Gastroprotective effect of carnosic acid γ-lactone derivatives

Article abstract of DOI:10.1021/np900822x

Carnosic acid (1) has been shown to possess gastroprotective activity in vitro and in vivo. However, little is known of the gastroprotective effect or cytotoxicity of carnosic acid γ-lactone (3). To determine structure-activity relationships, a series of 17 esters of 3 were prepared including aliphatic, aromatic, and heterocyclic derivatives. Also, two units of 3 were coupled with succinic and phthalic acid as linkers. The compounds were assessed for their gastroprotective effect in the HCl/EtOH-induced gastric lesions model in mice and for cytotoxicity in human lung fibroblasts, human adenocarcinoma AGS cells, and Hep G2 hepatocellular carcinoma cells. At a single oral dose of 40 mg/kg, the gastroprotective effect increased moderately with the length of the alkyl chain. The best effects were observed for the butyrate (9) and chloroacetate (6) derivatives. Activity of fatty acid esters increased with chain length but decreased with unsaturation. The best gastroprotective effect, with lowest cytotoxicity, was found for the palmitate (11) and oleate (12) derivatives.

Full text of DOI:10.1021/np900822x

J. Nat. Prod. 2010, 73, 639–643
639
Gastroprotective Effect of Carnosic Acid γ-Lactone Derivatives§
Mariano Walter Pertino,‡ Cristina Theoduloz,† Jaime A. Rodr´ıguez,† Tania Ya´n˜ez,† Viviana Lazo,‡ and
Guillermo Schmeda-Hirschmann*,‡
Laboratorio de CultiVo Celular, Facultad de Ciencias de la Salud, UniVersidad de Talca, Casilla 747, Talca, Chile, and Laboratorio de
Qu´ımica de Productos Naturales, Instituto de Qu´ımica de Recursos Naturales, UniVersidad de Talca, Casilla 747, Talca, Chile
ReceiVed December 16, 2009
Carnosic acid (1) has been shown to possess gastroprotective activity in vitro and in vivo. However, little is known of
the gastroprotective effect or cytotoxicity of carnosic acid γ-lactone (3). To determine structure-activity relationships,
a series of 17 esters of 3 were prepared including aliphatic, aromatic, and heterocyclic derivatives. Also, two units of
3 were coupled with succinic and phthalic acid as linkers. The compounds were assessed for their gastroprotective
effect in the HCl/EtOH-induced gastric lesions model in mice and for cytotoxicity in human lung fibroblasts, human
adenocarcinoma AGS cells, and Hep G2 hepatocellular carcinoma cells. At a single oral dose of 40 mg/kg, the
gastroprotective effect increased moderately with the length of the alkyl chain. The best effects were observed for the
butyrate (9) and chloroacetate (6) derivatives. Activity of fatty acid esters increased with chain length but decreased
with unsaturation. The best gastroprotective effect, with lowest cytotoxicity, was found for the palmitate (11) and oleate
(12) derivatives.
The Mediterranean medicinal and aromatic plants Rosmarinus
officinalis L. (rosemary) and SalVia officinalis L. (sage) are well
known for their biological effects, including chemopreventive and
antioxidant activities.1,2 The gastroprotective effect of sage and
rosemary and the main active constituents from the plant extracts
were reported previously.3,4 Rosemary and sage extracts contain
carnosic (1) and rosmarinic acids as the main bioactive constituents
that have been associated with several of the reputed therapeutic
actions of the crude drugs.
The abietane diterpenes from R. officinalis have shown cytotox-
icity to P388 murine leukemia cells,5 as well as antibacterial6,7 and
antioxidant effects.2 Carnosic acid is the main phenolic diterpene
found in R. officinalis leaves. It is usually isolated with variable
amounts of carnosol.8 Other phenolic diterpenes occurring in the
plant are rosmanol,9,10 epirosmanol,2,9 carnosaldehyde,2 and ros-
madial.11
Carnosic acid (1) has been proposed to be the gastroprotective
constituent of S. officinalis (sage). While 1 has been extensively
investigated for several biological effects, much less is known on
the effects and structure-activity relationship of the γ-lactone and
derivatives. The lactone carnosol has been described as an anti-
inflammatory,12 hepatoprotective,13 and antitumor14 compound. The
aim of the present study was to assess the effect of 3 and various
esters toward prevention of gastric ulcers.
