Design, synthesis and biological evaluation of benzamide and phenyltetrazole derivatives with amide and urea linkers as BCRP inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.09.009

Breast cancer resistant protein (BCRP/ABCG2), a 72 kDa plasma membrane transporter protein is a member of ABC transporter superfamily. Increased expression of BCRP causes increased efflux and therefore, reduced intracellular accumulation of many unrelated

Full text of DOI:10.1016/j.bmcl.2017.09.009

Accepted Manuscript
Design, synthesis and biological evaluation of benzamide and phenyltetrazole
derivatives with amide and urea linkers as BCRP inhibitors
Nehaben A. Gujarati, Leli Zeng, Pranav Gupta, Zhe-Sheng Chen, Vijaya L.
Korlipara
PII:
S0960-894X(17)30890-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.09.009
BMCL 25273
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 July 2017
1 September 2017
2 September 2017
Please cite this article as: Gujarati, N.A., Zeng, L., Gupta, P., Chen, Z-S., Korlipara, V.L., Design, synthesis and
biological evaluation of benzamide and phenyltetrazole derivatives with amide and urea linkers as BCRP inhibitors,
Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.09.009
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Bioorganic & Medicinal Chemistry Letters
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Design, synthesis and biological evaluation of benzamide and phenyltetrazole
derivatives with amide and urea linkers as BCRP inhibitors
Nehaben A. Gujarati, Leli Zeng, Pranav Gupta, Zhe-Sheng Chen and Vijaya L. Korlipara*
Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University, Queens, NY 11439
ARTICLE INFO
ABSTRACT
Article history:
Received
Breast cancer resistant protein (BCRP/ABCG2), a 72 kDa plasma membrane transporter protein
is a member of ABC transporter superfamily. Increased expression of BCRP causes increased
efflux and therefore, reduced intracellular accumulation of many unrelated chemotherapeutic
agents leading to multidrug resistance (MDR). A series of 31 benzamide and phenyltetrazole
derivatives with amide and urea linkers has been synthesized to serve as potential BCRP
inhibitors in order to overcome BCRP-mediated MDR. The target derivatives were tested for
their cytotoxicity and reversal effects in human non-small cell lung cancer cell line H460 and
mitoxantrone resistant cell line H460/MX20 using the MTT assay. In the benzamide series,
compounds 6 and 7 exhibited a fold resistance of 1.51 and 1.62, respectively at 10 µM
concentration which is similar to that of FTC, a known BCRP inhibitor. Compounds 27 and 31
were the most potent analogues in the phenyltetrazole series with amide linker with a fold
resistance of 1.39 and 1.32, respectively at 10 µM concentration. For the phenyltetrazole series
with urea linker, 38 exhibited a fold resistance of 1.51 which is similar than that of FTC and is
the most potent compound in this series. The target compounds did not exhibit reversal effect in
P-gp overexpressing resistant cell line SW620/Ad300 suggesting that they are selective BCRP
inhibitors.
Revised
Accepted
Available online
Keywords:
ABCG2
BCRP inhibitors
Multidrug resistance
Benzamide and phenyltetrazole derivatives
Cytotoxicity and reversal assay
2009 Elsevier Ltd. All rights reserved
.
Cancer is the second leading cause of death in the United
States.1 Although a wide variety of drugs are available to fight
cancer, Multidrug resistance (MDR) is a major contributor
towards the failure of cancer chemotherapy.2 Cancer cells can be
primarily resistant to chemotherapy or acquire MDR during
treatment, making chemotherapy ineffective.3 ATP-binding
cassette (ABC) proteins belong to a superfamily of proteins that
consists of 49 members classified from A through G subfamily.
ABC transporters use ATP hydrolysis to transport substrates
across cell membrane against their electrochemical gradient and
many of the human ABC proteins including P-glycoprotein (P-
which was isolated from Aspergillus fumigatus, is a highly potent
and specific inhibitor of BCRP. However, it is not suitable for
therapeutic use due to its neurotoxicity.10, 11
N
N
N
N
O
N
O
NH
O
O
O
HM30181: P-gp Inhibitor
N
gp/ABCB1)
and Breast
Cancer
Resistance
Protein
N
N
N
H
N
H
N
N
(BCRP/ABCG2) are efflux transporters.4 The decreased
accumulation of chemotherapeutic agents in the tumor cells is
one of the most common causes of MDR. BCRP (ABCG2) is the
second member of the subfamily G, consisting of 655 amino
acids, first identified in a multidrug resistant human breast cancer
O
O
NH
OCH3
NH
NH
O
O
O
NO2
N
Tariquidar analogue (XR9577)
NO2
Benzamide derivative (12)
15BCRP IC50= 0.94 µM
P-gp IC50 = n.e.
