One-pot synthesis of vinca alkaloids-phomopsin hybrids

Article abstract of DOI:10.1021/jm500530v

Hybrids of vinca alkaloids and phomopsin A have been elaborated with the aim of interfering with the vinca site and the peptide site of the vinca domain in tubulin. They were synthesized by an efficient one-pot procedure that directly links the octahydrophomopsin lateral chain to the velbenamine moiety of 7′-homo-anhydrovinblastine. In their modeled complexes with tubulin, these hybrids were found to superimpose nicely on the tubulin-bound structures of vinblastine and phomopsin A. This good matching can account for the fact that two of them are very potent inhibitors of microtubules assembly and are cytotoxic against four cancer cell lines.

Full text of DOI:10.1021/jm500530v

Brief Article
pubs.acs.org/jmc
One-Pot Synthesis of Vinca Alkaloids−Phomopsin Hybrids
Olga Gherbovet,† Claire Coderch,‡ María Concepcion García Alvarez,† Jerome Bignon,† Sylviane Thoret,†
́
́
̂
Franco
̧
ise Gueritte,† Federico Gago,‡ and Fanny Roussi*,†
́
†Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, UPR 2301 du CNRS, Avenue de la Terrasse, 91198,
Gif-sur-Yvette Cedex, France
‡
́
́ ́ ́
Area de Farmacología, Departamento de Ciencias Biomedicas, Universidad de Alcala, 28871 Alcala de Henares, Madrid, Spain
S
* Supporting Information
ABSTRACT: Hybrids of vinca alkaloids and phomopsin A
have been elaborated with the aim of interfering with the
“vinca site” and the “peptide site” of the vinca domain in
tubulin. They were synthesized by an efficient one-pot
procedure that directly links the octahydrophomopsin lateral
chain to the velbenamine moiety of 7′-homo-anhydrovinblas-
tine. In their modeled complexes with tubulin, these hybrids
were found to superimpose nicely on the tubulin-bound
structures of vinblastine and phomopsin A. This good
matching can account for the fact that two of them are very
potent inhibitors of microtubules assembly and are cytotoxic
against four cancer cell lines.
ancer is characterized by uncontrolled cell proliferation
C
and inappropriate cell survival. Microtubules play an
important role in these cellular processes and have been one of
the most efficacious targets for tumor chemotherapy. They are
dynamic polymers made of heterodimers of α and β tubulin.
Microtubule-binding molecules, a highly important class of
anticancer agents, are generally derivatives of intricate natural
products. They interfere with assembly or disassembly of
microtubules by suppressing microtubule dynamics required for
mitotic spindle function in rapidly dividing cells. In this way,
they induce a cascade of events causing cells to commit suicide,
that is, to undergo apoptosis or programmed cell death.1
Conventionally, microtubule-binding drugs are classified as
stabilizing or destabilizing agents.2 Among the assembly
inhibitors, the vinca alkaloids (Figure 1) are highly potent
antimitotic drugs that prevent tubulin polymerization into
microtubules. Vinblastine 1 and vincristine 2, isolated in the
late 50s, and their synthetic derivatives vindesine 3, vinorelbine
4, and vinflunine 5 are used extensively in cancer chemo-
therapy.
Figure 1. Vinca alkaloids currently used in chemotherapy.
the vinca alkaloids, and most of them are peptides, they are
considered to occupy the “‘peptide site”’ of the vinca domain.2
This is the case for the antimitotic cyclopeptide phomopsin A
6,4 a hexapeptide that contains a 13-membered cyclic moiety
and a lateral chain of three dehydroamino acids (Figure 2). 6
and unnatural rac-octahydrophomopsin A 75 are very potent
tubulin assembly inhibitors (IC50 of 0.56 and 0.40 μM,
respectively) but are relatively weak cytotoxic agents.
Vinca alkaloids bind near the GTP-binding site in the “vinca
site” of the so-called vinca domain. Atomic details about this
“vinca site” were gained when Knossow et al. published the X-
ray structure of vinblastine 1 bound to the tubulin-stathmin:
RB3-SLD complex ((Tc)2R).3 They showed that it is located at
the interface between two tubulin heterodimers. Vinblastine is
oriented so that velbenamine and vindoline moieties interact
with the β1 monomer and the α2 monomer of the next
heterodimer, velbenamine being close to the GTP/GDP
nucleotide exchange site of the β1 subunit.
Phomopsin A 6 was also cocrystallized with tubulin.6
Superimposition of 1 and 6 binding sites revealed that they
substantially overlap. The velbenamine moiety of 1 and the
macrocyle of 6 fill the same areas that define the “vinca site”,
Other molecules are known to bind within the tubulin vinca
Received: April 3, 2014
domain. Since their binding site partly coincides with that of
© XXXX American Chemical Society
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Figure 2. Phomopsin A 6 and rac-octahydrophomopsin A 7.
whereas the vindoline moiety of 1 and the lateral chain of 6
accommodate in opposite directions. In particular, phomopsin
A gives rise to multiple contacts with Tyrβ224, one of the
residues sandwiching the GDP/GTP nucleotide exchangeable
site (Figure 3).
