Improved synthesis of a [4.4]-spirolactam β-turn mimetic as surrogate of the didemnin side chain dipeptide Pro-N-Me-D-Leu

Article abstract of DOI:10.1016/j.tet.2004.04.052

An efficient synthesis of [4.4]-spirolactam restricted derivatives of the didemnin side chain dipeptide L-Pro-N-Me-D-Leu is described. This methodology involves: (a) peptide coupling of N-Boc-2-allylproline with D-Leu-OBn; (b) OsO₄/NaIO₄ mediated allyl oxidation and intramolecular cyclization to the corresponding cyclic hemiaminals; and (c) NaBH₄ mediated reduction of an intermediate N-acyliminium ion. This synthetic strategy gave significant better results than the previously reported strategies for the synthesis of [4.4]-spirolactam β-turn mimetics.

Full text of DOI:10.1016/j.tet.2004.04.052

Tetrahedron 60 (2004) 5177–5183
Improved synthesis of a [4.4]-spirolactam b-turn mimetic as
surrogate of the didemnin side chain dipeptide Pro-N-Me-D-Leu
*
´ ´ ´ ´
Marta Gutierrez-Rodrıguez, M. Teresa Garcıa-Lopez and Rosario Herranz
´ ´
Instituto de Quımica Medica (CSIC), Juan de la Cierva 3, E-28006 Madrid, Spain
Received 17 February 2004; revised 13 April 2004; accepted 22 April 2004
Abstract—An efficient synthesis of [4.4]-spirolactam restricted derivatives of the didemnin side chain dipeptide L-Pro-N-Me-D-Leu is
described. This methodology involves: (a) peptide coupling of N-Boc-2-allylproline with D-Leu-OBn; (b) OsO4/NaIO4 mediated allyl
oxidation and intramolecular cyclization to the corresponding cyclic hemiaminals; and (c) NaBH4 mediated reduction of an intermediate
N-acyliminium ion. This synthetic strategy gave significant better results than the previously reported strategies for the synthesis of
[4.4]-spirolactam b-turn mimetics.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
The didemnins are a family of macrocyclic depsipeptides,
isolated from several tunicates, which exhibit a wide variety
of biological activities, including antitumor,1,2 antiviral,2
and immunosupresive properties.2,3 These depsipeptides
contain a common 23-membered macrocycle, made up of
six subunits [(3S,4R,5S)-isostatine (Ist1), (2S,4S)-3-oxo-4-
hydroxy-2,5-dimethylhexanoic acid (Hip2), (Leu3), (Pro4),
[N,O-(Me)2-Tyr5], and (Thr6)], and differ in the side chain
attached to the threonine NH, through a N-Me-D-leucine
residue. Among these macrocyclic depsipeptides, didemnin
B (Fig. 1, 1), isolated in 1981 from the Caribbean tunicate
Trididemnum solidum,4 and aplidine (2), isolated in 1990
from the Mediterranean tunicate Aplidium albicans,5 have
stood out because of their potent antitumoral activity. The
good antitumoral therapeutic profile of aplidine in pre-
clinical and phase I clinical studies has facilitated its recent
entry into phase II clinical trials.1,2,6–9 Analysis of the X-ray
crystal structure of didemnin B,10 as well as conformational
studies on didemnin B11,12 and aplidine,13,14 have shown
that their side chain folds back toward the macrocycle into a
type II b-turn conformation. With the aim of studying the
contribution of this b II turn conformation to the bioactive
conformation responsible for the antitumoral activity, as a
Figure 1.
first step for the design of didemnin peptidomimetics, we
decided to synthesize didemnin B and aplidine analogues,
where the side chain dipeptide L-Pro8-N-Me-D-Leu7 was
it introduces only an additional methylene bridge, and
replaced by a b II turn mimetic.15 As shown in Figure 1, a
allows the essential side chain and configuration of the
[4.4]-spirolactam was selected for this replacement, because
(D-Leu7) residue at the iþ2 position of the b-turn to be
retained.16,17 Furthermore, according to the described data
Keywords: Didemnins; Peptidomimetics; b-Turn mimetics; Spirolactams;
for this type of b-turn mimetic,18 it would fix the dipeptide
Mitsunobu type reaction; Hemiaminal reduction.
backbone f, c torsion angles into values (250.9, 128.7;
91.1, and 25.48) very close to those observed in the crystal
*
Corresponding author. Tel.: þ34-91-5622900; fax: þ34-91-5644853;
e-mail address: rosario@iqm.csic.es
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.04.052

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5178
structure of didemnin B10 (265, 125; 103, and 2298) and
those calculated for aplidine (263, 131; 90, and 2388).13,14
Herein, we described our efforts to develop an efficient
synthesis of [4.4]-spirolactam pseudodipeptide derivatives
3, which would give access to the proposed conformation-
ally restricted didemnin analogues in acceptable yields.
