Synthesis, anticancer and MRP1 inhibitory activities of 4-alkyl/aryl-3,5-bis(carboethoxy/carbomethoxy)-1,4-dihydro-2,6-dimethylpyridines

Article abstract of DOI:10.1007/s00044-012-9994-0

Fourteen new 4-alkyl/aryl-3,5-bis-(carboethoxy/carbomethoxy)-1,4-dihydro-2, 6-dimethylpyridines (4a-4n) have been prepared by conventional and microwave irradiation (MWI) methods from a three component reaction mixture viz., alkyl acetoacetate (1), appropriate aldehyde (2) and ammonium acetate (3). The compounds prepared have been purified and characterized by their spectral (FTIR, ¹H NMR and MS) data. The two synthetic methods employed have been compared in terms of relative yields and reaction times. On comparison, the MWI method has been found to be easy, simple, eco-friendly, rapid and high yielding. The synthesized compounds have been evaluated for their cytotoxic activity against HT-29 (colon cancer) and MDA-MB (breast cancer) cell lines and MRP1 inhibitory activity using the insect cell membrane MRP 1 ATPase assay. Though some of the compounds could exhibit some degree of cytotoxicity it was found to be low in comparison to standard. Among the compounds tested 4g was relatively better in its MRP1 inhibitory action (IC₅₀ = 16 μM) but not comparable to that of benzbromarone (IC₅₀ = 4 μM).

Full text of DOI:10.1007/s00044-012-9994-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-9994-0
ORIGINAL RESEARCH
Synthesis, anticancer and MRP1 inhibitory activities
of 4-alkyl/aryl-3,5-bis(carboethoxy/carbomethoxy)-1,4-dihydro-2,
6-dimethylpyridines
•
Srinivas Nayak Amgoth Mahendar Porika
•
Sadanandam Abbagani Achaiah Garlapati
Malla Reddy Vanga
•
•
Received: 8 August 2011 / Accepted: 11 February 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Fourteen new 4-alkyl/aryl-3,5-bis-(carboethoxy/
carbomethoxy)-1,4-dihydro-2,6-dimethylpyridines (4a–4n)
have been prepared by conventional and microwave irradia-
tion(MWI) methods froma three component reactionmixture
viz., alkyl acetoacetate (1), appropriate aldehyde (2) and
ammonium acetate (3). The compounds prepared have been
purified and characterized by their spectral (FTIR, 1H NMR
andMS)data.Thetwosyntheticmethodsemployedhavebeen
compared in terms of relative yields and reaction times. On
comparison, the MWI method has been found to be easy,
simple, eco-friendly, rapid and high yielding. The synthesized
compounds have been evaluated for their cytotoxic activity
against HT-29 (colon cancer) and MDA-MB (breast cancer)
cell lines and MRP1 inhibitory activity using the insect cell
membrane MRP 1 ATPase assay. Though some of the com-
pounds could exhibit some degree of cytotoxicity it was found
to be low in comparison to standard. Among the compounds
tested 4g was relatively better in its MRP1 inhibitory action
(IC50 = 16 lM)butnotcomparabletothatofbenzbromarone
(IC50 = 4 lM).
Keywords 1,4-Dihydropyridines Á
Microwave irradiation Á Cytotoxicity Á
Multidrug resistance-associated protein Á
Thin layer chromatography
Introduction
Multidrug resistance associated protein 1 (MRP1) is a
membrane-bound, 1531 amino acid containing (190-kDa)
energy-dependent efflux transporter belonging to the super
family of ATP-binding cassette (ABC) transporters
involved in cancer cell multidrug resistance (MDR phe-
notype). It is a membrane-bound, energy-dependent efflux
pump which is structurally and functionally related to the
MDR protein, P-glycoprotein (P-gp/ABCB1) (Teodori
et al., 2006). MRP1 is over expressed in the membranes of
cancer cells and effectively extrudes various cytotoxic
agents out of tumor cells, leading to a decrease in cellular
drug concentrations. It is widely expressed in all tissues
except in the human liver, it plays a significant role in
tissue defense from toxic agents (Okamura et al., 2009;
Leslie et al., 2005) in combination with other ABC trans-
porters. Like P-gp, MRP1 confers resistance on cancer cells
by using the energy of ATP binding and hydrolysis to
efflux anticancer drugs.
