Naphthalenyl derivatives for hitting P-gp/MRP1/BCRP transporters

Article abstract of DOI:10.1016/j.bmc.2012.12.021

Substituted naphthalenyl derivatives bearing oxazole, or thiazole or furyl heteronuclei have been carried out as bioisosters of aryl-oxazoles and -thiazoles derivatives previously reported in order to investigate the role of the hindrance on the activity towards P-gp/BCRP/and MRP1 transporters. In addition, the role of naphthalenyl group to modulate P-gp intrinsic activity of these compounds was ascertained. The results demonstrated that all naphthalenyl derivatives displayed comparable P-gp activity with respect to lead compounds previously characterized in our SAR studies but were less active towards BCRP and MRP1 pumps. In terms of intrinsic activity, the replacement of aryl with naphthalenyl moiety led to P-gp inhibitors, unambiguous or ambiguous substrates on the base of the heteronucleus and the substituent on the naphthalenyl fragment. Indeed, oxazole derivatives were: inhibitors (R = H, F, OH), unambiguous substrates (R = OCH₃), or ambiguous substrate (R = Br); thiazole derivatives were: unambiguous substrates (R = OCH₃, Br), or ambiguous substrates (R = H, F). Finally furyl derivatives were ambiguous substrates.

Full text of DOI:10.1016/j.bmc.2012.12.021

Bioorganic & Medicinal Chemistry 21 (2013) 1324–1332
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Naphthalenyl derivatives for hitting P-gp/MRP1/BCRP transporters
a
Nicola A. Colabufo a, , Marialessandra Contino , Mariangela Cantore a,b, Elena Capparelli a,
⇑
Maria Grazia Perrone a, Giuseppe Cassano c, Giuseppe Gasparre c, Marcello Leopoldo a, Francesco Berardi a,
Roberto Perrone a
a Dipartimento Farmacia, Università degli Studi di Bari ALDO MORO, Via Orabona 4, 70125 Bari, Italy
b Clinical Experimental Oncology Laboratory, National Cancer Institute Giovanni Paolo II, Viale O. Flacco, 65, 70124 Bari, Italy
c Dipartimento di Bioscienze, Biotecnologie e Scienze Farmacologiche, Università di Bari, Università degli Studi di Bari ALDO MORO, Via Orabona 4, I-70125 Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Substituted naphthalenyl derivatives bearing oxazole, or thiazole or furyl heteronuclei have been carried
out as bioisosters of aryl-oxazoles and -thiazoles derivatives previously reported in order to investigate
the role of the hindrance on the activity towards P-gp/BCRP/and MRP1 transporters. In addition, the role
of naphthalenyl group to modulate P-gp intrinsic activity of these compounds was ascertained.
The results demonstrated that all naphthalenyl derivatives displayed comparable P-gp activity with
respect to lead compounds previously characterized in our SAR studies but were less active towards BCRP
and MRP1 pumps. In terms of intrinsic activity, the replacement of aryl with naphthalenyl moiety led to
P-gp inhibitors, unambiguous or ambiguous substrates on the base of the heteronucleus and the substi-
tuent on the naphthalenyl fragment. Indeed, oxazole derivatives were: inhibitors (R = H, F, OH), unambig-
uous substrates (R = OCH3), or ambiguous substrate (R = Br); thiazole derivatives were: unambiguous
substrates (R = OCH3, Br), or ambiguous substrates (R = H, F). Finally furyl derivatives were ambiguous
substrates.
Received 15 October 2012
Revised 7 December 2012
Accepted 13 December 2012
Available online 22 December 2012
Keywords:
P-glycoprotein
Naphthalenyl derivatives
BCRP
MRP1
MDR revertant agents
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
The most potent reported P-gp inhibitors, tariquidar and elacr-
idar (Fig. 1), entered clinical studies but recent PET studies employ-
MultiDrug Resistance (MDR) is a complex phenomenon mainly
due to the overexpression of some transmembrane proteins belong-
ing to the ABC (ATP Binding Cassette) transporter family.1 ABC trans-
porters use the energy from the ATP hydrolysis to efflux xenobiotics
out of cells exerting a physiological role of defense in several biolog-
ical barriers such as Blood Brain Barrier (BBB) and organs involved in
the absorption and elimination.2 The overexpression of ABC proteins
in tumour cells led to MDR that is the major cause of the failure of
antineoplastic treatment since ABC transporters are responsible
for the efflux of chemotherapeutic agents out of tumour cells.1–4
Among these transporters, P-glycoprotein (P-gp), Breast Cancer
Resistance Protein (BCRP) and MultiDrug Resistance-associated
Protein1 (MRP1) are the mostly involved in MDR.2–5 One of the
strategy approached to overcome MDR in cancer therapy is the
use of a P-gp inhibitor in co-administration with the classic anti-
neoplastic drugs to restore drug sensitivity of tumour cells.6–12
ing the corresponding 11C-radiolabelled ligand demonstrated that
both inhibitors displayed high activity towards the target (P-gp)
as well as moderate activity towards BCRP.13–17
Recently, we developed a potent P-gp inhibitor, MC18, and a P-gp
substrate, MC266 (Fig. 2), that have been [11C] radiolabeled and
tested in in vivo PET studies18–22 leading to significant and coherent
results in comparison with the data previously reported in vitro.23
Starting from these lead compounds, we developed biphenyl
derivatives obtaining P-gp potent inhibitors although displaying
moderate activity towards BCRP and MRP1.24 For improving the
P-gp selectivity of these ligands, a set of aryloxazole and arylthiaz-
ole derivatives have been developed.25 In these ligands aryloxazole
or arylthiazole fragment decreased P-gp activity with respect to
lead compound MC18, whereas showed an increase in BCRP and
MRP1 activities. In addition, the presence and/or the position of
different substituents on the aryl fragment modulated both the
interaction versus BCRP or MRP1 and the intrinsic P-gp activity.
In fact all aryloxazoles and arylthiazoles were found to be P-gp
substrates whereas MC18 and MC70 were P-gp inhibitors.
Abbreviations: MDR, MultiDrug Resistance; ABC, ATP Binding Cassette; P-gp, P-
glycoprotein; BCRP, Breast Cancer Resistance Protein; MRP1, MultiDrug Resistance-
associated Protein1; Papp, apparent permeability; Calcein-AM, Acetoxymethyl esters
of calcein; MDCK, Madin-Darby Canine Kidney; FTC, Fumitremorgin C.
Considering that the aryl fragment and the presence and posi-
tion of substituent play an important role on the activity towards
P-gp, BCRP, MRP1, and it address the P-gp intrinsic activity of
⇑
Corresponding author. Tel.: +39 080 5442747; fax: +39 080 5442231.
