Reversal of multidrug resistance in cancer cells by novel asymmetrical 1,4-dihydropyridines

Article abstract of DOI:10.1007/s12272-013-0149-8

Multidrug resistance (MDR) is an important obstacle that limits the efficacy of chemotherapy in many types of cancer. In this study, 14 novel asymmetrical DHPs possessing pyridyl alkyl carboxylate substitutions at C ₃ and alkyl carboxylate groups at C₅ in addition to a nitroimidazole or nitrophenyl moiety at C₄ position were synthesized. Calcium channel blocking (CCB) activity was measured in guinea pig ileal longitudinal smooth muscle. Cytotoxicity was tested on 4 human cancer cell lines, while MDR reversal capacity was examined on P-glycoprotein overexpressing doxorubicin resistant MES-SA-DX5 and compared with non-resistant MES-SA cells. Compounds showed different CCB (IC₅₀: 29.3 nM-4.75 μM) and cytotoxic activities (IC₅₀: 6.4 to more than 100 μM). Several compounds having nitrophenyl moiety at C₄, could significantly reverse resistance to doxorubicin at 0.5 and 1 μM. The most active ones were 7e and 7g containing ethyl carboxylate and isopropyl carboxylate at C ₅, respectively. CCB activity, which is considered an undesirable effect for these agents, of 7e and 7g were 33 and 20 times lower than nifedipine, respectively. In conclusion, the newly synthesized asymmetrical DHP compounds showed promising MDR reversal and antitumoral activities with low CCB effects and could be of therapeutic value in drug resistant cancer.

Full text of DOI:10.1007/s12272-013-0149-8

Arch. Pharm. Res.
DOI 10.1007/s12272-013-0149-8
RESEARCH ARTICLE
Reversal of multidrug resistance in cancer cells by novel
asymmetrical 1,4-dihydropyridines
•
Omidreza Firuzi Katayoun Javidnia
•
•
Elham Mansourabadi Luciano Saso
•
•
Ahmad Reza Mehdipour Ramin Miri
Received: 11 February 2013 / Accepted: 30 April 2013
Ó The Pharmaceutical Society of Korea 2013
Abstract Multidrug resistance (MDR) is an important
obstacle that limits the efficacy of chemotherapy in many
types of cancer. In this study, 14 novel asymmetrical DHPs
possessing pyridyl alkyl carboxylate substitutions at C3 and
alkyl carboxylate groups at C5 in addition to a nitroimidazole
or nitrophenyl moiety at C4 position were synthesized. Cal-
cium channel blocking (CCB) activity was measured in
guinea pig ileal longitudinal smooth muscle. Cytotoxicity
was tested on 4 human cancer cell lines, while MDR reversal
capacity was examined on P-glycoprotein overexpressing
doxorubicin resistant MES-SA-DX5 and compared with
non-resistant MES-SA cells. Compounds showed different
CCB (IC50: 29.3 nM–4.75 lM) and cytotoxic activities
(IC50: 6.4 to more than 100 lM). Several compounds having
nitrophenyl moiety at C4, could significantly reverse resis-
tance to doxorubicin at 0.5 and 1 lM. The most active ones
were 7e and 7g containing ethyl carboxylate and isopropyl
carboxylate at C5, respectively. CCB activity, which is
considered an undesirable effect for these agents, of 7e and
7g were 33 and 20 times lower than nifedipine, respectively.
In conclusion, the newly synthesized asymmetrical DHP
compounds showed promising MDR reversal and antitu-
moral activities with low CCB effects and could be of ther-
apeutic value in drug resistant cancer.
Keywords Cancer Á Multidrug resistance Á
Dihydropyridines Á Cytotoxicity Á Calcium channel
blocking
Abbreviations
DHPs Dihydropyridines
MDR Multidrug resistance
Introduction
O. Firuzi Á K. Javidnia Á E. Mansourabadi Á
A. R. Mehdipour Á R. Miri (&)
Cancer is one of the most important causes of death all over
the world among various racial and ethnic groups (King and
Robins 2006). Different therapeutic modalities such as sur-
gery, radiotherapy and chemotherapy are used for treatment
of cancer, among which chemotherapy is one of the most
common methods (Kasper et al. 2005). Successful treatment
of cancer by chemotherapy is to a large extent dependent on
the effectiveness of cytotoxic anticancer drugs, which are
used either alone or in combination with other methods.
Unfortunately, in the recent years, many types of cancer have
become either intrinsically resistant to all initial chemo-
therapeutic treatments or acquire resistance to a broad
spectrum of these agents over time, a phenomenon called
multidrug resistance (MDR) (Ejendal and Hrycyna 2002;
Szakacs et al. 2006; Borst et al. 2007).
Medicinal and Natural Products Chemistry Research Center,
Shiraz University of Medical Sciences, PO Box 3288, 71345
Shiraz, Iran
e-mail: mirir@sums.ac.ir; ramin.miri.15@gmail.com
K. Javidnia Á E. Mansourabadi Á R. Miri
Department of Medicinal Chemistry, Faculty of Pharmacy,
Shiraz University of Medical Sciences, Shiraz, Iran
L. Saso
Department of Physiology and Pharmacology ‘‘Vittorio
Erspamer’’, Sapienza University of Rome, Rome, Italy
Present Address:
A. R. Mehdipour
Computational Structural Biology Group, Max Planck Institute
of Biophysics, Max von laue-str 3, 60438 Frankfurt am Main,
Germany
123

O. Firuzi et al.
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 on the one hand and atypical MDR that
includes other mechanisms on the other hand (Teodori
et al. 2002; Zarrin et al. 2010). P-glycoprotein (P-gp) also
known as MDR protein 1 or ABC sub-family B member 1
is one of the main causes of typical MDR that limits the
effectiveness of chemotherapy (Stavrovskaya and Stroms-
kaya 2008).
were performed on a Burker-AdvanceDPX-500 MHz in d1-
chloroform. Tetramethylsilane was used as the internal
standard. The mass spectra were obtained with a Hewlett-
¨
Packard spectrometer [Hewlett-Packard (HP) 6890, Bob-
lingen, Germany] with a direct inlet system at 70 eV, The
IR spectra were measured with a Perkin-Elmer spectrom-
eter (KBr disk) (Perkin-Elmer, Waltham, MA). All spectra
confirmed the structure of the synthesized compounds.
Synthesis of aryl acetoacetate (3a–3c)
Because of the importance of MDR in failure of che-
motherapy in many instances, a number of studies have
focused on the development of MDR reversal agents (Miri
and Mehdipour 2008; Eid et al. 2012). 1,4-Dihydropyri-
dines (DHPs) have been introduced as a class of calcium
channel blockers (Edraki et al. 2009). In the beginning of
the 1980s, it was discovered that calcium channel blockers
are able to inhibit the process of MDR (Tsuruo et al. 1981;
Tsuruo et al. 1982). Several studies have been performed
since then in order to find new DHP derivatives for reversal
of MDR. For instance, some derivatives of niguldipine and
their pyridine counterparts (Zhou et al. 2005a, b) as well as
compounds bearing pyridyl groups on 3 and 5 positions of
DHP nucleus have been synthesized and tested (Tasaka
et al. 2001; Mehdipour et al. 2007; Foroughinia et al.
2008). Furthermore, Zhou et al. have introduced some DHP
derivatives, which are effective on typical MDR including
MDR-associated protein 1 and Breast cancer related pro-
tein-mediated MDR (Zhou et al. 2005a, b).