S. N. Amgoth Á A. Garlapati (&)
University College of Pharmaceutical Sciences, Kakatiya
University, Warangal 506009, Andhra Pradesh, India
e-mail: achaiah_g@yahoo.co.in
MDR is a tremendous problem in the treatment of many
types of cancer; the patients who overexpress multidrug
resistance proteins such as P-gp and MRP1 in their tumors
are usually not responsive to the treatment associated with
anticancer agents (Bellamy and Dalton, 1994). Thus, many
of these patients progress to an advanced stage of disease
and have poorer prognoses. It has been hypothesized that
inhibition of MDR transporters can restore sensitivity to
some oncolytics, allowing patients with drug-resistant
M. R. Vanga (&)
R &D Centre, Divis Laboratories Ltd., B-34, Industrial Estate,
Sanath Nagar, Hyderabad 500 018, Andhra Pradesh, India
e-mail: vmr@divislaboratories.com;
vangamallareddy@yahoo.co.in
M. Porika Á S. Abbagani
Department of Biotechnology, Kakatiya University,
Warangal 506009, Andhra Pradesh, India
123

Med Chem Res
tumors to become responsive to their drug therapy
(Avendano and Menendez, 2002). This hypothesis is
presently being tested in the clinic for P-gp (Coley, 2010).
However, the search for MRP1 inhibitors (or modulators/
chemosensitizers) to overcome MRP1/ABCC1-mediated
MDR began just a few years ago and few potent and
selective modulators have been investigated (Boumendjel
et al., 2005; Yang et al., 2010).
and oncologists to investigate further in view of the
growing resistance to chemotherapy by cancer cells.
A series of 4-phenyl-3,5-dibenzoyl-1,4-dihydropyri-
dines (Fig. 1, Structure 1) differently substituted at the
4-phenyl ring have been reported to show a varied cyto-
toxicity against two human oral tumor cell lines (HSC-2
and HSG) (Kawase et al., 2002). Among such compounds,
those substituted with 2-CF3 (IC50 = 8.7 lM) and 2-Cl
(IC50 = 7.0 lM) were reported to show the highest cyto-
toxic activity against HSC-2. On the other hand, cytotoxic
activity of 2-CF3 analog (IC50 = 8.7 lM) against HSG was
reported higher than 2-Cl analog (IC50 = 28 lM). These
data suggested that-3,5-dibenzoyl-1,4-dihydropyridines
can be considered as leads for the development of new drug
for cancer treatment.
1,4-Dihydropyridines (DHPs), a class of calcium chan-
nel blockers are used as therapeutic agents to treat angina
and hypertension (Bernard et al., 1974). Several of them
are also reported to exhibit a variety of biological and
pharmacological activities, viz., bronchodilatory (Suresh
et al., 2007), vasodilatory (Iwanami et al., 1979), hepato-
protective (Isaac et al., 1998), neuroprotectant (Klusa,
1995), platelet aggregation inhibitory (Carlos et al., 1988)
and cerebral anti-ischemic (Sircar et al., 1991). They are
also reported to possess potential anticancer properties
(Boer and Gekeler, 1995). Shortly after recognizing the
chemosensitizing properties of a calcium antagonist
verapamil, lead to a thinking allowed by finding that the
other calcium antagonists, the DHP derivatives were also
potent chemosensitizing compounds in vitro (Avendano
and Menendez, 2002). Hence, this field has attracted the
attention of several medicinal chemists, pharmacologists
Shigeyuki et al., (2001) reported the structure–activity
relationships of some newly synthesized 1,4-dihydropyri-
dine derivatives (Fig. 1, Structure 2). The compound having
a 1-pentyl group at the 4-position and 3-pyridylpropyl ester
groups at 3- and 5-positions was found to be effective in
overcoming P-glycoprotein-mediated multidrug resistance
(MDR) in cultured human cancer cells, in vitro.
Another member of dihydropyridine derivative B-859-
35 (Fig. 1, Structure 3) was reported to show a selective
carcinostatic effect on some tumors (Johann et al., 1991).
Fig. 1 Some of the 1,4-
dihydropyridine molecules as
anticancer agents
R
O
O
C6H5
C6H5
H3C
N
H
CH3
O
O
1
O
O
N
H3C
N
H
CH3
N
2
NO2
O
O
H3C
O
O
N
H3C
N
H
CH3
HCl
B-859-35
123

Med Chem Res
In order to evaluate whether the anticancer activity of
B-859-35 can be modulated, the new molecule was com-
bined with several established antitumor drugs. A combi-
nation of B-859-35 with VP-16 (etoposide) in MDR
expressing Walker rat carcinoma cells has been reported to
exhibits synergetic effect. A combination of B-859-35 with
doxorubicin was shown to result in stronger synergism than
verapamil/doxorubicin, especially at low concentrations of
B-859-35 (Johann et al., 1991). These findings indicated
that an antitumor agent B-859-35 reversed multidrug
resistance and thus this molecule could emerge as an
interesting drug for cancer treatment. Figure 1 has included
some more dihydropyridines as anticancer and MRP1
inhibitory agents.
their spectral and analytical properties and evaluated them
for their possible anticancer activity against human cancer
cell lines (HT-29 and MDA-MB) and MRP1 inhibitory
activity by standard experimental methods and the results
are presented in this communication.
