Total synthesis of aeruginosin 298-A analogs containing ring oxygenated variants of 2-carboxy-6-hydroxyoctahydroindole

Article abstract of DOI:10.1016/j.tet.2008.03.107

Aeruginosins are a family of naturally occurring oligopeptides sharing a common perhydroindole-2-carboxylic acid (l-Choi) core structure. Many aeruginosins exhibit inhibitory activity against serine proteases including thrombin. Aeruginosin 298-A is a tetrapeptide containing the l-Choi core structure and three other unusual amino acids or their derivatives; it is a thrombin and trypsin inhibitor. As part of our effort in finding effective thrombin inhibitors from natural product analogs and to understand the influence of the rigid bicyclic amino acid to the thrombin inhibition activities, we synthesized two aeruginosin 298-A analogs in which the l-Choi is replaced with ring oxygenated perhydroindole-2-carboxylic acids. The Choi variants are enantiomers synthesized from d- and l-glucose, respectively. The preparation of the aeruginosin 298-A analogs containing the ring oxygenated Choi variants and their inhibition activities toward thrombin and trypsin are reported.

Full text of DOI:10.1016/j.tet.2008.03.107

Tetrahedron 64 (2008) 5784–5793
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Total synthesis of aeruginosin 298-A analogs containing ring
oxygenated variants of 2-carboxy-6-hydroxyoctahydroindole
*
Xiaoping Nie, Guijun Wang
Department of Chemistry, University of New Orleans, New Orleans, LA 70148, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Aeruginosins are a family of naturally occurring oligopeptides sharing a common perhydroindole-
Received 21 November 2007
Received in revised form 11 March 2008
Accepted 31 March 2008
2-carboxylic acid (
L
-Choi) core structure. Many aeruginosins exhibit inhibitory activity against serine
proteases including thrombin. Aeruginosin 298-A is a tetrapeptide containing the
L-Choi core structure
and three other unusual amino acids or their derivatives; it is a thrombin and trypsin inhibitor. As part of
our effort in finding effective thrombin inhibitors from natural product analogs and to understand the
influence of the rigid bicyclic amino acid to the thrombin inhibition activities, we synthesized two
Available online 8 April 2008
aeruginosin 298-A analogs in which the
L-Choi is replaced with ring oxygenated perhydroindole-2-
carboxylic acids. The Choi variants are enantiomers synthesized from
D
- and -glucose, respectively. The
L
preparation of the aeruginosin 298-A analogs containing the ring oxygenated Choi variants and their
inhibition activities toward thrombin and trypsin are reported.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
their derivatives.1–10 More than 20 compounds in this family have
been isolated and identified so far.10 Typically, these compounds
Aeruginosins are a class of naturally occurring oligopeptides
containing common bicyclic core structure, 2-carboxy-6-
hydroxyoctahydroindole (Choi), and other unusual amino acids or
are tetrapeptides containing the 2-carboxy-6-hydroxyoctahy-
a
droindole core structure (L-Choi 1, Fig. 1), which has the configu-
ration of 2S,3aS,6R,7aS.11–13 While a majority of aeruginosins share
4
3
3a
7a
5
6
OH
HO
2
OH
OH
1
N
H
HO
O
N
H
N
H
HO
O
HO
O
7
2-carboxyl-6-hydroxy-octahydroindole
5β-hydroxy-L-Choi
3
3a, 7a-di-epi-L-Choi
2
(L-Choi)
1
HO
HO
O
HO
HO
L-Choi
D-Leu
HN
N
N
O
N
NH2
NH2
N
NH2
O
HN
D-Hpla
L-Argol
O
NH
O
O
O
NH
HN
O
O
H
N
OH
OH
NH2
NH
HO
H3CO
OSO3
OH
HO
Aeruginosin EI 461
5
Aeruginosin 298-A
4
Dysinosin A
6
Figure 1. Structures of several octahydroindole-2-carboxylic acids and aeruginosins.
* Corresponding author. Tel.: þ1 504 280 1258; fax: þ1 504 280 6860.
E-mail address: gwang2@uno.edu (G. Wang).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.03.107

X. Nie, G. Wang / Tetrahedron 64 (2008) 5784–5793
5785
the same Choi structure as in aeruginosin 298-A (4), several other
Choi structures with different stereochemistry or substituents have
important issue in developing effective thrombin inhibitor based
anticoagulants.
also been reported. These include the 3a,7a-diepi-
L
-Choi 2 in
In order to understand the function of the Choi core structures
and stereochemical effect of the P2 unit toward thrombin inhibition,
we synthesized novel aeruginosin 298-A analogs (I and II) in which
the P2 Choi structure is replaced with a ring oxygenated variant.
aeruginosin EI 461 (5)15 and the 5,6-dihydroxyl octahydroindole-
2-carboxylic acid 3 in dysinosins.9,10
Many aeruginosins have shown activities against serine pro-
teases such as trypsin, thrombin, and factor VIIa. Thrombin (factor
IIa) is the key enzyme in blood coagulation cascade; direct
inhibition of thrombin has been shown to be an important method
to treat thrombosis and other blood clotting disorders.16–21 Aeru-
ginosin 298-A (4) was the first compound in the family exhibiting
serine protease inhibitory activities. It contains the sequence of
Previously we had reported a general method for converting D-glu-
cose to the ring oxygenated variants (7, 8) of the Choi core structures
in which the methylene at C-4 position is replaced with an oxygen
atom and with an additional hydroxyl group at the C-7 position.27
O
OH
O
OH
D
-p-hydroxylphenyllactic acid
6-hydroxyotahydroinhole -Choi), and
through peptidic linkages. Aeruginosin 298-A inhibits thrombin at
(
D
-Hpla),
D
-leucine,
L
-2-carboxy-
N
H
O
(L
L-argininol
(L-Argol)
BnO
N
H
O
BnO
OBn
OBn
IC50¼0.5
m
M and trypsin at IC50¼1.7
mM. The X-ray crystal struc-
7
8
ture11 of the ternary complex of 298-A bound to thrombin–hirugen
indicated that its binding mode at the active site is similar to that of
Here we report the synthesis of the ring oxygenated variants of
Choi without the additional hydroxyl group, and the aeruginosin
298-A analogs I and II containing the corresponding O-Choi
variants and their activities toward thrombin and trypsin. These
tetrapeptides (I, II) have the same amino acid sequence as in
aeruginosin 298-A except that the Choi is replaced by a pair of
enantiomeric oxygenated Choi variants.
D
-Phe-Pro-Arg chloromethyl ketone and other serine protease in-
hibitors.21 The P1 residue Argol occupies the S1 binding site of
thrombin, and the -Leu and
-Hpla bind to the S3 subsite.11 The
D
D
five-membered ring of the Choi residue occupies the hydrophobic
S2 binding site, while its six-membered ring projects out and
loosely interacts with Tyr60A and Trp60D from thrombin. Dysi-
HO
HO
O
O
N
N
HN
HN
O
O
NH
O
NH
O
O
O
H
N
H
N
NH2
NH
OH
NH2
NH
OH
HO
HO
OH
OH
I
II
nosin A (6) is a factor VIIa inhibitor (Ki¼0.11
m
M) and thrombin
2. Results and discussion
inhibitor (Ki¼0.45
m
M).9 The X-ray crystal structure of 6 with
thrombin reveals that it occupies the S1–S2–S3 binding sites. The
Choi is underneath tryptophan 86 and it appears not to be hydro-
gen bonded with the S2 binding site.9
The Choi analogs in I and II can be prepared by a modified
method for the synthesis of 7 and 8. The preparation of the pro-
tected Choi analog in compound I is shown in Scheme 1. D-Glucose
The unusual structure and interesting biological activities of this
classof moleculeshaveattractedseveral researchgroups tocarryout
their total syntheses.10,12,13,22–24 Several methods are also available
for synthesizing the Choi core structure and its analogs.25–29
Moreover, there also have been some confusions about the structure
assignment for aeruginosins, several proposed structures of aeru-
ginosins including aeruginosin 298-A, EI 461 have been revised or
reassigned,12–14,15,24 there is also a proposed structure revision for
aeruginosin 205 based on synthesis.14 Besides the synthesis of the
aeruginosins, there is also a great interest in preparing analogs of the
aeruginosin natural products and understanding the structure–
activity relationship (SAR) pattern for these serine protease in-
hibitors. While several aeruginosin 298-A analogs have been
synthesized, most of the modifications or variations of structures are
on the units other than the Choi.25,26,30,31 These include the prepa-
ration of a combinatorial library and the study of stereochemical
influences of the amino acid residues at P3, P4 or P1 to the trypsin
inhibition activity30 and several other analogs of aeruginosins with
different amino acid residues.31 Many aeruginosin analogs have
exhibited trypsin inhibition potency and are not very selective
toward thrombin. Gaining selectivity among serine proteases is an
was converted to the diol 10 in 65% yield over four steps.27 Sub-
sequent mono-benzylation of the 2-hydroxyl group of 10 using
dibutyltin oxide and benzyl bromide gave 11 in 63% yield along
with 13% of 3-hydroxyl benzylated byproduct. The 3-benzyloxy
byproduct can be converted back to 10 by catalytic hydrogenolysis
and dimethoxyl benzylidene acetal protection and reused again.
