Synthesis and evaluation of substituted indolizidines as peptidomimetics of RGD tripeptide sequence

Article abstract of DOI:10.1016/j.tet.2008.12.015

The synthesis of seven peptidomimetics of RGD is presented. The indolizidine building block was obtained by condensation of allylglycine with dimethoxydihydrofuran followed by an intermolecular cyclization. The bicyclic ring was functionalised with a carb

Full text of DOI:10.1016/j.tet.2008.12.015

Tetrahedron 65 (2009) 1402–1414
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journal homepage: www.elsevier.com/locate/tet
Synthesis and evaluation of substituted indolizidines as peptidomimetics
of RGD tripeptide sequence
a
a
,
b,
Delphine Halie , Joe¨lle Perard-Viret , Sylvie Dufour y, Jacques Royer a
*
´
a UMR 8638,CNRS/Universite´ Paris Decsartes, Faculte´ des Sciences Pharmaceutiques et Biologiques, 4 avenue de l’observatoire, 75270 Paris Cedex 06, France
b UMR144, CNRS/Institut Curie, 26 rue d’Ulm, 75248 Paris cedex 05, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 October 2008
Received in revised form 4 December 2008
Accepted 5 December 2008
Available online 9 December 2008
The synthesis of seven peptidomimetics of RGD is presented. The indolizidine building block was ob-
tained by condensation of allylglycine with dimethoxydihydrofuran followed by an intermolecular cy-
clization. The bicyclic ring was functionalised with a carboxylic acid and a guanidinium appendage. The
seven peptidomimetics were evaluated by cell-adhesion assays.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
a scaffold, which could be converted into a potent integrin ligand by
introducing a carboxylic and a guanidine appendage (Fig. 1).
Integrins are transmembrane receptors, which regulate cell at-
tachment and response to the extracellular matrix. They are in-
volved in many physiopathological processes: coagulation of blood,
tumor-induced angiogenesis, osteoporosis, restenosis, acute renal
failure, ocular diseases, metastasis formation, and sickle cell anemia
and represent an interesting target for therapeutic.1
Among the various known integrins the avb3 and avb5 integrin
receptors are of great interest since they are involved in angio-
genesis, a target for cancer therapy by inhibiting vascularization of
tumors.2
2. Results and discussion
In this paper, we report the synthesis and the biological activity
of RGD peptidomimetics 1. On this bicyclic compound, the length
and the relative geometry of the two side chains bearing the acid
and basic functions can be adapted to optimize the affinity with the
receptor. The peptidomimetics were evaluated in vitro by cell-ad-
hesion assays.
The tripeptide Arg-Gly-Asp (RGD) is the motif of recognition of
most integrins on several matrix proteins (vitronectin (VN), fibro-
nectin (FN)). The specificity of each integrin depends on the con-
formation of the triad induced by the other residues.3
2.1. Synthesis of the indolizidine building block
The first step was the condensation of dimethoxydihydrofuran8
(2) with racemic allylglycine (3) in acidic medium (Scheme 1).
This condensation led to lactam 4, which was directly cyclized in
refluxing formic acid to give only two diastereoisomers at the C7 po-
sition 5 and 6 (60:40), which can be separated through column chro-
matography. Practically, the crude reaction mixture was used. The
formiate was hydrolyzed by KHCO3 (1.1 equiv) in MeOH to yield alco-
hols 7 and 8 in 86% yield. The hydroxyl group was protected as a benzyl
To date, several inhibitors have been imagined and prepared.4 A
cyclic pentapeptide (c(–RGDf[NMe]V–) cilengitide) was found quite
active while a lot of studies have emerged in order to access pep-
tidomimetics.5 In these series of compounds, azabicycloalkanes
have been developed as versatile scaffolds to mimic peptide con-
formation.6 Their bicyclic ring backbone gives geometry with fixed
dihedral angles, on which the acid (Asp) and basic (Arg) side chains
can be placed at an optimal distance of 13 Å allowing the
recognition.
O
R2
O
R1O
N
H
O
OH
n
We have been interested in developing the synthesis of several
new structures as integrin inhibitors since we recently developed
an efficient synthesis of indolizin-3-one.7 We envisaged it as
N
H
HN
R1 = H, Bn
2 = H, COOH
O
NH2
R
mNH
* Corresponding author. Tel.: þ33 1 5373 9748; fax: þ33 1 4329 1403.
O
n = 1,2
m = 0,1
´
E-mail addresses: joelle.perard@univ-paris5.fr (J. Perard-Viret), sylvie.dufour@
1
curie.fr (S. Dufour).
y
Figure 1.
Tel.: þ33 1 5624 6335; fax: þ33 1 5624 6349.
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.12.015

D. Halie et al. / Tetrahedron 65 (2009) 1402–1414
1403
O
O
O
O
O
O
HO
OMe
HO
HOCO
OMe
HOCO
ii
OMe
OMe
O
OMe
O
OMe
O
i
NH2, HCl
iii
N
N
N
N
3
N
+
+
O
+
O
O
O
H
O
H
H
H
5
7
8
6
2
4
dr = 40/60
v
O
O
O
N
R1O
R1O
R1O
R1O
OEt
OH
O
OMe
OMe
O
iv
N
N
+
+
N
O
O
H
H
H
H
9a R = Bn
10a R = Bn
12a R = Bn
11a R = Bn
9b R= MOM
10b R= MOM
12b R= MOM
11b R= MOM
Scheme 1. Synthesis of the indolizidine building block: (i) HCl 1 M; (ii) HCOOH, reflux, 60% (2 steps); (iii) KHCO3 (1.1 equiv), MeOH, 0 ꢀC, 86%; (iv) R1¼Bn, trichloroacetimidate
i
(3 equiv), TfOH (0.25 equiv), C6H12/CH2Cl2, 22 h, 76%; R1¼MOM, Pr2NEt (4.5 equiv), MOMCl (4.5 equiv), THF, rt, 2 days, 87%; (v) NaBH4, EtOH, reflux, R1¼Bn, 84%, R1¼MOM, 83%.
or a MOM ether in good yield to give 9a, 10a (76%) and 9b, 10b (87%),
respectively.
same intermediates. This transformation was attempted with
benzyl ether 16a. Actually, this synthetic way was not useful: the
yield was low (oxidation of the primary alcohol into acid) and
purification of the guanidine compound was difficult (several
chromatographies were needed). Consequently, we decided to
begin with the acid side chain.
In this sequence, we started working on the acid side chain (Scheme
3). Alcohol 16a was oxidized by Jones reagent to give the acid 18 in good
yield (67%). Coupling with b-alanine tert-butyl ester and L-aspartic acid
Their reaction with NaBH4 in refluxing ethanol allowed an easy
separation of the two diastereoisomers. Indeed isomer 10a and 10b,
which exhibited a cis group were not reduced but just trans-
esterified by ethanol. Compounds 12a, 12b and 11a, 11b were iso-
lated in 84% and 83% overall yield. NMR studies made it possible to
establish a cis-diaxial relationship between the hydroxyl and ester
groups in compound 10 (or 12) and could explain the chemo-
selectivity of the NaBH4 reduction.
di-tert-butyl ester furnished 19 (83%) and 20 (88%), respectively.
Hydroboration of 19 or 20 gave the alcohols 21 (71%) or 22
(68%). Contrary to several reports, substitution of the hydroxyl
group of 21 by a guanidine through a Mitsunobu reaction with
DIAD/PPh3 and di-Boc- or di-Cbz-guanidine was proved to be un-
successful.9 We thus investigated the more reactive reagents dipi-
peridinazodicarboxylate (ADDP) and tributylphosphine (Bu3P).10
Surprisingly, use of these reagents led to an unexpected compound:
the guanidine carbamate 23.11
2.2. Functionalization of the indolizidine building block
Having prepared the bicyclic skeleton, we had to introduce the
two side chains with an appropriate relative configuration. An allyl
group was first introduced (a to the carbonyl of the lactam) to be
transformed into the guanidine. This allylation was performed after
deprotonation using LDA in tetrahydrofuran and allyl bromide
(Scheme 2). Lactams 11a and 11b bearing the ether and hydroxyl
functions in a trans relationship were deprotonated with 2 equiv of
LDA to furnish, after treatment with allyl bromide, 16aþ17a (87:13,
46% yield) and 16bþ17b (71:29, 54%), respectively. When the
allylation was conducted with the diprotected derivative 13 (MOM
and TBDMS ether), 14 and 15 were isolated in improved yield and
diastereoselectivity (10:90, 88%). It is noteworthy that the diastereo-
selectivity was inverted compared to the free hydroxyl series. Thus
both isomer series can be easily attained by using protected or
unprotected alcohol. In all cases, the relative configuration of the
allyl group was assigned by NOE experiments.
4
Eventually, starting from compound 22, use of DEAD/PPh3 in-
stead DIAD/PPh3 permitted to access to the guanidilated product
25. Boc deprotection with trifluoroacetic acid gave two new mimics
24 and 26. Hydrogenation on Pd/C of 25 followed by trifluoroacetic
acid treatment also gave another analogue 27.
The same sequence was applied to the trans diastereoisomer
17b (Scheme 4).
Oxidation of alcohol 17b gave acid 28 in moderate yield (54%),
which was coupled with L-glutamic acid di-tert-butyl ester (90%).
Then after hydroboration of 29 (65%), subsequent reaction of thus
obtained 30 with di-Boc-guanidine led to 31 (carbamate) and 33
(guanidinium). TFA-cleavage of the tert-butoxy and tert-butoxy-
carbonyl groups of 31 and 33 afforded 32 and 34, respectively.
We investigated the transformation of compounds 12a.
When these esters reacted with strong bases, a carbanion was
The next task was to perform the functional group interchanges
to obtain the acidic and basic side chains.
We anticipated transforming the allyl group into guanidine.
Furthermore, different acid side chains could be obtained from the
MOMO
MOMO
MOMO
OTBDMS
O
OTBDMS
OTBDMS
N
ii
N
N
O
O
H
i
H
H
+
RO
OH
13
N
15
14
88%, dr 10 : 90
O
H
ii
RO
RO
iii
OH
OH
11a R = Bn
11b R= MOM
N
N
O
O
H
H
+
a: R = Bn, 46%, dr 87 : 13
b: R = MOM, 54% dr 71 : 29
17
16
Scheme 2. Allylation of the indolizidine: (i) Et3N, TBDMSOTf, CH2Cl2, ꢁ10 ꢀC, 88%; (ii) LDA, ꢁ78 ꢀC, allyl bromide, THF; (iii) Bu4NF, THF, 97%.

1404
D. Halie et al. / Tetrahedron 65 (2009) 1402–1414
O
R1
O
O
R1
BnO
COOtBu
BnO
BnO
BnO
COOtBu
N
H
O
OH
O
OH
O
N
H
O
N
N
N
N
iii
ii
H
H
H
i
H
21 R1 = COOtBu
22 R1 = H
18
16a
19 R1 = COOtBu
20 R1 = H
HO
COOH
COOH
COOtBu
O
O
BnO
COOtBu
BnO
H
N
N
H
O
H
O
N
N
H
iv
vi
21
23
24
BocN
HN
O
NH2
O
O
NH2
NH
NH
O
O
O
BnO
COOtBu
R2O
COOH
N
H
O
N
H
O
N
N
H
H
v
vii
viii
22
26 R2 = Bn
27 R2 = H
25
NBoc
NBoc
H2N
NH
NH
H2N
Scheme 3. Functionalization of the cis diastereoisomer: (i) CrO3, H2SO4, acetone, 0 ꢀC, 40 min, 67%; (ii) NMM, ClCOOiBu, L-BAla-OtBu or L-Asp(OtBu)-OtBu, CH2Cl2, R1¼H, 83%,
R1¼COOtBu, 88%; (iii) (a) 9-BBN; (b) H2O2, AcONa, R1¼COOtBu, 68%, R1¼H, 71%; (iv) ADDP, PBu3, di-Boc-guanidine, toluene, reflux, 2 h, 60%; (v) DEAD, PPh3, di-Boc-guanidine, THF,
rt, 64 h, 92%; (vi) TFA, CH2Cl2, 100%; (vii) R2¼Bn, TFA, CH2Cl2, 100%; (viii) R2¼H; (a) H2 Pd/C 10%, 72%; (b) TFA, CH2Cl2, 100%.
formed at carbon 5. First, this center was methylated by using
KHMDS and methyl iodide in good yield to obtain single dia-
stereoisomer 35 (Scheme 5). Then allylation was performed
with the same organic base and allyl bromide, the diastereo-
selectivity was low (de 20%) and the yield was moderate (55%)
(Scheme 5).
The double bond of 36 and 37 was converted into guanidinium
group with DEAD/PPh3 in good overall yield (54% and 87%, re-
spectively, for 3 steps). Saponification of ethyl ester was very dif-
ficult because of the steric hindrance. We checked several bases:
NaOH, KOH, KOtBu, NaSMe, LiSMe, leading to no reaction or deg-
radation of the starting material. Indeed, acidic conditions were
COOtBu
O
O
COOtBu
O
MOMO
COOtBu
MOMO
MOMO
ii
MOMO
N
H
O
COOtBu
OH
O
N
H
O
OH
O
N
N
N
N
iii
i
H
H
H
H
30
29
28
17b
HO
COOH
COOtBu
O
O
MOMO
HO
N
H
O
COOtBu
N
H
O
COOH
N
N
H
H
iv
vi
31
32
30
BocN
HN
O
NH2
O
O
NH2
NH
NH
O
COOtBu
COOH
O
O
MOMO
HO
N
H
O
COOtBu
N
COOH
H
O
N
N
H
H
v
vii
34
33
30
NBoc
NBoc
NH
NH
H2N
H2N
Scheme 4. Functionalization of the trans diastereoisomer: (i) CrO3, H2SO4, acetone, 0 ꢀC, 40 min, 54%; (ii) NMM, ClCOOiBu, L-Glu(OtBu)-OtBu, CH2Cl2, 90%; (iii) (a) 9-BBN; (b) H2O2,
AcONa, 65%; (iv) ADDP, PBu3, di-Boc-guanidine, THF, reflux, 2 h, 43%; (v) DEAD, PPh3, di-Boc-guanidine, THF, rt, 64 h, 60%; (vi) TFA, CH2Cl2, 100%; (vii) TFA, CH2Cl2, 100%.

