New functionalized aminofurazans as potential antimitotic agents in the sea urchin embryo assay

Article abstract of DOI:10.1016/j.mencom.2010.05.002

A series of new furazan (1,2,5-oxadiazole) derivatives based on structural overlap with combretastatin have been synthesized. Targeted molecules were evaluated using the sea urchin embryo assay; several agents demonstrated 1-4 μmol dm^-3 antipro

Full text of DOI:10.1016/j.mencom.2010.05.002

Mendeleev
Communications
Mendeleev Commun., 2010, 20, 132–134
New functionalized aminofurazans as potential
antimitotic agents in the sea urchin embryo assay
Aleksei B. Sheremetev,*a Dmitrii E. Dmitriev,a Nataliya K. Lagutina,a Mikhail M. Raihstat,a
Alex S. Kiselyov,b Marina N. Semenova,c Natalie N. Ikizalpd and Victor V. Semenov*a,d
a N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian
Federation. Fax: +7 499 135 5328; e-mail: sab@ioc.ac.ru, vs@zelinsky.ru
b deCODE Chemistry, Chicago, IL 60517, USA
c N. K. Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, 119334 Moscow,
Russian Federation
d Chemical Block Ltd., 3723, Limassol, Cyprus
DOI: 10.1016/j.mencom.2010.05.002
A series of new furazan (1,2,5-oxadiazole) derivatives based on structural overlap with combretastatin have been synthesized.
Targeted molecules were evaluated using the sea urchin embryo assay; several agents demonstrated 1–4 μmol dm–3 antiproliferative
activity in this in vivo model.
Targeting tubulin in rapidly dividing tumor cells has been a well
OH
validated strategy for cancer therapy.1,2 Screening of natural
products for cytotoxic activity yielded structurally diverse classes
MeO
MeO
MeO
B
of mitotic spindle poisons. Most of the known anti-mitotic
agents, including colchicine and Vinca alkaloids, inhibit tubulin
polymerization. In contrast, Taxol™ has an opposite action: it
stimulates polymerization in vitro and stabilizes spindle MT’s.3
High toxicity found for the mitotic poisons4–9 prompted scientists
to expand their search for the synthetic modulators of tubulin
that (a) mimic natural products, (b) result in the same anti-
mitotic effect and (c) display better efficacy/safety window. In
general, colchicine derivatives are structurally simpler than Vinca
alkaloids or Taxol™ derivatives, as exemplified by combreta-
statin A-4 (CA-4). This agent features a tilted ‘biaryl’ structural
motif (A and B rings) connected by a hydrocarbon bridge of
variable length. The linker provides for cis-configuration of the
biaryl template necessary for the efficient interaction of a mole-
cule with the colchicine binding site of tubulin.10
A
NHAc
O
MeO
MeO
MeO
C
A
OMe
Colchicine
OMe
Combretastatin A-4
CA-4
OR2
MeO
B
N
O
N
MeO
R1O
A
Note that five-membered heterocycle templates were reported
to provide both non-isomerizable and metabolically stable isosteric
replacement for the cis-styrene featured in combretastatins.11
For example, combretafurazans were reported to be more potent
in anti-mitotic agents compared to CA-4 in neuroblastoma cells.12
In our search for new synthetic anti-mitotic agents, we focused
on heterocyclic molecules with pharmacophore arrangement that
mimics combretastatin and related compounds. Initially, we studied
derivatives of both 1,3,4-oxadiazoles13,14 and 1,3,4-triazoles.15
The biaryl (1,2,4-oxadiazol-3-yl)furazan template was expected
to provide for the (i) isosteric replacement of the cis-styrene
bond while maintaining proper alignment of the aryl substituents
for the tubulin activity and (ii) yield better cell permeability
compared to the substituted phenyl group.13–15 Considering
complexity of both in vitro and ex vivo assays measuring tubulin
activity of an agent, we decided to profile newly synthesized
compounds in the sea urchin embryo model instead.16 This
phenotypic in vivo assay includes (i) a fertilized egg test for
antimitotic activity displayed by the cleavage alteration/arrest,
and (ii) the behavioral monitoring of a free-swimming blastulae
treated immediately after hatching.16,17
OMe
'Combretafurazans'
Commercially available furazan derivative 1 provided for a
good starting point in the synthesis. Key intermediate 218,19 was
prepared by diazotization of amidoxime 1 as described pre-
viously. Treatment of 2 with substituted amines in the presence
of Et3N yielded corresponding amidoximes 3 and 4. The sub-
sequent ring-to-ring interconversion reaction20–22 took place
with KOH in ethylene glycol at reflux to furnish 5 and 6.
Notably, this is the first example of the rearrangement applied
to the synthesis of a bicyclic (1,2,4-oxadiazol-3-yl)furazan ring
system. This step is both robust and high-yielding (~80% for
both 5 and 6).
In order to introduce a 1,2,4-oxadiazolyl-N-substituent into
the targeted molecules, we first prepared trichloromethyl deriva-
tive 723 from furazan derivative 124,25 using a modification of
a published procedure. Specifically, the nucleophilic displace-
ment of the CCl3 group26,27 in compound 7 was achieved with
amines in THF to afford desired molecules 8a–r.† A wide variety
of amine reagents were tolerated under the reaction conditions
(Table 1). The yields of 8a–r were 76–96%. Unfortunately, our
attempts to further modify the unsubstituted amino group in
these compounds via the reductive amination were unsuccessful
Our chemistry efforts were centered around structural modi-
fications of a 4-amino-3-(1,2,4-oxadiazol-3-yl)furazan template.
An overview of this approach is summarized in Scheme 1.
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© 2010 Mendeleev Communications. All rights reserved.

