Topical anti-inflammatory activity of boropinic acid and its natural and semi-synthetic derivatives

Article abstract of DOI:10.1016/j.bmcl.2010.11.107

Boropinic acid is a natural isopentenyloxycinnamic acid extracted from the aerial parts of Boronia pinnata Sm. (Rutaceae) with soybean 5-lipoxygenase inhibitory activity. In this paper the topical anti-inflammatory activity of boropinic acid and some of its natural and semi-synthetic derivatives was evaluated using the Croton oil ear test in mice as a model of acute inflammation. Some of the tested compounds (15, 17, 19, 20) revealed an effect comparable (ID₅₀ = 0.18 ÷ 0.72 μmol/cm²) to that of the reference drug indomethacin (ID₅₀ = 0.23 μmol/cm ²), a non-steroidal anti-inflammatory drug.

Full text of DOI:10.1016/j.bmcl.2010.11.107

Bioorganic & Medicinal Chemistry Letters 21 (2011) 769–772
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Topical anti-inflammatory activity of boropinic acid and its natural
and semi-synthetic derivatives
Francesco Epifano a, , Silvio Sosa b, Aurelia Tubaro b, M. Carla Marcotullio c, Massimo Curini c,
⇑
Salvatore Genovese a
a Dipartimento di Scienze del Farmaco, Università ‘G. D’Annunzio’ Chieti-Pescara, Via dei Vestini 31, Chieti Scalo (CH) 66013, Italy
b Dipartimento dei Materiali e delle Risorse Naturali, Università di Trieste, Via A. Valerio 6, 34127 Trieste, Italy
c Dipartimento di Chimica e Tecnologia del Farmaco, Sezione di Chimica Organica, Università degli Studi di Perugia, Via del Liceo, Perugia 06123, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Boropinic acid is a natural isopentenyloxycinnamic acid extracted from the aerial parts of Boronia pinnata
Sm. (Rutaceae) with soybean 5-lipoxygenase inhibitory activity. In this paper the topical anti-inflamma-
tory activity of boropinic acid and some of its natural and semi-synthetic derivatives was evaluated using
the Croton oil ear test in mice as a model of acute inflammation. Some of the tested compounds (15, 17,
Received 16 October 2010
Revised 22 November 2010
Accepted 23 November 2010
Available online 26 November 2010
19, 20) revealed an effect comparable (ID50 = 0.18 ꢀ 0.72
methacin (ID50 = 0.23
mol/cm2), a non-steroidal anti-inflammatory drug.
Ó 2010 Elsevier Ltd. All rights reserved.
l
mol/cm2) to that of the reference drug indo-
l
Keywords:
Anti-inflammatory activity
Boropinic acid
Croton oil ear test
Prenyloxycinnamic acids
Prenyloxyphenylpropanoids
3-(40-Isopentenyloxy-30-methoxyphenyl)-2-trans propenoic acid,
commonly known as boropinic acid, 1 is a secondary metabolite
biosynthetically related to ferulic acid in which an isopentenyl
chain is attached to the phenolic group. It has been isolated in
2000 by Ito et al. from the aerial parts of Boronia pinnata Sm., an
Australian shrub belonging to the family of Rutaceae.1 Since its dis-
covery, this natural compound showed valuable pharmacological
properties that have been recently reviewed.2 In particular,
boropinic acid showed valuable in vitro inhibitory effects on the
growth of Helicobacter pylori and soybean 5-lipoxygenase (5-LOX)
activity.3
our research aimed to better define the pharmacological profile
of 1 and its natural and semi-synthetic derivatives, we synthesized
a series of isopentenyloxycinnamic acids differently substituted in
position 30 and assessed their topical anti-inflammatory activity.
These compounds were drawn in such a structure in order to verify
the influence of different substituents located in the aromatic ring
on the biological activity.
Boropinic acid 1 and 40-isopentenyloxycinnamic acid 2, the lat-
ter a natural prenyloxyphenylpropanoid identified in Esenbeckia
hieronymi Engl. (Fam. Rutaceae),4 were synthesized as already
reported.5
The synthesis of 30-halo-40-isopentenyloxycinnamic acids was
accomplished starting from 3-halo-4-hydroxybenzaldehydes, that
in all cases were purified by crystallization in H2O. To this aim
3-fluoro-4-hydroxybenzaldehyde 4 was obtained in 50% yield
starting from commercially available 2-fluorophenol 3 by formyla-
tion with hexamethylenetetramine and CF3COOH at 100 °C for 24 h
(Scheme 1).6
3-Chloro-4-hydroxybenzaldehyde 6 was synthesized in 64%
yield by chlorination of commercially available 4-hydroxybenzal-
dehyde 5 with N-chlorosuccinimide (NCS) in CHCl3 at 60 °C for
12 h (Scheme 2).7
COOH
O
R
1 R = OMe
2 R = H
Inhibition of 5-lipoxygenase suggests a possible in vivo anti-
inflammatory activity of 1. Since inflammation is a universal and
physiological response involved in several diseases, compound 1
and a series of its novel natural and semi-synthetic derivatives
were investigated for their anti-inflammatory activity. Continuing
Finally 4-hydroxy-3-iodobenzaldehyde 7 was obtained in 72%
yield by iodination of 4-hydroxybenzaldehyde 5 with KI/I2 in
NH4OH 30% (aq) as the solvent at room temperature for 6 h
(Scheme 3).8
⇑
Corresponding author.
E-mail address: fepifano@unich.it (F. Epifano).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.107

