Application of acyl cyanophosphorane methodology to the synthesis of protease inhibitors: Poststatin, eurystatin, phebestin, probestin and bestatin

Article abstract of DOI:10.1016/S0040-4020(03)00860-3

Full details are given for the syntheses of the protease inhibitors, poststatin and eurystatin by the acyl cyanophosphorane coupling procedure used for the formation of α-keto amides. We have also extended this methodology to the syntheses of the related α-hydroxy amide natural products, phebestin, probestin and bestatin. The key step in the latter synthetic sequences involved diastereomeric selectivity in the reduction of the α-keto precursor to the corresponding α-hydroxy amide by the use of zinc borohydride.

Full text of DOI:10.1016/S0040-4020(03)00860-3

Tetrahedron 59 (2003) 6771–6784
Application of acyl cyanophosphorane methodology to the
synthesis of protease inhibitors: poststatin, eurystatin, phebestin,
probestin and bestatin
*
Harry H. Wasserman, Anders K. Petersen and Mingde Xia
Department of Chemistry, Yale University, P.O. Box 208107, New Haven, CT 06520-8107 USA
Received 28 April 2003; revised 22 May 2003; accepted 29 May 2003
Abstract—Full details are given for the syntheses of the protease inhibitors, poststatin and eurystatin by the acyl cyanophosphorane coupling
procedure used for the formation of a-keto amides. We have also extended this methodology to the syntheses of the related a-hydroxy amide
natural products, phebestin, probestin and bestatin. The key step in the latter synthetic sequences involved diastereomeric selectivity in the
reduction of the a-keto precursor to the corresponding a-hydroxy amide by the use of zinc borohydride.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
The present work describes the use of this acyl cyano-
phosphorane procedure in the formation of a series of
natural products containing a-keto or a-hydroxy amide
linkages which are of special interest as protease
inhibitors.4–14 The synthetic targets included poststatin
and eurystatin, inhibitors of prolyl endopeptidases. The
methodology was also applied to the preparation of the
aminopeptidase inhibitors, phebestin, probestin and bestatin
by stereocontrolled reduction of initially formed a-keto
amides to the corresponding a-hydroxy amides.
In the course of an extensive investigation of the chemistry
of vicinal tricarbonyls, we prepared a series of peptidyl
tricarbonyl derivatives which showed notable activity as
serine protease inhibitors. These tricarbonyl aggregates
were formed from orthogonally protected peptidic car-
boxylic acids by coupling with benzyl(triphenylphosphor-
anylidene)acetate in the presence of EDCI. The peptidyl
keto ylide esters thus formed were subsequently oxidized by
Oxonew, ozone or DMD to the corresponding tricarbonyl
esters (Scheme 1).1
2. Results and discussion
2.1. Poststatin (6)
More recently, we have modified this carbonyl-insertion
protocol using (cyanomethylene)triphenylphosphorane (2)
in the formation of stable acyl cyanophosphorane inter-
mediates 3 from carboxylic acids 1. The products were then
converted by ozone at low temperature to labile a,b-diketo
nitriles. These powerful electrophiles undergo rapid reac-
tion with nucleophiles such as alcohols and amines to yield
a-keto esters (5a) or amides (5b), respectively (Scheme 2).2,3
Poststatin (6) is a naturally-occurring pentapeptide isolated
from Streptomyces viridochromogenes. The sequence is
H-Val-Val-Pos-D-Leu-Val-OH,15 where Pos is the unusual
(S)-3-amino-2-oxopentanoic acid named L-postine. The
a-keto amide group appears to be essential for the biological
Scheme 1.
Keywords: poststatin; a-keto amides; acyl cyanophosphorane.
*
Corresponding author. Tel.: þ1-2034323973; fax: þ1-2034329990; e-mail: harry.wasserman@yale.edu
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00860-3

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H. H. Wasserman et al. / Tetrahedron 59 (2003) 6771–6784
Scheme 2.
activity of this oligopeptide as is the case for a variety of
cyclic peptides, such as the cyclotheonamides,5a orbi-
culamides6 and eurystatins,9 among other related bioactive
products.
Two pathways were originally considered for assembling
poststatin (6) which can be considered to be a postine
residue substituted at both ends by a dipeptide fragment. In
one route, the N-terminal would be coupled to N-protected
valylvaline, followed by a second-stage C-terminal reaction
with O-protected D-leucylvaline. In a second approach, the
order of coupling would be reversed. Our choice of the first
route (Scheme 3) was based on preference for a pathway
which would postpone the ozone oxidation of the acyl
cyanophosphorane intermediate to the penultimate step,
thereby minimizing any possible epimerization of the
postine residue.
The synthesis started with the acyl cyanophosphorane 8,
formed (88%) by the EDCI coupling of commercially
available Cbz-protected (S)-(þ)-2-aminobutanoic acid 7
with (cyanomethylene)triphenylphosphorane (2). After
removal of the Cbz-protecting group by hydrogenolysis,18
the amine 9 was treated with Cbz-protected valylvaline 10
under standard peptide-coupling conditions yielding the
tripeptide 11 as the sole epimer. Ozonolytic cleavage of the
An earlier synthesis of poststatin followed an established
route15d,f,16 for the formation of enzyme inhibitors in this
family, involving formation of a-hydroxy amide precursors,
which could then be oxidized to the final ketone-derived
products. The present procedure which has general
applicability and requires fewer steps follows the plan
outlined in Scheme 3.17
Scheme 3.

H. H. Wasserman et al. / Tetrahedron 59 (2003) 6771–6784
6773
Scheme 4.
carbon–phosphorus double bond in 11 generated a labile
a,b-diketo nitrile intermediate which was allowed to react
in situ with D-leucylvaline O-benzyl ester 12 to form the
protected pentapeptide 13. Essentially no epimerization of
the postine unit was observed at this stage.
mide thrombin inhibitors and the PED inhibitor, poststatin,
they contain an a-keto amide residue which is thought to
play a significant role in the enzyme inhibition.5 A previous
synthesis of 14a featured the formation of an a-hydroxy
amide precursor which was oxidized to the a-keto grouping
at a late stage of the procedure.15a,b,e
Hydrogenolysis of the benzylic protecting groups of 13
yielded crude product 6. NMR examination suggested the
presence of 15–20% of a poststatin epimer. Purification by
reverse-phase chromatography yielded the desired product,
which was identical in all respects to an authentic sample of
poststatin (6).19
Two routes were investigated for the synthesis of the
eurystatins, both of which generated the tripeptide 22 as a
key intermediate.21 The first synthesis (Scheme 4)
began with Cbz-protected alanine 15, which was converted
to the acyl cyanophosphorane 16 with (cyano-
methylene)triphenylphosphorane (2). Ozonolysis of 16
generated the corresponding diketo nitrile 17, which was
reacted in situ with leucine tert-butyl ester 18 to give the
dipeptide 19. Removal of the Cbz-protecting group from 19
yielded the a-amino vicinal dicarbonyl product 20.
Coupling with the carboxyl group of di-N-protected
ornithine 21 yielded the carbonyl-extended tripeptide 22.
2.2. Eurystatin A (14a)
Eurystatins A and B (14a and b), inhibitors of prolyl
endopeptidase, isolated from Streptomyces eurythermus,
incorporate (S)-3-amino-2-oxobutanoic acid, leucine and
ornithine in a 13-membered ring.20 Like the cyclotheona-
Scheme 5.

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H. H. Wasserman et al. / Tetrahedron 59 (2003) 6771–6784
In the second route (Scheme 5), featuring an ylide-stabilized
a-amino ketone 25, the ozonolysis was carried out at a later
stage in the synthesis. The required Fmoc-protected acyl
cyanophosphorane 24 was prepared from Fmoc alanine (23)
by our general coupling procedure.
The Fmoc protecting group was eliminated smoothly with
piperidine, and the crude amine 25 was coupled with the
ornithine derivative 21 affording the cyanophosphorane 26.
Ozonolysis yielded 27, which was trapped with leucine tert-
butyl ester (18) to form the tripeptide 22.
Of the two procedures for the preparation of 22, the route
given in Scheme 5 is preferred, since as in the poststatin
synthesis,17 the deferral of the ozonolysis until a late stage
in the sequence provides better protection for the vicinal
dicarbonyl system and the chiral centers during the
deprotection steps.
These products have been the object of studies leading to the
development of several synthetic approaches, including
Sharpless’s asymmetric aminohydroxylation of a,b-
unsaturated amides, Ojima’s ring-opening of b-lactams
and the asymmetric catalytic reduction of a-keto carboxylic
acids.25 Our success in preparing a-keto amides from
carboxylic acids by the facile procedure outlined in
Scheme 2 prompted us to explore the diastereoselective
reduction of these keto derivatives as a simple, direct route
to the a-hydroxy amide system.
Simultaneous removal of the protecting groups at the
d-amino and the carboxyl termini of tripeptide 22 to form 28
was accomplished with TFA (Scheme 6). Cyclization of 28
to 29 was performed in DMF under conditions of high
dilution using diphenyl phosphorylazide (DPPA) and
sodium hydrogen carbonate. Following hydrogenolysis of
the cyclic tripeptide 29, the amine 30 was coupled with
(E)-6-methyl-2-hepteneoic acid 3122 to yield eurystatin A
(14a) identical to the natural material.23,24
2.4. Phebestin
In the synthesis of phebestin, outlined in Scheme 7, it was
found that the best results were obtained by carrying out the
reduction at an early stage of the assembly of the tripeptide
framework. Accordingly, the synthesis began with N-Boc-
D-phenylalanine (32) which was coupled with (cyano-
methylene)triphenylphosphorane (2) to form the acyl
cyanophosphorane 33. Ozonolysis of 33 was followed by
2.3. a-Keto amides in the synthesis of hydroxy peptides
We next directed our attention to a group of hydroxy
peptides containing a b-amino a-hydroxy amide residue
exemplified by the natural products phebestin, probestin and
bestatin.
Scheme 6.

H. H. Wasserman et al. / Tetrahedron 59 (2003) 6771–6784
6775
Scheme 7.
coupling with the benzyl ester of L-valine at low
temperature to yield the a-keto amide 34. At this early
point, we explored the use of reducing agents for the
diastereoselective conversion of 34 to 35.
phebestin.26 The selectivity in the reduction of 34 to 35 may
be explained on the basis of chelation control in which the
two carbonyl groups coordinate with the zinc ion permitting
hydride attack at the less hindered side of the a-carbonyl
group.
Among the reducing agents which were examined,25
including complexes of rhodium with chiral ligands,
Dip-Cl, DIBAL-H, zinc borohydride, L-Selectridew,
K-selectridew and (ButO)3BH–Li, it was found that zinc
borohydride resulted in the highest diastereomeric
selectivity (92:8) in the reduction of the keto substrate to
the alcohol. In practice, the borohydride (0.15 M) in ether
(2 equiv.) was added with stirring to the solution of the
a-keto amide 34 in THF at 2788C under N2. After
separation of the desired diastereomer 35 by preparative
TLC, the product was deprotected by hydrogenolysis and
then coupled with the benzyl ester of L-phenylalanine to
yield the tripeptide 36. Removal of protecting groups from
36, using hydrogenolysis and TFA sequentially, yielded a
product 37 (80%) identical in every respect with natural
2.5. Probestin and bestatin
In the synthesis of probestin, Scheme 8, the unusual
b-amino-a-hydroxy amide linkage was introduced at the
dipeptide stage for further coupling with a second dipeptide
unit. Thus, the doubly protected dipeptide 38, containing the
a-keto amide residue, was subjected to reduction by zinc
borohydride to give the a-hydroxy product 39 with
diastereomeric selectivity 93:7 (85%). Further trans-
formation of 39, by hydrogenolysis to remove the benzyl
group followed by coupling with the benzyl ester of
prolylproline, gave 40. This was followed by sequential
deprotection with hydrogenation and TFA to yield probestin
(41).
Scheme 8.

