Diphenylamine-based retinoid antagonists: Regulation of RAR and RXR function depending on the N-substituent

Article abstract of DOI:10.1016/j.bmc.2011.03.026

Based upon the structure-activity relationships of diphenylamine derivatives with retinoid synergistic activity (RXR agonists), novel diphenylamine derivatives with a long alkyl chain (9a and 9b) or a benzyl group (10a-f) as the N-substituent were designe

Full text of DOI:10.1016/j.bmc.2011.03.026

Bioorganic & Medicinal Chemistry 19 (2011) 2501–2507
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Diphenylamine-based retinoid antagonists: Regulation of RAR and RXR
function depending on the N-substituent
Kiminori Ohta a, Emiko Kawachi b, Hiroshi Fukasawa c, Koichi Shudo d, Hiroyuki Kagechika b,
⇑
a Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
b Graduate School of Biomedical Science, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai,
Chiyoda-ku, Tokyo 101-0062, Japan
c Institute of Medicinal Molecular Design, Inc., 5-24-5 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
d Research Foundation Itsuu Laboratory, 2-28-10 Tamagawa, Setagaya-ku, Tokyo 158-0094, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Based upon the structure–activity relationships of diphenylamine derivatives with retinoid synergistic
activity (RXR agonists), novel diphenylamine derivatives with a long alkyl chain (9a and 9b) or a benzyl
group (10a–f) as the N-substituent were designed and synthesized. All the synthesized compounds dose-
dependently inhibited HL-60 cell differentiation induced by 3.3 ꢀ 10ꢁ10 M Am80. Among them, com-
pound 10f showed the most potent inhibitory activity, and the mechanism was shown, by means of
transactivation assay for RARs and RXRs, to involve antagonism against RARs. The N-substituent of the
diphenylamine skeleton plays an important role in determining the receptor selectivity for RARs or RXRs,
as well as the agonist or antagonist nature of the activity.
Received 11 February 2011
Revised 8 March 2011
Accepted 10 March 2011
Available online 15 March 2011
Keywords:
Retinoid
RAR
Antagonist
Diphenylamine
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Therefore, we have developed various RAR and RXR ligands con-
taining nitrogen atoms (Fig. 2).11 A heterocyclic RXR agonist HX600
Retinoids, that is, natural and synthetic analogues of all-trans-
retinoic acid (ATRA, 1), play an important role in cell differentia-
tion, proliferation and embryonic development in vertebrates,1
and are used as therapeutic agents in the fields of dermatology
and oncology.2 Their biological activities are mediated by binding
to and activating two types of specific nuclear receptors, retinoic
acid receptors (RARs) and retinoid X receptors (RXRs), each having
(6) was developed by mimicry of the bent structure of 2 with a
benzodiazepine structure.9 We also found potent RXR agonists
DA024 (7a) and PA024 (7b) bearing a nitrogen atom as the linking
group between two aromatic rings.12,13 The diphenylamine skele-
ton has some advantages for drug discovery, because the linker
nitrogen atom can be readily modified with various substituents.
Among the synthesized diphenylamine derivatives, DA010 (8)
showed dual RAR and RXR agonist activities. RAR agonist activity
was remarkably diminished by the introduction of an N-alkyl
substituent such as a cyclopropylmethyl group, which affected
the potency of RXR agonist activity, as well as discriminating the
two receptors in terms of affinity (Fig. 2b).
three subtypes, a . RARs and RXRs are ligand-inducible tran-
, b, c 3
scription factors and their endogenous ligands are 1 and 9-cis-ret-
inoic acid (2), respectively (Fig. 1).4 Major retinoidal activities are
elicited by RAR–RXR heterodimers. RXR is a silent partner of
RAR,5 and the heterodimers can be activated by an RAR agonist
such as TTNPB (3)6 and Am80 (4, Fig. 2a),7 but not by an RXR ago-
nist. On the other hand, an RXR agonist, such as LGD1069 (5) or
HX600 (6), acts as a retinoid synergist, since it dose-dependently
increases the activity of a low concentration of RAR agonist
(Fig. 1).8,9 The synthetic compounds 3 and 5 should have high sta-
bility as a result of transformation of the unstable polyene struc-
tures of 1 and 2 into benzene rings. Modifications of retinoid
structures with aromatic rings and the introduction of heteroatoms
have afforded potent RAR-selective agonists with remarkable
chemical stability and good bioavailability, such as Am80 (4).10,11
Figure 3 summarizes the structure–activity relationship for ret-
inoid synergy of DA010 derivatives bearing an N-alkyl group in the
presence of 1 ꢀ 10ꢁ10 M Am80.12 As the N-alkyl group was made
⇑
Corresponding author. Tel.: +81 3 5280 8032; fax: +81 3 5280 8127.
E-mail address: kage.omc@tmd.ac.jp (H. Kagechika).
