Novel C2-C3′ N-peptide linked macrocyclic taxoids. Part 2: Synthesis and biological activities of docetaxel analogues with a peptide side chain at C2 and their macrocyclic derivatives

Article abstract of DOI:10.1016/j.bmc.2006.09.030

The synthesis of a series of novel docetaxel analogues possessing a peptide side chain at the C2 position as well as peptide macrocyclic taxoids is described. These compounds were designed to mimic a region of the α-tubulin loop equivalent to the paclitax

Full text of DOI:10.1016/j.bmc.2006.09.030

Bioorganic & Medicinal Chemistry 15 (2007) 563–574
Novel C2–C30 N-peptide linked macrocyclic taxoids. Part 2:
Synthesis and biological activities of docetaxel analogues with a
peptide side chain at C2 and their macrocyclic derivatives
`
Anne-Laure Larroque, Joelle Dubois, Sylviane Thoret, Genevieve Aubert,
¨
*
`
´
´
Angele Chiaroni, Franc¸oise Gueritte and Daniel Guenard
Institut de Chimie des Substances Naturelles, CNRS, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France
Received 31 May 2006; revised 12 September 2006; accepted 15 September 2006
Available online 24 October 2006
Abstract—The synthesis of a series of novel docetaxel analogues possessing a peptide side chain at the C2 position as well as peptide
macrocyclic taxoids is described. These compounds were designed to mimic a region of the a-tubulin loop equivalent to the paclit-
axel binding pocket of b-tubulin. Fifteen new peptide taxoids were obtained and evaluated as inhibitors of microtubule disassembly
as well as cell proliferation. The relationships between these new taxoids and the tau protein motif interacting with microtubules are
discussed.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
In 1998, the tubulin structure was determined by elec-
tron crystallography on zinc-induced sheets of tubulin
´
The diterpenoid natural product Taxolꢁ (paclitaxel)1 1a
and its semi-synthetic analogue Taxotereꢁ (docetaxel)2
1b are two important chemotherapeutic drugs widely
used today for the treatment of various types of cancer.
They are known to exert their therapeutic effect, at least
in part, by promoting the assembly of tubulin into
microtubules3 and stabilizing the resulting microtubules.
They thereby inhibit microtubule disassembly4 and
normal dynamic reorganization of the microtubular
network required for mitosis.
stabilized with paclitaxel 1a.5,6 The 3.7 A resolution
˚
obtained not only provided information on the struc-
tural and functional composition of the a,b-tubulin di-
mer, but enabled localization of the paclitaxel 1a
binding site on b-tubulin. Moreover, it was shown that
the region of a-tubulin that is equivalent to the paclit-
axel binding pocket is occupied by an eight amino acid
peptide (Val362-Ala369) belonging to the loop connect-
ing the B9 and B10 strands (Fig. 1).7,8 It was proposed
that this loop could act as an endogenous microtubule-
stabilizing factor.7–10 Snyder et al.7 have compared the
electron density of the paclitaxel-binding pocket in the
tubulin map with all the known paclitaxel conforma-
tions. The T-shaped or butterfly-like structure with
the particular orientation of its C13 side chain and es-
ter groups at C2 and C4 was identified as the most
probable one.
Based on these results, we performed molecular model-
ing to study the superimposition of the a- and b-tubulin
structures in this region and we observed conformation-
al similarities between the octapeptide Val362-Val-
Pro-Gly-Gly-Asp-Leu-Ala369 and the structure of pac-
litaxel when it is bound to b-tubulin (Fig. 2).11 We noted
that six amino acids, Val363-Pro-Gly-Gly-Asp-Leu368,
of the a-tubulin octapeptide showed good insertion
Keywords: Microtubules; Docetaxel; Taxoids; Tau protein.
*
Corresponding author. Tel.: +33 1 69823121; e-mail: guenard@
icsn.cnrs-gif.fr
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.030

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A.-L. Larroque et al. / Bioorg. Med. Chem. 15 (2007) 563–574
Figure 1. (a) The paclitaxel binding site in b-tubulin and (b) the B9–B10 loop in a-tubulin.7
Figure 3. Macrocyclic peptide taxoids (AA, amino acids of the a-
tubulin region equivalent to the paclitaxel binding site).
of the interaction between tubulin and the peptide ana-
logues, we also synthesized three other compounds (4,
5a–5b) in which the amino acids at the C30 position
are different from those constituting the octapeptide res-
idue. Compounds 4 and 5b also interacted with microtu-
bules but to a lesser extent than the previous series
thereby showing some specificity of the amino acids of
the a-tubulin loop toward the interaction with b-tubulin.
Figure 2. Overlapping of paclitaxel (yellow) and octapeptide V362-
A369 (magenta) after manual superimposition of a- and b-tubulin
secondary structure (Sybyl software).
between positions 30 and 2 of paclitaxel 1a, while the
other amino acids mimic a part of the paclitaxel
skeleton.
From these observations, we designed new macrocyclic
analogues by insertion of specific amino acids of the
a-tubulin loop between the 2 and 30 positions of docet-
axel (Fig. 3) and, as reported in a previous communica-
tion, we synthesized taxoids 2a–2d and 3a–3d bearing
from one to four amino acids (Val-Pro-Gly-Gly) at the
C30 position.11 The new taxoids exhibited excellent
interaction with microtubules, showing IC50 values
similar to that of docetaxel. To confirm the specificity
These preliminary results encouraged us to continue our
investigation on the synthesis of taxoids bearing amino
acids at the C2 position (compounds 6a–6d) as well as
macrocyclic analogues with a peptide bridge between