Materials and methods
Chemistry
Fourteen new 4-alkyl/aryl-3,5-bis-(carboethoxy/carbomethoxy)-
1,4-dihydro-2,6-dimethylpyridines (4a–4n, Scheme 1) have
been synthesized by a modified Hantzsch one-pot synthesis
involving the three-component condensation reaction of
alkyl acetoacetate (1; 2 M) with appropriate aldehyde (2;
1 M) and ammonium acetate (3; 3 M) by conventional as
well as microwave-induced methods (Anniyappan and
Perumal, 2002). Completion of the reaction was monitored
by TLC. The title compounds 4a–4n were obtained in good
to excellent yields (62–90%). But however MWI method,
in comparison to conventional heating was simple,
easy, eco-friendly and the reactions were rapid and high
yielding.
Therefore, in view of growing incidents of different
cancer cells resistance to various potent anticancer agents
and DHPs exhibiting an ability to chemosensitize such
cells it has been thought worthwhile to synthesize some
new dihydropyridine derivatives bearing different alkyl
and aryl groups at 4-position and methyl/ethyl ester groups
at the 3- and 5-positions of the DHP system and evaluate
them for their possible anticancer as well as MRP1 inhib-
itory activities. The proposed compounds are synthesized
by two different synthetic methods and characterized by
Scheme 1 Synthesis of 4-alkyl/
aryl-3,5-bis (carboethoxy/
carbomethoxy)-1,4-dihydro-2,6-
dimethylpyridines
O
O
CH3COO-NH+
+
R-CHO
+
OR1
4
H3C
(2)
(3)
(1)
Method A: MeOH/
12-18hr
Method B: DMF/MWI
480W,1-6min
O
R
O
R1O
OR1
H3C
N
H
CH3
R= -n-C3H7, -n-C4H9,C H5,
R1 =CH 3,C H5
6
2
(4)
C6H4.NO2-3,C 6H4.CH3-4
C6H4.OCH3-4,C H4.Cl-4
6
123

Med Chem Res
Experimental procedures are given as general methods.
All M.P. were determined in open capillaries using
Toshnwal melting point apparatus. Infra red spectra of the
compounds were recorded in KBr pellet using Shimdzu
FTIR-8700 spectrophotometer, 1HNMR spectra were
recorded on Avance-300 MHz spectrophotometer using
TMS as an internal standard and Mass spectra by direct
inlet method on VG Micro Mass 7070 H spectrophotometer
operating at 70 eV. Elemental analyses of the compounds
were carried out Elementar instrument.
122–124°C; IR (KBr) cm-1: 3351 (–NH), 2956 (C–H,
aliphatic), 1644 (C=O, CO2 Et); 1HNMR (CDCl3): d (ppm)
0.85 (t, 3H, –CH2CH2CH3 at C4), 1.30 (m, 8H,
–CO2CH2CH3 at C3 & C5 and CH2CH2CH3 at C4), 2.32 (s,
6H, –CH3 at C2 & C6 of DHP), 3.94–4.22 (m, 6H,
–CH2CH2CH3 at C4 & –CO2CH2CH3 at C3, C5), 5.62 (s,
1H, –NH), 7.26 (s, 1H, –CH at C4); MS (m/z): 296 (M?);
Elemental analyses: Calcd. For C16H25N1O4: C, 65.06; H,
8.53; N, 4.74. Found C, 65.01; H, 8.42; N, 4.64.
(b) 4-n-Butyl-3,5-bis (carboethoxy)-1,4-dihydro-2,6-dimethyl-
pyridine (4b; R = -n-C4H9, R1 = C2H5) Yield: conven-
tional method 39%, microwave method 72%; m.p.
92–94°C; IR (KBr) cm-1: 3344 (–NH), 2932 (C–H, ali-
General procedure for the synthesis of 4-alkyl/aryl-
3,5-bis-(carboethoxy/carbomethoxy)-1,4-dihydro-2,
6-dimethylpyridines (4a–4n)
1
phatic), 1651 (C=O, CO2 Et); HNMR (CDCl3): d (ppm)
Conventional method
0.82 (t, 3H, –CH2CH2CH2CH3 at C4), 1.30 (m, 10H,
–CO2CH2CH3 at C3, C5 & –CH2CH2CH2CH3 at C4), 2.38
(s, 6H of –CH3 at C2 & C6), 4.22 (m, 6H, –CH2CH2
CH2CH3 at C4 & –CO2CH2CH3 at C3, C5), 5.52 (s, 1H,
–NH), 7.28 (s, 1H, –CH at C4); MS (m/z): 310 (M?);
Elemental analyses: Calcd. for C17H27N1O4: C, 65.99; H,
8.80; N, 4.53. Found C, 65.84; H, 8.72; N, 4.41.
