Synthesis and preliminary evaluation of 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones as angiogenesis inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.02.033

Sunitinib (Sutent) is a receptor tyrosine kinase (RTK) and angiogenesis inhibitor approved for the treatment of renal cell carcinomas, gastrointestinal stromal tumours and pancreatic neuroendocrine tumours. A key structural motif retained throughout medicinal chemistry efforts during sunitinib's development was the indoline-2-one group. In the search for new anti-angiogenic scaffolds, we previously reported that non-indoline-2-one-based derivatives of semaxanib (SU5416, a structurally simpler sunitinib predecessor that underwent Phase III trials) are active as angiogenesis inhibitors, indicating that the group is not essential for activity. This Letter describes the synthesis and structure–activity relationships of another class of non-indoline-2-one angiogenesis inhibitors related to sunitinib/semaxanib; the 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones. A focussed library of 19 analogues was prepared using a simple novel process, wherein commercially available substituted arylacetic acids activated with an amide coupling reagent (HBTU) were reacted with the potassium salt of 3,5-dimethyl-1H-pyrrole-2-carbaldehyde in one-pot. Screening of the library using a cell-based endothelial tube formation assay identified 6 compounds with anti-angiogenesis activity. Two of the compounds were advanced to the more physiologically relevant rat aortic ring assay, where they showed similar inhibitory effects to semaxanib at 10 μg/mL, confirming that 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones represent a new class of angiogenesis inhibitors.

Full text of DOI:10.1016/j.bmcl.2016.02.033

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 preliminary evaluation of 5,7-dimethyl-2-aryl-3H-
pyrrolizin-3-ones as angiogenesis inhibitors
Nicholas S. Kirk a, Anna Bezos b, Anthony C. Willis c, Pichit Sudta d,e, Sunit Suksamrarn e,
Christopher R. Parish b, Marie Ranson f, Michael J. Kelso a,
⇑
a Illawarra Health and Medical Research Institute, School of Chemistry, University of Wollongong, NSW 2522, Australia
b Cancer and Vascular Biology Group, Department of Cancer Biology and Therapeutics, John Curtin School of Medical Research, Australian National University, Canberra ACT
2601, Australia
c Single Crystal X-ray Diffraction Unit, Research School of Chemistry, Australian National University, Canberra ACT 2601, Australia
d Division of Chemistry, Phetchaburi Rajabhat University, Phetchaburi 76000, Thailand
e Department of Chemistry, Srinakharinwirot University, Bangkok 10110, Thailand
f Illawarra Health and Medical Research Institute, School of Biological Sciences, University of Wollongong, NSW 2522, Australia
a r t i c l e i n f o
a b s t r a c t
Sunitinib (SutentÒ) is a receptor tyrosine kinase (RTK) and angiogenesis inhibitor approved for the treat-
ment of renal cell carcinomas, gastrointestinal stromal tumours and pancreatic neuroendocrine tumours.
A key structural motif retained throughout medicinal chemistry efforts during sunitinib’s development
was the indoline-2-one group. In the search for new anti-angiogenic scaffolds, we previously reported
that non-indoline-2-one-based derivatives of semaxanib (SU5416, a structurally simpler sunitinib prede-
cessor that underwent Phase III trials) are active as angiogenesis inhibitors, indicating that the group is
not essential for activity. This Letter describes the synthesis and structure–activity relationships of
another class of non-indoline-2-one angiogenesis inhibitors related to sunitinib/semaxanib; the 5,7-
dimethyl-2-aryl-3H-pyrrolizin-3-ones. A focussed library of 19 analogues was prepared using a simple
novel process, wherein commercially available substituted arylacetic acids activated with an amide cou-
pling reagent (HBTU) were reacted with the potassium salt of 3,5-dimethyl-1H-pyrrole-2-carbaldehyde
in one-pot. Screening of the library using a cell-based endothelial tube formation assay identified 6 com-
pounds with anti-angiogenesis activity. Two of the compounds were advanced to the more physiologi-
cally relevant rat aortic ring assay, where they showed similar inhibitory effects to semaxanib at
Article history:
Received 9 December 2015
Revised 10 February 2016
Accepted 13 February 2016
Available online xxxx
Keywords:
Angiogenesis inhibitor
Knoevenagel
3H-Pyrrolizin-3-one
Semaxanib
SU5416
Sunitinib
10
lg/mL, confirming that 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones represent a new class of angiogen-
esis inhibitors.