Phenyltetrazole derivative (23)
16BCRP IC50= 0.078 µM
12BCRP IC50 = 0.70 µM
P-gp IC50 = 0.33 µM
MRP1 IC50 = 9.82 µM
6
cell line MCF-7/AdrVp.5, BCRP transports a wide variety of
molecules and plays a key role in protecting the body against
xenobiotics.7
R1
N
N
O
A
R1
N
A
N
N
B
H
B
BCRP actively transports many structurally unrelated
chemotherapeutic agents such as mitoxantrone, methotrexate,
topotecan, SN38 and flavopiridol out of the cells leading to the
development of multidrug resistance (MDR).8 Overexpression of
BCRP in cancer cells is a significant mechanism behind MDR.
Efforts have been made to design potential modulators in order to
overcome BCRP-mediated MDR.9 Fumitremorgin C (FTC)
NH
NH
R2
R2
(X)
C
O
(X)
C
O
Figure 1. SAR development of benzamide and phenyltetrazole
series.
Tariquidar, a third generation P-gp inhibitor is two times more
selective in inhibiting P-gp than BCRP.12 Removal of the

tetrahydroisoquinoline ring of tariquidar has been found to impart
selectivity towards BCRP inhibition.13 Various modifications
related to tarquidar and HM30181 have led to the identification
of the benzamide derivative 12 (IC50 = 0.94 µM in Hoechst assay
using MCF-7 MX cells) and the phenyltetrazole derivative 23
(IC50 = 0.078 µM in Hoechst assay using MDCKII BCRP cells)
(Scheme 2). The intermediate aryl diazonium salts were prepared
from substituted anilines using sodium nitrite and hydrochloric
acid in water and ethanol mixture.18 1,5-Dipolar cyclization of 16
with aryl diazonium salts yielded the tetrazole analogues 17 and
18. The amines 19 and 20 were obtained by Pd/C catalyzed
hydrogenation of 17 and 18 (Scheme 3) and were reacted with
various substituted benzoyl chlorides to obtain the amide
analgoues 21-26 and with various substituted aryl isocyanates to
obtain the urea analogues 32-36.
which have been used as the lead compounds in the present
study.13,
14, 15, 16
A series of benzamide and phenyltetrazole
derivatives with amide or urea linkers between rings B and C,
and various substitutions were introduced at R1 and R2 on rings A
and C, respectively to impart a range of steric, electronic and
solubility characteristics to further explore the SAR.
The target compounds 6-15 and 21-41 were synthesized as
illustrated in Schemes 1-4. 2-Nitrobenzoyl chloride 1 was
obtained by refluxing 2-nitrobenzoic acid and thionyl chloride in
DCM (Scheme 1).17 Reacting 2-nitrobenzoyl chloride 1 with 4-n-
propyl aniline or 4-(trifluoromethyl)aniline in presence of
triethylamine led to amide analogues 2 and 3, respectively.
Hydrogenation using 10% Pd/C resulted in reduction of the nitro
analogues 2 and 3 to the amines 4 and 5 in quantitative yields.
The amines were then reacted with various substituted aryl
isocyanates to obtain the urea analogues 6-11 and with various
substituted benzoyl chlorides to obtain the amide analgoues 12-
15.
Scheme 3. Synthesis of phenyltetrazole analogues 21-26 and 32-36. (a) H2,
10% Pd/C, 50 psi, reagent alcohol, 7 h; (b) Substituted benzoyl chloride,
Et3N, DCM, 0 °C to rt, overnight; (c) Substituted phenylisocyanate, DCM, rt,
3 h.
Scheme 4. Synthesis of phenyltetrazole analogues. (a) H2, Pd/C, 50 psi,
reagent alcohol, 7 h; (b) H2, Pd/C, 60 psi, DMF, reagent alcohol, overnight;
(c) Acetic anhydride, Et3N, DCM, rt; (d) Di-2-pyridyl thionocarbonate, DCM,
rt; (e) NaNO2, NaN3, HCl/H2O, 0 °C to rt; (f) Bromoacetylbromide, DCM, 0
°C to rt.
