Potent and orally bioavailable CDK8 inhibitors: Design, synthesis, structure-activity relationship analysis and biological evaluation

European Journal of Medicinal Chemistry 214 (2021) 1 13248

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Potent and orally bioavailable CDK8 inhibitors: Design, synthesis,

structure-activity relationship analysis and biological evaluation

Mingfeng Yu ^a, Theodosia Teo ^a, Yuchao Yang ^a, Manjun Li ^a, Yi Long ^a, Stephen Philip ^a,

Benjamin Noll ^a, Gary K. Heinemann ^a, Sarah Diab ^a, Preethi Eldi ^b, Laychiluh Mekonnen ^a,

Abel T. Anshabo ^a, Muhammed H. Rahaman ^a, Robert Milne ^a, John D. Hayball ^b,

,

*

Shudong Wang ^a

^aDrug Discovery and Development, Cancer Research Institute, Clinical and Health Sciences, University of South Australia, Adelaide, South Australia, 5000,

Australia

^bExperimental Therapeutics, 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 regulates transcription either by phosphorylation of transcription factors or, as part of a four-

subunit kinase module, through a reversible association of the kinase module with the Mediator com-

plex, a highly conserved transcriptional coactivator. Deregulation of CDK8 has been found in various

types of human cancer, while the role of CDK8 in supressing anti-cancer response of natural killer cells is

being understood. Currently, CDK8-targeting cancer drugs are highly sought-after. Herein we detail the

discovery of a series of novel pyridine-derived CDK8 inhibitors. Medicinal chemistry optimisation gave

rise to 38 (AU1-100), a potent CDK8 inhibitor with oral bioavailability. The compound inhibited the

proliferation of MV4-11 acute myeloid leukaemia cells with the kinase activity of cellular CDK8 damp-

ened. No systemic toxicology was observed in the mice treated with 38. These results warrant further

pre-clinical studies of 38 as an anti-cancer agent.

Received 21 December 2020

Received in revised form

23 January 2021

Accepted 24 January 2021

Available online 3 February 2021

Keywords:

CDK8 inhibitor

Structure-activity relationship

Drug-like properties

Pharmacokinetics

Toxicity

1. Introduction

comprises cyclin C, MED12 and MED13 [9], CDK8 regulates tran-

scription through a reversible association of the kinase module

Cyclin-dependent kinases (CDKs) in partnership with their

respective cognate cyclins orchestrate a wide range of cardinal

regulatory events, but particularly cell division and transcription

[1]. Originally discovered as a putative kinase partner of cyclin C

[2,3], CDK8 is a ubiquitously expressed, primarily transcriptional

member of the CDK family [4]. The kinase controls gene expression

by phosphorylation of transcription factors including neurogenic

locus notch homolog proteins (NOTCH) [5], mothers against

decapentaplegic homologs (SMADs) [6], sterol regulatory element-

binding protein 1C (SREBP-1C) [7] and signal transducer and acti-

vator of transcription 1 (STAT1) [8]; the phosphorylation either

directly tunes the activity of these transcription factors or primes

them for ubiquitin-mediated proteasomal degradation. Alterna-

tively, as part of a four-subunit kinase module that additionally

with the Mediator complex, a highly conserved transcriptional

coactivator that serves as a “molecular bridge” to transfer regula-

tory signals from DNA-binding transcription factors directly to the

RNA polymerase II preinitiation complex [10].

Given the pivotal regulatory role of CDK8 in transcription, it is

hardly surprising that its deregulation is implicated in multiple

types of human cancer. The CDK8-coding gene was conferred an

oncogene status ﬁrst in colorectal cancer in 2008 [1 1]. Since then,

the kinase activity of CDK8 has been found oncogenic in melanoma

[12], breast cancer [13], acute myeloid leukaemia (AML) [14],

pancreatic cancer [15], prostate cancer [16], and so forth [1 7]. In

addition, a previous study has shown that CDK8 kinase activity

attenuates the defence of natural killer cells against malignant cells

and restrains the tumour surveillance of the former cells [18]; this

concept has been further validated both genetically (via deletion of

the CDK8-coding gene in natural killer cells) [19] and pharmaco-

logically (by use of several exogenous small-molecule inhibitors of

CDK8) [20]. Taken together, these studies provide a rationale for the

* Corresponding author.

E-mail address: shudong.wang@unisa.edu.au (S. Wang).

https://doi.org/10.1016/j.ejmech.2021.113248

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

potential application of pharmacological inhibition of CDK8 as a

strategy for cancer therapy. Accordingly, there is a pressing demand

in discovering potent and selective small-molecule CDK8

inhibitors.

potent and selective CDK8 inhibitor [24]. This discovery in combi-

nation with our virtual screening results (vide supra) not only

boosted our conﬁdence in seeking pyridine-derived CDK8 in-

hibitors but also offered a reasonable starting point for medicinal

chemistry optimisation.

An analysis of the X-ray crystal structure of CCT251545 bound to

CDK8/cyclin C (Fig. 2) [24] assisted in our rational design of novel

CDK8 inhibitors: (I) The pyridine core acts as a hinge binder, with its

nitrogen atom forming a hydrogen bond with the NH of Ala100. An

interaction of this kind had been considered typical of CDK8 in-

hibitors yet with distinct chemical scaffolds [14,24,36], so the pyr-

idine core was retained in our structural optimisation. (II) The

proper size and orientation of the chlorine atom at the pyridine-C3

Since the report of cortistatin A (Fig. 1) as a high-afﬁnity ligand

of CDK8 in 2009 [21], diverse scaffolds, either naturally derived or

chemically synthesised, have been exploited in search of CDK8

inhibitors. The known CDK8 inhibitors include, but are not limited

to, senexin B [22], CCT251545 [23,24], MSC253818 [25], SEL120-

34A [26], W-34 [27], T-418 [28], and derivatives of steroid [29,30],

1,6-naphthyridine [31], thieno[2,3-c]pyridine [32], 4-(1H-pyrrol-4-

yl)pyridine [33], pyrido[2,3-b][1,5]benzoxazepin-5(6H)-one [34]

(Fig. 1). These compounds inhibit CDK8 at low nanomolar con-

centrations, and two of them have advanced into phase I clinical

trialsdsenexin B (BCD-115) and SEL120-34A (SEL120) for treat-

ments of local advanced and metastatic breast cancer with estrogen

receptor-positive/human epidermal growth factor receptor 2-

negative (NCT03065010, completed with no results released yet),

and of AML or high-risk myelodysplastic syndrome (NCT04021368,

recruiting patients), respectively (ClinicalTrials.gov, last accessed

21^stDecember 2020).

position (Fig. 1) permit a Cl-p interaction [37] with the gatekeeper

residue Phe97 (Fig. 2), and therefore this atom remained

Our earlier attempt to identify novel CDK8 inhibitor scaffolds

through virtual screening gave rise to a total of 476 hits prior to

visual inspection and subsequent selection for biological validation

[35]. Several classes of scaffolds dominated the results, including 4-

substituted quin(az)olines, 1,3-diarylureas, and multi-substituted

pyridines. While structural optimisation of the ﬁrst two chemical

series for potency, selectivity and drug-like properties is underway,

we report herein our progress on developing pyridine-derived

CDK8 inhibitors.

2. Results and discussion

Fig. 2. X-ray crystal structure of CCT251545 bound to CDK8/cyclin C (PDB ID: 5BNJ).

CCT251545 is engaged in three hydrogen bonds (black dotted lines) as well as

p-cation

and Cl- interactions (yellow dotted lines) with CDK8. The kinase is depicted as sky

p

2.1. Rational design of 3,4,5-trisubstituted pyridines

blue ribbons. Nitrogen atoms are shown in royal blue, oxygen atoms in red, receptor

carbon atoms in grey, ligand carbon atoms in green, and the chlorine atom is coloured

orange. The illustration was generated using PyMOL Molecular Graphics System

In 2015, an elegant study was published on the discovery of

CCT251545 (Fig. 1), a 3,4,5-trisubstituted pyridine derivative, as a

€

Version 2.4.0 Schrodinger, LLC.