Ó 2016 Elsevier Ltd. All rights reserved.
Sunitinib 1 (SutentÒ, Pfizer, Fig. 1)1 is an indolin-2-one-based
angiogenesis inhibitor approved for the treatment of vascularised
renal cell carcinomas,2 gastrointestinal stromal tumours3 and pan-
creatic neuroendocrine tumours.4 Its mechanism of action involves
inhibition of at least eight different receptor tyrosine kinases
(RTKs), including vascular endothelial growth factor receptors
1–3 (VEGFR1–VEGFR3), platelet-derived growth factor receptors
(PDGFRs)
a
and b, stem cell factor receptor (Kit), Fms-like tyrosine
kinase 3 (FLT-3) and colony-stimulating factor-1 receptor (CSF-
1R).5 The indolin-2-one (oxindole) portion was considered crucial
for activity of sunitinib 1 and was retained throughout develop-
ment. Indeed, the structurally simpler indolin-2-one predecessor
semaxanib (SU5416) 2 underwent a Phase III clinical trial for
advanced colorectal cancer.6 In recent work, we showed that in
spite of its perceived importance the indolin-2-one moiety is not
essential for anti-angiogenic activity in this class, demonstrating
Abbreviations: HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate; CSF-1R, colony-stimulating factor-1 receptor; DIPEA, N,N-
diisopropylethylamine; FLT-3, Fms-like tyrosine kinase 3; FOV, field of view;
HUVEC, human umbilical vein endothelial cell; PDGFR, platelet-derived growth
factor receptor; RTK, receptor tyrosine kinase; THF, tetrahydrofuran; VEGFR,
vascular endothelial growth factor receptor.
that ring-opened 3,5-dimethyl-1H-pyrrol-2-yl-2-arylacrylate
3
variants of semaxanib 2 inhibit angiogenesis in a rat aortic ring
model.7 We now report the discovery of a second class of non-
indolin-2-one-based angiogenesis inhibitors related to sunitinib
1/semaxanib 2; the 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones 4
(Fig. 1).
⇑
Corresponding author. Tel.: +61 2 4221 5085; fax: +61 2 4221 4287.
E-mail address: mkelso@uow.edu.au (M.J. Kelso).
http://dx.doi.org/10.1016/j.bmcl.2016.02.033
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kirk, N. S.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.02.033

2
N. S. Kirk et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
NEt2
NH
O
7
1
N
R
N
H
N
5
N
H
O
H
F
2
O
O
O
4'
2'
'
N
H
N
H
R
3'
R
®
1
2
Sunitinib (Sutent )
Semaxanib (SU5416)
3,5-Dimethyl-1H-pyrrol-2-yl- 5,7-Dimethyl-2-aryl-3H-
2-arylacrylates 3
pyrrolizin-3-ones 4
Figure 1. Angiogenesis inhibitors related to sunitinib 1.