Figure 4. Envisaged hybrids of 7′-homo-vinblastine and phomopsin 10.
11. So far, the 8′ position did not appear to tolerate bulky
groups: with a methyl ester at 8′, compounds are highly active
on tubulin but totally inactive if 8′ carries an ethyl or a benzyl
ester.9 Anyway, we postulated that using a more flexible linker
in 8′ could favor accommodation of the phomopsin lateral
chain in tubulin.
Our strategy relied on the elaboration of an aldehyde
intermediate 12, obtained in one step by an enlargement
reaction of vinorelbine 4, that could lead to the desired hybrids
13 by means of a reductive amination with peptide chains of
various lengths (ProOMe, ProIleOMe, and ProIleAsp(OBn)2)
corresponding to those of phomopsin A lateral chain (Scheme
1).
Figure 3. Superimposition of vinblastine (blue) and phomopsin A
(purple) in their respective binding sites in tubulin which define the
“vinca domain” (green, α2-subunit; pink, β1-subunit).
These noteworthy observations urged us to elaborate hybrids
of 1 and 6 with the idea of obtaining molecules that could
interfere with the “peptide” and the “vinca” sites simultaneously
and addressing the question of whether the biological activities
of the resulting compounds would be improved over those of
the parent molecules.
Scheme 1. Envisaged Synthetic Pathway
So far, we have reported the synthesis of two series of vinca−
phomopsin hybrids (Figure 4). Hybrids of the first generation 8
linked the phomopsin lateral chain to the tertiary amine of the
velbenamine moiety of anhydrovinblastine and vinorelbine.7
Although very active, molecular modeling showed that the
absolute configuration of the quaternary nitrogen forces the
phomopsin A lateral chain to orientate into a pocket different
from that of the native compound. The second generation of
vinca−phomopsin hybrids 9, with a correct orientation of the
phomopsin A lateral chain and a simplified velbenamine
moiety, was found to be totally inactive.8
In this paper, we report the synthesis of a new family of 7′-
homo-anhydrovinblastine and phomopsin hybrids 10 by means
of a one-step procedure saving the four-ring structure of the
velbenamine upper part and controlling the orientation of the
inserted lateral chain. To achieve this goal, we used an original
and highly selective insertion reaction of activated alkynes into
the gramine bridge of vinorelbine 4, which was recently worked
out in our group.9 This insertion reaction led to highly active
diester or ester amide 7′-homo-anhydrovinblastine derivatives
To obtain aldehyde 12, commercial 3-(trimethylsilyl)-
propynal was reacted with vinorelbine 4 in acetonitrile at
room temperature for 2 h. The insertion reaction occurs
probably by a Michael addition of the tertiary amine of 4 on the
acetylene, generating a zwitterion 14. It would lead to the
reactive alkylideneindoleninium ion 15 after a spontaneous
fragmentation of the gramine bridge. This ion could then be
trapped intramolecularly to give inserted 16. Nevertheless, this
compound was not observed and the deprotected 12 was
formed directly (20%). To increase the yield of this reaction,
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desilylation agents (TBAF, AgF) and various bases (DMA,
K2CO3) were screened to favor the in situ deprotection. Finally,
using 3 equiv of DMA at room temperature for 3 h increased
the yield to 36% (Scheme 2).
Scheme 3. Double Reduction of Iminium Species 23 and 24
to Compounds 20−22
a
Scheme 2. Synthesis of Aldehyde 12
a
Reagents and conditions: (a) 3-(trimethylsilyl)propynal, CH3CH, 2h,
rt, 20%; (b) 3-(trimethylsilyl)propynal, CH3CN, 2 h, rt, then DMA, 3
h, rt, 36%.
To avoid the purification of aldehyde 12, which is not very
stable on silica gel, a one-pot synthesis of hybrids 20−22 was
considered. Thus, 4 was reacted with 1.1 equiv of 3-
(trimethylsilyl)propynal at room temperature. After 2 h of
stirring necessary for the formation of aldehyde 12, an amount
of 1.75 equiv of peptide 17 or 18 or 19 was added, followed by
2.6 equiv of sodium triacetoxyborohydride in solution in
dichloromethane (Table 2). The diastereoisomeric ratio
Reductive aminations on 12 were then considered to obtain
the desired hybrids (Table 1). As rac-octahydrophomopsin A 7
a
Table 1. Synthesis of Hybrids 20−22
a
Table 2. One-Pot Synthesis of Hybrids 20−22
a
The asterisk (∗) indicates isolated yield of the major diastereomer.
