Discovery of a potent, highly selective, and orally bioavailable inhibitor of CDK8 through a structure-based optimisation

Article abstract of DOI:10.1016/j.ejmech.2021.113391

CDK8 is deregulated in multiple types of human cancer and is viewed as a therapeutic target for the treatment of the disease. Accordingly, the search for small-molecule inhibitors of CDK8 is being intensified. Capitalising on our initial discovery of AU1-100, a potent CDK8 inhibitor yet with a limited degree of kinase selectivity, a structure-based optimisation was carried out, with a series of new multi-substituted pyridines rationally designed, chemically prepared and biologically evaluated. Such endeavour has culminated in the identification of 42, a more potent CDK8 inhibitor with superior kinomic selectivity and oral bioavailability. The mechanism underlying the anti-proliferative effect of 42 on MV4-11 cells was studied, revealing that the compound arrested the G1 cell cycle and triggered apoptosis. The low risk of hepato- and cardio-toxicity of 42 was estimated. These findings merit further investigation of 42 as a targeted cancer therapeutic.

Full text of DOI:10.1016/j.ejmech.2021.113391

European Journal of Medicinal Chemistry 218 (2021) 113391
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of a potent, highly selective, and orally bioavailable inhibitor
of CDK8 through a structure-based optimisation
Mingfeng Yu, Yi Long, Yuchao Yang, Manjun Li, Theodosia Teo, Benjamin Noll,
Stephen Philip, Shudong Wang*
Drug Discovery and Development, Cancer Research Institute, Clinical and Health Sciences, University of South Australia, Adelaide, South Australia, 5000,
Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
CDK8 is deregulated in multiple types of human cancer and is viewed as a therapeutic target for the
treatment of the disease. Accordingly, the search for small-molecule inhibitors of CDK8 is being inten-
sified. Capitalising on our initial discovery of AU1-100, a potent CDK8 inhibitor yet with a limited degree
of kinase selectivity, a structure-based optimisation was carried out, with a series of new multi-
substituted pyridines rationally designed, chemically prepared and biologically evaluated. Such
endeavour has culminated in the identification of 42, a more potent CDK8 inhibitor with superior
kinomic selectivity and oral bioavailability. The mechanism underlying the anti-proliferative effect of 42
on MV4-11 cells was studied, revealing that the compound arrested the G1 cell cycle and triggered
apoptosis. The low risk of hepato- and cardio-toxicity of 42 was estimated. These findings merit further
investigation of 42 as a targeted cancer therapeutic.
Received 16 February 2021
Received in revised form
16 March 2021
Accepted 17 March 2021
Available online 26 March 2021
Keywords:
CDK8 inhibitor
Structure-based optimisation
Kinase selectivity
Oral bioavailability
Anti-proliferation mechanism
MV4-11
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
embryonic fibroblasts [11].
Deregulation of CDK8 has been considered as a driver lesion in
As a member of the cyclin-dependent kinase (CDK) family, CDK8
is typically involved in regulating transcription in two forms [1,2]:
(i) partnered with cyclin C, CDK8 phosphorylates a number of
transcription factors such as signal transducer and activator of
transcription 1 (STAT1) [3], neurogenic locus notch homolog pro-
teins (NOTCH) [4] and mothers against decapentaplegic homologs
(SMADs) [5], modulating their activity or priming them for
ubiquitin-mediated proteasomal degradation; or (ii) recruited to
form a four-subunit kinase module that additionally consists of
cyclin C, MED12 and MED13, CDK8 binds reversibly to the Mediator
complex, a central integrator as well as a signal processor and
transducer in the RNA polymerase II general transcriptional ma-
chinery [6e8]. In contrast to the well-studied role of CDK8 in
controlling gene expression, its function in cell division remains
much less understood [9], with related studies reported sporadi-
cally [10,11]. For instance, the Skp2-MacroH2A1-CDK8 axis has
been demonstrated to orchestrate the G2/M transition in mouse
various types of human cancer. Specifically, CDK8 was identified as
an oncogene in colorectal cancer [12], and amplification and/or
mutation of this gene were detected in melanoma [13], acute
myeloid leukaemia (AML) [14], and cancers of breast [11], pancreas
[15], prostate [16], gastrointestinal tract, bladder, and other organs
[17]. Silencing the expression of CDK8 with microRNAs, small
interfering RNAs or short hairpin RNAs inhibited in vitro cell pro-
liferation of breast [18e20], prostate [16], colorectal [12,21] and
pancreatic [15] cancers, and melanoma [13]. Correspondingly,
pharmacological suppression of the kinase activity of CDK8 by
exogenous small-molecule inhibitors shrank tumours not only in
xenograft models derived from cell lines of COLO205, HCT116,
LS513, LS1034 and SW620 colorectal [22e25], MCF7 and MDA-MB-
231 breast [26,27], and VCaP prostate [10] cancers, SET-2, KG-1 and
MV4-11 AML [14,28], and RPMI 8226 myeloma [29,30], but also in
patient-derived xenografts [24]. In addition, treatment of natural
killer cells with CDK8 inhibitors has been very recently found to
enhance the lysis of HL-60 AML cells and B-chronic lymphocytic
leukaemia cells in vitro, and to promote tumour surveillance in the
murine B16eF10-luc2 syngeneic melanoma model [31]. Taken
together, CDK8 represents a therapeutic target for validation,
* Corresponding author.
E-mail address: shudong.wang@unisa.edu.au (S. Wang).
https://doi.org/10.1016/j.ejmech.2021.113391
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
particularly in the treatment of human cancer, and consequently
exogenous small-molecule inhibitors of this kinase with adequate
potency, selectivity and drug-like properties are highly sought-
after.
hampering the understanding of underlying mechanisms of its
anti-proliferative effect on MV4-11 cells and its toxicity towards
murine long bones and lungs. Besides, the in vitro drug metabolism
and pharmacokinetics (DMPK) profile of AU1-100 was suboptimal.
For instance, the compound inhibited two cytochrome P450
(CYP450) enzymes moderately, increasing the likelihood of
inducing drug-drug interactions in combination therapy. Herein we
describe our endeavour in the structure-based optimisation of
AU1-100 for superior CDK8 inhibitory selectivity and drug-like
properties.
The initial discovery of some CDK8 inhibitors (Fig. 1) arose
serendipitously from the searches for inhibitors of alternative tar-
gets. Typical examples include (i) cortistatin A, a natural product
with anti-angiogenic activity [32,33]; (ii) senexin A, which was
developed based upon the results of a high-throughput screening
of more than 100,000 commercially available small molecules for
the inhibition of p21-induced transcription [34,35]; and (iii)
CCT251545 that was originally identified as an inhibitor of the WNT
signalling pathway from a high-throughput cell-based reporter
assay [25,36]. While structurally simpler steroidal inhibitors and
degraders of CDK8 were derived from cortistatin A using analogue-
based drug design [37,38], medicinal chemistry optimisation of
senexin A and CCT251545 gave rise to senexin B (also known as
BCD-115) [39] and CCT251921 [23], respectively, with improved
CDK8 inhibitory activity and specificity. Among them, BCD-115 has
been clinically trialled in combination with endocrine therapy in
women with estrogen receptor-positive/human epidermal growth
factor receptor 2-negative, locally advanced and metastatic breast
cancer (NCT03065010: phase I completed). The other CDK8 inhib-
itor that has entered a clinical trial is SEL120 (reported as SEL120-
34A in the literature [28]) for treating AML or high-risk myelo-
dysplastic syndrome (NCT04021368: phase I ongoing). Alternative
CDK8 inhibitors with diverse chemical scaffolds have been recently
reviewed [2,40e42].
2. Results and discussion
2.1. Structure-based design
To increase the kinase inhibitory selectivity for CDK8 over GSK-
3a/b requires, as a first step, a comprehension of the molecular
basis for the potency of AU1-100 towards the three kinases. The
compound was first docked into one of CDK8 crystal structures
(PDB ID: 5BNJ) using Glide, a rapid and accurate approach for the
ligand-receptor docking and the scoring of ligand poses [59,60].
The binding mode thus generated predicts multiple interactions of
AU1-100 with CDK8, which include, but are not limited to,
hydrogen bonds,
Intriguingly, the
onlydrather than the entire benzofuranyl moietydin interacting
with CDK8-R356. similar interaction was postulated as
p-cation stacking and Cl-p contact (Fig. 2).
p-cation stacking engages the furan component
A
a
contributor of the exquisite selectivity of CCT251545 for CDK8 due
to an uncommon insertion of C-terminal R356 into the hinge region
of the kinase [36]. Accordingly, we envisaged that replacement of
the benzofuranyl moiety with a smaller five-membered aromatic
ring at the pyridine-C5 position could hold promise of fine-tuning
the degree of CDK8 inhibitory selectivity (Fig. 3).
At the same time, our structural analysis of known CDK8 in-
hibitors derived from a pyridine core revealed that both ortho po-
sitions of the pyridine ring were scarcely exploited, with very few
examples demonstrated [23,30,61]. Among them is CCT251921
with a primary amino group at the pyridine-C2 position (Fig. 1);
We have ongoing interests in discovering and developing small-
molecule inhibitors of CDKs for cancer therapy [43e56], with our
recent attention turned to a hunt for potent and selective CDK8
inhibitors with favourable drug-like properties [2,57,58]. One of our
attempts has resulted in the identification of AU1-100 (Fig. 1) as a
potent CDK8 inhibitor [58]. However, AU1-100 displayed a mod-
erate inhibitory specificity for CDK8 in a screening against a se-
lection of 62 kinases, with the kinase activity of GSK-3
suppressed to the same magnitude as that of CDK8 (i.e., Ki ¼ 0.014,
0.013 and 0.004 M for CDK8, GSK-3 and GSK-3 , respectively),
a/b
m
a
b
Fig. 1. Chemical structures of selected CDK8 inhibitors.
2

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
Fig. 2. Predicted binding pose of AU1-100 (in gold sticks) in CDK8/cyclin C (in grey ribbons, with selected amino acid residues annotated). Hydrogen bonds are drawn as dashed
lines, and
p
-cation and Cl-
p
interactions as dotted and dash-dotted lines, respectively. Nitrogen atoms are shown in blue, oxygen atoms in red, and the chlorine atom is coloured
€
green. The illustration was generated using PyMOL Molecular Graphics System Version 2.4.0 Schrodinger, LLC.
Fig. 3. Design of novel CDK8 inhibitors.
this amino group was found to form a hydrogen bond with the
reported synthetic route [58]. Briefly, 3-bromo-5-chloropyridine 1
backbone carbonyl of CDK8-D98, contributing to the affinity for the
kinase [23]. The counterpart of CDK8-D98 in GSK-3a is E196 that
contains one more carbon atom in the side chain [62]. This residue
difference led us to wonder whether introduction of an
appropriate-size hydrogen bond donor onto the pyridine-C2 posi-
sequentially underwent (i) lithium-assisted chlorination with
hexachloroethane at the C4 position, (ii) nucleophilic aromatic
displacement of the newly introduced chlorine by tert-butyl 1-oxo-
2,8-diazaspiro[4.5]decane-8-carboxylate, and (iii) Pd(dppf)
Cl2$CH2Cl2-catalysed Suzuki coupling at the C5 position with
boronic acids or boronate esters of diverse five-membered heter-
oaromatic rings, giving rise to the desired compounds 4e32 in
varying yields (Scheme 1).
tion of AU1-100 would perturb the interaction with GSK-3
a but
maintain or even strengthen the contact with CDK8. Such mole-
cules as a 2-amino derivative of AU1-100 could be docked into a
GSK-3
crystal structure of this kinase was (and is) available in the Protein
Data Bank. Nevertheless, prototype molecules with 2-
aminopyridine scaffold were pursued to examine the hypothesis
experimentally (Fig. 3).
a
crystal structure to test our hypothesis, but regrettably no
Synthesis of 8-(2-amino-5-(benzofuran/benzothiophen-2-yl)-
3-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-ones 42 and 43
was carried out via multiple steps (Scheme 2). Detailedly, 4-
chloropyridin-2-amine 33 was successively halogenated at C5 and
C3 positions with N-bromosuccinimide (NBS) at room temperature
and with N-chlorosuccinimide (NCS) at reflux, respectively,
affording 5-bromo-3,4-dichloropyridin-2-amine 35 in an overall
yield of approximately 56%. To minimise the chance of the 2-amino
group interfering with the subsequent nucleophilic aromatic sub-
stitution and palladium-catalysed cross-coupling reaction, 35 was
a
2.2. Synthesis
Preparation of 8-(3-chloro-5-aryl-pyridin-4-yl)-2,8-diazaspiro
[4.5]decan-1-ones 4e32 was accomplished using our previously
3

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
Scheme 1. Synthesis of 8-(3-chloro-5-aryl-pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-ones 4e32. Reagents and conditions: (a) lithium diisopropylamide (2.0 M in THF/heptane/
ethylbenzene), hexachloroethane, THF, ꢁ78 ꢀC,
2 h, 65%; (b) tert-butyl 1-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate, triethylamine, 1-methoxy-2-propanol, microwave
150e300 W, 220 ꢀC, 2.5 h, 79%; (c) appropriate boronic acid or boronate ester, Pd(dppf)Cl2$CH2Cl2, 0.5 M Na2CO3, CH3CN, microwave 150e300 W, 120e160 ꢀC, 1e2 h, 10e67%.
Scheme 2. Synthesis of 8-(2-amino-5-(benzofuran/benzothiophen -2-yl)-3-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-ones 42 and 43. Reagents and conditions: (a) NBS,
CH3CN, rt, 3 h, 58%; (b) NCS, CH3CN, reflux, 3 h, 96%; (c) sodium hydride (60% dispersion in mineral oil), 4-methoxybenzyl chloride, DMF, 0 ꢀC to rt, 2.25 h, 36: 66%, 37: 28%; (d) tert-
butyl 1-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate, KF, triethylamine, N-methyl-2-pyrrolidone, microwave 150e300 W, 38: 220 ꢀC, 1 h, 48%, 39: 225 ꢀC, 2.5 h, 68%; (e) 2-
(benzofuran-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane or benzo[b]thiophen-2-ylboronic acid, Pd(dppf)Cl2$CH2Cl2, 0.5 M Na2CO3, CH3CN, microwave 150e300 W, 130 ꢀC, 1 h,
40: 44%, 41: 45%; (f) TFA, rt, o/n, 42: 75%, 43: 61%.
protected by 4-methoxybenzyl to give both mono- and di-
substituted forms in an excellent overall yield (94%), with the
latter being the major product (36: 66%). Fully and partially pro-
tected forms 36 and 37 were individually subjected to microwave-
assisted nucleophilic aromatic displacement of the chlorine at the
C4 position with tert-butyl 1-oxo-2,8-diazaspiro[4.5]decane-8-
carboxylate at very high temperature (220e225 ꢀC); potassium
fluoride (KF) was used to facilitate the displacement via the halide
exchange (Halex) process (i.e., conversion of 4-Cl to 4-F that served
as a better leaving group) [63]. Compounds 38 and 39 thus obtained
were coupled with their respective 2-(benzofuran-2-yl)-4,4,5,5-
2.3. Structureeactivity relationship analysis
Our previous studies showed that MV4-11 AML cells were the
most sensitive to AU1-100 in a screening against a panel of 19
human cancer cell lines with different origins [58]. Accordingly, this
cell line was chosen to assess the anti-proliferative effect of the
newly synthesised compounds using our in-house resazurin assay
[64]. In parallel, the inhibitory activity of these compounds towards
CDK8/cyclin C was evaluated externally with the 33PanQinase®
activity assayda radiometric kinase assay using [
As described in Section 2.1, docking results showed that only the
furan component of AU1-100 was projected to be involved in the p-
g-
33P]-ATP.
tetramethyl-1,3,2-dioxaborolane
and
benzo[b]thiophen-2-
ylboronic acid in the presence of Pd(dppf)Cl2$CH2Cl2, yielding the
corresponding adducts 40 and 41 in decent yields (40: 44% and 41:
45%). Removal of the 4-methoxybenzyl protecting group(s) from
each adduct was effected in trifluoroacetic acid (TFA) at room
temperature overnight, furnishing 42 and 43 in yields of 75% and
61%, respectively.
cation interaction with the positively charged guanidinium side
chain of CDK8-R356 (Fig. 2). To confirm this experimentally, 4 with
a simpler furan-2-yl substituent at the pyridine-C5 position was
first synthesised and tested (Table 1). This compound was just
slightly less active towards both CDK8 and MV4-11 cells when
compared with its benzofuran-2-yl counterpart AU1-100
4

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
Table 1
Chemical structures and biological activities of 4e8.
Compound
Ar
CDK8/cyclin C inhibition, Ki (
m
M)a
MV4-11 anti-proliferation, GI50
0.36 0.35
(m
M)b
AU1-100 (38c)
0.014
4
0.026
0.048
0.94 0.04
40c
29.16 1.19
5
0.042
0.014
0.75 0.02
0.58 0.14
39c
6
0.010
0.021
0.48 0.08
41c
0.35 0.22
(continued on next page)
5

