrical derivatives of DHP
28
Compounds were synthesized using modified Hantzsch reaction . Two mmol (0.23-0.32 g)
of appropriate alkyl acetoacetate IVa-d and 3 mmol (0.23 g) of ammonium acetate were
dissolved in appropriate alkyl alcohol. The reaction mixture was refluxed for 24 hrs. After
completion of the reaction as indicated by TLC, aryl aldehyde VIIa-d (2 mmol, 0.30 g) and
2-pyridyl methyl-3-oxobutanoate III (2 mmol, 0.38 g) were added to the solution. After
refluxing for further 24 hrs, the solvent was evaporated under reduced pressure. The products
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were purified by column chromatography and preparative TLC on silica gel, affording pure
materials 1a-d, 2a-d, 3a-d and 4a-d. The final products were recrystallized from diethyl ether
and petroleum ether 29. Characteristic data of synthesized compounds are as follows:
3-pyridin-2-yl methyl 5-methyl 2,6-dimethyl-4-(1-methyl-5-nitro-1H-imidazol-2-yl)-
1,4-dihydropyridine-3,5-dicarboxylate (1a)
1H NMR (500 MHz, CDCl3) δH 8.61 (1H, brs, NH), 8.53 (1H, d, C6-H-pyridyl, J=3.9
Hz), 7.92 (1H, s, C4-H-imidazol), 7.62 (1H, t, C4-H-pyridyl, J=7.3 Hz), 7.21 (1H, t, C5-H-
pyridyl, J=5.9 Hz), 7.08 (1H, d, C3-H-pyridyl, J=7.6 Hz), 5.35 and 5.09 (2H, dd, O-CH2-
pyridyl, J=12.8 Hz), 5.18 (1H, s, C4-H-DHP), 3.94 (3H, s, N1-CH3-imidazol), 3.66 (3H, s, O-
CH3), 2.29 and 2.23 (6H, 2s, C2-CH3 and C6-CH3). MS (EI): m/z 428 ([MH]+, 8), 410 (39),
335 (53), 291 (84), 259 (100), 192 (56), 93 (93). IR (KBr) 3264 (NH), 3056 (CH-aromatic),
2934 (CH-aliphatic), 1695 (CO), 1536, 1378 (NO2) cm-1.
3-pyridin-2-yl methyl 5-ethyl 2,6-dimethyl-4-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,4-
dihydropyridine-3,5-dicarboxylate (1b)
1H NMR (500 MHz, CDCl3) δH 8.94 (1H, brs, NH), 8.53 (1H, d, C6-H-pyridyl, J=4.0
Hz), 7.92 (1H, s, C4-H-imidazol), 7.61 (1H, t, C4-H-pyridyl, J=7.3 Hz), 7.21 (1H, t, C5-H-
pyridyl, J=5.9 Hz), 7.09 (1H, d, C3-H-pyridyl, J=7.7 Hz), 5.35 and 5.10 (2H, dd, O-CH2-
pyridyl, J=12.9 Hz), 5.19 (1H, s, C4-H-DHP), 4.12 (2H, q, O-CH2CH3, J=7.0 Hz), 3.97 (3H,
s, N1-CH3-imidazol), 2.22 and 2.27 (6H, 2s, C2-CH3 and C6-CH3), 1.22 (3H, t, O-CH2CH3,
J=7.0 Hz). MS (EI): m/z 442 ([MH]+, 29), 424 (56), 349 (84), 305 (80), 259 (100), 93 (96).
IR (KBr) 3270 (NH), 3058 (CH-aromatic), 2933 (CH-aliphatic), 1700 (CO), 1506, 1373
(NO2) cm-1.
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3-pyridin-2-yl methyl 5-isopropyl 2,6-dimethyl-4-(1-methyl-5-nitro-1H-imidazol-2-
yl)-1,4-dihydropyridine-3,5-dicarboxylate (1c)
1H NMR (500MHz, CDCl3) δH 8.60 (1H, brs, NH), 8.53 (1H, d, C6-H-pyridyl,
J=3.8Hz), 7.92 (1H, s, C4-H-imidazol), 7.62 (1H, t, C4-H-pyridyl, J=7.4Hz), 7.21 (1H, t, C5-
H-pyridyl, J=5.9 Hz), 7.09 (1H, d, C3-H-pyridyl, J=7.6 Hz), 5.35 and 5.11 (2H, dd, O-CH2-
pyridyl, J=12.9 Hz), 5.17 (1H, s, C4-H-DHP), 5.01 (1H, sep, O-CH(CH3)2, J=6.1 Hz), 3.99
(3H, s, N1-CH3-imidazol), 2.23 and 2.28 (6H, 2s, C2-CH3 and C6-CH3), 1.17and 1.20(6H, 2d,
OCH(CH3)2, J= 6.1 Hz). MS (EI): m/z 456([MH]+, 90), 438(20), 363(46), 329(64), 259(85),
233(68), 178(38), 93(100). IR (KBr) 3280(NH), 3135, 3051(CH-aromatic), 2982, 2930(CH-
aliphatic), 1707, 1658(CO), 1540, 1339(NO2) cm-1
3-pyridin-2-yl methyl 5-tert-butyl 2,6-dimethyl-4-(1-methyl-5-nitro-1H-imidazol-2-yl)-
1,4-dihydropyridine-3,5-dicarboxylate (1d)
1H NMR (500MHz,CDCl3) δH 8.88 (1H, brs, NH), 8.55 (1H, d, C6-H-pyridyl, J=3.8
Hz), 7.93 (1H, s, C4-H-imidazol), 7.65 (1H, t, C4-H-pyridyl, J=7.4 Hz), 7.25 (1H, t, C5-H-
pyridyl, J=5.9 Hz), 7.11 (1H, d, C3-H-pyridyl, J=7.6Hz), 5.35 and 5.11 (2H, dd, O-CH2-
pyridyl, J=13.0 Hz), 5.14 (1H, s, C4-H-DHP ), (1H, d, O-CH2-pyridyl, J=13.0 Hz),3.97 (3H,
s, N1-CH3-imidazol), 2.28 and 2.18(6H, 2s, C2-CH3 and C6-CH3), 1.41 (9H, s, O-C(CH3)3).
