Biologically active heteroarotinoids exhibiting anticancer activity and decreased toxicity

Article abstract of DOI:10.1021/jm970196m

A series of retinoids, containing heteroatoms in a cyclic ring and called heteroarotinoids, were synthesized, and their biological activity was evaluated using tissue culture lines that have measurable responses to trans- retinoic acid (t-RA). Transglutam

Full text of DOI:10.1021/jm970196m

J . Med. Chem. 1997, 40, 3567-3583
3567
Biologica lly Active Heter oa r otin oid s Exh ibitin g An tica n cer Activity a n d
Decr ea sed Toxicity
Doris M. Benbrook,† Matora M. Madler,‡ Lyle W. Spruce,‡ Paul J . Birckbichler,§ Eldon C. Nelson,|
Shankar Subramanian,‡ G. Mahika Weerasekare,‡ J onathan B. Gale,‡ Manford K. Patterson, J r.,
Binghe Wang,∇ Wei Wang,∇ Shennan Lu,† Tami C. Rowland,§ Paul DiSivestro,† Charles Lindamood III,#
Donald L. Hill,# and K. Darrell Berlin*,‡
Departments of Obstetrics & Gynecology, Urology, and Medicinal Chemistry and Pharmaceutics, University of Oklahoma
Health Sciences Center, P.O. Box 26901, Oklahoma City, Oklahoma 73l90, Departments of Chemistry and Biochemistry and
Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, The Samuel Roberts Noble Foundation, Inc.,
2510 Highway 199 East, Ardmore, Oklahoma 73402, and Kettering-Meyer Laboratories, Southern Research Institute,
P.O. Box 55305, Birmingham, Alabama 35255-5305
Received March 24, 1997X
A series of retinoids, containing heteroatoms in a cyclic ring and called heteroarotinoids, were
synthesized, and their biological activity was evaluated using tissue culture lines that have
measurable responses to trans-retinoic acid (t-RA). Transglutaminase (TGase) was assessed
in the human erythroleukemia cell line (GMO6141A) as an indicator of differentiation and
apoptosis. Proliferation was evaluated in a human cervical cell line, CC-1, which exhibits dose-
dependent alterations in growth rate in response to treatment with trans-retinoic acid.
Activation of nuclear retinoic acid receptors was determined in a reporter cell line established
from CC-1. The reporter line, called CC-B, contains a reporter gene controlled by a retinoic
acid responsive element (RARE) and a thymidine kinase (tk) promoter. Treatment of the CC-B
line with the heteroarotinoids resulted in a dose-responsive and retinoid-dependent regulation
of reporter gene expression. The heteroarotinoids exhibited activity in all assays and correlated
in a statistically significant manner between assays. RARE transactivation activity in CC-B
cells correlated with induction of TGase in GMO6141A (R ) 0.96) and with a decrease in the
growth rate of CC-1 cells (R ) -0.90). The ability of the selected heteroarotinoids to induce
differentation, inhibit proliferation, and activate nuclear receptors demonstrates the chemo-
therapeutic potential of these agents. In view of the biological activity cited, an in vivo toxicity
study was conducted on male B6D2F1 mice with three heteroarotinoids, namely 8 [(2E,4E,6E)-
3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimethylthiochroman-6-yl)-2,4,6-heptatrienoic acid], 10
[(2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimethylchroman-6-yl)-2,4,6-heptatrienoic
acid], and 13 [(E)-p-[2-(4,4-dimethylchroman-6-yl)propenyl]benzoic acid]. The mice were used
with gavage of heteroarotinoids in corn oil [0.1, 0.2, 0.4, or 0.8 mg/kg] and with 0.01 or 0.05
mg/kg of TTNPB (5) [(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-prope-
nyl]benzoic acid] as reference controls. The target organs affected in the mice by the three
heteroarotinoids were those typically associated with t-RA (1) toxicity. The maximum tolerated
dose (MTD) of 13 was 9.4 mg/kg/day, which was equal in toxicity to that of t-RA (1) and 1000-
fold less toxic than TTNPB (5). The MTDs of 8 and 10 were 34 and 32 mg/kg/day, respectively,
which is 3-fold less toxic than t-RA (1) and 3000-fold less toxic than TTNPB (5). The 3000-fold
reduced toxicity, compared with only a 27% reduction biological activity of 8 and 10 with respect
to that of TTNPB, observed in our assays indicates a good therapeutic ratio of these
heteroarotinoids over the parent compound. The biological activity and reduced toxicity of
these heteroarotinoids demonstrate the potential efficacy as anticancer agents.
In tr od u ction
demonstrated by the reversal of the premalignant
lesions, oral leukoplakia in patients treated with 13-
cis-RA, for example.7 In head and neck cancer, 13-cis-
RA (2) caused decreased incidence of second primary
tumors in patients who were free of the disease follow-
ing surgery or radiation therapy.8,9 Both t-RA (1) and
13-cis-RA (2) have clearly demonstrated potential as
chemotherapeutic agents in the treatment of a variety
of cancers.10 The most successful clinical effectivenensss
has been observed in the treatment of acute promyelo-
cytic leukemia (APL).11-15 A combination of 13-cis-RA
(2) with interferon R-2a had demonstrated activity in
patients with squamous cell carcinomas of the skin and
cervix.5 It is known that t-RA (1) is converted in vitro16
to 9-cis-retinoic acid (9-cis-RA, 3), which also has
anticancer activity.17
Investigations of the biology and chemistry of retin-
oids have become highly intensified with the recognition
that trans-retinoic acid (t-RA, 1)1-3 and 13-cis-retinoic
acid (13-cis-RA, 2)4-7 possess anticancer activity. Po-
tential use of these agents in chemoprevention has been
* To whom correspondence should be addressed. E-mail: kdberlin@
bmb-fs1.biochem.okstate.edu.
† Department of Obstetrics & Gynecology, University of Oklahoma
Health Sciences Center.
‡ Department of Chemistry, Oklahoma State University.
§ Department of Urology, University of Oklahoma Health Sciences
Center.
| Department of Biochemistry and Molecular Biology, Oklahoma
State University.
The Samuel Roberts Noble Foundation, Inc.
∇ Department of Medicinal Chemistry and Pharmaceutics, Univer-
sity of Oklahoma Health Sciences Center.
The clinical effectiveness of t-RA (1) is limited by
#
Kettering-Meyer Laboratories.
X Abstract published in Advance ACS Abstracts, October 1, 1997.
significant toxicity.10 Considerable effort has been
S0022-2623(97)00196-9 CCC: $14.00 © 1997 American Chemical Society

3568 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Benbrook et al.
Ta ble 1. Relative Transcriptional Activation Activities of 8-21
Relative to t-RA57
expended to develop synthetic mimics of 1 which will
exhibit similar biological effects as 1 but with less
toxicity. Fenretinide (4) is a synthetic retinoid that
possesses few side effects and is in clinical trials as a
chemopreventive agent for oral leukoplakia, breast,
bladder, and prostate cancer.18
Resu lts a n d Discu ssion
Ch em istr y. Arotinoids are a class of retinoids found
to modulate cellular differentiation and proliferation in
types of cells responsive to t-RA (1).10 A few retinoidal
amides46 have been diagnosed as being much more
active than t-RA (1) in terms of differentiation-inducing
activity on human promyelocytic leukemia cell line HL-
60. Conceivably, this may result from the increased
ability of amides to rotate and conform to both RAR and
RXR receptors. In the present work, the target com-
pounds were 8-21 (Table 1) where special cases 18-
21 have an internal ester linkage which has a lower
energy barrier to C-O bond rotation than the amides
and will allow greater flexibility and possibly a better
fit with the receptor. As a model system, the barrier to
rotation of the C-O bond in the ester methyl acetate
has been found to be 10-15 kcal/mol while the simple
amide counterpart, namely acetamide, has a barrier
near 23 kcal/mol.47
Selection of the heteroarotinoids 8-21 was done on
the following basis. Systems 8-12 allow a determina-
tion of the effect of the heteroatom and type of side
group on receptor specificity. Acids 9 and 11, as well
as 8 and 10, differ only in the heteroatom while 11 and
12 differ in the nature of the side group. Acid 13 and
esters 14 and 15 permit an assessment of receptor
activity by acid versus ester while esters 14 and 15
differ in heteroatoms. Systems 16 and 17 are void of a
geminal dimethyl group, but differences in activity due
to the size of the lipophilic group adjacent to the
heteroatom can be assessed. Heteroarotinoids 18-21
have flexible connecting ester linkages which may
enable the system to better “fit” the receptor. Esters
18 and 19 differ only in the heteroatom, while 18 and
20 have different terminal groups. Inclusion of the
thiosemicarbazone 20 resulted, in part, from prior
knowledge that certain thiosemicarbazones exhibited
good anticancer activity.48 The connecting linkage in
the novel 21 is highly hindered and reversed from that
in 18-20. Consequently, C-O rotation in 21 may be
somewhat restricted, and thus the receptor binding
Some arotinoids, defined as retinoids with at least one
aromatic ring (as 5-TTNPB), exhibited anticancer activ-
ity.19 However, 5 was extremely toxic. Heteroaroti-
noids have been defined as systems with one aromatic
ring and at least one heteroatom20 in the partially
saturated ring (as retinoids 6 and 7) with X ) O, S, NR,
etc. A few reported heteroarotinoids have anticancer
activity.20,21 We have prepared a series of heteroaroti-
noids 8-21 (Table 1) and have evaluated their activity
and the toxicity of three active agents.
Retinoid regulation of gene expression leads to several
biological activities with anticancer effects.22-31 For
example, retinoid induction of the transglutaminase
(TGase) gene is associated with induction of differentia-
tion and apoptosis.10,32-39 TGases are a group of Ca2+
-
dependent enzymes that catalyze the post-translational
modifications of proteins by introducing ꢀ-(γ-glutamyl)-
lysine isopeptide bonds.40,41 Both RAR- and RXR-
selective retinoids induce expression of the TGase gene,
implicating the RAR/RXR heterodimer in this ac-
tivity.42-44
The biological activities of the compounds herein were
tested in several in vitro assays to evaluate regulation
of gene expression through RARE’s, tumor cell growth,
and TGase expression. Three active agents (8, 10, and
13) were selected for toxicity studies in mice. Interest-
ingly, heteroarotinoid 14 displayed better ability than
t-RA (1) to reverse cornification in vaginal smears of
vitamin A-deficient, ovariectomized rats while 10 was
slightly less effective.45 This further supports our
hypothesis that heteroarotinoids can possess useful
anticancer activity.

Biologically Active Heteroarotinoids
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3569
Sch em e 1
Sch em e 2
Sch em e 3
capability might be altered in either a positive or
negative fashion.
The syntheses of 9-12 were developed as follows.
Acids 9 and 10 have been reported49 as have 11 and
certain relatives.50,51 However, unknown acid 12 was
prepared from 22 via the reaction sequence of 22 f 23
f 24 f 25 f 26 f 27 f 28 f 29 f 12 as shown
(Scheme 1). The yields for each step were average to
very good and were highly dependent upon conditions.
Acid 13 and esters 14 and 15 are known.20,21,50 Esters
16 and 17, which have no geminal dimethyl group, were
synthesized starting from 30 via the reaction sequence
as illustrated in Scheme 2, namely 30 f 31 f 32 f 33
f 34 f 35 f 36 f 37 f 17. The intermediates and
yields with respect to each step for R ) ethyl and
n-octyl, respectively, were 31 (89%), 32a /32b (80%;
40%), 33a /33b (98%; 98%), 34a /34b (72%, qt), 35a /35b
(86%; 80%), 36a /36b (96%; 98%), 37/16 (91%; 79%), and
17 (64%). Chiral 31 has been reported, but few proper-
ties were recorded.52,53 R-Alkylation to the chiral sul-
foxide center gave, after chromatography, light oils for
32a /32b (R ) ethyl and n-octyl, respectively). A modi-
fied Sharpless procedure54 for the oxidation of 37
afforded a good conversion to 17.
carbonyl group in 34a /34b to yield 35a /35b was also
facile under somewhat standard conditions with LiAlH4.
Acid-catalyzed phosphorylation as shown gave the cor-
responding phosphonium salt 36a /36b in high yields in
both examples. All intermediates 31-35 were utilized
as quickly as possible after preparation as some dete-
rioration occurred upon standing. Such degradation
was minimized by storage in a freezer. Salts 36a /36b,
although apparently stable, were also used at once and
subjected to the conditions outlined to generate the
esters 37/16 (R ) ethyl and n-octyl). These esters were
also best stored in the dark and in a freezer. The ethyl
analog 37 (R ) ethyl) was oxidized under our modified
Sharpless conditions to chiral sulfoxide 17, which is the
first chiral heteroarotinoid reported.
Flexible heteroarotinoids 18-21 required special
methods for each case. Synthon 38, in an ethanolic
solution, was oxidatively cleaved55 with Clorox to the
corresponding carboxylic acid 39 (Scheme 3).51,55 Es-
terification with ethyl 4-hydroxybenzoate, DCC, and a
catalytic amount of DMAP gave diester 18 (83%).
The preparation of the nitrogen analog 19 was
initially attempted via a Michael type addition with
4-bromoaniline (40, Scheme 4) and ethyl acrylate but
was unsuccessful, possibly due to the electron-with-
Experience with simple, related model systems strongly
inferred that removal of the oxygen from 32a /32b was
critical for later steps in the synthesis. Near quantita-
tive yields of both the ethyl and n-octyl derivatives 33a /
33b were realized when 32a /32b were treated with NaI/
trifluoroacetic anhydride in acetone. Acetylation of 33a /
33b to give 34a /34b was smooth with acetic anhydride
in preference to acetyl chloride. Reduction of the

3570 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Benbrook et al.
Sch em e 4
Sch em e 5
F igu r e 1. Dose-response transactivation of an RARE. The
CC-B cell line was treated with a range of concentrations of
the retinoids indicated. After 48 h of treatment, the CAT
activity was quantitated, and RARE transactivation was
derived as the ratio of activities in treated over control
cultures. The results are presented as transactivation relative
to that observed in CC-B cultures treated with t-RA.
Sch em e 6
barrier (∆G*) was determined, via a variable temper-
ature NMR analysis, to be between 15-16 kcal/mol at
26 °C (see the Experimental Section). This barrier is
sufficient to markedly restrict rotation around the C-O
ester bond but not large enough to prevent such rotation
at room temperature.
Sch em e 7
Biology. An in vitro model system which has dem-
onstrated a linear dose-dependent response to retinoids
was chosen to evaluate the biological activities of the
heteroarotinoids.57 This model uses a cervical carci-
noma cell line (CC-B) which contains integrated copies
of a reporter plasmid that is regulated by the retinoic
acid receptor element (RARE) from the RARâ gene. The
specific RARE has been shown to be regulated by the
RAR/RXR heterodimer and RXR/RXR homodimer in the
presence of t-RA.25 Treatment of CC-B with retinoids
or heteroarotinoids results in dose-responsive increased
expression of the reporter gene, chloramphenical acetyl
transferase (CAT). Figure 1 illustrates a dose-response
increase in CAT activity in the CC-B in response to
treatment with a range of concentrations of 8 and 10.
Since 8 and 10 were approximately 73-77% as active
as TTNPB (which also induced a dose-response effect),
the approach is an appropriate method to assess and
compare the biological activities.
drawing nature of the bromine substituent. N-Acylation
was achieved, however, with 40 and 3,3-dimethylacryl-
oyl chloride in chloroform at reflux and led to 41 (87%)
which required careful recrystallization to avoid poly-
merization.
Cyclization of 41 to the lactam 42 required an
unusually high temperature. Amide 41 was carefully
melted, and the temperature was raised to 130 °C at
which time solid, anhydrous AlCl3 was added portion-
wise over a 2-h period in the absence of solvent.
Recrystallization of the crude product gave 42 (65%).
Reduction of the carbonyl group in 42 was accomplished
with H3B:S(CH3)2 in boiling toluene with a high conver-
sion to 43 (90%). N-Methylation of 43 led to derivative
44 (86%) uder the conditions shown.
Transmetalation of 44 occurred with n-BuLi in ether,
and the salt was subsequently carbonated with solid
CO2 in ether to give the carboxylic acid 45 (30%)
(Scheme 5). Efforts to increase the yield of 45 included
the addition of TMEDA during transmetalation, but 19
was not obtained. Esterification of 45 gave 19 (43%).
The synthesis of thiosemicarbazone 20 (Scheme 6)
required the preparation of aldehyde 46 (43%), which
was obtained using the previously cited carboxylic acid
39 and 4-hydroxybenzaldehyde as illustrated. Conden-
sation of aldehyde 46 with thiosemicarbazide easily
afforded thiosemicarbazone 20 (83%).
The coumarol 4756 was esterified (Scheme 7) in the
manner similar to that used to obtain 18 and 19. The
two methyl groups on the benzene ring in 21 serve to
decrease the flexibility of the ester functional group by
hindering the rotation of the C-O bond. Because the
steric effect was expectedly large in 21, the rotational
The dose of 10-6 M was chosen for screening the other
heteroarotinoids since this concentration exhibited the
greatest differential in RARE transactivation by indi-
vidual retinoids and is pharmacologically relevant. All
agents tested did induce RARE transactivation (Table
1) in this model within the range 29-56% of that of t-RA
(1). Thioacid 11 exhibited the highest activity at 56%
of the t-RA (1) control. In general, compounds contain-
ing a sulfur heteroatom exhibited greater activities than
their oxygen-containing counterparts (compare 11 ver-
sus 9 and 15 versus 14 and 18 as well as 8 versus 10).
Systems with an aryl group in the side chain displayed
greater activity when associated with a sulfur hetero-
atom (compare 11 versus 8 and 12), but the reverse
situation seems to persist with the oxygen analogs
(compare 10 versus 9, 13, and 14). Interestingly, the
systems with a five-membered ring exhibited greater
activity than the six-membered compounds for both

