Asymmetric, stereocontrolled total synthesis of (+) and (-)-spirotryprostatin B via a diastereoselective azomethine ylide [1,3]-dipolar cycloaddition reaction

Article abstract of DOI:10.1016/S0040-4020(02)00630-0

The asymmetric, stereocontrolled total syntheses of (+) and (-)-spirotryprostatin B (2) are described. Formation of the core pyrrolidine ring was accomplished via a diastereoselective asymmetric [1,3]-dipolar cycloaddition reaction. Addition of 3-methoxy-

Full text of DOI:10.1016/S0040-4020(02)00630-0

Tetrahedron 58 (2002) 6311–6322
Asymmetric, stereocontrolled total synthesis of (1) and
(2)-spirotryprostatin B via a diastereoselective azomethine ylide
[1,3]-dipolar cycloaddition reaction
Paul R. Sebahar,a Hiroyuki Osada,b Takeo Usuib and Robert M. Williamsa
,
*
aDepartment of Chemistry, Colorado State University, Fort Collins, CO 80523, USA
bLaboratory of Antibiotics, RIKEN, 2-1 Wako-shi, Saitama 351-0198, Japan
Received 21 March 2002; accepted 22 April 2002
The authors wish to dedicate this paper to the myriad of impressive accomplishments of Professor Yoshito Kishi of Harvard University and
in recognition of his receiving the Tetrahedron Prize
Abstract—The asymmetric, stereocontrolled total syntheses of (þ) and (2)-spirotryprostatin B (2) are described. Formation of the core
pyrrolidine ring was accomplished via a diastereoselective asymmetric [1,3]-dipolar cycloaddition reaction. Addition of 3-methoxy-3-
methylbutanal to (5R,6S )-2,3,5,6-tetrahydro-5,6-diphenyl-1,4-oxazin-2-one generated an azomethine ylide that reacted with ethyl
oxindolylidene acetate to furnish the desired cycloadduct (11) that possessed the correct relative and absolute stereochemistry of natural
spirotryprostatin B. The key dipolar cycloaddition reaction sets four contiguous stereogenic centers. Reductive cleavage of the oxazinone
generated the spiro-oxindole pyrrolidine (19) that was coupled to D-proline benzyl ester and cyclized to the pentacyclic diketopiperazine 22.
A Barton-modified Hunsdiecker protocol effected oxidative decarboxylation to yield 12-epi-spirotryprostatin B (30). Thermodynamic
epimerization of the D-proline stereogenic center with sodium methoxide yielded spirotryprostatin B as the major product. The antipode of
the natural product, ent-spirotryprostatin B, was prepared from (5S,6R)-2,3,5,6-tetrahydro-5,6-diphenyl-1,4-oxazin-2-one. Several synthetic
intermediates and spirotryprostatin analogs were tested for their activity as G2/M phase cell cycle inhibitors and microtuble assembly against
3Y1 and tsFT210 mammalian cells. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
these screening technologies to identify a wide array of
interesting natural products from the fermentation broth of
the fungus Aspergillus fumigatus and other microbial
sources.3
Elucidating the regulatory machinery of the cell cycle is
crucial to understanding how defects in the regulatory
mechanism of the cell result in uncontrolled growth and
differentiation, such as cancer.1 Small-molecule natural
products are proving invaluable in contemporary studies of
cellular probes through their ability to specifically bind
target proteins that modulate signal transduction cascades.
Numerous examples exist in which the biological function
of a particular cellular factor have been investigated through
the use such compounds.2 Therefore, the development of
new and specific inhibitors of signal transduction cascade
pathways will continue to be extremely important in the
understanding of the regulatory mechanism of the cell cycle.
Included in the families of fungal metabolites identified in
this manner are the fumitremorgins,4 the tryprostatins,5 the
cyclotryprostatins4 and the spirotryprostatins (1 and 2, Fig.
1).6 The primary target of tryprostatin A and cyclo-
tryprostatins A and B are microtubules which induce
M-phase specific inhibition and microtubule disassembly.7
This family of prenylated, cyclo-L-Trp-L-Pro-derived poly-
cyclic alkaloids has received considerable attention recently
due to their unique biological activities and interesting
chemical structures. These substances have therefore
attracted considerable synthetic attention and individual
total syntheses of each representative class have been
reported.8–10
Recently, powerful bioassays have been developed to
specifically identify new natural products that inhibit the
progression of the cell cycle at distinct phases. Using
temperature-sensitive mammalian tsFT210 cells and rat
normal fibroblast 3Y1 cells, Osada et al., have exploited
The tryprostatin family of secondary metabolites are the
consequence of several modes of isoprenylation of the
tryptophan moiety of the simple cyclic dipeptide progenitor
cyclo-L-Trp-L-Pro.11 The structurally most interesting and
complex members of this family are the spirotryprostatins A
and B which, curiously display among the weakest
Keywords: cycloaddition reaction; azomethine; aldehydes.
*
Corresponding author. Tel.: þ1-970-491-6747; fax: þ1-970-491-5610;
e-mail: rmw@chem.colostate.edu
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)00630-0

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P. R. Sebahar et al. / Tetrahedron 58 (2002) 6311–6322
Scheme 1. General synthetic plan for the synthesis of 2.
With the construction of the desired framework represented
as in the pentacyclic substance 7, completion of the
synthesis would mandate judiciously timed oxidative
decarboxylation and installation of the isoprene-derived
unsaturation via elaboration of the pentacyclic substance 7.
The utility of [1,3]-dipolar cycloadditions is a well-
established synthetic method for the formation of variously
substituted pyrrolidine rings.13 Numerous methods exist,
including reaction of azomethine ylides with oxindolylidene
acetate dipolarophiles, for the construction of spiro-
oxindole systems related to that present in 1 and 2.14
However, the literature contains conflicting evidence as to
the regio- and diastereochemical outcome of such reactions.
Figure 1. Structures of the spirotryprostatins, tryprostatins, cyclotrypros-
tatins and fumitremorgins.
biological activity of this family of cell cycle inhibitors.
Isolated in 1996, from A. fumigatus, spirotryprostatin A (1)
and spirotryprostatin B (2) were shown to completely inhibit
the progression of cells at concentrations greater than 253
and 34.4 mM, respectively. Despite their relatively modest
biological activity relative to other members of this family,
the spirotryprostatins have nonetheless garnered the most
attention due to their intriguing molecular structures. The
detailed mechanism of action by which these substances
inhibit microtubule assembly is presently not known and
studies to discover the target of these natural products have
been hampered by the small quantities of these substances
that can be conveniently isolated from the producing
organism. Herein, we present a full account of our efforts
towards the total synthesis of both antipodes of spiro-
tryprostatin B and analogs.12
2. Results and discussion
At the outset, we focused on devising an efficient and
stereocontrolled method to construct the core spiro-
oxindole-containing pyrrolidine ring as the backbone to
our synthetic strategy as shown Scheme 1. It was envisioned
that an asymmetric [1,3]-dipolar cycloaddition between a
chiral azomethine ylide of the general type 4 and an
oxindolylidene acetate (3) could, in both a relative and
absolute sense, generate the desired spiro-amino acid 5. If
successful, the reaction would generate two of the
three necessary stereogenic centers contained in the
natural product. Coupling with a suitable proline
derivative (6) followed by cyclization would yield the
diketopiperazine 7.
Scheme 2.

P. R. Sebahar et al. / Tetrahedron 58 (2002) 6311–6322
6313
Azomethine ylides derived from our diphenyl oxazinone-
based glycine template,15 and related chiral glycine-based
azomethine ylide equivalents,16 reveal that the regio- and
stereochemistry of the resulting cycloadducts are dependent
upon both the nature of the aldehyde and the dipolarophile.
While simple symmetrical alkenyl dipolarophiles (i.e.
dimethylmaleate) usually proceed with a high degree of
endo-selectivity, there are few studies that address the
regiochemical aspects of asymmetrically substituted
dipolarophiles. It was therefore difficult to predict if the
amide or the ester moiety of the oxindolylidene acetate (3)
would dominate in directing the facial approach of the
dipole. In this particular instance, there are thus eight
possible diastereomeric transition state structures, and only
one of which culminates in the desired spirotryprostatin
stereostructure.
Scheme 3. Mechanism proposed for partitioning of 14 to cycloadducts 11
and 12.