A.-L. Larroque et al. / Bioorg. Med. Chem. 15 (2007) 563–574
565
the C30 and C2 positions (Fig. 3). The synthesis and bio-
logical evaluation of these novel taxoids are presented in
this paper as well as a discussion of the relationships be-
tween these new taxoids and the tau protein motif inter-
acting with microtubules.
2. Chemistry
2.1. Taxoids with a peptide side chain at C2
From a retrosynthetic point of view, compounds 6a–6d
may be obtained, after deprotection of the C7, C10, and
C20 hydroxyl groups and of the amine function of the
amino acids, from docetaxel 1b conveniently protected
after selective removal of benzoate at C2 and step by
step addition of the amino acids.
The protected docetaxel analogue 8 was chosen as start-
ing material.12 To mimic the desired a-tubulin loop, it
was necessary to have the C-terminal part of the peptide
linked to the 2 position of 8. Based on molecular mod-
eling (Fig. 2), leucine should be the first amino acid to
be linked. Unfortunately, the linkage of the protected
Z-leucine proved to be problematic. The failure of 8 to
undergo esterification with Z-protected leucine may be
attributed to steric hindrance. For that reason, we decid-
ed to esterify compound 8 with the less hindered Z-pro-
tected glycine and consequently, only the four amino
acids of the a-tubulin loop were used. Compounds 6a–
6d could thus be obtained from the protected docetaxel
analogue 8 after addition of the protected Z-amino acids
added step by step at the C2 position. The coupling with
Z-protected glycine was realized by esterification using
DCC and DMAP. Contrary to our previous results,13–15
the esterification of the C2 hydroxyl group with protect-
ed amino acids required careful control to avoid rear-
rangement of the oxetane ring.16 A large excess of the
protected amino acid and DCC (30 equiv) was needed
to achieve esterification, compound 7a being obtained
with a yield of 63% (Scheme 1). To lengthen the peptide
chain, we used the same strategy as for compounds 3a–
3d11: deprotection of the benzyloxycarbonyl group by
hydrogenolysis and formation of a peptide bond using
EDCI and 1-HOBt. Thus, compounds 7b–7d were ob-
tained with good yields ranging from 75% to 95%
(Scheme 1). All silyl protecting groups were removed
with HF/pyridine affording compounds 9a–9d, while
the terminal amino acids were all deprotected
by hydrogenolysis, leading to compounds 6a–6d
(Scheme 1).
Scheme 1. Reagents and conditions: (i) DCC (30 equiv), DMAP
(0.1 equiv), ZNH-Gly-OH (30 equiv), toluene, rt, 15 h (63%); (ii) Pd/C
10%, H2, MeOH, rt, 5 h and EDCI (5 equiv), HOBt (5 equiv), ZNH-
AA-OH (5 equiv), NMM, CH2Cl2, rt, 30 min (7b, 85%; 7c, 95%; 7d,
75%); (iii) HF/pyridine 70% (45 equiv), pyridine, CH3CN, 0 ꢂC and rt
(9a, 65%; 9b, 74%; 9c, 70%; 9d, 69%); (iv) Pd/C 10%, H2, MeOH, rt, 5 h
(6a, 67%; 6b, 84%; 6c, 51%; 6d, 36%).
developed by Boger et al.17 The hydroxyl groups at
C7, C10, and C20 of compound 15 were finally deprotec-
ted affording the expected macrocyclic compound 10
(Scheme 2).
Two other macrocyclic taxoids (compounds 21 and 26)
were also synthesized using the same strategy that led
to compound 10. Taxoid 21 (Scheme 3) bears a peptide
chain between the N30 position and the meta-position of
the C2 aromatic ring, while taxoid 26 (Scheme 4) bears a
peptide N30–C2 tether formed with amino acids of the
a-loop. The meta-position for the substitution on the
benzoate at C2 was chosen because meta-substituted
derivatives are generally more active as shown in a
previous study on other taxoid macrocycles.15
To synthesize 21, we first added glycine benzyl ester at
the C-terminal part of the tripeptide taxoid 13 after
hydrogenolysis (compound 16). Esterification of the
C2 hydroxyl group of 16 with 3-nitrobenzoic acid was
then realized to afford compound 17 in good yield. After
simultaneous hydrogenolysis of the benzyl ester and
hydrogenation of the nitro group, macrocyclization
was accomplished using Boger’s conditions.17 Com-
pound 21 was finally obtained by full deprotection of
the hydroxyl groups (Scheme 3).
2.2. Macrocyclic taxoids
To synthesize compound 10, we used previously adapted
reactions: introduction of urea function on the amino
group at C30 with CDI11 followed by the addition of
the tripeptide Val-Pro-Gly-OBn using classical coupling
reagents (compound 13) and esterification of the C2
hydroxyl group by Z-Gly as described above (com-
pound 11). After hydrogenolysis of the terminal amino
acid protecting groups, the resulting compound was
then subjected to macrocyclization using conditions
The synthesis of taxoid 26 started from 16 (Scheme 4).
After deprotection of the terminal amino acid at C30,
coupling with the amino acid H2N-Asp(Ot-Bu)-OBn
under the usual conditions led to compound 22. The

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A.-L. Larroque et al. / Bioorg. Med. Chem. 15 (2007) 563–574
Scheme 2. Reagents and conditions: (i) CDI (3 equiv), tripeptide H2N-Val-Pro-Gly-OBn (2 equiv), NMM (6.5 equiv), CH2Cl2/CH3CN 1:1, 60 ꢂC,
15 h (81%); (ii) DCC (30 equiv), DMAP (0.1 equiv), ZNH-Gly-OH (30 equiv), toluene, rt, 15 h (64%); (iii) Pd/C 10%, H2, MeOH, rt, 5 h and EDCI
(5 equiv), HOBt (5 equiv), NMM, CH2Cl2/DMF 5:1, 0 ꢂC, 2 h (58%); (iv) HF/pyridine 70% (45 equiv), pyridine, CH3CN, 0 ꢂC and rt (10, 53%; 14,
74%).
protected aspartic acid was prepared from commercially
available Fmoc-Asp(Ot-Bu)-OH by esterification to its
benzyl ester and removal of the Fmoc group. Introduc-
tion of the glycine residue at C2 led to compound 23
which after macrocyclization and final deprotection
afforded the macrocyclic taxoid 26. Attempted removal
of the t-butyl ester group of the aspartic acid residue
of 26 using a variety of methods proved unsuccessful,
only degradation products being obtained.18
group at C2 plays a major role in the interaction with
the amino acids of the binding site. We also noted that
this hydrophobic group needs to be directly linked to
the hydroxyl group at C2 since compound 9a bearing
a hydrophobic carbobenzoxy group exhibits no activity
on microtubule disassembly. This shows that an impor-
tant steric constraint exists in this area. Compounds 6a–
6d and 9b–9d show no activity against the KB cell line
while compound 9a, though inactive on microtubules,
exhibits significant cytotoxicity equivalent to that of
compound 5a. In the taxoid series, a few derivatives
have already been reported to be inactive toward tubulin
but still cytotoxic.13,21,22 This may be due to a different
cellular mode of action which would deserve further
investigation. Concerning the three macrocyclic taxoids
10, 21, 26 and their open-chain analogues 14, 18, 19, 24,
only compounds 18 and 19 exhibit very good interaction
with microtubules, demonstrating again the importance
of a hydrophobic group at the C2 position and that
macrocyclization induces a loss of activity on microtu-
bule disassembly. All the compounds are non-cytotoxic
toward the KB cell line.
To compare the biological activities of the macrocyclic
taxoids with their open-chain precursors, compounds
11, 17, and 23 were also subjected to HF/pyridine
deprotection to afford 14 (Scheme 2), 18 (Scheme 3),
and 24 (Scheme 4), respectively, while taxoid 19
was obtained by hydrogenolysis of compound 18
(Scheme 3).
3. Biological results and discussion
The new compounds 6a–6d, 9a–9d, 10, 14, 18, 19, 21, 24,
and 26 were evaluated for their inhibition of cold-in-
duced microtubule disassembly19 and for their cytotox-
icity against the KB cancer cell line.20 In Table 1, only
the active compounds together with analogues 2a–2d
and 3a–3d described previously are included. The com-
pounds of the 6a–6d and 9a–9d series are inactive on
microtubule disassembly except for analogue 6a which
presents only a weak inhibition. The difference between
these series and the first series 2a–2d and 3a–3d is the po-
sition of the peptide and the presence of benzoate at the
C2 position. These results confirm that a hydrophobic
To explain the inactivity of the macrocyclic taxoids,
molecular modeling studies were performed on com-
pounds 10, 21, and 26 which bear a peptide present in
the a-loop. At first, we verified whether the peptide
bonds, the urea and amide functions, of these macrocy-
clic analogues could be in a trans conformation as in the
natural peptide. NMR conformational analysis (rotat-
ing-frame Overhauser enhancement spectroscopy
(ROESY)) in DMSO-d6 was performed and the obser-
vation of exchange spots between some protons showed

