Synthesis and biological evaluation of analogs of the marine alkaloids granulatimide and isogranulatimide

Article abstract of DOI:10.1016/j.ejmech.2012.06.012

A series of pyrrolic analogs and two series of regioisomeric pyrazolic analogs of the marine alkaloids granulatimide and isogranulatimide were prepared. The synthesis of the two first ones was based on the condensation reaction of diversely 5-substituted 3-bromoindoles with pyrrole or pyrazole followed by addition of the intermediates on maleimide or dibromomaleimide, respectively, the so-obtained acyclic adducts being finally photocyclized to the desired analogs. Compounds of the last series were obtained by reacting different 5-substituted-indole-3-glyoxylates with N-Boc-pyrazole-3-acetamide and subsequent photochemical cyclization of the adducts. All the compounds were evaluated for their in vitro growth inhibitory properties toward eight cancer cell lines. Several compounds were also assayed for their ability to abrogate the G2-cell cycle checkpoint or to inhibit a panel of Ser/Thr kinases. Lastly, computer-assisted phase-contrast microscopy (quantitative videomicroscopy) revealed that the three most potent compounds (4a, 9a, 9e), with IC₅₀ growth inhibitory concentrations ranging between 10 and 20 μM, displayed cytostatic, not cytotoxic, anticancer effects.

Full text of DOI:10.1016/j.ejmech.2012.06.012

European Journal of Medicinal Chemistry 54 (2012) 626e636
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of analogs of the marine alkaloids
granulatimide and isogranulatimide
Sébastien Deslandes , Delphine Lamoral-Theys 1, Céline Frongia c , Stefan Chassaing ,d,
a
,
1
b
,
,
d
a,c
Céline Bruyère , Olivier Lozach , Laurent Meijer , Bernard Ducommun c , Robert Kiss ,
e
f
f
,
g
,
d
,
h
b
a,
*
Evelyne Delfourne
a
Laboratoire de Synthèse et Physicochimie de Molécules d’Intérêt Biologique, Université Paul Sabatier, UMR CNRS 5068, 118 route de Narbonne, 31062 Toulouse Cédex 9, France
Laboratoire de Chimie Analytique, Toxicologie et Chimie Physique Appliquée and Boulevard du Triomphe, 1050 Bruxelles, Belgium
ITAV-UMS3039, Université de Toulouse; F-31106 Toulouse, France
CNRS; ITAV-UMS3039, F-31106 Toulouse, France
Laboratoire de Toxicologie, Faculté de Pharmacie, Université Libre de Bruxelles, Bruxelles, Belgium
CNRS, ‘Protein Phosphorylation & Human Disease’ group, Station Biologique, 29680-Roscoff, France
ManRos Therapeutics, Centre de Perharidy, 29680-Roscoff, France
CHU de Toulouse, F-31059 Toulouse, France
b
c
d
e
f
g
h
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 January 2012
Received in revised form
A series of pyrrolic analogs and two series of regioisomeric pyrazolic analogs of the marine alkaloids
granulatimide and isogranulatimide were prepared. The synthesis of the two first ones was based on the
condensation reaction of diversely 5-substituted 3-bromoindoles with pyrrole or pyrazole followed by
addition of the intermediates on maleimide or dibromomaleimide, respectively, the so-obtained acyclic
adducts being finally photocyclized to the desired analogs. Compounds of the last series were obtained
by reacting different 5-substituted-indole-3-glyoxylates with N-Boc-pyrazole-3-acetamide and subse-
quent photochemical cyclization of the adducts. All the compounds were evaluated for their in vitro
growth inhibitory properties toward eight cancer cell lines. Several compounds were also assayed for
their ability to abrogate the G2-cell cycle checkpoint or to inhibit a panel of Ser/Thr kinases. Lastly,
computer-assisted phase-contrast microscopy (quantitative videomicroscopy) revealed that the three
most potent compounds (4a, 9a, 9e), with IC50 growth inhibitory concentrations ranging between 10 and
4
June 2012
Accepted 7 June 2012
Available online 21 June 2012
Keywords:
Marine alkaloid
Granulatimide
Isogranulatimide
Pyrrolic analogs
Pyrazolic analogs
G2/M checkpoint inhibitors
Protein kinases
20
m
M, displayed cytostatic, not cytotoxic, anticancer effects.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Quantitative videomicroscopy
1. Introduction
In this biological context, granulatimide 1 and isogranulatimide
, natural alkaloids isolated from the ascidian Didemnum gran-
2
During the cell division cycle, G1 and G2 checkpoints are acti-
ulatum, have raised considerable interest as cell cycle G2/M
checkpoint inhibitors (Fig. 1) [4e6]. From a mechanistic point of
view, they have been identified as selective inhibitors of the Chk1
kinase [7], a key enzyme involved in the G2/M checkpoint [8]. Since
this pioneering work, intensive structureeactivity relationship
studies have been carried out to investigate the structural param-
eters required for the biological activity [9,10]. In this way, the
importance of the maleimide moiety, which establishes the two
vated in response to DNA damage and consequently block the cycle
progression to allow time for DNA repair. Interestingly, in more
than 50% of cancer cells, the G1 checkpoint is impaired as a conse-
quence of p53 tumor suppressor mutation. Therefore, it has been
proposed that a combination of DNA-damaging agent with a G2/M
checkpoint inhibitor should force selectively cancer cells into
a premature and lethal mitosis, such a combination being currently
envisioned as an original and particularly promising chemothera-
peutic approach [1e3].
8
5
87
fundamental hydrogen bonds with Glu and Cys in the ATP
binding pocket of the enzyme, has been pointed out.
In continuation of previous studies in which the imidazole ring
of the natural compounds has been replaced by different aromatic
rings, we report here, on the synthesis, in vitro antitumor proper-
ties, kinase inhibitory properties and G2/M checkpoint abrogation
*
Corresponding author. Tel.: þ33 561556293.
E-mail address: delfourn@chimie.ups-tlse.fr (E. Delfourne).
The two first authors contributed equally to the article.
1
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.012

S. Deslandes et al. / European Journal of Medicinal Chemistry 54 (2012) 626e636
627
yield, respectively). These compounds were cyclized under our
conditions involving microwaves into the pyrrolic analogs 3b, 3c
and 3e in moderate yields, 49, 60 and 59%, respectively. Deme-
thylation of compound 3c by boron tribromide in dichloromethane
furnished quantitatively the hydroxy-derivative 3d previously
prepared by Hugon and co-workers from its corresponding
benzylic derivative (R ¼ OBn) [13].
The synthetic route used to obtain the pyrrolic analogs was
adapted to prepare the first series of pyrazolic analogs (Scheme 2).
Bromo derivatives 7aec were reacted with pyrazole, in the same
conditions as before, the only change being the time of reaction
which was longer (4h instead of 2h). N-indolylpyrazoles 10aec
were obtained in modest to correct yields (61, 30 and 55%,
respectively) through the formation of a CeN bond and not a CeC
bond as previously observed with pyrrole and indole [11,13]. The
conjugated base of these compounds generated in situ by action of
LiHMDS was added on N-t-butyldimethylsilyl (TBDMS)-dibromo-
maleimide [14] to give the acyclic intermediates 11aec in low
yields (24, 20 and 27%, respectively) resulting of the concomitant
deprotection of the TBDMS protective group. Photocyclization of
these last derivatives furnished the desired pyrazolic analogs 4aec
in 58, 63 and 78% yields. Ultimately, the hydroxy-analog 4d was
obtained in good yield by demethylation of the methoxy derivative
Fig. 1. Structures of the granulatimide and isogranulatimide natural alkaloids and
analogues.
evaluation, of three families of granulatimide or isogranulatimide
analogs. The first one 3aee consists in a completion of a pyrolic
series previously reported by Hugon and co-workers [11] whereas
in the two last ones, 4aed and 5aec, the imidazole ring of the
natural compounds has been replaced by a pyrazole ring.
4c with BBr
3
.
The synthetic route to the second series of regioisomeric pyr-
azolic analogs 5aec was based on the methodology reported by
Faul et al. to prepare bisindolylmaleimides [15] and later on used by
Piers et al. to publish an improved synthesis of isogranulatimide
(Scheme 3) [16].
2
. Chemistry
The synthesis of the already described compound 3a was real-
ized on the basis of the synthetic scheme previously proposed,
except for the last step for which the same reagents, Pd black and
nitrobenzene were involved, but with microwave irradiation
instead of heating (Scheme 1). Under these conditions, the yield
was improved from 16 to 44% [11].
The 5-methyl or 5-methoxy-indole were coupled with pyrrole
after prior transformation into 3-bromo derivatives 7b and 7c [12]
to give the indolopyrrolic compounds 8b and 8c, in respectively 39
and 58% overall yield for the two steps. Condensation of these two
compounds with maleimide, and in addition with methyl-
N-Boc-pyrazole-3-acetamide 14 was obtained by Boc-protection
of pyrazole-3-acetamide, itself prepared from R.G. Jones’ eight-step
procedure [17]. The ethyl-5-methoxy (or 5-methyl)-3-glyoxylates,
12b and 12c, were readily obtained in 97 and 93% yield, respec-
tively, from the corresponding indoles by using the two-step
reaction scheme previously reported to prepare 12a [18]. Following
N-BOC-protection, the so-obtained compounds 13aec were reac-
ted with compound 14 in the presence of tBuOK. The final photo-
chemical cyclization of the indolylpyrazolylmaleimides 15aec
under the same conditions as for the first series 3aec gave the N-
pyrazolic-BOC protected pentacyclic derivatives 16aec in about 3%
yield for the two steps. In a first approach, no attempt was done to
maleimide for compound 8c, by using catalytic amounts of SnCl
2
in
toluene, led to the Michael adducts 9b, 9c and 9e (28, 59 and 61%
Scheme 1.

628
S. Deslandes et al. / European Journal of Medicinal Chemistry 54 (2012) 626e636
pyrazolic 4aed and pyrazolic 5aec, the second group (including
mainly 4a and 4d) exhibited the highest growth inhibitory activity
with mean IC50 growth inhibitory concentrations of 11 and 16
respectively, e.g. similar to the activity displayed by granulatimide
Table 1). In contrast, isogranulatimide displayed weak in vitro
m
M
(
growth inhibitory activity only (Table 1). The open structures 9aec,
e and 11aec, precursors of pyrrolic 3 and pyrazolic 4 analogs, dis-
played in vitro antitumor activities of around 20
active compound of both series being the methoxy derivative 9c
M; Table 1). It is also interesting to
mM with the most
(
mean IC50 concentration ¼ 13
m
mention that excepted for 11a/4a, in main cases, the closure of the
open structure into the corresponding final product has resulted in
a lowering of the in vitro growth inhibition activity (Table 1).
