Synthesis, modeling, and RET protein kinase inhibitory activity of 3- and 4-substituted β-carbolin-1-ones

Article abstract of DOI:10.1021/jm8007823

A series of β-carbolin-2-ones and 3,10-dihydro-2H-azepino[3,4-b]indol- 1-ones have been designed, synthesized, and evaluated as RET protein kinase inhibitors on the basis of their structural similarity with the prototype indolin-2-one RPI-1. Some β-carbolin-2-ones (structure 2) showed an ability to inhibit RET enzymatic activity in vitro and proliferation of RETC634R oncogene-transformed NIH3T3 cells comparable to that of the reference compound. The docking analysis of the interaction of these compounds with the crystallographic structure of RET tyrosine kinase domain suggested a new binding interaction scheme different from the one proposed during their design. The rigid structure of the compounds of this series represents a new scaffold with potential advantages in the design of RET protein kinase inhibitors.

Full text of DOI:10.1021/jm8007823

J. Med. Chem. 2008, 51, 7777–7787
7777
Synthesis, Modeling, and RET Protein Kinase Inhibitory Activity of 3- and 4-Substituted
ꢀ-Carbolin-1-ones
Raffaella Cincinelli,§ Giuliana Cassinelli,¶ Sabrina Dallavalle,*,§ Cinzia Lanzi,*,¶ Lucio Merlini,§ Maurizio Botta,*,#
Tiziano Tuccinardi,† Adriano Martinelli,† Sergio Penco,§ and Franco Zunino¶
Dipartimento di Scienze Molecolari Agroalimentari, UniVersita` di Milano, Via Celoria 2, 20133 Milano, Italy, Unita` di Chemioterapia e
Farmacologia Antitumorale Preclinica, Dipartimento di Oncologia Sperimentale e Laboratori, Fondazione IRCCS Istituto Nazionale dei
Tumori, Via Venezian 1, 20133 Milano, Italy, Dipartimento Farmaco Chimico Tecnologico, UniVersita` degli Studi di Siena, Via De Gasperi 2,
53100 Siena, Italy, and Dipartimento di Scienze Farmaceutiche, UniVersita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy
ReceiVed June 27, 2008
A series of ꢀ-carbolin-2-ones and 3,10-dihydro-2H-azepino[3,4-b]indol-1-ones have been designed,
synthesized, and evaluated as RET protein kinase inhibitors on the basis of their structural similarity with
the prototype indolin-2-one RPI-1. Some ꢀ-carbolin-2-ones (structure 2) showed an ability to inhibit RET
enzymatic activity in vitro and proliferation of RETC634R oncogene-transformed NIH3T3 cells comparable
to that of the reference compound. The docking analysis of the interaction of these compounds with the
crystallographic structure of RET tyrosine kinase domain suggested a new binding interaction scheme different
from the one proposed during their design. The rigid structure of the compounds of this series represents a
new scaffold with potential advantages in the design of RET protein kinase inhibitors.
Chart 1. Configurational Equilibrium of RPI-1
Introduction
Gain-of-function mutations of the RET gene have been
implicated as driving oncogenic alterations in thyroid cancer.1,2
RET encodes the receptor tyrosine kinase for glial cell line-
derived neurotrophic family factors. Chromosomal rearrange-
ments known as RET/PTCs are specifically detected in a subset
of papillary thyroid carcinomas, whereas missense point muta-
tions are associated with the medullary variant (MTCa) occurring
either sporadically or in a familial pattern.3 In particular, somatic
mutations of RET are found in sporadic cases and germline
mutations have been identified as the cause of multiple endocrine
neoplasia type 2 (MEN 2) inherited cancer syndromes which
include MTC as a main component of the disease. Both germline
and somatic RET mutations produce dominant-acting oncogenic
proteins endowed with constitutive tyrosine kinase activity.1
Activated oncogenes involved in tumor pathogenesis are
attractive targets for new selective therapies.4 The potential role
of RET oncogenes and their products as targets for thyroid
cancer therapy has been supported by several preclinical studies
using both gene therapy and pharmacological approaches.5,6 In
such studies, the inhibition of RET activity has been shown to
cause in vitro reversion of the cell transformed phenotype and
to produce an antitumor effect against in vivo human MTC
xenografts. The occurrence of tumor regressions associated with
apoptosis and reduced angiogenesis in animals treated with anti-
Ret targeted therapy has also been documented.7-9 These effects
are indeed consistent with the concept of “oncogene addiction”
whereby RET oncogene-harboring thyroid cancers may depend
on RET oncogenic pathways for their sustained proliferation
and survival.10 RET targeting thus appears to be a promising
approach for the treatment of specific thyroid cancers, although
a clinically useful option is still not available.
Among the different strategies developed to abrogate RET
oncogenic activity, the use of small molecule inhibitors is
currently the most suitable for clinical testing. Several tyrosine
kinase inhibitors have been reported to inhibit RET activity
including natural11 and synthetic compounds belonging to
different structural classes.5,6 A few of these compounds, all
characterized by multikinase inhibitory activity, have recently
entered phase II clinical trials, such as the anilinoquinazoline
ZD6474 (vandetanib), the nicotinamide AMG706 (motesanib),
the bis-arylurea BAY43-9006 (sorafenib), and the indolinone
SU11248 (sunitinib).12-14
The indolinone-based structure represents a useful scaffold
for the design of tyrosine kinase inhibitors.15 We previously
reported the in vitro and in vivo activity profile of the indolin-
2-one compound RPI-1 (formerly Cpd1),7,9,16,17 an orally
available inhibitor of the RET and MET tyrosine kinases.18
Similar to other indolin-2-ones,19 it undergoes configurational
equilibrium. In polar solvents the RPI-1 E configuration (Chart
1) is favored. However, in a recent modeling study of the RET
TK binding domain,20 RPI-1 interacted with the model only in
the Z configuration. This finding is consistent with crystal-
lographic studies showing that other indolin-2-ones bind to ATP
* To whom correspondence should be addressed. For S.D.: phone,
+39(0)250316818; fax, +39(0)250316801; e-mail, sabrina.dallavalle@unimi.it.
For C.L.: phone, +39(0)223902627; fax, +39(0)223902692; e-mail:
cinzia.lanzi@istitutotumori.mi.it. For M.B.: phone, +39(0)577234306; fax,
+39(0)577234333; e-mail, botta@unisi.it.
§
Universita` di Milano.
Istituto Nazionale dei Tumori.
Universita` di Siena.
Universita` di Pisa.
¶
#
†
a Abbreviations: MTC, medullary variant carcinoma; MEN 2, multiple
endocrine neoplasia type 2; rmsd, root-mean-square deviation; MD,
molecular dynamics; MM-GBSA, molecular mechanics generalized Born
surface area; MBP, myelin basic protein; CG, conjugate gradient; PME,
particle mesh Ewald; GAFF, general Amber force field.
10.1021/jm8007823 CCC: $40.75
2008 American Chemical Society
Published on Web 11/19/2008

7778 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Chart 2. General Structure of the New Compounds
Cincinelli et al.
Scheme 1a,b
a Reagents and conditions: (a) ꢀ-alanine ethyl ester hydrochloride, WSC, HOBT, TEA, THF, room temp, 24 h, 96%; (b) Mel, CH3CN, K2C03, reflux,
30 h, 72%; (c) LiOH ·H2O, EtOH, room temp, 48 h, 89-94%; (d) PPA, 95 °C, 90 min to 6 h, 45-68%; (e) NaH, DMF, PMBCI, room temp, 4 h, 50%. (f)
For 8b: 7a, Mel, NaH, DMF, room temp, 2 h, 40%. For 8c: 7b, PMBCI, NaH, DMF, room temp, overnight, 33%. (g) (CF3SO2)2O, Na2CO3, CH2CI2, room
temp, 3-12 h, 26-75%; (h) p-HO-PhB(OH)2, Pd(PPh3)4, toluene, NaHCO3, EtOH, 60-90 min, 12-78%; (i) TFA, reflux, 5 h, 49%. b See Table 1.
binding site of some kinases (e.g., FGFR) in the Z configura-
tion.21 The equilibrium favoring the E configuration could
account for the low potency of RPI-1. In an attempt to improve
the inhibitory properties of indolinone-related compounds, this
study was undertaken to modify the indolinone scaffold
maintaining the critical interactions within the RET kinase
domain.
On these bases, we designed a series of 3,10-dihydro-2H-
azepino[3,4-b]indol-1-ones (1) and of 4- (2) and 3-substituted
ꢀ-carbolin-2-ones (3) that showed a satisfactory superimposition
of the structural features deemed important for activity with
those of both diastereoisomers of RPI-1, i.e., the 5,6-dimethoxy-
indole moiety and the carbonyl group. Furthermore, for the
compounds of the 1 and 2 series there was also a good
superimposition for the phenyl group. Some 3-aryl ꢀ-carbolin-
1-ones derivatives have already been reported by Wang and
co-workers as potent inhibitors of tumor cell proliferation, when
substituted with electron-withdrawing substituents in the indole
ring.22 The general structure of compounds in this series is
reported in Chart 2, and the three-dimensional models of
representative compounds appear in Figure 1. The new com-
pounds were evaluated as RET inhibitors using cell-based and
biochemical assays, and the docking mode of selected com-
pounds was investigated by molecular modeling.
Chemistry
For the synthesis of the 3,10-dihydro-2H-azepino[3,4-b]indol-
1-ones (1) we first followed a procedure described by Me´rour23
that was based on a palladium-mediated Suzuki reaction of an
appropriate arylboronic acid with the enol triflate of an aze-
pino[3,4-b]indole-1,5-dione (9) (Scheme 1). The latter was
obtained by PPA cyclization of the acid 6, in turn prepared by
amidation of a substituted indole-2-carboxylic acid (4) with
ꢀ-alanine ethyl ester hydrochloride, followed by hydrolysis with
lithium hydroxide.
Figure 1. Superimposition between RPI-1 (colored green) and the new
compounds (1a, 2a, and 3a, colored cyan, magenta, and gray,
respectively). The most important pharmacophoric features are also
reported (aromatic and hydrogen bond acceptor and donor features are
reported as orange, red, and cyan spheres, respectively).