D. Halie et al. / Tetrahedron 65 (2009) 1402–1414
1405
O
O
O
O
BnO
BnO
BnO
BnO
OEt
O
OEt
O
OEt
O
OEt
O
ii
5
N
i
N
N
N
+
H
H
H
H
37
12a
36
35
Scheme 5. Allylation of the trans diaxial diastereoisomer: (i) KHMDS, MeI, THF, 99%; (ii) KHMDS, allyl bromide, 55%, dr 60:40.
O
O
O
BnO
HO
BnO
OEt
O
OH
OEt
O
N
ii
ii
NH
i
OH
N
H
H
H
O
NBoc
NH2
NH
38
39
36
NH2
BocN
HN
O
O
O
BnO
BnO
HO
OEt
O
OEt
O
OH
OH
N
i
N
NH
H
H
H
O
NBoc
NH2
BocN
NH
37
40
41
NH2
HN
Scheme 6. Functionalization and hydrolysis of the trans diaxial diastereoisomer: (i) (a) 9-BBN; (b) H2O2, AcONa; (c) DEAD, PPh3, di-Boc-guanidine, THF, rt (38, 54%; 40, 87%);
(ii) EtOH/H2O, HCl 3 M, 100%.
needed: hydrolysis in aqueous ethanol with 3 M hydrochloric acid
at reflux for 20 h gave compounds 39 and 41 of which the amide
bond, the benzyl ether were also hydrolyzed (Scheme 6). These
compounds 39 and 41 had been also considered as potential pep-
tidomimetics and they have been tested in adhesion assays.
We synthesized seven potential peptidomimetics of RGD in ra-
cemic form, in 10 or 11 steps and in 0.6% to 3.4% total yield from
racemic allylglycine.
3. Conclusions
The scaffold of indolizin-3-one was used to prepare seven
structures as potential mimics of the RGD tripeptide sequence.
These compounds were evaluated in cell-adhesion test. Two of
them showed some activities: they possess the acid and guanidine
side chains in a cis relationship. This configuration corresponds to
the required geometry of a b-turn (type II). Optimization of the
length of the side chains and use of optically active allylglycine as
starting material are under investigation.
2.3. Biological test of the seven peptidomimetics
The compounds 24, 26, 27, 32, 34, 39, and 41 were evaluated in
vitro for their ability to inhibit cell adhesion. Their activity was
compared to that of c(RGDfV) and GRGES that were used as positive
and negative competitors for cell adhesion. We used two different
cell lines, mouse sarcoma S180 cells and human vascular endothelial
4. Experimental section
4.1. General experimental methods
All commercially available reagents were used without further
purification unless otherwise noted. Tetrahydrofuran was freshly
distilled from benzophenone ketyl radical under argon prior to use.
Column chromatography was performed with silica gel (35–70
mesh). Melting points are reported uncorrected.
Eahy926 cells. These cells express a5b1 and avb
3 integrins.12 The
c(RGDfV) peptide strongly inhibited S180 cell adhesion to VN
(Fig. 2A, black bars) with 19% and 5% of cell adhesion in the presence
of 10 mM and 200 mM of its competitor, respectively. This cyclic
peptide inhibited less efficiently S180 cell adhesion to FN-coated
surface (Fig. 2B, black bars) with 95% and 69% of adhesion in the
presence of 10 mM and 500 mM c(RGDfV), respectively. In contrast,
the control peptide GRGES has no inhibitory effect on the two types
of coated surfaces (Fig. 2, white bars). Among the peptidomimetics
tested, only compounds 26 and 27 showed inhibitory effect on S180
cell adhesion to VN but they are less efficient compared to the
c(RGDfV). We measured 65% and 74% of adhesion in the presence of
NMR spectra were recorded on 300 and 400 MHz Bruker Avance
spectrometers. Chemical shifts are reported in parts per million.
Coupling constants (J values) are reported in hertz. 13C and 1H peak
assignments were made based on DEPT, HMQC, HSQC, HSBC, and
NOESY data and IR spectra were recorded on a Perkin Elmer 1600
FT-IR spectrometer. MS experiments were performed on a Q-Tof
Micromass-1 spectrometer.
compound 26 or 27 at 200
m
M, respectively (Fig. 2A, dashed bars).
4.1.1. (5S
*
,7S
*
,8aR
*
)-7-Hydroxy-3-oxo-octahydro-indolizin-5-
,7R ,8aR )-7-hydroxy-
They did not interfere with S180 cell adhesion to FN-coated surfaces.
The c(RGDfV) peptide also interfered with Eahy926 adhesion to
VN but to a lesser extent compared to S180 cell adhesion, with
carboxylic acid methyl ester 7 and (5S
3-oxo-octahydro-indolizin-5-carboxylic acid methyl ester 8
To a solution of formiates 5 and 6 (10.91 g, 45.2 mmol, 1.0 equiv)
in 210 mL of anhydrous methanol was added solid KHCO3 (4.54 g,
45.4 mmol, 1.0 equiv) at 0 ꢀC and the reaction was stirred 40 min at
0 ꢀC. KHCO3 was filtered and the filtrate was concentrated in vacuo.
*
*
*
74.8ꢂ7.0% and 9.2ꢂ4.4% of adhesion measured at 10
200 M c(RGDfV). All compounds tested showed no inhibitory ef-
fect on Eahy926 adhesion to VN nor FN-coated surfaces.
mM and
m

1406
D. Halie et al. / Tetrahedron 65 (2009) 1402–1414
Compound 7: Rf¼0.3 (AcOEt/MeOH 90:10), mp: 94 ꢀC. 1H NMR
(CDCl3, 300 MHz): dH 1.1 (1H, ddd, J¼11.7, 11.7, 11.7 Hz, H8ax); 1.48
(1H, m, H6); 1.6 (1H, m, H1); 2.09 (1H, br d, J¼11.8 Hz, H8eq); 2.2 (1H,
m, H1); 2.38 (3H, m, 2H2, H6); 3.66 (4H, br s, OMe, H7); 3.75 (1H,
dddd, J¼7.5, 7.5, 7.5, 7.5 Hz, H8a); 4.8 (1H, d(l), J¼5.7 Hz, H5). 13C
NMR (75 MHz, CDCl3): dC 25.5 (C1); 30.4 (C2); 34.7 (C6); 41.8 (C8);
50.2 (C5); 52.6 (OMe); 53.9 (C8a); 65.2 (C7); 170.9, 174.8 (CO, C3). IR
(NaCl disc, cmꢁ1): nmax 3432, 2952, 1736, 1666, 1432, 1213. HRMS
(ESþ, [MNa]þ): calcd for C10H15NO4 236.0899, found 236. 0909.
4.1.2. (5S
*
,7S
*
,8aR
*
)-7-Benzyloxy-3-oxo-octahydro-indolizin-5-
carboxylic acid methyl ester 9a and (5S
*
,7R ,8aR )-7-benzyloxy-3-
*
*
oxo-octahydro-indolizin-5-carboxylic acid methyl ester 10a
Alcohols 7 and 8 (10.7 g, 50.4 mmol) were dissolved in 40 mL of
dichloromethane under argon, and 80 mL of cyclohexane
was added. At rt, 19 mL (102 mmol, 2 equiv) of benzyl-tri-
chloroacetimidate was added, followed by 440 mL (5 mmol,
0.1 equiv) of triflic acid. The mixture was refluxed for 16 h, 3 mL of
benzyl-trichloroacetimidate was further added and the reflux was
continued for 20 h. The mixture was filtered on silica gel and
washed with dichloromethane. The filtrate was evaporated and
purification by flash column chromatography on silica gel (EtOAc/
cyclohexane/MeOH¼80:15:5) gave 9a and 10a as a colorless oil
(13.6 g, 89%).
Compound 9a: Rf¼0.49 (AcOEt/cyclohexane/MeOH 80:15:5). 1H
NMR (CDCl3, 300 MHz): dH 1.16 (1H, ddd, J¼11.7, 11.7, 11.7 Hz, H8ax);
1.58 (2H, m, H1, H6ax); 2.2 (2H, m, H1, H8eq); 2.38 (2H, m, H2); 2.55
(1H, dm, J¼7, 5 Hz, H6eq); 3.44 (1H, dddd, J¼11.3, 11.3, 3.8, 3.8 Hz,
H7ax); 3.66 (3H, s, OMe); 3.73 (1H, m, H8a); 4.51 (2H, s, CH2Ph); 4.89
(1H, d, J¼5.6 Hz, H5); 7.26 (5H, m, Ar). 13C NMR (75 MHz, CDCl3): dC
25.2 (C1); 29.9 (C2); 31.7 (C6); 38.8 (C8); 49.8 (C5); 52.2 (OMe); 53.3
(C8a); 69.9 (CH2Ph); 72.0 (C7); 127.4 (3CAr); 128.1 (CAr); 137.8 (CAr-
quat); 170.9, 174.8 (CO, C3). IR (NaCl disc, cmꢁ1): nmax 3352, 2926,
1732, 1682, 1454, 1366, 1291, 1208, 1110. HRMS (ESþ, [MNa]þ): calcd
for C17H21NO4 326.1368, found 326.1365.
Compound 10a: Rf¼0.43 (AcOEt/cyclohexane/MeOH 80:15:5). 1H
NMR (CDCl3, 300 MHz): dH 1.29 (1H, ddd, J¼13.7, 12.0, 2.2 Hz, H8ax);
1.57 (1H, dddd, J¼12.4, 10.5, 10.5, 8.7 Hz, H1); 1.71 (1H, ddd, J¼14.3,
7.0, 2.1 Hz, H6ax); 2.19 (1H, dddd, J¼13.4, 13.4, 13.4, 13.4 Hz, H8eq);
2.28 (1H, m, H1); 2.40 (2H, m, 2H2); 2.71 (1H, dddd, J¼14.2, 3.3, 1.7,
1.7 Hz, H6eq); 3.51 (3H, s, OMe); 3.84 (1H, br s, H7eq); 4.11 (1H, dddd,
J¼11.8, 8.4, 8.4, 3.6 Hz, H8a); 4.41 (1H, d, J¼11.8 Hz, CH2Ph); 4.51
(1H, d, J¼11.8 Hz, CH2Ph); 4.74 (1H, d, J¼6.0 Hz, H5); 7.25 (5H, m,
HAr). 13C NMR (75 MHz, CDCl3): dC 25.9 (C1); 29.3 (C6); 30.1 (C2);
37.1 (C8); 48.2 (C5); 49.4 (C8a); 52.0 (OMe); 70.1 (CH2Ph); 70.7 (C7);
127.0 (2CAr); 127.4 (CAr); 128.2 (CAr); 138.2 (CArquat); 171.0, 174.6
(CO, C3). IR (NaCl disc, cmꢁ1): nmax 3448, 2951, 1747, 1696, 1417,
1359, 1293, 1209, 1093. MS (ESþ): m/z 326 ([MNa]þ).
Figure 2.
4.1.3. (5S
*
,7S
*
,8aR
*
)-7-Methoxymethoxy-3-oxo-octahydro-
indolizin-5-carboxylic acid methyl ester 9b and (5S
*
,7R ,8aR )-
*
*
Purification by flash column chromatography on silica gel (EtOAc/
MeOH¼95:5 then 90:10) gave 7 and 8 as a yellow solid (8.3 g, 86%).
A sample of each diastereoisomer was obtained.
7-methoxymethoxy-3-oxo-octahydro-indolizin-5-carboxylic
acid methyl ester 10b
To alcohols 7 and 8 (371 mg, 1.74 mmol) dissolved in 7 mL of
anhydrous THF under argon and cooled to 0 ꢀC, were successively
Compound 8: Rf¼0.3 (AcOEt/MeOH 90:10), mp: 127 ꢀC. 1H NMR
(CDCl3, 300 MHz): dH 1.30 (1H, ddd, J¼13.8, 12.0, 2.2 Hz, H8ax); 1.55
(1H, dddd J¼12.4, 9.8, 9.8, 8.5 Hz, H1); 1.76 (1H, ddd, J¼14.3, 7.0,
2.2 Hz, H6ax); 2.01 (1H, dm, J¼13.4 Hz, H8eq), 2.23 (1H, dddd, J¼12.3,
9.3, 7.1, 3.0 Hz, H1); 2.3–2.5 (3H, m, 2H2, H6); 3.67 (3H, s, OMe); 4.08
(1H, dddd, J¼11.8, 8.0, 8.0, 3.5 Hz, H8a); 4.19 (1H, m, H7eq); 4.68 (1H,
d(l), J¼6.0 Hz, H5). 13C NMR (75 MHz, CDCl3): dC 25.8 (C1); 30.2 (C2);
32.7 (C6); 38.8 (C8); 48.1 (C5); 49.2 (C8a); 52.4 (COMe); 63.7 (C7);
171.9, 175.3 (CO, C3). IR (NaCl disc, cmꢁ1): nmax 3388, 2962, 1749,
1665, 1441, 1313, 1215, 1090, 1071. MS (ESþ): m/z¼236 ([MNa]þ).
HRMS (ESþ, [MNa]þ): calcd for C10H15NO4 236.0899, found
236.0893.
added diisopropylethylamine (860
mL, 5.2 mmol, 3 equiv) and
chloromethoxymethane (395 L, 5.2 mmol, 3 equiv). The mixture
m
was stirred for 20 h, 1.5 equiv of each reagent was added again and
stirring was continued for 16 additional hours. After addition of
a saturated solution of NH4Cl, the ester was extracted by CH2Cl2, the
organic layers were dried (MgSO4), and concentrated under vac-
uum. Purification by flash column chromatography on silica gel
gave 388 mg (87%) of 9b and 10b.
Compound 10b: Rf¼0.39 (AcOEt/cyclohexane/MeOH 80:15:5). 1H
NMR (CDCl3, 300 MHz): dH 1.25 (1H, ddd, J¼13.6, 11.7, 1.9 Hz, H8ax);
1.52 (1H, m, H1); 1.66 (1H, ddd, J¼14.4, 7.0, 2.0 Hz, H6); 2.03 (1H,

