Discovery of dihydrofuranoallocolchicinoids - Highly potent antimitotic agents with low acute toxicity

Article abstract of DOI:10.1016/j.ejmech.2020.112724

Two series of heterocyclic colchicinoids bearing β-methylenedihydrofuran or 2H-pyran-2-one fragments were synthesized by the intramolecular Heck reaction. Methylenedihydrofuran compounds 9a and 9h were found to be the most cytotoxic among currently known

Full text of DOI:10.1016/j.ejmech.2020.112724

Journal Pre-proof
Discovery of dihydrofuranoallocolchicinoids - highly potent antimitotic agents with low
acute toxicity
Ekaterina S. Shchegravina, Elena V. Svirshchevskaya, Sebastien Combes, Diane
Allegro, Pascale Barbier, Benoit Gigant, Paloma F. Varela, Andrei E. Gavryushin,
Daria A. Kobanova, Andrey E. Shchekotikhin, Alexey Yu. Fedorov
PII:
S0223-5234(20)30696-6
DOI:
https://doi.org/10.1016/j.ejmech.2020.112724
EJMECH 112724
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 June 2020
Revised Date: 16 July 2020
Accepted Date: 3 August 2020
Please cite this article as: E.S. Shchegravina, E.V. Svirshchevskaya, S. Combes, D. Allegro, P. Barbier,
B. Gigant, P.F. Varela, A.E. Gavryushin, D.A. Kobanova, A.E. Shchekotikhin, A.Y. Fedorov, Discovery
of dihydrofuranoallocolchicinoids - highly potent antimitotic agents with low acute toxicity, European
Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112724.
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MeO
MeO
Heck MeO
reaction
MeO
MeO
Heck
reaction
NHAc
NHAc
NHAc
MeO
2 steps
1 step
MeO
R2
MeO
MeO
R3
OH
O
O
I
O
R1
8 examples
IC50 up to 0,01 nM
3 examples
IC50 up to 6 nM

Discovery of dihydrofuranoallocolchicinoids - highly potent antimitotic agents with
low acute toxicity
Ekaterina S. Shchegravina1, Elena V. Svirshchevskaya2, Sebastien Combes3, Diane Allegro,4 Pascale
Barbier,4 Benoit Gigant5, Paloma F. Varela5, Andrei E. Gavryushin 1,6, Daria A. Kobanova1, Andrey E.
Shchekotikhin1,7, Alexey Yu. Fedorov*1
1Department of Organic Chemistry, Nizhny Novgorod State University, Gagarina av. 23, Nizhny Novgorod 603950,
Russian Federation
2 Department of Immunology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, GSP-7, Miklukho-
Maklaya Street, 16/10, 117997 Moscow, Russian Federation
3CRCM, CNRS UMR7258, Laboratory of Integrative Structural and Chemical Biology (ISCB), NSERM, U1068,
Institut Paoli-Calmettes, Aix-Marseille Université, UM105, F-13009, Marseille, France
4Institute of NeuroPhysiopathology (INP) - CNRS UMR 7051, Aix-Marseille University, 27 Boulevard Jean
Moulin, 13385 Marseille Cedex 5, France
5Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette,
France.
6 Nanoscape AG, Am Klopferspitz 19, 82152 Planegg, Germany
7 Gause Institute of New Antibiotics, 11 B. Pirogovskaya Street, 119021 Moscow, Russian Federation
*corresponding author: Tel/Fax: +7 (831) 4623085. E-mail: afedorovnn@yandex.ru
Abstract: Two series of heterocyclic colchicinoids bearing β-methylenedihydrofuran or 2H-
pyran-2-one fragments were synthesized by the intramolecular Heck reaction.
Methylenedihydrofuran compounds 9a and 9h were found to be the most cytotoxic among
currently known colchicinoids, exhibiting outstanding antiproliferative activity on tumor cell
lines in picomolar (0.01-2.1 nM) range of concentrations. Compound 9a potently and
substoichiometrically inhibits microtubule formation in vitro, being an order of magnitude more
active in this assay than colchicine. Derivatives 9a and 9h revealed relatively low acute toxicity
in mice (LD50 ≥10 mg/kg i.v.). The X-Ray structure of colchicinoid 9a bound to tubulin
confirmed interaction of this compound with the colchicine binding site of tubulin.
Keywords: colchicine, tubulin, antiproliferative activity, antimitotics, X-ray structure
Introduction
Development of multidrug resistance (MDR) [1], and severe side effects of anticancer
agents are among the most significant drawbacks of modern anticancer therapy. . Emergence of
MDR is mostly associated with the drug efflux mediated by ABC-protein family [2,3].
Alterations of β3-tubulin isotype is one of the causes of MDR development [4]. Several modern
antimitotic agents such as taxanes and Vinca alkaloids are subject of MDR-mediating efflux

mechanisms [5–8]. Numerous studies in this area have revealed evidences that colchicine-
binding site ligands are able to overcome MDR mediated by ABCB1-protein (P-gp) [9–12]
.
Recently, colchicine has been approved by FDA for the treatment of patients with acute gout. It
has also been found promising in the therapy of amyloidosis [13], Behсеt’s disease [14], familial
Mediterranean fever [15] and some other autoimmune and inflammatory diseases [16–19].
However, unpredictable severe side effects occurring in some patients and significant systemic
toxicity prevent its application as a chemotherapeutic agent, where a higher dosage is required
[20,21].
Colchicine binds tubulin in a slow and poorly reversible manner. The half-live of tubulin-
colchicine complex was found to be about 12.5 hours [22]. An increase of affinity, or even
covalent binding to tubulin could be realized via Michael addition of nucleophilic groups in Cys,
Lys and Tyr residues to tubulin-binding molecules, possessing an activated double bonds
[23,24]. Covalent modifications of various approved drugs and clinical candidates [25] such as
telaprevir-based covalent HCV-protease inhibitor [26], irreversible EGFR inhibitors afatinib
[27,28], osimertinib [29] and rociletinib [30], covalent Btk and PI3Kα-inhibitors ibrutinib
[31,32] and CNX1351 [33] lead to an increase in their affinity to the target protein, and thus to
improved efficacy.
We considered that the introduction of an activated double bond into a molecule of a
colchicinoid may significantly facilitate and strengthen its binding to tubulin via irreversible
bond formation. Herein, we report a straightforward synthesis and biological evaluation of two
types of heterocyclic colchicinoids, potentially capable of covalent interactions with tubulin:
those possessing a 2H-pyran-2-one scaffold, and a series of compounds containing a
methylenedihydrofuran fragment with an aryl-conjugated exocyclic double bond [34]. Two of
them were active against tumor cell cultures in low picomolar concentrations, displaying at the
same time relatively low acute toxicity in mice.
Results and discussion
Chemistry
First, we attempted to synthesize colchicinoids of type 4, bearing a conjugated 5-
membered lactone ring (D) with an exo-double bond potentially active as a Michael acceptor
(Scheme 1). Such compounds were supposed to interact with cysteine residues in the colchicine
site of tubulin [34].

Pathway A
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
NHAc
NHAc
O
NHAc
a, b
NHAc
O
c
93%
MeO
R2
MeO
MeO
2
MeO
1
R1
R2
O
O
OH
4
I
I
OMe
O
Pathway C
OMe
R1
3a R1=R2=H,
3b R1=H, R2=Me, 75%
3c R1=R2=Me, 77%
3d R1=H, R2=Cl, 84%
3e R1=H, R2=CF3, 72%
87%
O
O
Pathway B
NHAc
OR3
MeO
MeO
f
d
R4 E-
isomer
7a R4 = Me
HO
7b R4 =
23%
27%
MeO
66%
MeO
MeO
MeO
MeO
NHAc
NHAc
OH
MeO
MeO
MeO
MeO
R4
O
OR3
OH
R4
O
O
7c R =
MeO
5a R3 = Me
5b R3 = H
4
e
7
O
Z-
6 - isomer
OMe
98%
MeO
(traces)
Scheme 1. Synthesis of allocolchicinoids bearing a conjugated lactone as a D-cycle. Reagents
and conditions: a) HCl, AcOH, 100 °C, 3 h, reflux; b) NaOH, I2, KI, H2O, 0-5 °C, 1 h; c) α,β-
unsaturated acid, DCC, DMAP, DCM, rt, overnight; d) methyl acrylate, Pd(OAc)2, Et3N, MeCN,
80 °C, 20 h; e) NaOH, H2O, rt, 2 h; f) methyl or ethyl ester of α,β-unsaturated acid acid,
Pd(OAc)2, Et3N, H2O, 80 °C, overnight.
Naturally occurring (aR,7S)-colchicine (1) was converted into iodocolchinol (2) [35]
using 2-step reaction sequence. It was acylated by several α,β-unsaturated acids under Steglich
reaction conditions (Scheme 1, Pathway A). Corresponding phenolic esters 3a-d were supposed
to undergo intramolecular Heck reaction leading to conjugated lactones 4. However, under the
tested conditions only hydrolysis of ester groups took place. When iodo-colchinol 2 was reacted
with E-forms of α,β-unsaturated esters, the formation of coumarine derivatives 7a-c was
observed, albeit in low yields (Scheme 1, Pathway C). Obviously, the Heck coupling afforded Z-
intermediate 6, which underwent in situ lactonization with formation of allocolchicinocoumarins
7 [36]. Several 4-arylcoumarins are known to exhibit potent antitumor activity [37]. Regarding
this, we have also synthesized 4-arylcoumarins 7b and 7c. On the other hand, our recent studies
[38–42] have shown that the size of substituents in cycle D of heterocyclic allocolchicinoids
dramatically affects their antiproliferative activity. Therefore, methyl crotonate was used as a
reagent, leading to the formation of methyl-substituted coumarin 7a. Synthesis of the
unsubstituted analog failed, as the cross-coupling of iodo-colchinol 2 with methyl acrylate lead
to product 5 possessing E-configuration that prevented its cyclization to coumarin (Scheme 1,
Pathway B).