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H. H. Wasserman et al. / Tetrahedron 59 (2003) 6771–6784
In an accompanying study during the probestin synthesis,
the b-amino-a-hydroxy intermediate 39 was deprotected.
The product was shown to be identical in every respect to an
authentic sample (Sigma) of bestatin (42).
(15.000 g, 49.8 mmol) was added at once. The temperature
rose to 30–358C for about 10 min. Stirring was continued at
rt for 4 h. Water (40 mL) was added, and the organic layer
was washed consecutively with water (40 mL), sodium
hydrogen carbonate (aq) (sat., 40 mL), and water (40 mL),
and dried with magnesium sulfate (1 h). Filtration and
concentration yielded a yellow–brown oil (26 g) which was
purified by flash chromatography (1000 g, EtOAc/
hex¼50:50, 140 mL fractions, 38–49) to yield the desired
3. Experimental
3.1. General methods
product (8) as
a solid off-white foam (17.539 g,
33.69 mmol, 88%): mp 50–608C; TLC (EtOAc/
hex¼50:50): Rf 0.30; [a]2D0¼þ26.18 (c 1.23, CHCl3); IR:
All reactions were carried out in oven-dried glassware under
an atmosphere of N2. Solvents and reagent solutions were
transferred using gastight syringes. Tetrahydrofuran (THF),
benzene (PhH), and dichloromethane (CH2Cl2) were
distilled from calcium hydride (CaH2) under dinitrogen.
Other solvents (A.C.S. spectrophotometric grade) were
3390, 2990, 2160, 1705, 1575, 1495, 1430, 1100, 685 cm21
;
1H NMR (CDCl3): d 7.80–7.45 (m, 15, Ph3P), 7.33 (m, 5,
Ph), 5.57 (d, J¼7.14 Hz, 1, NH), 5.10 (s (br), 2, CH2O), 4.92
(m, 1, a-H), 2.08 (m, 1, b-H), 1.82 (m, 1, b-H), 0.93 (t,
J¼7.28 Hz, 3, CH3); 13C NMR (CDCl3): d 193.8 (d,
JC–P¼2.47 Hz), 155.5, 136.5, 133.2 (d, JC–P¼10.49 Hz, 6),
133.0 (d, JC–P¼2.33 Hz, 3), 128.9 (d, JC–P¼12.60 Hz, 6),
128.1 (2), 127.5 (2), 127.5, 122.4 (d, JC–P¼93.90 Hz, 3),
120.7 (d, JC–P¼15.15 Hz), 66.0, 57.1 (d, JC–P¼9.08 Hz),
47.2 (d, JC–P¼126.55 Hz), 26.3, 9.1; MS: m/z 521 (100,
MþH), 328 (90, Ph3PC(CN)(CO)); HRMS: m/z calcd for
C32H30N2O3P (MþH)þ 521.1994, found 521.1985.
˚
dried over 3-A molecular sieves, when necessary, and used
without further purification. Commercially available
reagents were purchased from Aldrich Chemical Company,
Inc., Wisconsin. Ozone was generated from dry dioxygen
using a Welsbach ozonator.
Flash chromatography was conducted on silica gel (40–
63 mm, Merck Silica Gel 60) by the method of Still et al.27
All chromatographic purification was monitored by thin-
layer chromatography (TLC) on 250 mm 60 F254 silica-gel
plates from Merck. The compounds were visualized under
ultraviolet light (UV), iodine (I2), and/or by heating with
phosphomolybdic acid stain (PMA): 5% PMA in ethanol.
Melting intervals were determined with a Thomas–Hoover
capillary melting point apparatus. Optical rotations ([a]2D0)
were determined on a Perkin–Elmer 241 polarimeter.
Infrared spectra (IR) were measured in chloroform
(CHCl3) using a NaCl cuvette on a Perkin–Elmer IR 1420
3.2.2. (1-{1-[3-Cyano-1-ethyl-2-oxo-3-(triphenyl-l5-
phosphanylidene)-propylcarbamoyl]-2-methylpropyl-
carbamoyl]-2-methylpropylcarbamoyl}-2-methylpro-
pyl)-carbamic acid benzyl ester (11). Sodium carbonate
(4.866 g, 46 mmol) was dissolved in water (28 mL), and
H-Val-Val-OH (4.964 g, 22.95 mmol) was added. The
resulting clear solution was cooled in an ice–water bath.
A solution of benzyl chloroformate (3.6 mL, 4.3 g,
25 mmol) in dioxane (3.6 mL) was added drop-wise. After
18 h stirring at rt, the reaction mixture was extracted with
CH2Cl2 (2£80 mL). The clear aqueous phase was acidified
by the portion-wise addition of sodium hydrogen sulfate
(11.4 g, 95.0 mmol). The precipitated product was extracted
with EtOAc (4£100 mL). The combined extracts were
washed with water (2£40 mL) and dried (MgSO4, 1 h).
Filtration and concentration afforded a white solid (6.862 g,
19.58 mmol, 85%). The product (10) was recrystallized
from EtOAc/hex¼50:50 (20 mL/g): mp 134.5–137.08C;
TLC (EtOAc/hex/AcOH¼40:59:1): Rf 0.30; 1H NMR
(CDCl3): d 12.6 (s (br), 1, OH), 7.34 (m, 5, Ph), 6.64 (d,
J¼8.58 Hz, 1, NH), 5.56 (d, J¼8.53 Hz, 1, NH), 5.11 (s, 2,
CH2O), 4.56 (dd, J¼8.28 Hz, 4.80, 1, a-H), 4.07 (t,
J¼7.87 Hz, 1, a-H), 2.23 (m, 1, b-H), 2.08 (m, 1, b-H),
1.03–0.88 (m, 12, 4£CH3). Cyanophosphorane 8 (17.233 g,
33.10 mmol) was dissolved in EtOAc (400 mL), and Pd/C
(10% Pd, 34 g, 200% (w/w)) was added. The reaction
mixture was stirred vigorously for 5 h at rt under an
atmosphere of H2, followed by filtration through Celite filter
agent. Concentration furnished a pale-yellow crude product
as a 1:1:2 mixture of unconverted starting material, a
cyclized product and (9), which was used without further
purification. A sample (1.5 g) was chromatographed (250 g,
CH2Cl2/MeOH/Et3N¼95.3:3.7:1, 35 mL fractions, 23–27)
to yield the pure product: TLC (CH2Cl2/MeOH/
Et3N¼95.3:3.7:1): Rf 0.30; [a]2D0¼þ14.55 (c 1.01,
CHCl3); IR: 2990, 2950, 2155, 1575, 1475, 1425, 1210,
1
spectrophotometer. H NMR data were obtained using a
Bruker AM-500 spectrometer (500 MHz) with tetramethyl-
silane (TMS, d;0) as internal standard, or (methyl
sulfoxide)-d6 (DMSO-d6, d¼2.49 ppm for residual 1H).
Proton-decoupled 13C NMR spectra were recorded in
deuteriochloroform (CDCl3, d¼77.0 ppm), deuterio-
methylene chloride (CD2Cl2, d¼53.8 ppm), or (methyl
sulfoxide)-d6 (DMSO-d6, d¼39.5 ppm) on the same instru-
ment at 125 MHz. Data are reported as follows: chemical
shift in parts per million downfield from TMS (d),
multiplicity (s¼singlet, d¼doublet, t¼triplet, q¼quartet,
quint¼quintet, m¼multiplet, (br¼broad)), coupling con-
stants (J or kJl for an average value) in Hz, integration, and
assignments. Low-resolution mass spectra (MS) and high-
resolution mass spectra (HRMS) were done at the Mass
Spectrometry Facility at the Yale Medical School, Yale
University. Exact molecular masses are given for the
1
isotopes H, 12C, 14N, 16O, 19F, 31P. Elemental analyses
(EA) were obtained from Atlantic Microlab, Inc., Georgia.
3.2. Experimental procedures used in the synthesis of
poststatin
3.2.1. [(3-Cyano-1-ethyl-2-oxo-3-(triphenyl-l5-phos-
phanylidene)-propyl]-carbamic acid benzyl ester (8).
Cbz-Abu-OH (7) (9.071 g, 38.23 mmol) was dissolved in
CH2Cl2 (212 mL), and DMAP (0.461 g, 3.8 mmol) was
added followed by EDCI (9.529 g, 49.7 mmol). Within a
1
1100, 670 cm21; H NMR (CDCl3): d 7.80–7.47 (m, 15,
Ph3P), 4.00 (dd, J¼7.17, 5.33 Hz, 1, a-H), 1.89 (m, 1, b-H),
minute,
(cyanomethylene)triphenylphosphorane
(2)

