Total synthesis and stereochemistry of cytoblastin

Article abstract of DOI:10.1016/S0968-0896(98)00113-8

The first total synthesis and stereochemical assignment of cytoblastin were reported. Key steps included the palladium-mediated coupling of N-SEM-7-bromoindolactam V ((-)-11) with allylstannane c-13, and osmium tetroxide-mediated dihydroxylation of 14, both of which were stereoselective. The stereochemistry of cytoblastin was determined as 1A via spectroscopic analysis of the pentacyclic derivative 21 of cytoblastin. A connection was then made between the stereochemistry so elucidated and the Kishi/Rando hypothesis for the structural correlation between (S)-1,2-diacylglycerol and tumor promoters for the process of protein kinase C activation. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00113-8

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1243±1254
Total Synthesis and Stereochemistry of Cytoblastin
O®r A. Moreno y and Yoshito Kishi *
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA
Received 26 May 1998; accepted 5 June 1998
AbstractÐThe ®rst total synthesis and stereochemical assignment of cytoblastin were reported. Key steps included the
palladium-mediated coupling of N-SEM-7-bromoindolactam V (( )-11) with allylstannane c-13, and osmium tetr-
oxide-mediated dihydroxylation of 14, both of which were stereoselective. The stereochemistry of cytoblastin was
determined as 1A via spectroscopic analysis of the pentacyclic derivative 21 of cytoblastin. A connection was then
made between the stereochemistry so elucidated and the Kishi/Rando hypothesis for the structural correlation between
(S)-1,2-diacylglycerol and tumor promoters for the process of protein kinase C activation. # 1998 Elsevier Science
Ltd. All rights reserved.
Introduction
thus inhibiting catalytic function. An important func-
tion of the binding of (S)-DAG to the regulatory
domain is to relieve this inhibitory constraint, resulting
in the activation of PKC.
Protein kinase C (PKC) is a serine/threonine-speci®c
kinase, central to regulatory processes in all cells.1 PKC
actually refers to a family of kinases which are generally
activated by the simultaneous presence of (S)-1,2-di-
acylglycerol ((S)-DAG), Ca2+, and an acidic phospho-
lipid such as phosphatidylserine, although some isoforms
have been reported to be calcium-independent. Under
quiescent conditions, PKC resides in the cytoplasm,
where it is inactive. PKC is transiently activated by the
endogenous (S)-DAG, which is produced via the recep-
tor-mediated activation of a phospholipase C, which
cleaves phosphatidylinositol to generate (S)-DAG and
phosphoinositol. Phosphorylation of (S)-DAG by a
speci®c kinase, or hydrolysis at the 2-position then
removes (S)-DAG from circulation, allowing for tem-
poral regulation of PKC. PKC possesses a regulatory
domain and a protein kinase domain. (S)-DAG binds to
the regulatory domain of PKC, resulting in the trans-
location of the enzyme to the plasma membrane, where
the enzyme becomes catalytically active. The regulatory
domain contains a pseudosubstrate sequence which is
thought to bind to the active site of the kinase domain,
The regulatory domain of PKC is of exceptional interest
because not only does it speci®cally recognize (S)-
DAG's, but it binds to and is activated by an extremely
diverse group of non-DAG's. These molecules include
the phorbol esters, the ingenols, the aplysiatoxins, the
teleocidins, and the bryostatins. The central issue for
understanding the mechanism of PKC activation by
(S)-DAG and these tumor promoters is to relate the
structures of these dissimilar molecules to their identical
function; namely, the occupation of the same binding
site and the activation of PKC. A structural model in
which the pharmacophores of the various PKC activa-
tors were identi®ed had previously been presented by
Kishi and Rando2 (Fig. 1). This model was experi-
mentally developed through structure±activity studies on
debromoaplysiatoxin and its strategically-chosen analo-
gues in reference to (S)-DAG and its analogues. In this
model, the interaction of PKC with the hydrophilic
atoms correlated in Fig. 1 represents the sine qua non
for the activation of PKC. The circled hydrophobic
portions are not necessary, but they signi®cantly
increase activity.
Key words: Natural products; cytoblastin; protein kinase C;
tumor promoters.
*Corresponding author. Tel: (617) 495-4679; Fax: (617) 495-
5150; E-mail: kishi@chemistry.harvard.edu
yPresent address: Amgen, 1840 DeHavilland Drive, Thousand
Oaks, California 91320-1789, USA
Of particular interest to the current studies is the teleo-
cidin class of tumor promoters. The naturally occur-
ring ( )-indolactam V (IL-V; 2) contains the common
0968-0896/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00113-8

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O. A. Moreno, Y. Kishi/Bioorg. Med. Chem. 6 (1998) 1243±1254
Figure 1. The pharmacophores of various tumor promoters.
structural element of this class of tumor promoters. This
natural product contains the minimum structural
requirements for PKC activation.3 It is widely recog-
nized that the lactam ring of IL-V exists as an equili-
brium of two solution conformations with the major
conformer named TWIST and the minor conformer
SOFA. Although the exact ratio of the two conformers
varies with solvent and temperature, they typically exist
as roughly a 3:1 ratio in CDCl3 at room temperature.4
It is this conformational issue which makes IL-V and
the teleocidin class of tumor promoters a topic of
controversy with respect to their structural correlation
with (S)-DAG, and several di€erent models concerning
the IL-V pharmacophore have evolved.5
Cytoblastin6,7 (1) is a natural product isolated from
Streptoverticillium eurocidium, found in soil samples
collected in Yokohama City, Kanagawa Prefecture,
Japan. It was found to promote the proliferation of T
cells, while showing low cytotoxicity and no anti-
microbial activity. Although the gross structure of
cytoblastin was reported, the stereochemistry remained

O. A. Moreno, Y. Kishi/Bioorg. Med. Chem. 6 (1998) 1243±1254
1245
unknown. Interestingly, the upper half of cytoblastin
apparently corresponds to ( )-IL-V (2), and we felt that a
structural correlation between cytoblastin and (S)-DAG
might give further insights into the pharmacophore of the
PKC-based tumor promoters. Intriguingly, when the
structural correlation proposed by Kishi and Rando is
adopted and an imaginary acyl group is placed on the
C.27 alcohol, the lower half of cytoblastin also corre-
sponds to the pharmacophore of (S)-DAG. Thus, cyto-
blastin could be viewed as a pseudo-dimeric form of (S)-
DAG (Fig. 2). Clearly, the validity of this hypothesis
hinges on the stereochemistry of cytoblastin, and we
report the ®rst total synthesis and stereochemical
assignment of cytoblastin.8
the nine-membered lactam ring present in the teleocidin
family of natural products exists as a mixture of two
conformers (vide ante), whereas inversion of one of the
stereocenters on this ring results in a single conformer at
room temperature. Cytoblastin (1) was reported to exist
as a mixture of two conformers,6 suggesting that at least
the relative stereochemistry of the upper half of 1 cor-
responds to that of ( )-IL-V (2). Thus, ( )-IL-V (2) was
chosen as a starting point for the synthesis. Two di€er-
ent approaches were explored.
