Synthesis, structural characterisation, and preliminary evaluation of non-indolin-2-one-based angiogenesis inhibitors related to sunitinib (Sutent)

Article abstract of DOI:10.1071/CH13219

The indolin-2-one fused-ring system and the 2,4-dimethylpyrrole unit represent key structural motifs in the anticancer drug sunitinib (Sutent) and predecessor angiogenesis inhibitors that have undergone anticancer clinical trials (e.g. semaxanib, SU5416). In pursuit of novel anti-angiogenic scaffolds, we were interested in identifying whether the indolin-2-one group in these structures could be modified without losing activity. This paper describes novel condensation chemistry used to prepare a test series of (E)-and (Z)-alkenes related to SU5416 that retain the 2,4-dimethylpyrrole unit while incorporating ring-opened indolin-2-ones. Unique structural characteristics were identified in the compounds, such as intramolecular hydrogen bonds in the (Z)-alkenes, and several examples were shown to possess significant anti-angiogenic activity in a rat aorta in vitro model of angiogenesis. The work demonstrates that the indolin-2-one moiety is not an absolute requirement for angiogenesis inhibition in the sunitinib/SU5416 class.

Full text of DOI:10.1071/CH13219

RESEARCH FRONT
CSIRO PUBLISHING
Aust. J. Chem. 2013, 66, 864–873
Full Paper
http://dx.doi.org/10.1071/CH13219
Synthesis, Structural Characterisation, and Preliminary
Evaluation of Non-Indolin-2-one-based Angiogenesis
Inhibitors Related to Sunitinib (SutentÒ)
A F
,
B F
,
C
B
Pichit Sudta, Nicholas Kirk, Anna Bezos, Anthony Gurlica,
B
B
D
E
Rhys Mitchell, Thomas Weber, Anthony C. Willis, Samran Prabpai,
E
C
A
Palangpon Kongsaeree, Christopher R. Parish, Sunit Suksamrarn,
and Michael J. Kelso
B G
,
A
Department of Chemistry, Faculty of Science, Srinakharinwirot University,
Bangkok 10110, Thailand.
B
School of Chemistry, Faculty of Science, University of Wollongong, Wollongong,
NSW 2522, Australia.
C
Cancer and Vascular Biology Group, Department of Immunology, John Curtin School
of Medical Research, Australian National University, Canberra, ACT 2600, Australia.
D
Single-crystal X-ray Diffraction Unit, Research School of Chemistry,
Australian National University, Canberra, ACT 2600, Australia.
E
Centre for Excellence in Protein Structure and Function, Faculty of Science,
Mahidol University, Bangkok 10400, Thailand.
F
These authors contributed equally to this work.
G
Corresponding author. Email: mkelso@uow.edu.au
The indolin-2-one fused-ring system and the 2,4-dimethylpyrrole unit represent key structural motifs in the anticancer
drug sunitinib (SutentÒ) and predecessor angiogenesis inhibitors that have undergone anticancer clinical trials
(e.g. semaxanib, SU5416). In pursuit of novel anti-angiogenic scaffolds, we were interested in identifying whether the
indolin-2-one group in these structures could be modified without losing activity. This paper describes novel condensation
chemistry used to prepare a test series of (E)- and (Z)-alkenes related to SU5416 that retain the 2,4-dimethylpyrrole unit
while incorporating ring-opened indolin-2-ones. Unique structural characteristics were identified in the compounds, such
as intramolecular hydrogen bonds in the (Z)-alkenes, and several examples were shown to possess significant anti-
angiogenic activity in a rat aorta in vitro model of angiogenesis. The work demonstrates that the indolin-2-one moiety is
not an absolute requirement for angiogenesis inhibition in the sunitinib/SU5416 class.
Manuscript received: 29 April 2013.
Manuscript accepted: 24 May 2013.
Published online: 20 June 2013.
Introduction
inhibitors of angiogenesis have been intensely studied as anti-
cancer agents and several have been approved for clinical use.[4]
The first FDA-approved angiogenesis inhibitor was the
vascular endothelial growth factor (VEGF)-targeting monoclo-
nal antibody (Mab) bevacizumab (AvastinÒ; Genentech Inc.,
San Francisco, 2003), which continues to be used today in
combination therapies against metastatic colorectal cancer,[5]
non-small-cell lung cancer,[6] and metastatic breast cancer.[7]
Newer VEGF-targeting agents are in various stages of
clinical development, including, for example, VEGF-TrapR1R2
(Aflibercept, Regeneron Inc.), a chimeric soluble VEGF recep-
tor that binds to and neutralises VEGF.[8] In addition to these
biologics, small-molecule-receptor tyrosine kinase inhibitors
(RTKIs) targeting VEGF receptors (VEGFR1ꢀVEGFR3) and
Angiogenesis, the growth of new blood vessels from pre-
existing vascular networks, occurs only under certain conditions
in adults, such as during wound healing, pregnancy, and men-
struation, but can be much more prevalent in pathological
conditions, especially cancer.[1,2] Solid tumours that grow
beyond ,1–2 mm require angiogenesis to avoid becoming
hypoxic from the effects of growth factor mutation-induced
aberrant blood vessel formation and increased energy con-
sumption.[3] In the absence of angiogenesis, solid tumours enter
latent phases and rely on anaerobic metabolism for energy,
limiting their growth.[2,3] Angiogenesis is also a major con-
tributor to metastasis as the newly formed vessels provide a
route for tumour cells to enter the circulation.[3] Accordingly,
Journal compilation Ó CSIRO 2013
www.publish.csiro.au/journals/ajc

Angiogenesis Inhibitors Related to Sunitinib (SutentÒ)
865
other kinase signalling pathways have been extensively investi-
gated as anti-angiogenics and several have progressed to the
market. These include sunitinib 1 (SutentÒ, Pfizer),[9] pazopa-
nib (VotrientÒ, GSK),[10] sorafenib (NexavarÒ, Bayer),[11] and
vandetanib (CaprelsaÒ, AstraZeneca).[12]
by NMR spectroscopy and X-ray crystallography, and reports
that several members show significant anti-angiogenic activity
in a rat aorta in vitro model of angiogenesis.
Chemistry
Sunitinib 1 (Fig. 1) is approved for the treatment of highly
vascularised renal cell carcinomas,[13] gastrointestinal stromal
tumours,[14] and pancreatic neuroendocrine tumours.[15] Its
mechanism of action involves inhibition of at least eight differ-
ent receptor tyrosine kinases, including VEGF1–VEGF3,
platelet-derived growth factor receptors (PDGFRa and
PDGFRb), stem-cell factor receptor (Kit), Fms-like tyrosine
kinase 3 (FLT-3), and colony-stimulating factor-1 receptor
(CSF-1R).[16] During development, the indolin-2-one
(oxindole) and 2,4-dimethylpyrrole portions of Sunitinib 1 were
identified as key pharmacophores and retained throughout
med-chem optimisation efforts. Several other closely related
compounds that contain these groups were also (and continue to
be) evaluated in clinical trials.[17] For example, the structurally
simpler sunitinib predecessor semaxanib (SU5416) 2 (Fig. 1)
underwent a Phase III clinical trial for advanced colorectal
cancer.
In pursuit of novel and patentable anti-angiogenic scaffolds,
we hypothesised that in spite of its perceived importance, the
indolin-2-one moiety may not be essential for anti-angiogenic
activity in the sunitinib/SU5416 class. The hypothesis has been
explored in the current work using a test series of SU5416-like
alkenes that attach the 2,4-dimethylpyrrole unit to ring-opened
indolin-2-ones (i.e. compounds (Z)-3a, (E)-3bꢀ(Z)-10a,
(E)-10b, Fig. 1). This paper details the synthesis of the series
using novel condensation chemistry, describes some of the
unique structural characteristics of the alkenes, as identified
Initial synthetic efforts aimed to identify a general condensation
reaction that could provide access to the target alkenes. In the
reported syntheses of sunitinib 1, SU5416 2, and related indolin-
2-ones, the base indolin-2-ones and requisite N-unsubstituted
pyrrole-2-carboxaldehydes were condensed by refluxing the
two components in ethanol with piperidine.[18] Attempts to
adapt this procedure to the condensation of the model substrate
ethyl-2-(2-nitrophenyl)acetate 13 and 3,5-dimethylpyrrole-2-
carboxaldehyde 11 failed to yield any of the desired alkenes
(Z)-3a or (E)-3b. Other Knoevenagel or Perkin-type condensa-
tions attempted with 13 and 11 using various bases and solvents
and under a variety of conditions were similarly unsuccessful.