OH
We hypothesised that 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-
O
ones might possess anti-angiogenic properties due to their struc-
tural similarity to sunitinib 1, SU5416 2 and acrylates 3. The
hypothesis was tested by developing new chemistry to create a
small library of analogues carrying systematic variations at the
2-aryl ring and investigating their structure–activity relationships
(SAR). Sporadic reports of 3H-pyrrolizin-3-ones have appeared in
the literature but little remains known about their chemistry and
biological activity.8–13 Complex structures incorporating a 3H-pyr-
rolizin-3-one-type core have been reported, including tricyclic pyr-
rolo-indolones, pyrrolo-isoindolones, tetracyclic isoindolo-
indolones and other polycyclic heterocycles.14,15 A number of these
are marine natural products or their derivatives.16
O
R3
R1
N
H
R2
H
5
HBTU, DIPEA,
CH2Cl2, rt
KH, THF
0 o
Flask 1
Flask 2
C
O
N
N
O
H
O
N
R3
R1
N
R2
K
Combine contents
of Flasks 1 and 2
at 0 o
C
N
Divergent access to 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones
appeared achievable in one-pot from 3,5-dimethyl-1H-pyrrole-2-
carbaldehyde 5 and the many commercially available substituted
phenylacetic acids. We had previously shown that the K+ salt of
5 (generated using KH in THF) is smoothly N-acylated by methyl
chloroformate,7 leading us to consider that the same salt might
also undergo N-acylation reactions with phenylacetic acids pre-
activated as acid halides or with amide coupling reagents. A sim-
ple, divergent synthesis was envisaged wherein activated pheny-
lacetic acids generated in one flask are combined with the K+ salt
of 5, which had been freshly prepared in a separate flask. Mixing
of the flasks would initiate pyrrole N-acylation/amide formation
and ensuing benzylic proton abstraction under the basic reaction
conditions (excess KH) would trigger an intramolecular Knoeve-
nagel condensation to deliver 5,7-dimethyl-2-aryl-3H-pyrrolizin-
3-ones.
Preliminary synthetic efforts surveying a variety of amide cou-
pling reagents, procedures and rates and orders of addition in
model reactions with phenylacetic acid and pyrrole aldehyde 5
yielded 5,7-dimethyl-2-phenyl-3H-pyrrolizin-3-one 6 as the major
product in many instances, as observed by TLC analysis. The high-
est yield of 6 (48%, Scheme 1) was obtained when 1.1 mol equiv of
amide coupling reagent 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetram-
ethyluronium hexafluorophosphate (HBTU) and 2.0 equiv of N,N-
diisopropylethylamine (DIPEA) were stirred in CH2Cl2 at room
temperature with 1.0 equiv of phenylacetic acid to form the acti-
vated hydroxybenxzotriazoyl ester in one flask (Flask 1) and
2.0 mol equiv of KH was stirred in THF at 0 °C with 1.0 equiv of
aldehyde 5 in a separate flask (Flask 2) to generate the K+ pyrrolate
salt. Pre-cooling Flask 1 to 0 °C and pouring its contents into Flask 2
at 0 °C in one portion completed the procedure (Scheme 1).
Employing the method with a systematic series of mono-substi-
tuted phenylacetic acid derivatives carrying F, Cl, Br, Me, OCH3 and
NO2 groups at each of the 20-, 30- and 40-positions similarly pro-
vided analogues 7–24 (Scheme 1). While the yields were low to
moderate (4–48%) the simple procedure allowed rapid access to
useable quantities of pure analogues for the SAR study. 1H and
13C NMR data for all compounds were unambiguous and consistent
with 2-aryl-3H-pyrrolizin-3-ones. A single crystal X-ray structure
obtained for 20-NO2 derivative 22 further confirmed its structure
O
R3
R1
6-24
R3 Compound Yield %
R2
R1
R2
H
F
H
H
Cl
H
H
Br
H
H
Me
H
H
OMe
H
H
NO2
H
H
H
F
H
H
6
7
8
9
48
37
37
38
16
35
30
13
44
34
21
41
37
33
45
30
28
15
4
H
H
F
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Cl
H
Cl
H
H
Br
H
H
H
Br
H
H
Me
H
H
Me
H
H
OMe
H
OMe
H
H
H
NO2
H
H
NO2
H
Scheme 1. Divergent one-pot synthesis of 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-
ones 6–24.
