Synthesis, in vitro antiproliferative activities, and Chk1 inhibitory properties of indolylpyrazolones and indolylpyridazinedione

Article abstract of DOI:10.1016/j.ejmech.2006.06.012

The synthesis of 5-indolylpyrazol-3-one, 4-indolylpyrazol-3-one and 4-indolyl-pyridazin-3,6-dione is reported. Their Chk1 inhibitory properties have been evaluated and their in vitro antiproliferative activities toward three tumor cell lines: murine leukemia L1210, human colon carcinoma HT29 and HCT116 have been determined. 4-Indolyl-pyridazin-3,6-dione is inactive against Chk1 and exhibits weak cytotoxicities toward the tumor cell lines tested. The IC₅₀ values toward Chk1 of the two indolylpyrazolones are identical and are in the micromolar range, but the cytotoxicities of 4-indolylpyrazol-3-one are significantly stronger than those of 5-indolylpyrazol-3-one. Since 4-indolylpyrazol-3-one and 5-indolylpyrazol-3-one can present several conformers and tautomeric forms, molecular modelling in the ATP binding site of Chk1 has been carried out to investigate which form could induce the best stabilization in the active site of the enzyme. To get an insight into the kinase selectivity of these compounds, their inhibitory activities toward Src kinase were evaluated.

Full text of DOI:10.1016/j.ejmech.2006.06.012

European Journal of Medicinal Chemistry 41 (2006) 1470e1477
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, in vitro antiproliferative activities, and Chk1 inhibitory
properties of indolylpyrazolones and indolylpyridazinedione
a
Elisabeth Conchon , Bettina Aboab , Roy M. Golsteyn ,
a
b
b
Francisco Cruzalegui, Thomas Edmonds, St e´ phane L e´ once ,
b
a,
Bruno Pfeiffer , Michelle Prudhomme *
a
Laboratoire SEESIB, Universit e´ Blaise Pascal, UMR 6504 du CNRS, 63177 Aubi e` re Cedex, France
b
Institut de Recherches SERVIER, Division Recherche Canc e´ rologie, 125 Chemin de ronde, 78290 Croissy sur Seine, France
Received 21 June 2006; accepted 22 June 2006
Available online 22 September 2006
Abstract
The synthesis of 5-indolylpyrazol-3-one, 4-indolylpyrazol-3-one and 4-indolyl-pyridazin-3,6-dione is reported. Their Chk1 inhibitory prop-
erties have been evaluated and their in vitro antiproliferative activities toward three tumor cell lines: murine leukemia L1210, human colon car-
cinoma HT29 and HCT116 have been determined. 4-Indolyl-pyridazin-3,6-dione is inactive against Chk1 and exhibits weak cytotoxicities
toward the tumor cell lines tested. The IC50 values toward Chk1 of the two indolylpyrazolones are identical and are in the micromolar range,
but the cytotoxicities of 4-indolylpyrazol-3-one are significantly stronger than those of 5-indolylpyrazol-3-one. Since 4-indolylpyrazol-3-one and
5
-indolylpyrazol-3-one can present several conformers and tautomeric forms, molecular modelling in the ATP binding site of Chk1 has been
carried out to investigate which form could induce the best stabilization in the active site of the enzyme. To get an insight into the kinase se-
lectivity of these compounds, their inhibitory activities toward Src kinase were evaluated.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: Indolylpyrazolones; Indolyl-pyridazin-3,6-dione; Antitumor agents; Chk1 inhibitors
1
. Introduction
cytotoxicities toward three tumor cell lines: murine leukemia
L1210, human colon carcinoma HT29 and HCT116.
Staurosporine isolated from cultures of Streptomyces sp.
is a non-selective kinase inhibitor [4]. Among the kinases
that are inhibited by staurosporine, Chk1, which is a serine/
threonine kinase, plays a major role in the cell cycle arrest
in response to DNA damage [5].
