Total synthesis of a conformationally constrained didemnin B analog

Article abstract of DOI:10.1021/jo001640n

The total synthesis of a didemnin B analogue containing a conformationally constrained replacement for the isostatine moiety is reported. Synthetic highlights include an improved preparation of 2-hydroxy-3-cyclohexenecarboxylic acid and a new strategy for accessing the macrocycle.

Full text of DOI:10.1021/jo001640n

2734
J . Org. Chem. 2001, 66, 2734-2742
Tota l Syn th esis of a Con for m a tion a lly Con str a in ed Did em n in B
An a log
Dong Xiao, Matthew D. Vera, Bo Liang, and Madeleine M. J oullie´*
Department of Chemistry, University of Pennsylvania, 231 South 34th Street,
Philadelphia, Pennsylvania 19104-6323
Received November 20, 2000
The total synthesis of a didemnin B analogue containing a conformationally constrained replacement
for the isostatine moiety is reported. Synthetic highlights include an improved preparation of
2-hydroxy-3-cyclohexenecarboxylic acid and a new strategy for accessing the macrocycle.
In tr od u ction
The didemnins, natural products isolated from a
marine tunicate,1 exhibit a variety of biological activities.2
Most didemnins share a common 23-membered depsipep-
tide core and differ only in the side chains attached to
the nitrogen of threonine.
Investigations of the mechanism of action of didemnin
B (1) (Figure 1) have identified eEF-1R3 and palmitoyl
protein thioesterase4,5 as binding proteins. The actions
of didemnins A and B on protein biosynthesis in rabbit
reticulocyte lysates were found to involve both eEF-1R
and EF-2.6,7 Didemnin B was reported to induce apoptosis
in human cells (HL-60) at an extremely high rate.8-10
Although these results are significant, the understanding
of the mechanism of action of the didemnins is still
evolving.
Constrained analogues may be used to probe the
bioactive conformation of ligands as amply demonstrated
by earlier research.11,12 As the mode of action and bio-
logical receptors of the didemnins are still being inves-
tigated, a constrained analogue may serve the dual pur-
pose of probing the bioactive conformation and of provid-
ing structural information about the protein-ligand
complex. Although synthetic routes to a constrained ana-
logue were previously investigated, a stereocontrolled
F igu r e 1. Structure of didemnin B (1).
total synthesis could not be achieved, and the resulting
diastereomeric mixture could not be separated.13 At the
onset of this work, we realized that the low energy crystal
structure of didemnin B14 was an attractive target to
mimic. Therefore, we designed a constrained analogue
of didemnin B (2, Figure 2) to probe whether the crystal
structure was related to the bioactive conformation. In
the macrocycle salt of 1, which is inactive,15 the isostatine
region differs considerably from that of the solid-state
structure of didemnin B. Our interest focused on the
isostatine portion of the molecule, because the con-
strained fragment could also be useful to examine the
role of â-hydroxy-γ-amino acids present in several bio-
active natural products.
(1) Rinehart, K. L., J r.; Gloer, J . B.; Cook, J . C., J r.; Mizsak, S. A.;
Scahill, T. A. J . Am. Chem. Soc. 1981, 103, 1857-1859.
(2) Sakai, R.; Rinehart, K. L.; Kishore, V.; Kundu, B.; Faircloth, G.;
Gloer, J . B.; Carney, J . R.; Namikoshi, M.; Sun, F.; Hughes, J . R. G.;
Gravalos, G.; de Quesada, T. G.; Wilson, G. R.; Heid, R. M. J . Med.
Chem. 1996, 39, 2819-2834.
(3) Crews, C. M.; Collins, J . L.; Lane, W. S.; Snapper, M. L.;
Schreiber, S. L. J . Biol. Chem. 1994, 269, 15411-15414.
(4) Crews, C. M.; Lane, W. S.; Schreiber, S. L. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 4316-4319.
(5) Meng, L.; Sin, N.; Crews, C. M. Biochemistry 1998, 37, 10488-
10492.
Resu lts a n d Discu ssion
The design of an isostatine constrained analogue
utilized the X-ray structure of didemnin B as the starting
point.14 The dihedral angles of the isostatine fragment
derived from the X-ray structure of didemnin B were
matched with those of a hydroxycyclohexane amino acid
(3) with the stereochemistries shown in Figure 3.
The resulting constrained didemnin B analogue (2,
Figure 4) was investigated by molecular modeling tech-
niques. Using a combination of MM2 force field mechan-
ics as well as molecular dynamics, a minimized structure
of the constrained analogue was obtained. As a control,
calculations using Macromodel v3IX and Molecular Dy-
namics showed no major differences in energy for didem-
(6) Ahuja, D.; Vera, M. D.; SirDeshpande, B. V.; Morimoto, H.;
Williams, P. G.; J oullie´, M. M.; Toogood, P. L. Biochemistry 2000, 39,
4339-4346.
(7) SirDeshpande, B. V.; Toogood, P. L. Biochemistry 1995, 34,
9177-9184.
(8) Beidler, D. R.; Ahuja, D.; Wicha, M. S.; Toogood, P. L. Biochem.
Pharmacol. 1999, 58, 1067-1074.
(9) Grubb, D. R.; Wolvetang, E. J .; Lawen, A. Biochem. Biophys. Res.
Commun. 1995, 215, 1130-1136.
(10) J ohnson, K. L.; Lawen, A. Immunol. Cell Biol. 1999, 77, 242-
248.
(13) Mayer, S. C.; Pfizenmayer, A. J .; Cordova, R.; Li, W.-R.; J oullie´,
M. M. Tetrahedron: Asymmetry 1994, 5, 519-522.
(14) Hossain, M. B.; van der Helm, D.; Antel, J .; Sheldrick, G. M.;
Sanduja, S. K.; Weinheimer, A. J . Proc. Natl. Acad. Sci. U.S.A. 1988,
85, 4118-4122.
(15) Mayer, S. C.; Carroll, P. J .; J oullie´, M. M. Acta Crystallogr.
1995, C51, 1609-1614.
(11) Giannis, A.; Kolter, T. Angew. Chem., Int. Ed. Engl. 1993, 32,
1244-1267.
(12) Hruby, V. J . In Conformationally Directed Drug Design: Pep-
tides and Nucleotides as Templates or Targets; Vida, J . A., Gordon,
M., Eds.; American Chemical Society: Washington, D. C., 1983; pp
9-28.
10.1021/jo001640n CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/28/2001

Conformationally Constrained Didemnin B Analog
J . Org. Chem., Vol. 66, No. 8, 2001 2735
stereoisomers of the proposed cyclohexane amino acid
were incorporated into 1, none of their minimized struc-
tures gave a good overlay with the X-ray structure of 1.
Both chemical intuition and molecular modeling seemed
to indicate that this analogue was a good representation
of the crystal structure of didemnin B. It is important to
note the different configuration at the amine center,
which is S in the analogue and R in didemnin B. This
modification is the only way to preserve the conformation
of isostatine in the constrained analogue. However, by
doing so, the sec-butyl moiety was deleted from the
proposed analogue for synthetic feasibility. Based on the
structure-activity relationships of the didemnins, this
modification was believed to have only a minor effect on
the biological activity of didemnins.2
The initial retrosynthetic analysis (Figure 2) was
designed according to the strategy previously developed
in our group.17 The first disconnection was made between
the macrocycle and the side chain. Macrocyclization
would take place between the Hip acid and the leucine
amino group. An important feature of the previous
synthesis was the preservation of the â-ketoamide as a
protected Hip unit in order to control the stereochemistry
of the methyl group located between two carbonyls. The
acidity of the proton of the activated keto ester and that
of a protonated tertiary amine (which are typical acid
scavengers in peptide coupling) are within 2 orders of
magnitude. During the long reaction time needed under
high dilution cyclization, epimerization of the methyl
group was a possibility. Therefore, the length of the
synthesis of the Hip unit was justified due to the
necessity to secure a stereoselective synthesis. The next
disconnection was made between the cyclohexane amine
and the known tetrapeptide acid.17 The lower portion of
the molecule was further disconnected through the ester
bond to give the known Hip alcohol (Hip-OH) and a fully
protected 3-azido-2-hydroxycarboxylic acid.
F igu r e 2. Initial retrosynthesis of the isostatine-constrained
analogue (2).
The major challenge in the synthesis of 2 was to
develop an efficient preparation of the proposed (S,S,S)-
â-hydroxy-γ-aminocyclohexanecarboxylic acid (3, Fig-
ure 3).
The synthesis of the desired cyclohexane amino acid
began with an auxiliary type Diels-Alder reaction to
afford an enantiomerically pure cyclohexene intermedi-
ate, which could be manipulated further. Thus, the
camphorsultam18 derived dienophile 4 reacted with an
oxygenated diene, 1-acetoxy-1,3-diene, available as a
mixture (6:4 ratio, trans:cis). Initial thermal conditions
in refluxing toluene nonselectively gave several products
(5:5a :5b ) 48%:10%:30%) whose structures were char-
acterized by NMR and X-ray crystallography (Scheme
1).19,20 Attempts to use Lewis acids such as TiCl4 to
increase the diastereoselectivity were hampered by the
sensitive diene’s propensity to polymerize. However, it
was found that the desired Diels-Alder reaction could
be effected in 4 M lithium perchlorate in ether.21 Two
products were isolated in a ratio of 96:4, in which the
F igu r e 3. Dihedral angles of the isostatine fragment and
hydroxycyclohexane amino acid (3).
F igu r e 4. Comparison of the dihedral angles in didemnin B
and the proposed analogue.
nin B,16 which supports the calculations obtained for the
constrained analogue. The minimized structure over-
lapped well with the X-ray structure of 1. The calculated
dihedral angles of didemnin B and the analogue were
well matched. Remarkably, when the other seven possible
(17) Li, W.-R.; Ewing, W. R.; Harris, B. D.; J oullie´, M. M. J . Am.
Chem. Soc. 1990, 112, 7659-7672.
(18) Oppolzer, W.; Chapuis, C.; Bernardinelli, G. Helv. Chim. Acta
1984, 67, 1397-1401.
(19) Xiao, D. Ph.D. Thesis, University of Pennsylvania, Philadelphia,
1999.