Mendeleev Commun., 2010, 20, 132–134
OH
OH
OH
N
OH
N
N
N
i
iii
iv
H2N
NH2
H2N
Cl
H2N
NH
NH
N
NH2
n
OMe
n
N
N
N
N
N
N
N
O
O
O
O
MeO
1
2
3 n = 0
4 n = 1
5 n = 0
6 n = 1
v
ii
O
O
H
CCl3
NHR
O
O
N
N
N
CCl3
vi
N
N
N
N
NH
N
N
NH
N
vi
R
n
H2N
N
H2N
N
n
n
N
N
N
N
N
O
O
O
O
MeO
MeO
7
8a–r
9 n = 0
11a–h
10 n = 1
Scheme 1 Reagents and conditions: i, see refs. 18, 19; ii, see ref. 23; iii, p-MeOC6H4(CH2)nNH2/NEt3, EtOH or PriOH; iv, KOH, ethylene glycol, reflux;
v, CCl3COCl, BuOAc, reflux; vi, RNH2, THF.
under a variety of experimental conditions. Easy access to inter-
mediates 5 and 6 (vide supra) allowed us to address this issue.
Namely, ring-closing reactions of 5 and 6 to form targeted
1,2,4-oxadiazole derivatives 9 and 10 were accomplished with
CCl3COCl in butyl acetate at reflux. A final step towards desired
molecules 11a–h was accomplished via the nucleophilic displace-
ment of a CCl3 moiety with the respective amines in THF at
room temperature. The yields ranged from 72 to 86% (Table 1).
We further elaborated the stepwise nucleophilic displace-
ment of both NO2 groups in 3,4-dinitrofurazan 1228 with N-
and O-nucleophiles to access compound 14 as suggested by the
earlier experimental data on the tubulin activity within the furazan
series (Scheme 2).12,27,29 Synthetic protocols describing similar
transformations in the furazan series are scarce, primarily
due to the hazard of starting compound 12. In our hands, this
safety concern was successfully addressed by working with
dilute (< 0.35 mol dm–3) solutions of 12 in CH2Cl2 at room
temperature. While studying this conversion, we found that
the initial introduction of the N-substituent was critical to the
nucleophilic displacement of the remaining NO2 group with the
O-nucleophile. Following this protocol, we isolated compound
14 in 70.5% overall yield. Reversing the order of reactions,
namely reacting 12 with the O-nucleophile followed by the
N-nucleophile led to a complex mixture of products, none of
them major (Scheme 2).
Table 1 Synthesis of 4-amino-3-(1,2,4-oxadiazol-3-yl)furazans 8a–r and
11a–h.
Pro-
duct
Yield Pro-
(%)a duct
Yield
(%)a
n
R
n
R
OMe
8a
1
94
8n
1
79
S
OMe
N
N
8b
8c
1
1
89
90
8o
8p
1
1
93
92
Me
Et
N
OMe
N
Ph
N
8d
8e
2
1
96
88
8q
8r
1
1
79
78
OMe
OMe
N
H
N
N
Ph
N
OMe
O
8f
1
93
11a
1
H
73
O
Cl
We further tested the molecules of 8a–r, 11a–h and 12 in the
phenotypic sea urchin embryo assay in order to identify com-
pounds targeting tubulin.16,17 Combretastatin A-4 disodium
phosphate (CA-4P, OxiGene) served as a benchmark reference
compound.16 We monitored the effect of furazans 8, 11 and
14 on two specific developmental stages of the sea urchin
embryo, namely: (i) fertilized egg to assess antimitotic activity
and (ii) behavioral monitoring of a free-swimming blastulae to
detect changes in the embryo swimming pattern. In this assay,
76
84
8g
8h
1
1
85
76
11b
11c
1
0
OMe
OMe
OMe
F
CF3
8i
8j
1
1
91
87
11d
11e
0
0
86
80
OMe
O
N
†
1
Both H and 13C NMR
1H 4.5–4.7
13C 41–48
O
F
data confirmed structures
of the targeted furazans.
For example, 13C NMR
spectra of compounds
8a–r featured four signals
specific to quaternary car-
bons of the furazan-1,2,4-
oxadiazole core. The IR
13C ca. 137.4
1H 6.4–6.7
R
N
8k
8l
1
1
1
92
85
79
11f
11g
11h
0
0
0
81
83
76
O
N
N
1H 9.0–9.5
H
H2N
13C ca. 155.4
N
N
13C ca. 171.6
N
13C ca. 159
N
N
O
8m
N
spectrum of these molecules displayed intense aminofurazan-NH2 vibra-
O
1
tional bands at ca. 3440 and ca. 3320 cm–1. Typical ranges for the H
aYields refer to the isolated analytically pure materials.†
and 13C NMR shifts of compounds 8a–r are shown.
– 133 –

