Synthesis, kinase inhibitory potencies, and in vitro antiproliferative evaluation of new pim kinase inhibitors

Article abstract of DOI:10.1021/jm901018f

Members of the Pim kinase family have been identified as promising targets for the development of antitumor agents. After a screening of pyrrolo[2,3-a]- and [3,2-a]carbazole derivatives toward 66 protein kinases, we identified pyrrolo[2,3-a]carbazole as a

Full text of DOI:10.1021/jm901018f

J. Med. Chem. 2009, 52, 6369–6381 6369
DOI: 10.1021/jm901018f
Synthesis, Kinase Inhibitory Potencies, and in Vitro Antiproliferative Evaluation of New Pim Kinase
Inhibitors†
Rufine Akue-Gedu,‡,§ Emilie Rossignol,‡,§ Stephane Azzaro,‡,§ Stefan Knapp, ,^ Panagis Filippakopoulos, Alex N. Bullock,
Jenny Bain,# Philip Cohen,# Michelle Prudhomme,‡,§ Fabrice Anizon,*,‡,§ and Pascale Moreau‡,§
ꢀ
ꢀ
ꢀ
‡Clermont Universiteꢀ, Universiteꢀ Blaise Pascal, Laboratoire SEESIB, F-63177 Aubieꢁre, France, §CNRS, UMR6504, F-63177 Aubieꢁre, France,
Structural Genomics Consortium, University of Oxford, Old Road Campus Building, Roosevelt Drive, Oxford OX3 7DQ, U.K., ^Department of
Clinical Pharmacology, University of Oxford, Old Road Campus Building, Roosevelt Drive, Oxford OX3 7DQ, U.K., and #MRC Protein
Phosphorylation Unit, College of Life Sciences, Sir James Black Centre, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
Received May 12, 2009
Members of the Pim kinase family have been identified as promising targets for the development of
antitumor agents. After a screening of pyrrolo[2,3-a]- and [3,2-a]carbazole derivatives toward 66 protein
kinases, we identified pyrrolo[2,3-a]carbazole as a new scaffold to design potent Pim kinase inhibitors.
In particular, compound 9 was identified as a low nM selective Pim inhibitor. Additionally, several
pyrrolo[2,3-a]carbazole derivatives showed selectivity for Pim-1 and Pim-3 over Pim-2. In vitro anti-
proliferative activities of 9 and 28, the most potent Pim inhibitors identified, were evaluated toward
three human solid cancer cell lines (PA1, PC3, and DU145) and one human fibroblast primary culture,
revealing IC50 values in the micromolar range. Finally, the crystal structure of Pim-1 complexed
with lead compound 9 was determined. The structure revealed a non-ATP mimetic binding mode with
no hydrogen bonds formed with the kinase hinge region and explained the selectivity of pyrrolo-
[2,3-a]carbazole derivatives for Pim kinases.
Introduction
by the pim proto-oncogenes.1-3 The first member of the
family, pim-1, was originally identified as a common proviral
insertion site in Moloney murine leukemia virus-induced
T-cell lymphomas in mice.4 In humans, three Pim family
members (Pim-1, Pim-2, and Pim-3) have been identified
which share 60-70% sequence identity in their kinase
domains.5 Pim kinases have been reported to be implicated
in various signaling pathways and play key roles regulating
cell cycle progression and apoptosis. Pim kinases are over-
expressed in many cancer types and are believed to play a role
in the initiation and progression of the malignant phenotype.
In particular, Pim-1 and Pim-2 have been reported to be
highly expressed in leukemia, lymphoma, prostate cancer,
and multiple myeloma.6-14 Moreover, it has been shown that
Pim-2 overexpression promotes growth-factor independent
survival of hematopoietic cell lines.11 A number of studies also
reported aberrant expression of Pim-3 in pancreas and colon
cancer and suggested an involvement of Pim-3 in gastric
adenoma-adenocarcinoma transition and subsequent inva-
sion and metastasis.15-18 Furthermore, it has been established
that Pim kinases are implicated in the development of resi-
stance to tyrosine kinase inhibitors19 and to the mTOR
inhibitor rapamycin.20,21 These findings suggest that Pim
kinases may be relevant anticancer drug targets and esta-
blished synthesis of Pim inhibitors as an attractive area of
chemical biology. Only a few selective Pim kinase inhibitors,
targeting mainly Pim-1 and Pim-2, have been described in the
literature so far but an increasing number of recent publica-
tions reporting the identification of new and selective Pim
kinase inhibitors indicate a growing interest in this field. As
illustrated in Figure 1, these inhibitors present a high degree of
chemical diversity. Quercetagetin I is a flavonoid of submi-
cromolar inhibitory activity towardPim-1.22 Incontrast tothe
Serine/threonine kinases of the Pim family (Provirus inte-
gration site for Moloney murine leukemia virusa) are encoded
†The structure of the cocrystal 9/Pim-1 have been deposited in the
PDB with accession code 3JPV.
*To whom correspondence should be addressed. Phone: þ33 (0) 4 73
40 53 64. Fax: þ33 (0) 4 73 40 77 17. E-mail: Fabrice.ANIZON@
univ-bpclermont.fr.
a Abbreviations: AMPK, AMP-activated protein kinase; ATF2,
activating transcription factor 2; ATP, adenosine triphosphate; BRSK,
brain-specific kinase; CaMK, calmodulin-dependent kinase; BSA, bo-
vin serum albumin; CaMKK, CaMK kinase; CDK, cyclin-dependent
protein kinase; CHK, checkpoint kinase; CK, casein kinase; CSK, C-
terminal Src kinase; DDQ, 2,3-dichloro-5,6-dicyano-p-benzoquinone;
DMF, dimethylformamide; DTT, dithiothreitol; EF2K, elongation-
factor-2 kinase; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,
N0,N0-tetraacetic acid; ERK, extracellular-signal-regulated kinase;
GSK3, glycogen synthase kinase 3; GST, glutathione transferase;
HIPK, homeodomain-interacting protein kinase; His6, hexahistidine;
IKK, inhibitory κB kinase; JNK, c-Jun N-terminal kinase; Lck, lym-
phocyte cell-specific protein-tyrosine kinase; MAPK, mitogen-activated
protein kinase; MAPKAP-K, MAPK-activated protein kinase; MARK,
microtubule-affinity-regulating kinase; MBP, myelin basic protein;
MELK, maternal embryonic leucine-zipper kinase; MKK1, MNK,
MAPK-integrating protein kinase; MSK, mitogen- and stress-activated
protein kinase; MST, mammalian homologue Ste20-like kinase; NEK,
NIMA (never in mitosis in Aspergillus nidulans)-related kinase; PAK,
p21-activated protein kinase; PDK, 3-phosphoinositide-dependent pro-
tein kinase; PHK, phosphorylase kinase; PIM, provirus integration site
for Moloney murine leukemia virus; PKA, cAMP-dependent protein
kinase; PKB, protein kinase B (also called Akt); PKC, protein kinase C;
PKD, protein kinase D; PLK, polo-like kinase; PPSE, polyphosphoric
acid trimethylsilyl ester; PRAK, p38-regulated activated kinase; PRK,
protein kinase C-related kinase; ROCK, Rho-dependent protein kinase;
RSK, p90 ribosomal S6 kinase; S6K1, S6 kinase 1; Sf21, Spodoptera
frugiperda (fall armyworm) 21; SGK, serum- and glucocorticoid-in-
duced kinase; SmMLCK, smooth-muscle myosin light-chain kinase;
Src, sarcoma kinase; SRPK, serine-arginine protein kinase; mTOR,
mammalian target of rapamycin; TFA, trifluoroacetic acid.
r
2009 American Chemical Society
Published on Web 09/29/2009
pubs.acs.org/jmc

