Synthesis and preliminary chemotherapeutic evaluation of the fully C-linked glucuronide of N-(4-hydroxyphenyl)retinamide

Article abstract of DOI:10.1016/j.bmc.2005.12.044

All-trans retinoic acid analogues such as N-(4-hydroxyphenyl)retinamide (4-HPR) are effective chemopreventive and chemotherapeutic agents but their utility has been hampered by dose-limiting side effects. The glucuronide derivatives of 4-HPR, the oxygen-l

Full text of DOI:10.1016/j.bmc.2005.12.044

Bioorganic & Medicinal Chemistry 14 (2006) 3038–3048
Synthesis and preliminary chemotherapeutic evaluation of the fully
C-linked glucuronide of N-(4-hydroxyphenyl)retinamide
Joel R. Walker,a Galal Alshafie,b Nirca Nieves,c Jamie Ahrens,d Margaret Clagett-Dame,c,d
Hussein Abou-Issab and Robert W. Curley, Jr.a,*
aDivision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
bDepartment of Surgery, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210, USA
cPharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, WI 53706, USA
dDepartment of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA
Received 19 August 2005; revised 9 December 2005; accepted 13 December 2005
Available online 10 January 2006
Abstract—All-trans retinoic acid analogues such as N-(4-hydroxyphenyl)retinamide (4-HPR) are effective chemopreventive and
chemotherapeutic agents but their utility has been hampered by dose-limiting side effects. The glucuronide derivatives of 4-HPR,
the oxygen-linked 4-HPROG and the carbon-linked 4-HPRCG, have been found to be more effective agents. The synthetic route
to the fully C-linked analogue of 4-HPROG (4-HBRCG), which employs Suzuki coupling and Umpolung chemistries as key
methodologies, is shown. The results of this study show 4-HBRCG to be an effective chemotherapeutic agent in a rat mammary
tumor model while being devoid of classical retinoid toxicities.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
and dermatological disorders occurred in a substantial
number of subjects.5
Retinol (1) and its metabolites (Fig. 1) are involved in
regulating many biological processes including vision,
reproduction, cell differentiation, and growth. Besides
being essential to normal cell function, the retinol
metabolite all-trans retinoic acid (atRA, 2) also shows
antiproliferative action in cancer.1 Unfortunately at
pharmacologically effective doses, atRA causes severe
toxicity. Therefore, the development of retinoid ana-
logues possessing a higher therapeutic index is needed.
One of the most investigated synthetic retinoids is
N-(4-hydroxyphenyl)retinamide (4-HPR; 3), which has
been shown to be effective in numerous types of animal
tumor models and has been evaluated in a phase III clin-
ical trial.2 A possible benefit was reported for the pre-
vention of second breast malignancy in premenopausal
women with surgically removed stage I breast cancer
or ductal carcinoma in situ (DCIS). Although 4-HPR
was generally well tolerated, it resulted in a decrease in
plasma retinol levels3,4 and diminished dark adaptation,
Glucuronidation of drugs and natural products is a
common metabolic pathway that usually facilitates
excretion.6 An important metabolite of 3 is 4-HPR-
O-glucuronide (4-HPROG; 5) in which the phenolic
hydroxyl group is linked to the sugar. Subsequent to
its discovery, 5 was synthesized and evaluated for bio-
activity, and was shown to have excellent chemopre-
ventive and chemotherapeutic activities in
a rat
mammary tumor model.7 However, it was not deter-
mined if the glucuronide 5, which was shown to be
hydrolyzed to 3 via b-glucuronidase,8 was advanta-
geous due to improved bioavailability of 3 or had
activity in its own right as an intact 5. To study this
issue, an enzymatically stable glucuronide analogue
was synthesized by replacing the phenolic oxygen with
a
methylene group. The carbon-linked analogue
4-HPR-C-glucuronide (4-HPRCG; 6) was shown to
have excellent chemopreventive9 and chemotherapeu-
tic10 properties. Furthermore, much like 4-HPR, 5
and 6 show low affinity relative to atRA for binding
to the nuclear retinoic acid receptors (RARs), which
mediate most of the actions of natural retinoids.9
Although 4-HPR causes apoptosis in numerous cancer
cell lines,11 the precise mode of action of these
Keywords: Retinoids; Carbon-linked glucuronide; Chemoprevention;
Chemotherapy; Breast cancer; Synthesis.
*
Corresponding author. Tel.: +1 614 292 7628; fax: +1 614 292
2588; e-mail: curley.1@osu.edu
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.12.044

J. R. Walker et al. / Bioorg. Med. Chem. 14 (2006) 3038–3048
3039
4-HPR has been shown to be 100 times less teratogen-
ic than RA and this toxicity may also be caused by
the liberation of RA. For the effective antitumor agent
4-HPRCG, amide bond hydrolysis may still occur
in vivo liberating retinoic acid by similar mechanisms
as for 4-HPR. To eliminate this possibility, the fully
C-linked analogue (4-HBRCG; 7) was prepared by
replacing the amide bond of 4-HPRCG with a meth-
ylene group to give the fully C-linked derivative of
4-HPR-O-glucuronide, 7 (Fig. 1). While these com-
pounds are technically no longer glucuronides of
hydroxyl compounds, abbreviations have been as-
signed to convey the rationale behind their develop-
ment and to adhere to conventions in the field. The
synthesis and therapeutic evaluation of 4-HBRCG
are reported herein.
R
R = CH2OH
R = COOH
1
2
OH
O
X
X = NH
3
4
X = CH2
OH
HO
OH
COOH
Y
O
O
X
X = NH, Y = O
5
6
7
X = NH, Y = CH2
X = CH2, Y = CH2
2. Chemistry
The recently reported improved synthetic route to
4-HPRCG employs a Suzuki coupling reaction between
an exoanomeric methylene sugar and an aryl bromide.10
This methodology, originally developed by Johnson and
co-workers,14,15 gives ready access to b-arylmethyl-C-
glycosides. It was intended to use the same chemistries
in the synthetic design of 4-HBRCG, allowing us to ob-
tain the key benzyl bromide 14 (Scheme 1). Using a con-
vergent approach, an Umpolung derivative of retinal
was then alkylated with the benzyl bromide to obtain
the carbon skeleton of the final target, 4-HBRCG
(Scheme 2).
Figure 1. Natural and synthetic retinoids: (1) retinol; (2) atRA; (3) 4-
HPR; (4) 4-HBR; (5) 4-HPROG; (6) 4-HPRCG; and (7) 4-HBRCG.
synthetic retinoids remains unclear. While 4-HPR (3)
has been shown to be an effective chemopreventive
and therapeutic agent, some of its effects may be
attributed to in vivo hydrolysis of the amide bond,
liberating atRA.12 To investigate this possibility, an
unhydrolyzable analogue of 4-HPR, 4-hydroxybenzyl
retinone (4-HBR; 4), was synthesized. Both 3 and 4
were shown to be equipotent chemotherapeutics in
the dimethylbenz[a]anthracene (DMBA)-induced rat
mammary tumor model.13 In addition, both 3 and 4
cause apoptosis in cultured mammary tumor cells.12
In vitamin A-deficient rats, 3, but not 4, is hydrolyzed
to liberate retinoic acid.12 Furthermore, 4-HPR, but
not the C-linked analogue 4, induces CYP26B1
mRNA expression in a RA-like manner in the lungs
of vitamin A-deficient rats. Based on the positive
chemotherapeutic and apoptotic-inducing activity of
3 and 4, it appears that hydrolysis of 4-HPR is not
required for the therapeutic effect of this retinoid,
but rather, the liberation of RA may contribute to
its retinoid-based toxic side effects.
