Synthesis, checkpoint kinase 1 inhibitory properties and in vitro antiproliferative activities of new pyrrolocarbazoles

Article abstract of DOI:10.1016/j.bmc.2008.02.061

In the course of structure-activity relationship studies on granulatimide analogues, new pyrrolo[3,4-c]carbazoles have been synthesized in which the imidazole heterocycle was replaced by a five-membered ring lactam system or a dimethylcyclopentanedione. Moreover, the synthesis of an original structure in which a sugar moiety is attached to the indole nitrogen and to a six-membered D ring via an oxygen is reported. The inhibitory activities of the newly synthesized compounds toward checkpoint kinase 1 and their in vitro antiproliferative activities toward three tumor cell lines: murine leukemia L1210, and human colon carcinoma HT29 and HCT116 are described.

Full text of DOI:10.1016/j.bmc.2008.02.061

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4419–4430
Synthesis, checkpoint kinase 1 inhibitory properties and in vitro
antiproliferative activities of new pyrrolocarbazoles
Elisabeth Conchon,a Fabrice Anizon,a Bettina Aboab,a Roy M. Golsteyn,b
b
b
a,
*
´
´
Stephane Leonce, Bruno Pfeiffer and Michelle Prudhomme
a
`
Laboratoire SEESIB, Universite´ Blaise Pascal, UMR 6504 du CNRS, 63177 Aubiere, France
bInstitut de Recherches SERVIER, Division Recherche Cance´rologie, 125 Chemin de ronde, 78290 Croissy sur Seine, France
Received 17 December 2007; revised 13 February 2008; accepted 19 February 2008
Available online 5 March 2008
Abstract—In the course of structure–activity relationship studies on granulatimide analogues, new pyrrolo[3,4-c]carbazoles have
been synthesized in which the imidazole heterocycle was replaced by a five-membered ring lactam system or a dimethylcyclopentan-
edione. Moreover, the synthesis of an original structure in which a sugar moiety is attached to the indole nitrogen and to a six-mem-
bered D ring via an oxygen is reported. The inhibitory activities of the newly synthesized compounds toward checkpoint kinase 1
and their in vitro antiproliferative activities toward three tumor cell lines: murine leukemia L1210, and human colon carcinoma
HT29 and HCT116 are described.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
tent Chk1 inhibitor, is currently being tested in clinical
trials.9,10
Over the past decade, a great deal of interest has been
devoted to checkpoint kinase 1 (Chk1) as a therapeutic
target for cancer treatment.1–4 Chk1, a serine/threonine
kinase, is a key signal transducer in DNA-damage re-
sponse pathways. Normal cells respond to DNA dam-
age by activating cell cycle checkpoints in G1/S and
G2/M. When activated, these checkpoints prevent tem-
porarily the progression of the cell cycle to allow time
for DNA repair. In more than 50% of cancer cells, the
G1 checkpoint is lacking due to loss of p53 or Rb pro-
teins. Therefore, these cells are dependent upon the G2
checkpoint for DNA repair after damage. Chk1 is a ma-
jor regulator of the G2 checkpoint. When active, it
causes the inactivation of cyclin B/CDK1 and thereby
cell cycle arrest. Accordingly, a combination of a
DNA damaging agent and a G2 checkpoint abrogator
should drive selectively G1 checkpoint-deficient cells
into a mitotic catastrophe leading to cell death.5–8 Thus,
Chk1 inhibitors are expected to sensitize cancer cells,
especially p-53 deficient cells, to DNA damaging agents.
The combination of cisplatin, and topotecan, a well-
known topoisomerase I inhibitor, with UCN-01, a po-
Granulatimide and isogranulatimide (Fig. 1) are natural
products isolated from the ascidian Didemnum granula-
tum11,12 that have been previously identified as Chk1
inhibitors with IC50 values of 0.25 and 0.1 lM.13 Like
UCN-01, granulatimide, and isogranulatimide are
ATP competitive Chk1 inhibitors. Their crystal struc-
tures in complex with Chk1 have been determined.13,14
In the course of structure–activity relationship studies,
granulatimide analogues, with and without substituents
on the indole moiety, and in which the D and/or E rings
were modified, have been synthesized.15–30
In our previous studies, we observed that the imide D
ring of bis-imide A (Fig. 1) was sensitive to nucleophiles
and that the upper carbonyl of the D ring was particu-
larly important for the interaction with the ATP-binding
site of Chk1. Therefore, our aim was to synthesize new
bis-imide A analogues either with a lactam D ring in
which the carbonyl group was oriented toward the imide
E heterocycle, or with a five-membered D carbocycle
containing two carbonyl functions instead of an imide
D ring. Moreover, since in UCN-01, the sugar moiety
stabilizes the drug in the ATP-binding site of Chk1 by
additional hydrogen bonds, we wanted to attach a sugar
part to compound B (Fig. 1) which proved to be a strong
Chk1 inhibitor.29 In this paper, we describe the synthesis
Keywords: Granulatimide; Pyrrolo[3,4-c]carbazoles; Antitumor agents;
Chk1 inhibitors.
*
Corresponding author. Tel.: +33 4 73 40 71 24; fax: +33 4 73 40 77
17; e-mail: Michelle.PRUDHOMME@univ-bpclermont.fr
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.061

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E. Conchon et al. / Bioorg. Med. Chem. 16 (2008) 4419–4430
H
H
N
H
N
E
H
N
H
N
E
N
O
O
O
O
O
O
O
O
O
O
E
O
OH
O
C
C
NH
C
A
A
B
D
A
B
D
N
B
D
NH
N
N
N
H
N
N
H
N
H
N
O
H
O
H
HO
O
granulatimide
isogranulatimide
bis-imide A
B
C
H
N
H
N
H
H
N
H
N
2
N
O
O
O
O
3
O
X
Y
O
1
OH
O
4
O
O
NH
N
H
NH
N
N
N
N
O
5
N
H
6
1'
4
O
H3C
H
O
O
H
O
OH
2'
5 X,Y = O
11 X = Y = H
H3CO
NHCH3
staurosporine
13
6'
HO
3'
OH
OH
4'
Figure 1. Chemical structures of granulatimide, isogranulatimide, staurosporine, compounds A, B, C, and H previously described and newly
synthesized compounds 4, 5, 11, and 13.
of these new pyrrolo[3,4-c]carbazoles that can be consid-
ered as granulatimide analogues in which the imidazole
heterocycle has been replaced by a five-membered ring
system bearing one or two carbonyl functions, as well
as the synthesis of a compound bearing a sugar unit
linked to the indole nitrogen and to a diphenolic D ring.
The Chk1 inhibitory activities and the cytotoxicities of
the newly synthesized compounds toward three tumor
cell lines (murine leukemia L1210, and human colon car-
cinoma HT29 and HCT116) are reported.
tuent was born by C5 (its small chemical shift value
was due to the magnetic anisotropy of the triple bond)
and the ethoxycarbonyl group was attached to C4.
