Prenyloxyphenylpropanoids as novel lead compounds for the selective inhibition of geranylgeranyl transferase I

Article abstract of DOI:10.1016/j.bmcl.2007.01.097

In this study, we synthesized some natural and semisynthetic prenyloxyphenylpropanoids (e.g., coumarins and cinnamic acid derivatives) and we assessed their in vitro inhibitory activity against farnesyl transferase (FTase) and geranylgeranyl transferase I (GGTase I). No compound was an effective inhibitor of FTase, while farnesyloxycinnamic acids were shown to selectively inhibit GGTase I with IC₅₀ values ranging from 28 to 39 μM.

Full text of DOI:10.1016/j.bmcl.2007.01.097

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2639–2642
Prenyloxyphenylpropanoids as novel lead compounds
for the selective inhibition of geranylgeranyl transferase I
Francesco Epifano,a,* Massimo Curini,b Salvatore Genovese,b
Michelle Blaskovich,c Andrew Hamiltonc,d and Said M. Sebtic
aDipartimento di Scienze del Farmaco, Via dei Vestini 31, 66013 Chieti Scalo (CH), Italy
bDipartimento di Chimica e Tecnologia del Farmaco, Sezione di Chimica Organica, Via del Liceo, 06123 Perugia, Italy
cDrug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, Department of Interdisciplinary Oncology,
University of South Florida College of Medicine, Tampa, FL 33612, USA
dDepartment of Chemistry, Yale University, New Haven, CT 06520, USA
Received 20 December 2006; revised 29 January 2007; accepted 30 January 2007
Available online 2 February 2007
Abstract—In this study, we synthesized some natural and semisynthetic prenyloxyphenylpropanoids (e.g., coumarins and cinnamic
acid derivatives) and we assessed their in vitro inhibitory activity against farnesyl transferase (FTase) and geranylgeranyl transferase
I (GGTase I). No compound was an effective inhibitor of FTase, while farnesyloxycinnamic acids were shown to selectively inhibit
GGTase I with IC50 values ranging from 28 to 39 lM.
Ó 2007 Elsevier Ltd. All rights reserved.
Prenyltransferases such as farnesyltransferase (FTase)
and geranylgeranyltransferase I (GGTase I) are excel-
lent targets for designing novel anticancer drugs since
the small GTPases of the Ras superfamily are involved
in neoplastic transformation.1–5 For example, Ras pro-
teins which are farnesylated and Rho and Ral proteins
which are geranylgeranylated are found persistently acti-
vated in human cancers. Furthermore, a large number
of studies demonstrated the involvement of these GTP-
ases in uncontrolled cell division, resistance to apopto-
sis, angiogenesis, invasion and metastasis.1–6 The fact
that post-translational modifications of small GTPases
by FTase or GGTase I are required for their cancer
causing activity prompted us and others to design and
develop inhibitors of these two enzymes as novel anti-
cancer drugs. FTase and GGTase I transfer the 15-car-
bon farnesyl and the 20-carbon geranylgeranyl,
respectively, from farnesylpyrophosphate (FPP) and
geranylgeranylpyrophosphate (GGPP) to the cysteine
of proteins that end with CaaX sequence (C = cysteine,
a = aliphatic amino acid, and X = any amino acid) at
their carboxyl termini.1–6 FTase prefers when X is
methionine or serine, whereas GGTase I prefers when
X is leucine or isoleucine. To date most FTase and
GGTase I inhibitors have focused on the development
of inhibitors that compete with the CaaX binding site,
and only a few have targeted the FPP and GGPP bind-
ing sites.