Compound 11 was treated with TCDI to give the thiocarbamate 12,
which was then subjected to radical reduction using tributyltin
hydride, producing 3-deoxy acetal 13 in high yield. Hydrolysis of
acetal 13 with 80% acetic acid followed by dimesylation and
selective bromo displacement of the primary mesylate afforded
bromide 16 in good overall yield. The alkylation reaction of N-Boc
diethyl amino malonate with bromide 16 led to the cyclized
product 18 directly presumably via intermediate 17 by displace-
ment of mesylate. In an analog with an additional 3-hydroxy sub-
stituent, the open-chained alkylation product was obtained instead
and subsequent cyclization was accomplished by deprotecting the
Boc group to the free amine.27 The mono hydrolysis of 18, however,
proved not so straightforward. The exact condition applied
successfully to a similar but uncyclized acyclic amino acid diester
(1 N aq NaOH, EtOH, room temperature, 5 h)27 was not working in

5786
X. Nie, G. Wang / Tetrahedron 64 (2008) 5784–5793
HO
HO
HO
O
Ph
O
1.Bu2SnO/Tol Ph
O
O
ref. 27
65% over 4steps
O
O
O
2.BnBr, Tol
63% over 2 steps
HO
HO
11
OH
OH
OH
OBn
10
9
TCDI,
O
Ph
Bu3SnH, AIBN
Ph
HO
HO
DMAP
O
O
H
Ph
O
O
HOAc
O
O
S
90%
O
88%
OBn
N
90%
OBn
N
OBn
12
14
13
MsCl,Py
DCM
95%
BocHN CO2Et
MsO
MsO
Br
NaBr,TBAB,
DMSO
BocHNCH(CO2Et)2
NaH, TBAI, Tol
O
O
MsO
CO2Et
O
82%
52%
OBn
MsO
OBn
15
16
OBn
17
0.2 N LiOH, THF, 99%
CO2Et
CO2Et
1. aq 1N KOH
BocN
CO2Et
BocN
CO2H
O
EtOH, 40-50 °C BocN
2. Toluene, 110 °C
O
O
+
OBn
OBn
OBn
20 (15%)
18
19 (67%)
Scheme 1. Synthesis of protected Choi analog 20 from D-glucose.
this case. Unchanged starting material was found even after stirring
at room temperature for almost two days. The difference in
hydrolysis was understandable since the cis-fused ring system
would be more hindered for saponification than the acyclic chain
system. After several trials, we found that under conditions of
stronger base (1 N KOH) and elevated temperature (40–50 ꢀC),
compound 18 was hydrolyzed completely in less than 12 h. Sub-
sequent decarboxylation in refluxing toluene afforded both ester 19
and acid 20 in 67 and 15% yield, respectively, and the presence of
acid 20 is due to over hydrolysis of 18 to a diacid under those fairly
harsh conditions. Since compound 19 can be hydrolyzed to 20
almost quantitatively, the separation of the acid with ester is
unnecessary; the crude mixture of 19 and 20 obtained in the
decarboxylation reaction was subjected to hydrolysis directly
without separation, producing the acid 20 with 7:1 diastereomeric
selectivity in 85% yield from 18 over 3 steps.
The diastereomeric mixture, either 19a and 19b, or 20a and 20b,
could not be separated by chromatography efficiently, however, the
derivatives 21a and 21b from the mixture of 19a and 19b can be
separated from each other cleanly via flash chromatography. The
NMR studies of amines 21a and 21b further confirmed the above
structural assignment (Fig. 3).
For compound 21a, the signals corresponding to protons H-2
(3.74 ppm), H-9 (3.78 ppm), H-3cis (2.27 ppm), and H-3trans
(2.13 ppm), can be assigned unambiguously based on coupling
H3cis
H
H
3cis H9
O
H3trans
EtO
H3trans
H2
9O
EtO
O
H2
N
H
N
OBn
OBn
H
H
H
O
major isomer 21a
minor isomer 21b
The decarboxylation products 19 and 20 were obtained as
mixtures of diastereomers. The major isomers 19a and 20a are
assigned as R-amino acids based on the decarboxylation–tauto-
merization mechanism shown in Figure 2. The protonation of the
intermediate enediol would be on the more accessible convex face
(Re face) of the cis-fused ring system giving the R-isomer as the
major product. This assignment is confirmed by 2D NOESY NMR
study as well.
21a: (ppm): H2 3.74, H3trans 2.13, H3cis 2.27, H9 3.78.
J (Hz): J2-3cis= 10.2, J2-3-trans= 3.3, J9-3cis= 4.1, J9-3trans= 0
21b: (ppm): H2 4.00, H3trans 2.26, H3cis 2.04, H9 3.93.
J (Hz): J2-3cis= 7.7, J2-3trans= 8.5, J9-3cis= 4.7, J9-3trans= 0
Figure 3. Configurations and 1H NMR chemical shifts of the diastereomers 21a and
21b.
OH (Et)
O
O
(R)
N
H
O
Re face
O
H
O
H
OH
CO2H (Et)
CO2H
Si face
(Et) HO
O
O
H
O
BocN
(Et) HO
O
OBn
major isomer
O
-CO2
N
H
H
O
O
N
H
O
O
Tautomerization
OBn
OH (Et)
O
OBn
OBn
O
(S)
H
O
N
H
O
OBn
minor isomer
Figure 2. Stereochemistry outcome of the decarboxylation step.

X. Nie, G. Wang / Tetrahedron 64 (2008) 5784–5793
5787
OMe
O
H
N
O
CO2H
O
H
N
BocN
PyBOP
DIEA,DMF
OMe
NO2
O
H
N
N
H
BocN
H
N
+
NH
NO2
O
H2N
OBn
NH
20
OBn
23a (79%) + 23b (11%)
22
OMe
O
H
N
O
H
N
NO2
N
H
HN
TFA/DCM
23a
NH
O
OBn
24
Scheme 2. Synthesis of protected O-Choi-Arg dipeptide 24.
constants and COSY. The coupling constants J9-3cis¼4.1 Hz,
J9-3trans¼0 Hz, suggested that the dihedral angle between H-9 and
H-3trans is almost 90ꢀ, and the dihedral angle between H-9 and
H-3cis is either around 44ꢀ or 133ꢀ based on Karplus equations.32
Furthermore, strong NOE signal between H-9 and H-3cis and no
NOE signal between H-9 and H-3trans clearly demonstrate that the
dihedral angle between H-9 and H-3cis should be around 44ꢀ
rather than 133ꢀ. Therefore, H-3cis is cis to H-9, and H-3trans is
trans to H-9. For the same reason, H-2 is cis to H-3cis, and trans to
H-3trans because of much larger coupling constant and stronger
NOE signal between H-2 and H-3cis. Similarly, the stereochemistry
of 21b can also be elucidated based on coupling constants and NOE
signal strength. Therefore, the NMR studies proved that major
isomer 21a is R-amino acid and minor isomer 21b is S-amino acid.
With the stereochemistry of both major and minor isomer of
Choi analogs 21a and 21b confirmed by the NMR studies, we con-
tinued the synthesis with the peptide coupling reactions using the
inseparable mixture of 20a and 20b with commercially available
methyl ester of Nu-nitro-arginine hydrochloride salt 22 (Scheme 2).
The dipeptides 23a and 23b were separable by chromatography
and were obtained in 79 and 11% yield, respectively. The major
isomer compound 23a was then deprotected to give the TFA salt 24
and is used for the coupling reaction later on.
by literature procedure.23 It is noteworthy that the
group of HPLA 26 was not protected here and this has no influence
on the coupling reaction when PyBOP was used as the coupling
agent. Furthermore, the racemic O-benzyl D,L-HPLA could also be
a
-hydroxyl
used since the free hydroxyl group in 27 allowed easy separation of
the two diastereomers by chromatography. The dipeptide 27 was
hydrolyzed to the free acid and then coupled with the TFA salt 24,
affording tetrapeptide 28 in good yield. The presence of free
hydroxyl group in 27 did not affect the peptide coupling reaction
either. The reduction of the methyl ester group in 28 with LiBH4
gave compound 29 in fairly good yield providing that the reaction
was quenched immediately when only a small amount of starting
material was left. Finally Pd/C catalyzed hydrogenolysis of 29 in
methanol with the addition of 0.5% TFA yielded compound I, the
analog of aeruginosin 298-A, almost quantitatively (Scheme 3).
