Synthesis and RET protein kinase inhibitory activity of 3-arylureidobenzylidene-indolin-2-ones

Article abstract of DOI:10.1016/j.bmcl.2007.04.091

A novel series of 3-arylureidobenzylidene-indolin-2-ones was synthesized and their inhibitory activity against Ret tyrosine kinase investigated in comparison with the Ret inhibitor RPI-1 as a reference compound for this series. A few compounds were able t

Full text of DOI:10.1016/j.bmcl.2007.04.091

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3962–3968
Synthesis and RET protein kinase inhibitory activity of
3-arylureidobenzylidene-indolin-2-ones
a,
b
Eleonora Rizzi,a Giuliana Cassinelli,b,* Sabrina Dallavalle, Cinzia Lanzi,
*
Raffaella Cincinelli,a Raffaella Nannei,a Giuditta Cuccurub and Franco Zuninob
a
`
Dipartimento di Scienze Molecolari Agroalimentari, Universita di Milano, Via Celoria 2, 20133 Milano, Italy
b
`
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
Received 24 April 2007; accepted 25 April 2007
Available online 30 April 2007
Abstract—A novel series of 3-arylureidobenzylidene-indolin-2-ones was synthesized and their inhibitory activity against Ret tyrosine
kinase investigated in comparison with the Ret inhibitor RPI-1 as a reference compound for this series. A few compounds were able
to revert the RETC634R oncogene-transformed morphologic phenotype of NIH3T3MEN2A cells and showed a selective antiprolif-
erative activity against these cells as compared to parental NIH3T3 cells or NIH3T3 cells transformed with a non-tyrosine kinase
oncogene (NIH3T3H-RAS). Inhibition of Ret enzyme activity by effective derivatives was confirmed in a kinase assay. Structure–
activity relationship indicated a favourable activity for 5,6-dimethoxyindolinone derivatives with H, OH, or OMe in the para posi-
tion of the distal aryl ring.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The RET protooncogene is a typical example of a gene
encoding a receptor protein tyrosine kinase involved in
the aetiology of human tumours.1 Activating RET
mutations and rearrangements have been implicated in
the development of sporadic and inherited forms of thy-
roid cancer. In papillary carcinomas, RET oncogenes,
designated RET/PTCs, derive from somatic chromo-
somal rearrangements linking the promoter and N-ter-
minal domains of unrelated genes to the C-terminal
fragment of the RET protooncogene. As a result, chime-
ric, constitutively active, forms of Ret tyrosine kinase
are ectopically expressed in thyroid cells.2,3 In sporadic
cases of medullary thyroid carcinoma (MTC), RET mis-
sense mutations are present at a somatic level, whereas
RET mutations at a germline level are responsible for
the inherited type-2 Multiple Neoplasia (MEN2) syn-
dromes which include MTC.4,5 RET mutants identified
in thyroid carcinomas are dominantly acting oncogenes
which confer gain-of-function to the encoded proteins.
In fact, all RET oncoproteins are characterized by con-
stitutive ligand-independent tyrosine kinase activity that
is required for their transforming ability.6 RET onco-
gene transforming activity can be assessed by transfec-
tion assays on NIH3T3 cells and is typically associated
with constitutive tyrosine phosphorylation of Ret onco-
proteins, alteration of cell morphology, anchorage-inde-
pendent growth in vitro and tumorigenicity in vivo.7
Different strategies designed to block the oncogenic
activity of RET, including gene therapy approaches,
monoclonal antibodies, neutralizing aptamers and tyro-
sine kinase inhibitors, have provided evidence that RET
oncogenes and their products are potential targets for
selective thyroid cancer therapy.8,9 However, a clinically
useful therapeutic option for treating patients with
RET-associated cancers is still not available. Over the
past few years, a number of small-molecule tyrosine
kinase inhibitors have been shown to inhibit Ret activity.
