Heterocycle-containing retinoids. Discovery of a novel isoxazole arotinoid possessing potent apoptotic activity in multidrug and drug-induced apoptosis-resistant cells

Article abstract of DOI:10.1021/jm0010320

In a search for retinoic acid (RA) receptor ligands endowed with potent apoptotic activity, a series of novel arotinoids were prepared. Because the stereochemistry of the C9-alkenyl portion of natural 9-cis-RA and the olefinic moiety of the previously syn

Full text of DOI:10.1021/jm0010320

2308
J . Med. Chem. 2001, 44, 2308-2318
Heter ocycle-Con ta in in g Retin oid s. Discover y of a Novel Isoxa zole Ar otin oid
P ossessin g P oten t Ap op totic Activity in Mu ltid r u g a n d Dr u g-In d u ced
Ap op tosis-Resista n t Cells
Daniele Simoni,*,† Marinella Roberti,‡ Francesco Paolo Invidiata,§ Riccardo Rondanin,† Riccardo Baruchello,†
Cinzia Malagutti,† Angelica Mazzali,† Marcello Rossi,† Stefania Grimaudo,# Francesca Capone,#
Luisa Dusonchet, Maria Meli, Maria Valeria Raimondi,# Marilena Landino, Natale D’Alessandro,
Manlio Tolomeo,# Dhar Arindam,X Shennan Lu,X and Doris M. BenbrookX
Dipartimento di Scienze Farmaceutiche, Via Fossato di Mortara 17-19, Universita` di Ferrara, 44100 Ferrara, Italy;
Dipartimento di Scienze Farmaceutiche, Universita` di Bologna, Bologna, Italy; Dipartimento Farmacochimico Tossicologico e
Biologico, Universita` di Palermo, Palermo, Italy; Divisione di Ematologia e Servizio AIDS, Policlinico di Palermo, Palermo,
Italy; Dipartimento di Scienze Farmacologiche, Universita` di Palermo, Palermo, Italy; and Department of Obstetrics and
Gynecology and Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, P.O. Box
26901 WP2470, Oklahoma City, Oklahoma 73190
Received J uly 17, 2000
In a search for retinoic acid (RA) receptor ligands endowed with potent apoptotic activity, a
series of novel arotinoids were prepared. Because the stereochemistry of the C9-alkenyl portion
of natural 9-cis-RA and the olefinic moiety of the previously synthesized isoxazole retinoid 4
seems to have particular importance for their apoptotic activity, novel retinoid analogues with
a restricted or, vice versa, a larger flexibility in this region were designed and prepared. The
new compounds were evaluated in vitro for their ability to activate natural retinoid receptors
and for their differentiation-inducing activity. Cytotoxic and apoptotic activities were, in
addition, evaluated. In general, these analogues showed low cytotoxicity, with the restricted
structures being slightly more active than the more flexible ones. As an exception, however,
the isoxazole retinoid 15b proved to be particularly able to induce apoptosis at concentrations
<5 µM, showing a higher activity than the classical retinoids such as all-trans-RA, 13-cis-RA,
and 9-cis-RA and the previously described synthetic retinoid 4. 15b also exhibited a good affinity
for the retinoid receptors. Interestingly, another important property of 15b was its ability to
induce apoptosis in the HL60R multidrug-resistant (MDR) cell line, at the same concentration
as is effective in HL60. Therefore, 15b represents a new retinoid possessing high apoptotic
activity in an MDR cell line. The ability of 15b to act on K562 and HL60R cells suggests that
this compound may have important implications in the treatment of different leukemias, and
its structure could offer an interesting model for the design of new compounds endowed with
apoptotic activity on MDR- and retinoid-resistant malignancies.
In tr od u ction
genes designated R, â, and γ.1,4 These receptors form
RXR-RAR heterodimers that regulate transcription by
binding to RA response elements (RAREs) in the
promoters of retinoid-responsive genes.1 ATRA binds
and activates the RARs, whereas 9-cis-retinoic acid (9-
cis-RA) (2) binds and activates both RARs and RXRs.1
Owing to their ability to regulate aberrant cell growth,
retinoids are currently being evaluated as preventive
or therapeutic agents in a variety of human premalig-
nancies and cancer.5a-g Encouraging preliminary clini-
cal results have also demonstrated the importance of
retinoids in combination chemotherapy of cancer.5a,6
Indeed, retinoids may increase the activity of other
biologic or chemotherapeutic agents, thus offering new
opportunities for the development of effective combina-
tion regimens. Moreover, by inducing apoptosis, they
may overcome tumor resistance to conventional anti-
cancer agents. In current oncologic practice, ATRA is
being used to induce remission in acute promyelocytic
leukemia (APL) patients.7
Retinoids are a class of natural and synthetic vitamin
A analogues structurally related to all-trans-retinoic
acid (ATRA, 1) (Figure 1).1 They are involved in the
physiology of vision and as morphogenic agents during
embryonic development.1 They are known to play a
major role in regulating the growth and differentiation
of a wide variety of normal and malignant cell types
and can inhibit cell proliferation and induce differentia-
tion and apoptosis at the cellular level.2a-e The induction
of apoptosis is related to cell growth and differentiation
in various ways, depending on the cell type.3a,b Retinoids
exert most of their effects by binding to specific nuclear
retinoic acid receptors (RARs) and retinoid X receptors
(RXRs), each of which is encoded by three separate
* Corresponding author (telephone +39-0532-291291; fax +39-0532-
291296; e-mail smd@dns.unife.it).
† Universita` di Ferrara.
‡ Universita` di Bologna.
§ Dipartimento Farmacochimico Tossicologico e Biologico, Universita`
di Palermo.
The ability of retinoids to regulate cellular processes
in vivo is unfortunately associated with a high incidence
of undesiderable side effects. These include toxicity in
#
Policlinico di Palermo.
Dipartimento di Scienze Farmacologiche, Universita` di Palermo.
X University of Oklahoma Health Sciences Center.
10.1021/jm0010320 CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/02/2001

Heterocycle-Containing Retinoids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 14 2309
F igu r e 1. Natural and synthetic retinoids.
skin and mucous membranes, hyperlipidemia, skeletal
side effects, visual disturbances, and teratogenicity.2b,8-12
Thus, one way to expand the therapeutic use of retinoids
is not only to increase the efficacy but also to reduce
the toxicity of the compounds.13,14 In this regard, as
stated before, retinoids interact with a diverse range of
receptor subtypes to produce their wide spectrum of
effects. Therefore, it is reasonable to think that it may
be possible to design selective retinoid receptor subtype
agonists devoid of dangerous side effects. Moreover,
apoptosis is a major modality by which tumor cells can
be eliminated, so the identification of new retinoids able
to induce apoptosis in different tumor cell types, inde-
pendent or not of their differentiating activity, can be
an important goal in cancer therapy and may provide
new useful tools for the treatment of patients with
retinoid-resistant APL or other malignancies.
