Elaboration of vinblastine hybrids using a reactive in situ generated N-carboxyanhydride

Article abstract of DOI:10.1039/c0ob01065k

Hybrids of vinblastine and phomopsin, designed by a molecular modelling study, were elaborated in order to target tubulin. The key step of the synthesis (fragmentation and insertion of vindoline) was mediated by an internal N-carboxyanhydride (or O-acylca

Full text of DOI:10.1039/c0ob01065k

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PAPER
Elaboration of vinblastine hybrids using a reactive in situ generated
N-carboxyanhydride†
Claire Rannoux, Fanny Roussi,* Marie-The´re`se Martin and Franc¸oise Gue´ritte
Received 23rd November 2010, Accepted 7th April 2011
DOI: 10.1039/c0ob01065k
Hybrids of vinblastine and phomopsin, designed by a molecular modelling study, were elaborated in
order to target tubulin. The key step of the synthesis (fragmentation and insertion of vindoline) was
mediated by an internal N-carboxyanhydride (or O-acylcarbamate). This reaction was diastereospecific
and addition of silver salts could reverse the diastereoselectivity. Even if the synthesized compounds are
inactive, this synthesis represents an original example of a C–N fragmentation mediated by a
N-carboxyanhydride.
Introduction
Microtubules play a key role in many cellular functions such
as cell division, as they form the mitotic spindle along which
chromosomes migrate during mitosis. These polymers of a and b
tubulin heterodimers are constantly in dynamic instability. Some
drugs can alter this dynamic and tubulin binding molecules are
today one of the most important classes of anticancer agents.
Among these antimitotic drugs, vinca alkaloids1 are commonly
used in cancer chemotherapy, inhibiting tubulin polymerization
into microtubules.
Their precise binding site in the so-called vinca domain was
determined a few years ago2 and it was shown that it partially
overlaps with that of phomopsin A 43 (a cyclopeptide natural com-
pound). In order to explore the vinca domain and to elaborate new
acute derivatives, we have recently synthesized vinca–phomopsin
hybrids, linking the hexahydrophomopsin lateral chain to the
tertiary amine of the cleavamine moiety of anhydrovinblastine
and vinorelbine.4 Albeit very active, we showed that, in the tubulin
binding site, the phomopsin lateral chain of these hybrids was not
oriented like the native compound but was extending in another
direction.
So as to elaborate new hybrids with a correct orientation of
this peptide side chain, we designed, by molecular modelling
studies (SYBYL 7.3), new hybrids of type 5 (Fig. 1). These
modelling experiments showed that low energy conformers of 5
superimposed correctly with phomopsin and vinblastine in their
active conformations (Fig. 2).
In these compounds, a simplified cleavamine moiety (i.e.,
suppression of cycle D) is used as a template to keep the
vindoline and phomopsin side chain in a correct orientation.
Fig. 1 Some vinca alkaloids 1–3, phomopsin A 4 and target-simplified
vinca–phomopsin A hybrids 5.
These hybrids may be elaborated starting from (L)-tryptophan,
that could provide the desired (S) absolute configuration at
C-8. The formation of the central nine membered ring and the
stereoselective insertion of vindoline at C3 could be performed
simultaneously using a modified procedure of that developed by
Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, Av-
enue de la terrasse, 91198 Gif sur Yvette, France. E-mail: roussi@icsn.cnrs-
gif.fr; Fax: +33 (0)1 69 07 72 47; Tel: +33 (0)1 69 82 31 21
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra of all compounds. See DOI: 10.1039/c0ob01065k
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By analogy with these results, we reasoned that, starting from
compound 14, we could generate the transient iminium ion 12 from
intermediate 13, bearing a N-carboxyanhydride (NCA) function
(Fig. 4).
Fig. 2 Superimposition of phomospin A 4 (blue) and vinblastine 1
(magenta) in their active conformation in tubulin with a low energy
conformer of hybrid 5 (green) with R = H and X = CH2OCO.
Fig. 4 Envisaged retrosynthetic pathway.
To our knowledge, an N-carboxyanhydride has, so far, never
been used to induce a C–N fragmentation.7 The choice of this
group is, nevertheless, strategic in our case as it would play a triple
role and would serve:
Magnus and colleagues5,6 in their very elegant and powerful total
synthesis of vinblastine. Indeed, these authors have shown that
the coupling of vindoline to a precursor of cleavamine 6 could be
achieved, in a few days, by trapping an intermediate delocalized
cation, generated via a C–N fragmentation using p-nitrobenzyl
chloroformate. They nicely showed that the regiochemistry and
the S/R ratio of the formed bond is dependent on the rate of the
capture of the iminium ion before its racemization. This rate, in
turn, depends on the temperature and the dielectric constant of
the solvent in the reaction (Fig. 3).
- as a protecting group of the acid function,
- as an activating group to fragment the C3–N bond to generate
the macrocyclic cation 12 that could be captured by vindoline to
form the expected 11,
- as a template on compound 11 for the direct addition of the
peptide chain of phomopsin.
Results and discussion
Compound 14 was elaborated in four steps from L-tryptophan
benzyl ester (Scheme 1). Magnus et al. have already prepared the
first intermediate 16,8 as a mixture of two diastereomers (43%
and 7%) by a one pot Pictet–Spengler/lactamization reaction
with dimethyl a-ketoglutarate in refluxing THF for 72 h. In our
case, we found that using a two-step procedure (Pictet–Spengler
in refluxing dichloromethane, 48 h), then lactamization in toluene
(reflux, 24 h) allowed the selective formation of the cis-diaxial
adduct 16 (50% yield for two steps). The lactam function was thus
selectively reduced using 9 BBN9 and hydrogenolysis of the benzyl
ester furnished the corresponding acid 14 (Scheme 1).
With this compound in hand, the formation of the transient
intermediate 13 was next studied (Scheme 2). The formation of
the NCA turned out to be quite difficult because of the congested
environment around nitrogen: it was unable to react even with
small and reactive 1-chloroethylchloroformate. Using standard
procedures to generate this anhydride (triphosgene alone,10 or with
pyridine or triethylamine11) only resulted in recovering starting
Fig. 3 Magnus synthesis of vinblastine.
4874 | Org. Biomol. Chem., 2011, 9, 4873–4881
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Table 1
Entry Ar
Solvent Temp Additive Cpd
Yieldb
1
2
3
4
5
Vindoline CH2Cl2 0 ◦
C
C
/
/
17 (3R)
17 (3R)
17 (3R)
17 (3R)
50%
60%
ND
ND
Vindoline CH3CN 0 ◦
Vindoline CH2Cl2 -40 ◦C /
Vindoline CH3CN -40 ◦C /
TMBa
CH3CN 25 ◦
C
/
18 (3R), (3S) 0.4/1c 30%
a TMB = trimethoxybenzene. b Yield of isolated compounds after purifica-
tion by flash chromatography. c Absolute configuration of major and minor
isomers could not be determined because of absence of NOEs between
TMB and the macrocycle.
Scheme 1
Scheme 3
the reaction was performed at room temperature in acetonitrile, a
mixture of 3R and 3S 17 was formed. That result clearly indicates
that TMB can partially trap intermediate 12 from “below”, even
at room temperature.
Furthermore, the fast formation of a transient intermediate, that
was slowly replaced by final compound 17, could be detected when
the reaction, performed with vindoline, was followed by NMR.
These observations led us to assume the following hypothesis
(Scheme 4): highly reactive cation 12 may be trapped by chlorine
(derived from degradation of triphosgene) leading to unstable
1913 that could not be isolated. Compound 19 may then slowly
regenerate cation 12. While leaving, Cl- may form an intimate ion
pair14 with 12, and may block its a-face, which could allow the
b-face to be attacked by vindoline and could explain the observed
diastereoselectivity.
Scheme 2
material. Finally, a “pre-activation” of the acid function as a
cesium salt12 allowed a clean and total conversion of 14 when
reacted with 1/3 equivalent of triphosgene and vindoline. The use
of a NCA significantly enhanced the rate of this functionalization:
transformation of intermediate 14 was completed in a few hours.
After fast flash chromatography, an insertion compound 17 was
isolated (Scheme 2).
Complete NMR assignments confirmed that insertion of vindo-
line occurred at C3, thus validating the NCA strategy. In addition,
the insertion compound was obtained as only one diastereomer.
