Chemo- and Regioselective Functionalization of Nortrilobolide: Application for Semisynthesis of the Natural Product 2-Acetoxytrilobolide

Article abstract of DOI:10.1021/acs.jnatprod.5b00333

The difference in reactivity of the hexaoxygenated natural product thapsigargin (1) and the pentaoxygenated nortrilobolide (3) was compared in order to develop a chemo- and regioselective method for the conversion of nortrilobolide (3) into the natural product 2-acetoxytrilobolide (4). For the first time, a stereoselective synthesis of 2-acetoxytrilobolide (4) is described, which involves two key reactions: the first chemical step was a one-pot substitution-oxidation reaction of an allylic ester into its corresponding α,β-unsaturated ketone. The second process consisted of a stereoselective α′-acyloxylation of the key intermediate α,β-unsaturated ketone to afford its corresponding acetoxyketone, which was converted into 2-acetoxytrilobolide (4) in a few steps. This innovative approach would allow the synthesis of a broad library of novel and valuable penta- and hexaoxygenated guaianolides as potential anticancer agents.

Full text of DOI:10.1021/acs.jnatprod.5b00333

Article
pubs.acs.org/jnp
Chemo- and Regioselective Functionalization of Nortrilobolide:
Application for Semisynthesis of the Natural Product
‑Acetoxytrilobolide
2
†
†
‡
,†
Nhu Thi Quynh Doan, Franc o̧ is Crestey, Carl Erik Olsen, and Søren Brøgger Christensen*
†Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen,
Universitetsparken 2, 2100 Copenhagen Ø, Denmark
‡
Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, 1871
Frederiksberg C, Denmark
*
S Supporting Information
ABSTRACT: The difference in reactivity of the hexaoxy-
genated natural product thapsigargin (1) and the pentaoxy-
genated nortrilobolide (3) was compared in order to develop a
chemo- and regioselective method for the conversion of
nortrilobolide (3) into the natural product 2-acetoxytrilobolide
(
4). For the first time, a stereoselective synthesis of 2-
acetoxytrilobolide (4) is described, which involves two key reactions: the first chemical step was a one-pot substitution−oxidation
reaction of an allylic ester into its corresponding α,β-unsaturated ketone. The second process consisted of a stereoselective α′-
acyloxylation of the key intermediate α,β-unsaturated ketone to afford its corresponding acetoxyketone, which was converted into
2
-acetoxytrilobolide (4) in a few steps. This innovative approach would allow the synthesis of a broad library of novel and
valuable penta- and hexaoxygenated guaianolides as potential anticancer agents.
2
+
uaianolides are a class of biologically active sesquiter-
made thapsigargin (1) a major tool for investigation of the Ca
6
G
pernes that have been intensively studied during the last
homeostasis in cells. In addition, conjugating thapsigargin (1)
to peptides has provided recently several prodrugs as efficient
treatments of various cancer types such as prostate and liver
1
few decades. Among this large family of compounds,
hexaoxygenated guaianolides such as thapsigargin (1) isolated
2
7
from Thapsia garganica L. and pentaoxygenated guaianolides
cancer.
3
such as trilobolide (2) isolated from Laser trilobum (L.) Borkh,
Hexaoxygenated as well as pentaoxygenated guaianolides can
be found in umbelliferous plants (Apiaceae). Until recently
hexaoxygenated guaianolides had only been found in the genus
Thapsia, whereas pentaoxygenated guaianolides such a8s
nortrilobolide (3) were known from Thapsia and Laser.
as shown in Figure 1, are potent inhibitors of the sarco/
2
+
4
endoplasmic reticulum Ca -ATPase (SERCA).
Both types of guaianolides have the same binding site in the
SERCA pump, but trilobolide (2) has a lower affinity than
5
thapsigargin (1). The subnanomolar affinity for SERCA has
́
However, Zidek and co-workers have isolated hexaoxygenated
-acetoxytrilobolide (4) and 2-hydroxydeacetyltrilobolide (5)
2
from L. trilobum, revealing that both L. trilobum and T.
garganica can express the enzyme needed for producing
9
hexaoxygenated guaianolides.
Although biologists quickly acted on the unique properties of
thapsigargin (1), chemists and biochemists reacted more
slowly. Consequently, the guaianolide biosynthesis pathway
remains a subject of particular interest. At present, kunzeaol is
assumed to be the first monocyclic precursor for the
8
b,10
biosynthesis of the guainolide backbone of thapsigargins,
but no clear understanding of either formation of the tricyclic
guaianolide skeleton or the introduction of the many oxygen
atoms on the skeleton exists. In the absence of this significant
information on its biosynthesis, the possibility for genetically
modifying other organisms to produce the highly oxygenated
Figure 1. Absolute configuration of a number of penta- and
hexaoxygenated guaianolides.
Received: April 15, 2015
Published: June 16, 2015
©
2015 American Chemical Society and
American Society of Pharmacognosy
1406
DOI: 10.1021/acs.jnatprod.5b00333
J. Nat. Prod. 2015, 78, 1406−1414

Journal of Natural Products
Article
Scheme 1. Retrosynthesis of 2-Acetoxytrilobolide (4) from Nortrilobolide (3)
a
guaianolide does not yet exist. Several chemical studies have
allowed the preparation of thapsigargins, nortrilobolides, and
Scheme 2. Selective Angelate Cleavage
1
,11
related guaianolides,
but this has required a tremendous
synthetic effort, as exemplified by the total synthesis of
thapsigargin (1) in 42 steps from (S)-carvone published by
11c
Ley and co-workers.
However, a possible pathway for
accessing the hexaoxygenated guaianolides could be the use of
the pentaoxygenated guaianolides as starting materials. To the
best of our knowledge, there is no such precedent successful
synthetic investigation in the literature. In this present study,
the synthesis of 2-acetoxytrilobolide (4) from nortrilobolide
a
Reagents and conditions: (a) (1) KMnO (4 equiv), BnEt NCl (0.08
4
3
(
3) was chosen as a representative example. Although
equiv), toluene, H O, rt, 7 h; (2) MeOH, pyridine, H O, reflux
2
2
oxygenation of C-2 in nortrilobolide (3) is not feasible since
this methylene group is not activated, an appropriate
transformation of C-3 into a ketone as observed in derivative
conditions, 7 h; (b) (1) OsO (0.015 equiv), NMO (1.15 equiv),
4
acetone, H
O, rt, 4 h; (2) NaIO (3 equiv), rt, 16 h; (3) MeOH,
2
4
pyridine, H O, reflux conditions, 16 h.
2
6
could possibly allow a stereoselective oxidation of C-2, which
should provide 2-acetoxytrilobolide (4) after a few subsequent
chemical modifications (Scheme 1).
1
1c
3
-alcohol.
However, the same procedure performed on
nortrilobolide (3) resulted in the ring-opening of the five-
membered ring to afford diketone 9 and acetal 10 in a 1:1
mixture, as depicted in Scheme 3. According to the NMR
spectra, only one isomer of 10 was formed, the relative
configuration of which was not be determined. The lower
reactivity of the C-4 double bond in thapsigargin (1) is
probably caused by steric hindrance of the octanoyl group in
the 2-position, which is not the case in nortrilobolide (3).
Hydrazinolysis of angelate esters into their corresponding 3-
hydrazinopropionate derivatives followed by spontaneous
cyclization to afford the corresponding alcohols has successfully
been applied. Indeed, in the case of thapsigargin (1),
hydrazinolysis furnished the deacylated compound 11, as
shown in Scheme 4. In contrast, hydrazinolysis of nortrilobolide
(3) afforded a mixture of triol 12 (isomeric mixture of angelate
and tiglate ester) and tetraol 13 in a 5:1 ratio. In addition,
relactonized product 14 was also observed.
In order to see if tetraol 13 could be used as starting material
in an alternative synthetic route to yield 4, the 8-hydroxy group
had to be protected to enable selective oxidation of the 3-
hydroxy group. In the case of deacylated derivative 11,
treatment with 2,2-dimethoxypropane in acidified acetone led
to the corresponding acetonide in good yield. Unfortunately,
attempts to convert tetraol 13 into acetonide 15 by treatment
with 2,2-dimethoxypropane under acidic conditions were not
successful, as only the methoxy ketal 16 was isolated in 43%
yield, as observed in Scheme 5.
Since angelic acid is an α,β-unsaturated acid, this ester group
is the most resistant to saponification among the other esters in
thapsigargin (1) and trilobolide (2). However, several methods
have been developed for selective cleavage of this ester by
either potassium permanganate or osmium tetraoxide-periodate
oxidation in order to get the corresponding pyruvate ester,
which could be cleaved selectively by solvolysis in the presence
12
of pyridine in methanol to form the expected 3-alcohol. The
3
-alcohol can easily be oxidized into the corresponding 3-
12b
ketone.
A great number of procedures for α-oxygenation of a
18
carbonyl group have been described including, for example,
1
3
oxidation with manganese(III) species or other heavy
1
4
metals, sigmatropic rearrangement of the corresponding
1
5
16
acyloxyenamines, hypoiodite-catalyzed α-oxyacylation, or
11c
epoxidation of the corresponding trimethylsilyl enol ethers.