Figure 1. The structures of endogenous RAR and RXR ligands.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.026

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K. Ohta et al. / Bioorg. Med. Chem. 19 (2011) 2501–2507
Figure 3. Structure–activity relationship for retinoid synergy of diphenylamine
derivatives with various N-alkyl substituents. Each column indicates the activity of
the test compound at the concentration of 1 ꢀ 10ꢁ8
M in the presence of
1 ꢀ 10ꢁ10 M Am80 in HL-60 cell differentiation assay. The dashed line shows the
level of differentiated cells (%) induced by 1 ꢀ 10ꢁ10 M Am 80 alone. Abbreviations
are Pr: n-propyl, Bu: n-butyl, Pen: n-pentyl, and Hex: n-hexyl group as the
N-substituent.
Figure 2. (a) The structures of synthetic agonists of RARs and RXRs. (b) The activity
alterations caused by introduction of the N-cyclopropylmethyl substituent. EC50
means the concentration that induced differentiation of 50% of cells in the presence
of the compound alone in HL-60 cell differentiation assay. SEC50 (synergistic
effective concentration) means the concentration that induced differentiation of
50% of cells in the presence of both the compound and an RAR agonist (1 ꢀ 10ꢁ10
M
Am80).12
longer, the synergistic activity increased, and the N-n-propyl com-
pound showed the most potent synergistic activity. Introduction of
N-alkyl groups longer than n-propyl reduced the synergistic activ-
ity, and the activity was completely lost in the case of the N-n-hex-
yl compound. Based on the structure–activity relationships of RXR
ligands, we focused on further modification of the N-substituent on
the diphenylamine skeleton. Here, we describe the design, synthe-
sis and biological activities of novel diphenylamine-based retinoid
modulators bearing a long or large N-substituent.
Figure 4. Diphenylamine derivatives 9 and 10 with longer or larger N-substituent,
respectively.
nylamine skeleton was synthesized according to the reported pro-
cedure: Pd-catalyzed amination of aniline derivative (11) with
ethyl 4-iodobenzoate.13 Long alkyl chains, n-heptyl and n-octyl,
were introduced on the nitrogen atom of 12 by reaction with the
corresponding iodoalkane in the presence of NaH as a base. The es-
ter group of N-alkylated compounds (13) was hydrolyzed with
aqueous 20% KOH solution to give compounds 9. Compound 12
was treated with various benzyl halides in the presence of NaH
as a base to afford N-benzylated compounds (14), which were
hydrolyzed with 20% KOH aqueous solution to afford the corre-
sponding carboxylic acids 10a–e. An N-pentafluorobenzyl deriva-
tive (15) was synthesized similarly from the reaction of 12 with
the corresponding benzyl bromide. During the following hydrolysis
of the ester group, nucleophilic substitution reaction on the penta-
fluorophenyl ring of 15 by an ethoxide anion produced from 20%
KOH and EtOH also occurs to afford an N-4-ethoxytetrafluoroben-
zyl derivative 10f in quantitative yield.14 The structure of 10f was
identified by several analytical methods. Low-MS spectra showed a
2. Results
2.1. Chemistry
Our previous studies showed that introduction of a bulky alkyl
group of moderate size (3 or 4 carbons) into the diphenylamine
skeleton is effective for obtaining potent RXR agonist activity. In
order to understand the effects of various N-substituents on the
biological activities and also to obtain retinoid antagonists, we de-
signed N-substituted diphenylamine derivatives (9) and (10) with
various n-alkyl and benzyl groups as longer and larger N-substitu-
ents, respectively (Fig. 4). In terms of N-benzyl groups, we chose six
substituents with quite different structures and electric properties
to understand the steric and electric effects.
The divergent synthesis of the designed molecules 9 and 10 is
summarized in Scheme 1. The key intermediate (12) with a diphe-

K. Ohta et al. / Bioorg. Med. Chem. 19 (2011) 2501–2507
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Scheme 1. Divergent synthesis of diphenylamine derivatives 9 and 10. Reagents and conditions: (a) Pd2(dba)3, rac-BINAP, NaOtBu, ethyl 4-iodobenzoate, toluene, 48% yield;
(b) NaH, n-C7H15I or n-C8H15I, DMF, 96%-quantitative yields; (c) 20% KOH aq EtOH, 90%-quantitative yields; (d) NaH, benzyl halides, DMF, 93%-quantitative yields; (e) NaH,
pentafluorobenzyl bromide, DMF, 99% yield.
Figure 5. Inhibitory activity of (a) N-alkylated and (b) N-benzylated compounds toward HL-60 cell differentiation induced by 3.3 ꢀ 10ꢁ10 M Am80.
molecular ion peak at m/z 529 in high-resolution MS analysis, with
the chemical composition C30H31F4NO3. 1H NMR spectra indicated
the presence of an ethoxy group and 19F spectra showed two kinds
of peaks of AA0XX0 split type. In the 13C NMR spectra, we observed
four kinds of split aromatic carbon peaks caused by coupling with
fluorine atoms. Therefore, we identified the structure of compound
10f as 4-[N-(4-ethoxy-2,3,5,6-tetrafluoro)benzyl-N-(5,6,7,8-tetra-
hydro-5,5,8,8-tetramethylnaphthalene-2-yl)amino]benzoic acid.
