Eponemycin analogues: Syntheses and use as probes of angiogenesis

Article abstract of DOI:10.1016/S0968-0896(98)00089-3

Derivatives of the epoxy-β-aminoketone containing natural product eponemycin have been prepared in order to study the molecular mode of action of this anti-angiogenic compound. Synthesis and use of a biotinylated dihydroeponemycin analogue demonstrated th

Full text of DOI:10.1016/S0968-0896(98)00089-3

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1209±1217
Eponemycin Analogues: Syntheses and use as Probes of
Angiogenesis
Ny Sin, Lihao Meng, Hak Auth and Craig M. Crews*
Department of Molecular, Cellular and Developmental Biology, Yale University, 219 Prospect Street, New Haven,
CT 06520-8103, USA
Received 17 March 1998; accepted 5 May 1998
AbstractÐDerivatives of the epoxy-b-aminoketone containing natural product eponemycin have been prepared in
order to study the molecular mode of action of this anti-angiogenic compound. Synthesis and use of a biotinylated
dihydroeponemycin analogue demonstrated that dihydroeponemycin forms a covalent adduct with at least two intra-
cellular proteins in human endothelial cells. Pretreatment of cells with a ®ve equivalent excess of dihydroeponemycin
precluded biotin-dihydroeponemycin binding indicating a speci®c interaction between natural product and the target
proteins. This biotin-dihydroeponemycin derivative will prove useful in the puri®cation and identi®cation of epone-
mycin receptors. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
proven useful in our e€orts to use biologically active
natural products as molecular probes in the study of
angiogenesis. As part of this endeavor, a semi-synthetic
fumagillin biotinylated derivative was constructed and
demonstrated to bind covalently to the metalloprotease
methionine aminopeptidase 2 (MetAP-2).6 We and oth-
ers have further shown that fumagillin inhibits the in
vivo and in vitro enzymatic activity of MetAP-2,6,7
providing strong evidence that MetAP-2 is the physio-
logical target of fumagillin.
Tumor-induced new blood vessel formation (angiogen-
esis or neovascularization) plays a crucial role in both
tumor progression and metastases by supplying essential
nutrients to tumor cells within the cell mass and by
providing conduits for dislodged cancerous cells.1 Given
the critical role of angiogenesis in tumor progression,
signi®cant pharmaceutical interest has focused on the
identi®cation of novel anti-angiogenic compounds.2
Clearly, such compounds hold much clinical promise as
a means of limiting both the size and spread of primary
tumors. Despite the identi®cation and development of a
number of potential anti-angiogenic therapeutics, little
is known of the mechanisms of action of these com-
pounds at the molecular level. For example, TNP-470,
derived from the biologically active natural product
fumagillin,3 is the leading anti-angiogenic drug in
clinical trials.4,5 However, until recently, investigation
of this compound was limited primarily to its poten-
tial clinical applications. Because of its potency and
endothelial cell selectivity, TNP-470/fumagillin has
In our continuing development of angiogenic molecular
probes, we have recently focused on one of the most
promising angiostatic natural products, eponemycin (1).
Isolated from Streptomyces hygroscopicus No. P247-71,8
this microbial metabolite has proven to be very potent
in the inhibition of endothelial cell growth and migra-
tion in vitro and in the inhibition of neovascularization
in an animal model system.9 Speci®cally, eponemycin
was found to exhibit in vivo anti-angiogenic activity
(ID50=0.1 ng/egg) in the chorioallantoic membrane
(CAM) of growing chick embryos.9 Despite this potent
activity and potential clinical use, the mechanism of
action for eponemycin remains unknown.
Key words: Angiogenesis; natural product; mode of action;
anity reagent; adduct formation.
*Corresponding author. Fax: (203) 432-5713;
E-mail: craig.crews@yale.edu
In designing molecular probes based on biologically
active natural products, it is important not to compro-
mise the active pharmacophore of the original
0968-0896/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00089-3

1210
N. Sin et al./Bioorg. Med. Chem. 6 (1998) 1209±1217
compound. Fortunately, comparison of eponemycin
with a related natural product, epoxomicin, provides
insights into the critical structural determinants that
comprise eponemycin's pharmacophore. Epoxomicin
(2), isolated from the actinomycete strain, No. Q996-
17,10 also possesses an epoxy-b-aminoketone moiety.
Moreover, like eponemycin, epoxomicin is more active
against solid tumors in animals than leukemias, sug-
gesting that it too mediates its antitumor activity via an
anti-angiogenic mechanism.10 Since the two compounds
share several structure elements, we propose that the
common structural motifs may represent an anti-angio-
genic pharmacophore (3) (Figure 1).
Results and Discussion
Anity reagent synthesis
Given the similar biological activities between epone-
mycin and dihydroeponemycin,8 the latter was chosen
as the sca€olding for the design of an eponemycin-based
molecular probe. Our syntheses of 4 and 5 were in¯u-
enced by the previously reported strategies of Schmidt11
and Hoshi.12 Our approach to 4 and 5 began with the
addition of a vinyl lithium derived from treatment of 2-
bromo-1-hydroxy-2-propene (7)12,13 with t-butyl
lithium,14 to aldehyde 6, which a€orded a 1:1 insepar-
able mixture of diol 8 (Scheme 1). Selective protection of
the primary alcohol of 8 as a t-butyldimethylsilyl (TBS)
ether and a Swern oxidation of the remaining secondary
alcohol yielded the a,b-unsaturated ketone 9. Epoxida-
tion of 9 with hydrogen peroxide11 a€orded the epoxy-
ketone 10a and 10b as a 1.2:1 mixture which was readily
separated by ¯ash column chromatography. Final
removal of the benzyloxycarbonyl protecting group of
10a through a catalytic hydrogenation12 reaction gave
epoxy-b-aminoketone 11 in excellent yield.
Given the potency of eponemycin and the requirement
for the epoxide for biological activity,8 we hypothesized
that eponemycin may mediate its cytostatic activity via a
covalently interaction with a protein receptor. As the
®rst step towards the puri®cation and identi®cation of
an eponemycin receptor(s), an anity reagent, dihy-
droeponemycin-biotin (5), has been synthesized and
used to probe endothelial cellular proteins. Because
dihydroeponemycin (4) has comparable biological
activities to eponemycin,8 we also synthesized it for
use as a positive control for binding speci®city assays
(Fig. 2).
We next turned to the preparation of carboxylic acid 13
(Scheme 2), the left-hand fragment of 4. Coupling of
isooctanoic acid 12 (a generous gift from Exxon) with
Figure 1.
