Synthesis and biological evaluation of a new series of highly functionalized 7′- homo -anhydrovinblastine derivatives

Article abstract of DOI:10.1021/jm4004347

Sixteen new 7′-homo-anhydrovinblastine derivatives were prepared in one or two steps from vinorelbine by means of an original and regiospecific rearrangement and subsequent diastereoselective reduction. This strategy has allowed fast access to a family of vinca alkaloid derivatives with an enlarged and functionalized ring C′. Their synthesis and biological evaluation are reported. One compound (compound 35) is 1.7 times more active than vinorelbine as a tubulin assembly inhibitor. Moreover, some of these compounds are highly cytotoxic, and two of them are more potent than vinorelbine on HCT116 and K562 cell lines. Molecular modeling studies, carried out with two of the new vinca derivatives, provide useful hints about how a given functionalization introduced at positions 7′ and 8′ of the C′ ring results in improved binding interactions between one of the new derivatives and the interdimer interface when compared to the parent compound vinblastine.

Full text of DOI:10.1021/jm4004347

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of a New Series of Highly
Functionalized 7′-homo-Anhydrovinblastine Derivatives
Olga Gherbovet,† Claire Coderch,‡ María Concepcion García Alvarez,† Jerome Bignon,† Sylviane Thoret,†
́
́
̂
Marie-Therese Martin,† Francoise Gueritte,† Federico Gago,‡ and Fanny Roussi*,†
̧
̀
́
†Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, UPR 2301 du CNRS, LabEx Lermit, Avenue de la
Terrasse, 91198 Gif-sur-Yvette Cedex, France
‡Departamento de Farmacología, Universidad de Alcala,
28871 Alcala de Henares, Madrid, Spain
́ ́
S
* Supporting Information
ABSTRACT: Sixteen new 7′-homo-anhydrovinblastine deriv-
atives were prepared in one or two steps from vinorelbine by
means of an original and regiospecific rearrangement and
subsequent diastereoselective reduction. This strategy has
allowed fast access to a family of vinca alkaloid derivatives
with an enlarged and functionalized ring C′. Their synthesis
and biological evaluation are reported. One compound
(compound 35) is 1.7 times more active than vinorelbine as
a tubulin assembly inhibitor. Moreover, some of these compounds are highly cytotoxic, and two of them are more potent than
vinorelbine on HCT116 and K562 cell lines. Molecular modeling studies, carried out with two of the new vinca derivatives,
provide useful hints about how a given functionalization introduced at positions 7′ and 8′ of the C′ ring results in improved
binding interactions between one of the new derivatives and the interdimer interface when compared to the parent compound
vinblastine.
Performing selective modifications on these highly complex
molecules represents a challenge for organic chemists. For
almost 40 years, many derivatives have been elaborated by semi
or total synthesis.10 Nevertheless, most of them were obtained
by modifications of the lower vindoline part, as changes in the
velbanamine moiety are much less trivial. In addition, it is
known that even a slight change in the upper indole part
impacts the biological activities (two out of the five vinca
alkaloids used in the clinic come from modifications of the
velbenamine moiety, either on ring C′ or on rings C′ and D′,
see 7 and 8).
Chemical modifications of the velbanamine part, which do
not cause loss or induce an increase of activity, have been
reviewed.3,11,12 For example, small hydrophobic groups can be
introduced on C-12′ of ring A′.13 Ring D′ can also undergo
important modifications,14 but ring-opening results in total loss
of pharmacological activities. Thus, very recently, Boger et al.
have synthesized original C-20′ urea and thiourea derivatives of
vinblastine, some of which are more cytotoxic than vinblastine
on selected cancer cell lines.15 The C′ ring is clearly essential
for obtaining antimitotic properties: ring-opening dramatically
suppresses any pharmacological activity, whereas ring con-
traction from 9 to 8 centers, as for vinorelbine 7 and vinflunine
8, leads to vinca alkaloids exhibiting markedly efficacious
antitumor properties. In some cases, however, ring C′ can
withstand large changes. Thus, in the course of our work on
INTRODUCTION
■
Microtubules are major structural components in cells. They
play a crucial role in a number of cellular functions, such as cell
division, as key constituents of the mitotic spindle. Micro-
tubules are dynamic polymers of α,β-tubulin heterodimers that
assemble in a head to tail arrangement.1 Each monomer binds a
GTP molecule2 which is nonhydrolysable and nonexchangeable
at the α subunit but is hydrolyzed to GDP at the β subunit
during the polymerization reaction that leads to microtubule
formation.
Tubulin-binding molecules interfere with microtubule
assembly and functions, thus resulting in mitotic arrest of
eukaryotic cells. Among them, Vinca alkaloids3 (Figure 1)
represent one of the most important classes of anticancer
agents. Vinca alkaloids are a family of indole−indoline dimeric
natural compounds that, from a biogenetic point of view,
originate from the oxidative coupling of vindoline 2 to
catharanthine 1.4 Vinblastine (VLB) 3 and vincristine 4,
isolated at the end of the 1950s,5 as well as synthetic vindesine
5,6 vinorelbine (VLN) 7,7 and more recently vinflunine 88 are
widely used in cancer chemotherapy.
They inhibit the assembly of tubulin into microtubules by
binding in the so-called vinca domain9 and, consequently,
prevent cell division. This binding site is at the longitudinal
interface between two α,β-tubulin heterodimers and vinca
alkaloids are oriented in such a way that their velbanamine and
vindoline moieties interact with both heterodimers, with
velbenamine being next to the GTP/GDP nucleotide exchange
site of a β-subunit.
Received: March 25, 2013
Published: July 3, 2013
© 2013 American Chemical Society
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Figure 1. Vinblastine 3 and natural and unnatural congeners.
Scheme 1. Envisaged Synthetic Pathway
vinca−phomopsin hybrids,16 we linked various peptide chains
to the tertiary amine of the velbenamine moiety of
anhydrovinblastine 6 and vinorelbine 7 and found that some
of the resulting derivatives were very potent inhibitors of
tubulin polymerization despite their large size. Molecular
modeling showed that they could indeed be accommodated
at the interface between α- and β-tubulin. Even more
surprisingly, Kuehne et al. have shown that homologation
from 9 to 10 carbons on C′ leading to an active compound if it
takes place on the “upper” side (7′a-homo-vinblastine 9) but to
an inactive one (18′a-homo-vinblastine 10) when this is
performed on the “lower” side.17,18
All of these results prompted us to design and synthesize a
new series of vinca derivatives with an enlarged and
functionalized ring C′. We postulated that functionalization of
7′-homo-anhydrovinblastine on C-7′ and/or C-8′ might lead to
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Table 1. Synthesis of a Series of β-Enamino Esters 22−29
c
entry
alkyne
R1
R2
compd
yield (%)
a
1
14
15
16
17
18
19
20
21
CO2Me
CO2Et
H
H
H
22
23
24
25
26
27
28
29
36
33
32
33
50
52
50
52
a
2
3
4
CO2Bn
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
b
5
CONHBn
CONHiPr
CONHAII
CONHtBu
b
6
b
7
b
8
a
b
c
Typical conditions: 1.1 equiv of alkyne, rt for 2 h. Typical conditions: 2 equiv of alkyne, rt for 2 h. Isolated yield.
derivatives with increased potency. In fact, the added groups
should be positioned, within the vinca domain, in the vicinity of
the exchangeable GTP binding site, as this may reinforce their
biological activity.19
with one electron withdrawing carboxylate group (entries 2−3)
generated the desired products in moderate yields. When the
acetylenes were disubstituted with carboxylates, insertions were
successful (entries 4−8). The best results were obtained with
unsymmetrical ester amide alkynes (entries 5−8). In these
cases, using 2 equiv of alkyne increased significantly the yield of
this insertion. Remarkably, the regioselectivity was complete
and provided, for compounds 26−29, a perfect control of the
groups thus inserted at positions 7′ and 8′.
RESULTS AND DISCUSSION
■
Chemistry. The “naked” 7′-homo-vinblastine compound 9,
found to be as active as vinblastine 3,17 was prepared by total
synthesis by Kuehne et al.. We reasoned that we could
elaborate functionalized 7′-homo-anhydrovinblastine compound
11 in one step from vinorelbine 7, by a direct enlargement
reaction, using its gramine bridge as a reactive template
(Scheme 1). Indeed, in indole chemistry, gramine is known to
be a good precursor of alkylideneindolenine that can be trapped
by various nucleophiles.20 A few years ago, a facile coupling of
gramine and activated acetylene was shown to generate
alkylideneindolenines via elimination of an enamine species.21
Indeed, we postulated that, in our case, activated acetylenes
would play a double role by generating an alkylideneindolenine
that could be trapped intramolecularly by the transient enamine
intermediate.22 This reaction may occur via a conjugate Michael
addition of the tertiary nitrogen of vinorelbine 7 to acetylenes,
generating a zwitterion 12. Spontaneous fragmentation of the
gramine bridge would lead to the reactive alkylideneindoleni-
nium ion 13. This ion could then be trapped intramolecularly
to give inserted compounds 11.
Afterward, the reduction of these various β-enamino esters
22−29 was considered. Two different reductive systems were
tested in anhydrous CH2Cl2 (Table 2). All of them were
24
reduced by using either NaBH4/ZnI2 or NaBH(OAc)3,25 the
latter providing the best yields. Moreover, this reduction was
highly diastereoselective and up to two asymmetric centers
were controlled, leading to enantiopure 8′R or 7′S,8′R β-amino
esters 30−37.
Table 2. Synthesis of β-Amino Esters 30−37
a
This insertion on vinorelbine 7 was studied using methyl
propiolate as the Michael acceptor (Table 1, entry 1). Various
conditions were screened in order to perform this reaction in a
selective and mild way by preventing any over reactions of the
β-enaminone thus formed.23 The nature of the solvent proved
to be crucial for this rearrangement, which was complete in
polar aprotic solvents like DMF or CH3CN in only 2 h at room
temperature. Under the conditions of 1.1 equiv of methyl
propiolate, the expected compound 22 was isolated in 36%
yield. The scope of this insertion was then investigated using
various reactive acetylenes (entries 2−8). Overall, the expected
compounds were obtained in yields that were deemed to be
satisfactory in light of framework complexity. It should be
noted, however, that loss of material was observed during the
purification by flash chromatography. First, activated acetylenes
yield (%)
b
c
entry compd
R1
R2
NaBH4/Znl2
NaBH(OAc)3
1
2
3
4
5
6
7
8
30
31
32
33
34
35
36
37
CO2Me
CO2Et
H
H
H
52
51
86
33
69
95
90
CO2Bn
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CONHBn
CONHiPr
CONHAll
CONHtBu
95
81
78
72
34
a
b
Isolated yield. Typical conditions: anhydrous CH2Cl2, 3 equiv of
c
Znl2 at 0 °C for 1 h, then 5 equiv of NaBH4 overnight at rt. Typical
conditions: anhydrous CH2Cl2, 3 equiv of NaBH(OAc)3 for 4 h at 0
°C.
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The (8′R,7′S) stereochemistry in compounds 30−37 was
were totally inactive. On the contrary, C-7′ tolerates bulky
groups as exemplified by compound 27 that was very active,
inhibiting tubulin polymerization. Moreover, most of the
reduced compounds were much more active than their parent
β-enamino esters, except for compounds 33 and 36, whose
activities were comparable to those of 25 and 28.
determined by NOE experiments (Figure 2). For example, for
The activities of compounds 22, 25−29, 30, and 33−37 were
evaluated on human HCT-116 colorectal carcinoma, K562
leukemia, U87-MG glioblastoma, and HUVEC endothelial cells
(Table 4). Their activities were, in many cases, in a nanomolar
range. Once again, the β-amino esters were 10−500 times more
potent than their β-enaminone analogues (see, for example,
compounds 33 and 25) except for compounds 26 and 34,
which showed similar cytotoxicity, and compounds 29 and 37,
because in this case the reduced compound was less active.
Notably, compounds 30 and 33 were more active than
vinorelbine 7 on HCT-116 and K562 cells, and compound 33
was as active as 7 on U87 (1.5 nM) and HUVEC (2.5 nM) cell
lines. It should be pointed out that there was no clear
correlation between cytotoxicity and tubulin inhibition
assembly as the more potent compounds in the tubulin
polymerization assay (i.e., 27, 35, and 37) were not the most
cytotoxic.
The effects of the most cytotoxic compounds 30 and 33 were
then investigated on cell cycle distribution of K562 and U87-
MG cells by flow cytometry at various concentrations (Figure
3). As these compounds were highly cytotoxic, albeit 3−10
times less potent than vinorelbine 7 for inhibition of tubulin
polymerization, we intended to ascertain that they lead to cell
ring arrest in G2/M, thus confirming their biological target.
