Synthesis and evaluation of 7-azaindole derivatives bearing benzocycloalkanone motifs as protein kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2020.115468

Protein kinases are important drug targets, especially in the area of oncology. This paper reports the synthesis and biological evaluation of new 7-azaindole derivatives bearing benzocycloalkanone motifs as potential protein kinase inhibitors. Four compounds 8g, 8h, 8i, and 8l were discovered to inhibit cyclin-dependent kinase 9 (CDK9/CyclinT) and/or Haspin kinase in the micromolar to nanomolar range. 8l was identified as the most potent Haspin inhibitor (IC₅₀ = 14 nM), while 8g and 8h acted as dual inhibitors of CDK9/CyclinT and Haspin. These novel compounds constitute a promising starting point for the discovery of dual protein kinase inhibitors that have potential to be developed as anticancer agents, since both CDK9/CyclinT and Haspin are considered to be drug targets in oncology.

Full text of DOI:10.1016/j.bmc.2020.115468

Journal Pre-proofs
Synthesis and evaluation of 7-azaindole derivatives bearing benzocycloalka-
none motifs as protein kinase inhibitors
Malikotsi A. Qhobosheane, Lesetja J. Legoabe, Béatrice Josselin, Stéphane
Bach, Sandrine Ruchaud, Jacobus P. Petzer, Richard M. Beteck
PII:
S0968-0896(20)30282-0
https://doi.org/10.1016/j.bmc.2020.115468
BMC 115468
DOI:
Reference:
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Bioorganic & Medicinal Chemistry
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20 May 2019
22 March 2020
25 March 2020
Please cite this article as: M.A. Qhobosheane, L.J. Legoabe, B. Josselin, S. Bach, S. Ruchaud, J.P. Petzer, R.M.
Beteck, Synthesis and evaluation of 7-azaindole derivatives bearing benzocycloalkanone motifs as protein kinase
inhibitors, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115468
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Synthesis and evaluation of 7-azaindole derivatives bearing
benzocycloalkanone motifs as protein kinase inhibitors.
1,
Malikotsi A. Qhobosheane,1 Lesetja J. Legoabe *, Béatrice Josselin2,3, Stéphane Bach2,3,
Sandrine Ruchaud2, Jacobus P. Petzer1,4, Richard M Beteck1
1
Centre of Excellence for Pharmaceutical Sciences, North-West University, Private Bag X6001,
Potchefstroom 2520, South Africa
2
Sorbonne Université, CNRS, UMR 8227, Integrative Biology of Marine Models, Station Biologique
de Roscoff, CS 90074, 29688 Roscoff Cedex, France
3
Sorbonne Université, CNRS, FR2424, Plateforme de criblage KISSf (Kinase Inhibitor Specialized
Screening facility), Station Biologique de Roscoff, Place Georges Teissier, 29682 Roscoff, France
4
Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001,
Potchefstroom 2520, South Africa
Abstract
Protein kinases are important drug targets, especially in the area of oncology. This paper
reports the synthesis and biological evaluation of new 7-azaindole derivatives bearing
benzocycloalkanone motifs as potential protein kinase inhibitors. Four compounds 8g, 8h, 8i,
and 8l were discovered to inhibit cyclin-dependent kinase 9 (CDK9/CyclinT) and/or Haspin
kinase in the micromolar to nanomolar range. 8l is identified as the most potent Haspin
inhibitor (IC50 = 14 nM), while 8g and 8h are dual inhibitors of CDK9/CyclinT and Haspin.
These novel compounds constitute a promising starting point for the discovery of dual protein
kinase inhibitors that have potential to be developed as anticancer agents, since both
CDK9/CyclinT and Haspin are considered to be drug targets in oncology.
Key words: 7-azaindole, benzocycloalkanone, protein kinase, structure-activity relationship,
Haspin, CDK9/CyclinT, dual inhibitor, oncology.

Introduction
Protein kinases are one of the most crucial target classes for treating human disorders. These
enzymes catalyse the phosphorylation of proteins1 by transferring a γ-phosphate group from
a donor (usually ATP) to tyrosine, serine and threonine residues.2,3 It was discovered that the
human genome encodes for 518 protein kinases. This so-called “kinome” includes 478 typical
and 40 atypical enzymes that are involved in various biological processes within the cell,3,4
including cell fate and regulation of metabolism.5 Protein kinase domains contain a small β-
pleated N-lobe which is connected to a large α-helical C-lobe by a short hinge fragment.6 ATP
binds to the cleft between the N- and the C-terminal lobes of the kinase domain where the
adenine group of ATP is sandwiched between hydrophobic residues and makes contact with
the hinge region via hydrogen bonds.6 Within the ATP pocket lies a residue called the
“gatekeeper”, which controls access to the hydrophobic “back pocket” of the kinase.6
The dysregulation of kinase signaling pathway plays a major role in disease pathophysiology,
and this has been demonstrated in a wide variety of diseases including cancer,
neurodegenerative disorders, cardiovascular disorders, inflammatory disorders and metabolic
diseases.2,4,7 Protein kinases thus represent attractive drug targets for development of novel
drugs.8,9 As reported in literature10 20–33% of drug discovery efforts worldwide are directed at
the protein kinase superfamily. As a result of this intensive screening program, 48 small
molecule protein kinase inhibitors have been approved by US food and drug administration
(FDA) to date, most of which are for oncological indications.10 The majority of approved kinase
inhibitors are used in the treatment of malignancies (36 against solid tumors including
lymphomas and seven against non-solid tumors, e.g. leukemias),10 and a few of them are
used to treat non oncological disorders including rheumatoid arthritis, idiopathic pulmonary
fibrosis, cerebral vasospasm, glaucoma, Crohn’s disease, ulcerative colitis and organ
rejection.6,10 In addition, at least 175 kinase inhibitors are reported to be in clinical trials
worldwide.10
Targeting two or more protein kinases with a single inhibitor, coined as polypharmacology,
represents an effective strategy to cancer treatment.11 According to literature12, selective drugs
against cancer cells should eradicate tumours more specifically, reducing side effects in
normal cells. However, inhibition of a single target often shows transient efficacy due to the
development of resistance. Dual targeting agents are better at overcoming incomplete efficacy
and drug resistance, therefore they provide optimal effects in cancer treatment.12
Protein kinase inhibitors have successfully been discovered in previous studies, however, the
design of selective small-molecule kinase inhibitors proves challenging due of the high