In our hands, triflation of the azepinedione 7a gave a mixture
of compounds triflated on the amide and indole NHs. Therefore
compounds 1a-d were only obtained by protecting the amide

Substituted ꢀ-Carbolin-1-ones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7779
Scheme 2a
a For R1-R6, see Table 1. Reagents and conditions: (a) WSC, HOBT, THF, room temp; (b) IBX, EtOAc, 80°C or PCC, CH2Cl2, 40-50 °C; (c) TFA,
CH3CN, 80 °C.
Table 1. Structures of Compounds 1-3
compd
R1
R2
R3
R4
R5
R6
1a
1b
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
2f
2g
2h
2i
3a
3b
3c
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
Ph
Ph
4-OH-Ph
4-OH-Ph
4-OH-Ph
4-OH-Ph
H
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
CH3
CH3
CH3
CH3
CH3
CH3
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
PMBa
H
H
H
H
H
H
H
Figure 2. Inhibition of RET autophosphorylation and expression in
NIH3T3MEN2A cells treated with compound 2b or RPI-1 as reference
compound. Cells were exposed to vehicle (DMSO) or to the indicated
concentrations of the compounds for 24 h. Whole cell lysates were
analyzed by Western blotting with anti-p(Y1062)Ret and anti-Ret
antibodies. A cell extract from untreated parental NIH3T3 cells was
run in parallel. Anti-actin blot is shown as protein loading control.
CH3
4-OCH3-Ph OCH3 OCH3
4-OH-Ph
4-OH-Ph
4-OH-Ph
4-OH-Ph
H
CH3
Ph
3-OH-Ph
4-OH-Ph
H
OCH3 OCH3
OCH3 OCH3
H
OCH3
H
H
CH2-Ph
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
H
H
H
H
H
H
H
or the indole NH with a methyl or a 4-methoxybenzyl group
(8b,c). Because of these difficulties, we designed a more
straightforward and general method that could be applied also
to the synthesis of 5-alkyl substituted azepinoindol-1-ones and
to the synthesis of compounds 2 and 3. Appropriate indole-2-
carboxylic acids were coupled with ꢀ- or a γ-aminoalcohols to
obtain, after IBX oxidation, ꢀ- or a γ-oxoamides that were
cyclized via an intramolecular Friedel-Crafts reaction to give
directly the expected compounds 1-3 (Scheme 2). Alternatively,
condensation with ꢀ- or γ-aminoketones gave directly the
oxoamides ready for cyclization. This approach has been
recently disclosed and used for the synthesis of compounds 2
and 3.24 The synthesis of N-unsubsituted derivatives required
the use of an appropriate R1 protecting group.
CH2-Ph
H
CH3
H
H
Naf-2-CH2
H
H
a PMB ) 4-methoxybenzyl.
1 and 3 were characterized by a lower potency and by lack of
revertant activity. Therefore, only compounds of the series 2
were further subjected to a RET kinase assay to assess direct
enzyme inhibition. The inhibition of RET activation in treated
NIH3T3MEN2A cells was eventually examined by Western
blotting.
The results of the cell-based screening with the new com-
pounds are reported in Table 2, together with RET kinase
inhibition data for selected molecules.
Results and Discussion
Cellular and Biochemical Studies. To assess the effect of
the new compounds on RET-driven cellular transformation,
we tested their ability to affect the proliferation and/or the
transformed morphologic phenotype of NIH3T3 cells stably
transfected with the C634R mutant of RET associated with the
MEN2A syndrome (NIH3T3MEN2A).2,3 Compounds that showed
an antiproliferative activity comparable to that displayed by the
reference molecule RPI-1 or showed an ability to revert the
morphology of the transformed phenotype were further tested
on NIH3T3 and NIH3T3 cells transformed with a non-tyrosine
kinase oncogene (NIH3T3H-RAS) to assess the drug selectivity
against RET-oncogene-expressing cells. Compounds exhibiting
these features belong to the series 2. Compounds of the series
Compounds with structure 1, characterized by a seven-
membered azepinone ring fused to an indole moiety, showed
in general a lower antiproliferative activity than the reference
compound against NIH3T3MEN2A cells and no revertant capacity.
In contrast, compounds with structure 2, in particular 2a,b and
2f-h, appeared to be comparable to the reference compound
RPI-1 as for the activity on NIH3T3MEN2A cells. In addition,
some of them (2a and 2b) induced a partial or transient reversion
of the transformed morphology of these cells. Similar to RPI-
1, 2a and 2b showed a selective antiproliferative activity against
NIH3T3 cells expressing the RET mutant. Moreover, the
remarkable inhibitory activity in the RET kinase assay by both
compounds suggested that the lack of N-methyl in the indole

7780 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Cincinelli et al.
Table 2. In Vitro Activity of Compounds 1-3
antiproliferative activity (IC50 µM)a (72 h)
RET KAd
(IC50 µM)
compd NIH3T3MEN2A
NIH3T3
NIH3T3H-RAS
RPI-1
1a
1b
1c
1d
1e
3.6 ( 1.6b
19.8 ( 7.1
14.4 ( 5.5
18.4 ( 3
6.8 ( 2.5
7.8 ( 2
16.7 ( 5.4
9.9 ( 1.2
4.7 ( 0.3c
1.95 ( 0.2c
17.3 ( 1.1
12.2 ( 1c
15.9 ( 2.6
5.6 ( 0.04
3.3 ( 0.6
3.2 ( 0.3c
7.5 ( 1
16.3 ( 3.7
19.7 ( 2
0.16 ( 0.05
>0.6
1f
1g
2a
2b
2c
2d
2e
13.9 ( 2
7.2 ( 2.2
>30
13.5 ( 0.3
6.6 ( 2
>30
0.11 ( 0.04
0.06 ( 0.02
0.48 ( 0.12
0.60 ( 0.01
>0.6
>0.6
0.25 ( 0.1
0.09 ( 0.01
25 ( 8
>30
2f
10.6 ( 2.5
17.8 ( 0.1
6.7 ( 0.1
17.7 ( 0.6
2g
2h
2i
3a
3b
3c
13.8 ( 2.8
6.1 ( 2
Figure 3. Schematic representation of the main interactions of Z-RPI-1
into the RET binding site.
13.3 ( 1
34.4 ( 1.2
18.4 ( 0.8
energy, with a root-mean-square deviation (rmsd) between the
docked and crystallized geometries in the limit of the crystal-
lographic resolution (the rmsd was less than 1.4 Å, whereas
the crystallographic resolution was greater than 2.5 Å).
We have already reported the docking of the reference
compound RPI-1, and our previous studies suggested that the
active diastereoisomer has the Z configuration.20 Figure 3 shows
a schematic representation of the main interactions of this ligand
inside the RET ATP binding site, with the indolin-2-one central
scaffold that formed two H-bonds with the backbone of Glu805
and Ala807, respectively. These two interactions were supported
by the analysis of the X-ray complexes between different kinases
and inhibitors interacting in the ATP binding site28 and by
pharmacophoric studies.29 Such studies confirmed in fact that
the backbone portion of these two residues play a role in
inhibitor binding, as they make H-bonds with N-1 and N-6 of
the ATP adenine, as well as with most kinase inhibitors. With
regard to the lipophilic interactions, the indolin-2-one group
mainly interacted with Phe735, Val738, and Val804 whereas
the phenolic substituent interacted with Leu730.
Figure 4A shows the docking of compound 2a. Its interactions
were indeed completely different from those hypothesized in
the design step; the nitrogen and the oxygen of the pyridone
ring formed H-bonds with the backbone of key residues Glu805
and Ala807, respectively. The planar 2H-pyrido[3,4-b]indol-
1(9H)-one central scaffold mainly interacted with Leu730,
Val738, Ala756, and Leu881, while the nitrogen of the indole
moiety seemed devoid of important interactions. As for the 4-(p-
hydroxyphenyl) substituent, it was inserted into a pocket
delimited by Leu779, Ile788, Val804, and Ser891 and the
hydroxy group formed an H-bond with Asp892 that constitutes
an H-bond network with Lys758 and Glu775.
a Mean values ( SD from at least two independent experiments per-
formed in duplicate are reported. b Stable reversion of transformed morphol-
ogy of NIH3T3MEN2A cells exposed to 10 µM RPI-1. c Partial or transient
reversion of transformed morphology of NIH3T3MEN2A cells observed
during drug treatment. d Kinase assay using recombinant RET active protein
and MBP as a substrate.
moiety did not abrogate the enzyme inhibitory activity.
These findings were apparently (but see later) in contrast with
the results previously obtained in a series of analogues of the
indolin-2-one RPI-125 in which the presence of N-methyl in the
indole moiety was found to abrogate the inhibitory activity.
The low antiproliferative activity of compounds 2c,d could
be explained by the lack of one or both the methoxy groups on
the indole moiety, a detrimental effect already observed in the
indolinone series.25 Despite the deletion of the phenolic OH,
compound 2g maintained the antiproliferative effect with only
a slight reduction of the inhibitory activity on recombinant
enzyme. At variance, deletion of the whole phenolic ring (2f)
heavily impaired the enzyme inhibitory activity. Finally,
compounds of structure 3, intended to mimic the Z-isomer of
RPI-1, were much less active.
The RET inhibitory activity of compound 2b, identified as the
best inhibitor in these series, was further confirmed on
NIH3T3MEN2A intact cells. Accordingly to the relative activity in
the Ret kinase assay, 2b inhibited Ret tyrosine phosphorylation in
a dose-dependent way showing a slightly higher potency, compared
to RPI-1, upon 24 h of cell treatment. As previously reported for
RPI-1,7 RET kinase inhibition in treated cells was associated with
a reduction of Ret protein expression (Figure 2).
Molecular Modeling. Since the present results seemed in
apparent contrast with previous studies, suggesting a preferential
or exclusive role of the Z isomer of indolinone molecules for
the tyrosine kinase inhibitory activity,19-21 we investigated the
binding interactions between the new derivatives and the RET
tyrosine kinase domain. An automated docking analysis of
compounds reported in Table 1 was performed into the
crystallographic structure of RET kinase (PDB code 2ivu26)
using GOLD 3.2 software.27
The parameters chosen in GOLD (see Experimental Section)
were tested for their ability to reproduce the crystallized binding
geometry of the 4-anilinoquinazoline ZD6474. The docking
software easily found the binding geometry corresponding to
the crystallized complex among the solutions with the lowest
The docking studies highlighted that the 3-substitution
determined the weakening of the H-bond between the oxygen
of the pyridone ring and Ala807 and the loss of the H-bond
between the nitrogen of the pyridone ring and Glu805 (see
docking of compound 3a in Figure 4B). Finally the docking of
compound 1a (Figure 4C) revealed that the change of geometry
due to the seven-membered structure of the azepine ring fused
to the indole determined a completely different binding mode,
with the formation of two H-bonds with D892 and the loss of
the fundamental interactions with the key residues Glu805 and
Ala807.
