Synthesis and Quantitative Structure-Activity Relationships of Selective BCRP Inhibitors

Article abstract of DOI:10.1002/cmdc.201200377

The breast cancer resistance protein (BCRP/ABCG2) is a member of the ABC transporter superfamily. This protein has a number of physiological functions, including protection of the human body from xenobiotics. The overexpression of BCRP in certain tumor cell lines causes cross-resistance against various drugs used in chemotherapeutic treatment. In a previous work we showed that a new class of compounds derived from XR9576 (tariquidar) selectively inhibits BCRP. In this work we synthesized more members of this class, with modification on the second and third aromatic rings. The inhibitory activities against BCRP and P-gp were assayed using a Hoechst 33342 assay for BCRP and a calcein AM assay for P-gp. Finally, quantitative structure-activity relationships for both aromatic rings were established. The results obtained show the importance of the electron density on the third aromatic ring, influenced by substituents, pointing to interactions with aromatic residues of the protein binding site. In the second aromatic ring the activity of compounds is influenced by the steric volume of the substituents.

Full text of DOI:10.1002/cmdc.201200377

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DOI: 10.1002/cmdc.201200377
Synthesis and Quantitative Structure–Activity
Relationships of Selective BCRP Inhibitors
Federico Marighetti, Kerstin Steggemann, Markus Hanl, and Michael Wiese*[a]
The breast cancer resistance protein (BCRP/ABCG2) is
a member of the ABC transporter superfamily. This protein has
a number of physiological functions, including protection of
the human body from xenobiotics. The overexpression of
BCRP in certain tumor cell lines causes cross-resistance against
various drugs used in chemotherapeutic treatment. In a previ-
ous work we showed that a new class of compounds derived
from XR9576 (tariquidar) selectively inhibits BCRP. In this work
we synthesized more members of this class, with modification
on the second and third aromatic rings. The inhibitory activi-
ties against BCRP and P-gp were assayed using a Hoechst
33342 assay for BCRP and a calcein AM assay for P-gp. Finally,
quantitative structure–activity relationships for both aromatic
rings were established. The results obtained show the impor-
tance of the electron density on the third aromatic ring, influ-
enced by substituents, pointing to interactions with aromatic
residues of the protein binding site. In the second aromatic
ring the activity of compounds is influenced by the steric
volume of the substituents.
Introduction
A major problem in cancer chemotherapy is the development
of multidrug resistance (MDR) mediated by ABC transporters.
This class of membrane proteins uses the energy of ATP hy-
drolysis to actively pump metabolites or xenobiotics out of
cells against a concentration gradient. If members of this
family are overexpressed in chemotherapy-resistant tumor
cells, they cause a remarkable decrease in cytoplasmic concen-
trations of chemotherapeutic agents. The ABC transporters
that have been identified to play an important role in MDR are
P-glycoprotein (P-gp, ABCB1), the breast cancer resistance pro-
tein (BCRP, ABCG2), and the multidrug-resistance-associated
protein 1 (MRP1, ABCC1).[1]
longs to the ABCC subfamily, and like many other transporters
in this group, has an extra N-terminal transmembrane domain
with five transmembrane helices.[7]
P-gp is able to transport a large variety of chemically diverse
substrates. These are usually lipophilic and amphipathic com-
pounds with molecular weights between 400 and 4000 Da.[8]
Under physiological conditions BCRP transports conjugated
and unconjugated steroids and primary bile acids.[9] Like P-gp,
BCRP is also able to efflux a wide variety of chemotherapeutic
substances such as mitoxantrone, anthracyclines, antifolates,
and tyrosine kinase inhibitors.[10] MRP1 is involved in the trans-
port of bile salts and other organic anions.[11]
In humans, 48 ABC transporters have been identified and
classified into seven subfamilies (A–G).[2] P-gp belongs to the
ABCB subfamily. It has a molecular weight of ~170 kDa and
consists of 1280 amino acids. It has two transmembrane re-
gions with two nucleotide binding domains. Each transmem-
brane region consists of six transmembrane segments.[3,4] BCRP
is a member of the ABCG subfamily, and like all members of
the ABCG subfamily is a half transporter. BCRP has a molecular
weight of 72 kDa and is composed of 655 amino acids. While
most ABC transporters contain an N-terminal transmembrane
domain followed by the nucleotide binding domain, BCRP has
a reverted structure with an N-terminal nucleotide binding
domain followed by a transmembrane domain with six trans-
membrane helices. BCRP may function as a homodimer proba-
bly linked by a disulfide bond, or as a tetramer.[5,6] MRP1 be-
A possible strategy to reverse ABC-transporter-mediated
MDR is the inhibition of these proteins by inhibitors, also
called modulators. To date, many inhibitors of P-gp are known,
while for the other two ABC transporters the arsenal of inhibi-
tors is relatively small. GF120918 (elacridar) and XR9576 (tari-
quidar) are potent third-generation P-gp inhibitors that also in-
hibit BCRP.[12] Tyrosine kinase inhibitors such as imatinib and
gefitinib have been found to increase the accumulation of sub-
strates like Hoechst 33342 in cells expressing BCRP and P-
gp.[13–15]
Some natural products such as fumitremorgin C (FTC), try-
prostatin A, and novobiocin are found to be selective inhibitors
of BCRP. Ko143, a synthetic derivative of FTC, is a selective
BCRP inhibitor with low cytotoxicity and is 10-fold more
potent than its precursor.[16] Flavonoids, a large class of natural
compounds, are also able to inhibit BCRP, but they also show
inhibitory effects against P-gp and MRP1. We recently reported
the discovery of a new class of specific BCRP inhibitors pos-
sessing a scaffold consisting of three aromatic rings connected
by two amide bonds.[17] Herein we describe the further devel-
opment of this class of BCRP inhibitors. Most of the synthe-
[a] F. Marighetti,+ Dr. K. Steggemann,+ M. Hanl, Prof. Dr. M. Wiese
Pharmaceutical Institute, University of Bonn
An der Immenburg 4, 53121 Bonn (Germany)
E-mail: mwiese@uni-bonn.de
[+] These authors contributed equally to this work.
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sized compounds are selective for BCRP, whereas some inhibit
BCRP and P-gp. The results of biological testing were used to
build a structure–activity relationship (SAR) model that is
useful to understand the SAR for the second and third aromat-
ic rings of this compound class.
Results and Discussion
Chemistry
The basic scaffold of the investigated compounds consists of
two aromatic rings connected by an amide linker. The first aro-
matic ring has a hydroxyethyl group at the para position with
respect to the amide group. The second aromatic ring also
bears an amide function with various substituents such as
methyl, cyclohexyl, or substituted aromatic ring systems. The
two amide groups on the
Scheme 1. General strategy for the synthesis the new BCRP inhibitors. Re-
agents 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.
second aromatic ring are at po-
sitions ortho, meta, or para. The
Table 1. Structures and inhibitory activities of the investigated compounds.
studied compounds were pre-
pared by following the general
strategy shown in Scheme 1. In
the first step 2-(4-aminopheny-
l)ethanol 1 was allowed to react
with different substituted nitro-
IC50 [mm][a]
benzoyl
chlorides,
yielding
amides 2a–k. Afterward, the ar-
omatic nitro group was reduced
by catalytic hydrogenation with
palladium/charcoal to get sub-
stituted anilines 3a–k. The reac-
tion of these anilines with an
appropriate acid chloride yield-
ed the desired compounds 4–
28.
Compd
Subst.
R1
R2
R3
BCRP
P-gp
4
5
6
7
8
9
ortho
meta
para
H
H
H
OCH3
H
OCH3
H
CH3
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
4-Nitrophenyl
4-Nitrophenyl
4-Nitrophenyl
4-Nitrophenyl
4-Nitrophenyl
4-Nitrophenyl
4-Nitrophenyl
4-Nitrophenyl
4-Nitrophenyl
4-Nitrophenyl
Cyclohexyl
1.58ꢀ0.36
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
OCH3
OCH3
H
CH3
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
6.96ꢀ2.20
3.93ꢀ0.11
8.43ꢀ1.63
2.69ꢀ0.86
4.08ꢀ0.48
2.59ꢀ0.86
3.09ꢀ0.22
NE
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
NE
NE
NE
Biological activity and QSAR
Methyl
NE
NE
3-Nitrophenyl
3-Aminophenyl
4-Aminophenyl
4-Cyanophenyl
4-Chlorophenyl
3-Chlorophenyl
4-Trifluoromethylphenyl
4-Acetoxyphenyl
4-tert-Butylphenyl
4-Isopropoxyphenyl
4-Hydroxyphenyl
3,4-Dimethoxyphenyl
4-Methylphenyl
1.31ꢀ0.25
8.65ꢀ2.37
8.12ꢀ1.00
2.54ꢀ0.19
3.08ꢀ1.32
3.48ꢀ0.54
2.20ꢀ0.57
12.8ꢀ2.10
2.95ꢀ0.83
1.15ꢀ0.29
21.2ꢀ3.67
3.61ꢀ0.97
4.59ꢀ0.57
21.3ꢀ2.87
14.0ꢀ4.29
15.5ꢀ2.05
NE
The inhibitory activity of the in-
vestigated substances against
BCRP was determined in MCF-7
MX cells using the Hoechst
33342 assay, as described previ-
ously. The calcein AM assay was
used to measure the inhibitory
potency against P-gp in A2780
Adr cells. Table 1 lists the activity
data for 25 compounds against
BCRP and P-gp. Two of the in-
hibitors, compounds 4 and 28,
have been reported previous-
ly.[17] Furthermore, it was shown
that six members of this new
NE
NE
NE
NE
H
H
H
H
11.7ꢀ3.87
8.42ꢀ2.45
NE
7.75ꢀ1.19
NE
H
H
H
[a] Values are the mean ꢀSD from three independent experiments; NE: no effect up to a concentration of
10 mm.
class of BCRP inhibitors are also able to block the transport of
pheophorbide A in MDCK BCRP cells. As the pIC50 values from
both assays were linearly correlated, we concluded that the in-
hibitory effect of the studied substances is not specific for
Hoechst 33342 and could be extended to other BCRP sub-
strates.
Two of the newly synthesized compounds, 5 and 6, showed
no activity against BCRP in the Hoechst 33342 accumulation
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assay. These are the only compounds with meta or para substi-
tution on the second ring. In contrast, the ortho-substituted
compound 4 is a potent and selective BCRP inhibitor. There-
fore, the relative position of the two amide groups seems to
be crucial.
Owing to the different substitution patterns, the three com-
pounds have a considerable difference in their spatial position-
ing of the aromatic rings, pointing to some steric requirements
for BCRP inhibition. Furthermore, only ortho-substituted com-
pounds can establish a hydrogen bond between the NH and
the C=O of the amide groups on the second ring. This interac-
tion may be important for the active conformation of this com-
pound class.
In case of the second aromatic ring, the presence of any
substituent decreases the inhibitory potency of the com-
pounds relative to that of the reference inhibitor 4. The substi-
tution at position R2 causes a slight decrease in activity (com-
pounds 8, 10, and 12), whereas substitution at R1 leads to
a more significant decrease in activity (compounds 9, 11, and
13).