After 24 h treatment of K562 cells with 50 nM of 30 or 10 nM
of 33, a net increase in the number of cells arrested at the G2/
M growth phase was observed. A similar cell cycle profile was
obtained with U87-MG cells after 24 h treatment with 50 nM
of 30 or 5 nM of 33, and increasing the concentration of 30 or
Figure 2. Selected NOE for compound 35.
compound 35, correlations could be observed, on the one
hand, between H-20′β and H-5′β, H-5′β and NH-28′, NH-28′
and H-9′β, H-9′β and H1′β, CH3-26′ and CH3-30′ and, on the
other hand, between H-20′α and H-14 and H-5′α and H-7′.
This selectivity could be explained by the addition of the
hydride on the lower face of velbenamine through the
formation of an imino-boron-enol ester complex, as postulated
by Palmieri et al.25
Overall, this two-step procedure enabled us to elaborate an
original series of 16 new vinca alkaloids in a very efficient way.
Biological Activity. Compounds 22−37 were evaluated for
inhibition of tubulin polymerization. The results are summar-
ized in Table 3. All tested compounds except 23, 24, 31, and 32
displayed significant inhibitory activity on microtubule
assembly. Compound 35 was even more potent than
vinorelbine (IC50‑35/IC50‑VLN = 0.6), and compounds 27 and
37 were almost as active (IC50‑27/IC50‑VLN = 2.0 and IC50‑37
/
IC50‑VLN = 1.7). Position 8′ did not appear to tolerate large
substituents because compounds 23 (R = Et) and 24 (R = Bn)
Table 3. Inhibition of Tubulin Polymerization by Compounds 22−37
a
a
entry
R1
R2
compd
22
microtubule assembly inhibitory activity IC50 (μM)
compd microtubule assembly inhibitory activity IC50 (μM)
1
2
Me
Et
H
H
H
18.0
>100
>100
6.0
30
31
32
33
34
35
36
37
2.5
>100
>100
8.6
23
3
Bn
24
4
Me
Me
Me
Me
Me
CO2Me
25
5
CONHBn
CONHiPr
CONHAll
CONHtBu
26
10.4
1.6
2.7
0.5
4.9
1.1
6
27
7
28
3.7
8
29
10.4
0.7
9
7 (VLN)
3 (VLB)
10
2.1
a
IC50 is the concentration of a compound that inhibits 50% of the rate of microtubule assembly (concentration in tubulin = 3 mg/mL).
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Table 4. Cytotoxicity of Compounds 22, 25−29, 30, and 33−37
a
IC50 (nM)
R2 CONHBn
R2 H
R2 CO2Me
R2 CONHiPr
R2 CONHAll
R2 CONHtBu
7 (VLN)
3 (VLB)
22
30
25
33
26
34
27
35
28
36
29
37
HCT116
K562
35
20
2
3.5
7
500
500
700
250
18
15
7
500
1000
700
6
250
80
200
75
2500
1500
350
100
60
1800
1000
3000
2000
45
50
10
25
60
50
25
25
300
250
80
5
U87
1
1.5
2.5
10
70
20
HUVEC
2
0.8
8
100
40
60
700
150
70
a
IC50 measures the drug concentration required for the inhibition of 50% cell proliferation after 72 h of incubation.
Figure 3. Effect of compounds 30, 33, and vinorelbine on cell-cycle distribution in chronic myelogenous leukemia K562 and glioblastoma U87-MG
cell lines determined by flow cytometry analysis. DNA content was assessed via propidium iodide staining.
33 led to essentially the same results in K562 and U87-MG
cells (data not shown).
formation of a direct hydrogen bond between the charged
amino group N-6′ in the velbenamine moiety and the backbone
carbonyl of Valβ1177 instead of the water-bridged interaction
that is established in the vinblastine complex. This improved
interaction, along with the repuckering of the C′ ring brought
about by the increase in the number of carbons and the
position of the substituents at 7′ and 8′, also favors the
formation of two hydrogen bonds involving the side chain of
Asnα2329, one between the carboxamide oxygen and the indolic
NH-17′ (on ring B′) and another between the carboxamide
nitrogen and the ester carbonyl C-23′ on ring C′. Additionally,
the carbonyl ester at 8′ establishes a hydrogen bonding
interaction with the backbone NH of Tyrβ1224. Besides, the
binding of 35 is further stabilized by a van der Waals interaction
between the iPr moiety in the CONHiPr group at 7′ and the
side chain of Leuα2248 in the T7 loop of α2-tubulin.
Structural Interpretation. To provide a structural ration-
ale to the observed differences in the inhibition of tubulin
polymerization between β-enaminones 22, 25−29, and their β-
amino esters equivalents 30 and 33−37, we model-built the
complexes of 35 and 27 with a β2α1-tubulin interface and
carried out molecular dynamics (MD) simulations. Both 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 Mutual
adaptation between the ligands and the side chains of the
amino acids making up the binding site during the MD
trajectories improved the intermolecular interactions and
provided distinct details for each complex. The van der Waals
and hydrogen bonding interactions observed for 35 were
overall similar to those already reported for vinblastine,26 but
the modification of ring C′ favors the formation of additional
hydrogen bonds that contribute to tightening the interaction
with both tubulin subunits (Figure 4). Thus, the hydrogen
bond established between the NH-28′ of the CONHiPr moiety
at position 7′ and the side chain of Aspβ1179 favors the
On the other hand, the unsaturation between positions 7′
and 8′ in 27 does not favor a good orientation of the newly
introduced moieties (Figure 5) and the two substituents facing
the α2 subunit force a readjustment of the T7 loop of α2-
tubulin. The iPr moiety of the CONHiPr group still establishes
van der Waals interactions with Leuα2248 and Ala247, but its
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CONCLUSION
■
A series of 16 novel vinca derivatives enlarged and function-
alized on ring C′ was designed and synthesized from
vinorelbine 7 by means of a new and efficient two-step
procedure. First, eight β-enamino esters 22−29 were
synthesized by an insertion reaction that occurs under mild
conditions, with good regioselectivity using several mono or
disubstituted, asymmetric or nonasymmetric activated acety-
lenes. Subsequently, β-amino esters 30 and 33−37 were
obtained by reduction of these β-enamino esters with
NaBH(OAc)3 with a total diastereoselectivity in good to
excellent yields. Their ability to inhibit tubulin polymerization
was evaluated, and one disubstituted compound, 35, is shown
to be more active than vinorelbine 7. Interestingly, we
demonstrate that β-amino esters 30 and 33−37 are globally
more active than β-enamino esters 22 and 25−29 due to the
fact that their greater flexibility allows a better fit in the vinca
domain. In addition, position C-7′ is shown to tolerate large
substituents in contrast with position C-8′ because the
substituents located in the latter position are oriented toward
the α2-tubulin binding pocket, whereas the C-7′ substituents
are oriented at the interdimer interface space created by the
wedge that this type of antitumor drugs introduce in tubulin.
Notably, two β-amino esters, 30 and 33, proved to be highly
cytotoxic, with 33 being much more active against HCT116
and K562 cell lines than vinorelbine 7. Moreover, 30 and 33
appear to elicit their cytotoxicities in a fashion similar to
vinorelbine via inhibition of tubulin polymerization, which then
leads to cell cycle arrest in G2/M.
Figure 4. PyMOL cartoon and stick representation of 35 (yellow
sticks) bound at the β1−α2 interdimer interface near the GDP
molecule (cyan, green, and magenta, respectively). Only polar
hydrogens are shown for clarity. The residues lining the binding site
are shown as sticks, and those establishing a hydrogen bond (dashed
line) are labeled in bold.
Taken together, the present results demonstrate that
introduction of functionalities on the “upper side” of velben-
amine can lead to potent compounds, provided these groups
are oriented at the β1−α2 interdimer interface near the GDP
molecule, as it is known that this space is also occupied by
other tubulin binding molecules like phomopsin A and
soblidotin.27
EXPERIMENTAL SECTION
■
General Information. Anhydrous solvents and starting materials
were obtained from commercial suppliers and used as received, except
for vinorelbine, which was a gift from Institut de Recherche Pierre
Fabre, and alkynes 18−21, which were synthesized using a published
procedure.28 All reactions were performed in oven-dried glassware
under an argon atmosphere. Flash column chromatographies were
performed using normal phase silica gel (60 Å, 40−63 μm). Chemical
Figure 5. PyMOL cartoon and stick representation of 27 (yellow
sticks) bound at the β1−α2 interdimer interface near the GDP
molecule (cyan, green, and magenta, respectively). Only polar
hydrogens are shown for clarity. The residues lining the binding site
are shown as sticks, and those establishing a hydrogen bond (dashed
line) are labeled in bold.
1
shifts (δ) for H NMR and 13C NMR spectra are reported in ppm
1
relative to acetonitrile or chloroform resonances. Data for H NMR
are reported as follows: chemical shift (δ in ppm), multiplicity (s =
singlet, br s = broad singlet, d = doublet, t = triplet, q = quadruplet, m
= multiplet), coupling constant (Hz), integration. Data for 13C NMR
are reported in terms of chemical shift (δ in ppm). [α]D are expressed
in deg cm3 g−1 dm−1 for a concentration of compound in mg cm−3.
High-resolution mass spectra (HMRS) were recorded by electrospray
ionization (ESI). All the tested compounds 22−37 possess a purity
≥95% that was determined by UPLC using a C18 column (2.1 mm ×
50 mm) and a gradient of water/acetonitrile from 95:5 to 0:100.
General Procedure A for the Synthesis of Alkynes. A solution
of methyl propiolate (0.45 mL, 5 mmol) in anhydrous THF (25 mL)
was cooled to −78 °C, under argon, before a solution of n-BuLi (1.6 M
in hexanes, 3.3 mL, 5.25 mmol) was added dropwise. The mixture was
stirred at that temperature for 30 min before the isocyanate (5 mmol)
was added dropwise. The resulting mixture was stirred at −78 °C for
30 min. A saturated aqueous solution of ammonium chloride (20 mL)
was added, and the mixture was allowed to warm to room temperature.
The two phases were separated, and the aqueous layer was extracted
orientation prevents formation of a hydrogen bond with either
the carboxamide of Aspβ1179 or the backbone of Tyrβ1224.
This arrangement therefore results in a different orientation of
the vindoline group within the binding site that makes the
interaction between the charged amino group in the velben-
amine domain and the backbone carbonyl of Valβ1177 possible
only through a bridging water molecule. The only hydrogen
bonds that are maintained are those established with Asnα2329,
and these are favored by the puckering of the C′ ring.
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with ethyl acetate. The combined organic extracts were washed with a
saturated solution of sodium hydrogenocarbonate (20 mL), dried over
magnesium sulfate, filtered, and concentrated under reduced pressure
to give a brown oil that was purified by column chromatography on
silica gel using heptane/ethyl acetate 4:1.
Methyl 4-(Allylamino)-4-oxobut-2-ynoate 20. Following general
procedure A, methyl 4-(allylamino)-4-oxobut-2-ynoate was obtained as
a yellow oil (261 mg, 31%). 1H NMR (CDCl3, 500 MHz) δ 6.58 (br s,
1H), 5.81−5.73 (ddd, J = 17.0 and 10.2 and 5.4 Hz, 1H), 5.19 (d, J =
17.0 Hz, 1H), 5.14 (d, J = 10.2 Hz, 1H), 3.89 (t, J = 5.4 Hz, 1H), 3.78
(s, 3H). 13C NMR (CDCl3, 75 MHz) δ 152.9, 150.8, 132.6, 117.7,
77.6, 73.8, 53.5, 42.5.