As shown in Figure 5A, the docking of compound 2b, which
differs from 2a (Figure 4A) only for the methyl substitution on

Substituted ꢀ-Carbolin-1-ones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7781
Figure 4. Docking of compounds 2a (A), 3a (B), and 1a (C) into the RET binding site.
Figure 5. Docking of compounds 2b (A), 2h (B), and 2d (C) into the RET binding site.
the indole nitrogen, highlighted the same binding disposition
and the same interactions described above. Furthermore, the
N-methyl substituent showed lipophilic interactions with Leu730.
Figure 5B shows the docking of compound 2h which differs
from compound 2b (Figure 5A) for the 4-(m-hydroxyphenyl)
substituent. In agreement with a profile of activity in the
antiproliferative and biochemical tests very similar to that of
2b, it showed all the main interactions of 2b, in particular the
H-bond between the phenolic OH group and Asp892.
remaining simulation time was constant. Analyzing the rmsd
from the X-ray structure of all the heavy atoms of the complexes,
we observed that, after an initial increase, the rmsd did not
exceed the value of 1.4 Å (see Figure 6B), suggesting that our
MD procedure was correct.
As regard the two ligands, during the whole MD simulation
their relative position appeared to change only slightly, since
the rmsd of the heavy atoms of the ligands between the starting
and the final structures of the MD simulation was 1.2 Å for 2b
and 1.1 Å for 2d.
Finally, compounds 2c and 2d are both characterized by the
absence of the 7-methoxy substituent, and their low activity
seemed to suggest a role for this group. However, in all the
already mentioned compounds, this substituent did not show
any important interaction with the enzyme and was exposed to
the solvent. As shown in Figure 5C, compound 2d showed the
same interaction scheme; it possessed all the main interactions
of the other compounds, and the 7-methoxy substituent was
exposed to the solvent. Therefore, these data suggested that its
lower affinity could be determined by different solvation effects.
In order to investigate this hypothesis, compounds 2b and
2d were subjected to 1 ns of molecular dynamics (MD)
simulation with explicit water, followed by a molecular
mechanics generalized Born surface area (MM-GBSA) analy-
sis.30 This approach has been shown to be able to estimate the
ligand-receptor energy interaction.31-35 It averages contribu-
tions of gas-phase energies and solvation free energies calculated
for snapshots of the complex molecule as well as the unbound
components, extracted from MD trajectories according to the
procedure fully described in the Experimental Section.
As shown in Table 3, the two ligands possessed a very similar
binding interaction scheme: the nitrogen and the oxygen of the
pyridone ring of both ligands maintained the H-bond with the
backbone of Glu805 and Ala807, respectively, the 4-(p-
hydroxyphenyl) substituent maintained an H-bond with the
network constituted by Lys758 and Glu775 and Asp892, but
instead of interacting with the carboxylic group of Asp892, it
formed an H-bond with Glu775 and occasionally interacted with
the backbone of Asp892.
The two MD trajectories were then analyzed through the MM-
GBSA method. As shown in Table 4, the molecular mechanical
energy (∆EMM) of the interaction of the ligands with RET (i.e.,
the sum of the electrostatic and van der Waals energies) was
very similar for both compounds. However, the solvation free
energy (∆GBSol), which includes the polar solvation free energy
calculated with the generalized Born (GB) approximation model
and the nonpolar part obtained by fitting solvent accessible
surface area (SASA), was more favorable for compound 2b.
Therefore, even if the energy difference between the interaction
of the two compounds was in the same order of magnitude of
the standard deviation of error (see Table 4), the effect of the
As shown in Figure 6A, after 200 ps of MD, both complex
systems reached equilibrium, since the total energy for the

7782 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Cincinelli et al.
Table 4. MM-GBSA Results for Compounds 2b and 2da
2b (STD)
2d (STD)
Ele
VdW
Eint
-41.9 (4.1)
-38.0 (3.4)
0.0 (0.0)
-42.9 (3.8)
-36.5 (3.6)
0.0 (0.0)
∆EMM
GBSur
GB
∆GBSolv
∆GGBSA
-79.9 (3.9)
-5.8 (0.1)
41.1 (1.9)
35.3 (1.9)
-44.6 (3.1)
-79.4 (4.3)
-5.3 (0.1)
42.7 (2.3)
37.4 (2.3)
-42.0 (3.5)
a ∆EMM is the sum of the electrostatic (Ele), van der Waals (VdW), and
internal (Eint) energies. ∆GBSolv is the sum of the polar (GB) and nonpolar
(GBSur) solvation free energy. Finally ∆GGBSA is the sum of the molecular
mechanical and solvation free energy. STD means the standard deviation.
Data are expressed as kcal/mol.
activity against the RET protein kinase, which is correlated with
an antiproliferative activity on NIH3T3MEN2A cells comparable
to that of the reference compound RPI-1 which is correlated
with a revertant activity on NIH3T3MEN2A cells. No precise
correlations between inhibitory activity against RET kinase and
antiproliferative activity could be detected for compounds of
this series because it is likely that the cell growth inhibition
reflects inhibitory effects on multiple targets. Nonetheless,
compounds of series 2 showed a preferential antiproliferative
activity on RET mutant-expressing cells. As for SAR, it appears
that methylation of the indole nitrogen is not detrimental but
seems to increase the activity (2b vs 2a). The presence of the
two methoxy groups in positions 6 and 7 of the indole moiety
is important in maintaining the activity, and the presence of a
3- or 4-OH substituted phenyl ring in position 4 is also
beneficial, compound 2b being the most active, on both the
enzyme and cells. All these results are well explained by the
analysis of the docking of the compounds in the crystallographic
model of the enzyme. The same analysis also explains the lack
of activity of compounds with structure 3, where the ring
attached to position 3 seems devoid of favorable interactions.
For these reasons, this latter group of compounds was not
investigated further. The same could be possible for compounds
of structure 1, where most probably the change of geometry
due to the seven-membered structure of the azepine ring fused
to the indole moves the substituent in position 5 to another zone
of unfavorable interactions.
Figure 6. Results of the MD simulation of compounds 2b (black)
and 2d (red) complexed with RET: (A) total energy (kcal/mol) of the
system plotted versus time (ps); (B) rmsd in angstroms (Å), between
the protein and the starting X-ray structure for all the heavy atoms,
versus time (ps).
Table 3. Hydrogen Bonds Analysis of 2b and 2d with RET during the
Simulation of Both Complexes
The compounds structurally most similar to the isomer E of
RPI-1, i.e., 2a and 2b, are the most active of the series. At first
glance, this finding could have appeared surprising, since on
the basis of previous studies, the isomer Z is likely responsible
of the inhibitory effects.21 However, considering the results of
the present docking analysis, the strong H-bond interactions with
Glu805 and Ala807 of the enzyme ATP binding site do not
occur with the indole NH and the CO amide groups of 2, as we
hypothesized, but with the amide NH and CO groups of the
pyridone ring. The consequence is that the compounds dock
themselves in the binding site in a position flipped of roughly
180° with respect to RPI-1, and the phenolic OH in position 4
of 2a and 2b interacts with amino acids different from those
interacting with RPI-1. The docking analysis also explains the
slightly better inhibitory activity of 2b vs 2a in the kinase assay,
on the basis of the absence of H-bond interactions with the
indole NH and of the existence of lipophilic interactions with
Leu730. Notably, in spite of the different binding mode to the
RET kinase, compound 2b maintained the ability to inhibit RET
protein expression, in addition to tyrosine phosphorylation, as
previously described for RPI-1.7
donor
acceptor H
acceptor
distance (Å)
% occupied
2b (R ) OCH3)
E775@OE2
E805@O
LIG@O3
LIG@O1
LIG@H1
LIG@N2
A807@H
D892@H
LIG@O1
LIG@H2
A807@N
D892@N
2.63 (0.11)
2.70 (0.19)
2.83 (0.22)
3.10 (0.16)
97.0
94.6
94.1
54.4
2d (R ) H)
LIG@O3
E805@O
E775@OE2
LIG@O1
A807@H
LIG@N2
LIG@H1
D892@H
A807@N
LIG@H2
LIG@O1
D892@N
2.79 (0.09)
2.92 (0.12)
2.58 (0.10)
3.11 (0.16)
100
99.8
98.0
54.1
7-methoxy substituent on the activity could be explained in terms
of solvation free energy, in agreement with our hypothesis.
Conclusions
The results reported above indicate that compounds with
structure 2 (e,g, 2a, 2b, and 2h) exhibit a remarkable inhibitory
In conclusion, we have found a new series of inhibitors of
RET protein kinase with an inhibitory and antiproliferative

Substituted ꢀ-Carbolin-1-ones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7783
7,8-Dimethoxy-3,4-dihydro-2H,10H-azepino[3,4-b]indole-1,5-
dione (7a). A mixture of 6a(0.76 g, 2.6 mmol) and PPA (58 g)
was stirred at 95 °C for 6 h. After the mixture was cooled, iced
water was added and the aqueous phase was continuously extracted
with a solution CH2Cl2/MeOH 95:5. Purification by flash chroma-
tography (eluent, CH2Cl2/MeOH 95:5) afforded the title compound
activity comparable to that of RPI-1, which may provide a new
lead candidate. Since this series is characterized by lack of
changes of configuration, the rigid structure could provide
biochemical and pharmacological advantages over conventional
indolinones.
1
in a 45% yield. H NMR (DMSO-d6) δ: 12.20 (s, 1H, NH), 8.68
Experimental Section
(t, 1H, CONH, J ) 5.9 Hz), 7.75 (s, 1H, 1 Ar), 6.99 (s, 1H, 1Ar),
3.78 (s, 6H, 2OMe), 3.3-3.5 (m, 2H, CH2), 2,75-2.85 (m, 2H,
CH2). Anal. (C14H14N2O4) C, H, N.
General Chemical Methods. All reagents and solvents were
reagent grade or were purified by standard methods before use.
Melting points were determined in open capillaries on a Bu¨chi
melting point apparatus and are uncorrected. Column chromatog-
raphy was carried out on flash silica gel (Merck 230-400 mesh).
