Synthesis and biological activities of 4-substituted pyrrolo[2,3-a] carbazole Pim kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.08.029

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 and biological activities (Pim kinase inhibition and in vitro antiproliferative potency) of

Full text of DOI:10.1016/j.ejmech.2012.08.029

European Journal of Medicinal Chemistry 56 (2012) 225e236
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological activities of 4-substituted pyrrolo[2,3-a]carbazole Pim
kinase inhibitors
,
,
,
,
,d
Francis Giraud a b, Rufine Akué-Gédu a b, Lionel Nauton a b, Nicolas Candelon a b, Eric Debiton c
,
,
,
b,
a,b,
*
Vincent Thérya b, Fabrice Anizon a
, Pascale Moreau
**
a Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, F-63000 Clermont-Ferrand, France
b CNRS, UMR 6296, ICCF, BP 80026, 63171 Aubière, France
c Clermont Université, Université d’Auvergne, Imagerie Moléculaire et Thérapie Vectorisée, BP 10448, F-63000 Clermont-Ferrand, France
d Inserm, UMR 990, IMTV, F-63005 Clermont-Ferrand, France
a r t i c l e i n f o
a b s t r a c t
Article history:
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 and biological activities (Pim kinase inhibition
and in vitro antiproliferative potency) of new 4-substituted pyrrolo[2,3-a]carbazoles. The results
demonstrated that the Pim kinase inhibitory potency (especially Pim-3) can be conserved for pyrrolo
[2,3-a]carbazoles bearing a methoxycarbonyl group at the 4-position without a formyl at the 3-position.
Moreover, compound 27 that was found to be active against Pim-1 and Pim-3 kinases showed anti-
proliferative activities in the micromolar range.
Received 27 April 2012
Received in revised form
20 August 2012
Accepted 20 August 2012
Available online 30 August 2012
Keywords:
Pyrrolo[2,3-a]carbazole
Suzuki coupling
Ó 2012 Elsevier Masson SAS. All rights reserved.
Photo-induced cyclization
Pim kinase inhibition
In vitro antiproliferative activities
1. Introduction
groups at the 4-position, that could replace the formyl group at the
3-position of the parent series and possibly reinforce the interac-
As part of our studies directed toward the synthesis of biologi-
cally active heteroaromatic compounds, we were interested in the
preparation of 4-substituted pyrrolo[2,3-a]carbazoles as Pim kinase
inhibitors. Recently, we reported the synthesis and biological
tion between these compounds and Pim kinase ATP-binding site.
Then, the potencies of these new compounds toward the three Pim
isoforms were evaluated. In addition, as over-expression of Pim
kinases has been observed in various solid tumors such as prostate,
the in vitro antiproliferative activities of these compounds were
studied toward a human fibroblast primary culture and three
human solid cancer cell lines: PC3 and DU145 (prostatic carcinoma)
as well as PA1 (ovarian carcinoma).
activities of pyrrolo[2,3-a]carbazole-3-carbaldehyde
A that is
a potent and selective inhibitor of the Pim kinase family members
(Fig. 1) [1]. The structureeactivity relationship study achieved
around this scaffold showed that this series of compounds consti-
tute potent inhibitors of Pim kinases and attractive molecules for
drug development, for instance to inhibit the invasiveness of Pim-
overexpressing cancer cells [1e4]. To enlarge the structural diver-
sity introduced on this scaffold, we undertook the preparation of
pyrrolo[2,3-a]carbazoles bearing carboxy or methoxycarbonyl
2. Chemistry
To prepare the 6-amino derivatives, we first envisaged
a
synthetic pathway starting from commercially available
4-nitroindole-3-carbaldehyde (Scheme 1). Firstly, the indole
nitrogen atom was protected using benzenesulfonyl chloride and
cesium carbonate in refluxing acetonitrile leading to compound 1
in 94% yield. Methyl 2-bromoacrylate 2 was subsequently obtained
in 95% yield from a Wittig type reaction as reported by Bourderioux
et al. [5]. All our assays to achieve Suzuki coupling between 2 and
pinacol boronate ester 5 failed. Thus, the nitro group of compound
2 was reduced in the presence of iron powder and ammonium
* Corresponding author. Clermont Université, Université Blaise Pascal, Institut de
Chimie de Clermont-Ferrand, BP 10448, F-63000 Clermont-Ferrand, France.
Tel.: þ33 (0) 4 73 40 79 63; fax: þ33 (0) 4 73 40 77 17.
** Corresponding author. Clermont Université, Université Blaise Pascal, Institut de
Chimie de Clermont-Ferrand, BP 10448, F-63000 Clermont-Ferrand, France.
Tel.: þ33 (0) 4 73 40 53 64; fax: þ33 (0) 4 73 40 77 17.
E-mail
addresses:
Fabrice.ANIZON@univ-bpclermont.fr
(F.
Anizon),
Pascale.MOREAU@univ-bpclermont.fr (P. Moreau).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.08.029

226
F. Giraud et al. / European Journal of Medicinal Chemistry 56 (2012) 225e236
COOR
R'
4
CHO
3
R = H or Me
R' = H or NH2
N
H
N
H
N
H
N
H
A, pyrrolo[2,3-a]carbazole-3-carbaldehyde
4-substituted pyrrolo[2,3-a]carbazoles
Fig. 1. Structure of compound A and 4-substituted pyrrolo[2,3-a]carbazoles.
chloride to give compound 3 in 90% yield [6], and the amino group
was protected in the presence of Boc2O and DMAP leading to
compound 4 in 70% yield. Subsequent Suzuki cross-coupling
between bromo derivative 4 and compound 5 [7e12], using
potassium carbonate and tetrakis(triphenylphosphine)palla-
dium(0) [5] allowed the preparation of compound 6 in 76% yield.
Treatment of compound 6 with TBAF [13] led to compound 8 in 56%
yield. Unexpectedly, pyrrolocarbazole derivative 7 was also isolated
in 5% yield and led to the ring-opening product 9 in 73% yield when
the deprotection of the amino group was conducted in the presence
of TFA. When we carried on the synthesis from compound 8 in
order to cleave the benzenesulfonyl group using magnesium
turnings in methanol [14], the reaction yielded reduced compound
10 in 47% yield [15,16].
deprotection step was carried out for an extended time, a partial
hydrolysis of the methoxycarbonyl group was also observed,
leading to the corresponding carboxylic acid 26. Finally, cyclization
of compound 25 using a 400 W medium-pressure Hg lamp (pyrex
filter) in the presence of a catalytic amount of I2 led to compound 27
and side product 28 in 59% and 29% yields, respectively. As for
compound 19, the pyrrolo[2,3-a]carbazole structure of 27 was
confirmed by a 1He1H NOESY experiment (Fig. 2). Carboxylic acid
29 was prepared after saponification of the corresponding methyl
ester using sodium hydroxide.
3. Results and discussion
The kinase inhibitory potencies of compounds 19, 20, 27, and 29
Due to the difficulties encountered for the benzenesulfonyl
group cleavage of compound 8 and the amino deprotection of
compound 7, we decided to perform the synthesis using a tert-
butoxycarbonyl as indole nitrogen protecting group and an azido
group as precursor of the final amino function (Scheme 2). Pro-
tected derivative 11 was easily prepared according to previously
described procedures [17,18] and its methyl bromoacrylate deriv-
ative 12 was prepared as described above. Nitro group of compound
12 was then reduced to give compound 13 in 69% yield [6], and the
azido group was introduced by adding sodium azide to the diazo-
nium salt prepared from compound 13 in the presence of sodium
nitrite in acetic acid [19]. Suzuki cross-coupling between
compound 14 and boronate 5 led to product 15 in 60% yield using
the same procedure as the one described above. After several trials,
we noticed that the deprotection steps should be completed after
the reduction of the azido group to proceed in acceptable yields.
Therefore, 16 was prepared in 61% yield by reduction of 15 using
stannous chloride [20e22]. Subsequent deprotection of TIPS group
in the presence of TBAF afforded 17 in 89% yield. After tBoc cleavage
using K2CO3 in MeOH/H2O [23], photo-induced cyclization of
compound 18 was performed under irradiation using a 400 W
medium-pressure Hg lamp fitted with a Pyrex filter, in the presence
of a catalytic amount of I2 [24]. The expected pyrrolo[2,3-a]carba-
zole 19 was obtained, as confirmed by a 1He1H NOESY NMR
experiment showing NOE correlations between all neighboring
protons of the scaffold (Fig. 2). Finally, carboxylic acid 20 was
prepared in 60% yield after saponification of ester 19 using sodium
hydroxide.
were evaluated at 10 and 1 mM concentrations in duplicate assays
against all three Pim family kinases. Kinase assays were performed
by the International Centre for Kinase Profiling (Dundee, UK) as
previously described [25]. The percentages of residual kinase
activities are reported in Table 1. IC50 values were determined when
the remaining kinase activity was less than 60% when the
compounds were tested at 1 mM.
As evident from Table 1, the results obtained indicate that,
compared to parent compound A, these new derivatives were less
active toward Pim kinases. Nevertheless, it is noteworthy that
compound 27, bearing a methoxycarbonyl group at the 4-position
demonstrated inhibitory potencies toward Pim-1 and Pim-3 with
IC50 values of 3
sponding 6-amino analog 19 was also found to be active toward
Pim-3 with an IC50 value of 0.41 M. In both cases, the methyl ester
mM and 0.5 mM, respectively. Moreover, the corre-
m
derivatives were more efficient toward Pim kinases than the
carboxylic acid analogs, the 6-position being substituted or not by
an amino group. The putative binding mode between the ATP-
binding site of Pim-3 and compound 27 was examined by molec-
ular modeling experiments.