MS (EI): m/z 470([MH]+, 16), 377 (52), 352(34), 321(54), 259(62), 233(100), 93(78). IR
(KBr) 3298(NH), 3070(CH-aromatic), 2972, 2934(CH-aliphatic), 1679(CO), 1514, 1339
(NO2) cm-1.
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3-pyridin-2-yl methyl 5-methyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-
3,5-dicarboxylate (2a)
1H NMR (500 MHz, DMSO-d6) δH 9.07 (1H, s, NH), 8.44 (1H, d, C6-H-pyridyl,
J=4.4 Hz), 7.67 (1H, d, C3-H-phenyl, J=7.4 Hz), 7.61 (2H, m,C4-H-pyridyl and C5-H-
phenyl), 7.50 (1H, d, C6-H-phenyl, J=7.7 Hz), 7.37 (1H, t, C4-H-phenyl, J=7.6 Hz), 7.23 (1H,
m, C5-H-pyridyl), 6.82 (1H, d, C3-H-pyridyl, J=7.8 Hz), 5.60 (1H, s, C4-H-DHP), 5.04 (2H,
s, O-CH2-pyridyl), 3.42 (3H, s, O-CH3), 2.22 and 2.28 (6H, 2s, C2-CH3 and C6-CH3). MS
(EI): m/z 424 ([MH]+, 12), 406 (20), 331 (100), 284 (18),192 (16), 93 (25). IR (KBr) 3265
(NH), 3076 (CH-aromatic), 2947 (CH-aliphatic), 1713, 1698 (CO), 1530, 1351 (NO2) cm-1.
3-pyridin-2-yl methyl 5-ethyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-
dicarboxylate (2b)
1H NMR (500 MHz, CDCl3) δH 9.04 (1H, s, NH), 8.44 (1H, m, C6-H-pyridyl), 7.69
(1H, m, C3-H-phenyl), 7.60-7.64 (2H, m, C4-H-pyridyl and C5-H-phenyl), 7.50 (1H, m, C6-
H-phenyl), 7.38 (1H, m, C4-H-phenyl), 7.24 (1H, m, C5-H-pyridyl), 6.85 (1H, d, C3-H-
pyridyl, J=7.8 Hz), 5.66 (1H, s, C4-H-DHP), 5.04 and 5.02 (2H, dd, O-CH2-pyridyl, J=13.8
Hz), 3.81-4.00 (2H, m, O-CH2CH3), 2.23 and 2.26 (6H, 2s, C2-CH3 and C6-CH3), 1.04 (3H, t,
O-CH2CH3, J=7.0 Hz). MS (EI): m/z 438 ([MH]+, 39), 420 (15), 345 (100), 329 (20), 92
(40). IR (KBr) 3258 (NH), 3073 (CH-aromatic), 2984 (CH-aliphatic), 1700 (CO), 1531, 1350
(NO2) cm-1.
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3-pyridin-2-yl
methyl
5-isopropyl
2,6-dimethyl-4-(2-nitrophenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (2c)
1H NMR (500 MHz, CDCl3) δH 8.50 (1H, d, C6-H-pyridyl, J=3.6 Hz), 7.70 (1H, d,
C3-H-phenyl, J=8.0 Hz), 7.55 (2H, m, C6-H-phenyl and C4-H-pyridyl), 7.46 (1H, t, C5-H-
phenyl, J=7.3 Hz), 7.25 (1H, t, C4-H-phenyl, J=6.0 Hz), 7.14 (1H, t, C5-H-pyridyl, J=5.9
Hz), 7.00 (1H, d, C3-H-pyridyl, J=7.7 Hz), 5.94 (1H, s, C4-H-DHP), 5.76 (1H, s, NH), 5.24
and 5.13 (2H, dd, O-CH2-pyridyl, J=13.6 Hz), 4.95 (1H, m, O-CH(CH3)2), 2.29 and 2.33
(6H, 2s, C2-CH3 and C6-CH3), 1.21 and 0.97 (6H, 2d, O-CH(CH3)2, J=6.6 Hz). MS (EI): m/z
452 ([MH]+, 6), 434 (25), 359 (100), 317 (47), 270 (29), 92 (36). IR (KBr) 3267 (NH), 3067
(CH-aromatic), 2980 (CH-aliphatic), 1694 (CO), 1522, 1349 (NO2) cm-1.
3-pyridin-2-yl methyl 5-tert-butyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-
3,5-dicarboxylate (2d)
1H NMR (500 MHz, CDCl3) δH 8.53 (1H, d, C6-H-pyridyl, J=3.9 Hz), 7.65-7.68 (2H,
m, C3-H-phenyl and C4-H-pyridyl), 7.56 (1H, d, C6-H-phenyl, J=7.7 Hz), 7.48 (1H, t, C5-H-
phenyl, J=7.4 Hz), 7.27 (1H, m, C4-H-phenyl), 7.22 (1H, t, C5-H-pyridyl, J=5.9 Hz), 7.09
(1H, d, C3-H-pyridyl, J=7.6 Hz), 5.87 (1H, s, C4-H-DHP), 5.78 (1H, brs, NH), 5.31 and 5.16
(2H, dd, O-CH2-pyridyl, J=13.8), 2.31 and 2.28 (6H, 2s, C2-CH3 and C6-CH3), 1.36 (9H, s,
O-C(CH3)3). MS (EI): m/z 466 ([MH]+, 58), 392 (36), 373 (48), 317 (100), 287 (25), 93 (39),
57 (53). IR (KBr) 3227 (NH), 3080, 3024 (CH-aromatic), 2974 (CH-aliphatic), 1703, 1694
(CO), 1526, 1353 (NO2) cm-1.
