Allocolchicinoids bearing a Michael acceptor fragment for possible irreversible binding of tubulin

Article abstract of DOI:10.1039/d0md00060d

We describe an attempt to apply the concept of covalent binding towards the highly active allocolchicinoids selected on the basis of SAR analysis of previously synthesized molecules. To achieve the irreversible binding of the agent to the cysteine residues of the colchicine site of tubulin protein, we synthesized a number of new allocolchicinoids bearing the acceptor moiety. Some of the new derivatives possess cytotoxic activity against COLO-357, BxPC-3, HaCaT, and HEK293 cell lines in a low nanomolar range of concentrations. A substoichiometric mode of microtubule assembly inhibition was demonstrated. The most active compounds possess close to colchicine general toxicity on mice. This journal is

Full text of DOI:10.1039/d0md00060d

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RESEARCH ARTICLE
Allocolchicinoids bearing a Michael acceptor
fragment for possible irreversible binding of
tubulin†
Cite this: DOI: 10.1039/
d0md00060d
a
a
b
b
Ekaterina S. Sazanova, Iuliia A. Gracheva,
Diane Allegro, Pascale Barbier,
cd
e
and Alexey Yu Fedorov *a
Sébastien Combes, Elena V. Svirshchevskaya
We describe an attempt to apply the concept of covalent binding towards the highly active
allocolchicinoids selected on the basis of SAR analysis of previously synthesized molecules. To achieve the
irreversible binding of the agent to the cysteine residues of the colchicine site of tubulin protein, we
synthesized a number of new allocolchicinoids bearing the acceptor moiety. Some of the new derivatives
possess cytotoxic activity against COLO-357, BxPC-3, HaCaT, and HEK293 cell lines in a low nanomolar
range of concentrations. A substoichiometric mode of microtubule assembly inhibition was demonstrated.
The most active compounds possess close to colchicine general toxicity on mice.
Received 22nd February 2020,
Accepted 10th April 2020
DOI: 10.1039/d0md00060d
rsc.li/medchem
The activity and/or selectivity of low molecular weight
therapeutic molecules can be increased by introduction a
Introduction
Malignant neoplastic diseases have long been the second
most common cause of death worldwide, and the treatment
reactive functional group designed for covalent binding to
specific sites in the target.
1
9
of cancer is still a crucial aspect of modern medicine. In the
case of usage of chemotherapeutic small molecules in cancer
therapy, the main disadvantages include low selectivity of
biological action and, as a result, high systemic toxicity. The
decrease in therapeutic activity is also often associated with
the emergence of the resistant tumor cells due to the
Target covalent inhibitors (TCI) have gained popularity in
the pharmaceutical sector in recent years as potentially more
1
0
beneficial in efficiency and selectivity: due to the high
strength of the covalent bond, the inhibitor (electrophile)
irreversibly attach to the target (nucleophile), leading to its
inactivation. The most significant progress has been made in
2
11
activation of various anti-apoptotic pathways, drug target
targeting the cysteine residues due to their relatively low
3
mutations preventing drug binding, increased expression of
prevalence and tendency to act as a nucleophile. In recent
years, several anticancer drugs have been introduced to the
pharmacological market by FDA, the mechanism of action of
4
proteins that compensate for the loss of the drug target, and
in many cases drug resistance is a result of the functioning of
the ATP-binding cassette proteins (P-gp and BCRP membrane
which involves covalent binding to cysteine residues of
5–8
12–14
pumps) removing xenobiotics from the cells.
Thereby, the
various proteins
(Fig. 1).
search for the new synthetically available antimitotic agents
with a high therapeutic index and improved pharmacokinetic
parameters is a matter of current interests.
Structures shown in Fig. 1a contain a Michael acceptor
fragment (highlighted in green), due to which covalent
binding to the free SH-group of protein cysteine residues is
possible (Fig. 1b). Other functional groups capable of reacting
via this mechanism include small strain cycles (electrophilic
cyclopropanes, epoxides, four-membered β-lactams or
β-lactones), aldehyde functions, amines, acetals, and others.
Such approach enabled the creation of a number of drugs
used in clinical practice for the treatment of various diseases,
a
Department of Chemistry, N. I. Lobachevsky State University of Nizhny Novgorod,
2
3 Gagarin Avenue, 603950 Nizhny Novgorod, Russian Federation
b
Institute of NeuroPhysiopathology (INP) – CNRS UMR 7051, Aix-Marseille
University, 27 Boulevard Jean Moulin, 13385 Marseille, Cedex 5, France
c
CRCM, CNRS, Inserm, Institut Paoli-Calmettes, Aix-Marseille University, 232
1
5–19
as well as molecules currently undergoing clinical trials.
Boulevard de Sainte-Marguerite, 13009 Marseille, France
d
DOSynth Platform, CRCM, Faculté de Pharmacie, Aix-Marseille Université, 27
Herein we suggest a series of allocolchicine derivatives
bearing Michael acceptor units enabling the compounds to
be potentially capable of covalent interaction with tubulin,
the key protein of cell division. In this case, the creation of
Boulevard Jean Moulin, 13385 Marseille, Cedex 5, France
e
Laboratory of Cell Interactions, Shemyakin-Ovchinnikov Institute of Bioorganic
Chemistry RAS, 16/10 Miklukho-Maklaya Street, 117997 Moscow, Russian
Federation
covalent inhibitors may lead to
a progress in drugs
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0md00060d
application against drug-resistant cancers. In the vast
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Fig. 2 Tubulin covalent inhibitors.
Currently, the only examples of TCI among colchicine
ligands are 2-demethyl-2-chloroacetyl thiocolchicine (2CTC)
2
7
and 3-demethyl-3-chloroacetyl thiocolchicine (3CTC) (Fig. 2),
synthesized to study the morphology of the binding site.
Studies with these derivatives showed the presence of
interaction between colchicine and Cys241 at the boundary
Fig. 1 Examples of covalent inhibitors in clinical practice (a), and
illustration of Michael reaction with biological thiols (b).
2
8,29
of the α,β-tubulin heterodimer.
majority of cases, the interaction of tubulin with a ligand
molecule is realized due to the formation of hydrogen bonds,
ionic, Van der Waals and hydrophobic interactions at the
protein binding site. Nevertheless, in the last decades, several
molecules binding to tubulin via covalent bonds have been
Results and discussion
Synthesis and investigation of acceptor properties
Acting as a mitotic poison, colchicine prevents the mitotic
spindle assembly, that leads to a block in mitosis, and reduces
2
0,21
30
discovered
(Fig. 2). Withaferin A, a steroidal lactone
cell motility. Moreover, as an immunosuppressant, colchicine
found in plants of the Solanaceae family, was shown to
possess anticancer activity in a variety of human cancer cells
in vitro and in vivo, including due to the covalent binding to
accumulates in the immune system cells, which brings about
3
1,32
the suppression of inflammatory reactions.
As most other
colchicine site ligands, this compound exerts a destructive
effect on the blood supply to tumors, preventing the formation
2
2
Cys303 of β-tubulin. Pironetin, an α/β unsaturated lactone,
originally isolated from fermentation solutions of
Streptomyces species, contains the Michael system for binding
3
3
of new vessels or destroying already formed capillaries. In
addition, colchicine site ligands are a little susceptible to
multidrug resistance associated with the alteration of tubulin
isotypes. In the human body, there are 8 isoforms of α-tubulin
and 7 isoforms of β-tubulin, their expression is tissue-specific.
