Use of copper(I) catalyzed azide alkyne cycloaddition (CuAAC) for the preparation of conjugated pyrrolo[2,3-a]carbazole Pim kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.02.009

We have previously demonstrated that pyrrolo[2,3-a]carbazole-3- carbaldehydes are potent Pim kinase inhibitors with in vitro antiproliferative activities. In the present study, we report the synthesis of new pyrrolocarbazoles substituted at the N-10 position. When their ability to inhibit Pim kinase activities were evaluated in in vitro assays, we observed that this nitrogen atom can be substituted without loss of Pim-1 and Pim-3 inhibitory potencies. Moreover, when we added a fluorescent dansyl group (compound 13), we were able to show that 13 penetrates the plasma membrane and enters the cytoplasm.

Full text of DOI:10.1016/j.ejmech.2012.02.009

European Journal of Medicinal Chemistry 50 (2012) 304e310
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Use of copper(I) catalyzed azide alkyne cycloaddition (CuAAC) for the preparation
of conjugated pyrrolo[2,3-a]carbazole Pim kinase inhibitors
,
b
,
,
b
,
,
,
,b
Boris Letribot a 1, Rufine Akué-Gédu a 1, Niina M. Santio c d, Malika El-Ghozzi a b, Daniel Avignant a
,
Federico Cisnetti a b, Päivi J. Koskinen c d, Arnaud Gautier a b, Fabrice Anizon a b, Pascale Moreau a
,
,
,
,
,b,
*
a Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, 63000 Clermont-Ferrand, France
b CNRS, UMR 6296, ICCF, BP 80026, 63171 Aubière, France
c Turku Center for biotechnology, University of Turku and Abo Akademi University, Finland
d Department of Biology, University of Turku, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
We have previously demonstrated that pyrrolo[2,3-a]carbazole-3-carbaldehydes are potent Pim kinase
inhibitors with in vitro antiproliferative activities. In the present study, we report the synthesis of new
pyrrolocarbazoles substituted at the N-10 position. When their ability to inhibit Pim kinase activities were
evaluated in in vitro assays, we observed that this nitrogen atom can be substituted without loss of Pim-1
and Pim-3 inhibitory potencies. Moreover, when we added a fluorescent dansyl group (compound 13), we
were able to show that 13 penetrates the plasma membrane and enters the cytoplasm.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 20 January 2012
Received in revised form
2 February 2012
Accepted 3 February 2012
Available online 10 February 2012
Keywords:
Pyrrolo[2,3-a]carbazole-3-carbaldehyde
Pim kinase inhibition
CuAAC (Copper(I) Catalyzed Azide Alkyne
Cycloaddition)
1. Introduction
All these data indicate that Pim kinases are important signalling
proteins in tumour biology that could be targeted for the devel-
The Pim kinases (Pim-1, -2, -3) form a small sub-family of three
closely related mammalian serine/threonine protein kinases within
the CAMK (calcium/calmodulin-dependent protein kinase) group.
These enzymes are constitutively active due to a unique hinge
structure within their kinase domains [1] and are known to inhibit
apoptosis and promote cell cycle progression via phosphorylation of
multiple substrates [2]. Normally their expression levels are tightly
regulated, but both Pim-1 and Pim-2 are often found overexpressed
in human haematopoietic malignancies and in prostate cancer,
while elevated levels of Pim-3 have been shown to be associated
with several types of solid tumours [2]. The oncogenic properties of
Pim kinases were originally demonstrated in transgenic mice,
where their overexpression in B and T cells resulted in development
of lymphomas [3]. Similar results have also been observed from
a transgenic mouse model of human prostate cancer [4].
opment of new antitumour drugs [5].
Recently, we described the synthesis and biological activities of
pyrrolo[2,3-a]carbazole-3-carbaldehyde A [6]. When tested at
10
mM concentration towards a panel of 66 protein kinases,
compound A was found to be a potent and selective inhibitor of the
Pim kinase family members. We also revealed its crystal structure
in complex with Pim-1 (Fig. 1). The structureeactivity relationship
study undertaken with compound A and its derivatives demon-
strated that these compounds are potent inhibitors of Pim kinases
and attractive molecules for drug development, especially to inhibit
the recently demonstrated invasiveness of Pim-overexpressing
cancer cells [6e8].
As shown in Fig. 1, compound A was found to occupy the
ATP-binding site of Pim-1. However, in contrast to typical ATP-
mimetic inhibitors, no hydrogen bond was formed with the hinge
region. The planar pyrrolocarbazole scaffold was inserted into the
ATP-binding cleft with NH groups oriented away from the hinge
region. The only hydrogen bond involved the aldehyde and the
conserved active site lysine, Lys67. Moreover, the C2eC3 region of
the inhibitor formed an aromatic stacking interaction with the
P-loop phenylalanine Phe49.
* Corresponding author. CNRS, UMR 6296, ICCF, BP 80026, 63171 Aubière, France.
Tel.: þ33 0 4 73 40 79 63; fax: þ33 0 4 73 40 77 17.
E-mail address: Pascale.MOREAU@univ-bpclermont.fr (P. Moreau).
Both authors contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.009

B. Letribot et al. / European Journal of Medicinal Chemistry 50 (2012) 304e310
305
a
+
N
H
N
N
N
N
N
SO2Ph
SO2Ph
(CH2)4
2
Br(CH2)4
1
3 (84%)
d
b, c
CHO
N
N
R
N
N
(CH2)4
N3(CH2)4
4 (59%)
R = SO2Ph, 5 (82%)
R = H, 6 (84%)
e
f, c
CHO
Fig. 1. Binding of pyrrolocarbazole-3-carbaldehyde A to the ATP-binding pocket of
Pim-1. The hydrogen bond is indicated in broken blue line. (PDB coordinate file 3JPV).
Molecular graphics images were produced using UCSF Chimera [9]. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
N
N
H
N3(CH2)4
7 (92%)
Reagents and conditions: (a) 1,4-dibromobutane, NaH (b) POCl3, DMF,
MW (c) NaOH (d) NaN3, DMF (e) aq. NaOH, MeOH (f) POCl3, DMF
In this study, various substituents were introduced using copper (I)
catalyzed azide alkyne cycloaddition (CuAAC) ꢀ a so-called “click
chemistry” reaction ꢀ at theN-10 positionwhichis not involved in the
direct interaction between pyrrolo[2,3-a]carbazole-3-carbaldehydes
and the ATP-binding site of Pim kinases. These substituents are
either hydroxyl/ammonium groups to improve water solubility of
thesecompounds ora fluorescentmoiety to determine the subcellular
localisation of the corresponding conjugate.