Fig. 1. Chemical structures of selected CDK8 inhibitors.

2

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

unchanged. (III) The amide of the spirolactam moiety at the

pyridine-C4 position bridges Lys52 and Asp173 through two

hydrogen bonds. We envisaged that a replacement of this moiety

with a heteroaromatic ring appended with an amide arm could

maintain, if not strengthen, the contact with both amino acids. (IV)

literature survey of the chemical structures containing a

spirolactam-pyridine core unveiled that reversing the topology of

the phenyl-pyrazole pendant at the pyridine-C5 position remained

unexplored and, more importantly, that replacing this pendent

with fused ring systems was underexplored. Accordingly, we

explored chemical space along both lines, with the common aim of

While the phenyl ring at the pyridine-C5 position forms a p-cation

interaction with Arg356, the entire 4-(1-methyl-1H-pyrazol-4-yl)

phenyl moiety occupies the solvent-exposed region, an element of

the kinase structure that has the capacity to tolerate structural

modiﬁcations and is thus often exploited for structure-based drug

design [38]. As a result, our medicinal chemistry optimisation of

CCT251545 focused on C4 and C5 positions of the pyridine ring.

Modiﬁcation at the Pyridine-C4 Position. The hydrogen bonding of

the 2,8-diazaspiro[4.5]decan-1-one moiety to Lys52 and Asp173

contributes to the high afﬁnity of CCT251545 for CDK8 (Fig. 2). In

fact, both amino acids are conserved across the CDK family [39]. As

such, we hypothesised that the moieties at the pyrimidine-C4 po-

sition of our previously synthesised CDK inhibitors [40e48] that

were capable of interacting with either or both counterparts of

CDK8-Lys52/Asp173 in alternative CDKs could stand a chance of

replacing the 2,8-diazaspiro[4.5]decan-1-one group of CCT251545

at no cost of CDK8 inhibitory potency. With this in mind, we

revisited our in-house library of co-crystal structures of small-

molecule inhibitors in complex with CDKs, and found several

functionalised ﬁve-membered aromatic moieties formed hydrogen

bonds with CDK2-Asp145 or CDK9-Asp167 [41,42,45]; two equiv-

alents of CDK8-Asp173 [39]. One of such moieties was 1H-pyrrol-4-

yl, but a survey of the relevant literature revealed that this moiety

in the context of the pyridine core had been claimed in a patent

application [49] (Fig. 3). To circumvent this, we altered the topology

of the pyrrole-pyridine system, linking the former to the latter via

the N1⁰-C4 bond instead, with a view to contacting CDK8-Asp173 by

an exocyclic carboxylic acid or amide arm (part I, Fig. 3).

enhancing the p-cation interaction with CDK8-Arg356 by intro-

duction of electron-rich substituents (part II, Fig. 3).

2.2. Chemistry

Thesynthetic route deployed to prepare3-chloro-5-(4-(1-methyl-

1H-pyrazol-4-yl)phenyl)-4-(3-substituted-1H-pyrrol-1-yl)pyridines

8e10 is delineated in Scheme 1. In situ mono-lithiation at the C4 po-

sition of 3-bromo-5-chloropyridine 1 with lithium diisopropylamide

(LDA) at ꢀ78 ^ꢁC was followed by an exchange with a chlorine from

hexachloroethane, giving 3-bromo-4,5-dichloropyridine 2 in 65%

yield. This newly introduced chlorine was further displaced with

methyl 1H-pyrrole-3-carboxylate under microwave irradiation in the

presence of caesium carbonate to afford methyl 1-(3-bromo-5-

chloropyridin-4-yl)-1H-pyrrole-3-carboxylate 3 in a yield of 48%. In

parallel, starting from 1-chloro-4-iodobenzene 4, two halogen

atomsdiodine and chlorinedwere successively replaced with 1-

methyl-1H-pyrazol-4-yl and 4,4,5,5-tetramethyl-1,3,2-dioxaborolan

-2-yl, respectively, using two palladium-catalysed reactions: a

Suzuki coupling with the iodine using Pd(dppf)Cl₂$CH₂Cl₂, and a

Miyaura borylation of the chlorine employing a combination of

Pd₂(dba)₃withXPhos. Bothreactionswerecarriedoutinthepresence

of a base at an elevated temperature overnight, resulting in 1-methyl-

4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-pyr-

azole 6 in a good overall yield (5: 58% and 6: 83%). A second Suzuki

couplingofbromide3withboronateester6usingthesamepalladium

catalyst yet under microwave irradiation for a much shorter time (i.e.,

Optimisation at the Pyridine-C5 Position. The 4-(1-methyl-1H-

pyrazol-4-yl)phenyl pendant of CCT251545 nestles in the solvent

channel that typically tolerates structural modiﬁcations (Fig. 2). A

1

h) gave methyl 1-(3-chloro-5-(4-(1-methyl-1H-pyrazol-4-yl)

phenyl)pyridin-4-yl)-1H-pyrrole-3-carboxylate 7 in 67% yield, which

was subsequently subjected to saponiﬁcation in an alkaline

Fig. 3. Design of novel chemical scaffolds for CDK8 inhibitors.

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European Journal of Medicinal Chemistry 214 (2021) 113248

Scheme 1. Synthesis of 3-chloro-5-(4-(1-methyl-1H-pyrazol-4-yl)phenyl)-4-(3-substituted-1H-pyrrol-1-yl)pyridines 8e10. Reagents and conditions: (a) (1) LDA (2.0 M in THF/

heptane/ethylbenzene), THF, ꢀ78 ^ꢁC, 1 h; (2) hexachloroethane, THF, ꢀ78 ^ꢁC, 1 h, 65%; (b) methyl 1H-pyrrole-3-carboxylate, Cs₂CO₃, 1,4-dioxane, microwave 150e300 W, 200 ^ꢁC, 1 h,

48%; (c) 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, Pd(dppf)Cl₂$CH₂Cl₂, Na₂CO₃, THF/H₂O, 80 ^ꢁC, o/n, 58%; (d) bis(pinacolato)diboron, Pd₂(dba)₃, XPhos,

potassium acetate, 1,4-dioxane, 85 ^ꢁC, o/n, 83%; (e) Pd(dppf)Cl₂$CH₂Cl₂, 0.5 M Na₂CO₃, CH₃CN, microwave 150e300 W, 120 ^ꢁC, 1 h, 67%; (f) 1 M NaOH, CH₃OH, reﬂux, 2 h, 95%; (g)

NH₃(0.5 M in 1,4-dioxane) or CH₃NH₂$HCl, HATU, DIPEA, DMF, 0 ^ꢁC to rt, o/n, 9: 73%, 10: 70%.

methanolic solution at reﬂux to yield carboxylic acid 8 in a yield of

95%. Finally, amination of carboxylic acid 8 with either ammonia or

methylamine hydrochloride was effected by 1-[bis(dimethylamino)

methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexaﬂuoro

phosphate (HATU), a coupling reagent, in the presence of N,N-diiso-

propylethylamine (DIPEA) in anhydrous N,N-dimethylformamide

(DMF), giving the corresponding amides 9 and 10 in good yields (9:

73% and 10: 70%).

The above synthetic approach was successfully applied to the

preparation of 8-(3-chloro-5-substituted-pyridin-4-yl)-2,8-diazas

piro[4.5]decan-1-ones with a single exception of 8-(3-chloro-5-((1-

methyl-1H-indazol-5-yl)amino)pyridin-4-yl)-2,8-diazaspiro[4.5]

decan-1-one 12 that was synthesised via Pd₂(dba)₃/Xantphos-cata-

lysed Buchwald-Hartwig amination (Scheme 2). In brief, 3-bromo-

4,5-dichloropyridine 2 sequentiallyunderwentnucleophilic aromatic

substitution with tert-butyl 1-oxo-2,8-diazaspiro[4.5]decane-8-

carboxylate, and Pd(dppf)Cl₂$CH₂Cl₂-catalysed Suzuki coupling

with diverse aromatic boronic acids or boronate esters, affording

desired 8-(3-chloro-5-aryl-pyridin-4-yl)-2,8-diazaspiro[4.5]decan-

1-ones 13e34, 36 and 38e41 in varying yields (13e69%). Among

them, 34 and 36 were further deprotected using lithium hydroxide in

a mixture of tetrahydrofuran (THF) and methanol to yield the corre-

sponding (aza)indoles 35 and 37 with an intact NH site, respectively

(35: 80% and 37: 75%).