They include natural products such as the fungal metab-
olites clavilactones10 and several synthetic compounds
including indolocarbazoles (CEP-701, CEP-751),11,12
pyrazolopyrimidines (PP1, PP2),13–15 anilinoquinazo-
lines (ZD6474),16 pyrrolopyrimidines (AEE788),17
pyridinyloxyphenylureas (BAY 43-9006)18 and indolin-
2-one derivatives (RPI-12,19,20 and SU1124821).
Keywords: RET; Protein kinase; Antitumour; Thyroid cancer; Indoli-
none; Synthesis.
*
Corresponding authors. Tel.: +39 (0)223902627; fax: +39
(0)223902692 (G.C.); tel.: +39 (0)250316818; fax: +39 (0)250316801
(S.D.); e-mail addresses: giuliana.cassinelli@istitutotumori.mi.it;
sabrina.dallavalle@unimi.it
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.091

E. Rizzi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3962–3968
3963
In an effort to improve the Ret inhibitory activity of
RPI-1 (Chart 1), we designed a series of new compounds
derived by insertion of a diphenyl urea moiety on the ac-
tive nucleus of 5,6-dimethoxyindolinone. The impor-
tance of the diphenyl urea moiety for the inhibition of
a number protein kinases, including p38 MAPK,
RAF-1 and CDKs, is consistent with earlier observa-
tions regarding this structural motif.22 Recently, other
classes of diphenyl urea derivatives have been shown
to be potent inhibitors of KDR tyrosine kinase.23
favour strongly the E product. The assigned configura-
tion of the compounds was based upon the chemical
shifts of the protons at the C-20 and C-60 positions in
the phenyl ring of the benzylidene moiety (Table 1). It
has been demonstrated through NOE experiments that
the chemical shift of C-20 and C-60 protons is in the
range 7.45–7.84 ppm for the E isomers and 7.85–8.53
for the Z isomers.25
A cell-based screening26 was performed to assess inhibi-
tion of RET-driven transformation by the synthesized
compounds. The inhibition of RET transforming activ-
ity was evaluated by using NIH3T3 cells transformed by
the C634R mutant of RET associated with the MEN2A
syndrome (NIH3T3MEN2A) as a model system.15 Com-
pounds able to revert the oncogene-transformed mor-
phologic phenotype of NIH3T3MEN2A cells were further
Compounds 1–11 (Table 1) were synthesized24 by con-
densation of the appropriate indolin-2-one with the cor-
responding 4-arylureidobenzaldehyde in the presence of
piperidine. The aldehydes were obtained in turn by reac-
tion of 4-formylphenylisocyanate with the appropriate
arylamine (Scheme 1). Compound 12 was obtained by
the same sequence but using 4-methylpiperazine. In
the case of 13, the required aldehyde was prepared by
coupling 4-carboxybenzaldehyde with aniline.
tested on NIH3T3 cells and NIH3T3 cells transformed
15
with a non-tyrosine kinase oncogene (NIH3T3H-RAS
)
to compare the antiproliferative effect of each com-
pound on the three cell lines. Furthermore, the same
compounds selected as the most effective were subjected
to a Ret kinase assay to assess direct enzyme inhibition.
Ret inhibition in NIH3T3MEN2A cells was assessed by
the receptor autophosphorylation in Western blot anal-
ysis. In each test, RPI-1 [3-(4-hydroxybenzylidene)-5,6-
dimethoxyindolin-2-one], previously characterized as a
Ret inhibitor,19 was used as the reference compound.
Compound 14 was obtained by hydrogenation of 3 in
the presence of Pd/C (Chart 2). In general, the coupling
reaction gave the E isomer as the major product, except
in the case of compounds 1, 2 and 10. Therefore the
presence of the 5,6-dimethoxy substitution seems to
HO
The results of the cell-based screening and kinase assay
with the synthesized compounds are reported in Table 2.
