Daucane phytoestrogens: A structure-activity study

Article abstract of DOI:10.1021/np0201671

The estrogenic activity of a series of analogues of the daucane ester ferutinin (1a) modified at the acyl moiety was investigated in a yeast screen containing the human estrogen receptor α. Rather strict structure - activity relationships were observed. T

Full text of DOI:10.1021/np0201671

1612
J . Nat. Prod. 2002, 65, 1612-1615
Da u ca n e P h ytoestr ogen s: A Str u ctu r e-Activity Stu d y
Giovanni Appendino,*,† Paola Spagliardi,‡ Giancarlo Cravotto,‡ Victoria Pocock,§ and Stuart Milligan*,§
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universita` del Piemonte Orientale,
Viale Ferrucci 33, 28100 Novara, Italy, Dipartimento di Scienza e Tecnologia del Farmaco, Via Giuria 9, 10125 Torino, Italy,
and Endocrinology and Reproductive Research Group, Kings College, SE1 1 UL London, U.K.
Received April 10, 2002
The estrogenic activity of a series of analogues of the daucane ester ferutinin (1a ) modified at the acyl
moiety was investigated in a yeast screen containing the human estrogen receptor R. Rather strict
structure-activity relationships were observed. Thus, while the parent polyol (jaeschkeanadiol, 2a ) was
inactive, the presence of a p-hydroxybenzoyl moiety was necessary for activity in the yeast screen.
Homologation and vinylation were both detrimental for activity, as were methylation of the p-hydroxyl
substituent and the introduction of oxygen functions on the adjacent carbons.
Plants from the genus Ferula (family Umbelliferae) have
a long history of use in medicine, perfumery, and cuisine,1
and their hormonal effects, anecdotically documented in
medicinal and veterinarian practice,2 have been confirmed
by modern animal studies.3 The active constituents are
believed to be daucane alcohols esterified with aromatic
acids, as exemplified by ferutinin (1a ),4 the p-hydroxyben-
zoyl ester of jaeschkeanadiol () ferutinol, 2a ).5 Ferutinin
shows powerful estrogen activity both in binding and in
functional assays6 and has been developed in the former
USSR as a dietary supplement to treat hot flashes and
other menopausal-associated disorders.7 High concentra-
tions of ferutinin have also been reported in the roots of F.
hermonis Boiss. (“Zallouh”),8 a rare Middle-East species
currently being promoted as a natural alternative to
Viagra.9 While structure-activity studies have been per-
formed with various classes of naturally occurring estrogen
mimics,10 there has been no attempt to identify the
structural requirements responsible for the estrogenic
activity of daucane derivatives. This is most surprising,
since the reported affinity of ferutinin for the estrogen
receptors is higher than that of the well-known phytoestro-
gen genistein,11 while a large variety of daucane derivatives
is available from plants and microorganisms, providing
access to a series of diversely functionalized templates.12
We report here the first structure-activity study on the
estrogenic activity of daucane derivatives, taking ferutinin
(1a ) as a lead compound and focusing on its oxygenated
aromatic acyl group. Variation of this moiety underlies the
occurrence of a large variety of jaeschkeanadiol esters in
plants from the genus Ferula.13 Many of these compounds
co-occur with ferutinin, but are difficult to obtain in pure
form by isolation, and were therefore more conveniently
synthesized from the parent diol 2a .
aromatic acids was investigated under a variety of condi-
tions. Reaction with acyl chlorides was sluggish with both
methoxy- and acetoxy-substituted compounds. Several days
and a large excess of reagents were necessary for comple-
tion, as exemplified by the trimethoxybenzoyl derivative
1j () palliferidine15), whose synthesis required prolonged
heating of 2a (7 days at 40 °C) with a large excess (20
equiv) of trimethoxybenzoyl chloride. A more general
synthesis could be achieved directly from acetyl-protected
phenolic acids using a DCC-mediated coupling under forced
conditions (toluene, reflux). Chemoselective aminolysis of
the acetoxy esters with pyrrolidine in dichloromethane was
uneventful, eventually affording the final phenolic esters.
