Scaffold identification of a new class of potent and selective BCRP inhibitors

Article abstract of DOI:10.1002/cmdc.201402498

We recently reported the synthesis and quantitative structure-activity relationships of a new breast cancer resistance protein (BCRP) inhibitor class. In the study presented herein, we investigated the possibility to better define the scaffold of this compound class by removing or modifying the aromatic ring A with various substituents selected on the basis of their electronic and lipophilic properties. The results show that this aromatic ring is important, but not essential, for activity. Many of the selected substituents led to compounds with low activity, but in some cases activity was retained. Among these, a phenolic hydroxy group proved to impart as much potency to the molecule as a hydroxyethyl side chain, initially considered necessary for activity. This derivative is one of the most active compounds in this class, maintaining an inhibitory activity similar to that of the reference compound; it is also selective for BCRP.

Full text of DOI:10.1002/cmdc.201402498

DOI: 10.1002/cmdc.201402498
Full Papers
Scaffold Identification of a New Class of Potent and
Selective BCRP Inhibitors
Federico Marighetti, Kerstin Steggemann, Maria Karbaum, and Michael Wiese*[a]
We recently reported the synthesis and quantitative structure–
activity relationships of a new breast cancer resistance protein
(BCRP) inhibitor class. In the study presented herein, we inves-
tigated the possibility to better define the scaffold of this com-
pound class by removing or modifying the aromatic ring A
with various substituents selected on the basis of their elec-
tronic and lipophilic properties. The results show that this aro-
matic ring is important, but not essential, for activity. Many of
the selected substituents led to compounds with low activity,
but in some cases activity was retained. Among these, a phe-
nolic hydroxy group proved to impart as much potency to the
molecule as a hydroxyethyl side chain, initially considered nec-
essary for activity. This derivative is one of the most active
compounds in this class, maintaining an inhibitory activity sim-
ilar to that of the reference compound; it is also selective for
BCRP.
Introduction
ATP binding cassette (ABC) transporters are membrane pro-
teins that are able to translocate a wide variety of substrates
through the cell membrane using ATP hydrolysis as energy
source. The overexpression of certain poly-specific ABC trans-
porters in cancer cells is the most significant mechanism
behind multidrug resistance (MDR). ABC transporters involved
in MDR are P-glycoprotein (P-gp), breast cancer resistance pro-
tein (BCRP), and multidrug resistance protein 1 (MRP1).[1]
were found to be selective BCRP inhibitors. Ko143, a synthetic
analogue of fumitremorgin C, is a potent and selective inhibi-
tor of BCRP.[14] Tyrosine kinase inhibitors have also been found
to inhibit BCRP function.[15] It was recently reported that the ef-
ficacious third-generation P-gp modulator XR9576 (tariquidar)
is also a BCRP inhibitor, although less potent.[16] Structural
modification of XR9576, with deletion of the tetrahydroisoqui-
noline group, led to compounds that were shown to be selec-
tive BCRP inhibitors.[17] In our previous work[18] we investigated
the structure–activity relationships for compounds of this new
class with modifications on the aromatic rings B and C of the
original scaffold (Figure 1). The aim of this study is to investi-
gate the importance of modifications on aromatic ring A in
order to better define a minimum scaffold for of this new
BCRP inhibitor class. Herein we report the synthesis and bio-
BCRP is a member of the ABCG subfamily, consisting of one
N-terminal nucleotide binding domain followed by six trans-
membrane segments.[2,3] BCRP has been suggested to function
as a homodimer, with its two monomers connected by a disul-
fide bridge.[4] BCRP is expressed in many tissues of the human
body, particularly in brain, placenta, liver, intestine, kidney, and
stem cells.[5] Under physiological conditions, it plays a protec-
tive role against xenobiotics and is probably also involved in
the transport of several physiological substrates such as por-
phyrins and vitamins.[6] BCRP is nonselective and is able to
transport many kinds of molecules, both positively and nega-
tively charged. Cytotoxic drugs transported by BCRP include
mitoxantrone, topotecan, SN-38, camptothecin, flavopiridol,
and methotrexate.[7] A possible strategy to overcome BCRP-
mediated resistance would be the use of potent and selective
inhibitors or modulators. Among the limited number of BCRP
inhibitors currently known are natural or synthetic compounds,
and only a few of these are selective for this transporter.[8] Fla-
vonoids and derivatives thereof are natural compounds report-
ed to be potent but nonselective BCRP inhibitors.[9–11] Other
natural products such as fumitremorgin C[12] and novobiocin[13]
[a] F. Marighetti, Dr. K. Steggemann, M. Karbaum, Prof. Dr. M. Wiese
Pharmaceutical Institute, University of Bonn
An der Immenburg 4, 53121 Bonn (Germany)
E-mail: mwiese@uni-bonn.de
Figure 1. Structures of tariquidar (top), the initial series of derived BCRP in-
hibitors[18] (bottom left), and the current series of investigated compounds
(bottom right).
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402498.
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logical evaluation of 30 new derivatives with varying substitu-
ents on ring A and selected substituents on ring C.
Results and Discussion
The general outline of the synthesis of the derivatives is shown
in Scheme 1. As in our previous work[18] the presence of sub-
stituents on ring B (R’ and R’’; Figure 1) had been proven disad-
*
*
Figure 2. s–p diagram of selected active ( ) and inactive ( ) substituents.
The substituent constants were taken from Ref. [31].
assays are listed in Table 1. Compounds show no inhibitory
activity at concentrations up to 10 mm are reported as having
no effect (NE). Considering the activity data and the s–p dia-
gram of the substituents present on ring A (Figure 2), it can be
concluded that substituents with small s-Hammett values are
active, whereas there is no decisive influence of lipophilicity,
probably except for very polar groups such as amide and sul-
fonamide, which lead to inactive compounds. In comparing
the activity data and calculated logP values, there seems to be
a tendency of higher activity with greater lipophilicity. Com-
pounds 9, 14, and 27 are the most lipophilic and are among
the most active. However, a plot of calculated logP values
versus pIC50 values of the active derivatives does not confirm
this conclusion. Although the regression line points upward,
the squared correlation coefficient is close to zero (r2 =0.05),
both for all compounds and the subset of 4-nitro derivatives
(data not shown).
Scheme 1. Synthesis of the BCRP inhibitors. Reagents and conditions: a) THF,
Et3N, RT, 12 h; b) H2, Pd/C, 2 bar, RT, 12 h; c) R-COCl, THF, Et3N, RT, 12 h.
vantageous, only compounds unsubstituted at these positions
were synthesized in this study. The previously published com-
pound 4 was used as a reference compound in this study for
comparison. This compound has a hydroxyethyl group as R2,
initially thought to be important for activity, and a nitro group
as R1, which had been indicated in our precedent study as
most favorable at this position. For this reason, many of the
compounds in this study also have R1 as a nitro group. Howev-
er, we also explored several other substituents at this position
which had previously led to active compounds.
With regard to compounds with a nitro group at R1, com-
pound 5, in which the terminal hydroxy group at R2 is replaced
by a methyl ether, shows even slightly better inhibitory activity
To investigate the importance of the hydroxyethyl substitu-
ent on ring A it was replaced by several other groups. The se-
lection was based on synthetic feasibility and the physico-
chemical properties of the substituents. They were selected
from a s–p diagram so that at least two were present from
each quadrant (Figure 2). We also considered hydrogen bond
acceptor and donor properties as potentially important and in-
cluded them in the selection. In two compounds, phenyl
ring A was replaced by a 4-hydroxycyclohexyl group, which
had been proven to be beneficial for activity. In one com-
pound the entire phenyl ring A was removed and replaced by
a small ethyl group, leading to an inactive derivative.
(IC50 =1.23 mm) than the reference compound
4 (IC50 =
1.58 mm). Replacement of the hydroxy group with an acetyl
ester also retains activity (compound 6) to a high degree,
showing that a hydrogen bond donor is not necessary at this
position. Replacement of the hydroxyethyl group with a phe-
nolic hydroxy group (compound 8) leads to a derivative nearly
as potent as the reference compound. However, compound 7,
with a methoxy group at R2, is twofold less potent than com-
pound 8. Interestingly, a nonpolar propyl group at R2 (com-
pound 9) leads to the second most potent compound in the
series (IC50 =0.94 mm). This is unexpected, as derivatives con-
taining an nonpolar methyl group at this position (19, 25, 26)
show low activity or are even inactive. In contrast, compound
11, with no substituent at R2, has much lower inhibitory activi-
ty against BCRP (IC50 =13.4 mm). Substitution with a benzoyl
group (compound 14) gives a compound with an IC50 value of
1.63 mm. Compounds with bromine (10), polar amide (12), sul-
fonamide (13), or tertiary amine (15) substituents showed no
inhibition up to 10 mm.
The inhibitory activity of the compounds against BCRP was
determined by a Hoechst 33342 assay in BCRP-overexpressing
MCF-7 MX cells. To evaluate the selectivity as well, the inhibito-
ry activity against P-gp was initially screened at a concentration
of 10 mm using a calcein AM assay with A2780Adr cells. For
compounds showing >25% inhibition at 10 mm, full dose–re-
sponse curves were measured to determine IC50 values. The
structures of the derivatives and the results of the biological
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and 5).[18] A possible explanation might be solubility
problems due to the high lipophilicity of this com-
pound.
Table 1. Inhibitory activities of compounds 4–34.
In the series of compounds bearing a 3,4-dime-
thoxy substitution at R1, the activity trends differed
somewhat. Replacement of the hydroxyethyl side
chain at R2 by a phenolic hydroxy group increased
the inhibitory activity threefold (ref. [18] no. 27: IC50 =
3.61 mm, versus compound 17: IC50 =1.16 mm). Again,
compound 16, with a methoxy group at R2, is three-
fold less active than the corresponding phenol. In
contrast to compounds with a nitro group at position
R1, the absence of a substituent at position R2 did
not dramatically decrease activity, and compound 20
was found to be a BCRP inhibitor with an IC50 value
of 1.75 mm. Chlorine (18) or a methyl group (19) led
to derivatives with low potency.