E-mail address: nicolaantonio.colabufo@uniba.it (N.A. Colabufo).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.021

N. A. Colabufo et al. / Bioorg. Med. Chem. 21 (2013) 1324–1332
1325
Agilent 1100 LC/MSD trap system VL. Purity of final compounds
was established by combustion analysis of the corresponding
hydrochloride salts, confirming a purity >95%.
2.1. General procedure for the synthesis of 5-(4-aryl)furan-2-
carboxylic acids (3a,b)
A solution of arylboronic acid 1a,b (1 mmol), 5-bromo-2-fura-
noic acid (1 mmol) and K2CO3 (1 mmol) in EtOH (20 mL) was stir-
red under N2 for 30 min. Pd(PPh3)4 (0.1 mmol) was added and the
mixture was refluxed for 4–6 h. After cooling to room temperature,
the solvent was evaporated and the crude was dissolved in H2O
(50 mL). Aqueous solution was acidified with HCl 2 N and ex-
tracted with AcOEt (3 ꢀ 50 mL). Organic phases were washed with
NaHCO3 s.s. then acidified with HCl 3 N. Insoluble carboxylic acids
3a,b were separated and dried.
2.1.1. 5-Naphthalen-2-yl-furan-2-carboxylic acid (3a)
Yield: 28%. White solid. ESIꢁ/MS m/z: 237 [MꢁH]ꢁ. ESIꢁ/MS/MS
m/z: 193 (100). 1H NMR (CDCl3) d: 6.30–6.66 (m, 1H, furyl), 7.20–
7.89 (m, 8H, aromatic). 11.00 (br s, 1H, OH).
Figure 1. Elacridar and tariquidar structures.
2.1.2. 5-(6-Methoxynaphtalen-2-yl)-furan-2-carboxylic acid
(3b)
Yield: 30%. White solid. ESIꢁ/MS m/z: 267 [MꢁH]ꢁ. ESIꢁ/MS/MS
m/z: 223 (100). 1H NMR (CDCl3) d: 3.73 (s, 3H, CH3), 6.30–6.66 (m,
1H, furyl), 7.02–7.86 (m, 7H, aromatic). 11.00 (br s, 1H, OH).
2.2. General procedure for the synthesis of (6,7-dimethoxy-3,4-
dihydro-1H-isoquinolin-2-yl)-[5-(4-aryl)-2-furyl]methanones
(4a,b)
A solution of carboxylic acid 3a,b (1 mmol) and CDI (1.1 mmol)
in dry THF was stirred at room temperature overnight. A solution
of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (1 mmol) in THF
was added at the mixture and the solution was stirred at room
temperature for 6 h. The solvent was evaporated and the residue
was dissolved in H2O (50 mL). The aqueous phase was extracted
with AcOEt (3 ꢀ 50 mL). The collected organic solution was dried
over Na2SO4 and evaporated. The residue was purified on silica
gel column chromatography in specific condition below reported
for each compound.
Figure 2. MC18 and MC266 structures.
2.2.1. (6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-(5-
naphthalen-2-yl-furan-2-yl)-methanone (4a)
Yield: 83%. White solid. Eluted with CHCl3. ESI+/MS m/z: 436
[M+Na]+. ESI+/MS/MS m/z: 379 (73), 223 (43). 1H NMR (CDCl3) d:
2.95–3.05 (m, 2H, NCH2CH2), 3.86 (s, 3H, CH3), 3.88 (s, 3H, CH3),
4.02–4.12 (m, 2H, NCH2CH2), 4.85–4.98 (m, 2H, NCH2), 6.65–8.20
(m, 11H, aromatic). Mp: 121–123 °C.
ligands, in the present work we aimed to: (1) study the role of the
hindrance on the activity towards P-gp/BCRP/ and MRP1 replacing
the aryl group with substituted naphtalenyl fragments; (2) evalu-
ate if the naphthalenyl fragment modulated P-gp intrinsic activity.
For these purposes three heteronuclei: (i) oxazole; (ii) thiazole; (iii)
furyl have been evaluated (Fig. 3).
2.2.2. (6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-[5-(6-
methoxynaphthalen-2-yl)-furan-2-yl]-methanone (4b)
Yield: 76%. White solid. Eluted with CHCl3. The solid was crys-
tallized from CHCl3/petroleum ether. ESI+/MS m/z: 466 [M+Na]+.
ESI+/MS/MS m/z: 451 (7). 1H NMR (CDCl3) d: 2.89–2.99 (m, 2H,
NCH2CH2), 3.86 (s, 3H, CH3), 3.88 (s, 3H, CH3), 3.94 (s, 3H, CH3),
4.01–4.07 (m, 2H, NCH2CH2), 4.90–5.15 (m, 2H, NCH2), 6.49–8.12
(m, 10H, aromatic). Mp: 121–123 °C.
2. Materials and methods
Column chromathography was performed with 1:30 Merck sil-
ica gel 60 Å (63–200 lm) as the stationary phase. Melting points
were determined in open capillaries on a Gallenkamp electrother-
mal apparatus. 1H NMR spectra were recorded in CDCl3 or DMSO-
d6 at 300 MHz on a Varian Mercury-VX spectrometer. All spectra
were recorded on the free bases. All chemical shift values are re-
ported in ppm (d). Recording of mass spectra was done on an
HP6890-5973 MSD gas chromatograph/mass spectrometer; only
significant m/z peaks, with their percentage of relative intensity
in parentheses, are reported. All spectra were in accordance with
the assigned structures. ESI-MS analyses were performed on an
2.3. General procedure for the synthesis of 6,7-dimethoxy-2-[[5-
(4-aryl)-2-furyl]methyl]-3,4-dihydro-1H-isoquinolines (5a,5b)
A solution of the amide 4a,b (1 mmol, 10 mL dry THF) was
added dropwise to a suspension of LiAlH4 (2 mmol) in dry THF
(5 mL). The reaction was refluxed for 3 h. After cooling at room

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Figure 3. Development of P-gp ligands.
temperature, water (10 mL) was added. The aqueous phase was ex-
tracted with AcOEt (3 ꢀ 50 mL) and the collected solution was
dried over Na2SO4 and evaporated. The residue was purified on sil-
ica gel column chromatography in the condition below reported
for each compound.
(45 mL) of NH4OH (28%), H2O and CH2Cl2 (1:1:1). The mixture
was stirred at room temperature for 4 h. Insoluble amides 7a–d
were separated by filtration while the amide 7e was obtained
removing the solvent under vacuo. The crude was washed with
H2O (20 mL) and extracted with AcOEt (3 ꢀ 20 mL).
