Gastric cytoprotective activity of ilicic aldehyde: Structure-activity relationships

Article abstract of DOI:10.1016/j.bmcl.2005.05.053

A series of sesquiterpene compounds possessing both eudesmane and eremophilane skeletons were tested as gastric cytoprotective agents on male Wistar rats. The presence of an α,β-unsaturated aldehyde on the C-7 side chain together with a hydroxyl group at C-4 is the requirement for the observed antiulcerogenic activity. In an attempt to establish new molecular structural requirements for this gastric cytoprotective activity, a structure-activity study was performed.

Full text of DOI:10.1016/j.bmcl.2005.05.053

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3547–3550
Gastric cytoprotective activity of ilicic aldehyde:
Structure–activity relationships
a
b
b
Osvaldo J. Donadel,a, Eduardo Guerreiro, Alejandra O. Marıa, Graciela Wendel,
*
´
Ricardo D. Enriz,c Oscar S. Giordanoa and Carlos E. Tonna
a
´
´
INTEQUI-CONICET, Facultad de Quımica, Bioquımica y Farmacia, Universidad Nacional de San Luis,
Chacabuco y Pedernera, 5700 San Luis, Argentina
b
´
´
Departamento de Farmacia, Facultad de Quımica, Bioquımica y Farmacia, Universidad Nacional de San Luis,
Chacabuco y Pedernera, 5700 San Luis, Argentina
c
´
Departamento de Quımica, Facultad de Quımica, Bioquımica y Farmacia, Universidad Nacional de San Luis,
´
´
Chacabuco y Pedernera, 5700 San Luis, Argentina
Received 20 April 2005; revised 16 May 2005; accepted 17 May 2005
Available online 15 June 2005
Abstract—A series of sesquiterpene compounds possessing both eudesmane and eremophilane skeletons were tested as gastric cyto-
protective agents on male Wistar rats. The presence of an a,b-unsaturated aldehyde on the C-7 side chain together with a hydroxyl
group at C-4 is the requirement for the observed antiulcerogenic activity. In an attempt to establish new molecular structural
requirements for this gastric cytoprotective activity, a structure–activity study was performed.
Ó 2005 Elsevier Ltd. All rights reserved.
Antitumor, antimicrobial, antifeedant, cytotoxic, anti-
bacterial, antifungal, and allergenic contact dermatitic
activities of several sesquiterpene lactones has been pre-
viously reported.1
Previous studies have determined that dehydroleucodine
1 (Fig. 1), a sesquiterpene lactone of the guaianolide
type isolated from Artemisia douglasiana Besser, shows
a pharmacological cytoprotective effect and significantly
prevents the formation of gastric lesion induced by
several necrotizing agents. A structure–activity relation-
ship study reveals that the presence of a,b-unsaturated
carbonyl groups seems to be responsible for the
bioactivity.2
Figure 1. Structure of dehydroleucodine.
electrophilic acceptor and sulfhydryl-containing com-
pounds of the gastric mucosa.3 In this regard, a-methyl-
ene-c-lactone and conjugated cyclopentenone are
among the most active functional groups.4
It has been reported that gastric cytoprotection may be
mediated by at least two different mechanisms. The first
one concerning prostaglandins (PG) and the second one
involving sulfhydryl-containing compounds of the
mucosa. The mechanism of cytoprotection might be
mediated, at least in part, by the reaction between
The main objective of this paper was to investigate the
antiulcer activities of several sesquiterpenes (Fig. 2) that
were derived from the natural products ilicic acid 2 and
ilicic alcohol 3, both isolated from the aerial parts of
Flourensia oolepis Blake.5 In addition to the above-men-
tioned compounds, the eremophilane tessaric acid 4 was
recovered from Tessaria absinthioides H. et A. and its
derivatives were also tested.6 Both plant species were
grown in the semi-arid central-western region of Argen-
tina. Compounds 2–4 were used as starting materials to
prepare the derivatives 5–14.
Keywords: Gastric cytoprotection; Sesquiterpenes; Ilicic aldehyde;
Eudesmane; Eremophilane.
*
Corresponding author. Tel.: +54 2652 439909; fax: +54 2652
426711; e-mail: odonadel@unsl.edu.ar
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.053

3548
O. J. Donadel et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3547–3550
Figure 2. Natural sesquiterpenes from T. absinthioides and F. oolepis.