conservation of amino acids in the ATP-binding site of the majority of protein kinases.7,13 High
resolution structural studies may reveal unique kinase active site features, which can be
exploited to develop target specific inhibitors.14 This has proved to be an effective strategy in
developing selective protein kinase inhibitors in previous studies.15 For instance,15 structural
information of the proviral integration site for Moloney murine leukemia virus-1 (PIM1)
complexed with 1 led to the discovery of a selective PIM1 inhibitor, 2 (figure 1). Replacing the
homopiperidine ring of 1, which extends into the specificity area of PIM1 but forms no
significant binding interactions, with a piperazine ring (2) improves selectivity by formation of
additional hydrogen bonds with Asp-128 and Glu-171, while retaining key interactions.15 As a
result, the piperazine ring is reported to be a critical substituent for selectivity and potency in
this regard.15
The 7-azaindole scaffold is well known as a privileged fragment for kinase inhibition, and it
has been confirmed as an excellent hinge-binding element in many protein kinase
inhibitors.16,17 More than 90 human kinases have shown sensitivity to 7-azaindole based
inhibitors including compounds that have been developed to target Janus kinase 3 (JAK3),
aurora kinases (STK) rho-associated, coiled-coil-containing protein kinase 1 (ROCK1).16
7-Azaindole derivatives bearing benzocycloalkanone motifs have been developed in recent
studies and have demonstrated good potencies for protein kinase inhibition. A previous study18
reported 7-azaindole-benzofuranone hybrids as potent and selective inhibitors of the
mammalian target of rapamycin (mTOR). Several inhibitors were synthesised, and the
majority selectively inhibited mTOR with nanomolar potencies.18 The most potent inhibitor (3)
exhibited an IC50 value of 0.46 nM for mTOR. Furthermore, a 4-piperazinyl bearing compound
(4) was cocrystallised with phosphoinositide 3-kinase gamma (PI3Kƴ), which was used as a
substitute in crystallographic studies, owing to its high similarity to mTOR at the ATP binding
sites.18 The predicted binding mode revealed that the 7-N forms a hydrogen bond to Val-2240
in the hinge region of the ATP binding site of the protein.18 The 4-hydroxy group of the
benzofuranone ring of 4 forms a hydrogen bond to Lys-2187. Moreover, the 6-hydroxy group
forms a hydrogen bond to Asp-2195 and the backbone NH of Phe-2358.18 Additionally, a
series of substituted benzofuranone derivatives bearing azaindole moieties was
synthesised.15 The analogue bearing 7-azaindole (5) was among the most potent protein
kinase inhibitors reported in this study. Although it was non-selective, 5 inhibited PIM1, death-
associated protein kinase 1 (DAPK1), glycogen synthase kinase 3β (GSK3β), protein kinase
C α (PKCα), polycystic kidney disease 2 (PKD2) gene and ROCK1 with nanomolar
potencies.15

O
O
OH
O
O
O
N
N
N
NH
N
N
H
H
1 (PIM1 IC50 = 410 nM)
2 (PIM1 IC50 = 3 nM)
HO
HO
N
N
O
O
OH
OH
O
O
N
N
N
N
3 (mTOR IC50 = 0.46 nM)
4 (mTOR IC50 = 0.94 nM)
O
O
O
N
NH
N
N
H
5 (PIM1 IC50 = 2 nM)
Figure 1: 7-azaindole derivatives bearing benzocycloalkanone motifs reported in previous
studies.15,18
In the present study, we synthesised thirteen 7-azaindole derivatives bearing various
benzocycloalkanone motifs at position C3. Among others, α-tetralone and 1-indanone were
considered. The benzocycloalkanones were further substituted with polar and non-polar
functional groups to provide a wide range of compounds with varying activities.

Results and discussion
Chemistry
The target compounds were prepared following the synthetic route outlined in table 1. As key
intermediate, 7 was synthesised from 6 in a yield of 38%, employing the Duff reaction as
described in the experimental section.19,20 Formation of 7 was confirmed by 13C NMR analysis;
the signal at 185.8 is representative of a carbonyl carbon, and confirms successful formylation
of 6. The test compounds (8a-l) were subsequently synthesised by introducing a variety of
commercial benzocycloalkanones to 7 via an acid catalysed aldol condensation.21
Table 1: Synthesis of 7-azaindole derivatives bearing benzocycloalkanone motifs (8a-l)
O
O
O
R
R
(X)
(X)
a
b
N
H
N
H
N
H
N
N
N
6
7
8a-l: R = NH2, OH, OCH3
X = O, CH2, (CH2)2, CH2O, CH2S,
Compound
X
R
Yield (%)a
7
-
-
38
8a
8b
8c
8d
8e
8f
(CH2)2
CH2
6- OCH3
74
73
90
86
45
48
72
61
62
95
95
97
5- OCH3
(CH2)2
(CH2)2
CH2O
CH2S
(CH2)2
(CH2)2
CH2
6,7-di OCH3
7- OCH3
-
-
8g
8h
8i
-
6- OH
5,6-di OCH3
5,6-dioxole
-
8j
CH2
8k
8l
CH2
O
6- OH
a Isolated yields. Reaction conditions: (a) AcOH, H2O, hexamethylenetetramine, 120 ˚C, 1h;
(b) HCl/MeOH, 100 ˚C, 6h

