Synthesis and pharmacological activity of diterpenylnaphthoquinone derivatives

Article abstract of DOI:10.3390/molecules16108614

New diterpenylquinones, combining a diterpene diacid and a naphthoquinone, were prepared from junicedric acid and lapachol. The new derivatives were assessed as gastroprotective agents by the HCl-EtOH-induced gastric lesions model in mice as well as for basal cytotoxicity on the following human cell lines: Normal lung fibroblasts (MRC-5), gastric epithelial adenocarcinoma (AGS), and hepatocellular carcinoma (Hep G2). Several of the new compounds were significantly active as antiulcer agents and showed selective cytotoxicity against AGS cells.

Full text of DOI:10.3390/molecules16108614

Molecules 2011, 16, 8614-8628; doi:10.3390/molecules16108614
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Pharmacological Activity of
Diterpenylnaphthoquinone Derivatives
1
2
3
Mariano Walter Pertino , Cristina Theoduloz , Jose Antonio Palenzuela ,
3
4
2
2
Maria del Mar Afonso , Erdem Yesilada , Francisco Monsalve , Paulo González ,
2
1,
Daniel Droguett and Guillermo Schmeda-Hirschmann *
1
Instituto de Química de Recursos Naturales, Universidad de Talca, Casilla 747, Talca, Chile
2
Facultad de Ciencias de la Salud, Universidad de Talca, Casilla 747, Talca, Chile
3
Instituto Universitario de Bio-Orgánica Antonio Gonzalez G., Universidad de La Laguna,
Tenerife 38206, Spain
4
Faculty of Pharmacy, Yeditepe University, Istanbul 34755, Turkey
*
Author to whom correspondence should be addressed; E-Mail: schmeda@utalca.cl;
Tel.: +56-71-200288; Fax: + 56-71-201573.
Received: 13 September 2011; in revised form: 29 September 2011 / Accepted: 30 September 2011 /
Published: 13 October 2011
Abstract: New diterpenylquinones, combining a diterpene diacid and a naphthoquinone,
were prepared from junicedric acid and lapachol. The new derivatives were assessed as
gastroprotective agents by the HCl-EtOH-induced gastric lesions model in mice as well as
for basal cytotoxicity on the following human cell lines: Normal lung fibroblasts (MRC-5),
gastric epithelial adenocarcinoma (AGS), and hepatocellular carcinoma (Hep G2). Several
of the new compounds were significantly active as antiulcer agents and showed selective
cytotoxicity against AGS cells.
Keywords: labdane diterpenes; lapachol derivatives; diterpenylnaphthoquinones;
gastroprotection; basal cytotoxicity
1
. Introduction
In the year 2002 stomach cancer and peptic ulcer disease represented 1.5 and 0.5% of the total
causes of death worldwide, respectively [1]. Chronic gastric ulcers can lead to gastric and pancreatic
cancer [2,3]. Several natural products have been shown to display significant gastroprotective effects

Molecules 2011, 16
8615
in animal models of induced gastric ulcers. These agents include diterpenes with different structural
skeletons [4-11]. The labdane diterpenes from Araucaria araucana (Molina) Koch (Araucariaceae)
and their semisynthetic derivatives present gastroprotective activity with a wide range of basal
cytotoxicity on human cell lines [9,10]. According to Halle and Spielmann there is a significant
correlation between cytotoxicity of mammalian cell culture systems and acute oral toxicity (LD ) in
50
animals [12]. Basal cytotoxicity is a valid model to predict starting doses for in vivo lethality assays
in rodents.
Lapachol is a naphthoquinone that can be obtained in high yields from the wood or trunk bark of
Tabebuia species (Bignoniaceae), including Tabebuia heptaphylla (Vell. Conc.) Toledo [13]. Several
bioactivities have been described for lapachol and its semisynthetic derivatives, e.g., activation of the
Epstein-Barr virus [14], molluscicidal [15,16], trypanocidal [17], antiviral, antiproliferative [18], and
DNA-topoisomerase inhibitory activity [19].
A new approach to drug design is to link two molecules with individual intrinsic effect into a
single compound, named a “hybrid compound” [20]. A classification of “hybrid” molecules includes
conjugates, cleavage conjugates, fused hybrid molecules, and merged hybrids [20-23]. The use of
lapachol to obtain lapachol hybrid derivatives with diterpenes has not been explored previously, but
changes in the gastroprotective effect and basal cytotoxicity of the resulting products should be expected.
In this paper we describe the preparation of junicedric acid and lapachol derivatives and examine
the structure-activity relationships/trends of the new compounds as gastroprotective agents in mice.
The basal cytotoxicity of the new compounds was also assessed towards the following human cell
lines: normal lung fibroblasts (MRC-5), epithelial adenocarcinoma (AGS), and hepatocellular
carcinoma (Hep G2).
2
. Results and Discussion
The diterpene junicedric acid (I) (Scheme 1) was obtained by saponification and oxidation of a
mixture of labdane diterpenes from Araucaria araucana [9,10]. Isomerization of the double bond of I
to II was carried out by treating I in acetic acid (HOAc) with HBr. Reduction of the double bond of I
was carried out by catalytic hydrogenation of the terpene (Scheme 1). Lapachol (2-hydroxy-3-(3-
methyl-2-butenyl)-[1,4]-naphthoquinone) (IV, Scheme 2) was obtained from “lapacho” wood extract [13].
The hydrogenated products were obtained treating lapachol in ethyl acetate (EtOAc) with palladium on
activated carbon (Pd/C) (Scheme 2).
Scheme 1. Preparation of derivatives II and III from junicedric acid (I).
1
2
14
OH
13
15 OH
OH
1
1
2
0
1
7
16
O
O
O
1
4
9
a
2
3
10
8
b
5
7
6
O
19O
O
1
8
HO
HO
HO
III
JA (I)
II
2
Reagents and conditions: (a) H , Pd/C 10%, rt, EtOAc; (b) HOAc/HBr, rt, 24 h.

Molecules 2011, 16
Scheme 2. Preparation of derivatives V and VI from lapachol (IV).
8616
O
O
O
O
8
OH
8a
2
OH
OH
OH
a
7
6
1
4
b
2
'
3
4'
4a
5
1'
3'
O
O
5'
O
O
V
IV
VI
V
2 2
Reagents and conditions: (a) H , Pd/C 10%, EtOAc, rt; (b) H , Pd/C 10%, 4 bar, 2 h, 65 °C, HOAc.
Twelve esters combining a diterpene and lapachol or its derivatives were prepared in moderate to
good yields starting from the diterpene diacids I, II or III. The quinone moieties used included
2
-hydroxy-3-(3-methyl-2-butenyl)-[1,4]-naphthoquinone (lapachol) (IV), 2-hydroxy-3-(3-methyl-
butyl)-[1,4]-naphthoquinone (V) (dihydroprenyllapachol) and 2-hydroxy-3-(3-methyl-butyl)-5,6,7,8-
tetrahydro-[1,4]-naphthoquinone (VI) (dihydroprenyl-5,6,7,8-tetrahydrolapachol) (Scheme 2). All the
products were characterized by spectroscopic means. Compounds 1–12 (Figure 1) are described for the
first time.
Figure 1. Structures of compounds 1–12.
O
O
O
O
O
1
8
2
14
16
8
5
1
3
15
O
2
3
8a
4a
O
O
1
1
1
4
7
6
2
5
0
1
7
O 1'
O
O
1
4
9
2
3
10
7
4
'
6
2'
O
3
'
1
8
5'
O
9
O
O
1
RO
RO
RO
1
: R = H;
3: R = H;
5: R = H;
2
: R = CH
3
4: R = CH
3
6: R = CH
3
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
RO
RO
RO
7
: R = H;
9: R = H;
10: R = CH
11: R = H;
12: R = CH
3
8
: R = CH
3
3
The diterpene junicedric acid has two acid groups at C-15 and C-19. However, only the acid group at
C-15 is reactive enough to form esters or amides when the diacid is treated with N,N-dicyclohexyl-
carbodiimide (DCC)/dimethylaminopyridine (DMAP) and the alcohols or amines. Diesters or diamides
can be obtained via the acyl chloride [24].
Compounds 1–12 were evaluated for gastroprotective effect and basal cytotoxicity (Tables 1 and 2).
When assayed for gastroprotective effect, lapachol (IV) at 5, 10 and 50 mg/kg did not present a
statistically significant effect in the EtOH-HCl-induced gastric lesions model in mice when compared
with the vehicle control (Tween 80). Comparisons were made using Dunnett’s or Student-Newman-