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
Table 1 (continued)
Compound
Ar
CDK8/cyclin C inhibition, Ki (
m
M)a
MV4-11 anti-proliferation, GI50
0.19 0.09
(m
M)b
7
0.018
35c
0.056
0.014
8.77 2.04
8
1.05 0.67
a
Inhibition of CDK8/cyclin C was measured with the33PanQinase® activity assay at ProQinase GmbH, and the Km value used as the ATP concentration. Each apparent
inhibition constant (Ki) was calculated using its corresponding IC50 value and the Km (ATP) value.
b
GI50 values were determined in-house by the 72 h resazurin assay, and are presented as mean standard deviation derived from at least two replicates.
Compounds and their biological data were previously reported in Ref. [58].
c
(Ki ¼ 0.014
digit nanomolar kinase inhibition (Ki ¼ 0.026
micromolar anti-proliferation (GI50 ¼ 0.94 M). Encouraged by
m
M and GI50 ¼ 0.36
m
M), and maintained low double-
formyl or an acetyl group onto the furan-3-yl ring of 5 or the thi-
ophen-3-yl ring of 7 (Table 3), the first half further tested by
attaching an electron-donating phenyl to the furan-2-yl-C5 posi-
tion of 6 (Table 2). This sizeable attachment gave rise to single-digit
mM) and sub-
m
these data, compounds 5e8 with a furan-3-yl, a thiophen-2-yl, a
thiophen-3-yl and an 1H-pyrrol-3-yl, respectively, were further
prepared. All four compounds displayed higher inhibitory activities
against both CDK8 and MV4-11 cells than did their corresponding
benzo-fused counterparts 40, 39, 41 and 35 (reported previously
[58]), with Ki and GI50 values in ranges of 0.010e0.042 and
nanomolar CDK8 inhibition (19: Ki ¼ 0.005
mM), which stimulated
the pursuit of bulky pedants at the same position. However,
replacement of the phenyl with several azacycle-containing groups
including 5-(pyrrolidin-1-ylmethyl) (20), 5-(piperidin-1-ylmethyl)
(21), 5-morpholinomethyl (22), 5-((4-Boc-piperazin-1-yl)methyl)
(23) enfeebled the kinase inhibitory activity by up to 26.4-fold
0.19e1.05
mM, respectively. Collectively, both enzymatic and
cellular inhibitory activities of 5e8 were comparable, if not supe-
rior, to those of AU1-100. These results indicated that replacement
of [5,6]-fused aromatic moieties with their constitutive five-
membered rings was well tolerated at the pyridine-C5 position.
Given that all the five-membered aromatic rings introduced
thus far were not substituted elsewhere, it was deemed advisable
to functionalise them to explore the structureeactivity relationship
further (Tables 2 and 3). Introduction of a small-size methyl (9) or a
long, linear (ethoxymethoxy)methyl (11) onto the furan-2-yl-C5
(Ki ¼ 0.032e0.132
mM).
As expected, all the compounds with a formyl or an acetyl
substituent at the furan-3-yl or thiophen-3-yl ring were much less
potent against CDK8 than their respective parent molecules (25:
Ki
¼
0.185
m
M
versus 5: Ki
¼
0.042
mM; 26e29:
Ki ¼ 0.052e4.655
mM versus 7: Ki ¼ 0.018
mM) (Table 3). The 1H-
pyrrol-3-yl counterpart of furan-3-yl 5 or thiophen-3-yl 7 was a
more active inhibitor of CDK8 (8: Ki ¼ 0.014 M), but their 1H-
pyrazol-4-yl equivalent was far inferior (30: Ki ¼ 0.254 M).
Masking the NH site of the latter with a methyl or a difluoromethyl
partly restored kinase inhibitory potency (31: Ki ¼ 0.230 M and
32: Ki ¼ 0.093 M). Contrastingly, 24 with its pyrrol-2-yl-NH
covered by a methyl retained CDK8 inhibitory activity, with a Ki
value of 0.030 M (Table 2).
m
m
position of 4 (Ki ¼ 0.026
(9: Ki ¼ 0.022 M and 11: Ki ¼ 0.036
a formyl at the same site reduced CDK8 inhibitory activity by over
M) (Table 2). A similar phenomenon was
m
M) maintained the potency against CDK8
m
mM), whereas incorporation of
m
m
three times (Ki ¼ 0.082
m
observed in functionalisation of the thiophen-2-yl ring of 6. Sub-
stitution with a methyl at any available position or with a chlorine
atom at C5 caused the inhibition of CDK8 to a degree comparable to
m
In general, compounds with their five-membered aromatic rings
unsubstituted elsewhere or substituted by electron-donating
groups were typically one-digit or low two-digit nanomolar in-
that of the parent molecule (12e15: Ki ¼ 0.008e0.014
Ki ¼ 0.010 M). In contrast, 16 and 17 appended with a 5-acetyl and
a 5-formyl, respectively, were more than six-fold less active against
M). Di-
mM versus 6:
m
hibitors of CDK8 (i.e., Ki ¼ 0.005e0.042
was translated into sub-micromolar anti-proliferative effect on
MV4-11 cells (GI50 ¼ 0.19e0.96 M) (Tables 2 and 3) with a very few
exceptionsd8, 9 and 11 with Ki values of <0.050 M yet with GI50
values of >1 M.
Among all the compounds synthesised thus far (Tables 1e3), 19
with 5-(5-phenylthiophen-2-yl) arm displayed the highest
mM). Such kinase inhibition
the kinase (16: Ki ¼ 0.066
m
M and 17: Ki ¼ 0.067
m
m
substitution with a 4-methyl and a 5-formyl significantly weak-
ened the CDK8 inhibition, with the Ki value being one order of
m
m
magnitude higher (18: Ki ¼ 0.119
mM). These data suggested that
electron-donating groups like methyl and chlorine were tolerated
but electron-withdrawing groups such as formyl and acetyl
engendered detrimental effects. This postulation was not unex-
pected because these substituted five-membered aromatic rings
a
inhibitory potency against CDK8, and therefore was subjected to a
screening against a selection of five kinases, namely glycogen
synthase kinase (GSK)-3a, GSK-3b, protein kinase C (PKC)-q, CDK1/
were likely to be engaged in the
p
-cation interaction with CDK8-
cyclin B and CDK9/cyclin T1, all of which were identified as off-
targets of AU1-100 [58]. Upon treatment with 19 at a concentra-
R356, where electronic properties of their substituents could in-
fluence the strength of electrostatic attraction [65]. While the
second half of the postulation was confirmed by introduction of a
tion of 1
mM, kinase activities of CDK1/9 and PKC-
q
were either
deprived
hardly affected or slightly diminished, whereas GSK-3a/b
6

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
Table 2
Chemical structures and biological activities of 4, 6 and 9e24.
Compound
X
R
CDK8/cyclin C inhibition, Ki (
m
M)a
MV4-11 anti-proliferation, GI50 (m
M)b
4
9
10
11
6
O
O
O
O
S
S
S
S
S
S
S
S
S
S
S
S
e
0.026
0.022
0.082
0.036
0.010
0.008
0.010
0.010
0.014
0.066
0.067
0.119
0.005
0.132
0.106
0.032
0.046
0.030
0.94 0.04
4.95 5.07
5.19 0.06
2.97 0.38
0.48 0.08
0.91 0.01
0.65 0.13
0.65 0.08
0.56 0.10
1.44 0.67
0.75 0.08
2.53 0.13
0.96 0.05
3.37 2.25
4.01 0.84
2.96 2.86
5.15 0.38
0.90 0.06
5-CH3
5-CHO
5-(CH2O)2CH2CH3
e
12
13
14
15
16
17
18
19
20
21
22
23
24
3-CH3
4-CH3
5-CH3
5-Cl
5-COCH3
5-CHO
4-CH3-5-CHO
5-phenyl
5-(pyrrolidin-1-ylmethyl)
5-(piperidin-1-ylmethyl)
5-morpholinomethyl
5-((4-Boc-piperazin-1-yl)methyl)
e
S
NCH3
a
Inhibition of CDK8/cyclin C was measured with the33PanQinase® activity assay at ProQinase GmbH, and the Km value used as the ATP concentration. Each apparent
inhibition constant (Ki) was calculated using its corresponding IC50 value and the Km (ATP) value.
b
GI50 values were determined in-house by the 72 h resazurin assay, and are presented as mean standard deviation derived from at least two replicates.
of 62% and 74% kinase activity, respectively (Fig. 4). Both percentage
values were similar to those of AU1-100, indicating the comparable
potency of 19 against both GSK-3 isoforms to that of the control.
Given that the structural diversification at the pyridine-C5 po-
sition failed to return an improved inhibitory specificity for CDK8
assessments; the other single-digit nanomolar CDK8 inhibitor, 19,
was not chosen for its poor inhibitory selectivity over GSK-3
the preliminary screening (Fig. 4).
a/b in
Pharmacokinetic data stated in Table 5 were acquired by treat-
ing male Sprague Dawley rats and male BALB/c mice with the same
regimens as those for AU1-100 [58]. Briefly, rats were administered
42 or 43 via an intravenous (IV) injection at 5 mg/kg or per os (PO) at
20 mg/kg, whilst mice were given 42 or 43 IV at 2 mg/kg or PO at
10 mg/kg. After IV administration to rats, concentrations of 42 and
over GSK-3a/b, the effort was subsequently switched to the modi-
fication of the underexplored C2 site. Compounds 42 and 43 with a
common 2-amino group were then prepared and evaluated. Both
were more effective in inhibiting the kinase activity of CDK8 than
their 2-unsubstituted counterparts AU1-100 and 39 (reported
43 in plasma rose up to 9.36 and 11.10 mM within 2 and 9 min, and
previously [58]), with Ki values of 0.009 and 0.007
respectively (Table 4). These single-digit nanomolar inhibitory ac-
tivities towards CDK8 correlated with low sub-micromolar anti-
m
M for 42 and 43,
subsequently declined to half the levels at 1.45 and 4.41 h,
respectively, giving rise to their respective area under the plasma
concentration versus time curve (AUC) values of 8.92 and
proliferative effects on MV4-11 cells (42: GI50 ¼ 0.60
m
M and 43:
18.36 mM$h. The decline in the plasma concentration of each
GI50 ¼ 0.16
m
M). In comparison with CCT251921, 42 and 43 were
compound was attributed to its extravascular distribution (42:
Vdss ¼ 1.57 L/kg and 43: Vdss ¼ 1.32 L/kg) and its clearance from
plasma (42: Cl ¼ 1.43 L/h$kg and 43: Cl ¼ 0.79 L/h$kg). On the other
hand, following oral gavage of rats with 42 and 43 separately, their
respective concentrations in plasma reached maxima of 3.20 and
more potent antagonists of CDK8 but weaker inhibitors of MV4-
11 cells. Compound 42 was further tested against the same kinase
panel as that for 19, revealing that none of five off-target kinases
were targeted potently, with percentages of residual kinase activity
ranging from 61% to 97% (Fig. 4). The desired selectivity for CDK8
1.35
mM at 4 and 5 h, and then were halved approximately 3 and 5 h
over GSK-3
a/b
was eventually attained by introduction of a simple
later, resulting in AUC values of 25.09 and 19.46 mM$h, respectively.
amino group to the C2 position of the pyridine core.
In comparison with AU1-100, 42 showed an increased oral AUC
value, achieving a significantly improved oral bioavailability in rats
(42: F ¼ 70% versus AU1-100: F ¼ 38%). In contrast, 43 is less orally
bioavailable (F ¼ 26%). The same held true when the three com-
pounds were assessed in mice; 42 and AU1-100 had an identical F
value of 33% that was greater than that of 43 (F ¼ 28%). Only 42 was
advanced further by virtue of its superior oral bioavailability in
rodents, particularly in rats.
2.4. Pharmacokinetic profiling of 42 and 43 in murines
Preclinical pharmacokinetic evaluation is a pivotal component
of drug discovery and should be carried out at the very early stage
to prioritise the candidates with the best chance of success [66].
Accordingly, two out of the three most potent CDK8 inhibitors
(Ki < 0.010
mM), i.e., 42 and 43, were subjected to in vivo single-dose
pharmacokinetic studies prior to further in vitro biological
7

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
Table 3
Chemical structures and biological activities of 5, 7, 8 and 25e32.
Compound
Y/Z
R/R0
CDK8/cyclin C inhibition, Ki (
m
M)a
MV4-11 anti-proliferation, GI50 (m
M)b
5
25
7
26
27
28
29
O
O
S
S
S
e
0.042
0.185
0.018
4.655
1.745
0.845
0.052
0.75 0.02
5.15 0.18
0.19 0.09
>100
28.70 3.54
27.10 7.00
0.73 0.08
5-CHO
e
2-COCH3
2-CHO
4-CHO
5-CHO
S
S
8
C
H
H
CH3
CHF2
0.014
0.254
0.230
0.093
1.05 0.67
8.52 0.91
5.53 1.61
6.14 0.86
30
31
32
N
N
N
a
Inhibition of CDK8/cyclin C was measured with the33PanQinase® activity assay at ProQinase GmbH, and the Km value used as the ATP concentration. Each apparent
inhibition constant (Ki) was calculated using its corresponding IC50 value and the Km (ATP) value.
b
GI50 values were determined in-house by the 72 h resazurin assay, and are presented as mean standard deviation derived from at least two replicates.
Fig. 4. Kinase inhibition profiles of 19 and 42 (at 1 mM) over a selection of five off-target kinases of AU1-100dGSK-3a/b, PKC-q, CDK1/cyclin B and CDK9/cyclin T1. Percentages of
residual kinase activity of CDK8 and off-target kinases were acquired at ProQinase GmbH and Reaction Biology Corporation, respectively. All the kinase data of AU1-100 were
previously reported in Ref. [58], and are included in the figure for the comparison with the corresponding values of 19 and 42.
8

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
Table 4
Chemical structures and biological activities of 42 and 43.
Compound
X
R
CDK8/cyclin C inhibition, Ki (
m
M)a
MV4-11 anti-proliferation, GI50 (m
M)b
AU1-100
42
O
O
S
S
H
NH2
H
0.014
0.009
0.014
0.007
0.011
0.36 0.35
0.60 0.03
0.58 0.14
0.16 0.01
0.05 0.01
39c
43
NH2
CCT251921
structure in Fig. 1
a
Inhibition of CDK8/cyclin C was measured with the33PanQinase® activity assay at ProQinase GmbH, and the Km value used as the ATP concentration. Each apparent
inhibition constant (Ki) was calculated using its corresponding IC50 value and the Km (ATP) value.
b
GI50 values were determined in-house by the 72 h resazurin assay, and are presented as mean standard deviation derived from at least two replicates.
Compounds and their biological data were previously reported in Ref. [58].
c
Table 5
Pharmacokinetic properties of 42 and 43 in rats and micea.
Species
Compound
Route
Dose (mg/kg)
Vdss (L/kg)
Cl (L/h$kg)
Cmax
(m
M)
Tmax (h)
T1/2 (h)
AUC (
m
M$h)
F (%)
Rat
AU1-100b
IV
PO
IV
PO
IV
PO
5
20
5
20
5
20
1.11
e
1.39
e
14.40
2.77
9.36
3.20
11.10
1.35
0.04
2.75
0.03
4.00
0.15
5.00
0.90
2.24
1.45
6.99
4.41
10.09
9.70
14.58
8.92
25.09
18.36
19.46
-
38
-
70
-
26
42
43
1.57
e
1.43
e
1.32
e
0.79
e
Mouse
AU1-100b
IV
PO
IV
PO
IV
PO
2
10
2
10
2
10
1.99
e
3.78
e
3.44
5.01
4.16
2.23
4.96
1.68
0.04
0.42
0.05
0.34
0.05
0.18
0.34
1.11
0.37
3.44
0.88
1.67
1.39
2.30
1.55
2.56
2.40
3.40
-
33
-
33
-
28
42
43
1.24
e
3.25
e
1.60
e
2.00
e
a
Non-compartmental pharmacokinetic analysis was performed using Phoenix WinNonlin (Certara, St. Louis, MO, USA) for each curve of plasma concentration versus time,
and mean values derived from three rats or ten mice are presented.
b
Pharmacokinetic data of AU1-100 were previously reported in Ref. [58], and are included in the table for the comparison with those of 42 and 43.
2.5. Physicochemical characterisation of 42
solubility of the chemical through equilibrating the conversion
between its neutral and charged forms, and eventually affects its
oral absorption [67]. Compound 42 has a pKa value of 5.27 which is
greater than that of AU1-100 (pKa ¼ 4.90), suggesting that the
former would be more protonated than the latter at, for example,
gastric pH of 1.5e3.5 [68]. It is evident that a poor aqueous solu-
bility can limit the absorption of a drug from the gastrointestinal
tract, which in turn reduces its oral bioavailability. The
To better understand the pharmacokinetic behaviour of 42 in
rodents described above, the compound underwent a physico-
chemical profiling, with the negative log of the acid dissociation
constant (pKa), distribution coefficient (Log D), aqueous solubility,
and Caco-2 permeability measured (Table 6). The pKa value of a
chemical predicts its ionisation potential that influences the
Table 6
Physicochemical properties of 42.
Physicochemical propertya
AU1-100f
42
Negative log of the acid dissociation constant: pKab
4.90 0.02
10e30
4.21
22.9 0.6
23.4 3.2
1.02
5.27 0.01
3e20
4.08
29.7 1.9
29.8 2.9
1.00
Aqueous solubility (m
M)c
d
Distribution coefficient: Log D7.4
Caco-2 permeabilitye
AdB Papp ( ꢂ 10ꢁ6 cm/s)
BdA Papp ( ꢂ 10ꢁ6 cm/s)
Efflux ratio
a
Physicochemical properties were measured by Cyprotex Discovery Ltd.
Using a fast ultraviolet (UV) spectrometric titration.
By turbidimetry.
Using the shake flask assay with a buffer at pH 7.4.
Using Caco-2 cell line that was derived from a human colon carcinoma and that resembles intestinal epithelial cells. Transport of a compound across the cell monolayer
b
c
d
e
occurs in both directions: apical to basolateral (AdB) and basolateral to apical (BdA). Papp: apparent permeability coefficient; efflux ratio ¼ Papp(BdA)/Papp(AdB).
f
Physicochemical data of AU1-100 were previously reported in Ref. [58], and are included in the table for the comparison with those of 42.
9

