Synthesis and biological evaluation of selected 7H-pyrrolo[2,3-d]pyrimidine derivatives as novel CDK9/CyclinT and Haspin inhibitors

Article abstract of DOI:10.1016/j.cbi.2021.109643

Protein kinases, including CDK9/CyclinT and Haspin, are regarded as potential drug targets in cancer therapy. Findings from a previous study suggested 7-azaindole as a privileged scaffold for producing inhibitors of CDK9/CyclinT and Haspin. Inspired by these findings, the current study synthesised and evaluated thirteen (13) C6-substituted 7-azaindole and twenty (20) C4-substituted structurally related 7H-pyrrolo[2,3-d]pyrimidine derivatives against a panel of protein kinases, including CDK9/CyclinT and Haspin. Eleven of the 7H-pyrrolo[2,3-d]pyrimidine derivatives exhibited activity toward CDK9/CyclinT, while 4 of compounds had activity against Haspin. The best CDK9/CyclinT (IC₅₀ of 0.38 μM) and Haspin (IC₅₀ of 0.11 μM) activities were achieved by compounds 7d and 7f, respectively. Hence, these compounds may be valuable starting points for development of new anti-cancer drugs.

Full text of DOI:10.1016/j.cbi.2021.109643

Chemico-Biological Interactions 349 (2021) 109643
Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Synthesis and biological evaluation of selected 7H-pyrrolo[2,3-d]
pyrimidine derivatives as novel CDK9/CyclinT and Haspin inhibitors
,
Lianie Pietersea, Richard M. Betecka, Blandine Baratteb c, Omobolanle J. Jesumorotia,
,
c
b
a
,
b
,
,*
Thomas Robertb , Sandrine Ruchaud , Stephane Bach
c, Lesetja J. Legoabea
´
a Centre of Excellence for Pharmaceutical Sciences, North-West University, Private Bag X6001, Potchefstroom, 2520, South Africa
b
´
Sorbonne Universite, CNRS, UMR8227, Integrative Biology of Marine Models Laboratory (LBI2M), Station Biologique de Roscoff, 29680, Roscoff, France
c
´
Sorbonne Universite, CNRS, FR2424, Plateforme de Criblage KISSf (Kinase Inhibitor Specialized Screening Facility), Station Biologique de Roscoff, 29680, Roscoff
Cedex, France
A R T I C L E I N F O
A B S T R A C T
Keywords:
Protein kinases, including CDK9/CyclinT and Haspin, are regarded as potential drug targets in cancer therapy.
Findings from a previous study suggested 7-azaindole as a privileged scaffold for producing inhibitors of CDK9/
CyclinT and Haspin. Inspired by these findings, the current study synthesised and evaluated thirteen (13) C6-
substituted 7-azaindole and twenty (20) C4-substituted structurally related 7H-pyrrolo[2,3-d]pyrimidine de-
rivatives against a panel of protein kinases, including CDK9/CyclinT and Haspin. Eleven of the 7H-pyrrolo[2,3-d]
pyrimidine derivatives exhibited activity toward CDK9/CyclinT, while 4 of compounds had activity against
Protein kinase
7-Deazapurine
CDK9/CylinT
Haspin
Anticancer
Haspin. The best CDK9/CyclinT (IC50 of 0.38 μM) and Haspin (IC50 of 0.11 μM) activities were achieved by
compounds 7d and 7f, respectively. Hence, these compounds may be valuable starting points for development of
new anti-cancer drugs.
1. Introduction
appropriate PK. Nowadays, 52 PK inhibitor-based drugs are approved by
the US food and drugs administration, where 46 of the 52 is used in the
In 1955 Fischer and Krebs reported evidence that the conversion of
phosphorylase b to a, as observed in cell-free muscle extracts, is
dependent on nucleotides with high energy phosphates in the presence
of high energy divalent metal ions [1]. This opened a door into the world
of protein kinases (PKs) as phosphorylase became the first known PK to
be purified [2]. Since then, it has become apparent that the human
kinome consists of 518 PKs (equating to 1.7% of all human genes) and is
indispensable to normal cellular function [3]. PKs play a key part in
regulating signal transduction by promoting the transfer of phosphoryl
groups from phosphate donors, like adenosine triphosphate (ATP), to
receptor substrates [4]. The associated signal transduction pathways
significantly influence biological systems as they determine cellular re-
sponses to environmental stimuli and modify gene expression [5].
Erroneous signal transduction may lead to disruption of normal bio-
logical processes and could ultimately result in diseases such as, cancer,
inflammation, diabetes and neurodegenerative disease [6–9]. Thus, the
appeal of PK inhibitors as drug targets is rooted in the expectation that
erroneous signal transduction can be halted by inhibition of the
treatment of neoplastic diseases. Examples include abemaciclib, palbo-
ciclib and ricociclib (CDK4/6 inhibitors) [10].
Cyclin-dependant kinase CDK9/CyclinT complex is an example of
such a therapeutic target. CDK9 is a member of CMGC family of kinases:
for Cyclin-dependent kinase (CDK), Mitogen-activated protein kinase
(MAPK), Glycogen synthase kinase (GSK) and CDC-like kinase (CLK).
CMGC kinases are key regulators involved in the regulation of DNA
repair and cell fate (cell proliferation, differentiation, apoptosis) [11].
CDK9 is indispensable in the governance of non-ribosomal transcription,
which includes super-enhancers regulated gene expression.
Super-enhancers are clusters of DNA regulatory components that guide
transcription of genes of significance in cell-identity [12]. As abnor-
malities in the regulation of CDK9 activity have been described in acute
myeloid leukaemia (AML), other haematological malignancies and solid
tumours, the chemical inhibition of CDK9 is considered as a promising
strategy in cancer therapy [11].
Another kinase of interest is the Haploid germ cell-specific nuclear
protein kinase (Haspin). Haspin is one of the newer additions to the
* Corresponding author.
E-mail address: Lesetja.Legoabe@nwu.ac.za (L.J. Legoabe).
https://doi.org/10.1016/j.cbi.2021.109643
Received 30 May 2021; Received in revised form 7 August 2021; Accepted 6 September 2021
Available online 9 September 2021
0009-2797/© 2021 Elsevier B.V. All rights reserved.

L. Pieterse et al.
Chemico-Biological Interactions 349 (2021) 109643
kinome and remains relatively underexplored as a drug target ([13]. It is
present across a variety of eukaryotic species (animal, fungi and plants)
and was first detected in male germ cells [14,15]. This Ser/Thr kinase is
structurally unique as compared to other members of the PK family [16].
It displays essential function during cell division and is involved in
histone code printing [13,17,18]. Haspin depletion by CRISPR/Cas9 in
Breast cancer HeLa cells triggers severe mitotic defects and ultimately
cell death by mitotic catastrophe [19].
study, the substituents were chosen to match their counterparts in the
previous study to determine if results would mirror that of the previous
study. After the initial synthesis, it was decided to expand the series in
order to examine a more diverse set of compounds. The additional
substituents (7c–e, 7h–l) were selected to complement the existing
substituents (7a, 7b, 7f, 7g, 7m–p).
These compounds were evaluated in vitro against CDK9/CyclinT and
Haspin kinases (see Tables 1–3). Additionally, these compounds were
screened against other PKs: (i) CDK2/CyclinA, (ii) CDK5/p25, (iii) Pim-
In humans, Haspin mRNA expression has been marked in all prolif-
erating cell-lines such as, thymus, bone marrow and foetal liver cell-
lines. Haspin has also been found overexpressed in several highly pro-
liferative malignant tumours such as melanomas, myelomas, pancreatic
and bladder cancer [20–23]. The degree of Haspin expression in tissue
correlates with the degree of cellular proliferation and differentiation
each tissue experiences and often correlates with the most aggressive
stages of the cancers [18,24–27]. Furthermore, Haspin inhibition or
knockdown has been shown to slow down the development of several
cancers in animal models [20]; [22,28,29]. Consequently, Haspin could
prove to be a valuable target in mitosis and thus cancer therapy.
In a previous study [30], 7-azaindole derivatives were identified as
dual-inhibitors of CDK9/CyclinT and Haspin (see 1 and 2, Fig. 1). This
illustrates the ability of the 7-azaindole scaffold to produce potential
novel CDK9/CyclinT and Haspin inhibitors. In the previous study, the
substituents were selected based on their commercial availability,
relative affordability and the fact that the scaffold was novel to most of
the targets on the PK panel, thus allowing for simpler structures to be
examined whilst retaining novelty [30]. Prompted by the success of the
previous study, a series of selected 6-substituted 7-azaindole derivatives
1, (iv) CK1ε, (v) GSK-3β, (vi) LmCK1 (from the parasite Leishmania
major) and (vii) ABL1. The structures of the selected 7-azaindole and 7-
deazapurine derivatives were subjected to a SciFinder search in order to
establish the novelty of each compound. The structure search for com-
pounds 5b, 5c, 5f, 5g–k, 7c–e, 7g, 7h, 7l–n, and 8a – c produced no
former references and they were considered novel. Compounds 5a [31],
5d, 5e [32], 7a, 7b, 7f [33], 7i, 7j, 7k [34], 7o [35] and 7p [36] had
been referenced in previous literature works. Nevertheless, none of the
compounds had previously been examined as CDK9/CyclinT or Haspin
inhibitors. In this study, we described the inhibitory activity in the low
micromolar range of six compounds against CDK9/CyclinT and five
compounds against Haspin. Among these molecular hits, four com-
pounds are dual-target inhibitors.
2. Material and methods
The selected 7-azaindole and 7-deazapurine derivatives were syn-
thesised as described in the experimental section. Materials for synthesis
were sourced from AK Scientific (for 5a–k, 7a, 7b, 7e, 7f, 7g, 7l, 7m, 7n,
7o, 7p and 8a – c) and Ambeed (for 7c, 7d, 7h, 7j, and 7k), while CHR
6494 and flavopiridol were purchased from Sigma Aldrich. All materials
and
a series of structurally related 7H-pyrrolo[2,3-d]pyrimidine
(7-deazapurine) derivatives were synthesised. In the case of the current
Fig. 1. A depiction of the 7-azaindole and the 7-deazapurine scaffolds.
2