In addition, α-halogenation of a ketone followed by
1
7
17b
substitution with a carboxylate group might be a possibility.
Herein, we wish to report for the first time the synthesis of 2-
acetoxytrilobolide (4) from nortrilobolide (3). The procedure
uses a one-pot cleavage of an angelate ester and the oxidation of
the alcohol intermediate into its corresponding ketone as well
as a stereoselective α′-acyloxylation.
12a
RESULTS AND DISCUSSION
■
As seen in Scheme 2, attempts to selectively remove the
angelate ester of nortrilobolide (3) employing either the
Analogously, 17 formed by deacetylation of 3 by treatment
with triethylamine in methanol was converted into a mixture of
a minor amount of the 3-angelate ester 18 and a major amount
of the 3-methyl ether 16 (in a 1:9 ratio) after treatment with
2,2-dimethoxypropane and acetone under acidic conditions
(Scheme 6). The two epimeric methyl ethers 16 were formed
in the approximate ratio 1:1.
potassium permanganate (KMnO ) or osmium tetraoxide
4
12
(
OsO ) procedures, which were successful for thapsigargin
4
(1), failed due to extensive oxidation of the C-4 double bond in
nortrilobolide (3), affording a complex mixture.
A milder procedure for the removal of the angelate ester via
ozonolysis was then attempted. Ozonolysis of the angelate ester
of thapsigargin (1) proceeded fairly selectively to afford the
corresponding pyruvate ester, which was solvolyzed to give the
This unexpected outcome indicated that the substitution at
C-3 probably occurred by an S 1 reaction. Intriguing in this
N
1
407
DOI: 10.1021/acs.jnatprod.5b00333
J. Nat. Prod. 2015, 78, 1406−1414

Journal of Natural Products
Article
a
Scheme 3. Ozonolysis of Nortrilobolide (3) Yielding Diketone 9 and Acetal 10
a
Reagents and conditions: (a) (1) O , dichloromethane (DCM), −78 °C, 10 min then PPh (7 equiv), rt, 16 h; (2) MeOH, pyridine, H O, reflux
3
3
2
conditions, 6 h, 33% (in a 1:1 ratio).
a
Scheme 4. Hydrazinolysis of Nortrilobolide (3) Yielding Triol 12 and Tetraols 13 and 14
a
Reagents and conditions: NH NH ·H O (1.26 equiv), EtOH, reflux conditions, 16 h, 35% for 12, 9% for 13.
2
2
2
a
Scheme 5. Acetalation of 13 Yielding Methoxy Ketal 16
a
Reagents and conditions: (a) 2,2-dimethoxypropane (125 equiv), p-TsOH (2 mol %), acetone, 50 °C, 16 h, 43%, dr 1:1.
a
Scheme 6. Synthesis of Derivatives 16 and 18
a
Reagents and conditions: (a) TEA (20 equiv), MeOH, rt, 16 h; (b) 2,2-dimethoxypropane (82 equiv), p-TsOH (2 mol %), acetone, 50 °C, 16 h,
35% (over two steps, 9:1 ratio).
context is that the corresponding acetalation for O-8-
debutanoyl thapsigargin could run in excellent yield to give
nortrilobolide (3) into the corresponding 3-alcohol 19, it was
found that the presence of water was crucial (entry 1), while a
19
the corresponding angeloyl acetal. These surprising findings
suggested that the angelate ester of nortrilobolide (3) would be
prone to undergo substitution with a hydroxy group in an acidic
mixture of water and an organic aprotic solvent. As predicted,
treatment of nortrilobolide (3) with an acidic mixture of water
and acetonitrile did afford the desired 3-alcohol 19 as shown in
Table 1. The reactions were performed on a 0.1−1 mmol scale
weak acid (pK 4−5) required prolonged reaction time (entry
a
2
), and a stronger acid (pK −2−0.5) accelerated not only the
a
formation of the allylic alcohol 19 (entries 3−7) but also its
decomposition. The reaction was enhanced at high temperature
obtained either by heating or by the use of microwave (MW)
irradiations (entries 5, 6, and 9). Moreover, at elevated
temperatures only a catalytic amount of the acidic medium
was needed to achieve high conversion of nortrilobolide (3)
into the desired product (entry 7). However, the allylic alcohol
19 appeared to be unstable in an acidic medium, explaining the
20
and resulted in yields ranging from 30% to 35%. NMR
analysis showed the presence of two alcohols corresponding to
a mixture of epimers 19R and 19S in a 1.25:1 ratio. During the
optimization of the reaction conditions for the conversion of
1
408
DOI: 10.1021/acs.jnatprod.5b00333
J. Nat. Prod. 2015, 78, 1406−1414

Journal of Natural Products
Article
Table 1. Substitution of Angelate Ester of Nortrilobolide (3)
under Acidic Aqueous Conditions
Scheme 8. One-Pot Two-Step Substitution−Oxidation of 3
a
into Ketone 6
acid
temperature
reaction
time (h)
conversion
e
a
entry
1
(equiv)
(°C)
solvent
(%)
Reagents and conditions: (a) CrO (1.4 equiv), 1 M HF(aq) (2
3
equiv), CH CN, MW, 95 °C, 2 h, 74%.
AcOH
12)
60
2
18
18
6
CH CN
0
∼10
∼50
∼90
100
3
3
(
2
3
4
5
6
7
AcOH
3)
60
CH CN/
an α,β-unsaturated ketone. Furthermore, it was demonstrated
that a selective cleavage of the angelate ester over the remaining
ester functionalities in nortrilobolide (3) could be successfully
achieved by using this one-pot substitution−oxidation
procedure.
With the easy access to the α,β-unsaturated ketone 6, the
next challenge was to introduce an acetoxy group in the α′-
position. Preliminary attempts at α′-oxidation of ketone 6 to
afford α′-hydroxyketone 20 were performed using the
camphor-based oxaziridine methodology developed by Davis
3
c
(
H O
2
p-TsOH
2)
42
CH CN/
3
c
(
H O
2
p-TsOH
2)
60
CH CN/
3
d
(
H O
2
p-TsOH
2)
100
1
CH CN/
3
d
(
H O
2
b
TFA (5)
80
1
CH CN/
100
3
d
H O
2
TFA (0.4)
80
60
4
CH CN/
∼90
3
d
H O
2
21
a
and co-workers. Thus, treatment of ketone 6 with freshly
prepared lithium diisopropylamide (LDA) (3 equiv) followed
by the addition of camphorsulfonyl oxaziridine (2 equiv) did
not lead to formation of the expected α′-hydroxyketone 20.
Probably, the two hydroxy groups at the 7- and 11-positions
prevented the lithiation of the α′-position. Introduction of an
acetoxy group at the α′-position using a recently described
8
9
HF (0.5)
16
1
CH CN
∼60
∼95
3
a
b
HF (3)
85
CH CN
3
a
b
c
1
M aqueous solution. Under MW irradiations. Ratio CH CN−
3
d
e
H O was 5:1. Ratio CH CN−H O was 9:1. Reactions were
2
3
2
performed on a 0.1−1 mmol scale, and conversion was determined
1
by H NMR analysis of the crude material.
22
procedure on similar complex guaianolides also failed, as
shown in Scheme 9. The major difference between 6 and the
obtained poor yields although the starting material was fully
converted.
23
The epimeric alcohols 19 were successfully oxidized by
Dess−Martin periodinane in the presence of pyridine to the
corresponding ketone 6 in 87% yield (Scheme 7).
Scheme 9. Attempts for the Stereoselective α′-Hydroxylation
a
and α′-Acyloxylation of Ketone 6
Scheme 7. Oxidation of the Allylic Alcohol 19 into Key-
a
Intermediate Ketone 6
a
Reagents and conditions: (a) LDA (3 equiv), dry THF, −78 °C, 3 h,
then (1R)-(−)-(10-camphorsulfonyl)oxaziridine (2 equiv), −78 to
−
30 °C, 3 h; (b) KMnO (2.10 equiv), AcOH (35 equiv), Ac O (9.5
4
2
a
Reagents and conditions: (a) Dess−Martin periodinane (1.5 equiv),
equiv), dry benzene, 85 °C, 16 h.
pyridine (9 equiv), dry DCM, rt, 2 h, 87%.
model compounds used in the study mentioned is the presence
of an ester in the 8-position, which could explain the difference
of reactivity.
With the aim of limiting the degradation of the allylic alcohol
intermediate 19 during the angelate cleavage in the acidic
medium, a one-pot procedure was developed in order to
oxidize in situ the allylic alcohol intermediate 19 into the
corresponding ketone 6. After extensive investigation, it was
found that treatment of nortrilobolide (3) with chromium(VI)
oxide in a mixture of a 1 M aqueous hydrofluoric acid solution
and acetonitrile afforded the desired ketone 6 in 74% yield after
only 2 h at 95 °C under microwave conditions, as highlighted in
Scheme 8.