Thus, despite the failure to obtain conjugated lactones of type 4, we synthesized a number
of new colchicinoids 3, 5 and 7 as potential Michael acceptors, supposedly capable for covalent
binding to cysteine residues of the colchicine site of tubulin.
Further, iodo-colchinol 2 was subjected to the alkylation using various allyl bromides
(Scheme 2), followed by intramolecular Heck reaction leading to ethers 8a-h. According to this
method, dihydrofuranoallocolchicinoids 9a-h containing exo-double bond in D-ring were
obtained in only four preparative steps starting from commercially available colchicine with
conservation of the absolute configuration at C-7.
R1
R2
MeO
MeO
MeO
MeO
MeO
MeO
NHAc
NHAc
NHAc
NHAc
Br
Pd(dppf)Cl 2, AcOK
DMSO, 80 oC
MeO
MeO
K2CO3
+
MeO
R2
MeO
R2
MeO
DMF, 60 o
C
MeO
O
O
O
OH
I
I
R1
R2
R1
2
R1
8a R1=R2=H
60%
9a (66%)
9b (62%)
9c (86%)
9d (44%)
9e (0%)
9f (69%)
9g (63%)
9h (73%)
10a (23%)
10b (traces)
10c (0%)
10d (traces)
10e (68%)
10f (0%)
8b R1=H, R2=Me 71%
8c R1=R2=Me
8d R1=H, R2=Et
8e R1=H,
64%
62%
R2=CO2Me
8f R1=CH3
R2=
73%
55%
10g (traces)
10h (0%)
8g R1=H, R2=Ph
8h
73%
61%
O
O
F
I
F
F
F
9h
8h
Scheme 2. Synthesis of dihydrofuran-containing allocolchicinoids
Dihydrofuranoallocolchicinoids 9, containing exo-double C=C bond can easily undergo
acid-catalyzed isomerization into corresponding benzofurans 10 with aromatization (Scheme 3).
H+
O
- H+
O
O
R1
R1
H
R1
H
H
R2
R2
R2
10
9
Scheme 3. Acid-catalyzed isomerization of dihydrofuranoallocolchicinoids 9 into benzofurans
10.
Compound 9e completely isomerized into benzofuran 10e during the Heck reaction,
while colchicinoids 9a and 10a were initially obtained in 3:1 ratio. The degree of isomerization
of 9a into corresponding benzofuran 10a could not be reduced by temperature decrease,
shortening of the reaction time or varying of the amount of bases. Screening of additives to the

reaction mixture in order to suppress this undesired reaction revealed that the addition of B2Pin2
decreased the formation of 10a from 23% to 7%. We presume that the isomerization takes places
via the deprotonation of the allylic α-carbon in the dihydrofuran ring followed by the double
bond migration. In this case, B2Pin2 might act as a buffer, decreasing the basicity of acetate anion
in the reaction media by complexation. For the compounds 9b-d, and 9f-g the isomerization into
benzofuranoallocolchicinoids 10b-d and 10f-g was not observed under the Heck reaction
conditions. Compound 9h was designed to be incapable of isomerizing into 10h due to the
absence of α-hydrogen atoms on the dihydrofuran ring D.
Biology
Antiproliferative activity of New Compounds against Cancer Cell Lines
Antiproliferative activity of all synthesized compounds against human cell lines was
investigated by MTT assay. Among new colchicinoids 3, 5, and 7, containing fragments of
Michael acceptors, none exceeded the colchicine level of cytotoxicity (Table 1). Most of the
compounds exhibited activity in 2-4 orders of magnitude lower than colchicine. Compound 5a
with a tricyclic skeleton was found to be the least active. No correlation between the activity and
the electronic features of the substituents was observed.
*
Table 1. Antiproliferative activity of colchicinoids 3 and 7(IC50 , nM)
3a
150
30
3b
161
4
3c
160 810
32 800
3d
3e
30
6
5a
7a
7b
870
160
4000
837
Colchicine (1)
>4000 160
>4000 140
1.3
2.4
16
COLO357
SW620
158
32
>4000 805 810
139 160 825
170 >4000
180 >4000
32
16
HEK-293
Raji
16
*Data are shown as the averages of 5 experiments. Standard deviations were within 10% and not shown for
the easier reading of the results
The in vitro cytotoxic activity of the compounds 9a-d, 9f-h, 10a and 10e was tested on a
broader panel of tumor cell lines of different origin: pancreatic (PANC-1, COLO357), cervical
(HeLa) andcolon (SW620) cancers, Burkitt's lymphoma (Raji), immortalized kidney cells (HEK-
293), and keratinocytes (HaCaT). The calculated IC50 values are summarized in Table 2. Among
these compounds, 9a and fluorinated colchicinoid 9h with exo-double bonds exhibited the most
prominent antiproliferative activity known for colchicinoids, with IC50 values in the low
picomolar range for some cell lines. Methyl-substituted furan 10a, isomeric to dihydrofurane 9a,
was substantially less active, but also cytotoxic already in subnanomolar concentrations. The

cytotoxicity significantly decreases with an increase of a substituent size. Thus, colchicinoid 9b
with a monomethylated exo-double bond is drastically less cytotoxic than its analogs 9a and 9h.
A double-methylated analog 9c or ethyl-substituted colchicinoid 9d revealed the activity one or
two orders of magnitude lower than 9b (Table 2). Compounds 9f and 9g with more bulky
substituents, as well as 10e, were not cytotoxic. Therefore, the substitution of the exo-double
bond in the dihydrofuran fragment was found to be an important factor affecting the activity of
colchicinoids 9. However, it cannot be excluded that the presence of substituents on the
exocyclic double bond also facilitates its isomerization in the cell media to less cytotoxic
benzofurans.
Table 2. Antiproliferative activity of colchicinoids with dihydrofuran D-cycle (IC50* nM)
9a
9b
5
9c
9d
9f
9g
9h
10a*
10e
830
1
860
780
800
4330 >20000
1.3
1.3
2.4
COLO357 0.04
0.02
0.04
0.03
0.03
6
810 >20000 >20000
1.3
4100
SW620
HaCaT
HeLa
0.02
0.3
14
32
390
410
400
850
420
135
150
166
80
4500
4250
4370
4025
0.5
0.8
5
810
805
0.3
1.3
16
1.7
3.8
32
4280 >20000
4420
820
HEK-293
PANC-1
0.08
0.08
0.03
0.03
0.01
0.05
810
800
>20000
4600
2.1
1.3
0.1
16
3.8
90
3900
Raji
*
Compound 10a was obtained from 9a by the treatment with trifluoroacetic acid in DCM with a quantitative yield.
Most potent compounds 9a and 9h are shown in bold.
*
Data are shown as the averages of 4 experiments. Standard deviations were within 10% and not shown for the
easier reading of the results
Cell cycle analysis
The impact of colchicinoid 9a on cell cycle and its comparison with the action of
colchicine was established (Fig. 1). Both compounds decreased the population of cells in G1-
phase, accumulating cells in G2/M phase during incubation for 48 hours (Fig. 1). As expected,
both colchicinoids exhibited the same manner of cell cycle arrest and apoptosis induction (Fig.
1). Notably, in COLO357 culture the apoptotic cells were effectively eliminated, oppositely to
SW620, where percentage of apoptotic cells increased 5-10-fold over the control (Fig. 1).
Similar results were found for 9h (data not shown).

Figure 1. Cell cycle analysis in COLO357 and SW620 cells. COLO357 (a, b) or SW620 (c, d)
cells were incubated without (black lines) or with 5 nM of 9a or colchicine (1) for 48 h (grey
histograms), fixed, perforated, stained with propidium iodide (PI), and analyzed by flow
cytometry. SW620 cells formed diploids and tetraploids (arrows). M1, M2, M3 correspond to
G2/M, G1, and apoptosis (Apo) phases of cell cycle. Table data are shown as percentages of
cells in each phase.
Notably, 48 h incubation of these cells in the presence of the colchicinoids did not result
in significant apoptosis due to a low number of dying cells at this time point. In case of a longer
incubation, the number of apoptotic cells increases significantly (SI Figure 1) [38].
Destabilization of microtubules and inhibition of assembly
Compound 9a, as well as colchicine, disrupted the mitotic spindle and induced tubulin
dissipation and chromosomes disturbance (Fig. 2). In the control images (Fig. 2 a, d) a well-
developed microtubules network and mitotic spindles were observed. In contrast, total
microtubules depolymerization and chromosomes disordering were recognized after cells
incubation with 9a within 24 hours (Fig. 2 b, c, e, f). This clearly indicates that synthesized
compound 9a inhibits cell division by influencing the tubulin assembly.

Figure 2. Disruption of mitotic spindle by 9a and colchicine. SW620 (a-c) or COLO387 (d-f)
were incubated without (a, d) or with 5 nM 9a (b, e) or colchicine (c, f) for 24 h, fixed, and
stained with anti β-tubulin antibody (red). Note the loss of mitotic spindles as well as tubulin
disassembling, and chromosome scattering (b, c, e, f) after cell incubation with colchicinoids.
Nuclei are stained with Hoechst 33342 (blue). Objective bars correspond to 12-16 μM.
Figure 3 shows the effects of colchicine (panel A) as a positive control, and 9a (panel B),
on the turbidimetry time course of microtubule assembly from pure tubulin. 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 graphs A and B of Figure 3 show that the extent
of inhibition by colchicine and 9a, 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
ligand/tubulin mole ratio of 0.27 mol/mol for colchicine and 0.06 mol/mol for 9a.. Thus,
compound 9a exhibits more potent inhibition of tubulin polymerization as well as stronger
binding to colchicine site on microtubules than colchicine itself. . Interestingly, combretastatin
A-4 (CA4), similar to colchicine in the mode of action on tubulin, but possessing also
antivascular activity, revealed R=0.09 in similar experiments [38]. The difference in R is likely a
reflexion of a difference in binding affinity rather than kinetics parameters, suggesting that ring
D and/or exo-bond of compound 9a makes contribution to the induction of assembly inhibition.

Time, min
Time, min
Figure 3. Effect of ligands on the turbidity time course of in vitro microtubule assembly. The
aliquots were incubated for 40 min at 4 °C prior to start the reaction by warming the samples to
37 °C (A). Effect of various concentrations of colchicine (a: 0 μM; b: 1 μM; c: 3 μM; d: 5 μM;
and e: 7 μM) on tubulin aggregation at 18 μM in polymerization buffer. (B) Effect of various
concentrations of 9a (a: 0 μM; b: 0.5 μM; c: 0.75 μM; d: 1 μM; e: 2 µM, and f: 5 μM) on tubulin
aggregation at 18 μM in polymerization buffer. The inserts on the graphs represent the
percentage of assembly inhibition as a function of the mole ratio of the total ligand to total
tubulin in the solution (R).
In vivo toxicity of the compounds 9a and 9h
Determination of the acute in vivo toxicity was performed in C57BL/6, BALB/c, CBA,
A/Sn, and outbred CD1 mice (3 mice of each strain per group). Several genetically different
strains were used to cover a variety of genotypes [43]. Compounds 9a, 9h, and colchicine were
dissolved in DMSO to 7.9 and 8.6 mg/mL respectively, diluted by saline, and 200 µl of this
solution per mouse was injected intravenously. Pure DMSO was diluted in the same way and
was used as a control (detailed description is in Experimental Section). Preparations were
injected at 5 and 10 mg/kg of body weight. All the mice injected with 5 mg/kg colchicine died 14
h after the injections. None of the mice in DMSO, 9a, and 9h groups died at 5 mg/kg dose. When
the mice were injected with 10 mg/kg DMSO, 9a, and 9h, only one mouse of three C57BL/6
died in 9a group. Consequently, LD50 for 9a can be estimated as 10 mg/kg and LD50>10 mg/kg
for 9h. Estimated LD50 for colchicine (mice, i. v.) is 1.6 mg/kg [44], which corresponds well to
the numbers given above. Interestingly, structurally similar furan derivative of allocolchicine 11,
previously synthesized by us [38], demonstrated much higher acute toxicity (LD50 = 2 mg/kg for
C57BL/6 mice, i. v.).