For the preparation of analog II, L-glucose was used as starting
material to prepare the O-Choi variants. As shown in Scheme 4,
using exactly the same methods described in Scheme 1, the O-Choi
variant 30 was synthesized as an inseparable mixture of di-
astereomers in 7:1 ratio. The diastereomers 30 can be separated by
flash chromatography after removing the Boc group, similarly to
their enantiomers 21a and 21b. The major isomer has the same
stereochemistry as L-Choi in aeruginosin 298-A. To synthesize the
The dipeptide Leu-HPLA 27 was prepared by coupling reaction
tetrapeptide, the diastereomeric mixture 30 was used directly
without separation in the coupling reaction with compound 22 to
produce the dipeptide 31. However, unlike the dipeptide 23, the
of the commercially available
D-leucine ethyl ester 25 with
O-benzyl
D-HPLA 26, which was prepared from O-benzyl
D-tyrosine
PyBOP
1. 0.2 N LiOH, THF
O
DIEA
DCM
COOH
OH
2. 24, HATU, DIEA, DMF
OEt
N
+
BnO
OEt
77% over 2 steps, from 23a to 28
H
94%
H2N
O
OH
27
BnO
O
26
25
OH
OMe
O
H
H
N
O
O
O
O
N
NHNO2
NH
NHNO2
NH
N
H
N
H
N
N
N
H
N
H
LIBH4, THF
72%
O
OH
O
O
OH
O
BnO
BnO
OBn
OBn
28
29
OH
H
O
O
N
NH2
NH
Pd/C/H2
N
H
0.5% TFA in MeOH
N
N
H
98%
OH
O
O
HO
OH
Scheme 3. Synthesis of aeruginosin 298-A analog I.
I

5788
X. Nie, G. Wang / Tetrahedron 64 (2008) 5784–5793
MeO
H
O
HO2C
O
NBoc
22, PyBOP
DIEA,DMF
O2NHN
N
TFA/DCM
Scheme 1
N
H
NBoc
O
L-Glucose
NH
71%
O
BnO
31
BnO
30
MeO
MeO
H
N
O
O
O
H
N
O
O
O2NHN
O2NHN
N
27, HATU,
DIEA, DMF
N
H
N
H
NH
N
H
NH
O
OH
NH
O
OBn
O
BnO
BnO
32
33a (57%) + 33b (8%)
HO
H
N
O
O
O
Pd/C/H2
0.5% TFA in MeOH
O2NHN
LIBH4, THF
52%
N
N
N
H
33a
II
H
NH
O
OH
99%
OBn
BnO
34
Scheme 4. Preparation of aeruginosin analog II.
dipeptide 31 was an inseparable diastereomeric mixture, so the
mixture was carried forward in the synthesis. The Boc group was
removed to give compound 32, which was then coupled with the
dipeptide 27 in standard conditions to afford the tetrapeptide 33 as
a mixture of diastereomers. The diastereomeric tetrapeptides were
separated by flash chromatography to give the pure isomers 33a
(major) and 33b. After the reduction of the methyl ester with LiBH4
and global deprotection, we successfully synthesized compound II,
the exact analog of aeruginosin 298-A with one methylene group
replaced by an oxygen atom.
After completing the synthesis of the teterapeptide analogs, we
carried out enzyme assays for thrombin and trypsin by standard
enzymes assay conditions in the literature.30,33,34 Commercially
available thrombin inhibitor PPACK and trypsin inhibitor were used
to calibrate the enzyme concentrations and as standards. The IC50
coupling of the dipeptide formed from Choi variants and arginine
derivative with the dipeptide formed from the HPLA and -leucine
D
resulted in the tetrapeptides in good yields, which were then con-
verted to the target compounds I and II efficiently. We found that
compound I showed good selectivity to thrombin over trypsin. But
compound II, which contains the same configuration as in the nat-
ural product showed preference for trypsin instead. Although these
compounds showed some loss of activities, the SAR pattern obtained
here can help us to design more selective thrombin inhibitor. Further
structure optimization is necessary to obtain compounds with high
potency and good selectivity among serine proteases.
4. Experimental section
4.1. General methods
value for PPACK was determined as 0.018
the standard trypsin inhibitor had IC50 value of 1.3
pound I inhibited thrombin at <24 g/mL, it showed no inhibition
toward trypsin; analog II inhibited trypsin with IC50 at 2.0 g/mL
and thrombin at <22 g/mL. The P2 units in compounds I and II are
m
g/mL in this study and
m
g/mL. Com-
Reagents, solvents, and starting materials were purchased from
Aldrich, VWR, or Lancaster unless otherwise specified. Anhydrous
solvents were purchased from Aldrich in sure-seal bottles and used
directly without further treatment. 1H and 13C NMR spectra were
acquired on a Varian 400 MHz or 300 MHz NMR machine. Melting
point was measured using a Fisher-Johns melting point apparatus.
Typically thin layer chromatography (60 Å pore, UV254, phospho-
molybdic acid as the staining agent) was used to monitor reactions.
Silica gel (230–400 meshes) was used for flash chromatography.
Solvents were generally removed under reduced pressure using
Buchi rotavapor R200.
m
m
m
mirror images to each other, this is the only difference of the two
analogs. While compound II prefers trypsin over thrombin, it
inhibited trypsin with IC50 value slightly lower than that of aeru-
ginosin 298-A. Compound I has opposite configuration as aerugi-
nosin 298-A, it inhibited thrombin similarly to compound II, but
showed no inhibition toward trypsin. Gaining selectivity among
serine proteases is an important issue in developing effective oral
anticoagulants. This result indicated that compound I can be used
as a starting point to optimize the thrombin inhibition activity.
Further study modifying the P1 and P3 units will help us to obtain
thrombin inhibitors with improved potency and selectivity.
4.2. Synthesis of aeruginosin analog I
4.2.1. Preparation of 1,5-anhydro-2-O-benzyl-4,6-O-
benzylidene-
D-glucitol (11)
3. Conclusions
A solution of 1,5-anhydro-4,6-O-benzylidene-D
-glucitol 1027
(7.32 g, 29.0 mmol) and Bu2SnO (7.95 g, 31.9 mmol) in toluene
(100 mL) was heated to reflux for 3 h. Toluene was evaporated
under reduced pressure. Benzyl bromide (7.6 mL, 63.8 mmol) and
toluene (100 mL) were then added to the residue. The resulting
solution was heated to reflux overnight and then cooled to room
temperature, treated with mixture of saturated NaHCO3 (100 mL)
and EtOAc (100 mL), and filtered through a pad of CeliteÒ. The or-
ganic layer was separated and the aqueous layer was extracted with
We have developed an efficient synthesis for aeruginosin 298-A
analogs in which the P2 Choi unit is modified by replacing a methy-
lene group in the six-membered ring with an oxygen and different
configurations. The ring oxygenated Choi variants were synthesized
from D-glucose or L-glucose; the formation of the bicyclic Choi vari-
antsfeaturesalkylationandcyclizationstepsinaone-potfashionand
good diastereoselectivity in the decarboxylation step. Segment

X. Nie, G. Wang / Tetrahedron 64 (2008) 5784–5793
5789
EtOAc (200 mLꢁ3). The combined organic layers were dried
(Na2SO4) and concentrated under reduced pressure. Re-
crystallization of the crude with EtOAc/hexane gave 5.37 g com-
pound 11 as pure colorless crystals. Another 0.91 g of product was
obtained with column chromatography purification of the mother
liquid (DCM/EtOAc/hexane, 10:9:1). Totally 6.28 g, yield 63%, mp
After evaporation under reduced pressure to remove most solvent,
the crude was purified by recrystallization (EtOAc/hexane) to give
2.38 g of 14 as colorless crystals, 90% yield, mp 107–108 ꢀC, [
a]
D
þ7.7 (c 0.5, CHCl3). 1H NMR (400 MHz, CDCl3)
d 7.38–7.28 (m, 5H),
4.61 (d, 1H, J¼12.5 Hz), 4.55 (d, 1H, J¼12.5 Hz), 4.05 (dd, 1H, J¼12.5,
5.0 Hz), 3.84 (dd, 1H, J¼11.3, 5.0 Hz), 3.77 (dd, 1H, J¼11.3, 5.0 Hz),
3.64–3.59 (m, 1H), 3.55 (dt, 1H, J¼10.0, 5.0 Hz), 3.21 (dd, 1H, J¼11.3,
10.0 Hz), 3.16 (dd, 1H, J¼8.8, 5.0 Hz), 2.54–2.49 (m, 1H), 1.51 (q, 1H,
161–162 ꢀC, [
a]
ꢂ31.9 (c 1.7, CHCl3). 1H NMR (500 MHz, CDCl3)
D
d
7.54–7.51 (m, 2H), 7.41–7.31 (m, 8H), 5.53 (s, 1H), 4.79 (d, 1H,
J¼11.5 Hz), 4.70 (d, 1H, J¼11.5 Hz), 4.32 (dd, 1H, J¼10.3, 4.8 Hz), 4.03
(dd, 1H, J¼11.5, 5.5 Hz), 3.86 (t, 1H, J¼9.1 Hz), 3.67 (t, 1H, J¼10.3 Hz),
3.59 (ddd, 1H, J¼10.3, 9.1, 5.5 Hz), 3.48 (dd, 1H, J¼9.7, 9.1 Hz), 3.37
(dt, 1H, J¼9.7, 4.8 Hz), 3.32 (t, 1H, J¼10.9 Hz), 2.86 (s, 1H); 13C NMR
J¼11.3 Hz); 13C NMR (100 MHz, CDCl3)
d 137.9, 128.4, 127.8, 127.6,
81.2, 71.9, 71.0, 69.7, 65.7, 62.4, 38.8; HRMS calcd for C13H18O4þH,
239.1283; found, 239.1283.
