Conjugation of L-NAME to prenyloxycinnamic acids improves its inhibitory effects on nitric oxide production

Article abstract of DOI:10.1016/j.bmcl.2013.03.050

A series of 10 compounds resulting from the conjugation of O-prenylated naturally occurring benzoic and cinnamic acids to l-NAME were synthesized and tested together with the corresponding unprenylated parent molecule as anti-inflammatory agents for their inhibitory effects on NO production in LPS-stimulated RAW 264.7 macrophages. Results indicated that the coupling between O-geranyl and O-isopentenylcinnamic acids and l-NAME led to products with an enhanced activity when compared to the parent compounds.

Full text of DOI:10.1016/j.bmcl.2013.03.050

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2933–2935
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Conjugation of L-NAME to prenyloxycinnamic acids improves
its inhibitory effects on nitric oxide production
a
a
b
c
Salvatore Genovese a, , Francesco Epifano , Serena Fiorito , Massimo Curini , Mariangela Marrelli ,
⇑
Francesco Menichini c, Filomena Conforti c
a Dipartimento di Farmacia, Università ‘G. D’Annunzio’ Chieti-Pescara, Via dei Vestini 31, 66100 Chieti Scalo (CH), Italy
b Dipartimento di Chimica e Tecnologia del Farmaco, University of Perugia, Via del Liceo, 06132 Perugia, Italy
c Department of Pharmacy and Health and Nutrition Sciences, University of Calabria, I-87036 Rende (CS), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 10 compounds resulting from the conjugation of O-prenylated naturally occurring benzoic and
Received 30 January 2013
Revised 11 March 2013
Accepted 13 March 2013
Available online 21 March 2013
cinnamic acids to L-NAME were synthesized and tested together with the corresponding unprenylated
parent molecule as anti-inflammatory agents for their inhibitory effects on NO production in LPS-stimu-
lated RAW 264.7 macrophages. Results indicated that the coupling between O-geranyl and O-isopente-
nylcinnamic acids and
parent compounds.
L-NAME led to products with an enhanced activity when compared to the
Keywords:
Oxyprenylated natural cinnamic acids
Ó 2013 Elsevier Ltd. All rights reserved.
L
-NAME
Nitric oxide
Inflammation
The nitric oxide (NO) radical, synthesized in vivo by the oxida-
tion of -arginine catalyzed by either the inducible (i-) or constitu-
Oxyprenylated natural products (isopentenyloxy-, geranyloxy-,
and the less spread farnesyloxy-compounds) represent a family of
secondary metabolites that were previously considered as biosyn-
thetic intermediates of the most widespread C-prenylated deriva-
tives. However, recent evidence suggests that these natural
products are important biologically active phytochemicals. More
than 350 related molecules have been isolated from plants primar-
ily belonging to the families of Rutaceae, Apiaceae, and Composi-
tae, comprising common edible vegetables and fruits, as well as
from fungi and bacteria. A wide variety of compounds containing
a prenyloxy side chain were isolated and these comprise alkaloids,
coumarins, flavonoids, cinnamic and benzoic acids, phenols, alco-
hols, aldehydes, anthraquinones, chalcones, lignans, xanthones,
aceto- and benzophenones. Many of the isolated oxyprenylated
natural products were shown to have remarkable anti-cancer,
anti-inflammatory, anti-microbial, and anti-fungal effects using
in vitro and in vivo models. The chemistry and pharmacology of
this class of natural products has been recently reviewed.16 In this
context, one of these oxyprenylated secondary metabolites,
namely 40-geranyloxyferulic acid (1), was seen to exert in vivo ben-
eficial effects both as an anti-inflammatory agent, being an inhibi-
tor of COX-217 expression and i-NOS activity,18 and as a dietary
feeding colon cancer chemopreventer.19 The pharmacological
properties of compound (1) has been recently reviewed.20
L
tive (c-) isoforms of NO synthase (NOS), is involved in several
physiological and pathological processes in mammals, most of
which have been largely reviewed in the last decade.1 It is also well
known how an excessive production of NO by iNOS in macro-
phages is involved in various acute and chronic inflammatory dis-
eases.2–4 High levels of NO are implicated in carcinogenesis,5,6
either by causing mutagenesis, deamination of DNA bases7–9 or
promoting the formation of carcinogenic N-nitroso compounds in
vivo.10 This latter biotransformation implies the rapid and sponta-
neous reaction of NO with triplet O2 to form stable anions, like ni-
trite and nitrate,11,12 that in turn non-enzymatically N-nitrosylate
the primary and secondary amines to produce carcinogenic nitros-
amines.10 Moreover under inflammatory conditions, macrophages
can greatly increase their production of both NO and superoxide
anion simultaneously, which rapidly react with each other to form
peroxynitrite anion, thus playing an additional damaging role in
inflammation and also possibly in the multistage process of carci-
nogenesis.13 The peroxynitrite anion is then able to activate the
constitutive and inducible forms of cyclooxygenase (COX-1 and
COX-2, respectively), which are rate determining enzymes for
prostaglandin biosynthesis during the inflammatory process.14,15
Literature reports indicate that inflammation represents the
basis of severe acute and chronic syndromes like cancer, cardiovas-
cular diseases, neurological disorders, and several others.21 Thus
⇑
Corresponding author.
E-mail address: s.genovese@unich.it (S. Genovese).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.050