Starting from readily available d-D-gluconolactone (8),
hydroxyl protection using mild conditions with MOMCl
and diisopropylethylamine gives the protected lactone 9
(Scheme 1). Olefination using Petasis reagent16,17 gives
the known exoanomeric methylene sugar 10 in good
yield.14 Hydroboration of the exocyclic olefin with
9-BBN-H, followed by a Suzuki coupling with p-bro-
mobenzyl alcohol, gives exclusively the b-arylmethyl-
C-glucoside 11. Previous reports have shown this reac-
tion to be stereoselective.10,15,18 Due to subsequent
planned chemistry, the benzyl alcohol was easily pro-
tected to yield the methyl ether 12. In order to obtain
the glucuronide, the MOM groups were cleaved in acid
OMOM
OMOM
OH
OMOM
b
a
c
O
O
O
O
MOMO
MOMO
MOMO
MOMO
HO
HO
MOMO
MOMO
MOMO
MOMO
HO
MOMO
O
O
OH
11
8
9
10
OMe
OMe
OMOM
O
O
O
O
O
d
e
f
AcO
AcO
AcO
MOMO
MOMO
AcO
MOMO
AcO
AcO
OMe
OMe
Br
13
14
12
Scheme 1. Reagents and conditions: (a) MOMCl, (i-Pr)2NEt, Bu4NI, CH2Cl2, 48 h, 83%; (b) Cp2Ti(CH3)2, PhCH3, 70 ꢁC, 18 h, 87%; (c) i—9-BBN-
H, THF, reflux, 6 h; ii—PdCl2(dppf), 3 M K3PO4, DMF, p-bromobenzyl alcohol, 18 h, 67%; (d) i—NaH, THF, 1.5 h; ii—CH3I, 18 h, 90%; (e) i—6 N
HCl, MeOH, 18 h; ii—TEMPO, NaClO, KBr, NaHCO3, 0 ꢁC, 45 min; iii—CH3I, DMF, 20 h; iv—Ac2O, pyridine, DMAP, 18 h, 82%; (f) HBr,
AcOH, 18 h, 86%.

3040
J. R. Walker et al. / Bioorg. Med. Chem. 14 (2006) 3038–3048
CN
O
O
Si
a
b
H
16
15
OAc
AcO
OAc
COOMe
Si
O
O
c
CN
17
OAc
AcO
HO
OAc
O
O
COOMe
O
d
18
7
OH
OH
COOH
O
Scheme 2. Reagents and conditions: (a) TBDSMCN, Et3N, CH2Cl2, 20 h, 78%; (b) i—LiHMDS, THF, ꢀ78 ꢁC, 30 min; ii—compound 14, THF,
ꢀ78 ꢁC, 3 h, 47%; (c) TBAF, THF, 1 h, 75%; (d) i—K2CO3, MeOH, 4 ꢁC, 20 h; ii—KOH, MeOH, 4 ꢁC, 20 h, 82%.
and the primary alcohol was selectively oxidized to the
carboxylate using 2,2,6,6-tetramethyl-1-piperidinyloxy
free radical (TEMPO).19,20 Typically, TEMPO is used
catalytically. However, these conditions resulted in
non-selective oxidation, yielding mixtures of benzylic
ketones. Variations in the time, temperature, base,
amount of TEMPO, amount of sodium hypochlorite,
and order of addition were evaluated without any suc-
cess in cleanly generating 13. From past experience in
our laboratory, other oxidatively sensitive sugar-type
molecules can undergo selective oxidations when excess
TEMPO is used.21 When excess TEMPO, KBr, and
NaOCl are premixed in a NaHCO3 solution, deprotec-
ted 12 was added and selectively oxidized, efficiently
yielding the 6-position carboxylate. Methylation of the
carboxylate, followed by acetylation of the remaining
alcohols, gives the protected C-aryl-glucuronide 13 in
good yield over four steps. Benzylic methyl ethers can
be displaced by bromide using hydrobromic acid22,23
and when exposed to HBr in acetic acid, methyl ether
13 smoothly gave the key benzyl bromide C-glucuronide
intermediate 14. This surprisingly facile reaction yielded
a very stable benzyl bromide, which was isolated by
crystallization.
nohydrin was deprotonated with LiHMDS followed by
addition of bromide 14. Subsequent chromatography
of the alkylated TBDMS-protected product 17 allowed
for recovery of the valuable unreacted bromide. Treat-
ment of the alkylated product with fluoride unmasked
the ketone to give the penultimate material 18. Using
model reactions, substantial efforts were done to at-
tempt to improve the yield of the alkylation reaction
including comparing TMS- and TBDMS-silylcyanohy-
drin reactivities, employment of different bases, chang-
es in stoichiometry, temperature, and time. Even
though recovery of unreacted bromide 14 was impor-
tant, yields of 17 remained modest due to sensitivities
of the polyene retinoid reactant. Last, careful deprotec-
tion of the acetates and saponification of the methyl
ester gave the final target, 4-HBRCG (7). In the end,
more than 2 g of 4-HBRCG was produced to facilitate
the animal studies and in vitro assays.
3. Biological results and discussion
Preliminary evaluation of the chemotherapeutic activity
of 4-HBRCG (7) against mammary tumors was con-
ducted, and its toxicity profile was also assessed. As pre-
viously described,7,13 tumor-bearing female rats (treated
ca. 50 days earlier with DMBA) were fed the control or
retinoid-containing diets [RA (2), 4-HPR (3), or 4-
HBRCG(7)] at 2 mmol/kg diet for 22 days. As shown
in Table 1, 7 is as effective as 2 and 3 in reducing tumor
volume (30–40%), whereas control group tumor vol-
umes increased nearly 200% by 22 days. Figure 2 shows
that the time-course changes in tumor volumes was sim-
ilar for all three treatment retinoids. Likewise, the data
in Table 2 show that for 7, individual tumors in the
The next step in this route was the key alkylation of
electrophile 14 with a retinal anion equivalent (Scheme
2). The most suitable Umpolung strategy for the
chemically sensitive retinal is to employ the protected
cyanohydrin derivative,24 more particularly, the
silylcyanohydrin25 of retinal. The trimethylsilyl-
cyanohydrin of retinal was first revealed and used in
the synthesis of 4-HBR.12,26 Retinal (15) exposed to
tert-butyldimethylsilylcyanide
catalytic Et3N gave chromatographically stable cyano-
(TBDMSCN)
with
hydrin 16. In the alkylation reaction, the TBDMS-cya-

J. R. Walker et al. / Bioorg. Med. Chem. 14 (2006) 3038–3048
3041
Table 1. Effect of retinoid treatment on DMBA-induced rat mammary
tumor volumea
common sign of retinoid toxicity. Perhaps of even great-
er importance, 4-HBRCG caused a much smaller reduc-
tion than did either 4-HPR or RA in plasma retinol
levels (Fig. 3A). It is well known that both RA27 and
4-HPR3 can reduce circulating levels of blood retinol.