With respect to the relative stereochemistry of the prenyl
side-chain, the reaction was expected to be diastereo-
selective in the desired sense since, earlier studies in our
laboratories suggested that bulky aliphatic aldehydes
preferentially form the E-ylide.15 Assuming that the
E-ylide geometry would dominate in the present case, four
possible diastereomers could be reasonably expected to
result from the planned cycloaddition. As shown in Scheme
2, reaction of the azomethine ylide derived from oxazinone
817 and aldehyde 918 with ethyl oxindolylidene acetate 1019
in the presence of molecular sieves in hot toluene, resulted
in the formation of two cycloadducts 11 and 12 in a 1:2 ratio
and 86% combined yield. We were pleased to observe that
this initial set of reaction conditions indeed afforded the
desired cycloadduct as evidenced by 1H, 13C NMR and nOe
experiments.
Addition of the aldehyde 9 to oxazinone 8 should initially
generate the salt 14 which can then be deprotonated a- to the
lactone carbonyl or b- to the nitrogen atom to give the ylide
15 or the eneamine 16, respectively. Dipole 15 can then
condense with ethyl oxindolylidene acetate (10) to generate
the desired cycloadduct 11. If eneamine 16 is formed, then
under the thermal conditions of the reaction, nitrogen-
assisted extrusion of methoxide furnishes the thermodyna-
mically more stable (relative 14) conjugated iminium ion
species 17.
Deprotonation a- to the carbonyl would then generate the
azomethine ylide 18 that can suffer cycloaddition to yield
12. To minimize formation of the undesired cycloadduct,
the reaction was performed at 608C instead of at reflux
temperature, improving the yield of 11 to 82% with only 6%
of 12 being formed.
The relative and absolute stereochemistry of the desired
cycloadduct 11 was further secured through single-crystal
X-ray analysis as depicted in Scheme 2. This result
suggested that approach of the dipolarophile to the
azomethine ylide occurs with the carboethoxy group being
positioned opposite to the bulky phenyl groups in an
exo-fashion. The reaction must therefore proceed via an
E-beta-exo transition state20 and constructs the entire
prenylated tryptophyl moiety of spirotryprostatin B in a
single, simple operation. However, the yield was far from
ideal since 11 was isolated as a 1:2 mixture along with 12,
which results from the elimination of methanol from the
desired cycloadduct. Additionally, a small amount of a third
product 13 was produced and confirmed to be the reversed
regio- and stereoisomer of the desired cycloadduct. There-
fore, additional effort was directed at shifting the ratio of
cycloadducts towards compound 11.
With the key spiro-tetracyclic intermediate (11) in hand,
focus shifted to construction of the diketopiperazine as
detailed in Scheme 4. Reductive cleavage of the chiral
auxiliary afforded acid 19 which was esterified with
TMSCHN2 to yield the corresponding methyl ester 20 in
86%. Attempts to acylate the nitrogen of the pyrrolidine ring
We had not foreseen the possible loss of methanol from the
aldehyde progenitor as these conditions had heretofore
proven to be very mild and tolerated a wide range of
aromatic and aliphatic aldehydes.21 It was not clear whether
the elimination was occurring during the reaction or after
formation of the cycloadduct. Re-subjecting 11 to refluxing
toluene in the presence of molecular sieves did not afford
any of the eliminated cycloadduct 12 suggesting that a
distinct azomethine ylide was being formed in situ and a
proposed mechanism for the formation of 12 is illustrated in
Scheme 3.
Scheme 4.

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P. R. Sebahar et al. / Tetrahedron 58 (2002) 6311–6322
of 20 failed under a number of conditions. Only trace
amounts of product were ever obtained and were compli-
cated by acylation of the oxindole nitrogen. The decreased
nucleophilicity of the pyrrolidine nitrogen can be attributed
to the surrounding steric bulk. The ester and propylidene
groups which are a- to the amine, are in an anti-
configuration effectively blocking each face of the nitrogen
from electrophilic attack.
This initially discouraging result was eventually turned into
an asset since, it was soon realized that the steric hindrance
about the nitrogen might allow for direct coupling on the
free, zwitterionic amino acid without concomitant self-
condensation with the active ester. Thus, amino acid 19 was
taken on crude from the preceeding hydrogenation step and
directly coupled with L-proline benzyl ester with BOP22 as
the activating agent to give the dipeptide 21 in 74% for the
two steps. Reduction of the benzyl ester followed by BOP-
mediated cyclization afforded the desired diketopiperazine
(DKP) 22 in excellent yield. The stage was now set for
sequential installation of the two olefinic moieties.
Scheme 6.
Several strategies were examined for the installation of the
enamide functionality and the prenyl side-chain. The initial
plan was to first form the C-8/C-9 unsaturation and
subsequently secure the C-19/C-20 olefinic group since
the planned oxidative decarboxylation would involve an
alkyl radical that might react with a proximal prenyl group.
However, it was recognized that if an undesired intra-
molecular radical cyclization process were to occur, it
would have to occur via a stereoelectronically disfavored
5-endo-trig cyclization.23 With these considerations in mind
attempts to effect a radical-based oxidative decarboxylation
were pursued. Saponification of the ethyl ester of 22 was
attempted using LiOH in THF/MeOH/H2O, but failed to
give any of the desired carboxylic acid. After some
exploration, it was found that LiI in refluxing pyridine24
furnished the desired carboxylic acid (Scheme 5). However,
attempts to affect the oxidative decarboxylation either
through the use of Pb(OAc)425 or iodosobenzene diacetate26
were unsuccessful, apparently due to the lability of the
oxindole 28 amide. The oxindole nitrogen atom of 22 was
protected as the corresponding SEM derivative 23.
Cleavage of the ethyl ester with LiI in refluxing pyridine
furnished the corresponding acid that was subjected to
Kochi-type conditions generating the eneamide 24, albeit
only in poor yields (10–25%).
and acidic conditions, more vigorous conditions resulted in
decomposition. These results suggested that the isopropyl-
idene group needed to be in place prior to installation of the
C-8/C-9 unsaturation. To this end, diketopiperazine 22 was
subjected to treatment with TsOH in refluxing toluene
resulting in the formation of the desired olefin 25 in good
yield with only trace amounts of the isomeric disubstituted
olefin present (Scheme 6). As before, the SEM group was
used to protect the oxindole nitrogen (26).
Subjecting the carboxylic acid, resulting from saponifica-
tion of the ethyl ester 26, to a classical Kochi-type oxidative
decarboxylation protocol produced the over-oxidized triene
27. Despite extensive effort, we were unable to obviate
oxidation of the proline residue under a wide range of
Kochi-type conditions. Triene 27 provided an intriguing
analog of spirotryprostatin B once the protecting group was
removed. The free oxindole 28 was thus obtained using
dimethyl aluminum chloride followed by heating in
diisopropylethyl amine.27
We next turned to examining a Barton-modified
Hunsdiecker reaction as a possible solution to the oxidative
decarboxylation problem.28 This reaction has found utility
in a number of applications for the generation of alkyl
halides, however the application of this method for the
formation of a,b-unsaturated amino acid derivatives has
seldom been reported. The ethyl ester was converted to the
acid as above with lithium iodide in hot pyridine yielding
Unfortunately, all attempts to install the C-19/C-20
unsaturation with 24 as a substrate were uniformly
unsuccessful under a range of acidic elimination conditions.
While the enamide proved to be stable to both mildly basic
Scheme 5.
Scheme 7.

P. R. Sebahar et al. / Tetrahedron 58 (2002) 6311–6322
6315
carboxylic acid 29, (Scheme 7). Treatment of 29 with DCC,
DMAP and N-hydroxy pyridine-2-thione yielded a product
30 whose 1H NMR spectroscopic signatures closely
resembled those of the natural product with the exception
of some slight variations in the chemical shifts of several
resonances.
The Barton-modified Hunsdiecker protocol converts the
carboxylic acid, via radical decarboxylation of the
N-hydroxy pyridine-2-thione ester, into a secondary alkyl
radical that is quenched by the solvent, BrCCl3, into the
corresponding alkyl bromides, which under thermal con-
ditions, eliminate HBr to form the olefin. The overall yield
for this process was far from exceptional (34–43%) and, it
is possible that the formation and relative rates of
elimination of the two diastereomeric bromides, might
have contributed to the recovery of only moderate amounts
of the desired product. We suspect that only the bromide
that is positioned trans, antiperiplanar to the a-hydrogen,
suffers facile elimination to give 12-epi-spirotryprostatin
B. Our excitement that the reaction had occurred as planned
was tempered by the discrepancies observed in the 1H NMR
data between the natural spirotryprostatin B and product 30.
Scheme 9. Thermodynamic epimerization of 32 to 2.