Biologically Active Heteroarotinoids
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3571
F igu r e 3. Dose-dependent regulation of CC-B growth. The
percentage of cells counted in cultures treated with the
indicated retinoids for 7 days in relation to the number of cells
in the control cultures is plotted against the concentration of
agent utilized.
was true. The control TTNPB was the most effective
at all concentrations. Given the complexity of interac-
tion of the retinoid receptor dimers and the various
RAREs found in the genome, it is likely that the RARE
used in this study provides only a small indication of
the total effects induced by these heteroarotinoids.
Therefore, the reduced activities of the heteroarotinoids
in comparison to that of the natural retinoids is not
necessarily undesirable if the end result is increased
specificity. The specificity is under investigation.
Although there is a linear correlation between RARE
transactivation and growth inhibition in the cell line
described herein, other activities of these retinoids, such
as the anti-AP1 effects, may well contribute to growth.
Agents 8, 10, and 13 displayed higher growth inhibitory
activity then expected from the corresponding transac-
tiviation activity (Figure 2, panel A).
The other anticancer activity of the heteroarotinoids
screened in this study involved induction of TGase
activity in HEL cells.10,32-34,61,62 All of the agents
evaluated induced TGase activity in the range of 16-
52% relative to that of t-RA. A linear correlation (R )
0.96) between the degree of TGase inducation in HEL
cells and the degree of RARE transactivation in CC-B
was observed (Figure 1, panel B).
In summary, this study demonstrates for the first
time that certain heteroarotinoids transactivate the
RARE from the RARâ gene through endogenous recep-
tors. This transactivation correlates in a statistically
signficant manner with TGase induction in human
erythroleukemia cells and with growth inhibition in a
cervical carcinoma cell line. These assays clearly show
strong correlations between the profiles of heteroaroti-
noid activities. This is the first systematic study
involving only heteroarotinoids in such assays and
demonstrates that potentially useful chemotherapeutic
properties are present in such retinoids.
The uses of retinoids as preventive and therapeutic
agents for the treatment of various forms of cancer have
been delineated.10,48,58-68 However, toxicity remains a
serious problems with many retinoids, including syn-
thetics. In view of the above activity as well as the ODC
and TOC activity of 8,49 10,49 and 13,20 a second major
objective was to utilize rats to determine the maximum
tolerated doses (MTDs), to identify target organs for
F igu r e 2. Correlation of RARE transactivation with biological
activity. The degree of RARE transactivation is plotted against
the growth rate (panel A) and the induction of TGase activity
(panel B). In panel A, all values represent the percent increase
in activity above basal levels induced by retinoid and heteroa-
rotinoid treatment. In panel B, the results are presented as
values relative to t-RA (1). Linear regression analysis (Harvard
Graphics, version 3.0 software) was used to draw the lines and
to determine the correlation coefficients.
oxygen- and sulfur-containing analogs (compare 9 with
13 and 14 and compare 11 with 15). It is quite impor-
tant to note that sulfur-containing heteroarotinoids have
demonstrated greater activity over the oxygen counter-
parts both in the ornithine decarboxylase culture (ODC)
assay51 in mice as well as in the tracheal organ culture
(TOC) assay in hamsters.20
One property of retinoids which shows promise for
anticancer activity58-68 is the inhibition of tumor cell
growth.1,10 Generally, the effects of retinoids on such
growth is dependent upon the nature of the retinoid and
the cells involved. Both growth stimulation and growth
inhibition have been reported in the same cell line
depending upon the concentration of retinoid.57-61 The
CC-B cell line exhibited statistically significant growth
stimulation when treated with 1 × 10-9 M cis-9-RA but
marked growth inhibition at 10-6-10-5 M cis-9-RA.57
At 10-6 M used for screening purposes, both stimulation
and inhibition of growth were induced by heteroaroti-
noids. The degree of growth effects correlated linearly
(R ) -0.90) with the degree of activity observed in the
transactivation assay (Figure 2, panel A). In general,
agents which increased growth rate were poor RARE
transactivators and those which decreased growth rate
were better RARE transactivators.
The importance of concentration and type of retinoid
utilized in determining growth effects is further il-
lustrated in Figure 3. At concentrations of 10-6 M and
higher, 9-cis-RA was a more effective growth inhibitor
than agent 15, but at lower concentrations, the reverse

3572 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Benbrook et al.
and high-dose of 10, and high-dose of 8 groups. Onset
of 10% body weight reduction from vehicle 1 values were
days 61, 53, and 54 in the mid-dose 2, the high-dose of
10, and the high-dose of 8 groups, respectively. No body
weight reductions of e10% from vehicle 1 values oc-
curred in the remaining 10 and 8 groups.
toxicity, and to define toxicity relative to t-RA (1) and
TTNPB (5). In addition, an examination of agents 8,
10, and 13 allows an assessment of both the influence
of structural variation and the nature of the heteroatom
on toxicity in such heteroarotinoids. Such information
could be quite instructive in terms of improved agent
design and potential anticarcinogenic efficacy. Table 2
summarizes the toxicological effects observed for each
retinoid in this study. Overt toxicity, as evidenced by
rapid onset of clinical signs of hypervitaminosis A
syndrome (Table 3) with high incidence of mortality, was
observed in the 0.1, 0.2, 0.4, and 0.8 mg/kg TTNPB
groups. Incidence and latency to mortality was related
to dose. Fracture incidence and average number of
fractures per mouse decreased with increasing dose.
This was attributed to death from overt toxicity prior
to development of fractures. Three mortalities occurred
in the 0.05 mg/kg TTNPB group, with deaths from days
14 to 22. Fracture incidence was 7/8 in this group, with
clinical signs of factures (avoiding pressure on limbs)
beginning day 11 (Table 3). Fracture incidence was 1/7
for the 0.01 mg/kg TTNPB group. Clinical signs of
vitamin A skin toxicity (alopecia and/or skin scaling;
Table 3) were present at about day 7 in the 0.05 mg/kg
group and day 14 in the 0.01 mg/kg group. Final body
weights in the 0.01 and 0.05 mg/kg groups were
significantly reduced from vehicle 2 values at termina-
tion on day 25. Body weight reductions were dose-
related with 10% body weight reduction form vehicle 2
values at approximately day 8 in the 0.05 mg/kg TTNPB
group.
Table 4 summarizes the significant (p e 0.05 relative
to vehicle) effects of the three compounds, as revealed
by hematology and clinical chemistry. On day 8 in the
0.1, 0.2, 0.4, and 0.8 mg/kg TTNPB groups, large
increases in numbers of circulating white and red blood
cell counts, hematocrit, and hemoglobin and large
decreases in serum triglycerides and cholesterol were
observed. These changes reflected the overt toxicity in
these groups. The only significant clinical pathologic
effect in the 0.01 mg/kg TTNPB group was a 3-fold
increase in circulating white blood cells at study ter-
mination (day 25-6.58 ( 4.24 103/mm versus 2.10 (
0.47 for vehicle 2). Large decreases in serum trigly-
cerides and large increases in circulating white blood
cells in the 0.05 mg/kg TTNPB group at study termina-
tion were attributed to overt toxicity.
With the 8 and 10 compounds, elevations in serum
triglyceride were the primary alterations as detected by
clinical pathology. Effects were evident as early as day
8 and were seen in the low-dose and mid-dose groups.
Effects tended to be restricted to the first portion of the
study. Elevations, relative to vehicle 1, ranged from 35
to 116%. For 13, significant effects on serum trigly-
cerides were limited to day 29 in the mid-dose 1 and
high dose groups. Elevations were 82 and 100% higher
relative to vehicle 1, respectively.
Significant toxicity in the 13 groups did not occur until
the increases in dosages on day 30. Mortalities occurred
in the mid-dose 2 (4, days 57 to 63) and high-dose (8,
days 43 to 62) groups. Except for the high-dose group,
in which high mortality may have precluded the devel-
opment of fractures, fracture incidence, average number
of fractures per rat, and onset of fractures, as evidenced
by the clinical sign of avoiding pressure on limbs, was
related to dose. Clinical signs of skin toxicity were
present at high incidence in all groups. Final body
weights were significantly reduced from vehicle 1 values
of 8.8, 16.5, and 27.9%, respectively, in the low-dose,
mid-dose 1, and mid-dose 2 groups. Onset of 10% body
weight reduction from vehicle 1 values was dose-related,
with days of onset of 51, 41, and 36 in the mid-dose 1,
mid-dose 2, and high-dose groups, respectively. There
was no 10% body weight reduction from vehicle 1 values
in the low-dose group.
For all three compounds, elevations in serum alkaline
phosphatase occurred at day 29 and/or later in all dose
groups and in a dose-related manner. Elevations ranged
from 14 to 143% above vehicle 1 values.
Elevations in circulating white blood cell counts
occurred on day 8 in the low-dose 2 and high-dose of 10
and 8 groups (196, 45, 143, and 175% higher, respec-
tively). For 13, increased circulating white blood cells
occurred on day 8 in the mid-dose 2 group (76% higher
relative to vehicle 1) and in a dose-related fashion at
study termination (day 65, no significant elevation in
the high-dose group). Elevations were 180, 259, and
330% in the low- and mid-dose 1 and 2 groups, respec-
tively.
Organ weight effects of each retinoid have been
presented in Table 5. Testes-to-body weight ratios were
reduced, 24-55% from vehicle 1 values, in all dose levels
of the three compounds. Significant changes in organ-
to-body weight ratios relative to vehicle were also
observed for thymus in the low-dose of 8 group (+28.1%),
the mid-dose 2 of 13 group (-30.2%), and the 0.05 mg/
kg TTNPB group (-41.3%), adrenal in the 0.05 mg/kg
TTNPB group (+56.8%) and spleen in the mid-dose 2
and high-dose of 10 groups (+36.4 and 48.6%, respec-
tively), low and mid-dose 1 and 2 of 8 groups (+82.4,
+151.1, and +256.5%, respectively), and 0.01 and 0.05
mg/kg TTNPB groups (+124.6 and +335.7%, respec-
tively).
Significant toxicity was not observable in the 8 and
10 groups until the increases in dosage on day 51. The
toxicity was limited to the mid-dose 2 of 10 and high-
dose of 8 and 10 groups. There was only one mortality,
which was on day 57, in the mid-dose 1 of the 10 group.
A high incidence of fractures was noted in the mid-dose
2 and high-dose of 10 and high-dose of 8 groups, with
onset of fractures on day 62 in the high-dose of the 10
group. Since there were no clinical signs of fractures
in the mid-dose 2 of the 10 and high-dose of the 8
groups, one can conclude that onset of bone toxicity
occurred relatively near study termination on day 65.
Clinical signs of skin toxicity were present predomi-
nately in the high dose of the 10 group. Final body
weights were significantly reduced from vehicle 1 values
of 9.6, 17.4, and 9.6%, respectively, in the mid-dose 2
Table 6 summarizes the gross observations of patho-
logic efforts at necropsy for each retinoid. Enlarged
lymph nodes and spleens were observed in high inci-
dence in all 13 and TTNPB groups. (Enlarged spleens
were not observed in the 0.2, 0.4 and 0.8 mg/kg TTNPB

Biologically Active Heteroarotinoids
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3573

3574 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Benbrook et al.
Ta ble 3. Clinical Signs of Hypervitaminosis in the Toxicity
Study of 8, 10, and 13 in Male B6D2F1 Mice
avoiding
skin
pressure
group
dose
scalinga
alopeciab on limbs
vehicle 1
TTNPB (5)
0/16 (-)c
0/16 (-)
0/16 (-)
0.1 mg/kg/day
0.2 mg/kg/day
0.4 mg/kg/day
0.8 mg/kg/day
low-dose
mid-dose 1
mid-dose 2
High Dose
low-dose
14/16 (8) 10/16 (8)
14/16 (6) 10/16 (8)
0/16 (-)
0/16 (-)
0/16 (-)
0/16 (-)
0/16 (-)
0/16 (-)
0/16 (-)
0/16 (-)
0/16 (-)
0/16 (-)
0/16 (-)
15/16 (6)
12/16 (6)
8/16 (6)
3/16 (6)
8
0/16 (-) 1/16 (38)
1/16 (41) 1/16 (10)
0/16 (-) 1/16 (19)
0/16 (-) 1/16 (45)
10
0/16 (-)
0/16 (-)
mid-dose 1
mid-dose 2
high-dose
0/16 (-) 2/16 (38)
0/16 (-) 3/16 (41)
2/16 (57) 4/16 (36) 4/16 (62)
7/16 (61) 6/16 (26) 1/16 (65)
6/16 (57) 6/16 (43) 2/16 (62)
7/16 (38) 7/16 (45) 6/16 (45)
6/16 (36) 4/16 (41) 4/16 (36)
13
low-dose
mid-dose 1
mid-dose 2
high-dose
vehicle 2
TTNPB (5) 0.01 mg/kg/day
4/8 (14)
8/8 (9)
6/8 (16)
8/8 (7)
0/8 (-)
7/8 (11)
0.05 mg/kg/day
a
Sites of skin scaling were ears, mouth, nose, eyelids, feet, tail,
b
and/or ventral body. Sites of alopecia were face, ventral body,
and/or forlimbs. c Number of animals in group with clinical sign/
total animals in group (day of first observation).
groups.) No enlarged spleens were observed in the 8
group. There were only low incidences of enlarged
lymph nodes.
Calculations of 30-day maximally tolerated dose,
based upon total dose to 10% weight loss, show that the
three compounds have a favorable toxicologic profile
relative to the reference retinoids. Maximally tolerated
doses (Table 2) were approximately 34 mg/kg for 8, 24-
32 mg/kg for 10, 6-9 mg/kg for 13, and 0.01 mg/kg for
TTNPB. In male B6D2F1 mice, the 30-day maximally
tolerated dose of 10% weight loss for t-RA is ap-
proximately 10 mg/kg.69-71 With this relative measure
of toxicity, 10 and 8 are approximately 3-fold less toxic,
on a mg/kg basis, than t-RA (1) and 3000-fold less toxic
than TTNPB (5). In contrast, 13 is approximately equal
in toxicity to t-RA (1) and 630-940-fold less toxic than
TTNPB (5). This reduced toxicity of 13 is significant
compared to other arotinoids59,69 and suggests that
insertion of a heteroatom into the cyclohexenyl ring of
arotinoids will result in significantly reduced toxicity.
Thus, removal of the dimethyl group at the 4-position
from the cyclohexenyl ring in 5 results in reduced
toxicity of 13 relative to TTNPB (5).69 This lowered
toxicity does not appear to be due solely to reduced
absorption in vivo since elevated triglycerides were
observed as early as day 8 to 0.1 mg/kg for the 8 and
10 agents, and increased circulating white blood cell
counts were observed at day 8 at 0.4 mg/kg for 13.
Within the three compounds, 13 can be ranked as 3-4-
fold more toxic than 8 and 10, with 10 being more toxic
than 8 based upon the observance of fractures in the
mid-dose 2 group and skin scaling in the high-dose
group.
These data suggest that target organs for the three
compounds are those typically found for retinoids.10,62
In the absence of the hypervitaminosis A syndrome,
target organ effects of retinoids are in skin (epithelial
hyperplasia, hyperkeratoses and subacute inflamma-
tion), spleen (lymphoid hyperplasia, hematopoietic cell