Most studies on R. officinalis have been focused on antioxidant
activity of the extract and its constituents. The main compounds
with higher free radical scavenging/antioxidant effect were carnosic
acid and rosmarinic acid.15 Antibacterial activity of 20 diterpenes
isolated from SalVia species against Gram-positive and Gram-
negative bacteria was previously evaluated.16 The compounds
assessed included products with a lactone group between C-20 and
C-6 and between C-20 and C-7, carnosic acid derivatives, and other
diterpenes with various oxidation and substitution patterns. A
carnosic acid derivative and a C-20 and C-6 lactone with a quinoid
system in the aromatic ring gave MICs of 20-25 µg/mL against
Staphylococcus aureus and 8-10 µg/mL against Bacillus subtilis,
respectively. The isolation, structural modification, and cytotoxicity
of abietane diterpenes from PeroVskia abrotanoides on murine
leukemia cells P388 were reported recently.5 The authors presented
results relating to substitution at the C-20 carboxylic acid and
cytotoxicity toward the P388 leukemia cell line.
Herein, we describe the preparation of various semisynthetic
derivatives of carnosic acid γ-lactone (3) and examine the
structure-activity relationships of these compounds as gastropro-
tective agents and their cytotoxicity. The effects were compared
with those of the parent compound toward normal lung fibroblasts
MRC-5 (ATCC CCL-171), human gastric adenocarcinoma AGS
cells (ATCC CRL-1739), and human Hep G2 hepatocellular
carcinoma cells, as well as on the HCl/EtOH-induced gastric lesions
model in mice. The MRC-5 cells were used as a control of basal
cytotoxicity. Epithelial gastric AGS cells were selected because we
were looking for gastroprotective activity. Hepatocytes, Hep G2
(ATCC HB-8065), were included because they express the P450
cytochrome system, differing from the two previous cell lines, which
are unable to metabolize the compounds.
Results and Discussion
Carnosic acid γ-lactone (3) was prepared by an intramolecular
esterification of 1 using dicyclohexylcarbodiimide (DCC) and
dimethylaminopyridine (DMAP) in dry CH2Cl2. In the same way,
carnosic acid γ-lactone 12-methyl ether (4) was prepared from
carnosic acid 12-methyl ether (2). Compound 5 was obtained
by acetylation of 3. For the preparation of derivatives 6-20,
100 mg (0.3 mmol) of 3 was used for each reaction. DCC (80
mg, 0.39 mmol) and a catalytic amount of DMAP were employed
in all of the reactions. Esterification of 3 was performed using
chloroacetic (57 mg), isobutyric (53 mg), propionic (45 mg),
butyric (53 mg), lauric (120 mg), palmitic (154 mg), oleic (169
mg), or linoleic (168 mg) acid, with DCC/DMAP in CH2Cl2 for
2 h at room temperature, affording the corresponding chloro-
acetate 6, isobutyrate 7, propionate 8, butyrate 9, laurate 10,
palmitate 11, oleate 12, and linoleate 13 in 18-83% w/w yields.
Treatment of 3 with benzoic (73 mg), nicotinic (82 mg),
phenylacetic (74 mg), indoleacetic (106 mg), and indolebutyric
(122 mg) acid (Sigma Chemical Co.) using the same protocol
as described above afforded the benzoate 14, nicotinate 15,
phenylacetate 16, indoleacetate 17, and indolebutyrate 18 in
28-85% w/w yields. Treatment of 3 with succinic (100 mg)
and phthalic (71 mg) acid and DCC/DMAP in CH2Cl2 gave the
corresponding diesters (19 and 20) in 41% and 32% yield,
respectively. The purity of all derivatives was over 98%, as
§ Dedicated in memory of Prof. Jaime A. Rodr´ıguez.