The premises for our new work on retinoids were
formulated by preliminary studies carried out in our
laboratory. Indeed, we recently demonstrated that some
new isoxazole-containing retinoids are endowed with
interesting differentiating properties against human
APL HL60 cells.15,16 We have also evaluated many
synthetic heterocyclic isoxazole retinoids by cell growth
inhibition and differentiation assays and also with
respect to their effects on cell cycle progression.17 We
found that a novel G1 phase-targeting compound 4
(Figure 1), which is structurally related to arotinoids,
was endowed with apoptotic activity. This derivative
bearing the cis configuration at the double bond may
resemble 9-cis-RA but, unlike this natural compound,
does not bind the retinoid receptors. We found that 4
was able to induce apoptosis in HL60 cells after only
24 h of treatment. The percentages of apoptotic cells in
cultures treated with compound 4 were 23, 60, and 90%
after 24, 48, and 72 h, respectively. This apoptosis-
inducing activity was 6.5 and 4 times higher than those
of 13-cis-RA and 9-cis-RA, respectively, whereas ATRA
at the concentration of 50 µM was unable to induce
apoptosis. Interestingly, the trans isomer 5 was only a
poor inducer of apoptosis but retained an appreciable
differentiating activity in HL60 cells. Thus, we have
considered 4 as a possible new lead for the development
of novel apoptosis-inducing agents structurally related
to retinoids and targeted to the cell cycle. Here, we
describe our results on the synthesis and biological
activity of some new isoxazole-containing arotinoids
structurally related to the aromatic retinoid (E)-4-[2-
(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-
propenyl]benzoic acid (TTNPB or Ro13-7410, 3).1,5d All
of the new retinoids were tested for their differentiating,
cytotoxic, and apoptotic activities. In addition, the
abilities of the compounds to regulate the retinoid
receptors in vitro were evaluated using transcriptional
activation assays.
This study describes, for the first time, the novel
heterocyclic isoxazole retinoid 15b (Scheme 1) that
contains an isoxazole heterocycle replacing the alkenyl
portion of TTNPB and is able to induce apoptosis in
HL60 cells after only 24 h of treatment at a concentra-
tion of 10 µM. The compound 15b is also a potent
inducer of apoptosis in leukemia cell lines resistant to
drugs and to retinoic acid, such as the multidrug-
resistant variant of HL60, HL60R, the BCR-ABL onco-
gene-expressing K562 leukemia, and the HUT78 T-cell
lymphoma. Therefore, these findings further support
our hypothesis that heterocyclic analogues of arotinoids
such as 15b can possess apoptotic activity and may
provide an interesting new class of drugs for the therapy
of cancer, especially leukemias resistant to conventional
treatments.
Design a n d Ch em istr y
As our lead 4 may be considered to be a heterocyclic
analogue of TTNPB (3), we utilized the structure of this
known arotinoid as the basis for the design of novel
retinoids endowed with apoptotic activity.18 Because the
stereochemistry of the C9-alkenyl portion of 9-cis-RA,
which corresponds to the only olefinic moiety in 4,
seemed to be of particular importance for apoptotic
activity, we planned the synthesis of new retinoid
analogues having varied flexibility in this region. Thus,
the alkenyl motif of TTNPB was replaced by an isox-
azole (compounds 11b and 15b) or an isoxazoline ring
(compounds 10b and 14b) (Scheme 1), to restrict the
molecule, or by a flexible amino- or oxymethyl group,
to enable the system to better “fit” the receptor as in
analogues 17e-h and 20b (Scheme 2). Although com-
pounds 17e and 20b were known from patent litera-
ture,19 their biological activities were not described.
Because inclusion of a heteroatom in the arotinoid
ring reduces the toxicity 1000-fold and inclusion of a
heteroatom in the ring of trans-retinoic acid reduces the
toxicity 3-fold,13,14,20 we considered that introduction of
a heterocycle in place of the alkenyl portion of TTNPB,
such as in the isoxazole and the isoxazoline systems,
might similarly produce compounds endowed with re-
tinoid-like activity but with concomitant reduced toxic-
ity. In this regard it is worth noting that heteroaroti-

2310 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 14
Simoni et al.
Sch em e 1
noids, a class of TTNPB analogues containing at least
one heteroatom in the partially saturated ring, were less
toxic than arotinoids and have demonstrated promise
as anticancer agents.14 The reason for their reduced
toxicity appears to be due to a single structural alter-
ation, namely, inclusion of a heteroatom in the bicyclic
ring. Our heterocyclic retinoids 10b, 14b, 11b, and 15b,
however, retain some structural features of 4, that is,
the heterocycle and tetrahydrotetramethylnaphthalenyl
rings. On the other hand, analogues 17e-h and 20b
contain an acyclic heteroatom and the tetrahydrotet-
ramethylnaphthalenyl ring. We speculated that such
new retinoid analogues might retain the apoptotic
activity of 4.
The syntheses of the isoxazoline and isoxazole deriva-
tives 10b, 14b, 11b, and 15b are outlined in Scheme 1.
We took advantage of known procedures for the pro-
curement of aldehyde 6,21 whereas aldehyde 7 was
commercially available. Reaction of 6 and 7 with hy-
droxylamine in an MeOH/H2O (3:1) solution produced
the corresponding oximes 8 and 13 in high yields. Wittig
reaction between carbomethoxybenzaldehyde 7 and
methyltriphenylphosphonium bromide, in the presence
of NaH, readily produced olefin 9 in 80% yield. Olefin
12 was also easily obtained through a similar Wittig
reaction on aldehyde 6. The nitrile oxide generated from
oxime 8 following Torssell’s procedure22 underwent a
[3 + 2] regioselective cycloaddition with alkene 9 to
produce isoxazoline 10a in good yield. In the same
manner, the isoxazoline 14a was obtained in lower yield
from the nitrile oxide generated from oxime 13 and
alkene 12. Oxidation of the isoxazoline rings of 10a and
14a was easily accomplished by reaction with N-
bromosuccinimide to produce the corresponding 3-bromo
derivatives, which were then dehydrobrominated by
triethylamine in methylene chloride to the desired
isoxazoles 11a and 15a in 85% overall yield. Upon
treatment with lithium hydroxide in aqueous methanol,
isoxazolines 10a and 14a , as well as the isoxazoles 11a
and 15a , underwent facile hydrolysis of the ester
functions to give, after treatment with aqueous HCl, the
acids 10b, 14b, 11b, and 15b.

Heterocycle-Containing Retinoids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 14 2311
Sch em e 2
Ta ble 1. Cell Growth Inhibition Activity Expressed as IC50
and Apoptotic Activity Expressed as AC50 (Concentration Able
To Induce 50% Apoptosis) of Compounds 10b, 11b, 14b, 15b,
20b, and 17e-h in HL60 Cellsa
Preparation of flexible retinoids 17e-h and 20b used
standard procedures (Scheme 2). Acids 17e and 20b
were prepared as reported, and some of the steps for
17a -c and 20a followed known procedures.19 The
synthesis of amines 17a -c involved reductive amination
using sodium cyanoborohydride of the imine produced
by reaction of aminobenzoates 16a -c and aldehyde 6.
The N-methyl derivative 17d was prepared by reaction
of secondary amine 17a with formaldehyde and sodium
cyanoborohydride. Ether 20a was prepared by alkyla-
tion of the phenoxide of 4-hydroxybenzoate 19 by
benzylic bromide.
compound
IC50 (µM)
AC50 (µM)
10b
11b
14b
15b
20b
17h
17e
17g
17f
68 ( 15
80 ( 21
75 ( 8
>100
>100
>100
5 ( 2.6
>100
95 ( 13
70 ( 7.2
88 ( 6.7
99 ( 15
>100
1 ( 0.4
>100
58 ( 9.6
40 ( 2.9
49 ( 8.7
60 ( 6.9
18 ( 1.2
ATRA
Biologica l Resu lts a n d Discu ssion
aEvaluation after 48 h of treatment.