Due to the insolubility of 2, a direct hydrogenation did
not work. Protection of the imide nitrogen with a tert-
butyldimethylsilyl group led to the more soluble deriva-
tive 3, which was hydrogenated in the presence of Raney
Nickel to give lactam 4 with concomitant removal of the
tert-butyldimethylsilyl substituent. Deprotection of the
indole nitrogen could be due to the presence of alumi-
num hydroxide residues in Raney Nickel. The position
of the tert-butyldimethylsilyl group was determined
2. Results and discussion
2.1. Chemistry
2
from a HMBC NMR experiment (Fig. 3). Two J cou-
plings between the exchangeable proton at 9.43 ppm
and two quaternary carbons at 141.7 and 144.6 ppm
as well as 3J coupling between this proton and two qua-
ternary carbons at 120.5 and 121.0 ppm showed that the
TBDMS group was attached to the maleimide nitrogen.
The quaternary carbon at 120.5 ppm could be assigned
from its 3J coupling with two protons at 7.62 and
7.47 ppm. The quaternary carbon at 144.6 ppm could
Compound 4 with a lactam D heterocycle was prepared
as outlined in Scheme 1. A Diels–Alder reaction between
3-(indol-3-yl)-maleimide and ethyl cis-b-cyanoacrylate,
followed by the oxidation of the cycloadduct with
DDQ gave compound 2. 3-(Indol-3-yl)-maleimide was
previously synthesized by Bergman et al.31 and used as
a dienophile for Diels–Alder reactions.32,21 The struc-
ture of compound 2 was assigned from NMR experi-
ments on indole intermediate 1A before oxidation with
DDQ. Actually, the Diels–Alder reaction led to two
intermediate isomers indole 1A and indoline 1B that
could be separated by chromatography. In Diels–Alder
reactions with indolyl maleimide, we had previously ob-
served the formation of indole and indoline isomers
which could be identified from 1H NMR data: an indole
NH is shifted at about 11 ppm whereas an indoline NH
is shifted at about 7.5 ppm.23 All protons and carbons of
compound 1A were assigned from 1H–1H COSY,
1H–13C HSQC, and HMBC correlations (Fig. 2). The
C5 carbon (24.2 ppm) was much more shielded than
the C4 carbon (39.7 ppm). Therefore, the cyano substi-
2
be assigned from its unique J coupling with the indolic
NH at 9.43 ppm.
To obtain a five-membered D ring containing two car-
bonyl functions, a Diels–Alder reaction was first per-
formed between indolyl maleimide and cyclopent-4-en-
1,3-dione, but the cycloadduct was isolated as a mixture
containing several isomers. In the literature, a large
number of Diels–Alder cycloadditions are described
using cyclopent-4-en-1,3-dione leading to cycloadducts
in their enolic form.33–37 Oxidation of the mixture of iso-
mers either with DDQ or in TFA also led to a mixture of
isomers, probably containing carbonyl and enol forms.
To avoid the presence of enol isomers, the Diels–Alder
reaction was carried out with 2,2-dimethyl-cyclopent-4-

E. Conchon et al. / Bioorg. Med. Chem. 16 (2008) 4419–4430
4421
H
N
H
N
H
N
H
N
O
O
O
O
O
O
O
O
O
O
O
NC
DDQ
COOEt
OEt
OEt
+
OEt
CN
CN
CN
N
H
N
H
N
H
N
H
2
1A
1B
D
NaH
TBDMSCl
TBDMS
N
H
N
O
O
O
O
O
H2, Raney
nickel
O
OEt
NH
CN
N
H
N
H
4
3
Scheme 1. Synthesis of compound 4.
dione prepared via a Suzuki coupling between N-Boc in-
dole boronic acid and 4-bromo-2,2-dimethyl-cyclopent-
4-en-1,3-dione E38 affording compound 7 (Scheme 2).
A Diels–Alder reaction between 7 and maleimide gave
cycloadduct 8 as the indole isomer, which was further
oxidized into compound 5 in the presence of TFA.
11.33
H
N
O
O
177.1
7.84
177.1
H
H
40.4
H 4.22
7.12
H
4.41
125.5
40.7
39.7
169.6
120.2
CO2Et
105.2
119.4
122.5
Compound 11, analogue of compound 5 with a lactam E
heterocycle, was synthesized from 3-(1H-indol-3-yl)-1H-
2,5-dihydro-pyrrol-2-one F27 and 2,2-dimethylcyclopen-
tanedione as outlined in Scheme 2. Cycloadduct 9 was ob-
tained as the indole isomer. Oxidation of 9 into 11 was
difficult to achieve. Using DDQ, the lactam function of
the upper heterocycle was oxidized into imide. Intermedi-
ate 10 was obtained in the presence of TFA in the condi-
tions indicated in the experimental section after heating at
80 ꢁC for 48 h in 91% yield. When the mixture was heated
at 80 ꢁC for 3 days instead of 48 h, only the imide was iso-
lated. The positions of the double bonds in compound 10
were assigned from the 1H NMR spectrum showing a cou-
pling between the two protons of the lactam heterocycle
CH2 and a vicinal proton. After isolation, a solution of
compound 10 in THF was re-oxidized for 24 h at 80 ꢁC
to give the required compound 11 in 89% yield.
H 3.54
24.2
127.3
H
4.62
H
7.21
136.1
N
H
111.5
C
117.7
11.47
N
H
7.43
Figure 2. Chemical shifts of carbons (italics) and protons (bold) in 1A
assigned from 1H–1H COSY, 1H–13C HSQC, and HMBC NMR
correlations.
TBDMS
N
O
O
9.08
H
H
O
121.0
144.6
120.5
141.7
H
7.47
To enhance the solubility and/or modify the affinity for
the ATP-binding site of Chk1, and since in stauro-
sporine15 (Fig. 1), a potent indolocarbazole Chk1 inhib-
itor, a sugar moiety is attached to the indole nitrogens, a
glucosyl moiety was coupled to the indolyl maleimide
before reaction with benzoquinone (Scheme 3).
OEt
CN
H
N
H
7.69
9.43
7.62
Figure 3. Assignment of the position of the TBDMS group in
compound 3. Chemical shifts of protons in bold.