At this point, it was reasoned that the aldehyde in 11 was
unreactive owing to a combination of steric hindrance from the
neighbouring pyrrole 3-Me group and because electron donation
from the pyrrole nitrogen into the aldehyde was reducing its
electrophilic character. It was postulated that a small electron-
withdrawing group attached to the pyrrole nitrogen should
increase the aldehyde’s reactivity without increasing steric
hindrance. Attaching electron-withdrawing groups to pyrrole
nitrogens has previously been shown to activate aldehydes at the
2-position.[19,20] Accordingly, N-methylcarbamoyl pyrrole-2-
carboxaldehyde 12 was prepared in 86 % yield by acylating the
potassium salt of 11 (formed by N-deprotonation of 11 with KH
in THF at 08C) with methyl chloroformate (Scheme 1).
Stirring ethyl-2-(2-nitrophenyl)acetate 13 with 12 in reflux-
ing THF in the presence of K2CO3 and 18-crown-6 (Method a,
Rꢀ
N
H
O
N
H
R
O
R2
R1
N
H
O
R1 ꢁ NO2, R2 ꢁ OEt
R1 ꢁ NO2, R2 ꢁ OMe
R1 ꢁ NO2, R2 ꢁ OAllyl
(Z)-3a, (E)-3b
Sunitinib
1
NEt2
R ꢁ F, R
ꢀ ꢁ
N
H
(Z)-4a, (E)-4b
(Z)-5a, (E)-5b
(Z)-6a, (E)-6b
(Z)-7a, (E)-7b
(Z)-8a, (E)-8b
(Z)-9a, (E)-9b
(Z)-10a, (E)-10b
R ꢁ H, R
ꢀ
ꢁ H
Semaxanib (SU5416)
2
R1 ꢁ NO2, R2 ꢁ NHEt
R1 ꢁ NO2, R2 ꢁ NHPh
R1 ꢁ NO2, R2 ꢁ NHBn
R1 ꢁ H, R2 ꢁ OEt
R1 ꢁ Cl, R2 ꢁ OEt
Fig. 1. Structures of sunitinib 1, semaxanib (SU5416) 2, and alkenes (Z)-3a, (E)-3bꢀ(Z)-10a, (E)-10b.
(a)
O
N
O
N
H
OMe
H
H
O
11
12
Scheme 1. Synthesis of N-methylcarbamoyl pyrrole aldehyde 12. Reagents and conditions: (a) (i) KH, THF, 08C; (ii) CH3OCOCl, 2 h (86 %).

866
P. Sudta et al.
O
N
H
N
H
(a) or (b)
O
N
ꢂ
ꢂ
R2
O
O
R1
H
OMe
O
2
R2
R
1
R1
(Z)-3a−(Z)-10a
R
13–20
12
(E )-3b−(E)-10b
Phenylacetic
Reaction
conditions Product, yield [%]
R1
R2
Product, yield [%]
EsterB/AmideC
13
14
15
16
17
18
19
20
NO2
NO2
NO2
NO2
NO2
NO2
OEt
a
a
a
a
a
a
(Z)-3a, 42
(Z)-4a, 36
(Z)-5a, 41
(Z)-6a, 21
(Z)-7a, 22
(E)-3b, 35
(E)-4b, 40
(E)-5b, 33
(E)-6b, 0
OMe
OAll
NHEt
NHPh
NHBn
(E)-7b, ꢃ5A
(Z)-8a, 16
(Z)-9a, 0
(Z)-10a, 5
(E)-8b, ꢃ5A
(E)-9b, 32
(E)-10b, 41
H
OEt
OEt
b
b
Cl
ACompounds could not be isolated in pure form.
BEsters 13−15, 19, 20 were synthesised by refluxing the phenylacetic acids in the requisite alcohol
(as solvent) with H2SO4 (cat.).
CAmides 16−18 were obtained by stirring 2-nitrophenylacetic acid with the requisite amines
under standard solution-phase peptide coupling conditions (i.e. HBTU/DIPEA in CH2Cl2).
Fig. 2. Synthesis of alkenes (Z)-3a, (E)-3bꢀ(Z)-10a, (E)-10b. Reagents and conditions: (a) K2CO3, 18-crown-6,
THF, reflux, 16 h; (b) LDA, THF, ꢀ788C, 30 min.
Fig. 2) was found to directly afford the pyrrole N-deprotected
Knoevenagel condensation products (Z)-3a (42 %) and
(E)-3b (35 %). Similar yields of the desired N-deprotected (Z)-
and (E)-alkenes were obtained directly from reactions of 12 with
methyl-2-(2-nitrophenyl)acetate 14 and allyl-2-(2-nitrophenyl)
acetate 15 (i.e. (Z)-4a, (E)-4b and (Z)-5a, (E)-5b respectively).
The condensation reactions did not proceed when the N-methyl
carbamoyl group of 12 was replaced with either N-ethyl
or N-tbutyl carbamates, possibly owing to increased steric
hindrance around the aldehyde.
Use of N-alky- or N-aryl-2-(2-nitrophenyl)acetamides in
place of 2-(2-nitrophenyl)acetic esters led to reduced yields of
the Z-isomers and none or very low yields of the E-isomers.
Nevertheless, several amides were able to be isolated in
sufficient quantity and purity (.95 % by 1H NMR) for charac-
terisation and angiogenesis inhibition testing. Use of N-ethyl-
2-(2-nitrophenyl)acetamide 16 in the reaction afforded a 21 %
yield of (Z)-6a without formation of any (E)-6b. N-Phenyl-2-
(2-nitrophenyl)acetamide 17 and N-benzyl-2-(2-nitrophenyl)
acetamide 18 yielded 22 and 16 % yields respectively of
alkenes (Z)-7a and (Z)-8a, with less than 5 % of the trans
isomers (E)-7b and (E)-8b being isolated. All alkenes except
(E)-7b and (E)-8b were tested for angiogenesis inhibition.
In our structure–activity study, it was of interest to estab-
lish the importance of the 2-nitro group for angiogenesis
inhibition. Alkenes (Z)-9a, (E)-9b, (Z)-10a, and (E)-10b were
targeted for this purpose. However, attempts to perform the
condensation (Fig. 2, Method a) with 12 and either ethyl-2-
phenylacetate 19 or ethyl-2-(2-chlorophenyl)acetate 20 were
unsuccessful, thus highlighting the requirement for the ortho-
nitro group in this reaction. Switching to the stronger
base lithium-N,N-diisopropylamide (LDA) and carrying the
reaction out with ethyl-2-phenylacetate 19 in THF at ꢀ788C
(Fig. 2, Method b) afforded a 32 % yield of the trans alkene
(E)-9b whereas none of the cis alkene (Z)-9a was formed.
Applying the same procedure with 2-(2-chlorophenyl)acetate
20 yielded 41 % of (E)-10b and 5 % of the cis isomer (Z)-10a.
Cis-stereochemistry was confirmed in alkenes (Z)-3a–
(Z)-10a using 2-dimensional nuclear overhauser effect spectros-
copy (2D-NOESY) spectra, which in all cases showed NOE
crosspeaks between the vinylic proton and the ortho proton on
the phenyl ring (Fig. 3a). A second feature common to all cis
isomers was a downfield chemical shift of the pyrrole NH
signals (11.80–12.91 ppm) in their 1H NMR spectra relative to
the corresponding trans isomers (6.47–6.87 ppm, Fig. 3b).