The pyrazol-3-one heterocycle is found in many biologi-
cally active compounds. Some of them are inhibitors of tumor
necrosis factor-a production and of the JNK3 kinase [1].
Others were found to be antihyperglycemic agents and
were isolated as their hydroxy tautomers [2]. Very recently,
moderate in vitro antiproliferative activities were described
for N,N-dialkylaminoalkyl substituted bisindolyl and diphenyl
pyrazolone derivatives [3]. In the search for new Chk1 inhib-
itors, we were interested in 5-indolylpyrazol-3-one and 4-
indolylpyrazol-3-one (Fig. 1). We report here their synthesis
together with their Chk1 inhibitory activities and their
Staurosporine is an ATP-competitive Chk1 inhibitor. In the
structure of Chk1 in complex with staurosporine, two hydrogen
bonds are observed between the lactam heterocycle and the ATP
binding site of the enzyme: the first one between the lactam NH
8
5
and the carbonyl oxygen of Glu , and the second one between
the oxygen of the carbonyl group of the lactam function of the
8
7
drug and the amide nitrogen of Cys [6]. The structures of both
-indolylpyrazol-3-one and 5-indolylpyrazol-3-one could be
4
*
Corresponding author. Tel.: þ33 4 73 40 71 24; fax: þ33 4 73 40 77 17.
E-mail address: Michelle.PRUDHOMME@univ-bpclermont.fr (M.
compatible with the formation of these hydrogen bonds, the in-
dole moiety being oriented either toward the carbonyl group or
Prudhomme).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.06.012

E. Conchon et al. / European Journal of Medicinal Chemistry 41 (2006) 1470e1477
1471
Cys87
Glu85
H
N
H
N
H
N
HN NH
O
O
O
O
O
HN
HN
N
N
N
H
N
N
H
H
O
H C
3
5
-indolylpyrazol-3-one 2 4-indolylpyrazol-3-one 4 4-indolylpyridazine-3,6-dione 5
H CO
3
NHCH3
staurosporine
Fig. 1. Structures of 5-indolylpyrazol-3-one, 4-indolylpyrazol-3-one, 4-indolylpyridazine-3, 6-dione and staurosporine.
toward the opposite site like the two indole moieties of stauro-
sporine. Therefore, in spite of the structures of our pyrazolones,
less rigid than staurosporine and not necessarily planar, the in-
hibitory activities toward Chk1 have been investigated.
form was not observed in closely related 3-hydroxy-acrylates
[12,13]. Reaction of compound 3 with hydrazine hydrate in an
acidic medium as described in non-aromatic series [14] gave
pyrazolone 4 in 74% yield. As for compound 2, the two ex-
changeable protons of the pyrazolone 4 are broad signals
To get an insight into the influence of the size of the upper het-
erocycle, indolyl-pyridazin-3,6-dione was synthesized and its
biological properties were evaluated and compared with those
of the indolylpyrazolones. These compounds can present tauto-
meric forms that could bind differently in the ATP binding site of
Chk1. Accordingly, molecular modelling has been carried out to
get an insight into the stabilization of the different forms in the
active site of Chk1. Since, very recently several pyrazolones
were described as potent Src kinase inhibitors [7], the inhibitory
activities of 4-indolylpyrazol-3-one, 5-indolylpyrazol-3-one
and4-indolyl-pyridazin-3,6-dionetoward Src were also evaluated.
1
shifted at 10.00 and 11.36 ppm. The H NMR spectrum does
0
not allow to confirm a tautomeric equilibrium between 4, 4
0
0
and 4 . Compound 4 has also been obtained previously in
two steps from ethyl 3-indoleacetate via 3-dimethylamino-
2-(1H-indol-3-yl)propenoate [15].
4-(Indol-3-yl)-pyridazine-3,6-dione 5 (Scheme 1) was pre-
pared by the reaction of hydrazine hydrate with 3-(indol-
3-yl)-furane-2,5-dione according to a method described in
indolocarbazole series [16], followed by acidic treatment.