Compd
R1
R2
IC50 [mm][a]
logPcalcd
BCRP
P-gp
NE
NE
NE
4
5
6
7
8
9
4-NO2
CH2CH2OH
CH2CH2OCH3
CH2CH2OCOCH3
1.58Æ0.36[18]
1.23Æ0.40
1.32Æ0.28
3.56Æ0.84
1.76Æ0.18
0.94Æ0.33
NE
3.34
4.13
4.28
4.15
3.46
5.73
5.37
4.20
3.16
3.01
5.60
4.34
4.33
3.64
5.37
4.84
4.38
3.44
3.38
4.86
4.16
5.37
5.06
6.00
5.03
4.37
6.10
5.11
2.80
3.70
2.97
–
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
3,4-OCH3
3,4-OCH3
3,4-OCH3
3,4-OCH3
3,4-OCH3
4-NHCOCH3
4-NH2
4-Cl
OCH3
OH
NE
27.5Æ1.84
CH2CH2CH3
Br
NE
NE
NE
NE
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
tariquidar
H
13.4Æ4.2
NE
NE
Only a few derivatives were investigated that pos-
sess a chlorine or methyl substituent at R1, as these
were found to be less active in the precedent study.
In agreement with the positive contribution of a phe-
nolic hydroxy group, compound 24 was found to be
as active as the counterpart with a hydroxymethyl
side chain at this position (ref. [18] no. 20: IC50 =
3.08 mm). Whereas a methyl substituent at R2 led to
inactive (26) or poorly active (19) derivatives, a benzo-
yl group had a strong positive effect, and compound
27 was found to be the most active derivative in the
whole series, with an IC50 value of 0.56 mm.
Regarding compounds unsubstituted at R2, it is
possible to correlate their activity values expressed in
logarithmic form with van der Waals surface area
(VSA) of ring C containing the R1 substituent, calculat-
ed with the MOE software package.[19] Increasing the
surface area of the R1 group leads to increased BCRP
inhibitory activity. The two most active compounds
of this series without substituents at R2—compounds
20 and 21—have the highest VSA values (164.3 and
154.1 Š2, respectively), whereas compounds 11 and
22 have low respective VSA values: 132.4 and
125.0 Š2. The correlation between activity and VSA
suggests that a voluminous substituent at R1 increas-
CONH2
SO2NH2
(C=O)Ph
N(CH3)2
OCH3
OH
NE
1.63Æ0.33
NE
5.17Æ0.27
NE
3.34Æ0.25
1.16Æ0.21
8.29Æ3.10
22.8Æ2.8
1.76Æ0.64
2.09Æ0.20
23.3Æ8.9
NE
4.09Æ0.50
4.47Æ1.00
Cl
CH3
H
H
H
OCH3
OH
CH3
CH3
NE
13.5Æ2.6
3.98Æ0.84
NE
NE
NE
NE
NE
NE
4-Cl
4-Cl
2.91Æ0.88
NE
4-CH3
4-CH3
4-CF3
4-CF3
4-CF3
4-CF3
4-NO2
4-CF3
4-NO2
NE
(C=O)Ph
CH2CH2OCH3
OH
0.56Æ0.24
5.11Æ0.87
1.80Æ0.43
NE
6.07Æ1.48
NE
14.0Æ3.4
Cl
H
NE
NE
NE
NE
4-OH-cHex
4-OH-cHex
CH2CH3
6.11Æ2.58
3.35Æ1.06
NE
0.68[30]/1.45[16]
20.8Æ4.4
NE
–
[a] Determined by Hoechst 33342 assay in MCF-7 MX cells and by calcein AM assay in
A2780Adr cells; data are the meanÆSD (n=3); NE: no effect for concentrations up to
10 mm.
The trends observed for the compound series with a trifluo-
romethyl group at R1 are in agreement with those of the 4-
nitro series. This group was found to be slightly less active
than the nitro derivative in the previously investigated series
with a hydroxyethyl group at R2. Again, replacement of the hy-
droxyethyl group by a phenolic hydroxy group did not change
activity significantly (ref. [18] no. 22: IC50 =2.20 mm, versus
compound 29: IC50 =1.80 mm). If no substituent is present at
R2, activity is largely decreased with a nitro group at position
R1, and is abolished in the case of a trifluoromethyl substituent
(compound 31). The only exception in the activity trends is
compound 28, with an IC50 value of 5.11 mm, which was found
to be twofold less active than its analogue with a hydroxyethyl
group at R2 (ref. [18] no. 22: IC50 =2.20 mm), whereas the activi-
ties of both derivatives are identical in the 4-nitro series (nos. 4
es the BCRP inhibitory activity of this compound class; this
contribution to activity is increased by the absence of a sub-
stituent at R2. This observation is in agreement with what has
been suggested in our previous publication.[18]
The structure–activity relationship of the substituents on
ring A can be explained by SlogP, PM3 HOMO, and VSA de-
scriptors (Figure 3). For compounds with a 4-nitro group on
ring C, the active derivatives cluster in the descriptor space. As
apparent in Figure 3, active derivatives must have a VSA
2
>150 Š and a SlogP >1. These compounds must also have
values of PM3 HOMO (a descriptor highly correlated with s-
Hammett) for the aromatic ring A between À9 and À10 eV.
The two compounds (13 and 15) out of this range are not
active against BCRP. Compounds with a cyclohexyl ring (32
and 33) in place of the phenyl ring A are less potent against
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significant inhibitory potency against P-gp, with an IC50 value
of 5.17 mm. Given the presence of a benzophenone moiety, the
latter compound could be expected to be a P-gp inhibitor, as
other studies with compounds containing a benzophenone
group had shown this to be a structural element recognized
by P-gp.[20,21] This is substantiated by the finding that the other
derivative containing a benzophenone group (compound 27)
showed very similar inhibitory activity against P-gp, with an
IC50 value of 6.07 mm. As could be expected by the common
presence of dimethoxy substitution in P-gp inhibitors, com-
pounds with such a substitution pattern were found to be
mostly non-selective for BCRP, and their inhibitory potency
against P-gp was similar to that against BCRP. In particular,
compounds 16, 17, and 20 possess similar IC50 values for P-gp,
whereas compounds 18 and 19 show no or low activity.
To investigate the capacity of the compounds to reverse the
resistance of BCRP-expressing cells, the cytotoxicity of the
BCRP substrate SN-38, the active metabolite of irinotecan, was
determined in the presence and absence of a representative
from the series of compounds. Compound 8 was selected as
being equipotent to the reference 4 and with relatively good
water solubility. Figure 5 illustrates the effect of compound 8
Figure 3. 3D plot of the SlogP, PM3 HOMO, and VSA descriptors of com-
pounds with various substituents on aromatic ring A and a nitro group on
ring C. Active derivatives are shown as black spheres, and inactive ones are
in grey. Descriptor values were calculated for the substructure of a substitut-
ed benzene.
BCRP, but are still active, whereas an ethyl group at this posi-
tion leads to loss of activity (compound 34).
To investigate the possibility of a substrate-dependent pat-
tern of BCRP inhibition, 12 derivatives and tariquidar were also
tested with pheophorbide A as substrate. The inhibitory activi-
ties of the tested derivatives showed a high correlation, in
agreement with the results obtained for a smaller set of deriva-
tives containing a hydroxyethyl side chain.[17] The correlation
between IC50 values in both test systems is shown in Figure 4.
The calcein AM assay was used to investigate P-gp inhibi-
tion. Here compound 8 (IC50 =27.5 mm) showed only weak
inhibitory activity, whereas compound 14 was found to have
Figure 5. Representative shift in the dose–response curve of SN-38 cytotox-
icity caused by increasing inhibitor (compound 8) concentration. Compound
~
&
8 was investigated at 5 mm ( ) and 10 mm ( ). MDCK BCRP cells without in-
!
*
hibitor ( ) are more resistant than sensitive MDCK cells ( ). Shown is a rep-
resentative example from three independent experiments with two repli-
cates each.
on the EC50 of SN-38 in MDCK BCRP cells. Even at 5 mm the
concentration effect curve is largely shifted to the left with
a further shift occurring at the higher concentration of 10 mm.
The MTT assay clearly shows that compound 8 is able to com-
pletely reverse the resistance of MDCK BCRP cells against
SN-38.
Conclusions
In summary, it is possible to assert that the hydroxyethyl sub-
stituent present in the original scaffold of this class of BCRP in-
hibitors is not necessary for the BCRP inhibitory activity of
these compounds. Replacement of this substituent with a hy-
droxy group leads to selective BCRP inhibitors with IC50 values
Figure 4. Correlation of inhibitory potencies of selected compounds in the
Hoechst 33342 and pheophorbide A assays of BCRP inhibition (r2 =0.91).
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night at room temperature under 2 bar H2 atmosphere in a Paar
apparatus. The catalyst was filtered off, the solution was concen-
trated, and the product was dissolved in a little THF and precipitat-
ed with PE.
similar to those of compounds with a hydroxyethyl group at
the same position. Furthermore, the presence of a substituent
at R2 is not necessary if position R1 is occupied by a group
with a van der Waals surface area higher than ~150 Š2. Finally,
the aromatic ring A was identified to be important for the ac-
tivity of the compounds against BCRP, and its removal leads to
compounds with low or no inhibitory activity against BCRP.
Furthermore, methylation of the hydroxy group at R2 leads to
compounds that are less active than non-methylated com-
pounds. Compounds with a 3,4-dimethoxyphenyl group at R1
show a slightly different activity pattern from those with a 4-ni-
trophenyl substituent, and they also exhibit considerable inhib-
itory activity against P-gp.
