Acid-induced rearrangement of epoxygermacranolides: Synthesis of furanoheliangolides and cadinanes from nobilin

Article abstract of DOI:10.3390/molecules22122252

The acid-induced rearrangement of three epoxyderivatives of nobilin 1, the most abundant sesquiterpene lactone in Anthemis nobilis flowers, was investigated. From the 1, 10-epoxyderivative 2, furanoheliangolide 5 was obtained, while the 4, 5-epoxy group o

Full text of DOI:10.3390/molecules22122252

molecules
Article
Acid-Induced Rearrangement of
Epoxygermacranolides: Synthesis of
Furanoheliangolides and Cadinanes from Nobilin
Maria De Mieri 1,*, Martin Smieško 2 ID , Isidor Ismajili 1, Marcel Kaiser 3,4 and
Matthias Hamburger 1,
*
ID
1
Pharmaceutical Biology, Pharmazentrum, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland;
isidor.ismajili@gmail.com
Molecular Modeling, Pharmazentrum, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland;
martin.smiesko@unibas.ch
Department of Medical Parasitology & Infection Biology, Swiss Tropical and Public Health Institute,
Socinstrasse 57, 4000 Basel, Switzerland; marcel.kaiser@swisstph.ch
University of Basel, Petersplatz 1, 4001 Basel, Switzerland
2
3
4
*
Correspondence: mariademieri@gmail.com (M.D.M.); Matthias.Hamburger@unibas.ch (M.H.)
Received: 16 November 2017; Accepted: 13 December 2017; Published: 18 December 2017
Abstract: The acid-induced rearrangement of three epoxyderivatives of nobilin
sesquiterpene lactone in Anthemis nobilis flowers, was investigated. From the 1,10-epoxyderivative
furanoheliangolide was obtained, while the 4,5-epoxy group of did not react. Conversely, when
1, the most abundant
2,
5
3
the 3-hydroxy function of nobilin was acetylated (12), the 4,5-epoxy derivative did cyclize into
cadinanes (15 and 16) under Lewis acid catalysis. The reactivity of the 4,5- and 1,10-epoxy derivatives
of nobilin (2 and 3) was compared with that of parthenolide, and rationalized on the basis of quantum
chemical calculations. All isolated reaction products were fully characterized by spectroscopic and
computational methods, and their in vitro anti-protozoal activity was evaluated. The paper could
provide new insights into the biosynthesis of this class of natural products.
Keywords: sesquiterpene lactones; epoxygermacranolides; acid-catalyzed rearrangement; mechanism
of reaction; anti-protozoal activity
1. Introduction
Sesquiterpene lactones are the largest group of secondary metabolites, comprising more than
10,000 structures known up to now. They possess high structural diversity and are chemotaxonomic
markers of the Asteraceae and Compositae families [1]. Germacrene-type sesquiterpenoids possess
a cyclodecadiene ring in their scaffold, and are considered key precursors in the biosynthesis of other
natural sesquiterpenes. The two endocyclic double bonds, found in four possible geometrical isomers,
are responsible for the high reactivity of the cyclodecadiene moiety, which undergoes cyclization even
under mild conditions [
and biomimetic synthesis [
the spatial rearrangement of the cyclodecadiene ring and, therefore, on the stereochemistry of the
diene. We recently reported on the spontaneous rearrangement of nobilin , an E,Z-germacranolide
isolated from the flowers of Roman chamomile (Anthemis nobilis) in aqueous media under physiological
conditions [ ]. We then investigated the rearrangement of in aqueous and organic acidic media and
found a transformation into various cadinane derivatives [ ]. In the present study we investigated the
rearrangement of epoxy derivatives of nobilin ( ) under acidic conditions. We hypothesized that
the presence of an epoxy group, acting as a conformational anchor, would strongly affect the outcome of
2]. Therefore, germacrenes are ideal starting points for diversity-oriented
3–6]. It is well established that the outcome of the cyclization depends on
1
7
1
8
2
–4
1
Molecules 2017, 22, 2252; doi:10.3390/molecules22122252
www.mdpi.com/journal/molecules

Molecules 2017, 22, 2252
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the cyclization [
in feverfew (Parthenium integrifolium), which undergoes transannular cyclization to a guianolide
skeleton [10 12]. We here compare the reactivity of nobilin ( ) and of its epoxy derivatives and
9]. This has previously been shown for parthenolide, the major sesquiterpene lactone
–
1
2
3
with that of parthenolide, and rationalize the outcome on the basis of conformational analysis and
computational studies.
2. Results and Discussion
2.1. Chemical Studies
Nobilin
and were prepared according to reported procedures [13]. Treatment of nobilin
meta-chloroperoxybenzoic acid (m-CPBA) in dichlomethane afforded compounds in a 10:1:0.5 ratio.
showed identical spectroscopic data to those reported for the natural compound [14].
was here obtained for the first time by the Sharpless epoxidation method (Scheme 1) [15].
was inferred via J couplings and NOEs. The 3J
1
was isolated in large quantities from the Anthemis nobilis flower cones. Epoxides
2
3
1
with
2–4
Epoxide
Epoxide
2
3
The beta stereochemistry of the 4,5-epoxy group of
3
H-5/H-6 value of 9.7 Hz was indicative of the anti-diaxial orientation of H-5 and H-6, and key NOEs
between H-6/H-8 and H-6/H3-14 supported the cofacial orientation of H-6, H-8, and H3-14. Similarly,
key NOEs between H-5/H-7, H-5/H3-15, and H-1/H3-15 showed that H-7, H3-15, and H-1 were on
the same molecular plane, in agreement with the optimized structure of 3.
Scheme 1. Structures of nobilin
1 and epoxy derivatives 2–4. The carbon numbering follows the
semi-systematic nomenclature of sesquiterpenes.
We began our study by treating compound
that has been extensively used to induce transannular cyclization in germacradiene derivatives [
After 1 h at room temperature, compound (50%) and an inseparable mixture of and in a 3:2 ratio
(10%) were obtained (Scheme 2). Compound had the same molecular formula and degree of
unsaturation as
(molecular ion at m/z 385.1632 [M + Na]+, calculated for C20H26NaO6: 385.1622)
by analysis of its 13C-NMR and HRESIMS spectra. Analysis of COSY and HMBC spectra revealed
that the fragment C4–C9 of was as in the starting material. A significant downfield shift for C-1 and
C-10 (δC = 77.6, C-1; δC = 87.3, C-10) compared to those in δC = 60.7, C-1; δC = 57.7, C-10) suggested
that a rearrangement of the epoxy group has occurred, although both carbons still bore hydroxyl
groups. The downfield shift of C-3 (δC = 81.7 in vs. δC = 71.2 in ), along with the long-range HMBC
2
with p-toluensulfonic acid (p-TsOH
·
H2O), a mild acid
8,
16].
5
6
7
5
2
5
2
(
5
2