2.3.1. 6,7-Dimethoxy-2-(5-naphthalen-(2-yl-furan-2-yl-
methyl)-1,2,3,4-tetrahydro-1H-isoquinoline (5a)
2.4.1. 2-Naphthamide (7a)
Yield: 87%. White solid. GC–MS m/z: 171(M+, 86), 155 (100), 127
(97); 1H NMR (CDCl3) d: 6.00 (s, br, 2H, NH2 exchange with D2O),
7.32–8.36 (m, 7H, aromatic). Mp: 195–197 °C.
Yield: 67%. White solid. Eluted with CHCl3. Hydrochloride salt
crystallized from MeOH/Et2O. ESI+/MS m/z: 422 [M+Na]+. ESI+/
MS/MS m/z: 407 (100); 365 (78). 1H NMR (CDCl3) d: 2.80–2.95
(m, 4H, NCH2CH2), 3.68 (s, 2H, NCH2), 3.81 (s, 3H, CH3), 3.83 (s,
3H, CH3), 3.85 (s, 2H, furylNCH2), 6.40–8.15 (m, 11, aromatic).
Mp: 223–225 °C.
2.4.2. 6-Fluoronaphthalene-2-carboxamide (7b)
Quantitative yield. White solid. GC–MS m/z: 189 (M+, 77), 173
(100), 145 (86); 1H NMR (CDCl3) d: 6.05 (s, br, 2H, NH2, exchange
with D2O), 7.30–8.35 (m, 6H, aromatic). Mp: 211–214 °C.
2.3.2. 6,7-Dimethoxy-2-[5-(6-methoxynaphtalen-2-yl)-furan-2-
yl-methyl]-1,2,3,4-tetrahydro-1H-isoquinoline (5b)
2.4.3. 6-Bromonaphthalene-2-carboxamide (7c)
Yield: 33%. White solid. Eluted with CHCl3. Hydrochloride salt
crystallized from MeOH/Et2O. ESI+/MS m/z: 452 [M+Na]+. ESI+/
MS/MS m/z: 437 (100); 216 (49). 1H NMR (CDCl3) d: 2.80–2.94
(m, 4H, NCH2CH2), 3.63 (s, 2H, NCH2), 3.77 (s, 3H, CH3), 3.80 (s,
3H, CH3), 3.82 (s, 3H, CH3), 3.91 (s, 2H, furylNCH2), 6.30–7.72 (m,
10, aromatic). Mp: 234–237 °C.
Yield: 67%. White solid. GC–MS m/z: 252 (M+2, 6), 250 (M+, 6),
126 (100); 1H NMR (CDCl3) d: 6.03 (s, br, 2H, NH2, exchange with
D2O), 7.46–8.29 (m, 6H, aromatic). Mp: 223–224 °C.
2.4.4. 6-Methoxynaphthalene-2-carboxamide (7d)
Yield: 90%. White solid. GC–MS m/z: 201 (M+, 94), 185 (100),
157 (44). 1H NMR (CDCl3) d: 3.74 (s, 3H, CH3), 6.04 (s, br, 2H,
NH2), 7.04–8.32 (m, 6H, aromatic). Mp: 225–228 °C.
2.4. General procedure for the synthesis of naphthalene-2-
carboxamides (7a–e)
2.4.5. 6-Hydroxynaphthalene-2-carboxamide (7e)
Amides 7a–e were prepared from the corresponding commer-
cially available carboxylic acids (1 mmol) 6a–e treated with an ex-
cess of SOCl2 (7 mmol) in presence of Et3N (0.1 mmol) under reflux
for 1 h. The corresponding acylchloride was added to a mixture
Yield: 28%. Yellow solid. Eluted with CHCl3/AcOEt (7:3). GC–MS
m/z: 187 (M+, 86), 171 (100), 143 (45); 1H NMR (CDCl3) d: 6.63 (s,
br, 2H, NH2, exchange with D2O), 7.03–8.35 (m, 6H, aromatic), 9.52
(s, br, 1H, OH, exchange with D2O). Mp: 186–190 °C.

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1327
2.5. General procedure for the synthesis of naphthalene-2-
carbothioamide (8a–d)
179 (100). 1H NMR (CDCl3) d: 4.64 (s, 2H, CH2), 7.28–8.45 (m, 7H,
aromatic). Mp: 115–116 °C.
Thioamides 8a–d were prepared from the corresponding amides
7a–d (3 mmol) in the presence of Lawesson’s reagent (3.4 mmol) in
dry THF (25 mL) under reflux for 3 h. After evaporation of solvent,
the crude was partitioned between H2O (20 mL) and CH2Cl2
(20 mL) and the aqueous phase was extracted with CH2Cl2
(3 ꢀ 50 mL). The collected solutions were dried over Na2SO4 and
evaporated. The residue was purified on silica gel column chroma-
tography in specific condition below reported for each compound.
2.6.4. 4-(Chloromethyl)-2-(2-methoxynaphthalen-6-yl)oxazole
(9d)
Yield: 26%. Yellow solid. Eluted with petroleum ether/AcOEt
(8:2). GC–MS m/z: 275 (M++2, 20), 273 (M+, 59), 169 (100). 1H
NMR (CDCl3) d: 3.73 (s, 3H, CH3), 4.64 (s, 2H, CH2), 7.28–8.45 (m,
7H, aromatic). Mp: 111–112 °C.
2.6.5. 6-(4-(Chloromethyl)oxazol-2-yl)naphthalen-2-ol (9e)
Yield 47%. Yellow solid. Eluted with CHCl3. GC–MS m/z: 261
(M++2, 23), 259 (M+, 72), 169 (100). 1H NMR (CDCl3) d: 4.53 (s,
2H, CH2), 7.06–8.33 (m, 7H, aromatic), 9.30 (s, br, 1H, OH, exchange
with D2O).
2.5.1. Naphthalene-2-carbothioamide (8a)
Yield: 64%. Yellow solid. Eluted with CHCl3. ESI+/MS m/z: 188
[M+H]+; ESI+/MS/MS m/z: 171 (100), 129 (28). 1H NMR (CDCl3) d:
2.04 (s br, 2H, NH2), 7.30–7.70 (m, 7H, aromatic). Mp: 153–156 °C.
2.6.6. 4-(Chloromethyl)-2-(naphthalen-6-yl)thiazole (10a)
Quantitative yield. White solid. Eluted with CHCl3. GC–MS m/z:
261 (M++2, 39), 259 (M+, 100). 1H NMR (CDCl3) d: 4.69 (s, 2H, CH2),
7.00–7.90 (m, 8H, aromatic). Mp: 107–109 °C.
2.5.2. 6-Fluoronaphthalene-2-carbothioamide (8b)
Yield: 78%. Yellow solid. Eluted with CH2Cl2. ESI+/MS m/z: 206
[M+H]+; ESI+/MS/MS m/z: 189 (100), 147 (14). 1H NMR (CDCl3) d:
2.04 (s br, 2H, NH2), 7.00–7.70 (m, 6H, aromatic). Mp: 164–166 °C.
