4,5-Di-substituted benzyl-imidazol-2-substituted amines as the structure template for the design and synthesis of reversal agents against P-gp-mediated multidrug resistance breast cancer cells

Article abstract of DOI:10.1016/j.ejmech.2014.06.016

Over-expression of P-glycoprotein (P-gp), a primary multidrug transporter which is located in plasma membranes, plays a major role in the multidrug resistance (MDR) of cytotoxic chemotherapy. Naamidines are a class of marine imidazole alkaloids isolated from Leucetta and Clathrina sponges, possessing a Y-shaped scaffold. Based on the results previously obtained from the third-generation MDR modulator ONT-093 and other modulators developed in our group, we designed and synthesized a series of novel 4,5-di-substituted benzyl-1-methyl-1H-imidazol-2-substituted amines using the Naamidine scaffold as the structure template. Subsequently, their reversing activity for Taxol resistance has been evaluated in P-gp-mediated multidrug resistance breast cancer cell line MDA435/LCC6MDR. Compounds 12c with a Y-shaped scaffold, and compound 17c which is 'X-shaped' scaffold and possesses a 4-diethylamino group at aryl ring B, turned out to be the most potent P-gp modulators. It appears that compounds 12c and 17c at 1 μM concentration can sensitize LCC6MDR cells toward Taxol by 26.4 and 24.5 folds, with an EC₅₀ 212.5 and 210.5 nM, respectively. These two compounds are about 5-6 folds more potent than verapamil (RF = 4.5). Moreover, compounds 12c and 17c did not exhibit obvious cytotoxicity in either cancer cell lines or normal mouse fibroblast cell lines. This study has demonstrated that the synthetic Naamidine analogues can be potentially employed as effective, safe modulators for the P-gp-mediated drug resistance cancer cells.

Full text of DOI:10.1016/j.ejmech.2014.06.016

European Journal of Medicinal Chemistry 83 (2014) 74e83
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
4,5-Di-substituted benzyl-imidazol-2-substituted amines as the
structure template for the design and synthesis of reversal agents
against P-gp-mediated multidrug resistance breast cancer cells
Nan Zhang a, Zhaohui Zhang a, Iris L.K. Wong b, Shengbiao Wan a, Larry M.C. Chow b,
,
*
Tao Jiang a
a Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China
b Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Hong Kong, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Over-expression of P-glycoprotein (P-gp), a primary multidrug transporter which is located in plasma
membranes, plays a major role in the multidrug resistance (MDR) of cytotoxic chemotherapy. Naami-
dines are a class of marine imidazole alkaloids isolated from Leucetta and Clathrina sponges, possessing a
Y-shaped scaffold. Based on the results previously obtained from the third-generation MDR modulator
ONT-093 and other modulators developed in our group, we designed and synthesized a series of novel
4,5-di-substituted benzyl-1-methyl-1H-imidazol-2-substituted amines using the Naamidine scaffold as
the structure template. Subsequently, their reversing activity for Taxol resistance has been evaluated in
P-gp-mediated multidrug resistance breast cancer cell line MDA435/LCC6MDR. Compounds 12c with a Y-
shaped scaffold, and compound 17c which is ‘X-shaped’ scaffold and possesses a 4-diethylamino group at
aryl ring B, turned out to be the most potent P-gp modulators. It appears that compounds 12c and 17c at
Received 29 January 2014
Received in revised form
17 May 2014
Accepted 9 June 2014
Available online 10 June 2014
Keywords:
Naamidine analogues
Multidrug resistance
Structureeactivity relationship
P-glycoprotein
1 mM concentration can sensitize LCC6MDR cells toward Taxol by 26.4 and 24.5 folds, with an EC50 212.5
Modulator
Marine alkaloid
and 210.5 nM, respectively. These two compounds are about 5e6 folds more potent than verapamil
(RF ¼ 4.5). Moreover, compounds 12c and 17c did not exhibit obvious cytotoxicity in either cancer cell
lines or normal mouse fibroblast cell lines. This study has demonstrated that the synthetic Naamidine
analogues can be potentially employed as effective, safe modulators for the P-gp-mediated drug resis-
tance cancer cells.
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
alleviated [3]. Among the ABC transporters involved in MDR, the
membrane permeability glycoprotein, termed P-glycoprotein (P-
One of the major obstacles for the treatment of cancers with
chemotherapeutic agents is multidrug resistance (MDR) [1], which
could be correlated with a number of mechanisms such as drug
metabolism, p53 mutations, DNA repair capacity, hostile tumor
microenvironment and the modified apoptotic pathways [2]. Pri-
marily, the resistance to therapy is due to the over-expression of
ATP-binding cassette (ABC) transporters, which often result in the
increased efflux of chemotherapeutic drugs from cancer cells.
Consequently, the intracellular concentration of drugs is lowered
and their toxic effects towards malignant cells are ultimately
gp), plays the most critical role. Therefore, one would expect that
the inhibition of P-gp over-expression could effectively reverse the
multidrug resistance of tumor cells and restore their sensitivity to
drugs, eventually improving the outcome of chemotherapy.
Over the past two decades, three generations of P-gp inhibitors
have been developed, aiming to reverse the MDR in cancer cells.
Particularly, the first and the second generation of inhibitors, such
as Verapamil [4], Cyclosporine A [5], VX-710 [6] and PSC833 [7],
mainly act by competitively binding to the P-gp substrate sites
against the chemotherapy drugs, lacking sufficient affinity and
specificity. In addition, most of these compounds are P450 enzyme
inhibitors, which give rise to undesirable drug interactions;
because they are essentially competing against combination ther-
apy drugs, these compounds have exhibited inferior in vivo activity,
high toxicity and unpredictable pharmacokinetic interactions [1,8].
* Corresponding author. School of Medicine and Pharmacy, Ocean University of
China, 5 Yushan Road, Qingdao 266003, China.
E-mail address: jiangtao@ouc.edu.cn (T. Jiang).
http://dx.doi.org/10.1016/j.ejmech.2014.06.016
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

N. Zhang et al. / European Journal of Medicinal Chemistry 83 (2014) 74e83
75
Notably, the third-generation of P-gp modulators such as Tariquidar
(XR9576), Zosuquidar (LY335979), Elacridar (GF120918) and ONT-
093 (OC144-093), not only exhibited little competitive effect
against combination therapy drugs [9e12], but also showed less
impact on their pharmacokinetic properties, indicating broader
clinical applications. However, so far, none of the compounds re-
ported has been approved by regulatory agencies, thus the dis-
covery of novel MDR modulators with high activity and low toxicity
still remains a huge challenge to pharmaceutical chemists.
Recently, our group has disclosed a series of effective MDR
reversal agents, such as methylated Quercetin [13], permethyl
Ningalin B analogues [14,15] and (E,E)-4,6-bis(styryl)-2-O-gluco-
pyranosyl-pyrimidine [16], exhibiting remarkable modulating ac-
tivity to ABC drug efflux pumps with low toxicity (Fig. 1).
Specifically, it was found that Ningalin B derivatives 1a, 1b, 1c and
1d have displayed the best P-gp modulating activity, and they can
Comparing with the Y-shaped structure of ONT-093 (OC144-093),
the linkers between the benzene and imidazole rings in the Naa-
midine structure are longer and more flexible, which should pro-
vide more conformation options for the binding with P-gp. As
reported in the references [25e27], the introduction of lipophilic
groups and hydrogen bond acceptors seems to favor the binding
with P-gp, and the methylation of hydroxy groups, or removing
hydantoin group at the 2-amino position could effectively reduce
their cytotoxic activity [21]. Therefore, our design of the target
molecules is based on the principles described as follows: the C4
and C5 positions of the central 2-aminoimidazole ring would be
replaced with benzyl groups substituted with methoxy or dieth-
ylamino groups; by acylation reactions, the 2-amino group can be
linked with moieties containing aryl rings which have been
substituted with multiple methoxy groups. These novel Naamidine
analogues have been examined for their MDR-reversing activity in
the P-gp over-expressing cancer cell line MDA435/LCC6MDR.
sensitize LCC6MDR cells toward Taxol at 1 mM concentration by
18.2, 9.9, 42.7 and 42.7 folds, respectively. Even though a common
“pharmacophore” has not been identified in the inhibitors or sub-
strates of P-gp [6], for instance, the well-known third-generation
MDR modulator ONT-093 (OC144-093) [12], it appears that these
compounds do possess a Y-shaped scaffold composed of aromatic
rings A, B and C (Fig. 1). Therefore, we envisioned that this common
Y-shaped structure would be an important “pharmacophore”
scaffold for P-gp inhibiting activity. On the other hand, it was found
that the methoxy-substituted analogues, instead of the hydroxy-
substituted ones, could increase the MDR reversal activity and
reduce cytotoxicity [13,14,16]. Based on the results obtained from
structureeactivity relationship studies, we expect that another
class of Y-shaped marine natural alkaloids, Naamidines, could be
modified and developed into new P-gp modulators.
