New Macrocyclic Lathyrane Diterpenes, from Euphorbia lagascae ...

New Macrocyclic Lathyrane Diterpenes, from
Euphorbia lagascae, as Inhibitors of Multidrug
Resistance of Tumour Cells
NoØlia Duarte 1
Nora GyØmµnt 2
Pedro M. Abreu 3
Joseph Molnµr 2
Maria-JosØ U. Ferreira 1
Original Paper
Abstract
The new macrocyclic lathyrane diterpenes latilagascenes A and B
(1 and 2), the diacetylated derivative of 2, latilagascene C (3), and
the known diterpenes ent-16a,17-dihydroxyatisan-3-one (4) and
ent-16a,17-dihydroxykauran-3-one (5), isolated from the methanol
extract of Euphorbia lagascae, were examined for their effects
on the reversal of multidrug resistance (MDR) on mouse lymphoma
cells. Among the active lathyrane derivatives 1 ± 3, compound
2 displayed the highest inhibition of rhodamine 123 efflux
of human MDR1 gene transfected mouse lymphoma cells
when compared to the untreated cells or the positive control verapamil.
The new compounds are the first macrocyclic lathyrane
diterpenes showing oxidation at C-16, whose structures were
characterized by extensive spectroscopic methods, including 2D
NMR experiments ( 1 H- 1 H COSY, HMQC, HMBC and NOESY). The
known phenolic compounds vanillic acid (6), p-salicylic acid (7),
isofraxidin (8) and cleomiscosin A (9) were also isolated from
this species.
Key words
Euphorbia lagascae ´ lathyrane ´ diterpenes ´ phenolic compounds
´ multidrug resistance ´ P-glycoprotein
162
Introduction
The occurrence of resistance to anticancer agents is a major obstacle
for successful cancer chemotherapy. Broad-spectrum resistance
to chemotherapy in human cancer has been called multidrug
resistance (MDR). One of the most effective mechanisms
of MDR involves P-glycoprotein (Pgp), a glycosylated transmembrane
protein efflux pump encoded by the human MDR1 gene
that is greatly overexpressed in most cancer cells found to be resistant
to therapeutic agents [1]. It belongs to the ATP-binding
cassette (ABC) superfamily of transporter proteins and decreases
the intracellular drug accumulation, by an ATP-dependent efflux,
reducing its cytotoxic effect and enabling the tumour cells to survive.
Pgp-mediated resistance is generally extended to structurally
and functionally unrelated drugs. In fact, the spectrum of
drugs interacting with Pgp includes a large number of compounds
such as anthracyclines, Vinca alkaloids, taxanes and
other drugs widely used as anticancer agents. A promising strategy
to overcome drug resistance is to develop MDR modulators
that can inhibit Pgp activity. Therefore, in recent years several
natural and synthetic compounds have been reported as MDR
modulators. However, in spite of the great number of MDR inhibitors
known, no effective modulator is available for the clinical
practice [2], [3].
Euphorbia lagascae Spreng. (Euphorbiaceae) is a herb that has
been cultivated for the production of vernolic acid, an epoxidised
fatty acid with potential industrial value, which is found in high
levels in the seeds of this species [4]. Furthermore, Euphorbia
lagascae has been used in folk medicine to treat cancer, tumours
and warts [5]. Recent phytochemical studies on Euphorbia species
identified several macrocyclic jatrophane diterpenes and a
Affiliation
1 CECF, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal
2 Department of Medical Microbiology, University of Szeged, Szeged, Hungary
3 CQFB/REQUIMTE, Faculty of Sciences and Technology, New University of Lisbon, Caparica, Portugal
Correspondence
Prof. Maria JosØ Umbelino Ferreira ´ CECF ´ Faculty of Pharmacy ´ University of Lisbon ´ Av. das ForcË as Armadas ´
1600±083 Lisbon ´ Portugal ´ Fax: +351-21-794-6470 ´ E-mail: mjuferreira@ff.ul.pt
Received May 20, 2005 ´ Accepted June 20, 2005
Bibliography
Planta Med 2006; 72: 162±168 Georg Thieme Verlag KG Stuttgart ´ New York
DOI 10.1055/s-2005-873196 ´ Published online December 5, 2005
ISSN 0032-0943

lathyrane-type diterpenoid as powerful Pgp inhibitors [6], [7],
[8], [9]. The purpose of the present study was to search for new
novel MDR modulators from Euphorbia lagascae.
Materials and Methods
General experimental procedures
Melting points were determined on a Köffler apparatus and are
uncorrected. Optical rotations were obtained using a Perkin Elmer
241 polarimeter. IR spectra were determined on an FTIR Nicolet
Impact 400, and NMR spectra were recorded on a Bruker
ARX-400 NMR spectrometer ( 1 H 400 MHz; 13 C 100.61 MHz),
using CDCl 3 , MeOD or C 5 D 6 N as solvents. MS were taken on a Kratos
MS25RF spectrometer (70 eV) and HR-MS and HR-SIMS on a
Micromass Autospec spectrometer. Column chromatography
was carried out on SiO 2 (Merck 9385). TLC were performed on
precoated SiO 2 F 254 plates (Merck 5554 and 5744) and visualized
under UV light and by spraying with sulphuric acid-acetic acidwater
(1 : 20:4) or sulphuric acid-water (1 :1) followed by heating.