Scheme 1. Synthesis of benzamide analogues 6-15. (a) SOCl2, DCM, reflux,
3 h; (b) Substituted anilines, Et3N, DCM, 0 °C to rt, 16 h; (c) H2, Pd/C, 50
psi, reagent alcohol, 7 h; (d) Substituted phenylisocyanate, THF, rt, 3 h; (e)
Substituted benzoyl chloride, Et3N, DCM, 0 °C to rt, overnight.
Pd/C catalyzed reduction of nitro derivatives 23 and 29 led to
the amines 27 and 37, respectively which were acetylated using
acetic anhydride to obtain acetamide analogues 28 and 38.19
Isothiocyanate analogues 29 and 39 were synthesized from the
amino analogues 27 and 37 by reacting them with di-2-pyridyl
thionocarbonate.20 The azide analogues 30 and 40 were obtained
from amines 27 and 37 by diazotization and subsequent treatment
with sodium azide.21 Acylation of the amino analogues 27 and 37
using bromoacetylbromide yielded analogues 31 and 41.22
The human cancer cell lines, H460 (non-small cell lung
cancer) and its MX-selected derivative ABCG2-overexpressing
cell line H460/MX20 were used to determine the cytotoxicity as
well as reversal effects using the MTT assay. FTC was chosen as
a positive control and the reversal of mitoxantrone resistance was
calculated.23
Scheme
Benzenesulfonylhydrazine, EtOH , rt; (b) NaNO2, conc. HCl, EtOH/ H2O, < 0
−10 to −15 °C.
2.
Synthesis
of
compounds
17
and
18.
(a)
°C; (c) Pyridine,
Sulfonyl hydrazide 16 was prepared from commercially
available 2-nitrobenzaldehyde and benzenesulfonylhydrazine

In the benzamide series, all the target compounds with amide
or urea linkers 6-15 exhibited IC50 values greater than 100 µM in
both H460 and H460/MX20 cell lines (Table 1). In the
phenyltetrazole series, the target compounds with the amide
linker 21-31, the IC50 values of analogues 21, 23, 27, 28, and 31
were greater than 100 µM and the remaining analogues exhibited
IC50 values in the range of 20 and 60 µM in the two cell lines.
In the phenyltetrazole series, among the analogues containing
the urea linker 32-41, compound 32 with 4-OCH3 substitution at
both R1 and R2 positions exhibited IC50 values of 52.54 7.51
µM and 57.41 6.78 µM in H460 cells and H460/MX20 cells,
respectively (Table 2). Phenyltetrazole derivative 33 with 4-
OCH3 substitution at R1 and 4-NO2 substitution at R2 exhibited
IC50 values of 21.67 5.53 µM and 28.53 3.19 µM in H460
cells and H460/MX20 cells, respectively. All the other analogues
in the phenyltetrazole series containing the urea linker exhibited
an IC50 value greater than 100 µM.
Table 1. Cytotoxicity of benzamide derivatives using MTT assay
For the reversal assay, all the target compounds were tested at
10, 3, and 1 µM concentrations (Tables 3-5). The fold resistance
of mitoxantrone was found to be 28.66-fold against H460/MX20
cell line which was reduced to 1.49-fold in presence of 10 µM
FTC, a known BCRP inhibitor.
H460
(µM)
H460/MX20
(µM)
Cmpd #
X
R1
R2
QPlogP
NH
NH
NH
NH
NH
NH
˗
4-n-C3H7
4-n-C3H7
4-n-C3H7
4-n-C3H7
4-n-C3H7
4-CF3
4-NO2
3-NO2
2-NO2
4-Br
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
3.83
3.94
3.84
4.85
4.53
3.78
4.67
4.70
4.91
5.49
6
7
8
9
BCRP ATPase activity
4-OCH3
4-NO2
4-NO2
3-NO2
2-NO2
4-OCH3
400
10
11
12
13
14
15
350
300
250
4-n-C3H7
4-n-C3H7
4-n-C3H7
4-n-C3H7
˗
˗
˗
200
150
100
50
1 µM
aIC50 values are represented as mean ꢀ ꢀSD of three independent experiments.
QPlogP is the log Po/w value calculated using QikProp, Schrödinger.