770
F. Epifano et al. / Bioorg. Med. Chem. Lett. 21 (2011) 769–772
OH
COOMe
CHO
OH
a
F
F
a
HO
HO
X
X
8-13
I
CHO
b,c
3
4
Scheme 1. Reagents and conditions: (a) hexamethylenetetramine (1 equiv),
CF3COOH (1 equiv), 100 °C, 24 h.
COOH
O
OH
OH
X
14-19
Cl
a
Scheme 4. Reagents and conditions: (a) (MeO)3POCH2COOMe (3 equiv), Na
(3 equiv) MeOH, 70 °C, 24 h; (b) isopentenyl bromide (1 equiv), K2CO3 (1 equiv),
acetone , 80 °C, 2 h; (c) NaOH 2 N (aq), reflux, 30 min, acid/base workup
CHO
CHO
5
6
ears to quantify oedema as weight difference between the two
plugs. The anti-inflammatory activity was expressed as percent
of oedema reduction in mice treated with the compounds under
test with regard to control mice. Oedema values, expressed as
means standard error of the mean, were analysed by one way
analysis of variance followed by Dunnett’s test for multiple com-
parison of unpaired data. A probability level lower than 0.05 was
considered as significant. Experiments complied with the Italian
D.L. n. 116 of January 1992 and associated guidelines in the Euro-
pean Communities Council Directive of 24 November 1986 (86/609
ECC) concerning animal welfare and appendix A of the European
Convention ETS 123.
Scheme 2. Reagents and conditions: (a) NCS (1 equiv), CHCl3, 60 °C, 12 h.
Benzaldehydes substituted in position 3 with a CH3– and a
NO2– group were commercially available.
Synthesized and commercially available 3-substituted-4-hydro-
xybenzaldehyde were then submitted to a Wittig–Horner olefin-
ation9 by reaction with methyl trimethylphosphonacetate and Na
in MeOH at 70 °C for 24 h to obtain the corresponding cinnamic
acid methyl esters 8 (X = –F, 86% yield), 9 (X = –Cl, 99% yield), 10
(X = –Br, 99% yield), 11 (X = –I, 81% yield), 12 (X = –Me, 89% yield),
and 13 (X = –NO2, 99% yield).10 All these derivatives were in turn
alkylated in position 40 by reaction with isopentenyl bromide pro-
moted by K2CO3 as the base in acetone at 80 °C for 2 h, followed by
basic hydrolysis in the same vessel and acid/base workup to afford
pure isopentenyloxycinnamic acids 14 (X = –F, 76% yield), 15
(X = –Cl, 99% yield), 16 (X = –Br, 80% yield), 17 (X = –I, 71% yield),
18 (X = –Me, 87% yield), and 19 (X = –NO2, 97% yield) (Scheme 4).
Finally the last product, 3-amino-4-isopentenyloxycinnamic
acid 20 was obtained in 59% yield from the nitro-derivative 19
by reduction with SnCl2 in EtOH at 70 °C for 30 min. (Scheme 5).
All the semi-synthetic isopentenyloxycinnamic acids are re-
ported herein for the first time.
The results on the anti-inflammatory activity screening of com-
pounds 1, 2, and 14–20, administered at the dose of 0.3 l
mol/cm2,
are reported in the Table 1, in comparison to those of the same dose
of the non-steroidal anti-inflammatory drug (NSAID) indomethacin
(a cycloxygenases [COXs] inhibitor), and nordihydroguaiaretic acid
