Synthesis and biological activities of pyrazolo[3,4-g]quinoxaline derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.08.067

The synthesis of new pyrazolo[3,4-g]quinoxaline derivatives, as well as their Pim kinases (Pim-1, Pim-2, Pim-3) inhibitory potencies and in vitro antiproliferative activities toward a human fibroblast primary culture and three human solid cancer cell lines (PA1, PC3 and DU145) are described. The results obtained in this preliminary structure-activity relationship study have pointed out that most of the compounds in this series exhibited interesting in vitro Pim-3 kinase inhibitory potencies. Moreover, some of the tested compounds have demonstrated favorable antiproliferative potencies.

Full text of DOI:10.1016/j.ejmech.2010.08.067

European Journal of Medicinal Chemistry 45 (2010) 5520e5526
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and biological activities of pyrazolo[3,4-g]quinoxaline derivatives
,
b
,
,
b
,
,
,b,
Laurent Gavara a 1, Emmanuelle Saugues a 1, Georges Alves c d, Eric Debiton e, Fabrice Anizon a
**
,
*
Pascale Moreau a
,b,
a Clermont Université, Université Blaise Pascal, SEESIB, BP 10448, F-63000 Clermont-Ferrand, France
b CNRS, UMR 6504, SEESIB, F-63177 Aubière, France
c Clermont Université, Université Blaise Pascal, GReD, BP10448, F-63000 Clermont-Ferrand, France
d CNRS, UMR 6247, GReD, Aubière, France
e Clermont Université, Université d’Auvergne, UMR INSERM U990, BP 184, 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:
The synthesis of new pyrazolo[3,4-g]quinoxaline derivatives, as well as their Pim kinases (Pim-1, Pim-2,
Pim-3) inhibitory potencies and in vitro antiproliferative activities toward a human fibroblast primary
culture and three human solid cancer cell lines (PA1, PC3 and DU145) are described. The results obtained
in this preliminary structureeactivity relationship study have pointed out that most of the compounds in
this series exhibited interesting in vitro Pim-3 kinase inhibitory potencies. Moreover, some of the tested
compounds have demonstrated favorable antiproliferative potencies.
Received 15 July 2010
Received in revised form
30 August 2010
Accepted 31 August 2010
Available online 15 September 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Pyrazolo[3,4-g]quinoxaline
Kinase inhibition
In vitro antiproliferative activities
1. Introduction
more particularly kinase inhibitory potency. Thus, pyrazolo[3,4-g]
quinoxalines, substituted on the pyrazine moiety (C-6 and C-7
As part of our ongoing studies focused on the preparation of
potential biologically active compounds, and more particularly Pim
kinases inhibitors, we were interested in the synthesis of new pyr-
azolo[3,4-g]quinoxaline derivatives. We have previously reported
the preparation and biological activities of pyrrolocarbazole deriv-
atives that have shown potent inhibitory potencies toward the Pim
kinases [1,2]. These kinases have been described to play diverse
biological roles in cell survival, proliferation and differentiation
[1e3]. In this paper, we focus on the preparation of 1H-pyrazolo[3,4-
g]quinoxaline derivatives by condensation of 1H-indazole-5,6-
positions) by alkyl or aryl groups, were reported by Merck & Co. as
Akt inhibitors [4e8]. Other derivatives were also described as
tyrosine kinase or PKC inhibitors [9,10]. Two additional reports
accounted for the synthesis of substituted pyrazolo[3,4-g]quinoxa-
lines or pyrazolo[3,4-g]quinoxalin-4,9-diones [11,12]. Finally, this
aromatic scaffold was rarely found incorporated into a more
complex heteroaromatic structure [13e15]. Accordingly, we
synthesized new pyrazolo[3,4-g]quinoxaline derivatives in which
the pyrazine moiety was either substituted by ethyl groups or fused
to a non-aromatic 5- or 6-membered ring. Then, the inhibitory
potencies of these new compounds toward Pim-1, Pim-2 and Pim-3
kinases were evaluated. In addition, these pyrazoloquinoxaline
derivatives were assayed for their in vitro antiproliferative activities
toward a human fibroblast primary culture and three human solid
cancer cell lines: PA1 (ovarian carcinoma), PC3 and DU145 (prostatic
carcinoma).
diamine with a-chloroketones and 1,2-diketones. To the best of our
knowledge, there are only a few reports in the literature accounting
for the synthesis of these derivatives. It has been shown that this
family of compounds could display interesting biological activity,
* Corresponding author. Clermont Université, Université Blaise Pascal, SEESIB, BP
10448, F-63000 Clermont-Ferrand, France. Tel.: þ33 (0) 4 73 40 53 64; fax: þ33 (0)
4 73 40 77 17.
2. Chemistry
** Corresponding author. Clermont Université, Université Blaise Pascal, SEESIB, BP
10448, F-63000 Clermont-Ferrand, France. Tel.: þ33 (0) 4 73 40 79 63; fax: þ33 (0)
4 73 40 77 17.
The synthesis of compounds 11e17 was carried out starting from
1H-indazole-5,6-diamine 3. The preparation of 3 was initially
reported by Fries et al. from 6-nitroindazole 1 by nitration using
H2SO4/HNO3 and subsequent reduction with zinc dichloride in the
presence of hydrochloric acid [16]. Starting from 1, we applied other
E-mail addresses: fabrice.anizon@univ-bpclermont.fr (F. Anizon), pascale.
moreau@univ-bpclermont.fr (P. Moreau).
