Tethered lipids from Thapsia garganica

Article abstract of DOI:10.1021/np049781y

The two macrocyclic lipids 2-hydroxymethyl-1,4-dioxacycloicosane-5,20-dione (1) and 3-hydroxy-1,5-dioxacyclohenicosane-6,21-dione (2) have been isolated from the fruits of Thapsia garganica. Tethered lipids are unprecedented in the plant kingdom.

Full text of DOI:10.1021/np049781y

©
Copyright 2004 by the American Chemical Society and the American Society of Pharmacognosy
Volume 67, Number 9
September 2004
Rapid Communications
Sch em e 1
Teth er ed Lip id s fr om Th a psia ga r ga n ica
†
‡
Huizhen Liu, Carl Erik Olsen, and
,
†
S. Brøgger Christensen*
Department of Medicinal Chemistry, Danish University of
Pharmaceutical Sciences, Universitetsparken 2,
DK-2100 Copenhagen, Denmark, and Department of
Natural Sciences, Royal Veterinary and Agricultural
University, B u¨ lowsvej 17,
DK-1870 Frederiksberg C, Denmark
Received J uly 6, 2004
Abstr a ct: The two macrocyclic lipids 2-hydroxymethyl-1,4-
dioxacycloicosane-5,20-dione (1) and 3-hydroxy-1,5-dioxacy-
clohenicosane-6,21-dione (2) have been isolated from the fruits
of Thapsia garganica. Tethered lipids are unprecedented in
the plant kingdom.
of the obtained thapsigargin revealed the presence of
unexpected signals. Rechromatography afforded two im-
purities, compounds 1 and 2. The presence of the AB part
of an ABX patterns at 3.74 ppm, two double doublets at
4.38 and 4.26 ppm, and a complex signal at 5.13 ppm,
together with signals characteristic for R-protons in esters
and signals that could be assigned to ester carbonyl
carbons, strongly suggested compound 1 to be a 1-O,2-O-
diacylglycerol. The absence of a signal originating in a
terminal methyl group could only be explained by assuming
the compound to be a tethered lipid. A molecular formula
of C H O proved the compound to be a diester of hexa-
Tethered lipids are lipids in which two of the oxygens of
glycerol are included in a macrocyclic ring. The compounds
have pronounced effects on the membranes of cells afford-
ing modulation of the activities of enzymes such as protein
kinases and phospholipases. These properties have encour-
aged syntheses and testing of a number of such lipids.1
Tethered lipids have never been found in nature except for
archaebacteria, e.g., Methanospirillium hangatei.4 The
presence of terpenoid glycerol ethers in the membrane is
assumed to enable the bacteria to grow in hostile environ-
ments. No other living organism has previously been
reported to form tethered lipids. The present report,
however, documents the presence of this group of com-
pounds in the fruits of the Mediterranean plant Thapsia
garganica L. (Apiaceae).
-3
1
9
34
5
decanedioic acid (1). Compound 2 possessed the same
molecular formula, but the NMR spectrum revealed the
presence of only one AB part of an ABX spectrum.
Structure 2 explains this finding.
Compound 1 possessed a small positive specific rotation.
Synthesis of 1 (Scheme 1) established the absolute config-
uration at C-2 as R. An esterification of (R)-1-O-benzyl-
glycerol (3) with 8-nonenoic acid (4) facilitated by dicyclo-
hexylcarbodiimide (DCCD) afforded the diester 5, which
by treament with Grubb’s first-generation catalyst was
converted into the macrocyclic intermediate 6. Palladium
on charcoal catalyzed hydrogenation and hydrogenolysis
yielded S-1 with a specific rotation of -3.3°. The overall
yield of the three steps was 48%. The negative rotation of
Our attempt to develop thapsigargin into a drug for
treatment of prostate cancer5 has made T. garganica a
,6
7
focus for our research for some years. Encouraging devel-
opments in this project prompted us to isolate thapsigargin
in 50 g amounts. In-depth examination of the NMR spectra
*
To whom correspondence should be addressed. Tel: +45 3530 6253.
Fax: +45 3530 6041. E-mail: sbc@dfuni.dk.
†
Danish University of Pharmaceutical Sciences.
Royal Veterinary and Agricultural University.
‡
1
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© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/05/2004