A mixture of 16.67 mM of corresponding alcohol 1a–1c
(Sigma-Aldrich Chemie GmbH, Deisenhofon, Germany)
and 16.67 mmol 2,2,6-trimethyl-4H-1,3-dioxin-4-one
(Sigma-Aldrich Chemie GmbH, Deisenhofon, Germany)
were refluxed in 10 ml xylene and stirred vigorously at a
temperature of 150 °C for 45 min. The formation of final
product was confirmed using thin layer chromatography
(TLC) ([90 %). Afterwards, the reaction mixture was
cooled and the xylene removed. The product was purified
by TLC on silica gel with chloroform–methanol (90/10) as
the mobile phase to give pure compounds 3a–3c.
Pyridine-2-yl-propyl-3-oxobutanoate (3a) yield: 91 %
IR (KBr): t1743 (C=O, ester), 1716 (C=O, ketone),
2958 cm-1 (C–H aromatics).
Pyridine-3-yl-propyl-3-oxobutanoate (3b) yield: 93 %
We have recently synthesized some novel DHP ana-
logues that contain pyridyl group at C3 and C5 in addition
to a nitroimidazole moiety at C4 that show good MDR
reversal activity (Mehdipour et al. 2007; Foroughinia et al.
2008). All DHP derivatives tested in our laboratory so far
have had symmetrical structures, but in the present study
we decided to examine the effect of asymmetry at C3 and
C5 positions on MDR reversal activity of these compounds.
Besides, the cytotoxic effect of the newly synthesized
DHPs were measured on 4 different human cancer cell
lines, and their calcium channel antagonistic activity, was
also evaluated.
IR (KBr): t 1748 (C=O, ester), 1717 (C=O, ketone),
2958 cm-1 (C–H aromatics).
Pyridine-4-yl-propyl-3-oxobutanoate (3c) yield: 95 %
IR (KBr): t1742 (C=O, ester), 1716 (C=O, ketone),
2963 cm-1 (C–H aromatics).
Synthesis of alkyl 3-aminocrotonate (5a–5c)
A solution of alkyl acetoacetic esters 4a–4c (2 mmol)
(Merck, Darmstadt, Germany) and ammonium acetate
(3 mmol) in 5 ml ethanol were refluxed in 10 ml ethanol
and stirred vigorously at a temperature of 90 °C for 24 h.
Then, the reaction mixture was cooled and ethanol
removed. IR spectra of the compounds were recorded to
confirm the structure of 5a–5c. Then, the compounds were
immediately used in subsequent reactions.
Materials and methods
Physical measurements
The melting points were measured on a hot stage apparatus
(Electrothermal, Essex, UK) and were uncorrected. Ele-
mental analyses (C, H, N) were undertaken on 7a–7n by
the Microanalytical Department, Central Laboratories for
Research, Shiraz University of Medical Sciences and were
within 0.4 % of the calculated value. The 1H-NMR spectra
Methyl 3-aminocrotonate (5a)
IR (KBr): t 1716 (C=O, ester), 3511, 3333 cm-1 (NH2).
123

Multidrug resistance reversal by dihydropyridines
Ethyl 3-aminocrotonate (5b)
(m/z) 452 (7), 434 (4), 420 (6), 329 (33), 297 (72), 120 (22),
106 (13), 92 (50), 78 (7), 51 (5).
IR (KBr): t 1716 (C=O, ester), 3511, 3332 cm-1 (NH2).
Isopropyl 3-aminocrotonate (5c)
IR (KBr): t 3188 (NH), 3057 (CH-aromatic), 2925 (CH-
aliphatic), 1682 (CO), 1344, 1526 cm-1 (NO2).
3-Ethyl-5-(3-(pyridine-4-yl)propyl)-2,6-dimethyl-4-(4-
nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7c)
IR (KBr): t 1716 (C=O, ester), 3509, 3332 cm-1 (NH2).
1H-NMR (CDCl3, 500 Hz): d 1.23 (t, 3H, COOCH2CH3,
j = 7.1 Hz), 1.90–1.93 (m, 2H, COOCH2CH2CH2), 2.36 (2s,
6H, C2–CH3 and C6–CH3), 2.56 (t, 2H, COOCH2CH2CH2,
j = 7.7 Hz), 4.04–4.12 (m, 4H, COOCH2CH2CH2 and
COOCH2CH3), 5.11 (s, 1H, C4–H), 5.87 (brs, 1H, NH–DHP),
7.02 (d, 2H, C2,6H-pyridyl, j = 5.9 Hz), 7.45 (d, 2H, C2,6H-
phenyl, j = 8.7 Hz), 8.08 (d, 2H, C3,5H-phenyl, j = 8.7 Hz),
8.47 (d, 2H, C3,5H-pyridyl, j = 6.0 Hz).
General procedure for the synthesis of asymmetrical
derivatives of 1,4-DHP (7a–7n)
A solution of compounds 5a–5c, 3a–3c and aryl or
heteryl aldehydes were protected from light and refluxed
in ethanol (30 ml) for 24 h. after cooling, the solution
was concentrated under reduced pressure and purified by
TLC on silica gel with chloroform–ethanol (95–5 %).
The product was recrystallized from diethyl ether–
petroleum ether to give pure compounds 7a–7n as prism
crystals.
Found C, 64.41 H, 5.83; N, 9.06 %. Anal. (C25H27N3O6)
requires C, 64.50; H, 5.85; N, 9.03 %.
MS: (m/z) 466 (1), 448 (40), 343 (100), 329 (4), 297 (44),
271 (4), 196 (21), 136 (11), 120 (58), 106 (13), 93 (20), 77 (6).
IR (KBr): t 3194 (NH), 3075 (CH-aromatic), 2951 (CH-
aliphatic), 1688, 1703 (CO), 1343, 1513 cm-1 (NO2).
3-Methyl-5-(3-(pyridine-4-yl)propyl)-2,6-dimethyl-4-(4-
nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7a)
1H-NMR (CDCl3, 500 Hz): d 1.90–1.94 (m, 2H, CO-
OCH2CH2CH2), 2.34–2.37 (2s, 6H, C2–CH3 and C6–CH3),
2.55 (t, 2H, COOCH2CH2CH2, j = 7.2 Hz), 3.65
(s,3H,COOCH3), 4.03–4.09 (m, 2H, COOCH2CH2CH2),
5.10 (s, 1H, C4–H), 5.91 (brs, 1H, NH–DHP), 7.02 (d, 2H,
3-Methyl-5-(3-(pyridine-3-yl)propyl)-2,6-dimethyl-4-(2-
nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7d)
1H-NMR (CDCl3, 500 Hz): d 1.86–1.92 (m, 2H, CO-
OCH2CH2CH2), 2.34 (2s, 6H, C2–CH3 and C6–CH3), 2.47
(t, 2H, COOCH2CH2CH2, j = 7.8 Hz), 3.58 (s, 3H,
COOCH3), 3.99–4.12 (m, 2H, COOCH2CH2CH2), 5.78 (s,
1H, C4–H), 5.89 (brs, 1H, NH–DHP), 7.16–7.19 (dd, 1H,
C5H-pyridyl), 7.23 (t, 1H, C4H-phenyl, j = 8.6 Hz), 7.43
(d, 1H, C6H-phenyl, j = 8.4), 7.47 (t, 1H, C5H-phenyl,
j = 8.5 Hz), 7.53 (d, 1H, C6H-pyridyl, j = 1.3 Hz), 7.68
(d, 1H, C3H-phenyl, j = 7.0 Hz), 8.30 (s, 1H, C2H-pyri-
dyl), 8.40 (d, 1H, C4H-pyridyl, j = 4.8 Hz).
C2,6H-pyridyl, j = 5.8 Hz), 7.44 (d, 2H, C2,6H-phenyl,
j = 8.7 Hz), 8.07 (d, 2H, C3,5H-phenyl, j = 8.7 Hz), 8.47
(d, 2H, C3,5H-pyridyl, j = 6.0 Hz).
Found C, 63.91; H, 5.56; N, 9.30 %. Anal.
(C24H25N3O6) requires C, 63.85; H, 5.58; N, 9.31 %.