Figure 2.

N. Sin et al./Bioorg. Med. Chem. 6 (1998) 1209±1217
1211
Scheme 1.
Scheme 2.
serine benzylester using HBTU, followed by protection
of the serine moiety side chain as a TBDPS ether and
hydrogenalysis of the benzyl protecting group a€orded
13. An HATU and HAOt initiated coupling of car-
boxylic acid 13 with epoxy-b-aminoketone 11, followed
by removal of the silyl protecting groups accomplished
the synthesis of dihydroeponemycin 4. Correlation of
the spectroscopic data of 4 with the published data
established the stereochemistry of 4 and epoxy-b-amino-
ketone 11.
group was removed to give the carboxylic acid left hand
fragment 16. Coupling of the epoxy-b-amino-ketone 11
to 16 a€orded the tripeptide 17. The Fmoc and silyl
protecting groups of 17 were removed in a single step
using tetrabutylammonium ¯uoride. Finally, coupling
of the resulting amine with N-hydroxysuccinimide-bio-
tin reagent (Calbiochem) accomplished the synthesis of
the biotinylated dihydroeponemycin anity reagent (5).
Covalent and selective adduct formation with eponemycin
protein receptors
After the synthesis of 4, our attention focused on the
synthesis 5 (Scheme 3). Our approach to 5 began with
coupling of N-Fmoc-e-aminocaproic acid 14 to the ser-
ine benzylester which gave hydroxyester 15 in excellent
yield. The hydroxyl group of 15 was protected as a
TBDPS ether and subsequently the benzyl protecting
Given the importance of the epoxide moiety for biolo-
gical activity,8 we hypothesized that eponemycin may
form a covalent adduct with a protein receptor(s) via
nucleophilic attack on the epoxide. In order to test this
hypothesis, the biotinylated dihydroeponemycin anity
Scheme 3.

1212
N. Sin et al./Bioorg. Med. Chem. 6 (1998) 1209±1217
reagent 5 was incubated with cultured bovine aortic
endothelial cells (BAECs) in increasing concentrations
before examination of protein lysates for newly biotinyl-
ated proteins. Lysates from treated cells were analyzed
by denaturing sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS±PAGE), protein immobiliza-
tion onto polyvinylidene di¯uoride (PVDF) membrane
followed by biotinylated protein detection via avidin
conjugated horseradish peroxidase (HRP). As shown in
Figure 3, in the absence of added dihydroeponemycin-
biotin, only higher molecular weight biotinylated pro-
teins were detected. However, upon addition of increas-
ing concentrations of 5 to intact BAECs, a major
23 kDa protein and minor 25 kDa protein were biotinyl-
ated at concentration of 5 equal to the IC50 value for
BAEC proliferation inhibition (data not shown). A
much weaker biotinylated protein of approximately
30 kDa, which correlates with the addition of 5, was
detected upon overexposure of the enhanced chemi-
luminescence blot (data not shown).
Conclusions
Neovascularization (angiogenesis) is an attractive target
for pharmaceutical intervention since it plays an impor-
tant role in a variety of human pathologies. In addition
to tumor growth and metastatic control, diabetic reti-
nopathy and rheumatoid arthritis are also examples of
diseases currently being targeted using an anti-angio-
genic pharmacological approach. Despite the many
clinical applications of several of the leading anti-
angiogenic compounds, little is known of the molecular
mechanisms by which they act. In order to gain possible
insights into endothelial cell cycle control, we have
synthesized derivatives of the potent anti-angiogenic
natural product eponemycin. Addition of the cell-
permeable dihydroeponemycin-biotin anity reagent 5
to human endothelial cell cultures results in covalent
adduct formation between this natural product deriva-
tive and two 23±25 kDa proteins. Moreover, these pro-
tein±ligand interactions were shown to be speci®c
through the pretreatment of endothelial cells with excess
non-biotinylated compound 4 before challenging with
the biotinylated natural product derivative. Covalent
attachment to these protein receptors will greatly facil-
itate the puri®cation and identi®cation of these proteins
that are likely to mediate the anti-angiogenic activity of
eponemycin.
Given the high degree of reactivity of the epoxide moi-
ety, it was necessary to rule out the possibility that the
protein±natural product adduct formation is simply due
to non-speci®c association. To discount this possibility,
a
®ve molar excess of non-biotinylated dihydro-
eponemycin was added to BAECs for 30 min before the
cells were challenged with biotinylated dihydro-
eponemycin 5. As seen in Figure 3, such pretreatment
of the cells with 4 abolishes subsequent covalent
interaction with 5. This result demonstrates that 5 is
speci®cally and selectively interacting with at least two
protein receptors with molecular weights of 23 and
25 kDa. These proteins are presently being puri®ed in
sucient quantities for protein sequencing.
Experimental
General experimental methods
Solvent and reagent abbreviations used are Bn=
benzyl, Boc=tert-butyloxycarbonyl, DIEA=diisopropyl-
ethylamine, DMF=dimethylformamide, DMSO=
Figure 3. Biotinylated protein analysis from 12.5% SDS±PAGE of dihydroeponemycin-biotin (4) treated HUVEC lysates visualised
with ECL and avidin-HRP.

N. Sin et al./Bioorg. Med. Chem. 6 (1998) 1209±1217
1213
dimethylsulfoxide, ECL=enhanced chemiluminescence,
ECGS=endothelial cell growth supplement, Fmoc=
9-¯uorenylmethoxycarbonyl, HBTU=O-benzotriazo-1-
yl-N,N,N0,N0-tetramethyluronium hexa¯uorophosphate,
2-bromo-3-propen-1-ol (7) (2.73 g, 19.9 mmol) solution
in Et2O (10 mL) was added dropwise. The solution was
warm to 0 ꢀC immediately. After stirring for 4 h, the
solution was re-cooled to 78 ꢀC and was treated with
Z-leucinal (6) (1.22 g, 4.89 mmol) solution in Et2O
(10 mL) dropwise. After stirring at 78 ꢀC for 2.5 h, the
reaction was stirred at 0 ꢀC for an additional 1.5 h; sub-
sequently, it was quenched with brine (30 mL). The
organic layer was separated and the aqueous layer was
extracted with Et2O (3Â50 mL). The combined organic
layer was dried and concentrated. The product was puri-
®ed by ¯ash column chromatography (hexanes:EtOAc,
1:1) to give a mixture of diol 8 (807.4 mg, 54%): IR
HRP=horseradish
umbilical cord venous endothelial cells, NHS=N-
peroxidase,
HUVEC=human
hydroxyl succinimide, PMA=phosphomolybdic acid,
PVDF=polyvinylidene
di¯uoride,
SDS±PAGE=
sodium dodecyl sulfate polyacrylamide gel electrophor-
esis, TBS=tert-butyldimethylsilyl, TBDPS=tert-butyl-
diphenylsilyl, THF=tetrahydrofuran, TFA=tri¯uoro-
acetic acid, Z=benzyloxycarbonyl.