2. Results and discussion
Two alternative strategies have been described for the
synthesis of [4.4]-spirolactam structures, both from 2-allyl-
proline derivative 5 (retrosynthetic Scheme 1). The first,
reported by Ward et al.19 for the preparation of derivatives
of L-Pro-L-Leu, involves the reductive N-alkylation of the
C-terminal amino acid with N-protected 2-(formylmethyl)-
proline, followed by lactamization of the resulting second-
ary amine 4. Whereas the second, described for the synthesis
of L-Pro-L-Tyr,20,21 and L-Pro-Gly derivatives,18,22 involves
a first step of peptide coupling (6), followed by cyclization
via allyl group oxidation–reduction and a Mitsunobu type
N-alkylation.
Scheme 2. Reagents: (a) OsO4, NaIO4, (2:1) MeOH/H2O; (b) H-D-Leu-
OMe·PTSA, TEA, ZnCl2, NaBH3CN, CH2Cl2; (c) 2-chloro-1-methyl-
pyridinium iodide, TEA, CH2Cl2.
The low yield of the first strategy hampered the preparation
of our proposed didemnin analogues in acceptable amounts.
Therefore, we studied the application of the second
alternative synthetic strategy shown in Scheme 3. This
required, as first step, the peptide coupling of 5 with H-D-
Leu-OBn·PTSA, which initially was carried out using
1-hydroxy-benzotriazole (HOBt) and N,N0-dicyclohexyl-
carbodiimide (DCC) as activating reagent, in the presence
of N-methylmorpholine (NMM).27 These reaction con-
ditions led to the dipeptide 6a in a low yield (28%).
Therefore, to optimize yield, we carried out a study of the
coupling using other activating reagents and reaction
conditions. The results are shown in Table 1. Neither the
use of (benzotriazol-1-yloxy)tris(dimethylamino)-phos-
phonium hexafluoro-phosphate (BOP)/HOBt27 nor N,N0-
bis(2-oxo-3-oxazolidinyl)phosphinic choride (BOP-Cl),28,29
activating reagents for sterically hindered amino acids,
improved the yield of 6a. Moreover, when we used DCC/
HOBt or BOP/HOBt, the dipeptide was obtained mixed with
the 1-hydroxybenzotriazole ester derived from 5,† which
had identical Rf in TLC to that of 6a. Excellent yield
(.90%) of this dipeptide was obtained using 1-hydroxy-7-
azabenzotriazole (HOAt)27,30 and DCC or N-[(dimethyl-
amino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-
methylmethanaminium hexafluoro-phosphate N-oxide
(HATU),27 and 2 equiv. of H-D-Leu-OBn·PTSA.
Scheme 1. Retrosynthesis of spirolactam pseudodipeptides 3.
Initially, the building of our spirolactam skeleton was tried
by applying the first strategy, using O-Bn/N-Boc protecting
groups. The starting material, N-Boc-2-allyl-L-Pro-OH (5),
was prepared by applying the Khalil et al.23 procedure of
N-Boc-protection of sterically hindered amino acid to 2-
allyl-L-Pro-OH, which was obtained from L-Pro-OH
according to the Annunziata et al.24 improvement to the
Seebach’s methodology for amino acid a-alkylation with
self-reproduction of chirality.25 As shown in Scheme 2, the
oxidation of the double bond of the allyl group in (5), by
treatment with OsO4 and NaIO4, gave a (<1:1) mixture of
epimeric hemiacetals 8 (95%), which was not resolved. The
reductive amination of this mixture with H-D-Leu-
OBn·PTSA, using NaBH3CN/ZnCl2 as reducing agent, led
to the secondary amine 4a, whose zwitterionic character
hampered its purification. Consequently, the reaction crude
was directly used in the next step of g-lactamization. This
was carried out using 2-chloro-1-methylpyridinium
iodide as condensing agent,26 obtaining the desired N- and
C-protected spiropseudodipeptide 3a in a 24% overall yield
from 5.
The oxidation of the allyl group in the dipeptide 6a was
performed with OsO4 and NaIO4, and the resulting aldehyde
9a was reduced in situ with NaBH4 to the corresponding
alcohol 10a. Unlike the previously described synthesis of
other [4.4]-spirolactam dipeptide mimics,18,20–22 the treat-
ment of this alcohol with the Mitsunobu reagents,
triphenylphosphine and diethyl azodicarboxylate
(DEAD),31 led exclusively to the product of O-alkylation,
the cyclic imidate 11a, and the formation of the desired
g-lactam derivative 3a was not observed. The same result
was obtained when we tried to favour the N-alkylation
†
The 1H NMR spectrum of the mixture showed the presence of <30% of
this 1-hydroxybenzotriazole ester. The signals corresponding to this side
product, as well as its Rf in TLC, were identical to those of the product
resulting from the treatment of 5 with HOBt in the presence of DCC,
without H-D-Leu-OBn.