6370 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Akueꢀ-Geꢀdu et al.
Scheme 1. Synthesis of Pyrrolo[2,3-a]- and [3,2-a]carbazolesa
a Reagents and conditions: (a) PhNHNH2, AcOH, 4 = 24%, 6 =
17%; (b) NaOH, MeOH, 5 = 96%, 7 = 92%; (c) (i) (COCl)2, DMF,
CH2Cl2, (ii) aq NaOH, 8 = 66%, 9 = 65%; (d) (i) PhNHNH2, PPSE,
CH3NO2, (ii) DDQ, 36%; (e) (i) PhNHNH2, choline chloride-ZnCl2 1:2,
(ii) DDQ, EtOAc, 78%.
Figure 1. Some of the Pim kinase inhibitors described recently in
the literature.
moderate inhibitory activity of quercetagetin, a potent and
selective Pim-1 inhibitory activity of nanomolar potency was
identified in substituted imidazo[1,2-b]- and triazolo[4,3-b]-
pyridazines series23,24 (II) andhighly potent organoruthenium
derivatives III.25,26 In addition, isoxazolo[3,4-b]quinoline-
dione IV have been reported as a nanomolar Pim-1 and
Pim-2 inhibitor with a high degree of selectivity27 and the
thiazolidine derivative V has micromolar potency for these
two kinases.28 Finally, compound VI and its analogues have
recently been reported to be a new class of selective Pim-1 and
Pim-2 inhibitors with nanomolar potencies.29 As part of our
ongoing studies concerning the design of new anticancer
agents, we are particularly interested in novel polyheterocyclic
scaffolds with kinase inhibitory potencies and in vitro anti-
proliferative activities. Here, we focused on the synthesis of a
series of pyrrolocarbazoles and in particular the preparation
of pyrrolo[2,3-a]- and [3,2-a]carbazole derivatives. Apart
from recent work by Fousteris et al. that disclosed an example
of pyrrolo[2,3-a]carbazole derivative as a modest inhibitor of
CDK1,30 pyrrolocarbazoles have not been identified as potent
and selective kinase inhibitors so far. In this study, we report
our efforts adopting the pyrrolo[2,3-a]carbazole scaffold for
the development of potent and novel Pim kinase inhibitors.
Finally, an X-ray structure of a cocrystal of Pim-1 in complex
with our lead compound was solved, revealing its ATP
competitive but not mimetic binding mode.
Synthesis and Kinase Inhibitory Activity. To identify new
scaffolds for the design of new kinase inhibitors, we started
our study from the pyrrolo[3,2-a]- and [2,3-a]carbazoles 5
and 7 (Scheme 1), which were described for the first time
more than 20 years ago.31,32 Pyrrolo[3,2-a]carbazole 5 or
substituted derivatives were synthesized either by Pd cross-
coupling,33 Fischer indole synthesis,34 or using photochemi-
stry.35,36 Substituted pyrrolo[2,3-a]carbazoles have been
recently synthesized by Stamos and Nikolaropoulos.30,37
Our approach to access these structures was a Fischer
indolization from tetrahydroindolone 2 and phenylhydra-
zine in acetic acid (Scheme 1). Poor yield and purification
difficulties led us to protect the nitrogen atom of compound 2
with a benzenesulfonyl group. Starting from protected deri-
vative 1,38 pyrrolocarbazole 4 was isolated in 24% yield.
Deprotection using sodium hydroxide in methanol led to 5 in
96% yield. Comparatively, compound 6 and 7 were prepared
from 339 in 17% and 92% yields, respectively, for Fischer
indolization and deprotection steps. Formylation of com-
pounds 5 and 7 was carried out using oxalyl chloride and
DMF in CH2Cl2, leading to 8 and 9 in 66% and 65% yields,
respectively.
At this stage, we undertook a screen of compounds 5, 7, 8,
and 9 toward a panel of protein kinases. Compounds were
tested at 10 μM toward 66 protein kinases, using an assay
described by Bain et al.40 The results are shown as the
percentage of remaining activity in the presence of an inhibitor
compared with the control incubations in the absence of
inhibitor (Table 1). No significant activity was found for
pyrrolo[3,2-a]carbazoles 5 and 8. These two compounds in-
duced less than 30% of enzyme inhibition with all kinases
tested, except for compound 5, causing 53% inhibition of
ERK8 and 71% inhibition of Pim-1, and for compound 8,
inducing 64% inhibition of CaMK1. Conversely, we were
pleased to notice that compound 9 was a potent and selective
inhibitor of Pim kinases, with an inhibition of enzymatic
activity of 98% for Pim-1, 93% for Pim-2, and 99% for
Pim-3. Furthermore, unsubstituted compound 7 was slightly
active toward Pim-1 and Pim-3 kinases, with an inhibition of
enzymatic activity of 80% and 62%, respectively.
These preliminary results encouraged us to perform the
synthesis of new pyrrolo[2,3-a]carbazole derivatives. When
compared to 7, compound 9 is bearing a formyl group at the
C-3 position, which positively contributes to the Pim kinase
inhibition (compare 7 and 9). We expected that the introduc-
tion of various substituents at this position would lead to
compounds of increased inhibitory potency. Nevertheless,
the poor yield obtained for the synthesis of compound 7 was
detrimental to the overall synthesis efficiency. The methods
used to optimize the Fischer indolization step are outlined in
Scheme 1. We first tried the use of polyphosphoric acid
trimethylsilyl ester (PPSE) for the indolization followed by
the use of DDQ for the aromatization, as described by
Fousteris et al.37 for the preparation of pyrrolo[2,3-a]-
carbazole substituted at the C-2 position by an ethoxycar-
bonyl group. Using this method, the yield of preparation of 6
from 3 was increased 2-fold from 17% to 36%. Nevertheless,
this yield was not satisfying and the use of dangerous
nitromethane prompted us to find other conditions. Recent
work by Xu et al. has related the use of ionic liquid for
Fischer indole synthesis.41 We then decided to experiment
this ionic liquid method by the use of the easy accessible
choline chloride-zinc chloride 1:2 ionic liquid. A mixture of
phenyl hydrazine and 3 in this ionic liquid was heated at
120 ꢀC and subjected to DDQ oxidation in a one-pot
procedure, to give 6 in a higher 78% yield.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6371
Table 1. Residual Activities (%) at 10 μM for Compounds 5 and 7-9
compd
This optimized procedure for the synthesis of the pyrrolo-
[2,3-a]carbazole scaffold made easier the preparation of new
diversely substituted derivatives. We then followed up our
investigations by the synthesis of pyrrolo[2,3-a]carbazoles
diversely substituted at the C-3 position (Scheme 2). Reduc-
tion of 9 using NaBH4 led to 10 in 67% yield. Carboxamide
11 and trifluoroacetyl derivative 12 were prepared from 7
using chlorosulfonylisocyanate and trifluoroacetic anhy-
dride in 20% and 42% yields, respectively. Reaction of 7
with oxalyl chloride was carried out to prepare the acyl
chloride intermediate 13, which was directly used for the
following step. Treatment of 13 with ethanol or diethylamine
led respectively to the ester 14 in 73% yield and to amide 15 in
66% yield. Reduction of 14 was carried out with LiAlH4 to
give alcohol 16 in 72%. Reaction of 15 with hydroxylamine
led to oxime derivative 17 in 19% yield.
To get insight on the influence of the substituents intro-
duced at the C-3 position or the presence of a benzenesulfo-
nyl group at the N-1 position, compounds 6, 10-12, 16, and
17 were tested for their ability to inhibit Pim kinases at 10 and
1 μM (Table 2). Compared to compound 9, no increase of
inhibition was observed. Only compounds 11 and 12, bearing
carboxamido or trifluoroacetyl groups, exhibited a Pim
kinase inhibition percentage higher than 90% at 10 μM.
Compound 11 caused 91% inhibition of Pim-1 and Pim-3 at
10 μM. At the same concentration, compound 12 induced
93% inhibition of Pim-3 activity. The reduction of the formyl
to a hydroxymethyl group (compound 10) was detrimental
to the inhibition potency, with a residual activity in the same
range as the nonsubstituted pyrrolocarbazole 7. Moreover,
compound 6, bearing a benzenesulfonyl group at the N-1
position, was only slightly active, with an enzymatic inhibi-
tion <40% against the three Pim kinases. All together, this
first insight on the inhibition potencies of these derivatives
indicates that the presence of a free indole nitrogen and of a
strong hydrogen bond acceptor like a formyl group at the C-
3 position seem to be required for good activity.
To carry on with the synthesis of new pyrrolo[2,3-a]-
carbazole analogues, we prepared new derivatives substi-
tuted at the C-7 position. Substitution at this position was
easily accessible from p-bromophenylhydrazine. Reaction of
p-bromophenylhydrazine with ketone 3 in ionic liquid fol-
lowed by oxidation in the presence of DDQ led to compound
18 in 74% yield (Scheme 3). Deprotection was performed in
NaOH/MeOH, leading to 19 in 92% yield. Various substit-
uents were introduced via a Suzuki cross-coupling by reac-
tion of 19 with several aryl boronic acids in the presence of
Pd(PPh3)2Cl2 and Na2CO3. Compounds 20-27 were iso-
lated in 50-65% yields.
Formylation of 19 was carried out in the same conditions
as that for the preparation of compound 9, leading to 28 in
66% yield (Scheme 3). However, these conditions could not
be applied to 20-27 for solubility problems. Therefore, the
formylation of 20 was achieved at 120 ꢀC in Vilsmeier
conditions using POCl3 and DMF, leading to the isolation
of 29 in 45% yield. To shorten the reaction time, formylation
of compounds 21-27 was performed under microwave
irradiation. Carboxamides 29-35 were obtained in
30-67% yield. Unfortunately, a formylated analogue of 27
could not be isolated because of purification problems.
These new compounds 19, 28-35 were tested toward Pim
kinases at 10 and 1 μM (Table 2). Only three compounds
were potent inhibitors, causing more than 90% of enzyme
inhibition at 10 μM. At this concentration, when bromo
kinases
MKK1
ERK1
5
7
8
9
75 ( 6
97 ( 18
100 ( 10
116 ( 2
130 ( 9
111 ( 0.4
115 ( 14
91 ( 4
111 ( 7
89 ( 9
47 ( 16
99 ( 11
107 ( 0.8
82 ( 3
110 ( 8
107 ( 2
102 ( 1
87 ( 1
96 ( 7
89 ( 11
84 ( 8
87 ( 37
108 ( 9
94 ( 11
102 ( 11
118 ( 10
108 ( 3
108 ( 6
100 ( 13
111 ( 13
78 ( 4
104 ( 14
95 ( 9
94 ( 13
113 ( 2
111 ( 7
96 ( 4
108 ( 9
96 ( 4
109 ( 2
83 ( 4
112 ( 38
120 ( 22
78 ( 9
85 ( 10
112 ( 8
107 ( 16
105 ( 4
82 ( 10
83 ( 4
50 ( 7
87 ( 13
92 ( 8
102 ( 7
128 ( 8
113 ( 8
102 ( 23
80 ( 9
74 ( 10
92 ( 0.5
20 ( 2
38 ( 7
77 ( 5
76 ( 3
96 ( 1
98 ( 6
64 ( 9
38 ( 2
60 ( 12
25 ( 2
31 ( 4
84 ( 17
83 ( 1
28 ( 4
71 ( 1
27 ( 1
55 ( 2
84 ( 0.2
69 ( 0.5
72 ( 4
42 ( 3
56 ( 4
71 ( 4
60 ( 12
33 ( 8
97 ( 4
38 ( 4
88 ( 5
74 ( 9
91 ( 4
68 ( 2
79 ( 3
59 ( 3
88 ( 0.3
22 ( 18
61 ( 14
103 ( 3
62 ( 3
88 ( 3
78 ( 11
89 ( 2
71 ( 4
2 ( 0.4
7 ( 1
ERK2
JNK1
JNK2
JNK3
p38R MAPK
P38β MAPK
p38γ MAPK
p38δ MAPK
ERK8
55 ( 6
105 ( 3
99 ( 2
RSK1
RSK2
PDK1
PKBR
PKBβ
SGK1
86 ( 1
120 ( 4
107 ( 0.2
82 ( 13
89 ( 2
95 ( 2
86 ( 2
95 ( 2
111 ( 27
91 ( 7
83 ( 9
90 ( 2
69 ( 3
95 ( 6
118 ( 12
89 ( 3
97 ( 2
S6K1
PKA
ROCK2
PRK2
86 ( 4
101 ( 16
95 ( 10
91 ( 6
87 ( 2
84 ( 8
106 ( 6
109 ( 16
95 ( 5
90 ( 7
114 ( 7
90 ( 4
102 ( 0.2
36 ( 0.3
91 ( 5
88 ( 6
101 ( 7
89 ( 5
PKCR
103 ( 14
87 ( 2
71 ( 3
PKCζ
PKD1
MSK1
94 ( 5
MNK1
MNK2
MAPKAP-K2
MAPKAP-K3
PRAK
CAMKKR
CAMKKβ
CaMK1
smMLCK
PHK
101 ( 7
103 ( 14
95 ( 20
88 ( 7
114 ( 12
92 ( 8
87 ( 6
80 ( 5
82 ( 7
71 ( 6
112 ( 6
73 ( 5
102 ( 9
95 ( 2
105 ( 7
67 ( 0.1
99 ( 6
79 ( 6
94 ( 8
76 ( 8
82 ( 20
99 ( 1
76 ( 4
93 ( 4
29 ( 11
86 ( 12
90 ( 2
123 ( 2
58 ( 8
111 ( 2
97 ( 1
111 ( 12
67 ( 5
85 ( 1
80 ( 2
116 ( 12
58 ( 6
93 ( 7
CHK1
CHK2
GSK3β
CDK2-Cyclin A
PLK1
96 ( 8
109 ( 1
106 ( 6
92 ( 1
104 ( 0.3
85 ( 6
105 ( 5
101 ( 6
89 ( 19
113 ( 1
98 ( 17
100 ( 2
86 ( 6
95 ( 1
108 ( 14
79 ( 4
102 ( 1
103 ( 7
93 ( 16
96 ( 8
AURORA B
AURORA C
AMPK
MARK3
BRSK2
MELK
CK1
107 ( 3
91 ( 15
104 ( 7
90 ( 6
CK2
96 ( 21
96 ( 5
71 ( 3
NEK2a
NEK6
NEK7
92 ( 3
96 ( 1
IKKβ
Pim-1
110 ( 32
53 ( 0.1
77 ( 5
29 ( 3
94 ( 10
77 ( 1
106 ( 1
20 ( 0.1
62 ( 8
38 ( 2
90 ( 13
82 ( 5
Pim-2
Pim-3
1 ( 5
SRPK1
MST2
EF2K
84 ( 8
63 ( 4
90 ( 14
25 ( 4
64 ( 4
88 ( 13
87 ( 1
94 ( 10
85 ( 20
76 ( 1
74 ( 5
92 ( 7
101 ( 7
59 ( 7
97 ( 1
84 ( 13
82 ( 10
92 ( 3
105 ( 3
95 ( 18
100 ( 3
88 ( 13
96 ( 4
HIPK2
HIPK3
PAK4
72 ( 18
106 ( 4
84 ( 9
99 ( 3
91 ( 2
PAK5
PAK6
99 ( 8
Src
Lck
102 ( 23
81 ( 14
72 ( 17
98 ( 18
88 ( 14
98 ( 11
99 ( 11
114 ( 49
87 ( 8
CSK

6372 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Akueꢀ-Geꢀdu et al.
Scheme 2. Synthesis of Pyrrolo[2,3-a]carbazole Derivatives Substituted at the C-3 Positiona
a Reagents and conditions: (a) NaBH4, MeOH, 67%; (b) (i) ClSO2NCO, CH3CN; (ii) HCl, H2O/THF, 20%; (c) (CF3CO)2O, DMF, 42%; (d)
(COCl)2, Et2O; (e) EtOH, NEt3, 73% from 7; (f) LiAlH4, dioxane, 72%; (g) Et2NH, Et2O, 66% from 7; (h) NH2OH, pyridine, 19%.
Table 2. Residual Activities (%) at 10 and 1 μM for Compounds 7,
9-12, 15, 17, 19, and 28-35 (IC50 (μM) are Indicated in Parenthesis)
In Vitro Antiproliferative Activity. In vitro antiprolifera-
tive activities of 9 and 28 were examined by a fluorometric
assay (resazurin reduction test) using one human fibroblast
primary culture and three human solid cancer cell lines: PC3
and DU145 (prostate cancer cells) and PA1 (ovarian
carcinoma) (Table 3). Whereas compound 9 exhibited the
best inhibitory activity toward Pim kinases, compound 28
was found to be more active against the cell lines tested,
possibly due to its improved cellular penetration. The best in
vitro antiproliferative activity of compound 28 was found
against PA1 cells, with an IC50 value of 2.8 μM. In addition,
compound 28 exhibited an interesting potency against PC3
(IC50 value of 5.9 μM), which is known to express high level
of Pim-1 kinase. Toward DU145 and fibroblast cell lines, in
vitro antiproliferative activities of compound 28 were found
to be in the same range, with IC50 values of 12.2 μM toward
DU145 cell line and 11 μM toward fibroblast cells. In
comparison to 28, compound 9 was found to be less active.
As for compound 28, the higher potency of compound 9 was
determined against the PA1 cell line, with an IC50 value of
4.5 μM. Toward the other cell lines, IC50 values were found
to be in the range 10-30 μM, with the best potency against
PC3 (IC50 value of 9.5 μM).
Pim-1
compd 10 μM 1 μM
Pim-2
10 μM 1 μM
Pim-3
10 μM 1 μM
6
7
9
61 ( 7 83 ( 5
20 ( 0
2 ( 0
62 ( 13 65 ( 12
62 ( 8
7 ( 1
64 ( 3 102 ( 4
38 ( 2
1 ( 5
(0.12 ( 0.01)
(0.51 ( 0.23)
(0.01 ( 0.00)
10
11
28 ( 2
9 ( 0
72 ( 15 47 ( 18 46 ( 9
27 ( 2
18 ( 2
9 ( 7
68 ( 12
(0.78 ( 0.02)
(0.21 ( 0.06)
12
20 ( 3
61 ( 13 30 ( 1
54 ( 28
7 ( 3
20 ( 8
(0.17 ( 0.05)
16
17
19
35 ( 4
41 ( 6
12 ( 0
90 ( 5
80 ( 3
69 ( 4
50 ( 9
27 ( 2
46 ( 3
69 ( 4
62 ( 9
67 ( 15
21 ( 7
21 ( 3
8 ( 2
72 ( 8
74 ( 2
55 ( 11
(0.44 ( 0.01)
28
6 ( 2
31 ( 2
53 ( 7
82 ( 5
6 ( 0
11 ( 3
(0.57 ( 0.04)
(0.04 ( 0.01)
29
30
31
32
33
17 ( 3
16 ( 1
39 ( 7
15 ( 2
6 ( 1
64 ( 18 31 ( 3
60 ( 5 53 ( 1
69 ( 21 70 ( 8
65 ( 15 12 ( 1
46 ( 8
54 ( 6
77 ( 5
54 ( 9
26 ( 3
68 ( 16
67 ( 4
15 ( 0
48 ( 6
15 ( 1
6 ( 1
65 ( 3
39 ( 1
55 ( 2
32 ( 5
72 ( 10
59 ( 19
(0.66 ( 0.18)
28 ( 0 79 ( 1
29 ( 1
(0.20 ( 0.04)
X-ray Structure Analysis. All the structure-activity rela-
tionship studies presented above were established without
any structural insight on the mode of binding of this new
series of Pim kinase inhibitors. Therefore, after the identifi-
cation of our lead compound, we were interested to deter-
mine the binding mode and point out the interactions that
would explain the high selectivity of this inhibitor for Pim
kinases and its modest but significant selectivity for Pim-3.
To accomplish this, compound 9, the best active derivative in
this series, was cocrystallized with Pim-1 kinase and the
34
35
52 ( 6
49 ( 2
23 ( 1
31 ( 2
70 ( 8
77 ( 3
68 ( 10 70 ( 23 73 ( 11
analogues 19 and 28 were evaluated, the percentages of
residual enzymatic activity were found to be 12% and 6%
for Pim-1 and 8% and 6% for Pim-3, respectively. As for
compounds 7 and 9, the presence of a formyl group at the C-3
position was favorable to the inhibitory potency. Conver-
sely, the presence of an aryl group led to the smallest
inhibitory potencies when compared with compounds 9 or
28, except for compound 33 bearing a 2,4-difluorophenyl
group at the C-7 position, inhibiting Pim-1 and Pim-3
kinases with a percentage of inhibition of 94%. While
compounds 30 and 32, bearing respectively phenyl and
4-fluorophenyl groups, exhibited similar and mild potencies
toward the three Pim kinases, it appears that the increased
inhibitory activity for compound 33 is due to the extra
fluorine atom at the 2 position of the phenyl group.
Compounds that caused enzyme inhibition greater than
90% at 10 μM (compounds 9, 11, 12, 19, 28, and 33, Table 2)
were used for the determination of IC50 values toward corre-
sponding Pim kinases. All these compounds showed an IC50
value in the submicromolar range. The inhibitory potency was
different considering the kinase tested. Our results suggest
that the order of inhibitory potency for these new inhibitors
was Pim-3 > Pim-1> Pim-2. The bestcompoundof the series
against all the three Pim kinases was the formyl derivative 9,
with IC50 values of 0.01 μM for Pim-3, 0.12 μM for Pim-1, and
0.51 μM for Pim-2. Moreover, compound 28 exhibited a
nanomolar inhibitory potency for Pim-3, with an IC50 value
of 0.04 μM. Compound 28 was also active against Pim-1, with
an IC50 value of 0.57 μM.
˚
X-ray crystal structure was solved at 2.35 A resolution.
Statistics on data collection and refinement are summarized
in Table 4. Compound 9 occupied the ATP-binding site of
Pim-1. However, in contrast to typical ATP mimetic inhibi-
tors, the planar pyrrolocarbazole scaffold was inserted into
the ATP binding cleft with aromatic NH oriented away from
the hinge region not forming hydrogen bonds with Glu121 or
another backbone carbonyl of the Pim hinge region. Thus,
binding of compound 9 is ATP competitive but not ATP
mimetic (Figure 2). The aldehyde formed a hydrogen bond
with the conserved active site lysine, Lys67. On the basis of
the presence of a formyl group in the structure of these
compounds, one could envisage the formation of a Schiff
base with a primary amino group in the enzyme, stabilized by
conjugation with the aromatic pyrrolocarbazole nucleus.
Conversely, the carbonyl group could be considered to
belong to a vinylogous formamide system, with the conju-
gated N-1 electron pair decreasing the electrophilicity of the
carbonyl group. The electron density and its distance to the
lysine amino group indicated that, in the conditions used for
crystallization, no covalent bond was formed between the
inhibitor and the enzyme active site. The C2-C3 region of
the inhibitor formed an aromatic stacking interaction with