A Diels–Alder reaction between N-BOM indolylmalei-
mide bearing a glucosyl moiety (compound G)39 and
benzoquinone gave compound 12 in which the sugar
part was attached to the lower oxygen of the D cycle
(Scheme 3). The mechanism involves probably a transfer
of hydride from the sugar to the quinone oxygen, fol-
lowed by the transfer of a proton to the phenolate
en-1,3-dione, prepared from commercially available 2-
methyl-cyclopentan-1,3-dione as described by Agosta
and Smith,37 leading directly to oxidized compound 5
(Scheme 2). Compound 5 could also be obtained from
2,2-dimethyl-4-(2-N-Boc
indolyl)-cyclopent-4-en-1,3-

4422
E. Conchon et al. / Bioorg. Med. Chem. 16 (2008) 4419–4430
H
N
O
H
N
O
O
O
O
O
O
toluene
N
H
N
H
O
5
O
H
N
H
N
O
O
O
O
O
O
E
Br
B(OH)2
O
N
Boc
toluene
N
Pd(PPh3)4, benzene
EtOH, Na2CO3
Boc
N
H
O
6
7
8
O
TFA
dioxane
H
N
O
O
O
N
H
5
O
H
N
O
H
N
H
N
O
O
O
O
TFA
O
O
dioxane
toluene
N
H
N
H
N
H
F
O
9
O
10
TFA
dioxane
H
N
O
O
N
H
O
11
Scheme 2. Syntheses of compounds 5 and 11.
ion.40 Deprotection of the imide nitrogen and the hydro-
xy groups of the carbohydrate carried out by hydrogen-
olysis with Pd(OH)2, as the catalyst, then aminolysis
using NH4OH in THF/MeOH led to compound 13. In
mediate 12. As expected, compound 13 bearing a sugar
moiety was much more soluble than quinone B and
hydroquinone C (Fig. 1).
1
the H NMR spectrum of compound 13, the anomeric
2.2. Chk1 inhibitory activities
proton, a doublet, generally shifted about 7.5 ppm,
was absent. Only two exchangeable protons were ob-
served between 11 and 12 ppm (11.28 and 11.93 ppm).
Thirteen quaternary carbons were observed in the 13C
NMR spectrum. The structure of spiro compound 13
The inhibitory activities toward Chk1 of compounds 4,
5, 11, and 13 were evaluated and compared with those
of granulatimide, isogranulatimide, bis-imide A, com-
pound H (a regioisomer of 4 in which the D and E rings
are inverted), quinone B, and hydroquinone C (IC50 val-
ues in lM, Table 1). The percentages of Chk1 inhibition
at a compound concentration of 10 lM were determined
1
was confirmed by H–1H COSY, HMBC, and HSQC
NMR experiments. Moreover, only one exchangeable
proton was observed in the 1H NMR spectrum of inter-

E. Conchon et al. / Bioorg. Med. Chem. 16 (2008) 4419–4430
4423
O
BOM
N
BOM
N
H
N
O
O
O
O
O
O
E
OH
OH
O
1) H2, Pd(OH)2
C
A
B
D
toluene
N
2) NH4OH,
THF/MeOH
N
N
O
O
O
OBn
O
O
OBn
OH
BnO
BnO
HO
OBn
OBn
OBn
OBn
HO
OH
13
12
G
Scheme 3. Synthesis of compound 13.
Table 1. Percentages of Chk1 inhibition at a drug concentration of
10 lM
Protein Data Bank. Docking was performed with bis-
imide A and compounds 4, 5, and H (Fig. 4). With bis-
imide A, seven hydrogen bonds were observed. In addi-
tion to the two fundamental hydrogen bonds between
the carbonyl on the left and the imide NH of the E hetero-
cycle with Cys87 and Glu85, respectively, hydrogen bonds
were also observed between the indole NH and the car-
boxylate of the side chain of Glu91, and between the upper
carbonyl of the D heterocycle and the amine of the side
chain of Lys38. Moreover, three hydrogen bonds were
formed via a water molecule: between the carbonyl on
the right of the E heterocycle and the amine of Asp148, be-
tween the imide NH of the D heterocycle and the carbox-
ylate of the side chain of Asp148, and between the lower
carbonyl of the D heterocycle and the amine of the side
chain of Lys132 and the carboxylate of the side chain of
Glu91. This important hydrogen bond net is in agreement
with the strong inhibitory activity of bis-imide A toward
Chk1 (IC50 value: 20 nM). Several water molecules in this
area have been reported to make or not contacts with var-
ious families of Chk1 inhibitors.41–47 The docking of com-
pound 4 resulted in an orientation in the ATP-binding site
of Chk1 that was similar to that of bis-imide A. Compared
with bis-imide A, the same hydrogen bond net was found
except for the carbonyl of the D heterocycle oriented to-
ward the indole, which is missing in compound 4. Com-
pared with compound 4, compound H, in which
heterocycles D and E are inverted, made fewer hydrogen
bonds with the enzyme, in particular, on the upper right
side. The variations in the stabilization of the compounds
in the ATP-binding site of Chk1 via hydrogen bonds ap-
pear to be partially responsible for the different Chk1
inhibitory potencies of bis-imide A, compound 4 and
compound H.
Compound
% of Chk1 IC50 Chk1 L1210 HCT HT29
inhibition (lM)
at 10 lM
116
Granulatimide 93.9
Isogranulatimide 89.7
0.081
0.44
2.8
10
6.1
13
5.7
13.7
9.7
A
4
94.4
83
0.020
0.024
0.051
32.7 nd
>50
nd
8.54 0.58
H
5
85.4
63
nd
32.7 38.5
nd
14.5
nd
1.92
33
11
13
B
20
73
2.2
21.2 46.4 57.8
1.7
2.5
86
96
0.269
0.311
6.8
1.1
5.3 17.4
3.1 5.6
C
IC50 values (lM) toward Chk1. In vitro antiproliferative activities
against three tumor cell lines: murine leukemia L1210, and human
HT29 and HCT116 colon carcinoma (IC50 lM).
and the IC50 values in micromolar were measured for the
most efficient inhibitors. Compound 4 was a stronger
Chk1 inhibitor than granulatimide, isogranulatimide,
and its regioisomer H. The IC50 value of compound 4 to-
ward Chk1 (24 nM) was in the same range as that of bis-
imide A, suggesting that the lower carbonyl of the D ring
is not essential for Chk1 inhibition. Imide 5 and lactam 11
with a 2,2-dimethylcyclopenten-4-dione as the D ring
were weak Chk1 inhibitors, probably because of the two
methyl groups that could induce steric hindrance in the
ATP-binding site of Chk1.
The presence of the glycosyl moiety attached to both in-
dole nitrogen and phenolic oxygen, though inducing a
better solubility, is detrimental to Chk1 inhibition (com-
pare compounds 13 and C).
