Cytotoxic and multidrug resistance reversal activities of novel 1,4-dihydropyridines against human cancer cells

Article abstract of DOI:10.1016/j.ejphar.2014.10.058

Multidrug resistance (MDR) caused by P-glycoprotein (P-gp, ABCB1, MDR-1) transporter over-expression in cancer cells substantially limits the effectiveness of chemotherapy. 1,4-Dihydropyridines (DHPs) derivatives possess several pharmacological activities. In this study, 18 novel asymmetrical DHPs bearing 3-pyridyl methyl carboxylate and alkyl carboxylate moieties at C₃ and C₅ positions, respectively, as well as nitrophenyl or hetero aromatic rings at C₄ were synthesized and tested for MDR reversal with the aim of establishing a structure-activity relationship (SAR) for these agents. Effect of these compounds on P-gp mediated MDR was assessed in P-gp over-expressing MES-SA/DX5 doxorubicin resistant cells by flow cytometric detection of rhodamine 123 efflux. MDR reversal was further examined as the alteration of doxorubicin's IC₅₀ in MES-SA/DX5 cells in the presence of DHPs by MTT assay and was compared to nonresistant MES-SA cells. Direct anticancer effect was examined against 4 human cancer cells including HL-60, K562, MCF-7 and LS180. Calcium channel blocking (CCB) activity was also measured as a potential side effect. Most DHPs, particularly compounds bearing 3-nitrophenyl (A2B2 and A3B2) and 4-nitrophenyl (A3B1 and A4B1) moieties at C₄ significantly inhibited rhodamine 123 efflux at 5-25 μM, showing that the mechanism of MDR reversal by these agents is P-gp transporter modulation. Same derivatives were also able to selectively lower the resistance of MES-SA/DX5 to doxorubicin. A2B2 bearing ethyl carboxylate at C₅ had also high direct antitumoral effect (IC₅₀ range: 3.77-15.60 μM). Our findings suggest that SAR studies of DHPs may lead to the discovery of novel MDR reversal agents.

Full text of DOI:10.1016/j.ejphar.2014.10.058

European Journal of Pharmacology 746 (2015) 233–244
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Cytotoxic and multidrug resistance reversal activities of novel
1,4-dihydropyridines against human cancer cells
Farnaz Shekari a, Hossein Sadeghpour b, Katayoun Javidnia a, Luciano Saso c, Farhad Nazari a,
a,
Omidreza Firuzi a, , Ramin Miri
n
n
a Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
b Department of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
c Department of Physiology and Pharmacology “Vittorio Erspamer”, Sapienza University of Rome, Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Multidrug resistance (MDR) caused by P-glycoprotein (P-gp, ABCB1, MDR-1) transporter over-expression
in cancer cells substantially limits the effectiveness of chemotherapy. 1,4-Dihydropyridines (DHPs)
derivatives possess several pharmacological activities. In this study, 18 novel asymmetrical DHPs bearing
3-pyridyl methyl carboxylate and alkyl carboxylate moieties at C3 and C5 positions, respectively, as well
as nitrophenyl or hetero aromatic rings at C4 were synthesized and tested for MDR reversal with the aim
of establishing a structure–activity relationship (SAR) for these agents. Effect of these compounds on
P-gp mediated MDR was assessed in P-gp over-expressing MES-SA/DX5 doxorubicin resistant cells by
flow cytometric detection of rhodamine 123 efflux. MDR reversal was further examined as the alteration
of doxorubicin's IC50 in MES-SA/DX5 cells in the presence of DHPs by MTT assay and was compared to
nonresistant MES-SA cells. Direct anticancer effect was examined against 4 human cancer cells including
HL-60, K562, MCF-7 and LS180. Calcium channel blocking (CCB) activity was also measured as a potential
side effect. Most DHPs, particularly compounds bearing 3-nitrophenyl (A2B2 and A3B2) and
4-nitrophenyl (A3B1 and A4B1) moieties at C4 significantly inhibited rhodamine 123 efflux at 5–
25 mM, showing that the mechanism of MDR reversal by these agents is P-gp transporter modulation.
Same derivatives were also able to selectively lower the resistance of MES-SA/DX5 to doxorubicin. A2B2
Received 16 July 2014
Received in revised form
26 October 2014
Accepted 27 October 2014
Available online 14 November 2014
Keywords:
1,4-Dihydropyridine (CID 104822)
Multidrug resistance
Cytotoxicity
Rhodamine 123 (CID 65217)
P-glycoprotein
bearing ethyl carboxylate at C5 had also high direct antitumoral effect (IC50 range: 3.77–15.60 M). Our
μ
findings suggest that SAR studies of DHPs may lead to the discovery of novel MDR reversal agents.
& 2014 Elsevier B.V. All rights reserved.
1. Introduction
classes of chemotherapeutic drugs such as anthracycline deriva-
tives, vinca alkaloids or podophyllotoxins. It has been shown that a
Multidrug resistance (MDR), defined as resistance of tumor
cells to chemotherapeutic agents that are dissimilar in function
and structure, is one of the main obstacles in conventional
chemotherapy and an important cause of the failure of cancer
treatment (Gottesman, 1993; Liscovitch and Lavie, 2002). MDR is
linked with several different mechanisms. One important mechan-
ism is over-expression of P-glycoprotein (P-gp), a protein encoded
by the MDR-1 gene that belongs to ATP-binding cassette (ABC)
membrane transporters family. MRP1, MRP2 and BCRP are other
major ABC transporters associated with MDR (Ozben, 2006;
Palmeira et al., 2012). P-gp pump mediates the efflux of different
variety of compounds with diverse structures are able to inhibit
P-gp pump, one class of which are 1,4-dihydropyridines (1,4-DHPs)
(Liu, 2009; Viale et al., 2011; Voigt et al., 2007).
1,4-DHPs are a useful class of calcium channel blocking (CCB)
agents, commonly used for treatment of cardiovascular diseases
(Sica, 2006; Yamakage and Namiki, 2002). DHPs have also shown a
broad range of other pharmacological effects such as antioxidant,
anticonvulsant, anti-inflammatory, antidiabetic and antitubercular
properties (Edraki et al., 2009; Moghadam et al., 2006; Saini et al.,
2008). MDR reversal has also been reported as one of the
important pharmacological potentials of DHPs (Carosati et al.,
2012; Zarrin et al., 2010). In 1980, Tsuruo et al. (1983) reported
for the first time that DHPs demonstrated MDR reversal effects on
cancer cell lines. It has been shown that the main mechanism of
DHPs as MDR reversal agents is P-gp inhibition (Coburger et al.,
2010; Edraki et al., 2009; Firuzi et al., 2013; Hilgeroth et al., 2013).
However, detailed structure–activity relationship (SAR) studies of
Abbreviations: ABC, ATP-binding cassette; MDR, multidrug resistance; P-gp,
P-glycoprotein; DHP, dihydropyridine; CCB, calcium channel blocking; MTT, thia-
zolyl blue tetrazolium bromide; Rh123, rhodamine 123.
n
Corresponding authors. Tel.: þ98 711 230 3872; fax: þ98 711 233 2225.
E-mail addresses: firuzio@sums.ac.ir (O. Firuzi), mirir@sums.ac.ir (R. Miri).
http://dx.doi.org/10.1016/j.ejphar.2014.10.058
0014-2999/& 2014 Elsevier B.V. All rights reserved.