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6373
Scheme 3. Synthesis of Pyrrolo[2,3-a]carbazole Derivatives Substituted at the C-7 Positiona
a Reagents and conditions. (a) (i) 4-Br-PhNHNH2, choline chloride-ZnCl2 1:2, 120 ꢀC, (ii) DDQ, dioxane, 74%; (b) NaOH, MeOH, 92%;
(c) (i) ArB(OH)2, Pd(PPh3)2Cl2, aq Na2CO3, THF, (ii) aq NaOH, 50-65%; (d) (i) (COCl)2, DMF, CH2Cl2, 0 ꢀC, (ii) aq NaOH, rt, R = Br, 66%;
(e) (i) POCl3, DMF, 120 ꢀC, (ii) aq NaOH, rt, R = 3-CH3O-Ph, 45%; (f) (i) POCl3, DMF, 100 ꢀC, μw, (ii) aq NaOH, 30-67%.
Table 3. In Vitro Antiproliferative Activity (IC50 in μM) of Compounds
9 and 28
IC50 (μM)
compd
PC3
DU145
PA1
fibroblast
9
28
9.5 ( 0.5
5.9 ( 0.4
26 ( 2
12.2 ( 0.8
4.5 ( 0.4
2.8 ( 0.2
21 ( 1
11 ( 1
Table 4. Data Collection and Refinement of the Pim-1 Crystal Struc-
ture in Complex with Compound 9
Data Collection
PDB ID
space group
3JPV
P65
Figure 2. Binding of compound 9 to the ATP binding site of Pim-1.
(A) Residues in the vicinity of the inhibitor are shown in stick
representation and are labeled. Carbon atoms of the inhibitor are
shown in yellow and carbon atoms of the substrate peptide are
colored in white. (B) 2FoFc electron density map around the
inhibitor contoured at 2σ.
˚
cell dimensions: a, b, c (A)
97.82 97.82 80.74
2.35 (2.48-2.35)
18386 (2654)
99.9 (100.0)
4.6 (4.6)
a
˚
resolution (A)
unique observationsa
completenessa (%)
redundancya
a
kinase inhibitors can be efficiently anchored by polar con-
tacts with the active site lysine and by hydrophobic interac-
tion to Pim active site residues.
Rmerge
I/σIa
0.127 (0.763)
10.6 (2.0)
Refinement
Key residues interacting with compound 9 are conserved
within Pim family members. It is therefore surprising that
compound 9 and a number of similar compounds show
selectivity for Pim-1 and Pim-3 over Pim-2. Preferential
binding of inhibitors to Pim-1 over Pim-2 has been noted
before.23,42 Comparing the structures of Pim-1 with Pim-2
(PDB code 2IWI) revealedthatonly four residuesaredifferent
between the two active sites. Interestingly two residues are
located in the hinge region substituting the Pim-1 residue
Glu124 with a leucine and Val126 with an alanine in Pim-2.
The lack of polar interactions of Glu124 with Arg122 results
most likely in the observed disorder of the arginine side chain
in the Pim-2 crystal structure. Probably the destabilization of
the hinge region in Pim-2 caused by the Glu124Leu substitu-
tion influence hinge dynamics and consequently inhibitor
binding. Also the Val126/Ala substitution in Pim-2 may result
in slighly reduced hydrophobic interactions with the inhibitor.
In addition, substitutions in the glycine rich loop residues
located in a region that hinges the movement of Phe49 into the
active site (Ser51Thr; Ser46Lys) may influence the energy
barrier of this movement and may result in weaker inhibitor
binding for Pim-2. However, more detailed studies are neces-
sary to unravel differences of dynamic active site properties of
Pim family members.
˚
resolution (A)
2.35
Rwork/Rfree (%)
no. of atoms (protein/other/water)
17.5/23.1
2213/83/160
22.03/29.59/30.63
0.015
2
B factors (A ) (protein/other/water)
˚
˚
rmsd bonds (A)
rmsd angles (deg)
Ramachadran:
favored (%)
1.540
91.5
7.6
0.8
0.0
allowed (%)
generously allowed (%)
disallowed (%)
a Values in parentheses correspond to the highest resolution shell.
the P-loop phenylalanine Phe49, which is flipped into the
kinase active site as observed in a number of other Pim-1
inhibitor complexes.42-45
The presence of a proline residue in the Pim hinge region
does not permit the formation of two hydrogen bonds to
ATP or ATP mimetic inhibitors. However, a number of
inhibitors including flavonoid and bisindolylmaleimide
showed typical ATP mimetic hinge binding when crystallized
with Pim-1.29,42 The structure of an imidazo[1,2-b]pyridazine
showed recently that this inhibitor class interacts with Pim-1
in a non-ATP mimetic binding mode.23 In this structure, the
inhibitor formed similar interactions with Pim-1 as observed
for compound 9, including polar interaction with lysine 67,
aromatic interactions with the glycine rich loop residue
Phe49, and a number of hydrophobic contacts involving
Leu44, Ile104, and Leu120. Thus, it seems that for Pim
Conclusion
In conclusion, this work reports a structure-activity rela-
tionship studies of new pyrrolo[2,3-a]carbazoles as Pim kinase

6374 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Akueꢀ-Geꢀdu et al.
inhibitors. An improved synthesis of the pyrrolo[2,3-a]-
carbazole scaffold by Fischer indole synthesis in ionic liquid
conditions was achieved to get an easier access to diversely
substituted derivatives. Compound 9, substituted at the C-3
position by a formyl group, exhibited a potent and selective
inhibitory potency of Pim kinases when tested toward a panel
of 66 kinases, with a higher activity against Pim-3 kinase in the
nanomolar range. In addition, compound 9 and its C-7
brominated analogue 28 were evaluated for their in vitro
antiproliferative potencies toward three cancer cell lines and
a fibroblast primary culture. IC50 values in the micromolar
range were determined for compounds 9 and 28 toward PA1
and PC3 cell lines. Finally, the structure of a cocrystal 9/Pim-1
was resolved by X-ray analysis, showing the interaction
of 9 within the ATP binding site. This X-ray structure
provided valuable information that will facilitate further
development of the structure based design of new Pim kinase
inhibitors in this series, work that has already been initiated in
our laboratory.
and methanol was evaporated. Water was added, and the
mixture was extracted with EtOAc. The organic fractions were
washed with saturated aq NaHCO3, brine, and dried over
MgSO4. After evaporation, the residue was purified by flash
chromatography (cyclohexane/EtOAc, from 95:5 to 8:2) to give
5 (420 mg, 2.04 mmol, 96%) as a yellow solid. HPLC purity,
system I: >99%, λ=248 nm, tR =23.8 min. For spectral data of
compound 5, see ref 33.
1-Benzenesulfonyl-1,10-dihydropyrrolo[2,3-a]carbazole (6). Pre-
paration of the Ionic Liquid. A mixture of choline chloride (10.0 g,
71.6 mmol) and zinc chloride (19.5 g, 143 mmol) in toluene (200
mL) was refluxed in a Dean-Stark apparatus under vigorous
stirring for 15 h. After cooling and decantation, the ionic liquid
was obtained as a yellow oil, which can be directly used or conserved
under inert atmosphere in anhyd ether.
Step A. A mixture of phenylhydrazine (393 mg, 3.63 mmol), 3
(500 mg, 1.82 mmol), and ionic liquid (5.25 g, 12.7 mmol) was
stirred at 120 ꢀC for 2 h. After cooling, a 0.5 M aq HCl solution
was added before extraction with EtOAc (3 ꢀ 50 mL). The
organic fractions were washed with brine and then dried over
MgSO4. The solution contained the major indolization product
and the minor expected product.
Step B. DDQ (0.29 g, 1.28 mmol, necessary DDQ quantity
was determined from a 1H NMR spectrum of the intermediate
mixture) was added to this solution and the mixture was stirred
at room temperature for 15 h. The mixture was washed with
brine and dried over MgSO4. After evaporation, the residue was
purified by flash chromatography (pentane/EtOAc, 95:5 then
90:10) to give 6 (490 mg, 1.41 mmol, 78% yield) as a white solid;
mp 148 ꢀC. HRMS (ESIþ) calcd for C20H15N2O2S (M þ H)þ
347.0854, found 347.0861. IR (KBr): 3430, 1630, 1610 cm-1. 1H
NMR (400 MHz, DMSO-d6): 6.85 (1H, d, J = 3.5 Hz),
7.28-7.39 (4H, m), 7.44-7.52 (2H, m), 7.53 (1H, d, J =3.5
Hz), 7.64 (1H, d, J=8.0 Hz), 7.75-7.80 (2H, m), 8.01 (1H, d, J=
8.0 Hz), 8.12 (1H, d, J=8.0 Hz), 10.12 (1H, br s, NH). 13C NMR
(100 MHz, DMSO-d6): 111.4, 112.6, 113.0, 117.1, 119.9 (2C),
125.5, 126.1, 126.7 (2C), 129.5 (2C), 133.9 (CH), 120.4, 121.9,
123.8, 127.0, 130.6, 137.9, 138.8 (C). HPLC purity, system I:
>99%, λ =306 nm, tR =28.6 min.
1,10-Dihydropyrrolo[2,3-a]carbazole (7). A 5 M aq NaOH
solution (125 mL) was added to a suspension of 6 (3.12 g, 9.0
mmol) in MeOH (1.0 L). The mixture was refluxed for 12 h. The
reaction mixture was then concentrated under vacuum until
formation of a precipitate, which was collected by filtration. The
solid was washed with MeOH (10 mL) and then with Et2O (2 ꢀ
30 mL) to give 7 (1.71 g, 8.3 mmol, 92%) as an off-white powder;
mp 295 ꢀC. HRMS (ESIþ) calcd for C14H11N2 (M þ H)þ
207.0922, found 207.0925. IR (KBr): 3420, 3385, 1650, 1455,
1445, 1395, 1345, 1310 cm-1. 1H NMR (400 MHz, DMSO-d6):
6.61 (1H, dd, J1 =3.0 Hz, J2 =2.0 Hz), 7.18 (1H, ddd, J1 =8.0
Hz, J2 =7.0 Hz, J3 =1.0 Hz), 7.33 (1H, ddd, J1 =8.0 Hz, J2 =7.0
Hz, J3=1.0 Hz), 7.37 (1H, d, J=8.5 Hz), 7.42 (1H, t, J=3.0 Hz),
7.64 (1H, d, J=8.0 Hz), 7.75 (1H, d, J=8.5 Hz), 8.07 (1H, d, J=
7.5 Hz), 10.88 (1H, br s, NH), 10.95 (1H, br s, NH). 13C NMR
(100 MHz, DMSO-d6): 102.8, 111.2, 112.0, 112.1, 118.6, 119.0,
123.3, 123.6 (CH), 116.2, 121.8, 124.1, 126.2, 126.4, 138.1 (C).
HPLC purity, system I: >99%, λ =249 nm, tR =24.5 min.
Experimental Section
Chemistry. General. IR spectra were recorded on a Perkin-
Elmer Paragon 500 spectrometer (ν in cm-1). NMR spectra
were performed on a Bruker AVANCE 400 (1H, 400 MHz; 13C,
100 MHz) or AVANCE 500 (1H, 500 MHz; 13C, 125 MHz),
chemical shifts δ in ppm, the following abbreviations are used:
singlet (s), doublet (d), broad doublet (br d), triplet (t), quad-
ruplet (q), doublet of doublet (dd), doublet of doublet of doublet
(ddd), sextuplet (sx), multiplet (m), broad signal (br s). Mass
spectra (ESIþ) were determined on a high-resolution Micro Q-
Tof apparatus. Mass spectra (FABþ) were determined on a
high-resolution Fisons Autospec-Q spectrometer at CESAMO
(Talence, France). Chromatographic purifications were per-
formed by flash silica gel Geduran SI 60 (Merck) 0.040-0.063
mm column chromatography. Reactions were monitored by
TLC using fluorescent silica gel plates (60 F254 from Merck).
Experiments under microwave irradiation were performed
using a CEM Discover Benchmate apparatus. Melting points
were measured on a Reichert microscope and are uncorrected.
The purity of tested compounds was established to be g95% by
HPLC analysis using a Dionex liquid chromatograph (TCC-
100, 25 ꢀC; P580; UVD340U) and a C18 Dionex Acclaim 120
˚
column (4.6 mm ꢀ 250 mm, 5 μm, 120 A). Detection wavelength
is indicated for each compound. Solvents were (A) water, 0.1%
TFA; (B) acetonitrile; (C) water; system I: 95:5 A/B for 5 min
and then 95:5 A/B to 5:95 A/B in 30 min and then 5:95 A/B for 5
min; 0.8 mL/min; system II: 95:5 C/B for 5 min and then 95:5 C/
B to 5:95 C/B in 30 min and then 5:95 C/B for 5 min; 0.8 mL/min.
3-Benzenesulfonyl-3,10-dihydropyrrolo[3,2-a]carbazole (4).
Phenylhydrazine (1.73 mL, 17.6 mmol) was added to a solution
of 2 (2.42 g, 8.8 mmol) in glacial acetic acid (35 mL). The mixture
was refluxed for 38 h. After evaporation, the residue was
purified by flash chromatography (cyclohexane/EtOAc, from
99:1 to 8:2) to give 4 (740 mg, 2.14 mmol, 24%) as a light-yellow
solid; mp 185 ꢀC. HRMS (ESIþ) calcd for C20H15N2O2S (M þ
H)þ 347.0854, found 347.0855. IR (KBr): 3420 cm-1. 1H NMR
(400 MHz, DMSO-d6): 7.21 (1H, d, J =4.0 Hz), 7.22 (1H, t, J =
7.5 Hz), 7.40 (1H, t, J =7.5 Hz), 7.58 (1H, d, J =8.0 Hz), 7.61
(2H, t, J =8.0 Hz), 7.70 (1H, t, J =7.5 Hz), 7.82 (1H, d, J =8.5
Hz), 7.88 (1H, d, J =3.5 Hz), 8.03 (2H, d, J =7.5 Hz), 8.10 (1H,
3,10-Dihydropyrrolo[3,2-a]carbazole-1-carbaldehyde (8).
A
solution of oxalyl chloride (89 μL, 1.02 mmol) and DMF (86
μL, 1.11 mmol) in CH2Cl2 (25 mL) was stirred at 0 ꢀC for 20 min.
A solution of 5 (200 mg, 0.97 mmol) in anhyd CH2Cl2 (25 mL)
was then added dropwise. The mixture was stirred at 0 ꢀC for 20
min and then for 2.5 h at room temperature. After evaporation,
5% aq NaOH solution (50 mL) was added. The mixture was
stirred overnight and water was added. The mixture was ex-
tracted with EtOAc and the organic fractions were dried over
MgSO4. After evaporation, the residue was purified by flash
chromatography (cyclohexane/EtOAc, from 7:3 to 5:5) to give 8
(151 mg, 0.645 mmol, 66%) as a white solid; mp 218-220 ꢀC.
HRMS (FABþ) calcd for C15H10N2O (M)þ 234.0793, found
d, J =8.5 Hz), 8.13 (1H, d, J =8.0 Hz), 11.84 (1H, s, NH). 13
NMR (100 MHz, DMSO-d6): 104.7, 107.0, 111.1, 117.3, 119.0,
119.7, 124.6, 125.5, 126.6 (2C), 129.7 (2C), 134.5 (CH), 115.5,
117.3, 122.8, 132.6, 133.0, 137.1, 139.0 (C).
3,10-Dihydropyrrolo[3,2-a]carbazole (5). A 5 M aq NaOH
solution (18 mL) was added to a solution of 4 (738 mg, 2.13
mmol) in MeOH (180 mL). The mixture was refluxed for 15 h,
C

