Synthesis, biological evaluation and molecular modeling of a novel series of 7-azaindole based tri-heterocyclic compounds as potent CDK2/Cyclin E inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.12.023

From four molecules, inspired by the structural features of fascaplysin, with an interesting potential to inhibit cyclin-dependent kinases (CDKs), we designed a new series of tri-heterocyclic derivatives based on 1H-pyrrolo[2,3-b]pyridine (7-azaindole) an

Full text of DOI:10.1016/j.ejmech.2015.12.023

European Journal of Medicinal Chemistry 108 (2016) 701e719
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis, biological evaluation and molecular modeling of a novel
series of 7-azaindole based tri-heterocyclic compounds as potent
CDK2/Cyclin E inhibitors
,
,
**
,
, d
Christine B. Baltus a 1, Radek Jorda b 1, Christophe Marot a, Karel Berka c
,
b,
d
b
a, *
ꢀ
ꢁ
ꢀ a
Vaclav Bazgier
, Vladimír Krystof , Gildas Prie , Marie-Claude Viaud-Massuard
UMR 7292 GICC Equipe 4 Innovation Moleculaire et Therapeutique, Labex SYNORG, University of Tours, Faculty of Pharmacy, 31 Avenue Monge, 37200
a
ꢀ
ꢀ
Tours, France
b
ꢀ
Laboratory of Growth Regulators & Department of Chemical Biology and Genetics, Centre of the Region Hana for Biotechnological and Agricultural
ꢁ
ꢂ
Research, Palacký University and Institute of Experimental Botany AS CR, Slechtitelu 27, CZ-78371 Olomouc, Czech Republic
c Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacky University Olomouc, 17. Listopadu
12, 77146 Olomouc, Czech Republic
d Department of Physical Chemistry, Faculty of Science, Palacký University, 17. Listopadu 1192/12, 771 46 Olomouc, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
From four molecules, inspired by the structural features of fascaplysin, with an interesting potential to
inhibit cyclin-dependent kinases (CDKs), we designed a new series of tri-heterocyclic derivatives based
on 1H-pyrrolo[2,3-b]pyridine (7-azaindole) and triazole heterocycles. Using a Huisgen type [3 þ 2]
cycloaddition as the convergent key step, 24 derivatives were synthesized and their biological activities
were evaluated. Comparative molecular field analysis (CoMFA), based on three-dimensional quantitative
structureeactivity relationship (3D-QSAR) studies, was conducted on a series of 30 compounds from the
literature with high to low known inhibitory activity towards CDK2/cyclin E and was validated by a test
set of 5 compounds giving satisfactory predictive r2 value of 0.92. Remarkably, it also gave a good pre-
diction of pIC50 for our tri-heterocyclic series which reinforce the validation of this model for the pIC50
prediction of external set compounds. The most promising compound, 43, showed a micro-molar range
inhibitory activity against CDK2/cyclin E and also an antiproliferative and proapoptotic activity against a
panel of cancer cell lines.
Received 28 October 2015
Received in revised form
11 December 2015
Accepted 12 December 2015
Available online 19 December 2015
Keywords:
Cyclin-dependent kinase 2
Kinase inhibitors
Anti-tumor agent
1H-pyrrolo[2,3-b]pyridine
[3 þ 2] cycloaddition
1,4-Triazole
© 2015 Elsevier Masson SAS. All rights reserved.
1,5-Triazole
3D-QSAR CoMFA
1. Introduction
As a part of our research on medicinal chemistry, we are inter-
ested in developing new CDK inhibitors (CKIs), in particular to-
Cyclin-dependent protein kinases (CDKs) are universal eukary-
otic cell cycle regulators that promote the passage through the re-
striction point, initiation of DNA replication, and mitosis [1]. Given
the fact that CDKs have oncogenic potential and are amenable to
pharmacological inhibition, their inhibitors have therefore attracted
great interest as potential anticancer agents [2,3].
wards CDK4/cyclin D and CDK2/cyclin E, since they both participate
in the phosphorylation of the retinoblastoma protein (pRb). Each
member of the pRb pathway (such as CDK4(6)/cyclin D, p16 and
CDK2/cyclin E), which activates the transcription factors at the G1-S
transition phase, which in its turn regulates the expression of
several genes involved in DNA replication, can be deregulated in
cancers. In some human cancers (such as lung cancer or leukemia),
cyclin E (E1/E2) is over-expressed and CDK2 is hyper-activated [4].
Therefore, many CKIs have been developed and some of them are
undergoing clinical trials [5]. Most inhibitors that entered clinical
trials belong to the group of pan-selective CDK inhibitors [6], but
some highly selective CDK inhibitors like THZ1 [7] or PD-0332991
[8] were also described (Fig. 1). However, simultaneous targeting
of multiple CDKs (particularly CDK1 and 2) seems to be more
Abbreviation: CCR2, CC chemokine receptor 2; CCL2, CC chemokine ligand 2;
CCR5, CC chemokine receptor 5.
* Corresponding author.
** Corresponding author.
E-mail addresses: radek.jorda@upol.cz (R. Jorda), marie-claude.viaud-
massuard@univ-tours.fr (M.-C. Viaud-Massuard).
1
These authors contributed equally.
http://dx.doi.org/10.1016/j.ejmech.2015.12.023
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

702
C.B. Baltus et al. / European Journal of Medicinal Chemistry 108 (2016) 701e719
Fig. 1. Selected CDK inhibitors and their main CDK target.
advantageous in terms of anticancer activity, because many CDKs
can compensate for the lack of others in cancer cells [9,10]. In
addition, the knowledge about CDKs is growing constantly and it is
still unclear whether observed effects are due to the activity of any
single CDK or combinations of CDKs.
chemistry domain and by analogy with the structural features of a
M) (Fig. 1), as
other groups did with indole analogues [12e14], we designed and
synthesized four novel heterocyclic molecules (21, 22, 24 and 37)
(Figs. 2 and 3). 21 and 37 are composed of two 7-azaindole het-
erocycles linked each other by a 1,4- or 1,5-triazole type linker. The
known CKI, fascaplysin [11] (IC50 CDK4/D ¼ 0.35
m
Since our laboratory has an expertise in the heterocyclic
Fig. 2. 1,4-disubstituted triazole derivatives 21e36.
Fig. 3. 1,5-disubstituted triazoles derivatives 37e44.

C.B. Baltus et al. / European Journal of Medicinal Chemistry 108 (2016) 701e719
703
Table 1
Copper-catalyzed cycloaddition reaction.2
Entry
Azide
Alkyne
Additivea
Time (h)
Product
Yield (%)b
1
2
3
4
5
6
7
8
11a
11a
12a
12a
12a
11b
12b
12b
16
3a
4c
3a
4c
4a
3a
3a
4a
4a
3a
3b
4b
3b
4b
4b
3b
TBAF
e
24
16
24
26
21
20
16
72
16
18
18
16
16
16
16
16
21
22
23
24
23
25
26
26
83
80
TBAF
e
e
72
26
38
e
DMF
TBAF
TBAF
DMF
e
57
68
59
9
27
28
10
11c
12
13c
14
15
16
20
TBAF
TBAF
DMF
TBAF
DMF
e
11a
12a
11b
12b
16
29 and 30
31
32 and 33
34
35
36
43 and 39
84
47 and 37
57
89
53
20
TBAF
a
TBAF 3 equivalents; DMF 1 mL.
Isolated yields.
Products obtained as a mixture and separated by chromatography on silica gel column [30].
b
c
third ring in 22 and 24 was replaced by a phenyl group. Their
skeleton can be viewed as an open and non-planar structure of
fascaplysin, where the indole ring was replaced by a 7-azaindole.
This open form design gives a more flexible molecule which could
allow a better fitting into the binding site of the enzyme. Moreover,
contrary to fascaplysin, free rotation potential should avoid DNA
intercalation [15].
of these four compounds were found to be interesting since the IC50
values of 21 and 37 are in the micro molar range (experimental
pIC50: 5.1, 4.0, 4.3 and 5.1 respectively for 21, 22, 24 and 37)
(Table 4).
In parallel, 3D-QSAR CoMFA analyses on known CDK2/cyclin E
inhibitors were performed and the results allowed us to create and
validate a model, which will be presented later in this article.
Notably, the CDK2/cyclin E activity predictions obtained by this
model for 21, 22, 24 and 37 were in good correlation with the
experimental ones with a maximal difference of about 1 log (pre-
dicted pIC50: 5.2, 5.3, 5.3 and 5.8 respectively for 21, 22, 24 and 37)
The inhibitory activities of these four molecules were tested on
recombinant CDK2/cyclin
E and CDK4/cyclin D complexes.
Although the results obtained for CDK4/cyclin D with 21 and 22
were not satisfactory [16], the inhibitory activities on CDK2/cyclin E
Table 2
Ruthenium-catalyzed cycloaddition reaction.3
Entry
Azide
Alkyne
Time (h)
Product
Yield (%)a
1
2
3
4
5
6
7
8
11a
12a
11b
12b
11a
12a
11b
12b
4a
4a
4a
4a
4b
4b
4b
4b
60
20
20
20
20
20
20
20
37
38
39
40
41
42
43
44
41
90
73
18
62
97
18
34
a
Isolated yields.

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C.B. Baltus et al. / European Journal of Medicinal Chemistry 108 (2016) 701e719
Table 3
Values of CoMFA models.
2. Results and discussion
2.1. Chemistry
CoMFA
Partial charge
GasteigereHuckel
Tri-heterocyclic derivatives were synthesized via a key reaction,
q2a
0.49
0.99
0.10
a Huisgen type [3 þ 2] cycloaddition [17] between an alkyne and an
azide function substituted on a 7-azaindole heterocycle. The alkyne
function was introduced on the C-2 or the C-3 position of the 7-
azaindole moiety via an halogenation reaction followed by a
palladium-catalysed cross-coupling reaction (Sonogashira coupling
reaction) [18e20] (Scheme 1).
r2b
Sc
F valued
Ne
1021.002
3
Contributions
Steric
Electrostatic
Column filtering
0.42
0.58
2.0
In one hand, a simple iodation reaction on the commercially
available 7-azaindole 1 led to the derivative 2 bearing the iodine
atom on the C-3 position. The latter was treated with ethynyl-
trimethylsilane in a Sonogashira cross-coupling reaction catalyzed
by PdCl2(PPh3)2 and copper iodide in the presence of triethylamine.
The alkyne 3a could be used in the cycloaddition reaction either as
prepared, or after the removal of the trimethylsilyl group in the
presence of tetrabutylammonium fluoride (TBAF) as 4a (Scheme 1).
In the other hand, the introduction of the iodine atom at the C-2
position is described in Scheme 1. A benzenesulfonyl group was
introduced at the N-1 position of 1 using a classical procedure of
amine protection with benzenesulfonyl chloride [21]. The protected
derivative 5 was halogenated by iodine on the C-2 position, after
deprotonation with a strong base, here lithium diisopropylamide
(LDA) leading to the expected compound 6 (in a mixture with a
diiodinated derivative, see exp. part, 1H NMR ratio 1:0.34), result
also observed by Naka et al. [22] (Scheme 1, pathway A). Due to the
low yield obtained for 6, we then decided to perform a two steps
reaction at once, halogenation followed by deprotection of the
amine function, yielding 7 in a good yield for the two steps (Scheme
1, pathway B). To introduce the desired alkyne function, the
Sonogashira cross-coupling reaction was performed on 6 and 7
with ethynyltrimethylsilane. 3c was obtained with a much better
a
Cross-validated correlation coefficient.
b
Non-cross-validated correlation coefficient.
Standard error of estimate.
F test value.
c
d
e
Optimum number of components.
(Table 4).
Following these preliminary results, we decided to design a
series of 7-azaindole based tri-heterocyclic derivatives to evaluate
them towards CDK2/cyclin E, to predict their activity by our 3D-
QSAR CoMFA model and validate the model by comparison with the
experimental values, in order to develop new CDK2/cyclin E in-
hibitors as potential anti-cancer agents. The synthesis of these
derivatives was based on the pharmacomodulation of 21 and 37: i)
attachment of the triazole linker chain on both sides at the C-2 or C-
3 position of the 7-azaindole moiety, ii) variation of the linker chain
length, iii) carbonyl function group kept or not on the linker chain,
iv) 1,4- or 1,5-triazoles.
Herein is presented the synthesis of a new series of 7-azaindole
based tri-heterocyclic molecules as potent CDK2/cyclin E inhibitors,
a 3D-QSAR CoMFA model, and their biological evaluation.
Table 4
Antiproliferative, CDK2/E activity and CoMFA prediction data for studied compounds.
Cmpds
IC50
(
m
M)a
pIC50
CoMFA prediction pIC50
Cytotoxic activity in cancer cell lines
IC50
M)a
Activation of caspases (fold)b
(
m
CDK2/E
K562
MCF-7
G361
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
7.2
>50
29.3
>50
6.1
17.8
25.8
18.4
>25
30.0
9.2
18.0
8.7
3.6
>25
1.9
9.1
1.1
2.7
13.4
27.6
3.0
2.0
36.1
0.2
5.1
4.0
4.5
4.0
5.2
4.7
4.6
4.7
4.6
4.5
5.0
4.7
5.1
5.4
4.6
5.7
5.0
6.0
5.6
4.9
4.6
5.5
5.7
4.5
e
5.2
5.3
6.4
5.3
6.1
5.4
5.9
5.0
6.8
4.7
5.4
5.7
4.2
4.7
6.1
5.9
5.8
6.4
6.0
6.2
5.5
5.8
5.6
5.8
e
39.5
>50
87.1
>50
>25
>25
60.7
64.7
>25
47.4
>25
58.5
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
27.2
>10
44.2
33.6
>50
>50
36.9
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
33.4
>50
>50
>50
31.5
>50
20.8
n.d.
1.2
3.1
2.2
1.5
3.9
4.5
7.5
2.1
2.1
4.5
1.7
1.8
1.9
1.2
1.6
1.9
1.9
2.9
4.2
3.3
2.0
2.2
13.8
n.d.
n.d.
n.d.
>25
>25
66.6
44.27
>25
>50
>25
>50
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
39.9
>25
30.9
0.73
Roscovitine
Imatinib
>100
e
e
a
Average values from at least three determinations.
Relative caspase-3/7 activity in G361 melanoma cells after treatment with studied compounds at 50
b
mM concentration. Synthetic peptide Ac-DEVD-AMC was used as a
substrate and caspase-3/7 activity was measured after 6 h by microplate reader at 346 nm/442 nm (excitation/emission).

C.B. Baltus et al. / European Journal of Medicinal Chemistry 108 (2016) 701e719
705
Scheme 1. Synthesis of the alkyne derivatives 3 and 4.
yield than 3b (86% and 46% respectively). The trimethylsilyl group
respectively.
of 3c was removed with TBAF [23] leading to the derivative 4b. The
removal of the trimethylsilyl group in 3b was attempted without
success, as the reaction conditions led to the removal of both tri-
methylsilyl and benzenesulfonyl protecting groups, ending up to
the unprotected derivative 4b in 79% yield. The pathway B was
found to be the pathway of choice for the synthesis of 4b since it
gave a better overall yield compared to pathway A, 59% and 18%
In parallel, different length carbon chains bearing the desired
azide function were introduced at the C-3 and C-2 (Scheme 2)
positions of the 7-azaindole heterocycle.
First, concerning the compounds substituted at the C-3 position,
1 was reacted in a Friedel and Craft acylation reaction with selected
acid chlorides (8a and 8b) yielded the derivatives 9a and 9b con-
taining a 2 or 3 carbons chain. The carbonyl group in 9 could be kept
Scheme 2. Synthesis of the azide derivatives.