1
Both authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.067

L. Gavara et al. / European Journal of Medicinal Chemistry 45 (2010) 5520e5526
5521
Table 1
Reaction conditions and yields for the preparation of compounds 11e17. Conditions: (A) MeCN, PTSA; (B) THF, NEt3; (C) AcOH.
1,2-Diketone or
a
-chloroketone
Conditions
Yield
(%)
Product
N
N
O
N
4
A
32
22
11
12
N
H
Cl
N
O
N
5
6
B
N
N
Cl
H
O
Et
Et
N
Et
N
Et
A
50
13
N
N
H
O
H
N
N
N
N
O
14a
14b
N
N
Me
Me
7
A
35, 50
O
Me
N
H
N
N
N
O
O
Ph
N
Ph
8
C
33
15
N
H
Me
Me
H
N
N
Me
N
N
16a
16b
O
N
N
9
C
32
O
N
H
N
Me
Me
H
N
N
Me
Me
Me
N
N
17a
17b
O
O
N
N
Me
10
C
30
N
H
N

5522
L. Gavara et al. / European Journal of Medicinal Chemistry 45 (2010) 5520e5526
NOE
1C0.0H3
8.03
8.05
H2.86
H
H
15.0
13.30
1.33
1.41 H3C
2.06–2.18H
1.30 H3C
13.33
H
H
52.8/53.3
106.1
105.8
2.00–2.10
H
N
N
N
N
45.4
139.1
135.0
139.2
N
N
139.4
139.1
164.1
158.9
31.6
165.6
H
H
1.30–1.40
39.6
N
N
161.9
24.3
124.9
124.2
18.4
135.4
134.4
134.3
38.8
119.2
52.1
119.1
8.39
2.23–2.31
H
0.56/1.09
18.0/20.0
H
8.43
H
8.40
H
H
H
52.8/53.3
8.44
H
H
2.75
3.16
3.00
17a
14b
Fig. 1. 1H and 13C assignments of compounds 14b and 17a. Chemical shift values are given in ppm. Relevant 1He13C, 1He15N and NOE correlations are indicated by arrows.
reaction conditions for the preparation of compound 3, with
a substantial yield improvement (Scheme 1). Nitration of commer-
cially available 6-nitroindazole 1 was performed using H2SO4 and
potassium nitrate [17] to give 2 in 84% yield after recrystallization
from glacial acetic acid (in comparison, Fries et al., 70% [16]). Nitro
groups were subsequently reduced using ammonium formate in the
presence of 10% Pd/C togive 3 in 86% yield (in comparison, Fries et al.,
70% [16]). Diamine 3 was further used for condensation reactions
an acidic medium. The condensation with hexane-3,4-dione 6 was
performed in acetonitrile in the presence of a catalytic amount of
PTSA to give 13 in moderate yield. Using the same reaction proce-
dure, the condensation with camphorquinone
7 led to the
regioisomers 14a and 14b which were successfully separated by
chromatography to give 14a and 14b in 35% and 50% yields,
respectively. Unfortunately, the use of PTSA in acetonitrile with 5-
membered ring diketones 8 [19,20], 9 and 10 led to degradation
products. Therefore, the condensation reactions were performed in
acetic acid according to literature procedure [21]. Thus, compounds
15e17 were obtained in 30e33% yields. As in the case of cam-
with various 1,2-diketones or a-chloroketones to give 1H-pyrazolo
[3,4-g]quinoxaline derivatives 11e17 in moderate to good yields
(22e85%) (Scheme 1, Table 1). Condensation with 2-chlor-
α-chloroketones 4 and 5
O N
2
Pd/C, HCO NH
2 4
H N
2
or
H SO , KNO
MeOH
2
4
3
1,2 diketones 6–10
N
11 – 17
N
N
84%
86%
22–85%
N
H
N
H
N
H
O N
2
O N
2
H N
2
1
2
3
Scheme 1. Synthesis of 1H-pyrazolo[3,4-g]quinoxaline derivatives 11e17.
ocyclohexanone 4 was performed in acetonitrile in the presence of
p-toluenesulfonic acid (PTSA) as acidic catalyst [18] leading to 11 in
32% isolated yield. Unfortunately, when the same reaction condi-
tions were applied to 2-chlorocyclopentanone 5, the attempted
product could not be obtained. Accordingly, the condensation of 5
with diamine 3 was achieved under basic conditions using trie-
thylamine in THF to give 12 in 22% yield. The condensation reactions
using 1,2-diketones 6e10 and diaminoindazole 3 were carried out in
phorquinone, a mixture of regioisomers was obtained when the
condensation reactions were carried out with diones 9 and 10.
Despite our efforts, regioisomers 16a and 16b were not separable by
chromatography. Fortunately, regioisomers 17a and 17b were
partially isolated from the mixture.