TLC analysis was conducted on silica gel plates (Merck 60F254).
NMR spectra were recorded at 300 MHz with a Bruker instrument.
Chemical shifts (δ values) and coupling constants (J values) are
given in ppm and Hz, respectively. Compounds 2 and 3 were
prepared as already described.24
3-[(5,6-Dimethoxy-1H-indole-2-carbonyl)amino]propionic Acid
Ethyl Ester (5a). A solution of 5,6-dimethoxy-1H-indole-2-carboxylic
acid (1.46 g, 6.6 mmol), EDC (1.52 g, 7.9 mmol), and HOBt (1.07 g,
7.9 mmol) in 36 mL of THF was stirred 8 h at room temperature.
Then ꢀ-alanine ethyl ester hydrochloride (3.04 g, 19.8 mmol) and TEA
(2.74 mL, 19.8 mmol) were added. The mixture was stirred overnight
at room temperature. After partial evaporation of the solvent, water
and HCl 10% (12 mL) were added. The precipitate was filtered to
give 2.02 g of the title compound. Yield 96%. 1H NMR (300 MHz,
CDCl3) δ: 9.15 (1H, NH), 7.00 (s, 1H, 1 Ar), 6.82 (s, 1H, 1 Ar),
6.65-6.75 (m, 2H, CHd + NHCO), 4.20 (q, 2H, OCH2, J ) 7.1
Hz), 3.98 (s, 3H OMe), 3.95 (s, 3H, OMe), 3.65-3.75 (m, 2H, CH2N),
2.65 (t, 2H, CH2 J ) 6.9 Hz), 1.25 (t, 3H, CH3, J ) 7.1 Hz). Anal.
(C16H20N2O5) C, H, N.
7,8-Dimethoxy-10-methyl-3,4-dihydro-2H,10H-azepino[3,4-
b]indole-1,5-dione (7b). A mixture of 6b (324 mg, 1.06 mmol)
and PPA (13 g) was stirred at 90 °C for 90 min. After the mixture
was cooled, an amount of 50 mL of iced water was added and the
aqueous phase was extracted with CH2Cl2. The organic layer was
dried over Na2SO4 and evaporated in vacuo to afford 208 mg of
1
the title compound. Yield 68%. H NMR (DMSO-d6) δ: 8.65 (t,
1H, CONH, J ) 5.9 Hz), 7.74 (s, 1H, 1Ar), 7.19 (s, 1H, 1Ar),
3.99 (s, 3H, 2OMe), 3.87 (s, 3H, OMe), 3.80 (s, 3H, NMe),
3.45-3.50 (m, 2H, CH2), 2,72-2.78 (m, 2H, CH2). Anal.
(C15H16N2O4) C, H, N.
7,8-Dimethoxy-10-(4-methoxybenzyl)-3,4-dihydro-2H,10H-
azepino[3,4-b]indole-1,5-dione (7c). A suspension of 7a (0.843
g, 3.07 mmol) in DMF (15.2 mL) was cooled to 0 °C. Then NaH
(60% in mineral oil, 0.843 g, 7.37mmol) was added. The solution
was stirred for 1 h at room temperature. Then PMBCl (1.19 g, 7.37
mmol) was dropped and stirring was continued for further 3 h. The
mixture was added to water and extracted twice with dichlo-
romethane. The organic layer was dried over Na2SO4 and evapo-
rated in vacuo. Flash chromatography (eluent from ethyl acetate/
hexane 85:15 to ethyl acetate) afforded 7c. Yield 50%, mp 260
°C. 1H NMR (DMSO-d6) δ: 8.69 (t, 1H, CONH, J ) 5.9 Hz), 7.73
(s, 1H, 1 Ar), 7.18 (s, 1H, 1 Ar), 7.11 (d, 2H, 2Ar, J ) 8.6 Hz),
6.84 (d, 2H, 2Ar, J ) 8.6 Hz), 5.82 (s, 2H, CH2Ph), 3.79 (s, 6H,
2OMe), 3.69 (s, 3H, OMe), 3.3-3.4 (m, 2H, CH2), 2,74-2.82 (m,
2H, CH2). Anal. (C22H22N2O5) C, H, N.
7,8-Dimethoxy-2,10-dimethyl-3,4-dihydro-2H,10H-azepino[3,4-
b]indole-1,5-dione (8b). To a suspension of 7a (314 mg, 1.14 mmol)
in DMF (4.35 mL), NaH (60% in mineral oil, 65 mg, 2.69 mmol)
was added. The solution was stirred for 1 h at room temperature, and
then MeI (389 mg, 2.74 mmol) was dropped and stirring was continued
for 1 h. The mixture was added to water and extracted twice with
dichloromethane. The organic layer was dried over Na2SO4 and
evaporated in vacuo. Flash chromatography (eluent, CH2Cl2/MeOH
98:2) afforded 8b. Yield 40%, mp 182 °C. 1H NMR (CDCl3) δ: 7.82
(s, 1H, 1Ar), 6.82 (s, 1H, 1Ar), 4.02, 3.95, 3.95, (2s, 9H, 2OMe and
NMe), 3.7-3.8 (m, 2H, CH2), 3.25 (s, 3H, NMe), 2.8-2.9 (m, 2H,
CH2). Anal. (C16H18N2O4) C, H, N.
3-[(5,6-Dimethoxy-1-methyl-1H-indole-2-carbonyl)amino]pro-
pionic Acid Ethyl Ester (5b). To a solution of 5a (500 mg, 1.56
mmol) in 25 mL of acetonitrile, K2CO3 (970 mg, 7.02 mmol) and
MeI (554 mg, 3.9 mmol) were added. The solution was refluxed
for 30 h. Then the precipitate was filtered and the solvent
evaporated. The residue was dissolved in CH2Cl2 and washed with
40 mL of 1 N HCl. The organic layer was dried over Na2SO4 and
evaporated in vacuo. The crude residue was purified by column
chromatography (eluent, hexane/ethyl acetate 35:65) to afford 5b.
1
Yield 72%, mp 135 °C. H NMR (CDCl3) δ: 7.00 (s, 1H, 1 Ar),
6.76 (s, 1H, 1 Ar), 6.73 (s, 1H, 1 Ar), 4.17 (q, 2H, OCH2, J ) 7.1
Hz), 4.01 (s, 3H OMe), 3.96 (s, 3H, OMe), 3.90 (s, 3H, NMe),
3.64-3.70 (m, 2H, CH2), 2.60-2.68 (m. 2H, CH2), 1.27 (t, 3H,
CH3, J ) 7.1 Hz). Anal. (C17H22N2O5) C, H, N.
3-[(5,6-Dimethoxy-1H-indole-2-carbonyl)amino]propionic Acid
(6a). To a suspension of 5a (1 g, 3.12 mmol) in 55 mL of EtOH
was added LiOH ·H2O (0.262 g, 6.24 mmol), and the mixture was
stirred for 48 h at room temperature. After evaporation of the
solvent, the residue was dissolved in water and cold concentrated
HCl was dropped during 15 min under stirring. The precipitate was
filtered to obtain 810 mg of the title compound. Yield 89%, mp
7,8-Dimethoxy-2-(4-methoxybenzyl)-10-methyl-3,4-dihydro-
2H,10H-azepino[3,4-b]indole-1,5-dione (8c). A suspension of 7b
(205 mg, 0.71 mmol) in DMF (3.7 mL) was cooled to 0 °C, and
then NaH (60% in mineral oil, 20.5 mg, 0.85 mmol) was added.
The solution was stirred for 1 h at room temperature, and then
PMBCl (138 mg, 0.85 mmol) was dropped and stirring was
continued overnight. The mixture was added to water (6 mL) and
extracted twice with ethyl acetate. The organic layer was dried over
Na2SO4 and evaporated in vacuo. Flash chromatography (eluent,
hexane/ethyl acetate 45:55) afforded the title compound as a white
1
245 °C. H NMR (DMSO-d6) δ: 12.25 (br s, 1H, COOH), 11.26
(s, 1H, NH), 8.26 (t, 1H, NH, J ) 5.2 Hz), 7.05 (s, 1H, 1Ar), 6.96
(s, 1H, 1 Ar), 6.87 (s, 1H, 1Ar), 3.76 (s, 3H, OMe), 3.75 (s, 3H,
OMe), 3.4-3.5 (m, 2H, CH2), 2.45-2.55 (m, 2H, CH2). Anal.
(C14H16N2O5) C, H, N.
1
solid. Yield 33%, mp 167 °C. H NMR (CDCl3) δ: 7.80 (s, 1H,
1Ar), 7.30 (d, 2H, 2Ar, J ) 8.5 Hz), 6.88 (d, 2H, 2Ar, J ) 8.5
Hz), 6.79 (s, 1H, 1Ar), 4.77 (s, 2H, CH2Ph), 4.09 (s, 3H, OMe),
3.99 (s, 3H, OMe), 3.95 (s, 3H, OMe), 3.76 (s, 3H, NMe),
3.65-3.75 (m, 2H, CH2), 2.65-2.75 (m, 2H, CH2). Anal.
(C23H24N2O5) C, H, N.
Trifluoromethanesulfonic Acid 7,8-Dimethoxy-1-oxo-1,2,3,10-
tetrahydroazepino[3,4-b]indol-5-yl Ester (9a). To a suspension
of compound 7c (290 mg, 0.735 mmol) and Na2CO3 (109 mg, 1.03
mmol) in dry CH2Cl2 (7.5 mL) was added trifluoromethanesulfonic
anhydride (317 mg, 1.10 mmol). The mixture was stirred for 3 h
at room temperature. Then water was added and the aqueous phase
was extracted with CH2Cl2. The organic layer was dried over
3-[(5,6-Dimethoxy-1-methyl-1H-indole-2-carbonyl)amino]pro-
pionic Acid (6b). To a suspension of of 5b (378 mg, 1.13 mmol)
in 20 mL of EtOH was added LiOH ·H2O (96 mg, 2.26 mmol),
and the mixture was stirred for 48 h at room temperature. After
evaporation of the solvent, the residue was dissolved in water and
cold 6 N HCl was dropped during 15 min under stirring. The white
precipitate was filtered to obtain 324 mg of the title compound.