Scheme 1. Preparation of compounds 4, and 7.
and solvents (including hydrophobic media required to solubilise
substrates of pharmaceutical interest such 7) without the need of
reducing reagents [11,12]. We previously reported the preparation
and the reactivity of several complexes related to [CuCl(SIMes)(1,10-
Phen)] [11] and [CuCl(SIMes)(4,7-Cl-Phen)] [12]. Notwithstanding
their general and practical interest, only a few examples of struc-
tural characterisation of such complexes were reported to date [13].
As the three-dimensional arrangement of [CuCl(SIMes)(4,7-Cl-
Phen)] complex has not been described previously, and as we now
succeeded in getting X-ray diffraction-suitable monocrystals after
exhaustive crystallizations assays, we now report the structural
characterisation of this complex (Fig. 2, Table 1).
2. Chemistry
In order to synthesize the target molecules using a straightfor-
ward and modular strategy, we took advantage of CuAAC to connect
either hydrophilic substituents or a dansyl fluorescent label.
Consequently, we started the preparation of azide 7 (Scheme 1),
which was synthesized in four steps from compound 1. In an
initial attempt, compound 1 was allowed to react with 1,4-
dibromobutane in the presence of tBuOK. This led to a mixture of
products 2 and 3, which were isolated in 40% and 45% yields,
respectively. The formation of side-product 2 was probably due to
the nucleophilic attack of tBuOK or the generated tBuOH to the
benzenesulfonyl group, leading to a cleavage of the N1-protecting
group and subsequent cyclisation. Synthesis of compound 2 was
optimized (90% yield) by reacting 1 with 1,4-dibromobutane in the
presence of tBuONa in toluene at 100 ꢁC. Gratefully, the replacement
of alkoxydes by the non nucleophilic base NaH resulted in the
production of 3 in 84% yield. To get an insight into the effect on the
biological activity of the substitution of both nitrogen atoms by alkyl
chains, compound 4, the formylated analogue of 2, was prepared in
59% yield using a VilsmeiereHaack reaction. The azide derivative 7
was obtained by substitution of the bromine atom of 3 using sodium
azide and subsequent deprotection/formylation sequence.
As displayed in Table 1, the bond distances and angles fall into the
range of what was found for the related catalyst [CuCl(SIMes)(1,10-
Phen)]. The introduction of the two chlorine atoms on the
8 R = OH
9 R = N+(CH3)2 CH2CH2OH, Cl
CHO
+
-
R
N
N
H
10 R = NHdansyl
N3(CH2)4
7
a
c
b
CHO
CHO
CHO
N
N
H
N
N
H
N
N
H
(CH2)4
N
(CH2)4
N
(CH2)4
N
N
N
TFA
N
N
N
N
N
OH
Having azide 7 in hands, we examined its CuAAC reaction with
several alkynes bearing hydroxymethyl, choline (ammonium) and
dansyl groups (Scheme 2).
HN
11
SO2
OH
12
13
Room temperature reaction of 7 with propargyl alcohol 8
catalyzed by
5 mol% of Cu(I)/THPTA (tris(hydroxypropyl)tri-
NMe2
Reagents and conditions:
azolylmethyl-amine)/ascorbic acid in methanol was unsuccessful
[10]. Therefore, we used [CuCl(1,3-bis(2,4,6-trimethylphenyl)imi-
dazolidin-2-ylidene)(4,7-dichloro-1,10-phenanthroline)] ([CuCl(-
SIMes)(4,7-Cl-Phen)]), an air stable Cu(I) complex, that belongs to
the metaleNHC family. These copper(I) catalysts were introduced to
efficiently catalyse CuAAC reactions on a large range of temperature
(a) propargyl alcohol, [CuCl(SIMes)(4,7-Cl-Phen)] (7 mol%), MeOH, 92%
(b) N-(2-hydroxyethyl)-N,N-dimethylprop-2-yn-1-aminium chloride,
[CuCl(4,7-Cl-Phen)(SIMes)] (13 mol%), MeOH, 49%
(c) 5-(dimethylamino)-N-(2-propynyl)-1-naphthalenesulfonamide,
[CuCl(4,7-Cl-Phen)(SIMes)] (6 mol%), MeOH, 92 %.
Scheme 2. Preparation of compounds 11, 12 and 13.

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B. Letribot et al. / European Journal of Medicinal Chemistry 50 (2012) 304e310
Fig. 2. ORTEP view of [CuCl(SIMes)(4,7-Cl-Phen)] (nitrogen: blue, chloride: green,
copper: orange). CCDC ID: 46715. C33H32Cl3Cu1N4, monoclinic, space group C2/c,
Fig. 3. Fluorescence emission and excitation spectra of dansyl conjugate 13 recorded in
DMSO/0.05 mol Lꢀ1 pH ¼ 7.45 phosphate buffer v/v 9:1.
a
¼
31.832(3) Å,
b
¼
13.2828(13) Å,
c
¼
16.6462(16) Å,
s
b
¼
92.714(5)ꢁ,
V ¼ 7030.4(12) Å3, T ¼ 293(2) K, Z ¼ 8, Final R (I > 2
(I)): R1 ¼ 0.0492, wR2 ¼ 0.0463,
s ¼ 1.0978. (For interpretation of the references to colour in this figure legend, the
3. Results and discussion
reader is referred to the web version of this article.)
The kinase inhibitory potencies of compounds 4, 7, 11, 12, and 13
were evaluated at 10 mM concentrations in duplicate assays against
all three Pim family kinases, as previously described [15] and the
percentages of residual kinase activities are reported in Table 2. IC50
values were determined when the remaining kinase activity was
phenanthroline heterocycle resulted in the shortening of CueCl
distance, indicating a lower electron donation of 4,7-dichloro-1,10-
phenanthroline compared to that of 1,10-phenanthroline. The
latter distance is still longer than the one found in [CuCl(SIMes)].