MS: (m/z) 434 (5), 420 (4), 343 (11), 329 (100), 297 (6),
196 (3), 136 (8), 120 (22), 106 (13), 92 (14), 77 (4), 51 (2).
IR (KBr): t 3278 (NH), 3077 (CH-aromatic), 2947 (CH-
aliphatic), 1699 (CO), 1342, 1508 cm-1 (NO2).
Found C, 63.79; H, 5.57; N, 9.34 %. Anal.
(C24H25N3O6) requires C, 63.85; H, 5.58; N, 9.31 %.
MS: (m/z) 452 (1), 434 (36), 420 (2), 343 (1), 329 (16),
297 (31), 270 (55), 196 (13), 136 (8), 120 (87), 106 (23), 92
(100), 77 (13), 51 (8).
3-Methyl-5-(3-(pyridine-3-yl)propyl)-2,6-dimethyl-4-(3-
nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7b)
1H-NMR (CDCl3, 500 Hz): d 1.89–1.95 (m, 2H, CO-
OCH2CH2CH2), 2.36–2.38 (2s, 6H, C2–CH3 and C6–CH3),
2.57 (t, 2H, COOCH2CH2CH2, j = 7.7 Hz), 3.65 (s, 3H,
COOCH3), 4.01–4.12 (m, 2H, COOCH2CH2CH2), 5.10 (s,
1H, C4–H), 5.87 (brs, 1H, NH–DHP), 7.18–7.20 (dd, 1H,
C5H-pyridyl), 7.37 (t, 1H, C5H-phenyl, j = 7.9 Hz), 7.41
(d, 1H, C6H-phenyl, j = 7.8 Hz), 7.64 (d, 1H, C6H-pyridyl,
j = 7.6 Hz), 7.99 (d, 1H, C4H-phenyl, j = 8.2 Hz), 8.11 (s,
1H, C2H-phenyl), 8.37 (s, 1H, C2H-pyridyl), 8.43 (d, 1H,
C4H-pyridyl, j = 3.5 Hz).
IR (KBr): t 3188 (NH), 3067 (CH-aromatic), 2944 (CH-
aliphatic), 1699, 1688 (CO), 1342, 1508 cm-1 (NO2).
3-Ethyl-5-(3-(pyridine-4-yl)propyl)-2,6-dimethyl-4-(3-
nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7e)
1H-NMR (CDCl3, 500 Hz): d 1.23 (t, 3H, COOCH2CH3,
j = 7.11 Hz), 1.90–1.95 (m, 2H, COOCH2CH2CH2), 2.37
(2s, 6H, C2–CH3 and C6–CH3), 2.56 (t, 2H, CO-
OCH2CH2CH2, j = 7.7 Hz), 4.06–4.16 (m, 4H,
COOCH2CH2CH2 and COOCH2CH3), 5.11 (s, 1H, C4–H),
5.94 (brs, 1H, NH–DHP), 7.03 (d, 2H, C2,6H-pyridyl,
Found C, 63.78; H, 5.56; N, 9.29 %. Anal.
(C24H25N3O6) requires C, 63.85; H, 5.58; N, 9.31 %.MS:
123

O. Firuzi et al.
3-Isopropyl-5-(3-(pyridine-4-yl)propyl)-2,6-dimethyl-4-(4-
nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7h)
j = 5.9 Hz), 7.37 (t, 1H, C5H-phenyl, j = 7.9 Hz), 7.64 (d,
1H, C6H-phenyl, j = 7.7 Hz), 7.99 (d, 1H, C4H-phenyl,
j = 7.8 Hz), 8.14 (s, 1H, C2H-phenyl), 8.47 (d, 2H, C3,5H-
pyridyl, j = 5.9 Hz).
1H-NMR (CDCl3, 500 Hz): d 1.12–1.24 (2d, 6H, CO-
OCH(CH3)2, j = 6.2, 6.2 Hz), 1.88–1.94 (m, 2H, CO-
OCH2CH2CH2), 2.34–2.36 (2s, 6H, C2–CH3 and C6–CH3),
2.56 (t, 2H, COOCH2CH2CH2, j = 7.7 Hz), 4.03–4.09 (m,
2H, COOCH2CH2CH2), 4.95–4.98 (m, 1H, COOCH(CH3)2),
5.10 (s, 1H, C4–H), 5.86 (brs, 1H, NH–DHP), 7.02 (d, 2H,
Found C, 64.41; H, 5.83; N, 9.01 %. Anal.
(C25H27N3O6) requires C, 64.50; H, 5.85; N, 9.03 %.
MS: (m/z) 448 (35), 343 (100), 329 (4), 297 (50), 196
(16), 136 (9), 120 (18), 106 (13), 92 (12), 77 (2), 51 (1).
IR (KBr): t 3272 (NH), 3068 (CH-aromatic), 2957 (CH-
aliphatic), 1697 (CO), 1348, 1524 cm-1 (NO2).
C2,6H-pyridyl, j = 5.8 Hz), 7.45 (d, 2H, C2,6H-phenyl,
j = 8.7 Hz), 8.07 (d, 2H, C3,5H-phenyl, j = 8.7 Hz), 8.47 (d,
2H, C3,5H-pyridyl, j = 5.9 Hz).
3-Isopropyl-5-(3-(pyridine-2-yl)propyl)-2,6-dimethyl-4-(3-
nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7f)
Found C, 64.98; H, 6.11; N, 8.79 %. Anal.
(C26H29N3O6) requires C, 65.12; H, 6.10; N, 8.76 %.
MS: (m/z) 480 (2), 462 (39), 448 (2), 420 (10), 434 (4),
392 (8), 375 (2), 297 (46), 271 (18), 196 (19), 136 (8), 120
(30), 106 (8), 92 (24), 77 (8), 51 (5).
1H-NMR (CDCl3, 500 Hz): d 1.10–1.26 (2d, 6H, CO-
OCH(CH3)2, j = 6.2, 6.2), 2.02–2.07 (m, 2H, CO-
OCH2CH2CH2), 2.34–2.37 (2s, 6H, C2–CH3 and C6–CH3),
2.75 (t, 2H, COOCH2CH2CH2, j = 7.6 Hz), 4.04–4.12 (m,
2H, COOCH2CH2CH2), 4.93–4.98 (m, 1H, COOCH(CH3)2),
5.09 (s, 1H, C4–H), 5.72 (brs, 1H, NH–DHP), 7.04 (d, 1H,
C6H-pyridyl, j = 7.7 Hz), 7.09 (t, 1H, C4H-pyridyl,
j = 8.4 Hz), 7.35 (t, 1H, C5H-phenyl, j = 8.8 Hz), 7.56 (t,
1H, C5H-pyridyl, j = 7.0 Hz), 7.65 (d, 1H, C6H-phenyl,
j = 7.7 Hz), 7.98 (d, 1H, C4H-phenyl, j = 8.2 Hz), 8.14 (s,
1H, C2H-phenyl), 8.50 (d, 1H, C3H-pyridyl, j = 4.8 Hz).
Found C, 64.99; H, 6.13; N, 8.73 %. Anal.
(C26H29N3O6) requires C, 65.12; H, 6.10; N, 8.76 %.
MS: (m/z) 462 (3), 420 (3), 343 (3), 196 (5), 136 (5), 120
(100), 106 (13), 93 (10), 78 (2).
IR (KBr): t 3196 (NH), 3076 (CH-aromatic), 2971 (CH-
aliphatic), 1701, 1674 (CO), 1346, 1511 cm-1 (NO2).
