A convenient synthesis of cis-restricted combretastatin analogues with pyrazole and isoxazole cores

Article abstract of DOI:10.1016/j.mencom.2019.03.015

A series of combretastatin analogues, diarylpyrazoles and diarylisoxazoles, have been synthesized and evaluated for their antimitotic tubulin-binding activity using the phenotypic sea urchin (Paracentrotus lividus) embryo assay. One pyrazole analogue and

Full text of DOI:10.1016/j.mencom.2019.03.015

Mendeleev
Communications
Mendeleev Commun., 2019, 29, 163–165
A convenient synthesis of cis-restricted combretastatin
analogues with pyrazole and isoxazole cores
Dmitry V. Tsyganov,a Marina N. Semenova,b Leonid D. Konyushkin,a
Vladimir I. Ushkarov,a Mikhail M. Raihstata and Victor V. Semenov*a
a N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow,
Russian Federation. E-mail: vs@zelinsky.ru
b N. K. Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, 119334 Moscow,
Russian Federation
DOI: 10.1016/j.mencom.2019.03.015
O
X
N
A series of combretastatin analogues, diarylpyrazoles and
diarylisoxazoles, have been synthesized and evaluated for their
antimitotic tubulin-binding activity using the phenotypic sea
urchin (Paracentrotus lividus) embryo assay. One pyrazole
analogue and four isoxazole analogues have been identified
as potent antimitotic agents comparable with combretastatins
A-2 and A-4, with the lowest observable effective concentra-
tion of 1–10 nmol dm–3 for cleavage alteration of the test
embryos.
OAc
OMe
(RO)n
(RO)n
Sea urchin embryo test:
LOEC 1–10 nmol dm–3
OH
n = 1–4
X = NH, O
2 stages
OMe
N
(Combretastatin A-4:
O
LOEC 2 nmol dm–3
)
OAc
OMe
X
(RO)n
(RO)n
OH
OMe
Microtubule-targeting antimitotic compounds affect cell division
and cause apoptosis, thereby attracting a considerable attention
as potential antitumor agents. Unfortunately, clinical application
of these compounds is limited because of undesirable systemic
toxicity and multiple drug resistance acquired by malignant cells.
Therefore, there is an ongoing need to design molecules displaying
both better therapeutic window and an ability to overcome multi-
drug resistance.
are considered to be of particular interest for anticancer drug
design. The cis-configuration of the ethene linker in combreta-
statins is essential for the specific interaction with the colchicine
binding site of tubulin.1,2 Isosteric replacement of the labile cis-
double bond in the parent combretastatins by five-membered N-con-
taining heterocycle provides non-isomerizable and metabolically
stable combretazole structures with pronounced antimitotic tubulin-
binding activity both in vitro and in vivo.3–14 Among known
combretazoles 4,5(3,4)-diarylpyrazoles and 4,5-diarylisoxazoles
are the most potent.3
Conformationally restricted analogues of natural cytostatic
stilbens, combretastatin A-2 (CA2) and combretastatin A-4 (CA4),
The preparation of structural analogues of CA4, combreta-
pyrazoles 1a and 1b4 as well as combretaisoxazoles 2a5,6 and
2b5, has been published, but the reported synthetic routes are
rather complicated. In addition, the known data on compound
2b lack detailed experimental procedure, melting point value
and spectral characteristics.5 Furthermore, there exists a signi-
ficant difference (700-fold) in published values of cytotoxicity for
compounds 1a and 1b towards human umbilical vein endothelial
cells, HUVEC. Diarylpyrazole 1b inhibits the growth of HUVEC
with GI50 value of 7 nmol dm–3, whereas its close analogue 1a is
inactive up to 5 mmol dm–3 concentration.4 This difference does
not correspond to the structural similarity for compounds 1a/1b
towards analogous molecule of combretastatin A-4 containing a
double bond instead of pyrazole rings. Such variation of bio-
logical activity for 1a and 1b may be explained either by erroneous
determination of chemical structure(s) or by the presence of
bioactive impurities in the synthesized samples.4 To clarify this
matter, we analyzed both synthetic schemes and the analytical
data for these compounds. Specifically, we assumed that the
cycloaddition of sydnones to silylated acetylenes4 may yield an
alternative intermediate silylated pyrazole (or a mixture of
respective isomers) affording the inactive 3,5-diarylpyrazole
derivative.
Bridge
RO
A
RO
B
OMe
OH
OMe
CA2: R + R = CH2
CA4: R = Me
O
N
HN
N
MeO
MeO
MeO
MeO
OMe
OMe
OH
OH
OMe
OMe
2a
1a
N
N
O
NH
MeO
MeO
MeO
MeO
OMe
OMe
OH
OH
OMe
OMe
In general, the presence of OH group in the benzene ring
hampers the synthesis of target structures 1 and 2 and requires
introduction and removal of protecting benzylic group at the
2b
1b
Combretapyrazoles
Combretaisoxazoles
© 2019 Mendeleev Communications. Published by ELSEVIER B.V.
on behalf of the N. D. Zelinsky Institute of Organic Chemistry of the
Russian Academy of Sciences.
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Mendeleev Commun., 2019, 29, 163–165
O
O
R
R
OH
HO
Ac
ii
i
H
+
MeO
MeO
OMe
MeO
HN
N
R
R
R
iv
3a
4a,c
5a,c (56–61%)
MeO
R
OMe
OH
OH
O
O
OMe
R
R
OAc
OMe
iii
1a (72%), 1c (63%)
MeO
MeO
OMe
O
N
MeO
R
MeO
MeO
R
6a,c (crude)
5'a,c (72–81%)
v
a R = OMe
c R = H
OMe
OH
OMe
2a (55%)
Scheme 1 Reagents and conditions: i, NaOH, EtOH, room temperature, 24 h; ii, AcCl, pyridine, CH2Cl2, room temperature, 3 h; iii, PhI(OAc)2, MeOH,
H2SO4, room temperature, 8 h; iv, EtOH, N2H4 ·2HCl, reflux, 1 h; v, EtOH, NH2OH·HCl, reflux, 2 h.
hydrogenation stage. Accordingly, we proposed a convenient
synthetic protocol for the known and new pyrazole- and isoxazole-