If the dehydration step was the culprit in the loss of
stereochemical integrity of 22, then subjecting the two
substrates (22 and 31) separately to the elimination
conditions would yield the same product. This indeed
proved to be the case wherein it was observed that the
pentacyclic product 25 was formed exclusively from either
substrate when treated with TsOH in hot toluene. It is well-
known that cis-diketopiperazines are thermodynamically
more stable than the corresponding trans-isomers for cyclic
anhydrides of proline.29 In contrast, reported syntheses of
the fumetrimorgins have demonstrated the ability of
substrates with the 6-6-5-ring system to undergo epimeriza-
tion from the cis-configuration to the trans-configuration.30
The absolute stereochemistry of the L-proline residue was
not in doubt in the initial stages of the synthesis and both the
relative and absolute stereochemistry of the spiro-oxindole
moiety had been secured by X-ray crystallographic analysis
of 11. Thus, we suspected that an epimerization had
occurred in the proline ring either at the stage of the
elimination of methanol from 22, or during the ethyl ester
cleavage to ultimately give 12-epi-spirotryprostain B 30.
With the stereochemical issues clarified, we returned to the
task of converting 12-epi-spirotryprostatin B (30) into the
natural stereoisomer as shown in Scheme 9. Addition of
NaOMe in MeOH at 08C to 30 yielded an equilibrium
mixture of spirotryprostatin B (2): 12-epi-spirotryprostatin
B (30) in a 2:1 ratio. These diastereomers were easily
separated by chromatography and the recovered 30 could be
re-subjected to the epimerization protocol giving 2 in 62%
overall yield for the two cycles. The synthetic and natural
specimens of (2)-spirotryprostatin B displayed identical
spectroscopic data including optical rotation. In like
fashion, (þ)-ent-spirotryprostatin B was synthesized start-
ing with the opposite antipode of 8.17
To decipher at what stage the suspected epimerization
reaction was occurring, the complementary D-proline-
derived cis-diketopiperazine 31, was constructed as shown
in Scheme 8. This was accomplished in a similar fashion to
that utilized for the formation of the trans-diketopiperazine
22. Thus, coupling of amino acid 19 with D-proline benzyl
ester (74%) followed by hydrogenation of the benzyl ester
and cyclization (94% over two steps) afforded 31.
3. Biological activity
The effects of compounds 11, 19, 20, 21, 22, 25, 28, 29, 30,
their enantiomers, and ent-spirotryprostatin B on cell cycle
control and microtuble assembly were examined. Given the
moderate activities of the title compounds (IC50¼14.0 mM
for spirotryprostatin B), it was not surprising to find that all
of the spirotryprostatin analogs prepared in this study that
were tested had no effect on in vitro microtubule assembly
and had little or no effect on in vitro cell cycle inhibition.
Three compounds (30, ent-30, and ent-2) did however,
provide some intriguing results.
12-epi-Spirotryprostatin B (30) was shown to cause partial
accumulation of cells at the G2/M phase at concentrations of
125 mM but were toxic to 3Y1 and tsFT210 cells at 250 mM
or higher concentrations. The enantiomer of 30 was
however, neither toxic to the cells nor showed any activity
for cell cycle proliferation and microtuble assembly. Similar
results were seen in the testing of spirotryprostatin (2) and
ent-2. Spirotryprostatin B has been reported to inhibit
tubulin polymerization and to be cytotoxic to mammalian
cells6 whereas ent-2 had no effect on in vitro microtubule
Scheme 8.

6316
P. R. Sebahar et al. / Tetrahedron 58 (2002) 6311–6322
assembly or in vitro cell cycle inhibition but was toxic to
3Y1 and tsFT210 cells at 31.3 and 15.6 mM concentrations,
respectively. These data suggest that the molecular target of
ent-spirotryprostatin B is different from that of the natural
product. Further studies aimed at elucidating the cellular
target of these substances and the molecular mechanism by
which they arrest the cell cycle are currently under
investigation in these laboratories.
added dropwise over 15 min. 3-Methyl-3-methoxybutan-1-
ol (12.0 g, 100 mmol) along with pyridine (16.5 mL,
200 mmol) in 100 mL of CH2Cl2 was added dropwise
over 15 min. The reaction was stirred 15 min. more at
2788C and then Et3N (75 mL, 0.5 mol) in 75 mL of CH2Cl2
was added over 15 min with vigourous stirring. The solution
was kept at 788C for 15 min and then warmed to 48C and
stirred for another 15 min. 1N HCl was used to acidify to pH
24 and the layers separated. The aqueous layers were
extracted with three 50 mL portions of CH2Cl2. The organic
layers were combined, dried over Na2SO4, filtered and
evaporated. The crude product could be obtained by column
chromatography with 2:1 CH2Cl2/Et2O as the eluent to yield
11.5 g (97%). The product was further purified by distilla-
4. Conclusion
In summary, the synthesis of both antipodes of spiro-
tryprostatin B (2) has been achieved utilizing a diastereo-
selective, asymmetric [1,3]-dipolar cycloaddition reaction
as the key step. This strategy, which sets four contiguous
stereogenic centers in one step, also appears to be adaptable
to the synthesis of spirotryprostatin A and efforts are
underway in this regard. In addition, a tertiary methyl ether
was demonstrated to serve as a suitable progenitor of the
prenyl group providing an alternative method for the
introduction of the isopropylidene functionality. Moreover,
the inherent thermodynamic stability of diketopiperazines
with the 5-6-5 ring system have been shown to preferen-
tially favor the cis-configuration. Application of the
methodology developed in this work is being applied to
the synthesis of other potentially biologically useful
members of the spirotryprostatin structural class.
1
tion to remove any impurities. For 9: H NMR (300 MHz,
CDCl3) d CHCl3: 1.31 (s, 6H), 2.53 (d, J¼3.3 Hz, 2H),
3.26, (s, 3H), 9.84 (t, J¼3.3 Hz); 13C NMR (75 MHz,
CDCl3) d CDCl3: 18.6, 42.7, 46.6, 67.1, 195.4; IR
(NaCl/neat) 2974, 2937, 2828, 1732 cm21; LRMS (EIþ)
calcd for C6H13O2 (m/z) 117.1, found (m/z ) 117.1.
5.3. Cycloaddition
To a 500 mL round-bottom flask with stir bar was added
(5R,6S )-2,3,5,6-tetrahydro-5,6-diphenyl-1,4-oxazin-2-one
(5.0 g, 19.8 mmol), ethyl oxindolylidene acetate (10) (6.4 g,
˚
29.6 mmol) and 5.0 g of activated 3 A molecular sieves. An
oven-dried condenser was attached and the system was
flushed with argon. Freshly distilled toluene (250 mL) was
added followed by 3-methyl-3-methoxybutanal (9, 2.75 g,
23.7 mmol). The reaction was then heated to 608C and kept
at that temperature for 1 h at which time the heating mantle
was turned off. The reaction was allowed to cool to room
temperature and filtered through Celite to remove the sieves.
Concentration afforded an orange solid which was
chromatographed (SiO2, 4:1 hexanes/EtOAC!1:1 hexane-
s/EtOAc) to afford 9.2 g of 11 (82%) and 0.70 g of 12
(6.3%) and 0.12 g of 13 (1.1%). Analytical samples of 11
were generated by recrystallization from EtOH.
5. Experimental
5.1. Cell culture and proliferation assay
Rat normal fibroblast 3Y1 cells31 were grown in Dulbecco’s
modified MEM culture medium supplemented with 10%
fetal calf serum under a humidified atmosphere containing
5% CO2. Exponentially growing 3Y1 cells were treated with
the test compounds for 24 h. The distribution of DNA
content was determined by flow cytometry and relative cell
numbers (cell number at 24 h per initial cell number at
0 h£100) were counted. MTT assay is a colorimetric assay
using 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetra-
zolium bromide. The cell viability was determined by this
assay with minor modifications.32
5.3.1. Spiro[3H-indole-3,70 (60H )-[1H ]pyrrolo[2,1-
c ][1,4]oxazine]-80-carboxylic acid, 1,2,30,40,80,80a-hexa-
hydro-60-(2-methoxy-2-methylpropyl)-10,2-dioxo-30,40-
diphenyl, ethyl ester, (3S,30S,40R,60S,80R,80aR) (11).
