Synthesis and biological activity of pyrrole analogues of combretastatin A-4

Article abstract of DOI:10.1016/j.bmcl.2016.05.026

A series of pyrrole analogues of combretastatin (CA-4) were synthesized and tested for their anti-proliferative activity. The highly diastereoselective acyl-Claisen rearrangement was used to provide 2,3-syn disubstituted morpholine amides which were used as precursors for the various analogues. This synthesis allows for the preparation of 1,2- and 2,3-diaryl-1H-pyrroles which are both geometrically similar to CA-4. These pyrrolic analogues were tested for their anti-proliferative activity against two human cell lines, K562 and MDA-MB-231 with 2,3-diaryl-1H-pyrrole 35 exhibiting the most potent activity with IC₅₀ value of 0.07 μM against MDA-MB-231 cell line.

Full text of DOI:10.1016/j.bmcl.2016.05.026

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological activity of pyrrole
analogues of combretastatin A-4
Eun-Kyung Jung a, Euphemia Leung b, David Barker a,
⇑
a School of Chemical Sciences, University of Auckland, 23 Symonds St, Auckland, New Zealand
b Auckland Cancer Society Research Centre and Department of Molecular Medicine and Pathology, University of Auckland, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyrrole analogues of combretastatin (CA-4) were synthesized and tested for their anti-
proliferative activity. The highly diastereoselective acyl-Claisen rearrangement was used to provide
2,3-syn disubstituted morpholine amides which were used as precursors for the various analogues.
This synthesis allows for the preparation of 1,2- and 2,3-diaryl-1H-pyrroles which are both geometrically
similar to CA-4. These pyrrolic analogues were tested for their anti-proliferative activity against two
human cell lines, K562 and MDA-MB-231 with 2,3-diaryl-1H-pyrrole 35 exhibiting the most potent
Received 13 April 2016
Revised 8 May 2016
Accepted 9 May 2016
Available online xxxx
Keywords:
Pyrroles
Combretastatin A-4
Antiproliferative agents
Cancer
activity with IC50 value of 0.07
l
M against MDA-MB-231 cell line.
Ó 2016 Elsevier Ltd. All rights reserved.
Acyl-Claisen
Combretastatin A-4 (2), a natural product isolated from the bark
of the South African bush willow Combretum caffrum, is one of the
most potent antimitotic agents and disrupts microtubulin dynam-
ics.1–13 Combretastatin A-4 binds to the colchicine (1) domain of
tubulin and inhibits microtubule polymerisation, resulting in
destabilisation of the microtubule cytoskeleton and inhibition of
mitosis. The structural simplicity and potent cytotoxicity of CA-4
has led to the development of analogues of CA-4 as new anticancer
agents (Fig. 1).2,3,11–13
Structure–activity-relationship (SAR) studies with CA-4 have
indicated that the cis-configuration of the olefinic bridge between
the two aromatic rings A and B is essential. The 3,4,5-trimethoxy
group on ring A affects cytotoxic activity and 3-hydroxy-4-meth-
oxy groups on ring B affects binding to tubulin.1,4,5,8,13 In terms
of the spatial relationship, the cis-orientation of the two rings of
CA-4 is required for binding in the colchicine-binding site of
tubulin. Nevertheless, it is notable that the cis-olefin in CA-4 can
convert into the thermodynamically more stable, and inactive,
trans-olefin due to its metabolic instability. This cis–trans isomeri-
sation results in a loss of antimitotic activity and cytotoxic-
ity.1,2,5,7,9,14 The low water-solubility of CA-4 results in its low
efficacy in vivo and therefore the water-soluble sodium phosphate
prodrug of CA-4, fosbretabulin (CA-4P), was designed and is
currently in phase II/III clinical trials.5–11 Efforts to remove the
possibility of cis–trans isomerisation have been attempted by
replacing the cis-olefin with a five-membered heterocycle which
result in a cis-like constrained configuration. CA-4 analogues hav-
ing five-membered heterocyclic rings such as oxazoles, imidazoles
and triazoles have been reported to have increased potency and
bioactivity.2,3,13,14
Pyrrole-linked heterocyclic analogues have also been previously
synthesised and their antimitotic activity evaluated with
3,4-diaryl-1H-pyrrole-2-carboxylates,15 4,5-diaryl-1H-pyrrole-2-
carboxylates,7 3,5-diaryl-1H-pyrrole-2-carboxylates16 and 1,2-dia-
ryl-1H-pyrroles10 being reported.