Flourensia oolepis Blake was collected at Cuesta del
Gato, Juana Koslay, Departamento La Capital, San
Luis, Argentina. Its identification was confirmed by
Prof. L. A. Del Vitto and a voucher specimen was
deposited at the Universidad Nacional de San Luis
Herbarium under No. 2329. T. absinthioides H. et A.
(voucher specimen No. 0461) was collected from El Vol-
Scheme 2. Reagents and conditions: (a) Jones reagent (87%); (b)
˚
TsOH, C6H6, MS 4 A, 80 °C (93%); (c) POCl3, pyridine, ꢀ20 °C (56%);
(d) HMDS, TMSCl, CH2Cl2, rt (91%); (e) (F3CCO)2O, pyridine,
DMAP, rt (92%).
´
can, Departamento La Capital, San Luis.
chemical transformations were used to obtain 11 and
12, as shown in Scheme 2.
Air-dried aerial parts of F. oolepis (2.5 kg dry weight)
were extracted, as previously described.5,7 After several
chromatographic purifications, ilicic acid 2 (1.35 g)
and ilicic alcohol 3 (1.1 g) were obtained.7,8 From dried
aerial parts of T. absinthioides (5 kg dry weight), tessaric
acid 4 (25 g) was obtained, as previously reported.6,9
Compound 13 was prepared from tessaric acid 4 by
direct esterification with MeOH/HCl (Scheme 3).18 The
synthesis of 1(10),2,11(13)-eremophilatrien-12-oic acid
14 was carried out by treatment of 13 with AlCl3 and
LiAlH4 in THF. This compound was characterized by
1H NMR, 13C NMR, CGMS, and HRMS (Table 1).
As shown in Scheme 1, compounds 5 and 6 were pre-
pared in a one-pot reaction from compound 2.10
Photo-oxidation of compound 5 under a sunlight lamp
(1000 W) for 40 h with oxygen circulation through a
glass frit,11 followed by triphenylphosphine treatment,12
led to the formation of compound 7 in 92% yield.10
Male Wistar rats weighing 200–250 g were used for
acute gastric ulcerations induced by ethanol.19 The ani-
mals, randomly assigned into groups (n = 5–8), were de-
prived of food for 24 h prior to starting the experiments
and had free access to water.
Ilicic aldehyde 8 (Scheme 2) was prepared from alcohol
3 using the Jones reagent in the usual way.13 After silica
gel chromatography (n-hexane/EtOAc 1:9 as eluent), the
title compound (1.3 g, 87%) was obtained; 1H NMR and
13C NMR are shown in Table 1.5,14 c-Costic aldehyde
Briefly, absolute ethanol (1 mL/animal) was adminis-
tered intragastrically to control rats and rats pretreated
1 h before with compounds 1–14, respectively (100 mg/
kg, suspended in 0.4% carboxymethylcellulose, po).
One hour after ethanol administration, the animals were
sacrificed and the stomach was removed. The gastric
lesions formed were counted and the mean ulcerative
index was calculated, according to the method previ-
ously described.2 An index of 0 denoted no erosions,
while an index of 5 corresponded to maximal damage.
Control rats received absolute ethanol and vehicle
9
15 was obtained in good yield by dehydration of 8 with
p-TsOH in dry C6H6. Compound 8 was converted into
costic aldehyde 10,16 according to a previous method
using POCl3 in dry pyridine at ꢀ21 °C.17 Two diverse
(po). A proton pump inhibitor, omeprazole (Ulcozol
´
Ò
10 , Bago, 20 mg/kg), which is a commonly prescribed
drug for increased gastric acid secretion and gastric
ulcer, was used as a reference drug for comparison.
Omeprazole was also given 1 h before absolute ethanol
was administered.
The results are shown in Table 2. Statistical analysis of
data was performed using one-way analysis of variance
(ANOVA) with Tukey–Kramer multiple comparisons
method, with a level of significance of p < 0.05.
In previous papers, we have demonstrated the cytopro-
tective activities of 1 and other related sesquiterpene lac-
tones against the formation of gastric lesions induced by
various necrotizing agents.2,4
˚
Scheme 1. Reagents and conditions: (a) TsOH, C6H6, MS 4 A, 80 °C
(after silica gel chromatography 55% 5 and 46% 6); (b) (i) i-Pro, bengal
rose, sunlight, 48 h, (ii) MeOH, triphenylphosphine, rt, overnight;
(iii) Ac2O, pyridine, rt, overnight (72%).