In DMSO, the exchangeable protons gave three thin signals
at 10.58, 11.74 and 12.00 ppm. Two isomers could be ob-
tained in this reaction: a five-membered ring N-amino deriv-
ative [17e19] or a six-membered ring hydrazide [19,20]. In
a five-membered ring N-amino derivative, the two protons
of the amino group should be shifted at about 5 ppm. The ab-
sence of exchangeable protons at about 5 ppm confirms un-
equivocally the hydrazide form.
2
. Results and discussion
2
.1. Chemistry
The synthetic method for the preparation of compound 2 is
outlined in Scheme 1. Ethyl 1-chloro-malonate was coupled
to indole in the presence of diethylaluminium chloride. This
Lewis acid was successfully used for the acylation of indoles
in high yields [8]. Reaction of the resulting ethyl 3-oxo-3-
2.2. Chk1 inhibitory activities
(
1H-indol-3-yl)-propionate 1 with hydrazine hydrate according
To our knowledge, compound 4 has never been tested toward
kinases and only compounds bearing pyrazolinone heterocycles
different than the ones present in compound 2 are claimed in
two patents as serine/threonine and tyrosine kinase inhibitors
[21,22]. In Schemes 2A and 3A are shown the tautomeric forms
of compounds 2 and 4 together with the possible fundamental
to a method described for the preparation of polysubstitued pyr-
azolones [9] led to pyrazolone 2. Like pyrazol-3-ones without
substituents on the nitrogens, compound 2 could exist as three
tautomers 2, 2 and 2 (Scheme 2) [10]. In the H NMR spectrum
of compound 2 in DMSO, the two signals observed for the ex-
changeable protons of the pyrazolone heterocycle are broad sig-
0
00
1
8
5
87
hydrogen bonds with Glu and Cys in the ATP binding site
0 0 0
1
nals at 9.90 and 11.53 ppm. However, the H NMR spectrum
of Chk1. Conformers 2 a (conformer of 2 ), and 4 a (the same
0 ꢀ 00
molecule as 4 but with a rotation of 180 ) as well as 2 and
does not allow to confirm the presence of tautomeric forms.
For the two-step synthesis of compound 4, formylation of
commercial methyl 3-indolyl-acetate with methyl formate af-
ter deprotonation with sodium hydride according to a method
described with a phenyl substituent instead of an indole [11]
led to 3-hydroxy-acrylate 3 in a quantitative yield. The formyl
0
0
85
4 may be able to form similar hydrogen bonds with Glu
8
7
and Cys . These hydrogen bonds are also possible in com-
pound 5 (Scheme 4A). Molecular modelling has been carried
out using as model the complex structure of Chk1/staurosporine
[6] downloaded from the Protein Data Bank. Concerning

1472
E. Conchon et al. / European Journal of Medicinal Chemistry 41 (2006) 1470e1477
H
N
O
O
O
NH
O
O
Cl
Et AlCl
OEt
OEt
H N-NH , H O
2
2
2
2
N
H
N
H
1
2
N
H
H
N
HO
O
NH
OCH3
NaH
OCH3
H N-NH , H O
2
2
2
O
O
4
N
HCOOMe
N
EtOH, AcOH
3
H
H
N
H
HN NH
O
O
O
O
O
1
) H N-NH , H O
2
2
2
2) HCl
5
N
N
H
H
Scheme 1. Synthetic schemes for compounds 2, 4, and 5.
compound 2 (Scheme 2B), the same hydrogen bond net as ob-
served with staurosporine is only conserved with the carbonyl
inhibition at a drug concentration of 10 mM were determined
(Table 1). None of thethree compounds inhibits Srcsignificantly.