6.29 (s, 1H, H-17), 5.78 (dd, J = 10.2 and 4.8 Hz, 1H, H-7), 5.37 (s,
1H, H-3′), 5.28 (s, 1H, H-4), 5.22 (d, J = 10.2 Hz, 1H, H-6), 4.20 (q, J
= 6.9 Hz, 2H, H-26′), 4.11 (d, J = 15.5 Hz, 1H, H-9′α), 3.90 (d, J =
15.5 Hz, 1H, H-9′β), 3.72−3.70 (m, 1H, H-20′β), 3.68 (s, 3H, H-25),
3.66 (s, 3H, H-22), 3.62 (s, 1H, H-2), 3.58 (d, J = 16.3 Hz, 1H, H-
5′α), 3.52 (s, 3H, H-24′), 3.37 (d, J = 16.2 Hz, 1H, H-1′β), 3.34 (d, J =
16.3 Hz, 1H, H-5′β), 3.33 (d, J = 15.5, 1H, H-8α), 3.26 (td, J = 9.9 and
4.6 Hz, 1H, H-11a), 2.72 (d, J = 15.5 Hz, 1H, H-8β), 2.71 (s, 1H, H-
19), 2.68 (s, 3H, H-23), 2.52 (d, J = 13.1 Hz, 1H, H-20′α), 2.46 (td, J
= 19.9 and 6.9 Hz, 1H, H-11β), 2.18−2.08 (m, 1H, H-10β and H-
10α), 2.07 (d, J = 16.2 Hz, 1H, H-1′α), 1.94 (s,3H, H-27), 1.94 (s, 1H,
H-2′), 1.92−1.89 (m, 2H, H-21′), 1.63 (td, J = 14.4 and 7.0 Hz, 1H,
H-20β), 1.30−1.24 (m, 1H, H-20α), 1.27 (t, J = 6.9 Hz, 1H, H-27′),
0.94 (t, J = 7.4 Hz, 3H, H-22′), 0.56 (t, J = 7.0 Hz, 3H, H-21). 13C
NMR (CD3CN, 500 MHz) δ 175.3 (C-23′), 173.4 (C-24), 171.9 (C-
26), 171.5 (C-25′), 159.9 (C-16), 154.8 (C-18), 154.6 (C-7′), 140.2
(C-4′), 136.5 (C-16′), 132.6 (C-18′), 131.6 (C-6), 130.3 (C-11′),
125.8 (C-7), 125.7 (C-13), 125.3 (C-14), 125.0 (C-3′), 122.8 (C-14′),
121.8 (C-15), 121.3 (C-12′), 120.0 (C-13′), 118.7 (C-10′), 112.2 (C-
15′), 105.3 (C-8′), 95.8 (C-17), 84.8 (C-2), 80.9 (C-3), 77.8 (C-4),
67.5 (C-19), 60.9 (C-26′), 57.0 (C-22), 55.7 (C-12), 54.5 (C-19′),
54.5 (C-5′), 53.2 (C-24′), 53.0 (C-25), 52.5 (C-20′), 52.0 (C-10),
51.8 (C-8), 45.7 (C-11), 44.2 (C-5), 39.2 (C-23), 35.6 (C-2′), 35.6
(C-1′), 32.3 (C-20), 28.5 (C-21′), 24.5 (C-9′), 21.6 (C-27), 15.4 (C-
27′), 13.0 (C-22′), 9.0 (C-21). HRMS-ESI calcd for C50H61N4O10
877.4387, found 877.4388.
Methyl 4-(tert-Butylamino)-4-oxobut-2-ynoate 21. Following
general procedure A, methyl 4-(tert-butylamino)-4-oxobut-2-ynoate
1
was obtained as a yellow oil (302 mg, 33%). H NMR (CDCl3, 500
MHz) δ 3.81 (s, 3H), 1.37(s, 9H). 13C NMR (CDCl3, 75 MHz) δ
153.1, 149.9, 78.7, 71.9, 53.4, 53.3, 28.6.
General Procedure B for the Synthesis of the Enamino-
Esters 22−29. Method 1. Alkyne (0.071 mmol, 1.1 equiv) was added
to a solution of vinorelbine (50 mg, 0.064 mmol, 1 equiv) in CH3CN
(0.5 mL). The reaction 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
with ethyl acetate/acetone (1:0 to 4:1). The second fraction provided
the corresponding vinca derivatives 22, 23, or 24.
Method 2. Alkyne (0.128 mmol, 2 equiv) was added to a solution of
vinorelbine (50 mg, 0.064 mmol, 1 equiv) in CH3CN (0.5 mL). The
reaction 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 alumina with heptane/ethyl
acetate (4:1 to 0:1). The second fraction provided the corresponding
vinca derivatives 25−29.
Compound 24. Following the general procedure B (method 1) and
using benzyl propiolate 16, compound 24 was obtained as a white
25
powder by column chromatography on silica gel (19 mg, 32%); [α]D
1
−66 (c 1.00, CHCl3). H NMR (CD3CN, 500 MHz) δ 8.90 (s, 1H,
OH), 8.15 (s, 1H, H-17′), 7.60 (s, 1H, H-7′), 7.58 (d, J = 7.3 Hz, 1H,
H-12′), 7.41 (d, J = 7.3 Hz, 2H, H-28′), 7.34 (t, J = 7.3 Hz, 2H, H-
29′), 7.29 (dd, J = 14.6 and 7.3 Hz, 1H, H-30′), 7.12 (d, J = 7.3 Hz,
1H, H-15′), 6.98 (s, 1H, H-14), 6.96 (t, J = 7.3 Hz, 1H, H-14′), 6.82
(t, J = 7.3 Hz, 1H, H-13′), 6.29 (s, 1H, H-17), 5.79 (dd, J = 10.5 and
5.4 Hz, 1H, H-7), 5.35 (s, 1H, H-3′), 5.28 (s, 1H, H-4), 5.26 (d, J =
13.0 Hz, 1H, H-26′), 5.22 (d, J = 10.5 Hz, 1H, H-6), 5.19 (d, J = 13.0
Hz, 1H, H-26′), 4.13 (d, J = 15.4 Hz, 1H, H-9′α), 3.92 (d, J = 15.4 Hz,
1H, H-9′β), 3.74 (d, J = 13.0 Hz, 1H, H-20′ β), 3.68 (s, 3H, H-25),
3.65 (s, 3H, H-22), 3.61 (s, 1H, H-2), 3.59 (d, J = 4.0 Hz, 1H, H-5′α),
3.50 (s, 3H, H-24′), 3.35−3.33 (m, 2H, H-5′β and H-1′β), 3.31 (d, J =
5.4 Hz, 1H, H-8α), 3.25 (td, J = 9.3 and 4.8 Hz, 1H, H-10β), 2.72−
2.70 (m, 1H, H-8β), 2.70 (s, 1H, H-19), 2.67 (s, 3H, H-23), 2.54 (dd,
J = 13.0 and 4.3 Hz, 1H, H-20′α), 2.45−2.43 (m, 1H, H-10α), 2.16−
2.13 (m, 1H, H-11β), 2.11−2.09 (m, 1H, H-1′b), 2.09−2.07 (m, 1H,
H-11α), 1.94 (s, 3H, H-27), 1.93−1.91 (m, 2H, H-21′), 1.90 (s, 1H,
H-2′), 1.63 (dd, J = 14.1 and 7.4 Hz, 1H, H-20β), 1.28 (dd, J = 14.1
and 7.4 Hz, 1H, H-20α), 0.93 (t, J = 7.4 Hz, 3H, H-22′), 0.56 (t, J =
7.4 Hz, 3H, H-21). 13C NMR (CD3CN, 125 MHz) δ 174.9 (C-23′),
173.1 (C-24), 171.5 (C-26), 170.9 (C-25′), 159.6 (C-16), 154.7 (C-
7′), 154.4 (C-18), 139.8 (C-4′), 138.9 (C-27′), 136.2 (C-16′), 132.2
(C-18′), 131.2 (C-6), 129.9 (C-11′), 129.5 (C-29′), 129.0 (C-28′),
128.8 (C-30′), 125.5 (C-7), 125.4 (C-13), 125.0 (C-14), 124.6 (C-3′),
122.5 (C-14′), 121.4 (C-15), 121.0 (C-12′), 119.7 (C-13′), 116.6 (C-
10′), 111.9 (C-15′), 104.0 (C-8′), 95.4 (C-17), 84.5 (C-2), 80.6 (C-3),
77.5 (C-4), 67.1 (C-19), 66.3 (C-26′), 56.7 (C-22), 56.0 (C-19′), 54.3
(C-5′), 54.2 (C-12), 52.9 (C-24′), 52.7 (C-25), 52.1 (C-20′), 51.6 (C-
10), 51.5 (C-8), 45.4 (C-11), 43.8 (C-5), 38.9 (C-23), 35.2 (C-1′),
35.2 (C-2′), 32.0 (C-20), 28.2 (C-21′), 24.3 (C-9′), 21.3 (C-27), 12.7
(C-22′), 8.7 (C-21). HRMS-ESI calcd for C55H63N4O10 939.4544,
found 939.4561.
Compound 22. Following the general procedure B (method 1)
with methyl propiolate 14, compound 22 was obtained as a white
25
powder (20 mg, 36%); [α]D −55 (c 1.00, CHCl3). 1H NMR
(CD3CN, 300 MHz) δ 8.89 (s, 1H, OH), 8.15 (s, 1H, H-17′), 7.60 (d,
J = 7.9 Hz, 1H, H-12′), 7.52 (s, 1H, H-7′), 7.13 (d, J = 7.9 Hz, 1H, H-
15′), 6.99 (s, 1H, H-14), 6.98 (t, J = 7.9 Hz, 1H, H-14′), 6.89 (t, J =
7.9 Hz, 1H, H-13′), 6.29 (s, 1H, H-17), 5.76 (dd, J = 10.7 and 5.0 Hz,
1H, H-7), 5.38−5.36 (m, 1H, H-3′), 5.28 (s, 1H, H-4), 5.22 (d, J =
10.7 Hz, 1H, H-6), 4.11 (d, J = 15.1 Hz, 1H, H-9′α), 3.90 (d, J = 15.1
Hz, 1H, H-9′β), 3.72 (s, 3H, H-26′), 3.69 (s, 3H, H-25), 3.66 (s, 3H,
H-22), 3.62 (s, 1H, H-2), 3.58 (d, J = 16.1 Hz, 1H, H-5′α), 3.52 (s,
3H, H-24′), 3.35 (d, J = 16.1 Hz, 1H, H-5′β), 3.34 (d, J = 16.1 Hz, 1H,
H-8α), 3.32 (dd, J = 12.0 and 5.7 Hz, 1H, H-20′β), 3.27 (td, J = 9.5
and 6.0 Hz, 1H, H-10β), 2.74−2.70 (m, 1H, H-8b), 2.71 (s, 1H, H-
19), 2.68 (s, 3H, H-23), 2.52 (dd, J = 12.0 and 3.5 Hz, 1H, H-20′α),
2.46 (td, J = 9.5 and 5.0 Hz, 1H, H-10α), 2.16 (td, J = 13.8 and 6.0 Hz,
1H, H-11β), 2.12−2.10 (m, 2H, H-1′), 2.10 (s, 3H, H-27), 2.09−2.06
(m, 1H, H-11α), 1.94−1.93 (m, 1H, H-2′), 1.90 (dd, J = 15.2 and 7.6
Hz, 2H, H-21′), 1.63 (dd, J = 14.7 and 7.0 Hz, 1H, H-20β), 1.28 (dd, J
= 14.7 and 7.0 Hz, 1H, H-20α), 0.95 (t, J = 7.6 Hz, 3H, H-22′), 0.56
(t, J = 7.0 Hz, 3H, H-21). 13C NMR (CD3CN, 75 MHz) δ 175.0 (C-
23′), 173.1 (C-24), 171.9 (C-26), 171.5 (C-25′), 159.6 (C-16), 154.4
(C-18), 154.4 (C-7′), 139.8 (C-4′), 136.2 (C-16′), 132.2 (C-18′),
131.2 (C-6), 130.0 (C-11′), 125.5 (C-7), 125.4 (C-13), 125.0 (C-14),
124.7 (C-3′), 122.5 (C-14′), 121.4 (C-15), 121.0 (C-12′), 119.8 (C-
13′), 116.5 (C-10′), 111.9 (C-15′), 104.0 (C-8′), 95.4 (C-17), 84.5
(C-2), 80.5 (C-3), 77.4 (C-4), 67.1 (C-19), 56.6 (C-19′), 54.3 (C-12),
54.2 (C-5′), 52.9 (C-25), 52.7 (C-22), 52.1 (C-10), 51.8 (C-20′), 51.6
(C-26′), 51.5 (C-8), 45.3 (C-11), 43.8 (C-5), 38.9 (C-23), 35.2 (C-
2′), 35.2 (C-1′), 32.0 (C-20), 28.2 (C-21′), 24.2 (C-9′), 21.0 (C-27),
12.7 (C-22′), 8.7 (C-21). HRMS-ESI calcd for C49H59N4O10 863.4231,
found 863.4257.
