Synthesis, structure-activity relationship and crystallographic studies of 3-substituted indolin-2-one RET inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.01.011

The synthesis, structure-activity relationships (SAR) and structural data of a series of indolin-2-one inhibitors of RET tyrosine kinase are described. These compounds were designed to explore the available space around the indolinone scaffold within RET active site. Several substitutions at different positions were tested and biochemical data were used to draw a molecular model of steric and electrostatic interactions, which can be applied to design more potent and selective RET inhibitors. The crystal structures of RET kinase domain in complex with three inhibitors were solved. All three compounds bound in the ATP pocket and formed two hydrogen bonds with the kinase hinge region. Crystallographic analysis confirmed predictions from molecular modelling and helped refine SAR results. These data provide important information for the development of indolinone inhibitors for the treatment of RET-driven cancers.

Full text of DOI:10.1016/j.bmc.2010.01.011

Bioorganic & Medicinal Chemistry 18 (2010) 1482–1496
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, structure–activity relationship and crystallographic studies
of 3-substituted indolin-2-one RET inhibitors
a
b
c
c
Luca Mologni a, , Roberta Rostagno , Stefania Brussolo , Phillip P. Knowles , Svend Kjaer ,
Judith Murray-Rust c, Enrico Rosso b,d, Alfonso Zambon b, , Leonardo Scapozza d,
Neil Q. McDonald c, Vittorio Lucchini b, Carlo Gambacorti-Passerini a
*
a Department of Clinical Medicine and Prevention, University of Milano-Bicocca, via Cadore 48, 20052 Monza, Italy
b Department of Environmental Sciences, University of Ca’ Foscari, Venezia, Italy
c Structural Biology Laboratory, Cancer Research UK, London Research Institute, London, UK
d School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis, structure–activity relationships (SAR) and structural data of a series of indolin-2-one
inhibitors of RET tyrosine kinase are described. These compounds were designed to explore the available
space around the indolinone scaffold within RET active site. Several substitutions at different positions
were tested and biochemical data were used to draw a molecular model of steric and electrostatic inter-
actions, which can be applied to design more potent and selective RET inhibitors. The crystal structures of
RET kinase domain in complex with three inhibitors were solved. All three compounds bound in the ATP
pocket and formed two hydrogen bonds with the kinase hinge region. Crystallographic analysis con-
firmed predictions from molecular modelling and helped refine SAR results. These data provide impor-
tant information for the development of indolinone inhibitors for the treatment of RET-driven cancers.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 28 September 2009
Revised 5 January 2010
Accepted 6 January 2010
Available online 11 January 2010
Keywords:
RET
Kinase inhibitor
Crystal structure
QSAR
1. Introduction
leading to constitutive ligand-independent receptor dimerization,
or in the intracellular tyrosine kinase domain, causing constitutive
RET (REarranged during Transfection) is a transmembrane tyro-
sine kinase receptor essential for development of the sympathetic,
parasympathetic and enteric nervous systems and of the kidney.1
Its extracellular portion is composed of four cadherin-like domains,
a calcium binding site and a cysteine-rich domain; the intracellular
portion is a typical tyrosine kinase (TK) domain. RET is a compo-
nent of a signalling system for GDNF-family ligands, and its tyro-
sine kinase function is thought to be activated by trans-
phosphorylation events which follow ligand-induced receptor
dimerization. Under physiological conditions, the formation of a
activation of the enzyme, independently of its dimerization.1 Pap-
illary Thyroid Carcinoma (PTC), representing 80–90% of thyroid
carcinomas, is characterized by a genomic rearrangement (a chro-
mosomal inversion or translocation) in which the sequence of RET
gene encoding for the kinase domain fuses with portions of other
genes generating RET/PTC oncogenes. In such oncogenes, the cata-
lytic domain of RET is normally fused to the N-terminal region of
proteins which homodimerize in a ligand-independent manner,
so that the resulting oncogenic fusion protein is activated by con-
stitutive dimerization.1,2 The importance of the biological pro-
cesses in which RET is involved and the large variety of tumors
caused by its deregulation make RET a prime target for therapeutic
intervention.
Small-molecule inhibitors of oncogenic protein kinases repre-
sent an important strategy for anticancer therapy. These molecules
usually compete with ATP for binding in the enzyme active site and
prevent phosphate transfer from ATP to the appropriate residue
(Tyr, Ser, Thr), thus blocking auto- and substrate phosphorylation
and therefore signal transduction and oncogenic activity. Small
molecules, belonging to several chemical classes have been shown
to inhibit the oncogenic activity of RET:1 two pyrazolo-pyrimidine
derivatives, PP1 and PP2; the indolocarbazoles CEP-701 and CEP-
751; SU11248 and RPI-1, two 2-indolinone derivatives; and
complex with the ligand and a co-receptor (GFRa1–4) is needed
to trigger RET kinase activity. When deregulated, RET becomes a
potent oncogene. Oncogenic conversion can occur through either
genomic rearrangements or germline point mutations. Depending
on the type of mutations, RET induces different types of tumor.
Germline point mutations cause three hereditary cancer syn-
dromes: Multiple Endocrine Neoplasia type 2A (MEN2A), MEN2B,
and Familial Medullary Thyroid Carcinoma (FMTC). Point muta-
tions are localized either in the extracellular cysteine-rich domain,
* Corresponding author.
E-mail address: luca.mologni@unimib.it (L. Mologni).
Present address: The Institute of Cancer Research, London, UK.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.011

L. Mologni et al. / Bioorg. Med. Chem. 18 (2010) 1482–1496
1483
indolin-2-ones 8 and 9, respectively substituted with a methoxy
group at 5- and 7-position, were obtained from (3-methoxy-phe-
nyl)-acetonitrile by electrophilic nitration and subsequent hydro-
lysis of the nitrile group and reduction of the corresponding
carboxylic acids (Scheme 2).11–13 Indolin-2-ones 3 and 8–12 were
converted to 3-substituted indolin-2-ones 13–25 by Knoevenagel
aldolic condensation, employing available aldehydes in the pres-
ence of piperidine as the base catalyst (Scheme 3).8
O
R
R
5
5
i
3:
R5= OCF3
O
O
10:
11:
12:
R5 = CH3;
R5 = Br
N
H
N
H
R5 = NO2
3,10,11,12
Scheme 1. Preparation of 5-substituted-indolin-2-ones from 5-substituted isatins.
Reagents and conditions: (i) NH2NH2H2O, 140 °C, 4 h, ice water, pH 2, rt, 2 days.
The condensed compounds 18 and 19, with a reactive bromine
at position 5, could be further derivatized by Suzuki cross-coupling
with a series of boronic acids or esters (Scheme 4).14 Compound 25
with a reactive bromine on furan ring was further derivatized, by
Suzuki cross-coupling, with furan-3-ylboronic acid and thiophen-
3-ylboronic acid to obtain compounds 30 and 31 (Scheme 5).
The 5-nitro-3-substituted-indolin-2-ones (20, 21, 23–25 and
30) were converted to the corresponding 5-amino-derivatives
ZD6474, a 4-anilinoquinazoline. Among these inhibitors, ZD6474
(Vandetanib, Zactima™) is the most promising and is undergoing
phase II clinical trials.3
We previously described nanomolar anti-RET activity of five
small molecules belonging to different classes of kinase inhibitors:
PD173955 (a pyrido-[2,3-d]pyrimidine), AG1478 (a quinazoline)
and three compounds belonging to the 2-indolinone scaffold.4,5
In particular, we studied more in depth the activity of the indoli-
none compound SU5416, which potently inhibited RET in vitro
and in vivo.5 These results suggested that quinazolines and indoli-
nones could represent potential lead structures of RET inhibitors.
Recently, we solved the crystal structure of the RET-ZD6474 com-
plex;6 as expected, the planar quinazoline structure binds in the
ATP binding site and occupies the position of the adenine of ATP.
Similarly, 2-indolinone derivatives have been shown to bind in
the ATP site of FGFR.7 Sun et al.8 studied the structure–activity
relationship (SAR) of a large panel of 3-substituted indolin-2-one
compounds against various tyrosine kinases, showing the impor-
tance of substituents around the 2-indolinone core for potency
and specificity. Rizzi et al.9 reported the activity of a small series
of 2-indolinone compounds on RET kinase. According to their find-
ings, 5,6-dimethoxyindolinone derivatives should be favoured as
RET inhibitors.
Although the 2-indolinone structure appears as a promising
scaffold for the design of potent RET inhibitors, an accurate and
extensive structure–activity relationship analysis has not been at-
tempted thus far. In the present work, we designed and studied a
set of novel 3-substituted indolin-2-one derivatives as potential
RET inhibitors. In addition, nine commercially available com-
pounds were included in our analysis. The inhibitors were tested
to determine biochemical activity against RET, ALK, ABL and FLT3
kinases. Cellular activity was studied in RET-transformed and in
non-transformed cells. Using biochemical data, the compounds
were subjected to QSAR analysis. Finally, the crystal structures of
three RET-ligand complexes were solved.
(32–37) by
a modification of Bellamy’s reduction procedure
(Scheme 6).15 Finally, the amino group of 5-amino-3-(3,5-dime-
tyl-1H-pyrrol-2-ylmethylene)-indolin-2-one 33 was converted
into amide 38 by peptidic coupling16 with 4-((4-methylpipera-
zin-1-yl)methyl)benzoic acid using HBTU as the coupling agent
(Scheme 7). All tested compounds are reported in Table 1.
All compounds possess the 3-exocyclic double bond and may be
in principle present in the E (the substituent points into the direc-
tion of the aromatic ring) or the Z configuration (into the direction
of carboxylic oxygen). The two isomers were sometimes both de-
tected into the crude product of the Knoevenagel reaction, and
could be separated by flash chromatography and characterized
by NMR NOESY spectroscopy. Compounds with a pyrrolic (e.g.,
compound 33) or indolic (22) ring at position 3 revealed dipolar
interactions between the H4 proton of the oxindolic ring and the
vinyl proton (as described by Sun et al.8) indicating a Z configura-
tion. A second diagnostic feature is the intramolecular H-bond be-
tween pyrrol aminic hydrogen and the oxindole carboxylic oxygen.
These features were utilized for the assignment of the Z configura-
tion to all pyrrolic compounds. In compounds with the furanic ring,
NOESY spectra revealed dipolar interaction of the H4 proton of the
aromatic ring with protons of the primary furane ring (compounds
25 and 36) or with protons of the secondary furane or thiophene
ring (compounds 30, 31 and 37). These interactions assign to these
compounds the E configuration. This configurational preference is
due to the electronic repulsion, in configuration Z, between the fur-
anic and carboxylic oxygens.8 Compounds with the p-anisole ring
(23 and 34) or the p-N,N-diethylaniline ring (24 and 35) were pres-
ent as both E and Z isomers. Analysis of the interconversion pro-
cesses showed that the equilibrium constants are nearly unitary,
suggesting a minimal energy difference between the isomers (Sup-
plementary Table S1). In the case of the nitroderivatives 23 and 24,
the interconversion is very fast and the distribution between Z and
E isomers cannot be controlled. Therefore, these substances were
excluded from biochemical assays. Interconversions of the amin-
oderivatives 34 and 35 are 3 orders of magnitude slower (Supple-
mentary Table S1). Thus, the Z and E isomers of 34 were isolated
2. Chemistry
5-Substituted indolin-2-one compounds 3 and 10–12 were ob-
tained by reduction of the corresponding 5-substituted isatins
(indoline-2,3-dione) with hydrazine hydrate (Scheme 1).8 For the
synthesis of compound 3, the isatin was prepared from 4-trifluoro-
methoxy-phenylamine via Sandmeyer reaction (not shown).10 The
O
R
R
5
R
O
5
5
N
N
O
i
ii
OH
NO
iii
N
H
NO
2
2
R
R
7
R
7
7
6, 7
R5= OCH3 R7 = H
R5= H R7= OCH3
4, 5
8, 9
4,6,8:
5,7,9:
Scheme 2. Nitration of 2-(3-methoxyphenyl)acetonitrile and hydrolysis of nitrile group. Reagents and conditions: (i) CH3COOH, acetic anhydride, HNO3, 0 °C, 1 h; (ii)
HClconcd, reflux, 3 h; (iii) Pd/C 5%, H2, MeOH, 2 h rt.