234
F. Shekari et al. / European Journal of Pharmacology 746 (2015) 233–244
these compounds are scares and they are much needed in order to
establish the best structural features required for the MDR reversal
effect.
2.2.3. General procedure for the synthesis of asymmetrical
derivatives of 1,4-DHP (A1B1ꢁA3B5)
A
mixture of pyridin-3-yl-methyl-3-oxobutanoate III, alkyl
On the other hand, several studies have shown intrinsic cytotoxi-
city of DHPs (Firuzi et al., 2013; Hahn et al., 1997; Sarkarzadeh et al.,
2013), which appears to be stronger against tumor cell lines
compared to non-cancer cells (Laupeze et al., 2001; Morshed et al.,
2005).In continuation of our ongoing research programs focused on
the design of potent MDR-reversal agents of DHP prototype (Firuzi et
al., 2013; Foroughinia et al., 2008; Mehdipour et al., 2007), in this
study, we synthesized novel asymmetrical 1,4-DHP derivatives con-
taining 3-pyridyl methyl carboxylate at C3, aromatic or hetero
aromatic rings at C4, and alkyl carboxylate at C5 position and
evaluated their inhibition of P-gp transporter and resultant MDR
reversal activity on MES-SA/DX5 cell line using flow cytometry and
MTT assays with the main aim of establishing a SAR for these
compounds. Furthermore, cytotoxic activity evaluation of compounds
was performed on four cancer cell lines including HL-60, K562, MCF-
7 and LS180. When DHPs are designed for MDR reversal, their CCB
activity could have important side effects on the cardiovascular
system, hence in vitro CCB activity of synthesized compounds was
also investigated to rule this effect as a potential adverse effect
(Khoshneviszadeh et al., 2009).
3-aminocrotonate A1–4 and aryl or heteroaryl aldehydes B1–5
was refluxed in 15 ml ethanol or isopropanol or t-butanol (corre-
sponding to alkyl 3-aminocrotonate) overnight. The completion of
the reaction was monitored using thin layer chromatography (TLC).
After completion, the solvent was evaporated and the residue was
purified by preparative TLC on silica gel using chloroform–ethanol
(94ꢁ6) as mobile phase. The final product was recrystallized from
diethyl ether or a mixture of diethyl ether/petroleum ether. Because
of DHP derivatives sensitivity to light, all above procedures were
carried out under a sodium lamp to prevent degradation of
products by natural irradiation (Memarian and Mirjafari, 2005).
Melting points of synthetic compounds were determined on a
hot stage apparatus (Electro thermal, Essex, UK) and are uncor-
rected. 1H NMR spectra were performed by Burker-AdvanceDPX-
500 MHz in d1-chloroform and Tetramethylsilane (TMS) was used
as the internal standard. Mass spectra were recorded on an Agilent
spectrometer (Agilent technologies 9575c inert MSD, USA) and the
IR spectra were obtained using a Perkin-Elmer spectrometer (KBr
disk) (Perkin-Elmer, Waltham, MA).
2.2.3.1. 3-Pyridin-3-ylmethyl 5-methyl 2,6-dimethyl-4-(4-nitrophenyl)-
1,4-dihydropyridine-3,5-dicarboxylate (A1B1). 1H NMR (500 MHz,
CDCl3) δH (ppm): 2.35 and 2.37 (2s, 6H, C2–CH3 and C6–CH3), 3.63
(s, 3H, COOCH3), 5.04 and 5.15 (dd, 2H, COOCH2–pyridyl, J¼12.60 Hz),
5.08 (s, 1H, DHP–C4–H), 5.75 (brs, 1H, NH), 7.22–7.24 (m, 1H, pyridyl–
H5), 7.36(d, 2H, 4-nitro phenyl–H2,6, J¼8.75 Hz), 7.48 (d, 1H, pyridyl–
H4, J¼7.85 Hz), 8.03 (d, 2H, 4-nitro phenyl–H3,5, J¼8.75 Hz), 8.55 (s,
1H, pyridyl–H2), 8.56 (d, 1H, pyridyl–H6, J¼3.5 Hz).
2. Material and methods
2.1. Materials
Pyridine-3-yl-methanol, alkyl acetoacetate (alkyl: methyl,
ethyl, isopropyl, t-butyl) and DMSO were purchased from Merck,
Darmstadt, Germany. Rhodamine 123, thiazolyl blue tetrazolium
bromide (MTT) and 2,2,6-trimethyl-4H-1,3-dioxin-4-one was from
Sigma-Aldrich, Saint Louis, MO. RPMI 1640, Dulbecco's phosphate
buffered saline 10 ꢀ and penicillin-G/streptomycin were products
of Biosera, Ringmer, UK and FBS (Fetal Bovine serum) was from
Invitrogen, San Diego, CA, USA. Doxorubicin and cisplatin were
obtained from Ebewe Pharma, Unterach, Austria.
MS (EI), m/z (%): 423 (Mþ, 8), 392 (4), 364 (4), 331 (11), 301
(100), 287 (10), 210 (10), 165 (18), 150 (9), 92(42).
IR (KBr): 3281 (NH), 3056 (CH-aromatic), 2927 (CH-aliphatic),
1702, 1676 (C¼O), 1508, 1341 (NO2) cmꢁ1
.
2.2.3.2. 3-Pyridin-3-ylmethyl 5-ethyl 2,6-dimethyl-4-(4-nitrophenyl)-
1,4-dihydropyridine-3,5-dicarboxylate (A2B1). 1H NMR (500 MHz,
CDCl3) δH (ppm): 1.2 (t, 3H, COOCH2CH3, J¼6.85 Hz), 2.35 and 2.37
(2s, 6H, C2–CH3 and C6–CH3), 4.08 (q, 2H, COOCH2CH3, J¼6.85 Hz),
5.04 and 5.15 (dd, 2H, COOCH2–pyridyl, J¼12.55 Hz), 5.09 (s, 1H,
DHP–C4–H), 5.77 (brs, 1H, NH), 7.23–7.26 (m, 1H, pyridyl–H5), 7.37 (d,
2H, 4-nitro phenyl–H2,6, J¼8.15 Hz), 7.48 (d, 1H, pyridyl–H4), 8.03 (d,
2H, 4-nitro phenyl–H3,5, J¼8.15 Hz), 8.54 (m, 2H, pyridyl–H2,6).
MS (EI), m/z (%): 437 (Mþ, 5), 392 (3), 364 (4), 345 (6), 315
(100), 301 (4), 287 (4), 269 (4), 151 (5), 92 (21).
2.2. Chemistry
2.2.1. Synthesis of pyridin-3-yl-methyl-3-oxobutanoate (III)
A mixture of 20 mmol (3.86 g) of pyridine-3-yl-methanol I and
20 mmol (2.48 g) of 2,2,6-trimethyl-4H-1,3-dioxin-4-one II was
refluxed in 10 ml xylene at 150 1C for 45 min. The formation of
compound was confirmed by thin layer chromatography (TLC).
After cooling the mixture, xylene was removed under reduced
pressure and the product was purified by column chromatography
over silica gel using petroleum ether–ethyl acetate (Yield: 83%).
IR (KBr): 3297 (NH), 3084 (CH-aromatic), 2974 (CH-aliphatic),
1674 (C ¼O), 1516, 1348 (NO2) cmꢁ1
.
IR (KBr):
υ
2961(C–H aromatic), 1746 (CO, ester), 1715 (CO,
ketone) cmꢁ1
.
2.2.3.3. 3-Pyridin-3-ylmethyl 5-isopropyl 2,6-dimethyl-4-(4-nitroph-
enyl)-1,4-dihydropyridine-3,5-dicarboxylate (A3B1). 1H NMR (500 M-
Hz, CDCl3) δH (ppm): 1.10 (d, 3H, COOCH(CH3)2, J¼6.05 Hz), 1.22 (d,
3H,COOCH(CH3)2, J¼6.05 Hz), 2.34 and 2.36 (2s, 6H, C2–CH3 and C6–
CH3), 4.95 (m, 1H, COOCH(CH3)2), 5.05 and 5.15 (dd, 2H, COOCH2–
pyridyl, J¼12.65 Hz), 5.08 (s, 1H, DHP–C4–H), 5.78 (brs, 1H, NH), 7.21–
7.24 (m, 1H, pyridyl–H5), 7.37 (d, 2H, 4-nitro phenyl–H2,6, J¼ 8.3 Hz),
2.2.2. General synthesis of alkyl 3-aminocrotonate (A1–4)
A mixture of ammonium acetate (3 mmol) IV and alkylacetoa-
cetate a1–4 (2 mmol) were refluxed in 10 ml of corresponding
alkyl alcohol derivative for 24 h. Afterwards the product was
immediately used in subsequent reaction without further purifica-
tion. The formation of product was confirmed by IR spectra.
Methyl 3-aminocrotonate (A1)
7.49 (d, 1H, pyridyl–H4, J¼7.40 Hz), 8.02 (d, 1H, 4-nitro phenyl–H3,5
,
J¼ 8.3 Hz), 8.54 (m, 2H, 3-pyridyl–H2,6).
MS (EI), m/z (%): 451 (Mþ, 6), 408 (5), 392 (5), 359 (6), 329
(100), 287 (43), 273 (6), 92 (16.39).
IR (KBr): 1716 (C¼O, ester), 3511, 3333 (NH2) cmꢁ1
Ethyl 3-aminocrotonate (A2)
IR (KBr): 1716 (C¼O, ester), 3511, 3332 (NH2) cmꢁ1
Isopropyl 3-aminocrotonate (A3)
IR (KBr): 3292 (NH), 3081 (CH-aromatic), 2976 (CH-aliphatic),
1679 (C¼O), 1516, 1349 (NO2) cmꢁ1
.
IR (KBr): 1716 (C¼O, ester), 3509, 3332 (NH2) cmꢁ1
t-butyl 3-aminocrotonate (A4)
2.2.3.4. 3-Pyridin-3-ylmethyl 5-tert-butyl 2,6-dimethyl-4-(4-nitrophenyl)-
IR (KBr): 1717 (C¼O, ester), 3450, 3336 (NH2) cmꢁ1
1,4-dihydropyridine-3,5-dicarboxylate (A4B1). 1H NMR (500 MHz, CDCl3)

