Synthesis of Flexible Sulfur-Containing Heteroarotinoids That Induce Apoptosis and Reactive Oxygen Species with Discrimination between Malignant and Benign Cells

Article abstract of DOI:10.1021/jm030346v

Regulation of growth, differentiation, and apoptosis by synthetic retinoids can occur through mechanisms that are dependent and independent of their ability to bind and activate nuclear retinoic acid receptors. The objective of this study was to determine

Full text of DOI:10.1021/jm030346v

J . Med. Chem. 2004, 47, 999-1007
999
Syn th esis of F lexible Su lfu r -Con ta in in g Heter oa r otin oid s Th a t In d u ce
Ap op tosis a n d Rea ctive Oxygen Sp ecies w ith Discr im in a tion betw een
Ma lign a n t a n d Ben ign Cells
Shengquan Liu,† Chad W. Brown,† K. Darrell Berlin,*,† Aridam Dhar,‡ Suresh Guruswamy,‡ David Brown,‡
Ginger J . Gardner,§ Michael J . Birrer,§ and Doris M. Benbrook*,‡
Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078-3071, Department of Obstetrics &
Gynecology, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, Oklahoma 73190, and Cell and
Cancer Biology Department, Center for Cancer Research, National Cancer Institute, 9610 Medical Center Drive, Building C,
Rockville, Maryland 20850
Received J uly 16, 2003
Regulation of growth, differentiation, and apoptosis by synthetic retinoids can occur through
mechanisms that are dependent and independent of their ability to bind and activate nuclear
retinoic acid receptors. The objective of this study was to determine if increasing flexibility of
the heteroarotinoid structure would affect the specificity of the synthetic retinoids for the
receptors and for their regulation of cancerous and nonmalignant cells. Methods were developed
to produce the first examples of heteroarotinoids 15a -15h , which contain urea and/or thiourea
linking groups between two aryl rings. Substituents at the para position of the single phenyl
ring were either an ester, a nitro group, or a sulfonamide group. Ovarian cancer cell lines
Caov-3, OVCAR-3, SK-OV-3, UCI-101, and 222 were utilized, and the inhibitory prowess of
the heteroarotinoids was referenced to that of 4-HPR (25). Similar to 4-HPR (25), the
heteroarotinoids inhibited growth of all cell lines at micromolar concentrations. Although the
heteroarotinoids did not activate retinoic acid receptors, the agents induced potent growth
inhibition against the cancer cells with weak activity against normal and benign cells. The
growth inhibition was associated with cell loss and induction of reactive oxygen species.
In tr od u ction
cant anticancer properties, as has 13-cis-retinoic acid
(3).6 Some synthetic successor retinoids, such as aroti-
Retinoids (analogues of vitamin A) are known to
possess anticancer properties as well as provide useful
pharmaceutical responses in the treatment of various
other disorders and thus have received considerable
scientific attention during the past 2 decades.1 Retinoids
exhibit their therapeutic properties, at least in part, by
interaction with and activation of a specific group of
nuclear receptors, termed retinoid receptors.2 The ret-
inoid receptors are divided into two distinct types,
namely the retinoic acid receptors (RARs)3 and the
retinoid X receptors (RXRs).4 For each type, three
receptor subtypes, namely RARR, -â, and -γ and RXRR,
-â, and -γ, have been identified, for a total of six retinoid
receptors. These retinoid receptors belong to the hor-
mone receptor superfamily, and, as a group, this family
of proteins regulates the transcription of genes that
control a variety of metabolic, developmental, and
differentiation pathways. Because the misregulation of
any one of these pathways could enhance carcinogen-
esis, the hormones, or compounds such as retinoids, may
regulate these receptors and are therefore potentially
chemopreventive agents.5
noid TTNPB (4) and the clinically employed Etretinate
(5), have also shown potent anticarcinogenic character-
istics.6 However, all of these retinoids have found
limited application due to teratogenicity7 and/or toxic
manifestations such as diarrhea, hair loss, nausea, and
headaches.7,8 Thus, the study of heteroarotinoids origi-
nated9,10 as an attempt to mimic the anticarcinogenic
action of retinoids, while at the same time reducing
unwanted side effects. In fact, several heteroarotinoids
have demonstrated reduced toxicity compared to 1 and
49e and, in some examples, exhibited greater anticancer
Naturally occurring retinoids trans-retinoic acid (1)
and 9-cis-retinoic acid (2) are endogenous ligands for
the retinoid receptors and have demonstrated signifi-
* Corresponding authors. For biology (D.M.B.): e-mail, Doris-
Benbrook@ouhsc.edu. For chemistry (K.D.B.): e-mail, kdb@okstate.edu.
† Oklahoma State University.
‡ University of Oklahoma Health Sciences Center.
§ National Cancer Institute.
10.1021/jm030346v CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/09/2004

1000 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Liu et al.
activity than selected natural retinoids.9,10 Conse-
quently, heteroarotinoids are currently under extensive
investigation for the potential treatment of a variety of
disorders.9,10
Ta ble 1. Synthesis of Urea/Thiourea-Containing
Heteroarotinoids 15a -h from 18 or 24
Heteroarotinoids such as 6-10 constitute a class of
synthetic retinoids9,10 that structurally resemble aroti-
noids in that at least one aryl moiety is present within
the molecular structure. However, heteroarotinoids
entry
R
X
R
Z
product
yield (%)
1
2
3
4
5
6
7
8
H
H
H
H
O
S
S
S
S
O
S
S
H
H
H
H
CH3
H
H
CO2Et
CO2Et
NO2
SO2NH2
NO2
CO2Et
CO2Et
SO2NH2
15a
15b
15c
15d
15e
15f
15g
15h
64
92
83
87
82
43
66
56
H
CH3
CH3
CH3
H
the fixed ligands based upon X-ray diffraction studies
of RARγ t-RA where both ligand and receptor were in
rigid forms.9c Moreover, a urea or thiourea linker
between the aryl rings introduces marked flexibility in
the ligand and provides H-bonding capablility in a
strategic location within the molecule. Certain studies
had suggested12,13 that the presence of a three-atom
linker may increase specificity for the RARs. Others
demonstrated that a linker with an attached, nonbulky
functionality capable of hydrogen bonding via proton
donation (such as via an OH) enhanced RARγ selectiv-
ity.12,14 By constructing an agent possessing a semiflex-
ible, three-atom linker with self-capability of hydrogen
bonding through proton donation, it was reasoned that
possibly a higher RARγ specificity would be produced,
thus further increasing the efficacy of such retinoids.
