Synthesis and biological evaluation of selected 7-azaindole derivatives as CDK9/Cyclin T and Haspin inhibitors

Article abstract of DOI:10.1007/s00044-020-02560-1

The 7-azaindole scaffold attracts attention due to its value in the design of inhibitors of diseases related protein kinases. However, this scaffold has not been evaluated against Haploid germ cell-specific nuclear protein kinase (Haspin). Herein, we report the synthesis of a select set of 7-azaindole derivatives and their evaluation against Haspin. The compounds were also evaluated against CDK9/Cyclin T kinase. The synthesis of 7-azaindole derivatives was achieved through Suzuki coupling using appropriate halogenated 7-azaindole and boronic acids. Seven of the screened compounds exhibited activity as CDK9/Cyclin T and/or Haspin inhibitors in a nanomolar to low micromolar range. The most promising dual inhibiting compound 18c, exhibited an inhibitory potential of 0.206 μM against CDK9/Cyclin T and 0.118 μM against Haspin. The dual inhibition of CDK9/Cyclin T and Haspin could afford a potentially potent antimitotic agent of value in further anticancer studies.

Full text of DOI:10.1007/s00044-020-02560-1

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02560-1
ORIGINAL RESEARCH
Synthesis and biological evaluation of selected 7-azaindole
derivatives as CDK9/Cyclin T and Haspin inhibitors
1
Lianie Pieterse1 Lesetja J. Legoabe1 Richard M. Beteck
Sandrine Ruchaud2
Béatrice Josselin2,3 Stéphane Bach2,3
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Received: 28 March 2020 / Accepted: 7 May 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
The 7-azaindole scaffold attracts attention due to its value in the design of inhibitors of diseases related protein kinases.
However, this scaffold has not been evaluated against Haploid germ cell-specific nuclear protein kinase (Haspin). Herein, we
report the synthesis of a select set of 7-azaindole derivatives and their evaluation against Haspin. The compounds were also
evaluated against CDK9/Cyclin T kinase. The synthesis of 7-azaindole derivatives was achieved through Suzuki coupling
using appropriate halogenated 7-azaindole and boronic acids. Seven of the screened compounds exhibited activity as CDK9/
Cyclin T and/or Haspin inhibitors in a nanomolar to low micromolar range. The most promising dual inhibiting compound
18c, exhibited an inhibitory potential of 0.206 µM against CDK9/Cyclin T and 0.118 µM against Haspin. The dual inhibition
of CDK9/Cyclin T and Haspin could afford a potentially potent antimitotic agent of value in further anticancer studies.
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Keywords Protein kinases 7-Azaindole anticancer CDK9/ Cyclin T Haspin
Introduction
phosphate donors such as ATP, to receptor substrates that
range from small molecules to lipids and proteins (Lawson
et al. 2016). Signal transduction, in turn is linked to cell
growth, metabolism, differentiation and apoptosis (Liao
2007). Therefore, any dysregulation of PKs (such as over-
expression) could result in serious ramifications, the likes of
which include, cell transformation and cancer, inflamma-
tion, diabetes and neurodegenerative diseases like Alzhei-
mer’s disease (Flight 2013; Huse and Kuriyan 2002; Noh
and King 2007). Hence, PKs have become appealing targets
for drug discovery, even being touted as the second most
important drug targets after G-protein-coupled receptors
(Cohen 2002).
The majority of the manifold cellular processes in the
human body are vastly dependent on a superfamily of
homologous proteins, known as protein kinases (PKs)
(Hanks and Hunter 1995; Hunter 1995). The PKs that make
up the human kinome tally up to a total of 518 PKs, thus
accounting for 1.7% of human genes (Manning et al. 2002)
and are integrally linked to the process of protein phos-
phorylation which regulates signal transduction, as these
proteins facilitate the transfer of phosphoryl groups from
This forms the backdrop against which the discovery of
novel PK inhibitors plays out and indeed, a number of
kinase inhibitors are in clinical use or undergoing clinical
trials, like imatinib, for the treatment of chronic myelo-
genous leukaemia (CML) and trastuzumab in breast cancer
(Fedorov et al. 2010). Nevertheless, these inhibitors target
only a slight portion of the kinome and numerous clinically
relevant kinases still remain relatively underexplored
(Duong‐Ly and Peterson 2013).
One such PK is the Haploid germ cell-specific nuclear
protein kinase (Haspin). Haspin has only recently been
added to the ranks of known PKs and is thought to
phosphorylate histone H3 during mitosis (Higgins 2010).
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02560-1) contains supplementary
material, which is available to authorized users.
* Lesetja J. Legoabe
lesetja.legoabe@nwu.ac.za
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 Laboratory (LBI2M), Station Biologique de
Roscoff, CS 90074, 29680 Roscoff Cedex, France
3
Sorbonne Université, CNRS, FR2424, Plateforme de criblage
KISSf (Kinase Inhibitor Specialized Screening facility), Station
Biologique de Roscoff, CS90074, 29680 Roscoff Cedex, France

Medicinal Chemistry Research
It is believed that identification of Haspin inhibitors might
supply valuable candidates for biological studies and
cancer treatment. This belief stems from the following
factors: (i) Haspin is deemed important in the regulation of
mitosis and Haspin RNAi in tumour cell lines thwarts
chromosome alignment and completion of the normal
mitotic process (Dai et al. 2005; Dai et al. 2006). This
suggests that Haspin inhibitors have the potential to act as
novel antimitotic agents with an aptitude for halting the
proliferation of cancer cells (de Cárcer et al. 2007; Schmidt
and Bastians 2007). (ii) Haspin mRNA is predominantly
expressed in proliferative cells and not in non-dividing
cells, making it another attractive target for cancer treat-
ment (Higgins 2001a, b). (iii) Not only is the primary
sequence of Haspin structurally different to that of other
known kinases, but it also does not fit into any of the
known eukaryotic kinase families and humans appear to
have only one Haspin homologue, indicating that the
design of highly selective Haspin inhibitors is probable
(Higgins 2003; Higgins 2001a, b; Kannan et al. 2007).
Thus, designing a study that aims to explore the Haspin
target, may yield valuable novel knowledge in the field of
PK inhibition and treatment of disease.
In general, PK inhibitors are largely designed to bind to
the ATP binding site situated within the deep cleft between
the N- and C-terminal lobes of the kinase catalytic domain.
Binding is generally the product of hydrogen bond inter-
actions with the hinge region that connects the two lobes
(Liao 2007), and as such, the 7-azaindole scaffold (1) has
been highlighted as a prime candidate for targeting PKs
because of the scaffolds enhanced fit within the ATP
binding site:
the pyridine N serves as a hydrogen bond acceptor,
while the NH of the pyrrole ring acts as a hydrogen bond
donor, together they form bidentate hydrogen bonds with
the hinge region of the kinase (Irie and Sawa 2018; Mérour
et al. 2014; Zuccotto et al. 2009). Therefore, it is hardly
surprising that in recent years, the 7-azaindole scaffold
has been widely explored in terms of PK inhibition and a
few examples of PK successfully inhibited using the 7-
azaindole scaffold include: anaplasmic lymphoma kinase
(ALK) (Gummadi et al. 2013), the aurora kinases
(Bavetsias et al. 2013), cell division cycle 7 kinase (Cdc 7)
(Harrington et al. 2013), C-Met kinase (Kim et al. 2008),
DYRK1A (Gourdain et al. 2013), focal adhesion kinase
(FAK) (Heinrich et al. 2013), IκK kinase(IKK) (Liddle
et al. 2009), KIT/FMS dual kinase (Zhang et al. 2013),
p38α mitogen-activated protein kinase (p38α MAP)
(Mavunkel et al. 2010), proto-oncogene serine/threonine-
protein kinase 1 (PIM1) (Nakano et al. 2012), mammalian
target of rapamycin kinase (m-TOR) (Tsou et al. 2010),
tropomyosin-related kinase A (TrkA) (Hong et al. 2012)
(see Fig. 1).
It is important to note that the 7-azaindole scaffold has
never been explored for Haspin inhibition. Prompted by the
relative novelty of the Haspin target and the general success
of the 7-azaindole scaffold in PK inhibition, twenty-four 7-
azaindole derivatives (divided into two series: 4-substituted
7-azaindoles and 5-substituted 7-azaindoles, see Tables 1
and 2), were synthesised and evaluated in vitro as potential
Haspin inhibitors. The compounds were selected for their
simplicity and comparatively inexpensive method of
synthesis, while also being novel to the Haspin target.
A SciFinder search of each compound was conducted to
determine the novelty of each compound. Compounds 18c,
18f, 18h, 18i, 19e, 19l and 19o had no known references in
SciFinder and are deemed novel. In turn, compounds 18a
(Metz et al. 2011), 18b (Arnold et al. 2007), 18d (Ledeboer
et al. 2006), 18e (Allegretti et al. 2001) and 18g (Ibrahim
et al. 2007), 19a (Ibrahim et al. 2007), 19b (Huang et al.
2019), 19c (Ibrahim et al. 2007), 19d (Huang et al. 2019),
19f (Ibrahim et al. 2007), 19g (Huang et al. 2019), 19h
(Huang et al. 2019), 19i (Huang et al. 2019), 19j (Liu et al.
2019), 19k (Huang et al. 2019), 19m (Ibrahim et al. 2007)
and 19n (Ibrahim et al. 2007) have previously featured in
literature. Alongside Haspin, the derivatives were screened
against other PKs: (i) HsCDK2/Cyclin A, (ii) HsCDK5/p25,
(iii) HsCDK9/Cyclin T, (iv) HsPim-1, (v) SscCK1δ/ε, (vi)
SscGSK3α/β and (vii) LmCK1.
Materials and methods
All compounds were synthesised according to the general
methods discussed in the experimental section. Starting
materials were procured from AK Scientific, CHR-6494 and
flavopiridol were purchased from Sigma Aldrich and were
used without further purification, unless otherwise specified.
Proton (1H) and carbon (13C) nuclear magnetic resonance
(NMR) spectra were recorded by means of a Bruker Avance
III 600 spectrometer at frequencies of 600 MHz and
151 MHz, respectively, in deuterated dimethylsulfoxide
(DMSO-d6). The chemical shifts (δ) are reported in parts
per million (ppm) and internally referenced to the signal of
tetramethylsaline (TMS). Spin multiplicities are indicated as
follow: s (singlet), d (doublet), dd (doublet of doublets), td
(triplet of doublets), t (triplet), q (quartet) and m (multiplet).
Coupling constants (J) are reported in Hz. High resolution
mass spectra (HRMS) were recorded by means of a Bruker
micrOTOF-Q II mass spectrometer in atmospheric pressure
chemical ionisation (APCI) mode (positive ion polarity).
High performance liquid chromatography (HPLC) analyses
were performed by means of an Agilent 1100 HPLC sys-
tem, using a Venusil XBP C18(2) (4.6 mm ×150 mm) 5 µM
column with mobile phase A (water + 0.1% formic acid)
and mobile phase B (acetonitrile + 0.1% formic acid)