The structures of the regioisomers 14a/14b and 17a/17b were
determined using 1D 1H and 13C NMR spectra and 2D NMR
experiments (1He13C HSQC, 1He13C HMBC and 1He15N HMBC and
Table 2
Kinase inhibitory potencies (% of residual kinase activity) and antiproliferative activities of compounds 3, 11e15, 17a and 17b (IC50 in
caused cell proliferation inhibition less than 50% at 10 M when tested toward PA1 and PC3 cells.
mM). na: not active. nd: compounds that
m
Compounds
Kinase inhibition e % of residual kinase activity
Pim-1 Pim-2
10
Antiproliferative activity (IC50 in mM)
Pim-3
10
Fibro
PA1
PC3
DU145
m
M
1
m
M
10
m
M
1
m
M
m
M
1 mM
3
11
12
13
14a
14b
15
17a
17b
59 ꢀ 7
55.1 ꢀ 0.3
66.6 ꢀ 0.8
49 ꢀ 13
45 ꢀ 7
95 ꢀ 1
90 ꢀ 2
95 ꢀ 1
89 ꢀ 3
88 ꢀ 5
96 ꢀ 4
na
na
na
na
76 ꢀ 13
78 ꢀ 9
na
na
na
na
na
32 ꢀ 3
19.7 ꢀ 0.2
46.4 ꢀ 0.2
31 ꢀ 8
69 ꢀ 1
58 ꢀ 2
81 ꢀ 2
84 ꢀ 5
95 ꢀ 4
83 ꢀ 12
95 ꢀ 14
73 ꢀ 2
72 ꢀ 3
nd
>50
nd
>50
nd
nd
>50
>50
>50
nd
nd
>50
nd
>50
nd
nd
>50
>50
>50
nd
>50
nd
>50
nd
nd
35 ꢀ 4
>50
>50
16 ꢀ 1
nd
6.5 ꢀ 0.8
nd
nd
4.1 ꢀ 0.3
4.7 ꢀ 0.4
6.1 ꢀ 0.5
82 ꢀ 20
37 ꢀ 2
71 ꢀ 5
na
43 ꢀ 3
83 ꢀ 9
na
na
na
na
79 ꢀ 3
69 ꢀ 2
na
na
na
33 ꢀ 3
59 ꢀ 1
88.5 ꢀ 0.5
44 ꢀ 8

L. Gavara et al. / European Journal of Medicinal Chemistry 45 (2010) 5520e5526
5523
NOESY) (Fig. 1). According to a NOESY experiment performed with
one of the regioisomers 17 (finally identified as 17a), an NOE
correlation was observed between the two hydrogens at 8.05 ppm
and 13.33 ppm, leading in turn to the assignment of indazole
moiety hydrogens at 8.43 ppm and 8.44 ppm. Furthermore, 1He15N
HMBC experiment led to the observation of relevant 3J correlations
between two aromatic hydrogens (8.05, 8.44 ppm), three aliphatic
hydrogens (2.86 ppm, 2.75 ppm, 3.16 ppm) and the two nitrogens
of the pyrazine moiety allowing to unambiguous identification of
the regioisomer 17a. Final assignments were made from 1He13C
HMBC experiments based on the correlations observed between
the hydrogens at 2.86, 2.75 and 3.16 ppm and the aromatic carbons
at 135.4 and 139.2 ppm.
12, bearing a non-substituted 5-membered ring does not show any
significant activity toward PA-1 cells, showing that the substitution
of the non-aromatic ring plays an important role for the cytotoxic
potency in this series.
In conclusion, we have described the synthesis of new pyrazolo
[3,4-g]quinoxaline derivatives substituted at the C-6 and C-7 posi-
tions of the pyrazoloquinoxaline nucleus either by ethyl groups or
fused on the pyrazine moiety to a non-aromatic 5- or 6-membered
ring. The results obtained in this preliminary structureeactivity
relationship study have pointed out that in this series, except for
compound 15, all the derivatives exhibited interesting in vitro Pim-3
kinase inhibitory potencies. Moreover, some of the tested
compounds have demonstrated favorable antiproliferative poten-
cies. This suggests that these scaffolds could be of interest for the
development of new Pim kinase inhibitors with antiproliferative
activities. The preparation of a largerlibraryof analogues is currently
under investigation in our group.
Regarding regioisomers 14a/14b, 1He15N HMBC experiments
carried out on one of the regioisomers 14 did not lead to the
observation of correlations between aromatic or aliphatic hydro-
gens and the two pyrazine nitrogens. Fortunately, long range
1He13C coupling was observed between the hydrogen at 3.00 ppm
and a carbon at 135.0 ppm. Based on the NMR assignments deter-
mined for compound 17a (particularly the long range coupling
between the hydrogens at 2.75 ppm and 3.16 ppm and the equiv-
alent carbon at 135.4 ppm) the studied regioisomer was identified
as 14b. Final assignments were performed according to the 1He13C
HSQC and 1He13C HMBC experiments.
4. Experimental
4.1. Chemistry
4.1.1. General
IR spectra were recorded on Shimadzu FTIR-8400S or Per-
3. Results and discussion
kineElmer Spectrum 65 spectrometers (
n
in cmꢁ1). NMR spectra
were performed on a Bruker AVANCE 400 (1H: 400 MHz, 13C:
100 MHz) or Bruker AVANCE 500 (1H: 500 MHz, 13C: 125 MHz),
The biological activities (kinase inhibitory potency and in vitro
antiproliferative activity) were determined for compounds 11e15,
17a and 17b. Due to purification problems, isomers 16a and 16b
were not evaluated for their biological properties.