Apparently, the shift of small substituents from position 2 to position 3 of the furan or
dihydrofuran ring of heterocyclic allocolchicinoids has a profound effect on antiproliferative
activity and systemic toxicity. Lower acute toxicity can be translated into expansion of
therapeutic window of the novel colchicinoids. To better understand the structure-activity
relationship of allocolchicine derivatives, the structure of selected compound 9a in tubulin
complex was resolved by X-ray crystallography and is discussed below.
Crystal structure of 9a-T2R
To gain an insight into the mechanism of microtubule assembly inhibition by
colchicinoids 9, we determined the 2.3 Å resolution the crystal structure of compound 9a bound
to T2R, a complex of two tubulin heterodimers stabilized by the stathmin-like domain of the
RB3 protein [45] (Fig. 4). The structure shows that 9a binds to the colchicine site of tubulin in a
mode very similar to that of colchicine, interacting with the T5 loop of the α-subunit and with β-
strands S8 and S9, loop T7 and helices H7 and H8 of β-tubulin (Fig. 5). Therefore, the most
likely mechanism of action of 9a is preventing the curved-to-straight tubulin structural changes
that occur during microtubule assembly, same to colchicine [46].
The exo-C=C bond is directed toward a narrow pocket, in which bulky substituents on the
dihydrofuran fragment could not be easily accommodated, giving a reason why compounds with
larger substituents are less active than 9a and 9h (Table 2). This bond is located in the vicinity of
the Thr314 and Met259 residues of β-tubulin, but is too far for a covalent interaction (Fig. 4).
We concluded, therefore, that compound 9a, contrarily to our anticipations, does not bind
covalently with tubulin.

Figure 4. Tubulin binding of compound 9a. The T2R:9a complex comprises two αβ-tubulin
heterodimers stabilized by the stathmin-like domain of the RB3 protein (blue). Colchicinoid 9a
(cyan) binds to β-tubulin (green) at the interface with the α-subunit (pink).
A
B
Figure 5. A. Fobs-Fcalc electron density omit map contoured at the 3σ level of 9a bound to
tubulin. B. Close-up of 9a bound to tubulin and comparison with colchicine. The tubulin
secondary structural elements that contact 9a are labeled. Colchicine (yellow) from the
tubulin:colchicine complex [47] (pdb id 5EYP) is shown after superposition of β-tubulin from
that complex to that of tubulin:9a (root mean square deviation, 0.53 Å (423 Cαs compared)).
Only tubulin from T2R:9a is shown.
Conclusion
We synthesized a series of heterocyclic colchicinoids bearing dihydrofuran fragments of
type 9 as a novel class of antiproliferative analogs of the naturally occurring colchicine (1).
These compounds were obtained in four preparative steps starting from colchicine, under
conservation of the absolute configuration at C(7). Dihydrofuran 9a and its fluorinated analog 9h
exhibited cytotoxicity in the picomolar concentration range for all studied tumor cell lines.

Isomeric benzofuran 10a was 1-2 orders of magnitude less cytotoxic. Compared to colchicine
(1), and combretastatin A-4, allocolchicinoid 9a proved to be a more efficient tubulin assembly
inhibitor, acting in a substoichiometric mode. First in vivo experiments demonstrated significant
decrease in acute toxicity for the compounds 9a and 9h (an order of magnitude) compared to
colchicine, so that LD50 for 9a was estimated as 10 mg/kg and for 9h even higher, with LD50 =
1.6 mg/kg for colchicine under the same conditions. The localization of the compounds 9 in the
colchicine site of tubulin has been unambiguously proven by X-ray diffraction.
The synthetic availability and strong cytotoxic activity exhibited by the new colchicinoids
make them perspective objects for in-depth biological investigations. We consider the
compounds 9a and 9h as promising leads for future development of water-soluble prodrugs for
in vivo experiments.
Acknowledgements
This research was supported by Russian Science Foundation (project 19-13-00158). B.G.
thanks the “Fondation ARC pour la recherche sur le cancer”. Diffraction data were collected at
the Proxima1 and Proxima2 beamlines at SOLEIL synchrotron (Saint-Aubin, France). We are
most grateful to the machine and beamline groups for making these experiments possible. The
study was carried out on the equipment of the Collective Usage Center “New Materials and
Resource-saving Technologies” (Lobachevsky State University of Nizhny Novgorod)
Experimental part
Chemistry
General information.
13
1H NMR and C NMR spectra were recorded on Agilent DDR2 400 spectrometer at 25
°C. Chemical shifts (δ) are reported in ppm for the solution of compound in CD3OD, CDCl3 and
DMSO-d6 with internal reference TMS and J values in Hertz. Atomic numeration is given only
for NMR assignment. MALDI spectra were recorded on Bruker Microflex LT spectrometer.
Elemental analysis was performed using an Elementar (Vario Micro Cube) apparatus, all the
compounds were found to have purity >95%. Column chromatography was performed using
Merck Kieselgel 60 (70 – 230 mesh). All reactions were performed with commercially available
reagents. Solvents were purified according to standard procedures. The petroleum ether used
corresponds fraction 40 – 70 °C. Synthesis of iodocolchinol 2 was performed as described earlier
[35,38].
Synthesis of colchicinoids 3a-e.

General procedure for synthesis of esters 3a-e. Iodo-colchinol 2 (1.00 equiv.) was treated with
the corresponding α,β-unsaturated carboxylic acid (1.00 equiv.) and 4-DMAP (0.10 equiv.) in ca.
1 M dichloromethane solution under inert atmosphere. DCC (1.20 equiv.) was added to the
solution in portions, and the reaction was stirred at room temperature for 20 hours. Solvent was
removed under reduced pressure, and the residue purified by column chromatography.
(5S)-5-acetamido-2-iodo-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]cycloheptene-3-yl
acrylate (3a)
Eluent: petroleum ether-ethyl acetate-ethanol (5 : 1 : 1). Pale-yellow solid, yield 87%.
M.p. = 139 – 141 °C.
1H NMR (400 MHz, DMSO-d6): δ = 8.38 (d, J = 8.5 Hz, NH, 1H), 7.77 (s, C8-H, 1H), 7.17 (s,
C11-H, 1H), 6.82 (s, C4-H, 1H), 6.64 (dd, J = 17.2, 1.0 Hz, Cα-H, 1H), 6.48 (dd, J = 17.2, 10.4
Hz, Cβ-H, 1H), 6.25 (dd J = 10.4, 1.0 Hz, Cβ-H’, 1H), 4.55 – 4.45 (m, C7-H, 1H), 3.85 (s, OMe,
3H), 3.79 (s, OMe, 3H), 3.56 (s, OMe, 3H), 2.58 – 2.52 (m, С6-H, 1H), 2.21 – 2.13 (m, C5-H,
1H), 2.11 – 2.03 (m, C5-H, 1H), 1.93 – 1.87 (m, C6-H, 1H), 1.86 (s, COCH3, 3H).
13C NMR (101 MHz, DMSO -d6): δ = 168.50, 163.31, 152.99, 150.16, 149.71, 142.65, 140.48,
139.53, 134.90, 134.49, 133.64, 127.44, 122.15, 117.97, 108.22, 88.09, 60.78, 60.55, 55.86,
47.91, 38.30, 33.35, 22.64.
Elemental analysis: for C23H24INO6 calcld.: C, 51.41; H, 4.50; found: C, 51.53; H, 4.64.
MALDI: (pos. mode): 537.2 (M+) 68%.
(5S)-5-acetamido-2-iodo-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]cycloheptene-3-yl
(E)-but-2-enoate (3b)
Eluent: petroleum ether-ethyl acetate-ethanol (8 : 1 : 1). Pale-yellow solid, yield 75%.
M.p. = 130 – 131 °C.
1H NMR (400 MHz, DMSO-d6): δ = 8.38 (d, J = 8.1 Hz, NH, 1H), 7.76 (s, C8-H, 1H), 7.23 (dd,
J = 15.4, 6.9 Hz, Cα-H, 1H), 7.13 (s, C11-H, 1H), 6.82 (s, C4-H, 1H), 6.21 (d J = 15.5 Hz, Cβ-H,
1H), 4.57 – 4.42 (m, C7-H, 1H), 3.85 (s, OMe, 3H), 3.79 (s, OMe, 3H), 3.56 (s, OMe, 3H), 2.60
– 2.53 (m, C6-H, 1H), 2.19 (dd, J = 11.6, 6.2 Hz, C5-H, 1H), 2.09 (dd, J = 12.3, 7.2 Hz, C5-H,
1H), 1.99 (d, J = 6.7 Hz, Cβ-CH3, 3H), 1.92 – 1.86 (m, C6-H, 1H), 1.85 (s, COCH3, 3H).
13C NMR (101 MHz, DMSO -d6): δ = 168.47, 163.34, 152.96, 150.16, 149.88, 148.80, 142.55,
140.48, 139.45, 134.89, 133.45, 122.19, 121.27, 118.05, 108.22, 88.30, 60.77, 60.54, 55.85,
47.88, 38.30, 29.85, 22.63, 18.06.
Elemental analysis: for C24H26INO6 calcld.: C, 52.28; H, 4.75; found: C, 52.40; H, 4.53.