MeO
MeO
Two compounds in this series (3 and 4) induced a stable
reversion (up to 72 h) of the RET-induced transformed
phenotype of NIH3T3MEN2A cells, with IC50 compara-
ble to that of the reference compound RPI-1. Com-
pounds 6 and 7 induced a partial reversion of the
transformed cell morphology (cell flattening) associated,
O
N
H
RPI-1
Chart 1.
Table 1. Structure of new 3-arylureidobenzylidene-indolin-2-ones
O
NH
NH R
4
R
1
O
N
R
R
2
3
Compound
Config.
R1
R2
R3
R4
mp (ꢁC)
% Yield
da
1a
1b
2
Z
E
Z
E
E
E
E
E
E
E
Z
E
H
H
H
H
H
H
Ph
Ph
Ph
Ph
Ph
265–267
224–226
271–273
255–258
250–252
255–258
250–252
260 (dec)
248–250 (dec)
>200 (dec)
249–250
264–265
23
50
35
65
32
65
32
25
58
40
52
46
8.44
7.61
8.46
7.61
7.61
7.61
7.61
7.60
7.58
7.62
8.44
7.59
H
Cl
H
H
3
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
4
Me
H
5
4-F-Ph
4-OH-Ph
6
H
7
H
4-OMe-Ph
4-NMe2-Ph
3,4-(OCH2O)-Ph
8
H
9
H
10
11
OH
H
Ph
4-Py
OMe
OMe
a Chemical shift of the 20 and 60 protons on the proximal aryl ring.

3964
E. Rizzi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3962–3968
O
NH
NH R
4
R
R
1
O
R
O
4
N
R
NH
NH R
4
NCO
CHO
2
R
NH
1
2
3
O
N
R
R
2
a
b
3
CHO
Scheme 1. Reagents and conditions: (a) Toluene, reflux, 2–5 h; (b) EtOH, piperidine, 90 ꢁC, 3–7 h.
IC50 values were obtained for RPI-1 and 3, a lower
activity for 6, while 4 and 12 were inactive or almost
inactive, respectively. Compound 4 showed no selectiv-
ity among the three cell lines, suggesting a different
mechanism of action. Indeed, N-methylation resulted
in a loss of ability to inhibit Ret kinase activity as doc-
umented for compound 4 and the N-methyl derivative of
RPI-1 (not shown). The ability of 4 to induce reversion
of the transformed phenotype likely reflects modulation
of downstream events implicated in various survival
pathways. This interpretation is consistent with efficacy
of 4 in H-RAS transformed cells and in a partial rever-
sion of these cells (not shown).
Me
N
N
O
O
NH
HN
HN
HN
O
MeO
MeO
MeO
MeO
MeO
O
O
O
N
N
N
MeO
H
H
H
13
12
14
Chart 2.
particularly in the case of 7, with cell suffering (Fig. 1).
Compound 12 induced a transient reversion of the trans-
formed cell phenotype which was lost after 24 h of treat-
ment. Conversely, none of these compounds affected the
transformed morphology of NIH3T3H-RAS cells (Fig. 1).
Western blot analysis confirmed the ability of com-
pounds 3 and 6 of inhibiting Ret tyrosine kinase in
NIH3T3MEN2A cells as indicated by dose-dependent
inhibition of the receptor autophosphorylation. In con-
trast to the cell-free enzyme assay, compound 3 showed
higher efficacy than compound 6 in cell culture (Fig. 2).
As previously reported for RPI-1,20 Ret kinase inhibi-
tion in treated cells was associated with a reduction of
RET protein expression.