These strongly deshielded signals suggested the presence of
intramolecular hydrogen bonds in the cis alkenes between the
pyrrole NH and carbonyl oxygen atoms.[21] Furthermore, the cis
isomers were all observed to be significantly less polar than their
corresponding trans isomers (by TLC), which, in addition to
simplifying purification by silica gel column chromatography,
supported the presence of the hydrogen bonds. The presence of
an intramolecular hydrogen bond in allyl ester (Z)-5a was
eventually confirmed (in the solid state) by X-ray crystallogra-
phy (Fig. 4a). It is noteworthy that analogous hydrogen bonds
are found in SU5416 2 and related indolin-2-one-based angio-
genesis inhibitors that contain (Z)-alkenes.[22]
The 1H NMR spectrum of (E)-3b displayed an unusual pair of
doublet-of-quartet signals at 4.10 and 4.23 ppm (total integra-
tion 2H), suggesting the presence of diastereotopic ethyl ester
CH2 protons (Supplementary Material, Fig. S1b). In contrast,
the CH2 group of (Z)-3a displayed the expected first-order
quartet at 4.12 ppm. (Supplementary Material, Fig. S1 a).
Complex methylene signals were similarly observed for the

Angiogenesis Inhibitors Related to Sunitinib (SutentÒ)
867
(a)
NOE
(b)
cis-alkenes
Pyrrole NH
chemical shift [ppm]
trans-alkenes
Pyrrole NH
Compound chemical shift [ppm]
Compound
N
H
H
H
(Z)-3a
(Z)-3a
(Z)-5a
(Z)-6a
(Z)-7a
(Z)-8a
(Z)-9a
(Z)-10a
11.89
11.82
12.91
12.21
12.00
12.21
N/A
(E)-3b
(E)-4b
(E)-5b
(E)-6b
(E)-7b
(E)-8b
(E)-9b
(E)-10b
6.80
6.74
6.75
N/A
O
H-bond
R2
R1
(Z)-3a–(Z)-10a
6.47
N/A
6.84
6.75
11.80
N/A: not available
Fig. 3. (a) cis-Stereochemistry was confirmed in (Z)-3a–(Z)-10a by the presence of a nuclear overhauser effect
(NOE) between the vinylic proton and ortho proton on their respective phenyl rings. (b) Comparison of the pyrrole NH
chemical shift values in the 1H NMR spectra of cis-alkenes (Z)-3a–(Z)-10a and trans-alkenes (E)-3b–(E)-10b. The
downfield-shifted signals in the cis series are indicative of intramolecular hydrogen bonds between the pyrrole NH and
carbonyl oxygen atoms.
C3
C5
C3
C1 C2
C4
C4
C8
C2
C5
C1
N7
C6
C6
N7
O18
C8
C9
C16
O17
C14
Hydrogen
bond
C23
O21
C9
C10
C11
C10
C15
C19
O20
C19
O20
C24
C12
N16
C15
O18
C22
C13
C11
017
O21
C22
C14
C12
C13
C23
C24
Fig. 4. ORTEP plots of allyl esters (a) (Z)-5a; and (b) (E)-5b. An intramolecular hydrogen bond was observedin (Z)-5a between the pyrroleN–H and carbonyl
˚
oxygen atoms (N7 to O20 distance ¼ 2.7 A).
allyl ester methylene CH2 of (E)-5b (2 ꢁ doublet-of-doublets at
4.59 and 4.64 ppm) and the ethyl ester CH2 group of (E)-10b
(2 ꢁ quartets at 4.18 and 4.25 ppm). Such observations are
consistent with the disubstituted phenyl rings of these com-
pounds being unable to freely rotate about the ipso Ar–C bond
owing to a steric clash between the bulky ortho phenyl-ring
substituents (i.e. ortho-NO2 in (E)-3b and (E)-5b, and ortho-Cl
in (E)-10b) and the pyrrole ring located on the same side of the
alkene double bond (Supplementary Material, Fig. S1c). The
restricted rotation and resulting axial double-bond chirality in
these ‘overcrowded’ alkenes explains the presence of diaster-
eotopic CH2 signals in the 1H NMR spectra. That the CH2 group
of (E)-9b (which contains no aryl ortho substituent) produced
a simple quartet at 4.22 ppm supports this explanation, as does
the appearance of the CH2 groups of cis-alkenes (Z)-5a, (Z)-6a,
and (Z)-10a as first-order quartets (integration 2H), and the
benzyl CH2 group of (Z)-8a as a simple doublet (integration 2H).
Single-crystal X-ray structures were obtained for the pair of
allyl esters (Z)-5a and (E)-5b (Fig. 4). The solid-state structure
of (Z)-5a provided clear evidence for an intramolecular hydro-
gen bond stabilising a pseudo-7-membered ring within its
structure. The interatomic distance between the pyrrole nitrogen
and the carbonyl oxygens atom in the crystal structure was very
˚
short (2.7 A), suggesting that the hydrogen bond in (Z)-5a is
relatively strong.[21] The X-ray structure of (E)-5b showed no
intramolecular hydrogen bonds.
Angiogenesis Inhibition
The alkenes were tested for anti-angiogenic effects using our
previously reported rat aorta in vitro model of angiogenesis

868
P. Sudta et al.
100
90
80
70
60
50
40
30
20
10
0
1 µg mLꢄ1
10 µg mLꢄ1
100 µg mLꢄ1
Fig. 5. Inhibition of angiogenesis by alkenes (Z)-3a, (E)-3bꢀ(Z)-10a, (E)-10b relative to negative control (no compound) and positive controls PI-88[24]
(100 mg mLꢀ1) and SU5416 2. Female Fischer 344 rat thoracic aortic sections were cultured in a gel fibrin matrix in 48-well plates in the presence or absence of
compounds. Cultures were fed on Day 4 and vessel outgrowths measured on Day 5.[23] Wells were visualised under 40ꢁ magnification and the percentage
of the field of view (FOV) occupied by vessel outgrowths reported as FOV occupancy. Negative control (no compound, unshaded bar) showed 85.7 % FOV
occupancy. Compounds showing little or no activity at higher concentrations were not tested at lower concentration. Data represent the mean FOV occupancy
(%) generated from at least six replicate cultures of each test compound at each concentration tested (see Supplementary Material Table S1 for statistics).
(Fig. 5).[23] In this assay, thoracic aortic sections are excised
from female Fischer 344 rats and suspended in a fibrin gel matrix
in 48-well plates. A minimum of six replicate cultures for each
compound treatment (at each concentration) are prepared. The
cultures are fed on Day 4 and vessel outgrowths from the rings
measured on Day 5. Angiogenesis is visualised under a micro-
scope (40ꢁ magnification) and quantified manually as the
percentage of the field of view (FOV) around the vessel frag-
ment occupied by new vessel outgrowths (i.e. FOV %). Under
these conditions, absence of test compound produces 85.7 %
FOV occupancy (Fig. 5; control: no compound).
The cis-ethylamide (Z)-6a showed reduced effects (FOV
occupancy 51.7 % at 100 mg mLꢀ1, inactive at 10 mg mLꢀ1
)
relative to (Z)-3a, indicating that directly substituting the ester
group for an amide is detrimental for activity. Observing that
amides (Z)-7a and (Z)-8a were inactive at 100 mg mLꢀ1 sup-
ported this conclusion. Removal of the NO2 group from the
phenyl ring was found to completely abolish activity (i.e. (E)-9b
v. (E)-3b). Interestingly, replacing the NO2 group with Cl had no
effect on activity at 100 mg mLꢀ1, with both (E)-10b and (E)-3b
completely inhibiting angiogenesis, but at 10 mg mLꢀ1 (E)-10b
showed no activity.
Two positive controls PI-88 and SU5416 2 were included in
the assays. PI-88 is a highly sulfated oligosaccharide-based
angiogenesis inhibitor[24] currently undergoing Phase III clini-
cal trials as a single treatment following cancer surgery in
subjects with hepatitis virus-related hepatocellular carcino-
ma.[25] At 100 mg mLꢀ1, PI-88 reduced FOV occupancy to
39.2 %. SU5416 2 produced an unusual response where greater
inhibition of angiogenesis was observed at 10 mg mLꢀ1 (FOV
occupancy 9.7 %) than at 100 mg mLꢀ1 (FOV occupancy
30.6 %), whereas 61.4 % FOV occupancy was observed at
1 mg mLꢀ1. Small crystals observed in wells containing 100 mg
mLꢀ1 SU5416 2 suggested that its low solubility in the medium
may have caused the poor dose dependency.