Biological results
In an effort to discover new protein kinase inhibitors, we synthesised 12 novel compounds and
screened them against a small panel of disease-relevant protein kinases including various
cyclin-dependent kinases (CDKs): HsCDK2/CyclinA, HsCDK5/p25, HsCDK9/CyclinT,
HsHaspin, human Proto-oncogene serine/threonine-protein kinase PIM1, porcine casein
kinase 1 δ/ε (SscCK1δ/ε), LmCK1 (from the intracellular parasite Leishmania major) and
porcine Glycogen-Synthase Kinase 3 (SscGSK3α/β). All compounds were initially tested at
concentrations of 1 µM (table 2) and 10 µM (table 2a in supplementary data). Compounds that
inhibited more than 70% of the kinase activity at 1 µM inhibitor concentration were considered
active and were further tested over a wide range of concentrations. From the data obtained,
sigmoidal plots of kinase activity versus inhibitor concentration were constructed. As example,
the curve obtained for compound 8l is reported in figure 2. The IC50 values of the active
compounds were determined from these plots, and the results are reported in table 3.
Figure 2: Sigmoidal dose response curve of kinase activity versus the inhibitor concentration
for the inhibition of Haspin by 8l (IC50 = 14 nM). The ATP concentration used in the kinase
assays was 10 µmol/L. Values are means, n=2.
Four compounds showed promising activity for Haspin and CDK9/CyclinT (8g, 8h, 8i and 8l),
with IC50 values in the micromolar to nanomolar range.

Haspin is a serine/threonine kinase overexpressed in cancer cells,24 that specifically
phosphorylates histone H3 at threonine 3 during mitosis.22,23 As a result, inhibition of Haspin
activity is considered as a promising strategy for cancer treatment.24
Moreover, CDK9/CyclinT is the catalytic subunit of positive transcription elongation factor b
(p-TEFb) that has the ability to phosphorylate the C-terminal domain subunit of RNA
polymerase II, and reach RNA transcription elongation.25 In recent years, CDK9/CyclinT has
emerged as a druggable target for the development of cancer therapeutics.26 Inhibition of
CDK9/CyclinT associated with blocking RNA synthesis can lead to a reduction in levels of
short-lived pro-survival proteins and the induction of apoptosis, which provides an effective
way to control tumour growth.27
The 7-azaindole derivatives showed varied degrees of activity depending on the nature of the
substituents attached. While most compounds in this series were inactive against all tested
kinases, compounds 8i, 8g, 8h and 8l showed promising activity as inhibitors of Haspin and/or
CDK9/CyclinT. 8l was discovered as the most potent inhibitor (IC50 = 14 nM), and it displayed
high selectivity for Haspin. Moreover, the structurally similar analogue 8i also selectively
inhibited Haspin, with lower enzymatic potency (IC50 = 3305 nM) owing to the steric bulk of the
5,6-dimethoxy substituent on the indanone ring. The high potency of compounds 8h (IC50 =
77 nM) and 8l could be ascribed to the 6-hydroxy substituent attached to α-tetralone and 3-
coumaranone rings of 8h and 8l respectively, which may likely form hydrogen bonds with the
binding site amino acids, as seen in figure 3 and figure 4.
Furthermore, the bulkier α-tetralone analogues 8g and 8h are noteworthy as dual inhibitors of
CDK9/CyclinT and Haspin, contrary to the corresponding 1-indanone and 3-coumaranone
derivatives (8i and 8l), and they possess varying specificities for both kinases. The analogue
bearing α-tetralone with a 6-hydroxy substituent (8h) was more potent for Haspin than its
unsubstituted counterpart (8g, IC50 = 1321 nM). In addition, replacing the coumaranone ring
of 8l with α-tetralone, as exemplified by 8g and 8h created analogues with improved
CDK9/CyclinT potency (IC50 = 424 nM and 495 nM, respectively). The additional CH2 present
in tetralones could be actively involved in the orientation of the inhibitors in the binding pocket
of CDK9/CyclinT. Interestingly, 8g and 8h displayed similar CDK9/CyclinT potency, which
suggests that the 6-hydroxy substituent of 8h plays no significant role in CDK9/CyclinT activity.
The above observations suggest that α-tetralone may be the most optimal substituent for dual
inhibition of CDK9/CyclinT and Haspin among the compounds of this study. Furthermore,
conjugating coumaranone and 7-azaindole is a suitable strategy for developing potent and