3 µM
5 µM
10 µM
Table 2. Cytotoxicity of phenyltetrazole derivatives using MTT
assay
0
Basal
Activity
6
7
27
34
Compounds
H460/
MX20
(µM)
QPlogP
Cmpd
#
H460
(µM)
X
R1
R2
4-OCH3
aATPase activity is represented as mean SD of two independent
experiments
-
-
-
-
4-OCH3
4-OCH3
3-OCH3
3-OCH3
> 100
> 100
3.51
2.90
2.92
3.02
21
22
23
24
24.73
2.62
27.44
3.14
4-NO2
4-NO2
3-NO2
Figure 2. Effect of selected target compounds on ATPase
activity of ABCG2.
> 100
> 100
19.85
1.55
25.41
2.95
In the benzamide series, the lead compound 12 with amide
linker, 4-n-C3H7 at R1 and 4-NO2 at R2 exhibited fold resistance
of 2.76 at 10 µM concentration (Table 3). Compounds 6, 7, 11,
and 13 exhibited a fold resistance lower than that of the lead
compound 12. Among benzamide compounds possessing a urea
linker, compound 6 with 4-n-C3H7 at R1 and 4-NO2 at R2 and
compound 7 with 4-n-C3H7 at R1 and 3-NO2 at R2 exhibited a fold
resistance of 1.51 and 1.62 at 10 µM concentration which is
similar to that of FTC.
38.90
3.68
44.71
4.21
-
-
3-OCH3
3-OCH3
2-NO2
3.18
4.52
25
26
26.97
3.25
40.60
2.55
4-n-C3H7
-
-
3-OCH3
3-OCH3
4-NH2
> 100
> 100
> 100
> 100
2.80
3.08
27
28
4-NHCOCH3
44.70
6.67
59.01
6.19
-
3-OCH3
4-NCS
3.37
29
20.30
1.97
21.51
1.75
-
-
3-OCH3
3-OCH3
4-OCH3
4-N3
2.35
3.71
3.63
30
31
32
In the phenyltetrazole series with amide linker, compound 22
with 4-OCH3 at R1 and 4-NO2 at R2 and compound 23 with 3-
OCH3 at R1 and 4-NO2 at R2 were the lead molecules with a fold
resistance of 1.76 and 1.43, respectively (Table 4).16 Compounds
26, 27 and 31 exhibited a fold resistance lower than the lead
compounds 22 and 23 as well as FTC at all three concentrations.
Compound 26 with 3-OCH3 at R1 and 4-n-C3H7 at R2 exhibited
the lowest fold resistance, however, as previously described, in
the cytotoxicity assay it exhibited IC50 values of 26.97 and 59.01
µM, respectively in H460 and H460/MX20 cell lines (Table 2).
Compounds with lower IC50 values can have intrinsic cytotoxic
effect, which in turn can result in ostensibly lower fold
resistance. Compounds 27 with 3-OCH3 at R1 and 4-NH2 at R2
and 31 with 3-OCH3 at R1 and 4-NHCOCH2Br at R2 were the
4-NHCOCH2Br
4-OCH3
> 100
> 100
52.54
7.51
21.69
5.53
57.41
6.78
28.53
3.19
NH
NH
4-OCH3
4-NO2
2.30
33
NH
NH
NH
NH
NH
NH
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
4-NO2
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
2.16
2.95
2.65
2.50
2.25
3.01
34
35
36
37
38
39
3-NO2
2-NO2
4-NH2
4-NHCOCH3
4-NCS
NH
NH
3-OCH3
3-OCH3
4-N3
> 100
> 100
> 100
> 100
2.09
2.87
40
41
4-NHCOCH2Br
aIC50 values are represented as mean ꢀ ꢀSD of three independent experiments.
QPlogP is the log Po/w value calculated using QikProp, Schrödinger.

most potent analogues in this series with a fold resistance of 1.39
and 1.32, respectively at 10 µM concentration.
was observed at 3 µM and 1 µM concentrations for all three
series of analogues.