3.2. The most active compounds are cytostatic, not cytotoxic
Of the most active compounds under study and for which we had
sufficient amounts for further testing are included 4a, 9a and 9e,
which were assayed by means of quantitative videomicroscopy. The
analyses were carried out with the A549 NSCLC and the U373 GBM
cell lines, which both display various levels of resistance to pro-
apoptotic stimuli as mentioned above. While p53 is mutated in
U373 Glioblastoma cells, it is not in A549 NSCLC cells (Table 1).
Fig. 2A shows that 9e induced cytostatic effects on A549 NSCLC cells,
as did 4a and 9a (data not shown). The same features were observed
with 4a (data not shown), 9a (Fig. 2B) and 9e (data not shown).
The morphological illustrations reported at high magnification
in Fig. 2 further reveal that 9a and 9e induce cytostatic effects, i.e.
a decrease in cell proliferation (while no cytotoxicity is observed)
through marked enlargements of cancer cell sizes, a feature that
could reflect modifications in the organization of the actin cyto-
skeleton according to data we obtained previously with other
compound types of natural origin [26,27].
Scheme 2.
improve this low yield. The BOC group was finally cleaved to yield
quantitatively compounds 5aec.
3
. Pharmacology
3
.1. In vitro growth inhibition in cancer cell lines
Growth inhibitory activity was determined by means of the MTT
colorimetric assay in a panel of eight cancer cell lines including cell
lines that display actual sensitivity to pro-apoptotic stimuli (MCF-7
19], PC-3 [19], Hs683 [20,21], B16F10 [22]) versus cell lines that
display various levels of resistance to pro-apoptotic stimuli (A549
23], U373 [20,21], OE21 [24]). In addition, the p53 status (reported
[
3.3. G2/M checkpoint abrogation
[
as either mutated or non-mutated) is indicated for most cell lines in
Table 1.
We examined the ability of the synthesized compounds to
ꢀ/ꢀ
abrogate an activated G2/M checkpoint in HCT116 p53
cells. This
The data from Table 1 reveal that the in vitro growth inhibitory
activity associated with each compound under study is similar in (i)
p53 wild-type (non-mutated) or p53 mutated cancer cells and (ii)
in cancer cells displaying sensitivity versus a certain degree of
resistance to pro-apoptotic stimuli. In contrast, heterogeneous
patterns in terms of in vitro growth inhibitory activity have been
observed with respect to the 23 compounds under study. Among
the compounds of the three final series namely pyrrolic 3aee,
checkpoint was activated following etoposide (VP-16) treatment
and illegitimate entry into mitosis was monitored using an adap-
tation of the previously described mitotic trap assay [5,28]. Briefly,
cells were treated with etoposide for 1 h then released in drug free
media with or without inhibitor for 23 h. Nocodazole was added to
the media, thus trapping the cells that could overcome or exit the
G2/M checkpoint in a mitosis-like state. The percentage of mitotic
cells, reflecting the ability of the assayed compound to act as
a checkpoint abrogator, was evaluated by histone H3 phosphory-
lation labeling and DNA content was monitored using flow
cytometry.
As presented in Table 2 following etoposide treatment, cells
arrested their cell cycle at G2/M. In contrast, 75% of cells treated
with etoposide and the potent Chk1 inhibitor AZD7762 (300 nM)
did not arrest at G2/M, proceeded to mitosis, and were trapped in
mitosis by nocodazole. When isogranulatimide and granulatimide
were used in agreement with published results [5], moderate
checkpoint abrogating effect was observed with about 20% of cells
escaping the checkpoint and subsequently trapped in mitosis.
Compound 4a displayed a similar activity, while in contrast all
other derivatives had a weaker activity in this biological assay. This
result could not explain the cytotoxicity observed by 4a in the sense
that theoretically, G2 checkpoint inhibitors are not expected to be
cytotoxic by themselves but in the presence of a DNA-damaging
agent. It has to be pointed out that compound 3a reported by
Prudhomme and co-workers to be a potent Chk1 inhibitor (IC50
of 24 nM) did not display checkpoint abrogating effect in our
Scheme 3.

S. Deslandes et al. / European Journal of Medicinal Chemistry 54 (2012) 626e636
629
Table 1
In vitro growth inhibitory concentrations (IC50 indices).
Compounds
TP53 statusa
A549
Wt
U373
Mut
LoVo
Wt
MCF-7
Wt
HS683
Wt
PC-3
Mut
OE21
nd
B16F10
nd
Mean ꢂ SEM
Granulatimide
Isogranulatimide
18
79
18
14
26
>100
36
8
8
8
15
>100
>100
>100
55
8
20
6
>100
33
22
26
3
62
11
52
39
99
40
0.4
4
34
7
44
5
42
26
8
4
4
25
86
87
70
24
27
26
46
99
49
42
35
>100
36
25
23
>100
52
45
58
>100
72
3
64
22
23
93
>100
45
50
25
8
6
11
91
50
75
66
>100
64
22
75
>100
36
>100
>100
>100
26
9
11
6
37
>100
>100
>100
27
e
5
35
19
15
22
93
27
25
37
e
14 ꢂ 5
>82
e
3
3
3
3
3
4
4
4
4
5
5
5
8
9
9
9
9
1
1
1
1
1
1
a
b
c
d
e
a
b
c
d
a
b
c
c
a
b
c
75
38 ꢂ 8
>100
37
>100
>100
2
>46
39 ꢂ 6
>99
>50
11 ꢂ 4
32 ꢂ 10
>50
>100
28
0.2
57
>100
8
>100
>100
51
43
4
5
15
9
5
35
e
e
e
7
75
2
37
61
2
0.5
0.6
6
>100
>100
>100
13
8
16 ꢂ 5
e
e
>85
e
e
>68
>63
e
e
84
35
29
19
33
67
47 ꢂ 7
20 ꢂ 8
>22
13 ꢂ 6
20 ꢂ 5
>98
>98
>92
25 ꢂ 2
24 ꢂ 3
23 ꢂ 3
>100
53
4
7
e
10
7
28
42
0a
0b
0c
1a
1b
1c
>100
>100
>100
25
29
29
>100
>100
>100
27
27
27
>100
>100
>100
27
15
11
>100
>100
69
29
29
>100
>100
>100
27
26
26
28
30
30
8
The IC50 indices (
m
M) represent the in vitro growth inhibitory concentrations at 50% obtained after having cultured the cells for 3 days in presence of the drug of interest.
a
Whether each cancer cell line under study displays wild-type or mutated forms of TP53 is described in reference [25] (nd and e mean not determined).
experiment. Such a result was already observed by Golsteyn’s
group when testing different Chk1 inhibitors with IC50 ranging
from 2.8 to 400 nM, only one indolocarbazole compound (S27888)
scored positive in their checkpoint bypass assay [29].
5. Experimental section
5.1. Chemistry
1H and 13C NMR spectra were performed on Bruker Avance 300
1
13 31
3.4. Inhibition of kinases
or Avance 500 (cryoprobe TCI H{ C, P}) spectrometers with the
chemical shifts of the remaining protons of the deuterated solvents
serving as internal standards. IR spectra were obtained on a Perkin-
Elmer 883 spectrophotometer. Mass spectra were recorded on a
GCT premier waters mass spectrometer for CI and high-resolution
mass spectra (HRMS) were recorded on a UPLC Xevo G2 Q TOF
Waters spectrometer for ESI. Reagents were purchased
from commercial sources and used as received. Flash-
In order to get an insight into the kinase selectivity, the final
analogs 3e5 were tested on a selection of purified serine/threonine
kinases, namely CDK5/p25, CK1, CLK1, DYRK1A, GSK-3, as described
in Section 4. Doseeresponse curves allowed the determination of
IC50 values relating to kinase activity inhibition, which are reported
inTable 3. Results show weakto actual inhibitoryactivities, with poor
selectivity in this small kinase panel. No clear-cut correlation with
the anti-proliferative effects shown in Table 1 appeared except as in
the case of compound 4a and 4d, for which their capacity to inhibit
other kinases could be responsible for the cytotoxicity observed.
chromatography was performed on silica gel (15e40 mM) by
means of the solvent systems indicated below. The purity of tested
compounds was established to be >95% by HPLC analysis using
a Waters acquity liquid chromatograph and a BECH 18 column
(
2.1 mm ꢁ 50 mm ꢁ 1.7
mM). Detection wavelength was 318 nm.
4
. Conclusion
Solvents were (A) water, 0.1% TFA; (B) acetonitile, 0.1% TFA. The
method was A/B 80:20 for 0.2 min, then a gradient mode up to A/B
0:100 for 4 min, then A/B 0:100 for 1 min then a gradient up to A/B
80:20 for 0.2 min.
In conclusion, the current study reports about the in vitro
growth inhibitory activity of pyrazolic analogs of the marine
granulatimide alkaloid in a panel of eight cancer cell lines and
a pyrolic series was also completed. The data show that the in vitro
growth inhibitory activity of the most potent compounds was
5.1.1. 3-Bromo-5-methyl-1H-indole (7b)
A solution of 5-methylindole (200 mg, 1.52 mmol) in anhydrous
DMF anhydre (7.5 mL) was degased and added dropwise at room
w10 mM in the above-mentioned panel of cancer cell lines. The
most active compounds displayed similar growth inhibitory
activity whether the cancer cells (i) were p53 wild-type (non-
mutated) or p53 mutated cancer cells and (ii) displayed sensitivity
versus a certain degree of resistance to pro-apoptotic stimuli. In
term of cancer cell growth inhibition these compounds were
cytostatic, not cytotoxic, and one compound revealed a G2 check-
point abrogating effect similar to that observed with the natural
alkaloids. The most active compounds from the present study
therefore deserve further chemical derivatization and pharmaco-
logical characterization to bring them at a drugable level.
temperature to a solution of bromine (78 mL, 1.52 mmol) in anhy-
drous and degassed DMF (7.5 mL). After 2 h stirring in the dark, the
reaction was poured into ice-cold water (80 mL), aqueous ammonia
(1.4 mL) and Na
the organic layers were dried over MgSO
solvent, the crude product was used without further purification in
2
SO
3
(1 g). After extraction with CH
2
Cl
2
(3 ꢁ 60 mL),
4
. After removal of the
1
the next step. H NMR (acetone-d
6
) 2.87 (s, 3H); 6.54 (dd, 1H,
J ¼ 1.5 Hz and 8.3 Hz); 6.73 (d, 1H, J ¼ 0.6 Hz); 6.85 (d, 1H,
13
J ¼ 8.4 Hz); 7.01 (d, 1H, J ¼ 2.4 Hz); 10.87 (br. s, 1H). C NMR
(acetone-d 22.5, 91.0, 113.5, 119.6, 126.0, 126.2, 128.8, 131.0, 136.1
6
) d

630
S. Deslandes et al. / European Journal of Medicinal Chemistry 54 (2012) 626e636
Fig. 2. Quantitative videomicroscopy on A549 NSCLC and U373 GBM cell lines.