In the last five years, our research group studied chem-
ical and pharmacological properties of secondary
metabolites of phenylpropanoid biosynthetic origin con-
taining a sesquiterpenyl, monoterpenyl, and isopentenyl
chains attached to a phenol group, that represents quite
a rare group of natural products.7–11 Among these the
ethyl ester (2) of 3-(40-geranyloxy-30-methoxyphenyl)-
2-trans propenoic acid (1), the latter isolated in 1966
from the bark of Acronychia baueri Schott, an Austra-
lian small plant belonging to the family of Rutaceae,12
showed a series of interesting biological effects such as
cancer chemoprevention by dietary feeding in rats and
other effects closely related to cancer growth and devel-
opment, that were recently reviewed.13
In continuation of our studies aimed to evaluate phar-
macological properties of natural and semi-synthetic
prenyloxyphenylpropanoids, we wish to report herein
the activity of these compounds as in vitro inhibitors
of prenyl transferases, namely FTase and GGTase I.
In addition to compounds (1) and (2), we synthesized
Keywords: Prenyloxyphenylpropanoids; Prenyltransferase; Coumarins;
Cinnamic acid derivatives; Anticancer activity.
*
Corresponding author. Tel.: +39 08713555321; fax: +39
08713555315; e-mail: fepifano@unich.it
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.097

2640
F. Epifano et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2639–2642
and evaluated six natural prenyloxyphenylpropanoids,
namely 3-(40-geranyloxy-30-OH-phenyl)-2-trans prope-
noic acid (3), isolated from the same source of com-
pound (1),12 boropinic acid (5), isolated from Boronia
pinnata Sm.,14 valencic acid (9), isolated from Citrus sin-
ensis L. and Aegle marmelos (Fam. Rutaceae),15 4-iso-
COOH
COOEt
a
HO
HO
R
R
pentenyloxy-3-methoxy
benzoic
acid
(11),
4-
b,c
geranyloxy-3-methoxy benzoic acid (12), both isolated
as methyl esters from the liverwort Trichocolea lanata
(Ehrh.) Dumm. (Fam. Trichocolaceae),16 umbellipre-
nine (13), a farnesyloxycoumarin commonly found in
Ammi, Ruta and Citrus species,8 auraptene (14), the
most abundant geranyloxycoumarin extracted from
plants belonging to genus Citrus,8 collinin (15), isolated
from Zanthoxylum schinifolium,8 7-isopentenyloxy-
coumarin (16), extracted from plants belonging to genus
Ruta,8 and four semi-synthetic compounds, namely (4),
the ethyl esters of acid (3), 3-(40-isopentenyloxy-30-OH-
phenyl)-2-trans propenoic acid (6), 3-(40-farnesyloxy-30-
OH-phenyl)-2-trans propenoic acid (7), 3-(40-farnesyl-
oxy-30-methoxyphenyl)-2-trans propenoic acid (8) and
finally 40-geranyloxybenzoic acid (10).
COOEt
O
R
R = -H, OMe
Figure 1. Reagents and conditions: (a) EtOH, concd H2SO4 (cat.),
reflux 12 h; (b) geranyl bromide (1.2 equiv.), K2CO3 (1.2 equiv.),
acetone, reflux 2 h; (c) crystallization.
into ethyl esters by reaction in refluxing EtOH under
catalysis of concd H2SO4, alkylated with geranyl bro-
mide in refluxing acetone using dry K2CO3 as base
and finally purified by crystallization in n-hexane
(Fig. 1).18
R'
Acids (7) and (8) were obtained by the same procedure
reported for the synthesis of compounds (1), (3) and
(5)11 in 78% and 84% yield, respectively, and using all
trans-farnesyl bromide as alkylating agent.17 Finally
prenyloxycoumarins (13) and (16) were synthesized in
86% and 99% yield, respectively, by the same procedure
reported for the synthesis of auraptene and collinin,7
using all trans-farnesyl bromide and 4-bromo-2-meth-
yl-2-butene as alkylating agents.16
R'''O
OR''
3 R' =-CH=CH-COOH, R'' = -H, R''' = geranyl
4 R' = -CH=CH-COOEt, R'' = -H, R''' = geranyl
5 R' = -CH=CH-COOH, R'' = -OMe, R''' = isopentenyl
6 R' = -CH=CH-COOH, R'' = -H, R''' = isopentenyl
7 R' = -CH=CH-COOH, R'' = -H, R''' = farnesyl
8 R' = -CH=CH-COOH, R'' = -OMe, R''' = farnesyl
9 R' = -COOH, R'' = -H, R''' = isopentenyl
10 R' = -COOH, R'' = -H, R''' = geranyl
11 R' = -COOH, R'' = -OMe, R''' = isopentenyl
12 R' = -COOH, R'' = -OMe, R''' = geranyl
Compounds 1–16 were then evaluated for their ability to
inhibit in vitro FTase and GGTase I at a concentration
of 100 lM (Table 1).
