An improved synthesis of the C-linked glucuronide of N-(4-hydroxyphenyl)retinamide.

Article abstract of DOI:10.1016/S0960-894X(02)00427-4

Retinoic acid analogues such as N-(4-hydroxyphenyl)retinamide (4-HPR) are effective chemopreventatives and chemotherapeutics for numerous types of cancer. The C-linked analogue of the O-glucuronide of 4-HPR (4-HPRCG) has been shown to be a more effective agent. The synthetic route to this molecule has been significantly improved by access to a key C-benzyl-glucuronide intermediate through employment of a Suzuki coupling reaction between an exoanomeric methylene sugar and an aryl bromide. Preliminary evidence shows 4-HPRCG has chemotherapeutic activity.

Full text of DOI:10.1016/S0960-894X(02)00427-4

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2447–2450
An Improved Synthesis of the C-Linked Glucuronide of
N-(4-Hydroxyphenyl)retinamide
Joel R. Walker,a Galal Alshafie,b 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, The Ohio State University, Columbus, OH 43210, USA
Received 15March 2002; accepted 10 May 2002
Abstract—Retinoic acid analogues such as N-(4-hydroxyphenyl)retinamide (4-HPR) are effective chemopreventatives and
chemotherapeutics for numerous types of cancer. The C-linked analogue of the O-glucuronide of 4-HPR (4-HPRCG) has been
shown to be a more effective agent. The synthetic route to this molecule has been significantly improved by access to a key C-
benzyl-glucuronide intermediate through employment of a Suzuki coupling reaction between an exoanomeric methylene sugar and
an aryl bromide. Preliminary evidence shows 4-HPRCG has chemotherapeutic activity. # 2002 Elsevier Science Ltd. All rights
reserved.
Retinol (1; Fig. 1) and its metabolites are involved in
many biological processes including vision, cell differ-
entiation, and growth. Besides being essential to normal
cell function, the retinol metabolite retinoic acid (2) also
shows antiproliferative action in skin disease and can-
cer.1 Unfortunately, at pharmacological doses retinoic
acid causes severe toxicity. Therefore, development of
retinoid analogues possessing a higher therapeutic index
is of interest. 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 tumor models and has been involved in phase III
clinical trials.2
or had activity in its own right as intact 4. To study this
issue, an enzymatically stable glucuronide analogue was
synthesized replacing the phenolic oxygen with a meth-
ylene group. As summarized in Table 1, the carbon-
linked analogue 4-HPR-C-glucuronide (4-HPRCG; 5)
was shown to have chemopreventative qualities superior
to the O-linked 4.6 It has not yet been shown whether
5 shares the chemotherapeutic activity of 4 in the
Glucuronidation of drugs and natural products is a
common metabolic pathway that usually facilitates
excretion.3 An important metabolite of 3 is 4-HPR-O-
glucuronide (4), in which the phenolic hydroxyl group is
linked to the sugar. Subsequent to its discovery, 4 was
synthesized and evaluated for bioactivity, and was
shown to have excellent chemopreventative and chemo-
therapeutic activity in a rat mammary tumor model.4
However, it was not determined if the glucuronide 4, which
was shown to be hydrolyzed to 3 via b-glucuronidase,5
was advantageous due to improved bioavailability of 3
*Corresponding author. Tel.: +1-614-292-7628; fax: +1-614-292-
2435; e-mail: curley.1@osu.edu
Figure 1. Natural and synthetic retinoids.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00427-4