Similar to RPI-1, compounds 3, 6 and 7 showed a selec-
tive antiproliferative effect against NIH3T3MEN2A cells
as compared to that of NIH3T3H-RAS cells or the paren-
tal NIH3T3 cells, thus confirming that RET oncogene
expression could be a major determinant of cell sensitiv-
ity to drug treatment. Compounds 3, 4, 6, 7 and 12 were
tested for their inhibitory activity in a Ret kinase assay27
in comparison with RPI-1. The results indicated com-
pound 6 as the most effective inhibitor. Comparable
The results of this study allow some consideration as to
the structure-activity relationship. Comparison of the
biochemical and cellular effects of the compounds that
proved able to induce the reversion of the transformed
phenotype provides the following information:
Table 2. In vitro activity of the synthesized compounds
NIH3T3MEN2A reversion (10 lM)
NIH3T3MEN2A
Antiproliferative activity (IC50 in lM) (72 h)
Ret Kinase Assayb (IC50lM)
NIH3T3
NIH3T3H-RAS
RPI-1
1a
1b
2
YES (s)a
NO
NO
3.6 1.6
>40
16.3 3.7
19.7
2
0.17 0.01
10 0.7
>40
4.6 0.1
NO
3
YES (s)a
YES (s)a
NO
14 2.2
6.6 0.8
8.7 0.1
5.4 1.4
0.2 0.001
Inactivec
4
4.1
1
5
6.1 1.7
8.2 1.8
6
YES (p)a
YES (p)a
NO
24.4 0.1
14.7
22.2
12.3 1.3
3
0.05 0.02
0.58 0.22
7
3.6
>40
19.4
1
2
8
9
NO
NO
2
10
11
12
13
14
7.5 0.1
4.7 0.1
NO
YES (t)a
NO
NO
15.8 0.7
12.2 0.4
21.5 2.8
>3
a (s), stable; (p), partial; (t), transient.
b Phosphorylation of MBP as substrate.
c inactive up to 3lM.

E. Rizzi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3962–3968
3965
Figure 1. Effect of selected 3-arylureidobenzylidene-indolin-2-ones on the transformed morphologic cell phenotype of murine fibroblasts NIH3T3
transfected with RETC634R (NIH3T3MEN2A) or H-RAS oncogene (NIH-3T3H-RAS). Cells were treated with vehicle or 10 lM compound, then
photographed under a phase-contrast microscope after 24 h (NIH3T3MEN2A) or 72 h (NIH-3T3H-RAS) of treatment. Parental NIH3T3 cells are
shown for comparison. Original magnification 100·.
(a) 3, characterized by an unsubstituted distal aryl
group, retained activity features, including direct
Ret kinase inhibitory activity, comparable to those
of RPI-1.
(b) The presence of substituents in the distal aryl moi-
ety reduced the ability to inhibit Ret function in cell
culture, as indicated by the less persistent effects of
6 and 7 on transformed morphology. This is

3966
E. Rizzi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3962–3968
tics, Bresso, Italy) for the generous gift of 5,6-dim-
ethoxyindolin-2-one and RPI-1.
References and notes
1. Pasini, B.; Ceccherini, I.; Romeo, G. Trends Genet. 1996,
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Figure 2. Effect of treatment with compounds 3, 6 and RPI-1 reference
compound on Ret autophosphorylation and expression. NIH3T3MEN2A
cells were exposed to vehicle or to the indicated concentrations of
drugs for 24 h. Whole cell lysates were analyzed by Western blotting
with the indicated antibodies. pY, generic pTyr; pY1062, specific pRet-
Tyr1062.
9. Lanzi, C.; Cassinelli, G.; Cuccuru, G.; Zunino, F. In:
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consistent with the loss of activity of compounds
with bulkier substituents (e.g., derivatives 8 and
9). On the other hand, the presence of hydroxyl
group in the distal aryl (compound 6) conferred
the highest inhibitory activity on Ret enzyme in
cell-free system. In fact, the O-methyl derivative
of compound 6 (7) was found ten times less potent.
(c) Methylation of the indolin-2-one nitrogen of 3
causes loss of Ret inhibitory activity and specificity
against Ret-transformed cells.