Furthermore, due to a high expression of RARγ in both
skin15 and urogenital tissues,16 such an agent could be
useful for the specific treatment of disorders such as
melanoma or vulvarian carcinomas.
We now report the synthesis and biological activity
of sulfur-containing heteroarotinoids 15a -15h with
thiourea and urea linkers between the aryl rings. No
heteroarotinoids with this type of linker have been
reported.11a Interestingly, the heteroarotinoids described
did not activate retinoid receptors in two assays. How-
ever, the sulfur-containing heteroarotinoid systems
15a -h (Tables 1 and 2) demonstrated marked inhibi-
tory ability for growth of ovarian cancer cell lines Caov-
3, OVCAR-3, SK-OV-3, ICU-101, and 222. The EC50
values of the compounds were compared to that of
4-hydroxyphenylretinamide (4-HPR), a clinically used
retinoid,17 which has been shown to induce reactive
oxygen species (ROS). Growth inhibition by heteroaro-
tinoids 15a was associated with cell loss and induction
of ROS.
contain an aryl-fused heterocyclic ring as a modification,
and several studies have shown that some heteroaro-
tinoids demonstrate promising inhibition of various
cancers as well as reduced toxicity.2c,9,10 Recent inves-
tigations of heteroarotinoids focused on structures
containing a two-atom “linker” group (two atom moiety
connecting the two aryl rings),9 such as an amide (8),9b
ester (9),9c or N-methoxyamide (10)9b functionality,
among others.9e It logically followed that synthesis and
study of heteroarotinoids containing three-atom or even
four-atom linkers, similar to those in the two-atom
linker structures, would be worthwhile. Although a
search of the literature revealed some retinoids with
three- or more-atom linkers,11-13 structures with only
a few types of three-atom linker groups, such as chal-
cone (11), propenol (12), or R-hydroxyamide groups
(13),12 have been reported. Only one retinoid (14)
containing a 1,4-diketone group as a four-atom linker
was found.11b
The rationale for our present work is based upon the
above citations and on the following observations.
Utilizing a computer simulation program which, when
modified slightly, allowed the creation of flexible ligand/
receptor, or combinations thereof, it was discovered that
the flexible ligands were at a slightly lower energy than
Resu lts a n d Discu ssion
Ch em istr y. Due to the reduced toxicity and cancer
inhibition exhibited by some reported sulfur heteroarotin-
oids,2c,9a,9e a heterocyclic ring containing a gem-dimethyl
group adjacent to a sulfur atom was chosen as part of

Flexible Sulfur-Containing Heteroarotinoids
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 1001
Ta ble 2. Potencies and Efficacies of Growth Inhibition of Ovarian Cancer Cell Lines by Heteroarotinoids 15a -h
Caov-3 OVCAR-3 SK-OV-3
potencya (µM) potencya (µM) potencya (µM)
product
15a
efficacyb (%)
efficacyb (%)
efficacyb (%)
7.0 ( 0.3
4.6 ( 1.7
3.7 ( 0.3
7.0 ( 3.5
7.4 ( 0.7
10.2 ( 0.7
4.0 ( 2.8
0.9 ( 0.7
3.7 ( 0.2
63.7 ( 5.1
38.1 ( 5.4
89.2 ( 4.3
16.7 ( 4.0
52.1 ( 7.4
54.9 ( 7.4
37.4 ( 7.8
30.3 ( 7.4
100.0 ( 3.7
3.9 ( 0.2
3.9 ( 0.3
1.0 ( 0.1
4.4 ( 0.8
3.7 ( 0.2
4.7 ( 1.0
4.4 ( 0.4
4.4 ( 0.6
6.9 ( 0.3
160.4 ( 3.0
133.8 ( 5.4
155.2 ( 2.2
74.1 ( 6.7
149.5 ( 3.2
107.0 ( 7.0
114.4 ( 4.9
90.0 ( 6.2
100.0 ( 4.7
4.1 ( 0.8
3.9 ( 1.0
0.2 ( 0.5
7.8 ( 0.8
3.9 ( 1.2
7.2 ( 2.3
4.4 ( 2.3
6.9 ( 1.5
8.2 ( 0.6
71.8 ( 6.4
64.9 ( 6.9
69.1 ( 2.4
50.5 ( 8.4
78.4 ( 7.2
75.7 ( 16.4
85.4 ( 9.5
67.7 ( 13.7
100.0 ( 3.9
15b
15c
15d
15e
15f
15g
15h
25 (4-HPR)
a
The potency difference is the concentration that exerts half-maximal activity (EC50 value) derived from a graph of percent growth
b
inhibition versus concentration using GraphPad Software. The efficacy is the growth inhibition activity of the compound at 10 µM
concentration relative to the maximum activity observed for 4-HPR with the respective cell line. The maximal growth inhibition activity
of 10 µM 4-HPR (25) was 61.97% for Caov-3, 43.1% for OVCAR-3, and 64.9% for SK-OV-3. The results represent the average and standard
error of three experiments performed in triplicate.
Sch em e 1
the structural foundation for this series of compounds.
It was conceived that the conversion 16 f 17 f 18
Heteroarotinoid analogues 15f-h were envisioned to
be accessible from unknown amine 24. Thus, the
conversions of 19 f 20 f 21 f 22 f 23 f 24 (Scheme
1) was implemented and provided 24, which was then
used to generate the targets 15a -h (Table 1). The
sequence utilized 3-methylthiophenol in a Michael ad-
dition to mesityl oxide and yielded ketone 20 (87%).
Treatment of ketone 20 with excess MeMgBr gave
alcohol 21 (84%). Dehydration-cyclization of 21 was
then accomplished by the action of AlCl3 in CS2 to afford
thiochroman 22 (93%). Nitration of thiochroman 22 was
achieved using HNO3/Ac2O18 to afford the 6-isomer 23
in a modest yield (26%), with several byproducts being
formed, including some via oxidation of the sulfur atom.
Reduction of the nitro group in 23 occurred with Fe/
AcOH18 in refluxing EtOH to yield amine 24 (45%).
The thiourea or urea group (Table 1) was chosen as a
three-atom linker due to its somewhat flexible nature,
which might allow a good fit into the RARs and
therefore enhance activation. Moreover, a thiourea or
urea function provides two -NH- groups capable of
hydrogen bonding with the receptor, thus possibly
improving RARγ selectivity. To generate the desired
heteroarotinoids 15a -h possessing urea linking groups,
the conversions of 18 f 15a -e and 24 f 15f-h (Table
1) were performed. Amine 18 (or 24) was coupled with
isocyanates and/or isothiocyanates in dry THF. The
crude products were recrystallized from appropriate
solvent systems to afford the desired compound with
generally good yields.
would provide key intermediate amine 18, which could
be utilized to incorporate the desired structural char-
acteristics into the target heteroarotinoids 15a -e.