A.-L. Larroque et al. / Bioorg. Med. Chem. 15 (2007) 563–574
567
Scheme 3. Reagents and conditions: (i) Pd/C 10%, H2, MeOH, rt, 5 h and EDCI (5 equiv), HOBt (5 equiv), H2N-Gly-OBnÆHCl (5 equiv), NMM,
CH2Cl2, rt, 1.25 h (78%); (ii) DCC (30 equiv), DMAP (10 equiv), 3-nitrobenzoic acid (30 equiv), toluene, 60 ꢂC, 5 h (70%); (iii) Pd/C 10%, H2,
MeOH, rt, 5 h and DCC (10 equiv), HOBt (10 equiv), CH2Cl2, 0 ꢂC and rt, 5 h (44%); (iv) HF/pyridine 70% (45 equiv), pyridine, CH3CN, 0 ꢂC and rt
(18, 56%; 17, 70%); (v) Pd/C 10%, H2, MeOH, rt, 5 h (77%).
that the three taxoids 10, 21, and 26 were present in
solution as two major conformers which were not the
result of cis–trans isomerization of the urea or amide
functions. The correlations observed in the spectra
between groups of the molecule were very abundant
and did not permit us to conclude on the conformation
(T-shaped or ‘non-polar’ form) adopted by our com-
pounds. Molecular dynamic modeling studies in vacuum
media were performed on these macrocyclic taxoids
using Sybyl 6.9 software and Tripos force field. For each
compound, the lower energy conformers were deter-
mined and manually docked in the b-tubulin binding
site7 to observe the possible interactions. For derivative
10, most of the best conformers obtained were unable to
fit the b-tubulin binding pocket (Fig. 4).
T-shaped conformation of 1a, with a good superimposi-
tion of the 30-phenyl and 2-benzoate but like compound
10, the peptide chain is far from the taxane skeleton and
follows neither the 30-benzamide orientation nor the ori-
entation of the a-loop.
Thus, this peptide chain conformation induces unfavor-
able interactions within the b-tubulin binding site partic-
ularly at the His-229 level. On the other hand, the
peptide chain conformation of lower energy conformer
26 is very different from that of the previous macrocycles
and this chain seems to pass around His-229. However
the tert-butyl ester protecting group of the aspartic acid
moiety produces steric bulk and certainly prevents bind-
ing to b-tubulin (Fig. 6). The results obtained from
docking visualizations and supported by NMR and
X-ray experiments are in good agreement with the bio-
logical results and explain the inactivity of the three
macrocyclic taxoids. It can also be deduced that the con-
formation of these macrocycles is too rigid in water or in
the presence of tubulin, preventing these compounds
from adopting a favorable geometry for microtubule
interaction.
We noted that the peptide chain is far from the taxane
skeleton such that some amino acids are in close contact
with the binding site and there is an important overlap
with the His-229 environment thereby explaining the
inactivity of this compound. The tridimensional struc-
ture of 21 was determined by X-ray crystallography.
The good superimposition of this X-ray structure and
one of the lower energy conformers of 21 (Fig. 5) al-
lowed us to validate our molecular dynamic modeling
study of this compound. The superimposition of the
X-ray structure of 21 and paclitaxel 1a agreed with the
Concerning the good biological activity of compounds
18 and 19, some molecular modeling studies have been
carried out. Because of the many degrees of freedom,

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A.-L. Larroque et al. / Bioorg. Med. Chem. 15 (2007) 563–574
Scheme 4. Reagents and conditions: (i) Pd/C 10%, H2, MeOH, rt, 5 h and EDCI (5 equiv), HOBt (5 equiv), H2N-Asp(OtBu)-OBn (5 equiv), NMM,
CH2Cl2, rt, 5.75 h (57%); (ii) DCC (30 equiv), DMAP (0.1 equiv), ZNH-Gly-OH (30 equiv), toluene, rt, 15 h (60%); (iii) Pd/C 10%, H2, MeOH, rt, 5 h
and EDCI (5 equiv), HOBt (5 equiv), CH2Cl2/DMF 5:1, 0 ꢂC, 7.5 h (53%); (iv) HF/pyridine 70% (45 equiv), pyridine, CH3CN, 0 ꢂC and rt (26, 60%;
24, 73%).
Table 1. Biological activities of compounds 6a–6d, 9a–9d, 10, 14, 17–
19, 22, and 23 compared with docetaxel 1b and compounds 2a–2d, 3a–
3d, 4, and 5a–b11
Compound
Microtubule
Cytotoxicity against
b
KB IC50 (lM)
a
disassembly IC50 (lM)
Docetaxel 1b
0.5
0.8
0.0001
0.67
0.004
4.50
0.05
>10
0.10
>10
4.00
1.10
0.09
1.05
>10
0.09
>10
>10
2a
3a
2b
3b
2c
3c
2d
3d
4
0.8
1.7
0.5
0.6
0.4
0.5
0.3
2.6
5a
5b
6a
9a
18
19
Inactive
1.0
51
Inactive
0.5
1.0
Figure 4. Lower energy conformer of 10 (yellow) bound to b-tubulin in
globular form (green).
a IC50 is the concentration that inhibits 50% of the rate of microtubule
disassembly.
b IC50 measures the drug concentration required for the inhibition of
50% cell proliferation after 72 h incubation.
it was not possible to isolate one or two particular con-
formers of 18 and 19. Thus, the taxane moiety of these
molecules can easily be docked to the taxol site while
the peptide chain is located on the exterior and can
adopt multiple conformations whatever the nature of
the amino acids of the chain.

A.-L. Larroque et al. / Bioorg. Med. Chem. 15 (2007) 563–574
569
Figure 5. X-ray structure 21 (orange) (a) superimposed with lower energy conformer of compound 21 (yellow), (b) superimposed with T-shaped
paclitaxel 1a (green), and (c) bound to b-tubulin in globular form (green).
microtubule disassembly. The good interaction of these
derivatives can be compared with the results obtained by
Amos et al. on tau protein.10 This protein has a micro-
tubule binding domain containing several copies of a
conserved motif which binds to a site on b-tubulin that
overlaps with the paclitaxel binding site.23,10 Futher-
more, part of the tau repeat motif has the sequence
Thr-His-Val-Pro-Gly-Gly-Asn, resembling the con-
served sequence Thr-Val-Val-Pro-Gly-Gly-Asp-Leu in
the extended loop of a-tubulin.24 It has been proposed10
that this motif could bind close to the taxol site on b-tu-
bulin and our results strongly support this hypothesis
since, for instance, compounds 2d or 3d, as or more ac-
tive than docetaxel, possess the Val-Pro-Gly-Gly motif
with a favorable interaction at the taxol binding site.
These amino acids could occupy the same area as the
one taken by the corresponding peptide chain of tau in
its complex with tubulin. The successive addition of
the amino acids at N30 shows that, after a decrease of
activity for 2b, the addition of the following two glycines
Figure 6. Lower energy conformer of 26 (white) bound to b-tubulin in
globular form (green).
allows the peptide chain to recover a conformation for a
better interaction. Another peptide chain can be easily
placed in this area, but the lower activity of 4 and 5b
indicates a preference in favor of the a-loop-specific
amino acids, thereby confirming our working hypoth-
esis.
4. Conclusion
Twenty-six new taxoids bearing amino acids have been
synthesized including three macrocyclic derivatives.
Compared to the C30 analogues 2a–2d, 3a–3d, com-
pounds 6a–6d and 9a–9d having a peptide side chain
at C2 were devoid of any tubulin interaction as were
macrocyclic compounds 10, 21, 26 and the open-chain
compounds 14 and 24. Only acyclic taxoids 18 and 19
exhibit significant activity on microtubule disassembly.
These results confirm the importance of the C2-hydro-
phobic group for a good interaction with tubulin and
also the steric constraint in this area due to the presence
of histidine 229. The insertion of the a-loop peptide be-
tween the 30-NH and 2-C positions of docetaxel did not
improve the interactions with the b-tubulin binding site.
However, in this configuration, the peptide is perhaps
too constrained and cannot fit in the binding site.
On the contrary, the same peptide chain introduced at
the C2 position cannot adopt the geometry observed
in 2d or 3d and mainly interacts with His-229. Concern-
ing macrocyclic compound 21, the ortho position would
be worth further study in order to place the peptide
chain far from the histidine residue.
The results may lead to the design of new taxoid com-
pounds bearing a tau repeat motif at strategic positions
on docetaxel or paclitaxel in order to improve tubulin
interaction.
5. Experimental
5.1. General
However, taken as a whole, our studies show that specif-
ic amino acids of the a-loop as in compounds 2a–2d, 3a–
3d, 18, and 19 can maintain or increase the activity on
1H and 13C NMR spectra were recorded with a
Bruker AM 300 or 500 MHz. ROESY spectra were