The yield was sometimes poor due to the competitive
formation of jaeschkeanadiol acetate (2b), the result of an
acetyl-transfer reaction. The hindered nature of the sec-
ondary hydroxyl of 2a and its involvement in a strong
intramolecular hydrogen bonding with the tertiary hy-
droxyl presumably underlie the difficulties encountered in
the esterification reaction.16
The modifications pursued on the acyl moiety of ferutinin
were homologation, bis-homologation, and vinylation of the
benzoyl group (1b-d ), as well as methylation, acetylation,
and deletion of the phenolic hydroxyl (1e-g), and the
introduction of further oxygenated functions adjacent to it
(1h -j). Compounds combining modifications at the ipso-,
meta-, and para-carbons of the 4-hydroxybenzoyl moiety
of ferutinin were also prepared (1k -o). Table 1 sum-
marizes the preparation of the library of jaeschkeanadiol
esters and provides a key to the plethora of trivial names
that have been assigned to some of these compounds.
The estrogenic activity of jaeschkeanadiol (2a ) and its
various 6-acyl derivatives was investigated in a yeast
screen containing the human estrogen receptor alpha
(ERR). A number of compounds showed little or no activity,
including jaeschkeanadiol (2a ), jaeschkeanadiol benzoate
(1g), jaeschkeanadiol anisate (1e), and compounds 1i-o.
In contrast, ferutinin (1a ) showed a high estrogenic activity
Resu lts a n d Discu ssion
J aeschkeandiol (2a ) was obtained by hydrolysis of feru-
tinin (1a ), in turn isolated in ca. 1.9% yield from the roots
of the nonpoisonous chemotype of giant fennel (F. commu-
nis L.) from Sardinia.14 The acylation of 2a with oxygenated
* To whom correspondence should be addressed. Tel: +39 011 670 7684
(G.A.); +44 20 7848 6271 (S.M.). Fax:
+ 39 011 670 7687 (G.A.);
+44 20 7848 6280 (S.M.). E-mail: appendin@pharm.unito.it (G.A.);
Stuart.Milligan@kcl.ac.uk (S.M.).
† Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Far-
macologiche.
‡ Dipartimento di Scienza e Tecnologia del Farmaco.
§ Endocrinology and Reproductive Research Group, Kings College.
10.1021/np0201671 CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/18/2002

Daucane Phytoestrogens
J ournal of Natural Products, 2002, Vol. 65, No. 11 1613
Ta ble 1. Synthetic Yields, Trivial Names, and Estrogenic Activity Measured in an Estrogen-Inducible Yeast Assay for Ferutinin (1a )
and Its Analogues 1b-1oa
EC50 relative
to estradiol (%)
R1
R2
R3
X
yieldb
trivial name
ferutinin
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
H
H
H
H
H
H
H
H
H
OMe
H
H
H
OH
OH
OH
OH
OMe
OAc
H
OH
OH
OMe
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
OH
OMe
OMe
OMe
OH
OH
OMe
OMe
0.5
CH2
CH2-CH2
CHdCH (E)
89% (B)
75% (B)
42% (B)
79% (A)
c
69% (A)
7% (B)
10% (B)
78% (A)
57% (B)
48% (B)
66% (B)
29% (B)
51% (B)
<0.0001
<0.0001
<0.0001
<0.0001
0.2
inactive
<0.1
inactive
inactive
inactive
inactive
inactive
inactive
inactive
ferutidin25
fersorin26
teferidine27
akiferidin28
teferin29
palliferidin15
CH2
CH2-CH2
CHdCH (E)
CHdCH (E)
CHdCH (E)
H
OMe
a
b
Compounds failing to produce a response in the yeast bioassay corresponding to 0.2 nM estradiol are marked as inactive. Conditions
A: acyl chloride-pyridine; Conditions B: acid, DCC, DMAP, toluene, room temperature for 1b and 1c; 90 °C for the remaining compounds.
c Prepared by acetylation (Ac2O, pyridine, room temperature) of ferutinin.