(20) Xiao, D.; Carroll, P. J .; Mayer, S. C.; Pfizenmayer, A. J .; J oullie´,
M. M. Tetrahedron: Asymmetry 1997, 9, 47-53.
(21) Grieco, P. A.; Beck, J . P. Tetrahedron Lett. 1993, 34, 7367-
7370.
(16) Mayer, S. C. Ph.D. Dissertation, University of Pennsylvania,
1995.

2736 J . Org. Chem., Vol. 66, No. 8, 2001
Xiao et al.
Sch em e 1
Sch em e 3
Sch em e 2
conditions, such as Na2CO3 or NaH with iodine, did not
give rise to the desired transformation. We attributed this
lack of reactivity to an intramolecular hydrogen bond
between the acid and hydroxyl groups. Using a more
reactive electrophilic iodine species, iodonium biscollidine
perchlorate,28 we obtained the desired iodolactonization
product 12 in 70% yield (Scheme 3).
Subsequently, the iodine was removed under radical
conditions using tris(trimethylsilyl)silane,29 and the re-
sulting hydroxy group of 13 was then protected as its
benzyl ether 14 in 84% yield. The lactone was subjected
to transesterification with methanol and K2CO3 to free
the γ-hydroxyl group. However, very dilute potassium
carbonate was required to prevent epimerization of the
resulting ester, and under these conditions, methanolysis
was incomplete. In addition, the ester was unstable on
silica gel and reverted to lactone 14. Therefore, a mixture
of hydroxy ester and starting material was used without
purification in the next steps. Inversion of the γ-hydroxyl
group with azide was chosen to introduce the nitrogen
functionality. The reagents DPPA or Zn(N3)2Py2 under
Mitsunobu conditions did not afford a product. As the
corresponding mesylate was not effectively displaced by
azide ion even at elevated temperature, we synthesized
a triflate (15) which, when subjected to sodium azide in
DMF, gave the desired product (16) in 56% yield.
With the functionalities and desired stereochemistries
secured, synthesis of the required amino acid proceeded
uneventfully. The methyl ester 16 was hydrolyzed to
afford the corresponding free acid 17 in 97% yield. The
stereochemistry of the azido acid was confirmed by an
X-ray crystal structure analysis. Subsequently, 17 was
treated with hydrogen and Pd on carbon to yield the
corresponding amine and to deprotect the â-hydroxyl
group in one step to afford 18 in 81% yield (Scheme 3).
The synthesis of the Hip-azidocyclohexane ester frag-
ment utilized a protocol which was developed in the
synthesis of 1.17 The initial coupling between the pro-
tected Hip-OH and 17 was conducted under a variety of
standard coupling conditions. Unfortunately, DCC-
DMAP, acid chloride, mixed trichlorobenzoic anhydride,
1-methyl-2-chloropyridinium iodide, and tributylamine
all failed to give a useful yield of the desired product.
Realizing that the steric demands of both the acid and
the alcohol were exceedingly high, we decided to modify
the steric environment around the hydroxyl group of the
Hip-OH (Scheme 4) by replacing the MOM-protected
hydroxyl group with the epi-Hip-OH (22). As mentioned
desired endo product 5 predominated with a yield of 82%
based on the recovered starting material. No exo product
was detected but the minor isomer was identified to be
an endo 1,3-regioisomer 5a .
Although the Diels-Alder reaction was successful,
there were two significant drawbacks. The large scale use
of lithium perchlorate caused safety concerns, particu-
larly in light of the long reaction time of 10 days to two
weeks required to complete the reaction. An attempt to
use a catalytic amount of lithium perchlorate as reported
by Reetz et al. did not give a good yield.22 Therefore, an
alternative route was sought to provide the desired
intermediate (Scheme 2).
We adopted an aldol-metathesis protocol developed by
Crimmins and co-workers.23 An oxazolidinone derived
unsaturated imide 7 was chosen as a precursor to the
aldol substrate. Compound 7 was synthesized by convert-
ing the Evans auxiliary 6 to the corresponding acryloyl
derivative.24 Compound 7 was subjected to a titanium
tetrachloride mediated Sakuri reaction to afford the
elongated aldol substrate (8).25 The standard aldol condi-
tions26 afforded diene 9 with excellent stereoselectivity
in 79% yield. The cyclohexene ring was then closed (10)
using Grubbs catalyst to effect the diene metathesis in
high yield.27
The products from the Diels-Alder reaction or from
the aldol-metathesis approach (5 and 10) were hydrolyzed
to give the corresponding hydroxy acid 11 in nearly
quantitative yield (Scheme 3). Introduction of the γ-azido
group began with an iodolactonization reaction. Surpris-
ingly, treatment of the hydroxy acid under normal
(22) Reetz, R. T.; Fox, D. N. A. Tetrahedron Lett. 1993, 34, 1119-
1122.
(23) Crimmins, M. T.; King, B. W. J . Org. Chem. 1996, 61, 4192-
4193.
(24) Ho, G.-J .; Mathre, D. J . J . Org. Chem. 1995, 60, 2271-2273.
(25) Wu, M.-J .; Wu, C.-C.; Lee, P.-C. Tetrahedron Lett. 1992, 33,
2547-2548.
(26) Evans, D. A.; Bartroli, J .; Shih, T. L. J . Am. Chem. Soc. 1981,
103, 2127-2129.
(28) Lemieux, R. U.; Morgan, A. R. Can. J . Chem. 1965, 43, 2190-
2198.
(29) Chatgilialoglu, C. Acc. Chem. Res. 1992, 25, 188-194.
(27) Schwab, P.; Grubbs, R. H.; Ziller, J . W. J . Am. Chem. Soc. 1996,
118, 100-110.

Conformationally Constrained Didemnin B Analog
J . Org. Chem., Vol. 66, No. 8, 2001 2737
Sch em e 4
Sch em e 5
in the retrosynthetic analysis, oxidation to the ketone was
one of the last transformations planned after macrocycle
ring closure. Protection of this center as a MOM ether
was intended to prevent epimerization during cyclization.
Therefore, the chirality of the starred center was ir-
relevant and a change of stereochemistry might lead to
conformational changes and steric reduction of the
alcohol substrate. The desired epi-Hip alcohol (22) was
prepared by oxidation of Hip alcohol (19) and subsequent
reduction of the resultant ketone with LiAlH4 to give
alcohol 20. This alcohol was protected with MOMCl, and
the benzyl protecting group was then removed. This
intermediate 21 could then be coupled with 17 to give a
reasonable 47% yield of the desired product (22) (Scheme
4). The strikingly different reactivity between the two
epimeric alcohols was not completely understood. It
appeared that subtle steric match-up of alcohol sub-
strates with the cyclohexane acid played a role.
Sch em e 6
Sch em e 7
In an attempt to understand the different reactivities
of the epimeric alcohols, calculations using the CAChe
3.8 program30 suggested a possible reason. Molecular
dynamics simulation of the two alcohols using default
parameters gave three stable conformers for the bonds
examined. Three Newman projections corresponding to
these three conformers can be identified (Figure 5). Of
The TBS ether of 22 was removed using Amberlyst 15
to give primary alcohol 23 in 94% yield. This alcohol was
subjected to a two-step oxidation protocol: Dess-Martin
reagent31 to convert it to the corresponding aldehyde,
followed by a chlorite oxidation32 to the carboxylic acid
24. The acid was then esterified with DCC-DMAP and
benzyl alcohol to afford the desired benzyl ester (25). The
azide was reduced to the amine (26), using trimethyl
phosphine with 2 equiv of water in THF.33 The amine
(26) was subsequently coupled with the known tetrapep-
tide acid17 (27) using HATU to afford the desired coupled
product (28) in 70% yield (Scheme 6).
With the linear precursor (28) in hand, palladium-
catalyzed hydrogenation cleanly removed both the ben-
zyloxycarbonyl group and the benzyl ester leaving the
cyclohexyl benzyl ether intact (Scheme 7). The amino acid
was then cyclized to 29, using HATU at 0.01 M concen-
tration in 38-45% yield. The macrocycle (29) was then
F igu r e 5. Newman projections of (a) most favorable confor-
mation of epi-OH; (b) same conformation of Hip-OH; (c) desired
Hip-OH conformation for activated ester attack.
these, the most stable conformation should be the one in
which hydrogen bonding between the hydroxyl group and
the OMOM group is possible, which occurs when they
are in a gauche conformation. Examination of molecular
models indicated that for the incoming activated ester
to approach the hydroxyl group, it had to avoid the bulky
isopropyl and OMOM groups. Figure 5 shows the two
preferred conformations, when the hydroxyl group and
the OMOM group are in a gauche arrangement. While
in the case of the epi-Hip alcohol there is room for the
approach of the activated ester, the same conformation
in the Hip-alcohol shows the hydroxyl group to be
sterically hindered.
(30) 3.8 Ed.; CAChe Scientific Inc.: 1995.
(31) Dess, D. B.; Martin, J . C. J . Am. Chem. Soc. 1991, 113, 7277-
7287.
(32) Dalcanale, E.; Montanari, F. J . Org. Chem. 1986, 51, 567-569.
(33) Bosch, I.; Urpˆı, F.; Vilarrasa, J . J . Chem. Soc., Chem. Commun.
1995, 91-92.
The next target for the new synthetic strategy required
a benzyl-protected azido ester which was obtained as
shown in Scheme 5.

2738 J . Org. Chem., Vol. 66, No. 8, 2001
Xiao et al.
sultam 3 (124.5 mg, 0.46 mmol) and acetoxy-1,3-diene (260.0
mg, 2.3 mmol). The solution was heated under reflux for 6 h.
The solvent was evaporated and the residue was chromato-
graphed using 10-20% EtOAc-petroleum ether to afford 5
(84.6 mg, 48%), 5a (17.6 mg 10%), and 5b (52.7 mg, 30%) all
as colorless crystals.
treated with 10% HCl (aq)-ethyl acetate solution (1:1)
to removed both the MOM and Boc protecting groups.
The free amine was coupled to a benzyl-protected side
chain (30) to yield 31. Subsequent oxidation of 31 by
Dess-Martin reagent converted the macrocyclic hydroxyl
group to the ketone. Finally, the deprotection of both
benzyl ether groups was realized by using 2 equiv of
palladium black in 20% formic acid in methanol to afford
the desired isostatine-constrained didemnin B analogue
(2, Figure 2).
