3,5-Dibenzoyl-1,4-dihydropyridines: Synthesis and MDR reversal in tumor cells

Article abstract of DOI:10.1016/S0968-0896(01)00363-7

Fifteen 4-phenyl-3,5-dibenzoyl -1,4-dihydropyridines (BzDHPs) (1 15) substituted at the 4-phenyl ring were synthesized and compared to their cytotoxic activity and multidrug resistance (MDR)-reversing activity in in vitro assay systems. Among them, 2-CF

Full text of DOI:10.1016/S0968-0896(01)00363-7

Bioorganic & MedicinalChemistry 10 (2002) 1051–1055
3,5-Dibenzoyl-1,4-dihydropyridines:
Synthesis and MDR Reversal in Tumor Cells
Masami Kawase,a,* Anamik Shah,b Harsukh Gaveriya,b Noboru Motohashi,c
Hiroshi Sakagami,d Andreas Vargae and Joseph Molnarf
aFaculty of Pharmaceutical Sciences, Josai University, Saitama 350-0295, Japan
bDepartment of Chemistry, Saurashtra University, Rajkot-360 005, India
cMeiji Pharmaceutical University, Tokyo 204-8588, Japan
dDepartment of Dental Pharmacology, Meikai University School of Dentistry, Saitama 350-0283, Japan
eDepartment of Molecular Parasitology, Humboldt University, Berlin, Germany
fFaculty of Medicine, Institute of Microbiology, Albert Szent-Gyorgyi Medical University, Szeged, Hungary
¨
Received 6 August 2001; accepted 9 October 2001
Abstract—Fifteen 4-phenyl-3,5-dibenzoyl-1,4-dihydropyridines (BzDHPs) (1–15) substituted at the 4-phenylring were synthesized
and compared to their cytotoxic activity and multidrug resistance (MDR)-reversing activity in in vitro assay systems. Among them,
2-CF3 (5) (IC50=8.7 mM), 2-Cl( 11) (IC50=7.0 mM) and 3-Cl( 12) (IC50=7.0 mM) derivatives showed the highest cytotoxic activity
against human oral squamous carcinoma (HSC-2) cells. The activity of P-glycoprotein (Pgp) responce for MDR in tumor cells was
reduced by some of derivatives (3, 4, 8, 12), verapamil(VP) and nifedipine (NP). These data suggest that 3,5-dibenzoy-l4-(3-
chlorophenyl)-1,4-dihydro-2,6-dimethylpyridine (12) can be recommended as a new drug candidate for MDR cancer treatment.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
agents, such as vinca alkaloids or anthracyclines, and
VP, cardiovascular side effects were found.8 It is very
important finding that DHPs without calcium antag-
Multidrug resistance (MDR) of cancer cells has often
been correlated with the overexpression of P-glycopro-
tein (Pgp). An ABC transporter ATP-binding cassette
acts as a cellular pump membrane transporter by
extruding the anticancer agents and preventing their
antitumor effect.1 Therefore, it is important to develop
molecules that can inhibit Pgp activity.2,3 A variety of
compounds have been shown to inhibit Pgp-mediated
drug efflux.4 Characterizing common structuralfeatures
of Pgp-blocking agents is challenging. In general, MDR
active compounds are highly lipophilic and have aro-
matic ring systems in the molecule5 and a cationic or
dicationic side chain.4 Most compounds also possess a
tertiary nitrogen atom with positive charge at a physi-
ological pH.6 Among the possible resistance modifiers,
the dihydropyridines (DHPs), calcium antagonists, have
been studied extensively as the analogue of verapamil
(VP).7 In a combination treatment with antitumor
7
onistic activity possess MDR reversalactivity. Struc-
ture–activity relationship of DHP calcium channel
antagonists suggests that two acylsubstituents at the 3-
and 5-positions in DHP ring might affect the activity of
DHP calcium channel antagonists.9 Actually, the antago-
nist activity is optimized by ester substituents at the 3- and
5-positions and is reduced by their replacement with acetyl
group.9 By a rhodamine 123 fluorescent assay, we have
demonstrated that 3,5-diacetyl-1,4-dihydropyridines are
the efficient Pgp inhibitors.10 Studies of structure–activ-
ity relationships have demonstrated that the most
hydrophobic compound shows the highest MDR rever-
sing effect, but, the lipophilicity is not the only determi-
nant of MDR-modulating activity. Furthermore,
alterations in the substituents at 4-phenyl ring or sub-
stituent’s position reduced the activity. In this paper, we
investigated the MDR-reversalactivities of 3,5-diben-
zoyl-2,6-dimethyl-1,4-dihydro-4-phenylpyridine deriva-
tives (BzDHPs) (1–15) against mouse lymphoma cells
transfected with human MDR1 gene, and their cyto-
toxic activity against human oral tumor cell lines.