Molecules 2017, 22, 2252
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correlation from H-3 to C-10, suggested the presence of an ether bridge between carbons C-3 and
C-10. Hence, was a furanoheliangolide. The beta stereochemistry of the epoxy bridge, inferred on
the basis of geometrical and mechanistic considerations, was confirmed through key NOEs between
5
H-1/H-3, H-3/H-6, H3-14/H-8, and H3-14/H-6 that were in agreement with the 3D structure of
(Figures S22 and S23).
5
Scheme 2. Reactivity of 2 with acids.
Compounds 6 and 7 were obtained as an inseparable mixture in a ratio of 3:1. For both compounds,
a [M + Na]+ ion at m/z 385.1632 was observed, corresponding to a molecular formula of C20H26NaO6
(calculated for C20H26NaO6: 385.1622). Hence, the two molecules were isomers. Close inspection of
the NMR data (Tables 1 and 2) revealed that in both compounds the methylene group at C-9 had been
replaced by vinylic moieties (δH = 5.18 and 5.30 for
6 and 7, respectively), thereby suggesting that the
opening of the oxirane and subsequent formation of an allylic carbon had occurred. The differing
chemical shifts of the vinylic protons were attributed to the E- and Z-stereochemistry of the newly
formed double bond, respectively. The strong NOESY contact between H-9 and H-1 (δH = 5.18 and
4.05, respectively), and between H3-14 and H-6 (δH = 1.95 and 6.00, respectively), in the major isomer
6
of the mixture were in favor of a trans-oriented double bond. For the minor compound of the mixture,
7
a strong NOESY cross peak between H-9 and H3-14 (δH = 5.30 and 1.80, respectively) supported the cis
configuration of the double bond at C-9/C-10 (Figure S28). Interestingly, the C-1 epimer of compound
7 has recently been isolated from Anthemis nobilis [17].
A similar rearrangement to a furanoheliangolide was observed after treatment of
2
with BF3 Et2O
·
in anhydrous conditions, and
Surprisingly, epoxy derivative
by TLC and NMR (Figure S63).
5
3
was obtained as the major reaction product (50%) (Scheme 2).
did not react under the above reaction conditions, as confirmed
For further confirmation of the higher reactivity of the 1,10-epoxyde compared to the 4,5-epoxide,
the bisepoxy derivative was submitted to the same acidic treatment (Scheme 3). Similar to , treatment
of afforded C-3-C-10 furanoheliangolide derivatives (HRESIMS m/z 401.1591
4
2
4
8–10. In compound 8
[M + Na]+, calculated for C20H26NaO7 401.1571), which was obtained as a major reaction product
by treatment with both acid catalysts, the 4,5-epoxy group did not react. Conversely, the 4,5-epoxide
underwent nucleophilic addition of the catalyst and elimination in compounds
9 and 10, respectively.
Compound
9
, showed a molecular formula of C27H34O10S (HRESIMS m/z 573.1786 [M + Na]+,
1
calculated for C27H34NaO10S: 573.1765). In the H-NMR spectrum, resonances characteristic of
an AA0BB0 aromatic system were present (δH = 7.41 and 7.76 pseudo d, J = 8.3 Hz, 2 protons
each), together with an additional methyl group attached to an aromatic ring (δH = 2.45, s, 3H).
These spectroscopic data, together with the sulphonyl group indicated by the molecular formula,
showed that the p-toluensulphonate group has reacted with
located at C-5, as indicated by the downfield shift of H-5 (δH = 4.79 and δC = 89.3 in
4
. The site of the nucleophilic attach was
vs. δH = 2.85 and
9

Molecules 2017, 22, 2252
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3
δC = 63.4 in
of ≤1 Hz.
4), while its orientation was established as alpha on the basis of the JH-5/H-6 value
Scheme 3. Reactivity of 4 with acids.
Table 1. 1H-NMR spectroscopic data for compounds
δ in ppm).
5–
10 (CD3OD for 5–9, CDCl3 for 10; 500.13 MHz;
9 b
(mult, J in Hz)
δ
5 δ
6 δ
7 δ
8 δ
10 δ
H
(mult, J in Hz)
H
H
H
H
H
Position
(mult, J in Hz)
(mult, J in Hz)
(mult, J in Hz)
(mult, J in Hz)
1
3.85 (br d, 4.6)
4.05 (dd, 10.6, 6.5) 4.96 (dd, 10.2, 3.0)
3.90 (d, 4.3)
3.96 (dd, 5.2, 1.4)
6.22 (d, 5.7)
2.03 (ddd, 14.5,
1.82 (ddd, 13.2,
6.2, 1.4)
2.41 (ddd, 13.2,
11.4, 5.2)
1.94 a
2.12 (dd, 13.8, 6.8)
2.44 (ddd, 13.8,
11.0, 4.3)
3.8, 3.0)
2
2.32–2.20 a
2.36 (ddd, 14.5,
2.19 (ddd, 14.5,
6.6, 4.5)
5.89 (d, 5.7)
10.2, 3.7)
3
4
5
4.79 (dd, 8.6, 8.3)
4.49 a
-
4.49 a
4.63 (dd, 11.0, 6.8) 4.22 (dd, 11.4, 6.2)
-
-
-
-
-
-
5.26 (dq, 7.2, 1.5)
5.27 (dq, 10.5, 1.5) 5.39 (dq, 10.5, 1.5)
3.06 (d, 7.3)
4.79 (br s)
5.48 (dq, 5.8, 1.2)
5.95 (ddq, 5.8,
5.2, 1.2)
6
7
8
6.14 a
6.00 (dd, 10.5, 9.5) 6.38 (dd, 10.5, 9.2)
5.25 (dd, 7.3, 5.5)
5.63 (br d, 5.7)
3.31 (m)
3.08 (dddd, 9.0,
7.5, 3.0, 3.0)
3.09 (dddd, 9.5,
9.5, 3.5, 3.0)
3.02 (dddd, 9.5,
9.2, 3.5, 3.0)
3.35 (dddd, 9.0,
5.5, 2.6, 2.5)
3.26 (dddd, 10.5,
5.2, 2.8, 2.5)
5.06 (ddd, 10.0,
6.9, 1.2)
5.17 (ddd, 10.5,
5.0, 3.2)
5.33 (dd, 9.5, 9.0)
5.57 (dd, 10.2, 9.5) 6.00 (dd, 10.5, 9.5)
5.00 (dd, 9.8, 9.0)
1.87 a
2.05 (br dd,
14.6, 9.8)
β
1.91 a
2.13
(dd, 14.3, 10.3)
2.09 (dd, 14.8, 9.5)
1.70 (d, 14.8)
2.14 (dd, 15.4, 5.0)
2.22 (dd, 15.4, 3.2)
9
5.18 (dq, 10.2, 1.5) 5.30 (dq, 10.5, 1.5)
10
11
12
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
5.65 (dd, 3.0, 0.7)
6.17 a
5.71 (d, 3.0)
6.16 (d, 3.5)
5.85 (d, 3.0)
6.25 (d, 3.5)
5.74 (br d, 2.5)
6.23 (br d, 2.8)
5.41 (br d, 2.5)
5.86 (br d, 2.8)
5.89 (br d, 2.5)
6.22 br d, 2.8)
13
14
15
1.49 (s)
1.66 (br s)
-
1.95 (d, 1.5)
1.76 (d, 1.5)
-
1.80 (d, 1.5)
1.79 (d, 1.5)
-
1.37 (br s)
1.33 (s)
1.18 (br s)
-
1.53 (s)
1.73 (t, 1.2)
-
1.27 (s)
0
1
-
0
2
-
-
-
-
-
-
0
3
6.17 (qq, 7.0, 1.5)
1.96 (dq, 7.0, 1.5)
1.89 (quint, 1.5)
6.20 (qq, 7.0, 1.5)
1.95 (dq 7.0, 1.5)
1.89 (quint, 1.5)
6.20 (qq, 7.0, 1.5)
1.98 (dq 7.0, 1.5)
1.91 (quint, 1.5)
6.16 (qq, 7.0, 1.5)
1.94 (dq 7.0, 1.5)
1.88 (quint, 1.5)
6.15 (br q, 7.2)
1.91 (br d, 7.2)
1.84 (quint, 1.5)
6.16 (br q, 7.2)
2.00 (br d, 7.2)
1.91 (quint, 1.5)
0
4
0
5
a
b
Overlapped signals; p-toluenesulphonate group: H-2, H-20 δH = 7.76 pseudo d, J = 8.3 Hz; H-3, H-30 δH
=
7.41 pseudo d, J = 8.3 Hz; -CH3 δH = 2.45, s.