706
C.B. Baltus et al. / European Journal of Medicinal Chemistry 108 (2016) 701e719
or reduced with lithium aluminium hydride (LiAlH4) in the pres-
ence of aluminium chloride (AlCl3) [24] (leading to 10) before the
substitution of the chlorine atome by the azide group with sodium
azide was set up. The azide derivatives 11 and 12 were obtained in
moderate to good yields (Scheme 2).
whole series by presence of phenyl group instead of 7-azaindole
ring.
2.2. 3D-QSAR model
The 7-azaindole derivative 16, substituted in C-3 by a methylene
azide group, was obtained in a 4 steps synthetic procedure from 1
(Scheme 2). Bromination of 1 at the C-3 position by N-bromo-
succinimide (NBS) yielded 13, which was protected with a benze-
nesulfonyl group. The derivative 14 was methylated at the C-3
position with AlMe3 using tetrakis(triphenylphosphine)palladium
as catalyst [25,26] to afford 15 in a good yield. Bromination of the
methyl group with NBS and azobisisobutyronitrile (AIBN) [27]
followed by a nucleophilic substitution with sodium azide led to
16 in moderate yield for the two steps.
Alternatively, the methylene azide scaffold was introduced on
the C-2 position of the heterocycle (Scheme 2). The acid derivative
17 was obtained by deprotonation of 5 by LDA followed by the
addition of carbon dioxide. The compound 17 was esterified with
ethanol yielding 18, which was reduced to the alcohol 19 by using
LiAlH4 in dimethoxyethane (DME). The hydroxyl group newly
formed was substituted by a chlorine, in the presence of thionyl
chloride, which was itself substituted by the azide group leading to
the derivative 20 in good yield.
To rationalize the design and, thus, to understand physi-
ochemical properties and structural parameters of the pharmaco-
phore,
a
three-dimensional quantitative structure activity
relationship (3D-QSAR) study of numerous CKIs derivatives by
comparative molecular field analysis (CoMFA) [32] was performed.
Traditional QSAR models are unable to explain complex structure
activity data because of the extreme specificity of biological activity
described by 3D intermolecular forces and predicted on 3D mo-
lecular structures. Consequently, the most relevant QSAR model
would be shape-dependent and would describe steric and elec-
trostatic interactions with sufficient accuracy. Indeed, the
comparative molecular field analysis (CoMFA) method meets these
requirements and has become a powerful tool for studying 3D-
QSAR.
A CoMFA study starts by the examination of the differences in
targeted properties which are related to changes in the shape of the
steric and electrostatic fields surrounding the molecules. A QSAR
table is then used to accommodate the details of the shape of each
field by sampling their magnitudes at regular intervals throughout
a specified region of space [32]. Similarity indices are calculated at
regularly spaced grid points for the prealigned molecules. Instead
of the direct measurement of the similarity between all mutual
pairs of a molecule, indirect evaluation of the similarity of each
molecule in the data set with a common probe atom is calculated
[33]. A linear regression equation of similarity with biological ac-
tivities is finally derived.
The alkyne (3 and 4) and azide (11, 12, 16 and 20) derivatives
were then engaged in [3 þ 2] cycloaddition reactions. Depending
on the chosen catalyst, copper or ruthenium, the reaction led to 1,4-
or 1,5-disubstituted triazole respectively.
First, copper (I) iodide was used as catalyst for the copper-
catalyzed [3 þ 2] Huisgen cycloaddition reaction in the presence
of diisopropylethylamine (DIPEA) in THF (Table 1) [28]. An attempt
using copper sulfate and sodium ascorbate was made but it did not
give as good results as with copper iodide (results not shown). The
corresponding 1,4-disubstituted triazoles 21e36 (Fig. 2) were ob-
tained with moderate to good yields in 16 he72 h. When the non-
terminal alkyne derivatives 3a and 3b were used as starting ma-
terials, this reaction was carried out in a one pot procedure together
with the deprotection of the trimethylsilyl group by TBAF (Table 1,
entries 1, 3, 6, 7, 10, 11, 13 and 16) [29]. However, when this one pot
reaction was tested on the azide derivatives containing a carbonyl
function 12, the reaction did not occur (Table 1, entries 3 and 7). In
this case, terminal alkyne derivatives (4a, 4b or phenylacetylene 4c)
had to be used in order to obtain the expected tri-heterocycle de-
rivatives (Table 1, entries 4, 5, 8, 12 and 14). Moreover, due to the
poor solubility of 12a and 12b in THF, small amount of dime-
thylformamide (DMF) was added in the reaction mixture. This
copper-catalyzed reaction led to the obtention of a series of 1,4-
disubstituted triazole derivatives shown in Fig. 2.
Secondly, terminal alkyne (4) and azide (10 and 12) derivatives
were engaged in a ruthenium-catalyzed [3 þ 2] cycloaddition re-
action with pentamethylcyclopentadienylbis(triphenylphosphine)
ruthenium(II) chloride (Cp*RuCl(PPh3)2) in THF (Table 2) [31]. The
1,5-disubstituted triazole derivatives (37e44), obtained in low to
good yields in 20 he60 h, are presented in Fig. 3.
To conclude, all 24 synthesized compounds have consisted of
three parts: 7-azaindole, triazole and aryl part. The first hetero-
cycle, 7-azaindole, is linked by carbon C2 or C3 to nitrogen N1 of
triazole in all derivatives, third aryl is linked to triazole at position 4
or 5 resulting two series: 1,4-disubstituted (21e36) and 1,5-
disubstituted triazoles (37e44). Both series are subdivided to two
another libraries differing by binding of third aryl (mainly 7-
azaindole) to triazole. While compounds 29e36 and 41e44 bind
by carbon C2 of 7-azaindole to triazole, the rest of compounds
containing 7-azaindole (21, 23, 25e28, 37e40) bind through carbon
C3 to triazole. Only two derivatives (22, 24) have varied from the
This indirect ligand-based approach can assist in understanding
structureeactivity relationships (SARs) and can also serve as a tool
in designing more potent anticancer agents. We believe this study
provides useful information about the structural requirements of
CDK2 inhibitors, and expect the results will aid in the design of new
synthetic drugs.
2.3. Computational methods
2.3.1. Data sets
A total of 30 ligands from the literature, 25 for the training set
(L1-L25) and 5 for the test set (L26-L30), with known structures
and biological activities towards CDK2/cyclin E, were used in the
study (See Supp. Info. Tables S1 and S2) [34]. The IC50 values were
converted to the corresponding pIC50 (-logIC50) and used as
dependent variables in CoMFA analyses. The pIC50 values span a
range of 4.5 log units, providing a broad and homogenous data set
for the 3D-QSAR study. The initial structures of 30 compounds were
constructed using the Scketch Builder module on Sybyl X 2.1
(TRIPOS) [35]. Conformations of compounds in the training set and
test set were generated using the multisearch utility in Sybyl X 2.1.
The conformer with the lowest energy was extracted and energy
minimization was performed using the Tripos force field [36], with
a distance-dependent dielectric constant, and the Powell conjugate
gradient algorithm, with a convergence criterion of 0.01 kcal molꢀ1
.
2.3.2. Molecular alignment
Structural alignment is one of the most sensitive parameters in
3D-QSAR analyses. The accuracy of the prediction of CoMFA models
and the reliability of the contour models depend strongly on the
structural alignment of the molecules [37]. The molecular align-
ment of the training set was achieved by the Sybyl X 2.1 routine
docking (FlexX) with X-ray crystal structure of CDK2 (PDB: 2A4L)
(Fig. 4a). In order to investigate the interaction mechanism

C.B. Baltus et al. / European Journal of Medicinal Chemistry 108 (2016) 701e719
707
Fig. 4. Alignment in the active site of CDK2 (PDB: 2A4L) of a) training set ligands L1-L25, b) test set ligands L26-L30, c) our designed ligands 21e44.
q2 = 0.49
R² = 0.9245
9
8
7
6
5
4
3
9
8
7
6
5
4
3
r2 = 0.99
3
4
5
6
7
8
9
3
4
5
6
7
8
9
Actual pIC50
Actual pIC50
a)
b)
Fig. 5. CoMFA predicted and actual activities of ligands from a) the training set, b) the test sets.
between the ligand and the targeted enzyme, the molecular
docking analysis was performed based on the active site of the built
3D model of 2A4L. The FlexX docking methods [38] were used to
explore the reasonable binding mode of ligands in the active site of
2A4L. Once the molecules from the training set are docked, the
poses with the best docking score values were selected as the
aligned molecules used for the CoMFA study.
2.3.3. CoMFA studies
Sybyl X 2.1 was used for molecular modeling and 3D-QSAR
analysis on a PC workstation equipped with an Intel(R) Xeon(R)
CPU 2.67 GHz processor. Steric and electrostatic CoMFA fields were
calculated using the Lennard-Jones and the Coulomb potentials.32
The partial atomic charge was calculated by the Gasteiger-Hückel
method [39]. Default parameters (Tripos force field, dielectric dis-
tance 1/r2, steric and electrostatic cut-off 30 kcal molꢀ1, positively
charged sp3 hybridized carbon atom, grid spacing 2 Å) were used
unless stated otherwise. With standard options for scaling of vari-
ables, the regression analysis was carried out using the full cross-
validated partial least squares (PLS) method (leave one out) [40].
To derive 3D-QSAR models, the CoMFA descriptors were used as
independent variables and the pIC50 as the dependent variable.
Partial least squares (PLS) regression analysis was conducted with
the standard implementation in the Sybyl X 2.1 package. The pre-
dicted values of the models were evaluated by leave-one-out cross-
validation [41] (Table 3).
2.3.4. Predictive power of CoMFA models
To test the predictability of the analyses, the activities of training
set compounds were calculated from the best CoMFA model
considering Gasteiger-Hückel partial charges, steric and electro-
static field combinations. The correlation between experimental
and predicted training set activities is shown in Fig. 5a and in-
dicates a good agreement between experimental and predicted
values (See Supp. Info. Table S1).
A test set made of 5 compounds was used to verify the efficacy of
the CoMFA model (See Supp. Info. Table S2). These 5 molecules
were aligned in the active site of the enzyme in the same way as the
training set (Fig. 4b). Fig. 5b shows that the test set pIC50 pre-
dictions and the actual pIC50 were in a similar range of values (See
Supp. Info. Table S2), giving a predictive correlation coefficient r2 of
Fig. 6. Contour plot using CoMFA steric and electrostatic fields for CDK2 inhibitors
with 38. Green indicates regions where bulky groups increase activity; yellow indicates
regions where bulky groups decrease activity; blue indicates regions where positive
charges increase activity or negative charges decrease it; red indicates regions where
negative charges increase activity or positive charges decrease it. (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of
this article.)

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C.B. Baltus et al. / European Journal of Medicinal Chemistry 108 (2016) 701e719
0.92, which validates this model.
comparison with unmodified structures (pairs 30e31, 33e34,
37e38, 41e42), in some pairs the activity decreased (pairs 21e23,
25e26, 39e40, 43e44). Observed differences are probably related
to the orientation of the whole molecules in the active site of kinase
and with the possibility to use the carbonyl as a hydrogen bond
acceptor. The most active compounds from this library belong to
the group of 1,5-disubstituted triazoles (structural isomers 38e42,
Once the model was validated with the test set, our 24 tri-
heterocyclic derivatives were aligned in the active site of the
enzyme (Fig. 4c) and the CoMFA allowed us to obtain a prediction
for their inhibitory activities (pIC50) (Table 4).
2.3.5. CoMFA contour maps
The CoMFA steric and electrostatic fields from the final non-
cross-validated analysis were plotted as 3D colored contour maps.
In these contour maps, each colored contour represents particular
properties (Fig. 6): green for regions of high steric tolerance (80%
contribution), yellow for low steric tolerance (20% contribution),
red for regions of decreased tolerance for positive charge (20%
contribution) and, blue for regions of decreased tolerance for
negative charge (80% contribution). The larger size of the green-
eyellow region compared to the redeblue region indicates a
greater contribution of steric fields than electrostatic ones in
determining the biological activity. The derivative 38 was selected
to feature in the CoMFA contour map (Fig. 6), since it was the tri-
heterocyclic derivative having the best IC50 value on the recombi-
nant CDK2/cyclin E complex (Table 4). Indeed, the carbonyl group in
38 was located in a region of negatively charged electrostatic field
and the whole molecule seemed to meet the requirement of the
steric constraints.
39e43) and their IC50(CDK2) reaches 1.1 mM (Table 4).
2.5. Flexible docking
To further explore SAR of novel compounds we decided to use
our flexible docking procedure in conjunction with the Autodock
Vina molecular docking program to model binding interactions
with CDK2 (see the Experimental section for details). Crystal
structure (PDBID: 2A4L) reveals that the ATP binding groove is
formed by a planar, mainly hydrophobic, pocket between Ile10 and
Leu134. On the edge of this pocket are situated main chains of
Glu81 and Leu83 forming hydrogen bond acceptor-donor-acceptor
pattern for interaction with purine bases of roscovitine or ATP. The
pocket further tilts and extends towards the bay between two polar
residues His84 and Lys89. This bay is filled with phenyl moiety of
roscovitine in the crystal structure, but it can also accommodate
larger ligands, such as biaryls [42].
In this study, 3D-CoMFA QSAR analyses were used to predict the
CDK2/cyclin E inhibitory activity of a set of tri-heterocyclic com-
pounds. The QSAR model gave good statistical results in terms of q2
and r2 values, respectively 0.495 and 0.99. The CoMFA model pro-
vided the most significant correlation of steric and electrostatic
fields with the biological activities. Overall, the CoMFA method
provided a better statistical model, which implies the significance
of steric and electrostatic fields in the selectivity and activity of
these compounds. The statistical significance and robustness of the
3D-QSAR models generated were confirmed using a test set
(r2 ¼ 0.92). The effects of the steric, electrostatic fields around the
aligned molecules on their activities were clarified by analyzing the
CoMFA contour maps. Most of our compounds (~70%) were pre-
dicted with a maximum deviation of 1 log unit. The micromolar
range activity of our best compounds were correctly predicted (36,
38, 39, 42 and 43), since the predictive deviation is only about 0.5
log unit. Only a few inactive compounds were predicted with a
micromolar activity (however, this shows that our model do not
miss potent compounds).
Our molecular docking showed the binding mode of molecule
43 within the binding pocket of CDK2 kinase (Fig. 7; binding
energy ꢀ9.5 kcal/mol). The ligand forms a U-shape with two
branches formed by 7-azaindoles and turning loop formed by tri-
azole moiety. Triazole shows two interactions e directly with Thr14
and more distantly with Asp145 (possibly through water molecule).
First 7-azaindole branch interacts with the typical main chain in-
teractions e Glu81 and Leu83. Second branch seems to be more
movable as it has just intraction with main chain of Glu12, whereas
it is more exposed to the water.
2.6. Anticancer activity in vitro
Table 4 and Table S4 (See Supp. Inf.) summarized also anticancer
activity of prepared compounds towards three cancer cell lines.
Inhibition of CDKs in cells either by chemical or genetic deple-
tion lead to the i) reduction of cell proliferation, ii) changes in cell
cycle profile and iii) induction of apoptosis [43,44].
We therefore analyzed the antiproliferative and proapoptotic
activity of all derivatives to confirm reported inhibition of CDKs.
First, we treated a panel of cancer cell lines with increasing con-
centrations and determined the compounds cytotoxicity (IC50) after
72 h (Table 4). Unexpectedly, majority of compounds did not
display measurable IC50, due to the limited solubility in culture
medium. Otherwise, we determined the IC50 values of the most
active derivatives reaching up to mid-micromolar ranges (Table 4).
The most active compound 43 was further profiled on a addi-
tional cancer cell lines (See Supp. Info. Table S3).
2.4. CDK inhibition
The 24 tri-heterocyclic derivatives synthesized were tested for
CDK2/cyclin E kinase inhibition. The obtained data are presented in
Table 4.
The majority of these compounds displayed submicromolar
activities toward CDK2, what we consider as a good starting point
for further improvement of molecules. The structural diversity of
library allowed us to evaluate structureeactivity relationships. The
first observed trend is related to substitution of 7-azaindole at ni-
trogen N1 by bulky phenylsulphonyl moiety that leads to a decrease
of CDK2 activity of unmodified molecules (compared pairs 27e28,
29e30, 32e33, 35e36). No measurable CDK inhibition of two
compounds bearing phenyl group as a third aryl (compounds 22,
24) showed us that 7-azaindole as a third heterocycle is beneficial
(probably due to the presence of nitrogen N1 as a hydrogen donor).
In fact, CDK2 inhibition was also reduced mostly with the short-
ening of the alkyl chain between first 7-azaindole and triazole
(compared pairs 30e33, 37e39, 41e43). In addition, modification
of alkyl chain can also influence the activity; while the presence of a
keto group leads to increase of CDK2 inhibitory activity in
In parallel, we tested the ability of our compounds to activate
apoptosis in treated G361 cells after 24 h at a single dose of 50 mM.
Using the one-step cellular caspase-3/7 activity assay [45] we
determined that many compounds increased caspases activity over
untreated control cells (Table 4); compound 43 more than ten-
times and therefore we chose 43 as a candidate and evaluated its
biological effects on melanoma G361 cell line in more detail.
In addition, the selectivity profile of the compound 43 was
further characterized by assays on additional CDKs, including CDK1,
CDK4, CDK5, CDK7 and CDK9. The compound yielded IC50 values in
a mid-micromolar range (Table 5) and clearly showed that 43 is a
pan-specific inhibitor of CDKs.
Asynchronously growing cells were treated by increasing