1
440 J ournal of Natural Products, 2004, Vol. 67, No. 9
Rapid Communications
the S-form proved that 1 isolated from T. garganica is the
R-form. The considerably smaller numerical value of the
specific rotation is explained by facile acyl migration
between the oxygens on glycerol (Scheme 1). Leaving a
solution of 1 in toluene with some silica gel added further
support to this hypothesis. After stirring overnight, a
considerable amount of 2 was observed by NMR. A silica
gel-catalyzed migration of the acyl groups between O-1,
O-2, and O-3 apparently occurs, eventually establishing
equilibrium between (R)-1, 2, and (S)-1. During the re-
peated chromatographic procedures for isolating 3 no doubt
similar acyl migrations have occurred, affording some
racemization of 1 and formation of 2. The optical activity
of 1, although limited, reveals that R-1 must be genuine.
The possibility of 2 being an artifact formed during the
purification procedure cannot be excluded.
3-Hydr oxy-1,5-dioxacycloh en icosan e-6,21-dion e (2): vis-
1
cid oil; H NMR (CDCl
3
, 300 MHz) δ 4.15 (2H, dd, J ) 12.0,
3
.0 Hz, H-2 and H-4), 4.14 (2H, dd, J ) 12.0, 3.0 Hz, H-2′ and
H-4′), 4.04 (1H, quintet, J ) 3.0, H-3), 2.33 (4 H, t, J ) 7.2
Hz, H-7 and H-20), 1.62 (4H, m, H-8 and H-19), 2.27 (20H, m,
H-9-H-18); 13C NMR (CDCl
, 75 MHz) δ 174.1 (C, C-6 and
C-21), 68.5 (CH, C-3), 65.2 (CH , C-2 and C-4), 34.3 (CH , C-7
and C-20), 28.5, 28.4, 28.1, 25.0 (CH , C-8-C-19); HRFABMS
3
2
2
2
+
+
35 5
H O , 343.2484);
m/z 343.2506 [M + H] (calcd for C19
+
+
365.2319 [M + Na] (calcd for C H34NaO5 , 365.2304).
1
9
Syn th esis of Com p ou n d (S)-1. To a solution of (R)-3-
benzyloxy-1,2-propandiol (3, 200 mg, 1.1 mmol), 4 (400 mg,
2
.5 mmol), and 4-(dimethylamino)pyridine (1.20 g, 9.8 mmol)
in dry CH Cl (60 mL) was added at 0 °C a solution of DCCD
2.02 g, 9.8 mmol) in dry CH Cl (4 mL). The solution was
filtered after stirring at room temperature for 4-5 h, and the
precipitate rinsed with CH Cl . The solvent was evaporated
2
2
(
2
2
2
2
in vacuo. Compound (S)-5 (350 mg, 74%) was isolated by
column chromatography over silica gel eluted with toluene-
ethyl acetate (40:1). To a solution of (S)-5 (229 mg, 0.5 mmol)
Exp er im en ta l Section
P la n t Ma ter ia l. Fruits of Thapsia garganica L. were
collected on the island of Ibiza, Spain, in J uly 2002. A voucher
specimen is deposited at The Herbarium at the University of
Copenhagen DFHSBC1.
Extr a ction a n d Isola tion . The fruits (1 kg) were blended
with 3 L of ethanol, and the mixture was left overnight. The
mixture was filtered and the filtrate concentrated in vacuo.
The residue was dissolved in toluene (1 L) and the solution
washed three times with 330 mL of water. The organic phase
was concentrated to give approximately 60 g of an oily dark
residue. The residue was chromatographed over silica gel 60
in CH
2
Cl
2
(55 mL) was added over 1 h a solution of Grubb’s
Cl (27 mL) at room
first-generation catalyst (50 mg) in CH
2
2
temperature. After stirring for an additional 18 h at room
temperature, the solvent was removed in vacuo. Compound
(S)-6 (160 mg, 68%) was obtained as a viscid oil by column
chromatography of the residue over silica gel eluted with
toluene-ethyl acetate (50:1), [R]25
D
3
+17.7° (c 0.10 CHCl ). A
50 mL flask containing a solution of (S)-6 (194 mg, 0.45 mmol)