L. Pieterse et al.
Chemico-Biological Interactions 349 (2021) 109643
Table 1
A table depicting the synthesis of C6-substituted 7-azaindole derivatives, target compounds and yields achieved.
were used without further purification, unless otherwise specified.
Proton (1H) and carbon (13C) nuclear magnetic resonance (NMR) spectra
were recorded utilizing a Bruker Avance III 600 spectrometer at fre-
quencies of 600 MHz and 150 MHz, respectively. Deuterated dimethyl
sulfoxide (DMSO‑d6) served as the solvent for all NMR analysis. The
chemical shifts (δ) are reported in parts per million (ppm) and internally
referenced to the signal of tetramethyl silane (TMS). Spin multiplicities
are indicated as follow: s (singlet), d (doublet), t (triplet), q (quartet), dd
(doublet of doublets) and m (multiplet) and ddd (doublet of doublet of
doublets). 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
Chloro-7-deazapurine) were reacted with a variety of commercially
available boronic acids, by means of Suzuki-Miyaura coupling, to afford
a series of C6-substituted 7-azaindole derivatives (5a–k) and a series of
C4-substituted-7H-pyrrolo[2,3-d]pyrimidine
derivatives
(7a–p),
respectively. Furthermore, the 4,7-bis-substituted-7H-pyrrolo[2,3-d]
pyrimidine derivatives (8a–c) were derived from 4-chloro-7H-pyrrolo
[2,3-d]pyrimidine subjected to a combination of Suzuki-Miyaura
coupling (at the 4-Cl) and Chan-Lam coupling (at the NH) with the
appropriate boronic acids. Suzuki-Miyaura coupling promotes carbon-
carbon cross coupling between organoborons and organic halides in
the presence of a palladium catalyst [37,38]; whereas Chan-Lam
coupling refers to a reaction between aryl boronic acids and an alco-
hols or amines and is catalysed by copper complexes [39]. In the
instance of compounds 5a–k and 7a–p, tetrakis(triphenylphosphine)
palladium(0) (Pd(PPh3)4), served as catalyst, while compounds 8 –c,
were synthesised by means of cupric acetate Cu(OAc)2 in addition to Pd
(PPh3)4.
system, 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) against a gradient of 50% B (0–2min),
◦
100% B (2–8min), 100% B (8–11min), 50% B (11.1–13min) at 22 C.
Verification of the compounds were achieved by utilizing NMR and
HRMS analyses. In terms of HRMS, the MH+ for every compound was
determined and compared to the predicted MH+. All the determined
MH+ values corresponded to the predicted values, thus proving that
each compound exhibits the correct molecular weight for its structure.
Melting points (mp) were determined by a Buchi B545 melting point
apparatus and are uncorrected. Thin layer chromatography (TLC) was
performed using silica gel 60 TLC plates (Merck) and a UV254 fluores-
cent indicator to determine retention factor (Rf), with petroleum ether:
ethyl acetate (1:1) as eluent.
1
Concomitantly, the H and 13C NMR spectra revealed the anticipated
number of hydrogen and carbon atoms, respectively, for each com-
pound. In general, the 1H NMR spectra of all the 7-azaindole derivatives
depict a peak at approximately 6.40 that can be assigned to H3. In the
case of the 7H-pyrrolo[2,3-d]pyrimidine derivatives, a similar peak is
seen at approximately 6.80 and denotes the corresponding hydrogen H5.
Furthermore, the 7H-pyrrolo[2,3-d]pyrimidine derivatives possess a
3. Experimental
3.1. Chemistry
6-Chloro-7-azaindole and 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (6-
3

L. Pieterse et al.
Chemico-Biological Interactions 349 (2021) 109643
Table 2
A table depicting the synthesis of the C4-substituted 7H-pyrrolo[2,3-d]pyrimidine derivatives, target compounds and yields
achieved.
very distinct H2 singlet at approximately 8.80 ppm. Additionally, the 7-
azaindole and 7H-pyrrolo[2,3-d]pyrimidine derivatives produce a broad
singlet at approximately 12.20 ppm, in most cases. The absence of this
peak in the proton spectra of the 4,7-bissubstituted 7H-pyrrolo[2,3-d]
pyrimidine derivatives, could suggest that the Chan-Lam coupling has
been successful. All the characterization data can be found in the sup-
porting info.
solution and 6.092 mmol of the appropriate boronic acid in 25 mL of a
3:1 toluene: ethanol mixture was prepared. The reaction mixture was
stirred continuously under reflux at 120 ◦C for at least 24 h. TLC plates
were used to monitor the progress of the reaction. Upon completion of
the reaction, the reaction mixture was left to cool to room temperature
before the solvent was removed in vacuo. This yielded a residue that was
dissolved in dichloromethane and washed with distilled water (3 × 25
mL). The combined organic layers were dried over suitable drying agent
(MgSO4), filtered and evaporated in vacuo. The resulting product was
recrystallized from suitable solvents.
3.2. General synthetic procedure for the C6-substituted 7-azaindole
derivatives (5a–k)
Compounds 5a–k were synthesised under nitrogen atmosphere, in
accordance with required conditions for Suzuki-Miyaura coupling re-
action. For each compound, a mixture of 6-chloro-7-azaindole (1.523
mmol), Pd(PPh3)4 (0.0023 mmol), 1.6 mL of a 2 M K2CO3 aqueous
3.3. General synthetic procedure for the selected 4-substituted-7H-pyrrolo
[2,3-d]pyrimidine derivatives (7a–p)
Compounds 7a–p were synthesised under inert nitrogen atmosphere.
4

L. Pieterse et al.
Chemico-Biological Interactions 349 (2021) 109643
Table 3
A table depicting the synthesis of the 4, 7-bissubstituted 7H-pyrrolo[2,3-d]pyrimidine derivatives, target compounds and yields
achieved.
The preparation of each compound entailed mixing 4-chloro-7H-pyrrolo
[2,3-d]pyrimidine (6-Chloro-7-deazapurine) (1.95 mmol), Pd(PPh3)4
(0.05 mmol), 1.6 mL of a 2 M K2CO3 solution and 5.85 mmol of the
appropriate boronic acid in 50 mL of a 3:1 toluene: ethanol mixture. The
reaction mixture was stirred under reflux at 120 ◦C for a minimum
period of 24 h. Progress was monitored with TLC plates, using petroleum
ether: ethyl acetate (1:1 v/v) mixture as mobile phase. Upon completion
of the reaction, the solvent was removed under reduced pressure. This
yielded a residue that was dissolved in dichloromethane and washed
with distilled water (3 × 25 mL). The combined organic layers were
dried with MgSO4, filtered and subjected to reduced pressure. The
resulting residues were recrystallized from suitable solvents.
Hz, Ar–H), 7.51–7.46 (3H, m, Ar–H), 7.39 (1H, t, J = 7.3 Hz, Ar–H), 6.48
(1H, m, Ar–H). 13C NMR (DMSO‑d6, 150 MHz) δ 149.96, 149.06,
140.37, 129.40, 129.13, 128.53, 127.24, 126.89, 119.28, 113.08,
100.36. APCI-HRMS m/z: calcd for C13H10N2 (MH+), 195.0917, found:
195.0913; Purity (HPLC): 99%.
3.4.2. 6-(4-Methylphenyl)-1H-pyrrolo[2,3-b]pyridine (5b)
Compound was synthesised from 4-methylphenylboronic acid and 6-
chloro-7-azaindole in a yield of 94%, recrystallized from acetonitrile,
maroon coloured crystals, mp 218 ◦C, Rf = 0.67 (dichloromethane: ethyl
acetate; 4:1). 1H NMR (DMSO‑d6, 600 MHz) δ 11.66 (1H, s, NH),
8.01–7.95 (Ar–H), 7.61 (1H, d, J = 8.2 Hz, Ar–H), 7.47–7.44 (1H, m,
Ar–H), 7.28 (2H, d, J = 7.6 Hz, Ar–H), 6.46–6.43 (1H, m, Ar–H), 2.35
(3H, s, Me-4’). 13C NMR (DMSO‑d6, 150 MHz) δ 149.55, 148.54, 137.36,
137.14, 129.23, 128.82, 126.46, 126.28, 118.54, 112.30, 99.83, 20.76.
APCI-HRMS m/z: calcd. for C14H12N2 (MH+), 209.1073, found
209.1077; Purity (HPLC): 99%.
3.4. General synthetic procedure for the selected 4,7-bis-substituted-7H-
pyrrolo[2,3-d]pyrimidine derivatives (8a–c)
Compounds 8a–c were synthesised under inert nitrogen atmosphere.
To prepare each of the bis-substituted compounds, a mixture of 4-chloro-
7H-pyrrolo[2,3-d]pyrimidine (6-Chloro-7-deazapurine) (3.26 mmol),
Pd(PPh3)4 (0.10 mmol), Cu(OAc)2 (3.88 mmol), K2CO3 (17.37 mmol)
and 16.3 mmol of the appropriate boronic acid in 50 mL of a 1:1 water:
ethanol mixture was refluxed at 120 ◦C for a minimum of 24 h. Progress
was monitored with TLC plates using petroleum ether: ethyl acetate (1:1
v/v) as mobile phase. Upon completion of the reaction, the reaction
mixture was filtered through Celite® and the filtrate concentrated under
reduced pressure. The resulting residue was dissolved in dichloro-
methane and washed with distilled water (3 × 25 mL). The combined
organic layers were dried with MgSO4 and evapourated in vacuo. The
resulting product was recrystallized from a suitable solvent.
3.4.3. 6-(3-Methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (5c)
Compound was synthesised from 3-methoxyphenylboronic acid and
6-chloro-7-azaindole in a yield of 54%, recrystallized from acetonitrile,
brown coloured crystals, mp 139 ◦C, Rf = 0.67 (4 dichloromethane: 1
ethyl acetate). 1H NMR (DMSO‑d6, 600 MHz) δ 11.69 (1H, s, NH), 7.97
(1H, d, J = 8.2 Hz, Ar–H), 7.62–7.60 (3H, m, Ar–H), 7.45–7.43 (1H, m,
Ar–H), 7.35–7.32 (1H, m, Ar–H), 6.90 (1H, ddd, J = 8.1, 2.5, 0.8 Hz,
Ar–H), 6.42–6.41 (1H, m, Ar–H), 3.79 (3H, s, MeO-3′). 13C NMR
(DMSO‑d6, 150 MHz) δ 159.67, 149.25, 148.51, 141.43, 129.71, 128.88,
126.46, 126.85, 118.96, 118.84, 113.90, 112.81, 111.55, 99.91, 55.12
(MeO-3’). APCI-HRMS m/z: calcd. for C14H12N2O (MH+), 225.1022,
found 225.1020; Purity (HPLC): 92%.
3.4.1. 6-Phenyl-1H-pyrrolo[2,3-b]pyridine (5a)
3.4.4. 6-(4-Methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (5d)
Compound was synthesised from phenylboronic acid and 6-chloro-7-
azaindole in a yield of 26%, recrystallized from acetonitrile, gold-
Compound was synthesised from 4-methoxyphenylboronic acid and
6-chloro-7-azaindole in a yield of 72%, recrystallized from acetonitrile,
dark yellow-coloured crystals, mp 185 ◦C, Rf = 0.62 (dichloromethane:
ethyl acetate; 4:1). 1H NMR (DMSO‑d6, 600 MHz) δ 11.61 (1H, s, NH),
◦
coloured crystals, mp 189 C, Rf = 0.71 (dichloromethane: ethyl ace-
tate; 4:1). 1H NMR (DMSO‑d6, 600 MHz) δ 11.73 (1H, s, NH), 8.10 (2H,
d, J = 7.8 Hz, Ar–H), 8.03 (1H, d, J = 8.2 Hz, Ar–H), 7.66 (1H, d, J = 8.2
5