As shown in Fig. 3A, the sequence analysis of the three Pim
kinase isoforms Pim-1, Pim-2 and Pim-3 revealed a high degree of
homology. As no three dimensional structure of Pim-3 has been
solved so far, we decided to generate a model of Pim-3 active site by
sequence homology modeling from a known Pim-1 X-ray crystal
structure. Most of the Pim-1 structures available in the Protein Data
Bank (PDB) present a good resolution and can be used. We started
ꢀ
from the 2C3I model [27] that showed a 1.9 A resolution and a well
Finally, the preparation of 1,10-dihydropyrrolo[2,3-a]carbazole-
4-carboxylic acid 29 was achieved in seven steps from commer-
cially available indole-3-carbaldehyde using a similar synthetic
pathway (Scheme 3). Firstly, compound 22 was prepared as
described above after tBoc protection of the indole nitrogen atom
and introduction of the methyl bromoacrylate side chain [5].
Subsequent Suzuki cross-coupling with pinacol boronate ester 5
using potassium carbonate and tetrakis(triphenylphosphine)
palladium(0) led to compound 23 in 77% yield. Cleavage of the
triisopropylsilyl and tert-butoxycarbonyl groups in the presence of
TBAF and K2CO3/MeOH/H2O, respectively, led to compound 25 in
70% yield from compound 23. When the tert-butoxycarbonyl
defined gatekeeper loop containing the Phe49. The Pim-3 Q86V86
sequence available in UniProtKB database [28], presenting 73.6% of
sequence identity with Pim-1, was used for the construction of the
Pim-3 model. In order to validate our method, we also generated
a Pim-2 homology model from the Q9P1W9 sequence available in
UniProtKB (59.9% of sequence identity with Pim-1) [29] and the
2C3I crystal structure. The Pim-2 homology model was then
compared to the 2IWI Pim-2 crystal structure available in the PDB
ꢀ
[30]. As shown Fig. 3B, an RMSD of 1.917 A was found for all atoms
in the superimposition of the generated Pim-2 model and 2IWI
structure, thus validating our method. Docking experiments were
then performed for compound 27 using our Pim-3 model. The

F. Giraud et al. / European Journal of Medicinal Chemistry 56 (2012) 225e236
227
O
O
Br
O
O
NO2
NO2
NO2
H
H
a
b
N
N
N
H
SO2Ph
SO2Ph
4-nitroindole-3-carbaldehyde
1
2
c
O
B O
O
O
O
O
O
O
Br
5
Boc
Boc
Boc
Boc
N
N
N
NH2
TIPS
Br
e
d
N
N
N
N
TIPS
SO2Ph
SO2Ph
SO2Ph
6
4
3
O
O
O
O
Boc
Boc
Boc
Boc
N
N
f
6
+
N
H
N
N
N
H
SO2Ph
8
H
7
g
h
O
O
O
O
Boc
NH
NH2
N
N
H
H
N
H
NH2
9
10
(a) PhSO2Cl, Cs2CO3, CH3CN, reflux, 94% (b) Ph3P=CHCO2Me, NBS, K2CO3, CH2Cl2, –20 °C
to rt, 95% (c) Fe, NH4Cl, isopropanol/water, 60 °C, 90% (d) DMAP, Boc2O, CH2Cl2, rt, 70% (e)
K2CO3, Pd(PPh3)4, H2O/dioxane, 70°C, 76% (f) TBAF, THF, 0°C, 5% (7) and 56% (8) (g) TFA,
rt, 73% (h) Mg, I2, MeOH, reflux, 47%.
Scheme 1. Preparation of compounds 1e4 and 6e10.
structure corresponding to the best docking score (6.32) was not
retained in regards to the conformation of 27 and its location
outside of the hydrophobic ATP adenine binding cleft. Thus,
another complex (score of 5.27) showing 27 in a preferred
conformation and bound to the hydrophobic pocket was selected
and minimized. As shown in Fig. 3C, compound 27 interacts within
Pim-3 ATP-binding site via hydrophobic interactions between the
ester methyl group and Phe51 and two hydrogen bonds. The ester
carbonyl is H-bonded with Lys69 side chain, and the hydrogen at
the N1 position with Glu124 backbone carbonyl. Compared to

228
F. Giraud et al. / European Journal of Medicinal Chemistry 56 (2012) 225e236
O
O
O
O
NO2
NO2
H
NO2
H
Br
a
b
N
N
N
Boc
Boc
H
4-nitroindole-3-carbaldehyde
11
12
c
O
O
O
O
O
O
N3
NH2
N3
Br
Br
e
d
N
N
N
N
Boc
Boc
TIPS
Boc
15
14
13
f
O
O
O
O
O
O
NH2
NH2
NH2
g
h
N
N
H
N
H
N
N
N
H
TIPS
Boc
Boc
16
17
18
i
O
O
OH
O
NH2
NH2
j
N
H
N
H
N
H
N
H
20
19
(a) DMAP, Boc2O, CH3CN, rt, 99% (b) Ph3P=CHCO2Me, NBS, K2CO3, CH2Cl2, –20 °C to rt,
98% (c) Fe, NH4Cl, isopropanol/water, 60 °C, 69% (d) NaNO2, NaN3, H2O, AcOH, 0°C, 97%
(e) K2CO3, Pd(PPh3)4, H2O/dioxane, 5, 70°C, 60% (f) SnCl2, MeOH/THF, 0°C to reflux, 61%
(g) TBAF, THF, 0°C, 89% (h) K2CO3, MeOH/water, 70°C (i) I2 (cat), CH3CN, rt, 41% from 17
(j) NaOH 1N, MeOH/THF, reflux, 60%.
Scheme 2. Preparation of compounds 11e20.
compound A bound to Pim-1 (3JPV), due to the presence of the
methyl ester group at the 4-position, the pyrrolocarbazole scaffold
of 27 was flipped over, orienting the NH groups toward the bottom
of the pocket.
Finally, in vitro antiproliferative activities of compounds 19, 20,
27, and 29 were evaluated using the resazurin reduction test [31].
All the compounds were firstly tested toward PA1 (ovarian carci-
20 and 29, that were found to be inactive against Pim kinases, did
not show any antiproliferative activity when they were tested at
1 mM and 10 mM toward PA1 and PC3 cells. Compound 19 that was
found to inhibit Pim-3 was also inactive toward the cell lines tested.
Contrarily, methyl ester derivative 27, that was found to be active
against Pim-1 and Pim-3, showed interesting antiproliferative
activities in the micromolar range toward PA1 and PC3 cells as well
as against the human fibroblast primary culture tested.
In conclusion, new 4-substituted pyrrolo[2,3-a]carbazoles,
substituted or not at the 6-position by an amino group, were
synthesized and evaluated for their biological activities. The results
obtained in this preliminary relationship study demonstrated that
noma) and PC3 (prostate carcinoma) cancer cells at 1
concentrations. When the growth inhibition was found to be more
than 50% with compounds tested at 10 M, IC50 values were
mM and 10 mM
m
determined toward PA1, PC3, DU145, another prostatic carcinoma,
and a human fibroblast primary culture (Table 1). Carboxylic acids

F. Giraud et al. / European Journal of Medicinal Chemistry 56 (2012) 225e236
229
range. Finally, complementary molecular modeling studies are
currently undertaken in our group to understand why methyl
esters 19 and 27 are more active toward Pim kinases compared to
their carboxylic acid analogs 20 and 29.
O
CH3
O
CH3
H
H
O
H
O
NH2
H
H
H
H
4. Experimental
H
H
N
H
N
H
N
H
N
H
H
H
H
H
4.1. Chemistry
27
4.1.1. General
19
Starting materials were obtained from commercial suppliers
and used without further purification. IR spectra were recorded on
a Shimadzu FTIR-8400S spectrometer (v in cmꢀ1). NMR spectra,
performed on a Bruker AVANCE 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), triplet (t), septet
(sept), doublet of doublet (dd), doublet of doublet of doublet (ddd),
multiplet (m), broad signal (br s). High-resolution mass spectra
Fig. 2. 1He1H NOE correlations (indicated by arrows) determined for compounds 19
and 27. For details, see Supporting information.
the Pim kinase inhibitory potency (especially Pim-3) can be
conserved for a pyrrolo[2,3-a]carbazole bearing a methoxycarbonyl
group at the 4-position without a formyl at the 3-position. More-
over, compounds 27 that was found to be active against Pim-1 and
Pim-3 kinases showed antiproliferative activities in the micromolar
O
O
Br
O
H
O
H
a
b
N
H
N
N
Boc
Boc
22
Indole-3-carbaldehyde
21
c
O
O
O
O
O
O
R
e
d
N
N
N
H
N
H
H
N
N
TIPS
Boc
Boc
25, R = Me
26, R = H
24
23
f
O
O
O
O
R
N
+
H
N
N
H
H
NH2
27, R= Me
29, R = H
g
28
(a) DMAP, Boc2O, CH3CN, rt, 99% (b) Ph3P=CHCO2Me, NBS, K2CO3, CH2Cl2,
–20 °C to rt, 85% (c) 5, K2CO3, Pd(PPh3)4, H2O/dioxane, 70 °C, 77% (d) TBAF,
THF, 0 °C, 92% (e) K2CO3, MeOH/H2O, 70 °C, 76% of 25 (f) I2, CH3CN, rt, 59%
of 27, 29% of 28 (g) NaOH 1 N, MeOH, THF, Reflux, 76%.
Scheme 3. Preparation of compounds 21e29.

230
F. Giraud et al. / European Journal of Medicinal Chemistry 56 (2012) 225e236
Table 1
Kinase inhibitory potencies: % of residual kinase activity at 10 mM and 1 mM (IC50 (mM) in brackets when determined) and antiproliferative activities of compounds A, 19, 20, 27
and 29 (IC50 in
m
M). nd: Not determined.