(approximately 200-fold lower than that of estradiol),
closely followed by its acetate (1f, Table 1). Significant
estrogenic activity was also detected in compound 1h (<1/
1000 compared to estradiol), and an indication of estrogenic
activity was detected in compounds 1b, 1c, 1d , and 1e at
the highest dose levels, representing, however, an activity
< 1/106 compared to estradiol. Ferutinin (1a ) administered
orally has also been reported to increase uterine weight in
rats,11 and our findings suggest that this compound must
be considered as one of the most active phytoestrogens, on
a par with 8-prenylnaringenin and coumestrol.17
The ER is quite promiscuous in its binding, but a general
requirement for estrogen activity includes an aromatic ring
bearing a hydroxyl which can act both as a hydrogen donor
and a hydrogen acceptor.18 Methylation of the phenolic
hydroxyl significantly decreases the affinity for the ER, but
several antiestrogens such as tamoxifen and clomiphene
remain strong binders despite the lack of a phenolic moiety.
Furthermore, extensive studies on natural products and
xenochemicals have shown that two ring structures sepa-
rated by two carbon atoms (steroidal and synthetic estro-
gens and diphenyl ethanes) have higher binding affinity
than compounds with a single ring structure (e.g., alkyl-
phenols, parabens) or two rings separated by one carbon
atom alone.19 These suggestions have been fully supported
by crystallographic studies of ER-ligand complexes, which
have indicated that the critical spacing of hydrophobic and
hydrogen bond interactions plays a major role in binding
affinity.20 These structural requirements are consistent
with the low or negligible estrogenic activity of the p-O-
methylated analogues of ferutinin, but do not completely
explain the high potency of the parent compound, where
the phenolic moiety is connected by an ester, and not an
ethylene tether, to a cyclic element.
F igu r e 1. Competitive displacement of [2,4,6,7-3H] 17â-estradiol from
isolated ER alpha by 17â-estradiol, ferutinin (1a ), and compound 1f
(fersorin).
tion.21 The relative binding affinity of 1f was considerably
lower (<1% of that of estradiol), suggesting that its strong
activity in the yeast bioassay may reflect the conversion
to a more active metabolite by hydrolysis of the phenolic
acetate. In cotransfection experiments, ferutinin binding
could inhibit the interaction of ERâ with TRAP220, while
the interaction of ERR with this nuclear receptor coacti-
vator remained unaffected.21 The differential effect of
ferutinin (1a ) on ERR and ERâ may have a considerable
significance for its biological activity in vivo. As a subtype-
selective phytoestrogen, ferutinin may thus be useful as a
selective estrogen receptor modulator (SERM), providing
a new opportunity to build on natural product diversity to
develop hormonal agents.
Ferutinin (1a ) strongly interacted with ERR and ERâ
in competitive binding assays (Figures 1 and 2), showing
an affinity approximately 10% of that of estradiol on both
receptors and confirming a recent independent observa-
Taken together, the results of these structure-activity
studies show that the p-hydroxybenzoyl moiety is a key
element of the ferutinin (1a ) pharmacophore and that

1614 J ournal of Natural Products, 2002, Vol. 65, No. 11
Appendino et al.
enzymatic hydrolysis of chlorophenol red â-D-galactopyrano-
side by monitoring the absorbance at 540 nm.30
Recep tor Bin d in g Exp er im en ts. Estrogen receptor bind-
ing activity was studied using recombinant human estrogen
receptors R and â (ERR and ERâ) obtained from PanVera
Corporation (Madison, WI).17 Dilutions of the test compounds
were incubated in 100 µL of buffer (10 mM Trizma preset
crystals (pH 7.5), 10% glycerol, 2 mM D,L-dithiothreitol (DTT),
1 mg/mL bovine serum albumin) with 15 nM [2,4,6,7-3H]-l7â-
estradiol (84.0 Ci/mmol; Amersham Life Science, Amersham,
UK) and ER (1.5 nM). The mixture was incubated overnight
at 4 °C, and free and bound hormone were separated using
100 µL of 50% hydroxylapatite slurry (in 50 mM Tris-HCl, pH
7.4, 1 mM EDTA). After three washes in buffer (ERR: 40 mM
Tris-HCl, pH 7.5, 100 mM KCl, 1 mM EDTA, 1 mM EGTA;
ERâ: 40 mM Tris-HCl, pH 7.5), the slurry was extracted with
two washes of 200 µL of ethanol, and the radioactivity in the
extracts was determined.