(125 MHz, CDCl3)
d
138.0, 137.0, 129.1, 128.5, 128.2, 127.9, 127.8,
4.2.5. Preparation of 1,5-anhydro-2-O-benzyl-3-deoxy-4,6-di-O-
126.2, 101.8, 81.0, 77.7, 74.7, 73.4, 70.9, 68.7, 68.4; HRMS calcd for
20H22O5þH, 343.1545; found, 343.1538.
methylsulfonyl-D-ribo-hexitol (15)
C
A solution of diol 14 (1.93 g, 8.1 mmol) in DCM (10 mL) and pyri-
dine (7.5 mL) was treated with methanesulfonyl chloride (2.5 mL,
32.4 mmol). The mixture was left stirring under anhydrous condi-
tion till completion of the reaction (usually 12 h). The reaction
mixture was then poured into a beaker containing ice and saturated
aq NaHCO3 (20 mL). After stirring in ice bath for 1 h, the reaction
mixture was extracted with DCM (50 mLꢁ3), and washed with 1 N
HCl (30 mLꢁ3) and brine. The organic layer was dried and concen-
trated. Column chromatographic (EtOAc/hexane, 30:70) purification
4.2.2. Preparation of 1,5-anhydro-2-O-benzyl-4,6-O-benzylidene-
3-O-imidazolythiocarbonyl- -glucitol (12)
D
A solution of monoalcohol 11 (5.30 g, 15.50 mmol), TCDI (thio-
carbonyl diimidazole, 3.31 g, 18.60 mmol), and DMAP (2.27 g,
18.60 mmol) in chloroform (40 mL) was heated to refluxed over-
night. The reaction mixture was cooled to room temperature,
diluted with EtOAc (250 mL), washed with saturated aq NaHCO3
and brine, dried (Na2SO4), and concentrated under reduced pres-
sure. The crude material was purified by column chromatography
(hexane/EtOAc, 4:1) to produce compound 12 as a light yellow solid
of the residuegave 15 (3.46 g, 95%) as colorless thickoil, [
a
]D þ20.4 (c
3.6, CHCl3). 1H NMR (400 MHz, CDCl3)
d
7.37–7.28 (m, 5H), 4.60 (d,
1H, J¼11.3 Hz), 4.57 (dt, 1H, J¼11.3, 5.0 Hz), 4.51 (d, 1H, J¼11.3 Hz),
4.42 (d, 1H, J¼11.3 Hz), 4.35 (dd, 1H, J¼11.3, 5.0 Hz), 4.10 (ddd, 1H,
J¼11.3, 5.0, 2.5 Hz), 3.63 (ddd,1H, J¼16.3,10.0, 5.0 Hz), 3.52–3.48 (m,
1H), 3.22 (dd,1H, J¼11.3,10.0 Hz), 3.07 (s, 3H), 3.04 (s, 3H), 2.90–2.82
(6.30 g, 90%), mp 96–97 ꢀC, [
(400 MHz, CDCl3)
a
]
ꢂ3.5 (c 0.8, CHCl3). 1H NMR
D
d
8.21 (s,1H), 7.58 (s,1H), 7.40–7.38 (m, 2H), 7.33–
7.32 (m, 3H), 7.26–7.19 (m, 5H), 7.04 (s, 1H), 6.18 (dd, 1H, J¼10.0,
8.8 Hz), 5.47 (s, 1H), 4.65 (d, 1H, J¼12.5 Hz), 4.48 (d, 1H, J¼12.5 Hz),
4.37 (dd, 1H, J¼10.0, 5.0 Hz), 4.20 (dd, 1H, J¼11.3, 6.3 Hz), 3.88–3.82
(m, 1H), 3.75–3.69 (m, 2H), 3.57–3.49 (m, 2H); 13C NMR (100 MHz,
(m, 1H), 1.76 (q, 1H, J¼11.3 Hz); 13C NMR (100 MHz, CDCl3)
d 137.5,
128.4, 127.9, 127.5, 76.2, 71.9, 71.2, 71.0, 70.1, 67.4, 38.6, 37.4, 37.0;
HRMS calcd for C15H22O8S2þH, 395.0834; found, 395.0835.
CDCl3)
d 183.7, 136.9, 135.3, 130.6, 129.0, 128.4, 128.3, 128.2, 127.8,
126.0,118.2,101.3, 82.6, 79.0, 75.0, 72.9, 71.2, 68.6, 68.5; HRMS calcd
4.2.6. Preparation of 1,5-anhydro-2,3-di-O-benzyl-6-bromo-6-
for C24H24N2O5SþH, 453.1484; found, 453.1476.
deoxy-4-O-methylsulfonyl-D-glucitol (16)
A mixture of dimesylate 15 (3.46 g, 8.7 mmol), NaBr (5.41 g,
52.6 mmol), and TBAB (846 mg, 2.6 mmol) in DMSO (20 mL) was
stirred at 60 ꢀC for 12 h. The reaction mixture was cooled to room
temperature and then poured into water (200 mL). The water phase
was extracted with ethyl acetate five times, the combined organic
phase was washed with water, dried (sodium sulfate), and con-
centrated. Flash chromatography gave 2.72 g (82% yield) colorless
crystals of 16 (along with 0.29 mg of recovered starting material 15,
4.2.3. Preparation of 1,5-anhydro-4,6-O-benzylidene-3-
deoxy-2-O-benzyl-D-ribo-hexitol (13)
To a magnetically stirred solution of 12 (5.76 g, 12.7 mmol) and
AIBN(afewcrystals)intoluene(30 mL)wasaddedsolutionofBu3SnH
(6.9 mL, 12.7 mmol) in toluene (30 mL) dropwise under N2. The re-
action mixture was immersed immediately in a pre-heated oil-bath
(120 ꢀC) once the adding of Bu3SnH solution began. After stirring at
120 ꢀC for 2 h, the reaction was treated dropwise with another por-
tion of Bu3SnH (6.9 mL, 12.7 mmol) in toluene (30 mL) and refluxed
overnight. The mixture is then cooled down to room temperature,
quenchedwithsaturatedNaHCO3 (50 mL)and EtOAc(50 mL), filtered
through a pad of CeliteÒ, and extracted with EtOAc (100 mLꢁ3). The
combined organic layer was washed with brine, dried (Na2SO4), and
concentrated underreducedpressure. Recrystallizationof theresidue
with EtOAc/hexane gave 2.54 g compound 13 as pure colorless crys-
tals. Another 1.10 g of product was obtained with column chroma-
tography purification of the mother liquid (EtOAc/hexane, 1:20).
8.3% recovered), mp 63–65 ꢀC, [
(400 MHz, CDCl3)
a
]
þ34.0 (c 1.9, CHCl3). 1H NMR
D
d
7.38–7.31 (m, 5H), 4.61 (d, 1H, J¼11.3 Hz), 4.58–
4.53 (m, 1H), 4.48 (d, 1H, J¼11.3 Hz), 4.14 (dd, 1H, J¼10.0, 3.8 Hz),
3.69 (dd, 1H, J¼11.3, 2.5 Hz), 3.69–3.61 (m, 1H), 3.50 (dd, 1H, J¼11.3,
5.0 Hz), 3.46–3.42 (m, 1H), 3.26 (t, 1H, J¼11.3 Hz), 3.10 (s, 3H), 2.88–
2.83 (m, 1H), 1.78 (q, 1H, J¼11.3 Hz); 13C NMR (100 MHz, CDCl3)
d
137.4, 128.3, 127.8, 127.4, 76.5, 74.6, 71.2, 71.1, 70.1, 38.8, 36.9, 31.6;
HRMS calcd for C14H19BrO5SþH, 379.0215; found, 379.0212.