a
Reagents and conditions: (a) 3-(trimethylsilyl)propynal, CH3CN, 2
is as potent as phomopsin A 6 on tubulin and the synthesis of
the phomopsin unsaturated tripeptide side chain requires a
multistep procedure,10 we made peptide chains containing
natural L-proline, L-isoleucine, and L-aspartic acid. Commer-
cially available Pro-OMe 17 was used, whereas Pro-Ile-OMe 18
and Pro-Ile-Asp-(OBn)2 19 were synthesized by classical
peptide coupling.7
Three hybrids were obtained with a ratio of 80:20. Note that
calculated yields correspond solely to the major diaster-
eoisomer, as it was the only one that could be isolated by
column chromatography. As loss of material was observed
during the purification, yields were estimated satisfying with
regard to the molecular complexity of the synthesized
compounds.
h, rt; (b) AA, NaBH(OAc)3, DCM, rt, 2 h. The asterisk (∗) indicates
isolated yield of the major diastereomer.
remained the same and once again; only the major
diastereoisomer could be isolated. It is important to note that
five different reactions (insertion, silyl group deprotection,
amination, and double reduction of iminiums 23 and 24) take
place in the same flask.
The configuration of the new steric center for 20−22 was
determined by NOE experiments. For example, for 22,
correlations could be observed between H-12′ and H-9′α, H-
12′ and H-8′, H-1′α and H-2′ on the lower face and between
H-1′β and H-9′β on the upper face. In parallel, molecular
dynamics simulations were run for the two possible (8-R) and
(8-S) epimers of 22. They confirmed that such correlations are
only consistent with a (8-R) configuration (Figure 5). This R
configuration at C-8′ for the isolated hybrids 20−22
If 1.8 equiv of peptide could be used, an excess of sodium
triacetoxyborohydride (2.6 equiv) was employed, as a double
reduction of two iminium species 23 and 24 occurs during the
reaction (Scheme 3).
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corresponds to those essential for a correct orientation of the
lateral chain in the binding site of tubulin.
Molecular modeling was undertaken on 20 and 21 to
determine whether they occupy the entire vinca domain in
comparison with vinblastine and phomopsin A. The structures
of 20 and 21 were assembled in a potential binding
conformation at the β2α1-tubulin interface, and molecular
dynamics (MD) simulations were carried out. All ligands were
located between the GDP-bound β-tubulin subunit of the
“bottom” heterodimer (β1) and the GTP-Mg2+-bound α-
tubulin subunit of the “top” heterodimer (α2).3 Complex
relaxation and enhanced sampling over the course of the MD
trajectories resulted in improved adaptation of each ligand
within the binding site and provided details of the
intermolecular interactions.
As postulated, the R configuration at C-8′ for the hybrids
20−22 enables a correct orientation of the lateral chain in the
binding site of tubulin. Consequently, as for that of phomopsin
A, both are oriented in the direction of Tyrβ224 and GDP. In
addition, for 21, the carbonyl amide of proline establishes a
hydrogen bonding interaction with the OH of Tyrβ224 (Figure
6b, red circle) that pushes isoleucine out of the β2α1-tubulin
interdimer.
The superimposition of 20, 1, and 6 in their respective
binding sites clearly indicates that the hybrid occupies the
whole vinca domain, as its lateral chain superimposes perfectly
to that of 6. The only difference resides in the conformation of
the amino acids chain that is much flatter in the case of
phomopsin A, because of the presence of dehydroamino acids
(Figure 7).
Figure 5. NOE correlations for 22.
Compounds 20−22 were evaluated for inhibition of tubulin
polymerization (Table 3). Compounds 20 and 21 displayed
Table 3. Biological Activities of Hybrids 20−22
In conclusion, three new 7′-homo-anhydrovinblastine and
phomopsin hybrids carrying peptide chains of different lengths
at position 8′ were synthesized starting from vinorelbine 4
using a selective and efficient one-pot procedure. Compound
20 was shown to inhibit microtubule assembly with an IC50
similar to those of 1 and 4 and to display good cytotoxicity
against four cancer cell lines. Molecular modeling studies
indicated that the lateral chain of 20 is lodged within the same
pocket that is occupied by natural phomopsin A 6, but the
latter is less flexible because of the presence of unsaturated
amino acids. These results pave the way to the elaboration of
more potent vinca−peptide hybrids by varying the nature and
the bulk of the peptide chain. This is why, thereafter, the
synthesis of hybrids with a more rigid lateral chain is envisaged
to determine the contribution of the rigidity of the peptidic
chain to the biological activity. Likewise, it seems worthwhile,
according to molecular modeling studies, to use hydrophilic
amino acids at the end of the lateral chain to improve the
tubulin binding affinity. Additionally, it is known that a
simplification of the vindoline part of the vinca alkaloids
leads to inactive compounds.7b,11 The elaboration of simplified
vinblastine−phomospin hybrids using this insertion reaction
could be considered as a proof of concept: if the added peptide
chains have a real influence on activity, a new family of
antimitotic compounds could be elaborated. Contrariwise, if
they have no impact, a simplification of the vinblastine moiety
should induce loss of activity.
a
IC50‑tubulin is the concentration of a compound that inhibits 50% of
the rate of microtubule assembly (concentration in tubulin, 3 mg/mL).
b
IC50‑cell line measures the drug concentration required for the
inhibition of 50% cell proliferation after 72 h of incubation.
significant inhibitory activity on microtubule assembly.
