Potent and selective tariquidar bioisosters as potential PET radiotracers for imaging P-gp

Article abstract of DOI:10.1016/j.bmcl.2012.12.084

Compounds 8a-d have been designed as bioisosters of tariquidar for imaging P-gp expression and density by PET. The results displayed that compounds 8b and 8d could be considered potential P-gp/BCRP ligands suitable as ¹¹C and ¹⁸F radiotracers, respectively.

Full text of DOI:10.1016/j.bmcl.2012.12.084

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1370–1374
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent and selective tariquidar bioisosters as potential PET
radiotracers for imaging P-gp
Marialessandra Contino, Laura Zinzi, Maria Grazia Perrone, Marcello Leopoldo, Francesco Berardi,
⇑
Roberto Perrone, Nicola Antonio Colabufo
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari ‘A. Moro’, via Orabona, 4, 70125 Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Compounds 8a–d have been designed as bioisosters of tariquidar for imaging P-gp expression and density
by PET. The results displayed that compounds 8b and 8d could be considered potential P-gp/BCRP ligands
suitable as 11C and 18F radiotracers, respectively.
Received 15 November 2012
Revised 20 December 2012
Accepted 23 December 2012
Available online 5 January 2013
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Arylthiazoles
P-glycoprotein (P-gp)
MRP1
Tariquidar
The Adenosine triphosphate (ATP) Binding Cassette (ABC) trans-
porter P-glycoprotein (P-gp) is expressed in several tissues such as
small intestine, Blood Brain Barrier (BBB), Blood Cerebro Spinal
Fluid Barrier (BCSF), Blood Testis Barrier (BTB), hepatocytes and
kidneys.1 P-gp modulates both xenobiotics absorption and excre-
tion in these barriers and organs.2 Furthermore, P-gp overexpres-
sion is one of the main cause of the failure of chemotherapeutic
treatment as it modulated chemotherapeutic drugs efflux.3 In addi-
tion, since several drugs are P-gp substrates and the efflux pump
saturation could induce an alteration in gastrointestinal tract
absorption and in brain barrier permeation, there is a considerable
interest in the quantification of P-gp expression and function by
non-invasive imaging analysis such as PET.4 In this scenario, an
important goal is the development of P-gp PET radiotracers dis-
playing high affinity and selectivity towards the target.5,6
but it was unsuitable to visualize P-gp expression in murine tumor
model.
In order to potentiate the activity and selectivity of our lead
compounds (MC18, MC113), we developed bioisosters bearing
arylthiazole moiety11 as depicted in Figure 3 and among them
MC90 that displayed, with respect to our lead compounds, super-
imposed results. Comparing MC90 to tariquidar we hypothesized
that the arylthizole fragment could be considered as biosisoster
of 3-carboxamidequinoline (A) (Fig. 1). Since, tetrahydroisoquino-
line moiety is the same (C) and the N-phenylbenzamide fragment
(B) could be considered the pivotal part of the molecule useful to
increase P-gp activity in nanomolar range, we designed a bioequiv-
alent fragment inserting it between A and C of MC90 structure to
obtain a set of compounds belonging to general formula reported
in Figure 4. The substituents on arylthiazole moiety have been se-
lected taking into account the radiolabelling reaction for obtaining
11C- or 18F-PET probes. For this purpose, methoxy derivative (8b)
and the corresponding hydroxy derivative (8a) as precursor for
Among P-gp radiotracers, [11C]tariquidar has been the mostly
studied, as P-gp inhibitor (Fig. 1); on this radiotracer some PET
studies have been performed and recent results demonstrated that
it was dose-dependently transported by P-gp and BCRP pumps.7
In our laboratory, MC18 and MC113 have been designed as P-gp
ligands and their biological evaluation classified them as potent
and selective P-gp inhibitors;8,9 thus, these ligands have been
[
11C]8b and fluoroderivative (8d) and the corresponding nitrode-
rivative (8c) as precursor for [18F]8d have been planned. Com-
pounds 4 and 5 (amide and thioamide, respectively) have been
prepared to demonstrate the role of arylthiazole fragment in these
molecules since the basic moiety (C) was the same while central
nucleus (B) was our hypothesis. All compounds have been tested
in the three known biological assays for determining: (i) P-gp
activity; (ii) ATP-ase activity; (iii) Apparent Permeability (Papp).4
All these results permit to establish the potency and the P-gp
interacting mechanism (substrate or inhibitor or modulator). In
particular, substrates activate ATP-ase whereas inhibitors are
[
11C]-radiolabelled and tested in vivo by microPET analysis
(Fig. 2).6,10 11C]MC18 displayed in vivo and in vitro interesting
[
and consistent results while [11C]MC113 showed high brain uptake
⇑
Corresponding author. Tel.: +39 080 5442727; fax: +39 080 5442231.
E-mail address: nicolaantonio.colabufo@uniba.it (N.A. Colabufo).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.084