As shown in Table 1 a well defined and distinguished
pattern of results was recorded. None of the 16 com-
OR'
Table 1. Effects of prenyloxyphenylpropanoids 1–16 (100 lM) on
FTase and GGTase I inhibition in vitro
R''O
O
O
Compound
% Inhibition
FTase
GGTase I
13 R' = -H, R'' = farnesyl
14 R' = -H, R'' = geranyl
15 R' = -OMe, R'' = geranyl
16 R' = -H, R'' = isopentenyl
1
2
13.4 6.4
5.5 0.6
78.6 12.8
3.0 13.4
72.4 9.4
7.5 21.5
31.0 (n = 2)
46.4 (n = 2)
93.5 (n = 2)
83.9 (n = 2)
0 (n = 1)
3
12.7 23.0
ꢀ7.4 22.1
9.3 (n = 2)
43.2 (n = 2)
16.2 (n = 2)
11.0 (n = 2)
0 (n = 2)
4
5
6
7
8
Compounds (1), (3), (5), (9), (11), (12), (14), and (15)
were synthesized as already reported.11 The synthesis
of compounds (2), (4), (6), (7), (8), (10), (13), and (16)
was accomplished following an environmentally friendly
route similar to that already described.7,11 Ethyl esters
(2) and (4) were obtained in 89% and 92% overall yield,
respectively, starting from commercially available ferulic
and trans p-coumaric acids, that were first converted
9
10
11
12
13
14
15
16
17.6 (n = 2)
2.4 (n = 2)
0 (n = 2)
0 (n = 1)
0 (n = 1)
0 (n = 1)
12.5 4.8
7.9 9.9
13.4 6.4
18.6 6.1
34.2 6.9
ꢀ10.3 15.4
15.5 15.9
14.1 14.8

F. Epifano et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2639–2642
2641
Table 2. IC50 values for inhibition of GGTase I for compounds 1, 3, 7
and 8
Acknowledgments
Authors from Italy wish to acknowledge financial sup-
port from MIUR (Rome, Italy) National Project ‘Svi-
luppo di processi sintetici ecocompatibili nella sintesi
organica’, COFIN 2004.
Compound
IC50 (lM)
1
3
7
8
55 14
39 9.5
28 (n = 1)
66 (n = 1)
References and notes
1. Sebti, S. M.; Hamilton, A. D. Oncogene 2000, 19, 6584.
2. Russo, P.; Loprevite, M.; Cesario, A.; Ardizzoni, A. Curr.
Med. Chem. Anticancer Agents 2004, 4, 123.
3. Pais, J. E.; Bowers, K. E.; Stoddard, A. K.; Fierke, C. A.
Anal. Biochem. 2005, 345, 302.
4. Appels, N. M.; Beijinen, J. H.; Schellens, J. H. Oncologist
2005, 10, 565.
5. Basso, A. D.; Kirschmeier, P.; Bishop, W. R. J. Lipid Res.
2006, 47, 15.
6. El Oualid, F.; Cohen, L. H.; Van der Marel, G. A.;
Overhand, M. Curr. Med. Chem. 2006, 13, 2385.
7. Curini, M.; Epifano, F.; Maltese, F.; Marcotullio, M. C.;
Tubaro, A.; Altinier, G.; Prieto Gonzales, S.; Rodriguez,
J. C. Bioorg. Med. Chem. Lett. 2004, 14, 2241, and
references cited herein.
8. Curini, M.; Cravotto, G.; Epifano, F.; Giannone, G. Curr.
Med. Chem. 2006, 1, 199, and references cited herein.