HPLC was carried out on a Merck-Hitachi instrument, with
UV detection (254 nm), using a Merck LiChrospher 100 RP-18
(10 mm, 250 ”10 mm) column.
Plant material
Euphorbia lagascae Spreng. was collected in Cova da Beira, Coimbra,
Portugal, and identified by Dr. Teresa Vasconcelos of Instituto
Superior de Agronomia, University of Lisbon. A voucher specimen
(No. 323) has been deposited at the herbarium of Instituto
Superior de Agronomia.
Extraction and isolation
The air-dried powdered plant (5.9 kg) was extracted with methanol
(6 ” 12 L) at room temperature. Evaporation of the solvent
(under vacuum, 40 8C) from the crude extract yielded a residue
of 284 g which was suspended in boiling MeOH and frozen, to
give a precipitate (55 g) consisting mainly of waxes, that was
eliminated by filtration. The filtrate was evaporated, and the residue
(233 g) resuspended in a MeOH/H 2 O solution (1 :2, 3 L),
and extracted with Et 2 O (6 ”2 L). The ether extract was dried
(Na 2 SO 4 ) and evaporated under vacuum (40 8C), yielding a residue
(68.3 g) that was chromatographed over SiO 2 (8 ”120 cm, 2
kg), using mixtures of n-hexane/EtOAc (1 : 0, 4 L; 19 : 1 to 7: 3,
5 % gradient, 2.5 L each eluent; 13 :7 to 1:9, 5 % gradient, 1 L
each eluent; 0 :1, 8 L) and EtOAc/MeOH (19 : 1 to 17: 5, 5 % gradient,
1 L each eluent; 4 : 1 to 1 : 4, 10% gradient, 1 L each eluent) as
eluting solvents. The more apolar crude fractions containing
mainly waxes and triterpenes were discarded. The residue (7 g)
of the polar crude fraction eluted with EtOAc (8 L) was subjected
to CC on SiO 2 (5 ”80 cm, 250 g) with mixtures of n-hexane/EtOAc
(1 :1, 2 L; 2 : 3 to 0:1, 10 % gradient, 1 L each eluent) and EtOAc/
MeOH (19 : 1 to 4 : 1, 10 % gradient, 0.5 L each eluent; 1 : 1, 0.5 L).
After TLC monitoring the column chromatographic fractions
were combined into four fractions (F A to F D ).
The residue (1.55 g) of fraction F A (n-hexane/ EtOAc 1 : 1, 2 L) was
submitted to CC (3 ”80 cm, 80 g SiO 2 ; n-hexane/ EtOAc; 9 :1 and
4 :1, 0.5 L each eluent; 7:3 and 3:2, 0.75 L; 1 : 1 and 2 : 3, 0.5 L;
3 :7 and 1 :4, 0.25 L and 1 :9, 0.5 L). Fractions eluted with n-hexane/EtOAc
(7:3 to 1 : 1, 2 L) were associated (733 mg) and further
rechromatographed by CC (2”50 cm, 60 g SiO 2 , n-hexane/CH 2 Cl 2 ,
1 :9, 0.25 L; 0:1, 0.4 L and CH 2 Cl 2 /MeOH, 99 :1, 0.45 L; 39 :1, 0.75
L;19 : 1, 0.3 L; 9 :1, 0.3 L). Solvent evaporation from fractions
eluted with n-hexane/CH 2 Cl 2 (1 :9 and 0:1, 0.65 L) gave a mixture
(100 mg) that was purified by preparative TLC (CHCl 3 /
MeOH, 19 :1) yielding 7.2 mg of compound 8 (R f = 0.3, CHCl 3 /
MeOH, 19 : 1), and 7 mg of a compound with a strong absorption
at 254 nm, that was further purified by HPLC (MeOH/H 2 O, 3 : 1, 4
mL/min) to afford 4 mg of compound 1 (R t = 16 min; R f = 0.66,
CHCl 3 /MeOH, 19 : 1). Fractions eluted with CH 2 Cl 2 /MeOH (39 : 1,
0.75 L) were associated, and the residue (160 mg) chromatographed
on preparative TLC (CHCl 3 /MeOH, 9:1, 2 ”) to yield
35 mg of compound 6 (R f = 0.34, CHCl 3 /MeOH, 9 :1), and 10 mg
of compound 7 (R f = 0.25, CHCl 3 /MeOH, 9 :1).