1
Optical rotations were determined at the sodium D line
(589 nm) and recorded on a Perkin±Elmer 241 polari-
meter. IR spectra were recorded from neat samples
evaporated on a sodium chloride plate using a Perkin±
Elmer Paragon 1000 FT-IR instrument. NMR spectra
were recorded in CDCl3 using a Bruker AM-500
(500 MHz), except as noted. High and low resolution
mass spectra were obtained at the Mass Spectrometry
Laboratory of the University of Illinois at Urbana-
Champaign. High-pressure liquid chromatography
(HPLC) separations were performed on a Ranin Pre-
parative Chromatography System using an 8Â100 mm or
25Â100 mm Prep Nova-Pak HR silica (60 A, 6 mm)
column for analytical or preparative scale, respectively.
Flash column chromatography was performed follow-
ing the method of Still with Merck silica gel 60, 230±400
mesh and monitored by thin-layer chromatography
(TLC) using Merck silica gel 60 F254 precoated 0.25 mm
layer thickness plates. Hexanes:EtOAc or CH2Cl2:MeOH
solvent systems were used for elution and the TLC plates
were stained with anisaldehyde or PMA.
3336, 2956, 1693 cm 1; H NMR: d 7.36±7.33 (m, 5H),
5.16±5.06 (m, 5H), 4.25±4.24 (d, J=5.0 Hz, 1H), 4.20±
4.12 (m, 1H), 4.0±3.95 (m, 1H), 3.91±3.84 (m, 1H), 3.68±
3.61 (d, J=6.1 Hz, 1H), 3.38±3.37 (d, J=5.1 Hz, 1H),
3.09±3.07 (d, J=5.7 Hz, 1H), 1.71±1.65 (m, 2H), 1.47±
1.41 (m, 1H), 1.39±1.37 (m, 1H), 1.35±1.25 (m, 1H),
0.97±0.89 (m, 6H); 13C NMR: d 156.9, 156.8, 148.7,
147.3, 136.4, 136.3, 128.4, 128.4, 128.1, 128.0, 127.9,
127.8, 113.9, 113.1, 74.6, 66.8, 66.7, 64.5, 63.7, 52.7,
52.1, 41.3, 38.3, 24.9, 24.6, 23.1, 23.0, 21.9, 21.4;
HRFABMS calcd for C17H26NO4 308.1862, found
308.1863.
(3RS,4S,)-4-Benzyloxycarbonylamino-2-tert-butyldimethyl-
siloxymethyl-3-hydroxy-6-methyl-1-heptene. To a solu-
tion of 8 (807.4 mg, 2.63 mmol) was added imidazole
(196.7 mg, 2.89 mmol), and t-butyldimethylsilyl chloride
(407.8 mg, 2.71 mmol). After stirring at rt for 12 h, the
reaction was vacuum ®ltered. The mother liquor was
concentrated and puri®ed by ¯ash column chromato-
graphy (hexanes:EtOAc, 5;1) to give (3RS,4S,)-4-benzyl-
oxycarbonylamino-2-tert-butyldimethylsiloxymethyl-3-
hydroxy-6-methyl-1-heptene (880.7 mg, 80%): IR 3434,
Solvents were puri®ed before each reaction as described
below unless otherwise noted. THF and dichloro-
methane were distilled from sodium and benzophenone
ketyl and calcium hydride respectively. DMF was dis-
tilled from calcium hydride under reduced pressure and
stored over 4 A molecular sieves. DIEA and piperidine
were purchased from Aldrich and were used without
further puri®cation.
1
2955, 1704 cm 1; H NMR d: 7.32±7.28 (m, 5H), 5.28±
5.26 (d, J=9.7, 1H), 5.19±5.03 (m, 4H), 4.29±4.2 (m,
3H), 3.93±3.90 (p, J=4.7 Hz, 1H), 3.27±3.26 (d,
J=4.27 Hz, 1H), 1.70±1.65 (m, 1H), 1.56±1.51 (m, 1H),
1.38±1.32 (m, 1H), 0.97±0.91 (m, 15H), 0.11±0.09 (d,
J=5.9 Hz, 6H); 13C NMR: d 156.9, 148.3, 136.8, 128.5,
128.0, 128.0, 127.9, 111.8, 74.4, 66.6, 64.9, 51.8, 41.9,
26.0, 24.9, 23.4, 22.2, 18.3.
All anhydrous reactions were performed under a nitro-
gen atmosphere except as noted. The glassware for these
anhydrous reactions was ¯ame-dried and cooled under
nitrogen atmosphere prior to use. The progress of the
reactions was monitored by TLC unless otherwise
noted. Reactions were dried over MgSO4 or Na2SO4
before concentration which was performed under
reduced pressure using a Buchi roto-evaporator.
(4S)-4-Benzyloxycarbonylamino-2-tert-butyldimethyl-
siloxymethyl-3-oxo-6-methyl-1-heptene (9). To a 78 ꢀC
solution of oxalyl chloride (0.11 mL, 1.30 mmol) in
CH2Cl2 (1 mL) was added, in 8 min interval, DMSO
(0.18 mL, 2.59 mmol) in CH2Cl2 (1 mL), (3RS,4S,)-4-
benzyloxycarbonylamino-2-tert-butyldimethylsiloxy-
methyl-3-hydroxy-6-methyl-1-heptene
(182.1 mg,
0.432 mmol), and triethylamine (0.36 mL, 2.59 mmol).
