Regulation of retinoidal actions by diazepinylbenzoic acids. Retinoid synergists which activate the RXR-RAR heterodimers

Article abstract of DOI:10.1021/jm9704309

In human HL-60 promyelocytic leukemia cells, diazepinylbenzoic acid derivatives can exhibit either antagonistic or synergistic effects on the differentiation-inducing activities of natural or synthetic retinoids, the activity depending largely on the nature of the substituents on the diazepine ring. Thus, a benzolog of the retinoid antagonist LE135 (6), 4-(13H- 10,11,12,13-tetrahydro-10,10,13,13,15-pentamethyldinaphtho[2,3-b][1,2- e]diazepin-7-yl)benzoic acid (LE540, 17), exhibits a 1 order of magnitude higher antagonistic potential than the parental LE135 (6). In contrast, 4- [5H-2,3-(2,5-dimethyl-2,5-hexano)-5-methyldibenzo[b,e][1,4]diazepin-11-yl]- benzoic acid (HX600, 7), a structural isomer of the antagonistic LE135 (6), enhanced HL-60 cell differentiation induced by RAR agonists, such as Am80 (2). This synergistic effect was further increased for a thiazepine, HX630 (29), and an azepine derivative, HX640 (30); both synergized with Am80 (2) more potently than HX600 (7). Notably, the negative and positive effects of the azepine derivatives on retinoidal actions can be related to their RAR- antagonistic and RXR-agonistic properties, respectively, in the context of the RAR-RXR heterodimer.

Full text of DOI:10.1021/jm9704309

4222
J . Med. Chem. 1997, 40, 4222-4234
Regu la tion of Retin oid a l Action s by Dia zep in ylben zoic Acid s.1 Retin oid
Syn er gists Wh ich Activa te th e RXR-RAR Heter od im er s
Hiroki Umemiya,† Hiroshi Fukasawa,† Masayuki Ebisawa,† Laurence Eyrolles,† Emiko Kawachi,†
Ghislaine Eisenmann,‡ Hinrich Gronemeyer,‡ Yuichi Hashimoto,§ Koichi Shudo,† and Hiroyuki Kagechika*,†
Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, J apan, and Institute of
Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, J apan, and Institut de Ge´ne´tique
et de Biologie Mole´culaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Sante´ et de la
Recherche Me´dicale, 67404 Illkirch Cedex, Strasbourg, France
Received J une 30, 1997X
In human HL-60 promyelocytic leukemia cells, diazepinylbenzoic acid derivatives can exhibit
either antagonistic or synergistic effects on the differentiation-inducing activities of natural
or synthetic retinoids, the activity depending largely on the nature of the substituents on the
diazepine ring. Thus, a benzolog of the retinoid antagonist LE135 (6), 4-(13H-10,11,12,13-
tetrahydro-10,10,13,13,15-pentamethyldinaphtho[2,3-b][1,2-e]diazepin-7-yl)benzoic acid (LE540,
17), exhibits a 1 order of magnitude higher antagonistic potential than the parental LE135
(6). In contrast, 4-[5H-2,3-(2,5-dimethyl-2,5-hexano)-5-methyldibenzo[b,e][1,4]diazepin-11-yl]-
benzoic acid (HX600, 7), a structural isomer of the antagonistic LE135 (6), enhanced HL-60
cell differentiation induced by RAR agonists, such as Am80 (2). This synergistic effect was
further increased for a thiazepine, HX630 (29), and an azepine derivative, HX640 (30); both
synergized with Am80 (2) more potently than HX600 (7). Notably, the negative and positive
effects of the azepine derivatives on retinoidal actions can be related to their RAR-antagonistic
and RXR-agonistic properties, respectively, in the context of the RAR-RXR heterodimer.
Ch a r t 1
Retinoids, i.e., retinoic acid (all-trans, 1a , Chart 1)
and its analogues, modulate various biological functions,
such as cell differentiation, proliferation, and embryonic
development in vertebrates.2 Retinoids also have po-
tential chemopreventive and therapeutic applications
in the fields of dermatology and oncology.3 Retinoic acid
(1a ) has a remarkable remedial effect on acute promy-
elocytic leukemia (APL).4 Further, the inhibitory effect
of retinoids on IL-6 production suggests their possible
usefulness in various IL-6-associated diseases, including
psoriasis and rheumatoid arthritis.5 Some synthetic
retinoids have been successfully used in the treatment
of psoriasis.6 However, retinoid therapy is still re-
stricted by the wide range of undesirable side effects.
Therefore, it is important to understand the mechanistic
basis of the pleiotropic retinoidal activities to provide
the basis for the design of action-specific retinoids with
extended clinical applicability.
It is well established that retinoidal activities result
mainly from the transcriptional regulation of specific
gene programs. Retinoids bind to and transactivate two
classes of receptors, retinoic acid receptors (RARR, -â,
-γ) and retinoid X receptors (RXRR, -â, -γ), both of which
belong to the steroid/thyroid nuclear receptor super-
family.2,7 RARs bind retinoic acid (1a ) and its 9-cis-
isomer (1b ), while RXRs bind only 9-cis-retinoic acid
(1b). It is generally accepted that the biological activi-
ties of retinoids are mediated by RAR-RXR het-
erodimers whose subunits follow a defined hierarchy of
ligand responsiveness. Recent results have demon-
strated that, although both the RAR and RXR subunits
can efficiently bind their cognate ligands, only the RAR
† Graduate School of Pharmaceutical Sciences, University of Tokyo.
‡ Institut National de la Sante´ et de la Recherche Me´dicale.
§ Institute of Molecular and Cellular Biosciences, University of
Tokyo.
partner can autonomously respond to its cognate ligands.
In contrast, RXR ligands can contribute to the tran-
scriptional activity of the heterodimer only when the
X Abstract published in Advance ACS Abstracts, December 1, 1997.
S0022-2623(97)00430-5 CCC: $14.00 © 1997 American Chemical Society

Diazepinylbenzoic Acids as Retinoid Synergists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4223
Ch a r t 2
Ch a r t 3
Ch a r t 4
Ch a r t 5
RAR partner is liganded, thus giving rise to the concept
of RXR subordination.8
Importantly, recent efforts in the design of synthetic
retinoids and the availability of 3D structure informa-
tion9 have led to the identification of retinoids with
improved specificity and/or functionality. Various com-
pounds showing selective affinities for the receptors, i.e.,
RAR-, RXR-, or subtype-selective retinoids, have been
reported.10-12 For example, Am80 (2) and Am555S (3)
are retinoids which lack RARγ-activation potential10 but
nevertheless exhibit most of the retinoidal activities, like
retinoic acid (1a ).13 Ch55 (4) is an extraordinarily
potent synthetic RAR pan-agonist which does not bind
to CRABP (cellular retinoic acid-binding protein). In
support of these efforts, distinct roles of RAR- and RXR-
selective ligands in cell proliferation, differentiation, and
apoptosis were recently reported.14
In contrast to the structural variety of retinoid
agonists,2 only a few compounds are presently known
to display antagonistic activity; some of them exhibit
subtype-selectivity or affinities to RARs as high as those
of retinoid agonists.15-17 In structural terms most of
the retinoid antagonists consist of a bulky hydrophobic
group on an agonist skeleton and may be classified into
two types; type I antagonists (Chart 2) have a large
group in the hydrophobic region [for example, the 2,5-
dimethyl-2,5-hexano group of Am80 (2)] of retinoid
agonists, while the type II antagonists have a bulky
hydrophobic group in the middle of the molecule, around
the linking group between the alkylated aromatic and
the benzoic acid moieties.
promyelocytic leukemia cells HL-60.15c,18 On the other
hand, HX600 (7) enhances the differentiation-inducing
activities of retinoic acid (1a ) or Am80 (2) in HL-60
assay.19 Mechanistic investigation showed that HX600
(7) binds to RXR of RXR-RAR heterodimers to syner-
gize with RAR ligands.20 This positive regulation of
retinoidal actions by HX600 (7), which is completely
inactive alone, is a specific nuclear phenomenon, and
should be distinguished from the combination of retin-
oids with interferons, dibutyryl cAMP, and so on.21
Recently, RXR-selective ligands have been reported to
exhibit synergism with retinoid agonists,22 but the
activity of HX600 (7) is characteristically different from
that of RXR ligands such as LGD1069 (5).20 In this
paper, we describe the design, synthesis, and retinoid-
regulatory activities of various dibenzodiazepines and
related compounds.
Resu lts
Ch em ist r y. Various diaryldiazepinylbenzoic acids
(14-22) shown in Charts 3-5 were synthesized simi-
larly to LE135 (6).15c As an example, the synthetic
method for LE540 (17) is illustrated in Scheme 1.
Reaction of 5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-3-ni-
tro-2-naphthylamine (23) with 1-bromonaphthalene in
the presence of copper iodide, followed by methylation
with NaH and methyl iodide, gave the tertiary amine
25. When 25 was reduced with Fe under acidic condi-
tions, significant migration of the N-naphthyl group also
Previously, we reported the characterization of two
isomeric dibenzodiazepine derivatives, LE135 (6) and
HX600 (7), which exert opposite regulatory activities
when combined with “classical” retinoid agonists (Chart
1). LE135 (6) can bind selectively to RARR and RARâ,
with higher affinity to RARâ, and inhibits the retinoic
acid (1a ) or Am80 (2)-induced differentiation of human

4224 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Umemiya et al.