L. Pieterse et al.
Chemico-Biological Interactions 349 (2021) 109643
8.03 (2H, d, J = 8.0 Hz, Ar–H), 7.96 (1H, d, J = 8.2 Hz, Ar–H), 7.56 (1H,
d, J = 8.2 Hz, Ar–H), 7.43 (1H, s, Ar–H), 7.02 (2H, d, J = 8.0 Hz, Ar–H),
6.42 (1H, s, Ar–H), 3.80 (3H, s, MeO-4′). 13C NMR (DMSO‑d6, 150 MHz)
δ 159.41), 149.43, 148.54, 132.44, 128.83, 127.62, 126.16, 118.17,
114.02, 111.97, 99.82, 55.16 (MeO-4’). APCI-HRMS m/z: calcd. for
(1H, m, Ar–H), 8.01 (1H, d, J = 8.14 Hz, Ar–H), 7.67 (1H, d, J = 8.2 Hz,
Ar–H), 7.49–7.47 (1H, m, Ar–H), 7.44 (1H, dd, J = 7.8, 4.7 Hz, Ar–H),
6.45–6.43 (1H, m, Ar–H). 13C NMR (DMSO‑d6, 150 MHz) δ 148.91,
148.65, 147.70, 146.96, 135.24,133.69, 129.16, 127.32, 123.76,
119.39, 112.83, 100.04. APCI-HRMS m/z: calcd. for C12H9N3 (MH+),
196.0869, found 196.0886; Purity (HPLC): 91%.
C
14H12N2O (MH+), 225.1022, found 225.1035; Purity (HPLC): 97%.
3.4.5. 6-(4-Fluorophenyl)-1H-pyrrolo[2,3-b]pyridine (5e)
3.4.10. 6-(Pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine (5j)
Compound was synthesised from 4-fluorophenylboronic acid and 6-
chloro-7-azaindole in a yield of 68%, recrystallized from acetonitrile,
light brown coloured crystals, mp 217 ◦C, Rf = 0.89 (dichloromethane:
ethyl acetate; 4:1). 1H NMR (DMSO‑d6, 600 MHz) δ 11.69 (1H, s, NH),
8.16–8.09 (2H, m, Ar–H), 8.01 (1H, d, J = 8.1 Hz, Ar–H), 7.62 (1H, d, J
= 8.1 Hz, Ar–H), 7.47 (1H, s, Ar–H), 7.28 (2H, d, J = 8.3 Hz, Ar–H),6.45
(1H, s, Ar–H). 13C NMR (DMSO‑d6, 150 MHz) δ 163.08, 161.46 (s),
148.49 (d, J = 0.8 Hz), 136.38 (d, J = 3.0 Hz), 128.98 (s), 128.38 (d, J =
8.3 Hz), 126.77 (s), 118.73 (s), 115.42 (d, J = 21.4 Hz), 112.40 (s),
99.88 (s). APCI-HRMS m/z: calcd. for C13H9FN2 (MH+), 213.0823,
found 213.0824; Purity (HPLC): 99%.
Compound was synthesised from 4-pyridininylboronic acid and 6-
chloro-7-azaindole in a yield of 59%, recrystallized from acetonitrile,
yellow coloured powder, mp 167 ◦C, Rf = 0.28 (dichloromethane: ethyl
acetate; 4:1). 1H NMR (DMSO‑d6, 600 MHz) δ 11.85 (1H, s, NH), 8.64
(2H, dd, J = 4.5, 1.6 Hz, Ar–H), 8.08 (1H, d, J = 8.2 Hz, Ar–H), 8.05 (2H,
dd, J = 4.5, 1.6 Hz, Ar–H), 7.79 (1H, d, J = 8.2 Hz, Ar–H), 7.57 (1H, d, J
= 3.4 Hz, Ar–H), 6.50 (1H, d, J = 3.4 Hz, Ar–H). 13C NMR (DMSO‑d6,
150 MHz) δ 150.15, 148.55, 146.74, 146.42, 129.05, 128.11, 120.57,
120.31, 113.03, 100.11. APCI-HRMS m/z: calcd. for C12H9N3 (MH+),
196.0869, found 196.0875; Purity (HPLC): 91%.
3.4.11. 6-(6-Fluoropyridin-3-yl)-1H-pyrrolo[2,3-b]pyridine (5k)
3.4.6. 6-(3,4-Difluorophenyl)-1H-pyrrolo[2,3-b]pyridine (5f)
Compound was synthesised from 6-fluoro-3-pyridinylboronic acid
and 6-chloro-7-azaindole in a yield of 59%, recrystallized from aceto-
nitrile, yellow coloured powder, mp 194 ◦C, Rf = 0.28 (dichloro-
methane: ethyl acetate; 4:1). 1H NMR (DMSO‑d6, 600 MHz) δ 11.79 (1H,
s, NH), 8.91 (1H, d, J = 2.5 Hz, Ar–H), 8.62 (1H, d, J = 8.3, 2.6 Hz,
Ar–H), 8.06 (1H, d, J = 8.1 Hz, H-4), 7.71 (1H, d, J = 8.2 Hz, H-5), 7.52
(1H, d, J = 3.4 Hz, H-2), 7.28 (1H, d, J = 8.6, 2.7 Hz, Ar–H), 6.48 (1H, d,
J = 3.4 Hz, Ar–H). 13C NMR (DMSO‑d6, 150 MHz) δ 162.85 (d, J =
236.3 Hz), 148.51 (s), 145.85 (s), 145.44 (d, J = 15.4 Hz), 139.86 (d, J
= 8.1 Hz), 133.99 (d, J = 4.4 Hz), 129.18 (s), 127.34 (s), 119.36 (s),
112.65 (s), 109.47 (d, J = 37.7 Hz), 100.01 (s. APCI-HRMS m/z: calcd.
for C12H8FN3 (MH+), 214.0775, found 214.0781; Purity (HPLC): 93%.
Compound was synthesised from 3,4-difluorophenylboronic acid
and 6-chloro-7-azaindole in a yield of 13%, recrystallized from aceto-
◦
nitrile, dark green coloured crystals, mp 173 C, Rf = 0.85 (dichloro-
methane: ethyl acetate; 4:1). 1H NMR (DMSO‑d6, 600 MHz) δ 11.72 (1H,
s, NH), 8.07 (1H, ddd, J = 12.5, 8.0, 2.1 Hz, Ar–H), 7.99 (1H, d, J = 8.2
Hz, Ar–H), 7.92 (1H, ddd, J = 4.2, 3.5, 2.0 Hz, Ar–H), 7.65 (1H, d, J =
8.2 Hz, Ar–H), 7.50–7.45 (2H, m, Ar–H), 6.44–6.42 (1H, m, Ar–H). 13
C
NMR (DMSO‑d6, 150 MHz) δ 150.45 (dd, J = 45.3, 12.9 Hz), 148.82 (dd,
J = 47.5, 12.7 Hz), 148.42 (s), 147.08 (s), 137.63 (dd, J = 5.7, 3.5 Hz),
130.15 (dd, J = 411.0, 10.8 Hz), 129.10 (s), 127.32 (s), 123.00 (dd, J =
6.4, 3.1 Hz), 119.27 (s), 116.38 (dd, J = 398.9, 17.7 Hz), 112.57 (s),
100.01 (s). APCI-HRMS m/z: calcd. C13H8F2N2 (MH+), 231.0728, found
231.0736; Purity (HPLC): 96%.
3.4.12. 4-Phenyl-7H-pyrrolo[2,3-d]pyrimidine (7a)
Compound was synthesised from phenylboronic acid and 6-chloro-7-
deazapurine in a yield of 53%, recrystallized from methanol, green
coloured powder, mp 221 ◦C, Rf = 0.46 (hexane: ethyl acetate: 1:1). 1H
NMR (DMSO‑d6, 600 MHz) δ 8.82 (1H, s, Ar–H), 8.18–8.15 (2H, m,
Ar–H), 7.64 (1H, d, J = 3.5 Hz, Ar–H), 7.57 (2H, m, Ar–H), 7.52 (1H, t, J
= 7.2 Hz, Ar–H), 6.86 (1H, d, J = 3.5 Hz, Ar–H). 13C NMR (DMSO‑d6,
150 MHz) δ 155.47, 152.63, 150.88, 137.94, 129.95, 128.79, 128.52,
127.75, 114.50, 99.90. APCI-HRMS m/z: calcd. for C12H9N3 (MH+),
196.0869 found 196.0863; Purity (HPLC): 99%.
3.4.7. 6-[4-(Trifluoromethyl)phenyl]-1H-pyrrolo[2,3-b]pyridine (5g)
Compound was synthesised from 4-trifluoromethylphenylboronic
acid and 6-chloro-7-azaindole in a yield of 50%, recrystallized from
acetonitrile, gold-coloured crystals, mp 247 ◦C, Rf = 0.71 (dichloro-
methane: ethyl acetate; 4:1). 1H NMR (DMSO‑d6, 600 MHz) δ 11.80 (1H,
s, NH), 8.31 (2H, d, J = 7.9 Hz, Ar–H), 8.07 (1H, d, J = 7.2 Hz, Ar–H),
7.82 (2H, d, J = 7.8 Hz, Ar–H), 7.74 (1H, d, J = 8.0 Hz, Ar–H), 7.55 (1H,
s, Ar–H), 6.49 (1H, s, Ar-HAr-H). 13C NMR (DMSO‑d6, 150 MHz) δ
148.56 (s), 147.58 (s), 143.73 (s), 129.07 (s128.15 (d, J = 31.8 Hz),
127.65 (s), 126.97 (s), 125.54 (q, J = 3.6 Hz), 124.43 (q, J = 271.8 Hz),
119.63 (s), 113.05 (s), 100.01 (s). APCI-HRMS m/z: calcd. for C14H9F3N2
(MH+), 263.0791, found 263.0807; Purity (HPLC): 99%.
3.4.13. 4-(4-Methylphenyl)-7H-pyrrolo[2,3-d]pyrimidine (7b)
Compound was synthesised from 4-methylphenylboronic acid and 6-
chloro-7-deazapurine in a yield of 24%, recrystallized from methanol,
pale yellow crystals, mp 207 ◦C, Rf = 0.61 (hexane: ethyl acetate: 1:1).
1H NMR (DMSO‑d6, 600 MHz) δ 12.21 (1H, s, NH), 8.80 (1H, s, Ar–H),
8.08 (2H, d, J = 8.1 Hz, Ar–H), 7.63 (1H, d, J = 3.5 Hz, Ar–H), 7.38 (2H,
d, J = 8.0 Hz, Ar–H), 6.87 (1H, d, J = 3.6 Hz, Ar–H), 6.87 (3H, s, Me-4′).
13C NMR (DMSO‑d6, 150 MHz) δ 155.49, 152.52, 150.86, 139.73,
135.19, 129.38, 128.45, 127.41, 114.24, 99.99, 20.96 (Me-4’). APCI-
HRMS m/z: calcd. for C13H11N3 (MH+), 210.1026, found 210.1029;
Purity (HPLC): 94%.
3.4.8. 4-(1H-Pyrrolo[2,3-b]pyridin-6-yl)benzonitrile (5h)
Compound was synthesised from 4-cyanophenylboronic acid and 6-
chloro-7-azaindole in a yield of 58%, recrystallized from acetonitrile,
◦
gold-coloured crystals, mp 216 C, Rf = 0.65 (dichloromethane: ethyl
acetate; 4:1). 1H NMR (DMSO‑d6, 600 MHz) δ 11.80 (1H, s, NH),
8.27–8.24 (2H, m, H-2′, H-6’), 8.04 (1H, d, J = 8.2 Hz, H-4), 7.90–7.87
(2H, m, Ar–H), 7.74 (1H, d, J = 8.2 Hz, Ar–H), 7.54 (1H, d, J = 3.4 Hz,
Ar–H), 6.47 (1H, d, J = 3.4 Hz, Ar–H). 13C NMR (DMSO‑d6, 150 MHz) δ
148.57, 147.16, 144.18, 132.67, 129.12, 128.02, 127.03, 119.89,
119.04, 113.27, 110.31, 100.12. APCI-HRMS m/z: calcd. for C14H9N3
(MH+), 220.0869, found 220.0881; Purity (HPLC): 98%.
3.4.14. 3-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)phenol (7c)
Compound was synthesised from 3-hydroxyphenylboronic acid and
6-chloro-7-deazapurine in a yield of 35%, recrystallized from methanol,
1
◦
white crystals, mp 263 C, Rf = 0.57 (hexane: ethyl acetate: 1:1). H
NMR (DMSO‑d6, 600 MHz) δ 12.23 (1H, s, NH), 9.66 (1H, s, OH), 8.85
(1H, s, Ar–H), 7.69–7.67 (1H, m, Ar–H), 7.67–7.64 (2H, m, Ar–H), 7.42
(1H, t, J = 7.8 Hz, Ar–H), 6.98 (1H, dd, J = 8.1, 1.7 Hz, Ar–H), 6.88 (1H,
d, J = 3.5 Hz, Ar–H). 13C NMR (DMSO‑d6, 150 MHz) δ 157.70, 155.62,
152,66, 150.87, 139.28, 129.84, 127.56, 119.39, 117.10, 115.24,
3.4.9. 6-(Pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridine (5i)
Compound was synthesised from 3-pyridinylboronic acid and 6-
chloro-7-azaindole in a yield of 32%, recrystallized from acetonitrile,
pale orange coloured powder, mp 177 ◦C, Rf = 0.11 (dichloromethane:
ethyl acetate; 4:1). 1H NMR (DMSO‑d6, 600 MHz) δ 11.76 (1H, s, NH),
9.23 (1H, d, J = 1.5 Hz, Ar–H), 8.52 (1H, d, J = 5.6 Hz, Ar–H), 8.38–8.36
114.51, 100.00. APCI-HRMS m/z: calcd. for
C
12H9N3O (MH+),
6