3-Ethyl-5-(3-(pyridine-3-yl)propyl)-2,6-dimethyl-4-(3-
nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7i)
1H-NMR (CDCl3, 500 Hz): d 1.23 (t, 3H, COOCH2CH3,
j = 7.5 Hz), 1.90–1.93 (m, 2H, COOCH2CH2CH2), 2.34–2.38
(2s, 6H, C2–CH3 and C6–CH3), 2.57(t, 2H, COOCH2CH2CH2,
j = 7.7 Hz), 4.04–4.11 (m, 4H, COOCH2CH2CH2 and
COOCH2CH3), 5.11 (s, 1H, C4–H), 5.91 (brs, 1H, NH–DHP),
7.20 (t, 1H, C5H-pyridyl), 7.37 (t, 1H, C5H-phenyl,
j = 7.9 Hz), 7.41 (d, 1H, C6H-phenyl, j = 7.7 Hz), 7.64 (d,
1H, C6H-pyridyl, j = 7.5 Hz), 7.99 (d, 1H, C4H-phenyl,
j = 7.8 Hz), 8.13 (s, 1H, C2H-phenyl), 8.37 (s, 1H, C2H-pyr-
idyl), 8.44 (d, 1H, C4H-pyridyl).
IR (KBr): t 3207 (NH), 3089 (CH-aromatic), 2976 (CH-
aliphatic), 1695, 1670(CO), 1346, 1525 cm-1 (NO2).
3-Isopropyl-5-(3-(pyridine-3-yl)propyl)-2,6-dimethyl-4-(3-
nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7g)
Found C, 64.71; H, 5.90; N, 9.09 %. Anal.
(C25H27N3O6) requires C, 64.50; H, 5.85; N, 9.03 %.
MS: (m/z) 466 (2), 447 (45), 420 (3), 343 (44), 329 (6),
297 (91), 196 (29), 136 (13), 120 (25), 106 (21), 92 (56), 78
(8), 51 (5).
1H-NMR (CDCl3, 500 Hz): d 1.11–1.26 (2d, 6H, CO-
OCH(CH3)2, j = 6.2, 6.2 Hz), 1.88–1.94 (m, 2H, CO-
OCH2CH2CH2), 2.35–2.38 (2s, 6H, C2–CH3 and C6–CH3),
2.58 (t, 2H, COOCH2CH2CH2, j = 7.7 Hz), 4.01–4.11 (m,
2H, COOCH2CH2CH2), 4.94–4.99 (m, 1H, COOCH(CH3)2),
5.10 (s, 1H, C4–H), 5.80 (brs, 1H, NH–DHP), 7.18–7.21 (dd,
1H, C5H-pyridyl), 7.37 (t, 1H, C5H-phenyl, j = 8.8 Hz), 7.41
(d, 1H, C6H-phenyl, j = 7.7 Hz), 7.64 (d, 1H, C6H-pyridyl,
j = 7.7 Hz), 7.99 (d, 1H, C4H-phenyl, j = 8.3 Hz), 8.14 (s,
1H, C2H-phenyl), 8.37 (s, 1H, C2H-pyridyl), 8.43 (d, 1H, C4H-
pyridyl, j = 4.7 Hz).
IR (KBr): t 3206 (NH), 3081 (CH-aromatic), 2979 (CH-
aliphatic), 1692 (CO), 1342, 1522 cm-1 (NO2).
3-Methyl-5-(3-(pyridine-3-yl)propyl)-2,6-dimethyl-4-(1-
methyl-5-nitro-1-imidazole-2-yl)-1,4-dihydropyridine-3,5-
dicarboxylate (7j)
1H-NMR (CDCl3, 500 Hz): d 1.92–1.98 (m, 2H, CO-
OCH2CH2CH2), 2.25–2.26 (2s, 6H, C2–CH3 and C6–CH3),
2.63 (t, 2H, COOCH2CH2CH2, j = 7.7 Hz), 3.68 (s, 3H, N–
CH3), 4.08–4.16 (m, 2H, COOCH2CH2CH2), 4.21 (s, 3H,
COOCH3), 5.14 (s, 1H, C4–H), 7.19–7.21 (dd, 1H, C5H-
pyridyl), 7.45 (d, 1H, C6H-pyridyl, j = 7.7 Hz), 7.95 (s, 1H,
H–Imidazole), 8.41 (s, 1H, C2H-pyridyl), 8.45 (d, 1H, C4H-
pyridyl, j = 3.3 Hz), 8.45 (brs, 1H, NH–DHP).
Found C, 65.07; H, 6.12; N, 8.77 %. Anal.
(C26H29N3O6) requires C, 65.12; H, 6.10; N, 8.76 %.
MS: (m/z) 480 (2), 461 (46), 447 (3), 343 (10), 329 (1), 297
(100), 196 (25), 136 (15), 120 (40), 106 (26), 92 (77), 77 (10),
51 (6).
IR (KBr): t 3208 (NH), 3087 (CH-aromatic), 2979 (CH-
aliphatic), 1695 (CO), 1347, 1522 cm-1 (NO2).
123

Multidrug resistance reversal by dihydropyridines
Found C, 58.22; H, 5.55; N, 15.41 %. Anal.
(C22H25N5O6) requires C, 58.01; H, 5.53; N, 15.38 %.
MS: (m/z) 438 (33), 329 (21), 297 (41), 272 (7), 120
(29), 106 (25), 92 (50), 51 (4).
COOCH3), 5.14 (s, 1H, C4–H), 7.06 (d, 2H, C2,6H-pyridyl,
j = 6.0 Hz), 7.95 (s, 1H, H–imidazole), 8.25 (brs, 1H, NH–
DHP), 8.49 (d, 2H, C3,5H-pyridyl, j = 6.0 Hz).
Found C, 58.09; H, 5.56; N, 15.41 %. Anal.
(C22H25N5O6) requires C, 58.01; H, 5.53; N, 15.38 %.
MS: (m/z) 455 (2), 438 (100), 329 (37), 297 (28), 120
(26), 106 (11), 93 (11), 51 (2).
IR (KBr): t 3423(NH), 3081(CH-aromatic), 2979(CH-
aliphatic), 1700 (CO), 1375, 1501 cm-1 (NO2).
3-Methyl-5-(3-(pyridine-2-yl)propyl)-2,6-dimethyl-4-(1-
methyl-5-nitro-1-imidazole-2-yl)-1,4-dihydropyridine-3,5-
dicarboxylate (7k)
IR (KBr): t 3423 (NH), 3081 (CH-aromatic), 2924 (CH-
aliphatic), 1701 (CO), 1376, 1501 cm-1 (NO2).
3-Isopropyl-5-(3-(pyridine-2-yl)propyl)-2,6-dimethyl-4-(1-
methyl-5-nitro-1-imidazole-2-yl)-1,4-dihydropyridine-3,5-
dicarboxylate (7n)
1H-NMR (CDCl3, 500 Hz): d 2.07–2.10 (m, 2H, CO-
OCH2CH2CH2), 2.25–2.27 (2s, 6H, C2–CH3 and C6–CH3),
2.77 (t, 2H, COOCH2CH2CH2, j = 7.1 Hz), 3.68 (s, 3H, N–
CH3), 4.11–4.18 (m, 2H, COOCH2CH2CH2), 4.20 (s, 3H,
COOCH3), 5.13 (s, 1H, C4–H), 7.07 (d, 1H, C4H-pyridyl,
j = 7.8 Hz), 7.10 (d, 1H, C6H-pyridyl, j = 4.9), 7.57 (t, 1H,
C5H-pyridyl, j = 7.15 Hz), 7.94 (s, 1H, H-imidazole), 8.22
(brs, 1H, NH–DHP), 8.50 (d, 1H, C3H-pyridyl, j = 4.0 Hz).
Found C, 58.10; H, 5.51; N, 15.36 %. Anal.
(C22H25N5O6) requires C, 58.01; H, 5.53; N, 15.38 %.
MS: (m/z) 438 (6), 329 (21), 120 (100), 93 (10), 78 (5), 51
(2).