4.2.7. Preparation of the bicyclic compound 18
Totally 3.64 g, yield 88%, mp 105–107 ꢀC, [
NMR (400 MHz, CDCl3)
a
]
D ꢂ40.5 (c 0.6, CHCl3). 1H
A
stirred solution of diethyl 2-[(tert-butyloxycarbonyl)-
d
7.54–7.52 (m, 2H), 7.42–7.31 (m, 8H), 5.54 (s,
amino]malonate (4.00 g, 14.5 mmol) in dry toluene (25 mL) under
N2 was treated with NaH (60% in mineral oil, 680 mg, 17.0 mmol).
The reaction mixture was stirred at room temperature for 0.5 h
before compound 16 (1.59 g, 4.2 mmol) in 25 mL toluene was added
dropwise. The reaction mixture was stirred at room temperature
for 1 h before TBAI (465 mg, 1.26 mmol) was added. The resultant
reaction mixture was then heated to reflux for 17 h. The reaction
mixture was cooled to 0 ꢀC, quenched by 0.5 N HCl (25 mL),
extracted with EtOAc (100 mLꢁ3), washed with brine, dried over
Na2SO4, filtered, and concentrated, the residue was purified by
column chromatography (hexane/EtOAc, 9:1 to 7:1) to produce
1H), 4.79 (d, 1H, J¼12.5 Hz), 4.70 (d, 1H, J¼12.5 Hz), 4.34 (dd, 1H,
J¼10.0, 5.0 Hz), 4.12 (dd,1H, J¼10.0, 5.0 Hz), 3.74–3.65 (m, 2H), 3.55–
3.48 (m, 1H), 3.36–3.30 (m, 2H), 2.65–2.60 (m, 1H), 1.72 (q, 1H,
J¼11.3 Hz); 13C NMR (100 MHz, CDCl3)
d 137.9, 137.3, 129.0, 128.4,
128.2, 127.8, 127.6, 126.1, 101.6, 76.5, 73.3, 72.0, 71.0, 70.5, 69.2, 35.6;
HRMS calcd for C20H22O4þH, 327.1596; found, 327.1591.
4.2.4. Preparation of 1,5-anhydro-2,-O-benzyl-3-
deoxy-D-ribo-hexitol (14)
A suspension of 13 (3.61 g, 11.1 mmol) in 80% HOAc (75 mL) was
heated to 55 ꢀC with stirring till all starting material was consumed.
compound 18 as thick oil (1.03 g, 52%), [
a]
D þ30.4 (c 3.2, CHCl3). 1H

5790
X. Nie, G. Wang / Tetrahedron 64 (2008) 5784–5793
NMR (300 MHz, CDCl3) mixture of two conformers
d
7.34–7.24 (m,
TFA/DCM (1:4, 10 mL) at 0 ꢀC for 5 h. The solvent was evaporated
under reduced pressure and saturated NaHCO3 (15 mL) and EtOAc
(25 mL) were added to the residue. The aqueous layer was extracted
with EtOAc three times. The combined organic layers were dried over
Na2SO4. Solvent was removed under reduced pressure. The crude
was purified by column chromatography (hexane/EtOAc, 7:3) to
give 21b (12 mg, 16%) and 21a (56 mg, 75%), totally 91% conversion.
Major isomer 21a, colorless oil: 1H NMR (300 MHz, CDCl3)
5H), 4.64–4.48 (m, 2H), 4.25–4.10 (m, 4.6H), 4.10–4.01 (m, 1.4H),
3.94–3.81 (m, 1H), 3.78–3.59 (m, 1H), 3.25–3.19 (m, 1.6H), 2.77 (d,
1.4H, J¼12.9 Hz), 2.38 (d, 1H, J¼12.9 Hz), 1.93–1.72 (m, 1H), 1.50 (s,
3.5H),1.43 (s, 5.5H),1.30–1.26 (m, 3.5H),1.22–1.17 (m, 2.5H); 13C NMR
(62.5 MHz, CDCl3)
d 169.3, 168.7, 168.4, 153.7, 153.2, 138.2, 128.3,
127.5,127.4, 80.7, 80.6, 77.2, 75.6, 74.8, 72.7, 72.5, 70.6, 70.2, 69.1, 69.0,
68.3, 61.9, 61.3, 59.4, 58.6, 42.3, 41.1, 30.5, 29.3, 28.2, 27.9, 13.9, 13.8;
HRMS calcd for C25H35NO8þH, 478.2441; found, 478.2441.
d
7.43–7.24 (m, 5H), 4.60 (d, 1H, J¼11.5 Hz), 4.54 (d, 1H, J¼11.5 Hz),
4.20 (q, 2H, J¼7.1 Hz), 3.95 (ddd, 1H, J¼10.7, 4.1, 2.2 Hz), 3.86–3.77
(m, 2H), 3.74 (dd, 1H, J¼10.2, 3.3 Hz), 3.23 (m, 1H), 3.08 (dd, 1H,
J¼10.4, 10.2 Hz), 2.84 (br, 1H), 2.46 (dq, 1H, J¼14.0, 2.2 Hz), 2.27 (ddd,
1H, J¼14.3, 10.2, 4.1 Hz), 2.13 (dd, 1H, J¼14.3, 3.3 Hz), 1.74 (ddd, 1H,
J¼14.0, 11.0, 4.9 Hz), 1.26 (t, 3H, J¼7.1 Hz); 13C NMR (75 MHz, CDCl3)
4.2.8. Synthesis of the protected O-Choi variants, ester 19 and acid 20
A solution of 18 (400 mg, 0.84 mmol) in ethanol (10 mL) was
treated with 1 N KOH (10 mL) and the reaction mixture was stirred
at 40–50 ꢀC till all starting material was consumed (about 12 h).
The reaction mixture was washed with ether (10 mLꢁ3). The
combined ether solution then was washed with water (10 mLꢁ2).
The aqueous solution was combined, acidified with 1 N HCl to pH
2–3, extracted with chloroform (50 mLꢁ5), dried over Na2SO4, and
concentrated. The residue was subjected to decarboxylation in
refluxing toluene (25 mL, about 14 h). Evaporation of the toluene
under reduced pressure and chromatographic purification gave
three potions, an overlapping mixture of esters 19b and 19a
(100 mg, 29%), pure 19a (130 mg, 38%), and mixture of acid 20a and
20b (50 mg, 15%), totally 82% conversion. Major isomer ester 19a
(R-configuration), colorless oil gradually solidified: 1H NMR
d
174.5, 138.2, 128.3, 127.6, 127.5, 76.9, 71.1, 69.2, 69.1, 61.1, 59.3, 58.6,
38.0, 31.2, 14.1; HRMS calcd for C17H23NO4þH, 306.1705; found,
306.1700. Minor isomer 21b, colorless oil: 1H NMR (300 MHz, CDCl3)
d
7.39–7.25 (m, 5H), 4.59 (d, 1H, J¼11.8 Hz), 4.55 (d, 1H, J¼11.8 Hz),
4.18 (q, 2H, J¼7.1 Hz), 4.05–3.97 (m, 2H), 3.93 (dd, 1H, J¼4.7, 2.7 Hz),
3.82 (ddd, 1H, J¼15.1, 10.2, 4.7 Hz), 3.43–3.40 (m, 1H), 3.15 (dd, 1H,
J¼10.7, 10.2 Hz), 2.36 (dp, 1H, J¼13.7, 2.2 Hz), 2.26 (dd, 1H, J¼14.0,
8.5 Hz), 2.22 (br, 1H), 2.04 (ddd,1H, J¼14.0, 7.7, 4.7 Hz), 1.68 (ddd, 1H,
J¼13.7, 10.7, 4.4 Hz), 1.27 (t, 3H, J¼7.1 Hz); 13C NMR (75 MHz, CDCl3)
d
175.4, 138.3, 128.4, 127.6, 127.5, 77.8, 71.0, 69.3, 69.2, 61.0, 58.0, 57.9,
37.3, 32.2, 14.2; HRMS calcd for C17H23NO4þH, 306.1705; found,
(300 MHz, CDCl3, mixture of two conformers ratio 6:4)
d
7.37–7.24
306.1703.