Changes in the expression of tubulin isotypes are characteristic
of many types of cancer, and overexpression of βI, βII, βIII, βVIa
and βV-isoforms correlates with the aggressive course of the
disease, resistance to chemotherapy and, as a consequence,
with low patient survival. The effectiveness of colchicine site
ligands in turn does not depend on the composition of
2
3
to Cys316 of α-tubulin. Ottelion A (PRR 112378), first
isolated from the fresh water plant Ottelia alismoides, is a
highly cytotoxic compound with IC50 values in the pM–nM
range against a panel of 60 human cancer cell lines, and may
covalently bond with Cys241 residue by a 1,6-Michael
2
4
addition reaction. Batabulin (T138067) has long been a
promising inhibitor of tubulin, forming a covalent adduct by
interaction with Cys239 via the nucleophilic substitution
2
5
reaction in the pentafluorophenyl fragment, and reached
2
6
34,35
phase II clinical trials.
β-tubulin isotypes.
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Despite the unique combination of properties, colchicine
has not found so far an application in antitumor clinical
practice due to substantial and poorly controlled systemic
(Scheme 2). The choice of compound as an example 5c was
based on a compromise between the activity (Table 1) and
4
5
the double bond shielding. Under the given conditions, the
complete conversion of 5c to rac-13 was observed in 2 h. It
demonstrated electrophilic nature of the compound 5c.
3
6
toxicity even at therapeutic doses. Numerous attempts of
chemical modifications of colchicine aimed at reducing the
side effects have been realized. Rather successful examples
are presented mostly by synthetic allocolchicinoids made via
converting a seven-membered cycle C to a six-membered and/
Biological investigations
3
7–40
or creation a fused heterocyclic ring D.
Having this in
The in vitro cytotoxicity of the synthesized compounds 5a–j
and 12a, b towards the human epithelial cell lines (COLO-
357, BxPC-3, HaCaT, HEK293) and murine fibroblasts (L929)
was investigated. A tetrazolium-based assay was used to
determine the drug concentration required to inhibit cell
growth by 50% after incubation in the culture medium for 72
hours. The calculated IC50 values are summarized in Table 1.
The experimental data show that the ester derivatives (5a,
5c, 5e, 5g, 5i, and 12a) are more active than the compounds
where the acceptor fragment is attached to the core molecule
via the amide function (5b, 5d, 5f, 5h, 5j, and 12b). All
compounds obtained are active in the nanomolar
concentrations against epithelial cell lines, while the 5i
derivative is active in concentrations of hundreds of
picomoles.
Inhibition of the cell cycle in comparison with colchicine
(1) was studied for the most active compounds 5c and 5i in
COLO-357 and low sensitive L929 cells in the same
concentrations of 5 μM. Derivatives 5c and 5i as well as
colchicine (1) effectively induced cell accumulation in the G2/
M phase corresponding to non-dividing cells, which steadily
resulted in apoptosis (Fig. 4).
mind, and with the idea of the introduction of Michael
4
1–43
acceptor units,
we designed structures of potential
irreversible tubulin inhibitors with a selected number of
attached fragments allowing them to react with target
cysteine residues (Fig. 3).
For the synthesis of colchicinoid agents with expected
capability of covalent binding to tubulin, we used
4
4
colchicinoid intermediates previously described by us.
Commercial colchicine (1) was converted into
deacetylallocolchicine 2 by a three-step cleavage of the
acetamide group in 56% yield. In the next step, the amino
group in 2 was acylated with glycolic acid or protected glycine
under Steglich conditions to give allocolchicines 3 and 4 in
good yields. In another route, colchicine (1) was converted to
4
4
iodocolchinol 6 in 2 steps according to the known protocol
with 88% yield. Deacetylation of 6 by procedure similar to
the cleavage of Ac-group of colchicine (1) led to
deacetyliodocolchinol 7. After the acylation of the amino
group in 7 with glycolic acid or N-Boc-glycine under Steglich
conditions, derivatives 8 and 9 were subjected to catalytic
tandem
Sonogashira
coupling/Larock-type
cyclization
sequence with propargyl acetate and propargyl alcohol,
respectively, to give furano-allocolchicinoids 10 and, after
deprotection, 11. Finally, the α,β-unsaturated carbonyl
fragment was introduced into the structures of
allocolchicinoids by reactions with a number of unsaturated
acids under the Steglich conditions to afford the target
derivatives 5a–j and 12a, b (Scheme 1).
To test the Michael-type reactivity of the synthesized
compounds towards thiol nucleophiles, the reaction of
compound 5c with protected cysteine was performed
To visualize the full population of cells after 5c, 5i and
colchicine (1) treatment, monolayer COLO-357 cultures were
analyzed by confocal microscopy. All the studied compounds
were found to effectively disrupt the mitotic spindle, which
led to the scattering of chromosomes, and the inability of
cells to divide (Fig. 5).
To define the therapeutic range of the most promising
compounds, we determined intravenous LD50 and LD100
acute toxicity of 5c, 5i and 1 in C57BL/6 mice. The LD50/LD100
−
1
for 5c and 5i were 6.5/9.8 and 6.1/8.4 mg kg , respectively,
−
1
versus 8.5/10.9 mg kg for colchicine (1). Lethality was
observed in 40–48 h for 5c and in 24 h for 5i after injections.
Thus, the new compounds possess systemic toxicity close to
colchicine.
In order to investigate is the cytotoxic effect of compounds
of type
5 and 12 with Michael acceptor fragment a
consequence of their strong binding to tubulin, we studied
the effects of 5i on microtubule formation. Fig. 6 shows the
effects of colchicine (1) (panel A) as positive control, and 5i
(
panel B), on the turbidimetry time course of microtubule
assembly from pure tubulin. A clear inhibition was noted,
and the rate of assembly as well as the final amount of
microtubules was lower in the presence of ligands than in
the control experiment. When the samples were cooled to 15
°
C, the polymers depolymerized (data not shown). The insets
Fig. 3 Structure of colchicine and design of allocolchicine derivatives
bearing Michael acceptor units.
of panel A and B of Fig. 6 show that the extent of inhibition
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2 3 3
Scheme 1 Synthesis of target allocolchichine derivatives 5, 12. Reagents and conditions: (a) Boc O, DMAP, Et N, CH CN, reflux, 3 h; (b) MeONa,
MeOH, 40 °C, 1 h; (c) TFA, CH
f–j, 12a, b), DIC, NHS, Et N, CH
compd 5e; (e) 0.1 M HCl, AcOH, 100 °C, 3 h; (f) NaOH, I
CH CN, reflux, 26 h; (i) NaOMe (20 mol%), MeOH, rt, 1.5 h; (j) HCl, EtOH, rt, 20 h; (k) propargyl acetate (for 10) or propargyl alkohol (for 11),
PdIJOAc) (0.05 equiv.), CuI (0.1 equiv.), AcOK (3 equiv.), Ph P (0.15 equiv.), CH CN, 70 °C, 10 h.