Kinase inhibition ꢀ % of residual kinase activity
Pim-1 Pim-2
10 10
Cpds
Antiproliferative activity (IC50 in mM)
Pim-3
10
Fibro
PA1
PC3
DU145
m
M
1
m
M
m
M
1
mM
m
M
1 mM
A
2 ꢁ 0.4
(0.12 ꢁ 0.01)
39 ꢁ 1
(nd)
nd
7 ꢁ 1
nd
1 ꢁ 5
nd
21 ꢁ 1
nd
4.5 ꢁ 0.4
nd
9.5 ꢁ 0.5
nd
26 ꢁ 2
nd
(0.51 ꢁ 0.23)
77 ꢁ 4
(nd)
(0.01 ꢁ 0.00)
16 ꢁ 2
19
20
27
29
90 ꢁ 7
103 ꢁ 1
57 ꢁ 5
90 ꢁ 3
93 ꢁ 8
101 ꢁ 1
95 ꢁ 6
100 ꢁ 0
40 ꢁ 2
(0.41 ꢁ 0.06)
55 ꢁ 1
87 ꢁ 1
89 ꢁ 11
103 ꢁ 3
47 ꢁ 14
101 ꢁ 20
nd
nd
nd
nd
(nd)
(nd)
(nd)
15 ꢁ 1
(3 ꢁ 1)
70 ꢁ 18
(nd)
75 ꢁ 1
(nd)
24 ꢁ 2
8 ꢁ 4
nd
27 ꢁ 5
nd
6 ꢁ 4
nd
> 50
nd
(0.5 ꢁ 0.1)
65 ꢁ 13
(nd)
93 ꢁ 8
(nd)
Fig. 3. A) Human Pim kinases: alignment of amino acid sequences (from Swiss-Prot, accession numbers: Pim-1 (P11309-2), Pim-2 (Q9P1W9), Pim-3 (Q86V86)). The ATP-binding
ꢀ
domain, defined as amino acid residues located within a distance < 5 A from AMP-PNP bounded to Pim-1 (PDB code: 1YXT, 1XR1, 2BZK) is highlighted in gray. B) Superimposition of
Pim-2 model (blue) and 2IWI Pim-2 structure (green). C) Docking model of compound 27 bound to the Pim-3 ATP-binding site. Molecular graphics images were produced using
UCSF Chimera [26]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

F. Giraud et al. / European Journal of Medicinal Chemistry 56 (2012) 225e236
231
(ESIþ) were determined on a high-resolution Micro Q-Tof appa-
ratus (CRMP, Université Blaise Pascal, Clermont-Ferrand, France).
Chromatographic purifications were performed by column chro-
combined organic fractions were dried over MgSO4 and evaporated.
Residue was purified by column chromatography (EtOAc/cyclo-
hexane, from 1:4 to 1:3) to give 3 (280 mg, 0.64 mmol, 90%) as
a yellow powder. Mp ¼ 131e133 ꢂC; IR (ATR): 3352, 1718, 1609,
matography using 40e63 mm silica gel. Reactions were monitored
by TLC using fluorescent silica gel plates (60 F254 from Merck).
Melting points were measured on a Reichert microscope and are
uncorrected. Photochemical reactions were carried out at room
temperature in an annular reactor equipped with a water-cooling
circuit using a 400 W medium-pressure Hg lamp fitted with
a Pyrex filter. A solution of the reactant was deoxygenated for
45 min with an argon stream then irradiated for the appropriate
time indicated below.
1431, 1370, 1287, 1246, 1167 cmꢀ1 1H NMR (400 MHz, DMSO-d6):
;
3.83 (s, 3H, CH3), 5.24 (br s, 2H, NH2), 6.61 (d, 1H, J ¼ 8.0 Hz), 7.09
(t, 1H, J ¼ 8.0 Hz), 7.23 (d, 1H, J ¼ 8.0 Hz), 7.62 (t, 2H, J ¼ 8.0 Hz), 7.72
(t, 1H, J ¼ 8.0 Hz), 8.01 (d, 2H, J ¼ 8.0 Hz), 8.40 (s, 1H), 8.68 (s, 1H);
13C NMR (100 MHz, DMSO-d6): 53.4 (CH3), 102.5 (CH), 111.2 (CH),
112.4 (C), 116.5 (C), 117.0 (C), 125.3 (CH), 126.4 (CH), 126.8 (2CH),
129.9 (2CH), 133.7 (CH), 134.88 (CH), 134.94 (C), 136.2 (2C), 162.9
(C]O); HRMS (ESIþ) calcd for C18H1679BrN2O4S (M þ H)þ 435.0014,
found 435.0022.
4.1.2. 4-Nitro-1-(phenylsulfonyl)-1H-indole-3-carbaldehyde 1
To a solution of 4-nitro-1H-indole-3-carbaldehyde (150 mg,
0.79 mmol) in CH3CN (4 mL) was added Cs2CO3 (513 mg,
1.58 mmol). The suspension was refluxed under argon for 45 min.
Oil bath was removed before addition of benzenesulfonyl chloride
(0.11 mL, 0.87 mmol) and then reflux was continued for 1 h. The
reaction mixture was allowed to reach room temperature, filtered
and washed with CH2Cl2. Water was added and the product was
extracted with CH2Cl2. Combined organic fractions were dried over
MgSO4 and evaporated. The residue was triturated in small
volumes of cyclohexane and filtered off to give 1 (245 mg,
0.74 mmol, 94%) as a beige powder. Mp ¼ 154e156 ꢂC; IR (ATR):
1680, 1519, 1451, 1381, 1177, 1139 cmꢀ1; 1H NMR (400 MHz, DMSO-
d6): 7.65e7.70 (m, 3H), 7.81 (t,1H, J ¼ 7.6 Hz), 8.00 (d,1H, J ¼ 8.0 Hz),
8.22 (d, 2H, J ¼ 8.0 Hz), 8.39 (d, 1H, J ¼ 8.0 Hz), 9.01 (s, 1H), 10.05 (s,
1H, CHO); 13C NMR (100 MHz, DMSO-d6): 118.3 (CH), 120.1 (C),
120.3 (CH), 123.0 (C), 126.3 (CH), 127.5 (2CH), 129.9 (C), 130.3 (2CH),
135.6 (C), 135.8 (CH), 138.51 (CH), 138.53 (C), 185.5 (C]O); HRMS
(ESIþ) calcd for C15H10N2NaO5S (M þ Na)þ 353.0208, found
353.0225.
4.1.5. Methyl 3-{4-[bis(tert-butoxycarbonyl)amino]-1-(phenyl-
sulfonyl)-1H-indol-3-yl}-2-bromoprop-2-enoate 4
To a solution of compound 3 (1.22 g, 2.80 mmol) in anhydrous
CH2Cl2 (30 mL) at room temperature under argon were added
Boc2O (1.53 g, 7.00 mmol) and then DMAP (34 mg, 0.28 mmol).
This solution was stirred at room temperature for 48 h. Water
was added to the reaction medium and the product was extracted
with CH2Cl2. Combined organic fractions were dried over MgSO4
and concentrated. Residue was purified by column chromatog-
raphy (EtOAc/cyclohexane, 1:3) to give 4 (1.25 g, 1.96 mmol, 70%)
as a yellow oil. IR (ATR): 1726, 1448, 1368, 1260, 1238, 1153,
984 cmꢀ1 1H NMR (400 MHz, DMSO-d6): 1.27 (s, 18H, 6CH3 Boc),
;
3.82 (s, 3H, CH3), 7.21 (d, 1H, J ¼ 8.0 Hz), 7.45 (t, 1H, J ¼ 8.0 Hz),
7.63 (t, 2H, J ¼ 8.4 Hz), 7.74 (t, 1H, J ¼ 7.6 Hz), 8.01 (d, 1H,
J ¼ 8.0 Hz), 8.10 (d, 2H, J ¼ 8.0 Hz), 8.22 (s, 1H), 8.59 (s, 1H); 13C
NMR (100 MHz, DMSO-d6): 27.2 (6CH3 Boc), 53.5 (CH3 ester), 82.7
(2C Boc), 113.3 (CH), 114.1 (C), 114.8 (C), 125.2 (CH), 126.0 (CH),
126.3 (C), 127.0 (2CH), 128.2 (CH), 130.0 (2CH), 131.4 (CH), 131.8
(C), 134.3 (C), 135.3 (CH), 135.9 (C), 150.7 (2C]O Boc), 162.5 (C]O
ester); HRMS (ESIþ) calcd for C28H3179BrN2NaO8S (M þ Na)þ
657.0882, found 657.0896.
4.1.3. Methyl 2-bromo-3-[4-nitro-1-(phenylsulfonyl)-1H-indol-3-yl]
prop-2-enoate 2
To a solution of methyl(phosphoranylidene)acetate (377 mg,
1.13 mmol) in anhydrous CH2Cl2 (8 mL) at ꢀ20 ꢂC under argon
was added N-bromosuccinimide (221 mg, 1.24 mmol). The solu-
tion was stirred for 45 min before addition of compound 1
(186 mg, 0.56 mmol) and K2CO3 (257 mg, 1.86 mmol). The reac-
tion mixture was allowed to reach room temperature and stirring
was continued for 48 h. It was filtered through a short pad of
Celite and washed with CH2Cl2. Filtrate was concentrated under
reduced pressure. Residue was purified by column chromatog-
raphy (EtOAc/cyclohexane, from 1:3 to 1:1) to give 2 (250 mg,
0.54 mmol, 95%) as a yellow powder. Mp ¼ 162e163 ꢂC; IR (ATR):
1717, 1526, 1448, 1380, 1354, 1245, 1154 cmꢀ1; 1H NMR (400 MHz,
DMSO-d6): 3.85 (s, 3H, CH3), 7.61e7.67 (m, 3H), 7.77 (t, 1H,
J ¼ 7.2 Hz), 8.09 (d, 1H, J ¼ 8.0 Hz), 8.15 (d, 2H, J ¼ 8.0 Hz), 8.29 (s,
1H), 8.44 (d, 1H, J ¼ 8.0 Hz), 8.64 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): 53.6 (CH3), 113.6 (C), 114.4 (C), 119.1 (CH), 121.0 (C),
121.1 (CH), 125.4 (CH), 127.2 (2CH), 130.1 (2CH), 131.2 (CH), 135.0
(CH), 135.2 (C), 135.5 (CH), 135.8 (C), 142.4 (C), 162.6 (C]O);
HRMS (ESIþ) calcd for C18H1379BrN2NaO6S (M þ Na)þ 486.9575,
found 486.9588.