(m, 5H), 4.68–4.43 (m, 2.4H), 4.35 (d, 0.6H, J¼8.8 Hz), 4.19–4.07 (m,
2H), 4.05–3.98 (m, 1H), 3.97–3.70 (m, 3H), 3.22 (dd, 1H, J¼9.3,
9.0 Hz), 3.00 (d, 0.6H, J¼12.6 Hz), 2.66 (d, 0.4H, J¼12.6 Hz), 2.30 (m,
0.4H), 2.25 (m, 0.6H), 2.15 (dd, 0.6H, J¼9.3, 3.8 Hz), 2.10 (dd, 0.4H,
J¼9.3, 4.1 Hz), 1.76–1.93 (m, 1H), 1.49 (s, 3.6H), 1.44 (s, 5.4H), 1.17 (t,
4.2.11. Preparation of dipeptides 23a and 23b
At 0 ꢀC, a solution of acid 20 (mixture of diastereomers, 0.69 g,
1.83 mmol) and methyl ester of Nu-nitro-arginine$HCl 22 (0.49 g,
1.83 mmol) in DMF (5 mL) was treated subsequently with PyBOP
(0.95 g, 1.83 mmol) and DIEA (0.64 mL, 3.66 mmol). The reaction
was stirred at room temperature for 24 h before it was diluted with
EtOAc (200 mL), washed with saturated NaHCO3 and brine, dried
(Na2SO4), and concentrated. The residue was purified by column
chromatography (chloroform/hexane, 40:60 with addition of 3%
MeOH) to give pure dipeptides 23a (0.85 g, 79%) and 23b (0.12 g,
3H, J¼5.8 Hz); 13C NMR (75 MHz, CDCl3)
d 172.0, 171.8, 154.7, 154.2,
138.3, 128.2, 128.1, 127.5, 127.4, 80.1, 79.8, 76.0, 75.3, 70.6, 70.2, 69.5,
69.4, 68.7, 68.6, 60.6, 59.0, 57.5, 57.2, 35.2, 34.8, 30.5, 29.4, 28.2,
13.9; HRMS calcd for C22H31NO6þH, 406.2230; found, 406.2223.
4.2.9. Alternative method for synthesizing amino acid 20
Asolutionof18 (1.03 g, 2.16 mmol)inethanol (20 mL)wastreated
with 1 N KOH (20 mL) and the reaction mixture was stirred at
40–50 ꢀC till all starting material was consumed (about 12 h). The
reaction mixture was washed with ether (10 mLꢁ3). The combined
ether solution then was washed with water (10 mLꢁ2). The aqueous
solution was combined, acidified with 1 N HCl to pH 2–3, extracted
with chloroform (50 mLꢁ5), dried over Na2SO4, and concentrated.
The residue was subjected to decarboxylation in refluxing toluene
(25 mL, about 14 h). Evaporation of the toluene under reduced
pressure gave a crude mixture of ester 19 and acid 20, which was
dissolved in THF (25 mL), treated with 0.2 N LiOH (25 mL), and stir-
red at room temperature till all ester was consumed. The reaction
mixture was acidified with 1 N HCl (pH¼2–3). Extraction with
chloroform (30 mLꢁ5) followed by evaporation gave the acid 20
(diastereomers mixture ratio 7:1, 0.69 g, 85% yield for 3 steps), which
was used in the peptide coupling reaction without further purifica-
tion. Major isomer 20a can be obtained by purification of a small
amountofcrudethroughcolumnchromatography. Majorisomer20a
11%). Major isomer 23a: [
(300 MHz, CDCl3),
a
]
ꢂ13.7 (c 2.6, CHCl3). 1H NMR
D
d
8.82 (br, 1H), 7.80 (br, 2H), 7.35–7.26 (m, 5H),
7.12 (d, 1H, J¼8.3 Hz), 4.72–4.66 (m, 1H), 4.66 (d, 1H, J¼11.5 Hz),
4.58 (d, 1H, J¼11.5 Hz), 4.38 (d, 1H, J¼10.2 Hz), 4.10–3.84 (m, 4H),
3.76 (s, 3H), 3.62 (m, 1H), 3.36–3.25 (m, 1H), 3.21–3.13 (m, 1H), 2.85
(m, 1H), 2.39 (d, 1H, J¼13.7 Hz), 2.24–2.12 (m, 1H), 2.05–1.92 (m,
1H), 1.89–1.77 (m, 1H), 1.77–1.57 (m, 3H), 1.47 (s, 9H); 13C NMR
(62.5 MHz, CDCl3)
d 173.5, 172.4, 159.4, 156.3, 138.5, 128.4, 127.7,
127.6, 81.7, 77.2, 71.0, 69.5, 69.2, 62.1, 59.7, 52.6, 50.3, 40.2, 34.6,
31.2, 28.3, 24.3; HRMS calcd for C27H40N6O9þH, 593.2935; found,
593.2938. Minor isomer 23b: 1H NMR (300 MHz, CDCl3),
d 8.66 (br,
1H), 7.82 (br, 2H), 7.35–7.27 (m, 5H), 6.72 (d, 1H, J¼8.0 Hz),
4.66–4.60 (m, 1H), 4.59 (d, 1H, J¼11.5 Hz), 4.52 (d, 1H, J¼11.5 Hz),
4.31 (t, 1H, J¼7.6 Hz), 4.20–4.14 (m, 1H), 4.10–4.02 (m, 1H),
4.00–3.95 (m, 1H), 3.76 (s, 3H), 3.69–3.46 (m, 2H), 3.38–3.20 (m,
2H), 2.63–2.51 (m, 1H), 2.24–2.14 (m, 1H), 2.07–1.88 (m, 3H),
1.85–1.59 (m, 3H), 1.42 (s, 9H); 13C NMR (62.5 MHz, CDCl3)
d 173.3,
172.2, 159.4, 154.8, 138.1, 128.5, 127.8, 127.5, 81.1, 76.2, 70.8, 69.5,
68.4, 60.8, 57.0, 52.8, 51.0, 40.4, 34.1, 30.4, 30.1, 28.4, 23.8; HRMS
calcd for C27H40N6O9þH, 593.2935; found, 593.2930.
(R-configuration): 1H NMR (300 MHz, CDCl3)
d 7.37–7.28 (m, 5H),
4.60 (d, 1H, J¼11.5 Hz), 4.52 (d, 1H, J¼11.5 Hz), 4.53–4.47 (m, 1H),
4.12–4.06 (m, 1H), 4.00–3.88 (m, 2H), 3.84–3.75 (m, 1H), 3.38–3.22
(m,1H), 2.47–2.33 (m,1H), 2.26–2.14 (m,1H),1.93–1.84 (m,1H),1.58–
4.2.12. Synthesis of dipeptide 27
1.51 (m, 1H), 1.48 (s, 9H); 13C NMR (100 MHz, CDCl3)
d
176.4, 155.3,
PyBOP (1.56 g, 3.0 mmol) and DIEA (1.05 mL, 6.0 mmol) were
138.2, 128.3, 127.6, 127.5, 80.7, 75.7, 70.7, 69.3, 68.6, 59.0, 57.6, 34.6,
30.1, 28.3; HRMS calcd for C20H27NO6þH, 378.1917; found, 378.1916.
added subsequently to a stirred solution of (R)-
b
-[4-(benzyloxy)
phenyl]lactic acid 2623 (680 mg, 2.5 mmol) and
D-leucine ethyl
ester hydrochloride 25 (590 mg, 3.0 mmol) in DCM (25 mL) at 0 ꢀC.
After stirring for 24 h at room temperature, the reaction mixture
was diluted with EtOAc (100 mL), washed with saturated NaHCO3,
1 N HCl, water, and brine, dried over Na2SO4, and concentrated
4.2.10. Synthesis of amines 21a and 21b
The overlapping mixture of ester 19b and 19a (100 mg,
0.25 mmol) obtained from the previous step was treated with

X. Nie, G. Wang / Tetrahedron 64 (2008) 5784–5793
5791
under reduced pressure. The crude was purified by column chro-
13C NMR (75 MHz, CDCl3)
d
174.9, 173.8, 172.9, 171.8, 159.2, 157.7,
matography (hexane/EtOAc, 7:2) to get pure dipeptide 26 (970 mg,
138.3, 137.8, 136.8, 130.5, 130.3, 129.2, 128.8, 128.5, 128.3, 128.1,
127.9, 127.7, 127.4, 114.9, 114.7, 114.4, 77.1, 74.8, 72.7, 72.4, 70.4, 70.3,
69.8, 69.6, 69.3, 65.1, 60.7, 59.5, 50.7, 50.4, 40.7, 39.8, 39.5, 39.3, 36.3,
29.6, 29.4, 27.9, 25.1, 24.9, 24.6, 24.4, 23.4, 23.2, 21.4, 21.2, 20.7;
HRMS calcd for C43H57N7O10þH, 832.4245; found, 832.4255.