5
.1.2. 5-Methyl-2-(1H-pyrrol-2-yl)-1H-indole (8b)
To a solution of compound 7b (1.52 mmol) in distilled CH
15 mL) was added pyrrole (211 L, 3 mmol) and TFA (40 L). The
and 3.4 Hz); 6.54 (m, 1H); 6.86 (ddd, 1H, J ¼ 1.3 Hz, J ¼ 2.7 Hz and
5.2 Hz); 7.21 (d, 1H, J ¼ 8.2 Hz); 7.26 (d, 1H, J ¼ 0.7 Hz); 10.23 (br. s,
2
Cl
2
13
(
m
m
1H); 10.50 (br. s, 1H). C NMR (acetone-d
6
) 22.5, 97.0, 107.2, 110.7,
mixture was stirred at room temperature for 30 min. After addition
of aqueous ammonia up to alkaline pH, the reaction media was
concentrated and the crude product was purified by flash-
chromatography (petroleum ether/AcOEt 85:15) to give the
expected compound as a light-sensitive white solid (152 mg, 39%
112.1, 120.7, 121.1, 124.2, 127.4, 129.9, 131.6, 134.5, 136.9. IR (KBr)
ꢀ1
3359, 2924, 1775, 1707, 1353, 1184.799 cm
.
5.1.3. 5-Methoxy-2-(1H-pyrrol-2-yl)-1H-indole (8c)
Same procedure as for compound 8b involving 7c [12] (770 mg,
ꢃ
þ
yield), mp 168 C. HRMS (CIþ) [M þ H] calcd 197.1079, found
3.4 mmol), distilled CH
2
2
Cl (35 mL), pyrrole (475 mL, 6.8 mmol) and
1
1
97.1060. H NMR (acetone-d
6
) 2.36 (s, 3H); 6.17 (td, 1H, J ¼ 2.6 Hz
TFA (100 mL). The crude product was purified by flash-
Table 2
Table 3
Evaluation of the G2/M checkpoint abrogation.
Inhibition of kinases.
Compounds
Mitotic index
Compounds
CDK5
CK1
CLK1
0.52
1.5
10
0.26
0.3
3.5
>10
0.09
0.42
0.13
DYRK1A rat
GSK-3
3
4
4
4
5
8
8
1
a
a
9.6
23.9
13.9
3.9
0
5.0
8.3
5.5
17.2
19.4
75.0
0
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
2.1
7.5
>10
2.6
4.0
>10
>10
0.51
10
>10
>10
1.1
3.2
7.1
>10
0.16
2.6
1.3
8.3
>10
0.2
0.57
1.7
1.7
0.42
0.49
0.045
0.7
0.041
0.9
b
d
b
b
c
2.1
>10
>10
0.18
1.7
1b
Isogranulatimide
Granulatimide
AZD
0.91
0.31
0.23
1.4
0.17
IC50 values were determined from doseeresponse curves on purified protein
kinases assayed in the presence of various compounds.
Control

S. Deslandes et al. / European Journal of Medicinal Chemistry 54 (2012) 626e636
631
chromatography (petroleum ether/AcOEt 85:15) to give the
expected compound as a light-sensitive white solid (417 mg, 58%
5.1.7. 8-Methyl-11H-1,5,11,11b-tetraaza-benzo[a]trindene-4,6-
dione (3b)
ꢃ
þ
þ
yield), mp 147 C. HRMS (ESI ) [M þ H] calcd 213.1028, found
A suspension of 9b (50 mg, 0.17 mmol) and Pd black (18.1 mg,
0.171 mmol) in nitrobenzene (2.5 mL) was irradiated at 300 W for
30 min. After cooling, petroleum ether (5 mL) was added and the
mixture was filtered on silica gel to remove nitrobenzene (petro-
leum ether, then petroleum ether/AcOEt 90:10 and petroleum
ether/AcOEt 1/1) to give the expected compound as a dark red solid
1
2
13.1019. H NMR (acetone-d
and 3.3 Hz); 6.58 (m, 2H); 6.71 (dd, 1H, J ¼ 2.4 Hz and 8.7 Hz); 6.87
td, 1H, J ¼ 1.5 Hz and 2.7 Hz); 7.01 (d, 1H, J ¼ 2.4 Hz); 7.23 (d, 1H,
6
) 3.78 (s, 3H); 6.19 (td, 1H, J ¼ 2.7 Hz
(
1
3
J ¼ 8.7 Hz); 10.23 (br. s, 1H); 10.48 (br. s, 1H). C NMR (acetone-d
6.73, 97.41, 103.34, 107.17, 110.80, 112.62, 112.96, 120.68, 127.35,
31.73, 133.57, 135.02, 156.09. IR (KBr) 3417, 3386, 1608, 1489, 1450,
6
)
5
1
1
ꢃ
þ
þ
(24 mg, 49% yield), mp >260 C. HRMS (ESI ) [M þ H] calcd
ꢀ
1
1
241, 1226 cm
.
290.0930, found 290.0922. H NMR (acetone-d
d, 2H, J ¼ 2.1 Hz); 7.25 (td, 1H, J ¼ 6.6 Hz and 1.2 Hz); 7.48 (d, 1H,
J ¼ 8.4 Hz); 8.30 (t, 1H, J ¼ 2.1 Hz); 8.48 (dd, 1H, J ¼ 2.4 Hz and
6
) 2.52 (s, 3H); 7.05
(
5
2
.1.4. 3-(5-Methyl-2-(1H-pyrrol-2-yl)-1H-indol-3-yl)pyrrolidine-
,5-dione (9b)
13
0.9 Hz). C NMR (DMSO-d
6
) 22.7, 101.2, 106.2, 113.1, 116.2, 117.8,
A mixture of compound 8b (60 mg, 0.31 mmol), maleimide
120.3, 124.6, 126.8, 130.5, 132.2 (2C), 136.0, 139.3, 167.8, 170.5. IR
(KBr) 3443e3224, 1710, 1490, 1216, 1084, 1216 cm . HPLC: t
R
ꢀ
1
(
59 mg, 0.62 mmol), a catalytic amount of SnCl
2
in toluene (17 mL)
was refluxed for 24 h. After removal of the solvent, the residue was
purified by flash-chromatography (petroleum ether/AcOEt 90:10
then 70:30) to give the expected compound as a off-white solid
2.28 min.
5.1.8. 8-Methoxy-11H-3a,5,11-triaza-benzo-[a]-trindene-4,6-dione
(3c)
Same procedure as for compound 3b involving 9c (140 mg,
0.45 mmol) and Pd black (48 mg, 0.45 mmol) in nitrobenzene
(7 mL). The filtration on silica gel (petroleum ether/AcOEt 1:0 up to
0:1) gave the expected product as a dark red solid (82 mg, 60%
ꢃ
þ
þ
(
24.4 mg, 28% yield), mp 168 C. HRMS (ESI ) [M þ H] calcd
1
2
94.1243, found 294.1267. H NMR (acetone-d
dd, 1H, J ¼ 5.1 Hz and 18.3 Hz); 3.28 (dd, 1H, J ¼ 9.9 Hz and
8.3 Hz); 4.59 (dd, 1H, J ¼ 5.1 Hz and 9.9 Hz); 6.25 (td, 1H, J ¼ 2.4 Hz
and 3.6 Hz); 6.49 (ddd, 1H, J ¼ 1.5 Hz, 2.7 Hz and 3.0 Hz); 6.95 (dd,
H, J ¼ 1.5 Hz and 8.1 Hz); 6.98 (td, 1H, J ¼ 1.5 Hz and 2.7 Hz); 7.15
br. s, 1H); 7.29 (d, 1H, J ¼ 8.1 Hz); 10.19 (br. s, 1H); 10.33 (br. s, 1H);
0.41 (br. s, 1H). 13C NMR (acetone-d
) 22.73, 38.46, 41.43, 107.90,
10.39, 111.00, 113.05, 119.58, 121.4, 125.02, 125.35, 128.64, 130.20,
33.02, 136.69, 179.05, 182.01. IR (KBr) 3359, 2924, 1775, 1707, 1353,
6
) 2.36 (s, 3H); 3.04
(
1
ꢃ
þ
þ
1
(
1
1
yield), mp >260 C. HRMS (ESI ) [M þ H] calcd 306.0879, found
1
306.0884. H NMR (acetone-d
6
) 3.92 (s, 3H); 7.03 (dd, 1H, J ¼ 8.7 Hz
6
and 2.7 Hz); 7.05 (d, 1H, J ¼ 1.8 Hz); 7.05 (d, 1H, J ¼ 2.4 Hz); 7.50 (dd,
1H, J ¼ 8.7 Hz and 0.6 Hz); 8.25 (d, 1H, J ¼ 2.7 Hz); 8.30 (dd, 1H,
13
1
J ¼ 2.4 Hz and 1.8 Hz); 9.84 (br. s, 1H); 11.52 (br. s, 1H). C NMR
ꢀ1
1184, 799, 731 cm
.
(DMSO-d
6
) 55.96, 100.23, 104.26, 105.37, 112.92, 114.46 (2C), 116.40,
1
17.67, 121.31, 122.74, 125.02, 134.13, 134.64, 154.99, 166.91, 169.84.
ꢀ
1
5.1.5. 3-[5-Methoxy-2-(1H-pyrrol-2-yl)-1H-indol-3-yl]-
IR (KBr) 3316, 2923, 2852, 1747, 1707, 1486, 1459, 1208, 1025 cm
HPLC: t 2.05 min.
.
pyrrolidine-2,5-dione (9c)
R
Same procedure as for compound 9b involving 8c (320 mg,
1
.50 mmol), maleimide (290 mg, 3.00 mmol), a catalytic amount of
SnCl and toluene (90 mL) Purification of the crude product by
flash-chromatography (CH
5.1.9. 8-Hydroxy-11H-1,5,11,11b-tetraaza-benzo[a]trindene-4,6-
dione (3d)
2
2
Cl
2
/MeOH 98:2) gave the expected
To a solution of 3c (15 mg, 0.05 mmol) in CH
added BBr (1 M in CH Cl , 2 mL, 2 mmol). After 4 h stirring at room
temperature, the mixture was poured into a saturated solution of
NaHCO (20 mL). Filtration of the mixture gave quantitatively the
expected compound as a dark red solid (12.3 mg, 84% yield), mp
2 2
Cl (6 mL) was
ꢃ
compound as a pale green solid (276 mg, 59% yield), mp 142 C.
