Benzanilide-biphenyl replacement: A bioisosteric approach to quinoline carboxamide-type ABCG2 modulators

Article abstract of DOI:10.1021/ml4000832

Recently reported compounds such as UR-COP78 (6) are among the most potent and selective ABCG2 modulators known so far but are prone to rapid enzymatic cleavage at the central benzanilide moiety. In search for more stable analogues, according to a bioisosteric approach, a series of N-(biphenyl-3-yl)quinoline carboxamides was prepared by solid phase and solution phase synthesis. The biphenyl moiety was constructed by Suzuki coupling. Inhibition of ABCB1 and ABCG2 was determined in a calcein-AM and a Hoechst 33342 microplate assay, respectively. Most synthesized compounds selectively inhibited the ABCG2 transporter at submicromolar concentrations with a maximal inhibitory effect (I_max) over 90% (e.g., UR-COP228 (22a), IC₅₀ 591 nM, I_max 109%; UR-COP258 (31), IC₅₀ 544 nM, I_max 112%), though with lower potency and selectivity than 6. The biphenyl analogues are considerably more stable and demonstrate that the benzanilide core is not a crucial structural feature of quinoline carboxamide-type ABCG2 modulators.

Full text of DOI:10.1021/ml4000832

Letter
pubs.acs.org/acsmedchemlett
Benzanilide−Biphenyl Replacement: A Bioisosteric Approach to
Quinoline Carboxamide-Type ABCG2 Modulators
Cristian Ochoa-Puentes,†,‡,⊥ Stefanie Bauer,§,⊥ Matthias Kuhnle,§ Gunther Bernhardt,§
̈
̈
Armin Buschauer,*,§ and Burkhard Konig*,†
̈
†Institute of Organic Chemistry, University of Regensburg, D-93040 Regensburg, Germany
‡Departamento de Química, Universidad Nacional de Colombia, Bogota
́
D.C., Colombia
§Institute of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany
S
* Supporting Information
ABSTRACT: Recently reported compounds such as UR-COP78 (6) are among the most
potent and selective ABCG2 modulators known so far but are prone to rapid enzymatic
cleavage at the central benzanilide moiety. In search for more stable analogues, according to a
bioisosteric approach, a series of N-(biphenyl-3-yl)quinoline carboxamides was prepared by
solid phase and solution phase synthesis. The biphenyl moiety was constructed by Suzuki
coupling. Inhibition of ABCB1 and ABCG2 was determined in a calcein-AM and a Hoechst
33342 microplate assay, respectively. Most synthesized compounds selectively inhibited the
ABCG2 transporter at submicromolar concentrations with a maximal inhibitory effect (Imax
)
over 90% (e.g., UR-COP228 (22a), IC50 591 nM, Imax 109%; UR-COP258 (31), IC50 544
nM, Imax 112%), though with lower potency and selectivity than 6. The biphenyl analogues
are considerably more stable and demonstrate that the benzanilide core is not a crucial structural feature of quinoline
carboxamide-type ABCG2 modulators.