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L. Mologni et al. / Bioorg. Med. Chem. 18 (2010) 1482–1496
R
3
R
5
R CHO
R
3
5
O
O
N
H
i
N
H
R
7
R
7
3,8,9,10,11,12
13,14,15,16,17,18,19,20,21,22,23,24,25
20:
21:
R5 = NO2 R3=
R5= NO2 R3=
13:
R5= OCF3 R3=
R5= OCH3 R3=
3:
8:
R5= OCF3
R5 = OCH3
R7 = OCH3
R5 = CH3
R5 = Br
N
N
14:
15:
N
N
N
9:
R7 = OCH3 R3=
22:
23:
R5= NO2 R3=
10:
11:
N
OCH3
NEt2
R3=
16:
17:
R5= NO2
R5 = CH3 R3=
R5= CH3 R3=
N
N
12
: R5 = NO2
24:
25:
R5= NO2 R3=
R5= NO2 R3=
18:
19:
Br
R5= Br
R5= Br
R3=
N
O
R3=
N
Scheme 3. Condensation of 5-substituted indolin-2-ones. R5 and R7 = H unless specified. Reagents and condition: (i) piperidine, EtOH, reflux 4 h.
and subjected as such to the in vitro test. Separation of the isomers
of the aminoderivative 35 was unsuccessful and the compound
was tested as a mixture (Table 1).
concentration causing half-maximal inhibition of RET kinase activ-
ity. As shown in Table 1, most compounds having a pyrrol ring-
based substituent in position 3 were good RET inhibitors, with
IC50 values in the nanomolar to low micromolar range. Among
them, inhibitors with hydrophobic (methyl/methoxy) or bulky
groups in position 5 (such as 16, 17, 26, 27) tended to be less active.
Interestingly, dimethyl-pyrrol almost invariably conferred higher
potency than pyrrol alone (compare 17 vs 16 and SU5416 vs 45).
It is difficult to tell whether the difference between 28 and 26 lies
in the pyrrol ring or in the different position of the oxygen atom
within the furan ring at position 5. Inhibitor SU9516, with an imid-
azole ring, showed comparable anti-RET activity. Notably, com-
pounds SU5416 and SU11248 were previously shown to inhibit
RET kinase in cells and in mice.5,17 Compound 45 showed similar,
but less specific, activity in RET-positive cells (Table 2).
3. Results and discussion
3.1. Kinase inhibition
In order to determine inhibitory activity against RET kinase, the
compounds were tested on purified recombinant RET kinase do-
main as described.4 Non-linear regression of dose–response exper-
iments allowed calculation of IC50 value, defined as the inhibitor
Furan-substituted compounds were much less active compared
to pyrrol-substituted-indolin-2-ones. One reason for their poor
inhibition may be related to the predominant E configuration of
the double bond in position 3, while most of pyrrol-substituted
compounds are in the Z form, as described above. However, it can-
not be excluded that furan may be disadvantageous in terms of
interactions with the surrounding environment, compared to a
pyrrol, independently of the configuration.
O
R
R
9
9
B
R
5
O
or
R
R
N
H
N
H
9
9
Br
R
5
R B(OH)
5
2
O
O
N
H
N
H
i
18,19
26,27,28,29
18:
R9= H
26:
27:
R5=
O
; R9= H
; R9= H
19:
R9= CH3
OH
Br
B
HO
N
Y
R5=
O
N
O
O N
2
Y
O N
2
O
28:
29:
R5=
O
; R9= CH3
; R9= CH3
O
i
N
H
N
H
25
30:
31:
Y= O
Y= S
R5=
N
Scheme 4. Suzuki cross-coupling of 5-Br-substituted-indolin-2-one. Reagents and
conditions: (i) boronic acid/ester, Na2CO3(satd), Pd(PPh3)4, dioxane, 110 °C, 12 h.
Scheme 5. Suzuki cross-coupling on 5-bromofuran group. Reagents and condi-
tions: (i) 3-boronic acid, Na2CO3(satd), Pd(PPh3)4, dioxane, 110 °C, 12 h.

L. Mologni et al. / Bioorg. Med. Chem. 18 (2010) 1482–1496
1485
that are able to block VEGFR activity in addition to the primary tu-
mor target have gained popularity in recent years, due to the pos-
sibility of affecting tumor angiogenesis as well as tumor growth.19
Compounds 33 and SU11248 showed good cellular selectivity, de-
spite lack of RET specificity versus FLT3 in vitro. SU5416, while
showing excellent in vitro activity, was much less potent in cells,
possibly due to poor solubility in cell culture medium.
R
3
R
3
O N
2
H N
2
O
O
i
N
H
N
H
20,21,23,24,25,30
32,33,34,35,36,37
21,33:
R3=
20,32:
R3=
R3=
3.2. Quantitative structure–activity relationship (QSAR)
N
O
N
23,34:
25,36:
NEt2
O
QSAR analysis for RET inhibition by the indolin-2-one com-
pounds was performed in order to correlate the biochemical data
with compounds structures, and to identify positive and negative
structural features within this series of compounds. The analysis
was run by SYBYL v7.3 with standard parameters,20 using the Com-
parative Molecular Field Analysis (CoMFA) method. Only steric and
electrostatic properties of molecules were considered. Due to the
limited number of ligands, it was not possible to separate the data
into a training set and a test set. Because the indolin-2-one deriv-
atives can exist as alternative isomers, it was important to estab-
lish which configuration should be used for alignment. Based on
previous crystallographic studies of indolinone compounds7,21
and our experimental data with compound 34, it is likely that
the Z isomer is the active form. Therefore, CoMFA analysis was
run with all compounds in Z configuration. The activity data input
were represented by Log (1/IC50). Two alignment methods (data-
base and pharmacophoric) were carried out. The best results were
obtained using the database alignment and the corresponding data
are summarized in Table 3. A remarkable difference between cross-
validated q2 and non cross-validated r2 correlation coefficients was
evident, indicating that the data were over-fitted, as discussed by
Leach.22 In order to overcome the over-fitting problem, an analysis
of residual plot (predicted vs actual pIC50) was run to identify out-
liers, that is, the compounds lying especially far from the diago-
nal.20 Six compounds whose activity was badly predicted were
eliminated (28, 41, 43, 15, SU6656, VEGFR2KI-II) and CoMFA
was repeated, yielding more consistent results (Table 3).
OCH3
24,35:
30,37:
R3=
R3=
Br
R3=
O
Scheme 6. Preparation of 5-amino-substituted-indolin-2-one. Reagents and con-
ditions: (i) SnCl2ꢀ2H2O, 1% HCl(concd), EtOH, 150 °C, 4 h.
Compounds with a phenyl ring system as the 3-substituent
showed a heterogeneous behaviour. For example, the difference
between MAZ51 and 41 is striking. Again, stereochemistry may ex-
plain this apparent paradox: MAZ51 exists predominantly as the E
isomer on the double bond of the 3-group, because the naphthyl
substituent is quite large and there is repulsion with the 2-car-
bonyl group. However, the presence of a methyl group in position
4 of 41 forces the naphthyl moiety to adopt the Z configuration,
which is the more active of the two. This 4-methyl effect was not
observed for 42 and SU4984. In this case, the substituent group
in position 3 is less rigid compared to MAZ51/41 and may adopt
a favourable conformation more easily. Interestingly, compound
34, which is always synthesized as a mixture of isomers, co-crys-
tallized in the ATP pocket of RET in the Z form (see below), demon-
strating that this is the active configuration. When the pure Z
isomer was isolated, it showed higher potency compared to the
mixture (Table 1).
To analyze specificity and cellular activity of 3-substituted
indolin-2-ones,
a selected subset of compounds was tested
CoMFA analysis allowed the identification of specific features
around the inhibitor scaffold, that may influence inhibitor binding.
A graphical display of this analysis is depicted in Figure 1, as colour
coded contours around compound 33. Steric fields are indicated in
green and yellow (Fig. 1A), with green indicating areas in which
bulky groups are favoured, while yellow marks areas where bulky
groups are disfavoured. The analysis suggested that bulky groups
are preferred at R3 position. In fact, pyrrol is favoured due to its
interaction with the oxygen of the oxindole (SU5416, 32, 33, 45,
in vitro against three related tyrosine kinases, and in cell growth
assays using the IL3-dependent BaF3 cell system.4 The majority
of them showed good selectivity for RET versus ALK and ABL ki-
nases, indicating that they are not general tyrosine kinase inhibi-
tors (Table 2). On the contrary, most compounds inhibited RET
and FLT3 with similar potency. This result is not surprising, since
both RET and FLT3 belong to the same structural family of the split
tyrosine kinases, epitomized by VEGFR. However, there were
examples of preference for RET versus FLT3: the highest selectivity
was shown by compound SU6656, which is also a potent Src-fam-
ily kinases inhibitor,18 and therefore did not show cellular selectiv-
ity. Interestingly, compounds 29 and VEGFR2KI-I were potent and
specific RET inhibitors both in vitro (at least with regard to the tar-
gets tested here) and in cells, respectively showing 30- and 100-
fold more potent inhibition of RET-transformed BaPTC2 cells com-
pared to parental BaF3 cells. A previous report of compound VEG-
FR2KI-I showed that it is a potent VEGFR inhibitor (IC50 = 70 nM),
while it does not affect EGFR, IGF1R or PDGFR kinases.8 Inhibitors
29, and VEGFR2KI-I all have IC50 <1 lM). Accordingly, compounds
carrying a furan ring at that position are predicted to have negative
interaction with the oxygen, and showed higher IC50 (30 is the
most active compound with an IC50 of 12
small groups are favoured at position R5. Consistent with this find-
ing, compounds showing higher activity (IC50 <1 M) either carry
lM). On the contrary,
l
no substitution at that position, or have an amine or halogen group,
with the exception of compound 29. Compounds 26 and 27 bear
bulky groups in R5 that abrogate the effect of the pyrrol ring in
N
N
N
H
H2N
N
N
NH
HO
N
H
O
i
O
N
H
O
O
N
H
33
38
Scheme 7. Peptidic coupling. Reagents and conditions: (i) HBTU, DIEA, DMF, 12 h, rt.