Yield 94%, mp 200 °C. 1H NMR (DMSO-d6) δ: 12.23 (s, 1H,
COOH), 8.34 (t, 1H, NH, J ) 5.9 Hz), 7.07 (s, 1H, 1 Ar), 7.03 (s,
1H, 1Ar), 6.94 (s, 1H, 1Ar), 3.94 (s, 3H, OMe), 3.83 (s, 3H, OMe),
3.76 (s, 3H, NMe), 3.3-3.5 (m, 2H, CH2), 2.45-2.55 (m, 2H, CH2).
Anal. (C15H18N2O5) C, H, N.

7784 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Cincinelli et al.
1
Na2SO4 and evaporated in vacuo. Flash chromatography (eluent
CH2Cl2/MeOH 99.5:0.5) afforded 9a (26%), mp >250 °C (dec).
1H NMR (DMSO-d6) δ: 12.87 (s, 1H, NH), 7.21 (s, 1H, 1Ar), 6.98
(s, 1H, 1Ar), 6.56 (t, 1H, CHd, J ) 7.1 Hz), 4.5-4.6 (m, 2H,
CH2), 3.82 (s, 3H, OMe), 3.78 (s, 3H, OMe). Anal. (C15H13-
F3N2O6S) C, H, N.
Trifluoromethanesulfonic Acid 7,8-Dimethoxy-2,10-dimethyl-
1-oxo-1,2,3,10-tetrahydroazepino[3,4-b]indol-5-yl Ester (9b). To
a suspension of compound 8b (127 mg, 0.42 mmol) and Na2CO3
(63.7 mg, 0.60 mmol) in dry CH2Cl2 (1.6 mL) was dropped a
solution of trifluoromethanesulfonic anhydride (176 mg, 0.613
mmol) in CH2Cl2 (2.6 mL). The mixture was stirred at room
temperature overnight. Then water was added and the aqueous phase
was extracted with CH2Cl2. The organic layer was dried over
Na2SO4 and evaporated in vacuo. Flash chromatography (eluent
CH2Cl2/MeOH 99:1) afforded 9b (75%), mp 96 °C. 1H NMR
(CDCl3) δ: 7.31 (s, 1H, 1Ar), 6.80 (s, 1H, 1Ar), 6.09 (t, 1H, CHd,
J ) 7.1 Hz), 4.04 (s, 3H, OMe), 3.99 (s, 3H, OMe), 3.93 (s, 3H,
NMe), 3.83 (d, 2H, CH2N, J ) 7.1 Hz), 3.28 (s, 3H, NMe). Anal.
(C17H17F3N2O6S) C, H, N.
°C. H NMR (acetone-d6) δ: 7.20 (d, 2H, 2Ar, J ) 8.4 Hz), 7.08
(s, 1H, 1Ar), 7.85 (d, 2H, 2Ar, J ) 8.4 Hz), 6.17 (t, 1H, CHd, J
) 6.7 Hz), 6.11 (s, 1H 1 Ar), 4.01 (s, 3H, OMe), 3.89 (s, 3H,
OMe), 3.84 (d, 2H, CH2N, J ) 6.7 Hz), 3.40 (s, 3H, NMe), 3.15
(s, 3H, NMe). Anal. (C22H22N2O4) C, H, N.
5-(4-Hydroxyphenyl)-7,8-dimethoxy-2-(4-methoxybenzyl)-10-
methyl-3,10-dihydro-2H-azepino[3,4-b]indol-1-one (1d). A solu-
tion of 9c (30 mg, 0.055 mmol) and palladium tetrakis (5 mg) in
1.26 mL of toluene was stirred at room temperature for 30 min.
Then a solution of 4-hydroxybenzeneboronic acid (11.5 mg, 0.083
mmol) in EtOH (0.5 mL) and a saturated solution of Na2CO3 (0.5
mL) were added sequentially. After the mixture was stirred for 60
min at 80 °C, brine was added and the mixture was extracted with
CH2Cl2. The organic layer was dried over Na2SO4 and evaporated
in vacuo. Flash chromatography (eluent, hexane/ethyl acetate 50:
50) afforded 1d (78%), mp 193 °C. 1H NMR (acetone-d6) δ: 7.39
(d, 2H, 2 Ar, J ) 8.4 Hz), 7.18 (d, 2H, 2Ar, J ) 8.4 Hz), 7.08 (s,
1H, 1Ar), 6.80-6.90 (m, 4H, 4Ar), 6.09 (s, 1H, 1Ar), 5.95 (t, 1H,
CHd, J ) 6.9 Hz), 4.71 (s, 2H, CH2Ph), 4.06 (s, 3H, OMe), 3.90
(s, 3H, OMe), 3.75 (s, 6H, NMe and OMe), 3.38 (s, 3H, NMe).
Anal. (C29H28N2O5) C, H, N.
5,6-Dimethoxy-1-methyl-1H-indole-2-carboxylic Acid (4b). To
a solution of 5,6-dimethoxy-1H-indole-2-carboxylic acid ethyl ester
(500 mg, 2 mmol) in CH3CN (32 mL) were added K2CO3 (1.24 g,
9 mmol) and iodomethane (0.31 mL, 5 mmol), and the mixture
was stirred at reflux for 13 h. The mixture was cooled to room
temperature and filtered and the filtrate evaporated in vacuo. The
solid was then washed with 1 N HCl and extracted with CH2Cl2,
and the combined organic layers were dried over Na2SO4 and
evaporated to give 480 mg (93%) of 5,6-dimethoxy-1-methyl-1H-
indole-2-carboxylic acid ethyl ester, mp 148 °C. 1H NMR (CDCl3)
δ: 7.17 (s, 1H), 7.00 (s, 1H), 6.75 (s, 1H), 4.53 (q, J ) 7.5 Hz,
2H), 4.04 (s, 3H), 3.97 (s, 3H), 3.90 (s, 3H), 1.20 (q, J ) 7.5 Hz,
3H). 13C NMR (75 MHz, CDCl3) δ: 162.1, 149.8, 146.0, 134.9,
126.3, 118.6, 109.9, 102.4, 92.1, 60.2, 56.1, 56.0, 31.8, 14.4.
The above ester (0.48 mg, 1.8 mmol) was added to a solution of
NaOH (2.90 g, 72.4 mmol) in ethanol (31.8 mL), and the mixture
was refluxed for 2 h. Ethanol was evaporated to dryness in vacuo,
the residue was dissolved in water, and the solution was acidified
to pH 3 with 12 N HCl to form a yellow solid. The mixture was
allowed to stand at 0 °C for 15 min, and the product was isolated
Trifluoromethanesulfonic Acid 7,8-Dimethoxy-2-(4-methoxy-
benzyl)-10-methyl-1-oxo-1,2,3,10-tetrahydroazepino[3,4-b]indol-
5-yl Ester (9c). To a suspension of compound 8c (80 mg, 0.136
mmol) and Na2CO3 (29.1 mg, 0.274 mmol) in dry CH2Cl2 (1 mL)
was dropped trifluoromethanesulfonic anhydride (84.6 mg, 0.294
mmol). The mixture was stirred at room temperature overnight,
and then water was added and the aqueous phase was extracted
with CH2Cl2. The organic layer was dried over Na2SO4 and
evaporated in vacuo. Flash chromatography (eluent, hexane/ethyl
1
acetate 55:45) afforded 9c (28%), mp 190 °C. H NMR (acetone-
d6) δ: 7.32 (d, 2H, 2Ar, J ) 8.6 Hz), 7.29 (s, 1H, 1Ar), 7.18 (s,
1H, 1Ar), 6.89 (d, 2H, 2Ar, J ) 8.6 Hz), 5.99 (t, 1H, CHd, J )
7.5 Hz), 4.76 (s, 2H, CH2Ph), 4.06 (s, 3H, OMe), 3.94 (s, 3H, OMe),
3.89 (d, 2H, CH2N, J ) 7.5 Hz), 3.84 (s, 3H, NMe), 3.78 (s, 3H,
NMe). Anal. (C24H23F3N2O7S) C, H, N.
5-(4-Hydroxyphenyl)-7,8-dimethoxy-3,10-dihydro-2H-azepi-
no[3,4-b]indol-1-one (1a). A solution of 9a (375 mg, 0.222 mmol)
and palladium tetrakis (15 mg) in 5 mL of toluene was stirred at
room temperature for 3 h. Then a solution of 4-hydroxybenzenebo-
ronic acid (46 mg, 0.33 mmol) in EtOH (2 mL) and a saturated
solution of Na2CO3 (2 mL) were added. After the mixture was
stirred for 90 min at 80 °C, brine was added and the mixture was
extracted with ethyl acetate. The organic layer was dried over
Na2SO4 and evaporated in vacuo. Flash chromatography (eluent,
hexane/ethyl acetate 60:40) afforded 1a (12%), mp 159 °C. 1H
NMR (acetone-d6) δ: 11.25 (s, 1H, NH), 8.65 (s, 1H, CONH), 7.25
(d, 2H, 2Ar, J ) 8.4 Hz), 7.08 (s, 1H, 1Ar), 6.91 (d, 2H, 2Ar, J )
8.4 Hz), 6.30 (t, 1H, CHd, J ) 6.9 Hz), 6.01 (s, 1H, 1Ar),
4.45-4.55 (m, 2H, CH2), 3.89 (s, 3H, OMe), 3.42 (s, 3H, OMe).
Anal. (C20H18N2O4) C, H, N.
1
by filtration (330 mg, 85%), mp 221 °C. H NMR (DMSO-d6) δ:
12.6 (br s, 1H), 7.10 (s, 1H), 7.08 (s, 1H), 7.05 (s, 1H), 4.00 (s,
3H), 3.87 (s, 3H), 3.78 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ:
163.2, 149.9, 146.1, 135.1, 126.8, 118.4, 110.0, 103.0, 93.7, 56.1
(2C), 32.1. Anal. (C12H13NO4) C, H, N.
