Structure-activity relationship study on benzoic acid part of diphenylamine-based retinoids

Article abstract of DOI:10.1016/j.bmcl.2012.11.008

Based on structure-activity relationship studies of the benzoic acid part of diphenylamine-based retinoids, the potent RXR agonist 4 was derivatized to obtain retinoid agonists, synergists, and an antagonist. Cinnamic acid derivatives 5 and phenylpropionic acid derivatives 6 showed retinoid agonistic and synergistic activities, respectively. The difference of the activities is considered to be due to differences in the flexibility of the carboxylic acid-containing substituent on the diphenylamine skeleton. Compound 7, bearing a methyl group at the meta position to the carboxyl group, was an antagonist, dose-dependently inhibiting HL-60 cell differentiation induced by 3.3 × 10^-10 M Am80.

Full text of DOI:10.1016/j.bmcl.2012.11.008

Bioorganic & Medicinal Chemistry Letters 23 (2013) 81–84
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship study on benzoic acid part
of diphenylamine-based retinoids
Kiminori Ohta a, Emiko Kawachi b, Koichi Shudo c, Hiroyuki Kagechika b,
⇑
a Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
b Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
c 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 on structure–activity relationship studies of the benzoic acid part of diphenylamine-based reti-
noids, the potent RXR agonist 4 was derivatized to obtain retinoid agonists, synergists, and an antagonist.
Cinnamic acid derivatives 5 and phenylpropionic acid derivatives 6 showed retinoid agonistic and syner-
gistic activities, respectively. The difference of the activities is considered to be due to differences in the
flexibility of the carboxylic acid-containing substituent on the diphenylamine skeleton. Compound 7,
bearing a methyl group at the meta position to the carboxyl group, was an antagonist, dose-dependently
inhibiting HL-60 cell differentiation induced by 3.3 Â 10À10 M Am80.
Received 20 September 2012
Revised 1 November 2012
Accepted 6 November 2012
Available online 15 November 2012
Keywords:
Retinoid
Diphenylamine
Agonist
Ó 2012 Elsevier Ltd. All rights reserved.
Antagonist
Synergist
Retinoids, which are natural and synthetic analogues of all-
trans-retinoic acid (ATRA), play an important role in cell differenti-
ation, 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 of
into account the bent structure of 9CRA, we replaced the benzani-
lide skeleton with a diphenylamine structure to obtain RXR ago-
nists, such as DA124 (Fig. 1).10 The diphenylamine skeleton has
some advantages for drug discovery, because it is readily con-
structed by Pd-catalyzed coupling of aniline derivatives with aryl
halides, and the linker nitrogen atom can also be readily modified
with various substituents.11 Indeed, the diphenylamine skeleton
has been used as a central scaffold of selective nuclear receptor
modulators for estrogen receptor (ER),12 androgen receptor
which has three subtypes, a . The RARs and RXRs are li-
, b, and c 3
gand-inducible transcription factors, and their endogenous ligands
are ATRA and 9-cis-retinoic acid (9CRA), respectively (Fig. 1).4 Ma-
jor retinoidal activities are elicited by RXR–RAR heterodimers, in
which RXR is a silent partner.5 Thus, the heterodimers can be acti-
vated by an RAR agonist such as Am80 (Fig. 1),6 but not by an RXR
agonist alone. Structurally, a hydrophobic moiety and a polar car-
boxyl group are necessary for binding to RARs and RXRs. The posi-
tional and spatial relations between these parts are significant for
the activity. RAR ligands have a straight and planar skeleton, while
RXR ligands have a bent and twisted structure. As established dur-
ing the development of Am80, modification of the straight and pla-
nar ATRA structures with aromatic rings and the introduction of
heteroatoms affords highly active and selective RAR ligands with
remarkable chemical stability and good bioavailability.7,8
RXR agonists act as retinoid synergists, and dose-dependently
increase the activity of low concentrations of retinoids.9 Taking
⇑
Corresponding author. Tel.: +81 3 5280 8032; fax: +81 3 5280 8127.
E-mail addresses: kage.omc@tmd.ac.jp, kage.chem@tmd.ac.jp (H. Kagechika).
Figure 1. Structures of endogenous and synthetic RAR and RXR ligands.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.008