2934
S. Genovese et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2933–2935
H
the search for novel, alternative, and safer anti-inflammatory
agents as well as the better description of the mechanism of action
underlying the observed effects, is a field of research of current and
growing interest.
N
H
COOCH3
+
N
N
H
Ar
COOH
NO2
NH2
COOH
CH2Cl2
r.t.
EEDQ
Et3N
O
OCH3
H
N
(1)
H
COOCH3
As a continuation of our ongoing studies aimed to better depict
the phytochemical and pharmacological properties of natural and
semisynthetic oxyprenylated phenylpropanoids and polyketides,
also in this work we synthesized and investigated the effects of
10 compounds resulting from the conjugation via an amide bond
of selected oxyprenylated benzoic and cinnamic acids to
roarginine methyl ester ( -NAME) with the aim to analyze a poten-
tial synergism of action between the well known anti-NOS activity
N
N
H
NO2
HN
Ar
O
L-NAME adducts
L-nit-
Scheme 1. Synthesis of
L
of L-NAME and the anti-inflammatory effects of O-prenyl deriva-
(5), 61% (6), 62% (7), 54% (8), 56% (13), 56% (14), 62% (15), 60%
(16), 58% (19), and 61% (20).
All compounds were then assessed for their potential to inhibit
the LPS-induced production of NO in murine macrophage RAW
tives,16,17 and of their unbound counterparts on NO production in
bacterial lipopolysaccharide (LPS)-stimulated RAW 264.7
macrophages.
The chemical structures of the compounds we studied are illus-
trated in Figure 1.
264.7 cells pre-stimulated by LPS (1 lg/mL). Pro-inflammatory
agents, like LPS, can significantly increase NO production in macro-
phages through activation of i-NOS.26 The treatment of RAW 264.7
macrophages for 24 h induced NO production, which was then
quantified by the chromogenic Griess reaction, measuring the
accumulation of nitrite, a stable metabolite of NO. Table 1 reports
Chemical synthesis and natural sources of compounds (1–4), (
9–11), and (17 and 18) have been already reported,22–24 while
compound 9, recently isolated from Piper crassinervum H.B.K. (Pip-
eraceae),25 was synthesized from commercially available methyl p-
hydroxybenzoate and geranyl bromide in the presence of K2CO3 as
the base in acetone following the already reported scheme for the
geranylation of phenolic acids.22 Conjugated adducts were synthe-
sized by condensation between L-NAME hydrochloride and the
prenyloxy acid promoted by N-ethoxycarbonyl-2-ethoxy-1,2-dihy-
droquinoline (EEDQ) in the presence of Et3N (Scheme 1).
the level of inhibition of NO production expressed as IC50 (lM). L-
NAME and indomethacin were used as controls. For all compounds
we performed the MTT test using the same cell line. Results ob-
tained from this assay indicated that every adduct had virtually
no effects on cell viability (data not shown).
From results obtained and reported in the Table 1 all L-NAME
conjugated products, with the exception of compound (13), are
This brief and simple reaction provided the desired products
after crystallization (n-hexane) with the following yields: 55%
Table 1
Inhibitory effects of the synthetized compounds on
the LPS-induced NO production in RAW 264.7 cells
R1
Compound
1
2
IC50 (lM)
100.24 0.12g
>200
R2O
R3
3
>200
4
5
6
7
8
9
>200
64.19 0.13d
67.19 0.09e
62.61 0.04c
41.65 0.06a
>200
1 R1 = CH=CH-COOH, R2 = geranyl, R3 = OCH3
2 R1 = CH=CH-COOH, R2 = isopentenyl, R3 = OCH3
3 R1 = CH=CH-COOH, R2 = isopentenyl, R3 = H
4 R1 = CH=CH-COOH, R2 = geranyl, R3 = H
5 R1 = CH=CH-CO-LNAME, R2 = isopentenyl, R3
OCH3
=
10
>200
11
>200
6 R1 = CH=CH-CO-LNAME, R2 = geranyl, R3 = OCH3
7 R1 = CH=CH-CO-LNAME, R2 = isopentenyl, R3 = H
8 R1 = CH=CH-CO-LNAME, R2 = geranyl, R3 = H
9 R1 = COOH, R2 = isopentenyl, R3 = OCH3
10 R1 = COOH, R2 = geranyl, R3 = OCH3
12
>200
13
14
15
16
>200
53.12 0.21b
110.50 0.04i
81.18 0.05f
>210
11 R1 = COOH, R2 = isopentenyl, R3 = H
12 R1 = COOH, R2 = geranyl, R3 = H
17
13 R1 = CO-LNAME, R2 = isopentenyl, R3 = OCH3
14 R1 = CO-LNAME, R2 = geranyl, R3 = OCH3
15 R1 = CO-LNAME, R2 = isopentenyl, R3 = H
16 R1 = CO-LNAME, R2 = geranyl, R3 = H
17 R1 = CH2CH2COOH, R2 = isopentenyl, R3 = H
18 R1 = CH2CH2COOH, R2 = geranyl, R3 = H
19 R1 = CH2CH2COLNAME, R2 = isopentenyl, R3 = H
20 R1 = CH2CH2COLNAME, R2 = geranyl, R3 = H
18
19
20
>200
103.10 0.03h
125.00 0.17j
148.00 0.47k
170.04 0.37l
Indomethacinea
L-NAMEa
Data are expressed as mean S.E.M. (n = 4). Dif-
ferent letters along column indicate statistically
significant differences at P <0.05 (Tukey’s test).
a
Positive controls.
Figure 1.