In the phenyltetrazole series with the urea linker, compounds
32, 33, 34, 37, and 38 exhibited fold resistance lower or similar
to FTC (Table 5). However, as discussed previously compounds
32 with 4-OCH3 at R1 and 4-OCH3 at R2 and 33 with 4-OCH3 at
R1 and 4-NO2 at R2 had a lower IC50 value in the cytotoxicity
assay as compared to the other analogues (Table 2). Compound
38 with 3-OCH3 at R1 and 4-NHCOCH3 at R2 exhibited a fold
resistance of 1.51 which was found to be similar to that of FTC
and was the most potent member of this series followed by
compound 37 with 3-OCH3 at R1 and 4-NH2 at R2 and 34 with 3-
OCH3 at R1 and 4-NO2 at R2. A similar trend in the fold resistance
All the target compounds (6-15 and 21-41) were tested at
three different concentrations (10, 3 and 1 µM) for the reversal
assay in parental human colon cancer SW620 cells and paclitaxel
resistant SW620/Ad300 cells. Verapamil was chosen as the
positive control and the fold resistance of paclitaxel was
calculated. Verapamil was found to have a fold resistance of
2.31, 2.65 and 3.54 at 10, 3, and 1 µM concentrations,
respectively. All the target compounds exhibited a fold resistance
greater than 45 which is comparable to the fold resistance of
paclitaxel. They did not affect the reversal of paclitaxel in P-gp
overexpressing resistant cell line SW620/Ad300 suggesting that
these analogues are selective towards ABCG2-mediated MDR.
Table 3. Reversal effect of benzamide on mitoxantrone resistant ABCG2 overexpressing H460/MX20 cells
Conc.
(µM) IC 50 SD (µM) IC 50 SD (µM)
H460
H460/MX20
Fold
Resistance
Cmpd #
X
R1
R2
Mitoxantrone
0.215 0.011
0.199 0.023
0.211 0.017
0.209 0.014
0.201 0.054
0.205 0.021
0.206 0.011
0.212 0.062
0.225 0.016
0.221 0.017
0.214 0.025
0.217 0.015
0.217 0.021
0.211 0.026
0.218 0.014
0.221 0.016
0.222 0.013
0.221 0.023
0.217 0.011
0.213 0.051
0.218 0.015
0.212 0.015
0.205 0.021
0.209 0.012
0.211 0.025
0.221 0.012
0.206 0.011
0.215 0.012
0.211 0.062
0.224 0.016
0.209 0.016
0.148 0.058
0.168 0.013
0.186 0.007
6.161 0.172
0.325 0.024
0.482 0.053
0.622 0.031
0.348 0.042
0.501 0.072
0.651 0.022
1.120 0.077
2.563 0.137
3.662 0.214
0.952 0.142
2.121 0.161
2.655 0.117
0.985 0.072
2.173 0.112
3.371 0.356
0.452 0.058
0.605 0.045
0.711 0.055
0.594+0.057
0.783+0.075
0.833+0.043
0.512+0.064
0.727+0.022
0.785+0.072
1.713+0.044
2.151+0.132
2.814+0.252
1.656+0.11
28.66
1.51
2.24
2.89
1.62
2.33
3.03
5.21
11.92
17.03
4.43
9.87
12.35
4.58
10.12
15.68
2.12
2.81
3.31
2.76
3.64
3.87
2.38
3.38
3.65
7.70
9.36
11.75
7.97
10.00
13.10
1.49
2.10
2.85
10
3
6
7
NH 4-n-C3H7 4-NO2
NH 4-n-C3H7 3-NO2
NH 4-n-C3H7 2-NO2
1
10
3
1
10
3
8
1
10
3
NH 4-n-C3H7
4-Br
9
1
10
3
NH 4-n-C3H7 4-OCH3
10
11
12
13
14
15
1
10
3
NH
4-CF3
4-NO2
1
10
3
-
-
-
-
4-n-C3H7 4-NO2
4-n-C3H7 3-NO2
4-n-C3H7 2-NO2
4-n-C3H7 4-OCH3
FTC
1
10
3
1
10
3
1
10
3
2.012+0.11
1
2.526+0.173
0.321 0.052
0.451 0.031
0.612 0.034
10
3
1
aIC50 values are represented as meanꢀ ꢀSD of three independent experiments. Fold resistance was calculated as by dividing
the IC50 values of the substrate (MX) in presence or absence of inhibitor by the IC50 of parental cells without inhibitors.

Table 4. Reversal effect of phenyltetrazole derivatives with amide linker on mitoxantrone resistant ABCG2 overexpressing
H460/MX20 cells
H460
IC 50 SD
(µM)
H460/MX20
IC 50 SD
(µM)
Fold
Resistance
Conc.