Sch em e 1a
a
(a) 1-Bromonaphthalene/copper iodide/K2CO3/∆; (b) NaH; CH3I; (c) H2/Pd-C; (d) p-CH3OOCPhCOCl; (e) ∆/polyphosphoric acid; (f)
NaOH/EtOH; (g) KNO3/H2SO4.
Sch em e 2a
a
(a) 1-Chloro-2-nitrobenzene/KOH/DMSO; (b) Fe/HCl; (c) p-CH3OOCPhCOCl; (d) ∆/polyphosphoric acid; (e) NaOH/EtOH; (f) AlCl3/2-
nitrobenzoyl chloride; (g) LiAlH4; (h) Ph3PCH3Br/n-BuLi; (i) H2/Pd-C.
occurred to afford a mixture of the desired N-methyl-
N-(1-naphthyl)diamine 26 and the isomeric N-methyl-
N′-(1-naphthyl)diamine. Similar migration did not
occur when the N-aryl group was a 5,6,7,8-tetrahydro-
5,5,8,8-tetramethyl-2-naphthyl or 2-naphthyl group, as
in the intermediates for the synthesis of HX600 (7) or
LE550 (18), respectively. Compound 25 was success-
fully reduced by catalytic hydrogenation to give 26
(86%), which was acylated with terephthalic acid monom-
ethyl ester chloride to give the amide 27 in 95%.
Treatment of 27 with polyphosphoric acid, followed by
basic hydrolysis of the methyl ester, afforded LE540
(17). Introduction of a nitro group on the unsubstituted
benzo group was performed by nitration of the methyl
ester of LE135 (6) using KNO3 in sulfuric acid, to afford
LE531 (16, Scheme 1).
31) of HX600 (7) were prepared according to the method
shown in Scheme 2. 5,6,7,8-Tetrahydro-5,5,8,8-tetra-
methyl-2-naphthol (32) or the corresponding thiol (33)
was reacted with 1-chloro-2-nitrobenzene in the pres-
ence of KOH in DMSO, followed by reduction of the nitro
group with Fe under acidic conditions, to give the
diphenyl ether (34) or thioether derivative (35), respec-
tively. Friedel-Craft acylation of 1,2,3,4-tetrahydro-
1,1,4,4-tetramethylnaphthalene (36) with 2-nitrobenzoyl
chloride gave 37. Two-step reduction of the nitro group
with Fe/HCl/EtOH and then the keto group with LiAlH4
gave a diphenylmethane derivative 38. Wittig reaction
of 37 with methyltriphenylphosphonium bromide, fol-
lowed by catalytic hydrogenation, afforded a R-methyl-
diphenylmethane derivative, 39. Acylation of 34, 35,
38, and 39 with terephthalic acid monomethyl ester
chloride, followed by cyclization and ester hydrolysis,
The oxa-, thia-, and monoazepine derivatives (28-

Diazepinylbenzoic Acids as Retinoid Synergists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4225
Ta ble 1. Antagonistic Activity of Diaryldiazepine Derivatives
on Am80 (2)-Induced HL-60 Cell Differentiation
antagonistic activity was observed when the benzo
group of LE135 (6) was replaced with a naphtho group.
Thus, LE540 (17) is a more potent retinoid antagonist.
compound
IC50, Ma
compound
IC50, Ma
HL-60 cell differentiation induced by 3.0 × 10-9
M
LE135 (6)
1.5 × 10-7
LE531 (16)
LE540 (17)
LE550 (18)
LE560 (19)
LE570 (20)
LE590 (21)
2.8 × 10-7
3.6 × 10-8
1.2 × 10-7
3.5 × 10-7
3.2 × 10-7
8.3 × 10-7
inactiveb
Am80 (2) was completely inhibited by addition of 3.0 ×
10-7 M LE540 (17) and was reduced by half by 1.0 ×
10-7 M LE540 (17). LE550 (18), an isomer of LE540
(17), is as active as LE135 (6). Introduction of a bulkier
aromatic moiety such as a phenanthrene or anthracene
ring instead of the naphtho group seems to be ineffec-
tive, since both LE560 (19) and LE570 (20) were rather
weaker antagonists than LE135 (6). LE590 (21), pos-
sessing two 5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaph-
tho groups, also exhibited antagonistic activity in HL-
60 assay (Figure 1b). Interestingly, 1.0 × 10-6 M LE590
(21) decreased the potency of Am80 (2) only by up to
half, regardless of the concentration of Am80 (2), which
indicated a noncompetitive antagonistic character of
LE590 (21).
LE510 (14a )
LE511 (14b)
LE515 (14c)
LE523 (15a )
LE527 (15b)
> 1.0 × 10-6 b
inactive
1.3 × 10-7
2.1 × 10-7
a
IC50 was determined as the concentration of a test compound
which reduces by half the percentage of differentiated HL-60 cells
b
induced by 1 × 10-9 M Am80 (2). “Inactive” means there was no
activity at 1.0 × 10-6 M test compound. “> 1.0 × 10-6 M” means
there was slight inhibitory activity at 1.0 × 10-6 M test compound.
gave HX620 (28), HX630 (29), HX640 (30), and HX641
(31), respectively.
The 2-oxodiazepines HX800 (40) and HX801 (41) are
derivatives lacking one benzo group of HX600 (7).
Friedel-Craft acylation of 1,2,3,4-tetrahydro-1,1,4,4-
tetramethylnaphthalene (36) with terephthalic acid
monomethyl ester chloride gave 42. Nitration of 42 with
KNO3 in sulfuric acid afforded a single nitrated com-
pound 43 in 53% yield. After reduction of the nitro
group, 44 was treated with glycine methyl ester hydro-
chloride in pyridine to give a 2-oxodiazepine 45 in 33%
yield. HX801 (41) was obtained by N-methylation of 45
with NaH and methyl iodide, followed by hydrolysis of
the ester.
R et in oid -An t a gon ist ic Act ivit ies (LE Ser ies).
The activities of diazepine derivatives were evaluated
in terms of the ability to induce or to affect retinoid-
induced differentiation of human promyelocytic leuke-
mia cells HL-60.23,24 The morphological changes were
examined by microscopy after Wright-Giemsa staining,
and the differentiation state was determined by means
of nitro blue tetrazolium (NBT) reduction assay as a
functional marker of differentiation.25 These two in-
dexes of differentiation correlate well with each other.26
We initially evaluated the antagonistic activity of
LE135-related compounds functionally as the inhibitory
potency on the differentiation-inducing activity of Am80
in HL-60 cells.
All the azepine derivatives (14-22) were completely
inactive by themselves below 1 × 10-6 M in HL-60
assay. The IC50 values of the diazepine derivatives (LE
series) on 1 × 10-9 M Am80 (2)-induced HL-60 cell
differentiation are shown in Table 1. As reported
previously, LE135 (6) antagonized the differentiation-
inducing activity of Am80 (2),15c and the IC50 value is
1.5 × 10-7 for 1.0 × 10-9 M Am80 (2). Replacement of
the cyclic alkyl group of LE135 (6) with bulky acyclic
alkyl group(s) diminished the antagonistic activity, as
seen for 14a -c. The 2,5-dimethyl-2,5-hexano group of
LE135 (6) is important for the activity, as in the case of
aromatic retinoid agonists.13 Two diazepines with a
longer N-alkyl group, LE523 (15a ) and LE527 (15b),
were as active as LE135 (6), which has an N-methyl
group.
The hydrophobic benzo group (unsubstituted) of LE135
(6) seems to be essential for its antagonistic activity.15c
Therefore, several compounds with modifications of the
benzo group were synthesized, and their retinoid-
antagonistic activities were examined. Introduction of
a nitro group onto LE135 (6), yielding LE531 (16),
slightly decreased the activity (Table 1). Increase of the
Str u ctu r a l Evolu tion fr om Retin oid An ta gon ists
to Retin oid Syn er gists (HX Ser ies). A cyclic alkyl
moiety (2,5-dimethyl-2,5-hexano group) is necessary in
the dibenzodiazepine structure of the retinoid antago-
nist LE135 (6), possibly in order to bind to and antago-
nize RARs. However, the introduction of another 2,5-
dimethyl-2,5-hexano group on the unsubstituted benzo
group of LE135 (6) generated the noncompetitive an-
tagonist LE590 (21), although the hydrophobic aromatic
region is important, as seen in LE540 (17). The two
tetrahydrotetramethylnaphtho moieties of LE590 (21)
seem to have different roles in eliciting its biological
activity. This hypothesis led us to synthesize HX600
(7), an isomer of LE135 (6), with the cyclic alkyl group
on a different aromatic ring, i.e., structurally, HX600
(7) may lack the necessary hydrophobic group at the
proper position for binding to RARs.