Fraction F B eluted with n-hexane/EtOAc (2 :3, 1 L, 1.65 g) was
submitted to CC (3 ”80 cm, 100 g SiO 2 ; n-hexane/EtOAc; 7:3, 1
L; 3:2 and 1 :1, 0.5 L each eluent, 2 :3 and 3 : 7, 1 L each; 1 : 4
and 0:1 0.5 L each eluent and EtOAc/MeOH 19 :1 to 9 :1, 5 % gradient,
0.5 L each eluent). Fractions eluted with n-hexane/EtOAc
were pooled, and the mixture (1 g) rechromatographed twice by
CC (2 ”50 cm, 60 g SiO 2 ) using mixtures of n-hexane/CH 2 Cl 2 (1 : 9,
0.2 L; 0:1, 0.25 L) and CH 2 Cl 2 /MeOH (99 :1, 0.25 L, 49:1, 0.75 L;
19 :1, 0.25 L). The residue of the fractions eluted with CH 2 Cl 2 /
MeOH (49 :1 and 19 : 1, 1 L, 490 mg) was rechromatographed
twice by CC as above, yielding 162 mg of a compound that was
further purified by recrystallisation (CH 2 Cl 2 /n-hexane), affording
126 mg of compound 4 (R f = 0.37, CHCl 3 /MeOH, 19 : 1).
Fraction F C eluted with n-hexane/EtOAc (3:7, 1 L and 1 : 4, 1 L,
2.26 g) was submitted to CC (3 ”80 cm, 150 g SiO 2 ; n-hexane/
CH 2 Cl 2 , 1:1 to 0:1, 10 % gradient, 0.5 L each eluent; and CH 2 Cl 2 /
MeOH, 49 : 1, 0.5 L;19 :1, 1.5 L; 9 :1, 0.5 L; 17: 1, 0.3 L). The mixture
(1.5 g) of the fractions eluted with CH 2 Cl 2 /MeOH (19 : 1, 1.5 L) was
further rechromatographed by CC (2”50 cm, 70 g SiO 2 ; n-hexane/EtOAc;
2 :3, 1.2 L; 1:7, 0.9 L, 1 :4, 0.6 L, 0:1, 0.3 L and
EtOAc/MeOH 49 :1, 0.3 L). Solvent evaporation of fractions eluted
with n-hexane/EtOAc (2 :3 and 3:7, 2.1 L, 917 mg) gave a mixture
that was subject to CC (2 ”50 cm, 60 g SiO 2 ; n-hexane/CH 2 Cl 2 ,
1 :9, 0.6 L; 0 :1, 0.3 L and CH 2 Cl 2 /MeOH, 99 :1, 0.9 L, 97:3, 0.9 L;
19 :1, 0.3 L). Fractions eluted with CH 2 Cl 2 /MeOH (99 :1) were
associated and the residue (140 mg) rechromatographed by CC
as above, yielding 40 mg of a compound that was crystallised
from MeOH, to afford 16 mg of compound 9 (R f = 0.6, CHCl 3 /
MeOH, 17: 3). Fractions eluted with CH 2 Cl 2 /MeOH, 97: 3 and
19 :1 (1.2 L, 601.2 mg), were rechromatographed as above, affording
500 mg of an impure compound that was then purified
by HPLC (MeCN/H 2 O, 11 :9, 5 mL/min, 254 nm), yielding 287 mg
of compound 2(R t = 13 min; R f = 0.5, CHCl 3 /MeOH, 17:3). Purification
of the compound with R t = 6 min (20 mg) by preparative
TLC (CHCl 3 /MeOH, 9:1, 2 ”) afforded 10 mg of compound 5
(R f = 0.4, CHCl 3 /MeOH, 9 :1).
Latilagascene A [(2R*,3S*,4R*,5R*,6R*,9S*,11S*,15R*)-16-acetoxy-
15b-cinnamoyloxy-5a,6b-epoxy-3b-hydroxy-14-oxolathyr-12Eene,
1]: oil; [a] D 25 : ±113.08 (CHCl 3 , c 0.1); HR-MS: m/z = 522.2615
[M] + , (calcd. for C 31 H 38 O 7 : 522.2617); IR (film): n max = 3411, 2922,
2855, 1730, 1714, 1652, 1640, 1452, 1257, 1041, 853 cm ±1 ; EI-MS:
m/z (rel. int) = 522 [M] + (< 1), 504 [M ±H 2 O] + (< 1), 391 [M ±
C 9 H 7 O] + (1), 331 [M±C 9 H 7 O±CH 3 COOH] + (1) 313 [M ± C 9 H 7 O±
Original Paper
163
Duarte N et al. New Macrocyclic Lathyrane ¼ Planta Med 2006; 72: 162 ±168

Original Paper
CH 3 COOH ± H 2 O] + (1) 289 (7), 149 (24), 147 (31), 131 [C 9 H 7 O] +
(89), 103 (34), 69 (48), 57 (81), 55 (56), 43 (100); 1 H- and 13 C-
NMR see Table 1.
