Synthesis of a novel prodrug of 3-(4′-geranyloxy-3′- methoxyphenyl)-2-trans-propenoic acid for colon delivery

Article abstract of DOI:10.1016/j.bmcl.2005.07.088

A novel high-yielding and environment-friendly synthesis of the anticancer compound 3-(4′-geranyloxy-3′-methoxyphenyl)-2-trans-propenoic acid is described. This compound was conjugated to H₂N-Ala-Pro dipeptide to give a prodrug to be activated by intestinal ACE and to be used in the treatment of different forms of colon cancer. Data on the chemical and enzymatic stability of this novel prodrug are reported.

Full text of DOI:10.1016/j.bmcl.2005.07.088

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5049–5052
Synthesis of a novel prodrug of 3-(40-geranyloxy-30-
methoxyphenyl)-2-trans-propenoic acid for colon delivery
Massimo Curini,* Francesco Epifano and Salvatore Genovese
Dipartimento di Chimica e Tecnologia del Farmaco, Sezione di Chimica Organica, Via del Liceo, 06123 Perugia, Italy
Received 23 June 2005; revised 27 July 2005; accepted 29 July 2005
Available online 13 September 2005
Abstract—A novel high-yielding and environment-friendly synthesis of the anticancer compound 3-(40-geranyloxy-30-methoxyphe-
nyl)-2-trans-propenoic acid is described. This compound was conjugated to H2N-Ala-Pro dipeptide to give a prodrug to be activated
by intestinal ACE and to be used in the treatment of different forms of colon cancer. Data on the chemical and enzymatic stability of
this novel prodrug are reported.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Ferulic acid (1) is a widespread natural compound
belonging to the class of cinnamic acids.1,2 In the last
decade, ferulic acid and its derivatives have attracted
the attention of many researchers due to their valuable
pharmacological properties.
and by combined lipopolysaccharide and interferon-c,6
and suppression of inducible nitric oxide synthase5 and
cycloxygenase-2 as such and of its promoter activities;6,7
furthermore, some myo-inositol esters of (2) exerted a
good inhibitory activity on phorbol ester-induced super-
oxide generation and Epstein–Barr virus activation.8
These data were collected using whole cell systems so
it is conceivable that, under experimental conditions,
the above-reported esters could be hydrolyzed to
the parent acid once inside the cell, so the true active
principle exerting these biological effects would be (2).
Then 3-(40-geranyloxy-30-methoxyphenyl)-2-trans-prop-
enoic acid becomes a novel candidate as chemopreven-
tive drug for the cure of various types of cancer and
as anti-inflammatory compound. For these reasons the
design and synthesis of biologically active new ferulic
acid derivatives is a field of current and growing interest.
CO2H
RO
OCH3
1 R = H
2 R = geranyl
A biosynthetically related secondary metabolite of (1) is
3-(40-geranyloxy-30-methoxyphenyl)-2-trans-propenoic
acid (2), in which a geranyl chain is attached to the phe-
nolic group. It has been isolated in 1966 from the bark
of Acronychia baueri Schott, an Australian small tree
belonging to the family of Rutaceae.3
In the last three years, our work was devoted to the
design and synthesis of natural and semisynthetic gera-
nyloxy substituted compounds as colon cancer chemo-
preventive agents. One of the approaches for the
treatment of local diseases of colon is the use of pro-
drugs which optimizes drug delivery, improves its effica-
cy, and lowers the absorption and release of the drug in
the stomach and small intestine thereby facilitating
quantitative delivery to the colon and increasing the bio-
availability at the site of action. The different approach-
es for colonic drug delivery have been recently
exhaustively reviewed.9 Among these, a promising one
is represented by the selective cleavage of prodrugs con-
taining an oligopeptide chain by enzymes located in the
external side of the intestinal brush border membrane.10
Although known for four decades, only in the last five
years some of the pharmacological properties of (2)
and its synthetic derivatives began to be characterized.
In particular, the ethyl ester of (2) showed a series of
interesting biological effects such as colon and tongue
cancer chemoprevention by dietary feeding in rats,4,5
inhibition of cellular responses induced by phorbol ester
Keywords: Cinnamic acids; Colon delivery; Colon cancer; Ferulic acid
derivatives; Prodrug.