HX600 (7) itself showed absolutely no retinoidal
activity in the HL-60 assay, but dose-dependently
enhanced the differentiation-inducing activities of Am80
(2) as shown in Figure 2a.19 For example, the percent-
age of 1.0 × 10-9 M Am80 (2)-induced differentiated
cells (44%) was increased to 65% and 90% by addition
of 1.0 × 10-8 and 1.0 × 10-7 M HX600 (7), respectively.
Various azepine derivatives related to HX600 (7) were
examined for retinoid synergistic activities based on HL-
60 cell differentiation. The synergistic effective con-
centration (SEC50) value shown in Table 2 was defined
as the EC50 of the synergist in combination with 3.0 ×
10-10 M Am80 (2), which alone induces HL-60 cell
differentiation to the extent of ca. 20%. HX500 (22a )
with no alkyl group on the benzo groups was inactive
below 1.0 × 10-6 M. In contrast to the diminution of
the antagonistic activity of LE510 (14a ), HX610 (22f)
with two acyclic alkyl groups exhibited synergism with
Am80 (2), and its SEC50 value was 6.3 × 10-9 M in the
presence of 3.0 × 10-10 M Am80 (2). Among compounds
having one substituent (R1 in Chart 3), only HX515
(22e) exhibited synergistic activity with Am80 (2),
although its potency is weak.
HX620 (28), the oxazepine analogue of HX600 (7),
showed similar retinoid synergistic activity with Am80
(2). Improvement of the synergistic activity was ob-
served in HX630 (29) and HX640 (30), the thiazepine
and the monoazepine analogues of HX600 (7), respec-
tively. The activities of HX630 (29) and HX640 (30)

4226 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Umemiya et al.
F igu r e 1. Anatgonistic activities of diazepine derivatives LE540 (17, a) and LE590 (21, b) on Am80 (2)-induced HL-60 cell
differentiation. The vertical scale is the percentage of differentiated cells evaluated from NBT reduction assay, and the horizontal
scale is the molar concentration of Am80 (2). Concentrations of test compounds are 0 (O), 1.0 × 10-7 M (9), 3.0 × 10-7 M (4), and
1.0 × 10-6 M (b).
F igu r e 2. (a-c) Synergistic activities of HX600 (7), HX630 (29), or HX640 (30) with Am80 (2) on HL-60 cell differentiation
assay. The vertical scale is the percentage of differentiated cells evaluated from NBT reduction assay, and the horizontal scale
is the molar concentration of Am80 (2). Concentration of test compounds are 0 (O), 1.0 × 10-10 M (3), 1.0 × 10-9 M (2), 1.0 × 10-8
M (9), and 1.0 × 10-7 M (b). (d) Effects of HX600 (7, open symbol) or HX630 (29, closed symbol) on HL-60 cell differentiation
induced by various retinoids. Concentrations of added retinoids were 1.0 × 10-9 M Am555S (3, circular), 1.0 × 10-10 M Ch55 (4,
square), and 1.0 × 10-6 M LGD1069 (5, triangle).
were dose-dependent (Figure 2). The extent of dif-
that for HX600 (7, 3.2 × 10-9 M) by 1 order of
ferentiation (less than 10%) induced by 1.0 × 10-10
M
magnitude.
Am80 (2) was dramatically increased to 70-80% by the
addition of 1.0 × 10-7 M HX630 (29) or HX640 (30).
Even when 1.0 × 10-8 M HX630 (29) or HX640 (30) was
added, the EC50 value of Am80 (2) increased to 1.5 ×
10-10 or 1.6 × 10-10 M, respectively. The SEC50 values
of the azepines are 6.2 × 10-10 M for HX630 (29) and
5.5 × 10-10 M for HX640 (30), which are smaller than
HX641 (31), a methylated analogue of HX640 (30),
exhibited synergistic activity with Am80 (2) below 1.0
× 10-7 M, but it is particularly noteworthy that the
differentiation induced by 3.0 × 10-8 M Am80 (2) was
suppressed to the basal level by addition of 1.0 × 10-6
M HX641 (31). A similar but slight decrease of retinoid
synergism was also seen reproducibly in the case of

Diazepinylbenzoic Acids as Retinoid Synergists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4227
(29), and the IC50 value of HX600 (7) is 4.0 × 10-7
M
Ta ble 2. Synergistic Activities of Azepine Derivatives with
Am80 (2) on HL-60 Cell Differentiation
compared with a value of 1.0 × 10-6 M for LGD1069
compound
SEC50, Ma
compound
SEC50, Ma
(5).
Recep tor -Bin d in g Affin ities of Azep in e Der iva -
tives. The antagonistic or synergistic activities of the
azepine derivatives are specific, and their structure-
activity relationships indicated the possible participa-
tion of nuclear retinoid receptors. The relative binding
affinities to RARs and RXRs for selected compounds
were therefore examined by competition assays using
recombinant receptors. The corresponding Ki values27
are shown in Table 3 (some of these data was reported
previously15c,20,28). The retinoid antagonist LE135 (6)
binds selectively to RARR and RARâ; the binding
affinities correlate well with its antagonistic potential
in HL-60 assays. Interestingly, the binding affinities
of the more potent antagonist LE540 (17) to RARR and
RARâ are similar to those of LE135 (6), but LE540 (17)
binds to all RAR and RXR subtypes with Ki values of
the order of 1 µM.
Retinoid synergists (HX600 series) bind to the three
RXR subtypes, but their receptor binding affinities are
remarkably weaker than those of LGD1069 (5), and
HX801 (41) did not significantly bind to any receptor.
Furthermore, the Ki values do not correlate with their
synergistic activities in HL-60 assay. LGD1069 (5) was
reported to be an RXR-selective retinoid, but also binds
to RARs with Ki values of 50-180 nM in our competitive
binding experiments using recombinant receptors. HX
compounds bind to RARR and RARâ, besides RXRs, but
not to RARγ. Previously, we reported the dual func-
tionality of HX600 (6), RAR-antagonistic and RXR-
agonistic activities, based on the transactivation prop-
erties of HX600 (6).20 These data indicated that the
synergistic activities of HX compounds result from
binding to the RXR site of RXR-RAR heterodimers, and
the antagonistic activity at high doses of HX600 (6)
results from competition at the RAR site of the het-
erodimers.
HX600 (7)
3.2 × 10-9
HX620 (28)
HX630 (29)
HX640 (30)
HX641 (31)
HX800 (40)
HX801 (41)
LGD1069 (5)
5.8 × 10-9
6.2 × 10-10
5.5 × 10-10
2.8 × 10-9
inactive
HX500 (22a )
HX511 (22b)
HX513 (22c)
HX514 (22d )
HX515 (22e)
HX610 (22f)
inactive
inactive
inactive
inactive
1.0 × 10-7
4.6 × 10-7
6.3 × 10-9
3.1 × 10-10
a
SEC50 was determined as the concentration of a test compound
which induce HL-60 cell differentiation to the extent of 50% in
the presence of 3.0 × 10-10 M Am80 (2). This concentration of
Am80 (2) induced the differentiation of HL-60 cells by ca. 20%.
“Inactive” means there was no activity below 1.0 × 10-6 M test
b
compound.
HX600 (7), as reported previously,19 as well as HX515
(22e) or HX610 (22f) at high dose (> 1.0 × 10-6 M, data
not shown). At high concentrations, these compounds
seem to be antagonists to RAR agonists.
Some RXR-selective retinoids have been reported to
synergize with RAR ligands.22 Thus, we compared the
azepine derivatives with LGD1069 (5, Chart 1), a typical
RXR-selective retinoid.12 Though, in our experiments,
LGD1069 (5) alone exhibited differentiation-inducing
activity toward HL-60 cells with an EC50 of 2.1 × 10-7
M, LGD1069 (5) at lower concentration (below 1.0 ×
10-7 M) enhanced the activity of Am80 (2), and the
SEC50 value was 3.1 × 10-10 M. Thus, HX630 (29) and
HX640 (30) are retinoid synergists as potent as RXR-
selective retinoid LGD1069 (5).
To clarify the role of the unsubstituted benzo group
of HX600 (7), we designed 2-oxobenzodiazepine deriva-
tives HX800 (40) and HX801 (41, Scheme 3) in which
the anilino group of HX600 (7) was replaced with an
acetamido moiety. Only HX801 (41) exhibited signifi-
cant synergism with Am80 (2), although its potency
(SEC50: 4.6 × 10-7 M) is weaker than that of HX600
(7). This result indicated that the hydrophobic proper-
ties of the unsubstituted benzo group of HX600 (7) were
not necessary, but were important for the synergistic
activity.
Discu ssion
Effects of HX600 a n d HX630 on Va r iou s Retin -
oid s. Various azepine derivatives (HX series) enhance
the activity of Am80 (2). Previously, we reported that
HX600 (7) also synergizes with retinoic acid (1a ).19
Therefore, the effects of two typical synergistic azepines,
HX600 (7) and HX630 (29), on various retinoids were
examined using the HL-60 assay (Figure 2d). Am555S
(3) is a more RARR-selective retinoid than Am80 (2),
and Ch55 (4) binds strongly to all three subtypes of
RARs. HX600 (7) and HX630 (29) increased the potency
of both retinoids in a dose-dependent manner below 1.0
× 10-7 M. HX630 (29) is more potent than HX600 (7),
regardless of the retinoids.