[a ]2D5¼288.8 (c 1.0, CH2Cl2); melting point: 225–
2278C; 1H NMR (300 MHz, CDCl3) d CHCl3: 0.68 (t,
J¼6.9 Hz, 3H), 1.09 (s, 6H), 1.14 (dd, J¼3.6, 16.2 Hz, 2H),
1.70 (d, J¼3.3, 15.9 Hz, 2H), 3.08 (s, 3H), 3.63–3.85 (m,
2H), 3.95 (d, J¼7.5 Hz, 1H), 4.04 (t, J¼3.3 Hz, 1H), 4.65
(d, J¼7.5 Hz, 1H), 5.07 (d, J¼3.3 Hz, 1H), 6.40 (d,
J¼3.3 Hz, 1H), 6.91 (d, J¼7.5 Hz, 1H), 7.00 (dt, J¼0.9,
7.5 Hz, 1H), 7.15 (d, J¼7.5 Hz, 1H), 7.18–7.33 (m, 10H),
7.44 (d, J¼7.5 Hz, 1H), 8.09 (br s, 1H); 13C NMR (75 MHz,
CDCl3) d CDCl3: 23.6, 26.9, 45.4, 50.6, 53.3, 56.0, 57.1,
57.5, 61.5, 65.5, 74.5, 77.1, 110.8, 124.0, 126.2, 127.2, 128.3,
128.4, 128.5, 129.4, 129.5, 130.2, 130.4, 137.5, 138.3, 142.3,
5.2. Preparation of microtubule and turbidity assay (in
vitro microtubule assembly assay)
Calf brain microtubule protein was prepared by two cycles
of assembly-disassembly33 and stored at 2808C in Mes
buffer (100 mM 2-(N-morpholino)ethanesulfonic acid
(Mes), 1 mM EGTA and 0.5 mM MgCl2) at pH 6.8. Protein
concentrations were determined by using the Dc Protein
Assay (BioRad, Hercules, CA). Microtubule assembly was
monitored by the turbidity assay as described previously.34,
35
170.1, 173.0, 178.9; IR (NaCl/neat) 3308, 1734, 1618 cm21
.
ent-11: [a]2D5¼91.7 (c 1.0, CH2Cl2).
5.3.2. Spiro[3H-indole-3,70 (60H )-[1H ]pyrrolo[2,1-
c ][1,4]oxazine]-80-carboxylic acid, 1,2,30,40,80,80a-hexa-
hydro-60-(2-methyl-2-prop-ene-yl)-10,2-dioxo-30,40-
5.2.1. 3-Methyl-3-methoxybutanal (9). To an oven-dried
2000 mL three-neck round-bottom flask with stir bar was
added DMSO (15.8 mL, 22.3 mmol) in 50 mL of CH2Cl2.
The solution was cooled to 2788C under argon and oxalyl
chloride (10 mL, 112 mmol) in 250 mL of CH2Cl2 was

P. R. Sebahar et al. / Tetrahedron 58 (2002) 6311–6322
6317
diphenyl-, ethyl ester, (3S,30S,40R,60S,80R,80aR) (12).
5.3.5. Spiro[3H-indole-3,30-pyrrolidine]-40,50-dicar-
boxylic acid, 1,2-dihydro-20-(2-methoxy-2-methylpro-
pyl)-2-oxo-, 40-ethyl ester, 50-methyl ester, (20S,3S,
40R,50R ) (20). Recrystallized cycloadduct 11 (0.50 g,
0.88 mmol) was added to a sealable pressure tube and
dissolved in 10 mL of 1:1 THF/EtOH. The solvent was
purged with argon for 5 min and PdCl2 (155 mg,
0.88 mmol) was added. The tube was sealed and flushed
with H2 before finally pressurizing to 70 Psi. The reaction
was stirred for 36 h and then filtered through Celite to
remove the palladium catalyst. Concentration afforded a
viscous oil which was taken up 5 mL of 1:1 CH2Cl2/MeOH.
TMSCHN2 (21.0 mL of a 2.0 M solution in hexanes) was
added until a yellow color persisted. The reaction was
stirred 5 min. and then concentrated under reduced pressure.
Column Chromatography with 1:1 hexanes/EtOAc affored
325 mg (91%) of white solid 20. [a ]25¼227.3 (c 0.97,
1
[a]2D5¼52.8 (c 0.95, CHCl3); H NMR (400 MHz, CDCl3)
d CHCl3: 1.11 (t, J¼6.8 Hz, 3H), 1.16 (s, 3H), 1.19 (s, 3H),
1.80–1.94 (m, 2H), 3.18 (s, 3H), 3.41 (d, J¼6.0 Hz, 1H),
4.00–4.14 (m, 2H), 4.53 (m, 1H), 4.69 (d, J¼7.6 Hz, 1H),
5.0 (s, 1H), 6.41 (d, J¼2.8 Hz, 1H), 6.84 (d, J¼7.6 Hz,
2H), 7.01–7.35 (m, 12H), 7.62 (br s, 1H); 13C NMR
(100 MHz, CDCl3) d CDCl3: 13.5, 18.8, 26.2, 54.1, 57.3,
59.8, 60.2, 61.4, 68.7, 78.0, 109.8, 119.8, 122.7, 125.9,
126.1, 126.9, 127.8, 128.1, 128.3, 128.6, 129.0, 129.2,
136.0, 136.4, 141.1, 141.4, 168.6, 171.8, 177.6; IR
(NaCl/neat) 3305, 1730, 1618 cm21; HRMS (FABþ)
calcd for C23H33O5N2 (m/z ) 537.2389, found (m/z )
537.2383.
5.3.3. Spiro[3H-indole-3,70 (60H )-[1H ]pyrrolo[2,1-
c ][1,4]oxazine]-80-carboxylic acid, 1,2,30,40,80,80a-hexa-
hydro-60-(2-methoxy-2-methylpropyl)-10,2-dioxo-30,40-
diphenyl-, ethyl ester, (3S,30S,40R,60S,80R,80aR) (13).
[a]2D5¼118.1 (c 1.05, CHCl3); 1H NMR (400 MHz,
CDCl3) d CHCl3: 0.64 (t, J¼6.8 Hz, 3H), 1.42 (s, 3H),
1.67 (s, 3H), 3.46–3.68 (m, 1H), 3.78–3.83 (m, 1H), 4.04
(d, J¼7.6 Hz, 1H), 4.36 (d, J¼3.6 Hz, 1H), 4.50 (t,
J¼7.6 Hz, 1H), 4.51 (s, 1H), 4.87 (d, J¼7.6 Hz, 1H), 5.0
(s, 1H), 6.08 (d, J¼3.6 Hz, 2H), 6.97, (t, J¼6.8 Hz, 1H),
6.84, (d, J¼6.8 Hz, 1H), 7.16–7.28 (m, 12H), 7.62 (br s,
1H); 13C NMR (100 MHz, CDCl3) d CDCl3: 14.2, 24.7,
24.8, 43.0, 49.5, 56.1, 59.8, 60.4, 60.5, 60.7, 65.6, 73.7,
79.2, 110.3, 123.1, 124.7, 126.5, 127.4, 127.7, 127.8, 128.0,
128.3, 129.2, 129.4, 137.0, 137.1, 141.1, 169.5, 170.4,
179.4; IR (NaCl/neat) 3288, 1718, 1621 cm21; HRMS
(FABþ) calcd for C24H36O6N2 (m/z) 569.2651, found
(m/z) 569.2640.
1
CHCl3); H NMR (400 MHz, CDCl3) d CHCl3: 0.63 (t,
J¼6.8 Hz, 3H), 0.90 (dd, J¼1.6, 14.4 Hz, 1H), 0.99 (s, 3H),
1.10 (s, 3H), 1.19 (dd, J¼9.6, 14.4 Hz, 1H), 3.08 (s, 3H),
3.17 (br s, 1H), 3.58–3.66 (m, 1H), 3.70 (d, J¼8.8 Hz, 1H),
3.72–3.80 (m, 2H), 3.76 (s, 3H), 4.58 (d, J¼8.8 Hz, 1H),
6.82 (d, J¼7.6 Hz, 1H), 6.96 (dt, J¼0.8, 7.6 Hz, 1H), 7.18
(dt, J¼0.8, 7.6 Hz, 1H), 7.36 (d, J¼7.6 Hz, 1H), 7.98 (br s,
1H); 13C NMR (100 MHz, CDCl3) d CDCl3: 1.53, 24.4,
25.8, 40.6, 49.4, 52.8, 54.9, 59.1, 61.0, 61.1, 63.7, 74.4,
109.4, 122.7, 126.2, 127.8, 128.6, 140.9, 169.4, 175.2,
178.0; IR (NaCl/neat) 3244, 1734 cm21; HRMS (FABþ)
calcd for C21H29O6N2 (m/z) 405.2025, found (m/z)
405.2024.