Ester ifica tion of J a esch k ea n a d iol. (1) With Acyl Ch lo-
r id es. (Syn th esis of 1j a s a n Exa m p le). To a solution of
jaeschkeanadiol (2a , 256 mg, 1.1 molar equiv) in pyridine-
CH2Cl2 (3:1, 13 mL) were added trimethoxybenzoyl chloride
(4.97 g, 21.5 mmol, 20 molar equiv) and DMAP (ca. 10 mg).
After stirring for 7 days at 40 °C, the reaction was worked up
by dilution with CHCl3 and washing with 1 N HCl, saturated
aqueous NaHCO3, and brine. After drying (Na2SO4) and
removal of the solvent, the residue was purified by column
chromatography (petroleum ether-EtOAc 8:2) to afford 364
mg of 1j (78%).
(2) With Acetyla ted P h en olic Acid s (Syn th esis of 1m
a s a n Exa m p le). To a suspension of diacetylcaffeic acid (183
mg, 0.69 mmol, 2.9 molar equiv) in dry toluene (3 mL) were
sequentially added jaeschkeanadiol (2a , 60 mg; 0.24 mmol),
DCC (137 mg; 0.66 mmol; 2.8 mol), and DMAP (31 mg; 0.25
mmol; 1 molar equiv). The reaction mixture was heated at 90
°C for 3 h and then worked up by dilution with EtOAc and
washing with saturated aqueous NaHCO3 and brine. After
drying (Na2SO4) and removal of the solvent, the residue was
suspended in Et2O and left standing for 3 h at 5 °C. After
filtration of the copious precipitate of dicyclohexylurea and
evaporation of the solvent, the residue was purified by column
chromatography (petroleum ether-EtOAc gradient, from 9:1
to 8:2) to afford 75 mg of the protected ester. The latter was
dissolved in THF (1 mL) and treated with an excess of
pyrrolidine (ca. 250 µL). After stirring at room temperature
for 30 min, the reaction was worked up by dilution with EtOAc,
and the organic phase was sequentially washed with 2 N HCl,
saturated aqueous NaHCO3, and brine. Removal of the solvent
gave 48 mg (66%) of 1m as an amorphous foam.
F igu r e 2. Competitive displacement of [2,4,6,7-3H] 17â-estradiol from
isolated ER beta by 17â-estradiol, ferutinin (1a ), and compound 1f
(fersorin).
daucanes qualify as novel structure templates for estrogen
receptor binding. p-Hydroxybenzoic acid is structurally
related to preservatives used in food, cosmetics, and
pharmaceuticals and has been shown to exert a very weak
estrogenic activity.22 Thus, the spectacular increase in
estrogenic activity observed in ferutinin (1a ) compared to
p-hydroxybenzoic acid must be the result of the esterifi-
cation to a daucane alcohol. Investigations aimed at the
modification of the terpenoid moiety of ferutinin are in
progress and will be reported in due course.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. IR spectra were
recorded on a Shimadzu DR 8001 spectrophotometer. MS (EI,
1
70 eV) were taken on VG 7070 EQ spectrometers. H and 13C
NMR spectra were recorded with a Bruker DRX-300 spec-
trometer (300 and 75 MHz respectively). The solvent signals
(CHCl3/CDCl3 7.27/76.9 ppm) were used as internal reference.