The synergistic efficiency of HX600 (7) decreased
when HX600 (7) was used at higher concentration (1.0
× 10-6 M) or when a low concentration of retinoic acid
(1a ) or Am80 (2) was used, as previously reported.19 The
suppressive effect at high concentration was also ob-
served for the differentiation-inducing activity of Am555S
(3) or Ch55 (4). Interestingly, the weak differentiation-
inducing activity of LGD1069 (5) was dose-dependently
inhibited by HX600 (7) and also slightly by HX630 (29).
In contrast to its synergistic activity, the suppressive
effect of HX600 (7) is more potent than that of HX630
Various azepine derivatives were synthesized, and
their ability to regulate retinoidal activities was exam-
ined. LE135 (6) is a retinoid antagonist which was
designed on the basis of the ligand superfamily concept.15c
LE135 (6) is classified structurally as a type II antago-
nist (Chart 2) and is regarded as an analogue of Am80
(2), conformationally restricted by the bulky methyla-
nilino group. This central aromatic moiety is apparently
important for the antagonistic activity of this retinoid,
since the benzodiazepine derivative with an acetamido
group instead of the methylanilino group of LE135 (6)
had no effect on retinoidal activities.15c The structure-
activity relationships of LE135 (6) described above
confirm the importance of this region for antagonist
function, in addition to the necessary 2,5-dimethyl-2,5-
hexano group. Elongation of the N-alkyl group did not
change the antagonistic activity. The naphtho group
is most effective in this series, and LE540 (17) is the
most active retinoid antagonist among the diaryldiaz-
epine derivatives examined. The antagonistic activity
of LE540 (17) may be elicited through binding to RARs,
as is apparently the case for LE135 (6). The RAR pan-
antagonistic property of LE540 (17) is supported by the
results of transactivation assay using reporter cells

4228 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Umemiya et al.
Sch em e 3a
a
(a) AlCl3/p-CH3OOCPhCOCl; (b) KNO3/H2SO4; (c) Fe/HCl; (d) Cl-H3N+CH2COOCH3/pyridine; (e) NaOH/EtOH; (f) NaH; CH3I.
Ta ble 3. Receptor Binding Affinities of Retinoid Antagonists and Retinoid Synergists
Ki values, × 10-9
RARγa
M
compound
RARR
3.3
no data
6.5
RARâ
RXRR
RXRâ
RXRγ
Agonist
retinoic acid (1a )
9-cis-retinoic acid (1b)
Am80 (2)
4.1
no data
30
0.04
1.0
330
12
nb
54
3.8
nb
120
11
nb
nbb
RXR-Selective
LGD1069 (5)
180
50
130
16
5.9
8.3
Retinoid Antagonist
LE135 (6)
LE540 (17)
1400
1300
220
500
nb
490
nb
3800
nb
1700
nb
1500
Retinoid Synergist
HX600 (7)
nb
680
320
320
810
no data
nb
nb
nb
nb
nb
nb
nb
1900
3800
900
1800
900
640
1200
400
790
150
nb
1000
2100
620
1000
170
HX620 (28)
HX630 (29)
HX640 (30)
HX641 (31)
HX801 (41)
980
900
1600
2000
nb
nb
nb
a
b
See ref 27. nb (not bound) means that 1000-fold excess of a test compound did not affect the binding of the labeled compound to the
receptors.
stably transfected with GAL-RAR chimeras (data not
shown). The subtype selectivity (RARâ > RARR .
RARγ) of LE135 (6) was less pronounced for LE540 (17),
and the in vitro binding affinities to RARs could not
explain the difference in potency between LE135 (6) and
LE540 (17). The ability of LE540 (17) to bind to RXRs
might significantly contribute to its activity.
vivo receptor-binding affinities, as was recently sug-
gested for a mutant glucocorticoid receptor.29 Along the
same lines, it is possible that HX compounds interact
efficiently with RXRs of RXR-RAR heterodimers within
the transcriptionally active complex formed with coac-
tivators, while they may not, or very weakly, interact
with RXR monomers or the homodimers in the in vitro
binding assays.
Several azepines enhanced the activity of retinoids
in HL-60 assay. These compounds are regarded as pure
synergists, since they are completely inactive alone,
which contrasts with the agonistic/synergistic activity
of LGD1069 (5). In HX600 (7) the loss of the 2,5-
dimethyl-2,5-hexano group [giving HX500 (22a )] or the
carboxyl group (not shown) diminishes the synergistic
activity. The bulky hydrophobic group can be replaced
with acyclic alkyl group(s), such as diisopropyl groups,
as seen in HX610 (22f). Replacement of one of the two
nitrogen atoms of HX600 (7) with a nonpolar thio or
methylene group increased the synergistic activity.
The synergistic activities of HX600 (7) could be
attributed to its RXR-agonistic properties, as deduced
from the receptor-binding affinities and the results of
transactivation experiments.20 However, the in vitro
binding affinities of HX compounds to RXRs are very
weak, with Ki values of micromolar order, as shown in
Table 3. The order of the retinoid-synergistic potency
of HX compounds did not reflect their Ki values to
recombinant RXRs. HX630 (29) and HX640 (30) were
almost as active as LGD1069 (5) in HL-60 differentia-
tion assay, but their affinities to RXRs are lower than
those of LGD1069 (5) by 2 orders of magnitude. Some
ligands are reported to have different in vitro and in
HX600 (7) has a dual function, exerting both syner-
gistic and antagonistic activities. Some compounds,
especially those harboring an N-methyl group on the
diazepine ring, such as HX515 (22e) and HX610 (22f),
exhibited similar dual activities. This property is more
marked in HX641 (31), having a methyl group at the
corresponding benzylic position, than in HX640 (30)
without the methyl group. The antagonistic activity at
high concentrations may be mediated by binding to and
inactivating RARs. HX600 (7), and even HX630 (29),
suppressed the differentiation-inducing activity of the
RXR-selective retinoid LGD1069 (5). Notably, in our
experiments LGD1069 (5) did bind weakly to RARs.
Thus LGD1069 (5) acts as a weak RAR agonist whose
activity in the RXR-RAR heterodimer is further en-
hanced by its potent RXR agonistic activity. HX600 (7)
may antagonize LGD1069 (5) via the RAR subunit of
RXR-RAR heterodimers. The Ki values of HX com-
pounds to RARs seem not to be simply correlated with
their antagonistic activity (or reduced synergistic activ-
ity). HX630 (29) and HX640 (30) seem to be more
efficient synergists than HX600 (7, synergist/antagonist)
or even LGD1069 (5, synergist/agonist), considering
both the potency and the selectivity of the dual func-

Diazepinylbenzoic Acids as Retinoid Synergists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4229
was stirred for 24 h. The mixture was poured into 2 N
hydrochloric acid, and extracted with AcOEt. The organic
layer was dried over Na2SO4, and evaporated. The residue
was chromatographed on silica gel (flash column, AcOEt:n-
hexane, 1:20) to give 27 (95%) as colorless crystals. 27: 1H
NMR (CDCl3) δ 8.36 (s, 1 H), 8.22 (d, 2 H, J ) 8.4 Hz), 8.07 (s,
1 H), 7.85 (m, 1 H), 7.66 (d, 2 H, J ) 8.4 Hz), 7.57 (d, 1 H, J
) 8.1 Hz), 7.50 (m, 2 H), 7.33 (m, 2 H), 7.25 (s, 1 H), 7.02 (m,
3 H), 3.91 (s, 3 H), 3.33 (s, 3 H), 1.73 (s, 4 H), 1.34 (s, 6 H),
1.31 (s, 6 H).
4-(13H -10,11,12,13-Tet r a h yd r o-10,10,13,13,15-p en t a -
m et h yld in a p h t h o[2,3-b][1,2-e][1,4]d ia zep in -7-yl)b en zo-
ic Acid (LE540, 17). A solution of 27 (4.00 g, 7.69 mmol) in
a small amount of CH2Cl2 was added to polyphosphoric acid
(40 g), and the mixture was heated at 110 °C for 2 h and then
allowed to cool. Water was added, and the whole was
extracted with CH2Cl2. The organic layer was washed with
brine, dried over Na2SO4, and evaporated. The crude product
was purified by silica gel column chromatography (AcOEt:n-
hexane, 1:8) to give methyl 4-(13H-10,11,12,13-tetrahydro-
10,10,13,13,15-pentamethyldinaphtho[2,3-b][1,2-e][1,4]diazepin-
7-yl)benzoate (83%), which was hydrolyzed in 2 N NaOH/
ethanol at reflux for 30 min to afford LE540 (17, quant.).