Compound 20 was even more potent than 1 and slightly less
potent than 4, and 21 was almost as active as 1. It clearly
indicates that the phomopsin side chain has a synergistic effect
on activity because we have shown previously that insertion of
inappropriate bulky groups at 8′ lead to inactive compounds.9
Nevertheless, the size of the added groups should not be too
large in view of the total loss of activity observed for 22. These
results confirm that the 8′ position can tolerate bulky groups
when a methylene linker is used and go along the same line as
the molecular modeling results.
EXPERIMENTAL SECTION
■
General Information. Vinorelbine tartrate was a gift from the
Even if it is less cytotoxic than vinorelbine 4 and vinblastine
1, 20 was found very active against the four tested cell lines
mainly on U87 glioblastoma, K562 erythromyeloblastoid
leukemia, and MCF-7 breast cancer cells.
Institut de Recherche Pierre Fabre. All reactions were performed in
oven-dried glassware under an argon atmosphere. H NMR and 13C
1
NMR spectra were recorded on a Bruker ARX500 or Avance 600
spectrometer. Tested 20−22 possess a purity of ≥95% that was
D
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Figure 7. PyMOL cartoons and stick superimposition of 20 (yellow
sticks), vinblastine 1 (blue sticks), and phomopsin A 6 (magenta
sticks) in their “active conformation” within their respective binding
sites.
H-19), 2.70 (m, 1H, H-8α), 2.69 (s, 3H, H-23), 2.56 (dd, J = 14.5 and
4.3 Hz, 1H, H-20′α), 2.48 (m, 1H, H-10α), 2.22−2.15 (m, 2H, H-11α
and H-11β), 2.18 (m, 1H, H-1′β), 2.08 (m, 1H, H-2′), 2.01 (s, 3H, H-
27), 1.93 (m, 2H, H-21′), 1.73 (dd, J = 14.0 and 7.3 Hz, 1H, H-20α),
1.28 (dd, J = 14.0 and 7.3 Hz, 1H, H-20β), 0.95 (t, J = 7.3 Hz, 3H, H-
22′), 0.54 (t, J = 7.3 Hz, 3H, H-21). 13C NMR (125 MHz, CD3CN): δ
(ppm) 192.0 (C-25′), 174.7 (C-23′), 173.1 (C-24), 171.5 (C-26),
162.0 (C-7′), 159.8 (C-16), 154.5 (C-18), 139.8 (C-4′), 136.0 (C-
16′), 132.7 (C-18′), 131.2 (C-6), 129.9 (C-11′), 125.5 (C-7), 125.4
(C-13), 124.7 (C-14), 124.4 (C-3′), 122.6 (C-14′), 120.5 (C-12′),
120.5 (C-15), 120.0 (C-13′), 115.3 (C-10′), 114.0 (C-8′), 111.9 (C-
15′), 95.5 (C-17), 84.5 (C-2), 80.6 (C-3), 77.5 (C-4), 67.5 (C-19),
56.6 (C-22), 55.5 (C-19′), 55.1 (C-5′), 54.2 (C-12), 52.9 (C-24′),
52.7 (C-25), 51.8 (C-10), 51.6 (C-8), 50.7 (C-20′), 45.3 (C-11), 43.8
(C-5), 38.9 (C-23), 35.9 (C-1′), 34.7 (C-2′), 32.0 (C-20), 28.1 (C-
21′), 21.3 (C-27), 20.7 (C-9′), 12.6 (C-22′), 8.7 (C-21). HRMS
(ESI): calcd for C48H55N4O9 831.3969, found 831.3990. [α]D −60
(CHCl3, c 1.0).
General Procedure for the Synthesis of Hybrids 20−22.
Method 1. To a solution of amine (0.04 mmol, 1.75 equiv) and
aldehyde 12 (20 mg, 0.02 mmol, 1 equiv) in 0.2 mL of
dichloromethane was added NaBH(OAc)3 (13 mg, 0.06 mmol, 2.6
equiv) at room temperature. The mixture was stirred for 3 h at 0 °C.
The resulting mixture was diluted with dichloromethane and washed
with a saturated sodium carbonate solution. The organic layers were
combined, washed with brine, dried over magnesium sulfate, and
filtered. The solvents were removed under reduced pressure to provide
the crude compound. Purification was carried out on silica gel using
ethyl acetate/acetone (1:0 to 4:1) to afford desired compound 20 (3
mg, 16% yield) as a white solid.
Figure 6. PyMOL cartoons and sticks representation of (a) hybrid 20
(yellow) and (b) hybrid 21 (yellow), Tyr β224 (blue), and GDP
(green and orange) bound at the β1α2 tubulin interdimer interface.
determined by UPLC using a C18 column (2.1 mm × 50 mm) and a
gradient of water/acetonitrile from 95:5 to 0:100.