C.B. Baltus et al. / European Journal of Medicinal Chemistry 108 (2016) 701e719
709
Fig. 7. Molecular docking of 43 into CDK2 (PDBID: 2A4L). Docking pose of 43 is shown on the left with ball and stick representation with annotated hydrogen bonds and roscovitine
moiety in line representation. 2D interaction pattern of 43 is shown on the right. Amino acids within 3.5 Å from the ligand are shown whereas direct hydrogen bonds are shown
with arrows. Solvent exposed part of the ligand is shown with circles.
Table 5
concentrations of 43 for 24 h and then analyzed by flow cytometry.
Compound 43 potently reduced population of actively-replicating
cells (BrdU positive) in G361 (Fig. 8A) and MCF-7 (data not
shown) cells and accumulated cells in G1 and G2/M phases. We also
observed a dose-dependent increase in sub G1/apoptotic cells that
CDK selectivity profile for compound 43 and some known CDK inhibitors assayed as
a control.
Kinases
IC50
43
(m
M)a
Dinaciclib
BS-181
LY2835219
LDC000067
reached up 34% for 43 (80 mM).
CDK1
CDK2
CDK4
CDK5
CDK7
CDK9
11.5
2.0
9.0
10.7
>50
9.0
0.072
0.002
0.127
0.045
0.170
0.178
14.0
1.8
44.7
3.7
0.134
1.790
0.371
0.347
0.005
0.405
3.112
0.101
3.95
2.33
3.16
4.96
>20
The previous analysis was complemented by the monitoring of
the cellular expression of some cell cycle regulators in G361 cells
treated with 43 for 24 h. We observed a rapid dose-dependent
decrease of Rb level starting already after of 20
mM treatment.
0.23
This inhibitory pattern corresponds to reduction of number of cells
in S and G2/M phases upon treatment documented by decrease of
protein levels of cyclins A and B (Fig. 8B).
a
Average values from at least three determinations.
In addition, we evaluated the effect of 43 on the
Fig. 8. A) Effect of compound 43 on cell cycle in G361 cells treated for 24 h. Flow cytometric analysis (10 000 counts) of DNA stained by propidium iodide (FL3 signal) and 5-bromo-
20-deoxyuridine (FL1 signal). The number in the lower right corner refers to the percentage of apoptotic cells (subG1 population). B) Immunobloting analysis of cell cycle-related
proteins in G361 cells treated by compound 43 for 24 h. C) Compound 43 inhibits the phosphorylation of CDKs substrates. G361 cells were first synchronized with nocodazole (3 ng/
mL for 16 h) and then treated with 43 as indicated. Substrates of CDKs were detected by immunoblotting analysis and tubulin was included as a control for equal protein loading.

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C.B. Baltus et al. / European Journal of Medicinal Chemistry 108 (2016) 701e719
Fig. 9. A) Immunoblotting analysis of apoptosis-related proteins in G361 cells treated by compound 43 for 24 h. Tubulin level is included as a control for equal loading. B)
Fluorimetric caspase-3,7 activity assay in G361 cell lysates treated with varying doses of 43 for 24 h. The activities of caspases were measured using the fluorogenic substrate Ac-
DEVD-AMC and normalized against an untreated control.
phosphorylation of known CDK substrates. When G361 cells were
treated with 43 for 4 h, the abundance of the phospho-forms of Rb,
protein phosphatase PP1alpha and nucleophosmin (NPM)
decreased (Fig. 8c).
Among the most potent compounds, the derivative 43, con-
taining a 1,5-disubstituted triazole, was found to be a pan-specific
inhibitor of CDKs. The binding mode of 43 in CDK2 cavity have
been described by molecular docking study and revealed donor-
acceptor pattern of interaction of 7-azaindole ring with the main
chain of Glu81 and Leu83. Cellular inhibition of CDK2 was
confirmed by the monitoring of changes in the phosphorylation of
known CDK substrates, Rb, PP1alpha and NPM.
Antiproliferative activity of 43 was studied in detail on G361 and
MCF-7 cell lines and revealed a decrease of DNA replication and
proliferation and a cell accumulation in G1 and G2/M phases in
treated cells as it was previously described for numerous other pan-
selective CDK inhibitors. In parallel, compound 43 showed to
induce apoptosis in treated cells as it was documented by flow
cytometry (subG1 population), imunoblot analysis of cleaved PARP
and caspase activity assay.
2.7. Induction of apoptosis
Besides measuring antiproliferative activity of compounds 43
we also evaluated in a detail the ability of 43 to induce apoptosis in
G361 cells by imunoblotting and caspase activity assay. As shown in
Fig. 9A, compound 43 influenced mitochondrial pathway of
apoptosis in treated cells as documented by a decrease of pro-
survival protein Mcl-1 and an increase of proapoptotic proteins Bax,
Bak and Bid. Dose-dependent response has clearly correlated with
observations of i) cellular level of zymogenes of effector caspases 3
and 7, ii) their active fragments and iii) cleavage of substrate, PARP
(poly-ADP ribose polymerase). In addition, the activation of cas-
pases was confirmed by an enzyme activity assay using fluo-
rescently labeled substrates of caspases 3 and 7 (Fig. 9B).
4. Experimental section
4.1. Material and methods
3. Conclusion
All commercial materials were used without further purifica-
tion. For anhydrous and inert reactions, the glassware was dried in
an oven and several vacuumenitrogen cycles were performed be-
forehand. The thin layer chromatography (TLC) studies were per-
formed using commercial pre-coated aluminium sheets silica gel
(60 Å, F254) marketed by Merk. The purifications by chromatog-
raphy on silica gel columns were carried out on an ISCO purification
unit, Combi Flash RF 75 PSI, with Redisep flash silica gel columns
(60 Å, 230e400 mesh, grade 9385). Purities of compounds were
assessed by inspection of their proton and carbon nuclear magnetic
resonance spectroscopy (1H and 13C NMR) spectra and high reso-
lution mass spectrometry (HRMS) specta. NMR spectra were
measured on a Brücker Ultrashield 300 spectrometer, 300 MHz (1H)
and 75 MHz (13C) in deuterated chloroform (CDCl3) or dime-
In summary, thanks to a convergent synthesis pattern, 24 pyr-
rolo[2,3-b]pyridine based derivatives (21e44) were synthesized in
5e10 steps, with moderate to very good yields. The key step [3 þ 2]
cycloaddition reaction allowed us to enlarge our library of tri-
heterocycles by giving us the opportunity to make 1,4- and 1,5-
disubstituted triazoles. All the compounds were evaluated for
in vitro inhibitory activity against CDK2/cyclin
E and anti-
proliferative activity on cancer cell lines. Interestingly, some of
them showed a single digit micro-molar inhibitory activity towards
CDK2/cyclin E. From known CKIs from the literature, a 3D-QSAR
CoMFA model was built and validated with good statistical results.
This model gave us a correct prediction of the biological activity of
our potent compounds towards CDK2/cyclin E and the contour map
obtained will be of great use for improving our CKIs through a
pharmacomodulation process.
thylsulfoxide (DMSO-d6). The notations used are:
d: chemical shift
(ppm), s: singlet; d: doublet; dd: doublet of doublet; t: triplet; m:

C.B. Baltus et al. / European Journal of Medicinal Chemistry 108 (2016) 701e719
711
multiplet; br: broad signal; J: coupling constant (Hz). The HRMS
was performed by the mass spectrometry service on a Q-Exactive
spectrometer from Thermo Scientific using the electrospray ion-
isation (ESI) technique. From known CKIs from the literature, a 3D-
QSAR CoMFA model was built and validated with good statistical
results. This model gave us a correct prediction of the biological
activity of our potent compounds towards CDK2/cyclin E and the
contour map obtained will be of great use for improving our CKIs
through a pharmacomodulation process.
(1.02 g, 25.41 mmol) and CH2Cl2 (20 mL) were mixed, stirred and
cooled down to 0 ꢁC in an ice bath, then benzene sulfonylchloride
(1.35 mL, 10.59 mmol) was added slowly. The mixture was left to
warm up to room temperature and stirred at this temperature for
1 h. The reaction was hydrolysed with water (20 mL) and extracted
by CH2Cl2 (twice). The organic layer was washed with a saturated
sodium chloride solution, dried over magnesium sulfate and
concentrated under reduced pressure to give 2.38 g of a beige solid.
The crude product was purified by chromatography on silica gel
column, cyclohexane/EtOAc 7:3, to give 2.17 g of the pure expected
product as a white solid in 99% yield. 1H NMR (300 MHz, CDCl3)
4.2. Synthesis of compounds 2e20
d
(ppm): 8.45 (dd, J ¼ 1.5, 4.8 Hz, 1H), 8.20 (d, J ¼ 7.5 Hz, 2H), 7.86
4.2.1. 3-Iodo-1H-pyrrolo[2,3-b]pyridine (2)
(dd, J ¼ 1.5, 7.8 Hz, 1H), 7.73 (d, J ¼ 4.0 Hz, 1H), 7.57 (dd, J ¼ 7.4 Hz,
1H), 7.52e7.47 (m, 2H), 7.19 (dd, J ¼ 4.8, 7.8 Hz, 1H), 6.61 (d,
1H-Pyrrolo[2,3-b]pyridine 1 (1.00 g, 8.47 mmol) was dissolved
in ethanol (40 mL) then iodine (3.23 g, 12.71 mmol), potassium
iodide (2.11 g, 12.71 mmol) and a sodium hydroxide solution
(12.71 mL, 12.71 mmol, 1 M) were added. The mixture was stirred
overnight at room temperature. The reaction mixture was diluted
with water and extracted with ethyl acetate (3 times). The organic
layers were gathered, washed with a thiosulfate solution (w/w 5%)
(twice) and a saturated sodium chloride solution, dried over mag-
nesium sulfate, filtered and concentrated to give an orange solid.
The crude product was recrystallised in methanol and water to give
2.00 g of the pure expected product as an orange solid in 97% yield.
J ¼ 4.0 Hz, 1H). 13C NMR (75 MHz, CDCl3)
d (ppm): 144.7 (2C), 138.3,
134.1, 129.9, 129.1 (2C), 128.0 (2C), 126.5, 123.0, 119.0, 105.5.
4.2.5. 2-Iodo-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (6)
In a heat dried and nitrogen purged tricol round bottom flask, 5
(1.00 g, 3.88 mmol) was dissolved in THF (40 mL). The solution was
cooled down to ꢀ60 ꢁC and LDA (2.33 mL, 4.66 mmol, 2 M in THF)
was added. The mixture was left to stir at ꢀ60 ꢁC for 15 min.
Diiodine (1.97 g, 7.76 mmol) in solution in THF (10 mL) was added
slowly. The mixture was left to warm up to room temperature
overnight and the reaction was quenched with water (25 mL). The
mixture was diluted with EtOAc, washed with a thiosulfate solution
(w/w 5%), water and a saturated sodium chloride solution, dried
over magnesium sulfate, filtered and concentrated under reduced
pressure to give 1.8 g of a brown solid. The crude product, a mixture
of 6 and a diiodinated derivative 6′, was purified by flash chro-
matography on silica gel column, cyclohexane/EtOAc 85:15, to give
750 mg of the clean expected product 6 as a pale yellow solid in 50%
yield, 6′ described below and a mixed fraction, both not quantified.
1H NMR (300 MHz, DMSO-d6)
d (ppm): 12.11 (br, 1H), 8.26 (dd,
J ¼ 1.5, 4.8 Hz, 1H), 7.72 (d, J ¼ 2.7 Hz, 1H), 7.69 (dd, J ¼ 1.5, 7.8 Hz,
1H), 7.16 (dd, J ¼ 4.8, 7.8 Hz, 1H). 13C NMR (75 MHz, DMSO-d6)
d
(ppm): 148.5, 144.2, 130.9, 128.5, 122.4, 116.9, 54.8.
4.2.2. 3-((trimethylsilyl)ethynyl)-1H-pyrrolo[2,3-b]pyridine (3a)
General procedure for the Sonogashira cross-coupling reaction.
Compound 2 (1.5 g, 6.15 mmol), copper iodide (118 mg, 0.62 mmol),
bis(triphenylphosphine)palladium(II)
dichloride
(217
mg,
0.31 mmol), triethylamine (1.28 mL, 9.23 mmol), ethynyl(trimethyl)
silane (1.30 mL, 9.23 mmol) and THF (35 mL) were introduced in an
oven dried round bottom flask under inert atmosphere. The
mixture was stirred at room temperature for 20 h. The reaction
mixture was diluted with EtOAc and filtered on Dicalite. The filtrate
was washed with water and a saturated solution of sodium chlo-
ride, dried over magnesium sulfate, filtered and concentrated. The
crude product was purified by chromatography on silica gel col-
umn, cyclohexane/EtOAc 7:3, to give 1.099 g of the clean expected
product as a beige solid in 84% yield. 1H NMR (300 MHz, DMSO-d6)
1H NMR (300 MHz, CDCl3)
d
(ppm): 8.55 (dd, J ¼ 1.5, 4.8 Hz, 1H),
8.22 (d, J ¼ 7.2 Hz, 2H), 7.79 (dd, J ¼ 1.6, 7.8 Hz, 1H), 7.58 (dd,
J ¼ 7.3 Hz, 1H), 7.52e7.43 (m, 2H), 7.20 (dd, J ¼ 4.8, 7.8 Hz, 1H), 6.83
(s, 1H). 13C NMR (75 MHz, CDCl3)
d (ppm): 149.7, 144.8, 138.8, 134.3,
129.2 (2C), 128.3 (2C), 127.9, 124.0, 120.4, 119.5, 76.4.
4.2.6. 2-Iodo-1-((2-iodophenyl)sulfonyl)-1H-pyrrolo[2,3-b]
pyridine (60)
1H NMR (300 MHz, CDCl3)
d
(ppm): 8.55 (dd, J ¼ 1.6, 8.0 Hz, 1H),
8.07 (dd, J ¼ 1.6, 4.8 Hz, 1H), 7.93 (dd, J ¼ 1.2, 7.8 Hz, 1H), 7.74 (dd,
d
(ppm): 12.12 (br, 1H), 8.30 (dd, J ¼ 1.5, 4.7 Hz, 1H), 7.93 (dd, J ¼ 1.5,
7.8 Hz, 1H), 7.88 (d, J ¼ 2.7 Hz, 1H), 7.18 (dd, J ¼ 4.7, 7.8 Hz, 1H), 0.24
(s, 9H). 13C NMR (75 MHz, DMSO-d6)
(ppm): 148.0, 144.4, 131.4,
J ¼ 1.6, 7.8 Hz, 1H), 7.58 (ddd, J ¼ 1.2, 7.4, 8.0 Hz, 1H), 7.22 (ddd,
J ¼ 1.6, 7.8, 7.8 Hz, 1H), 7.08 (dd, J ¼ 4.8, 7.8 Hz, 1H), 7.05 (s, 1H). 13
C
d
NMR (75 MHz, CDCl3) d (ppm): 153.7, 149.3, 144.2, 142.6, 141.9,
127.6, 120.8, 117.0, 99.6, 95.5, 95.4, 0.7 (3C).
134.6, 134.2, 128.1, 127.8, 123.5, 119.5, 119.3, 92.1.
4.2.3. 3-Ethynyl-1H-pyrrolo[2,3-b]pyridine (4a)
4.2.7. 2-Iodo-1H-pyrrolo[2,3-b]pyridine (7)
Compound 3a (181 mg, 0.85 mmol) was dissolved in anhydrous
THF, TBAF in solution in THF (1.7 mL, 1.70 mmol, 1 M) was added
and the mixture was stirred at room temperature for 2 h. The re-
action mixture was diluted with EtOAc, washed with water and a
saturated sodium chloride solution, dried over magnesium sulfate,
filtered and concentrated under reduced pressure to give 125 mg of
the expected product as a pale brown solid in quantitative yield. 1H
In a heat dried and nitrogen purged round bottom flask, 5
(509 mg,1.97 mmol) was dissolved in THF (20 mL). The solution was
cooled down to ꢀ60 ꢁC and LDA (1.97 mL, 3.95 mmol, 2 M in THF)
was added. The mixture was left to stir at ꢀ60 ꢁC for 15 min the
diiodine (752 mg, 2.96 mmol) in solution in THF (10 mL) was added.
The mixture was left to warm up to room temperature overnight
and the reaction was quenched with water (5 mL). The mixture was
diluted with EtOAc, washed with a thiosulfate solution (w/w, 5%),
water and a saturated sodium chloride solution, dried over mag-
nesium sulfate, filtered and concentrated under reduced pressure.
The crude product was purified by flash chromatography on silica
gel column, cyclohexane/EtOAc 85:15. A mixture of 6 and 6′ was
obtained. This mixture was solubilised in dioxane (10 mL) and so-
dium tert-butoxide (380 mg, 3.95 mmol) was added. The mixture
was left under stirring and reflux for 2 h. After being cooled down
to room temperature, the reaction was quenched with water
NMR (300 MHz, DMSO-d6)
d
(ppm): 12.08 (br, 1H), 8.29 (dd, J ¼ 1.5,
4.9 Hz, 1H), 7.96 (dd, J ¼ 1.5, 7.8 Hz, 1H), 7.86 (br s, 1H), 7.17 (dd,
J ¼ 4.9, 7.8 Hz, 1H), 4.12 (s, 1H). 13C NMR (75 MHz, DMSO-d6)
d
(ppm): 148.0, 144.3, 131.3, 127.6, 120.8, 117.0, 94.8, 82.0, 77.8.
4.2.4. 1-(Phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (5)
In a heat dried and nitrogen purged round bottom flask, 1H-
pyrrolo[2,3-b]pyridine 1 (1.01 g, 8.58 mmol), tetrabutylammonium
bromide (81 mg, 0.25 mmol), finely grounded sodium hydroxide