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5179
Scheme 3. Reagents: (a) H-D-Leu-OMe·PTSA, HOAt, DCC, NMM, CH2Cl2; (b) OsO4, NaIO4, (2:1) MeOH/H2O; (c) NaBH4, EtOAc, 278 8C; (d) Ph3P,
DEAD, THF; (e) Ph3P, NBS, CH2Cl2; (f) NaBH4, TFA; (g) Boc2O, (CH3)4NOH·H2O, CH3CN.
product, via the corresponding bromo intermediate, using
1
triphenylphosphine/N-bromosuccinimide. In the H NMR
serine and allo-threonine derivatives, in general, have
shown selectivity for the O-alkylation, leading to peptide
oxazolines,33 while threonine derivatives have shown
selectivity for N-alkylation31c,33b or 1,2-dehydration.33a
spectrum of imidate 11a, the 8-H proton signals appeared at
0.7 ppm lower field relative to those of g-lactam isomer 3a.
Similarly, C8 of 11a appeared considerably deshielded
(79.3 ppm) with respect to C8 of 3a (39.57). The structural
assignment of imidate 11a was supported by its HMBC
spectrum which did not show correlation between 8-H and
Ca(Leu), and between C8 and a-H(Leu), observed in the
g-lactam 3a spectrum. Interestingly, none of the previously
described syntheses of spirolactams via a Mitsunobu
reaction has pointed out the formation of cyclic imidates,
resulting from O-alkylation.18,20–22,32 In our case, the
preference for the O-alkylation versus the N-alkylation
could be due to a higher steric hindrance of the D-Leu side
chain in comparison with those of the Gly18,22 and Tyr20,21
spirolactam analogues previously described. The regio-
selectivity of the Mitsunobu reaction in other substrates
where there is also a possible competition between N- and
O-alkylation products, such as in b-hydroxy-a-amino acid-
derived peptides, depends on subtle steric effects.31b,c Thus,
To overcome the difficulty of preparing the g-lactam
derivative via a Mitsunobu alkylation, as an alternative,
we carried out the cyclization simultaneously with the
oxidation, by allowing the intermediate aldehyde 9a to stand
in the osmylation reaction mixture at room temperature for
24 h. Cyclic hemiaminals 12a were obtained as a (1:1)
mixture of epimers,‡ that were chromatographically
1
resolved; however, their respective H NMR spectra did
not show relevant NOE effects for their absolute configur-
ation assignment. The epimeric mixture was reduced via the
N-acyliminium ion intermediate using NaBH4 in neat TFA,
because both epimers showed similar reactivity. Spiro-
lactam 3b was isolated as its trifluoroacetate in 73% overall
yield from 5. Attempts failed to maintain the Boc protection
and 12a was recovered unchanged by carrying out the
reduction in AcOH, and by using NaBH3CN in the presence
of ZnCl2 or AcOH.
Table 1. Study on the yield optimization for dipeptide 6a
Finally, the reaction of trifluoroacetate 3b with di(tert-butyl)
dicarbonate in dry acetonitrile, using tetramethylammonium
hydroxide as base,23 led directly to the N-Boc acylated and
C-deprotected pseudodipeptide 3c (70%). The 1H NMR
spectra of this pseudodipeptide in CDCl3 and DMSO-d6
showed the presence of two conformations in a (2:1)
ratio, corresponding to the cis and trans rotamers at the
Activating reagent
Base
H-D-Leu-Obn (equiv.) Solvent 6a (%)
DCC/HOBt
BOP/HOBt
BOP-Cl
HOAt/DCC
HOAt/DCC
HOAt/HATU
HOAt/HATU
NMM
NMM
NMM
NMM
NMM
DIEAb
DIEAb
2.5
1.6
2.5
1
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
THF
28a
30a
30
50
96
55
90
2
1
2
THF
a
‡
Dipeptide 6a impure with a variable quantity of the 1-hydroxy-
benzotriazole ester derived from N-Boc-2-allylproline (5).
N-Ethyldiisopropylamine.
Similar hemiaminals were obtained by Genin et al.18 as unwanted side
products from the silica gel chromatography of the Pro-Gly-derived
aldehyde analogue of 9a.
b

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M. Gutierrez-Rodrıguez et al. / Tetrahedron 60 (2004) 5177–5183
5180
Boc-spirolactam bond. Signals due to both conformations
coalesced at 90 8C in DMSO-d6. The selectively N- and
C-protected [4.4]-spirolactam pseudodipeptides 3b and 3c
give access to backbone C- and N-extensions and, therefore,
to the preparation of our proposed conformationally
constrained didemnin analogues.
Anal. Calcd for C12H19NO5: C, 56.02; H, 7.44; N, 5.44.
Found: C, 56.00; H, 7.48; N, 5.43.
