Identification of pyrrolo[2,3-g]indazoles as new Pim kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.02.074

The synthesis and Pim kinase inhibition potency of a new series of pyrrolo[2,3-g]indazole derivatives is described. The results obtained in this preliminary structure-activity relationship study pointed out that sub-micromolar Pim-1 and Pim-3 inhibitory potencies could be obtained in this series, more particularly for compounds 10 and 20, showing that pyrrolo[2,3-g]indazole scaffold could be used for the development of new potent Pim kinase inhibitors. Molecular modeling experiments were also performed to study the binding mode of these compounds in Pim-3 ATP-binding pocket.

Full text of DOI:10.1016/j.bmcl.2013.02.074

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2298–2301
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of pyrrolo[2,3-g]indazoles as new Pim kinase
inhibitors
⇑
Laurent Gavara, Virginie Suchaud, Lionel Nauton, Vincent Théry, Fabrice Anizon , Pascale Moreau
Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, 63000 Clermont-Ferrand, France
CNRS, UMR 6296, ICCF, BP 80026, 63171 Aubière, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and Pim kinase inhibition potency of a new series of pyrrolo[2,3-g]indazole derivatives is
described. The results obtained in this preliminary structure–activity relationship study pointed out that
sub-micromolar Pim-1 and Pim-3 inhibitory potencies could be obtained in this series, more particularly
for compounds 10 and 20, showing that pyrrolo[2,3-g]indazole scaffold could be used for the develop-
ment of new potent Pim kinase inhibitors. Molecular modeling experiments were also performed to
study the binding mode of these compounds in Pim-3 ATP-binding pocket.
Received 31 January 2013
Revised 12 February 2013
Accepted 14 February 2013
Available online 26 February 2013
Keywords:
Pyrrolo[2,3-g]indazole
Indazole
Ó 2013 Elsevier Ltd. All rights reserved.
Indole
Pim kinase inhibition
R1
R1
7
R2
R2
Indole and indazole ring systems are considered as highly valu-
able heterocyclic scaffolds in drug discovery. The chemistry of in-
dole nucleus has been abundantly described, as well as
numerous biological applications leading to pharmaceutically ac-
tive molecules. Indazole nucleus is an indole bio-isoster. Several
recent reports in organic synthesis and medicinal chemistry con-
firmed the great interest of this scaffold.1–8 Particularly, indazole
derivatives have been described as protein kinase inhibitors.9–12
Accordingly, in our ongoing research aiming at developing new
Pim kinase inhibitors,13–18 we were interested in the development
of novel scaffolds containing the indazole heterocyclic system. Pim
(Provirus integration site for Moloney murine leukaemia virus)
family is represented by three highly homologous Ser/Thr kinases
(Pim-1, Pim-2 and Pim-3) involved in cell survival and malignant
transformation.19–24 Accordingly, Pim kinases are considered as
important targets in the field of drug discovery against cancer.
Therefore, in continuation of our study on the use of indazole
derivatives as Pim inhibitors, we herein report the synthesis and
biological evaluation of a series of diversely substituted dihydro-
pyrrolo[2,3-g]indazoles as well as the evaluation of their potencies
toward the three Pim kinase isoforms.
8
THP
N
N
H
N
HN
HN
H+
N
Conditions A, B, C or D
5
O2N
O2N
R1
1–7
8–18
R2
R1
R2
Conditions Product
% Yield
Compd
1
TMS
A
8
TMS
52
48
97
22
42
41
38
55
86
62
88
H
H
H
9
H
2
Et
H
Ph
B
B
A
C
B
C
B
D
B
10
11
12
13
14
Et
Ph
3a
4
CH2OBn
CH(OEt)2
Me
CH2OEt
CHO
Me
H
H
H
5
H
CO2Et
CO2Et
H
Me
15b
16
6
7
CO2Me
CO2Et
n-Pr
Ph
n-Pr
n-Pr
Ph
CO2Me
H
17b
18
Ph
CO2Et
We recently reported the synthesis of N1-protected 1,6-dihy-
dropyrrolo[2,3-g]indazoles 1–7 (Scheme 1).25 These compounds
can be considered as valuable synthetic intermediates that could
easily lead to deprotected derivatives and their corresponding
amino analogues. Thus, compound 1 was treated by acetic acid
Scheme 1. Synthesis of compounds 8–18. Reagents and conditions: (A) AcOH, THF/
H2O, (B) PTSA, EtOH or MeOH, H2O, (C) concd HCl, (D) concd HBr. aCompound 3
contains ꢀ30% of the 7-CH2OBn regioisomer, bSee Ref. 24.