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C.B. Baltus et al. / European Journal of Medicinal Chemistry 108 (2016) 701e719
(20 mL) and extracted with EtOAc. The organic layer was washed
with a saturated sodium chloride solution, dried over magnesium
sulfate, filtered and concentrated under reduced pressure to give
418 mg of the expected product as a pale orange solid in 87% yield.
DMSO-d6)
d
(ppm): 12.68 (br, 1H), 8.60 (s, 1H), 8.46 (dd, J ¼ 1.8,
7.8 Hz,1H), 8.36 (dd, J ¼ 1.8, 4.8 Hz, 1H), 7.29 (dd, J ¼ 4.8, 7.8 Hz, 1H),
4.94 (s, 2H). 13C NMR (75 MHz, DMSO-d6)
d (ppm): 186.9, 149.4,
145.1, 135.6, 130.0, 118.9, 118.2, 112.8, 46.7.
1H NMR (300 MHz, DMSO-d6)
d (ppm): 12.19 (br, 1H), 8.28 (dd,
J ¼ 1.6, 4.7 Hz, 1H), 7.95 (dd, J ¼ 1.5, 7.9 Hz, 1H), 7.09 (dd, J ¼ 4.7,
4.2.12. 3-Chloro-1-(1H-pyrrolo[2,3-b]pyridin-3-yl)propan-1-one
(9b)
7.9 Hz, 1H), 6.77 (sd, J ¼ 1.6 Hz, 1H). 13C NMR (75 MHz, DMSO-d6)
d
(ppm): 155.7, 147.7, 131.7, 126.9, 121.1, 114.8, 85.9.
In a heat dried and nitrogen purged round bottom flask,
aluminium chloride (3.225 g, 25.40 mmol) and CH2Cl2 (100 mL)
were mixed and stirred at room temperature for 5 min then 1
(600 mg, 5.08 mmol) was added. The mixture was stirred for 1.5 h
then cooled in an ice bath and 3-chloropropionyl chloride (2.42 mL,
25.40 mmol) was added slowly. The reaction mixture was left to
warm up to room temperature and left to stir overnight (16 h) at rt
under nitrogen atmosphere. The mixture was cooled in an ice bath
and quenched by MeOH (15 mL). The solvents were removed under
reduced pressure and a saturated sodium bicarbonate solution
(120 mL) was added to the resulting mixture which was then
extracted three times by EtOAc. The gathered organic layers were
washed with a saturated sodium chloride solution, dried over
magnesium sulfate, filtered and concentrated under reduced
pressure to give 1.01 g of the expected product as a beige solid 95%
4.2.8. 1-(Phenylsulfonyl)-2-((trimethylsilyl)ethynyl)-1H-pyrrolo
[2,3-b]pyridine (3b)
Compound 3b was synthesized following the general procedure
for the Sonogashira coupling reaction from 6. The crude product
was purified by chromatography on silica gel column, cyclohexane/
EtOAc 8:2, to give 285 mg of the expected product as a yellow oil
(purity 70%), 46% calculated yield. 1H NMR (300 MHz, CDCl3)
d
(ppm): 8.53 (dd, J ¼ 1.5, 4.7 Hz, 1H), 8.21 (d, J ¼ 7.3 Hz, 2H), 7.79
(dd, J ¼ 1.5, 7.9 Hz, 1H), 7.58 (dd, J ¼ 7.3 Hz, 1H), 7.53e7.43 (m, 2H),
7.20 (dd, J ¼ 4.8, 7.9 Hz, 1H), 6.83 (s, 1H), 0.35 (s, 9H). 13C NMR
(75 MHz, DMSO-d6)
d (ppm): 148.3, 146.4, 139.3, 134.1, 129.4, 129.1
(2C), 128.0 (2C), 121.4, 121.3, 119.7, 114.2, 105.0, 94.6, ꢀ0.25 (3C).
4.2.9. 2-((Trimethylsilyl)ethynyl)-1H-pyrrolo[2,3-b]pyridine (3c)
The derivative 3c was synthesized following the general pro-
cedure for the Sonogashira coupling reaction from 7. The crude
product was purified by chromatography on silica gel column,
cyclohexane/EtOAc 98:2, to give 381 mg of the clean expected
product as an orange solid in 86% yield. 1H NMR (300 MHz, DMSO-
yield. 1H NMR (300 MHz, DMSO-d6)
d (ppm): 12.57 (br, 1H), 8.58 (br
s, 1H), 8.49 (dd, J ¼ 1.8, 7.8 Hz, 1H), 8.34 (dd, J ¼ 1.8, 4.8 Hz, 1H), 7.26
(dd, J ¼ 4.8, 7.8 Hz, 1H), 3.96 (t, J ¼ 6.3 Hz, 2H), 3.40 (t, J ¼ 6.3 Hz,
2H). 13C NMR (75 MHz, DMSO-d6)
d (ppm): 192.2, 149.5, 144.8,
135.4, 130.0, 118.7, 118.1, 115.5, 41.3, 40.6.
d6)
d
(ppm): 12.14 (brs, 1H), 8.27 (dd, J ¼ 1.5, 4.7 Hz, 1H), 7.93 (ddd,
4.2.13. 3-(2-Chloroethyl)-1H-pyrrolo[2,3-b]pyridine (10a)
J ¼ 0.6, 1.5, 7.9 Hz, 1H), 7.09 (dd, J ¼ 4.7, 7.9 Hz, 1H), 6.77 (sd,
DME (10 mL) was put in a heat dried and nitrogen purged round
bottom flask and cooled in a ice bath to 0 ꢁC, then aluminium
chloride (685 mg, 5.15 mmol) and lithium aluminium hydride
(1.08 mL, 2.58 mmol, 2.4 mol Lꢀ1) are added slowly. 9a (200 mg,
1.03 mmol) in solution in DME (10 mL) was added and the mixture
was stirred and left to warm up to room temperature then strirred
at room temperature for 18 h. The reaction is quenched with MeOH
(10 mL), concentrated under reduced pressure, mixed with a
saturated sodium hydrogenocarbonate solution and extracted with
EtOAc. The organic layer was washed with a saturated sodium
chloride solution, dried over magnesium sulfate and concentrated
under reduced pressure to give 182 mg of the expected product as
J ¼ 2.0 Hz, 1H), 0.27 (s, 9H). 13C NMR (75 MHz, DMSO-d6)
d (ppm):
148.5, 145.2, 129.1, 119.6, 119.2, 116.9, 107.4, 99.2, 98.0, 0.2 (3C).
4.2.10. 2-Ethynyl-1H-pyrrolo[2,3-b]pyridine (4b)
Compound 3c (331 mg, 1.55 mmol) was dissolved in THF (5 mL)
and tetrabutylammonium fluoride (3.1 mL, 3.10 mmol, 1 M) was
added. The mixture was stirred at room temperature for 2 h. The
reaction mixture was diluted with EtOAc, washed with water and a
saturated sodium chloride solution, dried over magnesium sulfate,
filtered and concentrated. The crude product was purified by
chromatography on silica gel column, cyclohexane/EtOAc 98:2e7:3,
to give 149 mg of the clean expected product as a beige solid in 68%
an orange solid in 98% yield. 1H NMR (300 MHz, DMSO-d6)
d (ppm):
yield. 1H NMR (300 MHz, DMSO-d6)
d
(ppm): 12.20 (br, 1H), 8.28
11.47 (br, 1H), 8.19 (dd, J ¼ 1.5, 4.8 Hz, 1H), 8.01 (dd, J ¼ 1.5, 7.8 Hz,
1H), 7.37 (d, J ¼ 2.4 Hz, 1H), 7.04 (dd, J ¼ 4.8, 7.8 Hz, 1H), 3.86 (t,
J ¼ 7.4 Hz, 2H), 3.15 (t, J ¼ 7.4 Hz, 2H). 13C NMR (75 MHz, DMSO-d6)
(dd, J ¼ 1.6, 4.7 Hz, 1H), 7.95 (dd, J ¼ 1.6, 7.9 Hz, 1H), 7.09 (dd, J ¼ 4.7,
7.9 Hz, 1H), 6.77 (s, 1H), 4.58 (s, 1H). 13C NMR (75 MHz, DMSO-d6)
d
(ppm): 148.5, 145.0, 129.0, 119.6, 118.8, 116.8, 107.0, 85.4, 76.9.
d (ppm): 148.9, 143.0, 127.1, 124.6, 119.6, 115.4, 110.2, 45.5, 28.9.
4.2.11. 2-Chloro-1-(1H-pyrrolo[2,3-b]pyridin-3-yl)ethanone (9a)
In a heat dried and nitrogen purged round bottom flask,
aluminium chloride (3.99 g, 30.0 mmol) and CH2Cl2 (300 mL) were
mixed and stirred at room temperature for 5 min then 1 (714 mg,
6.05 mmol) was added. The mixture was stirred for 1.5 h then
cooled in an ice bath and chloroacetyl chloride (2.39 mL,
30.0 mmol) was added slowly. The reaction mixture was left to
warm up to room temperature and left to stir overnight (16 h) at
room temperature under nitrogen atmosphere. The mixture was
cooled in an ice bath and quenched by MeOH (60 mL). The solvents
were removed under reduced pressure and a saturated sodium
bicarbonate solution (120 mL) was added to the resulting mixture
which was then extracted three times by EtOAc. The gathered
organic layers were washed with a saturated sodium chloride so-
lution, dried over magnesium sulfate, filtered and concentrated
under reduced pressure to give 1.01 g of a beige solid. The solid was
triturated in CH2Cl2 and filtered to give 648 mg of the pure ex-
pected product as a pale beige solid in 55% yield. 1H NMR (300 MHz,
4.2.14. 3-(3-Chloropropyl)-1H-pyrrolo[2,3-b]pyridine (10b)
DME (20 mL) was put in a heat dried and nitrogen purged round
bottom flask and cooled in a ice bath to 0 ꢁC, then aluminium
chloride (798 mg, 6.00 mmol) and lithium aluminium hydride
(1.25 mL, 3.00 mmol, 2.4 mol Lꢀ1) are added slowly. 9b (250 mg,
1.20 mmol) in solution in DME (20 mL) was added and the mixture
was stirred and left to warm up to room temperature then strirred
at room temperature for 20 h. The reaction is quenched with MeOH
(30 mL), concentrated under reduced pressure, mixed with a
saturated sodium hydrogenocarbonate solution and extracted with
EtOAc. The organic layer was washed with a saturated sodium
chloride solution, dried over magnesium sulfate and concentrated
under reduced pressure to give 237 mg of an orange oil. The crude
product was purified by chromatography on silica gel column,
cyclohexane/EtOAc 6:4, to give 218 mg of the expected product as a
pale yellow solid in 94% yield. 1H NMR (300 MHz, DMSO-d6)
d
(ppm): 11.38 (br, 1H), 8.19 (dd, J ¼ 1.5, 4.5 Hz, 1H), 7.95 (dd, J ¼ 1.5,
7.8 Hz, 1H), 7.27 (br s, 1H), 7.03 (dd, J ¼ 4.5, 7.8 Hz, 1H), 3.66 (t,