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
turbidimetric solubility of 42 was estimated in between 3 and
to 27-time higher than that of CDK8 (Ki ¼ 0.009
mM). Further me-
20
mM, a span that is similar to that of AU1-100 (10e30
mM). These
dicinal chemistry optimisation to refine the kinase inhibitory
selectivity might not be necessary because all three off-targets are
implicated in human cancer. For example, FLT3 is a validated
therapeutic target in FLT3 mutation-positive AML, with two small-
molecule inhibitorsdmidostaurin and gilteritinibdapproved for
this indication by the United States Food and Drug Administration
(US FDA) [69,70], while pathological roles of LRRK2 and FMS in
malignancies are emerging [71e75]. To the best of our knowledge,
the inhibitory selectivity of 42 for CDK8 is at least comparable to
those of known inhibitors of this kinase.
ranges indicate that both compounds have limited aqueous solu-
bility. Log D of a compound is a measure of its lipophilicity that is a
key determinant of the pharmacokinetic behaviour of the com-
pound [67]. The Log D7.4 value of 42 was determined to be 4.08,
which is below that of AU1-100 (Log D7.4 ¼ 4.21) and is closer to an
optimal range of 0e3 [67]. A compound with its Log D value within
this range is typically considered to have a good balance between
solubility and permeability as well as to have low metabolic lia-
bility. Despite its suboptimal solubility and lipophilicity, 42 pos-
sesses high permeability with a passive apparent permeability
coefficient (Papp(AdB)) value of 29.7 ꢂ 10ꢁ6 cm/s acquired with the
Caco-2 cell-based permeability assay, which compares favourably
with that of AU1-100 (Papp(AdB) ¼ 22.9 ꢂ 10ꢁ6 cm/s). Moreover, an
efflux ratio of 1.00 for 42 reflects that little or no active efflux
occurred in the assay.
2.7. Safety profile of 42
In addition to a limited degree of CDK8 inhibitory selectivity,
AU1-100 exhibited moderate inhibition of two CYP450 enzyme
isoforms (i.e., CYP1A: IC50 ¼ 7.7
mM and CYP3A4: IC50 ¼ 4.6
mM)
[58], which might potentially interact with co-administered drugs,
leading to adverse drug reactions or toxicity [76]. The specificity for
CDK8 was successfully maximised by installation of a primary
amino group at the pyridine-C2 position of the compound (Table 4
and Fig. 5). The molecule thus generated, i.e., 42, was further
evaluated in vitro for its toxicity potential (Table 7). In contrast to
AU1-100, 42 has no inhibitory activity towards CYP1A and CYP3A4
as well as the other three isoforms tested (i.e., CYP2C9, CYP2C19
2.6. Kinase selectivity profile of 42
Having achieved the selectivity for CDK8 over GSK-3
42 was further screened against a panel of 371 kinases at a com-
pound concentration of 1 M. This concentration is above 110 times
a/b (Fig. 4),
m
greater than CDK8 Ki value of the compound. It was found that only
three other kinases, namely leucine-rich repeat kinase 2 (LRRK2),
colony stimulating factor 1 receptor (CSF1R, colloquially known as
FMS), and FMS-like tyrosine kinase 3 (FLT3), were deprived of more
than 50% kinase activity by 42 (Fig. 5). Ki values for the three kinases
and CYP2D6), with all IC50 values > 25
mM, suggesting a little
likelihood of inducing CYP450-mediated drug-drug interactions by
42. This can be important as combination therapy is prevalent in
the current treatments of cancer with kinase inhibitors [70].
were later measured to be in a range of 0.051e0.242 mM, which is 5-
Fig. 5. Kinase inhibition profile of 42 at 1 mM in a screening against a panel of 371 kinases. The human kinome tree was annotated and generated using KinMapbeta (http://www.
kinhub.org/kinmap/index.html). Kinase residual activity (%) values were included in Supplementary Data in the form of a spreadsheet file. The Ki value for each kinase with a
residual kinase activity of <20% was determined and presented in the table. All the data were acquired at Reaction Biology Corporation and ProQinase GmbH.
10

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
Table 7
Pharmaceutical properties of 42.
Parametera
AU1-100d
42
CYP450 inhibition, IC50
(
m
M)b
CYP1A
7.7 1.6
>25
>25
>25
4.6 0.9
10.9 2.2
>25
>25
>25
>25
>25
>25
CYP2C9
CYP2C19
CYP2D6
CYP3A4
hERG potassium channel inhibition, IC50 (m
M)c
a
Inhibition data were acquired by Cyprotex Discovery Ltd.
b
Using human liver microsomes in the presence of a CYP450 isoform-specific probe substrate: ethoxyresorufin for CYP1A, tolbutamide for CYP2C9, (S)-mephenytoin for
CYP2C19, dextromethorphan for CYP2D6, and midazolam for CYP3A4. Typically, potent inhibition: IC50 < 1 M; weak or no inhibition:
M; moderate inhibition: IC50 ¼ 1e10
IC50 > 10 M.
Using CHO-hERG cells. Typically, highly potent inhibition: IC50 < 0.1
M; moderate inhibition: IC50 ¼ 1e10
IC50 > 10 M.
m
m
m
c
m
M; potent inhibition: IC50 ¼ 0.1e1
m
mM; weak or no inhibition:
m
d
Inhibition data of AU1-100 were previously reported in Ref. [58], and are included in the table for the comparison with those of 42.
ꢀ
Furthermore, 42 is incapable of inhibiting the human ether-a-go-go
pharmacological inhibition of CDK8 in MDA-MB-468 cells induced
apoptosis [78]. The other breast cancer cell lines were 7.5- to 16-
related gene (hERG) potassium channel (IC50 > 25 mM). Blockade of
this cardiac ion channel induces QT interval prolongation, which
causes potentially fatal ventricular tachyarrhythmia termed torsade
de pointes [77]. Taken together, 42 has a low risk of causing hepato-
and cardio-toxicity.
fold less sensitive to 42 (GI50 ¼ 4.90e10.37
468, and the rest solid tumour cell lines tested were highly viable
(GI50 ¼ 25.80e64.78 M), with the exception of A2780 ovarian
M). Our previous studies showed that the
sensitivity of U-937 and MOLM-13 AML cell lines was only sec-
ondary to that of MV4-11 in a screening of AU1-100 against 20
mM) than MDA-MB-
m
cancer cells (GI50 ¼ 5.31
m
2.8. Anti-proliferative effects of 42 on cancer cell lines
human cell lines (U-937: GI50
GI50 ¼ 6.18 M versus MV4-11: GI50 ¼ 0.36
both cell lines remained amenable to 42 at single-digit micromolar
concentrations (U-937: GI50 4.88 and MOLM-13:
GI50 ¼ 5.11 M).
¼
1.36
m
M
and MOLM-13:
m
mM) [58]. Similarly,
As described in Section 2.3, discrepancies between CDK8 inhi-
bition and MV4-11 anti-proliferation were noted for a small subset
of compounds. This observation called for an interrogation of an
appropriate incubation time for the cell viability assay. In that
regards, MV4-11 cells were treated with 42 for a period of 24, 48, 72
or 96 h, and GI50 values were determined (Fig. S1). The anti-
proliferative effect increased up to 72-h treatment, and thereafter
started to plateau out. As a result, a 72-h incubation was considered
adequate for the cell viability assay and was implemented for
screening of 42 against a panel of 12 additional cancer cell lines
originated from blood/bone marrow, breast, ovary, pancreas and
colon (Fig. 6). Among them, MDA-MB-468 breast cancer cells were
¼
mM
m
2.9. Cellular mechanism of anti-proliferative action of 42
MV4-11 cell line was selected to investigate the mechanism of
the anti-proliferative action of 42 by virtue of its highest level of
sensitivity towards the compound. Firstly, cellular inhibition of
CDK8 kinase activity by 42 was confirmed using western blotting;
phosphorylation of STAT1-S727, a documented phosphorylation
site for CDK8 [3,79], was dampened, with the total level of STAT1
unaffected, after 24-h incubation with the compound at both 3 and
most sensitive to 42, resulting in a GI50 ¼ 0.65
growth inhibition was similar to that of MV4-11 cells
(GI50 ¼ 0.60 M), and is in line with a recent observation that
mM; this degree of
m
10 mM (Fig. 7A).
Fig. 6. Anti-proliferative effects of 42 on a panel of 13 human cancer cell lines. GI50 values were determined in-house by the 72-h cell viability assay using MTT (adherent cell lines)
or resazurin (suspension cell lines).
11

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
Fig. 7. Cellular mechanism of the anti-proliferative action of 42. (A) Western blot analysis of MV4-11 cells incubated with 42 for 24 h. b-Actin antibody was used as an internal
loading control. (B) Induction of apoptosis by 42 in MV4-11 cells. Cells were exposed to the compound for 72 h and analysed by annexin V/PI staining. The percentage of cells
undergoing apoptosis is defined as a sum of early apoptotic (annexin Vþ/PI-) and late apoptotic (annexin Vþ/PIþ) cell percentages. (C) Cell cycle analysis of MV4-11 cells after
incubation with 42 for 72 h. In all experiments, DMSO diluent was employed as a control, and 42 tested at concentrations of 3 and 10 mM.
Programmed cell death takes place mainly through apoptosis
[80]. To gauge whether apoptosis contributes to the observed anti-
proliferative effect of 42, MV4-11 cells were double-stained with
annexin V and propidium iodide (PI) upon incubation with the
compound for 72 h, and the apoptotic subpopulation was detected
by flow cytometry (Fig. 7B). In comparison with DMSO diluent, 42
induced approximately 4% and 10% more apoptotic cells (annexin
downregulated expression of anti-apoptotic protein myeloid cell
leukaemia 1 (Mcl-1) in the presence of the compound (Fig. S2).
To further evaluate whether the anti-proliferative effect of 42
results from the blockade of cell cycle progression, MV4-11 cells
were incubated with the compound at 3 and 10 mM for 72 h, fol-
lowed by an flow cytometric analysis of subpopulations at sub-G1,
G1, S and G2/M phases of the cell cycle (Fig. 7C). The compound
increased the G1-cells at both concentrations (i.e., by 11e27%), in
Vþ/PI- and annexin Vþ/PIþ) at concentrations of 3 and 10
mM,
respectively; a finding that correlates with an observation of the
agreement with
a
reduction in the phosphorylation of
12

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
retinoblastoma (Rb) protein at serine 780 (Fig. 7A) because phos-
phorylated Rb controls the G1-to-S transition of the cell cycle [81].
carried out on a Shimadzu Prominence UltraFast Liquid Chro-
matograph (UFLC) system (Kyoto, Japan) equipped with a CBM-20A
communications bus module, a DGU-20A5R degassing unit, an LC-
20AD liquid chromatograph pump, an SIL-20AHT auto-sampler, an
SPD-M20A photo diode array detector, a CTO-20A column oven and
3. Conclusions
By means of structure-based rational design coupled with
modular synthetic approaches developed newly and previously, a
number of new tri-/tetra-substituted pyridine derivatives were
prepared as CDK8 inhibitors. Compound 42 has been identified as
the most promising drug candidate by virtue of its high potency,
selectivity and favourable pharmacokinetic properties. 42 inhibited
cellular CDK8 evinced by targeting p-STAT1S727, arrested G1 cell
cycle and induced apoptosis in MV4-11 cells. Overall, this work has
paved the way for further preclinical characterisation of 42 in the
context of anti-AML therapy.
a
Phenomenex Kinetex C18 column (5
mm, 100 Å,
250 mm ꢂ 4.60 mm). Method A (at a flow rate of 1 mL/min,
gradient 5%e95% CH3OH containing 0.1% formic acid (FA) over
7 min followed by 95% CH3OH containing 0.1% FA over 13 min),
method B (at a flow rate of 1 mL/min, gradient 5%e95% CH3CN
containing 0.1% FA over 7 min followed by 95% CH3CN containing
0.1% FA over 13 min), method C (at a flow rate of 1 mL/min, gradient
5%e95% CH3OH containing 0.1% FA over 20 min followed by 95%
CH3OH containing 0.1% FA over 5 min) and method D (at a flow rate
of 1 mL/min, gradient 5%e95% CH3CN containing 0.1% FA over
20 min followed by 95% CH3CN containing 0.1% FA over 5 min) were
used for analytic RP-HPLC, and data thus acquired were processed
using LabSolutions Analysis Data System. Analytical TLC was per-
formed on Merck silica gel 60 F254 pre-coated aluminium plates
(0.2 mm) and visualised under UV light (254 nm). Flash column
chromatography was carried out using either a conventional glass
column loaded with GRACE Davison DAVISIL® silica gel 60 Å
4. Experimental
4.1. Method for computational modelling
A crystal structure of CDK8/cyclin C in complex with CCT251545
(PDB ID: 5BNJ) was used for computational modelling. The crystal
€
structure was pre-processed and refined with Schrodinger’s Protein
(40e63 mm) or a fritted solid loader packed with the same silica gel
Preparation Wizard module (Schrodinger Release 2020-4: Glide,
Schrodinger, LLC, New York, NY, 2020) to optimise all atom bonds
and orientations. The docking grid was generated to encompass the
ATP-binding site. Schrodinger’s Extra Precision Glide protocol
[59,60,82] was used to dock AU1-100 into the refined protein
crystal structure, where the best pose of the compound was
generated and compared with that of CCT251545.
on a Biotage FlashMaster Personalþ flash chromatography system.
General synthetic procedure A: To a suspension of a bromide
(1.00 equiv.), a boronic acid or boronate ester (1.05e1.20 equiv.) and
Pd(dppf)Cl2$CH2Cl2 (0.05 equiv.) in CH3CN (150 mM in bromide)
was added 0.5 M Na2CO3 aqueous solution (1.40 equiv.). The reac-
tion mixture was heated under microwave irradiation at
120e135 ꢀC for 1 h, cooled down, diluted with distilled H2O, and
extracted with DCM (3 ꢂ ). The organic extracts were combined,
washed with distilled H2O (1 ꢂ ), and concentrated under reduced
pressure. The residue was purified by Biotage® FlashMaster Per-
sonalþ flash chromatography (and, when necessary, triturated or
crystallised) to give the desired 5-substituted pyridine.
€
€
€
4.2. Chemistry
All the reagents (including boronic acids and boronate esters)
and non-anhydrous solvents were purchased from Sigma-Aldrich,
Merck, Combi-Blocks, AK Scientific, Thermo Fisher Scientific or
Ajax Finechem, and were used as received. Anhydrous solvents (i.e.,
DMF and THF) were collected freshly from a PureSolv™ Micro
solvent purification system with activated alumina columns. All the
reactions except microwave-assisted syntheses were carried out
with continuous magnetic stirring in ordinary glassware. Heating of
reactions was conducted with a DrySyn® single- or multi-position
heating block (Isleham, UK), and cooling of reactions achieved us-
ing an ice-salt or dry ice-acetone bath. Microwave-assisted syn-
theses were performed in a 10 or 35 mL pressure vessel using a CEM
Discover SP and Explorer 48/72/96 microwave system (Matthews,
NC, USA) controlled by Synergy software (Firmware version
DSCA02.17). 1H and 13C NMR spectra were recorded at 298 K on a
General synthetic procedure B: To
chloropyridine (1.00 equiv.) in NMP (600 mM in 4-chloropyrdine)
were added tert-butyl 1-oxo-2,8-diazaspiro[4.5]decane-8-
a solution of 4-
carboxylate (1.00e1.10 equiv.), KF (2.00 equiv.) and triethylamine
(3.00 equiv.). The reaction mixture was heated under microwave
irradiation at 220e225 ꢀC for 1e2.5 h, cooled down, diluted with
distilled H2O, and extracted with EtOAc (3 ꢂ ). The organic extracts
were combined, washed with distilled H2O (3 ꢂ ), and concentrated
under reduced pressure. The residue was purified by Biotage®
FlashMaster Personalþ flash chromatography to give the desired
spiro compound.
General synthetic procedure C: 4-Methoxybenzyl-protected
amine (1.00 equiv.) was dissolved in TFA (50 mM in amine). The
reaction mixture was stirred at room temperature overnight and
concentrated under reduced pressure. The residue was taken to
pH > 11 with saturated aqueous Na2CO3 solution and extracted
with DCM (3 ꢂ ). The organic extracts were combined and
concentrated under reduced pressure. The residue was purified by
Biotage® FlashMaster Personalþ flash chromatography (and crys-
tallised when necessary) to give the desired unprotected form.
3-Bromo-4,5-dichloropyridine (2): To a solution of LDA (2.0 M
in THF/heptane/ethylbenzene, 6.25 mL, 12.5 mmol) in anhydrous
THF (30 mL) at ꢁ78 ꢀC was added a solution of 3-bromo-5-
chloropyridine (1, 962 mg, 5.00 mmol) in anhydrous THF (10 mL).
The reaction mixture was stirred for 1 h, and a solution of hexa-
chloroethane (2.37 g, 10.0 mmol) in anhydrous THF (10 mL) added.
The reaction mixture was stirred for 1 h, warmed up to room
temperature, quenched with saturated NH4Cl aqueous solution
(30 mL), and extracted with DCM (3 ꢂ 100 mL). The organic extracts
were combined and concentrated under reduced pressure, and the
€
Bruker AVANCE III HD 500 spectrometer (Fallanden, Switzerland)
(1H at 500.2 MHz and 13C at 125.8 MHz), and were analysed using
Bruker Topspin 3.2 software. 1H and 13C NMR spectra are referenced
to 1H signals of residual non-deuterated solvents and 13C signals of
deuterated solvents, respectively. 1H NMR signals are reported with
chemical shift values
d
(ppm), multiplicity (s ¼ singlet, d ¼ doublet,
t ¼ triplet, q ¼ quartet, dd ¼ doublet of doublets, dt ¼ doublet of
triplets, td ¼ triplet of doublets, m ¼ multiplet, br ¼ broad and
app ¼ apparent), relative integrals, coupling constants J (Hz) and
assignments. High resolution mass spectra were recorded on an AB
SCIEX TripleTOF 5600 mass spectrometer (Concord, ON, Canada),
and the samples subjected to electrospray ionisation (ESI). Melting
points were determined using an open capillary on a Stuart SMP10
melting point apparatus or a Mettler Toledo MP50 melting point
system, and are uncorrected. The purity of the compounds used for
biological evaluation was determined by analytic reversed-phase
high-performance liquid chromatography (RP-HPLC), which was
13