Compound 12. 3-(Trimethylsilyl)propynal (10 μL, 0.07 mmol, 1.1
equiv) and dimethylamine 2 M (56 μL) were added to a solution of
vinorelbine (50 mg, 0.065 mmol, 1 equiv) in 0.5 mL of anhydrous
acetonitrile. After the mixture was stirred for 2 h at room temperature,
the solvent was evaporated under reduced pressure. The resulting
residue was purified by column chromatography on silica gel using
ethyl acetate/acetone (1:0 to 4:1) to afford 12 (20 mg, 36% yield). 1H
NMR (500 MHz, CD3CN): δ (ppm) 8.99 (s, 1H, H-25′), 8.22 (s, 1H,
H-17′), 7.89 (s, 1H, OH), 7.54 (d, J = 7.8 Hz, 1H, H-12′), 7.16 (d, J =
7.8 Hz, 1H, H-15′), 7.03 (s, 1H, H-14), 7.00 (s, 1H, H-7′), 6.97 (t, J =
7.8 Hz, 1H, H-14′), 6.89 (t, J = 7.8 Hz, 1H, H-13′), 6.31 (s, 1H, H-
17), 5.79 (dd, J = 10.4 and 3.9 Hz, 1H, H-7), 5.44 (d, J = 2.6 Hz, 1H,
H-3′), 5.29 (s, 1H, H-4), 5.23 (d, J = 10.4 Hz, 1H, H-6), 4.2 (d, J =
15.8 Hz, 1H, H-9′β), 3.72 (d, J = 15.8 Hz, 1H, H-5′α), 3.68 (s, 3H, H-
25), 3.64 (s, 1H, H-2), 3.64 (s, 3H, H-22), 3.59 (d, J = 15.8 Hz, 1H,
H-9′α), 3.51 (s, 3H, H-24′), 3.48 (m, 1H, H-20′β), 3.47(d, J = 15.8
Hz, 1H, H-5′β), 3.36 (dd, J = 15.8 and 4.9 Hz, 1H, H-8β), 3.30 (m,
1H, H-10β), 3.09 (dd, J = 14.8 and 11,9 Hz, 1H, H-1′α), 2.72 (s, 1H,
Method 2. 3-(Trimethylsilyl)propynal (20 μL, 0.14 mmol, 1.1
equiv) was added to a solution of vinorelbine (100 mg, 0.13 mmol, 1
equiv) in 1 mL of anhydrous acetonitrile. After the mixture was stirred
for 2 h at room temperature, amine (0.23 mmol, 1.75 equiv) and
NaBH(OAc)3 (70 mg, 0.33 mmol, 2.6 equiv) in 1 mL of
dichloromethane were added at room temperature. The mixture was
stirred for 3 h at room temperature. The resulting mixture was diluted
with dichloromethane and washed with a saturated sodium carbonate
solution. The organic layers were combined, washed with brine, dried
over magnesium sulfate, and filtered. The solvents were removed
under reduced pressure to provide the crude compound. Purification
was carried out by column chromatography on silica gel using ethyl
acetate/acetone (1:0 to 4:1), affording 20 (12 mg, 10% yield).
Compound 20. White solid, 16% yield (3 mg) by method 1, 10%
1
yield (12 mg) by method 2. H NMR (500 MHz, CDCl3) δ (ppm)
7.93 (s, 1H, H-17′), 7.70 (d, J = 7.3 Hz, 1H, H-12′), 7.11−7.05 (m,
2H, H-14′ and H-15′), 7.04 (t, J = 7.3 Hz, 1H, H-13′), 6.84 (s, 1H, H-
14), 6.11 (s, 1H, H-17), 5.83 (dd, J = 10.4 and 4.4 Hz, 1H, H-7), 5.50
E
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(s, 1H, H-4), 5.28 (d, J = 10.4 Hz, 1H, H-6), 5.16 (d, J = 4.2 Hz, 1H,
H-3′), 3.78 (s, 3H, H-22), 3.77 (s, 3H, H-25), 3.72 (s, 1H, H-2), 3.70
(d, J = 10.2 Hz, 1H, H-9′β), 3.67 (s, 3H, H-32′), 3.55−3.49 (m, 1H,
H-1′β), 3.51 (s, 3H, H-24′), 3.43−3.36 (m, 2H, H-8β and H-30′),
3.32−3.26 (m, 2H, H-5′β and H-10α), 3.16−3.13 (m, 1H, H-27′α),
3.04−2.98 (m, 1H, H-20′β), 2.96 (d, J = 13.6 Hz, 1H, H-5′α), 2.84−
