A novel class of highly potent multidrug resistance reversal agents: Disubstituted adamantyl derivatives

Article abstract of DOI:10.1016/j.bmcl.2009.07.127

Novel disubstituted adamantyl derivatives were synthesized and evaluated in a P-glycoprotein dependent multidrug resistance cancer cell line. The hit to lead optimization provided potent MDR reversal agents. Some potent adamantyl derivatives were more than 10-fold more potent than verapamil without considerable intrinsic cytotoxicity. The 3-trifluorophenyl derivative 14f did not affect the metabolism of CYP450 3A4, whereas most of MDR revertants had a weak inhibitory effect.

Full text of DOI:10.1016/j.bmcl.2009.07.127

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5376–5379
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel class of highly potent multidrug resistance reversal agents:
Disubstituted adamantyl derivatives
Kyung Hoon Min a, , Yan Xia b,c, Eun Kyung Kim a, Yinglan Jin b, Navneet Kaur b, Eun Seon Kim a,
*
Dae Kyong Kim a, Hwa Young Jung d, Yongseok Choi d, Mi-Kyung Park e, Yong Ki Min e,
Kiho Lee b, Kyeong Lee b,c,
*
a College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea
b Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea
c Korea University of Science and Technology, Daejeon, 305-333, Republic of Korea
d School of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea
e Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel disubstituted adamantyl derivatives were synthesized and evaluated in a P-glycoprotein depen-
dent multidrug resistance cancer cell line. The hit to lead optimization provided potent MDR reversal
agents. Some potent adamantyl derivatives were more than 10-fold more potent than verapamil without
considerable intrinsic cytotoxicity. The 3-trifluorophenyl derivative 14f did not affect the metabolism of
CYP450 3A4, whereas most of MDR revertants had a weak inhibitory effect.
Received 23 March 2009
Revised 27 July 2009
Accepted 28 July 2009
Available online 6 August 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Multidrug resistance
MES-SA/DX5
ABC transporter
P-glycoprotein
Adamantane
CYP3A4
Multidrug resistance (MDR) disables many potent anticancer
drugs and is primarily responsible for the failure of cancer chemo-
therapy.1 Although many mechanisms have been reported, MDR
results mainly from the overexpression of ATP-binding cassette
(ABC) transporters, such as, ABCB1 (P-glycoprotein, P-gp), ABCC1-7
(multidrug resistant related protein 1-7, MRP1-7) and ABCG2
(breast cancer resistance protein, BCRP), which pump out a huge
number of chemically unrelated anticancer agents. P-gp is consid-
ered to be more clinically significant than the other transporters,
and therefore, is viewed as a therapeutic target for re-sensitizing
multidrug resistant cancer cells to anticancer agents. Despite inten-
sive efforts to develop P-gp modulators to overcome MDR, many
candidates have been dropped because of their intrinsic toxicities
and pharmacokinetic interactions. Some agents like Zosuquidar
and Tariquidar are currently under clinical trials, but research efforts
have not yielded a clinically useful drug as yet. Hence, the develop-
ment of small molecule sensitizers remains a high-level priority to
meet clinical needs. Improved clinical trial protocols utilizing func-
tional imaging technology based on the use of 99mTc-sestamibi in
conjunction with RNA or protein-based technologies have differen-
tiated responders and non-responders with respect to ABC trans-
porter inhibition.2 Herein, we describe a novel class of highly
potent MDR reversal compounds and structure–activity
relationships.
We initially screened thousands of small molecules from an in-
house chemical library by using an image-based high-throughput
screening assay in a P-gp overexpressing MDR sarcoma cell line,
MES-SA/DX5. A number of primary hits were obtained by image-
based efflux assay using 3-ethyl-2-[3-(3-ethyl-2(3H)-benzooxazol-
ylidene-1-propenyl) benzoxzolium iodide (DiOC2)3 in a 384-well
format, and these findings were confirmed by cytotoxicity assays.
Subsequently, the adamantyl compound (10a), which showed
good activity at 5
hit compound was re-synthesized and showed similar activity to
verapamil (EC50 = 8.48 vs 7.87 M, respectively), a well-known
lM, became a focus of attention (Fig. 1). The
l
P-gp inhibitor. We next have carried out hit to lead optimization
to increase the MDR reversal activities of hit compounds. We
designed and synthesized the adamantyl derivatives described in
Schemes 1 and 2 by incorporating the common features of P-gp
inhibitors, that is, high hydrophobicity, and the presence of aro-
matic rings and protonable tertiary amines.