The constrained analogue (2) was tested for protein
biosynthesis inhibition. The assay was conducted in a
rabbit reticulocyte lysate.7 The constrained analogue has
an IC50 of 99 mM, which is about 20-fold less than
didemnin B (IC50 ) 4.4 mM) in the same assay.
5: mp 188-190 °C; Rf 0.40 (20% EtOAc/petroleum ether);
1H NMR (500 MHz, CDCl3) δ 6.03 (m, 1H), 5.87 (m, 1H), 5.61
(m, 1H), 3.89 (dd, J ) 7.5, 5.0 Hz, 1H), 3.42 (d, J ) 14.0 Hz,
1H), 3.34 (d, J ) 14.0 Hz, 1H), 3.27 (m, 1H), 2.25 (m, 1H),
2.06-1.69 (m, 8H), 1.99 (s, 3H), 1.38 (m, 2H), 1.15 (s, 3H), 0.96
(s, 3H); 13C NMR (125 MHz, CDCl3) δ 171.7, 170.3, 133.5,
123.6, 66.6, 65.1, 53.1, 48.3, 47.8, 44.7, 44.6, 37.8, 32.8, 26.6,
24.6, 21.0, 20.4, 19.9, 18.8; IR(neat) 1734, 1700, 1238 cm-1
;
HRMS m/z calcd for C19H31N2O5S (M + NH4): 399.1954, found
399.1967; [R]2D0 -244 (c ) 2.25, CHCl3). Anal. Calcd for
C
19H27NO5S: C, 59.82; H, 7.13; N, 3.67. Found: C, 60.17; H,
7.08; N, 3.32.
Acetic a cid (5R)-(10,10-d im eth yl-3,3-d ioxo-3λ6-th ia -4-
a za -tr icyclo [5.2.1.0.1,5] d eca n e-4-ca r bon yl)-(1S)-cycloh ex-
2-en yl ester (5a ): mp 136-138 °C; Rf 0.37 (20% EtOAc/
Con clu sion s
An analogue of 1 in which the isostatine moiety was
replaced by a (S,S,S)-â-hydroxy-γ-aminocyclohexanecar-
boxylic acid (3) was synthesized to probe the biological
conformation of didemnin B. The synthesis of this
analogue (2) used an alternative strategy which resulted
in a shorter and more efficient approach to the analogue
macrocycle. Although changes in the side chains2,34-36 and
in the tyrosine residue37,38 of 1 resulted in analogues with
equal or superior bioactivity to that of the parent
compound, changes in the isostatine region reduced the
protein biosynthesis inhibition activity. This decrease in
potency suggests that the bioactive conformation of 1 may
differ from its crystal structure in the isostatine region.
1
petroleum ether); H NMR (500 MHz, CDCl3) δ 5.77 (m, 1H),
5.58 (m, 1H), 5.40 (m, 1H), 3.81 (dd, J ) 7.6, 5.0 Hz, 1H), 3.41
(d, J ) 14.0 Hz, 1H), 3.36 (d, J ) 14.0 Hz, 1H), 3.22 (m, 1H),
2.39 (m, 1H), 2.19 (m, 1H), 2.04 (m, 1H), 1.96 (m, 5H), 1.81
(m, 4H), 1.30 (m, 2H), 1.04 (s, 3H), 0.87 (s, 3H); 13C NMR (125
MHz, CDCl3) δ 173.5, 170.5, 128.4, 127.3, 69.6, 65.1, 53.0, 48.4,
47.8, 44.6, 39.4, 38.4, 32.8, 29.5, 29.0, 26.4, 21.2, 20.8, 19.8;
IR (neat) 2959, 1727, 1696, 1331, 1239; HRMS m/z calcd for
C
19H31N2O5S (M + NH4): 399.1954, found 399.1946; [R]20 -7
D
(c ) 1.5, CHCl3).
Acetic a cid (6R)-(10,10-d im eth yl-3,3-d ioxo-3λ6-th ia -4-
a za -tr icyclo[5.2.1.0.1,5]d eca n e-4-ca r bon yl)-(1S)-cycloh ex-
2-en yl ester (5b): mp 189-191 °C; Rf 0.26 (20% EtOAc/
1
petroleum ether); H NMR (500 MHz, CDCl3) δ 6.02 (m, 1H),
5.79 (m, 1H), 5.74 (m, 2H), 3.89 (dd, J ) 7.6, 5.1 Hz, 1H), 3.51
(d, J ) 2.2 Hz, 2H), 3.34 (m, 1H), 2.23-2.09 (m, 5H), 1.96-
1.84 (m, 4H), 1.47 (m, 1H), 1.37 (m, 1H), 2.08 (s, 3H), 1.19 (s,
1H), 1.00 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 171.3, 171.1,
132.7, 124.8, 66.0, 65.4, 53.5, 48.8, 48.2, 45.1, 43.9, 39.0, 33.2,
26.8, 24.8, 21.8, 21.7, 21.5, 20.3; IR (neat) 1734, 1699, 1329,-
1239 cm-1; HRMS m/z calcd for C19H27NO5SNa (M + Na):
404.1507, found 404.1512; [R]2D0 +38.0 (c ) 1.61, CHCl3).
(4S)-3-(1-Acr yloyl)-4-ben zyl-2-oxa zolid in on e (7). To a
solution of acrylic acid (0.634 mL, 9.2 mmol) in 30 mL of THF
at -20 °C was added triethylamine (2.39 mL, 17.1 mmol)
followed by acryloyl chloride (0.7 mL, 8.6 mmol). The mixture
was stirred at that temperature for 2 h. LiCl (0.336 mg, 7.9
mmol) was added followed by oxazolidinone 6 (1.17 g, 6.6
mmol). The mixture was allowed to warm to room temperature
and stirred for 8 h. The reaction was quenched by addition of
0.2 N HCl, and THF was removed in vacuo. The residue was
partitioned between EtOAc and 0.2 N HCl. The organic layer
was washed with half-saturated NaHCO3 and brine, dried over
Na2SO4, filtered, and concentrated in vacuo. Column chroma-
tography using 5-20% petroleum ether/EtOAc yielded the title
compound as colorless crystals (1.375 g, 90%). 7: mp 73-74
°C; Rf 0.46 (20% EtOAc/petroleum ether); 1H NMR (500 MHz,
CDCl3) δ 7.60 (m, 1H), 7.44-7.30 (m, 5H), 6.68, (dd, J ) 17.1,
1.8 Hz, 1H), 6.00 (dd, J ) 10.6, 1.8 Hz, 1H), 4.80 (m, 1H), 4.26
(m, 2H), 3.39 (dd, J ) 13.4, 3.3 Hz, 1H), 2.85 (dd, J ) 13.5,
9.6 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 164.9, 153.3, 135.2,
131.8, 129.4, 129.0, 127.45, 127.38, 66.2, 55.2, 37.8; HRMS m/z
calcd for C13H14NO3 (M + H): 232.0974, found 232.0970;
[R]2D0 +79.6 (c ) 1.14, CHCl3).
Exp er im en ta l Section
Acetic Acid (6S)-(10,10-Dim eth yl-3,3-d ioxo-3λ6-th ia -4-
a za -tr icyclo[5.2.1.0.1,5]d eca n e-4-ca r bon yl)-(1R)-cycloh ex-
2-en yl Ester (5). Method A: To a round-bottomed flask was
added anhydrous lithium perchlorate (Aldrich) (51 g, 0.48 mol).
The flask was heated at 120 °C under reduced pressure with
vigorous stirring for 8 h. The flask was then cooled, and a
stream of argon was introduced, followed by addition of 120
mL of freshly distilled ether (Fisher) via a syringe. The mixture
was stirred for 1 h. At that time lithium perchlorate dissolved
to afford a clear solution. To this solution was introduced
sultam 3 (5.9 g, 22 mmol). After stirring for 20 min, freshly
distilled acetoxy-1,3-diene (12.3 g, 109 mmol) was introduced
through a syringe. The mixture was allowed to stir under
argon for 10 days. The mixture was diluted with 600 mL of
EtOAc and washed with 200 mL saturated NaS2O3 solution
and brine. The organic layer was dried over Na2SO4, filtered,
and concentrated under reduced pressure. Column chroma-
tography using 10-20% EtOAc/petroleum ether gradient
yielded starting material (0.885 g) and the title compound 5
(5.81 g, 82% based on the recovered starting material) as well
as the minor product 5a (0.213 g, 3%).
Method B: A round-bottomed flask under argon was charged
with freshly distilled toluene (4.6 mL) followed by addition of
(34) J ouin, P.; Poncet, J .; Dufour, M.-N.; Aumelas, A.; Pantaloni,
A. J . Med. Chem. 1991, 34, 486-491.
(35) Mayer, S. C.; Ramanjulu, J .; Vera, M. D.; Pfizenmayer, A.;
J oullie´, M. M. J . Org. Chem. 1994, 59, 5192-5205.
(36) Schmidt, U.; Griesser, H.; Haas, G.; Kroner, M.; Riedl, B.;
Schumacher, A.; Sutoris, F.; Haupt, A.; Emling, F. J . Peptide Res. 1999,
54, 146-161.
(37) Pfizenmayer, A. J .; Ramanjulu, J .; Vera, M. D.; Ding, X.; Xiao,
D.; Chen, W.-C.; J oullie´, M. M.; Tandon, D.; Toogood, P. L. Bioorg. Med.
Chem. Lett. 1998, 8, 3653-3656.
(38) Tarver, J . E., J r.; Pfizenmayer, A. J .; Vera, M. D.; Ding, X.;
Liang, B.; J oullie, M. M. Presented at the 218th National Meeting of
the American Chemical Society, Aug 22, 1999, New Orleans, LA, 1999;
ORG 585.
(4S)-3-(1-Oxo-5-h exen yl)-4-ben zyl-2-oxa zolid in on e (8).