Molecules 2011, 16
8617
Keuls test (data not shown). Compound II reduced gastric lesions by 79% at 5 mg/kg. The diterpene-
lapachol hybrid derivatives were investigated for gastroprotective effect at a single oral dose of
5
mg/kg (Table 1).
Table 1. Gastroprotective effect of compounds 1–12 on the HCl-EtOH-induced gastric
lesion model in mice. All compounds were assessed at a single oral dose of 5 mg/kg.
Lesion index (mm)
a
Compound
Protection (%)
Protected stomachs
Mean ± SEM
27.2 ± 9.3
Tween
Δ8(9) junicedric acid (II)
5.3 ± 2.4 *
15.3 ± 5.0
79
44
4/9
1
Tween
25.0 ± 6.9
1/14
4/9
0/9
3/8
1/8
5/9
0/10
0/7
0/9
0/8
0/9
0/9
3/9
6/9
2
5
6
7
8
8.7 ± 3.9
65
18
74
54
84
6
20.4 ± 3.6
6.5 ± 2.7 *
11.5 ± 4.4
3.9 ± 1.9 *
23.5 ± 3.6
1
1
1
2
27.4 ± 6.8
-
Tween
43.3 ± 8.2
3
4
9
17.9 ± 4.6 **
19.3 ± 5.0 **
15.7 ± 4.5 **
10.2 ± 3.5 **
9.4 ± 1.2 **
59
55
64
76
1
0
Lansoprazole (20 mg/kg)
73
a
*
and **: P < 0.05 different from untreated control (Tween); Number of stomachs which were
completely protected from any visible bleeding. One-way ANOVA with Student-Newman-Keuls
post-hoc test.
Derivatives 6 and 8 were significantly active, protecting 3/8 and 5/9 animals, respectively, from
lesions or stomach bleeding. An important gastroprotective effect was seen with derivatives 3, 4, 9 and
0 compared to untreated controls. However, for compounds 3, 4 and 9 all the stomachs showed at
1
least some lesions or bleeding. Compound 10 was a very active gastroprotective agent. Compounds 8
and 10 are methyl esters of II and IV, the quinone moiety bearing either the prenyl side chain or the
reduced prenyl side chain. Compound 6 was built from I and VI. It showed very low basal cytotoxicity
(
>1 mM) and proved to be one of the best gastroprotective products found in this study. The most active
compounds are C19 methyl esters of the parent compounds. This fact shows that changes in the structure of
any of the moieties will lead to changes in activity of the new compounds.
The diterpenylnaphthoquinones as well as I, II and IV were evaluated for basal cytotoxicity using
MRC-5 fibroblasts, AGS and Hep G2 cells (Table 2). No significant differences in basal cytotoxicity
were found between derivatives 1 and 7, where the diterpene moiety presents the double bond either at
8
(17)-en- (compound 1) or at 8(9)-en- (compound 7).

Molecules 2011, 16
8618
Table 2. Basal cytotoxicity of the lapachoyl ester derivatives from labdane diterpenes 1–12
towards MRC-5 fibroblasts and AGS and Hep G2 cells.
a
IC50 ± SEM (µM)
AGS
Compound
Fibroblasts
>1000
Hep G2
55 ± 3
>1000
>1000
57.4 ± 4.4
208 ± 11
>1000
>1000
27 ± 1
>1000
96 ± 9
379 ± 21
>1000
>1000
54.5 ± 5
290 ± 9
-
Lapachol (IV)
382 ± 15
304 ± 18
343 ± 22
170 ± 9
Junicedric acid (I)
181 ± 9
214 ± 32
210 ± 13
741 ± 40
156 ± 9
>1000
Δ8(9) junicedric acid (II)
1
2
3
4
5
6
7
8
9
361 ± 19
89 ± 5
382 ± 26
40 ± 6
69 ± 3
>1000
>1000
190 ± 8
>1000
179 ± 9
721 ± 35
294 ± 15
162 ± 10
114 ± 7
341 ± 17
>1000
1
1
1
0
1
2
336 ± 15
926 ± 35
0.33 ± 0.02
306 ± 11
323 ± 16
0.58 ± 0.02
162 ± 6
Etoposide
b
Lansoprazole
221 ± 9
a
Confluent cultures were treated with the culture medium containing the compounds at
concentrations ranging between 0 and 1,000 µM for 24 h. Cell viability was determined by the
neutral red uptake assay. Data are expressed as arithmetic mean values of three different
b
experiments in quadruplicate ± SEM; Reference compound.
Cytotoxicity strongly decreases after methylation, showing the relevance of the COOH function at
C-19 for this effect. In a broad sense, all compounds displayed lower basal cytotoxicity against
fibroblasts than against AGS or Hep G2 cells. Some selectivity was seen for compounds 1, 5, 7 and 11
towards Hep G2 cells. The most cytotoxic compound of this series (5) was built with I and the
hydrogenation product of lapachol VI. The IC50 values of this compound towards fibroblasts, AGS and
Hep G2 cells were 69, 40 and 27 µM, respectively. The increase in cytotoxicity was associated with poor
selectivity. The effect is completely lost after methylation of the C-19 COOH function of the diterpene.
A similar approach, but starting from myrcecommunic acid and p-benzoquinone was used to
prepare new diterpenylnaphthoquinones through a Diels-Alder cycloaddition [25]. Several of the
diterpenylnaphthoquinones presented higher cytotoxicity than the quinone moiety itself, the
naphthoquinone (NQ) system showing better effect than benzoquinone or anthraquinone
derivatives [25,26]. The most promising compounds present variations in the decaline part of the
naphthohydroquinone (NHQ) of the NQ system and show GI values (growth inhibition values that
5
0
reduce cell growth by 50%) in the 0.12–0.50 µM range towards the tumour cell lines A-549 (lung
carcinoma), HT-29 (colon carcinoma), and MEL-28 (malignant melanoma) [25]. The main difference
with this report is that [25] formed C-C bonds by cycloaddition, while we prepared esters of
dicarboxylic diterpenes and napththoquinones.