The presence of a methoxy group at position R2 (compound
8) results in a slight decrease in activity (IC50 =3.93 mm), but at
position R1 the presence of the same substituent (in 9) de-
creases the inhibitory activity by roughly fivefold (IC50 =
8.43 mm). Substitution at both positions (compound 7) results
in an inhibitory effect similar to that of compound 9 (IC50 =
6.96 mm). The activity of compound 9 could be explained by
the predominance of the steric effect at position R1 over the
effect at position R2.
Figure 1. a) Plot of van der Waals volumes of the substituted second aromat-
ic ring versus pIC50 values for compounds with a substituent on the second
1
2
~
*
aromatic ring at position R ( ) and with substitution at position R ( ).
b) Plot of measured versus calculated potencies for inhibition of BCRP in
MCF-7 MX cells according to Equation (1) for compounds with variations on
the third aromatic ring.
With methyl groups as substituents the same trend is ob-
served: A methyl group at position R2 (compound 10) decreas-
es the activity to an IC50 value of 2.69 mm, whereas the same
substitution at position R1 (compound 11) gives a less active
compound, with IC50 =4.08 mm. Compounds with chloro sub-
stituents at positions R1 (13) or R2 (12) on the second ring
have similar inhibitory effects against BCRP (IC50 values of 3.09
and 2.59 mm, respectively).
As shown in Figure 1a the activity of the substituted com-
pounds decreases linearly with increasing van der Waals
volume of the substituted aromatic ring, as calculated with
MOE.[18] This unfavorable steric effect is more evident at posi-
tion R1 than at position R2. On the whole, the SAR of the
second aromatic ring may be explained by a disadvantageous
steric interaction of the substituents with the binding site of
the protein.
the amide group as reference position. Vsurf_S is the interac-
tion field surface area of the substituted aromatic ring calculat-
ed by MOE. The relative importance of descriptors is 1 for
s and 0.963 for Vsurf_S. Figure 1b shows a plot of observed
versus calculated pIC50 values.
pIC50 ¼ 0:503 ꢁ spþm þ 0:00932 ꢁ Vsurf S þ 3:010
ð1Þ
n ¼ 13, r2 ¼ 0:81, s ¼ 0:156, F ¼ 45:9
A possible explanation for this trend could be the interac-
tion of the ring system at position R3 with aromatic residues of
the protein binding site and the consequential p–p stacking.
Furthermore, the presence of bulky substituents with a large
contact surface area on this ring increases the affinity of the li-
gands and hence the inhibitory activity.
To investigate the importance of the aromatic ring at posi-
tion R3, this ring was replaced with an aliphatic cyclohexyl
group (in 14) or a small methyl group (in 15). The presence of
either group resulted in loss of activity. Thus for BCRP inhibito-
ry activity, position R3 must be occupied by an aromatic ring.
For compounds bearing an aromatic ring at position R3, ac-
tivity was observed to be correlated with the electronic effect
of the substituents present on this ring and with the lipophilic-
ity of the aromatic system. The highest correlation was found
with s-Hammett and the hydrophobic surface area of the sub-
stituted ring [Eq. (1)]. Included in the correlation are all com-
pounds with R1,R2 =H, and the s values were selected with
However, one compound (compound 23) was found to be
an outlier from the correlation, showing much less inhibition
than calculated from the equation. A hypothesis suggested by
the similar activity values of compounds 23 and 26 is a possible
hydrolysis of the ester group to a phenolic hydroxy group
under the assay conditions. To test this hypothesis, TLC was
performed with compound 23 after various incubation times
in assay buffer at 378C. After only 10 min of incubation, the
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original spot disappeared and a new spot was observed with
the same Rf value as that of compound 26. Thus it can be con-
cluded that compound 23 is not stable under the assay condi-
tions.
We also tested whether lipophilicity could replace the sur-
face area in the QSAR indicating that distribution into the lipo-
philic membrane would be important. However, the obtained
equation possessed much lower statistical significance, and
moreover, the descriptor used for lipophilicity, p, proved to be
insignificant at the 95% level. This points to the importance of
more specific interactions for the inhibitory activity of the com-
pounds.
To evaluate the selectivity of this class of compounds, the
activity against P-gp was investigated using the calcein AM
assay. Most of the tested compounds did not show inhibition
of P-gp at a fixed concentration of 10 mm. However, some un-
expected exceptions were observed, as previously it was
thought that the absence of the tetrahydroisoquinoline group
is responsible for the selectivity of those compounds.[17] In this
study we observed that the deletion of this group is necessary
for selectivity, but not sufficient to assure that compounds are
not P-gp inhibitors. Indeed, the highest inhibitory activity
against P-gp is shown by compound 27, which contains a di-
methoxy substituent,in agreement with P-gp inhibitors from
different structural classes, for which this substitution pattern
was found to be advantageous.[19] The presence of a volumi-
nous lipophilic group on the third aromatic ring, as present in
compounds 24 and 25, gives compounds with weak inhibitory
activity against P-gp. Interestingly, the amino derivatives 17
and 18 show some inhibition of P-gp, while from the corre-
sponding nitro compounds only the 3-nitrobenzene derivative
showed some, albeit low inhibitory activity against P-gp.
To investigate the reversal of BCRP-mediated resistance by
this new class of compounds, the toxicity of two cytotoxic
BCRP substrates, mitoxantrone and SN-38 (the active metabo-
lite of irinotecan), were determined in the presence and ab-
sence of two selected compounds. Cell viability was deter-
mined by MTT assay. The cytotoxicity of mitoxantrone and SN-
38 were determined in parental MCF-7 and resistant MCF-7 MX
cells, as well as in parental MDCK and resistant MDCK BCRP
cells. MDCK BCRP cells were included because in this transfect-
ed cell line the resistance can be solely attributed to the pres-
ence of BCRP. EC50 values were determined for parental and re-
sistant cells without the addition of inhibitor and in the pres-
ence of two different concentrations of inhibitor. For the MTT
assays we used the potent and selective compounds 4 and 25
as inhibitors. Both studied compounds were able to reverse
BCRP-mediated resistance. As the measured absorption values
in the wells containing only compounds 4 or 25 remained con-
stant for the sensitive cell lines, one can conclude that the
compounds showed no toxicity up to 10 mm. Figure 2 shows
the effect of compound 4 on the EC50 value of mitoxantrone in
MDCK BCRP cells. In the presence of an inhibitor concentration
of 5 mm, a significant decrease in resistance is observed, and in
the presence of compound 4 at 10 mm the resistance is fully re-
versed and the cytotoxicity of mitoxantrone is restored, lead-
ing to a sensitivity of the MCF-7 MX cell line almost identical
Figure 2. Effect of compound 4 on the EC50 value of mitoxantrone in MCF-7
(&) and MCF-7 MX (&) cells investigated at concentrations of 5 and 10 mm.
Cells in the absence of inhibitor were used as control. Data are the average
ꢀSD from three independent experiments.
to that of the parental MCF-7 cells. This result shows that this
compound is able to reverse the resistance of MCF-7 MX cells
against mitoxantrone completely. Figure 3 shows the dose–re-
sponse curves of SN-38 in the presence and absence of com-
pound 25. The shift in the dose–response curves of MCF-7 MX
Figure 3. Shift of the dose–response curve of SN-38 caused by increasing in-
*
hibitor concentrations. Parental MDCK cells ( ), resistant MDCK BCRP cells
&
*
(
), MDCK BCRP cells + 5 mm compound 25 ( ), MDCK BCRP cells + 10 mm
~
compound 25 ( ).
cells caused by progressively higher inhibitor concentrations of
compound 25 indicates that this compound also dose-depend-
ently inhibits BCRP-mediated resistance to SN-38. In summary,
these results prove the conclusions drawn from preliminary ex-
periments reported in reference [17] that the inhibitory effect
is not substrate dependent.
Conclusions
We have previously shown that these tariquidar analogues
lacking the tetrahydroisoquinoline group are selective inhibi-
tors of BCRP.[17] In this study we describe the synthesis of
a larger series of compounds and their inhibitory potency
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against BCRP and P-gp. The results obtained in the Hoechst
33342 assay in MCF-7 MX cells show that compounds 4,[17] 16,
and 25 are the most potent inhibitors of BCRP in this class.
However, whereas compound 4 is not a P-gp inhibitor, com-
pounds 16 and 25 are also weak inhibitors of this transporter.
SAR analyses were performed to rationalize the activity dif-
ferences within this class of compounds and to guide the syn-
thesis of further derivatives. The results show that an electron-
withdrawing substituent at position R3 of the third aromatic
ring is important for activity, probably because this part of the
molecule interacts via a p–p stacking interaction with an aro-
matic residue in the BCRP binding site. At positions R1 and R2
all substituents led to a decrease in activity, pointing to steric
hindrance in this region.
General procedure for the hydrogenation of an aromatic nitro
group (C)
The nitro compound was dissolved in THF, and Pd/C was added.
The suspension was stirred overnight at room temperature under
H2 (2 bar) in a Paar apparatus. The catalyst was filtered off, the so-
lution was concentrated, and the product was precipitated with
THF/PE.
N-[4-(2-Hydroxyethyl)phenyl]-4,5-dimethoxy-2-nitrobenzamide
(2a). According to method B, 4,5-dimethoxy-2-nitrobenzoic acid
(724 mg, 4 mmol) and 4-aminophenethyl alcohol (658 mg,
4.8 mmol) were reacted, and compound 2a was obtained as
a white powder (913 mg, 66%): Rf =0.58 (EtOAc/PE, 1:1); 1H NMR
(500 MHz, [D6]DMSO): d=10.41 (s, 1H), 7.68 (s, 1H), 7.55 (d, J=
8.47 Hz, 2H), 7.24 (s, 1H), 7.18 (d, J=8.48 Hz, 2H), 4.61 (s, 1H), 3.93
(s, 3H), 3.91 (s, 3H), 3.58 (t, J=7.06 Hz, 2H), 2.69 ppm (t, J=
7.10 Hz, 2H); 13C NMR (125 MHz, [D6]DMSO): d=164.03, 153.13,
149.05, 138.89, 137.13, 135.06, 129.17 (2C), 127.59, 119.67 (2C),
111.23, 107.41, 62.41, 56.74, 56.46, 38.66 ppm; Anal. calcd for
C17H18N2O6·0.33H2O: C 57.95, H 5.34, N 7.95, found: C 58.07, H 5.25,
N 8.01.
Experimental Section
General methods
Commercial reagents and starting materials were purchased from
Sigma–Aldrich, Fluka, Acros, or Alfa Aesar and 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 on the fol-
lowing instruments: 1H NMR, Bruker Advance 500 (500 MHz);
13C NMR, Bruker Advance 500 (125.8 MHz). Chemical shifts (d) are
expressed in ppm, using solvent as an internal standard. The multi-
plicity of resonance peaks is indicated as singlet (s), broad singlet
(bs), doublet (d), triplet (t), quartet (q), and multiplet (m). 13C NMR
signals are classified as primary (CH3), secondary (CH2), tertiary (CH
or Ar-CH), or quaternary (Ar-C) carbon atoms. The 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 instrument (Elemen-
tar). Found values were all within 0.4% of the theoretical values,
except where indicated.