First approach
The key bond formation in the ®rst approach was envi-
sioned to take place via the addition of a suitably pro-
tected organometallic derivative of the upper half to an
epoxide (Scheme 1). The obvious advantage of this
approach is prescience of the consequent relative
stereochemistry.
Total synthesis
A priori, the upper half of cytoblastin was assumed to
correspond structurally to ( )-IL-V (2). It is known that
The feasibility of this approach was ®rst examined by
using a model system (Scheme 2). Synthesis of the
epoxide was planned via the corresponding allylic alco-
hol, with the consideration that such an epoxide could
be prepared in an optically active form via its asym-
metric epoxidation. As the allylic alcohol and the epox-
ide might prove to be unstable due to conjugation to the
indole ring, an electron-withdrawing protecting group
was deemed appropriate for the indole nitrogen, and the
mesitylsulfonyl (Mts) was chosen.
The allylic alcohols were synthesized as outlined in
Scheme 2. Under phase-transfer conditions,10 3-formyl-
indole (3) was protected as its mesitylsulfonamide
and converted selectively to cis- and trans-a,b-unsatu-
rated esters, DIBAL reduction of which furnished the
cis- and trans-allylic alcohols. Although stable in solu-
tion, these allylic alcohols decomposed upon concentra-
tion, where the major decomposition product appeared
to be the dimeric ether. Protection as the t-butyl-
dimethylsilyl (TBS) ether resolved this problem. Attempts
to epoxidize the allylic alcohols resulted in either
Figure 2. The structure of cytoblastin (1), ( )-indolactam V
(2), and an (S)-diacylglycerol, and the proposed structural cor-
relation among them.
Scheme 1. The ®rst retrosynthetic analysis of cytoblastin (1).

1246
O. A. Moreno, Y. Kishi/Bioorg. Med. Chem. 6 (1998) 1243±1254
Scheme 2. Reagents and conditions. (a) 1. Mts-Cl, NaOH, Cetyltrimethylammonium chloride, CH2Cl2, 0 ꢀC; 2. Ph3P=CHCO2Et,
CH2Cl2, rt; 3. DIBAL, THF, 78 ꢀC; 4. TBS-Cl, imidazole, DMF, rt. (b) 1. same as (a) 1; 2. (CF3CH2O)2P(O)CH2CO2Me,9
KHMDS, 18-Crown-6, THF, 78 ꢀC; 3. same as (a) 3; 4. same as (a) 4. (c) I2, Ag2O, aq THF. (d) KHMDS, THF, 78 ꢀC, followed
by addition of PhMgBr, 78 ꢀC!rt.
decomposition (MCPBA) or apparently clean conver-
sion but followed by decomposition to the diol on
isolation (dimethyldioxirane). In spite of the electron-
withdrawing sulfonamide, it appeared that the epoxide
was too unstable to isolate. However, it was possible to
generate the intermediate iodohydrin via treatment of
the allylic alcohols with iodine and silver oxide in wet
THF. Interestingly, the TBS-trans-allylic alcohol t-4 led
exclusively to the iodohydrin t-5, which was assumed to
be trans, but the cis-ole®n c-4 led to a chromato-
graphically separable 5/3 mixture of cis and trans-iodo-
hydrins t- and c-5.
protecting group had to withstand the organometallic
environment. It was our hope that the 2-(trimethylsilyl)-
ethoxymethyl (SEM)12 group would not only meet these
requirements, but would pose the added potential bene-
®t of stabilizing the organometallic center via coordina-
tion of its oxygen. Obviously, this silicon-based
protecting group strategy should o€er the opportunity
of single-step global deprotection.
The Grignard reagent derived from N-SEM-7-bromo-
indole could be prepared either by lithium±halogen
exchange followed by transmetallation with magnesium
bromide, or by direct insertion of activated magnesium
metal. Unfortunately, it failed to add to the in situ
derived epoxide. Then the organozinc species, generated
via lithium±halogen exchange followed by transmetalla-
tion with zinc chloride, was examined. This method was
successful, but its reproducibility proved capricious,
depending on the source of zinc chloride.13
Treatment of the iodohydrin with KHMDS in THF
under anhydrous conditions at 78 ꢀC resulted in clean
conversion to a less polar compound by silica gel thin-
layer chromatography (TLC), which was presumed to
be the epoxide. Treatment of the putative epoxide gen-
erated in situ with phenylmagnesium bromide resulted
in successful coupling in 75% yield. Again, the trans-
iodohydrin t-5 led to a single adduct t-6, inferred to be
trans, whereas the cis-iodohydrin c-5 led to a chroma-
tographically separable equimolar mixture of cis- and
trans-products t- and c-6.
The scenario of batch-dependence of organometallic
reagents has been a familiar one to this laboratory.14 An
additive was sought that would bestow reproducibility
upon the arylzinc±epoxide coupling reaction. Ulti-
mately, cerium chloride provided such a result, a€ord-
ing reproducible coupling. As before, the trans-
iodohydrin t-7 led to a single product t-8 in 60±75%
yield, whereas the cis-iodohydrin c-7 led to a separable
1/1 mixture of cis- and trans-adducts t- and c-8 in com-
parable yields.