in ethanol-acetic acid (10:1 v/v, 14 mL) and 100 mg of
palladium on charcoal (10%) was sealed with a rubber stopper.
A syringe through the stopper connected 200 mL of hydrogen
in a balloon to the contents of the flask. After stirring for 2 h
(800 g, Merck 07734, 0.063-0.20 mm) and eluted with dichlo-
romethane-ethyl acetate (20:1) with 0.5% of acetic acid, to
which increasing amounts of ethyl acetate were added. The
fractions containing thapsigargin were combined and concen-
trated to give 15 g of a viscid oil. A part of the residue (10 g)
was rechromatographed over LiChroprep RP-18 (500 g, Merck
the mixture was diluted with CH
through Celite. The filtrate was concentrated in vacuo to give
). The
2 2
Cl (30 mL) and filtered
(S)-1 (194 mg) as a viscid oil, [R]25
-3.3° (c 0.91, CHCl
D
3
NMR spectra could be superimposed on those of (R)-1 isolated
from the plant material.
1
1
3900, 40-63 µm) and eluted with methanol-water (7:3) with
Ack n ow led gm en t. This work was supported by Apotek-
erfonden of 1992, The Danish Cancer Society, and Carlsberg-
fondet.
% of acetic acid, to which increasing amounts of methanol
1
were added, to give thapsigargin (4.0 g). The H NMR
spectrum contained some unexpected signals. Rechromatog-
raphy over silica gel 60 (75 g) eluted with toluene-ethyl
acetate (9:1) with 1% of acetic acid afforded compound 1 (200
mg, 300 ppm of plant material) and compound 2 (150 mg, 225
ppm of plant material).
Su p p or tin g In for m a tion Ava ila ble: Tabulated NMR spectra of
compounds 5 and 6. This material is available free of charge via the
Internet at http://pubs.acs.org.
Refer en ces a n d Notes
2
-Hydr oxym eth yl-1,4-dioxacycloicosan e-5,20-dion e (1):
(
1) H e´ bert, N.; Beck, A.; Lennox, R. B.; J ust, G. J . Org. Chem. 1992, 57,
2
5
1
viscid oil; [R]
D
3 3
+1.0° (c 0.097, CHCl ); H NMR (CDCl , 300
1
777.
MHz) δ 5.13 (1H, ddd, J ) 3.0, 5.1, 5.7, 6.5 Hz, H-2), 4.38 (1H,
dd, J ) 3.0, 12.3 Hz, H-3), 4.26 (1H, dd, J ) 6.5, 12.3 Hz, H-3′),
(2) Menger, F. M.; Chen, X. Y.; Brocchini, S.; Hopkins, H. P.; Hamilton,
D. J . Am. Chem. Soc. 1993, 115, 6600-6608.
3) Ghosh, S.; Easwaran, K. R. K.; Bhattacharya, S. Tetrahedron Lett.
(
3
1
.75 (1H, dd, J ) 5.7, 12.0 Hz, H-R), 3.72 (1H, dd, J ) 5.1,
2.0 Hz, H-R′), 2.34 and 2.32 (4H, two overlapping t, J ) 7.2
1
996, 37, 5769-5772.
(
4) Poulter, C. D.; Aoki, T.; Daniels, L. J . Am. Chem. Soc. 1988, 110,
2620-2624.
(5) J akobsen, C. M.; Denmeade, S. R.; Isaacs, J . T.; Gady, A.; Olsen, C.
Hz, H-6 and H-19), 1.64 (4H, m, H-7 and H-18), 1.30 (20H, m,
1
3
H-8-H-17); C NMR (CDCl
C-5 and C-20), 72.4 (CH, C-2), 62.9 (CH
4.6 and 34.4 (CH , C-6 and C-19), 28.5 and 28.4 (CH
and C-18), 28.2, 27.9, 27.7, 27.6, 27.5, 27.4, 27.1, 27.0, 25.0,
3
, 75 MHz) δ 174.0 and 173.7 (C,
, C-3), 61.5 (CH , C-R),
, C-7
E.; Christensen, S. B. J . Med. Chem. 2001, 44, 4696-4703.
2
2
(
6) Denmeade, S. R.; J akobsen, C. M.; J anssen, S.; Khan, S. R.; Garrett,
E. S.; Lilja, H.; Christensen, S. B.; Isaacs, J . T. J . Natl. Cancer Inst.
2003, 95, 990-1000.
3
2
2
+
(7) Christensen, S. B.; Andersen, A.; Smitt, U. W. Prog. Chem. Nat. Prod.
and 24.8 (CH
calcd for C19
2
, C-8-C-17); HRFABMS m/z 343.2514 [M + H]
1
997, 71, 129-167.
+
+
(
H
35
O
5
, 343.2484); 365.2328 [M + Na] (calcd for
+
C
19
H
34NaO
5
, 365.2304).
NP049781Y