N-[4-(2-Hydroxyethyl)phenyl]-5-methoxy-2-nitrobenzamide (2b).
According to method B, 5-methoxy-2-nitrobenzoic acid (946 mg,
4.8 mmol) and 4-aminophenethyl alcohol (548 mg, 4 mmol) were
reacted, and compound 2b was obtained as a white powder
(935 mg, 73%): Rf =0.54 (EtOAc/PE, 1:1); 1H NMR (500 MHz,
[D6]DMSO): d=10.52 (s, 1H), 8.16 (d, J=9.77 Hz, 1H), 7.55 (d, J=
8.45 Hz, 2H), 7.22–7.17 (m, 4H), 4.64 (s, 1H), 3.93 (s, 3H), 3.58 (t,
J=7.09 Hz, 2H), 2.69 ppm (t, J=7.08 Hz, 2H); 13C NMR (125 MHz,
[D6]DMSO): d=163.95, 163.57, 138.91, 137.01, 135.88, 135.20,
129.22 (2C), 127.06, 119.72 (2C), 115.36, 114.35, 62.42, 56.67,
38.68 ppm; Anal. calcd for C16H16N2O5·0.33H2O: C 59.62, H 5.21, N
8.69, found: C 59.77, H 5.19, N 8.39.
N-[4-(2-Hydroxyethyl)phenyl]-4-methoxy-2-nitrobenzamide (2c).
According to method B, 4-methoxy-2-nitrobenzoic acid (986 mg,
5 mmol) and 4-aminophenethyl alcohol (685 mg, 6 mmol) were re-
acted, and compound 2c was obtained as a yellow powder
(573 mg, 36%): Rf =0.09 (EtOAc/PE, 1:1); 1H NMR (500 MHz,
[D6]DMSO): d=10.46 (s, 1H), 7.70 (d, J=8.51 Hz, 1H), 7.60 (d, J=
2.55 Hz, 1H), 7.54 (d, J=8.41 Hz, 2H), 7.37 (dd, J=8.53, 2.54 Hz,
1H), 7.18 (d, J=8.42 Hz, 2H), 4.59 (t, J=5.22 Hz, 1H), 3.90 (s, 3H),
3.58 (dt, J=7.05, 5.32 Hz, 2H), 2.69 ppm (t, J=7.08, 7.08Hz, 2H);
13C NMR (125 MHz, [D6]DMSO): d=163.64, 160.59, 148.61, 137.02,
135.22, 130.71, 129.22 (2C), 124.80, 119.78 (2C), 118.92, 109.58,
62.42, 56.47, 38.67 ppm; Anal. calcd for C16H16N2O5·0.5H2O: C 59.07,
H 5.27, N 8.61, found: C 60.08, H 5.05, N 8.62.
General procedure for the synthesis of acid chlorides (A)
The aromatic carboxylic acid (1 equiv) was dissolved/suspended in
dry THF or CH2Cl2 and a catalytic amount of dry DMF. Oxalyl chlo-
ride (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 were removed under reduced pressure to yield the
desired acid chloride, which was immediately used for the synthe-
sis of amides.
N-[4-(2-Hydroxyethyl)phenyl]-5-methyl-2-nitrobenzamide (2d).
According to method B, 5-methyl-2-nitrobenzoic acid (906 mg,
5 mmol) and 4-aminophenethyl alcohol (823 mg, 6 mmol) were re-
acted, and compound 2d was obtained as a yellow powder
(1041 mg, 69%): Rf =0.71 (EtOAc); 1H NMR (500 MHz, [D6]DMSO):
d=10.48 (s, 1H), 8.04 (d, J=8.3, 1H), 7.54 (m, 4H), 7.18 (d, J=8.5,
2H), 4.59 (t, J=5.2, 1H), 3.58 (td, J=5.3, 7.1, 2H), 2.69 (t, J=7.1,
2H), 2.46 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=164.02,
145.19, 143.96, 136.76, 135.07, 132.98, 130.87, 129.46, 129.06 (2C),
124.24, 119.54 (2C), 62.22, 38.49, 20.76 ppm; Anal. calcd for
C16H16N2O4: C 63.99, H 5.37, N 9.33, found: C 64.23, H 5.43, N 9.16.
General procedure for the synthesis of the amides (B)
The aromatic amine (1 equiv) and Et3N (3 equiv) were dissolved in
dry THF and stirred at 08C. The acyl chloride (0.8 equiv) synthe-
sized as per general method was suspended/dissolved in dry THF
and added slowly. The solution was stirred for 12 h at room tem-
perature. After completion of the reaction, the solvent was evapo-
rated, and the residue was dissolved in H2O and extracted three
times with EtOAc. 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/petrole-
um ether (PE) and purified by recrystallization with CH2Cl2/PE (1:1)
or EtOH or by column chromatography with EtOAc/PE (1:1) as
eluent.
N-[4-(2-Hydroxyethyl)phenyl]-4-methyl-2-nitrobenzamide (2e).
According to method B, 4-methyl-2-nitrobenzoic acid (906 mg,
5 mmol) and 4-aminophenethyl alcohol (823 mg, 6 mmol) were re-
acted, and compound 2e was obtained as a yellow powder
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(972 mg, 65%): Rf =0.64 (EtOAc); 1H NMR (500 MHz, [D6]DMSO): d=
10.49 (s, 1H), 7.94 (s, 1H), 7.64 (m, 2H), 7.55 (d, J=8.5, 2H), 7.18 (d,
8.5, 2H), 4.59 (t, J=5.2, 1H), 3.58 (td, J=5.3, 7.1, 2H), 2.69 (t, J=
7.1, 2H), 2.46 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=
164.01, 146.87, 141.53, 136.95, 135.25, 134.27, 130.16, 129.22 (3C),
124.37, 119.75 (2C), 62.39, 38.66, 20.60 ppm; Anal. calcd for
C16H16N2O4·0.16H2O: C 63.36, H 5.43, N 9.24, found: C 63.52, H 5.34,
N 9.22.
(EtOAc); 1H NMR (500 MHz, [D6]DMSO): d=10.59 (s, 1H), 8.34 (d,
J=8.87 Hz, 2H), 8.19 (d, J=8.90 Hz, 2H), 7.67 (d, J=8.44 Hz, 2H),
7.20 (d, J=8.50 Hz, 2H), 4.65 (s, 1H), 3.59 (t, J=7.10 Hz, 2H),
2.70 ppm (t, J=7.09 Hz, 1H); 13C NMR (125 MHz, [D6]DMSO): d=
163.78, 149.23, 140.85, 136.79, 135.56, 129.32 (2C), 129.14 (2C),
123.61 (2C), 120.60 (2C), 62.33, 38.67 ppm; Anal. calcd for
C15H14N2O4·1H2O: C 59.21, H 5.30, N 9.21, found: C 59.17, H 4.95, N
9.09.
N-[4-(2-Hydroxyethyl)phenyl]-2-nitro-5-chlorobenzamide
(2 f).
2-Amino-N-[4-(2-hydroxyethyl)phenyl]-4,5-dimethoxybenzamide
(3a). According to method C, compound 2a (800 mg, 2.31 mmol)
was reacted, and compound 3a was obtained as a brown powder
(607 mg, 83%): Rf =0.21 (EtOAc/PE, 1:1); 1H NMR (500 MHz,
[D6]DMSO): d=9.64 (s, 1H), 7.54 (d, J=8.49 Hz, 2H), 7.23 (s, 1H),
7.15 (d, J=8.50 Hz, 2H), 6.36 (s, 1H), 6.27 (s, 2H2), 4.59 (s, 1H), 3.73
(s, 3H), 3.72 (s, 3H), 3.61–3.54 (m, 2H), 2.68 ppm (t, J=7.14,
7.14 Hz, 2H); 13C NMR (125 MHz, [D6]DMSO): d=167.39, 153.43,
146.87, 139.18, 137.37, 134.45, 128.86 (2C), 120.98 (2C), 113.40,
105.57, 100.08, 62.45, 57.01, 55.32, 38.67 ppm; Anal. calcd for
C17H20N2O4·0.33H2O: C 64.54, H 6.37, N 8.86, found: C 63.02, H 6.39,
N 8.47.
According to method B, 5-chloro-2-nitrobenzoic acid (1008 mg,
5 mmol) and 4-aminophenethyl alcohol (823 mg, 6 mmol) were re-
acted, and compound 2 f was obtained as a yellow powder
(1269 mg, 79%): Rf =0.52 (EtOAc/PE, 1:1); 1H NMR (500 MHz,
[D6]DMSO): d=10.60 (s, 1H), 8.16 (d, J=8.72 Hz, 1H), 7.90 (d, J=
2.25 Hz, 1H), 7.82 (dd, J=8.78, 2.27 Hz, 1H), 7.54 (d, J=8.47 Hz,
2H), 7.20 (d, J=8.50 Hz, 2H), 4.59 (t, J=5.20 Hz, 1H), 3.59 (dt, J=
7.02, 5.27 Hz, 2H), 2.70 ppm (t, J=7.07 Hz, 2H); 13C NMR (125 MHz,
[D6]DMSO): d=162.47, 145.22, 138.82, 136.66, 135.57, 134.49,
130.82, 129.31 (2C), 129.27, 126.44, 119.83 (2C), 62.38, 38.67 ppm;
Anal. calcd for C15H13ClN2O4: C 56.17, H 4.09, N 8.73, found: C
56.21, H 4.20, N 8.57.
2-Amino-N-[4-(2-hydroxyethyl)phenyl]-5-methoxybenzamide
(3b). According to method C, compound 2b (1.17 g, 3.7 mmol)
was reacted, and compound 3b was obtained as a brown powder
(794 mg, 75%): Rf =0.23 (EtOAc/PE, 1:1); 1H NMR (500 MHz,
[D6]DMSO): d=9.94 (s, 1H), 7.57 (d, J=8.46 Hz, 2H), 7.18–7.14 (m,
3H), 6.89 (dd, J=8.85, 2.88 Hz, 1H), 6.71 (d, J=8.86 Hz, 1H), 5.83
(s, 2H), 4.65 (s, 1H), 3.72 (s, 3H), 3.60–3.56 (m, 2H), 2.68 ppm (t, J=
7.13 Hz, 2H); 13C NMR (125 MHz, [D6]DMSO): d=167.51, 149.53,
143.89, 137.17, 134.79, 128.93 (2C), 120.79 (2C), 119.75, 117.84,
116.05, 112.94, 62.43, 55.80, 38.68 ppm; Anal. calcd for
C16H18N2O3·0.66H2O: C 64.41, H 6.53, N 9.39, found: 64.14, H 5.75, N
9.00.
N-[4-(2-Hydroxyethyl)phenyl]-2-nitro-4-chlorobenzamide
(2g).