F. Shekari et al. / European Journal of Pharmacology 746 (2015) 233–244
235
δ
H (ppm): 1.40 (s, 9H, COOC(CH3)3), 2.34 and 2.38 (2s, 6H, C2–CH3 and
MS (EI), m/z (%): 423 (Mþ 5), 406 (100), 375 (10), 361 (39), 345
(20), 331 (93), 315 (8), 301 (53), 284 (43), 268 (31), 210 (11),
92 (64).
C6–CH3), 5.05 (s, 1H, DHP–C4–H), 5.15 and 5.23 (dd, 2H, COOCH2–
pyridyl, J¼ 13.15 Hz), 6.07 (s, 1H, NH), 7.41 (d, 2H,
4-nitro phenyl–H2,6, J¼ 8.25 Hz), 7.57 (m, 1H, pyridyl–H5), 7.86 (d, 1H,
pyridyl–H4),8.07 (d, 2H, 4-nitro phenyl–H3,5, J¼ 8.25 Hz), 8.63 (m, 2H,
3-pyridyl–H2,6).
IR (KBr): 3286 (NH), 3083 (CH-aromatic), 2948 (CH-aliphatic),
1678 (C¼O), 1525, 1345 (NO2) cm-1
.
MS (EI), m/z (%): 465 (Mþ, 5), 408 (20), 392 (5.74), 364 (4.09),
343 (46), 287 (100), 273 (12), 92 (41).
2.2.3.9. 3-Pyridin-3-ylmethyl 5-ethyl 2,6-dimethyl-4-(2-nitrophenyl)-
1,4-dihydropyridine-3,5-dicarboxylate (A2B3). 1H NMR (500 MHz,
CDCl3) δH (ppm): 1.15 (t, 3H, COOCH2CH3, J¼6.90 Hz), 2.32 (s, 6H,
C2–CH3 and C6–CH3), 4.00 (m, 1H, COOCH2CH3,), 4.12 (m, 1H,
COOCH2CH3,), 5.03 and 5.10 (dd, 2H, COOCH2–pyridyl, J¼12.45 Hz),
5.70 (brs, 1H, NH), 5.82 (s, 1H, DHP–C4–H), 7.19 (m, 1H, 2-nitrophenyl–
H4), 7.26 (m, 1H, pyridyl–H5), 7.45 (t, 1H, 2-nitrophenyl–H5, J¼
7.25 Hz), 7.50–7.55 (m, 2H, 2-nitrophenyl–H6 and pyridyl–H4), 7.69
(d, 1H, 2-nitrophenyl–H3, J¼ 7.95 Hz), 8.43 (s, 1H, pyridyl–H2), 8.49
(rd, 1H, pyridyl–H6).
IR (KBr): 3191 (NH), 3075 (CH-aromatic), 2975 (CH-aliphatic),
1704 (C¼O), 1512, 1346 (NO2) cmꢁ1
.
2.2.3.5. 3-Pyridin-3-ylmethyl 5-methyl 2,6-dimethyl-4-(3-nitrophenyl)-
1,4-dihydropyridine-3,5-dicarboxylate (A1B2). 1H NMR (500 MHz,
CDCl3) δH (ppm): 2.36 and 2.38 (2s, 6H, C2–CH3 and C6–CH3), 3.63
(s, 3H, COOCH3), 5.03 and 5.16 (dd, 2H, COOCH2–pyridyl, J¼12.55 Hz),
5.08 (s, 1H, DHP–C4–H), 5.88 (s, 1H, NH), 7.23–7.26 (m, 1H, pyridyl–H5),
7.32 (t, 1H, 3-nitro phenyl–H5, J¼7.80 Hz), 7.50–7.55 (m, 2H, 3-nitro
phenyl–H6 and pyridyl–H4), 7.99 (d, 1H, 3-nitro phenyl–H4,
J¼7.80 Hz), 8.04 (s, 1H, 3-nitro phenyl–H2), 8.50 (s, 1H, pyridyl–H2),
8.55 (d, 1H, pyridyl–H6).
MS (EI), m/z (%): 437 (Mþ, 6), 420 (100), 361 (33), 345 (100),
331 (7), 315 (52), 298 (26), 284 (22), 270 (28), 254 (17), 226 (18),
92 (84).
IR (KBr): 3286 (NH), 3090 (CH-aromatic), 2969 (CH-aliphatic),
MS (EI), m/z (%): 423 (Mþ, 5), 392 (3), 331 (6), 315 (5), 301
(100), 287 (7), 210 (5), 165 (9.84), 150 (4), 92(20).
1698, 1678 (C¼O), 1528, 1348 (NO2) cmꢁ1
.
IR (KBr): 3298 (NH), 3087 (CH-aromatic), 2958 (CH-aliphatic),
2.2.3.10. 3-Pyridin-3-ylmethyl 5-isopropyl 2,6-dimethyl-4-(2-nitro-
phenyl)-1,4-dihydropyridine-3,5-dicarboxylate (A3B3). 1H NMR (500-
MHz, CDCl3) δH (ppm): 0.97 (d, 3H, COOCH(CH3)2, J¼6.15 Hz), 1.21
(d, 3H, COOCH(CH3)2, J¼6.15), 2.27 and 2.33 (2s, 6H, C2–CH3 and C6–
CH3), 4.93–4.96 (m, 1H, COOCH(CH3)2, J¼6.15 Hz), 4.99 and 5.12 (dd,
2H, COOCH2–pyridyl, J¼12.50 Hz), 5.70 (brs, 1 H, NH), 5.86 (s, 1H,
DHP–C4–H), 7.19–7.22 (m, 1H, 2-nitrophenyl–H4), 7.23–7.26 (m, 1H,
pyridyl–H5), 7.45 (t, 1H, 2-nitrophenyl–H5, J¼7.65 Hz), 7.51 (d, 1H,
2-nitrophenyl–H6, J¼7.65 Hz), 7.58 (d, 1H, pyridyl–H4, J¼7.55 Hz), 7.72
(d, 1H, 2-nitrophenyl–H3, J¼8.00 Hz), 8.47 (s, 1H, pyridyl–H2), 8.50 (d,
1H, pyridyl–H6, J¼3.85 Hz).
1671 (C ¼O), 1527, 1346 (NO2) cmꢁ1
.
2.2.3.6. 3-Pyridin-3-ylmethyl 5-ethyl 2,6-dimethyl-4-(3-nitrophenyl)-
1,4-dihydropyridine-3,5-dicarboxylate (A2B2). 1H NMR (500 MHz,
CDCl3) δH (ppm): 1.21 (t, 3H, COOCH2CH3, J¼6.55 Hz), 2.36 and 2.39
(2s, 6H, C2–CH3 and C6–CH3), 4.08 (q, 2H, COOCH2CH3, J¼6.55 Hz),
5.02 and 5.16 (dd, 2H, COOCH2–pyridyl, J¼12.50 Hz), 5.08 (s, 1H,
DHP–C4–H), 5.73 (s, 1H, NH), 7.26 (m, 1H, pyridyl–H5), 7.32 (t, 1H, 3-
nitro phenyl–H5, J¼7.65 Hz), 7.49 (d, 1H, 3-nitro phenyl–H6,
J¼7.65 Hz), 7.55 (d, 1H, pyridyl–H4, J¼7.10 Hz), 7.99 (d, 1H, 3-nitro
phenyl–H4, J¼7.65 Hz), 8.05 (s, 1H, 3-nitro phenyl–H2), 8.49 (s, 1H,
pyridyl–H2), 8.54 (brd, 1H, pyridyl–H6).
MS (EI), m/z (%): 451 (Mþ 7), 434 (100), 392 (28), 359 (68), 329
(28), 287 (38), 270 (59), 254 (22), 92 (92), 78 (9).
MS (EI), m/z (%): 437 (Mþ, 5), 392 (4), 364 (4), 345 (6), 315
(100), 301 (5), 287 (6), 269 (4), 151 (6), 92 (21).
IR (KBr): 3258(NH), 3064(CH-aromatic), 2982(CH-aliphatic),
1689(C¼O), 1530, 1347 (NO2) cm-1
.
IR (KBr): 3292 (NH), 3094 (CH-aromatic), 2982 (CH-aliphatic),
1682 (C¼O), 1522, 1343 (NO2) cmꢁ1
.
2.2.3.11. 3-Pyridin-3-ylmethyl 5-tert-butyl 2,6-dimethyl-4-(2-nitro-
phenyl)-1,4-dihydropyridine-3,5-dicarboxylate (A4B3). 1H NMR (500-
MHz, CDCl3) δH (ppm): 1.35 (s, 9H, COOC(CH3)3), 2.26 and 2.27 (2s,
6H, C2–CH3 and C6–CH3), 4.98 and 5.14 (dd, 2H, COOCH2–pyridyl,
J¼12.50 Hz), 5.59 (brs, 1H, NH), 5.79 (s, 1H, DHP–C4–H), 7.21 (m, 1H,
2-nitrophenyl–H4), 7.26 (m, 1H, pyridyl–H5), 7.44 (t, 1H,
2-nitrophenyl–H5, J¼7.60 Hz), 7.50 (d, 1H, 2-nitrophenyl–H6,
J¼7.60 Hz), 7.59 (d, 1H, pyridyl–H4, J¼7.50 Hz), 7.68 (d, 1H,
2-nitrophenyl–H3, J¼8.00 Hz), 8.48 (s, 1H, pyridyl–H2), 8.50 (brd,
1H, pyridyl–H6, J¼3.75 Hz).
2.2.3.7. 3-Pyridin-3-ylmethyl 5-isopropyl 2,6-dimethyl-4-(3-nitro-
phenyl)-1,4-dihydropyridine-3,5-dicarboxylate (A3B2). 1H NMR (500 -
MHz, CDCl3) δH (ppm): 1.09 (d, 3H, COOCH(CH3)2, J¼6.00 Hz), 1.24
(d, 3H, COOCH(CH3)2, J¼6.00 Hz), 2.35 and 2.38 (2s, 6H, C2–CH3 and
C6–CH3), 4.94 (m, 1H, COOCH(CH3)2), 5.03 and 5.16 (dd, 2H,
COOCH2–pyridyl, J¼12.60 Hz), 5.07 (s, 1H, DHP–C4–H), 5.75 (brs,
1H, NH), 7.23–7.26 (m, 1H, pyridyl–H5), 7.32 (t, 1H, 3-nitrophenyl–H5,
J¼7.60 Hz), 7.50 (d, 1H, 3-nitrophenyl–H6, J¼7.25 Hz), 7.55 (d, 1H,
pyridyl–H4, J¼7.30 Hz), 7.98 (d, 1H, 3-nitrophenyl–H4, J¼7.60 Hz),
8.05 (s, 1H, 3-nitrophenyl–H2), 8.49 (s, 1H, pyridyl–H2), 8.54 (d, 1H,
pyridyl–H6 ).
MS (EI), m/z (%): 465 (Mþ, 0.82), 448 (6), 434 (64), 420 (97),
392(40), 359 (47), 345 (100), 329 (23), 315 (74), 287 (40), 270 (69),
254 (30), 92 (91).
MS (EI), m/z (%): 451 (Mþ, 5), 408 (3), 392 (5), 364 (6), 329
(100), 287 (50), 273 (8), 243 (5.74), 151 (9), 108 (6), 92 (27).
IR (KBr): 3288 (NH), 3071 (CH-aromatic), 2973 (CH-aliphatic),
IR (KBr): 3183 (NH), 3067 (CH-aromatic), 2973 (CH-aliphatic),
1692 (C¼O), 1529, 1357 (NO2) cmꢁ1
.
1682 (C¼O), 1519, 1344 (NO2) cmꢁ1
.
2.2.3.12. 3-Pyridin-3-ylmethyl 5-methyl 2,6-dimethyl-4-(1-methyl-5-
nitro-1H-imidazol-2-yl)-1,4-dihydropyridine-3,5-dicarboxylate
(A1B4). 1H NMR (500 MHz, CDCl3) δH (ppm): 2.23 and 2.25 (2s,
6H, C2–CH3 and C6–CH3), 3.66 (s, 3H, N–CH3), 3.97 (s, 3H, COOCH3),
5.05 and 5.20 (dd, 2H, COOCH2–pyridyl, J¼12.55 Hz), 5.12 (s, 1H,
DHP–C4–H), 7.26 (m, 1H, pyridyl–H5), 7.53 (d, 1H, pyridyl–H4,
J¼7.30 Hz), 7.93 (s, 1 H, imidazole–H), 8.47 (brs, 1H, NH), 8.54 (s,
1H, pyridyl–H2), 8.58 (d, 1H, pyridyl–H6, J¼3.60 Hz).
2.2.3.8. 3-Pyridin-3-ylmethyl 5-methyl 2,6-dimethyl-4-(2-nitrophenyl)-
1,4-dihydropyridine-3,5-dicarboxylate (A1B3). 1H NMR (500 MHz,
CDCl3) δH (ppm): 2.31 and 2.34 (2s, 6H, C2–CH3 and C6–CH3), 3.56
(s, 3H, COOCH3), 5.04 and 5.07 (dd, 2H, COOCH2–pyridyl, J¼12.45 Hz),
5.76 (s, 1H, DHP–C4–H), 5.78 (brs, 1H, NH), 7.18 (m, 1H, 2-nitrophenyl–
H4), 7.26 (m, 1H, pyridyl–H5), 7.45–7.50 (m, 3H, 2-nitrophenyl–H5,6
and pyridyl–H4), 7.66 (d, 1H, 2-nitrophenyl–H3, J¼7.60 Hz), 8.40 (s, 1H,
pyridyl–H2), 8.48 (d, 1H, pyridyl–H6).
MS (EI), m/z (%): 427(Mþ, 32), 368(14), 301(41), 291(51), 259
(83), 165(16), 108(41), 92(100).