2. Results and discussion
2.1. Chemistry
A series of Naamidine analogues have been prepared, in which
the 2,4,5-positions of 1-methyl-1H-imidazole rings were
substituted with different functional groups. Initially, three key
intermediates 9aec containing multiple methoxy and dieth-
ylamino groups were prepared, as illustrated in Scheme 1. The
synthesis of 4,5-bis(4-methoxybenzyl)-1-methyl-1H-imidazol-2-
amine (9a) has been reported previously [28]. 4,5-Bis(3,4-
dimethoxybenzyl)-1-methyl-1H-imidazol-2-amine (9b) and 4-
(3,4-dimethoxybenzyl)-5-(4-(diethylamino)benzyl)-1-methyl-1H-
imidazol-2-amine (9c) were prepared by a strategy similar to the
reported one [28], employing 4,5-diiodo-1-methyl-1H-imidazole
(2) as the starting material. At first, 4,5-diiodo-1-methyl-1H-imid-
azole (2) was treated with EtMgBr in anhydrous THF to furnish the
desired 5-imidazolyl Grignard reagent, which was allowed to react
Naamidines (Fig. 1) are a class of marine imidazole alkaloids,
which have been successively isolated from Leucetta and Clathrina
sponges in the past thirty years [17,18]. It was found that these
compounds exhibited interesting biological activities, such as
moderate antitumor and antibacterial activities [17,19e24].
Fig. 1. Structures of compounds with Y-shaped scaffold.

76
N. Zhang et al. / European Journal of Medicinal Chemistry 83 (2014) 74e83
Scheme 1. Synthesis of intermediates 9aec. (Reagents and conditions: (i) EtMgBr, THF, 25 ꢀC, 0.5 h, then 4-methoxybenzaldehyde, 24 h (3a); EtMgBr, THF, 25 ꢀC, 0.5 h, then 3,4-
dimethoxybenzaldehyde, 24 h (3b); EtMgBr, THF, 25 ꢀC, 0.5 h, then 4-(diethylamino)benzaldehyde, 24 h (3c); (ii) Et3SiH, BF3$Et2O, THF, 0 ꢀCe60 ꢀC, 24 h; (iii) EtMgBr, THF, 25 ꢀC,
0.5 h, then N-methylformanilide, 12 h; (iv) 10, THF, 25 ꢀC, 12 h (6a); 11, THF, 25 ꢀC, 12 h (6b and 6c); (v) Pd/C, H2, MeOH, acetic acid, 40 atm, 50 ꢀC, 24 h; (vi) n-BuLi, THF, ꢁ78 ꢀC,
0.5 h, then TsN3, 25 ꢀC, 1 h; (vii) NaBH4, MeOH, 0 ꢀCe25 ꢀC, 12 h.)
with 4-methoxybenzaldehyde, 3,4-dimethoxybenzaldehyde and 4-
(diethylamino)benzaldehyde to afford the corresponding alcohols
3aec. Subsequent ionic reduction of the benzylic hydroxyl group
with Et3SiH and BF3$Et2O has provided the desired intermediates
4aec. These compounds were treated with EtMgBr in anhydrous
THF to afford 4-imidazolyl Grignard reagents, which were allowed
to react with N-methylformanilide to generate the corresponding
aldehydes 5aec. At this stage, the resulting aldehydes were treated
with p-methoxyphenylmagnesium bromide (10), or 1,2-
dimethoxyphenylmagnesium bromide (11), affording alcohols
6aec. As previously reported [29] in the synthesis of Naamine C,
treating these alcohols with Et3SiH and BF3$Et2O would lead to the
intramolecular FriedeleCrafts cyclization reaction, instead of the
reduction of benzylic hydroxyl groups. Even though we found that
NaBH4 and NiCl2 were possible reducing agents, difficult post-
reaction processing due to the excess amount of nickel mud has
hampered their suitability in this transformation. In order to
improve this step, this reaction was attempted using Pd/C as the
catalyst under hydrogen pressure (40 atm), and it appeared that the
hydroxyl group has been eliminated completely, affording the
desired products 7aec in an 81%, 72% and 77% yield, respectively.
Next, compound 7a was lithiated at the 2-position with stoichio-
metric amount of n-BuLi, and the resulting intermediate was
treated with TsN3 to afford azide product 8a. Surprisingly, when
this method was applied to substrate 7b, the desired product 8b
was not obtained and most of the starting material was recovered.
In Shunsaku Ohta's [30] study, this result has been attributed to the
fact that lithiation takes place at the benzyl methylene group of the
5-position rather than the 2-position, which would be difficult to
further react with TsN3 due to the steric hindrance. Therefore, in his
Naamine A synthesis, the 2-position of the imidazole ring was first
brominated with NBS, and the resulting bromide was lithiated with
t-BuLi, followed by TsN3 treatment, ultimately affording the cor-
responding azide in a 32.8% total yield. In our study, 7b and 7c were
treated with more than two equivalents of n-BuLi, thus the lith-
iation reaction takes place at both the 2-position of the imidazole
ring and the benzyl methylene position. Subsequently, the resulting
di-lithiated intermediate was treated with TsN3 to afford the 2-
substituted azide products 8b and 8c in a 51% and 42% yield,
respectively. With these materials in hand, key intermediates 9aec
can be readily prepared by NaBH4 reduction of 8aec in MeOH.
As shown in Scheme 2, the target products can be synthesized
by the acylation reaction of compounds 9aec with a series of acyl
chlorides. Indeed, after treated with one equivalent of n-BuLi,
compounds 9aec readily reacted with 3,4,5-trimethoxybenzoyl
chloride to afford the corresponding products 12aec. Compound

N. Zhang et al. / European Journal of Medicinal Chemistry 83 (2014) 74e83
77
Scheme 2. Synthesis of target products. (Reagents and conditions: (i) n-BuLi, THF, ꢁ78 ꢀC, 0.5 h, then acyl chlorides, 25 ꢀC, 2 h; (ii) benzoyl chlorides, DIPEA, THF, 0 ꢀCe60 ꢀC, 24 h;
(iii) (E)-3-(3,4,5-trimethoxyphenyl)acryloyl chloride, DIPEA, THF, 0 ꢀCe60 ꢀC, 24 h; (iv) 3-(3,4,5-trimethoxyphenyl)propanoyl chloride, DIPEA, THF, 0 ꢀCe60 ꢀC, 24 h.)
13a and 14a were also obtained by the same approach. Interest-
ingly, when excess DIPEA and benzoyl chloride were employed to
react with 9aec, the N,N-benzoylbenzamide products 15aeb have
been obtained. Using the same synthetic strategy, compounds
16aeb, 17aec and 18b were also prepared, containing more ben-
zene rings or methoxy groups.
control. In order to study the correlation between the compound
lipophilicity and MDR reversal activity, CLogPs of the Naamidine
analogues have been calculated by software Sybyl 2.0, as illustrated
in Table 1.
Notably, it appears that none of the intermediates 9aec has
exhibited pronounced reversal activity (RF z 1), in which the 2-
amino group was not substituted. One of the most experimentally
supported [33,34] mechanism for P-gp action is the ‘‘hydrophobic
vacuum cleaner” model [32], which suggests that enhancing the
lipophilicity of compounds should be essential for the improve-
ment of P-gp modulating activity [26,27]. Therefore, it would be
difficult for the Naamidine analogues 9aec to stay in the bilayer to
interact with P-gp, because of their poor lipophilicity.
2.2. Biological studies of Naamidine analogues
As demonstrated in the literature [14,31], the reversal effect of
Naamidine analogues on the Taxol resistance can be determined
using the MDA435/LCC6MDR cell line. The MDA435/LCC6MDR cell
line is an ABCB1-transfected derivative of MDA435/LCC6 cell line,
and the latter is an estrogen-independent breast cancer cell line.
Notably, it was found that LCC6MDR cells are about 57.4 folds more
As expected, the introduction of multi-methoxy-substituted aryl
groups to the 2-amino group of the imidazole ring has substantially
increased compound lipophilicity, which possibly could improve
the P-gp modulating activity of Naamidine analogues. Specifically,
resistant to Taxol treatment (IC50 ¼ 149.3
11.3 nM) than their
parental LCC6 cells (IC50 ¼ 2.6 0.5 nM), which presumably should
be due to the over-expression of P-gp. In order to measure the MDR
reversal activity of Naamidine analogues, relative fold (RF) was
used, which essentially is the ratio of the IC50 obtained without
compounds 12a (RF ¼ 4.7, CLogP
¼
4.38), 12b (RF
¼
4.0,
CLogP ¼ 3.85), 16a (RF ¼ 3.7, CLogP ¼ 6.31) and 17a (RF ¼ 5.0,
CLogP ¼ 7.08) have exhibited similar modulating activity, using
Verapamil (RF ¼ 4.5, CLogP ¼ 4.47) as the positive control. In
addition, the modulating activity of 12a (RF ¼ 4.7) is at least 2 folds
modulator to the one with modulator (1
mM) (Table 1). Verapamil, a
well-known P-gp modulator, has been employed as the positive

78
N. Zhang et al. / European Journal of Medicinal Chemistry 83 (2014) 74e83
Table 1
Table 2
P-gp modulating activity of Naamidine analogues.
EC50 and in vitro cytotoxicity of 12c and 17c.