The cold bath was removed and the reaction mixture
was stirred at ambient temperature for additional
10 min. The reaction mixture was poured into aqueous
(3RS,4S)-4-Benzyloxycarbonylamino-3-hydroxy-2-hydr-
oxymethyl-6-methyl-1-heptene (8). To
a solution of
t-BuLi (35.5 mL, 49.8 mmol) in Et2O (30 mL) at 78 ꢀC,

1214
N. Sin et al./Bioorg. Med. Chem. 6 (1998) 1209±1217
HCl (1.5 mL, 2 N) and shaken; the organic layer was
separated and the aqueous layer was extrated with
CH2Cl2 (2Â5 mL). The combined organic layer was
washed with saturated NaHCO3 (3 mL), brine (5 mL),
dried and concentrated. The crude product was puri®ed
by ¯ash column chromatography (hexanes:EtAOc, 10:1)
to give 9 (168.4 mg, 93%): 1H NMR: d 7.36±7.31 (m,
5H), 6.24±6.22 (d, J=6.6 Hz, 2H), 5.42±5.41 (d,
J=8.7 Hz, 1H), 5.15±5.13 (m, 1H), 5.10 (s, 2H), 4.43±
4.31 (dd, J=44.7 Hz, 15.6, 2H), 1.78±1.67 (m, 1H),
1.61±1.52 (m, 1H), 1.43±1.35 (m, 1H), 1.04±1.02 (d,
J=6.5 Hz, 3H), 0.93 (s, 9H), 0.92±0.91 (d, J=6.8 Hz,
3H); 13C NMR: d 200.3, 156.1, 145.3, 128.5, 128.1,
128.0, 124.8, 66.9, 61.1, 53.7, 43.1, 25.9, 24.9, 23.4, 21.7,
18.3; HRFABMS calcd for C23H38NO4Si 420.2570,
found 420.2569.
the catalyst was removed by vacuum ®ltration through a
celite pad. The crude product was stirred in vacuo for
0.5 h (18.4 mg, 97%) and was used immediately without
further puri®cation: IR 3378, 2955, 1715, 1103,
1
838 cm
;
1H NMR: d 4.43±4.41 (d, J=10.4 Hz, 1H),
3.89±3.75 (m, 1H), 3.63±3.61 (dd, J=11.7, 1.3 Hz, 2H),
3.58±3.57 (d, J=8.8 Hz, 1H), 3.00±2.99 (d, J=5.0 Hz,
1H), 2.83±2.82 (d, J=5.1 Hz, 1H), 1.87±1.83 (m, 1H),
1.69±1.64 (m, 1H), 1.33±1.22 (m, 2H), 0.97±0.88 (m, 15H),
0.07±0.06 (d, J=6.0 Hz, 6H); 13C NMR: d 210.8, 62.6,
53.3, 48.3, 42.7, 29.7, 25.9, 25.8, 25.0, 23.6, 21.0, 18.2.
(S)-N-(6-Methylheptanoyl)serine benzyl ester. To a solu-
tion S-serine benzyl ester hydrochloride (0.50 g,
2.16 mmol) in CH2Cl2 (15 mL), was added isooctanoic
acid (0.374 g, 2.59 mmol), HBTU (1.07 g, 2.81 mmol)
and triethylamine (1.3 mL, 8.64 mmol). The reaction
was stirred at rt for 8 h, after which it was poured into
aqueous HCl (3 mL, 2 N). The organic layer was sepa-
rated and the aqueous layer was extracted with CH2Cl2
(2Â10 mL). The combined organic layer was washed
with saturated aqueous NaHCO3 (3 mL), brine (5 mL),
dried and concentrated. The crude product was puri®ed
by ¯ash column chromatography (hexanes:EtOAc, 1:1)
to give (S)-N-(6-methylheptanoyl)serine benzyl ester
(2RS,4S)-4-Benzyloxycarbonylamino-2-tert-butyldimethyl-
siloxymethyl-6-methyl-1,2-oxiranylheptane (10a, 10b).
To a 0 ꢀC solution of 9 (146.3 mg, 0.35 mmol) was
added benzonitrile (0.18 mL, 1.74 mmol), H2O2
(0.10 mL, 50% solution in H2O, 1.74 mmol) and diiso-
propylethylamine (0.30 mL, 1.74 mmol). The reaction
was stirred at 4 ꢀC for 15 h. The solvent was removed
and the crude oil product was puri®ed by ¯ash column
chromatography (hexanes:EtOAc, 10:1) to give a 1.2:1
of 10a:10b (107.2 mg, 71%).
(0.569 g, 82%): [a]d +8.91ꢀ (c 1.75, CHCl3); IR 3364,
20
1
2958, 1744, 1648 cm 1; H NMR: d 7.38±7.32 (m, 5H),
6.50±6.49 (d, J=6.9 Hz, 1H), 5.25±5.18 (dd, J=16.3,
12.4 Hz, 2H), 4.75±4.71 (m, 1H), 4.0±3.97 (q, J=5.5 Hz,
1H), 3.92±3.91 (q, J=2.6 Hz, 1H), 2.85±2.84 (d,
J=3.1 Hz, 1H), 2.33±2.17 (m, 1H), 2.05±1.96 (m, 2H),
1.37±1.23 (m, 4H), 1.17±1.08 (m, 2H), 0.92±0.79 (m,
6H); 13C NMR: d 170.4, 135.1, 128.6, 128.5, 128.2, 67.5,
63.6, 63.5, 54.7, 46.3, 44.5, 28.4, 25.2, 23.3, 22.0, 19.6,
16.7.
10a. [a]d +34.84ꢀ (c 0.31, CHCl3); IR 3346, 2957,
20
1713, 1517, 1257, 838 cm 1; H NMR: d 7.38±7.32 (m,
1
5H), 5.10±5.04 (m, 3H), 4.45±4.43 (d, J=11.5 Hz, 2H),
3.59±3.57 (d, J=11.6 Hz, 1H), 3.18±3.17 (d, J=5.0 Hz,
1H), 3.03±3.02 (d, J=4.9 Hz, 1H), 1.78±1.74 (m, 1H),
1.74±1.60 (m, 2H), 1.16±1.10 (m, 1H), 1.00±0.99 (d,
J=6.4 Hz, 3H), 0.95±0.94 (d, J=6.5 Hz, 3H), 0.88 (s,
9H), 0.07±0.06 (d, J=7.2 Hz, 6H); 13C NMR: d 156.2,
136.2, 128.5, 128.1, 128.0, 66.9, 62.0, 52.7, 48.3, 39.5,
25.8, 25.1, 23.4, 21.1, 18.3.
(S)-O-tert-Butyldiphenylsiloxymethyl-N-(6-methylhept-
anoyl)serine benzyl ester. To a solution of (S)-N-(6-
methylheptanoyl)serine benzyl ester (0.77 g, 2.40 mmol)
was added imidazole (0.212 g, 2.88 mmol), and t-butyl-
diphenylsilyl chloride (0.75 mL, 2.88 mmol). After stir-
ring at rt for 24 h, the reaction mixture was vacuum
®ltered and the mother liquor was washed with brine.