4.1.6. Methyl 3-{4-[bis(tert-butoxycarbonyl)amino]-1-(phenyl-
sulfonyl)-1H-indol-3-yl}-2-[1-(triisopropylsilyl)-1H-pyrrol-3-yl]
prop-2-enoate 6
To a solution of compounds 4 (215 mg, 0.34 mmol) and 5
(165 mg, 0.47 mmol) in 1,4-dioxane (7 mL) at room temperature
under argon were added water (2.5 mL) and K2CO3 (187 mg,
1.35 mmol). The solution was degassed with argon for 20 min,
tetrakis(triphenylphosphine)palladium(0) (39 mg, 0.03 mmol) was
added. Reaction mixture was heated overnight at 70 ꢂC. Water and
EtOAc were added and the mixture was filtered through a short
pad of Celite which was next washed several times with EtOAc.
The filtrate was washed with a saturated aqueous NaCl solution
and the organic fraction was dried over MgSO4 and evaporated.
Residue was purified by column chromatography (EtOAc/cyclo-
hexane, 1:3 then 1:2) to give 6 (201 mg, 0.26 mmol, 76%) as a pale
yellow foam. Mp ¼ 69e70 ꢂC; IR (ATR): 2963, 2869, 1753, 1712,
1448, 1367, 1243, 1151 cmꢀ1 1H NMR (400 MHz, DMSO-d6): 1.04
;
(d, 18H, J ¼ 7.2 Hz, 6CH3 iPr), 1.30 (s, 18H, 6CH3 Boc), 1.45 (sept, 3H,
J ¼ 7.2 Hz, 3CH iPr), 3.70 (s, 3H, CH3), 5.96e5.97 (m, 1H), 6.76 (s,
1H), 6.88 (t, 1H, J ¼ 2.4 Hz), 7.14 (d, 1H, J ¼ 7.6 Hz), 7.24 (d, 1H,
J ¼ 1.0 Hz), 7.37 (t, 1H, J ¼ 8.0 Hz), 7.48 (d, 1H, J ¼ 1.0 Hz), 7.60 (t,
2H, J ¼ 8.0 Hz), 7.70 (t, 1H, J ¼ 7.6 Hz), 7.86 (d, 2H, J ¼ 7.6 Hz), 7.90
(d, 1H, J ¼ 8.4 Hz); 13C NMR (100 MHz, DMSO-d6): 10.7 (3CH iPr),
17.4 (6CH3 iPr), 27.2 (6CH3 Boc), 51.8 (CH3 ester), 82.4 (2C Boc),
110.9 (CH), 113.0 (CH), 116.9 (C), 118.4 (C), 124.1 (CH), 124.5 (CH),
124.6 (CH), 125.3 (CH), 126.6 (2CH), 127.2 (CH), 127.4 (C), 129.0 (C),
129.8 (3CH), 131.9 (C), 134.5 (C), 134.9 (CH), 136.2 (C), 150.9 (2C]O
Boc), 167.1 (C]O ester); HRMS (ESIþ) calcd for C41H55N3NaO8SSi
(M þ Na)þ 800.3377, found 800.3369.
4.1.4. Methyl 3-[4-amino-1-(phenylsulfonyl)-1H-indol-3-yl]-2-
bromoprop-2-enoate 3
To a solution of compound 2 (330 mg, 0.71 mmol) in isopropanol
(15 mL) were successively added iron powder (238 mg, 4.25 mmol),
ammonium chloride (15 mg, 0.28 mmol) and water (1.5 mL). The
mixture was vigorously stirred at 60 ꢂC for 2 h. The reaction mixture
was filtered through a short pad of Celite and was washed with
water and EtOAc. The product was extracted with EtOAc and the

232
F. Giraud et al. / European Journal of Medicinal Chemistry 56 (2012) 225e236
4.1.7. Methyl 6-[bis(tert-butoxycarbonyl)amino]-1,10-dihydropyrrolo
[2,3-a]carbazole-4-carboxylate 7 and methyl 3-{4-[bis(tert-butoxy-
carbonyl)amino]-1-(phenylsulfonyl)-1H-indol-3-yl}-2-(1H-pyrrol-3-
yl)prop-2-enoate 8
To a solution of compound 6 (180 mg, 0.23 mmol) in THF (4 mL)
at 0 ꢂC under argon was slowly added a 1 M solution of TBAF in THF
(2.3 mL, 2.31 mmol). The solution was stirred at 0 ꢂC for 10 min and
then refluxed for 2.5 h. A saturated aqueous NaCl solution was
added and the product was extracted with EtOAc. The combined
organic fractions were dried over MgSO4 and evaporated. Residue
was purified by column chromatography (CH2Cl2/EtOAc, from 98:2
to 90:10) to give 7 (5 mg, 0.01 mmol, 5%) as a pale yellow solid and 8
(80 mg, 0.13 mmol, 56%) as a yellow foam.
as a yellow solid. Mp ¼ 137e138 ꢂC; IR (ATR): 3351, 1712, 1680,
1441, 1367, 1163 cmꢀ1 1H NMR (400 MHz, DMSO-d6): 1.39 (s, 9H,
;
3CH3 Boc), 3.16 (dd, 1H, J ¼ 5.2 Hz, J ¼ 14.8 Hz), 3.28 (d, 1H,
J ¼ 14.8 Hz), 3.47 (s, 3H, CH3), 3.89 (dd, 1H, J ¼ 5.2 Hz, J ¼ 14.8 Hz),
6.04 (d, 1H, J ¼ 2.0 Hz), 6.64e6.65 (m, 2H), 6.78 (d, 1H, J ¼ 7.6 Hz),
6.92 (d, 1H, J ¼ 2.0 Hz), 6.97 (t, 1H, J ¼ 7.6 Hz), 7.16 (d, 1H,
J ¼ 7.6 Hz), 8.68 (br s, 1H, NHBoc), 10.61 (br s, 1H, NH), 10.78 (br s,
1H, NH); 13C NMR (100 MHz, DMSO-d6): 28.0 (3CH3 Boc), 39.4
(CH2), 45.6 (CH), 51.0 (CH3 ester), 78.0 (C Boc), 106.6 (CH), 109.2
(CH), 112.1 (C), 115.1 (CH), 116.9 (CH), 117.3 (CH), 120.5 (CH), 123.1
(C), 123.3 (CH), 130.1 (C), 137.6 (2C), 154.4 (C]O Boc), 174.7 (C]O
ester); HRMS (ESIþ) calcd for C21H25N3NaO4 (M þ Na)þ 406.1743,
found 406.1749.
7: Yellow solid. Mp ¼ 168e170 ꢂC; IR (ATR): 3385, 1762, 1699,
1559, 1431, 1384, 1109 cmꢀ1; 1H NMR (400 MHz, DMSO-d6): 1.40 (s,
18H, 6CH3 Boc), 3.90 (s, 3H, CH3), 7.03 (d, 1H, J ¼ 7.6 Hz), 7.14 (d, 1H,
J ¼ 1.2 Hz), 7.37 (t, 1H, J ¼ 8.0 Hz), 7.56 (d, 1H, J ¼ 1.2 Hz), 7.65 (d, 1H,
J ¼ 7.6 Hz), 8.44 (s, 1H), 11.18 (br s, 1H, NH), 11.49 (br s, 1H, NH); 13C
NMR (100 MHz, DMSO-d6): 27.4 (6CH3 Boc), 51.2 (CH3 ester), 82.3
(2C Boc), 104.0 (CH), 111.3 (CH), 112.9 (C), 114.1 (C), 118.2 (CH), 120.0
(CH), 121.3 (C), 121.4 (C), 124.3 (CH), 125.4 (C), 125.7 (CH), 129.8 (C),
132.2 (C), 139.8 (C), 151.7 (2C]O Boc), 167.3 (C]O ester); HRMS
4.1.10. Methyl 2-bromo-3-[4-nitro-1-(tert-butoxycarbonyl)-1H-
indol-3-yl]prop-2-enoate 12
To a solution of methyl(phosphoranylidene)acetate (345 mg,
1.03 mmol) in anhydrous CH2Cl2 (7 mL) at ꢀ20 ꢂC under argon was
added N-bromosuccinimide (202 mg, 1.14 mmol). The solution was
stirred for 45 min before addition of compound 11 (150 mg,
0.52 mmol) and K2CO3 (238 mg, 1.72 mmol). The reaction mixture
was allowed to reach room temperature and stirring was continued
for 48 h. It was filtered through a short pad of Celite and washed
with CH2Cl2. Filtrate was concentrated under reduced pressure.
Residue was purified by column chromatography (EtOAc/cyclo-
hexane, from 1:4) to give 12 (217 mg, 0.51 mmol, 98%) as a yellow
powder. Mp ¼ 128e129 ꢂC; IR (ATR): 1746, 1723, 1522, 1436, 1372,
(ESIþ) calcd for C26H29N3NaO6 (M
þ
Na)þ 502.1954, found
502.1942.
8: Yellow foam. Mp ¼ 77e78 ꢂC; IR (ATR): 1705,1424,1369,1277,
1245, 1151 cmꢀ1; 1H NMR (400 MHz, DMSO-d6): 1.30 (s, 18H, 6CH3
Boc), 3.70 (s, 3H, CH3), 5.79 (br s, 1H), 6.71 (br s, 1H), 6.87 (dd, 1H,
J ¼ 7.6 Hz, J ¼ 0.8 Hz), 7.14 (d, 1H, J ¼ 7.8 Hz), 7.25 (s, 1H), 7.37 (t, 1H,
J ¼ 8.0 Hz), 7.49 (s, 1H), 7.63 (t, 2H, J ¼ 7.8 Hz), 7.71 (t, 1H, J ¼ 7.8 Hz),
7.82 (d, 2H, J ¼ 7.8 Hz), 7.92 (d, 1H, J ¼ 7.6 Hz), 11.02 (br s, 1H, NH);
13C NMR (100 MHz, DMSO-d6): 27.4 (6CH3 Boc), 52.0 (CH3 ester),
82.0 (2C Boc), 109.5 (C), 112.1 (CH), 115.4 (CH), 118.9 (C), 120.2 (CH),
121.3 (CH), 122.0 (CH), 123.7 (C), 124.0 (C), 126.5 (2CH), 126.7 (2CH),
129.9 (2CH), 131.1 (C), 134.1 (CH), 134.6 (CH), 136.9 (C), 138.0 (C),
151.5 (2C]O Boc), 166.9 (C]O ester); HRMS (ESIþ) calcd for
C32H35N3NaO8S (M þ Na)þ 644.2043, found 644.2047.