This is of importance for a CuAAC process because the CueCl bond
needs to be broken to enter the catalytic cycle (see discussion in ref
[11]). On the other hand, the resulting lower electron density at
the metal centre could explain the very high stability of
[CuCl(SIMes)(4,7-ClePhen)] in the presence of air.
The coupling between 7 and propargyl alcohol 8 using 7 mol% of
[CuCl(SIMes)(4,7-ClePhen)] led to complete conversion of 7 into
the expected triazole 11 that was further isolated after column
chromatography in 92% yield (Scheme 2). When azide 7 was used
for the preparation of the choline derivative 12, the low solubility of
N-(2-hydroxyethyl)-N,N-dimethylprop-2-yn-1-aminium chloride 9
prompted us to perform the reaction in boiling methanol, leading
to a total conversion of the starting material e thus highlighting the
catalyst’s robustness. Compound 12 (trifluoroacetate salt) was
isolated in 49% yield after purification by reverse phase HPLC. In
both cases, the aqueous solubility of compounds 11 and 12 was
greatly increased as compared to A. Finally, pyrrolocarbazole 7 was
labelled with 10 in similar conditions, leading to compound 13 in
92% yield. Compound 13 displayed a near-UV excitation band at
lexc ¼ 322 nm and, under excitation at the latter wavelength, its
emission spectrum showed a maximum at lem ¼ 501 nm (Fig. 3).
These spectral features are typical of dansyl moieties in aqueous
solutions [14]. No significant fluorescent emission was observed
when A was studied in the same conditions.
less than 10% when the compounds were tested at 10 mM.
As evident from Table 2, the results obtained for compound 4
indicate that potent inhibition of Pim kinases is compatible with
the substitution of both nitrogen atoms. Whereas Pim-1 inhibitory
potency of compound 4 was greatly enhanced, the activities against
the two other isoforms were similar to those of compound A.
However, this feature should be verified with a larger panel of 1,10-
disubstituted analogues.
Regarding compound 7, interesting submicromolar inhibitory
potencies have been measured towards both Pim-1 and Pim-3.
Compounds bearing a 1,2,3-triazole moiety 11e13 exhibited
similar inhibitory potencies against both Pim-1 and Pim-3.
Compound 11e13 inhibited Pim-1 in the same range as compound
A whereas they were less active towards Pim-3. Nevertheless, the
dansyl derivative 13 was slightly less potent than the non-
fluorescent derivatives.
Except for compound 4, all the other tested derivatives were less
active towards Pim-2 than the reference compound A, suggesting
that the substitution of only one of the two nitrogen atoms
(N-10 position) of the pyrrolocarbazole scaffold is detrimental for
Pim-2 kinase inhibition.
Altogether, these results indicate that the N-10 position of the
pyrrolocarbazole scaffold can be substituted by various groups
without abolishing Pim-1 inhibition, but that the potency against
Table 1
Bond lengths and angles of interest.
Table 2
Bonds lengths (Å)
In vitro inhibitory potencies towards Pim kinases of compounds A, 4, 7, 11e13:
percentage of residual activities when tested at 10
Not determined.
mM (IC50 in mM in brackets). nd:
CueCl
CueN3
CueN4
CueC1
[CuCl(SIMes)(4,7-ClePhen)]
[CuCl(SIMes)(1,10-Phen)]
[CuCl(SIMes)]
2.320 (2)
2.347 (2)
2.099 (1)
2.114 (4)
2.114 (5)
2.127 (4)
2.128 (5)
1.905 (4)
1.916 (7)
1.882 (4)
Compounds % of Pim kinase residual activities
Pim-1
Pim-2
Pim-3
Bond angles (ꢁ)
A
4
7
11
12
13
2 ꢂ 0.4 (0.12 ꢂ 0.01) 7 ꢂ 1 (0.51 ꢂ 0.23) 1 ꢂ 5 (0.01 ꢂ 0.00)
7 ꢂ 1 (0.008 ꢂ 0.001) 6 ꢂ 2 (0.35 ꢂ 0.01) 1 ꢂ 0 (0.013 ꢂ 0.002)
N3eCueN4 N4eCueCl N3eCueCl C1eCueCl
9 ꢂ 4 (0.105 ꢂ 0.001) 22 ꢂ 1 (nd)
8 ꢂ 2 (0.315 ꢂ 0.154) 36 ꢂ 3(nd)
9 ꢂ 2 (0.327 ꢂ 0.01) 65 ꢂ 9 (nd)
14 ꢂ 4 (0.108 ꢂ 0.037)
23 ꢂ 9 (0.235 ꢂ 0.023)
28 ꢂ 4 (0.49 ꢂ 0.04)
38 ꢂ 6 (nd)
[CuCl(SIMes)(4,7-ClePhen)] 76.6 (2)
99.5 (1)
99.9 (1)
99.2 (1)
102.0 (2)
115.9 (2)
120.5 (2)
178.5 (3)
[CuCl(SIMes)(1,10-Phen)]
[CuCl(SIMes)]
77.4 (2)
17 ꢂ 3 (nd)
66 ꢂ 8 (nd)

B. Letribot et al. / European Journal of Medicinal Chemistry 50 (2012) 304e310
307
other Pim family members is often reduced by these substitutions.
More particularly, we have demonstrated that the introduction of
diversely substituted alkyl-1,2,3-triazole moieties is permitted for
Pim-1 inhibition.