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F. Shekari et al. / European Journal of Pharmacology 746 (2015) 233–244
IR (KBr): 3271 (NH), 3060 (CH-aromatic), 2943 (CH-aliphatic),
1H, thiophenyl–H4), 7.06 (d, 1H, thiophenyl–H5, J¼ 4.6 Hz), 7.23 (m,
1700 (C ¼O), 1500, 1375 (NO2) cm-1
.
1H, pyridyl–H5), 7.53 (d, 1H, pyridyl–H4, J¼7.45 Hz), 8.53 (s, 2H,
pyridyl–H2,6).
MS (EI), m/z (%): 398 (Mþ, 45), 353 (11), 325 (86), 306 (100),
278 (18), 262 (85), 234 (16), 218 (27), 150 (26), 92 (58).
IR (KBr): 3298 (NH), 3095 (CH-aromatic), 2979 (CH-aliphatic),
2.2.3.13. 3-Pyridin-3-ylmethyl 5-ethyl 2,6-dimethyl-4-(1-methyl-5-
nitro-1H-imidazol-2-yl)-1,4-dihydropyridine-3,5-dicarboxylate
(A2B4). 1H NMR (500 MHz, CDCl3) δH (ppm): 1.23 (t, 3H,
COOCH2CH3, J¼7.00 Hz), 2.23 and 2.24 (2s, 6H, C2–CH3 and C6–
CH3), 3.99 (s, 3H, N–CH3), 4.12 (q, 2H, COOCH2CH3, J¼7.00 Hz),
5.06 and 5.21 (dd, 2H, COOCH2–pyridyl, J¼12.55 Hz), 5.13 (s, 1H,
DHP–C4–H), 7.26 (m, 1H, pyridyl–H5), 7.53 (d, 1H, 1H, pyridyl–H4,
J¼7.50 Hz), 7.92 (s, 1 H, imidazole–H), 8.54 (brs, 2H, NH and
pyridyl–H2), 8.58 (d, 1H, pyridyl–H6, J¼3.80 Hz).
1676 (C ¼O) cmꢁ1
.
2.2.3.18. 3-Pyridin-3-ylmethyl 5-isopropyl 2,6-dimethyl-4-(thiophen-2-
yl)-1,4-dihydropyridine-3,5-dicarboxylate (A3B5). 1H NMR (500 MHz,
CDCl3) δH (ppm): 1.22 (d, 3H, COOCH(CH3)2, J¼6.10 Hz), 1.26 (d, 3H,
COOCH(CH3)2), J¼6.10 Hz), 2.32 and 2.36 (2s, 6H, C2–CH3 and C6–
CH3), 5.04 (m, 1H, COOCH(CH3)2), 5.13 and 5.23 (dd, 2H, COOCH2–
pyridyl, J¼12.95 Hz), 5.37 (s, 1H, DHP–C4–H), 5.87 (brs, 1H, NH), 6.76
(brd, 1H, thiophenyl–H3), 6.83 (t, 1H, thiophenyl–H4), 7.05 (d, 1H,
thiophenyl–H5, J¼4.60 Hz), 7.23 (m, 1H, pyridyl–H5),7.54 (d, 1H,
pyridyl–H4, J¼7.45 Hz), 8.53 (s, 2H, pyridyl–H2,6).
MS (EI), m/z (%): 441 (Mþ, 37), 424 (4.46), 396 (4), 368 (23), 305
(39), 287 (11), 259 (100), 92 (63).
IR (KBr): 3270 (NH), 3058 (CH-aromatic), 2973 (CH-aliphatic),
1697 (C ¼O), 1500, 1375 (NO2) cm-1
.
MS (EI), m/z (%): 412 ( Mþ, 41), 369 (14), 325 (100), 287 (38),
234 (46), 188 (12.29), 150 (19.67), 92 (32).
2.2.3.14. 3-Pyridin-3-ylmethyl 5-isopropyl 2,6-dimethyl-4-(1-methyl-
5-nitro-1H-imidazol-2-yl)-1,4-dihydropyridine-3,5-dicarboxylate
(A3B4). 1H NMR (500 MHz, CDCl3) δH (ppm): 1.18 (d, 3H, COOCH
(CH3)2, J¼6.10 Hz), 1.21 (d, 3H, COOCH(CH3)2, J¼6.10 Hz), 2.24 and
2.25 (2s, 6H, C2–CH3 and C6–CH3), 4.02 (s, 3H, N–CH3), 5.00 (m, 1H,
COOCH(CH3)2, J¼6.10 Hz), 5.06 and 5.21 (dd, 1H, COOCH2–pyridyl,
J¼12.55 Hz), 5.11 (s, 1H, DHP–C4–H), 7.26 (m, 1H, pyridyl–H5), 7.53
(d, 1H, pyridyl–H4, J¼7.45 Hz), 7.92 (s, 1H, imidazole–H), 8.18 (brs,
1H, NH), 8.54 (s, 1H, pyridyl–H2), 8.58 (d, 1H, pyridyl–H6,
J¼3.80 Hz).
IR (KBr): 3293 (NH), 3090 (CH-aromatic), 2981 (CH-aliphatic),
1688 (C¼O) cmꢁ1
.
2.3. Calcium channel blocking activity
In vitro CCB activity of synthesized compounds was evaluated
on guinea pig ileal longitudinal smooth muscle (GPILSM) as
previously described with some modifications (Mehdipour et al.,
2007; Miri et al., 2007). Male albino guinea pigs were obtained
from Animal House Department of Shiraz University of Medical
Sciences. The animals were fasted overnight before the test. They
were sacrificed and their intestines were removed above the
ileocecal junction. Ileum was cut into about 1 cm segments and
segments were kept at 4 1C for 6–16 h in oxygenated physiological
saline solution of the following compositions: NaCl 119 mM, KCl
2.70 mM, CaCl2 ꢂ 02H2O 2.00 mM, MgCl2 ꢂ 06H2o 0.88 mM,
NaH2PO4 0.36 mM, NaHCO3 12.00 mM and glucose 5.50 mM.
Afterwards, muscle segments were mounted in 5 ml Jacketed
organ bath (Letica, Spain) containing oxygenated physiological
saline solution at 37 1C, under a resting tension of 0.5 g. The ileal
muscle contractions were recorded with a forced displacement
transducer (Hugo Sachs, March-Hugstetten and Germany) on a
physiograph (Hugo Sachs). The contraction was induced with
40 mM KCl and the KCl-induced contraction was considered as
100% value. Synthesized compounds and nifedipine (the positive
MS (EI), m/z (%): 455 (Mþ 33), 396 (6), 368 (51), 319 (24), 287
(27), 259 (100), 233 (20), 92 (62).
IR (KBr): 3268 (NH), 3051 (CH-aromatic), 2977 (CH-aliphatic),
1700 (C ¼O), 1503, 1373 (NO2) cmꢁ1
.
2.2.3.15. 3-Pyridin-3-ylmethyl
methyl-5-nitro-1H-imidazol-2-yl)-1,4-dihydropyridine-3,5-
dicarboxylate (A4B4). 1H NMR (500 MHz, CDCl3)
H (ppm): 1.41 (s,
5-tert-butyl
2,6-dimethyl-4-(1-
δ
9H, COOC(CH3)3), 2.17 and 2.25 (2s, 6H, C2–CH3 and C6–CH3), 3.99
(s, 3H, N–CH3), 5.05 and 5.19 (dd, 2H, COOCH2–pyridyl,
J¼12.55 Hz), 5.09 (s, 1H, DHP–C4–H), 7.26 (m, 1H, pyridyl–H5),
7.52 (d, 1H, pyridyl–H4, J¼ 7.40 Hz), 7.92 (s, 1H, imidazole–H (, 8.33
(brs, 1H, NH), 8.57 (s, 1H, pyridyl–H2), 8.57 (d, 1H, pyridyl–H6,
J¼3.80 Hz).
MS (EI), m/z (%): 469 (Mþ, 16), 396 (8), 368 (69), 352 (24), 287
(42), 260 (78), 233 (63), 92 (76.23), 57 (100).
IR (KBr): 3278 (NH), 3056 (CH-aromatic), 2972 (CH-aliphatic),
1705, 1671 (C¼O), 1517, 1373 (NO2) cmꢁ1
.
control) were added at the final concentrations of 10ꢁ10, 10ꢁ9
,
10ꢁ8, 10ꢁ7, 10ꢁ6 and 10ꢁ5 M for evaluation of their inhibitory
effect on muscle contraction and after 10 min KCl was added to
induce contraction. The IC50 value of each compound was calcu-
lated from concentration–response curve.
2.2.3.16. 3-Pyridin-3-ylmethyl 5-methyl 2,6-dimethyl-4-(thiophen-2-yl)-
1,4-dihydropyridine-3,5-dicarboxylate (A1B5). 1H NMR (500 MHz,
CDCl3) δH (ppm): 2.33 and 2.36 (2s, 6H, C2–CH3 and C6–CH3), 3.71
(s, 3H, COOCH3), 5.10 and.25(dd, 2H, COOCH2–pyridyl, J¼13.00 Hz),
5.35 (s, 1H, DHP–C4–H), 6.02 (brs, 1H, NH), 6.75 (d, 1H, thiophenyl–H3),
6.84 (t, 1H, thiophenyl–H4), 7.06 (d, 1H, thiophenyl–H5, J¼ 4.60 Hz),
7.23 (m, 1H, pyridyl–H5), 7.52 (d, 1H, pyridyl–H4, J¼ 7.45 Hz), 8.26 (s,
2H, pyridyl–H2,6).
2.4. Cell lines
Six cell lines were used in this study including HL-60 (acute
promyelocytic leukemia), K562 (chronic myelogenous leukemia),
MCF-7 (human breast adenocarcinoma), LS180 (human colon
adenocarcinoma), MES-SA/DX-5 (human uterine sarcoma with
P-gp over-expression) and its parental cell line MES-SA. All cell
lines were obtained from the National Cell Bank of Iran, Pasteur
Institute, Tehran, Iran. Cells were cultured in RPMI 1640 supple-
mented with 10% FBS (20% FBS for HL-60 cell line) and 100 units/
ml penicillin-G and 100 ug/ml streptomycin at 37 1C in humidified
air containing 5% CO2. K562 and HL-60 cell lines were grown in
suspension, while MCF-7, LS180, MES-SA and MES-SA/DX5 cells
were grown in monolayer culture.
MS (EI), m/z (%): 384 (Mþ, 40), 353 (7), 325 (45), 292 (87), 248
(100), 218 (13), 165 (16), 150 (15), 108 (15), 92 (74).
IR (KBr): 3332 (NH), 3070 (CH-aromatic), 2947 (CH-aliphatic),
1698 (C¼O) cmꢁ1
.
2.2.3.17. 3-Pyridin-3-ylmethyl 5-ethyl 2,6-dimethyl-4-(thiophen-2-yl)-
1,4-dihydropyridine-3,5-dicarboxylate (A2B5). 1H NMR (500 MHz,
CDCl3) δH (ppm): 1.28 (t, 3H, COOCH2CH3, J¼7.15 Hz), 2.33 and 2.37
(2s, 6H, C2–CH3 and C6–CH3), 4.18 (q, 2H, COOCH2CH3, J¼7.15 Hz),
5.12 and 5.24 (dd, 2H, COOCH2–pyridyl, J¼12.90 Hz), 5.38 (s, 1H,
DHP–C4–H), 5.83 (brs, 1H, NH), 6.76 (brd, 1H, thiophenyl–H3), 6.84 (t,