Compounds (1
m
M)
CLogPa
Mean IC50 of taxol (nM)b
RFc
1.0
Compounds Cytotoxicity (IC50, m
EC50 (nM)a M)b
LCC6MDRd
LCC6e
Verapamil
9a
9b
9c
12a
12b
12c
13a
14a
15a
15b
16a
16b
17a
17b
17c
e
149.3 11.3
2.6 0.5
LCC6
LCC6MDR
L929c
e
e
12c
17c
212.5 22.6
210.5 15.0
45.2 4.65
13.7 2.10
38.3 10.75
9.1 1.85
47.4 6.75
18.2 6.90
4.47
3.39
2.87
4.44
4.38
3.85
5.42
5.14
5.05
6.77
6.25
6.31
5.79
7.08
6.56
8.14
5.20
32.9 3.5
120.1 9.4
164.1 11.4
147.3 3.3
30.1 4.6
37.9 2.7
5.8 0.6
100.7 16.3
66.4 1.1
58.7 4.0
118.0 10.9
40.9 2.6
89.8 9.4
30.3 0.0
92.1 2.5
6.2 1.5
4.5
1.2
0.9
1.0
4.7
4.0
26.4
1.5
2.3
2.6
1.3
3.7
1.7
5.0
1.6
24.5
1.1
a
EC50 was defined as the concentration of modulators required to reduce the IC50
by half.
b
IC50 values were determined for LCC6, LCC6MDR and L929 cells after they were
treated with a series of concentrations of 12c and 17c. Each experiment has been
repeated two to three times, and the data were presented as the mean standard
error of mean.
c
L929 is a mouse connective tissue fibroblast cell line.
the in vitro cytotoxicity tests described as follows, the IC50 values of
12c and 17c were determined using cancer cell lines LCC6 and
LCC6MDR as well as normal mouse fibroblast cell line L929. Spe-
cifically, compound 17c (IC50 z 10
cytotoxic activity for three cell lines, whereas compound 12c
(IC50 z 40 M) showed weaker cytotoxic activity, which clearly
mM) has exhibited moderate
18b
138.8 7.8
a
m
CLogP values were generated using software Sybyl 2.0.
b
IC50 values were determined using LCC6MDR cells after they were treated with
different concentrations of Taxol in the presence of 1 M Naamidine analogues or
suggests that 12c should be a safer MDR modulator.
m
Verapamil. Each experiment has been repeated two times and the data were pre-
sented as the mean standard error of mean.
3. Conclusions
c
RF ¼ (IC50 without modulator)/(IC50 with modulator).
d
No modulator was used in LCC6MDR cells.
In this study, a series of novel 4,5-di-substituted benzyl-1-
methyl-1H-imidazol-2-substituted amines have been designed,
synthesized and evaluated for their P-gp-mediated MDR reversal
activity. These synthetic compounds have exhibited moderate to
excellent P-gp modulating activity against P-gp over-expressing
breast cancer cell line LCC6MDR. Results obtained from pre-
liminary SAR study suggest that compound lipophilicity is a critical
factor for the P-gp modulating activity. Notably, increasing the
number of methoxy groups on the phenyl groups presiding at the
2-position of the imidazole ring seems to be beneficial. On the other
hand, it appears that altering the substituents of the benzyl groups
located at the 4,5-position of the imidazole ring can significantly
impact the reversal activity as well. To our delight, compounds 12c
and 17c, which possess 4-(diethylamino) benzyl group at the 5-
position of imidazole, have displayed the best P-gp modulating
e
LCC6 cells were used without adding modulator.
higher than 13a (RF ¼ 1.5) and 14a (RF ¼ 2.3); compounds 16a
(RF ¼ 3.7) and 16b (RF ¼ 1.7) also exhibited higher RF values than
15a (RF ¼ 2.6) and 15b (RF ¼ 1.3) respectively. These results suggest
that increasing the number of methoxy groups on the phenyl ring at
the 2-position of the imidazole ring is beneficial to the reversal
activity, in accordance with the results obtained from our previous
studies. Moreover, as disclosed recently [25,35], multiple methoxy
substituents usually provide additional hydrogen bond acceptors,
which should promote the binding to P-gp proteins.
Interestingly, we found that altering the substituents on the
benzyl groups at the 4,5-positions of the imidazole ring could also
significantly impact the reversal activity. Compared with the 4,5-
bis-4-methoxybenzyl Naamidine analogues, the 4,5-bis-3,4-
dimethoxybenzyl Naamidine analogues contain more methoxy
groups but possess similar lipophilicity. However, none of them has
exhibited decent reversal activity except compound 12b (RF ¼ 4.0).
Specifically, compounds 15b (RF ¼ 1.3, CLogP ¼ 6.25), 16b (RF ¼ 1.7,
CLogP ¼ 5.79) and 17b (RF ¼ 1.6, CLogP ¼ 6.56) have displayed
much weaker modulating activity than compounds 15a (RF ¼ 2.6,
CLogP ¼ 6.77), 16a (RF ¼ 3.7, CLogP ¼ 6.31) and 17a (RF ¼ 5.0,
CLogP ¼ 7.08). To our delight, when the 5-position of imidazole ring
was substituted with a 4-(diethylamino)benzyl group, the reversal
activity has been greatly improved. It appear that compounds 12c
(RF ¼ 26.4) and 17c (RF ¼ 24.5) have exhibited the best reversal
activity, about 5e6 folds more potent than Verapamil (RF ¼ 4.5).
These results clearly indicated that modifying the benzyl groups at
the 4,5-positions of the imidazole ring could be the most viable way
to improve the binding affinity.
activity at a 1 mM concentration. As a matter of fact, they can re-
sensitized LCC6MDR cells towards Taxol by 26.4 and 24.5 folds,
with a EC50 212.5 and 210.5 nM, respectively. Importantly, we found
that compound 12c displayed little in vitro cytotoxicity to either
cancer cell lines or normal mouse fibroblast cell lines, exhibiting a
better safety profile. In summary, this work has successfully
demonstrated that the synthetic Naamidine analogues can be
employed as effective, safe modulators for the P-gp-mediated MDR
in cancer cells.
4. Experimental section
4.1. General
All moisture sensitive reactions were conducted under a nitro-
gen atmosphere in anhydrous, freshly distilled solvents. Anhydrous
THF was distilled under nitrogen from Na. Starting materials and
reagents, unless otherwise stated, were commercial grades and
used without further purification. p-methoxyphenylmagnesium
bromide (10) and 1,2-dimethoxyphenylmagnesium bromide (11)
were prepared by literature procedures [36]. Acyl chlorides were
prepared by corresponding acids and oxalyl chloride [37].
We next examined the EC50 and in vitro cytotoxicity of com-
pounds 12c and 17c, in an effort to quantitatively evaluate their
reversal potency and safety profile, as shown in Table 2. The EC50 is
defined as the concentration of modulators required to reduce the
IC50 by half. Each experiment has been repeated two or three times,
and the data were presented as the mean standard error of mean.
It was found that both 12c (EC50 ¼ 212.5
22.6 nM) and 17c
(EC50 ¼ 210.5 15.0 nM) have produced a pronounced effect on
All reactions were monitored by thin-layer chromatography
(TLC), on aluminum sheets (Silica gel 60-F254, E. Merck). Com-
pounds were visualized by UV light. Flash chromatography was
reversing the Taxol cytotoxicity in the MDA435/LCC6 MDR cells. In

N. Zhang et al. / European Journal of Medicinal Chemistry 83 (2014) 74e83
79
carried out using silica-gel 60 (200e300 mesh). Silicagel chroma-
tography solvents were analytical grade. Melting points were
determined with an X-4 digital micro melting point tester (Taike
Ltd., Beijing, China) and were uncorrected. 1H NMR (600 or
500 MHz) and 13C NMR (150 or 126 MHz) spectra were measured
with TMS as an internal standard when CDCl3 or DMSO-d6 were
used as a solvent. Chemical shifts are expressed in d (ppm) and
coupling constants in J (Hz). High resolution (ESI) MS spectra were
recorded using a QTOF-2 Micromass spectrometer.
white solid; mp 96e98 ꢀC; 1H NMR (500 MHz, CDCl3)
d
7.39 (s, 1H),
6.78 (d, J ¼ 8.2 Hz, 1H), 6.68 (d, J ¼ 1.6 Hz, 1H), 6.64 (d, J ¼ 8.1 Hz,
1H), 3.92 (s, 2H), 3.85 (s, 3H), 3.83 (s, 3H), 3.45 (s, 3H); 13C NMR
(126 MHz, CDCl3)
d 149.25, 147.90, 139.38, 133.27, 129.69, 119.83,
111.26, 111.17, 84.75, 55.91, 55.86, 32.54, 30.26; HRMS calcd for
(C13H15N2O2I þ H)þ 359.0251, found 359.0245.