Molecules 2017, 22, 2252
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Table 2. 13C-NMR spectroscopic data for compounds
5
–8
(CD3OD for 5–9, CDCl3 for 10; 125.77 MHz;
δ in ppm).
a
c
a
Position
5 δC
6 δC
7 δC
8 δC
9 δC
10 δC
1
2
3
77.6, CH
41.8, CH2
81.7, CH
141.9, C
76.2, CH
38.9, CH2
74.1, CH
142.2, C
69.4, CH
37.0, CH2
74.9, CH
145.9, C
77.9, CH
39.0, CH2
78.6, CH
66.2, C
78.8, CH
36.6, CH2
85.3, CH
75.2, C
139.3, CH
128.3, CH
110.4, C
4
137.8, C
5
6
7
8
124.0, CH
78.5, CH
52.2, CH
74.1, CH
46.8, CH2
87.3, C
139.0, C
171.8, C
124.8, CH2
19.6, CH3
20.2, CH3
167.9, C
126.7, CH
76.8, CH
50.9, CH
74.0, CH
126.8, CH
143.2, C
140.1, C
172.0, C
121.8, CH2
11.7, CH3
22.8, CH3
168.2, C
125.5, CH
78.0, CH
52.1, CH
72.5, CH
121.9, CH
144.1, C
140.0, C
172.0, C
121.8, CH2
19.5, CH3
23.6, CH3
168.2, C
68.7, CH
82.7, CH
49.09, CH
73.3, CH
49.15, CH2
87.1, C
137.2, C
171.7, C
127.0, CH2
20.7, CH3
20.5 b, CH3
168.0, C
89.3, CH
82.5, CH
48.0, CH
74.0, CH
49.3, CH2
85.8, C
137.1, C
171.3, C
125.5, CH2
22.6, CH3
24.2, CH3
168.3, C
129.6, CH
76.4, CH
48.2, CH
71.5, CH
42.9, CH2
87.3, C
137.7, C
169.6, C
123.8, CH2
28.0, CH3
20.2, CH3
167.4, C
9
10
11
12
13
14
15
0
1
2
3
4
0
128.7, C
128.7, C
128.6, C
128.7, C
127.0, C
126.2, C
0
140.4, CH
16.1, CH3
20.7, CH3
139.9, CH
16.0, CH3
20.8, CH3
140.0, CH
16.0, CH3
20.9, CH3
14.03, CH
16.0, CH3
20.6 b, CH3
140.3, CH
16.0, CH3
20.5, CH3
139.4, CH
15.7, CH3
20.6, CH3
0
0
5
a Extracted from 1H-13C 2D inverse detected experiments due to low amount of sample; b interchangeable within the
same column; c p-toluensulphonate group: C-1 (134.7), C-2/C-20 (129.0), C-3/C-30 (131.4), C-4 (147.2), -CH3 (21.6).
Compound 10 showed a molecular formula of C20H24NaO6 (HRESIMS m/z 383.1500 [M + Na]+,
calculated for C20H24NaO6: 383.1465) with nine degrees of hydrogen deficiency, which, compared to
indicated the loss of an oxygen group and the formation of an additional double bond. The NMR data
revealed diagnostic differences in comparison to , including three olefinic protons in the low field
4,
4
region, two of which showed 3J coupling (δH = 5.48 dq, J = 5.8 and 1.2 Hz, δH = 5.89 d, J = 5.7 Hz, and
δH = 6.22 d, J = 5.7 Hz), absence of oxygenated protons at C-1, C-3 and C-5, absence of the methylene
group attached at C-2, and the presence of a quaternary carbon resonating at δC = 110.4, diagnostic
of a hemiacetal group. The latter carbon resonance exhibited HMBC correlations with the 3J coupled
olefin and the olefinic methyl group. All this spectroscopic evidence suggested that a dihydrofurane
had been formed via elimination of the hydroxyl group at C-1, and that the 4(5)-epoxide had rearranged
into an allylic hemiacetal. A speculative mechanism for the latter transformation would start with
the opening of the 4,5-epoxy group at C-4, subsequent formation of an allylic alcohol (double bond
between C-3 and C-4 and hydroxyl group at C-5), followed by transposition of the hydroxyl group to
C-3 and of the double bond between C-4 and C-5 [18,19]. The cis stereochemistry of the C-4/C-5 double
bond was assigned via the key NOE between H-5 and H3-15. The 8-epimer of compound 10 has been
previously isolated as a natural product from the sunflower cultivar cv. Peredovick [20].
Given that the postulated reactivity of the 1(10)-epoxy group towards a transannular cyclization
was precluded by the high reactivity of the hydroxyl group at C-3, we acetylated this moiety of
epoxy derivatives
a transannular cyclization.
The reaction of 11 with p-TsOH
in 10% and 5% yield, respectively (Scheme 4). Compound 13 (HRESIMS m/z 427.1749 [M + Na]+,
calculated for C22H28NaO7: 427.1727) showed NMR data virtually superimposable to those of , with
2 and 3 in order to prevent the formation of the furane ring and to facilitate
·
H2O proceeded slowly and afforded compounds 13 and 14
6
the difference of an additional 3-acetoxy moiety. The compound was isolated in higher yield (40%)
using BF3·Et2O as acid catalyst.

Molecules 2017, 22, 2252
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Scheme 4. Reactivity of 11 and 12 with acids.
Compound 14 showed the same molecular formula as 13 and was a positional isomer of the latter.
Compared to 13, the NMR spectra indicated the absence of a methyl group at C-10 and of an olefinic
proton at C-9, together with the appearance of an exocyclic methylene group (δH = 5.48 and 5.51, br s,
δC = 116.5). Hence, the position of the unsaturation was at C-10/C-14.
As for
3, derivative 12 was recovered unchanged after stirring at room temperature for 12 h in the
presence of p-TsOH
·
H2O. Conversely, treatment of 12 with BF3 Et2O yielded cadinane acids 15 and 16.
·
Compound 15 showed a molecular formula of C20H24O5 (HRESIMS m/z 367.1530 [M + Na]+; calculated
for C20H24NaO5: 367.1516). Close inspection of the NMR data revealed that 15 was the 3-hydroxy
derivative of a cadinane that had been previously obtained by a reaction of nobilin with acids, whereby
the transannular cyclization of the 1(10) double bond was facilitated by the alkyl–oxygen cleavage of
the lactone ring. The reaction led to the formation of a single bond, C-5/C-10, which transformed the
cyclodecadiene into a decaline ring system [7,8].
Compound 16 had a molecular formula of C15H14O2 (HRESIMS m/z 249.0899 [M + Na]+,
calculated for C15H14NaO2: 249.0886). Its 1H-NMR spectrum displayed resonances of seven olefinic
protons and two methyl groups (Tables 3 and 4), indicative of the loss of the angelate and hydroxyl
substituents, and the formation of a naphthalene ring bearing the acrylate group (Scheme 4).

Molecules 2017, 22, 2252
7 of 17
1
Table 3. H-NMR spectroscopic data for compounds 13–16 (CD3OD; 500.13 MHz; δ in ppm).
Position
13 δ (mult, J in Hz)
14 δ (mult, J in Hz)
15 δ (mult, J in Hz)
16 δ (mult, J in Hz)
H
H
H
H
1
4.11 (dd, 10.6, 6.5)
4.01 (dd, 11.7, 3.0)
-
-
1.91 a
2.17 a (ddd, 15.0, 5.0, 3.0)
2.47 (ddd, 15.0, 11.7, 2.5)
2
6.65 (s)
7.91 (d, 8.5)
2.43 (ddd, 15.5, 6.5, 6.0)
3
4
5
6
5.49 (br d, 6.0)
5.30 a
-
-
-
7.34 (br d, 8.5)
-
-
5.32 (dq, 8.5, 1.2)
5.32 (dq, 10.3, 1.2)
5.87 (dd, 10.3, 2.0)
3.20 (dddd, 10.7, 2.0, 1.7,
1.5)
6.65 (s)
-
7.58 (br s)
-
5.28 a
3.17 (dddd, 9.5, 8.5, 3.5,
3.0)
5.65 (dd, 10.5, 9.5)
7
8
3.97 (d, 5.8)
-
5.03 (td, 10.5, 4.0)
5.50 (ddd, 7.5, 5.7, 2.7)
7.19 (d, 7.2)
2.49 a
2.90 (br dd, 13.6, 4.2)
1.80 a
9
5.24 (dq, 10.5, 1.2)
7.20 (d, 7.2)
2.00 (ddd, 13.0, 7.5, 6.0)
10
11
12
-
-
-
-
-
-
3.97 (sextet, 7.0)
-
-
-
-
-
5.74 (d, 3.0)
6.17 (d, 3.5)
5.75 (d, 1.5)
6.23 (d, 1.7)
5.31 (br s)
6.33 (br s)
5.74 (d, 1.8)
6.51 (d, 1.8)
13
5.48 (br s)
5.51 (br s)
14
15
1.97 (d, 1.2)
1.34 (d, 7.0)
2.65 (s)
1.85 (d, 1.2)
1.85 (d, 1.2)
2.10 (s)
2.46 (br s)
0
1
2
3
4
-
-
-
-
-
-
-
-
-
-
-
-
-
0
0
6.19 (qq, 7.0, 1.5)
1.99 (dq, 7.0, 1.5)
1.92 (quint, 1.5)
-
6.15 (qq, 7.0, 1.5)
1.96 (dq, 7.0, 1.5)
1.90 (quint, 1.5)
-
6.03 (qq, 7.0, 1.5)
1.81 (dq, 7.0, 1.5)
1.79 (quint, 1.5)
0
0
5
1”
2”
-
-
2.02, (s)
2.04, (s)
a
Overlapped signals.
Table 4. 13C-NMR spectroscopic data for compounds 13–16 (CD3OD; 125.77 MHz; δ in ppm).
a
a
Position
13 δC
14 δC
15 a δC
16 a δC
1
2
3
4
5
6
7
8
9
75.4, CH
35.2, CH2
74.6, CH
137.9, C
127.0, CH
76.0, CH
50.0, CH
72.8, CH
127.6, CH
141.7, C
139.0, C
170.8, C
121.3, CH2
11.1, CH3
22.2, CH3
167.6, C
70.6, CH
34.5, CH2
73.6, CH
139.4, C
124.8, CH
75.0, CH
49.1, CH
73.6, CH
39.8, CH2
144.4, C
135.2, C
170.0, C
125.7, CH2
116.5, CH2
21.7, CH3
166.8, C
140.5, C
113.9, CH
155.0, C
124.0, C
132.0, CH
126.6, C
48.3, CH
71.9, CH
33.6, CH2
31.5, CH
145.1, C
170.1, C
129.1, CH2
22.9, CH3
15.6, CH3
169.4, C
129.5, C
138.8, CH
15.4, CH3
20.5, CH3
-
132.5, C
125.0, CH
128.4, CH
136.4, C
126.1, CH
128.6, CH
135.60, C
127.4, CH
126.0, CH
135.4, C
145.5, C
171.8, C
127.6, CH2
19.4, CH3
21.7, CH3
-
10
11
12
13
14
15
0
1
2
3
4
0
128.0, C
127.2, C
-
-
-
-
0
139.5, CH
15.6, CH3
20.3, CH3
169.2, C
138.5, CH
14.6, CH3
19.0, CH3
170.0, C
0
0
5
1”
-
2”
20.9, CH3
19.7, CH3
-
-
a
Extracted from 1H-13C 2D inverse detected experiments due to low amount of sample.