Although amine 18 is known,9b different conditions were
employed here as outlined above. The reaction sequence
began with the nitration of the thiochroman 1610a using
HNO3/Ac2O18 to afford the 6-isomer 17 in a modest yield
(26%) with many other products being formed. However,
in this case only 1 equiv of the nitrating agent was used,
as compared to 2 equiv in the reported procedure,9b and
with little loss in yield (27% being reported previously9b).
Reduction of the nitro group in 17 was accomplished
using iron/acetic acid19 in refluxing EtOH to yield amine
18 (40%). Although a previous method9b recorded a yield
of 50% for amine 18 from the reduction of the nitro
group in 17, it required the use of a very large excess of
expensive TiCl3/HCl. In addition, the reaction workup
was complicated and required the handling of titanium
salts. In contrast, the iron/acetic acid approach is low-
cost and the workup is relatively simple.
Biologica l Activity
It seemed reasonable to prepare analogues of 15a ,
15b, and 15d possessing a methyl group attached to the
fused ring system at the C7 position, a situation which
could assist in determining more precisely the ligand-
receptor interactions within the hydrophobic region of
the receptor’s ligand-binding pocket (LBP).9b,c,20
Cisplatin is used as the current standard of care
chemotherapy administered to patients with ovarian
cancer. Compounds 15a -h (Table 2) were evaluated for
their ability to inhibit the growth of three ovarian
carcinoma cell lines, Caov-3, OVCAR-4-, and SK-OV-3,

1002 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Liu et al.
F igu r e 1. Heteroarotinoids and 4-HPR exhibit greater activity against cancerous and borderline ovarian cells than against
benign ovarian cells and normal endometrial cells. Cultures of an ovarian cancer cell line (OVCAR-3), a borderline ovarian tumor
(O1), a benign ovarian cyst (O3), and normal endometrial cells were treated with a range of concentrations of the indicated retinoids
or the same volume of solvent as a control. The percentage of growth inhibition after 3 days of treatment was determined using
the MTS assay. The results represent the average and standard deviation of three experiments performed in triplicate.
which vary in their sensitivities to retinoids and cis-
platin. The OVCAR-3 is cisplatin resistant, while Caov-3
and SK-OV-3 are cisplatin-sensitive. The SK-OV-3 line
is resistant to classical retinoid receptor-active retinoids,
while Caov-3 and OVCAR-3 are sensitive to these
retinoids. The activities of the heteroarotinoids were
compared to that of the well-known retinoid 4-HPR
(25),17 which has demonstrated chemoprevention activ-
resistant SK-OV-3 cell line were sensitive to these
compounds. The individual substitutions induced slight
alterations in the efficacy in a cell-dependent manner.
Overall, compound 15c, containing an NO2 group,
exhibited the highest activities against all cell lines
tested in terms of strong efficacies and very good
potencies. Regarding potencies, 15c was actually as good
or more potent than the clinical agent 4-HPR (25).
Given the potential clinical application of these
compounds for prevention and treatment of ovarian
cancer, it was important to determine if the agents were
toxic to normal cells and to pre-neoplastic lesions that
had not yet become cancerous. To evaluate the thera-
peutic ratios, the growth inhibition activities of het-
eroarotinoids 15a -c, 9-cis-retinoic acid (9-c-RA, 2), and
4-HPR (25) in primary cultures derived from normal
endometrial cells were compared to the activities in
cultures of the OVCAR-3 ovarian cancer cell line, a
borderline ovarian tumor (O1), and a benign ovarian
tumor (O3) (Figure 1). The 9-cis-retinoic acid (9-c-RA,
2) did not inhibit growth of any of these cultures.
Heteroarotinoids 15a -c and 4-HPR (25) exhibited
ity against ovarian cancer in clinical trials. The poten-
cies and efficacies derived from the growth inhibition
curves are summarized in Table 2.
All three cell lines were growth inhibited by all of the
heteroarotinoids tested, with EC50 values in the micro-
molar range, similar to 4-HPR (25). Interestingly, the
cisplatin-resistant OVCAR-3 cell line and retinoic acid-

Flexible Sulfur-Containing Heteroarotinoids
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 1003
independent mechanisms only at concentrations above
those achieved in clinical chemoprevention trials.22 At
concentrations <1 µM, however, 25 appears to behave
similarly to classical retinoids by inducing differentia-
tion through a receptor-dependent mechanism.17,22 Since
15a -h did not exhibit RARγ receptor activation in the
assays employed, it is conceivable that these hetero-
arotinoids may act by way of a different receptor or by
an independent pathway.17,22
The efficacy of these heteroarotinoids against ovarian
cancer cell lines which are resistant to standard cis-
platin chemotherapy and to trans-retinoic acid (1)
suggests that these agents may possess a unique mode
of action. This is consistent with the lack of direct
activation of the retinoic acid receptors. The present
study demonstrates that the growth inhibition by 15a -c
is due to cell loss. In a related study,9a it was shown
that cell loss in the OVCAR-3 ovarian cancer cell line
was due to induction of apoptosis. Since 15c exhibited
the highest degree of growth inhibition in a variety of
cell lines, this compound was evaluated in more detail
for the mechanism of apoptosis. In a study of head and
neck cancer cell lines, specific inhibitors were used to
demonstrate that the induction of apoptosis by 15c was
mediated through induction of ROS, G2 cell cycle arrest,
alterations in mitochondrial membrane permeability
transition (MPT), release of cytochrome c from the
mitochondria to the cytosol, and activation of caspase
3.21 In addition, a specific antagonist of retinoic acid
receptors was utilized to confirm that 15c induced
apoptosis independent of the retinoic acid receptors.21
The current study clearly demonstrates that ovarian
cancer cell lines also generate ROS in response to
treatment with heteroarotinoid 15c.