2.6.7. 4-(Chloromethyl)-2-(2-fluoronaphthalen-6-yl)thiazole
(10b)
2.5.3. 6-Bromonaphthalene-2-carbothioamide (8c)
Quantitative yield. Yellow solid. Eluted with CHCl3/AcOEt (8:2).
GC–MS m/z: 267 (M++2, 100), 265 (M+, 95), 232 (77). 1H NMR
(CDCl3) d: 2.04 (s, br, 2H, NH2), 7.40–7.90 (m, 6H, aromatic). Mp:
158–160 °C.
Quantitative yield. White solid. Eluted with CHCl3. ESI+/MS m/z:
278 [M+H]+; ESI+/MS/MS m/z: 242 (100). 1H NMR (CDCl3) d: 4.70 (s,
2H, CH2), 7.03–8.12 (m, 7H, aromatic). Mp: 129–132 °C.
2.6.8. 2-(2-Bromonaphthalen-6-yl)-4-(chloromethyl)thiazole
(10c)
Yield: 61%. White solid. Eluted with CHCl3. GC–MS m/z: 341
(M++4, 36), 339 (M++2, 100), 337 (M+, 77). 1H NMR (CDCl3) d:
4.80 (s, 2H, CH2), 7.36–8.40 (m, 7H, aromatic). Mp: 103–105 °C.
2.5.4. 6-Methoxynaphthalene-2-carbothioamide (8d)
Yield: 59%. Yellow solid. Eluted with CHCl3/AcOEt (9:1). ESI+/MS
m/z: 218 [M+H]+, ESI+/MS/MS m/z: 201 (100), 159 (39); 1H NMR
(CDCl3) d: 3.94 (s, 3H, CH3), 7.13–8.34 (m, 6H, aromatic), 7.45 (s
br, 2H, NH2, exchange with D2O).
2.6.9. 4-(Chloromethyl)-2-(2-methoxynaphthalen-6-yl)thiazole
(10d)
2.6. General procedure for the synthesis of 4-(chloromethyl)-2-
(naphthalen-6-yl)oxazoles (9a–e) and of 4-(chloromethyl)-2-
(naphthalen-6-yl)thiazoles (10a–d)
Yield: 87%. White solid. Eluted with CHCl3. ESI+/MS m/z: 290
[M+H]+; ESI+/MS/MS m/z: 254 (100), 256 (6). 1H NMR (CDCl3) d:
3.82 (s, 3H, CH3), 4.80 (s, 2H, CH2), 7.36–8.40 (m, 7H, aromatic).
Mp: 109–111 °C.
Intermediates aryloxazoles 9a–e were prepared condensing the
corresponding amides 7a–e with 1,3-dichloroacetone (2 mmol)
heating at 200 °C for 5 h. Arylthiazoles 10a–d were prepared con-
densing 8a–d (1 mmol) with 1,3-dichloroacetone (2 mmol) in EtOH
(25 mL). After cooling at room temperature, water (10 mL) and
AcOEt (10 mL) were added. The aqueous phase was extracted with
AcOEt (3 ꢀ 50 mL) and the combined solution was dried over
Na2SO4 and evaporated. The residue was purified on silica gel col-
umn chromatography in specific condition below reported for each
compound.
2.7. General procedure for the synthesis of amines 1,2,3,4-
tetrahydro-6,7-dimethoxy-2-((2-(naphthalen-6-yl)oxazol-4-yl)
methyl)isoquinolines (11a–e) and of 1,2,3,4-tetrahydro-6,7-
dimethoxy-2-((2-(naphthalen-6-yl)thiazol-4-yl)methyl)
isoquinoline (12a–d)
Compounds 11a–e and 12a–d were prepared alkilating 6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline (1.2 mmol) in the pres-
ence of intermediates 9a–e or 10a–d (1 mmol) in DMF (20 mL)
and in presence of Na2CO3 (2 mmol). The mixture was refluxed
overnight. The DMF was evaporated and the residue was parti-
tioned between H2O (20 mL) and CHCl3 (20 mL). The organic phase
was separated and the aqueous phase was extracted with CHCl3
(3 ꢀ 50 mL) and the collected solutions were dried over Na2SO4
and evaporated. The residue was purified on silica gel column
chromatography in specific condition below reported for each
compound.
2.6.1. 4-(Chloromethyl)-2-(naphthalen-6-yl)oxazole (9a)
Yield: 77%. Yellow solid. Eluted with petroleum ether/AcOEt
(8:2). GC–MS m/z: 245 (M++2, 9), 243 (M+, 32), 139 (100). 1H
NMR (CDCl3) d: 4.64 (s, 2H, CH2), 7.32–7.89 (m, 8H, aromatic).
Mp: 109–111 °C.
2.6.2. 4-(Chloromethyl)-2-(2-fluoronaphthalen-6-yl)oxazole
(9b)
Yield: 54%. White solid. Eluted with petroleum ether/AcOEt
(9:1). GC–MS m/z: 263 (M++2, 18), 261 (M+, 50), 171 (100). 1H
NMR (CDCl3) d: 4.62 (s, 2H, CH2), 7.28–8.54 (m, 7H, aromatic).
Mp: 109–111 °C.
2.7.1. 1,2,3,4-Tetrahydro-6,7-dimethoxy-2-((2-(naphthalen-6-
yl)oxazol-4-yl)methyl)isoquinoline (11a)
Yield: 64%. Orange solid. Hydrochloride salt recrystallized from
MeOH/Et2O. Eluted with CHCl3. ESI+/MS m/z: 400 [M+H]+; ESI+/MS/
MS m/z: 208 (56), 153 (100). 1H NMR (CDCl3) d: 2.94 (s, 4H,
CH2NCH2), 3.75–3.79 (m, 4H, NCH2CH2), 3.82 (s, 3H, CH3), 3.88 (s,
3H, CH3), 6.52–8.57 (m, 10H, aromatic). Mp: 232–235 °C.
2.6.3. 2-(2-Bromonaphthalen-6-yl)-4-(chloromethyl)oxazole
(9c)
Yield: 56%. Yellow solid. Eluted with petroleum ether/AcOEt
(8:2). GC–MS m/z: 325 (M++4, 13), 323 (M++2, 47), 321 (M+, 33),

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2.7.2. 2-((2-(2-Fluoronaphthalen-6-yl)oxazol-4-yl)methyl)-
1,2,3,4-tetrahydro-6,7-dimethoxyisoquinoline (11b)
3.84 (s, 3H, CH3), 3.93 (s, 3H, CH3), 6.52–8.37 (m, 9H, aromatic).