The purity of all synthesized compounds was confirmed by ele-
mental analyses.
were identified by nitro blue tetrazolium (NBT) reduction assay.15
Compounds and 10 did not show differentiation-inducing
9
activity alone, which means these compounds did not act as RAR
agonists. Next, their inhibitory activity toward cell differentiation
induced by the RAR agonist Am80 was examined. In the presence
of 3.3 ꢀ 10ꢁ10 M Am80, which induced differentiation of about
60% of cells, compounds 9 and 10, except for 10a, dose-depen-
dently inhibited cell differentiation, and the number of differenti-
ated cells decreased to less than 10% at 1 ꢀ 10ꢁ6
M test
compound (Fig. 5). The inhibitory activity of 9a with a n-heptyl
group is similar to that of 9b with a n-octyl group. Compound
10a with an N-benzyl group showed weak inhibitory activity,
compared to those of 9a and 9b. Compounds 10b and 10c with
an N-4-methylbenzyl and N-4-trifluoromethylbenzyl group,
respectively, showed more potent inhibitory activity than 10a.
Since the inhibitory activities of 10b and 10c were similar, the
2.2. Cell differentiation assay using HL-60
The biological activity of the synthesized compounds 9 and 10
was examined in terms of the ability to induce differentiation of
human promyelocytic leukemia HL-60 cells.11 Differentiated cells

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Figure 6. Transactivation-inhibitory activity of compounds 9a and 10f. Transactivation assays were carried out using COS-1 cells transfected with hRAR (
a
, b, or
c) and
(TREpal)3-TKLUC. Receptor transactivation was induced with 3 ꢀ 10ꢁ9 M Am80 for RAR
a
and b, or 3 ꢀ 10ꢁ9 M ATRA for RAR
c. The vertical scale is the transactivation relative
to that with agonist only, taken as 100.
electronic properties of the aromatic ring appear not to be signifi-
cant for the inhibitory activity. Compounds with a bulkier substitu-
ent, such as biphenyl structure (compound 10d) or a naphthyl
group (compound 10e), showed higher activity, and inhibited the
Am80-induced cell differentiation to about 10% and 30% at
1 ꢀ 10ꢁ7 M test compound, respectively. Compound 10f was the
most potent inhibitor among the test compounds, and inhibited
the retinoidal effect of Am80 to below the 10% level at
1 ꢀ 10ꢁ7 M. The cell differentiation–inhibitory activity was af-
fected by the bulkiness of the N-benzyl substituent.
derivatives 9a and 10f binds to the RAR site of RXR–RAR heterodi-
mers and inhibits the cell differentiation-inducing activity of the
RAR agonist. Although compounds 9a and 10f have a similar skele-
ton to compounds PA452 (16) and 17, its receptor selectivity was
completely different (Fig. 7b). We speculate that the difference
arises from a difference in the conformations of the N-substituents
of these compounds. Sulfonamide generally exists in a gauche-like
conformation, and the torsion angles of C(Ar)–N–S–C(Ar) are large.
In the case of compounds 9a and 10f, the N-substituent (n-alkyl or
benzyl, respectively) would have little effect on the extended
conformation of the diphenylamine structure, and so compounds
9a and 10f bind preferentially to RARs over RXRs. As summarized
in Figure 7a, the diphenylamine skeleton is suitable for binding
to both receptors, RARs and RXRs, and the N-substituent deter-
mines the receptor selectivity and the biological activity of the
compounds. Similar findings were reported for diphenylamine
2.3. Transactivation assay of 10f for RARs and RXRs
In order to confirm the inhibitory mechanism of the diphenyl-
amine derivatives, transient transactivation assay for RARs and
RXRs was examined using two selected compounds, 9a with an
N-alkyl group, and 10f with an N-benzyl group that was the most
active compound in HL-60 cell differentiation assay.16 Unexpect-
edly, compounds 9a and 10f did not inhibit the transactivation of
RXRs activated with 1 ꢀ 10ꢁ8 M RXR agonist PA024 (7b) (data
not shown). On the other hand, the transactivation induced by
3 ꢀ 10ꢁ9 M Am80 (RAR
a and b) or ATRA (RARc) with all subtypes
of RARs was dose-dependently inhibited by 9a or 10f (Fig. 6). The
result indicated that the introduction of a bulky N-alkyl group into
the diphenylamine skeleton of an RXR agonist afforded an RAR
antagonist.