Molecules 2017, 22, 2252
8 of 17
2.2. Computational Studies
In order to rationalize the differing reactivity of epoxides
2
and
3
(Figures 1 and 2, respectively)
and compare it with that of parthenolide (Scheme 5), we performed theoretical studies. The aim was
to characterize, at the molecular level, transition states of intermediates and products and to predict
reaction diagrams. Furthermore, we postulated the conversion of
3 into its corresponding micheliolide
derivative following the same reaction pathway as reported for parthenolide Figure 3.
Figure 1. Energy profile of the transformation of
2 into 5. Relative energies are reported in kcal/mol.
The hydrogen bonding interaction O-3 . . . H-6 is represented with a dashed line.
Figure 2. Energy profile of the putative transannular cyclization of
3. Relative energies are reported in
kcal/mol. The hydrogen bonding interaction O-3 . . . H-6 is represented with a dashed line.

Molecules 2017, 22, 2252
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O
O
HO
a, b
O
X
O
O
3
1
a, b
1
5
H
5
O
O
OH O
O
O
parthenolide
micheliolide
.
(a) p-TsOH.H2O, dichloromethane, 20°; (b) BF3 Et2O, toluene, 20°.
Scheme 5. Reactivity of 3 and parthenolide with acids.
Figure 3. Energy profile of the transformation of parthenolide into micheliolide. Relative energies are
reported in kcal/mol.
Prior to reaction calculations, an exhaustive conformational analysis of
carried out, followed by optimization of the most stable conformer by density functional calculation at
the B3LYP/6-31+G(d,p) level [21]. Calculations were performed using the SMD solvation model [22
with dichloromethane as a solvent to mimic the reaction conditions (a). For compounds and 3,
2, 3, and parthenolide was
]
2
the angelate ester moiety was omitted since it had no influence on the conformation and, therefore,
on the reactivity of the germacranolide ring, as previously shown [23].
The global minimum structure of
hydrogen bond between O-3 and H-6 (distance O3-H6 2.23 Å). Upon treatment with acid, the epoxy
group was protonated leading to the protonated reactant . The intrinsic reaction coordinate of the
transition state involved the formation of a carbocation at C-10 requiring an activation energy of
5.03 kcal/mol. The occurrence of a transannular cyclization for was precluded by the long distances
of C10–C5 and C10–C4 (4.05 and 3.68 Å, respectively) due to the non-conventional intramolecular
2 (Figure 1) was stabilized by a non-conventional intramolecular
A
B
B

Molecules 2017, 22, 2252
10 of 17
hydrogen bond between O-3 and H-6 (2.08 Å). Conversely, the close distance of O3–C1 (2.83 Å) enabled
the formation of the protonated product C.
The reaction diagram of the conversion of
Figure 2, with the guaianolide scaffold resulting from the transannular cyclization. As in case of
the energetically optimized conformation of was stabilized by a non-conventional intramolecular
hydrogen bond between O-3 and H-6 (2.33 Å). In acidic media, would lead to the protonated epoxide
. However, evolution of into transition state bearing a C-5 carbocation would be prevented by the
too high energy barrier of 12.7 kcal/mol.
Theoretical studies on the conversion of parthenolide into micheliolide (Figure 3) showed that
the formation of the transition state bearing the carbocation at C-5 was energetically more favored.
Furthermore, key distances of C1–C5 (3.03 and 2.61 Å in and , respectively) allowed a transannular
3 into C, a congener of micheliolide is shown in
2,
3
3
A
3
B
B
A
B
cyclization of the C1(10) double bond into the new bond C1–C5.
2.3. In Vitro Anti-Protozoal Activity
Considering the antiprotozoal activity of other sesquiterpene lactones [24,25], compounds 1, 3–8,
and 10 15 were tested for their in vitro activity against Trypanosoma brucei rhodesiense, T. cruzi, Leishmania
–
donovani, and Plasmodium falciparum (Table 5). In parallel, their cytotoxicity in L6 cells was evaluated in
order to obtain a first appreciation of the selectivity. The tested compounds exhibited anti-protozoal
activity in the micromolar range. Among the tested parasites, T. b. rhodesiense was generally found to
be the most sensitive (IC50 0.47–13.4
nobilin ( ) against T. b. rhodesiense. The anti-trypanosomal activity of furanoheliangolides
varied depending on the substitution patterns. Decoration of the cyclodecadiene skeleton with more
polar functional groups, e.g., a hydroxyl group as in and caused a doubling of activity. Acetylation
µM, Selectivity Index (SI) 0.3–4). Epoxides
3–4
were as active as
1
5, 8, and 10
6
7
of the alcoholic function at C-3 increased activity as observed in compounds 11–14 (IC50 0.47–1.57 µM).
However, all compounds showed significant cytotoxicity in L6 cells.
Table 5. In vitro activity of compounds
1, 3–8, and 10–15 against T. b. rhodesiense (STIB 900), T. cruzi
(Tulahuen C4 LacZ), L. donovani (MHOM-ET-67/L82), P. falciparum (NF54), and cytotoxicity in L6 cells.
T. b. rhodesiense
T. cruzi
L. donovani
P. falciparum
L6 cells
Compound
IC50 (µM) a
IC50 (µM) a
IC50 (µM) a
IC50 (µM) a
IC50 (µM) a
2.08± 0.09 (2.4) b
1.96± 0.16 (2.04) b
2.18 ± 0.49 (2.6) b
0.91 ± 0.16 (3.7) b
1.21 ± 0.16 (3.9) b
3.25 ± 0.50 (3.5) b
13.4 ± 0.46 (1.51) b
1.57 ± 0.005 (0.28) b
0.47 ± 0.006 (2.19) b
0.54 ± 0.013 (3.46) b
1.20 ± 0.151 (4.4) b
11.3± 3.3 (0.4) b
2.69± 1.08 (1.8) b
13.19± 3.31 (0.3) b
28.67 ± 8.95 (0.2) b
8.28 ± 2.40 (0.4) b
12.88 ± 0.65 (0.4) b
66.8 ± 7.79 (0.17) b
3.12± 0.52 (1.6) b
1
3
4
5
6–7
8
10
11
12
13
14
15
4.91 ± 0.53
4.0 ± 0.03
5.56 ± 0.30
3.33 ± 0.28
4.74 ± 0.24
11.5 ± 0.72
4.24 (0.9) h
2.39 (1.7) h
8.38 (0.7) h
3.20 (1.7) h
6.35 (0.5) h
4.06 (0.8) h
16.00 ± 0.34 (0.3) b
13.82 ± 1.59 (0.3) b
31.3 ± 2.40 (0.4) b
79.13 ± 1.02 (0.26) b
3.93 ± 0.53 (0.59) b
1.55 ± 0.31 (0.66) b
9.17 ± 0.61 (0.20) b
8.80 ± 0.94 (0.68) b
35.66 ± 1.10 (0.63) b
0.006 ± 0.01 f
47.9 ± 1.11 (0.24) b
0.59 (0.15) b 35.96 ± 0.18 (0.56) b
20.20 ± 2.098
2.31 ± 0.196
1.03 ± 0.076
1.87 ± 0.026
3.95 ± 0.088
22.44 ± 0.157
0.016 ± 0.01 g
132.19
±
11.49 ± 0.68 (0.2) b
2.60 ± 0.69 (0.5) b
12.76 ± 1.08 (0.15) b
15.4 ± 1.48 (0.6) b
36.81 ± 7.76 (0.61) b
2.0 ± 0.2 d
4.20 ± 0.28 (0.55) b
1.30 ± 0.03 (0.79) b
3.26 ± 0.22 (0.57) b
4.97 ± 0.43 (7.2) b
5.86 ± 0.59 (3.83) b
0.13 ± 0.01 e
13.32
±
0.333 (1.68) b
Positive control
0.01 ± 0.01 c
Average of two independent assays in µ
g/mL; b Selectivity Index (SI): IC50 in L6 cells divided by IC50 in the titled
a
parasitic strain; c melarsoprol; d benznidazole; e miltefosine; f chloroquine; g podophyllotoxin; h one assay.
3. Experimental Section
3.1. General Information
Optical rotation was measured in MeOH (1 mg/mL) with a P-2000 digital polarimeter
(Jasco, Tokyo, Japan) using a 10-cm microcell. UV and CD spectra were recorded in MeOH (120 g/mL)
µ
on a Chirascan CD spectrometer, and data analyzed with Pro-Data V2.4 software. NMR spectra were
recorded on a Bruker Avance III spectrometer operating at 500.13 MHz for 1H, and 125.77 MHz for
1
◦
13C. H-NMR, COSY, HSQC, HMBC, and NOESY spectra were measured at 18 C in a 1 mm TXI