The differential effects of 15a -c on normal versus
cancer cells observed in this study clearly show that
such agents with urea and/or thiourea linkers have a
favorable therapeutic ratio in that cancer and precancer
cells were growth inhibited to much greater extents
than normal or benign cells. This favorable therapeutic
ratio was also observed in another study that illustrated
that 15b and 15c inhibited the growth of OVCAR-3
xenograph tumors in an animal model to similar degrees
as noted with 4-HPR (25) without any evidence of
toxicity.23 The lack of hypervitaminosis A toxicities of
15b and 15c in the animal model supports our observa-
tions that these compounds act independently of the
retinoic acid receptors.9a,23,24
F igu r e 2. Growth inhibition of ovarian cancer cell lines is
due to cell loss within 48 h. The optical density (OD) represents
the number of cells present at the indicated times of treatment
with 15c.
strong growth inhibition against the ovarian cancer cell
line and weak inhibition of the normal endometrial cells,
demonstrating good therapeutic ratios. The heteroaro-
tinoids exhibited strong growth inhibition against the
borderline tumor, demonstrating potential to prevent
progression of pre-neoplastic lesions to cancer, while
4-HPR (25) exhibited weak activity against the border-
line culture. Although benign ovarian cysts rarely
progress to cancer, they are often removed surgically
due to the deleterious effects of an increasing size.
Heteroarotinoid 15c exhibited the most potent growth
inhibition activity (4 µM) against the benign ovarian
culture.
The strong growth inhibition activity of the hetero-
arotinoids suggests that cell loss may be due to induc-
tion of apoptosis. In two related studies9a,21 with 15a -c
(previously labeled SHetA4, SHetA3, and SHetA2,
respectively),9a,21 it was demonstrated that these agents
induced apoptosis in ovarian and head/neck cancer cell
lines. To further evaluate the mechanism of growth
inhibition in ovarian cancer, the effects of 15c at two
different concentrations (5 and 10 µM) were evaluated
on two additional ovarian cell lines (UCI 101 and 222).
Within 24 h of treatment, the growth of both cell lines
were completely blocked by 15c at both concentrations
(Figure 2). Within 48 h, cell loss was apparent at the
10 µM concentration (Figure 2). To further investigate
the mechanism of this cell loss, ROS was measured in
both cell lines after treatment with 15c over a range of
concentrations for 24 h. At 1 µM concentration, 15c did
not induce ROS, while at 5 and 10 µM concentrations
of 15c (SHetA2), ROS was induced in a dose-response
manner (Figure 3).
Con clu sion s
In summary, we have developed procedures to obtain
a variety of heteroarotinoids with a urea or thiourea
linking group. The series of heteroarotinoids 15a -h
Chemoprevention activity by 4-HPR (25) is thought
to induce apoptosis through retinoic acid-receptor-
F igu r e 3. Generation of ROS in ovarian cancer cell lines. After 24 h of treatment with the indicated amount of 15c, the UCI 101
cell line (A) and the 222 cell line (B) were evaluated for ROS as indicated by fluoresence units.

1004 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Liu et al.
(195 mg, 1.02 mmol). Recrystallization (H2CCl2:pentane) gave
15a (263 mg, 64%) as a white solid: mp 200-201 °C; IR (KBr)
3342, 3190, 1720, 1652 cm-1; 1H NMR δ 1.32 (s, 6 H), 1.35 (t,
J ) 6.87 Hz, 3 H), 1.38 (s, 6 H), 1.89 (s, 2 H), 4.31 (q, J ) 7.0
Hz, 2 H), 6.91 (dd, J ) 11.20, 2.80 Hz, 1 H), 7.04 (d, J ) 8.38
Hz, 1 H), 7.26 (bs, 1 H), 7.35 (d, J ) 8.65 Hz, 2 H), 7.42 (d, J
contain the first such examples possessing flexible urea
or thiourea groups as three-atom linkers.11 The synthe-
ses involved a coupling reaction of various isocyanates
or isothiocyanates in THF with previously rare or
unknown amines 18 and 24, respectively. The hetero-
arotinoids described herein effectively inhibit growth of
a variety of ovarian cancer cell lines. Some of the agents
have demonstrated anticancer activities and a thera-
peutic window comparable to that of the clinical agent
4-HPR (25).The sensitivityofcisplatin-resistant OVCAR-3
cell lines suggests the potential use of these heteroaro-
tinoids in preventing recurrence of ovarian cancer in
patients who have received cisplatin chemotherapy.
Other biologicaleffects of15a-c haveappeared elsewhere.9a
Moreover, 15a and 15b act as antagonists of Mycobac-
terium bovis BCG, which suggests the agents inhibit
mycolic acid synthesis (see the companion paper).25
Results from the present work and related stud-
ies9,21,23 demonstrate that this new class of heteroaro-
tinoids induces apoptosis in cancer cells to a greater
extent than in normal cells through a mechanism that
is independent of retinoic acid receptor activation and
involves the generation of ROS.26 Compound 15c (la-
beled ShetA2 in the literature9a) is now in preclinical
development for cancer prevention and treatment
through the National Cancer Institutes (NCI) Rapid
Access to Intervention Development (RAID) program.
Thus, the present work provides a background for
additional exploration of the structures of heteroaroti-
noids as anticancer agents.
) 2.80 Hz, 1 H), 7.51 (bs, 1H), 7.91 (d, J ) 11.60 Hz, 2 H); 13
C
NMR ppm 14.20, 31.75, 32.13, 35.84, 42.03, 54.07, 61.87,
118.42, 120.02, 120.92, 124.78, 128.93, 129.02, 130.92, 134.28,
142.33, 144.09, 153.76, 166.86. Anal. (C23H28N2O3S) C, H, N,
S.
E t h yl 4-{[N-(2,2,4,4-Tet r a m et h ylt h ioch r om a n -6-yl)-
th ioca r ba m oyl]a m in o}ben zoa te (15b). The ester was pre-
pared from 18 (1.0 mmol) and 4-ethoxycarbonylphenyl isothio-
cyanate (211 mg, 1.02 mmol). Recrystallization (H2CCl2:
pentane) gave 15b (394 mg, 92%) as a white solid: mp 134-
135 °C; IR (KBr) 3285, 3138, 1688 cm-1; 1H NMR δ 1.35 (m, 9
H), 1.43 (s, 6 H), 1.95 (s, 2 H), 4.32 (q, J ) 7.14 Hz, 2 H), 7.03
(dd, J ) 6.18, 2.20 Hz, 1 H), 7.15 (d, J ) 8.24 Hz, 1 H), 7.37
(d, J ) 2.06 Hz, 1 H), 7.53 (d, J ) 8.65 Hz, 2 H), 8.01 (bs, 1H),
8.02 (d, J ) 6.87 Hz, 2 H), 8.35 (bs, 1 H); 13C NMR ppm 14.16,
31.46, 32.35, 35.66, 42.25, 53.72, 60.97, 122.95, 123.65, 124.04,
127.72, 129.26, 130.65, 132.97, 133.07, 141.86, 144.70, 165.96,
179.21. Anal. (C23H28N2O2S2) C, H, N, S.
[(4-Nitr op h en yl)a m in o][(2,2,4,4-tetr a m eth ylth ioch r o-
m a n -6-yl)a m in o]m eth a n e-1-th ion e (15c). The compound
was prepared from 18 (1.0 mmol) and 4-nitrophenyl isothio-
cyanate (184 mg, 1.02 mmol). Recrystallization (HCCl3:pen-
tane) gave 15c (333 mg, 83%) as a bright yellow solid: mp
1
153-155 °C; IR (KBr) 3219, 3189 cm-1; H NMR (DMSO-d6)
δ 1.39 (s, 6 H), 1.44 (s, 6 H), 1.97 (s, 2 H), 7.03 (dd, J ) 6.04,
2.20 Hz, 1 H), 7.19 (d, J ) 8.24 Hz, 1 H), 7.33 (d, J ) 2.20 Hz,
1 H), 7.73 (d, J ) 9.06 Hz, 2 H), 7.95 (bs, 1H), 8.18 (d, J ) 7.0
Hz, 2 H), 8.44 (bs, 1 H); 13C NMR (DMSO-d6) ppm 31.47, 32.83,
35.71, 42.35, 53.58, 122.98, 123.03, 124.18, 124.62, 129.58,
132.28,133.94,143.97,144.56,145.18,179.01.Anal.(C20H23N3O2S2)
C, H, N, S.
Exp er im en ta l Section
Commercial reagents and solvents were used as received
unless otherwise noted. Anhydrous THF was obtained by
distillation from a purple solution of sodium and benzophen-
one. All of the isocyanates and isothiocyanates were com-
mercially available and were obtained from Lancaster Syn-
thesis, Inc., Windham, NH. Unless otherwise indicated, all
reactions were run under dry N2 and all purification beyond
recrystallization was done via flash chromatography, utilizing
J . T. Baker flash chromatography silica gel packing, 40 µm
particle size. Melting points were determined using a Thomas-
Hoover melting point apparatus and were uncorrected. IR
spectra were obtained using a Perkin-Elmer 2000 FT-IR
spectrometer as films or KBr pellets. Unless stated otherwise,
4-({[(2,2,4,4-Tet r a m et h yl(3H -b en zo[3,4-e]t h ia n -6-yl))-
a m in o]th ioxom eth yl}a m in o)ben zen esu lfon a m id e (15d ).
The sulfamide was prepared from 18 (1.0 mmol) and 4-isothio-
cyanatobenzenesulfonamide (218 mg, 1.02 mmol). Recrystal-
lization (95% EtOH) gave 15d (378 mg, 87%) as a white solid:
mp 191-193 °C; IR (KBr) 3358, 3272, 1340, 1167 cm-1 1H
;
NMR δ 1.32 (s, 6 H), 1.37 (s, 6 H), 1.91 (s, 2 H), 7.00 (d, J )
6.12 Hz, 1 H), 7.21 (dd, J ) 8.67, 1.98 Hz, 1 H), 7.29 (bs, 2 H),
7.58 (d, J ) 2.03 Hz, 1 H), 7.65 (d, J ) 7.67 Hz, 2 H), 7.73 (d,
J ) 7.64 Hz, 2 H), 10.97 (bs, 1 H), 10.02 (bs, 1 H); 13C NMR
ppm 31.24, 32.25, 35.27, 42.00, 53.43, 122.02, 122.69, 126.33,
127.49, 128.14, 136.44, 139.16, 142.60, 142.86, 179.48. Anal.
(C20H25N3O2S3) C, H, N, S.
1
all H and 13C NMR spectra were performed using DCCl3 as
the solvent and obtained using a Varian UNITYINOVA 400 BB
NMR spectrometer operating at 399.99 and 100.01 MHz,
respectively. Signals were referenced to TMS. Elemental
analyses were performed by Atlantic Microlab, Inc., Norcross,
GA.
[(2-Met h yl-4-n it r op h en yl)a m in o][(2,2,4,4-t et r a m et h -
ylth ioch r om a n -6-yl)a m in o]m eth a n e-1-th ion e (15e). The
compound was prepared from 18 (1.0 mmol) and 2-methyl-4-
nitrophenyl isothiocyanate (198 mg, 1.02 mmol). Recrystalli-
zation (HCCl3:hexanes) gave 15e (340 mg, 82%) as a white
Although the intermediates and final products appeared to
be relatively stable in light, precautions were taken to
minimize exposure to any light source and to the atmosphere.
In the purified state, all products were stored in the cold and
dark without significant decomposition. Intermediates ap-
peared to be similar in sensitivity and were treated likewise.
Gen er a l P r oced u r e for t h e P r ep a r a t ion of H et er o-
a r otin oid s 15a -h fr om Am in e 18 or 24. To a solution of
amine 18 or 24 (1.0 mmol) in dry THF (4.5 mL) at 0 °C was
added dropwise the desired isocyanate or isothiocyanate (1.02
mmol) in dry THF (5 mL) over 3 min. After the addition, the
reaction mixture was allowed to warm to room temperature
and then was stirred for 24 h. The solvent was evaporated,
and the residue was recrystallized to give an analytically pure
sample of the corresponding heteroarotinoid.
1
solid: mp 155-156 °C; IR (KBr) 3344, 3120 cm-1; H NMR δ
1.39 (s, 6 H), 1.43 (s, 6 H), 1.97 (s, 2 H), 2.25 (s, 3 H), 7.05 (dd,
J ) 6.04, 2.20 Hz, 1 H), 7.21 (d, J ) 8.24 Hz, 1 H), 7.37 (d, J
) 2.06 Hz, 1 H), 7.47 (bs, 1 H), 8.02 (m, 4 H); 13C NMR ppm
17.91, 31.47, 32.37, 35.77, 42.40, 53.367, 122.10, 123.74,
124.80, 125.87, 126.44, 129.69, 132.32, 133.54, 134.41, 142.45,
145.31, 145.49, 179.74. Anal. (C21H25N3O2S2) C, H, N, S.