0
0
form 2. Conformers 2 and 2 a show only one hydrogen bond be-
8
tween the hydroxy group and the carbonyl of Glu , whereas
5
2.4. In vitro antiproliferative activities
0
0
with 2 two hydrogen bonds are observed but both with the hy-
droxy group. With compound 4 (Scheme 3B) molecular model-
ling gave similar results. Only compound 4 gives the same
hydrogen bond net as that observed with staurosporine. With
The cytotoxicities of compounds 2 and 4 toward three tu-
mor cell lines: murine leukemia L1210, human colon carci-
noma HT29 and HCT116 were determined (Table 2). The
most sensitive cells are HCT116 colon carcinoma cells. Com-
pound 4 is much more cytotoxic than compound 2 against the
three tumor cell lines tested. The inhibition of Chk1 is not ex-
pected by itself to lead to cytotoxic compounds. However, the
ATP binding site being similar in all the kinases, except the
surrounding pockets, a Chk1 inhibitor generally inhibits other
kinases which could be responsible for its cytotoxicity. Prob-
ably, the inhibition of targets other than Chk1 can explain
the cytotoxicity of compound 4. The in vitro antiproliferative
activities observed for compound 5 are very weak, this com-
pound is very likely a poor kinase inhibitor.
0
0
85
4
whereas with 4 two hydrogen bonds are observed but both
and 4 a, only one hydrogen bond is formed with Glu ,
0
0
8
with Glu . Finally, with compound 5 (Scheme 4B) only one hy-
5
8
drogen bond can be observed with Glu .
5
The Chk1 inhibitory activities of compounds 2, 4 and 5
were evaluated (Table 1). The IC50 values are identical for
compounds 2 and 4 (5 mM) suggesting that the two hydrogen
8
5
87
bonds with Glu and Cys are conserved. Compound 5 was
inactive toward Chk1, which is in agreement with the molec-
ular modelling results. The biological data suggest that a five-
membered ring upper heterocycle is necessary for Chk1
inhibition.
3. Conclusion
2
.3. Src kinase inhibitory activities
In conclusion, this work reports the synthesis of 4-indolyl-
pyrazol-3-one and 5-indolylpyrazol-3-one, their cytotoxicities
toward three murine and human tumor cell lines and their inhib-
itory potencies toward Chk1. In spite of the position of the in-
dole substituent in 4 or 5 of the pyrazolone moiety, the Chk1
inhibitory potencies are identical, suggesting an identical orien-
tation of the pyrazolone heterocycle within the ATP binding
pocket of Chk1. The differences observed in the cytotoxicities
suggest targets other than Chk1 for compound 4. These targets
To get a first insight into the kinase selectivity, the inhibitory
activities of compounds 2, 4 and 5 were evaluated toward the
tyrosine kinase Src. Indeed, a recent paper [7] reports Src inhib-
itory activities in the micromolar range of some pyrazolone
derivatives in which the carbonyl group and the adjacent NH
of the pyrazolones show hydrogen bond interactions with the
3
Glu
39
341
and Met
residues of Src. The percentages of Src

E. Conchon et al. / European Journal of Medicinal Chemistry 41 (2006) 1470e1477
1473
A
B
GLU 85
H
CYS 87
N
O
NH
2
2
N
H
A
B
GLU 85
CYS 87
H
O
N
N
HN
HO
NH
2’
2'a
GLU 85
HN
2'
N
H
CYS 87
2
conformers
2’a
A
B
GLU 85
H
CYS 87
N
HO
N
2''
N
H
2’’
8
Scheme 2. On the left, tautomeric forms for compound 2 are presented with possible hydrogen bonds between Glu and Cys residues in the ATP binding site of
5
87
Chk1; on the right, hydrogen bonds obtained after molecular modelling are presented.
could be various kinases. The inhibitory activities of com-
pounds 2, 4 and 5 have been evaluated toward the tyrosine ki-
nase Src, but none of the three compounds inhibits this
tyrosine kinase significantly. A recent work describes a buried
pocket in Chk1 at the periphery of the ATP binding site which
can be used for the conception of potent and selective Chk1 in-
8
4
59
hibitors [23]. In particular, Leu and Asn residues can be ex-
ploited to form possible supplementary hydrogen bonds with
substituents introduced on the indole moiety of compound 2.