The aqueous layer was extracted with EtOAc:Et2O (1:1,
50 mL). The combined organic layer was dried and
concentrated. The crude oily product was puri®ed by
¯ash column chromatography (hexanes:EtOAc, 5:1) to
give (S)-O-tert-butyldiphenylsiloxymethyl-N-(6-methyl-
13.33ꢀ (c 0.15, CHCl3); IR 3350, 2956,
20
10b. [a]d
1
1715, 1513, 1256, 838 cm 1; H NMR: d 7.36±7.32 (m,
5H), 5.23±5.22 (d, J=8.3 Hz, 1H), 5.13±5.06 (m, 3H),
4.68±4.65 (t, J=7.2 Hz, 1H), 4.29±4.27 (d, J=11.9 Hz,
1H), 3.79±3.77 (d, J=11.9 Hz, 1H), 3.01±3.00 (d,
J=4.9 Hz, 1H), 2.91±2.90 (d, J=4.8 Hz, 1H), 1.74±1.67
(m, 1H), 1.51±1.47 (m, 1H), 1.42±1.37 (m, 1H), 0.99±
0.98 (d, J=6.1 Hz, 3H), 0.93±0.92 (d, J=6.6 Hz, 3H),
0.88 (s, 9H), 0.07±0.06 (d, J=4.6 Hz, 6H); 13C NMR: d
206.2, 155.9, 136.3, 128.5, 128.1, 128.0, 67.0, 62.6, 62.5,
54.8, 49.0, 40.5, 29.7, 25.8, 24.7, 23.3, 21.5, 18.3.
20
heptanoyl)serine benzyl ester (1.23 g, 92%): [a]d
+17.36ꢀ (c 1.10, CHCl3); IR 3319, 2956, 1746,
1660 cm 1; 1H NMR: d 7.59±7.55 (dd, J=13.5, 7.31 Hz,
4H), 7.45±7.41 (m, 2H), 7.40±7.32 (m, 9H), 6.31±6.30
(d, J=4.9 Hz, 1H), 5.23±5.17 (dd, J=14.9, 12.5 Hz,
2H), 4.79±4.76 (t, J=7.4 Hz, 1H), 4.19±4.17 (dd,
J=10.3, 2.4 Hz, 1H), 3.91±3.90 (dd, J=10.3, 2.7 Hz,
1H), 2.23±2.14 (m, 2H), 1.63±1.58 (m, 2H), 1.37±1.31
(2R,4S)-4-Amino-2-tert-butyldimethylsiloxymethyl-6-
methyl-1,2-oxiranylheptane (11). To a solution of 10a
(27.3 mg, 0.063 mmol) in MeOH (1.5 mL) was added
palladium on activated carbon catalyst (7.0 mg). After
stirring at rt under a hydrogen atmosphere for 15 min,

N. Sin et al./Bioorg. Med. Chem. 6 (1998) 1209±1217
1215
(m, 3H), 1.18±1.14 (m, 2H), 1.03 (s, 9H), 0.90±0.83 (m,
6H); 13C NMR: d 170.5, 135.5, 132.6, 129.9, 128.6,
128.4, 128.3, 127.8, 127.8, 67.3, 64.5, 54.1, 34.7, 32.5,
29.5, 29.3, 26.7, 22.6, 20.1, 19.3, 15.1.
Dihydroeponemycin (4). To a solution of (4S)-2-tert-
butyldimethylsiloxymethyl-4-[(S)-O-tert-butyldiphenyl-
siloxymethyl-N-(6-methylheptanoyl)serylamino]-6-
methyl-1,2-oxiranylheptane (22.9 mg, 0.0304 mmol) in
THF (1 mL) was added tetrabutylammonium ¯uoride
(0.129 mL, 0.5 M in THF, 0.0638 mmol). After 1 h, the
solvent was removed and the crude product was puri®ed
by ¯ash column chromatography (CH2Cl2:MeOH,
(S)-O-tert-Butyldiphenylsiloxymethyl-N-(6-methylhept-
anoyl)serine (13). To a solution of (S)-O-tert-butyldi-
phenylsiloxymethyl-N-(6-methylheptanoyl)serine benzyl
ester (1.23 g, 2.17 mmol) in MeOH (10 mL) was added
palladium on activated carbon catalyst (0.308 g). After
stirring at rt under a hydrogen atmosphere for 2 h, the
catalyst was removed by vacuum ®ltration through a
celite pad. The crude product was stirred in vacuo for
12 h to give 13 (1.018 g, 99%) which was used without
97:3) to give dihydroeponemycin 4 (10.5 mg, 86%):
20
[a]d
+35.76ꢀ (c 0.425, CHCl3); IR 3304, 2958,
1721, 1644, 1536, 1048 cm 1; 1H NMR: d 7.18 (br, 1H),
6.60 (br, 1H), 4.53±4.49 (m, 2H), 4.24±4.22 (d,
J=12.7 Hz, 1H), 4.20±4.18 (d, J=12.6 Hz, 1H), 4.12±
4.10 (d, J=10.6 Hz, 1H), 4.05±4.04 (d, J=5.4 Hz,
1H), 3.75±3.72 (d, J=5.81 Hz, 1H), 3.62±3.60 (m,
2H), 3.32 (br, 1H), 3.10±3.09 (d, J=4.8 Hz, 1H),
2.35±2.19 (m, 1H), 2.11±1.95 (m, 1H), 1.37±1.23 (m,
2H), 1.40±1.10 (m, 4H), 0.97±0.96 (d, J=6.6 Hz, 3H),
0.96±0.94 (d, J=6.6 Hz, 3H), 0.90±0.88 (d, J=6.4 Hz,
3H), 0.84±0.83 (d, J=7.1 Hz, 3H); 13C NMR: d 207.6,
171.5, 62.6, 61.5, 53.5, 51.5, 49.4, 46.3, 38.7, 29.7, 28.4,
25.8, 25.2, 25.1, 23.3, 21.9, 21.1, 19.5, 14.2, 12.1;
HRFABMS calcd for C20H37 N2O6 401.2652, found
401.2652.
further puri®cation: [a]d +16.76ꢀ (c 1.05, CHCl3); IR
20
3335, 2957, 1736, 1629 cm 1; 1H NMR: d 7.63±7.61 (dd,
J=6.4, 1.4 Hz, 4H), 7.46±7.27 (m, 6H), 6.32±6.31 (d,
J=3.5 Hz, 1H), 4.71±4.70 (d, J=3.6 Hz, 1H), 4.20±4.17
(dd, J=10.4, 3.0 Hz, 1H), 3.93±3.91 (dd, J=10.3,
2.4 Hz, 1H), 2.28±2.14 (m, 2H), 1.71±1.51 (m, 2H), 1.45±
1.23 (m, 3H), 1.19±1.10 (m, 2H), 1.05 (s, 9H), 0.89±0.88
(d, J=4.4 Hz, 3H), 0.87±0.86 (d, J=7.4 Hz, 3H); 13C
NMR: d 174.0, 135.5, 135.4, 132.8, 132.4, 64.0, 54.0,
44.5, 34.6, 31.9, 29.3, 26.8, 22.6, 19.3, 17.9, 15.0;
HRFABMS calcd for C27H40NO4Si 470.2727, found
470.2722.