Medicinal Chemistry Research
Fig. 1 A depiction of several compounds bearing the 7-azaindole
structure known to inhibit various PKs. References: (2)(Gummadi
et al. 2013), (3) (Bavetsias et al. 2013), (4) (Harrington et al. 2013), (5)
(Kim et al. 2008), (6) (Gourdain et al. 2013), (7) (Heinrich et al. 2013),
(8) (Liddle et al. 2009), (9) (Zhang et al. 2013), (10) (Mavunkel et al.
2010), (11) (Nakano et al. 2012), (12) (Tsou et al. 2010), (13) (Hong
et al. 2012)
against a gradient of 50% B (0–2 min), 100% B (2–8 min),
100% B (8–11 min), 50% B (11–11.1 min) and 50% B
(11.1–13 min) at 22 °C. Melting points (mp) were
determined by a Buchi B545 melting point apparatus and
are uncorrected. Thin layer chromatography (TLC) was
accomplished using silica gel 60 TLC plates (Merck) and a

Medicinal Chemistry Research
Table 1 Table depicting synthesis of 4-substituted 7-azaindole derivative, target compounds, and yields achieved
Compound
Yield (%)
42
Compound
Yield (%)
38
Compound
Yield (%)
75
39
34
39
29
63
59
UV254 fluorescent indicator to determine retention factor
(Rf) using petroleum ether: ethyl acetate (1:1) as eluent.
azaindole derivatives) were obtained in varying yields of
11-82%. (see Table 1 and Table 3). Synthesis of the 5-
substituted 7-azaindole derivatives (20a – 20o) proceeded
smoothly as per standard literature methods (Littke and Fu
2002). Noteworthy, although the reaction for the 4-
substituted 7-azaindole derivatives was performed with
the same basic reagents, the reaction of 4-chloro-7-
azaindole and appropriate boronic acid required a greater
ratio of boronic acid (at least 4 equivalences of azaindole)
for successful coupling. Thus, a total of 24 compounds
were produced according to conditions discussed above.
The synthesised compounds were confirmed by NMR and
HRMS analysis.
Experimental
Chemistry
Numerous 4-, and 5-substituted 7-azaindole derivatives
were prepared by means of Suzuki-Miyaura cross coupling
between either 4-chloro-1H-pyrrolo[2,3-b]pyridine or
5-bromo-1H-pyrrolo[2,3-b]pyridine and an assortment of
commercially available boronic acids in a single step
reaction. Suzuki coupling affords an excellent platform for
carbon-carbon bond formation and entails a palladium-
catalysed cross-coupling reaction between organoboron
compounds and organic halides or triflates (Kim et al.
2002; Wolfe and Buchwald 1999). Tetrakis(triphenyl-
phosphine)palladium(0) (Pd(PPh3)4) served as the catalyst
and the two series (4-substituted and 5-substituted 7-
General synthetic procedure for the selected 7-azaindole
derivatives Compounds 18a–19o were synthesised by
means of Suzuki coupling (also known as Suzuki-Miyaura
coupling) under nitrogen atmosphere. To a stirred solution
of either 4-chloro-7-azaindole for series 1 or 5-bromo-7-

Medicinal Chemistry Research
Table 2 Percentage of residual
activity for compounds 18a–b
and 19a–m at 1 and 10 µM using
a representative kinase panel
Cpd Hs CDK2/
Hs CDK5/
p25
Hs CDK9/
CyclinT
Hs Haspin Hs PIM1
Ssc
Ssc
Lm CK1
CyclinA
CK1δ/ε GSK3α/
β
18a 64 (99)
18b 81 (97)
18c 73 (73)
18d 59 (99)
18e 53 (77)
18f 59 (≥100)
66 (91)
62 (≥100)
53 (82)
83 (≥100)
6 (78)
5 (10)
19 (95)
14 (29)
10 (21)
58 (63)
15 (30)
20 (47)
50 (81)
66 (99)
10 (35)
90 (98)
54 (83) 70 (89) 72 (80)
71 (86) 70 (91) 91 (96)
79 (89) 78 (91) 79 (≥100)
87 (86) 75 (91) 46 (62)
9 (26)
14 (30)
15 (44)
6 (11)
60 (≥100) 67 (95) 87 (93) 63 (85)
54 (75)
10 (26)
20 (39)
29 (99)
77 (87)
79 (95) 73
(≥100)
94 (99)
18g ≥100 (91)
71 (≥100)
24 (74)
18 (73)
82
(≥100)
73
(≥100)
70 (79)
18h 63 (71)
79 (96)
3 (15)
8 (28)
76 (86) 78 (86) 41 (56)
18i ≥100 (89)
98 (≥100)
70 (82)
73 (83)
≥100
(≥100)
93 (95) ≥100
19 (44)
(89)
19a 24 (77)
19b 63 (88)
45 (61)
75 (89)
31 (94)
54 (81)
41 (73)
50 (71)
80 (≥100) 27 (57) 51 (86) 37 (≥100)
≥100
55 (90) 68 (94) 66 (82)
(≥100)
19c 61 (≥100)
19d ≥100 (94)
19e 90 (≥100)
19f 78 (88)
68 (≥100)
78 (≥100)
89 (94)
44 (62)
78 (63)
47 (69)
60 (74)
74 (63)
32 (64)
50 (59)
85 (71)
71 (76)
49 (56)
59 (≥100) 47 (84) 89 (91) 99 (81)
46 (≥100) 68 (78) 96 (95) 56 (69)
≥100 (77) 66 (79) 86 (94) 75 (66)
31 (≥100) 59 (71) 88 (97) 52 (82)
50 (86)
19g 37 (79)
63 (93)
≥100
48 (67) 78 (71) 63 (≥100)
(≥100)
19h 79 (≥100)
19i 93 (≥100)
75 (≥100)
54 (88)
43 (53)
79 (81)
≥100
(≥100)
61 (77) 66 (98) 61 (≥100)
≥100 (≥100) 92 (≥100)
50 (78)
92 (99) 94
(≥100)
≥100 (≥100)
19j 65 (87)
19k 68 (93)
19l ≥100 (91)
64 (≥100)
≥100 (88)
84 (≥100)
71 (89)
89 (73)
72 (73)
≥100 (97) 83 (87)
72 (88) 71 (97) ≥100 (≥100)
77 (62)
73 (90)
≥100 (98) 71 (63) 93 (78) ≥100 (61)
≥100
(≥100)
69 (78) 83 (95) 36 (88)
19m 99 (≥100)
19n 61 (87)
77 (93)
20 (≥100)a
49 (81)
21 (56)
74 (81)
40 (64)
7 (29)
37 (58)
39 (55)
58 (89) 86 (92) 71 (83)
49 (73) 60 (92) 62 (90)
19o 79 (≥100)
98 (≥100)
47 (52)
56 (≥100) 64 (81) 77 (93) ≥100 (≥100)a
Values represent the percentage of residual kinase activity for compounds used at 10 µM and 1 µM
concentrations. The values obtained with compounds at 1 µM are in bracket. The most significant results are
presented in bold-italic. Activities were assessed in duplicate using 10 µM ATP.
azaindole for series 2 (1.523 mmol), in 25 mL of a 3:1
toluene: ethanol mixture, (Pd(PPh3)4) (0.023 mmol) and
1.6 mL of a 2 M K2CO3 solution, the appropriate boronic
acid (1.523 mmol for series 1 and 6.092 mmol for series 2)
was added. Followed by additional 0.5 mmol additions if
deemed necessary during monitoring of TLC plates. The
reaction mixture was stirred continuously under reflux
(120˚C) for at least 24 h and then monitored via TLC until
complete. Subsequently, the reaction mixture was allowed
to cool to room temperature and the solvent was removed
under reduced pressure. The resulting residue was extracted
with distilled water and dichloromethane (3 × 25 mL). The
organic layers were combined and dried with MgSO4. The
solvent was once again removed under reduced pressure
and the resulting product was recrystallized from a suitable
solvent.
spectra of 4/5-Phenyl-7-azaindole derivatives: the target
compounds bear no hydrogen on C4 in the case of 4-
substituted compounds, or C5 in the case of 5-substituted
compounds and the presence of additional aromatic signals
suggest that aromatic moieties were successfully coupled.
1
Furthermore, the singlet at approximately 12 ppm in H-
NMR is assignable to N-H, this signifies that no reaction
took place at NH-position (position 1). The NMR spectra
and other characterisation data can be found in the Sup-
porting information.
4-Phenyl-1H-pyrrolo[2,3-b]pyridine (18a): compound
was synthesised from phenylboronic acid and 4-chloro-7-
azaindole in a yield of 42.37%, recrystallized from
acetonitrile, dark beige crystals, mp 208 – 210 °C, Rf =
0.48 (1 petroleum ether: 1 ethyl acetate). 1H NMR (DMSO-
d6, 600 MHz) δ 11.86 (1H, s, NH), 8.33 (1H, d, J = 4.8 Hz,
H-6), 7.81 (2H, d, J = 7.6 Hz, H-2′,H-6′), 7.60 (3H, m, H-2,
H-3′, H-5′), 7.50 (1H, t, J = 7.3 Hz, H-4′), 7.23 (1H, d, J =
4.8 Hz, H-5), 6.68–6.63 (1H, m, H-3). 13C NMR (DMSO-
d6, 150 MHz) δ 149.6 (C-7a), 143.4 (C-6), 140.7 (C-1′),
Physical properties and characterization The NMR spec-
tra of 4/5-Halo-7-azaindole—starting material distinctly
bears 7 carbons and 5 hydrogens peaks. The proton NMR