To a solution of oxazolidinone 7 (1.1 g, 4.76 mmol) in 20 mL of
CH2Cl2 at -78 °C was added TiCl4 (1 M in CH2Cl2, 6.7 mL,
6.7 mmol). After stirring the mixture for 10 min, allyltri-
methylsilane (2.48 mL, 14.3 mmol) was added to the dark-
brown solution. The mixture was stirred at -78 °C for 3 h and
then poured into a saturated NaHCO3 solution. The organic
layer was separated, and the aqueous layer was extracted with
ether (25 mL × 3). The combined organic layers were washed

Conformationally Constrained Didemnin B Analog
J . Org. Chem., Vol. 66, No. 8, 2001 2739
with brine, dried over Na2SO4, filtered, and concentrated under
reduced pressure. Column chromatography using 0-10%
EtOAc/petroleum ether yielded the title compound as a color-
less oil (1.144 g, 88%). 8: Rf 0.51 (20% EtOAc/petroleum ether);
1H NMR (500 MHz, CDCl3) δ 7.33-7.19 (m, 5H), 5.79 (m, 1H),
5.03-4.94 (m, 2H), 4.62 (m, 1H), 4.11 (m, 2H), 3.23 (dd, J )
13.5, 3.3 Hz, 1H), 2.94-2.81 (m, 2H), 2.69 (dd, J ) 13.5, 9.3
Hz, 1H), 2.08 (m, 2H), 1.72 (m, 2H); 13C NMR (125 MHz,
CDCl3) δ 173.2, 153.5, 137.8, 135.3, 129.4, 129.0, 127.4, 115.3,
66.2, 55.1, 37.9, 34.8, 33.0, 23.4; IR (neat) 3064, 3029, 1772,
1700, 1640 cm-1; HRMS m/z calcd for C16H20NO3 (M + H):
274.1443, found 274.1432; [R]2D0 +58.7 (c ) 1.08, CHCl3).
(2S,3R,4S)-3-(3-Hyd r oxy-2-a llyl-1-oxo-5-h exen yl)-4-ben -
zyl-2-oxa zolid in on e (9). To a solution of oxazolidinone 8 (1.0
g, 3.66 mmol) in 9 mL of CH2Cl2 at -15 °C was added dropwise
Bu2BOTf (1 M in CH2Cl2, 4.03 mL, 4.03 mmol) followed by
dropwise addition of triethylamine (0.612 mL, 4.39 mmol). The
solution was stirred at 0 °C for 15 min and then cooled to -78
°C. The freshly distilled acrolein (0.269 mL, 4.03 mmol) was
then added through a syringe. After stirring at -78 °C for 2
h, the mixture was allowed to warm to 0 °C for 30 min. Then
a pH 7 buffer (6 mL) was added, followed by 20 mL of MeOH
and dropwise addition of 6 mL of 30% hydrogen peroxide while
maintaining the temperature at 0 °C. After stirring for 1 h,
the mixture was concentrated and extracted with EtOAc (20
mL × 4). The organic layer was washed with 20 mL of 10%
NaHSO3 and brine, dried over Na2SO4, filtered, and concen-
trated in vacuo. Further column chromatography using 5-20%
EtOAc/petroleum ether yielded the title compound as a color-
less oil (0.95 g, 79%). 9: Rf 0.37 (20% EtOAc/petroleum ether);
1H NMR (500 MHz, CDCl3) δ 7.34-7.20 (m, 5H), 5.89 (m, 1H),
5.78 (m, 1H), 5.30 (dd, J ) 17.2, 1.4 Hz, 1H), 5.20 (d, J ) 10.5
Hz, 1H), 4.98 (m, 2H), 4.70 (m, 1H), 4.38 (dd, J ) 9.4, 4.8 Hz,
1H), 4.18-4.12 (m, 3H), 3.35 (dd, J ) 13.3, 3.3 Hz, 1H), 2.68
(dd, J ) 13.2, 10.1 Hz, 1H), 2.31 (d, J ) 3.5 Hz, 1H), 2.10 (dd,
J ) 14.5, 7.1 Hz, 2H), 1.98-1.90 (m, 1H), 1.75-1.68 (m, 1H);
13C NMR (125 MHz, CDCl3) δ 174.7, 153.6, 137.8, 137.3, 135.2,
129.4, 129.0, 127.4, 116.7, 115.4, 73.8, 66.1, 55.6, 47.5, 38.1,
31.6, 26.7; IR (neat) 3488 (b), 1779 (s), 1692 cm-1; HRMS m/z
calcd for C19H24NO4 (M + H): 330.1705, found 330.1697;
[R]2D0 +35.7 (c ) 0.82, CHCl3).
(1R,2R,4S)-4-Ben zyl-3-[(2-h yd r oxy-3-cycloh exen -1-yl)-
ca r bon yl]-2-oxa zolid in on e (10). To a solution of oxazolidi-
none 9 (229 mg, 0.696 mmol) in 7 mL of CH2Cl2 was added
benzylenedine(bistricyclohexylphosphine)ruthenium(II) dichlo-
ride (28.6 mg, 0.035 mmol). The solution was stirred for 30
min and then air was bubbled through the mixture for 3 h to
oxidize the remaining catalyst. Concentration of the solution
followed by chromatography (20-40% EtOAc/petroleum ether
gradient) gave the title compound as a colorless crystals (198.1
mg, 95%). 10: mp 159-161 °C; Rf 0.16 (30% EtOAc/petroleum
ether); 1H NMR (500 MHz, CDCl3) δ 7.34-7.20 (m, 5H), 5.96-
5.86 (m, 2H), 4.72 (m, 1H), 4.45 (d, J ) 3.3 Hz, 1H), 4.21 (m,
2H), 3.75 (m, 1H), 3.29 (dd, J ) 13.3, 3.2 Hz, 1H), 2.81 (m,
2H), 2.19 (m, 2H), 2.08-2.00 (m, 1H), 1.88 (m, 1H); 13C NMR
(125 MHz, CDCl3) δ 175.2, 153.1, 135.1, 131.4, 129.4, 128.9,
127.41, 127.38, 66.3, 63.7, 55.3, 44.4, 37.9, 25.1, 19.8; IR (neat)
3487 (w), 1777 (s), 1699 cm-1; HRMS m/z calcd for C17H20NO4
(M + H): 302.1392, found 302.1382; [R]2D0 -48.95 (c ) 0.86,
CHCl3).
(1S,2R)-2-Hyd r oxy-3-cycloh exen eca r boxylic Acid (11).
Compound 5 (5.23 g, 13.7 mmol) was dissolved in freshly
distilled THF followed by addition of distilled water and cooled
to 0 °C. The biphasic mixture was treated with one portion of
LiOH‚H2O (8.6 g, 204.6 mmol). The reaction was allowed to
warm to room temperature and stirred for 2 days. The mixture
was concentrated to half of the volume and diluted with ethyl
acetate. The pH of the aqueous layer was adjusted to 1 with 1
N KHSO4. The layer was separated, and the aqueous layer
was extracted with 100 mL of EtOAc three times. The
combined organic solution was washed with brine, dried over
Na2SO4, filtered, and concentrated under reduced pressure.
Column chromatography (2%-10% CH2Cl2/MeOH) yielded the
title compound as a colorless oil (1.908 g, 98%). This identical
procedure was applied to compound 10 to obtain the same
product. 11: Rf 0.12 (5% MeOH/ CH2Cl2); 1H NMR (500 MHz,
CDCl3) δ 5.91-5.80 (m, 2H), 4.43 (t, ) 3.9 Hz, 1H), 2.57 (m,
1H), 2.14 (m, 1H), 1.98 (m, 1H), 1.85 (m, 2H); 13C NMR (125
MHz, CDCl3) δ 179.2, 131.6, 127.2, 63.9, 45.0, 24.8, 19.1; IR
(neat) 3407 (b), 1713 (s) cm-1; [R]D20 -270.3 (c ) 1.81, CHCl3).
(1R,2S,5S,8S)-8-H yd r oxy-2-iod o-7-oxa -b icyclo[3.2.1]-
octa n -6-on e (12). To Ag(collidine)2ClO4 28 (1.27 g, 2.83 mmol)
in 5 mL of CH2Cl2 was added iodine (0.717 g, 2.83 mmol) in
one portion with vigorous stirring. After 20 min, the iodine
was completely dissolved and a white precipitate formed. This
suspension was cooled to -20 °C, and then a solution of
compound 11 (287 mg, 2.02 mmol) in 10 mL of CH2Cl2 was
added slowly via cannula into the suspension. The reaction
was stirred at that temperature for 3 h. The mixture was
diluted with EtOAc and filtered through a pad of Celite. The
Celite was washed with EtOAc, and then the combined organic
solution was washed with 5% HCl solution, half-saturated
NaHCO3, and brine. The organic layer was dried with Na2-
SO4, filtered, and concentrated under reduced pressure. The
crude material was subject to column chromatography
(10-30% EtOAc/petroleum ether) to give rise to colorless
crystals (379 mg, 70%). 12: mp 109-111 °C; Rf 0.15 (20%
EtOAc/petroleum ether); 1H NMR (500 MHz, CDCl3) δ 4.87
(s, 1H), 4.65 (dd, J ) 4.5, 1.1 Hz, 1H), 4.47 (dd, J ) 5.4, 4.9
Hz, 1H), 2.80 (s, 1H), 2.63 (s, 1H), 2.22-2.14 (m, 1H), 1.95-
1.78 (m, 3H); 13C NMR (125 MHz, CDCl3) δ 176.6, 85.1, 75.6,
47.0, 28.1, 22.18, 22.16; IR (neat) 3473, 1755 cm-1; HRMS m/z
calcd for C7H13NO3I (M + NH4): 285.9940, found 285.9946;
[R]2D0 -64.9 (c ) 1.34, CHCl3).