*Corresponding author. Tel.:+81-49-286-2233x455; fax:+81-49-271-
7984; e-mail: kawasema@josai.ac.jp
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00363-7

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M. Kawase et al. / Bioorg. Med. Chem. 10 (2002) 1051–1055
Results and Discussion
than that at the 2- or 4-position. However, 3-bromo
derivative (14) was inactive. Two 2-methoxy (9) and 4-
methoxy (10) derivatives have comparable activity with
the parent DHP (1). The introduction of 2-nitro (3), or
4-methylthio (8) groups at 4-phenylring eld to much
more active derivatives when compared to 1.
Chemistry
BzDHP derivatives (1–15) were prepared by the varia-
tions of the Hantzch reaction (Scheme 1).11 Thus,
refluxing the mixture of aqueous ammonia (excess),
aldehyde and 2 equiv of benzoylacetone in MeOH gave
BzDHPs (1–15) in moderate yields.
Lipophilicity
Lipophilicity is one of the important parameters affect-
ing MDR-modulating efficiency in the structure–activity
relationship studies of MDR modulating drugs.4,6 The
log P values of 1–15 were calculated by CLOGP14 and
were found to be higher (log P=4.26–7.47) when com-
pared to VP or nifedipine (NP) (Table 1). However,
lipophilicity alone was not an essential parameter of
direct MDR-modulating activity of the present series of
compounds.
Cytotoxicity
Fifteen BzDHP derivatives (1–15) showed significantly
varied cytotoxic activity against two human oraltumor
cell lines (HSC-2 and HSG), depending on the sub-
stituents at the 4-phenylring and the substituent’s posi-
tion. As shown in Table 1, compounds 5 (IC50=8.7 mM),
11 (IC50=7.0 mM) and 12 (IC50=7.0 mM) showed the
highest cytotoxic activity against HSC-2. The cytotoxi-
city was nearly the same to that of doxorubicine
(IC50=4.1 mM). On the other hand, cytotoxic activity of
5 (IC50=8.7 mM) against HSG was higher than those of
11 (28 mM) or 12 (150 mM). Normalfibrobalsts (HGF)
were relatively resistant to 5, 11 and 12, as judged by the
higher selectivity index (SI=HGF/HSC-2) ratio (60–
>143) suggesting a tumor-selective cytotoxicity of these
compounds.
In conclusion, we synthesized and evaluated a new ser-
ies of MDR-modulators derived from BzDHPs. The
potency of MDR reversalwas dependent on the nature
of substituents and their positions at the 4-phenylring
of BzDHPs. Compound 12 with the 3-chloro group on
4-phenylring showed the high MDR-modual ting activ-
ity and the tumor-specific cytotoxicity, indicating a new
drug candidate for MDR cancer chemotherapy.