Compared with bis-imide A, compound 5 was less stabi-
lized by H-bonding inside the ATP-binding pocket,
which could explain its weaker Chk1 inhibitory poten-
cies (Fig. 4). Since the D ring is surrounded by a hydro-
philic pocket, the replacement of the D maleimide
heterocycle by a more hydrophobic cycle was unfavor-
able for the stabilization of the compound. It can be ob-
2.3. Molecular modeling
To try to rationalize the above-described results, molecu-
lar modeling was carried out using as model the complex
structure of Chk1/staurosporine15 downloaded from the

4424
E. Conchon et al. / Bioorg. Med. Chem. 16 (2008) 4419–4430
Figure 4. Docking of bis-imide A and compound 4, 5, and H displayed into the ATP-binding site of Chk1.
served that in the gem-dimethylated compound 5, in
contrast with bis-imide A, the upper carbonyl of the D
ring did not participate in any hydrogen bonding. The
superimposition of bis-imide A and compound 5 in the
ATP-binding site of Chk1 (Fig. 5) shows a slight moving
of Lys38 and Asp148 possibly due to the steric hindrance
of the two methyl groups and also due to the presence of
hydrophobic methyl groups in this hydrophilic pocket.
Docking was also performed with compound 13 and the
results were compared with those obtained for the par-
ent compound C29 without the sugar moiety (Fig. 6).
Compared with compound C, an identical hydrogen
bond net was found in the upper region and, of course,
a different hydrogen bond net was observed in the lower
region. Besides two intramolecular hydrogen bonds be-
tween the OH in 20 and the lower oxygen of the phenolic
moiety, and between the OH in 40 and the oxygen of the
alcohol in 60, the sugar part of compound 13 allowed
Figure 5. Superimposition of bis-imide A (in red) and compound 5 (in
blue) in the ATP-binding site of Chk1.
two hydrogen bonds with Leu15 and Glu91. But the pres-

E. Conchon et al. / Bioorg. Med. Chem. 16 (2008) 4419–4430
4425
Figure 6. Docking of compounds C and 13 displayed into the ATP-binding site of Chk1.
ence of the sugar moiety eliminated the hydrogen bond
net observed with the indolic NH and the lower phenolic
OH in compound C. So, it is not possible to conclude
from the molecular modeling results about the weaker
Chk1 inhibitory potency of compound 13 with respect
to compound C.
which have been tested toward 19 other kinases proved
to be a selective Chk1 inhibitor.29 To get an insight into
the kinase selectivity, Src tyrosine kinase inhibitory
activities were evaluated for compounds 11 and 13.
The percentages of Src inhibition at a drug concentra-
tion of 10 lM were 19% for compound 11 and 9% for
compound 13. Both were poor Src inhibitors, but the
percentages of Chk1 and Src inhibition for compound
11 were in the same range, suggesting that compound
11 is not selective. Its capacity to inhibit other kinases
than Chk1 could be responsible for the cytotoxicity of
this compound.
2.4. In vitro antiproliferative activities
The cytotoxicities of the newly synthesized compounds
were evaluated toward three tumor cell lines: murine
leukemia L1210, and human colon carcinoma HT29
and HCT116 and compared with those of granulatimide
and isogranulatimide, bis-imide A, and compounds H,
B, and C.
3. Conclusion
No parallelism was observed between the cytotoxicities
and the Chk1 inhibitory activities which is not surprising
because a Chk1 inhibitor is not expected to be cytotoxic
by itself. However, the cytotoxicities of the new com-
pounds were examined to get an insight into possible
other targets than Chk1. The absence of cytotoxicity
could also be due to a poor cell penetration. Compound
4, the strongest Chk1 inhibitor of the new compounds de-
scribed in this study, was not cytotoxic toward L1210 cells
but strongly cytotoxic toward HT29 cells. Lactam 11, that
could not be considered as a Chk1 inhibitor, exhibited a
similar significant cytotoxicity toward the three cell lines
tested, suggesting other targets than Chk1 for this com-
pound. Compound 13, that inhibited Chk1 significantly,
was not really cytotoxic. Compounds exhibiting signifi-
cant cytotoxicities could inhibit other kinases than
Chk1 which could account for their cytotoxic effects.
In conclusion, this work reports the synthesis of new
maleimide or maleamide carbazoles possessing various
D rings with one or two carbonyl groups. Two methods
have been developed for the synthesis of compound 5,
either by a Diels–Alder cycloaddition on indolylmalei-
mide or via a Suzuki coupling followed by a Diels–Alder
reaction with maleimide to introduce the E ring. By
analogy with staurosporine, carbazole 13, in which the
sugar moiety is attached to the indole nitrogen and to
an oxygen born by the D ring, was also synthesized.
Among the newly synthesized compounds, compound
4 with a lactam D ring was the strongest Chk1 inhibitor
with an IC50 value of 24 nM. In spite of its weaker inhib-
itory potency toward Chk1 compared to the parent
compound C, compound 13 represents an interesting
structure providing a new avenue to the design of solu-
ble Chk1 inhibitors. Actually, as it has been shown in
our previous works,29 it is easy to introduce various
groups which could highly enhance the Chk1 inhibitory
potency, for example, by forming additional hydrogen
bonds with Cys87 or hydrophobic interactions with
Gly90.
2.5. Src kinase inhibition
Staurosporine is a non-selective kinase inhibitor, UCN-
01 is more selective, in contrast, compound C (Fig. 1),

4426
E. Conchon et al. / Bioorg. Med. Chem. 16 (2008) 4419–4430
4. Experimental
1H NMR (400 MHz, DMSO-d6): 1.40 (3H, t,
J = 7.0 Hz ), 4.50 (2H, d, J = 7.0 Hz), 7.40–7.47 (1H,
m), 7.66–7.71 (2H, m), 8.84 (1H, d, J = 8.0 Hz), 11.80
(1H, br s, NH), 13.09 (1H, br s, NH). 13C NMR
(100 MHz, DMSO-d6): 13.8 (CH3), 62.6 (CH2), 94.0,
114.1, 119.4, 119.5, 120.4, 129.0, 130.0, 142.8, 144.5 (C
quat), 112.6, 121.8, 125.4, 129.9 (C tert arom), 164.1,
167.3, 168.3 (C@O).
4.1. Chemistry
IR spectra were recorded on a Perkin-Elmer 881 spec-
trometer (m in cmꢀ1). NMR spectra were performed on
a Bruker AVANCE 400 and AVANCE 500 (chemical
shifts d in ppm) the following abbreviations are used:
singlet (s), broad singlet (br s), doublet (d), doubled dou-
blet (dd), triplet (t), doubled triplet (dt), multiplet (m),
pseudo quadruplet (pq), quintuplet (quint), tertiary car-
bons (C tert), and quaternary carbons (C quat). The sig-
nals were assigned from 1H–1H COSY, HSQC, and
HMBC NMR correlations. Low resolution mass spectra
(ESI+) and HRMS were determined on a MS Hewlett–
Packard engine. Chromatographic purifications were
performed by flash silicagel Geduran SI 60 (Merck)
0.040–0.063 mm column chromatography.
4.1.2.