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K. Ohta et al. / Bioorg. Med. Chem. Lett. 23 (2013) 81–84
Figure 2. Enhancement of the potency and selectivity of retinoid synergistic activity by structural modifications of diphenylamine-based retinoids. EC50 and SEC50
(synergistic effective dose) mean half-maximal (50%) effective concentration of test compound alone and in the presence of the RAR agonist Am80 (3.3 Â 10À10 M).10
(AR),13 and thyroid hormone receptor (TR),14 and is considered as a
privileged structure for the development of specific nuclear recep-
tor modulators. Several diphenylamine-based retinoids have been
synthesized by using DA124 as a lead compound,10,15–17 and the
structure–activity relationships on the nitrogen atom and on the
hydrophobic benzene ring of the diphenylamine skeleton have
been well investigated (Fig. 2). Among the synthesized diphenyl-
amine derivatives, compound 1 showed dual RAR and RXR agonis-
tic activities, that is, compound 1 has both retinoid agonistic and
synergistic activities.9a Introduction of a medium-sized N-alkyl
substituent such as a n-propyl group into 1, yielding 2, remarkably
diminished the retinoidal activity and enhanced the retinoid syner-
gistic activity, which was further enhanced by introduction of a
methyl group on the hydrophobic benzene ring, as observed in
the RXR-selective compounds 3 and 4 (Fig. 2).9a On the other hand,
there is little information on the structure–activity relationship for
the benzoic acid part of diphenylamine-based retinoids, except in
the case of aza analogs, such as pyridine- and pyrimidinecarboxylic
acids. Therefore, we focused on structural modifications of the ben-
zoic acid part of compound 1, and designed cinnamic acid deriva-
tives 5 and phenylpropionic acid derivatives 6 (Fig. 3). Compound 7
bearing a methyl group ortho to the linking amino group on the
benzoic acid part was also designed with the aim of obtaining en-
hanced synergistic activity by twisting the conformation of the
diphenylamine skeleton, by analogy with compounds 3 and 4.
The cinnamic acid derivatives 5 and the phenylpropionic acid
derivatives were synthesized as illustrated in Scheme 1.
6
5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthylamine (8a) and
5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthylamine (8b)
were synthesized by the literature procedure,6a and then reacted
with methyl 4-bromocinnamate under the conditions of the Buck-
wald amination reaction to afford the key intermediates with a
diphenylamine skeleton.13 They were treated with iodoalkane
and NaH to obtain the N-alkylated diphenylamine derivatives 9.
The ester group of 9 was hydrolyzed with 20% potassium hydrox-
ide to afford the corresponding carboxylic acids 5. The unsaturated
double bond of compounds 9 was reduced by catalytic hydrogena-
tion with Pd/C, followed by hydrolysis, affording the phenylpropi-
onic acid derivatives 6.
Synthesis of compound 7 is summarized in Scheme 2. 3-
Methyl-4-nitrobenzoic acid (10) was esterified with methanol, fol-
lowed by reduction of the nitro group to afford an aniline deriva-
tive 11. Compound 11 was reacted with 2-bromo-5,6,7,8-
tetrahydro-5,5,8,8-tetramethylnaphthalene in the presence of Pd
catalyst to afford the diphenylamine derivative 12.11 N-Methyla-
tion of 12, followed by hydrolysis of the ester group, afforded the
desired carboxylic acid 7.
Biological activities of the newly prepared compounds were
determined by assay of activity to induce differentiation of human
acute promyelocytic leukemia cell line, HL-60.16a Differentiated
cells were evaluated from the morphological changes, and also
determined by nitro blue tetrazolium (NBT) reduction assay.18 Fig-
ure 4 summarizes the retinoidal activity of compounds 5–7. N-
methylated derivatives 5a, 5c, 6a, and 6c showed moderate or
weak differentiation-inducing activity, and have more potent reti-
noidal activity than the corresponding N-n-propylated derivatives
5b, 5d, 6b, and 6d, as observed in the lower activity of 2 compared
with 1. That is, retinoidal activity can tolerate N-methyl group of
the diphenylamine skeleton but can not N-n-propyl group. There
is a remarkable difference of retinoidal activity between 5 and 6;
the activity of the cinnamic acid derivatives 5a and 5c was much
greater than that of the corresponding phenylpropionic acid deriv-
atives 6a and 6c. Thus, flexibility and planarity around the carbox-
ylic acid group of the diphenylamine derivatives markedly
influence the retinoidal activity. The activities of 5a (EC50
:
2.2 Â 10À8 M) and 5c (EC50: 3.8 Â 10À8 M) are more active that
the corresponding benzoic acid derivatives 1 and 4, and the elonga-
tion in length between hydrophobic part and the polar carboxylic
acid would increase the retinoid agonistic activity. Compound 7
bearing a methyl group on the phenyl ring exhibited no retinoidal
activity.
Figure 3. Target molecules for the structure–activity relationship study of the
benzoic acid moiety.