5,6-Dimethoxy-1-methyl-1H-indole-2-carboxylic Acid (3-Hy-
droxypropyl)amide (11e). 3-Amino-1-propanol (121 µL, 1.59 mmol)
was dissolved in dry THF (5.8 mL), and then EDC (311 mg, 1.59
mmol), HOBt (220 mg, 1.59 mmol), and 4b (250 mg, 1.06 mmol)
were added sequentially at room temperature. After the resulting
mixture was stirred overnight, the solvent was removed and the crude
product was poured into saturated aqueous NaHCO3 and extracted with
ethyl acetate. The combined organic layers were washed with 1 N
HCl, saturated aqueous NaHCO3, brine, dried over Na2SO4, and
5-(4-Hydroxyphenyl)-7,8-dimethoxy-10-methyl-3,10-dihydro-
2H-azepino[3,4-b]indol-1-one (1b). A solution of 1d (130 mg, 0.27
mmol) in 10 mL of TFA was refluxed for 5 h. After evaporation
of the solvent, a saturated solution of NaHCO3 was added. The
precipitate was filtered to obtain a crude that was purified by flash
chromatography (eluent, CH2Cl2/MeOH 96:4). Yield 49%, mp
1
concentrated to give 210 mg (64%) of 11a, mp 154 °C. H NMR
1
>270 °C. H NMR (DMSO-d6) δ: 9.50 (s, 1H, OH), 8.18 (t, 1H,
(DMSO-d6) δ: 8.28 (t, J ) 5.58 Hz, 1H), 7.07 (s, 1H), 7.03 (s, 1H),
6.92 (s, 1H), 4.57-4.45 (m, 1H), 3.95 (s, 3H), 3.84 (s, 3H), 3.76 (s,
3H), 3.55-3.44 (m, 2H), 3.29 (q, J ) 6.70 Hz, 2H),1.78 (q, J ) 6.70
Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ: 162.4, 148.8, 145.8, 133.9,
130.8, 118.5, 104.5, 103.0, 93.8, 59.1, 56.2, 56.1, 36.5, 32.9, 32.0.
Anal. (C15H20N2O4) C, H, N.
5,6-Dimethoxy-1-methyl-1H-indole-2-carboxylic Acid (3-Hy-
droxybutyl)amide (11f). 4-Amino-2-butanol (206 µL, 2.12 mmol)
was dissolved in dry THF (5.77 mL), and then EDC (415 mg, 2.12
mmol), HOBt (365 mg, 2.12 mmol), and 4b (250 mg, 1.06 mmol)
were added sequentially at room temperature. After the resulting
mixture was stirred for 3 h under nitrogen, the solvent was removed
and the crude product was poured into saturated aqueous NaHCO3
and extracted with ethyl acetate. The combined organic layers were
washed with 1 N HCl, saturated NaHCO3, brine, dried over Na2SO4,
NHCO, J ) 5.9 Hz), 7.10 (d, 2H, 2Ar, J ) 8.4 Hz), 7.09 (s, 1H,
1Ar), 6.75 (d, 2H, 2Ar, J ) 8.4 Hz), 6.01 (t, 1H, CHd, J ) 6.9
Hz), 5.95 (s, 1H, 1Ar), 3.99 (s, 3H, OMe), 3.81 (s, 3H OMe), 3.45
(m, 2H, CH2), 3.30 (s, 3H, NMe). Anal. (C21H20N2O4) C, H, N.
5-(4-Hydroxyphenyl)-7,8-dimethoxy-2,10-dimethyl-3,10-dihy-
dro-2H-azepino[3,4-b]indol-1-one (1c). A solution of 9b (133 mg,
0.31 mmol) and palladium tetrakis (21 mg) in 7 mL of toluene
was stirred at room temperature for 30 min. Then a solution of
4-hydroxybenzeneboronic acid (63 mg, 0.46 mmol) in EtOH (2.8
mL) and a saturated solution of Na2CO3 (2.8 mL) were added. After
the mixture was stirred for 60 min at 80 °C, brine was added and
the mixture was extracted with CH2Cl2. The organic layer was dried
over Na2SO4 and evaporated in vacuo. Flash chromatography
(eluent, hexane/ethyl acetate 35:65) afforded 1c (49%), mp 228

Substituted ꢀ-Carbolin-1-ones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7785
1
and concentrated to give 321 mg (99%) of 11b, mp 162 °C. H
NMR (DMSO-d6) δ: 8.26 (t, 5.95 Hz, 1H), 7.07 (s, 1H), 7.03 (s,
1H), 6.92 (s, 1H), 4.54 (d, J ) 4.8 Hz, 1H), 3.94 (s, 1H), 3.83 (s,
1H), 3.76 (s, 1H), 3.74-3-62 (m, 1H), 3.30 (q, J ) 5.9 Hz, 2H),
1.66-1.50 (m, 2H), 1.09 (d, J ) 6.3 Hz, 3H). 13C NMR (75 MHz,
DMSO-d6) δ: 162.3, 148.8, 145.8, 133.9, 130.8, 118.5 104.5, 103.0,
93.8, 64.5, 56.2 (2C), 40.7, 36.5, 31.9, 24.1. Anal. (C16H22N2O4)
C, H, N.
7,8-Dimethoxy-5,10-dimethyl-3,10-dihydro-2H-azepino[3,4-
b]indol-1-one (1f). Trifluoroacetic acid (121 µL, 1.57 mmol) was
added to a solution of 12f (60 mg, 0.197 mmol) in CH3CN (3.62 mL),
and the mixture was refluxed for 18 h. The solvent was removed, and
the crude product was poured into saturated aqueous NaHCO3 and
extracted with ethyl acetate. The organic phase was dried over Na2SO4
and filtered and the solvent was evaporated to give 27 mg (47%) of
1f after crystallization from acetone, mp 238 °C. 1H NMR (DMSO-
d6) δ: 8.06 (t, J ) 5.6 Hz, 1H), 7.26 (s, 1H), 7.10 (s, 1H), 5.80 (t, J
) 6.7 Hz, 1H), 3.94 (s, 3H), 3.89 (s, 3H), 3.81 (s, 3H), 3.35-3.20
(m, 2H), 2.37 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 168.1, 154.1,
150.3, 140.3, 138.6, 135.1, 127.4, 123.6, 121.6, 108.8, 96.5, 61.1, 60.9,
42.7, 37.1, 25.9. Anal. (C16H18N2O3) C, H, N.
5,6-Dimethoxy-1-methyl-1H-indole-2-carboxylic Acid (3-
Formylpropyl)amide (12e). To a solution of IBX (371 mg, 1.37
mmol) in DMSO (1.85 mL) was added 11e (200 mg, 0.684 mmol),
and the solution was stirred for 2 h at room temperature under
nitrogen. The solution was diluted with water and extracted with
ethyl acetate. The combined organic layers were washed with
saturated aqueous NaHCO3, water, dried over Na2SO4, and evapo-
rated. The crude product was crystallized from ethyl acetate to give
120 mg (61%) of 12e, mp 102 °C. 1H NMR (acetone-d6) δ: 9.81(t,
J ) 1.5 Hz, 1H), 7.77-7.60 (br s, 1H), 7.05 (s, 1H), 7.02 (s, 1H),
6.91 (s, 1H), 4.03 (s, 3H), 3.91 (s, 3H), 3.80 (s, 3H), 3.70 (q, J )
6.7 Hz, 2H), 2.77 (td, J ) 6.7 Hz, J ) 1.5 Hz, 2H). 13C NMR (75
MHz, acetone-d6) δ: 201.1, 162.3, 153.8, 149.4, 146.2, 134.2, 118.8,
103.9, 102.9, 93.1, 55.5, 53.3, 43.7, 33.0, 31.0. Anal. (C15H18N2O4)
C, H, N.
7,8-Dimethoxy-5-(4-methoxyphenyl)-10-methyl-3,10-dihydro-
2H-azepino[3,4-b]indol-1-one (1g). Trifluoroacetic acid (80 µL,
1.04 mmol) was added to a suspension of 12g (42 mg, 0.106 mmol)
in CH3CN (1 mL), and the mixture was refluxed for 18 h. The
solvent was evaporated, and the residue was poured into saturated
aqueous NaHCO3 and extracted with ethyl acetate. The combined
organic layers were dried over Na2SO4 and filtered, and the solvent
was evaporated in vacuo. The crude product was purified by PLC
(dichloromethane/methanol 97:3) to give 18 mg (43%) of 1g, mp
198 °C. 1H NMR (acetone-d6) δ: 7.26 (t, J ) 6.5 Hz, 1H), 7.25 (d,
J ) 8.7 Hz, 1H), 7.05 (s, 1H), 6.92 (d, J ) 8.7 Hz, 2H), 6.10 (t,
J ) 6.5 Hz, 1H), 6.02 (s, 1H), 4.03 (s, 3H), 3.90 (s, 3H), 3.83 (s,
3H), 3.60 (t, J ) 6.51 Hz, 2H), 3.35 (s, 3H). 13C NMR (75 MHz,
acetone-d6) δ: 163.4, 159.6, 149.6, 145.3, 140.8, 133.8, 133.3, 131.0,
129.6 (2C), 121.9, 117.3, 117.0, 113.5 (2C), 104.4, 92.9, 55.3, 54.7,
38.1, 31.3.
Cell Culture and Antibodies. Cells were maintained in Dul-
becco’s modified Eagle medium supplemented with 5% serum for
NIH3T3MEN2A or 10% calf serum (Colorado Serum Company,
Denver, CO) for NIH3T3 and NIH3T3H-Ras and incubated at 37
°C in a 10% CO2 atmosphere. Antiproliferative activity was
evaluated by cell counting after 72 h of treatment with compounds
dissolved in DMSO or solvent (0.5% final concentration). IC50
values were calculated from dose-response curves. The effect of
selected compounds on tyrosine phosphorylation and expression
of the RET oncoprotein was assessed by Western blot analysis of
NIH3T3MEN2A cells after 24 h of treatment as previously de-
scribed.17 The following antibodies were used: monoclonal anti-
pTyr antibody clone 4610 (Upstate Biotechnology, Lake Placid,
NY), rabbit polyclonal anti-Ret H300 and antiphospho Ret (Y1062)
(Santa Cruz Biotechnology, SantaCruz, CA), and anti-actin (Sigma
Chemical Company, St. Louis, MO).
Kinase Assay. A nonradioactive kinase assay was performed
using recombinant Ret active protein (Upstate, Lake Placid, NY)
and myelin basic protein (MBP) (Sigma) as kinase substrate, as
previously described.19 Briefly, the kinase activity was assayed in
the presence of Ret (30 ng), 2 µM MBP, solvent, or inhibitor
(DMSO 4% final concentration), and 5 µM ATP. After 10 min of
incubation at 30 °C, the enzyme-catalyzed reaction was terminated
by addition of 3-fold concentrated Laemmli buffer. Samples were
subjected to SDS-PAGE and immunoblotted with antiphospho-
tyrosine antibody. Phosphorylated MBP was revealed by enhanced
chemioluminescence reaction (GE Healthcare, Little Chalf-ont,
U.K.). Signal intensity was detected by ChemiDoc XRS system,
PC, and analyzed with the interfaced software Quantity One 4.6.3
(Biorad, Hercules, CA).