According to method B, 4-chloro-2-nitrobenzoic acid (1612 mg,
8 mmol) and 4-aminophenethyl alcohol (1316 mg, 9.6 mmol) were
reacted, and compound 2g was obtained as a yellow powder
(2280 mg, 88%): Rf =0.66 (EtOAc/PE, 1:1); 1H NMR (500 MHz,
[D6]DMSO): d=10.60 (s, 1H), 8.24 (d, J=2.05 Hz, 1H), 7.95 (dd, J=
8.21, 2.11 Hz, 1H), 7.80 (d, J=8.21 Hz, 1H), 7.53 (d, J=8.47 Hz, 2H),
7.19 (d, J=8.47 Hz, 2H), 4.59 (s, 1H), 3.61–3.55 (m, 2H), 2.69 ppm
(t, J=7.06 Hz, 2H); 13C NMR (125 MHz, [D6]DMSO): d=162.94,
147.59, 136.68, 135.55, 135.06, 133.77, 131.33, 131.04, 129.30 (2C),
124.32, 119.81 (2C), 62.37, 38.65 ppm; Anal. calcd for C15H13ClN2O4:
C 56.17, H 4.09, N 8.73, found: C 55.92, H 4.28, N 8.41.
2-Amino-N-[4-(2-hydroxyethyl)phenyl]-4-methoxybenzamide
(3c). According to method C, compound 2c (531 mg, 1.68 mmol)
was reacted, and compound 3c was obtained as a yellow powder
(330 mg, 68%): Rf =0.25 (EtOAc/PE, 1:1); 1H NMR (500 MHz,
[D6]DMSO): d=9.68 (s, 1H), 7.61 (d, J=8.86 Hz, 1H), 7.56 (d, J=
8.48 Hz, 2H), 7.13 (d, J=8.51 Hz, 2H), 6.53 (s, 2H2), 6.27 (d, J=
2.54 Hz, 1H), 6.17 (dd, J=8.81, 2.54 Hz, 1H), 4.58 (t, J=5.23 Hz,
1H), 3.58 (dt, J=7.14, 5.25 Hz, 2H), 2.68 ppm (t, J=7.14 Hz, 2H);
13C NMR (125 MHz, [D6]DMSO): d=167.53, 162.51, 152.31, 137.45,
134.37, 130.46, 128.88 (2C), 120.67 (2C), 108.26, 102.51, 99.62,
62.46, 55.02, 38.67 ppm; Anal. calcd for C16H18N2O3·0.2H2O: C 66.28,
H 6.40, N 9.66, found: C 66.53, H 6.402, N 9.624.
N-[4-(2-Hydroxyethyl)phenyl]-2-nitrobenzamide (2h). According
to method B, 2-nitrobenzoic acid (3.71 g, 20 mmol) and 4-amino-
phenethyl alcohol (2.74 g, 20 mmol) were reacted, and compound
2h was obtained as a white powder (369 mg, 64%): Rf =0.57
1
(EtOAc); H NMR (500 MHz, [D6]DMSO): d=10.55 (s, 1H), 8.13 (dd,
J=8.59, 1.06 Hz, 1H), 7.87–7.83 (dt, J=15.01, 1.10 Hz, 1H), 7.76–
7.72 (m, 2H), 7.55 (d, J=8.47 Hz, 2H), 7.19 (d, J=8.44 Hz, 2H), 4.59
(t, J=5.23 Hz, 1H), 3.59 (dt, J=7.03, 5.31 Hz, 2H), 2.70 ppm (t, J=
7.08 Hz, 2H); 13C NMR (125 MHz, [D6]DMSO): d=164.02, 146.00,
136.91, 135.35, 134.15, 132.92, 131.01, 129.40, 129.27 (2C), 124.35,
119.77 (2C), 62.40, 38.67 ppm; Anal. calcd for C15H14N2O4·0.25H2O:
C 61.96, H 5.03, N 9.63, found: C 62.34, H 4.83, N 9.53.
2-Amino-N-[4-(2-hydroxyethyl)phenyl]-5-methylbenzamide (3d).
According to method C, compound 2d (901 mg, 3 mmol) was re-
acted, and compound 3d was obtained as a white powder
(778 mg, 96%): Rf =0.18 (EtOAc/PE, 1:1); 1H NMR (500 MHz,
[D6]DMSO): d=9.86 (s, 1H), 7.59 (d, J=8.4, 2H), 7.41 (s, 1H), 7.15
(d, J=8.4, 2H), 7.01 (dd, J=1.9, 8.3, 1H), 6.66 (d, J=8.3, 1H), 6.05
(s, 2H2), 4.59 (s, 1H), 3.58 (t, J=6.8, 2H), 2.69 (t, J=7.1, 2H),
2.20 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=167.84,
147.47, 137.29, 134.61, 132.91, 128.92 (2C), 128.60, 123.26, 120.59
(2C), 116.66, 115.65, 62.43, 38.66, 20.16 ppm; Anal. calcd for
C16H18N2O2·0.33H2O: C 69.54, H 6.81, N 10.14, found: C 69.30, H
6.58, N 9.82.
N-[4-(2-Hydroxyethyl)phenyl]-3-nitrobenzamide (2i). According
to method B, 3-nitrobenzoic acid (928 mg, 5 mmol) and 4-amino-
phenethyl alcohol (823 mg, 6 mmol) were reacted, and compound
2i was obtained as a yellow powder (988 mg, 69%): Rf =0.15
(EtOAc/PE, 1:1); 1H NMR (500 MHz, [D6]DMSO): d=10.49 (s, 1H),
8.77 (t, J=2.0, 1H), 8.44–8.37 (m, 2H), 7.83 (t, J=8.0, 1H), 7.66 (d,
J=8.5, 2H), 7.21 (d, J=8.5, 2H), 4.60 (t, J=4.9, 1H), 3.60 (td, J=
4.8, 7.0, 2H), 2.70 ppm (t, J=7.1, 2H); 13C NMR (125 MHz,
[D6]DMSO): d=163.16, 147.77, 136.64, 136.42, 135.50, 134.19,
130.24, 129.10 (2C), 126.13, 122.44, 120.63 (2C), 62.28, 38.60 ppm;
Anal. calcd for C15H14N2O4·0.167H2O: C 62.28, H 4.99, N 9.68, found:
C 62.32, H 4.88, N 9.64.
2-Amino-N-[4-(2-hydroxyethyl)phenyl]-4-methylbenzamide (3e).
According to method C, compound 2e (901 mg, 3 mmol) was re-
acted, and compound 3e was obtained as a white powder
(709 mg, 87%): Rf =0.49 (EtOAc/PE, 1:1); 1H NMR (500 MHz,
[D6]DMSO): d=9.78 (s, 1H), 7.58 (d, J=8.5, 2H), 7.54 (d, J=8.1,
N-[4-(2-Hydroxyethyl)phenyl]-4-nitrobenzamide (2k). According
to method B, 4-nitrobenzoic acid (329 mg, 2.4 mmol) and 4-amino-
phenethyl alcohol (371 mg, 2 mmol) were reacted, and compound
2k was obtained as a yellow powder (440 mg, 76%): Rf =0.68
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1H), 7.14 (d, J=8.5, 2H), 6.54 (d, J=0.6, 1H), 6.40 (dd, J=1.4, 8.2,
1H), 6.31 (s, 2H2), 4.59 (t, J=5.0, 1H), 3.58 (dd, J=7.1, 11.8, 2H),
2.68 (t, J=7.1, 2H), 2.19 ppm (s, 3H); 13C NMR (125 MHz,
[D6]DMSO): d=167.75, 150.08, 141.94, 137.33, 134.52, 128.89 (2C),
128.75, 120.66 (2C), 116.62, 116.02, 112.71, 62.43, 38.66, 21.24 ppm;
Anal. calcd for C16H18N2O2·0.33H2O: C 69.54, H 6.81, N 10.14, found:
C 69.37, H 6.41, N 9.97.
N-[4-(2-Hydroxyethyl)phenyl]-2-(4-nitrobenzamido)benzamide
(4). According to method B, compound 3h (153 mg, 0.6 mmol)
and 4-nitrobenzoyl chloride (92.8 mg, 0.5 mmol) were reacted, and
compound 4 was obtained as a yellow powder (199 mg, 98%): Rf =
0.58 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d=11.87 (s,
1H), 10.50 (s, 1H), 8.42–8.35 (m, 3H), 8.15–8.11 (m, 2H), 7.94 (dd,
J=7.79, 1.04 Hz, 1H), 7.65–7.57 (m, 3H), 7.32 (t, J=7.57 Hz, 1H),
7.19 (d, J=8.41 Hz, 2H), 4.60 (t, J=5.19 Hz, 1H), 3.58 (dt, J=7.06,
5.31 Hz, 2H), 2.69 ppm (t, J=7.08 Hz, 2H); 13C NMR (125 MHz,
[D6]DMSO): d=167.17, 163.24, 149.53, 140.36, 138.19, 136.51,
135.71, 132.23, 129.15, 129.10 (2C), 128.77 (2C), 124.19 (2C), 124.05,
123.92, 121.98, 121.30 (2C), 62.37, 38.68 ppm; Anal. calcd for
C22H19N3O5·0.5H2O: C 63.76, H 4.86, N 10.14, found: C 63.97, H 4.88,
N 9.75.
2-Amino-5-chloro-N-[4-(2-hydroxyethyl)phenyl]benzamide (3 f).
According to method C, compound 2 f (1181 mg, 3.6 mmol) was
reacted, and compound 3 f was obtained as a white powder
(945 mg, 90%): Rf =0.40 (EtOAc/PE, 1:1); 1H NMR (500 MHz,
[D6]DMSO): d=10.00 (s, 1H), 7.66 (d, J=2.5, 1H), 7.58 (d, J=8.5,
2H), 7.21 (dd, J=2.5, 8.8, 1H), 7.16 (d, J=8.5, 2H), 6.77 (d, J=8.8,
1H), 6.41 (s, 2H2), 4.23 (s, 1H), 3.59 (t, J=7.1, 2H), 2.69 ppm (t, J=
7.1, 2H); 13C NMR (125 MHz, [D6]DMSO): d=166.52, 148.66, 136.95,
134.98, 131.82, 128.96 (2C), 127.92, 120.75 (2C), 118.16, 118.02,
116.32, 62.39, 38.65 ppm; Anal. calcd for C15H13ClN2O2: C 61.97, H
5.20, N 9.64, found: C 61.95, H 5.30, N 9.25.
N-[4-(2-Hydroxyethyl)phenyl]-3-(4-nitrobenzamido)benzamide
(5). According to method B, compound 3i (123 mg, 0.48 mmol)
and 4-nitrobenzoyl chloride (74 mg, 0.4 mmol) were reacted, and
compound 5 was obtained as a yellow powder (31 mg, 19%): Rf =
0.39 (EtOAc/PE, 1:1); 1H NMR (500 MHz, [D6]DMSO): d=10.84 (s,
1H), 10.24 (s, 1H), 8.39–8.32 (m, 3H), 8.24 (d, J=8.73 Hz, 2H), 8.04
(d, J=8.01 Hz, 1H), 7.72 (d, J=7.63 Hz, 1H), 7.67 (d, J=8.35 Hz,
2H), 7.52 (t, J=7.90 Hz, 1H), 7.18 (d, J=8.34 Hz, 2H), 4.60 (s, 1H),
3.59 (t, J=6.85 Hz, 2H), 2.70 ppm (t, J=7.05 Hz, 2H); 13C NMR
(125 MHz, [D6]DMSO): d=165.32, 164.12, 149.38, 140.46, 139.08,
137.19, 135.86, 135.01, 129.41 (2C), 129.05 (2C), 128.82, 123.69 (2C),
123.47, 123.17, 120.48 (2C), 120.26, 62.39, 38.66 ppm; Anal. calcd
for C22H19N3O5·1.33H2O: C 61.53, H 5.09, N 9.79, found: C 61.17, H
5.11, N 9.66.