4.1.5. N,N-diethyl-4-((4-iodo-1-methyl-1H-imidazol-5-yl)methyl)
benzenamine (4c)
4c was prepared as described for the synthesis of 4b using
Et3SiH (32 ml, 200 mmol), BF3$Et2O (25.9 ml, 200 mmol), 3c (34.6 g,
90 mmol) and anhydrous THF (500 ml). 4c (28.5 g, 86%) was ob-
tained as a white solid; mp 115e117 ꢀC; 1H NMR (500 MHz, CDCl3)
4.1.1. 4,5-Bis(4-methoxybenzyl)-1-methyl-1H-imidazol-2-amine
(9a)
9a was prepared by literature procedures [28] as a white solid;
mp 162e163 ꢀC; 1H NMR (600 MHz, CDCl3)
d
7.12 (d, J ¼ 8.6 Hz, 2H),
d
7.36 (s, 1H), 6.95 (d, J ¼ 8.6 Hz, 2H), 6.60 (d, J ¼ 8.7 Hz, 2H), 3.85 (s,
6.95 (d, J ¼ 8.6 Hz, 2H), 6.81 (dd, J ¼ 6.8, 4.8 Hz, 2H), 6.79 (d,
2H), 3.44 (s, 3H), 3.31 (q, J ¼ 7.1 Hz, 4H), 1.13 (t, J ¼ 7.1 Hz, 6H); 13
C
J ¼ 8.6 Hz, 2H), 3.78 (s, 5H), 3.75 (s, 3H), 3.74 (s, 2H), 3.17 (s, 3H); 13
C
NMR (126 MHz, CDCl3) d 146.66, 139.16, 133.87, 128.86, 123.57,
NMR (151 MHz, CDCl3)
d
158.60, 158.38, 147.04, 130.66, 129.57,
112.07, 84.38, 44.34, 32.55, 29.69, 12.53; HRMS calcd for
129.10, 128.96, 126.58, 121.28, 114.33, 114.15, 55.43, 55.31, 30.62,
29.95, 28.16; HRMS calcd for (C20H23N3O2 þ H)þ 338.1863, found
338.1857.
(C15H20N3I þ H)þ 370.0775, found 370.0774.
4.1.6. 5-(3,4-Dimethoxybenzyl)-1-methyl-1H-imidazole-4-
carbaldehyde (5b)
4.1.2. (4-Iodo-1-methyl-1H-imidazol-5-yl)(3,4-dimethoxyphenyl)
methanol (3b)
EtMgBr (2.0 M solution in THF, 40 ml, 80 mmol) was added to a
solution of 4b (26.8 g, 75 mmol) in anhydrous THF (300 ml) at 25 ꢀC
under N2 atmosphere. The resulting mixture was stirred for 30 min
and N-methylformanilide (10.2 ml, 75 mmol) was added to this
solution. After stirred at 25 ꢀC for 12 h, sat. NH4Cl (30 ml) was added
to quench the reaction. Then the THF was removed under reduced
pressure and the resulting mixture was extracted with CH2Cl2
(100 ml) three times. The organic layer was dried by anhydrous
Na2SO4 and concentrated to give a yellow oil. The residue was
purified by chromatography (acetoacetate/MeOH ¼ 30/1, v/v) to
give 5b (16.9 g, 87%) as a light yellow solid; mp 90e92 ꢀC; 1H NMR
EtMgBr (2.0 M solution in THF, 55 ml, 110 mmol) was added to a
solution of 4,5-diiodo-1-methyl-1H-imidazole (35.1 g, 105 mmol)
in anhydrous THF (500 ml) at 25 ꢀC under N2 atmosphere. The
resulting mixture was stirred for 30 min and 3,4-
dimethoxybenzaldehyde (17.4 g, 105 mmol) was added to this so-
lution. After stirred at 25 ꢀC for 24 h, sat. NH4Cl (50 ml) was added
to quench the reaction and the THF was removed under reduced
pressure. Then the resulting suspension was filtered, washed by
H2O (300 ml) and Et2O (50 ml), and dried in vacuo to give 3b
(35.3 g, 90%) as a white solid; mp 193e195 ꢀC; 1H NMR (500 MHz,
(500 MHz, CDCl3)
6.72 (s, 1H), 6.65 (d, J ¼ 8.1 Hz, 1H), 4.34 (s, 2H), 3.84 (s, 3H), 3.80 (s,
3H), 3.49 (s, 3H); 13C NMR (126 MHz, CDCl3)
187.61,149.31,148.00,
d
10.01 (s, 1H), 7.44 (s, 1H), 6.76 (d, J ¼ 8.1 Hz, 1H),
DMSO-d6)
d
7.59 (s, 1H), 6.95 (d, J ¼ 1.4 Hz, 1H), 6.91 (d, J ¼ 8.3 Hz,
1H), 6.71e6.66 (m, 1H), 6.24e6.21 (m, 1H), 5.80 (d, J ¼ 3.9 Hz, 1H),
d
3.72 (s, 3H), 3.72 (s, 3H), 3.41 (s, 3H); 13C NMR (126 MHz, DMSO-d6)
138.74, 138.20, 137.51, 128.93, 120.09, 111.49, 111.27, 55.88, 55.83,
31.52, 28.81; HRMS calcd for (C14H16N2O3 þ H)þ 261.1234, found
261.1229.
d
149.00, 148.18, 141.75, 135.21, 134.77, 117.68, 112.08, 109.57, 85.56,
66.49, 55.94, 55.83, 33.11; HRMS calcd for (C13H15N2O3I þ H)þ
375.0200, found 375.0196.
4.1.7. 5-(4-(Diethylamino)benzyl)-1-methyl-1H-imidazole-4-
carbaldehyde (5c)
4.1.3. (4-(Diethylamino)phenyl)(4-iodo-1-methyl-1H-imidazol-5-
yl)methanol (3c)
5c was prepared as described for the synthesis of 5b using
EtMgBr (2.0 M solution in THF, 40 ml, 80 mmol), 4c (27.6 g,
75 mmol), N-methylformanilide (10.2 ml, 75 mmol) and anhydrous
THF (300 ml). 5c (17.7 g, 86%) was obtained as a white solid; mp
3c was prepared as described for the synthesis of 3b using
EtMgBr (2.0 M solution in THF, 55 ml, 110 mmol), 4,5-diiodo-1-
methyl-1H-imidazole (35.1 g, 105 mmol), 4-(diethylamino)benzal-
dehyde (18.6 g, 105 mmol) and anhydrous THF (500 ml). 3c (35.9 g,
89%) was obtained as a white solid; mp 176e178 ꢀC; 1H NMR
76e78 ꢀC; 1H NMR (500 MHz, CDCl3)
d 9.99 (s, 1H), 7.39 (s, 1H), 6.96
(d, J ¼ 8.6 Hz, 2H), 6.57 (d, J ¼ 8.7 Hz, 2H), 4.26 (s, 2H), 3.47 (s, 3H),
(500 MHz, DMSO-d6)
d
7.56 (s, 1H), 7.03 (d, J ¼ 8.6 Hz, 2H), 6.62 (d,
3.30 (q, J ¼ 7.1 Hz, 4H), 1.12 (t, J ¼ 7.0 Hz, 6H); 13C NMR (126 MHz,
J ¼ 8.8 Hz, 2H), 6.00 (d, J ¼ 4.0 Hz, 1H), 5.75 (d, J ¼ 3.9 Hz, 1H), 3.42
CDCl3) d 187.20, 146.71, 138.71, 138.63, 138.07, 129.13, 122.64, 112.12,
(s, 3H), 3.29 (q, J ¼ 7.0 Hz, 4H), 1.05 (t, J ¼ 7.0 Hz, 6H); 13C NMR
44.32, 31.56, 28.18, 12.49; HRMS calcd for (C14H16N2O3 þ H)þ
(126 MHz, DMSO-d6)
d
146.89, 141.57, 135.57, 128.48, 126.64, 111.69,
272.1757, found 272.1756.
85.17, 66.62, 44.04, 33.12, 12.85; HRMS calcd for (C15H20N3OI þ H)þ
386.0724, found 386.0717.
4.1.8. (5-(3,4-Dimethoxybenzyl)-1-methyl-1H-imidazol-4-yl)(3,4-
dimethoxyphenyl)methanol (6b)
4.1.4. 5-(3,4-Dimethoxybenzyl)-4-iodo-1-methyl-1H-imidazole
(4b)
10 (2.0 M solution in THF, 35 ml, 70 mmol) was added to a so-
lution of 5b (16.9 g, 65 mmol) in anhydrous THF (200 ml). After
stirred at 25 ꢀC for 12 h under N2 atmosphere, sat. NH4Cl (20 ml)
was added to quench the reaction. Then the THF was removed
under reduced pressure and the resulting mixture was extracted
with CH2Cl2 (100 ml) three times. The organic layer was dried by
anhydrous Na2SO4 and concentrated to give a brown semi solid.
The residue was purified by chromatography (acetoacetate/
MeOH ¼ 10/1, v/v) to give 6b (24.1 g, 93%) as a white solid; mp
Et3SiH (32 ml, 200 mmol) and BF3$Et2O (25.9 ml, 200 mmol)
were slowly added to a solution of 3b (33.7 g, 90 mmol) in anhy-
drous THF (500 ml) at 0 ꢀC under N2 atmosphere. Then the resulting
mixture was heated to 60 ꢀC. After stirred for 24 h, the reaction was
cooled to room temperature and quenched by the addition of
saturated aqueous solution of NaHCO3. Then the THF was removed
under reduced pressure and the resulting mixture was extracted
with CH2Cl2 (100 ml) three times. The organic layer was dried by
anhydrous Na2SO4 and concentrated to give 4b (29.6 g, 92%) as a
45e47 ꢀC; 1H NMR (500 MHz, CDCl3)
d
7.38 (s, 1H), 7.06 (s, 1H), 6.93
(d, J ¼ 8.0 Hz, 1H), 6.75 (d, J ¼ 8.2 Hz, 1H), 6.71 (d, J ¼ 8.2 Hz, 1H),

80
N. Zhang et al. / European Journal of Medicinal Chemistry 83 (2014) 74e83
6.52 (d, J ¼ 8.0 Hz, 1H), 6.45 (s, 1H), 5.77 (s, 1H), 3.84 (s, 2H), 3.83 (s,
51%) as a light brown solid; mp 71e74 ꢀC; 1H NMR (500 MHz,
CDCl3)
6H), 3.79 (s, 3H), 3.66 (s, 3H), 3.35 (s, 3H); 13C NMR (126 MHz,
d
6.89 (d, J ¼ 1.6 Hz, 1H), 6.79 (dd, J ¼ 8.2, 1.5 Hz, 1H),
CDCl3)
d
149.17, 148.89, 148.19, 147.69, 141.05, 136.84, 136.53, 130.31,
6.76e6.71 (m, 2H), 6.56 (dd, J ¼ 8.2, 1.4 Hz, 1H), 6.45 (d, J ¼ 1.6 Hz,
125.76, 119.87, 118.80, 111.23, 111.13, 110.76, 109.98, 69.74, 55.89,
55.87, 55.76, 55.70, 31.62, 28.56; HRMS calcd for (C22H26N2O5 þ H)þ
399.1914, found 399.1908.