D. Halie et al. / Tetrahedron 65 (2009) 1402–1414
1407
dm, J¼13.4 Hz, H8eq); 2.21 (1H, m, H1); 2.26–2.44 (2H, m, 2H2); 2.48
(1H, br d, J¼14.4 Hz, H6eq); 3.26 (3H, s, COOMe); 3.63 (3H, s, OMe);
4.0 (2H, m, H7, H8a); 4.41 (1H, d, J¼6.8 Hz, OCH2O); 4.55 (1H, d,
J¼6.8 Hz, OCH2O); 4.66 (1H, d, J¼6.6 Hz, H5). 13C NMR (75 MHz,
CDCl3): dC 25.9 (C1); 29.9 (C6); 30.2 (C2); 37.2 (C8); 48.2 (C5); 49.5
(C8a); 52.2 (CH2OMe); 55.5 (OMe); 67.7 (C7); 94.0 (OCH2O); 171.2,
174.9 (CO, C3). IR (NaCl disc, cmꢁ1): nmax 3478, 2950, 1747, 1682,
1417, 1209, 1038, 919. MS (ESþ): m/z 280 ([MNa]þ). HRMS (ESþ,
[MH]þ): calcd for C12H19NO5 258.1341, found 258.1351.
(189 mg, 5 mmol, 10 equiv) was added. The solution was refluxed
for 3 h. After cooling, the solution was concentrated under vacuum,
borane precipitates were filtrated, and washed with CH2Cl2. The
filtrate was concentrated under reduced pressure, the residue was
diluted with CH2Cl2 and 10 mL of H2O, extracted twice by CH2Cl2.
The organic phase was dried with MgSO4 and evaporated to dry-
ness, where after the residue was purified by flash chromatography
on silica gel (EtOAc/cyclohexane 95:5, 99:1 to EtOAc/MeOH 95:5) to
afford 86 mg of 12b as a colorless oil (63%, 86% from 10b) and 23 mg
of 11b as a colorless oil (20%, 77% from 9b).
Compound 9b:Rf¼0.46 (AcOEt/cyclohexane/MeOH 80:15:5).1HNMR
(CDCl3, 300 MHz): dH 1.19 (1H, ddd, J¼11.7, 11.7, 11.7 Hz, H8ax); 1.61 (2H,
m, H1, H6); 2.24 (2H, m, H1, H8); 2.3–2.5 (3H, m, H2, H6); 3.34 (3H, s,
OMe); 3.63 (1H, m, H7); 3.72 (3H, s, OMe); 3.80 (1H, m, H8a); 4.65 (2H, s,
OCH2O); 4.9 (1H, d, J¼5.7 Hz, H5).13CNMR(75 MHz, CDCl3): dC 25.5 (C1);
30.2 (C2); 32.5 (C6); 39.7 (C8); 50.1 (C5); 52.2 (OMe); 53.5 (C8a); 70.9 (C7);
95.0 (OCH2O); 170.9, 174.2 (CO, C3).
Rf¼0.38 (AcOEt/cyclohexane/MeOH 80:15:5). 1H NMR (CDCl3,
300 MHz): dH 1.10 (3H, t, J¼7.2 Hz, CH2CH3); 1.25 (1H, ddd, J¼13.8,
12.1, 2.2 Hz, H8ax); 1.43 (1H, dddd, J¼12.4, 9.8, 9.8, 8.9 Hz, H1); 1.58
(1H, ddd, J¼14.4, 7.1, 2.3 Hz, H6); 1.95 (1H, ddd, J¼13.4, 13.4, 13.4 Hz,
H8eq); 2.12 (1H, dddd, J¼12.3, 8.3, 5.4, 2.6 Hz, H1); 2.22 (1H, ddd,
J¼17.0, 9.7, 2.6 Hz, H2); 2.29 (1H, m, H2); 2.40 (1H, dddd, J¼14.4, 3.3,
1.7, 1.7 Hz, H6eq); 3.19 (3H, s, OMe); 3.89 (2H, m, H7, H8a); 3.97 (1H,
dq, J¼10.8, 7.1 Hz, CH2O); 4.03 (1H, dq, J¼10.8, 7.2 Hz, CH2O); 4.27
(1H, d, J¼6.8 Hz, OCH2O); 4.43 (1H, d, J¼6.7 Hz, OCH2O); 4.48 (1H,
dd, J¼6.6, 1.1 Hz, H5). 13C NMR (75 MHz, CDCl3): dC 14.1 (CH3CH2O);
26.0 (C1); 29.3 (C6); 30.3 (C2); 37.1 (C8); 48.3 (C5); 49.5 (C8a); 55.4
(CH2OMe); 61.1 (OCH2CH3); 67.7 (C7); 93.9 (OCH2O); 170.4, 174.8
(CO, C3). IR (NaCl disc, cmꢁ1): nmax 3468, 2938, 1744, 1694, 1417,
1292, 1199, 1038. MS (ESþ): m/z 294 ([MNa]þ). HRMS (ESþ, [MH]þ):
calcd for C13H21NO5 272.1498, found 272.1494.
4.1.4. (5S
*
,7R
*
,8aR )-7-Benzyloxy-3-oxo-octahydro-indolizin-
*
5-carboxylic acid ethyl ester 12a
To a mixture of 9a and 10a (4.7 g, 15.5 mmol, cis/trans 20:80) dis-
solved in 250 mL of absolute alcohol, sodium borohydride (6 g,
159 mmol, 10 equiv) was added. The mixture was refluxed for 3 h.
After cooling, the solution was concentrated under vacuum, borane
precipitates were filtrated and washed with CH2Cl2. The filtrate was
concentrated under reduced pressure and the residue was diluted with
CH2Cl2 and H2O, extracted twice by CH2Cl2. The organic phase was
dried with MgSO4 and evaporated to dryness, where after the residue
was purified by flash chromatography on silica gel (EtOAc/MeOH 99:1
to 90:10) to afford 780 mg of 11a as a light yellow oil (16%, 80% from
10a) and 2.9 g of 12a as a white solid (68%, 85% from 9a).
4.1.7. (5S
*
,7S
*
,8aR )-5-Hydroxymethyl-7-methoxymethoxy-
*
hexahydro-indolizin-3-one 11b
Rf¼0.09 (AcOEt/cyclohexane/MeOH 80:15:5). 1H NMR (CDCl3,
300 MHz): dH 1.13 (1H, ddd, J¼11.7, 11.7, 11.7 Hz, H8ax); 1.44 (1H,
ddd, J¼13.2, 13.2, 6.6 Hz, H6); 1.58 (1H, m, H1); 2.05 (1H, br d,
J¼13.2 Hz, H6); 2.18 (2H, m, H1, H8eq); 2.35 (2H, m, 2H2); 3.30 (3H, s,
OMe); 3.57 (2H, br d, J¼6.7 Hz, CH2OH); 3.67 (1H, m, H8a); 3.83 (1H,
m, H7); 4.30 (1H, ddd, J¼6.6, 6.6, 6.6 Hz, H5); 4.62 (2H, br s, OCH2O).
13C NMR (75 MHz, CDCl3): dC 25.4 (C1); 30.5 (C2); 31.6 (C6); 40.3
(C8); 49.7 (C5); 53.2 (C8a); 55.4 (CH2OMe); 62.5 (CH2OH); 70.4 (C7);
94.9 (OCH2O); 175.0 (C3). IR (NaCl disc, cmꢁ1): nmax 2943, 1672,
1656, 1421, 1287, 1111, 1064. HRMS (ESþ, [MH]þ): calcd for
C11H19NO4 252.1212, found 272.1205.
Rf¼0.28 (AcOEt). 1H NMR (CDCl3, 300 MHz): dH 1.11 (3H, t, J¼7.2,
CH3); 1.28 (1H, ddd, J¼13.8, 12.0, 2.3 Hz, H8ax); 1.56 (1H, dddd,
J¼12.4, 9.6, 9.6, 9.1 Hz, H1); 1.70 (1H, ddd, J¼14.4, 7.1, 2.2 Hz, H6ax);
2.10 (1H, ddd, J¼13.4, 13.4, 13.4 Hz, H8eq); 2.25 (1H, dddd, J¼12.2,
9.4, 6.8, 2.7 Hz, H1); 2.42 (2H, m, 2H2); 2.71 (1H, dddd, J¼14.3, 3.2,
1.6, 1.6 Hz, H6eq); 3.83 (1H, m, H7); 3.92 (1H, dq, J¼10.7, 7.2 Hz,
OCH2); 4.02 (1H, dq, J¼10.7, 7.2 Hz, OCH2); 4.11 (1H, dddd, J¼11.8,
8.5, 8.5, 3.4 Hz, H8a); 4.41 (1H, d, J¼11.9 Hz, CH2Ph); 4.52 (1H, d,
J¼11.9 Hz, CH2Ph); 4.72 (1H, br d, J¼5.9 Hz, H5); 7.27 (5H, m, Ar). 13
C
NMR (75 MHz, CDCl3): dC 14.0 (CH3); 26.1 (C1); 29.3 (C6); 30.4 (C2);
37.1 (C8); 48.4 (C5); 49.6 (C8a); 61.3 (CH2O); 70.2 (CH2Ph); 70.6 (C7);
94.0 (OCH2O); 127.1 (2CAr); 127.5 (CAr); 128.3 (2CAr); 138.4 (CArquat);
170.7, 174.9 (CO, C3). IR (NaCl disc, cmꢁ1): nmax 3468, 2935, 1742,
1694, 1417, 1292, 1198, 1093. MS (ESþ): m/z 340 ([MNa]þ). HRMS
(ESþ, [MNa]þ): calcd for C18H23NO4 340.1525, found 340.1534.
4.1.8. (2S
hexahydro-indolizin-3-one 16a and (2R
benzyloxy-5-hydroxymethyl-hexahydro-indolizin-3-one 17a
15 mL LDA solution in THF (from diisopropylamine,
*
,5S
*
,7S
*
,8aR
*
)-2-Allyl-7-benzyloxy-5-hydroxymethyl-
*
,5S ,7S ,8aR )-2-allyl-7-
*
*
*
A
22.1 mmol, 3.1 mL, 2.1 equiv) and BuLi (1.94 M) in hexane
(22.1 mmol, 11.4 mL) cooled to ꢁ78 ꢀC was cannulated to amide 11a
(10.5 mmol, 2.9 g) in THF (35 mL) maintained at ꢁ78 ꢀC under ar-
gon. The solution was stirred at the same temperature for 20 min
and allyl bromide (11.6 mmol, 1 mL, 1.1 equiv) was slowly added.
The mixture was stirred at ꢁ78 ꢀC for 2 h and then treated with an
aqueous saturated NH4Cl solution (20 mL). The aqueous phase was
extracted several times with CH2Cl2. The organic phase was then
dried with MgSO4 and the solvent removed in vacuo. The residue
was purified by flash chromatography (AcOEt/MeOH 99.5:0.5 to
95:5) to give the two diastereomeric compounds 16a and 17a in
a 87:13 ratio and a 46% overall yield (827 mg of pure 16a as a pale
yellow oil, 97 mg of pure 17a as a pale yellow oil and 778 mg of
a mixture were isolated).
4.1.5. (5S
*
,7S
*
,8aR )-7-Benzyloxy-5-hydroxymethyl-hexahydro-
*
indolizin-3-one 11a
Rf¼0.39 (AcOEt/MeOH 90:10), mp: 95 ꢀC. 1H NMR (CDCl3,
300 MHz): dH 1.17 (1H, ddd, J¼11.8, 11.8, 11.8 Hz, H8ax); 1.47 (1H,
ddd, J¼12.9, 11.6, 6.6 Hz, H6ax); 1.59 (1H, dddd, J¼12.5, 9.6, 9.6,
7.3 Hz, H1); 2.2 (3H, m, H6, H8eq, H1); 2.38 (2H, m, H2); 3.56 (2H, d,
J¼7.0 Hz, CH2O); 3.7 (2H, m, H7, H8a); 4.35 (1H, ddd, J¼6.7, 6.7,
6.7 Hz, H5); 4.54 (2H, s, OCH2Ph); 7.30 (5H, m, Ar). 13C NMR
(75 MHz, CDCl3): dC 19.7 (C1); 29.2 (C2); 29.9 (C6); 38.4 (C8); 48.5
(C5); 52.0 (C8a); 60.5 (CH2O); 68.7 (CH2Ph); 70.8 (C7); 126.2 (3CAr);
127.2 (2CAr); 137.6 (CArquat); 174.9 (C3). IR (KBr disc, cmꢁ1): nmax
3319, 2943, 1742, 1664, 1458, 1290, 1100, 1074, 740. HRMS (ESþ,
[MH]þ): calcd for C16H21NO3 276.1600, found 276.1602. Anal. Calcd
for C16H21NO3: C, 69.79; H, 7.69; N, 5.09. Found: C, 69.33; H, 7.74; N,
4.99%.
Compound 16a: Rf¼0.42 (AcOEt/MeOH: 98/2). 1H NMR (CDCl3,
400 MHz): dH 1.17 (1H, ddd, J¼11.6, 11.6, 11.6 Hz, H8ax); 1.51 (1H, m,
H6); 1.79 (1H, m, H1); 2.01 (1H, m, H1); 2.13–2.28 (3H, m, H6, H8,
CH2CH]CH2); 2.44 (1H, m, CH2CH]CH2); 2.59 (1H, m, H2); 3.1 (1H,
br s, H–O); 3.5–3.8 (4H, m, CH2OH, H8a, H7); 4.38 (1H, ddbr d, J¼6.8,
6.8, 6.8 Hz, H5); 4.55 (2H, s, CH2Ph); 5.06 (1H, br d, J¼9.1 Hz,
CH2]CH); 5.09 (1H, br d, J¼9.1 Hz, CH2]CH); 5.77 (1H, dddd,
J¼16.0, 9.0, 7.0, 7.0 Hz, CH2]CH); 7.27 (5H, m, Ar). 13C NMR
4.1.6. (5S
*
,7R
*
,8aR )-7-Methoxymethoxy-3-oxo-octahydro-
*
indolizin-5-carboxylic acid ethyl ester 12b
To a mixture of 9b and 10b 128 mg (0.5 mmol, cis/trans 74:26)
dissolved in 10 mL of absolute alcohol, sodium borohydride