Selective α′-acyloxylation of α,β-unsaturated ketones using
manganese(III) acetate with a Dean−Stark trap has been
13
reported previously by Demir and co-workers. Therein, it was
found that using acetic anhydride as a cosolvent instead of
acetic acid did enhance the conversion rates; however, in our
particular case acetic acid was much more effective. Thus,
treatment of 6 with manganese(III) acetate in a mixture of dry
benzene and glacial acetic acid using a Dean−Stark trap to
remove the water formed during the reaction resulted in the
exclusive formation of α′-acetoxyketone 21 with the desired
stereochemistry at C-2, as confirmed by NMR analysis. Indeed,
Importantly, this reaction could be easily performed on a
gram scale. To the best of our knowledge, this is an
unprecedented procedure for converting an allylic ester into
1
409
DOI: 10.1021/acs.jnatprod.5b00333
J. Nat. Prod. 2015, 78, 1406−1414

Journal of Natural Products
Article
a
Scheme 10. Formation of 23 from 6
a
Reaction conditions: (a) Mn(OAc) ·2H O (2.15 equiv), AcOH−dry benzene (1:5), Dean-Stark, reflux conditions, 6 h; (b) TEA (26.5 equiv),
3
2
MeOH, 60 °C, 30 min, 57% (over two steps); (c) (S)-2-methylbutyric anhydride (4 equiv), 4-dimethylaminopyridine (DMAP) (1 mol %), THF, rt,
h, 78%.
1
Table 2. Attempts at the Stereoselective Reduction of Acetoxyketone 23 into Alcohols 24R and 24S
conditions
CeCl ·7H O, MeOH then NaBH , −60 °C
dr (24S/24R)
yield (%)
NaBH , MeOH, 0 °C
1:2
37
60
61
4
1:8
3
2
4
Zn(BH ) , THF, −30 °C → 10 °C, 16 h (with EDTA workup)
1:1.85
4
2
ROESY experiments showed a correlation between H-2 and H-
4 (for more details, see the Supporting Information).
Methanolysis of α′-acetoxyketone 21 in the presence of
triethylamine in methanol was easily achieved to afford the
desired O-8-debutanoyl 2-acetoxyketone (22) (57% yield over
two steps from ketone 6), which was successfully esterified to
give the (S)-methylbutanoate derivative 23 at O-8 in 78% yield,
as seen in Scheme 10.
Scheme 11. Angeloylation of 24S to Afford 2-
Acetoxytrilobolide (4)
a
1
12b
Stereoselective reduction of similar ketones to give the α-
a
alcohols has previously been performed using zinc borohy-
Reagents and conditions: (a) 2,4,6-trichlorobenzoyl chloride (2
equiv), TEA (2 equiv), angelic acid (2 equiv), toluene, 75 °C, 48 h,
9%.
1
1c,24
dride.
Thus, the 2-acetoxyketone 23 was converted into
4
the two separable epimeric alcohols 24R and 24S after
treatment with zinc borohydride and the use of an ethyl-
enediaminetetraacetic acid (EDTA) workup. However, a
selectivity of approximately 2:1 toward the undesired syn-
diastereomer 24R (3β-alcohol) was obtained, as depicted in
Table 2.
Comparison of the synthesized 2-acetoxytrilobolide (4) with
the reported isolated sample of the natural product confirmed
the correct stereochemistry at C-2 and C-3 (for more details,
see the Supporting Information).
The functionalization of the hexaoxygenated thapsigargin (1)
has previously been intensively studied in our group.
Application of the same chemical conditions to nortrilobolide
A chelation of zinc to both the C-3 ketone and the carbonyl
group of the acetyl moiety might explain the observed outcome
of this reduction. Due to the flexibility of the acetyl group at C-
2, the hydride might approach preferentially from the α-face,
(
3) has resulted in a clear difference of reactivity between these
resulting in the undesired syn-product 24R as the major
product. The same unfavorable ratio of isomers was observed
when sodium borohydride was used. Premixing ketone 23 with
cerium(III) chloride prior to the addition of sodium
borohydride did not improve the selectivity in favor of the
desired 24S derivative; on the contrary, the selectivity was
enhanced toward the undesired 24R derivative. In spite of this
unwanted selectivity of the reaction, the procedure is still
attractive since the unwanted alcohol 24R can be oxidized to
ketone 23, which then can be recycled for preparation of the
two classes of derivatives. Based on these findings a protocol for
selective cleavage of the angelate ester in nortrilobolide (3) has
been successfully applied. Furthermore, a one-pot procedure
for the substitution−oxidation reaction for converting
nortrilobolide (3) into the key-intermediate ketone 6 has
been developed. This might be a general procedure for
converting allylic esters into their corresponding α,β-unsatu-
rated ketones. Combined with a highly stereoselective α′-
acetoxylation of 6 on C-2, a semisynthesis of 2-acetoxytrilobo-
lide (4) has been successfully completed in six steps from
nortrilobolide (3). These valuable outcomes could provide an
expedient access to a wide library of analogues of trilobolides
and related thapsigargins. The synthesis of new hexaoxygenated
2
5
3
α-alcohol 24S. Finally, angeloylation of alcohol 24S under
Yamaguchi conditions led to the desired 2-acetoxytrilobolide
4) in 49% yield (Scheme 11).
(
1
410
DOI: 10.1021/acs.jnatprod.5b00333
J. Nat. Prod. 2015, 78, 1406−1414

Journal of Natural Products
Article
guaianolides and their biological evaluations are currently in
progress in our laboratory.
CDCl
3
) δ 209.8 (C-4), 200.5 (C-5), 176.1 (C-12), 172.4 (butanoyl
CO), 170.7 (acetyl CO), 85.8 (C-6), 82.1 (C-10), 78.8 (C-11),
6.3 (C-7), 74.7 (C-3), 70.3 (C-8), 50.1 (C-1), 36.5 (C-9), 36.1
butanoyl C-2), 29.5 (C-2), 25.4 (C-15), 22.6 (acetyl CH ), 22.5 (C-
7
(
3
EXPERIMENTAL SECTION
■
14), 21.9 (C-13), 17.7 (butanoyl C-3), 13.7 (butanoyl C-4); HRMS
+
m/z 499.1794 [M + Na + H O] , calcd for C H O Na 499.1791.
An analytically pure sample of compound 10 was obtained as a pale
yellow oil: H NMR (400 MHz, CDCl ) δ 5.57 (1H, t, J = 3.8 Hz, H-
General Experimental Procedures. A crude extract containing
nortrilobolide (3) was received from GenSpera (San Antonio, TX,
USA) and purified by dry column vacuum chromatography on silica
2
21 32 12
1
3
8
), 5.23 (1H, s, H-6), 4.60 (1H, t, J = 8.7 Hz, H-3), 4.50 (1H, s, OH),
gel using DCM−EtOAc (5:1) as eluent (R = 0.23) prior to use. All
f
4
.15 (1H, s, OH), 3.40 (1H, t, J = 5.4 Hz, H-1), 3.04 (1H, s), 2.86
solvents and reagents were obtained from commercial suppliers and
used without further purification unless otherwise stated. All air- and
moisture-sensitive reactions were conducted under argon using oven-
or flame-dried glassware and in dried solvents according to standard
procedures. Reactions were followed by thin-layer chromatography
(1H, dd, J = 14.9, 3.3 Hz, H-9a), 2.52−2.39 (3H, m, H-2, H-9b),
2
1
1
.30−2.22 (5H, m, H-15, butanoyl H-2), 1.99 (3H, s, acetyl CH ),
3
.70−1.55 (2H, m, butanoyl H-3), 1.52 (3H, s, H-14), 1.47 (3H, s, H-
3), 0.93 (3H, t, J = 7.4 Hz, butanoyl H-4); 13C NMR (101 MHz,
(
TLC) using precoated aluminum plates and visualized using vanillin
CDCl
3
) δ 209.4 (C-4), 175.6 (C-12), 172.4 (butanoyl CO), 170.1
reagent (15 g of vanillin, 250 mL of EtOH, and 2.5 mL of conc
H SO ). Flash column chromatography was performed with silica gel
acetyl CO), 104.9 (C-5), 84.2 (C-10), 84.0 (C-3), 80.4 (C-6), 79.5
(C-11), 76.6 (C-7), 66.6 (C-8), 56.9 (C-1), 38.8 (C-9), 36.7 (butanoyl
2
4
(
35−75 μm). Dry column vacuum chromatography was carried out
C-2), 28.6 (C-2), 26.2 (C-15), 22.7 (acetyl CH
(butanoyl C-3), 16.56 (C-13), 13.9 (butanoyl C-4); HRMS m/z
499.1781 [M + Na + H O] , calcd for C21H O12Na 499.1791.
2 32
Hydrazinolysis of Nortrilobolide (3). To a solution of
nortrilobolide (3) (1.00 g, 1.97 mmol) in absolute EtOH (30 mL)
was added hydrazine hydrate (0.12 mL, 2.47 mmol) at room
temperature under an argon atmosphere. The reaction mixture was
stirred under reflux for 16 h, then cooled to room temperature. The
reaction was concentrated, and the resulting crude material was
purified by flash column chromatography on silica gel using gradient
3
), 22.1 (C-14), 18.1
with silica gel (20−45 μm). Optical rotations were measured as [α]
values (c in g/100 mL). Yields refer to isolated compounds estimated
to be >95% pure as determined by H NMR spectroscopy. NMR
D
+
1
spectra were recorded on 400 and 600 MHz instruments. The
chemical shifts (δ) are given in parts per million (ppm) relative to
residual signals of the solvent (CDCl3 and CD OD). Coupling
3
1
constants (J values) are given in hertz (Hz). Multiplicities of H NMR
signals are reported as follows: s, singlet; d, doublet; dd, doublet of
doublets; dt, doublet of triplets; ddd, doublet of doublets of doublets;
dtt, doublet of triplets of triplets; t, triplet; m: multiplet; q, quartet; dq,
doublet of quartets; qq, quartet of quartets; b, broad signal.