94%), mp 85–87 ꢀC. 1H NMR (300 MHz, CDCl3)
d 7.44–7.31 (m, 5H),
7.18 (d, 2H, J¼8.5 Hz), 6.92 (d, 2H, J¼8.5 Hz), 6.91 (d, 1H, J¼8.2 Hz),
5.04 (s, 2H), 4.61–4.54 (m, 1H), 4.34–4.29 (m, 1H), 4.17 (q, 2H,
J¼7.1 Hz), 3.14 (dd, 1H, J¼14.3, 4.1 Hz), 3.11 (d, 1H, J¼4.1 Hz), 2.89
(dd,1H, J¼14.0, 7.4 Hz),1.66–1.42 (m, 3H),1.28 (t, 3H, J¼7.1 Hz), 0.92
(d, 3H, J¼1.7 Hz), 0.90 (d, 3H, J¼1.4 Hz); 13C NMR (62.5 MHz, CDCl3)
4.2.15. Preparation of aeruginosin 298-A analog I
d
173.0,172.8,157.8,137.0,130.7,129.0,128.5,127.9,127.4,114.8, 72.7,
Compound 29 (27 mg, 0.032 mmol) in 2.5 mL methanol con-
69.9, 61.3, 50.3, 41.3, 39.6, 24.6, 22.8, 21.8, 14.1; HRMS calcd for
24H31N O5þH, 414.2280; found, 414.2256.
taining 12.5 mL TFA was treated with Pd/C (2.0 mg). The suspension
C
was sequentially evacuated and purged with H2, stirred under
atmosphere of H2 (1 atm) for 24 h. The reaction mixture was filtered
through a pad of Celite, which was washed with several portion of
methanol. The combined solutionwas concentratedand dried under
vacuum to afford aeruginosin 298-A analog I as colorless amorphous
4.2.13. Synthesis of tetrapeptide 28
A solution of 27 (145 mg, 0.35 mmol) in THF (7.5 mL) was
treated with 0.2 N LiOH (7.5 mL) at room temperature. The reaction
mixture was stirred overnight and quenched with 1 N HCl. The
mixture was extracted with EtOAc, washed with brine, and dried
with Na2SO4. After evaporation of the solvent, the crude acid 27 was
dried under vacuum for 2 h.
To a solution of compound 23a (170 mg, 0.29 mmol) in DCM
(8 mL) was added TFA (2 mL) at 0 ꢀC. The reaction mixture was
warmed up to room temperature and stirred for 2 h before the
solvent was evaporated under reduced pressure. The TFA salt 24
was dried under vacuum for 2 h.
solid (30 mg, 98%), [
MeOH-d4, mixture of conformers)
a
]
þ50.8 (c 0.4, MeOH). 1H NMR (300 MHz,
D
d
8.03 (d, 0.6H, J¼8.5 Hz),
7.96–7.79 (br, 0.6H), 7.59 (d, 0.4H, J¼8.2 Hz), 7.53–7.40 (br, 0.4H),
7.06 (d, 2H, J¼8.0 Hz), 6.69 (d, 2H, J¼8.2 Hz), 4.73–4.57 (m, 1H),
4.57–4.39 (m,1H), 4.39–4.11 (m, 3H), 4.10–3.98 (m, 2H), 3.96 (s,1H),
3.92–3.82 (m, 1H), 3.82–3.72 (m, 1H), 3.72–3.46 (m, 1H), 3.38 (d,
2.4H, J¼5.2 Hz), 3.27–3.08 (m, 2H), 3.03 (t,1.6H, J¼9.6 Hz), 2.90–2.63
(m, 2H), 2.54 (dd,1H, J¼8.5, 4.4 Hz), 2.43–2.05 (m, 2H),1.79–1.41 (m,
6H), 1.41–1.22 (m, 1H), 0.90 (d, 3H, J¼6.0 Hz), 0.85 (d, 3H, J¼6.3 Hz);
HATU (135 mg, 0.35 mmol) and DIEA (0.15 mL, 0.87 mmol) were
added subsequently to a stirred solution of acid 27 and TFA salt 24
in DMF (2.5 mL) at 0 ꢀC. After stirred for 24 h at room temperature,
the reaction was quenched with saturated NaHCO3, extracted with
EtOAc (20 mLꢁ5), washed with brine, dried over Na2SO4, and
concentrated. The residue was purified by column chromatography
(2% MeOH in chloroform) to get pure tetrapeptide 28 (192 mg, 77%),
13C NMR (75 MHz, CDCl3, major conformer)
d 175.0, 173.8, 171.8,
157.0, 155.5, 130.1, 127.8, 114.4, 74.5, 72.1, 70.2, 63.5, 61.4, 60.6, 59.9,
51.0, 50.4, 40.7, 39.2, 39.0, 35.4, 31.9, 27.7, 25.1, 24.1, 22.1,19.8; HRMS
calcd for C29H46N6O8þH, 607.3455; found, 607.3455.
4.3. Synthesis of aeruginosin 298-A analog II
[
a
]
þ36.6 (c 1.4, CHCl3). 1H NMR (300 MHz, CDCl3, mixture of
The synthesis followed the same methods described in Section
4.2, only characterization data are given here.
D
conformers) d 8.62 (br, 1H), 8.03 (br, 1H), 7.60 (br, 2H), 7.41–7.24 (m,
10H), 7.18 (br, 1H), 7.14 (d, 2H, J¼8.2 Hz), 6.91 (d, 2H, J¼8.2 Hz), 5.02
(s, 2H), 4.72–4.59 (m, 2H), 4.62 (d, 1H, J¼11.8 Hz), 4.50 (d, 1H,
J¼11.8 Hz), 4.42–4.36 (m, 1H), 4.33 (d, 1H, J¼8.8 Hz), 4.30–4.13 (m,
1H), 4.10 (d,1H, J¼2.7 Hz), 4.03–3.82 (m, 2H), 3.73 (s, 3H), 3.69–3.59
(m, 1H), 3.54–3.30 (m, 2H), 3.28–3.17 (m, 1H), 3.09 (dd, 1H, J¼13.7,
5.2 Hz), 2.85 (dd, 1H, J¼14.0, 7.7 Hz), 2.76–2.42 (m, 2H), 2.32–2.13
(m, 2H), 2.04–1.36 (m, 6H), 1.30–1.18 (m, 1H), 0.91 (complex, 6H,
4.3.1. Synthesis of O-Choi variant, compound 30
Under the same condition described for 20, compound 30 was
synthesized as an inseparable diastereomeric mixture in 85% yield.
Major isomer 30a (S-configuration) is the enantiomer of 20a
(R-configuration), thus has identical NMR spectrum as 20a.
J¼6.6, 6.0 Hz); 13C NMR (62.5 MHz, CDCl3)
d
175.0, 174.6, 173.7,
4.3.2. Synthesis of dipeptide 33
173.1, 172.2, 171.7, 171.2, 159.2, 157.7, 157.6, 138.4, 138.0, 136.9, 130.6,
130.5, 130.3, 129.2, 128.8, 128.4, 127.8, 127.5, 114.8, 114.6, 114.5, 77.2,
74.4, 72.6, 72.4, 70.5, 69.8, 69.3, 60.8, 60.4, 59.6, 52.5, 52.4, 51.3,
50.3, 48.2, 40.8, 40.5, 40.0, 39.6, 39.4, 36.2, 31.0, 29.6, 29.4, 29.1,
25.0, 24.4, 23.4, 23.2, 21.4, 21.1, 20.8; HRMS calcd for
Under the same condition described for 22, compound 31 was
synthesized from compound 30 as an inseparable diastereomeric
mixture in 71% yield. Then 31 was subjected to the same reaction
condition for 28, to give pure tetrapeptides 33b (8%) and 33a (57%).
For major isomer 33a: [
CDCl3, mixture of conformers) d 8.59 (br, 1H), 7.40–7.24 (m, 10H),
a
]
þ8.0 (c 0.6, CHCl3). 1H NMR (400 MHz,
D
C
44H57N7O11þH, 860.4194; found, 860.4199.
7.16 (d, 1.33H, J¼8.4 Hz), 7.11 (d, 0.67, J¼8.4 Hz), 6.90 (br, 1H), 6.87
(d, 2H, J¼8.4 Hz), 4.99 (s, 0.67H), 4.97 (s, 1.33H), 4.74–4.45 (m, 6H),
4.45–4.36 (m, 2H), 4.27–4.25 (m, 0.67H), 4.19–4.13 (m, 1.33H),
4.08–4.02 (m,1.33H), 3.93–3.84 (m, 0.67H), 3.75 (s,1H), 3.70 (s, 2H),
3.76–3.66 (m, 2H), 3.44–3.02 (m, 4H), 2.95–2.71 (m, 2H), 2.68–2.56
(m, 1H), 2.51–2.39 (m, 1H), 2.37–2.16 (m, 2H), 2.10–1.80 (m, 1H),
1.77–1.49 (m, 3H), 1.27–1.17 (m, 2H), 0.84 (complex, 6H, J¼6.2 Hz);
4.2.14. Synthesis of compound 29
Freshly prepared 0.2 N LiBH4 solution in THF (1.0 mL,
0.20 mmol) was added via syringe to a N2 protected solution of 28
(132 mg, 0.15 mmol) in dry THF (5 mL) at 0 ꢀC. The resulting solu-
tion was stirred at 0 ꢀC for 30 min, another 1.0 mL 0.2 N LiBH4
solution was added, and continued stirring at 0 ꢀC for 30 min. The
reaction was quenched with water followed by saturated NH4Cl.