KEYWORDS: ABC transporter, breast cancer resistance protein, MCF-7 cells, solid phase synthesis, Suzuki coupling
indicating that water solubility was limiting the efficacy in this
class of compounds. When characterized in more detail with
respect to studies on human tumor xenograft models in nude
mice, surprisingly, compounds 5 and 6 proved to be unstable in
mouse plasma, due to rapid enzymatic cleavage at the
benzanilide group, whereas the ester group remained
unchanged under these conditions.13
As part of a project aiming at more stable ABCG2 inhibitors,
the replacement of the labile benzanilide core in 5 and 6 was
considered to be a promising bioisosteric approach. Here, we
report on N-biphenylyl quinoline carboxamides, which were
synthesized by analogy with a previously developed solid phase
synthesis protocol.12
As shown in Scheme 1, 4-bromo-2-nitrobenzoic acid 7 was
attached to Wang resin using EDC·HCl. Reduction of the nitro
group and acylation with quinoline-2- or quinoline-6-carbonyl
chloride 10a,b (freshly prepared) led to the resin bound amides
11a,b. In the next step, the biphenyl system was constructed via
Suzuki coupling between 11a,b and commercially available 4-
(hydroxymethyl)phenylboronic acid. Biphenyl derivatives 12a,b
were mesylated at the hydroxy group and substituted by the
tetrahydroisoquinolines 14−18.12 Finally, cleavage of the resin
with TFA/DCM (1:1) and treatment of the obtained
xpression of ATP-binding cassette (ABC) transporters
Esuch as ABCB1 (p-glycoprotein, p-gp), ABCC1 (MRP1),
or ABCG2 (breast cancer resistance protein, BCRP) is
associated with multidrug resistance (MDR).1,2 Additionally,
as numerous cytostatics are substrates, the chemotherapy of
malignant brain tumors compromises efflux pumps at the
blood−brain barrier, especially ABCB1 and ABCG2. By
analogy with an approach described for ABCB1,3,4 coadminis-
tration of ABCG2 inhibitors and appropriate cytostatics might
be useful to overcome MDR and to improve the chemotherapy
of malignancies in the CNS.5
Compared to ABCB1 modulators, the number of reported
ABCG2 inhibitors is still limited. An analogue of the natural
compound fumitremorgin C (1),6 Ko143 (2),7 is known as one
of the most potent and selective ABCG2 inhibitors (Chart 1),
whereas elacridar (3, GF120918)8 and tariquidar (4,
XR9576)9,10 are dual ABCB1 and ABCG2 modulators.
Recently, we described the synthesis of a new class of potent
and selective ABCG2 modulators derived from 4 (Chart 1).11,12
Compound 5, in which the quinoline-carboxamido residue was
shifted to the meta-position at the benzamide core and the two
methoxy groups were replaced by a methyl carboxylate, is
highly selective for ABCG2 with an IC50 value in the low
nanomolar range (65 nM) and a maximal inhibitory effect of
63% (Hoechst 33342 assay) relative to fumitremorgin C
(100%).13 The maximal response increased to about 90% when
one of the methoxy groups on the tetrahydroisoquinoline
moiety was replaced by a triethylenglycol chain (6),12
Received: December 7, 2012
Accepted: March 4, 2013
Published: March 4, 2013
© 2013 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
as characteristic features of compounds 5 and 6.11,12
Compounds 28−31, in which the length of the linker between
the tetrahydroisoquinoline moiety and the biphenyl motif was
extended by one methylene group, were synthesized in solution
starting from methyl 2-amino-4-bromobenzoate 24 and 4-[2-
(tert-butyldimethylsilyloxy)ethyl]phenylboronic acid14
(Scheme 2).
Chart 1. Structures of ABCG2 Modulators
ABCB1 and ABCG2 inhibitory activity of compounds
19a,b−21a,b, 22a, 23a, and 28−31 as well as of reference
compounds were investigated in a calcein-AM (ABCB1)15 and
a Hoechst 33342 (ABCG2) microplate assay13 using ABCB1-
overexpressing Kb-V1 and ABCG2-overexpressing MCF-7/
Topo cells. The data are summarized in Table 1.
Elacridar strongly inhibited both transporters without
preference for one of the two targets, whereas tariquidar was
almost equipotent with elacridar at ABCB1, but about four
times less potent at ABCG2. The potent ABCG2 inhibitor
Ko143 (2)7 was inactive at the ABCB1 transporter and showed
an IC50 of 117 nM with a maximal inhibitory effect comparable
to that of fumitremorgin C (1). Compounds 5 (UR-ME22-1)
and 6 (UR-COP78) were comparable to Ko143 regarding
potency and selectivity but produced a lower maximal response,
especially in the case of the poorly soluble dimethoxytetrahy-
droisoquinoline 5.