Latilagascene B [(2R*,3S*,4R*,5R*,6R*,9S*,11S*,15R*)-15b-cinnamoyloxy-5a,6b-epoxy-3b,16-dihydroxy-14-oxolathyr-12E-ene,
2]: White amorphous solid; [a] 25 D : ±168.078 (CHCl 3 , c 0.1); HR-
SIMS: m/z = 481.2583 [M + 1] + , (calcd. for C 29 H 36 O 6: 481.2590);
IR (KBr): n max = 3425, 2919, 2855, 1715, 1633, 1653, 1448, 1258,
1165, 1117, 853, 767, 730, cm ±1 ; FAB-MS: m/z (rel. int) = 503 [M
+ Na] + (13), 481 [M + 1] + (1), 355 [M + Na ± C 9 H 8 O 2 ] + (1), 315 (1),
131 (100), 121 (10), 103 (26), 89 (20), 77 (34); 1 H- and 13 C-NMR
see Table 2.
Acetylation of compound 2: Compound 2 (50 mg) was suspended
in Ac 2 O (1 mL) and pyridine (1 mL). After stirring at room temperature
for 48 hours, the reaction was worked up as usual,
yielding 40 mg of compound 3 (R f = 0.64, CHCl 3 /MeOH, 19 : 1).
Latilagascene C [(2R*,3S*,4R*,5R*,6R*,9S*,11S*,15R*)-3b,16-diacetoxy-15b-cinnamoyloxy-5a,6b-epoxy-14-oxolathyr-12E-ene,
3]:
oil; [a] D 25 : ±130.438 (CHCl 3 , c 0.1); IR (film): n max = 2923, 2854,
1740, 1717, 1660, 1631, 1454, 1368, 1234, 1167, 1141, 1038, 855,
763 cm ±1 ; EIMS: m/z (rel. int) = 564 [M] + (< 1), 433 [M ±
C 9 H 8 O 2 ] + (4), 131 (96), 103 (48), 93 (22), 84 (25), 77 (26), 43
(100); 1 H- and 13 C-NMR see Table 2.
Assay for MDR reversal effect
Cells: The L5178 Y mouse T-lymphoma parental cell line was
transfected with the pHa MDR1/A retrovirus as previously described
[10]. The L5178 MDR cell line and the L5178 Y parental
cell line (obtained from Prof. M. Gottesmann, NCI and FDA,
USA), were grown in McCoy's 5A medium with 10% heat-inactivated
horse serum, D-glutamine and antibiotics. MDR1-expressing
cell lines were selected by culturing the infected cells with
60 ng/mL colchicine to maintain expression of the MDR phenotype.
Cell viability was determined by trypan blue.
Table 1
NMR data of compound 1 (J in Hz)
Position 1 H 13 C DEPT HMBC (C®H) NOESY
164
1a 3.48 dd (7.6; 13.3) 39.8 CH 2 ± H-1b,H-4
1b 1.79t (13.3) ± ± ± H-1a, H-16a, 3-OH
2 2.16 m 44.3 CH 1b, 16a H-1a, H-3, H-4
3 4.16 br s 74.7 CH 1a, 16a, 16b H-2, H-4,
4 1.54 dd (3.7; 9.4) 51.7 CH 1a,5 H-1a, H-2, H-3, Me-17
5 3.66 d (9.4) 58.0 CH 7a,4,17 H-7b/8b, H-12, 16-Oac, H-3¢
6 ± 63.9C 17 ±
7a* 2.03 m 38.7 CH 2 17, 9H-7b/8b
7b** 1.56 m ± ± H-7a/8a
8a* 2.03 m 23.3 CH 2 9H-7b/8b
8b** 1.56 m ± ± H-7a/8a
91.14 m 33.9CH 18, 19H-7a/8a, H-11, Me-20
10 ± 26.4 C 9, 18, 19 ±
11 1.49 dd (7.9; 11.0) 29.8 CH 18, 19 H-7a/8a, H-9, Me-20
12 7.02 brd (11.0) 144.7 CH 20 H-5, H-7b/8b, H-19
13 ± 133.9C 20 ±
14 ± 194.8 C 1a, 12, 20 ±
15 ± 91.2 C 1a,1b ±
16a 4.48 dd (10.8; 11.2) 62.8 CH 2 1b H-1b, H-16b, 16-OAc
16b 3.99 dd (4.7; 11.2) H-2, H-16a
17 1.17 s 20.0 CH 3 ± H-4, H-7a/8a,
18 1.10 s 29.0 CH 3 9, 11, 19 H-9, H-11
190.85 s 16.2 CH 3 18 H-12, H-2¢
20 1.87 s 12.3 CH 3 12 H-9, H-11
3-OH 3.09brs ± ± ± H-1b
15-OCin
1¢ ± 165.6 C 3¢
2¢ 6.45 d (16.0) 117.3 CH 3¢ Me-19, H-3¢, H-5¢,H-6¢, H-7¢,H-8¢, H-9¢
3¢ 7.69d (16.0) 146.7 CH 5¢,9¢ H-2¢,H-5
4¢ ± 133.9C 2¢,5¢,6¢,8¢,9¢
5¢,9¢ 7.47 m 128.2 CH 3¢,5¢,6¢,8¢,7¢,9¢ H-2¢
6¢,8¢ 7.38 m 130.8 CH 5¢,9¢ H-2¢
7¢ 7.38 m 129.0 CH 5¢,9¢ H-2¢
16-OAc 2.06 s 172.2 C 16a, 16b, OAc H-5, H-16
20.9CH 3 ±
*, ** Overlapped signals.