The absolute configuration of the new quaternary center C3
was determined using NOESY experiments. Particularly, NOEs
between H8, H6b, H5b and H4b, and between H4a and H14¢
accounted for a 3R configuration. This configuration was quite
surprising as it resulted from an attack of vindoline exclusively on
the more hindered side of intermediate cation 12, syn to the NCA
function. Various experimental parameters were screened, in order
to reverse this selectivity (Scheme 3, Table 1). Nevertheless, neither
modification of the solvent (entry 2), nor decreased temperature
(entries 3 and 4) could modify the selectivity of the attack, contrary
to Magnus’s observations. These very disappointing results were,
however, tempered by those observed when replacing vindoline
with more reactive trimethoxybenzene (TMB). In that case, when
Scheme 4
With more reactive trimethoxybenzene, a competition between
chlorine and TMB could account for the formation of both di-
astereomers. To validate this mechanistic hypothesis, the reaction
was performed with vindoline in acetonitrile and an excess of
AgOTF (3 eq.) in order to trap Cl- as silver salts (Scheme 5).
In these conditions, we were pleased to obtain exclusively the
desired compound 11 with 91% yield. Observed NOEs between
H8, H6 and H14¢ confirmed a 3S configuration resulting in a
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Scheme 5
direct insertion of vindoline on the a-face. The observed total
diastereoselectivity was based on the generation of a hyper-reactive
NCA intermediate that not only permitted induction of the C3–
N bond fragmentation but also ensured a total chirality transfer
from the substrate to the C3 center. That strategy represents an
improvement to the Magnus methodology.6 In addition, even if
only the 3S diastereomer was useful in our case, the easy switch
over of the coupling selectivity simply by adding silver salts makes
this route very attractive and conceptually interesting.
The ring opening of the N-carboxyanhydride was next per-
formed on compound 11, using a three amino acid peptide
chain corresponding to that of the protected lateral chain of
hexahydrophomopsin (Scheme 6). As expected, the peptide bond
was formed simply by stirring 11 and the NH free proline of the
small peptide in acetonitrile for a few hours. Nevertheless, the high
nucleophilicity of N7 led also to the formation of the undesired
lactam 21 by reaction with methyl ester on C3. This side reaction
occurred at room temperature, whereas the temperature had to be
raised to 35 ◦C to complete the opening of the NCA. Nevertheless,
the expected compound 20 could be isolated in a reasonable yield
(40%). Deprotection of aspartic acid furnished compound 22 in
a quantitative yield. In addition, in order to decrease the high
nucleophilicity of N7, that could affect the potential biological
activity of the hybrids, a reductive amination was performed on
20 with propionaldehyde, to provide 23 that was converted to
the quaternary ammonium salt 24. In parallel, compound 20 was
reacted with chloroacetyl chloride to provide carbamate 25.
NMR spectra clearly indicated that compounds 20, 22–25 were
in equilibrium between several conformers both at low and room
temperature. NOE experiments confirmed that these were due to
the flexibility of the macrocycle. For example, one conformer of
compound 23 presented NOEs between H8, H20, H9 and H12,
whereas another one showed NOEs between H14¢, H6, H5 and
H4. This high flexibility of the macrocycle may promote generation
of the conformers that were predicted by molecular modelling
experiments to superimpose with phomopsin and vinblastine in
their active conformation (Fig. 2).
Scheme 6
demonstrate, once again,15 that modifications on the cleavamine
moiety of vinblastine-type compounds can have a dramatic impact
on activity.
Conclusions
Based on molecular modelling studies, we have elaborated
phomopsine–vinblastine hybrids to target tubulin. The key step of
this synthesis, i.e., the insertion of vindoline, proceeds rapidly with
a complete diastereoselectivity due to an intramolecular activation
of a pyrrolo-b-carboline using a N-carboxyanhydride. Unfortu-
nately, these molecules are inactive, in spite of the flexibility of
the macrocycle of the resulting compounds, which is expected to
favour the interaction with tubulin. In turn, this work is, to our
knowledge, the first example of a use of a N-carboxyanhydride as
an activating group for a C–N fragmentation. This example offers
new prospects for the NCA chemistry beyond peptides synthesis.
The activity of compounds 20–25 on tubulin polymerization
assays, as well as their cytotoxicity on KB, MCF7 and MCF7R cell
lines, was evaluated. It was found that none of these compounds
showed a significant microtubule assembly inhibitory activity or
cytotoxicity. These biological results were very disappointing and
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stirred for 10 min and the solvent was removed under reduced
pressure. Ether was added to the residue, the resulting mixture
was stirred 30 min and filtered through Celite to remove the
boran complex insoluble in ether. After removal of the solvent,
the product was purified by flash chromatography on silica gel
(heptane/EtOAc: 50/50 → heptane/EtOAc: 30/70) to give the
expected compound (1.54 g, 60%); [a]2D2 +43 (c 0.5 in CHCl3);
Experimental section
Chemistry
General methods. Anhydrous acetonitrile was purchased from
Aldrich. Anhydrous THF was dried and deoxygenated by dis-
tillation from sodium/benzophenone under argon. CH2Cl2 was
distilled from P2O5 under argon. Reactions were monitored by
thin layer chromatography (TLC) with SDS 0.25 mm silica gel 60
F254. TLC plates were visualized by exposure to UV light (254
nm) and stained with Salkowski reagent or ammonium molybdate.
Flash column chromatographies were performed using normal
n
max(film)/cm-1 3384, 2949 and 1725; dH (300 MHz; CDCl3) 1.78
(m, 1H), 1.97 (m, 1H), 2.12 (m, 1H), 2.45 (m, 1H), 2.97 (m, 1H),
3.11 (dd, 1H, J 15.8 and 1.9 Hz), 3.17 (dd, 1H, J 15.9 and 5.7
Hz), 3.35 (m, 1H), 3.76 (s, 3H), 4.24 (dd, 1H, J 5.6 and 1.93 Hz),
4.89 (d, 1H, J 12.4 Hz), 4.97 (d, 1H, J 12.4 Hz), 7.20–6.97 (m,
7H), 7.33 (d, 1H, J 8.0 Hz), 7.45 (d, 1H, J 7.9), 8.65 (s, 1H); dC(75
MHz, CDCl3) 19.8, 23.9, 42.1, 55.9, 53.0, 58.2, 64.3, 66.6, 106.1,
111.4, 118.5, 119.5, 122.3, 126.6, 128.5, 128.1, 128.0, 134.5, 135.8,
136.9, 172.5, 176.2; m/z (ESI) 404.1816 (M + H+; C24H25N2O4
requires 405.1814).
10% Pd/Carbon (293 mg, 0.276 mmol), was added to a solution
of reduced compound (1.11 g, 2.76 mmol) in anhydrous THF. The
mixture was stirred for 3 h under an H2 atmosphere and filtered
through a pad of Celite to afford quantitatively compound 14 (867
mg, 100%); [a]D22 +38 (c 0.5 in CHCl3); nmax(film)/cm-1 3056, 2951,
2849 and 1729; dH (300 MHz; CDCl3) 2.04–1.78 (m, 3H), 2.69
(m, 1H), 2.97 (m, 1H), 3.07 (dd, 1H, J 15.6 and 6.4), 3.41 (dd,
1H, J 15.6 and 3.8 Hz), 3.51(m, 1H), 3.85 (s, 3H), 3.86 (m, 1H),
7.10 (t, 1H, J 7.6), 7.18 (t, 1H, J 7.7 Hz), 7.32 (d, 1H, J 8.0 Hz),
7.52 (d, 1H, J 7.9 Hz), 8.42 (s, 1H); dC(75 MHz, CDCl3) 19.5,
24.0, 41.2, 53.9, 55.2, 60.5, 66.0, 107.4, 111.3, 119.0, 120.0, 122.9,
126.4, 131.1, 136.4, 174.3, 175.3; m/z (ESI) 337.1164 (M + Na+;
C17H18N2O4Na requires 337.1164).
˚
phase silica gel (60 A, 40–63 mm) or neutral alumina (70–230
mesh ASTM). H NMR and 13C NMR spectra were recorded
1
on a Bruker AC300 or Avance 600 or ARX500 spectrometer.
Chemical shifts (d) are reported in ppm relative to chloroform
for 1H NMR (d = 7.24 ppm) and for 13C NMR (d = 77.26
ppm) or relative to acetonitrile for 1H NMR (d = 1.94 ppm)
and for 13C NMR (d = 1.40 ppm). NMR attributions were based
on HMQC, HMBC and COSY correlations. Data are reported
as follows: chemical shift, multiplicity (s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet), integration, coupling
constant in Hz, attribution. Infrared (IR) spectra were recorded
on a Perkin Elmer Spectrum BX FT-IR instrument at 22 ◦C. [a]D
were measured using a JASCO P-1010 instrument. HRMS were
recorded by ElectroSpray Ionisation (ESI) with a ThermoQuest
AQA Navigator spectrometer.
(+)-5-Benzyl-11b-methyl (5S,11bR)-3-oxo-2,3,6,11-tetrahydro-
1H-indolizino[8,7b]indole-5,11b(5H)-dicarboxylate 16. Dimeth-
yl-2-oxoglutarate (2.21 mL, 15.2 mmol) was added to a solution
of L-tryptophan benzyl ester (3.74 g, 12.7 mmol) in CH2Cl2.