1H), 3.85 (s, 2H), 3.83 (s, 3H), 3.83 (s, 3H), 3.82 (s, 2H), 3.81 (s, 3H),
3.67 (s, 3H), 3.09 (s, 3H); 13C NMR (126 MHz, CDCl3)
d 149.19,
148.83, 147.71, 147.32, 139.07, 136.09, 133.28, 130.54, 124.76, 120.27,
119.83, 112.01, 111.16, 111.10, 111.06, 55.90, 55.89, 55.78, 55.70, 33.14,
29.46, 29.11; HRMS calcd for (C22H25N5O4 þ H)þ 424.1979, found
424.1973.
4.1.9. (5-(4-(Diethylamino)benzyl)-1-methyl-1H-imidazol-4-
yl)(3,4-dimethoxyphenyl)methanol (6c)
6c was prepared as described for the synthesis of 6b using 11
(2.0 M solution in THF, 35 ml, 70 mmol) and 5c (17.6 g, 65 mmol). 6c
(22.9 g, 86%) was obtained as a white solid; mp 117e119 ꢀC; 1H NMR
4.1.13. 4-((4-(3,4-Bimethoxybenzyl)-2-azido-1-methyl-1H-
imidazol-5-yl)methyl)-N,N-diethylbenzenamine (8c)
(500 MHz, CDCl3)
d
7.35 (s, 1H), 7.00 (s, 1H), 6.94 (d, J ¼ 7.7 Hz, 1H),
8c was prepared as described for the synthesis of 8b using 7c
(5.89 g, 15 mmol), n-Butyl lithium (2.5 M solution in hexanes,
13.2 ml, 33 mmol) and TsN3 (10.2 g, 52 mmol). 8c (2.72 g, 42%) was
obtained as a white solid; mp 101e103 ꢀC; 1H NMR (500 MHz,
6.80 (d, J ¼ 8.2 Hz, 2H), 6.77 (d, J ¼ 8.2 Hz, 1H), 6.54 (d, J ¼ 8.5 Hz,
2H), 5.75 (s, 1H), 3.83 (s, 3H), 3.78 (s, 4H), 3.36 (s, 3H), 3.29 (q,
J ¼ 7.0 Hz, 4H), 1.12 (t, J ¼ 7.0 Hz, 6H); 13C NMR (126 MHz, CDCl3)
d
148.81, 148.09, 146.49, 136.69, 128.82, 126.35, 124.22, 118.75,
CDCl3)
d
6.85 (s, 1H), 6.83 (s, 2H), 6.80 (dd, J ¼ 8.2, 1.8 Hz, 1H), 6.76
112.07, 110.80, 109.92, 69.71, 55.87, 55.72, 44.31, 31.64, 27.85, 12.51;
(d, J ¼ 8.2 Hz, 1H), 6.56 (d, J ¼ 8.7 Hz, 2H), 3.83 (s, 5H), 3.79 (s, 3H),
HRMS calcd for (C24H31N3O3 þ H)þ 410.2438, found 410.2433.
3.77 (s, 2H), 3.30 (q, J ¼ 7.0 Hz, 4H), 3.11 (s, 3H), 1.12 (t, J ¼ 7.0 Hz,
6H); 13C NMR (126 MHz, CDCl3)
d 148.77, 147.23, 146.50, 138.75,
4.1.10. 4,5-Bis(3,4-dimethoxybenzyl)-1-methyl-1H-imidazole (7b)
6b (11.9 g, 30 mmol) was dissolved in MeOH (200 ml) and acetic
acid (20 ml). This reaction mixture was stirred under a hydrogen
atmosphere (40 atm) in the presence of 10% Pd/C (200 mg) at 50 ꢀC
for 24 h. The catalyst was filtered and the filtrate was concentrated.
Then the residue was neutralized by sat. Na2CO3 and extracted with
CH2Cl2 (100 ml) three times. The organic layer was dried by
anhydrous Na2SO4 and concentrated to give a brown oil. The res-
idue was purified by chromatography (acetoacetate/MeOH ¼ 10/1,
v/v) to give 7b (8.25 g, 72%) as a white solid; mp 73e76 ꢀC; 1H NMR
135.72, 133.37, 128.73, 125.45, 124.45, 120.36, 112.08, 112.00, 111.11,
55.88, 55.73, 44.32, 33.14, 29.49, 28.36, 12.50.
4.1.14. 4,5-Bis(3,4-dimethoxybenzyl)-1-methyl-1H-imidazol-2-
amine (9b)
To a stirred solution of 8b (2.12 g, 5 mmol) in MeOH (100 ml),
NaBH4 (1.89 g, 50 mmol) was slowly added at 0 ꢀC. Then the re-
action mixture was heated to 25 ꢀC. After stirred at 25 ꢀC for 12 h,
the reaction mixture was quenched by the addition of sat. NH4Cl
(20 ml). Then the MeOH was removed under reduced pressure and
the resulting mixture was extracted with CH2Cl2 (30 ml) three
times. The organic layer was dried by anhydrous Na2SO4 and
concentrated to give 9b (1.75 g, 88%) as a white solid; mp
(500 MHz, CDCl3)
d
7.36 (s, 1H), 6.84 (d, J ¼ 1.5 Hz, 1H), 6.80 (dd,
J ¼ 8.2, 1.6 Hz, 1H), 6.75 (d, J ¼ 4.2 Hz, 1H), 6.73 (d, J ¼ 4.2 Hz, 1H),
6.55 (dd, J ¼ 8.1, 1.2 Hz, 1H), 6.44 (d, J ¼ 1.6 Hz, 1H), 3.90 (s, 2H), 3.88
(s, 2H), 3.83 (s, 3H), 3.82 (s, 3H), 3.79 (s, 3H), 3.66 (s, 3H), 3.34 (s,
160e164 ꢀC; 1H NMR (600 MHz, CDCl3)
d 6.80 (s, 1H), 6.76 (d,
3H); 13C NMR (126 MHz, CDCl3)
d
149.16, 148.83, 147.64, 147.21,
J ¼ 8.2 Hz,1H), 6.74 (s, 2H), 6.55 (d, J ¼ 8.1 Hz,1H), 6.51 (d, J ¼ 1.8 Hz,
138.81, 136.66, 133.72, 130.81, 125.53, 120.32, 119.85, 111.98, 111.16,
111.12, 55.90, 55.75, 55.67, 33.45, 31.68, 28.70; HRMS calcd for
(C22H26N2O4 þ H)þ 383.1965, found 383.1959.
1H), 3.84 (s, 3H), 3.81 (s, 3H), 3.80 (s, 5H), 3.75 (s, 2H), 3.73 (s, 3H),
3.28 (s, 3H); 13C NMR (151 MHz, CDCl3)
d 149.53, 149.33, 148.30,
148.10, 146.92, 129.89, 128.62, 123.19, 121.14, 120.50, 119.94, 111.99,
111.59, 111.13, 56.15, 56.06, 56.01, 55.95, 30.23, 30.12, 28.32; HRMS
calcd for (C22H27N3O4 þ H)þ 398.2074, found 398.2068.
4.1.11. 4-((4-(3,4-Dimethoxybenzyl)-1-methyl-1H-imidazol-5-yl)
methyl)-N,N-diethylbenzenamine (7c)
7c was prepared as described for the synthesis of 7b using 6c
(12.3 g, 30 mmol) and 10% Pd/C (200 mg). 7c (9.1 g, 77%) was ob-
tained as a white solid; mp 108e120 ꢀC; 1H NMR (500 MHz, CDCl3)
4.1.15. 4-(3,4-Dimethoxybenzyl)-5-(4-(diethylamino)benzyl)-1-
methyl-1H-imidazol-2-amine (9c)
9c was prepared as described for the synthesis of 9b using 8c
(2.17 g, 5 mmol) and NaBH4 (0.95 g, 25 mmol). 9c (1.69 g, 83%) was
obtained as a white solid; mp 113e117 ꢀC; 1H NMR (600 MHz,
d
7.34 (s, 1H), 6.84 (d, J ¼ 8.7 Hz, 2H), 6.82e6.79 (m, 2H), 6.75 (d,
J ¼ 7.9 Hz, 1H), 6.56 (d, J ¼ 8.7 Hz, 2H), 3.87 (s, 2H), 3.85 (s, 2H), 3.82
(s, 3H), 3.78 (s, 3H), 3.36 (s, 3H), 3.30 (q, J ¼ 7.1 Hz, 4H), 1.12 (t,
CDCl3)
d
7.23 (s, 2H), 6.86 (d, J ¼ 8.7 Hz, 2H), 6.77 (s, 3H), 6.58 (d,
J ¼ 7.1 Hz, 6H); 13C NMR (126 MHz, CDCl3)
d
148.78, 147.14, 146.46,
J ¼ 8.7 Hz, 2H), 3.82 (s, 3H), 3.82 (s, 3H), 3.74 (s, 4H), 3.32 (q,
138.39, 136.41, 133.79, 128.78, 126.19, 124.75, 120.44, 112.09, 111.98,
111.20, 55.91, 55.70, 44.33, 33.44, 31.72, 27.96, 12.52; HRMS calcd
for (C24H31N3O2 þ H)þ 394.2489, found 394.2483.