To analyze the ability of the fluorescent dansyl conjugate 13 to
enter into the cells and to visualize its subcellular localization, PC-3
prostate cancer cells grown on coverslips were treated for 24 h with
400 (1H: 400 MHz, 13C: 100 MHz), or a Bruker AVANCE 500 (1H:
500 MHz), are reported in ppm using the solvent residual peak as an
internal standard; the following abbreviations are used: singlet (s),
doublet (d), broad doublet (br d), triplet (t), quintet (quint), doublet
of doublet (dd), doublet of doublet of doublet (ddd), multiplet (m),
broad signal (br s). High resolution mass spectra (ESIþ) were
determined on a high resolution Micro Q-Tof apparatus (CRMP,
Université Blaise Pascal, Clermont-Ferrand, France). Chromato-
graphic purifications were performed by column chromatography
either 10 mM compound A or 13 or with DMSO (0,1%). When cell
samples were analysed by fluorescence microscopy, both DMSO
and compound A displayed hardly any autofluorescence, while
a bright fluorescent signal was detected in cells treated with
compound 13 (Fig. 4A). Interestingly, 13 localized into the cyto-
plasm of the cells (Fig. 4B), suggesting that it could penetrate the
plasma membrane, but was unable to enter the nuclei.
using 40e63
HPLC system (Prepstar 218 pump, Propstar 335 PDA detector, C18
250 ꢃ 41.4 mm 8 m column). Reactions were monitored by TLC
mm silica gel or by preparative HPLC through a Varian
m
using fluorescent silica gel plates (60 F254 from Merck). Melting
points were measured on a Reichert microscope and are uncor-
rected. Fluorescence excitation and emission spectra were recorded
on a Cary eclipse spectrophotometer (sample concentration:
10ꢀ5 mol Lꢀ1, 5 nm emission and excitation slits). [CuCl(SIMes)(4,7-
ClePhen)] was prepared on a 10 g scale by scaling-up a previously
described method [12]. Single crystals (purple needles) were ob-
In
conclusion,
new
pyrrolocarbazole-3-carbaldehydes,
substituted at the N-10 position, were synthesized and evaluated
for their ability to inhibit Pim kinases in in vitro assays. The results
obtained indicate that this nitrogen atom can be substituted
without abolishing inhibitory potency towards Pim-1 and Pim-3.
More particularly, Pim-1/Pim-3 inhibition is conserved when the
N-10 position of the pyrrolocarbazole scaffold is substituted by
alkyl-1,2,3-triazole moieties obtained by CuAAC. Furthermore, the
results with the dansyl conjugate 13 indicate that fluorescent labels
can be added to the compounds without significant loss of inhibi-
tory potency against Pim-1. They can also be used to analyse
the membrane permeability and subcellular localisation of the
compounds. Interestingly, compound 13 was able to enter the
cytoplasm, but not the nuclei, suggesting that it might be a useful
research tool to distinguish between nuclear and cytoplasmic
functions of Pim. Overall, we can envisage to use the CuAAC for the
N-10 position functionalisation of this series in order to prepare
novel Pim inhibitors.
tained by slow evaporation of a concentrated (w0.05 g mLꢀ1
)
solution of this compound in dichloromethane. The X-ray diffrac-
tion data were recorded on a Bruker APEX-II CCD diffractometer
using MoKaradiation.
4.1.2. 4,5,6,7-Tetrahydroindolo[1,2,3-fg]pyrrolo[3,2,1-kl][1,6]
benzodiazocine 2
A mixture of compound 1 (50 mg, 0.144 mmol), tBuONa (69 mg,
0.72 mmol) and 1,4-dibromobutane (70 mL, 0.59 mmol) in toluene
(5 mL) was stirred in an oil bath heated at 100 ꢁC for 15 h. After
cooling, water was added and the mixture was extracted with
EtOAc. The assembled organic fractions were washed with brine
and dried over MgSO4. After evaporation, the residue was purified
by column chromatography (cyclohexane/EtOAc, 95:5) to give
34 mg of compound 2 (0.131 mmol, 90%) as a beige solid.
Mp ¼ 132e134 ꢁC; IR (ATR): 1620, 1602 cmꢀ1; 1H NMR (400 MHz,
DMSO-d6): 2.01e2.07 (4H, m), 4.52e4.59 (4H, m), 6.57 (1H, d,
J ¼ 3 Hz), 7.18 (1H, ddd, J1 ¼ 8 Hz, J2 ¼ 7 Hz, J3 ¼ 1 Hz), 7.35e7.39
(2H, m), 7.39 (1H, d, J ¼ 8.5 Hz), 7.64 (1H, d, J ¼ 8 Hz), 7.80 (1H,
d, J ¼ 8.5 Hz), 8.08 (1H, d, J ¼ 7.5 Hz); 13C NMR (100 MHz, DMSO-
d6): 25.6, 25.8, 42.1, 45.9 (CH2), 101.9, 109.4, 112.0, 113.0, 118.9 (2C),
123.8, 129.7 (CHarom), 117.4, 122.1, 123.6, 127.8, 128.8, 140.0 (Carom);
HRMS (ESIþ) calcd for C18H17N2 (M þ H)þ 261.1392, found 261.1403.
4. Experimental
4.1. Chemistry
4.1.1. General
Starting materials were obtained from commercial suppliers and
used without further purification. Solvents were distilled prior to
use. IR spectra were recorded on a Shimadzu FTIR-8400S spec-
trometer (in cmꢀ1). NMR spectra, performed on a Bruker AVANCE
Fig. 4. (A) PC-3 prostate cancer cells grown on coverslips were treated for 24 h with either 10 mM compound A or 13 or with DMSO (0,1%). Bright Field (BF) and fluorescent (DAPI)
pictures were taken from fixed cell samples. Mean intensity values were measured from unprocessed DAPI-filtered images. Shown are background-corrected means from three
regions in two parallel samples. (B) For visualization of the subcellular localization of fluorescence, the grey-scale images were recoloured with cyan. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)

308
B. Letribot et al. / European Journal of Medicinal Chemistry 50 (2012) 304e310
4.1.3. 10-(4-Bromobutyl)-1-(phenylsulfonyl)-1,10-dihydropyrrolo
[2,3-a]carbazole 3
HRMS (ESIþ) calcd for C24H21N5NaO2S (M þ Na)þ 466.1314, found
466.1320.
To a mixture of compound 1 (1.00 g, 2.89 mmol) and dibro-
mobutane (5.4 mL) was added sodium hydride (60% in mineral oil,
460 mg, 11.5 mmol). The mixture was stirred at 100 ꢁC for 4 h and
the mixture was poured into a 1 M aqueous HCl solution (100 mL).