The chemical shifts (δ) are given in ppm, and the coupling
constants (J ) in Hz. Silica gel 60 (70-230 mesh, Merck) was
used for open-column chromatography. Phenolic acids were
acetylated by treatment with Ac2O (5 molar equiv) in pyridine
and were purified by crystallization upon dilution of the
reaction mixture with water or by column chromatography
after extraction of the reaction mixture with EtOAc.23 The
known jaeskeanadiol esters 1d ,24 1e,25 1f,26 1g,27 1h ,281i,29 and
1j15 were identified by comparison of their spectroscopic data
J a esch k ea n a d iol p-h yd r oxyp h en yla ceta te (1b): amor-
phous foam; IR (liquid film) νmax 3375, 1710, 1615, 1597, 1516,
1447, 1379, 1262, 1150, 964 cm-1; 1H NMR (300 MHz, CDCl3)
δ 7.08 (2H, AA′, acyl), 6.72 (2H, BB′, acyl), 5.48 (1H, br s, H-9),
4.92 (1H, dt, J ) 10, 1 Hz, H-6), 3.52 (2H, AB, acyl), 2.35 (1H,
t, J ) 10 Hz, H-7a), 2.23 (1H, dd, J ) 10, 1 Hz, H-7b), 1.79
(3H, br s, H-14), 0.98 (3H, s, H-15), 0.80-0.71 (each 3H, d, J
) 6.7 Hz, H-12, H-13); HREIMS m/z 354.2186 [M - H2O]+ (5)
(calcd for C23H32O4 - H2O, 354.2195).
1
(IR, H NMR) with the reported data.
P la n t Ma t er ia l a n d Isola t ion of J a esk ea n a d iol. F.
communis L. used in this investigation was collected in
Senenghe (Oristano, Sardinia) in March 2000 and was identi-
fied by Prof. Mauro Ballero (Dipartimento di Scienze Botan-
iche, Universita` di Cagliari, Italy). A voucher specimen (612B)
is deposited at the Dipartimento di Scienze Botaniche, Uni-
versita` di Cagliari. Ferutinin (1a ) was isolated from an acetone
extract of F. communis14 and was hydrolyzed to jaeskeanadiol
(2a ) using the protocol developed by Prof. Sukh Dev and co-
workers.5
Deter m in a tion of th e Estr ogen ic Activity in Vitr o. All
compounds were dissolved in ethanol, and a number of serial
dilutions were prepared. Aliquots (20 µL) were added to
individual wells in a 96-well plate, and the solvent was
evaporated. Estrogenic activity was determined using an
estrogen-inducible yeast screen (Saccharomyces cerevisiae)
expressing the human estrogen receptor (ERR) and containing
expression plasmids carrying estrogen-responsive sequences
controlling the reporter gene lac-Z (encoding the enzyme
â-galactosidase). Estrogenic activity was determined from the
J a esch k ea n a d iol p -h yd r oxyp h en ylp r op ion a t e (1c):
amorphous foam; IR (liquid film) νmax 3384, 1709, 1615, 1597,
1
1516, 1265, 1225, 1170, 1152, 830 cm-1; H NMR (300 MHz,
CDCl3) δ 7.04 (2H, AA′, acyl), 6.76 (2H, BB′, acyl), 5.50 (1H,
br s, H-9), 4.95 (1H, dt, J ) 10, 1 Hz, H-6), 2.88 (2H, m, acyl),
2.59 (2H, m, acyl), 2.33 (1H, t, J ) 10 Hz, H-7R), 2.12 (1H, dd,
J ) 10, 1 Hz, H-7â), 1.79 (3H, br s, H-14), 0.99 (3H, s, H-15),
0.87-0.85 (each 3H, d, J ) 6.7, H-12, H-13); HREIMS m/z
368.2339 [M - H2O]+ (12) (calcd for C24H34O4 - H2O, 368.2351).