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3-pyridin-2-yl methyl 5-methyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-
3,5-dicarboxylate (3a)
1H NMR (500 MHz, CDCl3) δH 8.56 (1H, m, C6-H-pyridyl), 8.08 (1H, m, C2-H-
phenyl), 8.00 (1H, m, C4-H-phenyl), 7.58-7.63 (2H, m, C6-H-phenyl and C4-H-pyridyl), 7.34
(1H, t, C5-H-phenyl, J=7.9 Hz), 7.20 (1H, m, C5-H-pyridyl), 7.05 (1H, d, C3-H-pyridyl,
J=7.8 Hz), 5.86 (1H, s, NH), 5.30 and 5.14 (2H, dd, O-CH2-pyridyl, J=13.4 Hz), 5.18 (1H, s,
C4-H-DHP), 3.65 (3H, s, O-CH3), 2.42 and 2.38 (6H, 2s, C2-CH3 and C6-CH3). MS (EI): m/z
424 ([MH]+, 10), 331 (100), 301 (31), 192 (44), 93 (55). IR (KBr) 3263 (NH), 3072 (CH-
aromatic), 2949 (CH-aliphatic), 1695 (CO), 1529, 1348 (NO2) cm-1.
3-pyridin-2-yl methyl 5-ethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-
dicarboxylate (3b)
1H NMR (500 MHz, CDCl3) δH 8.56 (1H, d, C6-H-pyridyl, J=4.7 Hz), 8.09 (1H, s, C2-
H-phenyl), 8.00 (1H, d, C4-H-phenyl, J=8.1 Hz), 7.58-7.63 (2H, m, C6-H-phenyl and C4-H-
pyridyl), 7.34 (1H, t, C5-H-phenyl, J=8.1 Hz), 7.20 (1H, t, C5-H-pyridyl, J=6.1 Hz), 7.04
(1H, d, C3-H-pyridyl, J=7.7 Hz), 5.78 (1H, s, NH), 5.29 and 5.14 (2H, dd, O-CH2-pyridyl,
J=13.4 Hz), 5.18 (1H, s, C4-H-DHP), 4.10 (2H, m, O-CH2CH3), 2.42 and 2.38 (6H, 2s, C2-
CH3 and C6-CH3), 1.23 (3H, t, O-CH2CH3, J=7.0). MS (EI): m/z 438 ([MH]+, 44), 345 (100),
329 (27), 315 (40), 206 (39), 93 (87). IR (KBr) 3261 (NH), 3071 (CH-aromatic), 2979 (CH-
aliphatic), 1699 (CO), 1525, 1348 (NO2) cm-1.
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3-pyridin-2-yl
methyl
5-isopropyl
2,6-dimethyl-4-(3-nitrophenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (3c)
1H NMR (500 MHz, CDCl3) δH 8.54 (1H, d, C6-H-pyridyl, J=4.0 Hz), 8.08 (1H, s, C2-
H-phenyl), 7.98 (1H, d, C4-H-phenyl, J=7.8 Hz), 7.57-7.62 (2H, m, C6-H-phenyl and C4-H-
pyridyl), 7.33 (1H, t, C5-H-phenyl, J=7.8 Hz), 7.19 (1H, t, C5-H-pyridyl, J=5.7 Hz), 7.03
(1H, d, C3-H-pyridyl, J=7.6), 5.76 (1H, s, NH), 5.27 (1H, d, O-CH2-pyridyl, J=13.4 Hz),
5.13 (2H, m, O-CH2-pyridyl and C4-H-DHP), 4.95 (1H, sep, O-CH(CH3)2, J=6.2 Hz), 2.40
and 2.36 (6H, 2s, C2-CH3 and C6-CH3), 1.25 and 1.09 (6H, 2d, O-CH(CH3)2, J=6.0 Hz). MS
(EI): m/z 452 ([MH]+, 19), 359 (100), 317 (92), 287 (43), 178 (45), 93 (75). IR (KBr) 3269
(NH), 3068 (CH-aromatic), 2980 (CH-aliphatic), 1695 (CO), 1522, 1348 (NO2) cm-1.
3-pyridin-2-yl methyl 5-tert-butyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-
3,5-dicarboxylate (3d)
1H NMR (500 MHz, CDCl3) δH 8.56 (1H, d, C6-H-pyridyl, J=3.6 Hz), 8.09 (1H, s, C2-
H-phenyl), 8.00 (1H, d, C4-H-phenyl, J=7.8 Hz), 7.62-7.64 (m, 2H, C6-H-phenyl and C4-H-
pyridyl), 7.35 (1H, t, C5-H-phenyl, J=7.8 Hz), 7.23 (1H, t, C5-H-pyridyl, J=5.7 Hz), 7.09
(1H, d, C3-H-pyridyl, J=7.6 Hz), 5.83 (1H, s, NH), 5.29 and 5.18 (2H, dd, O-CH2-pyridyl,
J=13.6 Hz), 5.13 (1H, s, C4-H-DHP), 2.41 and 2.34 (6H, 2s, C2-CH3 and C6-CH3), 1.40 (9H,
s, O-C(CH3)3). MS (EI): m/z 466 ([MH]+, 5), 373 (20), 317 (100), 287 (48), 178 (32), 93
(42). IR (KBr) 3266 (NH), 3065, 3012 (CH-aromatic), 2980 (CH-aliphatic), 1693 (CO),
1521, 1348 (NO2) cm-1.