With the objective of synthesis of pyrrolic analogues of CA-4 in
mind, herein, we report the synthesis of novel 1,2-diaryl and
2,3-diaryl-1H-pyrrole analogues of CA-4.
To prepare the desired analogues the Paal–Knorr pyrrole
synthesis was chosen with an acyl-Claisen approach being used
to form the diketone precursors. The acyl-Claisen rearrangement
of acid chlorides with (E)-allylic morpholines is highly
diastereoselective giving 2,3-syn-disubstitued amides.17,18 The
2,3-syn arrangement of substituents in 1,4-dicarbonyl compounds
is the preferred arrangement for heterocycle formation in
Paal–Knorr synthesis.20 For 1,2-diarylpyrroles, the two aryl groups
found in CA-4, 3,4,5-trimethoxy on A and 4-methoxy-3-hydroxy
substitution on B, would be introduced. We envisaged that
previously prepared morpholine amide 7 would allow for the
change in substitution pattern, ring A or B, by addition of different
aryl organometallic reagents. The second aryl group would
be added using the appropriate aniline in the Paal–Knorr
condensation (Scheme 1).
⇑
Corresponding author. Tel.: +64 9 373 7599; fax: +64 64 373 7422.
E-mail address: d.barker@auckland.ac.nz (D. Barker).
http://dx.doi.org/10.1016/j.bmcl.2016.05.026
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Jung, E.-K.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.026

2
E.-K. Jung et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
HO
4
MeO
2
MeO
MeO
N
N
5
NHAc
O
MeO
1
MeO
MeO
OMe
MeO
OMe
OH
OMe
OMe
OMe
4
3
Colchicine 1
OH
MeO
OMe
MeO
MeO
MeO
MeO
A
3
B
4
5
MeO
OMe
2
OH
N
1
N
OMe
MeO
MeO
R
R
2
Combretastatin A-4
MeO
5
6
HO
3-6
1,2-diaryl and 2,3-diaryl pyrrole analogues of CA-4
.
Figure 1. Structure of colchicine 1, combretastatin A-4 2 and the target pyrrolic derivatives of the natural product.
R1
R2
Br
O
O
PdCl2, CuCl
air, DMF/H2O
O
R1
R2
R1
R2
2
R3
N
tBuLi, THF
-78 °C to r.t
O
20 h
O
R3
R3
7
1
3
8 R1=R2=R3=OMe, 76 %
10
11
R =R =R =OMe, 86 %
R =OPr, R =OMe, R =H, 96 %
1
i
2
3
1
2
3
i
9
R =OPr, R =OMe, R =H, 75 %
R4
R5
NH2
R1
R7
N
R2
R6
R3
NaOAc, AcOH
80 °C, in air, 18 h
R6
R4
R5
12 R1=R2=R3=R5=OMe, R4=H, R6= OTBDPS, R7=Me, 13 %
(i)
1
2
3
5
4
6
7
3
R =R =R =R =OMe, R =H, R = OH, R =Me, 55 %
13 R1=OiPr, R2=R4=R5=R6=OMe, R3=H, R7= CHO, 8 %
(ii)
14 R1=OH, R2=R4=R5=R6=OMe, R3=H, R= CHO, 56 %
15
1
i
2
5
3
4
6
7
R =O Pr, R =R =MeO, R =R =R =H, R = CHO, 13 %
(ii)
16 R1=OH, R2=R5=MeO, R3=R4=R6=H, R7= CHO, 68 %
R4
R5
NH2
R1
Me
N
R2
R6
R3
R6
R4
NaOAc, AcOH
80 °C, N2 circulation, 18 h
R5
1
2
3
5
4
6
12
R =R =R =R = OMe, R =H, R = OTBDPS, 74 %
(i)
3 R1=R2=R3=R5= OMe, R4=H, R6= OH, 85 %
1
i
2
4
5
6
3
17
R =OPr, R =R =R =R =OMe, R =H, 85 %
(ii)
4 R1=OH, R2=R4=R5=R6=OMe, R3=H, 59 %
Scheme 1. Synthesis of 1,2-diaryl-pyrrolic analogues of combretastatin A-4. (i) TBAF, THF, rt, 18 h; (ii) AlCl3, CH2Cl2, rt, 16 h.