* To whom correspondence should be addressed. Tel: + 56-71-200288.
Fax: + 56-71- 200448. E-mail: schmeda@utalca.cl.
† Laboratorio de Cultivo Celular.
‡ Instituto de Qu´ımica de Recursos Naturales.
10.1021/np900822x  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/01/2010

640 Journal of Natural Products, 2010, Vol. 73, No. 4
Scheme 1. Preparation of Compounds 3 and 4
Pertino et al.
Scheme 3. Preparation of Compounds 19 and 20a
assessed by 1H NMR spectroscopy. The syntheses are sum-
marized in Schemes 1-3. All of the compounds were character-
ized by spectroscopic means, and compounds 6-20 are described
for the first time. Details of the preparation of the above esters
1
and tables of their H and 13C NMR spectra are provided in the
Supporting Information. The compounds were then evaluated
for gastroprotective effect and cytotoxicity. The results are
summarized in Table 1.
Carnosic acid γ-lactone 12-methyl ether was previously isolated
from the aerial parts of flowering S. officinalis.17,18 In the present
study, the parent compound (3) was tested for gastroprotective effect
on the experimental model of HCl/EtOH-induced gastric lesions
in mice (Table 1). A significant, dose-dependent gastroprotective
effect was found for 3, reducing gastric lesions by 59.5% at an
oral dose of 40 mg/kg. Under the same experimental conditions
the reference drug lansoprazole, at 20 mg/kg, reduced the lesions
by 80%. For comparison purposes and to study structure-activity
relationships, the 17 derivatives of 3 were evaluated at a single
oral dose of 40 mg/kg in mice.
a Reagents and conditions: (a) succinic acid; DCC; DMAP; CH2Cl2; (b)
phthalic acid; DCC; DMAP; CH2Cl2. Reactions were carried out at room
temperature.
The gastroprotective effect increased moderately with the alkyl
chain length, as can be observed by comparing the effects of the
acetate (5) and propionate (8) with the butyrate (9). However, the
prevention of gastric lesions was lower for the C12 derivative (10,
36% reduction) and increased in the C16 and C18 esters (11 and 12)
to 65% and 70%, respectively. Among the fatty acid esters, activity
Scheme 2. Preparation of Compounds 5-18a
a Reagents and conditions: (a) acetic anhydride; pyridine or appropriate organic acid; DCC; DMAP; CH2Cl2; (b) appropriate fatty acid; DCC; DMAP; CH2Cl2; (c)
appropriate aromatic acid; DCC; DMAP; CH2Cl2; (d) appropriate indolic acid; DCC; DMAP; CH2Cl2. All reactions were carried out at room temperature.