5
elution (toluene−EtOAc, 2:1 to 1:2) to furnish triol 12 as a pale
yellow solid (0.31 g, 35%), tetraol 13 as a pale yellow solid (0.06 g,
9%), and relactonized tetraol 14 (0.01 g, 1%) as a pale yellow solid.
Compound 13: H NMR (600 MHz, CD
1
13
Assignments of the NMR signals were made using 1D ( H, C,
DEPTQ) and 2D (COSY, HSQC, HMBC, ROESY) spectra.
Microwave-assisted synthesis was carried out in a Biotage Initiator
apparatus operating in single mode; the microwave cavity produced
controlled irradiation at 2.45 GHz. The reactions were run in sealed
vessels. These experiments were performed by employing magnetic
stirring and a fixed hold time using variable power to reach the desired
temperature (for 1−2 min) and then maintained at the desired
temperature in the vessel for the programmed time period. The
temperature was monitored by an IR sensor focused on a point on the
reactor vial glass. The IR sensor was calibrated to internal solution
reaction temperature by the manufacturer. HRMS data were recorded
on a micrOTOF-Q instrument using electrospray (ESI) as ionization
method.
1
OD) δ 5.83−5.80 (1H, m,
3
H-6), 4.53−4.46 (1H, m, H-3), 4.33 (1H, t, J = 3.6 Hz, H-8), 4.21−
4.16 (1H, m, H-1), 2.93 (1H, dd, J = 14.0, 3.5 Hz, H-9a), 2.36−2.28
(2H, m, H-9b, H-2a), 1.99 (3H, s, acetyl CH ), 1.93 (3H, s, H-15),
3
1.58 (1H, ddd, J = 13.4, 8.0, 6.9 Hz, H-2b), 1.41 (3H, s, H-14), 1.40
(3H, s, H-13); 13C NMR (151 MHz, CD
OD) δ 177.6 (C-12), 172.12
3
(acetyl CO), 145.5 (C-5), 130.7 (C-4), 88.0 (C-10), 80.43 (C-7/C-
11), 80.37 (C-7/C-11), 79.4 (C-6), 78.2 (C-3), 70.2 (C-8), 51.5 (C-
1), 41.1 (C-9), 35.8 (C-2), 22.45 (bs, acetyl CH
, C-14), 16.4 (C-13),
12.8 (C-15); HRMS m/z 379.1366 [M + Na] , calcd for C17 Na
OD) δ 5.06 (1H,
3
+
H O
24 8
1
379.1363. Compound 14: H NMR (600 MHz, CD
3
dd, J = 13.4, 3.3 Hz, H-8), 4.68 (1H, s, H-6), 4.42 (1H, t, J = 6.9 Hz,
H-3), 3.56 (1H, t, J = 6.9 Hz, H-1), 2.62 (1H, dd, J = 15.5, 3.3 Hz, H-
9a), 2.29 (1H, dt, J = 13.2, 7.7 Hz, H-2a), 2.05−2.01 (1H, m, H-9b),
Ozonolysis of Nortrilobolide (3). Ozone was bubbled through a
solution of nortrilobolide (3) (0.30 g, 0.59 mmol) in dry DCM (100
mL) at −78 °C for 10 min until the solution turned pale blue. The
solution was first flushed with oxygen for 10 min, then flushed with
2.03 (3H, s, acetyl CH ), 1.95 (3H, s, H-15), 1.58 (3H, s, H-13), 1.50
3
1
3
(1H, ddd, J = 13.4, 7.5, 6.2 Hz, H-2b), 1.44 (3H, s, H-14); C NMR
(151 MHz, CD
5), 135.5 (C-4), 84.59 (C-8), 83.8 (C-10), 80.1 (C-7/C-11), 78.6 (C-
3), 77.2 (C-7/C-11), 70.8 (C-6), 49.2 (C-1), 40.4 (C-9), 35.5 (C-2),
24.4 (C-14), 23.6 (C-13), 22.2 (acetyl CH ), 12.7 (C-15); HRMS m/z
379.1361 [M + Na] , calcd for C17H O Na 379.1363.
24 8
Methoxy Ketal 16. To a solution of tetraol 13 (40 mg, 0.11
mmol) in dry acetone (2 mL) were added 2,2-dimethoxypropane (1.7
mL, 13.8 mmol) and p-TsOH (2 mg, 2 mol %) at room temperature
under an argon atmosphere. The reaction was stirred at 50 °C
overnight, cooled to room temperature, and quenched by the addition
of a saturated aqueous NaHCO
was extracted with EtOAc (3 × 20 mL), and the combined organic
phases were dried over MgSO , filtered, and concentrated under
reduced pressure. The crude material was purified by dry vacuum
column chromatography on silica gel using toluene−EtOAc (5:1) as
eluent to lead to a mixture of epimeric methyl ethers 16 (20 mg, 43%)
in a 1:1 (R/S) ratio as a pale yellow solid. An analytically pure sample
OD) δ 180.0 (C-12), 172.3 (acetyl CO), 143.9 (C-
3
nitrogen for 10 min before Ph P (1.10 g, 4.19 mmol) was added to the
3
solution and stirred at room temperature overnight. The solution was
concentrated under reduced pressure, and the crude product was
purified through a short plug of silica gel using toluene−EtOAc (3:1 to
3
+
2
:1) as eluent before it was dissolved in dry MeOH (50 mL) and
treated with pyridine (5 mL) and H O (5 mL). The reaction mixture
2
was stirred under reflux for 6 h, cooled to room temperature, and
quenched by the addition of a saturated aqueous NH Cl solution (100
4
mL). The aqueous phase was extracted with DCM (3 × 50 mL), and
the combined organic phases were dried over MgSO , filtered, and
3
solution (20 mL). The aqueous phase
4
concentrated under reduced pressure. The crude material was purified
by dry vacuum column chromatography on silica gel using toluene−
EtOAc (3:1 to 2:1) as eluent to afford diketone 9 and acetal 10 (0.09
g, 33%) in a 1:1 ratio as a pale yellow oil. An analytically pure sample
4
1
of compound 9 was obtained as a pale yellow oil: H NMR (600 MHz,
CDCl ) δ 5.45 (1H, d, J = 4.8 Hz, H-8), 5.06 (1H, s, OH), 4.96 (1H, s,
3
1
H-6), 4.15 (1H, d, J = 11.2 Hz, H-1), 4.08 (1H, dt, J = 8.3, 4.1 Hz, H-
of compound 16R was obtained as a pale yellow solid: H NMR (600
3
9
1
), 3.94 (1H, s, OH), 3.82 (1H, s, OH), 2.97 (1H, d, J = 15.1 Hz, H-
a), 2.67 (1H, dd, J = 15.4, 5.9 Hz, H-9b), 2.61 (1H, ddd, J = 13.4,
1.3, 4.4 Hz, H-2a), 2.23 (3H, s, H-15), 2.18−2.04 (2H, m, butanoyl
MHz, CDCl ) δ 5.82 (1H, s, H-6), 4.27 (1H, dd, J = 4.7, 2.9 Hz, H-8),
4.13 (1H, dt, J = 8.9, 4.5 Hz, H-3), 3.88 (1H, dtt, J = 9.3, 4.7, 2.3 Hz,
3
H-1), 3.38 (3H, s, OCH ), 2.99 (1H, dd, J = 14.8, 4.5 Hz, H-9a),
3
H-2), 2.00 (3H, s, acetyl CH ), 1.68 (1H, ddd, J = 13.6, 8.1, 2.1 Hz, H-
2
s, H-14), 0.87 (3H, t, J = 7.4 Hz, butanoyl H-4); C NMR (101 MHz,
2.35−2.31 (1H, m, H-9b), 2.31−2.25 (1H, m, H-2a), 1.98 (3H, s,
3
b), 1.56 (3H, s, H-13), 1.54−1.48 (2H, m, butanoyl H-3), 1.29 (3H,
acetyl CH ), 1.95 (3H, s, H-15), 1.55−1.53 (7H, m, H-2a, H-13,
3
13
C(CH )-(CH )), 1.41 (3H, s, C(CH )-(CH )), 1.35 (3H, s, H-14);
3
3
3
3
1
411
DOI: 10.1021/acs.jnatprod.5b00333
J. Nat. Prod. 2015, 78, 1406−1414

Journal of Natural Products
Article
1
3
1
C NMR (151 MHz, CDCl ) δ 173.2 (C-12), 170.8 (acetyl-CO),
yellow solid: H NMR (600 MHz, CDCl ) δ 5.69 (1H, s, H-6), 5.61
3
3
1
(
5
(
43.9 (C-5), 127.4 (C-4), 101.1 (C(CH )-(CH )), 86.3 (C-10), 86.1
C-3), 79.4 (C-7/C-11), 78.8 (C-6), 76.3 (C-7/C-11), 66.3 (C-8),
6.9 (OCH ), 50.8 (C-1), 38.5 (C-9), 32.3 (C-2), 30.7 (C(CH )-
CH )), 23.8 (C(CH )-(CH )), 22.7 (CH CO), 21.2 (C-14), 16.1 (C-
3), 12.7 (C-15); HRMS m/z 433.1826 [M + Na] , calcd for
(1H, t, J = 3.6 Hz, H-8), 4.59 (1H, t, J = 6.8 Hz, H-3), 4.16 (1H, t, J =
7.1 Hz, H-1), 3.08 (1H, dd, J = 15.0, 3.5 Hz, H-9a), 2.52 (1H, s, OH),
2.40 (1H, dt, J = 13.5, 8.2 Hz, H-2a), 2.27 (3H, t, J = 6.8 Hz, butanoyl
H-2), 2.22 (1H, dd, J = 14.8, 3.9 Hz, H-9b), 1.97 (3H, s, acetyl CH3),
1.95 (3H, s, H-15), 1.79 (1H, s), 1.69−1.53 (3H, m, H-2b, butanoyl
3
3
3
3
3 3 3 3
+
1
C H O Na 433.1833. An analytically pure sample of compound 16S