2-Amino-4-chloro-N-[4-(2-hydroxyethyl)phenyl]benzamide (3g).
According to method C, compound 2g (1084 mg, 3.38 mmol) was
reacted, and compound 3g was obtained as a yellow powder
(914 mg, 93%): Rf =0.66 (EtOAc/PE, 1:1); 1H NMR (500 MHz,
[D6]DMSO): d=10.13 (s, 1H), 9.18 (s, 1H), 8.83 (d, J=1.48 Hz, 1H),
7.68 (d, J=8.34 Hz, 1H), 7.57 (d, J=8.50 Hz, 2H), 7.20 (d, J=
2.17 Hz, 1H), 7.17 (d, J=8.56 Hz, 2H), 6.88 (dd, J=8.31, 2.19 Hz,
1H), 4.58 (t, J=5.19 Hz, 1H), 3.61–3.55 (m, 2H), 2.69 ppm (t, J=
7.10 Hz, 2H); 13C NMR (125 MHz, [D6]DMSO): d=166.10, 153.16,
137.12, 136.73, 135.27, 130.26, 129.05 (2C), 120.76 (2C), 117.49,
116.11, 112.74, 62.38, 38.66 ppm.
N-[4-(2-Hydroxyethyl)phenyl]-4-(4-nitrobenzamido)benzamide
(6). According to method B, compound 3k (154 mg, 0.6 mmol) and
4-nitrobenzoyl chloride (93 mg, 0.5 mmol) were reacted, and com-
pound 6 was obtained as a white powder (210 mg, 100%): Rf =
2-Amino-N-[4-(2-hydroxyethyl)phenyl]benzamide (3h). According
to method C, compound 2h (4.29 g, 15 mmol) was reacted, and
compound 3g was obtained as a white powder (2.69 g, 70%): Rf =
1
0.63 (EtOAc); H NMR (500 MHz, [D6]DMSO): d=10.89 (s, 1H), 10.12
1
0.70 (EtOAc); H NMR (500 MHz, [D6]DMSO): d=9.88 (s, 1H), 7.62–
(s, 1H), 8.37 (d, J=8.87 Hz, 2H), 8.24 (d, J=8.90 Hz, 2H), 8.01 (d,
J=8.82 Hz, 2H), 7.95 (d, J=8.82 Hz, 2H), 7.68 (d, J=8.46 Hz, 2H),
7.17 (d, J=8.47 Hz, 2H), 4.61 (t, J=5.18 Hz, 1H), 3.59 (dd, J=12.31,
7.07 Hz, 2H), 2.69 ppm (t, J=7.11, 7.11 Hz, 2H); 13C NMR (125 MHz,
[D6]DMSO): d=164.78, 164.34, 149.43, 141.78, 140.41, 137.30,
134.87, 130.33, 129.53 (2C), 129.02 (2C), 128.60 (2C), 123.67 (2C),
120.49 (2C), 119.81 (2C), 62.40, 38.67 ppm; HPLC (RP8, H2O/MeOH,
20:80) purity 99%.
7.56 (m, 3H), 7.22–7.13 (m, 3H), 6.74 (dd, J=8.28, 1.02 Hz, 1H),
6.58 (ddd, J=8.07, 7.16, 1.14 Hz, 1H), 6.28 (s, 2H), 4.58 (t, J=
5.21 Hz, 1H), 3.58 (dt, J=7.10, 5.22 Hz, 2H), 2.69 (t, J=7.12 Hz,
2H); 13C NMR (125 MHz, [D6]DMSO): d=167.82, 149.80, 137.26,
134.69, 132.10, 128.94 (2C), 128.74, 120.66 (2C), 116.48, 115.54,
114.82, 62.44, 38.67 ppm; Anal. calcd for C15H16N2O2·0.33H2O: C
68.68, H 6.40, N 10.68, found: C 68.96, H 6.15, N 10.60.
3-Amino-N-[4-(2-hydroxyethyl)phenyl]benzamide (3i). According
to method C, compound 2i (2577 mg, 9 mmol) was reacted, and
compound 3i was obtained as a gray powder (1779 mg, 77%): Rf =
0.48 (EtOAc); 1H NMR (500 MHz, [D6]DMSO): d=9.94 (s, 1H), 7.64
(d, J=8.5, 2H), 7.17–7.10 (m, 3H), 7.08–7.07 (m, 1H), 7.05–7.03 (m,
1H), 6.73 (ddd, J=1.0, 2.3, 7.9, 1H), 5.26 (s, 2H), 4.58 (t, J=5.2,
1H), 3.58 (td, J=5.2, 7.1, 1H), 2.68 ppm (t, J=7.1, 1H); 13C NMR
(125 MHz, [D6]DMSO): d=166.28, 148.83, 137.39, 136.14, 134.64,
128.95, 128.81, 120.29 (2C), 116.80, 114.82, 113.09, 62.41,
38.64 ppm.
N-[4-(2-Hydroxyethyl)phenyl]-4,5-dimethoxy-2-(4-nitrobenzami-
do)benzamide (7). According to method B, compound 3a
(152 mg, 0.48 mmol) and 4-nitrobenzoyl chloride (74 mg,
0.4 mmol) were reacted, and compound 7 was obtained as
a yellow powder (77 mg, 41%): Rf =0.1 (EtOAc/PE, 1:1); 1H NMR
(500 MHz, [D6]DMSO): d=12.35 (s, 1H), 10.33 (s, 1H), 8.38 (d, J=
8.68 Hz, 2H), 8.26 (s, 1H), 8.12 (d, J=8.69 Hz, 2H), 7.54–7.50 (m,
3H), 7.20 (d, J=8.29 Hz, 2H), 4.60 (s, 1H), 3.87 (s, 3H), 3.86 (s, 3H),
3.62–3.57 (m, 1H), 2.70 ppm (t, J=7.03 Hz, 2H); 13C NMR (125 MHz,
[D6]DMSO): d=167.12, 162.88, 151.71, 149.41, 144.43, 140.64,
136.29, 135.79, 134.26, 129.09 (2C), 128.55 (2C), 124.19 (2C), 121.89
(2C), 113.80, 112.21, 105.08, 62.35, 56.21, 55.75, 38.66 ppm; Anal.
calcd for C24H23N3O7·0.4H2O: C 60.99, H 5.08, N 8.89, found: C
61.25, H 5.44, N 8.46.
4-Amino-N-[4-(2-hydroxyethyl)phenyl]benzamide (3k). According
to method C, compound 2k (407 mg, 1.42 mmol) was reacted, and
compound 3k was obtained as a white powder (299 mg, 82%):
Rf =0.46 (EtOAc); 1H NMR (500 MHz, [D6]DMSO): d=9.65 (s, 1H),
7.70 (d, J=7.78 Hz, 2H), 7.62 (d, J=8.10 Hz, 2H), 7.13 (d, J=
8.37 Hz, 2H), 6.59 (d, J=8.41 Hz, 2H, Ar-CH), 5.68 (s, 2H), 4.58 (t,
J=5.12 Hz, 1H), 3.58 (dd, J=12.30, 6.95 Hz, 2H), 2.67 ppm (t, J=
7.13 Hz, 2H); 13C NMR (125 MHz, [D6]DMSO): d=165.27, 152.16,
137.81, 134.13, 129.40 (2C), 128.91 (2C), 121.41, 120.25 (2C), 112.70
(2C), 62.47, 38.67 ppm.
N-[4-(2-Hydroxyethyl)phenyl]-4-methoxy-2-(4-nitrobenzamido)-
benzamide (8). According to method B, compound 3c (172 mg,
0.6 mmol) and 4-nitrobenzoyl chloride (93 mg, 0.5 mmol) were re-
acted, and compound 8 was obtained as a yellow powder
(131 mg, 60%): Rf =0.26 (EtOAc/PE, 1:1); 1H NMR (500 MHz,
[D6]DMSO): d=12.59 (s, 1H), 10.32 (s, 1H), 8.40 (d, J=8.86 Hz, 2H),
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8.25 (d, J=2.63 Hz, 1H), 8.12 (d, J=8.95 Hz, 2H), 8.00 (d, J=
8.89 Hz, 1H), 7.56 (d, J=8.45 Hz, 2H), 7.20 (d, J=8.46 Hz, 2H), 6.87
(dd, J=8.84, 2.61 Hz, 1H), 4.59 (t, J=5.19 Hz, 1H), 3.87 (s, 3H), 3.59
(dt, J=7.07, 5.26 Hz, 2H), 2.70 ppm (t, J=7.07 Hz, 2H); 13C NMR
(125 MHz, [D6]DMSO): d=167.40 (1C,CO), 163.14, 162.42, 149.59,
141.09, 140.22, 136.32, 135.76, 130.78, 129.09 (2C), 128.62 (2C),
124.30 (2C), 121.70 (2C), 113.84, 109.11, 106.10, 62.36, 55.68,
38.66 ppm; Anal. calcd for C23H21N3O6: C 63.44, H 4.86, N 9.65,
found: C 63.42, H 4.88, N 9.28.
136.60, 136.29, 135.93, 130.87, 129.16 (2C), 128.84 (2C), 124.23 (2C),
123.70, 122.03, 121.37 (2C), 121.18, 62.35, 38.66 ppm; HPLC (RP8,
MeOH/H2O, 80:20) purity 99.9%.
5-Chloro-N-[4-(2-hydroxyethyl)phenyl]-2-(4-nitrobenzamido)ben-
zamide (13). According to method B, compound 3 f (174 mg,
0.6 mmol) and 4-nitrobenzoyl chloride (93 mg, 0.5 mmol) were re-
acted, and compound 13 was obtained as a yellow powder
(84 mg, 48%): Rf =0.34 (silica gel, EtOAc/PE (1:1)); 1H NMR
(500 MHz, [D6]DMSO): d=11.74 (s, 1H), 10.53 (s, 1H), 8.40–8.34 (m,
3H), 8.11 (d, J=8.85 Hz, 2H), 7.97 (d, J=2.44 Hz, 1H), 7.68 (dd, J=
8.86, 2.46 Hz, 1H), 7.57 (d, J=8.41 Hz, 2H), 7.19 (d, J=8.44 Hz, 2H),
4.59 (t, J=5.18 Hz, 1H), 3.58 (dt, J=7.03, 5.29 Hz, 2H), 2.69 ppm (t,
J=7.06 Hz, 2H, 7); 13C NMR (125 MHz, [D6]DMSO): d=165.70,
163.32, 149.59, 140.02, 136.93, 136.29, 135.91, 131.82, 129.13 (2C),
128.82 (2C), 128.72, 128.01, 125.83, 124.18 (2C), 123.84, 121.26 (2C),
62.35, 38.66 ppm; Anal. calcd for C22H18ClN3O5: C 60.07, H 4.12, N
9.55, found: C 59.94, H 4.36, N 9.42.