F. Shekari et al. / European Journal of Pharmacology 746 (2015) 233–244
237
2.5. Cytotoxicity assay
used with some modifications (Munić et al., 2010; Wesolowska
et al., 2005; Xi et al., 2010). Tubes containing MES-SA/DX5 cells
(5 ꢀ 105 cells/ml) in RPMI 1640 medium without phenol-red were
In vitro cytotoxic activity of synthesized compounds was
evaluated by MTT reduction assay (Jassbi et al., 2014; Mosmann,
1983). Cells at the logarithmic phase were seeded into 96-well
plates with 3–5 ꢀ 103 cells/well (depending on the cell line) and
incubated for 24 h at 37 1C. Then, four different concentrations of
synthesized compounds, cisplatin and doxorubicin (as positive
controls) were added in triplicate and plates were incubated at
37 1C for another 72 h. After the incubation period, 80
was removed from each well and 80 l of MTT solution in
phosphate-buffered saline at a concentration of 5 mg/ml was
added for MTT assay (Plates with suspension cells were centri-
fuged before the procedure). After 4 h incubation and formation of
treated with 5 M Rh123 for 30 min at 37 1C. Afterwards cells
μ
were centrifuged and washed twice with ice-cold PBS and resus-
pended in RPMI 1640 medium containing verapamil (positive
control) or synthesized compounds as P-gp inhibitors. Tubes were
incubated for 30 min at 37 1C.
The fluorescence emission caused by the presence of Rh123 in
cells was measured by a FACSCalibur flow cytometer (Becton
Dickenson, USA) with an excitation and emission wavelengths of
488 and 530 nm, respectively. Geometric mean (Gmean) of fluor-
escence intensity values was determined using WINMDI 2.9
(TSRI, USA).
μl of media
μ
formazan crystals, 200 l DMSO was added to dissolve crystals.
μ
Finally absorbance was measured at a wavelength of 570 nm with
background correction at 650 nm using a microplate reader
(model 680, Bio-Rad, Japan) and IC50 (concentration that results
in 50% inhibition of cell viability) for each compound was
calculated with Curve Expert version 1.34 for windows.
2.7. MDR reversal assay
MDR reversal activity of synthesized compound was evaluated on
MES-SA/DX5 cell line (MDR subline of MES-SA cell line) by MTT assay.
At first, 4 ꢀ 103 cells/well were seeded into 96-well plates and
incubated for 24 h at 37 1C. Afterwards, two different concentrations
of synthesized compounds, which had no cytotoxic activity (cells
viability490%), were added in triplicate followed by the addition of
3 different concentration of doxorubicin after 1 h. After 72 h of
incubation, MTT assay was performed as described in Section 2.5.
2.6. Measurement of rhodamine 123 efflux by flow cytometry
To measure the inhibition of rhodamine 123 (Rh123) efflux
from MES-SA/DX5 cells by synthesized compounds, as an index of
P-gp function modulation, a previously described method was
Scheme 1. Synthetic pathway of 1,4-DHP derivatives (A1B1-A3B5).

238
F. Shekari et al. / European Journal of Pharmacology 746 (2015) 233–244
Finally, percentage of IC50 reduction of doxorubicin was calculated
Chemical structures of synthesized compounds along with their
physical data are listed in Table 1.
À
Á
as 100 ꢀ IC50 doxorubicin ꢁIC50doxorubicin þDHP =IC50doxorubicin
.
2.8. Western blot analysis of P-gp expression
3.2. Calcium channel blocking activity
Protein extraction from MES-SA/DX5 and MES-SA cells was
carried out by RIPA buffer containing protease inhibitor cocktail
and PMSF (phenylmethylsulfonyl fluoride) 10 mM. Cell lysates were
centrifuged at 16,000g for 30 min and the supernatants were
collected and kept at ꢁ20 1C before analysis. Bradford assay was
used for the determination of protein concentrations in the protein
extracts. Samples were loaded onto SDS polyacrylamide gel (7.5%)
and separated by the electrophoresis process. Then the proteins
were transferred to polyvinylidene difluoride (PVDF) membranes
in a Mini-Protean III transfer system (Bio-Rad, CA, USA) and
immunoblotted with a mouse monoclonal anti P-gp antibody
(C219, Calbiochem, Germany). Afterwards, the membrane was
incubated with goat anti-mouse secondary antibody conjugated
to horseradish peroxidase (Santa Cruz Biotechnologies, USA).
Finally immunoreactions were detected by ECL (Pierce, Rockford,
USA) and the images were taken by a Syngene G: box gel doc.
1,4-DHP derivatives are a known class of CCB agents and are
among the most commonly used cardiovascular drugs. In this study,
the IC50 value of each synthesized DHP compound (concentration that
results in 50% inhibition of smooth muscle contractility) was deter-
mined in guinea pig ileal longitudinal smooth muscle (GPLISM) as a
potential side effect when these agents are applied as MDR reversal
and cytotoxic drugs (Table 2).
Based on our findings, compounds A1B4–A4B4 (bearing 1-methyl-
5-nitro-imidazole-2-yl moiety at C4 position) exhibited the weakest
activities (IC50¼10ꢁ5ꢁ10ꢁ6 M), while the highest CCB activity
belonged to A1B2, A3B2 and A3B3, which were comparable with
nifedipine as a reference drug (IC50¼6.89 ꢀ 10ꢁ8 M).
3.3. Cytotoxicity assay
The intrinsic cytotoxic activities of compounds were evaluated
on HL-60, K562, MCF-7 and LS180 cell lines. Doxorubicin and
cisplatin were also tested as positive controls. IC50 values are
shown in Table 3.
3. Results
3.1. Chemistry
Tested compounds seemed in general to have stronger cyto-
toxic effects on HL-60 and K562 cell lines, while their effects on
LS180 cell line appeared to be weaker. The lowest IC50 belonged to
Asymmetrical 1,4-dihydropyridine (1,4-DHP) derivatives A1B1-
A3B5 were synthesized as described in Section 2.2. (Scheme 1).
Pyridin-3-yl-methyl-3-oxobutanoate III was prepared through the
reaction of corresponding alcohol I with 2,2,6-trimethyl-4-H-1,3-
dioxin-4-one II. A mixture of ammonium acetate IV and alkylace-
toacetate a1–4 were refluxed in suitable solvent to produce alkyl
3-aminocrotonate A1–4. Final DHP derivatives were synthesized
via the reaction of pyridin-3-yl-methyl-3-oxobutanoate III, alkyl
3-aminocrotonate A1–4 and aryl or heteroaryl aldehydes B1–5.
A2B5 (IC50 range: 1.06–4.12
15.60 M), which had 2-thienyl and 3-nitrophenyl at C4 position,
respectively. A2B5 was stronger than cisplatin, which was tested
as a positive control in all cell lines (IC50 range: 2.93–14.67 M). In
μM) and A2B2 (IC50 range: 3.77–
μ
μ
all tested compounds, except for A3B2 and A3B5, the increase in
the size of alkyl chain at C5 position potentiated the cytotoxic
activity. A3B2 and compounds with 1-methyl-5-nitro-imidazole-
2-yl moiety at C4 position (A1B4 to A4B4) had mostly IC50 values of
Table 1
Chemical structures and physical properties of synthesized 1,4-dihydropyridine compounds.
Compound
R1
R2
Melting point (1C)
Yield (%)
Rf
A1B1
A2B1
A3B1
A4B1
A1B2
A2B2
A3B2
A1B3
A2B3
A3B3
A4B3
A1B4
A2B4
A3B4
A4B4
A1B5
A2B5
A3B5
Methyl
Ethyl
Isopropyl
t-Butyl
Methyl
Ethyl
Isopropyl
Methyl
Ethyl
Isopropyl
t-butyl
Methyl
Ethyl
4-nitrophenyl
4-nitrophenyl
4-nitrophenyl
4-nitrophenyl
3-nitrophenyl
3-nitrophenyl
3-nitrophenyl
2-nitrophenyl
2-nitrophenyl
2-nitrophenyl
2-nitrophenyl
1-methyl-5-nitro-imidazole-2-yl
1-methyl-5-nitro-imidazole-2-yl
1-methyl-5-nitro-imidazole-2-yl
1-methyl-5-nitro-imidazole-2-yl
2- thienyl
193–194
118–120
137–138
147–150
171–172
153–155
185–189
166–170
136–137
136–138
131–133
Decomposed at 208
Decomposed at 211
201–204
Decomposed at 208
86–89
21.2
13.1
10.1
7.0
12.2
17.5
14.1
9.7
7.1
19.5
3.8
6.2
7.1
0.32
0.32
0.36
0.34
0.36
0.40
0.38
0.30
0.28
0.30
0.28
0.28
0.26
0.28
0.26
0.24
0.28
0.28
Isopropyl
t-Butyl
Methyl
Ethyl
16.7
11.2
5.5
4.8
5.7
2- thienyl
2- thienyl
95–96
102–104
Isopropyl