3
2
2
þ
þ
1
HRMS (ESI ) [M þ H] calcd 310.1192, found 310.1167. H NMR
(
acetone-d
6
) 3.09 (dd, 1H, J ¼ 18.0 Hz and 5.1 Hz); 3.31 (dd, 1H,
3
J ¼ 18.0 Hz and 9.9 Hz); 3.76 (s, 3H); 4.63 (dd, 1H, J ¼ 4.8 Hz and
ꢃ
þ
þ
1
9
1
.9 Hz); 6.26 (td, 1H, J ¼ 2.4 Hz and 3.6 Hz); 6.50 (m, 1H); 6.79 (dd,
H, J ¼ 2.4 Hz and 8.7 Hz); 6.85 (d, 1H, J ¼ 2.4 Hz); 6.98 (td, 1H,
>260 C. SM (ESI ) [M þ H] calcd 292.0722, found 292.0706. H
NMR (DMSO-d
) 6.89 (dd, 1H, J ¼ 2.5 Hz and 8.6 Hz); 7.04 (s, 1H);
7.42 (d, 1H, J ¼ 8.7 Hz); 7.99 (d, 1H, J ¼ 2.4 Hz); 8.22 (dd, 1H,
6
J ¼ 1.5 Hz and 2.7 Hz); 7.30 (d, 1H, J ¼ 8.7 Hz); 10.15 (br. s, 1H); 10.39
br. s, 2H). 13C NMR (acetone-d
24.25, 126.20, 128.51, 137.7. IR (KBr) 3353, 2940, 2833, 1775, 1708,
) 91.41, 113.75, 120.04, 121.83,
J ¼ 1.4 Hz and 2.7 Hz). C NMR (DMSO-d
13
(
1
6
6
) 99.6, 103.5, 107.0, 112.1,
113.8,114.5, 115.8,117.4, 120.4, 122.5, 124.5, 132.8, 134.1,152.2, 166.4,
ꢀ
1
ꢀ1
1488, 1218, 1182 cm
169.4. IR (KBr) 1740, 1710 cm . HPLC: t
R
1.99 min.
5
.1.6. 3-(5-Methoxy-2-(1H-pyrrol-2-yl)-1H-indol-3-yl)-1-
5.1.10. 8-Methoxy-5-methyl-11H-3a,5,11-triaza-benzo[a]trindene-
4,6-dione (3e)
methylpyrrolidine-2,5-dione (9e)
Same procedure as for compound 9b involving compound 2b
120 mg, 0.56 mmol), N-methylmaleimide (124 mg, 1.12 mmol),
Same procedure as for compound 3b involving 9e (30 mg,
0.094 mmol), Pd black (10 mg, 0.094 mmol) and nitrobenzene
(2 mL), the mixture was irradiated at 300 W for 1.5 h. Purification
by filtration on silica gel gave the expected compound as a dark red
(
a catalytic amount of SnCl and toluene (36 mL). Purification by
2
flash-chromatography (petroleum ether/AcOEt 80:20) gave the
expected compound as a pale green solid (110 mg, 61% yield),
ꢃ
þ
þ
solid (17.7 mg, 59% yield), mp >260 C. MS (ESI ) [M þ H] calcd
ꢃ
þ
þ
1
mp 132 C. HRMS (ESI ) [M þ H] calcd 324.1348, found
320.1035, found 320.1040. H NMR (acetone-d
6
) 3.18 (s, 3H); 3.93 (s,
3
4
3
24.1351. 1H NMR (acetone-d
6
) 3.01 (dd, 1H, J ¼ 18.0 Hz and
3H); 7.04 (m, 3H); 7.50 (d, 1H, J ¼ 8.8 Hz); 8.26 (d, 1H, J ¼ 2.5 Hz),
13
.8 Hz); 3.04 (s, 3H); 3.27 (dd, 1H, J ¼ 9.6 Hz and 18.0 Hz); 3.74 (s,
H); 4.55 (dd, 1H, J ¼ 4.8 Hz and 9.6 Hz); 6.24 (td, 1H, J ¼ 2.7 Hz
6
8.29 (m, 1H). C NMR (acetone-d ) 23.95, 55.82, 100.22, 104.24,
104.88, 112.97, 114.37, 114.67, 116.50, 116.89, 120.79, 122.56, 124.89,
134.08, 134.46, 154.97, 165.67, 168.68. IR (KBr) 3297, 1682, 1436,
and 3.3 Hz,); 6.46 (m, 1H,); 6.65 (d, 1H, J ¼ 2.4 Hz); 6.77 (dd, 1H,
J ¼ 8.7 Hz and 2.4 Hz); 6.98 (dt, 1H, J ¼ 2.7 Hz and 1.5 Hz); 7.30
ꢀ
1
R
1377, 1222, 1043 cm . HPLC: t 2.65 min.
13
(
(
1
1
1
d, 1H, J ¼ 8.7 Hz); 10.18 (br. s, 1H); 10.42 (br.s, 1H). C NMR
acetone-d
09.16, 111.23, 112.11, 119.64, 123.40, 126.87, 131.48, 131.69,
54.19, 176.6, 179.3. IR (KBr) 3337, 1692, 1438, 1282, 1218, 1119,
6
) 24.19, 35.32, 38.12, 54.92, 100.23, 106.52, 108.63,
5.1.11. 2-Pyrazol-1-yl-1H-indole (10a)
Same procedure as for compound 8b involving bromoindole
(838.5 mg, 4.3 mmol), distilled CH
2 2
Cl (35 mL), pyrazole (576 mg,
ꢀ1
030 cm
.
8.6 mmol) and TFA (120 L). The mixture was stirred at room
m

632
S. Deslandes et al. / European Journal of Medicinal Chemistry 54 (2012) 626e636
temperature for 24 h. Purification by flash-chromatography
petroleum ether/AcOEt 85:15) gave the expected compound as
atmosphere, to a solution of EtMgBr (1 M in THF, 2.4 mL, 2.4 mmol)
and the mixture was stirred for 30 min. A solution of 3,4-dibromo-1-
(tert-butyldimethylsilyl)-1H-pyrrole-2,5-dione (220 mg, 0.61 mmol)
in THF (2 mL) was then added and stirring was continued for 12 h.
(
ꢃ
þ
þ
a white solid (480 mg, 61% yield), mp 147 C. HRMS (ESI ) [M þ H]
1
calcd 184.0875, found 184.0882. H NMR (acetone-d
J ¼ 1.8 Hz and 2.4 Hz); 6.59 (br. s, 1H); 7.05 (td, 1H, J ¼ 1.5 Hz and
.6 Hz); 7.13 (td, 1H, J ¼ 1.5 Hz and 7.6 Hz); 7.48 (dd, 1H, J ¼ 0.8 Hz
and 7.6 Hz); 7.55 (dd, 1H, J ¼ 0.6 Hz and 7.6 Hz); 7.71 (d, 1H,
6
) 6.53 (dd, 1H,
The reaction media was poured into NH
10 mL) and was extracted with AcOEt (2 ꢁ 15 mL). The combined
organic layers were dried over MgSO and the solvent was removed.
The crude product was purified by chromatography on alumina
4
Cl (10% aqueous solution,
7
4
13
J ¼ 1.8 Hz); 8.31 (d, 1H, J ¼ 2.4 Hz); 10.85 (br. s, 1H). C NMR
acetone-d ) 88.8, 109.5, 113.2, 121.8, 122.0, 123.3, 129.7, 129.9,
35.9, 138.0, 142.5. IR 3122, 1694, 1621, 1586, 1566, 1470, 1393, 1233,
(
6
(petroleum ether/CH
2
Cl
2
/MeOH 8:2:0 up to 0:95:5) to give the
ꢃ
1
expected compound as a red solid (40 mg, 20% yield), mp 148 C.
ꢀ
1
þ
þ
1
1046, 919, 749 cm
.
HRMS (ESI ) [M þ H] calcd 371.0144, found 371.0151. H NMR
acetone-d
) 2.42 (s, 3H); 6.50 (dd, 1H, J ¼ 4.5 Hz and 1.8 Hz); 7.10
(dd, 1H, J ¼ 8.04 Hz and 1.2 Hz); 7.38 (s, 1H); 7.43 (d, 1H, J ¼ 8.4 Hz);
(
6
5.1.12. 5-Methyl-2-pyrazol-1-yl-1H-indole (10b)
13
Same procedure as for compound 8b involving 7b (320.0 mg,
7.72 (d, 1H, J ¼ 1.2 Hz); 8.20 (d, 1H, J ¼ 2.7 Hz). C NMR (acetone-d
6
)
1.52 mmol), distilled CH
2
Cl
2
(15 mL), pyrazole (211
m
L, 3 mmol) and
20.93, 91.29, 108.02, 111.61, 120.47, 123.76, 124.43, 126.19, 130.15,
130.23, 131.99, 135.40, 141.66, 166.19, 168.48. IR (KBr) 3322-2927,
TFA (40
mL). Purification by flash-chromatography (petroleum
ꢀ
1
ether/AcOEt 85:15) gave the expected compound as a white light-
1718, 1619, 1496, 1396, 1336, 1031, 651 cm .
ꢃ
þ
sensitive solid (90 mg, 30% yield), mp 153 C. HRMS (ESI )
þ
1
[
M þ H] calcd 198.1031, found 198.1026. H NMR (acetone-d
6
) 2.39
5.1.16. 3-Bromo-4-(5-methoxy-2-(1H-pyrazol-1-yl)-1H-indol-3-
yl)-1H-pyrrole-2,5-dione (11c)
Same procedure as for compound 11a involving 10c (410 mg,
1.92 mmol), THF (7 mL), LiHMDS (1 M in THF, 5.76 mmol,
(
s, 3H); 6.50 (dd, 1H, J ¼ 0.8 Hz and 2.2 Hz); 6.52 (dd, 1H, J ¼ 1.8 Hz
and 2.5 Hz); 6.95 (dd, 1H, J ¼ 1.2 Hz and 8.2 Hz); 7.32 (dd, 1H,
J ¼ 0.7 Hz and 1.5 Hz); 7.35 (m, 1H); 7.69 (d, 1H, J ¼ 1.5 Hz); 8.29 (dd,
13
1
H, J ¼ 0.5 Hz and 2.5 Hz); 10.72 (s, 1H). C NMR (acetone-d
6
) 22.6,
5.76 mL),
3,4-dibromo-1-(tert-butyldimethylsilyl)-1H-pyrrole-
8
8.6, 109.4, 112.9, 121.6, 124.8, 129.6, 130.2, 130.8, 134.2, 138.0, 142.4.
2,5-dione (740 mg, 2.0 mmol), THF (10 mL), 10% HCl (35 mL).
After work-up, the crude product was purified by flash-chroma-
tography (petroleum ether/AcOEt 80/20 up to 50/50) to give the
expected compound as a red solid (200 mg, 27% yield), mp
ꢀ
1
IR (KBr) 3382, 1597, 1471, 1390, 1048, 798, 748 cm
.
5
.1.13. 5-Methoxy-2-pyrazol-1-yl-1H-indole (10c)
Same procedure as for compound 8b involving 7c (770.0 mg,
.4 mmol), distilled CH Cl (35 mL), pyrazole (460 mg, 6.8 mmol)
L). The mixture was stirred in the dark for 12 h.