The object of this work was to investigate the impor-
tance of the C9 double bond in conferring apoptotic
activity to synthetic retinoids. The main aim was to
develop retinoids endowed with potent anticancer activ-
ity. In this context, it is worth noting that interesting
results were recently described regarding structural
modifications at the C9 double bond in 9-cis-RA as well
as in TTNPB.23-25 We have prepared a novel class of
C9-C10 conformationally restricted TTNPB analogues
10b, 11b, 14b, and 15b (Scheme 1) bearing an isoxazole
or isoxazoline ring in place of the alkenyl portion of this
molecule. Moreover, 17e-h and 20b (Scheme 2) with a
more flexible moiety were also prepared. These retinoids
were assayed in vitro for cell growth inhibition and the
ability to induce apoptosis in HL60 cells.
activity (Table 1). Analogues 17e-h were able to induce
apoptosis at 40-60 µΜ. Interestingly, growth inhibitory
and apoptotic effects of 15b on HL60 cells were mark-
edly greater than those of the other analogues (Figures
2a,b and 3 and Table 1). It was able to induce apoptosis
at concentrations <10 µM (Figure 2b) and thus was also
more active than the classical retinoids ATRA, 13-cis-
RA, and 9-cis-RA and isoxazole 4 (Figure 4).
At a concentration of 1 µM, 15b was unable to induce
apoptosis after 24-48 h. However, after 6-7 days of
exposure, almost all of the cells showed morphological
aspects of apoptosis (data not shown). Thus, it is worth
noting that, by transposing aryl groups at the 3,5-
positions of the isoxazole ring from those of 11b to 15b,
we may greatly enhance activity. Another important
property of 15b was its activity against drug-resistant
cells and cells resistant to classical retinoids (ATRA, 13-
cis-RA, and 9-cis-RA). For example, at concentrations
Our experiments showed that the treatment of HL60
cells for 48 h with 10b, 11b, 14b, or 17e-h had only
low inhibitory effects, with IC50 values in the range of
40-80 µM, whereas oxymethylene 20b was devoid of

2312 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 14
Simoni et al.
F igu r e 2. Cytotoxic (a) and apoptotic (b) activities of 15b in
HL60 leukemia cells. Cytotoxicity was evaluated by the trypan
blue dye exclusion test and apoptosis by a morphological
analysis (see Material and Methods). Data are the mean (
SD of five independent experiments.
F igu r e 3. Flow cytometry analysis of apoptosis in HL60 cells
treated for 48 h with 10b, 11b, 14b, or 15b. The percentage
of apoptotic events accumulating under the marker in the pre-
G0G1 phase of the cell cycle was calculated using CellQuest
software (Becton Dickinson).
effective against HL60, 15b was able to induce apoptosis
in HL60R, an MDR cell line derived from HL60 that
overexpresses the multidrug efflux pump P-glycoprotein
(Table 2). In the same manner, 15b induced apoptosis
in K562, an apoptotic agent-resistant leukemia cell line
expressing the BCR-ABL oncogene, and in T-cell lym-
phoma HUT78 (Table 2).
Compounds 10b, 11b, 14b, and 17e were also evalu-
ated in vitro for their ability to activate natural retinoic
acid receptors and for their differentiation-inducing
activity. The ability of the compounds to activate en-
dogenous retinoid receptors was evaluated in a vulvar
carcinoma cell line, SW962, which expresses all of the
known human RARs and RXRs. Figure 5 demonstrates
the receptor expression profile of this cell line in
comparison to other squamous carcinoma cell lines. The
SW962 cell line expresses RARR, RARγ, RXRR, and
RXRâ at levels similar to those of the other cell lines.
RARâ, however, which is often lost or decreased during
tumorigenesis, is expressed at a low level in the SW962
cell line. The SW962 cell line was stably transfected
with the reporter plasmid, RARE-tk-CAT, which con-
tains an RARE linked to a thymidine kinase (tk)
promoter that drives a chloramphenicol acetyl trans-
ferase (CAT) gene. This RARE is found in the RARâ
gene promoter. The retinoid receptors bind to the RARE
and thereby regulate expression of the CAT gene.
Retinoids that activate a receptor will cause an increase
in CAT protein expression in a concentration-dependent
manner. These compounds induced a concentration-
dependent increase in CAT expression, demonstrating
that the endogenous receptors in the SW962 cell line
are activated to transactivate the RARE (Table 3).
F igu r e 4. Percent apoptosis induced by trans-retinoic acid,
13-cis-retinoic acid, 9-cis-retinoic acid, and isoxazole 4 or 15b
at the concentration of 30 µM in HL60 leukemia cells.
Evaluation was performed after 48 h of treatment. Data are
the mean ( SD of five independent experiments.
Transactivation efficacy was determined by dividing the
fold RARE induction in cultures treated with 10 µM
synthetic retinoids by the fold induction in cultures
treated with 10 µM 9-cis-RA and multiplied by 100.
Compound 15b induced the highest activity, which was
97% of that induced by 9-cis-RA. To achieve this high
level, however, micromolar concentrations were re-
quired. Their potency was derived from dose-response
curves by determining the concentration of each com-
pound that induced half-maximal activation (EC50). The
more potent compounds, therefore, exhibit lower EC50
values. The high EC50 value of 15b is due to the sudden
increase in activity between 1 and 10 µM (Figure 6). At

Heterocycle-Containing Retinoids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 14 2313
F igu r e 6. Activation of endogenous retinoic acid receptors
in a vulvar carcinoma cell line SW962: (9) 10b; (2) 11b; (1)
14b; ([) 15b; (b) 17e; (O) 9-cis-RA. Cultures of cell line SW962
containing the RARE-tk-CAT reporter plasmid were treated
with each concentration of compound for 24 h, and the amount
of CAT expression in lysates of treated cells was quantitated.
The fold RARE induction was determined by dividing the
amount of CAT protein in the treated cultures by the amount
in the control cultures.
F igu r e 5. Expression of retinoid receptors in tumor cell lines.
Reverse transcriptase Polymerase Chain Reaction (RT-PCR)
was performed on RNA isolated from two vulvar carcinoma
cell lines, SW962 (lane 1) and A431 (lane 2), and two head
and neck carcinoma cell lines, SCC-2 (lane 3) and SCC-38 (lane
4). Primers that specifically amplify the RARR, RARâ, RARγ,
RXRR, and RXRâ retinoid receptor genes or the â actin gene
as a control for RNA quantity and quality were used as
indicated above the lanes. The reaction products were elec-
trophoresed on 1.5% agarose gels containing ethidium bromide
as pictured above. Reactions performed in the absence of RNA
were also electrophoresed (lane C) as negative controls. Lane
M contains DNA molecular weight markers, with the highest
band on the RAR gels on the left being 201 base pairs and the
highest band in the RXR gels on the right being 505 base pairs
followed by 396, 344, 288, 220, 201, 154, 134, and 75. The
middle picture was overexposed to show the weak RARâ bands.