Carnosic Acid γ-Lactone DeriVatiVes
Journal of Natural Products, 2010, Vol. 73, No. 4 641
Table 1. Dose-Response Gastroprotective Effect of Carnosic Acid γ-Lactone (3), Gastroprotective Effect at 40 mg/kg, and Cytotoxicity
(IC50, µM) of Derivatives 1-20a
compound
lesion index (mm)
% lesion reduction
cytotoxicity (IC50, µM)
carnosic acid γ-lactone (3)
10 mg/kg
20 mg/kg
40 mg/kg
control
35.7 ( 11.3
31.3 ( 12.3
18.3 ( 6.1b
45.1 ( 8.2
20.8
30.6
59.5
MRC-5
293
AGS
246
Hep G2
240
1
2
4
5
6
7
8
9
10
11
12
13
14
15
16
17
nt
nt
21.8 ( 3.2
54.9 ( 3.9
31.9 ( 1.6
290 ( 11
130 ( 7
119 ( 7
125 ( 6
44.4 ( 2.1
>1000
89.8 ( 5.2
45.2 ( 3.1
26.7 ( 1.8
275 ( 19
103 ( 8
41.3 ( 2.6
43.5 ( 4.2
58.1 ( 4.1
294 ( 17
203 ( 12
112 ( 9
196 ( 15
56.1 ( 2.8
>1000
13.3 ( 7.2b
17.6 ( 9.8b
8.5 ( 5.1b
12.9 ( 8.0b
18.7 ( 5.5b
8.0 ( 3.6b
29.3 ( 10.5b
16.0 ( 7.0b
13.9 ( 4.4b
25.0 ( 13.3b
23.8 ( 11.4b
41.7 ( 14.1
25.6 ( 9.0b
22.1 ( 8.9b
23.0 ( 13.5b
17.3 ( 9.2b
38.6 ( 15.1
45.7 ( 9.6
9.1 ( 6.2b
71
62
81
72
59
82
36
65
70
45
48
9
44
51
50
62
15
105 ( 7
113 ( 6
99.5 ( 4.9
413 ( 25
>1000
>1000
>1000
>1000
822 ( 49
253 ( 18
503 ( 25
280 ( 22
133 ( 8
85.6 ( 5.1
46.8 ( 2.8
623 ( 43.6
375 ( 23
>1000
293 ( 21
512 ( 41
296 ( 22
516 ( 36
109 ( 5
54.5 ( 3.3
>1000
289 ( 20
510 ( 35
264 ( 13
522 ( 31
251 ( 21
120 ( 7
>1000
18
19
20
control
678 ( 42
611 ( 43
lansoprazole (20 mg/kg)
80
306 ( 16
162 ( 11
221 ( 13
a Results are expressed as means ( SD. n ) 7. ANOVA followed by Dunnett’s multiple comparison test. nt: not tested. b P < 0.01 compared to
control group. Each concentration was tested in quadruplicate together with the control and repeated three times in separate experiments.
increased with the alkyl chain length but decreased with unsatura-
tion, as can be seen by comparing the lesion reduction by the C18:1
and C18:2 derivatives 12 and 13, which was 70% and 45%,
respectively.
The highest gastric lesion prevention activity was elicited by the
butyrate (9, 82%) and chloroacetate (6, 81%). The relevance of
the chlorine atom can be deduced by comparing the effect of the
acetate 5 with that of compound 6, bearing the halogen. The effect
of the aromatic derivatives 14 and 16 as well as 17 and 18, differing
in the CH2 units between the carbonyl ester function and the
aromatic ring of the side chain, was similar. In the diesters with
succinic and phthalic acid (19 and 20), only the succinate presented
a significant gastroprotective effect (62%).
When the γ-lactone derivatives were tested against human cell
lines, the highest cytotoxicity was found for the more lipophilic
γ-lactone methyl ether (4), with low selectivity and IC50 values of
31.9, 26.7, and 58.1 µM for fibroblasts, AGS cells, and HepG2
cells, respectively. A C4 side chain such as the butyrate (9), or a
C4 side chain linked to a heterocycle as in the indolebutyrate (18),
increased toxicity when compared with the parent compound. While
the cytotoxicity of compound 9 was higher for fibroblasts and Hep
G2 cells (IC50 of 44.4 and 56.1 µM, respectively) than AGS cells
(IC50: 99.5 µM), and compound 18 was more toxic to fibroblasts
and AGS cells (IC50 of 54.5 and 46.8 µM, respectively) than against
Hep G2 hepatocytes (IC50: 120 µM), the selectivity was not
sufficient to be of significance.
The cytotoxicity of acetate 5 was similar to that of carnosic acid
γ-lactone itself, but increased when a chlorine atom was placed in
the ester chain as in the chloroacetate 6. A remarkable change in
the activity was found for the long-chain fatty acid esters 10-12,
being far less cytotoxic than the starting compound, with IC50 values
>1000 µM toward fibroblasts and Hep G2 cells. Cytotoxicity
increased with unsaturation, as can be seen comparing the IC50
values of the oleate (12) with that of the linoleate (13). For the
esters with heterocycles (17 and 18), differing in two CH2 units,
higher cell toxicity was displayed by the indolebutyrate (18), with
an IC50 value about half that of the indoleacetate (17). When two
units of 3 were coupled with a linker to form diesters, the
cytotoxicity was less pronounced for 19 than for 20, and both
compounds were less toxic than 3.