LE540 (17): yellow needles (aqueous ethanol); mp >300 °C;
1H NMR (CDCl3) δ 8.87 (d, 1 H, J ) 8.4 Hz), 8.20 (d, 2 H, J )
8.4 Hz), 7.95 (d, 2 H, J ) 8.8 Hz), 7.85 (d, 1 H, J ) 7.7 Hz),
7.65 (m, 3 H), 7.42 (s, 1 H), 7.30 (s, 1 H), 7.17 (d, 1 H, J ) 8.4
Hz), 3.08 (s, 3 H), 1.68 (s, 4 H), 1.38 (s, 3 H), 1.36 (s, 3 H), 1.22
(s, 3 H), 1.21 (s, 3 H). Anal. (C33H32N2O2) C, H, N.
tions. Further structure-activity investigation is needed
in order to separate RAR-antagonistic and RXR-ago-
nistic activities.
RXRs act as heterodimer partners not only with
RARs, but also with other nuclear receptors such as
vitamin D3 receptors, thyroid hormone receptors, and
peroxisome proliferative activated receptors (PPARs).7
The allosteric effects of RXR ligands in RXR-RAR
heterodimers cause unique modulation of retinoid ac-
tions depending on the combinations of RAR or RXR
ligands,8c,22,30 and some of the above hormonal activities
can be positively or negatively regulated by RXR
ligands.31 The biological effects of RXR-agonistic diaz-
epines are being further evaluated in our laboratory.
In conclusion, we have developed new RAR antago-
nists and RXR agonists (retinoid synergists). Since they
have weak in vitro binding affinities to recombinant
monomeric or homodimeric retinoid nuclear receptors,
but exhibit potent regulation of retinoids in HL-60
assay, HX600 (7) and the related synergists are RXR
agonists selective for RXR-RAR heterodimers. Among
retinoid synergists, HX630 (29) and HX640 (30) showed
weaker RAR-antagonistic and greater RXR-agonistic
activities than HX600 (7). These synergistic azepines
should be useful tools for the elucidation of RXR
functions in retinoidal and other hormonal actions.
Furthermore, they can potentiate retinoid action at low
concentration, and thus may find application in the field
of retinoid therapy.
Other diazepine derivatives (14, 15, 18-22) were synthe-
sized from the corresponding nitroanilines and aryl halides
according to the above method, and their chemical and physical
properties are listed in Table 4.
4-(5H -7,8,9,10-Tet r a h yd r o-5,7,7,10,10-p en t a m et h yl-2-
n it r ob en zo[e]n a p h t h o[2,3-b][1,4]d ia zep in -13-yl)b en zo-
ic Acid (LE531, 16). KNO3 (18.4 mg, 0.18 mmol) was added
to a solution of methyl 4-(5H-7,8,9,10-tetrahydro-5,7,7,10,10-
pentamethylbenzo[e]naphtho[2,3-b][1,4]diazepin-13-yl)ben-
zoate (methyl ester of LE135, 60 mg, 0.132 mmol) in sulfuric
acid (5 mL) at 0 °C, and the mixture was stirred at 0 °C for 1
h, then poured into ice water, and extracted with CH2Cl2. The
organic layer was washed successively with 1 N NaHCO3,
water, and brine and dried over Na2SO4. After evaporation,
the residue was chromatographed on silica gel (flash column,
AcOEt:n-hexane, 1:8) to give methyl 4-(5H-7,8,9,10-tetrahydro-
5,7,7,10,10-pentamethyl-2-nitrobenzo[e]naphtho[2,3-b][1,4]-
diazepin-13-yl)benzoate (56%), which was hydrolyzed in 2 N
NaOH/ethanol at reflux for 1 h to afford LE531 (16, 71%).
LE531 (16): orange needles (aqueous ethanol); mp >300 °C;
1H NMR (CDCl3) δ 8.27 (dd, 1 H, J ) 8.4, 2.0 Hz), 8.15 (d, 2
H, J ) 8.8 Hz), 7.83 (d, 1 H, J ) 8.4 Hz), 7.80 (d, 2 H, J ) 8.4
Hz), 7.25 (s, 1 H), 7.13 (d, 1 H, J ) 2.0 Hz), 6.84 (s, 1 H), 3.34
(s, 3 H), 1.67 (s, 4 H), 1.32 (s, 3 H), 1.26 (s, 9 H). Anal.
(C29H29N3O4‚1/3 H2O), C, H, N.
2-[(5,6,7,8-Tetr a h yd r o-5,5,8,8-tetr a m eth yl-2-n a p h th yl)-
oxy]a n ilin e (34). A mixture of 5,6,7,8-tetrahydro-5,5,8,8-
tetramethyl-2-naphthol (97 mg, 0.475 mmol), 1-chloro-2-
nitrobenzene (77 mg, 0.48 mmol), and potassium hydroxide
(27 mg, 0.48 mmol) in DMSO (5 mL) was heated at 90 °C for
17.5 h. The reaction mixture was poured into 2 N hydrochloric
acid and extracted with CH2Cl2. The organic layer was washed
with 2 N hydrochloric acid and brine and dried over Na2SO4.
After evaporation, the crude product was chromatographed on
silica gel (flash column, AcOEt:n-hexane, 1:30) to give 5,6,7,8-
tetrahydro-5,5,8,8-tetramethyl-2-(2-nitrophenoxy)naphtha-
lene (67%) as a colorless oil. Fe powder (220 mg) was added
to a suspension of 5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-(2-
nitrophenoxy)naphthalene (103 mg) in 2 N hydrochloric acid
(2.5 mL) and ethanol (6 mL), and the mixture was refluxed
for 30 min. The reaction mixture was filtered, and the filtrate
was extracted with AcOEt. The organic layer was washed with
water and brine, dried over Na2SO4, and evaporated to give
crude 34. 34: colorless oil; 1H NMR (CDCl3) δ 7.21 (d, 1 H, J
) 8.8 Hz), 6.97 (d, 1 H, J ) 2.9 Hz), 6.95 (m, 1 H), 6.85 (dd, 1
H, J ) 8.1, 1.5 Hz), 6.82 (dd, 1 H, J ) 7.7, 1.5 Hz), 6.70 (m, 2
H), 3.82 (brs, 2 H), 1.68 (s, 4 H), 1.26 (s, 6 H), 1.25 (s, 6 H).
Exp er im en ta l Section
Gen er a l. Melting points were determined by using a
Yanagimoto hot-stage melting point apparatus and are uncor-
rected. Elemental analyses were carried out in the Microana-
lytical Laboratory, Faculty of Pharmaceutical Sciences, Uni-
versity of Tokyo, and were within (0.3% of the theoretical
values. NMR spectra were recorded on a J EOL J NM-GX400
(400 MHz) spectrometer. Chemical shifts are expressed in
ppm relative to tetramethylsilane. Mass spectra were recorded
on a J EOL J MS-DX303 spectrometer.
N-Meth yl-N-(1-n a p h th yl)-5,6,7,8-tetr a h yd r o-5,5,8,8-tet-
r a m et h yln a p h t h a len e-2,3-d ia m in e (26). A mixture of
5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-3-nitro-2-naphthyl-
amine (23, 16.4 g, 66.1 mmol), 1-bromonaphthalene (23.2 mL),
potassium carbonate (9.13 g, 66.1 mmol), and copper iodide
(575 mg) in o-xylene (180 mL) was heated at 150 °C for 19 h.
After removal of the solvent, the residue was purified by silica
gel column chromatography (n-hexane, then AcOEt:n-hexane,
1:40) to give 24 (66%, orange crystals, mp 127 °C, Anal.
C24H26N2O2). A solution of 24 (24.14 g, 64.5 mmol) in DMF
(400 mL) was added to a suspension of NaH (60%, 4.9 g, 129
mmol) in DMF (15 mL). After 1 h, methyl iodide (16.3 mL)
was added, and the mixture was stirred for 1 h. Removal of
the solvent afforded a residue, which was taken up in water
and extracted with CH2Cl2. The organic layer was washed
with water and brine and dried over Na2SO4. Removal of the
solvent under vacuum and recrystallization from n-hexane
gave 25 (98%, mp 154 °C, Anal. C25H28N2O2). 25 (3.30 g, 8.51
mmol) was dissolved in 300 mL of ethanol and was hydroge-
nated over 10% Pd-C (400 mg) at 40 °C for 4 h. After
filtration and removal of the solvent, the residue was chro-
matographed on silica gel (AcOEt:n-hexane, 1:7) to give 26
(86%). 26: 1H NMR (CDCl3) δ 8.01 (d, 1 H, J ) 8.4 Hz), 7.83
(d, 1 H, J ) 7.3 Hz), 7.55 (d, 1 H, J ) 8.4 Hz), 7.40 (m, 3 H),
7.08 (d, 1 H, J ) 7.3 Hz), 6.85 (s, 1 H), 6.64 (s, 1 H), 3.60 (brs,
2 H), 3.25 (s, 3 H), 1.61 (m, 4 H), 1.24 (s, 6 H), 1.10 (s, 6 H).
Meth yl 4-[[5,6,7,8-Tetr a h yd r o-5,5,8,8-tetr a m eth yl-3-[N-
m eth yl-N-(1-n aph th yl)am in o]-2-n aph th yl]car bam oyl]ben -
zoa te (27). Terephthalic acid monomethyl ester chloride (1.88
g, 9.51 mmol) was added to a solution of 26 (2.60 g, 7.26 mmol)
in dry benzene (50 mL) and pyridine (2.8 mL), and the whole

4230 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Ta ble 4. Chemical and Physical Properties of the Azepine Derivatives
Umemiya et al.
