Synthesis of Bifunctional Furano-Allocolchicinoids

Article abstract of DOI:10.1055/s-0036-1589060

An efficient seven-step semisynthetic approach towards non-racemic bifunctional furano-allocolchicinoids, starting from naturally occurring colchicine is presented. The Pd-catalyzed domino Sonogashira coupling/5- endo - dig cyclization was employed as the key step. The prepared compounds exhibited substantial cytotoxicity against T3M4, MiaPaCa-2, Colo-357, and PANC-1 cell lines. The presence of two functionalities with different reactivity (hydroxyl and amino groups) in the target molecules allows for an easy conjugation of furano-allocolchicinoids with drug delivery carriers, and opens promising opportunities for their further exploitation in the search of therapeutics..

Full text of DOI:10.1055/s-0036-1589060

SYNTHESIS
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
©
Georg Thieme Verlag Stuttgart · New York
2017, 49, A–F
paper
A
en
Synthesis
Iu. A. Gracheva et al.
Paper
Synthesis of Bifunctional Furano-Allocolchicinoids
Iuliia A. Grachevaa
Elena V. Svirshchevskayab
Elena A. Zaburdaevaa
MeO
MeO
MeO
NH2
NHAc
7
steps
MeO
Alexey Yu. Fedorov*a
MeO
MeO
O
O
a
Department of Chemistry, N. I. Lobachevsky State University of
Nizhny Novgorod, 23 Gagarin Avenue, 603950 Nizhny Novgorod,
Russian Federation
afnn@rambler.ru
Laboratory of Cell Interactions, Shemyakin-Ovchinnikov Institute
OMe
–)-(aR,7S)-colchicine
R = H, Me
(
OH
R
b
of Bioorganic Chemistry RAS, 16/10 Miklukho-Maklaya Street,
117997 Moscow, Russian Federation
Received: 08.04.2017
Accepted after revision: 29.05.2017
Published online: 26.06.2017
noids may be effective in the case of autoimmune and
neurodegenerative diseases, as well as for the treatment of
chronic infections.8
DOI: 10.1055/s-0036-1589060; Art ID: ss-2017-z0234-op
Among the compounds, structurally related to colchi-
Abstract An efficient seven-step semisynthetic approach towards
non-racemic bifunctional furano-allocolchicinoids, starting from natu-
rally occurring colchicine is presented. The Pd-catalyzed domino Sono-
gashira coupling/5-endo-dig cyclization was employed as the key step.
The prepared compounds exhibited substantial cytotoxicity against
T3M4, MiaPaCa-2, Colo-357, and PANC-1 cell lines. The presence of two
functionalities with different reactivity (hydroxyl and amino groups) in
the target molecules allows for an easy conjugation of furano-allocol-
chicinoids with drug delivery carriers, and opens promising opportuni-
ties for their further exploitation in the search of therapeutics.
cine,9 allocolchicine (2)10 and its analogues proved to be
suitable candidates for further elaboration due to their
promising biological activities.11 Recently, our group de-
scribed the synthesis and biological evaluation of a series of
heterocyclic allocolchicine congeners – pyrrolo-allocolchi-
12
cines 3–5, with the different orientation of the pyrrole
fragment and the corresponding furano-allocolchicines 6
and 7.1
3,14
Derivatives 3–7, containing a hydroxyl group in
the benzylic or pseudobenzylic position demonstrated high
antimitotic and apoptosis-inducing activity in nano- and
subnanomolar concentrations, and promising in vivo activi-
ty with less pronounced toxic effects in comparison with
parent colchicine (1) (Figure 1).
Key words antitumor agents, tubulin, colchicine, allocolchicinoids,
heterocycles
The key role of the mitotic spindle in cell division has
made it a promising target for cancer chemotherapy.1
Colchicine (1), an alkaloid found in the plants of the genus
Colchicum, Merendera, Androcymbium, Gloriosa, and Litton-
One way to reduce the general toxicity of a drug and to
improve its biodistribution upon the systemic administra-
15
tion is the design of carrier-linked drug delivery systems.
2
ia, effectively inhibits microtubule polymerization via
Conjugation of therapeutic agents with a wide spectrum of
macromolecules16 including antibodies, polysaccharides,
lectins, serum proteins, peptides, growth factors, and syn-
thetic polymers, as well as their incorporation into micelles
or liposomes17 can seriously extend the therapeutic poten-
tial of the drug. Indeed, when colchicinoid 6 was linked to
chitosan, the resulting conjugate demonstrated significant-
ly better tumor growth inhibition than the intact molecule
6, possibly, as a result of better accumulation in the tu-
mor.18 On the other hand, the linkage of colchicinoid with a
target carrier via a very liable ester bond placed in the pseudo-
benzylic (in the case of 6) or in the benzylic (compound 7)
positions of the molecule led to instability of the obtained
conjugates during the storage and administration. Herein,
we report the synthesis of furano-allocolchicinoids 8, con-
binding to tubulin, one of the main constituent elements of
3
spindle microtubules. For a long time, colchicine was con-
sidered a perspective antitumor agent. However, its signifi-
cant general toxicity in therapeutic doses prevents its usage
4
in cancer therapy. Apart from functioning as a ‘mitotic poi-
son’, colchicine also inhibits neutrophile motility and activ-
5
ity, leading to a net anti-inflammatory effect. This unique
set of biological properties of colchicine can explain its effi-
cacy in clinical treatment of familial Mediterranean fever,
Behcet’s disease, acute gout, chondrocalcinosis, and other
6
types of microcrystalline arthritis. Recently, colchicine was
suggested as a drug for the treatment of cardiovascular dis-
eases such as acute pericarditis, atrial fibrillation caused by
7
inflammation, and ischemic diseases. Potentially, colchici-
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Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–F

B
Synthesis
Iu. A. Gracheva et al.