1H-NMR (CDCl3, 500 Hz): d 1.17–1.24 (2d, 6H, CO-
OCH(CH3)2, j = 6.2, 6.3 Hz), 2.07–2.10 (m, 2H, CO-
OCH2CH2CH2), 2.27–2.28 (2s, 6H, C2–CH3 and C6–CH3),
2.78 (t, 2H, COOCH2CH2CH2, j = 7.3 Hz), 4.14–4.17 (m,
2H, COOCH2CH2CH2), 4.22 (s, 3H, N–CH3), 5.01–5.03
(m, 1H, COOCH(CH3)2), 5.10 (s, 1H, C4–H), 7.08 (d, 1H,
C4H-pyridyl, j = 8.2 Hz), 7.10 (d, 1H, C6H-pyridyl,
j = 8.7 Hz), 7.57 (t, 1H, C5H-pyridyl, j = 8.5 Hz), 7.95 (s,
1H, H–imidazole), 8.50 (d, 1H, C3H-pyridyl, j = 4.1 Hz).
Found C, 59.58; H, 6.06; N, 14.49 %. Anal.
(C24H29N5O6) requires C, 59.62; H, 6.05; N, 14.48 %.
MS: (m/z) 466 (12), 196 (2), 136 (3), 120 (100), 106 (4),
92 (6), 78 (2).
IR (KBr): t 3423 (NH), 3185 (CH-aromatic), 2947 (CH-
aliphatic), 1703, 1667 (CO), 1374, 1508 cm-1 (NO2).
3-Ethyl-5-(3-(pyridine-3-yl)propyl)-2,6-dimethyl-4-(1-
methyl-5-nitro-1-imidazole-2-yl)-1,4-dihydropyridine-3,5-
dicarboxylate (7l)
IR (KBr): t 3428 (NH), 3081 (CH-aromatic), 2978 (CH-
aliphatic), 1700, 1671 (CO), 1373, 1508 cm-1 (NO2).
1H-NMR (CDCl3, 500 Hz): d 1.24 (t, 3H, COOCH2CH3,
j = 7.1 Hz), 1.93–1.98 (m, 2H, COOCH2CH2CH2), 2.27 (s,
6H, C2–CH3 and C6–CH3), 2.63 (t, 2H, COOCH2CH2CH2,
j = 7.7 Hz), 4.10–4.16 (m, 4H, COOCH2CH2CH2 and
COOCH2CH3), 4.22 (s, 3H, N–CH3), 5.14 (s, 1H, C4–H),
7.19–7.21 (dd, 1H, C5H-pyridyl), 7.44 (d, 1H, C6H-pyridyl,
j = 7.8 Hz), 7.95 (s, 1H, H–imidazole), 8.41 (s, 1H, C3H-
pyridyl), 8.44 (d, 1H, C4H-pyridyl, j = 3.4 Hz).
Pharmacology
Male albino guinea pigs (300–450 g) were purchased from
Shiraz University Animal House Department. They had free
access to standard rodent chow and tap water ad libitum and
were housed at 23 2 °C temperatures, 55 10 %
humidity, and on a 12 h dark/light cycle. The feeding was
discontinued 1 day before starting the in vitro tests. The
animals were sacrificed and their intestines were removed
above the ileocecal valve. Smooth muscle segments of about
1 cm length were mounted under a resting tension of 500 mg
and were maintained at 37 °C in a 20 ml Jacketed organ bath
containing oxygenated (95 % O2 and 5 % CO2) physiolog-
ical saline solution of the following compositions: NaCl
137 mM, CaCl2 1.8 mM, KCl 2.7 mM, MgSO4 1.1 mM,
NaHPO4 0.4 mM, NaHCO3 12 mM and glucose 5 mM. The
muscle was equilibrated for 1 h with a solution changing
every 15 min. The contractions were recorded with a forced
displacement transducer (Hugo Sachs, March-Hugstetten
and Germany) on a physiograph (Hugo Sachs). All com-
pounds were dissolved in DMSO and the same volume of
solvent was used as the negative control, while nifedipine
was used as the positive control. The contraction was elicited
Found C, 58.79; H, 5.82; N, 14.89 %. Anal.
(C23H27N5O6) requires C, 58.84; H, 5.80; N, 14.92 %.
MS: (m/z) 452 (46), 329 (6), 297 (33), 273 (2), 196 (11),
120 (15), 106 (13), 92 (22), 51 (2).
IR (KBr): t 3438 (NH), 3081 (CH-aromatic), 2933 (CH-
aliphatic), 1698, 1674 (CO), 1373, 1500 cm-1 (NO2).
3-Methyl-5-(3-(pyridine-4-yl)propyl)-2,6-dimethyl-4-(1-
methyl-5-nitro-1-imidazole-2-yl)-1,4-dihydropyridine-3,5-
dicarboxylate (7m)
1H-NMR (CDCl3, 500 Hz): d 1.93–1.99 (m, 2H, CO-
OCH2CH2CH2), 2.26 (s, 6H, C2–CH3 and C6–CH3), 2.61
(t, 2H, COOCH2CH2CH2, j = 7.7 Hz), 3.68 (s, 3H, N–
CH3), 4.07–4.17 (m, 2H, COOCH2CH2CH2), 4.21 (s, 3H,
123

O. Firuzi et al.
MDR reversal assay
with 80 mM KCl. The contractile response was taken as the
100 % value for the tonic (slow) component of the response.
Test compounds were added in cumulative doses after the
maximum response causedby KCl addition. Test compound-
induced relaxation of contraction was expressed as the per-
cent of the control and IC50 values were determined from the
contraction-response curves (Mehdipour et al. 2007; Fo-
roughinia et al. 2008).
MES-SA-DX5 cells that over-express P-gp were used as a
model of typical MDR, which are developed from their
parental drug sensitive MES-SA (uterine sarcoma) cells.
Cells were seeded in 96 well plates with a density of
3 9 104 cells/ml. Various concentrations of test com-
pounds were added in triplicate followed by different
concentrations of doxorubicin after 1 h. Cells were then
further incubated for 72 h and the viability was measured
with the MTT assay as described above.
Cell lines and cell culture
HeLa (human cervical adenocarcinoma), LS180 (human
colon adenocarcinoma), MCF-7 (human breast adenocar-
cinoma) and Raji (human B lymphoma) cells were
obtained from the National Cell Bank of Iran, Pasteur
Institute, Tehran, Iran. All cell lines were maintained in
RPMI 1640 supplemented with 10 % FBS, and 100 U/ml
penicillin-G and 100 lg/ml streptomycin. MES-SA and
MES-SA-DX5 cells were first grown in Opti-MEM media
and then gradually adapted to RPMI in the course of
4 weeks. Cells were grown in monolayer cultures, except
for Raji cells, which were grown in suspension, at 37 °C in
humidified air containing 5 % CO2.
Results and Discussion
Fourteen novel asymmetrical dihydropyridines (DHPs)
were synthesized and their calcium channel blocking
(CCB), cytotoxic and MDR reversal properties were
examined.
Chemistry
The synthesis of the 1,4-DHP derivatives 7a–7n was
achieved following the steps outlined in Scheme 1 based
on modified method of Dagnino et al. (1986). Reaction of
alcohol 1a–1c with 2,2,6-trimethyl-4-H-1,3-dioxin-4-one 2
afforded the corresponding acetoacetic esters 3a–3c
([90 % yield) (Hyatt et al. 1984). Reaction of acetoacetic
esters 4a–4c with ammonium acetate produced the corre-
sponding alkyl 3-aminocrotonate 5a–5c. The asymmetrical
analogues, 7a–7n, were synthesized by a modified Han-
stzch reaction (*5–30 % yield). These compounds were
purified by preparative thin-layer chromatography and re-
crystallized, and then characterized by mass spectroscopy,
IR and 1H NMR. The yield and melting point of final
compounds are summarized in Table 1.