1408
D. Halie et al. / Tetrahedron 65 (2009) 1402–1414
(75 MHz, CDCl3): dC 30.3 (C1); 30.8 (C6); 35.8 (CH2CH]CH2); 39.4
(C8); 40.9 (C2); 49.7 (C5); 51.4 (C8a); 62.4 (CH2OH); 70.1 (CH2Ph);
71.8 (C7); 117.3 (CH2]CH); 127.5; 127.6; 128.3 (2CAr, CAr, 2CAr);
135.0 (CH2]CH); 138.1 (CArquat); 176.4 (C3). IR (NaCl disc, cmꢁ1):
nmax 3391, 2942, 1681, 1650, 1454, 1367, 1280, 1094, 917, 739. MS
(ESþ): m/z 338 ([MNa]þ). HRMS (ESþ, [MNa]þ): calcd for C19H25NO3
338.1732, found 338.1717.
m, H8eq, CH2 CH]CH2); 2.45 (1H, m, CH2 CH]CH2); 2.57 (1H, dddd,
J¼4.3, 4.3, 8.8, 8.8 Hz, H2); 3.34 (3H, s, CH2OMe); 3.61 (2H, d,
J¼7.2 Hz, CH2OH); 3.65 (1H, m, H8a); 3.85 (1H, dddd, J¼11.3, 11.3,
4.2, 4.2 Hz, H7ax); 4.38 (1H, ddd, J¼6.7, 6.7, 6.7 Hz, H5eq); 4.65 (2H, d,
J¼1.2 Hz, OCH2O); 5.06 (2H, m, CH2]CH); 5.76 (1H, dddd, J¼17.0,
10.1, 7.0, 7.0 Hz, CH2]CH). 13C NMR (75 MHz, CDCl3): dC 30.3 (C1);
31.4 (C6); 35.8 (CH2–CH]CH2); 40.0 (C8); 40.9 (C2); 49.7 (C5); 51.4
(C8a); 55.4 (CH2OMe); 62.5 (CH2OH); 70.4 (C7); 94.8 (OCH2O); 117.3
(CH2]CH); 135.1 (CH2]CH); 176.4 (C3). IR (NaCl disc, cmꢁ1): nmax
3397, 2943, 1684, 1441, 1279, 1151, 1105, 1034, 917. MS (ESþ): m/z
270 ([MH]þ). HRMS (ESþ, [MNa]þ): calcd for C14H23NO4 292.1525,
found 292.1520.
Compound 17a: Rf¼0.5 (AcOEt/MeOH 98:2). 1H (CDCl3,
300 MHz): dH 1.11 (1H, ddd, J¼11.7, 11.7, 11.7 Hz, H8ax); 1.3 (1H, ddd,
J¼11.3, 11.3, 11.3 Hz, H1); 1.49 (1H, ddd, J¼13.1, 11.6, 6.6 Hz, H6); 2.1
(2H, m, H6, CH2CH]CH2); 2.3 (2H, m, H1, H8); 2.57 (2H, m, H2,
CH2CH]CH2); 3.1 (1H, br s, H–O); 3.57 (3H, m, H8a, CH2OH); 3.68
(1H, dddd, J¼11.2, 11.2, 4.0, 4.0 Hz, H7); 4.37 (1H, ddd, J¼6.7, 6.7,
6.7 Hz, H5); 4.52 (2H, s, CH2Ph); 5.02 (1H, dd, J¼10.1, 1.6 Hz, H14
,
4.1.10. (5S
*
,7S
*
,8aR )-5-(tert-Butyl-dimethyl-silyloxymethyl)-
*
CH2cis]CH); 5.05 (1H, dd, J¼17.1, 1.6 Hz, CH2trans]CH); 5.71 (1H,
dddd, J¼16.9, 10.1, 6.8, 6.8 Hz, CH2]CH); 7.3 (5H, m, Ar). 13C NMR
(100 MHz, CDCl3): dC 31.1 (C6); 32.4 (C1); 35.4 (CH2CH]CH2); 40.0
(C8); 41.3 (C2); 49.9 (C5); 51.5 (C8a); 62.5 (CH2OH); 70.4 (CH2Ph);
72.0 (C7); 117.0 (CH2]CH); 127.7 (2CAr); 127.8 (CAr); 128.5 (2CAr);
135.5 (CH2]CH); 138.4 (CArquat); 176.0 (C3). IR (NaCl disc, cmꢁ1):
nmax 3396, 2927, 2867, 1666, 1454, 1367. MS (ESþ): m/z 316 ([MH]þ),
338 ([MNa]þ), 354 ([MK]þ). HRMS (ESþ, [MNa]þ): calcd for
C19H25NO3 338.1732, found 338.1732.
7-methoxymethoxy-hexahydro-indolizin-3-one 13
Alcohol 11b (343 mg, 1.5 mmol) in CH2Cl2 (10 mL) was cooled to
ꢁ10 ꢀC and triethylamine (525
m
L, 3.75 mmol, 2.5 equiv) and tert-
butyl-dimethylsilyl triflate (444
mL, 1.95 mmol, 1.3 equiv) were
added. The solution was stirred between ꢁ5 and ꢁ10 ꢀC for 30 min
and an aqueous saturated NaHCO3 solution (3 mL) was added. The
aqueous solution was extracted with CH2Cl2. The organic phase was
washed with water (2 mL) and then NaCl solution, dried, and the
solvent removed in vacuo. The residue was purified by flash chro-
matography on silica gel (ether/MeOH 99.5:0.5). Silyl ether 13 was
obtained as a pale yellow oil in 88% yield (453 mg).
4.1.9. (2S
methoxymethoxy-hexahydro-indolizin-3-one 16b and
(2R ,5S ,7S ,8aR )-2-allyl-5-hydroxymethyl-7-methoxy-
*
,5S
*
,7S
*
,8aR
*
)-2-Allyl-5-hydroxymethyl-7-
Rf¼0.45 (ether/MeOH 99:1). 1H NMR (CDCl3, 300 MHz): dH 0.04
(3H, s, SiMe); 0.06 (3H, s, SiMe); 0.89 (9H, s, SitBu); 1.15 (1H, ddd,
J¼11.8, 11.8, 11.8 Hz, H8ax); 1.49 (1H, ddd, J¼13.2, 11.8, 6.7 Hz, H6ax);
*
*
*
*
methoxy-hexahydro-indolizin-3-one 17b
The same experimental procedure as described for allylation
was applied to lactam 11b (100 mg, 0.43 mmol) using LDA prepared
1.61 (1H, dddd, J¼12.4, 9.5, 9.5, 7.5 Hz, H1); 2.20 (3H, m, H6eq, H8eq
,
H1); 2.37 (2H, m, 2H2); 3.36 (3H, s, OMe); 3.61 (1H, dd, J¼10.0,
4.7 Hz, CH2O); 3.68 (1H, dd, J¼10.0, 6.0 Hz, CH2OH); 3.76 (1H, dddd,
J¼11.5, 7.3, 7.3, 3.1 Hz, H8a); 4.06 (1H, dddd, J¼11.3, 11.3, 4.2, 4.2 Hz,
H7ax); 4.30 (1H, ddd, J¼5.8, 5.8, 5.8 Hz, H5eq); 4.68 (2H, s, OCH2O).
13C NMR (CDCl3, 100 MHz): dC ꢁ5.49, ꢁ5.53 (2SiMe); 18.1
(SiC(CH3)3); 25.7 (C1); 25.9 (3SiC(CH3)3); 30.5 (C2); 32.1 (C6); 40.1
(C8); 49.0 (C5); 54.2 (C8a); 55.3 (OMe); 64.0 (CH2O); 70.5 (C7); 94.8
(OCH2O); 173.8 (C3). IR (NaCl disc, cmꢁ1): nmax 2886, 1689, 1416,
1257, 1154, 1103, 1041, 837, 777. MS (ESþ): m/z 366 ([MNa]þ). HRMS
(ESþ, [MNa]þ): calcd for C17H33NO4Si 366.2077, found 366.2072.
from diisopropylamine (152
lithium 2.28 M (471 L, 1.07 mmol, 2.5 equiv). Stirring was main-
tained 3 h after addition of allyl bromide (41 L, 0.47 mmol,
mL, 1.07 mmol, 2.5 equiv) and butyl-
m
m
1.1 equiv). Flash chromatography on silica gel (AcOEt/MeOH 95:5)
allowed the isolation of 16b and 17b as a 71:29 ratio in a 54% yield
(17b: 8 mg as a pale yellow oil, 16b: 22 mg as a pale yellow oil and
32 mg of a mixture).
Compound 16b: Rf¼0.38 (AcOEt/MeOH 90:10). 1H NMR (CDCl3,
300 MHz): dH 1.15 (1H, ddd, J¼11.7, 11.7, 11.7 Hz, H8ax); 1.33 (1H, m,
H1); 1.55 (1H, ddd, J¼13.2, 11.7, 6.7 Hz, H6ax); 2.11 (2H, m, H6eq, CH2
CH]CH2); 2.28 (1H, br d, J¼12.1 Hz, H8eq); 2.37 (1H, ddd, J¼12.6,
8.5, 6.7 Hz, H1); 2.61 (2H, m, H2, CH2 CH]CH2); 3.39 (3H, s,
CH2OMe); 3.62 (1H, m, H8a); 3.66 (2H, d, J¼6.9 Hz, CH2OH); 3.87
(1H, dddd, J¼11.4, 11.4, 4.3, 4.3 Hz, H7ax); 4.4 (1H, ddd, J¼6.7, 6.7,
6.7 Hz, H5eq); 4.7 (2H, s, OCH2O); 5.06 (1H, dd, J¼10.1, 1.6 Hz,
CH2]CH); 5.11 (1H, dd, J¼17.1, 1.6 Hz, CH2]CH); 5.27 (1H, dddd,
J¼17.0, 10.1, 6.8, 6.8 Hz, CH2]CH). 13C NMR (CDCl3, 100 MHz): dC
31.6 (C6); 32.6 (C1); 35.4 (CH2 CH]CH2); 40.4 (C8); 41.3 (C2); 49.8
(C5); 51.3 (C8a); 55.5 (CH2OMe); 63.5 (CH2OH); 70.5 (C7); 95.0
(OCH2O); 126.9 (CH2]CH); 135.5 (CH2]CH); 176.3 (C3). IR (NaCl
disc, cmꢁ1): nmax 3403, 2931, 1667, 1436, 1276, 1151, 1105, 1039, 916.
MS (ESþ): m/z 270 ([MH]þ), 292 ([MNa]þ). HRMS (ESþ, [MNa]þ):
calcd for C14H23NO4 292.1525, found 292.1534.
4.1.11. (2S
methyl)-7-methoxymethoxy-hexahydro-indolizin-3-one 14 and
(2R ,5S ,7S ,8aR )-2-allyl-5-(tert-butyl-dimethyl-silyloxy-
*
,5S
*
,7S
*
,8aR )-2-Allyl-5-(tert-butyl-dimethyl-silyloxy-
*
*
*
*
*
methyl)-7-methoxymethoxy-hexahydro-indolizin-3-one 15
The same experimental procedure as described for allylation
was applied to lactame 13 (120 mg, 0.35 mmol) using LDA prepared
from diisopropylamine (63
lithium 2.3 M (195 L, 0.45 mmol, 1.3 equiv). Stirring was main-
tained 3 h after addition of allyl bromide (33 L, 0.39 mmol,
mL, 0.45 mmol, 1.3 equiv) and butyl-
m
m
1.1 equiv). Flash chromatography on silica gel (ether) allowed the
isolation of 15 and 14 as a 90:10 ratio in a 88% yield (14: 12 mg as
a yellow oil, 15: 106 mg as yellow oil).
Compound (2R
*
,5S
*
,7R
*
,8aR
*
)-15: Rf¼0.66 (AcOEt). 1H NMR
From 15: to silyl compound 15 (35 mg; 0.87 mmol) dissolved in
14 mL of anhydrous THF was added slowly a solution 1 M of tet-
rabutylammonium fluoride in THF (2.6 mL, 2.6 mmol, 3 equiv) at
0 ꢀC. Stirring was continued 30 min after removal of the ice bath. At
0 ꢀC 7 mL of water was added and the mixture was extracted three
times with dichloromethane. The organic phase was dried with
MgSO4 and the solvent removed in vacuo. The residue was purified
by flash chromatography (AcOEt/MeOH 97:3) to give 17b as a pale
yellow oil (226 mg, 97%).
(CDCl3, 300 MHz): dH 0.04 (3H, s, SiMe); 0.05 (3H, s, SiMe); 0.87
(9H, s, SitBu); 1.1 (1H, ddd, J¼11.8 Hz, H8ax); 1.47 (1H, ddd, J¼13.6,
11.6, 6.9 Hz, H6ax); 1.73 (1H, ddd, J¼13.0, 11.6, 6.9 Hz, H1); 1.96 (1H,
ddd, J¼12.9, 7.6, 4.1 Hz, H1); 2.17 (3H, m, H6, CH2 CH]CH2 H8); 2.48
(1H, m, CH2 CH]CH2); 2.52 (1H, m, H2); 3.32 (3H, s, OMe); 3.60 (1H,
dd, J¼10.0, 4.7 Hz, CH2O); 3.65 (1H, dd, J¼10.0, 5.8 Hz, CH2O); 3.70
(1H, m, H8a); 4.05 (1H, dddd, J¼11.3, 11.3, 4.3, 4.3 Hz, H7ax); 4.29
(1H, ddd, J¼5.6, 5.6, 5.6 Hz, H5ax); 4.64 (2H, d, J¼0.6 Hz, OCH2O);
5.06 (2H, m, CH2]CH); 5.74 (1H, dddd, J¼16.9, 10.1, 6.8, 6.8 Hz,
CH2]CH). 13C NMR (75 MHz, CDCl3): dC ꢁ5.6 (2SiMe); 18.1
(SiC(CH3)3); 25.8 (SiC(CH3)3); 30.5 (C1); 32.1 (C6); 35.9 (CH2
CH]CH2); 39.8 (C8); 40.7 (C2); 49.0 (C5); 52.5 (C8a); 55.6 (OMe);
64.4 (CH2O); 70.6 (C7); 94.6 (OCH2O); 117.1 (CH2]CH); 135.2
Compound 17b: Rf¼0.44 (AcOEt/MeOH 90:10). 1H NMR (CDCl3,
300 MHz): dH 1.16 (1H, ddd, J¼11.6, 11.6, 11.6 Hz, H8ax); 1.48 (1H,
ddd, J¼13.1, 11.7, 6.7 Hz, H6ax); 1.77 (1H, ddd, J¼13.1, 9.5, 6.3 Hz, H1);
2.0 (1H, m, H1); 2.07 (1H, ddd, J¼13.3, 13.3, 13.3 Hz, H6eq); 2.2 (2H,

D. Halie et al. / Tetrahedron 65 (2009) 1402–1414
1409
(CH2]CH); 175.1 (C3). IR (NaCl disc, cmꢁ1): nmax 2953, 2856, 1694,
1422, 1257, 1151, 1106, 1037, 916, 837, 777. MS (ESþ): m/z 406
([MNa]þ). HRMS (ESþ, [MNa]þ): calcd for C20H37NO4Si 384.2570,
found 384.2575.
11.8 Hz, H8ax); 1.23–1.52 (20H, m, H1, H6, 2C(CH3)3); 2.11–2.5
(3H, m, H1, CH2CH]CH2, H8); 2.51–2.9 (5H, m, CH2CH]CH2,
CH2CO, H2, H6); 3.58–3.89 (2H, m, H7, H8a); 4.51–4.67 (3H, m,
CH2Ph, NHCHCO); 4.85 (1H, br d, J¼6.2 Hz, H5); 5.02–5.14
(2H, m, CH2]CH); 5.67–5.84 (1H, m, CH2]CH); 6.96/7.20
(1H, d/d, J¼8.0/8.6 Hz, NH); 7.23–7.37 (5H, m, Ar). 13C NMR
(75 MHz, CDCl3): dC 27.9 and 28.0 (C(CH3)3); 30.8/31.2, 32.2/
32.4, 35.1, 37.2/37.3 (C6, C1, CH2CH]CH2, CH2CO); 39.6/39.7
Compound (2S
*
,5S
*
,7R
*
,8aR
*
)-14: Rf¼0.86 (AcOEt). 1H NMR (CDCl3,
300 MHz): dH 0.04 (3H, s, SiMe); 0.06 (3H, s, SiMe); 0.89 (9H, s, SitBu); 1.1
(1H, ddd, J¼11.7, 11.7, 11.7 Hz, H8ax); 1.27 (1H, m, H1); 1.5 (1H, ddd, J¼11.8,
11.8, 6.9 Hz, H6ax); 2.07–2.28 (3H, m, CH2 CH]CH2, H8eq, H6eq); 2.33 (1H,
m, H1); 2.5 (1H, dddd, J¼9.9, 9.9, 9.9, 3.9 Hz, H2); 2.65 (1H, ddd, J¼14.1,
5.3, 5.3 Hz, CH2 CH]CH2); 3.36 (3H, s, OMe); 3.64 (3H, m, H8a, CH2
CH]CH2); 4.08 (1H, dddd, J¼11.2, 11.2, 4.0, 4.0 Hz, H7ax); 4.28 (1H, ddd,
J¼5.8, 5.8, 5.8 Hz, H5eq); 4.68 (2H, br s, OCH2O); 5.06 (2H, m, CH2]CH);
5.77 (1H, dddd, J¼17.0, 10.1, 6.9, 6.9 Hz, CH2]CH). 13C NMR (CDCl3,
100 MHz): dC ꢁ5.6 (2SiMe); 18.1 (SiC(CH3)3); 25.8 (SiC(CH3)3); 32.1 (C6);
32.9 (C1); 35.4 (CH2 CH]CH2); 40.2 (C8); 41.3 (C2); 49.1 (C5); 52.4 (C8a);
55.2 (OMe); 63.9 (CH2O); 70.6 (C7); 94.8 (OCH2O); 116.7 (CH2]CH);
135.7 (CH2]CH); 174.5 (C3). IR (NaCl disc, cmꢁ1): nmax 2930, 1691, 1419,
1255,1104,1042, 840. MS (ESþ): m/z¼384 ([MH]þ), 406 ([MNa]þ). HRMS
(ESþ, [MNa]þ): calcd for C20H37NO4Si 406.2390, found 406.2386.
(C8); 40.9/41.0 (C2); 49.1/49.3, 50.9, 52/52.1 (C5, C8a
,
NHCHCO); 70.5 (CH2Ph); 72.4 (C7); 81.6/81.9 and 82.2/82.4
(C(CH3)3); 117 (CH2]CH); 127.6; 128.3 (2CAr, CAr, 2CAr); 135.3
(CH2]CH); 138.1 (CArquat); 169.2/169.3, 169.5/169.5, 169.9/
170, 175.6/175.7 (3CO, C3). HRMS (ESþ, [MNa]þ): calcd for
C31H44N2O7 579.3046, found 579.3019.
*
4.1.14. 3-[(2S ,5S
*
,7S
*
,8aR )-(2-Allyl-7-benzyloxy-3-oxo-
*
octahydro-indolizin-5-carbonyl)-amino]propionic acid
tert-butyl ester 20
The same experimental procedure as described for peptide
coupling was applied to carboxylic acid 18 (136 mg, 0.41 mmol)
4.1.12. (2S
*
,5S
*
,7S
*
,8aR
*
)-2-Allyl-7-benzyloxy-3-oxo-octahydro-
using NMM (50
formiate (60 L, 0.45 mmol,1.1 equiv). Stirring was maintained 20 h
after addition of amino acid (82 mg, 0.45 mmol, 1.1 equiv) and
NMM (100 L, 0.9 mmol, 2.2 equiv). Flash chromatography on silica
gel (AcOEt/cyclohexane 50:50 to 70:30) allowed the isolation of
a colorless oil (83%, 154 mg).
mL, 0.45 mmol, 1.1 equiv) and isobutyl chloro-
indolizin-5-carboxylic acid 18
m
To alcohol 16a (757 mg, 2.4 mmol) dissolved in 22 mL in acetone
was added dropwise Jones reagent 2.67 M (1.1 mL, 2.9 mmol,
1.2 equiv) at 0 ꢀC. Stirring was continued for 40 min, 10 mL of iso-
propanol was added, and the mixture was stirred for 20 min at 0 ꢀC.
After addition of 0.5 mL of water and 5 mL of 1 M HCl, the mixture
was extracted twice with 100 mL of dichloromethane. The organic
phase was dried (MgSO4) and the solvent removed in vacuo. The
residue was purified by flash chromatography on silica gel (AcOEt/
MeOH/AcOH 96:2:2) and the carboxylic acid 23 was isolated as
a beige solid (67%, 530 mg).
m
Rf¼0.45 (AcOEt/cyclohexane 60:40). 1H NMR (CDCl3,
400 MHz): dH 1.17 (1H, ddd, J¼11.8, 11.8, 11.8 Hz, H8ax); 1.2–1.5
(11H, m, H1, H6, 2C(CH3)3); 2.18 (1H, m, H1); 2.29 (1H, br d,
J¼12 Hz, H8); 2.35 (1H, m, CH2CH]CH2); 2.41 (2H, t, J¼6 Hz,
NHCH2CH2CO); 2.52–2.7 (3H, m, H2, H6, CH2CH]CH2); 3.32–
3.52 (2H, m, NHCH2CH2CO); 3.57 (1H, m, H8a); 3.9 (1H, dddd,
J¼11, 11, 4.5, 4.5 Hz, H7); 4.57 (1H, d, J¼11.6 Hz, CH2Ph); 4.63
(1H, d, J¼11.6 Hz, CH2Ph); 4.78 (1H, d, J¼5.9 Hz, H5); 5.02–
5.12 (2H, m, CH2]CH); 5.77 (1H, dddd, J¼15, 12, 5.9, 5.9 Hz,
CH2]CH); 6.65 (1H, br s, NH); 7.25–7.4 (5H, m, Ar). 13C NMR
(75 MHz, CDCl3): dC 28.1 (C(CH3)3); 30.9 (C6); 32.3 (C1); 35.1
(CH2CH]CH2, NHCH2CH2CO); 39.5 (C8); 41.1 (C2); 50.9 (C5);
52.1 (C8a); 70.5 (CH2Ph); 72.4 (C7); 81.1 (C(CH3)3); 117.1
(CH2]CH); 127.6; 128.4 (2CAr, CAr, 2CAr); 135.2 (CH2]CH);
138.5 (CArquat); 169.9, 171.4, 176.4 (2CO, C3). IR (NaCl disc,
cmꢁ1): nmax 3307, 2929, 1727, 1682, 1427, 1278, 1156, 737. MS
(ESþ): m/z 479 ([MNa]þ).
Rf¼0.27 (AcOEt/MeOH/AcOH 96:2:2), mp: 146 ꢀC. 1H NMR
(CDCl3, 400 MHz): dH 1.18 (1H, ddd, J¼11.7, 11.7, 11.7 Hz, H8ax); 1.36
(1H, m, H1); 1.63 (1H, ddd, J¼12.1, 12.1, 6.7 Hz, H6); 2.18 (1H, m,
CH2CH]CH2); 2.27–2.4 (2H, m, H1, H8); 2.58–2.69 (3H, m, H6, H2,
CH2CH]CH2); 3.57 (1H, m, H7); 3.72 (1H, m, H8a); 4.54 (1H, d,
J¼9.1 Hz, CH2Ph); 4.59 (1H, d, J¼9.1 Hz, CH2Ph); 4.91 (1H, br d,
J¼6.2 Hz, H5); 5.03–5.13 (2H, m, CH2]CH); 5.75 (1H, dddd, J¼16.9,
10.1, 6.8, 6.8 Hz, CH2]CH); 7.31 (5H, m, Ar). 13C NMR (75 MHz,
CDCl3): dC 31.9 (C6); 32.4 (C1); 35.0 (CH2CH]CH2); 39.2 (C8); 41.4
(C2); 50.5 (C5); 52.4 (C8a); 70.6 (CH2Ph); 72.7 (C7); 117.3 (CH2]CH);
127.7; 127.9; 128.6 (2CAr, CAr, 2CAr); 135.2 (CH2]CH); 138.1 (CAr-
quat); 173.1 (COOH); 176.4 (C3). MS (ESþ): m/z 352 ([MNa]þ).
4.1.15. (S)-2-[[(2S
*
,5S
*
,7S
*
,8aR )-7-Benzyloxy-2-(3-hydroxy-
*
4.1.13. (2S)-2-[(2S
*
,5S
*
,7S
*
,8aR
*
)-(2-Allyl-7-benzyloxy-3-oxo-
propyl)-3-oxo-octahydro-indolizin-5-carbonyl]-amino]-
succinic acid di-tert-butyl ester 21
The same experimental procedure as described for hydro-
boration was applied to alkene 19 (81 mg, 0.14 mmol) using a 0.5 M
solution of 9-BBN (1.5 mL, 0.73 mmol, 5 equiv). Stirring was
octahydro-indolizin-5-carbonyl)-amino]succinic acid
di-tert-butyl ester 19
To carboxylic acid 18 (55 mg, 0.167 mmol) dissolved in 1.5 mL of
anhydrous dichloromethane was added N-methylmorpholine (NMM)
(20
m
L, 0.18 mmol,1.1 equiv) at ꢁ10 ꢀC underargon, and the mixturewas
maintained 15 h after addition of sodium acetate 5 M (570 mL,
stirred for 30 min at the same temperature. Isobutyl chloroformiate
2.8 mmol, 20 equiv) and a 30% H2O2 solution (0.160 mL, 22 equiv).
Flash chromatography on silica gel (AcOEt/cyclohexane 50:50 to
90:10) allowed the isolation of a yellow oil (68%, 57 mg).
(23 mL, 0.18 mmol, 1.1 equiv) was added dropwise and stirring was
continued for 1 h at ꢁ10 ꢀC. The amino acid (51 mg, 0.18 mmol,
1.1 equiv) was dissolved in 1.5 mL of anhydrous dichloromethane under
Two diastereoisomers: Rf¼0.16 (AcOEt/cyclohexane 90:10). 1H
NMR (CDCl3, 400 MHz): dH 1.13/1.15 (1H, ddd/ddd, J¼11.7, 11.7,
11.7 Hz, H8); 1.3–1.98 (23H, m, H6, 2C(CH3)3, CH2CH2CH2OH); 2.27
(1H, m, H8); 2.32–2.87 (5H, m, H1, CH2CO, H2, H6); 3.6–3.66 (3H, m,
CH2OH, H8a); 3.72–3.82 (1H, m, H7); 4.5–4.64 (3H, m, CH2Ph,
NHCHCO); 4.79/4.85 (1H, m, H5); 6.74/6.82 (1H, d/d, J¼8.0/8.6 Hz,
NH); 7.25–7.35 (5H, m, Ar). 13C NMR (75 MHz, CDCl3): dC 20.2/27.1
(CH2); 28.0 and 28.1 (C(CH3)3); 30.3, 31.0, 31.4, 36.4, 37.3 (CH2);
39.6/39.7 (C8); 41.2/41.3 (C2); 49.1/49.4 (NHCHCO); 51.0 (C5); 52.1/
52.3 (C8a); 62.2 (CH2OH); 70.6 (CH2Ph); 72.4/72.5 (C7); 81.7, 82.0,
and 82.3/82.5 (C(CH3)3); 127.6; 128.5 (2CAr, CAr, 2CAr); 138.1 (CArquat);
169.4/169.6, 170.1, 170.2, 176.7/176.8 (3CO, C3).
argon and NMM (40 m
L, 0.36 mmol, 2.2 equiv) was added at 0 ꢀC. This
solution was stirred for 30 min and added via a syringe to the mixed
anhydride solution. Stirring was continued for 20 h at rt. The residue was
taken off with 10 mL of ethyl acetate, washed with 5 mL of 10% Na2CO3
solution, 5 mL of 10% KHSO4 solution, and 5 mL of brine. The organic
phase was dried over Na2SO4 and the solvent removed in vacuo. The
residue was purified by flash chromatography on silica gel (AcOEt/cy-
clohexane 25:75 to 70:30) and the compound was isolated as a white
foam (88%, 65 mg).
Two diastereoisomers: Rf¼0.53 (AcOEt/cyclohexane 40:60).
1H NMR (CDCl3, 400 MHz): dH 1.16/1.17 (1H, ddd, J¼11.8, 11.8,