The feasibility of this approach was then tested by using
an N-protected 7-metalloindole species (Scheme 3). At
this point, it was desirable to select a more realistic
protecting-group scheme for synthetic purposes. Since
the mesitylsulfonamide provided adequate protection
for the lower indole but did not allow for facile depro-
tection, it was replaced with the 2-(trimethylsilyl)-
ethylsulfonamide (SES).11 For the upper indole, the
Not only had the protecting-group strategy proved
suitable for the organometallic coupling conditions, it

O. A. Moreno, Y. Kishi/Bioorg. Med. Chem. 6 (1998) 1243±1254
1247
Scheme 3. Reagents and conditions. (a) KHMDS, THF, 78 ꢀC, followed by addition of Ce reagent (prepared from N-SEM-7-bromo-
indole by treatment with n-Buli, THF, 78 ꢀC, ZnCl2, 78 ꢀC!rt, then CeCl3, rt), 78 ꢀC!rt. (b) TBAF, EDA, DMF, 70 ꢀC.
also proved suitable for facile single-step global depro-
tection. It had been hoped that the vicinal coupling
constants for the cis- and trans-adducts would be indi-
cative of the relative stereochemistries introduced dur-
ing the coupling, providing a method to assign the
relative stereochemistry between C.19 and C.27 of cyto-
blastin. Unfortunately, this turned out not to be the
case. Although the 1H NMR spectra of t-9 and c-9 were
di€erent from each other, the vicinal spin-coupling
constants for the C.19 and C.27 protons were found to
be 7.2 Hz for both isomers.
Among several synthetic routes studied, the synthesis of
N-SEM-7-bromo-IL-V (( )-11) was most e€ectively
achieved as shown in Scheme 5.18 Although bromination
of ( )-IL-V (2) itself was recorded in the literature, its
eciency was rather poor.19 However, with the speci®c
protecting groups indicated, bromination was accom-
plished in 90% yield. Although not necessary, it was
most convenient for purposes of post-coupling puri®ca-
tion to deprotect the alcohol at this stage.
A concise synthesis of both trans- and cis-allylstannanes
t- and c-13 was also developed (Scheme 6).
The Heck coupling of ( )-11 with c-13 was initially
e€ected under the standard conditions, yielding a 3:1
ratio of the desired and undesired regioisomers in
excellent yields. Since trans- and cis-allylstannanes t-13
and c-13 were shown to behave equivalently towards
( )-11, the more readily available cis-allylstannane c-13
was used for optimization studies.
After several successful runs, this palladium-catalyzed
coupling mysteriously ceased to function, leaving only
With the coupling and protecting-group strategies hav-
ing been validated by the second model system, the next
step was to extend this approach to the real system. For
this purpose, coupling of the in situ generated epoxide
with the iodide 10 was extensively studied. Unfortu-
nately, although almost any metal could be directly
inserted,15 none proved fruitful towards the coupling.
Second approach
Because of the diculty encountered in the ®rst
approach, we developed the revised strategy, involving
the Heck coupling16 of N-SEM-7-bromoindolactam V17
with an allylstannane, followed by dihydroxylation
(Scheme 4).
Scheme 4. The second retrosynthetic analysis of cytoblastin (1).

1248
O. A. Moreno, Y. Kishi/Bioorg. Med. Chem. 6 (1998) 1243±1254
inhibited the reaction after oxidative insertion, and (4)
introduction of an oxygen functionality at the a-position
of allylstannanes resulted in exclusive formation of the
undesired regioisomer.20
The coupling of ( )-11 with c-13 transpired most e€ec-
tively utilizing a stoichiometric quantity of tetrakis-
(triphenylphosphine)palladium(0) in re¯uxing benzene
in the presence of excess tetrabutylammonium iodide
(Scheme 7) at approximately 0.1 M concentration.
Under these conditions, the product distribution
favored the desired regioisomer by at least 10:1. The
coupling product 14 was isolated in 80% yield, along
with less than 10% of the regioisomer 15. Intriguingly,
1H NMR analysis indicated that the desired regioisomer
14 consisted primarily of one diastereomer.
Scheme 5. Reagents and conditions. (a) 1. TBS-Cl, imidazole,
DMF, rt; 2. SEM-Cl, NaH, THF, 15 ꢀC; 3. NBS, DMF,
15 ꢀC; 4. TBAF, THF, rt.
The next step in the synthesis was dihydroxylation of
the terminal ole®n of 14. Catalytic osmylation (OsO4/
NMMO) was ®rst attempted, but it was quickly realized
that the substrate was too sensitive to the oxidizing
environment required to regenerate the osmium tetr-
oxide. The method employed by Sakai and co-workers
(OsO4, THF, pyridine, followed by aq NaHSO3
workup)21 was tried, as it reportedly a€ected a relatively
hindered ole®n preferentially over the indole ring. This
method indeed resulted in the apparently desired pro-
duct in 30±40% yield, along with about 20% recovered
starting material. However, a serious diculty was
encountered with this method. The reproducibility in
cleavage of the osmate ester in the workup proved
capricious; addition of aq NaHSO3 to the reaction
sporadically generated the product but more typically
resulted in complete decomposition. Observations made
during repeated trials suggested that water might have
Scheme 6. Reagents and conditions. (a) 1. SES-Cl, NaOH,
cetyltrimethylammonium
chloride,
CH2Cl2,
0 ꢀC;
2.
CH2=CHMgBr, THF, 78 ꢀC; 3. Ac2O, Py, rt; 4. n-Bu3Sn-
Cu(n-Bu) (CN)Li2, THF, 78 ꢀC. (b) 1. same as (a) 1; 2. n-
Bu3SnCH2CH=PPh3, THF, 78 ꢀC!rt.
unreacted starting material. Repeated e€orts to restore
function, including doping with various transition metal
salts, proved fruitless. When a stoichiometric quantity
of metal complex was employed, again no coupling was
observed. However, some of the halide had been con-
sumed and promoted to a less polar spot on TLC (silica
gel). Chromatographic isolation (silica gel) and spectral
characterization (1H NMR, MS) revealed this com-
pound to be the aryl-palladium species. Treatment of
this compound with c-13 and TBAI in re¯uxing ben-
zene resulted in complete conversion to the coupled
products in a 1:1 ratio of the two regioisomers. This
experiment was repeated, without isolation of the aryl-
palladium intermediate, producing a 3:1 ratio of the
two regioisomers.
This study presented a unique opportunity to break the
catalytic cycle down into discrete steps, thereby allowing
multi-variable analysis of the reaction pathway. Exten-
sive e€orts were made to determine the e€ects of sol-
vents, ligands, and allylstannanes, demonstrating that (1)
polar solvents (DMF, CH3CN) resulted in virtually
exclusive formation of 15, (2) weakly coordinating
ligands [Ph3As, (2-furyl)3P, (o-Tol)3P] also resulted in
virtually exclusive formation of 15, (3) stronger coordi-
nating and bidentate ligands (PBu3, DPPE, DPPP)
Scheme 7. Reagents and conditions. (a) Pd(PPh3)4, TBAI,
PhH, 80 ꢀC.