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3-pyridin-2-yl methyl 5-methyl 2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridine-
3,5-dicarboxylate (4a)
1H NMR (500 MHz, CDCl3) δH 8.56 (1H, d, C6-H-pyridyl, J=3.6 Hz), 8.02 (2H, d,
C3-H-phenyl and C5-H-phenyl, J=8.2 Hz), 7.58 (1H, t, C4-H-pyridyl, J=7.2 Hz), 7.40 (2H, d,
C2-H-phenyl and C6-H-phenyl, J=8.2 Hz), 7.20 (1H, t, C5-H-pyridyl, J=5.9 Hz), 7.02 (1H, d,
C3-H-pyridyl, J=7.6 Hz), 5.72 (1H, s, NH), 5.30 and 5.13 (2H, dd, O-CH2-pyridyl, J=13.3
Hz), 5.17 (1H, s, C4-H-DHP), 3.63 (3H, s, O-CH3), 2.40 and 2.36 (6H, 2s, C2-CH3 and C6-
CH3). MS (EI): m/z 424 ([MH]+, 46), 331 (100), 301 (38), 192 (65), 93 (93). IR (KBr) 3259
(NH), 3066 (CH-aromatic), 2948 (CH-aliphatic), 1699 (CO), 1513, 1342 (NO2) cm-1.
3-pyridin-2-yl methyl 5-ethyl 2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridine-3,5-
dicarboxylate (4b)
1H NMR (500 MHz, CDCl3) δH 8.55 (1H, d, C6-H-pyridyl, J=4.4 Hz), 8.02 (2H, d,
C3-H-phenyl and C5-H-phenyl, J=8.8 Hz), 7.58 (1H, m, C4-H-pyridyl), 7.41 (2H, d, C2-H-
phenyl and C6-H-phenyl, J=8.8 Hz), 7.20 (1H, m, C5-H-pyridyl), 7.02 (1H, d, C3-H-pyridyl,
J=7.8 Hz), 5.80 (1H, s, NH), 5.29 and 5.13 (2H, dd, O-CH2-pyridyl, J=13.4 Hz), 5.17 (1H, s,
C4-H-DHP), 4.08 (2H, m, O-CH2CH3), 2.39 and 2.35 (6H, 2s, C2-CH3 and C6-CH3), 1.20
(3H, m, O-CH2CH3). MS (EI): m/z 438 ([MH]+, 7), 345 (100), 315 (34), 206 (30), 93 (59). IR
(KBr) 3258 (NH), 3066 (CH-aromatic), 2973, 2937 (CH-aliphatic), 1700 (CO), 1510, 1342
(NO2) cm-1.
12

Page 13 of 43
3-pyridin-2-yl
methyl
5-isopropyl
2,6-dimethyl-4-(4-nitrophenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (4c)
1H NMR (500 MHz, CDCl3) δH 8.55 (1H, d, C6-H-pyridyl, J=3.6 Hz), 8.02 (2H, d,
C3-H-phenyl and C5-H-phenyl, J=8.4 Hz), 7.57 (1H, t, C4-H-pyridyl, J=7.4 Hz), 7.41 (2H, d,
C2-H-phenyl and C6-H-phenyl, J=8.4 Hz), 7.19 (1H, t, C5-H-pyridyl, J=5.9 Hz), 7.03 (1H, d,
C3-H-pyridyl, J=7.7 Hz), 5.80 (1H, s, NH), 5.28 (1H, d, O-CH2-pyridyl, J=13.4 Hz), 5.14
(2H, m, O-CH2-pyridyl and C4-H-DHP), 4.94 (1H, sep, O-CHCH3, J=6.1 Hz), 2.38 and 2.35
(6H, 2s, C2-CH3 and C6-CH3), 1.23 and 1.09 (6H, 2d, O-CH(CH3)2, J=6.1 Hz). MS (EI): m/z
452 ([MH]+, 58), 343 (30), 317 (29), 178 (40), 93 (100). IR (KBr) 3264 (NH), 3072 (CH-
aromatic), 2971, 2930 (CH-aliphatic), 1693 (CO), 1508, 1342 (NO2) cm-1.
3-pyridin-2-yl methyl 5-tert-butyl 2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridine-
3,5-dicarboxylate (4d)
1H NMR (500 MHz, CDCl3) δH 8.57 (1H, d, C6-H-pyridyl, J=3.6 Hz), 8.04 (2H, d,
C3-H-phenyl and C5-H-phenyl, J=8.3 Hz), 7.63 (1H, t, C4-H-pyridyl, J=7.2 Hz), 7.42 (2H, d,
C2-H-phenyl and C6-H-phenyl, J=8.3 Hz), 7.25 (1H, t, C5-H-pyridyl, J=5.9 Hz), 7.08 (1H, d,
C3-H-pyridyl, J=7.6 Hz), 5.78 (1H, s, NH), 5.31 and 5.19 (2H, dd, O-CH2-pyridyl, J=13.5
Hz), 5.13 (1H, s, C4-H-DHP), 2.39 and 2.34 (6H, 2s, C2-CH3 and C6-CH3), 1.39 (9H, s, O-
C(CH3)3). MS (EI): m/z 466 ([MH]+, 30), 317 (100), 287 (45), 178 (32), 93 (77). IR (KBr)
3239 (NH), 3095 (CH-aromatic), 2976, 2933 (CH-aliphatic), 1701, 1659 (CO), 1512, 1342
(NO2) cm-1.