Medicinal Chemistry Research
Table 3 A table depicting 5-substituted 7-azaindole derivative synthesis, compounds synthesised and yields achieved
Compound
Yield (%)
63
Compound
Yield (%)
46
Compound
Yield (%)
34
60
63
53
43
20
45
83
11
53
42
45
61
139.0 (C-4), 129.5 (C-3′, C-5′), 128.8 (C-3a), 128.7 (C-2′,
C-6′), 127.1 (C-4′), 117.7 (C-2), 114.7 (C-5), 99.4 (C-3).
APCI-HRMS m/z: calc. for C13H11N2 (MH+), 195.0916,
found 195.0920; Purity (HPLC): 98% (column: Venusil
XBP C18(2) 4.6 mm ×150 mm; retention time: 15 min).
(Me-4′). APCI-HRMS m/z: calc. for C14H13N2 (MH+),
209.1073, found 209.1067; Purity (HPLC): 97% (column:
Venusil XBP C18(2) 4.6 mm ×150 mm; retention time:
9 min).
0.4-(4-Methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (18c):
compound was synthesised from 4-methoxyphenylboronic
acid and 4-chloro-7-azaindole in a yield of 29.33%,
recrystallized from acetonitrile, pale yellow powder, mp
212–212 °C, Rf = 0.18 (2 petroleum ether: 1 ethyl acetate).
1H NMR (DMSO-d6, 600 MHz) δ 11.75 (1H, s, NH), 8.25
(1H, d, J = 4.8 Hz, H-6), 7.73 (2H, d, J = 8.5 Hz, H-2′,
H-6′), 7.52 (1H, t, J = 2.8 Hz, H-2), 7.14 (1H, d, J = 4.4 Hz,
H-5), 7.12 (2H, d, J = 8.6 Hz, H-3′, H-5′), 6.61 (1H, d, J =
2.9 Hz, H-3), 3.84 (3H, s, MeO-4′). 13C NMR (DMSO-d6,
150 MHz) δ 159.9 (C-4′), 149.6 (C-7a), 143.4 (C-6), 140.5
(C-1′), 131.2 (C-4), 129.9 (C-2′, C-6′), 126.8 (C-3a), 117.5
(C-2), 114.9 (C-3′, C-5′), 114.3 (C-5), 99.5 (C-3), 55.7
(MeO-4′). APCI-HRMS m/z: calc. for C14H13N2O (MH+),
4-(4-Methylphenyl)-1H-pyrrolo[2,3-b]pyridine
(18b):
compound was synthesised from 4-methylphenylboronic
acid and 4-chloro-7-azaindole in a yield of 38.80%,
recrystallized from acetonitrile, yellow powder, mp
169–171 °C, Rf = 0.38 (4 petroleum ether: 1 ethyl acetate).
1H NMR (DMSO-d6, 600 MHz) δ 11.78 (1H, s, NH), 8.27
(1H, d, J = 4.7 Hz, H-6), 7.67 (2H, d, J = 7.9 Hz, H-2′, H-
6′), 7.53 (1H, d, J = 2.9 Hz, H-2), 7.36 (2H, d, J = 7.7 Hz,
H-3′-H-5′), 7.16 (1H, d, J = 4.7 Hz, H-5), 6.60 (1H, d, J =
2.9 Hz, H-3), 2.39 (3H, s, Me-4′). 13C NMR (DMSO-d6,
150 MHz) δ 149.6 (C-7a), 143.4 (C-6), 140.7 (C-4′), 138.3
(C-1′), 136.1 (C-4), 130.0 (C-3′, C-5′), 128.6 (C-2′, C-6′),
126.9 (C-3a), 117.7 (C-2), 114.5 (C-5), 99.5 (C-3), 21.3