9. Curini, M.; Epifano, F.; Genovese, S. Bioorg. Med. Chem.
Lett. 2005, 15, 5049.
10. Kohno, H.; Suzuki, R.; Curini, M.; Epifano, F.; Maltese,
F.; Prieto Gonzales, S.; Tanaka, T. Int. J. Cancer 2006,
118, 2936.
11. Epifano, F.; Menghini, L.; Pagiotti, R.; Angelini, P.;
Genovese, S.; Curini, M. Bioorg. Med. Chem. Lett. 2006,
16, 5523.
pounds evaluated inhibited FTase potently. Compound
(6) was the most potent and only inhibited FTase by
43% at 100 lM. In contrast, four compounds, (1), (3),
(7), and (8) namely acids containing a geranyl or farnes-
yl side chain attached to the phenol group, inhibited
GGTase I with values ranging from 72.4% to 93.5%.
For compounds (1), (3), (7), and (8) subsequent dose re-
sponse experiments were performed, and the corre-
sponding IC50 values are shown in Table 2.
The most potent compound was the farnesyloxy deriva-
tive of trans p-coumaric acid (7) that inhibited GGTase I
with an IC50 value of 28 lM. Substituting the farnesyl
side chain with a geranyl one as in (3) (IC50 = 39 lM)
or an isopentenyl one as in (6) (IC50 ꢁ 100 lM) decreas-
es the ability of the prenyloxy derivative to inhibit
GGTase I, suggesting that the length of the isoprenyl
moiety is crucial for fully occupying the 20-carbon
GGPP binding pocket of this enzyme. Addition to (7)
of a methoxy group ortho to the 4-prenyloxy chain as
in (8) decreased its potency from an IC50 value of 28–
66 lM. Similarly, addition of a methoxy to (3) as in
(1) also decreased its potency suggesting that a methoxy
group is not preferred by the GGTase I binding pocket.
Benzoic acids are totally inactive towards inhibition of
both enzymes suggesting that an a, b-unsaturated conju-
gated carbon–carbon double bond is a main structural
feature for compounds to be active as GGT-ase I inhib-
itors. Furthermore, free acids are by far better GGTase I
inhibitors than the corresponding ethyl esters, indicating
that the binding pocket in GGTase I requires a negative-
ly charged carboxylate anion that probably mimics the
negatively charged pyrophosphate of the GGPP mole-
cule. Finally from data reported herein, it is evident that
lactones are less efficient inhibitors of GGTase I than the
corresponding cinnamic acid derivatives.
12. Prager, R. H.; Thregold, H. M. Aust. J. Chem. 1966, 19,
451.
13. Curini, M.; Epifano, F.; Genovese, S.; Marcotullio, M. C.;
Menghini, L. Anticancer Agents Med. Chem. 2006, 6, 571.
14. Ito, C.; Itoigawa, M.; Otsuka, T.; Tokuda, H.; Nishino,
H.; Furukawa, H. J. Nat. Prod 2000, 63, 1344.
15. Ali, M. S.; Pervez, M. K. Nat. Prod. Res 2004, 18, 141.
16. Perry, N. B.; Foster, L. M.; Lorimer, S. D.; May, B. C.;
Weavers, R. T. J. Nat. Prod. 1996, 59, 729.
17. Experimental. For the synthesis of compounds 1–12 the
same general procedure as reported previously was
followed (see Refs. 7,11). 3-(40-Geranyloxy-30-methoxyphe-
nyl)-2-trans propenoic acid (1). White solid; yield 96%;
analytical data are in full agreement with those reported in
the literature.9 Ethyl 3-(40-geranyloxy-30-methoxyphenyl)-
2-trans propenoate (2). White solid; yield 89%; analytical
data are in full agreement with those reported in the
literature.16 3-(40-Geranyloxyphenyl)-2-trans propenoic
acid (3). White solid; yield 97%; analytical data are in
full agreement with those reported in the literature.11
Ethyl 3-(40-Geranyloxyphenyl)-2-trans propenoate (4).