The resulting mixture was extracted with THF/CHCl3 (10:90,
20 mLꢁ5). The extracted solution was dried over Na2SO4, concen-
trated, and the crude product was purified by chromatography
(3–5% MeOH in CHCl3) to give pure 29 (91 mg, 72% yield). 1H NMR
13C NMR (100 MHz, CDCl3)
d 174.5, 174.0, 173.0, 172.6, 172.4, 171.9,
159.6, 159.5, 158.0, 157.9, 138.3, 138.2, 137.3, 137.2, 131.0, 130.9,
129.5, 129.2, 128.8, 128.7, 128.6, 128.2, 128.0, 127.9, 127.8(2), 127.7,
114.9, 114.8, 76.0, 74.0, 72.9, 72.8, 71.1, 70.6(2), 70.1, 69.8, 69.4, 65.1,
62.3, 60.7, 59.5, 59.0, 53.0, 52.9, 52.8, 51.8, 49.6, 49.2, 41.6, 40.9, 40.7,
39.9, 39.8, 29.9, 29.7, 28.5, 24.6, 24.5, 23.6, 21.5, 21.3; HRMS calcd
for C44H57N7O11þH, 860.4194; found, 860.4199.
(300 MHz, CDCl3, mixture of conformers) d 8.63 (br,1H), 8.07 (d,1H,
J¼9.1 Hz), 7.74 (br, 2H), 7.39–7.25 (m, 10H), 7.18 (br, 1H), 7.15 (d, 2H,
J¼6.4 Hz), 6.89 (d, 2H, J¼8.2 Hz), 4.99 (m, 2H), 4.74–4.51 (m, 2H),
4.51–4.41 (m, 1H), 4.41–4.25 (m, 3H), 4.25–4.14 (m, 1H), 4.14–4.00
(m, 2H), 3.99–3.79 (m, 1H), 3.77–3.59 (m, 2H), 3.58–3.38 (m, 2H),
3.36–2.99 (m, 3H), 2.83 (br, 2H), 2.71–2.46 (complex, 1H), 2.42–1.97
(m, 3H), 1.82–1.11 (m, 7H), 1.00–1.73 (complex, 6H, J¼5.5, 5.0 Hz);
4.3.3. Synthesis of compound 34
Under the same condition described for 29, compound 33 was
reduced with LiBH4 to give pure compound 34 in 52% yield. 1H NMR
(400 MHz, CDCl3, mixture of conformers)
d 8.55 (br, 1H), 7.56 (br,

5792
X. Nie, G. Wang / Tetrahedron 64 (2008) 5784–5793
2H), 7.41–7.25 (m, 10H), 7.16 (d, 2H, J¼8.8 Hz), 7.15–7.05 (m, 2H),
6.89 (d, 2H, J¼8.4 Hz), 4.97 (s, 2H), 4. 56 (s, 2H), 4.43–4.25 (m, 4H),
4.16–4.05 (m, 1H), 3.95–3.71 (m, 3H), 3.71–3.41 (m, 4H), 3.27–3.01
(m, 3H), 2.88–2.84 (m, 1H), 2.77–2.49 (m, 3H), 2.45–2.33 (m, 1H),
2.29–2.17 (m, 1H), 2.12–2.04 (m, 1H), 1.75–1.42 (m, 4H), 1.41–1.19
(m, 2H), 0.87 (d, 3H, J¼6.2 Hz), 0.82 (d, 3H, J¼6.2 Hz); 13C NMR
Trypsin assays were run similarly; a commercial egg white
trypsin inhibitor was used as the standard for calibration of the
enzyme concentrations. Buffer solution is composed of 50 mM
Tris–HCL (pH¼8), 100 mM NaCl, 1 nM CaCl2 aqueous solution. A
mixture of 50
(0.05 mg/mL), and 40
then 100 L substrate N-benzoyl-DL-arginine-4-nitroanilide hy-
drochloride (0.43 mg/mL solution in 10%DMSO and water) was
added. The final volume was 200
L, this was incubated at 37 ꢀC for
m
L of inhibitor solution, 10
m
L of trypsin solution
mL buffer solution was incubated for 5 min,
(100 MHz, CDCl3)
d
175.8, 173.2, 172.5, 159.5, 158.0, 138.1, 137.2,
m
131.0, 129.4, 128.8, 128.7, 128.2, 128.0, 127.7(2), 114.8, 77.5, 74.8,
72.9, 70.9, 70.6, 70.2, 66.4, 59.9, 54.3, 50.7, 50.1, 41.2, 40.3, 39.8, 31.5,
29.4, 28.9, 24.8, 23.5, 21.5; HRMS calcd for C43H57N7O10þH,
832.4245; found, 832.4255.
m
30 min with slow shaking. The UV absorbance at 405 nm before
adding substrate was recorded as the blank. The absorbance at
405 nm was recorded immediately after incubation and IC50 value
was calculated as an average of duplicate experiment.
4.3.4. Synthesis of aeruginosin 298-A analog II
Compound 34 was subjected to the same reaction condition as
for aeruginosin 298-A analog I, to give aeruginosin 298-A analog II
Acknowledgements
in 99% yield, [
a]
þ8.9 (c 0.47, MeOH). 1H NMR (400 MHz, MeOH-
D
d4, mixture of conformers)
d 7.68–7.64 (m, 1H), 7.59–7.56 (m, 1H),
We are grateful to the financial support from American Heart
Association scientist development grant #0430285N. We also
thank Branden Hopkinson and Navneet Goyal for their help in
running the enzyme assays and Sherwin Cheuk for his help in
preparing some intermediates.
7.05 (d, 1H, J¼8.4 Hz), 7.00 (d, 1H, J¼8.4 Hz), 7.05 (d, 1H, J¼8.4 Hz),
6.67 (d, 1H, J¼8.8 Hz), 6.64 (d, 1H, J¼8.1 Hz), 4.98 (d, 1H, J¼8.1 Hz),
4.72–4.54 (m, 1H), 4.43–4.31 (m, 2H), 4.26–4.13 (m, 2H), 4.09–3.96
(m, 2H), 3.96–3.67 (m, 4H), 3.59–3.36 (m, 3H), 3.26–3.09 (m, 2H),
3.07–2.86 (m, 2H), 2.85–2.67 (m, 2H), 2.43–2.14 (m, 3H), 1.75–1.42
(m, 6H), 1.37–1.22 (m, 2H), 0.95–0.80 (m, 4H), 0.73 (d, 2H,
J¼6.2 Hz); 13C NMR (100 MHz, MeOH-d4, major conformer)
d 176.3,
Supplementary data
176.1, 175.9, 174.3, 174.0, 173.8, 158.8, 158.7, 157.4, 157.3, 132.1, 131.0,
129.3, 129.2, 116.1, 115.6, 76.6, 75.4, 73.9, 73.7, 71.8, 68.1, 65.1, 64.8,
64.4, 62.9, 62.6, 60.9, 60.5, 54.3, 52.5, 52.4, 52.3, 50.7, 42.5, 42.4,
42.3, 41.7, 40.9, 40.8, 37.9, 33.4, 33.3, 31.0, 29.6, 29.4, 26.7, 26.3, 25.7,
1H and 13C NMR spectra for compounds 11–16, 18, 19a, 20a, 21a,
21b, 23a, 23b, 27–29, I, 33a, 34, II are provided. The 2D COSY and
NOESY spectra for compounds 21a and 21b are also provided.
Supplementary data associated with this article can be found in the
online version, at doi:10.1016/j.tet.2008.03.107.
25.5, 24.0, 23.9, 22.0, 21.9; HRMS calcd for
607.3455; found, 607.3460.
C
29H46N6O8þH,
References and notes
4.4. General procedures for enzyme inhibition assays
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trypsin inhibitor from chicken egg white, and trypsin substrate
N-benzoyl-DL-arginine-4-nitroanilide hydrochloride were pur-
chased from Sigma; chromogenic thrombin substrate, H-D-HHT-
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a microtiter plate reader (Biotek Powerwave XS Spectrophotome-
ter). A mixture of 50
thrombin solution (0.25 NIH units/mL), and 30
solution was incubated at 37 ꢀC for 10 min, then 50
m
L of inhibitor solution, 20
L thrombin buffer
L of substrate
mL of bovine
m
m
Spectrozyme (0.032 mg/mL) was added to each cell, this final
mixture was then incubated for 30 min at 37 ꢀC with slow shaking.
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405 nm before adding substrate was recorded as the blank. The
absorbance at 405 nm was measured immediately after the in-
cubation. The IC50 value was obtained from the curve of inhibitor
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