(µM)
Cmpd #
R1
R2
Mitoxantrone
0.215 0.011
0.149 0.061
0.156 0.013
0.305 0.054
0.121 0.012
0.143 0.121
0.244 0.021
0.230 0.043
0.239 0.032
0.240 0.052
0.345 0.044
0.561 0.013
0.715 0.016
0.333 0.022
0.414 0.045
0.501 0.019
0.121 0.051
0.173 0.012
0.207 0.112
0.211 0.031
0.212 0.061
0.217 0.041
0.52 0.022
6.161 0.172
0.492 0.011
0.505 0.035
0.746 0.025
0.378 0.024
0.535 0.035
0.587 0.064
0.307 0.032
0.387 0.022
0.585 0.055
0.866 0.056
1.39 0.15
28.66
2.29
2.35
3.47
1.76
2.49
2.73
1.43
1.80
2.72
4.03
6.47
8.38
5.12
6.14
8.74
0.61
0.96
1.65
1.39
2.28
2.55
5.58
6.16
6.34
4.43
4.88
5.68
5.89
6.98
7.51
1.32
2.07
2.81
1.49
10
3
4-OCH3
4-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
4-OCH3
4-NO2
21
22
23
24
25
26
27
28
29
30
31
1
10
3
1
10
3
4-NO2
1
10
3
3-NO2
1
1.801 0.72
1.10 0.32
10
3
2-NO2
1.32 0.27
1
1.88 0.152
0.131 0.022
0.207 0.031
0.354 0.051
0.299 0.057
0.491 0.072
0.549 0.072
1.202 0.012
1.325 0.037
1.363 0.025
0.952 0.053
1.05 0.051
1.221 0.17
1.267 0.23
1.501 0.33
1.615 0.63
0.283 0.056
0.446 0.025
0.605 0.054
0.321 0.052
10
3
4-n-C3H7
4-NH2
1
10
3
1
10
3
3-OCH3
3-OCH3
3-OCH3
4-NHCOCH3
4-NCS
0.554 0.086
0.725 0.084
0.352 0.015
0.379 0.052
0.457 0.065
0.415 0.026
0.455 0.056
0.558 0.014
0.250 0.061
0.175 0.073
0.241 0.033
0.148 0.058
1
10
3
1
10
3
4-N3
1
10
3
3-OCH3 4-NHCOCH2Br
1
10
FTC
3
1
0.168 0.013
0.186 0.007
0.451 0.031
0.612 0.034
2.10
2.85
aIC50 values are represented as meanꢀ ꢀSD of three independent experiments. Fold resistance was calculated as by dividing
the IC50 values of the substrate (MX) in presence or absence of inhibitor by the IC50 of parental cells without inhibitors.
ABCG2 utilizes energy derived from the hydrolysis of ATP
to efflux their substrates across the membrane against a
concentration gradient, and thus ATP consumption reflects its
ATPase activity.24 To assess the effect of the target compounds
on the ATPase activity of ABCB2, ABCB2-mediated ATP
hydrolysis was measured at various concentrations. ATPase
assay was carried out for selected potent analogues 6, 7, 27, and
34 (Figure 2). Among these analogues 6, 7, and 34 were found to
stimulate the basal BCRP ATPase activity by 1.47, 3.55 and 1.94
folds, respectively at 10 µM concentration. Compound 27 was
found to inhibit the basal BCRP ATPase activity by 0.52-fold.
Increased ATPase activity suggests that the analogues 6, 7, and
34 may be competitively inhibiting ABCG2 while compound 27
may be serving as a non-competitive inhibitor of ABCG2.
Analogues 7, 27 and 34 showed dose dependent effect on
ATPase activity; however compound 6 showed an increase in
ATPase activity up to 3 µM concentration followed by decrease
in ATPase activity at higher concentrations.

Table 5. Reversal effect of phenyltetrazole derivatives with urea linker on mitoxantrone resistant ABCG2 overexpressing H460/MX20
cells
Fold
Resistance
Conc.