H. H. Wasserman et al. / Tetrahedron 59 (2003) 6771–6784
6777
1.61 (m, 1, b-H), 1.45 (s (br), 2, NH2), 1.00 (t, J¼7.42 Hz, 3,
CH3); 13C NMR (CDCl3):
199.1, 133.4 (d,
11.99 mmol), H-Val-OBn·TsOH (5.000 g, 13.18 mmol) and
HOBt (1.620 g, 11.99 mmol) were suspended in CH2Cl2
(60 mL) and cooled in an ice–water bath. Triethylamine
(2.00 mL, 1.45 g, 14.3 mmol) was added followed by EDCI
(3.221 g, 16.8 mmol). Stirring was continued for 2 h at 08C,
followed by 4 h at rt. Citric acid (aq) (1%, 100 mL) was
added, and the organic phase was washed consecutively
with water (100 mL), sodium hydrogen carbonate (aq) (sat.,
100 mL) and water (50 mL). The CH2Cl2 phase was dried
(MgSO4, 1 h), filtered, and concentrated to afford a clear
colorless oil (5.088 g) which was purified by flash
chromatography (250 g, EtOAc/hex¼15:85, 70 mL frac-
tions, 12–26). The resulting colorless oil (4.703 g,
11.18 mmol, 93%) solidified on standing: mp 81.5–
83.08C; TLC (EtOAc/hex¼15:85): Rf 0.25; 1H NMR
(CDCl3): d 7.36 (m, 5, Ph), 6.69 (s (br), 1, NH), 5.20 (d
(AB), J¼12.25 Hz, 1, CH2O), 5.12 (d (AB), J¼12.25 Hz, 1,
CH2O), 4.80 (s (br), 1, NH), 4.57 (dd, J¼8.70, 4.58 Hz, 1,
a-H), 4.15 (s (br), 1, a-H), 2.20 (m, 1, CH-Val), 1.69 (m, 3,
CH2CH-D-Leu), 1.44 (s, 9, But), 0.93 (m, 6, 2£CH3-Val),
0.92 (d, J¼6.92 Hz, 3, CH3-D-Leu), 0.85 (d, J¼6.92 Hz, 3,
CH3-D-Leu). Boc-D-Leu-Val-OBn (4.329 g, 10.29 mmol)
was dissolved in trifluoroacetic acid (47 mL) and stirred at rt
for 45 min. Concentration furnished a colorless oil which
was stripped with benzene (3£50 mL) to yield [(1R)-1-
[(((1S)-1-[(benzyloxy)carbonyl]-2-methylpropyl)amino)-
carbonyl]-3-methylbutyl]ammonium trifluoroacetate as a
white solid (4.641 g, 10.68 mmol, .100%): mp 95–968C;
TLC (EtOAc/hex¼75:25): Rf 0.10–0.20; [a]2D0¼225.74 (c
1.01, CHCl3); IR: 2950, 1775, 1725, 1665, 1535,
d
JC–P¼10.49 Hz, 6), 133.1 (d, JC–P¼2.54 Hz, 3), 129.1 (d,
JC–P¼12.65 Hz, 6), 123.0 (d, JC–P¼94.27 Hz, 3), 121.8 (d,
JC–P¼16.12 Hz), 57.8 (d, JC–P¼7.37 Hz), 46.9 (d,
JC–P¼125.04 Hz), 28.8, 10.1; MS: m/z 387 (100, MþH),
328 (20, Ph3PC(CN)(CO)); HRMS: m/z calcd for
C24H24N2OP (MþH)þ 387.1626, found 387.1625. Fractions
30–38 from the above flash chromatography were concen-
trated to yield a white solid foam of (2S)-2-ethyl-5-imino-4-
(triphenylphosphoranylidene)pyrrolidin-3-one, 9a:18 mp
70–808C; TLC (CH2Cl2/MeOH/Et3N¼95.3:3.7:1): Rf
0.10; [a]2D0¼25.54 (c 1.12, CHCl3); IR: 3470, 2950,
1
1655, 1620, 1560, 1505, 1430, 1100, 670 cm21; H NMR
(CDCl3): d 9.16 (s (br), 1, NH), 8.99 (s (br), 1, NH), 7.84–
7.62 (m, 15, Ph3P), 3.96 (dd, J¼6.28, 5.01 Hz, 1, a-H), 1.94
(m, 1, b-H), 1.77 (m, 1, b-H), 1.05 (t, J¼7.41 Hz, 3, CH3);
13C NMR (CDCl3): d 197.0 (d, JC–P¼5.32 Hz), 169.3 (d,
JC–P¼17.85 Hz), 134.4 (d, JC–P¼3.07 Hz, 3), 133.5 (d,
JC–P¼10.86 Hz, 6), 129.9 (d, JC–P¼12.80 Hz, 6), 120.0 (d,
JC–P¼92.05 Hz, 3), 63.7 (d, JC–P¼124.09 Hz), 63.5 (d,
JC–P¼9.06 Hz), 24.5, 9.1; MS: m/z 387 (100, MþH);
HRMS: m/z calcd for C24H24N2OP (MþH)þ 387.1626,
found 387.1627. Cbz-Val-Val-OH (10) (6.456 g,
18.42 mmol) was dissolved in EtOAc (100 mL), and
HOBt (4.980 g, 36.85 mmol) was added. To the resulting
suspension, triethylamine (5.1 mL, 3.7 g, 36.6 mmol) was
added, yielding a clear solution. The amino cyano-
phosphorane 9 (50%, 15.74 g, 20.4 mmol) was added as a
solution in EtOAc (10 mL) and CH2Cl2 (2 mL). The
mixture was cooled in an ice–water bath and EDCI
(4.945 g, 25.80 mmol) was added. The stirring was
continued at 08C for 90 min, followed by 40 min at rt.
Water (50 mL) was added. The organic layer was washed
consecutively with sodium hydrogen carbonate (aq) (sat.,
50 mL) and water (50 mL). The organic phase was dried
(MgSO4, 1 h) and filtered. Concentration yielded a semi-
solid material. Purification by flash chromatography (75 g,
EtOAc/hex¼75:25, 25 mL fractions, 8–33) furnished a
white solid which was recrystallized from EtOAc (375 mL)
to yield the pure product (11) (9.122 g, 12.69 mmol, 69%):
mp 202–2038C; TLC (EtOAc/hex¼75:25): Rf 0.40;
[a]2D0¼218.12 (c 1.08, CHCl3); IR: 3390, 2990, 2950,
1
1170 cm21; H NMR (CDCl3): d 8.00 (s (br), 3, NH3),
7.63 (d, J¼8.47 Hz, 1, NH-Val), 7.33 (m, 5, Ph), 5.18 (d
(AB), J¼12.12 Hz, 1, CH2O), 5.06 (d (AB), J¼12.12 Hz, 1,
CH2O), 4.53 (dd, J¼8.56, 4.98 Hz, 1, a-H-Val), 4.30 (t (br),
kJl¼7.1 Hz, 1, a-H-D-Leu), 2.19 (m, 1, CH-Val), 1.72 (m, 3,
CH2CH-D-Leu), 0.97 (d, J¼5.89 Hz, 3, CH3-Val), 0.93 (d,
J¼5.75 Hz, 3, CH3-Val), 0.88 (d, J¼6.86 Hz, 3, CH3-D-
Leu), 0.83 (d, J¼6.88 Hz, 3, CH3-D-Leu); 13C NMR
(CDCl3): d 171.7, 170.0, 161.3 (d, JC – F¼39.35 Hz),
134.8, 128.6 (2), 128.4 (2), 128.3, 115.4 (d,
JC–F¼288.95 Hz), 67.6, 57.9, 52.5, 40.7, 30.8, 24.5, 22.1,
21.7, 18.9, 17.4; MS: m/z 321 (100, MþH); HRMS: m/z
calcd for C18H29N2O3 (MþH)þ 321.2178, found 321.2177.
A solution of H-D -Leu-Val-OBn·TFA (4.585 g,
10.55 mmol) in CH2Cl2 (250 mL) was treated with sodium
hydrogen carbonate (aq) (sat., 2£100 mL) followed by
washing with water (2£100 mL). The organic phase was
dried (MgSO4, 1 h), filtered and concentrated to yield a
colorless oil (12) (2.883 g, 9.00 mmol, 85%): TLC (EtOAc/
hex¼75:25): Rf 0.15; [a]2D0¼þ5.12 (c 1.25, CHCl3); IR:
3340, 2945, 1730, 1650, 1505, 1185, 1145 cm21; 1H NMR
(CDCl3): d 7.73 (d, J¼8.97 Hz, 1, NH-Val), 7.35 (m, 5, Ph),
5.21 (d (AB), J¼12.26 Hz, 1, CH2O), 5.12 (d (AB),
J¼12.26 Hz, 1, CH2O), 4.56 (dd, J¼9.13, 4.81 Hz, 1,
a-H-Val), 3.42 (dd (br), kJl¼10.0, 3.2 Hz, 1, a-H-D-Leu),
2.23 (m, 1, CH-Val), 1.71 (m, 2, CHCH-D-Leu), 1.44 (s (br),
2, NH2), 1.35 (m, 1, CH-D-Leu), 0.95 (d, J¼6.30 Hz, 3,
CH3), 0.93 (d, J¼6.86 Hz, 3, CH3), 0.92 (d, J¼6.15 Hz, 3,
CH3), 0.88 (d, J¼6.89 Hz, 3, CH3-D-Leu); 13C NMR
(CDCl3): d 175.5, 171.7, 135.3, 128.4 (2), 128.2 (2),
128.1, 66.7, 56.6, 53.3, 43.7, 30.9, 24.6, 23.2, 21.1, 19.0,
17.5; MS: m/z 321 (100, MþH); HRMS: m/z calcd for
C18H29N2O3 (MþH)þ 321.2178, found 321.2180.
1
2170, 1705, 1650, 1575, 1490, 1425, 1210, 685 cm21; H
NMR (CDCl3): d 7.76–7.47 (m, 15, Ph3P), 7.32 (m, 5, Ph),
6.58 (d, J¼7.19 Hz, 1, NH-Val), 6.55 (d, J¼8.54 Hz, 1, NH-
Abu), 5.51 (d, J¼8.28 Hz, 1, NH-Val), 5.13 (q (br),
kJl¼7.5 Hz, 1, a-H-Abu), 5.09 (d (AB), J¼12.50 Hz, 1,
CH2O), 5.06 (d (AB), J¼12.50 Hz, 1, CH2O), 4.25 (t (br),
kJl¼7.6 Hz, 1, a-H-Val), 4.03 (t (br), kJl¼7.4 Hz, 1, a-H-
Val), 2.05 (m, 3, 2£b-H-Val, b-H-Abu), 1.77 (m, 1, b-H-
Abu), 0.98–0.81 (m, 15, 5£CH3); 13C NMR (CDCl3): d
193.7 (d, JC–P¼2.52 Hz), 171.1, 170.0, 156.3, 136.4, 133.4
(d, JC–P¼10.70 Hz, 6), 133.2 (d, JC–P¼2.39 Hz, 3), 129.1
(d, JC–P¼12.64 Hz, 6), 128.3 (2), 127.8 (2), 127.8, 122.6 (d,
JC–P¼94.25 Hz, 3), 120.7 (d, JC–P¼14.67 Hz), 66.6, 60.3,
58.3, 55.8 (d, JC–P¼9.00 Hz), 47.7 (d, JC–P¼125.68 Hz),
31.3, 31.2, 26.2, 19.0, 18.9, 18.2, 17.8, 9.4; MS: m/z 719 (100,
MþH), 328 (100, Ph3PC(CN)(CO)); HRMS: m/z calcd for
C42H48N4O5P (MþH)þ 719.3362, found 719.3356.
3.2.3. 2-(2-Amino-4-methyl-pentanoylamino)-3-methyl-
butyric acid benzyl ester (12). Boc-D-Leu-OH (2.774 g,