J a esch k ea n a d iol h om ova n illa te (1k ): amorphous foam;
IR (liquid film) νmax 3420, 1725, 1603, 1516, 1464, 1454, 1433,
1
1273, 1152 cm-1; H NMR (300 MHz, CDCl3) δ 6.85 (1H, d, J
) 8 Hz, acyl), 6.77 (1H, s, acyl), 6.73 (1H, d, J ) 8 Hz, acyl),
5.75 (1H, br s, OH), 5.49 (1H, br s, H-9), 4.95 (1H, dt, J ) 10,
1 Hz, H-6), 3.86 (3H, s, -OCH3), 3.53 (2H, AB, acyl), 2.34 (1H,
t, J ) 10 Hz, H-7R), 2.19 (1H, dd, J ) 10, 1 Hz, H-7â), 1.79
(3H, br s, H-14), 1.00 (3H, s, H-15), 0.79-0.71 (each 3H, d, J

Daucane Phytoestrogens
J ournal of Natural Products, 2002, Vol. 65, No. 11 1615
) 6.7, H-12, H-13); HREIMS m/z 384.2315 [M - H2O]+ (11)
(calcd for C24H34O5 - H2O, 384.2301).
(3) Prakash, A. O.; Pathak, S.; Shaiv, A.; Mathur, R. Phytotherapy Res.
1994, 8, 28-32, and references therein.
(4) Saidkhodzhaev, A. I.; Nikonov, G. K. Khim. Prir. Soedin. 1973, 9,
28-30. Apart from many Ferula species, ferutinin has also been
isolated from Blumea amplectens DC. var. arenaria, a Compositae
plant (Pathak, V. P.; J akupovic, J .; J ain, S.; Bhakuni, D. S. Planta
Med. 1987, 53, 103-104).
(5) Sriraman, M. C.; Nagasampagi, B. A.; Pandey, R. C.; Dev. S.
Tetrahedron 1973, 29, 985-991.
(6) Ignatkov, V. I.; Ahmedhodzjaeva, H. T.; Babichev, V. Farmakol.
Toksikol. 1990, 53, 37-38.
(7) Shakhidoyatov, Kh. M. Proceedings of Medicinal Raw Material and
Phytopreparations for Medicine and Agriculture; Book of Abstracts,
September 29-October 1, 1999, Karaganda, Republic of Kazakstan,
pp 31-34.
(8) Galal, A. Pharmazie 2000, 55, 961-962. (b) Diab, Y.; Dolmazon, R.;
Bessie´re, J .-M. Flavour Fragr. J . 2001, 16, 120-122. (c) Galal, A.
M.; Abopurashed, E. A.; Ross, S. A.; ElSohly, M. A.; Al-Said, M. S.;
El-Feraly, F. S. J . Nat. Prod. 2000, 64, 399-400.
(9) El-Thaher, T. S.; Matalka, K. Z.; Taha, H. A.; Badwan, A. A. Int. J .
Impot. Res. 2001, 13, 247-251. For a report on the booming mar-
ket of “Zallouh”, see: http://europe.cnn.com/HEALTH/9808/12/
natural.viagra/.
(10) Davis, S. R.; Dalais, F. S.; Simpson, E. R.; Murkies, A. L. Recent Prog.
Horm. Res 1999, 54, 185-211.
(11) Singh, M. M.; Agnihotri, A.; Garg, S. N.; Agarwal, S. K.; Gupta, D.
N.; Keshri, G.; Kamboj, V. P. Planta Med. 1988, 492-494.
(12) Ghisalberti, E. L. Phytochemistry 1994, 37, 597-623.
(13) Gonza´lez, A. G.; Bermejo Barrera, J . In Progress in the Chemistry of
Organic Natural Products; Herz, W., Kirby, G. W., Moore, R. E.,
Steglich, W., Tamm, Ch., Eds.; Springer-Verlag: New York, 1995;
Vol. 64, pp 1-92.