The kinase inhibitory potencies of compounds 11e15, 17a and
17b were evaluated in duplicate as already described in the litera-
ture by Cohen’s group [22]. The percentages of residual activity,
chemical shifts
d in ppm, the following abbreviations are used:
singlet (s), doublet (d), triplet (t), quadruplet (q), quintuplet (quint),
doublet of doublet (dd), multiplet (m), broad signal (br s). Mass
spectra (ESIþ) were determined on a high-resolution Micro Q-Tof
apparatus (CRMP, Université Blaise Pascal, Clermont-Ferrand,
France). Chromatographic purifications were performed by flash
silica gel Geduran SI 60 (Merck) 0.040e0.063 mm column chro-
matography. 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.
when the compounds were tested at 10 mM and 1 mM toward Pim
kinases (Pim-1, Pim-2 and Pim-3), are reported in Table 2.
According to these results, Pim-3 was the most inhibited protein
kinase. For all compounds, except 15, the percentages of Pim-3
residual activity were inferior to 50% when the compounds were
tested at 10
cyclohexyl derivative 11 which induced 80% Pim-3 inhibition at
10 M. Regarding Pim-1, the best inhibitory activities were found
for compounds 3, 11, 13, 14a and 17b with a percentage of residual
activity in the range of 45e60% when tested at 10 M. Finally, none
of the tested compounds have shown any significant inhibitory
potency toward Pim-2 (% of residual kinase activity > 75% when
tested at 10 mM).
In vitro antiproliferative activities of compounds 11e15, 17a and
17b were evaluated toward a human fibroblast primary culture and
three human solid cancer cell lines: PA1 (ovarian carcinoma), PC3
and DU145 (prostatic carcinoma). All the compounds were firstly
m
M. The most active compound toward Pim-3 was the
4.1.2. 5,6-Dinitro-1H-indazole 2
A mixture of 6-nitro-1H-indazole 1 (5 g, 31 mmol) in concen-
trated sulfuric acid (70 mL) was cooled to 0 ꢂC and was slowly
added into a stirred solution of potassium nitrate (3.44 g, 34 mmol)
in concentrated sulfuric acid (30 mL) at 0 ꢂC. The solution was
stirred for 16 h at room temperature and then was poured on ice
(800 g). The solid was filtered off, washed with water and recrys-
tallized from glacial acetic acid (50 mL) to give 2 (5.35 g, 26 mmol,
84%) as a yellow solid.
m
m
1H NMR (400 MHz, DMSO-d6): 8.45 (1H, s), 8.56 (1H, s), 8.84
(1H, s), 13.01 (1H, br s, NH); 13C NMR (100 MHz, DMSO-d6): 109.1,
121.3, 137.2 (CHarom), 122.2, 135.8, 138.4, 141.6 (Carom).
tested toward PA1 and PC3 at 1
only determined when the growth inhibition was found to be more
than 50% with compounds tested at 10 M. The antiproliferative
mM and 10 mM, and IC50 values were
4.1.3. 1H-indazole-5,6-diamine 3
m
To a mixture of 2 (200 mg, 0.96 mmol) and 10% Pd/C (200 mg,
0.19 mmol, 20 mol%) in methanol (6 mL) was added ammonium
formate (600 mg, 9.5 mmol). The mixture was refluxed for 1 h and
the catalyst was removed by filtration through a Celite pad, which
was subsequently washed with methanol (30 mL). The filtrate was
evaporated under reduced pressure to give 3 (122 mg, 0.82 mmol,
86%) as a grey solid. Compound 3 was used directly for the next step
without any further purification.
effect of the tested drug was assessed by the resazurin reduction test
[23]. Under the conditions used, none of the tested compounds have
shown any significant antiproliferative activities toward the fibro-
blast and the prostatic carcinoma cells used (PC3, DU145) (Table 2).
Conversely, compounds 13, 15, 17a and 17b, substituted at the C-6
and C-7 positions of pyrazolo[3,4-g]quinoxaline nucleus by ethyl
groups or fused at the same positions to 5-membered rings, have
demonstrated interesting inhibitory potencies toward PA1 cells,
with IC50 values in the micromolar range. Moreover, these
compounds have shown a favorable selectivity index for PA1 cells
compared tothe fibroblast primaryculture. Interestingly, compound
1H NMR (400 MHz, DMSO-d6): 4.29 (2H, br s, NH2), 4.80 (2H, br
s, NH2), 6.57 (1H, s), 6.72 (1H, s), 7.53 (1H, s), 12.02 (1H, br s, NH);
13C NMR (100 MHz, DMSO-d6): 91.6, 101.7, 131.3 (CHarom), 115.8,
132.1, 136.3, 138.4 (Carom).

5524
L. Gavara et al. / European Journal of Medicinal Chemistry 45 (2010) 5520e5526
4.1.4. 6,7,8,9-Tetrahydro-1H-pyrazolo[3,4-b]phenazine 11
8.39 (1H, s), 8.42 (1H, s), 13.29 (1H, br s, NH); 13C NMR (100 MHz,
DMSO-d6): 10.0, 18.0, 20.0 (CH3), 24.3, 31.6 (CH2), 52.3 (CH), 52.8,
53.0 (C), 106.0, 119.2, 134.3 (CH), 124.2, 135.0, 139.1 (2C) (Carom),
163.6, 164.0 (Carom).