O. J. Donadel et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3547–3550
Table 1. 1H (200 MHz), and 13C NMR (50.23 MHz) data of 8a and 14
3549
H
8dH
14 dH
C
8 dC
20.08*
25.86*
44.44*
71.21
14 dC
1H–13C long range correlation
1
—
—
5.99 br m
5.53 m
—
1
CH2
CH2
CH2
C
128.82
125.62
121.41
36.07
CH
CH
CH
C
H-3
2
20
2
3
—
—
H-2, H-4
H-3, H-4, H-15
3
5.53 m
—
4
30
4
—
—
5
54.94
76.99
CH
CH2
CH
CH2
CH2
C
36.86
39.25
C
1.65 m
—
—
6
CH2
CH
CH2
CH2
C
6
1.85 m
1.26 m
2.53 m
9.52 m
6.28 br s
5.97 br s
0.92 s
1.11 s
7
37.28
32.71
6b
7
8
43.43*
40.92*
34.61
32.90*
30.85*
143.47
144.49
172.32
125.40
20.23
H-13
2.73 m
—
9
12
13
130
14
15
10
11
12
13
14
15
H-14
H-13
6.34 m
5.65 m
0.9 m
0.88 m
154.98
194.70
133.03
18.68
C
C
CH
CH2
CH3
CH3
C
CH2
CH3
CH3
22.43
15.12
COLOC correlation of 14 (d, ppm, TMS, CDCl3).
a Colorless needles, mp 83–84 °C. [a]D ꢀ10.6 (c 5.7, Me2CO).
* Interchangeable in each column.
highest level of gastric protection from the series (Table
2). The cytoprotective effect of this compound was com-
parable to that previously reported for dehydroleucodin
1.2 Furthermore, compounds 6–7 showed statistically
significant bioactivity. It should be noted that all the ac-
tive molecules have an electrophilic unsaturated bond
conjugated to the carbonyl group. On the other hand,
the carbonyl group must be included in an aldehyde or
an a-methylene-d-lactone functional group. These re-
sults are in agreement with those reported for sesquiter-
pene lactones and structurally related compounds.2,4
Scheme 3. Reagents and conditions: (a) MeOH/HCl, reflux (92%);
(b) AlCl3, LiAlH4, THF, 0 °C (82%).
Table 2. Gastric cytoprotective effect of compounds 1–14
Compound
Ulcerative indexa
Inhibition of damageb
1
0.16 0.10
4.62 0.12
4.80 0.12
4.62 0.12
4.00 0.31
2.50 1.00***
3.37 0.23*
0.20 0.12***
4.62 0.12
3.75 0.28
2.50 0.28***
0.66 0.16***
0.16 0.10***
4.62 0.37
3.70 0.28
3.00 0.28***
4.85 0.15
97
5
Although the sesquiterpenes studied here possess similar
skeletons, the results indicate that our experimental
model responds quite differently to structurally related
compounds. Compounds 2 and 3 showing a C-4-a-hy-
droxyl group and carboxylic acid or hydroxymethylene
functional group at the C-7 side chain were inactive.
Similar results were observed for derivatives 5, 9, and
14, all of them showing endocyclic unsaturation at ring
A of the decaline moiety. Compound 10 possessing an
exocyclic C-4 unsaturation displays a very low cytopro-
tective effect. Similarly, d-lactones 6 and 7 displayed
only a moderate cytoprotective activity.
2
3
1
4
5
5
17
48
30
96
5
6
7
8
9
10
23
48
86
96
5
11
12 (1° h)
12 (2° h)
13
14
Positive controlc
Control
23
38
0
The above results have suggested that in the eudes-
mane skeleton an a,b-unsaturated-d-lactone or a,b-
unsaturated aldehyde appeared to play a determinant
role in the cytoprotective effect, aside from some dis-
criminating properties of the decaline moiety. This
observation has been supported by results obtained
when tessaric acid 4, its methyl ester 13, and the re-
duced-dehydrated derivative 14 were assayed. Com-
pounds 4, 13, and 14 that belong to the eremophilane
series did not show an statistically significant activity.
In an additional experiment, we found that the inhibi-
tion response was dose dependent in the range 25–100
mg/kg (8 and 9, p < 0.01).
a All values are means SEM.
b All values are % of inhibition.
c Omeprazole.
* Statistical significance from control at p < 0.05.