N-(4-(2-Methoxyethyl)phenyl)-2-nitrobenzamide (2a): According
to method B, 4-(2-methoxyethyl)aniline (1.51 g, 10 mmol) and 4-ni-
trobenzoyl chloride (1.86 g, 10 mmol) were reacted, and com-
pound 2a was obtained as a pale-yellow powder (2.04 g, 68%):
Rf =0.35 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d=10.58
(s, 1H), 8.13 (dd, J=8.54, 1.02 Hz, 1H), 7.87–7.83 (m, 1H), 7.76–7.72
(m, 2H), 7.56 (d, J=8.45 Hz, 2H), 7.21 (d, J=8.47 Hz, 2H), 3.51 (t,
J=6.85 Hz, 2H), 3.24 (s, 3H), 2.77 (t, J=6.85 Hz, 2H); 13C NMR
(125 MHz, [D6]DMSO): d=164.04, 146.67, 137.04, 134.88, 134.15,
132.89, 131.01, 129.39, 129.21 (2C), 124.34, 119.80 (2C), 72.98,
57.94, 34.97 ppm; Anal. calcd for C16H16N2O4: C 63.99, H 5.32, N
9.33, found: C 64.03, H 5.32, N 9.54.
Experimental Section
(4-(2-Nitrobenzamido)phenethyl acetate (2b): According to
method B, 2-amino-N-[4-(2-hydroxyethyl)phenyl]benzamide (1.43 g,
5 mmol) and acetyl chloride (471 mg, 6 mmol) were reacted, and
compound 2b was obtained as a beige powder (1.54 g, 94%): Rf =
0.16 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d=10.59 (s,
1H), 8.18–8.09 (m, 1H), 7.88–7.83 (m,1H), 7.76–7.72 (m, 2H), 7.59
(d, J=8.49 Hz, 2H), 7.23 (d, J=8.47 Hz, 2H), 4.19 (t, J=6.86 Hz,
2H), 2.86 (t, J=6.84 Hz, 2H), 1.98 ppm (s, 3H); 13C NMR (125 MHz,
[D6]DMSO): d=170.38, 164.05, 146.62, 137.31, 134.14, 133.69,
132.83, 131.01, 129.36, 129.24 (2C), 124.32, 119.85 (2C), 64.48,
33.92, 20.80 ppm; Anal. calcd for C16H16N2O3: C 67.59, H 5.67, N
9.85, found: C 67.25, H 5.64, N 9.79.
The logP values were calculated with ACD logP, ver. 5.09 (Ad-
vanced Chemistry Development, Toronto, ON, Canada).
Chemistry
General methods: Commercial reagents and starting materials
were purchased from Sigma–Aldrich, Fluka, Acros, or Alpha Aesar
and were used without further purification. Reaction progress was
monitored by TLC on alumina plates coated with silica gel (Merck
silica gel 60 F254) and visualized by UV light (l=254 nm). Spectral
data were obtained as follows: 1H NMR, Bruker Advance 500
(500 MHz); 13C NMR, Bruker Advance 500 (125.8 MHz). Chemical
shifts (d) are expressed in ppm by using solvent as an internal
standard. Multiplicity of resonance peaks is indicated as singlet (s),
doublet (d), triplet (t), quartet (q) and multiplet (m). J values are
given in Hz, and the relative number of protons was determined
by integration. The solvent used for each spectrum is reported. Ele-
mental analyses were performed on a Vario EL (Elementar). Values
were all within 0.4% of theoretical, except when indicated. Spec-
troscopic data of published compounds are given in the Support-
ing Information for convenience.
2-Nitro-N-(4-propylphenyl)benzamide (2e): According to meth-
od B, 4-propylaniline (676 mg, 5 mmol) and 4-nitrobenzoyl chloride
(1.11 g, 6 mmol) were reacted, and compound 2e was obtained as
a white powder (1.27 g, 89%): Rf =0.48 (EtOAc/PE (1:1)); 1H NMR
(500 MHz, [D6]DMSO): d=10.54 (s, 1H), 8.15–8.10 (m, 1H), 7.89–
7.82 (m), 7.77–7.71 (m, 2H), 7.56 (d, J=8.5, 2H), 7.16 (d, J=8.5 Hz,
2H), 2.55–2.50 (m, 2H), 1.66–1.50 (m, 2H), 0.88 ppm (t, J=7.3, 3H);
13C NMR (125 MHz, [D6]DMSO): d=163.96, 146.66, 137.88, 136.68,
134.11, 132.90, 130.97, 129.37, 128.68 (2C), 124.31, 119.80 (2C),
36.82, 24.24, 13.66 ppm; Anal. calcd for C16H16N2O3: C 67.59, H 5.67,
N 9.85, found: C 67.25, H 5.64, N 9.79.
General procedure for synthesis of acid chlorides (A): Aromatic
carboxylic acid (1 equiv) was dissolved/suspended in dry THF or
2-Amino-N-(4-(2-methoxyethyl)phenyl)benzamide (3a): Accord-
ing to method C N-(4-(2-methoxyethyl)phenyl)-2-nitrobenzamide
(1.50 g, 5 mmol) was hydrogenated. The product was directly used
for further synthesis. Rf =0.86 (EtOAc/PE (1:1)); 1H NMR (500 MHz,
[D6]DMSO): d=9.92 (s, 1H), 7.64–7.61 (m, 3H), 7.20 (ddd, J=1.9,
5.6, 8.6 Hz, 3H), 6.76 (dd, J=1.0, 8.3 Hz, 1H), 6.61–6.57 (m, 1H),
6.31 (s, 2H), 3.53 (t, J=6.9 Hz, 2H), 3.26 (s, 3H), 2.78 ppm (t, J=
6.9 Hz, 2H); 13C NMR (125 MHz, [D6]DMSO): d=167.84, 149.82,
137.40, 134.23, 132.11, 128.89, 128.74, 120.70, 116.49, 115.53,
114.82, 73.04, 57.93, 34.98 ppm.
CH2Cl2 and
a catalytic amount of dry DMF. Oxalyl chloride
(1.2 equiv) was added dropwise, and the reaction mixture was
stirred at room temperature for 1 h. Finally, the solvent and excess
oxalyl chloride was removed under reduced pressure to yield the
desired acid chloride, which was used immediately for the synthe-
sis of amides.
General procedure for synthesis of amides (B): Aromatic amine
(1.2 equiv) and Et3N (3 equiv) were dissolved in dry THF and stirred
at 08C. The acyl chloride (1.0 equiv) synthesized as per general
method was dissolved in dry THF and added slowly. The solution
was stirred for 12 h at room temperature. After completion of the
reaction, the solvent was evaporated, and the residue was dis-
solved in H2O and extracted with EtOAc (3”15 mL). The organic
phases were combined and washed with 1n NaOH, 1n HCl, and
saturated brine, dried over MgSO4, and concentrated. The product
was precipitated with THF/petroleum ether (PE) and purified by re-
crystallization with CH2Cl2/PE (1:1) or EtOH or by column chroma-
tography with EtOAc/PE (1:1) as eluent.
4-(2-Aminobenzamido)phenethyl acetate (3b): According to
method C
4-(2-nitrobenzamido)phenethyl
acetate
(1.37 g,
4.2 mmol) was hydrogenated, and compound 3b was obtained as
1
a beige powder (937 mg, 78%): Rf =0.83 (EtOAc/PE (1:1)); H NMR
(500 MHz, [D6]DMSO): d=9.92 (s, 1H), 7.65–7.59 (m, 3H), 7.21–7.16
(m, 3H), 6.74 (dd, J=8.28, 1.07 Hz, 1H), 6.58 (ddd, J=8.13, 7.21,
1.19 Hz, 1H), 6.28 (s, 2H), 4.19 (t, J=6.92 Hz, 2H), 2.84 (t, J=
6.90 Hz, 2H), 1.98 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=
170.42, 167.89, 149.84, 137.75, 133.05, 132.14, 128.96 (2C), 128.78,
120.78 (2C), 116.50, 115.50, 114.83, 64.57, 33.97, 20.85 ppm; Anal.
calcd for C17H18N2O3·0.33H2O: C 67.09, H 6.18, N 9.20, found: C
67.47, H 6.01, N 9.16.
General procedure for hydrogenation of an aromatic nitro
group (C): Nitro compound was dissolved in THF, and palladium
on activated charcoal was added. The suspension was stirred over-
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2-Amino-N-(4-benzoylphenyl)benzamide (3j): According to meth-
od C N-(4-benzoylphenyl)-2-nitrobenzamide (1.38 g, 4 mmol) was
hydrogenated, and compound 3j was obtained as a white powder
(850 mg, 69%): Rf =0.87 (EtOAc/PE (1:1)); 1H NMR (500 MHz,
[D6]DMSO): d=10.32 (s, 1H, 7.93 (d, J=8.76 Hz, 2H), 7.76 (d, J=
8.77 Hz, 2H), 7.72 (dd, J=8.18, 1.26 Hz, 2H), 7.68–7.63 (m, 2H),
7.55 (t, J=7.58 Hz, 2H), 7.22 (ddd, J=8.43, 7.22, 1.47 Hz, 1H), 6.78
(dd, J=8.26, 0.91 Hz, 1H), 6.60 (t, J=8.06 Hz, 1H), 6.37 ppm (s,
2H); 13C NMR (125 MHz, [D6]DMSO): d=194.76, 168.32, 150.14,
150.06, 143.81, 137.79, 132.64, 132.32, 131.53, 130.99 (2C), 129.49
(2C), 129.03, 128.60 (2C), 119.63 (2C), 116.63, 114.84 ppm; Anal.
calcd for C20H16N2O2: C 75.93, H 5.10, N 8.86, found: C 75.77, H
5.20, N 8.75.
