An unhydrolyzable analogue of N-(4-hydroxyphenyl)retinamide: Synthesis and preliminary biological studies

Article abstract of DOI:10.1016/S0960-894X(01)00230-X

The synthesis of a nonhydrolyzable, carbon-linked analogue (4-HBR) of the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) using Umpolung methods is described. Preliminary studies of biological activity show 4-HBR is similar to 4-HPR in its actions although a potentially relevant and desirable difference is its reduced suppression of plasma vitamin A levels. These results show that 4-HPR does not have to be hydrolyzed to retinoic acid to produce its chemotherapeutic effects. Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(01)00230-X

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1583–1586
An Unhydrolyzable Analogue of N-(4-Hydroxyphenyl)retinamide:
Synthesis and Preliminary Biological Studies
Kevin L. Weiss,a Galal Alshafie,b Jason S. Chapman,c Serena M. Mershon,a
a,
Hussein Abou-Issa,b Margaret Clagett-Damec and RobertW. Curley, Jr. *
aDivision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
bDepartment of Surgery, College of Medicine, The Ohio State University, Columbus, OH 43210, USA
cDepartment of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA
Received 28 December 2000; accepted 3 April 2001
Abstract—The synthesis of a nonhydrolyzable, carbon-linked analogue (4-HBR) of the retinoid N-(4-hydroxyphenyl)retinamide (4-
HPR) using Umpolung methods is described. Preliminary studies of biological activity show 4-HBR is similar to 4-HPR in its
actions although a potentially relevant and desirable difference is its reduced suppression of plasma vitamin A levels. These results
show that 4-HPR does not have to be hydrolyzed to retinoic acid to produce its chemotherapeutic effects. # 2001 Elsevier Science
Ltd. All rights reserved.
The synthetic retinoid N-(4-hydroxyphenyl)retinamide
(4-HPR; 1) was developed a number of years ago and
showed promise as, in particular, a breast cancer
chemopreventive agent in animals.1 While this analogue
is derived from the natural retinoid, retinoic acid (RA;
2), it is less toxic and substantially less teratogenic.2 Con-
tinued interest in this retinamide has led to its exploration
in a clinical trial as a breast cancer chemopreventive agent3
and in animal studies as an antitumor agent.4
RAR-independentand laet RAR-dependentacitons of
4-HPR in these cells.10
Certainly it is plausible that 4-HPR might serve as a
prodrug which liberates RA in vivo. There is limited
evidence that 4-HPR may be metabolized to RA in
vivo,11 but there is also in vivo and in vitro work where
hydrolysis could not be detected.8,12 Thus, some of the
RA-like effects of 4-HPR could be due to hydrolysis of
4-HPR. To explore this possibility we have prepared
and are studying the nonhydrolyzable, carbon-linked
4-HPR analogue 4-hydroxybenzylretinone (3; 4-HBR).
The mechanism through which 4-HPR functions
remains unclear. When prepared, itwas assumed htat
4-HPR was an amide analogue that acted in a RA-like
manner. Subsequently, nuclear retinoic acid receptors
(RARs), which bind RA, and retinoid X receptors
(RXRs), which bind 9-cis-retinoic acid, were discovered.
These RARs function as ligand dependent transcription
factors mediating most or all of the effects of RA.5
While some researchers have reported that 4-HPR can
activate RARs using transactivation assays,6 we find
that 4-HPR has very low affinity for RAR and RXR
proteins.7 4-HPR has also been shown to induce apop-
tosis in tumor cells that typically respond to RA by dif-
ferentiating,8 and this can even occur in RA-resistant
cells.9 Recently, based on studies in wild-type and
receptor knockout F9 murine teratocarcinoma cells,
Clifford and co-workers have suggested there are early
After exploring a number of approaches to 4-HBR, an
Umpolung, or dipole inversion strategy,13 was ultimately
used in which a retinoid acyl anion equivalent was
reacted with a suitable benzyl halide. As shown in
*Corresponding author. Fax: +1-614-292-2435; e-mail: curley.1@osu.
edu
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00230-X