Cytotoxicity assay
Cell viability following exposure to synthetic compounds
was estimated by using the MTT reduction assay (Mos-
mann 1983; Miri et al. 2011). MCF-7 and Raji cells were
plated in 96-well microplates at a density of 5 9 104 cells/
ml (100 ll per well). LS180 and HeLa cells were plated at
densities of 1 9 105 and 2.5 9 104 cells/ml, respectively.
Control wells contained no drugs and blank wells con-
tained only growth medium for background correction.
After overnight incubation at 37 °C, half of the growth
medium was removed and 50 ll of medium supplemented
with different concentrations of synthetic compounds dis-
solved in DMSO were added in triplicate. Plates with Raji
cells were centrifuged before this procedure. DMSO con-
centration in wells did not exceed 0.5 %. Cells were further
incubated for 72 h, except for HeLa cells, which were
incubated for 96 h. At the end of the incubation time, the
medium was removed and MTT was added to each well at
a final concentration of 0.5 mg/ml and plates were incu-
bated for another 4 h at 37 °C. Then formazan crystals
were solubilized in 200 ll DMSO. The optical density was
measured at 570 nm with background correction at 655 nm
using a Bio-Rad microplate reader (Model 680). The per-
centage of inhibition of viability compared to control wells
was calculated for each concentration of the compound and
IC50 values were calculated with the software CurveExpert
version 1.34 for Windows. Each experiment was repeated
four times. Data are presented as mean SD
Pharmacology
In vitro CCB activities of compounds were determined in
guinea pig ileal longitudinal smooth muscle (GPILSM) and
the molar concentration of the test compound required to
produce 50 % inhibition of GPILSM (IC50) was determined
(Table 1). This model has been used by several investigators
for measurement of CCB activity (Iman et al. 2011).
Synthesized compounds 7a–7n exhibited weak to mod-
erate CCB activity (IC50 range: 134.0 nM–4.8 lM) relative
to the reference drug nifedipine (IC50: 68.9 nM), except for
compound 7b, which was about twofolds stronger than
nifedipine (IC50: 29.3 nM). Considering that CCB activity is
an undesired effect as long as MDR reversal drug develop-
ment is concerned, it is supposed that 7b is not a good choice
for further development since its strong CCB activity would
123

Multidrug resistance reversal by dihydropyridines
Most of the DHP compounds demonstrated cytotoxicity
in all 4 different cancer cells, including solid tumor (HeLa,
LS180, MCF-7) and hematopoietic malignancy cells
(Raji). The lowest observed IC50 belonged to the com-
Xylene
R1OH
1a-c
+
2
R1OCCH2CCH3
150° C
pound 7c in Raji (human
B
lymphoma) cells
2
3a-c
(6.4 2.1 lM). Compound 7c had also the lowest IC50
against MCF-7 cell line (16.0 3.4 lM). Comparison of
the effect of DHP compounds on the 4 cell lines used in
this study, showed that they generally had the highest IC50
values on LS180 (human colon adenocarcinoma) cells
(except for compound 7e).
EtOH
80° C
CH3COONH4
R2OCCH2CCH3
4a-c
R2OCHCH
5a-c
CCH3
As for the structure–cytotoxic activity relationships of
studied compounds, 7a–7i that contain nitrophenyl moiety
at C4 position had IC50 values in the range between 6.4 and
64.4 lM against cancer cells. These were much more
active than compounds 7j–7n bearing imidazole ring at C4,
which mostly had IC50 values of higher than 100 lm
against cancer cells. Therefore, it is clear that the sub-
stituent at the C4 position has a great impact on the cyto-
toxic effect of these compounds.
R2OCHCH
5a-c
CCH3
R1OCCH2CCH3
3a-c
Aryl or Heteryl aldehyde
O2N
R3
N
N
Other substitutions at C3 and C5 positions had also some
effects on the cytotoxic activity; however, these effects
were smaller compared to the effect of C4 substitution.
Compounds 7c and 7h have low IC50 values against most
of the cell lines. Both of these compounds have 3-(pyri-
dine-4-yl)propyl at their C3 position and a 4-nitrophenyl
group at their C4 position. Therefore, it seems that the
combination of these 2 moieties confers high cytotoxic
activity to these compounds. On the other hand, compari-
son of the IC50 values of 7g and 7b compounds against
cancer cells shows that the former compound is more
active than the later in all cell lines. Since 7g differs from
7b in having an isopropyl ester instead of a methyl ester at
C5 position, the presence of a longer alkyl ester at C5 seems
to improve the activity. Indeed, all compounds possessing
this moiety at C5 position (7f, 7g and 7h) have low IC50
values against all cancer cell lines.
R1OOC
COOR2
R1OOC
COOR2
N
N
H
H
7a-i
7j-n
Scheme 1 Synthetic pathway of target compounds 7a–7n
certainly limit the dosing for MDR reversal. On the other
hand, from this point of view, compounds with CCB IC50
values in the micromolar range (7a, 7d, 7e, 7g, 7i, 7k) seem
to be good candidates for further development. Comparison
of these asymmetric compounds with previously synthe-
sized symmetric compounds (Mehdipour et al. 2007; Fo-
roughinia et al. 2008) shows that both symmetrical and
asymmetrical compounds have same range of CCB activity
(IC50 values % 10-7 M) and they are at least ten folds
weaker than nifedipine. Therefore, it seems that the presence
of at least one pyridyl group at C3 or C5 is sufficient for
reduction of CCB activity as a side effect in this context. No
evident difference could be observed between the CCB
activity of compounds bearing nitrophenyl moiety (7a–7i
compounds) or imidazole group (7j–7n compounds) at C4
position, suggesting that substitutions at this position do not
considerably alter the CCB activity.
MDR reversal
MDR reversal activity of DHP derivatives was assessed on
MES-SA-DX5 cells that over-express P-gp and are resis-
tant to doxorubicin compared to their non-resistant parental
MES-SA cells (Figs. 1, 2).
Over-expression of P-gp, which pumps the cytotoxic
agents out of the cell, is a well-known cause of MDR in
cancer cells (Chen and Sikic 2012). MES-SA-DX5 cells
over-express P-gp and hence are an established cell model
for typical MDR (Koo et al. 2008; Angelini et al. 2012).
The efficiency of the test compounds in increasing the
cytotoxicity of doxorubicin in MES-SA-DX5 cells was
therefore determined as an index of their MDR reversal
capacity. Most of DHP compounds bearing nitrophenyl
Cytotoxicity
The cytotoxic activities of synthesized compounds were
evaluated on four human cancer cell lines and IC50 values
were calculated (Table 2).