2448
J. R. Walker et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2447–2450
Table 1. Effects of retinoid glucuronides on DMBA-induced rat
mammary tumor developmenta,b
Table 2. Retinoid binding to RARsa
Retinoid
RAR (Ki, nM)
Dietary supplementc
Mean tumor Rats with
latency (days) tumors (%)
Mean number
of tumors/rat
a
b
g
Retinoic acid (2)
4-HPR (3)
4-HPROG (4)
4-HPRCG (5)
0.4
0.51.4
>2800
>2800
>2800
Control diet
4-HPROG (4)
4-HPRCG (5)
40
48
5
79
57
5 27
1.43ꢁ0.29
0.71ꢁ0.19e
0.36ꢁ0.20e
>2200
>2200
>2200
>6000
>6000
NDb
d,e
aAdapted from ref 6.
b15rats/group.
aAdapted from ref 6.
bND, not determined.
cRetinoid dose of 2 mmol/kg diet.
dUnderestimate of mean latency since not all rats developed tumors.
eP<0.05versus control.
tions and rearrangements.7,8 One important electro-
philic substitution reaction is the olefination of sugar
lactones. The resultant exoanomeric methylene sugars
can serve as C-glycoside precursors.9
carcinogen-induced rat mammary tumor model. Further-
more, much like 3 and 4, 5 shows very low affinity for
the nuclear retinoic acid receptors (RARs), thought to
mediate most of the actions of retinoids (Table 2). This
raises the question about the mode of action of these
synthetic retinoids.
The previous syntheses5,10 of 5 did just allow the gen-
eration of modest quantities required for chemopre-
ventative studies.6 However, improvements in the
synthetic route were needed to facilitate further studies.
Starting with a fairly expensive sugar (ꢀ$20/g), the tar-
get was made in an overall yield of 2.2% over 13 steps.
The key step involved reaction of a benzyl Grignard
reagent with a bromosugar to form the anomeric
carbon–carbon bond, which was followed by nitration
of the aromatic ring, resulting in a mixture of nitro
regioisomers. We now wish to report a much more effi-
cient and less expensive synthesis of the title compound.
C-Glycosides are stable, conformationally similar ana-
logues of the O-glycosides and are important com-
pounds that can be used to study the function of
carbohydrates in cellular processes or serve as stable
metabolite mimics. Efficient synthetic routes to the 1-
position C-glycosides have become of increasing interest
and these usually involve a carbon–carbon bond form-
ing reaction with the anomeric carbon. Many approaches
have been utilized including electrophilic and nucleo-
philic substitution reactions, transition metal mediated
glycosidations, anomeric radical reactions, cycloaddi-
The current approach (Scheme 1) to the C-glucuronide
of interest was adapted from the method of Johnson
Scheme 1. Reagents and conditions: (a) MOMCl, (i-Pr)2NEt, Bu4NI, CH2Cl2, 48 h, 83%; (b) Cp2Ti(CH3)2, PhCH3, 70 ꢂC, 18 h, 87%; (c) (i) 9-BBN-
H, THF, reflux, 6 h; (ii) PdCl2 (dppf), 3 M K3PO4, DMF, 1-bromo-4-nitrobenzene, 18 h, 54%; (d) (i) 6 N HCl, MeOH, 18 h; (ii) TEMPO, NaOCl,
KBr, NaHCO3, 2 h; (iii) HCl (g), MeOH, 40 ꢂC, 5h; (iv) Ac 2O, pyridine, DMAP, 18 h, 84%; (e) Pd/C, H2, 40 psi, EtOAc, 4 h, 98%; (f) retinoic acid,
SOCl2, pyridine, 4 h, 86%; (g) (i) K2CO3, MeOH, 18 h; (ii) 5N KOH, MeOH, 18 h, 86%.