4.1.2. Synthesis of (5R)-7-[(1R)-1-benzyloxycarbonyl-3-
methylbutyl]-1-(tert-butoxy-carbonyl)-6-oxo-1,7-diaza-
spiro[4.4]nonane (3a). Triethylamine (61 mL, 0.43 mmol)
was added to a suspension of H-D-Leu-OBn·PTSA (95 mg,
0.43 mmol) in MeOH (10 mL), and the mixture was stirred
at room temperature and under argon for 15 min. The
epimeric mixture of hemiacetals 8 (470 mg, 1.83 mmol),
ZnCl2 (124 mg, 0.91 mmol), and NaBH3CN (126 mg,
2.01 mmol) were successively added to the mixture, and
the stirring was maintained for 6 h. The solvent was
removed under vacuum, and the residue was dissolved in
EtOAc (20 mL). The solution was washed with H2O
(10 mL) and brine (10 mL), dried over Na2SO4, and
evaporated to dryness. The residue (716 mg, 1.32 mmol),
corresponding to the crude amino acid 4a, was dissolved in
dry CH2Cl2 (120 mL), and, to this solution, 2-chloro-1-
methylpyridinium iodide (372 mg, 1.52 mmol) and triethyl-
amine (0.41 mL, 2.92 mmol) dissolved in CH2Cl2 (3 mL)
were successively added, and the mixture was refluxed for 3
days. The solution was successively washed with 10% citric
acid solution (2£30 mL), saturated NaHCO3 (2£30 mL),
and brine (2£30 mL), dried over Na2SO4, and evaporated to
dryness. The residue was purified by flash chromatography,
using a 10–100% gradient of EtOAc in hexane as eluant.
The spirolactam pseudodipeptide 3a was obtained as a white
solid (200 mg, 24%). Mp 91 8C. [a]2D0 21.58 (c, 1 in
MeOH). HPLC [Novapack C18] (A/B, 50:50) tR 9.47 min.
1H NMR (300 MHz, CDCl3): d 0.91 and 0. 93 [2d, 6H,
J¼6 Hz, Me (Leu)], 1.32 [s, 9H, Me (Boc)], 1.40 [m, 1H,
4-H (Leu)], 1.60–2.00 [m, 7H, 3-H (Leu), 4-H, 3-H, 9-H],
2.50 [c, 1H, J¼3, 6 Hz, 9-H], 3.10 [m, 1H, J¼3, 9 Hz, 8-H),
3.50 (m, 2H, 1-H), 3.73 (t, 1H, J¼9 Hz, 8-H), 4.92 [dd, 1H,
J¼5, 9 Hz, 2-H (Leu)], 5.10 [s, 2H, CH2 (Bn)], 7.30 (m, 5H,
aromatics). 13C NMR (75 MHz, CDCl3): d 21.06, 22.48,
23.12, 24.91, 28.24, 30.49, 36.26, 38.28, 39.57, 47.71,
52.17, 66.62, 66.86, 80.21, 128.26, 128.39, 128.57, 135.08,
153.50, 171.24, 174.82. ESI-MS m/e 445.5 [Mþ1]þ. Anal.
Calcd for C25H36N2O5: C, 67.54; H, 8.16; N, 6.30. Found:
C, 67.42; H, 8.27; N, 6.27.
3. Conclusion
The herein reported synthesis of [4.4]-spirolactam restricted
L-Pro-D-Leu derivatives, which involves, as key steps, the
cyclization of an a-(formylmethyl)-carboxamide, followed
by NaBH4 mediated reduction of the resulting hemiaminals,
constitutes an advantageous efficient methodology to the
described strategies for the synthesis of [4.4]-spirolactams,
such as reductive amination of a-(formylmethyl)-proline
derivatives, followed by g-lactamization, or aldehyde
reduction, followed by a Mitsunobu type alkylation.
4. Experimental
4.1. General procedures
All reagents were of commercial quality. Solvents were
dried and purified by standard methods. Analytical TLC was
performed on aluminum sheets coated with a 0.2 mm layer
of silica gel 60 F254, Merck. Preparative radial chromato-
graphy was performed on 20 cm diameter glass plates
coated with a 1-mm layer of silica gel PF254 Merck. Silica
gel 60 (230–400 mesh), Merck, was used for flash
chromatography. Melting points were taken on a micro
hot stage apparatus and are uncorrected. Optical rotations
were measured on a Perkin–Elmer 141 polarimeter. 1H
NMR spectra were recorded at 300, 400 or 500 MHz, using
TMS as reference, and 13C NMR spectra were recorded at
50, 75 or 100 MHz. Elemental analyses were obtained on a
CH-O-RAPID apparatus. Analytical RP-HPLC was per-
formed on a Waters Novapak C18 (3.9£150 mm, 4 mm) or a
mBondapak C18 (3.9£300 mm, 4 mm) columns, with a flow
rate of 1 mL/min, and using a tunable UV detector set at
214 nm. Mixtures of CH3CN (solvent A) and 0.05% TFA in
H2O (solvent B) were used as mobile phases.