O. A. Moreno, Y. Kishi/Bioorg. Med. Chem. 6 (1998) 1243±1254
1249
been the problem. Thus, 1,3-propanedithiol was used to
cleave the osmate ester under anhydrous conditions,
resulting in reproducible results.
1 in excellent yields. This result may suggest that the
rate-determining step involves an equilibrium between
the hemiaminal and the free indole NH, where removal
of the formaldehyde accelerates the reaction and drives
the reaction to completion. This rationale is supported
by the report that addition of ethylenediamine to TBAF
substantially improves the yields of SEM deprotec-
tions.25
With this workup procedure developed, the ecacy of
the dihydroxylation remained to be addressed. It was
envisioned that this might be achieved by enhancing the
reactivity of OsO4, which could be counteracted by
cooling, allowing for ®ne-tuning. Thus, N,N0-tetra-
methyl-ethylenediamine (TMEDA)22 was used to e€ect
osmylation at 90 ꢀC, and 1,3-propanedithiol was used
under anhydrous conditions to cleave the osmate
ester. This method consistently produced the dihy-
droxylated products in nearly quantitative yield. Sur-
prisingly, a single product accounted for 70% of the
mixture, with the other three comprising the remaining
30%.
Fortuitously, the global deprotection of the major pro-
duct 16 furnished a single product which matched cyto-
blastin (1)26 in every analysis performed (TLC, 1H
NMR (Fig. 3), [a]d, CD). Deprotection of the other
three osmylation products led to three compounds
which were clearly distinct from the natural product.
Stereochemical Elucidation
These results show that both the palladium(0)-mediated
coupling and the osmylation proceeded in a stereo-
selective manner. The bias observed in the osmylation
could plausibly be attributed to complexation of
osmium to the aminoindole ring prior to osmaoxetane
formation, resulting in facial-selective attack on the
ole®n. This rationale is loosely analogous to the ready
complexation of osmium with pyrrole as exploited by
Harman.23 The stereoselectivity exhibited by the Heck
coupling, on the other hand, is perplexing. The steric
bias must have arisen from the chirality in the lactam
ring, and it is most likely that the transmetallation24
occurred with some facial selectivity with respect to the
planar allylstannane. However, any connectivity
between these two suppositions remains unclear at this
time.
The fact that the synthesis reported led to the natural
product demonstrated that the stereochemistry of the
upper half of cytoblastin indeed corresponds to that of
( )-IL-V (2). However, the stereochemistry of the lower
half remained to be elucidated. To this end, e€orts to
crystallize cytoblastin or its derivatives had thus far
proved fruitless. Therefore, an alternative approach was
explored.
Global deprotection was then studied. Under the stan-
dard TBAF conditions, removal of the SES protecting
group was facile, but cleavage of the SEM protecting
group was problematic. It was serendipitously found
that, when a DMF solution of 16 containing excess
TBAF was evaporated in vacuo and stored under high
vacuum, the deprotection smoothly took place, to yield
Scheme 8. Reagents and conditions. (a) OsO4, TMEDA,
CH2Cl2 90 ꢀC. (b) TBAF, neat at 0.1 mm Hg.
Figure 3. 1H NMR spectra (5000 MHz) of the synthetic (Panel
A) and natural (Panel B) cycloblastin (1) in acetone-d6.

1250
O. A. Moreno, Y. Kishi/Bioorg. Med. Chem. 6 (1998) 1243±1254
Determination of the relative con®guration at the C.19
and C.27 positions was sought via spectroscopic stu-
dies on a cyclic derivative involving the upper indole
nitrogen and the ethylene glycol moiety. To this end, it
was found that treatment of 16 with lithium tetra-
¯uoroborate (LiBF4) and camphorsulfonic acid (CSA)
in THF resulted in transketalization of the SEM group
with the secondary alcohol, to give the seven-membered
aminal 17. 1H NMR analysis of the diacetate of 17
clearly showed the vicinal spin-coupling constant of the
C.19 and C.27 protons to be 10.0 Hz. Coupled with the
lack of NOE between them, this experiment established
the C.19 and C.27 protons in the seven-membered ring
to be trans, which transposed to a syn relationship in the
natural product. Thus, the complete structure of cyto-
blastin should be represented by either 1A or 1B.
Analysis of molecular models suggested that roughly a
nine-carbon aliphatic chain should provide such a
tether. Indeed, the ole®n 14 was successfully converted
into the macrolactone 21 (Scheme 9). Upon treatment
with: 1. K2CO3/MeOH; 2. CSA/THF/H2O; and 3.
TBAF/THF, 21 gave cytoblastin (1), demonstrating that
its complete structure was preserved in 21.
2-D NOESY of 21 revealed that the lactam ring was
locked into its minor (SOFA) conformation; a strong
NOE was observed between the amide N-H and
C.12-H but no NOE was detected between C.12-H and
C.8-H. Importantly, distinct NOE's were detected
between the C.19 proton and the protons of the ali-
phatic chain but no NOE was detected between the
C.27 proton and the protons of the aliphatic chain
(Fig. 4). Since the C.19 and C.27 relative stereo-
chemistry was shown to be trans (vide supra), this NOE
experiment allowed to establish on which side of the
aminal ring the chain passes. With the lactam ring
having provided the absolute spatial relationship
between the chain and the upper half, and the
NOESY having provided the orientation of the chain
relative to the aminal ring, the absolute stereo-
chemistry was deduced to be 1A. This conclusion was
further supported by the additional data provided by
the 2D NOESY NMR experiment. The structural
information thus provided is in agreement with that
provided by the MacroModel molecular modelling
program27 (Fig. 5).
In order to di€erentiate 1A from 1B, a method of cor-
relating the stereocenters in the lower half with those of
the upper half was explored, which exploited the obser-
vation that the SOFA conformation orients the lactam
hydroxymethyl on one side of the plane de®ned by the
upper indole ring, whereas the TWIST conformation
orients it on the other side. Therefore, by tethering the
two primary hydroxyl groups of 17, one might expect to
deduce the relative spatial relationship between the
upper and lower halves, which could then be extrapo-
lated to the absolute con®guration of the lower
portion.
Scheme 10. Reagents and conditions. (a) 1. TBS-Cl, imidazole,
DMF, rt. 2. OsO4, TMEDA, CH2Cl2, 90 ꢀC. (b) Azelaic acid,
.
EDAC, DMAP, DMAP HC1, CH2Cl2 rt. (c) LiBF4 (MeCN),
.