1410
D. Halie et al. / Tetrahedron 65 (2009) 1402–1414
4.1.16. 3-[[(2S
*
,5S
*
,7S
*
,8aR
*
)-7-Benzyloxy-2-(3-hydroxy-propyl)-
m, H7, H8a); 4.12 (2H, t, J¼6.0 Hz, CH2OCO); 4.48–4.70 (3H, m,
NHCHCO, CH2Ph); 4.70 (1H, m, H5); 7.29 (5H, m, Ar). 13C NMR
(100 MHz, MeOD): dC 25.6 (CH2CH2CH2OH); 26.6 (CH2CH2CH2OH);
32.6 and 32.7 (C1); 34.4 and 34.5 (C6); 35.4 (CH2COOH); 41.0 (C8);
41.3 (C2); 48.9 (NHCHCO); 51 (C5); 52.7 and 52.9 (C8a); 64.2 (C7); 67.0
3-oxo-octahydro-indolizin-5-carbonyl]-amino]propionic acid
tert-butyl ester 22
The same experimental procedure as described for hydro-
boration was applied to alkene 20 (106 mg, 0.23 mmol) using
a 0.4 M solution of 9-BBN in hexane (1.5 mL, 0.58 mmol, 2.5 equiv).
Stirring was maintained 16 h after addition of sodium acetate 5 M
(550 mL, 2.76 mmol, 12 equiv) and a 30% H2O2 solution (0.280 mL,
12 equiv). Flash chromatography on silica gel (AcOEt/MeOH 100 to
99:1) allowed the isolation of a yellow oil (71%, 74 mg).
(CH2OCO); 70.3 (CH2Ph); 127.5; 127.7; 128.1 and 128.3 (2CAr, CAr
,
2CAr); 138.1 (CArquat); 153.0 and 155.4 (C]N, OCONH); 170.7; 170.8;
172.6; 173; 173.1 and 177.2 (2NHCO, 2COOH, C3). HRMS (ESþ,
[MH]þ): calcd for C25H33N5O9 548.2357, found 548.2339.
Rf¼0.3 (AcOEt/MeOH 95:5). 1H NMR (CDCl3, 300 MHz): dH 1.14
(1H, ddd, J¼11.8, 11.8, 11.8 Hz, H8ax); 1.22–1.52 (12H, m, H1, H6,
CH2CH2CH2OH, 2C(CH3)3); 1.6 (2H, q, J¼11.8 Hz, CH2CH2CH2OH);
1.93 (1H, m, CH2CH2–CH2OH); 2.27 (1H, br d, J¼12 Hz, H8); 2.39 (2H,
t, J¼6 Hz, NHCH2CH2CO); 2.41–2.54 (2H, m, H2, H1); 2.6 (1H, br d,
J¼12 Hz, H6); 3.04 (1H, s, OH); 3.2–3.5 (2H, m, NHCH2CH2CO); 3.63
(1H, m, H8a); 3.63 (2H, t, J¼6.1 Hz, CH2OH); 3.88 (1H, dddd, J¼12, 12,
4.5, 4.5 Hz, H7); 4.55 (1H, d, J¼11.7 Hz, CH2Ph); 4.62 (1H, d,
J¼11.7 Hz, CH2Ph); 4.74 (1H, d, J¼5.9 Hz, H5); 6.79 (1H, t, J¼6 Hz,
NH); 7.2–7.45 (5H, m, Ar). 13C NMR (75 MHz, CDCl3): dC 27.2
(CH2CH2CH2OH); 28.1 (C(CH3)3); 29.7 (CH2CH2CH2OH); 31.1 (C6);
33.3 (C1); 35.1 (NHCH2CH2CO); 39.5 (C8); 41.3 (C2); 50.9 (C5); 52.3
(C8a); 62.1 (CH2OH); 70.5 (CH2Ph); 72.3 (C7); 81.1 (C(CH3)3); 127.6;
128.4 (2CAr, CAr, 2CAr); 138.4 (CArquat); 169.9, 171.4, 176.4 (2CO, C3).
4.1.19. 3-[[(2S
*
,5S
*
,7S
*
,8aR )-7-Benzyloxy-2-(3-bis(tert-
*
butyloxycarbonyl)guanidino-propyl)-3-oxo-octahydro-indolizin-
5-carbonyl]-amino]propionic acid tert-butyl ester 25
Alcohol 22 (76 mg, 0.16 mmol) and 1,3-bis(tert-butoxycarbonyl)
guanidine (70 mg, 0.27 mmol, 2.1 equiv) were dried under vacuo,
and dissolved in 5 mL of anhydrous THF under argon. Triphenyl-
phosphine (87 mg, 0.33 mmol, 2 equiv) was added to the solution
at rt, then diethylazodicarboxylate (DEAD) (52 mL, 0.33 mmol,
2 equiv) at 0 ꢀC. The mixture was stirred for 60 h at rt. After addi-
tion of dichloromethane, the solvent was removed in vacuo. The
residue was purified by flash chromatography on silica gel (Et2O/
cyclohexane 90:10) to give a white foam (105 mg, 92%).
Rf¼0.22 (Et2O/cyclohexane 90:10). 1H NMR (CDCl3, 300 MHz):
dH 1.15 (1H, ddd, J¼11.7, 11.7, 11.7 Hz, H8); 1.18–1.4 (3H, m, H1, H6,
CH2CH2CH2N); 1.44 (9H, s, C(CH3)3); 1.49 (9H, s, C(CH3)3); 1.55 (9H,
s, C(CH3)3); 1.65 (2H, m, CH2CH2CH2N); 1.94 (1H, m, CH2CH2CH2N);
2.28 (1H, br d, J¼11.6 Hz, H8); 2.39 (2H, t, J¼6 Hz, NHCH2CH2CO);
2.43–2.49 (2H, m, H2, H1); 2.63 (1H, br d, J¼12.7 Hz, H6); 3.34–3.49
(2H, m, NHCH2CH2CO); 3.56 (1H, m, H8a); 3.7–4.05 (3H, m, H7,
CH2N); 4.55 (1H, d, J¼11.8 Hz, CH2Ph); 4.63 (1H, d, J¼11.8 Hz,
CH2Ph); 4.74 (1H, d, J¼5.6 Hz, H5); 6.68 (1H, t, J¼5.9 Hz, NH); 7.3
(5H, m, Ar); 9.1–9.4 (2H, br s, NH2). 13C NMR (75 MHz, CDCl3): dC
26.2 (CH2CH2CH2N); 28.1 and 28.3 (3C(CH3)3, CH2CH2CH2N); 30.8
(C6); 33.3 (C1); 35.2 (NHCH2CH2CO); 39.6 (C8); 41.1 (C2); 44.3
(CH2CH2CH2N); 50.9 (C5); 52.3 (C8a); 70.6 (CH2Ph); 72.4 (C7); 78.6
81.1 and 83.7 (3C(CH3)3); 127.6; 128.3 (2CAr, CAr, 2CAr); 138.5 (CAr-
quat); 155,160.5, and 163.8 (C]N, CON); 169.9,171.3,176.4 (2CO, C3).
IR (NaCl disc, cmꢁ1): nmax 3382, 2977, 1715, 1608, 1514, 1368, 1251,
1152, 737. MS (ESþ): m/z 739 ([MNa]þ).
4.1.17. Guanidine carbamate 23
Alcohol 21 (60 mg, 0.1 mmol) and 1,3-bis(tert-butoxycarbonyl)
guanidine (53 mg, 0.2 mmol, 2 equiv) were dried under vacuo, and
dissolved in 2.5 mL of anhydrous toluene under argon. Tribu-
tylphosphine (50 mL, 0.2 mmol, 2 equiv) was added to the solution
at rt, then dipiperidinazodicarboxylate (ADDP) (52 mg, 0.2 mmol,
2 equiv) at 0 ꢀC. The mixture was refluxed for 4 h. After removal of
the toluene under vacuo, the residue was purified by flash chro-
matography on silica gel (Et2O/cyclohexane/MeOH 85:14:1)
allowed the isolation of 23 as a colorless oil (46 mg, 60%).
Two diastereoisomers: Rf¼0.14 (Et2O/cyclohexane/MeOH
85:14:1). 1H NMR (CDCl3, 400 MHz): dH 1.01–1.4 (31H, m, H8, H6,
3C(CH3)3, CH2CH2CH2OH); 1.5–1.7 (2H, m, CH2CH2CH2OH); 1.5–1.7
(1H, m, CH2CH2CH2OH); 2.1–2.4 (2H, m, H1, H8); 2.4–2.88 (4H, m,
CH2CO, H2, H6); 3.48–3.62 (1H, m, H8a); 3.62–3.78 (1H, m, H7);
4.03 (2H, br s, CH2OH); 4.43–4.59 (3H, m, CH2Ph, NHCHCO); 4.76
(1H, d, J¼11.7 Hz, H5); 7.21/7.26 (1H, d/d, J¼8.1/8.6 Hz, NH); 7.15–
7.29 (5H, m, Ar). 13C NMR (75 MHz, CDCl3): dC 26.7, 27.6/27.7 (CH2-
CH2CH2OH); 28.0 and 28.1 (3C(CH3)3); 29.8/30.4, 30.9/31.3, 33.3/
33.4 (C6, C1, CH2CO); 39.6/39.8 (C8); 41.2/41.3 (C2); 49.1/49.4, 50.9/
51.0, 52.0/52.2 (C5, NHCHCO, C8a); 65.5 (CH2OH); 70.9 (CH2Ph);
72.4/72.5 (C7); 81.7, 81.9, 82.2, and 82.5 (C(CH3)3); 127.7, 127.8,
128.5 (2CAr, CAr, 2CAr); 138.6 (CArquat); 158.5, 169.4, 169.7, 169.8,
170, 170.1, 170.2, 176/176.2 (C]N, 5CO, C3). MS (ESþ): m/z 760
([MH]þ). HRMS (ESþ, [MNa]þ): calcd for C38H57N5O11 782.3952,
found 782.3969.
4.1.20. 3-[[(2S
*
,5S
*
,7S
*
,8aR )-7-Benzyloxy-2-(3-guanidino-propyl)-
*
3-oxo-octahydro-indolizin-5-carbonyl]-amino]propionic acid 26
The same protocol for deprotection with TFA was applied to
compound 25 (43 mg, 0.06 mmol). Guanidyl compound was
obtained as foam in a quantitative yield (28 mg).
1H NMR (D2O, 300 MHz): dH 1.04 (1H, ddd, J¼11.7, 11.7, 11.7 Hz,
H8); 1.28 (2H, m, H1, CH2CH2CH2NH); 1.48 (3H, m, CH2CH2CH2NH,
H6); 1.7 (1H, m, CH2CH2CH2NH); 2.21 (1H, br d, J¼11.7 Hz, H8);
2.27–2.48 (4H, m, NHCH2CH2CO, H6, H1); 2.55 (1H, m, H2); 3.06 (2H,
t, J¼6.0 Hz, CH2CH2CH2NH); 3.31 (2H, t, J¼5.7 Hz, NHCH2CH2CO);
3.51 (1H, m, H7); 3.65 (1H, m, H8a); 4.47 (1H, s, CH2Ph); 4.62 (1H, d,
J¼5.6 Hz, H5); 7.28 (5H, m, Ar). 13C NMR (75 MHz, D2O): dC 25.2
(CH2CH2CH2NH); 27.2 (CH2CH2CH2NH); 31.6 (C6); 31.9 (C1); 33.4
4.1.18. Peptidomimetic 24
TFA (0.5 mL, 6.5 mmol) was added to a cooled (0 ꢀC) solution of
compound 23 (46 mg, 0.06 mmol) dissolved in CH2Cl2 (2 mL) with
a droplet of water. The mixture was stirred at rt for 4 h. The mixture
was concentrated in vacuo after addition of toluene (2 mL). Two
more additions and evaporations of toluene were done and finally
the residue was taken in ether (1 mL) and extracted with water
(three times). The aqueous phase was lyophilized to leave 24 as
a foam in a quantitative yield (33 mg).
(NHCH2CH2CO);
35.2
(NHCH2CH2CO);
38.2
(C8);
40.8
(CH2CH2CH2NH); 41.0 (C2); 51.6 (C8a); 53.3 (C5); 70.2 (CH2Ph); 71.8
(C7); 128.3; 128.5 and 128.7 (2CAr, CAr, 2CAr); 137.1 (CArquat); 156.6
(C]N); 171.4, 175.8, 178.6 (2CO, C3). MS (ESþ): m/z 460 ([MH]þ).
HRMS (ESþ, [MH]þ): calcd for C23H33N5O5 460.2560, found
460.2552.
4.1.21. 3-[(2S
*
,5S
*
,7S
*
,8aR )-[2-(3-Guanidino-propyl)-7-hydroxy-
*
Two diastereoisomers: mp: 60 ꢀC. 1H NMR (D2O, 400 MHz): dH
1.07 (1H, ddd, J¼11.7, 11.7, 11.7 Hz, H8); 1.32 (2H, m, CH2CH2CH2O,
H1); 1.52 (1H, m, H6); 1.65 (2H, m, CH2CH2CH2O); 1.75 (1H, m,
CH2CH2CH2O); 2.1 (1H, br d, J¼10.3 Hz, H8); 2.24 (1H, m, H6); 2.4 (1H,
m, H1); 2.59 (1H, m, H2); 2.7–2.88 (2H, m, CH2COOH); 3.65–3.8 (2H,
3-oxo-octahydro-indolizin-5-carbonyl]-amino]propionic acid 27
To benzyl ether 25 (45 mg, 0.063 mmol) dissolved in 7 mL of
methanol was added 35 mg of palladium on activated carbon (10% Pd).
The mixture was put under hydrogen and stirred 24 h. After filtration on
Celite, washing with ethyl acetate, the solvents were removed under