The mixture was heated at reflux for 2 days. The solvent was
removed under reduced pressure and the crude Pictet–Spengler
product was dissolved in toluene and heated at reflux for one
more day. After removal of the solvent, the mixture was dissolved
in EtOAc and washed with saturated sodium bicarbonate solution.
The aqueous layer was extracted twice with EtOAc and the
combined organic layers were dried over MgSO4 and concentrated
in vacuo. The product was purified by flash chromatography on
silica gel (heptane/EtOAc: 50/50) to give compound 16 as the
only diastereoisomer (2.66 g, 50% over 2 steps). [a]2D2 +55 (c 0.5 in
Compound 17. Compound 14 (110 mg, 0.35 mmol), was
dissolved in a 9/1 mixture of methanol and water (650 mL/65 mL).
A 20% solution of cesium carbonate was added dropwise until the
pH reached 7. The solvent was removed under vacuum and the
residue was carefully dried by the addition of acetonitrile (3 ¥
1 mL) to form an azeotrope. The residue and vindoline (53 mg,
0.12 mmol, 1/3 equiv.), were dissolved in anhydrous acetonitrile
(1 mL) and cooled to 0 ◦C. A solution of triphosgene (35 mg,
0.12 mmol, 1/3 equiv.) in dry acetonitrile (600 mL), was added
dropwise to the mixture and the reaction was stirred for 5 h at
◦C. After removal of the solvent, the product was purified by
8
CHCl3), [a]D lit. +58 (c 1.0 in CHCl3); nmax(film)/cm-1 3376, 2952,
0
flash chromatography on silica gel (heptane/EtOAc: 2/8) to give
compound 17 as one diastereoisomer (55 mg, 0.07 mmol, 60%
yield); [a]2D2 +5.5 (c 0.1 in CHCl3); nmax(film)/cm-1 2921, 2852,
1839, 1774 and 1731; dH (600 MHz; CD3CN) 0.59 (t, 3H, J 7.3 Hz,
21¢-H), 0.99 (sextuplet, 1H, J 7.1 Hz, 20¢-H), 1.53 (m, 1H, 5-H),
1.55 (q, 1H, J 7.1 Hz, 20¢-H), 1.94 (s, 3H, 30¢-H), 2.12 (m, 1H, 5-
H), 2.18 (m, 2H, 11-H), 2.19 (m, 1H, 4-H), 2.44 (t, 1H, J 13.7 Hz,
6-H), 2.57 (s, 3H, 24¢-H), 2.69 (q, 1H, J 8.5 Hz, 10¢-H), 2.88 (d,
1H, J 16 Hz, 8¢-H), 2.94 (s, 1H, 19¢-H), 3.35 (m, 1H, 10¢-H), 3.39
(m, 1H, 9-H), 3.43 (m, 1H, 6-H), 3.46 (m, 1H, 9-H), 3.48 (m, 1H,
8¢-H), 3.55 (s, 1H, 2¢-H), 3.57 (m, 1H, 4-H), 3.67 (s, 3H, 27¢-H),
3.68 (s, 3H, 19-H), 3.70 (s, 3H, 23¢-H), 4.29 (d, 1H, J 10.9 Hz,
8-H), 5.17 (s, 1H, 4¢-H), 5.23 (d, 1H, J 10.4 Hz, 6¢-H), 5.86 (dd,
1H, J 10.3 and 4.4 Hz, 7¢-H), 6.11 (s, 1H, 17¢-H), 7.03 (t, 1H, J
7.7 Hz, 13-H), 7.12 (t, 1H, J 7.9 Hz, 14-H), 7.38 (d, 1H, J 7.7 Hz,
12-H), 7.41 (s, 1H, 14¢-H), 7.48 (d, 1H, J 7.9 Hz, 15-H), 9.91 (s, 1H,
1-H); dC(150 MHz; CD3CN) 8.1 (21¢-C), 21.2 (30¢-C), 24.0 (5-C),
1742 and 1685; dH (500 MHz; CDCl3) 2.20 (m, 1H), 2.49 (dd, 1H,
J 16.6 and 9.2 Hz), 2.65 (dd, 1H, J 12.8 and 8.4 Hz), 2.91 (m, 1H),
3.10 (dd, 1H, J 16.0 and 7.4 Hz), 3.32 (d, 1H, J 16.0 Hz), 3.73
(s, 3H), 5.00 (s, 2H), 5.54 (d, 1H, J 7.2 Hz), 7.09–7.39 (m, 7H),
7.37 (d, 1H, J 7.9 Hz), 7.46 (d, 1H, J 7.5 Hz), 8.60 (s, 1H); dC(75
MHz, CDCl3) 24.4, 30.9, 34.3, 49.6, 52.8, 63.9, 66.9, 105.7, 111.5,
118.6, 119.7, 122.6, 126.0, 127.8, 128.2, 128.5, 130.9, 135.1, 137.0,
170.2, 172.3, 175.1; m/z (ESI) 441.1421 (M + Na+; C24H22N2O5Na
requires 441.1426).
(+)-(5S,11bR)-11b-methoxycarbonyl-2,3,5,6,11,11b-hexahydro-
1H-indolizino[8,7b]indole-5-carboxylic acid 14. A flask was
charged with compound 16 (2.66 g, 6.36 mmol), 9-BBN (2.33 g,
9.53 mmol) and anhydrous THF (40 mL). The mixture was
heated at reflux. When a TLC plate indicated that compound 16
was consumed, the reaction mixture was cooled under argon and
ethanolamine (575 mL, 9.53 mmol) was added. The solution was
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27.8 (9-C), 31.8 (4-C), 31.9 (20¢-C), 39.1 (24¢-C), 42.8 (6-C), 43.9
(5¢-C), 44.9 (11¢-C), 51.8 (10¢-C), 51.8 (8¢-C), 52.6 (27¢-C), 53.1
(3-C), 53.1 (19-C), 54.1 (12¢-C), 56.6 (23¢-C), 62.2 (8-C), 66.8 (19¢-
C), 77.5 (4¢-C), 80.5 (3¢-C), 84.3 (2¢-C), 95.1 (17¢-C), 105.0 (10-C),
112.3 (15-C), 119.3 (12-C), 120.3 (13-C), 120.6 (14¢-C), 122.9 (14-
C), 125.3 (15¢-C), 125.6 (7¢-C), 125.7 (13¢-C), 127.5 (11-C), 131.4
(6¢-C), 135.4 (16-C), 137.3 (2-C), 152.8 (22-C), 153.5 (18¢-C), 158.2
(16¢-C), 170.5 (20-C), 171.5 (29¢-C), 173.0 (25¢-C), 176.1 (17-C);
m/z (ESI) 797.3427 (M + H+; C43H49N4O11 requires 797.3398).
3.63 (s, 3H, 27¢-H), 3.65 (s, 1H, 2¢-H), 3.66 (m, 2H, 9-H), 3.72 (s,
3H, 19-H), 3.75 (s, 3H, 23¢-H), 4.42 (m, 1H, 8-H), 5.25 (m, 1H,
6¢-H), 5.29 (s, 1H, 4¢-H), 5.83 (dd, 1H, J 9.9 Hz and 5.0 Hz, 7¢-
H), 6.29 (s, 1H, 17¢-H), 6.96 (s, 1H, 14¢-H), 7.07 (t, 1H, J 7.9 Hz,
13-H), 7.13 (t, 1H, J 7.8 Hz, 14-H), 7.32 (d, 1H, J 7.8 Hz, 15-
H), 7.51 (d, 1H, J 7.9 Hz, 12-H), 8.72 (s, 1H, 1-H); dC(75 MHz;
CD3CN) 8.7 (21¢-C), 21.4 (30¢-C), 25.3 (5-C), 29.6 (9-C), 32.2 (4-
C), 32.2 (20¢-C), 39.1 (24¢-C), 44.3 (6-C), 44.5 (5¢-C), 45.6 (11¢-C),
51.8 (10¢-C), 51.9 (8¢-C), 52.8 (27¢-C), 53.1 (19-C), 54.6 (12¢-C),
56.4 (3-C), 57.0 (23¢-C), 63.0 (8-C), 67.6 (19¢-C), 77.8 (4¢-C), 81.0
(3¢-C), 84.6 (2¢-C), 95.6 (17¢-C), 108.7 (10-C), 112.6 (15-C), 118.9
(12-C), 120.8 (13-C), 122.2 (15¢-C), 123.5 (14-C), 124.4 (14¢-C),
125.6 (7¢-C), 125.7 (13¢-C), 129.6 (11-C), 131.6 (6¢-C), 136.0 (2-C),
136.6 (16-C), 153.2 (22-C), 154.7 (18¢-C), 159.7 (16¢-C), 171.3 (20-
C), 171.7 (29¢-C), 173.2 (25¢-C), 175.5 (17-C); m/z (ESI) 797.3400
(M + H+; C43H49N4O11 requires 797.3398).