MDR reversal on tumor cells
Rhodamine 123 assay has been widely accepted as a
direct and reproducible assay for measuring Pgp-
dependent drug efflux.12,13 Therefore, a series of
BzDHPs (1–15) have been evaluated for the ability to
inhibit the Pgp-mediated drug-efflux by the rhodamine
123 fluorescent assay in the human MDR1 gene trans-
fected T cell mouse lymphoma cell. Among fifteen
BzDHPs (1–15), 2-nitro- (3), 3-phenoxy- (4), 4-methyl-
thio- (8) and 3-chloro- (12) derivatives were more potent
than that of VP. A recent study conducted with 3,5-
diacetyl-DHPs has also shown that the presence of 3-
phenoxy substituent on the 4-phenylring considerabyl
enhanced the MDR-reversing activity.10 Among three
2- (11), 3- (12), and 4-chloro (13) derivatives, compound
12 was found to be more active than 11 or 13, meaning
that chloro-substitution at 3 position on phenyl ring
brings a higher cytotoxic activity and MDR activity
Experimental
Melting points of BzDHPs (1–15) were determined in
open glass capillaries in a paraffin bath and are uncor-
rected. H NMR spectra were performed on a JEOL
1
JNM-GSX 500 (500 MHz) spectrophotometer using
tetramethylsilane as an internal standard. Mass spectra
(MS) were recorded on a JEOL-JMS-DX300 spectro-
photometer with direct inlet system at 70 eV. Combus-
tion analyses were carried out on Coleman elemental
analyser at Vadodara, India. Thin layer chromato-
graphy (TLC) was performed on a Merck Kieselgel 60
F254 (Merck 5549, USA).
The following chemicals were obtained from each
indicated company: VP (Aldrich Chem. Comp. Inc.,
Milwarkee, WI, USA); NP (Wako Pure Chem. Ind.,
Scheme 1.

M. Kawase et al. / Bioorg. Med. Chem. 10 (2002) 1051–1055
Table 1. Cytotoxic activity, MDR modulating activity and log P of BzDHPs (1–15)
1053
Compound
Cytotoxic activity (IC50 mM)
SI (HGF/HSC-2)
MDR modulating activitya
Fluoresence
activity ratio
Calcd log P
HSC-2
HSG
HGF
FSCb
SSCb
FL-1b
Par (control)c
—
—
—
—
424
636
397
390
315
722
—
—
—
—
513
658
266
269
233
1960
7006
167
42.0
1.0
3.71
—
—
—
MDR + R123d
(ꢁ)-Verapamil399
403
1.1
550
11.8
Nifedipine
1
543
265
103
285
639
890
420
>1000
929
>1000
978
397
>1000
>1000
>1000
>1000
421
>1000
716
>1000
>1000
>100
1.6
1.6
>9.7
3.3
>1.6
112
3.4
>4.2
> <1.0
> <1.0
> <1.0
60.1
>143
19.4
>2.3
> <1.0
>24.4
—
—
—
571
407
2792
2416
617
758
822
1660
607
538
565
1882
328
871
434
—
2.35
5.37
5.11
5.11
7.47
6.25
6.25
6.25
5.93
5.29
5.29
6.08
6.08
6.08
6.23
4.26
—
560
547
514
555
541
562
567
525
547
562
534
567
540
543
550
—
312
267
263
269
298
285
298
267
403
283
267
275
270
283
259
—
3.4
2.4
16.8
14.5
3.7
4.6
4.9
10.0
3.7
3.3
3.4
11.3
2.0
5.2
2.6
—
2
3
4
5
6
7
8
9
10
11
12
13
14
8.7
8.7
117
236
>1000
>1000
>1000
193
>1000
>1000
>1000
>1000
28
150
365
>1000
>1000
7.0
7.0
37
434
>1000
15
Doxorubicinꢂ HCl4.1
5.3
aThe finalconcentration of each compounds was 8 mg/mL in culture medium containing cell suspension and 0.4% DMSO.
bFSC, forward scatter count; SSC, side scatter count; FL-1, fluorescence intensity.
cPar, a parentalcelwithout transfection of MDR gene.
dMDR, a parentalceltransfected with MDR gene.
Ltd., Osaka, Japan); Dulbecco’s modified eagle medium
(DMEM) (Gibco BRL, Grand Island, NY, USA); fetal
bovine serum (FBS) (JRH Biosci., Lenexa, KS, USA).