All newly synthesized compounds showed a marked
preference for ABCG2 inhibition. Compounds 19a, 20a, 21a,
and 23a were even inactive at the ABCB1 transporter. Whereas
the IC50 values (∼600 nM,) of compounds 19b and 22a were
comparable to that of tariquidar, the maximal inhibitory effects
were significantly higher, achieving 94% and 109% relative to
fumitremorgin C (1). Except for 21b, all modulators showed a
maximal ABCG2 inhibition of about 90% or higher. Compound
22a, the most potent modulator in this series, only showed a
marginal maximum inhibition of 20% at the ABCB1 trans-
porter.
carboxylic acids with trimethylsilyldiazomethane (TMSCHN2)
led to the desired methyl esters 19a,b−21a,b, 22a, and 23a.
The synthesized compounds were obtained in acceptable to
low overall yields, mainly due to incomplete coupling between
11a,b and the boronic acid as confirmed by NMR analysis of
byproducts. All analogues bearing a tetrahydroisoquinoline with
a triethylenglycol chain showed increased water solubility.
In addition, several homologues were synthesized retaining
the methyl ester and the quinoline-2-carboxamido substituents
a
Scheme 1. Solid Phase Synthesis of N-(Biphenyl-3-yl)-Substituted Quinoline Carboxamides 19a,b−21a,b, 22a, and 23a
a
Reagents and conditions: (i) 4-bromo-2-nitrobenzoic acid 7, EDC·HCl, DMAP, DMF/DCM 1/1, rt, overnight; (ii) SnCl2·2H2O, DMF, 80 °C,
overnight; (iii) quinoline carbonyl chlorides 10a,b, DIPEA, DCM, rt, 12 h (twice); (iv) 4-(hydroxymethyl)phenylboronic acid, Pd(PPh3)4, K3PO4,
DME, 80 °C, 21 h; (v) MsCl, DIPEA, DCM, rt, 6 h; (vi) tetrahydroisoquinolines 14−18, THF, 80 °C, 21 h; (vii) TFA/DCM (1:1), rt, 30 min
(twice); (viii) TMSCHN2, PhH/MeOH (1:1), rt, 1 h.
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 2. Solution Phase Synthesis of the N-(Biphenyl-3-yl)quinoline Carboxamides 28−31
a
Reagents and conditions: (i) methyl 2-amino-4-bromobenzoate 24, 4-[2-(tert-butyldimethylsilyloxy)ethyl]phenylboronic acid, Pd(PPh3)4, K3PO4,
THF, 80 °C, 15 h; (ii) quinoline-2-carbonyl chloride 10a, TEA, DCM, 40 °C, overnight; (iii) TBAF, THF, rt, 3 h; (iv) MsCl, TEA, DCM, rt, 5 h;
(v) tetrahydroisoquinolines 14, 15, 17, and 18, CH3CN, 80 °C, overnight.
Compounds 22a, 30, and 31 showed higher IC50 values
compared to all the reference compounds, but with respect to
the maximal inhibitory effects, they were superior to tariquidar,
elaquidar, and compounds 5 and 6, and comparable to Ko143
(2) (Figure 1). In general, the addition of one or two
Table 1. Inhibition of ABC Transporters by Reference
Compounds 1-6 and Quinoline Carboxamides 19a,b−21a,b,
22a, 23a, and 28−31
a
b
ABCB1
ABCG2
c
c
d
compd
IC50 (nM)
n.d.