Docking Methods. The crystal structure of RET (PDB code
2ivu26) was taken from the Protein Data Bank.32 After addition of
hydrogen atoms and correction of the wrong residues, the protein
complexed with its reference inhibitor (i.e., ZD6474) was minimized
using AMBER 9 software33 and the parm03 force field at 300 K.
The complex was placed in a rectangular parallelepiped water box,
an explicit solvent model for water, TIP3P, was used, and the
complex was solvated with a 8 Å water cap. Chlorine ions were
added as counterions to neutralize the system. Two steps of
minimization were then carried out. In the first stage, we kept the
complex fixed with a position restraint of 500 (kcal/mol)/Å2 and
we solely minimized the positions of the water molecules. In the
5,6-Dimethoxy-1-methyl-1H-indole-2-carboxylic Acid (3-Ox-
obutyl)amide (12f). Compound 11f (178 mg, 0.58 mmol) was
suspended in ethyl acetate (19.7 mL), and IBX (473 mg, 1.74 mmol)
was added. The resulting mixture was refluxed for 3 h, and then
the reaction was cooled to room temperature and filtered. The solid
was washed with ethyl acetate and the combined filtrates were
1
concentrated to yield 150 mg (85%) of 12f, mp 135-136 °C. H
NMR (DMSO-d6) δ: 8.27 (t, J ) 5.9 Hz, 1H), 7.07 (s, 1H), 7.03
(s, 1H), 6.92 (s, 1H), 3.93 (s, 3H), 3.83 (s, 3H), 3.76 (s, 3H),
3.46-3-38 (m, 2H), 2.72 (t, J ) 7.1 Hz, 2H), 2.13 (s, 3H). 13C
NMR (75 MHz, DMSO-d6) δ: 207.9, 162.3, 146.9, 145.8, 134.9,
133.9, 131.5, 130.5, 118.5, 104.7, 103.0, 93.8, 56.2, 56.1, 43.1,
34.6, 32.0, 30.3. Anal. (C16H20N2O4) C, H, N.
5,6-Dimethoxy-1-methyl-1H-indole-2-carboxylic Acid [3-(4-
Methoxyphenyl)-3-oxopropyl]amide (12g). To a suspension of
4b (47.5 mg, 0.20 mmol) in dry THF (1.09 mL) were added EDC
(59.3 mg, 0.30 mmol) and HOBt (40.9 mg, 0.30 mmol). The
mixture was stirred at room temperature, under nitrogen, for 3 h.
3-Amino-1-(4-methoxyphenyl)propan-1-one trifluoromethanesulfonate
(133 mg, 0.404 mmol) was added followed by N-ethyldiisopropy-
lamine (69 µL, 0.4 mmol). After the mixture was stirred for 1 h at
room temperature, the solvent was evaporated and the crude product
was poured into saturated NaHCO3 and extracted with ethyl acetate.
The combined extracts were then washed with 1 N HCl, saturated
aqueous NaHCO3, dried over Na2SO4, and concentrated. The crude
product was purified by flash chromatography (ethyl acetate/hexane
60:40) to give 45 mg (56%) of 12g, mp 136 °C. 1H NMR (DMSO-
d6) δ: 8.35 (t, J ) 5.6 Hz, 1H), 7.98 (d, J ) 8.9 Hz, 2H), 7.07 (s,
1H), 7.05 (d, J ) 8.9 Hz, 2H), 7.02 (s, 1H), 6.92 (s, 1H), 3.93 (s,
3H), 3.83 (s, 6H), 3.75 (s, 3H), 3.55 (dt, J ) 7.07 Hz, J ) 5.6 Hz,
2H), 3.26 (t, J ) 7.1 Hz, 2H). Anal. (C22H24N2O5) C, H, N.
7,8-Dimethoxy-10-methyl-3,10-dihydro-2H-azepino[3,4-b]in-
dol-1-one (1e). (A) Trifluoroacetic acid (19.7 µL, 0.26 mmol) was
added to a solution of 12e (50 mg, 0.172 mmol) in CH3CN (3.16
mL), and the mixture was refluxed for 1 h. The solvent was
evaporated and the crude product was purified by preparative layer
chromatography (ethyl acetate/hexane 95:5) to give 22 mg (47%)
1
of 1e, mp 208 °C. H NMR (DMSO-d6) δ: 7.80 (t, J ) 5.6 Hz,
1H), 7.26 (s, 1H), 7.14 (d, J ) 9.7 Hz, 1H), 7.07 (s, 1H), 6.13-6.00
(m, 1H), 3.97 (s, 3H), 3.88 (s, 3H), 3.80 (s, 3H), 3.5-3.4 (m, 2H).
13C NMR (75 MHz, DMSO-d6) δ: 163.4, 149.3, 146.1, 133.7,
129.7, 125.7, 117.5, 117.2, 101.7, 101.5, 93.4, 56.4 (2C), 39.9 (one
peak missing due to overlapping with solvent signal), 32.8. Anal.
(C15H16N2O3) C, H, N. (B) InCl3 (15 mg, 0.068 mmol) was added
to 12e (40 mg, 0.14 mmol) in CH3CN (2.4 mL), and the mixture
was refluxed for 2 h. Evaporation and PLC (ethyl acetate/hexane
95:5) gave 12 mg (32%) of 1e.

7786 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Cincinelli et al.
second stage, we minimized the entire system through 5000 steps
of steepest descent followed by conjugate gradient (CG) until a
convergence of 0.05 kcal/(Å ·mol) was attained.
energies were determined using the MOLSURF program applying
the LCPO method.37
The ligands were built by means of Maestro34 and were then
minimized in a water environment (using the generalized Born/
surface-area model) by means of Macromodel.35 They were
minimized using the CG method until a convergence value of 0.05
kcal/(Å·mol) was attained, using the MMFFs force field and a
distance-dependent dielectric constant of 1.0.
Acknowledgment. We are indebted to Cell Therapeutics,
Bresso, Italy, for a generous gift of 5,6-dimethoxyindolin-2-
one and RPI-1 and to Laura Zanesi for aid in editing the
manuscript. This work was partially supported by Associazione
Italiana per la Ricerca sul Cancro, Milan, Italy, by Alleanza
Contro il Cancro, Italy, and by University of Milano (FIRST
funds). Molecular graphics images were produced using the
UCSF Chimera package from the Resource for Biocomputing,
Visualization, and Informatics at the University of California,
San Francisco (supported by NIH Grant P41 RR-01081).
The ligands minimized in this manner were docked into the RET
binding site by means of GOLD 3.2.27 The region of interest used
by GOLD was defined to contain the atoms that stayed within 10
Å of ZD6474 in the minimized receptor model. The “allow early
termination” command was deactivated. All the other parameters
were used as GOLD default values, and the ligands were submitted
to 30 genetic algorithm runs. The docking analysis was carried out
using the ChemScore kinase fitness scoring function, and the best
docked conformation was then analyzed.
Supporting Information Available: Elemental analysis of
analysis data of the compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
MD Simulations. All simulations were performed using AMBER
9.33 MD simulations were carried out using the parm03 force field
at 300 K. The complex was placed in a rectangular parallelepiped
water box, an explicit solvent model for water, TIP3P, was used,
and the complex was solvated with a 8 Å water cap. Chlorine ions
were added as counterions to neutralize the system. Prior to MD
simulations, two steps of minimization were carried out. In the first
stage, we kept the protein fixed with a position restraint of 500
(kcal/mol)/Å2, and we solely minimized the positions of the water
molecules. In the second stage, we minimized the entire system by
applying a constraint of 20 kcal/mol on the R carbon. The two
minimization stages consisted of 5000 steps of steepest descent
followed by CG until a convergence of 0.05 kcal/(Å · mol) was
attained.
References
(1) Arighi, E.; Borrello, M. G.; Sariola, H. RET tyrosine kinase signaling
in development and cancer. Cytokine Growth Factor ReV. 2005, 16,
441–467.
(2) Asai, N.; Jijiwa, M.; Enomoto, A.; Hawai, K.; Maeda, K.; Ichiahara,
M.; Murakumo, Y.; Takahashi, M. RET receptor signaling: dysfunction
in thyroid cancer and Hirschsprung’s disease. Pathol. Int. 2006, 56,
164–172.
(3) Murakumo, Y.; Jijiwa, M.; Asai, N.; Ichihara, M.; Takahashi, M. RET
and neuroendocrine tumors. Pituitary 2006, 9, 179–192.
(4) Rosa, D. D.; Ismael, G.; Dal Lago, L.; Awada, A. Molecular-targeted
therapies: lessons from years of clinical development. Cancer Treat.
ReV. 2008, 34, 61–80.
(5) Drosten, M.; Pu¨tzer, B. M. Mechanisms of disease: cancer targeting
and the impact of oncogenic RET for medullary thyroid carcinoma
therapy. Nat. Clin. Pract. 2006, 3, 564–574.
(6) De Groot, J. W. B.; Links, T. P.; Plukker, J. T. M.; Lips, C. J. M.;
Hofstra, R. M. W. RET as a diagnostic and therapeutic target in
sporadic and hereditary endocrine tumors. Endocr. ReV. 2006, 27, 535–
560.
(7) Cuccuru, G.; Lanzi, C.; Cassinelli, G.; Pratesi, G.; Tortoreto, M.;
Petrangolini, G.; Seregni, E.; Martinetti, A.; Laccabue, D.; Zanchi,
C.; Zunino, F. Cellular effects and antitumor activity of RET inhibitor
RPI-1 on MEN2A-associated medullary thyroid carcinoma. J. Natl.
Cancer Inst. 2004, 96, 1006–1014.
(8) Drosten, M.; Hilken, G.; Bo¨ckmann, M.; Ro¨dicker, F.; Mise, N.;
Cranston, A. N.; Dahmen, U.; Ponder, B. A. J.; Pu¨tzer, B. M. Role of
MEN2A-derived RET in maintenance and proliferation of medullary
thyroid carcinoma. J. Natl. Cancer Inst. 2004, 96, 1231–1239.