1354, 1150 cmꢀ1 1H NMR (400 MHz, DMSO-d6): 1.66 (s, 9H, 3CH3
;
Boc), 3.85 (s, 3H, CH3), 7.61 (t,1H, J ¼ 8.0 Hz), 8.05 (d, 1H, J ¼ 8.0 Hz),
8.32 (s, 1H), 8.53 (d, 1H, J ¼ 8.0 Hz), 8.57 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): 27.4 (3CH3 Boc), 53.5 (CH3 ester), 86.1 (C Boc), 112.3 (C),
112.6 (C), 120.3 (CH), 120.4 (C), 120.8 (CH), 124.9 (CH), 131.2 (CH),
134.8 (CH), 136.1 (C), 142.3 (C), 147.7 (C]O Boc), 162.7 (C]O ester);
HRMS (ESIþ) calcd for C17H1779BrN2NaO6 (M þ Na)þ 447.0168,
found 447.0182.
4.1.11. Methyl 2-bromo-3-[4-amino-1-(tert-butoxycarbonyl)-1H-
indol-3-yl]prop-2-enoate 13
4.1.8. Methyl 6-(2,6-diaminophenyl)-1H-indole-4-carboxylate 9
Compound 7 (21 mg, 0.04 mmol) was placed at room temper-
ature under argon and TFA (1 mL) was added. The solution became
rapidly red and orange and was stirred at room temperature for
2.5 h before addition of a 5% aqueous NaHCO3 solution to adjust the
pH to 7. After extraction with EtOAc, the combined organic fractions
were dried over MgSO4 and evaporated. The crude residue was
purified by column chromatography (EtOAc/cyclohexane, 1:1 and
2:1) to give 9 (9 mg, 0.03 mmol, 73%) as a yellow oil. IR (ATR): 3352,
To a solution of compound 12 (100 mg, 0.23 mmol) in iso-
propanol (6 mL) were successively added iron powder (79 mg,
1.41 mmol), ammonium chloride (5 mg, 0.09 mmol) and water
(0.6 mL). The mixture was vigorously stirred at 60 ꢂC for 4 h. The
reaction mixture was filtered through a short pad of Celite and was
washed with water and EtOAc. The product was extracted with
EtOAc and the combined organic fractions were dried over MgSO4
and evaporated. Residue was purified by column chromatography
(EtOAc/cyclohexane, from 1:4 to 1:3) to give 13 (64 mg, 0.16 mmol,
69%) as a green-yellow solid. Mp ¼ 47e48 ꢂC; IR (ATR): 1720, 1437,
1698, 1461, 1431, 1356, 1169 cmꢀ1 1H NMR (400 MHz, DMSO-d6):
;
3.88 (s, 3H, CH3), 4.14 (br s, 4H, 2NH2), 6.04 (d, 2H, J ¼ 8.0 Hz), 6.75
(t, 1H, J ¼ 8.0 Hz), 6.96e6.98 (m, 1H), 7.49 (t, 1H, J ¼ 1.6 Hz), 7.55e
7.57 (m, 2H), 11.49 (br s, 1H, NH); 13C NMR (100 MHz, DMSO-d6):
51.5 (CH3), 102.0 (CH), 103.7 (2CH), 111.7 (C), 118.6 (CH), 121.1 (C),
124.8 (CH), 126.1 (C), 127.7 (2C), 127.8 (CH), 128.0 (CH), 137.7 (C),
145.9 (C), 167.1 (C]O); HRMS (ESIþ) calcd for C16H16N3O2 (M þ H)þ
282.1243, found 282.1242.
1370, 1150 cmꢀ1 1H NMR (400 MHz, DMSO-d6): 1.63 (s, 9H, 3CH3
;
Boc), 3.83 (s, 3H, CH3), 5.18e5.19 (br s, 2H, NH2), 6.64 (d, 1H,
J ¼ 8.0 Hz), 7.10 (d, 1H, J ¼ 8.0 Hz), 7.44 (d, 1H, J ¼ 8.0 Hz), 8.48 (s,
1H), 8.82 (s, 1H); 13C NMR (100 MHz, DMSO-d6): 27.5 (3CH3 Boc),
53.3 (CH3 ester), 84.7 (C Boc), 108.8 (CH), 110.7 (C), 111.3 (CH), 114.7
(C), 116.8 (C), 126.0 (CH), 127.4 (CH), 134.2 (CH), 135.5 (C), 148.6 (C]
O Boc), 160.0 (C), 163.1 (C]O ester); HRMS (ESIþ) calcd for
C17H2079BrN2O4 (M þ H)þ 395.0606, found 395.0604.
4.1.9. Methyl 2-(1H-pyrrol-3-yl)-3-{4-(tert-butoxycarbonyl)amino]-
1H-indol-3-yl}propanoate 10
To a solution of 8 (69 mg, 0.11 mmol) in anhydrous MeOH
(4.5 mL) at 0 ꢂC under argon were added magnesium turnings
(13 mg, 0.55 mmol). The reaction mixture was stirred at room
temperature for 2.5 h. A saturated aqueous NH4Cl solution was
added and the product was extracted with EtOAc. The combined
organic fractions were dried over MgSO4 and evaporated. Crude
residue was purified by column chromatography (CH2Cl2 then
CH2Cl2/EtOAc, 95:5 and 90:10) to give 10 (20 mg, 0.05 mmol, 47%)
4.1.12. Methyl 2-bromo-3-[4-azido-1-(tert-butoxycarbonyl)-1H-
indol-3-yl]prop-2-enoate 14
To a solution of compound 13 (423 mg, 1.07 mmol) in AcOH
(15 mL) at 0 ꢂC was slowly added a cold solution of sodium nitrite
(81 mg, 1.18 mmol) in water (3 mL). The mixture was stirred at 0 ꢂC
for 10 min and a cold solution of sodium azide (76 mg,1.18 mmol) in
water (3 mL) was slowly added. This solution was then stirred at
0 ꢂC for 1.5 h. A saturated aqueous NaCl solution was added and the

F. Giraud et al. / European Journal of Medicinal Chemistry 56 (2012) 225e236
233
product was extracted with CH2Cl2. The combined organic fractions
4.1.15. Methyl 3-{4-amino-1-(tert-butoxycarbonyl)-1H-indol-3-
were dried over MgSO4 and evaporated under reduced pressure
(cold water bath to avoid possible explosion). Residue was purified
by column chromatography (CH2Cl2) to give 14 (439 mg,1.04 mmol,
97%) as a pale brown powder. Mp ¼ 150e151 ꢂC; IR (ATR): 2128,
1732, 1700, 1435, 1372, 1152, 759 cmꢀ1; 1H NMR (400 MHz, CDCl3):
1.69 (s, 9H, 3CH3 Boc), 3.94 (s, 3H, CH3), 7.09 (dd, 1H, J ¼ 0.8 Hz,
J ¼ 8.0 Hz), 7.37 (t, 1H, J ¼ 8.0 Hz), 8.00 (d, 1H, J ¼ 8.0 Hz), 8.81 (s,
1H), 9.15 (s, 1H); 13C NMR (100 MHz, CDCl3): 28.1 (3CH3 Boc), 53.5
(CH3 ester), 85.3 (C Boc), 111.0 (C), 111.9 (CH), 112.8 (CH), 120.6 (C),
125.8 (CH), 128.7 (CH), 133.3 (C), 133.8 (CH), 137.8 (C), 142.6 (C),
146.9 (C]O Boc), 163.9 (C]O ester); HRMS (ESIþ) calcd for
C17H1779BrN4NaO4 (M þ Na)þ 443.0331, found 443.0340.
yl}-2-(1H-pyrrol-3-yl)prop-2-enoate 17
To a solution of compound 16 (76 mg, 0.14 mmol) in THF (3 mL)
at 0 ꢂC under argon was slowly added a 1 M solution of TBAF in
THF (1.41 mL, 1.41 mmol). The solution was stirred at 0 ꢂC for
15 min. A saturated aqueous NaCl solution was added and the
product was extracted with EtOAc. The combined organic fractions
were dried over MgSO4 and evaporated. Residue was purified by
column chromatography (EtOAc/cyclohexane, 2:3) to give 17
(48 mg, 0.12 mmol, 89%) as a orange yellow. Mp ¼ 41e42 ꢂC; IR
(ATR): 3381, 1732, 1698, 1614, 1492, 1437, 1370, 1153 cmꢀ1; 1H NMR
(400 MHz, DMSO-d6): 1.54 (s, 9H, 3CH3 Boc), 3.72 (s, 3H, CH3), 5.04
(br s, 2H, NH2), 5.91e5.93 (m, 1H), 6.52 (d, 1H, J ¼ 8.0 Hz, H5),
6.71e6.72 (m, 1H), 6.76e6.78 (m, 1H), 7.01 (t, 1H, J ¼ 8.0 Hz), 7.08
(s, 1H), 7.35 (d, 1H, J ¼ 8.0 Hz), 7.92 (d, 1H, J ¼ 1.2 Hz), 10.92 (br s,
1H, NH); 13C NMR (100 MHz, DMSO-d6): 27.5 (3CH3 Boc), 51.7 (CH3
ester), 83.6 (C Boc), 104.1 (CH), 107.8 (CH), 109.6 (CH), 116.1 (2C),
116.9 (C), 117.4 (CH), 118.1 (CH), 123.5 (CH), 125.4 (CH), 127.5 (C),
129.7 (CH), 135.6 (C), 142.7 (C), 148.6 (C]O Boc), 168.1 (C]O
ester); HRMS (ESIþ) calcd for C21H24N3O4 (M þ H)þ 382.1767,
found 382.1776.