7.49 (d, J=8.82 Hz, 2H), 7.28 (t, J=7.28 Hz, 1H), 6.78 ppm (d, J=
8.84 Hz, 2H); 13C NMR (125 MHz, [D6]DMSO): d=167.00, 162.97,
154.54, 149.48, 140.29, 138.66, 132.23, 129.89, 128.93, 128.63 (2C),
124.14 (2C), 123.75, 123.41 (2C), 122.73, 121.38, 115.22 ppm (2C);
HPLC: k’=0.53 (RP8 column, CH3CN/H2O (80:20)), purity 97.64%
2-(4-Nitrobenzamido)-N-(4-propylphenyl)benzamide (9): Accord-
ing to method B, compound 3e (87 mg, 0.5 mmol) and 4-nitroben-
zoyl chloride (111 mg, 0.6 mmol) were reacted, and compound 9
was obtained as a yellow powder (187 mg, 93%): Rf =0.84 (EtOAc/
1
PE (1:1)); H NMR (500 MHz, [D6]DMSO): d=8.37–8.33 (m, 3H), 8.16
(d, J=8.9 Hz, 2H), 7.95 (d, J=6.5 Hz, 1H), 7.58–7.52 (m, 3H), 7.22
(t, J=7.3 Hz, 1H), 7.13 (d, J=8.5 Hz, 2H), 2.52–2.49 (m, 2H), 1.60–
1.51 (m, 2H), 0.87 ppm (t, J=7.3 Hz, 3H); 13C NMR (125 MHz,
[D6]DMSO): d=166.80, 163.63, 149.14, 137.82, 136.61, 131.91,
129.16, 128.80 (2C), 128.53 (2C), 124.00, 123.91 (3C), 122.49, 120.96
(2C), 36.83, 24.23, 13.68 ppm; Anal. calcd for C23H21N3O4: C 68.47, H
5.25, N 10.42, found: C 68.11, H 4.93, N 10.19.
N-(4-(2-Methoxyethyl)phenyl)-2-(4-nitrobenzamido)benzamide
(5): According to method B, compound 3a (405 mg, 1.5 mmol) and
4-nitrobenzoyl chloride (371 mg, 2 mmol) were reacted, and com-
pound 5 was obtained as a beige powder (262 mg, 42%): Rf =0.79
(EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d=11.84 (s, 1H),
10.51 (s, 1H), 8.40–8.36 (m, 3H), 8.13 (d, J=8.64 Hz, 2H), 7.93 (d,
J=7.66 Hz, 1H), 7.63–7.58 (m, 3H), 7.31 (t, J=7.50 Hz, 1H), 7.20 (d,
J=8.42 Hz, 2H), 3.51 (t, J=6.83 Hz, 2H), 3.23 (s, 3H), 2.77 ppm (t,
J=6.81 Hz, 2H); 13C NMR (125 MHz, [D6]DMSO): d=167.16, 163.26,
149.49, 136.66, 135.22, 132.22, 129.11, 129.06 (2C), 128.76 (2C),
127.97, 124.16 (2C), 123.88, 121.98, 121.27 (2C), 72.95, 57.93,
34.98 ppm; Anal. calcd for C23H21N3O5·0.5H2O: C 64.48, H 5.18, N
9.81, found: C 64.27, H 5.01, N 9.70.
N-(4-Bromophenyl)-2-(4-nitrobenzamido)benzamide (10): Ac-
cording to method B, 2-amino-N-(4-bromophenyl)benzamide
(146 mg, 0.5 mmol) and 4-nitrobenzoyl chloride (111 mg, 0.6 mmol)
were reacted, and compound 10 was obtained as a pale-yellow
1
powder (75 mg, 34%): Rf =0.74 (EtOAc/PE (1:1)); H NMR (500 MHz,
[D6]DMSO): d=8.35 (d, J=9.0 Hz, 2H), 8.26 (d, J=7.3 Hz, 1H), 8.14
(d, J=8.9 Hz, 2H), 7.91 (d, J=7.8 Hz, 1H), 7.65 (d, J=8.9 Hz, 2H),
7.56 (t, J=7.1 Hz, 1H), 7.50 (d, J=8.8 Hz, 2H), 7.25 ppm (t, J=
7.5 Hz, 1H); 13C NMR (125 MHz, [D6]DMSO): d=167.10, 163.81,
149.63, 138.51, 131.96, 131.55 (2C), 129.19, 128.84 (2C), 124.52,
123.92 (2C), 124.40, 122.74 (2C), 115.60 ppm; Anal. calcd for
C20H14BrN3O4·0.6H2O: C 53.23, H 3.42, N 9.29, found: C 53.23, H
3.48, N 8.85.
4-(2-(4-Nitrobenzamido)benzamido)phenethyl acetate (6): Ac-
cording to method B, compound 3b (119 mg, 0.4 mmol) and 4-ni-
trobenzoyl chloride (111 mg, 0.6 mmol) were reacted, and com-
pound 6 was obtained as a pale-yellow powder (120 mg, 67%):
Rf =0.74 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d=11.71
(s, 1H), 11.01 (s, 1H), 8.39–8.33 (m, 3H), 8.14 (d, J=8.86 Hz, 2H),
7.94 (dd, J=7.81, 1.17 Hz, 1H), 7.62–7.56 (m, 3H), 7.27 (t, J=
7.38 Hz, 1H), 7.21 (d, J=8.48 Hz, 2H), 4.18 (t, J=6.88 Hz, 2H), 2.85
(t, J=6.87 Hz, 2H), 1.97 ppm (s, 3H); 13C NMR (125 MHz,
[D6]DMSO): d=170.42, 167.03, 163.50, 149.34, 141,26, 139,44,
137.17, 133.83, 132.06, 129.16, 129.12 (2C), 128.81 (2C), 124.07,
124.05 (2C), 123.56, 122.32, 121.17 (2C), 64.52, 33.97, 20.84 ppm;
Anal. calcd for C24H21N3O6·0.5H2O: C 63.15, H 4.86, N 9.21, found: C
63.26, H 4.72, N 8.95.
2-(4-Nitrobenzamido)-N-phenylbenzamide (11): According to
method B, 2-amino-N-phenylbenzamide (318 mg, 1.5 mmol) and 4-
nitrobenzoyl chloride (334 mg, 1.8 mmol) were reacted, and com-
pound 11 was obtained as a cream-colored powder (310 mg,
1
57%): Rf =0.89 (EtOAc/PE (1:1)); H NMR (500 MHz, [D6]DMSO): d=
11.37 (s, 2H), 8.38–8.35 (m, 2H), 8.33 (dd, J=8.22, 0.84 Hz, 1H),
8.17–8.12 (m, 2H), 7.95 (dd, J=7.82, 1.29 Hz, 1H), 7.69 (d, J=
7.61 Hz, 2H), 7.58 (t, J=7.35 Hz, 1H), 7.35–7.31 (m, 2H), 7.27 (t, J=
7.45 Hz, 1H), 7.10 ppm (t, J=7.39 Hz, 1H); 13C NMR (125 MHz,
[D6]DMSO): d=167.05, 163.57, 149.28, 138.90, 132.00, 129.23,
128.83 (2C), 128.74 (2C), 124.30, 124.08, 123.99 (2C), 123.47, 122.50,
121.05 ppm (2C); Anal. calcd for C20H15N3O4: C 66.48, H 4.18, N
11.63, found: C 66.31, H 4.48, N 11.45.
N-(4-Methoxyphenyl)-2-(4-nitrobenzamido)benzamide (7): Ac-
cording to method B, 2-amino-N-(4-methoxyphenyl)benzamide
(111 mg, 0.6 mmol) and 4-nitrobenzoyl chloride (111 mg, 0.6 mmol)
were reacted, and compound 7 was obtained as a yellow powder
(113 mg, 58%): Rf =0.70 (EtOAc/PE (1:1)); 1H NMR (500 MHz,
[D6]DMSO): d=11.96 (s, 1H), 10.49 (s, 1H), 8.41 (d, J=8.14 Hz, 1H),
8.38 (d, J=8.82 Hz, 2H), 8.13 (d, J=8.87 Hz, 2H), 7.94 (dd, J=7.81,
1.05 Hz, 1H), 7.62–7.58 (m, 3H), 7.30 (t, J=7.41 Hz, 1H), 6.92 (d, J=
9.05 Hz, 2H), 3.74 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=
166.95, 163.23, 156.17, 149.48, 132.18, 131.58, 129.02, 128.73 (2C),
124.16 (2C), 123.51, 122.96, 121.84, 113.95 (4C), 55.37 ppm; Anal.
calcd for C21H17N3O5·0.33H2O: C 63.47, H 4.48, N 10.57, found: C
63.59, H 4.60, N 10.50.
N-(4-Carbamoylphenyl)-2-(4-nitrobenzamido)benzamide (12): Ac-
cording to method B, 2-amino-N-(4-carbamoylphenyl)benzamide
(128 mg, 0.5 mmol) and 4-nitrobenzoyl chloride (111 mg, 0.6 mmol)
were reacted, and compound 12 was obtained as a yellow powder
(173 mg, 79%): Rf =0.30 (EtOAc); 1H NMR (500 MHz, [D6]DMSO): d=
11.52 (s, 1H), 10.67 (s, 1H), 8.38 (d, J=8.7 Hz, 2H), 8.25 (d, J=
8.2 Hz, 1H), 8.13 (d, J=8.6 Hz, 2H), 7.92–7.83 (m, 4H), 7.78 (d, J=
8.6 Hz, 2H), 7.63 (t, J=7.7 Hz, 1H), 7.34 (t, J=7.5 Hz, 1H), 7.24 ppm
(s, 2H); 13C NMR (125 MHz, [D6]DMSO): d=167.48, 167.32, 163.43,
149.50, 141.51, 140.34, 137.74, 132.23, 129.72, 129.21, 128.85 (2C),
128.31 (2C), 125.00, 124.29, 124.10 (2C), 122.59, 120.00 ppm (2C);
Anal. calcd for C20H16N3O6S·0.66H2O: C 60.57, H 4.20, N 13.46,
found: C 60.17, H 3.97, N 13.07.