J ¼ 7.1 Hz, 4H), 3.29 (s, 3H), 1.14 (t, J ¼ 7.1 Hz, 6H); 13C NMR
(151 MHz, CDCl3)
d 149.28, 148.05, 146.96, 146.84, 129.71, 128.79,
122.29, 122.08, 121.77, 120.61, 112.26, 111.80, 111.52, 56.08, 55.99,
44.43, 30.26, 30.02, 27.62,12.59; HRMS calcd for (C24H32N4O2 þ H)þ
409.2598, found 409.2595.
4.1.12. 4,5-Bis(3,4-dimethoxybenzyl)-2-azido-1-methyl-1H-
imidazole (8b)
n-Butyl lithium (2.5 M solution in hexanes, 13.2 ml, 33 mmol)
was added dropwise to a stirred solution of 7b (5.73 g, 15 mmol) in
anhydrous THF (100 ml) at ꢁ78 ꢀC. Then the reaction was stirred for
0.5 h at the 25 ꢀC. Then the reaction was cooled to ꢁ78 ꢀC again and
TsN3 (10.2 g, 52 mmol) was added. After stirring for 1 h at 25 ꢀC, the
reaction mixture was quenched by the addition of sat. NH4Cl
(20 ml). Then the THF was removed under reduced pressure and
the resulting mixture was extracted with CH2Cl2 (50 ml) three
times. The organic layer was dried by anhydrous Na2SO4 and
concentrated to give a pale brown oil, which was purified by
chromatography (ligroin/acetoacetate ¼ 2/1, v/v) to give 8b (3.23 g,
4.1.16. N-(4,5-bis(4-methoxybenzyl)-1-methyl-1H-imidazol-2-yl)-
3,4,5-trimethoxybenzamide (12a)
n-Butyl lithium (2.5 M solution in hexanes, 0.4 ml, 1.0 mmol)
was added dropwise to a stirred solution of 9a (0.337 g, 1 mmol) in
anhydrous THF (20 ml) at ꢁ78 ꢀC under N2 atmosphere and the
reaction was stirred for 0.5 h at rt. Then the reaction mixture was
cooled to ꢁ78 ꢀC again and 3,4,5-Trimethoxybenzoyl chloride
(0.23 g, 1 mmol) dissolved in anhydrous THF (5 ml) was added.
After stirring for 2 h at 25 ꢀC, the reaction mixture was quenched by
the addition of sat. NH4Cl (20 ml). Then the THF was removed under
reduced pressure and the resulting mixture was extracted with

N. Zhang et al. / European Journal of Medicinal Chemistry 83 (2014) 74e83
81
CH2Cl2 (30 ml) three times. The organic layer was dried by anhy-
drous Na2SO4 and concentrated. The residue was purified by
chromatography (ligroin/EtOAc ¼ 1/1, v/v) to give 12a (0.351 g, 66%)
6.84 (t, J ¼ 1.8 Hz, 2H), 5.30 (s, 1H), 3.92 (s, 2H), 3.84 (s, 2H), 3.79 (s,
3H), 3.79 (s, 3H), 3.37 (s, 3H); 13C NMR (126 MHz, CDCl3)
174.55,
158.63, 158.60, 138.42, 130.49,129.27, 129.08, 128.83, 128.71,128.62,
127.77,120.73,114.41,114.31, 55.31, 55.29, 29.69, 28.91, 27.98; HRMS
calcd for (C27H27N3O3 þ H)þ 442.2125, found 442.2125.
d
as a white solid; mp 117e120 ꢀC; 1H NMR (600 MHz, CDCl3)
d 7.53
(s, 2H), 7.10 (d, J ¼ 8.6 Hz, 2H), 7.03 (d, J ¼ 8.6 Hz, 2H), 6.86e6.82 (m,
4H), 3.92 (s, 2H), 3.91 (s, 6H), 3.88 (s, 3H), 3.84 (s, 2H), 3.79 (s, 3H),
3.79 (s, 3H), 3.37 (s, 3H); 13C NMR (151 MHz, CDCl3)
d
158.76,158.72,
4.1.21. N-(4,5-bis(4-methoxybenzyl)-1-methyl-1H-imidazol-2-yl)-
N-benzoylbenzamide (15a)
152.66, 140.39, 134.06, 129.39, 129.10, 128.95, 128.77, 120.89, 114.53,
114.42, 105.85, 60.94, 56.15, 55.42, 55.40, 55.38, 29.78, 28.91, 28.07;
HRMS calcd for (C30H33N3O6 þ H)þ 532.2442, found 532.2430.
Benzoyl chloride (0.16 g, 1.1 mmol) dissolved in anhydrous THF
(5 ml) and DIPEA (1 ml) was added to a solution of 9a (0.169 g,
0.5 mmol) in anhydrous THF (10 ml) at 25 ꢀC under N2 atmosphere.
After stirred for 24 h under N2 atmosphere, the reaction was
quenched by the addition of sat.NaHCO3 (10 ml). Then the THF was
removed under reduced pressure and the resulting mixture was
extracted with CH2Cl2 (30 ml) three times. The organic layer was
dried by anhydrous Na2SO4 and concentrated. The residue was
purified by chromatography (ligroin/EtOAc ¼ 1/1, v/v) to give 15a
(0.179 g, 66%) as a white solid; mp 135e137 ꢀC; 1H NMR (600 MHz,
4.1.17. N-(4,5-bis(3,4-dimethoxybenzyl)-1-methyl-1H-imidazol-2-
yl)-3,4,5-trimethoxybenzamide (12b)
12b was prepared as described for the synthesis of 12a using 9b
(0.198 g, 0.5 mmol), n-Butyl lithium (2.5 M solution in hexanes,
0.2 ml, 0.5 mmol) and 3,4,5-Trimethoxybenzoyl chloride (0.12 g,
0.5 mmol). 12b (0.213 g, 72%) was obtained as a white solid; mp
120e122 ꢀC; 1H NMR (600 MHz, CDCl3)
d 7.53 (s, 2H), 6.79 (s, 1H),
6.78 (d, J ¼ 1.6 Hz, 1H), 6.75 (dd, J ¼ 8.2, 1.9 Hz, 1H), 6.65 (d,
J ¼ 1.9 Hz,1H), 6.63 (d, J ¼ 8.2 Hz,1H), 6.60 (d, J ¼ 1.8 Hz,1H), 3.94 (s,
2H), 3.91 (s, 6H), 3.88 (s, 3H), 3.86 (s, 3H), 3.85 (s, 5H), 3.80 (s, 3H),
CDCl3)
d
7.80 (d, J ¼ 7.4 Hz, 4H), 7.53 (t, J ¼ 7.4 Hz, 2H), 7.37 (t,
J ¼ 7.8 Hz, 4H), 6.89 (d, J ¼ 8.5 Hz, 2H), 6.69 (d, J ¼ 8.5 Hz, 2H), 6.65
(d, J ¼ 8.6 Hz, 2H), 6.54 (d, J ¼ 8.6 Hz, 2H), 3.81 (s, 2H), 3.78 (s, 2H),
3.77 (s, 3H), 3.76 (s, 3H), 3.10 (s, 3H); 13C NMR (151 MHz, CDCl3)
3.78 (s, 3H), 3.40 (s, 3H); 13C NMR (151 MHz, CDCl3)
d 152.69,
149.43, 148.24, 140.47, 129.65, 129.28, 120.40, 119.89, 111.66, 111.49,
111.13, 105.85, 60.95, 56.16, 56.06, 55.98, 30.31, 29.09, 28.54; HRMS
calcd for (C32H37N3O8 þ H)þ 592.2653, found 592.2643.
d 171.81, 158.18, 157.76, 138.07, 137.57, 133.62, 132.86, 132.80, 129.57,
129.31, 129.30, 128.62, 128.60, 126.80, 114.03, 113.64, 55.37, 55.34,
32.47, 30.26, 28.58; HRMS calcd for (C34H31N3O4 þ H)þ 546.2387,
found 546.2380.