Ethyl/methyl acetoacetate (1; 0.05 M) and an appropriate
aldehyde (2; 0.025 M) were taken into a RB flask and dis-
solved in ethanol (25 cm3) by shaking. Ammonium acetate
(3; 0.025 M) was added, stirred and the reaction mixture was
heated under reflux for 12–18 h, on a hot water-bath. After
completion of the reaction (monitored by TLC), ethanol was
removed to a possible extent by distillation and the residue
was cooled, triturated with crushed ice (*100 g). The
product was filtered, washed with small portions of cold
water (3 9 10 cm3) and dried. The isolated product was
purified by recrystallization from 90% aqueous ethanol.
(c) 4-Phenyl-3,5-bis (carboethoxy)-1,4-dihydro-2,6-dimeth-
ylpyridine (4c;R = C6H5,R1 = C2H5) Yield: conventional
method 52%, microwave method 93%; m.p. 150–152°C; IR
(KBr) cm-1: 3340 (–NH), 3068 (C–H, aromatic), 2985 (C–
H, aliphatic), 1659 (C=O, CO2Et), 1591 (C=C, aromatic);
1HNMR (CDCl3): d (ppm) 1.24 (t, 6H, –CO2CH2CH3 at C3 &
C5), 2.26(s, 6H, –CH3 atC2 & C6), 4.10(q, 4H, –CO2CH2CH3
at C3 & C5), 4.96 (s, 1H, –NH), 5.62 (s, 1H, –CH at C4), and
7.10 to 7.32 (m, 5H, Ar–H at C4); MS (m/z) 328 (M?); Ele-
mentalanalyses: Calcd. for C19H23N1O4: C, 69.28; H, 7.04; N,
4.25. Found C, 69.16; H, 6.98; N, 4.18.
Microwave irradiation method
Ethyl/methyl acetoacetate (1; 0.05 M) and an appropriate
aliphatic aldehyde or aromatic aldehyde (2; 0.025 M) were
taken into a beaker and dissolved in minimum quantity of
dimethylformamide (10 cm3). To this solution, ammonium
acetate (3; 0.025 M) was added while stirring. A funnel
was inversely hanged over the beaker and the reaction
mixture was subjected to microwave irradiation at 480 W
for 2–6 min, with a pulse rate of 60 s each in a microwave
oven. Solvent was removed by distillation under reduced
pressure after completion of the reaction (TLC). The resi-
due was cooled and triturated with crushed ice (*100 g).
The resultant product was filtered, washed with small
portions of cold water (3 9 10 cm3), dried and purified by
recrystallization from 90% aqueous ethanol.
(d) 4-(3-Nitrophenyl)-3,5-bis(carboethoxy)-1,4-dihydro-2,
6-dimethylpyridine (4d; R = C6H4ÁNO2-3, R1 = C2H5)
Yield: conventional method 54%, microwave method 92%;
m.p. 160–162°C; IR (KBr) cm-1: 3345 (–NH), 3080 (C–H,
aromatic), 2991 (C–H, aliphatic), 1645 (C=O, CO2Et),
1
1525 (C=C, aromatic); HNMR (CDCl3): d (ppm) 1.29 (t,
6H, –CO2CH2CH3 at C3 & C5), 2.28 (s, 6H, –CH3 at C2 &
C6), 4.20 (q, 4H, –CO2CH2CH3 at C3 & C5), 4.98 (s, 1H,
–NH), 5.87 (s, 1H, –CH at C4), and 7.62 to 8.12 (m, 5H,
Ar–H at C4); MS (m/z) 374 (M?); Elemental analyses:
Calcd. for C19H22N2O6: C, 60.95; H, 5.92; N, 7.48. Found
C, 60.76; H, 5.93; N, 7.39.
Adopting the above two procedures the following
fourteen different 4-alkyl/aryl-3,5-bis-(carboethoxy/carbo-
methoxy)-1,4-dihydro-2,6-dimethylpyridines (4a–4n) were
prepared and characterized:
(e)
4-(4-Methoxyphenyl)-3,5-bis(carboethoxy)-1,4-dihy-
dro-2,6-dimethylpyridine (4e; R = C6H4ÁOCH3-4, R1 =
C2H5) Yield: conventional method 41%, microwave
method 86%; m.p. 148–150°C; IR (KBr) cm-1: 3342
(a) 4-n-Propyl-3,5-bis (carboethoxy)-1,4-dihydro-2,
6-dimethylpyridine (4a; R = -n-C3H7, R1 = C2H5) Yield:
conventional method 47%, microwave method 90%; m.p.