(S)-N-[6-N-Fluorenylmethoxycarbonylamino)hexanoyl]
serine benzyl ester (15). To a solution S-serine benzyl
ester hydrochloride (0.50 g, 2.16 mmol) in CH2Cl2
(20 mL), was added N-Fmoc-e-aminocaproic acid
(0.84 g, 2.38 mmol), HBTU (1.20 g, 3.23 mmol) and tri-
ethylamine (1.2 mL, 8.64 mmol). The reaction was stirred
at rt overnight, after which it was poured into aqueous
HCl (3 mL, 2 N). The organic layer was separated and
the aqueous layer was extracted with CH2Cl2
(2Â10 mL). The combined organic layer was washed
with saturated aqueous NaHCO3 (3 mL), brine (5 mL),
dried and concentrated. The crude product was puri®ed
by ¯ash column chromatography (1:3, hexanes:EtOAc)
(4S)-2-tert-Butyldimethylsiloxymethyl-4-[(S)-O-tert-butyl-
diphenylsiloxymethyl-N-(6-methylheptanoyl)serylamino]-
6-methyl-1,2-oxiranylheptane. To a solution of (S)-O-
tert-butyldiphenylsiloxymethyl-N-(6-methylheptanoyl)
serine (58.2 mg, 0.124 mmol) in CH2Cl2 (1 mL), was
added HATU (49.5 mg, 0.130 mmol), HOAt (17.7 mg,
0.130 mmol) and diisopropylethylamine (70.2 mL,
0.40 mmol). After 15 min, a solution of epoxy-b-amino-
ketone 11 (18.7 mg, 0.062 mmol) in CH2Cl2 (2 mL) was
added and stirred at rt for 10 h. The reaction mixture
was concentrated and puri®ed by ¯ash column chroma-
tography (CH2Cl2:MeOH, 97:3) to give (4S)-2-tert-
butyldimethylsiloxymethyl-4-[(S)-O-tert-butyldiphenyl-
siloxymethyl-N-(6-methylheptanoyl)serylamino]-6-
to give 15 (1.10 g, 96%): [a]d +5.91ꢀ (c 1.10, CHCl3);
20
1
IR 3314, 2936, 1721, 1644, 1527, 844 cm 1; H NMR: d
7.77±7.75 (d, J=7.5 Hz, 2H), 7.60±7.58 (d, J=7.4 Hz,
2H), 7.41±7.30 (m, 9H), 6.60±6.58 (d, J=7.5 Hz, 1H),
5.20 (s, 2H), 4.96 (br, 1H), 4.73±4.70 (m, 1H), 4.40±4.38
(d, J=6.8 Hz, 2H), 4.22±4.19 (t, J=6.6 Hz, 1H), 4.01±
3.98 (t, J=6.3 Hz, 1H), 3.92±3.89 (t, J=10.7 Hz, 1H),
3.23±3.10 (m, 3H), 2.28±2.25 (t, J=6.8 Hz, 2H), 1.93 (s,
1H), 1.67±1.66 (m, 2H), 1.52±1.35 (q, J=7.20 Hz, 2H);
13C NMR: d 173.3, 170.4, 165.6, 143.9, 141.3, 135.1,
128.6, 128.5, 128.1, 127.6, 127.0, 125.0, 119.9, 67.4, 66.5,
63.3, 54.7, 47.4, 47.2, 40.7, 38.6, 36.1, 29.5, 26.0, 24.9,
8.7; HRFABMS calcd for C31H35N2O6 531.2495, found
531.2495.
20
methyl-1,2-oxiranylheptane (36.2 mg, 78%): [a]d
+13.25ꢀ (c 0.15, CHCl3); IR 3291, 3072, 2957, 1829, 1727,
1
1641, 1112 cm
;
1H NMR: d 7.73±7.72 (d, J=6.7 Hz,
1H), 7.67±7.62 (dd, J=16.9, 6.7 Hz, 2H), 7.45±7.37 (m, 8H),
6.16 (br, 1H), 4.72±4.69 (t, J=8.8 Hz, 1H), 4.64±4.59
(m, 1H), 4.48±4.42 (dd, J=19.0, 11.6 Hz, 1H), 4.07±4.02
(m, 2H), 3.74±3.70 (q, J=7.6 Hz, 1H), 3.59±3.55 (dd,
J=11.4, 8.8 Hz, 1H), 3.20±3.19 (dd, J=5.0 Hz, 1H),
3.16±3.15 (d, J=5.1 Hz, 1H), 3.01 (d, J=2.6 Hz, 1H),
2.17±2.11 (m, 2H), 1.71±1.55 (m, 2H), 1.33±1.21 (m,
2H), 1.08 (s, 18H), 0.98±0.82 (m, 12H), 0.08±0.06 (d,
J=11.3 Hz, 6H); 13C NMR: d 206.5, 171.0, 170.5, 135.5,
135.4, 130.0, 129.9, 127.9, 127.9, 63.7, 62.7, 54.0, 50.9,
48.4, 39.6, 28.6, 26.8, 26.8, 25.8, 25.2, 25.1, 23.3, 22.0,
21.0, 19.6, 19.2, 19.1, 18.2.