5.3.6. Spiro[3H-indole-3,30-pyrrolidine]-40-carboxylic
acid, 1,2-dihydro-20-(2-methoxy-2-methylpropyl)-2-oxo-
50-[[(2S )-2-[(phenylmethoxy)carbonyl]-1-pyrrolidinyl]-
carbonyl]-, ethyl ester, (20S,3S,40R,50R) 21. To a 200 mL
round-bottom flask that contained amino acid 19 (3.75 g,
8.8 mmol) and was placed under high vacuum for 24 h was
added BOP22 (4.25 g, 9.7 mmol) and L-proline benzyl ester
hydrochloride (2.35 g, 9.7 mmol). The flask was flushed
with argon, 100 mL of CH3CN was added and the reaction
mixture cooled to 08C. With stirring, triethylamine
(2.70 mL, 19.3 mmol) was added dropwise and the solution
allowed to warm to room temperature and stir for 8 h. The
solvent was then evaporated, replaced with 100 mL of
EtOAc, washed with 2£15 mL, 1N HCl, 1£15 mL H2O,
2£15 mL 5% NaHCO3, 1£10 mL sat. brine sol., dried over
Na2SO4, filtered and evaporated to yield 5.0 g of a brown
foam 21 which was taken on crude. An analytical sample of
21 was generated by column chromatography with 1:1
5.3.4. Spiro[3H-indole-3,30-pyrrolidine]-40,50-dicar-
boxylic acid, 1,2-dihydro-20-(2-methoxy-2-methyl-
propyl)-2-oxo-, 40-ethyl ester, monohydrochloride,
(20S,3S,40R,50R) (19). Recrystallized cycloadduct 11
(5.0 g, 8.8 mmol) was added to a sealable pressure tube
and dissolved in 200 mL of 1:1 THF/EtOH. The solvent was
purged with argon for 5 min and PdCl2 (1.55 g, 8.80 mmol)
was added. The tube was sealed and flushed with H2 before
finally pressurizing to 70 Psi. The reaction was stirred for
36 h and then filtered through Celite to remove the
palladium catalyst. Concentration afforded a viscous oil
which was triturated with 1£25 mL Et2O, 1£25 mL
EtOAc, and 1£25 mL Et2O to give 3.75 g (quant. yield)
of a white solid 19 upon drying under high vaccum.
[a]2D5¼214.0 (c 1.0, MeOH); 1H NMR (300 MHz, DMSO
d6) d HOD: 0.64 (t, J¼6.9 Hz, 3H), 0.96 (s, 3H), 1.02 (s,
3H), 1.14 (dd, J¼3.6, 14.7 Hz, 1H), 1.80 (dd, J¼8.4,
15.0 Hz, 2H), 2.93 (s, 3H), 3.61–3.73 (m, 3H), 4.22 (dd,
J¼4.2, 8.1 Hz, 1H), 4.85 (d, J¼11.4 Hz, 1H), 6.96 (t,
J¼7.5 Hz, 1H), 7.00 (d, J¼7.5 Hz, 1H), 7.27 (d, J¼7.5 Hz,
1H), 7.62 (d, J¼7.5 Hz, 1H), 11.1 (br s, 1H); 13C NMR
(75 MHz, DMSO d6) d HOD: 15.0, 24.8, 25.6, 41.9, 50.5,
55.0, 60.2, 61.1, 63.1, 63.9, 75.0, 112.3, 120.2, 123.6, 124.6,
125.2, 125.7, 127.7, 129.9, 130.9, 131.7, 144.3, 168.2,
169.3, 176.3; IR (NaCl/neat) 3444, 3098, 3058, 2977, 1746,
1771, 1634, 1568 cm21; HRMS (FABþ) calcd for
C20H27O6N2 (m/z ) 391.1869, found (m/z) 391.1866.
1
hexanes/EtOAc: [a]2D5¼275.3 (c 1.0, CH2Cl2); H NMR
(300 MHz, 1208C, DMSO) d DMSO: 0.60 (t, J¼7.2 Hz,
3H), 0.88 (d, J¼3.9 Hz, 2H), 0.90 (s, 6H), 1.84 (br s, 1H),
2.05–2.16 (m, 1H), 2.75 (br s, 2H), 2.85 (s, 3H), 3.47–3.66
(m, 3H), 4.00 (d, J¼7.2 Hz, 1H), 4.51 (d, J¼7.5 Hz, 1H),
5.06 (s, 1H), 6.77 (t, J¼7.5 Hz, 1H), 6.81 (d, J¼7.5 Hz,
1H), 7.08 (dt, J¼1.2, 7.5 Hz, 1H), 7.15–7.28 (m, 6H), 9.97
(br s, 1H); 13C NMR (75 MHz, DMSO d6) d DMSO d6:
13.8, 25.6, 25.7, 47.4, 48.7, 49.0, 55.9, 59.9, 60.2, 60.6,
60.8, 62.7, 64.5, 66.6, 74.2, 109.9, 121.5, 122.2, 1222.6,
125.5, 128.2, 128.3, 128.4, 128.5, 129.0, 143.4, 170.2,
171.2, 172.3, 177.8; IR (NaCl/neat) 3239, 1731, 1725, 1645,
1618 cm21; HRMS (FABþ) calcd for C32H40O7N3 (m/z)
578.2866, found (m/z) 578.2862.
ent-Amino acid 19: [a]2D5¼10.0 (c 1.0, MeOH).

6318
P. R. Sebahar et al. / Tetrahedron 58 (2002) 6311–6322
ent-21: [a]2D5¼81.5 (c 1.0, CH2Cl2).
J¼7.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) d CDCl3:
22.09, 13.0, 17.1, 20.9, 23.2, 23.8, 29.2, 38.8, 44.0, 48.2,
54.2, 56.2, 59.7, 60.2, 60.8, 65.6, 69.0, 73.7, 109.5, 121.9,
123.0, 126.4, 128.7, 142.1, 161.9, 164.9, 168.8, 178.6; IR
(NaCl/neat) 2971, 1724, 1668 cm21; HRMS (FABþ) calcd
for C31H46O7N3Si1 (m/z ) 600.3105, found (m/z) 600.3109.
5.3.7. Spiro[1H,5H-dipyrrolo[1,2-a:10,20-d ]pyrazine-
2(3H ),30-[3H ]indole]-1-carboxylic acid, 10,20,5a,6,
7,8,10,10a-octahydro-3-(2-methoxy-2-methylpropyl)-
20,5,10-trioxo-, ethyl ester, (1R,2S,3S,5aS,10aR) 22. To a
100 mL round-bottom flask that contained 21 (5.0 g,
8.7 mmol) was added a stir bar and 20 mL of EtOH.
Argon was bubbled through for 5 min. and 10% Pd/C
(0.5 g) was added. The system was flushed with H2 and a
balloon of H2 was attached. The solution was stirred
vigorously for 1.5 h and then filtered through Celite,
evaporated and placed on high vacuum overnight. To the
crude mixture was added a stir bar, BOP22 (3.83 g,
8.6 mmol) and 80 mL of CH3CN. Triethylamine (1.2 mL,
8.6 mmol) was added dropwise and the reaction was
allowed to stir for 8 h at which time the solvent was
evaporated. Purification via column chromatography with
75:20:5 CH2Cl2/EtOAc/i-PrOH afforded 2.75 g (68%) of
22 as a white solid. For 22: [a ]2D5¼292.0 (c 1.0,
5.3.9. Eneamide 24. To a flame-dried 10 mL round-bottom
flask with stir bar was added SEM protected diketopiper-
azine 23 (70 mg, 0.08 mmol) and LiI (110 mg, 0.80 mmol).
An oven dried condensor was attached and the system was
flushed with argon, freshly distilled pyridine (5 mL) was
added and the system heated to reflux for 48 h. The solvent
was evaporated and replaced with 10 mL of EtOAc,
extracted with 5£2 mL 5% NaHCO3 and the aqueous
layers combined. The solution was then saturated with
NaCl, acidified to pH 4 with 1N HCl and extracted with
5£5 mL EtOAc. The organic layers were combined, dried
over Na2SO4, filtered and evaporated to yield a white solid,
which was used without further purification. To the flask
which contained the crude carboxylic acid was added
Cu(OAc)2 (1 mg, 0.006 mmol) and an oven dried condensor
was attached. The system was flushed with Ar and distilled
DMF (1 mL) was added. The reaction was wrapped in tin
foil and stirred for 15 min. at which time Pb(OAc)4 (55 mg,
0.12 mmol) was added. The mixture was stirred (still in the
dark) for 15 min more and then heated to reflux for 1.5 h.