D. Halie et al. / Tetrahedron 65 (2009) 1402–1414
1411
vacuo. The residue was purified by flash chromatography on silica gel
(ethyl acetate) to give a colorless oil (28 mg, 71%).
using NMM (37
formiate (44
after addition of amino acid (100 mg, 0.34 mmol, 1.1 equiv) and
NMM (74 L, 0.68 mmol, 2.2 equiv). Flash chromatography on silica
gel (AcOEt/cyclohexane 80:20) allowed the isolation of a colorless
oil (90%, 146 mg).
m
L, 0.34 mmol, 1.1 equiv) and isobutyl chloro-
mL, 0.34 mmol,1.1 equiv). Stirring was maintained 20 h
Rf¼0.36 (AcOEt/MeOH 99:1). 1H NMR (CDCl3, 300 MHz): dH 1.14
(1H, ddd, J¼11.7, 11.7, 11.7 Hz, H8); 1.23–1.38 (2H, m, H1,
CH2CH2CH2N); 1.38–1.45 (28H, s, H6, 3C(CH3)3); 1.59–1.73 (2H, m,
CH2CH2CH2N); 1.95 (1H, m, CH2CH2CH2N); 2.21 (1H, dm, J¼12 Hz,
H8); 2.4 (2H, t, J¼6.2 Hz, NHCH2CH2CO); 2.43–2.63 (3H, m, H2, H1,
H6); 3.42 (2H, m, NHCH2CH2CO); 3.59 (1H, m, H8a); 3.8 (1H, m,
CH2N); 4.01 (1H, m, CH2N); 4.13 (1H, m, H7); 4.73 (1H, d, J¼5.7 Hz,
H5); 6.74 (1H, t, J¼4.5 Hz, NH); 9.26 (2H, br s, NH2). 13C NMR
(75 MHz, CDCl3): dC 26.2 (CH2CH2CH2N); 28.1 and 28.3 (3C(CH3)3);
33.3, 33.9, 35.1 (NHCH2CH2CO, C8, C6, C1); 41.1 (C2); 41.7, 44.3
(NHCH2CH2CO, CH2CH2CH2N); 50.9, 52.3 (C5, C8a); 65.0 (C7); 78.6,
81.1, and 83.7 (3C(CH3)3); 127.6; 128.3 (2CAr, CAr, 2CAr); 138.5
(CArquat); 155 (C]N); 160.5 and 163.8 (C]N, CON); 169.9, 171.4,
176.5 (2CO, C3). IR (NaCl disc, cmꢁ1): nmax 3383, 2978, 1715, 1608,
1515, 1251, 1151, 1101, 736. MS (ESþ): m/z 626 ([MH]þ).
m
Two diastereoisomers: Rf¼0.6 (AcOEt/cyclohexane 90:10). 1H
(CDCl3, 300 MHz): dH 1.12 (1H, ddd, J¼10.8, 10.8, 10.8 Hz, H8ax); 1.39
(19H, m, 2C(CH3)3, H6); 1.72–1.87 (2H, m, NHCHCH2CH2CO, H1);
1.98–2.07 (1H, m, NHCHCH2CH2CO, H1); 2.1–2.3 (4H, m,
NHCHCH2CH2CO, H8, CH2CH]CH2); 2.4–2.6 (3H, m, H2, H6,
CH2CH]CH2); 3.29 (3H, s, CH2OMe); 3.66 (1H, m, H8a); 3.82/3.92
(1H, dddd/dddd, J¼11.3, 11.3, 3.9, 3.9/11.2, 11.2, 3.9, 3.9 Hz, H7ax);
4.33 (1H, m, NHCHCH2CH2CO); 4.63 (2H, m, OCH2O); 4.77/4.80 (1H,
d/d, J¼5.7/6.2 Hz, H5eq); 5.06 (1H, br d, J¼17.1 Hz, CH2]CH); 5.04
(1H, m, CH2]CH); 5.73 (1H, m, CH2]CH); 6.66/6.73 (1H, d/d, J¼7.7/
8.0 Hz, NH). 13C NMR (75 MHz, CDCl3): dC 27.3/27.4 (2C(CH3)3); 30.2
(C1, NHCHCH2CH2CO); 31.2 (C6); 31.5 (NHCHCH2CH2CO); 35.4 (CH2–
The same protocol for deprotection with TFA was applied to
compound described above (28 mg, 0.045 mmol). Guanidyl com-
pound 27 was obtained as an oil in a quantitative yield (17 mg).
1H NMR (D2O, 400 MHz): dH 1.14 (1H, ddd, J¼11.7, 11.7, 11.7 Hz,
H8); 1.24–1.35 (2H, m, H1, CH2CH2CH2N); 1.45–1.55 (2H, m, H6,
CH2CH2CH2N); 1.69 (1H, m, CH2CH2CH2N); 2.12 (1H, dm, J¼12.1 Hz,
H8); 2.21 (1H, dm, J¼13.2 Hz, H6); 2.39 (1H, ddd, J¼12.6, 8.9, 7.1 Hz,
H1); 2.47 (1H, t, J¼6.4 Hz, NHCH2CH2CO); 2.58 (1H, dddd, J¼9.4, 9.4,
9.4, 4.2 Hz, H2); 3.08 (2H, t, J¼6.8 Hz, CH2CH2CH2N); 3.35 (2H, t,
J¼6.4 Hz, NHCH2CH2CO); 3.65–3.75 (2H, m, H7, H8a); 4.62 (1H, d,
J¼5.6 Hz, H5). 13C NMR (75 MHz, D2O): dC 25.2 (CH2CH2CH2N); 27.3
(CH2CH2CH2N); 31.5 (C1); 33.5 (NHCH2CH2CO); 34.1 (C6); 35.3
(NHCH2CH2CO); 40.5 (C8); 40.8 (CH2CH2CH2N); 41.0 (C2); 51.2 (C5);
53.3 (C8a); 64.3 (C7); 156.7 (C]N); 171.7, 176.1, 178.8 (2CO C3). MS
(ESþ): m/z 370 ([MH]þ), 392 ([MNa]þ). HRMS (ESþ, [MNa]þ): calcd
for C16H27N5O5 370.2090, found 370.2078.
CH]CH2); 39.3 (C8); 40.4 (C2); 50.0, 50.9, 52.2 (C5, C8a
,
NHCHCH2CH2CO); 55.3 (CH2OMe); 70.8 (C7); 80.65 (C(CH3)3); 82.1/
82.2 (C(CH3)3); 95.2 (OCH2O); 117.3:117.5 (CH2]CH); 135.0/035.3
(CH2]CH); 169.6/169.7, 170.4/170.6, 171.8 (3CO); 176.4/176.4 (C3).
MS (ESþ): m/z 547 ([MNa]þ).
4.1.24. (2S)-2-[[(2R
,5S
* *
,7S
*
,8aR )-2-(3-Hydroxy-propyl)-7-
*
methoxymethoxy-3-oxo-octahydro-indolizin-5-carbonyl]-
amino]pentanedioic acid tert-butyl ester 30
The same experimental procedure as described for hydro-
boration was applied to 29 (139 mg, 0.27 mmol) using a 0.5 M so-
lution of 9-BBN (1.6 mL, 0.81 mmol, 3 equiv). Stirring was
maintained 15 h after addition of sodium acetate 5 M (0.57 mL,
2.84 mmol, 10.5 equiv) and a 30% H2O2 solution (0.33 mL, 12 equiv).
Flash chromatography on silica gel (AcOEt/MeOH 98:2 to 95:5)
allowed the isolation of a colorless oil (65%, 93 mg).
4.1.22. (2R
*
,5S
*
,7S
*
,8aR
*
)-2-Allyl-7-methoxymethoxy-3-oxo-
Rf¼0.15 (AcOEt/MeOH 98:2). 1H (CDCl3, 300 MHz): dH 1.18 (1H,
ddd, J¼11.2, 11.2, 11.2 Hz, H8); 1.3–1.72 (23H, m, 2C(CH3)3, H6,
CH2CH2CH2OH); 1.78–1.99 (3H, m, NHCHCH2CH2CO, H1); 1.99–2.12
(1H, m, H1); 2.12–2.3 (3H, m, NHCHCH2CH2CO, H8); 2.45–2.64 (2H,
m, H2, H6); 3.35 (3H, s, CH2OMe); 3.62–3.78 (3H, m, CH2OH, H8a);
3.89/3.99 (1H, dddd/dddd, J¼11.2, 11.2, 3.9, 3.9/11.2, 11.2, 4, 4 Hz,
H7); 4.33–4.45 (1H, m, NHCHCH2CH2CO); 4.67/4.68 (1H, d/d, J¼6.8/
6.8 Hz, OCH2O); 4.72/4.73 (1H, d/d, J¼6.8/6.8 Hz, OCH2O); 4.83/4.86
octahydro-indolizin-5-carboxylic acid 28
To alcohol 17b (105 mg, 0.39 mmol) dissolved in 3.5 mL of ace-
tone was added dropwise Jones reagent 2.67 M (220 mL, 0.58 mmol,
1.5 equiv) at 0 ꢀC. Stirring was continued for 30 min, 3 mL of iso-
propanol was added, and the mixture was stirred for 10 min at 0 ꢀC.
After addition of 1 mL of water and 1 mL of a saturated solution of
KHSO4, the mixture was extracted twice with 100 mL of dichloro-
methane. The organic phase was dried (MgSO4) and the solvent
removed in vacuo. The residue was purified by flash chromatog-
raphy on silica gel (AcOEt/MeOH/AcOH 97:1:2) and the carboxylic
acid 28 was isolated as a white foam (54%, 60 mg).
(1H, d/d, J¼6/5.3 Hz, H5); 6.74/6.82 (1H, d/d, J¼8.1/7.7 Hz, NH). 13
C
NMR (75 MHz, CDCl3): dC 25.7, 27.4, 27.7 (CH2); 28.0 (2C(CH3)3);
30.1, 30.2, 31.5 (CH2); 31.1 (C6); 39.3/39.5 (C8); 40.5/40.6 (C2); 50.9
(C5); 52.3 (C8a NHCHCH2CH2CO); 55.4 (CH2OMe); 62.3/62.5
(CH2OH); 70.9 (C7); 80.8/81.1, 82.5 (C(CH3)3); 95.2:95.3 (OCH2O);
169.6/169.7, 170.5/171, 172/172.2 (3CO); 177.1/177.3 (C3). IR (NaCl
disc, cmꢁ1): nmax 3313, 2934, 1730, 1536, 1453, 1369, 1257, 1154. MS
(ESþ): m/z 565 ([MNa]þ). HRMS (ESþ, [MNa]þ): calcd for
C27H46N2O9 565.3101, found 565.3084.
Rf¼0.35 (AcOEt/MeOH/AcOH 97:1:2). 1H (CDCl3, 300 MHz): dH
1.19 (1H, ddd, J¼11.8, 11.8, 11.8 Hz, H8ax); 1.58 (1H, ddd, J¼12.9, 12.9,
6.8 Hz, H6); 1.79 (1H, ddd, J¼13.0, 9.2, 7.7 Hz, H1); 2.05 (1H, m, H1);
2.25 (2H, m, H8, CH2CH]CH2); 2.55 (3H, m, H2, H6, CH2CH]CH2);
3.38 (3H, s, CH2OMe); 3.70 (1H, dddd, J¼11.4, 11.4, 3.9, 3.9 Hz, H7ax);
3.82 (1H, dddd, J¼11.6, 8.0, 8.0, 3.8 Hz, H8a); 4.69 (2H, s, OCH2O); 4.95
(1H, d, J¼5.4 Hz, H5eq); 5.06 (1H, br d, J¼17.1 Hz, CH2]CH); 5.09 (1H,
dd, J¼7.0, 1.3 Hz, CH2]CH); 5.8 (1H, dddd, J¼17.1, 10.1, 7.0, 7.0 Hz,
CH2]CH). 13C NMR (75 MHz, CDCl3): dC 30.4 (C1); 32.3 (C6); 35.4
(CH2–CH]CH2); 39.4 (C8); 41.0 (C2); 50.0 (C5); 52.3 (C8a); 55.4
(CH2OMe); 70.8 (C7); 94.7 (OCH2O); 117.4 (CH2]CH); 135.3
(CH2]CH); 172.3 (COOH); 176.4 (C3). IR (NaCl disc, cmꢁ1): nmax 2936,
2368, 1735,1651, 1451, 1211, 1112,1039. MS (ESþ): m/z 306 ([MNa]þ).
HRMS(ESþ, [MNa]þ): calcdforC14H21NO5 306.1317, found:306.1306.
4.1.25. Guanidine carbamate 31
Alcohol 30 (67 mg, 0.127 mmol) and 1,3-bis(tert-butoxy-
carbonyl) guanidine (70 mg, 0.27 mmol, 2.1 equiv) were dried un-
der vacuo, and dissolved in 2.5 mL of anhydrous THF under argon.
Tributylphosphine (63 mL, 0.25 mmol, 2 equiv) was added to the
solution at rt, then dipiperidinazodicarboxylate (ADDP) (63 mg,
0.25 mmol, 2 equiv) at 0 ꢀC. The mixture was refluxed 2 h. After
addition of water and dichloromethane, the mixture was extracted.
The organic phase was dried (MgSO4) and the solvent removed in
vacuo. The residue was purified by flash chromatography on silica
gel (Et2O/MeOH 97:3) to give a white solid (40 mg, 43%).
4.1.23. (2S)-2-[(2R
oxo-octahydro-indolizin-5-carbonyl)-amino]pentanedioic acid
tert-butyl ester 29
The same experimental procedure as described for peptide
coupling was applied to carboxylic acid 28 (88 mg, 0.31 mmol)
*
,5S
*
,7S
*
,8aR )-(2-Allyl-7-methoxymethoxy-3-
*
Rf¼0.1 (Et2O/MeOH 98:2). 1H (CDCl3, 400 MHz): dH 1.18 (1H, ddd,
J¼11.7, 11.7, 11.7 Hz, H8); 1.27–1.48 (28H, m, 3C(CH3)3, H6); 1.5–1.63
(1H, m, CH2CH2CH2OH); 1.73–2.28 (10H, m, H8, NHCHCH2CH2CO,

