Total Syntheses and Biological Evaluation of Both Enantiomers of Several Hydroxylated Dimeric Nuphar Alkaloids

Article abstract of DOI:10.1002/anie.201503934

Herein, we describe the first total syntheses of five members of the dimeric nuphar alkaloids: (+)-6,6′-dihydroxythiobinupharidine (+)-1a, (+)-6-hydroxythiobinupharidine (+)-1-b, (-)-6,6′-dihydroxythionuphlutine (-)-2-a, (-)-6,6′-dihydroxyneothiobinupharidine (-)-3a, and (+)-6,6′-dihydroxyneothionuphlutine (+)-4-a. The latter two have not been found in nature. We have also made each of their enantiomers (-)-1a-b, (+)-2a, (+)-3a, and (-)-4a. The key step in these syntheses was the dimerization of an α-aminonitrile (a hydrolytically stable surrogate for its corresponding hemiaminal) with chiral Lewis acid complexes. We have also reassigned the literature structures of (+)-1-a-1b - for those instances in which the NMR spectra were obtained in CD₃OD - to their corresponding CD₃O-adducts. Our efforts provide for the first time apoptosis data for (-)-3a, (+)-4a, and all five non-natural enantiomers prepared. The data indicate high apoptotic activity regardless of the enantiomer or relative stereochemical configuration at C7 and C7′. Nuphars are speedy killers: For the first time several of the hydroxylated dimeric nuphar alkaloids and their unnatural enantiomers have been synthesized. First-ever apoptosis data for many of these compounds is provided.

Full text of DOI:10.1002/anie.201503934

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Angewandte
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International Edition: DOI: 10.1002/anie.201503934
German Edition: DOI: 10.1002/ange.201503934
Total Synthesis
Total Syntheses and Biological Evaluation of Both Enantiomers of
Several Hydroxylated Dimeric Nuphar Alkaloids**
Alexander Korotkov, Hui Li, Charles W. Chapman, Haoran Xue, John B. MacMillan,*
Alan Eastman,* and Jimmy Wu*
Abstract: Herein, we describe the first total syntheses of five
members of the dimeric nuphar alkaloids: (+)-6,6’-dihydroxy-
thiobinupharidine (+)-1a, (+)-6-hydroxythiobinupharidine
(+)-1b, (À)-6,6’-dihydroxythionuphlutine (À)-2a, (À)-6,6’-
dihydroxyneothiobinupharidine (À)-3a, and (+)-6,6’-dihy-
droxyneothionuphlutine (+)-4a. The latter two have not
been found in nature. We have also made each of their
enantiomers (À)-1a–b, (+)-2a, (+)-3a, and (À)-4a. The key
step in these syntheses was the dimerization of an a-amino-
nitrile (a hydrolytically stable surrogate for its corresponding
hemiaminal) with chiral Lewis acid complexes. We have also
reassigned the literature structures of (+)-1a–1b—for those
instances in which the NMR spectra were obtained in
CD3OD—to their corresponding CD3O-adducts. Our efforts
provide for the first time apoptosis data for (À)-3a, (+)-4a,
and all five non-natural enantiomers prepared. The data
indicate high apoptotic activity regardless of the enantiomer or
relative stereochemical configuration at C7 and C7’.
cated activation of caspases 8 and 3 (but not caspase 9).
Gopas and co-workers subsequently reported that partially
purified combination extracts of (+)-1b and (À)-2b inhibit
NFkB signaling.[2] Despite the unprecedented speed with
which the dimeric nuphar alkaloids are able to induce
apoptosis, it is surprising that no other studies on their
biological mechanism of action have been published. Previous
reports also show that (+)-1b is an effective antibacterial,[3]
antifungal,[4] and immunosuppressant agent.[5]
The dimeric nuphar alkaloids are structurally unique
sulfur-containing triterpenoids isolated from the yellow water
lilies, Nuphar pumilum, Nuphar japonicum, and Nuphar lutea,
and were first described by Achmatowicz and co-workers
(Figure 1).[6] Three series (i.e., thiobinupharidines (+)-1a–d,
thionuphlutines (À)-2a–d, and neothiobinupharidines 3b–d,
whose structures differ in the relative stereochemical config-
urations at C7 and C7’, are known.[7] However, neither (À)-3a
from the neothiobinupharidine series nor any of the members
of the neothionuphlutine series 4a–d have been found in
nature.