Compound 18. Compound 14 (80 mg, 0.25 mmol), was
dissolved in a 9/1 mixture of methanol/water (550 mL/55 mL).
A 20% solution of cesium carbonate was added dropwise until the
pH reached 7. The solvent was removed under vacuum and the
residue was dried by forming an azeotrope with acetonitrile. The
residue and trimethoxybenzene (43 mg, 0.25 mmol, 1 equiv.), were
dissolved in anhydrous acetonitrile (700 mL), and cooled to 0 ◦C.
A solution of triphosgene (25.2 mg, 0.085 mmol, 1/3 equiv.), in
acetonitrile (600 mL), was added dropwise to the mixture and the
reaction was stirred for 5 h at 0 ◦C. After removal of the solvent,
the product was purified by flash chromatography on silica gel
(CH2Cl2/EtOH 97/3 → 94/6) to give compound 18 as a mixture
of diastereoisomers (dr: 1/0.4, 39 mg, 0.08 mmol, 30% yield);
major diastereomer: nmax(film)/cm-1 3408, 2927 and 1727; dH (500
MHz; CD3CN) 1.22 (m, 1H, 5-H), 1.82 (m, 1H, 5-H), 2.08 (m,
1H, 4-H), 3.35 (m, 2H, 4-H, 6-H), 3.42 (s, 3H, 8¢-H or 12¢-H), 3.45
(br s, 3H, 19-H), 3.43 (m, 1H, 6-H), 3.54 (m, 1H, 9-H), 3.66 (m,
1H, 9-H), 3.78 (s, 3H, 8¢-H or 12¢-H), 3.87 (s, 3H, 10¢-H), 4.37
(d, 1H, J 11.8 Hz, 8-H), 6.34 (s, 2H, 3¢-H, 5¢-H), 7.07 (t, 1H, J
7.8 Hz, 13-H), 7.13 (t, 1H, J 8.0 Hz, 14-H), 7.32 (d, 1H, J 8.0 Hz,
15-H), 7.48 (d, 1H, J 7.8 Hz, 12-H), 9.01 (s, 1H, 1-H); dC(75
MHz; CD3CN)/3-C and 1¢-C not visible/25.3 (5-C), 28.9 (9-C),
31.1 (4-C), 43.9 (6-C), 53.2 (19-C), 56.5 (10¢-C), 56.7 (12¢-C and
8¢-C), 63.4 (8-C), 94.0 (3¢-C and 5¢-C), 106.4 (10-C), 112.0 (15-C),
118.8 (12-C), 120.4 (13-C), 123.4 (14-C), 129.0 (11-C), 129.3 (2-
C), 136.9 (16-C), 153.1 (22-C), 161.1 (4¢-C), 162.4 (2¢-C and 6¢-C),
171.2 (20-C), 175.0 (17-C); m/z (ESI) 496.2 (M + H+).
Compounds 20 and 21. Compound 11 (101 mg, 0.13 mmol),
the trifluoroacetate salt of the peptide ProIleAspOBzl2 (121 mg,
0.19 mmol, 1.5 equiv.) and N-methyl morpholine (21 mL, 0.
19 mmol, 1.5 equiv.) were stirred in acetonitrile at 35 ◦C for
48 h to give, after treatment and purification by preparative layer
chromatography (CH2Cl2/EtOH: 93/7), compounds 20 (65 mg,
0.05 mmol) and 21 (51 mg, 0.04 mmol) with 40% and 32%
yield, respectively. Compound 20: [a]2D2 +21 (c 0.5 in CHCl3);
n
max(film)/cm-1 3326, 2922, 2852 and 1737; dH (600 MHz; CD3CN)
0.51 (t, 3H, J 7.0 Hz, 21¢-H), 0.84 (t, 3H, J 7.3 Hz, 31-H), 0.91 (d,
1H, J 6.9 Hz, 32-H), 1.07 (m, 1H, 5-H), 1.16 (m, 1H, 30-H), 1.24
(m, 1H, 20¢-H), 1.47 (m, 2H, 5-H, 30-H), 1.60 (m, 1H, 20¢-H), 1.89
(m, 2H, 24-H, 29-H), 1.96 (s, 3H, 30¢-H), 2.02 (m, 1H, 24-H), 2.10
(m, 2H, 23-H), 2.21 (m, 2H, 11¢-H), 2.31 (m, 1H, 4-H), 2.48 (m,
1H, 10¢-H), 2.64 (m, 1H, 6-H), 2.69 (s, 3H, 24¢-H), 2.74 (m, 2H,
8¢-H, 19¢-H), 2.87 (m, 1H, 6-H), 2.92 (t, 1H, J 5.6 Hz, 36-H), 3.04
(m, 1H, 4-H), 3.25 (m, 2H, 9-H), 3.31 (m, 1H, 10¢-H), 3.37 (dd,
1H, J 16.1 and 4.8 Hz, 8¢-H), 3.56 (s, 3H, 19-H), 3.62 (s, 1H, 2¢-H),
3.66 (s, 3H, 27¢-H), 3.63 (m, 1H, 8-H), 3.66 (m, 1H, 25-H), 3.70 (s,
3H, 23¢-H), 4.29 (dd, 1H, J 8.6 and 5.3 Hz, 28-H), 4.47 (dd, 1H, J
7.7 and 3.9 Hz, 22-H), 4.84 (td, 1H, J 8.1 and 6.1 Hz, 35-H), 5.06
(s, 1H, 48-H), 5.09 (s, 1H, 39-H), 5.23 (d, 1H, J 10.2 Hz, 6¢-H),
5.27 (s, 1H, 4¢-H), 5.80 (dd, 1H, J 10.2 and 5.0 Hz, 7¢-H), 6.27 (s,
1H, 17¢-H), 6.97 (t, 1H, J 7.8 Hz, 13-H), 7.03 (t, 1H, J 8.0 Hz,
14-H), 7.04 (s, 1H, 14¢-H), 7.15 (d, 1H, J 8.6 Hz, 27-H), 7.23 (d,
1H, J 8.0 Hz, 15-H), 7.26–7.36 (m, 11H, 34-H, 41-H, 42-H, 43-H,
44-H, 45-H, 50-H, 51-H, 52-H, 53-H, 54-H), 7.48 (d, 1H, J 7.8 Hz,
12-H), 8.38 (s, 1H, 1-H); dC(150 MHz; CD3CN) 8.4 (21¢-C), 12.1
(31-C), 16.2 (32-C), 21.3 (30¢-C), 25.4 (30-C), 25.9 (24-C), 27.8 (5-
C), 29.1 (23-C), 31.2 (9-C), 32.0 (20¢-C), 33.4 (4-C), 37.0 (36-C),
38.2 (29-C), 39.0 (24¢-C), 43.9 (5¢-C), 45.3 (11¢-C), 46.9 (6-C), 48.5
(25-C), 50.0 (35-C), 51.6 (8¢-C, 10¢-C), 52.6 (27¢-C), 52.7 (19-C),
54.2 (12¢-C), 56.4 (3-C), 56.7 (23¢-C), 58.5 (28-C), 60.8 (8-C), 61.4
(22-C), 66.9 (19¢-C), 67.4 (48-C), 68.0 (39-C), 77.5 (4¢-C), 80.5 (3¢-
C), 84.5 (2¢-C), 95.4 (17¢-C), 112.0 (15-C), 112.16 (10-C), 118.9
(12-C), 119.9 (13-C), 122.4 (14-C), 122.8 (15¢-C), 124.6 (14¢-C),
125.5 (13¢-C), 125.5 (7¢-C), 129.1, 129.2, 129.22, 129.23, 129.56,
129.59 (41-C, 42-C, 43-C, 44-C, 45-C, 50-C, 51-C, 52-C, 53-C,
54-C), 130.3 (11-C), 131.3 (6¢-C), 135.6 (2-C), 135.9 (16-C), 137.0
(49-C), 137.2 (40-C), 154.2 (18¢-C), 159.6 (16¢-C), 171.3 (46-C),
171.4 (37-C), 171.5 (29¢-C), 172.0 (33-C), 172.6 (26-C), 173.1 (25¢-
C), 174.9 (20-C), 175.0 (17-C); m/z (ESI) 1276.6182 (M + H+;
Compound 11. Compound 14 (296 mg, 0.94 mmol), was
dissolved in a 9/1 mixture of methanol/water (200 mL/2 mL).