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6375
1
234.0791. IR (KBr): 3400, 3300-3150, 1625 cm-1. H NMR
(400 MHz, DMSO-d6): 7.22 (1H, ddd, J1 =8.0 Hz, J2 =7.0 Hz,
J3 =1.0 Hz), 7.37 (1H, d, J=8.5 Hz), 7.38 (1H, ddd, J1 =8.0 Hz,
J2 =7.0 Hz, J3 =1.0 Hz), 7.86 (1H, d, J =8.0 Hz), 8.05 (1H, d,
J =8.5 Hz), 8.13 (1H, d, J =8.0 Hz), 8.41 (1H, s), 10.0 (1H, s,
CHO), 10.70 (1H, s), 12.50 (1H, s). 13C NMR (100 MHz,
DMSO-d6): 104.5, 112.0, 116.5, 118.8, 119.0, 123.9, 136.7
(CH), 109.2, 116.1, 118.8, 123.3, 132.7, 136.0, 137.8 (C),
185.3 (CdO). HPLC purity, system I: >99%, λ = 241 nm,
tR =23.3 min.
1-(1,10-Dihydropyrrolo[2,3-a]carbazol-3-yl)-2,2,2-trifluoro-
ethanone (12). Trifluoroacetic anhydride (45 μL, 0.32 mmol) was
added at room temperature to a solution of 7 (52 mg, 0.25 mmol)
in DMF (10 mL). The mixture was stirred at room temperature
for 18 h and ice and then water (10 mL) were added. The
aqueous layer was extracted with EtOAc (3 ꢀ 10 mL). The
organic fractions were combined, washed with brine (15 mL),
dried over MgSO4, and evaporated. The residue was triturated
in Et2O (20 mL) and the mixture was filtered to give 12 (32 mg,
0.106 mmol, 42%) as a green powder; mp > 295 ꢀC. HRMS
(ESIþ) calcd for C16H10F3N2O (M þ H)þ 303.0745, found
1,10-Dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde (9).
A
solution of oxalyl chloride (19.5 μL, 0.224 mmol) and DMF
(19 μL, 0.245 mmol) in anhyd CH2Cl2 (6 mL) was stirred at 0 ꢀC
for 20 min. A solution of 7 (43.6 mg, 0.211 mmol) in anhyd
CH2Cl2 (6 mL) was then added dropwise. The solution was
stirred at 0 ꢀC for 20 min then for 2.5 h at room temperature.
After evaporation, 5% aq NaOH (12 mL) was added. The
mixture was stirred for 12 h. After addition of water, the mixture
was extracted with EtOAc and the organic fractions were dried
over MgSO4. After evaporation, the residue was purified by
flash chromatography (cyclohexane/EtOAc, from 7:3 to 5:5)
to give 9 (32.4 mg, 0.138 mmol, 65%) as a white solid; mp >
290 ꢀC. HRMS (ESIþ) calcd for C15H11N2O (M þ H)þ
235.0871, found 235.0882. IR (KBr): 3450-3150, 1630, 1615
cm-1. 1H NMR (500 MHz, DMSO-d6): 7.23 (1H, t, J =7.5 Hz),
7.40 (1H, t, J =7.5 Hz), 7.70 (1H, d, J =8.0 Hz), 7.96 (1H, d, J =
8.5 Hz), 8.00 (1H, d, J =8.5 Hz), 8.15 (1H, d, J =7.5 Hz), 8.33
(1H, d, J=2.0Hz), 10.07 (1H, s), 11.10(1H, brs), 11.93(1H, brs).
13C NMR (125 MHz, DMSO-d6): 111.5, 112.1, 115.0, 119.0,
119.5, 124.3, 136.7 (CH), 118.3, 119.5, 122.5, 122.8, 123.5, 126.0,
138.5 (C), 185.3 (CdO). HPLC purity, system I: >99%, λ =
247 nm, tR =21.7 min.
303.0732. IR (KBr): 3274, 1639 cm-1 1H NMR (400 MHz,
.
DMSO-d6): 7.22 (1H, t, J =7.5 Hz), 7.40 (1H, ddd, J1 =8.0 Hz,
J2 =7.0 Hz, J3 =1.0 Hz), 7.70 (1H, d, J =8.0 Hz), 8.01 (1H, d,
J =8.5 Hz), 8.07 (1H, d, J =8.5 Hz), 8.15 (1H, d, J =8.0 Hz),
8.49-8.53 (1H, br s), 11.03 (1H, s), 12.39-12.48 (1H, br s). 13
C
NMR (100 MHz, DMSO-d6): 111.7, 112.1, 116.3, 119.3,
119.7, 124.7, 135.8 (q, JCF = 5 Hz) (CH), 110.1, 117.0 (q,
JCF = 292 Hz), 119.1, 122.3, 123.2, 124.1, 126.0, 138.6 (C),
174.0 (q, JCF =34 Hz, CdO). HPLC purity, system I: >98%,
λ =245 nm, tR =25.6 min.
Ethyl 2-(1,10-Dihydropyrrolo[2,3-a]carbazol-3-yl)-2-oxoace-
tate (14). Step A. Oxalyl chloride (660 μL, 7.7 mmol) was added
to a suspension of 7 (825 mg, 4.00 mmol) in anhyd Et2O (40 mL)
at 0 ꢀC. The mixture was stirred at room temperature for 4 h.
After filtration, the solid was washed with Et2O (2 ꢀ 40 mL) to
give the acid chloride intermediate 13 (972 mg) as a red-brown
solid, which was used without further purification.
Step B. Triethylamine (275 μL, 1.97 mmol) was added to a
suspension of the acid chloride intermediate (486 mg) in EtOH
(10 mL). The mixture was stirred at room temperature for 4 h.
After filtration, the solid was washed with EtOH (10 mL) and
then with Et2O (2 ꢀ 20 mL) to give 14 (446 mg, 1.46 mmol, 73%
from 7) as a yellow-green powder; mp > 295 ꢀC. HRMS
(ESIþ) calcd for C18H14N2NaO3 (M þ Na)þ 329.0902, found
(1,10-Dihydropyrrolo[2,3-a]carbazol-3-yl)methanol (10). Na-
BH4 (320 mg, 8.46 mmol) was added to a solution of 9 (147 mg,
0.63 mmol) in MeOH (30 mL), and the mixture was stirred at
room temperature for 1 h. H2O (10 mL) was added, and the
precipitate was collected by filtration. The solid was washed
with MeOH (5 mL) and then with Et2O (2 ꢀ 20 mL) to give 10
(100 mg, 0.423 mmol, 67%) as a gray powder; mp > 295 ꢀC.
HRMS (ESIþ) calcd for C15H11N2 (M - OH)þ 219.0922, found
329.0907. IR (KBr): 3292, 1725, 1602 cm-1 1H NMR (400
.
MHz, DMSO-d6): 1.37 (3H, t, J =7.0 Hz), 4.39 (2H, q, J =7.0
Hz), 7.20 (1H, t, J =7.5 Hz), 7.38 (1H, ddd, J1 =8.0 Hz, J2 =7.0
Hz, J3 =1.0 Hz), 7.67 (1H, d, J =8.0 Hz), 7.98-8.03 (2H, m),
8.12 (1H, d, J =8.0 Hz), 8.47 (1H, d, J =3.0 Hz), 11.07 (1H, s),
12.09-12.20 (1H, br s). 13C NMR (100 MHz, DMSO-d6): 14.0
(CH3), 61.6 (CH2), 111.6, 112.3, 115.7, 119.1, 119.6, 124.5, 136.6
(CH), 113.7, 118.7, 122.4, 123.3, 123.8, 126.1, 138.5 (C), 163.7,
179.4 (CdO).
219.0922. IR (KBr): 3375, 1648, 1456 cm-1 1H NMR (400
.
MHz, DMSO-d6): 4.72 (2H, s), 4.54-4.96 (1H, br s), 7.14 (1H, t,
J =7.5 Hz), 7.26-7.31 (2H, m), 7.40 (1H, d, J =8.5 Hz), 7.58
(1H, d, J =8.0 Hz), 7.71 (1H, d, J =8.5 Hz), 8.03 (1H, d, J =7.5
Hz), 10.48-11.46 (2H, 2 br s). 13C NMR (100 MHz, DMSO-d6):
55.7 (CH2), 110.9, 111.1, 111.5, 118.5, 119.0, 121.8, 123.3 (CH),
116.3, 117.7, 122.1, 124.1, 125.1, 126.4, 138.1 (C). HPLC purity,
system II: >97%, λ =252 nm, tR =20.6 min.
2-(1,10-Dihydropyrrolo[2,3-a]carbazol-3-yl)-N,N-diethyl-2-
oxoacetamide (15). The acid chloride intermediate 13 was pre-
pared as described above for the preparation of compound 14.
Diethylamine (305 μL, 2.92 mmol) was added to a suspension of
the acid chloride intermediate (486 mg) in anhyd diethylether
(10 mL). The mixture was stirred at room temperature for 4 h.
After filtration, the solid was washed with MeOH (3 mL) and
then with Et2O (2 ꢀ 20 mL) to give 15 (439 mg, 1.32 mmol, 66%
from 7) as a pale-gray powder; mp > 295 ꢀC. HRMS (ESIþ)
calcd for C20H20N3O2 (M þ H)þ 334.1556, found 334.1543. IR
(KBr): 3450-3100, 1635, 1608, 1429 cm-1. 1H NMR (400 MHz,
DMSO-d6): 1.10 (3H, t, J=7.0 Hz), 1.22 (3H, t, J=7.0 Hz), 3.28
(2H, q, J =7.0 Hz), 3.47 (2H, q, J =7.0 Hz), 7.20 (1H, ddd, J1 =
8.0 Hz, J2 =7.0 Hz, J3 =1.0 Hz), 7.37 (1H, ddd, J1 =8.0 Hz, J2 =
7.0 Hz, J3 =1.5 Hz), 7.66 (1H, d, J=8.0 Hz), 7.93 (1H, d, J=8.5
Hz), 7.99 (1H, d, J =8.5 Hz), 8.07 (1H, d, J =3.0 Hz), 8.12 (1H,
d, J =8.0 Hz), 11.04 (1H, s, NH), 11.96-12.01 (1H, br s, NH).
13C NMR (100 MHz, DMSO-d6): 12.7, 14.1 (CH3), 38.1, 41.7
(CH2), 111.4, 112.1, 115.3, 119.0, 119.5, 124.4, 134.9 (CH),
114.3, 118.3, 122.7, 123.2, 123.4, 126.1, 138.4 (C), 167.2, 187.0
(CdO).
1,10-Dihydropyrrolo[2,3-a]carbazole-3-carboxamide (11).
Chlorosulfonylisocyanate (27 μL, 0.31 mmol) was added at
0 ꢀC to a solution of 7 (57.4 mg, 0.278 mmol) in anhyd
acetonitrile (10 mL). The mixture was stirred at 0 ꢀC for 1.5 h,
and a 1N aq hydrogen chloride solution (5 mL) and THF (2 mL)
were added. The mixture was stirred at room temperature for
36 h, and water was added. After extraction with EtOAc, the
combined organic fractions were washed with an aq saturated
NaHCO3 solution, brine, dried over MgSO4, and evaporated.
The residue was purified by flash chromatography (eluent:
THF) to give 11 (13.8 mg, 0.055 mmol, 20%) as a beige solid;
mp > 300 ꢀC. HRMS (ESIþ) calcd for C15H12N3O (M þ H)þ
250.0980, found 250.0966. IR (KBr): 3430, 3370, 3335, 1650
cm-1. 1H NMR (500 MHz, DMSO-d6): 6.86 (1H, br s), 7.20 (1H,
t, J =8.0 Hz), 7.36 (1H, ddd, J1 =8.0 Hz, J2 =7.0 Hz, J3 =1.0
Hz), 7.46 (1H, br s), 7.65 (1H, d, J =8.0 Hz), 7.84 (1H, d, J =8.5
Hz), 8.02 (1H, d, J =8.5 Hz), 8.08 (1H, d, J =3.0 Hz), 8.10 (1H,
d, J =8.0 Hz). 13C NMR (100 MHz, DMSO-d6): 111.3, 112.8,
113.2, 118.7, 119.2, 123.7, 126.8 (CH), 112.1, 116.8, 112.1, 123.8,
124.8, 126.2, 138.3 (C), 166.7 (CdO). HPLC purity, system I:
>99%, λ =248 nm, tR =19.2 min.
2-(1,10-Dihydropyrrolo[2,3-a]carbazol-3-yl)ethanol (16).
LiAlH4 (2.00 mL, 2.00 mmol, 1 M in THF) was added at room
temperature to a suspension of 14 (153 mg, 0.50 mmol) in
dioxane (10 mL). THF was distilled, and the mixture was
refluxed for 12 h. After cooling, water was added (5 mL) and