Paper
in 9 were protected with acetic anhydride and Boc O, re-
4
5
2
6
MeO
MeO
3
MeO
MeO
spectively, to afford colchicinoid 10 in 52% yield over two
steps. Simultaneous deacylation of two acetate groups in
compound 10 by NaOMe, followed by Boc-deprotection un-
der acidic conditions gave deacetyliodocolchinol 11 in good
yield. Unfortunately, we have not succeeded in using the in-
termediate 11 for the preparation of target furano-allocol-
NHAc
O
NHAc
2
7
1
8
9
MeO 12
MeO
1
1
10
CO2Me
OMe
(
–)-(aR,7S) colchicine (1)
(–)-allocolchicine (2)
MeO
chicinoids 8, as its reaction with propargylic alcohols 12a,b
MeO
MeO
X
under Sonogashira reaction conditions1
3,14
yielded a com-
X
N
X
plex and inseparable mixture of products. However, when
Boc-protected iodoallocolchinol 13, prepared in one step
from diacetate 10 was subjected to the cascade Sonogashira
cross-coupling/Larock-type cyclization sequence with cor-
responding alkynes 12, furans 14a and 14b were obtained
in 81 and 70% yield, respectively. The choice of alkynes was
based on our previous studies,13 which indicated that the
MeO
MeO
MeO
MeO
MeO
MeO
N
3
4
5
N
X = NHAc, O, OH, N3
MeO
MeO
NHAc
NH2
MeO
MeO
MeO
OH
presence of CH OH or CH(Me)OH groups in the side chain of
2
MeO
MeO
MeO
the heterocyclic ring leads to higher in vitro antitumor ac-
tivity. Finally, the cleavage of the Boc-function was achieved
by treatment of 14 with aqueous 3 M HCl in CH Cl in the
O
O
2
2
MeO
6
R
8
R
presence of a scavenger to give the target products 8 in
good yields (Scheme 1).
HO
HO
O
R = H (a), Me (b)
The in vitro cytotoxicity of the synthesized compounds
toward Colo-357, PANC-1, MiaPaCa-2, and T3M4 cell lines
was investigated. A tetrazolium-based assay was used to de-
termine 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 Ta-
ble 1. Compound 8a inhibited cell proliferation at low nano-
molar concentrations, while its analogue 8b demonstrated
a modest cytotoxic effect.
7
R
HO
Figure 1 Structures of colchicine (1), allocolchicine (2), pyrrolo-allocol-
chicinoids 3–5, furano-allocolchicinoids 6,7, and the target molecules 8
taining simultaneously two functionalities, that is, amino
and hydroxyl groups, which can simplify the conjugation
and improve the stability of the linker between the thera-
peutic molecule and the targeted vector or carrier.
Colchicine (1) underwent the two-step oxidative ring
contraction according to modified Windaus procedure11c to
afford iodocolchinol 9 in 91% yield (Scheme 1). Then, the
phenolic hydroxyl group and the NH fragment of the amide
In conclusion, we have reported a short synthetic route
to a new type of bifunctional furano-allocolchicinoids con-
taining two different reacting groups. The synthesized
compounds retain their antimitotic properties and can be
MeO
MeO
MeO
MeO
MeO
MeO
Boc
Ac
NHAc
O
NHAc
N
a,b
1%
c,d
9
52%
MeO
MeO
MeO
OH
OAc
I
I
OMe
–)-(aR,7S)-colchicine (1)
8a, b
9
10
R
(
e
97%
OH
12a,b
MeO
MeO
MeO
MeO
Boc
NH
R
NH2
MeO
MeO
Boc
NH
MeO
OH
NH2
h
12a,b
f
MeO
MeO
MeO
92%
g
MeO
MeO
O
O
OH
OH
R = H 8a 85%
Me 8b 70%
R = H 14a 81%
Me 14b 70%
I
I
13
11
OH
OH
R
R
Scheme 1 Synthesis of bifunctional furano-allocolchicinoids 8. Reagents and conditions: (a) 0.1 M HCl, AcOH, 100 °C, 3 h; (b) NaOH, I , KI, H O, 0–5 °C,
2
2
2
2
1
h; (c) Ac O, TBAH, NaOH, 1,4-dioxane, r.t., 1 h; (d) Boc O, DMAP, Et N, MeCN, reflux, 24 h; (e) NaOMe (20 mol%), MeOH, r.t., 1.5 h; (f) HCl, EtOH, r.t.,
2
2
3
0 h; (g) Pd(OAc) (5 mol%), CuI (10 mol%), Ph P (15 mol%), KOAc (3 equiv), MeCN, 70 °C, 12 h; (h) aq 3 M HCl in CH Cl , 1,3,5-trimethoxybenzene, r.t.,
2
3
2
2
h.
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Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–F

C
Synthesis
Iu. A. Gracheva et al.
Paper
a
+
Table 1 In Vitro Cytotoxic Activity (IC50, nM) of Furano-Allocolchici-
noids 8a,b (vs 7a,b)14
HRMS (ESI): m/z [M + H] calcd for C22H24INO + H: 526.0721; found:
6
+
526.0723; m/z [M + Na] calcd for C22H24INO + Na: 548.0541; found:
6
548.0539.