(m, 3H), 7.52–7.55 (m, 2H), 7.58 (d, 4H, J=7.6 Hz). MS
m/e 438 (M+, 6%), 421 (100%). Anal. calcd for
C27H22N2O4: C, 73.97; H, 5.02; N, 6.39. Found: C,
73.99; H, 5.05; N, 6.40.
General procedure for the preparation of BzDHPs (1–
15)
3,5-Dibenzoyl-1,4-dihydro-2,6-dimethyl-4-(30 -phenoxy-
phenyl)pyridine (4). Mp 190 ꢀC (MeOH), yield 32%. H
1
A solution of benzoylacetone (1.0 g, 10 mmol) and
liquid ammonia (sp. gr. 0.90) (0.32 mL, 20 mmol) in
MeOH (10 mL) was treated with respective aldehyde
(5 mmol), and the mixture was refluxed for 20–24 h.
The separated solid was collected by suction and
then, after charcoal treatment, recrystallized from
MeOH.
NMR (CDCl3) d 1.90 (s, 6H), 5.13 (s, 1H), 5.63 (s, 1H),
6.65 (t, 1H, J=2.1 Hz), 6.69 (dd, 2H, J=2.1, 7.6 Hz),
6.82–6.85 (m, 2H), 7.03 (t, 1H, J=7.4 Hz), 7.07 (t, 1H,
J=7.8 Hz), 7.24 (t, 2H, J=7.8 Hz), 7.33 (t, 4H,
J=7.4 Hz), 7.42 (dt, 2H, J=1.5, 7.4 Hz), 7.52 (dt, 4H,
J=1.5, 7.4 Hz). MS m/e: 485 (M+, 62%), 316 (100%).
Anal. calcd for C33H27NO3: C, 81.63; H, 5.60; N, 2.88.
Found: C, 81.67; H, 5.62; N, 2.85.
3,5-Dibenzoyl-1,4-dihydro-2,6-dimethyl-4-phenylpyridine
(1). Mp 220–222 ꢀC (MeOH) (lit.11 mp 229–231 ꢀC),
yield 28%. H NMR (CDCl3) d 1.94 (s, 6H), 5.14 (s,
1H), 5.67 (s, 1H), 6.94 (d, 2H, J=7.4 Hz), 7.05 (t, 1H,
J=7.4 Hz), 7.12 (t, 2H, J=7.4 Hz), 7.33 (t, 4H,
J=7.6 Hz), 7.42 (t, 2H, J=7.6 Hz), 7.53 (d, 4H,
J=7.6 Hz). MS m/e 393 (M+, 56%), 316 (100%).
3,5-Dibenzoyl-4-(20 -trifluoromethylphenyl)-1,4-dihydro-
2,6-dimethylpyridine (5). Mp 190–192 ꢀC (MeOH), yield
1
1
18%. H NMR (CDCl3) d 1.89 (s, 6H), 5.39 (s, 1H),
5.54 (s, 1H), 7.19 (t, 1H, J=7.6 Hz), 7.34 (t, 4H,
J=7.6 Hz), 7.45 (t, 2H, J=7.3 Hz), 7.52 (t, 1H,
J=7.6 Hz), 7.57 (d, 1H, J=7.6 Hz), 7.65 (dd, 4H,
J=1.2, 7.6 Hz), 7.79 (d, 1H, J=7.6 Hz). MS m/e 461
(M+, 14%), 316 (100%). Anal. calcd for C28H22F3NO2:
C, 72.88; H, 4.80; N, 3.04. Found: C, 72.89; H, 4.80; N,
3.06.