6376 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Akueꢀ-Geꢀdu et al.
the mixture was extracted with EtOAc (3 ꢀ 10 mL). The organic
fractions were combined, washed with H2O (10 mL), dried
over MgSO4, and evaporated. The residue was triturated in
Et2O (20 mL) and filtered to give 16 (90 mg, 0.36 mmol, 72%) as
a brown-gray powder; mp 269-271 ꢀC. HRMS (ESIþ) calcd
for C16H15N2O (M þ H)þ 251.1184, found 251.1177. IR (KBr):
(500 mg, 1.18 mmol) in methanol (100 mL). The mixture was
refluxed for 12 h. The reaction mixture was then concentrated
under vacuum until there was formation of a precipitate. The
mixture was neutralized by addition of concentrated HCl, and
the solid was filtered off and washed with water to give 19 (310
mg, 1.09 mmol, 92%) as a brown solid; mp 250 ꢀC (decom-
position). HRMS (ESIþ) calcd for C14H1079BrN2 (M þ H)þ
285.0027, found 285.0040. IR (KBr): 3400, 1648, 1438 cm-1. 1H
NMR (400 MHz, DMSO-d6): 6.59 (1H, dd, J1 =3.0 Hz, J2 =2.0
Hz), 7.35 (1H, d, J=8.5 Hz), 7.38-7.42 (2H, m), 7.58 (1H, d, J=
8.5 Hz), 7.74 (1H, d, J =8.5 Hz), 8.25 (1H, d, J =2.0 Hz),
10.88-10.92 (1H, br s, NH), 11.05-11.09 (1H, br s, NH). 13C
NMR (100 MHz, DMSO-d6): 102.9, 112.2, 112.6, 113.1,
121.5, 124.0, 125.6 (CH), 110.8, 115.3, 121.5, 126.1, 126.7,
127.1, 136.8 (C).
General Procedure for the Preparation of Compounds (20-27).
To a solution of 18 (425 mg, 1 mmol) in THF (5 mL) were
added a solution of sodium carbonate (212 mg, 2 mmol) in water
(1 mL), Pd(PPh3)2Cl2 (35.1 mg, 0,05 mmol) and the correspond-
ing boronic acid (1.1 equiv). The mixture was refluxed over-
night. After cooling, the mixture was filtered on celite and the
solid was washed with acetone. After evaporation of the filtrate,
methanol (40 mL) and a 5 M aq NaOH solution (20 mL) were
added and the mixture was refluxed for 12 h. After concentra-
tion under vacuum and neutralization with concentrated HCl,
EtOAc was added. The organic layer was collected, and the
aqueous phase was extracted with EtOAc (3 ꢀ 30 mL). The
combined organic phases were washed with brine, dried over
MgSO4, and evaporated. The residue was purified by flash
chromatography (pentane/EtOAc, from 9:1 to 7:3) to give the
attempted compound.
3416, 3382, 3234, 1649, 1457 cm-1 1H NMR (400 MHz,
.
DMSO-d6): 2.92 (2H, t, J = 7.5 Hz), 3.71 (2H, dt, J1 = 7.5
Hz, J2 =5.5 Hz), 4.63 (1H, t, J =5.5 Hz), 7.13 (1H, ddd, J1 =8.0
Hz, J2 =7.0 Hz, J3 =1.0 Hz), 7.19 (1H, d, J =2.0 Hz), 7.28 (1H,
ddd, J1 =8.0, J2 =7.0 Hz, J3 =1.0 Hz), 7.31 (1H, d, J =8.5 Hz),
7.58 (1H, d, J=8.0 Hz), 7.70 (1H, d, J=8.5 Hz), 8.02 (1H, d, J=
7.5 Hz), 10.53-10.57 (1H, br s), 10.86 (1H, s). 13C NMR
(100 MHz, DMSO-d6): 29.1, 61.8 (CH2), 110.4, 111.1, 111.3,
118.5, 119.0, 121.2, 123.2 (CH), 113.3, 116.2, 121.9, 124.1, 125.9,
126.5, 138.1 (C). HPLC purity, system I: >98%, λ =252 nm,
tR =21.4 min.
2-(1,10-Dihydropyrrolo[2,3-a]carbazol-3-yl)-N,N-diethyl-2-
hydroxyiminoethanamide (17). Hydroxylamine hydrochloride
(261 mg, 3.76 mmol) was added to a solution of 16 (167 mg,
0.50 mmol) in pyridine (5 mL) and the mixture was refluxed for
12 h. H2O (5 mL) was added, and pyridine was removed under
vacuum. The aqueous layer was extracted with EtOAc (3 ꢀ
10 mL), and the combined organic fractions were washed with
brine (10 mL), dried over MgSO4, and evaporated. The residue
was triturated in EtOAc (20 mL) and filtered to give 17 (33 mg,
0.095 mmol, 19%) as a pale-beige powder; mp 268-269 ꢀC.
HRMS (ESIþ) calcd for C20H21N4O2 (M þ H)þ 349.1665,
found 349.1675. IR (KBr): 3266, 1609 cm-1 1H NMR (400
.
MHz, DMSO-d6): 1.03 (3H, t, J =7.0 Hz), 1.19 (3H, t, J =7.0
Hz), 3.26 (2H, q, J=7.0 Hz), 3.39-3.59 (2H, m), 7.17 (1H, t, J=
7.5 Hz), 7.33 (1H, t, J =7.5 Hz), 7.36 (1H, d, J =2.5 Hz), 7.61
(1H, d, J =8.0 Hz), 7.83 (1H, d, J =8.5 Hz), 7.86 (1H, d, J =8.5
Hz), 8.07 (1H, d, J =7.5 Hz), 10.90 (1H, s), 11.03-11.07 (1H, br
s), 11.21-11.26 (1H, br s). 13C NMR (100 MHz, DMSO-d6):
12.8, 13.8 (CH3), 37.6, 41.7 (CH2), 111.3, 113.2, 113.3, 118.7,
119.3, 123.8, 125.7 (CH), 109.7, 117.3, 122.5, 122.7, 123.7, 126.2,
138.3 (C), 150.6 (CdN), 164.4 (CdO). HPLC purity, system
I: >99%, λ =251 nm, tR =21.5 min.
1-Benzenesulfonyl-7-bromo-1,10-dihydropyrrolo[2,3-a]carba-
zole (18). Step A. A suspension of 4-bromophenylhydrazine
hydrochloride (1.62 g, 7.3 mmol) and anhyd sodium acetate
(0.60 g, 7.3 mmol) in DME (20 mL) was stirred at room
temperature for 1 h. Compound 3 (1.00 g, 3.63 mmol) and ionic
liquid (10.0 g, 24.3 mmol, prepared as for the synthesis of
compound 6) were added. The solvent was evaporated, and
the mixture was stirred at 120 ꢀC for 2 h. After cooling, a 0.5 M
aq HCl solution was added before extraction with EtOAc. The
organic fractions were washed with brine, dried over MgSO4,
and evaporated. The residue contained the major indolization
product and the minor expected product.
7-(3-Methoxyphenyl)-1,10-dihydropyrrolo[2,3-a]carbazole (20).
Compound 20 (172 mg, 0.55 mmol, 55%) as a brown solid; mp
202-205 ꢀC. HRMS (ESIþ) calcd for C21H17N2O (M þ H)þ
313.1341, found 313.1343. IR (KBr): 3390, 1654, 1609, 1584, 1461
cm-1. 1H NMR (400 MHz, DMSO-d6): 3.86 (3H, s, CH3), 6.59
(1H, dd, J1 =3.0 Hz, J2 =2.0 Hz), 6.90 (1H, ddd, J1 =8.0 Hz, J2 =
2.5 Hz, J3 =1.5 Hz), 7.29-7.31 (1H, m), 7.34 (1H, dt, J1 =7.5 Hz,
J2 =1.5 Hz), 7.36 (1H, d, J=8.5 Hz), 7.39 (1H, t, J=7.5 Hz), 7.39
(1H, t, J =2.5 Hz), 7.61 (1H, dd, J1 =8.5 Hz, J2 =1.5 Hz), 7.67
(1H, d, J =8.5 Hz), 7.82 (1H, d, J =8.5 Hz), 8.36 (1H, d, J =1.5
Hz), 10.83-10.88 (1H, br s, NH), 10.95-11.00 (1H, br s, NH). 13
C
NMR (100 MHz, DMSO-d6): 55.1 (CH3), 102.8, 111.5, 111.8,
112.2 (3C), 117.3, 119.1, 122.6, 123.7, 129.8 (CH), 116.4, 121.8,
124.7, 126.4, 127.0, 131.0, 137.8, 143.2, 159.7 (C).
7-Phenyl-1,10-dihydropyrrolo[2,3-a]carbazole (21). Compo-
und 21 (169 mg, 0.60 mmol, 60%) as a gray solid; mp > 250 ꢀC.
HRMS (ESIþ) calcd for C20H15N2 (M þ H)þ 283.1235, found
283.1248. IR (KBr): 3434, 3368, 1652 cm-1. 1H NMR (400 MHz,
DMSO-d6): 6.59 (1H, dd, J1 =3.0 Hz, J2 =2.0 Hz), 7.30-7.35
(1H, m), 7.37 (1H, J1 =8.5 Hz, J2 =0.5 Hz), 7.40 (1H, dd, J1 =3.0
Hz, J2 =2.5 Hz), 7.45-7.50 (2H, m), 7.61 (1H, dd, J1 =8.5 Hz,
J2 =2.0 Hz), 7.68 (1H, dd, J1 =8.5 Hz, J2 =0.5 Hz), 7.76-7.79
(2H, m), 7.82 (1H, dd, J1 =8.5 Hz, J2 =0.5 Hz), 8.35 (1H, d, J =
2.0 Hz), 10.83-10.89 (1H, br s, NH), 10.96-11.00 (1H, br s, NH).
13C NMR (100 MHz, DMSO-d6): 102.9, 111.6, 112.2, 112.3,
117.2, 122.5, 123.7, 126.2, 126.7 (2C), 128.8 (2C) (CH), 116.4,
121.8, 124.8, 126.4, 127.0, 131.1, 137.8, 141.6 (C).
7-(4-Biphenyl)-1,10-dihydropyrrolo[2,3-a]carbazole (22). Com-
pound 22 (230 mg, 0.64 mmol, 64%) as a brown solid; mp >
250 ꢀC. HRMS (ESIþ) calcd for C26H18N2 (M)þ 358.1470, found
358.1492. IR (KBr): 3650-3100, 1698, 1648 cm-1. 1H NMR (400
MHz, DMSO-d6): 6.60 (1H, dd, J1 = 3.0 Hz, J2 = 2.0 Hz),
7.36-7.41 (3H, m), 7.47-7.52 (2H, m), 7.68 (1H, dd, J1 =8.5 Hz,
J2 =1.5 Hz), 7.71 (1H, dd, J1 =8.5 Hz, J2 =0.5 Hz), 7.73-7.76
(2H, m), 7.77-7.80 (2H, m), 7.83 (1H, d, J =8.5 Hz), 7.87-7.91
(2H, m), 8.41-8.43 (1H, m), 10.83-10.89 (1H, br s, NH),
10.98-11.02 (1H, br s, NH). 13C NMR (100 MHz, DMSO-d6):
102.8, 111.6, 112.2, 112.3, 117.1, 122.4, 123.7, 126.4 (2C), 127.0
Step B. A solution of the residue from step A and DDQ (0.7 g,
3.1 mmol, necessary DDQ quantity was determined from a 1H
NMR spectrum of the intermediate mixture) in dioxane (20 mL)
was stirred at room temperature for 15 h. After evaporation,
EtOAc was added and the mixture was washed with water, with
brine, dried over MgSO4, and evaporated. The residue was
purified by flash chromatography (pentane/EtOAc 9:1) to give
18 (1.15 g, 2.70 mmol, 74%) as a brown solid; mp 173-175 ꢀC.
IR (KBr): 3434, 1631 cm-1
.
HRMS (ESIþ) calcd for
C20H1479BrN2O2S (M þ H)þ 424.9959, found 424.9976. 1H
NMR (400 MHz, DMSO-d6): 7.04 (1H, d, J =3.5 Hz), 7.41
(1H, d, J =8.0 Hz), 7.49-7.55 (3H, m), 7.62 (1H, t, J =7.5 Hz),
7.84 (1H, d, J =3.5 Hz), 7.92-7.99 (3H, m), 8.12 (1H, d, J =8.0
Hz), 8.38 (1H, d, J =1.5 Hz), 10.81 (1H, s, NH). 13C NMR (100
MHz, DMSO-d6): 112.4, 113.2, 114.9, 117.4, 121.9, 126.7 (2C),
127.0, 127.5, 129.9 (2C), 134.6 (CH), 111.7, 119.4, 120.1, 124.7,
126.6, 130.8, 136.8, 137.7 (C).
7-Bromo-1,10-dihydropyrrolo[2,3-a]carbazole (19). A 5 M aq
NaOH solution (10 mL) was added to a suspension of 18