Compound 25. Following the general procedure B (method 2) and
using dimethyl acetylenedicarboxylate 17, compound 25 was obtained
as a white powder by column chromatography on silica gel (21.9 mg,
25
1
Compound 23. Following the general procedure B (method 1) and
37%); [α]D −23 (c 1.00, CHCl3). H NMR (CD3CN, 500 MHz) δ
8.41 (s, 1H, H-17′), 7.34 (d, J = 7.9 Hz, 1H, H-12′), 7.14 (d, J = 7.9
Hz, 1H, H-15′), 6.99 (t, J = 7.9 Hz, 1H, H-14′), 6.93 (s, 1H, H-14),
6.90 (t, J = 7.9 Hz, 1H, H-13′), 6.27 (s, 1H, H-17), 5.78 (dd, J = 10.1
and 5.4 Hz, 1H, H-7), 5.27 (d, J = 10.1 Hz, 1H, H-6), 5.25 (s, 1H, H-
3′), 5.23 (s, 1H, H-4), 4.45 (d, J = 15.0 Hz, 1H, H-9′α), 4.00 (d, J =
using ethyl propiolate 15, compound 23 was obtained as a white
1
powder (18.4 mg, 33%); [α]25 −66.0 (c 1.00, CHCl3). H NMR
D
(CD3CN, 500 MHz) δ 8.16 (s, 1H, H-17′), 7.63 (d, J = 7.7 Hz, 1H, H-
12′), 7.52 (s, 1H, H-7′), 7.13 (d, J = 7.7 Hz, 1H, H-15′), 6.98 (s, 1H,
H-14), 6.97 (t, J = 7.7 Hz, 1H, H-14′), 6.89 (t, J = 7.7 Hz, 1H, H-13′),
6094
dx.doi.org/10.1021/jm4004347 | J. Med. Chem. 2013, 56, 6088−6100

Journal of Medicinal Chemistry
Article
15.0 Hz, 1H, H-9′β), 3.72 (s, 3H, H-22), 3.71 (s, 3H, H-26′), 3.71−
3.67 (m, 1H, H-5′α), 3.69 (s, 3H, H-28′), 3.67 (s, 3H, H-25), 3.58 (s,
1H, H-2), 3.49 (s, 3H, H-24′), 3.48−3.44 (m, 1H, H-1′β), 3.34 (d, J =
15.0 Hz, 1H, H-20′β), 3.29 (dd, J = 16.0 and 5.4 Hz, 1H, H-8α),
3.24−3.17 (m, 1H, H-10β), 2.95 (d, J = 15.0 Hz, 1H, H-20′α), 2.80 (s,
1H, H-19), 2.73 (d, J = 16.0 Hz, 1H, H-8β), 2.70−2.67 (m, 1H, H-
5′β), 2.67 (s, 3H, H-23), 2.53−2.50 (m, 1H, H-10α), 2.15−2.11 (m,
1H, H-1′α), 2.10−2.04 (m, 1H, H-11β), 1.94 (s, 3H, H-27), 1.92−
1.87 (m, 1H, H-11α), 1.91−1.86 (m, 2H, H-21′), 1.61 (q, J = 7.2 Hz,
1H, H-20β), 1.61−1.57 (m, 1H, H-2′), 1.33 (q, J = 7.2 Hz, 1H, H-
20α), 0.92 (t, J = 7.5 Hz, 3H, H-22′), 0.63 (t, J = 7.2 Hz, 3H, H-21).
13C NMR (CD3CN, 125 MHz) δ 175.4 (C-23′), 173.3 (C-24), 172.0
(C-25′), 171.9 (C-26), 167.0 (C-27′), 159.7 (C-16), 154.7 (C-18),
152.5 (C-7′), 139.3 (C-4′), 136.7 (C-16′), 133.0 (C-18′), 131.7 (C-6),
130.4 (C-11′), 126.0 (C-14), 125.8 (C-7), 125.3 (C-13), 124.3 (C-3′),
123.2 (C-14′), 122.1 (C-15), 120.7 (C-12′), 120.3 (C-13′), 119.9 (C-
8′), 114.9 (C-10′), 112.5 (C-15′), 95.8 (C-17), 84.6 (C-2), 81.1 (C-3),
77.8 (C-4), 66.5 (C-19), 57.3 (C-19′), 57.1 (C-22), 55.2 (C-5′), 54.7
(C-12), 53.2 (C-26′), 53.1 (C-25), 53.0 (C-28′), 51.6 (C-8), 51.5 (C-
24′), 51.2 (C-10), 51.1 (C-20′), 46.1 (C-11), 44.1 (C-5), 39.1 (C-23),
36.0 (C-2′), 35.1 (C-1′), 32.3 (C-20), 28.4 (C-21′), 26.4 (C-9′), 21.6
(C-27), 13.0 (C-22′), 9.1 (C-21). HRMS-ESI calcd for C51H61N4O12
921.4286, found 921.4285.
H-1′β), 3.40 (d, J = 15.5 Hz, 1H, H-5′α), 3.29 (dd, J = 12.3 and 4.5
Hz, 1H, H-8α), 3.20 (td, J = 10.5 and 4.9 Hz, 1H, H-10β), 3.05 (d, J =
15.5 Hz, 1H, H-5′β), 2.76 (s, 1H, H-19), 2.72 (d, J = 13.6 Hz, 1H, H-
20′α), 2.71 (dd, J = 12.3 Hz, 1H, H-8β), 2.67 (s, 3H, H-23), 2.45 (td, J
= 10.5 and 6.0 Hz, 1H, H-10α), 2.12 (d, J = 15.3 Hz, 1H, H-1′α),
2.09−2.06 (m, 1H, H-11β), 1.94 (s, 3H, H-27), 1.92−1.88 (m, 1H, H-
11α), 1.91−1.87 (m, 2H, H-21′), 1.65−1.57 (m, 1H, H-20β), 1.58−
1.56 (m, 1H, H-2′), 1.37−1.30 (m, 1H, H-20α), 1.08 (t, J = 7.1 Hz,
6H, H-30′), 0.92 (t, J = 7.5 Hz, 3H, H-22′), 0.63 (t, J = 7.3 Hz, 3H, H-
21). 13C NMR (CD3CN, 125 MHz) δ 175.5 (C-23′), 173.3 (C-24),
172.6 (C-25′), 171.9 (C-26), 165.9 (C-27′), 159.6 (C-16), 157.4 (C-
7′), 154.7 (C-18), 139.6 (C-4′), 136.8 (C-16′), 132.7 (C-18′), 131.7
(C-6), 130.4 (C-11′), 126.0 (C-14), 125.9 (C-7), 125.3 (C-13), 124.3
(C-3′), 123.1 (C-14′), 122.2 (C-15), 121.0 (C-12′), 120.2 (C-13′),
116.1 (C-10′), 115.1 (C-8′), 112.4 (C-15′), 95.4 (C-17), 84.6 (C-2),
81.1 (C-3), 77.8 (C-4), 66.6 (C-19), 57.2 (C-22), 56.8 (C-19′), 55.5
(C-20′), 54.6 (C-12), 53.1 (C-24′), 53.0 (C-25), 52.8 (C-26′), 51.6
(C-8), 51.3 (C-10), 50.9 (C-5′), 46.0 (C-11), 44.2 (C-5), 42.7 (C-
29′), 39.1 (C-23), 35.9 (C-2′), 35.1 (C-1′), 32.3 (C-20), 28.6 (C-21′),
26.7 (C-9′), 22.9 (C-30′), 21.7 (C-27), 13.0 (C-22′), 9.1 (C-21).
HRMS-ESI calcd for C53H65N5O11 948.4759, found 948.4767.
Compound 28. Following the general procedure B (method 2)
using alkyne 20, compound 28 was obtained as a yellow powder (30.2
25
mg, 50%); [α]D −32 (c 1.00, CHCl3). 1H NMR (CD3CN, 500
Compound 26. Following the general procedure B (method 2)
MHz) δ 8.88 (s, 1H, OH), 8.39 (s, 1H, H-17′), 7.39 (d, J = 7.8 Hz,
1H, H-12′), 7.13 (d, J = 7.8 Hz, 1H, H-15′), 6.99 (t, J = 7.8 Hz, 1H, H-
14′), 6.90 (t, J = 7.8 Hz, 1H, H-13′), 6.89 (s, 1H, H-14), 6.62 (t, J =
6.0 Hz, 1H, H-28′), 6.28 (s, 1H, H-17), 5.86−5.80 (m, 1H, H-30′),
5.77 (dd, J = 10.3 and 5.0 Hz, 1H, H-7), 5.26 (d, J = 10.3 Hz, 1H, H-
6), 5.25−5.24 (m, 1H, H-3′), 5.23 (s, 1H, H-4), 5.17 (d, J = 17.3 Hz,
1H, H-31′), 5.07 (d, J = 10.2 Hz, 1H, H-31′), 4.36 (d, J = 14.6 Hz, 1H,
H-9′β), 4.03 (d, J = 14.6 Hz, 1H, H-9′α), 3.83 (dt, J = 15.5 and 6.0 Hz,
1H, H-29′), 3.74 (dt, J = 15.5 and 6.0 Hz, 1H, H-29′), 3.79 (d, J = 11.7
Hz, 1H, H-20′β), 3.73 (s, 3H, H-22), 3.68 (s, 3H, H-26′), 3.67 (s, 3H,
H-25), 3.57 (s, 1H, H-2), 3.50 (s, 3H, H-24′), 3.46 (d, J = 14.8 Hz,
1H, H-1′β), 3.39 (d, J = 15.5 Hz, 1H, H-5′α), 3.28 (dd, J = 16.1 and
5.0 Hz, 1H, H-8α), 3.20 (td, J = 10.0 and 5.0 Hz, 1H, H-10β), 3.04 (d,
J = 15.5 Hz, 1H, H-5′β), 2.76 (s, 1H, H-19), 2.73 (d, J = 11.7 Hz, 1H,
H-20′α), 2.70 (d, J = 16.1 Hz, 1H, H-8β), 2.67 (s, 3H, H-23), 2.44 (td,
J = 10.0 and 6.3 Hz, 1H, H-10α), 2.12 (d, J = 14.8 Hz, 1H, H-1′α),
2.08−2.04 (m, 1H, H-11β), 1.94 (s, 3H, H-27), 1.93−1.90 (m, 1H, H-
11α), 1.89 (q, J = 7.5 Hz, 2H, H-21′), 1.61 (td, J = 14.2 and 7.2 Hz,
1H, H-20β), 1.58−1.56 (m, 1H, H-2′), 1.33 (td, J = 14.2 and 7.2 Hz,
1H, H-20α), 0.92 (t, J = 7.5 Hz, 3H, H-22′), 0.63 (t, J = 7.2 Hz, 3H,
H-21). 13C NMR (CD3CN, 125 MHz) δ 175.5 (C-23′), 173.3 (C-24),
172.6 (C-25′), 171.9 (C-26), 166.7 (C-27′), 159.6 (C-16), 157.0 (C-
7′), 154.7 (C-18), 139.5 (C-4′), 136.8 (C-16′), 136.1 (C-30′), 132.7
(C-18′), 131.7 (C-6), 130.4 (C-11′), 126.0 (C-14), 125.9 (C-7), 125.3
(C-13), 124.4 (C-3′), 123.1 (C-14′), 122.2 (C-15), 121.0 (C-12′),
120.2 (C-13′), 117.0 (C-31′), 115.9 (C-8′), 115.8 (C-10′), 112.4 (C-
15′), 95.4 (C-17), 84.6 (C-2), 81.0 (C-3), 77.8 (C-4), 66.6 (C-19),
57.2 (C-22), 56.7 (C-19′), 55.4 (C-20′), 54.7 (C-12), 53.1 (C-24′),
53.0 (C-25), 52.9 (C-26′), 51.6 (C-8), 51.3 (C-10), 51.0 (C-5′), 46.0
(C-11), 44.1 (C-5), 42.8 (C-29′), 39.1 (C-23), 35.9 (C-2′), 35.1 (C-
1′), 32.3 (C-20), 28.5 (C-21′), 26.7 (C-9′), 21.7 (C-27), 13.0 (C-22′),
9.1 (C-21). HRMS-ESI calcd for C53H63N5O11 946.4602, found
946.4646.