2
Cl
2
, r.t., 1 h; (d) glycolic acid (for compds 3, 8) or N-Boc-glycine (for 4, 9), or appropriate unsaturated acid (for 5a–d,
, rt, 16 h; then HCl, CH Cl , rt, 1 h in case of 4 and 9; 3,3-dimethylacrylic acid, DCC, DMAP, CH Cl , rt, 16 h for
, KI, H O, 0–5 °C, 2 h; (g) MOMCl, DIPEA, CH Cl , 0–20 °C, 20 h; (h) Boc O, DMAP, Et N,
3
2
Cl
2
2
2
2
2
2
2
2
2
2
3
3
2
3
3
Scheme 2 Testing of reactivity of compd 5c towards thiol nucleophiles.
by colchicine (1) and 5i, respectively, increased monotonically
with the mole ratio of the total ligand to total tubulin in the
solution (R). In these figures, 50% inhibition occurred at a
mole ratio of 0.27 mol (IC50 = 4.89 μM) of colchicine (1) per
mol of tubulin and at a mole ratio of 0.37 mol (IC50 = 6.78
μM) of 5i per mol of tubulin. Hence, compound 5i
demonstrates similar to colchicine substoichiometric mode
of microtubule formation inhibition.
Summarizing the biological studies, it can be concluded
that
a
surprisingly high activity, comparable to
4
4
allocolchicine, suggests that the acceptor fragment might
perform its function in tubulin binding. However, non-
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Table 1 In vitro cytotoxicity of the target compounds (IC50, nM)a
Compound
COLO-357
BxPC-3
HaCaT
HEK293
L929
Colchicine (1)
16
16
400
16
80
16
80
16
80
3
16
16
80
16
80
3
16
16
16
3
3
16
16
400
3
80
16
16
16
80
3
2000
5
5
5
5
5
5
5
5
5
5
1
1
a
b
c
d
e
f
g
h
i
j
2a
2b
16
80
3
80
3
16
16
16
0.6
3
0.6
400
16
>10 000
>10 000
2000
>10 000
2000
2000
2000
2000
80
16
16
400
80
3
16
3
400
16
2000
16
400
16
10 000
2000
b
rac-13
—
a
b
IC50 concentration inducing 50% inhibition of cell growth. Not determined.
specific interactions with other proteins or binding to non- Experimental
protein targets might occur that causes significant systemic
toxicity.
General materials and methods
Commercially available reagents (Aldrich, Alfa Aesar, Acros,
ABCR) were used without additional purification. Column
chromatography was performed using Macherey-Nagel
Conclusions
1
13
19
Kieselgel 60 (70–230 mesh). All H, C and F NMR spectra
were recorded at 25 °C in DMSO-d6 on Agilent DD2 400
instrument. Chemical shifts (δ) are reported in parts per
million (ppm) from TMS using the residual solvent resonance
In this work, we investigated the strategy associated with the
insertion of Michael acceptor fragments into colchicinoids to
target an efficient binding of the agents to the cysteine
residues of the colchicine-binding site of tubulin protein.
1
13
(
DMSO-d : 2.50 ppm for H NMR, 39.52 ppm for C NMR).
6
Covalent binding to a CH SH-fragment of cysteine is possible
2
Standard abbreviations are used to indicate multiplicities.
at least in non-physiological conditions. For all compounds,
high tubulin binding activity and low nanomolar inhibition
concentration of epithelial cells proliferation, in the range of
the best-known tubulin inhibitors, was demonstrated.
Further work in this direction is currently underway.
Fig. 5 Effect of colchicine (1) and compounds (5c) and (5i) on mitotic
spindle formation and β-tubulin expression in pancreatic epithelial cells
COLO-357. COLO-357 cells (seeded at 10 /glass) were incubated for
72 h without (a) or with 5 μM of colchicine (1) (b), 5c (c), or 5c (d), fixed,
stained with anti-βtubulin antibody (red) and nuclei stain Hoechst
33342 (blue). Scale bars correspond to 16–18 μm.
Fig. 4 Induction of cell cycle arrest by colchicine (1) and compds 5c
and 5i. COLO-357 (column a) and L929 (column b) cells were treated
for 72 h with 5 μM of colchicine (1), 5c or 5i, respectively; trypsinized,
permeabilized, and stained with PI. The percentage distribution of cells
is indicated in the tables. Control cells are shown in grey.
5
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stirred for 16 h at room temperature under an inert
atmosphere. The crude product obtained after solvent
removal was purified by column chromatography using
CH Cl /MeOH or PE/EtOAc/EtOH as eluent.
2
2
Methyl
(5S,
aR)-5-(2-(acryloyloxy)acetamido)-9,10,11-
trimethoxy-6,7-dihydro-5H-dibenzoija,c]cyclohepten-3-
carboxylate (5a). Purified by column chromatography (PE/
1
EtOAc/EtOH 2 : 1 : 1); white solid (45%); m.p. 163 °C; H NMR
(
1
400 MHz, DMSO-d
H, C8–H), 7.12 (d, J = 12.7 Hz, 1H, C10–H), 7.03 (d, J = 10.8
Hz, 1H, C11–H), 6.77 (s, 1H, C4–H), 6.34 (dd, J = 17.3, 1.7 Hz,
6
) δ 8.80 (d, J = 7.5 Hz, 1H, NH), 7.13 (s,
1
=
1
3
H, C5′–H), 6.21 (dd, J = 17.3, 10.2 Hz, 1H, C4′–H), 5.97 (dd, J
10.2, 1.6 Hz, 1H, C5′–H), 4.61 (s, 2H, C2′H ), 4.35 (dt, J =
2
3.1, 7.0 Hz, 1H, C7–H), 3.87 (s, 3H, OMe), 3.83 (s, 3H, OMe),
.78 (s, 3H, OMe), 3.51 (s, 3H, OMe), 2.64–2.59 (m, 1H,
C5H ), 2.23 (td, J = 13.0, 7.0 Hz, 1H, C5H ), 2.03 (dt, J = 12.2,
2
2
1
3
6
.0 Hz, 1H, C6H ), 1.88 (m, 1H, C6H ); C NMR (101 MHz,
2 2
6
DMSO-d ) δ 177.96, 165.99, 164.89, 163.54, 152.97, 150.43,
1
1
5
4
50.13, 140.76, 134.97, 134.44, 134.12, 132.24, 130.41, 127.79,
25.33, 112.08, 107.78, 62.19, 60.82, 60.68, 56.05, 55.85,
+
1.21, 35.71, 29.14; MS (MALDI-TOF): m/z = 470.0 [M + H] ,
+
92.0 [M + Na] ; elemental analysis calcd (%) for C H NO :
25
27
8
C 63.96, H 5.80, N 2.98; found: C 64.19, H 6.03, N 3.10.