123

Med Chem Res
(–NH), 3064 (C–H, aromatic), 2984 (C–H, aliphatic), 1650
(C=O, CO2Et), 1491 (C=C, aromatic); 1HNMR (CDCl3): d
(ppm) 1.25 (t, 6H, –CO2CH2CH3 at C3 & C5), 3.83 (s, 3H,
–CH3 at C4), 2.26 (s, 6H, –CH3 at C2 & C6), 4.30 (q, 4H,
–CO2CH2CH3 at C3 & C5), 5.46 (s, 1H, –NH), 5.82 (s, 1H,
–CH at C4), and 6.87 to 7.12 (m, 4H, Ar–H at C4); MS
(m/z) 359 (M?); Elemental analyses: Calcd. for
C20H25N1O5: C, 66.83; H, 7.01; N, 3.90. Found C, 66.68;
H, 6.92; N, 3.84.
92–94°C; IR (KBr) cm-1: 3334 (–NH), 2927 (C–H,
aliphatic), 1652 (C=O, CO2Et), 1490 (C=C, aromatic);
1HNMR (CDCl3): d (ppm) 0.90 (t, 3H, –CH2CH2CH3 at
C4), 3.74 (s, 6H, –CO2CH3 at C3 & C5), 2.28 (s, 6H, –CH3
at C2 & C6), 1.10 (t, 3H, –CH2CH2CH2CH3 at C4), 1.28–
1.34 (q, 4H, –CH2CH2CH2CH3 at C4), 5.58 (s, 1H, –NH),
6.98 (s, 1H, –CH at C4); MS (m/z): 281 (M?); Elemental
analyses: Calcd. for C15H23N1O4: C, 64.03; H, 8.24; N,
4.98. Found C, 63.98; H, 8.17; N, 4.84.
(f) 4-(4-Methylphenyl)-3,5-bis(carboethoxy)-1,4-dihydro-
2,6-dimethylpyridine (4f; R = C6H4ÁCH3-4, R1 = C2H5)
Yield: conventional method 53%, microwave method 89%;
m.p. 140–142°C; IR (KBr) cm-1: 3360 (–NH), 3076 (C–H,
aromatic), 2987 (C–H, aliphatic), 1697 (C=O, CO2Et),
(j) 4-Phenyl-3,5-bis (carbomethoxy)-1,4-dihydro-2,
6-dimethylpyridine (4j; R = C6H5, R1 = CH3) Yield:
conventional method 61%, microwave method 78%; m.p.
170–172°C; IR (KBr) cm-1: 3346 (–NH), 3062 (C–H,
aromatic), 2951 (C–H, aliphatic), 1698 (C=O, CO2Et),
1490 (C=C, aromatic); 1HNMR (CDCl3): d (ppm) 2.28
(s, 6H, –CH3 at C2 & C6), 3.55 (s, 6H, –CO2CH3 at C3 &
C5), 4.98 (s, 1H, –NH), 5.42 (s, 1H, –CH at C4), and 7.17 to
7.37 (m, 4H, Ar–H at C4); MS (m/z): 301 (M?); Elemental
analyses: Calcd. for C17H19N1O4: C, 67.76; H, 6.36; N,
4.65. Found C, 67.68; H, 6.25; N, 4.52.
1
1487 (C=C, aromatic); HNMR (CDCl3): d (ppm) 1.54 (t,
6H, –CO2CH2CH3 at C3 & C5), 2.34 (s, 3H, –CH3 at C4),
2.90 (s, 6H, –CH3 at C2 & C6), 4.50 (q, 4H, –CO2CH2CH3
at C3 & C5), 5.06 (s, 1H, –NH), 5.92 (s, 1H, –CH at C4),
and 7.20 to 7.42 (m, 4H, Ar–H at C4); MS (m/z) 343 (M?);
Elemental analyses: Calcd. for C20H25N1O4: C, 69.95; H,
7.34; N, 4.08. Found C, 69.86; H, 7.23; N, 4.02.
(k) 4-(3-Nitrophenyl)-3,5-bis(carbomethoxy)-1,4-dihydro-
2,6-dimethylpyridine (4k; R = C6H4ÁNO2-3, R1 = CH3)
Yield: conventional method 63%, microwave method 89%;
m.p. 140–142°C; IR (KBr) cm-1: 3357 (–NH), 3062 (C–H,
aromatic), 2954 (C–H, aliphatic), 1652 (C=O, CO2Et),
(g) 4-(4-Chlorophenyl)-3,5-bis (carboethoxy)-1,4-dihydro-
2,6-dimethylpyridine (4g; R = C6H4ÁCl-4, R1 = C2H5)
Yield: conventional method 59%, microwave method 85%;
m.p. 144–146°C; IR (KBr) cm-1: 3343 (–NH), 3046 (C–H,
aromatic), 2982 (C–H, aliphatic), 1651 (C=O, CO2Et),
1591 (C=C, aromatic) and 1092 (C–Cl); 1HNMR (CDCl3):