3. Discussion
Various synthetic ligands for RARs and RXRs have been devel-
oped. Typical synthetic RAR agonists consist of two aromatic rings
with hydrophobic substituents or a carboxyl group linked by two
or three atoms, and have a rather planar extended structure, while
typical RXR agonists have a folded structure with two aromatic
moieties linked by one atom. DA010 (8) acted as dual, but weak
agonist of RARs and RXRs. The introduction of an N-alkyl group with
1–5 carbon atoms (R1) or a substituent at the ortho position to the
nitrogen atom on the hydrophobic aromatic ring (R2) increased the
RXR agonist potency with loss of RAR agonist activity (Fig. 7a).
Some aza derivatives, such as PA024 (7b), are more potent RXR ago-
nists. Introduction of an N-acyl group, such as an acetyl group, into
DA010 (8) was not effective for obtaining RXR agonist activity (our
unpublished results). Several RXR antagonists have been developed
based on the structures of DA024 (7a) and PA024 (7b) as lead com-
pounds. Thus, PA452 (16), with a long substituent on the aromatic
ring, and compound 17 showed RXR-selective antagonist activity
(Fig. 7b).17,18 The present study suggests that the diphenylamine
Figure 7. (a) The structure–activity relationships of N-substituents of diphenyl-
amine derivatives. (b) The structures of the RXR antagonists PA452 (16) and N-
sulfonylated compound 17.

K. Ohta et al. / Bioorg. Med. Chem. 19 (2011) 2501–2507
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derivatives showing agonist or antagonist activity for other nuclear
receptors, such as thyroid hormone receptors, estrogen receptor
and androgen receptor.19–21 Thus, the diphenylamine skeleton
seems to be a key basic structure to develop specific nuclear recep-
tor modulators.
1.70 (s, 4H), 3.66 (t, J = 7.7 Hz, 2H), 4.31 (q, J = 7.0 Hz, 2H), 6.64 (d,
J = 8.8 Hz, 2H), 6.92 (dd, J = 2.6 Hz, 8.1 Hz, 1H), 7.10 (d, J = 2.2 Hz,
1H), 7.31 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H). Anal. Calcd
for C30H43NO2: C, 80.13; H, 9.64; N, 3.12. Found: C, 80.17; H,
9.46; N, 3.11.
5.2.3. Ethyl 4-[N-n-octyl-N-(5,6,7,8-tetrahydro-5,5,8,8-tetra-
methylnaphthalene-2-yl)amino]benzoate
4. Conclusion
96% Yield; colorless powder (n-hexane); mp 70 °C; 1H NMR
(400 MHz, CDCl3) d (ppm) 0.87 (t, J = 7.0 Hz, 3H), 1.24 (s, 6H),
1.26 (br m 10H), 1.30 (s, 6H), 1.35 (t, J = 7.3 Hz, 3H), 1.68 (br m,
2H), 1.70 (s, 4H), 3.65 (t, J = 7.7 Hz, 2H), 4.31 (q, J = 7.3 Hz, 2H),
6.64 (d, J = 8.8 Hz, 2H), 6.91 (dd, J = 2.2 Hz, 8.4 Hz, 1H), 7.09 (d,
J = 2.2 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.82 (dd, J = 2.2 Hz, 9.2 Hz,
2H).
In conclusion, novel diphenylamine derivatives 9a, 9b, and
10a–f with a long alkyl chain or a benzyl group as an N-substitu-
ent, inhibited the differentiation of HL-60 cells induced by RAR
agonists. The inhibitory mechanism of 9a and 10f was shown, by
means of transactivation assays for RARs and RXRs, to be antago-
nism of the RAR agonists at the RAR site of RAR–RXR heterodimers.
Interestingly, diphenylamine derivatives with a shorter alkyl group
(C1–C5) as the N-substituent acted as RXR agonists, while the
derivatives with a benzyl group acted as RAR antagonists, not
RXR ligands. The N-substituent of diphenylamine derivatives plays
an important role in determining the receptor selectivity between
RARs and RXRs, as well as the agonist or antagonist nature of the
activity.
5.2.4. Ethyl 4-[N-benzyl-N-(5,6,7,8-tetrahydro-5,5,8,8-tetra-
methylnaphthalene-2-yl)amino]benzoate
Quantitative yield; colorless needles (n-hexane); mp 128.5 °C;
1H NMR (400 MHz, CDCl3) d (ppm) 1.21 (s, 6H), 1.28 (s, 6H), 1.33
(t, J = 7.0 Hz, 3H), 1.68 (s, 4H), 4.31 (q, J = 7.0 Hz, 2H), 4.99 (s,
2H), 6.74 (dd, J = 1.8 Hz, 8.8 Hz, 2H), 7.03 (dd, J = 2.6 Hz, 8.4 Hz,
1H), 7.20 (d, J = 2.6 Hz, 1H), 7.22–7.25 (m, 1H), 7.29 (d, J = 8.4 Hz,
1H), 7.31 (d, J = 4.4 Hz, 4H), 7.80 (dd, J = 1.8 Hz, 9.2 Hz, 2H).