1412
D. Halie et al. / Tetrahedron 65 (2009) 1402–1414
CH2CH2CH2OH, H1); 2.43–2.59 (2H, m, H2, H6); 3.33 (3H, s,
CH2OMe); 3.64–3.76 (1H, m, H8a); 3.85/3.93 (1H, dddd/dddd,
J¼11.2, 11.2, 4.0, 4.0/11.2, 11.2, 4.0, 4.0 Hz, H7); 4.05–4.15 (2H, m,
CH2OH); 4.32–4.43 (1H, m, NHCHCH2CH2CO); 4.65/4.66 (1H, d/d,
J¼6.8/6.8 Hz, OCH2O); 4.69/4.695 (1H, d/d, J¼6.8/6.8 Hz, OCH2O);
4.81/4.84 (1H, d/d, J¼6/5.8 Hz, H5); 6.74/6.82 (1H, d/d, J¼8.0/7.6 Hz,
NH). 13C NMR (75 MHz, CDCl3): dC 26.5, 26.6, 27.3, 27.4, 28.0
(3C(CH3)3); 29.6, 31.2, 31.5 (CH2); 39.0/39.3 (C8); 40.3 (C2); 50.9
(C5); 52/52.1, 52.2/52.4 (C8a, NHCHCH2CH2CO); 55.3 (CH2OMe);
64.9 (CH2OH); 70.9 (C7); 80.8, 82.2, 82.3, 82.7 (C(CH3)3); 95.2
(OCH2O); 158.7, 169.7, 169.8, 170.7, 172.0 (5CO, C]N); 176.4/176.8
(C3). IR (NaCl disc, cmꢁ1): nmax 3403, 2979, 1730, 1554, 1370, 1256,
1156, 1042, 733. MS (ESþ): m/z 750 ([MNa]þ). HRMS (ESþ, [MNa]þ):
calcd for C34H57N5O12 750.3901, found 750.3885.
NHCHCH2CH2CO); 4.63 (1H, m, H5). 13C NMR (100 MHz, D2O): dC
25.5, 25.6 (CH2CH2CH2NH NHCHCH2CH2CO); 27.7 (CH2CH2CH2NH);
30.1, 30.3 (NHCHCH2CH2CO, C1); 34.0 (C6); 39.8 (C8); 40.8 (C2); 40.9
(CH2CH2CH2NH); 51.0/51.1 (C5); 52.4 (NHCHCH2CH2CO); 53.7/53.9
(C8a); 64.6 (C7); 156.7 (C]N); 171.9, 172.1 (CONH); 175.1
(NHCHCOOH); 177.1 (COOH); 179.3 (C3). MS (ESþ): m/z 428
([MH]þ). HRMS (ESþ, [MH]þ): calcd for C18H29N5O7 428.2145, found
428.2146.
4.1.29. (5S
*
,7R
*
,8aR )-7-Benzyloxy-5-methyl-3-oxo-octahydro-
*
indolizin-5-carboxylic acid ethyl ester 35
To 12a (430 mg, 1.42 mmol) dissolved in 90 mL of anhydrous
THF under argon at ꢁ78 ꢀC was added 23.3 mL (11.7 mmol,
1.1 equiv) of KHMDS 0.5 M in toluene. After 30 min, methyl iodide
(filtered on neutral aluminum oxide; 725 mL, 11.65 mmol, 1.1 equiv)
4.1.26. Peptidomimetic 32
was added. Stirring was continued for 3 h at ꢁ78 ꢀC and the re-
action was quenched with a saturated solution of NH4Cl. The
product was extracted twice with CH2Cl2. The organic phase was
dried (MgSO4) and the solvent removed in vacuo. Flash chroma-
tography on silica gel (AcOEt/cyclohexane 50:50) allowed the iso-
lation of 35 as a pale yellow oil (3.5 mg, 99%).
The same protocol for deprotection with TFA was applied to
compound 31 (29 mg; 0.04 mmol). Guanidyl compound was
obtained as a foam in a quantitative yield (19 mg).
1H NMR (D2O, 400 MHz): dH 1.11 (1H, ddd, J¼11.7, 11.7, 11.7 Hz,
H8); 1.4–1.58 (2H, m, H6, CH2CH2CH2O); 1.58–1.7 (3H, m,
CH2CH2CH2O); 1.72–1.9 (2H, m, NHCHCH2CH2COOH, H1); 1.9–2.17
(5H, m, NHCHCH2CH2COOH, H8, H1); 2.27 (1H, m, H6); 2.53 (1H, m,
H2); 3.78–3.9 (2H, m, H7, H8a); 4.03 (1H, m, NHCHCH2CH2COOH);
4.11 (2H, br s, CH2CH2CH2O); 4.68 (1H, m, H4). HRMS (ESþ, [MNa]þ):
calcd for C19H29N5O9 494.1863, found 494.1860.
Rf¼0.37 (AcOEt/cyclohexane: 70/30). 1H NMR (CDCl3, 300 MHz):
dH 1.12 (3H, t, J¼7.1 Hz; CH3CH2); 1.36 (1H, ddd, J¼13.5, 12.2, 2.6 Hz,
H8ax); 1.50 (1H, dddd, J¼10.4, 10.4, 10.4, 10.4 Hz, H1); 1.56 (1H, dd,
J¼14.4, 2.5 Hz, H6ax); 1.89 (3H, s, Me); 2.12 (2H, m, H1, H8eq), 2.33
(2H, m, 2H2); 2.65 (1H, ddd, J¼14.4, 3.5, 2.2 Hz, H6eq); 3.77 (1H,
dddd, J¼2.9, 2.9, 2.9, 2.9 Hz, H7eq); 3.94 (1H, dq, J¼10.7, 7.1 Hz,
OCH2CH3); 3.94 (1H, m, H8a); 4.04 (1H, dq, J¼10.7, 7.1 Hz, OCH2CH3);
4.40 (1H, d, J¼11.9 Hz, CH2Ph); 4.51 (1H, d, J¼11.9 Hz, CH2Ph); 7.29
(5H, m, Ar). 13C NMR (75 MHz, CDCl3): dC 14.0 (CH3CH2); 26.0 (Me);
26.6 (C1); 31.9 (C2); 36.7 (C8); 40.4 (C6); 52.1 (C8a); 59.2 (C5); 61.3
(OCH2CH3); 70.1 (CH2Ph); 70.7 (C7); 127.2 (2CAr); 127.5 (CAr); 128.3
(CAr); 138.5 (CArquat); 173.1, 177.7 (CO, C3). IR (NaCl disc, cmꢁ1): nmax
3460, 2938, 1682, 1455, 1271, 1027, 731. HRMS (ESþ, [MNa]þ): calcd
for C19H25NO4 354.1681, found 354.1665.
4.1.27. (2S)-2-[[(2R
*
*
,5S ,7S
*
,8aR )-2-(3-Bis(tert-butyloxycarbonyl)-
*
guanidino-propyl)-7-methoxymethoxy-3-oxo-octahydro-indolizin-
5-carbonyl]-amino]pentanedioic acid tert-butyl ester 33
The same experimental procedure as described for 25 above was
applied to 30 (33 mg, 0.062 mmol) using and 1,3-bis(tert-butoxy-
carbonyl) guanidine (39 mg, 0.15 mmol, 2 equiv), triphenylphos-
phine (40 mg, 0.15 mmol, 2 equiv), and diethylazodicarboxylate
(DEAD) (24 mL, 0.15 mmol, 2 equiv). Stirring was continued for 3
days. Flash chromatography on silica gel (Et2O/cyclohexane 70:30
to 100:0) allowed the isolation of 33 as a colorless oil (29 mg, 60%)
(two diastereoisomers).
4.1.30. (2R
octahydro-indolizin-5-carboxylic acid ethyl ester 36 and
(2S ,5S ,7R ,8aR )-2-allyl-7-benzyloxy-5-methyl-3-oxo-octahydro-
*
,5S
*
,7R
*
,8aR
*
)-2-Allyl-7-benzyloxy-5-methyl-3-oxo-
Rf¼0.45 (Et2O). 1H (CDCl3, 400 MHz): dH 1.21 (1H, ddd, J¼11.7,
11.7, 11.7 Hz, H8); 1.35–1.6 (37H, m, 4C(CH3)3, H6); 1.62–2.12 (8H, m,
CH2CH2CH2NH, H8, NHCHCH2CH2CO, H1); 2.15–2.3 (3H, m,
NHCHCH2CH2CO, H8); 2.47–2.63 (2H, m, H6, H2); 3.36 (3H, s,
CH2OMe); 3.7–4.06 (4H, m, H8a, CH2CH2CH2NH, H7); 4.09 (1H, m,
NHCHCH2CH2CO); 4.67 (1H, d, J¼6.8 Hz, OCH2O); 4.71 (1H, d,
J¼6.8 Hz, OCH2O); 4.74 (1H, d, J¼5.4 Hz, H5); 6.72 (1H, d, J¼7.6 Hz,
NH); 9.26 (2H, br s, NH2). 13C NMR (75 MHz, CDCl3): dC 26.3, 26.5,
27.4 (CH2CH2CH2NH NHCHCH2CH2CO); 28.1, 28.3 (4C(CH3)3); 30.8,
31.5, 31.6 (CH2CH2CH2NH, NHCHCH2CH2CO, C6, C1); 38.9/39.2 (C8);
40.1 (C2); 44.2 (CH2CH2CH2NH); 51, 52, 52.1, 52.3, 55.4 (C8a, C5,
NHCHCH2CH2CO, CH2OMe); 64; 70.9, 71.0 (C7); 78.6, 80.8, 82.3, 83.7
(4C(CH3)3); 95.2 (OCH2O); 155.0, 160.5, and 163.8 (C]N, N–CO);
169.8, 170.5, 170.7, 172.0, 176.9, 177.2 (4CO, C3). IR (NaCl disc, cmꢁ1):
nmax 3382, 2977, 1715, 1609, 1513, 1368, 1251, 1152, 737. MS (ESþ):
m/z 784 ([MH]þ).
*
*
*
*
indolizin-5-carboxylic acid ethyl ester 37
The same experimental procedure as described for allylation
was applied to 35 (1.5 g, 4.53 mmol) using KHMDS 0.5 M (9.5 mL,
4.75 mmol, 1.01 equiv) in 40 mL of THF. After 20 min stirring at
–78 ꢀC, solution was cooled at –110 ꢀC before addition of distillated
allyl bromide (470 mL, 5.4 mmol, 1.2 equiv) stirring was continued
1 h, then 3h 30 at ꢁ78 ꢀC. Flash chromatography on silica gel
(AcOEt/cyclohexane 50:50) allowed the isolation of 36 as a pale
yellow oil (561 g, 33%) and 37 as a pale yellow oil (366 mg, 22%).
Starting material was also recovered (35%).
Compound 36: Rf¼0.33 (Et2O/cyclohexane 50:50). 1H NMR
(CDCl3, 300 MHz): dH 1.13 (3H, t, J¼7.1 Hz, CH3CH2); 1.20 (1H, q,
J¼10.9 Hz, H1); 1.32 (1H, ddd, J¼14.6, 12.2, 2.4 Hz, H8ax); 1.56 (1H,
dd, J¼14.4, 2.4 Hz, H6ax); 1.90 (3H, s, Me); 2.18 (3H, m, CH2CH]CH2,
H1, H8eq); 2.48 (1H, m, H2); 2.66 (1H, ddd, J¼14.4, 3.4, 1.8 Hz, H6equ);
2.68 (1H, m, CH2CH]CH2); 3.77 (1H, br s, H7equ); 3.88 (1H, m, H8a);
3.93 (1H, dq, J¼10.7, 7.1 Hz, OCH2CH3); 4.04 (1H, dq, J¼10.7, 7.1 Hz,
OCH2CH3); 4.40 (1H, d, J¼11.9 Hz, CH2Ph); 4.52 (1H, d, J¼11.9 Hz,
CH2Ph); 5.04 (1H, br d, J¼17 Hz, CH2CH]CH2); 5.09 (1H, br d,
J¼17 Hz, CH2CH]CH2); 5.80 (1H, dddd, J¼17, 10.1, 6.9, 6.9 Hz,
CH2CH]CH2); 7.28 (5H, m, Ar). 13C NMR (75 MHz, CDCl3): dC 13.9
(CH3CH2); 26.2 (Me); 32.8 (C1); 35.0 (CH2CH]CH2); 36.5 (C8); 40.2
(C6); 42.1 (C2); 49.9 (C8a); 59.1 (C5); 61.3 (OCH2CH3); 70.0 (CH2Ph);
70.6 (C7); 116.4 (CH2CH]CH2); 127.1 (2CAr); 127.4 (CAr); 128.2
(2CAr); 136.0 (CH2CH]CH2); 138.5 (CArquat); 173.1, 178.5 (CO, C3). IR
(NaCl disc, cmꢁ1): nmax 2925, 1737, 1698, 1454, 1396, 1307, 1274,
1207, 1130, 1028, 913, 736. MS (ESþ): m/z 394 ([MNa]þ). Anal. Calcd
4.1.28. (2S)-2-[[(2R
*
,5S ,7S
* *
,8aR )-2-(3-Guanidino-propyl)-
*
7-hydroxy-3-oxo-octahydro-indolizin-5-carbonyl]-
amino]pentanedioic acid 34
The same protocol for deprotection with TFA was applied to
compound 33 (46 mg, 0.06 mmol). Guanidyl compound was
obtained as a white solid in a quantitative yield (33 mg).
Mp: 60 ꢀC. 1H (D2O, 400 MHz): dH 1.1 (1H, m, H8); 1.35–1.53 (5H,
m, CH2CH2CH2NH, H6); 1.77–1.98 (3H, m, NHCHCH2CH2CO, H8);
1.98–2.15 (2H, m, H8); 2.21 (1H, m, H6); 2.31 (2H, br s,
NHCHCH2CH2CO); 2.52 (1H, br s, H2); 3.06 (2H, t, J¼5.9 Hz,
CH2CH2CH2NH); 3.71 (1H, m, H7); 3.79 (1H, m, H8a); 4.25 (1H, m,