d (ppm) 1.30 (t, 6H, –CO2CH2CH3 at C3 & C5), 2.34 (s,
6H, –CH3 at C2 & C6), 4.10 (q, 4H, –CO2CH2CH3 at C3 &
C5), 4.94 (s, 1H, –NH), 5.70 (s, 1H, –CH at C4), and 7.10 to
7.36 (m, 4H, Ar–H at C4); MS (m/z): 363 (M?); Elemental
analyses: Calcd. for C19H22N1O4Cl: C, 62.72; H, 6.09; N,
3.85. Found C, 62.68; H, 6.02; N, 3.74.
1
1485 (C=C, aromatic); HNMR (CDCl3): d (ppm) 2.30 (s,
6H, –CH3 at C2 & C6), 3.15 (s, 6H, –CO2CH3 at C3 & C5),
4.96 (s, 1H, –NH), 5.30 (s, 1H, –CH at C4), and 7.10 to
7.36 (m, 4H, Ar–H at C4); MS (m/z): 347 (M?); Elemental
analyses: Calcd. for C17H18N2O6: C, 58.96; H, 5.24; N,
8.09. Found C, 58.82; H, 5.18; N, 7.98.
(l) 4-(4-Methoxyphenyl)-3,5-bis(carbomethoxy)-1,4-dihydro-
2,6-dimethylpyridine (4l; R = C6H4ÁOCH3-4, R1 = CH3)
Yield: conventional method 58%, microwave method 84%;
m.p. 166–168°C; IR (KBr) cm-1: 3366 (–NH), 2951 (C–H,
aliphatic), 1682 (C=O, CO2Et), 1487 (C=C, aromatic);
1HNMR (CDCl3): d (ppm) 2.32 (s, 6H, –CH3 at C2 & C6),
3.24 (s, 6H, –CO2CH3 at C3 & C5), 3.83 (s, 3H,
–OCH3 at C4), 5.10 (s, 1H, –NH), 5.84 (s, 1H, –CH at C4),
and 6.87 to 7.12 (m, 4H, Ar–H at C4); MS (m/z): 331 (M?);
Elemental analyses: Calcd. for C18H21N1O5: C, 65.24; H,
6.39; N, 4.23. Found C, 65.18; H, 6.25; N, 4.18.
(h) 4-n-Propyl-3,5-bis (carbomethoxy)-1,4-dihydro-2,
6-dimethylpyridine (4h; R = -n-C3H7, R1 = CH3) Yield:
conventional method 53%, microwave method 72%; m.p.
118–120°C; IR (KBr) cm-1: 3363 (–NH), 2953 (C–H,
aliphatic), 1652 (C=O, CO2Et), 1490 (C=C, aromatic);
1HNMR (CDCl3): d (ppm) 0.90 (t, 3H of –CH2CH2CH3 at
C4), 3.74 (s, 6H of –CO2CH3 at C3 & C5), 2.28 (s, 6H of
–CH3 at C2 & C6), 1.08–1.31 (q, 4H of –CH2CH2CH3 at
C4), 5.62 (s, 1H of –NH), 7.26 (s, 1H of –CH at C4); MS
(m/z): 267 (M?); Elemental analysis: Calcd. for
C14H21N1O4: C, 62.90; H, 7.92; N, 5.24. Found C, 62.78;
H, 7.90; N, 5.18.
(m) 4-(4-Methylphenyl)-3,5-bis (carbomethoxy)-1,4-dihy-
dro-2,6-dimethylpyridine (4m; R = C6H4ÁCH3-4, R1 =
CH3) Yield: conventional method 46%, microwave
method 62%; m.p. 140–142°C; IR (KBr) cm-1: 3349 (–NH),
2949 (C–H, aliphatic), 1697 (C=O, CO2Et), 1490 (C=C,
aromatic); 1HNMR (CDCl3): d (ppm) 2.26 (s, 6H, –CH3 at C2
(i) 4-n-Butyl-3,5-bis (carbomethoxy)-1,4-dihydro-2,6-dimethyl-
pyridine (4i; R = -n-C4H9, R1 = CH3) Yield: conven-
tional method 43%, microwave method 76%; m.p.
123

Med Chem Res
& C6), 3.77 (s, 6H, –CO2CH3 at C3 & C5), 2.34 (s, 3H, –CH3
at C4), 4.98 (s, 1H, –NH), 5.67 (s, 1H, –CH at C4), and 7.02 to
7.12 (m, 4H, Ar–H at C4); MS (m/z): 315 (M?); Elemental
analyses: Calcd. for C18H21N1O4: C, 68.55; H, 6.71; N, 4.44.
Found C, 68.42; H, 6.68; N, 4.14.