N-[4-(2-Hydroxyethyl)phenyl]-5-methoxy-2-(4-nitrobenzamido)-
benzamide (9). According to method B, compound 3b (171 mg,
0.6 mmol) and 4-nitrobenzoyl chloride (93 mg, 0.5 mmol) were re-
acted, and compound 9 was obtained as a yellow powder (91 mg,
1
56%): Rf =0.56 (EtOAc/PE, 1:1); H NMR (500 MHz, [D6]DMSO): d=
11.44 (s, 1H), 10.52 (s, 1H), 8.36 (d, J=8.74 Hz, 2H), 8.16 (d, J=
8.95 Hz, 1H), 8.11 (d, J=8.89 Hz, 2H), 7.58 (d, J=8.72 Hz, 2H), 7.44
(d, J=3.27 Hz, 1H), 7.20–7.15 (m, 3H), 4.61 (s, 1H), 3.85 (s, 3H),
3.57 (t, J=7.18 Hz, 2H), 2.68 ppm (t, J=7.03 Hz, 2H); 13C NMR
(125 MHz, [D6]DMSO): d=166.56, 163.12, 155.56, 149.35, 140.62,
136.61, 135.57, 130.87, 129.06 (2C), 128.74 (2C), 126.51, 124.38,
124.04 (2C), 121.17 (2C), 117.41, 114.13, 62.37, 55.77, 38.66 ppm;
HPLC (RP8, MeCN/H2O, 80:20) purity 95.8%.
2-(Cyclohexancarboxamido)-N-[4-(2-hydroxyethyl)phenyl]benza-
mide (14). According to method B, compound 3h (154 mg,
0.6 mmol) and cyclohexanecarboxylic acid chloride (73 mg,
0.5 mmol) were reacted, and compound 14 was obtained as
a white powder (150 mg, 82%): Rf =0.60 (EtOAc/PE, 1:1); 1H NMR
(500 MHz, [D6]DMSO): d=10.68 (s, 1H), 10.37 (s, 1H), 8.28 (d, J=
8.18 Hz, 1H), 7.82 (dd, J=7.80, 1.26 Hz, 1H), 7.59 (d, J=8.39 Hz,
2H), 7.50 (t, J=8.59 Hz, 1H), 7.20–7.16 (m, 3H), 4.63 (t, J=5.19 Hz,
1H), 3.59 (dt, J=7.07, 5.41 Hz, 2H), 2.70 (t, J=7.10 Hz, 2H), 2.27 (tt,
J=11.38, 3.53 Hz, 1H), 1.84 (dd, J=12.92, 2.54 Hz, 2H), 1.73–1.67
(m, 2H), 1.64–1.58 (m, 1H), 1.37 (ddd, J=23.93, 12.29, 2.89 Hz, 2H),
1.31–1.21 (m, 2H), 1.16 ppm (m, 1H); 13C NMR (125 MHz,
[D6]DMSO): d=173.99, 167.12, 138.63, 136.63, 135.54, 131.90,
129.07 (2C), 128.89, 123.19, 122.89, 121.34, 121.09 (2C), 62.40,
45.56, 38.68, 29.19 (2C), 25.53, 25.23 ppm (2C); HPLC (RP8, MeCN/
H2O, 80:20) purity 99.5%.
N-[4-(2-Hydroxyethyl)phenyl]-4-methyl-2-(4-nitrobenzamido)-
benzamide (10). According to method B, compound 3e (162 mg,
0.6 mmol) and 4-nitrobenzoyl chloride (93 mg, 0.5 mmol) were re-
acted, and compound 10 was obtained as a yellow powder
1
(71 mg, 31%): Rf =0.74 (EtOAc); H NMR (500 MHz, [D6]DMSO): d=
12.10 (s, 1H), 10.39 (s, 1H), 8.38 (d, J=9.0, 2H), 8.33 (s, 1H), 8.11 (d,
J=8.9, 2H), 7.87 (d, J=8.0, 1H), 7.57 (d, J=8.5, 2H), 7.19 (d, J=
8.5, 2H), 7.13 (dd, J=1.0, 8.0, 1H), 4.59 (t, J=5.2, 1H), 3.59 (td, J=
5.2, 7.1, 2H), 2.70 (t, J=7.1, 2H), 2.40 ppm (s, 3H); 13C NMR
(125 MHz, [D6]DMSO): d=167.32, 162.99, 149.49, 142.67, 140.30,
138.66, 136.35, 135.73, 129.07 (2C), 128.99, 128.61 (2C), 124.44,
124.20 (2C), 121.78, 121.45 (2C), 119.96, 62.34, 38.64, 21.50 ppm;
Anal. calcd for C23H21N3O5: C 65.86, H 5.05, N 10.02, found: C 65.58,
H 5.09, N 9.98.
2-Acetamido-N-[4-(2-hydroxyethyl)phenyl]benzamide (15). Ac-
cording to method B, compound 3h (128,2 mg, 0.5 mmol) and
acetyl chloride (31.0 mg, 0.4 mmol) were reacted, and compound
15 was obtained as a white powder (23 mg, 20%): Rf =0.44
(EtOAc/PE, 1:1); 1H NMR (500 MHz, [D6]DMSO): d=10.43 (s, 1H),
10.33 (s, 1H), 8.15 (d, J=7.43 Hz, 1H), 7.74 (d, J=6.96 Hz, 1H), 7.59
(d, J=7.56 Hz, 2H), 7.49 (t, J=7.44 Hz, 1H), 7.22–7.17 (m, 3H), 4.59
(s, 1H), 3.58 (s, 2H), 2.69 (t, J=6.72 Hz, 2H), 2.05 ppm (s, 3H);
13C NMR (125 MHz, [D6]DMSO): d=168.38, 166.89, 138.05, 136.82,
135.39, 131.65, 129.06 (2C), 128.75, 124.36, 123.16, 121.79, 120.85
(2C), 62.43, 38.69, 24.56 ppm; HPLC (RP8, MeCN/H2O, 80:20) purity
98.9%.
N-[4-(2-Hydroxyethyl)phenyl]-5-methyl-2-(4-nitrobenzamido)-
benzamide (11). According to method B, compound 3d (162 mg,
0.6 mmol) and 4-nitrobenzoyl chloride (93 mg, 0.5 mmol) were re-
acted, and compound 11 was obtained as a yellow powder
(101 mg, 48%): Rf =0.76 (EtOAc); 1H NMR (500 MHz, [D6]DMSO): d=
11.73 (s, 1H), 10.41 (s, 1H), 8.37 (d, J=8.6, 2H), 8.26 (d, J=8.3, 1H),
8.10 (d, J=8.6, 2H), 7.74 (s, 1H), 7.58 (d, J=8.2, 2H), 7.42 (d, J=
8.1, 1H), 7.19 (d, J=8.3, 2H), 4.59 (t, J=5.1, 1H), 3.58 (dd, J=6.9,
12.3, 2H), 2.69 (t, J=7.0, 2H), 2.38 ppm (s, 3H); 13C NMR (125 MHz,
[D6]DMSO): d=167.18, 162.98, 149.18, 140.24, 136.46, 135.65,
133.27, 132.61, 129.29, 129.07 (2C), 128.65 (2C), 124.13 (2C), 123.79,
121.92, 121.21 (2C), 62.34, 38.64, 20.58 ppm; Anal. calcd for
C23H21N3O5·0.33H2O: C 64.93, H 5.13, N 9.88, found: C 64.54, H 5.12,
N 9.73.
N-[4-(2-Hydroxyethyl)phenyl]-2-(3-nitrobenzamido)benzamide
(16). According to method B, compound 3h (154 mg, 0.6 mmol)
and 3-nitrobenzoyl chloride (93 mg, 0.5 mmol) were reacted, and
compound 16 was obtained as a yellow powder (105 mg, 52%):
Rf =0.1 (EtOAc/cyclohexane, 1:2); 1H NMR (500 MHz, [D6]DMSO):
d=11.81 (s, 1H), 10.44 (s, 1H), 8.69 (t, J=1.91 Hz, 1H), 8.43 (dd, J=
8.20, 2.24 Hz, 1H), 8.33 (d, J=8.14 Hz, 1H), 8.30 (d, J=7.80 Hz, 1H),
7.91 (d, J=7.47 Hz, 1H), 7.86 (t, J=7.99 Hz, 1H), 7.64–7.59 (m, 3H),
7.32 (t, J=7.63 Hz, 1H), 7.19 (d, J=8.41 Hz, 2H), 4.59 (t, J=5.20 Hz,
1H), 3.59 (dt, J=7.06, 5.40 Hz, 1H), 2.70 ppm (t, J=7.07, 7.07 Hz,
1H); 13C NMR (125 MHz, [D6]DMSO): d=167.11, 162.79, 148.17,
137.98, 136.58, 136.17, 135.66, 133.25, 132.14, 130.82, 129.10 (2C),
129.05, 126.54, 124.42, 124.13, 122.26, 121.14 (2C), 62.37,
38.67 ppm; HPLC (RP8, MeCN/H2O 80:20) purity 97.1%.
4-Chloro-N-[4-(2-hydroxyethyl)phenyl]-2-(4-nitrobenzamido)ben-
zamide (12). According to method B, compound 3g (140 mg,
0.84 mmol) and 4-nitrobenzoyl chloride (74 mg, 0.4 mmol) were re-
acted, and compound 12 was obtained as a yellow powder
(36 mg, 19%): Rf =0.47 (EtOAc/PE (1:1)); 1H NMR (500 MHz,
[D6]DMSO): d=12.03 (s, 1H), 10.54 (s, 1H), 8.52 (d, J=1.02 Hz, 1H),
8.39 (d, J=8.40 Hz, 2H), 8.12 (d, J=8.51 Hz, 2H), 7.98 (d, J=
8.37 Hz, 1H), 7.56 (d, J=7.98 Hz, 2H), 7.42–7.36 (m, 1H), 7.19 (d,
J=8.01 Hz, 2H), 4.59 (t, J=5.02 Hz, 1H), 3.58 (dd, J=12.22,
6.87 Hz, 2H), 2.69 ppm (t, J=6.86 Hz, 2H); 13C NMR (125 MHz,
[D6]DMSO): d=166.33, 163.52–163.50, 163.57, 149.64, 139.92,
2-(3-Aminobenzamido)-N-[4-(2-hydroxyethyl)phenyl]benzamide
(17). According to method C, compound 16 (51.3 mg, 0.2 mmol)
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was reacted, and compound 17 was obtained as a white powder
(50 mg, 67%): Rf =0.89 (MeOH); H NMR (500 MHz, [D6]DMSO): d=
(125 MHz, [D6]DMSO): d=167.18, 163.46, 136.83, 136.58, 135.64,
133.78, 132.13, 131.86, 130.99, 129.19, 129.08 (2C), 127.31, 125.70,
123.92, 123.78, 121.98, 121.22, 62.36, 38.68 ppm; Anal. calcd for
C22H19ClN2O3·1.33H2O: C 63.08, H 5.21, N 6.69, found: C 63,49, H
5.11, N 6.92.