Compound
8a
160
8b
800
7a
16
7b
64
To a stirred mixture of N-[(5S)-2-iodo-9,10,11-trimethoxy-3-(acetyl-
oxy)-6,7-dihydro-5Н-dibenzo[a,c]cycloheptene-5-yl]acetamide (0.48
g, 0.914 mmol) and DMAP (0.112 g, 0.914 mmol) in MeCN (5.3 mL)
Colo-357
PANC-1
160
32
800
16
16
–
30
64
–
were added a half of the solution of Boc O (0.916 g, 4.203 mmol) in
2
–b
MeCN (5.3 mL) and Et N (0.255 mL) under an inert atmosphere. The
3
MiaPaCa-2
resulting solution was stirred at 100 °C for 1 h. Then, the second half
T3M4
32
–
of the solution of Boc O was added, the mixture was stirred for 2 h at
2
a
IC50 (nM) concentration inducing 50% inhibition of cell growth.
Not determined.
90 °C, and overnight at r.t. After completion of the reaction (TLC mon-
itoring), sat. aq solution of citric acid (50 mL) was added, and the mix-
ture was extracted with CH Cl . The organic layer was washed with
b
2
2
brine, dried (Na SO ), and the crude product obtained after solvent
removal was purified by column chromatography (eluent: PE–EtOAc–
EtOH, 6:1:1) to afford compound 10 as a reddish foam; yield: 0.340 g
2
4
conjugated with various substrates via the amine group on
the B-ring to modify their physicochemical and cytotoxicity
profiles. This opens new promising options for therapeutic
intervention in cancer chemotherapy. Further work in this
direction is currently underway.
(59%); mp 88 °C.
1
H NMR (DMSO-d , 400 MHz): δ = 7.76 (s, 1 H), 7.11 (s, 1 H), 6.82 (s, 1
6
H), 5.19–5.13 (m, 1 H), 3.84 (s, 3 H), 3.78 (s, 3 H), 3.49 (s, 3 H), 2.70–
.61 (m, 2 H), 2.34 (s, 3 H), 2.30 (s, 3 H), 2.21–2.13 (m, 1 H), 2.11–2.02
2
(
m, 1 H), 1.46 (s, 9 H).
1
3
Commercially available reagents were used without additional purifi-
cation. Column chromatography was performed using Macherey-
Nagel Kieselgel 60 (70–230 mesh). All 1H and C NMR spectra were
C NMR (DMSO-d , 101 MHz): δ = 168.34, 153.05, 153.03, 150.35,
6
149.76, 140.57, 140.10, 139.45, 134.79, 133.55, 122.19, 119.69,
108.11, 88.55, 83.89, 60.74, 60.57, 60.57, 55.83, 34.79, 29.97, 27.37,
21.00.
13
recorded at r.t. in DMSO-d6 or CD OD on Bruker Avance DRX 500 or
3
Agilent DD2 400 instruments. Chemical shifts (δ) are reported in
MS (EI, 70 eV): m/z (%) = 625 (12), 583 (32), 483 (66), 424 (100), 409
parts per million (ppm) from TMS using the residual solvent reso-
(22), 381 (10), 297 (22), 282 (16), 238 (12), 139 (12).
1
13
nance (DMSO-d : 2.50 ppm for H NMR, 39.52 ppm for C NMR;
6
1
Anal. Calcd for C27 : C, 51.85; H, 5.16. Found: C, 51.62; H, 5.31.
H32INO
8
CD OD: 3.31 ppm for H NMR). Standard abbreviations for multiplici-
3
ties are used. EI mass spectra (70 eV) were obtained on a DSQ II mass
spectrometer (Thermo Electron Corporation) with a quadrupole mass
analyzer. Combustion analysis was performed using an Elementar
tert-Butyl [(5S)-3-Hydroxy-2-iodo-9,10,11-trimethoxy-6,7-dihy-
dro-5H-dibenzo[a,c]cyclohepten-5-yl)carbamate (13)
(
Vario Micro Cube) apparatus. High-resolution mass spectra (HRMS)
were recorded on a Thermo Fisher LTQ Orbitrap XL – FTMS Analyzer
HR-ESI-MS). Solvents were purified according to the standard proce-
dures. Petroleum ether (PE) used had bp 40–70 °C. Iodocolchinol 9
MeOH (8.0 mL) and a 2 M solution of freshly prepared NaOMe in
MeOH (0.066 mL) were added to a flask containing compound 10
(0.395 g, 0.632 mmol) under an inert atmosphere. The resulting solu-
tion was stirred for 1.5 h at r.t. After evaporating the solvent, the
product was partitioned between CH Cl and H O (1:1, 70 mL), the or-
(
11c
was prepared according to the previously proposed procedure.
2
2
2
ganic layer was washed with brine, and dried (Na SO ). After removal
2
4
of all volatiles, the pure product 13 was obtained as yellow crystals
and was used directly in the next step; yield: 0.333 g (97%); mp
123 °C.