6778
H. H. Wasserman et al. / Tetrahedron 59 (2003) 6771–6784
3.2.4. 2-(2-{3-[2-(2-Benzyloxycarbonylamino-3-methyl-
butyrylamino)-3-methyl-butyrylamino]-2-oxo-penta-
noylamino}-4-methylpentanoylamino)-3-methylbutyric
acid benzyl ester (13). The cyanophosphorane 11 (3.942 g,
5.48 mmol) was ozonized in CH2Cl2 (110 mL) at 2788C for
34 min. The blue–green reaction mixture was purged with
O2 and N2 for 8 and 10 min, respectively. To the resulting
yellow mixture was added drop-wise a solution of H-D-Leu-
Val-OBn (12) (2.636 g, 8.23 mmol) in CH2Cl2 (10 mL).
The mixture was stirred for 1 h before the cooling was
discontinued. Concentration afforded a yellow oil which
was subjected to flash chromatography (250 gþ35 g for pre-
adsorption, EtOAc/hex¼40:60, 70 mL fractions, 16–25) to
yield an off-white solid (1.330 g, 1.74 mmol, 32%). An
aliquot (0.4 g) was re-chromatographed (20 g, CH2Cl2/
MeOH¼96:4, 4.5 mL fractions, 9–17) yielding 13 as a
white solid (0.3 g) (,5% of the Pos epimer): mp 196–
2038C; TLC (EtOAc/hex¼50:50): Rf 0.40; [a]2D0¼þ12.00
3.3. Experimental procedures used in the synthesis of
eurystatin A
3.3.1. {3-Cyano-3-[diphenyl-(1-propenyl-buta-1,3-di-
enyl-l5-phosphanylidene]-1-methyl-2-oxopropyl}-car-
bamic acid benzyl ester (16). Cbz-ala-OH (15) (10.8120 g,
48.43 mmol) was dissolved in CH2Cl2 (270 mL) and DMAP
(0.5916 g, 4.84 mmol) was added followed by EDCI
(12.0710 g, 62.96 mmol). Within a minute, (cyanomethyl-
ene)triphenylphosphorane (2) (15.3244 g, 50.86 mmol) was
added at once. The temperature rose to 30–358C for about
10 min. Stirring was continued at rt for 4 h. Water (15 mL)
was added, and the organic layer was washed consecutively
with water (15 mL), sodium hydrogen carbonate (aq) (sat.,
15 mL), water (15 mL), and dried (MgSO4, 1 h). Filtration
and concentration yielded a yellow–brown oil (30.9150 g)
which was purified by flash chromatography (1300 g,
EtOAc/hex¼50:50, 70 mL fractions, 58–90) to yield the
desired product (16) as a white solid foam (20.8615 g,
41.18 mmol, 85%): mp 71–758C; TLC (EtOAc/
hex¼50:50): Rf 0.33; [a]2D0¼þ18.96 (c 1.00, CHCl3); IR:
3390, 3000, 2170, 1710, 1585, 1495, 1435, 1105 cm21; 1H
NMR (CDCl3): d 7.82–7.45 (m, 15, Ph3P), 7.33 (m, 5, Ph),
5.67 (d, J¼6.14 Hz, 1, NH), 5.11 (d (AB), J¼13.65 Hz, 1,
CH2O), 5.08 (d (AB), J¼13.65 Hz, 1, CH2O), 4.94 (quint,
kJl¼6.7 Hz, 1, a-H), 1.52 (d, J¼6.68 Hz, 3, CH3); 13C NMR
(CDCl3): d 194.6 (d, JC–P¼3.59 Hz), 155.3, 136.8, 133.4 (d,
JC–P¼10.73 Hz, 6), 133.2 (3), 129.1 (d, JC–P¼13.09 Hz, 6),
128.3 (2), 127.7 (3), 122.5 (d, JC–P¼93.63 Hz, 3), 120.7 (d,
JC–P¼14.91 Hz), 66.2, 52.4 (d, JC–P¼8.99 Hz), 46.6 (d,
JC–P¼125.91 Hz), 19.6; MS: m/z 507 (85, MþH), 328 (100,
Ph3PC(CN)(CO)); HRMS: m/z calcd for C31H28N2O3P
(MþH)þ 507.1837, found 507.1834.
1
(c 1.03, CHCl3); H NMR (CDCl3): d 7.47 (d, J¼8.48 Hz,
1, NH), 7.34 (m, 10, 2£Ph), 7.07–6.57 (m, 3, 3£NH),
5.62 (d, J¼6.02 Hz, 1, NH), 5.30 (m, 1, NH), 5.16 (d
(AB), J¼12.25 Hz, 1, CH2O), 5.09 (m, 2, CH2O), 5.08
(d (AB), J¼12.25 Hz, 1, CH2O), 4.64–4.51 (m, 2, a-H),
4.32 (m, 1, a-H), 4.08 (m, 1, a-H), 2.30–1.50 (m, 8, 7£b-H,
g-H-D-Leu), 1.08–0.77 (m, 27, 9£CH3); 13C NMR
(CDCl3): d 196.1, 171.9, 171.8, 171.6, 171.2, 159.3,
156.5, 136.5, 135.2, 128.5, 128.4 (2), 128.3 (3), 127.9 (2),
127.8 (2), 67.0, 66.8, 60.5, 58.5, 57.3, 55.2, 52.0, 41.0,
31.2, 31.1, 31.0, 24.7, 24.7, 22.8, 22.1, 19.1, 19.0, 18.3,
18.2, 17.9, 17.6, 9.6; MS: m/z 766 (100, MþH); HRMS:
m/z calcd for C41H60N5O9 (MþH)þ 766.4391, found
766.4394.
3.2.5. 2-(2-{3-[2-(2-Amino-3-methylbutyrylamino)-3-
methylbutyryl)amino]-2-oxo-pentanoylamino}-4-
methylpentanoylamino]-3-methylbutyric acid (6). To a
solution of 13 (0.275 g, 0.36 mmol) in CH2Cl2/
MeOH¼95:5 (25 mL) was added 10% Pd/C (0.14 g). The
mixture was purged with N2, before it was stirred vigorously
for 24 h at rt under an atmosphere of H2. The reaction
mixture was filtered through Celite filter agent, which was
rinsed with additional MeOH. Concentration yielded an off-
white solid (0.167 g). Flash chromatography (5 g (C-18
SiO2), water/MeOH¼40:60, 1 mL fractions, 7–18) yielded
synthetic poststatin (6) (15–20% of the Pos epimer) as
an off-white solid (0.142 g, 0.26 mmol, 73%): mp
161–1658C; TLC (C-18 SiO2) (water/MeOH¼40:60):
Rf 0.25; [a]2D0¼þ13.41 (c 0.88, AcOH); IR: 3210, 2950,
3.3.2. 2-(3-Benzyloxycarbonylamino-2-oxobutyryl-
amino)-4-methylpentanoic acid tert-butyl ester (19).
The cyanophosphorane 16 (15.1871 g, 29.98 mmol) was
ozonized in CH2Cl2 (600 mL) at 2788C for 36 min (8 min
at 2.02 mmol scale). The blue–green reaction mixture was
purged with O2 and N2 for 4 and 8 min, respectively. To the
resulting yellow solution of a,b-diketo nitrile 17 was added
a solution of H-Leu-OBut (18) (5.3343 g, 28.48 mmol) in
CH2Cl2 (36 mL). The mixture was stirred for 1 h before the
cooling was discontinued. Concentration afforded a deep
yellow oil which was stirred at rt for 24 h with a solution of
silver nitrate in THF/water¼4:1 (1 M, 300 mL, 300 mmol).
Water (300 mL) was added to the dark greenish slurry, and
the THF layer was washed with water (2£50 mL). The
aqueous phases were extracted with CH2Cl2 (3£100 mL).
The combined organic phases were dried (MgSO4, 1 h),
filtered, and concentrated to afford a clear yellow oil which
was subjected immediately to flash chromatography (750 g,
EtOAc/hex¼25:75, 100 mL fractions, 17–50) to yield the
desired product (19) (,5% of the Ala epimer) as a yellow
oil (7.9877 g, 19.00 mmol, 67%): TLC (EtOAc/
hex¼25:75): Rf 0.30; [a]2D0¼þ11.49 (c 0.89, CHCl3); IR:
3400, 2950, 1715, 1685, 1500, 1365, 1220, 1145 cm21; 1H
NMR (CDCl3): d 7.36 (m, 5, Ph), 7.25 (d, J¼8–9 Hz, 1,
NH), 5.44 (d, J¼7.37 Hz, 1, NH), 5.20 (quint, kJl¼7.4 Hz,
1, a-H-Ala), 5.13 (d (AB), J¼11.57 Hz, 1, CH2O), 5.09 (d
(AB), J¼11.57 Hz, 1, CH2O), 4.48 (dd, J¼8.37 Hz, 5.49, 1,
a-H-Leu), 1.72–1.53 (m, 3, CH2CH-Leu), 1.48 (s, 9, But),
1.47 (d, J¼7.4 Hz, 3, CH3-Ala), 0.96 (d, J¼6.27 Hz, 3,
1
1660, 1535 cm21; H NMR (major isomer) (DMSO-d6):
d 8.55 (d, J¼8.73 Hz, 1, NH), 8.38 (d, J¼6.66 Hz, 1, NH),
8.12 (d, J¼8.80 Hz, 1, NH), 7.97 (d, J¼8.66 Hz, 1, NH),
4.95 (m, 1, a-H), 4.42 (dt, J¼9.34 Hz, 4.77, 1, a-H), 4.28
(t (br), J¼7.45 Hz, 1, a-H), 4.05 (dd, J¼8.35, 5.44 Hz, 1,
a-H), 3.54 (s (br), 3, NH3), 3.22 (d, J¼4.56 Hz, 1, a-H),
2.08–1.92 (m, 3, 3£b-H-Val), 1.75 (m, 1, b-H-Abu),
1.62 (m, 1, g-H-D-Leu), 1.58–1.43 (m, 3, b-H-D-Leu, g-H-
D-Leu, b-H-Abu), 1.00–0.71 (m, 27, 9£CH3); 13C
NMR (major isomer) (DMSO-d6): d 196.2, 173.0, 171.0,
171.0, 171.0, 160.5, 58.2, 57.5, 57.1, 54.8, 51.5, 40.8,
31.0, 30.8, 30.2, 24.3, 23.0, 22.7, 21.4, 19.2, 19.2, 19.2,
18.0, 17.9, 17.2, 10.4; MS: m/z 542 (100, MþH); HRMS:
m/z calcd for C26H48N5O7 (MþH)þ 542.3554, found
542.3549.