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A.-L. Larroque et al. / Bioorg. Med. Chem. 15 (2007) 563–574
performed on a Bruker AM 500 spectrometer. Chem-
ical shifts are given as d values (in parts per million:
ppm) and are referenced to tetramethylsilane or to
the residual solvent proton or carbon peak. Mass
and high-resolution mass spectra were obtained with
an LCT Micromass or performed with a Voyager-
DE STR (PerSeptive Biosystemsꢁ). SDS silica gel 60
(35–70lm) was used for flash chromatography or pre-
parative chromatography purification of some com-
pounds. LH-20 Sephadex (Pharmacia) was used to
purify some products synthesized with DCC. All
chemicals were purchased from Fluka, Aldrich or Ac-
ros and the protected amino acids used without fur-
ther purification from Senn Chemicals or Neosystem.
Solvents were purchased from SDS and were dried be-
fore use. Standard workup means extraction with a
suitable solvent (EtOAc unless otherwise specified),
washing the organic layer with a solution of saturated
aqueous sodium hydrogen carbonate solution and
H2O or brine, drying over Na2SO4, and evaporation
under reduced pressure. Docetaxel was a gift from
Dr. Alain Commerc¸on (Aventis-Pharma). Microtubu-
lar proteins were purified from mammalian brain as
previously described.25 Cytotoxicity and microtubule
disassembly inhibition were carried out according to
established literature protocols.19,20
The synthesis of compound 8 is described in Ref. 12.
Compounds 11, 17, and 23 were prepared according to
this method. For experimental details and spectral data,
see the Supporting Information.
5.3. General procedure for removal of the benzyloxycar-
bonyl and the benzyl ester protecting groups by
hydrogenolysis
A typical procedure is described for the synthesis of
compound 19. Compound 18 (20 mg, 0.01 mmol) was
stirred at room temperature with MeOH (2 mL) and
Pd on charcoal (10%) under H2 for 5 h. After filtration
and washing of the catalyst, the organic layer was con-
centrated and pure compound 19 was obtained (14 mg,
77%). 1H NMR (500 MHz, CD3OD) d: 0.90 (d,
JH,H = 6.5 Hz, 3H), 0.97 (d, JH,H = 6.5 Hz, 3H), 1.14
(s, 3H), 1.21 (s, 3H), 1.71 (s, 3H), 1.75–1.85 (m, 1H),
1.79–1.90 (m, 1H), 1.93 (s, 3H), 1.94–2.08 (m, 1H),
1.95–2.06 (m, 1H), 2.10–2.20 (m, 2H), 2.15–2.24 (m,
1H), 2.31–2.42 (m, 1H), 2.41–2.52 (m, 1H), 2.43 (s,
3H), 3.51–3.59 (m, 1H), 3.59 (d, JH,H = 17 Hz, 1H),
3.73–3.80 (m, 1H), 3.76 (d, JH,H = 17 Hz, 1H), 3.90 (d,
JH,H = 7 Hz, 1H), 3.97 (t, JH,H = 17 Hz, 2H), 4.18–4.27
(m, 3H), 4.29–4.37 (m, 2H), 4.61 (d, JH,H = 3 Hz, 1H),
5.02 (d, JH,H = 9.5 Hz, 1H), 5.29 (s, 1H), 5.38 (d,
JH,H = 3 Hz, 1H), 5.63 (d, JH,H = 7 Hz, 1H), 6.23 (t,
JH,H = 9 Hz, 1H), 6.95 (d, JH,H = 7 Hz, 1H), 7.23–7.28
(m, 2H), 7.32–7.44 (m, 5H), 7.47 (s, 1H). 13C NMR
(125 MHz, CD3OD) d: 10.5, 14.6, 18.1, 20.0, 21.7,
23.3, 26.2, 27.2, 30.4, 32.2, 37.0, 37.6, 43.8, 44.4, 44.6,
48.0, 48.9, 57.6, 57.9, 58.9, 62.1, 72.7, 72.9, 75.2, 75.7,
76.4, 77.7, 79.3, 82.4, 86.0, 117.5, 120.4, 121.0, 128.1,
128.5, 129.5, 130.2, 132.2, 138.0, 139.6, 141.3, 149.6,
159.9, 168.4, 171.3, 172.1, 173.7, 174.7, 175.2, 176.5,
211.3. HRMS: m/z calcd for C53H68N6O18ÆNa+
1099.4488; found 1099.4441 (D = ꢀ4.3 ppm).
5.2. General procedure for esterification of C2 hydroxyl
A typical procedure is described for the synthesis of
compound 7a. A solution of the amino acid ZNH-
Gly-OH (119.2 mg, 0.57 mmol, 30 equiv), DCC
(117.7 mg, 0.57 mmol, 30 equiv), and DMAP (0.23 mg,
0.0019 mmol, 0.1 equiv) in anhydrous toluene (1 mL)
was stirred at room temperature for 15 min before add-
ing taxoid 8 (20 mg, 0.019 mmol) in anhydrous toluene
(1 mL). The solution was stirred at room temperature
for 15 h and then filtered through a Celite gel column
using EtOAc and concentrated in vacuo. After standard
workup, the crude product was dissolved in petroleum
ether with a few drops of MeOH and stored at 4 ꢂC.
White crystals of N-acylurea were removed by filtration
and the filtrate was purified by preparative chromatog-
raphy (CH2Cl2/MeOH 95:5) to afford pure 7a (15 mg,
Compounds 6a–6d were prepared according to this
method. For experimental details and spectral data,
see the Supporting Information.
5.4. General procedure for peptide bond formation on
taxoids
63%) as
a
white amorphous solid. 1H NMR
(300 MHz, CDCl3) d: ꢀ0.33 (s, 3H), ꢀ0.11 (s, 3H),
0.54–0.70 (m, 12H), 0.75 (s, 9H), 0.93–1.03 (m, 18H),
1.16 (s, 3H), 1.19 (s, 3H), 1.41 (s, 9H), 1.64 (s, 3H),
1.70–1.80 (m, 1H), 1.78–1.96 (m, 1H), 1.82 (s, 3H),
2.02–2.20 (m, 1H), 2.32 (s, 3H), 2.46–2.58 (m,1H), 3.74
(d, JH,H = 7 Hz, 1H), 3.97 (d, JH,H = 5 Hz, 2H), 4.21
(dAB, JH,H = 8 Hz, 1H), 4.33–4.38 (q,JH,H = 7 Hz,
1H), 4.46 (br s, 1H), 4.47 (dAB, JH,H = 8 Hz, 1H),
4.91 (d, JH,H = 8 Hz, 1H), 5.07–5.13 (d, JH,H = 12 Hz,
2H), 5.19 (s, 1H), 5.23 (dl hidden, 1H), 5.46 (d,
JH,H = 7 Hz, 1H), 5.53 (m, 1H), 6.15 (t, JH,H = 9 Hz,
1H), 7.19–7.38 (m, 10H). 13C NMR (75 MHz, CDCl3)
d: ꢀ6.0, ꢀ5.4, 5.2, 5.9, 6.8, 6.9, 10.4, 13.7, 18.1, 20.7,
22.9, 25.4, 26.3, 28.2, 35.3, 37.2, 43.0, 43.3, 46.4, 56.7,
58.2, 67.3, 71.9, 72.6, 75.3, 77.2, 78.5, 80.0, 80.8, 84.0,
126.4, 127.6, 128.5, 128.1, 128.4, 134.4, 136.0, 137.5,
138.7, 155.2, 156.6, 169.9, 170.3, 171.6, 205.3. MS
(ESI+): m/z 1259.9 [M+Na+].
A typical procedure is described for the synthesis of
compound 7d. To a solution of the taxoid 7c obtained
after deprotection of the benzyl ester by hydrogenolysis
(215 mg, 0.154 mmol) in CH2Cl2 (12 mL) were added
the amino acid ZHN-Val-OH (193.5 mg, 0.77 mmol,
5 equiv), NMM (85 lL, 0.77 mmol, 5 equiv), EDCI
(147.6 mg, 0.77 mmol, 5 equiv), and HOBt (104 mg,
0.77 mmol, 5 equiv). The solution was stirred for 1 h
at room temperature with a CaCl2 drying tube. After
workup, the residue was purified by silica gel chroma-
tography (Et2O/MeOH 1:0 to 96:4) to afford pure 7d
1
(171 mg, 75%) as a white amorphous solid. H NMR
(300 MHz, CDCl3) d: ꢀ0.33 (s, 3H), ꢀ0.09 (s, 3H),
0.50–0.68 (m, 12H), 0.75 (s, 9H), 0.85–1.01 (m, 24H),
1.12 (s, 6H), 1.41 (s, 9H), 1.62 (s, 3H), 1.62–1.75 (m,
1H), 1.80–2.05 (m, 6H), 1.80 (s, 3H), 2.05–2.34 (m,
1H), 2.34 (s, 3H), 2.37–2.53 (m,1H), 3.45–3.58