MALDI (DCTB, pos. mode): 574.0 (M+Na+) 41%, 552.0 (M+H+) 20%, 590.0 (M+K+) 13%.
(5S)-5-acetamido-2-iodo-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]cycloheptene-3-yl
3-methylbut-2-enoate (3c)
Eluent: petroleum ether-ethyl acetate-ethanol (8 : 1 : 1). Beige solid, yield 77%.
M.p. = 118 – 120 °C.
1H NMR (400 MHz, DMSO-d6): δ = 8.37 (d, J = 8.5 Hz, NH, 1H), 7.75 (s, C8-H, 1H), 7.11 (s,
C11-H, 1H), 6.81 (s, C4-H, 1H), 6.05 (s, Cα-H, 1H), 4.50 (dd, J = 19.3, 8.0 Hz, C7-H, 1H), 3.84
(s, OMe, 3H), 3.79 (s, OMe, 3H), 3.55 (s, OMe, 3H), 2.55 (dd, J = 12.6, 5.5 Hz, C6-H, 1H), 2.20
(s, Cβ-CH3, 3H), 2.19 – 2.12 (m, C5-H, 1H), 2.12 – 2.04 (m, C5-H, 1H), 2.02 (s, Cβ-CH3, 3H),
1.89 (d, J = 12.1 Hz, C6-H, 1H), 1.85 (s, COCH3, 3H).
13C NMR (101 MHz, DMSO -d6): δ = 168.46, 163.31, 161.63, 152.93, 150.16, 149.92, 142.46,
140.48, 139.39, 134.89, 133.30, 122.25, 118.16, 114.35, 108.22, 88.61, 60.76, 60.54, 55.85,
47.86, 38.32, 33.34, 27.14, 22.63, 20.32.
Elemental analysis: для C25H28INO6 calcld.: C, 53.11; H, 4.99; found: C, 53.24; H, 4.86.
MALDI: (pos. mode): 588.0 (M+Na+) 28%, 603.9 (M+K+) 34%.
(5S)-5-acetamido-2-iodo-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]cycloheptene-3-yl
(E)-3-chloroacrylate (3d)
Eluent: petroleum ether-ethyl acetate-ethanol (10 : 1 : 1). Pale-yellow powder, yield 84%.
M.p. = 121 – 122 °C.
1H NMR (400 MHz, DMSO-d6): δ = 8.39 (d, J = 8.4 Hz, NH, 1H), 8.02 (d, J = 13.4 Hz, Cα-H,
1H), 7.77 (s, C8-H, 1H), 7.17 (s, C11-H, 1H), 6.82 (s, C4-H, 1H), 6.79 (d, J = 13.4 Hz, Cβ-H,
1H), 4.48 (dt, J = 12.1, 7.8 Hz, C7-H, 1H), 3.84 (s, OMe, 3H), 3.79 (s, OMe, 3H), 3.56 (s, OMe,
3H), 2.55 (dd, J = 12.8, 5.6 Hz, C6-H, 1H), 2.24 – 2.14 (m, C5-H, 1H), 2.11 – 2.03 (m, C5-H,
1H), 1.89 (dd, J = 11.5, 6.6 Hz, C6-H, 1H), 1.86 (s, COCH3, 3H).
13C NMR (101 MHz, DMSO-d6): δ = 168.50, 161.28, 156.59, 153.01, 150.15, 149.50, 142.72,
141.14, 140.48, 139.54, 134.90, 133.79, 123.93, 122.11, 117.97, 108.23, 88.11, 60.77, 60.55,
55.86, 47.49, 33.35, 25.32.
Elemental analysis: for C23H23ClINO6 calcld.: C, 48.31; H, 4.05; found: C, 48.39; H, 4.16.
(5S)-5-acetamido-2-iodo-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]cycloheptene-3-yl
(E)-4,4,4-trifluorobut-2-enoate (3e)
Eluent: petroleum ether-ethyl acetate-ethanol (9 : 1 : 1) Beige powder, yield 72%.
M.p. = 115 − 117 °C.

1H NMR (400 MHz, DMSO-d6): δ = 8.38 (d, J = 8.2 Hz, NH, 1H), 7.78 (s, C8-H, 1H), 7.45 (dd,
J = 15.9, 6.7 Hz, Cα-H, 1H), 7.23 (s, C11-H, 1H), 7.08 (dd, J = 15.9, 2.1 Hz, Cβ-H, 1H), 6.82 (s,
C4-H, 1H), 4.48 (dt, J = 12.0, 7.7 Hz, C7-H, 1H), 3.85 (s, OMe, 3H), 3.79 (s, OMe, 3H), 3.56 (s,
OMe, 3H), 2.56 (dd, J = 12.4, 5.4 Hz, C6-H, 1H), 2.18 (dd, J = 12.3, 6.4 Hz, C5-H, 1H), 2.09
(dd, J = 12.8, 6.7 Hz, C5-H, 1H), 1.91 (dd, J = 11.8, 6.9 Hz, C6-H, 1H), 1.86 (s, COCH3, 3H).
13C NMR (101 MHz, DMSO -d6): δ = 168.53, 161.53, 156.59, 153.04, 150.16, 149.41, 142.86,
140.48, 139.61, 134.91, 134.01, 132.69, 128.88, 122.07, 117.80, 108.22, 87.82, 60.78, 60.55,
55.86, 48.05, 33.35, 25.32, 22.64.
Elemental analysis: for C24H23F3INO6 calcld.: C, 47.62; H, 3.83; found: C, 47.74; H, 3.92.
MALDI: (pos. mode) 627.9 (M+Na+) 87%, 643.9 (M+K+) 73%.
Synthesis of colchicinoids 5a,b and 7a-c.
Methyl
(E)-3-((5S)-5-acetamido-3-hydroxy-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo-
[a,c]cycloheptene-2-yl)acrylate (5a)
To the mixture of iodocolchinol 2 (100.0 mg, 0.206 mmol, 1.00 equiv.) and Pd(OAc)2 (4.6 mg,
0.02 mmol, 0.10 equiv.), MeCN (10 mL) was added under inert atmosphere. Et3N (66 μL, 0.620
mmol, 3.00 equiv.) and methyl acrylate (90 μL, 0.309 mmol, 1.50 equiv.) were subsequently
added. The reaction was run at 80 °C for 20 hours, solvent removed under reduced pressure and
the product isolated by column chromatography (petroleum ether – ethyl acetate – ethanol 4 : 1 :
1). White powder, yield 66%.
M.p. = 186 – 188 °C
1H NMR (400 MHz, DMSO-d6): δ = 10.32 (s, OH, 1H), 8.40 (d, J = 7.9 Hz, NH, 1H), 7.87 (d, J
= 16.1 Hz, =CH-CO2Me, 1H), 7.47 (s, C8-H, 1H), 6.90 (s, C11-H, 1H), 6.76 (s, C4-H, 1H), 6.55
(d, J = 16.1 Hz, C10-CH, 1H), 4.41 (dt, J = 14.3, 7.2 Hz, C7-H, 1H), 3.82 (s, OMe, 3H), 3.78 (s,
OMe, 3H), 3.70 (s, CO2Me, 3H), 3.52 (s, OMe, 3H), 2.21 – 2.01 (m, C6-H, C5-H, 3H), 1.94 –
1.91 (m, C6-H, 1H), 1.88 (s, COCH3, 3H).
13C NMR (101 MHz, DMSO -d6): δ = 168.36, 167.20, 155.99, 152.17, 150.35, 144.89, 140.59,
140.41, 134.68, 129.86, 125.09, 123.88, 118.59, 116.22, 111.14, 108.00, 60.57, 60.51, 55.84,
51.28, 48.53, 37.77, 29.99, 22.63.
Elemental analysis: for C24H27NO7 calcld.: C, 65.29; H, 6.16; found: C, 65.43; H, 6.28.
(E)-3-((5S)-5-acetamido-3-hydroxy-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]-
cyclohepta-2-yl)acrylic acid (5b)

Compound 5a (200.0 mg, 0.453 mmol) was suspended in 2.5M NaOH (15 mL). The reaction
was carried out for 1 hour at 20 °C. The solution was then acidified to pH 2, extracted with
AcOEt (3x40 mL), organic layer dried over Na2SO4 and concentrated under reduced pressure.
The product 5b was obtained as white solid in 98% yield.
M.p. = 178-179 °C
1H NMR (400 MHz, DMSO-d6): δ 10.27 (br.s, OH, 1H), 8.42 (d, J = 8.0 Hz, NH, 1H), 7.79 (d, J
= 16.1 Hz, =CH-CO2H, 1H), 7.41 (s, C8-H, 1H), 6.90 (s, C11-H, 1H), 6.74 (s, C4-H, 1H), 6.43
(d, J = 16.1 Hz, C10-CH, 1H), 4.39 (dt, J = 14.0, 7.2 Hz, C7-H, 1H), 3.80 (s, OMe, 3H), 3.76 (s,
OMe, 3H), 3.49 (s, OMe, 3H), 2.55 – 2.49 (m, C6-H, 1H), 2.17 – 2.04 (m, C5-H, 2H), 1.96 –
1.87 (m, C6-H, 1H), 1.86 (s, COCH3, 3H).
13C NMR (101 MHz, DMSO-d6): δ 168.40, 168.11, 155.84, 152.14, 150.35, 144.53, 140.59,
139.86, 134.72, 129.66, 125.01, 123.97, 118.82, 117.64, 111.19, 108.02, 60.58, 60.53, 55.85,
48.52, 37.80, 30.03, 22.65.
Elemental analysis: for C23H25NO7 calcld.: C, 64.63; H, 5.90; found: C, 64.79; H, 6.18.
MALDI: (neg. mode) 426.3 (M-H) 100%, 449.2 (M+Na+) 76%, 465.1 (M+K+) 68%.
General procedure for synthesis of compounds 7a-b
A Schlenk flask was charged with iodo-colchinol 2 (100.0 mg, 0.207 mmol, 1.00 equiv.) and
Pd(OAc)2 (4.7 mg, 0.021 mmol, 0.10 equiv.) under inert atmosphere. Distilled water (5 mL) and
the corresponding unsaturated ester (1.50 equiv.) followed by Et3N (62.7 mg, 0.621 mmol, 86
μL, 3.00 equiv.) were added and the reaction was run for 30 hours at 85 °C. The crude product
was extracted with AcOEt and purified using column chromatography.
N-((7S)-1,2,3-trimethoxy-12-methyl-10-oxo-5,6,7,10-tetrahydrobenzo-[6,7]cyclohepta[1,2-
g]chromen-7-yl)acetamide (7a)
Eluent: petroleum ether-ethyl acetate-ethanol (4 : 1 : 1). White powder, yield 23%.
M.p. = 177 – 179 °C.
1H NMR (400 MHz, DMSO-d6): δ = 8.48 (d, J = 8.3 Hz, NH, 1H), 7.65 (s, C8-H, 1H), 7.29 (s,
C12-H, 1H), 6.81 (s, C4-H, 1H), 6.38 (d, J = 1.1 Hz, C10-H, 1H), 4.55 (dt, J = 11.8, 7.6 Hz, C7-
H, 1H), 3.83 (s, OMe, 3H), 3.78 (s, OMe, 3H), 3.58 (s, OMe, 3H), 2.54 (dd, J = 13.0, 6.0 Hz, C6-
H, 1H), 2.20 (dd, J = 12.5, 6.6 Hz, C5-H, 1H), 2.09 – 2.01 (m, C5-H, C6-H, 2H), 1.89 (s,
COCH3, 3H), 1.85 (br.s, C11-CH3, 3H).
13C NMR (101 MHz, DMSO-d6): δ = 168.62, 159.81, 153.10, 152.87, 152.03, 150.30, 145.56,
140.61, 134.74, 130.20, 126.05, 122.92, 117.76, 113.95, 111.20, 108.21, 60.85, 60.53, 55.90,
48.40, 37.97, 29.83, 22.61, 18.12.