Visual inspection of the assay wells on Day 5 revealed that
outgrowths sprouting from rings cultured in the presence of
SU5416 2 (100 mg mLꢀ1) differed markedly in morphology
from those sprouting from control rings (no compound) or from
rings cultured in the presence of PI-88 (100 mg mLꢀ1) or alkenes
(e.g. (Z)-3a, 100 mg mLꢀ1) (Fig. 6). Whereas the outgrowths
from the control rings and rings grown in the presence of PI-88
or alkenes showed normal morphology, outgrowths from the
SU5416 2-treated rings consisted of fine hair-like structures that
lacked a visible lumen. At lower concentrations of SU5416 2
(10 mg mLꢀ1), the outgrowths showed normal morphology.
Concluding Remarks
Ethyl and methyl esters (Z)-3a, (E)-3b, (Z)-4a, and (E)-4b
were found to completely inhibit angiogenesis at 100 mg mLꢀ1
.
In summary, a novel series of alkenes (Z)-3a, (E)-3bꢀ(Z)-10a,
(E)-10b was synthesised to test the hypothesis that anti-
angiogenic activity can be retained in structures related to
semaxanib (SU5416) 2, a structurally simpler predecessor of the
FDA-approved drug sunitinib, when the indolin-2-one moiety is
modified through ring-opening. The 2,4-dimethylpyrrole
portion of SU5416 2 was retained in the test compounds to
strengthen conclusions regarding the importance of the indolin-
2-one group. In vitro angiogenesis inhibition assays revealed
that several of the alkene esters, including both (Z)- and
(E)-isomers, showed significant anti-angiogenic effects. Com-
pound (E)-3b emerged as the most potent, showing complete
inhibition of angiogenesis at 10 mg mLꢀ1. The work provides
At 10 mg mLꢀ1, (E)-3b continued to show complete inhibition
whereas the other three esters all showed significantly dimin-
ished effects at this concentration. At 1 mg mLꢀ1, the four esters
showed either minimal effects (,20 % difference relative to
negative control) or no activity. Surprisingly, the cis-allyl ester
(Z)-5a showed the same level of inhibition at both 100 and
10 mg mLꢀ1 (FOV occupancy 28.3 and 29.3 % respectively) but
no activity at 1 mg mLꢀ1. For the trans-allyl ester (E)-5b, strong
activity was observed at 100 mg mLꢀ1 (FOV occupancy 8.3 %)
but this activity was completely lost at 10 mg mLꢀ1. We
conclude from the data that (E)-3b is the most active of the
2-nitrophenylacetic ester series.

Angiogenesis Inhibitors Related to Sunitinib (SutentÒ)
869
(a)
(b)
(c)
Fig. 6. Morphology of vessel outgrowths from rat aortic rings cultured in the presence of: (a) control (no compound); (b) PI-88 (100 mg mLꢀ1); and
(c) SU5416 2 (100 mg mLꢀ1). The outgrowths from control rings and rings grown in the presence of PI-88 (and (Z)-3a; image not shown) appeared normal
whereas rings cultured in the presence SU5416 2 produced fine hair-like outgrowths with no lumen.
evidence that the indolin-2-one moiety is not essential for
anti-angiogenic activity in the sunitinib/SU5416-type class.
The novel condensation chemistry developed to access the
target series has scope for wider exploration. For example,
whereas the ortho-NO2 group was found to be necessary for
successful reactions with phenylacetic esters and amides, it
would be of interest to explore whether this group can be moved
to the meta or para positions on the phenyl ring and whether it
can be replaced altogether with other electron-withdrawing
groups. Additionally, it is likely that 12 could be useful as
a 3,5-dimethylpyrrole-2-carboxaldehyde surrogate in conden-
sation reactions with carbanions derived from substrates other
than phenylacetic esters and amides. It is also tempting to
speculate that pyrrole N-methyl carbamoylation might be
a more generally useful tactic in condensation reactions
with other pyrrole-2-carboxaldehydes where either enhanced
aldehyde reactivity or pyrrole N-protection is required.
Methyl 2-Formyl-3,5-dimethyl-1H-pyrrole-
1-carboxylate (12)
3,5-Dimethylpyrrole-2-carboxaldehyde 11 (1.47 g, 11.9 mmol)
in anhydrous THF (10 mL) was added dropwise over 10 min to a
stirring suspension of potassium hydride (0.57 g, 14.3 mmol) in
dry THF (50 mL) under N2 at 08C. Following complete addition,
the mixture was stirred at 08C for a further 30 min. Methyl
chloroformate (2.48 g, 26.2 mmol) diluted in anhydrous THF
(10 mL) was then added dropwise to the stirring mixture at 08C
over 10 min. The reaction was allowed to warm slowly to room
temperature and stirred for a further 2 h while monitoring by
TLC analysis (petroleum spirit/acetone, 80 : 20). After complete
consumption of starting material, the reaction was quenched
with ice-cold H2O (30 mL) and stirred for 15 min. The crude
mixture was extracted with EtOAc (3 ꢁ 25 mL) and the com-
bined organic extracts washed with H2O (25 mL) and brine
(25 mL) before drying over anhydrous MgSO4 and concentrat-
ing under vacuum. The crude product was purified by silica gel
column chromatography using a gradient from 100 : 0 to 95 : 5
petroleum spirit/acetone to provide 12 (1.83 g, 85 %) as a white
crystalline solid. Rf 0.53 (80 : 20 petroleum spirit/acetone), mp
44–458C. dH (500 MHz, CDCl3) 2.34 (s, 3H), 2.42 (s, 3H), 4.00
(s, 3H), 5.93 (s, 1H), 10.03 (s, 1H). dC (125 MHz, CDCl3) 12.9,
15.2, 54.3, 115.9, 130.3, 135.8, 138.2, 151.5, 181.2. nmax
(neat)/cmꢀ1 1730, 1650, 1496, 1451, 1326, 1153, 765. m/z
(HRMS-ESI) 182.0817 [M þ H]þ; C9H11NO3 requires
182.0812.
Experimental
Chemistry
3,5-Dimethylpyrrole-2-carboxaldehyde 11 was purchased from
Sigma–Aldrich. Semaxanib (SU5416) 2 was synthesised
in-house using a novel (unpublished) procedure. The spectro-
scopic data for the synthesised Semaxanib was identical in all
respects to that reported in the literature.[22] Anhydrous tetra-
hydrofuran (THF) was freshly distilled from sodium benzo-
phenone ketyl. Anhydrous CH2Cl2 was freshly distilled from
CaH2. All other solvents were of analytical reagent (AR) grade
and used without further purification. The term petroleum spirit
refers to that within the boiling range 40–608C. Column chro-
matography was performed using silica gel 60 (230–400 mesh,
Merck). Reaction monitoring by TLC was carried out using
Merck silica gel 60 F254 (0.2 mm) plates. Compounds were
visualised by examination under UV light or by staining with
General Method for the Synthesis of 2-Phenylacetic
Esters 13–15, 19, 20
A solution of the requisite 2-phenylacetic acid (,3 g) in the
appropriate alcohol (40 mL) with 5 drops of concentrated
sulfuric acid added was stirred at reflux under N2. On complete
disappearance of starting material (TLC; petroleum spirit/
acetone, 70 : 30), the reaction mixture was allowed to cool to
room temperature before removing the alcohol by evaporation
under vacuum. The crude residue was diluted with EtOAc
(50 mL), added to a separating funnel and washed with saturated
aqueous Na2CO3 (30 mL), H2O (3 ꢁ 25 mL), and brine (30 mL).
The organic layer was then dried over anhydrous MgSO4 and
concentrated under vacuum.