A 20% solution of cesium carbonate was added dropwise until
the pH reached 7. The solvent was removed under vacuum and
the residue was dried by forming an azeotrope with acetonitrile.
The residue, vindoline (215 mg, 0.47 mmol, 1/2 equiv.) and silver
trifluoromethane sulfonate (726 mg, 2.82 mmol, 3 equiv.) were
dissolved in anhydrous acetonitrile (4 mL) and cooled to -20 ◦C.
A solution of triphosgene (93 mg, 0.31 mmol, 1/3 equiv.), in
acetonitrile (1 mL), was added dropwise to the mixture and the
reaction was stirred overnight at -20 ◦C. The mixture was filtered
through Celite to remove silver salts and the product was filtered
through silica gel (CH2Cl2/EtOH: 97/3) to give compound 11 as
only one diastereoisomer (319 mg, 0.40 mmol, 91% yield). [a]D22
+30 (c 0.1 in CHCl3); nmax(film)/cm-1 3450, 2921, 2851, 1838, 1760
and 1747; dH (500 MHz; CD3CN; T 333 K) 0.56 (t, 3H, J 6.8 Hz,
21¢-H), 1.21 (m, 1H, 20¢-H), 1.27 (m, 1H, 5-H), 1.56 (m, 1H, 20¢-
H), 1.84 (m, 1H, 5-H), 1.97 (s, 3H, 30¢-H), 2.16 (m, 2H, 11¢-H),
2.47 (m, 1H, 4-H), 2.54 (m, 1H, 10¢-H), 2.57 (s, 3H, 24¢-H), 2.76 (s,
1H, 19¢-H), 2.78 (d, 1H, J 16.9 Hz, 8¢-H), 2.89 (t, 1H, J 13.6 Hz,
6-H), 3.02 (t, 1H, J 14.1 Hz, 4-H), 3.33 (td, 1H, J 9.7 Hz, 5.0 Hz,
10¢-H), 3.41 (dd, 1H, J 16.9 and 5.3 Hz, 8¢-H), 3.52 (m, 1H, 6-H),
4878 | Org. Biomol. Chem., 2011, 9, 4873–4881
This journal is
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C71H86N6O15 requires 1276.6189). Compound 21: [a]2D2 -15.8 (c
0.5 in CHCl3); nmax(film)/cm-1 3292, 2960, 2923, 2853 and 1737;
dH(300 MHz, CD3CN) 0.05 (t, 3H, J 7.2 Hz, 21¢-H), 0.76–0.92 (m,
6H, 29-H, 30-H, 20¢-H), 1.11 (m, 1H, 28-H), 1.31 (m, 1H, 20¢-H),
1.42 (m, 1H, 28-H), 1.73 (m, 1H, 5-H), 1.90 (s, 3H, 30¢-H), 1.94
(m, 1H, 27-H), 1.91–2.09 (m, 5H, 22-H, 21-H, 5-H), 2.16 (d, 2H,
11¢-H), 2.17 (m, 1H, 10¢-H), 2.28 (s, 1H, 19¢-H), 2.45 (d, 1H, J
17.4 Hz, 8¢-H), 2.52 (m, 1H, 4-H), 2.63 (m, 1H, 4-H), 2.68 (s, 3H,
24¢-H), 2.84 (t, 1H, J 11.6 Hz, 6-H), 2.91 (dd, 2H, J 6.2 and 3.9 Hz,
34-H), 3.07 (dd, 1H, J 15.5 and 6.7 Hz, 9-H), 3.28-3.16 (m, 2H,
8¢-H, 10¢-H), 3.32 (d, 1H, J 11.5 Hz, 6-H), 3.43 (dd, 1H, J 15.5
and 10.7 Hz, 9-H), 3.54 (s, 1H, 2¢-H), 3.60 (m, 1H, 23-H), 3.68 (s,
3H, 27¢-H), 3.79 (m, 1H, 8-H), 3.83 (s, 3H, 23¢-H), 3.86 (m, 1H,
23-H), 4.23 (dd, 1H, J 8.6 and 5.6 Hz, 26-H), 4.45 (t, 1H, J 4.9 Hz,
20-H), 4.84 (dt, 1H, J 8.1 and 6.1 Hz, 33-H), 4.97–5.13 (m, 6H, 4¢-
H, 6¢-H, 37-H, 46-H), 5.64 (dd, 1H, J 10.2 and 5.1 Hz, 7¢-H), 6.31
(s, 1H, 17¢-H), 6.36 (s, 1H, 14¢-H), 6.91 (d, 1H, 25-H), 7.00 (t, 1H,
J 7.8 Hz, 13-H), 7.13 (t, 1H, J 7.8 Hz, 14-H), 7.25–7.39 (m, 11H,
32-H, 39-H, 40-H, 41-H, 42-H, 43-H, 48-H, 49-H, 50-H, 51-H,
52-H), 7.42 (d, 1H, J 7.9 Hz, 15-H), 7.54 (d, 1H, J 7.8 Hz, 12-H),
9.57 (s, 1H, 1-H); dC (75 MHz, CD3CN) 7.6 (21¢-C), 12.0 (29-C),
16.2 (30-C), 21.2 (30¢-C), 22.4 (9-C), 23.6 (5-C), 25.5 (28-C), 26.1
(21-C), 29.3 (22-C), 31.5 (20¢-C), 37.1 (34-C), 37.6 (27-C), 38.0 (4-
C), 39.5 (24¢-C), 43.7 (5¢-C), 44.3 (11¢-C), 48.2 (23-C), 49.9 (33-C),
51.3 (8¢-C), 51.6 (10¢-C), 52.4 (6-C), 52.6 (27¢-C), 53.9 (12¢-C), 56.5
(23¢-C), 57.9 (3-C), 58.6 (26-C), 62.5 (20-C), 65.2 (8-C), 66.2 (19¢-
C), 67.3 (37-C), 67.9 (46-C), 77.31 (4¢-C), 80.6 (3¢-C), 84.2 (2¢-C),
95.7 (17¢-C), 110.1 (10-C), 111.9 (15-C), 119.3 (12-C), 119.9 (13-
C), 122.7 (14-C), 123.7 (14¢-C), 123.85 (15¢-C), 123.9 (13¢-C), 125.4
(7¢-C), 129.02 (49, C51-C), 129.05 (40-C, C42-C), 129.12 (11-C),
129.15 (50-C), 129.2 (41-C), 129.5 (39, C43, C48, C52-C), 131.3
(6¢-C), 135.9 (2-C), 136.7 (16-C), 136.9 (38-C), 137.1 (47-C), 154.7
(18¢-C), 159.9 (16¢-C), 171.3 (35-C, C44-C), 171.4 (29¢-C), 171.6
(18-C), 172.1 (31-C), 173.0 (25¢-C), 173.2 (24-C), 182.6 (17-C);
m/z (ESI) 1244.5918 (M + H+; C70H81N7O14 requires 1244.5920).
56.5 (25-C), 59.6 (28-C), 61.5 (22-C), 77.1 (4¢-C), 81.2 (3¢-C), 83.5
(2¢-C), 95.7 (17¢-C), 112.4 (15-C), 121.0 (14-C), 123.5 (13-C), 124.5
(14¢-C), 170.4 (29¢-C), 173.2 (37-C, 39-C), 174.2 (17-C); m/z (ESI)
1096.5212 (M + H+; C57H74N7O15 requires 1096.5243).