1
11.62 (s, 1H), 10.44 (s, 1H), 8.54 (dd, J=8.34, 0.98 Hz, 1H), 8.07–
7.75 (m, 1H), 7.61–7.56 (m, 3H), 7.25 (dt, J=7.67, 1.15 Hz, 1H), 7.21
(d, J=8.49 Hz, 2H), 7.17 (t, J=7.82 Hz, 1H), 7.12 (t, J=1.99 Hz, 1H),
6.99 (d, J=8.40 Hz, 1H), 6.76 (ddd, J=7.90, 2.22, 0.67 Hz, 1H), 5.37
(s, 2H), 4.60 (t, J=5.22 Hz, 1H), 3.66–3.52 (m, 2H), 2.70 ppm (t, J=
7.08, 7.08 Hz, 2H); 13C NMR (125 MHz, [D6]DMSO): d=167.51,
165.42, 149.39, 139.21, 136.41, 135.80, 135.48, 132.39, 129.44,
129.17 (2C), 129.02, 123.00, 122.16, 121.37 (2C), 121.03, 117.41,
113.70, 112.61, 62.38, 38.69 ppm; Anal. calcd for C22H21N3O3·1.5H2O:
C 68.41, H 5.79, N 10.88, found: C 68.04, H 5.67, N 10.44.
N-[4-(2-Hydroxyethyl)phenyl]-2-[4-(trifluoromethyl)benzamido]-
benzamide (22). According to method A, 4-trifluoromethylbenzoic
acid (95 mg, 0.5 mmol) was reacted to 4-trifluoromethylbenzoyl
chloride and then, according to method B, with compound 3h
(154 mg, 0.6 mmol) to obtain compound 22 as a white powder
(74 mg, 34%): Rf =0.23 (EtOAc/PE, 1:1); 1H NMR (500 MHz,
[D6]DMSO): d=11.85 (s, 1H), 10.46 (s, 1H), 8.43 (dd, J=8.24,
0.79 Hz, 1H), 8.09 (d, J=8.09 Hz, 2H), 7.96–7.93 (m, 3H), 7.64–7.60
(m, 1H), 7.59 (d, J=8.44 Hz, 2H), 7.33–7.29 (m, 1H), 7.19 (d, J=
8.47 Hz, 2H), 4.59 (t, J=5.20 Hz, 1H), 3.58 (dt, J=7.05, 5.23 Hz, 2H),
2.70 ppm (t, J=7.08 Hz, 2H); 13C NMR (125 MHz, [D6]DMSO): d=
167.27, 163.63, 138.49, 138.41, 136.46, 135.75, 132.30, 132.20–
131.39 (m,1C), 129.12 (2C), 128.12 (2C), 126.09 (2C), 123.96 (q, J=
269.49 Hz, 1C), 123.84, 123.47, 121.71, 121.32 (2C), 62.37,
38.67 ppm; Anal. calcd for C23H19F3N2O3: C 64.48, H 4.47, N 6.54,
found: C 64.28, H 4.68, N 6.45.
2-(4-Aminobenzamido)-N-[4-(2-hydroxyethyl)phenyl]benzamide
(18). According to method C, compound 4 (324 mg, 0.8 mmol) was
reacted, and compound 18 was obtained as a white powder
(161 mg, 71%): Rf =0.55 (silica gel, EtOAc/PE (1:1)); 1H NMR
(500 MHz, [D6]DMSO): d=11.54 (s, 1H), 10.44 (s, 1H), 8.56 (d, J=
7.1, 8.4, 1H), 7.90 (d, J=7.1, 7.9, 1H), 7.61 (dd, J=7.1, 6.1, 8.4, 4H),
7.55 (t, J=7.1, 7.8, 1H), 7.20 (dd, J=7.1, 8.0, 15.0, 3H), 6.62 (d, J=
7.1, 8.7, 2H), 5.83 (s, 2H), 4.61 (t, J=7.1, 5.2, 1H), 3.59 (m, 2H),
2.71 ppm (t, J=7.1, 2H); 13C NMR (125 MHz, [D6]DMSO): d=167.73,
164.36, 152.68, 139.81, 136.39, 135.74, 132.30, 129.12 (2C), 128.96,
128.78 (2C), 122.28, 121.58, 121.36 (2C), 120.71, 120.65, 113.08 (2C),
62.34, 38.67 ppm; Anal. calcd for C22H21N3O3·0.33H2O: C 69.28, H
5.73, N 11.02, found: C 69.11, H 5.52, N 10.80.
4-{2-[4-(2-Hydroxyethyl)phenylcarbamoyl]phenylcarbamoyl}-
phenyl acetate (23). According to method A, 4-acetoxybenzoic
acid (216 mg, 1.2 mmol) was reacted to 4-acetoxybenzoyl chloride
and then, according to method B, with compound 3h (369 mg,
1.44 mmol) to obtain compound 23 as a white powder (60 mg,
2-(4-Cyanobenzamido)-N-[4-(2-hydroxyethyl)phenyl]benzamide
(19). According to method B, compound 3h (154 mg, 0.6 mmol)
and 4-cyanobenzoyl chloride (83 mg, 0.5 mmol) were reacted, and
compound 19 was obtained as a white powder (98 mg, 51%): Rf =
0.23 (EtOAc/PE, 1:1); 1H NMR (500 MHz, [D6]DMSO): d=11.83 (s,
1H), 10.45 (s, 1H), 8.40 (dd, J=8.26, 0.70 Hz, 1H), 8.06–8.02 (m,
4H), 7.92 (dd, J=7.85, 1.27 Hz, 1H), 7.63–7.59 (m, 1H), 7.58 (d, J=
8.44 Hz, 2H), 7.31 (dt, J=7.70, 1.06 Hz, 1H), 7.20 (d, J=8.46 Hz,
2H), 4.59 (t, J=5.20 Hz, 1H), 3.59 (dt, J=7.10, 5.26 Hz, 2H),
2.70 ppm (t, J=7.07 Hz, 2H); 13C NMR (125 MHz, [D6]DMSO): d=
167.06, 163.22, 138.49, 138.10, 136.28, 135.60, 132.9 (2C), 132.11,
128.96 (2C), 127.86 (2C), 123.79, 123.46, 121.61, 121.15 (2C), 118.15,
114.22, 62.20, 38.50 ppm; Anal. calcd for C23H19N3O3·0.8H2O: C
69.09, H 15.19, N 10.51, found: C 68,95, H 4.79, N 10.47.
1
12%): Rf =0.24 (EtOAc/PE, 1:1); H NMR (500 MHz, [D6]DMSO): d=
11.75 (s, 1H), 10.45 (s, 1H), 8.47 (d, J=7.4, 1H), 7.94 (m, 3H), 7.60
(m, 3H), 7.33 (d, J=8.7, 2H), 7.28 (td, J=1.2, 7.7, 1H), 7.20 (d, J=
8.5, 2H), 4.59 (t, J=5.2, 1H), 3.59 (td, J=5.3, 7.1, 2H), 2.70 (t, J=
7.1, 2H), 2.30 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=
168.99, 167.38, 163.97, 153.35, 138.81, 136.43, 135.74, 132.31,
132.31, 129.12 (2C), 129.05, 128.67 (2C), 123.42, 122.91, 122.49 (2C),
121.40, 121.33 (2C), 62.36, 38.68, 20.99 ppm; Anal. calcd for
C24H22N2O5·0.66H2O: C 66.97, H 5.46, N 6.51, found: C 66.95, H 5.30,
N 6.90.
2-(4-tert-Butylbenzamido)-N-[4-(2-hydroxyethyl)phenyl]benza-
mide (24). According to method A, 4-tert-butylbenzoic acid
(98 mg, 0.4 mmol) was reacted to 4-tert-butylbenzoyl chloride and
then, according to method B, with compound 3h (123 mg,
0.48 mmol) to obtain compound 24 as a white powder (22 mg,
2-(4-Chlorobenzamido)-N-[4-(2-hydroxyethyl)phenyl]benzamide
(20). According to method B, compound 3h (154 mg, 0.6 mmol)
and 4-chlorbenzoyl chloride (88 mg, 0.5 mmol) were reacted, and
compound 20 was obtained as a white powder (166 mg, 84%):
1
13%): Rf =0.44 (EtOAc/PE, 1:1); H NMR (500 MHz, [D6]DMSO): d=
11.82 (s, 1H), 10.49 (s, 1H), 8.54 (dd, J=8.29, 0.80 Hz, 1H), 7.94 (dd,
J=7.86, 1.40 Hz, 1H), 7.86–7.83 (m, 2H), 7.58 (ddd, J=6.44, 5.47,
1.98 Hz, 5H), 7.25 (t, J=7.61 Hz, 1H), 7.20 (d, J=8.46 Hz, 2H), 4.62
(s, 1H), 3.59 (t, J=7.12 Hz, 2H), 2.70 (t, J=7.08 Hz, 2H), 1.30 ppm
(s, 9H); 13C NMR (125 MHz, [D6]DMSO): d=167.49, 164.63, 155.10,
136.75, 136.44, 135.76, 132.36, 132.00, 129.12 (2C), 129.08, 126.98
(2C), 125.87 (2C), 123.05, 122.29, 121.39 (2C), 121.07, 62.36, 38.68,
34.86, 31.01 ppm (3C); Anal. calcd for C26H28N2O3·1.4H2O: C 70.69,
H 7.03, N 6.34, found: C 70.83, H 7.49, N 6.77.
1
Rf =0.32 (EtOAc/PE, 1:1); H NMR (500 MHz, [D6]DMSO): d=11.77 (s,
1H), 10.45 (s, 1H), 8.45 (dd, J=8.35, 0.82 Hz, 1H), 7.94–7.89 (m,
3H), 7.65–7.57 (m, 5H), 7.29 (dt, J=7.71, 1.09 Hz, 1H),7.20 (d, J=
8.48 Hz, 2H) 4.60 (t, J=5.21 Hz, 1H), 3.61–3.57 (m, 2H), 2.70 ppm
(t, J=7.07 Hz, 2H); 13C NMR (125 MHz, [D6]DMSO): d=167.37,
163.70, 138.66, 137.01, 136.42, 135.79, 133.41, 132.32, 129.15,
129.13 (2C), 129.06 (2C), 123.57 (2C), 123.06, 121.50, 121.37 (2C),
121.26, 62.37, 38.67 ppm; Anal. calcd for C22H19ClN2O3·0.33H2O: C
65.92, H 4.95, N 6.99, found: C 65.97, H 4.69, N 6.83.