2.3. Structure-activity relationship analysis

Pharmacological inhibition of CDK8 upregulated the super-

enhancer-associated genes with tumour-suppressing and lineage-

controlling functions in a range of AML cell lines, particularly

Scheme 2. Synthesis of 8-(3-chloro-5-substituted-pyridin-4-yl)-2,8-diazaspiro[4.5]

decan-1-ones 12e41. Reagents and conditions: (a) tert-butyl 1-oxo-2,8-diazaspiro[4.5]

decane-8-carboxylate, triethylamine, 1-methoxy-2-propanol, microwave 150e300 W,

MV4-11 [14]. Hence, this cell line was selected to evaluate the

cytotoxicity of our newly synthesised compounds. The evaluation

was conducted in-house by means of a cell viability assay using

resazurin [50], and CDK8 inhibitory potency of the compounds

220 ^ꢁC, 2.5 h, 79%; (b) 1-methyl-1H-indazol-5-amine, Pd₂(dba)₃, Xantphos, Cs₂CO₃, 1,4-

dioxane, microwave 150e300 W, 180 ^ꢁC, 1 h, 25%; (c) appropriate boronic acid or

boronate ester, Pd(dppf)Cl₂$CH₂Cl₂, 0.5 M Na₂CO₃, CH₃CN, microwave 150e300 W,

120e140 ^ꢁC, 1 h, 13e69%; (d) LiOH, THF/CH₃OH, 50 ^ꢁC, 1 h, 35: 80%, 37: 75%.

measured with a radiometric kinase assay using [g-

³³P]-ATP (also

known as ³³PanQinase® Activity Assay) at ProQinase GmbH.

Nanomolar inhibitors of CDK8 were successfully derived from

the pyrrole(C4⁰)-pyridine(C4) system (see an example of 4-(1H-

pyrrol-4-yl)pyridines in Fig. 1) [33]. However, in our work, three

skeleton failed to achieve nanomolar CDK8 inhibitory activity

(Table 1), which could be attributed to unfavourable orientations of

carboxylic acid/amide pendants for the binding to the kinase. As

prototype molecules 8e10 with

a

pyrrole(N1⁰)-pyridine(C4)

4

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

Table 1

Chemical structures and biological activities of 8e10.

Compound

R

CDK8/cyclin C inhibition, K_i(

m

M)^[a]

MV4-11 anti-proliferation, GI₅₀(m

M)^[b]

8

9

10

OH

NH₂

NHCH₃

4.19

1.09

2.03

87.07 9.02

9.58 1.12

34.96 6.94

^[a]Inhibition of CDK8/cyclin C was measured with a radiometric kinase assay using [

value used as the ATP concentration. Each apparent inhibition constant (K_i) was calculated using the corresponding IC₅₀value and the K_m(ATP) value.

g-

³³P]-ATP (also known as ³³PanQinase® Activity Assay) at ProQinase GmbH, and the K_m

[b]

GI₅₀values were

determined in-house by a 72-h resazurin assay, and are presented as mean standard deviation derived from at least two replicates.

Table 2

Chemical structures and biological activities of 13e16.

Compound

R

CDK8/cyclin C inhibition, K_i(

m

M)^[a]

MV4-11 anti-proliferation, GI₅₀

0.84 0.11

(m

M)^[b]

13

0.042

14

15

0.346

0.124

5.34 0.92

7.43 1.39

16

0.700

12.20 3.87

CCT251545

0.009

0.03 0.02

^[a]Inhibition of CDK8/cyclin C was measured with a radiometric kinase assay using [

value used as the ATP concentration. Each apparent inhibition constant (K_i) was calculated using the corresponding IC₅₀value and the K_m(ATP) value.

g-

³³P]-ATP (also known as ³³PanQinase® Activity Assay) at ProQinase GmbH, and the K_m

[b]

GI₅₀values were

determined in-house by a 72-h resazurin assay, and are presented as mean standard deviation derived from at least two replicates.

5

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

Table 3

Chemical structures and biological activities of 12 and 17e30.

Compound

R

CDK8/cyclin C inhibition, K_i(

m

M)^[a]

MV4-11 anti-proliferation, GI₅₀

1.71 0.96

(m

M)^[b]

12

0.670

17

18

0.458

0.052

27.66 5.07

5.26 0.54

19

20

0.019

0.066

1.91 1.35

7.55 0.76

21

22

23

24

0.138

0.022

0.052

3.140

53.46 10.33

3.65 0.74

62.31 3.31

64.17 8.08

25

26

27

0.114

0.016

0.022

9.15 0.86

0.82 0.09

0.71 0.12

6

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

Table 3 (continued)

Compound

R

CDK8/cyclin C inhibition, K_i(

m

M)^[a]

MV4-11 anti-proliferation, GI₅₀

33.16 2.29

(m

M)^[b]

28

0.026

29

30

0.016

0.024

0.009

6.68 0.18

6.35 0.35

0.03 0.02

CCT251545

^[a]Inhibition of CDK8/cyclin C was measured with a radiometric kinase assay using [

value used as the ATP concentration. Each apparent inhibition constant (K_i) was calculated using the corresponding IC₅₀value and the K_m(ATP) value.

g-

³³P]-ATP (also known as ³³PanQinase® Activity Assay) at ProQinase GmbH, and the K_m

[b]

GI₅₀values were

determined in-house by a 72-h resazurin assay, and are presented as mean standard deviation derived from at least two replicates.

expected, these three compounds exerted little anti-proliferative

effect on MV4-11 cells.

Our attention was then turned to diversiﬁcation of the substit-

uent at the pyridine-C5 position of CCT251545 (Tables 2e4). As a

positive control, CCT251545 was ﬁrst synthesised in-house and

evaluated, showing high inhibitory activity towards CDK8

compounds 17 and 18, with inhibition of CDK8 and MV4-11 cells

weakened to an extent that was contingent on the orientation of the

methyl group (17 [N(1)-methyl]: K_i¼ 0.458 M and GI₅₀¼ 27.66

M) (Table 3).

Bridging the N(1)-methylindazolyl pendant and the pyridine core

with a secondary amino group deteriorated the kinase inhibition (i.e.,

m

mM

versus 18 [N(2)-methyl]: K_i¼ 0.052

m

M and GI₅₀¼ 5.26

m

(K_i¼ 0.009

m

M) and potent anti-proliferative effect on MV4-11 cells

12: K_i¼ 0.670

mM), so no alternative single-heteroatom linkers were

further attempted. Replacing the N(2)-CH₃site of the N(2)-methyl-

indazol-4-yl with CH afforded 19 appended with a 1H-indol-4-yl, and

(GI₅₀¼ 0.03

m

M) (Table 2). The former value is in agreement with

the reported data [24,51], whereas there is, to the best of our

knowledge, no literature value of the latter.