Compound 23. Freshly distilled propionaldehyde (116 mL,
1.61 mmol, 10 equiv.) and sodium triacetoxyborohybride (68
mg, 0.32 mmol, 2 equiv.) were added to a solution of 20 (202
mg, 0.16 mmol, 1 equiv.) in anhydrous CH2Cl2 (2 mL). After
stirring for 3 h at room temperature, the solvent was evaporated
to dryness and the crude product was purified by preparative layer
chromatography (CH2Cl2/EtOH: 93/7) to give compound 23 (160
mg, 0.12 mmol, 75%); [a]2D2 +11 (c 0.1 in CHCl3); nmax(film)/cm-1
2959, 1739 and 1658; dH(600 MHz, CD3CN) 0.48 (t, 3H, J 7.3 Hz,
21¢-H), 0.59 (t, 3H, J 7.3 Hz, 22-H), 0.84 (m, 3H, 34-H), 0.87 (m,
3H, 35-H), 1.12 (m, 1H, 33-H), 1.18 (m, 2H, 21-H), 1.21 (m, 1H,
20¢-H), 1.44 (m, 1H, 33-H), 1.59 (m, 1H, 20¢-H), 1.83 (m, 1H,
32-H), 1.95 (m, 2H, 5-H), 1.97 (s, 3H, 30¢-H), 1.93 (m, 1H, 27-H),
1.97 (m, 1H, 26-H), 2.03 (m, 1H, 27-H), 2.10 (m, 1H, 26-H), 2.16
(m, 1H, 4-H), 2.17 (m, 2H, 11¢-H), 2.36 (m, 1H, 20-H), 2.45 (m,
1H, 10¢-H), 2.47 (m, 1H, 4-H), 2.69 (m, 1H, 19¢-H), 2.70 (m, 3H,
24¢-H), 2.72 (m, 1H, 8¢-H), 2.94 (dd, 1H, J 5.6 and 4.9 Hz, 39-H),
3.19 (m, 1H, 9-H), 3.24 (m, 1H, 6-H), 3.31 (m, 1H, 10¢-H), 3.37
(dd, 1H, J 16.7 and 4.6 Hz, 8¢-H), 3.48 (m, 2H, 6-H, 9-H), 3.56
(s, 3H, 19-H), 3.62 (s, 1H, 2¢-H), 3.71 (s, 3H, 27¢-H), 3.71 (m, 1H,
28-H), 3.73 (s, 3H, 23¢-H), 3.81 (m, 1H, 28-H), 3.86 (m, 1H, 8-H),
4.24 (m, 1H, 31-H), 4.47 (m, 1H, 25-H), 4.84 (m, 1H, 38-H), 5.07
(s, 2H, 51-H), 5.10 (s, 2H, 42-H), 5.24 (d, 1H, J 10.3 Hz, 6¢-H),
5.27 (s, 1H, 4¢-H), 5.81 (dd, 1H, J 10.2 and 4.6 Hz, 7¢-H), 6.30 (s,
1H, 17¢-H), 6.93 (s, 1H, 14¢-H), 6.98 (t, 1H, J 7.6 Hz, 13-H), 7.03
(t, 1H, J 7.6, 14-H), 7.21 (m, 1H, 15-H), 7.29–7.40 (m, 12H, 30-H,
37-H, 44-H, 45-H, 46-H, 47-H, 48-H, 53-H, 54-H, 55-H, 56-H, 57-
H), 7.49 (d, 1H, J 7.6 Hz, 12-H), 8.33 (s, 1H, 1-H); dC (150 MHz,
CD3CN) 8.5 (21¢-C), 12.0 (22-C), 12.1 (34-C), 16.2 (35-C), 21.2 (5-
C), 21.3 (30¢-C), 22.3 (21-C), 25.5 (33-C), 25.8 (27-C), 28.7 (9-C),
29.0 (26-C), 31.9 (20¢-C), 34.2 (4-C), 37.0 (39-C), 38.2 (32-C), 39.1
(24¢-C), 43.8 (5¢-C), 45.2 (11¢-C), 48.6 (28-C), 50.0 (38-C), 51.0
(6-C), 51.5 (8¢-C), 51.0 (10¢-C), 52.7 (27¢-C), 52.8 (19-C), 54.2 (12¢-
C), 56.6 (3-C), 56.6 (23¢-C), 57.9 (20-C), 58.7 (31-C), 61.4 (25-C),
64.9 (8-C), 66.8 (19¢-C), 67.4 (51-C), 68.0 (42-C), 77.4 (4¢-C), 80.7
(3¢-C), 84.4 (2¢-C), 95.1 (17¢-C), 112.0 (15-C), 112.6 (10-C), 119.0
(12-C), 119.9 (13-C), 122.3 (14-C), 123.2 (15¢-C), 124.7 (14¢-C),
125.0 (13¢-C), 125.4 (7¢-C), 129.1, 129.2, 129.5, 129.6 (44-C, 45-C,
46-C, 47-C, 48-C, 53-C, 54-C, 55-C, 56-C, 57-C), 130.3 (11-C),
131.3 (6¢-C), 134.9 (2-C), 135.9 (16-C), 136.9 (52-C), 137.1 (43-
C), 154.0 (18¢-C), 159.1 (16¢-C), 171.5 (40-C, 49-C), 171.6 (29¢-C),
172.7 (29-C), 173.1 (25¢-C), 175.0 (23-C), 175.8 (17-C); m/z (ESI)
1318.6621 (M + H+; C74H92N7O15 requires 1318.6645).
Compound 22. 10% Pd/carbon (8 mg, 0.008 mmol), was added
to a solution of compound 20 (55 mg, 0.04 mmol) in anhydrous
THF. The mixture was stirred for 2 days under an H2 atmosphere
and filtered through a pad of Celite to afford quantitatively
compound 22 (43 mg, 100%); Apparent peaks dH (600 MHz;
MeOD) 0.50 (m, 3H, 21¢-H), 0.97 (t, 3H, J 7.3 Hz, 31-H), 1.07
(d, 1H, J 6.9 Hz, 32-H), 1.41–1.30 (m, 3H, 5-H, 30-H, 20¢-H),
1.62–1.74 (m, 3H, 5-H, 30-H, 20¢-H), 1.93–2.05 (m, 2H, 24-H,
29-H), 2.07 (s, 3H, 30¢-H), 2.15–2.25 (m, 2H, 23-H, 24-H), 2.26
(m, 2H, 11¢-H), 2.32 (m, 1H, 23-H), 2.59 (m, 1H, 10¢-H), 2.77 (s,
3H, 24¢-H), 2.79 (m, 1H, 19¢-H), 2.88 (m, 1H, 36-H), 3.15 (m, 1H,
8¢-H), 3.27 (m, 2H, 6-H), 3.53 (m, 1H, 10¢-H), 3.61 (m, 1H, 8¢-H),
3.64 (m, 1H, 9-H), 3.70 (s, 1H, 2¢-H), 3.73 (s, 3H, 19-H), 3.82 (s,
3H, 27¢-H), 3.83 (m, 1H, 8-H), 3.84 (m, 2H, 25-H), 3.84 (s, 3H,
23¢-H), 4.42 (d, 1H, J 7.2 Hz, 28-H), 4.44–4.74 (m, 2H, 22-H,
35-H), 5.39 (m, 1H, 6¢-H), 5.44 (s, 1H, 4¢-H), 5.90 (dd, 1H, J 10.2
and 5.0 Hz, 7¢-H), 6.38 (s, 1H, 17¢-H), 6.99 (s, 1H, 14¢-H), 7.08 (t,
1H, J 8.0 Hz, 14-H), 7.15 (t, 1H, J 7.8 Hz, 13-H), 7.37 (d, 1H, J
8.0 Hz, 15-H), 7.71 (m, 1H, 12-H); dC(150 MHz; MeOD) 8.6 (21¢-
C), 11.9 (31-C), 16.2 (32-C), 20.9 (30¢-C), 23.9 (5-C), 26.0 (30-C),
26.3 (24-C), 27.9 (9-C), 30.8 (23-C), 32.3 (20¢-C), 37.6 (36-C), 38.6
(29-C), 39.0 (24¢-C), 44.2 (5¢-C), 44.8 (11¢-C), 49.3 (6-C), 49.3 (27-
C), 50.7 (35-C), 51.4 (8¢-C, 10¢-C), 53.2 (19-C, 23¢-C), 54.3 (12¢-C),
Compound 24. Methyl iodide (29 mL, 0.47 mmol, 10 equiv.)