selective Haspin inhibitors. It may therefore be concluded that substitution of the 7-azaindole
moiety with appropriate benzocycloalkanone rings is necessary for CDK9/CyclinT and Haspin
inhibition.
Molecular modelling
To gain insight into potential binding modes and interactions of the newly synthesized 7-
azaindole derivatives, the most potent haspin inhibitor, compound 8l, was docked into the
active sites of haspin and CDK9/CyclinT, respectively. For this purpose, the Discovery Studio
3.1 modelling software (Accelrys) was used, and molecular docking was carried out according
to a modification of a previously reported protocol with the CDOCKER application of the
Discovery Studio.28 The structures of Haspin co-crystallised with 5-iodotubercidin (PDB entry:
6G34)29 and CDK9/CyclinT co-crystallised with CAN508 (PDB entry: 3TN8)30 were used.
These models were selected based on the high resolution of the crystallographic structures.
The pKa values and protonation states of the models were calculated, followed by energy
minimization, removal of crystal waters and co-crystallised ligands. The structures of the
ligands were constructed in Discovery Studio, and after docking with CDOCKER, the docking
orientations were refined using in situ ligand minimization. For each ligand, the highest ranked
orientation was selected among the ten solutions generated.
The accuracy of the docking procedure was evaluated by redocking the co-crystallised
ligands, 5-iodotubercidin and CAN508, into the active sites of haspin and CDK9/CyclinT,
respectively. The root mean square deviation (RMSD) of the docked orientation from the
position of the co-crystallised ligand was subsequently calculated. The best ranked
orientations of 5-iodotubercidin and CAN508 exhibited RMSD values of 0.656 Å and 1.07 Å,
respectively, from the position of the co-crystallized ligand (Fig. 3 and 4). RMSD values <1.5
Å are considered to be successful, and this protocol was thus deemed appropriate for docking
experiments with haspin and CDK9/CyclinT.31 For the docking of 8l into the active site of
haspin (Fig. 3), the highest ranked solution shows that the 7-azaindole moiety of 8l binds in
the similar position as the pyrrolo[2,3-d]pyrimidine cycle of 5-iodotubercidin. Hydrogen
bonding occurs between the benzofuranone moiety and Glu-492 and Lys-584, and between
the 7-azaindole moiety and Gly-608 and Gly-609. For 5-iodotubercidin, hydrogen bonding
occurs between the ribofuranosyl and Asp-611 and Gly-653, and between the pyrrolo[2,3-
d]pyrimidine cycle and Glu-606 and Gly-608. π-π stacking with Phe-607 is likely an important
stabilizing interaction and occurs for both ligands. Based on the similarity between the binding
modes and interactions of 8l and 5-iodotubercidin it may be proposed that active haspin
inhibitors should contain an aromatic nitrogen-containing heterocycle (for hydrogen bonding

and π stacking) as well as an oxygen rich side chain that can undergo hydrogen bond
interactions towards the entrance of the active site.
For the docking of 8l into the active site of CDK9/CyclinT (Fig. 4), the highest ranked solution
shows that the benzofuranone moiety of 8l binds in the same position as the 4-hydroxyphenyl
of CAN508 where hydrogen bonding occurs with Lys-48 and the side chain of Asp-167. In this
region CAN508 is also stabilized by hydrogen bonding to Lys-48 as well as Glu-66 and the
backbone nitrogen of Phe168.30 The 7-azaindole moiety of 8l binds in a region distinct from
the pyrazole of CAN508 and is hydrogen bonded to Thr-29. Interestingly, 8g, the most potent
CDK9/CyclinT inhibitor, exhibits a reversed binding orientation to 8l, which suggests that the
hydrogen bonding by the hydroxyl group of 8l plays an important role in determining the
orientation of binding in the active site. Although the molecular basis for the higher
CDK9/CyclinT inhibition by 8g versus 8l is not apparent, the difference between the binding
of 8l in haspin compared to CDK9/CyclinT might arise as a result of stronger interaction
network between 8l and haspin, than that between 8l and CDK9/CyclinT.
.
A
B

Figure 3: The docked binding orientations of 5-iodotubercidin (cyan) compared to the
orientation of the co-crystallized ligand (magenta) in the X-ray crystal structure of haspin (A),
as well as selected interactions formed by 8l (green) in the haspin active site (B). (Cut-off
values for hydrogen bonding, 3.4 Å; π-π stacking, 6 Å)
A
B
C
Figure 4: The docked binding orientations of CAN508 (cyan) compared to the orientation of
the co-crystallized ligand (magenta) in the X-ray crystal structure of CDK9/CyclinT (A), as well
as selected interactions formed by 8l (green) and 8g (orange) in the CDK9/CyclinT active site
(B and C, respectively). (Cut-off value for hydrogen bonding, 3.4 Å)

NH2
I
N
N
N
H2N
HO
O
N
HO
N
N
OH
NH
HO
H2N
5-iodotubercidin
CAN508
Figure 5: Structures of 5- iodotubercidin and CAN508

Table 2: Percentage of kinase inhibition by compounds 7 and 8a-l. The table displays the
remaining activities detected after treatment with 1 µM of the tested compounds. 100% of
residual activity is measured in the absence of inhibitor. The ATP concentration used in the
kinase assays was 10 µmol/L (values are given as means, n=2). Kinases are from human
origin (Homo sapiens) unless specified: Ssc, Sus scrofa; Lm, Leishmania major.
Compoun
d
% residual activity measured at 1 µM of the test compounds
CDK2/Cycli CDK5/p2 CDK9/Cyclin HASPI
PIM
1
Ssc_CK1δ Ssc_GSK3α/ Lm_CK
/ε
n A
5
T
N
β
1
7
≥100
≥100
59
93
75
35
84
≥10 61
0
≥10 83
0
91
81
36
92
≥100
66
66
42
37
49
42
45
35
61
57
79
83
76
96
96
84
64
90
71
89
70
8a
8b
8c
8d
8e
8f
84
86
56
53
70
87
56
56
11
16
59
59
49
46
≥100
≥100
≥100
≥100
≥100
70
≥100
≥100
≥100
77
99
84
≥10 86
0
≥100
81
94
97
97
98
72
92
80
80
76
66
77
81
8g
8h
8i
85
33
94
89
16
≥100
≥100
82
≥100
89
21
≥10 68
0
8j
36
90
91
73
77
79
64
8k
8l
98
36
69
87
12

Table 3: Dose-dependent effect of 7-azaindole derivatives (8g-l) on the inhibition activity of
human CDK9/CyclinT and Haspin protein kinases.
a
Compound
Structure
IC50
Hs_CDK9/CyclinT Hs_HASPIN
8g
424
495
NAc
NAc
1321
O
N
N
H
8h
77
OH
O
N
H
N
O
O
8i
3305
O
N
H
N
O
8l
14
OH
O
N
N
H
aIC50 values are reported in nM. Values are given as means, n=2.
bATP concentration used in the kinase assays was 10 µmol/L.
cNo activity observed at a maximal tested concentration of 10 µM.
Conclusion
The current study evaluated the 7-azaindole derivatives bearing benzocycloalkanone moieties
as potential protein kinase inhibitors. The most potent inhibitor among the evaluated
compounds (8l) inhibited the human Haspin with an IC50 of 14 nM. We have identified two
compounds (8g and 8h) as dual inhibitors of Haspin and CDK9/CyclinT, with IC50 values in
the micromolar range. None of the tested compounds showed any activity against the other
six protein kinases. Because of their ability to inhibit both Haspin and CDK9/CyclinT with high
potencies, it may be concluded that 7-azaindole derivatives synthesised in this study are good
leads for the future development of Haspin and CDK9/CyclinT inhibitors, and potential
anticancer drugs.

