Enantioselective synthesis of stephacidin B

Article abstract of DOI:10.1021/ja0510616

We describe an enantioselective synthetic route to the antiproliferative alkaloid stephacidin B (1) proceeding in 18 steps and 4.0% yield from 4,4-(ethylenedioxy)-2,2-dimethylcyclohexanone (3). Key features of the synthetic sequence include the use of the Corey-Bakshi-Shibata (CBS) reduction to introduce asymmetry early in the synthetic route, use of the novel electrophile N-(tert-butoxycarbonyl)-5-(isopropylsulfonyloxymethyl)-2,3-dihydropyrrole in a stereoselective enolate alkylation, a diastereoselective Strecker-type addition of hydrogen cyanide to an N-Boc enamine substrate in the solvent hexafluoroisopropanol, platinum-catalyzed nitrile hydrolysis under neutral conditions, cyclization of an acylamino radical intermediate to form the diketopiperazine core of stephacidin B, and implementation of a convergent procedure for introduction of the key 3-alkylidene-3H-indole 1-oxide functional group in the final stage of the route to prepare the structure 2, previously proposed to be the fungal metabolite avrainvillamide (17 steps, 4.2% yield). We observed that synthetic (-)-2 dimerized in the presence of triethylamine to form (+)-stephacidin B (>95%). We also obtained evidence that 2 can form 1 under mild conditions, and that 2 reacts with nucleophiles, such as methanol, by conjugate addition. Copyright

Full text of DOI:10.1021/ja0510616

Published on Web 03/24/2005
Enantioselective Synthesis of Stephacidin B
Seth B. Herzon and Andrew G. Myers*
Department of Chemistry and Chemical Biology, HarVard UniVersity, Cambridge, Massachusetts 02138
Received February 18, 2005; E-mail: myers@chemistry.harvard.edu
Scheme 1
The complex alkaloid stephacidin B (1) was recently isolated
from a fungal culture by a multistep process.1,2 Ultimately,
crystallization and X-ray analysis established the structure shown
(1), save for absolute stereochemistry, which is unknown. It was
recognized that 1 is potentially formed by dimerization of 2. A
mechanism for the putative dimerization reaction was advanced
that involved protonation of 2 followed by formation of bonds b
and a (see structure 1), in that order, via cationic intermediates.1b
The structure 2 had previously appeared in the patent literature
as the antiproliferative fungal isolate “avrainvillamide” (where it
was depicted as ent-2; neither relative nor absolute stereochemical
assignments were discussed)3 and was later described by Sugie and
co-workers as “CJ-17,665”, an isolate from a different fungal strain
(neither relative nor absolute stereochemistry was defined).4 Both
stephacidin B and avrainvillamide are reported to inhibit the growth
of cultured human cancer cells (IC50 values ∼50-100 nM), but
side-by-side comparisons of these compounds have not been made,
so far as we are aware. We have previously described methodology
to synthesize the substructure depicted in red within structure 2
and found that the unsaturated nitrone (3-alkylidene-3H-indole
1-oxide) function within the model compound we synthesized
readily underwent reversible addition of oxygen- and sulfur-based
nucleophiles to the carbon labeled â, which suggested that the
putative dimerization of 2 to form 1 might be initiated by bond
formation to carbon â5 (see also, ref 6), and not carbon R as
originally proposed.1b Here, we describe an enantioselective
synthesis of structure 2 (levorotatory, vide infra) and observe that
(-)-2 undergoes spontaneous dimerization to form (+)-stephacidin
B (1) in the presence of triethylamine.7
Our synthetic route to 2 and stephacidin B (1) begins with the
known, achiral cyclohexanone derivative 3,8 which was transformed
via its trimethylsilyl enol ether into the corresponding R,â-
unsaturated ketone in small-scale reactions by palladium-mediated
oxidation (98% yield, 1.3-g scale, Scheme 1).9 In larger scale
preparations, 3 was oxidized directly with 2-iodoxybenzoic acid
in the presence of 4-methoxypyridine N-oxide (70% yield, 10.4-g
scale).10 Enantioselective reduction of the R,â-unsaturated ketone
produced by either method was achieved using the Corey-Bakshi-
Shibata (CBS) catalytic protocol.11 The stereochemistry of the single
stereogenic center introduced in the CBS reduction step was
subsequently relayed to all others within stephacidin B (1). Because
neither the chirality of 1 nor 2 was known, we randomly selected
the (S)-CBS catalyst to illustrate our enantioselective route to
stephacidin B (1), forming the (R)-allylic alcohol 4 in >95% ee
(96% yield).12 Silyl ether formation and ketal hydrolysis then gave
the R,â-unsaturated ketone 5 (98% yield, two steps).