(a [5-LOX] inhibitor), used as references. Each compound provoked
a significant oedema inhibition, which ranged from 30% (2) to 78%
(17 and 19). The natural compounds, boropinic acid 1 and 40-isopen-
tenyloxycinnamic acid 2 induced 38% and 30% oedema reduction,
respectively. While the semi-synthetic derivatives 16 and 18 pro-
voked less than 20% oedema reduction, the other semi-synthetic
compounds induced oedema reductions ranging from 46% (20) to
78% (17 and 19). The reference compounds indomethacin and
nordihydroguaiaretic acid induced 62% and 36% oedema inhibition,
respectively.
The topical anti-inflammatory activity of compound 1 and its
derivatives 2, and 14–20 was evaluated as inhibition of the Croton
oil-induced ear dermatitis in mice.11
Thus, the natural compounds 1 and 2 as well as the most active
semi-synthetic ones (15, 17, 19, 20) were further investigated for
their dose–activity relationship, in comparison to indomethacin
and nordihydroguaiaretic acid.
Male CD-1 mice (28–32 g, Harlan Laboratories, Udine, Italy)
were anaesthetised with ketamine hydrochloride (145 mg/kg,
intraperitoneally; Virbac, Milan, Italy). Inflammation was induced
on the right ear (surface: about 1 cm2) by application of 80
lg of
As reported in the Table 2, boropinic acid 1 and its derivatives
Croton oil (Sigma Chemical Co., St. Louis, USA) dissolved in acetone.
The left ear remained untreated as preliminary experiments
showed that the vehicle did not affect the inflammatory response
or induce irritation. Control mice received only the irritant solu-
tion, whereas the others received both the irritant and the com-
pounds under test dissolved in acetone. Six hours later, mice
were sacrificed and a plug (6 mm Ø) was excised from both the
15, 17, 19 and 20 (0.1–1 l
mol/cm2) exerted a dose-dependent
anti-inflammatory effect. The relevant ID50 values (dose inducing
50% oedema inhibition), as index of anti-inflammatory potency,
showed that the anti-inflammatory activity of compound
(ID50 = 0.72
mol/cm2) was slightly increased by its demethoxyla-
tion to 2 (ID50 = 0.50
1
l
l
mol/cm2). In addition, the semi-synthetic
derivatives of 2 substituted in position 30 with a chlorine (15) or
iodine (17) atoms, but also with a nitro (19) or amino (20) func-
tional groups, were about twice more active than 2 (ID50 ranging
from 0.18 to 0.33
potency of these compounds was comparable to that of indometh-
acin (ID50 = 0.23
mol/cm2) and about twice higher than that of
nordihydroguaiaretic acid (ID50 = 0.54
mol/cm2).
OH
I
l
mol/cm2). Moreover, the anti-inflammatory
a
5
l
CHO
l
Depicting preliminary structure–activity relationships (SAR),
we have already revealed by docking studies that an unfuctional-
ized isopentenyloxy side chain in position 4 of the aromatic ring
7
Scheme 3. Reagents and conditions: (a) KI/I2 (1 equiv), NH4OH 30%, rt, 6 h.