Eth yl 4-{[N-(2,2,4,4,7-P en ta m eth ylth ioch r om a n -6-yl)-
ca r ba m oyl]a m in o}ben zoa te (15f). The ester was prepared
from 24 (1.0 mmol) and 4-ethoxycarbonylphenyl isocyanate
(195 mg, 1.02 mmol). Recrystallization (H2CCl2:pentane) gave
15f (183 mg, 43%) as a white solid: mp 121-123 °C; IR (KBr)
3331, 3203, 1716, 1659 cm-1; 1H NMR δ 1.33 (s, 6 H), 1.35 (t,
J ) 6.72 Hz, 3 H), 1.38 (s, 6 H), 1.89 (s, 2 H), 2.12 (s, 3 H),
4.31 (q, J ) 7.0 Hz, 2 H), 6.86 (bs, 1 H), 6.95 (s, 1 H), 7.31 (d,
J ) 8.52 Hz, 2 H), 7.49 (s, 1 H), 7.53 (bs, 1H), 7.88 (d, J ) 8.79
Hz, 2 H); 13C NMR ppm 14.32, 17.16, 31.53, 32.58, 35.35, 42.16,
E t h yl 4-{[N-(2,2,4,4-Tet r a m et h ylt h ioch r om a n -6-yl)-
ca r ba m oyl]a m in o}ben zoa te (15a ). The ester was prepared
from 18 (1.0 mmol) and 4-ethoxycarbonylphenyl isocyanate

Flexible Sulfur-Containing Heteroarotinoids
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 1005
54.02, 60.82, 118.20, 124.08, 124.63, 129.90, 130.42, 130.78,
132.09,134.28,141.60,142.83,153.82,166.40.Anal.(C24H30N2O3S)
C, H, N, S.
was stirred (room temperature, 1 h). The resulting clear
solution was heated at reflux (24 h). The new solution was
allowed to cool to room temperature and then was washed with
10% NaOH (2 × 25 mL). Combined aqueous layers were
extracted (ether; 3 × 25 mL). Combined organic extracts were
washed with H2O (25 mL) and brine (25 mL) and then dried
(Na2SO4). The solution was filtered and concentrated. The
residual oil was vacuum distilled to give 20 (23.26 g, 87%) as
a pale yellow liquid: bp 112-115 °C (0.8 mm); IR (neat) 1710
cm-1; 1H NMR δ 1.39 (s, 6 H), 2.15 (s, 2 H), 2.36 (s, 3 H), 2.67
(s, 3 H), 7.21 (m, 2 H), 7.32 (m, 2 H); 13C NMR ppm 21.09,
28.06, 32.08, 46.86, 54.37, 128.51, 129.95, 131.15, 134.65,
138.29, 138.48, 206.98. Ketone 20 was used immediately to
prepare 21.
2,4-Dim eth yl-4-(3-m eth ylp h en ylth io)-2-p en ta n ol (21).
To a 3.0 M solution of MeMgBr (80.5 mL, 0.241 mol) was added
dropwise 20 (26.18 g, 0.118 mol) in ether (48 mL). The solution
was stirred at room temperature (6 h) and then was poured
slowly onto ice. The resulting mixture was neutralized (5% H2-
SO4, pH ∼6.5). The ether layer was separated, and the aqueous
layer was extracted (ether; 3 × 50 mL). Organic extracts were
combined and dried (Na2SO4). Evaporation of the solvent gave
a yellow oil, which vacuum distilled to give 21 (23.57 g, 84%)
as a light yellow liquid: bp 110-112 °C (0.5 mm); IR (neat)
3325 cm-1; 1H NMR δ 1.34 (s, 6 H), 1.36 (s, 6 H), 1.80 (s, 2 H),
2.36 (s, 3 H), 3.54 (s, 1 H), 7.19 (m, 3 H), 7.39 (m, 1 H); 13C
NMR ppm 20.56, 31.47, 32.64, 35.02, 41.88, 54.46, 126.02,
126.79, 128.37, 132.36, 135.59, 139.68. Alcohol 21 was con-
verted at once to 22.
2,2,4,4,7-P en ta m eth ylth ioch r om a n (22). To CS2 (185 mL)
was added AlCl3 (52.6 g, 0.39 mol). To the resulting stirred
suspension of AlCl3 was added dropwise a solution of 21 (23.5
g, 98.6 mmol) in CS2 (60 mL) at room temperature over 15
min. The resulting suspension was heated at reflux (10 h) with
stirring. After cooling to room temperature, the suspension was
poured onto ice, and the resulting mixture was stirred (5 min).
This mixture was separated (two layers), and the aqueous
layer was extracted (ether; 3 × 92 mL). Organic extracts were
combined, washed with H2O (60 mL) and brine (60 mL), and
then dried (Na2SO4). Evaporation of the solvent gave an orange
oil which vacuum distilled to give 22 (20.2 g, 93%) as a pale
yellow oil: bp 97-99 °C (0.5 mm); 1H NMR δ 1.36 (s, 6 H),
1.39 (s, 6 H), 1.93 (s, 2 H), 2.25 (s, 3 H), 6.89 (m, 2 H), 7.26 (m,
1 H); 13C NMR ppm 20.67, 31.58, 32.73, 35.11, 41.97, 54.54,
125.93, 126.71, 128.28, 132.11, 135.48, 139.57. Ether 22 was
used at once to prepare 23.
Eth yl 4-{[N-(2,2,4,4,7-P en ta m eth ylth ioch r om a n -6-yl)-
th ioca r ba m oyl]a m in o}ben zoa te (15g). The ester was pre-
pared from 24 (1.0 mmol) and 4-ethoxycarbonylphenyl isothio-
cyanate (211 mg, 1.02 mmol). Recrystallization (ether:pentane)
gave 15g (292 mg, 66%) as a white solid: mp 128-129 °C; IR
1
(KBr) 3210, 3117, 1730 cm-1; H NMR δ 1.35 (m, 9 H), 1.43
(s, 6 H), 1.94 (s, 2 H), 2.24 (s, 3 H), 4.31 (q, J ) 7.14 Hz, 2 H),
7.06 (s, 1 H), 7.33 (s, 1 H), 7.53 (d, J ) 8.51 Hz, 2 H), 8.70 (bs,
1H), 7.99 (d, J ) 8.52 Hz, 2 H), 8.13 (bs, 1 H); 13C NMR ppm
14.25, 17.19, 31.53, 32.63, 35.36, 42.32, 53.71, 60.94, 123.20,
126.02, 127.60, 130.39, 131.21, 132.98, 134.03, 141.92, 142.06,
144.70, 165.79, 179.65. Anal. (C24H30N2O2S2) C, H, N, S.
4-({[(2,2,4,4,7-P en ta m eth yl(3H-ben zo[3,4-e]th ia n -6-yl))-
a m in o]th ioxom eth yl}a m in o)ben zen esu lfon a m id e (15h ).
The sulfonamide was prepared from 24 (1.0 mmol) and
4-isothiocyanatobenzenesulfonamide (218 mg, 1.02 mmol).