(9) Petrangolini, G.; Cuccuru, G.; Lanzi, C.; Tortoreto, M.; Belluco, S.;
Pratesi, G.; Cassinelli, G.; Zunino, F. Apoptotic cell death induction
and angiogenesis inhibition in large established medullary thyroid
carcinoma xenografts by Ret inhibitor RPI-1. Biochem. Pharmacol.
2006, 72, 405–414.
(10) Sharma, S. V.; Settleman, J. Oncogene addiction: setting the stage
for molecularly targeted cancer therapy. Genes DeV. 2007, 21, 3214–
3231.
(11) Cassinelli, G.; Lanzi, C.; Pensa, T.; Gambetta, R. A.; Nasini, G.;
Cuccuru, G.; Cassinis, M.; Pratesi, G.; Polizzi, D.; Tortoreto, M.;
Zunino, F. Clavilactones, a novel class of tyrosine kinase inhibitors
of fungal origin. Biochem. Pharmacol. 2000, 59, 1539–1547.
(12) Kundra, P.; Burman, K. D. Thyroid cancer molecular signaling
pathways and use of targeted therapy. Endocrinol. Metab. Clin. N.
Am. 2007, 36, 839–853.
(13) Deshpande, H. A.; Gettinger, S. N.; Sosa, J. A. Novel chemotherapy
options for advanced thyroid tumors: small molecules offer great hope.
Curr. Opin. Oncol. 2008, 20, 19–24.
(14) Schlumberger, M.; Carlomagno, F.; Baudin, E.; Bidart, J. M.; Santoro,
M. New therapeutic approaches to treat medullary thyroid carcinoma.
Nat. Clin. Pract. 2008, 4, 22–32.
MD was done on a 1 CPU/node, 4 node, 1024 MB RAM/node
cluster Intel Core 2 Duo E6600, 2.4 GHz, and the average wall
clock time for a 1 ns simulation was about 33 h.
Particle mesh Ewald (PME)36 electrostatics and periodic bound-
ary conditions were used in the simulation. The MD trajectory was
run using the minimized structure as the starting conformation. The
time step of the simulations was 2.0 fs with a cutoff of 10 Å for
the nonbonded interaction, and SHAKE was employed to keep all
bonds involving hydrogen atoms rigid. Constant-volume periodic
boundary MD was carried out for 80 ps, during which the
temperature was raised from 0 to 300 K. Then 1 ns of constant-
pressure periodic boundary MD was carried out at 300 K using
the Langevin thermostat to maintain constant the temperature of
our system.
The R carbons of the receptor were blocked with a harmonic
force constant of 20 (kcal/mol)/Å2 during the simulation. General
Amber force field (GAFF) parameters were assigned to the ligands,
while partial charges were calculated using the AM1-BCC method
as implemented in the Antechamber suite of AMBER 9.
Energy Evaluation. We extracted from the last 800 ps of MD
of the 2b- and 2a-RET complexes 400 snapshots (at time intervals
of 2 ps) for each species (complex, receptor, and ligand). In general,
the binding free energy of a protein-ligand complex (∆Gbind) is
defined as
∆Gbind ) Gcomplex - (Gprotein + Gligand
)
where Gcomplex, Gprotein, and Gligand are the free energies of the
complex, protein, and the ligand respectively.
For each system the free energy can be estimated as
(15) Sun, L.; Tran, N.; Tang, F.; App, H.; Hirth, P.; McMahon, G.; Tang,
C. Synthesis and biological evaluations of 3-substituted indolin-2-
ones: a novel class of tyrosine kinase inhibitors that exhibit selectivity
toward particular receptor tyrosine kinases. J. Med. Chem. 1998, 41,
2588–2603.
(16) Lanzi, C.; Cassinelli, G.; Cuccuru, G.; Zaffaroni, N.; Supino, R.;
Vignati, S.; Zanchi, C.; Yamamoto, M.; Zunino, F. Inactivation of
Ret/Ptc1 oncoprotein and inhibition of papillary thyroid carcinoma
cell proliferation by indolinone RPI-1. Cell. Mol. Life Sci. 2003, 60,
1449–1459.
G ) EMM + Gsol - TS
Electrostatic, van der Waals energies, and internal energies (EMM
)
were obtained using the SANDER module in AMBER 9.0.33 The
solvation free energy (Gsol) was obtained from the sum of the polar
solvation and nonpolar solvation free energy. The polar energy was
obtained using the Sander module, applying dielectric constants of
1 and 80 for the solute and the solvent, respectively. Nonpolar

Substituted ꢀ-Carbolin-1-ones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7787
(17) Lanzi, C.; Cassinelli, G.; Pensa, T.; Cassinis, M.; Gambetta, R. A.;
Borrello, M. G.; Menta, E.; Pierotti, M. A.; Zunino, F. Inhibition of
transforming activity of the ret/ptc1 oncoprotein by a 2-indolinone
derivative. Int. J. Cancer 2000, 85, 384–390.
(18) Cassinelli, C.; Lanzi, C.; Petrangolini, G.; Tortoreto, M.; Pratesi, G.;
Cuccuru, G.; Laccabue, D.; Supino, R.; Belluco, S.; Favini, E.; Poletti,
A.; Zunino, F. Inhibition of c-Met and prevention of spontaneous
metastatic spreading by the 2-indolinine RPI-1. Mol. Cancer Ther.
2006, 5, 2388–2397.
(19) Sun, L.; Tran, N.; Tang, F.; App, H.; Hirth, P.; McMahon, G.; Tang,
C. Synthesis and biological evaluations of 3-substituted indolin-2-
ones: a novel class of tyrosine kinase inhibitors that exhibit selectivity
toward particular receptor tyrosine kinases. J. Med. Chem. 1998, 41,
2588–2603.
(20) Tuccinardi, T.; Manetti, F.; Schenone, S.; Martinelli, A.; Botta, M.
Construction and validation of a RET TK catalytic domain by
homology modeling. J. Chem. Inf. Model. 2007, 47, 644–655.
(21) Mohammadi, M.; McMahon, G.; Sun, L.; Tang, C.; Hirth, P.; Yeh,
B. K.; Hubbard, S. R.; Schlessinger, J. Structures of the tyrosine kinase
domain of fibroblast growth factor receptor in complex with inhibitors.
Science 1997, 276, 955–960.
(22) Wang, S.; Dong, Y.; Wang, X.; Hu, X.; Liu, J. O.; Hu, Y. 3-Aryl
beta-carbolin-1-ones as a new class of potent inhibitors of tumor cell
proliferation: synthesis and biological evaluation. Org. Biomol. Chem.
2005, 3, 911–916.
(23) Chacun-Lefe`vre, B.; Joseph, B.; Me´rour, J.-Y. Synthesis and reactivity
of azepino[3,4-b]indol-5-yl trifluoromethanesulfonate. Tetrahedron
2000, 56, 4491–4499.
(24) Cincinelli, R.; Dallavalle, S.; Merlini, L. Intramolecular Friedel-Crafts
reaction of indoles with carbonyl groups: a simple synthesis of 3- and
4-substituted ꢀ-carbolin-1-ones. Synlett 2008, 1309–1312.
(25) Rizzi, E.; Cassinelli, G.; Dallavalle, S.; Lanzi, C.; Cincinelli, R.;
Nannei, R.; Cuccuru, G.; Zunino, F. Synthesis and RET protein kinase
inhibitory activity of 3-arylureidobenzylidene-indolin-2-ones. Bioorg.
Med. Chem. Lett. 2007, 17, 3962–3968.
inhibition of the RET tyrosine kinase domain. J. Biol. Chem. 2006,
281, 33577–33587.
(27) Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R.
Development and validation of a genetic algorithm for flexible docking.
J. Mol. Biol. 1997, 267, 727–748.
(28) Toledo, L. M.; Lydon, N. B.; Elbaum, D. The structure-based design
of ATP-site directed protein kinase inhibitors. Curr. Med. Chem. 1999,
6, 775.
(29) Traxler, P.; Green, J.; Mett, H.; Sequin, U.; Furet, P. Use of a
pharmacophore model for the design of EGFR tyrosine kinase
inhibitors: isoflavones and 3-phenyl-4(1H)-quinolones. J. Med. Chem.
1999, 42, 1018–1026.
(30) Massova, I.; Kollman, P. A. Combined molecular mechanical and
continuum solvent approach (MM-PBSA/GBSA) to predict ligand
binding. Perspect. Drug DiscoVery Des. 2000, 18, 113–135.
(31) Rizzo, R. C.; Toba, S.; Kuntz, I. D. A molecular basis for the selectivity
of thiadiazole urea inhibitors with stromelysin-1 and gelatinase-A from
generalized Born molecular dynamics simulations. J. Med. Chem.
2004, 47, 3065–3074.
(32) http://www.pdb.org/.
(33) Case, D. A.; Darden, T. A., Cheatham, T. E., III; Simmerling, C. L.;
Wang, J.; Duke, R. E.; Luo, R.; Merz, K. M.; Pearlman, D. A.;
Crowley, M.; Walker, R. C.; Zhang, W.; Wang, B.; Hayik, S.;
Roitberg, A.; Seabra, G.; Wong, K. F.; Paesani, F.; Wu, X.; Brozell,
S.; Tsui, V.; Gohlke, H.; Yang, L.; Tan, C.; Mongan, J.; Hornak, V.;
Cui, G.; Beroza, P.; Mathews, D. H.; Schafmeister, C.; Ross, W. S.;
Kollman, P. A. AMBER 9; University of California: San Francisco,
CA, 2006.
(34) Maestro, version 7.5; Schro¨dinger, Inc.: Portland, OR, 1999.
(35) Macromodel, version 8.5; Schro¨dinger, Inc.: Portland, OR, 1999.
(36) Essman, U.; Perela, L.; Berkowitz, M. L.; Darden, T.; Lee, H.;
Pedersen, L. G. A smooth particle mesh Ewald method. J. Chem. Phys.
1995, 103, 8577–8592.
(37) Weiser, J.; Shenkin, P. S.; Still, W. C. Approximate atomic surfaces
from linear combinations of pairwise overlaps (LCPO). J. Comput.
Chem. 1999, 20, 217–230.
(26) Knowles, P. P.; Murray-Rust, J.; Kjaer, S.; Scott, R. P.; Hanrahan,
S.; Santoro, M.; Iba´n˜ez, C. F.; McDonald, N. Q. Structure and chemical
JM8007823