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2.3. Cell lines
Cancer cell lines including K562 (human chronic myelogenous leukemia), LS180 (human
colon adenocarcinoma), MCF-7 (human breast adenocarcinoma), and also cell lines used for
MDR reversal tests, MES-SA and MES-SA/DX5 (human uterine sarcoma), were maintained
in RPMI 1640 supplemented with 10% FBS, and 100 units/ml penicillin-G and 100 g/ml
streptomycin. The percentage of FBS for HL-60 (human acute promyelocytic leukemia) was
20%. For MES-SA/DX5 cells, 100 nM doxorubicin was added to the maintenance flasks and
it was removed 24 hrs before starting the tests. LS180, MCF-7, MES-SA and MES-SA/DX5
cells were grown in monolayer cultures, while K562 and HL-60 cells were grown in
suspension, at 37°C in humidified air containing 5% CO2.
2.4. Cytotoxicity assay
30,31
Cytotoxicity of synthesized compounds was measured by MTT reduction assay
.
Suspensions of HL-60, K562, LS180 and MCF-7 with appropriate densities were prepared
and seeded in 96-well plates. The cytotoxicity assay was performed as described in our
32
previous study . After adding DMSO to solubilize the produced formazan, the absorbance
of each well was read at 570 nm with background correction at 650 nm by a microplate
reader (model 680, Bio-Rad, Japan).
2.5. MDR reversal measured by alteration of sensitivity to doxorubicin in resistant cells
MES-SA and MES-SA/DX5 cells were seeded in 96-well plates and maintained overnight at
37°C. Then the compounds at different concentrations were added in the absence or presence
14

Page 15 of 43
of doxorubicin. There was only doxorubicin in reference wells. After 3 days of incubation at
37°C, MTT assay was conducted as described in our previous study 32,33
.
2.6. Rhodamine 123 efflux assay for measurement of P-glycoprotein inhibition
34
R123 is a selective substrate for P-gp . The assay that is used here was a modified version
of the assay that was described in a previous study 32,35
.
The MES-SA/DX5 cells were trypsinized, suspended in fresh medium and counted.
Nine-hundred µL of the suspension containing 2.5 x 105 cells was placed in micro tubes and
100 µL of 5 µM R123 was added. Cells were kept at 37°C for 30 min and then were
centrifuged and washed twice with ice-cold PBS. Five-hundred µL of three different
concentrations of the synthesized compounds (ranging from 2.5 to 25 µM, final
concentration) were added to the cells alongside with two concentrations of verapamil (2.5
and 10 µM), which was used as a reference P-gp inhibitor compound. The cells were
incubated at 37°C for another 30 min. Finally, suspensions (5.0 x 105cells/ml) were analyzed
by a flow cytometer (BD FACS Calibur, Becton Dickenson, USA). The number of cells
counted for each sample was 10,000.
2.7. CCB activity evaluation
The CCB evaluation was carried out as previously described 32,36. Male guinea pigs (300-
450g) were purchased from Animal House Department, Shiraz University of Medical
Sciences, fasted for 24 hrs and sacrificed by a blow on the neck. One-cm segments of ileum
32
were installed in oxygenated tyrode solution
in jacketed organ bath (Pan Lab (Letica),
Spain) at 37°C. KCl (40 mM) was added to the solution in presence or absence of the
15

Page 16 of 43
synthesized compounds, nifedipine and DMSO as blank, final concentrations of which were
10-10, 10-9, 10-8, 10-7, 10-6 and 10-5 M (x3, at 10 min intervals). The inhibitory effect of each
compound was measured by percentile of the heights of the corresponding peaks in contrast
to the peaks made by KCl in the absence of the compound. IC50 of each tested compound
were calculated based on these percentages.
2.8. P-gp docking analysis
In this study, we applied AutoDock 4.2 package software to inquire the affinity and binding
modes of the synthesized derivatives to the binding pocket of P-gp. Since the high resolution
crystal structure of P-gp is not available, we applied the 3D structure of human P-gp which
has been recently generated by homology modeling and MD simulation studies on X-ray
structure of Apo murine P-gp (PDB entry: 3G5U) by our research group 37,38. All the docked
compounds were sketched and minimized (by molecular mechanics, MM+, and semi-
empirical, AM1, methods) using HYPERCHEM 7.0 software. AutoDock Tools 4.2 was used
to prepare ligands and protein for docking process. The PDBQT files were generated by
adding charges and defining the degree of torsions. The grid dimensions were set to
60 × 60 × 60 Å with points separated by 0.375 Å and the grid was centered at 93.12, 68.17
and 124.91 Å (X, Y and Z) that involved the active site of DHPs. Docking was performed
using Lamarckian Genetic Algorithm as the docking algorithm with 100 runs. Other
parameters were left as default.
16

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3. Results and Discussion
3.1. Synthesis
Unsymmetrical DHP derivatives (1a-d, 2a-d, 3a-d and 4a-d) were synthesized based on the
described method in “Experimental” section. The step-by-step method is depicted in Fig. 1.
Reaction of 2-pyridyl methyl alcohol I with 2,2,6-trimethyl-4-H-1,3-dioxin-4-one II
produced 2-pyridyl methyl-3-oxobutanoate III in a good yield (95%). The final unsymmetric
DHPs were synthesized according to a modified Hanstzch reaction. Hanstzch reaction has
been one of the major methods of DHPs synthesis since its introduction in 1882 39. Chemical
reaction of different alkyl acetoacetates IVa-d and ammonium acetate V in refluxing alkyl
alcohol resulted in the formation of different aminocrotonates VIa-d. The aforementioned
aminocrotonates then reacted with different aryl aldehydes and 2-pyridyl methyl-3-
oxobutanoate III in order to produce the final unsymmetric DHPs. Synthesized compounds
were then purified by preparative TLC and the chemical structures of the desired compounds
1
were confirmed by H NMR and EI-MS. The structures and physical properties of the
synthesized compounds are summarized in Fig. 1 and Table 1, respectively.