Molecules 2017, 22, 2252
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probe with a z-gradient, using standard Bruker pulse sequences. 13C-NMR/DEPTQ spectra were
recorded at 23 ◦C in a 5 mm BBO probe with a z-gradient. Spectra were analyzed by Bruker TopSpin
3.0 (Bruker Biospin, Fällende, Zurich, Switzerland). The following abbreviations are used to describe
the signal multiplicities: s (singlet), d (doublet), t (triplet), q (quartet,) and m (multiplet).
High-resolution mass spectra (ESI-TOF-MS) were recorded in positive mode on a Bruker
microTOF ESIMS system. Nitrogen was used as a nebulizing gas at a pressure of 2.0 bar, and as
◦
drying gas at a flow rate of 9.0 L/min (dry gas temperature 240 C). Capillary voltage was set at
45,000 V, and the hexapole at 230.0 Vpp. Instrument calibration was done with a reference solution of
0.1% sodium formiate in 2-propanol/water (1:1) containing 5 mM NaOH.
HPLC-PDA-ESIMS spectra were obtained in positive mode on a Bruker Daltonics Esquire
3000 Plus ion trap MS system, connected via T-splitter (1:10) to an Agilent HP 1100 system consisting of
degasser, binary mixing pump, autosampler, column oven, and a diode array detector (G1315B). Data
acquisition and processing was performed with Hystar 3.0 software (Bruker Optics, Fällende, Zurich,
Switzerland). Semipreparative HPLC separations were carried out with an Agilent HP 1100 series
system consisting of a quaternary pump, autosampler, column oven, and diode array detector (G1315B).
Waters SunFire
™
(Petaluma, CA, USA) (C18, 3.5
µ
m, 3.0
×
150 mm i.d.) and SunFire™ Prep C18 (5 µm,
10 150 mm i.d) columns were used for analytical and semi-preparative separations, respectively.
×
HPLC-grade MeOH, acetonitrile, (Scharlau Chemie S.A., Barcelona, Spain) and water were used for
HPLC separations. HPLC solvents contained 0.1% of HCO2H for analytical and semi-preparative
separations. Dry toluene was obtained by passing commercially available analytical grade toluene
(Scharlau Chemie S.A, Barcelona, Spain) through an activated alumina column. NMR spectra were
recorded in methanol-d4 (Armar Chemicals). Technical-grade solvents purified by distillation were
used for extraction and open column chromatography. Silica gel (63–200
Kenilworth, NJ, USA) was used for open-column chromatography.
µm and 15–40 µm, Merck,
3.2. In Vitro Biological Testing
◦
Compounds were dissolved in DMSO (10 mg/mL) and stored at
−
20 C until testing. Fresh
dilutions in medium were prepared for each bioassay. The DMSO concentration in the assay did not
exceed 1%. All assays were performed in at least three independent experiments. The purity of all
compounds was >95% unless stated otherwise.
The in vitro activities against the protozoan parasites T. b. rhodesiense (STIB900) bloodstream forms,
T. cruzi (Tulahuen C4 LacZ) intracellular amastigotes, L. donovani (MHOM-ET-67/L82) axenically
grown amastigotes, and P. falciparum (NF54) erythrocytic stage and cytotoxicity in L6 cells (rat skeletal
myoblasts) were determined as reported elsewhere [26].
3.3. Isolation of Nobilin (1)
Nobilin (
1
) was isolated from flower cones of Anthemis nobilis (Dixa AG, St. Gallen, Switzerland).
5 L), followed by CH2Cl2 (2 5 L). The CH2Cl2
Flowers were milled and percolated with n-hexane (2
×
×
extract was dried and separated by CC on SiO2 (400–600 mesh), using a gradient of n-hexane/EtOAc
(90:10 to 50:50) as mobile phase. Nobilin-enriched fractions were combined and purified by CC on
SiO2 (400–150 mesh). A gradient of n-hexane/EtOAc 95:5 to 60:40 was used as mobile phase. After
recrystallization from EtOAc/n-hexane, 800 mg of white solid were obtained. Spectroscopic data
were identical to those reported in the literature. Rf = 0.80 (SiO2, n-hexane/EtOAc 60:40). [α]D25
=
1
−
21.67 (c = 0.44, CH3OH). H-NMR (CD3OD, 500 MHz):
δ
1.74 (d, J = 1.4 Hz, 3H, 15-CH3), 1.87
(quint, J = 1.5 Hz, 3H, 50-CH3), 1.89 (br s, 3H, 14-CH3), 1.93 (dq, J = 7.2 and 1.5 Hz, 3H, 40-CH3), 2.23
(ddd, J = 13.8, 7.2 and 3.5 Hz, 1H, 2-Hb), 2.46 (dd, J = 12.7 and 10.5 Hz, 1H, 9-Hb), 2.50–2.68 (mov, 2H,
2-Ha and 9-Ha), 3.05 (dq, J = 10.0, and 1.8 Hz, 1H, 7-H), 4.39 (dd, J = 3.5 and 2.8 Hz, 1H, 3-H), 5.03
(ddd, J = 10.5, 10.0 and 3.7 Hz, 1H, 8-H), 5.17 (dq, J = 10.4 and 1.4 Hz, 1H, 5-H) 5.37 (dd, J = 9.3 and
7.2 Hz, 1H, 1-H), 5.70 (br s, 1H, 13-Hb), 6.05 (dd, J = 10.4 and 1.8 Hz, 1H, 6-H), 6.11 (qq, J = 7.2 and
1.5 Hz, 1H, 30-H), 6.20 (br s, 1H, 13-Ha) ppm. 13C-NMR (CD3OD, 125 MHz):
δ
16.1 (CH3, 40-C), 18.6