A solution of 1H-indazole-5,6-diamine 3 (100 mg, 0.67 mmol),
2-chlorocyclohexanone 4 (116 mg, 0.87 mmol) and PTSA mono-
hydrate (10 mol%) in acetonitrile (8 mL) was refluxed for 2 h.
Solvent was evaporated and the residue was dissolved in EtOAc
(20 mL) and a saturated aqueous NaHCO3 solution (10 mL) was
added. The organic layer was separated and dried over anhydrous
MgSO4. The residue obtained upon evaporation was purified by
flash chromatography (cyclohexane to EtOAc) to give 11 (48 mg,
0.21 mmol, 32%) as a brown solid.
4.1.7.2. Compound 14b. Mp ¼ 208e209 ꢂC; HRMS (ESIþ) calcd for
C17H19N4 (M þ H)þ 279.1610 found 279.1622; IR (ATR): 3161, 1650,
1616, 1587 cmꢁ1 1H NMR (400 MHz, DMSO-d6): 0.56 (3H, s, CH3),
;
1.09 (3H, s, CH3), 1.33 (3H, s, CH3), 1.30e1.40 (2H, m), 2.00e2.10
(1H, m), 2.23e2.31 (1H, m), 3.00 (1H, d, J ¼ 4.5 Hz), 8.03 (1H, s), 8.39
(1H, s), 8.40 (1H, s), 13.30 (1H, br s, NH); 13C NMR (100 MHz, DMSO-
d6): 10.0, 18.0, 20.0 (CH3), 24.3, 31.6 (CH2), 52.1 (CH), 52.8, 53.3 (C),
106.1, 119.1, 134.3 (CHarom), 124.2, 135.0, 139.05, 139.13, 161.9, 165.6
(Carom).
Mp ¼ 174e175 ꢂC; HRMS (ESI þ ) calcd for C13H13N4 (M þ H)þ
225.1140, found 225.1141; IR (ATR): 3179, 1634, 1568 cmꢁ1; 1H NMR
(400 MHz, DMSO-d6): 1.94e2.00 (4H, m), 3.05e3.11 (4H, m), 7.99
(1H, s), 8.428 (1H, s), 8.433 (1H, s), 13.30 (1H, br s, NH); 13C NMR
(100 MHz, DMSO-d6): 22.27, 22.32 (CH2), 32.7, 32.9 (CH2), 104.7,
118.8, 134.5 (CHarom), 125.4, 134.9, 138.6, 139.5, 152.4, 154.7 (Carom).
4.1.8. (ꢀ)-7-Phenyl-1,6,7,8-tetrahydrocyclopenta[b]pyrazolo[3,4-g]
quinoxaline 15
4.1.5. 1,6,7,8-Tetrahydrocyclopenta[b]pyrazolo[3,4-g]quinoxaline 12
A mixture of 1H-indazole-5,6-diamine 3 (100 mg, 0.67 mmol),
triethylamine (0.19 mL, 138 mg, 1.36 mmol) and 2-chlorocyclo-
To
a
solution of 1H-indazole-5,6-diamine
3
(12.6 mg,
0.085 mmol) in glacial acetic acid (0.3 mL) was added a solution of
4-phenylcyclopentane-1,2-dione 8 (14.8 mg, 0.085 mmol) in glacial
acetic acid (0.3 mL). The mixture was stirred for 1 h in an oil bath
heated at 80 ꢂC. After cooling, the solution was poured into ice-
water (10 mL) and then neutralized with a 5 M aqueous NaOH
solution. After extraction with EtOAc, the assembled organic frac-
tions were dried over MgSO4 and evaporated. The brown oil was
purified by flash chromatography (cyclohexane/EtOAc, 7:3) to give
15 (8 mg, 0.03 mmol, 33%) as a light yellow solid.
pentanone 5 (76 mL, 90 mg, 0.76 mmol) in anhydrous THF (3 mL)
was refluxed overnight. After evaporation of the solvent, water was
added and the mixture was extracted with EtOAc. The assembled
organic fractions were dried over MgSO4, evaporated and the
residue was purified by flash chromatography (cyclohexane/EtOAc,
60:40e0:100) to give 12 (30.8 mg, 0.15 mmol, 22%) as a light yellow
solid.
Mp ¼ 139e144 ꢂC; HRMS (ESIþ) calcd for C12H11N4 (M þ H)þ
211.0984, found 211.0998; IR (ATR): 3468,1635,1597 cmꢁ1; 1H NMR
(400 MHz, DMSO-d6): 2.22 (2H, quint, J ¼ 7.5 Hz), 3.10 (2H, t,
J ¼ 7.5 Hz), 3.11 (2H, t, J ¼ 7.5 Hz), 8.02 (1H, s), 8.43 (1H, s), 8.44 (1H,
s), 13.31 (1H, se); 13C NMR (100 MHz, DMSO-d6): 20.9, 31.5, 31.8
(CH2), 105.6, 119.2, 134.4 (CHarom), 124.9, 135.1, 139.0, 139.4, 159.3,
161.5 (Carom).