CSA, THF, rt. (d) EDAC, DMAP, DMAP HCl, CH2Cl2, rt.
Scheme 9. Reagents and conditions. (a) LiBF4 (MeCN), CSA,
THF, rt.

O. A. Moreno, Y. Kishi/Bioorg. Med. Chem. 6 (1998) 1243±1254
1251
Figure 5. A partial stereoview of the lactam 21.
represent C, N, O, and H, respectively.
,
,
,
,
cytoblastin have shown an activity towards PKC that is
roughly equivalent to ( )-IL-V.28 Finally, the results
reported immediately suggest new types of PKC activa-
tors, including C.27 acyl cytoblastin as well as dimeric
and pseudo-dimeric (S)-DAG's and tumor promoters.
Experimental
General information
Figure 4. The NOSEY spectrum (CDCl3) of 21. The panel A
shows the region of ( 0.2±8.2 ppm)Â( 0.2±8.2 ppm), whereas
the panel B shows the region of (4.1±5.4 ppm)Â(0.5±2.3 ppm).
The chemical shift for the C.19±H, C.27±H, C.28±H, and C.28±
H are 4.99 ppm (d), 4.49 (ddd), 4.56 (dd), and 3.73 (dd),
respectively.
NMR spectra were recorded on a Bruker AM-500
(500 MHz) spectrometer. Chemical shifts are reported in
parts per million (ppm) relative to tetramethylsilane.
The residual solvent peaks were used as internal refer-
ences. Fast atom bombardment (FAB) mass spectra
were obtained on either a JOEL JMS-AX505H or a
JOEL JMS-SX102, using 3-nitrobenzyl alcohol or gly-
cerol as the matrix. Sodium iodide was added when
indicated. Chemical ionization (CI) mass spectra were
recorded on a Kratos MS-50L double focusing instru-
ment using ammonia as the reagent gas. Optical rota-
tions were measured on a Perkin±Elmer 241 Polarimeter
at room temperature using sodium D line.
Conclusions
The two stereochemical hypotheses proposed at the
outset have been con®rmed. The stereochemistry about
the lactam ring has been shown to be degenerate with
that of the naturally occurring ( )-IL-V (2). Also, the
chirality at the C.27 in cytoblastin (1) was shown to be
equivalent to the C.9 stereochemistry of ( )-IL-V as
well as the C.2 stereochemistry of (S)-DAG. Although it
is generally agreed that ( )-IL-V (2) possesses the mini-
mum structural requirements for PKC activation, it is
interesting to note that cytoblastin had originally been
reported to exhibit a complete lack of activity towards
PKC.6 However, assays of both natural and synthetic
Analytical TLC was performed with E. Merck pre-
coated TLC plates, silica gel 60F-254, layer thickness
0.25 mm. Flash chromatography was performed with E.
Merck Kieselgel 60 (230±400 mesh) silica gel.
Reagents and solvents were used as supplied, with the
following exceptions: benzene, distilled from sodium

1252
O. A. Moreno, Y. Kishi/Bioorg. Med. Chem. 6 (1998) 1243±1254
benzophenone ketyl; chlorotrimethylsilane, distilled
from calcium hydride; ether, distilled from sodium
benzophenone ketyl; methylene chloride, distilled from
phosphorous pentoxide; pyridine, stored over anhy-
drous potassium hydroxide; tetrahydrofuran, distilled
from sodium benzophenone ketyl; toluene, distilled
from sodium.
3.0 (1H, dd, J=3.6 and J=17.6 Hz), 2.88 (3H, s), 2.76
(1H, t. J=6.0 Hz), 2.57 (1H, m), 0.91 (5H, m, d, J=
6.4 Hz), 0.61 (3H, d, J=6.7 Hz), 0.05 (9H, m).
Synthesis of cis-13. To a stirring 0.2 M solution of 3-
formylindole, 2.5 equiv powdered NaOH, and 0.01
equiv cetyltrimethylammonium chloride in CH2Cl2 at
0 ꢀC was added a 1.7 M solution of SES-Cl in CH2Cl2
dropwise. After 2 h, the reaction was acidi®ed with 1 N
HCl, and the phases separated. The organic phase was
washed with sat'd NaHCO3 and water, dried over
MgSO4, ®ltered and concentrated. Flash chromato-
graphy (silica gel; CH2Cl2) a€orded a beige crystalline
solid in 90% yield.
All reactions were performed under argon. For moist-
ure-sensitive reactions, reaction vessels were oven- or
¯ame-dried and allowed to cool under vacuum after
purging with argon.
Synthesis of ( )-11. To a stirring 0.2 M solution of ( )-
IL-V (( )-2)18 in DMF was added 1.0 equiv TBS-Cl, 2.0
equiv imidazole, and 0.1 equiv TBAI. After 2 h, the
reaction was diluted with EtOAc and washed with
water, 5% KHSO4, sat'd NaHCO3, and brine. The
organic phase was dried over MgSO4, ®ltered through
silica, and concentrated in vacuo to yield a slightly yel-
low crystalline solid, which was carried on without fur-
ther puri®cation.
To a stirring 0.5 M solution of LDA in THF at 0 ꢀC was
added 1.0 equiv of (n-Bu)3SnH dropwise. After 30 min,
the yellow solution was cooled to 78 ꢀC and added
dropwise via cannula to 1.05 equiv of vinyl triphenyl-
phosphorane 0.3 M in ether at 78 ꢀC. After 15 min, to
the resulting blood-red suspension was titrated the pro-
tected formylindole, as a 1 M solution in THF, until the
red color was dissipitated. The reaction was allowed to
warm to rt, poured into satd NH4Cl and extracted
twice with EtOAc. The organic phase was washed with
water and brine, dried over MgSO4, ®ltered, and con-
centrated. Flash chromatography (silica gel; 2% ether/
hexanes) a€orded ( )-13 as a mobile colorless oil in 50±
60% yield. HRMS: calcd. for C28H49NO2SSiSnNa
To a stirring 0.1 M solution of the crude product in
THF at 15 ꢀC was added 3.0 equiv NaH. After 30 min,
1.2 equiv SEM-Cl was added, and stirring was con-
tinued for another 1 h at 15 ꢀC. The reaction was
poured into satd NH4Cl and extracted with EtOAc.
The organic phase was washed with water (2Â) and
brine, dried over MgSO4, ®ltered through silica gel, and
concentrated in vacuo to yield a yellow oil, which was
carried on without further puri®cation.