A.-L. Larroque et al. / Bioorg. Med. Chem. 15 (2007) 563–574
571
(m, 1H), 3.62–3.74 (m, 1H), 3.72 (d, JH,H = 7 Hz, 1H),
3.97–4.28 (m, 4H), 4.28–4.40 (m, 4H), 4.43 (s, 1H),
4.46 (d, JH,H = 8 Hz, 1H), 4.76 (d, JH,H = 9 Hz, 1H),
5.11 (s, 3H), 5.25 (d, JH,H = 10 Hz, 1H), 5.43 (d,
JH,H = 7 Hz, 1H), 5.58 (d, JH,H = 8 Hz, 1H), 6.10 (t,
JH,H = 8 Hz, 1H), 7.16 (d, JH,H = 7 Hz, 1H), 7.26–7.42
(m, 10H), 8.04 (d, JH,H = 5 Hz, 1H). 13C NMR
(75 MHz, CDCl3) d: ꢀ6.0, ꢀ5.3, 5.3, 6.0, 6.8, 6.9, 10.3,
13.7, 17.5, 18.1, 19.1, 20.6, 23.0, 25.0, 25.4, 26.2, 28.1,
28.5, 31.5, 35.4, 37.2, 43.0, 43.2, 46.4, 47.9, 50.4, 56.9,
57.5, 58.3, 61.1, 67.2, 72.3, 72.6, 75.1, 75.4, 75.5, 77.2,
80.6, 80.8, 84.0, 126.3, 127.9, 128.6, 128.3, 134.1,
136.0, 138.1, 138.5, 155.7, 156.3, 169.5, 169.7, 170.5,
171.5, 171.7, 172.3, 205.1. MS (ESI+): m/z 1513.1
[M+Na+].
5 equiv) in CH2Cl2/DMF 5:1 (2 mL) was added drop-
wise. The mixture was stirred at 0 ꢂC, for 2 h water was
added (15.8 mL), and the aqueous layer was extracted
three times with EtOAc. After standard workup, the
crude residue was purified by preparative chromatogra-
phy (CH2Cl2/MeOH 9:1) to afford pure 15 (41.5 mg,
1
58%) as a white amorphous solid. H NMR (300 MHz,
CD3OD) d (major compound): ꢀ0.32 (s, 3H), ꢀ0.27 (s,
3H), 0.58–0.73 (m, 12H), 0.76 (s, 9H), 0.99–1.11 (m,
24H), 1.19 (s, 3H), 1.27 (s, 3H), 1.66 (s, 3H), 1.66–1.80
(m, 2H), 1.80–2.03 (m, 2H), 1.88 (s, 3H), 2.03–2.35 (m,
4H), 2.51 (s, 3H), 2.51–2.72 (m, 1H), 3.46–3.62 (m,
2H), 3.73 (d, JH,H = 7 Hz, 1H), 3.74–4.26 (m, 4H),
4.27–4.54 (m, 5H), 4.82 (d, JH,H = 2 Hz, 1H), 5.06 (d,
JH,H = 9 Hz, 1H), 5.24 (s, 1H), 5.53 (d, JH,H = 6.5 Hz,
1H), 5.58 (d, JH,H = 2 Hz, 1H), 6.29 (t, JH,H = 9 Hz,
1H), 6.83 (d, JH,H = 7 Hz, 1H), 6.95 (d, JH,H = 10 Hz,
1H), 7.25–7.43 (m, 5H). d (minor compound): 1.32 (s,
3H), 2.55 (s, 3H), 4.98 (d, JH,H = 9 Hz, 1H), 5.21 (s,
1H). 13C NMR (75 MHz, CD3OD) d: ꢀ5.8, ꢀ4.8, 6.3,
7.0, 7.3, 7.4, 10.9, 14.8, 18.6, 19.1, 19.7, 22.5, 22.9, 26.1,
26.6, 27.4, 29.7, 30.1, 36.7, 37.1, 42.7, 43.2, 44.3, 47.2,
47.4, 54.8, 56.8, 58.7, 61.8, 72.2, 74.3, 75.3, 76.8, 77.2,
78.7, 82.4, 85.4, 127.9, 128.3, 129.5, 136.6, 138.5, 140.8,
160.3, 170.4, 171.3, 172.0, 173.4, 174.4, 177.7, 212.0.
MS (ESI+): m/z 1304.8 [M+Na+].
In the same manner, compounds 7b, 7c, 16 and 22 were
synthesized (see Supporting Information).
5.5. General procedure for insertion of the urea function
at NH-30 position
A typical procedure is described for the synthesis of
compound 13. CDI (129 mg, 0.79 mmol, 3 equiv) in
CH2Cl2/CH3CN 1:1 (2 mL) was stirred at room temper-
ature and a solution of tripeptide H2N-Val-Pro-Gly-
OBn (245 mg, 0.53 mmol, 2 equiv) with NMM
(189 lL, 1.71 mmol) in CH2Cl2/CH3CN 1/1 (4 mL)
was added dropwise and the reaction mixture was stir-
red at 60 ꢂC. After 5 min compound 12 (250 mg,
0.26 mmol) in CH2Cl2/CH3CN 1/1 (6 mL) was added
and the reaction mixture was stirred at 60 ꢂC for 1 h.
After workup, the crude product was purified by silica
gel chromatography (heptane/EtOAc 2:8) to afford pure
5.6.2. Compound 25. Compound 25 was synthesized in
the same manner. Application of the macrocyclic proce-