The sizes for the amplified bands are ∼200 base pairs except
for RXRR, which is 400 base pairs.
Ta ble 4. Differentiating Activity of Compounds 10b, 11b, 14b,
and 15b in HL60 Cellsa
compound
CD14b (%)
CD11bc (%)
morphology
10b
11b
14b
15b
42.8
38.6
32.5
0
0
0
monocytes
monocytes
monocytes
(apoptosis)
granulocytes
ATRA
0
98.2
a
Evaluation after 6 days of exposure to each retinoid at a
concentration of 5 × 10-5 M. Monocyte-associated antigen.
b
c Granulocyte-associated antigen.
ever, lower than that shown by ATRA or 9-cis-RA (Table
4). The compounds were completely inactive as dif-
ferentiating agents in RA-resistant K562 and HL60R
leukemia and in the HUT78 lymphoma cell lines (data
not shown).
Our results indicate that introduction of a flexible
moiety in place of the alkenyl portion of TTNPB
produces compounds of low interest as apoptosis induc-
ers in leukemia cells. A difference exists between the
aminomethyl and the oxymethyl moieties, in that the
ether 20b is devoid of growth inhibitory activity, whereas
the amines retain this activity. In contrast, we found
that one isoxazole analogue (15b) had high retinoid
transactivation activity accompanied by apoptotic activ-
ity. Because natural and synthetic retinoids contain an
sp2 hybridized side chain, we may argue that the
inactivity of the isoxazolines may be due to the partially
hydrogenated heterocyclic ring, and therefore it is likely
that more structural resemblance to natural retinoids
is needed for transactivation to occur. Moreover, the
location of the substituents on the isoxazole in 11b and
15b seems to be the basic premise in conferring activity.
Thus, 15b appears to be a useful tool to understand the
physiological role of retinoid receptors in induction of
apoptosis.
Ta ble 2. Cell Growth Inhibition Activity Expressed as IC50
and Apoptotic Activity Expressed as AC50 of 15b in Retinoic
Acid-Resistant Cell Lines HL60R, K562, and HUT78a
cell line
IC50 (µM)
AC50 (µM)
HL60R
K562
HUT78
1.4 ( 0.5
2.3 ( 1.2
0.8 ( 0.2
4.2 ( 2.2
4.8 ( 3.4
1.5 ( 0.8
a
Evaluation after 48 h of treatment; these cell lines were
resistant to ATRA (IC50 and AC50 > 100).
Ta ble 3. Efficacy and Potency of âRARE Transactivation by
10b, 11b, 14b, 15b, 17e, and 9-cis-RA in SW962 Cells
compound
efficacya (%)
potencyb (nM)
10b
11b
14b
15b
17e
9-cis-RA
46.5
48.0
26.3
97.6
64.5
100.0
2860
129
961
10000
671
16.9
a
The efficacy was derived by dividing the fold RARE induction
at 10 µM retinoid by the fold induction at 10 µM 9-cis-RA (8-fold
b
induction) and multiplying by 100. The potency or concentration
It will be important to determine if 15b exhibits
selective activation of RAR or RXR subtypes and to
identify the mechanisms through which it induces
apoptosis. This study demonstrates that 15b can regu-
late gene transcription through natural RA receptors.
Dawson et al. reported the biological activity and
receptor selectivity of 4,5-dihydroisoxazole 10b, which
activated RARs with EC50 values in the nanomolar or
required to induce half of the maximal activity (EC50) was derived
from the dose-response curves in Figure 6.
lower concentrations, 15b is equally as effective as the
other compounds.
We also evaluated the differentiation-inducing effects
of 10b, 11b, 14b, and 15b in different leukemia cell
lines. Their differentiating activity in HL60 was, how-

2314 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 14
Simoni et al.
less range.26 The structural similarity of 10b to the
RXR-selective methyloxime of 4-(4,5,6,7-tetrahydro-
5,5,8,8-tetramethyl-2-naphthalenylcarbonyl)benzoic acid
may be the reason for RXR activation.27 The signifi-
cantly lower EC50 values reported by Dawson et al. in
comparison to this study may be explained by the
methods used to evaluate receptor activation. Whereas
Dawson et al. used a reporter construct that contained
two copies of the TREpal DNA binding site, this study
used the âRARE DNA binding site. The type of DNA
binding site used in receptor transactivation studies has
been shown to strongly effect the results obtained.28
Evaluating transcriptional regulation by using endog-
enous receptors in reporter cell lines, as in this study,
differs significantly from the receptor co-transfection
assays performed to determine receptor selectivity. The
former method has the benefit of being more biologically
relevant, because the receptors studied are at natural
levels. The disadvantage of this method is that the
receptor selectivity cannot be determined without using
cell lines in which endogenous receptor subtype protein
levels vary. The disadvantages of the co-transfection
method are its greater expense and artifacts induced
by the unnaturally high receptor levels. The higher
levels of individual receptor expression in the co-
transfection assays result in EC50 values lower than
those observed for endogenously expressed receptors in
reporter cell line assays. For instance, in our studies,
the EC50 value of 9-cis-RA is 16.9 nM, which is ∼17-
fold higher than reported in the co-transfection assay.26
Furthermore, compounds with increased receptor se-
lectivity may exhibit EC50 values in reporter cell line
assays higher than strong pan-agonists such as 9-cis-
RA. Therefore, it is expected that receptor co-transfec-
tion studies of the compounds in this paper will have
lower EC50 values for individual receptors than those
observed for an endogenous receptor mixture in the
reporter cell line assay. Studies of the receptor specific-
ity of these compounds are proposed in our plans to
understand the molecular mechanism and to develop
15b for clinical applications.
sequence of the bcr gene on chromosome 22 is fused to
the second exon of the c-abl gene on chromosome 9. The
resulting BCR-ABL protein is an oncoprotein that
confers resistance to apoptosis induced by etoposide,
actinomycin D, cycloheximide, and dexamethasone.41
The ability of 15b to act in this cell line as in BCR-
ABL-negative cell lines implies that it may be an
effective treatment for chronic myelogenous leukemia
and acute lymphoblastic (ALL), as well as nonlympho-
blastic (ANLL) leukemias expressing the BCR-ABL
oncogene. Also, the ability of 15b to induce apoptosis
in HL60R cells represents an important indication for
its possible clinical use. We have previously observed
that HL60R is resistant to drug-induced apoptosis
independently of its expression of P-glycoprotein. This
resistance seems to be correlated to the presence of high
constitutive levels of activated transcription factor NF-
kB, which is not observed in the sensitive parental
HL60.42
In summary, isoxazole arotinoid 15b represents a
novel retinoid endowed with apoptotic activity in MDR
cells. Its ability to act in K562 and HL60R cell lines
suggests that it may have important implications in the
treatment of different leukemias. Thus, 15b may be a
drug useful in both MDR- and apoptosis-resistant
malignancies.