In a report on the semisynthesis, cytotoxicity, and antimicrobial
effect of aromatic abietanes, the authors presented the IC50 data
(in µM) of the compounds prepared and the reference compound
6-mercaptopurine.19 Several of the compounds described were more
toxic than the products described in this report.
The compound with the highest gastroprotective effect (81%)
was 6; however, it was more cytotoxic than compounds 11 and
12, which present IC50 values of >1000 µM against MRC-5 and
HepG2 cells and >1000 and 822 µM toward AGS cells, respectively.
Taking together both gastroprotective effect and cytotoxicity, the
best effect with the lowest toxicity was found for the fatty acid
derivatives C16 (11) and C18:1 (12).
Experimental Section
General Experimental Procedures. Melting points were determined
on a Koffler hot stage apparatus (Electrothermal 9100) and were
uncorrected. Optical rotations were measured on a Jasco DIP 370 (Jasco
Analytical Instruments, Easton, MD) polarimeter in CHCl3 at 20 °C.
IR spectra were recorded on a Nicolet Nexus 470 FT-IR instrument
(Thermo Electron Corporation, Waltham, MA). The NMR spectra were
recorded on a Bruker Avance 400 (Bruker, Rheinstetten, Germany)
spectrometer at 400 MHz for 1H and 100 MHz for 13C in CDCl3.
Chemical shifts are given in δ (ppm) with TMS as the internal standard.
Low-resolution mass spectra were run on a VG Micromass ZAB-2F
and high-resolution mass spectra on a VG Micromass ZAB-2F at 70
eV (Varian Inc., Palo Alto, CA). Merck silica gel (0.063-0.2) was
used for column chromatography; precoated silica gel plates (Merck,
Kieselgel 60 F254, 0.25 mm) were used for TLC analysis. TLC spots
were visualized by spraying the chromatograms with p-anisaldehyde/
ethanol/acetic acid/H2SO4 (2:170:20:10 v/v) and heating at 110 °C for
3 min. Dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine
(DMAP) were from Merck (Schuchardt, Germany).
Plant Material. Carnosic acid (1) and carnosic acid 12-methyl ether
(2) were isolated from the aerial parts of R. officinalis cultivated in
Curico´, VII Regio´n, Chile. A voucher herbarium specimen (Pertino
001/2007) has been kept at the Herbario de la Universidad de Talca.
Compound 1 was obtained after extraction of the air-dried and powdered
plant material with petroleum ether/ethyl acetate (EtOAc) (1:1) and

642 Journal of Natural Products, 2010, Vol. 73, No. 4
Pertino et al.
successive purification by column chromatography on silica gel,
followed by crystallization. Preparation of the following compounds
is described in the Supporting Information.
Carnosic acid γ-lactone 12-indolebutyrate (18): yellow oil; [R]D20
+47 (c 0.09; CHCl3); IR (film) νmax 3418, 2956, 1800, 1748, 1433,
1126 cm-1; EIMS m/z 499 [M]+ (5), 453 (22), 286 (71), 71 (12), 230
(15), 218 (10), 186 (100), 130 (19); HREIMS m/z 499.2741 [M]+ (calcd
for C32H37NO4, 499.2723).
Carnosic acid γ-lactone (3): colorless crystals; mp 106-109 °C;
[R]2D0 +79 (c 0.12, CHCl3); IR (film) νmax 3382, 2956, 1788, 1411 cm-1
;
EIMS m/z 286 [M - CO]+ (100), 271 (30), 243 (34), 230 (57), 218
(42), 204 (24), 69 (13); HREIMS m/z 286.1920 [M - CO]+ (calcd for
C19H26O2, 286.1933).