Medicinal Chemistry Research
225.1022, found 225.1017; Purity (HPLC): 99% (column:
Venusil XBP C18(2) 4.6 mm × 150 mm; retention time:
7 min).
114.8 (s, C-5), 99.2 (s, C-3). APCI-HRMS m/z: calc. for
C13H9F2N2 (MH+), 231.0728, found 231.0736; Purity
(HPLC): 98% (column: Venusil XBP C18(2) 4.6 mm
×150 mm; retention time: 4 min).
4-(2,3-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine
(18d): compound was synthesised from 2,3-dimethoxyphe-
nylboronic acid and 4-chloro-7-azaindole in a yield of
37.73%, recrystallized from acetonitrile, sand coloured
powder, mp 191–192 °C, Rf = 0.16 (4 petroleum ether: 1
ethyl acetate). 1H NMR (DMSO-d6, 600 MHz) δ 11.69
(1H, s, NH), 8.24 (1H, d, J = 4.8 Hz, H-6), 7.47–7.44
(1H, m, H-2), 7.18 (1H, t, J = 7.8 Hz, H-5′), 7.14 (1H, dd,
J = 8.6, 1.2 Hz, H-4′), 7.05 (1H, d, J = 5.1 Hz, H-5), 6.99
(1H, dd, J = 7.4, 1.0 Hz, H-6′), 6.25 (1H, 3, H-3), 3.87 (3H,
s, MeO-2′), 3.48 (3H, s, MeO-3′). 13C NMR (DMSO-d6,
150 MHz) δ 152.9 (C-3′), 148.8 (C-7a), 146.2 (C-6), 142.1
(C-2′), 137.9 (C-4), 132.4 (C-3a), 126.0 (C-5′), 124.0 (C-
6′), 122.2 (C-1′), 118.7(C-2), 116.0 (C-5), 112.9 (C-4′),
99.4 (C-3), 60.4 (C-11′), 55.8 (C-13′). APCI-HRMS m/z:
calc. for C15H15N2O2 (MH+), 255.1128, found 255.1129;
Purity (HPLC): 100% (column: Venusil XBP C18(2)
4.6 mm ×150 mm; retention time: 5 min).
4-[4-(Trifluoromethyl)phenyl]-1H-pyrrolo[2,3-b]pyridine
(18g): compound was synthesised from 4-trifluorophenyl
boronic acid and 4-chloro-7-azaindole in a yield of 75.44%,
recrystallized from acetonitrile, sand coloured crystals, mp
204–205 °C, Rf = 0.13 (4 petroleum ether: 1 ethyl acetate).
1H NMR (DMSO-d6, 600 MHz) δ 11.90 (1H, s, NH), 8.33
(1H, d, J = 4.8 Hz, H-6), 7.99 (2H, d, J = 7.9 Hz, H-3′, H-
5′), 7.91 (2H, d, J = 7.9 Hz, H-2′, H-6′), 7.62–7.58 (1H, m,
H-2), 7.26 (1H, d, J = 4.8 Hz, H-5), 6.65–6.62 (1H, m, H-
3). 13C NMR (DMSO-d6, 150 MHz) δ 149.6 (s, C-7a),
143.5 (s, C-6), 143.0 (s, C-1′), 139.0 (s, C-4), 129.5 (s, C-
3a), 129.1 (q, J = 31.7 Hz, C-4′), 127.7 (s, C-2′, C-6′),
126.3 (q, J = 3.6 Hz, C-3′, C-5′), 124.7 (d, J = 272.1 Hz,
CF3), 117.7 (s, C-2), 114.9 (s, C-5), 99.2 (s, C-3). APCI-
HRMS m/z: calc. for C14H10F3N2 (MH+), 263.0790, found
263.0798; Purity (HPLC): 99% (column: Venusil XBP C18
(2) 4.6 mm ×150 mm; retention time: 6 min).
4-(4-Fluorophenyl)-1H-pyrrolo[2,3-b]pyridine
(18e):
4-(4-Chlorophenyl)-1H-pyrrolo[2,3-b]pyridine
(18h):
compound was synthesised from 4-fluorophenylboronic
acid and 4-chloro-7-azaindole in a yield of 34.10%,
recrystallized from acetonitrile, pale yellow powder, mp
219.3–219.7 °C, Rf = 0.44 (2 petroleum ether: 1 ethyl
compound was synthesised from 4-chlorophenylboronic
acid and 4-chloro-7-azaindole in a yield of 38.51%,
recrystallized from acetonitrile, yellow powder, mp
222–223 °C, Rf = 0.24 (2 petroleum ether: 1 ethyl acetate).
1H NMR (DMSO-d6, 600 MHz) δ 11.81 (1H, s, NH), 8.29
(1H, d, J = 4.8 Hz, H-6), 7.79 (2H, d, J = 7.7 Hz, H-3′,
H-5′), 7.60 (2H, d, J = 7.6 Hz, H-2′, H-6′), 7.57–7.55 (1H,
m, H-2), 7.18 (1H, d, J = 4.8 Hz, H-2), 6.61–6.58 (1H, m,
H-3). 13C NMR (DMSO-d6, 150 MHz) δ 149.6 (C-7a),
143.4 (C-6), 139.3 (C-1′), 137.7 (C-4), 133.6 (C-4′), 130.4
(C-3′, C-5′), 129.4 (C-2′, C-6′), 127.3 (C-3a), 117.5 (C-2),
114.6 (C-5), 99.3 (C-3). APCI-HRMS m/z: calc. for
C13H10ClN2 (MH+), 229.0527, found 229.0537; Purity
(HPLC): 83% (column: Venusil XBP C18(2) 4.6 mm
×150 mm; retention time: 6 min).
1
acetate). H NMR (DMSO-d6, 600 MHz) δ 11.84 (1H, s,
NH), 8.28 (1H, d, J = 4.9 Hz, H-6), 7.82 (2H, dd, J = 8.5,
5.6 Hz, H-2′, H-6′), 7.56 (1H, t, J = 3.0 Hz, H-2), 7.42–7.36
(2H, m, H-3′, H-5′), 7.18 (1H, d, J = 4.9 Hz, H-5), 6.60
(1H, d, J = 3.3 Hz, H-3). 13C NMR (DMSO-d6, 150 MHz) δ
162.7 (d, J = 245.5 Hz, C-4′), 149.6 (s, C-7a), 143.4 (s, C-
6), 139.6 (s, C-5), 135.4 (d, J = 3.0 Hz, C-1′), 130.8 (d, J =
8.3 Hz, C-2′, C-6′), 127.2 (s, C-3a), 117.6 (s, C-2), 116.4 (d,
J = 21.4 Hz, C-3′, C-5′), 114.7 (s, C-5), 99.3 (s, C-3).
APCI-HRMS m/z: calc. for C13H10FN2 (MH+),
213.082253, found 213.0826; Purity (HPLC): 99% (col-
umn: Venusil XBP C18(2) 4.6 mm ×150 mm; retention
time: 5 min).
4-(3,4-Difluorophenyl)-1H-pyrrolo[2,3-b]pyridine (18f):
compound was synthesised from 3,4-difluorophenylboronic
acid and 4-chloro-7-azaindole in a yield of 62.68%,
recrystallized from acetonitrile, pale orange crystals, mp
172–174 °C, Rf = 0.20 (4 petroleum ether: 1 ethyl acetate).
(DMSO-d6, 600 MHz) δ 11.93 (1H, s, NH), 8.35 (1H, d,
J = 4.8 Hz, H-6), 7.90–7.84 (1H, m, H-5′), 7.71–7.65 (2H,
m, H-2′, H-4′), 7.64 (1H, t, J = 2.8 Hz, H-2), 7.26 (1H, d,
J = 4.8 Hz, H-5), 6.68 (1H, m, H-3). 13C NMR (DMSO-d6,
150 MHz) δ 150.9 (dd, J = 33.0, 12.7 Hz, C-4′), 149.6 (s,
C-7a), 149.3 (dd, J = 34.6, 12.5 Hz, C-3′), 143.4 (s, C-6),
138.4 (s, C-4), 136.5 (dd, J = 5.0, 4.3 Hz, C-1′), 127.6 (s,
C-3a), 125.7 (dd, J = 6.5, 3.2 Hz, C-2′), 118.6 (d, J =
17.2 Hz, C-6′), 117.7 (d, J = 17.6 Hz, C-5′), 117.5 (s, C-2),
4-(Biphenyl-4-yl)-1H-pyrrolo[2,3-b]pyridine (18i): com-
pound was synthesised from 4-biphenylboronic acid and 4-
chloro-7-azaindole in a yield of 58.74%, recrystallized from
acetonitrile, olive green powder, mp 266–267 °C, Rf = 0.43
1
(2 petroleum ether: 1 ethyl acetate). H NMR (DMSO-d6,
600 MHz) δ 11.81 (1H, s, NH), 8.30 (1H, d, J = 4.6 Hz, H-
6), 7.88 (2H, d, J = 8.1 Hz, C-3′, C11′), 7.85 (2H, d, J =
7.6 Hz, C-2′, C12′), 7.76 (2H, d, J = 7.5 Hz, H-6′, H-10′),
7.56 (1H, d, J = 2.8 Hz, H-2), 7.51 (2H, t, J = 7.4 Hz, H-7′,
H-9′), 7.41 (1H, t, J = 7.4 Hz, H-8′), 7.24 (1H, d, J =
4.6 Hz, H-5), 6.67 (1H, d, J = 2.2 Hz, H-3). 13C NMR
(DMSO-d6, 150 MHz) δ 149.7 (C-7a), 143.4 (C-6), 140.5
(C-4′), 140.2 (C-5′), 140.0 (C-1′), 138.0 (C-4), 129.5 (C-7′,
C-9′), 129.3 (C-3′, C-11′), 128.2 (C-3a), 127.7 (C2′, C12′),
127.3 (C8′), 127.2 (C-6′, C-10′), 117.7 (C-2), 114.6 (C-5),
99.5 (C-3). APCI-HRMS m/z: calc. for C19H15N2 (MH+),