1
=634.2177; found=634.2187. H NMR (CDCl3): 7.88
(1H, d, J=7.9 Hz), 7.65 (1H, d, J=7.2 Hz), 7.43 (1H,s),
7.33 (2H, m), 6.18 (1H, d, J=10.7 Hz), 6.07 (1H, m),
3.17 (2H, m), 2.12 (2H, d, J=9.6 Hz), 1.5±0.7 (20H, m),
0.05 (9H, m).
To a stirring 0.1 M solution of the crude product in
DMF at 15 ꢀC was added dropwise 1.5 equiv of a
1.0 M solution of NBS in DMF. After an additional 10
min of stirring, the reaction was poured into water and
extracted into EtOAc. The organic phase was then
washed with water, 5% KHSO4, satd NaHCO3, and
brine, dried over MgSO4, ®ltered through silica gel, and
concentrated in vacuo to yield a viscous green oil, which
was carried on without further puri®cation.
Heck coupling of ( )-11 with c-13. To a stirring 0.1 M
solution of ( )-11 in benzene under argon was added
1.0 equiv of tetrakis(triphenylphosphine)palladium(0),
and the suspension was heated to re¯ux, whereupon the
reaction homogenized. After 1 h, the reaction was allowed
to cool to rt and 1.5 equiv of (c)-13 was added, along with
5.0 equiv of TBAI, and re¯uxing was resumed overnight.
The homogenous reaction was cooled to rt, resulting in
a suspension, which was diluted with EtOAc and ®ltered
through celite. The ®ltrate was then concentrated in
vacuo, and ¯ash chromatographed (silica gel; 75%
EtOAc) a€ording 14 as a yellow amorphous solid in
80% yield. HRMS: calcd for C39H58N4O5SSi2Na
=773.3564; found=773.3591. 1H NMR (CDCl3) (major
conformer only): 7.86 (1H, d, J=8.3 Hz), 7.34 (1H, d,
J=7.8 Hz) 7.30 (2H, m), 7.15 (1H,m), 6.97 (1H, s), 6.89
(1H, d, J=8.1 Hz), 6.77 (1H, s), 6.51 (1H, d, J=8.2 Hz),
6.37 (1H, m), 5.82 (1H, d, J=4.9 Hz), 5.27 (2H, m), 5.16
(1H, d, J=11.4 Hz), 4.86 (1H, d, J=17.2 Hz), 4.35
(1H, m), 4.31 (1H, d, J=10.1 Hz), 3.72 (1H, m), 3.53
(3H, m), 3.17 (3H, m), 3.02 (1H, m), 2.86 (3H, s), 2.57
To a stirring 0.2 M solution of the crude product in
THF was added 1.5 equiv of a 1.0 M solution of TBAF
in THF. After 30 min, the reaction was diluted with
EtOAc, washed with water (2Â) and brine, dried over
MgSO4, ®ltered and concentrated. Flash chromato-
graphy (silica gel; EtOAc) a€orded ( )-11 as a yellow
foam in 80% yield over four steps. [a]d 150ꢀ (c 0.51,
EtOH). HRMS: calcd for C23H36N3O3BrSiNa
1
=532.1607; found=532.1603. H NMR (CDCl3): 7.26
(2H, m, (under CDCl3 peak), 6.89 (1H , s), 6.43 (1H, d,
J=8.4 Hz), 5.88 (1H, d, J=10.7 Hz), 5.72 (1H, d,
J=10.6 Hz), 4.25 (2H, m, d, J=9.9 Hz), 3.72 (1H, m),
3.55 (3H, m, t, J=8.2 Hz), 3.13 (1H, bd, J=17.6 Hz),

O. A. Moreno, Y. Kishi/Bioorg. Med. Chem. 6 (1998) 1243±1254
1253
(1H, m), 0.93 (5H, d, J=6.3 Hz), 0.78 (2H, m), 0.59 (3H,
d, J=6.7 Hz), 0.04 (18H, m).
a€orded TBS silyl ether as a yellow foam in 90%
yield.
Osmylation of 14. To a stirring 0.1 M solution of 14 in
CH2Cl2 was added 3 equiv of TMEDA, and the solu-
tion was cooled to 90 ꢀC. OsO4 (1.0 equiv, 0.75 M in
CH2Cl2) was added dropwise over 30 min. After 2.5 h,
10 equiv of 1,3-propanedithiol was added, and the
resulting dark-green mixture was allowed to warm to rt.
The reaction was then diluted with methanol, 50% by
volume, and allowed to stir overnight. The CH2Cl2 was
removed in vacuo, and the mixture was ®ltered through
celite, rinsing with methanol, and concentrated in
vacuo, to a€ord a viscous dark green oil. Flash chro-
matography (silica gel; 2%CH3OH/CHCl3) a€orded a
colorless amorphous solid in 70% yield. [a]d 140ꢀ (c
0.42, EtOH). HRMS: calcd for C45H74N4O7S-
Osmylation of TBS silyl ether conducted under the same
condition as the one given for osmylation of 14, to give
18.
To a stirring 0.01 M solution of 18 in CH2Cl2 was added
5
equiv azelaic acid, 2 equiv DMAP, 1.5 equiv
.
DMAP HCl, and 1.2 equiv EDAC. After 2 h, the reac-
tion was washed with H2O, dried over MgSO4, ®ltered
and concentrated in vacuo. Flash chromatography
(silica gel; 50% EtOAc/hexanes!EtOAC) a€orded a
colorless foam in 85% yield. [a]d 110ꢀ (c 0.36, EtOH).
1H NMR (CDCl3): 7.85 (1H, d, J=8.3 Hz), 7.56 (1H, d,
J=7.9 Hz), 7.43 (1H, s), 7.26 (2H, m), 7.14 (1H, d,
J=8.0 Hz), 6.78 (1H, s), 6.53 (1H, d, J=8.3 Hz), 6.23
(1H, d, J=1.6 Hz), 5.89 (1H, d, J=11.8 Hz), 5.39 (1H,
d, J=9.5 Hz), 5.32 (1H, d, J=11.8 Hz), 4.55 (1H, m),
4.47 (1H, dd, J=11.7 and 1.8 Hz), 4.23 (3H, m, d,
J=9.9 Hz), 3.63 (3H, m), 3.47 (1H, t, J=9.8 Hz), 3.19
(2H, t, J=8.9 Hz), 3.1 (1H, bd, J=14.5 Hz), 2.86 (4H,
m, s), 2.56 (1H, m), 2.32 (4H, m), 1.61 (4H, m), 1.30
(6H, m), 0.92 (16H, m), 0.47 (3H, d, J=6.8 Hz), 0.0
(24H, m).
