Kinase inhibitory potencies and in vitro antiproliferative activities of N-10 substituted pyrrolo[2,3-a]carbazole derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.03.098

Development of potent and selective Pim kinase inhibitors has recently emerged as an important field for the design of new anti-cancer drugs. We report the synthesis of new N-10-substituted pyrrolo[2,3-a]carbazole derivatives and their evaluation as Pim kinase inhibitors. Moreover, in vitro antiproliferative activity of these compounds was evaluated toward a human fibroblast primary culture and three human solid cancer cell lines (PA1, PC3 and DU145). Compounds 3, 7 and 10 showed inhibitory potencies toward Pim-1 and Pim-3 in the nanomolar range. Additionally, dimethylamino analog 10 also demonstrated interesting sub-micromolar antiproliferative activities toward the cell lines tested.

Full text of DOI:10.1016/j.bmcl.2012.03.098

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3807–3809
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Kinase inhibitory potencies and in vitro antiproliferative activities of N-10
substituted pyrrolo[2,3-a]carbazole derivatives
Rufine Akué-Gédu a,b, Boris Letribot a,b, Emmanuelle Saugues a,b, Eric Debiton c,d, Fabrice Anizon a,b,
,
⇑
Pascale Moreau a,b,
⇑
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, F-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:
Development of potent and selective Pim kinase inhibitors has recently emerged as an important field for
the design of new anti-cancer drugs. We report the synthesis of new N-10-substituted pyrrolo[2,3-a]car-
bazole derivatives and their evaluation as Pim kinase inhibitors. Moreover, in vitro antiproliferative activ-
ity of these compounds was evaluated toward a human fibroblast primary culture and three human solid
cancer cell lines (PA1, PC3 and DU145). Compounds 3, 7 and 10 showed inhibitory potencies toward Pim-
1 and Pim-3 in the nanomolar range. Additionally, dimethylamino analog 10 also demonstrated interest-
ing sub-micromolar antiproliferative activities toward the cell lines tested.
Received 14 February 2012
Revised 23 March 2012
Accepted 28 March 2012
Available online 3 April 2012
Keywords:
Pyrrolo[2,3-a]carbazoles
Pim kinase inhibitors
In vitro antiproliferative activities
Antitumor agents
Ó 2012 Elsevier Ltd. All rights reserved.
The oncogenic Ser/Thr protein kinases Pim (Provirus Insertion
site of Moloney murine leukemia virus), comprising three highly
homologous isoforms Pim-1, Pim-2 and Pim-3, are part of the
CAMK (calcium/calmodulin-dependent protein kinase) family.
Pim kinases control various proteins involved in significant biolog-
ical processes such as cell cycle progression and apoptosis. As Pim
kinases are constitutively active, their functions are principally
regulated at the expression level, especially by the JAK/STAT sig-
naling pathway. Over-expression of Pim kinases have been noticed
in human leukemia and lymphoma, as well as in various cases of
solid tumors such as prostate, pancreatic, oral, hepatic and colon
cancers. Pim kinases are implicated in many signaling pathways
contributing to tumorigenesis. For example, it has been shown that
Pim-2 over-expression favors hematopoietic cell line survival in a
growth-factor independent manner. Moreover, in hematological
cells, production of cytokines and various survival factors is im-
proved by Pim-1 stimulation of several transcription factors like
c-Myb, NFATc1 and the RUNX family proteins. Additionally, the
three Pim isoforms are able to phosphorylate the pro-apoptotic
protein Bad leading to its inactivation and apoptosis inhibition.1–
of cancer cell migration and invasion.5 All these data demonstrate
the major importance of Pim kinases as signaling proteins that
could be intended for the identification of new antitumour
drugs.6,7
Recently, the synthesis and biological activities of pyrrolo[2,3-
a]carbazole-3-carbaldehyde
A A
were reported.8 Compound
(Fig. 1) exhibited potent and selective inhibitory potencies toward
Pim kinase family members when tested against a panel of 66 pro-
tein kinases. Thus, in order to identify new Pim kinase inhibitors, a
large structure–activity relationship study was performed within
this pyrrolocarbazole family.8–10 Highly potent inhibitors were
identified, mostly targeting Pim-1 and Pim-3 isoforms. Unfortu-
nately, possibly due to limitations in the solubility of this relatively
hydrophobic scaffold, despite potent kinase inhibitions, most of
pyrrolocarbazole derivatives have shown only moderate in vitro
antiproliferative activities.