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L. Mologni et al. / Bioorg. Med. Chem. 18 (2010) 1482–1496
Table 1
3-Substituted indolin-2-one: isomeric configurations and inhibitory activities on RET TK
R4
R3
O
R5
N
H
R7
ID
R3
R4
H
R5
R7
H
Isomer
RET IC50
2.0 0.3
(lM)
13
OCF3
Z
N
H
14
H
OCH3
H
Z
4.1 0.9
N
H
15
16
17
H
H
H
H
OCH3
H
Z
Z
Z
5.5 1.6
66 10
8.0 1.6
N
H
CH3
CH3
N
H
H
N
H
19
22
H
H
Br
H
H
Z
Z
1.4 0.3
7.5 1.3
N
H
NO2
NO2
O
N
H
25
26
H
H
H
H
80%E
22
40
3
8
Br
O
Z
N
H
N
N
27
28
H
H
H
H
Z
Z
20
5
N
H
1.1 0.3
O
N
N
H
29
H
H
Z
0.58 0.07
N
H
30
31
O
S
H
H
NO2
NO2
H
H
E
E
12
1
O
O
78 10
32
H
NH2
H
Z
Z
0.46 0.09
N
H
33
34
H
H
NH2
NH2
H
H
0.30 0.04
5.3 1.3
N
H
Z
E
OCH3
63
9

L. Mologni et al. / Bioorg. Med. Chem. 18 (2010) 1482–1496
1487
M)
Table 1 (continued)
ID
R3
R4
H
R5
R7
H
Isomer
RET IC50
1.0 0.3
(l
35
36
NH2
70%E
NEt2
Br
O
H
H
NH2
NH2
H
H
E
E
66 14
O
37
38
34
5
O
N
H
N
N
H
H
Z
5.9 1.0
N
H
O
39
40
CH3
CH3
H
H
H
H
Z
100
23
4
N
O
n.d.
3
41
42
CH3
CH3
H
H
H
H
n.d.
0.21 0.03
3.7 0.5
N
N
O
H
N
Z
43
44
H
H
H
H
H
H
Z
E
>100
N
2.3 0.4
OH
45
H
H
H
H
H
H
H
H
H
H
H
Z
Z
E
Z
0.46 0.04
0.17 0.03
1.1 0.2
N
H
SU5416
SU4984
SU6656
N
H
O
H
N
N
SNO2(CH3)2
0.77 0.21
N
H
SU5614
SU9516
H
H
Cl
H
H
Z
Z
1.0 0.2
1.6 0.2
N
H
N
OCH3
N
H
N
O
SU11248
H
F
H
Z
1.3 0.3
NH
N
H
MAZ51
H
H
H
H
H
n.d.
Z
45
9
N
VEGFR2KI-II
Br
1.7 0.3
N
H
(continued on next page)

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L. Mologni et al. / Bioorg. Med. Chem. 18 (2010) 1482–1496
Table 1 (continued)
ID
R3
R4
H
R5
H
R7
H
Isomer
Z
RET IC50 (lM)
O
O
VEGFR2KI-I
0.76 0.19
N
H
Table 2
blue indicates areas in which negative charges are disfavoured.
This analysis suggested that less electronegative groups are fa-
voured near R5: accordingly, SU11248 and 13, carrying a fluorine
and a difluoromethoxy group, respectively, showed lower activity
compared to SU5416 (with a chlorine atom in R5), 32 and 33 (both
with an amino group) and 29 (pyridine). These observations will
help design new inhibitors with improved inhibitory activity.
Kinase specificity and cellular activity of selected 3-substituted indolin-2-one
inhibitors
Compd
RET
ALK
ABL
FLT3
BaPTC2
BaF3
29
32
33
41
44
45
0.58
0.46
0.30
0.21
2.3
0.46
0.17
0.77
1.3
3.8
11
17
2.3
2.6
0.079
0.26
0.16
10
16
8
2.3
0.35
1.3
10
>50
17
>50
0.18
1.2
2.6
>50
>50
>50
>50
>50
11
>50
>50
19
4.8
>50
>50
>50
1.2
18
0.63
0.40
1.8
0.35
0.16
8.7
3.3. X-ray crystallographic analysis
SU5416
SU6656
SU11248
VEGFR2KI-I
6
0.16
0.22
0.025
Three compounds were selected for structural studies, based on
their structure and biochemical activity. The aim of this analysis
was to confirm and extend previous QSAR analyses. SU5416 and
33 were among the most potent inhibitors in kinase assays. Com-
pound 34 was chosen as a representative of compounds with a
phenyl ring at position R3 and because it is likely to be a mixture
of isomers during the crystallization process. Therefore, we could
conclusively determine which of the two isomers is the actual li-
gand. The crystal structures for the phosphorylated RET kinase do-
main (RET-KD-P) in complex with inhibitors SU5416, 33 and 34
were solved and refined at resolutions of 2.5, 2.6 and 2.0 Å, respec-
tively (Table 4). In all three, the kinase domain forms the same
‘head-to-tail’ dimer as found previously;6 the complexes of 33
and 34 crystallized in space group C2, and thus the dimer is formed
by crystallographic symmetry, while for SU5416 in P1, the two
protomers are related by a non-crystallographic twofold axis.
Superpositions based on the C-lobes of RET-KD-P from the three
complexes show very similar orientations of the N-lobes, and with-
in the N-lobes the extremity of the nucleotide binding loop (resi-
dues 733–735) is disordered/poorly defined in each case. The
activation loops resemble those in previous RET-KD-P structures
(e.g., PDB codes 2ivt, 2ivu); each molecule shows clear electron
density for pTyr905, but no side chain density for Tyr900, and
therefore no indication of its phosphorylation state. The side chain
of Glu775 from the C-helix forms a hydrogen bond to the active
2.3
5.4
0.55
4.3
0.76
Average IC50 values (here reported in micromolar units) were obtained in in vitro
kinase assays with four tyrosine kinases and in cell growth assays with BaF3
parental and RET-transformed (BaPTC2) cells.
Table 3
Statistical summary of QSAR-CoMFA analysis with database alignment of all
compounds in Z configuration
Alignment
No.
r2
q2
F-values
Complete dataset
w/o Outliersa
10
6
0.992
0.971
0.280
0.598
299.757
132.153
a
Outliers: 15, 28, 41, 43, SU6656, VEGFR2KI-II.
q2: cross-validated correlation coefficient.
r2: non-cross-validated correlation coefficient.
No.: optimum number of components.
R3. These data also predict that there is space for substitution in
position 6. Interestingly, RPI-1, an indolinone compound previ-
ously characterized as a RET inhibitor,23 has a methoxyl group at
R6, confirming that it should be possible to build on this position.
Electrostatic fields are shown as red and blue contours in Figure
1B: red indicates areas in which negative charges are favoured and
Figure 1. Contour plots for the best CoMFA model around compound 33. Contours volumes were fixed at 80% for favoured and 20% for disfavoured levels for all fields. (A)
Steric field: green indicates areas in which bulky groups are favoured, yellow indicates areas in which bulky groups are disfavoured. (B) Electrostatic field: red indicates areas
in which negative charges are favoured and blue indicates areas in which negative charges are disfavoured.

L. Mologni et al. / Bioorg. Med. Chem. 18 (2010) 1482–1496
1489
Table 4
indolinone compounds SU5402 and SU4984 in complex with
FGFR17 and in our RET:PP1 co-crystal structure.6 While all three
inhibitors bind in the ATP site, there are minor differences in ori-
entation, as shown in Figure 3. Compounds 33 and SU5416
might be expected to have their rings close to coplanar, re-
stricted by the NHꢀ ꢀ ꢀO intramolecular hydrogen bond, while
there is potential steric hindrance between the O atom and the
closest –CH2 of the substituted phenyl ring in 34 that might lead
to out-of-plane twisting. The refinement protocol allowed for the
planes of the two ring systems in each ligand to rotate with re-
spect to each other. In SU5416 the rings are coplanar, while in
33 and 34 there are small (ca 5°) opposite rotations of the six-
membered ring (Fig. 3).
Compound 34 (the pure Z isomer) is approximately 1 log less
active than 33 and SU5416 (Table 1). The most obvious explana-
tion for the observed difference in potency stems from stereo-
chemistry: while 33 and SU5416 are exclusively Z isomers,
compound 34 can rapidly isomerize in solution. Therefore, even
the isolated Z isomer will tend toward equilibrium where a sig-
nificant fraction of molecules is in the inactive E configuration.
An additional possible reason for the difference in activity is pro-
vided by crystallographic data: there is H-bonding potential be-
tween the carbonyl oxygen atom of Ala807 and the nitrogen
atom of the pyrrol ring in 3-position in 33 and SU5416, whereas
34 has a phenyl ring here. However the N–O distances are rather
long (3.5 Å in 33, 3.3 Å in SU5416) and the geometry non-opti-
mal to assign hydrogen bonds in these crystal structures. Never-
theless, the N(ligand)–O(807) interaction may be advantageous
during the docking process.
Crystallographic data confirmed the conclusions from QSAR
analysis with regard to steric fields around positions 3, 5 and
6 of the oxindole. In particular, the substituent in R3 points to-
wards the solvent and there is space for large groups, which
are likely to establish favourable interactions. This feature may
also be exploited to improve solubility by adding hydrophilic
groups, as suggested by the increased cellular activity of VEG-
FR2KI-I compared to SU5416. By contrast, the amine in position
R5 of compounds 33 and 34 is in close vicinity with the sur-
rounding pocket, as indicated by hydrogen bonding with
Asp892; in addition, this 5-NH2 group is very close to the side
chain nitrogen of Lys758, although the angle is not favourable
for establishing a hydrogen bond. Thus, the structural data sup-
port the conclusion that bulky substituents are not favoured in
R5. Finally, we noted that the cavity guarded by the gatekeeper
residue Val804 is not occupied by the ligands; in two of them
(34 and SU5416) it contains water molecules, and there is cer-
tainly space for a bulkier substituent in position 6 on the indoli-
none ring, as anticipated by CoMFA analysis. These data will be
useful to design more potent and specific inhibitors by further
elaboration of this scaffold.
Data collection and refinement statistics for RET/SU5416, RET/33 and RET/34
complexes
Data collection
Ligand
Date
Short code
Spacegroup
Cell a, b, c (Å)
SU5416
030807
SU5416
33
34
290707
AM01
C2
270607
AM07
C2
ꢀ
P1
50.70, 50.76, 71.60,
74.59
71.99,
70.68,
71.57
90, 110.35,
90
70.91,
78.87
90, 101.68,
90
Cell
a
, b,
c
(°)
103.50,
99.66,89.92
2
19–2.5
(2.56–2.50)
ZA
1
1
Resolution (Å)
Beamline
21.6–2.6
(2.74–2.60) (2.11–
2.00)
67–2.00
In-house
In-house
ESRF ID14-
1
0.9340
ADSC Q210
Wavelength (Å)
Detector
1.5418
1.5418
mar345
image plate CCD
11,886
3.1 (3.0)
99.1 (100)
5.9 (23.2)
4.0 (15.7)
17.0 (5.2)
48
mar345
image plate
23,524
2.2 (2.2)
95.4 (93.9)
4.4 (11.3)
3.9 (10.2)
16.6 (6.1)
48
Unique reflections
Average redundancy
Completeness
20,421
3.5 (2.3)
89.5 (54.6)
6.2 (50.7)
3.7 (41.6)
13.4 (2.1)
30
a
Rmerge (%)
b
Rp.i.m. (%)
I/r
(I)c
Wilson B
Refinement
Resolution (Å)
30.0–2.50
(2.56–2.50)
20.5 (31.8)
24.4 (31.2)
4327, 36, 46 2239, 19,
29
30, 26, 26
0.022, 2.4
95, 4, 1
30.00–2.60
(2.67–2.60) (2.05–2.00)
19.1 (27.0)
25.6 (35.9)
67–2.0
Rwork (%)
Rfree (%)
Number of atoms (protein,
ligand, water)
Average B-factors (protein,
ligands, water)
rms deviation bonds (Å), angles
(°)
Ramachandran plot % allowed,
generous, disallowed
21.6 (29.9)
27.5 (31.9)
2224, 20,
71
41, 47, 35
0.016, 1.6
95, 4, 1
39, 45, 38
0.016, 1.6
94, 5, 1
a
Rmerge
=
RhRi |I(h,i) ꢁ hI(h)i|/RhRihI(h)i where I(h,i) is the intensity value of the i-
th measurement of reflection h.
b
Rp.i.m.
=
Rh(1/(nh ꢁ 1) Ri |I(h,i) ꢁ hI(h)i|/RhRi where nh is the number of
,
observations of reflection h.
c
I/r(I) = Mean (I)/r(I).
site lysine 758. The Glu775 side chain also contacts a water mole-
cule in the inner cavity in SU5416 and 34, but there is no equiva-
lent electron density for this water molecule in 33. This water
molecule also interacts with the main chain nitrogen atom of
Asp892 (from the DFG motif). The side chain of Asp892 tends to
be disordered; it has been modelled as a single conformer in 34,
dual conformer in 33 and truncated at Cb in SU5416. The position
of the side chain of Glu805 (from the linker region) allows interac-
tion with main chain nitrogen atoms of 789 and 889—interactions
that were disrupted when the larger inhibitor ZD6474 was bound
(see PDB code 2ivu).
As anticipated, all three inhibitors bind in the nucleotide
binding site (Fig. 2). Compound 34 was crystallized in its Z con-
figuration, confirming this as the actual ligand. The three com-
pounds form hydrogen bonds to main chain atoms of the
hinge/linker region of the kinase (O805 and N807). Refined val-
ues for these distances are as follows: N–O805 is 2.80 Å for
SU5416, 3.07 Å for 33 and 3.00 Å for 34; O–N807 is 2.89 Å for
SU5416, 3.12 Å for 33 and 2.77 Å for 34. In 33 and 34, the 5-
NH2 substituent makes a long contact to a side chain oxygen
atom of Asp892 (3.03 Å in 33, 3.33 Å in 34). Hydrogen bonding
to the hinge region conforms to the canonical pattern, mimicking
interactions established by ATP with tyrosine kinases.24 The
same hydrogen bonds were described in previous studies with
4. Experimental section
4.1. In vitro kinase assay
His-tagged recombinant RET, ALK, ABL and FLT3 kinase domains
were expressed and purified as described.4 Commercial inhibitors
were purchased from Calbiochem (SU5416, SU4984, SU6656,
SU5614, SU9516, MAZ51, VEGFR2KI-I, VEGFR2KI-II) or obtained
from Pfizer, Inc. (SU11248). Compounds 39–45 were kindly pro-
vided by Prof. Giuseppe Zanotti (University of Padua, Italy). All
inhibitors were dissolved in DMSO, aliquoted and stored at
ꢁ20 °C. Upon thawing, aliquots were used immediately and never
re-frozen. The ELISA-based kinase assay was performed as
described.4