compound
mp, °C
crystal form
recrystn solvent
anal. formula
LE510 (14a )
LE511 (14b)
LE515 (14c)
LE523 (15a )
LE527 (15b)
LE531 (16)
LE540 (17)
LE550 (18)
LE560 (19)
LE570 (20)
LE590 (21)
HX500 (22a )
HX511 (22b)
HX513 (22c)
HX514 (22d )
HX515 (22e)
HX610 (22f)
HX620 (28)
HX630 (29)
HX640 (30)
HX641 (31)
HX800 (40)
HX801 (41)
172
266
298
191
128
yellow needles
orange needles
yellow needles
orange needles
orange needles
orange needles
yellow needles
red cubes
yellow needles
orange needles
yellow needles
orange needles
orange needles
orange prisms
orange needles
orange needles
orange needles
yellow needles
yellow needles
yellow needles
yellow prisms
colorless needles
colorless needles
EtOH-H2O
EtOH-H2O
EtOH-H2O
EtOH-H2O
EtOH-H2O
EtOH-H2O
EtOH-H2O
EtOH-H2O
EtOH-H2O
EtOH-H2O
EtOH-H2O
EtOH-H2O
EtOH-H2O
MeOH
EtOH-H2O
EtOH-H2O
EtOH-H2O
EtOH-H2O
EtOH-H2O
EtOH-H2O
EtOH-H2O
MeOH-n-hexane
AcOEt-n-hexane
C27H28N2O2
C25H24N2O2
C31H30N2O2‚1/2H2O
C31H34N2O2‚1/2H2O
C35H42N2O2
>300
C29H29N3O4‚1/3H2O
C33H32N2O2
>300
>300
>300
C33H32N2O2‚1/3H2O
C37H34N2O2
283-285
289
246
249
294-295
247
208
263
289
299
>300
224-226
>300
>300
C37H34N2O2‚2/3H2O
C37H44N2O2
C21H16N2O2
C25H24N2O2
C21H15N2O2Br
C27H20N2O2
C31H30N2O2‚1/3H2O
C27H28N2O2
C28H27NO3
C28H27NO2S
C29H29NO2
C30H31NO2‚3/4H2O
C
C
24H26N2O3‚3/4H2O
25H28N2O3
4-[2,3-(2,5-D im e t h y l-2,5-h e x a n o )d ib e n z [b ,f][1,4]-
oxa zep in -11-yl]ben zoic Acid (HX620, 28). Terephthalic
acid monomethyl ester chloride (63 mg, 0.32 mmol) was added
to a solution of 34 (80.5 mg, 0.264 mmol) in dry benzene (5
mL) and pyridine (0.1 mL), and the whole was stirred for 16.5
h. The mixture was poured into 2 N hydrochloric acid and
extracted with AcOEt. The organic layer was dried over
Na2SO4 and evaporated. The residue was chromatographed
on silica gel (flash column, AcOEt:n-hexane 1:20) to give
methyl 4-[[2-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naph-
thyl)oxy]phenyl]carbamoyl]benzoate (94%). A mixture of meth-
yl 4-[[2-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-
oxy]phenyl]carbamoyl]benzoate (111 mg, 0.238 mmol) and
polyphosphoric acid (2.2 g) was heated at 100 °C for 1.5 h. After
cooling, the reaction mixture was poured into water and
extracted with CH2Cl2. The organic layer was dried over
Na2SO4 and evaporated. The residue was chromatographed
on silica gel (flash column, AcOEt:n-hexane, 1:40) to give
methyl 4-[2,3-(2,5-dimethyl-2,5-hexano)dibenz[b,f][1,4]oxazepin-
11-yl]benzoate (31%), which was hydrolyzed in 2 N NaOH/
ethanol for 40 min to afford HX620 (28, quant.). HX620 (28):
yellow needles (aqueous ethanol); mp 289 °C; 1H NMR (CDCl3)
δ 8.19 (d, 2 H, J ) 8.8 Hz), 7.97 (d, 2 H, J ) 8.8 Hz), 7.46 (m,
1 H), 7.22 (m, 3 H), 7.18 (s, 1 H), 7.02 (s, 1 H), 1.66 (s, 4 H),
1.31 (s, 6 H), 1.12 (s, 6 H). Anal. (C28H27NO3) C, H, N.
Hz), 6.77 (m, 3 H), 4.30 (brs, 2 H), 1.64 (s, 4 H), 1.22 (s, 6 H),
1.20 (s, 6 H). Anal. (C20H25NS) C, H, N.
4-[2,3-(2,5-D im e t h y l-2,5-h e x a n o )d ib e n zo [b ,f][1,4]-
t h ia zep in -11-yl]ben zoic Acid (HX630, 29). Terephthalic
acid monomethyl ester chloride (1.10 g, 5.52 mmol) was added
to a solution of 35 (1.32 g, 4.24 mmol) in dry benzene (265
mL) and pyridine (4.2 mL), and the mixture was stirred for 6
h. The reaction mixture was poured into 2 N hydrochloric acid
and extracted with AcOEt. The organic layer was dried over
Na2SO4 and evaporated. The residue was chromatographed
on silica gel (AcOEt:n-hexane, 1:8) to give methyl 4-[[2-
[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)thio]-
phenyl]carbamoyl]benzoate (91%, colorless prisms from n-
hexane, mp 137 °C, Anal. C29H31NO3S). A mixture of methyl
4-[[2-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)thio]-
phenyl]carbamoyl]benzoate (1.83 g, 3.87 mmol, dissolved in a
small amount of CH2Cl2) and polyphosphoric acid (12 g) was
heated at 110 °C for 50 min. After cooling, the reaction
mixture was poured into water and extracted with CH2Cl2.
The organic layer was dried over Na2SO4 and evaporated. The
residue was chromatographed on silica gel (AcOEt:n-hexane,
1:8) to give methyl 4-[2,3-(2,5-dimethyl-2,5-hexano)dibenzo-
[b,f][1,4]thiazepin-11-yl]benzoate (89%, yellow crystal from
EtOH-H2O, mp 221 °C, Anal. C29H29NO2S), which was
hydrolyzed in 2 N NaOH/ethanol for 40 min to afford HX630
(29, 97%). HX630 (29): yellow needles (EtOH-H2O); mp 299
°C; 1H NMR (CDCl3) δ 8.17 (d, 2 H, J ) 8.4 Hz), 7.94 (d, 2 H,
J ) 8.4 Hz), 7.48 (dd, 1 H, J ) 7.7, 1.1 Hz), 7.45 (s, 1 H), 7.37
(m, 2 H), 7.13 (m, 1 H), 7.04 (s, 1 H), 1.65 (m, 4 H), 1.31 (s, 3
2-[(5,6,7,8-Tetr a h yd r o-5,5,8,8-tetr a m eth yl-2-n a p h th yl)-
th io]a n ilin e (35). A mixture of 5,6,7,8-tetrahydro-5,5,8,8-
tetramethyl-2-naphthalenethiol (4.0 g, 18.2 mmol), 1-chloro-
2-nitrobenzene (4.30 g, 27.3 mmol), and potassium hydroxide
(1.0 g, 18.2 mmol) in DMSO (60 mL) was heated at 100 °C for
21 h. The reaction mixture was concentrated under vacuum
and then poured into 2 N hydrochloric acid and extracted with
CH2Cl2. The organic layer was washed with 2 N hydrochloric
acid and brine and dried over Na2SO4. After evaporation, the
crude product was chromatographed on silica gel (AcOEt:n-
hexane, 1:25) to give 5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-
2-[(2-nitrophenyl)thio]naphthalene (66%, orange crystals from
n-hexane, mp 107 °C, Anal. C20H23NO2S). Fe powder (2.9 g)
was added to a suspension of 5,6,7,8-tetrahydro-5,5,8,8-tet-
ramethyl-2-[(2-nitrophenyl)thio]naphthalene (3.85 g, 11.3 mmol)
in 2 N hydrochloric acid (18 mL) and ethanol (30 mL), and
the mixture was refluxed for 15 min. The reaction mixture
was filtered, and the filtrate was extracted with AcOEt. The
organic layer was washed with water and brine and then dried
over Na2SO4. After evaporation, the residue was chromato-
graphed on silica gel (AcOEt:n-hexane, 1:20) to give 35 (38%).
35: colorless prisms (EtOH-H2O); mp 114 °C; 1H NMR
(CDCl3) δ 7.43 (dd, 1 H, J ) 7.5, 1.8 Hz), 7.21 (dt, 1 H, J )
7.3, 1.4 Hz), 7.14 (d, 1 H, J ) 8.1 Hz), 7.10 (d, 1 H, J ) 2.2
H), 1.28 (s, 3 H), 1.15 (s, 3 H), 1.07 (s, 3 H). Anal. (C28H27
NO2S) C, H, N.