5-Cyano-1,3-dihydro-4-ethyloxycarbonyl-6-tert-
butyldimethylsilyl-2H-pyrrolo[3,4-c]carbazole-1,3-dione
(3). To a suspension of 2 (70 mg, 0.21 mmol) in THF
(4 mL) was added NaH (60% dispersion in oil, 52 mg,
0.31 mmol) at 0 ꢁC. After stirring for 10 min at 0 ꢁC,
TBDMSCl (0.52 mmol, 79 mg) was added. The mixture
was stirred at room temperature for 30 min then water
was added. After extraction with EtOAc, the organic
phase was dried over MgSO4 and the solvent was re-
moved. The residue was purified by flash chromatogra-
phy (eluent cyclohexane/EtOAc 7:3) to give 3 (90 mg,
0.20 mmol, 96% yield) as a yellow solid.
4.1.1. 1,3,3a,4,5,10c-Hexahydro-5-cyano-4-ethyloxycarbon-
yl-2H,6H-pyrrolo[3,4-c]carbazole-1,3-dione (1A), 1,3,3a,4,
5,5a-hexahydro-5-cyano-4-ethyloxycarbonyl-2H,6H-pyrrol-
o[3,4-c]carbazole-1,3-dione (1B) and 1,3-dihydro-5-cyano-4-
ethyloxycarbonyl-2H,6H-pyrrolo[3,4-c]carbazole-1,3-dione
(2). To a suspension of 3-(indol-3-yl)-maleimide D
(500 mg, 2.35 mmol) in toluene (30 mL), ethyl cis-b-cya-
noacrylate (24.0 mmol, 3.0 mL) was added. The mixture
was refluxed for 5 days. After filtration, the solid residue
was washed with CH2Cl2 to give a mixture of the Diels–
Alder adducts 1A and 1B (200 mg, 0.61 mmol, 26%
yield). A solution of the Diels–Alder adduct (200 mg)
in dioxane (14 mL) was refluxed for 20 h in the presence
of DDQ (295 mg, 1.29 mmol). The solvent was removed.
Water was added to the residue. After filtration, the so-
lid residue was washed with water and EtOAc to give 2
(177 mg, 0.53 mmol, 87% yield).
Mp 278 ꢁC. IR (KBr) tC@O 1706, 1737, 1761 cmꢀ1, tCN
2231 cmꢀ1, tNH 3273 cmꢀ1. HRMS (ESI+) [M+H]+ calcd
for C24H26N3O4Si 448.1693; found: 448.1674. H NMR
1
(400 MHz, CDCl3): 0.60 (6H, s), 1.04 (9H, s), 1.50 (3H,
dt, J1 = 7.0 Hz, J2 = 1.0 Hz), 4.63 (2H, dq, J1 = 7.0 Hz,
J2 = 1.0 Hz), 7.47 (1H, dt, J1 = 8.0 Hz, J2 = 1.0 Hz),
7.62 (1H, d, J = 8.0 Hz), 7.69 (1H, t, J1 = 8.0 Hz,
J2 = 1.0 Hz), 9.08 (1H, d, J = 8.0 Hz), 9.43 (1H, s, NH).
13C NMR (100 MHz, CDCl3): ꢀ4.0, 14.1, 26.5 (CH3),
19.1 (C quat), 63.3 (CH2), 111.6, 122.7, 126.8, 130.3 (C tert
arom), 95.8, 114.1, 120.5, 120.9, 122.5, 129.4, 131.3, 141.6,
144.5 (C quat), 164.0, 171.0, 172.3 (C@O).
4.1.3. 2H,5H,7H-1,3,4,6-Tetrahydrodipyrrolo[3,4-a:3,4-
c]carbazole-1,3,4-trione (4). A solution of 3 (69 mg,
0.15 mmol) in DMF (6 mL) was hydrogenated for 7 days
(50 psi) in the presence of Raney Ni (150 mg). After filtra-
tion over Celite, the solvent was removed. EtOH was
added to the residue. After filtration, compound 4
(10 mg, 0.034 mmol, 23% yield) was isolated as a pale yel-
low solid.
4.1.1.1. NMR data for compound 1A. 1H NMR
(500 MHz, DMSO-d6): 1.34 (3H, t, J = 7.0 Hz), 3.54 (1H,
t, J = 5.0 Hz), 4.22 (1H, dd, J1 = 8.5 Hz, J2 = 5.0 Hz),
4.29–4.34 (2H, m), 4.41 (1H, d, J = 8.5 Hz), 4.62 (1H,
J = 5.0 Hz), 7.12 (1H, t, J = 8.0 Hz), 7.21 (1H, t,
J = 7.5 Hz), 7.43 (1H, d, J = 8.0 Hz), 7.84 (1H, d,
J = 8.0 Hz), 11.33 (1H, br s, NH), 11.47 (1H, s, NH). 13C
NMR (100 MHz, DMSO-d6): 14.0 (CH3), 60.9 (CH2),
24.2, 39.7, 40.4, 40.7 (CH), 111.5, 119.4, 120.2, 122.5 (CH
arom), 117.7 (C„N), 105.2, 125.5, 127.3, 136.1 (C quat
arom), 169.6, 177.1 (2C) (C@O).
Mp >280 ꢁC. IR (KBr) tC@O 1674, 1710, 1761 cmꢀ1, tNH
2997–3643 cmꢀ1. HRMS (ESI+) [M+Na]+ calcd for
C16H9N3O3Na 314.0542; found: 314.0529. 1H NMR
(400 MHz, DMSO-d6): 4.78 (2H, s), 7.40 (1H, dt,
J1 = 7.0 Hz, J2 = 1.0 Hz), 7.64 (1H, dt, J1 = 8.0 Hz,
J2 = 1.0 Hz), 7.73 (1H, d, J = 8.0 Hz), 8.78 (1H, s, NH),
9.03 (1H, d, J = 8.0 Hz), 11.17 (1H, br s, NH), 12.66
(1H, s, NH). 13C NMR (100 MHz, DMSO-d6): 43.9
(CH2), 111.9, 120.6, 125.3, 128.5 (C tert ), 119.3, 120.2,
120.3, 126.8, 128.3, 133.2, 138.1, 142.0 (C quat), 167.4,
167.7, 170.2 (CO).
4.1.1.2. NMR data for compound 1B. 1H NMR
(400 MHz, DMSO-d6): 1.34 (3H, t, J = 7.0 Hz), 3.09–
3.14 (2H, m), 3.88 (1H, d, J = 6.0 Hz), 4.23–4.37 (2H,
m), 4.54–4.60 (1H, m), 6.84 (1H, t, J = 7.5 Hz), 6.88
(1H, d, J = 8.5 Hz), 7.35 (1H, t, J = 7.5 Hz), 7.56 (1H, d,
J = 3.5 Hz, NH), 8.50 (1H, d, J = 8.0 Hz), 11.36 (1H, s,
NH). 13C NMR (100 MHz, DMSO-d6): 14.0 (CH3),
61.5 (CH2), 35.0, 35.7, 43.8, 61.5 (CH), 111.1, 118.5,
127.4, 133.5 (CH arom.), 114.3, 118.8, 121.6, 147.3,
156.8 (C quat + C„N), 167.9, 172.0, 176.0 (C@O).