1490
L. Mologni et al. / Bioorg. Med. Chem. 18 (2010) 1482–1496
Figure 2. (A–C) Electron density for compounds 33, 34 and SU5416. REFMAC-calculated electron density maps with 2mFo-DFc contoured at 1
r in green and mFo-DFc
contoured at 3 in blue; (D–F) ligand molecular surfaces of the ligand-binding pockets in RET-KD-P. The solvent side of a surface is white and the inside of a surface blue-
r
green. Ligands are shown in stick form, with carbon atoms blue for 33, cyan for 34 and dark red for SU5416. The gatekeeper Val804 side chain is highlighted in yellow. (G–I)
Ligplot schematic diagrams of inhibitor–protein interactions.
Figure 3. Superposition of 33, 34 and SU5416 complexes: lateral view (A) and view from the N-lobe (B). The RET-KD-P proteins were superimposed using C atoms from the
a
C-lobe. Inhibitor carbon atoms are shown in blue for 33, cyan for 34 and red for SU5416, and the respective protein atoms in pale shades of these colours. The gatekeeper
Val804 side chain is highlighted in yellow. Water molecules in the inner cavity are shown as spheres, cyan for 34 and pink for SU5416.

L. Mologni et al. / Bioorg. Med. Chem. 18 (2010) 1482–1496
1491
4.2. Cell growth assay
into the cryoprotectant n-paratone and flash-frozen in a dry
nitrogen stream at 100 K.
BaPTC2 cells expressing the RET/PTC2 fusion oncogene were
grown as described.4 Proliferation assays were set up in 96-well
format incubating 104 cells per well with increasing concentrations
of inhibitor, for 72 h. During the last 8 h, the cells were pulsed with
4.5. Data collection and structure solution
Data were collected as shown in Table 4, integrated with the
program MOSFLM25 and processed with SCALA and TRUNCATE.26
Structures solved by molecular replacement using MOLREP,27 as
implemented in the CCP4 Suite,26 with a search model made from
the phosphorylated RET-KD-P protein (PDB code 2ivt) with flexible
regions removed. The structures were refined using REFMAC528
and rebuilt using Coot.29 Other calculations were made with pro-
grams from the CCP4 Suite unless otherwise stated. The protein
and more ordered water molecules were rebuilt and refined for
several cycles prior to introduction of ligand into the calculation.
Ligand libraries were generated with the PRODRG server.30 Refine-
ment statistics are given in Table 4, and coordinates and structures
factors have been deposited with PDB codes 2X2K (33 complex),
2X2L (34 complex) and 2X2M (SU5416 complex). Structural figures
were generated with PyMOL (DeLano Scientific, South San Fran-
cisco, CA) and LIGPLOT.31
3H-thymidine (1
lCi/well) and then harvested onto a glass fibre fil-
ter. Cell-associated radioactivity was measured by liquid scintilla-
tion using a MicroBeta TriLux counter (Wallac). Each data point
was done in triplicate.
4.3. QSAR analysis
Computation of steric and electrostatic fields were based on
Lennard-Jones and Coulomb potentials, respectively. All molecules
were charged and aligned according to SU5416 structure, using the
indole core as a template; a grid (with a default 2 Å spacing, cover-
ing all the aligned molecules and extending beyond them by 4 Å in
all directions) was created automatically. Steric and electrostatic
properties were calculated using as probe a carbon sp3 atom with
a +1 charge. Steric and electrostatic fields were scaled with a de-
fault cut-off energy of 30 kcal/mol.20 Molecules preparation for
QSAR were performed by SYBYL v7.3; the design step was performed
by the Sketch option; minimization was performed using standard
Tripos force fields, using steepest descents as the first method and
conjugate gradients as the second one, 1000 iterations and a con-
4.6. Chemistry
All the starting materials used for the synthesis were commer-
cially available and used without purification; solvents and re-
agents were dried prior to use as required. Chromatographic
purification was performed on Silica Gel 60 M, 230–400 mesh
ASTM. 1H and 13C NMR were measured at 400 MHz and
100 MHz, respectively, in DMSO-d6 or CDCl3 with Me4Si as the
internal standard. Gas-chromatographic analyses were performed
with a 30 m, 0.25 mmID, Rtx-5MS capillary columm (5:95 diphe-
nyl-/dimethyl-polysiloxane) on a GS–MS unity. ESI-MS analyses
were performed under, negative or positive ionisation mode on
API 150 EX Turbo ion spray Amplied Biosistem. Mass spectra HRMS
analyses were performed under ESI positive or EI ionization mode,
on a QSTAR XL-MS unity.
*
vergence criterion of 0.05 kcal/mol A. Finally, Gasteiger–Marsilii
charges were added to molecules.
For CoMFA studies, the Partial Least Square (PLS) analysis was
initially performed with leave-one-out cross-validation and a col-
umn filtering of 1.0 kcal/mol or SAMPLS method, as indicated by
SYBYL-QSAR tutorials20 in order to determine the cross-validated
correlation coefficient q2 and the optimum number of components
to build the model. Finally, a non cross-validated PLS was per-
formed with the optimum number of component obtained from
the previous calculation and with a column filtering of 1.0 kcal/
mol, yielding the correlation coefficient r2.
CoMFA contour maps were generated as the scalar product of
*
coefficients and standard deviation (stdev coeff) and volume of
4.6.1. NMR NOESY spectroscopy
contours were fixed in 80% for favoured and 20% for disfavoured
levels for all fields.
For the Z isomer the diagnostic dipolar interaction is between
the H4 proton of the oxindolic ring and the vinylic H8 proton (the
adopted atom numbering is shown in Supplementary Scheme
S1). In the case of the E isomer the H4 proton interacts with the
protons of the primary R3 ring ortho to the junction (H11 in struc-
ture a, or H10/H14 in structure b) or with the protons of the second-
ary ring (H15/H17 in structure a).
4.4. Production of RET-KD-P:inhibitor crystals
GST-tagged recombinant RET kinase domain RET-KD (residues
705–1013) was expressed in Sf9 cells as described previously.6
Inhibitors were introduced into the protein solution prior to re-
moval of the GST tag as follows. A pellet from 1.5 l of insect cells
was purified by incubation with glutathione sepharose 4B beads
(Amersham Bioscience) for 2 h at 4 °C. The gel was washed to re-
move unbound material and then incubated with 2.5 mM ATP
4.6.2. Synthesis of (2)
A typical procedure for the synthesis of isatin10 was used to pre-
pare 2-hydroxyimino-N-(4-trifluoromethyl-phenyl)-acetamide (1).
A solution of sodium sulfate (50 g) in 60 mL of water was stirred
until all the solids were dissolved. A solution of 4-trifluorometh-
oxy-phenylamine (3.5 g, 20 mmol) in 30 mL of 1 N aqueous HCl
and 6 mL of EtOH was added to the resulting aqueous solution.
The mixture was stirred and chloral hydrate (3.6 g, 22 mmol)
was added. A solution of hydroxylamine hydrochloride (4.6 g,
66 mmol) in 20 mL of water was then added to the solution. The
final mixture was heated with stirring to a gentle reflux until the
solids were dissolved, and heating was continued for an additional
15 min. The resultant solution was poured on to 300 g of ice, stir-
red, and the product was precipitated from solution, collected by
suction filtration, washed with water and dried to give 4.5 g
(90%) of (1): 1H NMR (DMSO-d6, 400 MHz), d (ppm): 12.21 (br s,
1H), 10.37 (br s, 1H), 7.78 (d, 2H, J = 9.3 Hz), 7.63 (s, 1H), 7.32 (d,
2H, J = 9.0 Hz). 13C NMR (DMSO-d6, 100 MHz), d (ppm): 161.0,
156.2, 149.9, 122.6, 121.7, 128.1, 114.5(2C). ESI-MS (m/z)
and 5 mM magnesium sulfate for 2 h at room temperature. 10 ll
of stock inhibitor (10 mg/ml in DMSO) was added to 1 ml total vol-
ume of glutathione sepharose suspension and incubated at 4 °C for
1 h. The gel was washed with Buffer A (20 mM Tris pH 8, 100 mM
NaCl, 1 mM DTT and 1 mM EDTA), and then treated with GST-
linked 3C protease (PreScission protease, Amersham Bioscience)
overnight at 4 °C. Protein–inhibitor complexes were concentrated
to 4.5 mg/ml in buffer A.
Crystals were grown at 16 °C in sitting drops containing 2
ll
of protein solution and 2 l of reservoir solution (reservoir vol-
l
ume 0.5 ml): for 33 and 34 the reservoir solution contained
1.85 M sodium formate, 0.2 M LiCl and 0.1 M sodium citrate
pH 5.5; and for SU5416 the reservoir solution contained
1.75 M sodium formate, 0.15 M potassium thiocyanate and
0.1 M sodium citrate pH 5.5. All crystals were harvested directly