The 2,3-syn-dimethyl morpholine amide 719 was substituted
DMF-water, open to the air gave diketones 10 and 11 in excellent
using lithiated arenes and gave ketones 8 and 9, containing
3,4,5-trimethoxy or 3-isopropyl-4-methoxy phenyl groups respec-
tively. Wacker–Tsuji oxidation of 8 and 9 using PdCl2 and CuCl in
yields. Condensation of diketone 10 with 3-TBDPSO-4-
methoxyaniline in air, gave a complex mixture of products from
which the expected pyrrole 12 was obtained in only 13% yield.
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E.-K. Jung et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Condensation of diketone 11 with 3,4,5-trimethoxyaniline and
4-methoxyaniline was undertaken in air and gave 5-formyl
pyrroles 13 and 15 in 8% and 13% yield, respectively. We have
previously17 found that the 5-methyl group in similar fully
substituted pyrroles is susceptible to oxidation in the presence
of oxygen under Paal–Knorr condition. It has been proposed that
use of electron rich anilines results in the formation of a reactive
enamine which reacts with oxygen giving the formyl group.
Therefore, we conducted the condensation reactions under nitro-
gen to avoid formation of aldehyde and to hopefully increase
yield. The reaction of diketones 10 and 11 with 3-TBDPSO-4-
methoxyaniline and 3,4,5-trimethoxyaniline under nitrogen gave
only fully substituted methyl pyrroles 12 and 17 in good yields
of 74% and 85%, respectively, showing that the removal of oxygen
minimises various side products and contributes to an increase in
pyrrole formation. Finally, removal of the TBDPS group in 12
using TBAF, or removal of the isopropyl group in 17 using AlCl3,
provided the pyrrolic derivatives of CA-4, 3 and 4, in 85% and
59%, respectively. Similarly removal of the isopropyl group in 5-
formylpyrroles 13 and 15 gave additional analogues 14 and 16,
respectively.
Following the successful synthesis of 1,2-diaryl-pyrroles, we
next sought to synthesise 2,3-diaryl-pyrroles to compare the activ-
ity of 1,2-diaryl and 2,3-diaryl-1H-pyrroles.
O
MeO
MeO
O
O
1. I2, THF/H2O
HO
N
Cl
O
2. Zn, AcOH, 80 o
67 % (2 steps)
C
O
N
19
OMe
iPr2NEt
MeO
18
OMe
MeO
OMe
AlCl3, CH2Cl2
OMe
OMe
24 h, 26 %
20
21
1. LiAlH4, THF
65 oC, 20 h
2. DMP, CH2Cl2
2 h,
iPrO
MeO
Br
O
OH
iPrO
H
MeO
75 % (2 steps)
×
tBuLi, THF
-78 oC to r.t
MeO
OMe
OMe
MeO
OMe
OMe
23
22
Scheme 2. Synthesis of aldehyde 22.
iPrO
MeO
Br
O
O
O
iPrO
O
Cl
N
N
O
iPr2NEt
AlCl3, CH2Cl2
20 h, 43 %
tBuLi, THF, -78 °C
MeO
25
18
18 h, 75 %
24
PdCl2, CuCl
air, DMF/H2O
22 h, 76 %
Br
O
NBS
THF
iPrO
MeO
27
iPrO
MeO
amine
iPrO
N
R
N
R
NaOAc, AcOH
80 °C, 18 h
N2 circulation
-78 °C
2 h
O
MeO
26
29 R= 4-methoxyphenyl, 56 %
R= 4-methoxybenzyl, 70 %
R= 4-methoxyphenyl, 41 %
28 R= 4-methoxybenzyl, 59 %
30
MeO
MeO
B(OH)2
Pd(PPh3)4, 2 M K2CO3
DME, 90 °C, 24 h
31
OMe
OMe
MeO
OMe
MeO
MeO
MeO
AlCl3, CH2Cl2
iPrO
HO
4 h
N
R
N
R
MeO
MeO
32 R= 4-methoxyphenyl, 33 %
34 R= 4-methoxyphenyl, 30 %
35
33
R= 4-methoxybenzyl, 44 %
R= 4-methoxybenzyl, 63 %
Scheme 3. Synthesis of 2,3-diaryl pyrrolic analogues of combretastatin A-4.