Molecules 2011, 16
8619
3
. Experimental
3
.1. General Experimental Procedures
Optical rotations were obtained for solutions in CHCl (concentrations expressed in g/100 mL) on a
3
Jasco DIP 370 polarimeter (Jasco Analytical Instruments, Easton, MD, USA). IR spectra were
recorded on a Nicolet Nexus FT-IR instrument (Thermo Electron Corporation, Whaltham, MA, USA).
1
13
H-NMR spectra were recorded at 400 or 500 MHz and C-NMR data were obtained at 100 or 125 MHz
on a Bruker Avance spectrometer (Bruker, Rheinstetten, Germany). Chemical shifts are given in
δ (ppm) with TMS as the internal standard. Mass spectra were measured with an EBE trisector VG
Autospec Micromass spectrometer operating at 70 eV and are presented as m/z (rel. int. %). Silica gel
6
0 (Merck, 63–200 µm particle size) was used for column chromatography, precoated silica gel plates
Merck, Kieselgel 60 F254, 0.25 mm) were used for thin layer chromatography (TLC). TLC spots were
visualized by spraying the chromatograms with p-anisaldehyde-ethanol-acetic acid-H SO4
(
2
(
2:170:20:10 v/v) and heating at 110 °C for 3 min. 1,3-Dicyclohexylcarbodiimide (DCC) and
dimethylaminopyridine (DMAP) were from Merck (Schuchardt, Germany).
3
.2. Plant Material
Lapachol was isolated from the wood of Tabebuia heptaphylla as described previously [13] and
purified by successive silica gel column chromatography, followed by crystallization. The diterpene
junicedric acid (I) was obtained by saponification and oxidation of a mixture of labdane diterpenes
from Araucaria araucana resin. The resin was collected from healthy trees in Conguillío, Araucanía
Region, Chile. Voucher specimens have been deposited at the Herbarium of the Universidad de Talca.
3
.3. General Procedure for the Synthesis of Compounds I–III and V–VI
The crude Araucaria araucana resin was worked-up as described previously [9,10] to obtain a
mixture of diterpenes bearing an alcohol, aldehyde, acid or ester (acetate) function at C-15 and/or
C-19. After saponification (KOH, methanol), the diterpene mixture was oxidized with CrO to yield I.
3
Isomerization of the double bond of I to II was carried out by treating I in acetic acid with 47% HBr,
stirring constantly for 24 h. The reaction product was purified by silica gel column chromatography
(
86% w/w yield). Reduction of the double bond of I and IV was carried out by catalytic hydrogenation
of the terpene dissolved in ethyl acetate with 10% Pd/C in a 1:10 molar ratio with respect to the
diterpene, stirring constantly for 24 h to yield III and V, respectively. Compound VI was obtained by
high pressure hydrogenation of compound IV.
Δ8(9) Junicedric acid (II). HBr (3 mL) was added to a solution of junicedric acid (I) (2.51 g, 7.47 mmol)
in acetic acid (HOAc, 20 mL). The mixture was stirred at room temperature for 24 h, cooled in an ice
bath, and after addition of water the aqueous phase was extracted with EtOAc (3 × 20 mL). The extract
was dried over anhydrous Na SO , and taken to dryness under reduced pressure. The residue was
2
4
purified by silica gel column chromatography, eluting with hexane/EtOAc (8:2), yielding 2.15 g (86%)
of compound II.

Molecules 2011, 16
8620
1
7β-Dihydrojunicedric acid (III). Junicedric acid (I) (1.10 g, 3.27 mmol) was dissolved in 30 mL ethyl
acetate. After the addition of Pd/C (10%) vacuum was made to eliminate air. Then, hydrogen (H ) was
2
bubbled under constant agitation for 24 h. The reaction mixture was filtered and taken to dryness under
reduced pressure. The residue was purified by silica gel column chromatography eluting with
hexane/EtOAc (8:2), yielding III (827 mg, 75%).
2
-Hydroxy-3-(3-methylbutyl)-[1,4]-naphthoquinone (V). Compound V (dihydroprenyl lapachol) was
synthesized from 2-hydroxy-3-(3-methyl-2-butenyl)-[1,4]-naphthoquinone (lapachol) (IV) as described
for compound III, yielding 306 mg (70%) of V.
2
-Hydroxy-3-(3-methyl-butyl)-5,6,7,8-tetrahydro-[1,4]-naphthoquinone (VI). A solution of lapachol
IV) (400 mg, 1.65 mmol) in HOAc (4 mL) and Pd/C (10%) was submitted to a pressure of 4 bar and
5 °C for 2 h. The reaction mixture was cooled in an ice-bath and after addition of water, the aqueous
phase was extracted with EtOAc (3 × 20 mL), the extracts were dried over anhydrous Na SO , and
(
6
2
4
taken to dryness under reduced pressure. The residue was purified by silica gel column chromatography
eluting with hexane/EtOAc (9:1), yielding dihydroprenyl-5,6,7,8-tetrahydrolapachol VI (300 mg,
7
3%) and V (40 mg, 10%).
3
.4. General Procedure for the Synthesis of Compounds 1–12
Compounds 1, 3, 5, 7, 9 and 11 were prepared by treating the corresponding diacids (1 mEq) in dry
2 2
CH Cl (DCM) with 1,3-dicyclohexylcarbodiimide (DCC) (1 mEq) at room temperature under
constant stirring. After 10 min, lapachol or its reduction products (1 mEq) dissolved in dry DCM, were
added together with a catalytic amount of dimethylaminopyridine (DMAP). After 2–4 h, the reaction
was stopped by adding water and extracted with DCM. The extract was dried over Na
purified by silica gel column chromatography, to afford compounds 1, 3, 5, 7, 9 and 11 in 33, 42, 35,
8, 52 and 42% w/w yields, respectively. The corresponding methyl esters 2, 4, 6, 8, 10 and 12 were
2 4
SO , and
4
obtained treating the above cited compounds with diazomethane in 92, 89, 88, 93, 88 and 90% w/w yields,
1
respectively. The purity of all the derivatives was over 95%, as determined by H-NMR spectroscopy.
Lapachoyl junicedrate (1). Junicedric acid (I) (170 mg, 0.506 mmol), DCC (104 mg, 0.506 mmol), a
catalytic amount of DMAP and lapachol (IV) (120 mg, 0.506 mmol), were stirred at room temperature
in dry CH
water, the aqueous phase was extracted with EtOAc (3 × 20 mL). The extract was dried over
anhydrous Na SO and taken to dryness under reduced pressure. The residue was purified by silica gel
column chromatography, eluting with hexane/EtOAc (8:2), yielding 1 (94 mg, 33%): brown oil; [α]
2 2
Cl (20 mL) for 2–4 h. The reaction mixture was cooled in an ice bath. After addition of
2
4
20
D
+
3
22 (c 0.24, CHCl ); IR νmax (film) 3398, 2928, 2840, 1768, 1692, 1672, 1641, 1445, 1286, 1170,
−1
1
13
1
4
3
1
1
3 3
075 cm ; H-NMR (CDCl ): see Table 3; C-NMR (CDCl ): 39.10 (C-1), 19.90 (C-2), 37.88 (C-3),
4.18 (C-4), 56.27 (C-5), 26.02 (C-6), 38.72 (C-7), 148.01 (C-8), 56.33 (C-9), 40.88 (C-10), 21.14 (C-11),
5.86 (C-12), 31.00 (C-13), 40.58 (C-14), 170.42 (C-15), 19.96 (C-16), 106.59 (C-17), 29.07 (C-18),
78.45 (C-19), 12.79 (C-20), Quinone: 184.39, 184.52 (C-1 and C-4), 150.87 (C-2), 138.31 (C-3), 130.84,
32.00 (C-4a, C-8a), 126.73, 126.59 (C-5 and C-8), 134.88, 134.13 (C-6 and C-7), 23.67 (C-1′), 118.45
+
(C-2′), 133.86 (C-3′), 18.05 (C-4′), 25.85 (C-5′); EIMS m/z 560 [M] (5), 290 (10), 244 (24), 243 (21),