Experimental section
Chemicals and instrumentation
Unless otherwise noted, all starting materials and reagents were obtained from Sigma-Aldrich
or AK Scientific and used without further purification. Proton (1H) and carbon (13C) nuclear
magnetic resonance (NMR) spectra were recorded on a Bruker Avance III 600 spectrometer
at frequencies of 600 MHz and 151 MHz, respectively, with DMSO-d6 serving as NMR solvent.
All chemical shifts are reported in parts per million (δ) and were referenced to the residual
solvent signal (DMSO-d6: 2.50 and 39.52 ppm for 1H and 13C, respectively). Spin multiplicities
are given as s (singlet), d (doublet), dd (doublet of doublets), t (triplet) and m (multiplet). High
resolution mass spectra (HRMS) were recorded on a Bruker micrOTOF-Q II mass
spectrometer in atmospheric-pressure chemical ionisation (APCI) mode.
Chemical purities were determined by HPLC (Agilent 1200 system) with a Venusil XBP C18
column (4.60 × 150 mm, 5 μm) and a mobile phase of 30% acetonitrile and 70% MilliQ water
at a flow rate of 1 mL/min. At the start of each injection, a solvent gradient program was
initiated by linearly increasing the percentage acetonitrile to 85% over a period of 5 min. Each
run lasted 15 min, 20 µL of the test compounds in acetonitrile (1 mM) was injected into the
HPLC system and the eluent was monitored at wavelengths of 210 and 254 nm. The melting
points (mp) were determined with a Buchi B-545 melting point apparatus and are uncorrected.
Thin layer chromatography (TLC) was carried out using silica gel 60 F254 pre-coated
aluminium sheets (0.25 mm, Merck), while column chromatography was carried out with silica
gel 60 (Fluka, particle size 0.063–0.200 mm). Full-length kinases were used unless specified.
Peptide substrates were obtained from Proteogenix (Schiltigheim, France).
Synthesis of 1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde (7)
To a solution of 7-azaindole (10.0 g, 85 mmol) in water/acetic acid (2:1 v/v) (100 ml/50 ml)
was added hexamethylenetetramine (HMTA) (23.7 g, 169 mmol) and the reaction was refluxed
at 100-120 ˚C for 1h. The work up was done by cooling the reaction mixture on an ice-bath,
and the resulting precipitate was collected by filtration and dried.19,20
O
N
N
H

1
Yield: 38%; white solid; mp 199-212 °C (reported mp 194-196 °C32). H NMR (600 MHz,
DMSO) δ 12.71 (s, 1H), 9.93 (s, 1H), 8.48 (s, 1H), 8.43 – 8.35 (m, 2H), 7.28 (dd, J = 7.8, 4.7
Hz, 1H).13C NMR (151 MHz, DMSO) δ 185.9, 149.9, 145.3, 139.2, 129.7, 118.9, 117.1, 116.9.
APCI-HRMS m/z: calcld for C8H7N2O (MH+), 147.0553, found 147.0550. Purity (HPLC): 98.4%
General procedure for the synthesis of 7-azaindole analogues
To a solution of (7) 1-(1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde (0.300 g, 2.053 mmol) in 32%
aqueous HCl/methanol (1.5:1 v/v) (4.5 ml/3 ml) was added a selected bicyclic scaffold
(tetralone, indanone, 3-coumaranone, 4-Chromanone and Thiochroman-4-one) (2.053 mmol)
and the reaction was refluxed for at least 6 hours at 60-80 ˚C. The progress of the reaction
was monitored by silica gel TLC with EtOAc as mobile phase. When the reaction reached
completion, the target products were isolated by precipitation with ice-cold water, after which
they were purified by recrystallisation with ethanol.
(E)-2-((1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)-6-methoxy-3,4-dihydronaphthalen-
1(2H)-one (8a)
O
O
N
N
H
Yield: 74%; yellow needles; mp 251-252 °C (ethanol). 1H NMR (600 MHz, DMSO) δ 12.95 (s,
1H), 8.51 (d, J = 7.5 Hz, 1H), 8.44 (s, 1H), 8.04 (s, 1H), 7.94 (s, 2H), 7.39 (s, 1H), 6.92 (s, 2H),
13
3.85 (s, 3H), 3.12 (s, 2H), 2.97 (s, 2H). C NMR (151 MHz, DMSO) δ 185.2, 163.5, 146.2,
144.8, 140.6, 132.7, 131.4, 130.2, 129.5, 127.1, 126.1, 122.5, 116.9, 114.0, 112.8, 111.3,
55.9, 28.3, 27.9. APCI-HRMS m/z: calcld for C19H17N2O2 (MH+), 305.1285, found 305.1278.
Purity (HPLC): 96.8%
(E)-2-((1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)-5-methoxy-2,3-dihydro-1H-inden-1-one
(8b)
O
O
N
N
H