4.1.13. Methyl 3-{4-azido-1-(tert-butoxycarbonyl)-1H-indol-3-yl}-
2-[1-(triisopropylsilyl)-1H-pyrrol-3-yl]prop-2-enoate 15
To a solution of compounds 14 (420 mg, 0.99 mmol) and 5
(485 mg, 1.39 mmol) in 1,4-dioxane (15 mL) at room temperature
under argon were added water (5 mL) and K2CO3 (547 mg,
3.96 mmol). The solution was degassed with argon for 20 min,
tetrakis(triphenylphosphine)palladium(0) (114 mg, 0.10 mmol) was
added. Reaction mixture was heated overnight at 70 ꢂC. Water and
EtOAc were added and the mixture was filtered through a short pad
of Celite which was next washed several times with EtOAc. The
filtrate was washed with a saturated aqueous NaCl solution and the
organic fraction was dried over MgSO4 and evaporated. Residue
was purified by column chromatography (Et2O/pentane, 1:6) to
give 15 (338 mg, 0.60 mmol, 60%) as a pale yellow solid. Mp ¼ 45e
46 ꢂC; IR (ATR): 2943, 2866, 2127, 1731, 1700, 1535, 1435, 1371, 1151,
779 cmꢀ1; 1H NMR (400 MHz, CDCl3): 1.07 (t, 18H, J ¼ 7.6 Hz, 6CH3
iPr), 1.42 (sept, 3H, J ¼ 7.6 Hz, 3CH iPr), 1.56 (s, 9H, 3CH3 Boc), 3.82
(s, 3H, CH3), 6.30 (d, 1H, J ¼ 1.6 Hz), 6.69 (d, 1H, J ¼ 1.6 Hz), 6.80 (d,
1H, J ¼ 1.6 Hz), 7.05 (dd, 1H, J ¼ 8.0 Hz, J ¼ 0.8 Hz), 7.30 (t, 1H,
J ¼ 8.0 Hz), 7.41 (d,1H, J ¼ 1.2 Hz), 7.91 (d,1H, J ¼ 8.0 Hz), 8.35 (d,1H,
J ¼ 1.2 Hz); 13C NMR (100 MHz, CDCl3): 11.6 (3CH iPr), 17.8 (6CH3
iPr), 28.0 (3CH3 Boc), 52.1 (CH3 ester), 84.2 (C Boc), 111.5 (CH), 111.8
(CH), 112.4 (CH), 115.8 (C), 119.6 (2C), 121.5 (C), 124.0 (CH), 124.1
(CH), 125.1 (CH), 126.4 (C), 126.7 (CH), 130.9 (CH), 136.4 (C), 148.9
(C]O Boc), 169.0 (C]O ester); HRMS (ESIþ) calcd for C30H42N5O4Si
(M þ H)þ 564.3006, found 564.2990.
4.1.16. Methyl 6-amino-1,10-dihydropyrrolo[2,3-a]carbazole-4-
carboxylate 19
To a solution of compound 17 (72 mg, 0.19 mmol) in a mixture
of MeOH/water 3:1 was added K2CO3 (77 mg, 0.56 mmol) at room
temperature. The reaction mixture was stirred under reflux for
25 min. Saturated NH4Cl solution and EtOAc were added and the
product was extracted at pH 7e8. Organic layers were dried over
MgSO4 and evaporated. Residue was purified by column chroma-
tography (EtOAc/cyclohexane, 1:1) and directly engaged in the
next step.
To a solution of this compound (36 mg, 0.13 mmol) in freshly
distilled CH3CN (150 mL) in a Pyrex reactor was added one crystal
of iodine. The solution was degassed for 45 min with argon and
irradiated for 20 min. The solvent was removed under reduced
pressure. Residue was purified by column chromatography
(MeOH/CH2Cl2, 1:99) to give 19 (21 mg, 0.08 mmol, 41% from 17)
as a light brown solid. Mp ¼ 217e218 ꢂC; IR (ATR): 3381, 1678,
1646, 1514, 1439, 1388, 1151 cmꢀ1 1H NMR (400 MHz, DMSO-d6):
;
3.92 (s, 3H, CH3), 5.40 (br s, 2H, NH2), 6.52 (d, 1H, J ¼ 7.2 Hz, H7),
6.91 (d, 1H, J ¼ 8.0 Hz, H9), 7.09 (t, 1H, J ¼ 7.8 Hz, H8), 7.11 (dd, 1H,
J ¼ 2.1 Hz, J ¼ 2.9 Hz, H3), 7.50 (t, 1H, J ¼ 2.4 Hz, H2), 8.58 (s, 1H,
H5), 10.95 (br s, 1H, NH1), 11.13 (br s, 1H, NH10); 13C NMR
(100 MHz, DMSO-d6): 51.2 (CH3), 100.4 (CH), 103.8 (CH), 106.0
(CH), 111.0 (C), 111.9 (C), 116.0 (C), 118.5 (CH), 121.3 (C), 124.6 (C),
125.1 (CH), 125.4 (CH), 128.9 (C), 140.2 (C), 143.4 (C), 166.8 (C]O);
HRMS (ESIþ) calcd for C16H14N3O2 (M þ H)þ 280.1086, found
280.1085.
4.1.14. Methyl 3-{4-amino-1-(tert-butoxycarbonyl)-1H-indol-3-
yl}-2-[1-(triisopropylsilyl)-1H-pyrrol-3-yl]prop-2-enoate 16
To a solution of 15 (128 mg, 0.23 mmol) in MeOH/THF 2:1
(3 mL) at 0 ꢂC under argon was added anhydrous SnCl2 (215 mg,
1.13 mmol). The suspension was stirred at room temperature for
45 min and finally refluxed for 2 h. After cooling to room
temperature, a 1 N aqueous NaOH solution was added and the
product was extracted with EtOAc. The combined organic fractions
were dried over MgSO4 and evaporated. Residue was purified by
column chromatography (EtOAc/cyclohexane, 1:3) to give 16
(75 mg, 0.14 mmol, 61%) as an orange yellow powder. Mp ¼ 92e
93 ꢂC; IR (ATR): 2945, 2866, 1739, 1691, 1478, 1426, 1370,
1154 cmꢀ1; 1H NMR (400 MHz, DMSO-d6): 0.95 (d, 18H, J ¼ 8.0 Hz,
6CH3 iPr), 1.33 (sept, 3H, J ¼ 8.0 Hz, 3CH iPr), 1.52 (s, 9H, 3CH3
Boc), 3.74 (s, 3H, CH3), 5.03 (br s, 2H, NH2), 6.21e6.22 (m, 1H), 6.50
(dd, 1H, J ¼ 8.0 Hz, J ¼ 1.2 Hz), 6.62 (t, 1H, J ¼ 2.0 Hz), 6.78 (t, 1H,
J ¼ 2.0 Hz), 7.01 (t, 1H, J ¼ 8.0 Hz), 7.13 (d, 1H, J ¼ 1.2 Hz), 7.33 (dd,
1H, J ¼ 8.0 Hz, J ¼ 0.8 Hz), 7.82 (d, 1H, J ¼ 2.0 Hz); 13C NMR
(100 MHz, DMSO-d6): 10.6 (3CH iPr), 17.4 (6CH3 iPr), 27.4 (3CH3
Boc), 51.8 (CH3 ester), 83.4 (C Boc), 103.9 (CH), 109.0 (CH), 111.6
(CH), 116.4 (C), 116.9 (C), 118.7 (C), 122.3 (CH), 123.8 (CH), 124.1
(CH), 125.5 (CH), 128.1 (C), 129.4 (CH), 135.8 (C), 142.6 (C), 148.6
(C]O Boc), 167.7 (C]O ester); HRMS (ESIþ) calcd for
C30H44N3O4Si (M þ H)þ 538.3101, found 538.3117.
4.1.17. 6-Amino-1,10-dihydropyrrolo[2,3-a]carbazole-4-carboxylic
acid 20
Compound 19 (9 mg, 0.03 mmol) was dissolved in MeOH
(1.6 mL) and THF (0.4 mL). A 1 N aqueous NaOH solution (1.6 mL)
was added and the mixture was refluxed for 5 h. After cooling to
room temperature, a saturated aqueous NH4Cl solution was added
to adjust the pH to 8e9, and the product was extracted with EtOAc.
The combined organic fractions were dried over MgSO4 and
evaporated. Residue was purified by column chromatography
(EtOAc/cyclohexane, 2:1) to give 20 (5 mg, 0.02 mmol, 60%) as
a dark green solid. Mp ¼ 224e225 ꢂC; IR (ATR): 3391, 1738, 1647,
1559,1464, 1377,1154 cmꢀ1; 1H NMR (400 MHz, DMSO-d6): 5.37 (br
s, 2H, NH2), 6.51 (dd, 1H, J ¼ 1.2 Hz, J ¼ 8.0 Hz), 6.90 (dd, 1H,
J ¼ 1.2 Hz, J ¼ 8.0 Hz), 7.08 (t, 1H, J ¼ 8.0 Hz), 7.12e7.14 (m, 1H),
7.46e7.48 (m, 1H), 8.55 (s, 1H), 10.96 (br s, 1H), 11.16 (br s, 1H), 12.20

234
F. Giraud et al. / European Journal of Medicinal Chemistry 56 (2012) 225e236
(br s, 1H, COOH); 13C NMR (100 MHz, DMSO-d6): 100.2 (CH), 104.0
(CH), 105.8 (CH), 111.1 (C), 113.2* (C), 115.9 (C), 118.5 (CH), 121.2 (C),
124.6 (CH), 124.8 (C), 125.1 (CH), 128.6 (C), 140.1 (C), 143.3 (C), 168.4
(C]O), *chemical shift measured from a 1He13C HMBC experi-
ment; HRMS (ESIþ) calcd for C15H12N3O2 (M þ H)þ 266.0930, found
266.0923.