in THF/H2O in order to perform THP cleavage. Two products were
obtained: the deprotected derivative 8 was isolated in 52% yield,
as well as its desilylated analogue 9 in 48% yield. Deprotection of
compound 2 needed stronger acidic conditions. By using p-tolu-
⇑
Corresponding author. Tel.: +33 (0) 4 73 40 53 64; fax: +33 (0) 4 73 40 77 17.
E-mail address: Fabrice.ANIZON@univ-bpclermont.fr (F. Anizon).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.074

L. Gavara et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2298–2301
2299
R1
HN
R1
7
R2
R2
8
H
N
H
N
HN
H2, PtO2, EtOAc
N
N
5
O2N
8–10, 13–18
Compd Product
H2N
19–27
% Yield
R1
R2
8
19
20
21
22
23
24
25
26
27
TMS
H
H
H
76
43
82
20
–
9
10
13
14
15
16
17
18
Et
Ph
H
Me
Me
n-Pr
n-Pr
Ph
CO2Et
H
63
–
CO2Me
H
78
82
CO2Et
Ph
R1O2C
HN
R1O2C
R1O2C
HN
R2
R2
R2
H2, PtO2, EtOAc
conc. HCl, reflux
THP
N
THP
N
H
N
HN
N
N
N
O2N
H2N
H2N
5, 6
28, 70%
29, 77%
23, 61%
25, 23%
5, 28, 23 R1 = Et, R2 = Me
6, 29, 25 R1 = Me, R2 = n-Pr
Scheme 2. Synthesis of compounds 19–29.
Table 1
Kinase inhibitory potencies: % of residual kinase activity at 10 and 1
l
M
R1
7
Kinase inhibition—% of residual kinase activity
Pim-2
R2
8
Pim-1
Pim-3
H
N
HN
Compd
N
R3
5
10
17
l
M
1
l
M
10
l
M
1
l
M
10
9
l
M
1
l
M
Compd
R1
R2
H
R3
8
0
63
2
64 13
103 18
0
51
4
8
TMS
NO2
(0.47 0.02)
9
10
30
2
102 33
80
84 11
2
92
77
5
6
18
36
2
5
78
31
5
1
9
10
H
Et
H
Ph
NO2
NO2
51 10
(0.23 0.03)
43
82
4
1
(0.119 0.006)
11
12
13
14
15
26
27
26
72
54
1
3
6
6
4
63
70 17
61
97
27 10
2
109 10
117 31
99
105
66
19
25
26
47
6
2
1
1
2
71 11
76
77 10
72
24
11
12
13
14
15
H
H
H
CO2Et
H
CH2OEt
CHO
Me
Me
nPr
NO2
NO2
NO2
NO2
NO2
97 25
2
68
78
4
6
2
6
1
3
9
7
4
78 11
0
(0.26 0.02)
24
(0.546 0.009)
21
16
17
39
2
110 37
85
70
8
7
113 52
82
0
50
38
3
5
16
17
CO2Me
H
nPr
NO2
NO2
28
3
52
6
8
1
Ph
(1.0 0.7)
55
52 10
17
(0.46 0.05)
(0.53 0.04)
90 23
18
19
20
1
86
94
63 19
7
6
113 21
111 16
92 15
93 27
99 11
73 11
67 13
18
19
20
CO2Et
TMS
H
Ph
H
H
NO2
NH2
NH2
24
4
1
2
104
8
0
20
2
(0.033 0.002)
21
22
23
24
25
26
27
44
63
61
57 15
26
51 10
29
4
1
3
92
91
93
104 17
71
4
3
4
75 11
88 13
97 10
91
105
102 17
90
105
103 20
101
9
8
23
17
26
39
12
26
20
0
4
4
2
1
1
3
72
65
93 17
79
82 14
3
2
21
22
23
24
25
26
27
Et
H
CO2Et
H
CO2Me
H
Ph
NH2
NH2
NH2
NH2
NH2
NH2
NH2
Me
Me
nPr
nPr
Ph
93
78 11
92
74 18
3
6
0
8
2
0
9
5
111
74
3
68
72
7
4
9
0
CO2Et
Ph
IC50 values (in brackets) were determined when the remaining kinase activity was found to be inferior to 50% when the compounds were tested at 1 lM.