Duarte N et al. New Macrocyclic Lathyrane¼ Planta Med 2006; 72 162 ± 168

Table 2
NMR data of compound 2 and 3 (J in Hz)
Position 2 3
1 H 13 C HMBC (C®H) NOESY 1 H 13 C
1a 3.42 dd (7.4; 13.3) 39.3 ± H-1b, H-2 3.62 dd (7.6; 13.6) 41.0
1b 1.91 t (13.2) ± ± H-1a, H-2 1.55 m ±
2 2.04 m 45.5 ± H-1a, H-3, H-4, H-16a 2.41 m 42.4
3 4.44 br s 76.4 1a H-2, H-4 5.61 t (3.6) 76.6
4 1.53 dd (3.5; 9.3) 51.9 1a, 5 H-2, H-3, H-7a, H-12, Me-17 1.72 dd (3.9; 9.1) 50.8
5 3.65 d (9.4) 58.4 4, 17 ± 3.32 d (9.2) 57.0
6 ± 64.4 5, 7a, 17 ± ± 63.4
7a 2.04 m 38.6 9, 17 H-4, H-7b, Me-17 2.03 m 38.4
7b 1.59m ± H-7a 1.74 m
8a 2.01 m 23.2 ± ± 2.06 m 23.4
8b 1.57 m 1.55 m
91.12 m 33.8 18, 19 H-11, H-3¢,H-5¢,H-6¢,H-7¢,H-8¢,H-9¢ 1.14 m 34.0
10 ± 26.4 9, 18, 19 ± ± 26.5
11 1.46, dd(7.8; 10.7) 29.7 9, 19 H-9, Me-20, H-6¢,H-7¢, H-8¢ 1.49, dd (7.9, 10.9) 29.8
12 6.98 d (10.9) 144.6 20 H-4, Me-19 7.00 d (11.1) 144.9
13 ± 133.920 ± ± 133.9
14 ± 195.1 1a, 1b, 4, 12, 20 ± ± 194.3
15 ± 91.6 1a, 1b ± ± 90.9
16a 3.85 dd (4.0; 11.0) 61.2 1b, 2 H-2 4.13 dd (8.7; 11.0) 62.3
16b 3.80 dd (7.4; 11.0) 3.97 dd (6.3; 11.1)
17 1.16 s 19.3 ± H-4, H-7a,H-3¢, H-5¢,H-9¢ 1.16 s 19.9
18 1.08s 29.0 11, 19 Me-19 1.11 s 29.0
190.82 s 16.2 9, 18 H-12, Me-18 0.86 s 16.2
20 1.85 s 12.3 ± H-11 1.88 s 12.3
15-OCin
1¢ ± 165.6 2¢,3¢ ± 165.4
2¢ 6.44 d (16.0) 117.3 ± H-3¢,H-5¢,H-9¢ 6.43 d (16.0) 117.0
3¢ 7.65 d (16.0) 146.5 5¢,9¢ H-2¢,H-5¢,H-9¢, H-9, Me-17 7.71 d (16.0) 147.0
4¢ ± 133.92¢,6¢,8¢ ± 133.8
5¢,9¢ 7.45 m 128.1 3¢,6¢,7¢,8¢ H-2¢,H-3¢, H-9, Me-17 7.49 m 128.4
6¢,8¢ 7.35 m 130.6 5¢,9¢ H-9, H-11 7.41 m 130.9
7¢ 7.35 m 128.9 ± H-9, H-11 7.41 m 129.0
3-OAc ± ± ± ± 2.15* 170.9*
20.8
16-OAc ± ± ± ± 2.01* 169.1*
20.8
Original Paper
165
* Interchangeable values.
Assay for rhodamine 123 accumulation test: The harvested cells
were resuspended in serum-free McCoy's 5A medium and distributed
into Eppendorf tubes at the density of 2 ”10 6 cell/mL.
Then, 2 to 20 mL of the stock solution (1 mg/mL in DMSO) of the
tested compounds were added and the samples were incubated
for 10 min at room temperature. Following the addition of 10 mL
of rhodamine 123 to the samples (5.5 mM final concentration),
the cells were further incubated for 20 min at 37 8C, washed
twice, and resuspended in 0.5 mL phosphate-buffered saline
(PBS) for analysis. The fluorescence uptake of the cells was measured
by flow cytometry using a Beckton Dickinson FACScan instrument
equipped with an argon laser. The fluorescence excitation
and emission wavelengths were 488 nm and 520 nm,
respectively. Verapamil was used as a positive control, and the
influence of DMSO on the cells was monitored. The mean fluorescence
intensity was calculated as a percentage of the control
for the parental (PAR) and MDR cell lines as compared to untreated
cells. An activity ratio (R) was calculated on the basis of
the measured fluorescence values (FL-1) measured via the following
equation [11], [12].