3.2. Cytotoxicity
In order to investigate the anticancer potential of the synthesized compounds, the cytotoxic
activity evaluations were performed against 4 human cancer cell lines including HL-60,
K562, LS180 and MCF-7 by using MTT assay. The results are presented as IC50 values in
Table 2.
In general, compounds bearing nitrophenyl moiety at C4 (2a-d, 3a-d and 4a-d)
showed cytotoxic activities, while the agents with nitroimidazole group at this position (1a-d)
had no considerable cytotoxicity against the cell lines.
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Page 18 of 43
As for the substituent at C5 position, the compounds that possess iso-propyl and tert-
butyl (2c-d, 3c-d and 4c-d) had lower IC50 values, i.e., higher cytotoxic effects. It should be
noted that since all compounds with nitrophenyl groups had similar IC50 values, it can be
concluded that the aromatic group at C4 position is a more influential factor compared to the
alkyl group at C5 position. An exception is 2d bearing 2-nitrophenyl and tert-butyl at C4 and
C5 positions, respectively, which demonstrated low cytotoxic activity. This is probably due to
the change in the orientation caused by NO2 group and the bulk of tert-butyl substitute that
probably prevents the molecule from approaching its target site.
The cytotoxic potency of the compounds synthesized in the present study, were far
better than the potencies of the symmetrical molecules containing pyridyl, reported in our
previous studies, where only a few compounds showed IC50 values of lower than 100 µM
40,41. Therefore, it can be stated that cytotoxicity will be improved by adding carbon to the
carboxylate moieties at C3 and C5 positions.
3.3. MDR reversal measured by alteration of sensitivity to doxorubicin in resistant cells
MDR reversal activity was tested in P-gp overexpressing MES-SA/DX5 cells and the results
were compared to those obtained in the parental cell line, MES-SA. The findings related to
all synthesized compounds are presented in Tables 3 and 4, while one representative agent
from each of the 4 groups of derivatives are shown in Figs. 2 and 3. P-gp expression in MES-
SA and MES-SA/DX5 cells was examined by western blot and it was observed that while
MES-SA-DX5 cells express a considerable amount of P-gp, the expression of this protein is
below the detection limit in the parental MES-SA cells (data not shown).
MDR reversal activity measurement in P-gp overexpressing MES-SA/DX5 cells
revealed that compounds with 4-nitrophenyl (4a-d) and 3-nitrophenyl (3a-d) at C4 position,
18

Page 19 of 43
generally possessed higher MDR reversal activities than their counterparts having
nitroimidazole (1a-d) and 2-nitrophenyl (2a-d) rings (Table 3). Some of these compounds
(3c, 3d, 4b, 4c and 4d) caused a significant reduction in the IC50 of doxorubicin at
concentrations as low as 1 µM. Therefore, considering Log P values in Table 1, it can be
deduced that less lipophilic nitroimidazole substituents were worse P-gp modulators than 3-
and 4-nitrophenyl substituents. Moreover, a significant reduction of doxorubicin IC50 was
observed in the presence of compounds 3a, 3b and 4a at 2.5 µM concentrations and above. In
our previous studies, compounds bearing nitroimidazole showed MDR reversal effect in
HL60/MX1 (acute promyelocytic leukemia) cell line as a cell model of atypical MDR, which
is a mitoxantrone resistant derivative of the human leukemia HL60 cell line with altered
topoisomerase II catalytic activity and reduced levels of topoisomerase II alpha and beta
proteins 40,41. So, it is possible that these compounds are only effective on topoisomerase II
and not P-gp.
Comparing the alkyl groups at C5 position shows that the more lipophilic tert-butyl
and iso-propyl substitutes confer higher MDR reversal activities than methyl and ethyl
substitutions (Log P values are reported in Table 1). As expected, none of the tested
compounds significantly increased the sensitivity of non-resistant MES-SA cells to
doxorubicin (Table 4).
3.4. Rhodamine 123 efflux assay for measurement of P-glycoprotein inhibition
The efflux of Rh123 in MES-SA/DX5 cells was measured by a flow cytometric method. One
representative diagram is depicted in Fig. 4 and the geometric means of all experiments are
shown in Fig. 5.
19

Page 20 of 43
Rh123 is selectively pumped out of the cell by P-gp, therefore, inhibitors of P-gp can
34
prevent this process and increase intracellular accumulation of this fluorescent probe
.
Rh123 efflux assay was used to confirm the results obtained from MTT method and to
specifically show that the compounds work through P-gp pump inhibition.
Flow cytometric determination of Rh123 efflux in MES-SA/DX5 cells showed that in
general, the compounds with 4-nitrophenyl (4a-d) or 3-nitrophenyl at C4 position (3a-d) were
better modulators of P-gp, because they promoted intracellular Rh123 retention more strongly
and at lower concentrations compared to compounds with nitroimidazole (1a-d) or 2-
nitrophenyl (2a-d). 3c, 4a, 4b and 4c were the most promising P-gp inhibitors which caused
significant Rh123 accumulation compared to control in MES-SA/DX5 cells at 5 μM
concentration.
Generally, the Rh123 efflux results were in agreement with the findings of MDR
reversal assay obtained by MTT reduction method. However, as these two techniques
measure cellular events at different incubation times, some dissimilarities were observed in
the order of compound activities; While, 3d and 4d demonstrated significant reduction in the
IC50 of doxorubicin at concentrations as low as 1 µM (Table 3), these compounds exhibited
lower P-gp inhibitory activities in flow cytometric Rh123 determination assay compared to
the other derivatives in their subgroups, 3-nitrophenyl and 4-nitrophenyl bearing derivatives
at C4, respectively (Fig. 5).