(d) The replacement of the distal aryl moiety by a basic
piperazine (12) did not allow persistent cellular
effect, probably as a consequence of a reduced
interaction with the target, as indicated by the Ret
kinase assay.
(e) Hydrogenation of the exocyclic double bond (14 vs.
3), with the consequent loss of coplanarity of the
rings, strongly depressed the activity.
(f) Finally, the presence of the two methoxy groups at
positions 5 and 6 in the indolin-2-one nucleus seems
to play an important role in the activity (compare 3
vs 1b).
14. Carlomagno, F.; Vitagliano, D.; Guida, T.; Basolo, F.;
Castellone, M. D.; Melillo, R. M.; Fusco, A.; Gazit, A.;
Santoro, M. J. Clin. Endocrinol. Metab. 2003, 88, 1897.
15. Carniti, C.; Perego, C.; Mondellini, P.; Pierotti, M. A.
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etta, R. A.; Borrello, M. G.; Menta, E.; Pierotti, M. A.;
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In conclusion, the present study has identified new
inhibitors of Ret kinase in a series of 3-arylureidobenzy-
lidene-indolin-2-ones. Structure–activity relationship
evidences a favourable biochemical and biological activ-
ity activity of 5,6-dimethoxyindolinone derivatives with
H, OH, or OMe in the para position of the distal aryl
ring. Further studies are needed to optimize chemical–
physical properties as well as the potency of these
inhibitors.
20. Cuccuru, G.; Lanzi, C.; Cassinelli, G.; Pratesi, G.;
Tortoreto, M.; Petrangolini, G.; Seregni, E.; Martinetti,
A.; Laccabue, D.; Zanchi, C.; Zunino, F. J. Natl. Cancer
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Acknowledgments
This research was supported by MIUR (FIRB project
2001). We are indebted to Dr. E. Menta (Cell Therapeu-
21. Kim, D. W.; Jo, Y. S.; Chung, H. K.; Song, J. H.; Park, K.
C.; Park, S. H.; Hwang, J. H.; Rha, S. Y.; Kweon, G. R.;

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(300 MHz, DMSO-d6) d 10.30 (s, 1H, NH indol), 9.80 (s,
1H, –NH), 9.02 (s, 1H, –NH), 7.60–7.75(m, 4H, 4Ar), 7.42
(d, J = 8.56 Hz, 2H, 2Ar), 6.82 (d, J = 8.56 Hz, 2H, 2Ar),
6.51 (s, 1H, 1Ar), 3.80 (s, 3H, –OMe), 3.75 (s, 3H,
–OMe)3.65 (s, 3H, –OMe). Compound 8: 1H NMR
(300 MHz, DMSO-d6) d 10.31 (s, 1H, NH indol), 8.86 (s,
1H, –NH), 8.40 (s, 1H, –NH), 7.67 (m, 2H, 2Ar), 7.58 (m,
2H, 2Ar), 7.37 (s, 1H, 1Ar), 7.27 (s, 1H, CH@), 7.25 (d,
J = 8.56 Hz, 2H, 2Ar), 6.70 (d, J = 8.56 Hz, 2H, 2Ar), 6.51
(s, 1H, 1Ar), 3.79 (s, 3H, –OMe), 3.62 (s, 3H, OMe), 2.84
(s, 6H, NMe2). Compound 9: 1H NMR (300 MHz,
DMSO-d6) d 10.30 (s, 1H, NH indol), 9.06 (s, 1H,
–NH), 8.78 (s, 1H, –NH), 7.67 (m, 2H, 2Ar), 7.62 (m,
2H, 2Ar), 7.37 (s, 1H, 1Ar), 7.28 (s, 1H, CH@), 7.22 (d,
J = 2.23 Hz, 1H, 1Ar), 6.84 (d, J = 8.56 Hz, 1H, 1Ar), 6.79
(dd, J = 2.23, 8.56 Hz, 1H, 1Ar), 6.51 (s, 1H, 1Ar), 5.98 (s,
2H, OCH2O), 3.79 (s, 3H, –OMe), 3.62 (s, 3H, OMe).