L. Pieterse et al.
Chemico-Biological Interactions 349 (2021) 109643
212.0818, found 212.0816; Purity (HPLC): 99%.
NMR (DMSO‑d6, 600 MHz) δ 12.19 (1H, s, NH), 8.71 (1H, s, Ar–H),
8.04–8.03 (1H, m, Ar–H), 7.62 (1H, d, J = 3.6 Hz, Ar–H), 7.43 (1H, d, J
= 3.4 Hz, Ar–H), 6.95 (1H, d, J = 3.5 Hz, Ar–H), 6.77–6.76 (1H, m,
Ar–H). 13C NMR (DMSO‑d6, 150 MHz) δ 152.81, 152.66, 150.85,
146.00, 145.92, 127.64, 112.64, 112.51, 111.77, 100.22. APCI-HRMS
m/z: calcd. for C10H7N3O (MH+), 186.0662, found 186.0655; Purity
(HPLC): 93%.
3.4.15. 4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)phenol (7d)
Compound was synthesised from 4-hydroxyphenylboronic acid and
6-chloro-7-deazapurine in a yield of 82%, recrystallized from methanol,
gold crystals, mp > 300 ◦C, Rf = 0.61 (hexane: ethyl acetate: 1:1). 1H
NMR (DMSO‑d6, 600 MHz) δ 12.13 (1H, s, NH), 9.98 (1H, s, OH), 8.74
(1H, s, Ar–H), 8.08 (2H, d, J = 8.3 Hz, Ar–H), 7.58 (1H, s, Ar–H), 6.95
(2H, d, J = 8.2 Hz, Ar–H), 6.87 (1H, s, Ar–H). 13C NMR (DMSO‑d6, 150
MHz) δ 159.45, 155.54, 152.44, 150.82, 130.20, 128.77, 126.87,
115.61, 113.61, 100.18. APCI-HRMS m/z: calcd. for C12H9N3O (MH+),
212.0818, found 212.0814; Purity (HPLC): 90%.
3.4.21. 4-(1-Benzofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidine (7j)
Compound was synthesised from 2-benzofuranylboronic acid and 6-
chloro-7-deazapurine in a yield of 23%, recrystallized from methanol,
white powder, mp 271–272 ◦C, Rf = 0.42 (hexane: ethyl acetate: 1:1). 1H
NMR (DMSO‑d6, 600 MHz) δ 12.31 (1H, s, NH), 8.86 (1H, s, Ar–H), 7.91
(1H, s, Ar–H), 7.83 (2H, m, Ar–H), 7.75 (1H, d, J = 3.5 Hz, Ar–H), 7.49
(1H, d, J = 7.8 Hz, Ar–H), 7.39 (1H, t, J = 7.5 Hz, Ar–H), 7.20 (1H, d, J
= 3.5 Hz, Ar–H). 13C NMR (DMSO‑d6, 150 MHz) δ 155.42, 154.69,
153.18, 151.10, 146.01, 128.58, 128.03, 126.40, 123.84, 122.46,
113.30, 111.96, 108.53, 100.72. APCI-HRMS m/z: calcd. for C14H9N3O
(MH+), 236.0818, found 236.0792; Purity (HPLC): 97%.
3.4.16. 4-(3-Methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (7e)
Compound was synthesised from 3-methoxyphenylboronic acid and
6-chloro-7-deazapurine in a yield of 62%, recrystallized from methanol,
gold crystals, mp 219–220 ◦C, Rf = 0.48 (hexane: ethyl acetate: 1:1). 1H
NMR (DMSO‑d6, 600 MHz) δ 12.27 (1H, s, NH), 8.84 (1H, s, Ar–H), 7.74
(1H, d, J = 7.8 Hz, Ar–H), 7.68 (1H, dd, J = 2.4, 1.6 Hz, Ar–H), 7.65 (1H,
d, J = 3.5 Hz, Ar–H), 7.50 (1H, t, J = 7.9 Hz, Ar–H), 7.13–7.10 (1H, m,
Ar–H), 6.68 (1H, d, J = 3.5 Hz, Ar–H), 3.86 (1H, s, MeO-3′). 13C NMR
(DMSO‑d6, 150 MHz) δ 159.51, 155.31, 152.59, 150.84, 139.36, 129.90,
127.72, 120.94, 115.98, 114.53, 113.25, 99.91, 55.18 (MeO-3’). APCI-
HRMS m/z: calcd. for C13H11N3O (MH+), 226.0975, found 226.0981;
Purity (HPLC): 99%.
3.4.22. 4-(Thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidine (7k)
Compound was synthesised from 2-thienylboronic acid and 6-chloro-
7-deazapurine in a yield of 44%, recrystallized from methanol, pale
1
◦
green powder, mp 266 C, Rf = 0.64 (hexane: ethyl acetate: 1:1). H
NMR (DMSO‑d6, 600 MHz) δ 12.25 (1H, s, NH), 8.74 (1H, s, Ar–H), 8.17
(1H, dd, J = 4.1, 1.3 Hz, Ar–H), 7.86 (1H, dd, J = 5.0, 06 Hz, Ar–H), 7.69
(1H, d, J = 3.6 Hz, Ar–H), 7.35–7.32 (1H, m, Ar–H), 7.07 (1H, d, J = 3.6
Hz, Ar–H). 13C NMR (DMSO‑d6, 150 MHz) δ 152.72, 150.69, 149.67,
142.96, 130.23, 129.14, 128.92, 127.90, 112.17, 99.86. APCI-HRMS m/
z: calcd. for C10H7N3S (MH+), 202.0433, found 202.0442; Purity
(HPLC): 97%.
3.4.17. 4-(4-Methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (7f)
Compound was synthesised from 4-methoxyphenylboronic acid and
6-chloro-7-deazapurine in a yield of 39%, recrystallized from methanol,
dark green crystals, mp 205–207 ◦C, Rf = 0.51 (hexane: ethyl acetate:
1:1). 1H NMR (DMSO‑d6, 600 MHz) δ 8.78 (1H, s, Ar–H), 8.20–8.17 (2H,
m, Ar–H), 7.61 (1H, d, J = 3.5 Hz, Ar–H), 7.15–7.11 (2H, m, Ar–H), 6.88
(1H, d, J = 3.6 Hz, Ar–H), 3.85 (1H, s, MeO-4′). 13C NMR (DMSO‑d6,
150 MHz) δ 160.78, 155.11, 152.53, 150.80, 130.37, 130.05, 127.27,
114.20, 113.84, 99.99, 55.30 (MeO-4’). APCI-HRMS m/z: calcd. for
3.4.23. 4-(1-Benzothiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidine (7l)
Compound was synthesised from benzo[b]thien-2-ylboronic acid and
6-chloro-7-deazapurine in a yield of 44%, recrystallized from methanol,
gold crystals, mp 287–289 ◦C, Rf = 0.31 (hexane: ethyl acetate: 1:1). 1H
NMR (DMSO‑d6, 600 MHz) δ 12.33 (1H, s, NH), 8.79 (1H, s, Ar–H), 8.54
(1H, s, Ar–H), 8.05–8.03 (1H, m, Ar–H), 8.03–8.01 (1H, m, Ar–H), 7.75
(1H, d, J = 3.6 Hz, Ar–H), 7.46–7.44 (2H, m, Ar–H), 7.21 (1H, d, J = 3.6
Hz, Ar–H). 13C NMR (DMSO‑d6, 150 MHz) δ 152.50, 150.39, 149.29,
142.86, 140.21, 139.86, 128.30, 125.88, 125.75, 124.71, 124.54,
122.33, 112.74, 99.68. APCI-HRMS m/z: calcd. C14H9N3S (MH+),
252.0590, found 252.0577; Purity (HPLC): 98%.
C
13H11N3O (MH+), 226.0975, found 226.0978; Purity (HPLC): 97%.
3.4.18. 4-(2,3-Dimethoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (7g)
Compound was synthesised from 2,3-dimethoxyphenylboronic acid
and 6-chloro-7-deazapurine in a yield of 57%, recrystallized from
methanol, yellow crystals, mp 140–142 ◦C, Rf = 0.52 (hexane: ethyl
acetate: 1:1). 1H NMR (DMSO‑d6, 600 MHz) δ 12.11 (1H, s, NH), 8.81
(1H, s, Ar–H), 7.53 (1H, d, J = 3.5 Hz, Ar–H), 7.20 (2H, d, J = 4.6 Hz,
Ar–H), 7.09 (1H, d, J = 4.6 Hz, Ar–H), 6.36 (1H, d, J = 3.5 Hz, Ar–H),
3.89 (3H, s, Me-2′), 3.57 (3H, s, Me-3′). 13C NMR (DMSO‑d6, 150 MHz) δ
155.78, 152.72, 151.60, 150.58, 146.68, 132.35, 126.67, 123.84,
122.18, 116.75, 113.80, 100.25, 60.83 (MeO-2′), 55.83 (MeO-3’). APCI-
HRMS m/z: calcd. for C14H13N3O2(MH+), 256.1081, found 256.1089;
Purity (HPLC): 99%.
3.4.24. 4-(4-Fluorophenyl)-7H-pyrrolo[2,3-d]pyrimidine (7m)
Compound was synthesised from 4-fluorophenylboronic acid and 6-
chloro-7-deazapurine in a yield of 23%, recrystallized from methanol,
dark green coloured crystals, mp 243 ◦C, Rf = 0.36 (hexane: ethyl ace-
tate: 1:1). 1H NMR (DMSO‑d6, 600 MHz) δ 8.82 (1H, s, Ar–H), 8.25 (2H,
dd, J = 8.6, 5.7 Hz, H-2′, H-6′), 7.66 (1H, d, J = 3.5 Hz, H-6), 7.40 (2H,
m, H-3′, H-5’), 6.88 (1H, d, J = 3.5 Hz, Ar-H13C NMR (DMSO‑d6, 150
MHz) δ 163.21 (d, J = 247.8 Hz), 154.30 (s), 152.67 (s), 150.82 (s),
134.41 (d, J = 2.9 Hz), 130.81 (d, J = 8.7 Hz), 127.94 (s), 115.78 (d, J =
21.6 Hz), 114.29 (s), 99.81 (s). APCI-HRMS m/z: calcd. for C12H8FN3
(MH+), 214.0775, found 214.0776; Purity (HPLC):99%
3.4.19. 4-(4-Phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (7h)
Compound was synthesised from 4-phenoxyphenylboronic acid and
6-chloro-7-deazapurine in a yield of 24%, recrystallized from methanol,
white powder, mp 209–210 ◦C, Rf = 0.76 (hexane: ethyl acetate: 1:1). 1H
NMR (DMSO‑d6, 600 MHz) δ 12.23 (1H, s, NH), 8.80 (1H, s, Ar–H), 8.23
(2H, d, J = 7.9 Hz, Ar–H), 7.64 (1H, s, Ar–H), 7.46 (2H, t, J = 7.1 Hz,
Ar–H), 7.22 (1H, t, J = 6.9 Hz, Ar–H), 7.15 (4H, t, J = 7.5 Hz, Ar–H),
6.90 (1H, s, Ar–H). 13C NMR (DMSO‑d6, 150 MHz) δ 158.67, 155.75,
154.73, 152.58, 150.85, 132.73, 130.45, 130.25, 127.52, 124.23,
119.57, 118.04, 114.08, 99.98. APCI-HRMS m/z: calcd. for C18H13N3O
(MH+), 288.1131, found 288.1129; Purity (HPLC): 97%.
3.4.25. 4-(3,4-Difluorophenyl)-7H-pyrrolo[2,3-d]pyrimidine (7n)
Compound was synthesised from 3,4-difluorophenylboronic acid
and 6-chloro-7-deazapurine in a yield of 23%, recrystallized from
methanol, pale green crystals, mp 245–246 ◦C, Rf = 0.56 (hexane: ethyl
acetate: 1:1). 1H NMR (DMSO‑d6, 600 MHz) δ 8.84 (1H, s, Ar–H),
8.19–8.13 (1H, m, H-Ar-H), 8.10–8.05 (1H, m, H-2’), 7.70 (1H, d, J =
3.5 Hz), 7.64 (1H, dd, J = 18.9, 8.6 Hz, Ar-H6.94 (1H, d, J = 3.6 Hz,
Ar–H). 13C NMR (DMSO‑d6, 150 MHz) δ 152.91 (d, J = 4.6 Hz), 152.78
(s), 152.01 (dd, J = 197.7, 25.8 Hz), 150.74 (s), 149.35 (d, J = 220.0,
80.9 Hz), 135.45 (dd, J = 10.5, 7.0 Hz), 128.37 (s), 125.69 (dd, J = 6.8,
3.4.20. 4-(Furan-2-yl)-7H-pyrrolo[2,3-d]pyrimidine (7i)
Compound was synthesised from 2-furanylboronic acid and 6-
chloro-7-deazapurine in a yield of 32%, recrystallized from methanol,
green crystals, mp 204–205 ◦C, Rf = 0.61 (hexane: ethyl acetate: 1:1). 1H
7