1
cerium ammonium molybdate. H and 13C NMR spectra were
recorded on a Varian Inova-500 MHz spectrometer. Chemical
shifts (d) are expressed in ppm relative to tetramethylsilane
(0 ppm). Signal splitting patterns are described as singlet (s),
broad singlet (br s), doublet (d), triplet (t), broad triplet (br t),
quartet (q), multiplet (m) or a combination of the above. IR
spectra were recorded using a Nicolet Avatar 360 Fourier-
transform (FT)-IR spectrometer with characteristic absorption
bands reported in wavenumbers (cmꢀ1). High-resolution elec-
trospray ionisation mass spectra (HRMS-ESI) were recorded
using a factory-modified Waters QToF Ultima Mass Spec-
trometer (Wythenshawe, UK). Melting points were
determined using a Reichert melting point apparatus and are
uncorrected.
Ethyl-2-(2-nitrophenyl)acetate (13)
Yield: 97 %, white crystalline solid; mp 61–638C. dH
(500 MHz, CDCl3) 1.16 (t, 3H, J 7.0), 3.93 (s, 2H), 4.07
(q, 2H, J 7.0), 7.28 (d, 1H, J 7.5), 7.38 (t, 1H, J 8.0), 7.50
(t, 1H, J 7.5), 7.99 (d, 1H, J 8.0). dC (125 MHz, CDCl3) 13.7,

870
P. Sudta et al.
39.4, 60.9, 124.9, 128.2, 129.5, 132.9, 133.2, 148.5, 170.0. m/z
(HRMS-ESI) 210.075 [M þ H]þ; C10H12NO4 requires
210.0761.
m/z (HRMS-ESI) 257.0929 [M þ H]þ; C14H13N2O3 requires
257.0921.
N-Benzyl-2-(2-nitrophenyl)acetamide (18)
Methyl-2-(2-nitrophenyl)acetate (14)
95 %, off-white amorphous solid; mp 136–1388C. dH
(500 MHz, CDCl3) 3.88 (s, 2H), 4.45 (d, 2H, J 6), 6.12 (br s,
1H), 7.26 (t, 3H, J 8.0), 7.31 (t, 2H, J 7.5), 7.45 (t, 1H, J 7.5), 7.51
(d, 1H, J 7.5), 7.60 (t, 1H, J 7.5), 8.04 (d, 1H, J 8.5). dC
(125 MHz, CDCl3) 40.9, 43.8, 125.1, 127.5, 127.6, 128.5,
128.7, 130.2, 133.5, 133.6, 137.9, 148.9, 169.0. m/z (HRMS-
ESI) 271.1065 [M þ H]þ; C15H15N2O3 requires 271.1077.
Yield: 97 %, pale yellow oil. dH (500 MHz, CDCl3) 3.68
(s, 3H), 4.00 (s, 2H), 7.34 (d, 1H, J 8.0), 7.45 (t, 1H, J 8.0), 7.57
(t, 1H, J 8.0), 8.08 (d, 1H, J 8.0). dC (125 MHz, CDCl3) 39.8,
52.5, 125.5, 128.9, 133.5, 133.6, 133.8, 148.9, 170.6. m/z
(HRMS-ESI) 196.0615 [M þ H]þ; C9H10NO4 requires
196.0604.
Allyl-2-(2-nitrophenyl)acetate (15)
General Method for Condensation Reactions to Produce
Yield: 88 %, pale yellow oil. dH (500 MHz, CDCl3) 4.04
(s, 2H), 4.60 (dd, 2H, J 1.5, 4.5), 5.21 (dd, 1H, J 1.5, 10.0), 5.28
(dd, 1H, J 1.5, 17.0), 5.89 (m, 1H), 7.35 (d, 1H, J 7.5), 7.46
(t, 1H, J 7.5), 7.58 (t, 1H, J 7.5), 8.09 (d, 1H, J 7.5). dC (125 MHz,
CDCl3) 39.8, 66.0, 118.7, 125.4, 128.8, 132.0, 133.6, 133.8,
148.5, 169.8. m/z (ESI) 222.3 [M þ H]þ. m/z (HRMS-ESI)
222.0733 [M þ H]þ; C11H12NO4 requires 222.0761.
Alkenes (Z)-3a, (E)-3b2(Z)-8a, (E)-8b: Method a
To a dry round-bottom flask was added anhydrous potassium
carbonate (1.205 g, 8.73 mmol) in dry THF (15 mL) under
N2followed by 18-crown-6 (0.575 g, 2.18 mmol). The mixture
was stirred at room temperature for 10 min before adding
a solution of the appropriate 2-(2-nitrophenyl)acetate or acet-
amide (4.36 mmol) in dry THF (10 mL) and heating at reflux for
3 h. Methyl 2-formyl-3,5-dimethyl-1H-pyrrole-1-carboxylate
12 (0.789 g, 4.36 mmol) was diluted in dry THF (8 mL) and
added dropwise over 15 min to the stirring solution. The mixture
was then heated at reflux for a further 16 h. The reaction was
quenched with water (30 mL), transferred to a separating funnel,
and extracted with EtOAc (3 ꢁ 40 mL). The combined organic
layers were washed with saturated aqueous Na2CO3
(2 ꢁ 50 mL) and brine (2 ꢁ 50 mL), dried over anhydrous
MgSO4, and concentrated under vacuum. The crude product was
purified by silica gel column chromatography using a petroleum
spirit/acetone gradient of 100 : 0 to 80 : 20 to provide mixtures of
the (Z)- and (E)-isomers.
Ethyl-2-phenylacetate (19)
Yield: 80 %, pale yellow oil. dH (500 MHz, CDCl3) 1.23
(t, 3H, J 7.0), 3.56 (s, 2H), 4.13 (q, 2H, J 7.0), 7.29 (m, 5H). dC
(125 MHz, CDCl3) 14.2, 41.4, 60.8, 128.0, 134.3, 171.6. m/z
(HRMS-ESI) 165.0914 [M þ H]þ; C10H13O2 requires
165.0910.
Ethyl-2-(2-chlorophenyl)acetate (20)
Yield: 71 %, pale yellow oil. dH (500 MHz, CDCl3) 1.20
(t, 3H, J 7.0), 3.71 (s, 2H), 4.12 (q, 2H, J 7.0), 7.23 (m, 4H). dC
(125 MHz, CDCl3) 14.0, 38.9, 60.7, 128, 132.4, 134.3, 170.5.
m/z (HRMS-ESI) 199.0526 [M þ H]þ; C10H12ClO2 requires
199.0520.
(Z)-Ethyl 3-(3,5-Dimethyl-1H-pyrrol-2-yl)-2-(2-
nitrophenyl)acrylate ((Z)-3a)
General Procedure for the Synthesis of N-Alkyl or
N-Aryl-2-(2-nitrophenyl)acetamides 16–18
42 %, red crystalline solid; mp 124–1268C. Rf 0.50 (70 : 30
hexane/acetone). dH (500 MHz, CDCl3) 1.13 (t, 3H, J 7.0), 2.17
(s, 3H), 2.35 (s, 3H), 4.12 (q, 2H, J 7.0), 5.90 (s, 1H), 6.82
(s, 1H), 7.43 (m, 1H), 7.62 (t, 2H, J 8.0), 8.02 (d, 1H, J 8.0),
11.89 (br s, 1H). dC (125 MHz, CDCl3) 13.7, 14.1, 29.9, 62.1,
111.5, 112.7, 124.1, 124.6, 127.8, 129.9, 131.5, 132.5, 133.5,
134.3, 137.5, 148.5, 163.8. nmax (neat)/cmꢀ1 3275, 2354, 1684,
1577, 1569, 1544, 1518, 1367, 1335, 1319, 1262, 1196, 1153,
1026, 830, 789, 710. m/z (HRMS-ESI) 315.1350 [M þ H]þ;
C17H19N2O4 requires 315.1339.
A dry round-bottom flask was charged under N2with 2-
nitrophenylacetic acid (324 mg, 1.79 mmol), amine (2.42 mmol),
O-(Benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluoro-
phosphate (HBTU, 709 mg, 1.87 mmol), and CH2Cl2 (10 mL)
before adding N,N-diisopropylethylamine (DIPEA, 3.85 mmol).
The solution was stirred at room temperature while monitoring
by TLC analysis (petroleum spirit/acetone, 80 : 20). On com-
plete consumption of starting material, the reaction was diluted
with CH2Cl2 (30 mL) and washed with 5 % HCl (3 ꢁ 25 mL),
saturated aqueous Na2CO3 (3 ꢁ 25 mL), and brine (30 mL).