Reversing the topology of the phenyl-pyrazole pendant of

CCT251545 gave rise to 13, which was slightly less inhibitory to

returned a higher afﬁnity for CDK8 (K_i¼ 0.019

mM) and a more potent

growth inhibition of MV4-11 cells (GI₅₀¼ 1.91

mM). Expanding the

indole ring with an additional carbon gave rise to a range of (iso)

quinoline-based pendants (i.e., 20e25) with varying degrees of CDK8

inhibitory potency, indicating the position of the only nitrogen atom

played an important role. Detailedly, 22 with a quinolin-5-yl was the

CDK8, but maintained a low-nanomolar potency (K_i¼ 0.042

(Table 2). The anti-proliferative effect of 13 was just within the sub-

M). Spacing the two

mM)

micromolar concentration range (GI₅₀¼ 0.84

m

components of the reversed pendant with a methylene (i.e., 14)

further reduced kinase inhibitory activity as well as cellular po-

tency, and so did replacing the phenyl component with a less rigid

aliphatic heterocycle (i.e., a tetrahydropyran in 15 and a piperidine

in 16). These three compounds showed high sub-micromolar and

micromolar inhibitory activities against CDK8 and MV4-11 cells,

most active against CDK8 (K_i¼ 0.022

quinolin-8-yl hardly suppressed the kinase activity (K_i¼ 3.140

The rest were sub-micromolar inhibitors of CDK8

(K_i¼ 0.052e0.138 M). All of the (iso)quinoline-containing com-

pounds 20e25 dampened the proliferation of MV4-11 cells at

M). Removal of

mM), whereas 24 with an iso-

mM).

m

micromolar concentrations (i.e., GI₅₀¼ 3.65e64.17

m

respectively (K_i¼ 0.124e0.700

m

M and GI₅₀¼ 5.34e12.20

mM).

the sole nitrogen atom from (iso)quinolines furnished 26 with a

naphthalen-1-yl, and further introduction of a methyl or methoxy

group at the naphthalene-C4 position gave 27 and 28, respectively.

These threemolecules inhibited CDK8kinase activityat low two-digit

These results suggested that an increased ﬂexibility of the sub-

stituent at the pyridine-C5 position could be detrimental to inhi-

bition of both CDK8 kinase activity and MV4-11 cell growth. To

diminish the ﬂexibility, we switched to incorporate a wide range of

fused aromatic rings onto this position. Among them were [5,6]-,

[6,5]-, [6,6]- and [6,5,6]-membered-membered bi-/tri-cyclic fused

moieties (Tables 3 and 4).

nanomolar concentrations (K_i¼ 0.016e0.026

mM), which were

translated into their sub-micromolar anti-proliferative effects on

MV4-11 cells with the exception of 28 (GI₅₀¼ 0.82, 0.71 and 33.16

mM

for 26, 27 and 28, respectively). Again, another poor correlation be-

tween CDK8 inhibition and MV4-11 anti-proliferation was found for

29 and 30 with bulkier, tricyclic fused pendants. Both compounds

Direct amalgamation of the phenyl with the N-methylpyrazolyl

within CCT251545 formed two N-methylindazol-4-yl-containing

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European Journal of Medicinal Chemistry 214 (2021) 113248

Table 4

Chemical structures and biological activities of 31e41.

Compound

R

CDK8/cyclin C inhibition, K_i(

m

M)^[a]

MV4-11 anti-proliferation, GI₅₀

6.84 0.90

(m

M)^[b]

31

0.156

32

33

0.079

0.058

10.06 3.78

30.59 8.63

34

0.645

28.44 4.52

35

36

0.056

1.300

8.77 2.04

40.16 2.50

37

0.050

7.82 1.20

38

39

40

0.014

0.048

0.36 0.35

0.58 0.14

29.16 1.19

41

0.021

0.009

0.35 0.22

0.03 0.02

CCT251545

^[a]Inhibition of CDK8/cyclin C was measured with a radiometric kinase assay using [

value used as the ATP concentration. Each apparent inhibition constant (K_i) was calculated using the corresponding IC₅₀value and the K_m(ATP) value.

g-

³³P]-ATP (also known as ³³PanQinase® Activity Assay) at ProQinase GmbH, and the K_m

[b]

GI₅₀values were

determined in-house by a 72-h resazurin assay, and are presented as mean standard deviation derived from at least two replicates.

8

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

suppressed CDK8 kinase activity potently (K_i¼ 0.016 and 0.024

m

M),

approximately one order of magnitude lower than those of CDK8

but induced marginal toxicity towards MV4-11 cells (GI₅₀¼ 6.68 and

(K_i¼ 0.109 and 0.134

mM versus 0.014 and 0.014 mM, respectively)

6.35

mM).

(Table 5). These three off-targets were expected because

Two out of three most potent CDK8 inhibitors identiﬁed thus far

were 19 and 29 (Tables 1e3), where a ﬁve-membered ring was

attached to the pyridine core through its fused six-membered ring

(i.e., phenyl in both cases). This observation led us to consider and

test the possibility of improving CDK8 inhibitory activity by

diverting a connecting site onto a fused ﬁve-membered aromatic

ring instead (Table 4). Two such counterparts of 19 were ﬁrst pre-

pared, with connecting sites of 31 and 35 located at their respective

C2 and C3 positions of the common indole ring. Both counterparts

were weaker inhibitors of CDK8 and MV4-11 cells than 19 (31:

CCT251545 had been found to inhibit all of them [52]dto a lesser

extent towards GSK-3a/b but to a higher degree towards PCK-q in

comparison with 38 and 39.

2.5. In vitro anti-proliferative spectra of 38 and 39

As described in Section 2.3, 38 and 39 exerted sub-micromolar

anti-proliferative effects on MV4-11 cells. To deﬁne the scope of

such action, both compounds were further screened against 18

human cancer cell lines (originating in blood/bone marrow, colon/

rectum, pancreas, ovary and breast) and one non-transformed cell

line (i.e., normal human diploid lung ﬁbroblasts WI-38) (Table 6).

Among them, U-937 and MOLM-13 AML cell lines were the most

sensitive to 38 and 39, with GI₅₀values varying from 1.36 to

K_i¼ 0.156

m

M and GI₅₀¼ 6.84

m

M; 35: K_i¼ 0.056

mM and

GI₅₀¼ 8.77

mM). As expected, protecting the indole-NH of 35 with a

sizeable benzenesulfonyl group greatly decreased inhibition of

both the kinase and cells (34: K_i¼ 0.645

m

M and GI₅₀¼ 28.44

mM).

The same held true for the pair of 7-azaindole (37: K_i¼ 0.050

mM

6.18

and 0.58 m

m

M, but both less amenable than MV4-11 cell line (GI₅₀¼ 0.36

and GI₅₀¼ 7.82

(36: K_i¼ 1.300

m

M) and its N(1)-benzenesulfonyl-protected form

M for 38 and 39, respectively). Greater anti-proliferative

M and GI₅₀¼ 40.16

m

M). These ﬁndings were

effects of both compounds on MV4-11 than on MOLM-13 were in

agreement with higher expression of CDK8 in the former cell line

(Fig. S1). While GI₅₀values for the majority of solid tumour cell

consistent with our previous observation that ﬂexible substituents

might not be favourable at the pyridine-C5 position (Table 2). In

contrast, masking the indole-NH of 35 with a large-size benzyl

lines spanned from 16.18 to 44.25

mM, those for the rest were all

group maintained CDK8 inhibitory activity (33: K_i¼ 0.058

versus 35: K_i¼ 0.056 M) (Table 4). Similarly, substitution of the

indole-NH of 31 with a small-size methyl group even ameliorated

the potency against the kinase (32: K_i¼ 0.079 M versus 31:

K_i¼ 0.156 M). Both cases implied that an intact indole-NH might

m

M

more than 50 M. These results indicated that the in vitro anti-

m

proliferative effect of 38 and 39 could be cell-type speciﬁc, in

particular towards some of AML cell lines like MV4-11. Importantly,

both compounds imposed little inhibitory effects on non-

transformed WI-38 lung ﬁbroblasts, and their GI₅₀values were

about two orders of magnitude higher than those for MV4-11 cells,

achieving a desirable anti-proliferative selectivity. CCT251545 dis-

played a similar anti-proliferation proﬁle to those of 38 and 39; the

single exception was the sub-micromolar suppression of the con-

trol compound on the growth of PANC-1 pancreatic cancer cells

m

not be essential for CDK8 inhibitory activity. As a result, benzofur-

anyl and benzothiophenyl moieties, where their respective oxygen

and sulphur atoms are isosteres of the indole-NH, were selected to

replace the indolyl pendant. Among the four compounds thus

synthesised, 38, 39 and 41 formed a group of inhibitors with the

highest afﬁnity for CDK8 as well as with the greatest effectiveness

against MV4-11 cells. Speciﬁcally, K_ivalues of 38, 39 and 41 were

(GI₅₀¼ 0.55

mM).

determined to be 0.014, 0.014, and 0.021

which were comparable to that of CCT251545 (K_i¼ 0.009

respective GI₅₀values of 0.36, 0.58, and 0.35 M were about one

order of magnitude higher than that of the control (GI₅₀¼ 0.03 M).