I
n 2006, Yoshikawa and co-workers reported that 6-hydroxy-
thiobinupharidine (+)-1b induces apoptosis in U937 human
leukemia cells within 1 h (2.5–10 mm).[1] To their knowledge,
this was the most rapid induction of apoptosis by a small
molecule ever reported. Additional experiments based on
incubation with 20 mm of specific caspase inhibitors impli-
LaLonde proposed a biosynthetic mechanism for the
formation of the gross structures of the dimeric nuphar
alkaloids beginning from elimination/oxidation of (À)-
[*] Dr. A. Korotkov, H. Li, Dr. H. Xue, Prof. J. Wu
Department of Chemistry
Dartmouth College
Hanover, New Hampshire 03755 (United States)
E-mail: jimmy.wu@dartmouth.edu
C. W. Chapman, Prof. A. Eastman
Department of Pharmacology and Toxicology
Geisel School of Medicine
Lebanon, New Hampshire 03756 (United States)
E-mail: alan.r.eastman@dartmouth.edu
Dr. H. Xue
Natural Products LINCHPIN Laboratory
Texas A&M University
College Station, Texas, 77840 (United States)
Prof. J. B. MacMillan
Department of Biochemistry
UT Southwestern Medical Center
Dallas, Texas, 75390 (United States)
E-mail: john.macmillan@utsouthwestern.edu
[**] J.W. and A.E. were supported, in part, by a Research Scholar Grant,
RSG-13-011-01-CDD from the American Cancer Society. J.B.M. was
supported, in part, by NIH R01 CA149833 and the Welch
Foundation I-1689.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201503934.
Figure 1. Dimeric nuphar alkaloids.
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Lewis acids to (À)-12 would generate a chiral ion-pair
intermediate 13 in which the stereochemical information of
counterion A*À could assist in controlling the facial selectivity
(Scheme 2).
We prepared both enantiomers of 14 using known
methods.[9,13,14] We then converted (À)-14 to the nitrile (À)-
12 by oxidation to the N-oxide, followed by Polonovski
rearrangement and quenching with KCN (Scheme 3).
Scheme 3. Synthesis of nitrile 14.
Scheme 1. LaLonde’s proposed biosynthesis.
With (À)-12 in hand, we surveyed an extensive set of
chiral and achiral Brønsted acids and Lewis acid complexes
for their ability to alter the intrinsic selectivity at C7/C7ꢀ in the
dimerization reactions with Na2S4. These included chiral
phosphoric acids and numerous permutations of Lewis acids
and ligands. Table 1 is an abbreviated summary of these
studies (for a complete list, see the Supporting Information,
SI). The products obtained are bis-nitriles 15–18. The assign-
ment of the stereochemical configuration at C7/C7’ was
accomplished by converting them to the corresponding fully
reduced compounds 1d, 2d, 3d, and 4d, which were then
compared with literature data. After considerable experi-
mentation, we identified In(OTf)3/(3R,8S)-22 as the optimal
Lewis acid–ligand[15–17] combination for generating (+)-15
(corresponds to the thiobinupharidine configuration;
entry 16), whereas Cu(OTf)2 alone gave predominantly (À)-
17 (corresponds to the neothiobinupharidine configuration;
entry 1). Under these conditions, we could obtain syntheti-
cally useful amounts of all stereoisomers 15–18. The use of
chiral phosphoric acids or catalytic amounts of Lewis acids
was not optimal. That there was no pronounced matched–
mismatched effect with opposite enantiomers of ligands
suggests that the mechanism of dimerization may be more
complex than simple substrate–ligand interaction.
nupharidine (Scheme 1).[8] Attack of ene-iminium 6 with an
ambiphilic sulfur reagent 7 would initiate dimerization
through formation of the central thiaspirane ring. Hydration
or reduction of bis-iminium 10 would result in compounds 1–
4. Shenvi and co-workers proved the feasibility of LaLondeꢀs
proposal by completing the first and only total synthesis of
a member of the dimeric nuphar alkaloids, (À)-neothiobinu-
pharidine (À)-3d,[9] which unfortunately is not active in
apoptosis assays.[1] Yoshikawa reported that at least one
hemiaminal is required to see substantial apoptotic activity.