dure to 23 (40 mg, 0.023 mmol) and purification by pre-
parative chromatography (CH2Cl2/MeOH 85:15)
afforded pure compound 25 (18.3 mg, 53%) as a white
amorphous solid. 1H NMR (300 MHz, CDCl3) d (major
compound): ꢀ0.40 (s, 3H), ꢀ0.08 (s, 3H), 0.50–0.69 (m,
12H), 0.78 (s, 9H), 0.83–1.01 (m, 24H), 1.16 (s, 3H), 1.18
(s, 3H), 1.38 (s, 9H), 1.63 (s, 3H), 1.81–2.24 (m, 8H),
1.82 (s, 3H), 2.40 (s, 3H), 2.39–2.67 (m, 1H), 2.65–2.87
(m, 2H), 3.52–3.69 (m, 2H), 3.73 (d, JH,H = 7 Hz, 1H),
3.77–4.22 (m, 6H), 4.23–4.37 (m, 5H), 4.41 (d,
JH,H = 1.5 Hz, 1H), 4.48–4.53 (m, 1H), 4.91 (d,
JH,H = 8 Hz, 1H), 5.10 (s, 1H), 5.11 (d hidden, 1H),
5.47 (d, JH,H = 7 Hz, 1H), 6.16 (t, JH,H = 9 Hz, 1H),
7.22–7.43 (m, 5H), 7.59 (br s, 1H), 7.73 (d, JH,H = 8 Hz,
1H). d (minor compound): ꢀ0.36 (s, 3H), 0.72 (s, 9H),
1.45 (s, 9H), 2.52 (s, 3H), 5.53 (d, JH,H = 7 Hz, 1H).
13C NMR (75 MHz, CDCl3) d: ꢀ6.0, ꢀ5.3, 5.3, 6.0,
6.9, 10.4, 13.8, 18.1, 19.0, 21.0, 23.3, 25.5, 26.4, 27.9,
28.7, 28.8, 36.3, 37.2, 42.8, 42.9, 43.8, 46.5, 47.8, 58.3,
61.0, 72.2, 72.5, 74.8, 75.3, 81.1, 82.2, 84.2, 126.7,
128.4, 128.7, 133.4, 138.4, 138.6, 157.0, 168.7, 168.8,
168.9, 169.7, 170.3, 171.1, 171.8, 171.9, 172.7, 204.9.
MS (ESI+): m/z 1532.6 [M+Na+].
1
compound 13 (white powder, 286 mg, 81%). H NMR
(300 MHz, CDCl3) d: ꢀ0.34 (s, 3H), ꢀ0.14 (s, 3H),
0.54–0.74 (m, 12H), 0.74 (s, 9H), 0.87–1.02 (m, 24H),
1.10 (s, 3H), 1.18 (s, 3H), 1.63 (s, 3H), 1.80 (s, 3H),
1.80–2.30 (m, 8H), 2.43 (s, 3H), 2.43–2.60 (m,1H), 3.51
(d, JH,H = 7 Hz, 1H), 3.52–3.61 (m, 1H), 3.62–3.78 (m,
1H), 3.92 (d, JH,H = 7 Hz, 1H), 4.01 (d, JH,H = 4 Hz,
2H), 4.29–4.44 (m, 2H), 4.44 (d, JH,H = 1.5 Hz, 1H),
4.49–4.53 ( m, 1H), 4.64 (q, JH,H = 9 Hz, 2H), 4.96 (d,
JH,H = 9 Hz, 1H), 5.09 (s, 1H), 5.14 (s, 2H), 5.36 (br s,
1H), 5.74 (br s, 1H), 6.14 (t, JH,H = 9 Hz, 1H), 7.13–
7.36 (m, 10H). 13C NMR (75 MHz, CDCl3) d: ꢀ6.0,
ꢀ5.5, 5.3, 6.0, 6.9, 7.0, 10.7, 13.7, 17.4, 18.2, 19.4,
20.8, 23.2, 25.0, 25.5, 26.2, 27.6, 31.3, 35.8, 37.4, 41.4,
42.8, 46.6, 47.7, 56.4, 58.3, 59.9, 60.0, 67.2, 72.8, 74.1,
74.8, 75.5, 77.2, 78.2, 82.6, 83.7, 126.5, 127.7, 128.3,
128.4, 128.5, 128.6, 133.9, 134.5, 135.1, 138.0, 156.7,
169.7, 171.4, 171.8, 172.9, 206.0. MS (ESI+): m/z
1356.0 [M+Na+].
5.6.3. Compound 20. A solution of taxoid 17 (183 mg,
0.119 mmol) previously deprotected by hydrogenolysis,
and HOBt (160.8 mg, 1.19 mmol, 10 equiv) in CH2Cl2
(33 mL) was stirred at 0 ꢂC for 15 min and a solution
of DCC (245.5 mg, 1.19 mmol, 10 equiv) in CH2Cl2
(10 mL) was added dropwise. The mixture was stirred
at 0 ꢂC for 1 h and then stirred at room temperature
for an additional 4 h. The solution was filtered through
a Celite gel column using EtOAc and concentrated in
vacuo. After standard workup, the crude residue was
Synthesis of compound 12 is described in Ref 13.
5.6. Synthesis of macrocyclic taxoids 15, 20, and 25
5.6.1. Compound 15. A solution of taxoid 11 (85 mg,
0.056 mmol) previously deprotected by hydrogenolysis,
containing HOBt (37.8 mg, 0.28 mmol, 5 equiv) in
CH2Cl2/DMF 5:1 (26 mL), was stirred at 0 ꢂC for
15 min and a solution of EDCI (53.7 mg, 0.28 mmol,