inactive
193 18
223
IC50 (nM)
Imax (%)
1, fumitremorgin C
731 92
117 53
127 41
526 85
100
e
f
2, Ko143
103
63
69
63
88
85
94
87
92
7
e
3, elacridar
7
5
2
3
7
2
5
6
e
4, tariquidar
8
g
e
5, UR-ME22-1
>29000
>50000
inactive
65
8
h
6, UR-COP78
130 29
f
19a
1461 169
641 175
943 79
19b
4490 160
f
20a
inactive
20b
10900 170
1540 110
1031 170
237 65
f
21a
inactive
114 20
21b
18000 1560
67
1
i
22a, UR-COP228
1230 30
591 87
109
103
90
8
3
f
23a
inactive
839 105
1100 156
3214 1050
760 67
f
28
inactive
2
f
29
inactive
126 15
98
112 19
j
Figure 1. Concentration dependent inhibition of the ABCG2
transporter in MCF-7/Topo cells (Hoechst 33342 assay) by Ko143
(2), tariquidar (4), UR-ME22-1 (5), UR-COP228 (22a), and UR-
COP258 (31). The inhibition is expressed as % relative to the maximal
inhibition of ABCG2 by 10 μM of fumitremorgin C (1).
30
5420 230
6
j
31, UR-COP258
5990 520
544 53
a
Calcein-AM microplate assay (unless otherwise indicated) using
b
ABCB1-overexpressing Kb-V1 cells. Hoechst 33342 microplate assay
using ABCG2-overexpressing MCF-7/Topo cells. Mean values
c
SEM calculated from two to three independent experiments
performed in triplicate, unless otherwise indicated; n.d.: not
d
determined. Maximal inhibitory effects are expressed as percental
inhibition caused by the highest concentration of the compound tested
triethylene glycol chains at the tetrahydroisoquinoline core
increased the inhibitory activity compared to tariquidar,
elacridar, and compound 5 due to higher water solubility.
Investigations on the stability under physiological conditions
(mouse plasma) revealed that compounds 513 and 6 were
enzymatically completely degraded at the benzanilide within 30
min. To increase the stability, the benzanilide was replaced by
the biphenyl system. In this new series of modulators, the ester
group is prone to enzymatic cleavage resulting in the inactive
carboxylic acid. However, hydrolysis of the ester occurs much
slower than cleavage of the benzanilide: regarding stability in
mouse plasma (cf. Supporting Information), all biphenyl-type
modulators had a half-life of about 24 h compared to 10 min in
the cases of compounds 5 and 6. Additionally, the quinoline
carboxamide group was cleaved, though to a very small extent.
(70 or 100 μM, respectively) relative to fumitremorgin C (1) at a
e
f
concentration of 10 μM (100% inhibition). Ref 13. No effect up to a
g
concentration of 100 μM. Data from flow cytometric calcein-AM
h
i
assay.13 Ref 12. Maximum inhibition corresponds to 20% of the
j
maximal response to tariquidar (1 μM). A 50% maximal inhibitory
effect relative to tariquidar (1 μM).
The potency of compounds 28−31 was not increased
compared to that of the lower homologue 22a. The results in
the Hoechst 33342 assay were comparable to those for
compounds 19a,b−21a,b, 22a, and 23a, suggesting that the
distance between the tetrahydroisoquinoline core and the biaryl
moiety may be varied within this class of ABCG2 modulators.
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ACS Medicinal Chemistry Letters
Letter
In conclusion, the presented solid and solution phase
syntheses are straightforward methods to give convenient
access to N-biphenylyl-substituted quinoline carboxamides.
Among the prepared compounds, 22a and 31 were the most
potent and selective ABCG2 inhibitors. A prominent feature of
these ABCG2 modulators is their higher maximal inhibitory
effect, by far surpassing that of tariquidar-related compounds
and even surmounting that of the reference substance
fumitremorgin C. This again supports the hypothesis that
water solubility is the limiting factor with respect to efficacy of
previously reported ABCG2 modulators. Additionally, taking
into consideration the improved stability, the results demon-
strate that the benzanilide core structure is not essential but can
be replaced by a biphenyl moiety. This suggests that
optimization of the drug-like properties is possible according
to bioisosteric concepts to obtain potent and selective ABCG2
modulators for coadministration with cytostatics in orthotopic
brain tumor xenograft models in nude mice.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic methods, analytical data for compounds 19a,b−
21a,b, 22a, 23a, and 28−31, investigations on the stability of 6,
22a, and 30 in mouse plasma, and methods for in vitro assays.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
(8) de Bruin, M.; Miyake, K.; Litman, T.; Robey, R.; Bates, S. E.