Medicinal Chemistry Research
271.1229, found 271.1224; Purity (HPLC): 89% (column:
Venusil XBP C18(2) 4.6 mm × 150 mm; retention time:
5 min).
5-(4-Methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (19d):
compound was synthesised from 4-methoxyphenylboronic
acid and 5-bromo-7-azaindole in a yield of 82.70%,
recrystallized from acetonitrile, mustard coloured crystals,
mp 227–228 °C, Rf = 0.23 (2 petroleum ether: 1 ethyl
5-Phenyl-1H-pyrrolo[2,3-b]pyridine (19a): compound
was synthesised from phenylboronic acid and 5-bromo-7-
azaindole in a yield of 62.93%, recrystallized from ethanol,
pale orange crystals, mp 160–162 °C, Rf = 0.57 (2
petroleum ether: 1 ethyl acetate). 1H NMR (DMSO-d6,
600 MHz) δ 11.71 (1H, s, NH), 8.52 (1H, d, J = 2.1 Hz, H-
6), 8.21 (1H, d, J = 2.0 Hz, H-4), 7.71 (2H, d, J = 7.3 Hz,
H-2′, H-6′), 7.53-7.51 (1H, m, H-2), 7.48 (2H, t, J = 7.6 Hz,
H-3′, H-5′), 7.36 (1H, t, J = 7.3 Hz, H-4′), 6.52-6.50 (1H,
m, H-3). 13C NMR (DMSO-d6, 150 MHz) δ 148.6 (C-7a),
142.0 (C-6), 139.6 (C-1′), 129.5 (C-5), 128.7 (C-3′, C-5′),
127.4 (C-4′), 127.4 (C-4), 127.3 (C-2′, C-6′), 126.6 (C-2),
120.2 (C-3a), 100.7 (C-3). APCI-HRMS m/z: calc. for
C13H11N2 (MH+), 195.0916, found 195.0918; Purity
(HPLC): 99% (column: Venusil XBP C18(2) 4.6 mm ×
150 mm; retention time: 14 min).
5-(4-Methylphenyl)-1H-pyrrolo[2,3-b]pyridine (19b): com-
pound was synthesised from p-tolylboronic acid and 5-bromo-
7-azaindole in a yield of 59.94%, recrystallized from
acetonitrile, brownish orange crystals, mp 182–184 °C, Rf =
0.71 (2 petroleum ether: 1 ethyl acetate). 1H NMR (DMSO-d6,
600 MHz) δ 11.71 (1H, s, NH), 8.49 (1H, d, J = 1.7 Hz, H-6),
8.16 (1H, d, J = 1.7 Hz, H-4), 7.59 (2H, d, J = 7.9 Hz, H-2′,
H-6′), 7.51 (1H, t, J = 2.8 Hz, H-2), 7.28 (2H, d, J = 7.9 Hz,
H-3′, H-5′), 6.52–6.47 (1H, m, H-3), 2.4 (3H, s, Me-4′). 13C
NMR (DMSO-d6, 150 MHz) δ 148.4 (C-7a), 141.8 (C-6),
136.7 (C-4′), 136.5 (C-1′), 130.1 (C-5), 128.6 (C-3′, C-5′),
127.4 (C-4), 127.2 (C-2′, C-6′), 126.3 (C-2), 120.2 (C-3a),
100.6 (C-3), 21.1 (Me-4′). APCI-HRMS m/z: calc. for
C14H13N2 (MH+), 209.1073, found 209.1071; Purity (HPLC):
87% (column: Venusil XBP C18(2) 4.6 mm × 150 mm;
retention time: 11 min).
1
acetate). H NMR (DMSO-d6, 600 MHz) δ 11.68 (1H, s,
NH), 8.47 (1H, d, J = 1.9 Hz, H-6), 8.13 (1H, d, J = 1.8 Hz,
H-4), 7.63 (2H, d, J = 8.1 Hz, H-3′, H-5′), 7.50 (1H, t, J =
3.1 Hz, H-2), 7.04 (2H, d, J = 8.1 Hz, H-2′, H-6′),
6.50–6.46 (1H, m, H-3), 3.80 (3H, s, Me-4′). 13C NMR
(DMSO-d6, 150 MHz) δ 159.0 (C-4′), 148.2 (C-7a), 141.7
(C-6), 132.0 (C-1′), 128.4 (C-5), 127.7 (C-2′, C-6′), 127.3
(C-4), 126.0 (C-2), 120.2 (C-3a), 114.9 (C-3′, C-5′), 100.5
(C-3), 55.6 (MeO-4′). APCI-HRMS m/z: calc. for
C14H13N2O (MH+), 225.1022, found 225.1023; Purity
(HPLC): 100% (column: Venusil XBP C18(2) 4.6 mm
×150 mm; retention time: 11 min).
5-(2,3-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine
(19e): compound was synthesised from 2,3-dimethoxyphe-
nylboronic acid and 5-bromo-7-azaindole in a yield of
11.11%, recrystallized from acetonitrile, white crystals, mp
136–138 °C Rf = 0.36 (2 petroleum ether: 1 ethyl acetate).
1H NMR (CDCl3, 600 MHz) δ 10.19 (1H, s, NH), 8.54 (1H,
d, J = 1.8 Hz, H-6), 8.15 (1H, d, J = 1.7 Hz, H-4),
7.39–7.37 (1H, m, H-2), 7.15 (1H, t, J = 7.9 Hz, H-5′),
7.02 (1H, dd, J = 7.7, 1.3 Hz, H-4′), 6.96 (1H, dd, J = 8.2,
1.2 Hz, H-6′), 6.56 (1H, dd, J = 3.3, 1.7 Hz, H-3), 3.94 (3H,
s, Me-2′), 3.6 (3H, s, Me-3′). 13C NMR (CDCl3,151 MHz) δ
153.4 (C-3′), 148.0 (C-7a), 146.9 (C-6), 144.0 (C-2′), 134.2
(C-5), 129.7 (C-5′), 126.6 (C-6′), 125.4 (C-4), 124.4 (C-1′),
123.1 (C-2), 120.0 (C-3a), 111.7 (C-4′), 101.4 (C-3), 60.7
(MeO-2′), 56.1 (MeO-3′). APCI-HRMS m/z: calc. for
C15H15N2O2 (MH+), 255.1128, found 255.1130; Purity
(HPLC): 99% (column: Venusil XBP C18(2) 4.6 mm ×
150 mm; retention time: 6 min).
5-(3-Methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (19c):
compound was synthesised from 3-methoxyphenylboronic
acid and 5-bromo-7azaindole in a yield of 63.05%,
recrystallized from acetonitrile, pale yellow crystals, mp
140–141 °C, Rf = 0.63 (2 petroleum ether: 1 ethyl acetate).
1H NMR (DMSO-d6, 600 MHz) δ 11.73 (1H, s, NH), 8.52
(1H, d, J = 2.1 Hz, H-6), 8.22 (1H, d, J = 2.1 Hz, H-4), 7.52
(1H, t, J = 2.9 Hz, H-2′), 7.39 (1H, td, J = 7.7, 08 Hz, H-5′),
7.28 (1H, dd, J = 5.8, 2.0 Hz, H-4′), 7.25–7.23 (1H, m, H-
2), 6.93 (1H, dd, J = 8.2, 2.4 Hz, H-6′), 6.52 – 6.50 (1H, m,
H-3), 3.8 (3H, s, Me-3′). 13C NMR (DMSO-d6, 150 MHz) δ
160.3 (C-3′), 148.6 (C-7a), 142.0 (C-6), 141.1 (C-1′), 130.5
(C-5), 128.5 (C-5′), 127.5 (C-6′), 126.7 (C-4), 120.1 (C-2),
119.7 (C-3a), 113.0 (C-4′), 112.7 (C-2′), 100.7 (C-3), 55.6
(Me-3′). APCI-HRMS m/z: calc. for C14H13N2O (MH+),
225.1022, found 225.1030; Purity (HPLC): 69% (column:
Venusil XBP C18(2) 4.6 mm × 150 mm; retention time:
8 min).
5-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (19f):
compound was synthesised from 3,4-dimethoxyphenylboronic
acid and 5-bromo-7-azaindole in a yield of 46.25%,
recrystallized from acetonitrile, yellow crystals, mp
210–211 °C, Rf = 0.18 (2 petroleum ether: 1 ethyl acetate).
1H NMR (DMSO-d6, 600 MHz) δ 11.68 (1H, s, NH), 8.51
(1H, d, J = 2.0 Hz, H-6), 8.18 (1H, d, J = 1.9 Hz, H-4), 7.50
(1H, t, J = 2.8 Hz, H-2), 7.27 (1H, d, J = 1.9 Hz, H-2′), 7.22
(1H, dd, J = 8.2, 2.0 Hz, H-5′), 7.05 (1H, d, J = 8.3 Hz, H-6′),
6.50–6.48(1H, m, H-3), 3.9 (3H, s, Me-3′), 3.8 (3H, s, Me-4′).
13C NMR (DMSO-d6, 150 MHz) δ 149.6 (C-7a), 148.5 (C-
4′), 148.3 (C-3′), 141.9 (C-6), 132.4 (C-1′), 128.7 (C-5), 127.3
(C-6′), 126.2 (C-4), 120.1 (C-2), 119.3 (C-3a), 112.8 (C-5′),
111.2 (C-2′), 100.5 (C-3), 56.1 (MeO-3′), 56.0 (MeO-4′).
APCI-HRMS m/z: calc. for C15H15N2O2 (MH+), 255.1128,
found 255.1124; Purity (HPLC): 100% (column: Venusil
XBP C18(2) 4.6 mm ×150 mm; retention time: 11 min).