R = (FL ± 1 MDRtreated /FL ± 1 MDRcontrol )/(FL ± 1 parentaltreated /FL ± 1 parentalcontrol)
Results and Discussion
The Et 2 O-soluble fraction of the methanol extract of the aerial
air-dried powdered plant of Euphorbia lagascae was submitted
to successive chromatographic fractionation and purification, as
described in the Materials and Methods section, to afford the
new diterpenes 1 and 2 along with the known compounds ent-
16a,17-dihydroxyatisan-3-one (4), ent-16a,17-dihydroxykauran-
3-one (5), vanillic acid (6), p-salicylic acid (7), isofraxidin (8), and
Duarte N et al. New Macrocyclic Lathyrane ¼ Planta Med 2006; 72: 162 ±168

Original Paper
166
cleomiscosin A (9). Acetylation of 2 yielded the diacetylated derivative
3.
Compound 1 was obtained as an oil, whose molecular formula
was determined as C 31 H 38 O 7 from its HR-MS, which showed a
molecular ion at 522.2615, indicative of thirteen unsaturations.
In the IR spectrum, characteristic absorption bands for ester carbonyl
groups, an a,b-unsaturated ketone, and a hydroxy group
were observed. The EI-MS of 1 displayed a strong (89 %) peak at
m/z =131[C 9 H 7 CO] + and a ion at 391 [M ± C 9 H 7 O] + suggesting
the existence of a cinnamoyl group whose presence was also deduced
from the 1 H- (d = 7.38±7.47, 5H; 6.45 and 7.69 doublets,
J = 16.0 Hz) and 13 C-NMR spectra (d C = 133.9, 2 ”130.8, 129.0,
2 ”128.2, aromatic ring; 146.7, 117.3, olefinic carbons; 165.6 carbonyl).
Besides, the EI-MS showed ions at m/z = 331 [M±
C 9 H 7 O±CH 3 COOH ] + and 313 [M ± C 9 H 7 O±CH 3 COOH±H 2 O] + resulting
from the sequential loss of one acetoxy group and one hydroxy
group. These structural features were also confirmed by
the NMR spectral data of 1, which revealed the presence of one
acetyl group (d = 2.06, d C = 172.2), and a proton signal at
d = 3.09 without correlation in the HMQC spectrum (Table 1).
Moreover, the 1 H NMR spectrum of 1 showed signals for four tertiary
methyl groups (d = 1.87, 1.17, 1.10, 0.85), two methine protons
(d = 4.16, brs; 3.66, d, J = 9.4 Hz) and one diastereotopic
methylene group (d = 4.48, dd, J = 10.8, 11.2 Hz and d = 3.99
dd, J = 4.7, 11.2 Hz) bearing oxygen, and one olefinic proton at
d = 7.02 (brd, J = 11.0 Hz). Besides the signals of the acyl groups,
the remaining 13 C-NMR and DEPT spectral data showed resonances
of twenty carbons corresponding to four CH 3 , four CH 2 (one
oxygen bearing carbon at d C = 62.8), seven CH (two oxygenated
at d C = 58.0 and 74.7 and one sp 2 at 144.7) and five quaternary
carbons (a carbonyl group at d C = 194.8, one olefinic carbon at
133.9 and two oxygenated carbon at 63.9 and 91.2). The relatively
high-field carbonyl signal (d C = 194.8) and the existence of a
remarkable downfield shifted vinylic proton signal at d = 7.02
were consistent with an enone system in the molecule. The
gem-dimethyl-substituted cyclopropane ring was indicated by a
pair of highfield methine signals (d = 1.14 and 1.49) associated to
a quaternary carbon at d C = 26.4. Furthermore, an oxacyclopropane
ring was evidenced by the chemical shifts of the oxygenated
carbons at d C = 58.0 (CH) and 63.9 (C) and the lowfield methine
proton at d = 3.66. The above structural features, mainly
the presence of a cyclopropane ring, pointed to a lathyrane skeleton
(C 20 H 30 O 5 ) for 1. Further structural details were obtained by
2D NMR measurements (COSY, HMQC and HMBC), which allowed
the assignment of the remaining NMR resonances. The
1 H- 1 HCOSY( 3 J couplings) and HMQC experiments revealed the
structure of two sequences of correlated protons: -CH 2 -
CH(CH 2 OR)-CH(OR)-CH(R)-CH(OR)- (A); -CH 2 -CH 2 -CH(R)-CH(R)-
CH = C(C)- (B). The location of the olefinic methyl group
(d = 1.87, Me-20) in fragment B was deduced from its 4 J H-H coupling
with the vinylic proton at d = 7.02 (H-12). Furthermore, the
heteronuclear 2 J C-H and 3 J C-H connectivities displayed in the
HMBC spectrum of 1 (see Table 1), between the quaternary carbons
and the protons of the A and B spin systems, allowed the
linkage of the referred fragments, the assignment of an epoxy
ring at C-5/C-6, and a ketone group at C-14 (d C = 194.8), as well
as the establishment of the cyclopropane ring. The 3 J C-H HMBC
correlations also led to the location of two ester functions. The
ester carbonyl carbon at d C = 172.2 is bound to C-16 since it correlates
with the methylenic protons at d = 3.99 and 4.48, and the
acetyl methyl group at d = 2.06. The placement of the second
acyl group (d = 6.45 ± 7.38, d C = 165.6) on the quaternary carbon
C-15 (d C = 91.2) followed from the observed 2 J C-H correlation
with a vinylic proton (d = 7.69) of the cinnamoyl group, and the
absence of 3 J C-H correlation with the oxymethine protons. Evidence
for the location of the hydroxy group at C-3, was provided
by the lack of any correlation of H-3 with the carbonyl carbons.