L. Pieterse et al.
Chemico-Biological Interactions 349 (2021) 109643
3.2 Hz), 118.00 (dd, J = 18.1, 4.9 Hz), 117.26 (dd, J = 18.9, 5.7 Hz),
114.34 (s), 99.72 (s). APCI-HRMS m/z: calcd. for C12H7F2N3 (MH+),
232.0681, found 232.0669; Purity (HPLC): 96%.
(HPLC): 92%.
3.5. Protein kinase assays
3.4.26. 4-[4-(Trifluoromethyl)phenyl]-7H-pyrrolo[2,3-d]pyrimidine (7o)
Compound was synthesised from 4-(trifluoromethyl)phenylboronic
acid and 6-chloro-7-deazapurine in a yield of 53%, recrystallized from
methanol, yellow crystals, mp 218 ◦C, Rf = 0.64 (hexane: ethyl acetate:
1:1). 1H NMR (DMSO‑d6, 600 MHz) δ 8.89 (1H, s, Ar–H), 8.38 (2H, d, J
= 8.1 Hz, Ar–H), 7.93 (2H, d, J = 8.3 Hz, Ar–H), 7.73 (1H, d, J = 3.6 Hz,
Ar–H), 6.93 (1H, d, J = 3.6 Hz, Ar–H).13C NMR (DMSO‑d6, 150 MHz) δ
153.68 (s), 152.81 (s), 150.88 (s), 141.73 (s), 129.88 (q, J = 31.9 Hz),
129.29 (s), 128.53 (s), 125.68 (q, J = 3.6 Hz), 124.15 (q, J = 272.3 Hz),
114.84 (s), 99.65 (s). APCI-HRMS m/z: calcd. for C13H8F3N3 (MH+),
264.0743, found 264.0759; Purity (HPLC): 97%.
All the PK assays were executed using a ADP-Glo™ kinase assay kit,
following methods described by Nguyen and co-workers, in duplicate
[40], apart from: (i) LmCK1 (from Leishmania major, recombinant,
expressed in bacteria) was assayed in buffer A with 170
μM of
CK1-specific peptide substrate, RRKHAAIGSpAYSITA (where “Sp” rep-
resents phosphorylated serine); (ii) HsCK1 (casein kinase 1 , human,
recombinant, expressed by baculovirus in Sf9 insect cells) was assayed in
ε
ε
buffer A with 170
μM of the CK1-specific peptide substrate; (iii)
HsGSK-3β (glycogen synthase kinase-3, human, recombinant, expressed
by baculovirus in Sf9 insect cells) isoforms were assayed in buffer A with
20
μM of GS-1 peptide, YRRAAVPPSPSLSRHSSPHQSpEDEEE, a
GSK-3-selective substrate. Buffer A is composed of 10 mM MgCl2, 1 mM
EGTA, 1 mM DTT, 25 mM Tris-HCl pH 7.5, 50 g/mL heparin. The
3.4.27. 4-(4-Chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidine (7p)
μ
Compound was synthesised from 4-chlorophenylboronic acid and 6-
chloro-7-deazapurine in a yield of 23%, recrystallized from methanol,
peptides were sourced from Proteogenix (Oberhausbergen, France). The
kinase activities were measured by means of the ADP-GLO™ assay kit
according to the manufacturer recommendations (Promega, Madison,
WI). The 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. The kinase reactions were performed in 384-well plates in a
1
◦
white powder, mp 255 C, Rf = 0.42 (hexane: ethyl acetate: 1:1). H
NMR (DMSO‑d6, 600 MHz) δ 12.27 (1H, s, NH), 8.84 (1H, s, Ar–H), 8.21
(2H, d, J = 8.5 Hz, Ar–H), 7.67 (1H, d, J = 3.5 Hz, Ar–H), 7.64 (2H, d, J
= 8.5 Hz, Ar–H), 6.89 (1H, d, J = 3.6 Hz, Ar–H). 13C NMR (DMSO‑d6,
150 MHz) δ 154.04, 152.61, 150.77, 136.64, 134.72, 130.18, 128.79,
127.89, 114.32, 99.71. APCI-HRMS m/z: calcd. for C12H8ClN3 (MH+),
230.0480, found 230.0467; Purity (HPLC): 96%.
final volume of 6
μ
L (initially, the compounds were diluted in DMSO to
◦
obtain 10 mM stock solutions) for 30 min at 30 C in an appropriate
kinase buffer, with either protein or peptide as substrate in the presence
3.4.28. 4,7-Bis(4-methylphenyl)-7H-pyrrolo[2,3-d]pyrimidine (8a)
Compound was synthesised from 4-methylphenylboronic acid and 6-
chloro-7-deazapurine in a yield of 23%, recrystallized from methanol,
of 10 μM ATP. Subsequently, 6 μL of ADP-Glo™ kinase reagent were
added to quench the kinase reaction, followed by a 50 min incubation
time at room temperature (RT). Hereafter, 12 μL of Kinase Detection
1
◦
white powder, mp 158 C, Rf = 0.42 (hexane: ethyl acetate: 1:1). H
NMR (DMSO‑d6, 600 MHz) δ 8.90 (1H, s, H-2), 8.11 (2H, d, J = 8.0 Hz,
Ar–H), 8.05 (1H, d, J = 3.7 Hz, Ar–H), 7.76 (2H, d, J = 8.2 Hz, Ar–H),
7.43 (2H, d, J = 7.9 Hz, Ar–H′), 7.39 (2H, d, J = 8.1 Hz, Ar–H), 7.12 (1H,
d, J = 3.7 Hz, Ar–H), 2.43 (3H, s, Me-4′), 2.40 (3H, s, Me-10′). 13C NMR
(DMSO‑d6, 150 MHz) δ 156.54, 151.50, 151.02, 140.12, 136.29, 134.74,
134.66, 129.95, 129.69, 129.50, 128.61, 123.71, 115.45, 101.46, 20.99
(Me-4′), 20.58 (Me-10’). APCI-HRMS m/z: calcd. for C20H18N3 (MH+),
300.1495, found 300.1504; Purity (HPLC): 96%.
Reagent were added and the mixture was left for an hour at RT. The
resulting signal was measured by the Envision microplate luminometer
(PerkinElmer, Waltham, MA) and expressed in Relative Light Unit
(RLU).
The effect of the 7-azaindole and 7-deazapurine derivatives on kinase
activity was evaluated as a two-step process: Step 1- served as the pri-
mary screening, where all the compounds were tested at 1 and 10 μM
against a panel of eight disease-related kinases and the activities were
expressed in % of maximal activity, i.e. measured with DMSO diluted at
1% v/v concentration; Step 2- The more active compounds (deemed
“hits”) were then tested in duplicate over a wide range of concentrations
3.4.29. 4,7-Bis(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (8b)
Compound was synthesised from 4-methoxyphenylboronic acid and
6-chloro-7-deazapurine in a yield of 23%, recrystallized from methanol,
(usually between 0.0003 and 10 μM) against the appropriate targets
identified in step 1 (here Haspin and CDK9/Cyclin T) to determine their
IC50 values. The results can be found in Table 4 and Table 5.
1
◦
white powder, mp 175 C, Rf = 0.42 (hexane: ethyl acetate: 1:1). H
NMR (DMSO‑d6, 600 MHz) δ 8.85 (1H, s, Ar–H), 8.21 (2H, d, J = 8.7 Hz,
H-2′, H-6′), 7.98 (1H, d, J = 3.7 Hz, H-6), 7.74 (2H, d, J = 8.8 Hz, H-8′,
H-12′), 7.16 (2H, d, J = 8.7 Hz, Ar–H), 7.14 (2H, d, J = 8.8 Hz, Ar–H),
7.11 (1H, d, J = 3.7 Hz, Ar–H), 3.88 (3H, s, MeO-4′), 3.84 (3H, s, MeO-
10′). 13C NMR (DMSO‑d6, 150 MHz) δ 161.13, 158.13, 156.20, 151.50,
151.12, 138.72, 130.31, 130.24, 130.05, 125.52, 114.91, 114.53,
114.41, 101.28, 55.57 (MeO-4′), 55.44 (MeO-10’). APCI-HRMS m/z:
calcd. for C20H18N3O2 (MH+), 332.1394, found 332.1403; Purity
(HPLC): 93%.
3.6. Cytotoxicity prediction
The cytotoxicty of the most promising compounds were predicted by
using a prediction tool known as, CLC-Pred: in silico prediction of
cytotoxicity for tumour and non-tumour cell-lines [41]. The tool was
developed by making use of the existing Prediction of Activity Spectra
for Substances (PASS) algorithm to create and validate the classification
structure-activity relationship (SAR) models for predicting cytotoxicity
of structures against numerous types of human cell-lines using ChEMBL
experimental data. The experimental data from ChEMBL (IG50, IC50 and
% inhibition values) forms the foundation 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 [41].
3.4.30. 4,7-Bis(4-fluorophenyl)-7H-pyrrolo[2,3-d]pyrimidine (8c)
Compound was synthesised from 4-fluorophenylboronic acid and 6-
chloro-7-deazapurine in a yield of 23%, recrystallized from methanol,
1
◦
white powder, mp 163 C, Rf = 0.42 (hexane: ethyl acetate: 1:1). H
NMR (DMSO‑d6, 600 MHz) δ 8.94 (1H, s, Ar–H), 8.28 (2H, m, H-2’,
Ar–H), 8.12 (1H, d, J = 3.8 Hz, Ar–H), 7.92 (2H, m, Ar–H), 7.45 (4H, m,
Ar–H), 7.17 (1H, d, J = 3.8 Hz, Ar–H). 13C NMR (DMSO‑d6, 150 MHz) δ
163.41 (d, J = 248.3), 160.58 (d, J = 244.0 Hz), 155.48 (s), 151.57 (s),
151.14 (s), 133.61 (dd, J = 72.2, 2.7 Hz), 131.03 (d, J = 8.8 Hz), 130.37
(s), 130.09 (dd, J = 411.9, 10.8 Hz), 126.06 (d, J = 8.6 Hz), 116.90 (d, J
= 22.9 Hz), 115.93 (d, J = 21.7 Hz), 115.43 (s), 101.51 (s). APCI-HRMS
m/z: calcd. for C18H12F2N3 (MH+), 308.0994, found 308.0976; Purity
3.7. SwissADME prediction
In addition to the cytotoxicity, the most promising compounds were
also evaulated using the SwissADME prediction tool [42]. SwissADME is
a free web tool that utilises a set of fast, but robust predictive models.
8