J. R. Walker et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2447–2450
2449
and co-workers.11,12 Starting with readily available d-d-
gluconolactone (ꢀ$0.70/g), hydroxyl group protection
was accomplished with chloromethyl methyl ether and
diisopropylethylamine to give 7. Petasis reagent
(Cp2TiMe2)13,14 was prepared and used to olefinate the
lactone to give the enol ether 8 in good yield. The exo-
cyclic olefin was then hydroborated with 9-BBN-H and
followed by a Suzuki coupling reaction with 1-bromo-4-
nitrobenzene to give the b-arylmethyl-C-glycoside 9 in
modest yield. Efforts were made to improve this coup-
ling yield including changes in reaction temperature,
reagent stoichiometry, and the bases employed; however,
the original conditions12 were found to be optimal. In
order to obtain the glucuronide, the MOM groups were
cleaved by acid and then the primary alcohol at the 6-
position was selectively oxidized to the carboxylic acid
using 2,2,6,6-tetramethyl-1-piperidinyloxy free radical
(TEMPO).10,15 Methylation of the carboxylic acid using
HCl gas in methanol and acetylation of the remaining
alcohols afforded the novel key intermediate, the pro-
tected C-benzyl-glucuronide 10, in good yield over four
steps.16 The stereochemical outcome of the hydrobora-
tion has previously been shown to proceed to give the b-
isomer exclusively.9,12 This was further confirmed to
also be the case here with intermediate 10 via NOE
experiments.17 Reduction of the nitro group was facile
with hydrogen over palladium catalyst to give aniline
11. To couple the retinoid to the sugar derivative, reti-
noyl chloride was generated, from the treatment of reti-
noic acid with thionyl chloride, and reacted with the
aniline 11 to yield the protected retinoid conjugate 12 in
good yield. Mild cleavage of the acetates was followed
by saponification of the methyl ester to give 5 in 24%
yield over 13 steps. Characterization of the final product
gave data identical in all respects to the previously made
material.5
shows the results of the pilot study, which indicate that
5 is effective at reducing tumor volume and therefore
warrants chemotherapeutic study of longer duration
and with a larger treatment group size.
In summary, the improved synthesis uses considerably
less expensive starting material, results in a ten times
higher yield of 5, and avoids the tedious separation of
nitration regioisomers. Preliminary results from the
pilot study suggest that 5 does show chemotherapeutic
activity. The current synthetic route will facilitate the
generation of quantities sufficient for further extensive
chemotherapeutic animal studies and for gaining insight
into the mechanism of action of this important retinoid
analogue. Also, it appears that the exoanomeric meth-
ylene sugar 8 is a good precursor that can provide ready
access to C-glycosides and C-glucuronides of other
drugs or natural products.
Acknowledgements
This work was supported by grant CA49837 from
the National Cancer Institute, which is gratefully
acknowledged.
References and Notes
1. Moon, R. C.; Mehta, R.; Rao, K. V. N.; Hong, W. K.; Itri,
L. M. In The Retinoids: Biology, Chemistry, and Medicine, 2nd
ed.; Sporn, M. B., Roberts, A. B., Goodman, D. S., Eds.;
Raven: New York, 1994; p 545.
2. Veronesi, U.; De Palo, G.; Marubini, E.; Costa, A.; For-
melli, F.; Mariani, L.; Decensi, A.; Camerini, T.; Del Turco,
M. R.; Di Mauro, M. G.; Muraca, M. G.; Del Vecchio, M.;
Pinto, C.; D’Aiuto, G.; Boni, C.; Campa, T.; Magni, A.;
Miceli, R.; Perloff, M.; Malone, W. F.; Sporn, M. B. J. Natl.
Cancer Inst. 1999, 91, 1847.
3. Mulder, G. J.; Coughtrie, M. W. H.; Burchell, B. In Con-
jugation Reactions in Drug Metabolism; Mulder, G. J., Ed.;
Taylor and Francis: London, 1990; p 52.
4. Abou-Issa, H. M.; Curley, R. W., Jr; Panigot, M. J.;
Tanagho, S. N.; Sidhu, B. S.; Alshafie, G. A. Anticancer Res.
1997, 17, 3335.
To further justify a full chemotherapeutic study of 5, a
pilot study of the relative antitumor activity was under-
taken using previously described methods.4,18 Female
rats treated ca. 50 days earlier with dimethylbenz[a]an-
thracene (DMBA) were fed for 10 days with the treat-
ment retinoid. Each treatment group consisted of three
tumor-bearing rats, which were killed after the 10 days
of feeding. Each tumor on each rat was measured to
estimate the mean tumor volume per group. Table 3
5. Panigot, M. J.; Humphries, K. A.; Curley, R. W. J. Car-
bohydr. Chem. 1994, 13, 303.
Table 3. Effect of retinoid treatment on DMBA-induced rat mam-
mary tumor volumea
6. Abou-Issa, H. M.; Alshafie, G. A.; Curley, R. W., Jr;
Wong, M. F.; Clagett-Dame, M.; Repa, J. J.; Sikri, V. Anti-
cancer Res. 1999, 19, 999.
7. Levy, D. E.; Tang, C. The Chemistry of C-Glycosides; Per-
gamon: Oxford, 1995.
Dietary
supplementb
Initial tumor
volumec
Final tumor
volumec
% Change
+224d
ꢃ40e
ꢃ25f
8. Postema, M. H. D. C-Glycoside Synthesis; CRC: London,
1995.
9. Rajanbabu, T. V.; Reddy, G. S. J. Org. Chem. 1986, 51,
5458.
10. Wong, M. F.; Weiss, K. L.; Curley, R. W., Jr. J. Carbo-
hydr. Chem. 1996, 15, 763.
11. Johns, B. A.; Pan, Y. T.; Elbein, A. D.; Johnson, C. R. J.
Am. Chem. Soc. 1997, 119, 4856.
Control
0.25ꢁ0.12
0.94ꢁ0.23
0.16ꢁ0.050.12
0.29ꢁ0.17
0.56ꢁ0.28
0.56ꢁ0.13
ꢁ0.03
Retinoic acid (2)
4-HPR (3)
4-HPRCG (5)
0.23ꢁ0.12
ꢃ21g
aAt 10 days of feeding diet to three rats.
bRetinoid dose of 2 mmol/kg diet.
cValues=mean ꢁSE (cm3).
dTwo new tumors formed during the 10 days, no regression in 14/14
tumors.
12. Johnson, C. R.; Johns, B. A. Synlett 1997, 1406.
13. Petasis, N. A.; Bzowej, E. I. J. Am. Chem. Soc. 1990, 112,
6392.
eNo new tumors, partial regression in 10/10 tumors.
fTwo new tumors, partial regression in 10/14 tumors.
gNo new tumors, partial regression in 6/8 tumors.
14. Csuk, R.; Glanzer, B. I. Tetrahedron 1991, 47, 1655.