4.1.3. Synthesis of N-[2-allyl-N-(tert-butoxycarbonyl)-L-
prolyl]-D-leucine benzyl ester (6a). HOAt (980 mg,
7.2 mmol), H-D-Leu-OBn·PTSA (3.983 g, 18 mmol),
NMM (1.820 g, 18 mmol), and DCC (1.485 g, 7.2 mmol)
were successively added under argon to a 0 8C cooled
solution of (R)-N-Boc-2-allylproline23 (5) (1.530 g,
6 mmol) in CH2Cl2 (33 mL). The stirring was maintained
at 0 8C for 2 h, and subsequently at room temperature for
15 h. The precipitated N,N0-dicyclohexylurea was filtered
off, and the solution was evaporated to dryness. The residue
was dissolved in EtOAc (30 mL), and the resulting solution
was successively washed with 10% citric acid solution
(2£25 mL), saturated NaHCO3 (2£25 mL), and brine
(25 mL), dried over Na2SO4, and evaporated to dryness.
The residue was purified by flash chromatography, using a
(6:1) hexane/EtOAc mixture as eluant, to give the dipeptide
6a as a syrup (2.638 g, 96%). [a]2D0 þ12.48 (c, 1 in MeOH).
HPLC [mBondapack C18] (A/B, 40:60) tR 9.08 min. 1H
NMR (300 MHz, DMSO-d6, 90 8C): d 0.85 and 0.88 [2d, 6H,
J¼6, 7 Hz, Me (Leu)], 1.37 [s, 9H, Me (Boc)], 1.55–1.72
4.1.1. Synthesis of (5R,8RS)-1-(tert-butoxycarbonyl)-8-
hydroxy-6-oxo-7-oxa-1-azaspiro[4.4]nonane (8). OsO4
(2.5%, w/w solution in tert-butanol, 1.9 mL, 0.15 mmol)
was added to a solution of (R)-N-Boc-2-allylproline23 (5)
(633 mg, 2.48 mmol) in (2:1) MeOH/H2O (45 mL), under
argon, which was stirred at room temperature for 10 min.
NaIO4 (1.612 g, 7.43 mmol) was slowly added. After 2 h of
stirring at room temperature, H2O (30 mL) was added, and
the mixture was extracted with EtOAc (3£60 mL). The
combined organic extracts were washed with brine (50 mL),
dried over Na2SO4, and evaporated to dryness, to yield the
1
epimeric hemiacetals 8 as white solid (638 mg, 100%). H
NMR (200 MHz, CDCl3): d 1.32 [s, 9H, Me(Boc)], 1.59–
1.90 (m, 4H, 3-H, 4-H), 2.35–2.50 (m, 2H, 9-H), 3.20–3.45
(m, 2H, 2-H), 6.03 (m, 1H, 8-H). 13C NMR (50 MHz,
CDCl3): d 21.11, 28.18, 28.53, 36.22, 39.57, 41.81, 66.65,
80.24, 97.38, 161.21, 174.82. ESI-MS m/e 258.3 [MþH]þ.

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5181
[m, 5H, 3-H (Leu), 2-H (Leu), 4-H (Pro)], 2.00 [m, 2H, 3-H
(Pro)], 2.64 [m, 1H, 1-H (allyl)], 2.86 [m, 1H, 1-H (allyl)],
3.15 [m, 1H, 5-H (Pro)], 3.55 [m, 1H, 5-H (Pro)], 4.41 [m,
1H, 2-H (Leu)], 5.05 [s, 2H, CH2 (Bn)], 5.10 [dd, 2H, J¼14,
6 Hz, 3-H (allyl)], 5.63–5.72 [m, 1H, 2-H (allyl)], 7.28–
7.35 (m, 5H, aromatics), 7.46 (bs, 1H, NH). 13C NMR
(75 MHz, CDCl3): d 21.65, 22.30, 22.87, 24.67, 28.32,
34.71, 38.26, 41.15, 49.41, 50.98, 66.76, 69.73, 80.14,
119.37, 128.34, 128.54, 132.77, 135.63, 153.90, 172.79,
173.92. ESI-MS m/e 459.3 [MþH]þ. Anal. Calcd for
C26H38N2O5: C, 68.10; H, 8.35; N, 6.11. Found: C, 68.02;
H, 8.47; N, 6.08.