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K. L. Weiss et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1583–1586
Scheme 1, reaction of retinal (4) with TMSCN/Et3N14
afforded the labile silylcyanohydrin 5, which was used
as obtained. Crude 5 was deprotonated (NaHMDS) and
the resulting anion was alkylated with benzyl bromide 6.
The resulting compound 7 is deprotected (TBAF) as
obtained to provide target 3. The benzyl bromide 6,
which was employed as the electrophile, was prepared
as shown in Scheme 2.15 Surprisingly, 3 shows no evi-
care to avoid exposure to excess acid or heat, or by
using preparative reversed-phase HPLC (85% metha-
nol/water), 4-HBR with no more than 5–10% cis isomer
was obtained.
To justify detailed biological studies of 4-HBR and its
use as a probe for the mechanism of action of 4-HPR,
preliminary investigations of its actions have been con-
ducted. Previously, we have found that 4-HPR and its
analogues can shrink preformed mammary tumors.4
Therefore, a pilot study of the relative antitumor activ-
ity of 4-HBR was undertaken in female rats treated ca.
50 days earlier with dimethylbenz[a]anthracene
(DMBA) using previously described methods.4 In this
pilot study, three mammary tumor bearing rats/group
were sacrificed after 1, 7, 14, and 21 days of consuming
diet mixed with vehicle control or 2 mmol/kg of test
retinoid. Blood and liver were collected at each time
point and tumor volumes measured. The time course of
the tumor volume changes for the three rats surviving
the full 21 day experiment are shown in Table 1. While
the sample size is small, it appears that 4-HPR and 4-
HBR are indistinguishable in their substantial anti-
1
dence of existing in the enol form as determined by H
NMR in CDCl3 or ethanol-d6. However, itshould be
noted that once prepared, ketone 3 was found to
undergo thermal and acid-catalyzed isomerization to
the 13-cis isomer much more easily than 4-HPR, pro-
ducing a 60:40 trans/cis mixture at equilibrium. With
Scheme 1. (a) TMSCN, TEA (cat), CH2Cl2 (quant); (b) NaHMDS,
THF then 6; (c) TBAF, 9:1 THF/H2O (65% from 5).
Figure 1. Plasma concentrations of: (a) retinol in control rats (*) and
rats treated with 4-HPR (&), 4-HBR (~) and RA (*) assessed by
modifications of the method in ref 18; and (b) treatment retinoid (4-
HPR, &; 4-HBR, ~; RA, *). Values=meanÆSE; N=3.
Scheme 2. (a) NaH; TBDMSCl, THF (quant); (b) NaBH4, EtOH
(94%); (c) TFAA, THF (95%); (d) LiBr, THF (73%).
Table 1. Effect of retinoids on total mammary tumor volumea
Diet additive (2 mmol/kg)
Mean tumor volume (cm3)b
Day 1
Day 7
Day 14
Day 21
% changec
Vehicle control
4-HBR (3)
4-HPR (1)
RA (2)
0.06Æ0.01
0.59Æ0.43
0.20Æ0.09
0.69Æ0.18
0.08Æ0.03
0.47Æ0.59
0.17Æ0.13
0.46Æ0.06
0.11Æ0.05
0.15Æ0.17
0.10Æ0.08
0.27Æ0.11
0.15Æ0.06
0.09Æ0.07
0.05Æ0.03
0.18Æ0.08
+257
À85
À77
À74
aAt 21 days of feeding diet to three rats.
bValues=meanÆSE. Initial mean tumor volumes in control rats are smaller due to requirements of our approved animal use protocol which man-
date that tumors not be necrotic nor too large by the experiment’s end.
cFor changes in tumor volume from day 1 to day 21, p<0.05 versus control.

K. L. Weiss et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1583–1586
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tumor activity in this model. In fact, compared to the
control group, each treatment group had a significantly
steep decline in tumor volume (all p values <0.05).
During the course of this experiment, the diet that had
been held for a maximum of 7 days in the animal room
was extracted and analyzed by HPLC and showed no
evidence of retinoid isomerization or decomposition
(93% trans at start; 92% trans atday 7).
resulting from displacement of vitamin A (retinol) from
its serum retinol binding protein (RBP),16 thereby
minimizing retinol delivery to the eye. As shown in
Figure 1, there is much less of a reduction in plasma
retinol concentration in animals fed 4-HBR when com-
pared to 4-HPR and RA.17 Although 4-HBR appears to
compete as effectively as 4-HPR for [3H]-retinol binding
to RBP (Fig. 2), 4-HBR may cause less effect on plasma
retinol levels in vivo because it achieves a lower con-
centration in the circulation than does 4-HPR (Fig. 1b).
Previously, we have found that the body weight of ani-
mals eating retinoid-containing diets reflects retinoid
toxicity4 as does liver weight(unpublished resuslt).
While we found no difference in the liver weights among
the treatment groups (data not shown), the body
weights of the RA and 4-HPR-fed groups showed a
substantial (5–6%) decline compared to the virtually
unaffected 4-HBR and control-fed groups. This indi-
cates that 4-HBR does not demonstrate any untoward
toxicity at this dose and duration of feeding. Of perhaps
greater importance, one of the major toxicities asso-
ciated with 4-HPR therapy in humans is night blindness
Finally, in preliminary studies, 4-HBR has been found
to bind poorly to RARs a, b, and g, with an affinity
similar to that of 4-HPR (for example, Ki’s of 4-HBR,
4-HPR, and RA for RARg are >4000, >4000, and
0.7 nM, respectively). In conclusion, 4-HBR appears to
share many of the biological properties of 4-HPR,
including its effectiveness as an antitumor agent. How-
ever, 4-HBR may have a significantadvanatge over
4-HPR since the nonhydrolyzable analogue causes a
much reduced decline in serum retinol concentration
which may lessen the risk of developing night blindness
at therapeutic doses. Details of the chemistry, biochem-
istry, and biological activity of 4-HBR will be reported
in due course as further studies are conducted.
Acknowledgements
Financial supportin hte form of a grant(CA49837)
from the National Cancer Institute is gratefully
acknowledged.
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