Elemental analysis: for C24H25NO6 calcld: C, 68.07; H, 5.95; found: C, 68.19; H, 6.04.
MALDI: (pos. mode) 446.1 (M+Na+) 40%, 462.0 (M+K+) 63%.
N-((7S)-12-(4-hydroxy-3-methoxyphenyl)-1,2,3-trimethoxy-10-oxo-5,6,7,10-tetrahydro-
benzo[6,7]cyclohepta[1,2-g]chromen-7-yl)acetamide (7b)
Eluent: petroleum ether-ethyl acetate-ethanol (4 : 1 : 1) White powder, yield 27%.
M.p. = 186 - 188°C
1H NMR (400 MHz, DMSO-d6): δ = 9.58 (s, OH, 1H), 8.57 (d, J = 8.2 Hz, NH, 1H), 7.62 (s,
C8-H, 1H), 7.40 (s, C12-H, 1H), 7.16 (d, J = 1.9 Hz, C5’-H, 1H), 7.00 (d, J = 2.0 Hz, C6-H, 1H),
6.94 (s, C10-H, 1H), 6.81 (s, C2’-H, 1H), 6.39 (s, C4-H, 1H), 4.57 (dt, J = 11.5, 7.6 Hz, C7-H,
1H), 3.87 (s, OMe, 3H), 3.82 (s, OMe, 3H), 3.76 (s, OMe, 3H), 3.45 (s, OMe, 3H), 2.57 – 2.53
(m, C6-H, 1H), 2.24 – 2.18 (m, C5-H, 1H), 2.12 – 2.05 (m, C6-H, C5-H, 2H), 1.93 (s, COCH3,
3H).
13C NMR (101 MHz, DMSO-d6) δ 168.69, 159.93, 154.92, 152.84, 152.80, 150.20, 148.34,
147.71, 145.83, 140.54, 134.84, 129.91, 128.00, 125.51, 122.83, 121.70, 116.58, 115.65, 113.12,
112.71, 111.73, 108.35, 62.79, 60.71, 60.59, 55.86, 55.54, 48.46, 37.93, 22.63.
Elemental analysis: for C30H29NO8 calcld.: C, 67.79; H, 5.50; found: C, 67.45; H, 5.67.
MALDI: 571.1 (M+K) 35%, 554.2 (M+Na) 15%, 532.2 (M+H) 13%
General procedure for synthesis of compounds 8a-h
Into a Schlenk flask under inert atmosphere were placed iodo-colchinol 2 (100.0 mg, 0.207
mmol, 1.00 equiv.) and Na2CO3 (65.8 mg, 0.620 mmol, 3.00 equiv.). Dry DMF (12 mL) was
added. The mixture was stirred at room temperature for 10 minutes and corresponding allyl
bromide (2.00 equiv.) was added dropwise. The resulting solution was stirred at 60° C for 20
hours. The solvent was removed under reduced pressure and the corresponding allyl ether was
isolated using column chromatography.
N-((5S)-3-(allyloxy)-2-iodo-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]cycloheptene-5-
yl)acetamide (8a)
Eluent: petroleum ether–ethyl acetate–ethanol (7 : 1 : 1). Pale-beige solid, yield 60%.
M.p. = 82 – 83 °C
1H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J = 8.5 Hz, NH, 1H), 7.68 (s, C8-H, 1H), 6.98 (s,
C11-H, 1H), 6.78 (s, C4-H, 1H), 6.08 (ddd, J = 15.5, 10.5, 5.1 Hz, OCH2-CH, 1H), 5.56 (d, J =
17.2 Hz, Cvinyl-H, 1H), 5.33 (d, J = 10.5 Hz, Cvinyl-H’, 1H), 4.66 (s, OCH2-CH, 2H), 4.49 (dt, J =
12.0, 7.9 Hz, C7-H, 1H), 3.83 (s, OMe, 3H), 3.78 (s, OMe, 3H), 3.50 (s, OMe, 3H), 2.55 – 2.50

(m, C6-H, 1H), 2.23 – 2.12 (m, C5-H, 1H), 2.12 – 2.00 (m, C5-H, 1H), 1.89 (s, COCH3, 3H),
1.87 – 1.76 (m, C6-H, 1H).
13C NMR (101 MHz, DMSO-d6) δ 168.48, 155.69, 152.46, 150.15, 142.51, 140.51, 139.43,
134.82, 133.20, 128.32, 122.85, 117.55, 108.20, 107.79, 83.24, 69.13, 60.60, 60.55, 55.82,
48.16, 38.39, 30.00, 22.64.
Elemental analysis: for C23H26INO5 calcd.: C, 52.78; H, 5.01; found: C, 52.55; H, 5.14.
MALDI (DCTB, pos. mode): 523.1 (M+) 100%, 332.2 (29%), 242.3 (23%).
N-((5S)-3-(((E)-but-2-en-1-yl)oxy)-2-iodo-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]-
cycloheptene-5-yl)acetamide (8b)
Eluent: petroleum ether–ethyl acetate–ethanol (8 : 1 : 1). Pale-beige solid, yield 71%.
M.p. = 124 - 126 °C
1H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J = 8.6 Hz, NH, 1H), 7.66 (s, C8-H, 1H), 6.95 (s,
C11-H, 1H), 6.78 (s, C4-H, 1H), 5.95 (dd, J = 15.3, 6.5 Hz, OCH2-CH=, 1H), 5.77 – 5.66 (m,
=CH-CH3, 1H), 4.58 (dd, J = 4.2, 1.6 Hz, OCH2, 2H), 4.53 – 4.44 (m, C7-H, 1H), 3.83 (s, OMe,
3H), 3.77 (s, OMe, 3H), 3.50 (s, OMe, 3H), 2.55 – 2.51 (m, C6-H, 1H), 2.22 – 2.11 (m, C5-H,
1H), 2.10 – 2.00 (m, C5-H, 1H), 1.89 (s, COCH3, 3H), 1.88 – 1.81 (m, C6-H, 1H), 1.79 – 1.71
(m, =CH-CH3, 3H).
13C NMR (101 MHz, DMSO-d6) δ 168.42, 155.82, 152.44, 150.13, 142.43, 140.50, 139.42,
134.81, 129.89, 128.15, 125.95, 125.38, 122.88, 108.20, 107.79, 83.33, 69.03, 60.61, 60.54,
55.82, 48.14, 38.46, 22.63, 17.63.
Elemental analysis: for C24H28INO5 calcld.: C, 53.64; H, 5.25; found: C, 53.49; H, 5.32.
MALDI: (pos. mode) 537.3 (M+) 100%, 596.1 (M+Na+) 26%.
N-((5S)-2-iodo-9,10,11-trimethoxy-3-((3-methylbut-2-en-1-yl)oxy)-6,7-dihydro-5H-dibenzo-
[a,c]cycloheptene-5-yl)acetamide (8c)
Eluent: petroleum ether–ethyl acetate–ethanol (8 : 1 : 1). White solid, yield 64%.
M.p. = 77 – 79 °C.
1H NMR (400 MHz, DMSO-d6) δ 8.39 (d, J = 8.5 Hz, NH, 1H), 7.65 (s, C8-H, 1H), 6.96 (s,
C11-H, 1H), 6.78 (s, C4-H, 1H), 5.47 (t, J = 6.7 Hz, Cvinyl-H, 1H), 4.62 (d, J = 6.5 Hz, OCH2,
2H), 4.48 (dt, J = 12.0, 7.9 Hz, C7-H, 1H), 3.83 (s, OMe, 3H), 3.77 (s, OMe, 3H), 3.50 (s, OMe,
3H), 2.16 (dt, J = 12.1, 8.6 Hz, C6-H, 1H), 2.10 – 2.01 (m, C5-H, 2H), 1.88 (s, COCH3, 3H),
1.88 – 1.80 (m, C6-H, 1H), 1.77 (s, Cvinyl-CH3, 3H), 1.76 (s, Cvinyl-CH3’, 3H).