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
residue was purified by Biotage® FlashMaster Personalþ flash
chromatography (petroleum benzine ramping to 50% DCM in pe-
troleum benzine) to give 2 as a yellow solid (735 mg, 65%). RF
Biotage® FlashMaster Personalþ flash chromatography (DCM
ramping to 2.5% CH3OH in DCM), triturated and washed with n-
hexane to give
6 as a pinkish solid (56 mg, 32%). RF
(DCM:petroleum benzine ¼ 1:4) 0.27. 1H NMR (CDCl3)
d
8.53 (s, 1H,
122.1,
(DCM:CH3OH ¼ 94:6) 0.36. m.p. 175e176 ꢀC. 1H NMR (DMSO‑d6)
pyridinyl-H), 8.62 (s, 1H, pyridinyl-H). 13C NMR (CDCl3)
d
d 1.32 (app d, 2H, J 12.5, CH2), 1.80e1.93 (m, 2H, CH2), 1.94 (t, 2H, J
131.8, 142.5, 148.5, 150.5.
6.5, CH2), 3.00 (app s, 4H, CH2), 3.16 (t, 2H, J 6.5, CH2), 7.18 (t, 1H, J
4.0, thiophenyl-H), 7.40 (s, 1H), 7.57 (s, 1H) (total 2H, CONH &
thiophenyl-H), 7.71 (d, 1H, J 5.0, thiophenyl-H), 8.45 (s, 1H,
8-(3-Bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-
1-one (3): To a solution of chloride 2 (1.36 g, 5.99 mmol) and tert-
butyl 1-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate (1.83 g,
7.20 mmol) in 1-methoxy-2-propanol (10 mL) was added triethyl-
amine (2.51 mL, 18.0 mmol). The reaction mixture was heated un-
der microwave irradiation at 220 ꢀC for 2.5 h, cooled down, diluted
with DCM (40 mL), washed with distilled H2O (2 ꢂ 20 mL), and
concentrated under reduced pressure. The residue was purified by
Biotage® FlashMaster Personalþ flash chromatography (DCM
ramping to 4% EtOH in DCM) to give 3 as a beige solid (1.63 g, 79%).
pyridinyl-H), 8.53 (s, 1H, pyridinyl-H). 13C NMR (DMSO‑d6)
d 30.4,
31.5, 38.0, 41.4, 46.2, 127.6, 127.9, 128.3, 128.4, 128.9, 136.5, 149.3,
150.0, 152.1, 180.3 (two carbon signals overlapping or obscured).
HRMS (ESI-TOF) 348.0933 [M(35Cl)þH]þ & 350.0900 [M(37Cl)þH]þ;
calcd. for C17H19ClN3OSþ 348.0932 [M(35Cl)þH]þ & 350.0902
[M(37Cl)þH]þ. Anal. RP-HPLC Method C: tR 19.30 min, purity >98%;
Method D: tR 12.69 min, purity >98%.
8-(3-Chloro-5-(thiophen-3-yl)pyridin-4-yl)-2,8-diazaspiro
RF (DCM:EtOH ¼ 96:4) 0.31. 1H NMR (CDCl3)
d
1.54 (d, 2H, J 13.0,
[4.5]decan-1-one (7): Bromide 3 (173 mg, 502
tetramethyl-2-(thiophen-3-yl)-1,3,2-dioxaborolane
528
m
mol) and 4,4,5,5-
CH2), 2.08e2.16 (m, 2H, CH2), 2.17 (t, 2H, J 7.0, CH2), 3.30e3.44 (m,
4H, 2 ꢂ CH2), 3.39 (t, 2H, J 7.0, CH2), 5.90 (br s, 1H, CONH), 8.35 (s,
(111 mg,
m
mol) were reacted at 120 ꢀC using general synthetic proced-
1H, pyridinyl-H), 8.49 (s, 1H, pyridinyl-H). 13C NMR (CDCl3)
d
31.5,
ure A. The residue was purified by Biotage® FlashMaster Personalþ
flash chromatography (DCM ramping to 4% CH3OH in DCM), tritu-
rated and washed with n-hexane to give 7 as a beige solid (82 mg,
47%). RF (DCM:CH3OH ¼ 95:5) 0.30. m.p. 212e213 ꢀC. 1H NMR
32.6, 39.0, 42.1, 47.0, 119.8, 129.2, 149.8, 151.6, 153.2, 182.1 (two
carbon signals overlapping or obscured). HRMS (ESI-TOF) 344.0167,
346.0149 & 348.0118 [MþH]þ; calcd. for C13H16BrClN3Oþ 344.0160,
346.0140 & 348.0110 [MþH]þ.
(DMSO‑d6)
d 1.25 (app d, 2H, J 13.0, CH2), 1.72 (td, 2H, J 12.5 & 4.0,
8-(3-Chloro-5-(furan-2-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]
CH2), 1.87 (t, 2H, J 7.0, CH2), 2.73 (t, 2H, J 11.5, CH2), 3.01 (app d, 2H, J
12.5, CH2), 3.14 (t, 2H, J 7.0, CH2), 7.19 (dd,1H, J 5.0 & 1.0, thiophenyl-
H), 7.54 (s, 1H, CONH), 7.60 (dd, 1H, J 2.5 & 1.5, thiophenyl-H), 7.68
(dd, 1H, J 5.0 & 3.0, thiophenyl-H), 8.23 (s, 1H, pyridinyl-H), 8.43 (s,
decan-1-one (4): Bromide 3 (173 mg, 502
ylboronic acid (62 mg, 554
mmol) and furan-2-
m
mol) were reacted at 120 ꢀC using
general synthetic procedure A. The residue was purified by Bio-
tage® FlashMaster Personalþ flash chromatography (DCM ramping
to 3.5% EtOH in DCM), triturated and washed with n-hexane to give
4 as a beige solid (55 mg, 33%). RF (DCM:EtOH ¼ 96:4) 0.23. m.p.
1H, pyridinyl-H). 13C NMR (DMSO‑d6)
d 30.5, 31.8, 37.9, 41.3, 46.9,
124.3, 126.5, 127.4, 129.1, 129.3, 137.0, 148.9, 150.5, 152.6, 180.0 (two
carbon signals overlapping or obscured). HRMS (ESI-TOF) 348.0937
[M(35Cl)þH]þ & 350.0909 [M(37Cl)þH]þ; calcd. for C17H19ClN3OSþ
348.0932 [M(35Cl)þH]þ & 350.0902 [M(37Cl)þH]þ. Anal. RP-HPLC
Method A: tR 10.66 min, purity >98%; Method B: tR 8.38 min, purity
>98%.
167e168 ꢀC. 1H NMR (DMSO‑d6)
d 1.33 (app d, 2H, J 12.0, CH2), 1.78
(td, 2H, J 11.0 & 2.5, CH2), 1.94 (t, 2H, J 6.0, CH2), 2.79 (t, 2H, J 11.5,
CH2), 3.04 (d, 2H, J 11.5, CH2), 3.17 (t, 2H, J 6.0, CH2), 6.67 (app s, 1H,
furanyl-H), 6.77 (app s, 1H, furanyl-H), 7.58 (s, 1H, CONH), 7.87 (app
s, 1H, furanyl-H), 8.41 (s, 1H, pyridinyl-H), 8.48 (s, 1H, pyridinyl-H).
8-(3-Chloro-5-(1H-pyrrol-3-yl)pyridin-4-yl)-2,8-diazaspiro
13C NMR (DMSO‑d6)
d
30.5, 31.8, 37.9, 41.3, 45.8, 109.9, 112.2, 123.7,
[4.5]decan-1-one (8): Bromide 3 (173 mg, 502
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-(triisopropylsilyl)-
1H-pyrrole (210 mg, 601
mol) were reacted at 135 ꢀC using general
mmol) and 3-
128.3, 143.9, 148.8, 149.6, 149.9, 152.5, 180.0 (two carbon signals
overlapping or obscured). HRMS (ESI-TOF) 332.1164 [M(35Cl)þH]þ
m
&
334.1132 [M(37Cl)þH]þ; calcd. for C17H19ClN3Oþ2 332.1160
synthetic procedure A, followed by microwave irradiation at 160 ꢀC
for 1 h. The residue was purified by Biotage® FlashMaster Personalþ
flash chromatography (DCM ramping to 3.5% CH3OH in DCM) to
give 8 as a yellow solid (38 mg, 23%). RF (DCM:CH3OH ¼ 96:4) 0.27.
[M(35Cl)þH]þ & 334.1131 [M(37Cl)þH]þ. Anal. RP-HPLC Method A:
tR 11.12 min, purity >95%; Method B: tR 8.83 min, purity >97%.
8-(3-Chloro-5-(furan-3-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]
decan-1-one (5): Bromide 3 (173 mg, 502
m
mol) and 2-(furan-3-
m.p. 225e226 ꢀC. 1H NMR (DMSO‑d6)
d 1.28 (app d, 2H, J 13.0, CH2),
yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (102 mg, 526
m
mol)
1.77 (td, 2H, J 12.0 & 2.5, CH2), 1.92 (t, 2H, J 7.0, CH2), 2.90 (t, 2H, J
11.5, CH2), 2.96 (d, 2H, J 12.0, CH2), 3.16 (t, 2H, J 7.0, CH2), 6.22 (s, 1H,
pyrrolyl-H), 6.85 (s, 1H, pyrrolyl-H), 7.02 (s, 1H, pyrrolyl-H), 7.54 (s,
1H, CONH), 8.26 (s, 1H, pyridinyl-H), 8.30 (s, 1H, pyridinyl-H), 11.05
were reacted at 120 ꢀC using general synthetic procedure A. The
residue was purified by Biotage® FlashMaster Personalþ flash
chromatography (DCM ramping to 4% CH3OH in DCM), triturated
and washed with n-hexane to give 5 as a beige solid (90 mg, 54%).
RF (DCM:CH3OH ¼ 95:5) 0.31. m.p. 167e168 ꢀC. 1H NMR (DMSO‑d6)
(s,1H, pyrrolyl-NH). 13C NMR (DMSO‑d6)
d 30.4, 31.8, 37.9, 41.4, 46.5,
109.1, 117.4, 118.1, 118.4, 128.2, 130.4, 147.1, 150.5, 152.0, 180.2 (two
carbon signals overlapping or obscured). HRMS (ESI-TOF) 331.1330
[M(35Cl)þH]þ & 333.1300 [M(37Cl)þH]þ; calcd. for C17H20ClN4Oþ
331.1321 [M(35Cl)þH]þ & 333.1291 [M(37Cl)þH]þ. Anal. RP-HPLC
Method C: tR 12.25 min, purity >98%; Method D: tR 9.10 min, purity
>98%.
d
1.32 (app d, 2H, J 12.5, CH2), 1.76 (td, 2H, J 12.0 & 4.0, CH2), 1.94 (t,
2H, J 7.0, CH2), 2.95 (t, 2H, J 11.5, CH2), 3.04 (dt, 2H, J 12.0 & 3.5, CH2),
3.16 (t, 2H, J 7.0, CH2), 6.72 (app s, 1H, furanyl-H), 7.56 (br s, 1H,
CONH), 7.81 (t, 1H, J 1.5, furanyl-H), 7.96 (app s, 1H, furanyl-H), 8.29
(s, 1H, pyridinyl-H), 8.42 (s, 1H, pyridinyl-H). 13C NMR (DMSO‑d6)
d
30.5, 31.8, 37.9, 41.3, 46.7, 112.2, 121.1, 125.9, 127.8, 141.1, 143.7,
8-(3-Chloro-5-(5-methylfuran-2-yl)pyridin-4-yl)-2,8-
149.0, 150.3, 152.5, 180.1 (two carbon signals overlapping or
diazaspiro[4.5]decan-1-one (9): Bromide 3 (173 mg, 502
m
mol)
obscured). HRMS (ESI-TOF) 332.1164 [M(35Cl)þH]þ & 334.1138
and
4,4,5,5-tetramethyl-2-(5-methylfuran-2-yl)-1,3,2-
[M(37Cl)þH]þ; calcd. for C17H19ClN3Oþ2 332.1160 [M(35Cl)þH]þ
&
dioxaborolane (125 mg, 601 m
mol) were reacted at 135 ꢀC using
334.1131 [M(37Cl)þH]þ. Anal. RP-HPLC Method A: tR 10.37 min,
general synthetic procedure A. The residue was purified by Bio-
tage® FlashMaster Personalþ flash chromatography (DCM ramping
to 2.5% CH3OH in DCM), and crystallised with DCM/n-hexane to
give 9 as yellow crystals (56 mg, 33%). RF (DCM:CH3OH ¼ 94:6)
purity >95%; Method B: tR 8.22 min, purity >96%.
8-(3-Chloro-5-(thiophen-2-yl)pyridin-4-yl)-2,8-diazaspiro
[4.5]decan-1-one (6): Bromide 3 (173 mg, 502
phen-2-ylboronic acid (71 mg, 554
mol) were reacted at 120 ꢀC
using general synthetic procedure A. The residue was purified by
mmol) and thio-
m
0.39. m.p. 178e179 ꢀC. 1H NMR (DMSO‑d6)
d 1.34 (app d, 2H, J 12.5,
CH2), 1.78 (td, 2H, J 11.5 & 1.5, CH2), 1.95 (t, 2H, J 6.5, CH2), 2.36 (s,
14