Biologically Active Heteroarotinoids
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3575
Ta ble 5. Effects of Retinoids on Organ-to-Body Weight Ratios in the Toxicity Study on 8, 10, and 13 in Male b6d2f1 Mice
thymus
right testicle
spleen
adrenal
group
dose
(ratio × 1000)
day 65
(ratio × 1000)
day 65
(ratio × 1000)
day 65
(ratio × 1000)
day 65
vehicle 1
TTNPB (5)
1.3 ( 0.1 (-)a
b
b
b
b
1.7 ( 0.2 (+28.1)
1.5 ( 0.3 (+14.9)
1.6 ( 0.2 (+16.7)
1.5 ( 0.3 (+13.1)
1.6 ( 0.3 (+17.1)
1.6 ( 0.3 (+20.8)
1.5 ( 0.5 (+13.7)
1.3 ( 0.4 (-0.1)
1.5 ( 0.3 (14.8)
1.3 ( 0.4 (-6.7)
0.9 ( 0.5 (-30.2)c
b
4.2 ( 0.3 (-)
b
b
b
b
2.0 ( 0.3 (-)
b
b
b
b
2.1 ( 0.6 (+3.3)
2.0 ( 0.3 (+1.0)
2.2 ( 0.5 (+8.0)
2.6 ( 0.3 (-37.1)c
2.3 ( 0.5 (+14.2)
2.5 ( 0.6 (+26.5)
2.7 ( 0.2 (+36.4)c
3.0 ( 0.4 (+48.6)c
3.6 ( 0.9 (+82.4)c
5.0 ( 1.4 (+151.1)c
7.1 ( 0.7 (+256.5)c
b
0.4 ( 0.2 (-)
b
b
b
b
0.3 ( 0.1 (-34.5)
0.3 ( 0.1 (-13.1)
0.3 ( 0.2 (-23.6)
2.6 ( 0.4 (-28.0)
0.3 ( 0.1 (-34.8)
0.3 ( 0.2 (-31.4)
0.3 ( 0.1 (-15.1)
0.4 ( 0.2 (-7.6)
0.3 ( 0.1 (-14.9)
0.4 ( 0.2 (-3.1)
0.3 ( 0.1 (-30.5)
b
0.1 mg/kg/day
0.2 mg/kg/day
0.4 mg/kg/day
0.8 mg/kg/day
low-dose
mid-dose 1
mid-dose 2
high-dose
8
2.6 ( 0.8 (-37.8)c
3.1 ( 0.3 (-26.5)c
2.8 ( 0.2 (-32.7)c
2.7 ( 0.3 (-37.1)c
3.2 ( 0.6 (-24.4)c
2.9 ( 0.6 (-32.5)c
3.3 ( 0.3 (-22.3)c
3.0 ( 0.5 (-28.9)c
1.9 ( 0.1 (-55.1)c
2.0 ( 0.2 (-53.5)c
1.9 ( 0.1 (-54.8)c
b
10
13
low-dose
mid-dose 1
mid-dose 2
high-dose
low-dose
mid-dose 1
mid-dose 2
high-dose
day 25
day 25
day 25
day 25
vehicle 2
TTNPB (5)
1.7 ( 0.2 (-)
4.0 ( 0.7 (-)
2.2 ( 0.5 (-)
0.3 ( 0.1 (-)
0.01 mg/kg/day
0.05 mg/kg/day
1.9 ( 0.3 (+11.8)
1.0 ( 0.4 (-41.3)c
3.9 ( 0.5 (-1.2)
4.0 ( 0.3 (+1.5)
4.9 ( 1.2 (+124.6)c
9.5 ( 1.7 (+335.7)c
0.3 ( 0.0 (-9.6)
0.4 ( 0.1 (+56.8)c
a
b
Mean ( SD for 4 to 8 animals (% difference from vehicle). All animals dead by day 65. c P less than 0.05 relative to appropriate
vehicle group.
Ta ble 6. Gross Pathological Observations Consistent with
Hypervitaminosis A in the Toxicity Study of 8, 10, and 13 in
the Male B6D2F1 Mice
cell count suggest higher selectivity for retinoid im-
munologic effects with TTNPB (5) and 13. It should be
stated, however, that organ weight determinations and
gross observations of pathologic effects require histo-
pathologic confirmation.
enlarged
spleen
lymph nodes
enlargedb
group
dose
vehicle 1
TTNPB (5)
0/16a
4/16
0/16
0/16
0/16
0/16
0/16
0/16
0/16
0/16
1/16
0/16
0/16
3/16
5/16
6/16
4/16
0/16
16/16
16/16
12/16
8/16
1/16
0/16
2/16
1/16
1/16
2/16
1/16
1/16
6/16
6/16
8/16
8/16
In summary, the heteroarotinoids cited have very
favorable toxicologic profiles relative to the reference
retinoids, t-RA (1) and TTNPB (5). The MTD of 13 was
9.4 mg/kg/day which is equal in toxicity to t-RA (1)70,71
and 1000-fold less than TTNPB (5) (0.001 mg/kg/day).
The MTD values for 8 and 10 are 34 and 32 mg/kg/day,
respectively, which is 3-fold less toxic than t-RA (1) and
3000-fold less toxic than TTNPB (5), suggesting the
presence of the polyene side chain may be more favor-
able than the aryl group. This 3000-fold reduced
toxicity, compared with only 27% reduction in biological
activity of 8 and 10 in relation to that of TTNPB
observed in our assays, indicates an improved thera-
peutic ration of the heteroarotinoids over the parent.
In view of the important receptor binding capabilities
and growth-promoting characteristics of heteroaroti-
noids 10 and 14,48 these systems deserve additional
study. The therapeutic ultility of these compounds
awaits data from in vivo efficacy and pharmacokinetic
studies.
0.1 mg/kg/day
0.2 mg/kg/day
0.4 mg/kg/day
0.8 mg/kg/day
low-dose
mid-dose 1
mid-dose 2
high-dose
8
10
13
low-dose
mid-dose 1
mid-dose 2
high-dose
low-dose
mid-dose 1
mid-dose 2
high-dose
vehicle 2
TTNPB (5)
0.01 mg/kg/day
0.05 mg/kg/day
5/8
5/8
6/8
8/8
a
Number of animals in group with gross observations/total
b
number of animals in group. Mesenteric, mandibular, inguinal,
iliac, renal, and/or axillary.
proliferation), thymus (cortical epithelial necrosis), ad-
renal (hypertrophy of zona fasciculata), liver (periportal
cytoplasmic vacuolization, hematopoietic cell prolifera-
tion), forestomach (squamous epithelial hyperplasia,
subacute inflammation), bone (osteodystrophy), and
lymph nodes (lymphoid hyperplasia).62,69
Exp er im en ta l Section
Ch em ica l Meth od s. Gen er a l. IR spectra were recorded
on a Perkin-Elmer 2000 FT-IR as films or as KBr pellets. Mass
spectral data (EI and FAB) were obtained from an HP GC-
MS ENGINE, Model 5989B, using 3-nitrobenzyl alcohol as the
1
matrix. All H and 13C NMR spectra were taken on a Varian
Some structure-activity relationships among retin-
oids are suggested from these studies. Agents 8 and
10 appear to have high selectivity for elevation of serum
triglycerides, and 13 has selectivity for elevation of
serum alkaline phosphatase. Organ weight data sug-
gest higher activity for adrenal effects for TTNPB (5)
and testicular effects for all three compounds. The high
incidence of enlarged lymph nodes and spleens, higher
spleen weights, and increases in circulating white blood
XL-400 BB NMR spectrometer operating at 399.99 and 100
Hz, respectively, and signals were referenced to TMS. Melting
points were determined with a Thomas-Hoover melting point
apparatus and were uncorrected. Syntheses were executed,
unless otherwise indicated, under N2 and with the aid of a
magnetic stir bar. Acids 8,49 9,49 10,49 11,51 and 1320 as well
as esters 14,20 15,20 and 2272 were prepared as reported.
Intermediates were used at once, but intermediates and
products could be safely stored in a dark freezer for several
weeks without appreciable decay.

3576 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Benbrook et al.
(2E,4E,6E)-7-(2,3-Dih yd r o-3,3-d im eth ylben zo[b]th ien -
5-yl)-3-m eth yl-2,4,6-octa tr ien oic Acid (12) w ith In ter m e-
d ia te 29. A solution of n-butyllithium (1.6 M, 3.4 mL, 5.4
mmol) in hexane was added to a stirred solution of 28 (3.00 g,
5.35 mmol) in dry ether (35 mL). After stirring (dark) at room
temperature (15 min), the Wittig reagent was cooled to -78
°C, and a solution of ethyl (E)-OHCC(CH3)dCHCO2Et (0.76
g, 5.35 mmol) in dry ether (10 mL) was added dropwise. The
mixture was allowed to warm to room temperature and then
was stirred (45 h). Hexane was added, and the mixture was
filtered. The filtered solid was rinsed with ether which was
added to the hexane solution. Chromatography on silica gel
(hexanes:ether, 20:1) yielded slightly crude ester 29 as a yellow
oil (0.67 g, 36.3%). This light-sensitive ester 29 (0.665 g, 1.94
mmol) was treated (dark) with 35% aqueous KOH (2.0 mL) in
absolute ethanol (8 mL) with stirring and at reflux (1 h). After
being cooled to room temperature, the solution was diluted
with ethyl acetate and water, followed by quenching with 50%
aqueous acetic acid. Extracts (EtOAc) of the solution were
dried (Na2SO4), filtered, and evaporated to a yellow solid.
Recrystallization (absolute ethanol), followed by filtration,
washing (chilled ethanol, then hexanes), and drying gave free
acid 12 as yellow flakes (0.424 g, 25% from 28): mp 209.5-
210.7 °C; IR (KBr) 1683 (CdO of CO2H) cm-1; 1H NMR (DCCl3)
δ 1.40 [s, 6 H, C(CH3)2], 2.24 [s, 3 H, H(11)], 2.39 [s, 3 H, H(16)],
3.20 [s, 2 H, H(2)], 5.85 [bs, 1 H, H(17)], 6.41 [d, J ) 14.9 Hz,
1 H, H(14)], 6.56 [d, J ) 11.3 Hz, 1 H, H(12)], 7.06 [dd, J )
14.9 Hz, J ) 11.2 Hz, 1 H, H(13)], 7.13-7.28 [m, 3 H, H(4,6,7)];
13C NMR (DCCl3) δ 14.1 [C(16)], 16.5 [C(11)], 27.4 [C(8,9)], 47.2
[C(3)], 47.5 [C(2)], 117.5 [C(17)], 119.9 [C(4)], 122.2 [C(7)], 125.2
[C(6)], 125.6 [C(12)], 132.1 [C(7a) and C(13)], 135.4 [C(14)],
139.0 [C(5)], 140.7 [C(10)], 148.2 [C(3a)], 155.2 [C(15)], 171.2
[C(18)]. Anal. (C19H22O2S) C, H.
) 7.4 Hz, 2 H, H(19)], 6.8 [s, 1 H, H(11)], 7.25-8.05 [m, 7 H,
Ar-H]; 13C NMR (DCCl3) δ 10.8 [CH2CH3]; aliphatic-C: 14.14,
17.39, 20.51, 21.48, 26.57, 59.53, 60.70; Ar-C and vinylic-C:
124.76, 126.89, 128.01, 128.40, 128.80, 129.06, 129.25, 135.43,
138.08, 138.35, 142.09, 145.83, 166.11 [CdO]; at 26 °C [R]D
)
+39.60° (acetone); mass spectral (EI) data calcd for C23H26O3S
m/z (M+) 382.1602, found 382.1600. Anal. (C23H26O3S) C, H,
S.
Eth yl 4-[[(4,4-Dim eth yl-3,4-d ih yd r o-2H-ben zo[b]p yr a n -
6-yl)ca r bon yl]oxy]ben zoa te (18). Carboxylic acid 39 (0.500
g, 2.45 mmol) and ethyl 4-hydroxybenzoate (0.610 g, 3.67
mmol) in CH2Cl2 (30 mL) were mixed. To this mixture was
added dicyclohexylcarbodiimide (∼1.52 g, 7.35 mmol) and a
catalytic amount of DMAP (20 mg). The resulting cloudy
solution was stirred at room temperature (24 h) and then
filtered. The solvent was evaporated in vacuo, and the heavy
oil was subjected to chromatography (CH2Cl2:MeOH, 200:1).
To the resulting oil was added 2 mL of a solution of hexane:
EtOAc (4:1). The solution was cooled at 0-4 °C for 15 h,
during which time crystallization took place. The solid 18 was
filtered (mp 108-110 °C): 0.721 g (83%); IR (KBr) 1730 (CdO)
cm-1; 1H NMR (DCCl3) δ 1.39 [s, 6 H, C(CH3)2], 1.43 [t, 3 H, J
) 7.2 Hz, CH3], 1.88 [t, 2 H, J ) 6 Hz, CCH2], 4.29 [t, 2 H, J
) 6 Hz, CH2O], 4.39 [q, 2 H, J ) 7.2, OCH2], 6.87 [d, 1 H,
Ar-H], 7.30 [m, 2 H, Ar-H], 7.90 [dd, 2 H, Ar-H], 8.10 [d, 1 H,
Ar-H]; 13C NMR (DCCl3) δ 14.29 [CH3], 30.60 [C(CH3)2], 36.89
[ArCCH2], 61.02 [OCH2], 63.51 [ArOCH2], 117.32-158.66 [Ar-
C], 164.64 [CdO], 165.98 [CdO]. Anal. (C21H22O5) C, H.
E t h yl 4-(N,4,4-Tr im et h yl-1,2,3,4-t et r a h yd r oq u in olin -
yl)ben zoa t e (19). To a mixture of the carboxylic acid 45
(0.174 g, 0.794 mmol) and ethyl 4-hydroxybenzoate (0.198 g,
1.08 mmol) in CH2Cl2 (6 mL) was added dicyclohexylcarbodi-
imide (∼0.025 g, 7.9 mmol) and a catalytic amount of DMAP
(7 mg). The resulting cloudy solution was stirred at room
temperature (48 h) and then filtered. The solvent was
evaporated in vacuo, and the product was chromatographed
(HCCl3:MeOH, 50:1) to give the solid 19 (0.105 g, 45%): mp
Eth yl (E)-4-[2-(3,4-Dih yd r o-2-n -octyl-2H-1-ben zoth io-
p yr a n -6-yl)-1-p r op en yl]ben zoa te (16, R ) n -octyl). To a
boiling mixture of phosphonium salt 36b (2.0 g, 3 mmol), K2-
CO3 (0.4 g, 3 mmol), and 18-C-6 (30 mg) in H2CCl2 (15 mL)
was added ethyl 4-formylbenzoate (0.5 g, 3 mmol) in H2CCl2
(10 mL) in a single portion. The mixture was boiled (12 h)
and was then concentrated to an orange oil which was treated
with hexane (150 mL). A suspension formed and was filtered.
The filtrate was washed with brine, dried (Na2SO4), and
concentrated to a yellow oil which was separated on a silica
gel column (eluent, hexane:ethyl acetate, 1:1) to give sulfide
16 (or 37b) (1.1 g, 79%) as a white solid: mp 72-73 °C; IR
1
116-118 °C; IR (KBr) 1720 (CdO) cm-1; H NMR (DCCl3) δ
1.32 [s, 6 H, C(CH3)2], 1.40 [t, 3 H, J ) 7.2 Hz, CH3], 1.76 [t,
2 H, J ) 6 Hz, CCH2], 3.03 [s, 3 H, NCH3], 3.39 [t, 2 H, J ) 6
Hz, CH2N), 4.38 [q, 2 H, J ) 7.2 Hz, NCH2], 6.56 [d, 1 H, Ar-
H], 7.27 [d, 2 H, Ar-H], 7.90 [dd, 2 H, Ar-H], 7.98 [d, 1 H, Ar-
H], 8.10 [dd, 1 H, Ar-H]; 13C NMR (DCCl3) δ 14.62 [CH3], 30.28
[C(CH3)2], 36.42 [ArCCH2], 39.31 [NCH3], 47.79 [NCH2], 61.27
[ArNCH2], 109.87-155.60 [Ar-C], 165.53 [CdO], 166.43 [CdO].
Anal. (C22H25NO4) C, H, N.
1
(neat) 1750 cm-1 (CdO); H NMR (DCCl3) δ 0.95 [t, J ) 7.5
Hz, 3 H, (CH2)7CH3], 1.1-1.7 [m, 17 H, (CH2)7CH3 and
OCH2CH3], 2.8-2.95 [m, 1 H, H(3)], 2.15-2.3 [m, 4 H, H(3)
and H(10)], 2.8-2.9 [m, 2 H, H(4)], 3.1-3.2 [m, 1 H, H(2)], 4.3
[q, J ) 7.5 Hz, 2 H, OCH2CH3], 6.8 [s, 1 H, H(11)], 7.0-8.1
[m, 7 H, ArH]; 13C NMR (DCCl3) δ 14.05 [(CH2)3CH3]; aliphatic-
C: 14.31, 17.46, 22.61, 25.55, 27.08, 29.26, 29.54, 29.63, 31.87,
36.83, 37.83, 42.31; 60.79 [C(19)]; ArC and vinylic C: 124.03,
125.63, 126.34, 127.39, 128.93, 129.36, 132.08, 148.01, 149.10,
149.32, 149.8; 168.82 [C(18)]; mass spectral (EI) data calcd for
C29H38O2S m/ z (M•+) 450.2593, found 450.2595. Anal.
(C29H38O2S) C, H.
Eth yl (E)-4-[2-(3,4-Dih yd r o-2-eth yl-2H-1-ben zoth iop y-
r a n -6-yl)-1-p r op en yl]ben zoa te (17). To a stirred mixture
of Ti(O-i-Pr)4 (1.44 g, 5 mmol) and (+)-diethyl L-tartrate (2.0
g, 10 mmol) in H2CCl2 (50 mL) was introduced water (88 µL
syringe) in a single portion. The mixture was stirred to a
homogeneous solution. To this solution was added sulfide 37a
(1.78 g, 5 mmol, R ) ethyl) in H2CCl2 (15 mL). To this cooled
(-20 °C) mixture was added tert-butyl hydroperoxide (TBHP,
0.5 g, 5.5 mmol) in H2CCl2 (1.6 mL). Stirring was continued
(4 h) at -20 °C, and water (50 mL) was then added over 10
min. Stirring was continued at -20 °C (1 h) and at room
temperature (1 h). A white gel formed and was filtered off,
and the filtrate was dried (Na2SO4) and evaporated to yield
sulfoxide 17 as a light yellow solid. Recrystallization (HCCl3
and then ethanol) gave 17 as a white solid (1.2 g, 64%): mp
88-89 °C; 1H NMR (DCCl3) δ 0.85 [t, J ) 7.3 Hz, 3 H,
CH2CH3], 1.05 [t, J ) 7.4 Hz, 3 H, H(20)], 1.2-1.4 [m, 1 H,
H(3)], 1.5-1.7 [m, 2 H, CH2CH3], 1.8-1.9 [s, 3 H, H(10)], 2.2-
2.3 [m, 1 H, H(3)], 2.5-2.8 [m, 4 H, H(2) and H(4)], 4.1 [q, J
4-[[(4,4-Dim eth yl-3,4-d ih yd r o-2H-ben zo[b]p yr a n -6-yl)-
car bon yl]oxy]ben zaldeh yde Th iosem icar bazon e (20). Thi-
osemicarbazide (0.017 g, 0.184 mmol) was dissolved in H2O (1
mL) and AcOH (1 drop). Aldehyde 46 (0.050 g, 0.161 mmol)
dissolved in hot EtOH (3 mL) was added hot to the thiosemi-
carbazide solution. Crystals formed after 10 s, and the mixture
was allowed to stand at room temperature (8 h) and then was
cooled (12 h). The crystals were filtered and dried to give 20
(0.50 g, 81%): mp 185-186 °C; IR (KBr) 3436, 3403, 3266,
1
3164 (N-H) cm-1; H NMR (DCCl3) δ 1.33 [s, 6 H, C(CH3)2],
1.82 [t, 2 H, J ) 5.6 Hz, CCH2], 4.26 [t, 2 H, J ) 5.6 Hz, CH2O],
6.90 [d, 1 H, Ar-H], 7.31 [d, 2 H, Ar-H], 7.82 [dd, 1 H, Ar-H],
7.84 [d, 2 H, Ar-H], 8.06 [d, 1 H, Ar-H], 8.21 [s, 2 H, NH2],
11.46 [s, 1 H, CdNH]. 13C NMR (DCCl3) δ 30.26 [C(CH3)2],
36.13 [ArCCH2], 38.87 [CH2C], 63.17 [ArOCH2], 117.25-158.30
[Ar-C], 164.23 [CdO], 178.04 [CdN]. Anal. (C20H21N3O4S) C,
H, N.
Meth yl 4-(4,4,5,7-Tetr am eth yl-6-cou m ar yl)ben zoate (21).
Phenol 4756 (0.125 g, 0.568 mmol) and monomethylterephtha-
late (0.122 g, 0.682 mmol) were added to CH2Cl2 (10 mL). To
this cloudy mixture were added dicyclohexylcarbodiimide
(∼0.352 g, 1.7 mmol) and a catalytic amount of DMAP (10 mg).
The resulting cloudy solution was stirred at room temperature
(36 h) and then filtered. The solvent was evaporated in vacuo,
and the heavy oil was subjected to chromatography (0.67:1.33:
15, hexane:EtOAc:CH2Cl2). The white solid ester 21 was
obtained in a yield of 51% (0.109 g): mp 174-175 °C; IR (KBr)
1788 (CdO), 1737 cm-1 1H NMR (DCCl3) δ 1.48 [s, 6 H,
;
C(CH3)2], 2.16 [s, 3 H, Ar-CH3], 2.29 [s, 3 H, Ar-CH3], 2.62 [s,
2 H, C(O)CH2], 3.99 [s, 3 H, OCH3], 6.87 [s, 1 H, Ar-H], 8.20