Table 1 Anticancer activity of 4-alkyl/aryl-3,5-bis(carboethoxy/car-
bomethoxy)1,4-dihydro-2,6-dimethylpyridines
O
R
O
R1O
OR1
(n) 4-(4-Chlorophenyl)-3,5-bis(carbomethoxy)-1,4-dihydro-
2,6-dimethylpyridine (4n; R = C6H4ÁCl-4, R1 = CH3)
Yield: conventional method 57%, microwave method 85%;
m.p. 190–192°C; IR (KBr) cm-1: 3340 (–NH), 3068 (C–H,
aromatic), 2984 (C–H, aliphatic), 1696 (C=O, CO2Et), 1498
(C=C, aromatic); 1HNMR (CDCl3): d (ppm) 2.24 (s, 6H of
–CH3 at C2 & C6), 3.77 (s, 6H, –CO2CH3 at C3 & C5), 4.84 (s,
1H, –NH), 5.32 (s, 1H, –CH at C4), and 7.12 to 7.40 (m, 4H,
Ar–H at C4); MS (m/z): 335 (M?); Elemental analyses:
Calcd. for C17H18N1O4Cl: C, 60.81; H, 5.40; N, 4.17. Found
C, 60.75; H, 5.48; N, 4.08.
N
CH3
H3C
H
4a-n
S. no. Compound –R
code
–R1
IC50 (lM)
MDA-
MB
HT-
29
1
2
4a
4b
-n-C3H7
-n-C4H9
–C2H5 500
NA
NA
–C2H5 350.1
3
4
5
6
7
4c
4d
4e
4f
–C2H5 NA
–C2H5 244.1
–C2H5 330.6
–C2H5 500
–C2H5 268.0
NA
Biological activities
Evaluation of cell cytotoxicity
Cell culture
NO2
500
NA
OMe
CH3
Cl
Colon cancer (HT-29) cells and breast cancer (MDA-MB)
cells were maintained as a monolayer culture in DMEM
supplemented with 10% heat-inactivated fetal bovine
serum, 100 units/ml penicillin and 100 lg/ml streptomycin
at 37°C under 5% CO2 atmosphere and sub-cultured after
trypsinization (0.5% trypsin/2.6 mM EDTA).
310.2
261.7
4g
8
9
4h
4i
-n-C3H7
-n-C4H9
–CH3
–CH3
NA
NA
NA
500
Cytotoxicity assay
Cell suspension (1 9 104 cells, counted by Tryphan blue
exclusion dye method) with series of concentrations tested
(10, 50, 100, and 200 lg/cm3 in DMSO such that the final
concentration of DMSO in media is less than 1%) were
placed in 96-well plates and incubated in carbon dioxide
incubator with 5% CO2 at 37°C for 48 h in DMEM with 10%
FBS medium. Control wells contain 1% DMSO and cell
suspension. Then, the above media was replaced with 90 ll
of fresh serum-free media and 10 llofMTT reagent(5 mg/cm3)
and plates were incubated at 37°C for 4 h. There after the
above media was replaced with 200 ll of DMSO and incu-
bated further at 37°C for 10 min. The absorbance at 570 nm
was measured on a spectrophotometer. The readings were
averaged and viability of the test samples was compared with
DMSO control. IC50 values were determined from plot:
%inhibition (from control) versus concentration and the
results are presented in Table 1.
10
11
12
13
14
15
4j
–CH3
–CH3
–CH3
–CH3
–CH3
–
NA
NA
NO2
4k
500
368.2
451.9
NA
4l
NA
OMe
CH3
Cl
4m
500
4n
460.2
14.70
NA
Cisplatin
–
6.56
NA no activity
123

Med Chem Res
MRP1 ATPase assay
based on the cell viability, whereas the compounds 4d
(R = C6H4ÁNO2-3 & R1 = C2H5), 4g (R = C6H4ÁCl-4 &
R1 = C2H5) and 4k (R = C6H4ÁNO2-3 & R1 = CH3) were
uniquely active against both the cell lines. The test com-
pounds 4a (R = n-C3H7 & R1 = C2H5), 4b (R = n-C4H9
& R1 = C2H5), 4e (R = C6H4ÁOCH3-4 & R1 = C2H5), 4m
(R = C6H4ÁCH3-4 & R1 = CH3) and 4n (R = C6H4ÁCl-4
& R1 = CH3) were selectively active against only the
breast cancer cell lines MDA-MB, while two of the test
The MRP1 ATPase studies were carried out using purified
vesicles from Sf9 (Spodoptera frugiperda) (Solvo Bio-
technology, Budapest, Hungary). The colorimetric assay
for measuring transporter-associated ATPase activity was
performed as per the instructions of the manufacturer, a
modification of the method of Sarkadi et al. (1992).