(
5S)-5-[N-(tert-Butoxycarbonyl)acetamido]-2-iodo-9,10,11-trime-
thoxy-6,7-dihydro-5Н-dibenzo[a,c]cyclohepten-5-yl] Acetate (10)
To a well-stirred mixture of iodocolchinol 911c (0.5 g, 1.035 mmol),
1
1H NMR (DMSO-d
, 500 MHz): δ = 10.26 (s, 1 H), 7.56 (s, 1 H), 7.41 (d,
,4-dioxane (3.0 mL), TBAH (0.014 g, 0.041 mmol), and powdered
6
NaOH (0.104 g, 2.588 mmol) was added dropwise a solution of Ac O
J = 8.3 Hz, 1 H), 6.91 (s, 1 H), 6.74 (s, 1 H), 4.12 (dt, J = 11.5, 7.7 Hz, 1
H), 3.82 (s, 3 H), 3.77 (s, 3 H), 3.52 (s, 3 H), 2.46 (dd, J = 12.5, 5.6 Hz, 1
H), 2.15–2.07 (m, 1 H), 2.07–2.00 (m, 1 H), 1.91 (dd, J = 16.9, 10.9 Hz, 1
H), 1.34 (s, 9 H).
2
(0.117 mL, 1.241 mmol) in 1,4-dioxane (3.0 mL) at r.t. The resulting
mixture was stirred for 1 h under an inert atmosphere, then filtered,
washed with 1,4-dioxane, and evaporated. The residue was dissolved
in CH Cl , washed with H O, and dried (Na SO ). The solvent was re-
13
2
2
2
2
4
C NMR (DMSO-d , 126 MHz): δ = 155.54, 154.57, 152.16, 150.04,
6
moved under reduced pressure. The pure N-[(5S)-2-iodo-9,10,11-tri-
methoxy-3-(acetyloxy)-6,7-dihydro-5Н-dibenzo[a,c]cycloheptene-5-
yl]acetamide was obtained as yellowish crystals, and was used direct-
ly in the following step; yield: 0.480 g (88%); mp 158 °C.
1
1
7
42.82, 140.48, 139.05, 134.72, 126.45, 123.12, 110.03, 108.03, 81.32,
7.70, 60.48, 60.44, 55.78, 49.85, 37.88, 29.94, 28.16.
HRMS (ESI): m/z [M + Na]+ calcd for C23H28INO6 + Na: 564.0854;
found: 564.0853.
H NMR (DMSO-d , 400 MHz): δ = 8.38 (d, J = 8.3 Hz, 1 H), 7.75 (s, 1
6
H), 7.12 (s, 1 H), 6.81 (s, 1 H), 4.52–4.44 (m, 1 H), 3.84 (s, 3 H), 3.79 (s,
(
5S)-6,7-Dihydro-3-hydroxy-2-iodo-9,10,11-trimethoxy-5H-
3
1
H), 3.56 (s, 3 H), 2.58–2.52 (m, 1 H), 2.36 (s, 3 H), 2.25–1.95 (m, 3 H),
.86 (s, 3 H).
dibenzo[a,c]cyclohepten-5-yl-amine (11)
Compound 13 (0.2 g, 0.369 mmol) was placed in a 50 mL flask and
dissolved in a minimum amount of EtOH. Concd HCl (10 mL) was add-
ed to the solution, and the reaction mixture was stirred at r.t. for 1 h.
Then, an additional amount of concd HCl (5 mL) was added, and the
temperature was raised to 40 °C. After completion of the reaction
13
C NMR (DMSO-d , 101 MHz): δ = 168.36, 168.29, 152.90, 150.08,
6
1
1
49.91, 142.55, 140.46, 139.38, 134.79, 133.44, 122.13, 117.93,
08.19, 88.04, 60.67, 60.45, 55.82, 47.88, 38.19, 29.76, 22.54, 20.91.
(TLC monitoring), the solution was neutralized with aq NaOH. Vacu-
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Synthesis
Iu. A. Gracheva et al.
Paper
1
um evaporation of the volatiles gave a dark-yellow oily residue that
was extracted with CH Cl , washed with brine, and dried (Na SO ).
After removal of the solvent, compound 11 was obtained as white
H NMR (DMSO-d , 400 MHz): δ (mixture of diastereomers) = 7.58 (d,
6
J = 8.2 Hz, 1 H), 7.49 (s, 1 H), 7.44 (s, 1 H), 6.78 (s, 1 H), 6.70 (s, 1 H),
5.49 (s, 1 H), 4.85 (dd, J = 11.8, 6.2 Hz, 1 H), 4.30 (dd, J = 18.3, 9.4 Hz, 1
H), 3.84 (s, 3 H), 3.79 (s, 3 H), 3.45 (s, 3 H), 2.22–1.80 (m, 4 H), 1.48 (d,
J = 6.2 Hz, 3 H), 1.35 (s, 9 H).
2
2
2
4
crystals; yield: 0.150 g (92%); mp 155 °C.
1
H NMR (DMSO-d , 400 MHz): δ = 7.57 (s, 1 H), 7.16 (s, 1 H), 6.75 (s, 1
6
13
H), 3.82 (s, 3 H), 3.75 (s, 3 H), 3.57 (dd, J = 11.3, 7.0 Hz, 1 H), 3.51 (s, 3
H), 2.30–2.25 (m, 2 H), 2.06 (td, J = 12.9, 7.4 Hz, 1 H), 1.69 (td, J = 11.8,
C NMR (DMSO-d , 101 MHz): δ (mixture of diastereomers) = 161.97,
6
161.90, 154.76, 153.45, 152.24, 150.29, 140.58, 137.69, 134.78,
128.53, 126.14, 124.68, 121.76, 107.97, 105.43, 101.26, 101.22, 77.81,
62.27, 60.56, 56.56, 55.82, 50.23, 38.32, 30.06, 28.22, 22.02.