Yield: 73%; yellow powder; mp 281-282 °C (ethanol). 1H NMR (600 MHz, DMSO) δ 12.82 (s,
1H), 8.54 (s, 1H), 8.40 (s, 1H), 8.07 (s, 1H), 7.78 (s, 1H), 7.70 (s, 1H), 7.52 – 6.76 (m, 3H),
3.93 (m, 5H). 13C NMR (151 MHz, DMSO) δ 191.6, 165.1, 146.9, 142.5, 131.4, 129.7, 129.6,
125.6, 122.9, 121.2, 117.2, 115.5, 113.9, 111.4, 110.6, 104.2, 56.2, 33.1. APCI-HRMS m/z:
calcld for C18H15N2O2 (MH+), 291.1128, found 291.1119. Purity (HPLC): 97.7%
(E)-2-((1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)-6,7-dimethoxy-3,4-dihydronaphthalen-
1(2H)-one (8c)
O
O
O
N
N
H
Yield: 90%; yellow powder; mp 292 °C (ethanol). 1H NMR (600 MHz, DMSO) δ 12.88 (s, 1H),
8.48 (d, J = 7.6 Hz, 1H), 8.43 (d, J = 4.7 Hz, 1H), 8.03 (s, 1H), 7.92 (s, 1H), 7.45 (s, 1H), 7.41
13
– 7.31 (m, 1H), 6.95 (s, 1H), 3.87 (s, 3H), 3.81 (s, 3H), 3.12 (s, 2H), 2.93 (s, 2H). C NMR
(151 MHz, DMSO) δ 185.1, 153.7, 148.3, 145.2, 140.9, 138.5, 132.5, 131.1, 129.4, 126.5,
125.9, 122.3, 116.9, 111.2, 111.1, 109.5, 56.3, 55.9, 28.2, 27.7. APCI-HRMS m/z: calcld for
C20H19N2O3 (MH+), 335.1390, found 335.1391. Purity (HPLC): 92.4%
(E)-2-((1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)-7-methoxy-3,4-dihydronaphthalen-
1(2H)-one (8d)
O
O
N
N
H
Yield: 86%; yellow powder; mp 263-264 °C (ethanol).
1H NMR (600 MHz, DMSO) δ 12.64 (s, 1H), 8.38 (d, J = 4.6 Hz, 1H), 8.34 (d, J = 7.8 Hz, 1H),
8.03 (s, 1H), 8.00 (s, 1H), 7.44 (d, J = 2.5 Hz, 1H), 7.37 – 7.22 (m, 2H), 7.15 (dd, J = 8.2, 2.7
Hz, 1H), 3.82 (s, 3H), 3.12 (t, J = 5.7 Hz, 2H), 2.92 (t, J = 6.2 Hz, 2H). 13C NMR (151 MHz,
DMSO) δ 186.2, 158.6, 147.4, 142.9, 135.9, 134.7, 131.4, 130.2, 129.34, 129.04, 127.7,

121.1, 120.7, 117.1, 110.80, 110.69, 55.7, 28.1, 27.1. APCI-HRMS m/z: calcld for C19H17N2O2
(MH+), 305.1285, found 305.1268. Purity (HPLC): 96.3%
(E/Z)-3-((1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)chroman-4-one (8e)
O
O
N
N
H
Yield: 45%; yellow needles; mp 231-233 °C (ethanol). 1H NMR (600 MHz, DMSO) δ 12.67 (s,
1H), 8.57 – 8.21 (m, 2H), 8.02 (s, 1H), 7.98 – 7.81 (m, 2H), 7.58 (t, J = 7.7 Hz, 1H), 7.27 (dd,
J = 7.5, 4.7 Hz, 1H), 7.20 – 6.96 (m, 2H), 5.50 (s, 2H). 13C NMR (151 MHz, DMSO) δ 180.7,
160.9, 148.6, 144.2, 136.1, 130.5, 128.1, 127.53, 127.47, 126.2, 122.17, 122.10, 120.4, 118.2,
117.5, 109.6, 68.9. APCI-HRMS m/z: calcld for C17H13N2O2 (MH+), 277.0972, found 277.0962.
Purity (HPLC): 96.5%
(E)-3-((1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)thiochroman-4-one (8f)
O
S
N
N
H
Yield: 48%; yellow plates; mp 238 °C (ethanol). 1H NMR (600 MHz, DMSO) δ 12.89 (s, 1H),
8.41 (d, J = 6.1 Hz, 2H), 8.21 (s, 1H), 8.05 (d, J = 7.0 Hz, 1H), 7.92 (s, 1H), 7.58 – 7.45 (m,
1H), 7.41 (d, J = 7.3 Hz, 1H), 7.38 – 7.24 (m, 2H), 4.38 (s, 2H). 13C NMR (151 MHz, DMSO)
δ 184.8, 146.5, 142.3, 140.8, 133.5, 132.7, 130.11, 130.07, 129.9, 128.60, 128.43, 128.28,
126.3, 121.5, 117.3, 110.2, 29.8. APCI-HRMS m/z: calcld for C17H13N2OS (MH+), 293.0743,
found 293.0734. Purity (HPLC): 96.3%