5. Experimental
5.1. General considerations
5.2.5. Ethyl 4-[N-(4-methylbenzyl)-N-(5,6,7,8-tetrahydro-
5,5,8,8-tetramethylnaphthalene-2-yl)amino]benzoate
Quantitative yield; colorless powder (n-hexane); mp 107–
108 °C; 1H NMR (400 MHz, CDCl3) d (ppm) 1.21 (s, 6H), 1.28 (s,
6H), 1.33 (t, J = 7.0 Hz, 3H), 1.68 (s, 4H), 2.32 (s, 3H), 4.29 (q,
J = 7.0 Hz, 2H), 4.60 (s, 2H), 6.74 (dd, J = 1.8 Hz, 9.2 Hz, 2H), 7.02
(dd, J = 2.2 Hz, 8.4 Hz, 1H), 7.11 (d, J = 7.7 Hz, 2H), 7.19 (d,
J = 8.4 Hz, 1H), 7.20 (d, J = 2.2 Hz, 1H), 7.28 (d, J = 8.4 Hz, 4H),
7.79 (d, J = 9.2 Hz, 2H).
Melting points were determined with a Yanaco micro melting
point apparatus and were not corrected. 1H NMR, 13C NMR,
and 19F spectra were recorded with JEOL JNM-GX-400 and
JNM-EX-270 spectrometers. Chemical shifts for 1H NMR spectra
were referenced to tetramethylsilane (0.0 ppm) as an internal
standard. Chemical shifts for 13C NMR spectrum were referenced
to residual 13C present in the deuterated solvent. Chemical shift
values for 19F spectra were referenced relative to external trifluo-
roacetic acid (CF3CO2H, 0.0 ppm, with negative values upfield).
The splitting patterns are designated as follows: s (singlet), d
(doublet), t (triplet) and m (multiplet). Elemental analyses were
carried out in the Microanalytical Laboratory, Faculty of Pharma-
ceutical Sciences, The University of Tokyo, and were within 0.3%
of the theoretical values. Column chromatography was carried out
5.2.6. Ethyl 4-[N-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-
naphthalene-2-yl)-N-(4-trifluoromethylbenzyl)amino]benzoate
93% Yield; colorless prisms (n-hexane); mp 97 °C; 1H NMR
(400 MHz, CDCl3) d (ppm) 1.22 (s, 6H), 1.29 (s, 6H), 1.33 (t,
J = 7.3 Hz, 3H), 1.69 (s, 4H), 4.30 (q, J = 7.3 Hz, 2H), 5.04 (s, 2H),
6.71 (d, J = 9.2 Hz, 2H), 7.01 (dd, J = 2.6 Hz, 8.4 Hz, 1H), 7.19 (d,
J = 2.2 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.43 (d, J = 8.1 Hz, 2H),
7.57 (d, J = 8.1 Hz, 2H), 7.82 (d, J = 9.2 Hz, 2H).
using Merck silica gel 60 (0.063–0.200 lm) and TLC was per-
formed on Merck silica gel F254. Reagents were purchased from
Wako Pure Chemical Industries, Ltd, Sigma–Aldrich Co., and To-
kyo Chemical Industry, Ltd (TCI). All solvents were commercial
products of reagent grade, and were used without further
purification.
5.2.7. Ethyl 4-[N-(biphen-2-ylmethyl)-N-(5,6,7,8-tetrahydro-
5,5,8,8-tetramethylnaphthalene-2-yl)amino]benzoate
Quantitative yield; colorless oil; 1H NMR (400 MHz, CDCl3) d
(ppm) 1.19 (s, 6H), 1.28 (s, 6H), 1.33 (t, J = 7.3 Hz, 3H), 1.68 (s,
4H), 4.29 (q, J = 7.3 Hz, 2H), 4.86 (s, 2H), 6.65 (dd, J = 1.8 Hz,
8.8 Hz, 2H), 6.98 (dd, J = 2.6 Hz, 8.4 Hz, 1H), 7.15 (d, J = 2.6 Hz,
1H), 7.23–7.32 (m, 6H), 7.35–7.45 (m, 3H), 7.48–7.52 (m, 1H),
7.77 (dd, J = 2.2 Hz, 9.2 Hz, 2H).
5.2. Synthesis
5.2.1. General procedure of N-alkylation or N-benzylation of
diphenylamine derivatives
A suspension of NaH in dry DMF was added to a solution of 11 in
dry DMF under an Ar atmosphere, and the mixture was stirred for
20 min. The corresponding iodoalkane or benzyl halide was added
to the mixture, and the whole was stirred at room temperature,
then poured into water, and extracted with ether. The organic layer
was dried over Na2SO4 and concentrated. The residue was purified
with flash column chromatography on silica gel (AcOEt/n-hex-
ane = 1:10) to afford the corresponding N-substituted compound.