7.8 Hz, 1 H).
13
C NMR (DMSO-d , 101 MHz): δ = 155.62, 152.15, 150.06, 140.42,
6
1
5
39.15, 135.10, 126.62, 123.09, 110.51, 107.96, 81.38, 60.52, 60.49,
5.82, 50.06, 40.45, 30.22.
MS (EI, 70 eV): m/z (%) = 483 (58), 427 (50), 383 (27), 382 (92), 366
(55), 354 (46), 338 (64), 335 (56), 308 (43), 293 (48), 292 (42), 265
(
(
40), 249 (34), 205 (34), 193 (26), 165 (34), 152 (34), 139 (20), 127
12), 115 (10), 57 (100).
MS (EI, 70 eV): m/z (%) = 441 (100), 424 (100), 409 (100), 393 (74),
366 (42), 314 (52), 297 (41), 282 (40), 267 (34), 254 (25), 212 (12),
07 (25), 181 (22).
2
Anal. Calcd for C27H33NO : C, 67.06; H, 6.88. Found: C, 67.29; H, 6.52.
7
Anal. Calcd for C18H20INO : C, 48.99; H, 4.57. Found: C, 50.26; H, 4.69.
4
Compounds 8a,b; General Procedure
Compounds 14a,b; General Procedure
Compound 14 (1 equiv) and 1,3,5-trimethoxybenzene (2 equiv) were
dissolved in aq 3 M HCl in СН Cl (24 mL per 1 mmol of 14). The mix-
Compound 13 (1 equiv), Pd(OAc)2 (5 mol%), CuI (10 mol%), PPh3 (15
mol%), and KOAc (3 equiv) were placed in a two-necked flask under
an inert atmosphere. Anhyd MeCN (16.2 mL per 1 mmol of 13) and
alkyne 12 (1 equiv) were added. The mixture was stirred at 60 °C for 1
h, then the temperature was raised to 80 °C, and stirring was contin-
ued until completion of the reaction (TLC monitoring). The solvent
was removed under reduced pressure and the solid residue was puri-
fied by column chromatography (eluent: PE–EtOAc–EtOH) to obtain
pure 14.
2
2
ture was stirred at r.t. for 1 h. After completion of the reaction, solid
NaOH was added till pH 8 and the product was partitioned between
CH Cl and H O. The organic layer was washed with brine and dried
2
2
2
(
Na SO ). The solvent was removed under reduced pressure and the
2 4
solid residue was purified by column chromatography (eluent: PE–
EtOAc–EtOH) to afford compound 8.
(1S)-[1′,2′,3′-Trimethoxybenzo[5′,6′:5,4]-1H-1-amino-6,7-dihydro-
cyclohepta[3,2-f]-2′′-(1-hydroxymethyl)]benzofuran (8a)
tert-Butyl N-[(1S)-2′′-(1-Hydroxymethyl)-1′,2′,3′-trimethoxy-6,7-
dihydro-1H-benzo[5′,6′:5,4]cyclohepta[3,2-f]benzofuran-1-yl]car-
bamate (14a)
Compound 8a was prepared according to the general procedure from
14a (0.055 g, 0.117 mmol). Column chromatography with PE–EtOAc–
EtOH (2:1:1) as eluent gave 8a as white crystals; yield: 0.037 g (85%);
mp 265 °C.
Compound 14a was prepared according to the general procedure
from 13 (0.035 g, 0.065 mmol) and propargyl alcohol (12a; 0.004 mL,
1
H NMR (DMSO-d , 400 MHz): δ = 7.79 (s, 1 H), 7.59 (s, 1 H), 7.04 (s, 1
6
0.065 mmol). Column chromatography with PE–EtOAc–EtOH (5:1:1)
H), 6.81 (s, 1 H), 5.01 (s, 2 H), 3.84 (s, 3 H), 3.82–3.80 (m, 1 H), 3.78 (s,
as eluent gave product 14a as yellow crystals; yield: 0.025 g (81%); mp
3 H), 3.49 (s, 3 H), 2.40 (m, 2 H), 2.02 (td, J = 12.4, 7.4 Hz, 2 H).
1
21 °C.
13
C NMR (DMSO-d , 101 MHz): δ = 153.66, 153.58, 152.78, 150.39,
6
1
H NMR (DMSO-d , 500 MHz): δ (mixture of conformers) = 7.54 (d, J =
140.79, 134.04, 132.75, 129.20, 126.59, 123.61, 123.18, 108.24,
106.76, 106.26, 60.70, 60.57, 55.91, 50.68, 37.94, 36.84, 29.31.
6
8.5 Hz, 1 H), 7.50 (s, 1 H), 7.45 (s, 1 H), 6.78 (s, 1 H), 6.75 (s, 1 H), 5.39
(
3
2
t, J = 5.8 Hz, 1 H), 4.57 (d, J = 5.8 Hz, 2 H), 4.34–4.26 (m, 1 H), 3.84 (s,
H), 3.80 (s, 3 H), 3.46 (s, 3 H), 2.49–2.45 (m, 1 H), 2.16 (m, 1 H),
.02–1.95 (m, 1 H), 1.89 (dd, J = 19.0, 11.9 Hz, 1 H), 1.35 (s, 9 H).
MS (EI, 70 eV): m/z (%) = 369 (56), 252 (46), 323 (100), 322 (58), 307
(
(
53), 293 (88), 291 (54), 262 (50), 248 (39), 235 (36), 209 (42), 207
63), 205 (46), 183 (38), 176 (30), 155 (24), 152 (36), 135 (34), 115
1
3
C NMR (DMSO-d , 126 MHz): δ (mixture of conformers) = 158.33,
(26), 83 (24).