Me (Leu)], 1.41 [s, 9H, Me (Boc)], 1.45–1.78 [m, 5H, 3-H
(Leu), 4-H (Leu), 4-H, 3-H), 1.84–1.91 (m, 2H, 9-H), 2.10
(m, 1H, 3-H), 2.20 (m, 1H, 4-H), 2.80 (m, 1H, 9-H), 3.38–
3.61 (m, 2H, 2-H), 3.92 (m, 1H, 8-H), 4.23 [m, 1H, 2-H
(Leu)], 4.41 (m, 1H, 8-H), 5.10 [m, 2H, CH2 (Bn)], 7.34 (m,
5H, aromatics). 13C NMR (75 MHz, CDCl3): d 21.15, 23.32,
23.42, 24.20, 28.40, 34.31, 39.42, 41.01, 42.39, 47.75,
57.58, 65.96, 66.34, 67.08, 79.32, 127.92, 128.00, 128.22,
128.35, 136.41, 153.14, 165.99, 173.80. ESI-MS m/e 445.3
[MþH]þ. Anal. Calcd for C25H36N2O5: C, 67.54; H, 8.16;
N, 6.30. Found: C, 67.40; H, 8.36; N, 6.27.
4.1.6. Synthesis of (5R,8RS)-7-[(1R)-1-benzyloxy-car-
bonyl-3-methylbutyl]-1-(tert-butoxycarbonyl)-8-
hydroxy-6-oxo-1,7-diazaspiro-[4.4]nonane (12a). OsO4
(2.5%, w/w solution in tert-butanol, 2.9 mL, 0.23 mmol)
was added to a solution of the dipeptide 6a (1.567 g,
3.42 mmol) in (2:1) MeOH/H2O (108 mL), under argon,
which was stirred at room temperature for 10 min. NaIO4
(2.195 g, 10.3 mmol) was slowly added. After 24 h of
stirring at room temperature, H2O (40 mL) was added, and
the mixture was extracted with EtOAc (3£60 mL). The
combined organic extracts were washed with brine (50 mL),
dried over Na2SO4, and evaporated to dryness. The residue
was purified by flash chromatography, using a 20–100%
gradient of EtOAc in hexane as eluant, to give the two
epimeric hemiaminals 12a, as white solids, whose absolute
configuration at C8 could not be assigned. Epimer A.
(584 mg, 38%). Mp 140–141 8C. [a]2D0 248 (c, 1 in MeOH).
HPLC [Novapack C18] (A/B, 40:60) tR 14.45 min. 1H NMR
(300 MHz, acetone-d6): d 0.88 and 0.91 [2d, 6H, J¼7, 6 Hz,
Me (Leu)], 1.29 [s, 9H, Me (Boc)], 1.64–2.30 [m, 9H, 3-H
(Leu), 4-H (Leu), 3-H, 4-H, 9-H], 2.68 (dd, 1H, J¼6, 13 Hz,
OH), 3.37 (m, 2H, 2-H), 4.51 [dd, 1H, J¼5, 11 Hz, 2-H
(Leu)], 5.13 [d, 2H, J¼15 Hz, CH2 (Bn)], 5.79 (t, 2H,
J¼5 Hz, 8-H), 7.40 (m, 5H, aromatics). 13C NMR (75 MHz,
acetone-d6): d 21.80, 23.10, 24.58, 24.87, 28.59, 39.57,
40.79, 41.65, 48.48, 53.42, 66.89, 67.06, 79.41, 81.24,
128.64, 128.99, 129.08, 129.29, 135.26, 153.74, 171.67,
172.01. ESI-MS m/e 483.4 [MþNa]þ. Anal. Calcd for
C25H36N2O6: C, 65.20; H: 7.88; N, 6.08. Found: C, 65.18;
H, 7.92; N, 6.06. Epimer B. (584 mg, 38%). Mp 134–
135 8C. [a]2D0 þ268 (c, 1.2 in MeOH). HPLC [Novapack
C18] (A/B, 40:60) tR 15.97 and 18.75 min. 1H NMR
(300 MHz, CDCl3): d 0.87 and 0.89 [2d, 6H, J¼7, 6 Hz,
Me (Leu)], 1.40 [s, 9H, Me (Boc)], 1.50–2.60 [m, 9H, 3-H
(Leu), 4-H (Leu), 3-H, 4-H, 9-H], 3.40 (m, 2H, 2-H), 4.20–
5.40 [m, 5H, 2-H (Leu), CH2 (Bn), 8-H], 7.40 (m, 5H,
aromatics). 13C NMR (75 MHz, acetone-d6): d 21.30, 23.47,
24.14, 24.93, 28.28, 38.93, 40.25, 42.55, 47.73, 53.21,
66.82, 67.74, 77.58, 80.66, 128.11, 128.38, 128.54, 128.64,
135.83, 154.14, 171.24, 174.16. ESI-MS m/e483.4
[MþNa]þ. Anal. Calcd for C25H36N2O6: C, 65.20; H,
7.88; N, 6.08.
4.1.4. Synthesis of N-[N-(tert-butoxycarbonyl)-2-(2-
hydroxyethyl)-L-prolyl]-D-leucine benzyl ester (10a).
OsO4 (2.5%, w/w solution in tert-butanol, 4.81 mL,
0.38 mmol) was added to a solution of the dipeptide 6a
(2.603 g, 5.68 mmol) in (2:1) MeOH/H2O (180 mL), under
argon, which was stirred at room temperature for 10 min.