(µM) IC 50 SD (µM) IC 50 SD (µM)
H460
H460/MX20
Cmpd #
R1
R2
Mitoxantrone
0.215 0.011
0.211 0.019
0.229 0.021
0.255 0.025
0.122 0.025
0.174 0.021
6.161 0.172
0.355 0.041
0.382 0.035
0.442 0.062
0.331 0.051
0.541 0.027
28.66
1.56
1.78
2.06
1.54
2.52
10
3
4-OCH3
4-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
4-OCH3
4-NO2
4-NO2
3-NO2
2-NO2
4-NH2
32
1
10
3
33
34
35
1
10
3
0.194 0.011
0.113 0.042
0.190 0.016
0.718 0.032
0.403 0.054
0.544 0.013
3.34
1.87
2.53
1
10
3
0.179 0.035
0.144 0.024
0.148 0.115
0.254 0.071
0.222 0.058
0.235 0.014
0.246 0.011
0.176 0.028
0.216 0.045
0.228 0.064
0.128 0.053
0.275 0.062
0.341 0.058
0.217 0.044
0.233 0.086
0.294 0.052
0.252 0.054
0.281 0.071
0.279 0.026
0.185 0.025
0.211 0.055
0.254 0.054
0.148 0.058
0.832 0.124
0.750 0.024
0.807 0.161
0.967 0.145
0.882 0.104
0.978 0.056
1.172 0.112
0.379 0.044
0.571 0.065
0.838 0.075
0.325 0.094
0.454 0.052
0.627 0.022
0.550 0.047
0.667 0.032
1.515 0.21
0.685 0.151
0.691 0.012
0.913 0.32
0.472 0.06
0.583 0.075
0.798 0.132
0.321 0.052
3.87
3.49
3.75
4.50
4.10
4.55
5.45
1.76
2.66
3.90
1.51
2.11
2.92
2.56
3.10
7.05
3.19
3.21
4.25
2.20
2.71
3.71
1.49
1
10
3
36
37
38
39
40
41
1
10
3
1
10
3
3-OCH3
3-OCH3
3-OCH3
4-NHCOCH3
4-NCS
1
10
3
1
10
3
4-N3
1
10
3
3-OCH3 4-NHCOCH2Br
1
10
FTC
3
1
0.168 0.013
0.186 0.071
0.451 0.031
0.612 0.034
2.10
2.85
aIC50 values are represented as meanꢀ ꢀSD of three independent experiments. Fold resistance was calculated as by
dividing the IC50 values of the substrate (MX) in presence or absence of inhibitor by the IC50 of parental cells without
inhibitors.
In conclusion, a series of novel benzamide (6-15) and
phenyltetrazole derivatives (21-41) consisting of urea and amide
linkers were designed and synthesized as potential and
BCRP/ABCG2 selective inhibitors. The compounds were
evaluated for their cytotoxicity and reversal effects on
mitoxantrone resistance in two cell lines, parental cell line H460
and BCRP overexpressing cell line H460/MX20, using the MTT
assay. In the benzamide series, compounds 6 and 7 which
exhibited QPlogP values of 3.83 and 3.94, respectively were
found to have reversal effects on the cytotoxicity of mitoxantrone
in H460/MX20 cells comparable to the known BCRP inhibitor,
FTC at three different concentrations. In the tetrazole series,
compounds 27, 31, 34, 37, and 38 which exhibited QPlogP
values in the range of 2.16 - 3.71 were found to have reversal
activity comparable to that of FTC. Compounds 6, 7, 27, and 34
were further evaluated using ATPase assay. Compounds 6, 7, and
27 were found to be stimulators of basal ATPase, while
compound 34 was found to be an inhibitor of ATPase. Target
compounds did not exhibit any reversal effect on paclitaxel
resistant P-gp overexpressing SW620/Ad300 cells, suggesting
that these analogues are selective BCRP (ABCG2) inhibitors.
The target compounds can serve as lead molecules for developing

inhibitors to overcome multi-drug resistance caused by
overexpression of BCRP in cancer cells.
Wurpel, J. N. D.; Chen, Z.; Guo, H. Oncotarget 2015, 6,
39276-39291.
24. Wang, Y.; Kathawala, R. J.; Zhang, Y.; Patel, A.; Kumar, P.;
Shukla, S.; Fung, K. L.; Ambudkar, S. V.; Talele, T. T.;
Chen, Z. Biochemical Pharmacology 2014, 90, 367-378.
Acknowledgments
The authors thank Dr. Tanaji Talele and Udaykiran
Velagapudi for assistance with the QPlogP calucaltions. Support
for this research was provided by Saint John’s University.
A. Supplementary data
All experimental procedures (for both synthesis and
biological assays) and full characterization of compounds.
Supplementary data associated with this article can be found, in
the online version, at
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Design, synthesis and biological evaluation of
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amide and urea linkers as BCRP inhibitors
Nehaben A. Gujarati, Leli Zeng, Pranav Gupta,
Zhe-Sheng Chen and Vijaya L. Korlipara*
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