3,5-Dibenzoyl-1,4-dihydro-2,6-dimethyl-4-(30-nitrophenyl)-
pyridine (2). Mp 210–212 ꢀC (MeOH) (lit.15 mp 205 ꢀC),
1
yield 32%. H NMR (CDCl3) d 1.91 (s, 6H), 5.28 (s,
1H), 5.90 (s, 1H), 7.27 (t, 1H, J=7.6 Hz), 7.32 (d, 1H,
J=7.3 Hz), 7.37 (t, 4H, J=7.6 Hz), 7.46 (t, 2H,
J=7.3 Hz), 7.54 (d, 4H, J=7.6 Hz), 7.93 (d, 1H,
J=7.3 Hz), 7.94 (s, 1H). MS m/e 438 (M+, 25%), 316
(100%).
3,5-Dibenzoyl-4-(30 -trifluoromethylphenyl)-1,4-dihydro-
2,6-dimethylpyridine (6). Mp 188–190 ꢀC (MeOH), yield
1
26%. H NMR (CDCl3) d 1.93 (s, 6H), 5.23 (s, 1H),
5.71 (s, 1H), 7.15 (br d, 1H, J=8.2 Hz), 7.19–7.23 (m,
2H), 7.26–7.33 (m, 1H), 7.35 (t, 4H, J=7.6 Hz), 7.44 (tt,
2H, J=1.5, 7.6 Hz), 7.52 (dd, 4H, J=1.2, 7.6 Hz). MS
m/e 461 (M+, 65%), 316 (100%). Anal. calcd for
C28H22F3NO2: C, 72.88; H, 4.80; N, 3.04. Found: C,
72.87; H, 4.82; N, 3.05.
3,5-Dibenzoyl-1,4-dihydro-2,6-dimethyl-4-(20-nitrophenyl)-
pyridine (3). Mp 192–194 ꢀC (dioxane), yield 30%. H
1
NMR (CDCl3) d 1.87 (s, 6H), 5.66 (s, 1H), 5.77 (s, 1H),
7.17–7.21 (m, 1H), 7.34 (t, 4H, J=7.3 Hz), 7.42–7.46

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M. Kawase et al. / Bioorg. Med. Chem. 10 (2002) 1051–1055
3,5-Dibenzoyl-4-(40 -trifluoromethylphenyl)-1,4-dihydro-
1H), 6.57 (d, 2H, J=8.5 Hz), 6.70 (d, 2H, J=8.5 Hz),
6.97 (t, 4H, J=7.8 Hz), 7.06 (tt, 2H, J=1.5, 7.6 Hz),
7.09–7.12 (m, 4H), 8.07 (s, 1H). MS m/e 427
(75%)+429 (23%) (3:1, M+), 316 (100%). Anal. calcd
for C27H22ClNO2: C, 75.78; H, 5.18; N, 3.27. Found: C,
75.80; H, 5.15; N, 3.26.
2,6-dimethylpyridine (7). Mp 226–227 ꢀC (MeOH), yield
1
20%. H NMR (CDCl3) d 1.94 (s, 6H), 5.21 (s, 1H),
5.67 (s, 1H), 7.09 (d, 2H, J=8.2 Hz), 7.30 (t, 1H,
J=7.9 Hz), 7.36 (t, 4H, J=7.6 Hz), 7.39 (d, 1H,
J=7.9 Hz), 7.45 (tt, 2H, J=1.5, 7.6 Hz), 7.54 (dd, 4H,
J=1.5, 7.6 Hz). MS m/e 461 (M+, 68%), 316 (100%).
Anal. calcd for C28H22F3NO2: C, 72.88; H, 4.80; N,
3.04. Found: C, 72.90; H, 4.78; N, 3.02.
3,5-Dibenzoyl-4-(30-bromophenyl)-1,4-dihydro-2,6-dime-
thylpyridine (14). Mp 182–184 ꢀC (Dioxane), yield 38%.
1H NMR (CDCl3) d 1.94 (s, 6H), 5.12 (s, 1H), 5.69 (s,
1H), 6.85 (dt, 1H, J=1.2, 7.6 Hz), 6.97 (t, 1H,
J=7.6 Hz), 7.10 (t, 1H, J=1.8 Hz), 7.18–7.19 (m, 1H),
7.30 (t, 1H, J=7.6 Hz), 7.35 (t, 3H, J=7.6 Hz), 7.42–
7.47 (m, 2H), 7.52–7.56 (m, 4H). MS m/e 471
(45%)+473 (30%) (1:1, M+), 316 (100%). Anal. calcd
for C27H22BrNO2: C, 68.65; H, 4.69; N, 2.97. Found: C,
68.66; H, 4.66; N, 2.94.