2300
L. Gavara et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2298–2301
enesulfonic acid (PTSA) in EtOH/H2O, 10 was isolated in 97% yield.
From compound 3, the use of PTSA and ethanol cleaved the benzyl-
oxy moiety and produced the ethoxy-substituted derivative 11.
Formyl derivative 12 was obtained from 4 by concomitant indazole
nitrogen deprotection and acetal hydrolysis (Scheme 1).
As previously described for compounds 6 and 7,25 compound 5
was simultaneously deprotected and decarboxylated in refluxing
concentrated HCl, leading to compound 13 in 41% yield. To avoid
the decarboxylation, 5–7 were treated by PTSA leading to THP-
deprotected indazole alkyl carboxylates 14, 16 and 18 in 38–88%
yields. Low yield observed for compound 14 could be explained
by purification difficulties due to low solubility (Scheme 1).
Amino analogues were next prepared by catalytic hydrogena-
tion of the nitro function. Thus, compounds 8–10 and 13–18 were
hydrogenated in the presence of PtO2. Due to the difficulty to iso-
late 11 in satisfactory quantity and due to the very poor solubility
of compound 12, the corresponding amino derivatives were not
synthesized. Compounds 19–22, 24, 26 and 27 were obtained from
their nitro analogues in 20–82% yields (Scheme 2). However, in the
case of alkyl indazole carboxylates 14 and 16, we met some diffi-
culties to proceed to this reduction step. Accordingly, the reduction
of the nitro function was performed prior to the THP-deprotection
step, leading to compounds 28 and 29 in 70% and 77% yield, respec-
tively (Scheme 2). Deprotection of N-1 indazole nitrogen in con-
centrated HCl finally led to expected compounds 23 and 25 with
moderate yields which could be explained by partial hydrolysis
of the ester function.
The kinase inhibitory potencies of pyrroloindazoles 8–27 were
evaluated at 10 and 1 lM concentrations in duplicate assays
against Pim-1, Pim-2 and Pim-3. Kinase assays were performed
by the International Centre for Kinase Profiling (Dundee, UK).26
The percentages of kinase residual activities are reported in Table 1.
When the remaining kinase activity was found to be inferior to 50%
when the compounds were tested at 1
determined.
lM, IC50 values were also
According to the results reported in Table 1, Pim-3 is the iso-
form that was the most inhibited by the tested compounds. In con-
trast, Pim-2 was poorly inhibited. Regarding Pim-1, the IC50 values
were determined only for three compounds (10, 17 and 20).
Compounds 17 and 20 were moderate Pim-1 inhibitors, with IC50
values of 1.0 and 0.46 lM, respectively. The best result toward
Pim-1 was found for compound 10, bearing a nitro group at the
C5 position, an ethyl group at the C7 position and a phenyl group
at the C8 position, with an IC50 value of 0.23 lM.
Regarding Pim-3 inhibition, compounds 8, 10, 15–17, and 20 are
the most potent with IC50 values in the sub-micromolar range (Ta-
ble 1). Interestingly, with the exception of compound 20, all these
active compounds were bearing a nitro group at the C5 position
and were either mono-substituted at the position C7 (8) or C8
(15, 17), or di-substituted at these positions (10, 16). Among the
nitro derivatives, 10 exhibited the best inhibitory potency toward
Pim-3, showing that in this nitro series C7 and C8 positions can
be substituted by hydrophobic groups to inhibit both Pim-1 and
Pim-3 kinases. Nevertheless, the best inhibitory potency toward
Pim-3 was found for C7/C8 unsubstituted amino derivative 20,
with an IC50 value of 33 nM, showing that contrarily to what was
observed for nitro analogues, the substitution of positions C7
and/or C8 was detrimental to the inhibitory activity. These results
suggest that the interaction of 5-nitro and 5-amino analogues with
Pim-3 ATP-binding site may involve different binding modes.