(Fig. 2). The X-ray structure revealed that the 20-nitrophenyl ring
was tilted 35.5° relative to the plane of the 3H-pyrrolizin-3-one
ring system and positioned the electron-deficient NO2 nitrogen in
close proximity to the electronegative carbonyl oxygen (2.8 Å),
suggesting a favourable interaction between these two atoms.
Compounds 6–24 were screened for in vitro angiogenesis inhi-
bitory activity at 10 lM using the human umbilical vein endothe-
lial cell (HUVEC)-based endothelial tube formation assay.17 Matrix-
cultured HUVECs differentiate in response to growth factors,
becoming elongated and motile and able to self-organise into cap-
illary-like structures over a 2–12 h period. This process is disrupted
in a dose-dependent manner by small molecule inhibitors of angio-
genesis.18 Quantification of angiogenesis and the inhibitory effects
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N. S. Kirk et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
of compounds uses the method of Aranda.17 Briefly, images are
captured and the cells tallied and categorised as ellipsoid (undiffer-
entiated), sprouting (differentiating) or connected to other cells
(Supporting information, Fig. S1). Total numbers of closed polyhe-
dra are also counted, along with ‘complex meshes’ or polyhedral
containing walls more than 2 cells thick. Input of the data into
an equation (Supporting information, Eq. S1) generates an angio-
genesis score, which is then expressed as a percentage relative to
the angiogenesis score of the vehicle control (100% angiogenesis).
Reductions in scores from 100% in the presence of added com-
pounds correspond to angiogenesis inhibition.
Figure 3a shows that 20-NO2 analogue 22 was the most potent
inhibitor in the series, reducing angiogenesis to 74.0% 12.6 of
vehicle at 1
inhibited angiogenesis at 10
l
M and 29.7% 4.1 at 10
lM. Sunitinib 1 completely
l
M. The 30-NO2 23 and 40-NO2 24 ana-
logues showed almost no activity. Indeed all 30 (i.e., 8, 11, 14, 17,
20) and 40 (i.e., 9, 12, 15, 18)-substituted compounds failed to show
notable activity, confirming that these positions do not tolerate
substitution. The unsubstituted derivative 6 showed modest activ-
ity (68.4% 6.6 of vehicle) and similar activities were observed
with the 20-fluoro 7, 20-bromo 13, 20-methyl 16 and 20-methoxy
19 analogues. 20-Chloro derivative 10 showed no activity. Com-
pound 22 was shown to be 4-fold less toxic than sunitinib 1
towards related SVEC4-10 murine endothelial cells (Supporting
information, Fig. S2), consistent with general cytotoxicity not being
responsible for the observed anti-angiogenic effects. We conclude
that while the 30- and 40-positions are intolerant of substitution,
20-substituents are tolerated to some extent and in some cases
(e.g., NO2 group) they can increase activity. This is in agreement
Figure 2. Single crystal X-ray structure (ORTEP) of 22. Anisotropic displacement
ellipsoids represent 30% probability levels. Hydrogen atoms are drawn as circles
with small radii. Crystallographic data are provided in the Supporting information.
Atomic coordinates, bond lengths and angles and displacement parameters have
been deposited at the Cambridge Crystallographic Data Centre (CCDC accession
number 1439414).
Figure 3. (a) Inhibition of angiogenesis by 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones 6–24 in HUVEC endothelial tube formation assay. Angiogenesis scores represent
percentages ( SEM) relative to the angiogenesis score of the vehicle (DMSO) control (100% angiogenesis). All compounds were present at 10 M data
M. * An additional 1
point is shown for the most potent inhibitor 22. (b) Representative images showing greater angiogenesis inhibition in the presence of 20-NO2 analogue 22 (right) relative to 20-
fluoro 7 (left, both compounds present at 10 M).