4.1.4.
2H,7H-1,3-Dihydro-5,5-dimethylcyclopenta[4,5-
a]pyrrolo[3,4-c]carbazole-1,3,4,6-tetraone (5). A mixture
of 3-(indol-3-yl)-maleimide (100 mg, 0.47 mmol) and
2,2-dimethylcyclopentenedione (350 mg, 2.83 mmol) in
toluene (12 mL) was stirred at 140 ꢁC in a sealed tube
for 7 days. After cooling, the mixture was filtered off
and the residue was washed with dichloromethane and
Data for compound 2: mp >300 ꢁC. IR (KBr) tC@O 1662,
1702 cmꢀ1, tNH 3356 cmꢀ1. HRMS (ESI+) [M+Na]+
calcd for C18H11N3O4Na 356.0647; found: 356.0661.

E. Conchon et al. / Bioorg. Med. Chem. 16 (2008) 4419–4430
4427
EtOAc to give 5 (30 mg, 0.09 mmol, 20% yield) as an or-
ange solid.
dioxane (3 mL) was refluxed for 48 h. After evaporation,
water was added to the residue. The mixture was filtered
off to give compound 5 (24 mg, 0.072 mmol, 90% yield).
Mp >280 ꢁC. IR (KBr) tC@O 1700, 1726, 1742, 1767 cmꢀ1
,
tNH 3257, 3340 cmꢀ1. HRMS (ESI)+ [M+H]+ calcd for
C19H13N2O4 333.0875; found: 333.0890. 1H NMR
(400 MHz, DMSO-d6): 1.35 (6H, s), 7.52 (1H, t,
J = 7.5 Hz), 7.75 (1H, t, J = 7.5 Hz), 7.91 (1H, d,
J = 8.0 Hz), 9.06 (1H, d, J = 8.0 Hz), 11.68 (1H, s, NH),
13.07 (1H, s, NH).
4.1.8. 2H,7H-5,5-Dimethyl-1,3,3a,3b,6a,11c-hexahydro-
cyclopenta[4,5-a]pyrrolo[3,4-c]carbazole-1,4,6-trione (9).
A mixture of 3-(1H-indol-3-yl)-1H-2,5-dihydro-pyrrol-
2-one F (219 mg, 1.11 mmol) and 2,2-dimethylcyclopen-
tanedione (789 mg, 6.36 mmol) in toluene (4 mL) was
stirred at 140 ꢁC in a sealed tube for 4 days. After cool-
ing, the mixture was filtered off and the residue was
washed with dichloromethane to give 9 (226 mg,
0.70 mmol, 63% yield) as a pale yellow solid.
4.1.5. 2,2-Dimethyl-4-(2-N-Boc-indolyl)-cyclopent-4-en-
1,3-dione (7). A solution of 2-N-Boc-indole-boronic acid
(670 mg, 2.57 mmol) in EtOH (18 mL) was added to a mix-
ture of 2,2-dimethyl-5-bromo-cyclopentanedione
E
Mp >280 ꢁC. IR (KBr) tC@O 1689, 1723, 1763 cmꢀ1, tNH
3346, 3376 cmꢀ1. HRMS (ESI+) [M+H]+ calcd for
C19H19N2O3 323.1396; found: 323.1409. 1H NMR
(400 MHz, DMSO-d6): 0.70 (3H, s), 1.19 (3H, s), 3.19
(1H, t, J = 7.5 Hz), 3.35–3.49 (3H, m), 3.79 (1H, m),
4.48 (1H, d, J = 8.5 Hz), 6.96 (1H, t, J = 7.5 Hz), 7.08
(1H, t, J = 7.5 Hz), 7.35 (1H, d, J = 8.0 Hz), 7.64 (1H, s,
NH), 7.86 (1H, d, J = 8.0 Hz), 11.00 (1H, s, NH). 13C
NMR (100 MHz, DMSO-d6): 20.9, 21.6 (CH3), 32.2,
38.2, 44.8, 46.6 (CH), 43.5 (CH2), 50.6, 105.3, 126.2,
126.3, 137.0 (C quat), 111.2, 118.5, 120.9, 121.6 (C tert
arom), 175.1, 212.6, 215.9 (C@O).
(437 mg, 2.15 mmol) and Pd(PPh3)4 (208 mg, 0.078 mmol)
inbenzene(36 mL). A solution ofNa2CO3 (1.32 g)inwater
(49 mL) was added and the mixture was refluxed for 1.5 h.
After cooling, water was added. After extraction with
EtOAc, the organic phase was dried over MgSO4, the sol-
vent was removed, and the residue was purified by flash
chromatography (eluent: cyclohexane/EtOAc from 95:5
to 8:2) to give 7 (157 mg, 0.46 mmol, 22% yield) as a yellow
solid.
Mp 77 ꢁC. IR (KBr) tC@O 1705, 1730 cmꢀ1. Mass (ESI+)
[MꢀBoc+H]+ 240, [MꢀBoc+Na]+ 262, [M+Na]+ 362.
HRMS (ESI+) [MꢀBoc+H]+ calcd for C15H14NO2
240.1025; found: 240.1041. 1H NMR (400 MHz,
DMSO-d6): 1.30 (6H, s), 1.67 (9H, s), 7.02 (1H, s), 7.09
(1H, s), 7.27 (1H, t, J = 7.5 Hz), 7.41 (1H, t, J = 8.0 Hz),
7.61 (1H, d, J = 8.0 Hz), 8.03 (1H, d, J = 8.0 Hz). 13C
NMR (100 MHz, DMSO-d6): 19.9, 28.1 (CH3), 47.6,
85.2, 128.7, 129.1, 137.6, 150.0 (C quat), 114.7, 115.2,
121.8, 123.3, 126.4, 138.0 (C tert), 151.7, 204.7, 206.1
(C@O).
4.1.9. 2H,7H-5,5-Dimethyl-1,3,3a,11c-tetrahydrocyclo-
penta[4,5-a]pyrrolo[3,4-c]carbazole-1,4,6-trione (10). A
mixture of 9 (30 mg, 0.093 mmol), TFA (72 lL) and diox-
ane (3 mL) was stirred at 80 ꢁC for 48 h. After evapora-
tion, water was added to the residue, and the mixture
was filtered off to give 10 (27 mg, 0.084 mmol, 91% yield)
as an orange solid.