1492
L. Mologni et al. / Bioorg. Med. Chem. 18 (2010) 1482–1496
[MꢁH]ꢁ, 247.1. 2-Hydroxyimino-N-(4-trifluoromethoxy-phenyl)-
acetamide (1) (4 g, 16 mmol) was slowly added to 40 mL of con-
centrated sulphuric acid at 100 °C. The reaction mixture was
heated for 1 h and the resultant solution was poured into 280 g
of ice and water, stirred for 1 h and filtered. The solid was washed
with water and dried to give 1.7 g (46%) of 5-trifluoromethoxy-1H-
indole-2,3-dione (2): 1H NMR (DMSO-d6, 400 MHz), d (ppm): 11.16
(br s, 1H), 7.57 (dd, 1H, J = 8.2, 2.2 Hz), 7.50 (s, 1H, J = 8.2 Hz), 6.97
(d, 1H, J = 8.2 Hz); 13C NMR (DMSO-d6, 100 MHz), d (ppm): 188.6,
164.6, 154.6, 148.6, 136.1, 123.8, 123.0, 118.6. ESI-MS (m/z)
[MꢁH]ꢁ, 229.9.
1H), 6.98 (t, 1H, J = 7.7), 6.85 (d, 1H, J = 7.7 Hz), 6.81 (d, 1H,
J = 7.7 Hz), 3.67 (s, 3H), 3.55 (s, 2H). 13C NMR (CDCl3, 100 MHz) d
(ppm): 176.5, 143.7, 131.2, 126.0, 122.7, 116.9, 110.1, 55.6, 36.7.
MS (EI, 70 eV) m/z: 163.2 (M+, 100), 148.2 (38), 134.2 (20), 107.2
(26), 106.2 (44), 92.2 (44).
4.6.3. General reduction procedure of 5-substituted isatin to 5-
substituted-1,3-dihydro-indol-2-one
A stirred solution of 5-substituted isatin (0.1 mol) in hydra-
zine hydrate (60 mL) was heated to 140 °C for 4 h. The reaction
mixture was cooled to room temperature, poured into 300 mL of
ice water, and acidified to pH 2 with 6 N hydrochloric acid. After
standing at room temperature for 2 days the precipitate was col-
lected by vacuum filtration, washed with water, and dried under
vacuum (50% yield).
4.6.2.1. (5-Methoxy-2-nitro-phenyl)-acetonitrile (4) and (3-me-
thoxy-2-nitro-phenyl)-acetonitrile (5). A solution of (3-meth-
oxy-phenyl)-acetonitrile (3 g, 20 mmol) in a 1:1 mixture of glacial
acetic acid (23 mL) and acetic anhydride (23 mL) was cooled at
0 °C, and concentrated nitric acid (3.3 mL) was added dropwise.
After 1 h water was added (20 mL) of, the precipitate was filtered
and chromatographed on silica gel (90:10, n-Esane/AcOEt) to ob-
tain (5-methoxy-2-nitro-phenyl)-acetonitrile (4) 1.2 g (32%) and
(3-methoxy-2-nitro-phenyl)-acetonitrile (5) 0.8 g (22%), both as
yellow solid. (4): 1H NMR (CDCl3, 400 MHz), d (ppm): 8.26 (d, 1H,
J = 9.1), 7.18 (d, 1H, J = 2.8), 6.98 (dd, 1H, J = 9.1, 2.8), 4.25 (s, 2H),
3.95 (s, 3H).13C NMR (CDCl3, 100 MHz) d (ppm): 164.0, 140.3(2C),
128.6, 116.4, 116.3, 113.8, 56.1, 23.3. MS (EI, 70 eV) m/z: 177
(M+ꢁCH3, 26), 149 (100), 105 (14), 76 (21). (5): 1H NMR (CDCl3,
400 MHz), d (ppm): 7.5 (t, 1H, J = 8.1), 7.2 (d, 1H, J = 7.8), 7.07 (d,
1H, J = 7.8), 3.92 (s, 3H), 3.77 (s, 2H). 13C NMR (CDCl3, 100 MHz)
d (ppm): 151.6, 132.0(2C), 123.5, 120.6, 115.6, 112.9, 56.6, 19.9.
MS (EI, 70 eV) m/z: 177.2 (M+ꢁCH3, 26), 149.2 (100), 105.2 (14),
76.2 (21).
4.6.3.1. 5-Trifluoromethoxy-1,3-dihydro-indol-2-one
(3). Compound 3 (93% yield); 1H NMR (DMSO-d6, 400 MHz), dd
(ppm): 10.57 (br s, 1H), 7.22 (d, 1H, J = 2.0 Hz), 7.15 (ddquart, 1H,
J = 8.4, 2.6 Hz, JHF = 0.9 Hz), 6.87 (d, 1H, J = 8.4 Hz), 3.52 (s, 2H).
13C NMR (DMSO-d6, 100 MHz), d (ppm): 176.2, 142.9, 142.8,
127.7, 120.5, 120.1 (quartet, JCF = 255.0 Hz), 118.1, 109.7, 36.0.
ESI-MS (m/z) [MꢁH]ꢁ, 216.1.
4.6.3.2. 5-Methyl-1,3-dihydro-indol-2-one (10). Compound 10
(45% yield); 1H NMR (DMSO-d6, 400 MHz), d (ppm): 10.24 (br s,
1H), 6.99 (s, 1H), 6.94 (d, 1H, J = 7.8 Hz), 6.67 (d, 1H, J = 7.8 Hz),
3.39 (s, 2H), 2.22 (s, 3H). 13C NMR (DMSO-d6, 100 MHz), d (ppm):
176.3, 141.2, 129.9, 127.6, 125.8, 125.1, 108.8, 35.2, 20.6. MS (EI,
70 eV) m/z: 147.2 (M+, 95), 119.2 (55), 118.2 (100).
4.6.3.3. 5-Bromo-1,3-dihydro-indol-2-one (11). Compound 11
(60% yield); 1H NMR (DMSO-d6, 400 MHz), d (ppm): 10.48 (br s,
1H), 7.36 (s, 1H), 7.32 (d, 1H, J = 8.2 Hz), 6.73 (d, 1H, J = 8.2 Hz),
3.48 (s, 2H). 13C (DMSO-d6, 100 MHz) d (ppm): 176.6, 140.1,
134.6, 130.7, 128.2, 124.3, 119.2, 39.6. MS (EI, 70 eV) m/z: 213.1
(M+, 100), 211.1 (M+, 100), 184.1 (65), 182.1 (65), 158.0 (15),
156.0 (15), 132.1 (25), 104 (37).
4.6.2.2. (5-Methoxy-2-nitro-phenyl)-acetic acid (6) and (3-me-
thoxy-2-nitro-phenyl)-acetic acid (7). A stirred solution of (4) or
(5), (42 mg, 2.1 mmol) in concentrated HCl was heated to reflux.
After 3 h the mixture was cooled to 0 °C and 6 mL of ice water
was added. The precipitate was filtered and dried to give the com-
pounds in 67% yield. Compound 6 was obtain as pink solid: 1H
NMR (DMSO-d6, 400 MHz), d (ppm): 8.22 (d, 1H, J = 9.2), 6.93
(dd, 1H, J = 9.2, 2.8), 6.80 (d, 1H, J = 2.8), 4.05 (s, 2H), 3.90 (s, 3H),
1.78 (br s, 1H). 13C NMR (DMSO-d6, 100 MHz) d (ppm): 171.2,
163.1, 141.4, 133.8, 127.5, 118.7, 113.1, 56.1, 38.9. MS (EI, 70 eV)
m/z: 211.1 (M+, 17), 165.1 (70), 150.1 (80), 122.1 (50), 95.1 (48),
77.1 (100), 51.1 (70). Compound 7 was obtain as yellow solid: 1H
NMR (DMSO-d6, 400 MHz), d (ppm): 7.51 (t, 1H, J = 8.4), 7.24 (d,
1H, J = 8.4), 7.04 (d, 1H, J = 8.2), 3.86 (s, 3H), 3.61 (s, 2H), 3.35
(1H, OH).13C NMR (DMSO-d6, 100 MHz) d (ppm): 170.7, 150.6,
140.8. 131.6, 128.2, 123.5, 112.3, 56.6, 36.4. MS (EI, 70 eV) m/z:
211.1 (M+, 17), 165.1 (70), 150.1 (80), 122.1 (50), 95.1 (48), 77.1
(100), 51.1 (70).
4.6.3.4. 5-Nitro-1,3-dihydro-indol-2-one (12). Compound 12
(60% yield); 1H NMR (DMSO-d6, 400 MHz), d (ppm): 11.00 (br s,
1H), 8.13 (dd, 1H, J = 8.6, 2.4 Hz), 8.07 (d, 1H, J = 2.4 Hz), 6.88 (d,
1H, J = 8.6 Hz), 3.61 (s, 2H). 13C NMR (DMSO-d6, 100 MHz) d
(ppm): 176.6, 150.4, 141.7, 127.1, 124.9, 120.0, 109.0, 35.6. MS
(EI, 70 eV) m/z: 178.2 (M+, 100), 148.2 (56), 132.2 (25), 120.2
(30), 104.2 (65).
4.6.4. General procedure for 3-substituted indolin-2-one
A
stirred solution of proper oxindole (1 equiv), aldehyde
(1.2 equiv), and piperidine (0.1 equiv), in EtOH 10 mL/1 mmol
was heated to 90 °C for 4 h. After cooling at room temperature,
the precipitate was filtered, washed with cold EtOH.
We reported here the NMR chemical shift for the major isomer.
Only for compounds 23, 24, 34, 35, we described the NMR analyt-
ical data for both isomers.
4.6.2.3. 5-Methoxy-1,3-dihydro-indol-2-one (8) and 7-meth-
oxy-1,3-dihydro-indol-2-one (9). To a stirred solution of (6) or
(7) (300 mg, 1.5 mmol) in acetic acid (4.5 mL) was added Pd/C 5%
(20 mg) as catalyst, the mixture was poured under hydrogen atmo-
sphere. After 2 h at room temperature CH2Cl2 (15 mL) was added,
the mixture was filtered through Celite. The organic layer was con-
centrated and the residue was chromatographed on silica gel
(80:20, n-hexane/AcOEt) to give (8) 98 mg (40%) as white solid
and (9) 102 mg (50%) as orange solid. Compound 8: 1H NMR
(CDCl3, 400 MHz), d (ppm): 8.50 (br s, 1H), 6.85 (d, 1H, J = 2.0),
6.79 (d, 1H, J = 8.4 Hz), 6.74 (dd, 1H, J = 8.0, 2.0 Hz), 3.77 (s, 3H),
3.52 (s, 2H).13C NMR (CDCl3, 100 MHz) d (ppm): 177.4, 155.6,
135.8, 126.6, 112.4, 111.8, 109.9, 55.8, 36.6. MS (EI, 70 eV) m/z:
163.2 (M+, 100), 148.2 (38), 134.2 (20), 107.2 (26), 106.2 (44),
92.2 (44). (9): 1H NMR (CDCl3, 400 MHz), d (ppm): 8.07 (br s,
4.6.4.1. 3-(3,5-Dimethyl-1H-pyrrol-2-ylmethylene)-5-trifluoro-
methoxy-1,3-dihydro-indol-2-one (13). Compound 13 (60%
yield); 1H NMR (DMSO-d6, 400 MHz), d (ppm): 13.41 (br s, 1H),
10.94 (br s, 1H), 7.87 (s, 1H), 7.71 (s, 1H), 6.99 (d, 1H, J = 8.0 Hz),
6.93 (d, 1H, J = 8.0 Hz), 6.02 (s, 1H), 2.16 (s, 3H), 2.10 (s, 3H). 13C
NMR (DMSO-d6, 100 MHz), d (ppm): 169.5, 147.5, 143.2, 137.1,
136.7, 133.4, 127,5, 126.9, 125.8, 118.1, 113.1, 111.8, 111.4,
110.1, 109.7, 13.6, 11.4. ESI-HRMS (m/z) [M+H]+ calcd, 323.1001;
obs, 323.0992.