Compound 10: 1H NMR (300 MHz, DMSO-d6) d 10.75 (s,
1H, NH indol), 9.07 (s, 1H, –NH), 8.83 (s, 1H, –NH), 8.44
(d, J = 8.56 Hz, 2H, 2Ar), 7.82 (s, 1H), 7.74 (d,
J = 7.44 Hz, 1H, 1Ar), 7.58 (d, J = 8.56 Hz, 2H, 2Ar),
7.47 (d, J = 7.82 Hz, 2H, 2Ar), 7.23–7.37 (m, 3H,
22. Dumas, J. Curr. Opin. Drug Discov. Dev. 2002, 5, 718.
23. (a) Curtin, M. L.; Frey, R. R.; Heyman, H. R.; Sarris, K. A.;
Steinman, D. H.; Holmes, J. H.; Bousquet, P. F.; Cunha, G.
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B.; Stewart, K. D.; Davidsen, S. K.; Michaelides, M. R.
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R.; Frey, R. R.; Bousquet, P. F.; Cunha, G. A.; Moskey, M.
D.; Ahmed, A. A.; Soni, N. B.; Marcotte, P. A.; Pease, L. J.;
Glaser, K. B.; Yates, M.; Bouska, J. J.; Albert, D. H.; Black-
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24. General procedure for the synthesis of compounds 1–14. The
appropriate aldehyde (1 equiv) was added to EtOH (3 ml/
0.2 mmol) and the mixture was stirred until complete
solution. The oxindole (1 equiv) and piperidine (0.1 equiv)
were added, and the mixture was heated to 90 ꢁC for 3–
7 h, and cooled. The resulting precipitate was filtered,
washed with cold ethanol and dried to give the pure
compound. General procedure for the synthesis of interme-
diate aldehydes. 4-Formylphenylisocyanate (1 equiv) was
suspended in toluene (2 M) and the appropriate aniline
(1 equiv) was added. The mixture was refluxed for 2–5 h,
then the solvent was removed by rotatory evaporation and
the residue purified by flash chromatography on silica gel
(CH2Cl2/MeOH). The compound (intermediate Schiff
base) was dissolved in THF and 1 M HCl was added to
the stirred solution. After 1 h at room temperature, THF
was removed and the resulting precipitate was filtered,
washed with water and dried to give the pure compound.
Spectral data for compounds 1–14. Compound 1a: 1H
NMR (300 MHz, DMSO-d6) d 10.60 (s, 1H, NH indol),
9.01 (s, 1H, –NH), 8.78 (s, 1H, –NH), 8.44 (d, J = 8.56 Hz,
2H, 2Ar), 7.60–7.75 (m, 2H, 2Ar), 7.50–7.60 (m, 2H, 2Ar),
7.45–7.50 (m, 2H, 2Ar), 7.25–7.40 (m, 2H, 1Ar + CH@),
7.15–7.25 (m, 1H, 1Ar), 6.90–7.10 (m, 2H, 2Ar), 6.75–6.80