The chemistry employed to design the new compounds
described here is shown in Schemes 1 and 2. Compound 2 was
* Corresponding authors.
E-mail addresses: khmin@cau.ac.kr (K.H. Min), kaylee@kribb.re.kr (K. Lee).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.127

K. H. Min et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5376–5379
5377
CO2Me
generated by removing the benzyl group of 8 by catalytic hydroge-
nation in the presence of palladium on carbon. Compound 9 has a
free carboxylic group, which is capable of reacting with amines. For
compounds 10a and b coupling was achieved by using coupling
agents, that is, EDC or PyBOP in the presence of an organic base,
whereas the other amide coupled products 10c–i were readily syn-
thesized in the presence of PPAA in good yields.5 Based on syn-
thetic profile of 10e, we replaced the ester moiety of the aryl
ring in 10e with different amides, as shown in Scheme 2. Hydrog-
enolysis of the benzyl group of 6 furnished 11, which was further
coupled with 6,7-dimethoxy-1,2,3,4-dihydroisoquinoline to give
12.
O
O
N
O
HN
N
H
10a
O
R2
O
R1
O
HN
Figure 1. Hit molecule 10a identified from HTS and the general structure of the
MDR revertants prepared in this Letter.
Subsequently, base mediated hydrolysis of 11 provided the free
acid 13. Finally, PPAA-mediated coupling with various commer-
cially available amines led to the corresponding desired amides
14a–g. Furthermore, saponification of the methyl ester intermedi-
ate provided the corresponding meta-substituted benzoic acid on
the B-ring of the target compound, which was subsequently cou-
pled with furfurylamine to afford 14d.
The ability of all synthesized compounds to reverse MDR was
determined using a P-gp overexpressing human sarcoma cell line,
MES-SA/DX5, in the presence of 100 nM Taxol (Table 1). Further-
more, the MDR reversing activity of selected compounds was mea-
sured by reversal index, the ratio of the IC50 for Taxol only divided
by the IC50 for Taxol in combination with test compounds in the
synthesized by brominating 1-adamantane carboxylic acid 1, and
the corresponding intermediate 2 was subjected to Friedel–Crafts
alkylation with anisole to obtain compound 3. Deprotection of
the phenolic –OH of 3 via the cleavage of its methyl ether under
Lewis acid conditions (BBr3)4 and the subsequent selective protec-
tion of 4 with benzyl bromide under basic conditions afforded 5,
which was subsequently alkylated with ethyl chloroacetate to
produce 6 in excellent yields. Compound 7 was obtained by
saponifying 6 with LiOH, and subsequent coupling with methyl
3-aminobenzoate produced the amide 8 in good yield. The precur-
sor for the synthesis of the target adamantyl motifs (10a–i) was
O
O
O
b
a
c
HO
HO
Br
OMe
HO
1
2
3
O
OEt
O
O
O
d
e
OH
O
BnO
OH
BnO
HO
4
5
6
O
CO2Me
O
O
O
O
H2N
CO2CH3
f
O
HN
O
OH
BnO
BnO
g
7
8
CO2Me
CO2Me
O
O
O
i
h
O
HN
O
HN
HO
R
9
10
H
N
H
N
OCH3
OCH3
OCH3
OCH3
N
H
N
NH2
R:
N
10d
10a
10f
10b
10c
10e
OCH3
N
N
N
N
N
N
N
N
F
CF3
OCH3
10g
10h
10i
Scheme 1. Reagents and conditions: (a) AlCl3, Br2, À5 °C to rt, 93%; (b) anisole, AlCl3, À10 °C to rt, 94%; (c) BBr3, À10 °C to rt, 84%; (d) BnBr, KHCO3, 40 °C, 85%; (e)
ethylchloroacetate, K2CO3, rt, 85%; (f) LiOH, THF/H2O, 97%; (g) PyBOP, DMAP, rt, 72%; (h) H2,Pd/C, 94%; (i) EDC, DIPEA, HOBt, rt, 55% for 10a or PyBOP, DMAP, rt, 94% for 10b; or
PPAA, Et3N, DCM, rt for 10c–10i (28–70%).