*** Statistical significance from control at p < 0.001.
Pretreatment with omeprazole (20 mg/kg) caused a sig-
nificant reduction in ethanol-induced necrotic damage
of gastric mucosa. Under appropriate conditions, the
gastroprotective effect of omeprazole is mainly attrib-
uted to an enhancement of the mucus barrier rather than
a reduction of acid secretion.20
Regarding the influence of the substituents on the flexi-
ble side chain and at C-4, an interesting structure–activ-
ity correlation can be observed: the presence of an a,
From the present work, results obtained in the ethanol-
induced ulcer indicate that ilicic aldehyde 8 gives the

3550
O. J. Donadel et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3547–3550
´
Guzman, J.; Marıa, A. O.; Wendel, G. J. Med. Chem.
1992, 35, 2452.
´
b-unsaturated aldehyde group on the side chain was
mandatory for an acceptable cytoprotective activity in
this series (compare activities of compounds 2 and 3
with that of 8). However, the lack of activity obtained
for compounds 9 and 10 clearly indicates that the pres-
ence of an a,b-unsaturated aldehyde would be a struc-
tural requirement but not by itself sufficient for the
cytoprotective activity.
´
3. Szabo, S.; Trier, J. S.; Frankel, P. W. Science 1981, 214,
200.
4. Rodrıguez, A. M.; Enriz, R. D.; Santagata, L. N.;
´
Jauregui, E. A.; Pestchanker, M. J.; Giordano, O. S.
´
´
J. Med. Chem. 1997, 40, 1827.
5. Guerreiro, E.; Kavka, J.; Giordano, O. S.; Gros, E.
Phytochemistry 1979, 18, 1235.
6. Kurina Sanz, M. B.; Donadel, O. J.; Rossomando, P. C.;
Tonn, C. E.; Guerreiro, E. Phytochemistry 1997, 44, 897.
7. Abu Zarga, M. H.; Hamed, E. M.; Sabri, S. S.; Voelter,
W.; Zeller, K. P. J. Nat. Prod. 1998, 61, 798.
To outline the role of this functional group, the trimeth-
ylsilyl derivative 11 was prepared. The bioassays carried
out with derivative 11 showed a dramatic decrease in
activity when compared to that of compound 8
(p < 0.001, 8 vs 11).
8. Ilicic acid 2, solid powder, mp 181–182 °C, [a]D ꢀ36.1
(c 1.53, CHCl3). IR spectra mmax (cmꢀ1): 3420, 2300, 1695,
1625, 945, 895. The H NMR spectra (200 MHz, CDCl3)
1
presented signals at d 0.93 (s, 3H, H14), d 1.11 (s, 3H, H15),
d 6.23 (s, 1H, H13), d 5.63 (s, 1H, H130), d 2.50 (s, 1H, H7),
d 1.95 (m, 1H, H6) and d 1.86 (m, 1H, H60). Ilicic alcohol 3,
solid powder, mp 134–135 °C, [a]D ꢀ43.2 (c 1.9, CHCl3).
IR spectra mmax (cmꢀ1): 3500–3100, 1170, 1050 (–OH),
Additionally, when the C-4a-hydroxyl group was con-
verted in the corresponding trifluoroacetyl to yield
derivative 12, the bioactivity was similar to that of the
parent compound 8. On account of the good leaving
property of trifluoroacetate in compound 12, its stability
under experimental conditions similar to those in the
gastric juice was evaluated.21 In this case, after 1 h of
contact with synthetic gastric juice nearly 50% of com-
pound 12 was hydrolyzed to its C-4-hydroxy derivative
8. It was not possible to detect if the recovered hydroxy-
aldehyde was a diastereomerically pure compound or a
mixture of diastereomers at C-4 (consequence of the sol-
volysis through an SN1 mechanism). We perceive that
compound 12 might not be active by itself, but perhaps
its hydrolyzed derivative 8 (probably as a diastereomeric
mixture). Under the same experimental conditions com-
pounds, 8 and 11 proved to be stable after 2 h.
1
1640, 890 (R2C@CH2). The H NMR spectra (200 MHz,
CDCl3) presented signals at d 0.90 (s, 3H, H14), d 1.10 (s,
3H, H15), d 2.13 (br s, 1H, HO), d 4.15 (br s, 2H, H12), d
4.92 (s, 1H, H13), d 5.05 (s, 1H, H130). EIMS [C15H22O2]
[M+, 28] 238, 223, 220, 205, 202, 187, 162, 147, 135.