(m, 1H, 1Ar). Compound 1b: 1H NMR (300 MHz,
DMSO-d6) d 10.55 (s, 1H, NH indol), 9.05 (s, 1H, –NH),
8.80 (s, 1H, –NH), 7.61–7.67 (m, 3H, 3Ar), 7.50–7.65
(m, 2H, 2Ar), 7.45–7.50 (m, 2H, 2Ar), 7.20–7.40(m, 4H,
3Ar + CH@), 6.85–7.10 (m, 3H, 3Ar). Compound 2: 1H
NMR (300 MHz, DMSO-d6) d 10.10 (s, 1H, NH indol),
9.15 (s, 1H, –NH), 8.80 (s, 1H, –NH), 8.46 (m, 2H, 2Ar),
7.78–7.95 (m, 2H, 2Ar), 7.40–7.65 (m, 4H, 4Ar), 7.10–7.35
(m, 3H, 2Ar + CH@), 6.95–7.10 (m, 1H, 1Ar), 6.75–6.82
(m, 1H, 1Ar). Compound 3: 1H NMR (300 MHz, DMSO-
d6) d 9.20 (s, 1H, NH indol), 8.42 (s, 1H, –NH), 8.21 (s,
1H, –NH), 7.60–7.75 (m, 4H, 4Ar), 7.50–7.54 (m, 2H,
2Ar), 7.41 (s, 1H, 1Ar), 7.35 (s, 1H, CH@), 7.22–7.26 (m,
2H, 2Ar), 6.98–7.01 (m, 1H, 1Ar), 6.60 (s, 1H, 1Ar), 3.82
(s, 3H, –OMe), 3.64 (s, 3H, OMe). Compound 4: 1H NMR
(300 MHz, DMSO-d6) d 9.01 (s, 1H, –NH), 8.76 (s, 1H,
–NH), 7.69 (m, 2H, 2Ar), 7.61 (m, 2H, 2Ar), 7.45–7.52 (m,
3H, 3Ar), 7.25–7.35(m, 3H, 2Ar + CH@), 6.95–7.01 (m,
1H, 1Ar), 6.52 (s, 1H, 1Ar), 3.80 (s, 3H, –OMe), 3.65 (s,
1
2Ar + CH@), 6.89-7.11 (m, 3H, 3Ar). Compound 11: H
NMR (300 MHz, DMSO-d6) d 10.40 (s, 1H, NH indol),
9.25 (brs, 2H, 2-NH), 8.46 (m, 2H, 2Ar), 7.72 (m, 2H,
2Ar), 7.59 (m, 2H, 2Ar), 7.45 (m, 2H, 2Ar), 7.37 (s, 1H,
1Ar), 7.25 (s, 1H, CH@), 6.51 (s, 1H, 1Ar), 3.78 (s, 3H,
–OMe), 3.62 (s, 3H, Ome). Compound 12:. 1H NMR
(300 MHz, DMSO-d6) d 10.20 (s, 1H, NH indol), 7.75 (m,
2H, 2Ar), 7.55 (m, 2H, 2Ar), 7.48 (s, 1H, 1Ar), 7.10 (s, 1H,
CH@), 6.50 (s, 1H, 1Ar), 3.78 (s, 3H, –OMe), 3.60 (s, 3H,
OMe), 3.40–3.70 (m, 4H), 2.18–2.45 (m, 4H), 2.15 (s, 3H, -
1
NMe). Compound 13: H NMR (300 MHz, DMSO-d6) d
10.42 (s, 1H, NH indol), 10.35(s, 1H, –NH), 8.09 (d,
J = 8.56 Hz, 2H, 2Ar), 7.84 (d, J = 8.56 Hz, 2H, 2Ar), 7.79
(d, J = 7.82 Hz, 2H, 2Ar), 7.47 (s, 1H, 1Ar), 7.35–7.39 (m,
2H, 2Ar), 7.12-7.18 (m, 2H, 1Ar + CH@), 6.53 (s, 1H,
1Ar), 3.80 (s, 3H, –OMe), 3.58 (s, 3H, OMe). Compound
1
14: H NMR (300 MHz, DMSO-d6) d 10.20 (s, 1H, NH
indol), 8.60 (s, 1H, –NH), 8.52 (s, 1H, –NH), 7.45 (m, 2H,
2Ar), 7.25–7.35 (m, 4H,4Ar), 7.00–7.15 (m, 2H, 2Ar),
6.80–6.95 (m, 1H, 1Ar), 6.55 (s, 1H, 1Ar), 6.45 (s, 1H,
1Ar), 3.70–3.90 (m, 1H), 3.75 (s, 3H, –OMe), 3.62 (s, 3H,
OMe), 3.40–3.55 (m, 1H), 2.75–2.90 (m, 1H).