The study of compound A crystal structure in complex with
Pim-1 revealed an unique H-bond established between the formyl
group at the 3-position of the pyrrolo[2,3-a]carbazole scaffold and
Lys67 side chain of the ATP binding site. Moreover, it was found
that none of the pyrrolocarbazole nitrogen atoms was involved in
the interaction between A and the Pim-1 ATP-binding site. Thus,
in order to improve the hydrophily/hydrophoby balance of this ser-
ies, the N-10 position was selected for the introduction of an amin-
oalkylated side chain, assuming that as the formyl group present at
the 3-position of the pyrrolo[2,3-a]carbazole moiety is part of a
4
Recently, Pim kinases have been described as potent stimulators
⇑
Corresponding authors. Tel.: +33 (0) 4 73 40 53 64; fax: +33 (0) 4 73 40 77 17
(F.A.); tel.: +33 (0) 4 73 40 79 63; fax: +33 (0) 4 73 40 77 17 (P.M.).
E-mail addresses: Fabrice.ANIZON@univ-bpclermont.fr (F. Anizon), Pascale.
MOREAU@univ-bpclermont.fr (P. Moreau).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.098

3808
R. Akué-Gédu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3807–3809
Lys67
H
NH2
O
5
4
6
3
H
7
8
2
N
H
N
H
10
1
9
A
Figure 1. Binding of compound A to Pim-1 ATP binding site (surfaces are colored in orange-red (hydrophobic) and in blue (hydrophilic)) [8, PDB 3JPV]. H-bond indicated in
dashed line.
vinylogous formamide system, the formyl carbonyl function
should be less prompt to react with primary or secondary amino
a
b
c
groups.8 Therefore, in this Letter, we report our efforts to synthe-
size new N-10 aminoalkylated substituted pyrrolo[2,3-a]carbazole
derivatives and their ability to inhibit Pim kinases. Moreover,
in vitro antiproliferative activity of these compounds was evalu-
ated by a fluorometric assay (resazurin reduction test) toward a
human fibroblast primary culture and three human solid cancer
cell lines (PA1, PC3 and DU145). The drug likeness of new com-
pounds was also estimated and compared to the one of the refer-
ence A.
N
H
N
N
N
N
N
N
H
SO2Ph
SO2Ph
3
3
1
O
NH2
O
4
5
CHO
d
N
N
H
N
N
H
3
3
In order to synthesize the target molecules, our initial approach
included the introduction of aminoalkyl side chains from the corre-
sponding nitrile analog (Scheme 1). Thus, compound 18 was first
alkylated with 5-bromopentanenitrile in the presence of potassium
tert-butoxide. Subsequent benzenesulfonyl group deprotection
using NaOH in H2O/MeOH solution led to compound 2 in 86% yield.
The Vilsmeier–Haack formylation step was performed under
microwave irradiation as already described8,9 to give compound 3
in 64% yield. Unfortunately, our attempts to reduce the nitrile group
under mild hydrogenation conditions (HCO2NH4, Pd/C or HCO2NH4,
PtO2 in MeOH) failed to afford the desired reduced product.
An alternative pathway was to prepare the amine function from
a phthalimide intermediate (Scheme 2). Therefore, compound 1
was allowed to react with N-(4-bromobutyl)phthalimide in the
presence of tBuOK leading to the corresponding alkylated deriva-
tive 4. Prior to the formylation step, the benzenesulfonyl group
was removed and the protecting phthalimide group was switched
to a Boc group after hydrazinolysis to give 6 in 88% yield from com-
pound 4 (3 steps). After formylation using oxalyl chloride/DMF in
MeOH, compound 7 was obtained in 55% yield. Unfortunately,
we did not manage to isolate the deprotected analog.