3,5-Dibenzoyl-1,4-dihydro-2,6-dimethyl-4-[40-(methylthio)-
phenyl]pyridine (8). Mp 144–146 ꢀC (MeOH), yield
1
29%. H NMR (CDCl3) d 1.94 (s, 6H), 2.48 (s, 3H),
5.09 (s, 1H), 5.66 (s, 1H), 6.75 (d, 1H, J=8.2 Hz), 6.85
(d, 1H, J=8.2 Hz), 6.87 (d, 1H, J=8.2 Hz), 7.02 (d, 1H,
J=8.2 Hz), 7.28 (t, 2H, J=7.6 Hz), 7.34 (t, 2H,
J=7.6 Hz), 7.41–7.45 (m, 2H), 7.52–7.56 (m, 4H). MS
m/e 439 (M+, 88%), 316 (100%). Anal. calcd for
C28H25NO2S: C, 76.51; H, 5.73; N, 3.19. Found: C,
76.54; H, 5.72; N, 3.16.
3,5-Dibenzoyl-1,4-dihydro-4-(30,40,50-trimethoxyphenyl)-
2,6-dimethylpyridine (15). Mp 186 ꢀC (MeOH), yield
1
17%. H NMR (CDCl3) d 1.96 (s, 6H), 3.63 (s, 6H),
3,5-Dibenzoyl-1,4-dihydro-4-(20 -methoxyphenyl)-2,6-di-
3.71 (s, 3H), 5.11 (s, 1H), 5.67 (s, 1H), 6.12 (s, 2H), 7.35
(t, 4H, J=7.6 Hz), 7.44 (tt, 2H, J=1.5, 7.3 Hz), 7.56–
7.58 (m, 4H). MS m/e 483 (M+, 74%), 316 (100%).
Anal. calcd for C30H29NO5: C, 74.52; H, 6.04; N, 2.90.
Found: C, 74.55; H, 6.01; N, 2.89.
methylpyridine (9). Mp 238–240 ꢀC (MeOH), yield
1
22%. H NMR (CDCl3/DMSO-d6, 4:1) d 1.38+1.39 (s,
6H), 2.67+2.68 (s, 3H), 4.58 (s, 1H), 5.96–6.10 (m, 1H),
6.15–6.25 (m, 1H), 6.33–6.40 (m, 1H), 6.45–6.50 (m,
1H), 6.73–6.82 (m, 4H), 6.82–6.92 (m, 2H), 6.96–7.05
(m, 4H), 7.98 (s, 1H). MS m/e 423 (M+, 7%), 392
(100%). Anal. calcd for C28H25NO3: C, 79.41; H, 5.95;
N, 3.31. Found: C, 79.42; H, 5.96; N, 3.33.
Calculation of distribution coefficient. The log P values
of 1–15 were calculated by CLOGP.14
Cell culture. Human oralsquamous celclarcinoma
(HSC-2) cells and human salivary gland tumor (HSG)
cells were maintained as a monolayer culture at 37 ꢀC in
DMEM supplemented with 10% heat-inactivated FBS
in a humidified 5% CO2 atmosphere, and subcultured
by trypsinization. Human gingivalfibrobalsts (HGF)
were isolated from healthy gingival biopsies of a 10-
year-old female, as described previously.16 Cells
between the fifth and seventh passages were used.