Mp >280 ꢂC; HRMS (ESIþ) calcd for C18H15N4 (M þ H)þ
287.1297, found 287.1290; IR (ATR): 3190, 1644, 1607, 1494,
1355 cmꢁ1; 1H NMR (400 MHz, DMSO-d6): 3.25e3.33 (2H, m), 3.51
(1H, dd, J1 ¼17.0 Hz, J2 ¼ 8.0 Hz), 3.52 (1H, dd, J1 ¼17.0 Hz,
J2 ¼ 8.0 Hz), 3.82e3.92 (1H, m), 7.27 (1H, t, J ¼ 7.5 Hz), 7.37 (2H, t,
J ¼ 7.5 Hz), 7.45 (2H, d, J ¼ 7.5 Hz), 8.06 (1H, s), 8.45 (1H, s), 8.48
(1H, s), 13.34 (1H, se); 13C NMR (100 MHz, DMSO-d6): 39.6 (CH2),
40.8 (CH), 105.7, 119.4, 126.6, 127.1 (2C), 128.5 (2C), 134.5 (CHarom),
125.0, 135.3, 139.2, 139.4, 143.7, 158.6, 160.8 (Carom).
4.1.6. 6,7-Diethyl-1H-pyrazolo[3,4-g]quinoxaline 13
The same procedure as for the preparation of compound 11 was
used with 1H-indazole-5,6-diamine 3 (142 mg, 0.96 mmol) and
hexane-3,4-dione 6 (164 mg, 1.44 mmol). Flash chromatography
(cyclohexane to EtOAc) provided 4 (108 mg, 0.48 mmol, 50%) as
a yellow solid.
4.1.9. (ꢀ)-8-Methyl-1,6,7,8-tetrahydrocyclopenta[b]pyrazolo[3,4-g]
quinoxaline 16a and (ꢀ)-6-methyl-1,6,7,8-tetrahydrocyclopenta[b]
pyrazolo[3,4-g]quinoxaline 16b
To a solution of 1H-indazole-5,6-diamine 3 (50 mg, 0.34 mmol)
in glacial acetic acid (1.5 mL) was added 3-methylcyclopentane-
1,2-dione 9 (37.8 mg, 0.34 mmol). The mixture was stirred for
30 min in an oil bath heated at 110 ꢂC. After cooling, the solution
was poured into ice-water (10 mL) and then neutralized with a 5 M
aqueous NaOH solution. After extraction with EtOAc, the assembled
organic fractions were dried over MgSO4 and evaporated. The
brown oil was purified by flash chromatography (cyclohexane/
EtOAc, 7:3e2:8) to give a mixture of regioisomers 16a and 16b
(24 mg, 0.11 mmol, 32%) as a light yellow solid. The regioisomers
ratio (81:19) was determined from the 1H NMR spectra on the
signals at 8.03 ppm (minor isomer) and 8.06 ppm (major isomer).
HRMS (ESIþ) calcd for C13H13N4 (M þ H)þ 225.1140, found
225.1144. 1H NMR (400 MHz, DMSO-d6): 1.41 (3Ha þ 3Hb, d,
J ¼ 7.0 Hz), 1.72e1.82 (1Ha þ 1Hb, m), 3.04e3.10 (2Ha þ 2Hb, m),
8.03 (1Hb, s), 8.06 (1Ha, s), 8.43 (1Ha þ 1Hb, s), 8.45 (1Ha, s), 8.48
(1Hb, s), 13.28e13.35 (1Ha þ 1Hb, se). 13C NMR (100 MHz, DMSO-
d6), major regioisomer: 17.6 (CH3), 30.2, 30.4 (CH2), 37.9 (CH), 105.8,
119.2, 134.4 (CHarom), 124.9, 135.2, 139.1, 139.4, 159.2, 164.1 (Carom).
Mp ¼ 147e149 ꢂC; HRMS (ESIþ) calcd for C13H15N4 (M þ H)þ
227.1297 found 227.1293. IR (ATR): 3178, 1690, 1636, 1573 cmꢁ1; 1H
NMR (400 MHz, DMSO-d6): 1.350 (3H, t, J ¼ 7.5 Hz, CH3), 1.354 (3H,
t, J ¼ 7.5, CH3), 3.02 (2H, q, J ¼ 7.5 Hz, CH2), 3.03 (2H, q, J ¼ 7.5 Hz,
CH2), 8.01 (1H, s), 8.42 (1H, s), 8.45 (1H, s), 13.30 (1H, br s, NH); 13
C
NMR (100 MHz, DMSO-d6): 11.4, 11.5 (CH3), 27.3, 27.5 (CH2), 105.1,
119.0, 134.4 (CHarom), 125.2, 134.5, 138.4, 139.5, 155.3, 157.5 (Carom).
4.1.7. (6R,9S)-6-Methyl-6,9-dimethylmethano-6,7,8,9-tetrahydro-
1H-pyrazolo[3,4-b]phenazine 14a and (6S,9R)-9-methyl-6,9-
dimethylmethano-6,7,8,9-tetrahydro-1H-pyrazolo[3,4-b]phenazine
14b
The same procedure as for the preparation of compound 11 was
used with 1H-indazole-5,6-diamine 3 (130 mg, 0.88 mmol) and
(1R)-(ꢁ)-camphorquinone 7 (218 mg, 1.31 mmol). Flash chroma-
tography (Et2O/cyclohexane, 85:15) provided 14a (85 mg,
0.31 mmol, 35%) and 14b (122 mg, 0.44 mmol, 50%) as yellow
solids.