Surprisingly, the remaining compound 40 was slightly less active

towards CDK8 (K_i¼ 0.048 M), but barely suppressed the prolif-

eration of MV4-11 cells (GI₅₀¼ 29.16 M).

m

M, respectively, all of

2.6. Cellular CDK8 inhibition of 38

mM); their

m

Given that MV4-11 cells showed the highest sensitivity towards

38, this AML cell line was chosen to assess the cellular CDK8

inhibitory potency of the compound. MV4-11 cells were incubated

with 38 or CCT251545 for 2 h, and analysed by western blotting

(Fig. 5). Treatment with either compound signiﬁcantly reduced the

phosphorylation of serine 727 on STAT1, a known substrate of CDK8

[18,53], at concentrations of their respective 1 ꢂ and 5 ꢂ GI₅₀

values, but barely affected the level of total STAT1. These results

conﬁrmed the inhibition of the kinase activity of cellular CDK8 by

38.

m

2.4. Kinase inhibitory selectivity of 38 and 39

To understand kinase inhibitory selectivity of newly synthesised

CDK8 inhibitors, the two most potent molecules 38 and 39 were

selected and tested against, as a ﬁrst step, a wide range of other CDK

family members at a compound concentration of 1

mM (Fig. 4,

2.7. In Vitro Drug-like properties of 38 and 39

below dotted lines). None of the CDKs tested were deprived of more

than 50% kinase activity by either compound, with the exceptions

of CDKs 1 and 9 in the presence of 38. Their respective K_ivalues

Prior to in vivo pharmacokinetic and toxicity studies, drug-like

properties of 38 and 39 were evaluated in vitro, including physi-

cochemical proﬁling and ADMET (absorption, distribution, meta-

bolism, excretion and toxicity) screening (Table 7). Lipophilicity of a

compound contributes to ADME characteristics of the compound,

and distribution coefﬁcient (Log D) is typically used as a measure of

lipophilicity [54]. Both 38 and 39 had a Log D_7.4value of > 4, sug-

gesting their high lipophilicity. The negative log of the acid disso-

ciation constant (pKa) of a molecule predicts the degree of its

ionisation at a particular pH, and affects its solubility and perme-

ability by modulating the distribution of neutral and charged spe-

cies of the compound [54]. A pKa value of 4.90 for 38 indicated that

the compound was 50% protonated at pH 4.90. The aqueous solu-

bility of a compound plays an essential role in determining its ab-

sorption from the gastrointestinal tract and ultimately its oral

were later determined to be 1.895 and 1.765

were more than two orders of magnitude higher than that of CDK8

(i.e., 0.014 M). These results demonstrated that both CDK8 in-

mM, both of which

m

hibitors were highly selective across the CDK family. Further

proﬁling of kinase inhibitory selectivity included a screening

against a wider panel of 47 additional human kinases known to be

frequently targeted by CDK inhibitors (Fig. 4, above dotted lines).

The activities of only three kinasesdthe two isoforms of glycogen

synthase kinase-3 (GSK-3

were inhibited more than 50% by both 38 and 39 at 1

ﬁnities of both compounds for GSK-3 were comparable to those

for CDK8, with K_ivalues ranging from 0.004 to 0.043 M, whereas

their inhibitory effects on the kinase activity of PCK- were

a

/b

) and protein kinase C-theta (PCK-

q)d

mM. The af-

a/b

m

q

9

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

Fig. 4. Kinase inhibition proﬁles of 38 and 39 (at 1

mM) over a panel of 62 kinases (including 15 CDK/cyclin complexes). The data were acquired at Euroﬁns Scientiﬁc, Reaction

Biology Corporation and ProQinase GmbH.

Table 5

Off-targets of 38 and 39.

with their respective solubility levels of 10e30 and < 20 mM were

considered partially soluble. This suboptimal solubility became less

concerned because both 38 and 39 displayed high permeability,

with their passive apparent permeability coefﬁcient (P_app(AdB))

values being more than 20 ꢂ 10^ꢀ6cm/s. Additionally, both efﬂux

ratios of around 1 suggested that neither 38 nor 39 underwent

active efﬂux.

Given that a large portion of kinase inhibitors approved by the

United States Food and Drug Administration (US FDA) are engaged

in combination cancer therapies [56], the liabilities of 38 and 39 for

drug-drug interactions were assessed using a range of cytochrome

P450 (CYP450) enzymes; a family of haemoproteins that are

involved in the metabolism of drugs [57]. Both compounds exerted

no inhibitory effects on CYP2C9, CYP2C19 and CYP2D6

Compound CDK8/cyclin C inhibition, K_i(

m

M)^[a]Off-target kinase inhibition,

K_i(

M)^[a]

m

GSK-3

a

GSK-3

b

PCK-q

38

39

0.014

0.009

0.013

0.043

0.004

0.018

0.109

0.134

CCT251545

0.231^[b]0.345^[b]0.061^[b]

^[a]Inhibition of CDK8/cyclin C was measured at ProQinase GmbH, and GSK-3

PCK- were tested at Reaction Biology Corporation. The K_mvalue was used as the

ATP concentration for each kinase. Each apparent inhibition constant (K_i) was

a/b and

q

[b]

calculated using the corresponding IC₅₀and K_m(ATP) values.

calculated using the IC₅₀values reported in reference [52].

K_ivalues were

(IC₅₀> 25

IC₅₀¼ 4.59

CYP1A was inhibited quite modestly by 38 (IC₅₀¼ 7.7

m

M in all cases), and suppressed CYP3A4 moderately (38:

M and 39: IC₅₀¼ 2.11 M). The remaining isoform

M), but very

bioavailability [55]. Although a precise measure of aqueous solu-

bility could not be provided by the turbidimetric assay, 38 and 39

m

10

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

Table 6

Anti-proliferative effects of 38 and 39 on a panel of 20 human cell lines.

Origin

Cancer Cell line

Anti-proliferation, GI₅₀(m

M)^[a]

38

39

CCT251545

Blood/Bone marrow

HL-60

Kasumi-1

KG-1

35.31 4.91

> 50

ND^[b]

ND

> 50

37.03^[c]

7.13 1.25

19.98 0.89

KG-1a

19.55^[c]

ND

32.71^[c]

MOLM-13

MV4-11

NB4

6.18 0.10

0.36 0.35

40.80^[c]

5.81 1.35

0.58 0.14

ND

2.80 0.86

0.03 0.02

> 50

PL-21

25.35^[c]

ND

13.76^[c]

THP-1

> 50

ND

> 50

U-937

1.36 0.21

> 50

40.55 0.59

44.25 13.18

> 50

2.69 0.78

> 50

42.67 6.50

29.28 7.84

> 50

22.44 0.50

37.73 8.53

46.16^[c]

48.59 5.77

0.55 0.07

35.85 21.58

> 50

Colon/Rectum

COLO 205

HCT 116

HT-29

PANC-1

A2780

Pancreas

Ovary

Breast

21.43 2.08

> 50

17.57 0.39

> 50

MCF7

MDA-MB-231

MDA-MB-453

T-47D

40.71 3.33

23.32 9.96

16.18^[c]

34.21 8.73

17.45 0.64

17.92^[c]

35.03 3.84

29.91 2.26

43.06 4.78

15.05^[c]

Lung

WI-38 (normal)

48.05 13.32

> 50

^[a]GI₅₀values were determined in-house by 72-h cell viability assays using MTT (adherent cell lines) or resazurin (suspension cell lines), and are presented as mean standard

deviation derived from at least two replicates (unless otherwise stated). ^[b]ND: GI₅₀values were not determined. ^[c]GI₅₀values were acquired without replication.

weakly by 39 (IC₅₀¼ 16.6

mM). These data showed a low potential

for both compounds to induce CYP450-mediated drug-drug in-

teractions if co-administered with other therapeutic agents. Car-

diovascular toxicity remains one of the main reasons for post-

approval withdrawal of drugs [58]. To understand the potential

ꢀ

cardiotoxicity of 38 and 39, their impacts on the human ether-a-go-

go related gene (hERG) potassium channel were investigated. Both

compounds had little inhibition on this cardiac ion channel,

implying a slim likelihood of causing cardiotoxicity in vivo.