Carnosic acid γ-lactone 12-succinate (19): pale yellow oil; [R]D20
+52 (c 0.08; CHCl3); IR (film) νmax 2956, 1804, 1764, 1445, 1126
cm-1; HREIMS m/z 733.3725 [M + Na]+ (calcd for C44H54O8Na,
733.3716).
Carnosic acid γ-lactone 12-methyl ether (4): colorless crystals;
mp 110-113 °C; [R]2D0 +69 (c 0.18, CHCl3); IR (film) νmax 2954, 1794,
1623, 1430 cm-1; HRMS m/z 323.1966 [M - CO + Na]+ (calcd for
C20H28O2Na: 323.1987).
Carnosic acid γ-lactone 12-phthalate (20): colorless powder; mp
234-237 °C; [R]2D0 +62 (c 0.10; CHCl3); IR (film) νmax 2956, 1856,
1792, 1772, 1744, 1433, 1126 cm-1; HREIMS m/z 781.3746 [M +
Na]+ (calcd for C48H54O8Na, 781.3716).
Carnosic acid γ-lactone 12-acetate (5): pale yellow oil; [R]2D0 +64
(c 0.27, CHCl3); IR (film) νmax 2952, 1808, 1760, 1437, 1190 cm-1
;
The H NMR and 13C NMR data of compounds 3-9 as well as
1
EIMS m/z 328 [M - CO]+ (40), 286 (100), 271 (15), 243 (19), 230
(36), 218 (19), 204 (14), 69 (10); HREIMS m/z 328.2061 [M - CO]+
(calcd for C21H28O3, 328.2049).
10-20 are presented in Tables S1 and S2 for 1H and Tables S3 and S4
for 13C, respectively (see the Supporting Information). For preparation
of the compounds, see the Supporting Information. The structures of
the compounds were authenticated by comparing spectroscopic data
with those published for carnosic acid γ-lactone 12-methyl ether (4)17,18
and related diterpenes.5,6,10
Carnosic acid γ-lactone 12-chloroacetate (6): pale yellow oil; [R]2D0
+70 (c 0.18; CHCl3); IR (film) νmax 2968, 1824, 1784, 1437, 1126
cm-1; EIMS m/z 362 [M - CO]+ (100), 347 (40), 319 (25), 306 (40),
286 (31), 269 (23), 230 (44), 218 (27), 149 (22), 77 (24), 69 (30);
HREIMS m/z 413.1475 [M + Na]+ (calcd for C22H27ClO4Na, 413.1496).
Carnosic acid γ-lactone 12-isobutyrate (7): pale yellow oil; [R]D20
+69 (c 0.19; CHCl3); IR (film) νmax 2957, 1809, 1765, 1435, 1122
cm-1; EIMS m/z 356 [M - CO]+ (32) 286 (100), 243 (15), 230 (28),
218 (12), 71 (12); HREIMS m/z 407.2178 [M + Na]+ (calcd for
C24H32O4Na, 407.2198).
HCl/EtOH-Induced Ulcer Model in Mice. The gastroprotective
effect of the compounds was assessed by the HCl/EtOH-induced gastric
lesions model in mice.20 Male Swiss albino mice weighing 30 ( 3 g
were used. Animals were purchased from the Instituto de Salud Pu´blica
de Chile, Santiago de Chile. Animals were fed on certified Champion
diet with free access to water under standard conditions of 12 h
dark-light period, 50% relative humidity, and 22 °C. Mice were
randomly distributed into groups of 7 animals each and fasted for 24 h
with free access to water prior to the experiment. For carnosic acid
γ-lactone, three different doses were used: 40, 20, and 10 mg/kg. For
dose-response studies, at least three doses should be used. Doses of
40, 20, and 10 mg/kg were selected on the basis of previous work on
gastroprotective diterpenes to find a dose at which gastroprotection was
about 50% for the starting compound (carnosic acid γ-lactone).