Methyl (5S, aR)-5-(2-acrylamidoacetamido)-9,10,11-
Fig. 6 Effect of ligands on the turbidity time course of in vitro
microtubule assembly. The reaction was started by warming the
solution from 15 to 37 °C. (A) Effect of various concentrations of
colchicine (1) (a: 0 μM; b: 1 μM; c: 3 μM; d: 5 μM; and e: 7 μM) on
tubulin at 18 μM in polymerization buffer. (B) Effect of various
concentrations of 5i (a: 0 μM; b: 2 μM; c: 4 μM; d: 6 μM; and e: 10 μM)
on tubulin at 18 μM in polymerization buffer. The insets represent the
percentage of assembly inhibition as a function of the mole ratio of
the total ligand to total tubulin in the solution (R).
trimethoxy-6,7-dihydro-5H-dibenzoija,c]cyclohepten-3-
carboxylate (5b). Purified by column chromatography (PE/
1
EtOAc/EtOH 2 : 1 : 1); white solid (42%); m.p. 254 °C; H NMR
(400 MHz, DMSO-d ) δ 8.72 (d, J = 7.3 Hz, 1H, C7–NH), 8.27
6
(
1
t, J = 5.8 Hz, 1H, C2′–NH), 7.12 (s, 1H, C8–H), 7.11 (d, J =
0.1 Hz, 1H, C10–H), 7.02 (d, J = 10.8 Hz, 1H, C11–H), 6.77
(
(
s, 1H, C4–H), 6.26 (dd, J = 17.1, 10.2 Hz, 1H, C4′–H), 6.05
dd, J = 17.1, 2.2 Hz, 1H, C5′–H), 5.56 (dd, J = 10.2, 2.3 Hz,
1
H, C5′–H), 4.34 (dt, J = 12.8, 7.0 Hz, 1H, C7–H), 3.87 (s, 3H,
OMe), 3.83 (s, 3H, OMe), 3.78 (s, 3H, OMe), 3.51 (s, 3H,
OMe), 2.60 (q, J = 5.5 Hz, 1H, C5H ), 2.22 (m, 1H, C5H ), 2.02
(dt, J = 12.8, 6.3 Hz, 1H, C6H ), 1.90–1.82 (m, 1H, C6H
Allocolchicine atom numbering was used for signal
assignment of allocolchicinoids. EI mass spectra (70 eV) were
obtained on a DSQ II mass spectrometer (Thermo Electron
Corporation) with a quadrupole mass analyzer. ESI mass
spectra were recorder on a Polaris Q (Thermo Finnigan) mass
spectrometer. MALDI mass spectra were obtained on a
MALDI-TOF mass spectrometer Bruker Microflex LT.
Combustion analysis was performed using an Elementar
2
2
1
3
2
2
);
C
NMR (101 MHz, DMSO-d ) δ 177.98, 168.12, 164.75, 163.53,
6
152.94, 150.50, 150.44, 140.74, 135.05, 134.36, 134.19, 131.45,
130.37, 125.41, 125.36, 112.07, 107.81, 60.79, 60.71, 56.05,
55.86, 51.42, 41.71, 35.71, 29.16; MS (MALDI-TOF): m/z =
+
491.0 [M + Na] ; elemental analysis calcd (%) for C25
28 2 7
H N O :
(
Vario Micro Cube) apparatus. Optical rotation [α]
D
was
C 64.09, H 6.02, N 5.98; found: C 64.31, H 6.30, N 5.86.
Methyl (5S, aR)-5-(2-(((E)-but-2-enoyl)oxy)acetamido)-
measured on JASCO P-2000 polarimeter at 20 °C and λ 589
nm (cuvette length: 1.0 dm, volume: 1.0 mL); concentration
is given in g/100 mL. Solvents were purified according to the
standard procedures. Petroleum ether (PE) used is the
fraction with bp 40–70 °C. Compounds 3, 4, 10 and 11 were
prepared according to the previously described procedures.
Atomic numeration is given only for NMR assignment, for
details see ESI.†
9,10,11-trimethoxy-6,7-dihydro-5H-dibenzoija,c]cyclohepten-3-
carboxylate (5c). Purified by column chromatography (PE/
1
EtOAc/EtOH 3 : 1 : 1); white solid (37%); m.p. 144 °C; H NMR
6
(400 MHz, DMSO-d ) δ 8.77 (d, J = 7.4 Hz, 1H, NH), 7.13 (s,
4
4
1H, C8–H), 7.11 (d, J = 11.0 Hz, 1H, C10–H), 7.02 (d, J = 10.8
Hz, 1H, C11–H), 6.92 (dd, J = 15.6, 6.9 Hz, 1H, C5′–H), 6.77
(s, 1H, C4–H), 5.93 (dd, J = 15.6, 1.8 Hz, 1H, C4′–H), 4.56 (s,
General procedure for the synthesis of Michael acceptors
2H, C2′H
OMe), 3.83 (s, 3H, OMe), 3.78 (s, 3H, OMe), 3.51 (s, 3H,
OMe), 2.64–2.59 (m, 1H, C5H ), 2.22 (td, J = 13.1, 7.1 Hz, 1H,
C5H ), 2.02 (dt, J = 12.6, 6.0 Hz, 1H, C6H ), 1.93–1.87 (m, 1H,
2
), 4.34 (dt, J = 13.7, 6.9 Hz, 1H, C7–H), 3.87 (s, 3H,
5
a–d, f–j and 12a, b. Compound 3 (4) or 10 (11) (1 equiv.),
corresponding α,β-unsaturated acid (1 equiv.), and NHS (1
equiv.) were dissolved in CH Cl (20 mL per 1 mmol of
starting materials). 1,3-Diisopropylcarbodiimide (DIC; 1.5
equiv.) and Et N (3 equiv.) were added, and the mixture was
2
2
2
2
2
1
3
C6H ), 1.85 (dd, J = 6.9, 1.7 Hz, 3H, C6′H ); C NMR (101
2
3
3
MHz, DMSO-d
6
) δ 177.97, 166.22, 164.94, 163.54, 152.96,
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1
1
5
4
50.43, 150.18, 146.12, 140.76, 134.98, 134.43, 134.14, 130.41,
25.35, 121.68, 112.09, 107.79, 61.89, 60.83, 60.70, 56.06,
5.86, 51.19, 35.69, 29.15, 17.75; MS (MALDI-TOF): m/z =
7.0 Hz, 1H, C5H ), 2.03 (tt, J = 12.3, 6.7 Hz, 1H, C6H ), 1.94–
2
2
1
3
1.86 (m, 1H, C6H2); C NMR (101 MHz, DMSO-d ) δ 177.94,
6
165.52, 163.54, 163.00, 152.97, 150.43, 150.02, 140.76, 134.93,
134.48, 134.10, 131.14, 130.80, 130.40, 129.20, 125.31, 112.10,
107.78, 63.01, 60.81, 60.68, 56.05, 55.86, 51.25, 35.68, 29.12;
+
+
84.0 [M + H] , 506.0 [M + Na] ; elemental analysis calcd (%)
for C H NO : C 64.59, H 6.05, N 2.90; found: C 64.29, H
26
29
8
1
9
6
.25, N 2.69.
F NMR (376 MHz, DMSO-d
6
) δ −64.05; MS (MALDI-TOF): m/
+
Methyl
(5S,
aR)-5-(2-((E)-but-2-enamido)acetamido)-
z = 537.9 [M + H] ; elemental analysis calcd (%) for
9
,10,11-trimethoxy-6,7-dihydro-5H-dibenzoija,c]cyclohepten-3-
C H F NO : C 58.10, H 4.88, N 2.61; found: C 58.42, H 5.17,
26
26
3
8
carboxylate (5d). Purified by column chromatography (PE/
EtOAc/EtOH 2 : 1 : 1); white solid (49%); m.p. 223 °C; H NMR
N 2.44.