5378
K. H. Min et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5376–5379
H CO
3
O
NH
O
O
H3CO
H3CO
O
H CO
3
O
OEt
a
N
6
O
OEt
HO
b
11
12
N
O
O
O
O
H3CO
H3CO
O
R'
H3CO
H3CO
d
c
O
OH
N
13
14
R':
H
N
O
OCH3
NH
O
O
H
H
H
H
N
H
N
N
CN
N
CF3
N
N
O
OCH3
NH2
N
H
O
14e
14f
14g
14d
14a
14b
14c
Scheme 2. Reagents and conditions: (a) Pd/C, MeOH, 99%; (b) PPAA, Et3N, NaCN, rt, 66%; (c) LiOH, THF/H2O, 97%; (d) PPAA, Et3N, NaCN, rt for 14a–c (32–40%) and 14e–f (42–
50%); or (i) PPAA, Et3N, NaCN, methyl 3-amino benzoate, rt, (ii) LiOH, THF/H2O, rt, (iii) EDC, DIPEA, HOBt, furfurylamine, rt for 14d (76%).
Table 1
Table 2
Reversal activities of synthesized compounds in MES-SA/DX5 sarcoma cells
Effects of adamantyl derivatives with a more potent effect than verapamil on Taxol
cytotoxicity in MES-SA/DX5 sarcoma cellsa
a
b
Compound EC50
(lM, 100 nM
Fold
Intrinsic cytotoxicity GI50
M)
Taxol)
increase
(
l
Compound
Concd (lM)
IC50 of Taxol (nM)
Reversal indexb
Taxol only
Verapamil
8
10a
10b
10c
10d
10e
10f
10g
10h
10i
14a
14b
14c
14d
14e
14f
—
7.87
>15
8.48
7.78
>15
2.74
1.13
1.58
0.58
0.74
1.10
1.10
1.24
>15
10.76
0.84
0.71
0.83
—
1.0
—
0.9
1.0
—
2.9
7.0
5.0
13.6
10.6
7.2
7.2
6.4
—
0.7
9.4
11.1
9.5
7.54
>20
>20
>20
14.3
>20
>20
>20
>20
>20
14.0
13.2
>20
14.3
16.3
>20
>20
12.1
>20
Taxol only
Verapamil
7538
257
>2500
34
1253
9
611
9
917
8
94
3
167
3
610
4
740
5
1083
13
200
7
107
5
1
29
—
222
6.0
836
12.3
836
8.2
5
1
5
1
5
1
5
1
5
1
5
1
5
1
5
1
5
1
5
1
5
1
5
1
10d
10e
10f
10g
10h
10i
942
80.2
>1000
45.1
>1000
12.4
>1000
10.2
>1000
7.0
14a
14b
14e
14f
14g
14g
580
a
EC50 values were determined in the presence of 100 nM Taxol.10
GI50 values are the intrinsic cytotoxicities of test compounds.
37.7
>1000
70.5
>1000
34.9
b
resistant cells (Table 2). Taxol had an IC50 value in micromolar
range against MES-SA/DX5 (IC50 = 7.54 M), whereas Taxol usually
216
l
a
IC50 values of taxol were obtained in the presence or absence of 1 or 5
l
M of the
has an IC50 value at the single-digit nanomolar level against MES-
SA, a sensitive wild-type cancer cell line. Table 1 summarizes
reversal activity of adamantyl derivatives. The hit to lead optimiza-
tion was commenced with diversifying R1. The removal of the
dimethylaminoethyl group (10b) was well tolerated, but caused
test compound.
b
Reversal index is defined as the quotient of the IC50 of Taxol in resistant cells
and the IC50 of Taxol in the presence of a modulator.
intrinsic cytotoxicity against the MDR cells (GI50 = 14.3
lM). The
14 times more potent than verapamil.6 In addition, it was found
ester 8 did not show any reversal activity, indicating that amide
group at R1 is important for this activity. Incorporation of an aro-
matic ring at the R1 position significantly increased activity,
whereas the introduction of 4-t-butylaniline (10c) led to a loss of
activity. Reversal activities of 10d–10i were more potent than that
of verapamil, an established MDR-reversing agent. A comparison of
10e and 10d revealed that a more lipophilic and conformationally
rigid substituent was slightly more effective. Furthermore, intro-
duction of substituted piperazine significantly improved reversal
activity. Best reversal activity was observed for the trifluoroben-
that 5
l
M of compound 10g almost completely restored the cyto-
M of 10g made the resistant
toxicity of Taxol (Table 2), and that 1
l
cancer cells about 80-fold more sensitive to Taxol. A number of
derivatives modified at the R2 position were also synthesized to
investigate structure–activity relationships (SAR). Transposition
of the meta-ester group (10e) to the ortho (14a) or para (14b) posi-
tions did not affect reversal activity, whereas replacement of the
ester with an amide group (14c) resulted in a loss of activity and
an increase in cytotoxicity. The furfuryl amido derivative 14d
displayed weaker activity than verapamil, but the nitrile 14e and
zyl-substituted compound 10g (EC50 = 0.58
l
M), which was about
trifluoromethyl 14f derivatives (EC50 = 0.84, 0.71 lM, respectively)

K. H. Min et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5376–5379
5379
Figure 2. Inhibition of DIOC2 efflux from MES-SA/DX5 cells by an adamantyl derivative 10h. The cells were treated with fluorescent P-gp substrate DiOC2, incubated for 1 h
and washed three times with PBS. (A) Control, (B) 5 lM verapamil, (C) 1 lM 10h.