4.1.18. N-(4-(3,4-dimethoxybenzyl)-5-(4-(diethylamino)benzyl)-1-
methyl-1H-imidazol-2-yl)-3,4,5-trimethoxybenzamide (12c)
4.1.22. N-(4,5-bis(3,4-dimethoxybenzyl)-1-methyl-1H-imidazol-2-
yl)-N-benzoylbenzamide (15b)
15b was prepared as described for the synthesis of 15a using 9b
(0.199 g, 0.5 mmol) and benzoyl chloride (0.16 g, 1.1 mmol). 15b
(0.180 g, 61%) was obtained as a white solid; mp 192e194 ꢀC; 1H
12c was prepared as described for the synthesis of 12a using 9c
(0.204 g, 0.5 mmol), n-Butyl lithium (2.5 M solution in hexanes,
0.2 ml, 0.5 mmol) and 3,4,5-Trimethoxybenzoyl chloride (0.12 g,
0.5 mmol). 12c (0.138 g, 46%) was obtained as a white solid; mp
132e135 ꢀC; 1H NMR (500 MHz, CDCl3)
d
7.53 (s, 2H), 6.94 (d,
NMR (600 MHz, CDCl3)
d
7.82e7.77 (m, 4H), 7.50 (t, J ¼ 7.5 Hz, 2H),
J ¼ 8.3 Hz, 2H), 6.80 (d, J ¼ 8.1 Hz, 1H), 6.76 (d, J ¼ 8.2 Hz, 1H), 6.63
(s, 1H), 6.60 (d, J ¼ 8.4 Hz, 2H), 3.91 (s, 6H), 3.88 (s, 5H), 3.85 (s, 3H),
3.84 (s, 2H), 3.78 (s, 3H), 3.42 (s, 3H), 3.32 (q, J ¼ 7.0 Hz, 4H), 1.14 (t,
7.35 (t, J ¼ 7.8 Hz, 4H), 6.72 (d, J ¼ 1.8 Hz,1H), 6.65 (d, J ¼ 8.1 Hz,1H),
6.61 (d, J ¼ 8.2 Hz, 1H), 6.56 (dd, J ¼ 8.1, 1.7 Hz, 1H), 6.26 (d,
J ¼ 1.8 Hz, 1H), 6.24e6.22 (m, 1H), 3.83 (s, 10H), 3.72 (s, 3H), 3.55 (s,
J ¼ 7.0 Hz, 6H); 13C NMR (126 MHz, CDCl3)
d
173.87, 152.54, 149.28,
3H), 3.15 (s, 3H); 13C NMR (151 MHz, CDCl3)
d 171.80, 149.24,148.83,
148.08, 146.78, 140.26, 134.00, 129.66, 128.68, 122.95, 121.43,
120.38, 112.15, 111.55, 111.46, 105.74, 60.82, 56.04, 55.93, 55.81,
44.35, 30.18, 29.01, 27.72, 12.46; HRMS calcd for (C34H42N4O6 þ H)þ
603.3177, found 603.3169.
147.67, 147.26, 138.08, 137.54, 133.52, 132.91, 130.16, 129.36, 128.62,
126.75, 120.22, 119.54, 112.01, 111.07, 55.93, 55.76, 33.04, 30.27,
29.16; HRMS calcd for (C36H35N3O6
606.2594.
þ
H)þ 606.2599, found
4.1.19. N-(4,5-bis(4-methoxybenzyl)-1-methyl-1H-imidazol-2-yl)-
4-methoxybenzamide (13a)
4.1.23. N-(4,5-bis(4-methoxybenzyl)-1-methyl-1H-imidazol-2-yl)-
N-3,4-dimethoxybenzoyl-3,4-dimethoxybenzamide (16a)
13a was prepared as described for the synthesis of 12a using 9a
(0.169 g, 0.5 mmol), n-Butyl lithium (2.5 M solution in hexanes,
0.2 ml, 0.5 mmol) and 4-methoxybenzoyl chloride (0.08 g,
0.5 mmol). 13a (0.092 g, 39%) was obtained as a white solid; mp
16a was prepared as described for the synthesis of 15a using 9a
(0.169 g, 0.5 mmol) and 3,4-dimethoxybenzoyl chloride (0.200 g,
1 mmol). 16a (0.146 g, 44%) was obtained as a white solid; mp
171e173 ꢀC; 1H NMR (600 MHz, CDCl3)
d
7.46 (dd, J ¼ 8.4, 2.0 Hz,
134e136 ꢀC; 1H NMR (600 MHz, CDCl3)
d
8.19 (d, J ¼ 8.6 Hz, 2H), 7.10
2H), 7.40 (d, J ¼ 2.0 Hz, 2H), 6.97 (d, J ¼ 8.6 Hz, 2H), 6.76 (d,
J ¼ 8.5 Hz, 2H), 6.71 (d, J ¼ 8.6 Hz, 2H), 6.65 (d, J ¼ 8.7 Hz, 2H), 6.58
(d, J ¼ 8.6 Hz, 2H), 3.92 (s, 6H), 3.83 (s, 2H), 3.81 (s, 6H), 3.80 (s, 2H),
3.76 (s, 3H), 3.75 (s, 3H), 3.07 (s, 3H); 13C NMR (151 MHz, CDCl3)
(d, J ¼ 8.5 Hz, 2H), 7.02 (d, J ¼ 8.5 Hz, 2H), 6.88 (d, J ¼ 8.7 Hz, 2H),
6.84 (d, J ¼ 3.2 Hz, 2H), 6.83 (d, J ¼ 3.2 Hz, 2H), 3.91 (s, 2H), 3.84 (s,
5H), 3.79 (s, 3H), 3.78 (s, 3H), 3.35 (s, 3H); 13C NMR (151 MHz,
CDCl3)
d
161.77, 158.70, 158.68, 130.51, 129.38, 129.28, 128.94,
d 171.37, 158.24, 157.85, 153.05, 148.94, 138.81, 137.42, 132.88,
128.90, 120.68, 114.49, 114.40, 113.11, 55.43, 55.41, 55.38, 29.80,
28.89, 28.07; HRMS calcd for (C28H29N3O4 þ H)þ 472.2231, found
472.2220.
129.60, 129.43, 128.62, 126.56, 125.84, 123.80, 113.94, 113.62, 112.15,
110.24, 56.09, 56.04, 55.27, 42.08, 32.61, 30.23, 28.59; HRMS calcd
for (C38H39N3O8 þ H)þ 666.2810, found 666.2814.
4.1.20. N-(4,5-bis(4-methoxybenzyl)-1-methyl-1H-imidazol-2-yl)
benzamide (14a)
14a was prepared as described for the synthesis of 12a using 9a
(0.169 g, 0.5 mmol), n-Butyl lithium (2.5 M solution in hexanes,
0.2 ml, 0.5 mmol) and benzoyl chloride (0.07 g, 0.5 mmol). 14a
(0.077 g, 35%) was obtained as a white solid; mp 103e106 ꢀC; 1H
4.1.24. N-(4,5-bis(3,4-dimethoxybenzyl)-1-methyl-1H-imidazol-2-
yl)-N-3,4-dimethoxybenzoyl-3,4-dimethoxybenzamide (16b)
16b was prepared as described for the synthesis of 15a using 9b
(0.199 g, 0.5 mmol) and 3,4-dimethoxybenzoyl chloride (0.200 g,
1 mmol). 16b (0.188 g, 52%) was obtained as a white solid; mp
138e142 ꢀC; 1H NMR (600 MHz, CDCl3)
d
7.49 (dd, J ¼ 8.4, 2.0 Hz,
NMR (600 MHz, CDCl3)
d
8.24e8.21 (m, 2H), 7.43e7.36 (m, 3H), 7.10
2H), 7.42 (d, J ¼ 2.0 Hz, 2H), 6.75 (d, J ¼ 8.5 Hz, 2H), 6.73 (d,
(d, J ¼ 8.5 Hz, 2H), 7.02 (d, J ¼ 8.5 Hz, 2H), 6.85 (t, J ¼ 1.9 Hz, 2H),
J ¼ 1.8 Hz, 1H), 6.66 (d, J ¼ 8.2 Hz, 1H), 6.60e6.57 (m, 2H), 6.35 (d,

82
N. Zhang et al. / European Journal of Medicinal Chemistry 83 (2014) 74e83
J ¼ 1.7 Hz, 1H), 6.12 (d, J ¼ 8.2 Hz, 1H), 3.91 (s, 6H), 3.85 (s, 2H), 3.84
J ¼ 1.7 Hz, 1H), 6.67 (dt, J ¼ 16.5, 5.0 Hz, 3H), 6.50 (dd, J ¼ 8.2, 1.7 Hz,
1H), 6.36 (d, J ¼ 3.9 Hz, 5H), 3.89 (s, 4H), 3.82 (s, 3H), 3.81 (s, 3H),
3.80 (s, 6H), 3.78 (s, 12H), 3.73 (s, 3H), 3.60 (s, 3H), 3.11e3.04 (m,
2H), 2.90 (s, 3H), 2.90e2.83 (m, 4H), 2.73e2.65 (m, 2H); 13C NMR
(s, 2H), 3.82 (s, 6H), 3.81 (s, 6H), 3.70 (s, 3H), 3.60 (s, 3H), 3.12 (s,
3H); 13C NMR (151 MHz, CDCl3)
d 171.37, 153.10, 149.19, 148.95,
148.80, 147.69, 147.29, 138.83, 137.41, 133.47, 130.12, 126.43, 125.77,
123.88, 120.31, 119.46, 112.17, 110.99, 110.26, 56.09, 56.01, 33.10,
30.25, 29.08; HRMS calcd for (C40H43N3O10 þ H)þ 726.3021, found
726.3031.