2.8. In Vivo Pharmacokinetic Properties of 38

Fig. 5. Western blot analysis of MV4-11 cells incubated with 38 or CCT251545 for 2 h.

DMSO diluent was used as a negative control, and -actin antibody as an internal

b

loading control. Both compounds were tested at the concentrations of their respective

1 ꢂ and 5 ꢂ GI₅₀values.

Pharmacokinetic studies carried out in animals are at the heart

of drug discovery programmes by virtue of their value of predicting

Table 7

In vitro drug-like properties of 38 and 39.

Parameter^[a]

38

39

[b]

Distribution coefﬁcient: Log D

4.21

4.90 0.02

10-30

22.9 0.6

23.4 3.2

1.02

> 4

ND^[h]

< 20

20.4 0.9

19.5 0.3

0.96

16.6 1.8

> 25

7.4

Negative log of the acid dissociation constant: pKa^[c]

Aqueous solubility (

m

M)^[d]

Caco-2 permeability^[e]

AdB P_app( ꢂ 10^ꢀ6cm/s)

BdA P_app( ꢂ 10^ꢀ6cm/s)

Efﬂux ratio

CYP1A

CYP2C9

Cytochrome P450 inhibition, IC₅₀

(

m

M)^[f]

7.7 1.6

> 25

CYP2C19

> 25

CYP2D6

> 25

CYP3A4

4.59 0.91

10.9 2.2

2.11 0.21

14.7 1.4

hERG potassium channel inhibition, IC₅₀(m

M)^[g]

[a]

[b]

All the data were acquired at Cyprotex Discovery Ltd.

Using the shake ﬂask assay with a buffer at pH 7.4.

sing a fas[c] Using a fast ultraviolet (UV) spectrometric titration.

[d]

By turbidimetry.

[e]

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

occurs in both directions: apical to basolateral (AdB) and basolateral to apical (BdA). P_app: apparent permeability coefﬁcient; efﬂux ratio ¼ P_app(BdA)/P_app(AdB).

[f]

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

IC₅₀> 10 M.

^[g]Using CHO-hERG cells. Typically, highly potent inhibition: IC₅₀< 0.1

IC₅₀> 10 M.

ND: pKa was not determined for 39.

m

M; moderate inhibition: IC₅₀¼ 1e10

m

M; weak or no inhibition:

m

M; potent inhibition: IC₅₀¼ 0.1e1

m

M; moderate inhibition: IC₅₀¼ 1e10

m

M; weak or no inhibition:

m

[h]

11

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

Table 8

2.9. In Vivo Toxicity of 38

In vivo pharmacokinetic properties of 38 in rats and mice.

Parameter^[a]

Species

CCT251545

Rat

38

We were aware of a report on severe systemic toxic effects of

CCT251921, a potent CDK8 inhibitor that is structurally analogous to

CCT251545 [51,52]. The observed toxicity was regarded as on-target

effects [52], which has been challenged recently, with an argument

that several off-target kinases could be responsible for the systemic

toxicity of CCT251921 [61]. Accordingly, it was deemed advisable to

conduct an initial assessment of the in vivo toxicity of 38. Female

BALB/c mice were orally administered 38 (70 mg/kg), CCT251545

(70 mg/kg), or vehicle (i.e., 1% carboxymethyl cellulose (CMC)

aqueous solution, 10 mL/kg) once daily for seven consecutive days,

and observed for a further seven days. Individual body weights

were recorded daily, showing little changes in mice treated with

either 38 or vehicle and a rise of approximately 12% in the mouse

following the seven-day administration of CCT251545 (Fig. S2). On

day 15, all the mice were humanely killed prior to a collection of

organs and tissues for histopathological examination or

ﬂuorescence-activated cell sorting (FACS) analysis.

Histopathological examination of long bones (left femora),

lungs, hearts, livers, kidneys, intestines, uteri, ovaries and brains

revealed pathological changes in the ﬁrst two types of organs only

after the treatment with 38 or CCT251545 (Fig. 6). The growth plate

of the left femur collected from the CCT251545-treated mouse

showed necrosis of chondrocytes, abnormal orientation of chon-

drocyte columns, and loss of ground substance, all of which led to

extensive splitting of the cartilage (top panels in Fig. 6). Only mildly

reduced ground substance and a few sightly disordered chon-

drocyte columns were found in 38-treated mice. No morphological

changes were apparent in the vehicle-dosed group. Interstitial

pneumonitis was observed in the lungs of the mouse treated with

CCT251545, with an increased number of alveolar macrophages

detected (bottom panels in Fig. 6). In addition, vasculitis was

developed in some of blood vessels, evidenced by inﬁltration of

inﬂammatory cells and destruction of vessel walls. These lesions

were much less severe in 38-treated mice, whereas morphological

changes in the vehicle-dosed group were very mild and were likely

incidental to inhalation of the anaesthetic gas. Collectively, with the

same dosage, 38 caused far less severe pathological alterations in

left femora and lungs of mice than did CCT251545.

Rat

Mouse

Route

IV

PO

IV

PO

IV

PO

Dose (mg/kg)

5

20

4.06

1.33

4.24

17.5

e

5

20

2.77

2.75

2.24

14.6

e

2

10

5.01

0.42

1.11

2.30

e

C_max

(

mM)

10.6

0.07

1.08

10.5

1.38

1.20

e

14.4

0.04

0.9

3.44

0.04

0.34

1.39

1.99

3.78

e

T_max(h)

T_1/2(h)

AUC (

m

M$h)

9.7

V_dss(L/kg)

Cl (L/h$kg)

F (%)

1.11

1.39

e

41

38

33

[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-to-four rats or ten mice are

presented.

the pharmacokinetics, efﬁcacy and safety of drugs in humans [59].

Inspired by the in vitro drug metabolism and pharmacokinetics

(DMPK) proﬁle of 38 (Table 7), single-dose pharmacokinetic studies

were performed with male Sprague Dawley rats by an intravenous

(IV) injection at 5 mg/kg or per os (PO) at 20 mg/kg, as well as with

male BALB/c mice administered intravenously at 2 mg/kg or via oral

gavage at 10 mg/kg (Table 8). For the sake of comparison,

CCT251545 was also given to the same rat species using the iden-

tical dosages and routes of administration to those of 38. Following

the IV administration of 38, volumes of distribution at steady state

(V_dss) for rats (1.11 L/kg) and mice (1.99 L/kg) were much larger than

total blood volumes of the respective murine species [60], indi-

cating an extravascular distribution in both rodents. Plasma clear-

ance (Cl) rates in rats and mice were found to be 1.39 and 3.78 L/

h$kg, respectively; both values were not high relative to hepatic

blood ﬂow rates of the corresponding rodent species [60]. In par-

allel, after oral dosing of 38, maximal plasma concentrations (C_max

)

of 2.77 M in rats and 5.01 M in mice were reached at 2.75 and

m

0.42 h, respectively, while values of half-life (T_1/2) were calculated

to be 2.24 h in rats and 1.11 h in mice. The oral bioavailability (F) of

38 was 38% in rats and 33% in mice, achieving an area under the

plasma concentration versus time curve (AUC) value of 14.6 and

2.30

mM$h, respectively. In comparison with 38, administration of

FACS analysis of splenocytes showed that no obvious changes in

the percentages of dead cells, B cells, dendritic cells, granulocytes,

monocytes/macrophages and neutrophils were detected when

CCT251545 to rats gave rise to a higher V_dss(1.38 L/kg) and a lower

Cl (1.20 L/h$kg) in the IV route, but a larger AUC (17.5

PO method, yielding a marginally higher F (41%).

mM$h) in the

Fig. 6. Pathological changes to growth plates of left femora and lungs in mice after the treatment of vehicle (1% CMC, 10 mL/kg), 38 (70 mg/kg), or CCT251545 (70 mg/kg).