based analogues of CA2 and CA4 that included iodosodiacetate-
mediated rearrangement of easily accessible chalcones 5a,c
into dimethylketals 6a,c followed by their in situ cyclization to
provide the target pyrazoles (Scheme 1).
The protective acetyl group was easily introduced (step ii)
and removed (step iii) during the rearrangement of chalcones 5
into dimethylketals 6. Importantly, it was not necessary to isolate
the intermediate compounds 6. Instead, the reaction mixture was
treated with NH2NH2 or NH2OH to result in pyrazoles 1a,c or
isoxazole 2a.
The corresponding isomeric diarylpyrazole 1b and its analogues
1d,e as well as isoxazole-based analogues 2b,d–f were obtained
via similar approach from the respective benzaldehydes and
3-acetoxy-4-methoxyacetophenone 4b, which in turn could be
readily synthesized from guaiacol.15 The protective acetate groups
were successfully removed during the isomerization of chalcone
acetates 5' (Scheme 2).†
In our experiments, melting points for compounds 1a (171–
173°C) and 1b (232–234°C) did not match the reported values
(198–200°C and 90–93°C, respectively).4 Furthermore, the
NMR signal of the NH group from pyrazole ring of product
1a in CDCl3 was absent in the published data.4 Conversely, our
1H NMR spectra of compound 1a in DMSO-d6 showed signals
for two chemically distinct NH groups in the pyrazole ring at
12.9 and 13.1 ppm, suggesting the presence of two NH-tauto-
mers. In addition, the positions of aryl rings in product 1a were
unambiguously determined by NOESY, COSY, HSQC, and
HMBC spectra (see Online Supplementary Materials).
The synthesized compounds were evaluated for their biological
activity using the phenotypic sea urchin embryo assay.3 This assay
allowed for rapid evaluation of both antiproliferative and micro-
tubule destabilizing effects. Specific change of embryo swimming
pattern, namely spinning on the bottom of the vessel instead of
normal forward movement near the surface of the seawater,
indicated microtubule destabilizing action.
As shown in Table 1, the majority of combretazoles caused
pronounced cleavage alteration/arrest and embryo spinning sug-
gesting strong antimitotic microtubule destabilizing activity.
Effects of 1a and 2a,b,d,f were comparable to those of the
parent combretastatins CA2 and CA4. Antiproliferative activity
†
General procedure for the synthesis of chalcones 5. NaOH (60 mmol)
was added to a vigorously stirred solution of benzaldehyde 3 (20 mmol)
and arylacetophenone 4 (20 mmol) in EtOH (150 ml) at +5°C (ice
bath). The reaction mixture was stirred at room temperature for 24 h,
concentrated in vacuo, the residue was treated with distilled water (130 ml),
neutralized with 15% HCl and extracted with CH2Cl2 (3×80 ml). The
combined organic extracts were washed with brine (2×80 ml), dried over
anhydrous Na2SO4 and concentrated in vacuo. The resulting residue was
recrystallized from EtOAc–light petroleum (1:1) and dried to afford
chalcone 5.
overnight. Then the reaction mixture was diluted with water (300 ml)
and extracted with CH2Cl2 (3×100 ml). The organic layer was washed
with brine (2×80 ml), water (50 ml) and dried over anhydrous Na2SO4.
The solvent was removed in vacuo and the resulting oil was purified using
flash-chromatography (ethyl acetate–hexane, 1:4) to afford product 6 that
was used without further purification.
General procedure for the synthesis of chalcone acetates 5'. A mixture
of acetyl chloride (1.18 g, 15 mmol) and pyridine (1.19 g, 15 mmol) in
absolute CH2Cl2 (15 ml) was added by small portions to a stirred solution
of chalcone 5 (10 mmol) and pyridine (0.79 g, 10 mmol) in absolute
CH2Cl2 (50 ml) at 20°C. The reaction mixture was stirred at room
temperature for 3 h, then diluted with water (50 ml), and the organic layer
was separated. The aqueous layer was extracted with CH2Cl2 (30 ml). The
combined organic extracts were washed with water (3×50 ml), dried over
anhydrous Na2SO4 and evaporated. The residue was recrystallized from
EtOAc–light petroleum (1:1) and dried to afford chalcone acetate 5'.
General procedure for the synthesis of dimethylketals 6 with simul-
taneous removal of acetyl group. A solution of H2SO4 (50%, 5 ml)
in methanol was added dropwise to a vigorously stirred suspension of
chalcone 5' (20 mmol) and PhI(OAc)2 (30 mmol) in methanol (100 ml).
The mixture was stirred at room temperature for 8 h and kept at 2–6°C
General procedure for the synthesis of 4,5-diarylpyrazoles 1. N2H4 ·2HCl
(0.410 g, 3.9 mmol) was added to a solution of dimethylketal 6 (3.6 mmol)
in ethanol (20 ml), the mixture was refluxed for 1 h, concentrated in vacuo
and the resulting residue was dissolved in ethyl acetate (50 ml). The organic
layer was washed with 5% aq. NaHCO3 (50 ml), distilled water (50 ml),
dried over MgSO4 and concentrated in vacuo to furnish crude pyrazole 1.
Meting points were measured after recrystallization from ethyl acetate.
General procedure for the synthesis of 4,5-diarylisoxazoles 2. 4,5-Di-
arylisoxazoles 2 were prepared according to a modified procedure.17
NH2OH·HCl (3.9 mmol) was added to a solution of dimethylketal 6
(3.6 mmol) in ethanol (20 ml). The resulting mixture was refluxed for 2 h,
concentrated in vacuo, the residue was treated with ethyl acetate (50 ml),
and the organic phase was washed with distilled water (2×25 ml), dried
over MgSO4 and concentrated to afford crude isoxazole 2. Melting points
were measured after recrystallization from ethyl acetate.
– 164 –