The 4-HPR-induced reduction in plasma retinol has
been shown to produce impaired dark adaptation and
is the single-most important factor limiting the doses
of 4-HPR that have been used in human clinical trials.28
Because 4-HBRCG (7), fed at 2 mmol/kg diet, did not
produce any significant reduction in blood retinol
whereas the same amount of 4-HPR (3) did, it should
be possible to use 7 at relatively higher doses compared
to 3 before the risk of night blindness is incurred. The
reason that 4-HBRCG (7) shows no significant lowering
of blood retinol levels may be related to the manner in
which it is distributed in vivo (Fig. 3B). 4-HPR (3) is
known to reduce blood retinol levels by competing for
binding to the serum retinol-binding protein (RBP).3,29
Interestingly, both 4-HPR and the related analogue, 4-
HBR (4), show equivalent binding to RBP, yet 4-HBR
does not lower blood retinol levels.13 However, it should
be noted that 4-HBR circulates in the blood at lower lev-
els compared to 4-HPR when administered in equimolar
quantities.13 Thus, at least with respect to 3 and 4, blood
Experimental
group
Initial tumor
volume (cm3)
Final tumor
volume (cm3)
% Changeb
Control
atRA (2)
0.10 0.05
0.08 0.03
0.12 0.03
0.12 0.06
0.29 0.12
0.05 0.02
0.08 0.02
0.08 0.05
+190c
ꢀ38c
ꢀ33c
ꢀ33c
4-HPR (3)
4-HBRCG (7)
a Value = means SEM.
b Change from baseline.
c Denotes statistical significance, p < 0.05.
A
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
a
Figure 2. Effect of retinoid treatment on time-course changes in tumor
volume. Control (d); atRA (Æ); 4-HPR (j); and 4-HBRCG (h).
a
Table 2. Effect of retinoid treatment on individual tumors
Experimental Total
group
Complete
Partial
New
No
number regressiona regressionb tumors effect
of
tumors
Control
atRA
4-HPR
4-HBRCG
Control
atRA (2)
4-HPR (3)
4-HBRCG
(7)
15
10
19
10
0
1
1
2
0
8
2
0
0
0
13
1
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
B
15
6
3
2
a Represents tumors that totally disappeared and could not be
palpated.
b Represents tumors that showed 25–75% decrease in volume.
group responded similarly to the tumors in the other
retinoid-treated groups.
Importantly, 4-HBRCG (7) showed evidence of greatly
reduced toxicity relative to both RA (2) and 4-HPR
(3). During the feeding period, only RA caused a signif-
icant (p < 0.05) reduction in normal body weight gain
(+0.2% vs +3% for control, 4-HPR, and 4-HBRCG), a
atRA
4-HPR
4-HBRCG
Figure 3. Effect of retinoid treatment on plasma retinol levels (A); the
plasma drug levels (B). (A) ap < 0.05 relative to the control group.

3042
J. R. Walker et al. / Bioorg. Med. Chem. 14 (2006) 3038–3048
retinol levels are inversely related to the concentration of
4-HPR or its analogue that is present in the blood. In
the present study, less 4-HBRCG (7) was also present
in the plasma at sacrifice compared to 4-HPR, suggest-
ing this may account for the lesser effect of the glucuro-
nide analogue 7 on the circulating blood retinol levels.
Thus, future studies of the serum protein binding and
tissue distribution of the novel retinoid analogue 7
appear warranted.
Skeletal abnormalities are another adverse side effect of
high-dose retinoid therapy.33–35 In order to determine
the extent of any similar effect for 4-HPR (3) and the
analogue 7, we measured the bone mineral content
(BMC) of the femur of animals at the end of the feeding
study. As expected, atRA produced a significant reduc-
tion in the femur BMC compared to control animals
(Fig. 4B), whereas the groups receiving 3 or 7 showed
no such effect. In an earlier clinical study of women with
early breast cancer receiving 4-HPR, a statistically insig-
nificant trend toward an increase in bone resorption
markers was noted.36 This suggests that a 4-HPR ana-
logue such as 4-HBRCG that cannot liberate RA might
be advantageous in minimizing the risk of bone
complications.
As shown in Figure 4A, treatment with RA dramatically
increased plasma triglyceride (TG) concentration,
whereas 4-HBRCG did not cause this undesirable effect.
An increase in plasma TG is a well-known side effect of
oral RA administration30,31 and is mediated by binding
to the RAR family.32 As borne out in human trials,
4-HPR is clearly less potent than RA in producing this
side effect.5 It is possible that hydrolysis of 4-HPR
may have accounted for the small but non-significant
increase upon feeding of this retinoid in the present
study, whereas the non-hydrolyzable 4-HBRCG showed
no propensity to increase triglyceride levels.
As with 4-HPR and other related analogues (5 and 6) we
have studied, 4-HBRCG binds poorly to the RARs
(Fig. 5). 4-HPR was nearly 3000 times less potent than
atRA in competing for [3H]-RA binding to RARb,
and 2500 times less able to compete for binding to the
RARc, confirming earlier work.9,37 In the present study,
4-HBRCG also showed only weak RAR binding (300
times and 1400 times less potent than atRA in binding
to RARb and RARc, respectively). Binding of
4-HBRCG was also tested at the RARa, and it was
only approximately 20-fold more effective than 4-HPR,
which also shows only weak binding at this receptor
(data not shown).9 Furthermore, 4-HBRCG at
3.0
A
a
2.5
2.
0
A
1.5
1.0
0.5
0.0
100
80
60
40
4-HPR
Control
atRA
4-HPR
4-HBRCG
4-HBRCG
20
atRA
0.48
0.46
0.44
0.42
0.40
0.38
0.36
0.34
B
0
-11
-10
-9
-8
-7
-6
-5
-4
B
100
a
80
60
40
20
0
Control
atRA
4-HPR
4-HBRCG
-11
-10
-9
-8
-7
-6
-5
-4
Figure 4. Effect of retinoid treatment on plasma triglyceride level (A)
and bone mineral content (B). Values are means SEM. (A) ap < 0.05
relative to control, 4-HPR, and 4-HBRCG groups. (B) ap < 0.05
relative to the control group.
Log [Drug] (M)
Figure 5. Competition of retinoids for [3H]all-trans RA binding to
RARb (A) and RARc (B); atRA (Ç); 4-HPR (j); and 4-HBRCG (h).

J. R. Walker et al. / Bioorg. Med. Chem. 14 (2006) 3038–3048
3043
concentrations up to 10ꢀ4.5 M showed almost no com-
petition for [3H]-9-cis RA binding to the RXRs (data
not shown).
and indicates that RA-mediated toxicities should be
less of a problem with this improved retinoid, com-
pared to 4-HPR.