using alkyne 18, compound 26 was obtained as a yellow powder (32.0
25
mg, 50%); [α]D −75 (c 1.00, CHCl3). 1H NMR (CD3CN, 700
MHz) δ 8.82 (s, 1H, OH), 8.34 (s, 1H, H-17′), 7.36 (d, J = 7.9 Hz,
1H, H-12′), 7.29−7.28 (m, 2H, H-32′ and H-34′), 7.28−7.26 (m, 1H,
H-33′), 7.22−7.18 (m, 2H, H-31′ and H-35′), 7.11 (d, J = 7.9 Hz, 1H,
H-15′), 6.97 (t, J = 7.9 Hz, 1H, H-14′), 6.90 (t, J = 6.4 Hz, 1H, H-28′),
6.88 (t, J = 7.9 Hz, 1H, H-13′), 6.87 (s, 1H, H-14), 6.25 (s, 1H, H-17),
5.75 (dd, J = 9.8 and 4.4 Hz, 1H, H-7), 5.24 (d, J = 9.8 Hz, 1H, H-6),
5.22 (s, 1H, H-4), 5.22−5.19 (m, 1H, H-3′), 4.40 (dd, J = 14.8 and 6.4
Hz, H-29′α), 4.35 (d, J = 14.8 Hz, 1H, H-9′β), 4.24 (dd, J = 14.8 and
6.4 Hz, H-29′β), 4.01 (d, J = 14.8 Hz, 1H, H-9′α), 3.77 (d, J = 13.3
Hz, 1H, H-20′β), 3.71 (s, 3H, H-22), 3.65 (s, 3H, H-25), 3.55−3.54
(s, 1H, H-2), 3.53 (s, 3H, H-26′), 3.48 (s, 3H, H-24′), 3.46−3.44 (m,
1H, H-1′β), 3.32 (d, J = 14.5 Hz, 1H, H-5′α), 3.26 (d, J J = 14.4 Hz,
1H, H-8α), 3.19−3.16 (m, 1H, H-10β), 2.98 (d, J = 14.5 Hz, 1H, H-
5′β), 2.72 (s, 1H, H-19), 2.70−2.67 (m, 1H, H-20′α), 2.67 (d, J = 14.5
Hz, 1H, H-8β), 2.64 (s, 3H, H-23), 2.42−2.38 (m, 1H, H-10α), 2.10
(d, J = 16.1 Hz, 1H, H-1′α), 2.08−2.02 (m, 1H, H-11β), 1.92 (s, 3H,
H-27), 1.90−1.86 (m, 1H, H-11α), 1.83 (q, J = 7.5 Hz, 2H, H-21′),
1.62−1.57 (m, 1H, H-20β), 1.56−1.54 (m, 1H, H-2′), 1.34−1.28 (m,
1H, H-20α), 0.87 (t, J = 7.5 Hz, 3H, H-22′), 0.61 (t, J = 6.7 Hz, 3H,
H-21). 13C NMR (CD3CN, 175 MHz) δ 175.5 (C-23′), 173.4 (C-24),
172.6 (C-25′), 171.9 (C-26), 166.3 (C-27′), 159.7 (C-16), 157.6 (C-
7′), 154.8 (C-18), 140.5 (C-30′), 139.6 (C-4′), 136.8 (C-16′), 132.6
(C-18′), 131.7 (C-6), 130.0 (C-11′), 129.9 (C-33′), 129.5 (C-32′ and
C-34′), 128.6 (C-31′ and C-35′), 126.0 (C-14), 125.9 (C-7 and C-13),
124.5 (C-3′), 123.2 (C-14′), 122.3 (C-15), 121.0 (C-12′), 120.3 (C-
13′), 116.0 (C-10′ and C-8′), 112.4 (C-15′), 95.5 (C-17), 84.7 (C-2),
81.1 (C-3), 77.9 (C-4), 66.8 (C-19), 57.4 (C-19′), 57.2 (C-22), 55.4
(C-20′), 54.7 (C-12), 53.2 (C-24′), 53.0 (C-25), 52.7 (C-26′), 51.7
(C-8), 51.4 (C-10), 51.1 (C-5′), 46.1 (C-11), 44.3 (C-29′), 44.2 (C-
5), 39.2 (C-23), 36.0 (C-2′), 35.2 (C-1′), 32.3 (C-20), 28.7 (C-21′),
26.8 (C-9′), 21.7 (C-27), 13.1 (C-22′), 9.2 (C-21). HRMS-ESI calcd
for C53H63N5O11 996.4759, found 996.4789.
Compound 29. Following the general procedure B (method 2)
using alkyne 21, compound 29 was obtained as a yellow powder (32.1
mg, 52%). 1H NMR (CD3CN, 500 MHz) δ 8.91 (s, 1H, OH), 8.45 (s,
1H, H-17′), 7.44 (d, J = 7.6 Hz, 1H, H-12′), 7.17 (d, J = 7.6 Hz, 1H,
H-15′), 7.02 (t, J = 7.6 Hz, 1H, H-14′), 6.94 (t, J = 7.6 Hz, 1H, H-13′),
6.92 (s, 1H, H-14), 6.31 (s, 1H, H-28′), 6.28 (s, 1H, H-17), 5.81 (dd, J
= 10.1 and 4.9 Hz, 1H, H-7), 5.30 (d, J = 10.1 Hz, 1H, H-6), 5.27 (s,
1H, H-4), 5.27−5.26 (m, 1H, H-3′), 4.37 (d, J = 14.8 Hz, 1H, H-9′β),
4.05 (d, J = 14.8 Hz, 1H, H-9′α), 3.81 (d, J = 12.7 Hz, 1H, H-20′β),
3.77 (s, 3H, H-22), 3.73 (s, 3H, H-26′), 3.71 (s, 3H, H-25), 3.61 (s,
1H, H-2), 3.53 (s, 3H, H-24′), 3.51−3.46 (m, 1H, H-1′β), 3.46 (d, J =
13.5 Hz, 1H, H-5′α), 3.32 (d, J = 14.9 Hz, 1H, H-8α), 3.26−3.19 (m,
1H, H-10β), 3.14 (d, J = 13.5 Hz, 1H, H-5′β), 2.78 (s, 1H, H-19), 2.73
Compound 27. Following the general procedure B (method 2) and
using alkyne 19, compound 27 was obtained as a yellow powder (31.8
25
mg, 52%); [α]D −28 (c 1.00, CHCl3). 1H NMR (CD3CN, 500
MHz) δ 8.88 (s, 1H, OH), 8.38 (s, 1H, H-17′), 7.39 (d, J = 7.9 Hz,
1H, H-12′), 7.13 (d, J = 7.9 Hz, 1H, H-15′), 6.99 (t, J = 7.9 Hz, 1H, H-
14′), 6.90 (t, J = 7.9 Hz, 1H, H-13′), 6.89 (s, 1H, H-14), 6.37 (d, J =
7.1 Hz, 1H, H-28′), 6.27 (s, 1H, H-17), 5.78 (dd, J = 8.9 and 4.5 Hz,
1H, H-7), 5.26 (d, J = 8.9 Hz, 1H, H-6), 5.25−5.24 (m, 1H, H-3′),
5.23 (s, 1H, H-4), 4.34 (d, J = 14.6 Hz, 1H, H-9′β), 4.03 (d, J = 14.0
Hz, 1H, H-9′α), 3.92 (sext, J = 7.1 Hz, H-29′), 3.79 (d, J = 13.6 Hz,
1H, H-20′β), 3.73 (s, 3H, H-22), 3.68 (s, 3H, H-26′), 3.67 (s, 3H, H-
25), 3.57 (s, 1H, H-2), 3.50 (s, 3H, H-24′), 3.46 (t, J = 15.3 Hz, 1H,
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(d, J = 12.7 Hz, 1H, H-20′α), 2.72−2.68 (m, 1H, H-8β), 2.71 (s, 3H,
H-23), 2.47 (dd, J = 16.9 and 8.4 Hz, 1H, H-10α), 2.16 (d, J = 16.3
Hz, 1H, H-1′α), 2.14−2.08 (m, 1H, H-11β), 1.98 (s, 3H, H-27),
1.95−1.92 (m, 2H, H-11α and H-21′), 1.65 (td, J = 14.6 and 6.7 Hz,
1H, H-20β), 1.60−1.58 (m, 1H, H-2′), 1.40−1.36 (m, 1H, H-20α),
0.96 (t, J = 7.2 Hz, 3H, H-22′), 0.66 (t, J = 6.7 Hz, 3H, H-21). 13C
NMR (CD3CN, 125 MHz) δ 175.5 (C-23′), 173.3 (C-24), 172.7 (C-
25′), 171.9 (C-26), 166.3 (C-27′), 159.6 (C-16), 157.6 (C-7′), 154.7
(C-18), 139.6 (C-4′), 136.7 (C-16′), 132.6 (C-18′), 131.7 (C-6),
130.5 (C-11′), 126.0 (C-14), 125.9 (C-7), 125.3 (C-13), 124.3 (C-3′),
123.1 (C-14′), 122.2 (C-15), 121.1 (C-12′), 120.2 (C-13′), 116.1 (C-
10′), 114.8 (C-8′), 112.4 (C-15′), 95.4 (C-17), 84.6 (C-2), 81.1 (C-3),
77.8 (C-4), 66.6 (C-19), 57.3 (C-19′), 57.2 (C-22), 55.5 (C-20′), 54.7
(C-12), 53.1 (C-24′), 53.0 (C-25), 52.9 (C-26′), 52.7 (C-29′), 51.6
(C-8), 51.2 (C-10), 50.9 (C-5′), 46.0 (C-11), 44.1 (C-5), 39.1 (C-23),
36.0 (C-2′), 35.1 (C-1′), 32.2 (C-20), 29.1 (C-30′), 28.6 (C-21′), 26.7
(C-9′), 21.7 (C-27), 13.0 (C-22′), 9.1 (C-21). HRMS-ESI calcd for
C54H67N5O11 962.4915, found 962.4957.
General Synthetic Procedure C for the Synthesis of Amino-
Esters 27−32. Method 1. To a solution of anhydrous zinc iodide (3
equiv) in anhydrous CH2Cl2 (0.2 mL) at 0 °C were added the
corresponding enamino-esters 22−29 (20 mg, 1 equiv). The resulting
mixture was stirred at the same temperature for 1 h, after which
NaBH4 (5 equiv) was added. The solution was allowed to reach room
temperature and stirred at room temperature overnight. The reaction
was then quenched with a saturated ammonium chloride solution, and
the reaction mixture was extracted with CH2Cl2. After usual
treatments, purification of the crude compound was carried out by
column chromatography on silica gel using ethyl acetate/acetone (1:0
to 4:1).
+28.0 (c 1.00, CHCl3). H NMR (CD3CN, 600 MHz) δ 8.89 (s, 1H,
OH), 8.47 (s, 1H, H-17′), 7.36 (d, J = 7.9 Hz, 1H, H-12′), 7.15 (d, J =
7.9 Hz, 1H, H-15′), 7.01 (t, J = 7.9 Hz, 1H, H-14′), 6.92 (t, J = 7.9 Hz,
1H, H-13′), 6.79 (s, 1H, H-14), 6.26 (s, 1H, H-17), 5.77 (dd, J = 10.3
and 5.3 Hz, 1H, H-7), 5.27 (d, J = 10.3 Hz, 1H, H-6), 5.19 (s, 1H, H-
4), 5.13 (d, J = 5.3 Hz, 1H, H-3′), 4.19 (dd, J = 10.3 and 7.2 Hz, 2H,
H-26′), 4.13 (ddd, J = 15.3 and 10.0 and 5.0 Hz, 1H, H-9′β), 3.76 (s,
3H, H-22), 3.66 (s, 3H, H-25), 3.52 (s, 1H, H-2), 3.49 (s, 3H, H-24′),
3.27 (dd, J = 15.5 and 5.3 Hz, 1H, H-8β), 3.23 (dd, J = 15.0 and 11.6
Hz, 1H, H-1′β), 3.16 (td, J = 10.0 and 5.8 Hz, 1H, H-10β), 3.10 (dd, J
= 15.3 and 5.0 Hz, 1H, H-9′α), 3.01 (d, J = 15.5 Hz, 1H, H-5′β), 2.97
(dd, J = 11.0 and 6.8 Hz, 1H, H-8′), 2.85 (d, J = 12.0 Hz, 1H, H-20′β),
2.82 (d, J = 15.5 Hz, 1H, H-5′α), 2.77 (s, 1H, H-19), 2.70 (dd, J = 11.0
and 6.8 Hz, 1H, H-7′β), 2.67 (d, J = 15.5 Hz, 1H, H-8α), 2.65 (s, 3H,
H-23), 2.59 (dd, J = 11.0 and 6.8 Hz, 1H, H-7′α), 2.43 (td, J = 10.0
and 5.8 1H, H-10α), 2.21 (d, J = 15.0 Hz, 1H, H-1′α), 1.99 (td, J =
13.5 and 5.8 Hz, 1H, H-11β), 1.94 (s, 3H, H-27), 1.88 (q, J = 7.4 Hz,
2H, H-21′), 1.82 (td, J = 12.6 and 5.8 Hz, 1H, H-11α), 1.68 (d, J =
12.0 Hz, 1H, H-20′α), 1.58 (q, J = 7.1 Hz, 1H, H-20β), 1.30 (q, J = 7.1
Hz, 1H, H-20α), 1.13−1.09 (m, 1H, H-2′), 0.93 (t, J = 7.4 Hz, 3H, H-
22′), 0.65 (t, J = 7.1 Hz, 3H, H-21). 13C NMR (CD3CN, 600 MHz) δ
177.9 (C-25′), 176.3 (C-23′), 173.2 (C-24), 171.9 (C-26), 159.7 (C-
16), 154.3 (C-18), 140.8 (C-4′), 136.8 (C-16′), 132.5 (C-18′), 131.8
(C-6), 130.0 (C-11′), 126.0 (C-14), 125.9 (C-7), 125.0 (C-13), 124.6
(C-3′), 123.4 (C-14′), 122.9 (C-15), 120.3 (C-12′), 120.0 (C-13′),
118.3 (C-10′), 112.3 (C-15′), 95.1 (C-17), 84.6 (C-2), 81.1 (C-3),
77.9 (C-4), 66.1 (C-19), 62.0 (C-26′), 57.8 (C-19′), 57.2 (C-22), 57.1
(C-5′), 56.5 (C-7′), 54.7 (C-12), 53.1 (C-24′), 52.9 (C-25), 51.5 (C-
8), 50.9 (C-10), 50.3 (C-20′), 48.1 (C-8′), 46.0 (C-11), 44.1 (C-5),
39.2 (C-23), 36.0 (C-2′), 35.3 (C-1′), 32.2 (C-20), 28.5 (C-21′), 22.0
(C-9′), 21.7 (C-27), 15.1 (C-27′), 13.2 (C-22′), 9.1 (C-21). HRMS-
ESI calcd for C50H63N4O10 879.4544, found 879.4547.