D. Halie et al. / Tetrahedron 65 (2009) 1402–1414
1413
for C22H29NO4: C, 71.13; H, 7.87; N, 3.77. Found: C, 71.52; H, 7.96; N,
3.77%.
26.1 (Me); 27.6 (CH2CH2CH2N); 28.0 and 28.3 (CH2CH2CH2N,
2C(CH3)3); 33.1 (C1); 36.5 (C8); 40.3 (C6); 42.0 (C2); 44.3
(CH2CH2CH2N); 49.9 (C8a); 59.0 (C5); 61.2 (OCH2CH3); 69.9 (CH2Ph);
70.5 (C7); 78.4 (C(CH3)3); 83.6 (C(CH3)3); 127.0 (2CAr); 127.3 (CAr);
128.2 (2CAr); 138.3 (CArquat); 155.0; 160.5; 163.8 (C]N, NCOO);
173.0, 179.1 (CO, C3). IR (NaCl disc, cmꢁ1): nmax 3390, 2881, 1714,
1608, 1271, 1150. MS (ESþ): m/z 653 ([MNa]þ).
Compound 37: Rf¼0.2 (Et2O/cyclohexane 50:50). 1H NMR (CDCl3,
300 MHz): dH 1.14 (3H, t, J¼7.2 Hz, CH3CH2); 1.34 (1H, ddd, J¼13.3
12.3, 2.5 Hz, H8ax); 1.54 (1H, dd, J¼14.4, 2.5 Hz, H6ax); 1.60 (1H, m,
H1); 1.89 (3H, s, Me); 1.97 (1H, dd, J¼12.7, 6.0 Hz, H1); 2.10 (1H, ddd,
J¼13.4, 13.4, 13.4 Hz, H8eq); 2.28 (1H, m, CH2CH]CH2); 2.45 (1H, m,
H2); 2.50 (1H, m, CH2CH]CH2); 2.64 (1H, ddd, J¼14.4, 3.3, 2.2 Hz,
H6equ); 3.76 (1H, q, J¼2.7 Hz, H7); 3.95 (1H, dq, J¼10.8, 7.1 Hz,
OCH2CH3); 3.98 (1H, m, H8a); 4.04 (1H, dq, J¼10.7, 7.1 Hz, OCH2CH3);
4.38 (1H, d, J¼11.9 Hz, CH2Ph); 4.52 (1H, d, J¼11.9 Hz, CH2Ph); 5.06
(1H, br d, J¼10.1 Hz, CH2CH]CH2); 5.09 (1H, dd, J¼17.1, 1.4 Hz,
CH2CH]CH2); 5.85 (1H, dddd, J¼17, 10.1, 7.8, 6.0 Hz, CH2CH]CH2);
7.28 (5H, m, Ar). 13C NMR (75 MHz, CDCl3): dC 14.0 (CH3CH2); 25.9
(Me); 30.7 (C1); 34.7 (CH2CH]CH2); 36.8 (C8); 40.2 (C6); 41.9 (C2);
50.0 (C8a); 59.0 (C5); 61.3 (OCH2CH3); 70.1 (CH2Ph); 70.6 (C7); 116.7
(CH2CH]CH2); 127.2 (2CAr); 127.5 (CAr); 128.3 (2CAr); 136.4
(CH2CH]CH2); 138.3 (CArquat); 173.0, 178.8 (CO, C3). IR (NaCl disc,
cmꢁ1): nmax 2937, 1736, 1694, 1446, 1396, 1276, 1208, 1132, 1067,
912, 729. MS (ESþ): m/z 394 ([MNa]þ).
4.1.32. (2S
*
,4R
*
,6R
*
)-6-[(2R )-2-Carboxy-5-guanidino-pentyl]-4-
*
hydroxy-2-methyl-piperidin-2-carboxylic acid 39
Compound 38 (70 mg, 0.11 mmol) was dissolved in 5 mL of HCl
3 M. The mixture was refluxed for 20 h. The solvent was evaporated
under vacuo. To the residue were added 1 mL of diethyl ether and
2 mL of water, insoluble solids were filtered and the product was
extracted three times with 2 mL of water. The aqueous phase was
lyophilized to leave an orange oil in a 100% yield (38 mg).
1H NMR (D2O, 400 MHz): dH 1.3–1.65 (8H, m, CH2CHCOOH, H5,
Me, CH2CH2CH2N); 1.67 (1H, br d, J¼15.0 Hz, H3); 1.75–1.95 (3H, m,
CH2CH2CH2N, CH2CHCOOH, H5); 2.3–2.5 (2H, m, H3, CH2CHCOOH);
3.0 (2H, br s, CH2CH2CH2N); 3.67 (1H, m, H6); 4.05 (1H, br s, H4). 13
C
NMR (75 MHz, D2O): dC 24.4 (Me); 25.4 (CH2CH2CH2N); 28.7
(CH2CH2CH2N); 33.2 (C5); 34.4 (CH2CHCOOH); 37.8 (C3); 40.7
(CH2CHCOOH, CH2CH2CH2N); 47.5 (C6); 59.2 (C2); 61.7 (C4); 156.6
(C]N); 173.8, 178.6 (CO, C3). MS (ESþ): m/z 313 ([MꢁH2O]þ), 331
([MꢁH]þ). HRMS (ESþ, [MH]þ): calcd for C14H26N4O5 331.1981,
found 331.1965.
4.1.31. (2R
*
,5S
*
,7R
*
,8aR )-7-Benzyloxy-2-(3-bis(tert-
*
butyloxycarbonyl)guanidino-propyl)-5-methyl-3-oxo-octahydro-
indolizin-5-carboxylic acid ethyl ester 38
trans intermediate alcohol: the same experimental procedure as
described for hydoboration was applied to alkene 36 (309 mg,
0.83 mmol) using
a 0.5 M solution of 9-BBN in THF (5 mL,
2.49 mmol, 3 equiv). Stirring was maintained 16 h after addition of
sodium acetate 5 M (2 mL, 9.9 mmol, 12 equiv) and a 30% H2O2
solution (1 mL, 12 equiv). Flash chromatography on silica gel
(AcOEt/cyclohexane 90:10) allowed the isolation of a yellow oil
(60%, 193 mg).
4.1.33. (2S
*
,5S
*
,7R
*
,8aR )-7-Benzyloxy-2-(3-bis(tert-
*
butyloxycarbonyl)guanidino-propyl)-5-methyl-3-oxo-octahydro-
indolizin-5-carboxylic acid ethyl ester 40
cis intermediate alcohol: the same experimental procedure as
described for hydroboration was applied to alkene 37 (178 mg,
0.48 mmol) using a 0.5 M solution of 9-BBN in THF (2 mL, 1 mmol,
2.1 equiv). Stirring was maintained 16 h after addition of sodium
acetate 5 M (1.15 mL, 5.76 mmol, 12 equiv) and a 30% H2O2 solution
(0.6 mL, 5.76 mmol, 12 equiv). Flash chromatography on silica gel
(AcOEt/Et2O 90:10) allowed the isolation of a yellow oil (87%,
162 mg).
Rf¼0.24 (AcOEt/cyclohexane 95:5). 1H NMR (CDCl3, 300 MHz):
dH 1.09 (3H, t, J¼7.1 Hz, CH3CH2); 1.17 (1H, ddd, J¼11.4, 11.4, 11.4 Hz,
H1); 1.31 (1H, ddd, J¼13.9, 11.4, 2.4 Hz, H8ax); 1.44 (1H, m,
CH2CH2CH2OH); 1.54 (1H, dd, J¼14.3, 2.3 Hz, H6); 1.6 (2H, m,
CH2CH2CH2OH); 1.78–1.95 (4H, s, Me, CH2CH2CH2OH); 2.14 (1H,
dm, J¼11.4 Hz, H8); 2.25 (1H, ddd, J¼11.5, 7.9, 5.4 Hz, H1); 2.38 (1H,
m, H2); 2.62 (1H, br d, J¼14.4 Hz, H6); 3.6 (2H, t, J¼6.3 Hz,
CH2CH2CH2OH); 3.75 (1H, m, H7); 3.85 (1H, m, H8a); 3.93 (1H, dq,
J¼10.7, 7.1 Hz, OCH2CH3); 3.97 (1H, dq, J¼10.7, 7.1 Hz, OCH2CH3);
4.38 (1H, d, J¼11.9 Hz, CH2Ph); 4.49 (1H, d, J¼11.9 Hz, CH2Ph); 7.29
(5H, m, Ar). 13C NMR (75 MHz, CDCl3): dC 13.9 (CH3CH2); 26.1 (Me);
26.6 (CH2CH2CH2OH); 30.1 (CH2CH2CH2OH); 33.3 (C1); 36.4 (C8);
40.2 (C6); 42.2 (C2); 50.0 (C8a); 59.1 (C5); 61.3 (OCH2CH3); 62.1
(CH2CH2CH2OH); 70.0 (CH2Ph); 70.5 (C7); 127.1 (2CAr); 127.4 (CAr);
128.2 (2CAr); 138.3 (CArquat); 173.0, 179.7 (CO, C3). IR (NaCl disc,
cmꢁ1): nmax 3412, 2928,1734, 1681, 1399,1307,1273,1210,1131, 735.
MS (ESþ): m/z 412 ([MNa]þ).
Rf¼0.18 (AcOEt/Et2O 90:10). 1H NMR (CDCl3, 300 MHz): dH 1.15
(3H, t, J¼7.1 Hz, CH3CH2); 1.34 (1H, ddd, J¼13.6 11.2, 2.4 Hz, H8); 1.57
(1H, dd, J¼14.4, 2.5 Hz, H6); 1.61–1.96 (9H, m, Me, CH2CH2CH2OH,
H1); 2.13 (1H, dm, J¼13.5 Hz, H8); 2.28 (1H, br s, OH); 2.46 (1H, m,
H2); 2.67 (1H, ddd, J¼14.4, 3.4, 2.1 Hz, H6); 3.7 (2H, m,
CH2CH2CH2OH); 3.78 (1H, m, H7); 3.93–4.09 (3H, m, H8a, OCH2CH3);
4.41 (1H, d, J¼11.9 Hz, CH2Ph); 4.53 (1H, d, J¼11.9 Hz, CH2Ph); 7.3
(5H, m, Ar). 13C NMR (100 MHz, CDCl3): dC 14.1 (CH3CH2); 26.0 (Me);
27.2 (CH2CH2CH2OH); 30.5 (CH2CH2CH2OH); 32.4 (C1); 34.7
(CH2CH]CH2); 36.9 (C8); 40.3 (C6); 41.9 (C2); 50.3 (C8a); 59.1 (C5);
61.5 (OCH2CH3); 62.6 (CH2CH2CH2OH); 70.2 (CH2Ph); 70.6 (C7);
127.3 (2CAr); 127.6 (CAr); 128.4 (2CAr); 138.4 (CArquat); 173.1 179.6
(CO, C3). IR (NaCl disc, cmꢁ1): nmax 3389, 2952, 1651, 1454, 1286,
1066, 910, 728. MS (ESþ): m/z 412 ([MNa]þ).
The same experimental procedure as described for 25 was ap-
plied to intermediate trans (300 mg, 0.77 mmol) using and 1,3-
bis(tert-butoxycarbonyl) guanidine (311 mg, 1.2 mmol, 1.5 equiv),
triphenylphosphine (315 mg, 1.2 mmol, 1.5 equiv), and diethyl-
The same experimental procedure as described for 25 was ap-
plied to cis intermediate (24 mg, 0.062 mmol) using and 1,3-bis-
(tert-butoxycarbonyl) guanidine (26 mg, 0.1 mmol, 1.6 equiv),
triphenylphosphine (26 mg, 0.1 mmol, 1.6 equiv), and diethyl-
azodicarboxylate (DEAD) (190 mL, 1.2 mmol, 1.5 equiv). Stirring was
continued for 24 h. Flash chromatography on silica gel (Et2O/cy-
clohexane 50:50) allowed the isolation of 38 as a white solid
(436 mg, 90%).
azodicarboxylate (DEAD) (16 mL, 0.1 mmol, 1.6 equiv). Stirring was
Rf¼0.27 (Et2O/cyclohexane 50:50), mp: 132 ꢀC. 1H NMR (CDCl3,
300 MHz): dH 1.04 (3H, t, J¼7.1 Hz, CH3CH2); 1.07 (1H, m, H1); 1.25
(2H, m, CH2CH2CH2N H8); 1.4–1.55 (19H, m, 2C(CH3)3 H6); 1.58 (2H,
m, CH2CH2CH2N); 1.81 (3H, s, Me); 1.85 (1H, m, CH2CH2CH2N); 2.08
(1H, br d, J¼12.5 Hz, H8); 2.34 (2H, m, H2, H1); 2.59 (1H, br d,
J¼14.2 Hz, H6); 3.62–3.75 (2H, m, H7, CH2CH2CH2N); 3.75–3.89 (2H,
H8a, OCH2CH3); 3.89–4.0 (2H, m, CH2CH2CH2N OCH2CH3); 4.32 (1H,
d, J¼11.9 Hz, CH2Ph); 4.45 (1H, d, J¼11.9 Hz, CH2Ph); 7.21 (5H, m,
Ar); 9.22 (2H, m, NH2). 13C NMR (75 MHz, CDCl3): dC 13.9 (CH3CH2);
continued for 44 h. Flash chromatography on silica gel (Et2O/cyclo-
hexane 70:30) allowed the isolation of 40 as a white solid (40 mg,
100%).
Rf¼0.31 (AcOEt/Et2O 70:30), mp >60 ꢀC. 1H NMR (CDCl3,
300 MHz): dH 1.13 (3H, t, J¼7.2 Hz, CH3CH2); 1.31 (1H, m, H8); 1.41–
1.62 (20H, m, CH2CH2CH2N, 2C(CH3)3, H6); 1.63–1.62 (4H, m,
CH2CH2CH2N, H1); 1.95 (4H, m, Me, H1); 2.12 (1H, br d, J¼13.4 Hz,
H8); 2.4 (1H, m, H2); 2.67 (1H, br d, H6); 3.78 (1H, br s, H7); 3.82–
4.09 (5H, m, CH2CH2CH2N, H8a, OCH2CH3); 4.39 (1H, d, J¼12 Hz,

1414
D. Halie et al. / Tetrahedron 65 (2009) 1402–1414
CH2Ph); 4.52 (1H, d, J¼12 Hz, CH2Ph); 7.3 (5H, m, Ar); 9.23 (2H, br s,
NH2). 13C NMR (75 MHz, CDCl3): dC 14.0 (CH3CH2); 25.9 (Me); 26.8
and 27.3 (CH2CH2CH2N, CH2CH2CH2N); 28.0 and 28.3 (2C(CH3)3);
31.6 (C1); 36.9 (C8); 40.2 (C6); 42.0 (C2); 44.5 (CH2CH2CH2N); 50.1
(C8a); 59.0 (C5); 61.3 (OCH2CH3); 70.0 (CH2Ph); 70.6 (C7); 78.6
(C(CH3)3); 83.7 (C(CH3)3); 127.2 (2CAr); 127.5 (CAr); 128.3 (2CAr);
138.3 (CArquat); 155.1, 156.8, 163.9 (C]N, NCOO); 173.0, 179.2 (CO,
C3). IR (NaCl disc, cmꢁ1): nmax 3382, 2930, 1714, 1614, 1454, 1359,
1255, 1152. MS (ESþ): m/z 631 ([MNa]þ).
Data are the meanꢂSD of triplicates and are expressed as the % of
cell adhesion obtained on FN in the absence of competitor. The
positive and negative controls for the inhibition of cell adhesion
were done using the cyclic c(RGDfV) and GRGES peptides, re-
spectively. Two independent experiments were done for each
competitor and for each cell type.
Acknowledgements
´
We thank the ministere de l’enseignement superieur et de la
4.1.34. (2S
*
,4R
*
,6R
*
)-6-[(2S )-2-Carboxy-5-guanidino-pentyl]-4-
*
recherche for a grant and the DRAB for a financial support. We
thank Drs. Isabelle Berque-Bestel and Sandrine Ongeri Sandrine for
a molecular modeling of a peptidomimetic model.
hydroxy-2-methyl-piperidin-2-carboxylic acid 41
The same experimental procedure as described for 39 was ap-
plied to 40 (67 mg, 0.1 mmol) in 5 mL of HCl 3 M. The aqueous
phase was lyophilized to leave brown oil in a 100% yield (44 mg).
1H NMR (D2O, 400 MHz): dH 1.45 (3H, s, Me); 1.46–1.6 (5H, m,
CH2CHCOOH, H5, CH2CH2CH2N); 1.75 (1H, br d, J¼14.4 Hz, H3);
1.78–1.93 (3H, m, CH2CH2CH2N, CH2CHCOOH, H5); 2.48 (1H, br d,
J¼15.1 Hz, H3); 2.58 (1H, q, J¼6.4 Hz, CH2CHCOOH); 3.09 (2H, t,
J¼6.5 Hz, CH2CH2CH2N); 3.75 (1H, m, H6); 4.09 (1H, br s, H4). 13C
NMR (75 MHz, D2O): dC 24.5 (Me); 25.2 (CH2CH2CH2N); 28.2
(CH2CH2CH2N); 33.5 (C5); 34.1 (CH2CHCOOH); 37.7 (C3); 40.7
(CH2CH2CH2N); 40.9 (CH2CHCOOH); 47.8 (C6); 59.4 (C2); 61.8 (C4);
156.6 (C]N); 174.1, 179.1 (CO, C3). MS (ESþ): m/z 331 ([MꢁH]þ).
HRMS (ESþ, [MH]þ): calcd for C14H26N4O5 331.1981, found 331.1965.
References and notes
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4.2. Cell culture and cell-adhesion assay
4. Kumar, S.; Wang, Q.; Sasaki, A. Tetrahedron 2007, 63, 2084–2090 and references
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S180 and Eahy926 cells were cultured in DMEMþ10% FCS and
DMEMþ20%FCSþHAT, respectively. For adhesion assays, cells were
harvested with cell dissociation non-enzymatic solution (SIGMA).
Cells were pelleted by centrifugation and resuspended at a density
of 1ꢃ105 cells/ml in PBS/CaMg. Assays of cell adhesion to coated
substrates were performed on non-tissue culture treated 96-well
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plates. The wells were incubated with FN or VN at 10 m
g mlꢁ1 in PBS
overnight at 4 ꢀC followed by a 30-min incubation with 3 mg mlꢁ1
bovine serum albumin in PBS (previously heat-inactivated for
3 min at 80 ꢀC). The substrates were thoroughly washed and
maintained in PBS until use. The peptidomimetics solutions were
´
7. Halie, D.; Perard-Viret, J.; Royer, J. Heterocycles 2006, 68, 2471–2476.
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This paper described the same reaction. But, we do not agree with the proposed
mechanism. We suggest a simple transesterification type reaction since we
obtained the same product in the absence of ADDP and Bu3P.
prepared either at 20
peptidomimetic solution were added to the well with 50
cell suspension reaching the final peptidomimetic solution to
10 M, 200 M or 500 M for the assay. Each test was done in
triplicate. The plates were incubated at 37 ꢀC for 1 h and washed
with PBS to remove the non-adherent cells. Then, the wells were
fixed in 5% glutaraldehyde in PBS, washed, stained with 1% crystal
violet in 200 mM MES, and washed in 200 mM MES buffer, pH 6.
m
M, 400
m
M or 1 mM in PBSCaMg. 50
ml of
ml of the
m
m
m
The crystal violet fixed to the cells was dissolved in 100
mL of 10%
12. Beauvais, A.; Erickson, A.; Goins, T.; Craig, S. E.; Suzan, E.; Humphries, J. P.;
Dufour, S. J. Cell Biol. 1995, 128, 699–713.
acetic acid and OD was read with a microplate reader at 570 nm.