The relative stereochemistry of 1 was deduced from the NOESY
spectrum. The strong nuclear Overhauser interactions of the a-
oriented H-4, taken as a reference point on a biogenetic basis
[13], with H-3, H-2, H-1a, indicated the a orientation for these
three protons. Further cross peaks between H-1b and H-16a, H-
5 and H-3, and H-5/16-OAc confirmed the stereochemistry at C-
2, and established the configuration at C-5 and C-15, as well as
the trans A/B ring junction. Besides, H-5 also exhibited a nuclear
Overhauser effect at H-12, thus suggesting that this proton is oriented
above the plane of the macrocycle. The NOE enhancements
of H-12 at Me-19, H-11 at Me-18, and H-9 at Me-18, indicated
that H-9 and H-11 have the same a configuration, and
thereby a cis macrocycle/cycloprane ring connection. Moreover,
the E configuration of the C-12/C-13 double bond was deduced
from the observed cross peaks between H-9/Me-20 and H-11/
Me-20, that evidenced the orientation of Me-20 below the plane
of the molecule. The absence of NOE correlations between H-5/
Me-17, and the NOE effect observed between Me-17 and H-4 indicated
the a configuration of Me-17. All the above data are in
agreement with structure 1, corresponding to a new lathyrane
diterpene, named as latilagascene A. Although a great number
of highly oxidised macrocyclic lathyrane and jatrophane diterpenes
has been previously isolated from Euphorbia species, this
is the first reported occurrence of a related derivative oxidised
at C-16. Lathyrane derivatives containing the rare 5,6-epoxy
function were previously found in the structure of jolkinol [14],
[15]. A similar derivative was recently isolated from E. villosa [16].
Compound 2 was obtained as a white amorphous powder. Its
molecular formula was established by HR-SIMS, whose spectrum
showed a quasi-molecular ion at m/z = 481.2583 [M + H] + . The
UV, MS and NMR data of 2 clearly resemble those found for lati-
Duarte N et al. New Macrocyclic Lathyrane¼ Planta Med 2006; 72 162 ± 168

lagascene A (1). As for that compound, the base peak at m/z =
131 [C 9 H 7 CO] + displayed by the FAB-MS, and its NMR data also
indicated the presence of the cinammate ester (d = 7.35± 7.45,
5H; 6.44 and 7.65 doublets, J = 16.0 Hz; d C = 133.9, 2 ”130.6,
128.9, 2”128.1, 146.5, 117.3 and 165.6), and the same number of
protons bounded to oxygenated carbons (d = 4.44, 1H, brs; 3.85,
1H, dd, J = 4.0, 11.0 Hz and 3.80, 1H, dd, J = 7.4, 11.0 Hz; 3.65, 1H,
d, J = 9.4 Hz), although there was no evidence for the presence of
acetyl resonances (see Table 2). In addition to the cinnamoyl
group, the 13 C-NMR spectrum of 2 also evidenced two methines
(d C = 76.4, 58.4), one methylene (d C = 61.2), two quaternary carbon
linked to oxygen (d C = 64.4, 91.6), a ketone (d C = 195.1), and
two vinylic carbons (d C = 144.6, CH; 133.9, C). The above data
suggest that, in comparison with latilagascene A, compound 2
bears another free hydroxy group, which was located at C-16 on
the basis of observed differences in carbon and proton chemical
shifts for both compounds. The diamagnetic effect of 1.6 ppm at
C-1 (a-carbon), and the downfield shift of 1.2 ppm at C-2 (b-carbon)
observed for compound 2 relatively to 1, are in agreement
with the effects expected for the substitution of an acetoxy by a
hydroxy group. The clear deshielding of C-3 (1.7 ppm; g carbon)
may be due to intramolecular hydrogen bonding between the
hydroxy groups at C-16 and C-3, which also corroborates the existence
of free hydroxy groups at these positions. The above data
confirmed compound 2 as a new lathyrane diterpene, named as
latilagascene B.