Evaporation of the solvent and purification via column
chromatography with 75:20:5 CH2Cl2/EtOAc/i-PrOH to
yielded 5 mg (11%) of a clear oil. For 24: [a ]25¼26.2 (c
0.16, CHCl3); 1H NMR (400 MHz, CDCl3) d CHCl3: 20.04
(s, 9H), 0.79 (s, 3H), 0.91 (t, J¼7.6 Hz, 3H), 1.15 (s, 3H),
1.22 (d, J¼8.4 Hz, 1H), 1.92–2.00 (m, 2H), 2.10–2.1 (m,
1H), 2.18 (dd, J¼10.8, 13.6 Hz, 1H), 2.40–2.50 (m, 1H),
2.62 (s, 3H), 3.40–3.48 (m, 1H), 3.49–3.54 (m, 1H), 3.60 (t,
J¼8.8 Hz, 1H), 4.21–4.25 (m, 1H), 3.78–3.81 (m, 1H),
4.97 (d, J¼10.0 Hz, 1H), 5.02 (1/2 ABq, J¼10.8 Hz, 1H),
5.24 (1/2 ABq, J¼10.8 Hz, 1H), 5.63 (s, 1H), 6.99–7.09
(m, H), 7.2 (d, J¼7.6 Hz, 1H); 13C NMR (100 MHz,
CDCl3) d CDCl3: 21.23, 18.0, 22.2, 22.9, 25.4, 29.7, 39.7,
44.7, 48.4, 61.9, 63.8, 66.5, 70.1, 70.6, 109.7, 110.3, 118.0,
122.7, 126.6, 128.8, 137.2, 142.3, 161.3, 164.6, 169.0; IR
(NaCl/neat) 2952, 1727, 1683, 1650 cm21; HRMS (FABþ)
calcd for C28H40O5N3Si1 (m/z) 526.2737, found (m/z )
527.2727.
1
CH2Cl2); H NMR (300 MHz, CDCl3) d CHCl3: 0.92 (t,
J¼7.2 Hz, 3H), 1.11 (s, 3H), 1.19 (s, 3H), 1.72 (dd,
J¼4.2, 14.4 Hz, 1H), 1.75–2.08 (m, 3H), 2.21 (dd,
J¼10.5, 14.4 Hz, 1H), 2.49 (h, J¼6.0 Hz, 1H), 3.0 (s,
3H), 3.42 (ddd, J¼3.9, 7.5, 9.9 Hz, 1H), 3.49 (d,
J¼9.3 Hz, 1H), 4.67 (d, J¼9.9 Hz, 1H), 3.84–4.07 (m,
3H), 4.31 (dd, J¼5.4, 9.9 Hz, 1H), 4.89 (dd, J¼3.9,
10.5 Hz, 1H), 5.13 (dd, J¼1.2, 9.6 Hz, 1H), 6.96 (t,
J¼7.5 Hz, 1H), 6.99 (d, J¼7.5 Hz, 1H), 7.17 (d,
J¼7.5 Hz, 1H), 7.25 (dt, J¼1.9, 7.5 Hz, 1H), 8.42 (br
s, 1H); 13C NMR (75 MHz, CDCl3) d CDCl3: 12.9, 20.7,
23.1, 23.7, 29.1, 38.4, 43.8, 47.9, 53.3, 56.1, 59.3, 59.6,
60.1, 60.6, 73.5, 109.6, 121.1, 123.6, 126.3, 128.5, 141.0,
161.8, 165.2, 168.8, 179.5; IR (NaCl/neat) 3244, 1763,
1667, 1665 cm21; HRMS (FABþ) calcd for C25H32O6N3
(m/z) 470.2291, found (m/z) 470.2280.
ent-22: [a]2D5¼95.8 (c 1.2, CH2Cl2).
5.3.8. N-SEM diketopiperazine 23. To a flame-dried
10 mL round-bottom flask with stir bar was added
diketopiperazine 22 (65 mg, 0.14 mmol). The system was
flushed with Ar, THF added and cooled to 2788C. KHMDS
(0.33 mL of a 0.5 M sol., 0.16 mmol) was added and stirred
for 15 min. SEMCl (0.03 mL, 0.16 mmol) was added
dropwise and the reaction allowed to warm to room
temperature and stirred for 8 h. Sat. NH4Cl was added and
the reaction mixture poured into 10 mL EtOAc. The aq.
layer was extracted 3£5 mL with EtOAc, the organic layers
combine, dried over Na2SO4, filtered, evaporated and
chromatographed with 75:20:5 CH2Cl2/EtOAc/i-PrOH to
yield 60 mg (72%) of the white solid 23: [a ]2D5¼22.4 (c
0.74, CHCl3); 1H NMR (400 MHz, CDCl3) d CHCl3: 20.04
(s, 9H), 0.8 (t, J¼6.8 Hz, 6H), 1.12 (s, 3H), 1.18 (s, 3H),
1.70 (dd, J¼4.0, 14.4 Hz, 1H), 1.81 (dt, J¼3.2, 11.6 Hz,
1H), 1.88–2.01 (m, 1H), 2.02–2.12 (m, 1H), 2.21 (dd,
J¼10.8, 14.4 Hz, 1H), 2.50 (quint, J¼6.0 Hz, 1H), 3.01 (s,
3H), 3.40–3.48 (m, 1H), 3.47 (d, J¼9.2 Hz, 1H), 3.54 (t,
J¼8.8 Hz, 2H), 3.83–3.90 (m, 1H), 3.92–3.96 (m, 1H),
3.97–4.04 (m, 1H), 4.32 (dd, J¼5.6, 11.6 Hz, 1H), 4.85
(dd, J¼4.0, 10.8 Hz, 1H), 5.05 (1/2 Abq, J¼11.2 Hz, 1H),
5.13, (dd, J¼1.2, 9.2 Hz, 1H), 5.20 (1/2 Abq, J¼11.2 Hz,
1H), 7.06–7.09 (m, 2H), 7.22 (d, J¼7.6 Hz, 1H), 7.33 (t,
5.3.10. Spiro[1H,5H-dipyrrolo[1,2-a:10,20-d ]pyrazine-
2(3H ),30-[3H ]indole]-1-carboxylic acid, 10,20,5a,6,7,
8,10,10a-octahydro-3-(2-methyl-1-propenyl)-20,5,10-
trioxo-, ethyl ester, (1R,2S,3S,5aR,10aR-25). To a flame-
dried 250 mL round-bottom flask with stir bar was added
˚
diketopiperazine 22 (2.70 g, 5.75 mmol), 4 A molecular
sieves (5.0 g) and TsOH (1.0 g, 5.75 mmol). An oven-dried
condensor was attached, the system was flushed with argon,
freshly distilled toluene (200 mL) was added and the system
heated to reflux temperature for 8 h. The solvent was
evaporated and replaced with 100 mL of EtOAc, washed
with 2£15 mL 5% NaHCO3, 1£10 mL sat. brine sol., dried
over Na2SO4, filtered, evaporated and chromatographed
with 75:20:5 CH2Cl2/EtOAc/i-PrOH to yield 1.75 g (70%)
1
of 25: [a]2D5¼78.5 (c 1.0, CH2Cl2); H NMR (300 MHz,
CDCl3) d CHCl3: 0.79 (t, J¼7.5 Hz, 3H) 1.48 (d,
J¼1.5 Hz, 3H), 1.61 (d, J¼1.5 Hz, 3H), 1.90–2.10 (m,

P. R. Sebahar et al. / Tetrahedron 58 (2002) 6311–6322
6319
2H), 2.20–2.40 (m, 2H), 3.50–3.70 (m, 2H), 3.74–3.92 (m,
2H), 3.97 (d, J¼10.2 Hz, 1H), 4.33 (t, J¼7.5 Hz, 1H), 4.78
(dt, J¼1.5, 9.6 Hz, 1H), 5.12 (d, J¼9.6 Hz, 1H), 5.21 (d,
J¼10.2 Hz, 1H), 6.86 (d, J¼7.5 Hz, 1H), 7.03 (dt, J¼1.9,
7.5 Hz, 1H), 7.13 (d, J¼7.5 Hz, 1H), 7.24 (dt, J¼1.9,
7.5 Hz, 1H), 7.97 (br s, 1H); 13C NMR (75 MHz, CDCl3) d
CDCl3: 7.27, 11.9, 17.4, 19.4, 20.8, 39.3, 45.2, 52.8, 54.3,
54.9, 55.3, 57.2, 103.8, 113.1, 116.0, 119.2, 119.3, 122.8,
130.8, 134.8, 158.5, 160.3, 161.5, 171.0; IR (NaCl/neat)
3219, 1723, 1663, 1648 cm21; HRMS (FABþ) calcd for
C24H28O5N3 (m/z ) 438.2029, found (m/z) 438.2017.
and purification via column chromatography with 75:20:5
CH2Cl2/EtOAc/i-PrOH to yielded 8 mg (20%) of 26 as a
1
clear oil. For 26: [a ]25¼260.0 (c 0.05, CHCl3); H NMR
(400 MHz, CDCl3) d CHCl3: 20.02 (s, 9H), 0.93 (t,
J¼7.6 Hz, 3H), (1.34 (s, 3H), 1.56 (s, 3H), 2.89 (dt, J¼2.4,
8.0 Hz, 2H), 3.57 (t, J¼7.6 Hz, 2H), 4.12 (t, J¼8.8 Hz,
2H), 5.13 (d, J¼10.8 Hz, 1H), 5.21 (t, J¼11.2 Hz, 2H),
5.20 (d, J¼8.0 Hz, 1H), 5.75 (s, 1H), 6.22 (t, J¼3.2 Hz,
1H), 7.04–7.11 (m, 3H), 7.31 (t, J¼7.6 Hz, 1H); 13C NMR
(100 MHz, CDCl3) d CDCl3: 21.2, 18.0, 18.5, 25.4, 28.7,
29.9, 45.4, 62.1, 64.5, 66.6, 70.2, 110.1, 116.2, 119.7, 119.9,
123.0, 126.7, 127.4, 129.4, 135.5, 138.1, 138.5, 142.0,
ent-25: [a ]2D5¼274.0 (c 1.0, CH2Cl2).