H. H. Wasserman et al. / Tetrahedron 59 (2003) 6771–6784
6779
CH3-Leu), 0.96 (d, J¼6.17 Hz, 3, CH3-Leu); 13C NMR
(CDCl3): d 196.1, 170.7, 158.3, 155.4, 136.2, 128.4 (2),
128.1 (2), 128.0, 82.4, 66.9, 51.9, 51.4, 41.6, 27.9 (3), 24.9,
22.6, 22.0, 17.9; MS: m/z 421 (30, MþH), 321 (100, MþH–
CH2CMe2–CO2); HRMS: m/z calcd for C22H33N2O6
(MþH)þ 421.2338, found 421.2339.
washed consecutively with water (15 mL), sodium hydro-
gen carbonate (aq) (sat., 15 mL), water (15 mL), and
(MgSO4, 1 h). Filtration and concentration yielded a
yellow–brown oil which was purified by flash chromato-
graphy (300 g, EtOAc/hex¼50:50, 70 mL fractions, 26–62)
to yield the desired product (24) as a white solid foam
(4.8394 g, 8.14 mmol, 54%): mp 100–1058C; TLC (EtOAc/
hex¼50:50): Rf 0.30; [a]2D0¼þ19.82 (c 1.13, CHCl3); IR:
3390, 3005, 2170, 1715, 1585, 1495, 1435, 1105 cm21; 1H
NMR (CDCl3): d 7.76 (d, J¼7.50 Hz, 2, Ar-Fmoc), 7.72–
7.45 (m, 17, Ph3P, Ar-Fmoc), 7.39 (t, J¼7.42 Hz, 2, Ar-
Fmoc), 7.29 (t, J¼7.43 Hz, 2, Ar-Fmoc), 5.78 (d,
J¼7.04 Hz, 1, NH), 4.98 (quint, kJl¼6.9 Hz, 1, a-H), 4.35
(quint, kJl¼6.7 Hz, 2, CH2O), 4.21 (t, kJl¼7.2 Hz, 1, CH-
Fmoc), 1.57 (d, J¼6.76 Hz, 3, CH3); 13C NMR (CDCl3): d
194.6, 155.3, 144.1, 143.9, 141.2 (2), 133.4 (d,
JC–P¼10.45 Hz, 6), 133.3 (d, JC–P¼2 Hz, 3), 129.2 (d,
JC–P¼12.72 Hz, 6), 127.5 (2), 126.9 (2), 125.2 (2), 122.5 (d,
JC–P¼93.76 Hz, 3), 120.7 (d, JC–P¼15.21 Hz), 119.8 (2),
66.5, 52.4 (d, JC–P¼9.21 Hz), 47.1, 46.7 (d,
JC–P¼125.56 Hz), 19.7; MS: m/z 595 (65, MþH), 328
(100, Ph3PC(CN)(CO)); HRMS: m/z calcd for
C38H32N2O3P (MþH)þ 595.2150, found 595.2146.
3.3.3. 2-[3-(2-Benzyloxycarbonylamino-5-tert-butoxy-
carbonylaminopentanoylamino)-2-oxo-butyrylamino]-
4-methylpentanoic acid tert-butyl ester (22). A solution of
a-keto amide 19 (0.1953 g, 0.46 mmol) in CH2Cl2 (10 mL)
was stirred vigorously with 10% Pd/C (98 mg) under an
atmosphere of H2. The stirring was continued for 40 min at
rt. A sample was filtered through Celite filter agent.
Concentration furnished the crude (20) as a yellow oil:
TLC (EtOAc/hex¼75:25): Rf 0.05; [a]2D0¼þ4.17 (c 1.32,
CH2Cl2); IR: 3370, 2950, 1718, 1665, 1510, 1365, 1240,
1145 cm21
;
1H NMR (CDCl3): complex; 13C NMR
(CDCl3): complex; MS: m/z 289 (100, Mþ3H); HRMS:
m/z calcd for C14H29N2O4 (Mþ3H)þ 289.2127, found
289.2125 (1 ppm). The above reaction mixture was cooled
in an ice–water bath and briefly purged with N2. HOBt
(0.1255 g, 0.93 mmol), Cbz-Orn(Boc)-OH (21) (0.1872 g,
0.51 mmol), and EDCI (0.1247 g, 0.65 mmol) were added
as solids in the order listed, and stirring was continued for
2.5 h at rt. The reaction mixture was filtered through Celite
before citric acid (aq) (1%, 10 mL) was added. The organic
layer was washed with water (2£10 mL), dried (MgSO4,
0.5 h), filtered, and concentrated to furnish a pale yellow oil.
The crude product was dissolved in CH2Cl2 (30 mL) and
concentrated with SiO2 (3 g). Flash chromatography (20 g,
EtOAc/hex¼50:50, 5 mL fractions, 15–30) yielded the
desired product (22) as a white solid foam (0.1791 g,
0.28 mmol, 61%): mp 58–628C; TLC (EtOAc/hex¼50:50):
Rf 0.40; [a]2D0¼21.65 (c 0.97, CHCl3); IR: 3420, 2980,
1740, 1720, 1690, 1515, 1380, 1235, 1165 cm21; 1H NMR
(CDCl3): d 7.33 (m, 5, Ph), 7.30 (d, J¼8–9 Hz, 1, NH-Leu),
7.01 (s (br), 1, NH-Ala), 5.64 (d, J¼8.12 Hz, 1, NH-Orn),
5.27 (quint, kJl¼7.1 Hz, 1, a-H-Ala), 5.09 (s (br), 2, CH2O),
4.85 (s (br), 1, NH-d-Orn), 4.45 (dt, J¼5.48 Hz, 8.43, 1,
a-H-Leu), 4.37 (s (br), 1, a-H-Orn), 3.27 (m, 1, CH2N), 3.08
(m, 1, CH2N), 1.86 (m, 1, b-H-Orn), 1.71–1.51 (m, 6,
CH2CH-Leu, CH2CH-Orn), 1.46 (s, 9, Boc), 1.43 (d, J¼6–
7 Hz, 3, CH3-Ala), 1.42 (s, 9, But), 0.95 (d, J¼6.10 Hz, 3,
CH3-Leu), 0.94 (d, J¼6.06 Hz, 3, CH3-Leu); 13C NMR
(CDCl3): d 195.7, 171.5, 170.9, 158.7, 156.4, 156.3, 136.2,
128.4 (2), 128.0, 127.9 (2), 82.3, 79.0, 66.8, 53.5, 51.3, 50.3,
41.4, 39.4, 30.2, 28.3 (3), 27.9 (3), 26.0, 24.9, 22.6, 22.0,
16.6; MS: m/z 635 (25, MþH), 435 (70, MþH–CH2CMe2–
CO2), 479 (100, MþH22£CH2CMe2–CO2); HRMS: m/z
calcd for C32H51N4O9 (MþH)þ 635.3656, found 635.3674.
The reaction was repeated on a 7.07 g (16.8 mmol) scale
yielding 43% of the purified tripeptide 22.
3.3.5. [4-tert-Butoxycarbonylamino-1-(1-{3-cyano-1-
methyl-3-[(1-methylene-hexa-2,4-dienyl)-(1-methyl-
hexa-1,3,5-trienyl)-phenyl-l5-phosphanylidene]-2-oxo-
propylamino}-ethyl)-butyl]-carbamic acid benzyl ester
(26). Cyanophosphorane 24 (2.0828 g, 3.50 mmol) was
stirred in piperidine (35 mL, 30.14 g, 354 mmol) for 15 min
at rt. Concentration afforded a white solid. The crude
material was concentrated twice from CH2Cl2 (35 mL) and
triethylamine (2 mL) to afford the piperidine-free 4-amino-
2-[(1-methylene-hexa-2,4-dienyl)-(1-methyl-hexa-1,3,5-tri-
enyl)-phenyl-l5-phosphanylidene]-3-oxo-pentanenitrile
(25): TLC (EtOAc/hex¼75:25): Rf 0.05; IR: 2950, 2870,
1
2110, 1555, 1410, 1080 cm21; H NMR (CDCl3): d 7.87–
7.40 (m, 15, Ph3P), 4.19 (q, J¼6.85 Hz, 1, a-H), 2.06 (s (br),
2, NH2), 1.41 (d, J¼6.87 Hz, 3, CH3); 13C NMR (CDCl3): d
199.7, 133.5 (d, JC–P¼9.48 Hz, 6), 133.2 (d, JC–P¼2 Hz, 3),
129.2 (d, JC–P¼12.59 Hz, 6), 123.1 (d, JC–P¼93.84 Hz, 3),
121.8 (d, JC–P¼16.08 Hz), 52.5 (d, JC–P¼8.18 Hz), 45.9 (d,
JC–P¼125.66 Hz), 22.0; MS: m/z 373 (70, MþH), 328 (30,
Ph3PC(CN)(CO)); HRMS: m/z calcd for C23H22N2OP
(MþH)þ 373.1470, found 373.1481 (3 ppm). Amine 25
was dissolved in CH2Cl2 (35 mL) along with HOBt
(0.9466 g, 7.01 mmol). Cbz-Orn(Boc)-OH (21) (1.4117 g,
3.85 mmol) and EDCI (0.9400 g, 4.90 mmol) were added.
The stirring was continued for 20 h at rt. The reaction
mixture was washed consecutively with citric acid (aq) (1%,
15 mL), water (15 mL), sodium hydrogen carbonate (aq)
(sat., 15 mL), and water (15 mL). The combined organic
phases were dried (MgSO4, 1 h), filtered, and concentrated
to yield a red–brown oil. The product (26) was isolated by
flash chromatography (60 g, EtOAc/hex¼75:25, 25 mL
fractions, 15–31) as an off-white solid foam (1.4045 g,
1.95 mmol, 56%): mp 91–978C; TLC (EtOAc/hex¼75:25):
Rf 0.25; [a]2D0¼þ2.27 (c 1.06, CHCl3); IR: 3400, 2995,
2165, 1700, 1665, 1580, 1495, 1430, 1220 cm21; 1H NMR
(CDCl3): d 7.75–7.45 (m, 15, Ph3P), 7.34 (m, 5, Ph), 6.62
(d, J¼6.68 Hz, 1, NH-Ala), 5.49 (d, J¼7.58 Hz, 1, NH-
Orn), 5.08 (m, 3, a-H-Ala, CH2O), 4.59 (s (br), 1, d-NH-
Orn), 4.17 (s (br), 1, a-H-Orn), 3.09 (m, 1, CH2N), 3.00 (m,
3.3.4. [3-Cyano-1-methyl-2-oxo-3-(triphenyl-l5-phos-
phanylidene)-propyl]-carbamic acid 9H-fluoren-9-
ylmethyl ester (24). Fmoc-ala-OH (23) (4.7013 g,
15.10 mmol) was suspended in CH2Cl2 (84 mL) and
DMAP (0.1845 g, 1.51 mmol) was added followed by
EDCI (3.7633 g, 19.63 mmol). (Cyanomethylene)triphenyl-
phosphorane (2) (4.7777 g, 15.86 mmol) was added at once.
The clear yellow reaction mixture was stirred at rt for 4 h.
Water (15 mL) was added, and the organic layer was