Methyl
1
(5S,
aR)-9,10,11-trimethoxy-5-(2-((E)-4,4,4-
(
(
400 MHz, DMSO-d ) δ 8.66 (d, J = 7.4 Hz, 1H, C7–NH), 8.04
t, J = 5.8 Hz, 1H, C2′–NH), 7.12 (s, 1H, C8–H), 7.11 (d, J = 8.5
trifluorobut-2-enamido)acetamido)-6,7-dihydro-5H-dibenzoija,
c]cyclohepten-3-carboxylate (5h). Purified by column
6
Hz, 1H, C10–H), 7.02 (d, J = 10.9 Hz, 1H, C11–H), 6.77 (s, 1H,
C4–H), 6.63–6.53 (m, 1H, C5′–H), 5.93 (dd, J = 15.3, 1.8 Hz,
chromatography (PE/EtOAc/EtOH 3 : 1 : 1); white solid (39%);
1
m.p. 191 °C; H NMR (400 MHz, DMSO-d ) δ 8.74 (t, J = 7.5,
6
1
H, C4′–H), 4.33 (dt, J = 13.2, 6.7 Hz, 1H, C7–H), 3.87 (s, 3H,
7.1 Hz, 2H, 2xNH), 7.12 (s, 1H, C8–H), 7.12 (d, J = 10.6 Hz,
1H, C10–H), 7.02 (d, J = 10.8 Hz, 1H, C11–H), 6.85 (dd, J =
15.7, 1.9 Hz, 1H, C4′–H), 6.78 (s, 1H, C4–H), 6.76 (dd, J =
15.8, 2.0 Hz, 1H, C5′–H), 4.34 (dt, J = 13.0, 6.8 Hz, 1H, C7–H),
OMe), 3.83 (s, 3H, OMe), 3.78 (s, 3H, OMe), 3.50 (s, 3H,
OMe), 2.63–2.57 (m, 1H, C5H ), 2.22 (td, J = 13.0, 7.0 Hz, 1H,
C5H ), 2.01 (dp, J = 19.1, 6.4 Hz, 1H, C6H ), 1.90–1.83 (m,
2 2
1
2
1
3
H, C6H ), 1.76 (dd, J = 6.9, 1.7 Hz, 3H, C6′H ); C NMR
3.90 (dd, J = 7.9, 5.9 Hz, 2H, C2′H ), 3.87 (s, 3H, OMe), 3.83
2
3
2
(101 MHz, DMSO-d
6
) δ 177.99, 168.34, 165.06, 163.53, 152.94,
(s, 3H, OMe), 3.78 (s, 3H, OMe), 3.51 (s, 3H, OMe), 2.60 (q, J
1
1
5
4
50.54, 150.44, 140.74, 138.03, 135.07, 134.35, 134.20, 130.39,
25.57, 125.42, 112.07, 107.80, 60.79, 60.72, 56.06, 55.87,
1.40, 41.66, 35.71, 29.17, 17.34; MS (MALDI-TOF): m/z =
2
= 8.3, 6.5, 4.9 Hz, 1H, C5H ), 2.23 (td, J = 13.1, 7.0 Hz, 1H,
C5H ), 2.03 (dp, J = 12.6, 6.3 Hz, 1H, C6H ), 1.87 (td, J = 12.2,
2
2
1
3
2 6
7.0 Hz, 1H, C6H ); C NMR (101 MHz, DMSO-d ) δ 178.41,
+
+
83.1 [M + H] , 505.1 [M + Na] ; elemental analysis calcd (%)
167.99, 163.98, 162.50, 153.39, 150.89, 150.82, 141.19, 135.45,
134.83, 134.61, 133.07, 133.01, 130.79, 125.84, 112.51, 108.25,
for C H N O : C 64.72, H 6.27, N 5.81; found: C 64.94, H
26
30 2 7
1
9
6
.58, N 5.65.
Methyl (5S, aR)-9,10,11-trimethoxy-5-(2-(3-methylbut-2-
enamido)acetamido)-6,7-dihydro-5H-dibenzoija,c]cyclohepten-
-carboxylate (5f). Purified by column chromatography (PE/
61.21, 61.15, 56.50, 56.30, 51.92, 42.46, 36.13, 29.59; F NMR
(376 MHz, DMSO-d ) δ −63.37; MS (MALDI-TOF): m/z = 558.9
[M + Na] ; elemental analysis calcd (%) for C H F N O : C
6
+
2
6
27 3 2 7
3
58.21, H 5.07, N 5.22; found: C 58.52, H 5.25, N 5.00.
Methyl (5S, aR)-5-(2-(((2E,4E)-hexa-2,4-dienoyl)oxy)-
acetamido)-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzoija,c]-
cyclohepten-3-carboxylate (5i). Purified by column
chromatography (CH Cl /MeOH 20 : 1); white solid (86%); m.
1
EtOAc/EtOH 2 : 1 : 1); white solid (51%); m.p. 159 °C; H NMR
(
(
400 MHz, DMSO-d ) δ 8.61 (d, J = 7.4 Hz, 1H, C7–NH), 7.86
t, J = 5.9 Hz, 1H, C2′–NH), 7.12 (s, 1H, C8–H), 7.11 (d, J =
6
1
0.0 Hz, 2H, C10–H), 7.01 (d, J = 10.8 Hz, 1H, C11–H), 6.77
s, 1H, C4–H), 5.66 (s, 1H, C4′–H), 4.33 (dt, J = 13.0, 6.9 Hz,
H, C7–H), 3.87 (s, 3H, OMe), 3.83 (s, 3H, OMe), 3.78 (s, 3H,
OMe), 3.51 (s, 3H, OMe), 2.60 (q, J = 7.4, 6.8 Hz, 1H, C5H ),
.22 (td, J = 13.0, 7.1 Hz, 1H, C5H ), 2.04 (s, 3H, C5′CH ),
2
2
1
(
1
p. 173 °C; H NMR (400 MHz, DMSO-d ) δ 8.77 (d, J = 7.4 Hz,
6
1H, NH), 7.27–7.19 (m, 1H, C5′–H), 7.13 (s, 1H, C8–H), 7.11
(d, J = 10.8 Hz, 1H, C10–H), 7.02 (d, J = 10.9 Hz, 1H, C11–H),
6.77 (s, 1H, C4–H), 6.35–6.24 (m, 1H, C6′H), 5.89 (d, J = 15.2
Hz, 1H, C7′–H), 5.56 (d, J = 7.9 Hz, 1H, C4′–H), 4.57 (s, 2H,
2
2
1
1
2
3
2
.99 (dd, J = 12.6, 6.4 Hz, 1H, C6H ), 1.86 (td, J = 11.9, 7.0 Hz,
H, C6H
1
3
2
), 1.75 (s, 3H, C5′CH
3
); C NMR (101 MHz, DMSO-
C2′H
3H, OMe), 3.78 (s, 3H, OMe), 3.51 (s, 3H, OMe), 2.60 (dd, J =
13.3, 5.9 Hz, 1H, C5H ), 2.22 (td, J = 13.1, 7.2 Hz, 1H, C5H ),
2.06–1.98 (m, 1H, C6H ), 1.93–1.86 (m, 1H, C6H ), 1.82 (d, J =
2
), 4.38–4.31 (m, 1H, C7–H), 3.87 (s, 3H, OMe), 3.83 (s,
d6) δ 177.97, 168.52, 166.06, 163.52, 152.93, 150.53, 150.43,
1
1
2
5
49.01, 140.73, 135.06, 134.31, 134.18, 130.40, 125.42, 118.70,
12.04, 107.78, 60.78, 60.70, 56.03, 55.85, 51.34, 41.43, 35.72,
9.17, 26.78, 19.29; MS (MALDI-TOF): m/z = 497.1 [M + H] ,
19.1 [M + Na] ; elemental analysis calcd (%) for C27
2
2
2
2
+
13
4.9 Hz, 3H, C8′H3); C NMR (101 MHz, DMSO-d ) δ 177.96,
6
+
32 2 7
H N O :
166.26, 165.70, 163.53, 152.96, 150.42, 150.17, 145.75, 140.75,
140.50, 134.98, 134.42, 134.13, 130.42, 129.58, 125.34, 118.00,
112.08, 107.77, 60.82, 60.68, 56.05, 55.85, 51.19, 35.68, 29.14,
18.46; MS (EI) m/z (%) = 509.6 (100), 508.2 (48), 480.3 (20),
368.4 (9), 338.2 (14), 327.3 (9); elemental analysis calcd (%)
for C H NO : C 66.00, H 6.13, N 2.75; found: C 65.72, H
C 65.31, H 6.50, N 5.64; found C 65.02, H 6.68, N 5.88.