After extraction with EtOAc (3 ꢃ 80 mL), the assembled organic
fractions were dried over MgSO4, evaporated and the residue was
purified by column chromatography (EtOAc/cyclohexane, from
0:100 to 2:98) to give 1.16 g of compound 3 (2.41 mmol, 83%) as
a grey solid, and trace amount of compound 2. Mp ¼ 122e124 ꢁC; IR
4.1.6. 10-(4-Azidobutyl)-1,10-dihydropyrrolo[2,3-a]carbazole 6
To a solution of compound 5 (45 mg, 0.101 mmol) in methanol
(14 mL) was added a 5 M aqueous NaOH solution (2 mL). The
mixture was refluxed for 4 h. After evaporation of methanol under
reduced pressure, the mixture was extracted with CH2Cl2
(3 ꢃ 20 mL). The assembled organic fractions were dried over
MgSO4 and evaporated under reduced pressure. The residue was
purified by column chromatography (EtOAc/cyclohexane 2:8) to
give 26 mg of compound 6 (0.086 mmol, 84%) as a beige solid.
Mp > 250 ꢁC (decomposition); IR (ATR): 3421, 3380, 2093, 1650,
1614, 1560 cmꢀ1; 1H NMR (400 MHz, DMSO-d6): 1.52e1.61 (2H, m),
1.77e1.86 (2H, m), 3.29 (2H, t, J ¼ 7 Hz), 4.75 (2H, t, J ¼ 7 Hz),
6.63e6.65 (1H, m), 7.17 (1H, t, J ¼ 7.5 Hz), 7.36 (1H, t, J ¼ 7.5 Hz), 7.38
(1H, d, J ¼ 8 Hz), 7.42 (1H, t, J ¼ 2.5 Hz), 7.64 (1H, d, J ¼ 8 Hz), 7.76
(1H, d, J ¼ 8 Hz), 8.07 (1H, d, J ¼ 7.5 Hz), 11.36 (1H, br s, NH); 13C
NMR (100 MHz, DMSO-d6): 25.7, 27.6, 43.3, 50.4 (CH2), 102.9, 109.3,
112.2, 112.4, 118.7, 119.1, 123.5, 124.2 (CH), 116.3, 120.9, 123.7, 127.2,
127.6, 138.7 (C); HRMS (ESIþ) calcd for C18H17N5Na(M þ Na)þ
326.1382, found 326.1395.
(ATR): 1470, 1448, 1431, 1379, 1368, 1337, 1325 cmꢀ1 1H NMR
;
(400 MHz, DMSO-d6): 1.23e1.32 (2H, m), 1.39e1.49 (2H, m), 3.25
(2H, t, J ¼ 6.5 Hz), 4.95 (2H, t, J ¼ 7 Hz), 6.93 (1H, d, J ¼ 3.5 Hz), 7.17
(2H, d, J ¼ 8 Hz), 7.22e7.31 (4H, m), 7.51 (2H, t, J ¼ 8 Hz), 7.53 (1H, d,
J ¼ 3.5 Hz), 7.79 (1H, d, J ¼ 8 Hz), 8.12 (1H, d, J ¼ 8 Hz), 8.21 (1H, d,
J ¼ 7.5 Hz); 13C NMR (100 MHz, DMSO-d6): 26.4, 29.4, 34.1, 45.1
(CH2), 111.7, 113.3, 118.4, 119.1, 120.0, 120.2, 125.7, 127.0 (2C), 128.5
(2C), 131.4, 134.2 (CHarom), 123.0, 123.4, 124.1, 132.8, 133.4, 133.5,
141.7 (Carom); HRMS (ESIþ) calcd for C24H2279BrN2O2SBr (M þ H)þ
481.0585, found 481.0602.
4.1.4. 4,5,6,7-Tetrahydroindolo[1,2,3-fg]pyrrolo[3,2,1-kl][1,6]
benzodiazocine-1-carbadehy-de 4
4.1.7. 10-(4-Azidobutyl)-1,10-dihydropyrrolo[2,3-a]carbazole-3-
carbaldehyde 7
POCl3 (3 equiv) was slowly added to anhydrous DMF (2 mL) at
0 ꢁC. The solution was stirred at room temperature for 45 min, and
then was added to a solution of compound 2 (100 mg, 0.38 mmol)
in DMF (1 mL), prepared in a 10 mL CEM reaction vessel and cooled
to 0 ꢁC. The tube was sealed and was heated at 100 ꢁC under
microwave irradiation for 20 min (Pmax ¼ 150 W). After cooling, the
mixture was poured into a saturated aqueous NaHCO3 solution
(20 mL). After 30 min of stirring, the solid was filtered off and a 5%
aqueous NaOH solution (20 mL) was added. The mixture was stir-
red at room temperature overnight. The solid was filtered off and
purified by column chromatography (cyclohexane/EtOAc, from 8:2
to 4:6) to give 65 mg of compound 4 (0.23 mmol, 59%) as a brown
solid. Mp ¼ 212e214 ꢁC (decomposition); IR (ATR): 1651, 1540,
1446, 1395 cmꢀ1; 1H NMR (400 MHz, DMSO-d6): 2.02e2.13 (4H, m),
4.59e4.63 (2H, m), 4.66e4.70 (2H, m), 7.24 (1H, t, J ¼ 7.5 Hz), 7.44
(1H, t, J ¼ 7.5 Hz), 7.69 (1H, d, J ¼ 8 Hz), 8.06 (2H, s), 8.16 (1H, d,
J ¼ 7.5 Hz), 8.33 (1H, s), 9.98 (1H, s); 13C NMR (100 MHz, DMSO-d6):
25.2, 25.4, 42.2, 46.9 (CH2), 109.7, 112.7, 115.4, 119.40, 119.41, 124.8,
141.3 (CHarom), 117.7, 119.6, 123.0, 123.5, 125.0, 127.4, 140.5 (Carom),
184.7 (C]O); HRMS (ESIþ) calcd for C19H17N2O (M þ H)þ 289.1341,
found 289.1339.