H-3), 1.49 (3H, s, H-13), 1.34 (3H, s, H-14), 0.95 (3H, t, J = 7.4 Hz,
butanoyl H-4); C NMR (151 MHz, CDCl ) δ 176.2 (C-12), 172.9
21
30
8
1
13
has been obtained as a pale yellow solid: H NMR (600 MHz, CDCl )
3
3
δ 5.77 (1H, s, H-6), 4.24 (1H, dd, J = 4.6, 2.9 Hz, H-8), 4.13−4.05
(butanoyl CO), 171.3 (acetyl CO), 146.4 (C-5), 129.1 (C-4),
86.3 (C-10), 78.9 (C-7/C-11), 78.2 (C-6), 77.7 (C-3), 66.7 (C-8),
50.1 (C-1), 38.8 (C-9), 36.8 (butanoyl C-2), 34.7 (C-2), 22.6 (acetyl
(
2H, m, H-3, H-1), 3.31 (3H, s, OCH ), 2.79 (1H, dt, J = 14.7, 3.8 Hz,
3
H-9a), 2.52 (1H, dt, J = 14.6, 2.1 Hz, H-9b), 1.99 (6H, s, H-15, acetyl
CH ), 1.98−1.94 (1H, m, H-2a), 1.85−1.76 (1H, 1H, H-2b), 1.53
CH ), 22.5 (C-14), 18.1 (butanoyl C-3), 16.3 (C-13), 13.9 (butanoyl
3
3
+
(
(
(
(
(
(
2
4
3H, s, H-13), 1.52 (3H, s, C(CH )-(CH )), 1.40 (3H, s, C(CH )-
C-4), 12.9 (C-15); HRMS m/z 449.1767 [M + Na] , calcd for
3
3
3
13
CH )), 1.30 (3H, s, H-14); C NMR (151 MHz, CDCl ) δ 173.3
C H O Na 449.1782. An analytically pure sample of compound 19S
3
3
21 30
9
1
C-12), 170.5 (acetyl-CO), 142.5 (C-5), 130.9 (C-4), 100.9
was obtained as a pale yellow solid: H NMR (600 MHz, CDCl ) δ
3
C(CH )-(CH ), 88.9 (C-3), 85.8 (C-10), 79.6 (C-7/C-11), 78.7
3
3
5.61 (1H, s, H-6), 5.59 (1H, t, J = 4.0 Hz, H-8), 4.56 (1H, d, J = 7.5
Hz, H-1), 4.51 (1H, bs, H-3), 3.48 (1H, s, OH), 3.09 (1H, dd, J = 14.8,
3.6 Hz, H-9a), 2.42 (1H, s, OH), 2.25 (2H, t, J = 7.6 Hz, butanoyl H-
C-6), 76.2 (C-7/C-11), 66.2 (C-8), 56.5 (OCH ), 53.8 (C-1), 39.1
3
C-9), 31.1 (C-2), 30.7 (C(CH )-(CH )), 23.8 (C(CH )-(CH )),
3
3
3
3
2.6 (CH CO), 20.3 (C-14), 16.2 (C-13), 14.5 (C-15); HRMS m/z
3
2), 2.19−2.09 (2H, m, H-9b, H-2a), 1.97 (6H, s, H-15, acetyl CH ),
3
+
33.1830 [M + Na] , calcd for C H O Na 433.1833.
Acetonide 18. To a solution of nortrilobolide (3) (1.00 g, 1.97
21
30
8
1.81 (1H, ddd, J = 14.7, 8.1, 2.5 Hz, H-2b), 1.67−1.58 (2H, m,
1
1c
butanoyl H-3), 1.50 (3H, s, H-13), 1.21 (3H, s, H-14), 0.94 (3H, t, J =
mmol) in dry MeOH (100 mL) was added TEA (5.5 mL, 39.5 mmol)
at room temperature under an atmosphere of argon. The reaction was
stirred at room temperature for 4 h before it was quenched by the
13
7
1
4
.4 Hz, butanoyl H-4); C NMR (151 MHz, CDCl ) δ 176.4 (C-12),
3
72.9 (butanoyl CO), 171.3 (acetyl CO), 147.0 (C-5), 130.8 (C-
), 86.5 (C-10), 80.1 (C-3), 79.0 (C7/C11), 78.8 (C7/C11), 78.2 (C-
addition of an aqueous saturated NH Cl solution (100 mL). The
4
6), 66.7 (C-8), 51.7 (C-1), 39.1 (C-9), 36.8 (butanoyl C-2), 35.1 (C-
aqueous phase was extracted with EtOAc (3 × 100 mL), and the
combined organic phases were washed with brine (150 mL), dried
2
1
), 22.5 (acetyl CH ), 21.9 (C-14), 18.1 (butanoyl C-3), 16.3 (C-13),
4.1 (butanoyl C-4), 13.9 (C-15); HRMS m/z 449.1784 [M + Na] ,
3
+
over MgSO , filtered, and concentrated under reduced pressure to
4
calcd for C H O Na 449.1782.
21
30
9
afford the crude triol 17, which was used in the next reaction without
Ketone 6. Procedure A. Dess−Martin periodinane (180 mg, 0.42
mmol) was added portionwise to a solution of allylic alcohol 19 (120
mg, 0.28 mmol) in dry DCM (10 mL) and pyridine (0.2 mL, 2.5
mmol) at room temperature under an argon atmosphere. The reaction
mixture, which immediately turned dark brown, was stirred for 2 h at
room temperature. The resulting yellow solution was quenched by
addition of a saturated aqueous Na S O solution (5 mL) and a
5
further purification. An analytically pure sample of compound 17 was
+
obtained as a white solid: HRMS m/z 461.1767 [M + Na] , calcd for
C H O Na 461.1782. To a solution of crude 17 (1.97 mmol) in dry
22
30
9
acetone (20 mL) were added 2,2-dimethoxypropane (20 mL, 163
mmol) and p-TsOH (cat.) at room temperature under an argon
atmosphere. The reaction was stirred at 50 °C overnight, cooled to
room temperature, and quenched by the addition of a saturated
2
2
3
saturated aqueous NaHCO solution (5 mL). The aqueous phase was
3
aqueous NaHCO solution (50 mL). The aqueous phase was extracted
3
extracted with EtOAc (3 × 20 mL), and the combined organic phases
with EtOAc (3 × 100 mL), and the combined organic phases were
were washed with brine (30 mL), dried over MgSO , filtered, and
4
washed with brine (100 mL), dried over MgSO , filtered, and
4
concentrated under reduced pressure. The crude material was purified
by dry vacuum column chromatography on silica gel using toluene−
EtOAc (2:1) as eluent to furnish ketone 6 (104 mg, 87%) as a white
solid: [α]22 −4 (c 1.0, CHCl ); H NMR (400 MHz, CDCl ) δ 5.81
concentrated under reduced pressure. The crude product was purified
by dry vacuum column chromatography on silica gel using toluene−
EtOAc (5:1) as eluent to provide a mixture of 16 and acetonide 18
1
D
3
3
(
0.33 g, 35%) in a 9:1 ratio as a pale yellow solid. An analytically pure
1
(1H, s, H-6), 5.71 (1H, t, J = 3.7 Hz, H-8), 4.76 (1H, bs, H-1), 4.13
1H, s, OH), 3.32 (1H, dd, J = 14.8, 3.7 Hz, H-9a), 3.13 (1H, s, OH),
.43 (1H, dd, J = 19.5, 6.3 Hz, H-2a), 2.34 (1H, dd, J = 19.1, 2.8 Hz,
H-2b), 2.27 (2H, t, J = 7.5 Hz, butanoyl H-2), 2.09 (1H, dd, J = 14.7,
.8 Hz, H-9b), 1.98 (3H, s, acetyl CO)), 1.92 (3H, dd, J = 2.2, 1.3
Hz, H-15), 1.69−1.54 (2H, m, butanoyl H-3), 1.50 (3H, s, H-13), 1.20
sample of compound 18 has been obtained as a pale yellow solid: H
(
2
NMR (600 MHz, CDCl ) δ 6.11 (1H, q, J = 7.3 Hz, angeoyl H-3),
3
5
.84 (1H, s, H-6), 5.57−5.51 (1H, m, H-3), 4.31−4.26 (1H, m, H-8),
4.03−3.96 (1H, m, H-1), 3.19 (1H, s, OH), 2.94 (1H, dd, J = 14.6, 4.7
3
Hz, H-9a), 2.54 (1H, dt, J = 13.5, 7.7 Hz, H-2a), 2.42 (1H, dd, J =
1
4.7, 2.9 Hz, H-9b), 2.03−1.99 (3H, m, angeoyl H-4), 1.95 (3H, s,
1
3
(
3H, s, H-14), 0.93 (3H, t, J = 7.4 Hz, butanoyl H-4); C NMR (151
angeoyl 2-CH ), 1.93−1.89 (6H, m, acetyl CH , H-15), 1.61−1.56
3
3
MHz, CDCl ) δ 207.3 (C-3), 174.8 (C-12), 172.7 (butanoyl CO),
(
(
1H, m, H-2b), 1.55 (3H, s, H-13), 1.53 (3H, s, C(CH )-(CH )), 1.42
3H, s, C(CH )-(CH )), 1.36 (3H, s, H-14); C NMR (151 MHz,
3
3
3
13
171.3 (acetyl CO), 159.4 (C-5), 145.0 (C-4), 85.3 (C-10), 79.3 (C-
3
3
7
/C-11), 78.8 (C-7/C-11), 77.8 (C-6), 66.4 (C-8), 46.2 (C-1), 39.1
CDCl ) δ 173.3 (C-12), 170.8 (angeoyl CO), 168.0 (acetyl CO),
3
(
(
(
C-9), 36.74 (C-2/butanoyl C-2), 36.69 (C-2/butanoyl C-2), 22.5
1
1
7
40.9 (C-4), 138.8 (angeoyl C-3), 129.9 (C-5), 127.9 (angeoyl C-4),
acetyl CH ), 22.1 (H-14), 18.1 (butanoyl C-3), 16.3 (C-13), 13.8
01.1 (C(CH )-(CH ), 85.7 (C-10), 79.9 (C-3), 79.5, 78.8 (C-6),
3
3
3
+
butanoyl C-4), 9.9 (C-15); HRMS m/z 425.1834 [M + H] , calcd for
6.2, 66.2 (C-8), 51.6 (C-1), 38.4 (C-9), 33.3 (C-2), 30.7 (C(CH )-
3
C H O 425.1806.