13C NMR (101 MHz, DMSO-d6) δ 168.47, 156.01, 152.44, 150.14, 142.46, 140.52, 139.42,
137.64, 134.83, 128.18, 122.91, 119.54, 108.21, 107.92, 83.57, 65.56, 60.61, 60.56, 55.83,
48.21, 39.52, 38.36, 30.01, 25.50, 22.65, 18.16.
Elemental analysis: for C25H30INO5 calcld.: C, 54.45; H, 5.48; found: C, 54.59; H, 5.37.
N-((5S)-2-iodo-9,10,11-trimethoxy-3-(((E)-pent-2-en-1-yl)oxy)-6,7-dihydro-5H-dibenzo-
[a,c]cycloheptene-5-yl)acetamide (8d)
Eluent: petroleum ether–ethyl acetate–ethanol (4 : 1 : 1). Pale-beige solid, yield 62%.
M.p. = 102 – 104 °C.
1H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 8.5 Hz, NH, 1H), 7.66 (s, C8-H, 1H), 6.96 (s,
C11-H, 1H), 6.78 (s, C4-H, 1H), 6.02 – 5.94 (m, OCH2-CH, 1H), 5.69 (dt, J = 15.4, 5.7 Hz, CH2-
CH=CH, 1H), 4.58 (d, J = 8.9 Hz, OCH2-CH, 2H), 4.53 – 4.44 (m, C7-H, 1H), 3.83 (s, OMe,
3H), 3.78 (s, OMe, 3H), 3.51 (s, OMe, 3H), 2.26 – 1.97 (m, C6-H, C5-H, CH2-CH3, 5H), 1.89 (s,
COCH3, 3H), 1.87 – 1.81 (m, C6-H, 1H), 0.99 (t, J = 7.5 Hz, CH2-CH3, 3H).
13C NMR (101 MHz, DMSO-d6) δ 168.47, 155.86, 152.46, 150.15, 142.44, 140.52, 139.43,
136.45, 134.83, 128.22, 123.60, 122.89, 108.20, 107.93, 83.41, 69.16, 60.61, 60.55, 55.83,
48.17, 38.42, 30.02, 24.77, 22.64, 13.17.
Elemental analysis: for C25H30INO5 calcld.: C, 54.45; H, 5.48; found: C, 54.61; H, 5.59.
Methyl (E)-4-(((5S)-5-acetamido-2-iodo-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]-
cycloheptene-3-yl)oxy)but-2-enoate (8e)
Eluent: petroleum ether–ethyl acetate–ethanol (8 : 1 : 1). White solid, yield 73%.
M.p. = 93 – 95 °C.
1H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 8.5 Hz, NH, 1H), 7.71 (s, C8-H, 1H), 7.05 (s,
C11-H, 1H), 6.79 (s, C4-H, 1H), 6.77 (dt, J = 6.0, 1.6 Hz, CH-CO2Me, 1H), 5.13 (dd, J = 13.1,
7.2 Hz, CH2CH=, 1H), 4.49 (dt, J = 12.1, 7.8 Hz, C7-H, 1H), 3.83 (s, OMe, 3H), 3.78 (s, OMe,
3H), 3.64 (s, CO2Me, 3H), 3.51 (s, OMe, 3H), 3.34 (s, OCH2, 1H), 3.32 (s, OCH2, 1H), 2.55 –
2.51 (m, C6-H, 1H), 2.17 (td, J = 12.2, 6.4 Hz, C5-H, 1H), 2.06 (dd, J = 12.6, 6.7 Hz, C5-H,
1H), 1.88 (s, COCH3, 3H), 1.84 (dd, J = 11.2, 4.7 Hz, C6-H, 1H).
13C NMR (101 MHz, DMSO-d6) δ 171.18, 168.53, 154.29, 152.69, 150.14, 142.92, 142.06,
140.51, 139.70, 134.84, 130.53, 122.48, 110.12, 108.24, 104.67, 83.55, 60.67, 60.56, 55.83,
51.67, 48.10, 39.52, 38.28, 29.89, 29.40, 24.77, 22.66.
Elemental analysis: for C25H28INO7 calcld.: C, 51.65; H, 4.85; found: C, 51.52; H, 4.77.
MALDI: (СНСА, pos. mode): 603.9 (M+Na+) 100%, 581.0 (M+) 14%.

N-((5S)-3-(((E)-2,7-dimethylocta-2,6-dien-1-yl)oxy)-2-iodo-9,10,11-trimethoxy-6,7-dihydro-
5H-dibenzo[a,c]cycloheptene-5-yl)acetamide (8f)
Eluent: petroleum ether–ethyl acetate–ethanol (5 : 1 : 1). Pale-yellow oil, yield 55%. Atomic
numeration for NMR assignment is given in SI.
1H NMR (400 MHz, DMSO-d6) δ 8.44 (d, J = 8.5 Hz, NH, 1H), 7.65 (s, C8-H, 1H), 6.98 (s,
C11-H, 1H), 6.78 (s, C4-H, 1H), 5.46 (t, J = 6.1 Hz, C3’-H, 1H), 5.11 – 5.04 (m, C6’-H, 1H),
4.65 (d, J = 6.4 Hz, C1’-H, 2H), 4.48 (dt, J = 11.9, 7.9 Hz, C7-H, 1H), 3.82 (s, OMe, 3H), 3.77
(s, OMe, 3H), 3.50 (s, OMe, 3H), 2.22 – 2.09 (m, C6-H, C5-H, 2H), 2.09 – 2.04 (m, C4’-H, C5’-
H, 4H), 2.02 – 1.90 (m, C6-H, C5-H, 2H), 1.88 (s, COCH3, 3H), 1.75 (s, C2’-CH3, 3H), 1.62 (s,
C7’-CH3, 3H), 1.56 (s, C7’-CH3, 3H).
13C NMR (101 MHz, DMSO-d6) δ 168.49, 155.99, 152.45, 150.16, 142.45, 140.96, 140.53,
139.40, 134.84, 131.06, 128.21, 123.71, 122.94, 119.26, 108.21, 108.15, 83.65, 65.59, 60.62,
60.56, 55.84, 48.23, 39.52, 38.98, 38.40, 30.02, 25.84, 25.51, 22.65, 17.60, 16.53.
Elemental analysis: for C30H38INO5 calcld.: C, 58.16; H, 6.18; found: C, 58.28; H, 6.31.
N-((5S)-3-(cinnamyloxy)-2-iodo-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]-
cycloheptene-5-yl)acetamide (8g)
Eluent: petroleum ether–ethyl acetate–ethanol (6 : 1 : 1). Beige solid, yield 73%.
M.p. = 181 – 183 °C.
1H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 8.5 Hz, NH, 1H), 7.69 (s, C8-H, 1H), 7.50 (d, J =
7.2 Hz, C2’-H, C6’-H, 2H), 7.37 (t, J = 7.5 Hz, C3’-H, C5’-H, 2H), 7.29 (t, J = 7.3 Hz, C4’-H,
1H), 7.04 (s, C11-H, 1H), 6.90 (d, J = 16.0 Hz, CH=CH-Ph, 1H), 6.78 (s, C4-H, 1H), 6.54 (dt, J
= 16.0, 5.6 Hz, CH=CH-Ph, 1H), 4.83 (d, J = 5.5 Hz, OCH2, 2H), 4.54 – 4.44 (m, C7-H, 1H),
3.83 (s, OMe, 3H), 3.78 (s, OMe, 3H), 3.51 (s, OMe, 3H), 2.55 – 2.51 (m, C6-H, 1H), 2.17 (dt, J
= 18.0, 5.8 Hz, C5-H, 1H), 2.06 (td, J = 12.5, 5.9 Hz, C5-H, 1H), 1.94 – 1.85 (m, C6-H, 1H),
1.83 (s, COCH3, 3H).
13C NMR (101 MHz, DMSO-d6) δ 168.47, 155.88, 152.47, 150.15, 142.56, 140.52, 139.48,
136.07, 134.82, 132.54, 128.69, 128.40, 127.96, 126.51, 124.52, 122.85, 108.22, 107.99, 83.45,
69.28, 60.62, 60.55, 55.83, 48.17, 38.41, 30.01, 22.61.
Elemental analysis: for C29H30INO5 calcld.: C, 58.10; H, 5.04; found: C, 58.23; H, 5.19.
N-((5S)-3-((1,1-difluoroallyl)oxy)-2-iodo-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]-
cycloheptene-5-yl)acetamide (8h)

The reaction was carried out in a closed vial due to the low boiling point of 3-bromo-3,3-
difluoropropene. Eluent: petroleum ether–ethyl acetate–ethanol (7 : 1 : 1). White solid, yield
61%.
M.p. = 109 – 111 °C.
1H NMR (400 MHz, DMSO-d6) δ 8.49 (d, J = 8.1 Hz, NH, 1H), 7.77 (s, C8-H, 1H), 7.28 (s,
C11-H, 1H), 6.81 (s, C4-H, 1H), 6.26 (ddt, J = 17.5, 10.8, 6.7 Hz, CF2-CH=, 1H), 6.04 (dd, J =
17.2, 2.1 Hz, -CH=CH2, 1H), 5.80 (d, J = 10.8 Hz, -CH=CH2,1H), 4.44 (dt, J = 12.0, 7.6 Hz,
C7-H, 1H), 3.84 (s, OMe, 3H), 3.78 (s, OMe, 3H), 3.55 (s, OMe, 3H), 2.55 (dd, J = 13.3, 5.9 Hz,
C6-H, 1H), 2.23 – 2.01 (m, C6-H, C5-H, 3H), 1.86 (s, COCH3, 3H).
13C NMR (101 MHz, DMSO-d6) δ 168.52, 152.99, 150.16, 148.73, 142.80, 140.48, 139.93,
134.91, 133.41, 128.67, 123.64, 122.04, 116.90, 108.25, 88.11, 76.38, 60.77, 60.55, 55.86,
48.17, 37.92, 29.83, 22.48.
19F NMR (377 MHz, DMSO-d6) δ -65.69
Elemental analysis: for C23H24F2INO5 calcld.: C, 49.39; H, 4.33; found: C, 49.56; H, 4.24.
General procedure for synthesis of compounds 9a-h, 10e
Corresponding allyl ether 8a-h (1.00 equiv.), Pd(dppf)Cl2 (0.05 equiv.) and KOAc (3.00 equiv.)
were placed into a Schlenk flask. The flask was filled with inert atmosphere and dry DMSO (15
mL) was added. The mixture was stirred for 18 hours at 80° C, poured into distilled water (100
mL), and extracted with AcOEt (3x100 mL). The extract was dried over Na2SO4 and
concentrated under reduced pressure. The crude product was purified using column
chromatography.
N-((7S)-1,2,3-trimethoxy-11-methylene-6,7,10,11-tetrahydro-5H-benzo[6,7]cyclohepta[1,2-
f]benzofuran-7-yl)acetamide (9a)
Eluent: petroleum ether–ethyl acetate–ethanol (8 : 1 : 1). Pale-beige solid, yield 66%.
M.p. = 198 – 200 °C.
1H NMR (400 MHz, DMSO-d6): δ = 8.37 (d, J = 8.4 Hz, NH, 1H), 7.41 (s, C8-H, 1H), 6.86 (s,
C11-H, 1H), 6.77 (s, C4-H, 1H), 5.46 (s, Cvinyl-H, 1H), 5.14 (s, CH2-O, 2H), 5.02 (s, Cvinyl-H,
1H), 4.51 – 4.43 (m, C7-H, 1H), 3.83 (s, OMe, 3H), 3.79 (s, OMe, 3H), 3.48 (s, OMe, 3H), 2.19
– 1.99 (m, C6-H, C5-H, 3H), 1.87 (s, COCH3, 3H), 1.85 – 1.78 (m, C6-H, 1H).
13C NMR (101 MHz, DMSO-d6): δ = 168.38, 162.52, 152.19, 150.31, 143.89, 143.25, 140.56,
134.74, 126.57, 124.31, 123.61, 122.08, 108.03, 105.23, 99.63, 74.91, 60.55, 55.82, 48.50,
38.16, 29.94, 22.62.
Elemental analysis: for C23H25NO5 calcld.: C, 69.86; H, 6.37; found: C, 69.73; H, 6.24.
MALDI: (СНСА, pos. mode) 394.3 (M+) 100%.