2450
J. R. Walker et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2447–2450
15. Davis, N. J.; Flitsch, S. L. Tetrahedron Lett. 1993, 34, 1181.
16. (Methyl 2,3,4-tri-O-acetyl-ꢀ-D-glucopyranosyluronate)-4-
nitrophenylmethane (10). Oil 9 (4.14 g, 8.71 mmol) was dis-
solved in 120 mL of methanol and placed in an rt water bath.
HCl (6 N, 80 mL) was added and the solution stirred for 18 h.
The mixture was concentrated to a paste. Saturated NaHCO3
solution (150 mL) was added along with KBr (200 mg) and
TEMPO (200 mg). Clorox1 (150 mL) was then added drop-
wise while monitoring the pH of the reaction. NaOH (aq) was
used to maintain the pH between 9 and 10. After the addition
of the Clorox1 was complete, the reaction was stirred for 2 h.
The contents were washed (3ꢄ) with CH2Cl2, the aqueous
layer acidified to pH 2 and concentrated to dryness. The
remaining solid was triturated with methanol, which was then
transferred to a two-neck flask. Next, HCl gas was bubbled
through the solution warmed to 40 ꢂC for 5h. The reaction
mixture was concentrated and placed in an rt water bath.
Acetic anhydride (100 mL), pyridine (100 mL), and DMAP
(75mg) were added and the mixture stirred for 18 h. The
contents were diluted with water and extracted (3ꢄ) with ethyl
acetate. The organic layers were washed with water, brine, and
dried (MgSO4). This suspension was filtered and concentrated
to a brown gum, dissolved in hot ethanol, and upon cooling
gave 3.33 g (84%) of white crystals, mp 133–134 ꢂC. IR (cmꢃ1
3078 (w), 2952 (w), 2850 (w), 1758 (s), 1603 (m), 1521 (s), 1346
)
1
(s), 1219 (s), 1036 (m); H NMR (400 MHz, DMK-d6) d 1.94
(s, 3H), 1.96 (s, 3H), 2.00 (s, 3H), 2.94 (dd, 1H, J=14.7, 8.6
Hz), 3.12 (dd, 1H, J=14.7, 3.1 Hz), 3.65(s, 3H), 4.07 (dt, 1H,
J=8.6, 3.1 Hz), 4.21 (d, 1H, J=9.8 Hz), 4.92 (t, 1H, J=9.8
Hz), 5.06 (t, 1H, J=9.8 Hz), 5.31 (t, 1H, J=9.8 Hz), 7.56 (d,
2H, J=8.7 Hz), 8.15(d, 2H, J=8.7 Hz); 13C NMR (100 MHz,
DMK-d6) d 20.38, 20.47, 20.62, 37.93, 52.72, 70.47, 72.33,
73.92, 76.30, 77.91, 123.92, 131.55, 146.37, 147.74, 168.26,
169.89, 170.29; HRMS (ES) calcd for C20H23NO11 (M+Na)
476.1169, found 476.1175.
17. Three steady-state NOE experiments were performed on
intermediate 10. Irradiation at H1 (see Scheme 1 for number-
ing) resulted in enhancement of H5(12%), H3 (9%), H2 0 (5%)
and H10 (4%) at 3.12 ppm. Irradiation at H3 resulted in
enhancement of H5(7%) and H1 (6%). Irradiation at H5
resulted in enhancement of H1 (10%) and H3 (8%).
18. Weiss, K. L.; Alshafie, G.; Chapman, J. S.; Mershon,
S. M.; Abou-Issa, H.; Clagett-Dame, M.; Curley, R. W., Jr.
Bioorg. Med. Chem. Lett. 2001, 11, 1583.