152.2, 152.7, 177.0; IR (NaCl/neat) 1727, 1683 cm21
;
HRMS (FABþ) calcd for C27H33O4N3Si1 (m/z) 491.2240,
5.3.11. N-SEM diketopiperazine 26. To a flame-dried
10 mL round-bottom flask with stir bar was added 25
(65 mg, 0.15 mmol). The system was flushed with Ar, THF
added and cooled to 2788C. KHMDS (0.35 mL of a 0.5 M
sol., 0.18 mmol) was added and stirred for 15 min. SEMCl
(0.035 mL, 0.18 mmol) was added dropwise and the
reaction allowed to warm to room temperature and stirred
for 8 h. Sat. NH4Cl was added and the reaction mixture
poured into 10 mL EtOAc. The aq. layer was extracted
3£5 mL with EtOAC, the organic layers combine, dried
over Na2SO4, filtered, evaporated and chromatographed
with 75:20:5 CH2Cl2/EtOAc/i-PrOH to yield 70 mg (84%)
found (m/z) 491.2226.
5.3.13. Triene 28. To a flame-dried 10 mL round-bottom
flask with stir bar was added 27 (8 mg, 0.017 mmol)
dissolved in CH2Cl2 (2 mL) and cooled to 2788C. A 1.0 M
hexane solution of Me2AlCl (0.086 mL, 0.086 mmol) was
added dropwise under Ar. The mixture was warmed to room
temperature and stirred for 15 min. The solution was cooled
to 08C and poured into a sat. Na/K tartrate solution (2 mL)
also at 08C. The mixture was allowed to warm to room
temperature and stirred vigorously for 1 h. The aq. layer was
then extracted 3£5 mL with EtOAc, the organic layers
combined, dried over Na2SO4, filtered and evaporated.
Purification was accomplished by PTLC (1/2 of a 250 mm
plate) with 75:20:5 CH2Cl2/EtOAc/i-PrOH as the eluent to
1
of the white solid 26: [a]2D5¼263.5 (c 0.97, CHCl3); H
NMR (400 MHz, CDCl3) d CHCl3: 20.04 (s, 9H), 0.71 (t,
J¼7.2 Hz, 3H), 0.86–1.00 (m, 2H), (1.46 (s, 3H), 1.55 (s,
3H), 1.95 – 2.02 (m, 2H), 2.24–2.40 (m, 2H), 3.52–3.65
(m, 3H), 3.68–3.76 (m, H), 3.82–3.90 (m, 1H), 3.97 (d,
J¼10.0 Hz, 1H), 4.32 (t, J¼8.0 Hz, 1H), 4.78 (d,
J¼14.8 Hz, 1H), 5.09 (1/2 ABq, J¼11.2 Hz, 1H), 5.10
(d, J¼10.0 Hz, 1H), 5.20 (1/2 ABq, J¼11.2 Hz, 1H), 7.04
(d, J¼8.0 Hz, 1H), 7.07 (t, J¼8.0 Hz, 1H), 7.15 (d,
J¼8.0 Hz, 1H), 7.30 (t, J¼8.0 Hz, 1H); 13C NMR
(100 MHz, CDCl3) d CDCl3: 21.16, 13.7, 17.9, 18.5,
23.8, 25.9, 27.2, 45.7, 51.9, 59.0, 60.7, 61.3, 61.8, 63.7,
66.4, 70.1, 110.0, 119.4, 122.9, 125.1, 125.5, 129.4, 137.5,
142.8, 165.0, 166.8, 167.9, 175.9; IR (NaCl/neat) 1728,
1678 cm21; HRMS (FABþ) calcd for C30H42O6N3Si1
(m/z) 568.2843, found (m/z) 568.2827.
1
yield 3 mg (48%) of 28 as a clear oil. For 28: H NMR
(400 MHz, CDCl3) d CHCl3: 1.22 (s, 3H), 1.35 (s, 3H), 2.85
(dt, J¼3.2, 8.0 Hz, 2H), 4.09 (t, J¼8.8 Hz, 2H), 5.17 (d,
J¼8.8 Hz, 1H), 5.51 (d, J¼8.8 Hz, 1H), 5.75 (s, 1H), 6.19
(t, J¼3.2 Hz, 1H), 6.82 (d, J¼7.6 Hz, 1H), 6.97–7.05 (m,
2H), 7.37 (t, J¼7.6 Hz, 1H), 7.85 (br s, 1H); IR (NaCl/neat)
1763, 1667 cm21; HRMS (FABþ) calcd for C21H20O3N3
(m/z) 362.1504, found (m/z) 362.1484.
5.3.14. Spiro[1H,5H-dipyrrolo[1,2-a:10,20-d ]pyrazine-
2(3H ),30-[3H ]indole]-1-carboxylic acid, 10,20,5a,6,7,
8,10,10a-octahydro-3-(2-methyl-1-propenyl)-20,5,10-
trioxo-, (1R,2S,3S,5aR,10aR) 29. To a flame dried 100 mL
round-bottom flask with stir bar was added olefin 25 (0.87 g,
2.0 mmol) and LiI (2.66 g, 20.0 mmol). An oven-dried
condensor was attached and the system was flushed with
argon, freshly distilled pyridine (50 mL) was added and the
system heated to reflux for 48 h. The solvent was evaporated
and replaced with 50 mL of EtOAc, extracted with
5£10 mL 5% NaHCO3 and the aqueous layers combined.
The solution was then saturated with NaCl, acidified to pH 4
with 1N HCl and extracted with 5£10 mL EtOAc. The
organic layers were combined, dried over Na2SO4, filtered
and evaporated to yield 0.58 g (71%) of 29. The organic
layer from the first extraction was dried over Na2SO4,
filtered, evaporated and purified via column chromat-
ography with 75:20:5 CH2Cl2/EtOAc/i-PrOH to recover
80 mg of unreacted starting material 25. For 29:
[a ]2D5¼73.0 (c 0.8, MeOH); 1H NMR (300 MHz,
CD3OD) d MeOH: 1.27 (s, 3H), 1.29 (s, 3H), 1.98–2.04
(m, 2H), 2.18–2.30 (m, 2H), 3.50–3.70 (m, 2H), 3.37–3.46
(m, 1H), 3.51–3.58 (m, 2H), 3.74 (d, J¼10.2 Hz, 1H), 4.52
(t, J¼7.5 Hz, 1H), 4.96 (d, J¼5.1 Hz), 5.32 (d, J¼9.3 Hz,
1H), 6.84 (d, J¼7.5 Hz, 1H), 6.98 (dt, J¼1.9, 7.5 Hz, 1H),
5.3.12. N-SEM triene 27. To a flame-dried 10 mL round-
bottom flask with stir bar was added 26 (70 mg, 0.12 mmol)
and LiI (165 mg, 1.2 mmol). An oven dried condensor was
attached and the system was flushed with argon, freshly
distilled pyridine (5 mL) was added and the system heated
to reflux for 48 h. The solvent was evaporated and replaced
with 10 mL of EtOAc, extracted with 5£2 mL 5% NaHCO3
and the aqueous layers combined. The solution was then
saturated with NaCl, acidified to pH 4 with 1N HCl and
extracted with 5£5 mL EtOAc. The organic layers were
combined, dried over Na2SO4, filtered and evaporated to
yield 45 mg (68%) a white solid, which was used without
further purification. To the flask which contained the crude
carboxylic acid was added Cu(OAc)2 (1.5 mg, 0.008 mmol)
and an oven dried condensor was attached. The system was
flushed with Ar and distilled DMF (1 mL) was added. The
reaction was wrapped in tin foil and stirred for 15 min. at
which time Pb(OAc)4 (55 mg, 0.12 mmol) was added. The
mixture was stirred (still in the dark) for 15 min. more and
then heated to reflux for 1.5 h. Evaporation of the solvent

6320
P. R. Sebahar et al. / Tetrahedron 58 (2002) 6311–6322
7.19 (dt, J¼1.9, 7.5 Hz, 1H), 7.24 (d, J¼7.5 Hz, 1H); 13C
NMR (75 MHz, CD3OD) d CD3OD: 12.5, 18.7, 19.9, 22.3,
40.7, 47.0, 54.8, 55.9, 57.1, 58.8, 105.2, 115.8, 117.1, 121.3,
121.5, 124.2, 131.1, 137.7, 161.3, 163.0, 164.9, 173.1; IR
(NaCl/neat 3248, 1731, 1678, 1668 cm21; HRMS (FABþ)
calcd for C22H24O5N3 (m/z) 410.1716, found (m/z)
410.1698.
epi-ent-Spirotyprostatin B: [a ]2D5¼242.5 (c 0.8, CH2Cl2).