6
1
1
1
2
54.70, 153.67, 152.18, 150.24, 140.56, 137.79, 134.69, 131.42,
31.34, 128.69, 128.60, 128.53, 126.10, 124.61, 121.70, 107.96,
05.34, 103.14, 77.74, 60.47, 60.43, 56.17, 55.79, 50.22, 38.23, 29.98,
8.16.
Anal. Calcd for C21H23NO : C, 68.28; H, 6.28. Found: C, 68.02; H, 6.01.
5
(1S)-[1′,2′,3′-Trimethoxybenzo[5′,6′:5,4]-1H-1-amino-6,7-dihydro-
cyclohepta[3,2-f]-2′′-(1-hydroxyethyl)]benzofuran (8b)
+
HRMS (ESI): m/z [M + Na] calcd for C26H31NO + Na: 492.1993; found:
7
Compound 8b was prepared according to the general procedure from
492.1990.
14b (0.25 g, 0.517 mmol). Column chromatography with the eluent
PE–EtOAc–EtOH (2:1:1) gave product 8b as yellowish crystals; yield:
tert-Butyl N-[(1S)-2′′-(1-Hydroxyethyl)-1′,2′,3′-trimethoxy-6,7-di-
hydro-1H-benzo[5′,6′:5,4]cyclohepta[3,2-f]benzofuran-1-yl]carba-
mate (14b)
0.139 g (70%); mp 252 °C.
1
H NMR (DMSO-d , 400 MHz): δ (mixture of diastereomers) = 7.77 (s,
6
1
3
1
H), 7.53 (s, 1 H), 6.84 (s, 1 H), 6.79 (s, 1 H), 4.65 (q, J = 6.6 Hz, 1 H),
.84 (s, 3 H), 3.78 (s, 3 H), 3.75–3.71 (m, 1 H), 3.47 (s, 3 H), 2.37 (td, J =
2.0, 5.9 Hz, 1 H), 1.91 (m, 3 H), 1.50 (d, J = 6.5 Hz, 3 H).
Compound 14b was prepared according to the general procedure
from 13 (0.2 g, 0.369 mmol) and but-3-yn-2-ol (12b; 0.029 mL, 0.369
mmol). Column chromatography with PE–EtOAc–EtOH (5:1:1) as elu-
ent gave 14b as yellowish crystals; yield: 0.125 g (70%); mp 145 °C.
13
C NMR (DMSO-d , 101 MHz): δ (mixture of diastereomers) = 158.51,
6
158.47, 153.53, 152.28, 150.25, 150.24, 140.54, 134.89, 128.81,
125.83, 124.43, 122.02, 107.95, 106.28, 103.64, 103.62, 70.02, 63.43,
60.51, 55.83, 50.66, 30.12, 19.81.
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–F

E
Synthesis
Iu. A. Gracheva et al.
Paper
MS (EI, 70 eV): m/z (%) = 383 (84), 366 (70), 351 (33), 339 (48), 338
(3) (a) Wood, K. W.; Cornwell, W. D.; Jackson, J. R. Curr. Opin. Phar-
macol. 2001, 1, 370. (b) Graening, T.; Schmalz, H.-G. Angew.
Chem. Int. Ed. 2004, 43, 3230. (c) Ravelli, R. B. G.; Gigant, B.;
Curmi, P. A.; Jourdain, I.; Lachkar, S.; Sobel, A.; Knossow, M.
Nature 2004, 428, 198.
(100), 323 (50), 292 (50), 264 (50), 236 (47), 235 (50), 220 (58), 208
(62), 193 (58), 181 (44), 165 (52), 154 (44), 152 (38), 129 (21), 115
(26), 105 (20), 85 (18), 83 (12).
Anal. Calcd for C22H25NO : C, 68.91; H, 6.57. Found: C, 69.22; H, 6.84.
5
(
4) (a) Putterman, C.; Benchetrit, E.; Caraco, Y.; Levy, M. Semin.
Arthritis, Rheum. 1991, 21, 143. (b) Wiesenfeld, P. L.; Garthoff, L.
H.; Sobotka, T. J.; Suagee, J. K.; Barton, C. N. J. Appl. Toxicol. 2007,
Cell Cultures
Pancreatic cells (T3M4, PANC-1, Colo-357, MiaPaCa-2) 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 de-
tached 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 plates), adjusted to 3 × 105
cells/mL, and incubated overnight to achieve standardized growth
conditions.
2
7, 421. (c) Finkelstein, Y.; Aks, S. E.; Hutson, J. R.; Juurlink, D.
N.; Nguyen, P.; Dubnov-Raz, G.; Pollak, U.; Koren, G.; Bentur, Y.
Clin. Toxicol. 2010, 48, 407.
(
(
5) Pope, R. M.; Tschopp, J. Arthritis Rheum. 2007, 56, 3183.
6) (a) Slobodnick, A.; Shah, B.; Pillinger, M. H.; Krasnokutsky, S.
Am. J. Med. 2015, 128, 461. (b) Wallace, S. L. Arthritis Rheum.
2
2
006, 2, 389. (c) Manna, R. Curr. Drug Targets Inflamm. Allergy
005, 4, 117. (d) Masuda, K.; Nakajima, A.; Urayama, A.; Nakae,
K.; Kogure, M.; Inaba, G. Lancet 1989, 1, 1093. (e) Nuki, G. Curr.