(N-(4-Hydroxyphenyl)-2-(4-nitrobenzamido)benzamide (8): Ac-
cording to method B, 2-amino-N-(4-hydroxyphenyl)benzamide
(137 mg, 0.6 mmol) and 4-nitrobenzoyl chloride (92.8 mg,
0.5 mmol) were reacted, and compound 8 was obtained as
2-(4-Nitrobenzamido)-N-(4-sulfamoylphenyl)benzamide (13): Ac-
cording to method B, 2-amino-N-(4-sulfamoylphenyl)benzamide
(146 mg, 0.5 mmol) and 4-nitrobenzoyl chloride (111 mg, 0.6 mmol)
were reacted, and compound 13 was obtained as a pale-yellow
powder (183 mg, 83%): Rf =0.84 (EtOAc); 1H NMR (500 MHz,
1
a yellow powder (143 mg, 76%): Rf =0.44 (EtOAc/PE (1:1)); H NMR
(500 MHz, [D6]DMSO): d=12.15 (s, 1H), 10.33 (s, 1H), 9.34 (s, 1H),
8.49 (d, J=8.16 Hz, 1H), 8.36 (d, J=8.81 Hz, 2H), 8.11 (d, J=
8.83 Hz, 2H), 7.95 (dd, J=7.72, 0.85 Hz, 1H), 7.61–7.56 (m, 1H),
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[D6]DMSO): d=8.37 (d, J=8.8 Hz, 2H), 8.21 (d, J=7.4 Hz, 1H), 8.13
(d, J=8.9 Hz, 2H), 7.90 (d, J=6.6 Hz, 1H), 7.86 (d, J=8.8 Hz, 2H),
7.78 (d, J=8.8 Hz, 2H), 7.61 (t, J=7.3 Hz, 1H), 7.32 (t, J=7.5 Hz,
1H), 7.24 ppm (s, 2H); 13C NMR (125 MHz, [D6]DMSO): d=167.35,
163.61, 149.47, 141.96, 139.09, 132.18, 129.25, 128.89 (2C), 126.61
(2C), 125.23, 124.03 (2C), 122.87, 120.38 ppm (2C); HPLC: k’=3.88
(RP18 column, CH3OH/H2O (70:30)), purity 99.8%.
(d, J=8.86 Hz, 2H), 3.83 (s, 3H), 3.83 ppm (s, 3H); 13C NMR
(125 MHz, [D6]DMSO): d=167.23, 164.23, 154.46, 152.11, 148.85,
139.32, 132.26, 129.95, 128.86, 126.98, 123.26 (2C), 122.92, 122.04,
120.78, 120.01, 115.19 (2C), 111.59, 110.74, 55.87, 55.63 ppm; Anal.
calcd for C22H20N2O5·0.66H2O: C 65.34, H 5.32, N 6.93, found: C
65.00, H 5.31, N 6.86.
N-(2-(4-Chlorophenylcarbamoyl)phenyl)-3,4-dimethoxybenz-
amide (18): According to method A, 3,4-dimethoxybenzoic acid
(109 mg, 0.6 mmol) was reacted to yield 3,4-dimethoxybenzoyl
chloride, which was then, according to method B, reacted with 2-
amino-N-(4-chlorophenyl)benzamide (123 mg, 0.5 mmol) to obtain
compound 18 as a beige powder (84 mg, 41%): Rf =0.59 (EtOAc/
N-(4-Benzoylphenyl)-2-(4-nitrobenzamido)benzamide (14): Ac-
cording to method B, compound 3j (158 mg, 0.5 mmol) and 4-ni-
trobenzoyl chloride (111 mg, 0.6 mmol) were reacted, and com-
pound 14 was obtained as a pale-yellow powder (177 mg, 76%):
Rf =0.86 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d=11.40
(s, 1H), 10.83 (s, 1H), 8.42–8.35 (m, 2H), 8.19 (d, J=8.27 Hz, 1H),
8.15–8.11 (m, 2H), 7.92–7.88 (m, 3H), 7.78–7.75 (m, 2H), 7.71 (td,
J=8.38, 1.78, 1.78 Hz, 2H), 7.68–7.62 (m, 2H), 7.55 (t, J=7.61 Hz,
2H), 7.36 ppm (dt, J=7.64, 1.02 Hz, 1H); 13C NMR (125 MHz,
[D6]DMSO): d=194.75, 167.43, 163.52, 149.49, 143.15, 140.30,
137.59, 137.50, 132.43, 132.24, 132.17, 130.97 (2C), 129.51 (2C),
129.25, 128.89 (2C), 128.61 (2C), 125.54, 124.43, 124.06 (2C), 122.86,
119.98 ppm (2C); Anal. calcd for C27H19N3O5: C 69.67, H 4.11, N 9.03,
found: C 69.34, H 3.91, N 8.83.
1
PE (1:1)); H NMR (500 MHz, [D6]DMSO): d=11.51 (s, 1H), 10.60 (s,
1H), 8.43 (dd, J=8.34, 0.93 Hz, 1H), 7.90 (dd, J=7.88, 1.42 Hz, 1H),
7.76 (d, J=8.88 Hz, 2H), 7.62–7.58 (m, 1H), 7.49 (td, J=3.58,
2.07 Hz, 2H), 7.42 (d, J=8.90 Hz, 2H), 7.26 (dt, J=7.70, 1.16 Hz,
1H), 7.12 (d, J=8.26 Hz, 1H), 3.83 (s, 3H), 3.82 ppm (s, 3H);
13C NMR (125 MHz, [D6]DMSO): d=167.65, 164.37, 152.11, 148.80,
138.96, 137.71, 132.45, 129.09, 128.68 (2C), 128.03, 126.91, 123.19,
122.87, 122.66 (2C), 121.41, 120.18, 111.53, 110.78, 55.87,
55.64 ppm; Anal. calcd for C22H19ClN2O4: C 64.31, H 4.66, Cl 8.63, N
6.82, found: C 64.01, H 4.77, N 7.04.
N-(4-(Dimethylamino)phenyl)-2-(4-nitrobenzamido)benzamide
(15): According to method B, 2-amino-N-(4-(dimethylamino)phe-
nyl)benzamide (128 mg, 0.5 mmol) and 4-nitrobenzoyl chloride
(111 mg, 0.6 mmol) were reacted, and compound 15 was obtained
as an orange powder (86 mg, 42%): Rf =0.75 (EtOAc/PE (1:1));
1H NMR (500 MHz, [D6]DMSO): d=12.18 (s, 1H), 10.30 (s, 1H), 8.49
(d, J=8.1 Hz, 1H), 8.39 (d, J=8.9 Hz, 2H), 8.12 (d, J=8.9 Hz, 2H),
7.95 (d, J=6.8 Hz, 1H), 7.61 (t, J=7.2 Hz, 1H), 7.49 (d, J=9.0 Hz,
2H), 7.30 (t, J=7.4 Hz, 1H), 6.72 (d, J=9.1 Hz, 2H), 2.87 ppm (s,
6H); 13C NMR (125 MHz, [D6]DMSO): d=166.78, 162.84, 149.22,
147.91, 140.29, 138.54, 132.14, 128.87, 128.63 (2C), 127.81, 124.19
(2C), 123.78, 123,28, 122.88 (2C), 121.35, 112.44 (2C), 40.47 ppm
(2C); Anal. calcd for C22H20N4O4: C 65.34, H 4.98, N 13.85, found: C
65.38, H 4.99, N 13.58.
3,4-Dimethoxy-N-(2-(p-tolylcarbamoyl)phenyl)benzamide
(19):
According to method A, 3,4-dimethoxybenzoic acid (656 mg,
3.6 mmol) was reacted to yield 3,4-dimethoxybenzoyl chloride,
which was then, according to method B, reacted with 2-amino-(N-
4-methylphenyl)benzamide (679 mg, 3.0 mmol) to obtain com-
pound 19 as a white powder (1077 mg, 92%): Rf =0.82 (EtOAc/PE
1
(1:1)); H NMR (500 MHz, [D6]DMSO): d=11.74 (s, 1H), 10.43 (s, 1H),
8.50 (dd, J=0.9, 8.3 Hz, 1H), 7.92 (dd, J=1.3, 7.9 Hz, 1H), 7.62–7.56
(m, 3H), 7.52–7.48 (m, 2H), 7.28–7.22 (m, 1H), 7.17 (d, J=8.2 Hz,
2H), 7.13 (d, J=8.8 Hz, 1H), 3.83 (s, 3H), 3.82 (s, 3H), 2.28 ppm (s,
3H); 13C NMR (125 MHz, [D6]DMSO): d=167.50, 164.28, 152.10,
148.82, 139.16, 136.04, 133.52, 132.34, 129.16 (2C), 129.0, 126.93,
123.03, 122.43, 121.31 (2C), 121.03, 120.09, 111.56, 110.72, 55.87,
55.62, 20.65 ppm; Anal. calcd for C23H22N2O4: C 70.75, H 5.68, N
7.17, found: C 70.42, H 5.59, N 7.12.