Recrystallization (95% EtOH) gave 15h (252 mg, 56%) as a
white solid: mp 190-192 °C; IR (KBr) 3366, 3267, 1346, 1161
cm-1; 1H NMR δ 1.31 (s, 6 H), 1.37 (s, 6 H), 1.90 (s, 2 H), 2.15
(s, 3 H), 6.93 (s, 1 H), 7.23 (s, 1 H), 7.35 (d, J ) 3.57 Hz, 2 H),
7.68 (d, J ) 8.79 Hz, 2 H), 9.53 (bs, 1 H), 9.92 (bs, 1 H), 10.02
(bs, 1 H); 13C NMR ppm 17.11, 31.20, 32.40, 35.84, 41.94, 53.42,
122.02, 122.61, 122.77, 126.02, 126.37, 128.66, 129.84, 132.51,
134.53, 139.95, 180.09. Anal. (C21H27N3O2S3) C, H, N, S.
2,2,4,4-Tetr a m eth yl-6-n itr oth ioch r om a n (17). The fol-
lowing procedure gave a more pure sample of 17 over that
reported.9b To a solution of thiochroman 1610a (15.00 g, 72.7
mmol) in freshly distilled Ac2O (14 mL) at -5 °C (ice/salt) was
added dropwise a mixture of cold, concentrated HNO3 (5.21
mL) and Ac2O (12.20 mL) over 10 min. After being stirred at
-5 °C for 90 min, the reaction mixture was poured into a
solution of saturated, aqueous NaHCO3 (140 mL), and the
resulting emulsion was extracted (H2CCl2; 3 × 83 mL). The
combined organic extracts were washed with H2O (45 mL) and
brine (45 mL) and were then dried (Na2SO4). Evaporation of
the solvent gave a dark brown liquid, which was subjected to
flash chromatography [hexanes:EtOAc (3:1)] to provide 179b
(4.80 g, 26%) as a light yellow solid: mp 105-108 °C (lit9b 103-
1
107 °C); IR (KBr) 1519, 1345 cm-1; H NMR δ 1.45 (s, 6 H),
1.46 (s, 6 H), 2.00 (s, 2 H), 7.21 (d, J ) 8.01 Hz, 1 H), 7.88 (dd,
J ) 7.01, 2.03 Hz, 1 H), 8.26 (d, J ) 2.01 Hz, 1 H); 13C NMR
ppm 31.64, 32.43, 35.85, 43.04, 53.24, 120.90, 122.10, 128.15,
131.51, 143.09, 143.48. The nitro compound 17 was used
immediately to prepare 18.
2,2,4,4,7-P en ta m eth yl-6-n itr oth ioch r om a n (23). To a
solution of thiochroman 22 (15.00 g, 68.1 mmol) in freshly
distilled Ac2O (13 mL) at -5 °C (ice/salt) was added dropwise
a mixture of cold, concentrated HNO3 (4.88 mL) and Ac2O
(11.43 mL) over 10 min. After being stirred at -5 °C for 90
min, the reaction mixture was poured into a solution of
saturated, aqueous NaHCO3 (130 mL), and the resulting
emulsion was extracted (H2CCl2, 3 × 78 mL). Combined
organic extracts were washed with H2O (42 mL) and brine (42
mL) and were then dried (Na2SO4). Evaporation of the solvent
gave a dark brown liquid, which was subjected to flash
chromatography [hexanes:EtOAc (3:1)] to provide 23 (4.70 g,
26%) as a light yellow solid: mp 160-162 °C; IR (KBr) 1516,
1349 cm-1; 1H NMR δ 1.46 (s, 6 H), 1.49 (s, 6 H), 2.03 (s, 2 H),
2.64 (s, 3 H), 7.78 (s, 1 H), 7.97 (s, 1 H); 13C NMR ppm 19.83,
25.98, 31.93, 34.81, 46.27, 54.02, 122.94, 131.33, 132.45,
143.26, 144.33, 150.45. The nitro compound 23 was used
immediately to prepare 24.
2,2,4,4,7-P en ta m eth yl-6-a m in oth ioch r om a n (24). A mix-
ture of 23 (5.40 g, 20.3 mmol), iron powder (0.802 g, 14.3 mmol,
Sigma-Aldrich Chemical Co.), glacial acetic acid (1.70 g, 28.3
mmol), and absolute EtOH (11 mL) was stirred at reflux (12
h). The reaction was allowed to cool to room temperature and
was poured into H2O (42 mL), and the resulting emulsion was
extracted with ether (2 × 38 mL) and HCCl3 (3 × 38 mL).
Combined organic extracts were washed with H2O (3 × 38 mL),
dried (Na2SO4), and concentrated to give a dark oil. This crude
mixture was then dissolved in ether (28 mL), and the resulting
2,2,4,4-Tetr a m eth yl-6-a m in oth ioch r om a n (18). The fol-
lowing procedure gave a more pure sample of 18 compared to
that reported.9b A mixture of 179b (5.40 g, 21.5 mmol), iron
powder (0.850 g, 15.2 mmol, Sigma-Aldrich Chemical Co.),
glacial acetic acid (1.80 g, 30 mmol), and absolute EtOH (11
mL) was stirred at reflux for 12 h. The reaction was allowed
to cool to room temperature and was poured into H2O (45 mL),
and the resulting emulsion was extracted with ether (2 × 40
mL) and HCCl3 (3 × 40 mL). The combined organic extracts
were washed with H2O (3 × 40 mL), dried (Na2SO4), and
concentrated to give a dark oil. This crude mixture was then
dissolved in ether (30 mL), and the resulting solution was
extracted with 2 N HCl (2 × 30 mL). The acid solution was
neutralized with 40% Na2CO3 (pH ∼8), and the resulting
solution was extracted (ether; 2 × 30 mL). The organic extracts
were dried (Na2SO4), and the solvent was evaporated to give
amine 189b (0.370 g, 42%) as a tan oil which crystallized upon
standing overnight in a freezer and was recrystallized (hex-
anes) to yield an off white solid: mp 58-60 °C (lit.9b mp 57-
59 °C). Spectral data for the product matched reported
values,9b and amine 18 was used immediately to prepare 15a -
e.