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6377
(2C), 127.1 (2C), 127.3, 128.9 (2C) (CH), 116.4, 121.8, 124.8,
126.5, 127.0, 130.4, 137.8, 137.9, 139.8, 140.6 (C).
112.5, 117.7, 122.6, 123.9, 126.5 (2C), 128.9 (2C) (CH), 116.3,
121.7, 124.9, 126.6, 127.0, 129.6, 134.5, 138.3, 146.1 (C), 197.4
(CdO).
7-(4-Fluorophenyl)-1,10-dihydropyrrolo[2,3-a]carbazole (23).
Compound 23 (150 mg, 0.50 mmol, 50%) as a gray-green solid;
mp > 250 ꢀC. HRMS (ESIþ) calcd for C20H14FN2 (M þ H)þ
301.1141, found 301.1154. IR (KBr): 3410, 3397, 1651, 1605,
1514 cm-1. 1H NMR (400 MHz, DMSO-d6): 6.59 (1H, dd, J1 =
3.0 Hz, J2 =2.0 Hz), 7.27-7.33 (2H, m), 7.36 (1H, d, J=8.5 Hz),
7.39 (1H, dd, J1 =3.0 Hz, J2 =2.5 Hz), 7.58 (1H, dd, J1 =8.5 Hz,
J2 =2.0 Hz), 7.68 (1H, dd, J1 =8.5 Hz, J2 =0.5 Hz), 7.77-7.83
(3H, m), 8.33 (1H, d, J =2.0 Hz), 10.84-10.87 (1H, br s, NH),
10.96-10.98 (1H, br s, NH). 13C NMR (100 MHz, DMSO-d6):
102.8, 111.6, 112.2, 112.3, 115.5 (2C, d, JCF =21 Hz), 117.2,
122.4, 123.8, 128.5 (2C, d, JCF =8 Hz) (CH), 116.3, 121.8, 124.8,
7-Bromo-1,10-dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde
(28). A mixture of oxalyl chloride (32.5 μL, 0.38 mmol) and
DMF (31 μL, 0.40 mmol) in dichloromethane (10 mL) was
stirred at 0 ꢀC for 20 min. A solution of 19 (100 mg, 0.35 mmol)
in dichloromethane (10 mL) was added dropwise. The mixture
was stirred at 0 ꢀC for 20 min before reaching room temperature.
The solvent was evaporated and a 5% aq NaOH solution
(20 mL) was added. The mixture was stirred for 12 h and then
extracted with EtOAc. The organic phase was washed with
water, brine, dried over MgSO4, and evaporated. The residue
was purified by flash chromatography (cyclohexane/EtoAc 7:3
then 5:5) to give 28 (72 mg, 0.230 mmol, 66%) as a gray solid; mp
> 250 ꢀC. HRMS (ESIþ) calcd for C15H1079BrN2O (M þ H)þ
312.9976, found 312.9979. IR (KBr): 3400-3100, 1630 cm-1. 1H
NMR (400 MHz, DMSO-d6): 7.48 (1H, dd, J1 =8.5 Hz, J2 =2.0
Hz), 7.65 (1H, d, J =8.5 Hz), 7.94 (1H, d, J =8.5 Hz), 8.00 (1H,
d, J =8.5 Hz), 8.31 (1H, d, J =3.0 Hz), 8.34 (1H, d, J =2.0 Hz),
10.04 (1H, s), 11.18-11.23 (1H, br s, NH), 11.89-11.96 (1H, br
s, NH). 13C NMR (100 MHz, DMSO-d6): 112.6, 113.5, 115.3,
122.0, 126.6, 137.0 (CH), 111.2, 117.4, 119.5, 122.5, 123.1, 125.5,
126.6, 137.2 (C), 185.4 (CdO). HPLC purity, system I: >96%,
λ =264 nm, tR =23.9 min.
7-(3-Methoxyphenyl)-1,10-dihydropyrrolo[2,3-a]carbazole-3-
carbaldehyde (29). POCl3 (90 μL, 0.97 mmol) was slowly added
to anhyd DMF (2 mL) at 0 ꢀC. The mixture was stirred at 0-
10 ꢀC for 45 min until a yellow solution was obtained. A solution
of 20 (100 mg, 0.320 mmol) in DMF (1 mL) was then added. The
mixture was heated at 120 ꢀC for 24 h. After cooling, a 5% aq
NaOH solution (20 mL) was added and the mixture was stirred
at room temperature overnight. After extraction with EtOAc,
the organic phase was dried over MgSO4, evaporated, and the
residue was purified by flash chromatography (pentane/EtOAc,
from 7:3 to 1:9) to give 29 (49 mg, 0.144 mmol, 45%) as a light-
brown solid; mp 215 ꢀC (decomposition). HRMS (ESIþ) calcd
for C22H17N2O2 (M þ H)þ 341.1290, found 341.1305. IR (KBr):
3325, 1619 cm-1. 1H NMR (400 MHz, DMSO-d6): 3.87 (3H, s,
CH3), 6.91 (1H, d, J =7.5 Hz), 7.31-7.42 (3H, m), 7.69 (1H, dd,
J1 =8.5 Hz, J2 =1.5 Hz), 7.74 (1H, d, J =8.5 Hz), 7.95 (1H, d,
J =8.5 Hz), 8.08 (1H, d, J =8.5 Hz), 8.31 (1H, d, J =3.0 Hz),
8.45 (1H, s), 10.04 (1H, s), 11.09-11.13 (1H, br s, NH),
11.88-11.92 (1H, br s, NH). 13C NMR (100 MHz, DMSO-
d6): 55.1 (CH3), 111.8, 112.0, 112.2 (2C), 115.3, 117.8, 119.1,
123.5, 129.8, 136.9 (CH), 118.6, 119.6, 122.8 (2C), 124.1, 126.5,
131.4, 138.2, 142.9, 159.7 (C), 185.3 (CdO). HPLC purity,
system I: >96%, λ =267 nm, tR =24.0 min.
126.5, 127.0, 130.1, 137.7, 138.1 (d, JCF =3 Hz), 161.2 (d, JCF
243 Hz) (C).
=
7-(2,4-Difluorophenyl)-1,10-dihydropyrrolo[2,3-a]carbazole (24).
Compound 24 (165 mg, 0.52 mmol, 52%) as a gray-green solid;
mp > 250 ꢀC. HRMS (ESIþ) calcd for C20H13F2N2 (M þ H)þ
319.1047, found 319.1058. IR (KBr): 3401, 1652, 1616, 1594,
1510 cm-1. 1H NMR (400 MHz, DMSO-d6): 6.59 (1H, dd, J1 =
3.0 Hz, J2 =2.0 Hz), 7.21 (1H, tdd, J1 =8.5 Hz, J2 =3.0 Hz, J3 =
1.0 Hz), 7.33-7.39 (2H, m), 7.40 (1H, t, J=2.5 Hz), 7.45 (1H, dt,
J1 =8.5 Hz, J2 =2.0 Hz), 7.67 (1H, td, J1 =9.0 Hz, J2 =6.5 Hz),
7.69 (1H, d, J =8.5 Hz), 7.78 (1H, d, J =8.5 Hz), 8.19 (1H, s),
10.85-10.88 (1H, br s, NH), 11.03-11.06 (1H, br s, NH). 13C
NMR (100 MHz, DMSO-d6): 102.9, 104.3 (dd, JCF1 =27 Hz,
JCF2 =26 Hz), 111.3, 111.8 (dd, JCF1 =21 Hz, JCF2 =4 Hz),
112.1, 112.4, 119.4 (d, JCF =3 Hz), 123.8, 124.3 (d, JCF =3 Hz),
132.1 (dd, JCF1 =10 Hz, JCF2 =5 Hz) (CH), 116.2, 121.7, 124.4,
124.8 (d, JCF =1 Hz), 126.3 (dd, JCF1 =14 Hz, JCF2 =4 Hz),
126.5, 126.9, 137.7, 159.1 (dd, JCF1 =247 Hz, JCF2 =12 Hz),
161.1 (dd, JCF1 =246 Hz, JCF2 =12 Hz).
7-(4-Trifluoromethylphenyl)-1,10-dihydropyrrolo[2,3-a]carba-
zole (25). Compound 25 (227 mg, 0.65 mmol, 65%) as a gray
solid; mp > 250 ꢀC. HRMS (ESIþ) calcd for C21H14F3N2 (M þ
H)þ 351.1109, found 351.1125. IR (KBr): 3390, 3362, 1654, 1614
cm-1. 1H NMR (400 MHz, DMSO-d6): 6.60 (1H, dd, J1 =3.0
Hz, J2 =2.0 Hz), 7.39 (1H, d, J =8.5 Hz), 7.41 (1H, dd, J1 =3.0
Hz, J2 =2.5 Hz), 7.69 (1H, dd, J1 =8.5 Hz, J2 =2.0 Hz), 7.73
(1H, dd, J1 =8.5 Hz, J2 =0.5 Hz), 7.81 (2H, d, J =8.0 Hz), 7.84
(1H, d, J =8.5 Hz), 8.01 (2H, d, J =8.0 Hz), 8.47 (1H, d, J =1.5
Hz), 10.86-10.92 (1H, br s, NH), 11.06-11.10 (1H, br s, NH).
13C NMR (100 MHz, DMSO-d6): 102.9, 111.8, 112.2, 112.5,
117.7, 122.6, 123.9, 125.6 (2C, q, JCF =4 Hz), 127.2 (2C) (CH),
116.3, 121.8, 124.6 (q, JCF =272 Hz), 124.9, 126.5 (q, JCF =32
Hz), 126.6, 127.1, 129.3, 138.3, 145.6 (C).
7-(4-Trifluoromethoxyphenyl)-1,10-dihydropyrrolo[2,3-a]carba-
zole (26). Compound 26 (226 mg, 0.62 mmol, 62%) as a gray
solid; mp > 250 ꢀC. HRMS (ESIþ) calcd for C21H14F3N2O
(M þ H)þ 367.1058, found 367.1044. IR (KBr): 3401, 1652,
1514, 1464 cm-1. 1H NMR (400 MHz, DMSO-d6): 6.60 (1H, dd,
J1=3.0 Hz, J2=2.0 Hz), 7.37 (1H, d, J=8.5 Hz), 7.40 (1H, t, J=
2.5 Hz), 7.46 (2H, d, J =8.0 Hz), 7.62 (1H, dd, J1 =8.5 Hz, J2 =
1.5 Hz), 7.70 (1H, d, J =8.5 Hz), 7.81 (1H, d, J =8.5 Hz),
7.87-7.91 (2H, m), 8.38 (1H, d, J=1.5 Hz), 10.85-10.90 (1H, br
s, NH), 11.00-11.05 (1H, br s, NH). 13C NMR (100 MHz,
DMSO-d6): 102.9, 111.7, 112.2, 112.4, 117.5, 121.4 (2C), 122.5,
123.8, 128.3 (CH), 116.3, 120.2 (q, JCF =256 Hz), 121.8, 124.8,
126.5, 127.0, 129.6, 138.0, 141.0, 147.0 (q, JCF =2 Hz) (C).
7-(4-Acetylphenyl)-1,10-dihydropyrrolo[2,3-a]carbazole (27).
Compound 27 (195 mg, 0.60 mmol, 60%) as a brown solid;
mp 185 ꢀC (decomposition). HRMS (ESIþ) calcd for
C22H17N2O (M þ H)þ 325.1341, found 325.1354. IR (KBr):
3700-3100, 1649, 1597 cm-1. 1H NMR (400 MHz, DMSO-d6):
2.63 (3H, s, CH3), 6.59-6.61 (1H, m), 7.38 (1H, d, J =8.5 Hz),
7.39-7.41 (1H, m), 7.71-7.73 (2H, m), 7.84 (1H, d, J =8.5 Hz),
7.96 (2H, d, J =8.5 Hz), 8.06 (2H, d, J =8.5 Hz), 8.48 (1H, s),
10.86-10.90 (1H, br s, NH), 11.05-11.08 (1H, br s, NH). 13C
NMR (100 MHz, DMSO-d6): 26.7 (CH3), 102.9, 111.8, 112.2,
General Procedure for the Preparation of Compounds (30-35).
POCl3 (3 equiv) was slowly added to anhyd DMF (2 mL) at 0 ꢀC.
The solution was stirred at 0-10 ꢀC for 45 min until a yellow
solution was obtained, and then was added to a solution of
compound 21-27 (100 mg) in DMF (1 mL) at 0 ꢀC prepared in a
10 mL CEM reaction vessel. The tube was sealed and was heated
at 100 ꢀC under microwave irradiation for 20 min (150 W). After
cooling, the mixture was poured into a saturated aq NaHCO3
solution (20 mL). After 30 min of stirring, the solid was filtered
off and a 5% aq NaOH solution (20 mL) was added. The
mixture was stirred at room temperature overnight. The solid
was filtered off and then purified by flash chromatography
(pentane/EtOAc, from 7:3 to 1:9).
7-Phenyl-1,10-dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde
(30). Compound 30 (57 mg, 0.184 mmol, 52%) as a dark-brown
solid; mp 205 ꢀC (decomposition). HRMS (ESIþ) calcd for
C21H15N2O (M þ H)þ 311.1184, found 311.1201. IR (KBr):
1
3260, 1622 cm-1. H NMR (400 MHz, DMSO-d6): 7.31-7.36
(1H, m), 7.46-7.51 (2H, m), 7.68 (1H, dd, J1 =8.5 Hz, J2 =2.0
Hz), 7.75 (1H, dd, J1 =8.5 Hz, J2 =0.5 Hz), 7.77-7.81 (2H, m),
7.95 (1H, d, J=8.5 Hz), 8.06 (1H, d, J=8.5 Hz), 8.31 (1H, d, J=
3.0 Hz), 8.43 (1H, d, J=2.0 Hz), 10.04 (1H, s), 11.08-11.14 (1H,