6780
H. H. Wasserman et al. / Tetrahedron 59 (2003) 6771–6784
1, CH2N), 1.81 (m, 1, b-H-Orn), 1.61 (m, 1, b-H-Orn), 1.53
(d, J¼6.85 Hz, 3, CH3), 1.45 (m, 2, g-CH2-Orn), 1.41 (s, 9,
Boc); 13C NMR (CDCl3): d 194.3 (d, JC–P¼2 Hz), 170.2,
155.9, 155.8, 136.3, 133.4 (d, JC–P¼9.93 Hz, 6), 133.3 (d,
JC–P¼2.3 Hz, 3), 129.2 (d, JC–P¼12.69 Hz, 6), 128.4 (2),
127.9 (2), 127.9, 122.5 (d, JC–P¼93.51 Hz, 3), 120.6 (d,
JC–P¼15.53 Hz), 78.8, 66.7, 54.2, 51.2 (d, JC–P¼9.05 Hz),
46.8 (d, JC–P¼126.89 Hz), 39.8, 30.4, 28.3 (3), 25.4, 18.9;
MS: m/z 721 (45, MþH), 328 (100, Ph3PC(CN)(CO));
HRMS: m/z calcd for C41H46N4O6P (MþH)þ 721.3155,
found 721.3156.
an off-white semisolid material which was subjected to flash
chromatography (500 g, CH2Cl2/MeOH/AcOH¼95:4:1,
175 mL fractions, 21–45) to give a white solid 29
(2.2192 g, 4.82 mmol, 89%). The acetic acid remaining in
the purified product was removed by filtration through a
second column (110 g, CH2Cl2/MeCN¼50:50) which was
packed using the eluent with 1% acetic acid added. The
column was purged thoroughly with CH2Cl2/MeCN¼50:50
before the product was loaded as a supersaturated solution
in MeCN/water¼75:25. Recrystallization was accom-
plished from MeCN/water¼75:25: mp 245–2488C; TLC
(CH2Cl2/MeOH¼95:5): Rf 0.25; [a]2D0¼2145.12 (c 0.25,
DMSO); IR (DMSO): 3475, 1725, 1695, 1685, 1675, 1665,
3.3.6. 2-[3-(2-Benzyloxycarbonylamino-5-tert-butoxy-
carbonylaminopentanoylamino)-2-oxobutyrylamino]-4-
methylpentanoic acid tert-butyl ester (22). Cyano-
phosphorane 26 (0.5562 g, 0.77 mmol) was ozonized in
CH2Cl2 (15 mL) at 2788C until the reaction mixture turned
blue–green (16 min). After purging with O2 and N2, the
yellow solution of a,b-diketo nitrile 27 was quenched with a
solution of H-Leu-OBut (18) (0.1373 g, 0.73 mmol) in
CH2Cl2 (1 mL). The cooling was discontinued after 1 h.
Concentration furnished a yellow oil which was stirred at rt
for 24 h with a solution of silver nitrate in THF/water¼4:1
(1 M, 7.7 mL, 7.7 mmol). Water (10 mL) was added, and
the THF layer was washed with water (2£2 mL). The
aqueous phases were extracted with CH2Cl2 (3£5 mL). The
combined organic phases were dried (MgSO4, 1 h), filtered,
and concentrated to afford a yellow oil which was
immediately flash chromatographed (30 g, EtOAc/
hex¼50:50, 10 mL fractions, 5–14) to yield the desired
product (22) (,5% of the Ala epimer) as a yellow oil
(0.3557 g, 0.56 mmol, 76%).
1545, 1250 cm21
;
1H NMR (DMSO-d6): d 8.80 (d,
J¼7.79 Hz, 1, NH-Leu), 8.21 (d, J¼8.12 Hz, 1, NH-Ala),
7.40 (d, J¼6.98 Hz, 1, a-NH-Orn), 7.35 (m, 6, Ph, d-NH-
Orn), 5.03 (d, J¼12.62 Hz, 1, CH2O), 4.98 (d, J¼12.62 Hz,
1, CH2O), 4.68 (quint, kJl¼7.12 Hz, 1, a-H-Ala), 4.10 (td
(br), kJl¼8.6, 5.4 Hz, 1, a-H-Leu), 3.86 (q (br), kJl¼5.5 Hz,
1, a-H-Orn), 3.04 (m, 2, CH2N), 1.64–1.42 (m, 7, CH2CH-
Leu, CH2CH2-Orn), 1.15 (d, J¼6.76 Hz, 3, CH3-Ala), 0.88
(d, J¼6.18 Hz, 3, CH3-Leu), 0.82 (d, J¼6.27 Hz, 3, CH3-
Leu); 13C NMR (DMSO-d6): d 197.9, 172.4, 171.4, 165.0,
155.9, 136.9, 128.3 (2), 127.8, 127.7 (2), 65.5, 53.3, 52.2,
49.6, 38.6, 36.5, 28.0, 24.5, 24.3, 22.7, 21.4, 15.1; MS: m/z
461 (80, MþH), 185 (100); HRMS: m/z calcd for
C23H33N4O6 (MþH)þ 461.2400, found 461.2399. Concen-
tration from methanol provided the corresponding methyl
hemiketal as a single diastereomer; HRMS: m/z calcd for
C24H37N4O7 (MþHþCH3OH)þ 493.2662, found 493.2655.
3.3.8. (E)-6-Methyl-2-heptenoic acid (31). To a vigorously
stirred suspension of pyridinium chlorochromate
(39.5906 g, 183.66 mmol) in CH2Cl2 (270 mL) was added
3.3.7. (3-Isobutyl-7-methyl-2,5,6,9-tetraoxo-1,4-8-triaza-
cyclotridec-10-yl)-carbamic acid benzyl ester (29). Tri-
peptide 22 (3.4554 g, 5.44 mmol) was dissolved in CH2Cl2
(136 mL) and TFA (136 mL) was added at rt. The solution
was stirred for 90 min and concentrated. The pale yellow oil
was dissolved and concentrated repeatedly from CH2Cl2
(3£136 mL) to yield (28) as a white solid foam (3.3225 g,
5.61 mmol, 103% (including solvent traces)): mp 50–578C;
TLC (EtOAc/hex¼75:25): Rf 0.00; [a]2D0¼228.98 (c 0.92,
CHCl3); IR: 2950, 1780, 1720, 1670, 1525, 1225,
a
solution of 4-methylpentanol (20) (12.5112 g,
122.44 mmol) in CH2Cl2 (30 mL) over a period of 45 min.
The temperature rose to 358C, and the reaction mixture
turned dark brown–orange. After a total reaction time of
6 h, ether (300 mL) was added. The resulting greenish
solution was passed through Florisil (100 g), and the
granulate from the ether precipitation was washed with
additional ether (3£30 mL) which was likewise filtered. The
resulting green–yellow solution of 4-methylpentanal was
concentrated to a volume of 350 mL and added drop-wise
over a 30 min period to a solution of (tert-butoxycar-
bonylmethylene)triphenylphosphorane in CH2Cl2 (350 mL)
at rt. After an additional 2 h stirring, the reaction mixture
was concentrated to a volume of 230 mL and used without
further purification. In a separate experiment the preparation
was repeated on the same scale, and the crude product
((E)/(Z)¼95:5) was purified by fractional distillation to
yield tert-butyl (E)-6-methyl-2-heptenoate (19.5365 g,
98.52 mmol, 80% (57% of the pure (E)-isomer in the late
distillate) as a colorless oil: bp 708C, 0.5 mmHg; TLC
(EtOAc/hex¼5:95): Rf 0.50; IR: 2940, 1695, 1640, 1460,
1
1175 cm21; H NMR (DMSO-d6): d 11.91 (s (br), 1, OH),
8.81 (d, J¼8.34 Hz, 1, NH-Leu), 8.35 (d, J¼6.48 Hz, 1, NH-
Ala), 7.75 (s (br), 3, NH3), 7.44 (d, J¼8.34 Hz, 1, NH-Orn),
7.36 (m, 5, Ph), 5.00 (m, 3, a-H-Ala, CH2O), 4.27 (dt,
J¼3.77 Hz, 8.35, 1, a-H-Leu), 4.08 (m, 1, a-H-Orn), 2.77
(m, 2, CH2N), 1.69 (m, 2, CH2-Leu), 1.67–1.48 (m, 5,
g-CH-Leu, b-CH2-Orn, g-CH2-Orn), 1.25 (d, J¼7.32 Hz, 3,
CH3-Ala), 0.86 (d, J¼5.98 Hz, 3, CH3-Leu), 0.83 (d,
J¼5.84 Hz, 3, CH3-Leu); 13C NMR (DMSO-d6): d 196.8,
173.2, 171.7, 160.9, 158.8 (q, JC–F¼36.64 Hz), 156.1,
137.1, 128.5 (2), 128.0, 127.9 (2), 115.8 (q,
JC–F¼290.87 Hz), 65.7, 53.7, 50.4, 49.8, 39.3, 38.7, 29.1,
24.6, 23.8, 22.9, 21.2, 15.5; MS: m/z 479 (100, MþH–
TFA); HRMS: m/z calcd for C23H35N4O7 (MþH–TFA)þ
479.2506, found 479.2501. The acid 28 was dissolved in
degassed DMF (1087 mL) and cooled to 2158C (ethylene
glycol/CO2 (s)). Diphenylphosphoryl azide (5.86 mL,
7.48 g, 27.2 mmol) and sodium hydrogen carbonate
(4.57 g, 54.4 mmol) were added, and the suspension was
stirred briskly for 40 h. Filtration and concentration yielded
1
1385, 1370, 1310, 1245, 1155 cm21; H NMR (CDCl3): d
6.84 (dt, J¼15.58 Hz, 6.94, 1, b-H), 5.72 (dt, J¼15.58 Hz,
1.50, 1, a-H), 2.15 (q, kJl¼8.4 Hz, 2, g-H), 1.56 (nonet,
kJl¼6.7 Hz, 1, 1-H), 1.46 (s, 9, But), 1.31 (q, kJl¼6.9 Hz, 2,
d-H), 0.88 (d, J¼6.62 Hz, 6, 2£CH3); 13C NMR (CDCl3): d
166.1, 148.2, 122.8, 79.8, 37.1, 29.9, 28.1 (3), 27.5, 22.3 (2);
MS: m/z 199 (10, MþH); EA: calcd for C12H22O2: C, 72.68;
H, 11.18, found: C, 72.96; H, 11.05. The red–pink solution

H. H. Wasserman et al. / Tetrahedron 59 (2003) 6771–6784
6781
of crude 23 from above was mixed with TFA (230 mL) and
stirred for 2 h at rt. Concentration furnished a green oil that
was subjected to fractional distillation. The product was
collected as a colorless oil (15.3868 g, 108.2 mmol, 88%,
72% of the pure (E)-isomer in the late distillate): mp
<108C; bp 948C, 0.5 mmHg; TLC (EtOAc/hex¼15:85): Rf
1H NMR (CDCl3): d 11.61 (s (br), 1, OH), 7.10 (dt,
J¼15.59 Hz, 6.96, 1, b-H), 5.84 (dt, J¼15.59 Hz, 1.56, 1,
a-H), 2.25 (dq, kJl¼7.0, 1.6 Hz, 2, g-H), 1.59 (nonet,
kJl¼6.7 Hz, 1, 1-H), 1.37 (q, kJl¼7.2 Hz, 2, d-H), 0.91 (d,
J¼6.55 Hz, 6, 2£CH3); 13C NMR (CDCl3): d 172.4, 152.7,
120.5, 36.9, 30.2, 27.5, 22.3 (2); MS: m/z 143 (100, MþH),
125 (60, MþH–H2O).
(d, J¼8.45 Hz, 1, NH-Ala), 8.00 (d, J¼7.46 Hz, 1, a-NH-
Orn), 7.41 (t (br), kJl¼6.4 Hz, 1, d-NH-Orn), 6.60 (dt,
J¼15.44, 6.98 Hz, 1, CHvCCvO), 6.02 (dt, J¼15.44,
1.45 Hz, 1, CvCHCvO), 4.72 (quint, kJl¼7.5 Hz, 1, a-H-
Ala), 4.19 (q (br), kJl¼6.1 Hz, 1, a-H-Orn), 4.10 (td (br),
kJl¼8.5, 5.7 Hz, 1, a-H-Leu), 3.09 (m, 1, CH2N), 3.03 (m,
1, CH2N), 2.12 (q, kJl¼7.0 Hz, 2, CvCCH2), 1.66–1.42
(m, 8, CH2CH-Leu, CH2CH2-Orn, CHMe2), 1.27 (q,
kJl¼7.4 Hz, 2, CvCCCH2), 1.14 (d, J¼6.81 Hz, 3, CH3-
Ala), 0.89 (d, J¼6.25 Hz, 3, CH3-Leu), 0.86 (d, J¼6.62 Hz,
6, 2£CH3), 0.82 (d, J¼6.34 Hz, 3, CH3-Leu); 13C NMR
(DMSO-d6): d 197.9, 172.2, 171.5, 165.2, 165.0, 143.0,
124.0, 52.3, 51.1, 49.5, 38.7, 36.9, 36.5, 29.1, 28.3, 26.9,
24.5, 24.2, 22.7, 22.3, 21.4, 15.1; MS: m/z 469 (50,
MþHþH2O), 451 (100, MþH); HRMS: m/z calcd for
C23H39N4O5 (MþH)þ 451.2920, found 451.2922. Concen-
tration from methanol provided the corresponding methyl
hemiketal as a single diastereomer; HRMS: m/z calcd for
C24H43N4O6 (MþHþCH3OH)þ 483.3182, found 483.3179.
0.15; IR: 2950, 1685, 1640, 1410, 1375, 1360, 1280 cm21
;
3.3.9. N-[(3S,7S,10S,E)-7-Methyl-3-(2-methylpropyl)-
2,5,6,9-tetraoxo-1,4,8-triazacyclotridec-10-yl]-6-methyl-
2-heptenamide (14a).
A solution of 29 (1.0489 g,
2.28 mmol) in CH2Cl2/MeOH¼90:10 (50 mL) was stirred
vigorously with 10% Pd/C (0.2621 g) for 15 h at rt under an
atmosphere of H2. A sample was filtered through Celite
filter agent. Concentration yielded a white solid consisting
of the methyl hemiketal of 10-amino-3-isobutyl-7-methyl-
1,4,8-triaza-cyclotridecane-2,5,6,9-tetraone (30) as a single
3.4. Experimental procedures used in the synthesis of
phebestin
3.4.1. 6-tert-Butoxycarbonylamino-2-isopropyl-4,5-
dioxo-7-phenylheptanoic acid benzyl ester (34). A
solution of 2.4 g (4.4 mmol) cyanophosphorane 33 in
100 mL CH2Cl2 at 2788C was ozonized for 15 min until
the solution turned deep yellow/green. Then the solution
was purged with N2 for 15 min until light yellow, and to this
reaction was added 1.09 g (5.26 mmol) H-Val-OBn in
10 mL CH2Cl2. The mixture was stirred for 1 h at 2788C
and then slowly warmed to rt. The solvent was removed by
rotary evaporation and the crude product was purified by
silica gel chromatography (EtOAc/hex¼1:1) to give 1.3 g
(62%) as a white solid (34): mp 110–1128C; [a]2D0¼239.05
(c 1.4, CHCl3); IR (CHCl3) 3417, 3325, 3250, 1702 and
diastereomer:
mp
226–2298C;
TLC
(CH2Cl2/
MeOH¼95:5): Rf 0.00; [a]2D0¼285.19 (c 0.22, CH2Cl2/
MeOH¼90:10); IR (DMSO): 3400, 1715, 1685, 1665, 1650,
1535, 1240 cm21; H NMR (CD2Cl2/CD3OD): d 4.37 (q,
1
J¼6.69 Hz, 1, a-H-Ala), 4.26 (s (br), 5, NH/H2O), 4.01 (q
(br), kJl¼2.8 Hz, 1, a-H-Leu), 3.88 (s (br), 1, a-H-Orn),
3.11 (m, 1, CH2N), 3.08 (m, 1, CH2N), 1.95 (m, 1, CH2-
Leu), 1.75–1.50 (m, 6, Me2CHCH-Leu, CH2CH2-Orn),
1.13 (d, J¼6.84 Hz, 3, CH3-Ala), 0.95 (d, J¼6.12 Hz, 3,
CH3-Leu), 0.87 (d, J¼6.05 Hz, 3, CH3-Leu); 13C NMR
(CD2Cl2/CD3OD): d 173.3, 171.3, 168.7, 99.1, 53.2, 52.3,
50.7, 49.0 (sept, JC–D¼21.48), 38.5, 37.0, 28.2, 25.2, 24.4,
23.0, 21.7, 13.4 (NMR data were obtained directly from the
reaction mixture of an experiment conducted in CD2Cl2/
CD3OD. The NMR spectra were in addition recorded in
DMSO-d6 displaying a 60:40 mixture of the free ketone and
its methyl hemiketal); MS: m/z 359 (100, MþHþCH3OH),
327 (15, MþH); HRMS: m/z calcd for C15H27N4O4
(MþH)þ 327.2032, found 327.2031, m/z calcd for
1501 cm21
;
1H NMR (CDCl3, 300 MHz) d 0.92 (d,
J¼6.9 Hz, 3H), 0.96 (d, J¼6.9 Hz, 3H), 1.41 (s, 9H), 2.30
(m, 1H), 3.04 (dd, J¼13.7, 6.6 Hz, 1H), 3.30 (dd, J¼13.8,
4.7 Hz, 1H), 4.58 (dd, J¼4.6, 4.3 Hz, 1H), 5.10 (m, 1H),
5.19 (d, J¼12.2 Hz, 1H), 5.26 (d, J¼12.2 Hz, 1H), 5.36 (m,
1H), 7.12 (m, 2H), 7.25–7.41 (m, 8H); 13C NMR (CDCl3,
75 MHz) d 17.7, 19.1, 28.3, 31.4, 37.6, 56.7, 57.4, 67.4,
80.1, 127.1, 128.5, 128.66, 128.74, 129.5, 135.2, 135.8,
136.2, 155.0, 159.1, 170.7 195.5; HRMS calcd for
C27H35N2O6 (MþH)þ 483.2495, found 483.2491.
C16H31N4O5
(MþHþCH3OH)þ
359.2294,
found
359.2287. The colorless reaction mixture was purged with
N2. EDCI (0.6549 g, 3.42 mmol) and (E)-6-methyl-2-
heptenoic acid (31) (0.4048 g, 2.85 mmol) were added,
and stirring was continued for 3 h at rt. The reaction mixture
was filtered through Celite and concentrated. The crude
product was subjected to flash chromatography (200 g,
CH2Cl2/MeOH/AcOH¼95:4:1, 70 mL fractions, 14–41) to
give eurystatin A (14a) as a white solid (0.5727 g,
1.27 mmol, 56%). The acetic acid remaining in the purified
product was removed by filtration through a second column
(30 g, CH2Cl2/MeCN¼50:50), which was packed using the
eluent with 1% acetic acid added. The column was purged
thoroughly with CH2Cl2/MeCN¼50:50 before the product
was loaded as a supersaturated solution in MeCN/
water¼75:25. Recrystallization was accomplished from
MeCN/water¼75:25: mp 291–2948C; TLC (CH2Cl2/
MeOH¼95:5): Rf 0.20; [a]2D0¼2132.54 (c 0.25, DMSO);
IR (DMSO): 3460, 3290, 1725, 1675, 1645, 1545 cm21; 1H
NMR (DMSO-d6): d 8.80 (d, J¼7.77 Hz, 1, NH-Leu), 8.19
3.4.2. 6-tert-Butoxycarbonylamino-5-hydroxy-2-iso-
propyl-4-oxo-7-phenylheptanoic acid benzyl ester (35).
Zinc borohydride in diethyl ether (4.34 mL, 0.64 mmol) was
added to a solution of compound 34 (0.150 g, 0.321 mmol)
in THF at 2788C and the resulting mixture was stirred for
1 h. The reaction mixture was then quenched with water,
neutralized with dilute acetic acid and extracted with
CH2Cl2. The organic extracts were washed with water and
brine, dried (MgSO4), and concentrated in vacuo. The
resultant residue (92:8 mixture of diastereomers) was
purified by PLC (EtOAc/hex¼1:1) affording 35 (0.131 g,
82%): mp 119.5–1218C; [a]2D0¼þ10.2 (c 1.0, CHCl3); IR
(film) nmax 2442, 3406, 3274, 1737,1681 and 1514 cm21
;
1H NMR (CDCl3, 300 MHz) d 0.84 (d, J¼6.9 Hz, 3H), 0.90
(d, J¼6.8 Hz, 3H), 1.39 (s, 9H), 2.20 (m, 1H), 3.05 (dd,
J¼13.6, 6.5 Hz, 1H), 3.20 (m, 1H), 3.96 (m, 1H), 4.18 (s