-
5,6,7,8-Tet r a h yd r o-5,5,8,8-t et r a m et h yl-2-(2-n it r ob en -
zoyl)n a p h th a len e (37). AlCl3 (14.3 g) was added portionwise
to a mixture of 1,2,3,4-tetrahydro-1,1,4,4-tetramethylnaph-
thalene (36, 10.0 g, 53.2 mmol) and 2-nitrobenzoyl chloride
(9.4 g, 50.5 mmol) in dry CH2Cl2 (50 mL), and the mixture
was refluxed for 1.5 h. The reaction mixture was poured into
water and extracted with CH2Cl2. The organic layer was dried
over Na2SO4 and evaporated. The crude product was purified
by silica gel column chromatography (flash column, AcOEt:n-
hexane, 1:10) to give 37 (42%). 37: colorless needles (n-
1
hexane); mp 122 °C; H NMR (CDCl3) δ 8.23 (d, 1 H, J ) 8.1
Hz), 7.84 (s, 1 H), 7.75 (t, 1 H, J ) 6.2 Hz), 7.69 (t, 1 H, J )
7.0 Hz), 7.48 (dd, 1 H, J ) 7.7, 1.5 Hz), 7.34 (m, 2 H), 1.69 (s,
4 H), 1.28 (s, 6 H), 1.26 (s, 6 H). Anal. (C21H23NO3) C, H, N.
4-[2,3-(2,5-Dim eth yl-2,5-h exa n o)d iben z[b,e]a zep in -11-
yl]ben zoic Acid (HX640, 30). Fe powder (313 mg) was added
to a suspension of 37 (262 mg, 0.78 mmol) in hydrochloric acid
(2 mL) and ethanol (10 mL), and the mixture was refluxed for

Diazepinylbenzoic Acids as Retinoid Synergists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4231
15 min. The reaction mixture was filtered, and the filtrate
was extracted with AcOEt. The organic layer was dried over
Na2SO4 and evaporated to give crude 2-(5,6,7,8-tetrahydro-
5,5,8,8-tetramethylnaphthoyl)aniline (quant.). A solution of
2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthoyl)aniline (67
mg, 0.22 mmol) in ether (2 mL) was added to a suspension of
LiAlH4 (41 mg, 1.09 mmol) in ether (8 mL), and the mixture
was refluxed for 19 h, then poured into water, and extracted
with ether. The organic layer was dried over Na2SO4 and
evaporated. The crude product was chromatographed on silica
gel (flash column, AcOEt:n-hexane, 1:40 then 1:20) to give 38
(54%). Terephthalic acid monomethyl ester chloride (74 mg,
0.37 mmol) was added to a solution of 38 (88.5 mg, 0.30 mmol)
in dry benzene (4 mL) and pyridine (0.2 mL), and the mixture
was stirred for 1.5 h, poured into 2 N hydrochloric acid, and
extracted with AcOEt. The organic layer was dried over
Na2SO4 and evaporated. The residue was chromatographed
on silica gel (flash column, AcOEt:n-hexane, 1:20) to give
methyl 4-[[2-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naph-
thyl)methyl]phenyl]carbamoyl]benzoate (84%). A mixture of
methyl 4-[[2-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naph-
thyl)methyl]phenyl]carbamoyl]benzoate (103 mg, 0.23 mmol)
and polyphosphoric acid (1.56 g) was heated at 110 °C for 45
min. After cooling, the reaction mixture was poured into water
and extracted with CH2Cl2. The organic layer was dried over
Na2SO4 and evaporated. The residue was chromatographed
on silica gel (flash column, AcOEt:n-hexane, 1:20) to give
methyl 4-[2,3-(2,5-dimethyl-2,5-hexano)dibenz[b,e]azepin-11-
yl]benzoate (79%), which was hydrolyzed in 2 N NaOH/ethanol
for 1 h to afford HX640 (30, 97%). HX640 (30): yellow needles
(EtOH-H2O); mp >300 °C; 1H NMR (DMSO-d6, 120 °C) δ 8.05
(d, 2 H, J ) 8.4 Hz), 7.89 (d, 2 H, J ) 8.4 Hz), 7.39 (s, 1 H),
7.33 (m, 2 H), 7.26 (td, 1 H, J ) 7.3, 1.5 Hz), 7.16 (td, 1 H, J
) 7.3, 1.5 Hz), 7.09 (s, 1 H), 3.69 (s, 2 H), 1.66 (m, 4 H), 1.32
(s, 6 H), 1.11 (s, 6 H). Anal. (C29H29NO2) C, H, N.
H, J ) 8.4 Hz), 7.99 (d, 1 H, J ) 8.4 Hz), 7.97 (d, 1 H, J ) 8.4
Hz), 7.46 (br, 1 H), 7.25 (m, 4 H), 7.10 (s, 0.5 H), 7.09 (s, 0.5
H), 4.03 (q, 0.5 H, J ) 7.3 Hz), 3.62 (q, 0.5 H, J ) 7.3 Hz),
1.85 (d, 1.5 H, J ) 7.3 Hz), 1.65 (m, 4 H), 1.37 (s, 1.5 H), 1.37
(s, 1.5 H), 1.34 (d, 1.5 H, J ) 7.3 Hz), 1.27 (s, 1.5 H), 1.26 (s,
1.5 H), 1.17 (s, 1.5 H), 1.16 (s, 1.5 H), 1.04 (s, 1.5 H), 1.03 (s,
1.5 H); HRMS calcd for C30H31NO2 437.2356, Found: 437.2385.
Anal. (C30H31NO2‚3/4H2O) C, H, N.
Meth yl 4-(5,5,8,8-Tetr ah ydr o-5,5,8,8-tetr am eth yl-2-n aph -
th oyl)ben zoa te (42). AlCl3 (14.3 g, 107.5 mmol) was added
portionwise to a mixture of 1,2,3,4-tetrahydro-1,1,4,4-tetra-
methylnaphthalene (36, 10.0 g, 53.2 mmol) and terephthalic
acid monomethyl ester chloride (10.0 g, 50.5 mmol) in CH2Cl2
(50 mL) at 0 °C, and the whole was refluxed for 1 h. The
reaction mixture was poured into ice water and extracted with
CH2Cl2. The organic layer was washed with brine and dried
over Na2SO4. After evaporation, the crude product was
recrystallized from AcOEt to give 42 (99%). 42: colorless
1
needles (AcOEt); mp 139 °C; H NMR (CDCl3) δ 8.15 (d, 2 H,
J ) 8.8 Hz), 7.83 (d, 2 H, J ) 8.4 Hz), 7.79 (d, 1 H, J ) 1.8
Hz), 7.54 (dd, 1 H, J ) 8.1, 1.8 Hz), 7.41 (d, 1 H, J ) 8.4 Hz),
3.97 (s, 3 H), 1.72 (s, 4 H), 1.32 (s, 6 H), 1.29 (s, 6 H). Anal.
(C23H26O3) C, H, N.
Meth yl 4-(5,5,8,8-Tetr a h yd r o-5,5,8,8-tetr a m eth yl-3-n i-
tr o-2-n a p h th oyl)ben zoa te (43). KNO3 (240 mg, 2.37 mmol)
was added to a solution of 42 (693 mg, 1.98 mmol) in sulfuric
acid (5 mL) at 0 °C, and the mixture was stirred for 1 h. The
reaction mixture was poured into ice water and extracted with
CH2Cl2. The organic layer was washed with 1 N NaHCO3 and
brine and dried over Na2SO4. After evaporation, the crude
product was recrystallized from AcOEt to give 43 (53%). 43:
colorless needles (AcOEt); mp 192 °C; 1H NMR (CDCl3) δ 8.16
(s, 1 H), 8.11 (d, 2 H, J ) 8.4 Hz), 7.81 (d, 2 H, J ) 8.4 Hz),
7.38 (s, 1 H), 3.94 (s, 3 H), 1.77 (s, 4 H), 1.39 (s, 6 H), 1.31 (s,
6 H). Anal. (C23H25NO5) C, H, N.
2-[1-(5,6,7,8-Tetr a h yd r o-5,5,8,8-tetr a m eth yl-2-n a p h th -
yl)eth yl]a n ilin e (39). n-BuLi (1.6 M in hexane, 1.25 mL) was
added to a suspension of methyltriphenylphosphonium bro-
mide (542 mg, 1.50 mmol) in THF (5 mL) at -50 °C. After 15
min, a solution of 37 (337 mg, 1.00 mmol) in THF (5 mL) was
added to the solution of the ylide, and the whole was held at
room temperature overnight, then poured into water, and
extracted with CH2Cl2. The organic layer was dried over
Na2SO4 and evaporated. The residue was chromatographed
on silica gel (flash column, AcOEt:n-hexane, 1:20) to give
5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-[1-(2-nitrophenyl)-
ethenyl]naphthalene (79%), which was hydrogenated over 10%
Pd-C in methanol for 2 h to afford 39 (78%). 39: 1H NMR
(CDCl3) δ 7.27 (d, 1 H, J ) 7.7 Hz), 7.17 (m, 2 H), 7.07 (td, 1
H, J ) 7.7, 1.5 Hz), 6.86 (m, 2 H), 6.65 (d, 1 H, J ) 7.7 Hz),
4.03 (q, 1 H, J ) 7.3 Hz), 3.60 (brs, 2 H), 1.65 (s, 4 H), 1.61 (d,
3 H, J ) 7.0 Hz), 1.25 (s, 3 H), 1.24 (s, 3 H), 1.23 (s, 3 H), 1.22
(s, 3 H).