POCl3 (1.1 mL, 11.8 mmol) was slowly added to anhydrous DMF
(12 mL) at 0 ꢁC. The solution was stirred at room temperature for
1 h and cooled to 0 ꢁC. A solution of compound 6 (1.18 g, 3.89 mmol)
in anhydrous DMF (7 mL) was added and the mixture was stirred at
room temperature for 1 h. The mixture was poured into a saturated
aqueous NaHCO3 solution (200 mL). After 1.5 h of stirring, the solid
was filtered off and a 5% aqueous NaOH solution (250 mL) was
added. The mixture was stirred for 16 h at 50 ꢁC. The solid was
filtered off and then purified by column chromatography (CH2Cl2/
MeOH, from 98:2 to 85:15) to give 1.18 g of compound 7
(3.56 mmol, 92%) as yellow-brown solid. Mp ¼ 186e188 ꢁC; IR
(ATR): 3303, 2086, 1632 cmꢀ1 1H NMR (400 MHz, DMSO-d6):
;
1.50e1.59 (2H, m), 1.77e1.87 (2H, m), 3.29 (2H, t, J ¼ 7 Hz), 4.78
(2H, t, J ¼ 7 Hz), 7.22 (1H, t, J ¼ 7.5 Hz), 7.43 (1H, t, J ¼ 7.5 Hz), 7.70
(1H, d, J ¼ 8.5 Hz), 8.02 (2H, s), 8.15 (1H, d, J ¼ 7.5 Hz), 7.39 (1H, d,
J ¼ 3 Hz), 10.07 (1H, s), 12.34 (1H, br d, J ¼ 2.5 Hz); 13C NMR
(100 MHz, DMSO-d6): 25.6, 27.4, 43.3, 50.4 (CH2), 109.5, 112.4, 115.3,
119.18, 119.6, 124.5, 137.4 (CHarom), 118.5, 119.17, 121.9, 123.1, 123.8,
126.7, 139.2 (Carom), 185.3 (C]O); HRMS (ESIþ) calcd for C19H18N5O
(M þ H)þ 332.1511, found 332.1528.
4.1.5. 10-(4-Azidobutyl)-1-(phenylsulfonyl)-1,10-dihydropyrrolo
[2,3-a]carbazole 5
4.1.8. N-(2-hydroxyethyl)-N,N-dimethylprop-2-yn-1-aminium
chloride 9
To a solution of compound 3 (100 mg, 0.21 mmol) in DMF
(4 mL) was added NaN3 (67 mg, 1.0 mmol). The mixture was
stirred at 80 ꢁC for 5 h. After cooling, the mixture was poured
into water (100 mL) and extracted with EtOAc (3 ꢃ 50 mL). The
assembled organic fractions were washed with water, with brine
and dried over MgSO4. After evaporation under reduced pressure,
the residue was purified by column chromatography (EtOAc/
cyclohexane, 1:9) to give 76 mg of compound 5 (0.171, 82%) as
a brown solid. Mp ¼ 132e134 ꢁC; IR (ATR): 2088, 1470, 1447,
To a solution of propargyl chloride (70% w/w in toluene, 25 mL,
0.2 mol) in toluene (30 mL) was added dropwise a solution of
N,N-dimethylaminoethanol (17 mL, 0.17 mol) in toluene (30 mL). The
reaction was maintained at room temperature with a water bath
during the addition and the reaction mixture was stirred for 3 days.
The precipitate was filtered off and washed with pentane to give
23.8 g of 9 (0.15 mol, 86%) as an off-white hygroscopic solid. IR (ATR):
3254, 3216, 2124, 1458, 1441, 1376, 1149, 1085, 1026, 1002, 995, 917,
874 cmꢀ1; 1H NMR (400 MHz, CD3OD): 3,28 (6H, s), 3.55 (1H, s), 3.61
(2H, br s), 4.03 (2H, br s), 4.46 (2H, s); 13C RMN (100 MHz, CD3OD):
52.0 (2CH3), 56.5, 57.0, 66.6 (CH2), 72.7 (CH), 83.0 (C); HRMS (ESIþ)
calcd for C7H14NO (Mþ) 128.1075, found 128.1078.
1361 cmꢀ1 1H NMR (400 MHz, DMSO-d6): 0.95e1.04 (2H, m),
;
1.32e1.42 (2H, m), 3.07 (2H, t, J ¼ 7 Hz), 4.94 (2H, t, J ¼ 7 Hz),
6.94 (1H, d, J ¼ 3.5 Hz), 7.16 (2H, d, J ¼ 8 Hz), 7.23e7.32 (4H, m),
7.51 (2H, t, J ¼ 7.5 Hz), 7.53 (1H, d, J ¼ 3.5 Hz), 7.79 (1H, d,
J ¼ 8 Hz), 8.12 (1H, d, J ¼ 8 Hz), 8.21 (1H, d, J ¼ 7.5 Hz); 13C NMR
(100 MHz, DMSO-d6): 24.9, 25.4, 45.4, 49.9 (CH2), 111.7, 113.3,
118.4, 119.1, 120.0, 120.2, 125.6, 127.0 (2C), 128.5 (2C), 131.3, 134.2
(CHarom), 123.0, 123.4, 124.1, 132.9, 133.4, 133.5, 141.7 (Carom);
4.1.9. 10-{4-[4-(Hydroxymethyl)-1H-1,2,3-triazol-1-yl]butyl}-1,10-
dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde 11
To a solution of compound 7 (50 mg, 0.15 mmol) and prop-
argyl alcohol (34.6
mL, 0.60 mmol) in methanol (5 mL) was

B. Letribot et al. / European Journal of Medicinal Chemistry 50 (2012) 304e310
309
added [CuCl(SIMes)(4,7-ClePhen)] (7.1 mg, 0.011 mmol). The
4.2. Kinase assays
reaction mixture was stirred at room temperature for 20 h. After
evaporation under reduced pressure, the residue was purified by
column chromatography (EtOAc/MeOH, from 10:0 to 8:2) to give
54 mg of compound 11 (0.139 mmol, 92%) as a light brown solid.
4.2.1. In vitro kinase inhibition assays
The procedures for the in vitro protein kinase assays and for the
expression and activation of the protein kinases have been
described previously [15].