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(CH3, 14-C), 20.6 (CH3, 50-C), 23.6 (CH3, 15-C), 32.5 (CH2, 2-C), 49.0 (CH2, 9-C), 52.2 (CH, 7-C), 71.3
(CH, 8-C), 77.4 (CH, 3-C), 79.4 (CH, 6-C), 126.2 (CH, 5-C), 127.0 (CH, 1-C), 127.3 (CH2, 13-C), 128.9 (C,
20-C), 134.6 (C, 10-C), 137.3 (C, 11-C), 139.9 (CH, 30-C), 142.4 (C, 4-C), 168.3 (C, 10-C), 172.4 (C, 12-C)
ppm. HR-ESIMS calculated for C20H26O5Na [M + Na]+, 369.1672; found, 369.1672. The CD spectrum
(Figure S62) was in agreement with the literature [14].
3.4. Synthesis of Compounds 2 and 4
To a stirred solution of 1 (200 mg; 0.58 mmol) in DCM (10 mL), a solution of mCPBA (77%; 207 mg;
0.93 mmol) in DCM (1 mL) was added under cooling with ice. The reaction mixture was then allowed
to stand at room temperature for 4 h. On the basis of a TLC analysis, the reaction was quenched by the
addition of 10% Na2S2O3 (2
phase was then dried under Na2S2O4 and the solvent removed under reduced pressure. The obtained
×
5 mL). The absence of peroxides was tested with KI paper. The organic
residue was purified by chromatography (SiO2; n-hexane/AcOEt) to afford
2
(140 mg; 0.39 mmol;
70%), 3 (10 mg; 0.028 mmol; 7%), and 4 (9 mg; 0.024 mmol; 4%) as white solids.
(
−
)-(1R,10R)-1,10-Epoxynobilin (
2
) Rf = 0.5 (SiO2, hexane/EtOAc 60:40). [α]D25
1.48 (dd, J = 13.0, and 11.5 Hz, 1H, 9-Hax), 1.53 (s, 3H, 14-CH3), 1.63
=
−
16.9 (c = 0.1, CH3OH).
1H-NMR (CD3OD, 500 MHz):
δ
(ddd, J = 14.5, 10.2 and 2.6 Hz, 1H, 2-Hax), 1.80 (br s, 3H, 15-CH3), 1.85 (quint, J = 1.5 Hz, 3H, 50-CH3),
1.92 (dq, J = 7.0 and 1.5 Hz, 3H, 4 -CH3), 2.48 (ddd, J = 14.5, 4.5 and 4.3 Hz, 1H, 2-Heq), 2.50 (dd, J = 13.0,
0
and 3.0 Hz, 1H, 9-Heq), 2.94 (dd, J = 10.2, and 4.5 Hz, 1H, 1-H), 3.04 (dq, J = 10.0, and 1.6 Hz, 7-H), 4.40
(dd, J = 4.3 and 2.6 Hz, 1H, 3-H), 5.08 (ddd, J = 11.5, 10.0, and 3.0 Hz, 1H, 8-H), 5.27 (dq, J = 10.8, and
1.4 Hz, 1H, 5-H), 5.71 (br s, 1H, 13-Hb), 6.12 (qq, J = 7.0 and 1.5 Hz, 1H, 30-H), 6.22 (br m, 1H, 13-Ha),
6.38 (dd, J = 10.8, and 1.6 Hz, 1H, 6-H) ppm. 13C-NMR (CD3OD, 1205 MHz, extracted from HSQCC
0
and HMBC spectra):
δ
14.5 (CH3, 4 -C), 17.2 (CH3, 14-C), 19.1 (CH3, 5 -C), 21.9 (CH3, 15-C), 32.7 (CH2,
2-C), 46.8 (CH2, 9-C), 50.7 (CH, 7-C), 57.7 (C, 10-C), 60.70(CH, 1-C), 69.4 (CH, 8-C), 71.2 (CH, 3-C), 76.1
0
(CH, 6-C), 124.3 (CH, 5-C), 126.0 (CH2, 13-C), 127.3 (C, 2 -C), 135.4 (C, 11-C), 138.4 (CH, 3 -C), 142.1 (C,
4-C), 166.8 (C, 10-C), 170.3 (C, 12-C) ppm. HR-ESIMS calculated for C20H26O6Na [M + Na]+, 385.1622;
found, 385.1637.
The ECD spectrum of 2 (Figure S62) was in agreement with the literature [14].
(
−
)-(1R,4S,5R,10R)-1(10),4(5)-Diepoxynobilin (
4
): Rf = 0.30 (SiO2, n-hexane/EtOAc 60:40). [α]D25
1.40 (s, 3H, 15-CH3), 1.45 (overlapped, 1H, 9-Hax),
=
−
16.2
(c = 0.1, CH3OH). 1H-NMR (CDCl3, 500 MHz):
δ
1.46 (br s, 3H, 14-CH3), 1.80 (overlapped, 1H, 2-Hax), 1.82 (quint, J = 1.5 Hz, 3H, 50-CH3), 1.92 (dq,
J = 7.0 and 1.5 Hz, 3H, 40-CH3), 2.52 (dt, J = 15.0 and 4.2 Hz, 1H, 2-Heq), 2.60 (dd, J = 13.2, and 2.5 Hz,
1H, 9-Heq), 2.85 (d, J = 9.7 Hz, 1H, 5-H), 2.96 (dd, J = 10.6, and 4.2 Hz, 1H, 1-H), 3.07 (br d, J = 9.4 Hz,
7-H), 4.28 (dd, J = 4.2 and 3.0 Hz, 1H, 3-H), 5.02 (ddd, J = 12.0, 9.4 and 2.5 Hz, 1H, 8-H), 5.36 (br d,
J = 9.7 Hz, 1H, 6-H), 5.73 (br m, 1H, 13-Hb), 6.06 (qq, J = 7.2 and 1.5 Hz, 1H, 30-H), 6.34 (br m, 1H,
13-Ha) ppm. 13C-NMR (CDCl3, 125 MHz):
δ
15.8 (CH3, 40-C), 17.7 (CH3, 14-C), 20.0 (CH3, 15-C), 20.3
0
(CH3, 5 -C), 30.6 (CH2, 2-C), 47.4 (CH2, 9-C), 49.2 (CH, 7-C), 57.8 (C, 10-C), 58.9 (CH, 1-C), 62.4 (C, 4-C),
63.4 (CH, 5-C), 68.5 (CH, 8-C), 69.1 (CH, 3-C), 75.8 (CH, 6-C), 126.9 (C, 20-C), 128.1 (CH2, 13-C), 133.3
0
0
(C, 11-C), 140.0 (CH, 3 -C), 166.1 (C, 1 -C), 168.9 (C, 12-C) ppm. HR-ESIMS calculated for C20H26O7Na
[M + Na]+, 401.1571; found, 401.1588.
3.5. Synthesis of Compound 3
To a stirred solution of
and tBHP (solution 5.5 M in decane; 42
The reaction mixture was then allowed to stand at room temperature for 12 h. After TLC monitoring,
the reaction was quenched by adding 10% Na2S2O3 (2 5 mL). The absence of peroxides was tested
1
(60 mg; 0.17 mmol) in DCM (3 mL), VO(acac)2 (14 mg; 0.05 mmol)
µL; 0.2 mmol) were added under ice and argon atmosphere.
×
with KI paper. The organic phase was dried under Na2S2O4, the solvent was removed under reduced
pressure, and the obtained residue was purified by chromatography (SiO2; n-hexane/AcOEt 70:30) to
give 3 (54 mg; 0.15 mmol; 88%) as a white solid.