L. Pieterse et al.
Chemico-Biological Interactions 349 (2021) 109643
Table 4
Percentage of residual activity for compounds 5a–m, 7a–q and 8a-c at 10 μM and 1 μM using a representative kinase panel.
Cpd
Hs CDK2/CyclinA
Hs CDK5/p25
Hs CDK9/CyclinT
Hs Haspin
Hs PIM 1
Hs CK1
ε
Hs GSK3β
Lm CK1
Hs ABL1
5a
5b
5c
5d
5e
5f
73 (88)
≥100 (95)
68 (67)
60 (88)
84 (81)
67 (72)
65 (69)
100 (93)
73 (78)
94 (87)
83 (80)
87 (79)
≥100 (≥100)
70 (68)
46 (70)
72 (68)
65 (92)
36 (74)
16 (60)
14 (38)
29 (44)
10 (24)
83 (≥100)
99 (98)
11 (62)
14 (21)
10 (57)
19 (56)
58 (58)
58 (67)
19 (74)
28 (67)
39 (56)
88 (83)
85 (69)
83 (87)
81 (95)
84 (80)
92 (90)
76 (80)
95 (79)
100 (83)
93 (88)
≥100 (≥100)
83 (≥100)
≥100 (99)
88 (99)
≥100 (91)
82 (≥100)
80 (79)
73 (92)
68 (94)
32 (93)
51 (77)
47 (76)
65 (76)
88 (97)
98 (95)
38 (97)
60 (76)
22 (88)
65 (86)
84 (84)
79 (79)
64 (88)
72 (78)
69 (75)
90 (99)
81 (88)
80 (94)
92 (71)
59 (84)
51 (62)
≥100 (88)
≥100 (99)
≥100 (86)
99 (96)
97 (85)
82 (95)
96 (95)
≥100 (97)
≥100 (84)
≥100 (94)
86 (97)
74 (92)
86 (94)
84 (93)
67 (89)
77 (≥100)
82 (69)
93 (71)
97 (88)
71 (82)
87 (89)
55 (85)
84 (86)
95 (90)
87 (94)
77 (74)
90 (81)
94 (91)
98 (85)
59 (81)
87 (98)
NP
81 (92)
97 (82)
82 (≥100)
97 (98)
≥100 (≥100)
83 (93)
76 (84)
NP
90 (≥100)
94 (94)
≥100 (≥100)
89 (≥100)
77 (82)
61 (61)
65 (64)
100 (88)
87 (73)
62 (78)
43 (60)
74 (80)
77 (81)
66 (65)
74 (62)
56 (63)
72 (70)
66 (74)
32 (72)
36 (74)
47 (56)
40 (56)
86 (67)
96 (93)
56 (67)
61 (59)
34 (57)
62 (70)
76 (76)
70 (56)
55 (55)
61 (76)
80 (70)
76 (82)
80 (≥100)
65 (74)
90 (≥100)
92 (84)
92 (93)
≥100 (87)
96 (93)
97 (≥100)
≥100 (96)
98 (94)
≥100 (≥100)
≥100 (≥100)
≥100 (≥100)
94 (≥100)
100 (93)
99 (93)
83 (96)
86 (96)
5g
5h
5i
≥100 (≥100)
95 (≥100)
87 (≥100)
91 (93)
89 (≥100)
92 (98)
≥100 (≥100)
82 (89)
93 (95)
≥100 (90)
≥100 (≥100)
93 (96)
≥100 (98)
92 (93)
87 (≥100)
≥100 (≥100)
82 (≥100)
74 (≥100)
80 (81)
5j
≥100 (≥100)
≥100 (≥100)
≥100 (≥100)
≥100 (≥100)
≥100 (≥100)
≥100 (≥100)
63 (≥100)
99 (≥100)
92 (92)
5k
5l
94 (≥100)
72 (≥100)
≥100 (≥100)
11 (40)
≥100 (81)
98 (≥100)
90 (≥100)
92 (≥100)
55 (≥100)
50 (95)
76 (≥100)
83 (96)
5m
7a
7b
7c
7d
7e
7f
89 (≥100)
56 (98)
≥100 (≥100)
85 (89)
28 (73)
5 (40)
48 (≥100)
41 (91)
55 (77)
76 (≥100)
0 (22)
54 (86)
98 (99)
≥100 (≥100)
≥100 (≥100)
≥100 (88)
17 (55)
93 (≥100)
60 (≥100)
100 (89)
91 (89)
66 (76)
88 (87)
19 (60)
66 (90)
7g
7h
7i
≥100 (99)
≥100 (93)
75 (96)
50 (83)
100 (93)
100 (81)
96 (89)
86 (85)
93 (86)
≥100 (≥100)
≥100 (≥100)
66 (≥100)
37 (82)
90 (94)
7j
93 (92)
53 (86)
36 (≥100)
50 (≥100)
22 (≥100)
57 (94)
73 (87)
7k
7l
57 (≥100)
94 (≥100)
100 (≥100)
≥100 (≥100)
71 (≥100)
99 (≥100)
97 (98)
3 (53)
49 (76)
≥100 (≥100)
≥100 (≥100)
≥100 (≥100)
≥100 (≥100)
97 (≥100)
72 (76)
71 (79)
7m
7n
7o
7p
7q
8a
8b
8c
6 (33)
100 (85)
81 (80)
7 (40)
64 (≥100)
73 (≥100)
30 (≥100)
33 (≥100)
≥100 (92)
49 (49)
61 (72)
70 (≥100)
74 (76)
24 (72)
≥100 (≥100)
≥100 (≥100)
80 (≥100)
18 (61)
68 (74)
97 (99)
≥100 (100)
≥100 (100)
≥100 (100)
(82 (87)
≥100 (89)
89 (82)
98 (77)
99 (97)
88 (≥100)
≥100 (≥100)
Values represent the percentage of residual kinase activity for compounds used at 10
μ
M and 1
μ
M concentrations, respectively. The values in brackets signify the
M ATP. NP not performed.
results obtained with 1
μ
M. The most significant results are presented in bold italics. Activities were assessed in duplicate using 10 μ
scaffold are likely to be PK inhibitors [30]. In this study, thirteen (13)
7-azaindole and twenty (20) 7-deazapurine derivatives were synthesised
and screened against the CDK9/CyclinT and Haspin kinases. In addition,
the compounds were also screened against HsCDK2/CyclinA,
Table 5
IC50 values and selectivity ratios of the most promising compounds (com-
pounds inhibiting >70% of the kinase activity at 1
were assessed in duplicate using 10 M ATP.
Cpd
μM, see Table 4). Activities
μ
HsCDK5/p25, HsPim-1, HsCK1
ε
, HsGSK3β, LmCK1 and HsABL1.
M concentrations (diluted in
Hs CDK9/Cyclin T
M)
Hs Haspin
M)
Selectivity Ratio
Screenings were performed at 10 and 1
μ
(
μ
(
μ
CDK9/CyclinT
(
)
1% DMSO v/v). The most promising compound were subjected to
further testing at a wider range of concentrations. The IC50 values were
determined from dose-response curves (Prism-GraphPad). The results of
the screening and IC50 values for the most promising compounds can be
found in Tables 4 and 5, respectively.
Haspin
7a
0.94
1.39
0.59
0.38
1.26
1.24
0.67
0.62
0.72
1.44
1.10
0.015
NP
NP
–
7b
NP
–
7c
0.19
0.42
NP
3.11
7d
0.90
7e
–
In general, the results of this study echo those of a previous study. It
was observed that the position of substituents on the pyridine ring of the
7-azaindole scaffold, greatly affects a compound’s ability to exhibit PK
inhibition. The 4-position was deemed optimal for CDK9/CyclinT and
Haspin inhibition [30]. Like the 5-substituted 7-azaindole derivatives in
the previous study, the 6-substituted 7-azaindole derivatives display a
lack of inhibitory activity towards PKs deployed in this study (at the
concentrations tested). Additionally, the structurally related
4-substituted 7-deazapurines derivatives (substituted on the equivalent
to the 4-position on the 7-azaindole scaffold) displayed a greater incli-
nation toward PK inhibition.
7f
0.11
0.21
NP
11.27
7k
3.19
7m
–
–
–
–
–
–
7n
NP
7p
NP
7q
NP
Flavopiridol
CHR-6494
NP
0.055
IC50 values (in μM) were determined from dose-response curves by using Prism-
GraphPad (GraphPad software, San Diego, CA, USA). NP Not Performed. Results
obtained for dual-target inhibitors are presented in bold.
The screening against CDK9/CyclinT revealed 11 active compounds
(7a–f, 7m, 7n, and 7p) and 9 relatively inactive compounds (7g–7j, 7l,
These models enable the user to generate physiochemical properties,
lipophilicity, water solubility, pharmacokinetic properties, druglikeness
and qualities useful in medicinal chemistry for the desired compounds.
The complete results for compounds 7a, 7b, 7c, 7d, 7e, 7f, 7k, 7m, 7n,
and 7p can be found in the supplementary information.
7o and 8a–c). IC50 values for the active compounds ranged from 0.38 μM
to 1.44
hydroxyphenyl substituted derivative (7d) exhibiting an IC50 value of
0.38 M, followed by the C3-hydroxyphenyl substituted derivative (7c)
with an IC50 value of 0.59 M against CDK9/CyclinT. Interestingly, the
C3-methoxyphenyl (7e) and the C4-methoxyphenyl substituted deriva-
tive (7f) performed equivalently, with 1.24 M and 1.26 M IC50 values,
μM. The most active compound proved to be the C4-
μ
μ
4. Results and discussion
μ
μ
Previously, it has been determined that derivatives of the 7-azaindole
9