Molecules 2011, 16
8621
2
42 (100), 228 (14), 227 (85), 225 (14), 123 (10), 121 (27), 109 (13), 105 (13), 95 (10), 81 (15), 55
(
13); HREIMS m/z 560.3168 (calcd for C H O , 560.3138).
3
5
44
6
1
Table 3. Selected H-NMR data of compounds 1–6.
H
1
2
3
4
5
6
1
1
1
1
1
1
2
3
2.10 m
2.73 dd
2.11 m
2.73 dd
2.10 m
2.71 dd
2.08 m
2.71 dd
2.52 m
1.10 d (6.6)
2.05 m
2.06 m
4 α
4 β
6
2
.65 dd
2.66 dd
2.41-2.46 m 2.42-2.47 m 2.52 m
1.13 d (6.6) 1.13 d (6.6) 1.10 d (6.6)
1.07 d (6.6)
1.08 d (6.6)
7
4.86 s; 4.54 s 4.87 s; 4.54 s 4.85 s; 4.52 s 4.84 s; 4.51 s 4.84 s; 4.51 s 4.86 s; 4.51 s
8
1.26 s
0.63 s
-
1.20 s
0.54 s
3.64 s
1.23 s
0.61 s
-
1.17 s
0.51 s
3.61 s
1.24 s
0.60 s
-
1.20 s
0.59 s
3.63 s
0
OMe
Quinone
5
6
1
2
3
4
5
and 8
and 7
8.10 m
7.74 m
3.27 br d
5.07 br t
-
8.10 m
7.74 m
3.28 br d
5.09 br t
-
8.08 m
8.08 m
2.42 m
2.44 m
7.71 m
7.71 m
1.69 m
1.70 m
’
2.41 m
2.41 m
2.40 m
2.38 m
’
’
’
’
1.34 m
1.36 m
1.33 m
1.32 m
1.59 m
1.58 m
1.57 m
1.57 m
1.77 s
1.69 s
1.77 s
1.69 s
0.93 d (6.6)
0.93 d (6.6)
0.93 d (6.6)
0.93 d (6.6)
0.91 d (6.6)
0.91 d (6.6)
0.92 d (6.6)
0.92 d (6.6)
J (Hz): 14α, 14β = 15.3; 13, 14α = 5.5; 13, 14β = 8.1.
Lapachoyl junicedrate methyl ester (2). Compound 1 (50 mg, 0.089 mmol), was methylated with a
2
D
0
solution of CH
2
N
2
in ethyl ether, yielding 47 mg (92%) of 2: brown oil; [α] +24 (c 0.29, CHCl
3
3
);
):
−1 1
IR νmax (film) 2948, 2844, 1766, 1720, 1676, 1641, 1449, 1290, 1178, 1079 cm ; H-NMR (CDCl
1
3
see Table 3; C-NMR (CDCl
3
): 39.21 (C-1), 19.94 (C-2), 38.28 (C-3), 44.33 (C-4), 56.38 (C-5), 26.28
(
C-6), 38.79 (C-7), 148.11 (C-8), 56.45 (C-9), 40.87 (C-10), 21.15 (C-11), 35.90 (C-12), 30.98 (C-13),
4
0.39 (C-14), 170.24 (C-15), 19.99 (C-16), 106.46 (C-17), 28.84 (C-18), 177.77 (C-19), 12.59 (C-20),
1.14 (OMe), Quinone: 178.37, 184.44 (C-1 and C-4) 150.95 (C-2), 138.28 (C-3), 130.93, 132.09 (C-4a,
5
C-8a), 126.68, 126.53 (C-5 and C-8), 134.73, 134.00 (C-6 and C-7), 23.65 (C-1′), 118.53 (C-2′), 133.74
+
(
(
(
C-3′), 17.97 (C-4′), 25.73(C-5′); EIMS m/z 574 [M] (2), 350 (6), 291 (17), 290 (30), 258 (16), 256
11), 243 (10), 242 (67), 227 (24), 200 (10), 180 (11), 161 (13), 159 (10), 123 (14), 122 (15), 121
100), 109 (23), 107 (21), 105 (20), 95 (16), 93 (16), 91 (11), 81 (20), 79 (12), 67 (12), 55 (18);
HREIMS m/z 574.3308 (calcd for C H O , 574.3294).
3
6
46
6
Dihydroprenyl lapachoyl junicedrate (3). Compound 3 was synthesized as described for compound 1,
2
0
using terpene I and quinone V, yielding 105 mg (42%) of 3: pale yellow oil; [α] +49 (c 0.14,
D
−1 1
3
CHCl ); IR νmax (film) 2952, 2872, 1772, 1700, 1680, 1641, 1469, 1290, 1186, 1079 cm ; H-NMR
1
3
(
CDCl ): see Table 3; C-NMR (CDCl ): 39.18 (C-1), 19.97 (C-2), 37.97 (C-3), 44.24 (C-4), 56.40 (C-5),
3
3
2
6.07 (C-6), 38.77 (C-7), 148.01 (C-8), 56.46 (C-9), 40.61 (C-10), 21.19 (C-11), 35.93 (C-12), 31.02
(
C-13), 40.89 (C-14), 170. 36 (C-15), 19.96 (C-16), 106.60 (C-17), 29.06 (C-18), 178.16 (C-19), 12.79
C-20), Quinone: 184.10, 184.57 (C-1 and C-4) 151.14 (C-2), 140.17 (C-3), 130.96, 132.16 (C-4a, C-8a),
(