Rheumatol. Rep. 2008, 10, 218.
MTT-Assay
(7) Imazio, M.; Gaita, F. Future Cardiol. 2016, 12, 9.
Cytotoxic effect of the furano-allocolchicinoids 8a,b was estimated by
a standard 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
(8) (a) Peña-Altamira, E.; Prati, F.; Massenzio, F.; Virgili, M.;
Contestabile, A.; Bolognesi, M. L.; Monti, B. Expert Opin. Ther.
Targets 2016, 20, 627. (b) Zhang, J.; Rane, G.; Dai, X.;
Shanmugam, M. K.; Arfuso, F.; Samy, R. P.; Lai, M. K. P.; Kappei,
D.; Kumar, A. P.; Sethi, G. Ageing Res. Rev. 2016, 25, 55.
(9) (a) Marrelli, M.; Conforti, F.; Statti, G. A.; Cachet, X.; Michel, S.;
Tillequin, F.; Menichini, F. Curr. Med. Chem. 2011, 18, 3035.
(b) Shan, Y.; Zhang, J.; Liu, Z.; Wang, M.; Dong, Y. Curr. Med.
Chem. 2011, 18, 523. (c) Rajak, H.; Dewangan, P. K.; Patel, V.;
Jain, D. K.; Singh, A.; Veerasamy, R.; Sharma, P. C.; Dixit, A. Curr.
Pharm. Des. 2013, 19, 1923. (d) Brancale, A.; Silvestri, R. Med.
Res. Rev. 2007, 27, 209. (e) Combes, S.; Barbier, P.; Douillard, S.;
McLeer-Florin, A.; Bourgarel-Rey, V.; Pierson, J.-T.; Fedorov, A.
Yu.; Finet, J.-P.; Boutonnat, J.; Peyrot, V. J. Med. Chem. 2011, 54,
3153. (f) Ganina, O. G.; Daras, E.; Bourgarel-Rey, V.; Peyrot, V.;
Andresyuk, A. N.; Finet, J.-P.; Fedorov, A. Yu.; Beletskaya, I. P.;
Combes, S. Bioorg. Med. Chem. 2008, 16, 8806.
19
bromide (MTT, Sigma) test as described earlier. 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 was added to each well. Plates were
incubated by 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 (Microsoft).
Cytotoxic concentration giving 50% of the maximal toxic effect (IC50
)
was calculated from the titration curves. The inhibition of prolifera-
tion (inhibition index, II) was calculated as [1 – (ODexperiment/ODcontrol)],
where OD was MTT optical density.
(10) For a review on allocolchicines, see: Sitnikov, N. S.; Fedorov, A.
Yu. Russ. Chem. Rev. 2013, 82, 393.
(
11) (a) Boye, O.; Brossi, A. The Alkaloids: Chemistry and Pharmacol-
Funding Information
ogy;
4
V1o.l
Brossi, A.; Cordell, G. A., Eds.; Academic Press: San Diego,
1992, 125. (b) Boyer, F.-D.; Dubois, J.; Thoret, S.; Dau, M.-E. T. H.;
We thank the Russian Science Foundation (Project 16-13-10248) for
Hanna, I. Bioorg. Chem. 2010, 38, 149. (c) Nicolaus, N.; Reball, J.;
Sitnikov, N.; Velder, J.; Termath, A.; Fedorov, A. Yu.; Schmalz, H.-
G. Heterocycles 2011, 82, 1585. (d) Jain, N.; Yada, D.; Shaik, T. B.;
Vasantha, G.; Reddy, P. S.; Kalivendi, S. V.; Sreedhar, B.
ChemMedChem 2011, 6, 859. (e) Lu, Y.; Chen, J. J.; Xiao, M.; Li,
W.; Miller, D. D. Pharm. Res. 2012, 29, 2943. (f) Chosson, E.;
Santoro, F.; Rochais, C.; Santos, J. S.-d. O.; Legay, R.; Thoret, S.;
Cresteil, T.; Sinicropi, M. S.; Besson, T.; Dallemagne, P. Bioorg.
Med. Chem. 2012, 20, 2014.
financial support.
R
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asin
S
ecince
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1(6-13-1
0
2
4
8)
Supporting Information
Supporting information for this article is available online at
https://doi.org /10.1055/s-0036-1589060.
S
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p
p
ortioIgnfrm oaitn
S
u
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p
ortioIgnfrm oaitn
References
(
12) (a) Sitnikov, N.; Velder, J.; Abodo, L.; Cuvelier, N.; Neudorfl, J.-
M.; Prokop, A.; Krause, G.; Fedorov, A. Yu.; Schmalz, H.-G. Chem.
Eur. J. 2012, 18, 12096. (b) Sitnikov, N. S.; Kokisheva, A. S.; Fukin,
G. K.; Neudorfl, J.-M.; Sutorius, H.; Prokop, A.; Fokin, V. V.;
Schmalz, H.-G.; Fedorov, A. Yu. Eur. J. Org. Chem. 2014, 29, 6481.
(
1) (a) Dumontet, C.; Jordan, M. A. Nat. Rev. Drug Discov. 2010, 9,
90. (b) Stanton, R. A.; Gernert, K. M.; Nettles, J. H.; Aneja, R.