2.80 (m, 2H, H-8α and H-25′β), 2.74−2.71 (m, 1H, H-9′α), 2.70 (s,
1H, H-19), 2.68 (s, 3H, H-23), 2.69−2.64 (m, 1H, H-25′α), 2.63 (d, J
= 9.9 Hz, H-7′β), 2.59 (d, J = 10.0 Hz, H-27′β), 2.44−2.38 (m, 2H, H-
7′α and H-10β), 2.36−2.32 (m, 1H, H-8′), 2.22−2.17 (m, 2H, H-11),
2.14 (d, J = 14.0 Hz, 1H, H-1′α), 2.09 (s, 3H, H-27), 2.12−2.07 (m,
1H, H-29′α), 2.02−1.94 (m, 3H, H-20′α, H-28′β and H-29′β), 1.91−
1.73 (m, 4H, H-20β, H-21′ and H-28′α), 1.41−1.31 (m, 1H, H-20α),
1.25−1.22 (m, 1H, H-2′), 0.94 (t, J = 7.0 Hz, 3H, H-22′), 0.83 (t, J =
7.0 Hz, 3H, H-21). 13C NMR (125 MHz, CDCl3) δ (ppm) 175.4 (C-
31′), 174.5 (C-23′), 171.9 (C-24), 171.1 (C-26), 158.5 (C-16), 153.2
(C-18), 140.2 (C-4′), 135.3 (C-16′), 130.6 (C-18′), 130.3 (C-6),
130.3 (C-11′), 125.2 (C-14), 124.7 (C-7), 123.6 (C-3′), 123.5 (C-13),
122.0 (C-14′), 121.4 (C-15), 120.1 (C-12′), 118.8 (C-13′), 117.3 (C-
10′), 110.6 (C-15′), 94.7 (C-17), 83.9 (C-2), 79.8 (C-3), 76.7 (C-4),
66.7 (C-30′), 66.4 (C-19), 61.0 (C-25′), 58.2 (C-5′), 56.5 (C-19′),
56.2 (C-22), 55.8 (C-7′), 54.3 (C-27′), 53.6 (C-12), 53.5 (C-20′),
52.5 (C-25), 52.4 (C-24′), 52.3 (C-32′), 51.1 (C-10), 50.7 (C-8), 45.0
(C-11), 43.1 (C-5), 41.1 (C-8′), 38.6 (C-23), 35.6 (C-2′), 34.6 (C-1′),
31.2 (C-20), 29.8 (C-29′), 27.8 (C-21′), 26.4 (C-9′), 24.0 (C-28′),
21.4 (C-27), 12.6 (C-22′), 8.8 (C-21). HRMS (ESI) calcd for
C54H70N5O10 948.5117, found 948.5223. [α]D +6 (CHCl3, c 0.1).
Compound 21. White solid, 13% yield (3 mg) by method 1, 11%
6.97 (t, J = 8.6 Hz, 1H, H-14′), 6.91 (t, J = 8.6 Hz, 1H, H-13′), 6.25 (s,
1H, H-17), 5.76 (dd, J = 10.3 and 4.6 Hz, 1H, H-7), 5.26 (d, J = 10.3
Hz, 1H, H-6), 5.23 (s, 1H, H-4), 5.09 (d, J = 4.7 Hz, 1H, H-3′), 5.00
(s, 2H, H-43′), 4.94 (d, J = 4.3 Hz, 2H, H-51′), 4.81 (q, J = 6.7 Hz,
1H, H-40′), 4.12 (dd, J = 8.3 and 6.1 Hz, 1H, H-33′), 3.69 (s, 3H, H-
22), 3.66 (s, 3H, H-25), 3.64 (d, J = 13.3 Hz, 1H, H-9′β), 3.52 (s, 1H,
H-2), 3.43 (d, J = 10.3 Hz, 1H, H-1′β), 3.41 (s, 3H, H-24′), 3.30 (t, J =
6.8 Hz, 1H, H-7′α), 3.23 (dd, J = 16.4 and 4.9 Hz, 1H, H-8β), 3.13
(td, J = 9.3 and 4.4 Hz, 1H, H-10β), 3.08 (dd, J = 9.5 and 3.2 Hz, 1H,
H-30′), 2.98 (d, J = 15.1 Hz, 1H, H-5′α), 2.91 (d, J = 11.6 Hz, 1H, H-
20′β), 2.89−2.84 (m, 1H, H-5′β), 2.83 (d, J = 12.0 Hz, 1H, H-25′β),
2.80 (dd, J = 17.7 and 5.5 Hz, 1H, H-41′β), 2.78 (s, 1H, H-19), 2.76−
2.74 (m, 1H, H-9′α), 2.72 (dd, J = 17.7 and 5.5 Hz, 1H, H-41′α),
2.66−2.64 (m, 1H, H-8α), 2.63 (s, 3H, H-23), 2.56 (dd, J = 12.0 and
6.7 Hz, 1H, H-25′α), 2.50−2.44 (m, 2H, H-7′β and H-27′β), 2.42−
2.36 (m, 2H, H-10α and H-27′α), 2.31−2.26 (m, 1H, H-8′), 2.13−
2.09 (m, 1H, H-29′β), 2.07 (d, J = 10.3 Hz, 1H, H-1′α), 2.05−2.00
(m, 1H, H-11β), 1.94 (s, 3H, H-27), 1.87−1.77 (m, 8H, H-11α, H-
20′β, H-21′, H-28′α, H-29′α, H-34′), 1.74−1.68 (m, 1H, H-28′β),
1.66−1.58 (m, 1H, H-20α), 1.42−1.35 (m, 1H, H-35′α), 1.34−1.28
(m, 1H, H-20β), 1.20−1.15 (m, 1H, H-2′), 1.05−0.98 (m, 1H, H-