ꢃ
þ
þ
1
105 C. HRMS (ESI ) [M þ H] calcd 387.0093, found 387.0087. H
3
2
2
NMR (acetone-d
6
) 3.83 (s, 3H); 6.51 (dd, 1H, J ¼ 1.8 Hz and
and TFA (100
m
2.6 Hz); 6.91 (dd, 1H, J ¼ 2.5 Hz and 8.8 Hz); 7.12 (d, 1H,
J ¼ 2.5 Hz); 7.46 (dd, 1H, J ¼ 0.6 Hz and 8.8 Hz); 7.73 (dd, 1H,
Purification by flash-chromatography (petroleum ether/AcOEt 8:2)
gave the expected product as a white solid (396 mg, 55% yield), mp
J ¼ 0.5 Hz and 1.8 Hz); 8.20 (dd, 1H, J ¼ 0.6 Hz and 2.6 Hz); 10.11
ꢃ
þ
þ
1
13
1
17 C. HRMS (ESI ) [M þ H] calcd 214.0980, found 214.0979. H
(br. s, 1H); 11.36 (br. s, 1H). C NMR (acetone-d
6
) 56.8, 93.7, 104.8,
NMR (acetone-d ) 3.80 (s, 3H, OMe); 6.51 (m, 2H); 6.78 (dd, 1H,
6
109.8, 114.5, 114.7, 125.2, 128.3, 130.3, 132.0, 137.3, 141.1, 143.5,
J ¼ 2.4 Hz and 8.7 Hz); 7.06 (d, 1H, J ¼ 2.4 Hz); 7.37 (d, 1H,
J ¼ 8.7 Hz); 7.70 (d, 1H, J ¼ 1.8 Hz); 8.26 (d, 1H, J ¼ 2.4 Hz); 10.75 (br.
157.0, 167.9, 170.2. IR (KBr), 3435e3213, 1717, 1617, 1491, 1329,
ꢀ1
1211, 654 cm
.
13
6
s, 1H). C NMR (acetone-d ) 56.78, 89.02, 103.89, 109.42, 113.42,
1
13.88, 129.60, 130.48, 130.85, 138.38, 142.41, 156.62. IR (KBr) 3192,
5.1.17. 11H-1,5,11,11b-tetraaza-benzo[a]trindene-4,6-dione (4a)
A solution of 11a (18 mg, 0.05 mmol) in CH CN (11 mL) was
ꢀ1
1592, 1571, 1499, 1452, 1397, 1236, 1226 cm
.
3
irradiated at 350 nm for 5 h. Filtration of the mixture gave the
expected compound as an orange solid (8.1 mg, 58% yield), mp
5
.1.14. 3-(2-(1H-Pyrazol-1-yl)-1H-indol-3-yl)-4-bromo-1H-
ꢃ
þ
þ
1
pyrrole-2,5-dione (11a)
>260 C. HRMS (ESI ) [M þ H] calcd 277.0726, found 277.0714. H
NMR (DMSO-d
) 7.11 (d, 1H, J ¼ 2.2 Hz); 7.40 (t, 1H, J ¼ 7.5 Hz); 7.49
(t, 1H, J ¼ 7.6 Hz); 7.67 (d, 1H, J ¼ 8.2 Hz); 8.45 (d, 1H, J ¼ 2.2 Hz);
To a solution of 10a (65 mg, 0.36 mmol) in THF (1 mL) was added
6
ꢃ
dropwise at ꢀ15 C, LiHMDS (1 M solution in THF, 1.1 mL,
13
1.08 mmol). After 45 min stirring, a solution of compound 3,4-
8.60 (d, 1H, J ¼ 7.3 Hz); 11.26 (br. s, 1H); 13.66 (br. s, 1H). C NMR
(DMSO-d ) 98.54, 100.56, 112.31, 113.22, 121.26, 122.76, 123.70,
126.52, 130.64, 132.82, 137.59, 139.59, 146.30, 168.02, 169.65. IR
dibromo-1-(tert-butyldimethylsilyl)-1H-pyrrole-2,5-dione
[13]
6
(
140 mg, 0.38 mmol) in THF (1 mL) was added and the mixture
ꢀ
1
was let to come back up to rt for 12 h. The reaction media was then,
poured into ice-cold 10% HCl and it was extracted with AcOEt
(KBr) 3452e3148, 1755, 1707, 1635, 1588, 1467, 1351, 750 cm
HPLC: t 1.69 min.
.
R
(
3 ꢁ 20 mL). The organic layers were washed first, with a saturated
solution of NaHCO and then with brine. After drying over MgSO
3
4
5.1.18. 8-Methyl-11H-1,5,11,11b-tetraaza-benzo[a]trindene-4,6-
and removal of the solvent, the crude product was purified by flash-
dione (4b)
chromatography (petroleum ether/AcOEt 80:20 up to 50:50) to give
the expected compound as a red solid (30 mg, 24% yield), mp 114 C.
A solution of 11b (20 mg, 0.054 mmol) in toluene (11 mL) was
irradiated at 350 nm for 8 h. After removal of the solvent, the
crude product was purified by flash-chromatography (CH Cl /
2 2
MeOH 95:5 then MeOH/AcOH 99:1) to give the expected
ꢃ
þ
þ
1
HRMS (ESI ) [M þ H] calcd 356.9987, found 356.9998. H NMR
(
acetone-d
6
) 6.51 (dd, 1H, J ¼ 1.8 Hz and 2.5 Hz); 7.18 (m, 1H); 7.26
ꢃ
(
m, 1H); 7.57 (t, 2H, J ¼ 9.0 Hz), 7.73 (d, 1H, J ¼ 1.8 Hz); 8.24 (d, 1H,
compound as an orange solid (10 mg, 63% yield), mp >260 C.
13
þ
þ
1
J ¼ 2.5 Hz); 10.15 (br. s,1H); 11.57 (br. s,1H). C NMR (DMSO-d
6
) 91.2,
HRMS (ESI ) [M þ H] calcd 291.0882, found 291.0886. H NMR
1
1
08.1,111.9,120.4,122.5,123.0,125.2,130.6,133.2,134.9,138.5,141.8,
(DMSO-d
6
) 7.09 (d, 1H, J ¼ 2.0 Hz); 7.31 (ddd, 1H, J ¼ 8.3 Hz and
ꢀ
1
67.0, 169.1. IR (KBr) 3304, 1723, 1628, 1564, 1460, 1331, 1022 cm
.
2.1 Hz); 7.55 (d, 1H, J ¼ 8.3 Hz); 8.40 (dd, 1H, J ¼ 2.1 Hz); 8.43 (d,
13
1
H, J ¼ 2.0 Hz); 11.24 (br. s, 1H); 13.54 (br. s, 1H). C NMR (DMSO-
5
.1.15. 3-Bromo-4-(5-methyl-2-(1H-pyrazol-1-yl)-1H-indol-3-yl)-
H-pyrrole-2,5-dione (11b)
A solution of compound 10b (120 mg, 0.61 mmol) in THF (8 mL)
was added dropwise at room temperature, under nitrogen
d
6
) 21.84, 98.43, 99.91, 112.55, 112.80, 121.59, 123.10, 127.45,
1
128.90, 131.47, 133.77, 135.58, 139.41, 145.10, 169.45, 170.11. IR
ꢀ1
(KBr) 3446, 3250, 1751, 1701, 1629, 1590, 1488 cm . HPLC: t
R
1.90 min.

S. Deslandes et al. / European Journal of Medicinal Chemistry 54 (2012) 626e636
633
ꢃ
þ
þ
5
.1.19. 8-Methoxy-11H-1,5,11,11b-tetraaza-benzo[a]trindene-4,6-
dione (4c)
Same procedure as for compound 4b involving 11c (20 mg,
.052 mmol) in toluene (11 mL) and irradiation for 5 h. Filtration of
the mixture gave the expected compound as an orange solid (12.4
yield), mp 130 C. HRMS (ESI ) [M þ H] calcd 317.1263, found
1
317.1265. H NMR (CDCl
3
) 1.45 (t, 3H, J ¼ 7.1 Hz); 1.71 (s, 9H); 4.44
(q, 2H, J ¼ 7.1 Hz); 7.40 (m, 1H); 8.17 (m, 1H); 8.39 (m, 1H); 8.79 (s,
13
0
1H). C NMR (CDCl ) 13.6, 27.4 (3C), 62.1, 86.0, 114.97, 115.00, 121.5,
3
124.6, 126.0, 126.7, 134.6, 137.1, 148.0, 161.5, 179.2. IR (KBr) 1747,
1723, 1659, 1543, 1481, 1365, 1258, 1141 cm
ꢃ
þ
þ
ꢀ1
mg, 78% yield), mp >260 C. HRMS (ESI ) [M þ H] calcd 307.0831,
.
found 307.0819. 1H NMR (DMSO-d
6
) 3.38 (s, 3H); 7.09 (d, 1H,
J ¼ 2.5 Hz); 7.12 (dd, 1H, J ¼ 8.5 Hz and 2.5 Hz); 7.57 (d, 1H,
J ¼ 8.5 Hz); 8.15 (d, 1H, J ¼ 2.5 Hz); 8.42 (d, 1H, J ¼ 2.5 Hz); 11.25 (s,
5.1.24. tert-Butyl-3-(2-ethoxy-2-oxoacetyl)-5-methyl-1H-indole-1-
carboxylate (13b)
1
H); 13.51 (s, 1H). 13C NMR (DMSO-d
6
) 56.00, 98.52, 100.20, 106.22,
Same procedure as for compound 13a involving 12c (530 mg,
112.36, 113.89, 114.80, 122.09, 128.98, 131.92, 133.76, 139.52, 145.06,
2 2
2.3 mmol), anhydrous CH Cl (3 mL), di-tert-butyl-di-carbonate
,
1
155.65, 169.47, 170.17. IR (KBr) 3446, 3250, 1751, 1701, 1629, 1590,
(1.01 g, 4.6 mmol), DMAP (5.6 mg, 0.046 mmol), 1 N HCl (3 mL). The
expected compound was obtained quantitatively as a white solid
ꢀ
1
488 cm . HPLC: t
R
1.72 min.
ꢃ
þ
þ
(
760 mg), mp 186 C. HRMS (ESI ) [M þ H] calcd 332.1498, found
1
5
.1.20. 8-Hydroxy-11H-1,5,11,11b-tetraaza-benzo[a]trindene-4,6-
332.1485. H NMR (acetone-d
6
) 1.41 (t, 1H, J ¼ 7.7 Hz); 1.73 (s, 9H);
dione (4d)
Same procedure as for compound 3d involving 4c (50 mg,
.16 mmol), CH Cl (15 mL) BBr (1 M) in CH Cl (6 mL, 6 mmol),
stirring for 4 h and NaHCO
2.47 (s, 3H); 4.43 (q, 2H, J ¼ 7.1 Hz); 7.29 (dd, 1H, J ¼ 1.7 Hz and
13
8.6 Hz); 8.07 (d, 1H, J ¼ 8.6 Hz); 8.12 (m, 1H); 8.69 (s, 1H). C NMR
(CDCl ) 15.3, 28.4 (3C tBu), 57.0, 63.8, 87.6, 106.0, 116.6, 117.9, 130.2,
0
2
2
3
2
2
3
3
(20 mL). Filtration gave the expected
131.7, 139.0, 148.6, 150.1,159.5,164.1,181.6. IR (KBr) 1745, 1725, 1657,
ꢃ
ꢀ1
compound as a red solid (39 mg, 84% yield), mp >260 C. HRMS
(
d
1479, 1457, 1365, 1264, 1150 cm
.