C.B. Baltus et al. / European Journal of Medicinal Chemistry 108 (2016) 701e719
713
J ¼ 6.5 Hz, 2H), 2.82 (dd, J ¼ 6.9, 7.5 Hz, 2H), 2.03e2.12 (m, 2H). 13
C
4.2.19. 3-Bromo-1H-pyrrolo[2,3-b]pyridine (13)
NMR (75 MHz, DMSO-d6)
d
(ppm): 149.1, 142.9, 126.9, 123.5, 119.7,
In a heat dried and nitrogen purged round bottom flask, 1
(1.00 g, 8.46 mmol) was dissolved in CH2Cl2 (20 mL) and cooled in
an ice bath, then N-bromosuccinimide (1.66 g, 9.31 mmol) was
added. The mixture was left to warm up to room temperature
overnight under nitrogen atmosphere. The reaction mixture was
diluted with CH2Cl2 and washed with a saturated sodium hydro-
genocarbonate solution. The aqueous layer was extracted three
times with EtOAc and the gathered organic layers were washed
with a saturated sodium chloride solution, dried over magnesium
sulfate, filtered and concentrated to give 1.79 g of a brown solid. The
crude product was purified by chromatography on silica gel col-
umn, cyclohexane/EtOAc 1:1, to give 1.56 g of the expected product
as a light brown solid in 93% yield. 1H NMR (300 MHz, DMSO-d6)
115.3, 112.4, 45.5, 33.1, 22.3.
4.2.15. 3-(2-Azidoethyl)-1H-pyrrolo[2,3-b]pyridine (11a)
Compound 10a (178 mg, 0.99 mmol), sodium azide (322 mg,
4.95 mmol), DMF (5 mL) and water (2.5 mL) were mixed and stirred
at 60 ꢁC for 16 h. The mixture was cooled to room temperature,
diluted with water and extracted three times with EtOAc. The
organic layer was washed three times with a saturated sodium
chloride solution, dried over magnesium sulfate, filtered and
concentrated to give 190 mg of a brown oil. The crude product was
purified by chromatography on silica gel column, cyclohexane/
EtOAc 1:1, to give 141 mg of the expected product as a beige solid in
71% yield. 1H NMR (300 MHz, DMSO)
d (ppm): 11.46 (br, 1H), 8.20
d
(ppm): 11.45 (br, 1H), 8.38 (dd, J ¼ 1.2, 5.1 Hz, 1H), 8.06 (dd, J ¼ 1.2,
7.8 Hz, 1H), 7.48 (s, 1H), 7.28 (dd, J ¼ 5.1, 7.8 Hz, 1H). 13C NMR
(dd, J ¼ 1.8, 4.5 Hz, 1H), 8.01 (dd, J ¼ 1.8, 7.8 Hz, 1H), 7.35 (d,
(75 MHz, DMSO-d6)
d (ppm): 145.0, 140.9, 130.2, 125.6, 121.5, 116.3,
J ¼ 2.4 Hz, 1H), 7.04 (dd, J ¼ 4.5, 7.8 Hz, 1H), 3.60 (t, J ¼ 7.0 Hz, 2H),
2.97 (t, J ¼ 7.0 Hz, 2H). 13C NMR (75 MHz, DMSO)
d (ppm): 149.2,
89.9.
143.0, 127.1, 124.3, 119.7, 115.4, 110.2, 51.4, 25.0.
4.2.20. 3-Bromo-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine
(14)
4.2.16. 3-(3-Azidopropyl)-1H-pyrrolo[2,3-b]pyridine (11b)
In a heat dried and nitrogen purged round bottom flask, 13
(175 mg, 0.89 mmol), tetrabutylammonium bromide (10 mg,
0.03 mmol), finely grounded sodium hydroxide (107 mg,
2.67 mmol) and CH2Cl2 (5 mL) were mixed, stirred and cooled
down to 0 ꢁC in an ice bath, then benzene sulfonyl chloride
(0.142 mL, 1.11 mmol) was added slowly. The mixture was left to
warm up to room temperature and was stirred at this temperature
for 1 h. The reaction was hydrolyzed with water (6 mL) and
extracted by CH2Cl2 (twice). The organic layer was washed with
water and a saturated sodium chloride solution, dried over mag-
nesium sulfate and concentrated under reduced pressure to give an
orange solid. The crude product was triturated in pentane, filtered,
washed with pentane and dried to give 294 mg of the expected
product as an orange solid in 98% yield. 1H NMR (300 MHz, CDCl3)
Compound 10b (195 mg, 1.00 mmol), sodium azide (325 mg,
5.00 mmol), DMF (5 mL) and water (2.5 mL) were mixed and stirred
at 60 ꢁC for 16 h. The mixture was cooled to room temperature,
diluted with water and extracted three times with EtOAc. The
organic layer was washed three times with a saturated sodium
chloride solution, dried over magnesium sulfate, filtered and
concentrated to give 198 mg of the expected product as a yellow oil
99% yield. 1H NMR (300 MHz, DMSO-d6)
d (ppm): 11.37 (br,1H), 8.18
(dd, J ¼ 1.5, 4.7 Hz, 1H), 7.94 (dd, J ¼ 1.5, 7.8 Hz, 1H), 7.27 (br s, 1H),
7.03 (dd, J ¼ 4.7, 7.8 Hz, 1H), 3.37 (t, J ¼ 6.9 Hz, 2H), 2.75 (dd, J ¼ 7.2,
7.8 Hz, 2H), 1.85e1.95 (ddd, J ¼ 6.6, 6.9, 7.2, 7.8 Hz, 2H). 13C NMR
(75 MHz DMSO-d6)
d (ppm): 149.1, 142.9, 126.9, 123.4, 119.7, 115.3,
112.7, 50.8, 29.4, 22.3.
d
(ppm): 8.48 (dd, J ¼ 1.5, 4.8 Hz, 1H), 8.24e8.17 (m, 2H), 7.82 (dd,
4.2.17. 2-Azido-1-(1H-pyrrolo[2,3-b]pyridin-3-yl)ethanone (12a)
9a (50 mg, 0.26 mmol), sodium azide (85 mg, 1.30 mmol), DMF
(2 mL) and water (1 mL) were mixed and stirred at room temper-
ature for 36 h. The mixture was diluted with water (20 mL) and
extracted three times by EtOAc. The gathered organic layers were
washed twice with a saturated sodium chloride solution, dried over
magnesium sulfate, filtered and concentrated under reduced
pressure to give 51 mg of the pure expected product as a white solid
J ¼ 1.5, 8.0 Hz, 1H), 7.79 (s, 1H), 7.60 (dd, J ¼ 7.5 Hz, 1H), 7.54e7.46
(m, 2H), 7.27 (dd, J ¼ 4.8, 8.0 Hz, 1H). 13C NMR (75 MHz, CDCl3)
d
(ppm): 146.0 (2C), 138.0, 134.3, 129.2 (2C), 128.6, 128.1 (2C), 125.0,
122.5, 119.5, 95.6.
4.2.21. 3-Methyl-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine
(15)
In a heat dried and nitrogen purged round bottom flask, 14
(1.064 g, 3.16 mmol) and tetrakis triphenylphosphine palladium
(183 mg, 0.16 mmol) were dissolved in anhydrous THF (20 mL) then
trimethylaluminium (3.17 mL, 6.32 mmol, 2.0 mol Lꢀ1) was added
slowly. The mixture was stirred at 60 ꢁC for 16 h then cooled down
to room temperature, diluted with a saturated sodium hydro-
genocarbonate solution and extracted by EtOAc three times. The
organic layers were gathered, washed with a saturated sodium
chloride solution, dried over magnesium sulfate, filtered and
concentrated to give 1.157 g of a red solid. The crude product was
purified by chromatography on silica gel column, cyclohexane/
EtOAc 8:2, to give 782 mg of the expected product as a yellow solid
in 98% yield. 1H NMR (300 MHz, DMSO-d6)
d (ppm): 12.66 (br, 1H),
8.53 (s,1H), 8.47 (dd, J ¼ 1.8, 7.8 Hz,1H), 8.36 (dd, J ¼ 1.8, 4.8 Hz,1H),
7.29 (dd, J ¼ 4.8, 7.8 Hz, 1H), 4.66 (s, 2H). 13C NMR (75 MHz, DMSO-
d6) d (ppm): 189. 8,149.4,145.0,135.2,129.9,118.9, 118.0, 112.7, 54.2.
4.2.18. 3-Azido-1-(1H-pyrrolo[2,3-b]pyridin-3-yl)propan-1-one
(12b)
Compound 9b (238 mg, 1.14 mmol) was dissolved in DMF (5 mL)
then sodium azide (371 mg, 5.70 mmol) in solution in water (2.5 mL)
was added. The mixture was stirred at 60 ꢁC overnight (16 h) then
poured onto ice and extracted three times with EtOAc. The gathered
organic layers were washed twice with a saturated sodium chloride
solution, dried over magnesium sulfate, filtered and concentrated
under reduced pressure to give 282 mg a yellow solid. The crude
product was purified by chromatography on silica gel column,
CH2Cl2/EtOAc 7:3, to give 146 mg of the expected product as a white
in 91% yield. 1H NMR (300 MHz, CDCl3)
d
(ppm): 8.43 (dd, J ¼ 1.5,
4.8 Hz, 1H), 8.20e8.12 (m, 2H), 7.78 (dd, J ¼ 1.5, 7.8 Hz, 1H), 7.55 (dd,
J ¼ 7.1, 7.5 Hz, 1H), 7.50e7.42 (m, 4H), 7.18 (dd, J ¼ 4.8, 7.8 Hz, 1H),
2.25 (sd, J ¼ 1.3 Hz, 3H). 13C NMR (75 MHz, CDCl3)
d (ppm): 144.7
(2C), 138.6, 133.8, 129.0 (2C), 128.0, 127.8 (2C), 124.1, 123.2, 118.6,
115.1, 9.8.
solid in 60% yield. 1H NMR (300 MHz, DMSO-d6)
d (ppm): 12.56 (br,
1H), 8.57 (s, 1H), 8.48 (dd, J ¼ 1.8, 7.8 Hz,1H), 8.33 (dd, J ¼ 1.8, 4.8 Hz,
1H), 7.26 (dd, J ¼ 4.8, 7.8 Hz, 1H), 3.68 (t, J ¼ 6.3 Hz, 2H), 3.22 (t,
4.2.22. 3-(Azidomethyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]
pyridine (16)
J ¼ 6.3 Hz, 2H). 13C NMR (75 MHz, DMSO-d6)
d (ppm): 193.1, 149.4,
144.8, 135.2, 130.0, 118.6, 118.0, 115.3, 46.7, 37.9.
In a heat dried and nitrogen purged round bottom flask, 15

714
C.B. Baltus et al. / European Journal of Medicinal Chemistry 108 (2016) 701e719
(574 mg, 2.11 mmol), N-bromosuccinimide (413 mg, 2.32 mg),
azobisisobutyronitrile (13 mg, 0.08 mmol) and carbon tetrachloride
(10 mL) were mixed and stirred under reflux for 16 h. The reaction
mixture was cooled to room temperature, and concentrated under
reduced pressure. The crude product (brown oil), sodium azide
(686 mg, 10.55 mmol), DMF (15 mL) and water (7.5 mL) were mixed
and stirred at 60 ꢁC for 16 h. The mixture was cooled to room
temperature, diluted with water and extracted three times with
EtOAc. The organic layer was washed three times with a saturated
sodium chloride solution, dried over magnesium sulfate, filtered
and concentrated to give a yellow oil. The crude product was pu-
rified by chromatography on silica gel column, cyclohexane/EtOAc
8:2 to give 377 mg of the expected product as a white solid in 57%
residue was solubilized in EtOAc, washed with a saturated sodium
hydrogenocarbonate solution, water and a saturated sodium chlo-
ride solution, dried over magnesium sulfate, filtered and concen-
trated under reduced pressure to give 113 mg of the expected
product as a pale yellow solid in 94% yield. 1H NMR (300 MHz,
DMSO-d6)
d
(ppm): 11.51 (br, 1H), 8.13 (dd, J ¼ 1.5, 4.7 Hz, 1H), 7.85
(dd, J ¼ 1.5, 7.8 Hz,1H), 7.00 (dd, J ¼ 4.7, 7.8 Hz,1H), 6.29 (s, 1H), 5.28
(t, J ¼ 5.0 Hz, 1H), 4.60 (d, J ¼ 5.0 Hz, 2H). 13C NMR (75 MHz, DMSO-
d6)
d (ppm): 149.2, 142.3, 141.6, 127.9, 120.5, 115.8, 97.5, 57.4.
4.2.26. 2-(Azidomethyl)-1H-pyrrolo[2,3-b]pyridine (20)
In a heat dried and nitrogen purged round bottom flask, 19
(311 mg, 2.10 mmol) and CH2Cl2 (10 mL) were mixed then thionyl
chloride (0.184 mL, 2.52 mmol) was added. The mixture was stirred
at room temperature for 1 h then concentrated under reduced
pressure. The crude product was mixed with sodium azide (683 mg,
10.50 mmol), DMF (10 mL) and water (5 mL). The mixture was
stirred at 60 ꢁC overnight. The reaction mixture was poured onto
ice and extracted three times with EtOAc. The organic layers were
gathered and washed three times with a saturated sodium chloride
solution, dried over magnesium sulfate, filtered and concentrated.
The crude product was purified by chromatography on silica gel
column, cyclohexane/EtOAc 7:3, to give 252 mg of the expected
product as an off white solid in 69%. 1H NMR (300 MHz, DMSO-d6)
yield. 1H NMR (300 MHz, CDCl3)
d
(ppm): 8.48 (dd, J ¼ 1.6, 4.8 Hz,
1H), 8.23e8.16 (m, 2H), 7.91 (dd, J ¼ 1.6, 7.9 Hz,1H), 7.76 (s, 1H), 7.59
(dd, J ¼ 7.5 Hz, 1H), 7.54e7.45 (m, 2H), 7.23 (dd, J ¼ 4.8, 7.9 Hz, 1H),
4.46 (s, 2H). 13C NMR (75 MHz, CDCl3)
d (ppm): 147.4, 145.7, 138.1,
134.2, 129.1 (2C), 128.3, 128.1 (2C), 124.9, 121.6, 119.4, 113.5, 46.2.
4.2.23. 1-(Phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylic
acid (17)
In a heat dried and nitrogen purged tricol round bottom flask, 5
(1.05 g, 4.07 mmol) was solubilized in dry THF (15 mL), cooled
to ꢀ35 ꢁC then lithium diisopropylamide (5.1 mL, 10.16 mmol, 2 M)
was added slowly. The mixture was then cooled to ꢀ55 ꢁC and dry
ice was added slowly. The mixture was left to warm up to room
temperature under stirring overnight. The reaction was quenched
with water and extracted with EtOAC. The aqueous layer was
acidified by the addition of a 6 N hydrochloric acid solution to
pH ¼ 1 and extracted with EtOAC. The latter organic layer was
washed with a saturated sodium chloride solution, dried over
magnesium sulfate, filtered and concentrated under reduced
pressure to give 1.09 g of the expected product as a beige solid in
d
(ppm): 11.86 (br, 1H), 8.21 (dd, J ¼ 1.6, 4.7 Hz, 1H), 7.94 (dd, J ¼ 1.5,
7.8 Hz,1H), 7.06 (dd, J ¼ 4.7, 7.8 Hz,1H), 6.49 (sd, J ¼ 1.9 Hz,1H), 4.58
(s, 2H). 13C NMR (75 MHz, DMSO-d6)
d (ppm): 149.3, 143.5, 134.2,
128.7, 120.2, 116.3, 100.6, 47.6.
4.3. Synthesis of compounds 21e33. General procedure for the
copper-catalyzed [3 þ 2] cycloaddition reaction
4.3.1. 3-(2-(4-(1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-1,2,3-triazol-1-
yl)ethyl)-1H-pyrrolo[2,3-b]pyridine (21)
88% yield. 1H NMR (300 MHz, DMSO-d6)
d (ppm): 13.87 (br, 1H),
8.51 (dd, J ¼ 1.6, 4.7 Hz, 1H), 8.26 (d, J ¼ 7.0 Hz, 2H), 8.13 (dd, J ¼ 1.6,
7.9 Hz, 1H), 7.77 (dd, J ¼ 7.2 Hz, 1H), 7.72e7.65 (m, 2H), 7.39 (dd,
J ¼ 4.7, 7.9 Hz, 1H), 7.28 (s, 1H). 13C NMR (75 MHz, DMSO-d6)
In a heat dried and nitrogen purged round bottom flask, 3a
(58 mg, 0.27 mmol), 11a (50 mg, 0.27 mmol), copper iodide (1.4 mg,
0.03 mmol) and THF (5 mL) were mixed and stirred then tetrabu-
tylammonium fluoride (0.81 mL, 0.81 mmol, 1 M) and diisopro-
pylethylamine (0.092 mL, 0.54 mmol) were added. The mixture was
stirred at 40 ꢁC for 16 h. The reaction mixture was cooled to room
temperature, concentrated and purified by chromatography on
silica gel column, CH2Cl2/MeOH 95:5, to give 125 mg of a brown oil
which was triturated in MeOH/diethylether to give 61 mg of the
expected product as a beige solid in 69% yield. 1H NMR (300 MHz,
d
(ppm): 164.4, 149.5, 147.3, 138.5, 135.1, 133.3, 132.1, 129.8 (2C),
128.3 (2C), 121.1, 120.7, 112.5.
4.2.24. Ethyl 1H-pyrrolo[2,3-b]pyridine-2-carboxylate (18)
Compound 17 (302 mg, 1.00 mmol), EtOH (20 mL) and
concentrated sulfuric acid (0.2 mL) were mixed in a round bottom
flask and stirred under reflux for 20 h. The reaction mixture was
cooled to room temperature and concentrated under reduced
pressure. The residue was solubilized in EtOAc and washed three
times with a saturated sodium hydrogenocarbonate solution,
washed with water and a saturated sodium chloride solution, dried
over magnesium sulfate, filtered and concentrated under reduced
pressure. The crude product was purified by chromatography on
silica gel column, CH2Cl2/EtOAc 8:2, to give 178 mg of the expected
product as a white solid in 94% yield. 1H NMR (300 MHz, DMSO-d6)
DMSO-d6)
d (ppm): 11.87 (br, 1H), 11.42 (br, 1H), 8.47 (s, 1H), 8.37
(dd, J ¼ 1.2, 8.0 Hz, 1H), 8.28 (dd, J ¼ 1.5, 4.7 Hz, 1H), 8.18 (dd, J ¼ 1.5,
4.7 Hz, 1H), 7.99 (dd, J ¼ 1.1, 7.9 Hz, 1H), 7.85 (sd, J ¼ 2.6 Hz, 1H), 7.25
(d, J ¼ 2.4 Hz, 1H), 7.16 (dd, J ¼ 4.7, 8.0 Hz, 1H), 7.03 (dd, J ¼ 4.7,
7.9 Hz, 1H), 4.70 (t, J ¼ 7.3 Hz, 2H), 2.34e2.21 (m, 2H). 13C NMR
(75 MHz, DMSO-d6) d (ppm): 149.0, 148.9, 143.6, 143.0, 142.2, 128.7,
126.9, 124.2, 123.5, 120.2, 119.7, 117.5, 116.5, 115.5, 109.6, 105.7, 50.4,
26.4. HRMS-ESI (m/z): found 330.14636, calcd for
C18H16N7
d
(ppm): 12.49 (br, 1H), 8.41 (dd, J ¼ 1.7, 4.6 Hz, 1H), 8.11 (dd, J ¼ 1.6,
[M þ H]þ 330.14617.
8.0 Hz, 1H), 7.17 (s, 1H), 7.16 (dd, J ¼ 4.6, 8.0 Hz, 1H), 3.34 (q,
Compounds 22e36 were synthesized following the general
procedure for the copper-catalysed cycloaddition reaction (with or
without additives).
J ¼ 7.1 Hz, 2H), 1.34 (t, J ¼ 7.1 Hz, 3H). 13C NMR (75 MHz, DMSO-d6)
d
(ppm): 161.5, 149.3, 147.2, 131.2, 128.3, 119.5, 117.3, 106.9, 61.1, 14.7.
4.2.25. (1H-Pyrrolo[2,3-b]pyridin-2-yl)methanol (19)
4.3.2. 3-(2-(4-Phenyl-1H-1,2,3-triazol-1-yl)ethyl)-1H-pyrrolo[2,3-
b]pyridine (22)
Dimethoxyethane (10 mL) was placed in a dry round bottom
flask under nitrogen atmosphere and cooled to 0 ꢁC. Lithium
aluminium hydride (1.53 mL, 3.66 mmol, 2.4 M) then 18 (348 mg,
1.83 mmol) in solution in dimethoxyethane (10 mL) were added.
The mixture was left to slowly warm up to room temperature under
stirring overnight. The reaction was quenched with MeOH (15 mL)
and the mixture was concentrated under reduced pressure. The
Purification by chromatography on silica gel column, CH2Cl2/
MeOH 98:2, to give 123 mg of the expected product as an off white
solid in 80% yield. 1H NMR (300 MHz, DMSO-d6)
d (ppm): 11.42 (br,
1H), 8.57 (s, 1H), 8.20 (br, 1H), 7.97 (d, J ¼ 7.2 Hz, 1H), 7.80 (d,
J ¼ 7.2 Hz, 2H), 7.44 (dd, J ¼ 7.2 Hz, 1H), 7.32 (dd, J ¼ 7.2 Hz, 1H), 7.24
(d, 1H, J ¼ 2.1 Hz), 7.03 (dd, J ¼ 4.8, 7.8 Hz, 1H), 4.69 (t, J ¼ 7.2 Hz,