Thus, we next performed molecular modeling experiments in
order to study the putative binding mode of compounds 10 and
20 in the ATP-binding pocket of Pim-3. These two pyrroloindazoles
are the best Pim-3 inhibitors in the series and are bearing a nitro or
an amino group at the C5 position, respectively. We also studied
the Pim-3 binding modes of their amino/nitro counterparts;
Figure 1. Docking models of compounds 10 (A), 21 (B) and 20 (C) bound to the Pim-
3 ATP binding site. Hydrogen bonds are indicated as dashed lines. Molecular
graphics images were produced using UCSF Chimera.27
compounds 21 and 9, respectively. To the best of our knowledge,
no Pim-3 X-ray crystal structure has been solved so far. Therefore,
as Pim-3 presents a high sequence identity with Pim-1, we gener-
ated a Pim-3 homology model using the same method we previ-
ously reported.13 Thus, we generated
a Pim-3 model using
Modeller9V11 and UCSF Chimera,27–30 Sybylx2.031 software and
Pim-1 1XWS crystal structure available in the Protein Data Bank
(PDB). Docking experiments were then performed using Sybylx2.0
for compounds 9, 10, 20 and 21, and the best docking solution for
each compound was minimized. The docking solutions found for

L. Gavara et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2298–2301
2301
compounds 10, 20 and 21 are depicted in Figure 1. Concerning
Supplementary data
compound 9, docking experiments did not allow us to observe
any major interaction with Pim-3 ATP-binding pocket.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.02.
074.
As shown in Figure 1A and B, compounds 10 and 21, bearing
ethyl and phenyl substituents at the C7 and C8 positions, are in-
serted in Pim-3 ATP-binding cleft but showed different binding
modes. Compound 10 is placed deep in the pocket and interacts
via its nitro group with protonated Lys69 side chain, as well as
with a conserved water molecule. However, this compound does
not establish hydrogen bond with the hinge region. On the other
hand, the amino function of compound 21 is turned toward the
Glu124 backbone carbonyl leading to the formation of a weak
hydrogen bond (2.24 Å) with the hinge. An additional hydrophobic
interaction was found for 21 between the methyl group of C7 ethyl
substituent and Phe51 side chain. Regarding compound 20
(Fig. 1C), a similar orientation of the pyrroloindazole scaffold to
that of 21 was found. However, in this case, a strong hydrogen
bond can be established between the amino group of the inhibitor
and Glu124 backbone carbonyl (1.84 Å). This strong interaction
might explain the potent inhibitory potency of compound 20 to-
ward Pim-3. This interaction is weaker in the case of compound
21 probably due to the presence of the substituents at the C7
and C8 positions that prevent the amino group to get closer to
Glu124 backbone carbonyl. Finally, this molecular modeling study
showed that a different binding mode could probably be involved
in the interaction with Pim-3 of pyrroloindazoles substituted at the
C5 position by nitro or amino groups. In the amino series, a hydro-
gen bond with the hinge region is possible but is strongly influ-
enced by the substitution at the C7 and C8 positions. These
results will be used to further optimize the biological profile of this
1,6-dihydropyrrolo[2,3-g]indazole series.
In conclusion, a structure–activity relationship study was per-
formed on a series of 1,6-dihydropyrrolo[2,3-g]indazoles regarding
their Pim kinase inhibitory potencies. These results identified 1,6-
dihydropyrrolo[2,3-g]indazole as a promising scaffold for the
development of new potent Pim kinase inhibitors. We found that
compounds 10 and 20 exhibited interesting Pim-1 and Pim-3
inhibitory properties. More particularly, compound 20 demon-
strated a nanomolar activity against Pim-3. We also performed
molecular modeling experiments that enable us to propose a bind-
ing mode of compounds 10 and 20 in Pim-3 ATP-binding pocket.
Due to its high inhibitory potency toward Pim-3, compound 20
could be used as an interesting tool to study the biological role
of Pim-3 compared to the one of Pim-1 and Pim-2.
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The authors are grateful for the financial support of ANR (ANR-
08-JCJC-0131-CSD 3) and thank Bertrand Légeret for mass spectra
analysis.