In order to evaluate the ability of retinoids to activate
RAR-mediated gene transcription in vivo, CYP26
was measured at sacrifice in rat liver and lung. atRA
has been shown to induce the atRA-metabolizing
CYP26A1 mRNA via binding to RARs and direct
interaction of the liganded RAR/RXR heterodimer
with retinoic acid response element in the promoter re-
gion of this RA-responsive gene.38,39 The atRA was
highly effective in inducing CYP26A1 mRNA in liver
(67-fold above control; Fig. 6) and CYP26B1 in the
lung (46-fold above control; data not shown). 4-HPR
also showed significant activity in inducing CYP26
mRNAs in liver and lung (37- and 20-fold for
CYP26A1 and CYP26B1, respectively, compared to
control), whereas 4-HBRCG did not induce these cyto-
chrome p450 mRNAs. We showed previously that
atRA (2), and to a lesser extent 4-HPR (3), induces
the expression of CYP26B1 mRNA in lungs of vitamin
A-deficient rats.12 The fact that neither 3 nor 7 shows
particularly strong binding to the RARs coupled with
our finding that 4-HBRCG (7) actually shows slightly
enhanced affinity compared to 4-HPR for the RARs
yet does not induce CYP26 gene expression argues
against a direct interaction of 4-HPR with the receptor
as a mechanism to explain its ability to induce these
mRNAs. Rather, the hydrolysis of 4-HPR to atRA
may account for this induction, and as we have shown
previously, 4-HPR given orally to vitamin A-deficient
rats generates atRA in plasma that is detectable by
HPLC.12 The lack of induction of RAR-mediated gene
transcription by 4-HBRCG in vivo supports the con-
clusion that direct binding of 7 to RARs does not
occur at the retinoid levels fed in the present study,
The toxicity of 4-HPR has been reported to be reduced
compared to atRA, however the spectrum of toxicities
encountered is similar.40 4-HBRCG shows significant
improvement when compared to the natural hormone,
atRA, for all the measures of toxicity studied here
(weight loss, elevation of plasma TG, reduction in
BMC, and reduced blood retinol). When compared to
4-HPR, 4-HBRCG also shows advantage as it does
not reduce blood retinol to the same extent.
In conclusion, an efficient strategy was developed to
synthesize 4-HBRCG which was found to share the
ability with 4-HPR and atRA to reduce the size and
number of rat mammary tumors. However, a number
of toxic effects shown by the parent retinoids are
reduced or eliminated with 4-HBRCG. Thus, this
fully unhydrolyzable analogue may have a significant
advantage as a low toxicity chemotherapeutic agent.
Further studies will be required to investigate the
chemopreventive potential of 4-HBRCG and to more
clearly define its pharmacokinetic profile and mecha-
nism of action.
4. Experimental
4.1. Synthesis
4.1.1. General methods. Anhydrous THF and CH2Cl2
were obtained using distillation from sodium benzo-
phenone ketyl and calcium hydride, respectively.
Sigma–Aldrich (Milwaukee, WI) supplied starting
materials and reagents. Cambridge Isotopes Laborato-
ries (Cambridge, MA) supplied isotope labeled
solvents. All reactions and handling of retinoid-con-
taining compounds were done under gold fluorescent
lights. TLC was performed on Merck (Gibbstown,
NJ) silica gel 60 F254 aluminum plates. Column chro-
matography was performed with Merck silica gel 60
and reverse-phase flash chromatography with Merck
Lichroprepꢂ RP-18. Analytical HPLC was done on a
Beckman Instruments (San Ramon, CA) unit, with
model 127 pump and detector module 166, unless
otherwise noted, equipped with a Metachem Polaris
(Varian), 5 lm C-18, 250· 4.6 mm column. All reti-
noids were detected at a wavelength of 350 nm.
Melting points were determined with a Thomas–Hoo-
ver (Philadelphia, PA) capillary apparatus and are
uncorrected. Optical rotations were measured on a
Perkin-Elmer (Wellesley, MA) 241 polarimeter and
reported in mol dmꢀ1 gꢀ1. Ultraviolet spectra were
recorded on a Beckman Instruments DU-40 spectro-
photometer. Infrared spectra were recorded as films
on silver chloride plates using a Nicolet (Madison,
90
a
80
70
60
50
a,b
40
30
20
10
0
atRA
4-HPR
4-HBRCG
´ ´
WI) Protege 460 spectrophotometer. NMR spectra
were recorded on a Bruker (Billerica, MA) DRX 400
spectrometer. Mass spectra were recorded on a Micro-
mass (Milford, MA) QTOF Electrospray (ES) mass
spectrometer.
Figure 6. Fold induction of CYP26A1 mRNA in liver of retinoid-fed
a
rats relative to the control group. Values are means SEM. p < 0.05
relative to control and 4-HBRCG groups; bp < 0.05 relative to the
atRA-fed group.

3044
J. R. Walker et al. / Bioorg. Med. Chem. 14 (2006) 3038–3048
4.2. 2,3,4,6-Tetra-O-(methoxymethyl)-D-gluconic acid-d-
lactone (9)
through Celite. The supernatant was concentrated to
yield a red oil, which was chromatographed on silica
gel (4:1 then 2:1, hexanes/ethyl acetate) to afford 8.66 g
(87%) of yellowish oil. [a]D 46.8 (c 2.33, CH2Cl2); IR
(cmꢀ1) 2940 (m), 2895 (m), 1750 (w), 1440 (w), 1154
To a flame dried flask under argon atmosphere was sus-
pended d-gluconolactone (8) (7.38 g, 41.4 mmol) in
CH2Cl2 (400 mL). Upon cooling in an ice bath, diiso-
propylethylamine (57.6 mL, 331 mmol) was added drop-
wise, followed by careful addition of chloromethyl
methyl ether (50 g, 621 mmol). A significant amount of
white smoke formed in the reaction vessel. Solid tetrabu-
tylammonium iodide (50 g, 134 mmol) was added and
the solution was allowed to warm to rt. The reaction
mixture was stirred in the dark for 48 h during which
the solution gradually turned red. After cooling to
0 ꢁC, saturated aqueous NH4Cl (75 mL) was added,
the contents diluted with water and the layers separated.
The organic layer was washed with brine and the com-
bined aqueous layers were extracted with CH2Cl2 (3·).
The combined organic layers were dried (MgSO4), fil-
tered, and concentrated. The solids were then triturated
with ether (4·) and the ether was concentrated. The
resultant oil was chromatographed on silica gel (1:1,
hexanes/ethyl acetate) to afford 12.04 g (83%) of clear
oil. [a]D 118.4 (c 2.15, CH2Cl2); IR (cmꢀ1) 2948 (s),
2885 (s), 1757 (s), 1464 (m), 1443 (m), 1213 (s), 1150
1
(s), 1032 (s), 918 (m); H NMR (DMK-d6) d 3.31–3.37
(m, 12H), 3.64–3.71 (m, 2H), 3.78–3.83 (m, 2H), 3.88–
3.89 (m, 1H), 4.12 (d, 1H, J = 5.4 Hz), 4.35 (s, 1H),
4.51 (s, 1H), 4.62 (s, 2H), 4.66–4.84 (m, 6H); 13C
NMR (DMK-d6) d 55.15, 55.87, 56.04, 56.19, 67.50,
75.42, 76.68, 77.36, 81.08, 93.43, 95.35, 97.23, 97.64,
97.81, 156.39; HRMS (ES) calcd for C15H28O9
(M+Na): 375.1631. Found: 375.1628.