N-((7S,E)-11-ethylidene-1,2,3-trimethoxy-6,7,10,11-tetrahydro-5H-benzo[6,7]-
cyclohepta[1,2-f]benzofuran-7-yl)acetamide (9b)
Eluent: petroleum ether–ethyl acetate–ethanol (7 : 1 : 1). Pale-beige solid, yield 62%.
M.p. = 163 – 165 °C.
1H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J = 8.6 Hz, NH, 1H), 7.66 (s, C8-H, 1H), 6.96 (s,
C11-H, 1H), 6.75 (s, C4-H, 1H), 5.95 – 5.88 (m, Cvinyl-H, 1H), 4.64 – 4.54 (m, CH2-O, 2H), 4.49
(dd, J = 7.6, 3.7 Hz, C7-H, 1H), 3.83 (s, OMe, 3H), 3.78 (s, OMe, 3H), 3.50 (s, OMe, 3H), 2.48 –
2.44 (m, C6-H, 1H), 2.19 – 2.10 (m, C5-H, 2H), 2.06 (dd, J = 13.0, 7.6 Hz, C6-H, 1H), 1.89 (s,
COCH3, 3H), 1.74 (d, J = 6.2 Hz, Cvinyl-CH3, 3H).
13C NMR (101 MHz, DMSO-d6) δ 168.30, 158.72, 152.01, 150.31, 141.29, 140.55, 138.28,
134.75, 126.89, 126.11, 125.76, 124.61, 116.36, 108.06, 104.29, 75.74, 60.56, 60.50, 55.81,
48.25, 45.75, 38.49, 30.09, 22.63.
Elemental analysis: for C24H27NO5 calcld: C, 70.40; H, 6.65; found: C, 70.53; H, 6.72.
MALDI: (СНСА, pos. mode): 409.4 (M+) 100%.
N-((7S)-1,2,3-trimethoxy-11-(propan-2-ylidene)-6,7,10,11-tetrahydro-5H-benzo[6,7]-
cyclohepta[1,2-f]benzofuran-7-yl)acetamide (9c)
Eluent: petroleum ether–ethyl acetate–ethanol (7 : 1 : 1). Yellow solid, yield 86%.
M.p. = 166 – 167 °C.
1H NMR (400 MHz, DMSO-d6) δ 8.39 (d, J = 8.5 Hz, NH, 1H), 7.65 (s, C8-H, 1H), 6.96 (s,
C11-H, 1H), 6.78 (s, C4-H, 1H), 4.62 (d, J = 6.5 Hz, CH2-O, 2H), 4.48 (dd, J = 7.9, 4.1 Hz, C7-
H, 1H), 3.83 (s, OMe, 3H), 3.77 (s, OMe, 3H), 3.50 (s, OMe, 3H), 2.21 – 2.01 (m, C6-H, C5-H,
3H), 1.88 (s, COCH3, 3H), 1.87 – 1.84 (m, C6-H, 1H), 1.77 (s, Cvinyl-CH3, 3H), 1.75 (s, Cvinyl
CH3, 3H).
-
13C NMR (101 MHz, DMSO-d6) δ 168.47, 156.01, 152.45, 150.15, 142.46, 140.52, 139.43,
137.65, 134.83, 128.18, 122.91, 119.54, 108.21, 107.93, 83.58, 65.57, 60.62, 60.56, 55.83,
48.21, 38.37, 30.01, 25.50, 22.65, 18.17.
Elemental analysis: for C25H29NO5 calcld.: C, 70.90; H, 6.90; found: C, 70.76; H, 6.81.
MALDI: (pos. mode): 423.2 (M+) 100%.
N-((7S)-1,2,3-trimethoxy-11-propylidene-6,7,10,11-tetrahydro-5H-benzo[6,7]-
cyclohepta[1,2-f]benzofuran-7-yl)acetamide (9d)
Eluent: petroleum ether–ethyl acetate–ethanol (4 : 1 : 1). Beige solid, yield 44%.
M.p. = 125 – 127 °C.

1H NMR (400 MHz, DMSO-d6) δ 8.34 (d, J = 3.4 Hz, NH, 1H), 7.31 (s, C8-H, 1H), 6.99 (s,
C11-H, 1H), 6.77 (s, C4-H, 1H), 5.89 – 5.78 (m, Cvinyl-H, 1H), 5.15 (br.s., CH2-O, 2H), 4.49 –
4.46 (m, C7-H, 1H), 4.17 (q, J = 7.5 Hz, CH2CH3, 2H), 3.82 (s, OMe, 3H), 3.77 (s, OMe, 3H),
3.44 (s, OMe, 3H), 2.48 – 2.46 (m, C6-H, 1H), 2.14 – 2.09 (m, C5-H, 2H), 2.03 – 2.01 (m, C6-H,
1H), 1.86 (s, COCH3, 3H), 1.02 (t, J = 7.5 Hz, CH2CH3, 3H).
13C NMR (101 MHz, DMSO-d6) δ 168.36, 161.90, 158.71, 152.02, 150.32, 142.51, 141.12,
140.58, 134.78, 131.05, 127.63, 126.82, 126.07, 124.69, 104.25, 76.25, 60.60, 60.51, 55.84,
48.27, 38.51, 30.12, 22.65, 17.61, 13.61.
Elemental analysis: for C25H29NO5 calcld.: C, 70.90; H, 6.90; found: C, 70.78; H, 7.03.
MALDI (DCTB, pos. mode): 423.2 (M+) 85%.
Methyl
2-((7S)-7-acetamido-1,2,3-trimethoxy-6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-
f]benzofuran-11-yl)acetate (10e)
Eluent: petroleum ether–ethyl acetate–ethanol (7 : 2 : 2). Yellow solid, yield 68%.
M.p. = 151 – 152 °C.
1H NMR (400 MHz, CD3OD) δ 7.72 (s, C8-H, 1H), 7.62 (s, C11-H, 1H), 7.46 (s, Cfuran-H, 1H),
6.77 (s, C4-H, 1H), 4.77 (dd, J = 11.9, 6.5 Hz, C7-H, 1H), 3.92 (s, CO2Me, 3H), 3.91 (s, OMe,
3H), 3.78 (d, J = 0.5 Hz, CH2CO2Me, 2H), 3.73 (s, OMe, 3H), 3.49 (s, OMe, 3H), 2.54 (dd, J =
12.1, 5.0 Hz, C6-H, 1H), 2.29 (ddd, J = 16.0, 11.8, 6.2 Hz, C5-H, 2H), 2.05 (s, COCH3, 3H),
2.00 – 1.94 (m, C6-H, 1H).
13C NMR (101 MHz, CD3OD) δ 173.08, 172.48, 156.32, 154.11, 152.20, 144.49, 142.43,
138.67, 136.62, 130.48, 127.42, 126.51, 122.33, 114.81, 109.01, 106.67, 61.61, 61.44, 56.59,
52.60, 50.75, 39.83, 31.42, 30.04, 22.64.
Elemental analysis: for C25H27NO7 calcld.: C, 66.21; H, 6.00; found: C, 66.45; H, 6.13.
N-((7S)-1,2,3-trimethoxy-11-(6-methylhept-5-en-2-ylidene)-6,7,10,11-tetrahydro-5H-
benzo[6,7]cyclohepta[1,2-f]benzofuran-7-yl)acetamide (9f)
Eluent: petroleum ether–ethyl acetate–ethanol (4 : 1 : 1). Pale-beige oil, yield 69%. For atomic
numeration in NMR assignment see SI.
1H NMR (400 MHz, DMSO-d6) δ 8.34 (d, J = 8.5 Hz, NH, 1H), 6.98 (s, C8-H, 1H), 6.78 (s,
C11-H, 1H), 6.75 (s, C4-H, 1H), 4.99 (t, J = 6.4 Hz, C4’-H, 1H), 4.84 (d, J = 9.2 Hz, OCH2, 2H),
4.50 – 4.47 (m, C7-H, 1H), 3.81 (s, OMe, 3H), 3.76 (s, OMe, 3H), 3.44 (s, OMe, 3H), 2.47 – 2.42
(m, C5-H, 1H), 2.13 – 2.01 (m, C5-H, C6-H, C2’-H, C3’-H, 7H), 1.87 (s, COCH3, 3H), 1.63 (s,
C1’-CH3, 3H), 1.55 (s, C5’-CH3, 3H), 1.46 (s, C5’-CH3, 3H).