Exp er im en ta l Section
Ch em istr y. Gen er a l Meth od s a n d Ma ter ia ls. Melting
points were obtained in open capillary tubes and are uncor-
rected. Reactions and product mixtures were routinely moni-
tored by thin-layer chromatography (TLC) on Merck silica gel
precoated F254 plates. Infrared spectra (IR, cm-1) were mea-
sured on a Perkin-Elmer 257 instrument. Nuclear magnetic
resonance (1H NMR, δ) spectra were determined, when not
specified, in CDCl3 solution using a Bruker AC-200 spectrom-
eter, and peak positions are given in parts per million
downfield from tetramethylsilane as the internal standard; J
values are expressed in hertz. Light petroleum ether refers to
the 40-60 °C boiling range fractions. Column chromatogra-
phies were performed with Merck 60-200 mesh silica gel. All
drying operations were performed over anhydrous magnesium
sulfate. Column chromatography (medium pressure) was
carried out by using the “flash” technique. Microanalysis of
all new synthesized compounds agreed within (0.4% of
calculated values.
Gen er a l P r oced u r e for Oxim es 8 a n d 13. To a solution
of hydroxylamine hydrochloride (250 mg, 3.7 mmol) dissolved
in water (7 mL) was added NaHCO3 (470 mg, 5.6 mmol)
portionwise at 0 °C, and the mixture was stirred for 30 min
at room temperature. The appropriate aldehyde 6 or 7 (3.1
mmol), dissolved in methanol (5 mL), was then added to the
solution, and stirring was continued for an additional 6 h.
Methanol was evaporated in vacuo and the residue extracted
with diethyl ether. The organic extracts were washed with
brine, dried, and evaporated under reduced pressure. The
residue was chromatographed on silica gel (eluent, diethyl
ether/light petroleum).
The roles of individual RA receptors observed in
apoptosis have varied depending on the retinoids and
cell lines studied.29,30 In support of this theory, Fenre-
tinide (4-HPR) can induce differentiation in F9 mouse
teratocarcinoma cells in a receptor-dependent manner
at 1 µM,31 whereas at 3-12.5 µM, this compound
induces apoptosis in a variety of cell lines.31-37 The
finding that 4-HPR can induce apoptosis in RAR-null
cells indicates that its molecular mechanism of apoptosis
induction is independent of RA receptor activation.31
Other promising novel retinoids, such as CD437, and
some heteroarotinoids also induce apoptosis through
receptor-independent mechanisms in addition to their
receptor-dependent activities.38-40 The ability of the
compounds in this paper to induce apoptosis, but not
differentiation, in retinoid-resistant cell lines also sug-
gests that their mechanism of apoptosis may be receptor
independent, whereas their mechanism of differentia-
tion is receptor dependent.
5,6,7,8-Tetr a h yd r o-5,5,8,8-tetr a m eth yln a p h th a len e-2-
ca r ba ld eh yd e oxim e (8): yield 67%; mp 123-126 °C (diethyl
ether/light petroleum); 1H NMR 1.29 (s, 6H), 1.30 (s, 6H), 1.70
(s, 4H), 7.34 (s, 3H), 7.50 (s, 1H), 8.11 (s, 1H). Anal. (C15H21
NO): C, H, N.
-
4-(Hyd r oxyim in om eth yl)ben zoic a cid m eth yl ester
(13): yield 80%; mp 119-122 °C (diethyl ether/light petro-
leum); IR (KBr) 1726, 1609, 1438, 1284, 1110, 966, 763; 1H
NMR 3.9 (s, 3H), 7.27 (s, 1H), 7.69 (d, 2H, J ) 8.4), 8.06 (d,
2H, J ) 8.3), 8.17 (s, 1H). Anal. (C9H9NO3): C, H, N.
K562 is a cell line expressing the Philadelphia chro-
mosome, which is the product of a reciprocal exchange
between the long arms of chromosome 9 and 22, result-
ing in a hybrid gene, in which the amino-terminal
Gen er a l P r oced u r e for Olefin s 9 a n d 12. NaH (80%) (145
mg, 6 mmol), previously washed with dry hexane, was added

Heterocycle-Containing Retinoids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 14 2315
to a stirred suspension of methyltriphenylphosphonium bro-
mide (1.07 g, 3 mmol) in dry tetrahydrofuran (15 mL) contain-
ing the appropriate aldehyde 6 or 7 (3 mmol). After 5 h of
stirring at room temperature, diethyl ether (30 mL) was added,
and the mixture was poured into ice-water and extracted with
Et2O. The combined organic extracts were dried and evapo-
rated, and the residue was chromatographed on silica gel
(eluent, diethyl ether/light petroleum).
4-Vin ylben zoic a cid m eth yl ester (9): yield 60%; oil; IR
(neat) 1724, 1608, 1436, 1278, 1107, 782; 1H NMR 3.91 (s, 3H),
5.38 (d, 1H, J ) 10), 5.96 (d, 1H, J ) 17.6), 6.75 (dd, 1H, J )
17.6, J ) 10), 7.46 (d, 2H, J ) 8), 7.99 (d, 2H, J ) 8). Anal.
(C10H10O2): C, H.
The mixture was stirred at room temperature for 30 min and
then concentrated. The residue was treated with aqueous 5%
NaHCO3 (10 mL) and extracted with ethyl acetate. The
combined organic extracts were washed with brine, dried, and
evaporated under reduced pressure. The residue was purified
by chromatography (eluent, diethyl ether/light petroleum).
4-(5,6,7,8-Tetr ah ydr o-5,5,8,8-tetr am eth yl-2-n aph th alen -
2-ylm eth yla m in o)ben zoic a cid eth yl ester (17a ): yield
60%; mp 58-60 °C (diethyl ether/light petroleum); IR (KBr)
1
1691, 1611, 1520, 1455, 1286, 1178, 1107, 843, 769; H NMR
1.30 (s, 12H), 1.37 (t, 3H, J ) 7), 1.70 (s, 4H), 3.5 (br, 1H),
4.29-4.35 (m, 4H), 6.62 (d, 2H, J ) 8.8), 7.12 (dd, 1H, J )
8.4, J ) 1.4), 7.28-7.33 (m, 2H), 7.89 (d, 2H, J ) 8.7). Anal.
(C24H31NO2): C, H, N.
1,2,3,4-Tetr a h yd r o-1,1,4,4-tetr a m eth yl-6-vin yln a p h th a -
1
len e (12): yield 67%; oil; H NMR 1.27 (s, 6H), 1.29 (s, 6H),
3-(5,6,7,8-Tetr ah ydr o-5,5,8,8-tetr am eth yl-2-n aph th alen -
1.68 (s, 4H), 5.17 (d, 1H, J ) 10.8), 5.69 (d, 1H, J ) 17.6), 6.68
(dd, 1H, J ) 17.6, J ) 10.8), 7.23-7.31 (m, 3H). Anal.
(C16H22): C, H.
2-ylm eth yla m in o)ben zoic a cid eth yl ester (17b): yield
80%; mp 45-46 °C (diethyl ether/light petroleum); H NMR
1.29 (s, 12H), 1.39 (t, 3H, J ) 7), 1.70 (s, 4H), 3.50 (br, 1H),
4.29 (s, 2H), 4.36 (q, 2H, J ) 7), 6.81-6.88 (m, 1H), 7.16-7.39
(m, 6H). Anal. (C24H31NO2): C, H, N.