Table 3
optimization led to the elucidation of SAR for both terminal side
chains. Many of the adamantyl derivatives produced were found
to have a reversal activity greater than verapamil, and restore com-
CYP3A4 inhibition assay findings for compounds 10g–i, 14a, and 14e–g
a
Compound
IC50
(lM)
pletely cytotoxicity of Taxol against the MDR cancer cells at 5 lM.
10g
2.03
The EC50 value of 10g displayed 14-fold increase versus verapamil
without intrinsic cytotoxicity. On the other hand, 14f did not affect
CYP3A4 at all, and thus, it is unlikely to influence the plasma phar-
macokinetics of anticancer agents. SAR studies on reversal activity
and CYP3A4 would provide useful information for the design and
development of more selective and potent MDR reversal agents.
The present work suggests that this new class of highly potent
and selective compounds deserves further investigation.
10h
10i
14a
14e
14f
14g
ketoconazole
0.89
0.82
3.79
>5.00
No inhibition
2.84
0.004
a
IC50 values against human CYP3A4 were determined using a CYP3A4/BFC high-
throughput inhibitor screening kit purchased from BD Gentest.
Acknowledgements
had higher activity than the corresponding ester 10e. On the other
hand, weak cytotoxicity was observed for 14f7 (GI50 = 12.1
lM).
This study was supported by KRIBB Research Initiative Program
and Korea Science and Engineering Foundation (KOSEF) Grants
(2008-05676 and RO1-2008-000-11354-0), South Korea.
Five micromolar of the quinoline 14g also completely restored
the cytotoxic effect of Taxol. As illustrated in Table 2, selected
derivatives completely restored the cytotoxic effect of Taxol
against the resistant cancer cells at 5 lM, while verapamil only
partially enhanced the effect of Taxol at this concentration. DiOC2
efflux assay images indicate that the reversal activities of these
adamantyl derivatives resulted from P-gp inhibition (Fig. 2).
With regard to the development of MDR reversal agents, phar-
macokinetic interactions have been considered as an important
factor, because MDR modulators can change the pharmacokinetic
properties of anticancer drugs by inhibition of CYP450 system.1
Over half of all drugs are metabolized by CYP3A4, which is one
of the most important human metabolic enzymes, and the inhibi-
tion of CYP3A4 has been reported to alter the plasma pharmacoki-
netics of anticancer agents, and to be able to lead to unpredictable
side effects. In the present Letter, we evaluated the inhibitory
activities of selected compounds on CYP3A4 (Table 3).
References and notes
1. For recent reviews, see: (a) Szakacs, G.; Paterson, J. K.; Ludwig, J. A.; Booth-
Genthe, C.; Gottesman, M. M. Nat. Rev. Drug Disc. 2006, 5, 219; (b) Curr. Opin.
Pharmacol. 2006, 6, 350.; (c) Ozben, T. FEBS Lett. 2006, 580, 2903; (d) Longley, D.
B.; Johnston, P. G. J. Pathol. 2005, 205, 275; (e) Bisi, A.; Meli, M.; Gobbi, S.;
Rampa, A.; Tolomeo, M.; Dusonchet, L. Bioorg. Med. Chem. 2008, 16, 6474.
2. Pusztai, L.; Wagner, P.; Ibrahim, N.; Rivera, E.; Theriault, R.; Booser, D.;
Symmans, F. W.; Wong, F.; Blumenschein, G.; Fleming, D. R.; Rouzier, R.;
Boniface, G.; Hortobagyi, G. N. Cancer 2005, 104, 682.