F. Shekari et al. / European Journal of Pharmacology 746 (2015) 233–244
239
higher than 100
cancer cell lines.
μ
M and were the weakest agents against all
compounds. Compound A2B2 (with Gmeans of 56.9775.9 and
62.90711.42 at 10 and 25 M, respectively), was more active than
verapamil (with Gmeans of 43.5074.71 and 48.5470.89 at 10
and 25 M, respectively) as a reference P-gp inhibitor compound.
Similarly, A3B2 and A3B1, at the concentration of 10 and 25 M,
μ
μ
3.4. Flow cytometric measurement of rhodamine 123 efflux
μ
were more effective than verapamil at the same concentrations.
Generally, the compounds with 3-nitrophenyl (A1B2 to A3B2) or
4-nitrophenyl (A1B1 to A4B1) at C4 position showed greater P-gp
inhibitory activities, while derivatives with 1-methyl-5-nitro-imi-
dazole-2-yl moiety at C4 position (A1B4 to A4B4) showed the
lowest activities.
Rh123 is a known fluorescent substrate for P-gp and is pumped
out of the cell by this transporter (Twentyman et al., 1994). The
capacity of synthesized compounds to inhibit P-gp pump was
evaluated by Rh123 efflux in MES-SA/DX5 cells and results are
shown in Figs. 1 and 2.
According to the obtained data, most of the DHP compounds,
especially those possessing nitrophenyl at C4 position (A1B1 to
A4B3), induced a significant increase in Geometric mean (Gmean)
of fluorescence intensity compared to control at 5–25 mM (Fig. 2
A–C, P valueo0.05). Gmean of fluorescence intensity corresponds
to the amount of Rh123 retained inside the cells and is directly
correlated with P-gp inhibition. The highest Gmean values
belonged to A2B2, A3B2, A3B1 and A4B1 were the most effective
Table 2
Calcium channel blocking activity of synthesized 1,4-DHP derivatives.
Compound
IC507S.E.M. (M)
P value
A1B1
A2B1
A3B1
A4B1
A1B2
A2B2
A3B2
A1B3
A2B3
A3B3
A4B3
A1B4
A2B4
A3B4
A4B4
A1B5
A2B5
A3B5
Nifedipine
(3.572.8) ꢀ 10ꢁ7
(5.173.4) ꢀ 10ꢁ7
(8.272.9) ꢀ 10ꢁ7
(3.071.4) ꢀ 10ꢁ6
(4.972.0) ꢀ 10ꢁ8
(5.574.7) ꢀ 10ꢁ7
(2.971.1) ꢀ 10ꢁ8
(1.570.6) ꢀ 10ꢁ7
(1.670.8) ꢀ 10ꢁ7
(6.072.2) ꢀ 10ꢁ8
(8.073.8) ꢀ 10ꢁ7
(1.270.9) ꢀ 10ꢁ5
(1.670.9) ꢀ 10ꢁ6
(9.674.0) ꢀ 10ꢁ6
(4.771.9) ꢀ 10ꢁ6
(2.871.8) ꢀ 10ꢁ6
(9.673.4) ꢀ 10ꢁ7
(2.971.5) ꢀ 10ꢁ7
(6.972.8) ꢀ 10ꢁ8
0.99
0.87
0.04a
0.00a
1.00
0.78
0.97
0.99
0.98
1.00
0.10
0.00a
0.01a
0.00a
0.00a
0.01a
0.05a
0.57
Fceigll.s1. .REhf1fe2c3t 5ofμcMomwpoasunadddAe2dB2toontutbheesrectoennttaiionninogf r5hꢀod1a0m5 iMneES1-2S3Ai/nDMX5ESc-eSlAls/DaXnd5
incubated for 30 min at 37 1C. Afterwards cells were centrifuged and washed twice
with ice-cold PBS and resuspended in RPMI 1640 medium containing A2B2 as P-gp
inhibitor. After 30 min of incubation at 37 1C, 10,000 cells were counted by a flow
cytometer with excitation and emission wavelengths of 488 nm and 530 nm,
respectively. Finally histograms were drawn by WINMDI version 2.9.
a
Difference between the IC50 value of the compound and nifedipine (as
positive control) was significant (Po0.05). n¼4.
Table 3
Cytotoxic activities of synthesized 1,4-DHP derivatives against human cancer cell lines.
IC50 (μM)7S.E.M. (n¼4)
Compound
HL-60
K562
MCF-7
LS180
A1B1
A2B1
A3B1
A4B1
A1B2
A2B2
A3B2
45.0873.0
30.0670.6
26.5172.0
23.7571.8
8.3470.5
3.7770.4
4100
40.3474.5
21.1572.4
17.4071.8
15.4971.2
9.1172.2
8.2971.9
4100
51.8073.2
34.3072.5
26.4572.4
21.47 70.7
16.4271.3
15.6072.1
4100
71.2871.4
37.0173.1
34.7172.7
27.6572.6
18.6772.6
11.3971.5
4100
A1B3
A2B3
A3B3
A4B3
64.4472.7
55.9274.74
33.6570.4
27.4770.8
4100
70.6477.5
47.9074.6
38.2676.2
26.2573.1
4100
71.61 78.1
54.2976.4
27.8671.3
26.1372.4
4100
61.22710.8
57.93710.3
49.2272.6
41.8670.1
4100
A1B4
A2B4
4100
4100
4100
4100
A3B4
4100
4100
4100
4100
A4B4
A1B5
A2B5
A3B5
Cisplatin
Doxorubicin
59.3077.07
16.2070.3
1.06 70.0
13.6570.4
2.9370.2
(5.870.3) ꢀ 10ꢁ3
68.8777.7
15.3871.9
2.6170.8
7.8970.4
11.8471.3
(63.073.5) ꢀ 10ꢁ3
62.8573.4
23.6274.4
4.1270.7
18.8172.4
11.94 72.2
(45.775.1) ꢀ 10ꢁ3
4100
50.3974.5
3.3770.1
26.2270.6
14.6772.7
(85.1722.1) ꢀ 10ꢁ3

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F. Shekari et al. / European Journal of Pharmacology 746 (2015) 233–244
Fig. 2. Flow cytometric detection of rhodamine 123 efflux from MES-SA/DX5 cells. Retention of Rh123 in MES-SA/DX5 cells was measured by flow cytometry. Cells were
exposed to 5 M Rh123 for 30 min at 37 1C and then centrifuged and washed twice with ice-cold PBS and resuspended in RPMI 1640 containing verapamil (positive control)
μ
or synthesized compounds as P-gp inhibitors. After 30 min of incubation at 37 1C, the fluorescence caused by the presence of Rh123 in cells was measured by a flow
cytometer with excitation and emission wavelengths of 488 nm and 530 nm, respectively. Effects of synthesized compounds with 4-nitrophenyl (A), 3-nitrophenyl (B),
n
2-nitrophenyl (C), 1-methyl-5-nitro-imidazole-2-yl (D) and 2-thienyl (E) at C4 position and verapamil as a positive control are shown. VRP: verapamil, shows that the
difference between Gmean of fluoroscence intensities in the absence and presence of the test compound is significant (Po0.05).
3.5. MDR reversal assay
under study had no inherent cytotoxicity at the tested concen-
trations (cell viability490%, data not shown). Results are pre-
sented in Figs. 3–5.
A3B2 which has 3-nitrophenyl at C4 was the most effective
compound, significantly lowering the IC50 of doxorubicin in MES-
MDR reversal activity of synthesized compounds was evalu-
ated in MES-SA/DX5 cells. The compounds were also tested in
MES-SA parental non-resistant cell line for comparison. DHPs