4-Meth yl-4-(3-m eth ylp h en ylth io)-2-p en ta n on e (20). To
a solution of 3-methylthiophenol (19, 15.00 g, 0.121 mol),
mesityl oxide (11.42 g, 0.116 mol), and HCCl3 (47 mL) at 0 °C
was added 0.7 mL of triethylamine. The cold bath was removed
after the addition (0.25 h) of triethylamine, and the solution

1006 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Liu et al.
solution was extracted with 2 N HCl (2 × 28 mL). The acid
solution was neutralized (40% Na2CO3, pH ∼8), and the
resulting solution was extracted with ether (2 × 28 mL). The
combined extracts were dried (Na2SO4), and the solvent was
evaporated to afford amine 24 (2.16 g, 45%) as a tan oil, which
crystallized upon standing overnight in the freezer and was
recrystallized (hexanes) to give an off white solid: mp 61-63
°C; IR (KBr) 3459, 3366 cm-1; 1H NMR δ 1.34 (s, 6 H), 1.38 (s,
serial washes with assay buffer by centrifugation. The assay
buffer contained 10 µM glucose, 1 µM calcium chloride, and 2
µM magnesium chloride. Carboxy-H2DCFDA (50 µM) was then
added, and the assay for ROS production was initiated as
previously described.26 Fluorescence was measured on a Wal-
lace Victor-2 96-well plate reader (excitation at 485 nm and
emission at 538 nm).
Retin oic Acid Recep tor Activa tion . The activation of
endogenous receptors in cancer cells was evaluated as previ-
ously described.9b,c,27 Briefly, cultures of SW962 cells and CC-1
cells that had been previously transfected with RARE-RARâ-
tk-CAT reporter plasmid were treated with 15a -c, 9-c-RA
(positive control), or the same volume of control solvent
6 H), 1.89 (s, 2 H), 2.09 (s, 3 H), 6.74 (s, 1 H), 6.83 (s, 1 H); 13
C
NMR ppm 16.60, 31.41, 32.37, 35.25, 41.76, 54.72, 113.63,
121.04, 121.27, 129.98, 141.80, 142.22. Amine 24 was used
immediately to prepare 15f-h .
Gr ow th In h ibition Assa ys. The OVCAR-3, Caov-3, and
SK-OV-3 ovarian carcinoma cell lines were obtained from
American Type Culture Collection. The OVCAR-3 cultures
were maintained in RPMI media supplemented with 10% fetal
bovine serum (FBS). The Caov-3 and SK-OV-3 cultures were
maintained in MEM media supplemented with 10% FBS,
nonessential amino acids, sodium pyruvate, antibiotics, and
antimycotics (complete MEM). A primary culture of a benign
ovarian cyst and one of a borderline ovarian tumor were
established from specimens obtained from two consenting
patients undergoing surgery at the University of Oklahoma
Health Sciences Center. The specimens were cut into two
fragments, vortexed vigorously, and plated onto 100 mm tissue
culture dishes containing complete MEM media. Monolayer
cultures that developed were passaged once before cryogenic
preservation. All experiments were performed on cultures at
passages 3 or 4 in which the majority of cells were epithelial.
Fibroblasts began to outgrow the epithelial cells after passage
4. Only lots of FBS with undetectable levels (<10-8 M) of
retinol and retinoic acid isomers were used, as determined by
high performance liquid chromatography.
All compounds were dissolved in dimethyl sulfoxide (DMSO)
and stored as aliquots of 1000× stock solutions at -70 °C.
Since the retinoids appeared to be slightly light sensitive, all
manipulations involving retinoids were performed under
subdued lighting. Each experiment included control cultures
that received the same volume of DMSO solvent that was
administered in the treatment. The concentration of DMSO
in each experiment was always equal to or less than 0.01%, a
concentration which was not toxic and which did not induce
differentiation.
(DMSO-negative control) at concentrations of 10-9, 10-8, 10-7
,
10-6, and 10-5 M for 48 h. Activation of the endogenous
receptors in the SW962 and CC-1 cell lines was determined
by measuring the level of CAT (chloramphenicol acetyl trans-
ferase) protein present in each treated and untreated culture.
The ability of these compounds and concentrations to activate
each individual retinoic acid receptor (RAR) and retinoic X
receptor (RXR) were evaluated as previously cited.9b,9c
Ack n ow led gm en t. We gratefully acknowledge par-
tial support of this work by the National Institutes of
Health through grants from the National Cancer Insti-
tute (CA-73639 and F3CA83168), through the NRSA-
Fellowship (IF31 CA83168 to C.W.B.) from the National
Cancer Institute, and the National Cancer Institute
(CA-077711 to D.M.B.). Special thanks is also extended
to the National Institutes of Health and the Oklahoma
State University Department of Biochemistry and Mo-
lecular Biology for early support from the Minorities in
Biomedical Research Fellowship (5 R25 GM55244-02 to
C.W.B.). In addition, we are pleased to acknowledge
funding for the Varian Inova 400 MHz NMR spectrom-
eter in the Oklahoma Statewide Shared NMR Facility
by the National Science Foundation (BIR-9512269), the
Oklahoma State Regents for Higher Education, the W.
M. Keck Foundation, and Conoco, Inc. Salary support
(K.D.B.) by the College of Arts and Sciences is gratefully
acknowledged.
Cultures were plated in 96-well microtiter plates in volumes
of 150 µL at a concentration of 1000 cells/well. The following
day, retinoids were added at 4× concentrations in 50 µL of
media resulting in final concentrations of 1, 4, 7, and 10 µM
of each compound. After 3 days of treatment, the number of
living cells was quantitated using the MTS [3-(4,5-dimeth-
ylthiazol-2-ol)-5-(3-carboxymethoxyphenyl)-2H-tetrazolium in-
ner salt] assay (Promega Life Science, Madison WI). Meta-
bolically active cells reduce the MTS compound to an aqueous
soluble formazan, resulting in an absorbance measurable at
490 nm (Promega Technical Bulletin No.169). A mixture of
MTS solution and PMS (phenazine methosulfate) in Dulbecco’s
phosphate-buffered saline was added to each well. The plate
was wrapped with foil and shook at 180 rpm for 5 min on an
orbital shaker (Fisher Scienctific, Hanover Park, IL) before
incubating at 37 °C in 5% CO2. The plate was removed at
various intervals, shook for 5 min, and then read at OD490 on
a microtiter plate reader until the readings were in the linear
range (0.1-0.6). Each experiment was performed in triplicate,
and the three values for each treatment were averaged. To
determine the percent growth inhibition, the average OD of
the untreated cultures was divided by the average OD of the
control cultures treated with solvent alone and multiplied by
100.
Refer en ces
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M.; J ong, L.; Fanjul, A.; Cameron, J . F.; Lu, X. P.; Haefner, P.;
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acetate (carboxy-H2DCFDA) was obtained from Molecular
Probes (Eugene, OR), and 96-well black isoplates were ob-
tained from Perkin-Elmer Life Sciences (Boston, MA). Cells
were plated in 96-well black isoplates and treated with the
compounds. After the treatment interval, cells underwent

Flexible Sulfur-Containing Heteroarotinoids
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 1007
Acid Receptor Gamma Expressed in the Mouse Embryo. Mol.
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