We are now investigating this hypothesis. The poor Chk1

1474
E. Conchon et al. / European Journal of Medicinal Chemistry 41 (2006) 1470e1477
A
B
GLU 85
H
N
O
NH
CYS 87
N
H
4
4
A
B
GLU 85
CYS 87
N
H
O
HO
NH
N
HN
4
’
N
H
GLU 85
4'a
N
CYS 87
4
'
H
4
’a
A
B
GLU 85
H
CYS 87
N
HO
N
N
H
4
’’
4''
8
Scheme 3. On the left, tautomeric forms for compound 4 are presented with possible hydrogen bonds between Glu and Cys residues in the ATP binding site of
5
87
Chk1; on the right, hydrogen bonds obtained after molecular modelling are presented.

E. Conchon et al. / European Journal of Medicinal Chemistry 41 (2006) 1470e1477
1475
A
B
HN
NH
GLU 85
O
O
CYS 87
N
H
5
5
8
Scheme 4. After molecular modelling, only one hydrogen bond is observed between compound 5 and Glu residue in the ATP binding site of Chk1.
5
inhibitory properties of indolylpyridazin-3,6-dione seem to in-
dicate that a five-membered ring upper heterocycle is necessary
for an efficient Chk1 inhibition.
The residue was purified by flash chromatography (eluent
EtOAc/C H from 1:9 to 4:6) to give compound 1 (370 mg,
1.60 mmol, 38% yield) as a white solid. Mp. 37e39 C. IR
6
6
ꢀ
ꢁ
1
ꢁ1
(
[
KBr) n
1740 cm , nNH 3220 cm . Mass (ESIþ)
3
C]O
þ
1
M þ Na] 254. H NMR (400 MHz, CDCl ): 1.13 (3H, t,
4
. Experimental section
J ¼ 7.0 Hz), 3.77 (2H, s), 4.08 (2H, q, J ¼ 7.0 Hz), 7.15 (1H,
dt, J ¼ 7.0 Hz, J ¼ 1.5 Hz), 7.18 (1H, dt, J ¼ 7.0 Hz,
4
.1. Chemistry
1
2
1
J ¼ 1.5 Hz), 7.33 (1H, dd, J ¼ 7.0 Hz, J ¼ 2.0 Hz), 7.75
2
1
2
(
9
1H, d, J ¼ 3.5 Hz), 8.27 (1H, dd, J ¼ 7.0 Hz, J ¼ 1.5 Hz),
IR spectra were recorded on a PerkineElmer 881 spectrom-
1
2
1
3
ꢁ
1
.96 (1H, s). C NMR (100 MHz, CDCl ): 14.1 (CH ), 47.1
3 3
eter (n in cm ). NMR spectra were performed on a Bruker
AVANCE 400 (chemical shifts d in parts per million, the fol-
lowing abbreviations are used: singlet (s), broad singlet (br s),
doublet (d), doubled doublet (dd), triplet (t), doubled triplet
(CH ), 61.5 (CH O), 111.9, 122.1, 122.9, 123.9, 133.3 (C
2 2
tert arom), 117.1, 124.7, 136.6 (C quat arom), 168.4, 187.4
C]O).
(
(dt), multiplet (m), quadruplet (q), tertiary carbons (C tert),
quaternary carbons (C quat)). Low resolution mass spectra
4.1.2. 5-(3-Indolyl)-1,2-dihydro-1H,2H-pyrazol-3-one 2
To a solution of hydrazine hydrate (223 mL, 4.68 mmol) in
(
ESIþ) and HRMS were determined on an MS Hewlett Pack-
ethanol (1 mL) heated to reflux was added dropwise a solution
of compound 1 (50 mg, 0.216 mmol) in ethanol (2.5 mL). The
mixture was refluxed for 24 h. After evaporation, water was
added. After extraction with EtOAc, the organic phase was
dried over MgSO and the solvent was removed. CH Cl was
ard engine. Chromatographic purifications were performed by
flash silicagel Geduran SI 60 (Merck) 0.040e0.063 mm col-
umn chromatography.