1
Gen er a l P r oced u r e for Isoxa zolin es 10a a n d 14a . A
mixture of N-chlorosuccinimide (174 mg, 1.3 mmol), pyridine
(2 drops), and oxime 8 or 13 (1.3 mmol) in anhydrous CHCl3
(15 mL) was stirred for 1 h at 50-60 °C. Olefin 9 or 12 (1.4
mmol) was then added followed by triethylamine (0.27 mL,
1.95 mmol) in CHCl3 (5 mL). After 20 min of stirring at 25 °C,
water was added, and the organic phase was washed with 2.5%
HCl and water, then dried, and evaporated under reduced
pressure. The residue was chromatographed on silica gel
(eluent, ethyl acetate/light petroleum).
2-(5,6,7,8-Tetr ah ydr o-5,5,8,8-tetr am eth yl-2-n aph th alen -
2-ylm eth yla m in o)ben zoic a cid eth yl ester (17c): yield
1
67%; mp 40-42 °C (diethyl ether/light petroleum); H NMR
1.27 (s, 12H), 1.38 (t, 3H, J ) 7.1), 1.70 (s, 4H), 3.25 (br, 1H),
4.26-4.37 (m, 4H), 6.06-6.73 (m, 2H), 7.10-7.16 (m, 1H),
7.28-7.34 (m, 3H), 7.94 (dd, 1H, J ) 8.0, J ) 1.5). Anal.
(C24H31NO2): C, H, N.
4-(N-Meth yl-5,6,7,8-tetr ah ydr o-5,5,8,8-tetr am eth yln aph -
th a len -2-ylm eth yla m in o)ben zoic a cid eth yl ester (17d ).
Sodium cyanoborohydride (50 mg, 0.8 mmol) was added to a
stirred solution of 17a (183 mg, 0.5 mmol) in acetonitrile (5
mL), containing 37% aqueous formaldehyde (0.22 mL, 2.7
mmol). After 15 min of stirring at room temperature, the
reaction was treated dropwise with glacial AcOH until the
solution tested neutral. Stirring was continued for an ad-
ditional 45 min, with occasional addition of glacial AcOH to
maintain the pH near neutrality. The solvent was evaporated
at reduced pressure, and 2 N KOH (10 mL) was added to the
residue. The resulting mixture was extracted with diethyl
ether. The combined ether extracts were washed with 0.5 N
KOH, dried, and evaporated in vacuo. The residue was
chromatographed on silica gel (eluent, diethyl ether/light
petroleum): yield 80%; oil; 1H NMR 1.22 (s, 6H), 1.26 (s, 6H),
1.36 (t, 3H, J ) 7), 1.67 (s, 4H), 3.10 (s, 3H), 4.32 (q, 2H, J )
7), 4,57 (s, 2H), 6.71 (d, 2H, J ) 9), 6.92 (dd, 1H, J ) 8.2),
7.10 (s, 1H), 7.24 (d, 1H, J ) 8.3), 7.9 (d, 2H, J ) 9). Anal.
(C25H33NO2): C, H, N.
4-(5,6,7,8-Tet r a h yd r o-5,5,8,8-t et r a m et h yln a p h t h a len -
2-ylm eth oxy)ben zoic a cid m eth yl ester (20a ). To a stirred
solution of 19 (0.17 g, 1.1 mmol) in anhydrous N,N-dimethyl-
formamide (10 mL) was added sodium hydride (0.1 g, 3.85
mmol) in a single portion at room temperature. The mixture
was stirred for 10 min at room temperature, and then benzyl
bromide 18 (0.62 g, 2.2 mmol) was added. Stirring was
continued for another 2 h, diethyl ether (15 mL) was added,
and the mixture was poured into ice-water. The extract was
washed with brine, dried, and concentrated. The crude product
was purified by silica gel column chromatography (eluent,
diethyl ether/light petroleum): yield 56%; mp 152-154 °C
(methanol); IR (KBr) 1717, 1608, 1510, 1263, 1166, 1106, 1043,
847, 766; 1H NMR 1.25 (s, 6H), 1.29 (s, 6H), 1.69 (s, 4H), 3.89
(s, 3H), 5.04 (s, 2H), 7.01 (d, 2H, J ) 8.9), 7.22 (dd, 1H, J ) 8,
J ) 2.5), 7.33-7.35 (m, 2H), 8.00 (d, 2H, J ) 8.8). Anal.
(C23H28O3): C, H.
4-[3-(5,6,7,8-Tetr ah ydr o-5,5,8,8-tetr am eth yln aph th alen -
2-yl)-4,5-d ih yd r oisoxa zol-5-yl]ben zoic a cid m eth yl ester
(10a ): yield 63%; mp 138-140 °C (ethyl acetate/light petro-
leum); 1H NMR 1.22 (s, 6H), 1.24 (s, 6H), 1.63 (s, 4H), 3.24
(dd, 1H, J ) 16.6, J ) 7.6), 3.77 (dd, 1H, J ) 16.6, J ) 11.1),
3.85 (s, 3H), 5.71 (dd, 1H, J ) 11, J ) 7.8), 7.20-7.42 (m, 4H),
7.60 (d, 1H, J ) 1.5), 7.98 (dd, 2H, J ) 6.7, J ) 1.7). Anal.
(C25H29NO3): C, H, N.
4-[5-(5,6,7,8-Tetr ah ydr o-5,5,8,8-tetr am eth yln aph th alen -
2-yl)-4,5-d ih yd r oisoxa zol-3-yl]ben zoic a cid m eth yl ester
(14a ): yield 75%; mp 171-173 °C (ethyl acetate/light petro-
leum); IR (KBr) 1718, 1587, 1435, 1283, 1112, 916, 772; 1H
NMR 1.27 (s, 6H), 1.29 (s, 6H), 1.66 (s, 4H), 3.38 (dd, 1H, J )
16.5, J ) 8.9), 3.76 (dd, 1H, J ) 16.5, J ) 11), 3.94 (s, 3H),
5.68-5.78 (m, 1H), 7.15 (d, 1H, J ) 8.3), 7.31-7.35 (m, 2H),
7.77 (d, 2H, J ) 8.4), 8.08 (d, 2H, J ) 8.4). Anal. (C25H29NO3)
C, H, N.
Gen er a l P r oced u r e for Isoxa zoles 11a a n d 15a . To a
solution of the appropriate isoxazoline 10a or 14a (0.25 mmol)
in carbon tetrachloride (10 mL) was added NBS (66 mg, 0.37
mmol), and the mixture was gently refluxed for 3 h. Hydrogen
bromide was slowly liberated. The cooled solution was filtered
from the precipitated succinimide and washed with 5% aque-
ous sodium hydroxide and then with water until the organic
phase became clear. The organic layer was dried and the
solvent removed in vacuo. The residue was chromatographed
on silica gel (eluent, ethyl acetate/light petroleum).
4-[3-(5,6,7,8-Tetr ah ydr o-5,5,8,8-tetr am eth yln aph th alen -
2-yl)isoxa zol-5-yl]ben zoic a cid m eth yl ester (11a ): yield
80%; mp 137-140 °C (ethyl acetate/light petroleum); IR (KBr)
1716, 1594, 1424, 1280, 1106, 771; 1H NMR 1.33 (s, 6H), 1.36
(s, 6H), 1.74 (s, 4H), 3.97 (s, 3H), 6.94 (s, 1H), 7.42 (d, 1H, J )
8.1), 7.62 (dd, 1H, J ) 8.1, J ) 1.2), 7.83 (d, 1H, J ) 1.2), 7.93
(d, 2H, J ) 8.3), 8.16 (d, 2H, J ) 8.3). Anal. (C25H27NO3): C,
H, N.