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
3H, CH3), 2.80 (t, 2H, J 11.5, CH2), 3.02 (d, 2H, J 12.0, CH2), 3.18 (t, 2H,
J 6.5, CH2), 6.26 (s, 1H, furanyl-H), 6.66 (s, 1H, furanyl-H), 7.57 (s, 1H,
CONH), 8.39 (s, 1H, pyridinyl-H), 8.44 (s, 1H, pyridinyl-H). 13C NMR
[M(35Cl)þH]þ & 364.1059 [M(37Cl)þH]þ; calcd. for C18H21ClN3OSþ
362.1088 [M(35Cl)þH]þ & 364.1059 [M(37Cl)þH]þ. Anal. RP-HPLC
Method C: tR 19.56 min, purity >97%; Method D: tR 12.75 min,
purity >97%.
(DMSO‑d6)
d 13.6, 30.6, 31.9, 38.0, 41.3, 45.9, 108.2, 110.5, 124.3,
128.6, 147.3, 149.1, 149.6, 152.2, 152.7, 180.1 (two carbon signals
8-(3-Chloro-5-(4-methylthiophen-2-yl)pyridin-4-yl)-2,8-
overlapping or obscured). HRMS (ESI-TOF) 346.1321 [M(35Cl)þH]þ
diazaspiro[4.5]decan-1-one (13): Bromide 3 (173 mg, 502
mmol)
&
348.1289 [M(37Cl)þH]þ; calcd. for C18H21ClN3Oþ2 346.1317
and
4,4,5,5-tetramethyl-2-(4-methylthiophen-2-yl)-1,3,2-
[M(35Cl)þH]þ & 348.1287 [M(37Cl)þH]þ. Anal. RP-HPLC Method C:
tR 18.56 min, purity >97%; Method D: tR 13.09 min, purity >96%.
5-(5-Chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)pyridin-
dioxaborolane (124 mg, 553 m
mol) were reacted at 120 ꢀC using
general synthetic procedure A. The residue was purified by Bio-
tage® FlashMaster Personalþ flash chromatography (DCM ramping
to 3% CH3OH in DCM), triturated and washed with n-hexane to give
13 as a beige solid (65 mg, 36%). RF (DCM:CH3OH ¼ 94:6) 0.41. m.p.
3-yl)furan-2-carbaldehyde (10): Bromide 3 (173 mg, 502
mmol)
and (5-formylfuran-2-yl)boronic acid (77 mg, 552 mol) were
m
reacted at 120 ꢀC using general synthetic procedure A. The residue
was purified by Biotage® FlashMaster Personalþ flash chromatog-
raphy (DCM ramping to 3% CH3OH in DCM), and crystallised with
DCM/n-hexane to give 10 as yellow crystals (32 mg, 18%). RF
(DCM:CH3OH ¼ 94:6) 0.34. m.p. 195e196 ꢀC. 1H NMR (DMSO‑d6)
161e162 ꢀC. 1H NMR (DMSO‑d6)
d 1.33 (app d, 2H, J 12.5, CH2),
1.80e1.93 (m, 2H, CH2), 1.94 (t, 2H, J 6.5, CH2), 2.26 (s, 3H, CH3), 2.99
(app s, 4H, 2 ꢂ CH2), 3.16 (t, 2H, J 6.5, CH2), 7.21 (s, 1H, thiophenyl-
H), 7.28 (s, 1H, thiophenyl-H), 7.57 (s, 1H, CONH), 8.43 (s, 1H,
pyridinyl-H), 8.49 (s, 1H, pyridinyl-H). 13C NMR (DMSO‑d6)
d 15.5,
d
1.33 (app d, 2H, J 12.5, CH2), 1.78 (td, 2H, J 12.0 & 2.5, CH2), 1.90 (t,
30.3, 31.4, 37.9, 41.3, 46.1, 123.5, 128.4, 128.8, 129.9, 136.3, 137.3,
149.1, 149.8, 152.0, 180.1 (two carbon signals overlapping or
obscured). HRMS (ESI-TOF) 362.1090 [M(35Cl)þH]þ & 364.1060
2H, J 6.0, CH2), 2.77 (t, 2H, J 12.0, CH2), 3.07 (d, 2H, J 12.0, CH2), 3.16
(t, 2H, J 6.0, CH2), 7.09 (d, 1H, J 2.5, furanyl-H), 7.58 (s, 1H, CONH),
7.72 (d, 1H, J 2.0, furanyl-H), 8.53 (s, 1H, pyridinyl-H), 8.57 (s, 1H,
[M(37Cl)þH]þ; calcd. for C18H21ClN3OSþ 362.1088 [M(35Cl)þH]þ
&
pyridinyl-H), 9.68 (s, 1H, CHO). 13C NMR (DMSO‑d6)
d
30.2, 31.7,
364.1059 [M(37Cl)þH]þ. Anal. RP-HPLC Method A: tR 12.03 min,
37.9, 41.3, 46.2, 112.2, 121.9, 125.1, 128.3, 150.4, 150.7, 152.4, 153.0,
154.6, 178.3, 179.9 (two carbon signals overlapping or obscured).
HRMS (ESI-TOF) 360.1121 [M(35Cl)þH]þ & 362.1085 [M(37Cl)þH]þ;
purity >95%; Method B: tR 9.81 min, purity >97%.
8-(3-Chloro-5-(5-methylthiophen-2-yl)pyridin-4-yl)-2,8-
diazaspiro[4.5]decan-1-one (14): Bromide 3 (173 mg, 502
mmol)
calcd. for
C
18H19ClN3Oþ3 360.1109 [M(35Cl)þH]þ
&
362.1080
and
4,4,5,5-tetramethyl-2-(5-methylthiophen-2-yl)-1,3,2-
[M(37Cl)þH]þ. Anal. RP-HPLC Method C: tR 16.24 min, purity >96%;
dioxaborolane (124 mg, 553 m
mol) were reacted at 120 ꢀC using
Method D: tR 11.64 min, purity >98%.
general synthetic procedure A. The residue was purified by Bio-
tage® FlashMaster Personalþ flash chromatography (DCM ramping
to 3% CH3OH in DCM), triturated and washed with n-hexane to give
14 as a beige solid (110 mg, 60%). RF (DCM:CH3OH ¼ 96:4) 0.26.
8-(3-Chloro-5-(5-((ethoxymethoxy)methyl)furan-2-yl)pyr-
idin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (11): Bromide
(173 mg, 502 mol) and 2-(5-((ethoxymethoxy)methyl)furan-2-
yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (170 mg, 602 mol)
3
m
m
m.p. 172e173 ꢀC. 1H NMR (DMSO‑d6)
d 1.34 (app d, 2H, J 12.5, CH2),
were reacted at 135 ꢀC using general synthetic procedure A. The
residue was purified by Biotage® FlashMaster Personalþ flash
chromatography (DCM ramping to 3% CH3OH in DCM) to give 11 as
a beige solid (90 mg, 43%). RF (DCM:CH3OH ¼ 94:6) 0.38. m.p.
1.82e1.93 (m, 2H, CH2), 1.95 (t, 2H, J 7.0, CH2), 2.51 (s, 3H, CH3), 3.01
(app s, 4H, 2 ꢂ CH2), 3.18 (t, 2H, J 7.0, CH2), 6.87 (d, 1H, J 2.0,
thiophenyl-H), 7.16 (app s, 1H, thiophenyl-H), 7.59 (s, 1H, CONH),
8.43 (s, 1H, pyridinyl-H), 8.47 (s, 1H, pyridinyl-H). 13C NMR
137e138 ꢀC. 1H NMR (DMSO‑d6)
d
1.13 (t, 3H, J 7.0, CH2CH3), 1.32
(DMSO‑d6) d 15.0, 30.4, 31.4, 37.9, 41.2, 46.1,125.8,127.7,128.4,128.7,
(app d, 2H, J 12.5, CH2), 1.78 (td, 2H, J 12.5 & 3.5, CH2), 1.96 (t, 2H, J
6.5, CH2), 2.78 (t, 2H, J 12.0, CH2), 3.03 (d, 2H, J 12.0, CH2), 3.17 (t, 2H,
J 6.5, CH2), 3.54 (q, 2H, J 7.0, CH2CH3), 4.55 (s, 2H), 4.68 (s, 2H) (total
4H, furanyl-CH2OCH2), 6.60 (d, 1H, J 2.5, furanyl-H), 6.72 (d, 1H, J
2.5, furanyl-H), 7.57 (s, 1H, CONH), 8.39 (s, 1H, pyridinyl-H), 8.47 (s,
134.1, 141.5, 148.9, 149.9, 151.8, 180.0 (two carbon signals over-
lapping or obscured). HRMS (ESI-TOF) 362.1090 [M(35Cl)þH]þ
&
364.1059 [M(37Cl)þH]þ; calcd. for C18H21ClN3OSþ 362.1088
[M(35Cl)þH]þ & 364.1059 [M(37Cl)þH]þ. Anal. RP-HPLC Method A:
tR 12.01 min, purity >95%; Method B: tR 9.73 min, purity >97%.
8-(3-Chloro-5-(5-chlorothiophen-2-yl)pyridin-4-yl)-2,8-
1H, pyridinyl-H). 13C NMR (DMSO‑d6)
d 15.1, 30.5, 31.8, 38.0, 41.5,
46.0, 60.6, 62.8, 93.7, 110.1, 111.7, 123.5, 128.4, 149.2, 149.6, 150.0,
152.3, 152.6, 180.1 (two carbon signals overlapping or obscured).
HRMS (ESI-TOF) 420.1691 [M(35Cl)þH]þ & 422.1657 [M(37Cl)þH]þ;
diazaspiro[4.5]decan-1-one (15): Bromide 3 (173 mg, 502
mmol)
and (5-chlorothiophen-2-yl)boronic acid (90 mg, 554 mol) were
m
reacted at 120 ꢀC using general synthetic procedure A. The residue
was purified by Biotage® FlashMaster Personalþ flash chromatog-
raphy (DCM ramping to 3% CH3OH in DCM), and crystallised with
DCM/n-hexane to give 15 as yellow crystals (21 mg, 11%). RF
(DCM:CH3OH ¼ 94:6) 0.41. m.p. 195e196 ꢀC. 1H NMR (DMSO‑d6)
calcd. for
C
21H27ClN3Oþ4 420.1685 [M(35Cl)þH]þ
& 422.1655
[M(37Cl)þH]þ. Anal. RP-HPLC Method C: tR 18.91 min, purity >95%;
Method D: tR 13.41 min, purity >96%.
8-(3-Chloro-5-(3-methylthiophen-2-yl)pyridin-4-yl)-2,8-
diazaspiro[4.5]decan-1-one (12): Bromide 3 (173 mg, 502
mmol)
d 1.41 (app d, 2H, J 13.0, CH2), 1.92e2.10 (m, 2H, CH2), 2.02 (t, 2H, J
and
4,4,5,5-tetramethyl-2-(3-methylthiophen-2-yl)-1,3,2-
7.0, CH2), 2.98 (d, 2H, J 10.5, CH2), 3.20 (t, 2H, J 6.5, CH2), 3.23e3.39
(m, 2H, CH2), 7.19 (d, 1H, J 4.0, thiophenyl-H), 7.53 (s, 1H), 7.61 (s,
1H) (total 2H, thiophenyl-H & CONH), 8.46 (s, 1H, pyridinyl-H), 8.79
dioxaborolane (124 mg, 553 m
mol) were reacted at 120 ꢀC using
general synthetic procedure A. The residue was purified by Bio-
tage® FlashMaster Personalþ flash chromatography (DCM ramping
to 2.5% CH3OH in DCM), and triturated with n-hexane to give 12 as
an off-white solid (80 mg, 44%). RF (DCM:CH3OH ¼ 94:6) 0.45. m.p.
(s, 1H, pyridinyl-H). 13C NMR (DMSO‑d6)
d 30.2, 31.0, 32.2, 38.0, 41.1,
45.3, 46.9, 126.8, 127.0, 128.5, 129.7, 130.9, 134.4, 148.4, 149.4, 150.7,
180.1. HRMS (ESI-TOF) 382.0544 [M(35Cl/35Cl)þH]þ, 384.0514
202e203 ꢀC. 1H NMR (DMSO‑d6)
d
1.26 (app d, 2H, J 12.5, CH2), 1.70
[M(35Cl/37Cl)þH]þ
&
386.0489 [M(37Cl/37Cl)þH]þ; calcd. for
(td, 2H, J 12.0 & 4.0, CH2), 1.83 (t, 2H, J 6.5, CH2), 2.12 (s, 3H, CH3),
2.67 (t, 2H, J 12.0, CH2), 3.07 (app d, 2H, J 12.0, CH2) 3.12 (t, 2H, J 6.5,
CH2), 7.03 (d,1H, J 5.0, thiophenyl-H), 7.53 (s,1H, CONH), 7.60 (d,1H,
J 5.0, thiophenyl-H), 8.17 (s, 1H, pyridinyl-H), 8.47 (s, 1H, pyridinyl-
C
17H18Cl2N3OSþ
382.0542
[M(35Cl/35Cl)þH]þ,
384.0513
[M(35Cl/37Cl)þH]þ & 386.0484 [M(37Cl/37Cl)þH]þ. Anal. RP-HPLC
Method C: tR 21.27 min, purity >95%; Method D: tR 15.82 min,
purity >96%.
H). 13C NMR (DMSO‑d6)
d
14.2, 30.5, 31.8, 37.9, 41.3, 46.6, 125.5,
8-(3-(5-Acetylthiophen-2-yl)-5-chloropyridin-4-yl)-2,8-
125.9, 127.1, 130.2, 131.7, 135.6, 149.6, 151.7, 154.2, 179.9 (two carbon
diazaspiro[4.5]decan-1-one (16): Bromide 3 (173 mg, 502
mmol)
signals overlapping or obscured). HRMS (ESI-TOF) 362.1089
and (5-acetylthiophen-2-yl)boronic acid (94 mg, 552 mol) were
m
15

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
reacted at 120 ꢀC using general synthetic procedure A. The residue
was purified by Biotage® FlashMaster Personalþ flash chromatog-
raphy (DCM ramping to 3% CH3OH in DCM) to give 16 as a tan solid
(25 mg, 13%). RF (DCM:CH3OH ¼ 94:6) 0.38. m.p. 189e190 ꢀC. 1H
2H, CH2), 3.05 (app d, 2H, J 12.0, CH2) 3.10e3.25 (m, 2H, CH2), 3.17 (t,
2H, J 7.0, CH2), 7.34 (t, 1H, J 7.5, phenyl-H), 7.44 (t, 2H, J 7.5,
2 ꢂ phenyl-H), 7.47 (s, 1H), 7.51e7.62 (m, 2H) (total 3H, CONH &
2 ꢂ thiophenyl-H), 7.73 (d, 2H, J 7.5, 2 ꢂ phenyl-H), 8.45 (s, 1H,
NMR (DMSO‑d6)
d
1.35 (app d, 2H, J 13.0, CH2), 1.85 (td, 2H, J 11.5 &
pyridinyl-H), 8.65 (s, 1H, pyridinyl-H). 13C NMR (DMSO‑d6)
d 30.5,
1.5, CH2), 1.94 (t, 2H, J 6.5, CH2), 2.57 (s, 3H, CH3), 2.97 (t, 2H, J 11.0,
CH2), 3.07 (d, 2H, J 12.0, CH2), 3.16 (t, 2H, J 6.5, CH2), 7.45 (app s, 1H,
thiophenyl-H), 7.56 (s, 1H, CONH), 7.96 (d, 1H, J 3.5, thiophenyl-H),
8.52 (s, 1H, pyridinyl-H), 8.53 (s, 1H, pyridinyl-H). 13C NMR
31.4, 38.1, 41.4, 46.2, 123.9, 125.5, 128.1, 128.5, 128.9, 129.4, 133.6,
135.6, 145.2, 149.3, 149.4, 151.7, 180.3 (five carbon signals over-
lapping or obscured). HRMS (ESI-TOF) 424.1239 [M(35Cl)þH]þ
&
426.1212 [M(37Cl)þH]þ; calcd. for C23H23ClN3OSþ 424.1245
[M(35Cl)þH]þ & 426.1215 [M(37Cl)þH]þ. Anal. RP-HPLC Method C:
tR 22.92 min, purity >95%; Method D: tR 17.65 min, purity >97%.
8-(3-Chloro-5-(5-(pyrrolidin-1-ylmethyl)thiophen-2-yl)pyr-
(DMSO‑d6)
d 26.7, 30.5, 31.5, 37.9, 41.2, 46.4, 127.0, 128.4, 129.2,
133.9, 144.8, 145.0, 150.1, 150.3, 152.3, 179.9, 191.0 (two carbon
signals overlapping or obscured). HRMS (ESI-TOF) 390.1034
[M(35Cl)þH]þ & 392.1000 [M(37Cl)þH]þ; calcd. for C19H21ClN3O2Sþ
390.1038 [M(35Cl)þH]þ & 392.1008 [M(37Cl)þH]þ. Anal. RP-HPLC
Method C: tR 17.97 min, purity >96%; Method D: tR 13.09 min,
purity >96%.
idin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (20): Bromide
(173 mg, 502 mol) and 1-((5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)thiophen-2-yl)methyl)pyrrolidine (162 mg,
3
m
552 m
mol) were reacted at 120 ꢀC using general synthetic proced-
5-(5-Chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)pyridin-
ure A. The residue was purified by Biotage® FlashMaster Personalþ
flash chromatography (DCM ramping to 9% CH3OH in DCM), and
crystallised with DCM/n-hexane to give 20 as yellow crystals
(22 mg, 10%). RF (DCM:CH3OH ¼ 9:1) 0.36. m.p. 180e181 ꢀC. 1H
3-yl)thiophene-2-carbaldehyde (17): Bromide
3
(173 mg,
502
552
mmol) and (5-formylthiophen-2-yl)boronic acid (86 mg,
m
mol) were reacted at 120 ꢀC using general synthetic proced-
ure A. The residue was purified by Biotage® FlashMaster Personalþ
flash chromatography (DCM ramping to 3.5% CH3OH in DCM), and
crystallised with DCM/n-hexane to give 17 as orange crystals
(30 mg, 16%). RF (DCM:CH3OH ¼ 94:6) 0.39. m.p. 215e216 ꢀC. 1H
NMR (DMSO‑d6) d 1.32 (app d, 2H, J 13.0, CH2), 1.71 (app s, 4H,
2 ꢂ pyrrolidinyl-CH2), 1.83 (td, 2H, J 11.5 &1.5, CH2), 1.90 (t, 2H, J 6.5,
CH2), 2.52 (app s, 4H, 2 ꢂ pyrrolidinyl-CH2), 2.90 (t, 2H, J 11.0, CH2),
3.03 (d, 2H, J 12.0, CH2), 3.16 (t, 2H, J 6.5, CH2), 3.80 (s, 2H, thio-
phenyl-CH2-pyrrolidinyl), 6.97 (s, 1H, thiophenyl-H), 7.13 (s, 1H,
thiophenyl-H), 7.55 (s, 1H, CONH), 8.42 (s, 1H, pyridinyl-H), 8.44 (s,
NMR (DMSO‑d6) d 1.35 (app d, 2H, J 12.5, CH2), 1.85 (td, 2H, J 11.0 &
2.0, CH2), 1.94 (t, 2H, J 6.5, CH2), 2.90 (t, 2H, J 10.5, CH2), 3.07 (app d,
2H, J 11.5, CH2), 3.16 (t, 2H, J 6.5, CH2), 7.57 (s, 1H), 7.58 (s, 1H) (total
2H, CONH & thiophenyl-H), 8.07 (d, 1H, J 3.5, thiophenyl-H), 8.53 (s,
1H, pyridinyl-H), 8.58 (s, 1H, pyridinyl-H), 9.96 (s, 1H, CHO). 13C
1H, pyridinyl-H). 13C NMR (DMSO‑d6)
d 23.2, 30.4, 31.5, 37.9, 41.3,
46.3, 53.3, 54.0, 125.4, 127.0, 128.3, 128.5, 135.9, 145.3, 149.1, 150.2,
152.3, 180.0 (four carbon signals overlapping or obscured). HRMS
(ESI-TOF) 431.1675 [M(35Cl)þH]þ & 433.1636 [M(37Cl)þH]þ; calcd.
for C22H28ClN4OSþ 431.1667 [M(35Cl)þH]þ & 433.1637 [M(37Cl)þ
H]þ. Anal. RP-HPLC Method C: tR 14.47 min, purity >95%; Method
D: tR 10.22 min, purity >95%.
NMR (DMSO‑d6)
d 30.5, 31.5, 37.9, 41.2, 46.3, 126.8, 128.5, 129.4,
138.0, 144.1, 146.1, 150.0, 150.5, 152.4, 180.0, 184.6 (two carbon
signals overlapping or obscured). HRMS (ESI-TOF) 376.0885
[M(35Cl)þH]þ & 378.0856 [M(37Cl)þH]þ; calcd. for C18H19ClN3O2Sþ
376.0881 [M(35Cl)þH]þ & 378.0852 [M(37Cl)þH]þ. Anal. RP-HPLC
Method C: tR 18.06 min, purity >96%; Method D: tR 12.91 min,
purity >95%.
8-(3-Chloro-5-(5-(piperidin-1-ylmethyl)thiophen-2-yl)pyr-
idin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (21): Bromide
(173 mg, 502 mol) and 1-((5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)thiophen-2-yl)methyl)piperidine (170 mg,
3
m
5-(5-Chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)pyridin-
3-yl)-3-methylthiophene-2-carbaldehyde (18): Bromide
(173 mg, 502 mol) and (5-formyl-4-methylthiophen-2-yl)boronic
acid (94 mg, 552
mol) were reacted at 120 ꢀC using general syn-
3
553 m
mol) were reacted at 120 ꢀC using general synthetic proced-
m
ure A. The residue was purified by Biotage® FlashMaster Personalþ
flash chromatography (DCM ramping to 7% CH3OH in DCM) to give
21 as a tan solid (125 mg, 56%). RF (DCM:CH3OH ¼ 9:1) 0.35. m.p.
m
thetic procedure A. The residue was purified by Biotage® Flash-
Master Personalþ flash chromatography (DCM ramping to 3%
CH3OH in DCM), and crystallised with DCM/n-hexane to give 18 as a
yellow solid (30 mg, 15%). RF (DCM:CH3OH ¼ 94:6) 0.39. m.p.
198e199 ꢀC. 1H NMR (DMSO‑d6)
d 1.31 (app d, 2H, J 12.0, CH2), 1.38
(app s, 2H, piperidinyl-CH2),1.42e1.60 (m, 4H, 2 ꢂ piperidinyl-CH2),
1.84 (td, 2H, J 12.0 & 2.0, CH2), 1.90 (t, 2H, J 6.5, CH2), 2.41 (app s, 4H,
2 ꢂ piperidinyl-CH2), 2.88 (t, 2H, J 11.0, CH2), 3.04 (d, 2H, J 11.5, CH2),
3.15 (t, 2H, J 6.5, CH2), 3.67 (s, 2H, thiophenyl-CH2-piperidinyl), 6.96
(d, 1H, J 3.0, thiophenyl-H), 7.14 (d, 1H, J 2.5, thiophenyl-H), 7.55 (s,
1H, CONH), 8.41 (s, 1H, pyridinyl-H), 8.44 (s, 1H, pyridinyl-H). 13C
233e234 ꢀC. 1H NMR (DMSO‑d6)
d 1.36 (app d, 2H, J 12.5, CH2), 1.84
(td, 2H, J 11.0 & 1.0, CH2), 1.95 (t, 2H, J 6.5, CH2), 2.60 (s, 3H, CH3),
3.00 (t, 2H, J 10.0, CH2), 3.07 (d, 2H, J 12.0, CH2), 3.16 (t, 2H, J 6.5,
CH2), 7.38 (s, 1H), 7.57 (s, 1H) (total 2H, CONH & thiophenyl-H), 8.52
(s, 1H, pyridinyl-H), 8.54 (s, 1H, pyridinyl-H), 10.07 (s, 1H, CHO). 13C
NMR (DMSO‑d6)
d 24.0, 25.5, 30.4, 31.5, 37.8, 41.2, 46.3, 53.7, 57.3,
NMR (DMSO‑d6)
d
13.8, 30.5, 31.5, 37.9, 41.1, 46.3,126.8,128.4,132.6,
126.0, 127.1, 128.2, 128.4, 136.2, 144.5, 149.1, 150.2, 152.3, 179.9 (four
carbon signals overlapping or obscured). HRMS (ESI-TOF) 445.1827
[M(35Cl)þH]þ & 447.1781 [M(37Cl)þH]þ; calcd. for C23H30ClN4OSþ
445.1823 [M(35Cl)þH]þ & 447.1794 [M(37Cl)þH]þ. Anal. RP-HPLC
Method C: tR 14.64 min, purity >97%; Method D: tR 10.64 min,
purity >97%.
138.1, 144.9, 147.9, 149.9, 150.4, 152.3, 179.9, 183.7 (two carbon
signals overlapping or obscured). HRMS (ESI-TOF) 390.1040
[M(35Cl)þH]þ & 392.1006 [M(37Cl)þH]þ; calcd. for C19H21ClN3O2Sþ
390.1038 [M(35Cl)þH]þ & 392.1008 [M(37Cl)þH]þ. Anal. RP-HPLC
Method C: tR 18.33 min, purity >95%; Method D: tR 13.61 min,
purity >96%.
8-(3-Chloro-5-(5-(morpholinomethyl)thiophen-2-yl)pyr-
8-(3-Chloro-5-(5-phenylthiophen-2-yl)pyridin-4-yl)-2,8-
idin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (22): Bromide
(173 mg, 502 mol) and 4-((5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)thiophen-2-yl)methyl)morpholine (171 mg,
3
diazaspiro[4.5]decan-1-one (19): Bromide 3 (173 mg, 502
mmol)
m
and (5-phenylthiophen-2-yl)boronic acid (113 mg, 554 mol) were
m
reacted at 120 ꢀC using general synthetic procedure A. The residue
was purified by Biotage® FlashMaster Personalþ flash chromatog-
raphy (DCM ramping to 2.5% CH3OH in DCM), and crystallised with
DCM/n-hexane to give 19 as a yellow solid (40 mg, 19%). RF
(DCM:CH3OH ¼ 94:6) 0.48. m.p. 174e175 ꢀC. 1H NMR (DMSO‑d6)
553 m
mol) were reacted at 120 ꢀC using general synthetic proced-
ure A. The residue was purified by Biotage® FlashMaster Personalþ
flash chromatography (DCM ramping to 5% CH3OH in DCM) to give
22 as a tan solid (100 mg, 45%). RF (DCM:CH3OH ¼ 94:6) 0.40. m.p.
231e232 ꢀC. 1H NMR (DMSO‑d6)
d 1.31 (app d, 2H, J 12.5, CH2), 1.84
d
1.37 (app d, 2H, J 12.5, CH2), 1.98 (t, 2H, J 7.0, CH2), 2.00e2.10 (m,
(td, 2H, J 11.5 & 1.5, CH2), 1.90 (t, 2H, J 6.5, CH2), 2.44 (app s, 4H,
16