4
2
2
4
.1.1. Ethyl 3-oxo-3-(1H-indol-3-yl)-propionate 1
To a solution of indole (500 mg, 4.27 mmol) in CH Cl
added to the solid residue. After filtration, the residue was
washed with CH Cl to give 2 (20 mg, 0.100 mmol, 46% yield)
as a white solid. Mp. 148e149 C. IR (KBr) nC]O 1670 cm
2
2
ꢀ
2
2
(
20 mL) at 0 C was added AlClEt2 (820 mL, 964 mg,
ꢀ
ꢁ1
ꢀ
,
6
1
.40 mmol). The mixture was stirred at 0 C for 30 min. A
M solution of ethyl 1-chloro-malonate in hexane (6.50 mL)
ꢁ
1
þ
nNH 3100e3600 cm . HRMS (ESIþ) [M þ H] calcd for
1
ꢀ
ꢀ
C H N O: 200.0824, found 200.0832. H NMR (400 MHz,
11 10 3
was added dropwise at 0 C. After stirring at 0 C for 2 h, water
20 mL) was added. After extraction with EtOAc, the organic
phase was dried over MgSO and the solvent was removed.
DMSO-d ): 5.82 (1H, s), 7.14 (1H, t, J ¼ 7.0 Hz), 7.17 (1H,
6
(
t, J ¼ 7.0 Hz), 7.46 (1H, d, J ¼ 8.0 Hz), 7.71 (1H, d,
J ¼ 2.0 Hz), 7.83 (1H, d, J ¼ 8.0 Hz), 9.90 (1H, br s), 11.34
4
1
3
(
1H, s), 11.53 (1H, br s). C NMR (100 MHz, DMSO-d ):
Table 1
6
IC50 values (mM) toward Chk1, percentages of Src inhibition at a drug concen-
tration of 10 mM, and in vitro antiproliferative activities against three tumor
cell lines: murine leukemia L1210, human HT29 and HCT116 colon carci-
noma (IC50 mM)
86.5, 111.8, 119.3, 119.6, 121.6, 122.9 (C tert), 106.2, 124.4,
136.2, 138.9 (C quat), 161.3 (C]O).
Table 2
IC50 values (mM) toward Chk1 and in vitro antiproliferative activities against
three tumor cell lines: murine leukemia L1210, human HT29 and HCT116
colon carcinoma (IC50 mM)
Compounds IC50 Chk1 Percentage of Src
L1210 HCT116 HT29
(mM)
inhibition at a
drug concentration
of 10 mM
Compounds
IC50 Chk1 (mM)
L1210
HCT116
HT29
2
4
5
5
7.5
5.5
0
49.1
8.2
40.4
2.2
61.4
18.4
84.3
2
4
5
5
49.1
8.2
40.4
2.2
61.4
18.4
84.3
5
Inactive
5
Inactive
74.3
60.1
74.3
60.1

1476
E. Conchon et al. / European Journal of Medicinal Chemistry 41 (2006) 1470e1477
4
.1.3. Methyl 3-hydroxy-2-indolyl-acrylate 3
solution of methyl 3-indolyl-acetate (100 mg,
.53 mmol) in methyl formate (1.08 mL, 17.5 mmol) was
122.0, 130.8 (C tert), 124.9, 136.4, 136.5 (C quat), 153.6,
158.6 (C]O).
A
0
added to NaH (60% in oil, 103 mg, 2.65 mmol) in Et O
2
4.2. Chk1 inhibitory assays
(
2
2 mL). The mixture was stirred at room temperature for
4 h. MeOH (1 mL) was added to eliminate the excess of
Human Chk1 full-length enzymewith an N-terminal GST se-
quence was either purchased from Upstate Biochemicals (no.