NHBoc
NHBoc
6
7
Scheme 2. Synthesis of compound 7. Reagents and conditions: (a) N-(4-bromobu-
tyl)phthalimide, tBuOK, 60%; (b) (i) H2N–NH2, H2O, EtOH, (ii) NaOH, 94%; (c) Boc2O,
NaHCO3, H2O, THF, 94%; (d) (i) (COCl)2, DMF, CH2Cl2, (ii) NaOH, 55%.
a
b
N
N
N
H
N
N
N
H
SO2Ph
SO2Ph
Br(CH2)4
Me2N(CH2)4
1
8
9
CHO
c
N
N
H
Me2N(CH2)4
10 (67%)
Scheme 3. Preparation of compound 10. Reagents and conditions: (a) 1,4-dibro-
mobutane, NaH, 84%; (b) (i) Me2NHÁHCl, K2CO3, KI, THF, (ii) NaOH, MeOH, 57%; (c)
(i) (COCl)2, DMF, CH2Cl2, (ii) NaOH, 67%.
Based on these results, we directed our synthetic efforts toward
the preparation of a tertiary amino analog. Dimethylamino deriva-
tive 10 was synthesized in four steps from compound 1 (Scheme
3). Brominated derivative 8 was prepared by reaction of 1 with
1,4-dibromobutane in the presence of NaH.10 Tertiary amine 10
was successfully obtained in 67% yield by treatment of 8 with
dimethylamine, deprotection and formylation.
In vitro kinase inhibitory potencies of compounds 3, 7 and 10
were evaluated toward the three Pim kinase isoforms in duplicate
as already described in the literature by Cohen’s group.11 The per-
centages of residual activity, when the compounds were tested at
10 lM and 1 lM toward Pim kinases (Pim-1, Pim-2 and Pim-3),
are reported in Table 1. IC50 values were determined when the per-
centages of residual activity were inferior to 25% when the com-
pounds were tested at 1 lM.
CHO
a
b
N
H
N
N
N
H
N
N
H
Compounds 3 and 10 have shown similar inhibitory profile to-
ward the three Pim kinases. Compared to reference compound A,
both of them are more active against Pim-1 with IC50 in the nano-
molar range (46 nM for 3 and 75 nM for 10). Regarding Pim-3,
compounds 3 and 10 were slightly less active than reference A.
In contrast, no potent activity was measured toward Pim-2 for
SO2Ph
NC(CH2)4
NC(CH2)4
1
2
3
Scheme 1. Synthesis of nitrile derivative 3. Reagents and conditions: (a) (i) 5-
bromopentanenitrile, tBuOK, DMF, (ii) NaOH, MeOH, 86%; (b) POCl3, DMF, MW, 64%.

R. Akué-Gédu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3807–3809
3809
Table 1
Kinase inhibitory potencies: % of residual kinase activity at 10 and 1
(IC50 in M)
lM (IC50 in lM in brackets when determined) and antiproliferative activities of compounds A, 3, 7 and 10
l
Compd
Kinase inhibition—% of residual kinase activity
Pim-2
Antiproliferative activity (IC50 in lM)
Pim-1
Pim-3
Fibro
1
PA1
PC3
DU145
10
2
l
M
1
l
M
10
7
l
M
1
l
M
10
1
l
M
1
l
M
A
0.4
nd
1
nd
32
97
5
nd
21
4.5 0.4
9.5 0.5
26
20
2
1
(0.12 0.01)
0.1
(0.51 0.23)
40
(nd)
54
(0.01 0.00)
0.5
3
1
3
0.8
1
3
3
1
7
2
3
1
14.5 0.8
17.3 0.9
0.63 0.02
8
2
16
13.1 0.6
0.65 0.02
2
(0.046 0.003)
(0.041 0.009)
3
7
4
1
23
1
1
16
5
14.9 0.5
12.2 0.3
(0.47 0.05)
10
(nd)
22
0.20 0.01
10
1
24
4
74 15
1
0
7
0
0.486 0.003
0.96 0.06
(0.075 0.015)
(nd)
(0.08 0.00)
nd: not determined.
these two compounds. tButoxycarbamate derivative 7 was the less
active of the series with IC50 values of 0.47 M and 0.20 M toward
Pim-1 and Pim-3, respectively. The results obtained demonstrated
that Pim-1 inhibitory potency of this series could be improved by
the substitution of the N-10 position with a cyanobutyl or a dime-
thylaminobutyl chain.