þ
þ
1
ESI ) [M þ H] calcd 293.0675, found 293.0677. H NMR (DMSO-
) 6.88 (dd, 1H, J ¼ 2.4 Hz and 8.4 Hz); 6.97 (d, 1H, J ¼ 2.4 Hz); 7.42
d, 1H, J ¼ 8.4 Hz); 8.00 (d, 1H, J ¼ 2.4 Hz); 8.29 (d, 1H, J ¼ 2.4 Hz).
6
5.1.25. tert-Butyl-3-(2-ethoxy-2-oxoacetyl)-5-methoxy-1H-indole-
1-carboxylate (13c)
(
1
3
C NMR (DMSO-d
6
) 94.73, 98.14, 108.29 (2C), 113.93, 115.26, 119.99,
Same procedure as for compound 13a involving 12b (190 mg,
1
22.51, 129.06, 130.76, 133.72, 144.60, 153.29, 169.51, 170.52. IR
2 2
0.84 mmol), anhydrous CH Cl (2 mL), di-tert-butyl-di-carbonate
(370 mg, 1.68 mmol), DMAP (2 mg, 0.017 mmol), 1 N HCl (2 mL).
The expected compound was obtained as a yellow solid (270 mg,
ꢀ1
(
1
KBr) 3500-2976, 1701, 1636, 1585, 1483, 1193, 771 cm . HPLC: t
.15 min.
R
ꢃ
þ
þ
9
3% yield), mp 164 C. HRMS (ESI ) [M þ H] calcd 347.1369,
1
5.1.21. Ethyl 2-(5-methyl-1H-indol-3-yl)-2-oxoacetate (12b)
found 347.1374. H NMR (acetone-d
6
) 1.41 (t, 3H, J ¼ 7.1 Hz); 1.72
To a solution of 5-methylindole (200 mg, 1.52 mmol) in Et
2
O
(s, 9H); 3.88 (s, 3H); 4.43 (q, 2H, J ¼ 7.1 Hz); 7.06 (dd, 1H,
J ¼ 2.6 Hz and 9.1 Hz); 7.82 (d, 1H, J ¼ 2.6 Hz); 8.08 (d, 1H,
(
7 mL) was added dropwise at room temperature under nitrogen
13
atmosphere, oxalyl chloride (0.17 mL, 1.97 mmol) and the mixture
was stirred for 4 h. After removal of the solvent, EtOH (3.5 mL) and
Et N (2.4 mL, 1.82 mmol) were added to the residue. The mixture
3
J ¼ 9.1 Hz); 8.70 (s, 1H). C NMR (CDCl
3
) 15.3, 28.4 (3C), 57.0,
63.8, 87.6, 106.0, 116.6, 117.9, 130.2, 131.7, 139.0, 148.6, 150.1, 159.5,
164.1, 181.6. IR (KBr) 1742, 1662, 1611, 1587, 1481, 1454, 1368,
ꢀ
1
was refluxed for 5 h and then stirred at room temperature for 12 h.
1269, 1147, 1101 cm .
Filtration gave the expected compound as a white solid (340 mg,
ꢃ
þ
þ
9
2
4
1
7% yield), mp 202 C. HRMS (ESI ) [M þ H] calcd 232.0974, found
5.1.26. tert-Butyl 3-(2-amino-2-oxoethyl)-1H-pyrazole-1-
carboxylate (14)
Same procedure as for compound 13a involving 2-(1H-Pyrazol-
2 2
3-yl)-acetamide [17] (750 mg, 6 mmol), anhydrous CH Cl (20 mL),
di-tert-butyl-di-carbonate (2.8 g, 13 mmol), DMAP (15 mg,
0.12 mmol). After 4 h stirring, the solvent was removed to give the
1
32.0977. H NMR (acetone-d
6
) 1.37 (t, 3H, J ¼ 7.1 Hz); 2.46 (s, 1H);
.38 (q, 2H, J ¼ 7.2 Hz); 7.14 (dd, 1H, J ¼ 1.5 Hz and 8.3 Hz); 7.45 (d,
13
H, J ¼ 8.3 Hz); 8.12 (m, 1H); 8.38 (s, 1H); 11.24 (br. s, 1H). C NMR
) 13.9, 21.2, 61.5, 111.9, 112.3, 120.8, 125.2, 125.7, 131.8,
34.9,138.0,163.6,178.9. IR (KBr) 3236,1723,1626,1610,1503,1429,
(
DMSO-d
6
1
ꢀ
1
1266, 1135 cm
.
expected compound (1.5 g), which was used in the next step
1
without further purification. H NMR (DMSO-d
6
) 1.57 (s, 9H); 3.44
13
5
.1.22. Ethyl 2-(5-methoxy-1H-indol-3-yl)-2-oxoacetate (12c)
Same procedure as for 12b involving 5-methoxy-indole
(s, 2H); 6.42 (d, 1H, J ¼ 2.8 Hz); 8.15 (d, 1H, J ¼ 2.8 Hz). C NMR
(DMSO-d ) 27.4, 35.2, 84.5, 109.4, 131.6, 147.1, 152.1, 170.4.
6
(
1
150 mg, 1.02 mmol), Et
.32 mmol), EtOH (2.5 mL) and Et
gave the expected compound as a pale pink solid (235 mg, 93%
2
O (2.5 mL), oxalyl chloride (0.12 mL,
3
N (0.17 mL, 1.22 mmol) Filtration
5.1.27. tert-Butyl-3-(4-(1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-
pyrrol-3-yl)-1H-pyrazole-1-carboxylate (15a) and tert-Butyl-4,6-
dioxo-4,5,6,11-tetrahydro-2H-pyrazolo[4,3-a]pyrrolo[3,4-c]
carbazole-2-carboxylate (16a)
ꢃ
þ
þ
yield), mp 218 C. HRMS (ESI ) [M þ H] calcd 248.0923, found
1
2
4
1
48.0923. H NMR (acetone-d
6
) 1.38 (t, 3H, J ¼ 7.1 Hz); 3.86 (s, 1H);
.38 (q, 2H, J ¼ 7.1 Hz); 6.93 (dd, 1H, J ¼ 2.5 Hz and 8.8 Hz); 7.47 (d,
H, J ¼ 8.9 Hz); 7.83 (d, 1H, J ¼ 2.5 Hz); 8.37 (t, 1H, J ¼ 1.6 Hz); 11.23
To a solution of 14 (225 mg, 1.0 mmol) and 13a (350 mg,
ꢃ
1.1 mmol) in THF (5 mL) was added at 0 C, tBuOK (336 mg,
(
br. s, 1H). 13C NMR (acetone-d
6
) 13.9, 55.2, 61.5, 103.0, 112.2, 113.3,
3.0 mmol). After 12 h stirring, 37% HCl (1.2 mL) and AcOEt (10 mL)
were added. The organic layer was separated, washed with water
1
134,126.3, 131.3,138.0,156.0,163.3, 178.9. IR (KBr) 3159,1721,1621,
ꢀ
1
1475, 1208, 1129, 1025 cm
.
4
and dried over MgSO . Purification of the crude product by flash-
chromatography (CH
2
Cl
2
/MeOH 95:5) gave the expected
þ
5.1.23. tert-Butyl-3-(2-ethoxy-2-oxoacetyl)-1H-indole-1-
compound as a yellow solid (38 mg, 10% yield). MS (CI) [M þ H]
1
carboxylate (13a)
6
379.1 (100%) 279.1 (13.5%). H NMR (acetone-d ) 1.69 (s, 9H); 6.79
To a solution of ethyl 2-(1H-indol-3-yl)-2-oxoacetate [17]
(m, 1H); 6.95 (d, 1H, J ¼ 8.3 Hz); 7.09 (t, 1H, J ¼ 7.5 Hz); 7.33 (t, 1H,
J ¼ 7.2 Hz); 7.84 (s, 1H); 8.10 (d, 1H, J ¼ 9.0 Hz); 8.20 (s, 1H); 11.25
(br. s, 1H); 13.26 (br. s, 1H). IR (KBr) 3322, 1730, 1701, 1642, 1542,
(
500 mg, 2.30 mmol) anhydrous CH
tert-butyl-di-carbonate (990 mg, 4.60 mmol) and DMAP (5.6 mg,
.46 mmol). After 30 min stirring, 1 N HCl (2 mL) was added. The
organic layer was separated, dried over MgSO and concentrated to
give the expected compound as a pale pink solid (670 mg, 92%
2 2
Cl (2 mL) were added di-
ꢀ1
0
1454, 1350, 1150, 1090 cm . A solution of compound 15a (35 mg,
0.092 mmol) in CH CN (20 mL) was irradiated at 350 nm for 8 h.
After removal of the solvent, the residue was used without further
4
3

634
S. Deslandes et al. / European Journal of Medicinal Chemistry 54 (2012) 626e636
þ
þ
purification in the next step. HRMS (ESI ) [M þ H] calcd 377.1250,
6.25 (d, 1H, J ¼ 2.4 Hz); 6.91 (dd, 1H, J ¼ 9 Hz and 2.4 Hz); 7.96 (d,
13
found 377.1238.
1H, J ¼ 9 Hz); 8.13 (s, 1H); 11.24 (br. s, 1H); 13.27 (br. s, 2H). C NMR
DMSO-d ) 56.00, 106.94, 107.44, 110.87, 112.74 (2C), 113.34, 115.13,
122.47, 128.14, 133.11, 134.92, 138.16, 154.67, 169.96, 171.61. IR (KBr)
(
6
5
.1.28. tert-Butyl-3-(4-(5-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-
ꢀ
1
dihydro-1H-pyrrol-3-yl)-1H-pyrazole-1-carboxylate (15b) and tert-
Butyl-8-methyl-4,6-dioxo-4,5,6,11-tetrahydro-2H-pyrazolo[4,3-a]
pyrrolo[3,4-c]carbazole-2-carboxylate (16b)
3436, 3307, 1742, 1694, 1646, 1600, 1483, 1340, 1209 cm . HPLC: t
R
1.72 min.