4.1.7.1. Compound 14a. Mp ¼ 198e201 ꢂC; HRMS (ESIþ) calcd for
C17H19N4 (M þ H)þ 279.1610 found 279.1618; IR (ATR): 3155, 1647,
1615, 1588 cmꢁ1; 1H NMR (400 MHz, DMSO-d6): 0.56 (3H, s, CH3),
1.09 (3H, s, CH3), 1.33 (3H, s), 1.30e1.40 (2H, m), 2.00e2.10
(1H, m), 2.24e2.31 (1H, m), 3.01 (1H, d, J ¼ 4.5 Hz), 8.00 (1H, s),
4.1.10. (ꢀ)-7,8-Dimethyl-1,6,7,8-tetrahydrocyclopenta[b]pyrazolo
[3,4-g]quinoxaline 17a and (ꢀ)-6,7-dimethyl-1,6,7,8-
tetrahydrocyclopenta[b]pyrazolo[3,4-g]quinoxaline 17b
To a solution of 1H-indazole-5,6-diamine 3 (200 mg, 1.35 mmol)
in glacial acetic acid (5 mL) was added 3,4-dimethylcyclopentane-

L. Gavara et al. / European Journal of Medicinal Chemistry 45 (2010) 5520e5526
5525
1,2-dione 10 (170 mg, 1.35 mmol). The mixture was stirred for
30 min in an oil bath heated at 80 ꢂC. After cooling, the solution was
poured into ice-water (40 mL) and then neutralized with a 5 M
aqueous NaOH solution. After extraction with EtOAc, the assembled
organic fractions were dried over MgSO4 and evaporated. The
brown oil was purified by flash chromatography (Et2O/cyclo-
hexane, 6:4) to give a mixture of regioisomers 17a and 17b (96 mg,
0.40 mmol, 30%) as a yellow solid. Another chromatography led to
a partial separation of the mixture with 17a (9.6 mg, 0.04 mmol, 3%)
and 17b (12 mg, 0.05 mmol, 4%) as yellow solids.
The inhibition profile of the tested compounds was expressed as
the percentage of the residual kinase activity for an inhibitor
concentration of 1 or 10 mM. The IC50 values of inhibitors were
determined after carrying out assays at ten different concentrations
of each compound.
4.3. Antiproliferative activities
4.3.1. 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.
4.1.10.1. Compound 17a. Mp ¼ 183e189 ꢂC; HRMS (ESIþ) calcd for
C14H15N4 (M þ H)þ 239.1297, found 239.1303; IR (ATR): 3179, 1635,
1570 cmꢁ1
;
1H NMR (400 MHz, DMSO-d6): 1.30 (3H, d, J ¼ 6.5 Hz),
1.41 (3H, d, J ¼ 7.0 Hz), 2.06e2.18 (1H, m), 2.75 (1H, dd,
J1 ¼17.0 Hz, J2 ¼10.5 Hz), 2.86 (1H, dq, J1 ¼10.0 Hz, J2 ¼ 7.0 Hz), 3,16
(1H, dd, J1 ¼17.0 Hz, J2 ¼ 7.5 Hz), 8.05 (1H, s), 8.43 (1H, s), 8.44
(1H, s), 13.33 (1H, se); 13C NMR (100 MHz, DMSO-d6): 15.0, 18.4
(CH3), 38.8 (CH2), 39.6, 45.4 (CH), 105.8, 119.2, 134.4 (CHarom), 124.9,
135.4, 139.2, 139.4, 158.9, 164.1 (Carom).
4.3.2. Survival assays
Cells were plated at a density of 5 ꢃ103 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 at ꢁ20 ꢂC
4.1.10.2. Compound 17b. Mp ¼ 206e216 ꢂC; HRMS (ESIþ) calcd for
C14H15N4 (M þ H)þ 239.1297, found 239.1295; IR (ATR): 3142, 1638,
untiluse. Then 50 mLofeachtested solutionwasadded tothecultures.
A 48 h continuous drug exposure protocol was used. The anti-
proliferative effect of the tested drug was assessed by the resazurin
reduction test.
1601 cmꢁ1
;
1H NMR (400 MHz, DMSO-d6): 1.30 (3H, d, J ¼ 6.5 Hz),
1.41 (3H, d, J ¼ 7.0 Hz), 2.06e2.18 (1H, m), 2.77 (1H, dd,
J1 ¼17.0 Hz, J2 ¼ 10.5 Hz), 2.86 (1H, dq, J1 ¼10.0 Hz, J2 ¼ 7.0 Hz), 3.17
(1H, dd, J1 ¼17.0 Hz, J2 ¼ 7.5 Hz), 8.03 (1H, s), 8.42 (1H, s), 8.47 (1H,
s), 13.31 (1H, se); 13C NMR (100 MHz, DMSO-d6): 15.1, 18.4 (CH3),
39.0 (CH2), 39.6, 45.1 (CH), 105.7, 119.4, 134.4 (CHarom), 124.8, 135.3,
139.3, 139.4, 161.0, 161.9 (Carom).
4.3.3. Resazurin reduction test
Plates were rinsed with 200
overturning on absorbent towelling. 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
4.2. In vitro kinase inhibition assays
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 detailed
previously [21].
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
experimental wells compared with fluorescence in control wells,
after subtraction of the blank values.