M. Contino et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1370–1374
1371
O
*
O
O
O
O
N
N
H
N
O
N
H
A
B
C
Figure 1. [11C]tariquidar.
O
O
O
O
N
N
O
*
O
*
[11C]MC18
[11C]MC113
Figure 2. P-gp radiotracers.
Apparent Permeability so that BA/AB ratio is P2 for substrates
while it is 62 for P-gp inhibitors.
O
O
N
N
The synthesis of arylthiazole derivatives 8a–d, 4 and 5 is de-
picted in Scheme 1. The carboxamide 3 was prepared with the
commercially available 4-hydroxybenzaldehyde (1) and 6-chloro-
nicotinamide (2) in DMF. Compound 4 was obtained by condensing
compound 312 with 6,7-dimethoxy-1,2,3,4-tetrahyidroisoquinoline
in dry CH2Cl2 and the intermediate was reduced in the presence of
NaBH4. The amide function was treated with Lawesson’s reagent in
dry THF to give the corresponding thioamide (5). The crude 5 was
condensed with the appropriate arylbromoketone 7a–d, synthe-
sized starting from appropriate alkyketone 6a–d, leading to aryl-
S
O
MC90
Figure 3. Arylthiazole derivative MC90.
unable to stimulate this site. Furthermore, the Apparent Perme-
ability (Papp) represents the contribution of two different fluxes:
the first, from basolateral to apical is representative for passive
transport, while the flux from apical to basolateral is representa-
tive for active transport. Both fluxes contributed to define the
13
thiazoles 8a–d.
The biological results were listed in Table 1 where reference
compounds MC18, MC113 and MC90 are reported. All arylthiazoles
S
O
N
N
R
N
O
O
A
B
C
Figure 4. General formula of arylthiazole derivatives 8a–d. R = 3-OH, 3-OCH3, 4-NO2, 4-F.

1372
M. Contino et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1370–1374
O
O
O
O
H2N
H
H
NH2
A
+
N
O
HO
Cl
N
3
1
2
B
O
O
O
H2N
N
+
R
N
O
O
6a-d
4
D
C
S
O
O
Br
H2N
N
R
N
O
O
E
5
7a-d
S
O
O
N
N
R
N
O
8a-d
Scheme 1. Reagents and conditions: K2CO3, DMF (A); 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, NaBH4, CH2Cl2 (B); Lawesson’s reagent THF (C); Br2ÁCHCl3 (D); EtOH (E).
8a–d displayed submicromolar P-gp activity ranging from 0.25 to
0.95 M. Moreover, all ligands were inactive towards MRP1 (EC50
>100 M). Surprisingly, compound 8a was more active towards
BCRP (EC50 = 0.018 M) than towards P-gp (EC50 = 0.25 M). Com-
pound 8b displayed superimposed P-gp/BCRP activities whereas
compounds 8c and 8d were moderately more potent towards
P-gp than towards BCRP.
With respect to lead compounds MC18 (P-gp inhibitor), MC113
recently studied as [11C]MC11310 and MC90, thiazole derivatives
8a–d displayed comparable P-gp activity but were inactive to-
wards MRP1 pump. However, other substituents are needed to bet-
ter investigate their role in determining P-gp intrinsic activity.
These preliminary results indicated that compound 8b could be
considered P-gp/BCRP ligand tariquidar-like although less potent
towards P-gp with respect to reference compound. Compound 8d
could be considered potential P-gp/BCRP radiotracer tariquidar-
like even if it was less potent than 8a and tariquidar.7 Since P-gp
density at the human blood-brain barrier is in nanomolar range,14
it is need a PET radiotracer displaying inhibitory activity in the
same range to visualize P-gp expression. By contrast, in order to
measure P-gp activity are sufficient radiotracers, as verapamil
l
l
l
l
The derivatives 4 and 5, lacking arylthiazole nucleus, were
about 100-fold less potent than thiazoles 8a–d confirming that
this moiety plays
Permeability (Papp
a
crucial role in P-gp activity. Apparent
)
and ATP-ase activity for each compound
indicated that compounds 8a–d could be considered as P-gp
transported substrates. The same results have been obtained
for tariquidar.