3. Wadkins, R. M.; Houghton, P. J. Biochemistry 1995, 34, 3858.
4. Colotta, V.; Catarzi, D.; Varano, F.; Calabri, F. R.; Lenzi, O.; Filacchioni, G.;
Martini, C.; Trincavelli, L.; Deflorian, F.; Moro, S. J. Med. Chem. 2004, 47, 3580.
5. Ech-Chahad, A.; Minassi, A.; Berton, L.; Appendino, G. Tetrahedron Lett. 2005, 46,
5113.
6. Spectral data for 10g: 1H NMR (CDCl3, 300 MHz) d 8.38 (1H, s), 8.07 (1H, s),
8.00–8.03 (1H, m), 7.83 (1H, d, J = 7.8 Hz), 7.56–7.60 (2H, m), 7.43–7.48 (3H,
m), 7.33 (2H, d, J = 8.7 Hz), 6.96 (2H, d, J = 8.7 Hz), 4.61 (2H, s), 3.93 (3H, s), 3.71
(4H, m), 3.55 (2H, s), 2.43 (4H, m), 2.25 (2H, m), 2.00–2.09 (6H, m), 1.87 (4H,
m), 1.73 (2H, m); MS (EI) m/z 689 (M+); HRMS (EI+) m/z calcd for C39H42F3N3O5
689.3077, found 689.3077.
Of these, 14f did not inhibit CYP3A4 like third-generation MDR
modulators, that is, Tariquidar. Compound 14e exhibited a very
weak inhibitory effect (IC50 >5 lM). In this series, electron with-
7. Spectral data for 14f: 1H NMR (CDCl3, 300 MHz) d 8.41 (s, 1H), 7.89 (s, 1H), 7.83
(d, J = 7.2 Hz, 1H), 7.49 (d, J = 7.5 Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.36 (d,
J = 8.7 Hz, 2H), 6.96 (d, J = 8.7 Hz, 2H), 6.60 (m, 2H), 4.71 (s, 2H), 4.62 (s, 2H),
3.91 (t, J = 5.1 Hz, 2H), 3.85 (s, 6H), 2.80 (t, J = 5.1 Hz, 2H), 2.28 (b, 2H), 1.63–
2.15 (m, 12H); MS (EI) m/z 648 (M+); HRMS (EI+) m/z calcd for C37H39F3N2O5
648.2811, found 648.2811.
drawing groups seemingly attenuate interaction of modulators with
CYP3A4. Although the activities of 10h and 10i toward CYP3A4 were
stronger than the other compounds, their activities were more than
200-fold weaker than that of ketoconazole. The expressional level
and activity of CYP3A4 is known to depend on tumoral tissue type
and on genetic polymorphisms, which contribute to inter-individual
variations.8 The inhibitions of both P-gp and drug metabolism could
be advantageous during the treatment of MDR in specific types of
cancers, in which CYP3A4 is expressed and co-localized with P-gp.
In such a case, patients might also benefit from this type of dual inhi-
bition as long as the modulators do not have unexpected intrinsic
toxicity. Therefore, dual inhibitors, such as, 10g and 14g, as well as
P-gp selective inhibitors like 14f might be of therapeutic interest
in terms of the personalizing cancer therapy.8,9
8. Michael, M.; Doherty, M. M. J. Clin. Oncol. 2005, 23, 205.
9. (a) Zhou, S. F.; Di, Y. M.; Chan, E.; Du, Y. M.; Chow, V. D.; Xue, C. C.; Lai, X.;
Wang, J. C.; Li, C. G.; Tian, M.; Duan, W. Curr. Drug Metab. 2008, 9, 738; (b) van
Schaik, R. H. Drug Resist. Updat. 2008, 11, 77.
10. MDR reversal assay. The ability of compounds to potentiate the cytotoxicity of
Taxol was evaluated in MES-SA/DX5 cells, which was obtained from ATCC.
Cells were plated in 96-well plates at 1.2 Â 104 cells/well in 100
ll of medium
and incubated for 24 h at 37 °C. The cells were then treated with varying
concentrations of a test compound in the presence or absence of 100 nM Taxol
for 60 h. Then, cell survival was assayed using Cell Counting Kit-8 (dojindo).
Reversal of MDR is indicated if the compound enhances the toxicity of Taxol
against MES-SA/DX5 cells.
In summary, we synthesized and evaluated a novel class of
disubstituted adamantane-based MDR reversal agents. Hit to lead