*
Corresponding author. Tel.: +390755855106; fax: +390755855116;
e-mail: curmax@unipg.it
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.088

5050
M. Curini et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5049–5052
Different classes of brush border enzymes have been
identified, among which are peptidases, characterized
by different selectivity and distribution along the small
and large intestines.11,12 In particular, angiotensin con-
verting enzyme (ACE, dipeptidyl carboxypeptidase,
E.C. 3.4.15.1) hydrolyzes oligopeptides in which the ter-
minal residue is proline and has alanine or glycine in the
penultimate position.13
(2) and other prenyloxy-substituted cinnamic acid deriv-
atives by means of a short and environment-friendly
procedure, simple purification after standard work-up,
not needing chromatographic separation, and very high
yield with respect to those reported in the literature.3,5,14
Coupling of (2) with commercially available L-alanine
methyl ester hydrochloride in the presence of 1,3-
dicyclohexylcarbodiimide (DCC) and triethyl amine in
Et2O at room temperature gave ester (5), which was
subsequently transformed into 3-(40-geranyloxy-
30-methoxyphenyl)-2-trans-propenoyl-L-alanine (6) by
hydrolysis with hydroalcoholic NaOH 0.5 N. The same
two-step reaction sequence repeated for the coupling
of acid (6) with commercially available L-proline methyl
ester hydrochloride and subsequent hydrolysis gave
the desired 3-(40-geranyloxy-30-methoxyphenyl)-2-trans-
propenoyl-L-alanyl-L-proline (3).15 The overall yield of
the ꢀferulic acid to (3)ꢁ process was 56.6%.
The aim of our study was to synthesize a properly struc-
tured prodrug of (2) that could be selectively cleaved by
intestinal ACE so reaching in high concentration the
large bowel. Basing on the selectivity of this enzyme
(2) must be incorporated to H2N-Ala-Pro dipeptide,
such as in (3).
O
N
N
CO2H
H
O
O
The chemical stability of compound (3) was assayed by
incubating it at 37 ꢁC for 2 h in aqueous solutions at dif-
ferent pH values ranging from 0.5 to 9.5:16 at any pH
value tested (3) was absolutely stable and has been
recovered in quantitative yield from all reaction media.
The degree of enzymatic hydrolysis by large bowel exo-
peptidases and commercially available purified ACE
was assessed using the method reported by Kim and
co-workers13 after incubation of (3) for 30 min at
37 ꢁC with isolated intestinal brush border membranes
or purified ACE, the parent acid (2) was detected as
the only hydrolytic product.
OCH3
3
The synthesis of (3) was accomplished using commer-
cially available ferulic acid (1) as starting material
(Scheme 1). Initially, the acid (1) was easily transformed
into the corresponding methyl ester (4) by treatment
with refluxing MeOH in the presence of catalytic
amount of concd H2SO4. The 40-geranyloxy derivative
(2) was obtained by a two-step one-pot procedure: ger-
anylation of (4) with geranyl bromide and anhydrous
K2CO3 in acetone at 60 ꢁC and subsequent treatment
in the same vessel with a basic medium. The described
procedure represents a new entry to the synthesis of
In conclusion, the sequence described here provides
a new high-yielding synthesis of a novel prodrug of
3-(40-geranyloxy-30-methoxyphenyl)-2-trans-propenoic
acid, a compound subject of more growing interest as
potential drug for cancer treatment. Moreover this is,
to the best of our knowledge, the first example of pro-
drug activation by intestinal ACE, so that it could be
a novel approach of enzyme targeting for drug specific
colon delivery. Its mechanism of activation would en-
sure chemical and enzymatic stability while passing
through the stomach and small intestine and a selective
release in near proximity of the large bowel. Results ob-
tained from in vitro tests prompted us to carry out in vi-
vo pharmacological studies on (3) as a novel colon
cancer chemopreventive agent by dietary feeding in rats
that are now in progress.
CO2CH3
a
b,c
d,e
2
1
HO
4
OCH3
O
N
H
CO2R
GerO
5 R = CH3
6 R = H
OCH3
f,g
Acknowledgments
O
N
O
N
Financial support from MIUR National Project ꢀSvilup-
po di processi sintetici ecocompatibili nella sintesi
organica,ꢁ COFIN 2004 and from University of
Perugia, Italy, is gratefully acknowledged.