L. Pieterse et al.
Chemico-Biological Interactions 349 (2021) 109643
respectively. Additionally, the results show that the C4-hydroxyphenyl
is preferred over the C4-methoxyphenyl and C4-methylphenyl
Table 6
Predicted cytotoxicity of the most promising compounds (compounds inhibiting
>70% of kinase activity at 1 M).
μ
substituted derivatives (IC50 values: 0.38 μM (7d) > 1.24 μM (7f) >
1.39 μM (7b). Considering the halogen-substituted compounds, com-
Compound
Probability of
activity (Pa)
Cell-line
code
Cell-line full name
pound 7m (4-fluorophenyl substituted derivative) was 2.32 times more
active than compound 7p (4-chlorophenyl substituted derivative), with
7a
0.581
0.480
0.468
0.485
0.450
0.430
0.577
0.452
0.410
0.614
0.455
0.418
0.464
0.433
0.406
0.503
0.435
0.415
0.430
0.426
0.408
0.669
0.463
0.426
0.499
0.498
0.485
0.460
0.457
Hs 683
Oligodendroglioma
a 1.44
μM IC50 value. Furthermore, the C3,4-difluorophenyl substituted
NCI–H295R
A-375
Adrenal cortex carcinoma
Malignant melanoma
Oligodendroglioma
derivative (7n: IC50 value of 0.72) exhibits a slight decrease in activity
when compared to the C4-fluorophenyl monosubstituted derivative
(7m: IC50 value of 0.62), while the 4-trifluoromethylphenyl substituted
derivative (7o) was deemed relatively inactive. Noteworthy, compound
7k, which bears a 2-thiophene ring, is the only one of the compounds
with an alternative ring structure to exhibit activity for CDK9/CyclinT,
while the bis-substitued compounds (8a–c) were all deemed to be
relatively inactive.
7b
7c
7d
7e
7f
Hs 683
NCI–H295R
A-375
Adrenal cortex carcinoma
Malignant melanoma
Oligodendroglioma
Hs 683
NCI–H295R
NCI–H23
Hs 683
Adrenal cortex carcinoma
Non-small cell lung carcinoma
Oligodendroglioma
NCI–H295R
NCI–H23
Hs 683
Adrenal cortex carcinoma
Non-small cell lung carcinoma
Oligodendroglioma
Haspin screening data revealed four promising compounds (7c, 7d,
NCI–H295R
DU-145
Hs 683
Adrenal cortex carcinoma
Prostate carcinoma
7f and 7k). Here the IC50 values ranged from 0.11 μM to 0.42 μM. In
contrast to the CDK9/CyclinT results, the 4-methoxy substituted 7-dea-
Oligodendroglioma
zapurine derivative (7f) proved to be the most potent Haspin inhibitor
NCI–H295R
A-375
Adrenal cortex carcinoma
Malignant melanoma
Oligodendroglioma
(IC50 value of 0.11 μM), while the C4-hydroxy substituted 7-deazapurine
7g
7h
7i
Hs 683
derivative was 4-fold less potent (IC50 value of 0.42 μM). Additionally,
DMS-114
DU-145
Hs 683
Lung carcinoma
the C3-hydroxy substituted 7-deazapurine derivative seems to be fav-
oured over the C4-hydroxy substituted 7-deazapurine derivative (IC50
values of 0.19 and 0.42, respectively). Once again, compound 7k is the
only compound bearing an alternative ring that possessed activity to-
ward Haspin, at the concentrations tested. Interestingly, three of the four
compounds favoured Haspin over CDK9/CyclinT, with compounds 7c
and 7k being approximately 3-fold more selective toward Haspin, while
compound 7f was approximately 11 times more active.
Prostate carcinoma
Oligodendroglioma
A-375
Malignant melanoma
Adrenal cortex carcinoma
Non-small cell lung carcinoma
Cervical adenocarcinoma
Papillary renal carcinoma
Cervical adenocarcinoma
Acute T-lymphoblastic
leukeamia
NCI–H295R
NCI–H23
HeLa
ACHN
7j
HeLa
MOLT-4
Regarding the cytotoxicity, the prediction was achieved by using the
CLC-Pred prediction platform (see the “Cytotoxicity prediction”of the
material and methods section) and illustrated that most of the 7-deaza-
purine derivatives are expected to have a cytotoxicity score higher than
at least 0.400 for the given cell-lines (see Table 6). The score indicates
the percentage of compounds in the training data set that will likely be
less cytotoxic than the tested compound for the given cell-line. Based on
the results, compound 7m exhibited the highest cytotoxicity score of all
the derivatives for any given cell-line (cytotoxicity score of 0.672 against
Hs 683 (Oligodendroglioma). Translated this means that compound 7m
is 67.2% more likely than the known compounds in the training data set
to be cytotoxic against the Hs 683 cell line. In addition to the cytotox-
icity prediction, the SwissADME tool revealed that these compounds are
likely to cross the blood-brain barrier, thus highlighting their potential
use in brain tumours (see Fig. 2). The most promising compounds are
also expected to have high gastrointestinal absorption. Unfortunately,
the predicted bioavailability for these compounds are moderate
(achieving a bioavailability score of 0.55) and could benefit from
investigating options for increasing drug-likeness. The full set of prop-
erties and values predicted by the SwissADME tool can be found in the
supplementary information.
0.433
0.523
0.491
0.438
0.672
0.462
0.438
ACHN
Papillary renal carcinoma
Non-small cell lung carcinoma
Adrenal cortex carcinoma
Cervical adenocarcinoma
Oligodendroglioma
7k
NCI–H23
NCI–H295R
HeLa
7m
Hs 683
A-375
Malignant melanoma
Childhood acute myeloid
leukaemia with maturation
Oligodendroglioma
Kasumi 1
7n
7o
0.596
0.485
Hs 683
Kasumi 1
Childhood acute myeloid
leukaemia with maturation
Malignant melanoma
Oligodendroglioma
0.451
0.437
0.390
0.359
A-375
Hs 683
NCI–H295R
NALM-6
Adrenal cortex carcinoma
Adult B acute lymphoblastic
leukaemia
7p
7q
8a
8b
8c
0.663
0.447
0.414
0.512
0.465
0.426
0.464
0.421
0.418
0.449
0.427
0.390
0.518
0.447
0.420
Hs 683
Oligodendroglioma
NCI–H23
A-375
Non-small cell lung carcinoma
Malignant melanoma
Adrenal cortex carcinoma
Prostate carcinoma
NCI–H295R
DU-145
Hs 683
Oligodendroglioma
NCI–H295R
DMS-114
YAPC
Adrenal cortex carcinoma
Lung carcinoma
Pancreatic carcinoma
Non-small cell lung carcinoma
Lung carcinoma
NCI–H23
PC-9
5. Conclusion
HCT-15
Hs 683
Colon carcinoma
Oligodendroglioma
This study evaluated the inhibitory potential of two structurally
related chemical series against selected disease-related protein kinases.
The first series consisted of C6-substituted 7-azaindole derivatives while
second series is constituted by C4-substitued 7H-pyrrolo[2,3-d]pyrimi-
dine derivatives. The kinase-based screening assays performed against
several PKs, revealed that compounds belonging to the 7-deazapurine
scaffold and substituted on the C4-position, exhibited activity toward
the CDK9/CyclinT and Haspin kinases. On the other hand, the C6-
substituted 7-azaindole derivatives lacked activity at the concentra-
tions tested. In conjunction with previous results, it is postulated that the
substituents at C4-position play a key role in activity against CDK9/
CyclinT and Haspin. The most active compound against CDK9/CyclinT
proved to be the C4-hydroxyphenyl substituted 7H-pyrrolo[2,3-d]
NCI–H23
Kasumi 1
Non-small cell lung carcinoma
Childhood acute myeloid
leukaemia with maturation
pyrimidine derivative (7d) with an IC50 value of 0.38
μM. On the
other hand, the C4-methoxyphenyl substituted 7H-pyrrolo[2,3-d]pyrim-
idine derivative (7f) exhibited the best activity against Haspin (with IC50
value of 0.11 μM), making it the most potent hit compound of this study.
CRediT authorship contribution statement
Lianie Pieterse: Investigation, Formal analysis, Writing – original
10