35′β), 0.86 (t, J = 7.6 Hz, 3H, H-22′), 0.80 (d, J = 6.5 Hz, 3H, H-37′),
0.73 (t, J = 7.5 Hz, 3H, H-21), 0.72 (t, J = 7.5 Hz, 3H, H-36′). 13C
NMR (150 MHz, CD3CN) δ (ppm) 176.0 (C-38′), 175.7 (C-31′),
173.2 (C-23′), 172.0 (C-24), 171.9 (C-50′), 171.7 (C-42′), 171.5 (C-
26), 159.6 (C-16), 154.3 (C-18), 141.4 (C-4′), 137.3 (C-52′), 137.1
(C-44′), 136.6 (C-16′), 133.2 (C-18′), 131.6 (C-6), 131.3 (C-11′),
129.8 (C-46′, C-48′, C-54′ and C-56′), 129.5 (C-45′, C-49′, C-53′ and
C-55′), 129.4 (C-47′ and C-55′), 125.8 (C-14), 125.7 (C-7), 125.0
(C-13), 124.6 (C-3′), 122.9 (C-14′), 122.3 (C-15′ and C-15), 120.7
(C-12′), 119.8 (C-13′), 117.0 (C-10′), 95.4 (C-17), 84.6 (C-2), 80.9
(C-3), 77.7 (C-4), 69.7 (C-30′), 68.2 (C-43′), 67.7 (C-51′), 66.4 (C-
19), 64.2 (C-25′), 58.8 (C-5′), 58.4 (C-33′), 57.5 (C-22), 57.3 (C-
19′), 57.1 (C-27′), 56.3 (C-7′), 54.8 (C-20′), 54.5 (C-12), 52.9 (C-
25), 52.9 (C-24′), 51.5 (C-8), 51.1 (C-10), 49.9 (C-40′), 45.9 (C-11),
44.0 (C-5), 42.3 (C-8′), 39.2 (C-23), 38.7 (C-34′), 37.3 (C-41′), 36.8
(C-2′), 35.9 (C-1′), 32.2 (C-20), 31.6 (C-29′), 28.4 (C-21′), 27.3 (C-
9′), 25.9 (C-35′), 25.6 (C-28′), 21.6 (C-27), 16.3 (C-37′), 13.1 (C-
22′), 12.1 (C-36′), 9.2 (C-21). HRMS (ESI) calcd for C77H96N7O14
1342.7010, found 1342.7078; [α]D +12 (CHCl3, c 1.0.)
1
yield (3 mg) by method 2. H NMR (500 MHz, CD3CN) δ (ppm)
8.30 (s, 1H, H-17′), 7.78 (d, J = 8.6 Hz, 1H, H-32′), 7.59 (d, J = 6.8
Hz, 1H, H-12′), 7.18 (d, J = 7.6 Hz, 1H, H-15′), 7.10 (s, 1H, H-14),
7.04 (t, J = 7.6 Hz, 1H, H-14′), 6.97 (t, J = 7.6 Hz, 1H, H-13′), 6.29 (s,
1H, H-17), 5.83 (dd, J = 9.9 and 4.7 Hz, 1H, H-7), 5.32 (d, J = 9.9 Hz,
1H, H-6), 5.25 (s, 1H, H-4), 5.19 (d, J = 5.6 Hz, 1H, H-3′), 4.30 (dd, J
= 8.6 and 5.0 Hz, 1H, H-33′), 3.75 (s, 3H, H-39′), 3.69 (s, 3H, H-22),
3.68−3.64 (m, 1H, H-9′β), 3.58 (s, 3H, H-25), 3.56 (s, 1H, H-2), 3.49
(dd, J = 14.2 and 12.1 Hz, 1H, H-1′β), 3.44 (s, 3H, H-24′), 3.45−3.40
(m, 1H, H-7′α), 3.32 (dd, J = 15.8 and 5.3 Hz, 1H, H-8β), 3.20 (td, J
= 9.3 and 4.7 Hz, 1H, H-10β), 3.10 (dd, J = 10.0 and 2.9 Hz, 1H, H-
30′), 3.04 (d, J = 15.8 Hz, 1H, H-5′β), 2.98 (d, J = 12.2 Hz, 1H, H-
20′β), 2.94−2.89 (m, 2H, H-5′α and H-25′β), 2.88 (s, 1H, H-19),
2.89−2.85 (m, 1H, H-9′α), 2.76 (d, J = 15.9 Hz, 1H, H-8α), 2.67 (s,
3H, H-23), 2.58 (dd, J = 12.5 and 5.5 Hz, 1H, H-25′α), 2.51−2.45 (m,
4H, H-7′β, H-27′, H-10α), 2.43−2.38 (m, 1H, H-8′), 2.19−2.15 (m,
1H, H-29′α), 2.09 (d, J = 14.2 Hz, 1H, H-1′α), 2.08−2.02 (m, 1H, H-
11β), 1.98 (s, 3H, H-27), 1.96−1.92 (m, 1H, H-20′α), 1.93−1.88 (m,
3H, H-21′ and H-34′), 1.88−1.83 (m, 2H, H-11α and H-28′α), 1.83−
1.78 (m, 2H, H-28′β and H-29′β), 1.68−1.63 (m, 1H, H-20α), 1.43−
1.39 (m, 1H, H-35′α), 1.40−1.35 (m, 1H, H-20β), 1.21−1.17 (m, 1H,
H-2′), 1.13−1.09 (m, 1H, H-35′β), 0.94 (t, J = 7.4 Hz, 3H, H-22′),
0.86 (d, J = 6.3 Hz, 3H, H-37′), 0.81−0.78 (m, 6H, H-36′ and H-21).