NaH, then 50% aqueous acetic acid (2 mL) was added.
After extraction with EtOAc, the organic phase was washed
with saturated aqueous NaHCO then was dried over MgSO
14-346) or purified from extracts of Sf9 cells infected with a ba-
3
4
culovirus encoding GST-Chk1. Assays for compound testing
were based upon the method described by Davies et al. [24].
to give 3 (114 mg, 0.53 mmol, quantitative yield) as an orange
ꢁ1
ꢀ
solid. Mp. 70 C. IR (KBr) n
ꢁ
1652 cm , nCH 2924e
þ
CO
1
ꢁ1
2
855 cm , nNH 3401 cm . Mass (ESIþ) [M] 217,
4.3. Src inhibitory assays
þ
þ
1
[
M þ Na] 240, [M þ K] 256. H NMR (400 MHz,
DMSO-d ): 3.59 (3H, s), 6.92 (1H, dt, J ¼ 8.0 Hz,
6
1
Inhibitors were diluted with a Tecan Evo150 robot. The ki-
nase assay was performed with 4 mL of inhibitor (10%DMSO),
J ¼ 1.0 Hz), 7.03 (1H, dt, J ¼ 8.0 Hz, J ¼ 1.0 Hz), 7.23
2
1
2
(
1H, d, J ¼ 2.5 Hz), 7.27 (1H, d, J ¼ 8.0 Hz), 7.34 (1H, d,
J ¼ 8.0 Hz), 7.89 (1H, s), 10.62 (1H, br s), 11.01 (1H, s).
1
2
0 mL of kinase assay buffer 4ꢂ concentrated (80 mM MgCl ,
2
00 mM HEPES, 0.4 mM EDTA, 2 mM DTT), 10 mL of sub-
1
3
C NMR (100 MHz, DMSO-d ): 50.8 (CH ), 102.7, 106.6,
6
3
strate peptide (KVEKIGEGYYGVVYK, 370 nM) and 6 mL
of Src kinase (stock GTP purified diluted with 1ꢂ kinase assay
buffer to 200 nM). Ten microlitres of co-substrate (40 mM ATP
1
1
25.2, 135.7 (C quat), 111.2, 118.2, 120.3, 120.5, 124.3,
55.1 (C tert), 168.6 (C]O).
3
3
with 0.2 mCi P -g-ATP) was added with a Precision 2000 (Bi-
otek Robotic). The assay mixture was incubated for 20 min at
4
.1.4. 2H,3H-4-(1H-Indol-3-yl)-pyrazolin-3-one 4
A solution of hydrazine hydrate (4 mL) in a mixture of ace-
ꢀ
3
0
0 C and then the reaction was stopped by adding 200 mL of
.85% orthophosphoric acid, and then transferred to a phospho-
tic acid/MeOH (10 mL, 1:1 v/v) was heated to reflux. A solu-
tion of compound 3 (570 mg, 2.62 mmol) in acetic acid (5 mL)
was added dropwise. The mixture was refluxed for 24 h. After
evaporation, water was added to the solid residue, the mixture
was filtered off and the residue was washed with EtOAc to
give 4 (386 mg, 1.94 mmol, 74% yield) as a white solid.
cellulose filter microplate (Whatman e P81). The plate was
washed 3 times with 200 mL of 0.85% orthophosphoric acid
and dried with 200 mL of acetone. The remaining activity is
measured on a Topcount with 25 mL of scintillation solution
(Packard UltimaGold).
ꢀ
ꢁ1
ꢁ1
Mp. 240 C. IR (KBr) n
C]O
þ
1670 cm , nNH 3395 cm .