The organic layer was dried over anhydrous MgSO4 and con-
centrated to afford the pure amide.
(E)-Ethyl 3-(3,5-Dimethyl-1H-pyrrol-2-yl)-2-(2-
nitrophenyl)acrylate ((E)-3b)
35 %, red crystalline solid; mp 117–1198C. Rf 0.26 (70 : 30
hexane /acetone). dH (500 MHz, CDCl3) 1.19 (t, 3H, J 7.0), 1.95
(s, 3H), 2.19 (s, 3H), 4.10 (dq, 1H, J 4.0, 7.0), 4.23 (dq, 1H, J 3.7,
7.0), 5.74 (s, 1H), 6.80 (br s, 1H), 7.49 (t, 1H, J 7.5), 7.59 (t, 1H,
J 7.5), 7.67 (t, 1H, J 7.5), 7.75 (s, 1H), 8.19 (d, 1H, J 7.5). dC
(125 MHz, CDCl3) 11.3, 13.2, 14.1, 60.8, 110.7, 116.6, 123.4,
125.6, 127.2, 129.2, 130.1, 132.5, 133.3, 133.4, 136.7, 149.2,
166.6. nmax (neat)/cmꢀ1 3447, 3421, 1700, 1618, 1607, 1564,
1518, 1338, 1180, 1147, 1093, 795. m/z (HRMS-ESI) 315.1337
[M þ H]þ; C17H19N2O4 requires 315.1339.
N-Ethyl-2-(2-nitrophenyl)acetamide (16)
57 %, white amorphous solid; mp 137–1398C. dH (500 MHz,
CDCl3) 1.13 (t, 3H, J 7.0), 3.29 (q, 2H, J 7.0), 3.81 (s, 2H), 5.80
(br s, 1H), 7.45 (t, 1H, J 7.5), 7.50 (d, 1H, J 7.5), 7.60 (t, 1H,
J 7.5), 8.02 (d, 1H, J 8.5). dC (125 MHz, CDCl3) 15.0, 35.0, 41.1,
125.3, 128.6, 130.7, 133.6, 133.8, 149.1, 169.0. m/z (HRMS-
ESI) 209.0924 [M þ H]þ; C10H13N2O3 requires 209.0921.
N-Phenyl-2-(2-nitrophenyl)acetamide (17)
95 %, off-white amorphous solid; mp 141–1438C. dH
(500 MHz, CDCl3) 4.03 (s, 2H), 7.08 (t, 1H, J 7.5), 7.28
(t, 2H, J 7.5), 7.47 (m, 3H), 7.54 (d, 1H, J 7.0), 7.61 (t, 1H,
J 7.5), 7.82 (br s, 1H), 8.05 (d, J 8.5). dC (125 MHz, CDCl3) 41.9,
119.9, 124.4, 125.2, 128.6, 128.9, 130.0, 133.4, 133.7, 167.2.
(Z)-Methyl 3-(3,5-Dimethyl-1H-pyrrol-2-yl)-2-
(2-nitrophenyl)acrylate ((Z)-4a)
36 %, red crystalline solid; mp 66–688C. Rf 0.47 (80 : 20
petroleum spirit/acetone). dH (500 MHz, CDCl3) 2.14 (s, 3H),

Angiogenesis Inhibitors Related to Sunitinib (SutentÒ)
871
2.32 (s, 3H), 3.61 (s, 3H), 5.88 (s, 1H), 6.79 (s, 1H), 7.40 (d, 1H,
J 7.5), 7.41 (t, 1H, J 7.5), 7.59 (t, 1H, J 7.5), 8.00 (d, 1H, J 7.5),
11.82 (br s, 1H). dC (125 MHz, CDCl3) 11.8, 13.9, 52.0, 111.6,
114.0, 124.5, 124.6, 128.0, 131.5, 134.4, 137.3, 148.5, 168.2.
nmax (neat)/cmꢀ1 3455, 1691, 1603, 1555, 1522, 1430, 1343,
1222, 1190, 1172, 1149, 789, 718. m/z (HRMS-ESI) 301.1185
[M þ H]þ. C16H17N2O4 requires 301.1183.
(Z)-3-(3,5-Dimethyl-1H-pyrrol-2-yl)-2-(2-nitrophenyl)-
N-phenylacrylamide ((Z)-7a)
22 %, red crystalline solid; mp 177–1798C. Rf 0.36 (80 : 20
petroleum spirit/acetone). dH (500 MHz, CDCl3) 2.12 (s, 3H),
2.32 (s, 3H), 5.86 (s, 1H), 6.56 (s, 1H), 6.87 (br s, 1H), 7.12
(t, 1H, J 7.5), 7.32 (t, 2H, J 7.5), 7.37 (t, 2H, J 7.5), 7.56 (t, 1H,
J 8.0), 7.60 (d, 1H, J 8.0), 7.71 (t, 1H, J 8.0), 8.03 (d, 1H, J 8.0),
12.00 (br s, 1H). dC (125 MHz, CDCl3) 11.4, 13.6, 111.1, 115.6,
121.4, 124.1, 124.9, 129.0, 129.1, 130.1, 133.5, 133.5, 133.8,
135.8, 137.4, 149.7, 166.2. nmax (neat)/cmꢀ1 3400, 1650, 1595,
1592, 1518, 1498, 1436, 1362, 1352, 1318, 1229, 1188, 1151,
1004, 802, 790, 772, 759, 739, 718. m/z (HRMS-ESI) 362.1521
[M þ H]þ; C21H20N3O3 requires 362.1499.
(E)-Methyl 3-(3,5-Dimethyl-1H-pyrrol-2-yl)-2-
(2-nitrophenyl)acrylate ((E)-4b)
40 %, red crystalline solid; mp 90–938C. Rf 0.36 (80 : 20
petroleum spirit/acetone). dH (500 MHz, CDCl3) 1.95 (s, 3H),
2.20 (s, 3H), 3.70 (s, 3H), 5.74 (s, 1H), 6.74 (br s, 1H), 7.50 (d,
1H, J 7.5), 7.61 (t, 1H, J 7.5), 7.68 (t, 1H, J 7.5), 7.75 (s, 1H),
8.20 (d, 1H, J 7.5). dC (126 MHz, CDCl3) 11.6, 13.4, 52.3, 111.0,
116.3, 123.6, 125.5, 127.6, 129.4, 130.6, 132.7, 133.4, 133.8,
134.1, 149.4, 167.4. nmax (neat)/cmꢀ1 3293, 1689, 1570, 1544,
1518, 1432, 1365, 1334, 1317, 1263, 1195, 1180, 1151, 788,
709. m/z (HRMS-ESI) 301.1182 [M þ H]þ; C16H17N2O4
requires 301.1183.
(E)-3-(3,5-Dimethyl-1H-pyrrol-2-yl)-2-(2-nitrophenyl)-
N-phenylacrylamide ((E)-7b)
,5 %, red amorphous solid (impure, not tested in angiogen-
esis inhibition assay). dH (500 MHz, CDCl3) 1.92 (s, 3H), 2.13
(s, 3H), 5.70 (s, 1H), 6.47 (br s, 1H), 7.04 (t, 1H, J 7), 7.14 (br s,
1H), 7.24 (t, 2H, J 7.5), 7.43 (d, 2H, J 8.0), 7.57 (d, 1H, J 7.5),
7.68 (s, 1H), 7.75 (t, 1H, J 7.5), 7.66 (m, 1H), 8.13 (d, 1H, J 8.0).
dC (125 MHz, CDCl3) 11.2, 13.1, 110.6, 118.9, 120.4, 123.3,
124.1, 125.1, 125.4, 128.7, 129.6, 130.3, 131.5, 133.5, 133.8,
134.3, 138.0, 149.4, 165.0. m/z (HRMS-ESI) 362.1503
[M þ H]þ; C21H20N3O3 requires 362.1499.