4-[5-(5,6,7,8-Tetr ah ydr o-5,5,8,8-tetr am eth yln aph th alen -
2-yl)isoxa zol-3-yl]ben zoic a cid m eth yl ester (15a ): yield
60%; mp 140-143 °C (ethyl acetate/light petroleum); 1H NMR
1.32 (s, 6H), 1.36 (s, 6H), 1.73 (s, 4H), 3.96 (s, 3H), 6.82 (s,
1H), 7.43 (d, 1H, J ) 8.3), 7.58 (dd, 1H, J ) 8.3, J ) 1.7), 7.80
(d, 1H, J ) 1.7), 7.96 (d, 2H, J ) 8.4), 8.15 (d, 2H, J ) 8.4).
Anal. (C25H27NO3): C, H, N.
Gen er a l P r oced u r e for Am in es 17a )c. Sodium cyano-
borohydride (0.31 g, 5 mmol) was added at 0 °C to a solution
of the aldehyde 6 (1.08 g, 5 mmol) and the appropriate amine
16a -c (5 mmol) in MeOH (20 mL) containing AcOH (0.2 mL).
Gen er a l P r oced u r e for Ca r boxylic Acid s 10b, 11b, 14b,
15b, 17e)h , a n d 20b. A mixture of ester 10a , 11a , 14a , 15a ,
17a -d , or 20a (1 mmol), methanol (10 mL), water (6-7 mL),
and lithium hydroxide (40 mg, 1.5 mmol) was allowed to stand
at 50-60 °C for 24 h. The solution was concentrated in vacuo
to remove methanol, and the remaining aqueous solution was
extracted with diethyl ether to separate trace amounts of
unreacted ester. The aqueous solution was acidified with 1 M
hydrochloric acid and extracted with three portions of ethyl
acetate. [In the case of esters 17a -d , the aqueous solution

2316 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 14
Simoni et al.
was neutralized (pH 7) with 1 M hydrochloric acid and
extracted with dichloromethane.] The combined organic ex-
tracts were washed with saturated aqueous sodium chloride
and dried. Removal of the solvent under reduced pressure
afforded a residue, which was chromatographed on silica gel
(eluent, ethyl acetate/light petroleum).
4-[3-(5,6,7,8-Tetr ah ydr o-5,5,8,8-tetr am eth yln aph th alen -
2-yl)-4,5-d ih yd r oisoxa zol-5-yl]b en zoic a cid (10b ): yield
84%; mp 129-132 °C (ethyl acetate); IR (KBr) 1686, 1612,
1458, 1420, 1285, 896; 1H NMR 1.30 (s, 6H), 1.32 (s, 6H), 1.71
(s, 4H), 3.33 (dd, 1H, J ) 16.6, J ) 7.6), 3.87 (dd, 1H, J )
16.5, J ) 11.1), 5.81 (dd, 1H, J ) 11, J ) 7.6), 7.34-7.54 (m,
4H), 7.68 (d, 1H, J ) 1.1), 8.14 (d, 2H, J ) 8.2), 11.44 (br, 1H).
Anal. (C24H27NO3): C, H, N.
RPMI 1640 (Gibco, Grand Island, NY) containing 10% FCS
(Gibco), 100 units/mL penicillin (Gibco), 100 mg/mL strepto-
mycin (Gibco), and 2 mM L-glutamine (Sigma Chemical Co.,
St. Louis, MO) in a 5% CO2 atmosphere at 37 °C. SW962 cells
were obtained from ATCC and were grown in Cellgro L15
media (Fisher Scientific, Pittsburgh, PA) containing 10% FBS
(Sigma Chemical Co.) and antibiotic/antimycotic (Sigma Chemi-
cal Co.) in a 5% CO2 atmosphere at 37 °C. Only lots of FBS
containing undetectable (<8 nM) vitamin A, as determined by
HPLC, were used.
Cell Gr ow th In h ibition Assa ys. To determine the growth
inhibitory activity of the drugs tested, 2 × 105 cells were plated
into 25-mm wells (Costar, Cambridge, MA) in 1 mL of complete
medium and treated with different concentrations of each drug.
After 48 h of incubation, the number of viable cells was counted
on a hemocytometer after staining with trypan blue to exclude
dead cells and expressed as percent of control cell proliferation.
IC50 (drug concentration able to induce a 50% cell growth
inhibition) and AC50 (drug concentration able to induce apo-
ptosis in 50% of cells) were calculated after exposure of cells
to increasing concentrations of each drug for 48 h.
An a lysis of Cellu la r Differ en tia tion . Cells (2 × 105) were
exposed to each retinoid and after 6 days were examined for
induction of differentiation by morphology and flow cytometry.
The morphology of the cells was evaluated from cytospin slide
preparations stained with May Grunwald-Giemsa stain solu-
tions. The expression of monocytic (CD14) or granulocytic
(CD11c) cell surface antigens was studied by a two-color direct
immunofluorescence staining technique. Cells were stained
using FITC-conjugated mouse anti-human CD14 and PE-
conjugated anti-human CD11c mouse monoclonal antibodies
(both from ORTHO Diagnostic System, Raritan, NJ ). Control
studies were performed with negative murine isotype antibod-
ies. Analysis of fluorescence was performed using a FACSort
instrument (Becton Dickinson, Mountain View, CA).
Mor p h ologica l Eva lu a tion of Ap op tosis a n d Necr osis.
Apoptosis and necrosis were determined morphologically by
fluorescent microscopy after labeling with acridine orange and
ethidium bromide. Cells (2 × 105) were centrifuged (300g), and
the pellet was resuspended in 25 µL of the dye mixture. The
suspensions of 10 µL were placed on a microscope slide and
covered with a 22-mm2 coverslip and examined in oil immer-
sion with a 100× objective using a fluorescent microscope. Live
cells were determined by the uptake of acridine orange (green
fluorescence) and exclusion of ethidium bromide (red fluores-
cence). Live and dead apoptotic cells were identified by
perinuclear condensation of chromatin stained by acridine
orange or ethidium bromide, respectively, and by the formation
of apoptotic bodies. Necrotic cells were identified by uniform
labeling of the cells with ethidium bromide.
4-[5-(5,6,7,8-Tetr ah ydr o-5,5,8,8-tetr am eth yln aph th alen -
2-yl)-4,5-d ih yd r oisoxa zol-3-yl]b en zoic a cid (14b ): yield
1
88%; mp 219-221 °C (methanol); H NMR 1.28 (s, 6H), 1.29
(s, 6H), 1.69 (s, 4H), 3.39 (dd, 1H, J ) 16.7, J ) 8.9), 3.77 (dd,
1H, J ) 16.7, J ) 11), 5.70-5.80 (m, 1H), 7.15 (dd, 1H, J )
8.2, J ) 1.3), 7.31-7.35 (m, 2H), 7.80 (d, 2H, J ) 8.4), 8.15 (d,
2H, J ) 8.4), 12.15 (br, 1H). Anal. (C24H27NO3): C, H, N.