Methyl (5S, aR)-9,10,11-trimethoxy-5-(2-(((E)-4,4,4-
trifluorobut-2-enoyl)oxy)acetamido)-6,7-dihydro-5H-dibenzoija,
c]cyclohepten-3-carboxylate (5g). Purified by column
chromatography (PE/EtOAc/EtOH 3 : 1 : 1); white solid (49%);
m.p. 150 °C; H NMR (400 MHz, DMSO-d
2
8
31
8
1
6
) δ 8.81 (d, J = 7.5
6.25, N 2.98.
Hz, 1H, NH), 7.16 (d, J = 6.8 Hz, 1H, C5′–H), 7.13 (s, 1H, C8–
H), 7.12 (d, J = 10.3 Hz, 1H, C10–H), 7.03 (d, J = 10.8 Hz, 1H,
C11–H), 6.78 (dd, J = 15.9, 2.1 Hz, 1H, C4′–H), 6.78 (s, 1H,
Methyl
(5S,
aR)-5-(2-((2E,4E)-hexa-2,4-dienamido)-
acetamido)-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzoija,c]-
cyclohepten-3-carboxylate (5j). Purified by column
C4–H), 4.68 (s, 2H, C2′H
H), 3.87 (s, 3H, OMe), 3.83 (s, 3H, OMe), 3.78 (s, 3H, OMe),
.51 (s, 3H, OMe), 2.63–2.58 (m, 1H, C5H ), 2.24 (td, J = 13.0,
2
), 4.36 (dt, J = 13.0, 6.9 Hz, 1H, C7–
chromatography (PE/EtOAc/EtOH 3 : 1 : 1); white solid (69%);
1
m.p. 164 °C; H NMR (400 MHz, DMSO-d ) δ 8.67 (d, J = 7.3
6
3
2
Hz, 1H, C7–NH), 8.12 (t, J = 5.7 Hz, 1H, C3′–NH), 7.13 (s, 1H,
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C8–H), 7.11 (d, J = 10.9 Hz, 1H, C10–H), 7.01 (d, J = 11.5 Hz,
55.83, 48.69, 42.11, 38.09, 29.96, 18.26. MS (ESI): m/z (%) =
1
6
H, C11–H), 6.99–6.92 (m, 1H, C5′–H), 6.77 (s, 1H, C4–H),
.22–6.03 (m, 2H, C6′–H, C7′–H), 5.93 (d, J = 15.2 Hz, 1H,
520.2 (30), 380.3 (30), 368.3 (100), 352.3 (70), 337.3 (28), 321.3
(16), 250.3 (15); elemental analysis calcd (%) for C29
32 2 7
H N O :
C4′–H), 4.33 (dt, J = 12.8, 6.9 Hz, 1H, C7–H), 3.87 (s, 3H,
C 66.91, H 6.20, N 5.38; found: C 66.62, H 6.35, N 5.14.
OMe), 3.83 (s, 3H, OMe), 3.78 (s, 3H, OMe), 3.50 (s, 3H,
Synthesis of methyl (5S, aR)-9,10,11-trimethoxy-5-(2-((3-
methylbut-2-enoyl)oxy)acetamido)-6,7-dihydro-5H-dibenzoija,
c]cyclohepten-3-carboxylate (5e). Compound 3 (60.0 mg, 0.144
mmol), DCC (59.4 mg, 0.288 mmol), and DMAP (43.9 mg,
OMe), 2.61 (d, J = 6.2 Hz, 1H, C5H
2
), 2.22 (td, J = 13.1, 7.2 Hz,
1
1
H, C5H ), 2.01 (tt, J = 12.7, 6.3 Hz, 1H, C6H ), 1.86 (td, J =
2
2
1
3
2.0, 7.2 Hz, 1H, C6H ), 1.77 (d, J = 6.4 Hz, 3H, C8′H );
C
2
3
NMR (101 MHz, DMSO-d
6
) δ 177.99, 168.34, 165.50, 163.53,
2 2
0.360 mmol) were dissolved in CH Cl (14 mL), then 3,3-
1
1
6
52.94, 150.52, 150.44, 140.75, 139.46, 136.75, 135.06, 134.34,
34.19, 130.39, 129.89, 125.42, 122.60, 112.06, 107.80, 60.78,
0.71, 56.05, 55.86, 51.41, 41.79, 35.72, 29.17, 18.25; MS (EI):
dimethylacrylic acid (14.4 mg, 0.144 mmol) was added and
the mixture was stirred for 16 h at room temperature under
an inert atmosphere. The crude product obtained after
solvent removal was purified by column chromatography,
m/z (%) = 508.9 (52), 508.1 (100), 507.3 (70), 479.7 (38), 355.6
18), 328.2 (34), 313.5 (16), 223.6 (13), 209.4 (12); elemental
analysis calcd (%) for C28 : C 66.13, H 6.34, N 5.51;
found: C 66.37, H 6.61, N 5.66.