Biologically Active Heteroarotinoids
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3577
[d, 2 H, Ar-H], 8.30 [d, 2 H, Ar-H]; 13C NMR (DCCl3) δ 15.10
[CH3], 35.50 [C(CH3)2], 45.64 [ArCCH2], 52.56 [CH2], 117.53-
148.98 [Ar-C], 163.76 [CdO], 166.12 [CdO], 168.07 [CdO]; MS
(EI) calcd for 382.1416, found 382.1411. Anal. (C22H22O6) C,
H.
An NMR variable-temperature study was made with 21 in
DCCl3 over a range of 0-30 °C in a 5 mm tube. The equation
utilized to calculate the ∆G* was ∆G* ) 4.58Tc[9.97 + log(Tc/
∆ν].73 Two peaks for the gem-dimethyl groups appeared at δ
1.46 and δ 1.49 at 26 °C and coalesced at 27 °C. The value
for ∆G* was determined to be 15.72 kcal/mol (∆ν ) 9.3 Hz) at
27 °C.
NMR (DCCl3) δ 26.4 [CH3], 27.6 [C(CH3)2], 47.3 [C(CH3)2], 47.7
[SCH2], Ar-C [122.7, 122.9, 129.2, 134.9, 149.0, 149.5], 198.3
[CdO]. Anal. (C12H14OS) C, H.
2-(2,3-Dih ydr o-3,3-dim eth ylben zo[b]th ien -5-yl)-3-bu ten -
2-ol (27). To a fresh solution of H2CdCHMgBr (26, 33 mmol)
in dry THF (25 mL) was slowly added a solution of ketone 25
(2.00 g, 9.69 mmol) in dry THF (20 mL). After being stirred
at room temperature (3 h) and at reflux (1 h), the mixture was
cooled and quenched by the dropwise addition of saturated
aqueous NH4Cl solution. Ether extracts of the mixture were
washed with 5% NaHCO3 and brine and then dried (Na2SO4),
filtered, and evaporated to a yellow oil (2.35 g, qt). The alcohol
27 appeared to become colored quickly and was used im-
2-Meth yl-1-p h en ylp r op a n ol (23). To a cooled (0 °C)
solution of methylmagnesium iodide (0.17 mol) in 100 mL of
dry ether was added slowly a solution of ester 22 (10 g, 54.9
mol) in dry ether (15 mL). The resulting mixture was stirred
at 0-25 °C for 1 h and then at reflux (18 h). The final mixture
was diluted with ether, cooled (0 °C), and quenched with water,
20% NH4Cl, and finally with 10% H2SO4 in that order. Ether
extracts were washed with 5% NaHCO3 and dried (Na2SO4).
Evaporation of the solvent gave an oil which contained an
impurity that was detrimental to the next cyclization step.
Removal of the impurity was effected by adding a solution of
I2 and NaI in water to the oil which was suspended in a 6%
solution of KOH in H3COH. The resulting mixture was stirred
for 5 min with gentle warming and 5 min at room temperature.
Ether extracts of the resulting mixture were dried (Na2SO4)
and evaporated to an oil which was vacuum distilled with the
main fraction distilling at 80-85 °C/0.12 mm [lit.51,74 136-
137 °C/12 mm]. Chromatography of the oil over silica gel (3:1
hexanes:ether) gave 22 as a yellow oil (5.6 g, 56%). Alcohol
23 has not been well characterized to date. Thus, we obtained
the following data to substantiate the structure; n26.8 1.5582
[lit.74 n23 1.5609]; IR (neat) 3650-3150 cm-1 (O-H); 1H NMR
(DCCl3) δ 1.29 [s, 6 H, C(CH3)2], 2.31 [bs, 1 H, O-H], 3.11 [s, 2
H, CH2], 7.13-7.21 [m, 1 H, Ar-H], 7.22-7.32 [m, 2 H, Ar-
H], 7.41 [m, 2 H, Ar-H]; 13C NMR (DCCl3) δ 28.6 [C(CH3)2],
48.2 [CH2], 70.6 [C(CH3)2], Ar-C [125.9, 128.7, 129.2, 136.8].
2,3-Dih yd r o-3,3-d im eth ylben zo[b]th iop h en e (24). Al-
cohol 23 (9.60 g, 52.7 mmol) in CS2 (55 mL) was added
dropwise to a stirred suspension of AlCl3 (25.0 g, 0.18 mol) in
CS2 (50 mL). The resulting orange-red suspension was stirred
at reflux (3 h). The cooled mixture was diluted (H2CCl2) and
quenched with 5% HCl. Extracts (H2CCl2) were washed
(saturated NaHCO3, brine), dried (MgSO4), and evaporated to
an oil. Vacuum distillation (56-58 °C/0.37 mm) gave a pale
yellow oil (6.97 g, 80.5%) which was further purified over silica
gel (hexanes); n25.6 1.5757. The compound 24 has not been
well characterized, but certain derivatives have been re-
ported.74 Our spectral data are as follows: 1H NMR (DCCl3)
δ 1.35 [s, 6 H, C(CH3)2], 3.16 [s, 2 H, SCH2], 7.02-722 [m, 4
H, Ar-H]; 13C NMR (DCCl3) δ 27.2 [C(CH3)2], 46.9 [SCH2], 46.9
[C(CH3)2], Ar-C [122.0, 122.3, 124.1, 127.0, 140.1, 147.5]. Anal.
(C10H12S) C, H.
1
mediately to make 28: IR (neat) 3600-3150 cm-1 (O-H); H
NMR (DCCl3) δ 1.35 [s, 6 H, C(CH3)2], 1.60 [s, 3 H, CH3], 2.32
[bs, 1 H, OH], 3.15 [s, 2 H, SCH2], 5.11 [dd, J cis ) 10.6 Hz,
J gem ) 1.1 Hz, CHdC(H)H], 5.26 [dd, J trans ) 17.2 Hz, J gem
)
1.1 Hz, CHdC(H)H], 6.12 [dd, J trans ) 17.2 Hz, J cis ) 10.6 Hz,
1 H, CHdCH2], 7.08-7.22 [m, 3 H, Ar-H].
[3-(2,3-Dih ydr o-3,3-dim eth ylben zo[b]th ien -5-yl)-2-bu ten -
1-yl]tr ip h en ylp h osp h on iu m Br om id e (28). A solution of
alcohol 27 (2.00 g, 8.53 mmol) was added slowly to a stirred
and cooled (0 °C) mixture of Ph3PH+Br- (2.90 g, 8.45 mmol)
in H3COH (10 mL). An immediate blue-green-colored mixture
appeared which turned to bright yellow after 14 h of stirring.
The mixture was evaporated to about 7 mL, and dry ether was
added. Trituration caused the salt 28 to precipitate which was
recrystallized (H3COH). An analytical sample was obtained
by allowing a methanol solution of the salt to stand in a
diffusion chamber with an adjacent container of ether whose
vapors induced precipitation of white, crystalline 28 (3.53 g,
74.7%): mp 236-238 °C; 1H NMR (DCCl3) δ 1.32 [s, 6 H,
5
C(CH3)2], 1.61 [dd, J PH ) 4.4 Hz, J ) 1 Hz, 3 H, H3-
3
CCdCHCH2P], 3.14 [s, 2 H, SCH2], 4.91 [dd, J PH ) 15.2 Hz,
J ) 8.0 Hz, 2 H, CH2P], 5.55-5.65 [m, 1 H, H3CCdCHCH2P],
6.85 [d, J ) 8.0 Hz, 1 H, H(4)], 6.90 [dd, J ) 8.0 Hz, J ) 1.8
Hz, 1 H, H(6)], 7.06 [d, J ) 8.0 Hz, 1 H, H(7)], 7.6-8.0 [m, 15
H, P(C6H5)3]. Anal. (C32H32BrPS) C, H.
3,4-Dih yd r o-2H-1-ben zoth iop yr a n (30). Thiochroman-
4-one (3 g, 18 mmol), toluene (75 mL), water (120 mL),
concentrated HCl (60 mL), and the Clemmenson-Martin75
amalgam [50 g, prepared by shaking for 5 min a mixture of
mossy Zn (50 g, 765 g atom), mercuric chloride (5 g, 18 mmol),
concentrated HCl (2.5 mL), and water (75 mL)] were mixed.
The heterogeneous mixture was boiled and stirred for 72 h,
adding 20-mL portions of concentrated HCl at intervals of
about 6 h to maintain a total volume of 500 mL. The mixture
was allowed to cool to room temperature (1 h) and was then
gravity filtered. Toluene extracts of the aqueous layer and
the original organic layer were combined and washed with
saturated NaHCO3, water, and brine. The solution was dried
(MgSO4) and evaporated to give 30 as a yellow oil (2.7 g, 98%),
which was used directly to prepare sulfoxide 31: IR (neat)
1680 (CdO, weak) cm-1; 1H NMR (DCCl3) δ 2.1 [quintet, 3J HCCH
) 6.1 Hz, 2 H, H(3)], 2.8 [t, 3J HCCH ) 6.2 Hz, 2 H, H(4)], 3.0 [t,
3J HCCH ) 6.0 Hz, 2 H, H(2)], 7.05-7.2 [m, 4 H, ArH]; 13C NMR
(DCCl3) δ 22.75 [C(3)], 27.46 [C(4)], 29.56 [C(2)]; ArC: 123.79,
126.28, 126.45, 129.87, 132.77, 133.73. Reported properties:
1-(2,3-Dih yd r o-3,3-d im et h ylb en zo[b]t h ien -5-yl)et h a -
n on e (25). A solution of thioether 24 (8.00 g, 48.7 mmol) and
freshly distilled acetyl chloride (4.40 g, 56.1 mmol) in CS2 (65
mL) was added dropwise to a stirred suspension of AlCl3 (9.8
g, 73 mmol) in CS2 (70 mL). Stirring was continued at room
temperature (2 h) after which the mixture was cooled (0 °C)
and quenched (H2O). Ether extracts of the resulting mixture
were washed with 5% NaHCO3 and then brine. After drying
(Na2SO4), the solution was evaporated to an oil which was
chromatographed over silica gel (hexanes:ether, 4:1). The oil
dissolved in a minimum of hexane and was cooled to -78 °C
which resulted in an initial precipitation of a light yellow solid.
Intermittent cooling and warming from -78 to -60 °C
produced a crystalline, very lightly colored solid. Immediate
removal of the crystals, followed by vacuum drying (room
temperature/0.3 mm), produced off-white crystals of 25 (8.28
g, 82.4%), mp 20.1-21.4 °C. Concentration of the mother
liquors gave an additional 0.65 g of the same crystals for a
total yield of 8.93 g (88.9%). Other properties of 25 were n26
53
1
bp 81.5-82.5 °C/1.2 mm; H NMR (DCCl3) δ 1.90 [m, 2 H,
H(3)], 2.92 [quintet, 4 H, H(2) and H(4)], 6.90 [s, 4 H, ArH].
3,4-Dih yd r o-2H-1-ben zoth iop yr a n 1-Oxid e (31).54 To a
solution of Ti(i-OPr)4 (43 mL, 40.6 g, 143 mmol) and (+)-diethyl
L-tartrate (49 mL, 58.9 g, 286 mmol) in H2CCl2 (250 mL) was
added water (2.6 mL) via a syringe. The resulting mixture
was stirred (20 min) to a homogeneous solution. To this
solution was added sulfide 30 (21.45 g, 143 mmol). The
mixture was cooled to -20 °C, and a 3.1 M solution of TBHP
(180 mmol) in H2CCl2 (51 mL) was introduced dropwise.
Stirring was continued at -20 °C (4 h), and 25 mL of water
was then added dropwise. Stirring was continued at -20 °C
(1 h) and then at room temperature (1 h). A white gel formed
and was filtered off, and the filtrate was evaporated and dried
(Na2SO4). The resulting mixture was separated on silica gel
(column chromatography; ethyl acetate:hexane ) 85:15, 100%
MeOH). The fraction from methanol was evaporated to yield
31 (19 g, 82%) as a yellow oil. The system has not been well
1
1.6048; IR (neat) 1683 cm-l (CdO); H NMR (DCCl3) δ 1.39
[s, 6 H, C(CH3)2], 2.57 [s, 3 H, CH3], 3.24 [s, 2 H, SCH2], 7.25
[d, 1 H, Ar-H], 7.67 [d, 1 H, Ar-H], 7.73 [dd, 1 H, Ar-H]; 13C