N-[4-(2-Hydroxyethyl)phenyl]-2-(4-isopropoxybenzamido)benza-
mide (25). According to method A, 4-isopropoxybenzoic acid
(98 mg, 0.4 mmol) was reacted to 4-isopropoxybenzoyl chloride
and then, according to method B, with compound 3h (154 mg,
0.6 mmol) to obtain compound 25 as a white powder (132 mg,
2-(3-Chlorobenzamido)-N-[4-(2-hydroxyethyl)phenyl]benzamide
(21). According to method B, compound 3h (154 mg, 0.6 mmol)
and 3-chlorobenzoyl chloride (88 mg, 0.5 mmol) were reacted, and
compound 21 was obtained as a white powder (1974 mg, 100%):
1
1
Rf =0.21 (EtOAc/PE, 1:1); H NMR (500 MHz, [D6]DMSO): d=11.72 (s,
63%): Rf =0.73 (EtOAc/PE, 1:1); H NMR (500 MHz, [D6]DMSO): d=
1H), 10.53 (s, 1H), 8.35 (dd, J=8.25, 0.69 Hz, 1H), 7.96–7.91 (m,
2H), 7.86–7.83 (m, 1H), 7.67 (ddd, J=7.98, 2.00, 0.89 Hz, 1H), 7.62–
7.56 (m, 4H), 7.28 (t, J=7.50 Hz, 1H), 7.19 (d, J=8.50 Hz, 2H), 4.64
(m, 1H), 3.62–3.54 (m, 2H), 2.69 ppm (t, J=7.09 Hz, 2H); 13C NMR
11.74 (s, 1H), 10.45 (s, 1H), 8.54 (m, 1H), 7.92 (dd, J=1.4, 7.9, 1H),
7.85 (d, J=8.9, 2H), 7.58 (m, 3H), 7.23 (m, 3H), 7.06 (d, J=8.9, 2H),
4.75–4.67 (m, 1H), 4.60 (t, J=5.2, 1H), 3.60 (td, J=5.3, 7.1, 2H),
2.71 (t, J=7.1, 2H), 1.28 ppm (d, J=6.0, 6H); 13C NMR (125 MHz,
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[D6]DMSO): d=167.59, 164.13, 160.71, 139.32, 136.37, 135.82,
132.38, 129.13 (2C), 129.05 (2C), 129.02, 126.25, 122.92, 122.10,
121.43 (2C), 120.99, 115.58 (2C), 69.75, 62.36, 38.67, 21.84 ppm;
Anal. calcd for C25H26N2O4·0.25H2O: C 70.99, H 6.31, N 6.62, found:
C 70.63, H 6.18, N 6.53.
penicillin G. Cells were incubated in a humidified atmosphere con-
taining 5% CO2 at 378C.
Hoechst 33342 assay
2-(4-Hydroxybenzamido)-N-[4-(2-hydroxyethyl)phenyl]benza-
mide (26). Compound 23 was suspended in MeOH (10 mL) and
NaOH (3.3 mmol). The reaction proceeded at 608C. Compound 26
was extracted with EtOAc into the organic phase and then precipi-
tated as a white powder (102 mg, 54%) with THF/PE: Rf =0.71
(EtOAc/PE, 1:1); 1H NMR (500 MHz, [D6]DMSO): d=11.66 (s, 1H),
10.44 (s, 1H), 10.19 (s, 1H), 8.53 (dd, J=1.0, 8.4, 1H), 7.91 (dd, J=
1.4, 7.9, 1H), 7.77 (d, J=8.8, 2H), 7.58 (m, 3H), 7.23 (m, 3H), 6.90
(d, J=8.8, 2H), 4.60 (s, 1H), 3.60 (t, J=7.0, 2H), 2.71 ppm (t, J=7.1,
2H); 13C NMR (125 MHz, [D6]DMSO): d=167.61, 164.34, 161.11,
139.41, 136.39, 135.80, 132.36, 129.15 (4C), 129.02, 125.09, 122.81,
122.04, 121.42 (2C), 120.97, 115.62 (2C), 62.37, 38.69 ppm; Anal.
calcd for C22H20N2O4·0.5H2O: C 68.56, H 5.49, N 7.27, found: C
68.18, H 5.38, N 7.11.
For investigation of BCRP activity the Hoechst 33342 assay was
used as described previously, applying a Hoechst 33342 concentra-
tion of 1 mm in the assay.[14,18] In brief, the breast cancer resistant
cell line MCF-7 MX and the parental cell line MCF-7 were grown in
RPMI-1640 medium supplemented with 20% fetal bovine serum,
50 mgmLꢂ1 streptomycin, and 50 UmLꢂ1 penicillin G, in a 5% CO2
atmosphere at 378C. After reaching a confluence of 80–90%, cells
were harvested with trypsin–EDTA (0.05% trypsin, 0.02% EDTA),
transferred to a 50 mL tube, centrifuged (266 g, 48C, 4 min) and re-
suspended in fresh culture medium. The cell density was measured
with a Casy I Modell TT cell counter device (Schaerfe System
GmbH, Reutlingen, Germany). Cells were again centrifuged and re-
suspended in Krebs–HEPES buffer. Cells were seeded into black 96-
well plates at a density of 27000 cells per well in a volume of
90 mL. Test compounds (10 mL) at various concentrations were
added to give a total volume of 100 mL. The prepared plates were
then incubated under 5% CO2 atmosphere and 378C for 30 min.
After this pre-incubation period, Hoechst 33342 solution (20 mL,
6 mm) was added to each well. Fluorescence was measured imme-
diately at constant intervals of 60 s, at an excitation wavelength of
355 nm and an emission wavelength of 460 nm, using a BMG PO-
LARstar microplate reader held at 378C.
N-{2-[4-(2-hydroxyethyl)phenylcarbamoyl]phenyl}-3,4-dimethox-
ybenzamide (27). According to method A, 3.4-dimethoxybenzoic
acid (94 mg, 0.5 mmol) was reacted to 3,4-dimethoxybenzoyl chlo-
ride and then, according to method B, with compound 3h
(154 mg, 0.6 mmol) to obtain compound 27 as a white powder
(135 mg, 64%): Rf =0.05 (EtOAc/PE, 1:1); 1H NMR (500 MHz,
[D6]DMSO): d=11.74 (s, 1H), 10.45 (s, 1H), 8.51 (d, J=7.67 Hz, 1H),
7.92 (dd, J=7.86, 1.27 Hz, 1H), 7.63–7.57 (m, 3H), 7.50 (dd, J=6.53,
2.06 Hz, 2H), 7.25 (dt, J=7.72, 1.05 Hz, 1H), 7.21 (d, J=8.45 Hz,
2H), 7.13 (d, J=8.97 Hz, 1H), 4.60 (t, J=5.19 Hz, 1H), 3.83 (s, 3H),
3.82 (s, 3H), 3.59 (dt, J=7.08, 5.30 Hz, 2H), 2.70 ppm (t, J=7.07 Hz,
2H); 13C NMR (125 MHz, [D6]DMSO): d=167.53, 164.28, 152.11,
148.83, 139.17, 136.47, 135.78, 132.36, 129.12 (2C), 129.01, 126.90,
123.03, 122.43, 121.20 (2C), 121.03, 120.06, 111.58, 110.75, 62.36,
55.87, 55.63, 38.67 ppm; Anal. calcd for C24H24N2O5·0.33H2O: C
67.59, H 5.83, N 6.57, found: C 67.99, H 6.01, N 6.39.
Calcein AM assay
The inhibitory activity of studied compounds against P-gp was
tested in A2780 Adr cells using the calcein AM assay as described
previously.[20] In brief, the cells were prepared as described for the
Hoechst 33342 assay, but seeded into clear 96-well plates. To 90-mL
cell suspensions each containing 27000 cells, test compounds
(10 mL) at varying concentration were added, and the cells were
pre-incubated for 30 min. After the pre-incubation period, calcein
AM solution (33 mL, 1.25 mm) was added to each well. The fluores-
cence of each well was detected at constant intervals (60 s) up to
60 min using an excitation wavelength of 485 nm and an emission
wavelength of 520 nm, applying a 378C tempered BMG POLARstar
microplate reader. 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 non-
linear regression using the four-parameter logistic equation with
variable Hill slope (GraphPad Prism 5.0 software, San Diego, CA,
USA).
N-[4-(2-Hydroxyethyl)phenyl]-2-(4-methylbenzamido)benzamide
(28). According to method B, compound 3h (154 mg, 0.6 mmol)
and 4-methylbenzoyl chloride (77 mg, 0.5 mmol) were reacted, and
compound 28 was obtained as a white powder (170 mg, 91%):
1
Rf =0.53 (EtOAc/PE, 1:1); H NMR (500 MHz, [D6]DMSO): d=11.80 (s,
1H), 10.50 (s, 1H), 8.51 (dd, J=8.34, 0.98 Hz, 1H), 7.93 (dd, J=7.85,
1.36 Hz, 1H), 7.81 (d, J=8.20 Hz, 2H), 7.58 (dd, J=11.99, 4.96 Hz,
3H), 7.36 (d, J=7.92 Hz, 2H), 7.27–7.22 (m, 1H), 7.20 (d, J=8.53 Hz,
2H), 4.64 (s, 1H), 3.59 (t, J=7.10 Hz, 2H), 2.70 (t, J=7.08 Hz, 2H),
2.37 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=167.50,
164.68, 142.23, 139.33, 136.49, 135.75, 132.34, 132.01, 129.58 (2C),
129.15 (2C), 129.07, 127.16 (2C), 123.10, 122.52, 121.37 (2C), 121.24,
62.39, 38.69, 21.15 ppm; Anal. calcd for C23H22N2O3·0.33H2O: C
72.61, H 6.01, N 7.36, found: C 72.69, H 5.92, N 7.24.
MTT cytotoxicity assay
The influence of the new BCRP inhibitors on the cytotoxicity of mi-
toxantrone and SN-38 was investigated in MCF-7 MX and MDCK
BCRP cells by MTT cytotoxicity assay. Assays were performed as de-
scribed previously, with minor modifications.[19] Cells were seeded
into 96-well tissue-culture plates at a density of 10000 cells per
well for MCF-7 MX cells, or 1250 in case of MDCK BCRP cells, in
a total volume of 80 mL and kept at 378C under 5% CO2 for 6 h.
After cells had attached to the well bottom, 10 mL test compound
and 10 mL mitoxantrone solution were added. Cells were incubated
for 72 h, and then the MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; 20 mL of a 5 mgmLꢂ1 solution) was
added to each well. After 1 h incubation with MTT, the supernatant
Cell culture
The breast cancer resistant cell line MCF-7 MX and the parental cell
line MCF-7 were kindly provided by Dr. E. Schneider (Wadsworth
Center, Albany, NY, USA). Parental MDCK and MDCK BCRP cell lines
were kindly provided by Dr. A. H. Schinkel (NCI, Amsterdam). The
human ovarian carcinoma cell line A2780 and the corresponding
P-gp-overexpressing cell line A2780 Adr were purchased from the
European collection of animal cell cultures (ECACC, Nos. 93112519
[A2780] and 93112520 [A2780 Adr], UK). The cell lines were grown
in RPMI-1640 medium supplemented with 10% fetal bovine serum
(20% for MCF-7 cells), 50 mgmLꢂ1 streptomycin, and 50 UmLꢂ1
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Keywords: ABC transporter · antitumor agents · BCRP · drug
design · inhibitors · QSAR
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Received: August 10, 2012
Revised: October 22, 2012
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Published online on November 13, 2012
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