7
Med. Res. Rev. 2011, 31, 443. (c) Stanton, R. A.; Gernert, K. M.;
Nettles, J. H.; Aneja, R. Med. Res. Rev. 2011, 31, 443. (d) Aeluri,
M.; Chamakuri, S.; Dasari, B.; Guduru, S. K. R.; Jimmidi, R.;
Jogula, S.; Arya, P. J. Med. Chem. 2014, 114, 4640.
(c) Sitnikov, N. S.; Sintsov, A. V.; Allegro, D.; Barbier, P.; Combes,
S.; Onambele, L. A.; Prokop, A.; Schmalz, H.-G.; Fedorov, A. Yu.
MedChemComm 2015, 6, 2158.
(
2) Ghosh, S.; Jha, S. Colchicine – An Overview for Plant Biotechnolo-
gists, In Bioactive Molecules and Medicinal Plants; Ramawat, K.
G.; Mérillon, J. M., Eds.; Springer-Verlag: Berlin, 2008, 215.
(
13) Voitovich, Yu. V.; Shegravina, E. S.; Sitnikov, N. S.; Faerman, V. I.;
Fokin, V. V.; Schmalz, H.-G.; Combes, S.; Allegro, D.; Barbier, P.;
Beletskaya, I. P.; Svirshchevskaya, E. V.; Fedorov, A. Yu. J. Med.
Chem. 2015, 58, 692.
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–F

F
Synthesis
Iu. A. Gracheva et al.
Paper
(
14) Gracheva, Iu. A.; Voitovich, Iu. V.; Faerman, V. I.; Sitnikov, N. S.;
Takahashi, K.; Nohmi, K.; Izutani, N.; Kanda, M.; Suzuki, R. Biorg.
Med. Chem. 2009, 17, 4647. (e) Moulari, B.; Béduneau, A.;
Pellequer, Y.; Lamprecht, A. J. Controlled Release 2014, 188, 9.
(f) Ringhieri, P.; Diaferia, C.; Galdiero, S.; Palumbo, R.; Morelli,
G.; Accardo, A. J. Pept. Sci. 2015, 21, 415. (g) Chen, L. Q.; Huang,
W.; Gao, Z. G.; Fang, W. S.; Jin, M. J. Int. J. Nanomed. 2016, 11,
5457. (h) Lee, H. J.; Park, H. H.; Sohn, Y.; Ryu, J.; Park, J. H.; Rhee,
W. J.; Park, T. H. Appl. Microbiol. Biotechnol. 2016, 100, 10395.
(i) Jaferian, S.; Soleymaninejad, M.; Negahdari, B.; Eatemadi, A.
Biomed. Pharmacother. 2017, 88, 1046. (j) Zhang, M.; Yuan, P.;
Zhou, N.; Su, Y.; Shao, M.; Chi, C. RSC Adv. 2017, 7, 9347.
(17) Kuznetsova, N. R.; Svirshevskaya, E. V.; Sitnikov, N. S.; Abodo, L.;
Sutorius, H.; Zapke, J.; Velder, J.; Tomopoulou, P.; Oschkinat, H.;
Prokop, A.; Schmalz, H.-G.; Fedorov, A. Yu.; Vodovozova, E. L.
Russ. J. Bioorg. Chem. 2013, 39, 543.
Myrsikova, E. V.; Schmalz, H.-G.; Svirshevskaya, E. V.; Fedorov,
A. Yu. Eur. J. Med. Chem. 2017, 126, 432.
(15) (a) Ho, R. J. Y.; Chien, J. J. Pharm. Sci. 2014, 103, 71. (b) Dai, L.;
Liu, J.; Luo, Z.; Li, M.; Cai, K. J. Mater. Chem. B 2016, 4, 6758.
(
(
c) Luhmann, T.; Meinel, L. Curr. Opin. Biotechnol. 2016, 39, 35.
d) Hsu, H.-J.; Bugno, J.; Lee, S.; Hong, S. WIREs Nanomed. Nano-
biotechnol. 2017, 9, 1409. (e) Vahed, S. Z.; Salehi, R.; Davaran, S.;
Sharifi, S. Mater. Sci. Eng. C 2017, 71, 1327.
(
16) (a) Hamann, P. R.; Hinman, L. M.; Hollander, I.; Beyer, C. F.;
Lindh, D.; Holcomb, R.; Hallett, W.; Tsou, H. R.; Upeslacis, J.;
Shochat, D.; Mountain, A.; Flowers, D. A.; Bernstein, I. Bioconju-
gate Chem. 2002, 13, 47. (b) Gabizon, A.; Shmeeda, H.;
Barenholz, Y. Clin. Pharmacokinet. 2003, 42, 419. (c) Bagnato, J.
D.; Eilers, A. L.; Horton, R. A.; Grissom, C. B. J. Org. Chem. 2004,
6
9, 8987. (d) Homma, A.; Sato, H.; Okamachi, A.; Emura, T.;
(18) Svirshchevskaya, E. V.; Gracheva, Iu. A.; Kuznetsov, A. G.;
Myrsikova, E. V.; Grechikhina, M. V.; Zubareva, A. A.; Fedorov, A.
Yu. Med. Chem. (Los Angeles) 2016, 6, 571.
Ishizawa, T.; Kato, T.; Matsuura, T.; Sato, S.; Tamura, T.; Higuchi,
Y.; Watanabe, T.; Kitamura, H.; Asanuma, K.; Yamazaki, T.;
Ikemi, M.; Kitagawa, H.; Morikawa, T.; Ikeya, H.; Maeda, K.;
(19) Mosmann, T. J. Immunol. Methods 1983, 65, 55.
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Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–F