9. Tessaric acid 4, white solid, mp 154–155 °C, [a]D ꢀ143.4
(c 1.7, CHCl3). UV spectra kmax: 215 nm (e, 8500) and
243 nm (e, 14,000). IR spectra 2680, 1695, 1660, 1620,
1
1600 and 950 cmꢀ1. H NMR (200 MHz, CDCl3) d 6.33
(br s, 1H, H13), d 5.66 (br s, 1H, H130), d 5.88 (br s, 1H,
H1), d 0.98 (d, J = 7 Hz, 3H, H15), d 1.10 (s, 3H, H14), d
2.58 (m, 1H, H7); 13C NMR (50.23 MHz, CDCl3), 125.75,
149.13, 42.04, 35.84, 40.29, 39.43, 32.39, 29.26, 28.86,
173.59, 143.81, 171.64, 125.30, 19.08, 15.35.
´
10. (a) Donadel, O. J.; Garcıa, E. E.; Guerreiro, E.; Tonn, C.
E. An. Asoc. Quim. Argent. 1998, 86, 90; (b) Donadel, O.
In conclusion, the above-described results are an addi-
tional support for our hypothesis suggesting that the po-
lar function at C-4 in compound 8 plays a determinant
role in the cytoprotective effect reported here.
´
J.; Guerreiro, E.; Ardanaz, C. E. Rapid Commun. Mass
Spectrom. 2001, 15, 164.
11. Denny, R. W.; Nickon, A. In Organic Reactions, Wiley:
New York, 1973; Vol. 20, pp 133–336.
12. Zdero, C.; Bohlmann, F.; Anderberg, A.; King, R. M.
Phytochemistry 1991, 30, 2643.
13. Fieser, L. F.; Fieser, M. In Reagents for Organic Synthesis,
Wiley: New York, 1967; Vol. 1, pp 142–144.
The results of the present study proved that in the ses-
quiterpene series evaluated here, some structural
requirements are necessary to elicit cytoprotective effect.
Thus, the presence of an electrophilic center (i.e., a,b-
unsaturated carbonyl group) together with a second
polar group seems to be determinant, whereas confor-
mational requirements from decaline system seem to
play only a secondary role.
´
´
14. Gonzalez, A. G.; Bermejo Barrera, J.; Mendez, J. T.;
Lopez Sanchez, M.; Eiroa Martınez, J. L. Phytochemistry
´
´
´
1992, 31, 1816.
15. Bohlmann, F.; Ates (Go¨ren), N.; Jakupovic, J. Phyto-
chemistry 1983, 22, 1159.
¨
16. Okuz, S.; Topc¸u, G. A. Phytochemistry 1992, 31, 195.
¨
17. Urones, J. G.; Marcos, I. S.; Basabe, P.; Gimenez, A.;
´
Gonzalez, A.; Lithgow, A. M. Phytochemistry 1995, 38, 443.
´
18. Giordano, O. S.; Guerreiro, E.; Romo, J.; Jimenez, M. I.
Acknowledgments
´
Rev. Latinoamer. Quim. 1979, 6, 131.
19. Robert, A.; Nezamis, J. E.; Lancaster, C.; Hencher, A.
J. Gastroenterol. 1979, 77, 433.
The financial support from CONICET (PIP 5031) and
UNSL is gratefully acknowledged. We thank Professors
L. A. del Vitto for plant identification, and P. C. Rosso-
mando and E. Garcia for NMR measurements. C.E.T.
and R.D.E. are members of the researcher group of
CONICET-Argentina. This work is a part of the doc-
toral thesis of O.J.D.
20. Blandazzi, C.; Gherardi, G.; Marveggio, C.; Natale, G.;
Carignani, D.; Del Tacca, M. Digestion 1995, 56, 220.
21. Chemical stability of compound 12. To 50 ml of aqueous
solution containing NaCl (34.2 mM) and HCl (84.4 mM)
(final pH of 1.2), 150 mg of compound 12 was added. The
mixture was stirred at 35 °C, and aliquots of 10 ml were
removed every 30 min. Each individual fraction was
neutralized with solid NaHCO3 and extracted with Et2O.
The organic layers were dried, evaporated at low temper-
ature, and subjected to analysis by GC–MS. The results
showed that the quantitative hydrolysis of compound 12
to 8 was completed after 2 h.
References and notes
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´
J. R.; Enriz, R. D.; Rodrıguez, A. M.; Jauregui, E. A.;