(151 MHz, CDCl3)
d 174.38, 153.27, 149.33, 148.92, 147.80, 147.40,
137.29, 137.24, 136.50, 136.22, 133.08, 129.92, 127.06, 120.12, 119.60,
111.76, 111.26, 111.15, 110.71, 105.45, 100.00, 60.92, 60.90, 56.15,
55.98, 39.71, 33.12, 30.92, 29.85, 29.14; HRMS calcd for
(C46H55N3O12 þ H)þ 842.3859, found 842.3854.
4.1.25. (2E)-N-(4,5-bis(4-methoxybenzyl)-1-methyl-1H-imidazol-
2-yl)-3-(3,4,5-trimethoxyphenyl)-N-((E)-3-(3,4,5-
trimethoxyphenyl)acryloyl)acrylamide (17a)
4.2. Material for biological studies
17a was prepared as described for the synthesis of 15a using 8a
(0.169 g, 0.5 mmol) and (E)-3-(3,4,5-trimethoxyphenyl)acryloyl
chloride (0.256 g, 1 mmol). 17a (0.210 g, 54%) was obtained as a
DMSO and taxol were purchased from SigmaeAldrich. Dulbec-
co's
modified
Eagle's
medium
(DMEM),
white solid; mp 77e80 ꢀC; 1H NMR (600 MHz, CDCl3)
d 7.71 (d,
trypsineethylenediaminetetraacetic acid (EDTA), and penicillin/
streptomycin were from Gibco BRL. Fetal bovine serum (FBS) was
from Hyclone Laboratories. 2-(4, 5-Dimethylthiazol-2-yl)-5-[3-
(carboxymethoxy)phenyl]-2-(4-sulfophenyl)-2H-tetrazolium
(MTS) and phenazine methosulfate (PMS) were purchased from
Promega. Human breast cancer cell lines LCC6 and LCC6MDR were
kindly provided by Dr. Robert Clarke (Georgetown University,
Washington, DC). L929, a mouse connective tissue fibroblast, was
purchased from ATCC [14].
J ¼ 15.5 Hz, 2H), 7.18 (d, J ¼ 8.6 Hz, 2H), 6.85 (d, J ¼ 8.6 Hz, 2H), 6.76
(d, J ¼ 15.5 Hz, 2H), 6.73e6.69 (m, 6H), 6.59 (d, J ¼ 8.7 Hz, 2H), 3.94
(s, 2H), 3.93 (s, 2H), 3.89 (s, 6H), 3.82 (s, 12H), 3.73 (s, 3H), 3.67 (s,
3H), 3.14 (s, 3H); 13C NMR (126 MHz, CDCl3)
d 167.83, 158.29, 157.86,
153.40, 146.12, 140.70, 137.64, 137.17, 132.54, 129.82, 129.48, 129.29,
128.49, 127.15, 118.13, 114.06, 113.77, 105.83, 60.97, 56.20, 55.13,
32.68, 30.13, 28.68; HRMS calcd for (C44H47N3O10 þ H)þ 778.3334,
found 778.3334.
4.1.26. (2E)-N-(4,5-bis(3,4-dimethoxybenzyl)-1-methyl-1H-
imidazol-2-yl)-3-(3,4,5-trimethoxyphenyl)-N-((E)-3-(3,4,5-
trimethoxyphenyl)acryloyl)acrylamide (17b)
4.3. Cell culture
LCC6, LCC6MDR and L929 cells were maintained in DMEM
media supplemented with 10% (v/v) heat-inactivated FBS and 100
17b was prepared as described for the synthesis of 15a using 8b
(0.199 g, 0.5 mmol) and (E)-3-(3,4,5-trimethoxyphenyl)acryloyl
chloride (0.256 g, 1 mmol). 17b (0.183 g, 43%) was obtained as a
units/ml penicillin and 100
mg/mL streptomycin. Cells were
cultured at 37 ꢀC in a humidified atmosphere with 5% CO2. When
the cells reached confluence, they were detached by 0.05% trypsin-
EDTA [14,38].
white solid; mp 89e93 ꢀC; 1H NMR (600 MHz, CDCl3)
d 7.75 (s, 1H),
7.72 (s, 1H), 6.88 (d, J ¼ 1.9 Hz, 1H), 6.82 (s, 1H), 6.80 (s, 1H),
6.78e6.75 (m, 1H), 6.72 (s, 4H), 6.66 (d, J ¼ 8.2 Hz, 1H), 6.53 (d,
J ¼ 1.8 Hz, 1H), 6.46 (d, J ¼ 8.2 Hz, 1H), 6.38 (dd, J ¼ 8.2, 1.8 Hz, 1H),
3.97 (s, 2H), 3.94 (s, 2H), 3.89 (s, 6H), 3.83 (s, 12H), 3.81 (s, 3H), 3.75
(s, 3H), 3.73 (s, 3H), 3.68 (s, 3H), 3.16 (s, 3H); 13C NMR (151 MHz,
4.4. P-gp modulating activity
Six-thousand cells of LCC6MDR and anticancer drug taxol were
mixed with or without modulators to a final volume of 200 mL in
each well of 96-well plates. The plates were then incubated for 5
days at 37 ꢀC. The Cell Titer 96 aqueous assay was used to measure
the cell proliferation according to the manufacturer's instructions.
MTS (2 mg/mL) and PMS (0.92 mg/mL) were mixed in a ratio of
CDCl3)
d 167.90, 153.52, 149.25, 148.89, 147.78, 147.34, 146.30,
140.75, 137.68, 137.35, 133.22, 130.05, 129.93, 127.05, 120.13, 119.18,
118.24, 111.87, 111.07, 105.85, 61.08, 56.26, 55.76, 33.24, 30.27, 29.16;
HRMS calcd for (C46H51N3O12 þ H)þ 838.3546, found 838.3548.
4.1.27. (2E)-N-(4-(3,4-dimethoxybenzyl)-5-(4-(diethylamino)
benzyl)-1-methyl-1H-imidazol-2-yl)-3-(3,4,5-trimethoxyphenyl)-
N-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)acrylamide (17c)
17c was prepared as described for the synthesis of 15a using 9c
(0.204 g, 0.5 mmol) and (E)-3-(3,4,5-trimethoxyphenyl)acryloyl
chloride (0.256 g, 1 mmol). 17c (0.212 g, 50%) was obtained as a
20:1. An aliquot (10 mL) of the freshly prepared MTS/PMS mixture
was added to each well and the plate was incubated for 2 h at 37 ꢀC.
Optical absorbance at 490 nm was then recorded with a microplate
absorbance reader (Bio-Rad). IC50 values were calculated from the
doseresponse curves of MTS assays (Prism 4.0). All experiments
were performed in triplicate and repeated at least thrice, and the
results were represented as mean standard error of mean [14,39].
white solid; mp 99e101 ꢀC; 1H NMR (600 MHz, CDCl3)
d 7.73 (d,
J ¼ 15.5 Hz, 2H), 6.88 (d, J ¼ 1.9 Hz, 1H), 6.81 (dd, J ¼ 8.1, 1.9 Hz, 1H),
6.78 (s, 2H), 6.76 (d, J ¼ 6.8 Hz, 2H), 6.71 (s, 4H), 6.67 (d, J ¼ 8.2 Hz,
1H), 6.35 (d, J ¼ 8.7 Hz, 2H), 3.93 (s, 4H), 3.89 (s, 6H), 3.82 (s, 12H),
3.81 (s, 3H), 3.71 (s, 3H), 3.20 (q, J ¼ 7.0 Hz, 4H), 3.16 (s, 3H), 1.05 (t,
4.5. Cytotoxicity assay
J ¼ 7.0 Hz, 6H); 13C NMR (151 MHz, CDCl3)
d 167.97, 153.49, 148.83,
Ten-thousand cells of LCC6-MDR or LCC6 or L929 were mixed
with a series of concentrations of naamidine analogues to a final
147.25, 146.57, 146.21, 140.69, 137.48, 133.44, 129.91, 128.46, 127.68,
123.77, 120.22, 118.19, 111.97, 111.04, 105.78, 100.00, 61.07, 56.23,
55.77, 44.29, 33.24, 30.26, 28.54, 12.49; HRMS calcd for
(C48H56N4O10 þ H)þ 849.4069, found 849.4079.
volume of 100 mL in each well of 96-well plates. The plates were
then incubated for 3 days at 37 ꢀC. Then 50% inhibitory concen-
tration (IC50) of naamidine analogues was determined using MTS
proliferation assay as described previously [14].
4.1.28. N-(3-(3,4,5-trimethoxyphenyl)propanoyl)-N-(4,5-bis(3,4-
dimethoxybenzyl)-1-methyl-1H-imidazol-2-yl)-3-(3,4,5-
trimethoxyphenyl)propanamide (18b)
Acknowledgment
18b was prepared as described for the synthesis of 15a using 9b
(0.199 g, 0.5 mmol) and 3-(3,4,5-trimethoxyphenyl)propanoyl
chloride (0.258 g, 1 mmol). 18b (0.155 g, 37%) was obtained as a
This work was supported by grants from National Natural Sci-
ence Foundation of China (NSFC) (81373322), NSFC-Shandong Joint
Fund (U1406402) and a Special Fund for Marine Scientific Research
in the Public Interest of China (201005024).
white solid; mp 103e105 ꢀC; 1H NMR (600 MHz, CDCl3)
d 6.76 (d,

N. Zhang et al. / European Journal of Medicinal Chemistry 83 (2014) 74e83
83
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.06.016.
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