L. Mologni et al. / Bioorg. Med. Chem. 18 (2010) 1482–1496
1493
4.6.4.2. 3-(3,5-Dimethyl-1H-pyrrol-2-ylmethylene)-5-methoxy-
1,3-dihydro-indol-2-one (14). Compound 14 (50% yield); 1H
NMR (DMSO-d6, 400 MHz), d (ppm): 13.43 (br s , 1H), 10.57 (br s,
1H), 7.57 (s, 1H), 7.39 (d, 1H, J = 2.5 Hz), 6.74 (d, 1H, J = 8.4 Hz),
6.65 (dd, 1H, J = 8.4, 2.5 Hz), 5.99 (d, 1H, J = 2.5 Hz), 3.75 (s, 3H),
2.30 (s, 3H), 2.30 (s, 3H). 13C NMR (DMSO-d6, 100 MHz), d (ppm):
169.5, 154.8, 135.6, 132.1, 131.7, 126.9, 126.6, 123.7, 113.3,
112.5, 111.9, 109.7, 104.1, 55.7, 13.5, 11.4. ESI-HRMS (m/z)
[M+H]+ calcd, 269.1284; obs, 269.1297.
(DMSO-d6, 100 MHz), d (ppm): 169.6, 144.0, 142.2, 129.6, 129.7,
127.6, 126.1, 122.9, 122.6, 114.1, 114.0, 112.22, 109.4. ESI-MS (m/
z) [M+H]+, 253.9.
4.6.4.9. 3-(3,5-Dimethyl-1H-pyrrol-2-ylmethylene)-5-nitro-1,3-
dihydro-indol-2-one (21). Compound 21 (65% yield); 1H NMR
(DMSO-d6, 400 MHz), d (ppm): 13.32 (br s, 1H). 11.41 (br s, 1H),
8.75 (d, 1H, J = 2.3 Hz), 8.01 (dd, 1H, J = 8.5, 2.2 Hz), 7.92 (s, 1H),
7.02 (d, 1H, J = 8.6 Hz), 6.08 (bd, 1H, J = 2.4 Hz), 2.36 (s, 3H), 2.34
(s, 3H). 13C NMR (DMSO-d6, 100 MHz), d (ppm): 169.8, 142.9,
142.1, 138.4, 134.9, 127.3, 126.8, 126.4, 121.6, 113.8, 113.6,
109.8, 109.0, 13.6, 11.4. ESI-MS (m/z) [M+H]+, 284.0.
4.6.4.3. 3-(3,5-Dimethyl-1H-pyrrol-2-ylmethylene)-7-methoxy-
1,3-dihydro-indol-2-one (15). Compound 15 (60% yield); 1H
NMR (DMSO-d6, 400 MHz), d (ppm): 13.42 (br s, 1H), 10.81 (br s,
1H), 7.51 (s, 1H), 7.32 (d, 1H, J = 7.8 Hz), 6.92 (t, 1H, J = 7.8 Hz),
6.80 (d, 1H, J = 8.0 Hz), 5.96 (d, 1H, J = 2.5 Hz), 3.82 (s, 3H), 2.31
(s, 3H), 2.28 (s, 3H). 13C NMR (DMSO-d6, 100 MHz), d (ppm):
169.3, 143.6, 135.7, 131.6, 126.8, 126.7, 126.6, 123.8, 121.4,
113.2, 112.5, 111.0, 108.8, 55.6, 13.5, 11.3. ESI-HRMS (m/z)
[M+H]+ calcd, 269.1284; obs, 269.1297.
4.6.4.10. 3-(1H-Indol-3-ylmethylene)-5-nitro-1,3-dihydro-ind-
ol-2-one (22). Compound 22 eluted with CHCl3/EtOH/Et3N
(94:5:1) (85% yield); Z Isomer: 1H NMR (DMSO-d6, 400 MHz), d
(ppm): 12.17 (br s, 1H), 11.15 (br s, 1H), 9.49 (d, 1H, J = 3.1 Hz),
8.90 (d, 1H, J = 2.2 Hz), 8.53 (s, 1H), 8.34 (m, 1H), 8.07 (dd, 1H,
J = 8.3, 2.2 Hz), 7.52 (m, 1H), 7.25 (m, 1H), 7.00 (d, 1H, J = 8.6 Hz).
NOESY: dipolar interaction detected between H4 (d 8.90) and H8
(d 8.53). 13C NMR (DMSO-d6, 100 MHz), d (ppm): 168.3, 144.3,
142.0, 136.1, 135.0, 131.5, 128.5, 126.6, 123.0, 122.9, 121.2,
119.1, 116.5, 114.6, 112.4, 111.8, 108.8. EI-HRMS (m/z) [M]+ calcd,
305.0800; obs, 305.0801.
4.6.4.4. 5-Methyl-3-(1H-pyrrol-2-ylmethylene)-1,3-dihydro-in-
dol-2-one (16). Compound 16 (70% yield); 1H NMR (DMSO-d6,
400 MHz), d (ppm): 13.31 (br s, 1H), 10.77 (br s, 1H), 7.68 (s,
1H) 7.43 (m, 1H), 7.31 (m, 1H), 6.93 (m, 1H, J = 7.8 Hz), 6.78 (m,
1H), 6.74 (d, 1H, J = 7.8 Hz), 6.32 (m, 1H), 2.27 (s, 3H). NOESY:
dipolar interaction detected between H4 (d 7.43) and H8 (d
7.68). 13C NMR (DMSO-d6, 100 MHz), d (ppm): 169.3, 136.8,
130.0, 129.6, 127.4, 126.1, 125.5, 125.2, 120.1, 119.1, 117.1,
111.4, 109.3, 20.94; ESI-HRMS (m/z) [M+H]+ calcd, 225.1022;
obs, 225.1014.
4.6.4.11. 3-(4-Methoxybenzylidene)-5-nitroindolin-2-one (23 )
(70% yield); E isomer.1H NMR (DMSO-d6, 400 MHz), d (ppm):
11.30 (br s, 1H), 8.46 (d, 1H, J = 2.3 Hz), 8.17 (dd, 1H, J = 8.6, 2.3 Hz),
7.78 (part of anAA0BB0 system, 2H, ‘J’ = 9.0 Hz), 7.78 (s, 1H), 7.13 (part
of an AA0BB0 system, 2H, ‘J’ = 9.0 Hz), 7.05 (d, 1H, J = 8.6 Hz), 3.86 (s,
3H). NOESY: dipolar interaction detected between H4 (d 8.46) and
H10/H14 (d 7.78). 13C NMR (DMSO, 100 MHz), d (ppm): 169.1, 161.3,
148.2, 141.5, 139.6, 132.0, 126.2, 126.0, 125.9, 121.5, 117.0, 114.5,
109.9, 55.5. Z Isomer: 1H NMR (DMSO-d6, 400 MHz), d (ppm): 11.30
(br s, 1H), 8.66 (d, 1H, J = 2.3 Hz), 8.54 (part of an AA0BB0 system, 2H,
‘J’ = 9.1 Hz), 8.16 (s, 1H), 8.13 (dd, 1H, J = 8.6, 2.3 Hz), 7.07 (part of an
AA0BB0 system, 2H, ‘J’ = 9.1 Hz), 6.99 (d, 1H, J = 8.6 Hz), 3.85 (s, 3H).
NOESY: dipolar interaction detected between H4 (d 8.66) and H8 (d
8.16). 13C NMR (DMSO-d6, 100 MHz), d (ppm): 167.6, 162.0, 145.6,
142.0, 140.8, 135.2, 126.7, 124.5, 123.7, 121.6, 115.0, 114.0, 109.1,
55.5. ESI-MS (m/z) [M+H]+, 297.3.
4.6.4.5. 3-(3,5-Dimethyl-1H-pyrrol-2-ylmethylene)-5-methyl-1,
3-dihydro-indol-2-one (17). Compound 17 (60% yield); 1H NMR
(DMSO-d6, 400 MHz), d (ppm): 13.34 (br s, 1H), 10.65 (br s, 1H),
7.52 (d, 1H, J = 1.4 Hz), 7.50 (s, 1H), 6.88 (dd, 1H, J = 7.9, 1.6 Hz),
6.73 (d, 1H, J = 7.8 Hz), 5.97 (d, 1H, J = 2.6 Hz), 2.29 (s, 3H), 2.28
(s, 3H), 2.27 (s, 3H). 13C NMR (DMSO-d6, 100 MHz), d (ppm):
169.5, 136.0, 135.4, 131.3, 129.6, 126.6, 125.9, 123.2, 123.1,
118.6, 118.6, 113.0, 112.4, 20.9, 13.5, 11.3; ESI-HRMS (m/z)
[M+H]+ calcd, 253.1335; obs, 253.1327.
4.6.4.6. 5-Bromo-3-(1H-pyrrol-2-ylmethylene)-1,3-dihydro-in-
dol-2-one (18). Compound 18 (44% yield); 1H NMR (DMSO-d6,
400 MHz); signals broadened by radicals; d (ppm): 13.27 (br s,
1H), 10.99 (br s, 1H), 7.86 (br s, 2H), 7.37 (br s, 1H), 7.25 (br s,
1H), 6.82 (br s, 2H), 6.35 (br s, 1H), 13C NMR (DMSO-d6,
100 MHz), d (ppm): 174.1, 143.0, 134.7, 134.0, 133.2, 132.8,
131.7, 126.5, 126.4, 120.5, 118.5, 116.9, 116.5. ESI-HRMS (m/z)
[M+H]+ calcd, 288.9976, obs, 288.9980.
4.6.4.12. 3-(4-Diethylamino-benzylidene)-5-nitro-1,3-dihydro-
indol-2-one (24) (75% yield); E isomer. 1H NMR (DMSO-d6,
400 MHz), d (ppm): 11.16 (br s, 1H), 8.60 (d, 1H, J = 2.2 Hz), 8.11
(dd, 1H, J = 8.6, 2.2 Hz), 7.67 (part of an AA0BB0 system, 2H,
‘J’ = 8.8 Hz), 7.66 (s, 1H), 7.01 (d, 1H, J = 8.6 Hz), 6.80 (part of an
AA0BB0 system, 2H, ‘J’ = 8.8 Hz), 3.45 (q, 4H, J = 6.9 Hz), 1.13 (t, 6H,
J = 7.0 Hz). NOESY: dipolar interaction detected between H4 (d
8.60) and H10/H14 (d 7.67). 13C NMR (DMSO-d6, 100 MHz), d (ppm):
169.7, 149.8, 147.4, 141.4, 140.9, 133.1 (2C), 124.7, 122.4, 119.6,
118.9, 116.3, 111.0 (2C), 109.5, 43.7 (2C), 12.4 (2C). Z isomer: 1H
NMR (DMSO-d6, 400 MHz), d (ppm): 1H NMR (DMSO, 400 MHz), d
(ppm): 11.13 (br s, 1H), 8.55 (d, 1H, J = 2.2 Hz), 8.50 (part of an AA0BB0
system, 2H, ‘J’ = 8.9 Hz), 8.04 (dd, 1H, J = 8.6, 2.3 Hz), 7.97 (s, 1H),
6.94 (d, 1H, J = 8.6 Hz), 6.75 (part of an AA0BB0 system, 2H,
‘J’ = 8.9 Hz), 3.45 (q, 4H, J = 6.7 Hz), 1.13 (t, 6H, J = 6.8 Hz). NOESY:
dipolar interaction detected between H4 (d 8.55) and H8 (d 7.97).
13C NMR (DMSO-d6, 100 MHz), d (ppm): 167.9, 150.3, 144.5, 141.9,
141.8, 136.1 (2C), 127.2, 122.9, 121.2, 116.6, 113.7, 110.7 (2C),
108.6, 43.9 (2C), 12.5 (2C). ESI-MS (m/z) [M+H]+, 338.3.
4.6.4.7. 5-Bromo-3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,
3-dihydro-indol-2-one (19). Compound 19 (50% yield); 1H NMR
(DMSO-d6, 400 MHz); signals broadened by radicals; d (ppm):
13.35 (br s, 1H), 10.87 (br s, 1H), 7.99 (br s, 1H), 7.66 (br s, 1H),
7.18 (br s, 1H), 6.79 (br s, 1H), 6.00 (br s, 1H), 2.30 (br s, 3H),
2.29 (br s, 3H), 13C NMR (DMSO-d6, 100 MHz), d (ppm): 174.3,
142.1(2C), 138.4, 133.5, 132.7, 132.1, 130.2, 125.9, 118.3, 118.2,
116.3, 116.1, 18.7, 16.5. ESI-HRMS (m/z) [M+H]+ calcd, 317.0284;
obs, 317.0270.
4.6.4.8. 5-Nitro-3-(1H-pyrrol-2-ylmethylene)-1,3-dihydro-ind-
ol-2-one (20). Compound 20 (70% yield); 1H NMR (DMSO-d6,
400 MHz), d (ppm): 13.14 (br s, 1H). 11.51 (br s, 1H), 8.56 (d, 1H,
J = 2.3 Hz), 8.14 (s, 1H), 8.06 (dd, 1H, J = 8.7, 2.3 Hz), 7.45 (m, 1H),
7.03 (d, 1H, J = 8.6 Hz), 6.95 (m, 1H), 6.41 (m, 1H). 13C NMR
4.6.4.13. 3-(5-Bromo-furan-2-ylmethylene)-5-nitro-1,3-dihy-
dro-indol-2-one (25). Compound 25 eluted with CHCl3/EtOH
(98:2) (75% yield); E isomer: 1H NMR (DMSO-d6, 400 MHz), d
(ppm): 11.30 (br s, 1H), 9.12 (d, 1H, J = 2.3 Hz) 8.20 (dd, 1H, J = 8.6,