C.B. Baltus et al. / European Journal of Medicinal Chemistry 108 (2016) 701e719
715
2H), 3.33 (t, J ¼ 7.2 Hz, 2H). 13C NMR (75 MHz, DMSO-d6)
d
(ppm):
[M þ H]þ 358.14108.
148.9, 145.6, 143.0, 131.3, 129.4 (3C), 128.4, 126.9, 125.5 (2C), 124.3,
121.8, 109.5, 50.6, 26.3. HRMS-ESI (m/z): found 290.14018, calcd for
C
4.3.7. 3-((4-(1H-Pyrrolo[2,3-b]pyridin-3-yl)-1H-1,2,3-triazol-1-yl)
methyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (27)
17H16N5 [M þ H]þ 290.14002.
Purification by chromatography on silica gel column, CH2Cl2/
MeOH 98:2, to give 71 mg of the expected product as a white solid
4.3.3. 2-(4-(1H-Pyrrolo[2,3-b]pyridin-3-yl)-1H-1,2,3-triazol-1-yl)-
1-(1H-pyrrolo[2,3-b]pyridin-3-yl)ethanone (23)
in 68% yield. 1H NMR (300 MHz, DMSO-d6)
d (ppm): 11.88 (br, 1H),
Purification by chromatography on silica gel column, CH2Cl2/
MeOH 9:1e8:2, to give 123 mg of the expected product as a brown
solid not pure. The solid was tritutated in water, filtered, washed
with diethyl ether and dried to give 44 mg of the expected product
as a brown solid in 26% yield. 1H NMR (300 MHz, DMSO-d6)
8.54 (s, 1H), 8.45e8.35 (m, 2H), 8.27 (dd, J ¼ 1.6, 4.8 Hz, 1H), 8.19 (s,
1H), 8.15 (d, J ¼ 7.2 Hz, 2H), 8.02 (dd, J ¼ 1.6, 8.0 Hz, 1H), 7.88 (d,
J ¼ 2.6 Hz, 1H), 7.73 (dd, J ¼ 7.5 Hz, 1H), 7.67e7.59 (m, 2H), 7.33 (dd,
J ¼ 4.8, 8.0, Hz, 1H), 7.16 (dd, J ¼ 4.8, 7.9, Hz, 1H), 5.84 (s, 2H). 13
C
NMR (75 MHz, DMSO-d6)
d (ppm): 149.0, 147.1, 145.7, 143.7, 142.9,
d
(ppm): 12.80 (br, 1H),11.93 (br, 1H), 8.80 (d, J ¼ 3.0 Hz,1H), 8.50 (s,
137.8, 135.4, 130.1 (2C), 129.4, 128.8, 128.1 (2C), 126.5, 123.9, 121.7,
120.2, 120.0, 117.4, 116.5, 114.64, 105.4, 44.8. HRMS-ESI (m/z): found
456.12390, calcd for C23H18N7O2S [M þ H]þ 456.12372.
1H), 8.59e8.42 (m, 2H), 8.42e8.20 (m, 2H), 7.97 (d, J ¼ 2.1 Hz, 1H),
7.30 (dd, J ¼ 4.8, 7.8 Hz,1H), 7.19 (dd, J ¼ 4.2, 7.8 Hz,1H), 6.05 (s, 2H).
13C NMR (75 MHz, DMSO-d6)
d (ppm): 187.1, 149.4, 149.1, 145.2,
143.6, 142.5, 135.7, 134.5, 129.9, 128.8, 123.7, 122.1, 119.0, 118.1, 112.7,
105.7, 55.6. HRMS-ESI (m/z): found 344.12582, calcd for C18H14N7O
[M þ H]þ 344.12543.
4.3.8. 3-(1-((1H-Pyrrolo[2,3-b]pyridin-2-yl)methyl)-1H-1,2,3-
triazol-4-yl)-1H-pyrrolo [2,3-b]pyridine (28)
Purification by chromatography on silica gel column, CH2Cl2/
MeOH 95:5, to give 110 mg of the expected product as an orange
solid not pure. The solid was triturated in water, filtered, washed
with diethyl ether and dried to give 74 mg of the clean expected
product as a brown solid in 59% yield. 1H NMR (300 MHz, DMSO-d6)
4.3.4. 2-(4-Phenyl-1H-1,2,3-triazol-1-yl)-1-(1H-pyrrolo[2,3-b]
pyridin-3-yl)ethanone (24)
Purification by chromatography on silica gel column, CH2Cl2/
MeOH 98:2, to give 52 mg of the expected product as a white solid
d
(ppm): 11.96 (br, 1H), 11.89 (br, 1H), 8.52 (s, 1H), 8.44 (dd, J ¼ 1.3,
in 68% yield. 1H NMR (300 MHz, DMSO-d6)
d
(ppm): 12.80 (br, 1H),
7.9 Hz, 1H), 8.34e8.16 (m, 2H), 7.97e7.89 (m, 2H), 7.18 (dd, J ¼ 4.7,
8.79 (sd, J ¼ 3.0 Hz, 1H), 8.61 (s, 1H), 8.43 (dd, J ¼ 1.5, 7.8 Hz, 1H),
8.38 (dd, J ¼ 1.5, 4.8 Hz, 1H), 7.89 (d, J ¼ 7.6 Hz, 2H), 7.48 (dd, J ¼ 7.3,
7.6 Hz, 2H), 7.35 (dd, J ¼ 7.6 Hz, 1H), 7.30 (dd, J ¼ 4.8, 7.8 Hz, 1H),
7.9 Hz,1H), 7.06 (dd, J ¼ 4.7, 7.8 Hz,1H), 6.50 (sd, J ¼ 1.7 Hz,1H), 5.82
(s, 2H). 13C NMR (75 MHz, DMSO-d6)
d (ppm): 149.3, 149.1, 143.6,
143.5, 142.8, 134.3, 128.8, 123.8, 120.3, 117.5, 116.5, 116.4, 105.5,
100.6, 47.2. HRMS-ESI (m/z): found 316.13090, calcd for C17H14N7
[M þ H]þ 316.13052.
6.05 (s, 2H). 13C NMR (75 MHz, DMSO-d6)
d (ppm): 186.9, 149.4,
146.7, 145.2, 135.8, 131.3, 129.9, 129.4 (2C), 128.3, 125.6 (2C), 123.7,
119.0, 118.1, 112.6, 55.7. HRMS-ESI (m/z): found 304.11961, calcd for
C
17H14N5O [M þ H]þ 304.11929.
4.3.9. 2-(1-(2-(1H-Pyrrolo[2,3-b]pyridin-3-yl)ethyl)-1H-1,2,3-
triazol-4-yl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (29) and
3-(2-(4-(1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-1,2,3-triazol-1-yl)
ethyl)-1H-pyrrolo[2,3-b]pyridine (30)
4.3.5. 3-(3-(4-(1H-Pyrrolo[2,3-b]pyridin-3-yl)-1H-1,2,3-triazol-1-
yl)propyl)-1H-pyrrolo[2,3-b]pyridine (25)
Purification by chromatography on silica gel column, CH2Cl2/
MeOH 9:1, to give 49 mg of the expected product as a beige solid
not pure. The solid was triturated in THF, filtered, washed with THF
and dried to give 30 mg of the expected product as a brown solid in
Purification by chromatography on silica gel column, CH2Cl2/
MeOH 95:5, to give two fractions. Fraction 1: 87 mg of a brown solid
not pure. The solid was triturated in diethyl ether, filtered, washed
with diethyl ether and dried to give 80 mg of the expected product
29 as a brown solid in 43% yield. Fraction 2: 176 mg of a brown
solid/oil not pure. The solid/oil was triturated in EtOAc/CH2Cl2/
cyclohexanne, filtered, washed with CH2Cl2 and dried to give 51 mg
of the product 30 as a pale pink solid in 39% yield.
38% yield. 1H NMR (300 MHz, DMSO-d6)
d (ppm): 11.88 (br, 1H),
11.39 (br, 1H), 8.52 (s, 1H), 8.43 (dd, J ¼ 1.2, 7.8 Hz, 1H), 8.28 (dd,
J ¼ 1.5, 4.8 Hz, 1H), 8.19 (dd, J ¼ 1.5, 4.5 Hz, 1H), 7.95 (dd, J ¼ 1.2,
7.8 Hz, 1H), 7.89 (d, J ¼ 2.8 Hz, 1H), 7.32 (d, J ¼ 2.1 Hz, 1H), 7.17 (dd,
J ¼ 4.8, 8.1 Hz, 1H), 7.03 (dd, J ¼ 4.8, 7.8 Hz, 1H), 4.46 (t, J ¼ 7.3 Hz,
2H), 2.74 (t, J ¼ 7.3 Hz, 2H), 2.28 (td, J ¼ 7.3 Hz, 2H). 13C NMR
4.3.10. 2-(1-(2-(1H-Pyrrolo[2,3-b]pyridin-3-yl)ethyl)-1H-1,2,3-
(75 MHz, DMSO-d6)
d
(ppm): 149.2, 149.1, 143.6, 142.9, 142.4, 128.8,
triazol-4-yl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (29)
127.0, 123.6 (2C), 120.2, 119.7, 117.5, 116.4, 115.3, 112.5, 105.7, 49.6,
30.7, 22.1. HRMS-ESI (m/z): found 344.16204, calcd for C19H18N7
[M þ H]þ 344.16182.
1H NMR (300 MHz, DMSO-d6)
d (ppm): 12.19 (br, 1H), 8.45 (s,
1H), 8.34 (dd, J ¼ 1.5, 4.8 Hz,1H), 8.29e8.15 (br, 1H), 8.10 (dd, J ¼ 1.5,
7.9 Hz, 1H), 7.99e7.89 (m, 3H), 7.72 (s, 1H), 7.60 (dd, J ¼ 7.5 Hz, 1H),
7.47 (dd, J ¼ 7.5, 8.0 Hz, 2H), 7.30 (dd, J ¼ 4.8, 7.9 Hz, 1H), 7.08 (dd,
J ¼ 4.8, 7.9 Hz, 1H), 6.80 (s, 1H), 4.82 (t, J ¼ 6.8 Hz, 2H), 3.40e3.30
4.3.6. 3-(4-(1H-Pyrrolo[2,3-b]pyridin-3-yl)-1H-1,2,3-triazol-1-yl)-
1-(1H-pyrrolo[2,3-b]pyridin-3-yl)propan-1-one (26)
(m, 2H). 13C NMR (75 MHz, DMSO-d6)
d (ppm): 149.9, 149.4, 147.1,
Purification by chromatography on silica gel column, CH2Cl2/
MeOH 9:1e85:15, to give 93 mg of the expected product as an or-
ange solid not pure. The solid was triturated in CH2Cl2, filtered,
washed with diethyl ether and dried to give 77 mg of the expected
product as a beige solid in 57% yield. 1H NMR (300 MHz, DMSO-d6)
145.3, 143.3, 140.5, 137.8, 135.0, 130.4, 129.9 (2C), 129.2, 128.4, 128.3,
127.6 (2C),124.8,122.8,122.3,119.7,116.0, 97.3, 49.4, 25.7. HRMS-ESI
(m/z): found 470.13994, calcd for C24H20N7O2S [M þ H]þ 470.13937.
4.3.11. 3-(2-(4-(1H-Pyrrolo[2,3-b]pyridin-2-yl)-1H-1,2,3-triazol-1-
d
(ppm): 12.56 (br, 1H), 11.87 (br, 1H), 8.58 (s, 1H), 8.50 (s, 1H), 8.47
yl)ethyl)-1H-pyrrolo[2,3-b]pyridine (30)
(dd, J ¼ 1.5, 7.8 Hz, 1H), 8.39 (dd, J ¼ 1.5, 7.8 Hz, 1H), 8.32 (dd, J ¼ 1.5,
4.8 Hz,1H), 8.27 (dd, J ¼ 1.5, 4.8 Hz,1H), 7.88 (sd, J ¼ 2.8 Hz,1H), 7.25
(dd, J ¼ 4.8, 7.8 Hz, 1H), 7.16 (dd, J ¼ 4.2, 7.8 Hz, 1H), 4.84e4.74 (m,
1H NMR (300 MHz, DMSO-d6)
d (ppm): 12.13 (br, 1H), 11.43 (br,
1H), 8.41 (s, 1H), 8.19 (dd, J ¼ 1.5, 4.8 Hz, 2H), 7.97 (dd, J ¼ 1.5, 7.9 Hz,
1H), 7.93 (dd, J ¼ 1.5, 7.9 Hz, 1H), 7.24 (sd, J ¼ 2.4 Hz, 1H), 7.09e7.00
(m, 2H), 6.78 (sd, J ¼ 2.0 Hz, 1H), 4.75 (t, J ¼ 7.0 Hz, 2H), 3.39e3.29
2H), 3.72e3.60 (m, 2H). 13C NMR (75 MHz, DMSO-d6)
d (ppm):
192.5, 149.4, 149.0, 144.8, 143.6, 142.3, 135.3, 130.0, 128.7, 123.6,
120.5, 118.7, 118.0, 117.5, 116.4, 115.2, 105.6, 45.5, (1C in the DMSO
peak). HRMS-ESI (m/z): found 358.14171, calcd for C19H16N7O
(m, 2H). 13C NMR (75 MHz, DMSO-d6)
d (ppm): 149.5, 149.0, 143.2,
143.0, 140.4, 130.6, 128.3, 126.9, 124.4, 122.2, 120.9, 119.6, 116.4,
115.5, 109.4, 97.2, 50.6, 26.4. HRMS-ESI (m/z): found 330.14637,