6782
H. H. Wasserman et al. / Tetrahedron 59 (2003) 6771–6784
(br), 1H), 4.60 (dd, J¼9.2, 4.8 Hz, 1H), 4.98 (m, 1H), 5.15
(d, J¼12.1 Hz, 1H), 5.21 (d, J¼12.1 Hz, 1H), 5.79 (s (br),
1H), 7.22–7.29 (m, 10H); 13C NMR (CDCl3, 75 MHz) d
17.5, 19.2, 28.3, 31.4, 36.1, 56.0, 56.8, 67.1, 74.6, 80.6,
126.7, 128.4, 128.5, 128.6, 129.3, 135.4, 138.2, 157.7,
171.5, 172.7; HRMS calcd for C27H37N2O6 (MþH)þ
485.2651, found 485.2657. Anal. calcd for C27H36N2O6:
C, 66.92; H, 7.49; N, 5.78. Found: C, 66.84; H, 7.36; N,
5.74.
7.1 Hz, 1H), 2.84 (m, 2H), 3.03 (dd, J¼13.9, 5.2 Hz, 1H),
3.26 (m, 1H), 3.86 (d, J¼2.4 Hz, 1H), 4.13 (m, 1H), 4.33 (m,
1H), 7.10–7.32 (m, 10H), 7.79 (d, J¼8.3 Hz, 1H), 8.21 (d,
J¼7.1 Hz, 1H); 13C NMR (DMSO-d6, 125 MHz) d 17.8,
19.2, 30.7, 36.8, 38.9, 53.8, 54.6, 57.2, 69.9, 126.2, 126.4,
128.0, 128.4, 129.1, 129.3, 137.7, 137.8, 170.4, 171.4,
172.8; HRMS calcd for C24H32N3O5: (MþH)þ 442.2342,
found 442.2340.
3.5. Experimental procedures used in the synthesis of
probestin
3.4.3. 2-(6-tert-Butoxycarbonylamino-5-hydroxy-2-iso-
propyl-4-oxo-7-phenylheptanoylamino)-3-phenylpro-
pionic acid benzyl ester (36). A solution of 35 (0.100 g,
0.201 mmol) in anhydrous CH3OH (40 mL) was treated
with 10% Pd/C (15 mg, 0.15 wt equiv.) and stirred at 208C
under H2 (1 atm) for 2 h. The reaction mixture was filtered
through a pad of Celite, washed with 10% CH3OH/CH2Cl2
and concentrated in vacuo to afford the carboxylic acid as a
crude solid, which was immediately used in the next step
without further purification. A solution of the crude acid,
H-Phe-OBn (0.227 mmol) and 1-hydroxybenzotriazole
(30.7 mg, 0.23 mmol) in anhydrous CH2Cl2 (50 mL) at
08C was treated with triethylamine (46.0 mg, 0.45 mmol)
and the resultant mixture stirred to homogeneity. EDCI
(44.0 mg, 0.227 mmol) was then added and the solution was
allowed to warm to rt and stirred overnight. The reaction
was quenched with sat. NH4Cl, washed with H2O, NaHCO3,
brine, dried (MgSO4, 1 h), and the solvent removed. The
crude solid was purified by silica gel chromatography (5%
CH3OH in CH2Cl2) to give 36 (0.105 g, 83% for 2 steps):
mp 134–1368C; [a]D20¼26.18 (c 3.4, CHCl3); IR (film) nmax
3439, 3088, 2970, 1739, 1682, 1512 cm21 1H NMR
;
3.5.1. [1-Benzyl-3-cyano-2-oxo-3-(triphenyl-l5-phos-
phanylidene)-propyl]-carbamic acid tert-butyl ester
(33). To a solution of 0.4 g (3.3 mmol) DMAP and 6.4 g
(33 mmol) EDCI in 120 mL dry CH2Cl2 was added 8.2 g
(31 mmol) N-Boc-D-phenylalanine and 11.1 g (36.8 mmol)
(cyanomethylene)triphenylphosphorane (2). The reaction
was stirred for 5 h at rt and then quenched with 50 mL H2O,
washed with 50 mL sat. NaCl, and dried (MgSO4, 1 h). The
solvent was removed in vacuo and the crude solid purified
by silica gel column chromatography (EtOAc/hex¼1.5:1) to
give 14.9 g (88%) of 33 as a white solid: mp 185–1878C;
[a]2D0¼232.8 (C 2.0, CHCl3); IR (CHCl3) 3435, 3025, 2190,
1
1715, 1590 cm21. H NMR (CDCl3, 300 MHz) d 1.39 (s,
9H), 3.05 (m, 1H), 3.36 (m, 1H), 5.15 (m, 2H), 7.23 (m, 5H),
7.48–7.65 (m, 15H); 13C NMR (CDCl3, 75 MHz) d 28.4,
38.8, 57.1, 79.0, 121.2 (d, J¼14.7 Hz), 122.7 (d,
J¼93.6 Hz), 126.5, 128.2, 129.3 (d, J¼13.0 Hz), 129.9,
133.3, 133.7 (d, J¼10.4 Hz), 137.2, 155.3, 193.7; HRMS
calcd for C34H33N2O3P: (MþH)þ 549.2307, found
549.2303.
(CDCl3, 300 MHz) d 0.84 (d, J¼6.7 Hz, 3H), 0.89 (d,
J¼6.7 Hz, 3H), 1.37 (s, 9H), 2.11 (m, 1H), 3.06 (m, 4H),
4.07 (m, 2H), 4.3 (m, 1H), 4.92 (dd, J¼13.7, 6.3 Hz, 1H),
5.07 (d, J¼12.5 Hz, 1H), 5.14 (d, J¼12.5 Hz, 1H), 5.25 (m,
1H), 5.90 (s (br), 1H), 7.02 (m, 2H), 7.15–7.35 (m, 13H),
7.51 (d, J¼8.7 Hz, 1H); 13C NMR (CDCl3, 75 MHz) d 18.0,
19.4, 28.3, 30.8, 36.7, 37.8, 53.4, 55.6, 58.1, 67.3, 73.8,
80.2, 126.6, 127.1, 128.2, 128.3, 128.5, 128.58, 128.64,
129.3, 129.5, 135.1, 135.7, 138.3, 157.2, 171.0, 171.2 172.9;
HRMS calcd for C36H46N3O7 (MþH)þ 632.3336, found
632.3336.
3.5.2. 2-(3-tert-Butoxycarbonylamino-2-oxo-4-phenyl-
butyrylamino)-4-methylpentanoic acid benzyl ester
(38). A solution of 0.55 g (1.0 mmol) cyanophosphorane
33 in 20 mL CH2Cl2 at 2788C was ozonolyzed for 15 min
until the solution turned deep yellow/green. The solution
was purged with N2 for 5 min until light yellow and then
treated with H-Leu-OBn (0.22 g, 1.0 mmol) in 10 mL
CH2Cl2. The mixture was stirred for 1 h at 2788C and
then slowly warmed to rt. The solvent was removed, and the
crude product was purified by silica gel chromatography
(EtOAc/hex¼1:1) to give 38 (0.29 g, 58%) as a white solid:
mp 96–988C; [a]2D0¼249.7 (C 1.4, CHCl3); IR (CHCl3)
3.4.4. Phebestin, 2-[2-(3-amino-2-hydroxy-4-phenyl-
butyrylamino)-3-methylbutyrylamino]-3-phenylpro-
pionic acid (37). A solution of 36 (44 mg, 0.070 mmol) in
anhydrous CH3OH (5 mL) was treated with 5% Pd/C (7 mg,
0.15 wt equiv.) and stirred at 208C under H2 (1 atm) for 2 h.
The reaction mixture was filtered through a pad of Celite,
washed with 10% CH3OH/CH2Cl2 and concentrated in
vacuo to afford the carboxylic acid as a crude solid, which
was immediately used in the next step without further
purification. The crude solid was then treated at rt with 50%
TFA in CH2Cl2 (2 mL) for 15 min and the solvent was
removed immediately under reduced pressure to afford a
white salt. The salt was treated with NH4OH followed by
decantation to yield phebestin (37) (24.6 mg, 80% for 2
steps) as a white solid: mp 188–1918C, (lit. mp 190–
1928C); [a]2D0¼211.9 (c 1.0, HOAc), (lit. [a]2D7¼212.6 (c
3420, 3020, 2985, 1715, 1505 cm21 1H NMR (CDCl3,
;
300 MHz) d 0.94 (d, J¼5.6 Hz, 6H), 1.41 (s, 9H), 1.65 (m,
3H), 3.08 (dd, J¼13.0, 6.1 Hz, 1H), 3.30 (dd, J¼13.0,
4.5 Hz,1H), 4.68 (m, 1H), 5.04 (m, 1H), 5.22 (s, 2H), 5.38
(m, 1H), 7.10 (m, 2H), 7.25 (m, 3H), 7.37 (m, 5H); 13C
NMR (CDCl3, 75 MHz) d 21.8, 22.8, 24.9, 28.3, 37.6, 41.2,
51.0, 56.7, 67.4, 80.1, 127.1, 128.4, 128.6, 128.7, 129.5,
135.2, 135.7, 155.0, 158.9, 171.6, 195.4; HRMS calcd for
C28H36N2O6: (MþK)þ 535.2210, found 535.2203.
3.5.3. 2-(3-tert-Butoxycarbonylamino-2-hydroxy-4-phe-
nylbutyrylamino-4-methylpentanoic acid benzyl ester
(39). Zinc borohydride in diethyl ether (2.7 mL, 0.4 mmol)
was added to the solution 38 (0.1 g, 0.2 mmol) in THF at
2788C and the resulting mixture stirred for 1 h. The
reaction mixture was quenched with water, neutralized with
dilute acetic acid and extracted with CH2Cl2. The organic
extracts were washed with water and brine, dried (MgSO4),
0.54, HOAc)); IR (film) nmax 3380, 3220, 2960, 1655 cm21
;
1H NMR (DMSO-d6, 500 MHz) d 0.78 (d, J¼6.3 Hz, 3H),
0.81 (d, J¼6.4 Hz, 3H), 1.99 (m, 1H), 2.67 (dd, J¼13.4,