1
Si3Na=921.4484; found=921.4474. H NMR (CDCl3)
(major conformer only): 7.83 (1H, d, J=8.3 Hz), 7.69
(1H, d, J=7.9 Hz), 7.27 (2H, m), 7.19 (2H, m), 6.77 (1H,
s), 6.57 (1H, d, J=8.3 Hz), 6.16 (1H, s), 5.63 (1H, d,
J=11.5 Hz), 5.40 (1H, d, J=11.3 Hz), 5.38 (1H, d,
J=8.9 Hz), 4.39 (1H, m), 4.22 (2H, m, d, J=10.0 Hz),
3.85 (1H, m), 3.72 (1H, m), 3.6 (3H, m), 3.46 (1H, dd,
J=9.8 and 9.2 Hz), 3.1 (4H, m), 2.88 (3H, s), 2.55
(1H, m), 2.33 (1H, d, J=2.7 Hz), 2.22 (1H, m), 0.89
(16H, m), 0.50 (3H, d, J=6.7 Hz), 0.02 (24H, m).
To a stirring 0.03 M solution of this product in THF
was added 5 equiv of LiBF4, 1.0 M in CH3CN, followed
by 5 equiv of CSA, and the reaction was allowed to stir
overnight. The reaction was diluted with EtOAc,
washed with H2O (2Â) and brine, dried over MgSO4,
®ltered, and concentrated in vacuo. Flash chromato-
graphy (silica gel; 9/0.2/0.2 CH2Cl2/CH3OH/HOAc)
a€orded 20 as a colorless foam in 84% yield. [a]d 110ꢀ
(c 0.41, EtOH). HRMS: calcd for C43H60N4O9S-
SiNa=859.3748; found=859.3766. 1H NMR (CDCl3)
(major conformer only): 7.95 (1H, d, J=8.3 Hz), 7.47
(1H, d, J=8.0 Hz), 7.38 (1H, t, J=8.0 Hz), 7.30 (1H,
m), 7.18 (2H, m), 6.79 (1H, s), 6.49 (1H, d, J=8.1 Hz),
6.30 (1H, d, J=8.3 Hz), 5.91 (1H, d, J=10.7 Hz), 5.33
(1H, d, J=10.6 Hz), 4.88 (1H, d, J=9.3 Hz), 4.65 (1H,
br), 4.34 (1H, m), 4.28 (1H, d, J=10.1 Hz), 4.22 (1H,
m), 4.05 (1H, br m), 3.76 (1H, br), 3.59 (1H, br), 3.22
(2H, m), 3.10 (2H, br m), 2.83 (3H, s), 2.56 (1H, br),
2.25 (4H, br m), 1.60 (4H, br), 1.31 (6H, br), 0.87 (5H,
m), 0.58 (3H, d, J=6.5 Hz), 0.03 (9H, m).
Synthetic cytoblastin (1). To a solution of the triol in
DMF (arbitrary concentration) was added solid TBAF
(6 equiv), and the resulting red solution was con-
centrated in vacuo and placed on a vacuum line
(ꢁ0.1 mm Hg) overnight. The residue was partitioned
between 5% KHSO4 and EtOAc, and the phases were
separated. The organic phase was washed with H2O and
brine, dried over MgSO4, ®ltered and concentrated in
vacuo. Flash chromatography (silica gel; 10% CH3OH/
CHCl3) a€orded 1 as a colorless amorphous solid in
90% yield. [a]d 110ꢀ (c 0.30, MeOH). HRMS: calcd
for C28H34N4O4Na=513.2478; found=513.2473. 1H
NMR (acetone-d6) (major conformer only): 10.14 (1H,
br), 10.02 (1H, br), 7.45 (2H, m), 7.32 (1H, d,
J=8.1 Hz), 7.10 (1H, d, J=7.9 Hz), 7.00 (1H, t,
J=8.1 Hz), 6.96 (1H, s), 6.86 (1H, t, J=8.1 Hz), 6.41
(1H, d, 7.9 Hz), 6.28 (1H, s), 4.77 (1H, d, J=5.3 Hz),
4.56 (1H, m), 4.35 (1H, d, J=10.2 Hz), 4.12 (1H, m),
3.63 (1H, m), 3.50 (3H, m), 3.08 (1H, br d, J=17.6 Hz),
3.0 (1H, dd, J=3.6 and 17.2 Hz), 2.85 (3H, s), 2.52 (1H,
m), 0.85 (3H, d, J=6.4 Hz), 0.55 (3H, d, J=6.8 Hz).
To a stirring 1 mM solution of 20 in DMF was added 6
.
equiv DMAP, 4 equiv DMAP HCl, and 4 equiv EDAC.
After 5 days, the reaction was concentrated in vacuo,
the residue was dissolved in EtOAc and washed with
H2O, 5% KHSO4, satd NaHCO3, H2O, and brine, dried
over MgSO4, ®ltered, and concentrated in vacuo. Flash
chromatography (silica gel; CH2Cl2!20% EtOAC/
CH2Cl2), followed by size exclusion chromatography,
a€orded 21 as a colorless, amorphous solid in 15%
Preparation of 21 from 14. To a stirring 0.2 M solution
of 14 in DMF was added 2 equiv imidazole, 1.2 equiv
TBS-Cl, and 0.1 equiv TBAI. After 2 h, the reaction
was diluted with EtOAc, washed with H2O (2Â) and
brine, dried over MgSO4, ®ltered and concentrated.