3578 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Benbrook et al.
characterized previously, and the following data were thus
taken: 1H NMR (DCCl3) δ 2.0-2.2 [m, 1 H, H(3)], 2.4-2.7 [m,
1 H, H(3)], 2.8-3.3 [m, 4 H, H(2) and H(4)], 7.2-7.9 [m, 4 H,
ArH]; 13C NMR (DCCl3) δ 14.12 [C(3)], 28.43 [C(4)], 46.37
[C(2)]; ArC: 127.42, 130.51, 130.81, 131.65, 136.01, 138.04.
The optical rotation of 31 was taken in cells (l cm × 10 cm) on
a Perkin-Elmer 241 polarimeter. At 26 °C, [R]D ) -114.15°
(acetone). Reported54 properties: bp 117-120 °C/0.04 mm; 1H
NMR δ 1.09-3.50 [m, 6 H], 7.20-7.90 [m, 4 H]. The reported52
specific rotation was [R]D ) -21.8° (acetone) at 25 °C.
CH3 and H(3)], 2.2-2.3 [m, 1 H, H(3)], 2.75-2.95 [m, 2 H,
H(4)], 3.2-3.3 [m, 1 H, H(2)], 6.9-7.1 [m, 4 H, ArH]; 13C NMR
(DCCl3) δ 11.4 [CH3]; aliphatic-C: 29.36, 29.41, 29.43, 43.79;
ArC: 123.74, 126.32, 126.38, 129.51, 133.58, 133.83. Re-
corded53 properties: 1H NMR (DCCl3) δ 1.04 [t, 3 H], 1.40-
3.45 [m, 7 H], 6.83-7.60 [m, 4 H, ArH]. No other properties
of 33a have been reported. Sulfide 33a was used immediately
to prepare ketone 34a .
3,4-Dih yd r o-2-n -octyl-2H-1-ben zoth iop yr a n (33b, R )
n -octyl).76 To a stirred mixture of the sulfoxide 32b (1.7 g, 6
mmol) and NaI (2.2 g, 15 mmol) in acetone at 0 °C (ice-water
bath) was added slowly trifluoroacetic anhydride (2.5 mL, 18
mmol) in acetone. The reaction mixture was stirred (1 h) at 0
°C. Acetone was evaporated, water was added, and the
mixture was extracted (ether). The ether extracts were
3,4-Dih ydr o-2-eth yl-2H-1-ben zoth iopyr an 1-Oxide (32a,
R ) eth yl).54 To a cooled (-78 °C) solution of diisopropy-
lamine (6.0 mL, 43 mmol) in THF (50 mL) was added a
solution of n-butyllithium (26 mL, 1.6 M) in hexanes. The
reaction mixture was allowed to warm to room temperature
and was stirred for 1 h and then was again cooled to -78 °C.
The sulfoxide 31 (6.5 g, 39 mmol) in THF (50 mL) was added
to this solution. The reaction mixture was allowed to warm
to -30 °C (1 h) and was again cooled to -78 °C, after which
ethyl iodide (3.1 mL, 43 mmol) was added. Stirring continued
for 12 h, after which time 5% hydrochloric acid (50 mL) was
added, and the resulting solution was then extracted (HCCl3).
Combined extracts were washed with water, 5% NaHCO3,
water, and brine. When dried (Na2SO4), the solution was
concentrated to a brown oil which was separated on a silica
gel column (hexane:HCCl3:ethyl acetate ) 4:1:1). The second
fraction gave the alkylated product 32a (4.2 g, 80%) as a light
yellow oil which was used immediately: 1H NMR (DCCl3) δ
washed (water, saturated Na2
S2O3, water, and brine). After
drying (MgSO4), the solvent was evaporated, and the red oil
obtained was passed through a short silica gel column (hex-
ane). Solvent evaporation gave sulfide 33b (1.5 g, 95%) as a
light yellow oil which was used directly to make ketone 34b:
3
1H NMR (DCCl3) δ 0.88 [t, J HCCH ) 0.7 Hz, 3 H, CH3], 1.2-
1.5 [bs, 12 H, (CH2)6CH3], 1.6-1.8 [m, 3 H, CH2(CH2)6 and
H(3)], 2.2-2.3 [m, 1 H, H(3)], 2.75-2.9 [m, 2 H, H(4)], 3.25-
3.57 [m, 1 H, H(2)], 6.9-7.1 [m, 4 H, ArH]; 13C NMR (DCCl3)
δ 13.67 [CH3]; aliphatic-C: 22.22, 22.37, 25.40, 26.70, 28.88,
29.13, 29.27, 31.43, 33.92, 37.21; ArC: 123.14, 123,31, 125.89,
125.92, 126.05, 129.44. Recorded53
properties: 1H NMR
(DCCl3) δ 1.03 [t, 3 H, CH3], 1.43 [m, 12 H, (CH2)6CH3], 1.8-
3
1.13 [t, J HCCH ) 7.5 Hz, 3 H, CH3], 1.47 [m, 1 H, H(3)], 1.94
2.62 [m, 4 H, CH2(CH2)6 and H(4)], 2.80-3.10 [dd, 2 H, H(3)],
[m, 2 H, CH2CH3], 2.5 [m, 1 H, H(3)], 2.92-3.25 [m, 3 H, H(2)
and H(4)], 7.21-7.85 [m, 4 H, ArH]; 13C NMR (DCCl3) δ 10.97
[C(3)], 20.67 [CH2CH3], 21.27 [CH2CH3], 26.6 [C(4)], 59.7 [C(2)];
ArC: 127.2, 129.1, 129.6, 130.9, 136.6, 138.1. Recorded52
properties: 1H NMR (DCCl3) δ 1.09 [t, 3 H, CH3], 1.40-3.45
[m, 7 H], 6.83-7.08 [m, 4 H]. No other properties of 32a were
revealed in a search of the literature.
3.15-3.57 [bs, 1 H, H(2)], 6.97-7.31 [m, 4 H, ArH]. No other
properties of 33b have been reported.
6-Acetyl-2-eth ylth ioch r om a n (34a , R ) eth yl). To a
stirred suspension of AlCl3 (2.9 g, 22 mmol) in nitromethane
(50 mL) at 0 °C was added acetic anhydride (0.9 mL, 10 mmol).
To the mixture was added a solution of thiochroman 33a (1.8
g, 10 mmol) in CS2:nitromethane (1:5, 25 mL). The solution
was stirred at 0 °C (1 h) and then allowed to warm to room
temperature. Stirring was continued (48 h), and then the
solution was cooled (0 °C) and slowly quenched (H2O). The
aqueous layer was extracted (HCCl3), and the combined
organic extracts were washed (saturated NaHCO3, water, and
brine). After drying (Na2SO4), the solution was evaporated to
a red oil which was separated on a silica gel column (hexane:
ether, 20:80) to give the ketone 34a (1.5 g, 72%) as an orange
oil. Ketone 34a was used at once to prepare alcohol 35a : IR
3,4-Dih yd r o-2-n -oct yl-2H -1-b en zot h iop yr a n 1-Oxid e
(32b, R ) n -octyl).53,54 To a cooled (-78 °C) solution of
diisopropylamine (4.8 mL, 34 mmol) in THF (50 mL) was
added dropwise n-butyllithium (22 mL, 1.6 M in hexanes) over
a period of 1 h. The resulting solution was stirred at room
temperature for 1 h. After the solution was again cooled to
-78 °C, the sulfoxide 31 (5.72 g, 30 mmol) in THF (50 mL)
was added (15 min). The solution was then allowed to warm
to -30 °C (1 h) and again cooled to -78 °C. Then n-octyl
bromide (5.4 mL, 34 mmol) was added via syringe in a single
portion. Stirring was continued for another 12 h, after which
5% hydrochloric acid (50 mL) was added, and the solution was
extracted (HCCl3). Combined extracts were washed with
water, 5% NaHCO3, water, and brine. When dried (Na2SO4),
the solution was concentrated to a brown oil which was
separated on a silica gel column (hexane:HCCl3:ethyl acetate,
4:1:1). The second fraction gave the alkylated product 32b
(3.2 g, 40%) as a light yellow oil which was used at once: 1H
1
3
(neat) 1680 (CdO) cm-1; H NMR (DCCl3) δ 1.05 [t, J HCCH
)
7.4 Hz, 3 H, CH3], 1.6-1.8 [m, 3 H, H(3) and CH2CH3 ], 2.2-
2.3 [m, 1 H, H(3)], 2.3 [m, 1 H, H(3)], 2.5-2.6 [s, 3 H, CH3C-
(O)], 2.65-3.0 [m, 2 H, H(4)], 3.2-3.3 [m, 1 H, H(2)], 7.2-7.7
[m, 3 H, ArH]; 13C NMR (DCCl3) δ 11.25 [CH3]; aliphatic-C:
26.26, 28.75, 29.24, 29.82, 44.0; ArC: 126.01, 126.22, 129.23,
132.70, 133.41, 141.27; 197.25 [C(O)].
6-Acetyl-2-n -octylth ioch r om a n (34b, R ) n -octyl). Ace-
tic anhydride (0.5 mL, 6 mmol) was added to a stirred
3
NMR (DCCl3) δ 0.9 [t, J HCCH ) 0.7 Hz, 3 H, CH3], 1.2-1.4
(1.4 g, 9 mmol) in nitromethane (50 mL,
suspension of AlCl3
0 °C). To the stirred mixture was added a solution of 2-n-
:nitromethane (1:
[bs, 12 H, (CH2)6CH3], 1.4-1.7 [m, 2 H, CH2(CH2)6], 1.85 [m,
1 H, H(3)], 2.45 [m, 1 H, H(3)], 2.8-3.1 [m, 3 H, H(2) and H(4)],
7.1-7.8 [m, 4 H, ArH]; 13C NMR (DCCl3) δ 14.06 [C(3)];
aliphatic-C: 20.75, 22.54, 26.32, 26.53, 28.31, 29.08, 29.25,
29.40, 31.71; 57.99 [C(2)]; ArC: 127.2, 129.18, 129.46, 130.67,
135.47, 140.0. Recorded53 properties: 1H NMR (DCCl3) δ 0.88
[t, 3 H], 1.1-3.10 [m, 22 H], 7.24-8.10 [m, 4 H]. No other
properties of 32b have been reported.
octylthiochroman 33b (1.5 g, 6 mmol) in CS2
5, 25 mL). The solution was stirred at 0 °C (1 h) and then
allowed to warm to room temperature; stirring was continued
(48 h). The solution was cooled (0 °C) and then was quenched
(H2O). The aqueous layer was extracted (HCCl3). The extracts
and the original organic layer were combined and washed
(saturated NaHCO3, water, and brine). After drying (Na2SO4),
the solution was evaporated to a red oil which was separated
on a silica gel column (hexane:ether, 20:80) to give the ketone
34b (R ) n-octyl, quantitative yield) as an orange oil. Ketone
34b was used directly to make alcohol 35b. IR (neat) 1680
3,4-Dih yd r o-2-et h yl-2H -1-b en zot h iop yr a n (33a , R )
eth yl).53 To a stirred mixture of sulfoxide 32a (2.0 g, 10 mmol)
and NaI (3.7 g, 25 mmol) in acetone at 0 °C was added slowly
trifluoroacetic anhydride (4.2 mL, 30 mmol) in acetone (25 mL).
The reaction mixture was stirred at 0 °C (1 h) after which time
acetone was evaporated. Water was added to the resulting
mixture which was then extracted with ether. The ether
extracts were washed (water, saturated Na2S2O3, water, and
brine). After drying (MgSO4), the solvent was evaporated, and
the dark red oil obtained was subjected to flash chromatog-
raphy (silica gel, hexane). Hexane was evaporated to give the
sulfide 33a (1.8 g, 98%) as a light yellow oil: 1H NMR (DCCl3)
δ 1.07 [t, 3J HCCH ) 7.3 Hz, 3 H, CH3], 1.65-1.85 [m, 3 H, CH2-
1
3
(CdO) cm-1; H NMR (DCCl3,) δ 0.9 [t, J HCCH ) 0.7 Hz, 3 H,
CH3], 1.35-1.45 [m, 14 H, (CH2)7CH3 ], 2.0 [m, 1 H, H(3)], 2.3
[m, 1 H, H(3)], 2.7 [s, 3 H, CH3C(O)], 2.9-3.3 [m, 3 H, H(2)
and H(4)], 7.2-7.7 [m, 3 H, ArH]; 13C NMR (DCCl3) δ 14.07
[CH3]; aliphatic-C: 22.62, 22.90, 25.11, 27.99, 29.37, 29.42,
29.51, 29.62, 31.82, 34.19, 37.67; ArC: 126.29, 126.34, 129.56,
132.67, 137.75, 139.89; 197.42 [C(O)].
2-E t h yl-r-m et h ylt h ioch r om a n -6-m et h a n ol (35a , R )
eth yl). To a stirred suspension of LiAlH4 (0.39 g, 10 mmol)