4-[3-(5,6,7,8-Tetr ah ydr o-5,5,8,8-tetr am eth yln aph th alen -
2-yl)isoxa zol-5-yl]ben zoic a cid (11b): yield 80%; mp 239-
1
241 °C (methanol); IR (KBr) 1689, 1598, 1424, 1292, 772; H
NMR (acetone-d6) 1.33 (s, 6H), 1.37 (s, 6H), 1.76 (s, 4H), 5.63
(s, 1H), 7.51 (d, 1H, J ) 8.2), 7.75 (dd, 1H, J ) 8.2, J ) 1.9),
7.95(d, 1H, J ) 1.9), 8.08 (d, 2H, J ) 8.4), 8.21 (d, 2H, J )
8.4), 11.21 (br, 1H). Anal. (C24H25NO3): C, H, N.
4-[5-(5,6,7,8-Tetr ah ydr o-5,5,8,8-tetr am eth yln aph th alen -
2-yl)isoxa zol-3-yl]ben zoic a cid (15b): yield 74%; mp 218-
220 °C (methanol); 1H NMR 1.33 (s, 6H), 1.36 (s, 6H), 1.74 (s,
4H), 6.85 (s, 1H), 7.44 (d, 1H, J ) 8.4), 7.60 (dd, 1H, J ) 8.4,
J ) 1.2), 7.81 (d, 1H, J ) 1.2), 8.02 (d, 2H, J ) 8.2), 8.25 (d,
2H, J ) 8.2), 11.58 (br, 1H). Anal. (C24H25NO3): C, H, N.
4-(5,6,7,8-Tet r a h yd r o-5,5,8,8-t et r a m et h yln a p h t h a len -
2-ylm eth oxy)ben zoic a cid (20b): yield 80%; mp 199-201
°C (methanol); IR (KBr) 1681, 1605, 1512, 1427, 1253, 1167,
1
1043, 772; H NMR 1.30 (s, 12H), 1.70 (s, 4H), 5.07 (s, 2H),
7.04 (d, 2H, J ) 8.8), 7.21-7.24 (m, 1H), 7.34-7.36 (m, 2H),
8.08 (d, 2H, J ) 8.8), 11.97 (br, 1H). Anal. (C22H26O3): C, H.
4-(5,6,7,8-Tet r a h yd r o-5,5,8,8-t et r a m et h yln a p h t h a len -
2-ylm eth yla m in o)ben zoic a cid (17e): yield 90%; mp 207-
209 °C (water); IR (KBr) 1661, 1603, 1530, 1457, 1280, 1181,
838, 774; 1H NMR 1.28 (s, 6H), 1.29 (s, 6H), 1.70 (s, 4H), 3.52
(br, 1H), 4,33 (s, 2H), 6.63 (d, 2H, J ) 8.3), 7.11-7.16 (m, 1H),
7.28-7.36 (m, 2H), 7.95 (d, 2H, J ) 8.6), 12.55 (br, 1H). Anal.
(C22H27NO2): C, H, N.
3-(5,6,7,8-Tet r a h yd r o-5,5,8,8-t et r a m et h yln a p h t h a len -
2-ylm eth yla m in o)ben zoic a cid (17f): yield 80%; mp 151-
153 °C (methanol); 1H NMR 1.28 (s, 6H), 1.29 (s, 6H), 1.69 (s,
4H), 3.15 (br, 1H), 4,30 (s, 2H), 6.89 (dd, 1H, J ) 7.5, J ) 2.5),
7.15 (dd, 1H, J ) 7.5, J ) 2.5), 7.29-7.33 (m, 3H), 7.40-7.41
F low Cytom etr y. Cells were washed twice with ice-cold
PBS. To determine apoptosis and cell-cycle phase distribution,
the cells were resuspended at 1 × 106/mL of a hypotonic
fluorochrome solution containing propidium iodide [50 mg/mL
in 0.1% sodium citrate plus 0.03% (v/v) Nonidet P-40]. After 1
h of incubation, the samples were filtered through 40-µm nylon
mesh cloth, and fluorescence was analyzed as single-parameter
frequency histograms using a FACSort instrument. Apoptosis
was determined by evaluating the percentage of events ac-
cumulating in the pre-G0G1 position.
(m, 1H), 7.45 (d, 1H, J ) 7.5), 12.45 (br, 1H). Anal. (C22H27
NO2): C, H, N.
-
2-(5,6,7,8-Tet r a h yd r o-5,5,8,8-t et r a m et h yln a p h t h a len -
2-ylm eth yla m in o)ben zoic a cid (17g): yield 70%; mp 197-
199 °C (water); 1H NMR 1.28 (s, 6H), 1.29 (s, 6H), 1.69 (s, 4H),
3.77 (br, 1H), 4.42 (s, 2H), 6.60-6.70 (m, 2H), 7.12 (dd, 1H, J
) 7.3, J ) 2.2), 7.30-7.31 (m, 1H), 7.37 (dd, 1H, J ) 7.3, J )
2), 7.98 (dd, 2H, J ) 7.5, J ) 1.9), 12.62 (br, 1H). Anal. (C22H27
NO2): C, H, N.
-
Recep tor Activa tion . Cultures of the SW962 cell line that
had been stably transfected with the RARE-tk-CAT reporter
4-(N-Meth yl-5,6,7,8-tetr ah ydr o-5,5,8,8-tetr am eth yln aph -
th a len -2-ylm eth yla m in o)ben zoic a cid (17h ): yield 80%;
mp 261-263 °C (water); 1H NMR (dimethyl sulfoxide-d6) 1.16
(s, 6H), 1.19 (s, 6H), 1.60 (s, 4H), 3.07 (s, 3H), 4.59 (s, 2H),
6.74 (d, 2H, J ) 8.9), 6.87 (dd, 1H, J ) 8.1, J ) 1.1), 7.12 (s,
1H), 7.23 (d, 1H, J ) 8.1), 7.72 (d, 2H, J ) 8.8), 12.10 (br, 1H).
Anal. (C23H29NO2): C, H, N.
Biology. Cell Cu ltu r es. HL60 and K562 cell lines were
obtained from ATCC (Rockville, MD). HUT78 cells were
obtained as gifts from Dr. Giovina Ruberti (CNR, Rome, Italy).
HL60R were selected for multidrug resistance (MDR) by
exposure to gradually increasing concentrations of daunoru-
bicin and express P-glycoprotein. HL60 cells were grown in
gene were treated with each compound at 10-9, 10-8, 10-7, 10-6
,
and 10-5 M in dimethyl sulfoxide (DMSO). A control culture
was treated with the same volume of DMSO that was used in
the treated cultures. After 24 h of treatment, cellular extracts
were prepared and assayed for CAT protein using an ELISA
kit (Roche Molecular Biochemicals, Indianapolis, IN). The
amount of CAT in each extract was normalized by dividing by
the protein concentration in each extract that was determined
using the Bio-Rad protein assay (Bio-Rad, Richmond, CA).
Each treatment was performed in triplicate, and the normal-
ized CAT values were averaged and used to determine the fold
RARE induction. The fold RARE induction was derived by

Heterocycle-Containing Retinoids
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