(1R ,5S ,8S )-8-H y d r o xy -7-o xa -b ic yc lo[3.2.1]o c t a n -6-
on e (13). To compound 12 (100 mg, 0.37 mmol) in 8 mL of
benzene at 60 °C was added tris(trimethylsilyl)silane (0.173
mL, 0.56 mmol) through a syringe followed by addition of
AIBN (3.07 mg, 0.019 mmol) in one portion. After refluxing
the mixture for 4h, the benzene was removed under reduced
pressure, and the resultant oil was dissolved in acetone-water
(1:1), followed by treatment with KF (50 mg). After stirring
for 20 min, the solution was concentrated down to half of its
volume and extracted with EtOAc. The organic layer was
washed with water and brine, dried over Na2SO4, filtered, and
concentrated in vacuo. The crude material was subject to
column chromatography using 20-40% EtOAc/petroleum ether
to yield title compound as colorless crystals (44.5 mg, 84%)
13: mp 140-142 °C; Rf 0.16 (5% acetone/CHCl3); 1H NMR (500
MHz, CDCl3) δ 4.64 (d, J ) 5.2 Hz, 1H), 3.92 (s, 1H), 3.19 (s,
1H), 2.56 (d, J ) 4.6, 1H), 2.01-1.83 (m, 2H), 1.60-1.42 (m,
4H); 13C NMR (125 MHz, CDCl3) δ 178.3, 83.7, 77.9, 47.4, 27.3,
24.9, 16.7; IR (neat) 3416, 2933, 1770 cm-1; HRMS m/z calcd
for C7H14NO3 (M + NH4): 160.0973, found 160.0971; [R]D20
-21.8 (c ) 2.54, CHCl3). Anal. Calcd for C7H10O3: C, 59.14;
H, 7.09. Found, C, 59.25; H, 7.04.
(1R,5S,8S)-8-Be n zyloxy-7-oxa -b icyclo[3.2.1]oct a n -6-
on e (14). To compound 13 (1.05 g, 7.4 mmol) in 75 mL of THF
at 0 °C was added NaHMDS (1 M in toluene, 8.1 mL, 8.1
mmol). After stirring for 10 min, distilled BnBr (1.3 mL, 11
mmol) was added dropwise, followed by addition of tetrabu-
tylammonium iodide (2.73 g, 7.4 mmol) in one portion with
vigorous stirring. After warming the reaction mixture to room
temperature, it was stirred for 12 h and then quenched with
saturated ammonium chloride. The layers were separated, and
the aqueous layer was extracted with EtOAc. The organic layer
was washed with half-saturated NaHCO3 and brine, dried over
Na2SO4, filtered, and concentrated in vacuo. The crude mate-
rial was subject to column chromatography (10-30% EtOAc/
petroleum ether) to afford 14 as colorless crystals (1.458 g,
85%). 14: mp 82-83 °C; Rf 0.28 (20% EtOAc/petroleum ether);
1H NMR (500 MHz, CDCl3) δ 7.34-7.26 (m, 5H), 4.76 (m, 1H),
4.52 (s, 2H), 3.62 (s, 1H), 2.72 (t, J ) 2.2, 1H), 2.04-1.89 (m,
2H), 1.61-1.42 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 177.1,
137.3, 128.5, 127.9, 127.5, 84.2, 80.3, 70.2, 44.7, 27.6, 25.2, 17.1;
IR (neat) 3020, 1790 cm-1; HRMS m/z calcd for C14H20NO3 (M
+ NH4): 250.1443, found 250.1442; [R]2D0 -30.8 (c ) 0.79,
CHCl3).

2740 J . Org. Chem., Vol. 66, No. 8, 2001
Xiao et al.
(1S,2S,3S)-3-Azid o-2-ben zyloxycycloh exa n eca r boxyl-
ic Acid Met h yl E st er (16). To compound 14 (1.071 g, 4.6
mmol) in 150 mL of MeOH (HPLC grade) was added K2CO3
(1.27 g, 9.2 mmol) in three portions with vigorous stirring.
After 36 h, the reaction mixture was treated with 10% HCl to
adjust pH to 7. The methanol was removed on a rotary
evaporator, and the residue was dissolved in EtOAc followed
by washes with water and brine. The organic layer was dried
with Na2SO4, filtered, concentrated in vacuo, and pumped
under reduced pressure (<5 mmHg). The crude material was
dissolved in 70 mL of CH2Cl2 and cooled to 0 °C. Subsequent
treatment with pyridine (11 mL, 18.4 mmol), trifluoromethane-
sulfonic anhydride (1.15 mL, 5 mmol) for 2 h at 0 °C yielded
the crude triflate solution. This solution was diluted with
ether, washed with 5% HCl solution and half-saturated
NaHCO3, and brine. The organic layer was dried with
Na2SO4, filtered, and concentrated under reduced pressure.
The residue (15) was dissolved in 100 mL of DMF, followed
by addition of NaN3 (0.3 g, 4.6 mmol) in one portion. After
stirring overnight, another 1 equiv of NaN3 (0.3 g, 4.6 mmol)
was added and the reaction mixture stirred further for 8 h.
The solvent was distilled, and the crude product was treated
with 40 mL of THF and 20 mL of 10% HCl and stirred for 30
min. The aqueous solution was neutralized with 10% KOH and
concentrated to half of its original volume. The solution was
extracted with 50 mL of EtOAc three times, the organic layers
were washed with water and brine, dried over Na2SO4, filtered,
and concentrated under reduced pressure. Column chroma-
tography using 0-5% EtOAc/petroleum ether yielded the title
compound (447.8 mg, 56% based on the recovered starting
material). Further elution with 20-30% EtOAc/petroleum
ether afforded recovered starting material as a colorless oil
as an off-white powder (18.8 mg, 81%). 18: mp 242 °C (dec);
1H NMR (500 MHz, CD3OD) δ 3.36 (dd, J ) 10.2, 5.2 Hz, 1H),
3.12 (m, 1H), 2.59 (d, J ) 3.3 Hz, 1H), 1.88-1.85 (m, 1H), 1.78
(d, J ) 2.6, 1H), 1.39-1.01 (m, 4H); 13C NMR (125 MHz,
CD3OD) δ 172.5, 71.6, 53.3, 45.4, 28.4, 26.7, 20.3; IR (KBr):
2965 (b), 1580 cm-1; HRMS m/z calcd for C7H14NO3 (M + H):
160.0973, found 160.0968; [R]2D0 +72.5 (c ) 1.22, CHCl3).
(2R,3R,4S)-4-(Ben zyloxy)-1-[(ter t-bu tyld im eth ylsilyl)-
oxy]-2,5-d im eth yl-3-h exa n ol (20). Step 1: To compound 19
(2.296 g, 6.26 mmol) in 62 mL of CH2Cl2 was added Dess-
Martin reagent (3.19 g, 7.5 mmol) with vigorous stirring. After
2 h, the reaction mixture was diluted with 240 mL of ether
and then poured into 120 mL of saturated NaHCO3 containing
2 g of NaS2O3‚4H2O. The mixture was shaken gently until the
ether layer became clear. The organic layer was separated,
washed with brine, dried with Na2SO4, filtered, and concen-
trated under reduced pressure. Column chromatography using
0-5% EtOAc/petroleum ether yielded the title compound as a
colorless oil (2.18 g, 96%). The title compound: Rf 0.64 (5%
1
EtOAc/petroleum ether); H NMR (500 MHz, CDCl3) δ 7.35-
7.25 (m, 5H), 4.66 (d, J ) 11.9 Hz, 1H), 4.26 (d, J ) 11.9 Hz,
1H), 3.82 (dd, J ) 9.6, 8.3 Hz, 1H), 3.60 (d, J ) 5.3 Hz, 1H),
3.44 (dd, J ) 9.7, 5.7 Hz, 1H), 3.07 (m, 1H), 2.06 (m, 1H), 0.89-
0.84 (m, 9H), 0.77 (s, 9H), -0.08 (d, J ) 6.3 Hz, 6H); 13C NMR
(125 MHz, CDCl3) δ 214.5, 138.1, 128.3, 127.9, 127.7, 89.0,
72.5, 65.0, 44.2, 30.0, 25.9, 19.5, 17.6, 13.9, -5.5, -5.6; IR
(neat) 2934, 1716, 1471, 1095 cm-1; HRMS m/z calcd for
C
21H37O3Si (M + H): 365.2512, found 365.2497; [R]20 -48.7 (c
D
) 2.2, CHCl3).
Step 2: To a suspension of lithium aluminum hydride (193
mg, 5.08 mmol) in 10 mL of Et2O at -10 °C was added slowly
via cannula into a precooled solution (-10 °C) of the above
ketone (1.85 g, 5.08 mmol) in 20 mL of Et2O with vigorous
stirring. After 2 h, the reaction temperature was maintained
at 0 °C, and H2O (0.2 mL), 15% NaOH (0.2 mL), and H2O (0.4
mL) were added dropwise (Caution: reaction is exothermic).
The mixture was allowed to stir for 1 h at room temperature
and then dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was chromatographed three
times using 0-5% EtOAc/petroleum ether to provide the title
compound as a colorless oil (819.1 mg, 44%). The (2R,3S,4S)
isomer (856 mg, 46%) was recovered and recycled. 20: Rf 0.48
(5% EtOAc/petroleum ether); 1H NMR (500 MHz, CDCl3) δ
7.33-7.24 (m, 5H), 4.52 (d, J ) 12.1 Hz, 1H), 4.47 (d, J ) 12.1
Hz, 1H), 3.62 (dd, J ) 8.9, 3.8 Hz, 1H), 3.55 (dd, J ) 9.0, 5.9
Hz, 1H), 3.50 (dd, J ) 5.0, 2.7 Hz, 1H), 3.46 (m, 1H), 2.81 (d,
J ) 4.5 Hz, 1H), 1.97-1.89 (m, 2H), 1.02-0.90 (m, 9H), 0.89
(s, 9H), 0.05-0.02 (d, J ) 9.2, 6H); 13C NMR (125 MHz, CDCl3)
δ 138.5, 128.3, 127.6, 127.5, 77.6, 76.8, 73.3, 73.2, 35.0, 29.4,
26.0, 21.0, 17.2, 14.6, -4.1, -4.4; IR (neat) 3504, 2957, 1090,
1505 cm-1; HRMS m/z calcd for C21H39O3Si (M + H): 367.2668,
found 367.2673; [R]2D0 +2.2 (c ) 1.5, CHCl3).