Biologically Active Heteroarotinoids
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3579
in dry ether (10 mL) was added a solution of ketone 34a (1.5
g, 7 mmol) in dry ether (15 mL). The resulting mixture was
boiled (6 h). It was then cooled (0 °C), and ethyl acetate (25
mL) was slowly added followed by 5% HCl (10 mL). The new
mixture was stirred (5 min). The aqueous layer was extracted
with ether (3 × 25 mL). Combined organic layers were washed
with saturated NaHCO3, water, and brine. After drying
(MgSO4), the solvent was evaporated to an orange oil which
was separated on a silica gel column (hexane:ether, 1:1) to give
alcohol 35a (1.3 g, 86%) as a yellow oil. Alcohol 35a was used
give sulfide 37a as a yellow solid. Recrystallization (ether)
produced 37a as a white solid (2.3 g, 91%): mp 63-64 °C; IR
1
(neat) 1750 cm-1 (CdO); H NMR (DCCl3) δ 1.05 [t, J ) 7.4
Hz, 3 H, CH2CH3], 1.4 [t, J ) 7.3 Hz, 3 H, OCH2CH3], 1.6-
1.85 [m, 3 H, CH2CH3 and H(3)], 2.25-2.35 [m, 4 H, H(3) and
H(10)], 2.8-2.9 [m, 2 H, H(4)], 3.2-3.3 [m, 1 H, H(2)], 4.4 [q,
J ) 7.3 Hz, 2 H, OCH2CH3], 6.8 [s, 1 H, H(11)], 7.05-8.15[m,
7 H, ArH]; 13C NMR (DCCl3) δ 11.49 [CH2CH3]; aliphatic-C:
14.39,17.54, 29.43, 29.47, 29.66, 43.98, 60.90 [C(19)]; ArC and
vinylic C: 124.11, 125.80, 126.38, 127.16, 127.33, 127.72,
129.09, 129.46, 133.18, 133.65, 139.02, 143.10, 166.14 [C(18)];
mass spectral (EI) data calcd for C23H26O2S m/ z 366.1653,
found 366.1650. Anal. Calcd for C23H26O2S: C, 75.38; H, 7.16.
Anal. (C23H26O2S‚0.2H2O) C, H.
1
directly to prepare salt 36a : IR (neat) 3350 (O-H) cm-1; H
3
NMR (DCCl3) δ 1.05 [t, J HCCH ) 7.4 Hz, 3 H, CH3], 1.4-1.5
3
[d, J HCCH ) 6.4 Hz, 3 H, CH3C(OH)], 1.6-1.8 [m, 3 H, H(3)
and CH3CH2 ], 1.9-2.05 [s, 1 H, OH], 2.2-2.3 [m, 1 H, H(3)],
2.8-2.9 [m, 2 H, H(4)], 3.15-3.25 [m, 1 H, H(2)], 4.75-4.85
[m, 3J HCCH ) 6.4 Hz, 1 H, CH3CH(OH)], 6.9-7.1 [m, 3 H, ArH];
13C NMR (DCCl3) δ 11.39 [CH3]; aliphatic-C: 24.92, 29.32,
29.36, 29.46, 43.76; 69.97 [CH3C(OH)]; ArC: 123.51, 123.59,
126.42, 126.61, 133.82, 141.72.
4,4-Dim eth yl-3,4-d ih yd r o-2H-1-ben zop yr a n -6-ca r boxy-
lic Acid (39). Ketone 3820,55 (2.0 g, 0.01 mol), ethanol (35 mL),
and Clorox (100 mL) were mixed, and the resulting mixture
was boiled for 24 h. The solution was cooled (ice bath), and
then Na2S2O5 (75 mL) was added cautiously, followed by
concentrated HCl (20 mL). The resulting mixture was vacuum
filtered and the white solid was recrystallized (95% ethanol)
to give 39 (1.31 g, 65%): mp 227.5-228.5 °C [lit.55 mp 227-
2-n -Octyl-r-m eth ylth ioch r om a n -6-m eth a n ol (35b, R )
n -octyl). A solution of the ketone 34b (1.5 g, 6 mmol) in dry
ether (15 mL) was added to a stirred suspension of LiAlH4
(0.38 g, 9 mmol) in dry ether (10 mL). This mixture was
heated at reflux (6 h). It was then cooled to 0 °C, and ethyl
acetate (25 mL) was added slowly followed by 5% HCl (10 mL).
The mixture was stirred (5 min), and the aqueous layer was
extracted (ether). The organic layer and extracts were com-
bined and washed (saturated NaHCO3, water, and brine). After
drying (MgSO4), the solvent was evaporated to an orange oil
which was separated on a silica gel column (hexane:ether, 1:1)
to give alcohol 35b (1.2 g, 80%) as a yellow oil and which was
1
229 °C]; IR (KBr) 3600-2595 (CO2H), 1685 (CdO) cm-1; H
NMR (DCCl3) δ 1.38 [s, 6 H, C(CH3)2], 1.86 [t, 2 H, J ) 6 Hz,
CCH2], 4.27 [t, 2 H, J ) 6 Hz, CH2O], 6.83 [d, 1 H, Ar-H], 7.82
[dd, 1 H, Ar-H], 8.07 [d, 1 H, Ar-H]. 13C NMR (DCCl3) δ 30.59
[(CH3)2], 30.75 [ArCCH2], 36.49 [CCH2], 63.49 [CH2O], 117.1-
158.5 [Ar-C], 171.8 [CdO].
N-(4-Br om op h en yl)-3-m eth yl-2-bu ten a m id e (41). To
4-bromoaniline (40) (6.13 g, 0.035 mol) in 225 mL of HCCl3
was added slowly 3,3-dimethylacryloyl chloride (2.06 g, 0.017
mol) in HCCl3 (10 mL). The resulting cloudy, tan mixture was
then boiled for 5 h. After the mixture was cooled to room
temperature, the precipitate that formed was removed (vacuum
filtration). The solution was then washed (2 M HCl, saturated
NaHCO3, and brine). After drying (MgSO4), the solution was
concentrated in vacuo. Recrystalliztion (95% ethanol) of the
product gave off-white, flaky crystals of 41 (7.7 g, 87%): mp
118-119 °C; IR (KBr) 3320 (N-H), 1680 (CdO) cm-1; 1H NMR
(DCCl3) δ 1.88 [s, 3 H, CH3 (cis)], 2.21 [s, 3 H, CH3 (trans)],
5.69 [t, 1 H, J ) 1.2 Hz, CdCH], 7.26-7.45 [m, 4 H, Ar-H];
13C NMR (DCCl3) δ 19.99 [CH3 (cis)], 27.41 [CH3 (trans)],
116.40-137.30 [Ar-C, 118.29 [CdCH], 154.22 [(CH2CdCH)],
165.00 [CdO]; MS (FAB) calcd for C11H13BrNO 254, found 255
(MH+). Anilide 41 was used at once to make lactam 42.
6-Br om o-4,4-d im et h yl-2-oxo-1,2,3,4-t et r a h yd r oq u in o-
lin e (42). Anilide 41 (2.01 g, 7.94 mmol) was placed in a flask
which was then heated to 130-140 °C. Then AlCl3 (1.57 g
0.012 mol) was added portionwise over 1 h. The flask was
allowed to cool to 80 °C, and one final portion of AlCl3 (0.2 g)
was slowly added. The mixture was then stirred at that
temperature (0.5 h). After the mixture was cooled to room
temperature, 20 mL of ice water was cautiously added to the
dark brown solid. Chloroform (50 mL) was then added to
dissolve the solid. The solution was then stirred (15 min), and
10 mL of 2 M HCl was added. The aqueous layer was
extracted with HCCl3. The combined organic layers were
washed (saturated NaHCO3 and brine). After drying (MgSO4),
the solution was concentrated in vacuo to give 42 (1.57 g, 78%)
as a crude orange solid. Recrystallization (95% ethanol) gave
white crystals of 42 (1.31 g, 65%): mp 174-175 °C; IR (KBr)
3240 (N-H), 1700 (CdO) cm-1; 1H NMR (DCCl3) δ 1.6 [s, 6 H,
(CH3)2], 2.48 [s, 2 H, CH2CdO], 6.7-7.40 [m, 3 H, Ar-H]. 13C
NMR (DCCl3) δ 27.47 [CH3], 27.50 [CH3], 34.11 [ArCCH2],
44.86 [CH2], 116.15-134.99 [Ar-C], 171.15 [CdO]; MS (FAB)
calcd for C11H13BrNO 254, found 255 (MH+). Lactam 42 was
reduced at once to 43.
converted immediately to salt 36b: IR (neat) 3350 (O-H) cm-1
;
1H NMR (DCCl3) δ 0.9 [t, J HCCH ) 0.7 Hz, 3 H, CH3], 1.1-1.6
[m, 17 H, (CH2)7CH3 and CH3C(OH)], 1.75-1.95 [m, 2 H, OH
and H(3)], 2.0-2.2 [m, 1 H, H(3)], 2.8 [m, 2 H, H(4)], 3.1-3.15
[m, 1 H, H(2)], 4.65-4.8 [m, 1 H, CH3CH(OH ], 6.9-7.1 [m, 3
H, ArH]; 13C NMR (DCCl3) δ 14.07 [CH3]; aliphatic-C: 22.62,
22.70, 25.68, 25.70, 27.11, 29.27, 29.55, 29.64, 31.83, 34.34,
37.75; 70.05 [CH3C(OH)]; ArC: 123.540, 123.62, 126.50,
126.92, 126.98, 139.08, 141.01.
3
1-[(2-Eth ylth ioch r om a n -6-yl)eth yl]tr ip h en ylp h osp h o-
n iu m Br om id e (36a , R ) eth yl). A solution of alcohol 35a
(1.3 g, 6 mmol) and triphenylphosphine hydrobromide (2.0 g,
6 mmol) in H2CCl2 (50 mL) was stirred (room temperature,
24 h). After concentration, the resulting orange oil was
triturated (dry ether) to give phosphonium salt 36a (3.0 g, 96%)
as a slightly hygroscopic, white solid which was used at once
to prepare 37a .
1-[(2-n -Oct ylt h ioch r om a n -6-yl)et h yl]t r ip h en ylp h os-
p h on iu m Br om id e (36b, R ) n -octyl). A solution of alcohol
35b (4.4 g, 14 mmol) and triphenylphosphonium hydrobromide
(5.0 g, 14 mmol) in H2CCl2 (50 mL) was stirred (room
temperature, 24 h). The solvent was evaporated, and the oil
obtained was triturated (room temperature, dry ether) to
obtain 36b as a white solid (9.0 g, 98%), mp 67-68 °C. Salt
36b was used directly to prepare heteroarotinoid 37b: 1H
NMR (DCCl3) δ 0.9 [t, 3J HCCH ) 6.6 Hz, 3 H, CH3], 1.1-1.6 [m,
14 H, (CH2)7CH3], 1.7-1.95 [m, 4 H, CH3CH and H(3)], 2.0-
2.2 [m, 1 H, H(3)], 2.5-2.7 [m, 1 H, H(4)], 2.8 [m, 1 H, H(4)],
3.0-3.1 [m, 1 H, H(2)], 6.55 [m, 1 H, CH3CH], 6.9-7.1 [m, 3
H, ArH]; 13C NMR (DCCl3) δ 14.15 [CH3]; aliphatic-C: 16.89,
17.29, 22.68, 26.74, 26.95, 29.36, 29.44, 29.50, 29.74, 31.9,
34.12, 37.55 [CH3C(H)]; ArC: 117.42, 117.52, 118.24, 118.33,
126.88, 128.55, 128.67, 129.26, 129.36, 130.04, 130.08, 130.12,
130.16, 130.21, 130.24, 132.11, 132.21, 133.82, 133.97, 134.64,
134.67, 134.73, 134.76, 134.82.
Eth yl (E)-4-[2-(3,4-Dih ydr o-2-eth yl-1-oxy-2H-1-ben zoth i-
op yr a n -6-yl)-1-p r op en yl]ben zoa te (37a ). A mixture of 36a
(3.9 g, 11 mmol), K2CO3 (1.5 g, 11 mmol), and 18-C-6 (30 mg)
in H2CCl2 (25 mL) was boiled (2 h). Ethyl 4-formylbenzoate
(1.7 g, 10 mmol) was added rapidly to the mixture. After
boiling (12 h), the resulting mixture was concentrated to an
orange oil which was diluted with hexane. A suspension
formed and was filtered, and the filtrate was washed (brine)
and dried (Na2SO4). Concentration left a yellow oil which was
chromatographed on silica gel (hexane:ethyl acetate, 1:1) to
6-Br om o-4,4-d im eth yl-1,2,3,4-tetr a h yd r oqu in olin e (43).
Lactam 42 (1.0 g, 3.95 mmol) in 10 mL of dry, distilled toluene
was cooled (ice bath; 0-4 °C). The borane-dimethyl sulfide
(0.31 g, 4.10 mmol, 5% excess) complex was added dropwise.
The reaction mixture was stirred at 0 °C (15 min) and then
was boiled (5 h). After being cooled to room temperature, the
solution was treated with 10% Na2CO3 (15 mL). The solution
was then stirred at room temperature (30 min). The organic
layer was separated, dried (MgSO4), and concentrated in vacuo

3580 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Benbrook et al.
to give 43 (0.85 g, 90%, light yellow oil) which was used
immediately to make 44: IR (neat) 3420 (N-H) cm-1; 1H NMR
(DCCl3) δ 1.27 [s, 6 H, (CH3)2], 1.70 [t, 2 H, J ) 1.2 Hz, CCH2],
3.29 [t, 2 H, J ) 1.2 Hz, CH2N], 6.33 [d, 1 H, Ar-H], 7.01 [dd,
1 H, Ar-H], 7.24 [d, 1 H, Ar-H]; 13C NMR (DCCl3) δ 30.71 [CH3],
30.75 [CH3], 31.92 [ArCCH2], 36.65 [CCH2], 38.24 [CH2N],
108.39-142.53 [Ar-C]; MS (FAB) calcd for C11H14BrN 239,
found 239.
RARE-tk-CAT reporter plasmid, was used to evaluate activa-
tion of the heteroarotinoids via endogenous nuclear receptors.
A concentration of 4 × 105 cells per well was used to inoculate
6-well culture dishes containing Eagles Minimal Essential
Media (contains Earle’s Salts and L-glutamine via Cellgro
Mediatech, Herndon, VA) supplemented with nonessential
amino acids, sodium pyruvate, and 10% fetal bovine serum
(FBS). Only lots of FBS that contained negligible quantities
of t-RA (1), as determined by HPLC analysis, were used.
Triplicate cultures were treated with 1000× stocks (DMSO)
of the agents. The final concentration in DMSO in control and
treated cultures was 0.1%. After 48 h, cell extracts were
prepared and CAT activity was assayed as previously de-
scribed57 with the exception that [3H]acetyl-CoA was used
instead of [14C]acetyl-CoA. Protein concentrations of the
extracts were determined using the Bio-Rad protein assay.77
Transactivation activities were derived by dividing the average
of the CAT activity per milligram of protein in the drug-treated
culture by that in the control culture. Relative transactivation
activity was derived by dividing the activities observed in the
heteroarotinoid-treated cultures by that in cultures treated
with t-RA (1) (Table 1). The results, presented as percentages
in Table 1, are the average of three experiments.
6-Br om o-N,4,4-t r im et h yl-1,2,3,4-t et r a h yd r oq u in olin e
(44). The quinoline 43 (2.5 g 0.01 mol), NaHCO3 (1.51 g 0.018
mol), and H2O (2 mL) were mixed in a flask and cooled (15-
18 °C). Dimethyl sulfate (1.64 g 0.013 mol) was added
dropwise. The cloudy light yellow mixture was stirred at room
temperature until the evolution of CO2 ceased (∼1 h). The
solution was then heated to and maintained at 50 °C until all
CO2 ceased to be released (∼1 h). After being cooled to room
temperature, the solution was diluted with chloroform (25 mL),
and the layers were separated. Chloroform extracts of the
aqueous layer and the organic layer were combined and then
washed (H2O and brine). After drying (MgSO4), the solution
was concentrated in vacuo. The product was then cooled to 0
°C overnight during which time solid impurities formed. The
mixture was vacuum filtered and washed with hexane. Con-
centration in vacuo gave the N-methylated product 44 as a
yellow oil (2.18 g, 86%), which was used directly to make 45:
1H NMR (DCCl3) δ 1.25 [s, 6 H, C(CH3)2], 1.73 [t, 2 H, J ) 6
Hz, CCH2], 2.86 [s, 3 H, NCH3], 3.20 [t, 2 H, J ) 6 Hz, CH2N],
6.42 [d, 1 H, Ar-H], 7.13 [dd, 1 H, Ar-H], 7.23 [d, 1 H, Ar-H];
13C NMR (DCCl3) δ 30.70 [(CH3)2], 32.30 [ArCCH2], 36.88
[CCH2], 39.23 [CH2N], 47.48 [NCH3], 107.94-144.29 [Ar-C];
MS (FAB) calcd for C12H16BrN 253, found 253.
Gr ow th Ra te. Six-well tissue culture dishes were inocu-
lated with a concentration of 4 × 104
CC-B cells per well.
Twenty-four hours after plating, the media was replenished,
and triplicate cultures were treated with retinoids and het-
eroarotinoids as described above. The media and agents were
replenished every 2 days. After 7 days, the number of cells
per well in the treated and control cultures were determined
using a Particle Counter (Coulter ZM). Percent growth rate
was determined by dividing the average of the number of cells
in the treated culture by that in the control culture and
multiplying by 100. The results presented in Figure 1 (panel
A) are the average of three separate experiments.
N,4,4-Tr im eth yl-1,2,3,4-tetr a h yd r oqu in olin e-6-ca r box-
ylic Acid (45). To 1.6 M n-BuLi (4.90 mL, 784 mmol) in THF
was added dropwise the tetrahydroquinoline 44 (1.0 g, 4.11
mmol) in ether (5 mL). The solution was stirred at room
temperature (24 h). The cloudy orange mixture was then
poured over solid CO2 in excess ether. The mixture was stirred
until it warmed to room temperature (∼2 h), and then H2O
(20 mL) was added. The layers were separated, and the
aqueous layer was acidified (2 M HCl, pH ∼3). The resulting
mixture was extracted (CH2Cl2), and the extracts and organic
layer were combined and dried (MgSO4). The solution was
concentrated in vacuo, and the resulting solid was recrystal-
lized (95% ethanol). White flaky crystals (0.270 g, 30%) of the
carboxylic acid 45 were obtained: mp 223-225 °C; IR (KBr)
Gen er a l Meth od for Assa yin g Tr a n sglu ta m in a se Ac-
tivity. Human erythroleukemia cells (GMO6141A, Human
Genetic Mutant Cell Respository, Camden, NJ ) were inocu-
lated into T-25 flasks (Corning, Corning, NY) containing
McCoy’s Medium 5a (GIBCO/BRL, Grand Island, NY) supple-
mented with 10% fetal bovine serum (Intergen, Purchase, NY)
at a density of 2 × 105 cells/mL. The heteroarotinoids were
dissolved either in ethanol or DMSO. Twenty-four hours after
subculture, the cells were treated with a 10 µM solution of
either t-RA (1), the vehicle alone (control), or the heteroaro-
tinoid. The t-RA (1; Sigma, St. Louis, MO) was added to one
flask in all experiments to ensure that transglutaminase
induction occurred, and it was also used as the standard to
which the heteroarotinoid induction was compared. Cultures
were covered with aluminum foil to protect them from light
and then incubated at 37 °C (48 h). Cells ((10-40) × 106) were
collected by centrifugation at 2000 rpm (5 min). The resulting
cell pellet was resuspended in 10 mL of Ca,Mg-free Earles
solution, and the cell suspension was centrifuged at 2000 rpm
(3 min). These cells were twice washed again. The final
washed cell pellet was resuspended in 1 mL of Tris-buffered
saline (150 mM NaCl-1 mM EDTA-20 mM Tris-HCl, pH 7.4),
and the cell suspension was disrupted by a 10-s sonication.
Cell lysates were quantitated for trans-glutaminase activity
3500-2400 (CO2H) cm-1 1H NMR (DCCl3) δ 1.30 [s, 6 H,
;
C(CH3)2], 1.75 [t, 2 H, J ) 6 Hz, CCH2], 3.00 [s, 3 H, NCH3],
3.70 [t, 2 H, J ) 6 Hz, CH2N], 6.53 [d, 1 H, Ar-H], 7.82 [dd, 1
H, Ar-H], 7.92 [d, 1 H Ar-H]; 13C NMR (DCCl3) δ 30.03
[C(CH3)2], 31.88 [ArCCH2], 36.22 [CCH2], 38.99 [CH2N], 47.49
[NCH3], 109.44-149.25 [Ar-C], 171.95 [CdO]; MS (FAB) calcd
for C13H17NO2 219, found 219. Acid 45 was converted at once
to ester 19.
4-[[(4,4-Dim eth yl-3,4-d ih yd r o-2H-ben zo[b]p yr a n -6-yl)-
ca r bon yl]oxy]ben za ld eh yd e (46). To a mixture of the
carboxylic acid 39 (0.300 g, 1.45 mmol) and 4-formylphenol
(0.265 g, 2.17 mmol) in CH2Cl2 (20 mL) was added dicyclo-
hexylcarbodiimide (∼0.895 g, 4.33 mmol) and a catalytic
amount of DMAP (10 mg). The resulting cloudy solution was
stirred at room temperature (48 h) and then filtered. The
filtrate was cooled to 0 °C (24 h) and filtered again. Evapora-
tion of the solvent in vacuo gave an oil which was allowed to
stand at room temperature (24 h), during which time crystal-
lization took place. Recrystallization (hexane:EtOAc, 3:1) gave
solid 46 (0.195 g, 43%): mp 132-133.5 °C; IR (KBr) 2850
by measuring the covalent incorporation of
14C-labeled pu-
trescine into dimethylcasein in a calcium-dependent manner
as previously described.78 Protein was estimated using the
Bio-Rad procedure.77 The values for the TGase activity were
determined as the activity/mg of protein in the treated cultures
divided by that in solvent control cultures and represent the
mean of duplicate assays performed on replicate samples.
Figure 1 plots such activity versus RARE transactivation
(panel B).
1
((O)C-H), 2724, 1700, 1730 (CdO) cm-1; H NMR (DCCl3) δ
1.40 [s, 6 H, C(CH3)2], 1.87 [t, 2 H, J ) 5.6 Hz, CCH2], 4.30 [t,
2 H, J ) 5.6 Hz, CH2O], 6.88 [d, 1 H, Ar-H], 7.39 [d, 2 H, Ar-
H], 7.91 [d, 1 H, Ar-H], 7.97 [d, 2 H, Ar-H], 8.13 [d, 1 H, Ar-
H], 10.02 [s, 1 H, C(O)H]; 13C NMR (DCCl3) δ 30.61 [(CH3)2],
36.86 [ArCCH2], 63.54 [ArOCH2], 117.39-158.81 [Ar-C], 164.47
[CdO], 191.07 [CdO]; MS (FAB) calcd for C15H19O4 310, found
311 (MH+). The aldehyde 46 was converted immediately to
the corresponding thiosemicarbazone 20.
Ma ter ia ls a n d Meth od s for th e Toxicity Stu d ies. The
syntheses of 8, 10, and 13 were recorded previously.20,49
A
sample of TTNPB (5) was kindly provided by Dr. Peter F.
Sorter, Hoffmann-La Roche, Inc. Qualitative checks for purity
using high-pressure liquid chromatography (column, Spher-
isorb, ODS, 5 µm; Solvents, acetonitrile; 1% ammonium acetate
buffer, 85:15; wavelength, 254 and 340 nm; flow, 1 mL/min;
concentration, 1 mg/mL H3CCN) and melting point determina-
Tr a n sa ctiva tion Assa y. The CC-B cervical tumor cell
line,57 which contains stabilized integrated copies of RARâ-