NaIO4 (3.645 g, 17.11 mmol) was slowly added. After 2 h
of stirring at room temperature, H2O (40 mL) was added,
and the mixture was extracted with EtOAc (3£80 mL). The
combined organic extracts were washed with brine (50 mL),
dried over Na2SO4, and evaporated to dryness. The residue
was dissolved into dry EtOAc (180 mL), and the solution
was cooled to 278 8C. To this solution, NaBH4 (215 mg,
5.68 mmol) dissolved into MeOH (13 mL) was added, the
mixture was stirred at 278 8C for 1 h, and then 30 min at
room temperature. H2O (50 mL) was added, and the organic
phase was washed with brine (50 mL), dried over Na2SO4,
and evaporated to dryness. The residue was purified by flash
chromatography, using a 20–100% gradient of EtOAc in
hexane as eluant, to give the alcohol 10a as a syrup (1.200 g,
50%). [a]2D0 21.88 (c, 1.5 in MeOH). HPLC [mBondapack
C18] (A/B, 37:63) tR 9.98 min. 1H NMR (300 MHz, DMSO-
d6, 90 8C): d 0.89 and 0.86 [2d, 6H, J¼7, 6 Hz, Me (Leu)],
1.38 [s, 9H, Me (Boc)], 1.55–1.73 [m, 5H, 3-H (Leu), 4-H
(Leu), 4-H (Pro)], 1.90–2.08 [m, 4H, 3-H (Pro), 1-H
(hydroxyethyl)], 3.13–3.63 [m, 4H, 5-H (Pro), 2-H (hydro-
xyethyl)], 4.35 [m, 1H, 2-H (Leu)], 5.11 [s, 2H, CH2 (Bn)],
7.34 (m, 5H, aromatics), 7.80 (bs, 1H, NH). 13C NMR
(75 MHz, CDCl3): d 21.58, 22.77, 24.59, 28.27, 36.44,
38.22, 41.13, 48.57, 51.01, 58.78, 60.29, 66.87, 80.10,
128.21, 128.32, 128.51, 135.01, 153.51, 172.51, 174.54.
ESI-MS m/e 463.3 [MþH]þ. Anal. Calcd for C25H38N2O6:
C, 64.91; H, 8.28; N, 6.06. Found: C, 64.79; H, 8.45; N,
6.03.
4.1.5. Synthesis of (5R)-6-[(1R)-1-benzyloxycarbonyl-3-
methylbutyl]imino-1-(tert-butoxycarbonyl)-7-oxa-1-
azaspiro-[4.4]nonane (11a). Triphenylphosphine (1.348 g,
5.35 mmol) and DEAD (588 mg, 3.38 mmol) were added to
a solution of the alcohol 10a (1.200 g, 2.6 mmol) in dry THF
(20 mL), and the mixture was stirred at room temperature
for 14 h. The solvent was evaporated, and the residue was
treated with ethyl ether (20 mL), to precipitate the formed
triphenylphosphine oxide, which was filtered off. The
solution was evaporated to dryness, and the residue was
purified by flash chromatography, using a 15–50% gradient
of EtOAc in hexane as eluant, to give the cyclic imidate 11a
as a syrup (700 mg, 61%). [a]2D0 238.38 (c, 1.4 in MeOH).
HPLC [mBondapack C18] (A/B, 37:63) tR 9.51 min. 1H
NMR (300 MHz, CDCl3): d 0.84 and 0.87 [2d, 6H, J¼6 Hz,
4.1.7. Synthesis of (5R)-7-[(1R)-1-benzyloxycarbonyl-3-
methylbutyl]-6-oxo-1,7-diaza-spiro[4.4]nonane tri-
fluoroacetate (3b). NaBH4 (106 mg, 2.8 mmol) was
added to a solution of the epimeric mixture of hemiaminals
12a (430 mg, 0.93 mmol) in neat TFA (10 mL), and the
mixture was stirred at room temperature for 2 h. The solvent
was evaporated to dryness, and the residue was dissolved in
CH2Cl2 (20 mL). This solution was washed with H2O

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M. Gutierrez-Rodrıguez et al. / Tetrahedron 60 (2004) 5177–5183
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6. Raymond, E.; Paz-Ares, L.; Izquierdo, M.; Belanger, K.;
Maroun, J.; Bowman, A.; Anthoney, A.; Jodrell, D.; Armand,
J.-P.; Cortes-Funes, H.; Germa-Lluch, J.; Twelves, C.; Celli,
N.; Guzman, C.; Jimeno, J. Eur. J. Can. 2001, 37(Suppl. 6),
S32.
(5 mL), dried over Na2SO4, and evaporated to dryness. The
residue was dissolved in H2O (3 mL), and the resulting
solution was lyophilized, to give the title compound as a
yellow syrup (414 mg, 100%). [a]2D0 þ158 (c, 1 in MeOH).