123

O. Firuzi et al.
Table 1 Physical properties and calcium channel antagonist activities of synthetic dihydropyridines
O2N
R3
N
N
R1OOC
COOR2
R1OOC
COOR2
N
N
H
H
7j-n
7a-i
Compound
R1
R2
R3
Mp (°C)
Yield
(%)
IC50 SEM (M)a
n
7a
3-(Pyridine-4-yl)propyl
3-(Pyridine-3-yl)propyl
3-(Pyridine-4-yl)propyl
3-(Pyridine-3-yl)propyl
3-(Pyridine-4-yl)propyl
3-(Pyridine-2-yl)propyl
3-(Pyridine-3-yl)propyl
3-(Pyridine-4-yl)propyl
3-(Pyridine-3-yl)propyl
3-(Pyridine-3-yl)propyl
3-(Pyridine-2-yl)propyl
3-(pyridine-3-yl)propyl
3-(Pyridine-4-yl)propyl
3-(Pyridine-2-yl)propyl
Methyl
Methyl
Methyl
Ethyl
4-Nitro
3-Nitro
4-Nitro
2-Nitro
3-Nitro
3-Nitro
3-Nitro
4-Nitro
3-Nitro
–
152–154
138–140
114–118
178–180
167–170
120–122
114–116
180–182
116–118
158–162
150–152
166–168
174–178
138–140
14.4
5.3
(4.23 1.72) 9 10-6
(2.93 2.28) 9 10-8
(4.57 1.76) 9 10-7
(1.08 0.68) 9 10-6
(2.26 0.89) 9 10-6
(2.32 1.18) 9 10-7
(1.39 0.52) 9 10-6
(1.34 0.32) 9 10-7
(1.30 0.73) 9 10-6
(2.74 1.77) 9 10-7
(4.75 1.62) 9 10-6
(1.61 0.36) 9 10-7
(2.79 1.03) 9 10-7
(3.75 0.24) 9 10-7
(6.89 2.00) 9 10-8
3
4
4
3
4
4
4
4
4
3
4
4
4
3
3
7b
7c
29.4
24.543
24.7
19.7
13.2
29.4
26.5
15.0
9.7
7d
Methyl
Ethyl
7e
7f
Isopropyl
Isopropyl
Isopropyl
Ethyl
7g
7h
7i
7j
Methyl
Methyl
Ethyl
7k
–
7l
–
7.8
7m
Methyl
Isopropyl
Methyl
–
18.7
9.7
7n
–
Nifedipine
2-Nitro
a
Calcium channel antagonist activity was measured in guinea pig ileal longitudinal smooth muscle
Table 2 Cytotoxic activity of
synthetic dihydropyridines on
human cancer cell lines
Compound
IC50 (lM)
Hela
LS180
MCF-7
Raji
7a
13.2 2.6
24.8 6.4
27.2 2.4
26.5 8.5
33.5 9.5
14.8 6.6
20.0 3.5
12.6 1.1
22.4 5.2
[100
26.9 10.0
40.9 7.0
31.8 5.9
41.9 5.4
63.2 14.1
39.1 11.8
26.0 8.2
28.4 9.2
39.1 11.5
[100
24.3 2.9
37.5 3.4
16.0 3.4
30.9 5.8
37.6 10.2
24.3 7.1
20.3 5.5
18.7 3.0
18.1 5.0
[100
24.7 8.7
19.5 11.5
6.4 2.1
18.4 13.8
64.4 34.4
19.3 10.0
18.5 8.3
21.7 6.4
17.8 10.9
[100
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
[100
[100
[100
[100
7l
[100
[100
[100
[100
7m
60.4 21.2
[100
[100
[100
[100
7n
[100
88.9 55.3
0.237 0.182
[100
Values are presented as mean SD
of 3–4 experiments
Doxorubicin
0.511 0.112
0.061 0.011
0.184 0.187
123

Multidrug resistance reversal by dihydropyridines
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
Fig. 1 Effect of newly
synthesized dihydropyridine
derivatives (7a–7n) on
resistance to doxorubicin in
MES-SA-DX5 cells.
7c
7a
7d
7g
7b
*
*
Doxorubicin-resistant cells were
preincubated alone (open circle)
or with DHP compounds [7a–7i
at 0.5 (filled square) and 1 lM
(filled triangle); 7j–7n at 5
(filled diamond) and 10 lM
(filled inverted triangle)] for 1 h
and then exposed to different
concentrations of doxorubicin
for 72 h. Cell viability was
measured with the MTT assay
and expressed as percentage
compared to untreated control
cells. Significant versus
*
100
300
1000
100
300
1000
100
300
1000
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
7e
7f
*
*
*
*
*
100
300
1000
100
300
1000
100
300
1000
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
7i
7h
7k
doxorubicin alone (in the
absence of test compound) at
*p \ 0.05
*
*
*
*
*
1000
*
100
300
1000
100
300
1000
100
300
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
7l
7j
*
*
*
*
*
1000
100
300
1000
100
300
1000
100
300
Concentration of doxorubicin (nM)
100
80
60
40
20
0
100
80
60
40
20
0
7n
7m
*
*
*
100
300
1000
100
300
1000
Concentration of doxorubicin (nM)
Concentration of doxorubicin (nM)
moiety at C4 position (including 7a, 7c, 7e, 7g, 7h and 7i)
were able to reverse MDR in resistant cells at the con-
centrations of 0.5 and/or 1 lM, while the same concen-
trations were ineffective on non-resistant cells. The lack of
effect in non-resistant cells suggests that the mechanism of
action of these compounds is probably through interference
with P-gp.
of 1 lM, while compounds 7b and 7d were not effective
either on resistant or non-resistant cells. Concentrations
higher than 1 lM of compounds 7a–7i induced direct
cytotoxicity on cell lines (data not shown), therefore these
concentrations were not tested for MDR reversal, as the
direct cytotoxic effect of the test compound would have
been confounded with the MDR reversal effect.
The most effective compounds were 7e and 7g, which at
the concentration of 1 lM significantly increased the effect
of 300 nM doxorubicin and also at the concentrations of
0.5 and 1 lM augmented the effect of 1 lM doxorubicin.
In the order of efficiency, these derivatives were followed
by compounds 7c, 7f and 7h, which significantly reversed
the resistance to 1 lM doxorubicin at the concentrations of
0.5 and 1 lM. Compounds 7i and 7a reversed the resis-
tance to 1 lM doxorubicin only at the higher concentration
On the other hand, compounds possessing nitroimidaz-
ole at C4 position (7j–7n) were not effective on resistant
cells at 0.5 and 1 lM concentrations, however some of
them were selectively effective on MES-SA-DX5 cells at 5
and 10 lM. Compound 7n appeared to be the most effec-
tive DHP in this subgroup, since at the concentration of
5 lM it was able to significantly reverse the effect of 1 lM
doxorubicin. Compounds 7j and 7l at the concentration of
10 lM were able to significantly reverse the effect of
123

O. Firuzi et al.
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
Fig. 2 Effect of newly
synthesized dihydropyridine
derivatives (7a–7n) on
7b
7c
7a
resistance to doxorubicin in
MES-SA cells. Nonresistant
cells were incubated alone
(open circle) or with DHP
compounds [7a–7i at 0.5 (filled
square) and 1 lM (filled
triangle); 7j–7n at 5 (filled
diamond) and 10 lM (filled
inverted triangle) for 1 h and
then exposed to different
concentrations of doxorubicin
for 72 h. Cell viability was
measured with the MTT assay
and expressed as percentage
compared to untreated control
cells. Significant versus
10
30
10
30
10
30
100
80
60
40
20
0
7e
100
80
60
40
20
0
100
80
60
40
20
0
7f
7d
7g
*
10
30
10
30
10
30
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
7h
7i
doxorubicin alone (in the
absence of test compound) at
*p \ 0.05
10
30
10
30
10
30
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
7j
7l
7k
*
*
10
30
10
30
10
30
Concentration of doxorubicin (nM)
100
80
60
40
20
0
100
80
60
40
20
0
7m
7n
10
30
10
30
Concentration of doxorubicin (nM)
Concentration of doxorubicin (nM)
300 nM and 1 lM doxorubicin. Compound 7k was also
able to selectively reverse resistance to 1 lM doxorubicin
at the concentration of 10 lM. None of these compounds
had any direct cytotoxic effect on cell lines at the tested
concentrations (data not shown).
activity, with the latest being considered a side effect in
this case. Based on the obtained results, it appears that
compounds 7e and 7g possess high MDR reversal and
cytotoxic activities and at the same time low CCB prop-
erties. These characteristics make these compounds very
good candidates for MDR reversal and antitumoral drug
development.
In conclusion, MDR is one of the main mechanisms that
limits the efficacy of chemotherapeutic agents in many
types of cancer, therefore MDR reversal agents could be of
great help in management of drug resistant cancer. 1,4-
DHPs have been in clinical use as CCB agents for a long
time, but they are also able to reverse MDR in cancer cells.