′,2′,3′-Trimethoxybenzoij5′,6′:5,4]1H-(aR, 1S)-1-((((2‴E,4‴E)-
(
eluent PE/EtOAc/EtOH 3 : 1 : 1, to afford 5e as a white solid
1
H N O
32 2 7
(47.0 mg, 65%); m.p. 175 °C; H NMR (400 MHz, DMSO-d
6
) δ
8.75 (d, J = 7.4 Hz, 1H, NH), 7.13 (s, 1H, C8–H), 7.11 (d, J =
11.5 Hz, 1H, C10–H), 7.02 (d, J = 10.8 Hz, 1H, C11–H), 6.77
(s, 1H, C4–H), 5.74 (m, 1H, C4′–H), 4.51 (d, J = 3.1 Hz, 2H,
1
hexa-2‴,4‴-dienoyl)oxy)acetamido)-6,7-dihydrocycloheptaij3,2:
f ]-2″-acetoxymethylbenzofuran (12a). Purified by column
C2′H ), 4.35 (dt, J = 11.4, 6.9 Hz, 1H, C7–H), 3.87 (s, 3H,
2
chromatography (PE/EtOAc/EtOH 8 : 1 : 1); white solid (31%);
OMe), 3.83 (s, 3H, OMe), 3.78 (s, 3H, OMe), 3.51 (s, 3H,
OMe), 2.60 (dd, J = 13.3, 6.0 Hz, 1H, C5H), 2.22 (td, J = 12.8,
1
m.p. 139 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.68 (d, J = 8.0
Hz, 1H, NH), 7.56 (s, 1H, C4″–H), 7.50 (s, 1H, C7″–H), 7.25
7.1 Hz, 1H, C5H), 2.08 (d, J = 1.3 Hz, 3H, C5′–CH ), 2.01 (dt, J
3
(
(
m, 1H, C3‴–H), 7.00 (s, 1H, C3″–H), 6.80 (s, 1H, C4′–H), 6.30
dd, J = 6.2, 2.9 Hz, 2H, C4‴–H, C5‴–H), 5.92 (d, J = 15.3 Hz,
= 12.5, 6.2 Hz, 1H, C6H, the other C6H-signal is under
DMSO), 1.88 (d, J = 1.4 Hz, 3H, C5′–CH ); C NMR (101
3
1
3
1
H, C2‴–H), 5.22 (s, 2H, 2″–CH OAc), 4.57 (dd, J = 11.2, 6.5
MHz, DMSO-d ) δ 177.96, 166.41, 164.91, 163.53, 158.10,
2
6
Hz, 1H, C1–H), 3.84 (s, 3H, OMe), 3.79 (s, 3H, OMe), 3.51 (s,
H, 1-NHC(O)C H_ ), 3.39 (s, 3H, OMe), 2.56–2.52 (m, 1H, C6–
H), 2.21–2.13 (m, 1H, C6–H), 2.09 (s, 3H, OAc), 2.03–1.91 (m,
152.96, 150.42, 150.20, 140.75, 134.99, 134.41, 134.13, 130.44,
125.35, 114.91, 112.08, 107.78, 61.40, 60.82, 60.69, 56.05,
55.85, 51.14, 35.68, 29.16, 26.89, 19.97; MS (ESI): m/z (%) =
497.2 (29), 414.2 (19), 369.3 (42), 338.3 (62), 312.3 (100), 281.3
(59), 254.3 (28), 239.3 (18), 208.3 (11); elemental analysis
calcd (%) for C H NO : C 65.18, H 6.28, N 2.82; found: C
2
2
1
3
2
H, C7H
2
), 1.82 (d, J = 5.2 Hz, 3H, C6‴H
3
); C NMR (101
MHz, DMSO-d
6
) δ 169.96, 166.23, 165.90, 153.96, 152.37,
1
1
6
1
5
52.17, 150.39, 145.67, 140.64, 140.44, 137.98, 134.66, 129.60,
29.02, 125.80, 124.44, 122.31, 118.13, 108.08, 107.08, 105.91,
9.79, 62.25, 60.62, 60.50, 57.97, 55.84, 37.93, 29.86, 20.58,
8.46; MS (EI): m/z (%) = 564.1 (27), 563.3 (64), 562.6 (100),
61.0 (74), 450.0 (94), 449.0 (54), 408.4 (33), 392.2 (45), 377.6
2
7
31
8
65.34, H 6.47, N 3.07.
Synthesis of methyl (5S, aR)-5-(2-((3-((2-acetamido-3-
methoxy-3-oxopropyl)thio)butanoyl)oxy)acetamido)-9,10,11-
trimethoxy-6,7-dihydro-5H-dibenzoija,c]cyclohepten-3-
carboxylate (rac-13). A solution of 5c (70.0 mg, 0.145 mmol)
and N-acetyl-L-cysteine methyl ester (28 μL, 0.159 mmol) in
dry methanol (1 mL) was cooled to 0 °C. DIPEA (28 μL, 0.159
mmol) was added, and the reaction was stirred for 2 h. TLC
indicated full conversion. The crude product obtained after
solvent removal was purified by column chromatography,
eluent PE/EtOAc/EtOH 3 : 1 : 1, to afford rac-13 as a yellowish
(34), 333.6 (26), 319.4 (14), 303.4 (11), 263.9 (10); elemental
analysis calcd (%) for C H NO : C 66.06, H 5.90, N 2.49;
31
33
9
found: C 65.91, H 5.71, N 2.32.
′,2′,3′-Trimethoxybenzoij5′,6′:5,4]1H-(aR, 1S)-1-((((2‴E,4‴E)-
1
hexa-2‴,4‴-dienoyl)amido)acetamido)-6,7-dihydrocyclohepta-
ij3,2:f ]-2″-hydroxymethylbenzofuran (12b). Purified by column
chromatography (PE/EtOAc/EtOH 4 : 1 : 1); white solid (30%);
1
m.p. > 250 °C; H NMR (400 MHz, DMSO-d ) δ 8.55 (d, J =
solid (12.0 mg, 13%); m.p. 172 °C; [α] −60.0° (c 0.5000 in
6
D
1
8
1
6
1
1
4
3
2
.2 Hz, 1H, C1–H), 8.17 (t, J = 5.9 Hz, 1H, 1‴(O)–NH), 7.51 (s,
H, C4″–H), 7.47 (s, 1H, C7″–H), 7.04–6.97 (m, 1H, C3‴–H),
.79 (s, 1H, C3″–H), 6.75 (s, 1H, C4′–H), 6.20 (dd, J = 15.5,
0.3 Hz, 1H, C4‴–H), 6.13–6.04 (m, 1H, C5‴–H), 5.99–5.94 (m,
H, C2‴–H), 5.45 (t, J = 5.9 Hz, 1H, OH), 4.58 (m, 1H, C1–H),
.57 (d, J = 5.8 Hz, 2H, C2″–CH ), 3.91–3.84 (m, 2H, NHC H_ ),
CHCl
3
, 20 °C); H NMR (400 MHz, DMSO-d
6
) δ 8.73 (d, J = 7.5
Hz, 1H, N H_ Ac), 8.35 (d, J = 7.8 Hz, 1H, C7–NH), 7.12 (s, 1H,
C8–H), 7.11 (d, J = 8.5 Hz, 1H, C10–H), 7.03 (d, J = 10.8 Hz,
1H, C11–H), 6.77 (s, 1H, C4–H), 4.53 (s, 2H, C2′H ), 4.43–4.32
2
(m, 2H, C7–H, C2″–H), 3.87 (s, 3H, OMe), 3.83 (s, 3H, OMe),
3.78 (s, 3H, OMe), 3.62 (s, 3H, OMe), 3.52 (s, 3H, OMe), 3.18–
3.06 (m, 2H, C5′–H, C3″–H), 2.91–2.77 (m, 2H, C3″–H, C5–H),
2.63 (m, 2H, C5–H, C4′–H), 2.23 (td, J = 13.0, 7.2 Hz, 1H, C4′–
H), 2.02 (dt, J = 12.6, 6.3 Hz, 1H, C6–H), 1.90 (dd, J = 12.1, 7.2
Hz, 1H, C6–H), 1.83 (s, 3H, Ac), 1.23 (d, J = 6.8 Hz, 3H, C6′
2
2
.84 (s, 3H, OMe), 3.79 (s, 3H, OMe), 3.38 (s, 3H, OMe), 2.60–
.52 (m, 1H, C6–H), 2.16 (tt, J = 12.0, 6.1 Hz, 1H, C6–H), 2.06
(
dd, J = 12.5, 6.7 Hz, 1H, C7–H), 1.99–1.90 (m, 1H, C7–H),
1
3
1
d
1
1
.79 (d, J = 6.5 Hz, 3H, C6‴H
3
); C NMR (101 MHz, DMSO-
1
3
6
): δ = 168.25, 165.63, 158.42, 153.76, 152.24, 150.41, 140.61,
3 6
H ); C NMR (101 MHz, DMSO-d ) δ 177.94, 171.13, 170.21,
39.41, 137.15, 136.73, 134.73, 129.91, 128.62, 126.22, 124.74,
22.70, 121.76, 108.04, 105.73, 103.21, 60.63, 60.41, 56.23,
169.36, 166.03, 163.53, 152.97, 150.41, 150.10, 140.75, 134.97,
134.46, 134.11, 130.42, 125.32, 112.09, 107.77, 62.20, 60.80,
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6
3
0.68, 56.05, 55.85, 52.34, 52.02, 51.16, 45.65, 41.17, 36.16,
5.71, 31.32, 22.23, 20.99; MS (ESI): m/z (%) = 660 (1), 516
medium in 6-well plates (Costar). Colchicine (1), compd 5c or
5i (5 μM) was dissolved in 4 mL of complete medium and
added to the wells. Cells were cultivated for 72 h. After
incubation, cells were fixed with 1% paraformaldehyde,
permeabilized by 0.1% Triton X100 in PBS, washed, and
treated with Mowiol 4.88 medium (Calbiochem, Germany).