(Z)-Allyl 3-(3,5-Dimethyl-1H-pyrrol-2-yl)-2-
(2-nitrophenyl)acrylate ((Z)-5a)
41 %, deep-red crystalline solid; mp 70–728C. Rf 0.50
(80 : 20 petroleum spirit/acetone). dH (500 MHz, CDCl3) 2.17
(s, 3H), 2.35 (s, 3H), 4.56 (d, 2H, J 5.5), 5.10 (dd, 1H, J 1.5,
13.0), 5.13 (dd, 1H, J 1.5, 6.5), 5.79 (m, 1H), 5.90 (s, 1H), 6.82
(s, 1H), 7.43 (d, 1H, J 7.5), 7.44 (t, 1H, J 7.5), 7.62 (t, 1H, J 7.5),
8.04 (d, 1H, J 7.5), 12.91 (br s, 1H). dC (125 MHz, CDCl3) 11.7,
13.8, 65.7, 111.6, 114.2, 118.1, 124.6, 124.7, 127.9, 131.5,
131.6, 132.9, 133.5, 134.4, 137.4, 148.9, 167.3. nmax
(neat)/cmꢀ1 3300, 2360, 1685, 1577, 1570, 1542, 1517, 1507,
1364, 1312, 1182, 1144, 966, 932, 858, 790. m/z (HRMS-ESI)
327.1342 [M þ H]þ; C18H19N2O4 requires 327.1339.
(Z)-N-Benzyl-3-(3,5-dimethyl-1H-pyrrol-2-yl)-2-
(2-nitrophenyl)acrylamide ((Z)-8a)
16 %, red crystalline solid; mp 140–1428C. Rf 0.36 (80 : 20
petroleum spirit/acetone). dH (500 MHz, CDCl3) 2.09 (s, 3H),
2.31 (s, 3H), 4.50 (d, 2H, J 6), 5.50 (br s, 1H), 5.83 (s, 1H), 6.48
(s, 1H), 7.20 (d, 2H, J 8.0), 7.22 (m, 1H, J 7.0), 7.28 (t, 2H, J 7.5),
7.47 (m, 1H, J 8.0), 7.49 (m, 1H, J 6.5), 7.61 (t, 1H, J 7.5), 7.94
(d, 1H, J 8.5), 12.21 (br s, 1H). dC (125 MHz, CDCl3) 11.3, 13.7,
43.9, 110.7, 115.8, 124.0, 124.7, 127.3, 127.3, 128.3, 128.6,
128.9, 129.2, 133.1, 133.3, 133.4, 136.0, 138.0, 167.6. nmax
(neat)/cmꢀ1 3435, 1643, 1570, 1543, 1507, 1453, 1362, 1341,
1316, 1237, 1217, 1150, 786, 728, 709. m/z (HRMS-ESI)
376.1668 [M þ H]þ; C22H22N3O3 requires 376.1656.
(E)-Allyl 3-(3,5-Dimethyl-1H-pyrrol-2-yl)-2-
(2-nitrophenyl)acrylate ((E)-5b)
33 %, red crystalline solid; mp 102–1058C. Rf 0.40 (80 : 20
petroleum spirit/acetone). dH (500 MHz, CDCl3) 1.95 (s, 3H),
2.19 (s, 3H), 4.59 (dd, 1H, J 5.5, 13.5), 4.64 (dd, 1H, J 5.5, 13.5),
5.15 (dd, 1H, J 1.5, 10.0), 5.18 (dd, 1H, J 1.5, 18.5), 5.74 (s, 1H),
5.85 (m, 1H), 6.75 (br s, 1H), 7.50 (d, 1H, J 8.0), 7.60 (t, 1H,
J 8.0), 7.68 (t, 1H, J 8.0), 7.77 (s, 1H), 8.19 (d, 1H, J 8.0). dC
(125 MHz, CDCl3) 11.5, 13.4, 65.6, 111.0, 116.3, 117.7, 123.6,
125.4, 127.6, 129.5, 130.6, 132.5, 133.3, 133.7, 134.1, 149.4,
166.5. nmax (neat)/cmꢀ1 3455, 1687, 1610, 1557, 1524, 1343,
1257, 1232, 1068, 869, 733. m/z (HRMS-ESI) 327.1333
[M þ H]þ; C17H19N2O4 requires 327.1339.
(E)-N-Benzyl-3-(3,5-dimethyl-1H-pyrrol-2-yl)-2-
(2-nitrophenyl)acrylamide ((E)-8b)
,5 %, red amorphous solid (impure, not tested in angiogen-
esis inhibition assay). dH (500 MHz, CDCl3) 2.09 (s, 3H), 2.31
(s, 3H), 4.50 (d, 2H, J 6), 5.50 (br s, 1H), 5.83 (s, 1H), 6.48 (s,
1H), 7.20 (d, 2H, J 8.0), 7.22 (m, 1H, J 7.0), 7.28 (t, 2H, J 7.5),
7.47 (m, 1H, J 8.0), 7.49 (m, 1H, J 6.5), 7.61 (t, 1H, J 7.5), 7.94
(d, 1H, J 8.5). Note: owing to impurities, the chemical shift of
the pyrrolic NH signal could not be assigned unambiguously. dC
(125 MHz, CDCl3) 11.3, 13.6, 43.9, 110.7, 115.8, 124.0, 124.7,
127.3, 127.3, 128.4, 128.6, 128.9, 129.3, 133.088, 133.3, 133.4,
136.0, 137.974, 167.6. m/z (HRMS-ESI) 376.1675 [M þ H]þ;
C22H22N3O3 requires 376.1656.
(Z)-3-(3,5-Dimethyl-1H-pyrrol-2-yl)-N-ethyl-2-
(2-nitrophenyl)acrylamide ((Z)-6a)
21 %, red crystalline solid; mp 96–998C. Rf 0.17 (80 : 20
petroleum spirit/acetone). dH (500 MHz, CDCl3) 1.07 (t, 3H,
J 7.0), 2.09 (s, 3H), 2.31 (s, 3H), 3.31 (q, 2H, J 7.0), 5.18 (br s,
1H), 5.82 (s, 1H), 6.46 (s, 1H), 7.52 (m, 2H), 7.65 (t, 1H, J 8.0),
7.98 (d, 1H, J 8.0), 12.21 (br s, 1H). dC (125 MHz, CDCl3) 11.3,
13.5, 14.6, 34.8, 110.6, 116.4, 124.0, 124.7, 128.0, 128.7, 128.8,
132.9, 133.3, 133.4, 136.3, 149.5, 167.5. nmax (neat)/cmꢀ1 3408,
1639, 1576, 1569, 1517, 1457, 1350, 1224. m/z (HRMS-ESI)
314.1497 [M þ H]þ; C17H20N3O3 requires 314.1499.
General Method for Condensation Reactions to Produce
Alkenes (Z)-9a, (E)-9b and (Z)-10a, (E)-10b: Method b
Dry N,N-diisopropylamine (4.60 mmol) was dissolved in dry
THF (7 mL) under Ar in a three-neck 100-mL round-
bottom flask. The flask was cooled to ꢀ788C and n-butyllithium
(4.60 mmol) was added and stirred for 20 min. Ethyl-2-pheny-
lacetate 19 or ethyl-2-(2-chlorophenyl)acetate 20 (4.28 mmol)

872
P. Sudta et al.
˚
˚
was then added dropwise as a solution in dry THF (1 mL) and the
reaction mixture was stirred for a further 30 min. Methyl 2-
formyl-3,5-dimethyl-1H-pyrrole-1-carboxylate 12 (0.757 g,
4.18 mmol) was subsequently added dropwise as a solution in
dry THF (1 mL) and the reaction stirred for a further 30 min,
with monitoring by TLC (75 : 25 petroleum spirit/EtOAc). The
reaction was quenched with saturated aqueous NH4Cl (30 mL)
and the solvent removed under vacuum. EtOAc (30 mL) was
added to the residue and the organic layer was washed with
saturated aqueous NH4Cl (3 ꢁ 25 mL), saturated aqueous
Na2CO3 (2 ꢁ 25 mL), and brine (30 mL), before being dried
over anhydrous MgSO4 and concentrated under vacuum. The
crude product was purified by silica gel chromatography column
with a gradient from 100 : 0 to 70 : 30 petroleum spirit/EtOAc to
provide mixtures of the (Z)- and (E)-isomers.
b ¼ 10.2147(4) A, c ¼ 11.0598(6) A; a ¼ 72.485(3)8, b ¼
3
˚
76.605(2)8, g ¼ 71.312(3)8; V ¼ 850.89(7) A ; Dx ¼ 1.274 g
cmꢀ3; 7615 reflections measured (2y ¼ 4–54.28) merged to
3674 unique data R ¼ 0.064 (for 2849 data with I . 2s(I)),
Rw ¼ 0.187 (all data); S ¼ 0.99.