In a key carbon-carbon bond-forming reaction, the ketone 5
was deprotonated with potassium hexamethyldisilazide (KHMDS)
and the resulting enolate was trapped with the novel electrophile 6
[synthesized from N-(tert-butoxycarbonyl)-2,3-dihydropyrrole by
a sequence involving R-lithiation,13 formylation, reduction (boro-
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C O M M U N I C A T I O N S
hydride), and iso-propylsulfonylation], producing the trans-coupling
product 7 as a single diastereomer (70%, 4.4-g scale). Use of the
methanesulfonate ester corresponding to 6 in the alkylation gave 7
in lower yield (50%), presumably due to competitive proton transfer
from the methanesulfonate group. In a second critical transforma-
tion, the alkylation product 7 was found to undergo Strecker-like
addition of hydrogen cyanide, but only in the solvent hexafluoro-
isopropanol (HFIPA, 0 °C, 4 days), forming the N-Boc amino nitrile
8 (65%) and 16% of the diastereomeric amino nitrile (not shown,
yields of pure diastereomers, separated by flash-column chroma-
tography). We know of no close precedence for Strecker-like
additions to N-Boc enamine substrates such as 7. To establish the
stereorelationships required for synthesis of stephacidin B, the
R-carbon of the ketone 8 was epimerized by deprotonation with
KHMDS followed by quenching of the resultant enolate with pivalic
acid (88%, 487-mg scale). The platinum catalyst 9 of Ghaffar and
Parkins14 then served to transform the nitrile group of the epimerized
product into the corresponding primary amide (10, 85%). The latter
transformation was conducted under essentially neutral conditions;
its success within a complex substrate suggests that the method
may be of value in extension to the hydrolysis of other Strecker-
derived addition products (typically conducted at the extremes of
pH).15 Treatment of the primary amide 10 with thiophenol and
triethylamine led to conjugate addition of thiophenol as well as
cyclic hemiaminal formation, giving the tricyclic product 11 (95%).
A strictly analogous transformation occurred when p-methoxy-
thiophenol was used as nucleophile, giving a crystalline product,
whose structure (including all relative stereochemical assignments)
was solved by X-ray analysis (see Supporting Information).
Dehydration of the cyclic hemiaminal 11 in the presence of
trimethylsilyl triflate and 2,6-lutidine was accompanied by cleavage
of the N-Boc protective group; acylation of the pyrrolidinyl amino
group that was liberated with 1-methyl-2,5-cyclohexadiene-1-
carbonyl chloride, an acyl radical precursor developed by Jackson
and Walton,16 then formed the amide 13 (90% yield, two steps).
Heating of rigorously deoxygenated solutions of 13 and tert-amyl
peroxybenzoate in tert-butyl benzene as solvent at 119 °C produced
the bridged diketopiperazine core of stephacidin B in the form of
the tetracyclic product 14 (62% yield, 144-mg scale). This key
transformation (13 f 14) is believed to involve the formation of
an aminoacyl radical intermediate, as would be expected based on
precedent,16 followed by attack of that aminoacyl radical upon the
more substituted carbon of the enamide C-C double bond and
expulsion of phenylthiyl radical, events that were less predictable.
All efforts to prepare 14 using cyanide as the source of the final
(bridging) carbon atom and intermediates such as 10, 11, or their
derivatives as starting materials were unsuccessful.
Figure 1. 1H NMR spectra at 23 °C (500 MHz, 1:1 DMSO-d6-CD3CN)
of (a) synthetic 2, (b) synthetic 1, (c) stephacidin B from a fungal source.1a
The latter is a scanned spectrum from ref 1a. Note the peaks at δ 3.14,
2.49, and 1.99 in each spectrum correspond to residual water, DMSO-d5,
and HCD2CN, respectively.
to structure 2 (ent-avrainvillamide, based on the structure depicted
in ref 3, vide supra), spectroscopic data for avrainvillamide have
not been published, and thus we cannot claim that our synthetic
material and avrainvillamide are the same. While natural avrain-
villamide is reportedly dextrorotatory (+10.6°, CHCl3),3 synthetic
2 was found to be levorotatory (-35.1°, CHCl3, 25 °C). We draw
no conclusions from the deviations in sign and magnitude of the
two measurements, however, for the temperature of the rotational
measurement of natural avrainvillamide was not reported and, more
importantly, (-)-2 and (+)-1 are readily interconverted in solution
(vide infra). Comparison spectra (1H and 13C NMR) of CJ-17,665,
kindly provided by Dr. Yutaka Sugie (Pfizer Inc.), were similar in
many respects to spectra we obtained from synthetic 2 (notably,
all carbon resonances corresponded, within 0.6 ppm), but there was
1
a lack of correspondence in the H NMR spectra in the region δ
2.45-2.60, which is concerning. Because there are internal
1
inconsistencies in this region in H NMR spectra of CJ-17,665
provided to us, we cannot conclude at this time if synthetic 2 and
CJ-17,665 are the same or different.