716
C.B. Baltus et al. / European Journal of Medicinal Chemistry 108 (2016) 701e719
calcd for C18H16N7 [M þ H]þ 330.14617.
(d, J ¼ 7.8 Hz, 1H), 7.26 (dd, J ¼ 4.8, 7.8 Hz, 1H), 7.13e6.99 (m, 1H),
6.81 (s, 1H), 4.83 (t, J ¼ 6.4 Hz, 2H), 3.65 (t, J ¼ 6.4 Hz, 2H). 13C NMR
4.3.12. 2-(4-(1H-Pyrrolo[2,3-b]pyridin-2-yl)-1H-1,2,3-triazol-1-
yl)-1-(1H-pyrrolo [2,3-b]pyridin-3-yl)ethanone (31)
(75 MHz, DMSO-d6) d (ppm): 192.4, 149.4, 144.8, 140.4, 135.7, 135.3,
130.6, 130.0, 122.6, 118.9, 115.1, 97.3, 45.5, 38.8. HRMS-ESI (m/z):
The reaction mixture was cooled to room temperature and
filtered. The solid was washed with THF and dried under reduced
pressure to give 115 mg of the expected product as an off white
found 358.14135, calcd for C19H16N7O [M þ H]þ 358.14108.
4.3.17. 3-((4-(1H-Pyrrolo[2,3-b]pyridin-2-yl)-1H-1,2,3-triazol-1-yl)
methyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (35)
solid in 84% yield. 1H NMR (300 MHz, DMSO-d6)
d (ppm): 12.81 (br,
1H), 12.26 (br, 1H), 8.80 (sd, J ¼ 3.1 Hz, 1H), 8.55 (s, 1H), 8.44 (dd,
J ¼ 1.2, 7.9 Hz, 1H), 8.39 (d, J ¼ 3.9 Hz, 1H), 8.32e8.15 (br, 1H), 7.97
(d, J ¼ 7.9 Hz, 1H), 7.31 (dd, J ¼ 4.8, 7.9 Hz, 1H), 7.15e7.02 (m, 1H),
The reaction mixture was cooled to room temperature and
filtered. The solid was washed with MeOH and diethylether, and
dried to give 72 mg of the expected product as a beige solid in 89%
6.89 (s, 1H), 6.13 (s, 2H). 13C NMR (75 MHz, DMSO-d6)
d
(ppm):
yield. 1H NMR (300 MHz, DMSO-d6)
d (ppm): 12.09 (br, 1H), 8.54 (s,
186.9, 149.4 (2C), 145.2 (2C), 140.5, 135.8 (2C), 130.6, 129.9, 128.3,
124.0 (2C), 119.0, 118.1, 112.6, 97.4, 55.7. HRMS-ESI (m/z): found
344.12578, calcd for C18H14N7O [M þ H]þ 344.12543.
1H), 8.41 (dd, J ¼ 1.2, 4.6 Hz, 1H), 8.23 (s, 1H), 8.21e8.11 (m, 3H),
8.03 (dd, J ¼ 1.4, 7.9 Hz, 1H), 7.93 (d, J ¼ 7.9 Hz, 1H), 7.74 (dd,
J ¼ 7.4 Hz, 1H), 7.64 (dd, J ¼ 7.4, 7.8 Hz, 2H), 7.35 (dd, J ¼ 4.8, 7.9 Hz,
1H), 7.05 (dd, J ¼ 4.8, 7.9 Hz, 1H), 6.82 (sd, J ¼ 1.8 Hz, 1H), 5.90 (s,
4.3.13. 2-(1-(3-(1H-Pyrrolo[2,3-b]pyridin-3-yl)propyl)-1H-1,2,3-
triazol-4-yl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (32) and
3-(3-(4-(1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-1,2,3-triazol-1-yl)
propyl)-1H-pyrrolo[2,3-b]pyridine (33)
2H). 13C NMR (75 MHz, DMSO-d6)
d (ppm): 149.5,147.1,145.8,143.3,
140.9, 137.8, 135.4, 130.3, 130.2 (2C), 129.4, 128.4, 128.1 (2C), 126.8,
122.1, 121.7, 120.9, 120.0, 116.5, 114.2, 97.5, 44.9. HRMS-ESI (m/z):
found 456.12450, calcd for C23H18N7O2S [M þ H]þ 456.12372.
The reaction mixture was cooled to room temperature and
concentrated. The residue was purified by chromatography on silica
gel column, CH2Cl2/MeOH 95:5, to give two fractions. Fraction 1:
146 mg of an orange solid which was triturated in EtOAc/CH2Cl2/
cyclohexane, filtered, washed with water and diethyl ether, and
dried to give 107 mg of the clean expected product 32 as a light
brown solid in 47% yield. Fraction 2: 188 mg of a brown solid/oil
which was triturated in EtOAc/CH2Cl2/cyclohexane, filtered,
washed with water and diethylether, and dried to give 59 mg of the
clean expected product 33 as a light brown solid in 37% yield.
4.3.18. 2-((4-(1H-Pyrrolo[2,3-b]pyridin-2-yl)-1H-1,2,3-triazol-1-yl)
methyl)-1H-pyrrolo[2,3-b]pyridine (36)
The reaction mixture was cooled to room temperature and
concentrated. The residue was purified by chromatography on silica
gel column, CH2Cl2/MeOH 95:5, to give 97 mg of a light brown solid.
The solid was triturated in MeOH and diethylether, filtered and
dried. 64 mg of the expected product was obtained as a light brown
solid in 53% yield. 1H NMR (300 MHz, DMSO)
d (ppm): 12.14 (br,
1H), 11.98 (br, 1H), 8.51 (s, 1H), 8.30e8.12 (br, 2H), 8.00e7.90 (m,
2H), 7.13e7.02 (m, 2H), 6.85 (s, 1H), 6.55 (s, 1H), 5.85 (s, 2H). 13C
4.3.14. 2-(1-(3-(1H-Pyrrolo[2,3-b]pyridin-3-yl)propyl)-1H-1,2,3-
NMR (75 MHz, DMSO) d (ppm): 149.4, 149.3, 143.6, 143.2, 140.7,
triazol-4-yl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (32)
133.7, 130.4, 128.9, 128.3, 122.2 (2C), 116.8, 116.7, 116.5, 101.1, 97.4,
47.4. HRMS-ESI (m/z): found 316.13073, calcd for C17H14N7 [M þ H]þ
316.13052.
1H NMR (300 MHz, DMSO-d6)
d (ppm): 12.19 (br, 1H), 8.56 (s,
1H), 8.37 (dd, J ¼ 1.5, 4.8 Hz, 1H), 8.26e8.15 (br, 1H), 8.12e8.05 (m,
3H), 7.95 (d, J ¼ 7.8 Hz, 1H), 7.79 (s, 1H), 7.70 (dd, J ¼ 7.5 Hz, 1H),
7.66e7.56 (m, 2H), 7.32 (dd, J ¼ 4.8, 7.8 Hz, 1H), 7.07 (dd, J ¼ 4.8,
7.8 Hz, 1H), 6.83 (s, 1H), 4.52 (t, J ¼ 7.0 Hz, 2H), 2.79e2.70 (m, 2H),
4.4. Synthesis of compounds 38e44. General procedure for the
ruthenium-catalyzed [3 þ 2] cycloaddition reaction
2.35e2.22 (m, 2H). 13C NMR (75 MHz, DMSO-d6)
d (ppm): 147.4,
145.2 (2C), 143.2, 140.7, 138.1, 137.1, 135.1, 130.6, 130.0 (2C), 129.2,
128.3, 127.8 (2C), 123.8, 123.0, 122.2, 119.6, 119.0 (2C), 97.3, 49.6,
29.5, 21.7. HRMS-ESI (m/z): found 484.15571, calcd for C25H22N7O2S
[M þ H]þ 484.15502.
4.4.1. 3-(2-(5-(1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-1,2,3-triazol-1-
yl)ethyl)-1H-pyrrolo[2,3-b]pyridine (37)
In a heat dried and nitrogen purged round bottom flask, 11a
(64 mg, 0.34 mmol), 4a (61 mg, 0.43 mmol), Cp*RuCl(PPh3)2
(20 mg, 0.03 mmol) and THF (4 mL) were mixed and stirred under
reflux for 60 h. The reaction mixture was cooled to room temper-
ature, concentrated and purified by chromatography on silica gel
column, CH2Cl2/MeOH 95:5e9:1, to give 46 mg of the expected
product as an orange solid in 41% yield. 1H NMR (300 MHz, DMSO)
4.3.15. 3-(3-(4-(1H-Pyrrolo[2,3-b]pyridin-2-yl)-1H-1,2,3-triazol-1-
yl)propyl)-1H-pyrrolo[2,3-b]pyridine (33)
1H NMR (300 MHz, DMSO-d6)
d (ppm): 12.18 (br, 1H), 11.41 (br,
1H), 8.55 (s, 1H), 8.23e8.17 (m, 2H), 7.97 (dd, J ¼ 1.2, 3.5 Hz, 1H),
7.94 (dd, J ¼ 1.2, 3.5 Hz,1H), 7.33 (sd, J ¼ 2.3 Hz,1H), 7.07 (dd, J ¼ 4.8,
7.9 Hz, 1H), 7.04 (dd, J ¼ 4.8, 7.9 Hz, 1H), 6.83 (sd, J ¼ 2.0 Hz, 1H),
4.50 (t, J ¼ 7.0 Hz, 2H), 2.74 (t, J ¼ 7.2 Hz, 2H), 2.26 (td, J ¼ 7.0, 7.2 Hz,
d
(ppm): 12.22 (br, 1H), 11.36 (br, 1H), 8.31 (dd, J ¼ 1.5, 4.7 Hz, 1H),
8.12 (dd, J ¼ 1.5, 4.7 Hz, 1H), 7.93 (s, 1H), 7.87 (dd, J ¼ 1.2, 7.8 Hz, 1H),
7.68 (d, J ¼ 2.7 Hz, 1H), 7.50 (dd, J ¼ 1.2, 7.8 Hz, 1H), 7.11e7.15 (m,
2H), 6.89 (dd, J ¼ 4.7, 7.8 Hz, 1H), 4.70 (t, J ¼ 7.2 Hz, 2H), 3.20 (t,
2H). 13C NMR (75 MHz, DMSO-d6)
d (ppm): 149.5, 149.2, 143.2,
142.9,140.6,130.6,128.3,127.0,123.6,122.2,121.0,119.7,116.5,115.3,
112.4, 97.3, 49.8, 30.7, 22.1. HRMS-ESI (m/z): found 344.16199, calcd
for C19H18N7 [M þ H]þ 344.16182.
J ¼ 7.2 Hz, 2H). 13C NMR (75 MHz, DMSO)
d (ppm): 148.8, 148.7,
144.2,142.9,132.8,131.6, 127.6, 126.4,126.1,124.2, 119.5,118.3, 116.9,
115.3, 109.5, 100.0, 49.0, 25.9. HRMS-ESI (m/z): found 330.14622,
calcd for C18H16N7 [M þ H]þ 330.14617.
4.3.16. 3-(4-(1H-Pyrrolo[2,3-b]pyridin-2-yl)-1H-1,2,3-triazol-1-
yl)-1-(1H-pyrrolo [2,3-b]pyridin-3-yl)propan-1-one (34)
Compounds 38e44 were synthesized following the general
procedure for the ruthenium-catalysed [3 þ 2] cycloaddition
reaction.
The reaction mixture was cooled to room temperature and
filtered. The filtrated was concentrated and triturated with cyclo-
hexane/EtOAc. The two solids were gathered and washed with
water and diethylether to give 78 mg of the expected product as a
4.4.2. 2-(5-(1H-Pyrrolo[2,3-b]pyridin-3-yl)-1H-1,2,3-triazol-1-yl)-
1-(1H-pyrrolo [2,3-b]pyridin-3-yl)ethan-1-one (38)
The reaction mixture was cooled to room temperature and
filtered. The solid was washed with CH2Cl2, EtOAc and diethylether,
and dried. 106 mg of the expected product was obtained as a brown
brown solid in 57% yield. 1H NMR (300 MHz, DMSO-d6)
d
(ppm):
12.57 (br,1H),12.18 (br,1H), 8.58 (d, J ¼ 2.8 Hz,1H), 8.55 (s,1H), 8.48
(dd, J ¼ 0.8, 7.8 Hz, 1H), 8.40e8.27 (br, 1H), 8.25e8.10 (br, 1H), 7.93