1
HPLC [Novapack C18] (A/B, 60:40) tR 1.45 min. H NMR
7. Raymond, E.; Ady-Vago, N.; V, .; Ribrag, V.; Faivre, S.;
´
(300 MHz, acetone-d6): d 0.91 and 0.92 [2d, 6H, J¼6 Hz,
Me (Leu)]; 1.50 [m, 1H, 4-H (Leu)]; 1.66–1.94 [m, 2H, 3-H
(Leu)]; 2.11–2.72 (m, 6H, 3-H, 4-H, 9-H); 3.35–3.72 (m,
4H, 2-H, 8-H); 4.74 [dd, 1H, J¼6, 15 Hz, 2-H (Leu)]; 5.18
[s, 2H, CH2 (Bn)]; 7.37 (m, 5H, aromatics). 13C NMR
(75 MHz, acetone-d6): d 21.21, 23.26, 23.89, 30.24, 34.61,
30.01, 42.24, 46.65, 53.93, 67.59, 67.99, 69.58, 129.07,
129.35, 129.45, 136.77, 161.12, 161.57, 170.95, 172.65.
ESI-MS m/e 443.2 [Mþ1]þ. Anal. Calcd for
C22H29F3N2O4: C, 59.72; H, 6.61; N, 6.33. Found: C,
59.86; H, 6.65; N, 6.29.
´ ´
Lecot, F.; Wright, T.; Lopez-Lazaro, L.; Guzman, C.; Jimeno,
J.; Armand, J. P. Ann. Oncol. 2000, 11(Suppl. 4), 134.
´
8. Izquierdo, M. A.; Bowman, A.; Martınez, M.; Cicchella, B.;
´
´ ´
Lopez-Lazaro, L.; Guzman, C.; Germa, J.; Smyth, J. Ann.
´
Oncol. 2000, 11(Suppl. 4), 134.
9. Maroun, J.; Belanger, K.; Seymour, L.; Soulieres, D.;
Charpentier, D.; Goel, R.; Stewart, D.; Eva Tomiak,; Jimeno,
J.; Matthews, S. Ann. Oncol. 2000, 11(Suppl. 4), 134.
10. Hossain, M. B.; van der Helm, D.; Antel, J.; Sheldrick, G. M.;
Sanduja, S. K.; Weinheimer, A. J. Proc. Natl. Acad. Sci. USA
1988, 85, 4118–4122.
4.1.8. Synthesis of (5R)-1-(tert-butoxycarbonyl)-7-[(1R)-
1-carboxy-3-methylbutyl]-6-oxo-1,7-diazaspiro[4.4]-
nonane (3c). Tetramethylammonium hydroxide penta-
hydrate (158 mg, 0.87 mmol) and di(tert-butyl) dicarbonate
(144 mg, 0.66 mmol) were added to a solution of the
trifluoroacetate 3b (150 mg, 0.44 mmol) in acetonitrile
(5 mL). After 48 h of stirring at room temperature, the
solvent was evaporated to dryness, and the residue was
dissolved in CH2Cl2 (3 mL). The solution was extracted
with H2O (5 mL), and the extracts were lyophilized. The
resulting residue was purified by flash chromatography,
using 8–40% gradient of MeOH in CH2Cl2 as eluant, to
give the title compound 3c as a white solid (110 mg, 70%).
Mp 192–194 8C. [a]D20 238 (c, 1 in MeOH). HPLC
[Novapack C18] (A/B, 50:50) tR 13.41 min. 1H NMR
(300 MHz, DMSO-d6, T¼90 8C): d 0.87 and 0.91 [2d, 6H,
J¼7 Hz, Me (Leu)], 1.36 [s, 9H, Me (Boc)], 1.40 [m, 1H,
4-H (Leu)], 1.64 [t, 2H, J¼8 Hz, 3-H (Leu)], 1.70–1.90 (m,
5H, 3-H, 4-H, 9-H), 2.50 (m, 1H, 9-H), 3.10 (c, 1H, J¼9 Hz,
8-H), 3.30 (m, 2H, 2-H), 3.70 (t, 1H, J¼10 Hz, 8-H), 4.39 [t,
1H, J¼8 Hz, 2-H (Leu)]. 13C NMR (75 MHz, CDCl3): d
21.05, 22.60, 23.40, 25.24, 28.54, 30.44, 36.87, 38.20,
40.59, 48.19, 54.32, 67.29, 81.37, 154.09, 174.97, 176.01.
ESI-MS m/e 353.4 [M21]2. Anal. Calcd for C18H30N2O5:
C, 61.00; H, 8.53; N, 7.90. Found: C, 59.93; H, 8.59; N,7.88.
11. Kessler, H.; Will, M.; Antel, J.; Beck, H.; Sheldrick, G. M.
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