5.2.8. Ethyl 4-[N-(naphthalene-2-ylmethyl)-N-(5,6,7,8-tetra-
hydro-5,5,8,8-tetramethylnaphthalene-2-yl)amino]benzoate
99% Yield; 1H NMR (400 MHz, CDCl3) d (ppm) 1.22 (s, 6H), 1.28
(s, 6H), 1.32 (t, J = 7.3 Hz, 3H), 1.68 (s, 4H), 4.29 (q, J = 7.3 Hz, 2H),
5.15 (s, 2H), 6.80 (dd, J = 2.2 Hz, 9.2 Hz, 2H), 7.09 (dd, J = 2.2 Hz,
8.4 Hz, 1H), 7.28 (d, J = 2.6 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H),
7.42–7.47 (m, 3H), 7.73–7.82 (m, 4H), 7.81 (dd, J = 2.2 Hz, 9.2 Hz,
2H).
5.2.2. Ethyl 4-[N-n-heptyl-N-(5,6,7,8-tetrahydro-5,5,8,8-tetra-
methylnaphthalene-2-yl)amino]benzoate
5.2.9. Ethyl 4-[N-pentafluorobenzyl-N-(5,6,7,8-tetrahydro-
5,5,8,8-tetramethylnaphthalene-2-yl)amino]benzoate
Quantitative yield; colorless powder (n-hexane); mp 65.5 °C; 1H
NMR (400 MHz, CDCl3) d (ppm) 0.87 (t, J = 7.3 Hz, 3H), 1.24 (s, 6H),
1.27 (br m 8H), 1.30 (s, 6H), 1.35 (t, J = 10.0 Hz, 3H), 1.64 (br m, 2H),
99% Yield; colorless powder (n-hexane); mp 116–120 °C; 1H
NMR (400 MHz, CDCl3) d (ppm) 1.17 (s, 6H), 1.26 (s, 6H), 1.35

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K. Ohta et al. / Bioorg. Med. Chem. 19 (2011) 2501–2507
(t, J = 7.3 Hz, 3H), 1.67 (s, 4H), 4.32 (q, J = 7.0 Hz, 2H), 4.95 (s, 2H),
6.75 (dd, J = 2.2 Hz, 9.2 Hz, 2H), 6.85 (dd, J = 2.2 Hz, 8.4 Hz, 1H),
6.98 (d, J = 2.2 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H), 7.87 (dd, J = 1.8 Hz,
8.8 Hz, 2H).
5.2.16. 4-[N-(Biphen-2-ylmethyl)-N-(5,6,7,8-tetrahydro-5,5,8,8-
tetramethylnaphthalene-2-yl)amino]benzoic acid (10d)
97% Yield; colorless prisms (CH2Cl2–n-hexane); mp 237–239 °C;
1H NMR (400 MHz, CDCl3) d (ppm) 1.19 (s, 6H), 1.28 (s, 6H), 1.68 (s,
4H), 4.87 (s, 2H), 6.64 (d, J = 9.2 Hz, 2H), 6.98 (dd, J = 2.2 Hz, 8.8 Hz,
1H), 7.15 (d, J = 2.6 Hz, 1H), 7.23–7.31 (m, 6H), 7.35–7.44 (m, 4H),
7.48–7.79 (m, 1H), 7.80 (d, J = 8.8 Hz, 2H); Anal. Calcd for
5.2.10. General procedure of hydrolysis of ethyl ester
A mixture of the ester compound 12, 13 or 14 in EtOH and 20%
aqueous solution of KOH was stirred at room temperature. The
reaction mixture was poured into 1 M hydrochloric acid, and the
solution was extracted with CH2Cl2. The organic layer was dried
over Na2SO4 and concentrated. The residue was recrystallized from
appropriate solvents to afford the corresponding carboxylic acid
derivatives.
C34H35NO2: C, 83.40;H, 7.21;N, 2.86. Found:C, 83.11; H, 7.50;N, 2.75.
5.2.17. 4-[N-(Naphthalen-2-ylmethyl)-N-(5,6,7,8-tetrahydro-
5,5,8,8-tetramethylnaphthalene-2-yl)amino]benzoic acid (10e)
Quantitative yield; colorless powder (CH2Cl2–n-hexane); mp
233 °C; 1H NMR (400 MHz, CDCl3) d (ppm) 1.22 (s, 6H), 1.28 (s,
6H), 1.68 (s, 4H), 5.15 (s, 2H), 6.79 (dd, J = 2.2 Hz, 9.2 Hz, 2H),
7.09 (dd, J = 2.2 Hz, 8.4 Hz, 1H), 7.27 (d, J = 2.6 Hz, 1H), 7.31 (d,
J = 8.4 Hz, 1H), 7.44 (m, 3H), 7.78 (m, 4H), 7.84 (dd, J = 2.2 Hz,
9.2 Hz, 2H); Anal. Calcd for C32H33NO2: C, 82.90; H, 7.18; N, 3.02.
Found: C, 82.66; H, 7.48; N, 2.73.