13C NMR (125 MHz, CD3CN) δ (ppm) 175.9 (C-38′), 175.5 (C-
31′), 174.8 (C-23′), 173.0 (C-24), 171.8 (C-26), 159.4 (C-16), 154.8
(C-18), 141.4 (C-4′), 136.6 (C-16′), 133.2 (C-18′), 131.5 (C-6),
131.1 (C-11′), 125.7 (C-14), 125.7 (C-7), 124.8 (C-13), 124.4 (C-3′),
122.7 (C-14′), 122.3 (C-15), 120.5 (C-12′), 119.6 (C-13′), 116.6 (C-
10′), 112.1 (C-15′), 95.1 (C-17), 84.4 (C-2), 80.8 (C-3), 77.5 (C-4),
69.5 (C-30′), 66.0 (C-19), 64.2 (C-25′), 58.8 (C-5′), 57.5 (C-27′),
57.4 (C-19′), 57.0 (C-33′), 56.4 (C-22), 55.8 (C-7′), 55.3 (C-20′),
54.5 (C-12), 52.9 (C-25), 52.8 (C-24′), 52.7 (C-39′), 51.3 (C-8), 50.7
(C-10), 45.9 (C-11), 44.0 (C-5), 42.5 (C-8′), 39.1 (C-23), 38.2 (C-
34′), 36.9 (C-2′), 35.7 (C-1′), 32.0 (C-20), 31.5 (C-29′), 28.3 (C-
21′), 27.0 (C-9′), 26.0 (C-35′), 25.5 (C-28′), 21.5 (C-27), 16.4 (C-
37′), 12.9 (C-22′), 12.1 (C-36′), 9.0 (C-21). HRMS (ESI) calcd for
C60H81N6O11 1061.5958, found 1061.6012. [α]D +4 (CHCl3, c 0.1).
Compound 22. White solid, 26% yield (8 mg) by method 1, 27%
Modeling. Molecular modeling studies were performed using
crystal structures 1Z2B3a and the 3DU76 (PDB codes).
ASSOCIATED CONTENT
* Supporting Information
Procedures for the molecular dynamics simulation of 22,
■
S
docking experiments, inhibition of tubulin assembly measure-
1
ment, cell culture and proliferation assay, H and 13C NMR
spectra for 12, 20−22, NOE spectrum for 22, and full
minimized structures of both epimers of 22. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: + 0033 (0)1 69 82 31 21. Fax: + 0033 (0)1 69 07 72
47. E-mail: fanny.roussi@cnrs.fr.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Jacques Fahy (Pierre Fabre Laboratories) is gratefully acknowl-
edged for a generous gift of vinorelbine. O.G. gratefully
1
acknowledges the ED425 “Innovation Ther
́
apeutique, du
l Foundation,
yield (24 mg) by method 2. H NMR (600 MHz, CD3CN) δ (ppm)
Fondamental a for a grant and L’Orea
̀
l′Applique”
́
́
8.30 (s, 1H, H-17′), 7.78 (d, J = 8.6 Hz, 1H, H-32′), 7.59 (d, J = 6.8
Hz, 1H, H-12′), 7.30−7.23 (m, 10H, H-45′−H-49′ and H-53′−H-
57′), 7.13 (d, J = 8.6 Hz, 2H, H-15′ and H-39′), 7.02 (s, 1H, H-14),
UNESCO, and the French Academy of Science for her “Pour
les Femmes et la Science” prize.
F
dx.doi.org/10.1021/jm500530v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
ABBREVIATIONS USED
VLB, vinblastine; VLN, vinorelbine; TBAF, tributylammonium
fluoride; DMA, dimethylamine
■
REFERENCES
■
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