(S)-[O-tert-Butyldiphenylsiloxymethyl-N-(6-N-¯uorenyl-
carbonylamino)hexanoyl]serine benzyl ester. To a solu-
tion of 15 (427.8 mg, 0.806 mmol) was added imidazole

1216
N. Sin et al./Bioorg. Med. Chem. 6 (1998) 1209±1217
(78.8 mg, 0.97 mmol), and t-butyldiphenylsilyl chloride
(0.30 mL, 0.97 mmol). After stirring at rt for 15 h, the
reaction mixture was poured into brine (10 mL) and
extracted with EtOAc:Et2O (1:1, 3Â50 mL). The com-
bined organic layer was dried and concentrated. The
crude oily product was puri®ed by ¯ash column chro-
matography (hexanes:EtOAc, 1:1) to give (S)-[O-tert-
butyldiphenylsiloxymethyl-N-(6-N-¯uorenylcarbonyl-
amino)hexanoyl]serine benzyl ester (0.429 g, 69%):
(2 mL) was added and stirred at rt for 8 h. The reaction
mixture was concentrated and puri®ed by ¯ash column
chromatography (CH2Cl2:MeOH, 97:3) to give 17
(45.3 mg, 75%): [a]d +12.35ꢀ (c 0.58, CHCl3); IR
20
1
3304, 2930, 1723, 1649, 1514, 1113 cm
;
1H NMR
(300 MHz): d 7.77±7.71 (m, 2H), 7.67±7.62 (m, 2H),
7.56±7.28 (m, 14H), 7.05±7.03 (d, J=9 Hz, 1H) 6.80±
6.77 (d, J=8.2 Hz, 1H), 6.21±6.19 (d, J=6.6 Hz, 1H),
4.87 (br, 1H), 4.70±4.64 (t, J=9.8 Hz, 1H), 4.61±4.56
(m, 1H), 4.50±4.41 (t, J=12.2 Hz, 1H), 4.40±4.38 (d,
J=6.9 Hz, 1H), 4.23±4.19 (t, J=6.6 Hz, 1H), 4.06±3.99
(m, 2H), 3.75±3.67 (m, 1H), 3.56±3.55 (d, J=4.5 Hz,
1H), 3.53±3.49 (t, J=4.6 Hz, 1H), 3.19±3.13 (q,
J=5.2 Hz, 2H), 3.01±2.99 (d, J=4.8 Hz, 1H), 2.16±2.12
(t, J=7.0 Hz, 2H), 1.71±1.60 (m, 8H), 1.58±1.48 (m,
2H), 1.39±1.28 (m, 4H), 1.22±1.20 (d, J=6.0 Hz, 6H),
1.06 (s, 9H), 1.0±0.98 (d, J=6.0 Hz, 3H), 0.98±0.94 (t,
J=5.5, 6H), 0.87 (s, 9H), 0.06±0.04 (d, J=3.8 Hz, 6H);
13C NMR (75 MHz): d 206.4, 172.8, 170.3, 156.5, 144.1,
141.4, 135.6, 135.5, 130.1, 128.0, 127.9, 127.9, 127.7,
127.1, 125.1, 120.0, 66.5, 63.7, 63.0, 62.0, 54.1, 51.1,
48.5, 47.3, 40.8, 39.6, 38.7, 36.3, 29.7, 26.9, 25.8, 25.1,
23.4, 21.1, 19.3; HRFABMS calcd for C55H76N3O8Si2
962.5171, found 962.5171.
[a]d +11.5ꢀ (c 1.05, CHCl3); IR 3332, 3068, 2932,
20
1
1719, 1663, 1520, 1112 cm
;
1H NMR: d 7.78±7.52
(d, J=7.5 Hz, 2H), 7.60±7.54 (m, 6H), 7.45±7.31 (m,
10H), 6.29±6.28 (d, J=7.9 Hz, 1H), 5.19 (s, 2H), 4.87
(br, 1H), 4.77±4.74 (m, 1H), 4.71±4.00 (d, J=6.9 Hz,
2H), 4.24±4.22 (d, J=6.7 Hz, 1H), 4.20±4.17 (dd,
J=10.3, 2.6 Hz, 1H), 3.22±3.18 (q, J=6.5 Hz, 2H), 2.20±
2.18 (q, J=3.5Hz, 2H), 1.67±1.64 (m, 3H), 1.54±1.51
(m, 2H), 1.39±1.36 (m, 2H), 1.03 (s, 9H); 13C NMR:
d 172.4, 170.4, 156.4, 144.0, 141.3, 135.5, 128.6, 128.4,
128.3, 127.8, 127.8, 127.6, 127.0, 125.0, 119.9, 67.4,
66.5, 64.4, 54.1, 47.3, 40.7, 36.2, 29.6, 26.7, 26.2, 25.0,
19.3.
(S)-[O-tert-Butyldiphenylsiloxymethyl-N-(6-N-¯uorenyl-
methoxycarbonylamino)hexanoyl]serine (16). To a solu-
tion of (S)-[O-tert-butyldiphenylsiloxymethyl-N-(6-N-
¯uorenylcarbonylamino)hexanoyl]serine benzyl ester
(0.407 mg, 0.529 mmol) in MeOH (5 mL) was added
palladium on activated carbon catalyst (0.102 g). After
stirring at rt under a hydrogen atmosphere for 1 h and
15 min, the catalyst was removed by vacuum ®ltration
through a celite pad. The solvent was removed and
the crude product was stirred in vacuo to give 16
(0.336 g, 94%) as a white foam material and was used
(2R,4S)-2-Hydroxymethyl-4-[(S)-N-(6-aminohexanoyl)
serylamino]-6-methyl-1,2-oxiranylheptane. To a solution
of 17 (11.8 mg, 0.0122 mmol) in THF (0.5 mL) was
added tetrabutylamonium ¯uoride (76.0 mL, 0.5 M in
THF, 0.0378 mmol). After 1 h, the solvent was
removed and crude product was stirred in vacuo for 2 h
to give (2R,4S)-2-hydroxymethyl-4-[(S)-N-(6-amino-
hexanoyl)serylamino]-6-methyl-1,2-oxiranylheptane and
was used without further puri®cation.
without further puri®cation: [a]d +5.36ꢀ (c 1.18,
20
1
CHCl3); IR 3320, 2932, 1722, 1656 cm
;
1H NMR: d
Dihydroeponemycin-biotin (5). To a solution of (2R,4S)-
2-hydroxymethyl-4-[(S)-N-(6-aminohexanoyl)seryl-
amino]-6-methyl-1,2-oxiranylheptane (0.0122 mmol) in
DMSO (0.5 mL) and CH2Cl2 (0.1 mL) was added N-
hydroxysuccinimide-biotin reagent (8.8 mg, 0.018 mmol).