3,4-Dimethoxy-N-(2-(4-methoxyphenylcarbamoyl)-phenyl)benz-
amide (16): According to method A, 3,4-dimethoxybenzoic acid
(109 mg, 0.6 mmol) was reacted to yield 3,4-dimethoxybenzoyl
chloride, which was then, according to method B, reacted with 2-
amino-N-(4-methoxyphenyl)benzamide (123 mg, 0.5 mmol) to
obtain compound 16 as a cream-colored powder (103 mg, 51%):
Rf =0.65 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d=11.84
(s, 1H), 10.41 (s, 1H), 8.53 (d, J=8.05 Hz, 1H), 7.93 (d, J=7.08 Hz,
1H), 7.65–7.56 (m, 3H), 7.51–7.48 (m, 2H), 7.27–7.22 (m, 1H), 7.13
(d, J=8.98 Hz, 1H), 6.94 (d, J=9.02 Hz, 2H), 3.83 (s, 3H), 3.82 (s,
3H), 3.75 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=167.33,
164.25, 156.23, 152.11, 148.84, 139.26, 132.32, 131.53, 128.92,
126.96, 122.97 (2C), 122.23, 122.16, 120.89, 120.04, 113.95 (2C),
111.57, 110.74, 55.87, 55.64, 55.38 ppm; Anal. calcd for
C23H22N2O5·0.25H2O: C 67.22, H 5.52, N 6.82, found: C 66.82, H 5.23
N 7.07
3,4-Dimethoxy-N-(2-(phenylcarbamoyl)phenyl)benzamide (20):
According to method A, 3,4-dimethoxybenzoic acid (109 mg,
0.6 mmol) was reacted to yield 3,4-dimethoxybenzoyl chloride,
which was then, according to method B, reacted with 2-amino-N-
phenylbenzamide (106 mg, 0.5 mmol) to obtain compound 20 as
1
a white powder (127 mg, 67%): Rf =0.62 (EtOAc/PE (1:1)); H NMR
(500 MHz, [D6]DMSO): d=11.66 (s, 1H), 10.51 (s, 1H), 8.49 (dd, J=
8.36, 0.83 Hz, 1H), 7.93 (dd, J=7.85, 1.42 Hz, 1H), 7.73 (d, J=
7.51 Hz, 2H), 7.60 (t, J=7.07 Hz, 1H), 7.52–7.49 (m, 2H), 7.39–7.33
(m, 2H), 7.26 (dt, J=7.66, 1.19 Hz, 1H), 7.15–7.12 (m, 2H), 3.83 (s,
3H), 3.82 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=167.65,
164.32, 152.11, 148.82, 139.12, 138.65, 132.38, 129.08, 128.76 (2C),
126.94, 124.40, 123.09, 122.65, 121.25 (2C), 121.18, 120.11, 111.56,
110.76, 55.86, 55.63 ppm; Anal. calcd for C22H20N2O4: C 70.20, H
5.36, N 7.44, found: C 69.99, H 5.46, N 7.37.
N-(2-(4-Hydroxyphenylcarbamoyl)phenyl)-3,4-dimethoxybenz-
amide (17): According to method A, 3,4-dimethoxybenzoic acid
(73 mg, 0.4 mmol) was reacted to yield 3,4-dimethoxybenzoyl chlo-
ride, which was then, according to method B, reacted with 2-
amino-N-(4-hydroxyphenyl)benzamide (114 mg, 0.5 mmol) to
obtain compound 17 as a cream-colored powder (40 mg, 25%):
Rf =0.79 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d=11.91
(s, 1H), 10.31 (s, 1H), 9.31 (s, 1H), 8.54 (dd, J=8.30, 0.62 Hz, 1H),
7.92 (dd, J=7.88, 1.18 Hz, 1H), 7.60–7.56 (m, 1H), 7.51–7.47 (m,
4H), 7.23 (dt, J=7.74, 1.10 Hz, 1H), 7.13 (d, J=8.98 Hz, 1H), 6.76
2-(4-Acetamidobenzamido)-N-phenylbenzamide (21): Compound
22 (99 mg, 0.3 mmol) was dissolved in dry THF and 10 drops of
Et3N were added. A solution of acetyl chloride (39 mg, 0.5 mmol) in
THF was added dropwise. The solution was stirred for 3 h and af-
terward the solvent was evaporated under reduced pressure.
Water was added to the residue, followed by extraction with
EtOAc (3”10 mL). The organic phase was washed with 1n NaOH
and 1n HCl and evaporated under reduced pressure. The crude
compound was dissolved in THF and precipitated with PE. Com-
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Full Papers
pound 21 was obtained as a cream-colored powder (84 mg, 74%):
Rf =0.54 (EtOAc); 1H NMR (500 MHz, [D6]DMSO): d=10.34 (s, 1H),
8.46 (dd, J=8.28, 0.76 Hz, 1H), 7.94 (dd, J=7.84, 1.34 Hz, 1H), 7.86
(d, J=8.73 Hz, 2H), 7.76–7.69 (m, 4H), 7.60–7.55 (m, 1H), 7.38–7.33
(m, 2H), 7.23 (t, J=7.43 Hz, 1H), 7.12 (t, J=7.39 Hz, 1H), 2.08 ppm
(s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=168.96, 167.54, 164.34,
142.74, 138.85, 132.21, 128.97 (2C), 128.76, 128.12 (2C), 124.23,
122.93, 122.89, 121.49, 121.28 (2C), 118.67 (2C), 24.24 ppm; HPLC:
k’=2.34 (RP18 column, CH3OH/H2O (80:20)), purity 98.0%.
0.5 mmol) and 4-methylbenzoyl chloride (73 mg, 0.6 mmol) were
reacted, and compound 26 was obtained as a white powder
(139 mg, 80%): Rf =0.86 (EtOAc/PE (1:2)); 1H NMR (500 MHz,
[D6]DMSO): d=11.77 (s, 1H), 10.70 (s, 1H), 8.49 (d, J=8.23 Hz, 1H),
7.94 (dd, J=7.78, 1.02 Hz, 1H), 7.82 (d, J=8.13 Hz, 2H), 7.57 (dd,
J=11.69, 8.55 Hz, 3H), 7.35 (d, J=7.97 Hz, 2H), 7.22 (t, J=7.50,
7.50 Hz, 1H), 7.15 (d, J=8.25 Hz, 2H), 2.37 (s, 3H), 2.28 ppm (s,
3H); 13C NMR (125 MHz, [D6]DMSO): d=167.38, 164.78, 141.99,
136.26, 133.30, 132.15, 129.48 (2C), 129.15 (2C), 129.11, 127.20 (2C),
122.86, 122.74, 121.42, 121.34 (2C), 21.13, 20.65 ppm; Anal. calcd
for C22H20N2O2·1.4H2O: C 71.49, H 6.22, N 7.58, found: C 71.36, H
5.72, N 7.35.
2-(4-Aminobenzamido)-N-phenylbenzamide (22): Compound 11
0.6 (217 mg, 0.6 mmol) was reacted according to method C to
yield compound 22 as a white powder (119 mg, 60%): Rf =0.77
(EtOAc); 1H NMR (500 MHz, [D6]DMSO): d=11.47 (s, 1H), 10.49 (s,
1H), 8.54 (d, J=8.35 Hz, 1H), 7.91 (dd, J=7.85, 1.43 Hz, 1H), 7.72
(d, J=8.61 Hz, 2H), 7.61 (d, J=8.66 Hz, 2H), 7.58–7.54 (m, 1H),
7.40–7.35 (m, 2H), 7.20 (dt, J=1.11 Hz, 1H), 7.14 (t, J=7.39 Hz, 1H),
6.62 (d, J=8.65 Hz, 2H), 5.83 ppm (s, 2H); 13C NMR (125 MHz,
[D6]DMSO): d=167.81, 164.61, 152.77, 139.78, 138.58, 132.35,
129.03, 128.81 (2C), 128.78 (2C), 124.39, 122.35, 121.72, 121.35 (2C),
120.84, 120.67, 113.08 ppm (2C); HPLC: k’=1.63 (RP18 column,
CH3OH/H2O (80:20)); purity 98.4%
N-(4-Benzoylphenyl)-2-(4-methylbenzamido)benzamide (27): Ac-
cording to method B, 2-amino-N-(4-benzoylphenyl)benzamide
(158 mg, 0.5 mmol) and 4-methylbenzoyl chloride (93 mg,
0.6 mmol) were reacted, and compound 27 was obtained as
1
a white powder (170 mg, 78%): Rf =0.58 (EtOAc/PE (1:1)); H NMR
(500 MHz, [D6]DMSO): d=11.42 (s, 1H), 10.87 (s, 1H), 8.40 (dd, J=
8.32, 0.97 Hz, 1H), 7.94–7.90 (m, 3H), 7.82 (d, J=8.19 Hz, 2H), 7.80–
7.76 (m, 2H), 7.74–7.71 (m, 2H), 7.68–7.64 (m, 1H), 7.64–7.59 (m,
1H), 7.55 (t, J=7.65 Hz, 2H), 7.35 (d, J=7.91 Hz, 2H), 7.29 (dt, J=
7.67, 1.12 Hz, 1H), 2.37 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO):
d=194.77, 167.89, 164.81, 143.18, 142.23, 138.77, 137.63, 132.44
(2C), 132.24, 131.89, 130.98 (2C), 129.55 (2C), 129.50 (2C), 129.29,
128.62 (2C), 127.26 (2C), 123.55, 123.41, 121.82, 120.21 (2C),
21.13 ppm; Anal. calcd for C28H22N2O3: C 77.40, H 5.10, N 6.45,
found: C 76.98, H 5.24, N 6.34.
2-(4-Chlorobenzamido)-N-(4-methoxyphenyl)benzamide (23): Ac-
cording to method B, 2-amino-N-(4-methoxyphenyl)benzamide
(123 mg, 0.5 mmol) and 4-chlorobenzoyl chloride (88 mg,
0.6 mmol) were reacted, and compound 23 was obtained as
a light-grey powder (113 mg, 58%): Rf =0.74 (EtOAc/PE (1:1));
1H NMR (500 MHz, [D6]DMSO): d=11.88 (s, 1H), 10.41 (s, 1H), 8.48
(d, J=8.19 Hz, 1H), 7.92 (t, J=9.13 Hz, 3H), 7.65–7.57 (m, 5H), 7.28
(t, J=7.40 Hz, 1H), 6.94 (d, J=8.94 Hz, 2H), 3.74 ppm (s, 3H);
13C NMR (125 MHz, [D6]DMSO): d=167.17, 163.64, 156.23, 138.76,
136.99, 133.42, 132.27, 131.45, 129.13 (2C), 129.02 (2C), 128.96,
123.48, 123.08 (2C), 122.73, 121.34, 113.94 (2C), 55.36 ppm; Anal.
calcd for C21H17ClN2O3: C 66.23, H 4.50, N 7.36, found: C 66.53, H
4.91, N 6.98.