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
2 ꢂ morpholinyl-CH2), 2.91 (t, 2H, J 11.5, CH2), 3.03 (d, 2H, J 12.0,
CH2), 3.16 (t, 2H, J 6.5, CH2), 3.60 (app s, 4H, 2 ꢂ morpholinyl-CH2),
3.71 (s, 2H, thiophenyl-CH2-morpholinyl), 7.00 (d, 1H, J 2.5,
thiophenyl-H), 7.17 (app s, 1H, thiophenyl-H), 7.55 (s, 1H, CONH),
8.43 (s, 1H, pyridinyl-H), 8.44 (s, 1H, pyridinyl-H). 13C NMR
1H), 8.36 (s, 1H) (total 2H, furanyl-H & pyridinyl-H), 8.48 (s, 1H,
pyridinyl-H), 9.68 (s, 1H, CHO). 13C NMR (DMSO‑d6)
d 30.6, 31.8,
37.9, 41.3, 46.9, 123.6, 123.9, 124.1, 127.7, 147.0, 149.8, 150.4, 152.5,
152.6, 178.8, 180.0 (two carbon signals overlapping or obscured).
HRMS (ESI-TOF) 360.1098 [M(35Cl)þH]þ & 362.1062 [M(37Cl)þH]þ;
(DMSO‑d6)
d
30.4, 31.5, 37.9, 41.3, 46.3, 53.0, 56.9, 66.2, 126.5, 127.1,
calcd. for
C
18H19ClN3Oþ3 360.1109 [M(35Cl)þH]þ
& 362.1080
128.3, 128.6, 136.5, 143.6, 149.2, 150.1, 152.2, 180.0 (four carbon
signals overlapping or obscured). HRMS (ESI-TOF) 447.1599
[M(35Cl)þH]þ & 449.1568 [M(37Cl)þH]þ; calcd. for C22H28ClN4O2Sþ
447.1616 [M(35Cl)þH]þ & 449.1587 [M(37Cl)þH]þ. Anal. RP-HPLC
Method C: tR 14.03 min, purity >98%; Method D: tR 10.07 min,
purity >97%.
[M(37Cl)þH]þ. Anal. RP-HPLC Method C: tR 14.19 min, purity >95%;
Method D: tR 10.20 min, purity >97%.
8-(3-(2-Acetylthiophen-3-yl)-5-chloropyridin-4-yl)-2,8-
diazaspiro[4.5]decan-1-one (26): Bromide 3 (173 mg, 502
mmol)
and (2-acetylthiophen-3-yl)boronic acid (102 mg, 600 mol) were
m
reacted at 135 ꢀC using general synthetic procedure A. The residue
was purified by Biotage® FlashMaster Personalþ flash chromatog-
raphy (DCM ramping to 3.5% EtOH in DCM) to give 26 as a straw
solid (130 mg, 66%). RF (DCM:EtOH ¼ 94:6) 0.42. m.p. 209e210 ꢀC.
Tert-butyl
decan-8-yl)pyridin-3-yl)thiophen-2-yl)methyl)piperazine-1-
carboxylate (23): Bromide 3 (173 mg, 502 mol) and tert-butyl 4-
((5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiophen-2-yl)
4-((5-(5-chloro-4-(1-oxo-2,8-diazaspiro[4.5]
m
1H NMR (DMSO‑d6)
d 1.10e1.30 (m, 2H, CH2), 1.45e1.70 (m, 2H,
methyl)piperazine-1-carboxylate (246 mg, 602
m
mol) were reacted
CH2), 1.70e1.90 (m, 2H, CH2), 2.31 (s, 3H, CH3), 2.50e2.60 (m, 2H,
CH2), 2.60e2.75 (m, 1H, CHH), 2.85e3.05 (m, 1H, CHH), 3.10 (t, 2H, J
6.5, CH2), 7.16 (d,1H, J 5.0, thiophenyl-H), 7.51 (s,1H, CONH), 8.02 (d,
1H, J 5.0, thiophenyl-H), 8.14 (s, 1H, pyridinyl-H), 8.46 (s, 1H,
at 135 ꢀC using general synthetic procedure A. The residue was
purified by Biotage® FlashMaster Personalþ flash chromatography
(DCM ramping to 4% CH3OH in DCM), and crystallised with DCM/n-
hexane to give 23 as
a
yellowish solid (112 mg, 41%). RF
pyridinyl-H). 13C NMR (DMSO‑d6)
d 29.3, 30.6, 31.8, 31.9, 37.9, 41.2,
(DCM:CH3OH ¼ 96:4) 0.32. m.p. 184e185 ꢀC. 1H NMR (DMSO‑d6)
46.7, 47.2, 126.6, 128.6, 132.2, 133.1, 138.7, 141.0, 149.6, 150.0, 153.0,
d
1.32 (app d, 2H, J 12.5, CH2), 1.38 (s, 9H, C(CH3)3), 1.86 (td, 2H, J 11.5
179.9, 190.2. HRMS (ESI-TOF) 390.1047 [M(35Cl)þH]þ & 392.1014
& 1.5, CH2), 1.91 (t, 2H, J 6.5, CH2), 2.41 (app s, 4H, 2 ꢂ piperazinyl-
CH2), 2.92 (t, 2H, J 11.0, CH2), 3.02 (d, 2H, J 12.0, CH2), 3.16 (t, 2H, J
6.5, CH2), 3.74 (s, 2H, thiophenyl-CH2-piperazinyl), 6.99 (d, 1H, J 3.0,
thiophenyl-H), 7.18 (app s, 1H, thiophenyl-H), 7.56 (s, 1H, CONH),
8.44 (s, 2H, 2 ꢂ pyridinyl-H) (four proton signals overlapping or
[M(37Cl)þH]þ; calcd. for C19H21ClN3O2Sþ 390.1038 [M(35Cl)þH]þ
&
392.1008 [M(37Cl)þH]þ. Anal. RP-HPLC Method C: tR 13.78 min,
purity >98%; Method D: tR 10.21 min, purity >98%.
3-(5-Chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)pyridin-
3-yl)thiophene-2-carbaldehyde (27): Bromide
3
(173 mg,
obscured). 13C NMR (DMSO‑d6)
d
28.1, 30.3, 31.5, 37.9, 41.2, 46.3,
502
553
mmol) and (2-formylthiophen-3-yl)boronic acid (86 mg,
52.2, 56.4, 78.8, 126.4, 127.1, 128.3, 128.6, 136.3, 143.6, 149.1, 150.0,
152.1, 153.9, 180.0 (seven carbon signals overlapping or obscured).
HRMS (ESI-TOF) 546.2301 [M(35Cl)þH]þ & 548.2252 [M(37Cl)þH]þ;
calcd. for C27H37ClN5O3Sþ 546.2300 [M(35Cl)þH]þ & 548.2271
[M(37Cl)þH]þ. Anal. RP-HPLC Method C: tR 17.50 min, purity >99%;
Method D: tR 12.45 min, purity >99%.
m
mol) were reacted at 120 ꢀC using general synthetic proced-
ure A. The residue was purified by Biotage® FlashMaster Personalþ
flash chromatography (DCM ramping to 3% CH3OH in DCM), and
crystallised with DCM/n-hexane to give 27 as a yellow solid (60 mg,
32%). RF (DCM:CH3OH ¼ 94:6) 0.41. m.p. 229e230 ꢀC. 1H NMR
(DMSO‑d6) d 1.23 (app d, 2H, J 12.5, CH2), 1.64 (app s, 2H, CH2), 1.82
8-(3-Chloro-5-(1-methyl-1H-pyrrol-2-yl)pyridin-4-yl)-2,8-
(t, 2H, J 6.0, CH2), 2.55e2.80 (m, 2H, CH2), 2.95e3.25 (m, 2H, CH2),
3.11 (t, 2H, J 6.5, CH2), 7.28 (d, 1H, J 4.5, thiophenyl-H), 7.52 (s, 1H,
CONH), 8.21 (d, 1H, J 4.5, thiophenyl-H), 8.28 (s, 1H, pyridinyl-H),
8.52 (s, 1H, pyridinyl-H), 9.67 (s, 1H, CHO). 13C NMR (DMSO‑d6)
diazaspiro[4.5]decan-1-one (24): Bromide 3 (173 mg, 502
and 1-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
pyrrole (125 mg, 604
mol) were reacted at 135 ꢀC using general
mmol)
m
synthetic procedure A. The residue was purified by Biotage®
FlashMaster Personalþ flash chromatography (DCM ramping to
2.5% CH3OH in DCM) to give 24 as a yellow solid (80 mg, 46%). RF
(DCM:CH3OH ¼ 96:4) 0.31. m.p. 188e189 ꢀC. 1H NMR (DMSO‑d6)
d 30.6, 31.9, 37.9, 41.2, 47.1, 125.9, 127.0, 132.4, 135.4, 139.0, 145.6,
150.4, 150.9, 153.5, 180.0, 184.0 (two carbon signals overlapping or
obscured). HRMS (ESI-TOF) 376.0879 [M(35Cl)þH]þ & 378.0848
[M(37Cl)þH]þ; calcd. for C18H19ClN3O2Sþ 376.0881 [M(35Cl)þH]þ
&
d
1.24 (app d, 2H, J 12.5, CH2), 1.68 (td, 2H, J 12.0 & 4.0, CH2), 1.83 (t,
378.0852 [M(37Cl)þH]þ. Anal. RP-HPLC Method C: tR 15.93 min,
2H, J 6.5, CH2), 2.59 (t, 2H, J 12.0, CH2), 3.04 (d, 2H, J 12.5, CH2), 3.12
(t, 2H, J 6.5, CH2), 3.46 (s, 3H, CH3), 6.01 (app s, 1H, pyrrolyl-H), 6.12
(app s, 1H, pyrrolyl-H), 6.88 (app s, 1H, pyrrolyl-H), 7.53 (s, 1H,
CONH), 8.17 (s, 1H, pyridinyl-H), 8.44 (s, 1H, pyridinyl-H). 13C NMR
purity >97%; Method D: tR 11.05 min, purity >98%.
4-(5-Chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)pyridin-
3-yl)thiophene-3-carbaldehyde (28): Bromide
3
(173 mg,
502
602
mmol) and (4-formylthiophen-3-yl)boronic acid (94 mg,
(DMSO‑d6)
d
30.6, 31.9, 34.1, 37.8, 41.3, 46.5,107.6,110.7,123.2,124.6,
m
mol) were reacted at 135 ꢀC using general synthetic procedure
126.8, 127.1, 149.3, 151.8, 154.2, 179.9 (two carbon signals over-
A. The residue was purified by Biotage® FlashMaster Personalþ
flash chromatography (DCM ramping to 4% EtOH in DCM) to give 28
as a beige solid (70 mg, 37%). RF (DCM:EtOH ¼ 94:6) 0.40. m.p.
lapping or obscured). HRMS (ESI-TOF) 345.1475 [M(35Cl)þH]þ
&
347.1441 [M(37Cl)þH]þ; calcd. for C18H22ClN4Oþ 345.1477
[M(35Cl)þH]þ & 347.1447 [M(37Cl)þH]þ. Anal. RP-HPLC Method C:
tR 13.84 min, purity >98%; Method D: tR 10.45 min, purity >96%.
4-(5-Chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)pyridin-
208e209 ꢀC. 1H NMR (DMSO‑d6)
d 1.18 (app d, 2H, J 12.5, CH2), 1.55
(app t, 2H, J 10.0, CH2), 1.79 (t, 2H, J 6.5, CH2), 2.61 (t, 2H, J 10.5, CH2),
3.00 (d, 2H, J 10.0, CH2), 3.10 (t, 2H, J 6.5, CH2), 7.51 (s, 1H, CONH),
7.65 (d, 1H, J 2.5, thiophenyl-H), 8.12 (s, 1H, pyridinyl-H), 8.46 (s, 1H,
pyridinyl-H), 8.70 (d, 1H, J 2.0, thiophenyl-H), 9.79 (s, 1H, CHO). 13C
3-yl)furan-2-carbaldehyde (25): Bromide 3 (173 mg, 502
mmol)
and
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)furan-2-
carbaldehyde (123 mg, 554
m
mol) were reacted at 120 ꢀC using
NMR (DMSO‑d6) d 30.5, 31.8, 37.8, 41.1, 46.9, 126.7, 127.8, 128.4,
general synthetic procedure A. The residue was purified by Bio-
tage® FlashMaster Personalþ flash chromatography (DCM ramping
to 4% EtOH in DCM) to give 25 as a yellow solid (38 mg, 21%). RF
(DCM:CH3OH ¼ 94:6) 0.40. m.p. 184e185 ꢀC. 1H NMR (DMSO‑d6)
135.7, 139.9, 140.1, 149.6, 150.4, 153.4, 179.9, 185.8 (two carbon
signals overlapping or obscured). HRMS (ESI-TOF) 376.0875
[M(35Cl)þH]þ & 378.0837 [M(37Cl)þH]þ; calcd. for C18H19ClN3O2Sþ
376.0881 [M(35Cl)þH]þ & 378.0852 [M(37Cl)þH]þ. Anal. RP-HPLC
Method C: tR 12.74 min, purity >97%; Method D: tR 9.52 min, purity
>98%.
d
1.33 (app d, 2H, J 12.5, CH2), 1.75 (td, 2H, J 11.5 & 1.5, CH2), 1.95 (t,
2H, J 6.5, CH2), 2.95 (t, 2H, J 11.5, CH2), 3.08 (d, 2H, J 12.5, CH2), 3.16
(t, 2H, J 6.5, CH2), 7.57 (s, 1H, CONH), 7.76 (s, 1H, furanyl-H), 8.34 (s,
4-(5-Chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)pyridin-
17