Mendeleev Commun., 2019, 29, 163–165
R1
O
R1
O
R2
R3
R2
R3
OH
Ac
OAc
OMe
i
ii
H
+
R1
N
OMe
R2
R3
NH
R4
R4
iv
3b,d–f
4b
5b,d–f
R2
R4
R3
R4
R1
OH
R1
O
O
OMe
R2
R3
OAc
OMe
iii
OH
1b (12%), 1d (48%), 1e (18%)
R1
N
O
MeO
OMe
OMe
R2
R4
6b,d–f (crude)
5'b,d–f (88%)
v
R3
R4
b R1 = H, R2 = R3 = R4 = OMe
d R1 = R2 = R4 = H, R3 = OMe
OH
e R1 = R4 = OMe, R2 + R3 = OCH2O
OMe
f
R1 = H, R4 = OMe, R2 + R3 = OCH2O
2b (26%), 2d (52%), 2e (23%), 2f (18%)
Scheme 2 Reagents and conditions: i, NaOH, EtOH, room temperature, 24 h;3,16 ii, AcCl, pyridine, CH2Cl2, room temperature, 3 h;3,16 iii, PhI(OAc)2,
MeOH, H2SO4, room temperature, 8 h; iv, EtOH, N2H4·2HCl, reflux, 1 h; v, EtOH, NH2OH·HCl, reflux, 2 h.
Table 1 Effect of diarylpyrazoles 1 and diarylisoxazoles 2 on sea urchin
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microtubules, because this compound failed to induce embryo
spinning or formation of tuberculate arrested eggs typical of
microtubule destabilizing agents.
In summary, we have synthesized a series of cis-restricted
combretastatin analogues, diarylpyrazoles and diarylisoxazoles,
and demonstrated that in the sea urchin embryo model, diaryl-
isoxazoles were more potent than the respective 4,5-diaryl-
pyrazoles. Isoxazoles 2a,b,d,f exhibited antimitotic effect
comparable to that for combretastatins A-2 and A-4.
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Online Supplementary Materials
Supplementary data associated with this article can be found
in the online version at doi: 10.1016/j.mencom.2019.03.015.
Received: 25th September 2018; Com. 18/5698
– 165 –