12

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

either drug-treated group was compared to the vehicle-dosed

group, and that a slight increase in the percentage of CD3 T cells

was observed in the CCT251545-treated mouse in comparison with

vehicle-dosed mice (Fig. S3). Flow cytometric assessment of bone

marrows revealed that there were no apparent differences in the

percentages of dead cells, B cells and CD3 T cells among three

treatment groups (Fig. S4). Overall, the treatment of 38 elicited little

changes in immune cell subpopulations.

and a solution of hexachloroethane (2.37 g, 10.0 mmol) in anhy-

drous THF (10 mL) added. The reaction mixture was stirred for 1 h,

warmed up to room temperature, quenched with saturated

aqueous NH₄Cl solution (30 mL), and extracted with DCM

(3 ꢂ 100 mL). The organic extracts were combined and concen-

trated under reduced pressure, and the residue was puriﬁed by

Biotage® FlashMaster Personal^þﬂash chromatography (petroleum

benzine ramping to petroleum benzine:DCM ¼ 1:1) to give 2 as a

yellow solid (735 mg, 65%). R_F(DCM:petroleum benzine ¼ 1:4) 0.27.

3. Conclusions

¹H NMR (CDCl₃)

¹³C NMR (CDCl₃)

d

8.53 (s, 1H, pyridinyl-H), 8.62 (s, 1H, pyridinyl-H).

122.1, 131.8, 142.5, 148.5, 150.5.

1-(3-bromo-5-chloropyridin-4-yl)-1H-pyrrole-3-

d

Capitalising on our in-house virtual screening results and the

external discovery of CCT251545, a series of new multi-substituted

pyridines was chemically synthesised and biologically evaluated. A

close analysis of the structure-activity relationship of these com-

pounds disclosed multiple potent CDK8 inhibitors, with 38 and 39

topping the rest. Their capability of inhibiting the kinase activity of

cellular CDK8 was exempliﬁed by the use of 38 in MV4-11 AML

cells. While 38 and 39 displayed the selectivity for CDK8 in a

screening against a panel of 62 kinases, both compounds exerted

their potent anti-proliferative effects selectively on MV4-11 cell

line. In vivo pharmacokinetic and toxicity studies demonstrated

that 38 was not only orally bioavailable in rodents but also non-

toxic towards the majority of murine organs and tissues.

Methyl

carboxylate (3)

To a solution of chloride 2 (227 mg, 1.00 mmol) and methyl 1H-

pyrrole-3-carboxylate (126 mg, 1.01 mmol) in 1,4-dioxane (10 mL)

was added Cs₂CO₃(652 mg, 2.00 mmol). The reaction mixture was

heated under microwave irradiation at 200 ^ꢁC for 1 h, cooled down,

diluted with distilled H₂O (30 mL), and extracted with DCM

(3 ꢂ 30 mL). The organic extracts were combined and concentrated

under reduced pressure, and the residue was puriﬁed by Biotage®

FlashMaster Personal^þﬂash chromatography (petroleum benzine

ramping to petroleum benzine:EtOAc ¼ 85:15) to give 3 as a white

solid (152 mg, 48%). R_F(EtOAc:petroleum benzine ¼ 1:4) 0.41. ¹H

NMR (CDCl₃)

6.82 (dd, 1H, J 3.0 & 1.5, pyrrolyl-H), 7.37 (app t, 1H, J 1.5,

pyrrolyl-H), 8.68 (s, 1H, pyridinyl-H), 8.78 (s, 1H, pyridinyl-H). ¹³

NMR (CDCl₃) 51.5, 111.6, 118.5, 120.5, 122.4, 126.4, 130.6, 144.1,

d 3.85 (s, 3H, CH₃), 6.69 (app t, 1H, J 3.0, pyrrolyl-H),

4. Experimental

C

d

4.1. Chemistry

149.7, 151.6, 164.7. HRMS (ESIþ) 314.9540, 316.9519 & 318.9489

[MþH]^þ; calcd. for C₁₁H₉BrClN₂O^þ₂314.9531, 316.9510 & 318.9481

[MþH]^þ.

General synthetic procedure A

To a suspension of a bromide (1.00 equiv.), a boronic acid or a

boronate ester (1.05e1.50 equiv.) and Pd(dppf)Cl₂$CH₂Cl₂(0.05

equiv.) in CH₃CN (150 mM in bromide) was added 0.5 M aqueous

Na₂CO₃solution (1.40 equiv.). The reaction mixture was heated

under microwave irradiation at 120e140 ^ꢁ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 Personal^þﬂash chromatography (and,

when necessary, triturated or crystallised) to give the desired 5-

substituted pyridine.

4-(4-Chlorophenyl)-1-methyl-1H-pyrazole (5)

To a suspension of 1-chloro-4-iodobenzene (4, 7.16 g, 30.0 mmol)

and 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-

pyrazole (6.25 g, 30.0 mmol) in THF/H₂O (98 mL/33 mL) were added

Na₂CO₃(7.00 g, 66.0 mmol) and Pd(dppf)Cl₂$CH₂Cl₂(2.45 g,

3.00 mmol). The reaction mixture was heated at 80 ^ꢁC overnight,

cooled down and concentrated under reduced pressure. The residue

was puriﬁed by ﬂash column chromatography (petroleum benzine

ramping to petroleum benzine:EtOAc ¼ 1:1), and crystallised with

DCM/n-hexane to give 5 as a brown solid (3.37 g, 58%). R_F(EtOAc:-

petroleum benzine ¼ 1:1) 0.33. ¹H NMR (CDCl₃)

d 3.93 (s, 3H, CH₃),

7.30 (d, 2H, J 8.5, 2 ꢂ phenyl-H), 7.37 (d, 2H, J 8.5, 2 ꢂ phenyl-H), 7.57

General synthetic procedure B

To a solution of a carboxylic acid (1.00 equiv.) in anhydrous DMF

(100 mM in carboxylic acid) on an ice-salt bath were added DIPEA

(3.00 equiv.) and HATU (1.10 equiv.). The reaction mixture was

stirred for 10 min, and an amine (5.00 equiv.) added. The reaction

mixture was warmed up to room temperature, stirred overnight,

and diluted with distilled H₂O. The precipitate was collected by

centrifugation, puriﬁed by Biotage® FlashMaster Personal^þﬂash

chromatography, and triturated or crystallised to give the desired

amide.

(s,1H, pyrazolyl-H), 7.72 (s,1H, pyrazolyl-H).¹³C NMR (CDCl₃)

d 39.2,

122.3, 126.8, 127.1, 129.1, 131.2, 132.0, 136.8 (two carbon signals

overlapping or obscured). HRMS (ESIþ) 193.0528 [M(³⁵Cl)þH]^þ

&

195.0498 [M(³⁷Cl)þH]^þ; calcd. for C₁₀H₁₀ClN^þ₂193.0528 [M(³⁵Cl)þ

H]^þ& 195.0498 [M(³⁷Cl)þH]^þ.

1-Methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)

phenyl)-1H-pyrazole (6)

To

a suspension of chloride 5 (3.37 g, 17.5 mmol) and

4,4,4⁰,4⁰,5,5,5⁰,5⁰-octamethyl-2,2⁰-bi(1,3,2-dioxaborolane) (5.33 g,

21.0 mmol) in 1,4-dioxane (35 mL) were added AcOK (5.15 g,

General synthetic procedure C

To a suspension of LiOH (5.00 equiv.) in THF/CH₃OH (1:1,

250 mM in LiOH) was added a phenylsulfonyl-protected compound

(1.00 equiv.). The reaction mixture was heated at 50 ^ꢁC for 1 h,

cooled down, diluted with distilled H₂O, and extracted with DCM

(2 ꢂ). The organic extracts were combined and concentrated under

reduced pressure, and the residue was puriﬁed by Biotage®

FlashMaster Personal^þﬂash chromatography to give the desired

unprotected form.