(
CH )), 23.8 (C(CH )-(CH )), 22.6 (angeoyl 2-CH ), 20.9 (C-14),
21 29
9
3
3
3
3
Procedure B. To a MW vial containing a solution of nortrilobolide
3) (1.05 g, 2.07 mmol) was successively added a 1 M aqueous
2
0.8 (acetyl CH ), 16.1 (C-13), 12.8 (C-15); HRMS m/z 501.2101
3
+
(
[
M + Na] , calcd for C H O Na 501.2095.
25 34 9
solution of hydrogen fluoride (4.14 mL, 4.14 mmol) and chromium-
(VI) oxide (290 mg, 1.40 mmol) at room temperature. The MW vial
was sealed and heated under MW irradiation for 2 h at 95 °C. After
cooling to room temperature, the reaction mixture was diluted with
water (70 mL) and extracted with EtOAc (60 mL). The organic layer
was successively washed with water, a 2 M aqueous solution of
Allylic Alcohol 19. To a solution of nortrilobolide (3) (100 mg,
0
.2 mmol) in a mixture of MeCN−H O (6 mL, 5:1) was added p-
2
TsOH (60 mg, 0.32 mmol) at room temperature. The reaction
mixture was stirred at 60 °C for 6 h, cooled to room temperature,
diluted with EtOAc (30 mL), and washed with water (until pH ∼6).
The organic phase was then washed with brine (30 mL), dried over
MgSO , filtered, and concentrated under reduced pressure. The crude
NaHCO , and brine, dried over MgSO , filtered, and concentrated
3
4
4
product was purified by flash column chromatography on silica gel
using toluene−EtOAc (4:1) as eluent to afford a mixture of epimeric
alcohols 19 (63 mg, 75%) in a 1.25:1 (R/S) ratio as a pale yellow solid.
An analytically pure sample of compound 19R was obtained as a pale
under reduced pressure. The resulting off-white solid was purified by
column chromatography on silica gel using EtOAc−heptane (1:1) as
eluent to afford ketone 6 (651 mg, 74%) as a white solid with
spectroscopic data in accordance with previous characterizations.
1
412
DOI: 10.1021/acs.jnatprod.5b00333
J. Nat. Prod. 2015, 78, 1406−1414

Journal of Natural Products
Article
O-8-Debutanoyl 2-Acetoxyketone (22). A solution of ketone 6
230 mg, 0.54 mmol) and manganese triacetate dihydrate (310 mg,
26.3 (2-methyl butanoyl C-3), 23.2 (C-14), 22.6 (acetylC‑10 CH ), 20.7
3
(
(acetylC‑2 CH ), 16.4 (2-methyl butanoyl 2-CH ), 16.2 (C-13), 11.8
3
3
1
.16 mmol) in dry benzene−glacial acetic acid (45 mL, 5:1) was
(2-methyl butanoyl C-4), 10.4 (C-15); HRMS m/z 519.1843 [M +
+
stirred under reflux using a Dean−Stark apparatus. After 6 h, the dark
color of the solution disappeared and the reaction mixture was diluted
with EtOAc (30 mL) and washed with brine (30 mL). The separated
Na] , calcd for C H O Na 519.1837.
24
32 11
Alcohol 24S. To a solution of (S)-methylbutanoate (23) (24 mg,
0.048 mmol) in freshly distilled THF (2 mL) was cannulated a
precooled solution of zinc borohydride (3.5 mL of a 0.5 M solution in
organic phase was dried over MgSO , filtered, and concentrated under
4
reduced pressure to afford the crude 2-acetoxyketone 21 as a yellow
solid, which was used in the subsequent reaction without any further
purification. An analytically pure sample of compound 21 was obtained
as a pale yellow solid: [α]22 − 91.4 (c 0.35, CHCl ); H NMR (600
Et O, 1.75 mmol) at −30 °C. After stirring the solution for a further 2
2
h at −30 °C, an additional quantity of zinc borohydride (1 mL of a 0.5
M solution in Et O, 0.5 mmol) solution was added. The reaction
2
1
mixture was allowed to warm to 10 °C and stirred overnight. The
reaction mixture was diluted with EtOAc (30 mL) and quenched by
the slow addition of an aqueous EDTA solution (30 mL, 30% w/w).
The biphasic system was vigorously stirred at room temperature for 2
h. The separated aqueous phase was extracted with EtOAc (3 × 30
mL). The combined organic phases were washed with brine (50 mL),
D
3
MHz, CDCl ) δ 5.82 (1H, 1H, H-6), 5.68 (1H, t, J = 3.8 Hz, H-8),
3
5
.18 (1H, d, J = 3.6 Hz, H-2), 4.60−4.56 (1H, m, H-1), 4.09 (1H, s,
OH), 3.23 (1H, dd, J = 14.9, 3.7 Hz, H-9a), 3.12 (1H, s, OH), 2.27
2H, t, J = 7.3 Hz, butanoyl H-2), 2.23 (1H, dd, J = 14.7, 3.9 Hz, H-
b), 2.09 (3H, s, acetylC‑2 CH ), 2.01−1.97 (3H, m, H-15), 1.94 (3H,
(
9
3
s, acetylC‑10 CH ), 1.66−1.58 (2H, m, butanoyl H-3), 1.47 (3H, s, H-
1
dried over MgSO , filtered, and concentrated under reduced pressure.