572
A.-L. Larroque et al. / Bioorg. Med. Chem. 15 (2007) 563–574
purified on a LH-20 Sephadex column (Ø 2.5 cm · H
72 cm, bed: 353 mL, flux: 2.35 mL/min, CH2Cl2/MeOH
1:1) followed by preparative chromatography (EtOAc/
MeOH 95:5) to afford pure compound 20 (73.6 mg,
1.90 (s, 3H), 1.94–2.15 (m, 1H), 2.05–2.25 (m, 1H),
2.10–2.27 (m, 2H), 2.28 (s, 3H), 2.34–2.56 (m,1H),
3.47–3.55 (m, 1H), 3.65–3.70 (m, 1H), 3.77 (d,
JH,H = 6.5 Hz, 1H), 3.78–4.07 (m, 4H), 4.18–4.25 (m,
3H), 4.33–4.37 (m, 1H), 4.52 (br s, 1H), 4.60 (d,
JH,H = 8.5 Hz, 1H), 4.98 (d, JH,H = 9.5 Hz, 1H), 5.09
(br s, 2H), 5.15 (s, 1H), 5.26 (s, 1H), 5.41 (d,
JH,H = 6.5 Hz, 1H), 6.14 (t, JH,H = 8 Hz, 1H), 7.30–
7.42 (m, 10H). 13C NMR (125 MHz, CD3OD) d: 10.4,
14.5, 18.7, 19.7, 21.5, 23.2, 26.2, 27.1, 28.8, 30.3, 31.9,
37.0, 37.6, 42.8, 43.7, 44.3, 47.9, 49.7, 58.3, 59.0, 59.7,
62.2, 67.8, 72.7, 72.9, 75.2, 75.7, 77.6, 77.9, 78.8, 80.7,
82.1, 86.0, 128.2, 128.7, 129.6, 128.9, 129.1, 129.5,
137.9, 138.2, 139.5, 140.6, 157.7, 158.7, 171.2, 172.0,
172.6, 173.4, 174.5, 175.0, 211.5. HRMS: m/z calcd for
44%) as
a
white amorphous solid. 1H NMR
(500 MHz, CD3OD) d (major compound): ꢀ0.36 (s,
3 H), ꢀ0.04 (s, 3H), 0.60–0.75 (m, 12H), 0.82 (s, 9H),
0.95 (d, JH,H = 7 Hz, 3H), 1.00–1.06 (m, 21H), 1.25 (s,
3H), 1.32 (s, 3H), 1.70 (s, 3H), 1.83–1.93 (m, 3H), 1.94
(s, 3H), 1.94–2.12 (m, 1H), 2.14–2.22 (m, 3H), 2.41–
2.50 (m, 1H), 2.56–2.70 (m, 1H), 2.63 (s, 3H), 3.18 (d,
JH,H = 16 Hz, 1H), 3.53–3.81 (m, 2H), 3.63–3.70 (m,
2H), 3.85 (d, JH,H = 6.5 Hz, 1H), 4.08 (d, JH,H = 16 Hz,
1H), 4.16 (dAB, JH,H = 8 Hz, 1H), 4.23 (dAB,
JH,H = 8 Hz, 1H), 4.25–4.36 (m, 1H), 4.43–4.57 (m,
2H), 4.74 (br s, 1H), 4.99–5.05 (m, 1H), 5.26 (s, 1H),
5.45 (br s, 1H), 5.75 (d, JH,H = 6.5 Hz, 1H), 6.54 (t,
JH,H = 9 Hz, 1H), 7.25–7.47 (m, 7H), 7.80 (t,
JH,H = 8 Hz, 1H), 8.94 (s, 1H). d (minor compound):
ꢀ0.33 (s, 3H), ꢀ0.07 (s, 3H), 0.77 (s, 9H), 0.80 (d hid-
den, 3H), 0.87 (d, JH,H = 7 Hz, 3H), 1.17 (s, 3H), 1.18
(s, 3H), 1.71 (s, 3H), 1.81–1.96 (m, 1H), 1.91 (s, 3H),
2.00–2.06 (m, 2H), 2.13–2.21 (m, 1H), 2.16–2.26 (m,
2H), 2.56–2.70 (m, 1H), 2.61–2.70 (m, 1H), 2.71 (s,
3H), 3.44–3.53 (m, 2H), 3.94 (d, JH,H = 7 Hz, 1H),
4.25–4.36 (m, 2H), 4.43–4.57 (m, 2H), 4.69 (d,
JH,H = 2 Hz, 1H), 4.99–5.05 (m, 1H), 5.24 (s, 1H), 5.56
(d, JH,H = 7 Hz, 1H), 5.59 (br s, 1H), 6.17 (t,
JH,H = 9 Hz, 1H), 7.23–7.47 (m, 5H), 8.75 (s, 1H). 13C
NMR (125 MHz, CD3OD) d (major compound): ꢀ5.7,
ꢀ5.1, 6.3, 7.0, 7.3, 7.4, 11.1, 15.1, 19.2, 19.8, 22.0,
23.3, 24.0, 24.1, 26.2, 27.5, 29.7, 33.7, 36.6, 36.9, 43.8,
44.5, 44.8, 47.9, 48.1, 57.2, 57.6, 60.1, 63.5, 72.1, 74.5,
76.0, 76.4, 77.0, 77.8, 78.8, 82.4, 85.3, 125.3, 126.5,
128.0, 129.5, 129.0, 130.0, 131.8, 132.1, 136.3, 139.2,
140.3, 142.3, 160.0, 167.6, 171.7, 173.1, 174.1, 174.4,
175.3, 207.9. d (minor compound): 6.4, 11.2, 14.9,
19.1, 19.6, 21.7, 26.1, 27.0, 33.0, 43.5, 45.0, 47.7, 57.3,
57.9, 59.6, 62.4, 73.1, 74.2, 76.9, 77.1, 77.9, 79.7, 81.9,
85.4, 122.6, 128.2, 129.7, 136.2, 139.5, 172.6. MS
(ESI+): m/z 1423.78 [M+Na+].
C58H77N5O19ÆNa+ 1170.5110;
(D = ꢀ0.6 ppm).
found 1170.5104
Compounds 9a–9c, 10, 14, 21, 18, 26, and 24 were pre-
pared according to this method. For experimental de-
tails and spectral data, see the Supporting Information.
5.8. X-ray crystal structure analysis for compound 21
A small prismatic colorless crystal of (0.35 · 0.35 · 0.20)
mm, obtained from a mixture (methanol:dioxane:ether),
was chosen. To prevent crystal decay during data collec-
tion, the crystal was placed in a Linderman capillary
with the mother liquor. The X-ray structure analysis
showed that the crystal compound formula was
C53H66N6O17 + CH3OH with Mw = 1091.16 (Fig. 7).
The compound crystallizes in the monoclinic system,
space group P21, with two identical molecules (Z = 2)
in the unit-cell, of parameters: a = 13.888(6),
˚
b = 15.741(5), c = 16.009(6) A, b = 110.97(3)ꢂ, V =
3
3268 A ; dcal = 1.109 g cmꢀ3
, F(000) = 1160, (k(Mo-
˚
Ka) = 0.71073 A), l = 0.084 mmꢀ1. Refinement of 688
parameters converged to R1(F) = 0.0535 for the 5090
observed reflections and wR2(F2) = 0.2054 for all the
6682 unique data with a goodness-of-fit S factor of
1.003 (Fig. 7). CCDC 607125 contains the supplementa-
ry crystallographic data for this paper. These data can
be obtained free of charge at //www.ccdc.cam.ac.uk/de-
posit [or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB 1EZ, UK].
˚
5.7. General procedure for removal of the silyl protecting
groups
A typical procedure is described for the synthesis of
compound 9d. To a cooled solution of 7d (160 mg,
0.107 mmol) in pyridine–acetonitrile (6:93, 7 mL) was
added dropwise HF/pyridine (70%, 625 lL, 4.86 mmol,
45 equiv.) at 0 ꢂC, and the mixture was stirred at room
temperature for 4h. HF/pyridine (625 lL, 70%,
4.86 mmol, 45 equiv.) was added dropwise at 0 ꢂC. The
solution was stirred at room temperature for additional
3.5 h. The reaction was quenched by addition of saturat-
ed aqueous sodium hydrogencarbonate solution and the
aqueous layer was extracted three times with EtOAc.
After standard workup, the crude residue was purified
by preparative chromatography (CH2Cl2/MeOH 9:1)
to afford pure 9d (85 mg, 69%) as a white amorphous
solid. 1H NMR (500 MHz, CD3OD) d: 0.94 (d,
JH,H = 6.5 Hz, 3H), 0.99 (d, JH,H = 6.5 Hz, 3H), 1.07
(s, 3H), 1.19 (s, 3H), 1.45 (s, 9H), 1.66 (s, 3H), 1.73–
1.95 (m, 1H), 1.83–2.00 (m, 1H), 1.87–2.05 (m, 2H),
5.9. General procedure for peptide synthesis
A typical procedure is described for the synthesis of
dipeptide BocNH-Val-Pro-OBn. To a solution of the
amino acid BocNH-Val-OH (500 mg, 2.3 mmol) in
CH2Cl2 (15 mL) were added the amino acid H2N-Pro-
OBnÆHCl (556 mg, 2.3 mmol, 1 equiv), NMM (253 lL,
2.3 mmol, 1 equiv), EDCI (529 mg, 2.76 mmol,
1.2 equiv), and HOBt (373 mg, 2.76 mmol, 1.2 equiv).
The solution was stirred for 3.5 h at room temperature
with a CaCl2 drying tube. After workup, the residue
was purified by silica gel chromatography (heptane/
EtOAc 65:35) to afford pure BocNH-Val-Pro-OBn
1
(913 mg, 99%) as a colorless oil. H NMR (300 MHz,
3
CDCl3) d: 0.90 (d, JH,H = 7 Hz, 3H), 1.00 (d,
3JH,H = 7 Hz, 3H), 1.42 (s, 9H), 1.91–2.09 (m, 4H),
2.14–2.29 (m, 1H), 3.60–3.70 (m, 1H), 3.73–3.84 (m,