In vitro antiproliferative activities of compounds 3, 7 and 10
were evaluated toward a human fibroblast primary culture and
three human solid cancer cell lines: PA1 (ovarian carcinoma),
PC3 and DU145 (prostatic carcinoma). The antiproliferative effect
of the tested drug was assessed by the resazurin reduction test.12
As shown Table 1, compounds 3 and 7 have demonstrated antipro-
Overall, compound 10, that has shown improved Pim-1 kinase
l
l
inhibitory potency as well as enhanced in vitro antiproliferative
activity and favorable calculated drug-like properties, is particu-
larly attractive for the development of this series.
In conclusion, the synthesis of three new N-10-substituted pyr-
ro[2,3-a]carbazole derivatives was successfully achieved. The re-
sults obtained have shown that the improvement of the drug
likeness by introducing a N,N-dimethylaminobutyl chain at the
N-10 position of the pyrrolocarbazole scaffold (compound 10)
could improve both Pim-1 inhibition and antiproliferative activi-
ties toward the cancer cell line tested.
liferative activities in the range of 8–20 lM without significant
Acknowledgments
variation as compared to reference A. In contrast, the dimethyl-
amino derivative 10 exhibited interesting sub-micromolar antipro-
liferative activities toward all the cell line tested.
CNRS Valorisation (R.A.-G.) and French Ministère de l’Enseigne-
ment Supérieur et de la Recherche (E.S.) are greatly acknowledged
for financial support. The authors are grateful to Bertrand Légeret
for mass spectrometry analysis.
Finally, the physico-chemical properties (cLogP, solubility, drug
likeness, drug score) of compounds A, 3, 7 and 10 were evaluated
using Osiris property explorer (Table 2).13,14 Compounds logP val-
ues depict their partition coefficient between n-octanol and water,
measuring their hydrophylicity. Usually compounds with logP val-
ues inferior to 5.0 are supposed to present good absorption and
permeation properties. This was the case for compounds A, 3, 7
and 10 with calculated logP values ranging from 3.19 to 4.62. LogS
value is directly related to compounds aqueous solubility. More
than 80% of the marketed drugs have estimated logS values supe-
rior to À4. In this series, only dimethylamino derivative 10 has met
this criteria with an estimated logS value of À3.86 (compared to
À4.49 for A). Finally, the drug likeness and drug scores, that eval-
uate the overall potential of a compound to become a drug, were
calculated for each compound. As shown Table 2, the drug likeness
scores of compounds 3 and 7 were not very good with values of
À16.4 and À57, respectively. Compound 10 has shown the best
drug likeness (À0.04) and drug score (0.45) of the series, compared
to À3.57 and 0.3 for reference compound A. The Osiris molecular
properties were also calculated for doxorubicin, a well-known
anti-cancer agent, used for the treatment of solid tumors. As indi-
cated in Table 2, the global estimated drug score of compound 10 is
in the same range as the one of doxorubicin.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.
2012.03.098.
References and notes
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Table 2
Osiris molecular properties predictions for compounds A, 3, 7, 10 and doxorubin
Compd
MW
cLogP
LogS
DL
DS
11. Bain, J.; Plater, L.; Elliott, M.; Shpiro, N.; Hastie, J.; McLauchlan, H.; Klervernic,
I.; Arthur, S. C.; Alessi, D. R.; Cohen, P. Biochem. J. 2007, 408, 297.
12. O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J. Biochem. 2000, 267, 5421.
13. Sander, T.; Freyss, J.; Von Korff, M.; Reich, J. R.; Rufener, C. J. Chem. Inf. Model.
2009, 49, 232.
A
3
7
10
234
315
405
333
543
3.19
4.32
4.62
3.56
0.48
À4.49
À5.12
À5.57
À3.82
À4.51
À3.57
À16.4
À57
0.3
0.14
0.19
0.45
0.55
À0.04
7.19
14. Osiris molecular properties calculations program is freely available online at
the following site: http://www.organic-chemistry.org/prog/peo/
Doxorubicin
MW: molecular weight in g/mol; S: solubility; DL: Drug likeness; DS: drug score.