3,5-Dibenzoyl-1,4-dihydro-4-(40 -methoxyphenyl)-2,6-di-
methylpyridine (10). Mp 191–192 ꢀC (MeOH), yield
1
20%. H NMR (CDCl3) d 1.93 (s, 6H), 3.68 (s, 3H),
5.07 (s, 1H), 5.63 (s, 1H), 6.42+6.65 (d, 2H, J=8.8 Hz),
6.85+6.87 (d, 2H, J=8.8 Hz), 7.27 (t, 1H, J=7.6 Hz),
7.33 (t, 3H, J=7.6 Hz), 7.39–7.44 (m, 2H), 7.52–7.55
(m, 4H). MS m/e 423 (M+, 90%), 316 (100%). Anal.
calcd for C28H25NO3: C, 79.41; H, 5.95; N, 3.31. Found:
C, 79.43; H, 5.94; N, 3.29.
Cytotoxic activity
3,5-Dibenzoyl-4-(20-chlorophenyl)-1,4-dihydro-2,6-dime-
thylpyridine (11). Mp 216–218 ꢀC (MeOH), yield 31%.
1H NMR (CDCl3) d 1.90 (s, 6H), 5.62 (s, 2H), 6.97 (dt,
1H, J=1.5, 7.6 Hz), 7.06–7.10 (m, 2H), 7.18 (dd, 1H,
J=1.5, 7.8 Hz), 7.28–7.35 (m, 4H), 7.40–7.47 (m, 2H),
7.55–7.62 (m, 4H). MS m/e 427 (3.2%)+429 (1.2%) (3:1,
M+), 316 (100%). Anal. calcd for C27H22ClNO2: C,
75.78; H, 5.18; N, 3.27. Found: C, 75.80; H, 5.19; N, 3.25.
Cells were incubated for 24 h with the indicated con-
centrations of test samples in culture medium, and the
viable cell number was determined by 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
method.16 The A540 values of control HSC-2, HSG and
HGF cells were 0.805, 0.623 and 0.252, respectively.
Cell and fluorescence uptake. MDR1/A expressing cell
lines were selected by culturing the infected cells with
60 ng/mL colchicine to maintain the expression of the
MDR phenotype.17 The L5178 MDR cell line and the
L5178 Y parent cell line were grown in McCoy’s 5A
medium supplemented with 10% heat-inactivated horse
serum, l-glutamine and antibiotics. The cells were
adjusted to a concentration of 2 ꢃ 106/mL and resus-
pended in serum-free McCoy’s 5A medium, and 0.5 mL
aliquots of the cell suspension were distributed into each
Eppendorf centrifuge tube. Then, 2 mL of 2 mg/mL test
compounds were added and incubated for 10 min at
room temperature. Then, 50 mL rhodamine 123 (R123)
as indicator was added to the samples (5.2 mM final
3,5-Dibenzoyl-4-(30-chlorophenyl)-1,4-dihydro-2,6-dime-
thylpyridine (12). Mp 184–186 ꢀC (MeOH), yield 35%.
1H NMR (CDCl3) d 1.92 (s, 6H), 5.13 (s, 1H), 5.78 (s,
1H), 6.79–6.82 (m, 1H), 6.95–6.97 (m, 1H), 7.02–7.05
(m, 2H), 7.35 (t, 4H, J=7.6 Hz), 7.44 (tt, 2H, J=1.5,
7.6 Hz), 7.51–7.54 (m, 4H). MS m/e 427 (56%)+429
(17%) (3:1, M+), 316 (100%). Anal. calcd for
C27H22ClNO2: C, 75.78; H, 5.18; N, 3.27. Found: C,
75.79; H, 5.20; N, 3.29.
3,5-Dibenzoyl-4-(40-chlorophenyl)-1,4-dihydro-2,6-dime-
thylpyridine (13). Mp 228–230 ꢀC (MeOH), yield 28%.
1H NMR (CDCl3/DMSO-d6, 4:1) d 1.49 (s, 6H), 4.66 (s,

M. Kawase et al. / Bioorg. Med. Chem. 10 (2002) 1051–1055
3. Lehne, G. Current Drug Targets 2000, 1, 85.
1055
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20 min at 37 ꢀC, washed twice and resuspended in
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analysis. The fluorescence of cell population was mea-
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This study was supported by the Foundation for Cancer
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