Medicinal Chemistry Research
5-(4-Fluorophenyl)-1H-pyrrolo[2,3-b]pyridine (19g): com-
pound was synthesised from 4-fluorophenylboronic acid and
5-bromo-7-azaindole in a yield of 52.63%, recrystallized from
acetonitrile, dark yellow crystals, mp 182–184 °C, Rf = 0.48
5-(4-Chlorophenyl)-1H-pyrrolo[2,3-b]pyridine (19j): com-
pound was synthesised from 4-chlorophenylboronic acid and
5-bromo-7azaindole in a yield of 70.40%, recrystallized from
acetonitrile, gold crystals, mp 195-197 °C, Rf = 0.41 (2
petroleum ether: 1 ethyl acetate). 1H NMR (DMSO-d6,
600 MHz) δ 11.77 (1H, s, NH), 8.52 (1H, d, J = 2.2 Hz, H-
6), 8.22 (1H, d, J = 2.0 Hz, H-4), 7.76–7.73 (2H, m, H-2′,
H-6′), 7.54–7.51 (3H, m, H-2, H-3′, H-5′), 6.52–6.50 (1H, m,
H-3). 13C NMR (DMSO-d6, 150 MHz) δ 148.6 (C-7a), 141.9
(C-6), 138.5 (C-1′), 132.2 (C-4′), 129.4 (C-5), 129.1 (C-3′, C-
5′), 127.6 (C-2′, C6′), 127.3 (C-4), 126.6 (C-2), 120.1 (C-3a),
100.7 (C-3). APCI-HRMS m/z: calc. for C13H10ClN2 (MH+),
229.0527, found 229.0536; Purity (HPLC): 100% (column:
Venusil XBP C18(2) 4.6 mm × 150 mm; retention time:
8 min).
1
(2 petroleum ether: 1 ethyl acetate). H NMR (DMSO-d6,
600 MHz) δ 11.74 (1H, s, NH), 8.49 (1H, d, J = 2.2 Hz, H-6),
8.19 (1H, d, J = 2.1 Hz, H-4), 7.76–7.73 (2H, m, H-2′, H-6′),
7.53 (1H, t, J = 2.8 Hz, H-2), 7.32–7.29 (2H, m, H-3′, H-5′),
6.52–6.49 (1H, m, H-3). 13C NMR (DMSO-d6, 150 MHz) δ
162.1 (d, J = 243.6 Hz, C-4′), 148.5 (s, C-7a), 141.9 (s, C-6),
136.1 (d, J = 2.9 Hz, C-1′), 129.3 (d, J = 8.1 Hz, C-2′, C-6′),
127.7 (s, C-5), 127.5 (s, C-4), 126.6 (s, C-2), 120.1 (s, C-3a),
116.2 (d, J = 21.3 Hz, C-3′, C-5′), 100.6 (s, C-3). APCI-
HRMS m/z: calc. for C13H10FN2 (MH+), 213.0822, found
213.0823; Purity (HPLC): 100% (column: Venusil XBP C18
(2) 4.6 mm × 150 mm; retention time: 8 min).
4-(1H-Pyrrolo[2,3-b]pyridin-5-yl)benzonitrile (19k): com-
pound was synthesised from 4-cyanoboronic acid and 5-
bromo-7-azaindole in a yield of 33.53%, recrystallized from
acetonitrile, pale yellow crystals, mp 287–293 °C, Rf = 0.38
5-(3,4-Difluorophenyl)-1H-pyrrolo[2,3-b]pyridine (19h):
compound was synthesised from 3,4-difluorophenylboronic
acid and 5-bromo-7-azaindole in a yield of 43.02%,
recrystallized from acetonitrile, mustard coloured crystals,
mp 194–196 °C, Rf = 0.47 (2 petroleum ether: 1 ethyl
1
(2 petroleum ether: 1 ethyl acetate). H NMR (DMSO-d6,
600 MHz) δ 11.85 (1H, s, NH), 8.61 (1H, d, J = 2.2 Hz, H-6),
8.33 (1H, d, J = 2.1 Hz, H-4), 7.95 (2H, d, J = 8.5 Hz, H-3′,
H-5′), 7.93 (2H, d, J = 8.5 Hz, H-2′, H6′), 7.56 (1H, t, J =
3.3 Hz, H-2), 6.55–6.53 (1H, m, H-3). 13C NMR (DMSO-d6,
150 MHz) δ 149.0 (C-7a), 144.3 (C-6), 142.1 (C-1′), 133.3 (C-
3′, C-5′), 128.0 (C-2′, C-6′), 127.9 (C-5), 127.2 (C-4), 126.7
(C-2), 120.2 (C-3a), 119.5 (CN), 109.8 (C-4′), 101.0 (C-3).
APCI-HRMS m/z: calc. for C14H10N3 (MH+), 220.086924,
found 220.0872; Purity (HPLC): 99% (column: Venusil XBP
C18(2) 4.6 mm × 150 mm; retention time: 5 min).
5-(Biphenyl-4-yl)-1H-pyrrolo[2,3-b]pyridine (19l): com-
pound was synthesised from 4-biphenylboronic acid and 5-
bromo-7-azaindole in a yield of 19.90%, recrystallized from
acetonitrile, white powder, mp 264–265 °C, Rf = 0.36 (2
petroleum ether: 1 ethyl acetate). 1H NMR (DMSO-d6,
600 MHz) δ 11.76 (1H, s, NH), 8.59 (1H, d, J = 2.1 Hz,
H-6), 8.27 (1H, d, J = 2.0 Hz, H-4), 7.82 (2H, d, J = 8.3 Hz,
H-2, H-12′), 7.78 (2H, d, J = 8.4 Hz, H-3′, H-11′), 7.74
(2H, d, J = 7.3 Hz, H-6′, H-10′), 7.55–7.53 (1H, m, H-2),
7.50 (2H, t, J = 7.7 Hz, H-7′, H-9′), 7.39 (1H, t, J = 7.4 Hz,
H-8′), 6.53 (1H, m, H-3). 13C NMR (DMSO-d6, 150 MHz)
δ 148.6 (C-7a), 141.9 (C-6), 140.2 (C-4′), 139.0 (C-5′),
138.6 (C-1′), 129.5 (C-7′, C-9′), 128.1 (C-5), 127.9 (C-8′),
127.8 (C-3′, C-11′), 127.7 (C-2′, C-12′), 127.5 (C-4), 127.0
(C-6′, C10′), 126.5 (C-2), 120.2 (C-3a), 100.7 (C-3). APCI-
HRMS m/z: calc. for C19H15N2 (MH+), 271.1229, found
271.1222; Purity (HPLC): 73% (column: Venusil XBP C18
(2) 4.6 mm × 150 mm; retention time: 7 min).
1
acetate). H NMR (DMSO-d6, 600 MHz) δ 11.79 (1H, s,
NH), 8.53 (1H, d, J = 2.2 Hz, H-6), 8.24 (1H, d, J = 2.2 Hz,
H-4), 7.83 (1H, ddd, J = 12.2, 7.8, 2.2 Hz, H-2′), 7.59–7.56
(1H, m, H-5′), 7.55–7.53 (1H, m, H-2), 7.53–7.49 (1H, m,
H-6′), 6.52–6.50 (1H, m, H-3). 13C NMR (DMSO-d6,
150 MHz) δ 150.5 (dd, J = 157.2, 12.7 Hz, C-4′), 148.9 (dd,
J = 157.2, 12.7 Hz, C-3′), 148.7 (s, C-7a), 141.9 (s, C-6),
137.3 (dd, J = 6.3, 3.6 Hz, C-1′), 127.7 (s, C-5), 126.8 (s,
C-4), 126.5 (s, C-2), 123.9 (dd, J = 6.2, 3.1 Hz, C-6′), 120.1
(s, C-3a), 118.4 (d, J = 17.0 Hz, C-5′), 116.3 (d, J =
17.6 Hz, C-2′), 100.7 (s, C-3). APCI-HRMS m/z: calc. for
C13H9F2N2 (MH+), 231.0728, found 231.0724; Purity
(HPLC): 100% (column: Venusil XBP C18(2) 4.6 mm ×
150 mm; retention time: 9 min).
5-[4-(Trifluoromethyl)phenyl]-1H-pyrrolo[2,3-b]pyridine
(19i): compound was synthesised from 4-trifluorophenyl
boronic acid and 5-bromo-7-azaindole in a yield of 52.88%,
recrystallized from acetonitrile, pale yellow crystals, mp
236–237 °C, Rf = 0.50 (2 petroleum ether: 1 ethyl acetate).
1H NMR (DMSO-d6, 600 MHz) δ 11.84 (1H, s, NH), 8.59
(1H, d, J = 1.8 Hz, H-6), 8.31 (1H, d, J = 1.8 Hz, H-4), 7.96
(2H, d, J = 8.1Hz, H-3′, H-5′), 7.82 (2H, d, J = 8.1 Hz, H-
2′, H-6′), 7.56 (1H, d, J = 3.2 Hz, H-2), 6.54 (1H, d, J =
3.3 Hz, H-3). 13C NMR (DMSO-d6, 150 MHz) δ 148.9 (s,
C-7a), 143.7 (s, C-6), 142.1 (s, C-1′), 128.0 (s, C-5), 127.8
(s, C-4), 127.7 (d, J = 31.9 Hz, C-4′), 127.1 (d, J = 6.1 Hz,
C-2′, C-6′), 126.3 (d, J = 6.1 Hz, C-3′, C-5′), 126.2 (s, C-2),
125.0 (d, J = 271.7 Hz, CF3), 120.2 (s, C-3a), 100.9 (s, C-
3). APCI-HRMS m/z: calc. for C14H10F3N2 (MH+),
263.0790, found 263.0787; Purity (HPLC): 100% (column:
Venusil XBP C18(2) 4.6 mm × 150 mm; retention time:
10 min).
5-(Pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridine (19m): com-
pound was synthesised from pyridin-3-ylboronic acid and
5-bromo-7-azaindole in a yield of 44.78%, recrystallized
from acetonitrile and ethanol, yellow crystals, mp
176–177 °C, Rf = 0.07 (2 petroleum ether: 1 ethyl acetate).