l
l
l
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4
N. S. Kirk et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
pounds 6 and 22 were advanced to a rat aortic ring angiogenesis
assay to corroborate their anti-angiogenic properties in a more
physiologically relevant context. Both compounds showed robust
inhibitory effects at 10 lg/mL, confirming 5,7-dimethyl-2-aryl-
3H-pyrrolizin-3-ones as a new class of angiogenesis inhibitors. Fur-
ther structure–activity studies aimed at identifying more potent
inhibitors are warranted. Key targets to pursue would include suni-
tinib-like analogues, where the basic N,N-diethylethylenediamine
side chain amide of sunitinib is appended at the 6-position of
5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones. Profiling the class for
activity against a panel of RTKs should provide insights into the
mechanism of action of these compounds.
Acknowledgments
We thank the universities involved for supporting this work. P.
Sudta and S. Suksamrarn acknowledge financial support from the
Thailand Research Fund through the Royal Golden Jubilee PhD Pro-
gram. N. Kirk acknowledges support from the Australian Govern-
ment for an Australian Postgraduate Award. M. Ranson and M.
Kelso acknowledge partial financial support from University of
Wollongong Small Grant Schemes. A. Bezos and C. Parish acknowl-
edge support from an Australian National Health and Medical
Research Council (NHMRC) Program Grant.
Figure 4. Inhibition of angiogenesis by 6 and 22 in rat aortic ring assay. Data for
positive controls PI-88 (100 lg/mL) and SU5416 2 and negative control (DMSO
vehicle, no compound) are shown for comparison. Data represent the mean % FOV
occupancy ( SEM) generated from at least six replicate cultures of each compound.
with structure–activity conclusions from our previous work show-
ing that ortho-nitro groups contribute favourably to anti-angiogen-
esis activity in the 3,5-dimethyl-1H-pyrrol-2-yl-2-arylacrylate
series (Fig. 1, compound 3 R = NO2).7
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.02.
033.
Compounds 6 and 22 were examined next in a rat aortic ring
assay to confirm their anti-angiogenic properties in a more physi-
ologically relevant context. In this model, 1 mm rings sectioned
from female rat aortas are suspended in a fibrin gel matrix and
exposed to growth media. The aortic sections sprout new vessels
over 5 days, which are scored manually as the % field of view
(FOV) occupied (by vessels) under a light microscope (40X).7,19
Inhibitors of angiogenesis produce a quantifiable reduction in %
FOV occupancy (see Supporting information for further details).
The vehicle control (DMSO, no compound) showed 86% FOV occu-
pancy under the assay conditions (Fig. 4). Two positive control
compounds PI-88 and SU5416 2 were included in the study. PI-
88 is a highly sulfated oligosaccharide-based angiogenesis inhibi-
tor undergoing Phase III clinical trials as a treatment following can-
cer resection in patients with hepatitis virus-related hepatocellular
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SU5416 2 (synthesised in-house using the literature method)21
reduced % FOV occupancy to 61% at 1 g/mL and 10% at 10 g/
mL. Compound 22 produced no reduction in % FOV occupancy at
g/mL but showed a marked effect at 10 g/mL (4% FOV occu-
lg/mL PI-88 reduced % FOV occupancy to 39%.
l
l
1
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In summary, a divergent one-pot synthesis of 5,7-dimethyl-2-
aryl-3H-pyrrolizin-3-ones was developed using commercially
available mono-substituted phenylacetic acids and pyrrole alde-
hyde 5. Usable quantities of all analogues for the SAR study were
obtained but scope remains for improving the yields. Nineteen
analogues were synthesised and screened for anti-angiogenesis
activity in an endothelial tube formation assay. The 20-NO2 ana-
logue 22 was identified as the most potent inhibitor and the
unsubstituted parent compound 6 and 20-fluoro 7, 20-bromo 13,
20-methyl 16 and 20-methoxy 19 derivatives showed activity. In
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Please cite this article in press as: Kirk, N. S.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.02.033