Mp >280 ꢁC. IR (KBr) tC@C 1619, 1635 cmꢀ1, tC@O 1690,
1723, 1763 cmꢀ1, tNH 3370–3565 cmꢀ1. HRMS (ESI+)
[M+H]+ calcd for C19H17N2O3 321.1239; found:
4.1.6. 2H,7H-5,5-Dimethyl-1,3,3a,3b,6a,11c-hexahydro-
cyclopenta[4,5-a]pyrrolo[3,4-c]carbazole-1,3,4,6-tetraone
(8). A solution of compound 7 (157 mg, 0.46 mmol) and
maleimide (223 mg, 2.3 mmol) in toluene (3 mL) was
stirred at 140 ꢁC for 48 h in a sealed tube. The solvent
was removed and the residue was purified by flash chro-
matography (eluent: EtOAc/cyclohexane from 3:7 to
7:3) to give 8 (75 mg, 0.22 mmol, 48% yield) as a yellow
solid.
1
321.1241. H NMR (400 MHz, DMSO-d6): 1.16 (3H, s),
1.17 (3H, s), 3.30 (1H, pt, J = 8.0 Hz), 3.75 (1H, pt,
J = 9.0 Hz), 4.06 (1H, m), 4.21 (1H, d, J = 11.0 Hz), 7.09
(1 H, t, J = 7.0 Hz), 7.25 (1H, t, J = 7.5 Hz), 7.56 (1H, d,
J = 8.0 Hz), 8.07 (1H, d, J = 8.0 Hz), 8.11 (1H, s, NH),
11.64 (1H, s, NH). 13C NMR (100 MHz, DMSO-d6):
19.9, 20.0 (CH3), 31.8, 41.2 (CH), 45.4 (CH2), 47.2, 114.1,
123.8, 125.7, 139.8, 140.7, 147.1 (C quat), 112.7, 120.0,
122.2, 124.4 (C tert arom), 175.4, 203.6, 203.8 (C@O).
Mp >280 ꢁC. IR (KBr) tC@C 1616, 1639 cmꢀ1, tC@O
1704, 1720, 1768 cmꢀ1, tNH 3400–3600 cmꢀ1. HRMS
(ESI+) [M+H]+ calcd for C19H19N2O3 323.1396; found:
323.1410. 1H NMR (400 MHz, DMSO-d6): 1.09 (6H, s),
3.46 (2H, d, J = 8.0 Hz), 3.86 (2H, d, J = 8.0 Hz), 7.03
(1H, d, J = 7.5 Hz), 7.13 (1H, dt, J1 = 7.5 Hz,
J2 = 1.0 Hz), 7.30 (1H, dt, J1 = 7.5 Hz, J2 = 1.0 Hz),
7.79 (1H, d, J = 7.0 Hz), 11.21 (1H, s, NH), 11.69 (1H,
s, NH). 13C NMR (100 MHz, DMSO-d6): 22.2 (2
CH3), 46.2 (2 CH), 46.3 (2 CH), 111.3, 121.9, 125.8,
128.4 (C tert arom), 53.7, 94.0, 128.7, 146.2, 152.0 (C
quat), 174.2, 176.3, 195.6, 214.0 (C@O).
4.1.10. 2H,7H-5,5-Dimethyl-1,3-dihydrocyclopenta[4,5-
a]pyrrolo[3,4-c]carbazole-1,4,6-trione (11). A mixture of
10 (37 mg, 0.016 mmol) and TFA (50 lL) in dioxane
(4 mL) was stirred at 80 ꢁC for 24 h. After evaporation,
water was added to the residue. The mixture was filtered
off to give 11 (33 mg, 0.10 mmol, 89% yield) as a yellow
solid.
Mp >280 ꢁC. IR (KBr) tC@C 1619, 1634 cmꢀ1, tC@O
1694, 1722 cmꢀ1, tNH 3400–3565 cmꢀ1. HRMS (ESI)+
[M+H]+ calcd for C19H15N2O3 319.1083; found:
1
319.1084. H NMR (400 MHz, DMSO-d6): 1.38 (6H,
4.1.7.
2H,7H-1,3-Dihydro-5,5-dimethylcyclopenta[4,5-
s), 5.10 (2H, s), 7.43 (1H, dt, J1 = 7.5 Hz, J2 = 0.5 Hz),
7.70 (1H, dt, J1 = 8.0 Hz, J2 = 1.0 Hz), 7.86 (1H, d,
J = 8.0 Hz), 9.03 (1H, d, J = 8.0 Hz), 9.37 (1H, s, NH),
a]pyrrolo[3,4-c]carbazole-1,3,4,6-tetraone (5). A solution
of compound 8 (28 mg, 0.08 mmol) in TFA (64 lL) and

4428
E. Conchon et al. / Bioorg. Med. Chem. 16 (2008) 4419–4430
12.83 (1H, s, NH). 13C NMR (100 MHz, DMSO-d6):
20.3 (CH3), 45.7 (CH2), 49.9, 119.6, 125.2, 126.3,
130.0, 130.6, 130.7, 133.2, 143.5 (C quat), 112.9, 121.1,
125.5, 129.4 (C tert arom), 168.2, 203.2, 203.6 (C@O).
4.2. Molecular modeling
For docking experiments, all molecular mechanics cal-
culations were performed by the Macromodel48 molecu-
lar modeling software. We used as model the complex
structure of Chk1/staurosporine15 downloaded from
the Protein Data Bank (1NVR file).
4.1.11. 1,3-Dihydro-2-benzyloxymethyl-7,8-(1,1-(2,3,4,6-
tetra-O-benzyl-D-glucopyranosyl))-4-hydroxybenzo[1,2-
a]pyrrolo[3,4-c]carbazole-1,3-dione (12). A solution of
compound G (450 mg, 0.53 mmol) in toluene (30 mL)
was refluxed in the presence of p-benzoquinone
(1.73 g, 16.0 mmol). The solvent was removed and the
residue was purified by flash chromatography (eluent:
cyclohexane/EtOAc/toluene 10:4:2) to give 12 (170 mg,
0.177 mmol, 33% yield) as a red solid.
Energy minimization was done with AMBER force
field49,50 using the Truncated Newton Conjugate Gradi-
ent method (TNCG).
4.3. Chk1 inhibitory assays
Human Chk1 full-length enzyme with an N-terminal
GST sequence was either purchased from Upstate Bio-
chemicals (No. 14-346) or purified from extracts of Sf9
cells infected with a baculovirus encoding GST-Chk1.