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
3-yl)thiophene-2-carbaldehyde (29): Bromide
3
(173 mg,
2.5, CH2), 1.93 (t, 2H, J 6.5, CH2), 2.90 (t, 2H, J 10.0, CH2), 3.02 (d, 2H, J
12.5, CH2), 3.16 (t, 2H, J 6.5, CH2), 7.56 (s, 1H, CONH), 7.87 (t, 1H, J
59.0, CHF2), 8.02 (s, 1H), 8.34 (s, 1H), 8.44 (s, 1H), 8.50 (s, 1H) (total
502
553
mmol) and (5-formylthiophen-3-yl)boronic acid (86 mg,
m
mol) were reacted at 120 ꢀC using general synthetic proced-
ure A. The residue was purified by Biotage® FlashMaster Personalþ
flash chromatography (DCM ramping to 3% CH3OH in DCM), and
crystallised with DCM/n-hexane to give 29 as a yellowish solid
(110 mg, 58%). RF (DCM:CH3OH ¼ 94:6) 0.29. m.p. 203e204 ꢀC. 1H
4H, 2 ꢂ pyrazolyl-H & 2 ꢂ pyridinyl-H). 13C NMR (DMSO‑d6)
d 30.6,
31.8, 38.0, 41.3, 46.8, 110.5 (t, JC-F 247.0), 119.2, 124.9, 127.9, 128.0,
142.5, 149.3, 150.5, 152.6, 180.1 (two carbon signals overlapping or
obscured). HRMS (ESI-TOF) 382.1234 [M(35Cl)þH]þ & 384.1217
NMR (DMSO‑d6)
d
1.27 (app d, 2H, J 12.5, CH2), 1.70 (dt, 2H, J 11.5 &
[M(37Cl)þH]þ; calcd. for C17H19ClF2N5Oþ 382.1241 [M(35Cl)þH]þ
&
1.5, CH2), 1.89 (t, 2H, J 6.5, CH2), 2.80 (t, 2H, J 11.5, CH2), 3.06 (app d,
2H, J 12.5, CH2), 3.14 (t, 2H, J 6.5, CH2), 7.54 (s, 1H, CONH), 8.12 (s, 1H,
thiophenyl-H), 8.18 (s, 1H, thiophenyl-H), 8.30 (s, 1H, pyridinyl-H),
8.48 (s, 1H, pyridinyl-H), 10.00 (s, 1H, CHO). 13C NMR (DMSO‑d6)
384.1211 [M(37Cl)þH]þ. Anal. RP-HPLC Method C: tR 15.04 min,
purity >99%; Method D: tR 10.72 min, purity >99%.
5-Bromo-4-chloropyridin-2-amine (34): To a suspension of 2-
amino-4-chloropyridine (33, 7.72 g, 60.1 mmol) in anhydrous
CH3CN (120 mL) was added NBS (11.3 g, 63.5 mmol). The reaction
mixture was stirred at room temperature under N2 for 3 h and
concentrated under reduced pressure. The residue was purified by
flash column chromatography (DCM ramping to 4% CH3OH in DCM)
to give 34 as an orange solid (6.91 g, 58%). RF (EtOAc:petroleum
d
30.6, 31.8, 37.9, 41.3, 47.1, 127.4, 127.9, 134.0, 138.4, 139.2, 143.5,
149.6, 150.3, 152.7, 180.0, 184.6 (two carbon signals overlapping or
obscured). HRMS (ESI-TOF) 376.0877 [M(35Cl)þH]þ & 378.0848
[M(37Cl)þH]þ; calcd. for C18H19ClN3O2Sþ 376.0881 [M(35Cl)þH]þ
&
378.0852 [M(37Cl)þH]þ. Anal. RP-HPLC Method C: tR 16.40 min,
purity >96%; Method D: tR 10.80 min, purity >97%.
benzine ¼ 1:1) 0.45. 1H NMR (DMSO‑d6)
d 6.42 (br s, 2H, NH2), 6.67
8-(3-Chloro-5-(1H-pyrazol-4-yl)pyridin-4-yl)-2,8-diazaspiro
[4.5]decan-1-one (30): Bromide 3 (345 mg, 1.00 mmol) and tert-
(s, 1H, pyridinyl-H), 8.09 (s, 1H, pyridinyl-H). 13C NMR (DMSO‑d6)
d
104.6, 108.7, 142.3, 150.4, 159.9. HRMS (ESI-TOF) 206.9323,
butyl
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyr-
208.9299 & 210.9268 [MþH]þ; calcd. for C5H5BrClN2þ 206.9320,
azole-1-carboxylate (324 mg, 1.10 mmol) were reacted at 120 ꢀC
using general synthetic procedure A. The residue was purified by
Biotage® FlashMaster Personalþ flash chromatography (DCM
ramping to 7% CH3OH in DCM), and crystallised with DCM to give
30 as white crystals (145 mg, 44%). RF (DCM:CH3OH ¼ 94:6) 0.15.
208.9299 & 210.9270 [MþH]þ.
5-Bromo-3,4-dichloropyridin-2-amine (35): To 34 (6.85 g,
33.0 mmol) in CH3CN (125 mL) was added NCS (4.41 g, 33.0 mmol)
in portions. The reaction mixture was heated at reflux under N2 for
3 h, cooled down, and filtered. The solid was washed with CH3CN
(3 ꢂ 15 mL) and dried to give one portion of 35 as a tan powder
(5.55 g). The filtrate and CH3CN washings were combined and
concentrated under reduced pressure, and the residue was purified
by flash column chromatography (DCM ramping to 2% CH3OH in
DCM) to give a second portion of 35 as a beige solid (2.09 g) (total
7.64 g, 96%). RF (DCM:CH3OH ¼ 98:2) 0.49. 1H NMR (DMSO‑d6)
m.p. 233e234 ꢀC. 1H NMR (DMSO‑d6)
d 1.30 (app d, 2H, J 13.0, CH2),
1.76 (td, 2H, J 10.5 & 5.0, CH2), 1.94 (t, 2H, J 6.5, CH2), 2.84e3.02 (m,
4H, 2 ꢂ CH2), 3.16 (t, 2H, J 6.5, CH2), 7.56 (s, 1H), 7.87 (app s, 2H),
8.32 (s, 1H), 8.36 (s, 1H) (total 5H, CONH & 2 ꢂ pyrazolyl-H &
2 ꢂ pyridinyl-H), 13.09 (br s,1H, pyrazolyl-NH). 13C NMR (DMSO‑d6)
d
30.5, 31.8, 37.9, 41.3, 46.6, 115.7, 127.0, 128.3, 148.2, 150.4, 152.1,
180.1 (four carbon signals overlapping or obscured). HRMS (ESI-
d
d
6.83 (br s, 2H, NH2), 8.12 (s, 1H, pyridinyl-H). 13C NMR (DMSO‑d6)
105.1, 112.1, 140.4, 147.8, 156.1. HRMS (ESI-TOF) 240.8929,
TOF) 332.1272 [M(35Cl)þH]þ & 334.1242 [M(37Cl)þH]þ; calcd. for
C
16H19ClN5Oþ 332.1273 [M(35Cl)þH]þ & 334.1243 [M(37Cl)þH]þ.
242.8905 & 244.8876 [MþH]þ; calcd. for C5H4BrCl2Nþ2 240.8930,
Anal. RP-HPLC Method C: tR 13.74 min, purity >99%; Method D: tR
8.54 min, purity >99%.
242.8909 & 244.8880 [MþH]þ.
5-Bromo-3,4-dichloro-N,N-bis(4-methoxybenzyl)pyridin-2-
amine (36) & 5-bromo-3,4-dichloro-N-(4-methoxybenzyl)pyr-
idin-2-amine (37): To a solution of amine 35 (2.42 g, 10.0 mmol) in
anhydrous DMF (25 mL) on an ice-salt bath was added NaH (~60%
suspension in mineral oil, 1.60 g, 40.0 mmol) in portions. The re-
action mixture was stirred for 15 min, and a solution of 4-
methoxybenzyl chloride (3.26 mL, 24.0 mmol) in anhydrous DMF
(1 mL) was added. The reaction mixture was warmed up to room
temperature, stirred for 2 h, quenched with saturated NH4Cl
aqueous solution (50 mL), and extracted with EtOAc (3 ꢂ 50 mL).
The organic extracts were combined and concentrated under
reduced pressure. The residue was purified by Biotage® Flash-
Master Personalþ flash chromatography (petroleum benzine
ramping to 40% DCM in petroleum benzine) to give 36 as a col-
ourless, clear oil (3.18 g, 66%) and 37 as a white solid (1.01 g, 28%).
8-(3-Chloro-5-(1-methyl-1H-pyrazol-4-yl)pyridin-4-yl)-2,8-
diazaspiro[4.5]decan-1-one (31): Bromide 3 (190 mg, 551
and 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
pyrazole (121 mg, 582
mol) were reacted at 120 ꢀC using general
mmol)
m
synthetic procedure A. The residue was purified by Biotage®
FlashMaster Personalþ flash chromatography (DCM ramping to 7%
EtOH in DCM), and triturated with n-hexane to give 31 as a beige
solid (80 mg, 42%). RF (DCM:EtOH ¼ 93:7) 0.22. m.p.182e183 ꢀC. 1H
NMR (DMSO‑d6) d 1.31 (app d, 2H, J 13.0, CH2), 1.76 (td, 2H, J 11.5 &
4.0, CH2), 1.95 (t, 2H, J 7.0, CH2), 2.85e3.00 (m, 4H, 2 ꢂ CH2), 3.17 (t,
2H, J 7.0, CH2), 3.89 (s, 3H, CH3), 7.56 (s, 1H, CONH), 7.64 (s, 1H,
pyrazolyl-H), 7.96 (s, 1H, pyrazolyl-H), 8.27 (s, 1H, pyridinyl-H), 8.35
(s, 1H, pyridinyl-H). 13C NMR (DMSO‑d6)
d 30.6, 31.8, 38.1, 38.8, 41.4,
46.8, 116.5, 126.7, 128.3, 130.3, 138.7, 148.3, 150.3, 152.1, 180.3 (two
carbon signals overlapping or obscured). HRMS (ESI-TOF) 346.1430
[M(35Cl)þH]þ & 348.1398 [M(37Cl)þH]þ; calcd. for C17H21ClN5Oþ
346.1429 [M(35Cl)þH]þ & 348.1400 [M(37Cl)þH]þ. Anal. RP-HPLC
Method A: tR 9.73 min, purity >99%; Method B: tR 7.62 min, purity
>99%.
36: RF (DCM:petroleum benzine ¼ 2:1) 0.38. 1H NMR (CDCl3)
d 3.79
(s, 6H, 2 ꢂ CH3), 4.40 (s, 4H, 2 ꢂ CH2), 6.83 (d, 4H, J 8.5, 4 ꢂ phenyl-
H), 7.18 (d, 4H, J 8.5, 4 ꢂ phenyl-H), 8.24 (s, 1H, pyridinyl-H). HRMS
(ESI-TOF) 481.0069, 483.0047 & 485.0020 [MþH]þ; calcd. for
C
21H20BrCl2N2Oþ2 481.0080, 483.0060 & 485.0030 [MþH]þ. 37: RF
8-(3-Chloro-5-(1-(difluoromethyl)-1H-pyrazol-4-yl)pyridin-
4-yl)-2,8-diazaspiro[4.5]decan-1-one (32): Bromide 3 (173 mg,
(DCM:petroleum benzine ¼ 2:1) 0.44. 1H NMR (CDCl3)
d 3.80 (s, 3H,
CH3), 4.58 (d, 2H, J 5.0, CH2), 5.34 (br s, 1H, NH), 6.88 (d, 2H, J 8.0,
2 ꢂ phenyl-H), 7.27 (d, 2H, J 8.0, 2 ꢂ phenyl-H), 8.16 (s, 1H,
pyridinyl-H). HRMS (ESI-TOF) 360.9494, 362.9481 & 364.9442
502
mmol) and 1-(difluoromethyl)-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole (148 mg, 606
m
mol) were reacted
at 135 ꢀC using general synthetic procedure A. The residue was
purified by Biotage® FlashMaster Personalþ flash chromatography
(DCM ramping to 3.5% CH3OH in DCM) to give 32 as a pinkish solid
(128 mg, 67%). RF (DCM:CH3OH ¼ 94:6) 0.26. m.p. 196e197 ꢀC. 1H
[MþH]þ; calcd. for C13H12BrCl2N2Oþ 360.9505, 362.9485
&
364.9455 [MþH]þ.
8-(2-(Bis(4-methoxybenzyl)amino)-5-bromo-3-
chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (38): Chlo-
ride 36 (3.24 g, 6.72 mmol) and tert-butyl 1-oxo-2,8-diazaspiro[4.5]
NMR (DMSO‑d6) d 1.31 (app d, 2H, J 12.5, CH2), 1.74 (td, 2H, J 11.5 &
18