White solid; yield 92%; mp: 121–124 °C; IR (KBr):
In conclusion, the findings described in this paper indi-
cate that farnesyloxy- and geranyloxycinnamic acids
are potential lead compounds of a novel class of selec-
tive GGTase I inhibitors. It is noteworthy that the inter-
est towards molecules having this kind of mechanism of
inhibition as potential cancer therapeutic agents has
greatly increased over the past few years, and some com-
pounds having GGTase I inhibitory effects have been
reported.6 Considering that all compounds tested have
been easily synthesized from widely available and non-
toxic starting materials by a high-yielding, environmen-
tally friendly and cheap synthetic route, these results
provide further insights into the mechanism of action
and help to better define the pharmacological profile
of these secondary metabolites and related semi-synthet-
ic derivatives.
1
1685 cmꢀ1; H NMR (200 MHz, CDCl3 d): 1.33 (t, 3 H,
J = 7.2 Hz), 1.59 (s, 3H), 1.66 (s, 3H), 1.72 (s, 3H), 2.09–
2.25 (m, 4H), 4.25 (q, 2H, J = 7.2 Hz), 4.45–4.48 (m, 2H),
5.05–5.12 (m, 1H), 5.46–5.51 (m, 1H), 6.41 (d, 1H,
J = 3.6 Hz), 6.86–6.91 (m, 2H), 7.39–7.44 (m, 2H), 7.62
(d, 1H, J = 3.6 Hz); 13C NMR (50 MHz, CDCl3 d) 14.9,
16.1, 17.5, 25.6, 26.2, 39.4, 60.9, 64.9, 115.0, 118.8, 119.7,
123.8, 127.9, 130.1, 131.3, 141.6, 144.7, 156.9, 167.4; Anal.
Calcd for C21H28O3: C, 76.79; H, 8.59; O, 14.61. Found C,
76.77; H, 8.60; O, 14.59. Boropinic acid (5). White solid;
yield 96%; analytical data are in full agreement with those

2642
F. Epifano et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2639–2642
reported in the literature.11 3-(40-Isopentenyloxyphenyl)-2-
(11): White solid; yield 97%; analytical data are in full
agreement with those reported in the literature.11 4-
geranyloxy-3-methoxy benzoic acid (12): White solid;
yield 95%; analytical data are in full agreement with those
reported in the literature.11 Umbelliprenine (13). White
solid; yield 86%; analytical data are in full agreement with
those reported in the literature.8 Auraptene (14). White
solid; yield 99%; analytical data are in full agreement with
those reported in the literature.8 Collinin (15). White solid;
yield 74%; analytical data are in full agreement with those
reported in the literature.8 7-Isopentenyloxycoumarin
(16). White solid; yield 99%; analytical data are in full
agreement with those reported in the literature.8 Prenyl-
transferase activity assays. Inhibition studies were per-
trans propenoic acid (6). White solid; yield 97%; mp: 164-
165 °C; IR (KBr): 1690 cmꢀ1; 1H NMR (200 MHz, CDCl3
d): 1.71 (s, 3H), 1.77 (s, 3H), 4.54–4.56 (m, 2H), 5.44–5.47
(m, 1H), 6.38 (d, 1H, J = 3.7 Hz), 6.79–6.82 (m, 2H), 7.58–
7.61 (m, 2H), 7.74(d, 1H, J = 3.7 Hz); 13C NMR (50 MHz,
CDCl3 d) 18.3, 25.9, 65.1, 115.3, 117.6, 119.9, 128.3, 129.3,