An unequivocal link between synthetic and natural materials was
established when we observed that pure synthetic (-)-2 was
transformed into stephacidin B (1) in the presence of triethylamine
at 23 °C (eq 1). Stirring a solution of (-)-2 and a large excess of
triethylamine (15 vol %, 22 mM in 2) in acetonitrile at 23 °C led
to gradual bleaching of the initially bright yellow solution with
concomitant formation of a new, more polar material (TLC
analysis). Concentration of the reaction mixture after 3.5 h and
dissolution of the white solid residue obtained in a 1:1 mixture of
DMSO-d6-CD3CN provided a nearly pure solution of stephacidin
With the development of an efficient synthetic sequence to the
tetracyclic product 14, completion of the synthesis of 2 and 1 was
straightforward. First, 14 was transformed into the R-iodoenone
15 in a three-step sequence (72% yield, Scheme 1). Next, the
R-iodoenone 15 was coupled in a Suzuki reaction with the
arylboronic acid derivative 1617 (56% yield) or, more efficiently,
by an Ullmann-like coupling18 with the aryl iodide 1719 (10 mol %
Pd2dba3, Cu powder, 72% yield). Finally, the nitroarene coupling
product (18) was reduced in the presence of activated zinc
powder,5,20 forming the heptacyclic unsaturated nitrone 2 as a yellow
solid in 49% yield (scale: 5-10 mg, 17 steps, 4.2% yield from 3)
after purification by flash-column chromatography. The chromato-
graphically purified product 2 provided a clean 1H NMR spectrum
(Figure 1a) that was distinct from a published spectrum of
stephacidin B in the same solvent (Figure 1c). Although there is
no doubt that the synthetic material we have prepared corresponds
1
B (1, H NMR analysis, est. >95%, Figure 1b).
1H NMR spectra of synthetic and natural stephacidin B (the latter
from published data)1a corresponded exactly (cf., Figure 1b,c).
Synthetic stephacidin B was found to be dextrorotatory ([R]24
)
D
+91.0°, c 0.25, CH3CN). Rotations of natural 1 have not been
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methoxyphenylthio adduct corresponding to 11 (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
reported; thus, the absolute configuration of stephacidin B remains
unknown at this time.
Our preliminary studies leave little doubt that 1 and 2 are readily
interconverted in solution. For example, concentration of an
acetonitrile-water solution of pure synthetic stephacidin B (1) at
38 °C afforded a 2:1 mixture of 2 and 1, as well as unidentified
decomposition products. Also, whereas solutions of pure 1 in 50%
DMSO-d6-CD3CN appeared to be stable for at least 48 h at 23
°C,21 addition of powdered 3 Å molecular sieves led to partial
retrodimerization, giving a 2:1 mixture of 1 and 2 within 1 h at 23
°C. We also observed partial transformation of 1 to form 2 upon
exposure to silica gel (2D TLC analysis). From the data thus far,
it is clear that (-)-2 and (+)-1 readily interconvert under mild
conditions. This suggests that it is possible that the observed
biological activity of stephacidin B may be attributable to 2 formed
from 1 in vivo. In theory, the converse may be true, though this
seems less likely, simply upon consideration of concentration
effects.22
Finally, we have observed that solutions of 2 in pure methanol-
d4 rapidly (<10 min, 23 °C) form the diastereomeric products of
1,5-addition of methanol-d4 (eq 2). The ratio of diastereomeric
methanol-d4 adducts was ∼15:1 (stereochemistry not assigned). The
ratio of these diastereomeric adducts combined to 2 remaining in
solution suggests an equilibrium constant of 7.7 at 23 °C, although
this value must be regarded as tentative for we have not yet
conducted the experiments to establish that a true equilibrium exists
(the solution decomposed upon concentration). The value 7.7 is
somewhat larger than the equilibrium constant we had measured
for the model unsaturated nitrone previously prepared (K ) 2, 23
°C; the rate of methanol-d4 addition was also faster: t1/2 , 10 min
at 23 °C for 2 vs t1/2 ) 5 h at 23 °C in the model system),5 but
these differences are not surprising given the structural dissimilari-
ties of the two systems.
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Acknowledgment. Financial support from the National Institutes
of Health is gratefully acknowledged. S.B.H. acknowledges a
National Science Foundation predoctoral fellowship. We thank Dr.
Matthew Zajac for his assistance in the preparation of 3, Dr. Andrew
Haidle for X-ray analysis, and Dr. Yutaka Sugie for kindly
providing NMR spectra of CJ-17,665.
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(21) The merits of the DMSO-d6-CD3CN solvent system in stabilizing
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(22) Our results also leave open the possibility that stephacidin B is an artifact
of the isolation of 2; the converse may be true instead, though this would
appear to be less likely.
Supporting Information Available: Detailed experimental pro-
cedures and tabulated spectroscopic data (1H and 13C NMR, FT-IR,
and HRMS) for all new compounds, X-ray analysis of the p-
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