HRMS (ESIþ) [M þ H] calcd for C H N O: 200.0824,
1
1 10 3
1
4.4. Growth inhibition assays
found 200.0838. H NMR (400 MHz, DMSO-d ): 7.06 (1H,
6
t, J ¼ 7.0 Hz), 7.13 (1H, t, J ¼ 7.0 Hz), 7.41 (1H, t,
J ¼ 8.0 Hz), 7.56 (1H, s), 7.82 (1H, d, J ¼ 8.0 Hz), 7.92 (1H,
s), 10.00, 10.98, 11.36 (3H, 3br s, exchangeable protons).
Tumor cells were provided by American Type Culture Col-
lection (Frederik, MD, USA). They were cultivated in RPMI
1640 medium (Life Science Technologies, Cergy-Pontoise,
France) supplemented with 10% fetal calf serum, 2 mM of
L-glutamine, 100 units/mL of penicillin, 100 mg/mL of strepto-
mycin, and 10 mM of HEPES buffer (pH ¼ 7.4). Cytotoxicity
was measured by the microculture tetrazolium assay as de-
scribed in Ref. [25]. Cells were continuously exposed to
graded concentrations of the compounds for four doubling
times, then 15 mL of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide were added to each well
1
3
C NMR (100 MHz, DMSO-d ): 99.5, 106.9, 125.3, 135.9
6
(
1
C quat), 111.3, 118.6, 119.7, 120.9, 121.5, 135.9 (C tert),
58.3 (C]O).
4.1.5. 4-(1H-Indol-3-yl)-1H,2H-3,6-dihydro-
pyridazin-3,6-dione 5
A solution of 3-(indol-3-yl)-furane-2,5-dione (100 mg,
.46 mmol) in hydrazine hydrate (25 mL) was stirred at
0 C for 17 h. Water (10 mL) was added, then 12 N HCl
0
6
ꢀ
ꢀ
and the plates were incubated for 4 h at 37 C. The medium
was then aspirated and the formazan solubilized with 100 mL
of DMSO. Results are expressed as IC , concentration which
50
(6 mL) was added dropwise. After stirring for 30 min at
room temperature, then extraction with EtOAc, the organic
phase was dried over MgSO . The solvent was removed and
4
reduced by 50% the optical density of treated cells with re-
spect to untreated controls.
the residue was purified by flash chromatography (eluent:
EtOAc 100% to EtOAc/MeOH 90:10) to give 5 (21 mg,
ꢀ
0
(
.09 mmol, 20% yield) as a yellow solid. Mp. 45 C. IR
ꢁ
1
KBr) nC]O 1651 cm , nNH 3000e3396 cm . HRMS
ꢁ1
4.5. Molecular modelling
þ
(
2
ESIþ) [M þ H] calcd for C H N O : 228.0773, found
1
2 10 3 2
1
28.0786. H NMR (400 MHz, DMSO-d ): 7.19 (1H, t,
All molecular mechanics calculations were performed by
the Macromodel [26] molecular modelling software. We
used as model the complex structure of CHK1/STAURO-
SPORINE [6] downloaded from the Protein Data Bank
(1NVR file).
6
J ¼ 7.5 Hz), 7.21 (1H, t, J ¼ 7.5 Hz), 7.23 (1H, s), 7.51 (1H,
d, J ¼ 7.0 Hz), 7.92 (1H, d, J ¼ 7.0 Hz), 8.64 (1H, s), 10.58
1
3
(
NMR (100 MHz, DMSO-d ): 112.4, 116.0, 119.6, 120.7,
1H, s, NH), 11.74 (1H, s, NH), 12.00 (1H, s, NH).
C
6

E. Conchon et al. / European Journal of Medicinal Chemistry 41 (2006) 1470e1477
1477
Energy minimisation was done with AMBER force field
27,28] using the Truncated Newton Conjugate Gradient
method (TNCG).
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[
The authors are grateful to Bertrand L e´ geret, University
Blaise Pascal, Clermont-Ferrand, for recording the mass spec-
tra and Florence Sancier, Institut de Recherches Servier for the
setting of the Src enzyme assay.
[
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