(3S,4R,5R)-6-[(ter t-Bu t yld im et h ylsilyl)oxy]-4-(m et h -
oxym eth oxy)-2,5-d im eth yl-3-h exa n ol (21). Step 1: To com-
pound 20 (0.82 g, 2.2 mmol) in 6.9 mL of CH2Cl2 at 0 °C was
added sequentially diisopropylethylamine (1.76 mL, 10.1
mmol) and chloromethyl methyl ether (0.515 mL, 6.4 mmol)
dropwise through two separate syringes. The mixture was
allowed to warm to room temperature and was stirred over-
night. The reaction was then diluted with ether followed by
washes with 5% HCl solution, half-saturated NaHCO3, and
brine. The organic layer was dried with Na2SO4, filtered, and
concentrated under reduced pressure. Column chromatogra-
phy using 0-5% EtOAc/petroleum ether yielded the title
compound as a colorless oil (0.9 g, 98%). Rf 0.55 (5% EtOAc/
petroleum ether); 1H NMR (500 MHz, CDCl3) δ 7.32-7.30 (m,
5H), 4.72 (d, J ) 6.4 Hz, 1H), 4.59 (d, J ) 6.4 Hz, 1H), 4.47 (s,
2H), 3.70 (dd, J ) 8.9, 3.5 Hz, 1H), 3.51 (dd, J ) 5.2, 2.4 Hz,
1H), 3.47 (dd, J ) 7.0, 2.4 Hz, 1H), 3.37 (m, 1H), 3.32 (s, 3H),
2.04 (m, 1H), 1.84 (m, 1H), 1.05 (d, J ) 6.9 Hz, 3H), 0.94-
0.89 (m, 15H), 0.05-0.02 (m, 6H); 13C NMR (125M Hz, CDCl3)
δ 139.0, 128.3, 127.5, 127.4, 97.2, 82.7, 78.9, 73.0, 72.9, 56.0,
35.4, 30.3, 26.1, 21.1, 18.6, 15.9, -3.7, -4.8; IR (neat) 2923,
1
(429 mg). 16: Rf 0.36 (5% EtOAc/petroleum ether); H NMR
(500 MHz, CDCl3) δ 7.34-7.25 (m, 5H), 4.53 (s, 2H), 3.89 (dd,
J ) 9.3, 5.5 Hz, 1H), 3.71 (t, J ) 4.2, 1H), 3.58 (s, 3H), 2.77
(m, 1H), 1.86-1.38 (m, 6H); 13C NMR (125 MHz, CDCl3) δ
173.6, 138.0, 128.4, 127.8, 127.7, 77.4, 72.3, 59.0, 51.5, 42.5,
25.9, 23.0, 19.2; IR (neat) 2947, 2100, 1738 cm-1; HRMS m/z
calcd for C15H23N4O3 (M + NH4): 307.1770, found 307.1776;
[R]2D0 +43.9 (c ) 1.06, CHCl3).
(1S,2S,3S)-3-Azid o-2-b en zyloxy-cycloh exa n eca r b oxy-
lic Acid (17). Compound 16 (220 mg, 0.76 mmol) was
dissolved in 40 mL of freshly distilled THF, followed by
addition of 20 mL of distilled water. The reaction mixture was
cooled to 0 °C. The biphasic mixture was treated with one
portion of LiOH‚H2O (366.6 mg, 8.74 mmol). The reaction was
allowed to warm to room temperature and stirred for 2 days.
The mixture was concentrated to half its volume and diluted
with ethyl acetate. The pH of aqueous layer was adjusted to 2
with 1 N KHSO4. The layers were separated and the aqueous
layer was extracted with EtOAc three times. The combined
organic solution was washed with brine, dried over Na2SO4,
filtered, and concentrated under reduced pressure. Further
column chromatography (0-20% EtOAc/petroleum ether)
yielded the title compound as colorless crystals (204.1 mg,
97%). 17: mp 79-81 °C; Rf 0.23 (10% EtOAc/petroleum ether);
1H NMR (500 MHz, CDCl3) δ 7.31-7.24 (m, 5H), 4.58 (s, 2H),
3.84 (dd, J ) 9.3, 5.5 Hz, 1H), 3.74 (t, J ) 4.1, 1H), 2.80 (m,
1H), 1.86-1.41 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 179.0,
137.6, 128.4, 127.9, 127.8, 77.4, 72.7, 59.1, 42.6, 26.0, 22.9, 19.2;
IR (neat) 3031 (b), 2099, 1704 cm-1; HRMS m/z calcd for
C
14H21N4O3 (M + NH4): 293.1613, found 293.1619; [R]D20
+44.7 (c ) 2.46, CHCl3).
(1S,2S,3S)-3-Am in o-2-h yd r oxy-cycloh exa n eca r b oxyl-
ic Acid (18). In a Parr hydrogenation tube filled with argon
was added compound 17 (40 mg, 0.15 mmol) in 3.5 mL of
absolute ethanol followed by addition of 10% Pd on carbon (100
mg). The mixture was subjected to hydrogenation at 50 psi.
After 8 h, the reaction mixture was filtered through a pipet
plugged with an inert material, and the Pd/C residue was
washed with absolute ethanol. The combined ethanol solution
was concentrated in vacuo. The crude amino acid was dissolved
in 10 mL of distilled water then extracted with 10 mL of EtOAc
three times. The aqueous solution was liophilized to yield 18

Conformationally Constrained Didemnin B Analog
J . Org. Chem., Vol. 66, No. 8, 2001 2741
1464 cm-1; HRMS m/z calcd for C23H46NO4Si (M + NH4):
428.3196, found 428.3191; [R]2D0 -49.1 (c ) 1.19, CHCl3).
Martin reagent (128.4 mg, 0.3 mmol) with vigorous stirring.
After 4 h, the rectum mixture was diluted with ether and then
poured into 20 mL of saturated NaHCO3 containing 1.7% (w/
v) NaS2O3‚4H2O. The mixture was shaken gently until the
ether layer became clear. The organic layer was separated,
washed with brine, dried with Na2SO4, filtered, and concen-
trated under reduced pressure to afford a colorless oil. The oil
was dissolved in 3 mL of DMSO-MeCN-5% NaH2PO4 (1:1:1)
followed by addition of solid NaClO2 (280 mg, 3.1 mmol). After
stirring overnight, the reaction was concentrated in vacuo to
two-thirds of its original volume. The solution was diluted with
ether, washed with water and brine, dried over Na2SO4, and
filtered. The filtrate was concentrated under reduced pressure
to afford an oil, which was used without further purification.
The resultant oil was dissolved in 1.4 mL of CH2Cl2, followed
by sequential addition of benzyl alcohol (45 µL, 0.43 mmol),
DCC (85.8 mg, 0.42 mmol), and DMAP (14 mg, 0.11 mmol).
The reaction was allowed to stir overnight, at which time it
was diluted with ether and filtered to remove a white
precipitate. The filtrate was concentrated in vacuo, and the
resultant oil was chromatographed using 0-10% acetone/
hexanes to yield a colorless oil (94.1 mg, 64%). 25: Rf 0.48 (20%
acetone/hexane); 1H NMR (500 MHz, CDCl3) δ 7.37-7.23 (m,
10H), 5.16 (d, J ) 12.3 Hz, 1H), 5.07 (d, J ) 12.3 Hz, 1H),
4.92 (m, 1H), 4.68 (d, J ) 11.5 Hz, 1H), 4.59-4.56 (m, 2H),
4.51 (d, J ) 6.7 Hz, 1H), 4.04 (d, J ) 5.4 Hz, 1H), 3.93 (d, J )
3.6 Hz, 1H), 3.78 (s, 1H), 3.28 (s, 3H), 2.97 (m, 1H), 2.88 (m,
1H), 1.95-1.83 (m, 3H), 1.76 (m, 1H), 1.65-1.63 (m, 1H), 1.57-
1.48 (m, 2H), 1.14 (d, J ) 7.0 Hz, 3H), 0.90 (d, J ) 6.8 Hz,
3H), 0.87 (d, J ) 6.8 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ
174.1, 172.5, 138.2, 136.0, 128.5, 128.3, 128.2, 128.1, 127.6,
127.5, 98.3, 79.4, 78.2, 77.7, 72.6, 66.5, 59.2, 56.2, 43.1, 41.4,
28.7, 26.4, 23.7, 19.5, 19.4, 17.2, 11.8; IR (neat) 2940, 2099,
1734 cm-1; HRMS m/z calcd for C31H42N3O7 (M + H): 540.2961,
found 540.2940; [R]2D0 +8.5 (c ) 0.6, CHCl3).