Table 3
Compound
Effect of compounds 1±5 a on reversal of multidrug resistance
(MDR) on human MDR1 gene transfected mouse lymphoma
cells
Concentration
(mg/mL)
FL-1 b
Fluorescence
activity ratio
PAR + R123 c ± 914.8 ±
MDR + R123 d ± 21.8 ±
Verapamil 10 212.5 9.72
1 4 284.4 13.01
40 1006.1 46.04
2 4 206.6 28.11
40 750.3 102.07
3 4 266.2 12.18
40 300.8 13.76
4 4 13.1 0.60
40 12.0 0.55
5 4 7.4 1.00
40 8.1 1.09
DMSO 20 ml 6.8 0.7
a The results of compounds 2 and 5 were obtained from a different assay (PAR + R123: FL-
1 = 924.5; MDR + R123: FL-1 = 7.4; Verapamil: FL-1 = 137.3, FAR = 18.7).
b FL-1: Fluorescence intensity.
c Par: a parental cell without MDR gene.
d MDR: a parental cell line transfected with human MDR1 gene.
Original Paper
Acetylation of 2 afforded the corresponding diacetate derivative
3, named latilagascene C, whose EI-MS displayed a weak molecular
ion at m/z = 564. As expected, the main differences observed
in the NMR data of 3, when compared with those of 2 (Table 2),
were in proton and carbon chemical shifts of ring A and B. However,
it is interesting to note that no remarkable change was observed
at a-carbon C-3 (g-carbon relative to C-16).
The diterpenes ent-16a,17-dihydroxyatisan-3-one (4) {[a] D 25 :±388
(CHCl 3 , c 0.14)}, ent-16a,17-dihydroxykauran-3-one (5) {[a] D 25 :±978
(CHCl 3 , c 0.13)}, and the phenolic compounds vanillic acid (6), p-salicylic
acid (7), isofraxidin (8), and cleomiscosin A (9) {[a] D 25 :
08(CHCl 3 , c 0.10}, were identified by comparison of their spectral
data with those reported in the literature [17], [18], [19], [20], [21],
[22].
Diterpenes 1 ±5 were investigated for their MDR-reversing activity
on L5178 mouse lymphoma cells, using the rhodamine 123
exclusion test. As can be observed by the results summarized in
Table 3, compounds 1±3 were shown to enhance drug retention
in the cells by inhibiting the efflux-pump activity mediated by P-
glycoprotein, whereas compounds 4 and 5 were inactive. These
results showed concentration dependence for latilagascenes A
(1) and B (2). Latilagascene B was found to be a highly potent
inhibitor [fluorescence activity ratios R = 28.1 at 4 mg/mL and
102.1 at 40 mg/mL concentration], stronger than the positive control
verapamil (R = 18.7 at 10 mg/mL concentration). From the
above data, a structure-activity relationship between compounds
1 ± 3 can be suggested, since they only differ in the substitution
pattern of ring A. Compound 2 has two free hydroxy
groups at C-16 and C-3, the hydroxy at C-16 being acetylated in
compound 1, and both OH groups acetylated in compound 3.
The marked decrease of activity observed upon acetylation of
the hydroxy group at C-3, in compound 3 highlights the importance
of a free hydroxy at this position. They are lipophilic compounds
with calculated log P = 4.7 (1) 4.3 (2) and 5.2 (3), which
reinforce the importance of lipophilicity as a property of primary
importance in MDR reversers [23]. However, compound 3, which
is the most lipophilic, showed the lowest activity, thus confirming
[24] that other factors such as the presence of hydrogen bond
donor/acceptors seem to be decisive to the interaction of inhibitors
with Pgp. The present data lead us to conclude that ring A
plays a significant role in the modulation of MDR. Previous studies
on structure-activity relationships of macrocyclic diterpenes
from Euphorbia species, including several jatrophanes with closely
related structures, evidenced the importance of specific
moieties of the molecule in the interaction with Pgp [8], [25],
[26], [27]. However, the structural requirements for the Pgp
modulation of macrocyclic diterpenes are not completely elucidated
yet. This difficulty may arise from the high conformation
flexibility that characterises the macrocycle ring, in both jatrophanes
and lathyranes, which depends strongly on their substitution
pattern. Therefore, further SAR studies are needed in order
to improve our knowledge on Pgp binding.
Acknowledgements
Science and Technology Foundation, Portugal (FCT) supported
this work. The authors thank Dr. Teresa Vasconcelos (ISA, University
of Lisbon, Portugal) for identification of the plant, Prof. M.
Gottesmann and Prof. A. Aszalos (Food and Drug Administration,
USA) for cell lines and Szeged Foundation for Cancer Research.
167
Duarte N et al. New Macrocyclic Lathyrane ¼ Planta Med 2006; 72: 162 ±168

Original Paper
168
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