6378 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Akueꢀ-Geꢀdu et al.
br s, NH), 11.86-11.93 (1H, br s, NH). 13C NMR (100 MHz,
DMSO-d6): 111.9, 112.3, 115.3, 117.7, 123.4, 126.4, 126.7 (2C),
128.8 (2C), 136.9 (CH), 118.6, 119.6, 122.8 (2C), 124.2, 126.5,
131.5, 138.1, 141.4 (C), 185.4 (CdO). HPLC purity, system
I: >96%, λ =268 nm, tR =24.8 min.
3276, 1620 cm-1. 1H NMR (500 MHz, DMSO-d6): 7.46 (2H, d,
J =8.5 Hz), 7.69 (1H, dd, J1 =8.5 Hz, J2 =2.0 Hz), 7.75 (1H, d,
J =8.5 Hz), 7.88-7.92 (2H, m), 7.95 (1H, d, J =8.5 Hz), 8.06
(1H, d, J =8.5 Hz), 8.31 (1H, d, J =3.0 Hz), 8.46 (1H, d, J =2.0
Hz), 10.03 (1H, s), 11.15-11.18 (1H, br s, NH), 11.93 (1H, br d,
J=2.5 Hz, NH). 13C NMR (125 MHz, DMSO-d6): 112.1, 112.5,
115.4, 118.0, 121.5 (2C), 123.6, 128.5 (2C), 137.0 (CH), 118.6,
119.7, 120.3 (q, JCF =256 Hz), 122.9, 123.0, 124.3, 126.7, 130.1,
138.4, 140.8, 147.2 (q, JCF =2 Hz) (C), 185.5 (CdO). HPLC
purity, system I: >95%, λ =270 nm, tR =24.9 min.
7-(4-Biphenyl)-1,10-dihydropyrrolo[2,3-a]carbazole-3-carbal-
dehyde (31). Compound 31 (32 mg, 0.083 mmol, 30%) as a dark-
brown solid; mp > 250 ꢀC. HRMS (ESIþ) calcd for C27H19N2O
(M þ H)þ 387.1497, found 387.1492. IR (KBr): 3436, 1622
cm-1 1H NMR (400 MHz, DMSO-d6): 7.36-7.41 (1H, m),
.
7.47-7.52 (2H, m), 7.73-7.81 (6H, m), 7.88-7.92 (2H, m), 7.96
(1H, d, J =8.5 Hz), 8.09 (1H, d, J =8.5 Hz), 8.31 (1H, d, J =3.0
Hz), 8.51 (1H, s), 10.05 (1H, s), 11.12-11.16 (1H, br s, NH),
11.90 (1H, br d, J =2.5 Hz, NH). 13C NMR (100 MHz, DMSO-
d6): 112.0, 112.3, 115.3, 117.5, 123.3, 126.5 (2C), 127.1 (4C),
127.3, 129.0 (2C), 136.9 (CH), 118.6, 119.6, 122.8 (2C), 124.2,
126.5, 130.9, 138.0, 138.2, 139.8, 140.4 (C), 185.3 (CdO). HPLC
purity, system I: >97%, λ =310 nm, tR =27.8 min.
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.40
Source and Purification of Kinases. All protein kinases were of
human origin and encoded full-length proteins except for
RSK1, ROCK2, CK1δ, and AMPK (rat), ERK2 and lck
(mouse), and MKK1 (rabbit). Apart from the AMPK (AMP-
activated protein kinase) complex, which was purified from rat
liver, all other 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 glutathione-Sepharose, and
His6-tagged proteins on nickel/nitrilotriacetate-agarose.
Protein Kinase Assays. All assays (25.5 μL volume) were
carried out robotically at room temperature (21 ꢀC) and were
linear with respect to time and enzyme concentration under the
conditions used. Assays were performed for 30 min using Multi-
drop Micro reagent dispensers (Thermo Electron Corporation,
Waltham, MA) in a 96-well format. The concentration of
magnesium acetate in the assays was 10 mM and [γ-33P]ATP
(800 cpm/pmol) was used at 5, 20, or 50 μM as indicated in order
to be at or below the Km for ATP for each enzyme. Protein
kinases assayed at 5 μM ATP were: MKK1, ERK1, p38γ
MAPK, p38δ MAPK, ERK8, PKBR, PKCζ, PRK2, GSK3β,
CK2, MARK3, IKKβ, PIM2, EF2K, PLK1, Aurora C, HIPK2,
and PAK4. Protein kinases assayed at 20 μM ATP were: JNK1,
JNK2, p38β MAPK, PDK1, SGK1, S6K1, PKA, ROCK2,
PKCR, MSK1, MAPKAP-K2, MAPKAP-K3, PRAK,
CaMKKR, CaMKKβ, CHK1, CHK2, CDK2, Aurora B,
CK1, PIM1, PIM3, NEK7, MST2, HIPK3, PAK5, PAK6,
CSK. Protein kinases assayed at 50 μM ATP were: ERK2,
JNK3, p38R MAPK, RSK1, RSK2, PKBβ, PKD1, MNK1,
MNK2, AMPK, CaMK1, smMLCK, PHK, BRSK2, MELK,
NEK2a, NEK6, SRPK1, Src, Lck.
7-(4-Fluorophenyl)-1,10-dihydropyrrolo[2,3-a]carbazole-3-car-
baldehyde (32). Compound 32 (55 mg, 0.168 mmol, 50%) as a
light-brown solid; mp > 250 ꢀC. HRMS (ESIþ) calcd for
C21H14N2OF (M þ H)þ 329.1090, found 329.1101. IR (KBr):
3436, 3297, 1619 cm-1 1H NMR (400 MHz, DMSO-d6):
.
7.27-7.34 (2H, m), 7.65 (1H, dd, J1 =8.5 Hz, J2 =2.0 Hz),
7.74 (1H, d, J =8.5 Hz), 7.78-7.84 (2H, m), 7.95 (1H, d, J =8.5
Hz), 8.06 (1H, d, J =8.5 Hz), 8.31 (1H, s), 8.41 (1H, d, J =1.5
Hz), 10.04 (1H, s), 11.10-11.15 (1H, br s, NH), 11.88-11.95
(1H, br s, NH). 13C NMR (100 MHz, DMSO-d6): 112.0, 112.3,
115.3, 115.6 (2C, d, JCF =21 Hz), 117.7, 123.4, 128.5 (2C, d,
JCF =8 Hz), 136.9 (CH), 118.6, 119.6, 122.8 (2C), 124.2, 126.6,
130.6, 137.9 (d, JCF =3 Hz), 138.1, 161.3 (d, JCF =243 Hz),
185.4 (CdO). HPLC purity, system I: >96%, λ =267 nm, tR
25.0 min.
=
7-(2,4-Difluorophenyl)-1,10-dihydropyrrolo[2,3-a]carbazole-
3-carbaldehyde (33). Compound 33 (48 mg, 0.139 mmol, 44%)
as a beige solid; mp 220 ꢀC (decomposition). HRMS (ESIþ)
calcd for C21H13F2N2O (M þ H)þ 347.0996, found 347.0996. IR
(KBr): 3264, 1645, 1616 cm-1. 1H NMR (400 MHz, DMSO-d6):
7.22 (1H, td, J1 =8.5 Hz, J2 =2.5 Hz), 7.37 (1H, ddd, J1 =11.5
Hz, J2 =9.5 Hz, J3 =2.5 Hz), 7.52 (1H, dt, J1 =8.5 Hz, J2 =2.0
Hz), 7.69 (1H, td, J1 =9.0 Hz, J2 =7.0 Hz), 7.76 (1H, d, J =8.5
Hz), 7.95 (1H, d, J =8.5 Hz), 8.02 (1H, d, J =8.5 Hz), 8.27 (1H,
s), 8.31 (1H, s), 10.04 (1H, s), 11.19-11.26 (1H, br s, NH),
11.90-12.01 (1H, br s, NH). 13C NMR (100 MHz, DMSO-d6):
104.3 (dd, JCF1 =27 Hz, JCF2 =26 Hz), 111.6, 111.8 (dd, JCF1
=
21 Hz, JCF2 =4 Hz), 112.4, 115.2, 119.9 (d, JCF =2 Hz), 125.2,
132.1 (dd, JCF1 =9 Hz, JCF2 =5 Hz), 136.9 (CH), 118.3, 119.6,
122.8 (2C), 123.8, 125.2, 126.1 (dd, JCF1 =14 Hz, JCF2 =4 Hz),
126.5, 138.1, 159.1 (dd, JCF1 =247 Hz, JCF2 =12 Hz), 161.1 (dd,
JCF1 =246 Hz, JCF2 =12 Hz (C), 185.3 (CdO). HPLC purity,
system I: >97%, λ =264 nm, tR =25.2 min.
The assays were initiated with MgATP, stopped by the
addition of 5 μL of 0.5 M orthophosphoric acid, and spotted
on to P81 filter plates using a unifilter harvester (PerkinElmer,
Boston, MA).
Kinase substrates were: PKA, (single-letter code for amino
acids) LRRASLG (300 μM); PKCR, protein histone H1 (0.1
mg/mL); PHK, KRKQISVRGL (300 μM); CK1, RRKDLH-
DDEEDEAMSITA (0.5 mM); NEK2a, RFRRSRRMI (300
μM); NEK6 and NEK7, FLAKSFGSPNRAYKK (300 μM),
ROCK2 and PRK2, KEAKEKRQEQIAKRRRLSSL-
RASTSKSGGSQK (peptide corresponding to the C-terminal
region of ribosomal protein S6; 30 μM); Aurora B and Aurora
C, LRRLSLGLRRLSLGLRRLSLGLRRLSLG (300 μM);
ERK1, ERK2, ERK8, HIPK2, HIPK3 and MST2, p38R
MAPK, p38β MAPK, p38γ MAPK, and p38δ MAPK, MBP
(myelin basic protein; 0.33 mg/mL); IKKβ, LDDRHDSGL-
DSMKDEEY (300 μM); JNK1, JNK2, and JNK3, ATF2-
[19-96] (activating transcription factor 2[19-96]; 3 μM);
MARK3, KKKVSRSGLYRSPSMPENLNRPR (300 μM);
RSK1, RSK2, MAPKAP-K3, and PKD1, KKLNRTLSVA
(30 μM); MNK1 and MNK2, eIF4E (eukaryotic translation
initiation factor 4E) protein (0.5 mg/mL); EF2K,
RKKFGESKTKTKEFL (300 μM); Pim-1, Pim-2, and Pim-3,
RSRHSSYPAGT (300 μM); SGK1 and PKBβ, GRPRT-
SSFAEGKK (30 μM); PLK1, ISDELMDATFADQEAKKK
7-(4-Trifluoromethylphenyl)-1,10-dihydropyrrolo[2,3-a]carba-
zole-3-carbaldehyde (34). Compound 34 (70 mg, 0.185 mmol,
65%) as a light-brown solid; mp > 250 ꢀC. HRMS (ESIþ) calcd
for C22H14F3N2O (M þ H)þ 379.1058, found 379.1059. IR
1
(KBr): 3460, 3294, 1617 cm-1. H NMR (400 MHz, DMSO-
d6): 7.76 (1H, dd, J1 =8.5 Hz, J2 =1.5 Hz), 7.80 (1H, dd, J1 =8.5
Hz, J2 =1.0 Hz), 7.83 (2H, d, J =8.0 Hz), 7.97 (1H, d, J =8.5
Hz), 8.03 (2H, d, J =8.0 Hz), 8.09 (1H, d, J =8.5 Hz), 8.32 (1H,
s), 8.56 (1H, s), 10.04 (1H, s), 11.18-11.25 (1H, br s, NH),
11.88-11.97 (1H, br s, NH). 13C NMR (100 MHz, DMSO-d6):
112.2, 112.5, 115.3, 118.2, 123.5, 125.7 (2C, q, JCF =4 Hz), 127.3
(2C), 137.0 (CH), 118.5, 119.6, 122.8, 122.9, 124.3, 124.6 (q,
JCF =272 Hz), 126.6, 126.7 (q, JCF =32 Hz), 129.7, 138.7, 145.4
(C), 185.4 (CdO). HPLC purity, system I: >95%, λ =287 nm,
tR =26.5 min.
7-(4-Trifluoromethoxyphenyl)-1,10-dihydropyrrolo[2,3-a]carba-
zole-3-carbaldehyde (35). Compound 35 (72 mg, 0.183 mmol,
67%) as a beige solid; mp > 250 ꢀC. HRMS (ESIþ) calcd for
C22H14F3N2O2 (M þ H)þ 395.1007, found 395.1017. IR (KBr):

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6379
(300 μM); Src, KVEKIGEGTYGVVYK (300 μM), CaMK1,
YLRRRLSDSNF (300 μM); smMLCK, KKRPQRATSNVFA
(300 μM); SRPK1, RSRSRSRSRSRSRSR (300 μM); PAK4, 5,
and 6, RRRLSFAEPG (300 μM); CaMKKR and CaMKKβ,
AKPKGNKDYHLQTCCGSLAYRRR (300 μM); MELK and
BRSK2, KKLNRTLSFAEPG (300 μM); PKCζ, ERMRPRK-
RQGSVRRV (300 μM); CHK1 and CHK2, KKKVSRSGL-
YRSPSMPENLNRPR (200 μM); MAPKAP-K2, KKLNR-
TLSVA (30 μM); PRAK, KKLRRTLSVA (30 μM); PDK1,
(5 mg/mL) with an equal volume of reservoir solution contain-
ing 0.2 M NaF, 100 mM bistrispropane pH 8.5, 20% PEG3350,
and 10% ethylene glycol. Crystals grew to diffracting quality
within a few days. Crystals were cryoprotected using the well
solution supplemented with additional ethylene glycol and were
flash frozen in liquid nitrogen. Data were collected at a Rigaku
˚
FRE Superbright equipped with an RAXIS IV detector at 1.5 A.
Indexing and integration was carried out using MOSFLM,47
and scaling was performed with SCALA.48 Initial phases were
calculated by molecular replacement with PHASER49 using
2C3I as a starting model. The model was completed manu-
ally in COOT50 and was refined with REFMAC5.51 Thermal
motions were analyzed using TLSMD52 and hydrogen atoms
were included in late refinement cycles. Data collection and
refinement statistics can be found in Table 4. The model
and structure factors have been deposited with PDB accession
code: 3JPV.
against
KTFCGTPEYLAPEVRREPRILSEEEQEMFRDF-
DYIADWC (PDKtide; 0.1 mM); PKBR against GRPRTS-
SFAEG (30 μM); S6K1, KKRNRTLTV (100 μM); MSK1,
GRPRTSSFAEG (30 μM); AMPK, HMRSAMSGLHLVKRR
(0.2 mM); CK2, RRRDDDSDDD (165 μM); Lck, KVEKIGEG-
TYGVVYK (250 μM); CDK2, protein histone H1 (1 mg/mL);
GSK3β, YRRAAVPPSPSLSRHSSPHQS(PO4)-EDEEE (20
μM); CSK, KVEKIGEGTYGVVYK (0.25 mM); MKK1 was
assayed via its ability to activate ERK2 (0.07 mg/mL).
In Vitro Antiproliferative Assays. 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 100X nonessential amino
acids and 2 mg gentamicin base. Cells were grown at 37 ꢀC in a
humidified incubator under an atmosphere containing 5% CO2.
Survival Assays. Cells were plated at a density of 5 ꢀ 103 cells
in 150 μL of 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 ꢀC until use. Then 50 μL 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.
Resazurin Reduction Test. Plates were rinsed with 200 μL of
PBS at 37 ꢀC and emptied by overturning on absorbent
toweling. Then 150 μL of a 25 μg/mL solution of resazurin
in MEM without phenol red was added to each well. Plates
were incubated for 1 h at 37 ꢀC in a humidified atmosphere
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 wavelength of 590 nm. Under the conditions used,
fluorescence was proportional to the number of living cells in
the well. The IC50, defined as the drug concentration required
to inhibit cell proliferation by 50%, was calculated from the
curve of concentration-dependent survival percentage, de-
fined as fluorescence in experimental wells compared with
fluorescence in control wells, after subtraction of the blank
values. The results are shown as standard errors for duplicate
determinations.
Unless stated otherwise, 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. For CaMK1 and CaMKK isoforms,
the assay mixtures also contained 0.5 mM CaCl2 and 0.3 μM
calmodulin. PKCR was diluted into 20 mM Hepes (pH 7.4)/0.03
Triton X-100 and assayed in the same buffer containing 0.1 mg/
mL phosphatidylserine, 10 μg/mL diacylglycerol, and 0.1 mM
CaCl2. PHK (5-20 m-units) was diluted in 50 mM sodium
β-glycerophosphate (pH 7.0)/0.1% 2-mercaptoethanol and as-
sayed in a buffer comprising 50 mM Tris/HCl, 50 mM sodium
β-glycerophosphate, pH 8.2, and 0.04 mM CaCl2. EF2K
(5-20 m-units) was diluted into 50 mM Hepes (pH 6.6)/0.1%
2-mercaptoethanol/1.0 mg/mL BSA and assayed in the same
buffer containing 0.2 mM CaCl2 and 0.3 μM calmodulin.
smMLCK (5-20 m-units) was diluted in 50 mM Hepes (pH
7.5)/0.1 mM EGTA/1.0 mg/mL BSA/0.1% 2-mercaptoethanol
and assayed in the same buffer containing 5 mM CaCl2 and
10 μM calmodulin. PKA (5-20 m-units) was diluted in 20 mM
Mops (pH 7.5)/1 mM EGTA/0.01% Brij-35/1.0 mg/mL BSA/
0.1% 2-mercaptoethanol and assayed in 8 mM Mops (pH 7.5)/
0.2 mM EDTA. MKK1 was assayed via its ability to activate
ERK2 in incubations containing 25 mM Tris/HCl, pH 7.5, 0.1
mM EGTA, 0.1% 2-mercaptoethanol, 0.01% Brij-35 and
MgATP.46 After incubation for 15 min at 30 ꢀC, activated
ERK2 was assayed as described above.
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 μM. The IC50 values of inhibitors were
determined after carrying out assays at 10 different concentra-
tions of each compound. The results are shown as standard
errors for duplicate determinations.
Crystallography. Protein Purification. Pim-1 was prepared as
previously described.42 The protein was >95% pure as judged
by SDS-PAGE. The purified protein was homogeneous and had
an experimental mass of 35545 Da as expected from its primary
structure. Masses were determined by LC-MS, using an Agilent
LC/MSD TOF system with reversed-phase HPLC coupled to
electrospray ionization and an orthogonal time-of-flight mass
analyzer. Proteins were desalted prior to mass spectrometry by
rapid elution off a C3 column with a gradient of 5-95%
acetonitrile in water with 0.1% formic acid.
Acknowledgment. We are grateful to the European Union
ProKinase Research Consortium and CNRS Valorisation for
financial support. We thank the Kinase Profiling Service of
the Division of Signal Transduction Therapy, MRC Protein
Phosphorylation Unit, University of Dundee (managed by
Jenny Bain), for performing the protein kinase assays. We
ꢀ
also thank Bertrand Legeret for mass spectra analysis and
Aurelie Job for technical assistance for HPLC analysis. The
ꢀ
Structural Genomics Consortium is a registered charity (no.
1097737) that receives funds from the Canadian Institutes for
Health Research, the Canadian Foundation for Innovation,
Genome Canada, through the Ontario Genomics Institute,
GlaxoSmithKline, Karolinska Institutet, the Knut and Alice
Wallenberg Foundation, the Ontario Innovation Trust, the
Ontario Ministry for Research and Innovation, Merck & Co.
Inc., the Novartis Research Foundation, the Swedish Agency
for Innovation Systems, the Swedish Foundation for Strategic
Research, and the Wellcome Trust.
Crystallization, Data Collection, and Structure Solution. Crys-
tallization was carried out using the sitting drop vapor diffusion
method at 4 ꢀC. A 6 mL solution with 1 mg of PIM1 buffered in
50 mM Hepes pH 7.5, 250 mM NaCl, 10 mM DTT was prepared
containing 2 μL of inhibitor (from a 50 mM stock in DMSO)
and 20 μL of a 5 mM solution of consensus peptide
(ARKRRRHPSGPPTA-amide). This sample was then concen-
trated to 100 μL to yield a 10 mg/mL crystal sample. Crystals
of the complex were grown by mixing 150 nL of the protein