S. Genovese et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2933–2935
2935
able to decrease to different extents more efficiently than L-NAME
Acknowledgments
and indomethacine, the over-production of NO in LPS-stimulated
RAW 264.7 macrophages. The more significant result to this con-
cern was obtained with the conjugated compound (8) resulting
Authors from Chieti wish to acknowledge financial support to
the research from the University ‘G. D’Annunzio’ of Chieti-Pescara.
from the coupling of p-coumaric acid with
L-NAME
(IC50 = 41.65 0.06 M). Other prenyloxy cinnamic acid deriva-
tives performed slightly worse but recording an homogeneity in
l
Supplementary data
obtained data, being the values of inhibition comprised in the
range 62.61–67.19 lM. This pattern of results was different for
oxyprenylated benzoic acid derived products (13–16) for which
only one compound, namely (14), provide an excellent decrease
in NO production by macrophages, with an IC50 value of
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.03.
050.
References and notes
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a
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activity, being clear that cinnamic acid derivative conjugates are
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compounds (6), (8), (14), and (16) render the molecule more active
than their O-isopentenylated counterparts. This last observation is
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activity.17 Finally the OCH3 group in position 3 of the aromatic ring
seem not to play a definitive role in this context.
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In conclusion, exploiting peptide-like derivatives resulting from
the coupling of
acids, we have disclosed herein that the NO production inhibitory
activity of -NAME can be greatly improved by its linkage to other
L-NAME and oxyprenylated naturally occurring
L
known anti-inflammatory agents like prenyloxycinnamic acids.
Stating the well known importance of inflammation in the patho-
genesis of several severe diseases, the pivotal role played by COX
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and NOS in several types of cancer,27 and the efficacy of
L-NAME
and oxyprenylated secondary metabolites against severe syn-
dromes, for example colon cancer,28 the findings described herein
will be of certain interest in the next future to the development of a
novel class of prodrugs able to generate two or more biologically
sinergically active portions able to efficiently reduce the inflamma-
tory process acting on different endocellular targets.
´
27. Paduch, R.; Kandefer-Szerszen, M. Cancer Microenviron. 2011, 4, 187.
28. Kawamori, T.; Takahashi, M.; Watanabe, K.; Ohta, T.; Nakatsugi, S.; Sugimura,
T.; Wakabayashi, K. Cancer Lett. 2000, 148, 33.