N-(4-(2-Methoxyethyl)phenyl)-2-(4-(trifluoromethyl)benzamido)-
benzamide (28): According to method A, 4-trifluoromethylbenzoic
acid (228 mg, 1.2 mmol) was reacted with 4-trifluoromethylbenzoyl
chloride and then, according to method B, with compound 3a
(203 mg, 0.75 mmol), and compound 28 was obtained as a beige
1
powder (96 mg, 29%): Rf =0.81 (EtOAc/PE (1:1)); H NMR (500 MHz,
[D6]DMSO): d=11.83 (s, 1H), 10.47 (s, 1H), 8.43 (d, J=8.19 Hz, 1H),
8.11–8.07 (m, 2H), 7.96–7.91 (m, 3H), 7.64–7.57 (m, 3H), 7.31 (t, J=
7.46 Hz, 1H), 7.21 (d, J=8.43 Hz, 2H), 3.51 (t, J=6.84 Hz, 2H), 3.23
(s, 3H), 2.77 ppm (t, J=6.82 Hz, 2H); 13C NMR (125 MHz,
[D6]DMSO): d=167.28, 163.63, 138.49, 138.42, 136.60, 135.28,
132.29, 131.85, 129.08, 129.06 (2C), 128.12 (2C), 126.08 (2C), 123.97,
123.83, 123.48, 121.72, 121.33 (2C), 72.95, 57.93, 34.98 ppm; Anal.
calcd for C21H21F3N2O3·0.25H2O: C 64.50, H 4.85, N 6.27, found: C
64.61, H 4.80, N 6.27.
2-(4-Chlorobenzamido)-N-(4-hydroxyphenyl)benzamide (24): Ac-
cording to method B, 2-amino-N-(4-hydroxyphenyl)benzamide
(114 mg, 0.5 mmol) and 4-chlorobenzoyl chloride (70 mg,
0.4 mmol) were reacted, and compound 24 was obtained as
1
a white powder (140 mg, 95%): Rf =0.28 (EtOAc/PE (1:1)); H NMR
(500 MHz, [D6]DMSO): d=11.97 (s, 1H), 10.32 (s, 1H), 9.33 (s, 1H),
8.50 (d, J=8.17 Hz, 1H), 7.94–7.89 (m, 3H), 7.64 (d, J=8.53 Hz, 2H),
7.61–7.57 (m, 1H), 7.46 (d, J=8.80 Hz, 2H), 7.27 (t, J=7.62 Hz, 1H),
6.76 ppm (d, J=8.85 Hz, 2H); 13C NMR (125 MHz, [D6]DMSO): d=
167.09, 163.63, 154.49, 138.87, 137.00, 133.46, 132.25, 129.86,
129.17 (2C), 129.02 (2C), 128.93, 123.39 (3C), 122.53, 121.20,
115.20 ppm (2C); Anal. calcd for C21H17ClN2O3·0.66H2O: C 63.41, H
4.35, N 7.40, found: C 63.37, H 4.27, N 7.22.
N-(4-Hydroxyphenyl)-2-(4-(trifluoromethyl)benzamido)benz-
amide (29): According to method A, 4-trifluoromethylbenzoic acid
(133 mg, 0.7 mmol) was reacted with 4-trifluoromethylbenzoyl
chloride and then, according to method B, with 2-amino-N-(4-hy-
droxyphenyl)benzamide (114 mg, 0.86 mmol), and compound 29
was obtained as a beige powder (179 mg, 64%): Rf =0.99 (EtOAc);
1H NMR (500 MHz, [D6]DMSO): d=12.06 (s, 1H), 10.33 (s, 1H), 9.30
(s, 1H), 8.50 (d, J=7.8 Hz, 1H), 8.09 (d, J=8.2 Hz, 2H), 7.98–7.90
(m, 3H), 7.61 (t, J=7.8 Hz, 1H), 7.47 (d, J=8.9 Hz, 2H), 7.29 (t, J=
7.6 Hz, 1H), 6.76 ppm (d, J=8.8 Hz, 2H); 13C NMR (125 MHz,
[D6]DMSO): d=166.99, 163.50, 154.47, 138.63, 138.49, 132.23,
132.20–131.28 (m),129.87, 128.93, 128.06 (2C), 126.09 (2C), 123.68,
123.34 (2C), 127.31–120.58 (m), 122.81, 121.34, 115.18 ppm (2C);
Anal. calcd for C21H15F3N2O3·0.25H2O: C 62.30, H 3.86, N 6.92,
found: C 62.29, H 4.03, N 6.97.
2-(4-Chlorobenzamido)-N-p-tolylbenzamide (25): According to
method B,
2-amino-(N-4-methylphenyl)benzamide
(113 mg,
0.5 mmol) 4-chlorobenzoyl chloride (94 mg, 0.6 mmol) were react-
ed, and compound 25 was obtained as a white powder (51 mg,
1
28%): Rf =0.90 (EtOAc/PE (1:1)); H NMR (500 MHz, [D6]DMSO): d=
11.79 (s, 1H), 10.53 (s, 1H), 8.43 (d, J=8.18 Hz, 1H), 7.94–7.90 (m,
3H), 7.62 (d, J=8.49 Hz, 2H), 7.58 (m, 3H), 7.26 (t, J=7.48 Hz, 1H),
7.16 (d, J=8.19 Hz, 2H), 2.28 ppm (s, 3H); 13C NMR (125 MHz,
[D6]DMSO): d=167.27, 163.77, 136.87, 136.09, 133.41, 132.20,
129.14 (2C), 129.08 (5C), 123.40, 123.18, 121.63, 121.34 (2C),
20.63 ppm; Anal. calcd for C21H17ClN2O2·0.33H2O: C 68.02, H 4.80, N
7.55; found: C 67.91, H 4.63, N 7.31.
N-(4-Chlorophenyl)-2-(4-(trifluoromethyl)benzamido)benzamide
(30): According to method A, 4-trifluoromethylbenzoic acid
(114 mg, 0.6 mmol) was reacted with 4-trifluoromethylbenzoyl
chloride and then, according to method B, with 2-amino-N-(4-chlo-
2-(4-Methylbenzamido)-N-p-tolylbenzamide (26): According to
method B,
2-amino-(N-4-methylphenyl)benzamide
(113 mg,
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rophenyl)benzamide (123 mg, 0.5 mmol), and compound 30 was
obtained as a cream-colored powder (56 mg, 23%): Rf =0.85
(EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d=11.57 (s, 1H),
10.61 (s, 1H), 8.33 (d, J=7.7 Hz, 1H), 8.09 (d, J=8.1 Hz, 2H), 7.93
(d, J=8.3 Hz, 2H), 7.89 (d, J=6.6 Hz, 1H), 7.74 (d, J=8.9 Hz, 2H),
7.62 (m, 1H), 7.40 (d, J=8.9 Hz, 2H), 7.32 ppm (td, J=1.0, 7.7 Hz,
1H); 13C NMR (125 MHz, [D6]DMSO): d=167.31, 163.78, 138.50,
138.07, 137.74, 132.28, 129.12, 128.66 (2C), 128.18 (2C), 127.95,
125.99 (2C), 124.02, 123.96, 122.61 (2C), 122.20 ppm; Anal. calcd for
C21H14ClF3N2O2: C 60.23, H 3.37, N 6.69, found: C 60.29, H 3.26, N
6.62.
1H), 8.89 (s, 1H), 8.59 (dd, J=8.35, 1.04 Hz, 1H), 8.41 (d, J=
8.85 Hz, 2H), 8.14 (d, J=8.87 Hz, 2H), 7.85 (dd, J=7.94, 1.43 Hz,
1H), 7.59–7.55 (m, 1H), 7.23 (dt, J=7.82, 1.15 Hz, 1H), 3.33 (dq, J=
7.22, 5.63 Hz, 2H), 1.14 ppm (t, J=7.22 Hz, 3H); 13C NMR (125 MHz,
[D6]DMSO): d=168.37, 162.83, 149.57, 140.32, 139.14, 132.34,
128.59 (2C), 128.31, 124.28 (2C), 123.50, 120.76, 120.66, 34.36,
14.54 ppm; Anal. calcd for C16H15N3O4·0.2H2O: C, 60.64; H, 4.90; N,
13.26, found: C, 60.90; H, 4.84; N, 13.13.
Biological studies
Materials: All chemicals were purchased from Sigma–Aldrich Chem-
icals (Taufkirchen, Germany) unless otherwise specified. For cell-
based assays, 10 mm stock solutions of the compounds were pre-
pared in DMSO. Various dilutions of the test compounds were pre-
pared in sterile filtered Krebs-HEPES buffer (KHB). For some com-
pounds with low solubility, methanol (50%) was used for preparing
1 mm concentrations. The amount of methanol did not exceed
0.5% at the highest concentration assayed. The highest concentra-
tion of DMSO present was not more than 0.1%.
N-Phenyl-2-(4-(trifluoromethyl)benzamido)benzamide (31): Ac-
cording to method A, 4-trifluoromethylbenzoic acid (91 mg,
0.48 mmol) was reacted with 4-trifluoromethylbenzoyl chloride and
then, according to method B, with 2-amino-N-phenylbenzamide
(85 mg, 0.4 mmol), and compound 31 was obtained as a white
1
powder (81 mg, 42%): Rf =0.63 (EtOAc/PE (1:1)); H NMR (500 MHz,
[D6]DMSO): d=11.74 (s, 1H), 10.51 (s, 1H), 8.40 (dd, J=8.31,
0.87 Hz, 1H), 8.10 (d, J=8.10 Hz, 2H), 7.96–7.91 (m, 3H), 7.70 (dd,
J=8.55, 1.02 Hz, 2H), 7.65–7.60 (m, 1H), 7.37–7.30 (m, 3H),
7.13 ppm (m, 1H); 13C NMR (125 MHz, [D6]DMSO): d=167.36,
163.68, 138.64, 138.48, 138.28, 132.29, 133.00–130.10 (m), 129.14,
128.75 (2C), 128.14 (2C), 126.06 (2C), 124.37, 127.01–123 (m),
123.92, 123.80, 121.89, 121.27 ppm (2C); Anal. calcd for
C21H15F3N2O2: C 65.62, H 3.93, N 7.29, found: C 65.38, H 4.27, N
7.42.