Structure Determination
Images were measured on Nonius Kappa CCD diffract-
ometers (Mo Ka radiation, graphite monochromator,
l
˚
0.71073 A) and data were extracted using the DENZO pack-
age.[26] For (Z)-5a, structure solution was by direct methods
(SIR92)[27] and the structure was refined using the CRYSTALS
program package.[28] For (E)-5b, structure solution was by direct
methods (SIR97)[29] and the structure was refined using the
SHELXL-97 program package.[30] Atomic coordinates, bond
lengths and angles, and displacement parameters have been
deposited atthe Cambridge Crystallographic DataCentre(CCDC
accession numbers 905343 and 905344 respectively). These data
can be obtained free of charge via www.ccdc.cam.ac.uk/data_
requerst/cif, by emailing data_request@ccdc.cam.ac.uk, or by
contacting The Cambridge Crystallographic Data Centre, 12
Union Road, Cambridge CB2 1EZ, UK; fax: þ44 1223 336033.
(E)-Ethyl 3-(3,5-Dimethyl-1H-pyrrol-2-yl)-
2-phenylacrylate ((E)-9b)
32 %, yellow crystalline solid; mp 62–648C. Rf 0.43 (75 : 25
petroleum spirit/EtOAc). dH (500 MHz, CDCl3) 1.26 (t, 3H,
J 7.0), 1.91 (s, 3H), 2.19 (s, 3H), 4.22 (q, 2H, J 7.0), 5.69 (s, 1H),
6.84 (br s, 1H), 7.32 (d, 2H, J 7.0), 7.43 (t, 1H, J 7.0), 7.46 (t, 2H,
J 7.0), 7.75 (s, 1H). dC (125 MHz, CDCl3) 11.2, 13.1, 14.4, 60.5,
110.0, 120.5, 124.1, 127.4, 127.9, 128.8, 129.0, 130.2, 132.9,
137.3, 169.2. nmax (neat)/cmꢀ1 3442, 1697, 1607, 1560, 1497,
1227. m/z (HRMS-ESI) 270.1488 [M þ H]þ; C17H20NO2
requires 270.1489.
Angiogenesis Inhibition Assay
Compounds were tested for angiogenesis inhibition in 48-well
culture plates using the previously reported in vitro assay with
some modifications.[23] Briefly, the thoracic aortas from female
Fischer 344 rats were excised and the 2.5-cm-long vessels
placed in Hank’s buffered salt solution (HBSS) media con-
taining 2.5 mg mLꢀ1 amphotericin B and cross-sectioned at
1-mm intervals. In the angiogenesis assay, 15 mL of bovine
thrombin (50 NIH unit mLꢀ1 in 0.15 M NaCl) was added to each
well, followed by 0.5 mL per well of 3 mg mLꢀ1 bovine
fibrinogen in Medium 199. The thrombin and fibrinogen were
mixed rapidly and one vessel section was quickly placed in
the centre of the well before clot formation. Fibrin gel formation
usually occurred within 0.5 min, leaving the vessel fragment
suspended in the gel. On gel formation, 0.5 mL per well of
Medium 199 supplemented with 20 % foetal calf serum (FCS),
0.1 % 6-aminohexanoic acid, L-glutamine, and antibiotics
(gentamicin sulfate and amphotericin B), and test compound
were added. Six replicate cultures were examined for each
concentration of test compound. Vessels were cultured at 378C
in 5 % CO2 in a humidified environment for 7 days, with the
medium being changed on Day 4. On Day 5, the percentage of
the field of view occupied by vessel outgrowths (FOV occu-
pancy %) was used as a quantitative measure of angiogenesis
(inhibition) relative to control (no compound added).
(Z)-Ethyl 3-(3,5-Dimethyl-1H-pyrrol-2-yl)-
2-(2-chlorophenyl)acrylate ((Z)-10a)
5 %, yellow crystalline solid; mp 124–1268C. Rf 0.71 (70 : 30
petroleum spirit/EtOAc). dH (500 MHz, CDCl3) 1.20 (t, 3H,
J 7.0), 2.14 (s, 3H) 2.33 (s, 3H), 4.20 (q, 2H, J 7.0), 5.87 (s, 1H),
6.72 (s, 1H), 7.25 (m, 2H), 7.31 (d, 1H, J 7.0), 7.38 (d, 1H, J 7.0),
11.80 (br s, 1H). dC (125 MHz, CDCl3) 11.6, 13.8, 14.3, 61.0,
111.3, 116.1, 124.4, 126.9, 128.3, 129.2, 130.6, 131.8, 132.2,
133.6, 135.1, 141.2, 168.9. nmax (neat)/cmꢀ1 3273, 1680, 1576,
1545, 1465, 1188, 736. m/z (HRMS-ESI) 304.1099 [M þ H]þ;
C17H19NO2Cl requires 304.1099.
(E)-Ethyl 3-(3,5-Dimethyl-1H-pyrrol-2-yl)-
2-(2-chlorophenyl)acrylate ((E)-10b)
41 %, yellow amorphous solid; mp 70–748C. Rf 0.39 (75 : 25
petroleum spirit/EtOAc). dH (CDCl3, 500 MHz) 1.24 (t, 3H,
J 7.0), 1.93 (s, 3H), 2.20 (s, 3H), 4.18 (q, 1H, J 7.0), 4.25 (q, 2H,
J 7.0), 5.74 (s, 1H,), 6.75 (br s, 1H), 7.3 (m, 3H), 7.54 (d, 1H, J 8),
7.78 (s, 1H). dC (125 MHz, CDCl3) 11.2, 13.1, 14.3, 60.6, 110.3,
117.7, 123.9, 127.3, 128.1, 129.5, 129.5, 130.1, 131.9, 133.5,
135.1, 136.3, 167.4. nmax (neat)/cmꢀ1 3447, 1697, 1603, 1558,
1473, 1226, 745. m/z (HRMS-ESI) 304.1092 [M þ H]þ;
C17H19NO2Cl requires 304.1099.
Supplementary Material
Supplementary material consisting of 1H NMR spectra for
(Z)-3a and (E)-3b and angiogenesis inhibition assay statistics is
available on the Journal’s website.
X-Ray Crystallography
Crystal Data
Acknowledgements
Compound (Z)-5a. C18H18N2O4, M ¼ 326.35, T ¼ 200 K;
P. Sudta and S. Suksamrarn acknowledge financial support from the
Thailand Research Fund through the Royal Golden Jubilee PhD Program.
S. Prapbai and P. Kongsaeree thank the National Research University
Project through Mahidol University for financial support. M. Kelso
acknowledges partial financial support from the University of Wollongong
University Research Council (URC) Small Grants Scheme. A. Bezos and
C. R. Parish acknowledge support from an Australian National Health and
Medical Research Council (NHMRC) Program Grant.
˚
monoclinic, space group P21/c, Z ¼ 4; a ¼ 12.5669(4) A, b ¼
˚
˚
7.9086(3) A, c ¼ 17.6200(4) A; b ¼ 103.730(2)8 V ¼ 1701.15
3
(6) A ; Dx ¼ 1.274 g cmꢀ3; 32664 reflections measured (2y ¼
˚
5–558) merged to 3903 unique data; R ¼ 0.053 (for 2656 data
with I . 2s(I)), Rw ¼ 0.147 (all data); S ¼ 0.95.
Compound (E)-5b. C18H18N2O4, M ¼ 326.35, T ¼ 298(2) K;
˚
triclinic, space group P-1, Z ¼ 2; a ¼ 8.4326(4) A,

Angiogenesis Inhibitors Related to Sunitinib (SutentÒ)
873
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