Molecules 2011, 16
8622
1
2
26.68, 126.57 (C-5 and C-8), 134.01 133.78 (C-6 and C-7), 22.47 (C-1′), 37.51 (C-2′), 28.41 (C-3′),
+
2.35 (C-4′), 22.35 (C-5′); HREIMS m/z: 533.0547 [M-H-CO] (calcd for C H O : 533.3267).
3
4
45
5
Dihydroprenyl lapachoyl junicedrate methyl ester (4). Compound 3 (60 mg, 0.106 mmol) was
2
D
0
2 2
methylated with a solution of CH N in ethyl ether, yielding 55 mg (89%) of 4: pale yellow oil; [α]
−1
+
62 (c 0.22, CHCl ); IR ν (film) 2928, 2872, 1768, 1724, 1676, 1637, 1461, 1298, 1146, 1,083 cm ;
3
max
1
13
3 3
H-NMR (CDCl ): see Table 3; C-NMR (CDCl ): 39.23 (C-1), 19.97 (C-2), 37.51 (C-3), 44.35 (C-4),
5
3
1
1
6.40 (C-5), 26.29 (C-6), 38.81 (C-7), 148.11 (C-8), 56.46 (C-9), 40.41 (C-10), 21.19 (C-11), 35.93 (C-12),
1.02 (C-13), 40.89 (C-14), 170. 36 (C-15), 20.01 (C-16), 106.50 (C-17), 28.87 (C-18), 178.16 (C-19),
2.61 (C-20), 51.17 (OMe), Quinone: 184.10, 184.57 (C-1 and C-4) 151.11 (C-2), 140.17 (C-3), 130.96,
32.16 (C-4a, C-8a), 126.68, 126.56 (C-5 and C-8), 134.02, 133.78 (C-6 and C-7), 22.47 (C-1′), 37.51
+
(
C-2′), 28.41 (C-3′), 22.35 (C-4′), 22.35 (C-5′); HREIMS m/z: 577.1957 [M+H] (calc for C H O ,
3
6
49
6
5
77.3529).
Dihydroprenyl-5,6,7,8-tetrahydrolapachoyl junicedrate (5). Compound 5 was synthesized as described
20
for compound 1, using terpene I and quinone VI, yielding 87 mg (35%) of 5: pale yellow oil; [α] +35
D
−
1
(
c 0.55, CHCl
3
); IR νmax (film) 3338, 2924, 2860, 1768, 1692, 1661, 1621, 1473, 1206, 1134, 1083 cm ;
1
13
H-NMR (CDCl
3
): see Table 3; C-NMR (CDCl
3
): 39.10 (C-1), 19.88 (C-2), 37.89 (C-3), 44.17 (C-4),
5
3
1
6.33 (C-5), 26.00 (C-6), 38.71 (C-7), 147.93 (C-8), 56.38 (C-9), 40.54 (C-10), 21.03 (C-11), 35.84 (C-12),
0.94 (C-13), 40.77 (C-14), 170.46 (C-15), 19.88 (C-16), 106.53 (C-17), 28.99 (C-18), 180.25 (C-19),
2.70 (C-20), Quinone: 184.24, 186.91 (C-1 and C-4) 148.60 (C-2), 140.85 (C-3), 136.89, 142.76 (C-4a,
C-8a), 22.30, 21.03 (C-5 and C-8), 21.85, 21.11 (C-6 and C-7), 22.79 (C-1′), 37.46 (C-2′), 28.23 (C-3′),
+
2
1
2.26 (C-4′), 22.26 (C-5′); EIMS m/z 566 [M] (2), 273 (12), 251 (17), 250 (100), 248 (15), 193 (11),
50 6
92 (21), 121 (12). HREIMS m/z 566.3633 (calcd for C35H O , 566.3607).
Dihydroprenyl-5,6,7,8-tetrahydrolapachoyl junicedrate methyl ester (6). Compound 5 (40 mg, 0.071 mmol)
was methylated with a solution of CH in ethyl ether, yielding 36 mg (88%) of 6: pale yellow oil,
2
N
2
2
D
0
[
α] +28 (c 0.20, CHCl ); IR ν
(film) 2948, 2872, 1764, 1720, 1661, 1621, 1457, 1214, 1134,
3
max
−1
1
13
1
4
3
1
083 cm ; H-NMR (CDCl
3
): see Table 3; C-NMR (CDCl
3
): 39.43 (C-1), 20.12 (C-2), 38.50 (C-3),
4.55 (C-4), 56.61 (C-5), 26.49 (C-6), 39.01 (C-7), 148.32 (C-8), 56.66 (C-9), 40.60 (C-10), 22.11 (C-11),
6.13 (C-12), 31.20 (C-13), 41.04 (C-14), 170.69 (C-15), 20.21 (C-16), 106.66 (C-17), 29.06 (C-18),
77.98 (C-19), 12.79 (C-20), 51.34 (OMe), Quinone: 180.52, 187.19 (C-1 and C-4) 148.86 (C-2), 141.11
(
C-3), 137.15, 143.02 (C-4a, C- 8a), 23.04, 22.49 (C-5 and C-8), 21.28, 21.28 (C-6 and C-7), 21.37 (C-1′),
+
3
7.71 (C-2′), 28.49 (C-3′), 22.48 (C-4′), 22.48 (C-5′); EIMS m/z 580 [M] (2), 333 (13), 273 (19), 251
(
17), 250 (100), 121 (22); HREIMS m/z 580.3844 (calcd for C36
52 6
H O , 580.3764).
Lapachoyl Δ8(9) junicedrate (7). Compound 7 was synthesized as described for compound 1, using
2
D
0
diterpene II and quinone IV, yielding 124 mg (48%) of 7: pale yellow oil; [α] +72 (c 0.15, CHCl
3
);
−1
1
IR νmax (film) 3378, 2932, 2864, 1772, 1728, 1692, 1684, 1461, 1294, 1170, 1071 cm ; H-NMR
13
(CDCl
3 3
): see Table 4; C-NMR (CDCl ): 37.21 (C-1), 19.52 (C-2), 37.44 (C-3), 43.78 (C-4), 53.55
(
C-5), 20.73 (C-6), 34.26 (C-7), 126.82 (C-8), 139.89 (C-9), 39.76 (C-10), 25.65 (C-11), 34.20 (C-12),
3
1.35 (C-13), 41.03 (C-14), 170.19 (C-15), 19.72 (C-16), 19.61 (C-17), 28.65 (C-18), 183.77 (C-19),
7.97 (C-20), Quinone: 178.35, 184.50 (C-1 and C-4) 150.96 (C-2), 138.28 (C-3), 130.93, 132.09 (C-4a,
1