K. Ohta et al. / Bioorg. Med. Chem. Lett. 23 (2013) 81–84
83
Scheme 1. Syntheses of the cinnamic acids 5 and phenylpropionic acids 6. Reagents and conditions: (a) Pd2(dba)3, rac-BINAP, NaOtBu, methyl 4-bromocinnamate, toluene;
(b) NaH, iodoalkane, DMF; (c) 20% KOH aq, EtOH; (d) Pd/C, H2, MeOH.
Scheme 2. Synthesis of 7. Reagents and conditions: (a) c.H2SO4, MeOH; (b) Pd/C, H2, MeOH; (c) Pd2(dba)3, rac-BINAP, NaOtBu, 2-bromo-5,6,7,8-tetrahydro-5,5,8,8-
tetramethylnaphthalene, toluene; (d) NaH, iodomethane, DMF; (e) 20% KOH aq, MeOH.
Figure 4. Retinoidal activities of diphenylamine derivatives 5–7 in HL-60 cell assay.
Figure 5. Retinoid synergistic activity of compounds 5–7 in the presence of 1 Â 10À10 M Am80 in HL-60 cell differentiation assay. 1 Â 10À10 M Am80 alone (control) induced
differentiation of less than 10% cells.
Next, the retinoid synergistic activity of the synthesized com-
pounds was evaluated in the presence of 1 Â 10À10 M Am80. At this
concentration, Am80 induced differentiation of 5–10% of the cells
(Fig. 5).12a Compounds 5 showed potent differentiation-inducing
activity, but this appeared to be mostly due to their own intrinsic
activity (compare Fig. 4 with Fig. 5). Phenylpropionic acid deriva-
tives 6 showed little or no activity alone, but they remarkably en-
hanced the retinoidal activity of 1 Â 10À10 M Am80, although the

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K. Ohta et al. / Bioorg. Med. Chem. Lett. 23 (2013) 81–84
group onto the benzoic acid moiety generated a retinoid antagonist
7. Overall, the present and previous results indicate that diphenyl-
amine-based retinoids change their mode of action in a substitu-
ent-dependent manner. The structural information obtained here
and the active compounds that we have obtained should be useful
to develop novel modulators of retinoid actions with potential clin-
ical applications.
Acknowledgment
The work described in this paper was partially supported by
Grants-in-Aid for Scientific Research from The Ministry of Educa-
tion, Science, Sports and Culture, Japan (Grant No. 22136013).
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