In this assay two variants were employed. The activation
assay measured the increase in vanadate-sensitive ATPase
activity in the presence of a range of test compound con-
centrations through spectrophotometric quantification of
the amount of inorganic phosphate generated as a product
of ATPase-mediated conversion of ATP to ADP. This
assay gave a measure of the ability of an agent to stimulate
transporter activity (from basal levels), a characteristic
common to substrates of such ATPase transporters.
compounds 4i (R = n-C4H9
& R1 = CH3), and 4l
(R = C6H4ÁOCH3-4 & R1 = CH3) were selective against
the colon cancer cell lines HT-29. But, however, none of
the present series of dihydropyridines were comparable in
their potency with Cisplatin, the standard.
But, among these compounds, compound 4d with
m-nitrophenyl and ethyl ester groups was found to be rel-
atively more potent (IC50 = 244.1 lM) particularly against
the breast cancer cell lines (MDA-MB) and close to it was
the compound 4g with 4-chlorophenyl and ethyl ester
groups (IC50 = 268.2 lM) as the substituents. Similarly,
the test compound 4g was also relatively more potent
against the colon cancer cell lines (HT-29) with an IC50
value of 261.7 lM, whereas reference drug Cisplatin was
many times more potent against both the types of cancer
cell lines with the IC50 values 14.70 and 6.56 lM,
respectively.
In the inhibition assay, ATPase activity is maximally
stimulated using a saturating concentration of an activator of
the transporter being studied. MRP-1 ATPase activity stim-
ulated by 10 mM N-ethyl-maleimide-glutathione (NEM-GS)
mix, and the decrease in this maximum vanadate-sensitive
ATPase activity was measured in the presence of a range of
test compound concentrations. Agents which inhibit MRP-1
function will typically reduce the ATPase activity of the
pump, leading to a decrease in the activator-stimulated pro-
duction of inorganic phosphate. All MRP-1 ATPase studies
were carried out in the presence of 2 mM glutathione.
Benzbromarone(3,5-dibromo-4-hydroxyphenyl)-(2-ethyl-
3-benzofuranyl) methanone, aknownspecific MRP1inhibitor
was used as a positive control (Bobrowoska et al., 2003).
Positive control was performed in the presence of an ATPase
activator, NEM-GS (10 mM). In the activation assay, the
maximal stimulatory effectofthe testcompoundsisexpressed
on a scale where 0% is the basal ATPase activity and 100% is
the activity in the presence of the reference activator, NEM-
GS. In the ATPase inhibition assays, membrane was incu-
bated with NEM-GS in the presence or absence of increasing
concentrations of test compounds (1–1000 lM). Here, IC50 is
defined as the concentration of the test compounds that
inhibits by 50% the maximally stimulated ATPase.
Only eight new DHPs which showed anticancer activity
were investigated for their MRP1 inhibitory activity. In the
screening program, the capacity of the compounds either to
activate or to inhibit the ATPase activity on isolated Sf9
cell membranes expressing human MRP1 transporter were
studied. In the activation study, it was observed that only
one test compound, 4g induced inhibition on MRP1 basal
ATPase activity at concentrations above 5 lM. In theory, it
is assumed that good substrates activate transporter’s basal
ATPase activity, whereas slowly transported substrates
rather inhibit it. In agreement with this information, the
compound 4g has been shown to be slowly transported
substrates of MRP1. In addition, in the present study, 4g
was able to inhibit the NEM-GS-activated MRP1 ATPase
with respective IC50 value of 16 lM (mean of three
experiments) as comparable to a standard MRP1 inhibitor,
benzbromarone (IC50 = 4 lM). However, the rest of the
seven molecules failed to show inhibition at the concen-
trations tested.
Results and discussion
The new DHPs (4a–4n) were evaluated for cytotoxic
activity against both the cell lines: colon cancer (HT-29)
and breast cancer (MDA-MB) using the MTT assay
method. The inhibitory potencies (IC50) of the new DHPs
are listed in Table 1. The data indicates clearly that three
of the compounds: 4c (R = C6H5 & R1 = C2H5), 4h
The inhibition data indicates the % of MRP1 inhibited by
the test compound at a given concentration. The percentage
of MRP1 inhibition observed with compound 4g at 10 lM
concentration was found to be 47 (Fig. 2); and its maxi-
mum% inhibition of MRP1 was observed to be 65 at 50 lM
concentration. These results clearly indicate that the test
compound 4g specifically interact with MRP1 to inhibit, in
comparison to the reference drug benzbromarone.
(R = n-C3H7 & R1 = CH3) and 4j (R = C6H5 &
R1 = CH3) were inactive against both the cell lines tested,
123

Med Chem Res
70
60
50
40
30
20
10
0
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Acknowledgments The authors are grateful to the authorities of
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form of Rajiv Gandhi National Fellowship.
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123