After stirring at rt for 8 h, the solvent was removed
in vacuo and the crude product was puri®ed by ¯ash
column chromatography (CH2Cl2:MeOH, 80:20) to give
7.77±7.76 (d, J=7.5 Hz, 2H), 7.62±7.56 (m, 8H), 7.43±
7.29 (m, 8H), 6.37±6.36 (d, J=7.7 Hz, 1H), 4.89 (br s,
1H), 4.72±4.69 (dd, J=11.2, 3.4 Hz, 2H), 4.41±4.17 (m,
2H), 3.93±3.91 (d, J=10.3 Hz, 1H), 3.18±3.15 (t,
J=6.4 Hz, 2H), 2.22±2.19 (t, J=7.4 Hz, 2H), 1.66±1.62
(m, 2H), 1.52±1.49 (m, 2H), 1.36±1.33 (m, 2H), 1.05 (s,
9H); 13C NMR: d 173.3, 173.1, 144.0, 141.3, 135.5,
135.4, 130.0, 127.8, 127.6, 127.0, 125.0, 119.9, 66.5, 63.9,
53.9, 47.3, 40.8, 36.1, 29.6, 26.8, 26.1, 25.0, 19.3;
HRFABMS calcd for C40H47N2O6Si 679.3203, found
679.3201.
5 (7.20 mg, 81% over two steps): [a]d +17.2ꢀ (c 0.25,
20
CHCl3); IR 3307, 2925, 2479, 1717, 1684, 1653, 1646,
1
1457, 1260 cm 1; H NMR (300 MHz, CDCl3 with 7%
CD3OD): d 7.11±7.10 (br, 1H), 4.22±4.38 (t, J=5.9,
1H), 4.37±4.38 (m, 1H), 4.23±4.18 (t, J=5.7 Hz, 1H),
3.76±3.69 (m, 1H), 3.53±3.46 (t, J=10.9 Hz, 1H), 3.28±
3.24 (d, J=12.0 Hz, 1H), 3.14±3.04 (m, 2H), 2.96±2.94
(d, J=4.8 Hz, 1H), 2.84±2.78 (dd, J=13.0, 4.8 Hz, 1H),
2.64±2.60 (d, J=12.4 Hz, 1H), 2.22±2.11 (m, 1H), 2.09±
2.03 (t, J=6.1 Hz, 1H), 1.53±1.50 (m, 6H), 1.41±1.22 (m,
4H), 1.20-1.10 (m, 4H), 0.84±0.82 (d, J=5.9 Hz, 3H),
0.76±0.74 (d, J=6.9 Hz, 3H); HRFABMS calcd for
C34H59N6O9S 727.4064, found 727.4042.
(2R,4S)-2-tert-Butyldimethylsiloxymethyl-4-[(S)-O-tert-
butyldiphenylsiloxymethyl-N-(6-N-¯uorenylmethoxycarb-
onylserylamino]-6-methyl-1,2-oxiranylheptane (17). To a
solution of 16 (63.8 mg, 0.094 mmol) in CH2Cl2 (2 mL),
was added HATU (50.0 mg, 0.130 mmol), HOAt
(18.0 mg, 0.130 mmol) and diisopropylethylamine
(71.0 mL, 0.41 mmol). After 12 min, a solution of epoxy-
b-aminoketone 11 (18.9 mg, 0.063 mmol) in CH2Cl2

N. Sin et al./Bioorg. Med. Chem. 6 (1998) 1209±1217
1217
HUVEC cell culture and dihydroeponemycin-biotin
References
addition. Human umbilical cord venous endothelial
cells (HUVECs) were kindly supplied by J. Pober,
(Yale Boyer Center for Molecular Medicine) and
grown on collagen-coated plastic culture plates in
Dulbecco's Modi®ed Eagle's Medium (DMEM) plus
75 mg/mL Endothelial Cell Growth Supplement
(ECGS, Sigma Chemicals), 50 Units/L of penicillin and
50 Units/L streptamicin at 5% CO2, 3 ꢀC. Con¯uent cells
were routinely split 1:3 every three days. HUVECs
grown to 70% con¯uency were treated for 6 h with
di€erent concentrations of dihydroeponemycin-biotin.
In parallel HUVECs were preincubated for 1 h with
25 mM of dihydroeponemycin 4 before a subsequent
6 h incubation with 5. Treated and control cells were
lysed in sodium dodecyl sulfate (SDS) poly-
acrylamide gel sample bu€er and boiled before
separation by 12% SDS±PAGE. Gels were electro-
phoretically transferred to PVDF membrane and probed
using enhanced chemiluminescence (ECL, Amersham)
and streptavidin-conjugated horseradish peroxidase
(Sigma).
1. Folkman, J. Adv. Cancer Res. 1974, 19, 331.
2. Folkman, J.; Ingber, D. Semin. Cancer Biol. 1992, 3, 89.
3. Ingber, D.; Fujita, T.; Kishimoto, S.; Sudo, K.; Kanamaru,
T.; Brem, H.; Folkman, J. Nature 1990, 348, 555.
4. Fujimoto, J.; Hori, M.; Ichigo, S.; Hirose, R.; Sakaguchi,
H.; Tamaya, T. Cancer Lett. 1997, 113, 187.
5. Mohamed, E.; Piamsomboom, S.; Kudelka, A. P.; Kava-
nagh, J. Int. J. Gynecol. Cancer 1996, 6, 18.
6. Sin, N.; Meng, L.; Wang, M. Q. W.; Wen, J. J.; Bornmann,
W. G.; Crews, C. M. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 6099.
7. Grith, E. C.; Zhuang, S.; Turk, B. E.; Chen, S.; Chang, Y.-
H.; Wu, Z.; Biemann, K.; Liu, J. O. Chem. Biol. 1997, 4, 461.
8. Sugawara, K.; Hatori, M.; Nishiyama, Y.; Tomita, K.;
Kamei, H.; Konishi, M.; Oki, T. J. Antibiotics 1990, XLIII, 8.
9. Oikawa, T.; Hasegawa, M.; Shimamura, M.; Ashino, H.;
Murota, S.-I.; Morita, I. Biochem. Biophys. Res. Com. 1991,
181, 1070.
10. Hanada, M.; Sugawara, K.; Kaneta, K.; Toda, S.; Nish-
iyama, Y.; Tomita, K.; Yamamoto, H.; Konishi, M.; Oki, T. J.
Antibiotics 1992, 45, 1746.
11. Schmidt, U.; Schmidt, J. Synthesis 1994, 300.
12. Hoshi, H.; Ohnuma, T.; Aburaki, S.; Konishi, M.; Oki, T.
Tetrahedron Lett. 1993, 34, 1047.
Acknowledgement
13. Hatch, L. F.; Alexander, H. E.; Randolph, J. D. J. Org.
Chem. 1950, 15, 654.
The authors wish to thank the NIH for support of this
work through research grant 1-ROI-CA74967.
14. Corey, E. J.; Widiger, G. N. J. Org. Chem. 1975, 40, 2975.