Mp ¼ 183e185 ꢁC; IR (ATR): 3420e3040, 1627 cmꢀ1
;
1H NMR
(400 MHz, DMSO-d6): 1.70e1.89 (4H, m), 4.29 (2H, t, J ¼ 6.5 Hz),
4.44 (2H, d, J ¼ 5.5 Hz), 4.77 (2H, t, J ¼ 6.5 Hz), 5.10 (1H, t,
J ¼ 5.5 Hz), 7.22 (1H, t, J ¼ 7.5 Hz), 7.42 (1H, t, J ¼ 7.5 Hz), 7.69
(1H, d, J ¼ 8 Hz), 7.83 (1H, s), 8.01 (2H, s), 8.15 (1H, d,
J ¼ 7.5 Hz), 8.38 (1H, s), 10.06 (1H, s), 12.32 (1H, br s); 13C NMR
(100 MHz, DMSO-d6): 27.2, 27.3, 43.2, 48.9, 55.0 (CH2), 109.6,
112.4, 115.3, 119.2, 119.6, 122.6, 124.5, 137.5 (CHarom), 118.5, 119.2,
121.9, 123.1, 123.9, 126.7, 139.2, 147.9 (Carom), 185.3 (C]O); HRMS
Source and purification of kinases: All protein kinases were of
human origin and encoded full-length proteins. All proteins were
either expressed as GST (glutathione transferase) fusion proteins in
Escherichia coli or as hexahistidine (His6)-tagged proteins in Sf21
(Spodoptera frugiperda 21) insect cells. GST fusion proteins were
purified by affinity chromatography on glutathioneeSepharose,
and His6-tagged proteins on nickel/nitrilotriacetateeagarose.
Protein kinase assays: All assays (25.5 mL volume) were carried
(ESIþ) calcd for C22H21N5NaO2 (M
þ
Na)þ 410.1593, found
out robotically at room temperature (21 ꢁC) and were linear with
respect to time and enzyme concentration under the conditions
used. Assays were performed for 30 min using Multidrop Micro
reagent dispensers (Thermo Electron Corporation, Waltham, MA,
U.S.A.) in a 96-well format. The concentration of magnesium
410.1577.
4.1.10. N-({1-[4-(3-Formylpyrrolo[2,3-a]carbazol-10(1H)-yl)butyl]-
1H-1,2,3-triazol-4-yl}methyl)-2-hydroxy-N,N-
dimethylethanaminium trifluoroacetate 12
acetate in the assays was 10 mM and [
33P]ATP (800 c.p.m./pmol)
was used at 5 M for Pim-2 and 20 mM for Pim-1 and Pim-3, in
g
-
To a solution of compound 7 (200 mg, 0.60 mmol) and N-(2-
m
hydroxyethyl)-N,N-dimethylprop-2-yn-1-aminium
chloride
order to be at or below the Km for ATP for each enzyme.
(49 mg, 0.30 mmol) in methanol (12 mL) was added
[CuCl(SIMes)(4,7-ClePhen)] (25 mg, 0.038 mmol). The mixture was
stirred at 60 ꢁC for 20 h. After evaporation under reduced pressure,
water was added and the mixture was washed with EtOAc. Water
was azeotroped with ethanol and the residue was purified by
preparative RPHPLC (water 0.1% TFA/acetonitrile 55:45,
The assays were initiated with MgATP, stopped by the addition
of 5
plates using a unifilter harvester (PerkinElmer, Boston, MA, U.S.A.).
Kinase substrate was RSRHSSYPAGT (300 M) for Pim-1, Pim-2
mL of 0.5 M orthophosphoric acid and spotted on to P81 filter
m
and Pim-3. The enzymes were diluted in a buffer consisting of
50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 1 mg/mL BSA and 0.1%
2-mercaptoethanol and assayed in a buffer comprising 50 mM Tris/
HCl, pH 7.5, 0.1 mM EGTA and 0.1% 2-mercaptoethanol.
flow ¼ 40 mL/min, detection at
l
¼ 250 nm) to give 84 mg
of compound 12 (0.15 mmol, 49%) as
a
brownish solid.
Mp ¼ 87e89 ꢁC; IR (ATR): 3224, 1675, 1653, 1201, 1130 cmꢀ1 1H
NMR (500 MHz, DMSO-d6): 1.71e1.80 (2H, m), 1.85e1.94 (2H, m),
2.95 (6H, s), 3.27e3.31 (2H, m), 3.83e3.89 (2H, m), 4.39 (2H, t,
J ¼ 7 Hz), 4.60 (2H, s), 4,78 (2H, t, J ¼ 7 Hz), 5.20e5.45 (1H, br s), 7.22
(1H, t, J ¼ 7.5 Hz), 7.41 (1H, t, J ¼ 7.5 Hz), 7,67 (1H, d, J ¼ 8 Hz), 8.01
(2H, s), 8.15 (1H, d, J ¼ 7.5 Hz), 8.22 (1H, s), 8.37 (1H, d, J ¼ 3 Hz),
10.07 (1H, s),12.35 (1H, br d, J ¼ 2.5 Hz); 13C NMR (100 MHz, DMSO-
d6): 50.2 (2C) (CH3), 26.9, 27.2, 43.2, 49.4, 55.0, 58.4, 64.6 (CH2),
109.5, 112.4, 115.3, 119.2, 119.6, 124.5, 128.2, 137.5 (CHarom), 118.5,
119.2, 121.9, 123.2, 123.9, 126.8, 135.2, 139.1 (Carom), 185.4 (C]O);
HRMS (ESIþ) calcd for C26H31N6O2 (Mþ) 459.2508, found 459.2492.
The inhibition profile of the tested compounds was expressed as
the percentage of the residual kinase activity for an inhibitor
concentration of 10 mM. The IC50 values of inhibitors were deter-
mined after carrying out assays at 10 different concentrations of
each compound.
4.3. Cellular localisation assays
4.3.1. Cell culture
The human PC-3 prostate cancer cell line was cultured in RPMI-
1640 medium supplemented with 10% foetal bovine serum, 2 mM l-
glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin.
4.1.11. 5-(Dimethylamino)-N-({1-[4-(3-formylpyrrolo[2,3-a]
carbazol-10(1H)-yl)butyl]-1H-1,2,3-triazol-4-yl}methyl)
4.3.2. Subcellular localization assay
naphthalene-1-sulfonamide 13
PC-3 cells were seeded onto coverslips on 6-well plates and
incubated overnight for proper attachment. Thereafter cells were
treated with 10 mM compounds (A or 21) dissolved in DMSO or with
To a solution of compound 7 (50 mg, 0.15 mmol) and 10
[16] (45 mg, 0.156 mmol) in methanol (3 mL) was added
[CuCl(SIMes)(4,7-ClePhen)] (6.1 mg, 0.0093 mmol). The mixture
was stirred at 50 ꢁC for 16 h. After evaporation under reduced
pressure, the residue was purified by column chromatography
(EtOAc/cyclohexane, from 7:3 to 10:0, then EtOAc/MeOH, from
100:0 to 95:5) to give 86 mg of compound 13 (0.139 mmol, 92%) as
a yellow-brown solid. Mp ¼ 200e202 ꢁC; IR (ATR): 3450e3030,
0,1% DMSO. After 24 h, cells were washed with PBS, fixed for 15 min
with 4% paraformaldehyde and washed three times with PBS.