Biologically Active Heteroarotinoids
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3581
tions indicated 10 to be 99.8-100% pure and 8 and 13 to be
100% pure. When not in use, the compounds were stored in
sealed amber vials under a nitrogen atmosphere at -70 °C.
All compounds were weighed, and dosage formulations were
prepared in subdued light. After the compounds were formu-
lated in corn oil for dosing, the solutions were stored at room
temperature in sealed amber vials under a nitrogen atmo-
sphere.
Male B6D2F1 mice, 4 weeks of age, were obtained from
Charles River Laboratories (Raleigh, NC). Mice were quar-
antined for a minimum of 7 days prior to initiation of dosing.
Mice were also housed individually in suspended 73/4 in. × 105/8
in. × 51/16 in. solid-bottom polycarbonate cages fitted with
spun-bonded polyester fiber filter. Heat-treated hardwood
ships were used as bedding. Feed (NIH-07 open formula) and
city tap water (Edstrom automatic watering system) were
administered ad libitum. A light/dark cycle of 12 h light and
12 h dark was used. The optimal temperature and relative
humidity ranges for the animal room were 69-75 °F and 35-
65%, respectively. All animal care procedures conformed to
the guidelines found in Guide for the Care and Use of
Laboratory Animals, DHEW Publication No. (NIH)85-23 (Re-
vised 1985).
Agricultural Experiment Station, and supported under
project H-1250. This investigation was also supported
in the prior years by contract N01 CP 41005, National
Cancer Institute, National Institutes of Health, DHS,
in a contract to the Southern Research Institute (C.L.,
D.L.H.). We gratefully acknowledge the encouragement
and support of Dr. Carl Smith of the National Cancer
Institute of the National Institutes of Health. Funds for
the 400 MHz NMR spectrometer of the Oklahoma
Statewide Shared NMR Facility were provided by the
National Science Foundation (BIR-9512269), the Okla-
homa State Regents for Higher Education, the W. M.
Keck Foundation, and Conoco, Inc.
Refer en ces
(1) Moon, R. C.; Itri, L. M. Retinoids and Cancer. In The Retinoids;
Sporn, M. B., Roberts, A. B., Goodman, D. S., Eds.; Academic
Press: Orlando, FL, 1984; Vol. 2, pp 327-371. See also Volume
1.
(2) Hill, D. L.; Grubbs, C. J . Retinoids and Cancer Prevention. Annu.
Rev. Nutr. 1992, 12, 161-181.
Groups of 16 animals each were dosed daily by gavage (10
mL/kg) with either corn oil (vehicle 1) or 0.1, 0.2, 0.4 or 0.8
mg/kg of each retinoid. Due to overt toxicity at these dose
levels in the TTNPB groups, groups of eight animals each were
added to the study and dosed with either corn oil (vehicle 2),
0.01 or 0.05 mg/kg of TTNPB (5). Due to the lack of toxicity,
dose levels in the 8, 10, and 13 groups were increased 4-fold
on day 15, 10-fold on day 29, and 2-fold (8 and 10 only) on day
51. The study was terminated on day 65 for all three
compounds and on day 25 for the vehicle 2, 0.01 and 0.05 mg/
kg TTNPB groups. The toxicity data and effects induced by
t-RA (1) have been recorded independently on many occasions,
are well-known69-71 to be approximately 10 mg/kg/day for
many organs, are entirely reproducible, and are thus not
included herein.
Body weights and clinical signs were measured three times
weekly. On study days 8, 15, 29, 36, and at terminal sacrifice
(day 8 and at terminal sacrifice for the vehicle 2, 0.01 and 0.05
mg/kg groups), blood was collected from each animal from the
retro-orbital sinus under CO2 anesthesia. Portions were
placed in tubes containing potassium EDTA for hematology
analyses or in tubes without anticoagulant for alkaline phos-
phatase, cholesterol, and triglyceride analyses. Serum was
collected after centrifugation at 2000g for 2 min. Hematology
analyses were performed with an Ortho ELT-8 automated
hematology analyzer, which uses a laser light source to detect
RBC, WBC, and platelets. Hematocrit values were based upon
erythrocyte counts, and hemoglobin was measured by the
colorimetric determination of cyanomethemoglobin. Serum
chemistries were performed with a Centrifichem 500 auto-
mated centrifugal analyzer, which used test-specific reagent
kits obtained from the manufacturer and provided quantita-
tive, spectrophotometric analysis.
(3) Nadzan, A. M. Retinoids for the Treatment of Oncological
Diseases. Annu. Rep. Med. Chem. 1995, 30, 119-128.
(4) Motzer, R. J .; Schwartz, L.; Murray-Law, T.; Murphy, B. A.;
Hoffman, A. D.; Albino, A. P.; Vlamis, V.; Nanus, D. M.
Interferon Alfa-2a and 13-cis-Retinoic Acid in Renal Cell
Carcinoma: Antitumor Activity in Phase II Trial and Interac-
tions in Vitro. J . Clin. Oncol. 1995, 13, 1950-1957.
(5) Lippman, S. M.; Heyman, R. A.; Kurie, J . M.; Benner, S. E.;
Hong, W. K. Retinoids and Chemo-prevention: Clinical and
Basic Studies. J . Cell. Biochem. 1995, 22(S), 1-10.
(6) Kramer, K. H.; Digiovanna, J . J .; Moshell, A. N.; Tarone, R. E.;
Peck, G. L. Prevention of Skin Cancer in Xeroderma Pigmento-
sum with the Use of Oral Isotretinion. N. Engl. J . Med. 1988,
318, 1633-1637.
(7) Hong, W. K.; Endicott, J .; Itri, L. M.; Doos, W.; Batsakis, J . G.;
Bell, R.; Fofonoff, S.; Byers, R.; Atkinson, E. N.; Vaughan, C.;
Toth, B. B.; Kramer, A.; Dimery, I. W.; Skipper, I. W. S.; Strong,
S. 13-cis-RA in the Treatment of Oral Leukoplakia. New Engl.
J . Med. 1986, 315, 1501-1505.
(8) Hong, W. K.; Lippman, S. M.; Itri, L. M.; Karp, D. D.; Lee, J . S.;
Byers, R. M.; Schantz, S. P.; Kramer, A. M.; Lotan, R.; Peters,
L. J .; Dimery, I. W.; Brow, B. W.; Goefert, H. Prevention of
Secondary Tumors with Isotretinoin in Squamous Cell Carci-
noma in the Head and Neck. N. Engl. J . Med. 1990, 323, 795-
801.
(9) Kim, Y.-H.; Dohi, D. F.; Han, G. R.; Zou, C.-P.; Oridate, N.;
Walsh, G. L.; Nesbitt, J . C.; Xu, X. C.; Hong, W. K.; Lotan, R.;
Kurie, J . M. Retinoid Refractoriness Occurs During Lung
Carcinogenesis Despite Functional Retinoid Receptors. Cancer
Res. 1995, 55, 5603-5610.
(10) Sporn, M. B., Roberts, A. B., Goodman, D. S., Eds. The Retinoids-
Biology, Chemistry, and Medicine, 2nd ed.; Raven Press: New
York, 1994.
(11) Huang, M. E.; Ye, Y. C.; Chen, S. R.; Chair, J . R.; Lu, J . X.; Zhoa,
L.; Gu, L. J .; Wang, Z. Y. Use of All-trans-Retinoic Acid in the
Treatment of Acute Promyelocytic Leukemia. Blood 1988, 72,
567-572.
(12) Castaigne, S.; Chomienne, C.; Daniel, M. T.; Ballerini, P.; Berger,
All animals were necropsied, with 43 tissues from each being
animal examined for gross observations of pathologic effects.
Each carcass was X-rayed (40 kV, 3 mA, 2 min). At study
termination, the organs weighed included brain, liver, right
kidney, thymus, heart, lungs, right testicle, and spleen. The
method of euthansia was CO2 asphyxiation. Analysis of
variance, with comparisons of means by Students’ t-test, was
performed. Differences were considered significant if the value
for p was e0.05.
R.; Fenaux, P.; Degos, L. All-trans-Retinoic Acid as
a Dif-
ferentiation Therapy for Acute Promyelocytic Leukemia. I.
Clinical results. Blood 1990, 76, 1704-1709.
(13) Warrell, R. P., J r.; Frankel, S. R.; Miller, W. H., J r., Scheinberg,
D. A.; Itri, L. M.; Hittleman, W. N.; Vyas, R.; Andreff, M.; Tafuri,
A.; J akubowski, A. Differentiation Therapy of Acute Promyelo-
cytic Leukemia with Tretinoin (All-trans-Retinoic acid). N. Engl.
J . Med. 1991, 324, 1385-1393.
(14) Chen, A. X.; Xue, Y. Q.; Zhang, R.; Tao, R. F.; Xia, X. M.; Li, C.;
Wang, W.; Zu, W. Y.; Yao, X. Z.; Ling, B. J . A Clinical and
Experimental Study on All-trans-Retinoic Acid-Treated Acute
Promyelocytic Leukemia Patients. Blood 1991, 78, 1413-1419.
(15) Sun, G. L.; Huang, Y. G.; Chang, X. F.; J iang, G. S.; Zhang, T.
Clinical Study on All-trans-Retinoic Acid in Treatment of 544
Cases of Acute Promyelocytic Leukemia Treated. Chin. J .
Hematol. 1992, 13, 135-137.
(16) Heyman, R. A.; Mangelsdorf, D. J .; Dyck, J . A.; Stein, R. B.;
Eichele, G.; Evans, R. M. 9-cis-Retinoic Acid is a High Affinity
Ligand for the Retinoid X Receptor. Cell 1992, 68, 397-406.
(17) Miller, V. A.; Rigas, J . R.; Benedetti, F. M.; Verret, A. L.; Tong,
W. P.; Kris, M. G.; Gill, G. M.; Loewen, G. R.; Truglia, J . A.;
Ulm, E. H.; Warrell, R. P., J r., Initial Clinical Trials of the
Ack n ow led gm en t. We (E.C.N., K.D.B., D.M.B.,
P.J .B.) gratefully acknowledge partial support by a
grant from the National Institutes of Health, National
Cancer Institute (CA 73639-01), a grant (94A63) from
the American Institute for Cancer Research (DMB), by
the College of Arts and Sciences (K.D.B.), and by
Oklahoma Agricultural Experiment Station at Okla-
homa State University (E.C.N.). The research was
approved for publication by the Director, Oklahoma