Reversal of resistance by GF120918 in cell lines expressing the ABC
half-transporter, MXR. Cancer Lett. 1999, 146 (2), 117−126.
(9) Robey, R. W.; Steadman, K.; Polgar, O.; Morisaki, K.; Blayney,
M.; Mistry, P.; Bates, S. E. Pheophorbide a is a specific probe for
ABCG2 function and inhibition. Cancer Res. 2004, 64 (4), 1242−1246.
(10) Kannan, P.; Telu, S.; Shukla, S.; Ambudkar, S. V.; Pike, V. W.;
Halldin, C.; Gottesman, M. M.; Innis, R. B.; Hall, M. D. The ″specific″
P-glycoprotein inhibitor tariquidar is also a substrate and an inhibitor
for breast cancer resistance protein (BCRP/ABCG2). ACS Chem.
Neurosci. 2011, 2 (2), 82−89.
*E-mail: armin.buschauer@chemie.uni-regensburg.de (A.B.);
burkhard.koenig@chemie.uni-regensburg.de (B.K.).
Present Address
∥
́
(C.O.-P.) Departamento de Quimica, Universidad Nacional de
Bogota
́
Colombia Carrera 45 No 26-85, Edificio Uriel Gutierrez
́
DC, Colombia.
Author Contributions
⊥These authors (C.O.-P. and S.B.) contributed equally to this
work. All authors have given approval to the final version of the
manuscript.
(11) Kuhnle, M.; Egger, M.; Muller, C.; Mahringer, A.; Bernhardt, G.;
̈
̈
Fricker, G.; Konig, B.; Buschauer, A. Potent and selective inhibitors of
̈
breast cancer resistance protein (ABCG2) derived from the p-
glycoprotein (ABCB1) modulator tariquidar. J. Med. Chem. 2009, 52,
1190−1197.
Notes
The authors declare no competing financial interest.
(12) Puentes, C. O.; Hocherl, P.; Kuhnle, M.; Bauer, S.; Burger, K.;
̈
̈
̈
ACKNOWLEDGMENTS
Bernhardt, G.; Buschauer, A.; Konig, B. Solid phase synthesis of
■
̈
tariquidar-related modulators of ABC transporters preferring breast
cancer resistance protein (ABCG2). Bioorg. Med. Chem. Lett. 2011, 21,
3654−3657.
We are grateful to Manuel Bause for the synthesis of compound
25, to Maria Beer-Kron for excellent technical assistance, to Dr.
̈
Susan Bates from the NIH (Bethesda, MD) for the reference
compound 1 (fumitremorgin C), and to Dr. A. H. Schinkel,
Netherlands Cancer Institute (Amsterdam), for kindly
providing 2 (Ko143). C.O.P. thanks the German Academic
Exchange Service (DAAD) for a graduate fellowship.
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̈
Investigation of Nanoparticles, ABCG2 Modulators and Optical Imaging
of Intracerebral Xenografts. Doctoral thesis, University of Regensburg,
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ABBREVIATIONS
■
ABCB1, ATP-binding cassette transporter, subfamily B,
member 1; ABCC1, ATP-binding cassette transporter,
subfamily C, member 1; ABCG2, ATP-binding cassette
transporter, subfamily G, member 2; BCRP, breast cancer
resistance protein (= ABCG2); CNS, central nervous system;
IC50, concentration of inhibitor required to give 50% inhibition
of activity; MDR, multidrug resistance; MRP1, multidrug
resistance associated protein 1 (= ABCC1); p-gp, p-
glycoprotein (= ABCB1); SEM, standard error of the mean
(15) Hocherl, P. New Tariquidar-Like ABCB1 Modulators in Cancer
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Chemotherapy: Preclinical Pharmacokinetic/Pharmacodynamic Investiga-
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