Medicinal Chemistry Research
1H NMR (DMSO-d6, 600 MHz) δ 11.81 (1H, s, NH), 8.95
(1H, d, J = 1.9 Hz, H-6), 8.58–8.55 (2H, m, C-2′, C-4′),
8.30 (1H, d, J = 1.9 Hz, H-4), 8.15–8.12 (1H, m, H-6′),
7.55 (1H, t, J = 2.8 Hz, H-2), 7.51–7.48 (1H, m, H-5′),
6.55–6.52 (1H, m, H-3). 13C NMR (DMSO-d6, 150 MHz) δ
100.8 (C-7a), 120.2 (C-2′), 124.4 (C-4′), 125.5 (C-6), 126.9
(C-6′), 127.7 (C-5), 134.7 (C-1′), 135.1 (C-4), 141.9 (C-2),
148.2 (C-5′), 148.4 (C-3a), 148.8 (C-3). APCI-HRMS m/z:
calc. for C12H10N3 (MH+), 196.0869, found 196.0882;
Purity (HPLC): 100% (column: Venusil XBP C18(2)
4.6 mm × 150 mm; retention time:7 min).
5-(Pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine (19n): com-
pound was synthesised from pyridin-4-ylboronic acid and
5-bromo-7-azaindole in a yield of 45.12%, recrystallized
from acetonitrile, yellow powder, mp 211.3–214.0 °C, Rf =
0.07 (2 petroleum ether: 1 ethyl acetate). 1H NMR (DMSO-
d6, 600 MHz) δ 11.88 (1H, s, NH), 8.67 (1H, d, J = 2.1 Hz,
H-6), 8.63 (2H, dd, J = 5.3, 1.3 Hz, H-3′, H-5′), 8.40 (1H, d,
J = 2.1 Hz, H-4), 7.79 (2H, dd, J = 5.6, 1.5 Hz, H-2′, H-6′),
7.58–7.56 (1H, m, H-2), 6.57–6.54 (1H, m, H-3). 13C NMR
(DMSO-d6, 150 MHz) δ 150.6 (C-7a), 149.3 (C-3′-C-5′),
146.6 (C-6), 141.9 (C-1′), 128.0 (C-5), 127.00 (C-4), 125.4
(C-2), 121.7 (C-2′, C-6′), 120.2 (C-3a), 101.0 (C-3). APCI-
HRMS m/z: calc. for C12H10N3 (MH+), 196.0869, found
196.0886; Purity (HPLC): 99% (column: Venusil XBP C18
(2) 4.6 mm × 150 mm; retention time: 5 min).
serine); (ii) SscCK1δ/ε (casein kinase 1δ/ε, porcine brain,
native, affinity purified) was assayed in buffer A with
170 µM of the CK1-specific peptide substrate; (iii) SscGSK-
3α/β (glycogen synthase kinase-3, porcine brain, native,
affinity purified) isoforms were assayed in buffer A with
20 µM of GS-1 peptide, a GSK-3-selective substrate
(YRRAAVPPSPSLSRHSSPHQSpEDEEE). Composition
of buffer A: 10 mM MgCl2, 1 mM EGTA, 1 mM DTT,
25 mM Tris-HCl pH 7.5, 50 µg/mL heparin. The peptides
were obtained from Proteogenix (Oberhausbergen, France).
To measure the kinase activities, we used the ADP-
GloTM assay kit according to the recommendations of the
manufacturer (Promega, Madison, WI). This assay is a
luminescent ADP detection assay that provides an
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 384-well plates 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
10 µM 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).
The compounds were first diluted at 10 mM in DMSO
(stock solution) before to be analyzed as described here-
after. In this study, we used a two-step process to evaluate
the effect of the 7-azaindole derivatives on kinase activity:
(Step 1) As primary screening, the compounds were tested
at 10 and 1 µM against a panel of eight disease-related
kinases. Activities were expressed in % of maximal
activity, i.e. measured with dimethyl sulfoxide (DMSO)
diluted at 1% (v/v) concentration; (Step 2) The more
active compounds (dubbed “hits”) were next tested in
duplicate over a wide range of concentrations (usually
from 0.0003 to 10 μM) against the targets identified during
“step 1”, here Haspin and CDK9/CyclinT, and IC50 values
were determined from the dose response curves using
Prism-GraphPad (GraphPad Software, San Diego,
CA, USA).
5-(6-Fluoropyridin-3-yl)-1H-pyrrolo[2,3-b]pyridine
(19o): compound was synthesised from 2-fluoropyridine-5-
boronic acid and 5-bromo-7azaindole in a yield of 60.62%,
recrystallized from acetonitrile, gold powder, mp
204–205 °C, Rf = 0.26 (2 petroleum ether: 1 ethyl acetate).
1H NMR (DMSO-d6, 600 MHz) δ 11.82 (1H, s, NH), 8.59
(1H, d, J = 2.4 Hz, H-6), 8.55 (1H, d, J = 2.2 Hz, H-4′),
8.36–8.32 (1H, m, H-5′), 8.28 (1H, t, J = 2.3 Hz, H-2), 7.56
(1H, s, H-2′), 7.30 (1H, dd, J = 8.8, 2.9 Hz, H-6′), 6.52 (1H,
m, H-3). 13C NMR (DMSO-d6, 150 MHz) δ 162.8 (d, J =
235.3 Hz, C-4′), 148.8 (s, C-7a), 145.8 (d, J = 15.0 Hz, C-
2′), 141.9 (s, C-6), 140.9 (d, J = 7.9 Hz, C-1′), 133.7 (d,
J = 4.5 Hz, C-6′), 127.8 (s, C-5), 126.9 (s, C-4), 124.4 (s, C-
2), 120.1 (s, C-), 110.1 (d, J = 37.7 Hz, C-5′), 100.7 (s, C-
3). APCI-HRMS m/z: calc. for C12H9FN3 (MH+),
214.077502, found 214.0791; Purity (HPLC): 99% (col-
umn: Venusil XBP C18(2) 4.6 mm × 150 mm; retention
time: 4 min).
Cytotoxicity prediction
Protein kinase assays
The cytotoxicity for the most promising compounds were
predicted using CLC-Pred: in silico prediction of cytotoxi-
city for tumour and non-tumour cell-lines (Lagunin et al.
2018). CLC-Pred was developed by using the previously
developed Prediction of Activity Spectra for Substances
(PASS) algorithm to create and validate the classification
structure-activity relationship (SAR) models for predicting
Kinase activities were assayed in duplicate and described in
Nguyen et al (Nguyen et al. 2019) except for the following
enzymes: (i) LmCK1 (from Leishmania major, recombi-
nant, expressed in bacteria) was assayed in buffer A with
170 µM of the following CK1-specific peptide substrate:
RRKHAAIGSpAYSITA (“Sp” stands for phosphorylated