Molecules 2017, 22, 2252
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(
−
)-(4S,5R)-4,5-Epoxynobilin (
3
δ
): Rf = 0.70 (SiO2, n-hexane/EtOAc 60:40). [α]D25
1.34 (br s, 3H, 15-CH3), 1.82 (br s, 3H, 14-CH3), 1.84 (quint, J = 1.5 Hz,
=
−
6.19 (c = 0.1, CH3OH).
1H-NMR (CDCl3, 500 MHz):
3H, 50-CH3), 1.93 (dq, J = 7.0 and 1.5 Hz, 3H, 40-CH3), 2.23 (ddd, J = 14.4, 7.0 and 3.6 Hz, 1H, 2-Heq),
2.37 (dd, J = 12.0, and 11.0 Hz, 1H, 9-Hax), 2.59 (dd, J = 12.0, and 3.7 Hz, 1H, 9-Heq), 2.76 (dd, J = 14.4,
10.2, and 2.6 Hz, 1H, 2-Hax), 2.69 (br d, J = 9.7 Hz, 5-H), 3.05 (dddd, J = 9.7, 1.5,. 1.0, and 1.0 Hz, 7-H),
4.20 (dd, J = 3.6, and 2.6 Hz, 1H, 3-H), 4.92 (dd, J = 9.7, and 1.0 Hz, 1H, 6-H), 5.01 (ddd, J = 11.0, 9.7, and
3.7 Hz, 1H, 8-H), 5.40 (ddd, J = 10.2, 7.0, and 1.0 Hz, 1H, 1-H), 5.70 (dd, J = 1.5, and 0.8 Hz, 1H, 13-Hb),
6.07 (qq, J = 7.0 and 1.5 Hz, 1H, 30-H), 6.33 (br m, 1H, 13-Ha) ppm. 13C-NMR (CDCl3, 125 MHz):
δ
15.7 (CH3, 40-C), 18.0 (CH3, 14-C), 20.0 (CH3, 15-C), 20.2 (CH3, 50-C), 28.4 and 28.3 (CH2, 2-C), 46.8
(CH2, 9-C), 48.5 (CH, 7-C), 62.9 (C, 4-C), 63.8 (CH, 5-C), 69.3 (CH, 8-C), 73.2 and 73.1 (CH, 3-C), 77.4
0
(CH, 6-C), 122.9 (CH, 1-C), 127.1 (C, 2 -C), 127.4 (CH2, 13-C), 133.8 (C, 11-C), 135.0 (C, 10-C), 139.2 (CH,
30-C), 166.2 (C, 10-C), 169.1 (C, 12-C) ppm HR-ESIMS calculated for C20H26O6Na [M + Na]+, 385.1622;
found, 385.1639. The ECD spectrum is reported in the Supplementary Materials (Figure S62).
3.6. General Procedure for Degradation with p-TsOH·H2O
p-TsOH
0.055 mmol for
·
H2O (1–2 mg) was added to a solution of epoxy derivatives
2
–
4
and 11
–12 (20 mg;
2
and
3
; 0.053 mmol for
4
; 0.050 mmol for 11 and 12, respectively) in dichloromethane
◦
(3 mL), and stirred at 20 C. After the starting material (TLC on SiO2, n-hexane/EtOAc 40:60)
disappeared, the reaction was quenched with aqueous Na2HPO4 0.5 N (2 mL). The layers were
separated, and the organic layer dried over Na2SO4 and concentrated in vacuo to give a brownish
oil. The major reaction products were isolated by semi-preparative HPLC (gradient of H2O/CH3CN;
detection at 220 nm).
(+)-(4Z,1R,3R,6R,7R,8S,10S)-8-Angeloxy-3,10-epoxy-1-hydroxy-germacra-4(5),11(13)-dien-6,12-olide (5):
white solid (10 mg, 0.027 mmol, 50%); [α]2D5 = +6.85 (c 0.1, MeOH); for 1H and 13C-NMR data, see
Tables 1 and 3, respectively. HRESIMS m/z 385.1632 [M + Na]+ (calculated for C20H26NaO6: 385.1622).
(4Z,9E,1R,3S,6R,7S,8S)-8-Angeloxy-1,3-dihydroxy-germacra-4(5),9(10),11(13)-trien-6,12-olide and (4Z,9Z,1R,
3S,6R,7S,8S)-8-Angeloxy-1,3-dihydroxy-germacra-4(5),9(10),11(13)-trien-6,12-olide (6 and 7): white solid
(2 mg, 0.006 mol, 11% in a ratio of 3:1); for 1H and 13C-NMR data, see Tables 1 and 3, respectively.
HRESIMS m/z 385.1644 [M + Na]+ (calculated for C20H26NaO6: 385.1622).
(+)-(1R,3R,4S,5R,6S,7R,8S,10S)-8-Angeloxy-3(10),4(5)-diepoxy-germacra-11(13)-en-6,12-olide (8): white
solid (8 mg, 0.02 mol, 40%); [α]2D5 = +3.75 (c 0.1, MeOH); for 1H and 13C-NMR data, see Tables 1 and 3,
respectively. HRESIMS m/z 401.1591 [M + Na]+ (calculated for C20H26NaO7: 401.1571).
(
−
)-(1R,3R,4S,5S,6S,7R,8S,10S)-8-Angeloxy-3,10-epoxy-4-hydroxy-5-tosyloxygermacra-11(13)-en-6,12-olide
): white solid (2.7 mg, 0.005 mol, 10%); [α]D25
=
−
2.9 (c 0.2, MeOH); for H and 13C-NMR
1
(
9
data, see Tables 1 and 3, respectively. HRESIMS m/z 573.1786 [M + Na]+ (calculated for
C27H34NaO10S: 573.1765).
(+)-(1Z,4Z,3S,6R,7R,8S,10S)-8-Angeloxy-3,10-epoxy-3-hydroxy-germacra-1(2),4(5),11(13)-trien-6,12-olide
(
10): white solid (2.2 mg, 0.06 mol, 12%); [α]2D5 = +2.87 (c 0.1, MeOH); for 1H and 13C-NMR data, see
Tables 1 and 3, respectively. HRESIMS m/z 383.1500 [M + Na]+ (calculated for C20H24NaO6: 383.1465).
(+)-(4Z,9E,1R,3S,6R,7S,8S)-3-Acetoxy-8-angeloxy-1-hydroxy-germacra-4(5),9(10),11(13)-trien-6,12-olide (13):
white solid (2 mg, 0.005 mol, 10%); [α]D25 = +78.66 (c 0.5, MeOH); for 1H and 13C-NMR data, see Tables 2
and 4, respectively. HRESIMS m/z 427.1749 [M + Na]+ (calculated for C22H28NaO7: 427.1727).
(+)-(4Z,1R,3S,6R,7R,8S)-3-Acetoxy-8-angeloxy-1-hydroxy-germacra-4(5),10(14),11(13)-trien-6,12-olide (14):
white solid (1 mg, 0.002 mol, 5%); [α]2D5 = +51.75 (c 0.1, MeOH); for 1H and 13C-NMR data, see Tables 2
and 4, respectively. HRESIMS m/z 427.1749 [M + Na]+ (calculated for C22H28NaO7: 427.1727).