Molecules 2011, 16
8623
C-8a), 126.69, 126.53 (C-5 and C-8), 134.74, 134.01 (C-6 and C-7), 23.65 (C-1′), 118.51 (C-2′), 133.76
+
(
C-3′), 17.97 (C-4′), 25.73 (C-5′); EIMS m/z 560 [M] (3), 244 (45), 243 (14), 242 (73), 228 (16), 227
(
100), 225 (14), 221 (31), 175 (17), 173 (13), 109 (11), 105 (16), 95 (10), 81 (11), 55 (14); HREIMS
m/z 560.3135 (calcd for C35
H
44
O
6
, 560.3138).
1
Table 4. Selected H-NMR Data of compounds 7–12.
H
7
8
9
10
11
12
1
1
1
1
1
1
2
3
2.12 m
2.72 dd
2.50 dd
2.12 m
2.72 dd
2.50 dd
2.10 m
2.72 dd
2.53 m
2.10 m
2.70 dd
2.53 m
2.12 m
2.74 dd
2.46 dd
2.11 m
2.72 dd
2.45 dd
4 α
4 β
6
1.15 d (6.6) 1.15 d (6.5) 1.12 d (6.6) 1.12 d (6.6) 1.15 d (6.6) 1.14 d (6.6)
7
1.54 s
1.28 s
0.91 s
-
1.62 s
1.22 s
0.80 s
3.65 s
1.59 s
1.25 s
0.88 s
-
1.59 s
1.19 s
0.77 s
3.62 s
0.95 d (7.5) 0.93 d (7.5)
8
1.27 s
0.83 s
-
1.19 s
0.70 s
3.66 s
0
OMe
Quinone
5
6
1
2
3
4
5
and 8
and 7
8.11 m
7.75 m
3.29 br d
5.09 br t
-
8.11 m
7.75 m
3.28 br d
5.09 br t
-
8.09 m
7.72 m
2.46 m
1.35 m
1.58 m
8.09 m
7.72 m
2.46 m
1.36 m
1.58 m
8.10 m
7.74 m
3.28 br d
5.08 br t
-
8.10 m
7.74 m
3.28 br d
5.09 br t
-
’
’
’
’
’
1.78 s
1.70 s
1.78 br s
1.70 br s
0.93 d (6.6) 0.93 d (6.6) 1.78 br s
0.93 d (6.6) 0.93 d (6.6) 1.70 br s
1.78 br s
1.69 br s
J (Hz): 14α, 14β = 15.3; 13, 14α = 5.5; 13, 14β = 8.1.
Lapachoyl Δ8(9) junicedrate methyl ester (8). Compound 7 (60 mg, 0.107 mmol) was methylated with
2
D
0
a solution of CH
2 2
N in ethyl ether, yielding 57 mg (93%) of 8: pale yellow oil; [α] +57 (c 0.10,
−1 1
CHCl ); IR νmax (film) 2924, 2876, 1768, 1720, 1688, 1637, 1461, 1290, 1138, 1075 cm ; H-NMR
3
13
(CDCl
3 3
): see Table 4; C-NMR (CDCl ): 37.28 (C-1), 19.52 (C-2), 37.76 (C-3), 43.91 (C-4), 53.58
(
C-5), 20.87 (C-6), 34.34 (C-7), 126.80 (C-8), 139.86 (C-9), 39.59 (C-10), 25.73 (C-11), 34.24 (C-12),
3
1
1
1.35 (C-13), 41.03 (C-14), 170.21 (C-15), 19.74 (C-16), 19.61 (C-17), 28.47 (C-18), 178.12 (C-19),
7.79 (C-20), 51.08 (OMe), Quinone: 178.37, 184.76 (C-1 and C-4) 150.94 (C-2), 138.24 (C-3), 130.93,
32.11 (C-4a, C-8a), 126.69, 126.53 (C-5 and C-8), 134.74, 134.01 (C-6 and C-7), 23.65 (C-1′), 118.52
+
(
C-2′), 133.75 (C-3′), 17.97 (C-4′), 25.73 (C-5′); EIMS m/z 574 [M] (5), 256 (15), 245 (15), 244 (91),
2
43 (23), 242 (100), 235 (25), 227 (52), 175 (25), 173 (22), 121 (13), 107 (11), 105 (12), 55 (11);
HREIMS m/z 574.3307 (calcd for C36 , 574.3294).
46 6
H O
Dihydroprenyl lapachoyl Δ8(9) junicedrate (9). Compound 9 was synthesized as described for
20
compound 1, using terpene II and quinone V, yielding 91 mg (52%) of 9: pale yellow oil; [α] +41
D
−
1
(
1
c 0.18, CHCl ); IR (film) νmax 2936, 2876, 1768, 1696, 1680, 1637, 1465, 1294, 1150, 1079 cm ;
3
13
H-NMR (CDCl
3
): see Table 4; C-NMR (CDCl
3
): 37.25 (C-1), 19.55 (C-2), 37.51 (C-3), 43.79 (C-4),
5
3.55 (C-5), 20.76 (C-6), 34.27 (C-7), 126.84 (C-8), 140.18 (C-9), 39.78 (C-10), 25.78 (C-11), 34.20
(
(
C-12), 31.41 (C-13), 41.06 (C-14), 170.32 (C-15), 19.75 (C-16), 19.56 (C-17), 28.68 (C-18), 178.17
C-19), 17.98 (C-20), Quinone; 183.70, 184.58 (C-1 and C-4) 151.11 (C-2), 139.02 (C-3), 130.97,

Molecules 2011, 16
8624
1
32.17 (C-4a, C-8a), 126.69, 126.57 (C-5 and C-8), 134.04, 133.80 (C-6 and C-7), 22.47 (C-1′), 37.51
+
(
C-2′), 28.41 (C-3′), 22.36 (C-4′), 22.36 (C-5′); HREIMS m/z: 533.1329 [M–H–CO] (calcd for
: 533.3267).
34 45 5
C H O
Dihydroprenyl lapachoyl Δ8(9) junicedrate methyl ester (10). Compound 9 (40 mg, 0.071 mmol), was
2
D
1
0
methylated with a solution of CH N in ethyl ether, yielding 36 mg (88%) of 10: pale yellow oil; [α]
2
2
−
+
39 (c 0.24, CHCl
3
); IR (film) νmax 2928, 2876, 1768, 1724, 1668, 1637, 1461, 1290, 1154, 1071 cm ;
1
13
H-NMR (CDCl
3
): see Table 4; C-NMR (CDCl
3
): 37.30 (C-1), 19.57 (C-2), 37.51 (C-3), 43.92 (C-4),
5
3.59 (C-5), 20.89 (C-6), 34.36 (C-7), 126.83 (C-8), 139.08 (C-9), 39.60 (C-10), 25.77 (C-11), 34.24
(
C-12), 31.41 (C-13), 41.06 (C-14), 170.31 (C-15), 19.77 (C-16), 19.62 (C-17), 28.49 (C-18), 178.15
C-19), 17.80 (C-20), 51.14 (OMe), Quinone: 183.67, 184.50 (C-1 and C-4) 151.11 (C-2), 139.08 (C-3),
(
1
30.97, 132.17 (C-4a, C-8a), 126.69, 126.57 (C-5 and C-8), 134.03, 133.79 (C-6 and C-7), 22.46 (C-1′),
+
3
7.51 (C-2′), 28.41 (C-3′), 22.36 (C-4′), 22.36 (C-5′); HREIMS m/z: 577.1957 [M+H] (calcd for
C
36
H
49
O
6
: 577.3529).
Lapachoyl 17β-dihydrojunicedrate (11). Compound 11 was prepared as described for compound 1,
2
D
0
from diterpene III and quinone IV, yielding 85 mg (42%) of 11: brown oil; [α] +20 (c 0.09, CHCl
3
);
−1
1
IR (film) νmax 3398, 2956, 2848, 1772, 1692, 1676, 1637, 1457, 1290, 1170, 1075 cm ; H-NMR
13
(
CDCl
3
): see Table 4; C-NMR (CDCl
3.07 (C-6), 35.00 (C-7), 29.35 (C-8), 57.50 (C-9), 39.74 (C-10), 18.85 (C-11), 35.23 (C-12), 30.88 (C-13),
0.84 (C-14), 170.32 (C-15), 20.15 (C-16), 15.00 (C-17), 29.04 (C-18), 182.00 (C-19), 14.43 (C-20),
3
): 39.83 (C-1), 19.01 (C-2), 37.96 (C-3), 43.82 (C-4), 53.06 (C-5),
2
4
Quinone: 178.42, 184.48 (C-1 and C-4) 150.98 (C-2), 138.32 (C-3), 130.97, 132.13 (C-4a, C-8a),
1
1
2
26.71, 126.57 (C-5 and C-8), 133.78, 134.04 (C-6 and C-7), 23.68 (C-1′), 118.55 (C-2′), 134.78 (C-3′),
+
7.99 (C-4′), 25.76 (C-5′); EIMS m/z 562 [M] (5), 500 (20), 374 (10), 244 (43), 243 (18), 242 (100),
27 (64), 221 (16), 168 (15), 141 (16), 123 (15), 109 (18), 95 (13), 83 (12), 81 (14), 55 (19); HREIMS
m/z 562.3280 (calcd for C35
46 6
H O , 562.3294).
Lapachoyl 17β-dihydrojunicedrate methyl ester (12). Compound 11 (40 mg, 0.071 mmol) was
2
D
0
methylated with a solution of CH N in ethyl ether, yielding 37 mg (90%) of 12: brown oil; [α] +24
2
2
−1
(
1
c 0.34, CHCl
3
); IR (film) νmax 2944, 2844, 1764, 1724, 1676, 1637, 1457, 1290, 1146, 1079 cm ;
13
H-NMR (CDCl
3
): see Table 4; C-NMR (CDCl
3
): 39.85 (C-1), 19.09 (C-2), 37.27 (C-3), 43.93 (C-4),
5
3
1
1
2.99 (C-5), 23.06 (C-6), 34.98 (C-7), 29.30 (C-8),57.52 (C-9), 38.71 (C-10), 18.89 (C-11), 35.26 (C-12),
0.87 (C-13), 40.81 (C-14), 170.26 (C-15), 20.15 (C-16), 14.95 (C-17), 28.86 (C-18), 178.07 (C-19),
4.95 (C-20), 51.11 (OMe), Quinone: 178.36, 184.41 (C-1 and C-4) 150.95 (C-2), 138.27 (C-3),130.93,
32.09 (C-4a, C-8a), 126.68, 126.53 (C-5 and C-8), 134.74, 134.00 (C-6 and C-7), 23.66 (C-1′), 118.53
+
(
C-2′), 133.74 (C-3′), 17.98 (C-4′), 25.74 (C-5′); EIMS m/z 576 [M] (2), 293 (17), 244 (20), 243 (20),
2
42 (100), 227 (27), 123 (28), 109 (14), 95 (10), 81 (11), 55 (11); HREIMS m/z 576.3580 (calcd for
, 576.3451).
36 48 6
C H O
3
.5. HCl-EtOH-Induced Ulcer Model in Mice [7-10,27]
The gastroprotective activity of the compounds was assessed in the EtOH-HCl-induced gastric
lesion model. Male Swiss albino mice weighing 30 ± 3 g were used. The animals were fed on certified