Mp: 250 °C (dec)
Yield: 77%. White solid. Hydrochloride salt crystallized from
MeOH/Et2O. Eluted with CHCl3/MeOH (19:1). GC–MS m/z: 418
(M+, 1), 192 (100), 171 (18). 1H NMR (CDCl3) d: 2.87 (s, 4H,
CH2NCH2), 3.70–3.75 (m, 4H, NCH2CH2), 3.81 (s, 3H, CH3), 3.84 (s,
3H, CH3), 6.51–8.56 (m, 9H, aromatic). Mp: 231–234 °C.
2.8. Biology
2.8.1. Materials
Cell culture reagents were purchased from Celbio s.r.l. (Milano,
Italy). CulturePlate 96/wells plates were purchased from Perkin-
Elmer Life Science; Calcein-AM, MTT (3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyltetrazoliumbromide) were obtained from
Sigma–Aldrich (Milan, Italy).
2.7.3. 2-((2-(2-Bromonaphthalen-6-yl)oxazol-4-yl)methyl)-
1,2,3,4-tetrahydro-6,7-dimethoxyisoquinoline (11c)
Yield: 89%. Brown oil. Hydrochloride salt crystallized from
MeOH/Et2O. Eluted with CHCl3/MeOH (19:1). GC–MS m/z: 479
(M+, 1), 192 (100). 1H NMR (CDCl3) d: 2.87 (s, 4H, CH2NCH2),
3.70–3.75 (m, 4H, NCH2CH2), 3.81 (s, 3H, CH3), 3.85 (s, 3H, CH3),
6.45–8.56 (m, 9H, aromatic). Mp: 235–238 °C.
2.8.2. Cell cultures
MDCK-MDR1, MDCK-BCRP and MDCK-MRP1 cell lines are a gift
of Professor P. Borst, NKI-AVL Institute, Amsterdam, Netherland.
All the MDCKcells were grownin DMEMhigh glucose supplemented
with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin,
2.7.4. 1,2,3,4-Tetrahydro-6,7-dimethoxy-2-((2-(2-
methoxynaphthalen-6-yl)oxazol-4-yl)methyl)isoquinoline
(11d)
Yield: 57%. Yellow solid. Hydrochloride salt recrystallized from
MeOH/Et2O. Eluted with CHCl3. GC–MS m/z: 430 (M+, 0.70), 192
(100). 1H NMR (CDCl3) d: 2.81 (s, 4H, CH2NCH2), 3.70–3.78 (m,
4H, NCH2CH2), 3.81 (s, 3H, CH3), 3.85 (s, 3H, CH3) 3.93 (s, 3H,
CH3), 6.51–8.48 (m, 9H, aromatic). Mp: 223–225 °C (dec).
100 lg/mL streptomycin, in a humidified incubator at 37 °C with a
5% CO2 atmosphere. Caco-2 cells were a gift of Dr. Aldo Cavallini
and Dr. Caterina Messa from the Laboratory of Biochemistry,
National Institute for Digestive Diseases, ‘S. de Bellis’, Bari (Italy).
2.8.3. Calcein-AM experiment
These experiments were carried out as described by Feng et al.
with minor modifications.26 Each cell line (50,000 cells per well)
was seeded into black CulturePlate 96/wells plate with 100
lL med-
2.7.5. 6-(4-((3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1H)-
yl)methyl)oxazol-2-yl)naphthalen-2-ol (11e)
ium and allowed to become confluent overnight. 100 L of test com-
l
pounds were solubilized in culture medium and added to
monolayers. 96/Wells plate was incubated at 37 °C for 30 min.
Yield: 50%. Brown oil. Hydrochloride salt recrystallized from
MeOH/Et2O. Eluted with CHCl3/MeOH (19:1). ESI+/MS m/z 417
[M+H]+; ESI+/MS/MS m/z: 224 (97), 169 (100). 1H NMR (CDCl3) d:
2.81 (s, 4H, CH2NCH2), 3.25–3.35 (m, 4H, NCH2CH2), 3.39 (s, 3H,
CH3), 3.45 (s, 3H, CH3), 6.15–8.01 (m, 9H, aromatic), 9.4 (s, br s,
1H, exchange with D2O, OH). Mp: 249–252 °C (dec).
Calcein-AM was added in 100
lL of Phosphate Buffered Saline
(PBS) to yield a final concentration of 2.5
lM and plate was incu-
bated for 30 min. Each well was washed 3 times with ice cold PBS.
Saline buffer was added to each well and the plate was read to Victor
3 (PerkinElmer) at excitation and emission wavelengths of 485 and
535 nm, respectively. In these experimental conditions Calcein cell
accumulation in the absence and in the presence of tested com-
pounds was evaluated and fluorescence basal level was estimated
by untreated cells. In treated wells the increase of fluorescence with
respect to basal level was measured. EC50 values were determined
by fitting the fluorescence increase percentage versus log[dose].
2.7.6. 1,2,3,4-Tetrahydro-6,7-dimethoxy-2-((2-(naphthalen-6-
yl)thiazol-4-yl)methyl)isoquinoline (12a)
Yield: 38%. Brown solid. Eluted with CHCl3. Hydrochloride salt,
crystallized from MeOH/Et2O. ESI+/MS m/z: 417 [M+H]+. ESI+/MS/
MS m/z: 400 (47), 224 (100). 1H NMR (CDCl3) d: 2.88 (s, 4H,
CH2NCH2), 3.75–3.98 (m, 4H, NCH2CH2), 3.82 (s, 3H, CH3), 3.84 (s,
3H, CH3), 6.52–8.46 (m, 10H, aromatic). Mp: 243–246 °C.
2.8.4. Antiproliferative assay
Determination of cell growth was performed using the MTT assay
at 24 and 48 h. On day 1, 30,000 cells/well were seeded into 96-well
2.7.7. 2-((2-(2-Fluoronaphthalen-6-yl)thiazol-4-yl)methyl)-
1,2,3,4-tetrahydro-6,7-dimethoxyisoquinoline (12b)
Yield 50%. Brown oil. Hydrochloride salt, crystallized from
MeOH. Eluted with CH2Cl2/AcOEt (8:2). ESI+/MS m/z: 435 [M+H]+;
ESI+/MS/MS m/z: 418 (59), 242 (100), 192 (88). 1H NMR (CDCl3)
d: 2.88 (s, 4H, CH2NCH2), 3.72–3.96 (m, 4H, NCH2CH2), 3.82 (s,
3H, CH3), 3.84 (s, 3H, CH3), 6.52–8.44 (m, 9H, aromatic). Mp:
248–250 °C.
plates in a volume of 100
lL. On day 2, the various drugs concentra-
tion (0.1–100 M) were added. In all the experiments, the various
l
drug-solvents (ethanol, DMSO) were added in each control to evalu-
ate a possible solvent cytotoxicity. After the established incubation
time with drugs, MTT (0.5 mg/mL) was added to each well, and after
3 h incubation at 37 °C, the supernatant was removed. The formazan
crystals were solubilized using 100 lL of DMSO and the absorbance
values at 570 and 630 nm were determined on the microplate read-
er Victor 3 from PerkinElmer Life Sciences.