H
CO2R
GerO
OCH3
7 R = CH3
3 R = H
Scheme 1. Reagents and conditions: (a) MeOH, concd H2SO4, reflux,
overnight, 98% yield; (b) K2CO3, geranyl bromide, acetone, 60 ꢁC, 2 h;
(c) NaOH 2 N, 60 ꢁC, 3 h, 99% yield; (d) L-AlaOMeÆHCl, DCC, Et3N,
Et2O, rt, 6 h, 91% yield; (e) NaOH 0.5 N, EtOH/H2O 1:1 30 min, 88%
yield; (f) L-ProOMeÆHCl, DCC, Et3N, Et2O, rt, 6 h, 81% yield; (g)
NaOH 0.5 N, EtOH/H2O 1:1 30 min, 90% yield.
Supplementary material
Supplementary data associated with this article can be
found in the online version at doi:10.1016/j.bmcl.2005.
07.088.

M. Curini et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5049–5052
5051
phenyl)-2-trans- propenoic acid (2) (1.87 g, 5.7 mmol)
was dissolved in 57 mL of anhydrous Et2O and to this
solution were added triethyl amine (0.87 mL, 6.2 mmol)
and L-AlaOMeÆHCl (0.87 g, 6.2 mmol). A solution of
DCC (1.29 g, 6.2 mmol) in Et2O (25 mL) was then added
dropwise over a period of 30 min and the resulting
suspension was stirred at room temperature for 6 h and
filtered under vacuum. The ethereal solution was washed
in turn with 5% aq NaHCO3 (2· 50 mL) and 5% aq citric
acid (2· 50 mL), dried over Na2SO4, and evaporated to
dryness giving (5) as a pale yellow solid (2.15 g, 91%
yield). [a] (c 1, CH2Cl2) = +21.5; mp: 95–97 ꢁC; IR
(cmꢀ1): 1710, 1670; 1H NMR (200 MHz, CDCl3, d)
1.49 (d, 3H, J = 7.1 Hz), 1.61 (s, 3H), 1.68 (s, 3H), 1.75
(s, 3H), 1.95–2.12 (m, 4H), 3.79 (s, 3H), 3.90 (s, 3H),
4.64–4.67 (m, 2H), 4.81 (q, 1H, J = 7.1 Hz) 5.04–5.17 (m,
1H), 5.48–5.55 (m, 1H), 6.40 (d, 1H, J = 16.0 Hz), 6.83–
7.18 (m, 3H), 7.58 (d, 1H, J = 16.0 Hz); 13C NMR
(50 MHz, CDCl3, d) 16.7, 17.6, 18.3, 24.9, 25.3, 41.4,
48.1, 52.4, 56.5, 65.7, 109.8, 112.5, 117.8, 122.2, 123.7,
127.0, 131.7, 141.0, 141.6, 149.3, 149.8, 166.2, 173.8;
HRMS (EI) Calcd for, C24H33NO5: 415.2358. Found:
415.2352.
References and notes
1. Clifford, M. N. J. Sci. Food Agric. 1999, 79, 362.
2. Kroon, P. A.; Garcia-Conesa, M. T.; Fillingham, I. J.;
Hazlewood, G. P.; Williamson, G. J. Sci. Food Agric.
1999, 79, 428.
3. Prager, R. H.; Thregold, H. M. Aust. J. Chem. 1966, 19,
451.
4. Han, B. S.; Park, C. B.; Takasuka, N.; Naito, A.; Sekine,
K.; Nomura, E.; Taniguchi, H.; Tsuno, T.; Tsuda, H. Jpn.
J. Cancer Res. 2001, 92, 404.
5. Tanaka, T.; Kohno, H.; Nomura, E.; Taniguchi, H.;
Tsuno, T.; Tsuda, H. Oncology 2003, 64, 166, and
references cited herein.
6. Murakami, A.; Kadota, M.; Takahashi, D.; Taniguchi,
H.; Nomura, E.; Hosoda, A.; Tsuno, T.; Maruta, Y.;
Ohigashi, H.; Koshimizu, K. Cancer Lett. 2000, 157, 77.
7. Hosoda, A.; Ozaki, Y.; Kashiwada, A.; Mutoh, M.;
Wakabayashi, K.; Mizuno, K.; Nomura, E.; Taniguchi, H.
Bioorg. Med. Chem. 2002, 10, 1189.
8. Hosoda, A.; Nomura, E.; Murakami, A.; Koshimizu, K.;
Ohigashi, H.; Mizuno, K.; Taniguchi, H. Bioorg. Med.