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L. Mologni et al. / Bioorg. Med. Chem. 18 (2010) 1482–1496
2.3 Hz), 7.42 (d, 1H, J = 3.5 Hz), 7.41 (s, 1H), 7.03 (d, 1H, J = 8.6 Hz),
7.01 (d, 1H, J = 3.5). NOESY: detection of the dipolar interaction of
H4 (d 7.51) hampered by the isochronicity of H8 (d 7.41) and H11 (d
7.42). 13C NMR (DMSO-d6, 100 MHz) d (ppm): 174.6, 157.4, 153.3,
147.2, 133.4, 131.3, 130.0, 126.5, 125.9, 125.6, 124.8, 121.4, 114.9.
ESI-HRMS (m/z) [M+H]+ calcd, 333.9589; obs, 333.9580.
n-hexane/EtOH (74:20:5:1); (45% yield). 1H NMR (DMSO-d6,
400 MHz) d (ppm): 11.30 (br s, 1H), 9.10 (d, 1H, J = 2.4 Hz), 8.50 (m,
1H), 8.19 (dd, 1H, J = 8.6, 2.3 Hz), 7.89 (m, 1H), 7.50 (d, 1H,
J = 3.7 Hz), 7.44 (s, 1H), 7.13 (m, 1H), 7.10 (d, 1H, J = 3.6 Hz), 7.08 (d,
1H, J = 8.7 Hz). NOESY: detected dipolar interaction of H4 (d 9.10)
with H15 (d 8.50) and H17 (d 7.13). 13C NMR (DMSO-d6, 100 MHz) d
(ppm): 174.95, 158.00, 154.66, 153.39, 150.35, 146.93, 145.71,
131.31, 130.99, 126.98, 126.36, 123.89, 123.72, 121.80, 115.71,
114.92, 113.12. ESI-HRMS (m/z) [M+H]+ calcd, 322.0590; obs,
322.0560.
4.6.5. General procedure for 5-substituted-3-(1H-pyrrol)-1,3-
dihydro-indol-2-one, and 5-nitro-3-(5-substituted-3-yl-furan-
2-ylmethylene)-1,3-dihydro-indol-2-one by Suzuki cross-
coupling
To a stirred solution of the proper boronic acid/ester (0.1 mmol)
in dioxane (10 mL), was added a saturated solution of Na2CO3
(5 mL), the mixture was degassed and then added [Pd(P(Ph3)4)]
(10 mg). The reaction mixture was heated to 110 °C under argon
atmosphere for 12 h, cooled to room temperature and concen-
trated; CH2Cl2 (10 mL) was then added and the solution filtered
through Celite. The organic layer was concentrated and the residue
was chromatographed on silica gel.
4.6.5.6. 5-Nitro-3-(5-thiophen-3-yl-furan-2-ylmethylene)-1,3-
dihydro-indol-2-one (31) E isomer. Compound 31 eluted with
CH2Cl2/AcOEt/n-hexane/EtOH (74:20:5:1) (35% yield); 1H NMR
(DMSO-d6, 400 MHz), d (ppm): 11.30 (br s, 1H), 9.25 (d, 1H,
J = 2.3 Hz), 8.31 (dd, 1H, J = 2.7, 1.4 Hz), 8.22 (dd, 1H, J = 8.7,
2.4 Hz), 7.79 (d, part of AB system, 1H, J = 5.1, 2.8 Hz), 7.77 (d, part
of AB system, 1H, J = 5.1, 1.5 Hz), 7.56 (d, 1H, J = 3.8 Hz), 7.49 (s,
1H), 7.24 (d, 1H, J = 3.7 Hz), 7.06 (d, 1H, J = 8.7 Hz). NOESY: detected
dipolar interaction of H4 (d 9.25) with H15 (d 8.31) and H17 (d 7.79 or
7.77). 13C NMR (DMSO-d6, 100 MHz), d (ppm): 169.7, 155.5, 149.6,
147.8, 141.9, 130.6, 128.2, 126.6, 125.9, 125.3, 122.8, 121.9, 121.4,
118.6, 118.4, 110.4, 109.6. ESI-HRMS (m/z) [M+H]+ calcd,
338.0361; obs, 338.0350.
4.6.5.1. 5-Furan-2-yl-3-(1H-pyrrol-2-ylmethylene)-1,3-dihydro-
indol-2-one (26). Compound 26 eluted with CHCl3/Et3N (99:1)
(65% yield); 1H NMR (DMSO-d6, 400 MHz), d (ppm): 13.29 (br s,
1H), 10.99 (br s, 1H), 7.99 (d, 1H, J = 1.5 Hz), 7.87 (s, 1H), 7.68 (d,
1H, J = 1.5 Hz), 7.47 (dd, 1H, J = 8.2, 1.5 Hz), 7.35 (s, 1H), 6.89 (d,
1H, J = 8.2 Hz), 6.84 (m, 1H), 6.80 (d, 1H, J = 3.3 Hz), 6.55 (m, 1H),
6.35 (m, 1H). 13C NMR (DMSO-d6, 100 MHz), d (ppm): 169.3,
153.8, 142.0, 138.4, 129.6, 127.0, 126.0, 125.8, 124.2, 122.4,
120.7, 116.4, 113.9, 112.0, 111.6, 109.8, 104.0. ESI-HRMS (m/z)
[M+H]+ calcd, 277.0971; obs, 277.0969.
4.6.6. General procedure for 5-amino-3-substituted-1,3-
dihydro-indol-2-one
The reaction mixture of the proper 5-nitro-3-substituted-1,3-
dihydro-indol-2-one (1 mmol) and SnCl2ꢀ2H2O (10 mmol) in EtOH
(8 mL) and 1% HCl (0.08 mL) was heated at 150 °C for 4 h. After
cooling to room temperature, the mixture was evaporated and
the residue was chromatographed on silica gel, to give the target
compounds.
4.6.5.2. 5-(1-Methyl-1H-pyrazol-4-yl)-3-(1H-pyrrol-2-ylmethyl-
ene)-1,3-dihydro-indol-2-one (27). Compound 27 eluted with
CHCl3/Et3N, (99:1) (70% yield); 1H NMR (DMSO-d6, 400 MHz), d
(ppm): 13.33 (br s, 1H), 10.87 (br s, 1H), 8.03 (s. 1H), 7.87 (d, 1H,
J = 1.9 Hz), 7.81 (s, 1H), 7.78 (s, 1H), 7.35 (s, 1H), 7.32 (dd, 1H,
J = 8.3, 2.0 Hz), 6.83 (d, 1H, J = 8.2 Hz), 6.80 (m, 1H), 6.35 (m, 3H),
3.48 (s, 3H). 13C NMR (DMSO-d6, 100 MHz), d (ppm): 169.3,
137.3, 135.6, 129.6, 127.1, 126.4, 126.1, 125.8, 125.7, 123.7,
122.5, 120.2, 117.0, 115.4, 111.5, 109.8. ESI-HRMS (m/z) [M+H]+
calcd, 291.1245; obs, 291.1237.
4.6.6.1. 5-Amino-3-(1H-pyrrol-2-ylmethylene)-1,3-dihydro-in-
dol-2-one (32). Compound 32 eluted with AcOEt/EtOH/Et3N
(98:1:1) (70% yield); 1H NMR (DMSO-d6, 400 MHz), d (ppm):
13.42 (br s, 1H), 10.46 (br s, 1H), 7.28 (s, 1H), 7.28 (m,
1H), 6.81 (d, 1H, J = 2.2 Hz), 6.79 (m, 1H), 6.56 (d, 1H,
J = 8.1 Hz) 6.41 (dd, 1H, J = 8.1, 2.2 Hz), 6.30 (m, 1H), 4.66
(br s, 2H). 13C NMR (DMSO-d6, 100 MHz),
d (ppm): 174.2,
148.6, 135.2, 134.7, 130.9, 130.3, 130.1, 124.8, 123.3, 118.4,
116.3, 115.1, 109.9. ESI-HRMS (m/z) [M+H]+ calcd, 226,0980;
obs, 226.0966.
4.6.5.3. 3-(3,5-Dimethyl-1H-pyrrol-2-ylmethylene)-5-furan-3-
yl-1,3-dihydro-indol-2-one (28). Compound 28 eluted with
CHCl3/Et3N (99:1) (60% yield); 1H NMR (DMSO-d6, 400 MHz), d
(ppm): 13.37 (br s, 1H), 10.82 (br s, 1H), 8.11 (m, 1H), 7.97 (d, 1H,
J = 1.6 Hz), 7.71 (m, 1H), 7.64 (s, 1H), 7.33 (dd, 1H, J = 8.0, 1.6 Hz),
6.99 (m, 1H), 6.84 (d, 1H, J = 8.0 Hz), 6.01 (d, 1H, J = 2.5 Hz), 2.32 (s,
3H), 2.31 (s, 3H). 13C NMR (DMSO-d6, 100 MHz), d (ppm): 164.6,
143.9, 138.2, 137.2, 135.9, 131.9, 126.8, 126.5 (2C), 125.0, 123.7,
123.2, 115.5, 112.7, 112.6, 109.5, 109.0, 13.5, 11,4. ESI-HRMS (m/z)
[M+H]+ calcd, 305.1290; obs, 305.1275.
4.6.6.2. 5-Amino-3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,
3-dihydro-indol-2-one (33). Compound 33 eluted with AcOET/
EtOH/Et3N (98:1:1) (70% yield); 1H NMR (DMSO-d6, 400 MHz), d
(ppm): 13.38 (br s, 1H), 10.28 (br s, 1H), 7.28 (s, 1H), 6.89 (d, 1H,
J = 2.0 Hz), 6.56 (d, 1H, J = 8.1 Hz) 6.38 (dd, 1H, J = 8.1, 2.2 Hz),
5.96 (d, 1H, J = 2.2 Hz), 4.53 (s, 2H), 2.30 (s, 3H), 2.26 (s, 3H).
NOESY: dipolar interaction detected between H4 (d 6.89) and H8
(d 7.28). 13C NMR (DMSO-d6, 100 MHz), d (ppm): 169.3, 143.2,
134.7, 130.3, 129.6, 126.5, 126.4, 122.0, 114.2, 112.5, 112.1,
109.7, 104.4, 13.5, 11.2. ESI-HRMS (m/z) [M+H]+ calcd, 254.1293;
obs, 254.1284.
4.6.5.4. 3-(3,5-Dimethyl-1H-pyrrol-2-ylmethylene)-5-pyridin-
3-yl-1,3-dihydro-indol-2-one (29). Compound 29 eluted with
CHCl3/Et3N (99:1) (70% yield); 1H NMR (DMSO-d6, 400 MHz), d
(ppm): 13.37 (br s, 1H), 10.90 (br s, 1H), 8.95 (d, 1H, J = 1.8 Hz),
8.50 (d, 1H, J = 4.5 Hz), 8.18 (s, 1H), 8.09 (m, 1H), 7.76 (s, 1H),
7.44 (m, 1H), 6.96 (d, 1H, J = 8.1 Hz), 6.01 (s, 1H), 2.32 (s, 3H),
2.31 (s, 3H). 13C NMR (DMSO-d6, 100 MHz), d (ppm): 169.4,
147.5, 147.3, 137.9, 136.03, 136.0, 133.4, 132.1, 129.8, 126.75,
126.72, 124.1, 123.5, 116.5, 112.5, 112.1, 109.6, 13.4, 11.28. ESI-
HRMS (m/z) [M+H]+ calcd, 316.1449; obs, 316.1439.
4.6.6.3. 5-Amino-3-(4-methoxybenzylidene)indolin-2-one
(34). Compound 34 eluted with CH2Cl2/AcOEt/EtOH/Et3N
(77:20:2:1) (70% yield); E isomer: 1H NMR (DMSO-d6, 400 MHz),
d (ppm): 10.07 (br s, 1H), 7.67 (part of an AA0BB0 system, 2H,
‘J’ = 8.8 Hz), 7.44 (s, 1H), 7.06 (part of an AA0BB0 system, 2H,
‘J’ = 8.8 Hz), 7.02 (d, 1H, J = 2.0 Hz), 6.55 (d, 1H, J = 8.2 Hz), 6.46
(dd, 1H, J = 8.2, 2.0 Hz), 4.67 (br s, 2H), 3.83 (s, 3H). NOESY: dipolar
interaction detected between H4 (d 7.02) and H10/H14 (d 7.67). 13C
4.6.5.5. 3-[2,30]Bifuranyl-5-ylmethylene-5-nitro-1,3-dihydro-in-
dol-2-one(30) E isomer. Compound30eluting with CH2Cl2/AcOEt/