2.7.8. 2-((2-(2-Bromonaphthalen-6-yl)thiazol-4-yl)methyl)-
1,2,3,4-tetrahydro-6,7-dimethoxyisoquinoline (12c)
Yield: 30%. Brown oil. Hydrochloride salt, crystallized from
MeOH/Et2O. Eluted with CHCl3. ESI+/MS m/z: 495 [M+H]+; ESI+/
MS/MS m/z 304 (84), 192 (100). 1H NMR (CDCl3) d: 2.88 (s, 4H,
CH2NCH2), 3.72–3.96 (m, 4H, NCH2CH2), 3.82 (s, 3H, CH3), 3.88 (s,
3H, CH3), 6.51–8.42 (m, 9H, aromatic). Mp: 221–224 °C.
2.8.5. ATPlite assay
The MDCK-MDR1 cells were seeded into 96-well microplate in
100
l
L of complete medium at a density 2 ꢀ 104 cells/well.27 The
plate was incubated overnight in a humidified atmosphere 5% CO2
at 37 °C. The medium was removed and 100 lL of complete medium
2.7.9. 1,2,3,4-Tetrahydro-6,7-dimethoxy-2-((2-(2-
methoxynaphthalen-6-yl)thiazol-4-yl)methyl)isoquinoline
(12d)
Yield: 55%. Brown oil. Eluted with CHCl3. Hydrochloride salt,
crystallized from MeOH/Et2O. ESI+/MS m/z: 447 [M+H]+. ESI+/MS/
MS m/z: 430 (33), 254 (100), 192 (46). 1H NMR (CDCl3) d: 2.88 (s,
4H, CH2NCH2), 3.75–3.95 (m, 4H, NCH2CH2), 3.81 (s, 3H, CH3),
in the presence or absence of different concentrations of test com-
pounds was added. The plate was incubated for 2 h in a humidified
atmosphere 5% CO2 at 37 °C. Fifty microlitres of mammalian cell ly-
sis solution was added to all wells and the plate shaked for 5 min in
an orbital shaker. Fifty microlitres of substrate solution was added to
all wells and the plate shaked for 5 min in an orbital shaker. The plate
was dark adapted for 10 min and the luminescence was measured.

N. A. Colabufo et al. / Bioorg. Med. Chem. 21 (2013) 1324–1332
1329
2.8.6. Permeability experiments
2.8.6.1. Preparation of Caco-2 monolayer.
Each experiment was repeated at least with 3 different cell
preparations. In Figure 5 and 100% is the mean of the k values cal-
culated from efflux data under control conditions.
Caco-2 cells were
seeded onto a MillicellÒ assay system (Millipore), where a cell
monolayer is set in between a filter cell and a receiver plate,
at a density of 10,000 cells/well.28 The culture medium was re-
placed every 48 h and the cells kept for 21 days in culture. The
Trans Epithelial Electrical Resistance (TEER) of the monolayers
was measured daily, before and after the experiment, using an
epithelial voltohometer (MillicellÒ-ERS). Generally, TEER
3. Results
3.1. Chemistry
The syntheses of final compounds are depicted in Schemes 1
and 2.
values greater than 1000
optimal.
X for a 21 day culture, are considered
Naphthalenyl-furyl derivatives 5a,5b (Scheme 1) have been pre-
pared by intermediates 3a,3b synthesized by Suzuki reaction be-
tween arylboronic acids 1a,1b and 5-bromo-2-furanoic acid 2,
commercially available. Amides 4a,4b derived by condensation of
carboxylic acids (3a,3b) and 6,7-dimethoxy-1,2,3,4-tetrahydroiso-
quinoline employing 1,10-carbonildiimidazole as coupling agent.
Amines 5a,5b have been obtained by reduction with LiAlH4 of
the corresponding amides.
Naphthalen-oxazoles 11a–e and naphthalen-thiazoles 12a–d
have been synthesized as shown in Scheme 2. The amides 7a–e
were prepared starting from the acyl chloride of the corresponding
carboxylic acids 6a–e. Thioamides 8a–d were prepared by reaction
of the corresponding amides with Lawesson’s reagent. Intermedi-
ates 9a–e, 10c have been prepared condensing amides 7a–e or thi-
oamide 8c, by homogeneous reaction, with 1,3-dichloroacetone.
Intermediates 10a,b,d have been synthesized by reaction between
thioamides 8a,b,d with 1,3-dichloroacetone in EtOH. The amines
11a–e, 12a–d have been obtained by condensation between
the alkyl chlorides 9a–e, 10a–d and 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline.
2.8.6.2. Drug transport experiment.
After 21 days of Caco-2
cell growth, the medium was removed from filter wells and from
the receiver plate, which were filled with fresh HBSS buffer (Invit-
rogen). This procedure was repeated twice, and the plates were
incubated at 37 °C for 30 min. After incubation time, the HBSS
buffer was removed and drug solutions and reference compounds,
were added to the filter well at various concentrations (1–100 lM),
while fresh HBSS was added to the receiver plate. The plates were
incubated at 37 °C for 120 min. Afterwards, samples were removed
from the apical (filter well) and basolateral (receiver plate) side of
the monolayer to measure the permeability.
The apparent permeability (Papp), in units of nm/s, was calcu-
lated using the following equation:
ꢀ
ꢁ
ꢀ
ꢁ
½drugꢂacceptor
½drugꢂinitial
VA
Papp
¼
ꢀ
Area ꢀ Time
VA = the volume (in mL) in the acceptor well;
Area = the surface area of the membrane (0.11 cm2 of the well);
Time = the total transport time in seconds (7200 s);
[drug]acceptor = the concentration of the drug measured by UV
spectroscopy;
3.2. Biology
[drug]initial = the initial drug concentration (1 ꢀ 10ꢁ4 M) in the
apical or basolateral wells.