4.5. 2,6-Anhydro-1-deoxy-1-[4-(hydroxymethyl)phenyl]-
3,4,5,7-tetra-O-(methoxymethyl)-D-glycero-D-gulo-hepti-
tol (11)
To a flame dried flask under argon atmosphere was
added the exocyclic olefin 10 (3.75 g, 10.6 mmol) dis-
solved in dry THF (100 mL). 9-BBN-H (53.2 mL,
26.6 mmol, 0.5 M) was added dropwise. The mixture
was then refluxed for 4.5 h, cooled to rt, then K3PO4
(10 mL, 3 M) was added and allowed to stir for
10 min. p-Bromobenzyl alcohol (3.98 g, 21.3 mmol)
and PdCl2(dppf) (0.686 g, 0.85 mmol) dissolved in
DMF (100 mL) were added dropwise and stirred for
18 h. The reaction was diluted with water and ether,
and the layers separated. The organic layer was washed
with water and brine. The combined aqueous layers
were extracted with ether (3·). The organic layers were
combined, dried (MgSO4), concentrated, and chro-
matographed (1:1 then 1:2, hexanes/ethyl acetate) to af-
ford 3.29 g (67%) of orange oil. [a]D ꢀ26.2 (c 1.15,
DMK); IR (cmꢀ1) 3470 (w), 2932 (m), 2887 (m),
1692 (m), 1444 (w), 1150 (s), 1101 (s), 1024 (s), 918
(m); 1H NMR (DMK-d6) d 2.60 (dd, 1H, J = 9.4,
14.4 Hz), 3.18–3.42 (m, 5H), 3.25 (s, 3H), 3.35 (s,
3H), 3.40 (s, 3H), 3.44 (s, 3H), 3.54–3.61 (m, 2H),
3.73 (dd, 1H, J = 1.8, 11.3 Hz), 4.51–4.58 (m, 4H),
4.70 (d, 1H, J = 6.5 Hz), 4.77–4.85 (m, 4H), 4.93 (d,
1H, J = 6.5 Hz), 7.25 (s, 4H); 13C NMR (DMK-d6) d
38.35, 55.04, 56.45, 56.55, 64.44, 64.57, 67.42, 77.97,
79.07, 80.32, 81.63, 84.83, 97.20, 99.01, 99.19, 99.32,
127.15, 130.11, 138.75, 141.03; HRMS (ES) calcd for
C22H36O10 (M+Na): 483.2206. Found: 483.2188.
1
(s), 1035 (s), 912 (m); H NMR (CDCl3) d 3.36–3.42
(m, 12H), 3.77 (dd, 1H, J = 3.8, 11.3 Hz), 3.82 (dd,
1H, J = 2.8, 11.3 Hz), 3.99–4.05 (m, 2H), 4.29 (d, 1H,
J = 6.6 Hz), 4.55–4.56 (m, 1H), 4.65 (s, 2H), 4.69-4.92
(m, 7H); 13C NMR (CDCl3) d 55.42, 56.05, 56.11,
56.22, 66.12, 73.69, 74.77, 78.43, 96.56, 96.66, 96.78,
96.91, 97.13, 168.70; HRMS (ES) calcd for C14H26O10
(M+Na): 377.1424. Found: 377.1408.
4.3. Dimethyl titanocene, Cp2Ti(CH3)2 (Petasis’ reagent)
To a flame dried flask under argon atmosphere were
added titanocene dichloride (14.63 g, 58.8 mmol) and
absolute ether (300 mL), which was cooled to 10 ꢁC.
Methyl lithium (100 mL, 140 mmol, 1.4 M) was careful-
ly added dropwise in the dark. The cooling bath was re-
moved and the red solution was allowed to stir for
10 min. The solution was then cooled to 0 ꢁC and ice
water (25 mL) was carefully added to quench the unre-
acted methyl lithium. The layers were separated and
the aqueous layer was extracted with ether (2·). The
combined organic layers were dried (Na2SO4) under
argon for 1 h and concentrated in the dark at 20 ꢁC to
give 12.4 g of orange solid. Dry toluene (100 mL) was
added and the reagent was stored at 4 ꢁC and used
without characterization.
4.6. 2,6-Anhydro-1-deoxy-1-[4-(methoxymethyl)phenyl]-
3,4,5,7-tetra-O-(methoxymethyl)-D-glycero-D-gulo-hepti-
tol (12)
To a flame dried flask under argon atmosphere was add-
ed the C-glycoside benzyl alcohol 11 (2.44 g, 5.3 mmol)
dissolved in dry THF (100 mL). Sodium hydride
(0.63 g, 26.5 mmol) was added to the flask and the
suspension was stirred for 1.5 h. Iodomethane (4.5 g,
31.7 mmol) dissolved in THF (10 mL) was cannulated
into the reaction mixture and allowed to stir for 18 h.
After cooling in an ice bath, water was added carefully
to quench excess NaH. The mixture was extracted with
ether (3·), the organic layers combined, washed with
brine, dried (MgSO4), concentrated, and then chromato-
graphed (1:1 then 1:2, hexanes/ethyl acetate) to give
4.4. 2,6-Anhydro-1-deoxy-3,4,5,7-tetra-O-(methoxymeth-
yl)-D-gluco-hept-1-enitol (10)
To a flame dried flask under argon atmosphere was add-
ed the sugar lactone 9 (10.05 g, 28.4 mmol) dissolved in
dry toluene (140 mL). The toluene solution of dimethyl
titanocene (12.4 g, 59 mmol) was then added dropwise
to give a red solution. The mixture was then heated to
70 ꢁC and let stir in the dark for 18 h. The resultant
black solution was cooled and poured into hexanes
(ꢁ500 mL). A precipitate formed and was filtered

J. R. Walker et al. / Bioorg. Med. Chem. 14 (2006) 3038–3048
3045
2.37 g (94%) of clear oil. [a]D ꢀ27.0 (c 4.70, DMK); IR
(cmꢀ1) 2981 (s), 2883 (s), 1701 (w), 1513 (m), 1444
(m), 1378 (m), 1301 (m), 1158 (s), 1105 (s), 1028 (s),
4.8. 2,6-Anhydro-7-deoxy-7-[4-(bromomethyl)phenyl]-
3,4,5-tri-O-acetyl-L-glycero-L-gulo-heptinoic acid methyl
ester (14)
1
918 (s); H NMR (DMK-d6) d 2.61 (dd, 1H, J = 9.4,
14.4 Hz), 3.19–3.42 (m, 5H), 3.24 (s, 3H), 3.30 (s, 3H),
3.35 (s, 3H), 3.40 (s, 3H), 3.44 (s, 3H), 3.54–3.64 (m,
2H), 3.73 (dd, 1H, J = 2.6, 13.5 Hz), 4.38 (s, 2H), 4.50
(d, 1H, J = 6.4 Hz), 4.54 (d, 1H, J = 6.4 Hz), 4.70 (d,
1H, J = 6.5 Hz), 4.77–4.85 (m, 4H), 4.93 (d, 1H,
J = 6.5 Hz), 7.21 (d, 2H, J = 8.0 Hz), 7.28 (d, 2H,
J = 8.0 Hz); 13C NMR (DMK-d6) d 33.39, 55.05,
56.47, 56.49, 56.57, 57.97, 67.46, 74.84, 78.00, 79.10,
80.23, 81.66, 84.86, 97.20, 99.01, 99.21, 99.32, 128.15,
130.19, 137.23, 139.45; HRMS (ES) calcd for
C23H38O10 (M+Na): 497.2363. Found: 497.2384.