Method 2. NaBH(OAc)3 (3 equiv) was added to a solution of the
corresponding enamino-ester 22−29 (20 mg, 1 equiv) in CH2Cl2 (0.2
mL) at 0 °C. The reaction mixture was stirred for 4 h at 0 °C. The
resulting mixture was diluted with CH2Cl2 and washed with a
saturated sodium carbonate solution. Evaporation of the solvent
provided pure amino-esters 30−37.
Compound 32. Following the general procedure C (method 2)
compound 32 was obtained as a white powder (19 mg, 90%); [α]D
25
+31 (c 1.00, CHCl3). 1H NMR (CD3CN, 500 MHz) δ 8.47 (s, 1H, H-
17′), 7.40 (d, J = 7.5 Hz, 2H, H-28′), 7.36 (t, J = 7.5 Hz, 2H, H-29′),
7.34−7.31 (m, 1H, H-30′), 7.28 (d, J = 7.8 Hz, 1H, H-12′), 7.14 (d, J
= 7.8 Hz, 1H, H-15′), 6.99 (t, J = 7.8 Hz, 1H, H-14′), 6.84 (t, J = 7.8
Hz, 1H, H-13′), 6.78 (s, 1H, H-14), 6.24 (s, 1H, H-17), 5.76 (dd, J =
10.2 and 4.7 Hz, 1H, H-7), 5.27 (d, J = 10.2 Hz, 1H, H-6), 5.25 (d, J =
12.2 Hz, 1H, H-26′), 5.19 (s, 1H, H-4), 5.15 (d, J = 12.2 Hz, 1H, H-
26′), 5.12 (d, J = 5.0 Hz, 1H, H-3′), 4.15 (dd, J = 13.3 and 4.6 Hz, 1H,
H-9′β), 3.75 (s, 3H, H-25), 3.66 (s, 3H, H-22), 3.52 (s, 1H, H-2), 3.50
(s, 3H, H-24′), 3.25 (dd, J = 16.2 and 4.7 Hz, 1H, H-8β), 3.23 (t, J =
14.7 Hz, 1H, H-1′β), 3.16−3.13 (m, 1H, H-10β), 3.11 (dd, J = 13.3
and 4.6 Hz, 1H, H-9′α), 3.05 (td, J = 6.7 and 4.6 Hz, 1H, H-8′), 2.95
(d, J = 15.5 Hz, 1H, H-5′β), 2.83 (d, J = 11.4 Hz, 1H, H-20′β), 2.79
(d, J = 15.5 Hz, 1H, H-5′α), 2.76 (s, 1H, H-19), 2.72 (t, J = 11.5 Hz,
1H, H-7′β), 2.65 (s, 3H, H-23), 2.63 (d, J = 16.2 Hz, 1H, H-8α), 2.62
(dd, J = 11.5 and 6.7 Hz, 1H, H-7′α), 2.40 (td, J = 10.2 and 5.6 Hz,
1H, H-10α), 2.20 (d, J = 14.7 Hz, 1H, H-1′α), 2.01−1.96 (m, 1H, H-
11β), 1.94 (s, 3H, H-27), 1.93−1.90 (m, 1H, H-11α), 1.84 (q, J = 7.5
Hz, 2H, H-21′), 1.67 (d, J = 11.4 Hz, 1H, H-20′α), 1.58 (dd, J = 14.1
and 7.4 Hz, 1H, H-20β), 1.29 (dd, J = 14.1 and 7.4 Hz, 1H, H-20α),
1.12−1.08 (m, 1H, H-2′), 0.91 (t, J = 7.5 Hz, 3H, H-22′), 0.63 (t, J =
7.4 Hz, 3H, H-21). 13C NMR (CD3CN, 125 MHz) δ 177.7 (C-25′),
176.3 (C-23′), 173.2 (C-24), 171.9 (C-26), 159.7 (C-16), 154.3 (C-
18), 140.8 (C-4′), 137.9 (C-27′), 136.7 (C-16′), 132.6 (C-18′), 131.8
(C-6), 130.9 (C-11′), 130,0 (C-29′), 130.0 (C-28′), 129.6 (C-30′),
126.0 (C-7), 125.8 (C-14), 125.0 (C-13), 124.6 (C-3′), 123.3 (C-15),
122.9 (C-14′), 120.3 (C-12′), 120.1 (C-13′), 118.1 (C-10′), 112.3 (C-
15′), 95.1 (C-17), 84.5 (C-2), 81.1 (C-3), 77.8 (C-4), 67.8 (C-19),
66.1 (C-26′), 57.8 (C-19′), 57.2 (C-22), 57.1 (C-5′), 56.9 (C-7′), 54.7
(C-12), 53.1 (C-24′), 52.9 (C-25), 51.5 (C-8), 50.8 (C-10), 50.4 (C-
20′), 48.1 (C-8′), 46.0 (C-11), 44.1 (C-5), 39.3 (C-23), 36.0 (C-2′),
35.3 (C-1′), 32.2 (C-20), 28.5 (C-21′), 22.1 (C-9′), 21.7 (C-27), 13.3
(C-22′), 9.1 (C-21). HRMS-ESI calcd for C55H65N4O10 941.4700,
found 941.4730.
Compound 30. Following the general procedure C (method 2)
25
compound 30 was obtained as a white powder (14 mg, 69%); [α]D
+30 (c 1.00, CHCl3). 1H NMR (CD3CN, 600 MHz) δ 8.51 (s, 1H, H-
17′), 7.33 (d, J = 7.6 Hz, 1H, H-12′), 7.16 (d, J = 7.6 Hz, 1H, H-15′),
7.01 (t, J = 7.6 Hz, 1H, H-14′), 6.92 (t, J = 7.6 Hz, 1H, H-13′), 6.80 (s,
1H, H-14), 6.25 (s, 1H, H-17), 5.77 (dd, J = 10.7 and 5.1 Hz, 1H, H-
7), 5.26 (d, J = 10.7 Hz, 1H, H-6), 5.18 (s, 1H, H-4), 5.13 (d, J = 4.6
Hz, 1H, H-3′), 4.14 (d, J = 14.5 Hz, 1H, H-9′β), 3.75 (s, 3H, H-22),
3.71 (s, 3H, H-26′), 3.66 (s, 3H, H-25), 3.52 (s, 1H, H-2), 3.49 (s, 3H,
H-24′), 3.26 (dd, J = 14.9 and 5.1 Hz, 1H, H-8β), 3.21 (dd, J = 16.0
and 11.4 Hz, 1H, H-1′β), 3.16 (td, J = 9.9 and 5.6 Hz, 1H, H-10β),
3.10 (d, J = 14.5 Hz, 1H, H-9′α), 3.02−3.00 (m, 1H, H-8′), 2.99 (d, J
= 15.6 Hz, 1H, H-5′β), 2.84 (d, J = 11.3 Hz, 1H, H-20′β), 2.82 (d, J =
15.6 Hz, 1H, H-5′α), 2.80 (s, 1H, H-19), 2.70 (d, J = 12.2 Hz, 1H, H-
7′β), 2.68 (d, J = 14.9 Hz, 1H, H-8α), 2.65 (s, 3H, H-23), 2.60−2.56
(m, 1H, H-7′α), 2.44 (td, J = 9.9 and 5.6 Hz, 1H, H-10α), 2.20 (d, J =
16.0 Hz, 1H, H-1′α), 2.01−1.97 (m, 1H, H-11β), 1.94 (s, 3H, H-27),
1.87 (q, J = 7.6 Hz, 2H, H-21′), 1.80 (td, J = 12.2 and 5.6 Hz, 1H, H-
11α), 1.68 (d, J = 11.3 Hz, 1H, H-20′α), 1.57 (q, J = 7.6 Hz, 1H, H-
20β), 1.30 (q, J = 7.6 Hz, 1H, H-20α), 1.08 (s, 1H, H-2′), 0.92 (t, J =
7.6 Hz, 3H, H-22′), 0.64 (t, J = 6.9 Hz, 3H, H-21). 13C NMR
(CD3CN, 150 MHz) δ 178.3 (C-25′), 176.3 (C-23′), 173.1 (C-24),
171.8 (C-26), 159.6 (C-16), 154.2 (C-18), 140.8 (C-4′), 136.7 (C-
16′), 132.4 (C-18′), 131.7 (C-6), 130.9 (C-11′), 125.9 (C-14), 125.8
(C-7), 124.9 (C-13), 124.4 (C-3′), 123.3 (C-14′), 122.7 (C-15), 120.2
(C-12′), 120.0 (C-13′), 118.0 (C-10′), 112.3 (C-15′), 94.9 (C-17),
84.4 (C-2), 81.1 (C-3), 77.7 (C-4), 65.8 (C-19), 57.7 (C-19′), 57.1
(C-22), 57.0 (C-5′), 56.3 (C-7′), 54.6 (C-12), 53.0 (C-24′), 53.0 (C-
26′), 52.9 (C-25), 51.4 (C-8), 50.7 (C-10), 50.3 (C-20′), 47.8 (C-8′),
45.9 (C-11), 44.0 (C-5), 39.1 (C-23), 35.9 (C-2′), 35.2 (C-1′), 32.1
(C-20), 28.4 (C-21′), 22.1 (C-9′), 21.6 (C-27), 13.1 (C-22′), 9.0 (C-
21). HRMS-ESI calcd for C49H61N4O10 865.4387, found 865.4398.
Compound 31. Following the general procedure C (method 2)
Compound 33. Following the general procedure B (method 1)
compound 31 was obtained as a white powder (18.9 mg, 95%); [α]25
compound 33 was obtained as a white powder (7 mg, 33%); [α]D25 +4
D
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(c 1.00, CHCl3). H NMR (CD3CN, 600 MHz) δ 8.96 (s, 1H, OH),
17′), 7.39 (d, J = 7.9 Hz, 1H, H-12′), 7.14 (d, J = 7.9 Hz, 1H, H-15′),
6.99 (t, J = 7.9 Hz, 1H, H-14′), 6.90 (t, J = 7.9 Hz, 1H, H-13′), 6.73 (s,
1H, H-14), 6.45 (d, J = 6.9 Hz, 1H, H-28′), 6.25 (s, 1H, H-17), 5.76
(dd, J = 10.4 and 4.2 Hz, 1H, H-7), 5.27 (d, J = 10.4 Hz, 1H, H-6),
5.19 (s, 1H, H-4), 5.10 (d, J = 5.0 Hz, 1H, H-3′), 4.04 (d, J = 15.2 Hz,
1H, H-9′β), 3.97 (q, J = 6.9 Hz, 1H, H-29′), 3.77 (s, 3H, H-22), 3.69
(s, 3H, H-26′), 3.66 (s, 3H, H-25), 3.53 (s, 1H, H-2), 3.53 (s, 3H, H-
24′), 3.29 (t, J = 14.8 Hz, 1H, H-1′β), 3.28 (dd, J = 13.9 and 4.2 Hz,
1H, H-8β), 3.25 (dd, J = 15.2 and 6.5 Hz, 1H, H-9′α), 3.20 (m, 1H, H-
8′), 3.17 (td, J = 10.1 and 5.3 Hz, 1H, H-10β), 3.12 (d, J = 6.5 Hz, 1H,
H-7′), 3.09 (d, J = 12.3 Hz, 1H, H-20′β), 3.05 (d, J = 15.5 Hz, 1H, H-
5′β), 2.77 (s, 1H, H-19), 2.67 (d, J = 15.5 Hz, 1H, H-5′α), 2.66 (d, J =
13.9 Hz, 1H, H-8α), 2.66 (s, 3H, H-23), 2.46 (td, J = 10.1 and 5.3 Hz,
1H, H-10α), 2.23 (d, J = 14.8 Hz, 1H, H-1′α), 2.00 (dq, J = 12.9 and
5.3 Hz, 1H, H-11β), 1.94 (s, 3H, H-27), 1.87 (q, J = 7.7 Hz, 2H, H-
21′), 1.81 (td, J = 12.9 and 5.3 Hz, 1H, H-11α), 1.74 (dd, J = 12.3 and
2.6 Hz, 1H, H-20′α), 1.57 (dd, J = 14.2 and 7.3 Hz, 1H, H-20β), 1.29
(dd, J = 14.2 and 7.3 Hz, 1H, H-20α), 1.20−1.16 (m, 1H, H-2′), 1.12
(d, J = 6.9 Hz, 3H, H-30′), 1.09 (d, J = 6.9 Hz, 3H, H-31′), 0.91 (t, J =
7.7 Hz, 3H, H-22′), 0.61 (t, J = 7.4 Hz, 3H, H-21). 13C NMR
(CD3CN, 150 MHz) δ 176.3 (C-23′), 176.2 (C-25′), 173.2 (C-24),
171.9 (C-26), 170.3 (C-27′), 159.6 (C-16), 154.3 (C-18), 140.4 (C-
4′), 136.8 (C-16′), 132.4 (C-18′), 131.8 (C-6), 130.8 (C-11′), 126.3
(C-14), 125.8 (C-7), 125.0 (C-13), 124.6 (C-3′), 123.4 (C-14′), 123.2
(C-15), 120.5 (C-12′), 120.1 (C-13′), 118.5 (C-10′), 112.3 (C-15′),
95.0 (C-17), 84.5 (C-2), 81.1 (C-3), 77.8 (C-4), 69.7 (C-7′), 66.0 (C-
19), 58.1 (C-19′), 57.2 (C-22), 54.7 (C-12), 54.4 (C-5′), 53.1 (C-24′),
53.1 (C-25), 52.9 (C-26′), 51.6 (C-8), 50.8 (C-10), 50.3 (C-8′), 49.5
(C-20′), 46.0 (C-11), 44.1 (C-5), 42.1 (C-29′), 39.1 (C-23), 35.7 (C-
2′), 35.1 (C-1′), 32.2 (C-20), 28.7 (C-21′), 23.3 (C-30′), 23.0 (C-
31′), 21.7 (C-27), 19.8 (C-9′), 13.3 (C-22′), 9.0 (C-21). HRMS-ESI
calcd for C53H68N5O11 950.4915, found 950.4939.