Molecules 2011, 16
8625
Champion diet with free access to water under standard conditions of 12 h dark-light period, 50%
relative humidity, and 22 °C room temperature. The mice were randomly distributed into groups of
7
–14 animals each and fasted for 24 h with free access to water before the oral administration of test
samples by gavage. The purity of the tested compounds was higher than 95% by NMR analysis. To
keep the animal numbers to a minimum, dose-response studies were performed with lapachol at 5, 10
and 50 mg/kg to set the conditions for single-dose comparison of gastroprotective effect. The tested
compounds, lansoprazole (20 mg/kg) or the vehicle (12% Tween 80, 10 mL/kg) were administered
orally by gavage. After 50 min, all groups were orally treated with 0.2 mL of a solution containing
6
0% EtOH-0.3 M HCl for gastric lesion induction. Animals were sacrificed by cervical dislocation 1 h
after the administration of EtOH-HCl, and the stomachs excised and inflated by injection of 5%
formalin (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 expressed as the
sum of the length of all lesions. Based on previous studies on the parent diterpenes, comparison of the
new compounds 1–12 was carried out at a single oral dose of 5 mg/kg. 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. Tween 80 and lansoprazole (>98% purity
by HPLC) were purchased from Sigma-Aldrich.
3
.6. Cytotoxicity Assay [28]
The human cell lines MRC-5 normal lung fibroblasts (ATCC CCL-171), AGS gastric adenocarcinoma
cells (ATCC CRL-1739), and Hep G2 hepatocellular carcinoma cells (ATCC HB-8065) were obtained
from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were grown as
monolayers in the following media: MRC-5 and Hep G2 in minimum essential Eagle’s medium
(
(
MEM), with Earle’s salts, 2.0 mM L-glutamine (Sigma Chemical Co.) and 2.2 g/L sodium bicarbonate
Sigma Chemical Co.), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU/mL
penicillin, and 100 µg/mL streptomycin in a humidified incubator with 5% CO in air at 37 °C. Cell
2
passage was maintained between 10 and 16 for MRC-5 and between 79 and 82 for Hep G2 cells. The
medium was changed every 2 days. Culture media, antibiotics, and FBS were obtained from Invitrogen
Corp. Human gastric adenocarcinoma cells AGS (ATCC CRL-1739) were grown as monolayers in
Ham F-12 medium containing 1.0 mM L-glutamine and 1.5 g/L sodium bicarbonate, supplemented
with 10% heat-inactivated FBS, 100 IU/mL penicillin, and 100 µg/mL streptomycin in a humidified
2
incubator with 5% CO in air at 37 °C. Cell passage was maintained between 42 and 48. The medium
was changed every 2 days.
Confluent cultures of the different cell lines were treated with medium containing the compounds at
concentrations ranging from 1 up to 1,000 µM. The antisecretory drug lansoprazole was used as
reference compound. The substances were first dissolved in DMSO and then in the corresponding
culture medium supplemented with 2% FBS. The final concentration of DMSO in the test medium and
controls was 1%. The cells were exposed for 24 h to test medium with or without the compound
(
control). Each concentration was tested in quadruplicate together with the control and repeated three
times in separate experiments. At the end of the incubation the neutral red uptake assay was performed.

Molecules 2011, 16
.7. Statistical Analysis
Results were expressed as the mean ± SEM. In all experiments, statistical differences between
8626
3
several treatments and their respective control were determined by one-way ANOVA with Student-
Newman-Keuls post-hoc test. The level of significance was set at P < 0.05.
4
. Conclusions
The aim of this work was to synthesize diterpenylnaphthoquinones combining diterpene and
quinone moieties. To determine structure-activity relationships/trends, 12 esters were prepared. The
diterpenes used as building blocks comprised the diacids I, II and III. The naphthoquinone moieties
included lapachol, dihydroprenyl lapachol, and dihydroprenyl-5,6,7,8-tetrahydrolapachol. Compounds
6
, 8 and 10 were significantly active as antiulcer agents, protecting against induced gastric lesions in
mice by 74, 84 and 76%, respectively. Basal cytotoxicity of the compounds was determined on the
following human cell lines: normal lung fibroblasts (MRC-5), gastric epithelial adenocarcinoma
(
AGS) and hepatocellular carcinoma (Hep G2). Some compounds showed selective cytotoxicity
against AGS cells. Derivative 5 was the most cytotoxic product, with IC values of 69, 40 and 27 µM
5
0
for fibroblasts, AGS and Hep G2 cells, respectively. Derivative 6, with strong gastroprotective activity,
was devoid of cytotoxic effect towards the selected cell lines. The new compounds had different
biological effects than the building blocks and some of them were less toxic than the starting molecules.
Acknowledgments
Financial support of this work under FONDECYT Project 1085306 and Programa de Investigación
en Productos Bioactivos, Universidad de Talca is gratefully acknowledged. M.W.P. thanks the PBCT
PSD50 for a postdoctoral grant. The skillful technical work of Irene Manriquez is gratefully acknowledged.
Conflict of Interest
The authors declare no conflict of interest.
References and Notes
1.
WHO. The World Health Report; WHO: Geneva, Switzerland, 2003; p. 154-159.
2
Bahmanyar, S.; Ye, W.; Dickman, P.W.; Nyren, O. Long-term risk of gastric cancer by subsite in
operated and unoperated patients hospitalized for peptic ulcer. Am. J. Gastroenterol. 2007, 102,
1
185-1191.
3
4
.
Luo, J.; Nordenvall, C.; Nyren, O.; Adami, H.O.; Permert, J.; Ye, W. The risk of pancreatic cancer
in patients with gastric or duodenal ulcer disease. Int. J. Cancer 2007, 120, 368-372.
Areche, C.; Rodríguez, J.A.; Razmilic, I.; Yañez, T.; Theoduloz, C.; Schmeda-Hirschmann, G.
Gastroprotective and cytotoxic effect of semisynthetic ferruginol derivatives. J. Pharm.
Pharmacol. 2007, 59, 289-300.