For comparison purposes, the other compounds were assessed at 40
mg/kg. Compounds were suspended in 12% Tween 80 and administered
orally. Fifty minutes after treatment, all groups received orally 0.2 mL
of a solution containing 0.3 M HCl/60% EtOH (HCl/EtOH) for gastric
lesion induction. The antisecretory drug lansoprazole was used as the
reference compound. The control group received 12% Tween 80.
Animals were sacrificed by cervical dislocation 1 h after the administra-
tion of HCl/EtOH, and the stomachs were excised and inflated by
injection of saline (1 mL). The ulcerated stomachs were fixed in 5%
formalin for 30 min and opened along the greater curvature. The length
(mm) of each lesion was measured, and the lesion index was expressed
as the sum of the length of all lesions.20,21 The protocols were approved
by the Universidad de Talca Institutional Animal Care and Use
Committee, which follows the recommendations of the Canadian
Council on Animal Care.22
Cytotoxicity Assay. The human cell lines MRC-5 normal lung
fibroblasts (CCL-171), AGS gastric adenocarcinoma cells (CRL-1739),
and Hep G2 hepatocellular carcinoma cells (HB-8065) were obtained
from the American Type Culture Collection (ATCC, Manassas, VA).
The cells were grown as monolayers in the following media: MRC-5
and Hep G2 in MEM and AGS in Ham F-12. The MEM medium
contained 2 mM L-glutamine, 1 mM sodium pyruvate, and 1.5 g/L
sodium bicarbonate. Ham F-12 was supplemented with 2 mM
L-glutamine and 1.5 g/L sodium bicarbonate. All media were supple-
mented with 10% heat-inactivated fetal bovine serum, 100 IU/mL
penicillin, and 100 µg/mL streptomycin in a humidified incubator with
5% CO2 in air at 37 °C. For the experiments, cells were plated at a
density of 25 000 cells/mL in 96-well plates. Confluent cultures of the
different cell lines were treated with medium containing the compounds
at concentrations ranging from 0 up to 1000 µM. Compounds were
first dissolved in DMSO and then in medium. The final concentration
of DMSO in the test medium and controls was 1%. Cells were exposed
to test medium for 24 h, with or without the compound (control). Each
concentration was tested in quadruplicate together with the control and
repeated three times in separate experiments. Cell viability was
determined at the end of the incubation by means of the neutral red
uptake (NRU) assay.23 Results were converted to percentage of controls,
and the IC50 values were graphically obtained from the dose-response
curves.
Carnosic acid γ-lactone 12-propionate (8): pale yellow oil; [R]D20
+46 (c 0.08; CHCl3); IR (film) νmax 2957, 1804, 1770, 1439, 1130
cm-1; EIMS m/z 342 [M - CO]+ (30), 286 (100), 243 (20), 230 (38),
218 (19), 69 (8); HREIMS m/z 393.2013 [M + Na]+ (calcd for
C23H30O4Na, 393.2042).
Carnosic acid γ-lactone 12-butyrate (9): pale yellow oil; [R]2D0 +73
(c 0.08; CHCl3); IR (film) νmax 2961, 1800, 1765, 1443, 1126 cm-1
;
EIMS m/z 356 [M - CO]+ (21), 286 (100), 243 (15), 230 (27), 218
(13), 71 (9); HREIMS m/z 407.2212 [M + Na]+ (calcd for C24H32O4Na,
407.2198).
Carnosic acid γ-lactone 12-laurate (10): colorless oil; [R]2D0 +57
(c 0.14; CHCl3); IR (film) νmax 2956, 1816, 1764, 1437, 1126 cm-1
;
EIMS m/z 468 [M - CO]+ (18), 450 (8), 323 (10), 286 (100), 271
(12), 230 (16), 218 (9); HREIMS m/z 491.3526 [M - CO + Na]+
(calcd for C31H48O3Na, 491.3501).