L. Mologni et al. / Bioorg. Med. Chem. 18 (2010) 1482–1496
1495
NMR (DMSO-d6, 100 MHz),
d
(ppm): 13C NMR (DMSO-d6,
(0.2 mL, 1 mmol) was added at 0 °C. After 10 min, 5-amino-3-
(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one
(24) (50 mg, 0.2 mmol), and HBTU (77 mg, 0.2 mmol) were added.
The reaction mixture was stirred under argon atmosphere for 12 h
and concentrated under reduced pressure. The residue was chro-
matographed on silica gel and eluted with CHCl3/EtOH/Et3N,
(97:2:1) to give 20 mg (21%). 1H NMR (DMSO-d6, 400 MHz), d
(ppm): 13.36 (br s, 1H), 10.77 (br s, 1H), 10.08 (br s, 1H), 7.92
(m, 2H), 7.41 (m, 3H), 6.83 (d, 1H), 6.0 (s, 1H), 3.5 (s, 2H), 2.35 (s,
3H), 2.30 (s, 3H), 2.27 (s, 3H), 2.13 (s, 3H). 13C NMR (DMSO-d6,
100 MHz), d (ppm): 169.3, 166.4, 143.2, 140.0, 134.7, 130.3,
130.6, 129.8, 129.6, 127.8, 126.5, 126.4, 122.0, 114.2, 112.5,
112.1, 109.7, 104.4, 58.4, 54.5, 52.4, 45.4, 13.5, 11.2. ESI HRMS
(m/z) [M+H]+ calcd, 470.2556; obs, 470.2556.
100 MHz), d (ppm): 168.8, 160.2, 143.1, 134.8, 133.2, 131.3,
126.8, 126.7, 121.7, 115.5, 114.2, 110.2, 108.4, 55.3. Z isomer: 1H
NMR (DMSO-d6, 400 MHz), d (ppm): 10.12 (br s, 1H), 8.44 (part
of an AA0BB0 system, 2H, ‘J’ = 8.9 Hz), 7.46 (s, 1H), 7.00 (part of an
AA0BB0 system, 2H, ‘J’ = 8.9 Hz), 6.87 (d, 1H, J = 2.1 Hz), 6.49 (d,
1H, J = 8.1 Hz), 6.43 (dd, 1H, J = 8.1, 2.1 Hz), 4.55 (br s, 2H), 3.82
(s, 3H). NOESY: dipolar interaction detected between H4 (d 7.46)
and H8 (d 6.87). 13C NMR (DMSO-d6, 100 MHz), d (ppm): 167.3,
160.9, 143.1, 135.4, 134.2, 131.2, 127.1, 125.9, 125.3, 114.3,
113.6, 109.6, 105.6, 55.3. ESI-HRMS (m/z) [M+H]+ calcd,
267.1128; obs, 267.1140.
4.6.6.4. 5-Amino-3-(4-diethylamino-benzylidene)-1,3-dihydro-
indol-2-one (35). Compound 35 eluted with CH2Cl2/AcOEt/EtOH/
Et3N (77:20:2:1) (70% yield);
E
isomer: 1H NMR (DMSO-d6,
Acknowledgements
400 MHz), d (ppm): 9.96 (br s, 1H), 7.60 (part of an AA0BB0 system,
2H, ‘J’ = 9.0 Hz), 7.38 (s, 1H), 7.20 (d, 1H, J = 2.2 Hz), 6.74 (part of an
AA0BB0 system, 2H, ‘J’ = 9.0 Hz), 6.56 (d, 1H, J = 8.1 Hz), 6.45 (dd, 1H,
J = 8.1, 2.2 Hz), 4.85 (br s, 2H), 3.41 (q, 4H, J = 7.0 Hz), 1.13 (t, 6H,
J = 7.0 Hz). NOESY: dipolar interaction detected between H4 (d
7.20) and H10/H14 (d 7.60). 13C NMR (DMSO-d6, 100 MHz), d
(ppm): 169.5, 148.7, 142.5, 136.3, 133.0, 132.2 (2C), 122.8, 122.5,
120.6, 114.7, 110.9 (2C), 109.9, 109.1, 43.8 (2C), 12.5 (2C). Z iso-
mer: 1H NMR (DMSO-d6, 400 MHz), d (ppm): 9.99 (s, 1H), 8,38
(part of an AA0BB0 system, 2H, ‘J’ = 9.1 Hz), 7.32 (s, 1H), 6.85 (d,
1H, J = 2.2 Hz), 6.69 (part of an AA0BB0 system, 2H, ‘J’ = 9.1), 6.50
(d, 1H, J = 8.1 Hz), 6.41 (dd, 1H, J = 8.1, 2.2 Hz), 4.85 (br s, 2H),
3.41 (q, 4H, J = 7.0 Hz), 1.11 (t, 6H, J = 7.0 Hz). NOESY: dipolar inter-
action detected between H4 (d 6.85) and H8 (d 7.32). 13C NMR
(DMSO-d6, 100 MHz) d (ppm): 167.7, 149.1, 142.2, 136.6, 134.9
(2C), 130.9, 126.8, 121.5, 121.1, 113.5, 110.5 (2C), 109.2, 105.2,
43.8 (2C), 12.5 (2C). ESI-HRMS (m/z) [M+H]+ calcd, 308.1762; obs,
308.1762.
We thank Oriano Marin at University of Padua, Italy, for pep-
tide synthesis; Michela Viltadi, Sara Cazzaniga and Elisa Sala for
technical assistance in enzymatic assays; Luca Menilli, Davide
Pasin, Pietro Marafini, Camilla Brogiato, Giulia De Biasi and An-
drea Mirarco for technical help in compound synthesis. This
work was supported by the Italian Association for Cancer Re-
search (AIRC; grant number: IG-4637); the Italian Government
MIUR-COFIN program; EU Protein Kinase Research network
(Grant number: 503467).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.01.011.
References and notes
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4.6.6.6. 5-Amino-3-[2,30]bifuranyl-5-ylmethylene-1,3-dihydro-
indol-2-one (37). Compound 37 eluted with CH2Cl2/AcOEt/n-
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127.6, 127.18, 123.3, 121.9, 121.3, 116.6, 115.1, 115.0, 113.3. ESI-
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