Molecules 2011, 16
8627
5
6
7
8
9
1
1
Pertino, M.; Schmeda-Hirschmann, G.; Rodríguez, J.; Theoduloz, C. Gastroprotective effect and
cytotoxicity of terpenes from the Paraguayan crude drug “yagua rova” (Jatropha isabelli).
J. Ethnopharmacol. 2007, 111, 553-559.
.
.
.
.
Rodríguez, J.; Theoduloz, C.; Yáñez, T.; Becerra, J.; Schmeda-Hirschmann, G. Gastroprotective
and ulcer healing effect of ferruginol in mice and rats: Assessment of its mechanism of action
using in vitro models. Life Sci. 2005, 78, 2503-2509.
Rodríguez, J.A.; Theoduloz, C.; Sánchez, M.; Razmilic, I.; Schmeda-Hirschmann, G.
Gastroprotective and ulcer-healing effect of new solidagenone derivatives in human cell cultures.
Life Sci. 2005, 77, 2193-2205.
Schmeda-Hirschmann, G.; Rodríguez, J.A.; Astudillo, L. Gastroprotective activity of the diterpene
solidagenone and its derivatives on experimentally induced gastric lesions in mice.
J. Ethnopharmacol. 2002, 81, 111-115.
Schmeda-Hirschmann, G.; Astudillo, L.; Sepúlveda, B.; Rodríguez, J.; Theoduloz, C.; Yáñez, T.;
Palenzuela, J.A. Gastroprotective effect and cytotoxicity of natural and semisynthetic labdane
diterpenes from Araucaria araucana resin. Z. Naturforsch. C 2005, 60, 511-522.
0. Schmeda-Hirschmann, G.; Astudillo, L.; Rodríguez, J.; Theoduloz, C.; Yáñez, T. Gastroprotective
effect of the Mapuche crude drug Araucaria araucana resin and its main constituents.
J. Ethnopharmacol. 2005, 101, 271-276.
1. Sepúlveda, B.; Astudillo, L.; Rodríguez, J.; Yáñez, T.; Theoduloz, C.; Schmeda-Hirschmann, G.
Gastroprotective and cytotoxic effect of dehydroabietic acid derivatives. Pharmacol. Res. 2005,
5
2, 429-437.
1
1
2. Halle, W.; Spielmann, H. Two procedures for the prediction of acute toxicity (LD50) from
cytotoxicity data. ATLA 1992, 20, 40-49.
3. Schmeda Hirschmann, G.; Papastergiou, F. Naphthoquinone derivatives and lignans from the
Paraguayan crude drug "tayï pytá" (Tabebuia heptaphylla, Bignoniaceae). Z. Naturforsch. C 2003,
5
8, 495-501.
1
1
1
1
1
4. Pérez, S.; Estevez-Braun, E.; Ravelo, A.; Ferro, A.G.; Tokuda, E.A.; Mukainaka, H.; Nishino, T.
Inhibitory effects of lapachol derivatives on Epstein-Barr virus activation. Bioorg. Med. Chem.
2
003, 11, 483-488.
5. Barbosa, T.P.; Câmara, C.A.; Silva, T.M.S.; Martins, R.M.; Pinto, A.C.; Vargas, M.D. New
,2,3,4-tetrahydro-1-aza-anthraquinones and 2-aminoalkyl compounds from norlapachol with
1
molluscicidal activity. Bioorg. Med. Chem. 2005, 13, 6464-6469.
6. Silva, T.M.S.; Camara, C.A.; Barbosa, T.P.; Soares, A.Z.; da Cunha, L.C.; Pinto, A.C.;
Vargas, M.D. Molluscicidal activity of synthetic lapachol amino and hydrogenated derivatives.
Bioorg. Med. Chem. 2005, 13, 193-196.
7. Silva, R.S.F.; Costa, E.M.; Trindade, U.L.T.; Teixeira, D.V.; Pinto, M de C. F.R.; Santos, G.L.;
Malta, V.R.S.; De Simone, C.A.; Pinto, A.V.; de Castro, S.L. Synthesis of naphthofuranquinones
with activity against Trypanosoma cruzi. Eur. J. Med. Chem. 2006, 41, 526-530.
8. da Silva, A.J.M.; Buarque, C.D.; Brito, F.V.; Aurelian, L.; Macedo, L.F.; Malkas, L.H.;
Hickey, R.J.; Lopes, D.V.S.; Noel, F.; Murakami, Y.L.B.; et al. Synthesis and preliminary
pharmacological evaluation of new (+/−) 1,4-naphthoquinones structurally related to lapachol.
Bioorg. Med. Chem. 2002, 10, 2731-2738.

Molecules 2011, 16
8628
1
9. Esteves-Souza, A.; Figueiredo, D.V.; Esteves, A.; Câmara, C.A.; Vargas, M.D.; Pinto, A.C.;
Echevarría, A. Cytotoxic and DNA-topoisomerase effects of lapachol amine derivatives and
interactions with DNA. Braz. J. Med. Biol. Res. 2007, 40, 1399-1402.
2
2
2
2
0. Muregi, F.W.; Ishih, A. Next-generation antimalarial drugs: Hybrid molecules as a new strategy
in drug design. Drug Devel. Res. 2010, 71, 20-32.
1. Morphy, R.; Rankovic, Z. Designed multiple ligands. An emerging drug discovery paradigm.
J. Med. Chem. 2005, 48, 6523-6543.
2. Morphy, R.; Rankovic, Z. Fragments, network biology and designing multiple ligands.
Drug Discov. Today 2007, 12, 156-160.
3. Morphy, R.; Rankovic, Z. Multi-target drugs: Strategies and challenges for medicinal chemists.
The Practice of Medicinal Chemistry, 3rd ed.; Wermuth, C.G., Ed.; Elsevier-Academic Press:
San Diego, CA, USA, 2008; pp. 549-571.
2
2
2
4. Schmeda-Hirschmann, G.; Pertino, M.; Rodriguez, J.A.; Monsalve, F.; Droguett, D.; Theoduloz, C.
Synthesis, gastroprotective effect and cytotoxicity of new amino acid diterpene monoamides and
diamides. Molecules 2010, 15, 7378-7394.
5. del Corral, J.M.M.; Castro, M.A.; Rodríguez, M.L.; Chamorro, P.; Cuevas, C.; San Feliciano, A.
New cytotoxic diterpenylnaphthohydroquinone derivatives obtained from a natural diterpenoid.
Bioorg. Med. Chem. 2007, 15, 5760-5774.
6. Molinari, A.; Oliva, A.; Ojeda, C.; Escobar, J.; Gallardo, C.; del Corral, J.M.; Castro M.A.;
Cuevas, C.; San Feliciano, A. Synthesis, characterisation and cytotoxicity of chloro derivatives of
prenylnaphthohydroquinone. Bioorg. Med. Chem. 2005, 13, 3841-3846.
2
7. Olfert, E.D.; Cross, B.M.; McWilliam, A.A. Guide to the Care and Use of Experimental Animals;
Canadian Council on Animal Care: Ottawa, Ontario, Canada, 1993; Volume 1, pp. 1-213.
8. Rodríguez, J.A.; Haun, M. Cytotoxicity of trans-dehydrocrotonin from Croton cajucara on V79
cells and rat hepatocytes. Planta Med. 1999, 65, 522-526.
2
Sample Availability: Samples of the compounds lapachol, junicedric acid and some derivatives are
available from the authors.
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