Flash chromatography (silica gel; 20% EtOAc/hexanes)

1254
O. A. Moreno, Y. Kishi/Bioorg. Med. Chem. 6 (1998) 1243±1254
yield. [a]d 41ꢀ (c 0.39, CHCl3). HRMS: calcd for
C43H58N4O8SSiNa=841.3642; found=841.3632. 1H
NMR (CDCl3): 7.96 (1H, d, J=8.2 Hz), 7.39 (2H, s),
7.38 (1H, t, J=8.5 Hz), 7.32 (1H, d, J=7.9 Hz), 7.20
(1H, t, J=8.1 Hz), 6.85 (1H, s), 6.82 (1H, d, J=7.9 Hz),
6.64 (1H, d, J=7.9 Hz), 5.88 (1H, d, J=10.3 Hz), 5.36
(1H, d, J=10.3 Hz), 5.07 (1H, d, J=10.6 Hz), 4.99 (1H,
d, J=10.0 Hz), 4.66 (1H, dd, J=1.4 and 11.6 Hz), 4.56
(1H, m), 4.56 (1H, dd, J=2.2 and 12.2 Hz), 4.49 (1H,
ddd, J=2.2 and 10.3 Hz), 3.88 (1H, dd, J=4.4 and
11.4 Hz), 3.73 (1H, dd, J=2.4 and 11.7 Hz), 3.25 (2H,
m), 3.16 (1H, dd, J=4.7 and 14.7 Hz), 3.00 (1H, d,
J=10.9 Hz), 2.76 (1H, dd, J=1.9 and 14.5 Hz), 2.71
(3H, s), 2.36 (2H, m), 1.85±1.20 (12H, m), 1.14 (3H, d,
J=6.5 Hz), 0.92 (3H, d, J=6.5 Hz), 0.83 (2H, m), 0.02
(9H, s).
12. Lipshutz, B. H.; Pegram, J. J. Tetrahedron Lett. 1980, 21,
3343.
13. When the addition failed, the epoxide was quantitatively
rearranged to the homobenzylic ketone.
14. Jin, H. ; Uenishi, J.-I.; Christ, W. J.; Kishi, Y. J. Am.
Chem. Soc. 1986, 108, 5644. Takai, K.; Tagashira, M.; Kuroda,
T.; Oshima, K.; Utimoto, K.; Nozaki, H. J. Am. Chem. Soc.
1986, 108, 6048.
15. The organometallic derivatives prepared and tested for the
coupling included: MgCl2, MgCl2/CuX, Zn/Cu, Zn/Cu/
.
CuCN 2LiCl, ZnCl2/CeCl3, CuCN, and MnCl2.
16. For a review on Heck reactions, see for example: Hegedus,
L. S. Transition Metals in the Synthesis of Complex Organic
Molecules; University Science Books: Mill Valley, 1994; pp
103±113.
17. N-SEM-7-bromo-IL-V was chosen because we had pre-
viously encountered diculty isolating the corresponding N-
SEM-7-iodo-IL-V.
18. (±)-IL-V was prepared from l-tryptophan methyl ester,
according to the reported method: Kogan, T. P.; Somers, T. C.;
Venuti, M. C. Tetrahedron 1990, 46, 6623.
Acknowledgement
Financial support from the National Institutes of
Health (CA-22215) is gratefully acknowledged.
19. Irie, K.; Hagiwara, N.; Koshimizu, K. Tetrahedron 1987,
43, 5251.
20. The electronic and steric e€ects of allylstannanes were tes-
ted; the coupling with a-acetoxy, a-methoxy, and a-t-butyldi-
methylsilyloxyallylstannanes resulted in exclusive formation of
15, whereas tri-sec-butylstannane resulted in a 5/1 mixture of
14 and 15.
References and Notes
1. For recent reviews on PKC, see for example: Protein Kinase
C: Current Concepts and Future Perspectives; Ellis Horwood,
1992.
21. Takayama, H.; Yuhshin, T.; Kitajima, M.; Aimi N.; Sakai,
S. J. Org. Chem. 1994, 59, 4381.
2. Nakamura, H.; Kishi, Y.; Pajares, M. A.; Rando, R. R.
Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 9672. For reviews on
this work, see: Rando, R. R.; Kishi, Y. Biochemistry 1992, 31,
2211; Kishi, Y.; Rando, R. R. Acc. Chem. Res. 1998, 31, 163.
22. (a) For diamine acceleration of osmylations: Corey, E. J.;
DaSilva, J. P.; Virgil, S.; Yuen, P. W.; Connell, R. D. J. Am.
Chem. Soc. 1989, 111, 9243. (b) For examples for the use of
TMEDA or related diamines for low-temperature osmylation,
see: Wang, Y.; Babirad, S. A.; Kishi, Y. J. Org. Chem. 1992, 57,
468.
3. Fujiki, H.; Suganuma, M.; Tahira, T.; Yoshioka, A.;
Nakayasu, M.; Endo, Y.; Shudo, K.; Takayama, S.; Moore, R.
E.; Sugimura, T. Cellular Interactions by Environmental Tumor
Promoters; Japan Sci. Soc.: Utrecht, 1984; pp 37±45.
23. Hodges, L. M.; Gonzalez, J.; Koontz, J. I.; Myers, W. H.;
Harman, W. D. J. Org. Chem. 1993, 58, 4788 and references
cited therein.
4. Endo, Y.; Hasegawa, M.; Itai, A.; Shudo, K.; Tori, M.;
Asakawa, Y.; Sakai, S. Tetrahedron Lett. 1985, 26, 1069.
24. The extensive mechanistic studies conducted suggest that
the observed regioselectivity of the coupling towards the
desired adduct arises during the initial transmetallation step,
whereas net leakage through a p-allylpalladium intermediate
goes unilaterally towards the undesired adduct (ref 1b).
5. These models are quoted in reviews cited under reference 2.
6. Kumagai, H.; Iijima, M.; Dobashi, K.; Naganawa, H.;
Sawa, T.; Hamada, M.; Ishizuka, M.; Takeuchi, T. J. Anti-
biotics 1991, 44, 1029.
7. The numbering system adopted in this paper corresponds to
that of cytoblastin.6
25. Muchowski, J. M.; Solas, D. R. J. Org. Chem. 1984, 49,
203.
8. (a) Preliminary results were communicated in J. Am. Chem.
Soc. 1996, 118, 8180. (b) This paper is largely based on the
Dissertation of Moreno, O. A., Harvard University, July, 1996.
26. We are indebted to Drs. Kumagai and Naganawa at
Microbial Chemistry Research Foundation for a sample of
natural cytoblastin.
9. Still, W. C.; Gennari, C. Tetrahedron Lett. 1983, 24, 4405.
27. MacroModel version 45 provided courtesy of Professor
Still, Columbia University Minimizations were performed on a
Silicon Graphics Indigo workstation.
10. Fujii, N.; Futaki, S.; Yasamura, K.; Yajima, H. Chem.
Pharm. Bull. 1984, 32, 2660.
11. Weinreb, S. M.; Demko, D. M.; Lessen, T. A. Tetrahedron
Lett. 1986, 27, 2099.
28. Assays performed by Professor Rando at Harvard Medical
School and by Dr Gusovsky at Eisai Research Institute.