Chem. Lett. 2002, 10, 1855.
3-(40-Geranyloxy-30-methoxyphenyl)-2-trans-propenoyl-
L-alanine (6). 3-(40-Geranyloxy-30-methoxyphenyl)-2-
trans-propenoyl-L-alanine methyl ester (5) (0.68 g,
1.6 mmol) was dissolved in EtOH (5 mL) and to this
solution 5 mL of 1 N aq NaOH was added. The resulting
mixture was stirred for 1 h at room temperature, acidified
to pH 1 with 10% HCl, and extracted with Et2O (3·
30 mL). The collected organic phases were dried over
Na2SO4 and evaporated to dryness giving (6) as a pale
yellow solid (0.57 g, 88% yield). [a] (c 1,
CH2Cl2) = ꢀ50.6; mp: 113–115 ꢁC; IR (cmꢀ1): 3500
(br), 1710, 1670; 1H NMR (200 MHz, CDCl3, d) 1.52
(d, 3H, J = 7.1 Hz), 1.61 (s, 3H), 1.69 (s, 3H), 1.74 (s,
3H), 2.02–2.12 (m, 4H), 3.84 (s, 3H), 4.61–4.64 (m, 2H),
4.75 (q, 1H, J = 7.1 Hz) 4.99–5.09 (m, 1H), 5.45–5.52 (m,
1H), 6.43 (d, 1H, J = 15.8 Hz), 6.80–7.15 (m, 3H), 7.74
(d, 1H, J = 15.8 Hz); 13C NMR (50 MHz, CDCl3, d)
16.7, 17.7, 18.0, 25.7, 26.2, 39.5, 48.6, 55.8, 65.7, 109.8,
111.8, 117.2, 119.2, 122.3, 123.7, 127.3, 131.8, 141.2,
142.3, 149.3, 150.1, 166.9, 176.0; HRMS (EI) Calcd for,
C23H31NO5: 401.2202. Found: 401.2209.
3-(40-Geranyloxy-30-methoxyphenyl)-2-trans-propenoyl-
L-alanyl-L-proline methyl ester (7). The same procedure to
obtain compound (5) was followed. (81% yield). [a] (c 1,
CH2Cl2) = ꢀ15.2; mp: 143–146 ꢁC; IR (cmꢀ1): 1725, 1710,
1670; 1H NMR (200 MHz, CDCl3, d) 1.48 (d, 3H,
J = 7.1 Hz), 1.60 (s, 3H), 1.68 (s, 3H), 1.73 (s, 3H), 1.88–
2.37 (m, 10H), 3.53–3.61 (m, 1H), 3.72 (s, 3H), 3.91 (s, 3H),
4.60–4.63 (m, 1H), 4.65–4.68 (m, 2H), 4.99– 5.02 (m, 1H),
5.47–5.51 (m, 1H), 6.39 (d, 1H,J = 15.9 Hz), 6.82–7.18 (m,
3H), 7.51 (d, 1H, J = 15.9 Hz); 13C NMR (50 MHz, CDCl3,
d) 16.5, 17.7, 18.1, 24.6, 25.2, 25.6, 26.1, 33.0, 39.0, 47.4,
49.0, 54.8, 55.8, 65.8, 112.0, 119.1, 121.5, 122.0, 122.4, 123.7,
127.6, 131.2, 133.7, 141.0, 141.2, 149.4, 157.1, 166.2, 173.9;
HRMS (EI) Calcd for, C29H40N2O6: 512.2886. Found:
512.2880.
9. Sinha, V. R.; Kumria, R. Pharm. Res. 2001, 18, 557.
10. Pellicciari, R.; Garzon-Aburbeh, A.; Natalini, B.; Mari-
nozzi, M.; Clerici, C.; Gentili, G.; Morelli, I. J. Med.
Chem. 1993, 36, 4201, and references cited herein.
11. Skowbjerg, H. Clin. Chim. Acta 1981, 112, 205.
12. Auricchio, S.; Greco, L.; DeVizia, B.; Buonocore, V.
Gastroenterology 1978, 75, 1073.
13. Yoshioka, M.; Erickson, R. H.; Kim, Y. S. J. Clin. Invest.
1988, 81, 1090.
14. Hosoda, A.; Miyake, Y.; Nomura, E.; Mizuno, K.; Tanig-
uchi, H. ITE Lett. Batt. New Tech. Med. 2001, 2, 659.