Tubulin was identified by anti-tubulin antibody (Santa Cruz,
USA) followed by anti-mouse IgG-AlexaFluor555 (Molecular
Probes, Invitrogen, USA). Hoechst 33342 (Sigma) was used to
visualize nuclei. Slides were analyzed using Eclipse TE2000
confocal microscope (Nikon, Japan).
(
2
10), 455 (22), 416 (12), 369 (26), 354 (55), 312 (100), 297 (55),
54 (24), 239 (17), 211 (13); elemental analysis calcd (%) for
C H N O S: C 58.17, H 6.10, N 4.24; found: C 58.41, H
3
2
40 2 11
5
.97, N 4.35.
Cell cultures
Cells were grown in DMEM medium supplemented with 10%
fetal calf serum (FCS), pen–strep–glut (all from PanEco,
Moscow, Russian Federation). All cell lines used were
routinely tested for mycoplasma. Adherent cells were
detached using 0.05% trypsin–EDTA (PanEco, Moscow),
counted and sub-cultured. Twenty-four hours before assays,
cells were seeded in the appropriate plates (96- or 24-well
Animals
C57BL/6 mice were purchased from Pushchino Affiliation of
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
RAS, Moscow. All mice were 6–8 weeks old and maintained in
a minimal pathogen animal facility at the Shemyakin-
Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow.
All studies were conducted in an AAALAC accredited facility
in compliance with the PHS Guidelines for the Care and Use
of Animals in Research. The range of lethal concentrations
was predetermined in the preliminary experiments. To
conduct acute toxicity experiments mice (10 per group)
received intravenously a single dose of colchicine or its
derivatives. Four different doses were used: 4, 7, 10 and 13
5
plates), adjusted to 3 × 10 cells per mL, and incubated
overnight to achieve standardized growth conditions.
MTT-assay
Cytotoxic effect of the allocolchicinoids was estimated by a
standard
tetrazolium bromide (MTT, Sigma) test as described earlier.
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
4
6
All the compounds were dissolved in DMSO to 20 mM
concentration and stored at −20 °C until the assay. Different
dilutions of the new compounds from 20 μM to 0.1 nM were
prepared separately and transferred in 100 μL to the plates
with the cells. Non-treated cells served as controls. Plates
were incubated for 72 h. For the last 6 h, 5 mg mL of MTT
were added in the amount of 10 μL to each well. After the
incubation, culture medium was removed and 100 μL of
DMSO were added to each well. Plates were incubated at
shaking for 15 min to dissolve the formed formazan product.
Optical density was read on spectrophotometer Titertek (UK)
at 540 nm. Results were analyzed by Excel package
−1
mg kg . Toxic effect was evident 24 post injection (ruffled
fur, poor mobility, a decrease in body temperature). The
lethality was registered 24 h post injection in 10 and 13 mg
−
1
−1
−1
kg groups and some lethality at 48 h for 4 and 7 mg kg .
Mice survived 48 h were alive 1 week after.
Preparation of lamb brain tubulin
Tubulin was extracted from lamb brains by ammonium
sulfate fractionation and ion exchange chromatography and
4
7
stored in liquid nitrogen. Before use, aliquots of protein
were chromatographed in drained spin columns (1 cm × 5
cm) of Sephadex G25, equilibrated with polymerization buffer
(Microsoft). Cytotoxic concentration giving 50% of the
maximal toxic effect (IC ) was calculated from the titration
5
0
curves. The inhibition of proliferation (inhibition index, II)
was calculated as [1 − (ODexperiment/ODcontrol)], where OD was
MTT optical density.
(
20 mM sodium phosphate, 3.4 M glycerol, EGTA 1 mM,
MgCl 10 mM, 0.1 mM GTP, pH 6.5), followed by passage
2
through a gravity column of Sephadex G25 (1 cm × 10 cm)
equilibrated with the same buffer. Protein concentration was
Cell cycle analysis by flow cytometry
measured spectrophotometrically with
a
Perkin-Elmer
Cell cycle was analyzed using PI-stained DNA. COLO-357 and
L929 cells were collected at indicated time, trypsinized,
washed in ice-cold PBS, fixed by the addition of 70% ethanol
and left for 2 h at −20 °C. Thereafter, the cells were washed
spectrophotometer Beckman DU70 at 275 nm with an
−
1
−1
extinction coefficient of 1.09 L g
cm
in guanidine
hydrochloride in neutral aqueous buffer.
−
1
twice in PBS, stained with 50 μg mL of propidium iodide
Microtubule assembly assay
−
1
(
Sigma Chemical Co) in PBS, 10 μg mL of DNAse and
Microtubule assembly was performed with 18 μM tubulin in
polymerization buffer. The aliquots were incubated for 40
min at 4 °C prior to start the reaction by warming the
samples at 37 °C in thermostated cuvettes. The mass of
polymer formed was monitored by turbidimetry at 350 nm
with a Jasco V-750 spectrophotometer. Samples containing
the compound and their controls had less than 3% residual
DMSO.
analyzed by flow cytometry using FACScan device (BD, USA).
Total 2000 events were collected. The results were analyzed
using Flowing 2.5.1 software (Finland).
Confocal analysis
5
For confocal analysis, COLO-357 cells (10 /glass) were grown
overnight on sterile cover slips in 200 μL of complete culture
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Conflicts of interest
N. A. Yates, G. Romero, S. N. Sarkar and S. V. Singh, J. Biol.
Chem., 2014, 289, 1852–1865.
3 P. K. Naik, S. Santoshi, A. Rai and H. C. Joshi, J. Mol.
Graphics Modell., 2011, 29, 947–955.
4 C. Combeau, J. Provost, F. Lancelin, Y. Tournoux, F.
Prod'homme, F. Herman, F. Lavelle, J. Leboul and M.
Vuilhorgne, Mol. Pharmacol., 2000, 57, 553–563.
5 B. Shan, J. C. Medina, E. Santha, W. P. Frankmoelle, T.-C.
Chou, R. M. Learned, M. R. Narbut, D. Stott, P. Wu, J. C.
Jaen, T. Rosen, P. B. M. W. M. Timmermans and H.
Beckmann, Proc. Natl. Acad. Sci. U. S. A., 1999, 96,
5686–5691.
There are no conflicts to declare.
2
2
Acknowledgements
This work was supported by the Russian Science Foundation
(project 19-13-00158).
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