Coverslip samples were mounted on glass slides with Mowiol and
examined by LeicaDMRE fluorescent microscope. Pictures were
taken with Display Image command by Hamamatsu C4742-95 CCD
camera and HCImage software (Hamamatsu Corporation, NJ, USA).
Mean intensity values were analysed from 8-bit tif files and cyan
colour was added by ImageJ software (Wayne Rasband, NIH, USA).
For background correction, the average mean intensity value of
DMSO samples was counted and subtracted from the values of each
analyzed image.
;
1653, 1144 cmꢀ1 1H NMR (400 MHz, DMSO-d6): 1.63e1.77
(4H, br s), 2.74 (6H, s), 4.02 (2H, d, J ¼ 5.5 Hz), 4.11e4.18 (2H, m),
4.71e4.78 (2H, m), 7.14 (1H, d, J ¼ 7.5 Hz), 7.23 (1H, t, J ¼ 7.5 Hz),
7.39e7.51 (4H, m), 7.67 (1H, d, J ¼ 8 Hz), 7.99e8.05 (3H, m), 8.16
(1H, d, J ¼ 7.5 Hz), 8.21 (1H, d, J ¼ 8.5 Hz), 8.32 (1H, d, J ¼ 8.5 Hz),
8.37e8.42 (2H, m), 10.07 (1H, s), 12.31 (1H, br d, J ¼ 3 Hz); 13C
NMR (100 MHz, DMSO-d6): 44.9 (2C) (CH3), 26.9, 27.1, 37.8,
43.2, 48.8 (CH2); 109.5, 112.4, 115.0, 115.3, 119.1, 119.2, 119.6, 122.8,
123.4, 124.5, 127.7, 128.2, 129.4, 137.4 (CHarom), 118.5, 119.2, 121.9,
123.1, 123.8, 126.7, 128.9, 129.0, 135.9, 139.1, 143.4, 151.2 (Carom),
185.2 (C]O). HRMS (ESIþ) calcd for C34H33N7NaO3S (M þ Na)þ
642.2263, found 642.2258.
Acknowledgements
CNRS Valorisation (R A-G), Academy of Finland (PJK) and Drug
Discovery Graduate School (NMS) are greatly acknowledged for
financial support. The authors are grateful to Bertrand Légeret for
mass spectrometry analysis, Aurélie Job for preparative RPHPLC

310
B. Letribot et al. / European Journal of Medicinal Chemistry 50 (2012) 304e310
[6] R. Akué-Gédu, E. Rossignol, S. Azzaro, S. Knapp, P. Filippakopoulos,
A.N. Bullock, J. Bain, P. Cohen, M. Prudhomme, F. Anizon, P. Moreau, J. Med.
Chem. 52 (2009) 6369e6381.
purification of compound 12, and Pauline Labaume for the
synthesis of [CuCl(SIMes)(4,7-ClePhen)].
[7] R. Akué-Gédu, L. Nauton, V. Théry, J. Bain, P. Cohen, F. Anizon, P. Moreau,
Bioorg. Med. Chem. 18 (2010) 6865e6873.
[8] N.M. Santio, R.L. Vahakoski, E.-M. Rainio, J.A. Sandholm, S.S. Virtanen,
Appendix. Supplementary material
M. Prudhomme, F. Anizon, P. Moreau, P. Koskinen, Mol. Cancer
(2010) 279.
[9] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt,
E.C. Meng, T.E. Ferrin, J. Comput. Chem. 25 (2004) 1605e1612.
[10] V. Hong, S.I. Presolski, C. Ma, M.G. Finn, Angew. Chem. Int. Ed. 48 (2009)
9879e9883.
9
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2012.02.009.
References
[11] M.-L. Teyssot, A. Chevry, M. Traïka, M. El-Ghozzi, D. Avignant, A. Gautier,
Chem. Eur. J. 15 (2009) 6322e6326.
[12] M.-L. Teyssot, L. Nauton, J.-L. Canet, F. Cisnetti, A. Chevry, A. Gautier, Eur. J.
Org. Chem. (2010) 3507e3515.
[13] V.A. Krylova, P.I. Djurovich, M.T. Whited, M.E. Thompson, Chem. Commun. 46
(2010) 6696e6698.
[1] K.C. Qian, L. Wang, E.R. Hickey, J. Studts, K. Barringer, C. Peng, A. Kronkaitis,
J. Li, A. White, S. Mische, B. Farmer, J. Biol. Chem. 280 (2005) 6130e6137.
[2] L. Brault, C. Gasser, F. Bracher, K. Huber, S. Knapp, J. Schwaller, Haematologica
95 (2010) 1004e1015.
[14] Y. Liu, C.-N. Lok, BC.-B. Ko, TY.-T. Shum, M.-K. Wong, C.-M. Che, Org. Lett. 12
(2010) 1420e1423.
[3] H. Mikkers, J. Allen, P. Knipscheer, L. Romeyn, A. Hart, E. Vink, A. Berns, Nat.
Genet. 32 (2002) 153e159.
[15] J. Bain, L. Plater, M. Elliott, N. Shpiro, J. Hastie, H. McLauchlan, I. Klervernic,
S.C. Arthur, D.R. Alessi, P. Cohen, Biochem. J. 408 (2007) 297e315.
[16] A. Deiters, T.A. Cropp, M. Mukherji, J.W. Chin, J. C Anderson, P.G. Schultz, J. Am.
Chem. Soc. 125 (2003) 11782e11786.
[4] K. Ellwood-Yen, T.G. Graeber, J. Wongvipat, M.L. Iruela-Arispe, J. Zhang,
R. Matusik, G.V. Thomas, C.L. Sawyers, Cancer Cell 4 (2003) 223e238.
[5] F. Anizon, A.A. Sthil, V.N. Danilenko, P. Moreau, Curr. Med. Chem. 17 (2010)
4114e4133.