Considering 1) the unprecedented speed at which the
dimeric nuphar alkaloids are able to induce apoptosis, 2) the
lack of information regarding their biological mechanism of
action, and 3) their complex and exquisite chemical structure,
we decided to pursue the total syntheses of the biologically
active hydroxylated dimeric nuphar alkaloids. In the absence
of any catalysts or promoters, Shenvi reports that dimeriza-
tion favors the formation of the stereochemical relationship
corresponding to the neothiobinupharidine architecture (4:1
series 3 versus all others).[9] Thus, the principle challenge we
faced was how to override the inherent diastereochemical
outcome at C7 and C7’ during the formation of the
thiaspirane ring.
Although the requisite ene-iminium 6 (Scheme 1) can be
derived from hemiaminal 11 (Scheme 2), 11 is a hydrolytically
labile compound that cannot be purified by chromatography
and must be freshly made before each use. We therefore
elected to pursue a strategy employing nitrile (À)-12 as
a “masked” hemiaminal that is both chromatographically and
hydrolytically stable.[10–12] We further reasoned that promot-
ing the dimerization by application of chiral Brønsted or
We then converted bis-nitrile (+)-15 to the corresponding
bis-hemiaminal (+)-1a by treatment with AgNO3 in MeCN/
H2O at 458C (85% yield, Scheme 4).[18] The preparation of
Scheme 2. Strategy for controlling facial selectivity in thiaspirane
formation.
Scheme 4. Completion of synthesis.
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Table 1: Optimization of dimerization.
nonnatural) enantiomers (À)-1a–b, (+)-2a, (+)-3a, and (À)-
4a.
1
The H and 13C NMR spectra of synthetic (+)-1a–1b in
both CD3OD and CDCl3 were identical to both reported
literature values and to the spectra of materials isolated from
Nuphar lutea carried out by our group (see SI for isolation/
purification details) on the condition that the NMR experi-
ment in CDCl3 occurred first.[20–23] If the data acquisition for
the NMR spectrum in CD3OD preceded that of the NMR
experiment in CDCl3, then noticeable differences between
synthetic and literature values were apparent in the CDCl3
1H NMR spectra, while no differences were observed in the
CD3OD spectrum. Under these circumstances, if we removed
the CD3OD in vacuo and allowed the residue to age in air, the
material slowly converted back to (+)-1a–1b as judged by
1H NMR spectroscopy in CDCl3. The conversion was greatly
facilitated if the residue was washed with saturated aq.
NaHCO3. We speculated that in CD3OD, (+)-1a–1b is
converted to the CD3O-adducts 23a and 24a (Figure 2).
Entry[a]
1
Metal
Ligand[b]
none
15/16/17/18
Cu(OTf)2
0.2:0.2:1.0:0.3
7%:6%:51%:10%[c]
0.3:0.3:1.0:0.2
0.3:0.2:1.0:0.2
0.4:0.2:1.0:0.3
0.4:0.2:1.0:0.2
0.3:0.2:1.0:0.2
0.4:0.2:1.0:0.2
0.9:0.2:1.0:0.2
0.8:0.3:1.0:0.2
0.9:0.3:1.0:0.3
1.1:0.3:1.0:0.2
1.1:0.3:1.0:0.2
1.0:0.3:1.0:0.3
1.0:0.3:1.0:0.2
0.2:0.2:1.0:0.3
1.9:0.4:1.0:0.3
42%:8%:23%:6%[c]
1.6:0.3:1.0:0.3
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Sc(OTf)3
In(OTf)3
Cu(OTf)2
Sc(OTf)3
In(OTf)3
Cu(OTf)2
In(OTf)3
In(OTf)3
In(OTf)3
In(OTf)3
In(OTf)3
In(OTf)3
In(OTf)3
Cu(OTf)2
In(OTf)3
none
none
(S)-19a
(S)-19a
(S)-19a
(R)-19a
(R)-19a
(R)-19b
(R)-19c
(R)-19d
(4R,5S)-20a
(4R,5S)-20c
(4R,5R)-21
none
Figure 2. Revised structures.