A.-L. Larroque et al. / Bioorg. Med. Chem. 15 (2007) 563–574
573
Figure 7. X-ray structure of compound 21.
22
484.2424; found 484.2403 (D = ꢀ4.2 ppm). ½aꢁ ꢀ86.7
1H), 4.24–4.32 (m, 1H), 4.56–4.63 (m, 1H), 5.16 (q,
JH,H = 12 Hz, 2H), 5.22 (d, JH,H = 9 Hz, 1H), 7.30–
7.40 (m, 5H). 13C NMR (75 MHz, CDCl3) d: 17.4,
19.3, 25.0, 28.3, 29.0, 31.4, 47.1, 56.8, 58.9, 66.9, 79.5,
128.2, 128.3, 128.5, 135.6, 155.9, 171.2, 171.9. MS
(ESI+): m/z 427.1251 [M+Na+].
D
(c 1.0, MeOH).
5.10. Protection of amino acid FmocNH-Asp(OtBu)-OH
To a cooled solution of FmocNH-Asp(OtBu)-OH
(500 mg, 1.2 mmol) and NMM (134 lL, 1.2 mmol,
1 equiv) in distilled THF (6 mL) was added dropwise
158.5 lL of isobutylchloroformate (1.2 mmol, 1 equiv)
at ꢀ15 ꢂC. The mixture was stirred for 5 min and a solu-
tion of benzyl alcohol (252 lL, 2.43 mmol, 2 equiv) in
THF (1 mL) was added dropwise. The solution was stir-
red at room temperature for 50 min, filtered through a
Celite gel column, and then concentrated in vacuo.
The crude residue was purified by flash chromatography
(heptane/EtOAc 8:2) and then purified by crystallization
in ether with drops of EtOAc, to afford pure compound
FmocNH-Asp(OtBu)-OBn (469 mg, 77%) as a white
In the same manner, the tripeptide BocNH-Val-Pro-
Gly-OBn was synthesized: application of the general
procedure using dipeptide BocNH-Val-Pro-OBn
(600 mg, 1.9 mmol), after hydrogenolysis, with H2N-
Gly-OBnÆHCl (396 mg, 1.9 mmol, 1 equiv) in DMF
(4 mL) and purification by flash chromatography
(CH2Cl2/MeOH 95:5) afforded pure tridipeptide
BocNH-Val-Pro-Gly-OBn (721.7 mg, 82%) as a white
amorphous solid. 1H NMR (300 MHz, CDCl3) d:
0.90 (d, JH,H = 7 Hz, 3H), 0.97 (d, JH,H = 7 Hz, 3H),
1.43 (s, 9H), 1.80–2.16 (m, 4H), 2.33–2.43 (m, 1H),
3.52–3.63 (m, 1H), 3.65–3.78 (m, 1H), 4.08 (d,
JH,H = 5 Hz, 2H), 4.28 (q,JH,H = 6 Hz, 1H), 4.65 (dd,
1
amorphous solid. H NMR (300 MHz, CDCl3) d: 1.41
(s, 9H), 2.75–3.00 (m, 2H), 4.23 (t, JH,H = 7 Hz, 1H),
JH,H = 7.5 Hz,J0H,H = 2 Hz,
1H),
5.15
(q,
4.30–4.45 (m, 2H), 4.65 (td, JH,H = 4.5 Hz, J0H,H
=
JH,H = 12 Hz, 2H), 5.22 (d, JH,H = 9 Hz, 1H), 7.30–
7.42 (m, 5H). 13C NMR (75 MHz, CDCl3) d: 17.3,
19.5, 25.1, 28.3, 28.4, 31.3, 41.4, 47.5, 56.8, 59.6,
67.1, 79.6, 128.4, 128.5, 128.6, 135.2, 155.8, 169.4,
171.2, 171.8. HRMS: m/z calcd for C24H35N3O6ÆNa+
9 Hz, 1H), 5.20 (q, JH,H = 12 Hz, 2H), 5.84 (d,
JH,H = 9 Hz, 1H), 7.25–7.42 (m, 9H), 7.59 (d,
JH,H = 7.5 Hz, 2H), 7.76 (d, JH,H = 7.5 Hz, 2H). 13C
NMR (75 MHz, CDCl3) d: 28.0, 37.7, 47.1, 50.7, 67.3,
67.5, 81.9, 120.0, 125.1, 125.2, 125.2, 125.2, 128.2,

574
A.-L. Larroque et al. / Bioorg. Med. Chem. 15 (2007) 563–574
´
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22
D
524.2049; found 524.2039 (D = ꢀ1.9 ppm). ½aꢁ ꢀ15.9
20
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