J aesch kean adiol dih ydr ocaffeate (1l): amorphous foam;
IR (liquid film) νmax 3402, 1709, 1607, 1520, 1449, 1283, 1192,
1
1152, 1111 cm-1; H NMR (300 MHz, CDCl3) δ 6.75 (1H, d, J
) 8 Hz, acyl), 6.73 (1H, s, acyl), 6.60 (1H, d, J ) 8 Hz, acyl),
6.41 (1H, br s, OH), 6.22 (1H, br s, OH), 5.49 (1H, br s, H-9),
4.94 (1H, dt, J ) 10, 1 Hz, H-6), 2.85 (2H, m, acyl), 2.58 (2H,
m, acyl), 2.33 (1H, t, J ) 10 Hz, H-7R), 2.12 (1H, dd, J ) 10,
1 Hz, H-7â), 1.79 (3H, br s, H-14), 0.98 (3H, s, H-15), 0.87-
0.82 (each 3H, d, J ) 6.7, H-12, H-13); HREIMS m/z 402.2400
[M - H2O]+ (6) (calcd for C24H34O5 - H2O, 402.2406).
J a esch k ea n a d iol ca ffea te (1m ): amorphous foam; IR
(liquid film) νmax 3370, 1682, 1632, 1603, 1516, 1447, 1383,
1275, 1181, 1115 1111 cm-1; 1H NMR (300 MHz, CDCl3) δ 8.67
(1H, br s, OH), 7.68 (1H, d, J ) 16 Hz, acyl), 7.17 (1H, s, acyl),
7.01 (1H, d, J ) 8 Hz, acyl), 6.90 (1H, d, J ) 8 Hz, acyl), 6.47
(1H, d, J ) 16 Hz, acyl), 5.56 (1H, br s, H-9), 5.13 (1H, dt, J )
10, 1 Hz, H-6), 2.64 (1H, t, J ) 10 Hz, H-7a), 2.27 (1H, dd, J
) 10, 1 Hz, H-7â), 1.84 (3H, br s, H-14), 1.10 (3H, s, H-15),
0.93-0.91 (each 3H, d, J ) 6.7, H-12, H-13); HREIMS m/z
382.2154 [M - H2O]+ (12) (calcd for C24H32O5 - H2O, 382.2144).
J a esch k ea n a d iol fer u la te (1n ): amorphous foam; IR
(liquid film) νmax 3345, 1688, 1630, 1592, 1514, 1269, 1175,
1159, 1121, 1034 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.63 (1H,
d, J ) 16 Hz, acyl), 7.10 (1H, dd, J ) 8, 1 Hz, acyl), 7.04 (1H,
s, acyl), 6.94 (1H, d, J ) 8 Hz, acyl), 6.25 (1H, d, J ) 16 Hz,
acyl), 6.09 (1H, s, OH), 5.55 (1H, br s, H-9), 5.15 (1H, dt, J )
10, 1 Hz, H-6), 3.95 (3H, s, -OCH3), 2.50 (1H, t, J ) 10 Hz,
H-7R), 2.28 (1H, dd, J ) 10, 1 Hz, H-7â), 1.83 (3H, br s, H-14),
1.13 (3H, s, H-15), 0.95-0.92 (each 3H, d, J ) 6.7, H-12, H-13);
HREIMS m/z 396.2309 [M - H2O]+ (3) (calcd for C25H34O5 -
H2O, 396.2301).
(14) Appendino, G.; Cravotto, G.; Sterner, O.; Ballero, M. J . Nat. Prod.
1991, 64, 393-395.
(15) Kushmuradov, A. Y.; Saidkhodzhaev, A. I.; Kadyrov, A. S. Khim. Pri.
Soedin. 1981, 400.
(16) This intramolecular hydrogen bonding was detected in the X-ray
analysis of the 8,9-dichloromethylene derivative of jaeschkeanadiol,
the 4-hydroxyl acting as a donor and the 6-hydroxyl as an acceptor
(Garg, S. N.; Agarwal, S. K.; Fidelis, K.; Hossain, M. B.; Van Der
Helm, D. J . Nat. Prod. 1993, 56, 539-544). The remarkably easy
formation of a six-membered cyclic carbonate by reaction of jaesch-
keanadiol (2a ) with phosgene5 or carbonyldiimidazole (Tolstikov, G.