8.54 (s, 1H, H-17′), 7.38 (d, J = 7.7 Hz, 1H, H-12′), 7.17 (d, J = 7.7
Hz, 1H, H-15′), 7.02 (t, J = 7.7 Hz, 1H, H-14′), 6.94 (s, 1H, H-14),
6.93 (t, J = 7.7 Hz, 1H, H-13′), 6.25 (s, 1H, H-17), 5.78 (dd, J = 10.2
and 5.1 Hz, 1H, H-7), 5.29 (d, J = 10.2 Hz, 1H, H-6), 5.18 (s, 1H, H-
4), 5.15 (d, J = 5.5 Hz, 1H, H-3′), 4.10 (dd, J = 15.1 and 5.7 Hz, 1H,
H-9′β), 3.75 (s, 3H, H-22), 3.69 (s, 3H, H-26′), 3.66 (s, 3H, H-25),
3.59 (s, 3H, H-28′), 3.52 (s, 1H, H-2), 3.48 (s, 3H, H-24′), 3.47 (m,
1H, H-8′), 3.41 (d, J = 5.7 Hz, 1H, H-7′), 3.27 (dd, J = 16.3 and 5.1
Hz, 1H, H-8β), 3.23 (dd, J = 15.4 and 11.7 Hz, 1H, H-1′β), 3.16 (td, J
= 9.9 and 5.5 Hz, 1H, H-10β), 3.13 (dd, J = 15.1 and 5.7 Hz, 1H, H-
9′α), 3.05 (d, J = 16.0 Hz, 1H, H-5′β), 2.98 (d, J = 16.0 Hz, 1H, H-
5′α), 2.95 (d, J = 12.2 Hz, 1H, H-20′β), 2.85 (s, 1H, H-19), 2.68 (d, J
= 16.3 Hz, 1H, H-8α), 2.65 (s, 3H, H-23), 2.44 (td, J = 9.9 and 5.5 Hz,
1H, H-10α), 2.12 (d, J = 15.4 Hz, 1H, H-1′α), 2.02 (d, J = 12.2 Hz,
1H, H-20′α), 1.98 (td, J = 10.6 and 5.5 Hz, 1H, H-11β), 1.94 (s, 3H,
H-27), 1.85 (q, J = 7.5 Hz, 2H, H-21′), 1.78 (td, J = 10.6 and 5.5 Hz,
1H, H-11α), 1.57 (td, J = 14.4 and 7.2 Hz, 1H, H-20β), 1.32 (td, J =
14.4 and 7.2 Hz, 1H, H-20α), 1.10−1.07 (m, 1H, H-2′), 0.91 (t, J =
7.5 Hz, 3H, H-22′), 0.69 (t, J = 7.2 Hz, 3H, H-21). 13C NMR
(CD3CN, 150 MHz) δ 176.2 (C-25′), 176.1 (C-23′), 173.4 (C-27′),
173.1 (C-24), 171.8 (C-26), 159.5 (C-16), 154.2 (C-18), 140.4 (C-4′),
136.8 (C-16′), 133.0 (C-18′), 131.7 (C-6), 130.7 (C-11′), 126.0 (C-
14), 125.8 (C-7), 124.9 (C-13), 124.1 (C-3′), 123.3 (C-14′), 122.2
(C-15), 120.2 (C-13′), 120.1 (C-12′), 116.1 (C-10′), 112.5 (C-15′),
94.9 (C-17), 84.3 (C-2), 81.0 (C-3), 77.7 (C-4), 67.5 (C-7′), 65.7 (C-
19), 57.6 (C-19′), 57.1 (C-22), 54.6 (C-12), 53.1 (C-26′), 53.0 (C-
24′), 52.8 (C-25), 52.7 (C-5′), 52.5 (C-28′), 51.5 (C-8), 51.4 (C-20′),
50.6 (C-10), 50.0 (C-8′), 45.9 (C-11), 44.0 (C-5), 39.1 (C-23), 35.9
(C-2′), 35.2 (C-1′), 32.1 (C-20), 28.4 (C-21′), 22.5 (C-9′), 21.6 (C-
27), 13.1 (C-22′), 9.1 (C-21). HRMS-ESI calcd for C51H63N4O12
923.4442, found 923.4360.
Compound 34. Following the general procedure C (method 2)
Compound 36. Following the general procedure C (method 2)
compound 36 was obtained as a yellow powder (16 mg, 78%); [α]D
25
compound 34 was obtained as a yellow powder (19.1 mg, 95%);
25
1
[α]D −4 (c 1.00, CHCl3). H NMR (CD3CN, 500 MHz) δ 8.57 (s,
1H, H-17′), 7.43 (d, J = 8.1 Hz, 1H, H-12′), 7.37−7.33 (m, 2H, H-32′
and H-34′), 7.33−7.31 (m, 1H, H-33′), 7.26−7.23 (m, 2H, H-31′ and
H-35′), 7.18 (d, J = 8.1 Hz, 1H, H-15′), 7.03 (t, J = 8.1 Hz, 1H, H-
14′), 7.01 (t, J = 7.7 Hz, 1H, H-28′), 6.94 (t, J = 8.1 Hz, 1H, H-13′),
6.77 (s, 1H, H-14), 6.28 (s, 1H, H-17), 5.79 (td, J = 10.3 and 5.1 Hz,
1H, H-7), 5.30 (d, J = 10.3 Hz, 1H, H-6), 5.21 (s, 1H, H-4), 5.10 (d, J
= 4.5 Hz, 1H, H-3′), 4.38 (d, J = 6.3 Hz, 1H, H-29′), 4.08−4.04 (m,
1H, H-9′β), 3.79 (s, 3H, H-22), 3.69 (s, 6H, H-26′ and H-25), 3.55 (s,
1H, H-2), 3.54 (s, 3H, H-24′), 3.35 (d, J = 15.1 Hz, 1H, H-1′β), 3.34−
3.29 (m, 1H, H-8β), 3.31 (d, 1H, J = 17.5 Hz, H-9′α), 3.32−3.27 (m,
1H, H-8′), 3.22−3.19 (m, 1H, H-10β), 3.15 (d, J = 12.0 Hz, 1H, H-
20′β), 3.29−3.24 (m, 1H, H-7′), 3.00 (d, J = 15.2 Hz, 1H, H-5′β),
2.78 (s, 1H, H-19), 2.71 (d, J = 15.2 Hz, 1H, H-5′α), 2.69 (dd, J = 13.2
and 5.1 Hz, 1H, H-8α), 2.68 (s, 3H, H-23), 2.46 (td, J = 10.6 and 7.0
Hz, 1H, H-10α), 2.23 (d, J = 15.1 Hz, 1H, H-1′α), 2.13 (s, 3H, H-27),
2.04−1.99 (m, 1H, H-11β), 1.87−1.83 (m, 1H, H-11α), 1.81 (q, J =
7.8 Hz, 2H, H-21′), 1.78 (d, J = 12.0 Hz, 1H, H-20′α), 1.59 (dd, J =
14.2 and 7.6 Hz, 1H, H-20β), 1.35−1.30 (m, 1H, H-20α), 1.21 (t, J =
7.6 Hz, 1H, H-2′), 0.87 (t, J = 7.8 Hz, 3H, H-22′), 0.64 (t, J = 7.6 Hz,
3H, H-21). 13C NMR (CD3CN, 125 MHz) δ 176.3 (C-25′), 176.2 (C-
23′), 173.2 (C-24), 171.9 (C-26), 171.5 (C-27′), 159.6 (C-16), 154.4
(C-18), 141.0 (C-30′), 140.4 (C-4′), 136.8 (C-16′), 132.5 (C-18′),
131.8 (C-6), 130.8 (C-11′), 129.8 (C-33′), 129.3 (C-32′ and C-34′),
128.4 (C-31′ and C-35′), 126.4 (C-14), 125.8 (C-7), 125.1 (C-13),
124.4 (C-3′), 123.4 (C-14′), 123.2 (C-15), 120.5 (C-12′), 120.2 (C-
13′), 118.3 (C-10′), 112.4 (C-15′), 95.0 (C-17), 84.5 (C-2), 81.2 (C-
3), 77.9 (C-4), 70.1 (C-7′), 66.0 (C-19), 58.2 (C-19′), 57.2 (C-22),
54.7 (C-12), 53.2 (C-5′), 53.1 (C-24′), 53.0 (C-25), 52.9 (C-26′),
51.6 (C-8), 50.8 (C-10), 50.3 (C-8′), 49.6 (C-20′), 46.0 (C-11), 44.1
(C-5), 43.8 (C-29′), 39.2 (C-23), 35.8 (C-2′), 35.0 (C-1′), 32.2 (C-
20), 28.6 (C-21′), 21.7 (C-27), 20.0 (C-9′), 13.1 (C-22′), 9.1 (C-21).
HRMS-ESI calcd for C53H65N5O11 998.4915, found 998.4922.
+11 (c 1.00, CHCl3). 1H NMR (CD3CN, 500 MHz) δ 8.54 (s, 1H, H-
17′), 7.38 (d, J = 7.7 Hz, 1H, H-12′), 7.14 (d, J = 7.7 Hz, 1H, H-15′),
7.00 (t, J = 7.7 Hz, 1H, H-14′), 6.90 (t, J = 7.7 Hz, 1H, H-13′), 6.81 (t,
J = 5.7 Hz, 1H, H-28′), 6.75 (s, 1H, H-14), 6.25 (s, 1H, H-17), 5.83
(m, 1H, H-30′), 5.76 (dd, J = 10.3 and 4.8 Hz, 1H, H-7), 5.28 (d, J =
14.0 Hz, 1H, H-6), 5.19 (s, 1H, H-4), 5.18 (dd, J = 14.0 and 1.6 Hz,
1H, H-31′), 5.09 (d, J = 5.1 Hz, 1H, H-3′), 5.06 (dd, J = 10.6 and 1.6
Hz, 1H, H-31′), 4.02 (d, J = 12.7 Hz, 1H, H-9′β), 3.79−3.75 (m, 2H,
H-29′), 3.77 (s, 3H, H-22), 3.68 (s, 3H, H-26′), 3.66 (s, 3H, H-25),
3.53 (s, 1H, H-2), 3.52 (s, 3H, H-24′), 3.30 (dd, J = 10.9 and 4.8 Hz,
1H, H-8β), 3.29 (t, J = 14.7 Hz, 1H, H-1′β), 3.25 (d, J = 12.7 Hz, 1H,
H-9′α), 3.24−3.22 (m, 1H, H-8′), 3.20−3.19 (m, 1H, H-7′), 3.14 (dd,
J = 9.9 and 5.4 Hz, 1H, H-10β), 3.11 (d, J = 12.1 Hz, 1H, H-20′β),
3.05 (d, J = 15.4 Hz, 1H, H-5′β), 2.79 (s, 1H, H-19), 2.69 (d, J = 15.4
Hz, 1H, H-5′α), 2.68 (d, J = 16.0 Hz, 1H, H-8α), 2.66 (s, 3H, H-23),
2.46 (td, J = 9.9 and 5.4 Hz, 1H, H-10α), 2.22 (d, J = 14.7 Hz, 1H, H-
1′α), 2.00 (td, J = 9.9 and 5.4 Hz, 1H, H-11β), 1.94 (s, 3H, H-27),
1.86 (q, J = 7.4 Hz, 2H, H-21′), 1.82 (td, J = 10.9 and 5.4 Hz, 1H, H-
11α), 1.77 (dd, J = 12.1 and 2.8 Hz, 1H, H-20′α), 1.57 (td, J = 14.5
and 7.2 Hz, 1H, H-20β), 1.30 (td, J = 14.5 and 7.2 Hz, 1H, H-20α),
1.20−1.16 (m, 1H, H-2′), 0.91 (t, J = 7.4 Hz, 3H, H-22′), 0.61 (t, J =
7.2 Hz, 3H, H-21). 13C NMR (CD3CN, 125 MHz) δ 176.3 (C-25′),
176.2 (C-23′), 173.2 (C-24), 171.9 (C-26), 171.3 (C-27′), 159.6 (C-
16), 154.3 (C-18), 140.4 (C-4′), 136.8 (C-16′), 136.6 (C-30′), 132.5
(C-18′), 131.8 (C-6), 130.8 (C-11′), 126.4 (C-14), 125.7 (C-7), 125.0
(C-13), 124.5 (C-3′), 123.4 (C-14′), 123.2 (C-15), 120.5 (C-12′),
120.2 (C-13′), 118.3 (C-10′), 116.6 (C-31′), 112.4 (C-15′), 95.0 (C-
17), 84.4 (C-2), 81.2 (C-3), 77.8 (C-4), 70.0 (C-7′), 66.1 (C-19), 58.2
(C-19′), 57.2 (C-22), 54.7 (C-5′), 54.7 (C-12), 53.2 (C-24′), 53.1 (C-
25), 53.0 (C-26′), 51.6 (C-8), 50.8 (C-10), 50.3 (C-8′), 49.6 (C-20′),
46.0 (C-11), 44.1 (C-5), 42.4 (C-29′), 39.2 (C-23), 35.8 (C-2′), 35.1
(C-1′), 32.2 (C-20), 28.7 (C-21′), 21.7 (C-27), 19.9 (C-9′), 13.2 (C-
22′), 9.0 (C-21). HRMS-ESI calcd for C53H65N5O11 948.4759, found
948.4766.