Same procedure as for compound 15a involving 14 (171 mg,
6. Pharmacology
0
.76 mmol), 13b (171 mg, 0.84 mmol), tBuOK (255 mg, 2.28 mmol),
THF (5 mL), MS (DCI-NH ) of 15b 392.0 (45%), 359.0 (100%). A
solution of the crude compound in CH CN (15 mL) was irradiated at
50 nm for 8 h. After removal of the solvent, the crude product was
purified by flash-chromatography (CH Cl /MeOH 98:2) to give the
expected compound as a yellow solid (8.0 mg, 3% yield from 15b),
3
6.1. Determination of in vitro growth inhibition
3
3
The influence of each compound under study on the in vitro
growth rates of various cancer cell lines was determined using the
colorimetric MTT ((3-[4,5-dimethylthiazol-2yl])-diphenyl tetrazo-
lium bromide, Sigma, Belgium) assay as detailed previously
[19,21,22,24]. The use of the MTT colorimetric assay is based on the
capability of living cells to reduce the yellow product MTT (3-(4,5)-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a blue
product, formazan, by a reduction reaction occurring in the mito-
chondria. The number of living cells after 72 h of culture in the
presence (or absence: control) of the various compounds is directly
proportional to the intensity of the blue, which is quantitatively
measured by spectrophotometry (Biorad Model 680XR; Biorad,
Nazareth, Belgium) at a 570 nm wavelength (with a reference of
630 nm). Each experiment was carried out in six replicates.
The cell lines were incubated for 24 h in 96-microwell plates (at
a concentration of 10,000 to 40,000 cells/mL culture medium
depending on the cell type) to ensure adequate plating prior to cell
growth determination. The cells were cultured in RPMI (Invitrogen,
Merelbeke, Belgium) media supplemented with 10% heat inacti-
vated fetal calf serum (Invitrogen). All culture media were sup-
2
2
ꢃ
þ
mp >260 C. MS (CI) [M þ H] 408.1 (22%), 391.1 (70%), 308.0
1
(
100%), 291.1 (70%). H NMR (DMSO-d
J ¼ 9.0 Hz); 8.03 (d, 1H, J ¼ 8.1 Hz); 8.79 (s, 1H); 8.91 (s, 1H); 11.37
br. s, 1H); 14.20 (br. s, 1H). IR (KBr) 3348, 1757, 1740, 1708, 1636,
6
) 1.79 (s, 9H); 7.40 (d, 1H,
(
1
ꢀ
1
599, 1492, 1309, 1223, 1079, 800, 760 cm
.
5.1.29. tert-Butyl-3-(4-(5-methoxy-1H-indol-3-yl)-2,5-dioxo-2,5-
dihydro-1H-pyrrol-3-yl)-1H-pyrazole-1-carboxylate (15c) and tert-
Butyl-8-methoxy-4,6-dioxo-4,5,6,11-tetrahydro-2H-pyrazolo[4,3-a]
pyrrolo[3,4-c]carbazole-2-carboxylate (16c)
Same procedure as for compound 15a involving 14 (131 mg,
0
.58 mmol), 13C (222 mg, 0.64 mmol), tBuOK (195 mg, 1.74 mmol)
þ
and THF (2 mL). MS (CI) [M þ H] of 15c 409.0 (100%). A solution of
the crude product in CH
h. After removal of the solvent, this new crude product was used
in the next step.MS (DCI-NH ) of 16c 407.1 (100%); 124.1 (23%).
3
CN (12 mL) was irradiated at 350 nm for
8
3
5
.1.30. 3H-Pyrazolo[4,3-a]pyrrolo[3,4-c]carbazole-4,6(5H,11H)-
dione (5a)
To a solution of crude 16a (0.092 mmol) in CH
added TFA (4 mL, 51.6 mmol). After 4 h stirring, the solvent was
removed and a saturated solution of NaHCO (8 mL) was added to
the residue. Filtration gave quantitatively the expected compound
plemented with 4 mM glutamine, 100
m
g/mL gentamicin, and
penicillinestreptomycin (200 U/mL and 200
mg/mL; Invitrogen).
2
Cl
2
(4 mL) was
We have made use of seven human and one mouse cancer cell
lines. The seven human cancer cell lines include the OE21 esoph-
ageal cancer (obtained from the European Collection of Cell Culture
(ECACC); code 96062201), the A549 non-small-cell lung cancer
(obtained from the Deutsche Sammlung von Mikroorganismen und
Zellkulturen (DSMZ); code ACC107), the HS683 oligodendroglioma
(obtained from the American Type Culture Collection (ATCC); code
HTB-138), the U373 glioblastoma (ECACC; code 89081403), the PC-
3 prostate cancer (DSMZ; code ACC465), the MCF-7 breast cancer
(DSMZ; code ACC115) and the LoVo colon cancer (DSMZ; code
ACC350) cell lines, while the mouse cancer cell line relates to the
B16F10 melanoma (ATCC; code CRL-6475).
3
ꢃ
þ
þ
as a yellow solid (25.4 mg), mp >260 C. HRMS ((ESI ) [M þ H] )
1
calcd 276.2496, found 276.2492. H NMR (acetone-d
J ¼ 7.8 Hz); 7.51 (d, 1H, J ¼ 7.8 Hz); 7.69 (d, 1H, 7.8 Hz); 8.55 (s, 1H);
.90 (d, 1H, 7.8 Hz). 13C NMR (DMSO-d
) 108.20, 110.67, 112.12 (2C),
13.41, 120.85, 122.05, 124.05, 126.07, 132.32, 133.50, 137.81, 140.42,
6
) 7.34 (t, 1H,
8
1
6
167.12, 171.20. IR (KBr) 3437, 1687, 1642, 1438, 1208, 1139, 844,
ꢀ
1
R
725 cm . HPLC: t 1.49 min.
5
4
.1.31. 8-Methyl-3H-pyrazolo[4,3-a]pyrrolo[3,4-c]carbazole-
,6(5H,11H)-dione (5b)
Same procedure as for compound 5a involving 16b (5 mg,
6.2. Computer-assisted phase-contrast microscopy (quantitative
videomicroscopy)
0
2 2 3
.013 mmol), CH Cl (1 mL), TFA (1 mL,12.9 mmol), NaHCO (2 mL).
Filtration gave quantitatively the expected compound as a yellow
Quantitative videomicroscopy was employed to determine as
whether 4a, 9a and 9e displayed cytotoxic or rather cytostatic
effects in the human A549 NSCLC and the U373 GBM cell lines, as
detailed elsewhere [30,31]. Cancer cells were monitored for 72 h in
the absence (control) or the presence of each compound analyzed
at its MTT test-related IC50 growth inhibitory concentrations.
Movies were built on the obtained time-lapse image sequences and
enabled a rapid screening for cell viability [30,31]. Experiments
were conducted in triplicate.
ꢃ
þ
þ
solid (3.8 mg), mp >260 C. HRMS ((ESI ) [M þ H] ) calcd
1
2
91.0882, found 291.0888. H NMR (DMSO-d
6
) 2.50 (s, 3H); 7.31 (d,
1
H, J ¼ 8.7 Hz); 7.56 (d, 1H, 8.7 Hz); 8.53 (s, 1H); 8.67 (s, 1H); 11.06
13
(
2
(
br. s, 1H); 12.76 (br. s, 1H); 14.07 (br. s, 1H). C NMR (DMSO-d
1.85, 107.46, 110.61, 111.79 (2C), 122.03 (2C), 123.80, 127.61, 129.79
2C),132.99,137.91, 138.49,169.96, 171.53. IR (KBr) 3429,1739,1699,
6
)
ꢀ1
1638, 1478, 1370, 1207, 1141, 802 cm . HPLC: t
R
1.69 min.
5
4
.1.32. 8-Methoxy-3H-pyrazolo[4,3-a]pyrrolo[3,4-c]carbazole-
,6(5H,11H)-dione (5c)
6.3. Cell culture and checkpoint abrogation evaluation
Same procedure as for compound 5a involving crude 16c
ꢀ/ꢀ
(
(
(
0.58 mmol), CH
3
CN (15 mL). Purification by flash-chromatography
HCT116 p53
cells (a generous gift of B. Vogelstein) were
grown in DMEM (Invitrogen) supplemented with 10% FCS. Etopo-
side (Sigma) was applied to the cells for 1 h at a concentration of
CH Cl
2
2
/MeOH 95:5) gave the expected product as a yellow solid
13
ꢃ
þ
þ
5.0 mg, 3% yield from C), mp >260 C. HRMS (ESI ) [M þ H]
1
calcd 307.0831, found 307.0846. H NMR (DMSO-d
6
) 3.82 (s, 3H);
40 mM, before being washed extensively. Checkpoint recovery

S. Deslandes et al. / European Journal of Medicinal Chemistry 54 (2012) 626e636
635
experiment was performed essentially as described previously [6].
The AZD7762 Chk1 inhibitor was generously provided by Dr Sonya
Zabludoff (AstraZeneca). Cells were harvested and fixed with cold
inhibitors and identification of the structurally novel compound iso-
granulatimide, Cancer Res. 58 (1998) 5701e5706.
6] R.J. Andersen, M. Roberge, J. Sanghera, D. Leung, Preparation of granulatimide
derivatives for cancer treatment. International Patent (1999) WO99/47522, CA
131: 243451.
7] X. Jiang, B. Zhao, R. Britton, L.Y. Lim, D. Leong, J.S. Sanghera, B.B.S. Zhou, E. Piers,
R.J. Andersen, M. Roberge, Inhibition of Chk1 by the G2 DNA damage checkpoint
inhibitor isogranulatimide, Mol. Cancer. Ther. 3 (2004) 1221e1227.
[
[
7
0% ethanol. They were subsequently stained with either anti-
phospho H3 Ser-10 antibodies or the monoclonal 3.12.I.22 [28]
that were revealed using Alexa Fluor 488 conjugated antibodies.
DNA was stained with propidium iodide (Sigma). DNA content and
mitotic index were determined using an Accuri C6 flow cytometer.
[8] R. Boutros, V. Lobjois, B. Ducommun, CDC25 phosphatases in cancer cells: key
players? Good targets? Nat. Rev. Cancer 7 (2007) 495e507.
[
9] H. Hénon, E. Conchon, B. Hugon, S. Messaoudi, R.M. Golsteyn, M. Prudhomme,
Pyrrolocarbazoles as checkpoint 1 kinase inhibitors, Anti-Cancer Agents Med.
Chem. 8 (2008) 577e597.
6.4. Inhibition of kinases
[
10] S. Deslandes, S. Chassaing, E. Delfourne, Marine pyrrolocarbazoles and
analogues: synthesis and kinase inhibition, Mar. Drugs 7 (2009) 754e786.
Kinase activities were assayed in triplicate using Buffer D
[11] B. Hugon, B. Pfeiffer, P. Renard, M. Prudhomme, Synthesis of isogranulatimide
analogues possessing a pyrrole moiety instead of an imidazole heterocycle,
Tetrahedron Lett. 44 (2003) 3927e3930.