25. Sun, L.; Tran, N.; Tang, F.; App, H.; Hirth, P.; McMa-
hon, G.; Tang, C. J. Med. Chem. 1998, 41, 2588.
26. Cell culture and antibodies. Cells were maintained in
Dulbecco’s modified Eagle medium (DMEM) supple-
mented with 5% serum for NIH3T3MEN2A or 10% calf
serum (Colorado Serum Company, Denver, CO) for
NIH3T3 and NIH3T3H-RAS cells and incubated at 37 ꢁC
in a 10% CO2 atmosphere. Compounds, dissolved in
DMSO, were diluted in cell culture medium (0.5% solvent
final concentration). IC50s were calculated from dose-
response curves obtained by cell counting after 72 h of
treatment. 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 described.20 The
following antibodies were used: monoclonal anti-pTyr
antibody clone 4G10 (Upstate Biotechnology, Lake
Placid, NY); rabbit polyclonal anti-Ret H300 and anti-
pRet (Tyr1062) (Santa Cruz Biotechnology, Santa Cruz,
CA) and anti-tubulin (Sigma Chemical Company, St.
Louis, MO).
1
3H, –OMe), 3.20 (s, 3H, -NMe). Compound 5: H NMR
(300 MHz, DMSO-d6) d 10.30 (s, 1H, NH indol), 9.00 (s,
1H, –NH), 8.81 (s, 1H, –NH), 7.67 (m, 2H, 2Ar), 7.61 (m,
2H, 2Ar), 7.48–7.52 (2H, m, 2Ar), 7.40 (s, 1H, 1Ar), 7.28
(s, 1H, CH@), 7.10–7.15 (m, 2H, 2Ar), 6.51 (s, 1H, 1Ar),
1
3.80 (s, 3H, –OMe), 3.62 (s, 3H, OMe). Compound 6: H
NMR (300 MHz, DMSO-d6) d 10.20 (s, 1H, NH indol),
9.10 (s,1H, –OH), 8.89 (s, 1H, –NH),8.44 (s, 1H, –NH),
7.67 (m, 2H, 2Ar), 7.61 (m, 2H, 2Ar), 7.37 (s, 1H, 1Ar),
7.28 (s, 1H, CH@), 7.23 (d, J = 8.56 Hz, 2H, 2Ar), 6.70 (d,
J = 8.56 Hz, 2H, 2Ar), 6.51 (s, 1H, 1Ar), 3.78 (s, 3H,
–OMe), 3.62 (s, 3H, OMe). Compound 7: 1H NMR
27. Kinase assay. A non-radioactive kinase assay was per-
formed using recombinant Ret active protein (Upstate,
Lake Placid, NY) and Myelin Basic Protein (MBP)
(Sigma) as kinase substrate following the Upstate protocol

3968
E. Rizzi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3962–3968
with some modifications. Briefly, Ret protein (30 ng), was
concentrated Laemmli buffer, then samples were subjected
to SDS–PAGE and immunoblotted with anti-pTyr anti-
body. Phosphorylated MBP was revealed by chemiolumi-
nescence reaction ECL (GE Healthcare, Little Chalfont,
UK) and detected by ChemiDoc XRS System, PC. Signal
intensity was analyzed with the interfaced software
Quantity One 4.6.3 (Bio-Rad, Hercules, CA).
incubated for 10 min at 30 ꢁC in kinase buffer (11.6 mM
MOPS, pH 7, 0.2 mM EDTA, 0.9 mM EGTA, 13.5 mM
MgCl2, 4.5 mM b-glycerophosphate, 0.18 mM Na3VO4
and 0.18 mM dithiothreitol) with solvent or inhibitor
(DMSO 4% final concentration) in the presence of 2 lM
MBP and 5 lM ATP. The reaction was stopped by 3-fold