5.3.17. Spirotyprostatin B (2). To a flame-dried 10 mL
round-bottom flask with stir bar was added 12-epi-
spirotyprostatin
B (32) (0.95 g, 0.26 mmol), MeOH
(2 mL) was added and the system cooled to 08C. A
1 M solution of NaOMe in MeOH (0.26 mL) was added
dropwise and the mixture was stirred at 08C for 2 h at
which time 5 mL of saturated aqueous NH4Cl was added
along with 5 mL of EtOAc. The aqueous layer was
extracted with EtOAc (3£5 mL) and the organic layers
combined, dried over Na2SO4, filtered, evaporated and
purifed by chromatography (silica gel, eluted with
75:20:5 CH2Cl2/EtOAc/i-PrOH) to yield 0.044 g (46%)
of 2 and 0.28 g (30%) of 32. For 2: [a]2D5¼2151.1 (c
0.45, CHCl3); 1H NMR (400 MHz, CDCl3) d CHCl3:
1.28 (d, J¼0.9 Hz, 3H), 1.57 (d, J¼0.9 Hz, 3H), 1.94–
2.05 (m, 2H), 2.08–2.16 (m, 1H), 2.46–2.53 (m, 1H),
3.58 (ddd, J¼2.9, 9.3, 12.2 Hz, 1H), 3.84 (dt, J¼8.3,
12.2 Hz, 1H), 4.35 (dd, J¼6.1, 10.5 Hz, 1H), 5.22 (dt,
J¼1.2, 8.8 Hz, 1H), 5.4 (d, J¼8.8), 5.79 (s, 1H), 6.89
(d, J¼7.6 Hz, 1H), 6.99 (dt, J¼1.0, 7.6 Hz, 2H), 7.06
(dt, J¼1.0, 7.6 Hz, 1H), 7.23 (dt, J¼1.0, 7.6 Hz, 1H)
7.77 (br s, 1H); 13C NMR (75 MHz, CDCl3) d CDCl3:
18.4, 22.3, 25.5, 29.5, 45.0, 61.8, 61.9, 64.3, 110.0,
116.4, 120.7, 122.5, 127.4, 128.1, 129.3, 138.4, 138.5,
140.6, 155.2, 162.7, 178.1; IR (NaCl/neat) 3235, 1718,
1677, 1690 cm21; HRMS (EI) calcd for C21H21O3N3
(m/z ) 363.1583, found (m/z ) 363.1584.
ent-29: [a]2D5¼275.0 (c 1.0, MeOH).
5.3.15. Spiro[1H,5H-dipyrrolo[1,2-a:10,20-d ]pyrazine-
2(3H ),30-[3H]indole]-1-carboxylic acid, 5a,6,7,8,10,10a-
hexahydro-3-(2-methoxy-2-methylpropyl)-20,5,10-
trioxo-, ethyl ester, (1R,2S,3S,5aR,10aR-31). Compound
31 was generated in an identical fashion to diketopiper-
azine 22 yet afforded a higher yield (94%). For 31:
[a ]2D5¼81.7 (c 1.0, CH2Cl2); 1H NMR (300 MHz,
CDCl3) d CHCl3: 0.85 (s, 3H), 0.87 (t, J¼7.2 Hz, 3H),
1.24, (s, 3H), 1.77 (dd, J¼9.9, 14.1 Hz, 1H), 1.90–2.11
(m, 2H), 2.25–2.33 (m, 2H), 2.52 (d, J¼13.8 Hz, 1H),
2.79 (s, 3H), 3.56–3.65 (m, 2H), 3.73–3.81 (m, 3H),
4.31 (t, J¼7.5 Hz, 1H), 4.67 (d, J¼9.9 Hz, 1H), 5.09 (d,
J¼9.9 Hz, 1H), 6.86 (d, J¼7.5 Hz, 1H), 7.01 (t, J¼
7.5 Hz, 1H), 7.09 (d, J¼7.5 Hz, 1H), 7.23 (t, J¼7.5 Hz,
1H), 8.26 (br s, 1H); 13C NMR (75 MHz, CDCl3) d
CDCl3: 7.27, 15.8, 17.0, 19.2, 21.0, 33.5, 39.1, 41.3,
48.1, 52.7, 54.1, 54.7, 55.0, 55.5, 67.3, 103.2, 115.2,
119.8, 120.2, 122.3, 135.3, 158.0, 159.7, 161.0, 171.5; IR
(NaCl/neat) 3268, 1729, 1671, 1669 cm21
; HRMS
(FABþ) calcd for C25H32O6N3 (m/z) 470.2291, found
(m/z) 470.2296.
ent-Spirotryprostatin B: [a]2D5¼155.1 (c 0.33, CH2Cl2).
ent-Diketopiperazine 31: [a ]2D5¼281.2 (c 1.0, CH2Cl2).
5.3.16. Spiro[3H,5H-dipyrrolo[1,2-a:10,20-d ]pyrazine-
2(10H ),30-[3H ]indole]-20,5,10(10H )-trione, 5a,6,7,8-tet-
rahydro-3-(2-methyl-1-propenyl)-, (2S,3S,5aR ) (12-epi-
spirotyprostatin B) (30). To a flame-dried 100 mL round-
bottom flask with stir bar was added carboxylic acid 31
(0.29 g, 0.26 mmol), DCC (0.22 g, 1.06 mmol), DMAP
(0.13 g, 1.06 mmol) and 2-mercaptopyridine N-oxide
(0.112 g, 0.88 mmol). An oven-dried condenser was
attached and the system was flushed with argon and
wrapped in tin foil. Freshly distilled BrCCl3 (25 mL) was
added and the system was heated to 608C for 1 h. The
foil was then removed and the reaction heated to reflux
for 1.5 h. The solvent was evaporated and the resulting
oil was purified by chromatography (silica gel, eluted
with 75:20:5 CH2Cl2/EtOAc/i-PrOH) to yield 0.095 g
(37%) of 32. [a]2D5¼41.3 (c 0.8, CH2Cl2); 1H NMR
(300 MHz, CDCl3) d CHCl3: 1.50 (d, J¼1.5 Hz, 3H),
1.54 (d, J¼1.5 Hz, 3H), 1.90–2.16 (m, 3H), 2.18–2.30
(m, 2H), 3.40–3.48 (m, 1H), 3.52–3.60 (m, 1H), 3.81–
3.92 (m, 2H), 4.36 (dd, J¼6.9, 10.5 Hz, 1H), 5.13 (dt,
J¼1.5, 8.1 Hz, 1H), 5.54 (d, J¼9.3 Hz), 5.83 (s, 1H),
6.87 (d, J¼7.5 Hz, 1H), 7.01–7.09 (m, 2H), 7.19 (dt,
J¼1.9, 7.5 Hz, 1H), 7.22–7.27 (m, 1H) 7.69 (br s, 1H);
13C NMR (100 MHz, CDCl3) d CDCl3: 18.6, 22.2, 25.7,
29.3, 45.3, 62.0, 62.1, 64.8, 110.1, 115.8, 119.3, 121.8,
122.8, 127.2, 128.6, 129.3, 155.7, 162.5, 178.2; IR
Acknowledgments
We thank the National Science Foundation (Grant No. CHE
9731947) for financial support. The authors are also grateful
to Professor Raymond L. Funk of Pennsylvania State
University with helpful discussions concerning the mech-
anism of formation of compound 12. The authors also thank
Professor Larry E. Overman, of the University of California,
Irvine, and Professor Samuel J. Danishefsky of the Sloan-
Kettering Institute for communicating their results on
spirotryprostatin B prior to publication. RMW wishes to
acknowledge Professor Kishi’s passion for natural products
chemistry as this has provided the inspiration for this
work.36
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