F. Shekari et al. / European Journal of Pharmacology 746 (2015) 233–244
241
Fig. 3. Measurement of doxorubicin resistance reversal in MES-SA/DX5 and MES-SA cells. Reduction of doxorubicin's IC50 by synthesized compounds was evaluated in MES-
SA/DX5 (A) and MES-SA cell lines (B). Cells were seeded in 96-well microplates and after overnight incubation; they were consecutively treated with two different
concentrations of DHPs followed by doxorubicin. Plates were further incubated for 72 h, MTT assay was carried out and IC50 of doxorubicin was calculated in the absence or
n
presence of synthesized compounds and percent reduction of IC50 values were calculated. shows that the difference between the IC50 values of doxorubicin in the absence
and presence of the test compounds is significant (Po0.05).
SA/DX5 cells at 1 mM (Fig. 3A and Fig. 4D). This compound reduced
the IC50 of doxorubicin to 21.8 mM (data not shown), a similar
value to MES-SA cells, which could be interpreted as complete
reversal of MDR in resistant cells. Most of the synthesized
compounds possessing nitrophenyl at C4 position (A1B1 to A4B3)
significantly increased the cytotoxicity of doxorubicin on MES-SA/
DX5 cells at 1–5 mM, while they had no significant effect on
MES-SA cells at similar concentrations. Among the nitrophenyl
bearing compounds at C4, the presence of 4-nitrophenyl seemed to
confer a higher activity as seen in A2B1, A3B1 and A4B1 (Fig. 3A
and Fig. 4). On the other hand, replacement of nitrophenyl with
hetero aromatic moieties at C4 (in A1B4 to A3B5) caused a
reduction of MDR reversal activity.
MES-SA/DX5 cells over-express P-gp, while the level of P-gp in
MES-SA was not detectable (Fig. 6).
4. Discussion
In this study, 18 novel asymmetrical 1,4-DHP derivatives were
synthesized and assessed for cytotoxicity in 4 human cancer cell
lines and MDR reversal activity through P-gp modulation in
doxorubicin resistant cells and their SAR was examined. SAR
studies are of utmost importance for drug discovery in order to
determine the important structural features required for higher
pharmacological activities (Kumar et al., 2014; Singhuber et al.,
2011).
CCB activity of synthesized DHPs was determined as a potential
cardiovascular side effect of these compounds. Among compounds
bearing nitrophenyl moiety at C4 position, compounds with
4-nitrophenyl were weaker CCB agents compared to those having
2-nitrophenyl or 3-nitrophenyl. These findings are in accordance
3.6. Western blot analysis of P-gp expression
P-gp expression in MES-SA/DX5 and its parental cell line MES-
SA was examined by immunoblottiong. The results showed that

242
F. Shekari et al. / European Journal of Pharmacology 746 (2015) 233–244
A
B
100
80
60
40
20
0
0 uM
1 uM
2.5 uM
100
80
60
40
20
0
0 uM
1 uM
2.5 uM
*
*
*
*
*
*
*
*
*
0
200
400
600
800
1000
1200
0
200
400
600
800
1000
1200
Concentration of doxorubicin (nM)
Concentration of doxorubicin (nM)
C
D
100
80
60
40
20
0
100
80
60
40
20
0
0 uM
1 uM
2.5 uM
0 uM
1 uM
2.5 uM
*
*
*
*
*
*
*
*
*
*
*
0
200
400
600
800
1000
1200
0
200
400
600
800
1000
1200
Concentration of doxorubicin (nM)
Concentration of doxorubicin (nM)
Fig. 4. Multidrug resistance reversal in MES-SA/DX5 cells. Effect of doxorubicin on the viability of MES-SA/DX5 cells (doxorubicin-resistant) in the absence or presence of the
n
most potent MDR reversal compounds A2B1 (A), A3B1 (B), A4B1 (C) and A3B2 (D) was measured by MTT assay as described in Fig. 3. significant versus doxorubicin
treatment alone (Po0.05).
with previous studies of 1,4-DHPs (Foroughinia et al., 2008;
Mehdipour et al., 2007). Also there was no relationship between
alkyl chain size (R1) and CCB activity.
hetero aromatic moieties at this position (A1B4 to A3B5). On the
other hand, synthesized DHPs did not increase cytotoxicity of
doxorubicin on non-resistant MES-SA cells, which rules out a non-
specific synergistic effect of these derivatives with doxorubicin.
The findings on the alteration of doxorubicin's IC50 in MES-SA/
DX5 cells generally confirmed the results of Rh123 reflux. In both
methods, replacement of nitrophenyl with hetero aromatic moi-
eties at C4 (as in A1B4 to A3B5) leads to a reduction of MDR
reversal activity. Further, DHPs with 2-nitrophenyl as aromatic
ring were weaker than those with 3 or 4-nitrophenyl at C4
position. The compound A2B2 could not be properly evaluated
for MDR reversal effect on MES-SA/DX5 cells by MTT assay at
The cytotoxicity results of 1,4-DHPs studied in this project as
well as our previous observations (Firuzi et al., 2013; Foroughinia
et al., 2008) indicate that the anticancer effect is highly dependent
on the substituent at the C4 position. The presence of 1-methyl-5-
nitro-imidazole-2-yl moiety at this position considerably lowers
the cytotoxic effect, while nitrophenyl moiety seems to boost the
activity. In addition, asymmetrical molecules are much better
cytotoxic agents compared to symmetrical DHPs (Firuzi et al.,
2013; Foroughinia et al., 2008; Mehdipour et al., 2007).
P-gp transporter inhibition and the resultant MDR reversal
effect of the synthesized DHPs was evaluated by Rh123 efflux and
MTT reduction assays in MES-SA/DX5 cells. Results of Rh123 efflux
assay indicated that most of DHP compounds, especially those
bearing nitrophenyl at C4 position (A1B1 to A4B3), significantly
decreased Rh123 efflux compared to control and had a dose
dependent P-gp inhibitory effect. Therefore, it appears that altera-
tions of the substituent at C4 position have also considerable
effects on the P-gp inhibitory activity. Although the presence of
longer alkyls at C5 improved P-gp inhibition (except for A4B1,
A3B2 and A4B3), the aromatic ring at C4 position seems to be a
more important factor influencing Rh123 efflux inhibition.
Also based on the results obtained from the alteration of
doxorubicin's IC50 value in MES-SA/DX5 cells, DHPs with nitro-
phenyl at C4 position had higher activities compared to those with
concentrations higher than 1 M, because it had a high cytotoxi-
city on these cells, which would have been confounded with MDR
reversal effect.
μ
In conclusion, DHPs seem to provide a promising scaffold for
the discovery of P-gp modulators and cytotoxic agents and SAR
studies should be performed to determine the important struc-
tural features required for higher activity and lower potential side
effects. Compounds A2B2 and A3B2 (bearing 3-nitrophenyl at C4
position) showed the strongest P-gp inhibitory activity in Rh123
efflux assay, therefore, it appears that the presence of 3-
nitrophenyl ring at C4 position increases the P-gp mediated MDR
reversal activity. While CCB activity of A3B2 did not differ from
that of nifedipine, compound A2B2 was the most promising agent,
as it was the most potent P-gp inhibitor, had a high direct
anticancer activity against
4 cancer cells (IC50 range: 3.77–

F. Shekari et al. / European Journal of Pharmacology 746 (2015) 233–244
243
100
100
80
60
40
20
0
0 uM
2.5 uM
5 uM
0 uM
1 uM
2.5 uM
80
60
40
20
0
0
10
20
30
40
50
60
0
10
20
30
40
50
60
Concentration of doxorubicin (nM)
Concentration of doxorubicin (nM)
100
80
60
40
20
0
100
80
60
40
20
0
0 uM
1 uM
2.5 uM
0 uM
2.5 uM
5 uM
0
10
20
30
40
50
60
0
10
20
30
40
50
60
Concentration of doxorubicin (nM)
Concentration of doxorubicin (nM)
Fig. 5. Effect of synthesized derivatives on the response to doxorubicin in MES-SA cells. Effect of doxorubicin on the viability of MES-SA (non-resistant) cells in the absence or
presence of the most potent MDR reversal compounds A2B1 (A), A3B1 (B), A4B1 (C) and A3B2 (D) was measured by MTT assay as described in Fig. 3.
alkyl chain (ethyl and isopropyl) at C4 and C5 positions, respec-
tively (Firuzi et al., 2013). On the other hand, compound A2B5 with
2-thienyl at C4 position, showed the highest cytotoxicity (stronger
than cisplatin) and low CCB properties but it had no MDR reversal
effect. This compound could be suggested for further cytotoxic
assessments.
Acknowledgments
The authors wish to thank the financial support of the Vice-
Provost for Research, Shiraz University of Medical Sciences, Iran.
The results reported in this paper were part of the Pharm.D. Thesis
of Farnaz Shekari (Thesis Number: 447).
Fig. 6. P-gp expression in MES-SA/DX5 and MES-SA cell lines. Proteins were
extracted from cells and loaded onto SDS polyacrylamide gel (7.5%) and analyzed
for P-gp and β-actin expression using specific antibodies.
References
Carosati, E., Ioan, P., Micucci, M., Broccatelli, F., Cruciani, G., Zhorov, B.S., Chiarini, A.,
Budriesi, R., 2012. 1,4-Dihydropyridine scaffold in medicinal chemistry, the
story so far and perspectives (part 2): action in other targets and antitargets.
Curr. Med. Chem. 19, 4306–4323.
Coburger, C., Wollmann, J., Krug, M., Baumert, C., Seifert, M., Molnár, J., Lage, H.,
Hilgeroth, A., 2010. Novel structure–activity relationships and selectivity
15.60 M) and was also a weaker CCB agent compared to nifedi-
μ
pine. These findings are in agreement with the results of our
previous study, which showed that the most active MDR reversal
DHPs with high cytotoxicity, possessed 3-nitrophenyl ring and