To a dry flask equipped with a CaSO4 drying tube was
added the C-glucuronide methyl ether 13 (462 mg,
1.02 mmol) along with 30% HBr in acetic acid (5 mL,
25 mmol) at 0 ꢁC. The mixture was stirred for 30 min
and then for 18 h at rt. The mixture was diluted with
CH2Cl2 and then carefully washed with water and satu-
rated NaHCO3 solution. The organic layer was dried
(MgSO4), concentrated, and chromatographed (2:1 then
1:1, hexanes/ethyl acetate) to give 440 mg (86%) of white
foam, which was crystallized with ether, mp 116–117 ꢁC.
[a]D ꢀ12.03 (c 5.57, DMK); IR (cmꢀ1) 3026 (w), 2952
(w), 1754 (s), 1440 (m), 1370 (m), 1215 (s), 1101 (m),
1
4.7. 2,6-Anhydro-7-deoxy-7-[4-(methoxymethyl)phenyl]-
3,4,5-tri-O-acetyl-L-glycero-L-gulo-heptinoic acid methyl
ester (13)
1036 (m); H NMR (DMK-d6) d 1.93 (s, 3H), 1.94 (s,
3H), 1.95 (s, 3H), 2.76–2.83 (m, 1H), 2.92 (dd, 1H,
J = 3.5, 7.3 Hz), 3.64 (s, 3H), 3.96–3.99 (m, 1H), 4.20
(d, 1H, J = 9.7 Hz), 4.62 (s, 2H), 4.90 (t, 1H,
J = 9.7 Hz), 5.05 (t, 1H, J = 9.7 Hz), 5.29 (t, 1H,
J = 9.7 Hz), 7.25 (d, 2H, J = 8.2 Hz), 7.36 (d, 2H,
J = 8.2 Hz); 13C NMR (DMK-d6) d 20.40, 20.52,
20.63, 34.37, 38.12, 52.69, 70.58, 72.52, 74.04, 76.35,
78.43, 129.88, 130.68, 137.26, 138.67, 168.39, 169.91,
170.09, 170.29; HRMS (ES) calcd for C21H25BrO9
(M+Na): 523.0580. Found: 523.0602.
The MOM-protected glucoside 12 (2.43 g, 5.12 mmol)
dissolved in methanol (500 mL), aqueous HCl (6 N,
26 mL) was added, and the solution was stirred for
18 h at rt after which the mixture was concentrated
to dryness. In
a
separate flask, KBr (2.42 g,
20.38 mmol) and TEMPO (3.19 g, 20.41 mmol) were
added to a saturated NaHCO3 solution (400 mL)
and stirred for 20 min at 0 ꢁC. Aqueous NaOCl
(11.2 mL, 1.6–2.0 M) was then added and stirred
for 10 min. The deprotected sugar from above was
dissolved in saturated NaHCO3 solution (100 mL)
and added to the flask with the TEMPO mixture.
The mixture was stirred for 45 min at 0 ꢁC. The
reaction was quenched with EtOH (50 mL) and the
contents were washed with ether in a separatory fun-
nel. The aqueous layer was concentrated to dryness
and the remaining solid was exhaustively triturated
with methanol. The methanol was concentrated and
the dried residue was suspended in DMF (180 mL)
and then iodomethane (6.4 g) dissolved in DMF
(10 mL) was added and allowed to stir for 20 h un-
der argon at rt. Acetic anhydride (40 mL), pyridine
(20 mL), and DMAP (15 mg) were then added and
allowed to stir for 18 h. The reaction mixture was
diluted with water and extracted (3·) with ethyl ace-
tate. The organic layers were washed with water,
brine, dried (MgSO4), concentrated, and chromato-
graphed (2:1 then 1:1, hexanes/ethyl acetate) to give
1.90 g (82%) of clear oil that solidified upon stand-
ing, mp 84–86 ꢁC. [a]D ꢀ13.04 (c 1.15, DMK); IR
(cmꢀ1) 2956 (w), 2818 (w), 1750 (s), 1440 (m), 1370
(m), 1211 (s), 1105 (m), 1028 (m); 1H NMR
4.9. tert-Butyl-dimethylsilylcyanohydrin of retinal (16)
To a flame dried flask under argon atmosphere was add-
ed retinal (15) (1.03 g, 3.62 mmol) dissolved in dry
CH2Cl2 (50 mL). A catalytic amount of Et3N (0.1 mL)
was added, then tert-butyldimethylsilylcyanide (1.0 g,
7.08 mmol) dissolved in CH2Cl2 (10 mL) was added by
cannulation. The reaction mixture was stirred for 20 h
after which the solution was concentrated, chromato-
graphed (95:5, hexanes/ethyl acetate), dried (Na2SO4)
under argon, and subjected to vacuum overnight to give
1.20 g (78%) of orange oil. UV
kmax = 329 nm
(e = 49462); IR (cmꢀ1) 3042 (w), 2960 (s), 2928 (s),
2850 (s), 2239 (w), 1586 (w), 1472 (m), 1358 (m), 1256
(m), 1105 (s), 963 (s), 832 (s), 775 (m); 1H NMR
(DMK-d6) d 0.16 (s, 3H), 0.20 (s, 3H), 0.90 (s, 9H),
1.02 (s, 6H), 1.45–1.48 (m, 2H), 1.58–1.63 (m, 2H),
1.70 (s, 3H), 1.99 (s, 6H), 5.57–5.61 (m, 2H), 6.13–6.23
(m, 3H), 6.38 (d, 1H, J = 15.2 Hz), 6.86 (dd, 1H,
J = 11.3, 15.2 Hz); HRMS (ES) calcd for C27H43NOSi
(M+Na): 448.3012. Found: 448.2982.
4.10. 2,6-Anhydro-7-deoxy-7-[4-(retinoylmethyl)-phenyl]-
3,4,5-tri-O-acetyl-L-glycero-L-gulo-heptinoic acid methyl
ester (18)
(DMK-d6)
d 1.94 (s, 3H), 1.94 (s, 3H), 1.95
(s, 3H), 2.74–2.81 (m, 1H), 2.90 (dd, 1H, J = 3.4,
7.3 Hz), 3.30 (s, 3H), 3.65 (s, 3H), 3.94–3.99
(m, 1H), 4.18 (d, 1H, J = 9.8 Hz), 4.38 (S, 2H),
4.90 (t, 1H, J = 9.8 Hz), 5.05 (t, 1H, J = 9.8 Hz),
5.29 (t, 1H, J = 9.8 Hz), 7.22 (s, 4H); 13C NMR
(DMK-d6) d 20.39, 20.52, 20.60,38.12, 52.67, 58.03,
70.62, 72.53, 74.09, 74.73, 76.41, 78.62, 128.25,
130.16, 137.43, 137.76, 168.40, 169.89, 170.07,
170.30; HRMS (ES) calcd for C22H28O10 (M+Na):
475.1580. Found: 475.1577.
To a flame dried flask under argon atmosphere was add-
ed THF (40 mL) along with LiHMDS (1.0 M in hex-
anes, 3.8 mL, 3.8 mmol). The mixture was cooled to
ꢀ78 ꢁC and the silyl cyanohydrin of retinal 16 (1.08 g,
2.54 mmol) in THF (15 mL) was added by cannulation.