N-[N-[N-[(Ben zyloxycar bon yl]-L-leu cyl]-L-pr olyl]-3-(N,O-
d im eth yl-tyr osyl Ester w ith (1S,2S)-3[(2S,3R)-2-[(ter t-
Bu t oxyca r b on yl)a m in o]-3-h yd r oxyb u t yr a m id o]-2-b en -
zyloxycycloh exa n eca r boxylic Acid , (1S,2R,3S)-1-Isop r o-
p yl-2-(m eth oxym eth oxy)-3-m eth ylbu ta n oic Acid Ben zyl
Ester (28). To compound 25 (63 mg, 0.11 mmol) in 2.5 mL of
THF was added one drop (16 µL) of distilled water followed
by addition of trimethylphosphine (1 M in toluene, 252 mL,
0.252 mmol). After 1 h, air was bubbled through the reaction
mixture for 1 h. The reaction mixture was concentrated under
reduced pressure on a rotary evaporator and then pumped
under high vacuum (2 mmHg). The crude amine (26) was then
dissolved in 2 mL of DMF, followed by addition of tetrapeptide
acid (27, 126 mg, 0.17 mmol), collidine (36 µL, 0.252 mmol)
and HATU (67.8 mg, 0.18 mmol). After stirring overnight,
DMF was distilled under reduced pressure (2 mmHg). The
residue was dissolved in ethyl acetate and washed with 5%
HCl, half-saturated NaHCO3, and brine. The organic layer was
dried over Na2SO4, filtered, and concentrated under reduced
pressure. Further column chromatography using 10-30%
acetone/hexanes yielded the title compound as a colorless oil
(95 mg, 70%). 28: Rf 0.49 (30% acetone/hexane); 1H NMR (500
MHz, CDCl3) δ 8.39 (d, J ) 6.8 Hz, 1H), 7.62-7.79 (m, 1H),
7.36-7.18 (m, 15H), 7.03 (d, J ) 8.5 Hz, 2H), 6.81(d, J ) 8.4
Hz, 2H), 6.68 (d, J ) 9.0 Hz, 1H), 5.16 (dd, J ) 12.3, 2.9 Hz,
2H), 5.03 (m, 2H), 4.93-4.83 (m, 2H), 4.73-4.41 (m, 6H), 4.32
(d, J ) 3.1 Hz, 1H), 4.22 (d, J ) 7.9 Hz, 1H), 4.06-4.00 (m,
2H), 3.78 (S, 3H), 3.67-3.64, (m, 2H), 3.60 (m, 1H), 3.25 (s,
3H), 3.23 (s, 3H), 2.88 (m, 1H), 2.75 (d, J ) 11.5 Hz, 1H), 2.58
(m, 1H), 2.12 (m, 2H), 2.13 (m, 2H), 2.01-1.31 (m, 12H), 1.40
(s, 3H), 1.35 (s, 9H), 1.14-1.08 (m, 3H), 0.98-0.77 (m, 12H);
13C NMR (125 MHz, CDCl3) δ 174.1, 173.9, 173.3, 172.6,
(dialkylamide rotamer) 171.8, 171.3, 169.5, 168.9, 158.5, 156.7,
155.3, 138.4, 136.8, 136.0, 130.4, 130.2, 130.0, 128.5, 128.4,
128.3, 128.2, 128.18, 128.14, 128.03, 128.01, 127.8, 127.7,
127.6, 127.5, 127.4, 127.3, 127.2, 114.3, 114.0, 98.1, 80.3, 78.9,
78.0, 71.8, 71.6, 66.5, 66.4, 66.1, 57.3, 56.1, 56.0, 55.9, 55.4
(rotamer), 55.2, 54.3 (rotamer), 51.8, 51.0, 46.8, 45.7, 43.4, 43.3
(rotamer), 41.6, 41.4 (rotamer), 38.6, 38.2 (rotamer), 33.6, 28.8,
Step 2: To the above protected alcohol (0.858 g, 2.09 mmol)
in 21 mL of MeOH was added formic acid (0.84 mL) through
a syringe, followed immediately by addition of Pd black (0.224
g, 2.1 mmol) in one portion. After stirring for 20 min, the
reaction was filtered through a short column contained a plug
of inert material. (Caution: Pd black is highly flammable. Care
must be taken that the plug is kept moist). The filtrate was
added to 20 mL of saturated NaHCO3 and concentrated under
reduced pressure. The aqueous solution was extracted with
ether, washed with brine, dried over Na2SO4, filtered, and
concentrated in vacuo. Further column chromatography using
0-5% EtOAc/petroleum ether yielded the title compound as a
colorless oil (669.7 mg, 89%). 21: Rf 0.63 (10% EtOAc/petroleum
ether); 1H NMR (500 MHz, CDCl3) δ 4.70 (d, J ) 6.6 Hz, 1H),
4.65 (d, J ) 6.6 Hz, 1H), 3.93 (dd, J ) 10.0, 4.3 Hz, 1H), 3.66
(dd, J ) 10.0, 4.0 Hz, 1H), 3.59 (dd, J ) 11.4, 5.7 Hz), 3.49 (d,
J ) 6.0 Hz, 1H), 3.37 (s, 3H), 2.06-2.02 (m, 1H), 1.90 (m, 1H),
1.04 (d, J ) 7.1 Hz, 3H), 0.98 (d, J ) 3.4 Hz, 3H), 0.96 (d, J )
3.5 Hz, 3H), 0.88 (s, 9H), 0.05 (s, 6H); 13C NMR (125 MHz,
CDCl3) δ 97.4, 83.0, 75.6, 64.2, 56.0, 36.6, 29.9, 25.8, 20.2, 17.3,
15.1, -5.58, -5.62; IR (neat) 3430, 2956, 1036 cm-1; HRMS
m/z calcd for C16H37O4Si (M + H): 321.2461, found 321.2464;
[R]2D0 +11.1 (c ) 1.5, CHCl3).
(1S,2R,3R)-4-[(ter t-Bu tyldim eth ylsilyl)oxy]-1-isopr opyl-
2-(m eth oxym eth oxy)-3-m eth ylbu tyl (1S,2S,3S)-3-Azid o-
2-ben zyloxy-cycloh exa n eca r boxyla te (22). To azido acid
17 (53.8 mg, 0.2 mmol) and alcohol 21 (202 mg, 0.63 mmol) in
0.49 mL of toluene was added sequentially DCC (67.3 mg, 0.33
mmol) and DMAP (24.9 mg, 0.2 mmol). The mixture was
allowed to stir overnight. The reaction was first diluted with
ether and then filtered to remove a white precipitate (presum-
ably DCU). The filtrate was concentrated under reduced
pressure. After column chromatography (0-5% EtOAc/petro-
leum ether), the title compound was obtained as a colorless
oil (55.5 mg, 47%, based on the azido acid). Further elution
recovered the alcohol 21 (158.9 mg). 22: Rf 0.72 (10% EtOAc/
petroleum ether); 1H NMR (500 MHz, CDCl3) δ 7.30-7.24 (m,
5H), 5.01 (dd, J ) 7.1, 4.5 Hz, 1H), 4.66-4.53 (m, 4H), 3.96
(d, J ) 4.0 Hz, 1H), 3.81 (s, 1H), 3.76 (dd, J ) 7.1, 3.2 Hz,
1H), 3.34 (d, J ) 6.7 Hz, 2H), 3.29 (s, 3H), 2.86 (t, J ) 4.7 Hz,
1H), 1.91-1.48 (m, 8H), 0.87-0.84 (m, 18H), 0.02 (s, 6H); 13
C
NMR (125 MHz, CDCl3) δ 172.6, 138.2, 128.2, 127.7, 127.6,
98.5, 78.7, 78.4, 77.5, 72.8, 65.0, 59.2, 55.9, 43.3, 37.4, 28.8,
25.8, 23.5, 19.8, 19.5, 18.2, 16.2, 10.9, -5.46, -5.54; IR (neat)
2933, 2099, 1728, 1093, 1038 cm-1; HRMS m/z calcd for
C
30H51N3O6SiNa (M + Na): 600.3445, found 600.3462; [R]20
D
-0.67 (c ) 0.6, CHCl3).
(1S,2R,3R)-1-Isop r op yl-2-(m et h oxym et h oxy)-3-m et h -
ylbu ta n -4-ol (1S,2S,3S)-3-a zid o-2-Ben zyloxycycloh exa n e
ca r boxyla te (23). To compound 22 (64.7 mg, 0.11 mmol) in
1.2 mL of MeOH was added Amberlyst-15 (120 mg). After
stirring for 20 h, the reaction was filtered and the Amberlyst-
15 residue was washed thoroughly with 20 mL of MeOH. The
combined methanol solution was concentrated under reduced
pressure. Column chromatography using 10-30% EtOAc/
petroleum ether yielded the title compound as a colorless oil
(51.9 mg, 94%). 23: Rf 0.4 (20% acetone/hexane); 1H NMR
(500 MHz, CDCl3) δ 7.34-7.25 (m, 5H), 5.13 (dd, J ) 8.9, 2.9
Hz, 1H), 4.66-4.60 (m, 3H), 4.42 (d, J ) 6.8 Hz, 1H), 4.04 (d,
J ) 3.4 Hz, 1H), 3.76 (m, 2H), 3.45 (s, 2H), 3.44 (s, 2H), 3.28
(s, 3H), 2.97 (s, 1H), 2.88 (dd, J ) 8.6, 4.2 Hz, 1H), 1.98-1.50
(m, 8H), 0.86 (d, J ) 6.8 Hz, 3H), 0.84 (d, J ) 6.9 Hz, 3H),
0.81 (d, J ) 6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 172.5,
138.1, 128.3, 127.72, 127.69, 98.8, 78.6, 78.5, 78.1, 73.0, 64.3,
59.3, 56.3, 43.5, 36.3, 28.4, 26.6, 24.1, 19.9, 19.4, 15.5, 9.9; IR
(neat) 3495, 2949, 2101, 1727 cm-1; HRMS m/z calcd for
C
24H37N3O6Na (M + Na): 4.86.2580, found 486.2570; [R]D20
-16.9 (c ) 0.91, CHCl3).
(1S,2R,3R)-1-Isop r op yl-2-(m et h oxym et h oxy)-3-m et h -
ylbu ta n oic Ben zyl Ester , Ester w ith (1S,2S,3S)-3-Azid o-
2-ben zyloxycycloh exa n eca r boxyla te (25). To compound 23
(120 mg, 0.26 mmol) in 2.3 mL of CH2Cl2 was added Dess-

2742 J . Org. Chem., Vol. 66, No. 8, 2001
Xiao et al.
28.7, 28.3, 28.1 (rotamer), 25.4, 24.9, 23.4, 21.0, 19.40, 19.36,
2H), 7.12 (m, 1H), 6.91 (d, J ) 8.2 Hz, 2H), 5.16 (m, 2H), 5.13
(m, 1H), 4.93-4.67 (m, 6H), 4.61-4.48 (m, 4H), 4.25 (t, J )
6.4 Hz, 1H), 4.16 (m, 1H), 3.86 (s, 3H), 3.82 (s, 3H), 3.69 (m,
4H), 3.24 (m, 2H), 3.09 (s, 3H), 3.01 (m, 2H), 2.79 (m, 2H),
2.20-1.90 (m, 4H), 1.87-1.81 (m, 4H), 1.70-1.54 (m, 13H),
1.47-1.28 (m, 9H), 1.03-0.81 (m, 18H); 13C NMR (125 MHz,
CDCl3) δ 174.4, 171.3, 170.2, 168.1, 165.8, 161.8, 159.0, 158.6,
(dialkylamide rotamer) 156.7, 154.5, 150.4, 149.4, 138.3, 137.9,
137.3, 132.1, 130.3, 130.2, 129.0, 128.96, 128.5, 128.2, 128.0,
127.8, 127.6, 126.0, 121.5, 114.4, 114.1, 78.6, 75.3, 74.0, 72.6,
71.8, 71.0, 70.4, 61.5, 57.2, 56.4, 55.4, 52.4, 48.4, 47.5, 45.3,
44.5, 44.2, 43.6, 43.2, 36.5, 36.3, 34.9, 29.6, 29.1, 28.8, 28.5,
28.1, 26.0, 25.1, 24.7, 24.2, 23.4, 23.2, 22.4, 21.5, 20.7, 19.5,
19.3, 18.7, 17.9, 17.1, 16.6, 14.6; IR (neat) 3311, 2958, 1734,
1635 cm-1; HRMS m/z calcd for C70H99N7O15Na (M + Na):
1300.7097, found 1300.7072; [R]2D0 -10.4 (c ) 0.14, CHCl3).