Carnosic acid γ-lactone 12-palmitate (11): colorless oil; [R]2D0 +37
(c 0.11; CHCl3); IR (film) νmax 2952, 1804, 1764, 1441, 1134 cm-1
;
EIMS m/z 524 [M - CO]+ (13), 506 (8), 286 (100), 230 (12), 98 (7);
HREIMS m/z 575.4058 [M + Na]+ (calcd for C36H56O4Na, 575.4077).
Carnosic acid γ-lactone 12-oleate (12): colorless oil; [R]2D0 +48 (c
0.12; CHCl3); IR (film) νmax 2956, 1812, 1764, 1433, 1118 cm-1; EIMS
m/z 550 [M - CO]+ (9), 286 (100), 271 (7), 230 (13), 218 (7), 69 (6);
HREIMS m/z 601.8582 [M + Na]+ (calcd for C38H58O4Na, 601.8570).
Carnosic acid γ-lactone 12-linoleate (13): pale yellow oil; [R]D20
+32 (c 0.09; CHCl3); IR (film) νmax 2956, 1809, 1757, 1435, 1117
cm-1; EIMS m/z 548 [M - CO]+ (8), 323 (7), 286 (100), 271 (11),
230 (14), 218 (10), 69 (7); HREIMS m/z 548.4251 [M - CO]+ (calcd
for C37H56O3, 548.4229).
Carnosic acid γ-lactone 12-benzoate (14): colorless powder; mp
152-153 °C; [R]2D0 +48 (c 0.10; CHCl3); IR (film) νmax 2964, 1800,
1744, 1441, 1051 cm-1; EIMS m/z 390 [M - CO]+ (34), 375 (9), 285
(16), 105 (100), 77 (8); HREIMS m/z 441.2063 [M + Na]+ (calcd for
C27H30O4Na, 441.2042).
Carnosic acid γ-lactone 12-nicotinate (15): colorless powder; mp
155-157 °C; [R]2D0 +48 (c 0.10; CHCl3); IR (film) νmax 2964, 1796,
1744, 1437, 1083 cm-1; EIMS m/z 391 [M - CO]+ (76), 376 (18),
285 (35), 269 (10), 106 (100), 78 (12); HREIMS m/z 391.2126 [M -
CO]+ (calcd for C25H29NO3, 391.2147).
Carnosic acid γ-lactone 12-phenylacetate (16): pale yellow oil;
[R]2D0 +59 (c 0.20; CHCl3); IR (film) νmax 2956, 1800, 1760, 1441,
1119 cm-1; EIMS m/z 404 [M - CO]+ (26), 286 (100), 243 (12), 230
(27), 218 (13), 91 (19), 69 (8); HREIMS m/z 404.2367 [M - CO]+
(calcd for C27H32O3, 404.2351).
Carnosic acid γ-lactone 12-indoleacetate (17): yellow oil; [R]D20
+45 (c 0.10; CHCl3); IR (film) νmax 3418, 2964, 1804, 1752, 1445,
1114 cm-1; EIMS m/z 443 [M - CO]+ (15), 425 (13), 286 (42), 230
(12), 157 (36), 130 (100); HREIMS m/z 494.2330 [M + Na]+ (calcd
for C30H33NO4Na, 494.2307).
Statistical Analysis. Results were expressed as the mean ( SD.
Statistical differences between treatments and their respective control

Carnosic Acid γ-Lactone DeriVatiVes
Journal of Natural Products, 2010, Vol. 73, No. 4 643
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the F value was significant, post hoc differences were determined by
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set at P < 0.01. All statistical analyses were performed using the
software Statistica 5.1 (StatSoft, Inc.) and Statistical Package S-Plus
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Acknowledgment. This work was supported by FONDECYT (Grant
No. 1060841) and Programa de Investigacio´n en Productos Bioactivos,
Universidad de Talca. M.W.P. thanks the PBCT PSD50 for a post-
doctoral grant.
Supporting Information Available: Preparation of compounds
3-20 and the NMR data of compounds are presented in Tables S1
and S2 for 1H and S3 and S4 for 13C, respectively. This information is
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