Cell culture: The BCRP-expressing cell line MCF-7 MX and the pa-
rental cell line MCF-7 were grown in RPMI-1640 medium supple-
mented with 10% fetal bovine serum (FBS), 50 mgmLÀ1 streptomy-
cin, 50 UmLÀ1 penicillin G, and 2 mm l-glutamine. MDCK wild-type
and MDCK BCRP cell lines were received as a generous gift from
Dr. A. Schinkel (The Netherlands Cancer Institute, Amsterdam).
MDCK BCRP cells were generated by transfection of the canine
kidney epithelial cell line MDCKII with the human wild-type BCRP
cDNA C-terminally linked to the cDNA of green fluorescent protein
(GFP). These cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) with 10% FBS, 50 mgmLÀ1 streptomycin,
50 UmLÀ1 penicillin G, and 2 mm l-glutamine.
N-(4-Hydroxycyclohexyl)-2-(4-nitrobenzamido)benzamide (32):
According to method B, 2-amino-N-(4-hydroxycyclohexyl)benz-
amide (94 mg, 0.5 mmol) and 4-nitrobenzoyl chloride (93 mg,
0.5 mmol) were reacted, and compound 32 was obtained as
a pale-yellow powder (31 mg, 20%): Rf =0.52 (EtOAc); 1H NMR
(500 MHz, [D6]DMSO): d=12.68 (s, 1H), 8.59–8.53 (m, 2H), 8.42 (d,
J=8.82 Hz, 2H), 8.13 (d, J=8.83 Hz, 2H), 7.85 (dd, J=7.93, 1.33 Hz,
1H), 7.59–7.55 (m, 1H), 7.25–7.19 (m, 1H), 4.54 (d, J=4.41 Hz, 1H),
3.82–3.72 (m, 1H), 3.42–3.34 (m, 1H), 1.88–1.78 (m, 4H), 1.44–1.34
(m, 2H), 1.28–1.19 ppm (m, 2H); 13C NMR (125 MHz, [D6]DMSO):
d=167.85, 162.89, 149.58, 140.33, 138.92, 132.27, 128.61 (3C),
124.32 (2C), 123.50, 121.21, 120.74, 68.38, 48.17, 34.25 (2C),
30.09 ppm (2C); Anal. calcd for C20H21N3O5·0.66H2O: C 60.75, H
5.69, N 10.63, found: C 60.59, H 5.50, N 10.39.
The human ovarian carcinoma cell line A2780 and the correspond-
ing MDR1-overexpressing doxorubicin-resistant A2780Adr cell line
were purchased from ECACC (nos. 93112519 and 93112520). The
cell lines were grown in RPMI-1640 medium supplemented with
10% FBS, 50 mgmLÀ1 streptomycin, 50 UmLÀ1 penicillin G, and
2 mm l-glutamine. To maintain P-gp overexpression in the
A2780Adr cell line, periodically (every 5 to 7 passages) cells were
incubated with 1 mm doxorubicin for one passage. All cells were
maintained under a 5% CO2 humidified atmosphere at 378C. After
reaching a confluence of 80–90%, cells were harvested with tryp-
sin-EDTA (0.05% trypsin/0.02% EDTA), centrifuged (266 g, 48C,
4 min) and re-suspended in fresh culture medium. Cell counting
was performed with a CASY1 model TT cell counter (Schaerfe
System GmbH, Reutlingen, Germany) with 150 mm capillary to
determine constant cell densities in the various cell-based assays.
N-(4-Hydroxycyclohexyl)-2-(4-(trifluoromethyl)benzamido)benz-
amide (33): According to method A, 4-trifluoromethylbenzoic acid
(95 mg, 0.5 mmol) was reacted with 4-trifluoromethylbenzoyl chlo-
ride and then, according to method B, 2-amino-N-(4-hydroxycyclo-
hexyl)benzamide (94 mg, 0.5 mmol), and compound 33 was ob-
tained as a beige powder (26 mg, 16%): Rf =0.14 (EtOAc/PE (1:1));
1H NMR (500 MHz, [D6]DMSO): d=12.64 (s, 1H), 8.59–8.56 (m, 2H),
8.11 (d, J=8.10 Hz, 2H), 7.98 (d, J=8.23 Hz, 2H), 7.84 (dd, J=7.93,
1.35 Hz, 1H), 7.58–7.54 (m, 1H), 7.21 (dt, J=7.89, 1.09 Hz, 1H), 4.54
(d, J=4.35 Hz, 1H), 3.81–3.73 (m, 1H), 3.42–3.34 (m, 1H), 1.83 (dd,
J=10.85, 7.90 Hz, 4H), 1.44–1.34 (m, 2H), 1.28–1.19 ppm (m, 2H);
13C NMR (125 MHz, [D6]DMSO): d=167.91, 163.36, 139.08, 138.55,
132.27, 132.16–131.42 (m), 128.59, 128.02 (2C), 126.22 (2C), 125.08,
123.33, 121.08, 120.66, 68.39, 48.17, 34.27 (2C), 30.11 ppm (2C);
Anal. calcd for C21H21F3N2O3·H2O: C 59.43, H 5.46, N 6.60, found: C
59.51, H 5.29, N 6.55.
Hoechst 33342 accumulation assay: To investigate the inhibitory
effect of the synthesized compounds on BCRP, a Hoechst 33342 ac-
cumulation assay was performed as described earlier with
a
Hoechst 33342 concentration of 1mm in the fluorescence
assay.[16,22–26] The increase in cellular fluorescence due to Hoechst
33342 accumulation was determined in the absence and presence
of various inhibitor concentrations. Briefly, after reaching a conflu-
ence of 80–90%, cells were harvested by gentle trypsin treatment
(0.05% trypsin/0.02% EDTA) and then transferred to a 50 mL tube
followed by centrifugation (266 g, 48C, 4 min). The cell pellet ob-
tained was re-suspended in fresh culture medium, and the cell
density was determined with a CASY1 model TT cell counter
(Schaerfe System GmbH). Cells were again centrifuged and re-sus-
pended in KHB to yield a cell density of 3”106 cells per mL; 90 mL
of this final suspension, containing ~27000 cells, were seeded into
N-Ethyl-2-(4-nitrobenzamido)benzamide (34): According to meth-
od B, 2-amino-N-ethylbenzamide (82 mg, 0.5 mmol) and 4-nitro-
benzoyl chloride (130 mg, 0.7 mmol) were reacted, and compound
34 was obtained as a cream-colored powder (105 mg, 67%): Rf =
0.65 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d=12.84 (s,
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black 96-well plates (Greiner, Frickenhausen, Germany). Test com-
pounds (10 mL) at various concentrations were added to a total
volume of 100 mL. The prepared 96-well plate was incubated under
5% CO2 at 378C for 30 min. After this pre-incubation period, a solu-
tion of Hoechst 33342 (20 mL, 6 mm; protected from light) was
added to each well. Fluorescence was measured immediately at
constant intervals of 60 s up to 120 min at an excitation wave-
length of 355 nm and an emission wavelength of 460 nm using
a BMG POLARstar microplate reader (BMG Labtech, Offenburg, Ger-
many) held at 378C. For assay data analysis, the fluorescence from
KHB was initially subtracted from the fluorescence reading ob-
tained from MCF-7 MX cells. The upper plateau value (ymax) of each
fluorescence–time curve was determined based on a one-phase ex-
ponential association curve fit. From these data, concentration–
response curves were generated by nonlinear regression using the
four-parameter logistic equation with variable Hill slope (Prism 5.0,
GraphPad Software Inc., San Diego, CA, USA).
centration in a final volume of 200 mL. After incubation for 72 h,
MTT was added (20 mL of a 5 mgmLÀ1 solution) to each well. Plates
were further incubated for 1 h and then the assay was terminated
by removing supernatants and lysing the cells with 100 mL DMSO
per well. Cell viability was determined spectrophotometrically by
measuring absorbance at 544 nm and background correction at
710 nm using a BMG POLARstar microplate reader.
Keywords: ABCG2 · ABC transporter · BCRP · inhibitors ·
multidrug resistance
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Calcein AM assay: To determine the selectivity of compounds
toward BCRP, they were further tested for their P-gp inhibition by
calcein AM assay using A2780Adr cells, as described previous-
ly.[16,28] After reaching a confluence of 80–90%, cells were harvested
with trypsin-EDTA (0.05% trypsin/0.02% EDTA), centrifuged (266 g,
48C, 4 min) and re-suspended in fresh culture medium. Cells were
prepared as described above and seeded into clear 96-well plates
(Greiner) at a density of ~30000 cells in a volume of 90 mL per
well. Test compounds (10 mL) were then added, resulting in a final
volume of 100 mL per well. The prepared 96-well plates were pre-
incubated for 30 min. After this pre-incubation period, calcein AM
(33 mL, 1.25 mm; protected from light) was added to each well. The
fluorescence was measured immediately at constant time intervals
(60 s) up to 60 min using an excitation wavelength of 485 nm and
an emission wavelength of 520 nm with a BMG POLARstar micro-
plate reader held at 378C. The slope of the initial linear portion of
each fluorescence–time curve was calculated by linear regression.
From the slopes, concentration–response curves were generated
by nonlinear regression using the four-parameter logistic equation
with variable Hill slope (GraphPad Prism 5.0).
MTT cytotoxicity assay: To investigate the MDR-reversal capacity of
selected compounds, the MTT cytotoxicity assay was used. The
assay was performed as described earlier with minor modifica-
tions.[26,27,29] In brief, MDCK BCRP and parental MDCK cells were
seeded into 96-well tissue culture plates (Sarstedt, Newton, USA) at
a density of 2500 cells per well in a volume of 160 mL and kept
under 5% CO2 at 378C for 6 h. Attachment of cells was monitored
by microscope, and SN-38 (20 mL), the active metabolite of irinote-
can, was added at various concentrations. Finally, test compounds
(20 mL) were added to the cells to achieve the required final con-
Received: November 21, 2014
Revised: January 26, 2015
Published online on March 3, 2015
ChemMedChem 2015, 10, 742 – 751
www.chemmedchem.org
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