C.B. Baltus et al. / European Journal of Medicinal Chemistry 108 (2016) 701e719
717
solid in 90% yield. 1H NMR (300 MHz, DMSO)
d
(ppm): 12.79 (br,
6.71 (sd, J ¼ 2.0 Hz, 1H), 6.31 (s, 2H). 13C NMR (75 MHz, DMSO)
1H), 12.13 (br, 1H), 8.75 (sd, J ¼ 2.0 Hz, 1H), 8.38 (dd, J ¼ 1.5, 4.2 Hz,
1H), 8.36 (s, 1H), 8.31 (dd, J ¼ 1.2, 4.6 Hz, 1H), 8.15 (s, 1H), 8.13 (d,
J ¼ 7.8 Hz, 1H), 7.69 (sd, J ¼ 2.6 Hz, 1H), 7.26 (dd, J ¼ 5.1, 7.5 Hz, 1H),
7.19 (dd, J ¼ 4.7, 7.8 Hz, 1H), 6.12 (s, 2H). 13C NMR (75 MHz, DMSO)
d (ppm): 186.6, 149.4, 149.3, 145.2, 144.8, 136.0, 133.1, 131.8, 129.9,
129.3, 124.9, 120.5, 119.0, 118.0, 116.8, 112.5, 100.2, 55.3. HRMS-ESI
(m/z): found 344.12580, calcd for C18H14N7O [M þ H]þ 344.12543.
d
(ppm): 187.4, 149.4, 148.7, 145.2, 144.3, 135.9, 132.8, 132.1, 129.9,
4.4.7. 3-(3-(5-(1H-Pyrrolo[2,3-b]pyridin-2-yl)-1H-1,2,3-triazol-1-
yl)propyl)-1H-pyrrolo[2,3-b]pyridine (43)
The reaction mixture was cooled to room temperature and
concentrated. The residu was purified by chromatography on silica
gel column, CH2Cl2/MeOH 95:5, to give 20 mg of the expected
product as a brown solid in 18% yield. 1H NMR (300 MHz, DMSO)
128.0, 125.4, 119.0, 118.2, 118.0, 117.0, 112.7, 100.2, 54.4. HRMS-ESI
(m/z): found 344.12590, calcd for C18H15N7O [M þ H]þ 344.12543.
4.4.3. 3-(3-(5-(1H-Pyrrolo[2,3-b]pyridin-3-yl)-1H-1,2,3-triazol-1-
yl)propyl)-1H-pyrrolo[2,3-b]pyridine (39)
The reaction mixture was cooled to room temperature and
concentrated. The residu was purified by chromatography on silica
gel column, CH2Cl2/MeOH 95:5e9:1, to give 80 mg of the expected
product as an orange solid in 73% yield. 1H NMR (300 MHz, DMSO)
d
(ppm): 12.30 (br, 1H), 11.41 (br, 1H), 8.29 (dd, J ¼ 1.6, 4.7 Hz, 1H),
8.21e8.17 (m, 2H), 7.91e7.83 (m, 2H), 7.29 (sd, J ¼ 2.4 Hz, 1H), 7.12
(dd, J ¼ 4.7, 7.9 Hz, 1H), 6.98 (dd, J ¼ 4.7, 7.9 Hz, 1H), 6.54 (dd,
J ¼ 2.0 Hz, 1H), 4.65 (t, J ¼ 7.3 Hz, 2H), 2.78 (t, J ¼ 7.3 Hz, 2H), 2.19
d
(ppm): 12.29 (br, 1H), 11.30 (br, 1H), 8.34 (dd, J ¼ 1.5, 4.7 Hz, 1H),
(td, J ¼ 7.3 Hz, 2H). 13C NMR (75 MHz, DMSO)
d (ppm): 149.4, 149.2,
8.14 (dd, J ¼ 1.5, 4.7 Hz,1H), 8.03 (dd, J ¼ 1.5, 7.8 Hz,1H), 8.01 (s, 1H),
7.91 (sd, J ¼ 2.7 Hz, 1H), 7.72 (dd, J ¼ 1.2, 7.8 Hz, 1H), 7.16 (dd, J ¼ 4.7,
7.8 Hz, 1H), 7.16 (s, 1H), 6.93 (dd, J ¼ 4.7, 7.8 Hz, 1H), 4.52 (t,
144.8, 142.9, 133.3, 130.2, 129.17, 126.9, 124.7, 123.8, 120.5, 119.7,
116.8, 115.3, 112.3, 100.7, 48.7, 30.1, 22.0. HRMS-ESI (m/z): found
344.16226, calcd for C19H18N7 [M þ H]þ 344.16182.
J ¼ 7.0 Hz, 2H), 2.63 (t, J ¼ 7.4 Hz, 2H), 2.12 (td, J ¼ 7.0, 7.4 Hz, 2H). 13
C
NMR (75 MHz, DMSO)
d
(ppm): 149.1, 148.8, 144.3, 142.8, 132.8,
4.4.8. 3-(5-(1H-Pyrrolo[2,3-b]pyridin-2-yl)-1H-1,2,3-triazol-1-yl)-
1-(1H-pyrrolo [2,3-b]pyridin-3-yl)propan-1-one (44)
131.5, 127.7, 126.7, 126.3, 123.3, 119.5, 118.3, 117.0, 115.2, 112.5, 100.0,
48.1, 30.1, 22.1. HRMS-ESI (m/z): found 344.16184, calcd for
C
The reaction mixture was cooled to room temperature and
concentrated. The residu was purified by chromatography on silica
gel column, CH2Cl2/MeOH 95:5, to give 52 mg of the expected
product as a beige solid in 34% yield. 1H NMR (300 MHz, DMSO)
19H18N7 [M þ H]þ 344.16182.
4.4.4. 3-(5-(1H-Pyrrolo[2,3-b]pyridin-3-yl)-1H-1,2,3-triazol-1-yl)-
1-(1H-pyrrolo [2,3-b]pyridin-3-yl)propan-1-one (40)
The reaction mixture was cooled to room temperature and
concentrated. The residu was purified by chromatography on silica
gel column, CH2Cl2/MeOH 95:5e9:1, to give 24 mg of the expected
product as a yellow solid in 18% yield. 1H NMR (300 MHz, DMSO)
d
(ppm): 12.55 (br,1H),12.36 (br,1H), 8.56 (s,1H), 8.45 (d, J ¼ 7.9 Hz,
1H), 8.36e8.30 (m, 2H), 8.17 (s, 1H), 8.06 (d, J ¼ 7.9 Hz, 1H), 7.24 (dd,
J ¼ 4.8, 7.9 Hz, 1H), 7.15 (dd, J ¼ 4.8, 7.9 Hz, 1H), 7.02 (s, 1H), 4.95 (t,
J ¼ 6.6 Hz, 2H), 3.70 (t, J ¼ 6.6 Hz, 2H). 13C NMR (75 MHz, DMSO)
d
(ppm): 192.3, 149.5, 149.4, 144.8, 135.3, 133.3, 130.7, 130.0, 129.3,
d
(ppm): 12.53 (br, 1H), 12.33 (br, 1H), 8.52 (s, 1H), 8.40 (dd, J ¼ 1.4,
124.8, 120.7, 118.6, 118.1, 117.9, 116.9, 115.0, 101.3, 44.8, 37.8. HRMS-
8.0 Hz, 1H), 8.34 (dd, J ¼ 1.1, 4.6 Hz, 1H), 8.31 (dd, J ¼ 1.2, 4.7 Hz, 1H),
8.08 (dd, J ¼ 1.0, 7.8 Hz, 1H), 8.05 (sd, J ¼ 2.5 Hz, 1H), 8.00 (s, 1H),
7.23 (dd, J ¼ 4.8, 7.9 Hz, 1H), 7.19 (dd, J ¼ 4.8, 8.0 Hz, 1H), 4.78 (t,
J ¼ 6.7 Hz, 2H), 3.64 (t, J ¼ 6.7 Hz, 2H). 13C NMR (75 MHz, DMSO)
ESI (m/z): found 358.14136, calcd for
C
19H16N7O [M þ H]þ
358.14108.
4.5. Cell maintenance and cytotoxicity assays
d
(ppm): 192.5, 149.4, 148.8, 144.7, 144.3, 135.2, 132.5, 131.8, 130.0,
127.8, 126.4, 118.6, 118.4, 118.0, 117.0, 115.1, 100.1, 44.0, 38.1. HRMS-
The cytotoxicity of the studied compounds was determined
using cell lines of different histological origin as described earlier
[46]. Briefly, compounds in three-fold dilutions were added to the
cells in triplicate. The treatment lasted for 72 h, after which Calcein
AM solution was added, and the fluorescence of live cells at
485 nm/538 nm (excitation/emission) was measured with a Fluo-
roskan Ascent microplate reader (Labsystems). IC50 (the drug con-
centration that reduced the number of viable cells to 50%) values
were determined from the doseeresponse curves.
ESI (m/z): found 358.14135, calcd for
C
19H16N7O [M þ H]þ
358.14108.
4.4.5. 3-(2-(5-(1H-Pyrrolo[2,3-b]pyridin-2-yl)-1H-1,2,3-triazol-1-
yl)ethyl)-1H-pyrrolo[2,3-b]pyridine (41)
The reaction mixture was cooled to room temperature and
concentrated. The residu was purified by chromatography on silica
gel column, CH2Cl2/MeOH 95:5, to give 64 mg of the expected
product as a brown solid in 62% yield. 1H NMR (300 MHz, DMSO)
d
(ppm): 12.30 (br, 1H), 11.41 (br, 1H), 8.31 (dd, J ¼ 1.6, 4.7 Hz, 1H),
4.6. One-step cellular caspase-3/7 activity assay
8.14 (dd, J ¼ 1.6, 4.7 Hz,1H), 8.13 (s, 1H), 8.00 (dd, J ¼ 1.6, 7.9 Hz, 1H),
7.73 (dd, J ¼ 1.2, 7.9 Hz, 1H), 7.21 (sd, J ¼ 2.4 Hz, 1H), 7.14 (dd, J ¼ 4.7,
7.9 Hz, 1H), 6.94 (dd, J ¼ 4.6, 7.9 Hz, 1H), 6.77 (s, 1H), 4.89 (t,
J ¼ 7.2 Hz, 2H), 3.26 (t, J ¼ 7.2 Hz, 2H). 13C NMR (75 MHz, DMSO)
The activity of cellular caspases-3/7 was measured according to
published procedures [45]. Briefly, G361 cells were incubated in a
density of 20,000 cells/well in a 96-well plate overnight. Next day,
d
(ppm): 149.5, 148.9, 144.7, 143.0, 133.5, 130.4, 129.2, 126.6, 124.7,
the compounds were added (the final concentration reached 2 mM)
124.3,120.6,119.6,116.9,115.4,109.2,101.1, 49.8, 25.6. HRMS-ESI (m/
and the cells were incubated for the next 24 h. After incubation, 3x
caspase-3/7 assay buffer (150 mM HEPES pH 7.4, 450 mM NaCl,
150 mM KCl, 30 mM MgCl2, 1.2 mM EGTA, 1.5% Nonidet P40, 0.3%
z): found 330.14608, calcd for C18H16N7 [M þ H]þ 330.14617.
4.4.6. 2-(5-(1H-Pyrrolo[2,3-b]pyridin-2-yl)-1H-1,2,3-triazol-1-yl)-
1-(1H-pyrrolo [2,3-b]pyridin-3-yl)ethan-1-one (42)
CHAPS, 30% sucrose, 30 mM DTT, 3 mM PMSF) with 37.5 mM Ac-
DEVD-AMC as a substrate (SigmaeAldrich) was added to the
wells and plates were incubated at 37 ꢁC. The caspase-3/7 activity
was measured after 4 h using Fluoroskan Ascent microplate reader
(Labsystems) at 346 nm/442 nm (excitation/emission).
The reaction mixture was cooled to room temperature and
filtered. The solid was washed with CH2Cl2, EtOAc and diethylether,
and dried. 113 mg of the expected product was obtained as a brown
solid 97% yield. 1H NMR (300 MHz, DMSO)
d (ppm): 12.83 (br, 1H),
12.37 (br, 1H), 8.84 (sd, J ¼ 2.8 Hz, 1H), 8.37 (s, 1H), 8.35 (dd, J ¼ 1.6,
4.7 Hz,1H), 8.29 (s,1H), 8.27 (dd, J ¼ 1.6, 4.7 Hz,1H), 7.91 (dd, J ¼ 1.5,
7.9 Hz, 1H), 7.26 (dd, J ¼ 5.0, 7.7 Hz, 1H), 7.05 (dd, J ¼ 4.7, 7.9 Hz, 1H),
4.7. Immunoblotting and antibodies
Immunoblotting analysis was performed as described earlier

718
C.B. Baltus et al. / European Journal of Medicinal Chemistry 108 (2016) 701e719
[46]. Briefly, cellular lysates were prepared by harvesting cells in
Laemmli sample buffer. Proteins were separated on SDS-
polyacrylamide gels and electroblotted onto nitrocellulose mem-
branes. After blocking, the membranes were incubated with spe-
cific primary antibodies overnight, washed and then incubated
with peroxidase-conjugated secondary antibodies. Finally, peroxi-
dase activity was detected with ECL þ reagents (AP Biotech) using a
CCD camera LAS-4000 (Fujifilm). Specific antibodies were pur-
4.11. Molecular docking study
3D structures of compounds were prepared with Marvin, a
software used for drawing, displaying, and characterizing chemical
structures, substructures, and reactions [Marvin 14.7.7, 2014,
ChemAxon (http://www.chemaxon.com)]. 3D structures of ligands
were optimized and all hydrogens were added within the Mar-
vinSketch 14.7.7 program. All nonaromatic and nonring bonds were
set as rotatable within AutoDock Tools 1.5.4 program [47]. The
crystal structure for CDK2 with roscovitin (PDBID: 2A4L) was used
as the protein docking template with a docking grid of 16 Å around
the center of the ligand in the crystal structure, which was deleted
prior to docking. Polar hydrogens were added to receptor or
selected for all ligands with the AutoDock Tools program prior to
docking with the Autodock Vina 1.1.2 program [48].
chased from SigmaeAldrich (anti-a-tubulin, clone DM1A;
peroxidase-labeled secondary antibodies), Santa Cruz Biotech-
nology (anti-cyclin B, clone GNS1; anti-Mcl-1, clone S-19; anti-
PARP, clone F-2), Cell Signaling (anti-cyclin A, clone BF683; anti-
Rb, clone 4H1; anti-pRb antibodies phosphorylated at S780 and
S807/811; anti-pPP1a (T320); anti-PP1a; anti-pNPM (T199); anti-
NPM; anti-caspase-7; anti-caspase-3, clone 3G2; anti-Bax, clone
D2E11; anti-Bak, clone D4E4; anti-Bid), eBioscience (anti-5-bromo-
20-deoxyuridine-fluorescein, clone BU20A) or were a generous gift
Author contributions
ꢀ
from prof. Bartek from Danish Cancer Society Research Center,
Copenhagen, Denmark (anti-cyclin D1).
The manuscript was written through contributions of all au-
thors. All authors have given approval to the final version of the
manuscript.
4.8. Kinase inhibition assays
Acknowledgments
CDK2/Cyclin E and CDK1/Cyclin B kinases were produced in Sf9
insect cells via baculoviral infection and purified on a Ni-NTA col-
umn (Qiagen). CDK4/Cyclin D1, CDK5/p35NCK, CDK7/Cyclin H/
MAT1 and CDK9/Cyclin T1 were purchased from ProQinase GmbH.
The kinases were assayed with 1 mg/mL histone H1 (for CDK1/2/5)
or (YSPTSPS)2 KK peptide (for CDK7/CDK9) or RPPTLSPIPHIPR
This work was supported by the European funds for the
economical and regional development (FEDER), the Laboratory of
excellence Labex SYNORG, ANR-11-LABX-0029 and the Medical
Research Fondation (FRM: SPF20130526815). The authors also
gratefully acknowledge support through the projects LO1305 and
LO1204 from the Ministry of Education, Youth and Sports of the
Czech Republic. The work was also supported by the Czech Science
Foundation (grant 15-17282Y) and by student projects
IGA_PrF_2015_027 and IGA_PrF_2015_024 of Palacky University in
Olomouc.
peptide (for CDK4) in the presence of 15/15/15/0.15/1.5/1.5
(for CDK1/2/4/5/7/9), 0.05 Ci [
33P]ATP and of the test compound
in a final volume of 10 L, all in a reaction buffer (60 mM HEPES-
NaOH, pH 7.5, mM MgCl2,
orthovanadate, 1.2 mM DTT, 2.5
were stopped by adding 5
mM ATP
m
g-
m
3
3
mM MnCl2,
3 mM Na-
m
g/50
ml PEG20.000). The reactions
mL of 3% aq. H3PO4. Aliquots were spotted
onto P-81 phosphocellulose (Whatman), washed 3 times with 0.5%
aq. H3PO4 and finally air-dried. Kinase inhibition was quantified
using a FLA-7000 digital image analyzer (Fujifilm). The concen-
tration of the test compounds required to decrease the CDK activity
by 50% was determined from doseeresponse curves and desig-
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.12.023.
nated as IC50
.
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