(E)-2-((1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)-3,4-dihydronaphthalen-1(2H)-one (8g)
O
N
N
H
Yield: 72%; yellow needles; mp 257 °C (ethanol). 1H NMR (600 MHz, DMSO) δ 13.06 (s, 1H),
8.56 (d, J = 7.6 Hz, 1H), 8.45 (d, J = 4.5 Hz, 1H), 8.07 (s, 1H), 8.04 – 7.87 (m, 2H), 7.56 (t, J
= 7.1 Hz, 1H), 7.49 – 7.26 (m, 3H), 3.14 (s, 2H), 2.99 (s, 2H). 13C NMR (151 MHz, DMSO) δ
186.4, 144.5, 143.6, 140.3, 133.70, 133.61, 132.6, 131.8, 129.9, 128.9, 127.66, 127.37, 126.8,
122.7, 117.0, 111.3, 27.93, 27.87. APCI-HRMS m/z: calcld for C18H15N2O (MH+), 275.1179,
found 275.1168. Purity (HPLC): 97.3%
(E)-2-((1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)-6-hydroxy-3,4-dihydronaphthalen-
1(2H)-one (8h)
OH
O
N
N
H
Yield: 61%; light orange powder; mp 268-270 °C (ethanol). 1H NMR (600 MHz, DMSO) δ 12.99
(s, 1H), 8.54 (d, J = 7.5 Hz, 1H), 8.45 (s, 1H), 7.96 (d, J = 74.1 Hz, 2H), 7.85 (d, J = 8.4 Hz,
1H), 7.56 – 7.30 (m, 1H), 6.80 (d, J = 8.2 Hz, 1H), 6.72 (s, 1H), 3.17 (s, 1H), 3.10 (s, 2H), 2.89
(s, 2H). 13C NMR (151 MHz, DMSO) δ 185.0, 162.6, 146.2, 144.2, 140.0, 133.2, 131.9, 130.5,
129.5, 125.8, 125.5, 122.8, 116.9, 115.1, 114.4, 111.4, 28.3, 27.9. APCI-HRMS m/z: calcld for
C18H15N2O2 (MH+), 291.1128, found 291.1122. Purity (HPLC): 96.4%

(E)-2-((1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)-5,6-dimethoxy-2,3-dihydro-1H-inden-1-
one (8i)
O
O
O
N
N
H
Yield: 62%; yellow powder; mp 282 °C (ethanol). 1H NMR (600 MHz, DMSO) δ 12.80 (s, 1H),
8.55 (d, J = 7.7 Hz, 1H), 8.40 (d, J = 4.4 Hz, 1H), 8.04 (s, 1H), 7.75 (s, 1H), 7.33 (dd, J = 7.6,
4.9 Hz, 1H), 7.20 (d, J = 8.9 Hz, 2H), 3.91 (m, 6H), 3.84 (s, 2H). 13C NMR (151 MHz, DMSO)
δ 191.9, 155.4, 149.7, 144.5, 142.3, 132.8, 131.3, 129.83, 129.81, 129.45, 122.4, 121.3,
117.2, 111.4, 108.5, 105.0, 56.41, 56.12, 32.7. APCI-HRMS m/z: calcld for C19H17N2O3 (MH+),
321.1234, found 321.1236. Purity (HPLC): 97.7%
(E)-2-((1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)-6-amino-3,4-dihydronaphthalen-1(2H)-
one (8j)
O
O
O
N
N
H
Yield: 95%; yellow powder; mp 301 °C (ethanol). 1H NMR (600 MHz, DMSO) δ 12.82 (s, 1H),
8.53 (d, J = 7.4 Hz, 1H), 8.40 (s, 1H), 8.04 (s, 1H), 7.74 (s, 1H), 7.53 – 7.21 (m, 1H), 7.15 (d,
J = 5.3 Hz, 2H), 6.18 (s, 2H), 3.90 (s, 2H). 13C NMR (151 MHz, DMSO) δ 191.3, 173.2, 153.9,
148.4, 146.9, 142.4, 133.2, 132.5, 129.77, 129.63, 122.7, 117.25, 117.21, 111.3, 106.3,
102.80, 102.3, 32.9. APCI-HRMS m/z: calcld for C18H13N2O3 (MH+), 305.0921, found
305.0904. Purity (HPLC): 95.5%

(E)-2-((1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)-2,3-dihydro-1H-inden-1-one (8k)
O
N
N
H
Yield: 95%; light orange powder; mp 283 °C (ethanol). 1H NMR (600 MHz, DMSO) δ 13.01 (s,
1H), 8.61 (s, 1H), 8.42 (s, 1H), 8.14 (s, 1H), 7.88 (s, 1H), 7.77 (d, J = 5.5 Hz, 1H), 7.68 – 7.57
(m, 2H), 7.47 (s, 1H), 7.37 (s, 1H), 4.04 (s, 2H). 13C NMR (151 MHz, DMSO) δ 191.6, 165.1,
152.6, 142.3, 132.5, 131.9, 129.71, 129.66, 125.67, 125.61, 122.8, 117.2, 115.5, 111.4, 110.6,
56.2, 33.1. APCI-HRMS m/z: calcld for C17H13N2O (MH+), 261.1022, found 261.1012. Purity
(HPLC): 96.9%
(Z)-2-((1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)-6-hydroxybenzofuran-3(2H)-one (8l)
O
OH
O
N
N
H
Yield: 97%; light orange powder; mp 361 °C (ethanol). 1H NMR (600 MHz, DMSO) δ 12.97 (s,
1H), 11.24 (s, 1H), 8.78 (d, J = 7.7 Hz, 1H), 8.43 (d, J = 4.1 Hz, 1H), 8.31 (s, 1H), 7.60 (d, J =
8.3 Hz, 1H), 7.52 – 7.30 (m, 1H), 7.20 (s, 1H), 6.91 (s, 1H), 6.75 (d, J = 8.3 Hz, 1H). 13C NMR
(151 MHz, DMSO) δ 180.9, 167.5, 166.6, 146.3, 145.9, 141.4, 132.6, 131.8, 126.0, 120.9,
117.3, 114.1, 113.3, 108.4, 104.2, 99.2. APCI-HRMS m/z: calcld for C16H11N2O3 (MH+),
279.0764, found 279.0755. Purity (HPLC): 99.0%
Kinase inhibition assay
Kinase enzymatic activities were assayed in 384-well plates using the ADP-GloTM assay kit
(Promega, Madison, WI) according to the recommendations of the manufacturer. This assay
is a luminescent ADP detection assay that provides a homogeneous and high-throughput
screening method to measure kinase activity by quantifying the amount of ADP produced
during a kinase reaction. Briefly, the reactions were carried out in a final volume of 6 µl for 30
min at 30°C in appropriate kinase buffer, with either protein or peptide as substrate in the
presence of 10µM ATP. After that, 6 µl of ADP-GloTM Kinase Reagent was added to stop the
kinase reaction. After an incubation time of 50 min at room temperature (RT), 12 µl of Kinase