H. H. Wasserman et al. / Tetrahedron 59 (2003) 6771–6784
6783
and concentrated in vacuo. The residue which was a 93:7
mixture of diastereomers was purified by PLC (EtOAc/
hex¼1:1) affording 0.085 g (85%) of 39 as a white solid: mp
140–1428C; [a]2D0¼þ2.97 (c 2.2, CHCl3); IR (CHCl3) 3400,
1H NMR (CDCl3, 300 MHz) d 0.9 (m, 6H), 1.40 (s, 9H),
1.64 (m, 3H), 3.03 (m, 1H), 3.17 (m, 1H), 3.98 (m, 1H), 4.17
(dd, J¼6.8, 2.7 Hz, 1H), 4.67 (m, 1H), 5.00 (d, J¼7.6 Hz,
1H), 5.17 (s, 2H), 5.67 (m, 1H) and 7.21–7.40 (m, 11H); 13C
NMR (CDCl3, 75 MHz) d 21.7, 23.1, 24.8, 28.3, 36.7, 41.2,
50.4, 50.5, 55.5, 67.2, 73.4, 80.0, 126.5, 127.0, 127.5, 128.2,
128.5, 128.7, 129.4, 135.4, 138.3, 157.0, 172.8 and 172.9;
HRMS calcd for C28H38N2O6: (MþH)þ 499.2808, found
499.2806.
treated at rt with 50% TFA in CH2Cl2 (1 mL) for 15 min and
the solvent removed immediately in vacuo affording a white
salt. The salt was treated with NH4OH followed by
decantation to yield probestin (41) as a white solid
(17 mg, 84% for 2 steps): mp 167–1708C, (lit. mp 168–
1708C); [a]2D5¼2117 (c 1.0, CH3OH), (lit. [a]2D0¼2112 (c
0.9, CH3OH)); IR (CHCl3) nmax 3398, 2959, 1640 cm21; 1H
NMR (CD3OD, 300 MHz) d 0.98 (d¼6.4 Hz, 6H), 1.61 (m,
2H), 1.75 (m, 1H), 2.01 (m, 6H), 2.25 (m, 2H), 2.91 (dd,
J¼13.8, 6.9 Hz, 1H), 3.10 (dd, J¼13.8, 8.0 Hz, 1H), 3.30
(br, s, 1H), 3.63 (m, 2H), 3.75 (m, 2H), 3.86 (m, 1H), 4.10
(d, J¼3.3 Hz, 1H), 4.40 (m, 1H), 4.63 (m, 2H) and 7.31 (m,
5H); 13C NMR (CD3OD, 75 MHz) d 22.0, 23.6, 25.8, 25.9,
29.1, 30.0, 36.4, 40.8, 48.2, 51.3, 56.2, 59.7, 60.1, 69.7, 128.5,
130.0, 130.6, 136.8, 172.3, 172.7, 173.4, 175.2; HRMS calcd
for C26H38N4O6 (MþH)þ 503.2869, found 503.2872.
3385, 3235, 2900, 1740, 1693, 1650, 1528 and 1500 cm21
;
3.5.4. 1-{1-[2-(3-tert-Butoxycarbonylamino-2-hydroxy-
4-phenylbutyrylamino)-4-methyl-pentanoyl]-pyrroli-
dine-2-carbonyl}-pyrrolidine-2-carboxylic acid benzyl
ester (40). A solution of 39 (0.33 g, 0.66 mmol) in
anhydrous CH3OH (40 mL) was treated with 10% Pd/C
(50 mg, 0.15 wt equiv.) and stirred at 208C under H2 (1 atm)
for 2 h. The reaction mixture was filtered through a pad of
Celite, washed with 10% CH3OH/CH2Cl2 and concentrated
in vacuo to afford the carboxylic acid as a white solid
(0.257 g), which was immediately used in the next step
without further purification. A solution of the crude acid
(0.257 g), H-Pro-Pro-Obn (0.235 g, 0.692 mmol) and
1-hydroxybenzotriazole (94 mg, 0.692 mmol) in anhydrous
CH2Cl2 (50 mL) at 08C was treated with triethylamine
(140.1 mg, 1.28 mmol) and the resultant mixture stirred to
homogeneity. EDCI (133 mg, 0.69 mmol) was then added
and the solution was allowed to warm to rt and stirred
overnight. The reaction was quenched with sat. NH4Cl,
washed with H2O, NaHCO3, brine, dried (MgSO4, 1 h), and
the solvent was removed. The crude solid was purified by
silica gel chromatography (5% CH3OH in CH2Cl2) to give
40 (0.391 g, 89% for two steps): mp 80–838C; [a]2D0¼278.3
(c 1.2, CHCl3); IR (CHCl3) 3425, 3380, 3290, 2950, 1740,
3.6. Experimental procedures used in the synthesis of
bestatin
3.6.1. Bestatin, 2-(3-amino-2-hydroxy-4-phenylbutyryl-
amino)-4-methylpentanoic acid (42). Compound 39
(63 mg, 0.13 mmol) in a solution of 2 M HCl in EtOAc
(15 mL) was stirred at rt for 2.5 h. The resulting precipitate
was filtered and washed with EtOAc and then hexanes
yielding a white salt. A solution of this salt in anhydrous
CH3OH (10 mL) was treated with 10% Pd/C (9 mg,
0.15 wt equiv.) and stirred at 208C under H2 (1 atm) for
2.5 h. The reaction mixture was filtered through a pad of
Celite, washed with CH3OH and concentrated in vacuo to
afford a white solid which was crystallized from a mixture
of 5% hexanes in EtOAc to yield bestatin hydrochloride
(42)·HCl (40 mg, 92%): mp 210–2148C, (lit. mp 212–
2188C); [a]2D0¼215.2 (c 0.83, 1 M HCl), (lit. [a]2D2¼215.5
(c 1.0, 1 M HCl)); IR (KBr) nmax 3650–2250, 1723, 1660,
1604, 1532 and 1498 cm21; 1H NMR (CD3OD, 300 MHz) d
0.93 (d, J¼5.5 Hz, 6H), 1.69 (m, 3H), 2.91 (dd, J¼13.5,
6.6 Hz, 1H), 3.10 (dd, J¼13.5, 8.6 Hz, 1H), 3.74 (m, 1H),
4.04 (d, J¼2.4 Hz, 1H), 4.18 (m, 1H) and 7.2–7.4 (m, 5H).
1705, 1651, 1635 and 1495 cm21 1H NMR (CDCl3,
;
300 MHz) d 0.95 (d, J¼6.0 Hz, 3H), 0.97 (d, J¼5.2 Hz,
3H), 1.37 (s, 9H), 1.55–1.76 (m, 3H), 1.77–2.03 (m, 6H),
2.03–2.21 (m, 3H), 2.95 (m, 2H), 3.55 (m, 1H), 3.70 (m,
2H), 3.80 (m, 1H), 4.05 (m, 1H), 4.11 (m, 1H), 4.55 (m, 1H),
4.65 (m, 1H), 4.83 (dd, J¼14.0, 8.8 Hz, 1H), 5.00 (d,
J¼12.2 Hz, 1H), 5.06 (m, 1H), 5.22 (d, J¼12.2 Hz, 1H),
5.92 (d, J¼4.7 Hz, 1H), 7.14–7.43 (m, 10H) and 7.57 (d,
J¼8.8 Hz, 1H); 13C NMR (CDCl3, 75 MHz) d 21.5, 23.6,
24.6, 24.8, 28.0, 28.1, 28.3, 28.5, 37.0, 40.7, 46.5, 47.3,
48.7, 55.2, 58.1, 58.7, 66.9, 72.2, 126.3, 126.9, 128.2, 128.3,
128.4, 128.5, 128.6, 129.6, 135.6, 138.4, 155.9, 169.9,
171.6, 171.9, 172.3 and 174.9; HRMS calcd for
C38H52N4O8: (MþH)þ 503.2869, found 503.2872.
Acknowledgements
This work was supported by grants from the National
Institutes of Health, the National Science Foundation and
Pfizer, Inc. We thank Dr’s Erin Curtis and Michael
Jorgensen for technical assistance and Dr Walter McMurray
of the Yale Cancer Center Mass Spectrometry Resource for
determining HRMS spectra. We are pleased to acknowledge
valuable contributions by Mary-Lou Oates and Dr Jonathan
Parr.
3.5.5. Probestin, 1-{1-[2-(3-amino-2-hydroxy-4-phenyl-
butyrylamino)-4-methylpentanoyl]-pyrrolidine-2-car-
bonyl}-pyrrolidine-2-carboxylic acid (41). A solution of
40 (28 mg, 0.041 mmol) in anhydrous CH3OH (2 mL) was
treated with 5% Pd/C (4 mg, 0.15 wt equiv.) and stirred at
208C under H2 (1 atm) for 2 h. The reaction mixture was
filtered through a pad of Celite, washed with 10% CH3OH/
CH2Cl2 and concentrated in vacuo to afford the carboxylic
acid as a crude solid, which was immediately used in the
next step without further purification. The crude solid was
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1
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