L. Pieterse et al.
Chemico-Biological Interactions 349 (2021) 109643
Fig. 2. A BOILED-egg representation of the SwissADME predictions for the most promising compounds.
[5] D.K. Pokholok, J. Zeitlinger, N.M. Hannett, D.B. Reynolds, R.A. Young, Activated
signal transduction kinases frequently occupy target genes, Science 313 (2006)
533–536, https://doi.org/10.1126/science.1127677.
draft. Richard M. Beteck: Supervision, Reviewing and Editing. Blan-
dine Baratte: Investigation. Omobolanle J. Jesumoroti: Reviewing
and Editing. Thomas Robert: Writing – review & editing. Sandrine
[6] M.H. Flight, Neurodegenerative diseases: new kinase targets for Alzheimer’s
disease, Nat. Rev. Drug Discov. 12 (2013) 739, https://doi.org/10.1038/nrd4132.
[7] M. Huse, J. Kuriyan, The conformational plasticity of protein kinases, Cell 109
(2002) 275–282, https://doi.org/10.1016/s0092-8674(02)00741-9.
[8] H. Noh, G. King, The role of protein kinase C activation in diabetic nephropathy,
Kidney Int. 72 (2007) S49–S53, https://doi.org/10.1038/sj.ki.5002386.
[9] R. Sever, J.S. Brugge, Signal transduction in cancer, Cold Spring Harbor
perspectives in medicine 5 (2015) a006098.
´
Ruchaud: Writing – review & editing. Stephane Bach: Formal analysis,
Writing – review & editing. Lesetja J. Legoabe: Conceptualization,
Supervision, Reviewing and Editing.
Declaration of competing interest
[10] R. Roskoski Jr., Properties of FDA-approved small molecule protein kinase
inhibitors: a 2020 update, Pharmacol. Res. 152 (2020) 104609.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[11] L.C. Franco, F. Morales, S. Boffo, A. Giordano, CDK9: a key player in cancer and
other diseases, J. Cell. Biochem. 119 (2018) 1273–1284, https://doi.org/10.1002/
jcb.26293.
´
[12] D. Hnisz, B.J. Abraham, T.I. Lee, A. Lau, V. Saint-Andre, A.A. Sigova, R.A. Young,
Super-enhancers in the control of cell identity and disease, Cell 155 (2013)
934–947, https://doi.org/10.1016/j.cell.2013.09.053.
Acknowledgements
[13] J.M. Higgins, Haspin: a newly discovered regulator of mitotic chromosome
behavior, Chromosoma 119 (2010) 137–147, https://doi.org/10.1007/s00412-
009-0250-4.
The authors are grateful to Dr. Otto and Dr. Jordaan of the SASOL
Centre for Chemistry, North-West University, for recording the NMR and
[14] N.G. Amoussou, A. Bigot, C. Roussakis, J.-M.H. Robert, Haspin: a promising target
for the design of inhibitors as potent anticancer drugs, Drug Discov. Today 23
(2018) 409–415, https://doi.org/10.1016/j.drudis.2017.10.005.
[15] H. Tanaka, Y. Yoshimura, Y. Nishina, M. Nozaki, H. Nojima, Y. Nishimune,
Isolation and characterization of cDNA clones specifically expressed in testicular
germ cells, FEBS Lett. 355 (1994) 4–10, https://doi.org/10.1016/0014-5793(94)
01155-9.
´
ˆ
MS spectra, respectively. The authors also thank the Canceropole Grand
Ouest (3 MC network - Marine Molecules, Metabolism and Cancer), GIS
´
IBiSA (Infrastructures en Biologie Sante et Agronomie) and Biogenouest
(Western France life science and environment core facility network) for
supporting KISSf screening facility.
[16] J. Eswaran, D. Patnaik, P. Filippakopoulos, F. Wang, R.L. Stein, J.W. Murray,
S. Knapp, Structure and functional characterization of the atypical human kinase
haspin, Proc. Natl. Acad. Sci. Unit. States Am. 106 (2009) 20198–20203.
[17] J. Dai, J.M. Higgins, Haspin: a mitotic histone kinase required for metaphase
chromosome alignment, Cell Cycle 4 (2005) 665–668.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.cbi.2021.109643.
[18] J. Dai, B.A. Sullivan, J.M. Higgins, Regulation of mitotic chromosome cohesion by
Haspin and Aurora B, Dev. Cell 11 (2006) 741–750, https://doi.org/10.1016/j.
devcel.2006.09.018.
References
[19] D. Huertas, M. Soler, J. Moreto, A. Villanueva, A. Martinez, A. Vidal, J. McDermott,
Antitumor activity of a small-molecule inhibitor of the histone kinase Haspin,
Oncogene 31 (2012) 1408–1418.
[1] E.H. Fischer, E.G. Krebs, Conversion of phosphorylase b to phosphorylase a in
muscle extracts, J. Biol. Chem. 216 (1955) 121–132.
[20] Y. Chen, D. Fu, H. Zhao, W. Cheng, F. Xu, GSG2 (Haspin) promotes development
and progression of bladder cancer through targeting KIF15 (Kinase-12), Aging 12
(2020).
[2] E.G. Krebs, D.J. Graves, E.H. Fischer, Factors affecting the activity of muscle
phosphorylase b kinase, J. Biol. Chem. 234 (1959) 2867–2873.
[3] G. Manning, D.B. Whyte, R. Martinez, T. Hunter, S. Sudarsanam, The protein
kinase complement of the human genome, Science 298 (2002) 1912–1934, https://
doi.org/10.1126/science.1075762.
[21] L. Han, P. Wang, Y. Sun, S. Liu, J. Dai, Anti-melanoma activities of haspin inhibitor
CHR-6494 deployed as a single agent or in a synergistic combination with MEK
inhibitor, J. Canc. 8 (2017) 2933.
[22] X. Han, T. Kuang, Y. Ren, Z. Lu, Q. Liao, W. Chen, Haspin knockdown can inhibit
progression and development of pancreatic cancer in vitro and vivo, Exp. Cell Res.
385 (2019) 111605.
´
[4] M. Lawson, J. Rodrigo, B. Baratte, T. Robert, C. Delehouze, O. Lozach, M. Alami,
Synthesis, biological evaluation and molecular modeling studies of imidazo [1, 2-a]
pyridines derivatives as protein kinase inhibitors, Eur. J. Med. Chem. 123 (2016)
105–114, https://doi.org/10.1016/j.ejmech.2016.07.040.
[23] J.C. Melms, S. Vallabhaneni, C.E. Mills, C. Yapp, J.-Y. Chen, E. Morelli, N. Moret,
Inhibition of haspin kinase promotes cell-intrinsic and extrinsic antitumor activity,
Canc. Res. 80 (2020) 798–810.
11

L. Pieterse et al.
Chemico-Biological Interactions 349 (2021) 109643
´
ˇ
ˇ
ˇ
´
[24] G. de Carcer, I. Perez de Castro, M. Malumbres, Targeting cell cycle kinases for
[34] N. Sabat, P. Naus, J. Matyasovský, D. Dziuba, L.P. Slavetínska, M. Hocek, Synthesis
of fluorescent 2-substituted 6-(het) aryl-7-deazapurine bases {4-(Het) aryl-pyrrolo
[2, 3-d] pyrimidines} by aqueous suzuki–miyaura cross-coupling reactions,
Synthesis 48 (2016) 1029–1045, https://doi.org/10.1002/chin.201633165.
[35] S. Kashyap, J. Sandler, U. Peters, E.J. Martinez, T.M. Kapoor, Using ‘biased-
privileged’scaffolds to identify lysine methyltransferase inhibitors, Bioorg. Med.
Chem. 22 (2014) 2253–2260, https://doi.org/10.1016/j.bmc.2014.02.024.
[36] N. Bhagirath, R. Dominique, J. Kennedy-Smith, F.J. Lopez-Tapia, E. Mertz, Q. Qiao,
S.-S. So, Inhibitors of bruton’s tyrosine kinase. Google Patents, 2015.
[37] S.-W. Kim, M. Kim, W.Y. Lee, T. Hyeon, Fabrication of hollow palladium spheres
and their successful application to the recyclable heterogeneous catalyst for Suzuki
coupling reactions, J. Am. Chem. Soc. 124 (2002) 7642–7643, https://doi.org/
10.1021/ja026032z.
cancer therapy, Curr. Med. Chem. 14 (2007) 969–985, https://doi.org/10.2174/
092986707780362925.
[25] J. Higgins, Structure, function and evolution of haspin and haspinrelated proteins,
a distinctive group of eukaryotic protein kinases, Cellular and Molecular Life
Sciences CMLS 60 (2003) 446–462, https://doi.org/10.1007/s000180300038.
[26] J.M. Higgins, The Haspin gene: location in an intron of the Integrin αE gene,
associated transcription of an Integrin αE-derived RNA and expression in diploid as
well as haploid cells, Gene 267 (2001) 55–69, https://doi.org/10.1016/S0378-
1119(01)00387-0.
[27] F. Villa, P. Capasso, M. Tortorici, F. Forneris, A. De Marco, A. Mattevi,
A. Musacchio, Crystal structure of the catalytic domain of Haspin, an atypical
kinase implicated in chromatin organization, Proc. Natl. Acad. Sci. Unit. States Am.
106 (2009) 20204–20209, https://doi.org/10.1073/pnas.0908485106.
[28] H. Tanaka, M. Wada, J. Park, HASPIN kinase inhibitor CHR-6494 suppresses
intestinal polyp development, cachexia, and hypogonadism in Apcmin/+ mice,
Eur. J. Canc. Prev. 29 (2020) 481.
[38] J.P. Wolfe, S.L. Buchwald, A highly active catalyst for the room-temperature
amination and Suzuki coupling of aryl chlorides, Angew. Chem. Int. Ed. 38 (1999)
2413–2416, https://doi.org/10.1002/(SICI)1521-3773(19990816)38:16<2413::
AID-ANIE2413>3.0.CO;2-H.
[29] F. Yu, Y. Lin, X. Xu, W. Liu, D. Tang, X. Zhou, A. Xie, Knockdown of GSG2 inhibits
prostate cancer progression in vitro and in vivo, Int. J. Oncol. 57 (2020) 139–150.
[30] L. Pieterse, L.J. Legoabe, R.M. Beteck, B. Josselin, S. Bach, S. Ruchaud, Synthesis
and biological evaluation of selected 7-azaindole derivatives as CDK9/Cyclin T and
Haspin inhibitors, Med. Chem. Res. (2020), https://doi.org/10.1007/s00044-020-
02560-1.
[39] J.X. Qiao, P.Y. Lam, Recent advances in chan–lam coupling reaction: copper-
promoted C–heteroatom bond cross-coupling reactions with boronic acids and
derivatives. Boronic acids: Preparation and Applications in organic synthesis, Medicine
and Materials 1 (2011) 315–361, https://doi.org/10.1002/9783527639328.ch6.
´
[40] T.-N.-D. Nguyen, O. Feizbakhsh, E. Sfecci, B. Baratte, C. Delehouze, A. Garcia,
M. Mehiri, Kinase-based screening of marine natural extracts leads to the
identification of a cytotoxic high molecular weight metabolite from the
mediterranean sponge crambe tailliezi, Mar. Drugs 17 (2019) 569, https://doi.org/
10.3390/md17100569.
[31] M. Allegretti, A. Arcadi, F. Marinelli, L. Nicolini, Palladium-catalysed
functionalisation at 4-and 6-position of the 7-azaindole system, Synlett (2001),
https://doi.org/10.1055/s-2001-13366, 2001, 0609-0612.
[32] R. Cincinelli, L. Musso, L. Merlini, G. Giannini, L. Vesci, F.M. Milazzo, R. Artali, 7-
Azaindole-1-carboxamides as a new class of PARP-1 inhibitors, Bioorg. Med. Chem.
22 (2014) 1089–1103, https://doi.org/10.1016/j.bmc.2013.12.031.
[33] Q. Li, L. Persoons, D. Daelemans, P. Herdewijn, Iron/copper Co-catalyzed cross-
coupling reaction for the synthesis of 6-substituted 7-deazapurines and the
corresponding nucleosides, J. Org. Chem. 85 (2019) 403–418, https://doi.org/
10.1021/acs.joc.9b02414.
[41] A.A. Lagunin, V.I. Dubovskaja, A.V. Rudik, P.V. Pogodin, D.S. Druzhilovskiy, T.
A. Gloriozova, V.V. Poroikov, CLC-Pred: a freely available web-service for in silico
prediction of human cell line cytotoxicity for drug-like compounds, PloS One 13
(2018), e0191838, https://doi.org/10.1371/journal.pone.0191838.
[42] A. Daina, O. Michielin, V. Zoete, SwissADME: a free web tool to evaluate
pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small
molecules, Sci. Rep. 7 (2017) 42717, https://doi.org/10.1038/srep42717.
12