138.4, 144.2, 157.7, 168.8; Anal. Calcd for C14H16O3: C,
72.39; H, 6.94; O, 20.63. Found C, 72.41; H, 6.93; O,
20.61. 3-(40-Farnesyloxyphenyl)-2-trans propenoic acid
(7). White solid; yield 78%; mp: 199–202 °C; IR (KBr):
1688 cmꢀ1 1H NMR (200 MHz, CDCl3 d):1.60 (s, 3H),
;
1.62 (s, 3H), 1.67 (s, 3H), 1.80 (s, 3H), 2.03–2.14 (m, 8H),
4.47–4.50 (m, 2H), 5.06–5.13 (m, 2H), 5.47–5.52 (m, 1H),
6.40 (d, 1H, J = 3.6 Hz), 6.78 – 6.82 (m, 2H), 7.57–7.61 (m,
2H), 7.73 (d, 1H, J = 3.6 Hz); 13C NMR (50 MHz, CDCl3
d) 13.7, 16.1, 17.7, 25.7, 25.9, 26.7, 39.6, 64.9, 115.1, 117.4,
119.8, 123.9, 124.3, 128.5, 129.2, 131.3, 135.3, 141.6, 144.9,
157.2, 168.7; Anal. Calcd for C24H32O3: C, 78.22; H, 8.75;
O, 13.02. Found C, 78.24; H, 8.73; O, 13.00. 3-(40-
Farnesyloxy-30-methoxyphenyl)-2-trans propenoic acid
(8). White solid; yield 84%; mp: 216–218 °C; IR (KBr):
formed
by
determining
the
ability
of
prenyloxyphenylpropanoids to inhibit the transfer of
[3H]farnesyl and [3H]geranylgeranyl from [3H]farnesylpy-
rophosphate ([3H]FPP, Amersham Biosciences, Piscata-
way,
NJ)
and
[3H]geranyl-geranylpyrophosphate
([3H]GGPP, Perkin Elmer, Wellesley, MA) to recombi-
nant H-Ras-CVLL and H-Ras-CVLS, respectively, as
described previously.19 Briefly, 60,000g supernatants of
human Burkitt’s lymphoma (Daudi) cells (American Type
Culture Collection, Rockville, MD) were incubated for
30 min at 37 °C in the presence of either vehicle control or
prenyloxyphenylpropanoids, H-Ras substrate, and
0.5 lCi/sample of [3H]-FPP or [3H]GGPP. Samples were
denatured with SDS and precipitated with TCA then
filtered onto glass fiber filters. Unbound [3H]FPP or
[3H]GGPP was washed through the filters. Samples were
counted on a scintillation counter and activity of com-
pounds were compared to vehicle control to obtain
prenyltransferase inhibition values and IC50s.
1
1687 cmꢀ1; H NMR (200 MHz, CDCl3 d): 1.61 (s, 3H),
1.63 (s, 3H), 1.67 (s, 3H), 1.81 (s, 3H), 2.02–2.14 (m, 8H),
3.82 (s, 3H), 4.70–4.74 (m, 2H), 5.07–5.13 (m, 2H), 5.47–
5.51 (m, 1H), 6.42 (d, 1H, J = 3.6 Hz), 6.73–6.76 (m, 1H),
7.02–7.06 (d, 1H, J = 3.6 Hz), 7.19–7.22 (m, 2H); 13C
NMR (50 MHz, CDCl3 d) 13.7, 16.2, 17.6, 25.5, 25.8, 26.4,
39.8, 55.8, 65.9, 110.8, 114.6, 117.1, 119.8, 121.0, 123.8,
124.1, 127.9, 129.2, 131.2, 135.4, 141.9, 144.3, 145.3, 150.9,
168.1; Anal. Calcd for C25H34O4: C, 75.34; H, 8.60; O,
16.06. Found C, 75.33 H, 8.62; O, 16.04. Valencic acid (9):
White solid; yield 94%; analytical data are in full agree-
ment with those reported in the literature.11 40-geranyl-
oxybenzoic acid (10): White solid; yield 92%; analytical
data are in full agreement with those reported in the
literature.19 4-isopentenyloxy-3-methoxy benzoic acid
18. Hosoda, A.; Miyake, Y.; Nomura, E.; Mizuno, K.;
Taniguchi, H. ITE Lett. 2001, 2, 659.
19. Vogt, A.; Qian, Y.; McGuire, T. F.; Hamilton, A. D.;
Sebti, S. M. Oncogene 1996, 13, 1991.