The dark red solution was allowed to stir for 30 min at
ꢀ78 ꢁC. The crystalline bromoglucuronide 14 (2.78 g,
5.56 mmol) in THF (15 mL) was cannulated into the
flask and the mixture was stirred for 3 h at ꢀ78 ꢁC after

3046
J. R. Walker et al. / Bioorg. Med. Chem. 14 (2006) 3038–3048
which the solution changed to light red. The reaction
was removed from the cold bath and quenched with
1 M NH4Cl (10 mL). The mixture was extracted with
ethyl acetate (3·) and the organic layers were combined,
washed with brine, dried (Na2SO4), filtered, concentrat-
ed, and chromatographed (2:1, hexanes/ethyl acetate) to
give 1.0 g (47%) of yellow foam 17 and 1.7 g of recov-
ered bromide 14. The alkylated product was taken up
in 1% aqueous THF (200 mL) and chilled to 0 ꢁC.
TBAF (309 mg, 1.18 mmol) was added and the dark-
ened solution was stirred for 1 h. The reaction was dilut-
ed with water and extracted with ethyl acetate (3·). The
organic layers were combined, washed with brine, dried
(NaSO4), filtered, concentrated, and chromatographed
(2:1, hexanes/ethyl acetate) to give 628 mg (35% over
1.58 (m, 2H), 1.61 (s, 3H), 1.91 (s, 3H), 1.93–1.96 (m,
2H), 2.20 (s, 3H), 2.60 (dd, 1H, J = 8.7, 14.4 Hz),
3.03–3.08 (m, 2H), 3.21–3.29 (m, 2H), 3.37 (t, 1H,
J = 9.5 Hz), 3.53 (d, 1H, J = 9.5 Hz), 3.62 (s, 2H),
6.03–6.25 (m, 5H), 7.02–7.16 (m, 5H); 13C NMR
(MeOH-d4) d 12.88, 14.45, 20.29, 21.04, 21.93, 29.40,
34.00, 35.25, 38.34, 40.76, 52.11, 73.44, 74.63, 79.28,
80.36, 82.36, 124.37, 126.26, 129.92, 130.19, 130.86,
131.01, 131.12, 134.00, 134.23, 136.82, 138.70, 138.91,
139.04, 141.12, 154.22, 173.26, 201.21; HRMS (ES)
calcd for C34H44O7 (M+Na): 587.2985. Found:
587.2989.
5. Biology
two steps) of yellow foam. UV
(e = 36182); HPLC tR = 24.0 min, 1 mL/min (85:15,
MeOH/H2O both with 10 mM NH4OAc); IR (cmꢀ1
kmax = 379 nm
5.1. Animal studies
)
2956 (w), 2924 (w), 2863 (w), 1754 (s), 1672 (w), 1554
(m), 1436 (w), 1362 (w), 1215 (s), 1081 (w), 1028 (w),
971 (w); H NMR (DMK-d6) d 1.02 (s, 6H), 1.45–1.48
Mammary tumors were induced by intragastric intuba-
tion of 50-day-old female Sprague–Dawley rats (Harlan
Industries, Indianapolis, IN) with a single dose of 15 mg
DMBA in 1.0 mL of sesame oil per rat. The rats were
then maintained on a powdered Teklad 22/5 rodent
chow diet (W): 8640 (Harlan Industries, Indianapolis,
IN), and allowed food and water ad libitum. Four
months later, rats which had developed palpable tumors
were randomly assigned to the experimental groups
(four rats/group). The retinoid-treated groups were fed
diets supplemented with 2 mmol/kg diet atRA, 4-HPR,
or 4-HBRCG, respectively. The retinoids were added
to the diet in a vehicle consisting of 25 mL of ethanol–
tricaprylin (1:4, v/v) plus 2% (w/v) of a-tocopherol as
previously described.9 This vehicle was also added to
the control diet. The additives were blended into the
chow diets using a Hobart food mixer. The diets were
fed in stainless steel feeders designed with food hoppers.
The food was replaced weekly with freshly prepared
diets. Food consumption was determined once weekly,
and from that the average daily consumption/rat was
estimated. These diets were continuously fed for 22
days. Animals were also weighed weekly and monitored
for general health status and signs of possible toxicity
due to treatment.
1
(m, 2H), 1.58–1.62 (m, 2H), 1.69 (s, 3H), 1.90 (s, 3H),
1.93 (s, 3H), 1.95 (s, 3H), 2.01 (s, 3H), 2.03–2.05 (m,
2H), 2.28, (s, 3H), 2.75–2.89 (m, 2H), 3.64 (s, 3H),
3.71 (s, 2H), 3.95–3.98 (m, 1H), 4.19 (d, 1H,
J = 9.8 Hz), 4.90 (t, 1H, J = 9.8 Hz), 5.05 (t, 1H,
J = 9.8 Hz), 5.29 (t, 1H, J = 9.8 Hz), 6.15–6.35 (m,
5H), 7.13–7.20 (m, 5H); 13C NMR (DMK-d6) d 13.45,
14.68, 20.41, 20.96, 21.08, 21.15, 22.47, 34.15, 35.41,
38.76, 40.86, 52.25, 53.23, 71.18, 73.09, 74.63, 76.93,
79.13, 126.89, 129.86, 130.73, 130.93, 131.01, 131.47,
133.69, 134.89, 135.11, 137.14, 137.26, 138.95, 139.09,
140.96, 152.68, 168.97, 170.45, 170.64, 170.84, 198.78;
HRMS (ES) calcd for C41H52O10 (M+Na): 727.3458.
Found: 727.3456.
4.11. 2,6-Anhydro-7-deoxy-7-[4-(retinoylmethyl)-phenyl]-
L-glycero-L-gulo-heptinoic acid (7)
To a flask was added protected 18 (1.15 g, 1.64 mmol)
dissolved in methanol (500 mL) and chilled to 4 ꢁC.
Potassium carbonate (136 mg, 0.98 mmol) was added
and allowed to stir for 20 h. The reaction mixture was
concentrated at 25–30 ꢁC to ꢁ200 mL. Adjustment to
the original volume with methanol was followed by
addition of 1 N KOH (14 mL, 14 mmol). After stirring
for 20 h at 4 ꢁC, the reaction mixture was warmed and
allowed to stir for 5 h at rt. It was then cooled to 0 ꢁC
and carefully adjusted to pH 7 with 4 N HCl. The reac-
tion mixture was concentrated at 25–30 ꢁC to ꢁ100 mL,
cooled back to 0 ꢁC, and the pH carefully adjusted to 3
with 1 N HCl. The suspension was extracted with ethyl
acetate and the organic layers were combined, dried
(Na2SO4) under argon for 2 h, and carefully concentrat-
ed. The residue was chromatographed on reverse-phase
silica gel (gradient 70:30 to 85:15, methanol/water) to
yield 759 mg (82%) of yellow foam, which was stored
at ꢀ80 ꢁC until needed. UV kmax = 382 nm (e = 30019);
HPLC tR = 9.2 min (1 mL/min, 85:15 MeOH:H2O both
with 10 mM NH4OAc); IR (cmꢀ1) 3384 (br), 2920 (s),
1721 (m), 1664 (s), 1550 (s), 1427 (m), 1362 (m), 1232
Baseline measurement of initial tumor sizes, numbers,
and rat body weights was determined immediately be-
fore commencement of treatments, and final measure-
ments were recorded just prior to sacrifice of the
animals. Animals were palpated for tumors twice weekly
and tumor diameters were measured weekly by a
micrometer caliper. Tumor volumes were calculated
using the formula ½V ¼ 34 pr3ꢂ, where r is one-half the
mean of the sum of the largest diameter and the axis
at right angle to it. All tumors as well as lungs, liver,
and femur were excised at the end of the experiment
for chemical and histopathological evaluation. Blood
samples were also taken from each animal for determi-
nation of plasma retinol and triglyceride levels.