Molecules 2017, 22, 2252
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3.7. General Procedure for Degradation with BF3·Et2O
To a solution of epoxy derivatives
2
–
4
and 11
–
12 (20 mg; 0.055 mmol for
2
and
3
; 0.053 mmol
for
4
; 0.050 mmol for 11 and 12, respectively) in anhydrous toluene (5 mL), BF3 Et2O (5 µL) was
·
added under cooling in ice, and stirred at 20 ◦C. After disappearing of the starting material (TLC on
SiO2, n-hexane/EtOAc 40:60) the reaction was quenched with aqueous Na2HPO4 0.5 N (10 mL).
The layers were separated, and the organic layer dried over Na2SO4 and concentrated to dryness under
reduced pressure. The major reaction products were isolated by semi-preparative HPLC (gradient of
CH3CN/H2O; detection at 220 nm). Yields for individual compounds are reported in Schemes 2–4.
(+)-(7R,8S,10S)-8-Angeloxy-3-hydroxy-7,8,9,10-tetrahydro-cadalen-11(13)-en-12-oic acid (15): white
solid (6.8 mg, 0.02 mol, 40%); [α]2D5 = +82.95 (c 0.1, MeOH); for 1H and 13C-NMR data, see Tables 2
and 4, respectively. HRESIMS m/z 367.1530 [M + Na]+ (calculated for C20H24NaO5: 367.1516).
Cadalen-11(13)-en-12-oic acid (16): white solid (1.1 mg, 0.005 mol, 10%); for 1H and 13C-NMR data, see
Tables 2 and 4, respectively. HRESIMS m/z 249.0899 [M + Na]+ (calculated for C15H14NaO2: 249.0886).
3.8. General Procedure for Synthesis of Compounds 11 and 12
To a stirred solution of epoxy derivatives
2
and
3
(20 mg: 0.055 mmol) in dichlomethane (2 mL),
L; 0.12 mmol) and DMAP (cat.) were added.
acetic anhydride (35 L; 0.37 mmol), Hünig’s base (20
µ
µ
After disappearance of the starting material (TLC on SiO2, n-hexane/EtOAc 70:30) the reaction was
quenched by adding HCl 0.5 M (2 mL). The layers were separated and the organic layer dried over
Na2SO4 and concentrated to dryness under reduced pressure. Compounds 11 (17.8 mg; 80%) and
12 (15.5 mg; 70%) were isolated by chromatography on SiO2 (n-hexane/EtOAc 70:30; detection with
phosphomolybdic acid).
(+)-(1R,10R)-3-Acetoxy-1,10-epoxy nobilin (11): Rf = 0.3 (SiO2, hexane/EtOAc 60:40). [α]2D5 = +13.12
1
(c = 0.1, CHCl3). H-NMR (CDCl3, 500 MHz):
δ 1.36 (dd, J = 13.2, and 11.3 Hz, 1H, 9-Hax), 1.52 (s,
3H, 14-CH3), 1.66 (br dd, J = 10.2, and 4.5 Hz, 1H, 2-Hax), 1.81 (quint, J = 1.5 Hz, 3H, 50-CH3), 1.85 (d,
J = 1.5 Hz, 3H, 15-CH3), 1.91 (dq, J = 7.0 and 1.5 Hz, 3H, 40-CH3), 2.08 (s, 3H,2”), 2.53 (ddd, J = 15.5,
5.0 and 4.5 Hz, 1H, 2-Heq), 2.58 (dd, J = 13.2, and 3.0 Hz, 1H, 9-Heq), 2.83 (br dd, J = 10.2, and 4.5 Hz,
1H, 1-H), 2.85 (dq, J = 10.5, and 1.5 Hz, 7-H), 5.06 (ddd, J = 11.3, 10.5, and 3.0 Hz, 1H, 8-H), 5.20 (dd,
J = 5.0, and 2.2 Hz, 1H, 3-H), 5.22 (dq, J = 11.0, and 1.5 Hz, 1H, 5-H), 5.65 (dd, J = 11.0, and 1.5 Hz,
1H, 6-H), 5.66 (br m, 1H, 13-Hb), 6.05 (qq, J = 7.0 and 1.5 Hz, 1H, 30-H), 6.29 (br m, 1H, 13-Ha), ppm.
13C-NMR (Extracted from HSQC and HMBC spectra; CDCl3, 125 MHz):
δ
15.6 (CH3, 40-C), 17.2 (CH3,
14-C), 20.2 (CH3, 50-C), 21.0 (CH3, 2”), 22.8 (CH3, 15-C), 30.2 (CH2, 2-C), 46.8 (CH2, 9-C), 50.7 (CH,
7-C), 57.7 (C, 10-C), 60.3 (CH, 1-C), 69.0 (CH, 8-C), 72.6 (CH, 3-C), 75.2 (CH, 6-C), 125.0 (CH, 5-C), 127.0
(CH2, 13-C), 127.4 (C, 20-C), 134.0 (C, 11-C), 139.0 (CH, 30-C), 139.0 (C, 4-C), 166.4 (C, 10-C), 168.9 (C,
12-C), 169.3 (C, 1”3) ppm. HR-ESIMS calculated for C22H28O7Na [M + Na]+, 427.1727; found, 427.1753.
(+)-(4S,5R)-3-Acetoxy-4,5-epoxy nobilin (12): Rf = 0.2 (SiO2, n-hexane/EtOAc 60:40). [α]2D5 = +94.97
1
(c = 0.1, CHCl3). H-NMR (CDCl3, 500 MHz):
δ 1.36 (br s, 3H, 15-CH3), 1.79 (br s, 3H, 14-CH3), 1.83
(quint, J = 1.5 Hz, 3H, 50-CH3), 1.92 (dq, J = 7.0 and 1.5 Hz, 3H, 40-CH3), 2.02 (s, 3H,2”), 2.37 (ddd,
J = 15.0, 7.0 and 3.0 Hz, 1H, 2-Heq), 2.40 (dd, J = 12.8, and 11.0 Hz, 1H, 9-Hax), 2.56 (br d, J = 9.7 Hz,
5-H), 2.58 (dd, J = 12.8, and 3.6 Hz, 1H, 9-Heq), 2.76 (dd, J = 15.0, 10.0, and 2.5 Hz, 1H, 2-Hax), 3.06 dq,
J = 9.9, 1.5 Hz, 7-H), 4.61 (dd, J = 9.7, and 1.5 Hz, 1H, 6-H), 5.03 (ddd, J = 11.0, 9.9, and 3.6 Hz, 1H, 8-H),
5.36 (br t, J = 3.0 Hz, 1H, 3-H), 5.48 (ddd, J = 10.0, 7.0, and 1.0 Hz, 1H, 1-H), 5.69 (br m, 1H, 13-Hb),
6.05 (qq, J = 7.0 and 1.5 Hz, 1H, 30-H), 6.31 (br m, 1H, 13-Ha) ppm. 13C-NMR (Extracted from HSQC
0
0
and HMBC spectra; CDCl3, 125 MHz):
δ 15.7 (CH3, 4 -C), 18.0 (CH3, 14-C), 20.2 (CH3, 5 -C), 20.5 (CH3,
15-C), 20.9 (CH3,2”), 27.6 (CH2, 2-C), 46.8 (CH2, 9-C), 48.7 (CH, 7-C), 60.5 (C, 4-C), 62.4 (CH, 5-C), 69.4
(CH, 8-C), 72.9 (CH, 3-C), 77.6 (CH, 6-C), 122.8 (CH, 1-C), 127.0 (C, 20-C), 127.3 (CH2, 13-C), 134.0 (C,
11-C), 135.0 (C, 10-C), 139.2 (CH, 30-C), 166.1 (C, 10-C), 169.0 (C, 12-C), 169.2 (C,2”) ppm. HR-ESIMS
calculated for C22H28O7Na [M + Na]+, 427.1727; found, 427.1748.

Molecules 2017, 22, 2252
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3.9. Computational Methods
Conformational analysis of parthenolide,
2,
3, and
5
was performed with Schrödinger MacroModel
9.8 (Schrödinger, LLC, New York, NY, USA) employing the OPLS_2005 (Optimized Potentials for
Liquid Simulations) force field. For parthenolide, and , the calculations were done with the
2
3
chloroform solvation model (N.B. the dichloromethane solvation model is not available within
MacroModel). Conformers within a 2.0 kcal/mol energy window from the global minimum were
selected for geometrical optimization and energy calculation. Structural optimizations, transition state
(TS) searches, and vibrational calculations were carried out by applying DFT with the Becke’s nonlocal
three-parameter exchange and correlation functional, and the Lee–Yang–Parr correlation functional
level (B3LYP) using the 6-31+G(d,p) basis set in combination with the SMD solvation model [21
]
(solvent = dichloromethane), as implemented in the Gaussian 09 program package [27]. At optimized
geometries, vibrational analysis was performed, yielding one imaginary frequency for the transition
states and no imaginary frequencies for other molecular species.
4. Conclusions
A study of the reactivity of nobilin epoxy derivatives in acidic media has been carried out
through chemical and theoretical analysis. In agreement with what has been reported for similar
epoxy heliangolides [28], the type of rearrangement that compounds 2–4 undergo under acidic
catalysis depends solely on their conformation, which activates or deactivates the latent reactivity
of functional groups towards a structural rearrangement. Provided that the epoxy group and olefin,
can provide an nucleophilic site that can trigger a subsequent rearrangement, the reactivity of
the 1,10-epoxide towards electrophiles, similar to what has been observed for the corresponding
1(10)-olefin of 1, is much higher than that of the 4,5-epoxide. A key role in deactivating the reactivity
of the latter epoxide could be played by the 3-hydroxy group, which stabilizes the germacranolide
skeleton through non-conventional hydrogen bonding with H-6. This assumption would explain the
different reactivity of parthenolide compared to that of compounds 3 and 13. Nevertheless, the high
reactivity of the hydroxyl function at C-3 towards the C-10 carbocation provides easy access to the
class of furanoheliangolides with good yield. Since many of the isolated products are structurally
related to natural products, the present study provides new insight into the mechanism of natural
sesquiterpene-forming reactions.
Supplementary Materials: Supplementary data associated with this article can be found in the online version.
Acknowledgments: ECD spectra were measured at the Biophysics Facility, Biozentrum, University of Basel.
Maria De Mieri gratefully acknowledges Francesco Barbieri for proofreading the manuscript.
Author Contributions: M.D.M. and M.H. conceived and designed the experiments; I.I. performed the chemical
experiments; M.D.M. performed the collection and analysis of spectroscopic data; M.S. designed, performed, and
interpreted computational studies; M.K. designed and interpreted in vitro biological experiments; M.D.M., M.S.,
M.K., and M.H. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).