13C NMR (100 MHz, CDCl3): 28.0 (3CH3 Boc), 52.1 (CH3 ester), 83.8
(C Boc), 111.3 (CH), 112.1 (C), 113.6 (CH), 118.7 (CH), 119.1 (CH), 120.0
(C), 120.4 (CH), 120.9 (CH), 122.3 (C), 122.9 (CH), 126.6 (CH), 127.6
(C), 133.2 (CH), 135.2 (C), 148.7 (C]O Boc), 168.6 (C]O ester);
HRMS (ESIþ) calcd for C21H23N2O4 (M þ H)þ 367.1658, found
367.1662.
4.1.18. 1-(tert-Butoxycarbonyl)-1H-indole-3-carbaldehyde 21
4.1.21. Methyl 3-(1H-indol-3-yl)-2-(1H-pyrrol-3-yl)prop-2-enoate
25
To
a
solution of 1H-indole-3-carbaldehyde (500 mg,
3.44 mmol) in anhydrous acetonitrile (12 mL) at room tempera-
ture was added DMAP (84 mg, 0.69 mmol) and then Boc2O (1.13 g,
5.17 mmol). The mixture was stirred at room temperature over-
night and then solvent was removed under reduced pressure.
Residue was purified by column chromatography (EtOAc/cyclo-
hexane, 1:1) to give 21 (840 mg, 3.42 mmol, 99%) as a beigeꢀs1olid.
To a solution of compound 24 (257 mg, 0.70 mmol) in MeOH
(9 mL) at room temperature were added water (3 mL) and then
K2CO3 (291 mg, 2.10 mmol). The solution was stirred at 70 ꢂC for
50 min. After cooling to room temperature, a 1 N aqueous HCl
solution (5 mL) was added and the product was extracted with
EtOAc. The combined organic fractions were dried over MgSO4 and
evaporated. Residue was purified by column chromatography
(EtOAc/cyclohexane 1:1) to give 25 (143 mg, 0.54 mmol, 76%) as
a yellow powder. Mp ¼ 172e173 ꢂC; IR (ATR): 3375,1691,1516,1457,
1438, 1071 cmꢀ1; 1H NMR (400 MHz, DMSO-d6): 3.68 (s, 3H, CH3),
5.97 (dd, 1H, J ¼ 2.4 Hz, J ¼ 4.0 Hz), 6.67 (dd, 1H, J ¼ 2.0 Hz,
J ¼ 4.0 Hz), 6.85 (dd, 1H, J ¼ 2.4 Hz, J ¼ 4.4 Hz), 6.90 (d, 1H,
J ¼ 2.4 Hz), 7.05 (ddd, 1H, J ¼ 1.2 Hz, J ¼ 7.2 Hz, J ¼ 8.4 Hz), 7.12 (ddd,
1H, J ¼ 1.2 Hz, J ¼ 7.2 Hz, J ¼ 8.4 Hz), 7.36 (d, 1H, J ¼ 8.4 Hz), 7.51 (d,
1H, J ¼ 7.6 Hz), 7.97 (s, 1H), 10.88 (br s, 1H, NH), 11.42 (br s, 1H, NH);
13C NMR (100 MHz, DMSO-d6): 51.4 (CH3), 108.2 (CH), 110.9 (C),
111.7 (CH), 116.7 (CH), 117.4 (C), 117.9 (CH), 118.0 (CH), 119.9 (CH),
120.9 (C),121.8 (CH),127.0 (C),127.2 (CH),131.5 (CH),135.4 (C),168.4
(C]O); HRMS (ESIþ) calcd for C16H15N2O2 (M þ H)þ 267.1134,
found 267.1137.
Mp ¼ 116e117 ꢂC; IR (ATR): 1741, 1676, 1451, 1397, 1134 cm
;
1H
NMR (400 MHz, DMSO-d6): 1.66 (s, 9H, 3CH3 Boc), 7.38e7.47
(m, 2H), 8.14 (2d, 2H, J ¼ 7.6 Hz), 8.67 (s, 1H), 10.08 (s, 1H,
CHO); 13C NMR (100 MHz, DMSO-d6): 27.4 (3CH3 Boc), 85.4
(C Boc), 114.9 (CH), 120.6 (C), 121.3 (CH), 124.4 (CH), 125.5 (C),
125.8 (CH), 135.2 (C), 138.2 (CH), 148.2 (C]O Boc), 187.0 (C]O
aldehyde); HRMS (ESIþ) calcd for C14H15NNaO3 (M
þ
Na)þ
268.0950, found 268.0963.
4.1.19. Methyl 3-[1-(tert-butoxycarbonyl)-1H-indol-3-yl]-2-[1-
(triisopropylsilyl)-1H-pyrrol-3-yl]prop-2-enoate 23
To a solution of compounds 22 (350 mg, 0.92 mmol) and 5
(549 mg, 1.57 mmol) in 1,4-dioxane (15 mL) at room temperature
under argon were added water (5 mL) and K2CO3 (509 mg,
3.68 mmol). The solution was degassed with argon for 20 min,
tetrakis(triphenylphosphine)palladium(0) (106 mg, 0.10 mmol)
was added. Reaction mixture was heated overnight at 70 ꢂC. Water
and EtOAc were added and the mixture was filtered through a short
pad of Celite which was next washed several times with EtOAc. The
filtrate was washed with a saturated aqueous NaCl solution and the
organic fraction was dried over MgSO4 and evaporated. Residue
was purified by column chromatography (CH2Cl2/cyclohexane, 1:2,
1:1, 2:1, and pure CH2Cl2) to give 23 (370 mg, 0.71 mmol, 77%) as
pale yellow solid. Mp ¼ 112e113 ꢂC; IR (ATR): 1741,1708,1456,1370,
1217 cmꢀ1; 1H NMR (400 MHz, CDCl3): 1.06 (d, 18H, J ¼ 7.2 Hz, 6CH3
iPr), 1.38 (sept, 3H, J ¼ 7.2 Hz, 3CH iPr), 1.61 (s, 9H, 3CH3 Boc), 3.82
(s, 3H, CH3), 6.33 (s, 1H), 6.75 (d, 2H, J ¼ 6.0 Hz), 7.17 (t, 1H,
J ¼ 6.8 Hz), 7.28 (t, 1H, J ¼ 6.8 Hz), 7.48 (d, 1H, J ¼ 8.0 Hz), 7.56 (s,
1H), 7.86 (s, 1H), 8.08 (d, 1H, J ¼ 8.0 Hz); 13C NMR (100 MHz, CDCl3):
11.5 (3CH iPr), 17.7 (6CH3 iPr), 28.0 (3CH3 Boc), 52.1 (CH3 ester), 83.8
(C Boc), 108.0 (C), 111.8 (CH), 115.0 (CH), 116.5 (C), 119.6 (C), 119.8
(CH), 122.8 (CH), 123.9 (CH), 124.5 (CH), 124.6 (CH), 126.7 (CH),
126.8 (C), 128.8 (CH), 129.7 (C), 149.3 (C]O Boc), 169.0 (C]O ester);
HRMS (ESIþ) calcd for C30H42N2NaO4Si (M þ Na)þ 545.2812, found
545.2833.
4.1.22. 3-(1H-Indol-3-yl)-2-(1H-pyrrol-3-yl)prop-2-enoic acid 26
If the previous experiment was performed for a longer period
(1.5e2 h), some hydrolysis of ester 25 was observed and 26 was
isolated in 12e15% yield as a yellow powder. Mp ¼ 191e192 ꢂC; IR
(ATR): 3376, 1653, 1507, 1458, 1417, 1072 cmꢀ1; 1H NMR (400 MHz,
DMSO-d6): 5.97 (d, 1H, J ¼ 1.2 Hz), 6.65 (d, 1H, J ¼ 2.4 Hz), 6.82
(d, 1H, J ¼ 2.0 Hz), 6.93 (d, 1H, J ¼ 2.4 Hz), 7.04 (dd, 1H, J ¼ 0.4 Hz,
J ¼ 7.6 Hz), 7.11 (dd,1H, J ¼ 0.4 Hz, J ¼ 7.6 Hz), 7.35 (d,1H, J ¼ 8.0 Hz),
7.51 (d, 1H, J ¼ 8.0 Hz), 7.93 (s, 1H), 10.83 (br s, 1H, NH), 11.36 (br s,
1H, NH), 11.93 (br s, 1H, COOH); 13C NMR (100 MHz, DMSO-d6):
108.3 (CH), 111.0 (C), 111.7 (CH), 116.8 (CH), 118.0 (CH), 118.7 (CH),
119.8 (CH), 121.7 (CH), 122.0 (C), 124.4 (C), 126.8 (CH), 127.1 (C),
130.7 (CH), 135.4 (C), 169.4 (C]O); HRMS (ESIþ) calcd for
C15H12N2NaO2 (M þ Na)þ 275.0796, found 275.0797.
4.1.23. Methyl 1,10-dihydropyrrolo[2,3-a]carbazole-4-carboxylate
27 and methyl 6-(2-aminophenyl)-1H-indole-4-carboxylate 28
To a solution of compound 25 (44 mg, 0.17 mmol) in freshly
distilled CH3CN (150 mL) in a Pyrex reactor was added one crystal
of iodine. The solution was degassed for 45 min with argon and
irradiated for 20 min. The solvent was removed under reduced
pressure and residue was purified by column chromatography
(MeOH/CH2Cl2, 1:99 to 5:95) to give 27 (26 mg, 0.10 mmol, 59%) as
black powder and 28 (13 mg, 0.05 mmol, 29%) as a brown-pink
solid.