was added to a solution of 23 (60 mg, 0.05 mmol) in anhydrous
acetonitrile (1 mL). After stirring for 48 h at room temperature,
the solvent was evaporated and the crude product was purified
by preparative layer chromatography (CH2Cl2/EtOH: 93/7) to
give compound 24 (33 mg, 0.025 mmol, 53%) and some recovered
starting material (14 mg, 0.01 mmol); [a]2D2 -37 (c 0.1, CHCl3);
n
max(film)/cm-1 3454, 2963, 1737 and 1653; dH(300 MHz, CD3CN)
0.46–0.55 (m, 3H, 21¢-H), 0.55–0.64 (m, 3H, 22-H), 0.89 (t, 1H,
J 7.3 Hz, 35-H), 0.93 (d, 3H, J 6.9 Hz, 36-H), 1.14–1.27 (m, 3H,
5-H, 34-H, 20¢-H), 1.44–1.69 (m, 5H, 5-H, 21-H, 34-H, 20¢-H),
This journal is
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Org. Biomol. Chem., 2011, 9, 4873–4881 | 4879
©

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1.88 (m, 1H, 33-H), 1.98 (s, 3H, 30¢-H), 2.01–2.35 (m, 6H, 11¢-
H, 27-H, 28-H), 2.60 (m, 1H, 10¢-H), 2.77 (m, 1H, 8¢-H), 2.75 (s,
3H, 24¢-H), 2.84 (s, 1H, 19¢-H), 2.91 (m, 1H, 4-H), 2.96 (d, 1H, J
5.9 Hz, 40-H), 3.07 (m, 2H, 20-H), 3.14 (s, 3H, 23-H), 3.26–3.43
(m, 3H, 4-H, 8¢-H, 10¢-H), 3.53 (d, 1H, J 17.5 Hz, 9-H), 3.65 (s,
3H, 19-H), 3.66 (s, 1H, 2¢-H), 3.73 (s, 3H, 27¢-H), 3.80 (s, 3H,
23¢-H), 3. 58–3.85 (m, 4H, 6-H, 29-H), 3.91 (dd, 1H, J 17.7 and
8.7 Hz, 9-H), 4.30 (m, 1H, 32-H), 4.59–4.72 (m, 2H, 8-H, 26-H),
4.88 (m, 1H, 39-H), 5.11 (d, 2H, J 4.4 Hz, 52-H), 5.13 (d, 2H, J
3.8 Hz, 43-H), 5.22 (s, 1H, 4¢-H), 5.27 (d, 1H, J 10.4 Hz, 6¢-H),
5.83 (dd, 1H, J 10.2 and 5.0 Hz, 7¢-H), 6.34 (s, 1H, 17¢-H), 6.84
(s, 1H, 14¢-H), 7.07–7. 22 (m, 3H, 13-H, 14-H, 31-H), 7.30 (d, 1H,
J 8.0 Hz, 38-H), 7. 32–7. 49 (m, 11H, 15-H, 45-H, 46-H, 47-H,
48-H, 49-H, 54-H, 55-H, 56-H, 57-H, 58-H), 7.80 (d, 1H, J 7.9 Hz,
12-H), 8.84 (s, 1H, 1-H); dC (75 MHz, CD3CN)/4-C, 10-C, 11-C
not visible/8.4 (21¢-C), 10.7 (22-C), 12.0 (35-C), 1626.2 (9-C),.0
(36-C), 17.1 (21-C), 21.2 (30¢-C), 22.1 (5-C), 25.7 (28-C, 34-C),
30.1 (27-C), 31.8 (20¢-C), 37.1 (40-C), 38.2 (33-C), 38.9 (24¢-C),
43.7 (5¢-C), 45.4 (11¢-C), 49.9 (39-C, 29-C), 51.0, 51.4 (8¢-C, 10¢-
C), 52.7 (27¢-C), 53.2 (19-C), 54.3 (12¢-C), 56.1 (3-C), 56.6 (23¢-C),
58.8 (32-C), 61.6 (26-C), 63.0 (6-C), 66.1 (19¢-C), 67.4 (52-C), 68.0
(20-C, 43-C), 71.6 (8-C), 77.5 (4¢-C), 80.7 (3¢-C), 84.2 (2¢-C), 95.1
(17¢-C), 112.6 (15-C), 119.2 (12-C), 120.9 (13-C), 121.0 (15¢-C),
123.7 (14-C), 124.1 (14¢-C), 125.3 (13¢-C), 125.5 (7¢-C), 129.15,
129.24, 129.3, 129.6 (45-C, 46-C, 47-C, 48-C, 49-C, 54-C, 55-C,
56-C, 57-C, 58-C), 131.3 (6¢-C), 134.5 (2-C), 136.5 (16-C), 136.9
(53-C), 137.1 (44-C), 154.4 (18¢-C), 159.0 (16¢-C), 166.6, 171.4,
171.47, 171.53, 171.9, 172.9 (24-C, 30-C, 37-C, 41-C, 50-C, 25¢-
C, 29¢-C), 175.0 (17-C); m/z (ESI) 1332.6791 (M+; C75H94N7O15
requires 1332.6808).
9.2 (21¢-C), 12.0 (35-C), 16.2 (36-C), 21.2 (30¢-C), 22.0 (5-C), 25.2
(23-C), 25.6 (34-C), 25.9 (28-C), 27.7 (9-C), 29.4 (27-C), 32.0 (20¢-
C), 33.1 (4-C), 37.1 (40-C), 37.8 (33-C), 39.2 (24¢-C), 43.9 (5¢-C),
45.2 (11¢-C), 48.9 (6-C), 48.0 (29-C), 50.0 (39-C), 50.9, 51.0 (8¢-C,
10¢-C), 52.6 (19-C, 27¢-C), 54.5 (12¢-C), 56.7 (23¢-C), 57.2 (3-C),
57.4 (8-C), 58.7 (32-C), 62.2 (26-C), 66.1 (19¢-C), 67.4 (52-C), 68.0
(43-C), 77.5 (4¢-C), 80.7 (3¢-C), 83.3 (22-C), 85.0 (2¢-C), 94.9 (17¢-
C), 111.1 (10-C), 112.1 (15-C), 118.3 (12-C), 118.9 (13-C), 122.5
(15¢-C), 123.0 (14-C), 124.5 (13¢-C), 125.5 (14¢-C), 126.9 (7¢-C),
129.1, 129.2, 129.3, 129.6 (45-C, 46-C, 47-C, 48-C, 49-C, 54-C,
55-C, 56-C, 57-C, 58-C), 130.6 (11-C), 131.4 (6¢-C), 133.8 (2-C),
135.7 (16-C), 137.0 (53-C), 137.1 (44-C), 153.0 (20-C), 154.1 (18¢-
C), 158.9 (16¢-C), 170.5 (24-C), 171.3, 171.5, 171.6 (29¢-C, 41-C,
50-C), 172.0 (37-C), 172.6 (30-C), 173.0 (25¢-C), 175.5 (17-C); m/z
(ESI) 1382.6017 (M+; C74H89N7O17Cl requires 1382.6003). Minor
diastereomer : [a]D22 -60 (c 0.1, CHCl3); nmax(film)/cm-1 2927, 1740,
1365, 1217 and 768; dH(300 MHz, CD3CN) 0.57–0.67 (m, 6H,
23-H, 21¢-H), 0.89 (t, 1H, J 7.4 Hz, 35-H), 0.92 (d, 3H, J 6.9 Hz,
36-H), 1.19 (m, 1H, 34-H), 1.31–1.42 (m, 2H, 5-H, 20¢-H), 1.49 (m,
1H, 34-H), 1.65 (m, 1H, 20¢-H), 1.78 (m, 1H, 5-H), 1.89 (m, 1H,
28-H), 1.91–1.99 (m, 2H, 28-H, 33-H), 1.99 (s, 3H, 30¢-H), 2.03
(m, 2H, 27-H), 2.13 (m, 1H, 11¢-H), 2.36–2.50 (m, 1H, 4-H, 10¢-H,
11¢-H), 2.58 (s, 1H, 19¢-H), 2.60 (d, 1H, J 15.4 Hz, 8¢-H), 2.71 (s,
3H, 24¢-H), 2.89 (m, 1H, 4-H), 2.95 (m, 1H, 40-H), 2.96 (m, 1H,
6-H), 3.16 (d, J 15.7 Hz, 1H, 9-H), 3.25 (m, 1H, 29-H), 3.36 (m,
1H, 10¢-H), 3.41 (dd, 1H, J 15.8 and 5.6 Hz, 8¢-H), 3.49 (s, 3H,
19-H), 3.49 (m, 1H, 6-H), 3.61 (m, 1H, 9-H), 3.62 (s, 1H, 2¢-H),
3.71 (s, 3H, 27¢-H), 3.75 (s, 3H, 23¢-H), 3.78 (m, 1H, 29-H), 4.25
(dd, 1H, J 8.3 and 5.9 Hz 32-H), 4.38 (m, 1H, 26-H), 4.85 (m, 1H,
39-H), 5.06 (s, 2H, 52-H), 5.10 (s, 2H, 43-H), 5.19 (m, 1H, 8-H),
5.27 (s, 1H, 4¢-H), 5.33 (d, 1H, J 10.3 Hz, 6¢-H), 5.84 (dd, 1H, J
10.2 and 5.3 Hz, 7¢-H), 5.98 (m, 1H, 22-H), 6.27 (s, 1H, 17¢-H),
6.63 (s, 1H, 14¢-H), 6.95–7.08 (m, 2H, 13-H, 14-H), 7.17 (d, 1H, J
7.7 Hz, 15-H), 7.27–7.37 (m, 10H, 45-H, 46-H, 47-H, 48-H, 49-H,
54-H, 55-H, 56-H, 57-H, 58-H), 7.61 (d, 1H, J 7.6 Hz, 12-H), 8.32
(s, 1H, 1-H); dC (150 MHz, CD3CN) 8.7 (21¢-C), 12.0 (35-C), 16.2
(36-C), 21.3 (30¢-C), 22.2 (5-C), 24.3 (23-C), 25.6 (34-C, 28-C), 27.5
(9-C), 29.5 (27-C), 31.8 (20¢-C), 33.5 (4-C), 37.1 (40-C), 37.8 (33-
C), 38.7 (24¢-C), 43.7 (5¢-C), 44.9 (11¢-C), 48.7 (6-C), 48.2 (29-C),
50.0 (39-C), 51.8, 51.9 (8¢-C, 10¢-C), 52.7 (19-C, 27¢-C), 54.4 (12¢-
C), 56.7 (23¢-C), 57.2 (3-C), 57.7 (8-C), 58.7 (32-C), 62.2 (26-C),
67.1 (19¢-C), 67.4 (52-C), 68.0 (43-C), 77.4 (4¢-C), 80.8 (3¢-C), 83.9
(22-C), 84.5 (2¢-C), 94.7 (17¢-C), 111.1 (10-C), 112.1 (15-C), 119.2
(12-C), 120.1 (13-C), 122.0 (15¢-C), 122.9 (14-C), 124.2 (13¢-C),
125.4 (7¢-C), 126.3 (14¢-C), 129.1, 129.2, 129.3, 129.6 (45-C, 46-C,
47-C, 48-C, 49-C, 54-C, 55-C, 56-C, 57-C, 58-C), 130.5 (11-C),
131.3 (6¢-C), 134.8 (2-C), 137.3 (16-C), 137.0 (53-C), 137.2 (44-
C), 153.2 (20-C), 154.0 (18¢-C), 159.1 (16¢-C), 170.2 (24-C), 171.3,
171.5, 171.6 (29¢-C, 41-C, 50-C), 172.1 (37-C), 172.6 (30-C), 172.8
(25¢-C), 175.4 (17-C); m/z (ESI) 1382.6025 (M+; C74H89N7O17Cl
requires 1382.6003).