3582 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Benbrook et al.
Retinoid Receptor Panagonist 9-cis-Retinoic Acid. Clin. Cancer
Res. 1996, 2, 471-475.
(18) Costa, A.; Formelli, F.; Chiesa, F.; Decensi, A.; De Palo, G.;
Veronesi, U. Prospects of Chemoprevention of Human Cancers
with the Synthetic Retinoid Fenretinide. Cancer Res. 1994, 54
(S), 2032s-2037s.
(19) (a) Loeliger, P.; Bollag, W.; Mayer, H. Arotinoids, A New Class
of Highly Active Retinoids. Eur. J . Med. Chem. Chim. Ther.
1980, 15, 9-15. (b) Bollag, W. Retinoids and Cancer. Cancer
Chemother. Pharmacol. 1979, 3, 207-215.
(37) Davies, P. J . A.; Chiocca, E. A.; Stein, J . P. Retinoid-regulated
Expression of Tissue Transglutaminase in Normal and Leukemic
Myeloid Cells. Adv. Exp. Med. Biol. 1988, 231, 63-71.
(38) Yuspa, S. H.; Brent, T.; Lichti, U. Regulation of Epidermal
Transglutaminase Activity and Terminal Differentiation by
Retinoids and Phorbol Esters. Cancer Res. 1983, 43, 5707-5712.
(39) Leu, R. W.; Herriott, M. J .; Moore, P. E.; Orr, G. R.; Birckbichler,
P. J . Enhanced Transglutaminase Activity Associated with
Macrophage Activation. Possible Role in Fc-Mediated Phagocy-
tosis. Exp. Cell Res. 1982, 141, 191-199.
(20) Waugh, K. M.; Berlin, K. D.; Ford, W. T.; Holt, E. M.; Carrol, J .
P.; Schomber, P. R.; Thompson, M. D.; Schiff, L. J . Synthesis
and Characterization of Selected Heteroarotinoids. Pharmaco-
logical Activity as Assessed in Vitamin A Deficient Hamster
Tracheal Organ Cultures. Single Crystal X-Ray Diffraction
Analysis of 4,4-Dimethylthiochroman-6-yl Methyl Ketone l,l-
Dioxide and Ethyl (E)-p-[2-(4,4-Dimethylthiochroman-6-yl)pro-
penyl]benzoate. J . Med. Chem. 1985, 28, 116-124.
(21) Dawson, M. I.; Hobbs, P. D.; Derdzinski, K.; Chan, R. L.-S.;
Gruber, J .; Chao, W.-u.; Smith, S.; Thies, R. W.; Schiff, L. J .
Conformatinally Restricted Retinoids. J . Med. Chem. 1984, 27,
1516-1531.
(22) Giguere, V. Retinoic Acid Receptors and Cellular Retinoid
Dinding Proteins: Complex Interplay in Retinoid Signaling.
Endocrin. Rev. 1994, 15, 61-79.
(23) Petkovich, M. Regulation of Gene Expression by Vitamin A: The
Role of Nuclear Retinoic Acid Receptors. Annu. Rev. Nutr. 1992,
12, 443-471.
(24) Levin, A.; Sturzenbecker, L.; Kazmer, S.; Bosakowski, T.;
Huselton, C.; Allenby, G.; Speck, J .; Kratzeisen, C.; Rosenberger,
M.; Lovey, A.; Grippo, J . 9-cis-Retinoic Acid Stereoisomer Binds
and Activates the Nuclear Receptor RXRR. Nature 1992, 355,
359-361.
(25) Zhang, X.-K.; Lehmann, J .; Hoffman, B.; Dawson, M. I.; Cam-
eron, J .; Graupner, G.; Hermann, T.; Tran, P.; Pfahl, M.
Homodimer Formation of Retinoid X Receptor Induced by 9-cis-
Retinoic Acid. Nature 1992, 358, 587-591.
(26) Palmer, C. M.; Hsu, M. H.; Muerhoff, A. S.; Griffin, K. J .;
J ohnson, E. F. Interaction of Peroxisome Proliferatior-Activated
Receptor Alpha with the Retinoid X Receptor Alpha Unmasks a
Cryptic Peroxisome Proliferator Response Element That Over-
laps ARP-1 Binding Site in the CYP4A6 Promoter. J . Biol. Chem.
1994, 269, 18083-18089.
(27) Apfel, R.; Benbrook, D.; Lernhardt, E.; Ortiz, M. A.; Salbert, G.;
Pfahl, M. A Novel Orphan Receptor Specific for a Subset of
Thyroid Hormone-Responsive Elements and Its Interaction with
the Retinoid/Thyroid Hormone Receptor Subfamily. Mol. Cell.
Endocrin. 1994, 14, 7025-7035.
(28) Kliewer, S. A.; Umesono, K.; Heyman, R.; Mangelsdorf, D. J .;
Dyck, J . A.; Evans, R. M. Retinoid X Receptor-Coup-TF Interac-
tions Modulate Retinoic Acid Signaling. Proc. Natl. Acad. Sci.
U.S.A. 1992, 89, 1448-1452.
(29) Rosen, J .; Day, A.; J ones, T. K.; J ones, E. T. T.; Nadzan, A. M.;
Stein, R. B. Intracellular Receptors and Signal Transducers and
Activators of Transcription Superfamilies: Novel Targets for
Small-Molecule Drug Discovery. J . Med. Chem. 1995, 38, 4855-
4874.
(30) Boylan, J . F.; Lohnes, D.; Taneja, R.; Chambon, P.; Gudas, L. J .
Loss of Retinoic Acid Receptor Gamma Function in F9 Cells by
Gene Disruption Results in Aberrant Hoxa-1 Expression and
Differentiation Upon Retinoic Acid Treatment. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 9601-9605.
(31) Gianni, M.; Zanotta, S.; Terao, M.; Garattinni, S.; Garattini, E.
Effects of Synthetic Retinoids and Retinoic Acid Isomers on the
Expression of Alkaline Phosphatase in F9 Teratocarcinoma
Cells. Biochem. Biophys. Res. Commun. 1993, 196, 252-259.
(32) Suedhoff, T.; Birckbichler, P. J .; Lee, K. N.; Conway, E.;
Patterson, M. K., J r. Differential Expression of Transglutami-
nase in Human Erythroleukemia Cells in Response to Retinoic
Acid. Cancer Res. 1990, 50, 7830-7834.
(40) Birckbichler, P. J .; Patterson, M. K., J r. Modulation of Trans-
glutaminase-Catalyzed Isopeptide Bonds in Human Cells. In
Hemostasis and Cancer; Muszbek, L., Ed.; CRC Press: Boca
Raton, FL, 1987; pp 167-175.
(41) Greenberg, C. S.; Birckbichler, P. J .; Rice, R. H. Transglutami-
nases: Multifunctional Cross-Linking Enzymes that Stabilize
Tissues. FASEB J . 1991, 5, 3071-3077.
(42) Dawson, M. I.; Elstner, E.; Masahiro, K.; Chen, D.-L.; Pakkala,
S.; Kerner, B.; Koeffler, H. P. Myeloid Differentiation Mediated
Through Retinoic Acid Receptor/Retinoic X Receptor (RXR) not
RXR/RXR Pathway. Blood 1994, 84, 446-452.
(43) Zhang, L.-X.; Mills, K. J .; Dawson, M. I.; Collins, S. J .; J etten,
A. M. Evidence for the Involvement of Retinoic Acid Receptor
RARR-Dependent Signaling Pathway in the Induction of Tissue
Transglutaminase and Apoptosis by Retinoids. J . Biol. Chem.
1995, 270, 6022-6029.
(44) Boehm, M. F.; Zhang, L.; Zhi, L.; McClurg, M. R.; Berger, E.;
Wagoner, M.; Mais, D. E.; Suto, C. M.; Davies, P. J . A.; Heyman,
R. A.; Nadzan, A. M. Design and Synthesis of Potent Retinoid X
Receptor Selective Ligands that Induce Apoptosis in Leukemia
Cells. J . Med. Chem. 1995, 38, 3146-3155.
(45) Thorne, R. L. Z.; Rolf, R. L., J r.; Sunthankar, P.; Subramanian,
S.; Berlin, K. D.; Nelson, E. C.; Benbrook, D. M.; Birckbichler,
P. J . The Vitamin A Activity of Heteroarotinoids in Promoting
Epithelial Cell Differentiation as Evaluated by Vaginal-Smear
Assay in Rats. Unpublished results.
(46) Kagechika, H.; Himi, T.; Kawachi, E.; Shudo, K. Retinobenzoic
acids. 4. Conformation of Aromatic Amides with Retinoidal
Activity. Importance of trans-Amide Structure for the Activity.
J . Med. Chem. 1989, 32, 2292-2296.
(47) Eliel, E. L.; Wilen, S. H. Stereochemistry of Organic Compounds;
Wiley-Interscience: New York, 1994; pp 619-626.
(48) Berlin, K. D.; Subramanian, S.; Spruce, L. W.; Nelson, E. C.;
Thorne, R. L. Z.; Benbrook, D.; Birckbichler, P. J . Unpublished
results.
(49) Spruce, L. W.; Rajadhysksha, S. N.; Berlin, K. D.; Gale, J . B.;
Miranda, E. T.; Ford, W. T.; Blossey, E. C.; Verma, A. K.;
Hossain, M. B.; van der Helm, D.; Breitman, T. R. Heteroaro-
tinoids. Synthesis, Characterization, and Biological Activity in
Terms of an Assessment of These Systems to Inhibit the
Induction of Ornithine Decarboxylase Activity and to Induce
Terminal Differentiation of HL-60 Cells. J . Med. Chem. 1987,
30, 1474-1482.
(50) Subramanian, D. M.S. Thesis. Heteroarotinoids: Mimics of
Trans-Retinoic Acid; Oklahoma State University, 1990.
(51) Spruce, L. W.; Gale, J . B.; Berlin, K. D.; Verma, A. K.; Breitman,
T. R.; J i, X.; van der Helm, D. Novel Heteroarotinoids: Synthesis
and Biological Activity. J . Med. Chem. 1991, 34, 430-439.
(52) Collins, S. J .; Ruscetti, F. W.; Gallagher, R. E.; Gallo, R. C.
Normal Functional Characteristics of Cultured Human Promy-
elocytic Leukemia Cells (HL-60) After Induction of Differentia-
tion by Dimethyl Sulfoxide. J . Med Chem. 1979, 149, 969-974.
(53) Takata, T.; Mayuni, Y.; Fujimori, K.; Young, H. K.; Iyangi, T.;
Oae, S. Enzymatic Oxygenation of Sulfides with Cytochrome
P-450 from Rabbit Liver. Stereochemistry of Sulfoxide Forma-
tion. Bull. Chem. Soc. J pn. 1983, 56, 2300-2310.
(54) Pitchen, P.; Dunach, E.; Deshmukh, M. An Efficient Asymmetric
Oxidation of Sulfides to Sulfoxides. J . Am. Chem. Soc. 1984, 106,
8188-8193.
(55) Sunthankar, P. S.; Berlin, K. D.; Nelson, E. C.; Thorne, R. L.;
Geno, P. W.; Archer, J . C.; Rolf, L. L., J r.; Bartels, K. E.
Synthesis of Potential Metabolites of Ethyl (E)-4[2-(3,4-dihydro-
4,4-dimethyl-2H-benzopyran-6-yl)-1-propenyl]benzoate. J . Pharm.
Sci. 1993, 82, 543-545.
(33) Chiocca, E. A.; Davies, P. J .; Stein, J . P. The Molecular Basis of
Retinoic Acid. Transcriptional Regulation of Tissue Trans-
glutaminase Gene Expression in Macrophages. J . Biol. Chem.
1988, 263, 11584-11589.
(34) Davies, P. J . A.; Murtaugh, M. T.; Moore, W. T., J r.; J ohnson,
G. S.; Lucas, D. Retinoic Acid-Induced Expression of Tissue
Transglutaminase in Human Promyelocytic Leukemia (HL-60)
cells. J . Biol. Chem. 1985, 269, 5166-5174.
(35) Beard, R. L.; Chandraratna, R. A. S.; Colon, D. F.; Gillett, S. J .;
Henry, E.; Marler, D. K.; Song, T.; Denys, L.; Garst, M. E.;
Arefieg, T.; Klein, E.; Gil, D. W.; Wheeler, L.; Kochhar, D. M.;
Davies, P. J . A. Synthesis and StructuresActivity Relationships
of Stilbene Retinoid Analogs Substituted with Heteroaromatic
Carboxylic Acids. J . Med. Chem. 1995, 38, 2820-2829.
(36) Fesus, L.; Thomazy, V.; Falus, A. Induction and Activation of
Tissue Transglutaminase During Programmed Cell Death.
FEBS Lett. 1987, 224, 104-108.
(56) Wang, B.; Liu, S.; Borchardt, R. T. Development of a Novel
Redox-Sensitive Protecting Group for Amines Which Utilize a
Facilitated Lactonization Reaction. J . Org. Chem. 1995, 60, 539-
543.
(57) Benbrook, D. M.; Lu, S.; Flanagan, C.; Shen-Gunther, J .; Angros,
L. H.; Lightfoot, S. A. A Biological Assay for Activity and
Molecular Mechanism of Retinoids in a Cervical Tumor Cells.
Gyn. Oncol. 1997, 66, 114-121.
(58) Fong, C. J .; Sutkowski, D. M.; Braun, E. J .; Bauer, K. D.;
Sherwood, E. R.; Lee, C.; Koslowski, J . M. Effect of Retinoic Acid
on the Proliferation and Secretory Activity of Androgen-
Responsive Prostate Carcinoma Cels. J . Urol. 1993, 149, 1190-
1194.

Biologically Active Heteroarotinoids
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3583
(59) Lippman, S. M.; Meyskens, F. L. Retinoids in Nutrition and
Cancer Prevention: The Role of Micronutrients; Moon, T. E.,
Micozzi, M. S., Eds.; Marcel Dekker: New York, 1989; pp 243-
271.
(60) Pirisi, L.; Batova, A.; J enkins, G. R.; Hodam, J . R.; Creek, K. E.
Increased Sensitivity of Human Keratinocytes Immortalized by
Human Papillomavirus Type 16 DNA to Growth Control by
Retinoids. Cancer Res. 1992, 52, 187-193.
(71) Lindamood, C., III; Dillehay, D. L.; Lamon, E. W.; Giles, H. D.;
Shealy, Y. F.; Sani, B. P.; Hill, D. L. Toxicologic and Immunologic
Evaluations of N-(All-trans-Retinoyl)-DL-leucine and N-(All-
trans-Retinoyl)glycine. Toxicol. Appl. Pharamcol. 1988, 96, 279-
295.
(72) Wilson, S. R.; Phillips, L. R.; Pelister, Y.; Huffman, J . C.
Cyclobutene Derivatives as Isoprene Equivalents in Terpene
Synthesis. 3. Bicyclo[2.2.0]hexanes. J . Am. Chem. Soc. 1979, 101,
7373-7379.
(73) (a) Pople, J . A.; Schneider, W. G.; Bernstein, H. J . High
Resolution Nuclear Magnetic Resonance; Mc-Graw-Hill: New
York, 1959. (b) Ramarajan, K.; Berlin, K. D. A DNMR Study of
the Ring Reversal Process in 9-Methylene-7-oxa-1-thiaspiro[4.5]-
decan-8-one Derivatives. Proc. Okla. Acad. Sci. 1979, 59, 70-
73.
(74) (a) Daniele, R.; Montanari, F.; Hogeveen, H.; Maccagnani, G.
Neighboring Group Participation by Sulfinyl-Oxygen. Tetrahe-
dron Lett. 1964, 2685-2689. (b) Thompson, C. J .; Coleman, H.
J .; Hopkins, R. L.; Rall, H. T. Identification of Thiaindans in
Crude Oil by Gas-Liquid Chromatography, Desulfurization, and
Spectral Techniques. Anal. Chem. 1966, 38, 1562-1566.
(75) Martin, E. L. A Modification of the Clemmenson Method of
Reduction. J . Am. Chem. Soc. 1936, 58, 1438-1442.
(76) Drabowicz, J .; Oae, S. Mild Reduction of Sulfoxides with Trif-
luoroacetic Anhydride-Sodium Iodide System. Synthesis 1977,
404-405.
(61) Strickland, S.; Breitman, T. R.; Frickel, F.; Nurrenbach, A.;
Hadicke, E.; Sporn, M. B. Structure-Activity Relationships of a
New Series of Retinoidal Benzoic Acid Derivatives as Measured
by Induction of Differentiation of Murine F9 Teratocarcinoma
Cells and Human HL-60 Promyelocytic Leukemia Cells. Cancer
Res. 1983, 43, 5268-5272.
(62) Dawson, M. I., Okamura, W. H., Eds. Chemistry and Biology of
Synthetic Retinoids; CRC: Boca Raton, FL, 1990.
(63) Bollag, W.; Geiger, J .-M. In Retinoid Therapy; Cunliffe, W. J .,
Miller, A. J ., Eds.; MTP Press LTD: Lancaster, 1984; pp 1-7.
(64) Peck, G. L. Retinoids in Dermatology: An Interim Report. Arch.
Der. 1980, 116, 283-284.
(65) Hersh, J . H.; Danhauser, D. E.; Hand, M. E. Retinoic Acid
Embryopathy: Timing of Exposure and Effects on Fetal Devel-
opment. J . Am. Med. Assoc. 1985, 254, 909-910.
(66) Roenigk, H. H., J r. Retinoids. Cutis 1987, 39, 301-305.
(67) Steele, C. E.; Marlow, R.; Turton, J .; Hicks, R. M. In vitro
Teratogenicity of Retinoids. Br. J . Exp. Pathol. 1987, 68, 215-
223.
(68) Kochhar, D. M.; Penner, J . D. Developmental Effects of Isotre-
tinoin and 4-Oxoisotretinoin: The Role of Metabolism in Ter-
atogenicity. Teratology 1987, 36, 67-75.
(69) Lindamood, C., III; Giles, H. D.; Hill, D. L. Preliminary Toxicity
Profile of Arotinoids SMR-2 and SMR-6 in Male B6D2F1 Mice.
Fundam. Appl. Toxicol. 1987, 8, 517-530.
(70) Lindamood, C., III; Cope, F. O.; Dillehay, D. L.; Everson, M. P.;
Giles, H. D.; Lamon, E. W.; McCarthy, D. J .; Sartin, J . L.; Hill,
D. L. Pharmacological and Toxicological Properties of Arotinoids
SMR-2 and SMR-6 in Mice. Fund. Appl. Toxic. 1990, 14, 15-
29.
(77) Lowry, O. H.; Rosebrough, N. J .; Farr, A. L.; Randall, R. J .
Protein Measurement with the Folin Phenol Reagent. J . Biol.
Chem. 1951, 193, 265-275.
(78) Birckbichler, P. J .; Orr, G. R.; Patterson, M. K., J r.; Conway,
E.; Carter, H. A. Increase in Proliferative Markers After Inhibi-
tion of Transglutaminase. Proc. Natl. Acad. Sci. U.S.A. 1981,
78, 5005-5008.
J M970196M