(
10 mM MgCl
2
, 1 mM EGTA (ethyleneglycoltetraacetic acid), 1 mM
ꢃ
DTT (dithiothreitol), 25 mM Tris/HCl, 50 mg/ml Heparin) at 30 C,
at a final ATP concentration of 15 mM. Blank values were subtracted
and activities expressed as % of the maximal activity in the absence
of inhibitors. Controls were performed with appropriate dilutions
of DMSO. IC50 values were calculated from doseeresponse curves.
The kinase peptide substrates were obtained from Proteogenix
[
12] V. Bocchi, G. Palla, High yield selective bromination and iodination of indoles
in N, N-dimethylformamide, Synthesis (1982) 1096e1097.
[13] B. Hugon, F. Anizon, C. Bailly, R.M. Golsteyn, A. Pierré, S. Léonce, J. Hickman,
B. Pfeiffer, M. Prudhomme, Synthesis and biological activities of iso-
granulatimide analogues, Bioorg. Med. Chem. 15 (2007) 5965e5980.
[
[
[
14] M. Murase, K. Watanabe, T. Kurihara, S. Tobinaga, Total synthesis of
(ꢂ)-plumbazeylanone, Chem. Pharm. Bull. 46 (1998) 889e892.
15] M.M. Faul, L.L. Winneroski, C.A. Krumrich, A new efficient method for the
synthesis of bisindolylmaleimides, J. Org. Chem. 63 (1998) 6053e6058.
16] E. Piers, R. Britton, R.J. Andersen, Improved synthesis of isogranulatimide, a G2
checkpoint inhibitor. Synthesis of didemnimide C, isodidemnimide A, neo-
didemnimide A, 17-methylgranulatimide and isogranulatimide A-C, J. Org.
Chem. 65 (2000) 530e535.
(
Oberhausbergen, France).
DYRK1A (rat, recombinant, expressed in E. coli as a GST fusion
protein) was purified by affinity chromatography on glutathione-
agarose and assayed using Woodtide (KKISGRLSPIMTEQ) (1.5 g/
P] ATP (3000 Ci/
l. After 30 min incuba-
m
3
3
assay) as a substrate, in the presence of 15 mM [g-
[
17] R.G. Jones, M.J. Mann, New methods of synthesis of
b-aminoethylpyrazoles,
mmol; 10 mCi/ml) in a final volume of 30
tion at 30 C, the reaction was stopped by harvesting onto P81
phosphocellulose papers (Whatman) using a FilterMate harvester
m
J. Am. Chem. Soc. 71 (1953) 4048e4052.
ꢃ
[18] M.-O. Contour-Galcéra, A. Sidhu, P. Plas, P. Roubert, 3-Thio-1,2,4-triazoles, novel
somatostatin sst /sst agonists, Bioorg. Med. Chem. Lett. 15 (2005) 3555e3559.
2
5
[
[
19] P. Dumont, L. Ingrassia, S. Rouzeau, F. Ribaucour, S. Thomas, I. Roland, F. Darro,
F. Lefranc, R. Kiss, The Amaryllidaceae isocarbostyril narciclasine induces
apoptosis by activation of the death receptor and/or mitochondrial pathways
in cancer cells but not in normal fibroblasts, Neoplasia 9 (2007) 766e776.
20] F. Branle, F. Lefranc, I. Camby, J. Jeuken, A. Geurts-Moespot, S. Sprenger,
F. Sweep, R. Kiss, I. Salmon, Evaluation of the efficiency of chemotherapy in
in vivo orthotopic models of human glioma cells with and without 1p19q
deletions and in C6 rat orthotopic allografts serving for the evaluation of
surgery combined with chemotherapy, Cancer 95 (2002) 641e655.
21] L. Ingrassia, F. Lefranc, J. Dewelle, L. Pottier, V. Mathieu, S. Spiegl-Kreinecker,
S. Sauvage, M. El Yazidi, M. Dehoux, W. Berger, E. Van Quaquebeke, R. Kiss,
Structure-activity relationship analysis of novel derivatives of narciclasine (an
Amaryllidaceae isocarbostyril derivative) as potential anticancer agents,
J. Med. Chem. 52 (2009) 1100e1114.
[22] V. Mathieu, C. Pirker, E. Martin de Lassalle, M. Vernier, T. Mijatovic, N. De
Neve, J.F. Gaussin, M. Dehoux, F. Lefranc, W. Berger, R. Kiss, The sodium pump
alpha1 subunit: a disease progression-related target for metastatic melanoma
treatment, J. Cell. Mol. Med. 13 (2009) 3960e3972.
[23] T. Mijatovic, V. Mathieu, J.F. Gaussin, N. De Neve, F. Ribaucour, E. Van Qua-
quebeke, P. Dumont, F. Darro, R. Kiss, Cardenolide-induced lysosomal
membrane permeabilization demonstrates therapeutic benefits in experi-
mental human non-small cell lung cancers, Neoplasia 8 (2006) 402e412.
(
Packard) and were washed in 1% phosphoric acid. Scintillation
fluid was added and the radioactivity measured in a Packard
counter.
CLK1 (mouse, recombinant, expressed in E. coli as a GST fusion
protein) was assayed with RS peptide (GRSRSRSRSRSR) (1
assay).
mg/
CDK5/p25 (human, recombinant) was prepared as previously
described [32]. Its kinase activity was assayed in buffer C, with 1 mg
histone H1/ml.
[
GSK-3
using
a/
b
(porcine brain, native) was assayed in Buffer A and
GSK-3 specific substrate (GS-1:
a
YRRAAVPPSPSLSRHSSPHQSpEDEEE) (pS stands for phosphorylated
serine) [33].
CK1d/ε (porcine brain, native) was assayed in three-fold diluted
buffer C, using 25
mM CKS peptide (RRKHAAIGpSAYSITA), a CK1-
specific substrate [34]
[
24] C. Bruyère, C. Lonez, A. Duray, S. Cludts, J.M. Ruysschaert, S. Saussez, P. Yeaton,
R. Kiss, T. Mijatovic, Considering temozolomide as a novel potential treatment
for esophageal cancer, Cancer 117 (2011) 2004e2016.
Acknowledgments
[
25] http://www.sanger.ac.uk/perl/genetics/CGP/core_line_viewer?action¼cell_lines.
This research was supported by project grants from the Can-
céropole Grand-Ouest, the Association pour la Recherche sur le
Cancer (ARC-1092), the Ligue Nationale contre le Cancer (Comité
Grand-Ouest) (L.M.).
[26] G. Van Goietsenoven, A. Andolfi, B. Lallemand, A. Cimmino, D. Lamoral-Theys,
T. Gras, A. Abou-Donia, J. Dubois, F. Lefranc, V. Mathieu, A. Kornienko, R. Kiss,
A. Evidente, Amaryllidaceae alkaloids belonging to different structural
subgroups display activity against apoptosis-resistant cancer cells, J. Nat. Prod.
73 (2010) 1223e1227.
[
27] G. Van Goietsenoven, V. Mathieu, A. Andolfi, A. Cimmino, F. Lefranc, R. Kiss,
A. Evidente, In vitro growth inhibitory effects of cytochalasins and derivatives
in cancer cells, Planta Med 77 (2011) 711e717.
References
[
28] C. Didier, M. Quaranta, C. Cavelier, M.-O. Galcera, C. Demur, G. Laurent,
S. Manenti, B. Ducommun, G2/M checkpoint stringency is a key parameter in
the sensitivity of AML cells to genotoxic stress, Oncogene 27 (2008)
3811e3820.
[1] K. Vermeulen, D.R. Van Bockstaele, Z.N. Berneman, The cell cycle: a review of
regulation, deregulation and therapeutic target in cancer, Cell Prolif. 36 (2003)
131e149.
[
[
[
2] A.R. Cuddihy, M.J. O’Connell, Cell cycle responses to DNA damage in G2,
[29] G. Ferry, A. Studeny, C. Bossard, P.M. Kubara, D. Zeyer, J.P. Renaud, P. Casara,
G. De Nanteuil, M. Wierzbicki, B. Pfeiffer, M. Prudhomme, S. Leonce, A. Pierré,
J.A. Boutin, R.M. Golsteyn, Characterization of novel kinase 1 inhibitors by
in vitro assays and in human cancer cells treated with topoisomerase inhib-
itors, Life Sci. 89 (2011) 259e268.
[30] C. Decaestecker, O. Debeir, P. Van Ham, R. Kiss, Can anti-migratory drugs be
screened in vitro? A review of 2D and 3D assays for the quantitative analysis
of cell migration, Med. Res. Rev. 27 (2007) 149e176.
Cytology 222 (2003) 99e140.
3] C.H. McGowan, Running into problems: how cells cope with replicating
damaged DNA, Mutat. Res. 532 (2003) 75e84.
4] R.G.S. Berlinck, R. Britton, E. Piers, L. Lim, M. Roberge, R. Moreira da Rocha,
R. Andersen, Granulatimide and isogranulatimide, aromatic alkaloids with G2
checkpoint inhibition activity isolated from the brazilian ascidian Didemnum
granulatum: structure elucidation and synthesis, J. Org. Chem. 63 (1998)
9
850e9856.
[31] N. Belot, R. Pochet, C.W. Heizmann, R. Kiss, C. Decaestecker, Extracellular
S100A4 stimulates the migration rate of astrocytic tumor cells by modifying
the organization of their actin cytoskeleton, Biochim. Biophys. Acta 1600
(2002) 74e83.
[
5] M. Roberge, R.G.S. Berlinck, L. Xu, H.J. Anderson, L.Y. Lim, D. Curman,
C.M. Stringer, S.H. Friend, P. Davies, I. Vincent, S.J. Haggarty, M.T. Kelly,
R. Britton, E. Piers, R.J. Andersen, High-throughput assay for G2 checkpoint

636
S. Deslandes et al. / European Journal of Medicinal Chemistry 54 (2012) 626e636
[
32] S. Leclerc, M. Garnier, R. Hoessel, D. Marko, J.A. Bibb, G.L. Snyder, P. Greengard,
[33] A. Primot, B. Baratte, M. Gompel, A. Borgne, S. Liabeuf, J.L. Romette,
F. Costantini, L. Meijer, Purification of GSK-3 by affinity chromatography on
immobilized axin, Protein Expr. Purif. 20 (2000) 394e404.
J. Biernat, E.-M. Mandelkow, G. Eisenbrand, L. Meijer, Indirubins inhibit
glycogen synthase kinase-3b and CDK5/P25, two protein kinases involved in
abnormal tau phosphorylation in Alzheimer’s disease: a property common to
most cyclin-dependent kinase inhibitors ? J. Biol. Chem. 276 (2001) 251e260.
[34] J. Reinhardt, Y. Ferandin, L. Meijer, Purification of CK1 by affinity chroma-
tography on immobilised axin, Protein Expr. Purif 54 (2007) 101e109.