3
4
13
3), 1.38 (3H, s, H-14), 0.94 (3H, t, J = 7.4 Hz, butanoyl H-4);
C
The crude product was purified by dry vacuum column chromatog-
raphy on silica gel using toluene−EtOAc (2:1) as eluent to afford a
mixture of epimeric alcohols 24 (14.6 mg, 61%) in a 1.85:1 (R/S) ratio
NMR (151 MHz, CDCl ) δ 201.5 (C-3), 175.0 (C-12), 172.8
3
(
1
butanoyl CO), 171.2 (acetyl
CO), 170.2 (acetyl CO),
C‑10
C‑2
56.7 (C-5), 142.1 (C-4), 84.1 (C-10), 79.1 (C-7/C-11), 78.7 (C-7/
as a white solid. An analytically pure sample of compound 24S was
1
C-11), 78.0 (C-6), 73.5 (C-2), 66.2 (C-8), 51.8 (C-1), 38.8 (C-9),
obtained as a white solid: H NMR (600 MHz, CDCl ) δ 5.67−5.61
3
3
6.7 (butanoyl C-2), 23.0 (C-14), 22.6 (acetylC‑10 CH ), 20.7
(2H, m, H-6, H-8), 4.92 (1H, dd, J = 4.8, 3.3 Hz, H-2), 4.46−4.44
(1H, m, H-3), 4.34−4.32 (1H, m, H-1), 3.44 (1H, s, OH), 3.21 (1H,
dd, J = 14.8, 3.6 Hz, H-9a), 2.70 (1H, s, OH), 2.39−2.29 (2H, m, 2-
methyl butanoyl H-2, OH), 2.24 (1H, dd, J = 14.8, 3.9 Hz, H-9b), 2.10
3
(
acetylC‑2 CH ), 18.1 (butanoyl C-3), 16.2 (C-13), 13.8 (butanoyl
3
+
C-4), 10.4 (C-15); HRMS m/z 505.1682 [M + Na] , calcd for
C H O Na 505.1680. To a solution of crude 2-acetoxyketone 21
23
30 11
(
0.54 mmol) in dry MeOH (20 mL) was added TEA (2 mL, 14.3
(3H, s, acetyl CH ), 1.96−1.94 (3H, m, H-15), 1.93 (3H, s, acetyl
3
mmol) at room temperature under an atmosphere of argon. The
reaction mixture was stirred at 60 °C for 30 min before it was
concentrated under reduced pressure. The crude product was purified
by flash column chromatography on silica gel using toluene−EtOAc
CH ), 1.75−1.64 (1H, m, 2-methyl butanoyl H-3a), 1.50 (3H, s, H-
3
13), 1.46−1.41 (1H, m, 2-methyl butanoyl H-3b), 1.40 (3H, s, H-14),
1.14 (3H, d, J = 7.0 Hz, 2-methyl butanoyl 2-CH ), 0.91 (3H, t, J = 7.5
3
Hz, 2-methyl butanoyl H-4); 13C NMR (151 MHz, CDCl ) δ 175.5
3
(
2:1) to afford the O-8-debutanoyl 2-acetoxyketone (22) (63 mg, 57%
(2-methyl butanoyl CO), 175.1 (C-12), 172.9 (acetyl CO), 170.3
(acetyl CO), 144.8 (C-5), 126.6 (C-4), 84.9 (C-10), 84.6 (C-2),
83.7 (C-3), 79.0 (C-7/C-11), 78.7 (C-7/C-11), 77.1 (C-6), 66.4 (C-
8), 55.6 (C-1), 41.6 (2-methyl butanoyl C-2), 38.5 (C-9), 26.3 (2-
1
over two steps from 6) as a white solid: H NMR (600 MHz,
CD OD) δ 6.07 (1H, s, H-6), 5.22 (1H, d, J = 3.5 Hz, H-2), 4.67−4.64
3
(
1H, m, H-1), 4.38 (1H, t, J = 3.6 Hz, H-8), 3.13 (1H, dd, J = 14.3, 3.7
Hz, H-9a), 2.36 (1H, dd, J = 14.3, 3.5 Hz, H-9b), 2.11 (3H, s, acetylC‑2
methyl butanoyl C-3), 23.6 (C-14), 22.6 (acetyl CH ), 21.1 (acetyl
3
CH ), 1.99−1.98 (3H, m, H-15), 1.97 (3H, s, acetyl CH ), 1.47
CH ), 16.5 (C-13), 16.3 (2-methyl butanoyl 2-CH ), 13.1 (C-15), 11.8
(2-methyl butanoyl C-4); HRMS m/z 521.1996 [M + Na] , calcd for
3
C‑10
3
3
3
13
+
(
3H, s, H-14), 1.44 (3H, s, H-13); C NMR (151 MHz, CD OD) δ
3
2
03.8 (C-3), 176.5 (C-12), 172.0 (acetylC‑10 CO), 171.4 (acetyl
C H O Na 521.1993.
2-Acetoxytrilobolide (4). 2,4,6-Trichlorobenzoyl chloride (9.4
C‑2
24 34 11
9
CO), 160.6 (C-5), 141.1 (C-4), 85.9 (C-10), 80.8 (C-7/C-11), 80.2
(C-7/C-11), 79.1 (C-6), 75.2 (C-2), 69.8 (C-8), 53.0 (C-1), 40.8 (C-
μL, 0.06 mmol) and TEA (8.4 μL, 0.06 mmol) were added
successively to a solution of angelic acid (6 mg, 0.06 mmol) in dry
toluene (100 μL) at room temperature under an argon atmosphere.
The resulting mixture was stirred for 2 h and then treated with alcohol
24S (15 mg, 0.03 mmol). The reaction mixture was stirred at 75 °C for
2 days, then cooled to room temperature and quenched by the
addition of a saturated aqueous NH Cl solution (3 mL). The aqueous
phase was extracted with EtOAc (2 × 5 mL), and the combined
organic phases were dried over MgSO , filtered, and concentrated
9
1
), 23.1 (C-14), 22.6 (acetylC‑10 CH ), 20.6 (acetyl CH ), 16.3 (C-
3 C‑2 3
+
3), 10.1 (C-15); HRMS m/z 435.1264 [M + Na] , calcd for
C H O Na 435.1262.
19
24 10
(
S)-Methylbutanoate (23). To a solution of O-8-debutanoyl 2-
acetoxyketone (22) (70 mg, 0.17 mmol) in dry THF (1 mL) were
added successively (S)-(+)-2-methylbutyric anhydride (130 mg, 0.7
mmol) in dry THF (0.5 mL) and DMAP (2 mg, 0.016 mmol), at
room temperature under an argon atmosphere. The reaction mixture
was stirred for 1 h at room temperature, then diluted with EtOAc (10
mL) and washed successively with a 2 M aqueous H SO solution (5
4
4
under reduced pressure. The crude product was purified by dry
vacuum column chromatography on silica gel using toluene−EtOAc
2
4
mL), a saturated aqueous NaHCO solution (10 mL), and brine (10
mL). The organic phase was dried over MgSO , filtered, and
(3:1) as eluent to lead to the desired natural product 4 (8.5 mg, 49%)
3
1
as a colorless oil: H NMR (600 MHz, CD OD) δ 6.19 (1H, qq, J =
4
3
concentrated under reduced pressure. The crude product was purified
by dry vacuum column chromatography on silica gel using toluene−
EtOAc (2:1) as eluent to furnish the (S)-methylbutanoate (23) (65.5
7.2, 1.5 Hz, angeoyl H-3), 5.72−5.69 (2H, m, H-3, H-6), 5.62 (1H, t, J
= 3.7 Hz, H-8), 5.51 (1H, dd, J = 4.1, 2.9 Hz, H-2), 4.40−4.36 (1H, m,
H-1), 3.01 (1H, dd, J = 14.6, 3.7 Hz, H-9a), 2.36 (1H, q, J = 6.9 Hz, 2-
methyl butanoyl H-2), 2.31 (1H, dd, J = 14.6, 3.9 Hz, H-9a), 2.09 (3H,
26
22
1
mg, 78%) as a white solid: [α] −40 (c 0.3, CHCl ); H NMR
D
3
(
600 MHz, CDCl ) δ 5.80 (1H, s, H-6), 5.69 (1H, t, J = 3.7 Hz, H-8),
s, acetyl CH ), 2.01 (3H, dq, J = 7.2, 1.5 Hz, angeoyl H-4), 1.94 (3H,
3
3
5
.18 (1H, d, J = 3.4 Hz, H-2), 4.64−4.60 (1H, m, H-1), 4.26 (1H, s,
OH), 3.27 (1H, dd, J = 14.8, 3.7 Hz, H-9a), 3.24 (1H, s, OH), 2.33
1H, q, J = 6.9 Hz, 2-methyl butanoyl H-2), 2.18 (1H, dd, J = 14.7, 3.7
p, J = 1.5 Hz, angeoyl 2-CH ), 1.91 (3H, s, acetyl CH ), 1.88−1.86
3
3
(3H, m, H-15), 1.76−1.71 (1H, m, 2-methyl butanoyl H-3a), 1.52−
(
1.46 (1H, m, 2-methyl butanoyl H-3b), 1.45 (3H, s, H-14), 1.38 (3H,
Hz, H-9b), 2.09 (3H, s, acetylC‑2 CH ), 1.98 (3H, s, H-15), 1.94 (3H,
s, H-13), 1.17 (3H, d, J = 7.1 Hz, 2-methyl butanoyl 2-CH ), 0.95 (3H,
3
3
s, acetylC‑10 CH ), 1.73−1.64 (1H, m, 2-methyl butanoyl H-3a), 1.47
t, J = 7.5 Hz, 2-methyl butanoyl H-4); 13C NMR (151 MHz, CD OD)
3
3
(
3H, s, H-13), 1.46−1.40 (1H, m, 2-methyl butanoyl H-3b), 1.38 (3H,
δ 178.1 (C-12), 176.2 (2-methyl butanoyl CO), 171.9 (acetyl C
O), 171.7 (acetyl CO), 168.7 (angeoyl CO), 141.0 (C-5), 139.4
(angeoyl C-3), 133.3 (C-4), 128.8 (angeoyl C-2), 85.9 (C-10), 85.6
(C-3), 79.6 (C-2), 79.5 (C-11), 79.4 (C-7), 78.0 (C-6), 67.4 (C-8),
58.9 (C-1), 42.7 (2-methyl butanoyl C-2), 39.5 (C-9), 27.3 (2-methyl
butanoyl C-3), 23.5 (C-14), 22.6 (acetyl CH ), 21.1 (acetyl CH ), 20.7
s, H-14), 1.13 (3H, d, J = 7.0 Hz, 2-methyl butanoyl 2-CH ), 0.90 (3H,
3
t, J = 7.5 Hz, 2-methyl butanoyl H-4); 13C NMR (151 MHz, CDCl ) δ
3
2
01.5 (C-3), 175.6 (2-methyl butanoyl CO), 175.1 (C-12), 171.2
(
acetyl
CO), 170.2 (acetyl CO), 156.9 (C-5), 142.1 (C-4),
C‑10 C‑2
8
4.2 (C-10), 79.1 (C-7/C-11), 78.6 (C-7/C-11), 78.0 (C-6), 73.4 (C-
3
3
2), 66.2 (C-8), 51.7 (C-1), 41.6 (2-methyl butanoyl C-2), 38.8 (C-9),
(angeoyl 2-CH ), 16.7 (2-methyl butanoyl 2-CH ), 16.0 (angeoyl C-
3
3
1
413
DOI: 10.1021/acs.jnatprod.5b00333
J. Nat. Prod. 2015, 78, 1406−1414

Journal of Natural Products
Article
4
), 15.9 (C-13), 13.0 (C-15), 12.0 (2-methyl butanoyl C-4); HRMS
(15) (a) Beshara, C. S.; Hall, A.; Jenkins, R. L.; Jones, K. L.; Jones, T.