Mendeleev Commun., 2010, 20, 132–134
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the molecules of 8b,c and 11d,g displayed EC50 values of about
1–4 μmol dm–3 suggesting an antimitotic tubulin destabilizing
effect (Figure 1).
In summary, a series of furazan derivatives based on struc-
tural overlap with combretastatin have been designed, syn-
thesized and tested in the phenotypic sea urchin embryo in vivo
assay. The targeted molecules were prepared via a synthetic
sequence involving the formation of key chloroamidoxime 2.
Compound 2 was converted to trichloromethyl 1,2,4-oxadiazoles
7, 9 and 10 via a two-step protocol, namely, the ring-to-ring
interconversion reaction of amidoximes 3 and 4 to yield com-
pounds 5 and 6 with their subsequent cyclization. A nucleophilic
displacement of the CCl3 group in 7, 9 and 10 with a series of
amines furnished targeted furazans 8a–r and 11a–h in high
yields. A furazan analogue of combretastatin 14 was prepared
from 3,4-dinitrofurazan 12 via stepwise nucleophilic displacement
of nitro groups with N- and O-nucleophiles. The order of reac-
tions for 12 was discovered to be critical to the outcome of this
conversion. All targeted furazan derivatives were conveniently
purified via a straightforward recrystallization to furnish analyti-
cally pure materials immediately suitable for biological assays.
Online Supplementary Materials
Supplementary data associated with this article can be found
in the online version at doi:10.1016/j.mencom.2010.05.002.
Received: 1st December 2009; Com. 09/3428
– 134 –