15. Experimental. Methyl ferulate (4). Ferulic acid (1)
(15.0 g, 77.2 mmol) was dissolved in 300 mL of dry
MeOH and to this solution few drops of concd H2SO4
were added. The resulting mixture was stirred at reflux
overnight, then basified with aq 5% NaHCO3 and
extracted with EtOAc (3· 200 mL). The collected organic
phases were dried over Na2SO4 and evaporated to
dryness giving (4) (16.0 g, 98% yield) as brownish solid.
This compound was identified by comparison with a
commercially available sample.
3-(40-Geranyloxy-30-methoxyphenyl)-2-trans-propenoic
acid (2). Methyl ferulate (4) (1.26 g, 3.0 mmol) was
dissolved in dry acetone (15 mL) and to this solution
were added anhydrous K2CO3 (0.50 g, 3.6 mmol) and
geranyl bromide (0.65 mL, 3.3 mmol). The resulting
mixture was stirred at 60 ꢁC for 2 h, then 2 N NaOH
(15 mL) was added and the reaction mixture was stirred
at 90 ꢁC for additional 3 h. The cooled solution was
diluted with H2O (50 mL) and extracted twice with Et2O
(30 mL). The aqueous phase was acidified to pH 2 with
10% HCl and extracted with Et2O (3· 50 mL). The
collected organic phases were dried over Na2SO4 and
evaporated to dryness giving (2) as a white solid (0.33 g,
1
99% yield). Mp: 60–61 ꢁC; IR (cmꢀ1): 3500 (br), 1710, H
NMR (200 MHz, CDCl3, d) 1.63 (s, 3H), 1.70 (s, 3H),
1.77 (s, 3H), 2.04–2.24 (m, 4H), 3.94 (s, 3H), 4.68–4.72
(m, 2H), 5.06– 5.12 (m, 1H), 5.50–5.56 (m, 1H), 6.35 (d,
1H, J = 16.2 Hz), 6.88–7.30 (m, 3H), 7.76 (d, 1H,
J = 16.2 Hz); 13C NMR (50 MHz, CDCl3, d) 16.7, 17.7,
25.7, 26.2, 39.5, 55.9, 65.8, 109.9, 112.5, 114.7, 119.1,
123.0, 123.7, 126.8, 131.8, 141.2, 147.0, 149.5, 150.8,
172.7; HRMS (EI) Calcd for, C20H26O4: 330.1831.
Found: 330.1828.
3-(40-Geranyloxy-30-methoxyphenyl)-2-trans-propenoyl-
L-alanyl-L-proline (3). The same procedure to obtain
compound (6) was followed (90% yield). Pale yellow
solid; [a] (c 1, CH2Cl2) = ꢀ38.8; mp: 62–63 ꢁC; IR
(cmꢀ1): 3500 (br), 1739, 1658, 1655; 1H NMR
(200 MHz, CDCl3, d) 1.41 (d, 3H, J = 6.9 Hz), 1.61 (s,
3H), 1.68 (s, 3H), 1.74 (s, 3H), 1.89–2.39 (m, 10H), 3.52–
3.64 (m, 1H), 3.90 (s, 3H), 4.63–4.66 (m, 1H), 4.67–4.70
(m, 2H), 4.98–5.01 (m, 1H), 5.48–5.52 (m, 1H), 6.38 (d,
1H, J = 15.9 Hz), 6.83–7.30 (m, 3H), 7.55 (d, 1H,
3-(40-Geranyloxy-30-methoxyphenyl)-2-trans-propenoyl-
L-alanine methyl ester (5). 3-(40-Geranyloxy-30-methoxy-

5052
M. Curini et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5049–5052
J = 15.9 Hz); 13C NMR (50 MHz, CDCl3, d) 16.6, 17.8,
18.2, 24.8, 25.4, 25.6, 26.2, 33.1, 39.4, 47.5, 49.4, 55.8,
65.8, 112.4, 119.2, 121,4, 122.0, 122.6, 123.7, 127.6, 131.5,
133.9, 141.0, 141.3, 149.4, 157.7, 166.8, 174.1; HRMS
(EI) Calcd for, C28H38N2O6: 498.2729. Found: 498.2721.
For experiments aimed to evaluate the chemical and
enzymatic stability of compound (3), see Refs. 13, 16.
16. Landon, M. Methods Enzymol. 1977, 47, 145.