Assays for compound testing were based upon the meth-
od described by Davies et al.51
Mp 55 ꢁC. IR (KBr) tC@O 1685, 1758 cmꢀ1, tNH
3444 cmꢀ1. Mass (ESI+) [M+Na]+ 981. 1H NMR
(400 MHz, CDCl3): 2.96 (1H, d, J = 11.5 Hz), 3.92 (1H,
dd, J1 = 11.0 Hz, J2 = 2.0 Hz), 4.02 (1H, dd,
J1 = 11.0 Hz, J2 = 2.0 Hz), 4.06 (1H, d, J = 11.0 Hz), 4.11
(1H, d, J = 9.0 Hz), 4.19–4.29 (2H, m), 4.53 (1H, d,
J = 10.0 Hz), 4.57 (1H, d, J = 12.0 Hz), 4.67 (1H, d,
J = 12.0 Hz), 4.73–4.77 (4H, m), 4.85 (1H, d,
J = 11.0 Hz), 4.97 (1H, d, J = 11.0 Hz), 5.25 (2H, d,
J = 1.5 Hz), 5.91 (2H, d, J = 7.5 Hz), 6.71 (2H, t,
J = 8.0 Hz), 6.86 (1H, t, J = 7.5 Hz), 7.07 (1H, d,
J = 8.5 Hz), 7.10 (1H, d, J = 8.5 Hz), 7.16–7.45 (22 H,
m), 7.85 (1H, d, J = 8.0 Hz), 8.76 (1H, d, J = 8.0 Hz),
10.80 (1H, s, exchangeable proton). 13C NMR
(100 MHz, CDCl3): 66.9, 67.9, 71.7, 73.5, 74.5, 75.5, 75.8
(CH2), 74.1, 76.9, 82.0, 82.8, 112.8, 113.1, 114.6, 122.9,
125.1, 126.0, 126.9, 127.3, 127.4, 127.6, 127.7, 127.8,
128.0, 128.4, 128.5, 128.6 (C tert), 107.2, 109.5, 111.4,
115.3, 118.8, 123.0, 136.1, 137.3, 137.9, 138.0, 138.1,
138.4, 142.2, 148.2 (C quat), 168.3, 173.4 (C@O).
4.4. Growth inhibition assays
Tumor cells were provided by American Type Culture
Collection (Frederik, MD, USA). They were cultivated
in RPMI 1640 medium (Life Science technologies, Cer-
gy-Pontoise, France) supplemented with 10% fetal calf
serum, 2 mM L-glutamine, 100 U/mL penicillin,
100 lg/mL streptomycin, and 10 mM Hepes buffer (pH
7.4). Cytotoxicity was measured by the microculture tet-
razolium assay as described.52 Cells were continuously
exposed to graded concentrations of the compounds
for four doubling times, then 15 lL of 5 mg/mL 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
were added to each well and the plates were incubated
for 4 h at 37 ꢁC. The medium was then aspirated and
the formazan solubilized by 100 lL of DMSO. Results
are expressed as IC50, concentration which reduced by
50% the optical density of treated cells with respect to
untreated controls.
4.1.12. 2H-1,3-Dihydro-7,8-(1,1-D-glucopyranosyl)-4-
hydroxybenzo[1,2-a]pyrrolo[3,4-c]carbazole-1,3-dione (13).
A solution of 12 (43 mg, 0.045 mmol) in a mixture THF/
MeOH (2.0/0.5 mL) was hydrogenated (1 bar) in the pres-
ence of Pd(OH)2 (22 mg) at room temperature for 12 h.
The mixture was filtered over Celite and the solvents were
removed. A solution of the residue in THF/NH4OH (4.5/
9.0 mL) was stirred at room temperature for 4.5 h then the
solvents were removed. The residue was purified by flash
chromatography (eluent EtOAc) to give 13 (18 mg,
0.038 mmol, 84% yield) as a red solid.
4.5. Src inhibition assays
Inhibitors were diluted with a Tecan Evo150 robot.
The kinase assay was performed with 4 lL of inhibitor
(10% dimethylsulfoxide (DMSO)), 10 lL of kinase
assay buffer 4· concentrated (80 mM MgCl2,
200 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (Hepes), 0.4 mM ethylenediamine-tetraacetic acid
(EDTA), 2 mM DL-dithiothreitol (DTT)), 10 lL sub-
strate peptide (KVEKIGEGYYGVVYK, 370 nM),
and 6 lL Src kinase (stock GTP purified diluted with
1· kinase assay buffer to 200 nM). 10 lL co-substrate
(40 lM ATP with 0.2 lCi P33-c-ATP) was added with
a Precision 2000 (Biotek Robotic). The assay was
incubated for 20 min at 30 ꢁC, then stopped by adding
200 lL of 0.85% orthophosphoric acid, and trans-
ferred to a phosphocellulose filter microplate (What-
man-P81). The plate was washed three times with
200 lL of 0.85% orthophosphoric acid and dried with
200 lL acetone. The remaining activity was measured
on a Topcount with 25 lL scintillation solution (Pack-
ard UltimaGold).
Mp 240–242 ꢁC. IR (KBr) tC@O 1687, 1757 cmꢀ1
,
tNH,OH 3300–3600 cmꢀ1. HRMS (ESI+) [M+H]+ calcd
for C24H19N2O9 479.1091; found: 479.1103. H NMR
1
(400 MHz, DMSO-d6): 3.61–3.78 (3H, m), 3.88–3.91
(1H, m), 4.09–4.16 (2H, m), 4.79 (1H, t, J = 5.5 Hz,
OH), 5.30 (1H, d, J = 5.5 Hz, OH), 5.41 (1H, d,
J = 5.5 Hz, OH), 5.52 (1H, d, J = 5.5 Hz, OH), 7.10
(1H, d, J = 8.0 Hz), 7.27 (1H, d, J = 8.0 Hz), 7.55 (1H,
dt, J1 = 7.0 Hz, J2 = 1.0 Hz), 7.67 (1H, dt, J1 = 7.0 Hz,
J2 = 1.0 Hz), 7.98 (1H, d, J = 8.0 Hz), 8.80 (1H, d,
J = 8.0 Hz), 11.28 (1H, s), 11.93 (1 H, s). 13C NMR
(100 MHz, acetone-d6): 61.1 (CH2), 70.0, 74.1, 74.6,
76.8, 112.8, 113.6, 114.2, 122.4, 124.6, 127.0 (C tert),
106.9, 108.3, 109.0, 111.6, 120.1, 123.2, 128.6, 138.4,
138.5, 141.7, 148.4 (C quat), 169.4, 175.1 (C@O).

E. Conchon et al. / Bioorg. Med. Chem. 16 (2008) 4419–4430
4429
B.; Prudhomme, M. Eur. J. Pharmacol. 2007, 554, 106–
112.
Acknowledgment
´
26. Conchon, E.; Anizon, F.; Golsteyn, R. M.; Leonce, S.;
´
The authors are grateful to Bertrand Legeret, University
Blaise Pascal, Clermont-Ferrand, for recording the mass
spectra.
Pfeiffer, B.; Prudhomme, M. Tetrahedron 2006, 62, 11136–
11144.
27. Conchon, E.; Anizon, F.; Aboab, B.; Golsteyn, R. M.;
´
Leonce, S.; Pfeiffer, B.; Prudhomme, M. Eur. J. Med.
Chem. 2008, 43, 282–292.
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