13C NMR (101 MHz, DMSO-d6) δ 168.35, 159.24, 152.02, 150.29, 149.01, 141.25, 140.58,
134.77, 131.03, 126.63, 126.12, 126.05, 124.72, 123.91, 123.86, 108.05, 104.20, 75.82, 60.57,
60.50, 55.83, 48.53, 48.28, 38.49, 32.58, 25.98, 25.35, 23.30, 22.66, 17.46.
Elemental analysis: for C30H37NO5 calcld.: C, 73.29; H, 7.59; found: C, 73.47; H, 7.75.
MALDI (pos. mode): 491.2 (M+) 83%.
N-((7S)-11-((Z)-benzylidene)-1,2,3-trimethoxy-6,7,10,11-tetrahydro-5H-benzo[6,7]-
cyclohepta[1,2-f]benzofuran-7-yl)acetamide (9g)
Eluent: petroleum ether–ethyl acetate–ethanol (4 : 1 : 1). Light-brown solid, yield 63%.
M.p. = 135 – 137 °C.
1H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 8.4 Hz, NH, 1H), 7.58 (s, C8-H, 1H), 7.41 (t, J =
7.6 Hz, C2Ar-H, C6Ar-H, 2H), 7.32 (d, J = 7.4 Hz, C3Ar-H, C5Ar-H, 2H), 7.25 (t, J = 7.3 Hz, C4Ar-
H, 1H), 6.99 (t, J = 2.9 Hz, Cvinyl-H, 1H), 6.93 (s, C11-H, 1H), 6.79 (s, C4-H, 1H), 5.53 (qd, J =
16.3, 3.0 Hz, CH2-O, 2H), 4.52 (dd, J = 17.0, 9.5 Hz, C7-H, 1H), 3.84 (s, OMe, 3H), 3.81 (s,
OMe, 3H), 3.50 (s, OMe, 3H), 2.56 – 2.51 (m, C6-H, 1H), 2.19 – 2.06 (m, C5-H, 2H), 1.89 (s,
COCH3, 3H), 1.87 – 1.83 (m, C6-H, 1H).
13C NMR (101 MHz, DMSO-d6) δ 168.43, 161.42, 152.21, 150.37, 143.77, 140.60, 136.72,
136.27, 134.78, 128.73, 127.99, 126.87, 126.51, 124.91, 124.39, 121.52, 115.95, 108.07, 105.02,
74.69, 60.62, 60.61, 55.83, 48.56, 38.17, 29.97, 22.64.
Elemental analysis: for C29H29NO5 calcld.: C, 73.87; H, 6.20; found: C, 73.72; H, 6.34.
MALDI (DCTB, pos. mode): 471.1 (M+) 87%.
N-((7S)-10,10-difluoro-1,2,3-trimethoxy-11-methylene-6,7,10,11-tetrahydro-5H-benzo-
[6,7]cyclohepta[1,2-f]benzofuran-7-yl)acetamide (9h)
Eluent: petroleum ether–ethyl acetate–ethanol (6 : 1 : 1). White solid, yield 73%.
M.p. = 126 – 128 °C
1H NMR (400 MHz, DMSO-d6) δ 8.47 (d, J = 8.1 Hz, NH, 1H), 7.67 (s, C8-H, 1H), 7.11 (s,
C11-H, 1H), 6.81 (s, C4-H, 1H), 6.35 (br.s, Cvinyl-H, 1H), 5.93 (br.s, Cvinyl-H’, 1H), 4.50 (dt, J =
14.6, 7.4 Hz, C7-H, 1H), 3.84 (s, OMe, 3H), 3.80 (s, OMe, 3H), 3.50 (s, OMe, 3H), 2.59 – 2.52
(m, C6-H, 1H), 2.19 – 2.05 (m, C5-H, 2H), 1.97 – 1.93 (m, C6-H, 1H), 1.89 (s, COCH3, 3H).
13C NMR (101 MHz, DMSO-d6) δ 168.64, 154.71, 152.66, 150.26, 145.59, 140.55, 135.98 (J C-
F= 25.5 Hz), 134.80, 129.87, 127.82, 123.34, 123.24, 119.30, 113.93, 108.14, 105.52, 60.68,
60.59, 55.84, 48.75, 37.97, 29.73, 22.60.
19F NMR (377 MHz, DMSO) δ -66.04 (J H, F= 372.5, J H’, F= 165.2 Hz).
Elemental analysis: for C23H23F2NO5 calcld: C, 64.03; H, 5.37; found: C, 64.12; H, 5.45.

MALDI: (pos. mode): 431.1 (M+) 74%, 454.1 (M+Na+) 11%, 470.0 (M+K+) 20%.
N-((7S)-1,2,3-trimethoxy-11-methyl-6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-
f]benzofuran-7-yl)acetamide (10a)
Compound 9a (100.0 mg, 0.253 mmol) was dissolved in dry DCM (15 mL). Trifluoroacetic acid
(3 mL) was added, the mixture was stirred at room temperature for 1 hour, neutralized with sat.
aq. NaHCO3, and extracted with AcOEt (50 mL x 3). The extract was dried over Na2SO4 and
evaporated under reduced pressure. Yellow solid, yield 97.7 mg (98%).
M.p. = 202-204 °C
1H NMR (400 MHz, DMSO-d6) δ 8.45 (d, J = 8.5 Hz, NH, 1H), 7.74 (d, J = 1.3 Hz, C8-H, 1H),
7.49 (s, C11-H, 1H), 7.47 (s, C9-H, 1H), 6.79 (s, C4-H, 1H), 4.62 (dt, J = 11.9, 7.8 Hz, C7-H,
1H), 3.84 (s, OMe, 3H), 3.80 (s, OMe, 3H), 3.45 (s, OMe, 3H), 2.21 (d, J = 1.1 Hz, C10-CH3,
3H), 2.19 – 2.11 (m, C6-H, 1H), 2.02 (td, J = 12.6, 6.9 Hz, C5-H, 1H), 1.90 (s, COCH3, 3H),
1.89 – 1.82 (m, C6-H, 1H).
13C NMR (101 MHz, DMSO-d6) δ 168.40, 154.00, 152.28, 150.38, 141.94, 140.60, 137.67,
134.75, 128.54, 126.78, 124.74, 120.36, 115.19, 108.00, 105.78, 60.59, 60.57, 55.83, 48.37,
38.44, 30.69, 22.71, 7.66.
Elemental analysis: for C23H25NO5 calcld.: C, 69.86; H, 6.37; found: C, 69.59; H, 6.24.
MALDI: (pos. mode): 395.2 (M+) 89%, 418.1 (M+Na+) 14%.
Biology
Cell cultures
Human cell lines: pancreatic (PANC-1, COLO357), cervical (HeLa), colon (SW620) cancers,
Burkitt's lymphoma (Raji), and immortalized kidney (HEK-293) and keratinocytes (HaCaT) cells
were grown in DMEM medium supplemented with 10% fetal calf serum (FCS), pen-strep-glut
(all from PanEco, Moscow, Russian Federation). Adherent cells were detached using 0.05%
trypsin-EDTA (PanEco, Moscow), counted and sub-cultured. For the assay cells were seeded in
the appropriate plates (96- or 24-well plates), adjusted to 3x105 cells/mL.
MTT-assay
Cytotoxic effect of colchicine and the compounds was estimated by a standard 3-(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma) test. All the compounds were
dissolved in dimethyl sulfoxide to 20 mM concentration and stored at -20oC until use. Different
dilutions of the new compounds from 20 µM to 0.001 nM were prepared separately and
transferred in 100 µl to the plates. Cell were added at 5-10*103 per well. Non-treated cells served

as controls. Plates were incubated for 72 hrs. For the last 4 hours, 10 µl MTT (5 mg/ml) were
added to each well. After the incubation, culture medium was removed and 100 µl dimethyl
sulfoxide was added to each well. Plates were incubated in a shaker 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 (Microsoft). Cytotoxic concentration giving 50%
of the maximal toxic effect (IC50) was calculated from the titration curves. The inhibition of
proliferation (inhibition index, II) was calculated as [1 – (ODexperiment / ODcontrol)], where OD was
MTT optical density.
Confocal analysis
For confocal analysis, cells were grown overnight on sterile cover slips immersed into 6-well
plates (Costar) in 200 µl of complete culture medium. Colchicine or new compounds 9a and 9h
were dissolved in 4 ml of complete medium and added to the wells. β-tubulin was identified by
anti-β-tubulin antibody (SantaCruz, USA) followed by anti-mouse IgG-AlexaFluor555
(Molecular Probes, Invitrogen, USA) in cells permeabilized by 0.1% Triton X100 in PBS. Slides
were analyzed using Eclipse TE2000 confocal microscope (Nikon, Japan).
Flow cytometry
Cell cycle was analyzed using PI-stained DNA. Cells from colchicinoid-treated cultures were
collected at indicated time, trypsinized, washed in ice-cold PBS, fixed by the addition of 70%
o
ethanol and left for 2 hours at -20 C. Thereafter, the cells were washed twice in PBS, stained
with 50 μg/ml of propidium iodide (Sigma, USA) in PBS, treated with 10 µg/ml of RNAse and
analyzed by flow cytometry using FACScan device (BD, USA). Total 2000 events were
collected. The results were analyzed using WinMDI 2.8.
Acute toxicity in vivo
Determination of the acute in vivo toxicity was performed in C57BL/6, BALB/c, CBA, A/Sn,
and outbred CD1 mice (6 mice of each strain per group). All experimental procedures were
approved by Animal Care and Use Committee of Nizhny Novgorod State University, Russia.
Several genetically different strains were used to cover various genotypes. Preparations 9a, 9h,
and colchicine were dissolved in DMSO to 20 mM, diluted with saline, and injected
intravenously in 200 µl per mouse. Total volume of stack solution of colchicinoids in DMSO
varied from 30 to 43 µl depending on the mouse body weight. DMSO was diluted in the same
way (40 µl of DMSO in 160 µl saline) and was used as a control. Preparations were injected 5
(n=3 per strain, 15 total mice for preparation) and 10 mg/kg (n=3 per strain, 15 total mice for

preparation) of body weight. Mice were monitored during a week, lethality was detected only at
days 1-2. All studies were conducted in an AAALAC accredited facility in compliance with the
PHS Guidelines for the Care and Use of Animals in Research.
.
Microtubule assembly assay
Tubulin was purified from the lamb brain by ammonium sulfate fractionation and ion exchange
chromatography. The pure protein was stored in liquid nitrogen and prepared as described[38].
Protein concentrations were determined spectrophotometrically with a PerkinElmer
spectrophotometer Lambda 800 and an extinction coefficient at 275 nm of 1.07 L·g−1·cm−1 in
0.5% SDS in neutral aqueous buffer or 1.09 L·g−1·cm−1 in 6 M guanidine hydrochloride.
Microtubule assembly was performed in 20 mM sodium phosphate buffer, 1 mM EGTA, 10 mM
MgCl2, 3.4 M glycerol, and 1 mM GTP at pH 6.5. The aliquots were incubated for 40 min at 4
°C prior to start the reaction by warming the samples to 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.
Crystallization, data collection, and structure refinement of 9a bound to the T2R complex.
Compound 9a was added to the T2R complex using a 5-fold excess of the ligand over tubulin
(purified from ovine brain). Crystals were grown from T2R seeds obtained from subtilisin-treated
tubulin[45]. X-ray data were collected on the PROXIMA 1 beamline at SOLEIL Synchrotron
(Saint-Aubin, France), and were processed with autoPROC[48] which implements the
STARANISO treatment for anisotropy (http://staraniso.globalphasing.org/). Molecular
replacement was done with Phaser[49] using the T2R complex (pdb id 3RYC) as the search
model. The structural model was refined by BUSTER[50] with iterative model building in
Coot[51]. Data collection and refinement statistics are reported in Table 1 in Supplementary
Information. The atomic coordinates and structure factors have been deposited in the Protein
Data Bank under accession code 6TDE. Figures of structural models were generated with
PyMOL (www.pymol.org).
Associated content
Supporting Information
Supplementary data to this article can be found online at www.sciencedirect.com.
Accession Codes
PDB code for T2R with 9a is 6TDE.

Author information
Corresponding author
E-mail: afedorovnn@yandex.ru (A. Yu. F)
Notes
The authors declare no competing financial interest.
Author contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
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