Compound 35. Following the general procedure C (method 2)
25
compound 35 was obtained as a yellow powder (16 mg, 81%); [α]D
Compound 37. Following the general procedure C (method 2)
compound 37 was obtained as a yellow powder (14.5 mg, 72%);
+12 (c 1.00, CHCl3). 1H NMR (CD3CN, 600 MHz) δ 8.54 (s, 1H, H-
6097
dx.doi.org/10.1021/jm4004347 | J. Med. Chem. 2013, 56, 6088−6100

Journal of Medicinal Chemistry
Article
25
1
[α]D +1 (c 1.00, CHCl3). H NMR (CD3CN, 500 MHz) δ 8.55 (s,
1H, H-17′), 7.44 (d, J = 7.8 Hz, 1H, H-12′), 7.17 (d, J = 7.8 Hz, 1H,
H-15′), 7.03 (t, J = 7.8 Hz, 1H, H-14′), 6.94 (t, J = 7.8 Hz, 1H, H-13′),
6.78 (s, 1H, H-14), 6.34 (s, 1H, H-28′), 6.28 (s, 1H, H-17), 5.80 (td, J
= 9.9 and 5.2 Hz, 1H, H-7), 5.30 (d, J = 9.9 Hz, 1H, H-6), 5.22 (s, 1H,
H-4), 5.25−5.14 (d, J = 5.2 Hz, 1H, H-3′), 3.81 (t, J = 14.3 Hz, 1H, H-
9′β), 3.80 (s, 3H, H-22), 3.74 (s, 3H, H-26′), 3.70 (s, 3H, H-25), 3.61
(s, 1H, H-2), 3.56 (s, 3H, H-24′), 3.39 (d, J = 14.3 Hz, 1H, H-9′α),
3.35 (d, J = 13.2 Hz, 1H, H-8β), 3.22 (t, J = 13.4 Hz, 1H, H-1′β),
3.22−3.21 (m, 1H, H-8′), 3.21−3.18 (m, 1H, H-10β), 3.14 (d, J = 11.6
Hz, 1H, H-20′β), 3.12 (d, J = 6.5 Hz, 1H, H-7′), 3.10 (d, J = 14.5 Hz,
1H, H-5′β), 2.79 (s, 1H, H-19), 2.73 (d, J = 14.5 Hz, 1H, H-5′α), 2.69
(s, 3H, H-23), 2.68 (dd, J = 13.2 and 5.2 Hz, 1H, H-8α), 2.46 (td, J =
10.4 and 5.2 Hz, 1H, H-10α), 2.26 (d, J = 13.4 Hz, 1H, H-1′α), 2.06−
1.99 (m, 1H, H-11β), 1.97 (s, 3H, H-27), 1.95−1.93 (m, 2H, H-21′),
1.88−1.84 (m, 1H, H-11α), 1.81 (dd, J = 11.6 and 4.1 Hz, 1H, H-
20′α), 1.60 (dd, J = 13.6 and 7.0 Hz, 1H, H-20β), 1.36−1.33 (m, 1H,
H-20α), 1.32 (s, 9H, H-30′), 1.24−1.22 (m, 1H, H-2′), 0.96 (t, J = 7.7
Hz, 3H, H-22′), 0.64 (t, J = 7.0 Hz, 3H, H-21). 13C NMR (CD3CN,
125 MHz) δ 176.2 (C-25′), 176.0 (C-23′), 173.2 (C-24), 171.9 (C-
26), 170.6 (C-27′), 159.6 (C-16), 154.4 (C-18), 140.6 (C-4′), 136.8
(C-16′), 132.5 (C-18′), 131.8 (C-6), 130.8 (C-11′), 126.4 (C-14),
125.8 (C-7), 125.1 (C-13), 124.8 (C-3′), 123.4 (C-14′), 123.1 (C-15),
120.5 (C-12′), 120.2 (C-13′), 118.4 (C-10′), 112.3 (C-15′), 95.0 (C-
17), 84.5 (C-2), 81.2 (C-3), 77.9 (C-4), 69.9 (C-7′), 66.1 (C-19), 58.1
(C-19′), 57.2 (C-22), 54.7 (C-12), 54.5 (C-5′), 53.1 (C-24′), 53.1 (C-
25), 52.9 (C-26′), 52.0 (C-29′), 51.6 (C-8), 50.8 (C-10), 50.1 (C-8′),
49.6 (C-20′), 46.0 (C-11), 44.1 (C-5), 39.2 (C-23), 35.8 (C-2′), 35.3
(C-1′), 32.2 (C-20), 29.3 (C-30′), 28.7 (C-21′), 21.7 (C-27), 19.9 (C-
9′), 13.5 (C-22′), 9.1 (C-21). HRMS-ESI calcd for C54H69N5O11
964.5072, found 964.5109.
nM. For the U87 cell line, compound 30 was incubated at 10 and 50
nM, compound 33 at 1 and 5 nM, and vinorelbine at 1 and 5 nM.
After 24 h, all the cells were harvested, dissociated, and washed with 2
mL of phosphate buffer saline (PBS). The cells were then fixed at +4
°C with cold 70% ethanol for 30 min and stored at −20 °C overnight.
Ethanol was removed by centrifugation, and 2 mL of PBS were added
to wash the pellets. The cellular DNA was stained with propidium
iodide (PI, 50 mg/mL, Sigma) and RNase (100 mg/mL, Sigma) for 30
min. The cell-cycle profiles were determined by flow cytometry on a
FC500 flow cytometer (Beckman-Coulter, France).
Molecular Modeling. Compounds 27 and 35 were model-built
using vinblastine as a template, essentially as reported earlier for other
vinca derivatives.26 Point charges for the energy-minimized geometries
were assigned by fitting the quantum mechanically calculated (RHF/6-
31G*//RHF/3-21G*) molecular electrostatic potential (MEP) using
Gaussian 03 (Gaussian, Inc., Wallingford, CT). Consistent bonded and
nonbonded AMBER parameters for these ligands were assigned by
analogy or through interpolation from those already present in the
AMBER database for protein atoms (ff03). 27 and 35 were manually
docked at the longitudinal interface between two tubulin heterodimers
by superimposing them onto the vinblastine structure in the tubulin−
vinblastine complex.3 The resulting complexes were first energy-
minimized in vacuo to remove any steric clashes within the binding
site and then immersed in a truncated octahedron containing ∼32300
TIP3P water molecules and 23 Na+ ions. The sander and pmemd
modules from the AMBER12 suite of programs (http://ambermd.org/
) were used for the restrained and unrestrained MD simulations,
respectively. Periodic boundary conditions were applied, and electro-
static interactions were treated using the smooth particle mesh Ewald
method with a grid spacing of 1 Å. The cutoff distance for the
nonbonded interactions was 9 Å, the SHAKE algorithm was applied to
all bonds, and an integration step of 2.0 fs was used throughout. After
an initial energy minimization of the water molecules and counterions,
the system was heated to 300 K in 25 ps, after which the solvent was
allowed to redistribute around the positionally restrained solute for
220 ps. After this time, the restraints were removed and the system was
further simulated for 20 ns. Snapshots from each 10 ns MD trajectory
were collected every 20 ps for structural and energetic analyses. The
ptraj module was used to assess the stability of the complexes by
calculating the root-mean-square deviations (RMSD) from the initial
geometries and for calculating a representative average structure for
each complex from the unrestrained MD simulations after removal of
the water molecules for visualization purposes. The molecular graphics
program PyMOL version 0.99 (DeLano Scientific, LLC, Palo Alto,
CA) was employed for visualization and model building.
Inhibition of Tubulin Assembly. The drug, dissolved in DMSO
at different concentrations, was added to a solution of free tubulin
(obtained from sheep brain and prepared according to a published
procedure29) at 0 °C. Then the solution was placed in a temperature
controlled cell at 37 °C (microtubule assembly), and the increase of
the optical density was monitored in a UV spectrophotometer at 350
nm (the maximum was reached in about 1 min). The maximum rate of
assembly was recorded and compared to a drug-free sample. The IC50
of the compound was calculated from the effect of several
concentrations and compared to the IC50 of vinorelbine obtained
within the same day with the same tubulin preparation.
Cell Culture and Proliferation Assay. Cancer cell lines were
obtained from the American Type Culture Collection (Rockville, MD,
USA) and were cultured according to the supplier’s instructions.
Human K562 leukemia cells and HCT116 colorectal carcinoma cells
were grown in RPMI 1640 supplemented with 10% fetal calf serum
(FCS) and 1% glutamine. U87-MG human glioblastoma cells were
grown in Dulbecco’s Minimal Essential Medium (DMEM) containing
10% FCS and L-glutamine. Human umbilical vein endothelial cells
(HUVECs) were obtained from Lonza (Walkersville, MD, USA) and
cultured according to the supplier’s instructions in endothelial cell
growth medium (EGM2) containing growth factors and 2% FCS. Cell
lines were maintained at 37 °C in a humidified atmosphere containing
5% CO2. Cell viability was assessed using Promega CellTiter-Blue
reagent (Promega, Madison, WI, USA) according to the manufac-
turer’s instructions. Briefly, the cells were seeded in 96-well plates (2.5
× 103 cells/well) containing 50 μL of growth medium. After 24 h of
culture, the cells were supplemented with 50 μL of medium containing
different concentrations of the tested compound dissolved in DMSO
(less than 0.1% in each preparation). After 72 h of incubation, 20 μL of
resazurin was added for 1.5 h before recording fluorescence (λex = 560
nm, λem = 590 nm) using a microtiter plate fluorimeter. The IC50
corresponds to the concentration of compound that induced a 50%
decrease in fluorescence of drug-treated cells compared with untreated
cells. Experiments were performed in triplicate.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra for compounds 20−37. 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@icsn.cnrs-gif.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. Marie Elise Tran Huu
Dau is also acknowledged for fruitful discussions. O.G.
gratefully acknowledges the ED425 “Innovation Ther
́
apeutique,
du Fondamental a for a grant and L’Oreal
̀
l′Applique”
́
́
Cell Cycle Analysis. Exponentially growing cancer cells were
incubated with compound 30, 33, or vinorelbine at two concentrations
or DMSO. For the K562 cell line, compound 30 was incubated at 10
and 50 nM, compound 33 at 1 and 10 nM, and vinorelbine at 5 and 10
Foundation, UNESCO, and the French Science Academy for
her “Pour les Femmes et la Science” prize. F.G. thankfully
acknowledges financial support from Comunidad Autonoma de
́
6098
dx.doi.org/10.1021/jm4004347 | J. Med. Chem. 2013, 56, 6088−6100

Journal of Medicinal Chemistry
Madrid (S-BIO/0214/2006) and Comision
Article
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́
Interministerial de
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VLB, vinblastine; VLN, vinorelbine; AVLB, anhydrovinblastine
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