A.; Akbutina, F. A.; Dembitskii, A. D.; Valeev, F. A.; Miftakhov, M.
S. Zh. Org. Khim. 1992, 28, 2081-2090) supports the close spatial
relationship between the two hydroxyls of jaesekanadiol.
(17) Milligan, S.; Kalita, J .; Pocock, V.; Heyerick, A.; De Cooman, L.; Rong,
H.; De Keukeleire, D. Reproduction 2002, 123, 235-242.
(18) Anstead, G. M.; Carlson, K. E.; Katzenellenbogen, J . A. Steroids 1997,
62, 268-303.
J a esch k ea n a d iol sin a p in a te (1o): amorphous foam; IR
(liquid film) νmax 3465, 1697, 1632, 1601, 1514, 1283, 1258,
1175, 1154, 1117 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.61 (1H,
d, J ) 16 Hz, acyl), 6.85 (2H, s, acyl), 6.26 (1H, d, J ) 16 Hz,
acyl), 5.84 (1H, s, OH), 5.55 (1H, br s, H-9), 5.16 (1H, dt, J )
10, 1 Hz, H-6), 3.94 (3H, s, -OCH3), 2.51 (1H, t, J ) 10 Hz,
H-7a), 2.23 (1H, dd, J ) 10, 1 Hz, H-7â), 1.83 (3H, br s, H-14),
1.08 (3H, s, H-15); 0.94-0.92 (each 3H, d, J ) 6.7, H-12, H-13);
HREIMS m/z 426.2411 [M - H2O]+ (9) (calcd for C26H36O6 -
H2O, 426.2406).
(19) Blair, R. M.; Fang, H.; Branham, W. S.; Hass, B. S.; Dial, S. L.;
Moland, C. L.; Tong, W.; Shi, L.; Perkins, R.; Sheehan, D. M. Toxicol.
Sci. 2000, 54, 138-153.
(20) Brzozowski, A. M.; Pike, A. C. W.; Dauter, Z.; Hubbard, R. E.; Bonn,
T.; Engstrom, O.; Ohman, L.; Greene, G. L.; Gustafsson, J . A.;
Carlquist, M. Nature 1997, 389, 753-758.
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Kayama F. Biochem. Biophys. Res. Commun. 2002, 29, 354-60.
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Martinez, M.; Hernandez, A.; Rubio-Poo, C.; Lara, B. C.; Valenzuela,
F. Environ. Res. 1997, 75, 130-134.
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526.
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106.
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83, 2249-2252.
Ack n ow led gm en t. We are grateful to Prof. Mauro Ballero
(Universita` di Cagliari, Italy) for a generous gift of the
nonpoisonous chemotype of giant fennel from Sardinia, to Prof.
J . Sumpter (Brunel University, Uxbridge, UK) for providing
the estrogen-responsive yeast screen, and to Prof. D. De
Keukeleire (University of Ghent, Ghent, Belgium) for useful
discussions. Financial support from EU (Contract ERB FAIR
CT 1781) and MURST (Fondi ex-40%, Progetto Sostanze
Naturali e Analoghi Sintetici con Attivita` Antitumorale) is
gratefully acknowledged.
Su p p or tin g In for m a tion Ava ila ble: 13C NMR data for the un-
natural jaeskeanadiol esters 1b, 1c, 1k , 1l, 1m , 1n , and 1o. This ma-
terial is available free of charge via the Internet at http://pubs.acs.org.
Refer en ces a n d Notes
(1) Appendino, G. The Toxins of Ferula communis. In Virtual Activity,
Real Pharmacology; Verotta, L., Ed.; Research Signpost: Trivandrum;
1997; pp 1-15.
(2) For a historical account of the use of plants from the genus Ferula
as antifertility agents, see: Riddle, J . M. Eve’s Herbs; Harvard
University Press: Cambridge, 1997; pp 44-46.
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