Medicinal Chemistry Research
Table 4 Kinase inhibitory activity of compounds selected after
primary screening (compounds inhibiting >70% of the kinase
activity at 1 µM). Activities were assessed in duplicate using
10 µM ATP
the cytotoxicity of structures against various types of human
cell lines using ChEMBL experimental data. The experi-
mental data from ChEMBL (IG50, IC50 and % inhibition
values) forms the basis for a training set of 59882 structures
of compounds and the average accuracy of predicting area-
under-the-curve (AUC) is given as 0.930 and 0.927 for 278
cancer cell-lines, when calculated by leave-one-out and 20-
fold cross-validation procedure, respectively (Lagunin et al.
2018).
Cpd
HsCDK9/CyclinT
HsHaspin
18a
0.081
0.481
0.206
0.063
0.137
1.200
NP
NP
18b
0.221
0.118
1.016
NP
18c
18e
18f
18h
0.284
0.664
NP
Results and discussion
19n
Flavopiridol
CHR-6494
0.015
NP
7-Azaindole derivatives have been investigated against
numerous PKs wherein they have exhibited good inhibition
potentials (see Fig. 1). The inhibitory potential of this
compound class against Haspin, a kinase with less similarity
to those mentioned in Fig. 1, has not been investigated
(Amoussou et al. 2018). Hence, in the current study a
library of twenty-four 7-azaindole derivatives was synthe-
sised and screened against Haspin. The compounds were
also screened against a panel of other PKs. The screening
panel consisted of kinases: HsCDK2/CyclinA, HsCDK5/
p25, HsCDK9/CyclinT, HsHaspin, HsPIM1, SscCK1δ/ε,
SscGSK3α/β and LmCK1. Haspin is of particular interest
as a novel biological target with regards to these
compounds.
Initially, the compounds were screened at concentrations
of 10 and 1 µM (diluted in 1% DMSO v/v). Only the
compounds exhibiting inhibition greater than 70% at 1 µM
(as arbitrary threshold) underwent more thorough testing (at
a wide range of concentrations, usually 0.0003 µM–10 µM)
and IC50 values were determined by means of dose-
response curves (Prism-GraphPad). The results of the
in vitro kinase assays can be found in Table 3 and Table 4.
The 7-azaindole derivatives of this study proved to
exhibit inhibitory activity against two of the eight PKs in
the panel, namely, CDK9/Cyclin T and Haspin. Interest-
ingly, the compounds that were deemed active (18a, 18b,
18c, 18e, 18f, 18h, & 19n) all belong to the 4-substituted
7-azaindole derivatives, with the exception of one
5-substituted 7-azaindole derivative, 19n. Their 5-substituted
7-azaindole counterparts 19a, 19b, 19d, 19g, 19h and 19j
lacked significant activity, thus, it can be postulated that
substitution on the 4-position may greatly improve inhibi-
tion of CDK9/Cyclin T and Haspin. Moreover, active
compounds seem to favour inhibition of CDK9/Cyclin T
over Haspin with inhibition ranging from 0.063 µM (18e) ≤
IC50 ≤ 1.2 µM (18h) for CDK9/Cyclin T and 0.118 µM
(18c) ≤ IC50 ≤ 1.016 µM (18e) for Haspin.
0.055
Values represent IC50 reported in µM. Kinase activities were assessed
in duplicate
NP Not performed
substituent (18e), which exhibited inhibitory activity against
CDK9/CyclinT of 0.063 µM, followed closely by substitu-
tion with an unsubstituted phenyl ring (18a), which also
achieved sub-micromolar activity (0.081 µM). Comparison
of the active 4-substituted 7-azaindoles with the 4-
substituted compounds that did not exhibit significant
activity, revealed that the molecular weight of the sub-
stituent (in addition to position of the substituent) may have
an impact of inhibitory activity against CDK9/Cyclin T.
The compounds deemed active all bear a substituent of low
molecular weight (< 130 g/mol), where 130 g/mol per sub-
stituent is surpassed (18d, 18g, 18i), the compound has
lower or insignificant inhibitory CDK9/Cyclin T activity in
comparison to the active 4-substituted 7-azaindole deriva-
tives. In conjunction with molecular weight, inhibitory
activity for CDK9/Cyclin T may also be governed by the
electronegativity of the substituent. Three compounds
bearing comparable electronegative para-substituted phenyl
rings were assessed, it was found that the more electro-
negative the atom (electronegativity: F > OCH3 > CH3), the
better the inhibitory activity of the compound (CDK9/
CyclinT activity: 18e > 18c > 18b) see Fig. 2. However, the
impact exacted by molecular weight of substituents seems
to outweigh the impact exacted by the electronegativity of
substituents, as both 18g and 18h are highly electro-
negative, but exhibit only a poor activity and relatively low
inhibitory activity (e.g. IC50 of 1.2 µM for 18 h). Haspin,
like CDK9/Cyclin T, seems to favour the lower molecular
weight 4-substituted 7-azaindole derivatives (18b, 18c, 18e,
18h) with regards to inhibitory activity, with the exception
of 18a (substituted with an unsubstituted phenyl ring) which
exhibits no significant inhibitory activity.
Considering solely the inhibitory activity for CDK9/
CyclinT, the best results were achieved by substituting the
4-position of the 7-azaindole with a 4′-fluorophenyl
Regarding the cytotoxicity of the active compounds, the
CLC-Pred prediction platform was used to predict the

Medicinal Chemistry Research
Table 5 Predicted cytotoxicity most promising compounds
(compounds inhibiting >70% of the kinase activity at 1 µM)
Cpd
Probability to Cell-line
be active (Pa)
Cell-line full name
18a
18b
18c
18e
18f
0.721
0.636
0.652
0.759
0.734
0.787
Hs 683
Hs 683
Hs 683
Hs 683
Hs 683
Hs 683
Oligodendroglioma
Oligodendroglioma
Oligodendroglioma
Oligodendroglioma
Oligodendroglioma
Oligodendroglioma
18h
19n
Fig. 2 Examples of 7-azaindole derivatives showing the significance
of substitution with a more electronegative substituent in the para-
position of the phenyl ring
0.638
0.545
Hs 683
A-375
Oligodendroglioma
Malignant melanoma
Flavopiridol 0.509
MRC5
Embryonic lung
fibroblast (non-tumour
cell-line)
cytotoxicity of these compounds (Lagunin et al. 2018). All
of the active compounds (18a, 18b, 18c, 18e, 18f, 18h, &
19n) were predicted to have a cytotoxicity score higher than
at least 60% of the compounds in the training set for oli-
godendrogliomas (Hs 683). Compound 18h, exhibited the
highest predicted cytotoxicity score (0.787), meaning that
18h can be expected to be more cytotoxic against oligo-
dendrogliomas than 78.7% of the compounds in the training
set. Remarkably, the only active compound from the second
series 19n, is also the only active compound that is pre-
dicted to have cytotoxic activity against more than one cell-
line, oligodendroglioma and malignant melanoma (Hs 683:
0.683 and A-375: 0.545) (see Table 5).
CHR-6494
0.385
0.328
0.319
0.317
0.338
0.368
0.357
0.303
0.307
A-375
PA-1
SJSA-1
RKO
Malignant melanoma
Ovarian carcinoma
Osteosarcoma
Colon carcinoma
Kasumi 1 Childhood acute
DMS-114 myeloid leukaemia with
NCI-H838 maturation
SK-MEL- Lung carcinoma
1
Non-small cell
lung cancer
Metastatic melanoma
Breast carcinoma
MDA-
MB-453
Pa values represent the percentage of active compounds (from the
training set) that is predicted to have a lower cytotoxicity for a specific
cell-line than the investigated compound (Lagunin et al. 2018)
Overall, 7 active inhibitors of CDK9/Cyclin T and/or
Haspin have been identified (18a, 18b, 18c, 18e, 18f, 18h,
& 19n) with a high likelihood of being active against oli-
godendrogliomas. Thus, these compounds may have value
as potential antimitotic agents in cancer therapy.
proliferation and have been identified as targets in cancer
therapy, thus compounds 18a, 18b, 18c, 18e, 18f, 18h and
19n may potentially be of value as novel anti-proliferative
agents (Huertas et al. 2012; Walsby et al. 2014). Further-
more, it is postulated that compound 18c will make an
excellent lead compound for further exploration of
7-azaindole derivatives as Haspin inhibitors.
Conclusions
This article describes the synthesis of selected 4-, and 5-
substituted azaindole derivatives, formed by the combina-
tion of the 7-azaindole scaffold and appropriate boronic
acids. Protein kinase assays unveiled seven promising
compounds; two compounds exhibited selective activity
against CDK9/Cyclin T and one compound selectively
against Haspin, while four compounds were identified as
dual-target inhibitors of both CDK9/Cyclin T and Haspin,
all with IC50 values in a nanomolar to low micromolar
range. Furthermore, compound 18c not only proved to be
the most potent of the target inhibitors, but also, when
considered separately, was identified as the compound with
the best Haspin inhibitory activity (IC50 value of 0.118 µM
against Haspin and 0.206 µM against CDK9/Cyclin T).
Regarding CDK9/Cyclin T, compound 18e reigned
supreme with an IC50 value of 0.063 µM. Both Haspin and
CDK9/Cyclin T, have been connected to normal cell
Acknowledgements The authors are grateful to Dr. D.Otto and Dr. J.
Jordaan of the SASOL Centre for Chemistry, North-West University,
for recording the MS and NMR spectra, respectively. The authors
thank the “Ligue contre le Cancer du Grand-Ouest” comity, (districts:
29, 22, 56, 35, 45, and 79), Cancéropôle Grand Ouest (axis: natural sea
products in cancer treatment network), IBiSA (French Infrastructures
en sciences du vivant: biologie, santé et agronomie) and Biogenouest
(Western France life science and environment core facility network)
for supporting the KISSf screening facility (FR2424, CNRS and
Sorbonne Université), Roscoff, France.
Compliance with ethical standards
Conflict of interest Stéphane Bach is a founder and member of the
SAB of SeaBeLife Biotech (Roscoff, France). This company is
developing novel therapies for treating liver and kidney acute dis-
orders. This work was conducted in the absence of any commercial or

Medicinal Chemistry Research
financial relationships that could be construed as a potential conflict of
interest. The authors declare that they have no conflict of interest
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Higgins JM (2001b) Haspin‐like proteins: a new family of evolutio-
narily conserved putative eukaryotic protein kinases. Protein Sci
10:1677–1684
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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