4-[2,3-(2,5-Dim eth yl-2,5-h exa n o)-5-m eth yld iben z[b,e]-
a zep in -11-yl]ben zoic Acid (HX641, 31). Terephthalic acid
monomethyl ester chloride (97 mg, 0.49 mmol) was added to
a solution of 39 (121 mg, 0.40 mmol) in dry benzene (8 mL)
and pyridine (0.2 mL), and the mixture was stirred for 15 h.
The reaction mixture was poured into 2 N hydrochloric acid
and extracted with AcOEt. The organic layer was dried over
Na2SO4 and evaporated. The residue was chromatographed
on silica gel (flash column, AcOEt:n-hexane, 1:20) to give
methyl 4-[[2-[1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naph-
thyl)ethyl]phenyl]carbamoyl]benzoate (78%). A mixture of
methyl 4-[[2-[1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naph-
thyl)ethyl]phenyl]carbamoyl]benzoate (140 mg, 0.30 mmol) and
polyphosphoric acid (1.20 g) was heated at 150 °C for 40 min.
After cooling, the reaction mixture was poured into water and
extracted with CH2Cl2. The organic layer was dried over
Na2SO4 and evaporated. The residue was chromatographed
on silica gel (flash column, AcOEt:n-hexane, 1:40) to give
methyl 4-[2,3-(2,5-dimethyl-2,5-hexano)-5-methyldibenz[b,e]-
azepin-11-yl]benzoate (49%), which was hydrolyzed in 2 N
NaOH/ethanol for 3 h to afford HX641 (31, 91%). HX641
(31): yellow needles (aqueous ethanol); mp >300 °C; 1H NMR
(CDCl3, two conformers) δ 8.19 (d, 1 H, J ) 8.4 Hz), 8.18 (d, 1
Meth yl 4-(3-Am in o-5,5,8,8-tetr ah ydr o-5,5,8,8-tetr am eth -
yl-2-n a p h th oyl)ben zoa te (44). Fe powder (317 mg) was
added to a suspension of 43 (318.5 mg, 0.81 mmol) in 2 N
hydrochloric acid (6 mL) and ethanol (10 mL), and the mixture
was refluxed for 50 min. The reaction mixture was filtered,
and the filtrate was extracted with AcOEt. The organic layer
was washed with water and brine, dried over Na2SO4, and
evaporated to afford a crude product, which was recrystallized
from AcOEt:n-hexane to give 44 (95%). 44: yellow needles
1
(AcOEt:n-hexane); mp 162 °C; H NMR (CDCl3) δ 8.14 (d, 2
H, J ) 8.4 Hz), 7.69 (d, 2 H, J ) 8.8 Hz), 7.31 (s, 1 H), 6.67 (s,
1 H), 5.90 (brs, 1 H), 3.97 (s, 3 H), 1.65 (m, 4 H), 1.28 (s, 6 H),
1.11 (s, 6 H). Anal. (C23H27NO3), C, H, N.
4-[1,3-Dih yd r o-7,8-(2,5-d im eth yl-2,5-h exa n o)-2-oxo-2H-
1,4-ben zod ia zep in -5-yl]ben zoic Acid (HX800, 40). A mix-
ture of 44 (70 mg, 0.19 mmol) and glycine methyl ester
hydrochloride (38 mg, 0.31 mmol) in pyridine (5 mL) was
refluxed for 16 h. The reaction mixture was poured into 2 N
hydrochloric acid and extracted with CH2Cl2. The organic
layer was washed with water and brine, dried over Na2SO4,
and evaporated to afford a residue, which was chromato-
graphed on silica gel (flash column, AcOEt:n-hexane, 1:4) to
give methyl 4-[1,3-dihydro-7,8-(2,5-dimethyl-2,5-hexano)-2-oxo-
2H-1,4-benzodiazepin-5-yl]benzoate (45%). This product was
hydrolyzed in 2 N NaOH/ethanol for 20 min to afford HX800
(40, 83%). HX800 (40): yellow needles (MeOH-n-hexane); mp
1
>300 °C; H NMR (DMSO-d6, 120 °C) δ 8.05 (d, 2 H, J ) 8.4
Hz), 7.89 (d, 2 H, J ) 8.4 Hz), 7.39 (s, 1 H), 7.33 (m, 2 H), 7.26
(td, 1 H, J ) 7.3, 1.5 Hz), 7.16 (td, 1 H, J ) 7.3, 1.5 Hz), 7.09
(s, 1 H), 3.69 (s, 2 H), 1.66 (m, 4 H), 1.32 (s, 6 H), 1.11 (s, 6 H).
Anal. (C24H26N2O3‚3/4H2O) C, H, N.
4-[1,3-Dih yd r o-7,8-(2,5-d im eth yl-2,5-h exa n o)-1-m eth yl-
2-oxo-2H -1,4-b en zod ia zep in -5-yl]b en zoic Acid (H X801,
41). A solution of 45 (36 mg, 0.089 mmol) in dry DMF (4 mL)
was added to a suspension of NaH (60%, 7 mg, 0.18 mmol) in
DMF (1 mL). After 10 min, methyl iodide (0.02 mL) was added
to the mixture, and the whole was stirred for 2.5 h and then
evaporated. The residue was taken up in water and extracted
with CH2Cl2. The organic layer was washed with water and
brine and dried over Na2SO4. After evaporation, the residue
was chromatographed on silica gel (AcOEt:n-hexane, 1:1) to

4232 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Umemiya et al.
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give methyl 4-[1,3-dihydro-7,8-(2,5-dimethyl-2,5-hexano)-1-
methyl-2-oxo-2H-1,4-benzodiazepin-5-yl]benzoate (59%), which
was hydrolyzed in 2 N NaOH/ethanol for 40 min to afford
HX801 (41, 83%). HX801 (41): colorless needles (AcOEt:n-
hexane); mp >300 °C; 1H NMR (CDCl3) δ 8.13 (d, 2 H, J ) 8.8
Hz), 7.77 (d, 2 H, J ) 8.4 Hz), 7.22 (s, 1 H), 7.14 (s, 1 H), 4.84
(d, 1 H, J ) 10.6 Hz), 3.88 (d, 1 H, J ) 10.6 Hz), 3.41 (s, 3 H),
1.72 (m, 4 H), 1.39 (s, 3 H), 1.32 (s, 3 H), 1.21 (s, 3 H), 1.15 (s,
3 H). Anal. (C25H28N2O3) C, H, N.
Cells a n d Cu ltu r e. The human promyelocytic leukemia
cell line HL-6023,24 was provided by Prof. F. Takaku (Faculty
of Medicine, University of Tokyo) in 1980, and has been
maintained in continuous suspension culture.32 The cells are
cultured in plastic flasks in RPMI1640 medium, supplemented
with 5% fetal bovine serum (FBS, not delipidized) and anti-
biotics (penicillin G and streptomycin), in a humidified atmo-
sphere of 5% CO2 in air at 37 °C.
Differ en tia tion -In d u cin g Assa y. 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
concentration was kept below 0.5%. Control cells were given
only the same volume of ethanol. Am80 (2), as a positive
control, was always assayed at the same time. The cells were
incubated for 4 days and stained with Wright-Giemsa in order
to check for morphological change. The percentages of dif-
ferentiated cells were determined by nitro-blue tetrazolium
(NBT) reduction assay as described.25 Cells were incubated
for 20 min at 37 °C in RPMI1640 medium (5% FBS) and an
equal volume of phosphate-buffered saline (PBS) containing
NBT (0.2%) and 12-O-tetradecanoylphorbol 13-acetate (TPA;
200 ng/mL). The percentage of cells containing blue-black
formazan was determined on a minimum of 200 cells. The
evaluation of the differentiation from NBT reduction assay was
always consistent with the morphological result.26
Modification of the differentiation-inducing activity of Am80
(2) was examined in the presence of a suitable concentration
of the test compound according to the method described above.
In this experiment, the independent effects of Am80 (2) 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. Synergistic effective concentration (SEC50) value was
defined as the EC50 of the synergist in combination with 3.0
× 10-10 M Am80 (2). IC50 and SEC50 values of active
compounds were calculated from the NBT reduction assay data
by means of the van der Waerden method.33
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RARs and RXRs were measured by the nitrocellulose filter
binding assay method as described previously.15c Recombinant
receptors were prepared as fusion proteins with the maltose-
binding protein (MBP) for RARR and -â (ligand-binding
domain), or with glutathione-S-transferase (GST) for RXRs.
Recombinant RARγ was prepared as described.28 Competitive
binding assays were carried out using 8 nM [3H]Am80 (65 Ci/
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competing compounds required to decrease specific binding of
the labeled compound by 50% and [L] and Kd are the
concentration and the dissociation constant of the labeled
compound, respectively.27,34
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