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4
E.-K. Jung et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 1
Antiproliferative activity (IC50 values) of pyrrole compounds against K562 and MDA-MB-231 cell lines
a
a
Compounds
K562 IC50
(lM)
MDA-MB-231 IC50 (lM)
HO
N
MeO
P10
P10
MeO
OMe
O
OMe
4
HO
N
MeO
P10
P10
MeO
OMe
OMe
14
MeO
N
MeO
5.40 ( 0.02)
1.66 ( 0.02)
MeO
OH
O
OMe
3
HO
N
MeO
P10
8.43 ( 1.57)
OMe
16
OMe
MeO
MeO
HO
N
3.82 ( 0.49)
1.74 ( 0.14)
MeO
OMe
34
OMe
MeO
MeO
HO
0.21 ( 0.02)
0.07 ( 0.02)
N
MeO
OMe
35
Combretastatin A-4 (1)
0.003 ( 0.0005)
0.03 ( 0.0001)
a
Experiments were performed in triplicate; stated values are the averages of three independent determinations.
See Supplementary data for details.
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E.-K. Jung et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
Acyl-Claisen rearrangement of crotyl morpholine 18 and acid
Acknowledgements
chloride 19 gave 2-aryl-3-methylamide 20 (Scheme 2), however,
addition of lithiated arenes to amide 20 gave no ketone, presum-
ably due to steric hindrance. It was therefore decided to convert
amide 20 into a more reactive aldehyde, and this was achieved
utilising iodolactonisation of amide 20 followed by reductive ring
opening with zinc in acetic acid, giving acid 21. Acid 21 was then
reduced, to the primary alcohol followed by oxidation to give alde-
hyde 22. Unfortunately lithiated arene addition to aldehyde 22 was
also unsuccessful and it was again assumed that the aryl group at
Funding for this work was obtained from the Faculty Research
Development Fund from University of Auckland (grant numbers
3706773 and 3706824). This work is also supported by the Auck-
land Cancer Society and the Auckland Cancer Society Research
Centre.
Supplementary data
Supplementary data (experimental procedures, 1H and 13C NMR
spectra and cell proliferation assay information) associated with
this article can be found, in the online version, at http://dx.doi.
org/10.1016/j.bmcl.2016.05.026.
the
a-position was again hindering the addition.
The synthesis was then revised to introduce aromatic ring B
after pyrrole formation. The synthesis began from acyl-Claisen
rearrangement of crotylmorpholine 18 and acetyl chloride, giving
morpholine amide 24 in 43% yield (Scheme 3). Reaction of amide
24 with the lithiate of 3-isopropyl-4-methoxy bromobenzene gave
ketone 25 in good yield. Alkene oxidation of 25 using the Wacker–
Tsuji oxidation gave diketone 26 in 75% yield. Condensation of
diketone 26 with 4-methoxyaniline and 4-methoxybenzylamine
gave pyrroles 27 and 28 in 41% and 59% yields, respectively. To pre-
vent oxidation at the 5-methyl group, pyrrole formation was again
conducted under a nitrogen atmosphere. Bromination of pyrroles
27 and 28 using NBS in THF at À78 °C gave 3-bromo pyrroles 29
and 30, with no poly-brominated products obtained. Suzuki
coupling reaction between 3-bromopyrroles 29 and 30 and 3,4,5-
trimethoxyphenylboronic acid 31 provided the desired arylated
pyrroles 32 and 33. Finally, removal of isopropyl protecting groups
of 32 and 33 using AlCl3 provided the desired pyrrolic derivatives
of CA-4 34 and 35.
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active than combretastatin 2 itself with combretastatin being
twice as active as compound 35 against the MDA-MB-231 cell line.
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appropriate aniline gave the desired pyrrole analogues. 2,3-Diaryl
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active than 1,2-diaryl analogues. The route used to prepare these
analogues is easily adaptable to the preparation of other analogues
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