Regarding the substitution at C5 position, it is noticeable that in the most potent
compound sets, the iso-propyl containing compounds (3c and 4c) showed greater P-gp
inhibitory activities compared to other C5 substituents, but in the case of the least potent
series, the compounds containing tert-butyl at C5 position (1d and 2d) had better effects.
20

Page 21 of 43
In our previous studies, 3-nitrophenyl and 4-nitrophenyl at C4 position of the DHP
ring resulted in slightly higher activity compared to the compounds with nitroimidazole as the
aromatic ring at C4 position. Moreover, as noted in our former studies, the presence of larger
alkyl groups such as iso-propyl and tert-butyl at C5 position leads to more active compounds
32. These consequences are consistent with the results of the current study.
3.5. CCB activity evaluation
The CCB activity of synthesized compounds, as a potential side effect of compounds with
DHP structure, was evaluated on guinea pig ileal longitudinal smooth muscle (GPLISM) and
the IC50 related to each compound was calculated. The results are shown in Table 5.
Among the synthesized compounds, those bearing nitroimidazolyl or 4-nitrophenyl
group at C4 (4a-d and 1a-d) demonstrated the lowest CCB activity. Compound 1a with an
IC50 value of 8.1 x 10-6 M was 76 times less active than nifedipine as the reference CCB
agent. On the other hand, compounds possessing 2-nitrophenyl or 3-nitrophenyl at C4 (2a-d
and 3a-d) had the lowest IC50 values. Some of these compounds (3a-c and 2a) were even
stronger than nifedipine. These structure-activity relationship findings are in agreement with
the outcomes of our previous study 41.
In addition, introducing iso-propyl or tert-butyl groups at C5 position reduced the
CCB activity, while the opposite happened by methyl or ethyl substitutions. For example, 4c-
d blocked the calcium channels less than 4a-b and similarly 2c-d had higher IC50 values than
2a-b. Although C5 moiety had some effects on the IC50 values, the most influential factor to
determine the CCB activity appeared to be the aromatic group at C4 position. This is in line
with the previous studies and modeling of the 1,4-dihydropyridines as calcium channel
blockers 42,43
.
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Page 22 of 43
3.6. Molecular docking study
It has been reported that DHPs bind near the Nucleotide Binding Domain (NBD) of P-gp.
Borchers and colleagues determined the active site of DHPs by photo affinity labeling and
mass spectrometry 44. They suggested that DHPs bind to the P-gp 468 –527 residues. Szabon-
Watola and coworkers predicted the binding location of the DHPs resides near the conserved
Val907 of coupling helix 2, and an essential Mg2+ binding residue Gln475 of the Walker
45
domain . They suggested that a ligand binding in this location would be expected to limit
conformational reorganization necessary for xenobiotic efflux. The molecular docking
analysis of 1c, 2c, 4a and 4c suggested that the most active derivative, 4c, possessed the
lowest estimated free energy of binding (-10.59 kcal/mol) and estimated inhibition constant
(17.30 nM) (Table 6). Oxygen atoms of NO2 group on phenyl ring made two strong hydrogen
bond interactions with Arg543 and Ser474 and a key hydrogen bond could be seen between
NH of DHP ring and carbonyl side chain of Gln441. Carbonyl functions of 2-pyridyl methyl
carboxylate and iso-propyl carboxylate moieties involved in hydrogen bond interactions with
hydroxyl groups of Ser474 and Ser909, respectively. Additionally, isopropyl appeared to be
well accommodated into the hydrophobic pocket surrounded by Il470, Val471 and Tyr490
(Fig. 6. A). Compound 4a, having methyl carboxylate moiety, demonstrated the second best
estimated free energy of binding (-9.74 kcal/mol) and estimated inhibition constant (71.97
nM) (Table 6). It seems that the binding pose of 4c (containing iso-propyl carboxylate at C5)
is slightly different from that of compound 4a (containing methyl carboxylate at C5) (Fig. 6.
B). Consequently, 4c established hydrogen bond interaction between nitrogen atom of
pyridine ring and Asn903 but in the case of 4a, this interaction was not observed. Moreover,
unlike the bulky iso-propyl group in 4c, the small methyl function in 4a did not well orientate
22

Page 23 of 43
toward the mentioned hydrophobic residues. Compounds 1c and 2c, bearing nitroimidazole
and 2-nitrophenyl at C4, respectively, showed the lowest estimated free energies of binding (-
9.67 and kcal/mol, respectively) (Table 6). As illustrated in Fig. 6. C and Fig. 6. D, the
binding orientation of 2c and 1c is totally different from that of 4a and 4c. Therefore, for
compound 2c, 2-pyridyl methyl carboxylate moiety did not have any interaction with the
receptor, while NH of DHP ring involved in a hydrogen bond interaction with Ser909 and
NO2 formed two hydrogen bonds with Arg547 and Val472. Another hydrogen bond
interaction was established between Arg543 and carbonyl group of the methyl carboxylate
moiety Fig. 6. C. Binding interactions of compound 1c is illustrated in Fig. 6 .D.
Generally, molecular docking analysis of the compounds in the active site of P-gp
indicated that substitutions on C4 and C5 positions of DHP ring played an important role in
binding orientations of the tested compounds. The presence of 4-nitrophenyl (as in 4c) at C4
is more favored than 2-nitrophenyl (as in 2c) and nitroimidazole (as in 1c) and could
probably lead to more favorable interactions and better orientations. According to docking
results compound 4c, bearing iso-propyl carboxylate substitution at C3, might have better
binding affinity to P-gp active site than its counterpart, 4a, with methyl carboxylate moiety
due to the bulk of iso-propyl group, which provided better orientation of 4c in the pocket
comprising some lipophilic residues.