Compound 25. 1-Chloroethylchloroformate (mixture of enan-
tiomers) (34 mL, 0,31 mmol, 5 equiv.) was added to a solution
of 20 (78 mg, 0.063 mmol) in anhydrous dichloromethane (0,7
mL). After stirring for 3 h at room temperature, the solvent was
evaporated and the crude product was purified by preparative layer
chromatography (CH2Cl2/EtOH: 93/7) to give compound 25 as
two diastereomers (rd : 1/0.4) (66%); Major diastereomer :[a]D22
-34 (c 0.1, CHCl3); nmax(film)/cm-1 2927, 1740, 1365, 1217 and
768; dH(300 MHz, CD3CN) 0.73 (m, 3H, 21¢-H), 0.87 (t, 1H, J
7.4 Hz, 35-H), 0.91 (d, 3H, J 6.9 Hz, 36-H), 1.18 (m, 2H, 5-H,
34-H), 1.27 (m, 1H, 20¢-H), 1.31 (d, 1H, J 5.7 Hz, 23-H), 1.47
(m, 1H, 34-H), 1.62 (m, 1H, 20¢-H), 1.72 (m, 1H, 5-H), 1.89 (m,
1H, 28-H), 1.95 (m, 2H, 28-H, 33-H), 1.97 (s, 3H, 30¢-H), 2,02
(m, 2H, 27-H), 2.10 (m, 1H, 11¢-H), 2.41 (m, 1H, 4-H), 2.50 (td,
1H, J 12.4 and 5.2 Hz, 11¢-H), 2.65 (m, 1H, 10¢-H), 2.67 (s, 3H,
24¢-H), 2.79 (d, 1H, J 15.4 Hz, 8¢-H), 2.86 (s, 1H, 19¢-H), 2.93
(m, 1H, 4-H), 2.93 (m, 1H, 40-H), 3.05 (m, 1H, 6-H), 3.14 (m,
1H, 9-H), 3.25 (m, 2H, 29-H, 10¢-H), 3.31 (dd, 1H, J 16.1 and
5.1 Hz, 8¢-H), 3.41 (m, 1H, 6-H), 3.48 (s, 3H, 19-H), 3.58 (s, 1H,
2¢-H), 3.63 (m, 1H, 9-H), 3.70 (s, 3H, 27¢-H), 3.72 (m, 1H, 29-H),
3.76 (s, 3H, 23¢-H), 4.24 (dd, 1H, J 8.3 and 5.9 Hz 32-H), 4.39
(m, 1H, 26-H), 4.84 (m, 1H, 39-H), 5.05 (s, 2H, 52-H), 5.09 (s,
2H, 43-H), 5.23 (m, 1H, 8-H), 5.25 (s, 1H, 4¢-H), 5.29 (d, 1H, J
10.3 Hz, 6¢-H), 5.81 (dd, 1H, J 10.2 and 5.3 Hz, 7¢-H), 6.15 (d, 1H,
J 5.6 Hz, 22-H), 6.26 (s, 1H, 17¢-H), 6.92 (s, 1H, 14¢-H), 7.03 (m,
2H, 13-H, 14-H), 7.11 (d, 1H, J 7.7 Hz, 15-H), 7.30 (m, 10H, 45-H,
46-H, 47-H, 48-H, 49-H, 54-H, 55-H, 56-H, 57-H, 58-H), 7.59 (d,
1H, J 7.6 Hz, 12-H), 8.21 (s, 1H, 1-H); dC (150 MHz, CD3CN)
Computational procedures. All calculations were performed on
a PC workstation. The modeling study was performed using Sybyl
7.3 software. The MMFF94 force field was used for minimization
and partial charge calculations, a dielectric constant of 1.0 being
employed. Compound 5 was subjected to an unrestrained molec-
ular dynamics simulation at 1600 K for 20 000 fs. Conformations
were sampled every 100 fs during the simulation resulting in 200
4880 | Org. Biomol. Chem., 2011, 9, 4873–4881
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randomized structures. Each of these conformers was minimized
3 A. Cormier, M. Marchand, R. G. B. Ravelli, M. Knossow and B.
Gigant, EMBO Rep., 2008, 9, 1101.
4 Q. A. Ngo, F. Roussi, A. Cormier, S. Thoret, M. Knossow, D. Gue´nard
and F. Gue´ritte, J. Med. Chem., 2009, 52, 134; Q. A. Ngo, F. Roussi, S.
Thoret and F. Gue´ritte, Chem. Biol. Drug Des., 2010, 75, 284.
5 P. Magnus, J. S. Mendoza, A. Stamford, M. Ladlow and P. Willis, J.
Am. Chem. Soc., 1992, 114, 10232.
˚
and compared with others with a RMS of 0.3 A. The obtained
structures were ranked according to energy. They were analyzed
using Sybyl 7.3 software and superimposition of conformers was
based on the backbone atoms of vinblastine.
6 P. Magnus, A. Stamford and M. Ladlow, J. Am. Chem. Soc., 1990, 112,
8210.
7 For a general review see: H. Weyerstahl and H. Marschall, Compre-
hensive Organic Synthesis, B. M. Trost and I. Fleming, eds, 1991, 6,
1041.
8 P. Magnus, M. Ladlow, C. S. Kim and P. Boniface, Heterocycles, 1989,
28, 951.
Acknowledgements
We thank ICSN and CNRS for the grant that supported this
work for one of us (CR). We also thank Sylviane Thoret and
Genevie`ve Aubert (ICSN, CNRS) for biological assays. Jacques
Fahy and Philippe Maillas (Pierre Fabre Laboratories) are grate-
fully acknowledged for a generous gift of vindoline.
9 C. J. Collins, M. Lanz and B. Singaram, Tetrahedron Lett., 1999, 40,
3673.
10 W. H. Daly and D. Poche´, Tetrahedron Lett., 1988, 29, 5859.
11 R. Wilder and S. Mobashery, J. Org. Chem., 1992, 57, 2755.
12 S.-S. Wang, B. F. Gisin, D. P. Winter, R. Makofske, I. D. Kulesha, C.
Tzougraki and J. Meienhofer, J. Org. Chem., 1977, 42, 1287.
13 The generation of such an intermediate has also been reported by
Magnus, but in his case, it was formed when no nucleophile was present
in the reaction: P. Magnus, M. Giles, R. Bonnert, G. Johnson, L.
McQuire, M. Deluca, A. Merrit, C. S. Kim and N. Vicker, J. Am.
Chem. Soc., 1993, 115, 8116.
Notes and references
1 F. Roussi, F. Gue´ritte and J. Fahy, The Vinca alkaloids, in G. M.
Gragg, D. G. I. Kingston, D. J. Newman, editors. Anticancer Agents
from Natural Products, 2nd Ed., Boca Raton, FL: Taylor and Francis;
in press.
14 S. Winstein, E. Clippinger, A. H. Fainberg, R. Heck and G. C.
Robinson, J. Am. Chem. Soc., 1956, 78, 328.
15 J. Fahy, Curr. Pharm. Des., 2001, 7, 1181.
2 B. Gigant, C. Wang, B. G. R. Ravelli, F. Roussi, M. O. Steinmetz,
P. A. Cumi, A. Sobel and M. Knossow, Nature, 2005, 435,
519.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 4873–4881 | 4881
©