244
F. Shekari et al. / European Journal of Pharmacology 746 (2015) 233–244
profiling of cage dimeric 1, 4-dihydropyridines as multidrug resistance (MDR)
modulators. Bioorg. Med. Chem. 18, 4983–4990.
catalyzed by a new polystyrene-bound Mn(TPP)Cl. Bioorg. Med. Chem. Lett. 16,
2026–2030.
Edraki, N., Mehdipour, A.R., Khoshneviszadeh, M., Miri, R., 2009. Dihydropyridines:
evaluation of their current and future pharmacological applications. Drug
Discov. Today 14, 1058–1066.
Firuzi, O., Javidnia, K., Mansourabadi, E., Saso, L., Mehdipour, A.R., Miri, R., 2013.
Reversal of multidrug resistance in cancer cells by novel asymmetrical 1,4-
dihydropyridines. Arch. Pharm. Res. 36, 1392–1402.
Morshed, S.R.M., Hashimoto, K., Murotani, Y., Kawase, M., Shaha, A., Satoh, K.,
Kikuchi, H., Nishikawa, H., Maki, J., Sakagami, H., 2005. Tumor-specific cyto-
toxicity of 3, 5-dibenzoyl-1, 4-dihydropyridines. Anticancer Res. 25, 2033–2038.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J. Immunol. Methods 65,
55–63.
Munić, V., Kelnerić, Ž., Mikac, L., Eraković Haber, V., 2010. Differences in assessment
of macrolide interaction with human MDR1 (ABCB1, P-gp) using rhodamine-
123 efflux, ATPase activity and cellular accumulation assays. Eur. J. Pharm. Sci.
41, 86–95.
Ozben, T., 2006. Mechanisms and strategies to overcome multiple drug resistance
in cancer. FEBS Lett. 580, 2903–2909.
Palmeira, A., Vasconcelos, M.H., Paiva, A., Fernandes, M.X., Pinto, M., Sousa, E., 2012.
Dual inhibitors of P-glycoprotein and tumor cell growth: (re)discovering
thioxanthones. Biochem. Pharmacol. 83, 57–68.
Saini, A., Kumar, S., Sandhu, J.S., 2008. Hantzsch reaction: recent advances in
Hantzsch 1,4-dihydropyridines. J. Sci. Ind. Res. 67, 95–111.
Sarkarzadeh, H., Miri, R., Firuzi, O., Amini, M., Razzaghi-Asl, N., Edraki, N., Shafiee,
A., 2013. Synthesis and antiproliferative activity evaluation of imidazole-based
indeno[1,2-b]quinoline-9,11-dione derivatives. Arch. Pharm. Res. 36, 436–447.
Sica, D.A., 2006. Pharmacotherapy review: calcium channel blockers. J. Clin.
Hypertens. 8, 53–56.
Singhuber, J., Baburin, I., Ecker, G.F., Kopp, B., Hering, S.J., 2011. Insights into
structure-activity relationship of GABAA receptor modulating coumarins and
furanocoumarins. Eur. J. Pharmacol. 668, 57–64.
Foroughinia, F., Javidnia, K., Amirghofran, Z., Mehdipour, A., Miri, R., 2008. Design
and synthesis of new symmetrical derivatives of dihydropyridine containing a
pyridyl group on the 3, 5-positions and evaluation of their cytotoxic and
multidrug resistance reversal activity. J. Pharm. Pharmacol. 60, 1481–1489.
Gottesman, M.M., 1993. How cancer-cells evade chemotherapy—16th Richard-and-
Hinda-Rosenthal-Foundation Award Lecture. Cancer Res. 53, 747–754.
Hahn, K.A., Legendre, A.M., Schuller, H.M., 1997. Amputation and dexniguldipine as
treatment for canine appendicular osteosarcoma. J. Cancer Res. Clin. Oncol. 123,
34–38.
Hilgeroth, A., Baumert, C., Coburger, C., Seifert, M., Krawczyk, S., Hempel, C.,
Neubauer, F., Krug, M., Molnár, J., Lage, H., 2013. Novel structurally varied N-
Alkyl 1,4-dihydropyridines as ABCB1 inhibitors: structure-activity relationships,
biological activity and first bioanalytical evaluation. Med. Chem. 9, 487–493.
Jassbi, A.R., Miri, R., Asadollahi, M., Javanmardi, N., Firuzi, O., 2014. Cytotoxic,
antioxidant and antimicrobial effects of nine species of woundwort (Stachys)
plants. Pharm. Biol. 52, 62–67.
Khoshneviszadeh, M., Edraki, N., Javidnia, K., Alborzi, A., Pourabbas, B., Mardaneh, J.,
Miri, R., 2009. Synthesis and biological evaluation of some new 1,4-dihydro-
pyridines containing different ester substitute and diethyl carbamoyl group as
anti-tubercular agents. Bioorg. Med. Chem. 17, 1579–1586.
Kumar, P., Kumar, A., Song, Z.-H., 2014. Structure–activity relationships of fatty acid
amide ligands in activating and desensitizing G protein-coupled receptor 119.
Eur. J. Pharmacol. 723, 465–472.
Tsuruo, T., Iida, H., Nojiri, M., Tsukagoshi, S., Sakurai, Y., 1983. Circumvention of
vincristine and adriamycin resistance in vitro and in vivo by calcium influx
blockers. Cancer Res. 43, 2905–2910.
Twentyman, P.R., Rhodes, T., Rayner, S., 1994. A comparison of rhodamine 123
accumulation and efflux in cells with P-glycoprotein-mediated and MRP-
associated multidrug resistance phenotypes. Eur. J. Cancer 30, 1360–1369.
Viale, M., Cordazzo, C., De Totero, D., Budriesi, R., Rosano, C., Leoni, A., Ioan, P.,
Aiello, C., Croce, M., Andreani, A., Rambaldi, M., Russo, P., Chiarini, A., Spinelli,
D., 2011. Inhibition of MDR1 activity and induction of apoptosis by analogues of
nifedipine and diltiazem: an in vitro analysis. Investig. New Drugs 29, 98–109.
Voigt, B., Coburger, C., Monar, J., Hilgeroth, A., 2007. Structure-activity relationships
of novel N-acyloxy-1,4-dihydropyridines as P-glycoprotein inhibitors. Bioorg.
Med. Chem. 15, 5110–5113.
Wesolowska, O., Paprocka, M., Kozlak, J., Motohashi, N., Dus, D., Michalak, K., 2005.
Human sarcoma cell lines MES-SA and MES-SA/Dx5 as a model for multidrug
resistance modulators screening. Anticancer Res. 25, 383–389.
Xi, G.M., Sun, B., Jiang, H.H., Kong, F., Yuan, H.Q., Lou, H.X., 2010. Bisbibenzyl
derivatives sensitize vincristine-resistant KB/VCR cells to chemotherapeutic
agents by retarding P-gp activity. Bioorg. Med. Chem. 18, 6725–6733.
Yamakage, M., Namiki, A., 2002. Calcium channels-basic aspects of their structure,
function and gene encoding; anesthetic action on the channels—a review. Can.
J. Anesth. 49, 151–164.
Laupeze, B., Amiot, L., Bertho, N., Grosset, J., Lehne, G., Fauchet, R., Fardel, O., 2001.
Differential expression of the efflux pumps P-glycoprotein and multidrug
resistance-associated protein in human monocyte-derived dendritic cells.
Hum. Immunol. 62, 1073–1080.
Liscovitch, M., Lavie, Y., 2002. Cancer multidrug resistance: a review of recent drug
discovery research. Investig. Drugs J. 5, 349–355.
Liu, F.S., 2009. Mechanisms of chemotherapeutic drug resistance in cancer therapy
—a quick review. Taiwan. J. Obstet. Gynecol. 48, 239–244.
Mehdipour, A.R., Javidnia, K., Hemmateenejad, B., Amirghofran, Z., Miri, R., 2007.
Dihydropyridine derivatives to overcome atypical multidrug resistance: design,
synthesis, QSAR studies, and evaluation of their cytotoxic and pharmacological
activities. Chem. Biol. Drug Des. 70, 337–346.
Memarian, H.R., Mirjafari, A., 2005. Solid state photochemistry of 1, 4-
dihydropyridines. Bioorg. Med. Chem. Lett. 15, 3423–3425.
Miri, R., Javidnia, K., Mirkhani, H., Hemmateenejad, B., Sepeher, Z., Zalpour, M.,
Behzad, T., Khoshneviszadeh, M., Edraki, N., Mehdipour, A.R., 2007. Synthesis,
QSAR and calcium channel modulator activity of new hexahydroquinoline
derivatives containing nitroimidazole. Chem. Biol. Drug Des. 70, 329–336.
Moghadam, M., Nasr-Esfahani, M., Tangestaninejad, S., Mirkhani, V., 2006. Mild and
efficient oxidation of Hantzsch 1,4-dihydropyridines with sodium periodate
Zarrin, A., Mehdipour, A.R., Miri, R., 2010. Dihydropyridines and multidrug
resistance: previous attempts, present state, and future trends. Chem. Biol.
Drug Des. 76, 369–381.