4. Conclusions
In conclusion, 16 novel DHP derivatives were synthesized and examined for cytotoxicity,
MDR reversal capacities and also CCB activity as a potential side effect. The compounds
with noticeable inherent cytotoxicity, remarkable MDR reversal and low CCB effects were
identified. Consequently, 4a and 4c (both possessing 4-nitrophenyl at C4 and methyl (4a) and
23

Page 24 of 43
iso-propyl carboxylates (4c) at C5) were recognized as the most promising compounds with
low CCB activity which had moderate inherent cytotoxicity against cancer cells and
significantly increased the sensitivity of P-gp overexpressing MES-SA-DX5 cells to
doxorubicin at the low concentrations of 1 and 2.5 µM (4c and 4a, respectively).
Furthermore, the two compounds remarkably increased the intracellular accumulation of
Rh123 in MES-SA-DX5 cells comparable to that of verapamil.
Compound 3c (bearing 3-nitrophenyl at C4 and iso-propyl carboxylate at C5) was the
most effective MDR reversal derivative; however, its CCB activity did not differ much from
that of nifedipine. Compounds with nitroimidazole as the aromatic moiety at C4, showed
neither cytotoxicity nor MDR reversal effect.
The binding modes of potent compounds, 4a and 4c inside the P-gp active site
indicated that these compounds could be well accommodated in the active site and key
hydrogen bond interactions were observed between ligands and Gln441, Ser474, Arg543,
Ser909 and Asn903 of the active site.
Some of the synthesized DHP derivatives presented in this study, have cytotoxic and
MDR reversal effects, while possessing low CCB activity, and could represent promising
compounds for further development of novel agents useful for treatment of resistant types of
cancer.
Acknowledgements
The authors wish to thank the support of the Vice-Chancellor for Research, Shiraz University
of Medical Sciences.
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Table and figure caption list:
Table 1. Physical properties of synthesized unsymmetrical dihydropyridine compounds
Table 2. Cytotoxic activity of synthesized dihydropyridine compounds on various human
cancer cell
Table 3. Effects of synthesized dihydropyridine compounds on the sensitivity of multidrug-
resistant MES-SA/DX5 cells to doxorubicin assessed by MTT assay.
Table 4. Effects of synthesized dihydropyridine compounds on the sensitivity of non-
resistant MES-SA cells to doxorubicin assessed by MTT assay.
Table 5. Calcium channel blocking activities of synthesized unsymmetrical dihydropyridine
compounds.
Table 6. Docking results of selected compounds with P-gp.
Fig. 1. Synthesis procedure of 1,4-dihydropyridine derivatives.
Fig. 2. Effects of representative synthesized 1,4-dihydropyridine compounds on the
sensitivity of multidrug-resistant MES-SA/DX5 cells to doxorubicin examined by MTT
assay. MES-SA/DX5 cells were seeded in 96-well plates and compounds 1d (a), 2d (b), 3c (c)
and 4c (d) were added at 0.5–5 µM concentrations after 24 hrs. Doxorubicin was also added
at 100-1000 nM concentrations right after the test compound and cells were further incubated
for 72 hrs. MTT test was performed and the data were presented as % viability compared to
control (non-treated) wells. Compounds 3c (c) and 4c (d) caused significant changes at the
concentration of 1 µM, while compounds 1d (a) and 2d (b) did not differ from control
(doxorubicin alone) at 1 µM (data not shown). Data are mean ± S.E.M. of 3-5 independent
experiments and differences were analysed by using the one-way ANOVA followed by LSD
test.
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Fig. 3. Effects of representative synthesized compounds against non-resistant parental MES-
SA cells to doxorubicin measured by MTT assay. MES-SA cells were seeded in 96-well
plates after 24 hrs of incubation the compounds were added at 0.5–5 µM concentrations
immediately followed by doxorubicin addition at 100-1000 nM. After further incubation for
72 hrs, MTT test was performed and the data were presented as %viability compared to
control (non-treated) wells. Data are mean ± S.E.M. of 3-5 independent experiments.
Differences were analysed by using the one-way ANOVA followed by LSD test.
Fig. 4. Effect of compound 4c on the fluorescence intensity of Rh123 in MES-SA/DX5 cells.
MES-SA/DX5 cells were trypsinized and resuspended in fresh medium. 2.5 x 105 cells in a
micro tube were treated with Rh123 (5 µM, final concentration) and incubated at 37 °C for 30
min. The cells were centrifuged and washed twice with ice-cold PBS and then treated with 3
different concentrations of the synthesized compounds (5, 10 and 25 µM). The cells were
incubated at 37°C for another 30 min and finally, 10,000 of them were injected to a flow
cytometer.
Fig. 5. Inhibition of Rh123 efflux shown as mean fluorescence intensity in MES-SA/DX5
cells in the presence or absence of synthesized compounds and verapamil. MES-SA/DX5
cells were trypsinized and resuspended in fresh medium. 2.5 × 105 cells in a micro tube were
treated with Rh123 (5 µM, final concentration) and incubated at 37 °C for 30 min. The cells
were centrifuged and washed twice with ice-cold PBS and then treated with 3 different
concentrations of the synthesized compounds (ranging from 2.5 to 25 µM). The cells were
incubated at 37°C for another 30 min and finally, 10,000 of them were injected to a flow
cytometer. Ctrl: Control sample, VP: verapamil. Data are mean ± S.E.M. of 3-5 independent
experiments, and differences were analyzed by using the one-way ANOVA followed by LSD
30