Each compound has been tested to define the P-gp interacting
mechanism and the selectivity towards the other efflux pumps in-
volved in MDR, MRP1 and BCRP. In order to classify all compounds
as inhibitors or substrates towards P-gp, the following assays have
been performed: (i) apparent permeability determination (Papp) in
Caco-2 cells monolayer;25 (ii) inhibition of the Acetoxymethyl es-
ters of calcein (Calcein-AM) transport in Madin-Darby Canine Kid-
ney (MDCK) cells;23 (iii) ATP cell depletion in MDCK cells.24
The first assay allows to predict if the studied compound is a P-
gp inhibitor (Papp <2) or a P-gp substrate (Papp >2) studying the two
fluxes, from Basolateral to Apical (BA) and from Apical to
Basolateral (AB) compartments through the cell monolayer. ATPase
activation assay has been also performed to deepen the different
2.8.6.3. Efflux of Rhodamine-123.
glass coverslips at a density of 15,000/cm2.29,30 After 2 days,
cells were incubated for 10 min in the presence of 2.7
Cells were seeded onto
lM
Rhodamine-123. The coverslip was then mounted in a specially
constructed open chamber (0.5 mL), fixed on the stage of an in-
verted Nikon TE2000-S microscope, and then washed 4 times with
a medium containing 120 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2,
1.6 mM MgCl2, 11 mM glucose, 25 mM HEPES (pH 7.4). Excitation
wavelength (488 nm) was generated by Polychrome IV (Till
Photonics; Gräfelfing, Germany). Emission (525 nm, XF3017 filter
by Omega Optical, Brattleboro, VT, USA) was detected by a Micro-
max camera (RS Princeton Instruments; Trenton, NJ, USA) inter-
faced to a personal computer. The Workbench 2.2 software by
Axon Instruments (FHC Inc., Bowdoin, ME, USA) was used either
to drive the light generation and the camera or to quantify the
fluorescence values from a number of regions of interest. The
experiment was run at 22 °C. The efflux of Rhodamine-123 from
intracellular volume is quantitatively described by the following
equation:
F ¼ F0 ꢃ eꢁkt þ background
which can be written
ln ðF ꢁ backgroundÞ ¼ ln F0 ꢁ kt
where F0 is F at t = 0, k is the rate constant for Rhodamine-123
decay.27,28
Figure 4. Efflux of Rhodamine-123 from MDCK-BCRP cells decreased by 200 nM
FTC. Cells, loaded 10 min with 2.7 lM Rhodamine-123, were washed 4 times with
an incubation medium without Rhodamine-123. In this plot, the calculated values
belong to a line whose slope represents the rate constant (k). The mean of values
from the cytoplasm of 10 cells, and only one in four S.E.M. values are shown.
As an estimate of background we used the fluorescence value re-
corded from unloaded cells. In Figure 4, the k parameter was calcu-
lated with a least squares regression program.

1330
N. A. Colabufo et al. / Bioorg. Med. Chem. 21 (2013) 1324–1332
OH
B
OH
OH
O
A
OH
+
Br
O
O
R
R
O
2
1a,b
3a,b
R
a
H
B
b
OCH3
O
O
O
O
C
N
N
O
O
O
R
R
4a,b
5a,b
Scheme 1. Synthesis of naphthalenyl-furyl derivatives 5a,b. Reagents and conditions: Pd(PPh3)4, EtOH, K2CO3 s.s. (A); 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, CDI, dry
THF (B); LiAlH4, dry THF (C).
O
OH
R
A;B
A,B,C
R
S
6a-e
O
NH2
NH2
R
R
8a-d
7a-e
a
b
c
d
e
H
F
D
D
Br
OCH3
N
Cl
N
Cl
OH
O
S
R
R
10a-d
9a-e
E
E
O
O
O
O
N
N
N
N
S
O
R
R
11a-e
12a-d
Scheme 2. Synthesis of naphthalen-oxazoles 11a–e and naphthalen-thiazoles 12a–d. Reagents and conditions: Et3N, MsCl, ꢁ10 °C (A); NH4OH 10%, CH2Cl2 (B); Lawesson’s
reagent, dry THF (C); 1,3-dichloroacetone, homogeneous reaction for naphtyloxazoles or EtOH for naphtylthiazoles (D); Na2CO3, DMF, 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline (E).
induced mechanism since only substrates lead to a decrease of the
ATP in cells. The inhibition of Calcein-AM transport in MDCK cells
is useful to establish the potency of the interaction towards P-gp
and also the potential interference with BCRP and MRP1.
3.2.1. P-gp and MRP1 activity
Oxazole derivatives displayed P-gp activity comparable to that
of the corresponding thiazole derivatives and in detail the meth-
oxy-substituted compounds (11d and 12d) showed the highest

N. A. Colabufo et al. / Bioorg. Med. Chem. 21 (2013) 1324–1332
1331
Table 1
Biological evaluation of naphtalenyl series
A:
O
O
O
B:
N
Hetero
N
N
O
R
S
C:
a
Compd
R
Hetero
EC50
(lM)
Papp
ATPase
Intrinsic activity
P-gp
MRP1
5a
5b
11a
11b
11c
11d
11e
12a
H
OCH3
H
F
Br
OCH3
OH
H
F
Br
A
A
B
B
B
B
B
C
C
C
C
7.1
2.9
2.3
5.2
1.8
0.6
2.8
3.5
4.8
3.7
1.0
6.35
0.69
1.50
>100
>100
33.2
31.8
>100
14.7
>100
3.2
3.3
4.08
0.38
0.82
3.2
3.8
1.57
23.7
No
No
No
No
No
Yes
No
No
No
Yes
Yes
Ambiguous substrate
Ambiguous substrate
Inhibitor
Inhibitor
Ambiguous substrate
Unambiguous substrate
Inhibitor
Ambiguous substrate
Ambiguous substrate
Unambiguous substrate
Unambiguous substrate
Unambiguous substrate
Inhibitor
12b
12c
12d
8.8
9.5
5.9
6.0
>100
>100
>100
9.30
2.80
OCH3
MC266b
MC70b
MC18b
Inhibitor
a
Data represent the mean value of three independent determinations performed in duplicate.
Data reported in Ref. 25.
b
potency towards P-gp (EC50 = 0.6
l
M and EC50 = 1.0
lM, respec-
whereas were inactive towards MRP1(EC50 >100 lM). Moreover
tively) (Table 1). In term of selectivity, the thiazole 12d was found
more selective than the corresponding oxazole 11d since 12d dis-
these ligands could be considered as atypical P-gp substrates be-
cause they showed Papp values >2 but were unable to induce ATP
cell depletion.
played poor activity towards MRP1 (EC50 >100
moderate activity towards the same pump (EC50 = 14.7
l
M) whereas 11d
M).
l
In the permeability experiment, only compounds, 11a,b and 11e
showed Papp values <2 while all the other compounds displayed a
Papp values >2. In ATPase assay, these ligands displaying a Papp val-
ues <2 did not exert ATP cell depletion acting as P-gp inhibitors.