M. Contino et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1370–1374
1373
Table 1
Biological evaluation of arylthiazole 8a–d, amide (4) and thioamide (5) derivatives
S
O
O
N
N
R
N
O
Compound
R
P-gp
BCRP
EC50 (lM)
MRP1
Apparent Permeability BA/AB
ATP-ase
a
8a
8b
8c
8d
3-OH
3-OCH3
4-NO2
4-F
0.25 0.02
0.95 0.04
0.51 0.03
0.41 0.02
1.50
0.6 0.03
1.94
0.044 0.001
0.018 0.005
0.50 0.02
10 0.5
1.0 0.2
90
>100
>100
>100
>100
2.80
0.9
2.5
2.7
4.5
1.64
2.6
6.1
22
Y (20%)b
Y (20%)
Y (16%)b
Y (18%)b
MC18c
MC113
MC90c
Tariquidar
15
30
3
21
2
2.7
Not tested
0.010 0.005
Y (30%)b
X
O
H2N
N
N
O
O
Compound
X
P-gp
MRP1
Apparent Permeability BA/AB
ATP-ase
EC50 l
Ma
4
5
O
S
19 2.5
11 1.2
>100
>100
NT
NT
NT
NT
a
b
c
The result is the mean of three independent experiments sample in duplicate.
The percentage at 50 M of the effect is in parenthesis.
See Ref. 8 for MC18 and Ref. 11 for MC18 and MC90.
l
6. van Waarde, A.; Ramakrishnan, N. K.; Rybczynska, A. A.; Elsinga, P. H.; Berardi,
F.; de Jong, J. R.; Kwizera, C.; Perrone, R.; Cantore, M.; Sijbesma, J. W.; Dierckx,
R. A.; Colabufo, N. A. J. Med. Chem. 2009, 52, 4524.
7. Wanek, T.; Kuntner, C.; Bankstahl, J. P.; Mairinger, S.; Bankstahl, M.; Stanek, J.;
Sauberer, M.; Filip, T.; Erker, T.; Müller, M.; Löscher, W.; Langer, O. J. Cereb.
Blood Flow Metab. 2002, 2012, 32.
8. Colabufo, N. A.; Berardi, F.; Cantore, M.; Perrone, M. G.; Contino, M.; Inglese, C.;
Niso, M.; Perrone, R.; Azzariti, A.; Simone, G. M.; Porcelli, L.; Paradiso, A. Bioorg.
Med. Chem. Lett. 2008, 16, 362.
9. Colabufo, N. A.; Berardi, F.; Cantore, M.; Perrone, M. G.; Contino, M.; Inglese, C.;
Niso, M.; Perrone, R.; Azzariti, A.; Simone, G. M.; Paradiso, A. Bioorg. Med. Chem.
Lett. 2008, 16, 3732.
and desmethylloperamide, displaying P-gp activity in micromolar
range.
Moreover, these results demonstrated that the B fragment,
present in derivatives 8a–d, increased P-gp activity 10-fold with
respect to MC90, lacking this fragment. However, compounds
8a–d were an order of magnitude less active than tariquidar. In
conclusion, derivatives 8a–d could be considered tariquidar bioiso-
sters where arylthiazole moiety A mimicked the quinoline-3-
carboxamide and the inserted B fragment, present in 8a–d, could
be considered
fragment belonging to tariquidar.
a bioequivalent moiety of the corresponding
10. Mairinger, S.; Wanek, T.; Kuntner, C.; Doenmez, Y.; Strommer, S.; Stanek, J.;
Capparelli, E.; Chiba, P.; Müller, M.; Colabufo, N. A.; Langer, O. Nucl. Med. Biol.
2012, 39, 1219.
11. Colabufo, N. A.; Berardi, F.; Perrone, M. G.; Cantore, M.; Contino, M.; Inglese, C.;
Niso, M.; Perrone, R. ChemMedChem 2009, 4, 188.
12. Mitch, C. H.; Quimby, S. J.; Diaz, N.; Pedregal, C.; de la Torre, M. G.; Jimenez, A.;
Shi, Q.; Canada, E. J.; Kahl, S. D.; Statnick, M. A.; McKinzie, D. L.; Benesh, D. R.;
Rash, K. S.; Barth, V. N. J. Med. Chem. 2011, 54, 8000.
Supplementary data
Supplementary data (the synthesis and characterization of