4.1.20. Methyl 3-[1-(tert-butoxycarbonyl)-1H-indol-3-yl]-2-(1H-
pyrrol-3-yl)prop-2-enoate 24
To a solution of compound 23 (147 mg, 0.28 mmol) in THF (4 mL)
at 0 ꢂC under argon was slowly added a 1 M solution of TBAF in THF
(2.81 mL, 2.81 mmol). The solution was stirred at 0 ꢂC for 10 min
and then refluxed for 2.5 h. A saturated aqueous NaCl solution was
added and the product was extracted with EtOAc. The combined
organic fractions were dried over MgSO4 and evaporated. Residue
was purified by column chromatography (EtOAc/cyclohexane, 1:2)
to give 24 (95 mg, 0.26 mmol, 92%) as a pale yellow solid.
Mp ¼ 135e136 ꢂC; IR (ATR): 3248, 1742, 1684, 1399, 1372,
1154 cmꢀ1; 1H NMR (400 MHz, CDCl3): 1.62 (s, 9H, 3CH3 Boc), 3.85
(s, 3H, CH3), 6.24 (s, 1H), 7.06 (s, 1H), 7.26e7.28 (m, 3H), 7.37 (d, 2H,
J ¼ 7.2 Hz), 7.80 (d, 1H, J ¼ 6.4 Hz), 8.23 (s, 1H), 8.47 (br s, 1H, NH);
Compound 27: Mp ¼ 246e247 ꢂC; IR (ATR): 3324, 2919, 1644,
1557, 1509, 1437, 1156 cmꢀ1 1H NMR (500 MHz, DMSO-d6): 3.92
;
(s, 3H, CH3), 7.15 (t,1H, J ¼ 2.5 Hz, H3), 7.21 (t,1H, J ¼ 7.4 Hz, H7), 7.37
(t, 1H, J ¼ 7.5 Hz, H8), 7.53 (t, 1H, J ¼ 2.7 Hz, H2), 7.66 (d, 1H,
J ¼ 8.0 Hz, H9), 8.16 (d, 1H, J ¼ 7.6 Hz, H6), 8.56 (s, 1H, H5), 11.13 (br s,
1H, NH1), 11.31 (br s, 1H, NH10); 13C NMR (100 MHz, DMSO-d6): 51.2
(CH3), 104.0 (CH), 111.5 (CH), 112.6 (C), 115.8 (C), 116.9 (CH), 119.4
(CH), 119.6 (CH), 121.5 (C), 123.9 (C), 124.3 (CH), 125.3 (CH), 125.4
(C), 129.6 (C), 138.8 (C), 167.5 (C]O ester); HRMS (ESIþ) calcd for
C16H12N2NaO2 (M þ Na)þ 287.0796, found 287.0800.

F. Giraud et al. / European Journal of Medicinal Chemistry 56 (2012) 225e236
235
Compound 28: Mp ¼ 70e71 ꢂC; IR (ATR): 3359, 2925, 1694,
1559, 1495, 1437, 1171 cmꢀ1; 1H NMR (400 MHz, DMSO-d6): 3.89 (s,
3H, CH3), 4.77 (br s, 2H, NH2), 6.65 (t, 1H, J ¼ 7.6 Hz), 6.77 (d, 1H,
J ¼ 8.0 Hz), 6.95 (s, 1H), 7.02e7.07 (m, 2H), 7.56 (s, 1H), 7.70 (s, 1H),
7.77 (s, 1H), 11.48 (br s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): 51.5
(CH3), 101.9 (CH), 115.0 (CH), 116.5 (CH), 116.6 (CH), 120.3 (C), 123.2
(CH), 125.8 (C), 127.9 (CH), 128.0 (CH), 130.1 (C), 131.6 (CH), 137.1
(2C), 145.1 (C), 167.1 (C]O); HRMS (ESIþ) calcd for C16H15N2O2
(M þ H)þ 267.1134, found 267.1135.
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. Molecular modeling experiments
Modeller9V10 software [32] from the module included in UCSF
Chimera molecular modeling system [26,33] was used to generate
the 3D model of Pim-3. The model was generated using two
program options taking into account the water molecules of the
model and introducing a refinement on the possible loops. Before
docking experiments, the Pim-3 model obtained by this method
was processed under Sybylx2.0 software [34], using the biopolymer
module, to add properly all hydrogen atoms and to verify that none
of the residues were subjected to steric clashes. An energy mini-
mization calculation was then performed under Tripos force field,
with Gasteiger-Hückel charges, and finally water molecules were
removed from the model. For docking experiments in Sybylx2.0,
the threshold and bloat values were respectively set to 0.05 and 9.
The surflex module was used with GEOM X options allowing H and
heavy atom movement.
4.1.24. 1,10-Dihydropyrrolo[2,3-a]carbazole-4-carboxylic acid 29
To a solution of compound 27 (32 mg, 0.12 mmol) in MeOH
(8 mL) and THF (2 mL) was added at room temperature a 1 N
aqueous NaOH solution (6 mL) and the mixture was refluxed for
2.5 h. After cooling to room temperature, the mixture was acidified
with a 1 N aqueous HCl solution and the product was extracted
with EtOAc. The combined organic fractions were extracted with
a 1 N aqueous NaOH solution (3 ꢃ 20 mL), and the aqueous phase
was acidified to pH 4 with a 1 N aqueous HCl solution and extracted
with EtOAc. The combined organic fractions were dried over MgSO4
and evaporated. Residue was purified by column chromatography
(EtOAc/cyclohexane, 1:1) to give 29 (23 mg, 0.09 mmol, 76%) as
a beige powder. Mp ¼ 234e235 ꢂC; IR (ATR): 3401, 2925, 1653,
1558, 1506, 1457, 1165 cmꢀ1 1H NMR (400 MHz, DMSO-d6): 7.15
;
4.4. Antiproliferative activities
(t, 1H, J ¼ 2.8 Hz), 7.21 (t, 1H, J ¼ 7.2 Hz), 7.36 (t, 1H, J ¼ 7.2 Hz), 7.49
(t,1H, J ¼ 2.8 Hz), 7.64 (d, 1H, J ¼ 8.0 Hz), 8.14 (d, 1H, J ¼ 8.0 Hz), 8.52
(s, 1H), 11.05 (br s, 1H, NH), 11.24 (br s, 1H, NH), 12.26 (br s, 1H,
COOH); 13C NMR (100 MHz, DMSO-d6): 104.3 (CH), 111.5 (CH), 113.7
(C), 115.7 (C), 117.0 (CH), 119.3 (CH), 119.5 (CH), 121.5 (C), 124.0 (C),
124.2 (CH), 125.0 (CH), 125.7 (C), 129.4 (C), 138.8 (C), 168.8 (C]O);
HRMS (ESIþ) calcd for C15H10N2NaO2 (M þ Na)þ 273.0640, found
273.0653.
4.4.1. Cell cultures
Stock cell cultures were maintained as monolayers in 75-cm2
culture flasks in Glutamax Eagle’s minimum essential medium
(MEM) with Earle’s salts supplemented with 10% fetal calf serum,
5 mL 100 mM sodium pyruvate, 5 mL of 100ꢃ non-essential amino
acids and 2 mg gentamicin base. Cells were grown at 37 ꢂC in
a humidified incubator under an atmosphere containing 5% CO2.
4.4.2. Survival assays
4.2. Kinase assays
Cells were plated at a density of 5 ꢃ 103 cells in 150
mL culture
medium in each well of 96-well microplates and were allowed to
adhere for 16 h before treatment with tested drug. A stock solution
20 mM of each tested drug was prepared in DMSO and kept
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 [25].
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.
at ꢀ20 ꢂC until use. Then 50
mL of each tested solution were added
to the cultures. A 48 h continuous drug exposure protocol was used.
The antiproliferative effect of the tested drug was assessed by the
resazurin reduction test.
4.4.3. Resazurin reduction test
Plates were rinsed with 200
overturning on absorbent toweling. Then 150
m
L PBS at 37 ꢂC and emptied by
L of a 25 g/mL
m
m
solution of resazurin in MEM without phenol red was added to each
well. Plates were incubated for 1 h at 37 ꢂC in a humidified atmo-
sphere containing 5% CO2. Fluorescence was then measured on an
automated 96-well plate reader (Fluoroscan Ascent FL, Labsystem)
using an excitation wavelength of 530 nm and an emission wave-
length of 590 nm. Under the conditions used, fluorescence was
Protein kinase assays: All assays (25.5 mL volume) were carried
out robotically at room temperature (21 ꢂC) 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
acetate in the assays was 10 mM and [
was used at 5 M for Pim-2 and 20
g-
33P]ATP (800 c.p.m./pmol)
mM for Pim-1 and Pim-3, in
proportional to the number of living cells in the well. The IC50
,
defined as the drug concentration required to inhibit cell prolifer-
ation by 50%, was calculated from the curve of concentration-
dependent survival percentage, defined as fluorescence in experi-
mental wells compared with fluorescence in control wells, after
subtraction of the blank values.
m
order to be at or below the Km for ATP for each enzyme.
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 and
mL of 0.5 M orthophosphoric acid and spotted on to P81 filter
m
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.
The inhibition profile of the tested compounds was expressed as
the percentage of the residual kinase activity for an inhibitor
Acknowledgments
CNRS Valorisation (R. A.-G.) and “Région Auvergne/FEDER”
(Synbio Project) (N. C.) are greatly acknowledged for financial
support. We are grateful to Bertrand Légeret for mass spectrometry
analysis.

236
F. Giraud et al. / European Journal of Medicinal Chemistry 56 (2012) 225e236
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(2002) 589e591.
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[16] K. Singh, K. Singh, Tetrahedron Lett. 40 (2009) 2219e2221.
[17] S. Lee, S.B. Park, Org. Lett. 11 (2009) 5214e5217.
[18] G. Bringmann, S. Tasler, H. Endress, K. Peters, E.-M. Peters, Synthesis (1998)
1501e1505.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.ejmech.2012.
08.029.
[19] A. Friedrich, S. Bräse, S.E. O’Connor, Tetrahedron Lett. 50 (2009) 75e76.
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