The compounds 11d, 12c,d could be considered P-gp substrates
since they induced ATP cell depletion and displayed Papp values
>2. The other compounds belong to different substrates categories
reported in previous papers.15 Furyl derivatives, 5a,b displayed
3.2.2. BCRP activity
Compounds displaying the best P-gp activity and selectivity
such as 11d, 12c, 12d, have been tested for determining in single
cell BCRP activity. In this assay was evaluated their ability to inter-
fere with Rhodamine-123 efflux in MDCK-BCRP single cell.26,27 As
depicted in Figure 4, Fumitremorgin C (FTC), claimed BCRP inhibi-
tor, in this assay modified the slope of the curve (dotted line) with
respect the control (solid line). The dose–response results demon-
strated that these compounds at 200 nM were unable to interfere
with the Rhodamine efflux as reported in Figure 5. Indeed, tested
compounds did not exert the same effect of FTC on the MDCK cells
overexpressing the BCRP transporter.
moderate P-gp activiy (EC50 = 7.1 and 2.9 lM, respectively)
4. Discussion and conclusions
Starting from the two lead compounds, MC18 and MC266, pre-
viously characterized as P-gp inhibitor and substrate, respectively,
by phenyl and naphthalenyl series have been designed in order to
deepen the structural requirements useful for developing new po-
tent and selective P-gp ligands. These two series have been de-
signed take into account the pivotal role of the conformational
restriction of the spacer to obtain P-gp inhibitors (as for MC18).
In the biphenyl series the best compound was MC70 (Fig. 3) that
showed high P-gp activity (EC50 = 0.69 lM) but moderate activity
towards BCRP and MRP1 transporters (EC50 = 73 and EC50 = 9.3
lM,
for BCRP and MRP1, respectively).
Figure 5. The efflux of Rhodamine-123 is reduced by FTC in MDCK-BCRP cells. FTC
does not exert this effect in MDCK wild type cells. For each experiment, the value of
the rate constant (k), measured after adding the drug under evaluation, was
normalized to that obtained under control conditions.
In order to overcome MC70 limitation, bioisosterism has been
performed on the biphenyl fragment leading to aryloxazole and
arylthiazole derivatives (Fig. 3). These compounds were found less

1332
N. A. Colabufo et al. / Bioorg. Med. Chem. 21 (2013) 1324–1332
active towards P-gp and more active towards the other MDR
pumps, BCRP and MRP1.
References and notes
1. Gottesman, M. M.; Fojo, T.; Bates, S. E. Nat. Rev. Cancer 2002, 2, 48.
2. Leslie, E. M.; Deeley, R. G.; Cole, S. P. Toxicol. Appl. Pharmacol. 2005, 204, 216.
3. Tan, B.; Piwnica-Worms, D.; Ratner, L. Curr. Opin. Oncol. 2000, 12, 450.
4. Szakács, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.; Gottesman, M. M.
Nat. Rev. Drug Disc. 2006, 5, 219.
5. Borst, P.; Evers, R.; Kool, M.; Wijnholds, J. J. Natl. Cancer Inst. 2000, 92, 1295.
6. Avendano, C.; Menendez, J. C. Curr. Med. Chem. 2002, 9, 159.
7. Pérez-Tomás, R. Curr. Med. Chem. 2006, 13, 1859.
Since this aryl fragment and the presence and position of substi-
tuent addressed the activity towards P-gp or BCRP or MRP1, in the
present work the effect of the hindrance on the activity towards P-
gp/BCRP/and MRP1 replacing the aryl group with substituted
naphthalenyl fragments was evaluated. The employed heteronu-
clei: (i) oxazole; (ii) thiazole; (iii) furyl, could play an important
role in terms of selectivity.
8. Modok, S.; Mellor, H. R.; Callaghan, R. Curr. Opin. Pharmacol. 2006, 6, 350.
9. Teodori, E.; Dei, S.; Martelli, S.; Scapecchi, F.; Gualtieri, F. Curr. Drug Targets
2006, 7, 893.
Aryloxazole and arythiazole derivatives, previously developed,
displayed with respect to MC18 and MC70, a decreased P-gp activity
and selectivity.22 In addition, both aryl-oxazoles and -thiazoles were
P-gp substrates whereas MC18 and MC70 showed inhibitory activ-
ity. As listed in Table 1, compounds 11a,b and 11e displayed P-gp
inhibitory activity although less potent than MC18 and MC70. More-
over, 11d, 12c,d were found unambiguous substrates while all the
other compounds could be considered as ambiguous substrates.15
In terms of potency towards P-gp, the best results have been ob-
tained with methoxy-naphathalenyl substituted derivatives 11d
and 12d bearing oxazole and thiazole nuclei, respectively. Among
them, only 12d was inactive towards MRP1 and BCRP (Fig. 5) while
11d was inactive toward BCRP and displayed moderate MRP1
activity. These results lead to consider the thiazole nucleus as
pivotal to hit P-gp activity while oxazole nucleus compromised
the P-gp selectivity because 11d was active also towards MRP1.
Moreover, also 6-Br-substituted derivative 12c was inactive to-
wards BCRP and therefore the substituent unaffected BCRP activity.
Finally, furyl derivatives 5a,b displayed moderate P-gp activity and
good selectivity but they were ambiguous substrates.
In conclusion, the replacement of aryl with naphthalenyl
moiety led to obtain P-gp inhibitors, unambiguous or ambiguous
substrates. These different activities were modulated both by het-
eronucleus and substituent on the naphthalenyl fragment. Indeed,
oxazole derivatives were inhibitors when R = H, F, OH, unambigu-
ous substrates when R = OCH3, ambiguous substrate when
R = Br. Thiazole derivatives were unambiguous substrates when
R = OCH3, Br, while when R = H, F were ambiguous substrates.
Finally furyl derivatives were ambiguous substrates. These conclu-
sions in terms of intrinsic activity should be corroborated by other
characterizations such as the contribution of passive diffusion to
perform acceptable predictions of in vivo biodistribution.31
10. Thomas, H.; Coley, H. M. Cancer Control 2003, 10, 159.
11. Nobili, S.; Landini, I.; Giglioni, B.; Mini, E. Curr. Drug Targets 2006, 7, 861.
12. Palmeira, A.; Sousa, E.; Vasconcelos, M. H.; Pinto, M.; Fernandes, M. X. Curr.
Pharm. Des. 2012, 18, 4197.
13. Kannan, P.; Telu, S.; Shukla, S.; Ambudkar, S. V.; Pike, V. W.; Halldin, C.;
Gottesman, M. M.; Innis, R. B.; Hall, M. D. ACS Chem. Neurosci. 2011, 2, 82.
14. Dörner, B.; Kuntner, C.; Bankstahl, J. P.; Stanek, J.; Wanek, T.; Stundner, G.;
Mairinger, S.; Löscher, W.; Müller, M.; Langer, O.; Erker, T. J. Med. Chem. 2009,
52, 6073.
15. Wanek, T.; Kuntner, C.; Bankstahl, J. P.; Bankstahl, M.; Stanek, J.; Sauberer, M.;
Mairinger, S.; Strommer, S.; Wacheck, V.; Löscher, W.; Erker, T.; Müller, M.;
Langer, O. Eur. J. Nucl. Med. Mol. Imaging 2012, 39, 149.
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.12.021.