intermediates depicted in Scheme 1 are reported. Elemental anal-
yses for compounds 4, 5, 8a–d are included. Moreover, the biolog-
ical protocols and the corresponding references) associated with
this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmcl.2012.12.084.
13. Experimental section: 3-(2-(6-(4-((3,4-Dihydro-6,7-dimethoxyisoquinolin-2-
(1H)-yl)methyl)phenoxy)pyridin-3-yl)thiazol-4-yl)phenol (8a). Yellow oil, 60%
yield from column chromatography (CHCl3). ESI+/MS m/z 552 (M++1, 8) 549
(13), 359 (100). 1H NMR
d 2.78–2.84 (m, 4H, NCH2CH2), 3.60 (s, 2H,
OC6H4CH2N), 3.70 (s, 2H, CH2CH2NCH2), 3.82 (s, 3H, OCH3), 3.84 (s, 3H,
OCH3), 6.50–8.80 (m, 15H, aromatic and OH). Anal. (C32H29N3O4SÁ2HCl) C, H, N
(hydrochloride salt, white solid).
2-(4-(5-(4-(3-Methoxyphenyl)thiazol-2-yl)pyridin-2-yloxy)benzyl)-1,2,3,4-
tetrahydro-6,7-dimethoxyisoquinoline (8b). Yellow oil, 65% yield from column
chromatography (eluent CHCl3/MeOH 19:1). ESI+/MS m/z 566 (M++1, 40) 562
References and notes
(82) 551 (100). 1H NMR
d 2.65–2.95 (m, 4H, NCH2CH2), 3.58 (s, 2H,
1. Colabufo, N. A.; Berardi, F.; Contino, M.; Niso, M.; Perrone, R. Curr. Top. Med.
Chem. 2009, 9, 119.
2. Ohtsuki, S.; Terasaki, T. Pharm. Res. 2007, 24, 1745.
OC6H4CH2N), 3.75 (s, 2H, CH2CH2NCH2), 3.82 (s, 3H, OCH3), 3.84 (s, 3H,
OCH3), 3.88 (s, 3H, OCH3), 6.75–8.83 (m, 14H, aromatic). (C33H31N3O4SÁ2HCl) C,
H, N (hydrochloride salt, white solid).
3. Borst, P.; Evers, R.; Kool, N.; Wijnholds, J. J. Nat. Cancer Inst. 2000, 92, 1295.
4. Colabufo, N. A.; Berardi, F.; Perrone, M. G.; Capparelli, E.; Cantore, M.; Inglese,
C.; Perrone, R. Curr. Top. Med. Chem. 2010, 10, 1703.
5. Kannan, P.; John, C.; Zoghbi, S. S.; Halldin, C.; Gottesman, M. M.; Innis, R. B.;
Hall, M. D. Clin. Pharmacol. Ther. 2009, 86, 368.
2-(4-(5-(4-(4-Nitrophenyl)thiazol-2-yl)pyridin-2-yloxy)benzyl)-1,2,3,4-
tetrahydro-6,7-dimethoxyisoquinoline (8c). Yellow oil, 32% yield from column
chromatography (eluent CHCl3 ESI+/MS m/z 581 (M++1, 7) 579 (74) 491 (100).
1H NMR d 2.68–2.84 (m, 4H, NCH2CH2), 3.60 (s, 2H, OC6H4CH2N), 3.70 (s, 2H,
CH2CH2NCH2), 3.82 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.40–8.83 (m, 14H,

1374
M. Contino et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1370–1374
OC6H4CH2N), 3.70 (s, 2H, CH2CH2NCH2), 3.82 (s, 3H, OCH3), 3.84 (s, 3H,
aromatic). Anal. (C32H28N4O5SÁ2HClÁH2O) C, H, N (hydrochloride salt, white
solid).
OCH3), 6.51-8.80 (m, 14H, aromatic). Anal. (C32H28FN3O3SÁ2HCl) C, H,
N
2-(4-(5-(4-(4-Fluorophenyl)thiazol-2-yl)pyridin-2-yloxy)benzyl)-1,2,3,4-tetra-
hydro-6,7-dimethoxyisoquinoline (8d). Yellow oil, 20% yield from column
chromatography (eluent CHCl3/MeOH 19:1). ESI+/MS m/z 554 (M++1, 8) 390
(hydrochloride salt, white solid).
14. Uchida, Y.; Ohtsuki, S.; Katsukura, Y.; Ikeda, C.; Suzuki, T.; Kamiie, J.; Terasaki,
T. J. Neurochem. 2011, 117, 333.
(39) 539 (100). 1H NMR
d 2.75–2.84 (m, 4H, NCH2CH2), 3.58 (s, 2H,