F. Epifano et al. / Bioorg. Med. Chem. Lett. 21 (2011) 769–772
771
COOH
COOH
a
O
O
NH2
NO2
Scheme 5. Reagents and conditions: (a) SnCl2 (5 equiv), EtOH, 70 °C, 0.5 h.
Table 1
Anti-inflammatory activity of boropinic acid 1 and its derivatives
Substance
Nr. animals
Dose (
l
mol/cm2)
Oedema (mg) [Mean S.D.]
% Reduction
Controls
1
2
14
15
16
17
18
10
10
10
10
10
10
10
10
9
—
6.9 0.3
4.3 0.2*
4.8 0.4*
4.2 0.3*
3.5 0.3*
5.6 0.3*
1.5 0.3*
5.3 0.3*
1.5 0.4*
3.7 0.3*
2.6 0.4*
4.4 0.4*
—
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
38
30
39
49
19
78
23
78
46
62
36
19
20
9
10
10
Indomethacin
Nordihydroguaiaretic acid
*
p <0.05 at the analysis of variance, as compared to controls.
Table 2
Dose-dependent anti-inflammatory activity of boropinic acid 1 and its derivatives
Substance
Nr. animals
Dose (
l
mol/cm2)
Oedema (mg) [Mean S.D.]
% Reduction
DI50 (l
moli/cm2)
Controls
1
19
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
—
6.9 0.2
6.1 0.3*
4.4 0.2*
3.1 0.3*
6.0 0.2*
4.4 0.2*
2.3 0.2*
5.2 0.3*
3.4 0.3*
0.6 0.1*
4.8 0.2*
2.3 0.3*
0.8 0.3*
4.6 0.3*
2.2 0.2*
0.6 0.1*
5.4 0.3*
3.9 0.2*
1.3 0.2*
5.1 0.3*
2.6 0.4*
0.9 0.2*
5.6 0.3*
4.6 0.4*
2.4 0.3*
—
—
0.72
0.1
0.3
1.0
0.1
0.3
1.0
0.1
0.3
1.0
0.1
0.3
1.0
0.1
0.3
1.0
0.1
0.3
1.0
0.1
0.3
1.0
0.1
0.3
1.0
12
36
55
13
36
67
25
51
91
30
67
88
33
68
91
22
43
81
26
62
87
19
33
65
2
0.50
0.26
0.20
0.18
0.33
0.23
0.54
15
17
19
20
Indomethacin
Nordihydroguaiaretic acid
*
p <0.05 at the analysis of variance, as compared to controls.
is an essential feature for the inhibition of a key pro-inflammatory
enzyme like 5-LOX.12 The presence of the above cited moiety in all
compounds tested could partly account also for the anti-inflamma-
tory effect of the tested compounds. Also the cinnamic acid moiety
probably plays a key role in the inhibition of 5-LOX as previously
reported by Koshihara et al. in 1983.13 Anyway, from the obtained
results, the inhibition of 5-LOX seems not to be the main mecha-
nism by which the most active compounds (15, 17, 19, 20) reduce
the oedema formation induced by Croton oil, being their activity
about twice higher than that of the 5-LOX inhibitor nord-
ihydroguaiaretic acid and comparable to that of the dual COX
inhibitor indomethacin. So it could be hypothesised that, besides
an activity against 5-LOX, these semi-synthetic compounds could
trigger their action on other targets involved in inflammation. It
seems that the presence of an isopentenyloxy side chain is still a
structural requirement for the observed anti-inflammatory activ-
ity, but the introduction of bulky substituent like a iodine atom
(17) is a feature that led to an increased activity as compared to
the parent compound 2. The same hypothesis can be made for elec-
tron withdrawing groups like the –NO2 moiety, although in this
case also steric hindrance has to be considered to observe efficacy.
In fact the compound 14 having a fluorine atom in position 3, shar-
ing the same electronic properties with –NO2 derivative 19, is by
far less active than the latter. Also the presence of electron donat-

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F. Epifano et al. / Bioorg. Med. Chem. Lett. 21 (2011) 769–772
ing groups like the –NH2 moiety (20), or sterically medium sized
electron withdrawing groups like chlorine (15), led to an increased
activity, even though of lower extent.
References and notes
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In conclusion, the findings described herein indicate that some
semi-synthetic isopentenyloxycinnamic acids, structurally derived
from the natural product boropinic acid 1, could be regarded as po-
tential novel and effective anti-inflammatory agents, the potency
of which is comparable to the well known NSAID indomethacin.
All tested compounds were easily synthesized starting from widely
available starting materials, by good yielding and cheap synthetic
routes. Four of these derivatives showed a very interesting topical
anti-inflammatory activity in the Croton oil-induced ear dermatitis
test in mice. For these reasons the present study could be consid-
ered as a topic for future studies aimed at better define the
pharmacological profile of semi-synthetic isopentenyloxycinnamic
acids. To this aim, studies to get further insights into their mecha-
nism of action as well as to investigate the biological activity of
compounds substituted in positions other than 30 on the aromatic
ring, as well as the effects of functionalized O-side chains,
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a,b-unsaturated double bonds and isosters of the carboxylic groups
are now ongoing in our laboratories.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.11.107.