C.; Killeen, N. M.; Taylor, P. H.; Thomas, S. P.; Tomkinson, N. C. O.
Org. Lett. 2005, 7, 5729−5732. (b) Smithen, D. A.; Mathews, C. J.;
Tomkinson, N. C. O. Org. Biomol. Chem. 2012, 3756−3762.
+
m/z 603.2394 [M + Na] calcd for C H O Na 603.2412.
29
40 12
ASSOCIATED CONTENT
■
(16) Uyanik, M.; Suzuki, D.; Yasui, T.; Ishihara, K. Angew. Chem., Int.
*
S
Supporting Information
Ed. 2011, 50, 5331−5334.
Copies of H, 13C, HCOSY, HSQC, HMBC, and ROESY
NMR spectra of selected compounds. The Supporting
Information is available free of charge on the ACS Publications
website at DOI: 10.1021/acs.jnatprod.5b00333.
1
(17) (a) Smith, M. B.; March, J. G. Advanced Organic Chemistry, 5th
ed.; John Wiley and Sons: Toronto, 2001. (b) Erian, A. W.; Sherif, S.
M.; Gaber, H. M. Molecules 2003, 8, 793−865.
(18) Arentzen, R.; Reese, C. B. J. Chem. Soc., Chem. Commun. 1977,
2
(
70−272.
19) Paulsen, E. S.; Villadsen, J.; Tenori, E.; Liu, H.; Bonde, D. F.;
AUTHOR INFORMATION
■
Lie, M. A.; Bublitz, M.; Olesen, C.; Autzen, H. E.; Dach, I.; Sehgal, R.;
Nissen, P.; Møller, J. V.; Schiott, B.; Christensen, S. B. J. Med. Chem.
Corresponding Author
2
(
013, 56, 3609−3619.
*
Tel: +45 3533 6253. E-mail: soren.christensen@sund.ku.dk.
20) All attempts to cleave the angelate moiety under acidic
Notes
conditions provided the two epimeric alcohols 19 with 30−35% yields
when the conversion was above 50%. Only once was a 75% yield
obtained after treatment of nortrilobolide (1) with p-TsOH at 60 °C
for 6 h. Unfortunately, we were not able to reproduce this result under
the exact same conditions. See the Experimental Section for more
details.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Danish Council for Strategic
Research (grant 06-03-00296B) and GenSpera. We thank
GenSpera for crude nortrilobolide (3) isolated from Thapsia
garganica. H. Liu is gratefully thanked for assistance.
(
21) (a) Davis, F. A.; Haque, M. S. J. Org. Chem. 1986, 51, 4083−
085. (b) Davis, F. A.; Towson, J. C.; Weismiller, M. C.; Lal, S.;
Carroll, P. J. J. Am. Chem. Soc. 1988, 110, 8477−8482.
22) Marín-Barrios, R.; García-Cabeza, A. L.; Moreno-Dorado, F. J.;
Guerra, F. M.; Massanet, G. M. J. Org. Chem. 2014, 79, 6501−6509.
4
(
REFERENCES
■
1
(
23) A trace amount of the 2-acetoxyketone 21 was observed by H
NMR analysis. The starting material was recovered in 35% yield.
24) (a) Nakata, T.; Tanaka, T.; Oishi, T. Tetrahedron Lett. 1983, 24,
(
1) Santana, A.; Molinillo, J. M. G.; Macías, F. A. Eur. J. Org. Chem.
015, 2093−2110.
2) (a) Rasmussen, U.; Christensen, S. B.; Sandberg, F. Acta Pharm.
2
(
(
2
3
653−2656. (b) Oishi, T.; Nakata, T. Acc. Chem. Res. 1984, 17, 338−
44.
Suec. 1978, 15, 133−140. (b) Christensen, S. B.; Norup, E.
Tetrahedron Lett. 1985, 26, 107−111.
(25) For similar oxidation of allylic alcohol on related structures, see
̌
3) (a) Holub, M.; de Groote, R.; Herout, V.; Sorm, F. Collect. Czech.
(
ref 11c.
Chem. Commun. 1968, 33, 2911−2917. (b) Holub, M.; Samek, Z.; de
(26) A trace amount of (S)-(+)-2-methylbutyric anhydride was
̌
Groote, R.; Herout, V.; Sorm, F. Collect. Czech. Chem. Commun. 1973,
8, 1551−1562.
4) (a) Inesi, G.; Sagara, Y. Arch. Biochem. Biophys. 1992, 298, 313−
17. (b) Witcome, M.; Holub, M.; East, J. M.; Lee, A. G. Biochem.
Biophys. Res. Commun. 1994, 199, 916−921.
5) Winther, A.-M. L.; Liu, H.; Sonntag, Y.; Olesen, C.; le Maire, M.;
observed in the NMR spectra.
3
(
3
(
Soehoel, H.; Olsen, C. E.; Christensen, S. B.; Nissen, P.; Møller, J. V. J.
Biol. Chem. 2010, 285, 28883−28892.
(
6) Treiman, M.; Caspersen, C.; Christensen, S. B. Trends Pharmacol.
Sci. 1998, 19, 131−135.
(
7) Doan, N. T. Q.; Paulsen, E. S.; Sehgal, P.; Møller, J. V.; Nissen,
P.; Denmeade, S. R.; Isaacs, J. T.; Dionne, C. A.; Christensen, S. B.
Steroids 2015, 97, 2−7.
(
8) (a) Christensen, S. B.; Andersen, A.; Smitt, U. W. Progr. Chem.
Natl. Prod. 1997, 71, 131−167. (b) Drew, D. P.; Krichau, N.;
Reichwald, K.; Simonsen, H. T. Phytochem. Rev. 2009, 8, 581−599.
(
9) Harmatha, J.; Bude
Kmoníckova,
10) Pickel, B.; Drew, D. P.; Manczak, T.; Weitzel, C.; Simonsen, H.
T.; Ro, D. K. Biochem. J. 2012, 448, 261−271.
11) (a) Kaliappan, K. P.; Nandurdikar, R. S. Org. Biomol. Chem.
005, 3, 3613−3614. (b) Manzano, F. L.; Guerra, F. M.; Moreno-
Dorado, F. J.; Jorge, Z. D.; Massanet, G. M. Org. Lett. 2006, 8, 2879−
882. (c) Andrews, S. P.; Ball, M.; Wierschem, F.; Cleator, E.; Oliver,
sí
́
̌ ̌ ́ ̌ ́
nsky, M.; Vokac, K.; Kostecka, P.;
̌
́
E.; Zídek, Z. Fitoterapia 2013, 89C, 157−166.
(
(
2
2
S.; Hoegenauer, K.; Simic, O.; Antonello, A.; Huenger, U.; Smith, M.
D.; Ley, S. V. Chem.−Eur. J. 2007, 13, 5688−5712 and references
therein.
(
12) (a) Skytte, D. M.; Møller, J. V.; Liu, H.; Nielsen, H. O.;
Svenningsen, L. E.; Jensen, C. M.; Olsen, C. E.; Christensen, S. B.
Bioorg. Med. Chem. 2010, 18, 5634−5646. (b) Andersen, A.; Cornett,
C.; Lauridsen, A.; Olsen, C. E.; Christensen, S. B. Acta Chem. Scand.
1
(
3
(
994, 48, 340−346.
13) Demir, A. S.; Reis, O.; Cigdem Igdir, A. Tetrahedron 2004, 60,
427−3432.
14) Rawlinson, D. J.; Sosnovsky, G. Synthesis 1973, 567−603.
1
414
DOI: 10.1021/acs.jnatprod.5b00333
J. Nat. Prod. 2015, 78, 1406−1414