5.2.11. 4-[N-n-Heptyl-N-(5,6,7,8-tetrahydro-5,5,8,8-tetra-
methylnaphthalene-2-yl)amino]benzoic acid (9a)
99% Yield; colorless powder (CH2Cl2–n-hexane); mp 168 °C; 1H
NMR (400 MHz, CDCl3) d (ppm) 0.87 (t, J = 6.6 Hz, 3H), 1.25 (s, 6H),
1.25–1.31 (br m 8H), 1.31 (s, 6H), 1.70 (br m, 2H), 1.70 (s, 4H), 3.66
(t, J = 7.7 Hz, 2H), 6.63 (dd, J = 8.8 Hz, 2H), 6.93 (dd, J = 2.2 Hz,
8.4 Hz, 1H), 7.10 (d, J = 1.8 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.86
(d, J = 9.2 Hz, 2H). Anal. Calcd for C28H39NO2: C, 79.76; H, 9.32; N,
3.32. Found: C, 79.80; H, 9.62; N, 3.06.
5.2.18. 4-[N-(4-Ethoxy-2,3,5,6-tetrafluoro)benzyl-N-(5,6,7,8-
tetrahydro-5,5,8,8-tetramethylnaphthalene-2-
yl)amino]benzoic acid (10f)
Quantitative yield; colorless powder (CH2Cl2–n-hexane); mp
227–229 °C; 1H NMR (400 MHz, CDCl3) d (ppm) 1.16 (s, 6H), 1.25
(s, 6H), 1.36 (t, J = 7.3 Hz, 3H), 1.66 (s, 4H), 4.22 (q, J = 7.0 Hz,
2H), 4.92 (s, 2H), 6.75 (dd, J = 2.2 Hz, 9.2 Hz, 2H), 6.87 (dd,
J = 2.2 Hz, 8.4 Hz, 1H), 6.98 (d, J = 2.2 Hz, 1H), 7.26 (d, J = 8.4 Hz,
1H), 7.90 (dd, J = 1.8 Hz, 8.8 Hz, 2H); 13C NMR (100 MHz, CDCl3) d
(ppm) 15.0, 31.2, 31.4, 33.7, 33.9, 34.4, 42.8, 70.8, 113.1, 119.3,
124.9, 125.4, 127.9, 130.8, 136.0 (t), 138.6 (d), 141.0, 142.2 (d),
142.8, 143.2 (t), 145.8, 151.7, 167.2; 19F NMR (376 MHz, CDCl3) d
(ppm) ꢁ31.82 (AA’XX’), ꢁ17.37 (AA’XX’); MS (EI) m/z 529 (M+),
514 (100%); HRMS Calcd for C30H31NO3F4: 529.2240, Found:
529.2239; Anal. Calcd for C30H31NO3F4: C, 68.04; H, 5.90; N, 2.65.
Found: C, 67.78; H, 5.89; N, 2.61.
5.2.12. 4-[N-n-Octyl-N-(5,6,7,8-tetrahydro-5,5,8,8-tetra-
methylnaphthalene-2-yl)amino]benzoic acid (9b)
98% Yield; colorless cotton (CH2Cl2–n-hexane); mp 160 °C; 1H
NMR (400 MHz, CDCl3) d (ppm) 0.87 (t, J = 6.6 Hz, 3H), 1.25 (s,
6H), 1.26 (br m 10H), 1.31 (s, 6H), 1.70 (br m, 2H), 1.70 (s, 4H),
3.66 (t, J = 8.1 Hz, 2H), 6.63 (d, J = 9.2 Hz, 2H), 6.92 (dd, J = 2.2 Hz,
8.4 Hz, 1H), 7.10 (d, J = 2.2 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.87
(dd, J = 2.2 Hz, 9.2 Hz, 2H); Anal. Calcd for C29H41NO2: C, 79.95;
H, 9.49; N, 3.22. Found: C, 79.92; H, 9.54; N, 3.18.
5.2.13. 4-[N-Benzyl-N-(5,6,7,8-tetrahydro-5,5,8,8-tetra-
methylnaphthalene-2-yl)amino]benzoic acid (10a)
5.3. HL-60 cell differentiation-inducing assay
Quantitative yield; colorless powder (CH2Cl2–n-hexane); mp
272–273 °C; 1H NMR (400 MHz, CDCl3) d (ppm) 1.22 (s, 6H), 1.29
(s, 6H), 1.69 (s, 4H), 5.00 (s, 2H), 6.73 (dd, J = 1.8 Hz, 9.2 Hz, 2H),
7.04 (dd, J = 2.2 Hz, 8.4 Hz, 1H), 7.21 (d, J = 2.2 Hz, 1H), 7.23–7.25
(m, 1H), 7.30 (d, J = 7.3 Hz, 1H), 7.32 (d, J = 4.8 Hz, 4H), 7.84 (dd,
J = 2.2 Hz, 9.2 Hz, 2H); Anal. Calcd for C28H31NO2: C, 81.32; H,
7.56; N, 3.39. Found: C, 81.05; H, 7.49; N, 3.57.