5.2. Plasma triglyceride measurement
1
(w), 1089 (m), 1052 (m), 1102 (s), 967 (w); H NMR
(MeOH-d4) d 0.94 (s, 6H), 1.38–1.41 (m, 2H), 1.54–
Bloods were drawn from anesthetized animals in the
presence of EDTA as an anticoagulant, and the

J. R. Walker et al. / Bioorg. Med. Chem. 14 (2006) 3038–3048
3047
resulting plasma was used for the measurement of
plasma ‘true’ triglyceride levels using a kit from
Sigma–Aldrich (Saint Louis, MO). Briefly, the total
plasma triglyceride and glycerol concentrations were
determined, and the glycerol component was subtracted
from the total plasma triglyceride measurement to
obtain the ‘true’ serum triglyceride concentration.
of competing ligands at 4 ꢁC for 3 h. A hydroxylapatite
(HAP) assay was used to separate ligand bound to
receptor from that free in solution, and the radioactivity
associated with the HAP pellet was measured by scintil-
lation counting.
5.6. Isolation of RNA and quantitative PCR
5.3. Plasma retinoid assay
Total and poly(A)+ RNA was isolated as described.43
Briefly, lung and liver tissue was collected and flash-
frozen in liquid nitrogen until use. Tissue (0.5–1.0 g)
was homogenized in buffer (1:10, w/v), and total RNA
was isolated according to the method of Chomczynski
and Sacchi.44 A rat CYP26A1 partial cDNA was gener-
ated by PCR amplification from E10.5 day rat embryo
cDNA. The upstream (50-GCA GAT GAA GCG
CAG GAA ATA CG-30) and downstream (50-CCC
ACG AGT GCT CAA TCA GGA-30) primers were de-
signed based on the murine cDNA (gi: 668110). The 635
bp cDNA was subcloned into pGEM-Teasy (Promega,
Madison, WI) and sequenced. Similarly, a rat CYP26B1
partial cDNA was generated by PCR amplification from
E11.5 day rat embryo cDNA. The upstream (50-GCT
ACA GGG TTC CGG CTT CCA GTC-30) and down-
stream (50-TCC AGG GCG TCC GAG TAG TCT
TTG-30) primers were designed based on the murine
cDNA (gi: 31341987), and the 606 bp control cDNA
was subcloned and sequenced. The quantitative poly-
merase chain reaction (Q-PCR) assay was performed
using the real-time LightCycler system (Roche, India-
napolis, IN, USA) with LightCycler faststart DNA mas-
ter SYBR green1 kit (Roche, Indianapolis, IN, USA)
according to the manufacturer’s protocols. Poly(A)+
RNA (0.5–1.0 lg) was reverse transcribed (RT) using
AMV enzyme (Promega, Madison, WI, USA) and ran-
dom hexamers. The following primer sets were used for
Q-PCR: CYP26A1, upstream 50-ATG ATT CCT CGC
ACA AGC AG-30, downstream 50-GCT CCA GAC
AAC CGC TCA CT-30; CYP2B1, upstream 50-AGG
CCC AGC GAC TTA CCT TC-30, downstream
50-AGG GCG TCC GAG TAG TCT TT-30; and GAP-
DH, upstream 50-TGA AGG TCG GTG TGA ACG
GAT TTG GC-30, downstream 50-CAT GTA GGC
CAT GAG GTC CAC CAC-30. The primer sets for
CYP26A1, CYP26B1, and GAPDH amplify 409–519
bp (gi: 18426827), 708–957 bp (gi: 31220748), and
854–1836 bp (gi: 31377487), respectively.
To 500 lL of plasma was added 150 lL of ethanol
containing 0.75 lg of internal standard (N-(4-chlorophe-
nyl)retinamide). After mixing 30 s, 500 lL of ethyl
acetate was added followed by 1 min of mixing and
centrifugation for 5 min at 1000 rpm in an IEC CL
centrifuge. The ethyl acetate layer was removed and syr-
inge filtered through a 0.45 lm filter. The ethyl acetate
extraction was repeated two more times. The combined
extracts were evaporated and the residue reconstituted
in 100 lL of methanol. The methanol extract (20 lL)
was analyzed by HPLC. Chromatography was done
on a precolumn equipped 250· 4.6 mm Beckman Ultra-
sphere ODS column with an 85% methanol/water
mobile phase (both containing 10 mM ammonium ace-
tate) flowing at 1 mL/min. Analysis for both internal
standard and retinol was conducted at 350 nm, and
internal standard recoveries and retinol levels were
determined by comparison with standard curves, with
adjustment of the retinol level based on recovery.
Recoveries of internal standard averaged ca. 78%. Previ-
ous extraction of plasma from vitamin A deficient rats
showed no substances interfering with the elution
position of the retinol or internal standard. In the 4-
HPR-treated group, plasma levels of this retinoid were
evaluated simultaneously in the same samples as above.
In order to avoid interfering substances, plasma treat-
ment retinoid levels for RA and 4-HBRCG were mea-
sured using the above system and a step gradient of 75%
methanol for 15 min followed by 85% methanol for
40 min.
5.4. Bone mineral content measurement
The femur was disarticulated from the leg, and the
adhering soft tissue was removed by dissection. Femurs
were scanned using the Lunar PIXImus 2 system (Model
X2608, General Electric using LUNAR software version
1.45), and control measurements were made using the
small animal quality control phantom. Femurs were
scanned five times each with repositioning at each mea-
sure. The average value of the bone mineral content
(BMC) in grams for each animal is reported as one inde-
pendent measure.
5.7. Statistical analysis
Descriptive statistics on tumor volumes, tumor numbers,
retinol, triglyceride levels, and BMC were examined and
compared among the experimental groups. The statistical
significance of the groups’ comparisons was obtained
using analysis of variance (ANOVA), ANOVA with
repeated measures, and nonparametric tests.45–47 Values
were considered significant when the p < 0.05.
5.5. Nuclear retinoid receptor binding assay
Competition of 3 and 7 with [3H]-all-trans-RA (4.2–
4.6 nM) for binding to RARb and RARc, and with
[3H]-9-cis RA (1.9 nM) for binding to RXRc was deter-
mined using an in vitro ligand binding assay.41,42 [3H]-
All-trans-RA (40.5 Ci/mmol) or [3H]-9-cis RA (69.4 Ci/
mmol) was added to receptor containing extracts in
the absence and presence of increasing concentrations
Acknowledgments
This work was supported by Grant CA49837 from
the National Cancer Institute, which is gratefully

3048
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