6380 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Akueꢀ-Geꢀdu et al.
References
(22) Holder, S.; Zemskova, M.; Zhang, C.; Tabrizizad, M.; Bremer, R.;
Neidigh, J. W.; Lilly, M. B. Characterization of a potent and
selective small-molecule inhibitor of the PIM1 kinase. Mol. Cancer
Ther. 2007, 6, 163–172.
(23) Pogacic, V.; Bullock, A. N.; Fedorov, O.; Filippakopoulos, P.;
Gasser, C.; Biondi, A.; Meyer-Monard, S.; Knapp, S.; Schwaller, J.
Structural analysis identifies imidazo[1,2-b]pyridazines as PIM
kinase inhibitors with in vitro antileukemic activity. Cancer Res.
2007, 67, 6916–6924.
(24) Grey, R.; Pierce, A. C.; Bemis, G. W.; Jacobs, M. D.; Moody, C. S.;
Jajoo, R.; Mohal, N.; Green, J. Structure-based design of 3-aryl-6-
amino-triazolo[4,3-b]pyridazine inhibitors of Pim-1 kinase. Bioorg.
Med. Chem. Lett. 2009, 19, 3019–3022.
(25) Anand, R.; Maksimoska, J.; Pagano, N.; Wong, E. Y.; Gimotty,
P. A.; Diamond, S. L.; Meggers, E.; Marmorstein, R. Toward the
development of a potent and selective organoruthenium mamma-
lian sterile 20 kinase inhibitor. J. Med. Chem. 2009, 52, 1602–1611.
(26) Meggers, E.; Atilla-Gokcumen, G. E.; Bregman, H.; Maksimoska,
J.; Mulcahy, S. P.; Pagano, N.; Williams, D. S. Exploring chemical
space with organometallics: ruthenium complexes as protein kinase
inhibitors. Synlett 2007, 1177–1189.
(27) Tong, Y.; Stewart, K. D.; Thomas, S.; Przytulinska, M.; Johnson,
E. F.; Klinghofer, V.; Leverson, J.; McCall, O.; Soni, N. B.; Luo,
Y.; Lin, N.-H.; Sowin, T. J.; Giranda, V. L.; Penning, T. D.
Isoxazolo[3,4-b]quinoline-3,4(1H,9H)-diones as unique, potent
and selective inhibitors for Pim-1 and Pim-2 kinases: chemistry,
biological activities, and molecular modelling. Bioorg. Med. Chem.
Lett. 2008, 18, 5206–5208.
(28) Xia, Z.; Knaak, C.; Ma, J.; Beharry, Z. M.; McInnes, C.; Wang,
W.; Kraft, A. S.; Smith, C. D. Synthesis and evaluation of novel
inhibitors of Pim-1 and Pim-2 protein kinases. J. Med. Chem. 2009,
52, 74–86.
(29) Qian, K.; Wang, L.; Cywin, C. L.; Farmer, B. T.; Hickey, E.;
Homon, C.; Jakes, S.; Kashem, M. A.; Lee, G.; Leonard, S.; Li, J.;
Magboo, R.; Mao, W.; Pack, E.; Peng, C.; Prokopowicz, A.;
Welzel, M.; Wolak, J.; Morwick, T. Hit to lead account of the
discovery of a new class of inhibitors of Pim kinases and crystal-
lographic studies revealing an unusual kinase binding mode.
J. Med. Chem. 2009, 52, 1814–1827.
(30) Fousteris, M. A.; Papakyriakou, A.; Koutsourea, A.; Manioudaki,
M.; Lampropoulou, E.; Papadimitriou, E.; Spyroulias, G. A.;
Nikolaropoulos, S. S. Pyrrolo[2,3-a]carbazoles as potential cyclin
dependent kinase 1 (CDK1) inhibitors. Synthesis, biological eva-
luation, and binding mode through docking simulations. J. Med.
Chem. 2008, 51, 1048–1052.
(1) Saris, C. J.; Domen, J.; Berns, A. The pim-1 oncogene encodes two
related protein-serine/threonine kinases by alternative initiation at
AUG and CUG. EMBO J. 1991, 10, 655–664.
(2) Hoover, D.; Friedmann, M.; Reeves, R.; Magnuson, N. S. Re-
combinant human pim-1 protein exhibits serine/threonine kinase
activity. J. Biol. Chem. 1991, 266, 14018–14023.
(3) van der Lugt, N. M.; Domen, J.; Verhoeven, E.; Linders, K. van der
Gulden, H.; Allen, J.; Berns, A. Proviral tagging in E mu-myc
transgenic mice lacking the Pim-1 proto-oncogene leads to com-
pensatory activation of Pim-2. EMBO J. 1995, 14, 2536–2544.
(4) Cuypers, H. T.; Selten, G.; Quint, W.; Zijlstra, M.; Maandag, E. R.;
Boelens, W.; van Wezenbeek, P.; Melief, C.; Berns, A. Murine
leukemia virus-induced T-cell lymphomagenesis: integration of
proviruses in a distinct chromosomal region. Cell 1984, 37, 141–
150.
(5) Mikkers, H.; Nawijn, M.; Allen, J.; Brouwers, C.; Verhoeven, E.;
Jonkers, J.; Berns, A. Mice Deficient for all PIM kinases display
reduced body size and impaired responses to hematopoietic growth
factors. Mol. Cell. Biol. 2004, 24, 6104–6115.
(6) Shah, N.; Pang, B.; Yeoh, K.-G.; Thorn, S.; Chen, C. S.; Lilly,
M. B.; Salto-Tellez, M. Potential roles for the PIM1 kinase
in human cancer - a molecular and therapeutic appraisal. Eur.
J. Cancer 2008, 2144–2151.
(7) Morishita, D.; Katayama, R.; Sekimizu, K.; Tsuruo, T.; Fujita, N.
Pim kinases promote cell cycle progression by phosphorylating and
down-regulating p27Kip1 at the transcriptional and posttranscrip-
tional levels. Cancer Res. 2008, 68, 5076–5085.
(8) Peng, C.; Knebel, A.; Morrice, N. A.; Li, X.; Barringer, K.; Li, J.;
Jakes, S.; Werneburg, B.; Wang, L. Pim Kinase Substrate Identi-
fication and Specificity. J. Biochem. 2007, 141, 353–362.
(9) Bachmann, M.; Kosan, C.; Xing, P. X.; Montenarh, M.;
Hoffmann, I.; Moroy, T. The oncogenic serine/threonine kinase
Pim-1 directly phosphorylates and activates the G2/M specific
phosphatase Cdc25C. Int. J. Biochem. Cell Biol. 2006, 38, 430–443.
(10) Bachmann, M.; Moroy, T. The serine/threonine kinase Pim-1. Int.
J. Biochem. Cell Biol. 2005, 37, 726–730.
(11) Fox, C. J.; Hammerman, P. S.; Cinalli, R. M.; Master, S. R.;
Chodosh, L. A.; Thompson, C. B. The serine/threonine kinase
Pim-2 is a transcriptionally regulated apoptotic inhibitor. Genes
Dev. 2003, 17, 1841–1854.
(12) Aho, T. L.; Sandholm, J.; Peltola, K. J.; Ito, Y.; Koskinen, P. J.
Pim-1 kinase phosphorylates RUNX family transcription factors
and enhances their activity. BMC Cell Biol. 2006, 7, 21.
(13) Peltola, K. J.; Paukku, K.; Aho, T. L.; Ruuska, M.; Silvennoinen,
O.; Koskinen, P. J. Pim-1 kinase inhibits STAT5-dependent tran-
scription via its interactions with SOCS1 and SOCS3. Blood 2004,
103, 3744–3750.
(14) Aho, T. L.; Sandholm, J.; Peltola, K. J.; Mankonen, H. P.; Lilly,
M.; Koskinen, P. J. Pim-1 kinase promotes inactivation of the pro-
apoptotic Bad protein by phosphorylating it on the Ser112 gate-
keeper site. FEBS Lett. 2004, 571, 43–49.
(15) Zheng, H.-C.; Tsuneyama, K.; Takahashi, H.; Miwa, S.; Sugiya-
ma, T.; Popivanova, B. K.; Fujii, C.; Nomoto, K.; Mukaida, N.;
Takano, Y. Aberrant Pim-3 expression is involved in gastric
adenoma-adenocarcinoma sequence and cancer progression.
J. Cancer Res. Clin. Oncol. 2008, 134, 481–488.
(31) Sikharulidze, M. I.; Khoshtariya, T. E.; Kurkovskaya, L. N.;
Tret’yakova, L. G.; Efimova, T. K.; Suvorov, N. N. Pyrrolocarba-
zoles. 3. Synthesis and spectral properties of 1H-pyrrolo[2,3-a]-
carbazole. Chem. Heterocycl. Compd. (Engl. Transl.) 1979, 16,
1097–1100.
(32) Khoshtariya, T. E.; Sikharulidze, M. I.; Kurkovskaya, L. N.;
Suvorov, N. N. Soobsh. Akad. Nauk Gruz. SSR 1986, 124, 97–100.
ꢀ
ꢀ
(33) Pudlo, M.; Csanyi, D.; Moreau, F.; Hajos, G.; Riedl, Z.; Sapi, J.
First Suzuki-Miyaura type cross-coupling of ortho-azidobromo-
benzene with arylboronic acids and its application to the synthesis
of fused aromatic indole-heterocycles. Tetrahedron 2007, 10320–
10329.
(34) Dagher, K.; Terent’ev, P. B.; Kulikov, N. S. Tetracyanoethylation
and Fischer rearrangement of some 4-oxo-4,5,6,7-tetrahydroin-
doles. Chem. Heterocycl. Compd. (Engl. Transl.) 1986, 22, 172–175.
(35) Rawal, V. H.; Joes, R. J.; Cava, M. P. Palladium mediated
dehydrogenation in the photochemical cyclization of heterocyclic
analogs of stilbene. Tetrahedron Lett. 1985, 26, 2423–2426.
(36) Beccalli, E. M.; Marchesini, A.; Pilati, T. Synthesis of [a]annulated
carbazoles from indol-2(3H)-one. Synthesis 1992, 891–894.
(37) Fousteris, M. A.; Koutsourea, A. I.; Arsenou, E. S.; Leondiadis,
A. L.; Nikolaropoulos, S. S.; Stamos, I. K. Pyrrolocarbazole
analogs of aromatic skeleton of indolocarbazole natural products.
J. Heterocycl. Chem. 2004, 41, 349–353.
(38) Remers, W. A.; Roth, R. H.; Gibs, G. J.; Weiss, M. J. Synthesis of
indoles from 4-oxo-4,5,6,7-tetrahydroindoles. II. Introduction of
substituents into the 4 and 5 positions. J. Org. Chem. 1971, 36,
1232–1240.
(39) Kakushima, M.; Hamel, P.; Frenette, R.; Rokach, J. Regioselective
synthesis of acylpyrroles. J. Org. Chem. 1983, 48, 3214–3219.
(40) Bain, J.; Plater, L.; Elliott, M.; Shpiro, N.; Hastie, J.; McLauchlan,
H.; Klervernic, I.; Arthur, S. C.; Alessi, D. R.; Cohen, P. The
selectivity of protein kinase inhibitors: a further update. Biochem.
J. 2007, 408, 297–315.
(16) Li, Y.-Y.; Wu, Y.; Tsuneyama, K.; Baba, T.; Mukaida, N. Essen-
tial contribution of Ets-1 to constitutive Pim-3 expression in human
pancreatic cancer cells. Cancer Sci. 2009, 100, 396–404.
(17) Popivanova, B. K.; Li, Y.-Y.; Zheng, H.; Omura, K.; Fujii, C.;
Tsuneyama, K.; Mukaida, N. Proto-oncogene, Pim-3 with serine/
threonine kinase activity, is aberrantly expressed in human colon
cancer cells and can prevent Bad-mediated apoptosis. Cancer Sci.
2007, 98, 321–329.
(18) Li, Y.-Y.; Popivanova, B. K.; Nagai, Y.; Ishikura, H.; Fujii, C.;
Mukaida, N. Pim-3, a proto-oncogene with serine/threonine kinase
activity, is aberrantly expressed in human pancreatic cancer and
phosphorylates Bad to block Bad-mediated apoptosis in human
pancreatic cancer cell lines. Cancer Res. 2006, 66, 6741–6747.
(19) Adam, M.; Pogacic, V.; Bendit, M.; Chappuis, R.; Nawijn, M. C.;
Duyster, J.; Fox, C. J.; Thompson, C. B.; Cools, J.; Schwaller, J.
Targeting PIM kinases impairs survival of hematopoietic cells
transformed by kinase inhibitor-sensitive and kinase inhibitor-
resistant forms of Fms-like tyrosine kinase 3 and BCR/ABL.
Cancer Res. 2006, 66, 3828–3835.
(20) Hammerman, P. S.; Fox, C. J.; Birnbaum, M. J.; Thompson, C. B.
Pim and Akt oncogenes are independent regulators of hemato-
poietic cell growth and survival. Blood 2005, 105, 4477–4483.
(21) Giles, F. A Pim kinase inhibitor, please. Blood 2005, 105, 4158–
4159.
(41) Xu, D.-Q.; Yang, W.-L.; Luo, S.-P.; Wang, B.-T.; Wu, J.; Xu, Z.-Y.
Fischer indole synthesis in Bronsted acidic ionic liquids: A green,
e

Article
mild, and regiospecific reaction system. Eur. J. Org. Chem. 2007,
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6381
(46) Davies, S. P.; Reddy, H.; Caivano, M.; Cohen, P. Specificity and
mechanism of action of some commonly used protein kinase
inhibitors. Biochem. J. 2000, 351, 95–105.
(47) Leslie, A. G. W.; Powell, H. MOSFLM; MRC Laboratory of
Molecular Biology: Cambridge, 2007.
(48) Evans, P. SCALA;Scale Together Multiple Observations of Re-
flections; MRC Laboratory of Molecular Biology: Cambridge, 2007.
(49) McCoy, A. J.; Grosse-Kunstleve, R. W.; Storoni, L. C.; Read, R. J.
Likelihood-enhanced fast translation functions. Acta Crystallogr.,
Sect. D: Biol. Crystallogr. 2005, 61, 458–464.
(50) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular
graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60,
2126–2132.
(51) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of
macromolecular structures by the maximum-likelihood method.
Acta Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240–255.
(52) Painter, J.; Merritt, E. A. Optimal description of a protein structure
in terms of multiple groups undergoing TLS motion. Acta Crystal-
logr., Sect. D: Biol. Crystallogr. 2006, 62, 439–450.
1007–1012.
(42) Bullock, A. N.; Debreczeni, J. E.; Fedorov, O. Y.; Nelson, A.;
Marsden, B. D.; Knapp, S. Structural Basis of Inhibitor Specificity
of the Human Protooncogene Proviral Insertion Site in Moloney
Murine Leukemia Virus (PIM-1) Kinase. J. Med. Chem. 2005, 48,
7604–7614.
(43) Debreczeni, J. E.; Bullock, A. N.; Atilla, E.; Williams, D. S.;
Bregman, H.; Knapp, S. Meggers, E. Ruthenium Half-Sandwich
Complexes Bound to Protein Kinase Pim-1. Angw. Chem., Int. Ed.
2006, 45, 1580–1585.
(44) Qian, K. C.; Wang, L.; Hickey, E. R.; Studts, J.; Barringer, K.;
Peng, C.; Kronkaitis, A.; Li, J.; White, A.; Mische, S.; Farmer, B.
Structural basis of constitutive activity and a unique nucleotide
binding mode of human Pim-1 kinase. J. Biol. Chem. 2005, 280,
6130–6137.
(45) Holder, S.; Lilly, M.; Brown, M. L. Comparative molecular field
analysis of flavonoid inhibitors of the PIM-1 kinase. Bioorg. Med.
Chem. 2007, 15, 6463–6473.