The human promyelocytic leukemia cell line HL-60 was pro-
vided by Prof. F. Takaku (Faculty of Medicine, The University of To-
kyo) in 1980 and has been maintained in continuous suspension
culture. The cells are cultured in plastic flasks in RPMI1640 med-
ium, supplemented with 5% fetal bovine serum (FBS, not delipi-
dized), and antibiotics (penicillin
G and streptomycin) in a
humidified atmosphere of 5% CO2 in air at 37 °C. Test compounds
were dissolved in ethanol at 2 mM and added to the cells, which
were seeded at about 8 ꢀ 104 cells/mL; the final ethanol concentra-
tion was kept below 0.5%. Control cells were given only the same
volume of ethanol. Am80, as a positive control, was always assayed
at the same time. The cells were incubated for four days and
stained with Wright–Giemsa in order to check for morphological
change. The percentages of differentiated cells were determined
by NBT reduction assay. Cells were incubated for 20 min at 37 °C
in RPMI1640 medium (5% FBS) and an equal volume of phos-
phate-buffered saline (PBS) containing NBT (0.2%) and 12-O-tetra-
decanoylphorbol-13-acetate (TPA; 200 ng/mL). The percentage of
cells containing blue-black formazan was determined using a min-
imum of 200 cells. The evaluation of the differentiation from NBT
reduction assay was always consistent with the morphological re-
sult. Synergistic activity with Am80 was examined in the presence
of a suitable concentration of test compound according to the
method described above. In this experiment, the independent ef-
fects of Am80 and the test compound were always assayed, and
the percentages of differentiated cells were determined by NBT
reduction assay. The assays of test compounds were performed
at least twice, and the average of the two experiments was taken.
5.2.14. 4-[N-(4-Methylbezyl)-N-(5,6,7,8-tetrahydro-5,5,8,8-tetra-
methylnaphthalene-2-yl)amino]benzoic acid (10b)
Quantitative yield; colorless powder (CH2Cl2–n-hexane); mp
246–248 °C; 1H NMR (400 MHz, CDCl3) d (ppm) 1.22 (s, 6H), 1.29
(s, 6H), 1.68 (s, 4H), 2.32 (s, 3H), 4.96 (s, 2H), 6.73 (dd, J = 2.9 Hz,
9.2 Hz, 2H), 7.03 (dd, J = 2.2 Hz, 8.4 Hz, 1H), 7.12 (d, J = 8.1 Hz,
2H), 7.20 (d, J = 6.6 Hz, 1H), 7.21 (d, J = 2.2 Hz, 1H), 7.30 (d,
J = 8.4 Hz, 4H), 7.83 (d, J = 2.2 Hz, 9.2 Hz, 2H); Anal. Calcd for
C29H33NO2: C, 81.46; H, 7.78; N, 3.28. Found: C, 81.28; H, 7.82;
N, 3.44.
5.2.15. 4-[N-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethylnaphtha-
lene-2-yl)-N-(4-trifluoromethylbenzyl)amino]benzoic acid
(10c)
90% Yield; colorless powder (n-hexane); mp 209–210 °C; 1H
NMR (400 MHz, CDCl3) d (ppm) 1.22 (s, 6H), 1.29 (s, 6H), 1.69 (s,
4H), 5.04 (s, 2H), 6.70 (d, J = 8.8 Hz, 2H), 7.02 (dd, J = 2.2 Hz,
8.4 Hz, 1H), 7.19 (d, J = 2.2 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.44
(d, J = 8.4 Hz, 2H), 7.58 (d, J = 8.1 Hz, 2H), 7.86 (d, J = 2.2 Hz,
9.2 Hz, 2H); Anal. Calcd for C29H30NO2F3: C, 72.33; H, 6.28; N,
2.91. Found: C, 72.15; H, 6.41; N, 2.94.

K. Ohta et al. / Bioorg. Med. Chem. 19 (2011) 2501–2507
2507
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5.4. Transactivation assay for RARs and RXRs
Transient transactivation assays were carried out using COS-1
cells transfected with hRAR (
a, b, or c) and (TREpal)3-TKLUC or
mRXR ( , b, or ) and (DR1)5-pGL-TK. The COS-1 cells were ob-
a
c
tained from the Japanese Cancer Resources Bank (JCRB) and were
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DMEM and transfection was performed by use of the transfection
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Acknowledgments
The authors are grateful to Prof. P. Chambon, INSERM, France,
for his generous supply of RXR-expression vectors, and to Prof.
M. Yamamoto, Institute of Medical Science, The University of To-
kyo, for his generous supply of hTR-expression vector. The work
described in this paper was partially supported by Grants-in-Aid
for Scientific Research from The Ministry of Education, Science,
Sports and Culture, Japan (Grand No. 22136013).
18. Morishita, K.; Yakushiji, N.; Ohsawa, F.; Takamatsu, K.; Matsuura, N.;
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Bioorg. Med. Chem. Lett. 2009, 19, 1001.
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