(3R,8S)-22
Indeed, when we dissolved (+)-1a–b in CH3OH, then
removed the excess methanol and obtained the 1H NMR
spectra in CDCl3, new singlet resonances integrating to 3H
each were present. We assigned these resonances to the
methoxy protons in 23b–24b. From these data, we believe it
necessary to revise those literature structures of (+)-1a–b—in
which NMR experiments were carried out in CD3OD—to the
corresponding CD3O-adducts 23a and 24a. It is likely that an
analogous revision of 2a is also warranted. The ethoxylated
versions of some dimeric nuphar alkaloids have been noted,
but there is some disagreement as to whether these were
artifacts of the isolation/purification process.[24,25]
17
In(OTf)3
(3S,8R)-22
[a] Yields of isolated products for selected entries.
We then assayed both enantiomers of 1a–1b, 2a, 3a, 4a,
as well as bis-nitriles 15–18 for their apoptotic properties
against the human U937 cell line as judged by caspase
cleavage of poly(ADP-ribose) polymerase (PARP), a marker
of apoptosis (Table 2 and Figure 3). The activity of synthetic
(+)-1a–1b and (À)-2a were similar to that of the correspond-
ing materials we isolated from Nuphar lutea (see SI for full
data set). Moreover, the mono- and dihydroxylated dimeric
nuphar alkaloids all exhibited nearly the same activity,
regardless of their absolute or relative stereochemical con-
figurations. It also appears that (À)-3a, which is not found in
nature, seems to be the most potent at the 1 h time point
(entry 7). Bis-nitriles 16–18 exhibited no apoptotic activity.
2a–4a was carried out in a similar fashion and in comparable
yields. Selective monoreduction of the C6ꢀ hemiaminal over
the one at C6 in 1a could be accomplished to give 1b.[19] This
type of chemoselective reduction appears limited to 1a as
similar attempts on 2a--4a resulted in numerous reduction
products. The absolute stereochemical configurations as
depicted in Figure 1 are the naturally occurring ones, but we
also completed the syntheses of the nonnatural (or predicted
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How to cite: Angew. Chem. Int. Ed. 2015, 54, 10604–10607
Table 2: Apoptosis assays (PARP cleavage).
Angew. Chem. 2015, 127, 10750–10753
Entry
Compound
Enantiomer
1 h[a]
6 h[a]
1
2
3
4
5
6
7
8
9
10
(+)-1a
(À)-1a
(+)-1b
(À)-1b
(À)-2a
(+)-2a
(À)-3a
(+)-3a
(+)-4a
(À)-4a
natural
unnatural
natural
unnatural
natural
unnatural
predicted natural
predicted unnatural
predicted natural
predicted unnatural
–
–
–
–
–
–
5
10
–
–
5.0
2.5
5.0
5.0
2.5
5.0
1.25
2.5
2.5
2.5
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In conclusion, we have successfully completed the first
syntheses of several hydroxylated nuphar alkaloids. This was
accomplished by means of dimerization of nitrile (À)-12,
promoted by chiral Lewis acid complexes. Chemoselective
mono-reduction of (+)-6,6’-dihydroxythiobinupharidine
(+)-1a furnished (+)-6-hydroxythiobinupharidine (+)-1b.
Moreover, for the first time, apoptosis data is available for
these compounds. PARP cleavage assays confirm their ability
to induce very rapid apoptosis in human U937 cells (within
1 h). We anticipate that these compounds will serve as useful
tools for dissecting an important, but as yet undefined step in
the regulating apoptosis. Studies to clarify the biological
mechanism by which they operate are ongoing.
Keywords: apoptosis · cancer · caspases · nuphar alkaloids ·
total synthesis
Received: April 29, 2015
Published online: July 16, 2015
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