In this project, in continuation of our previous studies, we
synthesized novel asymmetrical derivatives of DHP and
measured their cytotoxic, MDR reversing and also CCB
Acknowledgments Financial support of the Shiraz University of
Medical Sciences, vice-chancellor of research is acknowledged. The
cytotoxicity section of this project was a part of Pharm. D thesis of E.
Mansourabadi (Thesis Number: 401). Also, the financial support from
the Italian Ministry for Education, University and Research, General
Management for the internationalization of scientific research is
gratefully acknowledged. The authors also wish to thank Dr. Sandra
123

Multidrug resistance reversal by dihydropyridines
Incerpi, University of Rome Tor Vergata, Italy, for her kind help in
setting up MES-SA and MES-SA-DX5 cells experiments.
Koo, J.-S., W.-C. Choi, Y.-H. Rhee, H.-J. Lee, E.-O. Lee, K.S. Ahn,
H.-S. Bae, K.-S. Ahn, J.-M. Kang, S.-U. Choi, M.O. Kim, J. Lu,
and S.-H. Kim. 2008. Quinoline derivative KB3-1 potentiates
paclitaxel induced cytotoxicity and cycle arrest via multidrug
resistance reversal in MES-SA/DX5 cancer cells. Life Sciences
83: 700–708.
Conflict of interest Authors declare that they have no conflict of
interest.
Mehdipour, A.R., K. Javidnia, B. Hemmateenejad, Z. Amirghofran,
and R. Miri. 2007. Dihydropyridine derivatives to overcome
atypical multidrug resistance: Design, synthesis, QSAR studies,
and evaluation of their cytotoxic and pharmacological activities.
Chemical Biology & Drug Design 70: 337–346.
References
Angelini, A., R. Di Pietro, L. Centurione, M.L. Castellani, P. Conti, E.
Porreca, F. Cuccurullo, and C. Di Ilio. 2012. Inhibition of
p-glycoprotein-mediated transport by s-adenosylmethionine and
cynarin in multidrug-resistant human uterine sarcoma MES-SA/
DX5 cells. Journal of Biological Regulators and Homeostatic
Agents 26: 495–504.
Miri, R., and A. Mehdipour. 2008. Dihydropyridines and atypical
MDR: A novel perspective of designing general reversal agents
for both typical and atypical MDR. Bioorganic & Medicinal
Chemistry 16: 8329–8334.
Miri, R., R. Motamedi, M.R. Rezaei, O. Firuzi, A. Javidnia, and A.
Shafiee. 2011. Design, synthesis and evaluation of cytotoxicity
of novel chromeno[4,3-b]quinoline derivatives. Archiv der
Pharmazie 344: 111–118.
Borst, P., J. Jonkers, and S. Rottenberg. 2007. What makes tumors
multidrug resistant? Cell Cycle 6: 2782.
Chen, K.G., and B.I. Sikic. 2012. Molecular pathways: Regulation
and therapeutic implications of multidrug resistance. Clinical
Cancer Research 18: 1863–1869.
Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and
survival: Application to proliferation and cytotoxicity assays.
Journal of Immunological Methods 65: 55–63.
Dagnino, L., M.C. Li-Kwong-Ken, M.W. Wolowyk, H. Wynn, C.R.
Triggle, and E.E. Knaus. 1986. Synthesis and calcium channel
antagonist activity of dialkyl 1,4-dihydro-2,6-dimethyl-4-(pyrid-
inyl)-3,5-pyridinedicarboxylates. Journal of Medicinal Chemis-
try 29: 2524–2529.
Edraki, N., A.R. Mehdipour, M. Khoshneviszadeh, and R. Miri. 2009.
Dihydropyridines: Evaluation of their current and future phar-
macological applications. Drug Discovery Today 14:
1058–1066.
Eid, S.Y., M.Z. El-Readi, and M. Wink. 2012. Carotenoids reverse
multidrug resistance in cancer cells by interfering with ABC-
transporters. Phytomedicine 19: 977–987.
Ejendal, K.F., and C.A. Hrycyna. 2002. Multidrug resistance and
cancer: The role of the human ABC transporter ABCG2. Current
Protein and Peptide Science 3: 503–511.
Foroughinia, F., K. Javidnia, Z. Amirghofran, A. Mehdipour, and R.
Miri. 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. Journal of Pharmacy and Pharma-
cology 60: 1481–1489.
Hyatt, J.A., P.L. Feldman, and R.J. Clemens. 1984. Ketenes. 20.
Thermal decomposition of 2,2,6-trimethyl-4H-1,3-dioxin-4-one
and 1-ethoxybutyn-3-one. Acetylketene. Journal of Organic
Chemistry 49: 5105–5108.
Iman, M., A. Davood, A.R. Nematollahi, A.R. Dehpoor, and A.
Shafiee. 2011. Design and synthesis of new 1,4-dihydropyridines
containing 4(5)-chloro-5(4)-imidazolyl substituent as a novel
calcium channel blocker. Archives of Pharmacal Research 34:
1417–1426.
Kasper, D.L., E. Braunwald, A.S. Fauci, S.L. Hauser, D.L. Longo,
J.L. Jameson, and K.J. Isselbacher. 2005. Harrison’s principles
of internal medicine. New York: McGraw-Hill.
Stavrovskaya, A.A., and T.P. Stromskaya. 2008. Transport proteins of
the ABC family and multidrug resistance of tumor cells.
Biochemistry 73: 592–604.
Szakacs, G., J.K. Paterson, J.A. Ludwig, C. Booth-Genthe, and M.M.
Gottesman. 2006. Targeting multidrug resistance in cancer.
Nature Reviews Drug Discovery 5: 219–234.
Tasaka, S., H. Ohmori, N. Gomi, M. Iino, T. Machida, A. Kiue, S.
Naito, and M. Kuwano. 2001. Synthesis and structure–activity
analysis of novel dihydropyridine derivatives to overcome
multidrug resistance. Bioorganic & Medicinal Chemistry Letters
11: 275–277.
Teodori, E., S. Dei, S. Scapecchi, and F. Gualtieri. 2002. The
medicinal chemistry of multidrug resistance (MDR) reversing
drugs. Farmaco 57: 385–415.
Tsuruo, T., H. Iida, S. Tsukagoshi, and Y. Sakurai. 1981. Overcoming
of vincristine resistance in P388 leukemia in vivo and in vitro
through enhanced cytotoxicity of vincristine and vinblastine by
verapamil. Cancer Research 41: 1967–1972.
Tsuruo, T., H. Iida, S. Tsukagoshi, and Y. Sakurai. 1982. Increased
accumulation of vincristine and adriamycin in drug-resistant
P388 tumor cells following incubation with calcium antagonists
and calmodulin inhibitors. Cancer Research 42: 4730–4733.
Zarrin, A., A.R. Mehdipour, and R. Miri. 2010. Dihydropyridines and
multidrug resistance: Previous attempts, present state, and future
trends. Chemical Biology & Drug Design 76: 369–381.
Zhou, X., R.A. Coburn, and M.E. Morris. 2005a. Effects of new
4-aryl-1,4-dihydropyridines and 4-arylpyridines on drug efflux
mediated by multidrug resistance-associated protein 1. Journal
of Pharmaceutical Sciences 94: 2256–2265.
Zhou, X.F., X. Yang, Q. Wang, R.A. Coburn, and M.E. Morris.
2005b. Effects of dihydropyridines and pyridines on multidrug
resistance mediated by breast cancer resistance protein: In vitro
and in vivo studies. Drug Metabolism and Disposition 33:
1220–1228.
King, R.J.B., and M.W. Robins. 2006. Cancer biology. New York:
Prentice Hall.
123