Detection Reagent was added for one hour at RT. The transmitted signal was measured using
the Envision (PerkinElmer, Waltham, MA) microplate luminometer and expressed in Relative
Light Unit (RLU). In order to determine the half maximal inhibitory concentration (IC50), the
assays were performed in duplicate in the absence or presence of increasing doses of the
tested compounds. Kinase activities are expressed in % of maximal activity, i.e. measured in
the absence of inhibitor.
Buffer:
(A) 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 25 mM Tris-HCl pH 7.5, 50 µg/ml heparin
Kinases:
HsCDK2/CyclinA (cyclin-dependent kinase-2, human, kindly provided by Dr. A. Echalier-
Glazer, Leicester, UK) was assayed in buffer A with 0.8 µg/µl of histone H1 as substrate;
HsCDK5/p25 (human, recombinant, expressed in bacteria) was assayed in buffer A with 0.8
µg/µl of histone H1 as substrate;
HsCDK9/CyclinT (human, recombinant, expressed by baculovirus in Sf9 insect cells) was
assayed
in
buffer
A
with
0.27
µg/µl
of
the
following
peptide:
YSPTSPSYSPTSPSYSPTSPSKKKK, as substrate;
HsHaspin-kd (human, kinase domain, amino acids 470 to 798, recombinant, expressed in
bacteria) was assayed in buffer A with 0.007 µg/µl of Histone H3 (1-21) peptide
(ARTKQTARKSTGGKAPRKQLA) as substrate;
HsPim-1 (human proto-oncogene, recombinant, expressed in bacteria) was assayed in buffer
A with 0.8 µg/µl of histone H1 (Sigma #H5505) as substrate;
LmCK1 (from Leishmania major, recombinant, expressed in bacteria) was assayed in buffer
A with 0.028 µg/µl of the following peptide: RRKHAAIGSpAYSITA as CK1-specific substrate;
SscCK1δ/ε (casein kinase 1δ/ε, porcine brain, native, affinity purified) was assayed in buffer
A with 0.022 µg/µl of the following peptide: RRKHAAIGSpAYSITA (“Sp” stands for
phosphorylated serine) as CK1-specific substrate;
SscGSK-3α/β (glycogen synthase kinase-3, porcine brain, native, affinity purified) isoforms
were assayed in buffer A with 0.010 µg/µl of GS-1 peptide, a GSK-3-selective substrate
(YRRAAVPPSPSLSRHSSPHQSpEDEEE).
Molecular docking
Modelling calculations were carried out with Discovery Studio 3.1. All applications in Discovery
Studio were set to their default values, and X-ray crystal structures were obtained from the
Protein Data Bank. The pKa values and protonation states of the protein models were firstly

calculated and hydrogen atoms were added to the amino acid residues at pH 7.4. A fixed atom
constraint was applied to the protein backbone, and the models were energy minimized using
the Smart Minimizer algorithm (maximum steps, 50000) with the implicit generalized Born
solvation model with molecular volume. The co-crystallized ligands, waters and the backbone
constraints were removed and the binding sites were identified. The structures of the test
ligands were drawn in Discovery Studio and submitted to the Prepare Ligands protocol. Atom
potential types and partial charges were assigned with the Momany and Rone CHARMm
forcefield, and docking of the ligands was carried out with the CDOCKER algorithm. For this
purpose, ten random ligand conformations were generated, the heating target temperature
was set to 700 K and full potential mode was employed. The docking solutions were finally
refined using in situ ligand minimization with the Smart Minimizer algorithm. Illustrations were
prepared with PyMOL.33
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The NMR and MS spectra were recorded by Daniel Otto and Johan Jordaan of the SASOL
Centre of Chemistry, North-West University. This work is based on the research supported by
the National Research Foundation of South Africa (Grant specific unique reference numbers
(UID) (96135). The authors also thank the Cancéropôle Grand-Ouest (axis: Nature sea
products in cancer treatment), GIS IBiSA (Infrastructures en Biologie Santé et Agronomie,
France), Biogenouest (Western France life science and environment core facilty network) for
supporting KISSf screening facility (Roscoff, France) and “Ligue Contre le Cancer Grand
Ouest” comity CIRGO (districts 29, 35, 22, 56 and 79). The Grant holders acknowledge that
opinions, findings and conclusions or recommendations expressed in any publication
generated by the NRF supported research are that of the authors, and that the NRF accepts
no liability whatsoever in this regard.

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Figures
O
O
OH
O
O
O
N
N
N
NH
N
N
H
H
1 (PIM1 IC50 = 410 nM)
2 (PIM1 IC50 = 3 nM)
HO
HO
N
N
O
O
OH
OH
O
O
N
N
N
N
3 (mTOR IC50 = 0.46 nM)
4 (mTOR IC50 = 0.94 nM)
O
O
O
N
NH
N
N
H
5 (PIM1 IC50 = 2 nM)
Fig. 1. 7-Azaindole benzocycloalkanones with reported protein kinase activity.

Fig. 2. Sigmoidal dose response curve of kinase activity versus the inhibitor concentration for
the inhibition of Haspin by 8l (IC50 = 14 nM).
NH2
I
N
N
N
H2N
HO
O
N
HO
N
N
OH
NH
HO
H2N
5-iodotubercidin
CAN508
Figure 5: Structures of 5- iodotubercidin and CAN508

Graphical abstract
O
N
O
NH
HO
IC50 (Haspin) = 14 nM