4.2.2. Source and purification of kinases
All protein kinases were of human origin and encoded full-
length proteins. All proteins were either expressed as GST (gluta-
thione 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.
Acknowledgements
The French Ministère de l’Enseignement Supérieur et de la
Recherche (ES), the ANR (ANR-08-JCJC-0131-CSD 3) (LG) and
Région Auvergne «Synbio Project» (GA) are greatly acknowledged
for financial support. The authors are grateful to Bertrand Légeret
for mass spectra analysis.
4.2.3. 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, USA) in a 96-well
format. The concentration of magnesium acetate in the assays was
References
[1] 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.
[2] R. Akué-Gédu, L. Nauton, V. Théry, J. Bain, P. Cohen, F. Anizon, P. Moreau,
Bioorg. Med. Chem. 18 (2010) 6865e6873.
[3] N. Shah, B. Pang, K.G. Yeoh, S. Thorn, C.S. Chen, M.B. Lilly, M. Salto-Tellez,
Eur. J. Cancer 44 (2008) 2144e2151.
[4] Z. Zhao, D.D. Wisnoski, S.E. Wolkenberg, W.H. Leister, Y. Wang, C.W. Lindsley,
Tetrahedron Lett. 45 (2004) 4873e4876.
10 mM and [
2 and 20 M for Pim-1 and Pim-3, in order to be at or below the Km
g- mM for Pim-
33P]ATP (800 c.p.m./pmol) was used at 5
m
for ATP for each enzyme.
The assays were initiated with MgATP, stopped by the addition
of 5
plates using a unifilter harvester (PerkineElmer, Boston, MA, USA).
Kinase substrates were RSRHSSYPAGT (300 M) for Pim-1, Pim-2
mL of 0.5 M orthophosphoric acid and spotted on to P81 filter
[5] C.W. Lindsley, Z. Zhao, W.H. Leister, R.G. Robinson, S.F. Barnett, D. Defeo-Jones,
R.E. Jones, G.D. Hartman, J.R. Huff, H.E. Huber, M.E. Duggan, Bioorg. Med.
Chem. Lett. 15 (2005) 761e764.
m
[6] S.F. Barnett, D.D. Defeo-Jones, G.D. Harman, H.E. Huber, S.M. Stirdivant, D.C.
Heimbrook, U.S. Pat. Appl. US 2004102360 A1, 2004. Chem. Abstr. 141 (2004)
7131.
[7] C.W. Lindsey, Z. Zhao, PCT Int. Appl. WO 2003086404 A1, 2003. Chem. Abstr.
139 (2003) 350755.
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.

5526
L. Gavara et al. / European Journal of Medicinal Chemistry 45 (2010) 5520e5526
[8] S.F. Barnett, D. Defeo-Jones, K.M. Haskell, H.E. Huber, E. Hans, D.D. Nahas, C.W.
Lindsey, Z. Zhao, G.D. Hartman, PCT Pat. Appl. WO 2003084473 A2, 2003.
Chem. Abstr. 139 (2003) 323527.
[9] M.R. Myers, P.E. Persons, C.Q. Ly, A.P. Spada, U.S. Pat. Appl. US 5476851 A,
1995. Chem. Abstr. 124 (1996) 232489.
[16] von K. Fries, K. Fabel, H. Eckhardt, Justus Liebigs Ann. Chem. 550 (1942)
31e49.
[17] G.-D. Zhu, J. Gong, V.B. Gandhi, K. Woods, Y. Luo, X. Liu, R. Guan, V. Klinghofer,
E.F. Johnson, V.S. Stoll, M. Mamo, Q. Li, S.H. Rosenberg, V.L. Giranda, Bioorg.
Med. Chem. 15 (2007) 2441e2452.
[10] K. Karabelas, P. Sjo, PCT Pat. Appl. WO 9946264 A1, 1999. Chem. Abstr 131
(1999) 228732.
[18] B. Das, K. Venkateswarlu, K. Suneel, A. Majhi, Tetrahedron Lett. 48 (2007)
5371e5374.
[11] B. Venugopalan, C.P. Bapat, P.J. Kamik, N.J. De Souza, Indian J. Chem. B 29B
(1990) 364e365.
[12] B. Venugopalan, S.S. Iyer, P.J. Kamik, N.J. De Souza, Heterocycles 26 (1987)
3173e3180.
[13] G. Saint-Ruf, J.C. Bourgeade, Chim. Ther 8 (1973) 447e450.
[14] N.P. Buu-Hoi, G. Saint-Ruf, J.C. Arcos, Bull. Soc. Chim. Fr. (1969) 838e842.
[15] R.R. Davies, J. Chem. Soc. (1955) 2412e2423.
[19] H. Muxfeldt, M. Weigele, V. Van Rheenen, J. Org. Chem. 30 (1965) 3573e3574.
[20] A. Salgado, Y. Dejaegher, G. Verniest, M. Boeykens, C. Gauthier, C. Lopin,
K.A. Tehrani, N. De Kimpe, Tetrahedron 59 (2003) 2231e2239.
[21] R.W. Bost, E.E. Towell, J. Am. Chem. Soc. 70 (1948) 903e905.
[22] 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.
[23] J. O’Brien, I. Wilson, T. Orton, F. Pognan, Eur. J. Biochem 267 (2000) 5421e5426.