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
decane-8-carboxylate (1.71 g, 6.72 mmol) were coupled at 220 ꢀC
for 1 h according to general synthetic procedure B. The residue was
purified by Biotage® FlashMaster Personalþ flash chromatography
(DCM ramping to 4% CH3OH in DCM) to give 38 as a white foam
NMR (CDCl3)
d
31.2, 32.2, 38.7, 42.0, 45.5, 46.8, 55.5, 114.2, 122.3,
122.4, 123.4, 124.2, 124.5, 129.2, 131.5, 140.1, 140.7, 148.4, 153.8,
155.3, 159.1, 181.7 (seven carbon signals overlapping or obscured).
HRMS (ESI-TOF) 533.1782, 534.1796 & 535.1754 [MþH]þ; calcd. for
(580 mg, 48%). RF (DCM:CH3OH ¼ 97:3) 0.34. 1H NMR (CDCl3)
d
1.51
C
29H30ClN4O2Sþ 533.1773, 534.1807 & 535.1743 [MþH]þ.
(d, 2H, J 12.5, spiro-CH2), 2.05e2.30 (m, 2H, spiro-CH2), 2.17 (t, 2H, J
7.0, spiro-CH2), 3.15e3.45 (m, 4H, spiro-CH2), 3.38 (t, 2H, J 7.0, spiro-
CH2), 3.78 (s, 6H, 2 ꢂ CH3), 4.35 (s, 4H, 2 ꢂ NCH2-phenyl), 5.82 (br s,
1H, CONH), 6.81 (dd, 4H, J 8.5 & 3.0, 4 ꢂ phenyl-H), 7.18 (dd, 4H, J 8.5
& 2.0, 4 ꢂ phenyl-H), 8.13 (s, 1H, pyridinyl-H). HRMS (ESI-TOF)
599.1417 & 601.1409 [MþH]þ; calcd. for C29H33BrClN4Oþ3 599.1419 &
601.1399 [MþH]þ.
8-(2-Amino-5-(benzofuran-2-yl)-3-chloropyridin-4-yl)-2,8-
diazaspiro[4.5]decan-1-one (42): Compound 40 (166 mg,
260 mmol) was de-protected using general synthetic procedure C.
The residue was purified by Biotage® FlashMaster Personalþ flash
chromatography (DCM ramping to 5% CH3OH in DCM), and crys-
tallised with CH3OH/DCM/EtOAc to give 42 as a white solid (77 mg,
75%). RF (DCM:CH3OH ¼ 94:6) 0.32. m.p. 234e235 ꢀC. 1H NMR
8-(5-Bromo-3-chloro-2-((4-methoxybenzyl)amino)pyridin-
4-yl)-2,8-diazaspiro[4.5]decan-1-one (39): Chloride 37 (1.65 g,
4.58 mmol) and tert-butyl 1-oxo-2,8-diazaspiro[4.5]decane-8-
carboxylate (1.28 g, 5.03 mmol) were coupled at 225 ꢀC for 2.5 h
according to general synthetic procedure B. The residue was puri-
fied by Biotage® FlashMaster Personalþ flash chromatography
(DCM ramping to 2% CH3OH in DCM) to give 39 as a yellowish foam
(DMSO‑d6) d 1.28 (app d, 2H, J 12.5, CH2), 1.77 (td, 2H, J 11.5 & 1.5,
CH2), 1.86 (t, 2H, J 6.5, CH2), 2.81 (t, 2H, J 11.0, CH2), 3.02 (d, 2H, J
12.0, CH2), 3.12 (t, 2H, J 6.0, CH2), 6.48 (s, 2H, NH2), 6.98 (s, 1H,
benzofuranyl-H), 7.25 (t, 1H, J 7.5, benzofuranyl-H), 7.30 (t, 1H, J 7.0,
benzofuranyl-H), 7.53 (s,1H, CONH), 7.59 (d,1H, J 8.0, benzofuranyl-
H), 7.64 (d, 1H, J 7.0, benzofuranyl-H), 8.00 (s, 1H, pyridinyl-H). 13C
NMR (DMSO‑d6)
112.9, 121.0, 123.1, 124.2, 129.1, 148.6, 153.8, 154.1, 154.3, 157.7, 180.3
d 30.2, 31.9, 38.0, 41.6, 46.0, 103.6, 109.3, 111.1,
(1.48 g, 68%). RF (DCM:CH3OH ¼ 97:3) 0.30. 1H NMR (CDCl3)
d
1.50
(d, 2H, J 10.5, spiro-CH2), 2.06e2.26 (m, 2H, spiro-CH2), 2.16 (t, 2H, J
6.5, spiro-CH2), 3.27 (app br s, 2H, spiro-CH2) 3.29e3.36 (m, 2H,
spiro-CH2), 3.37 (t, 2H, J 6.5, spiro-CH2), 3.80 (s, 3H, CH3), 4.55 (d,
2H, J 4.5, NHCH2-phenyl), 5.25 (br s, 1H), 5.66 (br s, 1H) (total 2H,
CONH & CH2eNH-pyridinyl), 6.88 (d, 2H, J 8.0, 2 ꢂ phenyl-H), 7.28
(d, 2H, J 8.5, 2 ꢂ phenyl-H), 8.06 (br s, 1H, pyridinyl-H). HRMS (ESI-
(two carbon signals overlapping or obscured). HRMS (ESI-TOF)
397.1421 [M(35Cl)þH]þ
&
399.1426 [M(37Cl)þH]þ; calcd. for
C
21H22ClN4O2þ 397.1426 [M(35Cl)þH]þ & 399.1396 [M(37Cl)þH]þ.
Anal. RP-HPLC Method C: tR 17.51 min, purity >97%; Method D: tR
12.46 min, purity >97%.
8-(2-Amino-5-(benzo[b]thiophen-2-yl)-3-chloropyridin-4-
yl)-2,8-diazaspiro[4.5]decan-1-one (43): Compound 41 (140 mg,
TOF) 479.0838, 481.0825
C
&
483.0791 [MþH]þ; calcd. for
21H25BrClN4Oþ2 479.0844, 481.0824 & 483.0794 [MþH]þ.
263 mmol) was de-protected using general synthetic procedure C.
8-(5-(Benzofuran-2-yl)-2-(bis(4-methoxybenzyl)amino)-3-
The residue was purified by Biotage® FlashMaster Personalþ flash
chromatography (DCM ramping to 3.5% CH3OH in DCM), and
crystallised with CH3OH/DCM/EtOAc to give 43 as a white solid
(66 mg, 61%). RF (DCM:CH3OH ¼ 94:6) 0.38. m.p. 264e265 ꢀC. 1H
chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (40): Bro-
mide 38 (365 mg, 608 mol) and 2-(benzofuran-2-yl)-4,4,5,5-
tetramethyl-1,3,2-dioxaborolane (178 mg, 729 mol) were reacted
m
m
at 130 ꢀC using general synthetic procedure A. The residue was
purified by Biotage® FlashMaster Personalþ flash chromatography
(DCM ramping to 1.5% CH3OH in DCM) to give 40 as a yellowish glue
NMR (DMSO‑d6) d 1.28 (app d, 2H, J 12.5, CH2), 1.75e2.05 (m, 4H,
2 ꢂ CH2), 3.04 (app s, 4H, 2 ꢂ CH2), 3.12 (t, 2H, J 6.0, CH2), 6.39 (s,
2H, NH2), 7.33 (t, 1H, J 7.5, benzothiophenyl-H), 7.37 (t, 1H, J 7.5,
benzothiophenyl-H), 7.44 (br s, 1H), 7.52 (s, 1H) (total 2H, CONH &
benzothiophenyl-H), 7.82 (d, 1H, J 7.5, benzothiophenyl-H), 7.93 (d,
1H, J 7.5, benzothiophenyl-H), 8.00 (br s, 1H, pyridinyl-H). 13C NMR
(170 mg, 44%). RF (DCM:CH3OH ¼ 96:4) 0.44. 1H NMR (CDCl3)
d 1.39
(d, 2H, J 12.5, spiro-CH2), 2.04 (t, 2H, J 6.5, spiro-CH2), 2.10 (app t,
2H, J 12.0, spiro-CH2), 2.95 (t, 2H, J 11.0, spiro-CH2), 3.23 (d, 2H, J
12.5, spiro-CH2), 3.29 (t, 2H, J 11.0, spiro-CH2), 3.79 (s, 6H, 2 ꢂ CH3),
4.47 (s, 4H, 2 ꢂ NCH2-phenyl), 5.55 (br s, 1H, CONH), 6.83 (d, 4H, J
8.0, 4 ꢂ phenyl-H), 6.84 (s, 1H, benzofuranyl-H), 7.21 (d, 4H, J 8.0,
4 ꢂ phenyl-H), 7.24e7.35 (m, 2H, 2 ꢂ benzofuranyl-H), 7.52 (d, 1H, J
8.0, benzofuranyl-H), 7.63 (d, 1H, J 6.5, benzofuranyl-H), 8.24 (s, 1H,
(DMSO‑d6)
d 30.3, 31.5, 38.0, 41.5, 46.3, 122.2, 123.5, 124.3, 124.6,
139.7, 139.9, 148.2, 153.5, 157.3, 180.3 (six carbon signals over-
lapping or obscured). HRMS (ESI-TOF) 413.1195 [M(35Cl)þH]þ
&
415.1165 [M(37Cl)þH]þ; calcd. for C21H22ClN4OSþ 413.1197
[M(35Cl)þH]þ & 415.1168 [M(37Cl)þH]þ. Anal. RP-HPLC Method C:
tR 17.66 min, purity >98%; Method D: tR 12.59 min, purity >99%.
1-Methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
1H-indazole (44): To a suspension of 5-bromo-1-methyl-1H-
indazole (2.11 g, 10.0 mmol) and 4,4,40,40,5,5,50,50-octamethyl-2,20-
bi(1,3,2-dioxaborolane) (3.81 g, 15.0 mmol) in DMSO (25 mL) were
added potassium acetate (2.95 g, 30.1 mmol) and Pd(dppf)
pyridinyl-H). 13C NMR (CDCl3)
d 32.3, 38.7, 41.9, 46.5, 53.2, 55.4,
104.1, 111.2, 113.8, 117.0, 118.6, 121.2, 123.1, 124.3, 129.4, 129.5, 130.8,
147.6, 153.7, 155.0, 155.6, 158.7, 160.5, 181.6 (thirteen carbon signals
overlapping or obscured). HRMS (ESI-TOF) 637.2575, 638.2596 &
639.2553 [MþH]þ; calcd. for C37H38ClN4Oþ4 637.2577, 638.2610 &
639.2547 [MþH]þ.
8-(5-(Benzo[b]thiophen-2-yl)-3-chloro-2-((4-
Cl2$CH2Cl2 (409 mg, 501 mmol). The reaction mixture was degassed
methoxybenzyl)amino)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-
with N2, heated at 80 ꢀC for 20 h, cooled down, and partitioned
between distilled H2O (200 mL) and EtOAc (200 mL). The mixture
was filtered through a pad of Celite®, and the solids were washed
with EtOAc (100 mL). The filtrate and washing were combined, and
the organic layer was separated. The aqueous layer was extracted
with EtOAc (2 ꢂ 100 mL). The organic layer and extracts were
combined, dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by Biotage® FlashMaster Per-
sonalþ flash chromatography (petroleum benzine ramping to 2%
CH3OH in DCM) to give 44 as a white solid (1.68 g, 65%). RF (DCM)
1-one (41): Bromide 39 (288 mg, 600
mmol) and benzo[b]thiophen-
2-ylboronic acid (128 mg, 719
m
mol) were reacted at 130 ꢀC using
general synthetic procedure A. The residue was purified by Bio-
tage® FlashMaster Personalþ flash chromatography (DCM ramping
to 2% CH3OH in DCM) to give 41 as a yellowish foam (145 mg, 45%).
RF (DCM:CH3OH ¼ 94:6) 0.61. 1H NMR (CDCl3)
d 1.37 (d, 2H, J 13.0,
spiro-CH2), 2.03 (t, 2H, J 6.5, spiro-CH2), 2.10 (td, 2H, J 11.5 & 3.5,
spiro-CH2), 3.03 (app br s, 2H, spiro-CH2), 3.21 (d, 2H, J 12.0, spiro-
CH2), 3.28 (t, 2H, J 6.5, spiro-CH2), 3.81 (s, 3H, CH3), 4.64 (d, 2H, J 5.0,
NHCH2-phenyl), 5.40 (t, 1H, J 4.5, CH2eNH-pyridinyl), 5.54 (br s, 1H,
0.21. 1H NMR (CDCl3)
d
1.37 (s, 12H, 4 ꢂ dioxaborolanyl-CH3), 4.07
CONH), 6.90 (d, 2H,
J
8.0,
2
ꢂ
phenyl-H), 7.24 (s, 1H,
(s, 3H, NCH3), 7.37 (dt, 1H, J 8.5 & 1.0, indazolyl-H), 7.80 (dd, 1H, J 8.5
& 0.5, indazolyl-H), 7.99 (d, 1H, J 0.5, indazolyl-H), 8.26 (s, 1H,
benzothiophenyl-H), 7.32 (d, 2H, J 8.0, 2 ꢂ phenyl-H), 7.27e7.42 (m,
2H, 2 ꢂ benzothiophenyl-H), 7.79 (d, 1H, J 7.5, benzothiophenyl-H),
7.85 (d, 1H, J 7.5, benzothiophenyl-H), 8.10 (s, 1H, pyridinyl-H). 13C
indazolyl-H). 13C NMR (CDCl3)
d
25.0, 35.6, 83.9, 108.3, 124.1, 129.6,
131.9, 133.7, 141.5 (five carbon signals overlapping or obscured).
19

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
HRMS (ESI-TOF) 259.1613 [MþH]þ; calcd. for C14H20BN2Oþ2
259.1613 [MþH]þ.
were calculated using Quattro Workflow V3.1.1 (Quattro Research
GmbH, Münich, Germany; www.quattro-research.com/). The fitting
model used for the IC50 determination was sigmoidal dose-
response (variable slope) with the parameters of TOP and BOT-
TOM fixed at 100% and 0%, respectively. The method of least squares
was used for regression analysis. Percentages of residual kinase
activity of the kinases (except CDK8) in the preliminary screening
8-(2-(Bis(4-methoxybenzyl)amino)-3-chloro-5-(1-methyl-
1H-indazol-5-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one
(45): Bromide 38 (510 mg, 850
mmol) and boronate ester 44
(230 mg, 891
m
mol) were reacted at 120 ꢀC using general synthetic
procedure A. The residue was purified by Biotage® FlashMaster
Personalþ flash chromatography (DCM ramping to DCM/EtOAc/
EtOH ¼ 50:45:5) to give 45 as a yellowish glue (90 mg, 16%). RF
in the presence of 1
mM 19 or 42 and in the near-kinome-wide
profiling with 1 M 42, and IC50 values of 42 against LRRK2, FMS
m
(DCM:EtOAc:EtOH ¼ 50:46:4) 0.17. 1H NMR (CDCl3)
d
1.29 (d, 2H, J
and FLT3 were measured using the HotSpot™ radioisotope-based
kinase assay at Reaction Biology Corporation (the assay protocol
is available at https://www.reactionbiology.com/services/target-
specific-assays/kinase-assays/kinase-screening). All the IC50
values determined by two contract research organisations were
13.0, spiro-CH2), 1.93 (t, 4H, J 7.0, 2 ꢂ spiro-CH2), 2.74 (app br s, 2H,
spiro-CH2), 3.12 (dt, 2H, J 12.5 & 1.0, spiro-CH2), 3.24 (t, 2H, J 7.0,
spiro-CH2), 3.79 (s, 6H, 2 ꢂ OCH3), 4.12 (s, 3H, NCH3), 4.43 (s, 4H,
2 ꢂ NCH2-phenyl), 5.49 (br s, 1H, CONH), 6.84 (d, 4H, J 8.5,
4 ꢂ phenyl-H), 7.25 (d, 4H, J 8.0, 4 ꢂ phenyl-H), 7.33 (d, 1H, J 8.5,
indazolyl-H), 7.46 (d, 1H, J 8.5, indazolyl-H), 7.61 (s, 1H, indazolyl-
H), 7.96 (s, 1H), 8.02 (s, 1H) (total 2H, indazolyl-H & pyridinyl-H).
HRMS (ESI-TOF) 651.2851, 652.2893 & 653.2840 [MþH]þ; calcd.
for C37H40ClN6Oþ3 651.2845, 652.2879 & 653.2816 [MþH]þ.
converted to Ki values using the Cheng-Prusoff equation: Ki ¼ IC50
/
(1 þ ([ATP]/Km(ATP))), where [ATP] is the concentration of ATP used
for the IC50 determination and Km (ATP) for each kinase is deter-
mined experimentally [83].
8-(2-Amino-3-chloro-5-(1-methyl-1H-indazol-5-yl)pyridin-
4-yl)-2,8-diazaspiro[4.5]decan-1-one (CCT251921): Compound
45 (80 mg, 0.12 mmol) was de-protected using general synthetic
procedure C. The residue was purified by Biotage® FlashMaster
Personalþ flash chromatography (DCM ramping to 7% CH3OH in
DCM), and washed with EtOAc (10 mL) to give CCT251921 as a
white solid (44 mg, 88%). RF (DCM:CH3OH ¼ 94:6) 0.34. m.p.
4.3.2. Cell culture
All the cell lines were obtained from the cell bank at Drug Dis-
covery and Development, University of South Australia. Each of
them was cultured, according to the American Type Culture
Collection (ATCC) recommendation, in Roswell Park Memorial
Institute (RPMI)-1640, Dulbecco’s Modified Eagle’s Medium
(DMEM), or Minimum Essential Media (MEM), with 10% foetal
bovine serum (FBS, Sigma-Aldrich, Castle Hill, NSW, Australia)
within a humidified incubator at 37 ꢀC in the presence of 5% CO2. No
mycoplasma contamination [84] was detected in cell culture.
266e267 ꢀC. 1H NMR (DMSO‑d6)
d 1.16 (app d, 2H, J 13.0, CH2), 1.65
(app t, 2H, J 10.0, CH2), 1.75 (t, 2H, J 7.0, CH2), 2.60 (app br s, 2H,
CH2), 2.95 (app d, 2H, J 12.5, CH2), 3.06 (t, 2H, J 7.0, CH2), 4.06 (s, 3H,
CH3), 6.07 (s, 2H, NH2), 7.27 (dd, 1H, J 8.5 & 1.5, indazolyl-H), 7.47 (s,
1H, CONH), 7.62 (s, 1H, indazolyl-H), 7.65 (s, 1H, indazolyl-H), 7.65
(d, 1H, J 8.5, indazolyl-H), 8.05 (s, 1H, pyridinyl-H). 13C NMR
4.3.3. Cell viability assays
MTT (Life Technologies, Mulgrave, Vic, Australia) and resazurin
(Sigma-Aldrich) assays were performed with adherent (solid
tumour) and suspension (leukaemia) cell lines, respectively, as re-
ported previously [44,64]. Compound concentrations required to
inhibit 50% of cell growth (GI50) were calculated using nonlinear
regression analysis.
(DMSO‑d6)
d 30.3, 31.9, 35.5, 37.9, 41.5, 47.4, 108.9, 109.3, 120.4,
123.8, 124.3, 128.5, 130.7, 132.6, 138.8, 148.2, 153.7, 156.3, 180.2 (two
carbon signals overlapping or obscured). HRMS (ESI-TOF) 411.1695
[M(35Cl)þH]þ & 413.1674 [M(37Cl)þH]þ; calcd. for C21H24ClN6Oþ
411.1695 [M(35Cl)þH]þ & 413.1665 [M(37Cl)þH]þ. Anal. RP-HPLC
Method A: tR 9.12 min, purity >95%; Method B: tR 7.82 min, purity
>98%.
4.3.4. Determination of in vivo pharmacokinetic properties
Healthy male Sprague-Dawley rats (250e350 g) or male adult
BALB/c mice (20e25 g) were allocated into IV- and PO-dosing
groups; n ¼ 3 per group for rats, and n ¼ 10 per group for mice.
Rodents were administered an IV injection of 42 or 43 via the tail
vein (5 mg/kg for rats, and 2 mg/kg for mice), or a single oral dose
by gavage (20 mg/kg for rats, and 10 mg/kg for mice; 2-h prior
fasting was fulfilled). IV formulations consisted of 42 or 43 dis-
solved in a mixture of 0.1 M sodium acetate buffer (pH 4.5):PEG
400:DMSO (9:9:2, v/v/v), and oral formulations were comprised of
42 or 43 dispersed in 1% (w/v) carboxymethyl cellulose aqueous
solution. Blood samples were collected from animals by jugular
vein cannula (rats) or cheek bleeding and cardiac puncture under
anaesthesia (mice) at time zero and at intervals up to 24 h.
Collected blood samples were centrifuged at 7000 g for 3 min to
separate plasma samples which were stored at ꢁ20 ꢀC until anal-
ysis. Concentrations of 42 or 43 in plasma samples were quantified
using a validated LC/MS/MS method with the AB SCIEX TripleTOF
5600 mass spectrometer. Briefly, plasma samples were supple-
mented with an internal standard (IS) and extracted with EtOAc,
and organic phases separated and dried in a Genevac HT-4X HCL
centrifugal vacuum evaporator (SP Scientific, Ipswich, UK). The
residues were reconstituted in acetonitrile/water (50:50), injected
into a Shimadzu Nexera HPLC system, and resolved on a Phenom-
4.3. Biology
4.3.1. Kinase assays
At ProQinase GmbH, the 33PanQinase® activity assay, a radio-
metric protein kinase assay, was used to measure the residual ki-
nase activity of CDK8/cyclin C. The assay was performed in a 96-
well FlashPlate™ from PerkinElmer (Boston, MA, USA) in a reac-
tion volume of 50
A reaction cocktail was formed by pipetting the following four
solutions in an order of [i] 20 L of assay buffer (standard buffer),
[ii] 5 L of ATP solution (in H2O), [iii] 5 L of a test compound (in 10%
DMSO) [iv] 10 L of enzyme (i.e., CDK8), solution & 10 L of sub-
strate (i.e., RBER-IRStide) solution (premixed). Each well contained
70 mM HEPES-NaOH pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 M so-
dium orthovanadate, 1.2 mM dithiothreitol (DTT), 50 g/mL poly-
ethylene glycol (PEG) 20000, 1.0 M ATP (the concentration
33P]-ATP
L CDK8 (ProQinase lot: 004), and
mL on a Beckman Coulter/SAGIAN™ Core System.
m
m
m
m
m
m
m
m
corresponds to the apparent Km (ATP) value of CDK8), [g-
(4-9 ꢂ 105 cpm per well), 1 ng/
m
20 ng/mL RBER-IRStide (ProQinase lot: 019 or 023). The reaction
cocktails were incubated at 30 ꢀC for 60 min. The reactions were
stopped with addition of 50 L of 2% (v/v) H3PO4, and plates aspi-
rated and washed twice with 200 L of 0.9% (w/v) NaCl. Incorpo-
m
m
ration of 33Pi was determined with a microplate scintillation
counter (Microbeta, Wallace). A residual kinase activity (%) at each
compound concentration and an IC50 value of a test compound
enex Kinetex Biphenyl column (1.7
a mobile phase flow rate of 0.5 mL/min. Mobile phase A was 5%
m
m, 100 Å, 50 mm ꢂ 2.1 mm) at
methanol and 0.1% FA in water, and mobile phase B (MPB) 95%
20

M. Yu, Y. Long, Y. Yang et al.
European Journal of Medicinal Chemistry 218 (2021) 113391
methanol and 0.1% FA in water. The mobile phase timetable was set
as a linear gradient starting from 2% MPB for 0.5 min, followed by
ramping to 100% MPB over 2.3 min and maintaining at 100% MPB
for 1.5 min before returning to 2% MPB for 0.5 min in preparation
for the next sample. Ratios of signal areas of compound/IS were
obtained from known concentrations of calibrators and were used
to construct a calibration curve which was used to determine the
plasma concentrations of unknown samples. The limit of quantifi-
cation was 5 ng/mL. The intraday and interday variability for each
compound was within 15%. Non-compartmental analyses of
plasma concentration versus time were performed using Phoenix
WinNonlin (Certara, St. Louis, MO, USA). These experiments were
carried out under the approval of the University of South Australia
Animal Ethics Committee (Animal Ethics Number: U21-16).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113391.
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