Cyclo-[N-(N-L-la ctyl-L-p r olyl-N-m eth y-D-leu cyl)-O-[[N-
[(2S,3R,4S)-4-[(1S,2S,3S)-3-a m in o-2-ben zyloxycycloh ex-
a n eca r bon yl]oxy-3-oxo-2,5-d im eth ylh exa n oyl]-L-leu cyl]-
L-p r olyl-N,O-d im et h yl-L-t yr osyl]-L-t h r eon yl] (2). To a
solution of 31 (2.5 mg, 0.002 mmol) in CH2Cl2 (0.3 mL) was
added Dess-Martin reagent (1.6 mg, 0.003 mmol) with vigor-
ous stirring. After 1 h, the reaction mixture was diluted with
2 mL of ether then poured into 1 mL of saturated NaHCO3
containing 19.2 mg of NaS2O3‚4H2O. The mixture was shaken
until the ether layer became clear. The organic layer was
separated, and the aqueous layer was extracted with 2 mL of
EtOAc. The combined organic layers were washed with brine,
dried with Na2SO4, filtered, and concentrated under reduced
pressure to afford a colorless thin film. The residue was
dissolved in HPLC grade MeOH (0.3 mL). Formic acid (15 mL)
was added through a syringe followed immediately by addition
of Pd black (3.0 mg, 0.028 mmol) in one portion. After stirring
for 20 min, the reaction was diluted with absolute ethanol and
filtered through a short column with a plug of inert material.
(Caution: Pd black is highly flammable; care should be taken
to ensure that the plug will remain moist.) The filtrate was
concentrated under reduced pressure. The residue was dis-
solved in chloroform and applied to a short (1 cm) silica gel
column. Elution with ethyl acetate afforded the title compound
as a semisolid (1.4 mg, 64%). 2: Rf 0.51 (10% MeOH/ CH2Cl2);
1H NMR (500 MHz, CDCl3) δ 8.34 and 8.08 (m, 1H), 7.99 (s,
1H), 7.48-7.44 (m, 1H), 7.03 (d, J ) 8.6 Hz, 2H), 6.81 (d, J )
8.5 Hz, 2H), 5.64 (m, 1H), 5.25 (s, 1H), 5.21 (m, 1H), 4.80 (m,
1H), 4.74 (d, J ) 6.4 Hz, 1H), 4.64 (m, 2H), 4.53 (dd, J ) 9.7,
4.7 Hz, 1H), 4.32 (m, 1H), 4.26 (m, 1H), 3.83 (m, 1H), 3.74 (s,
3H), 3.71 (m, 2H), 3.55 (m, 4H), 3.27 (m, 1H), 3.11 (m, 1H),
3.01 (s, 3H), 2.76 (s, 3H), 2.11 (m, 2H), 1.96-1.82 (m, 4H),
1.80-1.57 (m, 4H), 1.55-1.39 (m, 4H), 1.35 (m, 3H), 1.24-
0.98 (m, 19H), 0.84-0.71 (m, 12H); IR (neat) 3311, 2926, 1734,
1636, 1458 cm-1; HRMS m/z calcd for C56H85N7O15Na (M +
Na): 1118.6038, found 1118.6001; [R]2D0 -6.7 (c ) 0.06, CHCl3).
19.2, 16.8, 14.3, 11.6, 11.3; IR (neat) 3323, 2960, 1725 cm-1
;
HRMS m/z calcd for C70H95N5O17Na (M + Na): 1300.6623,
found 1300.6601; [R]2D0 -53.6 (c ) 0.63, CHCl3).
Cyclo-[N-(ter t-bu tyloxyca r bon yl)-O-[[N-[(2S,3R,4S)-4-
[(1S,2S,3S)-3-a m in o-2-ben zyloxycycloh exa n eca r bon yl]-
oxy-3-(m eth oxym eth oxy)-2,5-dim eth ylh exan oyl]-L-leu cyl]-
L-p r olyl-N,O-d im eth yl-L-tyr osyl]-L-th r eon yl] (29). In
a
Parr hydrogenation tube filled with argon was added com-
pound 28 (17.5 mg, 0.014 mmol) in 0.8 mL of absolute ethanol,
followed by addition of 10% Pd on carbon (16.2 mg). The
mixture was subjected to hydrogenation at 45-50 psi. After
4-6 h, the reaction mixture was filtered through a column
with a plug of inert material, and the Pd/C residue was washed
with absolute ethanol. (Care should be taken that the plug
will stay moist.) The combined ethanol solution was concen-
trated in vacuo. The crude amino acid was then dissolved in
1.3 mL of DMF, followed by addition of diisopropylethylamine
(7.2 mL, 0.041 mmol) and HATU (6.3 mg, 0.016 mmol). After
24 h, another portion of HATU (3.2 mg, 0.008 mmol) was
added, and the reaction was allowed to stir overnight. DMF
was distilled under reduced pressure. The residue was dis-
solved in ethyl acetate and washed with 5% HCl, half-
saturated NaHCO3, and brine. The organic layer was dried
over Na2SO4, filtered, and concentrated under reduced pres-
sure. Column chromatography using 20-40% acetone/hexanes
yielded the title compound as a clear thick oil (6.4 mg, 45%).
29: Rf 0.23 (30% acetone/hexane); 1H NMR (500 MHz, CDCl3)
δ 8.04 (m, 1H), 7.36-7.15 (m, 5H), 7.05 (d, J ) 8.7 Hz, 2H),
7.00 (d, J ) 8.6 Hz, 1H), 6.81 (d, J ) 8.6 Hz, 2H), 6.05 (d, J )
9.0 Hz, 1H), 5.31 (d, J ) 4.9 Hz, 1H), 5.11 (t, J ) 5.9 Hz, 1H),
4.73 (m, 2H), 4.65-4.61 (m, 2H), 4.58-4.42 (m, 4H), 4.31 (m,
1H), 3.80 (m, 1H), 3.71 (s, 3H), 3.55 (m, 2H), 3.34 (s, 3H), 3.26
(s, 1H), 3.16-3.12 (m, 1H), 2.98 (s, 3H), 2.87 (m, 1H), 2.36 (m,
1H), 2.11 (m, 1H), 1.96 (m, 2H), 1.84-1.07 (m, 15H), 1.01 (d,
J ) 6.5 Hz, 3H), 0.88 (d, J ) 6.6 Hz, 3H), 0.82 (m, 9H), 0.69
(d, J ) 6.8 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 174.2, 173.6,
172.9, 172.6, 171.1, 168.6, 168.2, 159.0, 137.5, 130.3, 128.8,
128.3, 128.1, 127.6, 114.3, 98.8, 81.0, 79.1, 78.7, 78.2, 73.8, 71.0,
61.4, 60.3, 56.3, 55.6, 55.3, 52.1, 48.2, 47.4, 43.9, 43.0, 42.7,
34.8, 29.5, 28.9, 28.4, 28.2, 28.1, 27.2, 24.6, 24.0, 22.9, 22.3,
20.5, 19.0, 18.6, 16.6, 14.1; IR (neat) 3318, 2959, 1723, 1631
cm-1; HRMS m/z calcd for C55H81N5O14Na (M + Na):1058.5678,
found 1058.5665; [R]2D0 -30.6 (c ) 0.18, CHCl3).
Cyclo-[N-(N-L-O-ben zyloxyla ctyl-L-p r olyl-N-m eth yl-D-
leu cyl)-O-[[N-[(2S,3R,4S)-4-[(1S,2S,3S)-3-a m in o-2-ben zy-
loxy-cycloh exa n eca r bon yl]oxy-3-h yd r oxy-2,5-d im eth yl-
h exa n oyl]-L-leu cyl]-L-p r olyl-N,O-d im et h yl-L-t yr osyl]-L-
th r eon yl] (31). To a solution of 29 (6.4 mg, 0.006 mmol) in
ethyl acetate (0.6 mL) was added a freshly prepared 10% HCl
solution (0.6 mL). After stirring for 2 days, the pH of the
reaction mixture was adjusted to 9 by addition of 3 N NaOH
solution. The resultant solution was extracted with ethyl
acetate three times. The combined organic layers were washed
with brine, dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residual amine was dissolved in 0.3 mL
of DMF followed by addition of the side chain acid 30 (4.1 mg,
0.01 mmol), collidine (3.6 µL, 0.027 mmol), and HATU (4.2 mg,
0.011 mmol). The solution was allowed to stir overnight. The
solvent (DMF) was distilled under reduced pressure, and the
crude product was dissolved in 10 mL of ethyl acetate. The
organic solution was washed with 5% HCl, half-saturated
NaHCO3, and brine, dried over Na2SO4, filtered, and concen-
trated under reduced pressure. Further column chromatog-
raphy using 50-80% EtOAc/petroleum ether yielded the title
compound as a colorless oil (2.7 mg, 34%). 31: Rf 0.28 (70%
EtOAc/petroleum ether); 1H NMR (500 MHz, CDCl3) δ 7.85
(m, 1H), 7.49 (s, 1H), 7.47-7.31 (m, 10H), 7.16 (d, J ) 8.5 Hz,
Ack n ow led gm en t. We gratefully acknowledge the
National Institute of Health (CA 40081), the NSF (CHE
99-01449), and the University of Pennsylvania for
financial support. We also thank Dr. Peter Toogood and
Dr. Deepika Auja at the University of Michigan for the
biological testing.
Su p p or tin g In for m a tion Ava ila ble: General procedures,
1H and/or 13C NMR spectra of compounds 2, 5, 5a , 5b, 7-14,
16-23, 25, 28, 29 and 31, and the X-ray data of compound
17. This material is available free of charge via the Internet
at http://pubs.acs.org.
J O001640N