52.5 mmol), Pd₂(dba)₃(320 mg, 350 mmol) and XPhos (667 mg,

1.40 mmol). The reaction mixture was heated at 85 ^ꢁC overnight,

cooled down, and partitioned between distilled H₂O (250 mL) and

EtOAc (250 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 Personal^þﬂash chromatography (DCM

ramping to DCM:EtOAc ¼ 9:1) to give 6 as a white solid (4.14 g,

83%). R_F(EtOAc:petroleum benzine ¼ 1:1) 0.50. ¹H NMR (CDCl₃)

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,

d

1.35 (s, 12H, 4 ꢂ dioxaborolanyl-CH₃), 3.94 (s, 3H, NCH₃), 7.47 (d,

13

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

2H, J 8.5, 2 ꢂ phenyl-H), 7.65 (s, 1H, pyrazolyl-H), 7.79 (d, 2H, J 8.0,

[M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method A: t_R10.73 min, purity >98%;

Method B: t_R9.13 min, purity >99%.

1-(3-Chloro-5-(4-(1-methyl-1H-pyrazol-4-yl)phenyl)pyridin-

4-yl)-N-methyl-1H-pyrrole-3-carboxamide (10)

2 ꢂ phenyl-H), 7.80 (s, 1H, pyrazolyl-H). ¹³C NMR (CDCl₃)

d 25.0,

39.2, 83.9, 123.2, 124.8, 127.4, 135.5, 135.6, 137.1 (seven carbon

signals overlapping or obscured). HRMS (ESIþ) 285.1772 [MþH]^þ;

calcd. for C₁₆H₂₂BN₂O^þ₂285.1769 [MþH]^þ.

Carboxylic acid 8 (152 mg, 401 mmol) and CH₃NH₂$HCl (136 mg,

Methyl 1-(3-chloro-5-(4-(1-methyl-1H-pyrazol-4-yl)phenyl)

pyridin-4-yl)-1H-pyrrole-3-carboxylate (7)

2.01 mmol) were coupled using general synthetic procedure B. The

precipitate was collected by centrifugation, puriﬁed by Biotage®

FlashMaster Personal^þﬂash chromatography (DCM ramping to

DCM:EtOH ¼ 94:6), and crystallised with DCM/n-hexane to give 10

as white crystals (110 mg, 70%). R_F(DCM:EtOH ¼ 94:6) 0.39. m.p.

Bromide 3 (140 mg, 444

468

mmol) and boronate ester 6 (133 mg,

m

mol) were reacted at 120 ^ꢁC using general synthetic proced-

ure A. The residue was puriﬁed by Biotage® FlashMaster Personal^þ

ﬂash chromatography (DCM ramping to DCM:EtOAc ¼ 7:3) to give 7

as a yellowish glue (117 mg, 67%). R_F(DCM:EtOAc ¼ 3:2) 0.28. ¹H

210e211 ^ꢁC. ¹H NMR (DMSO‑d₆)

d 2.66 (d, 3H, J 4.0, NHCH₃), 3.84 (s,

3H, pyrazolyl-CH₃), 6.54 (app s,1H, pyrrolyl-H), 6.73 (app t,1H, J 2.5,

pyrrolyl-H), 7.13 (d, 2H, J 8.0, 2 ꢂ phenyl-H), 7.30 (app s, 1H,

pyrrolyl-H), 7.52 (d, 2H, J 8.0, 2 ꢂ phenyl-H), 7.85 (q, 1H, J 4.5,

NHCH₃), 7.88 (s, 1H, pyrazolyl-H), 8.16 (s, 1H, pyrazolyl-H), 8.72 (s,

1H, pyridinyl-H), 8.85 (s, 1H, pyridinyl-H). ¹³C NMR (DMSO‑d₆)

NMR (CDCl₃)

d

3.76 (s, 3H), 3.91 (s, 3H) (total 6H, 2 ꢂ CH₃), 6.47 (app

t, 1H, J 2.0, pyrrolyl-H), 6.61 (dd, 1H, J 2.5 & 1.0, pyrrolyl-H), 7.02 (d,

2H, J 8.0, 2 ꢂ phenyl-H), 7.28 (app t, 1H, J 1.5, pyrrolyl-H), 7.38 (d,

2H, J 8.0, 2 ꢂ phenyl-H), 7.59 (s, 1H, pyrazolyl-H), 7.73 (s, 1H,

pyrazolyl-H), 8.63 (s, 1H, pyridinyl-H), 8.71 (s, 1H, pyridinyl-H). ¹³

C

d 25.6, 38.8, 108.9,121.1, 121.7, 123.2, 124.3, 125.0, 128.2, 128.7, 128.8,

NMR (CDCl₃)

d

39.2, 51.3, 111.2, 118.0, 122.2, 123.2, 125.9, 127.0,

130.9, 132.9, 135.3, 136.3, 142.1, 149.0, 150.0, 163.6 (two carbon

signals overlapping or obscured). HRMS (ESIþ) 392.1280 [M(³⁵Cl)þ

H]^þ& 394.1255 [M(³⁷Cl)þH]^þ; calcd. for C₂₁H₁₉ClN₅O^þ392.1273

[M(³⁵Cl)þH]^þ& 394.1244 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method A:

t_R10.93 min, purity >98%; Method B: t_R9.40 min, purity >99%.

8-(3-Bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-

1-one (11)

127.2,128.6,129.0,131.1,133.3,135.4,136.9,142.0,149.6,150.0,164.8

(two carbon signals overlapping or obscured). HRMS (ESIþ)

393.1112 [M(³⁵Cl)þH]^þ

&

395.1085 [M(³⁷Cl)þH]^þ; calcd. for

C

₂₁H₁₈ClN₄O₂^þ393.1113 [M(³⁵Cl)þH]^þ& 395.1084 [M(³⁷Cl)þH]^þ.

1-(3-Chloro-5-(4-(1-methyl-1H-pyrazol-4-yl)phenyl)pyridin-

4-yl)-1H-pyrrole-3-carboxylic acid (8)

To a solution of ester 7 (110 mg, 280

mmol) in CH₃OH (2.8 mL)

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 triethylamine (2.51 mL,

18.0 mmol). The reaction mixture was heated under 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 Personal^þﬂash chromatography (DCM ramping to

DCM:EtOH ¼ 96:4) to give 11 as a beige solid (1.63 g, 79%). R_F

was added 1 M aqueous NaOH solution (2.80 mL, 2.80 mmol). The

reaction mixture was heated at reﬂux for 2 h, cooled down, diluted

with distilled H₂O (30 mL), washed with EtOAc (15 mL), taken to pH

2 with 2 M aqueous HCl solution, and extracted with DCM

(3 ꢂ 15 mL). The organic extracts were combined and concentrated

under reduced pressure, and the residue was puriﬁed by Biotage®

FlashMaster Personal^þﬂash chromatography (DCM ramping to

DCM:CH₃OH ¼ 91:9) to give 8 as a white solid (101 mg, 95%). R_F

(DCM:CH₃OH ¼ 94:6) 0.27. m.p. 253e254 ^ꢁC. ¹H NMR (DMSO‑d₆)

(DCM:EtOH ¼ 96:4) 0.31. ¹H NMR (CDCl₃)

d 1.54 (d, 2H, J 13.0, 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, 1H,

d

3.84 (s, 3H, CH₃), 6.46 (dd, 1H, J 2.5 & 1.5, pyrrolyl-H), 6.80 (app t,

1H, J 3.0, pyrrolyl-H), 7.14 (d, 2H, J 8.0, 2 ꢂ phenyl-H), 7.43 (app t,1H,

J 1.5, pyrrolyl-H), 7.53 (d, 2H, J 8.0, 2 ꢂ phenyl-H), 7.88 (s, 1H,

pyrazolyl-H), 8.16 (s, 1H, pyrazolyl-H), 8.72 (s, 1H, pyridinyl-H), 8.85

(s, 1H, pyridinyl-H) (one carboxylic acid proton signal (COOH) not

pyridinyl-H), 8.49 (s, 1H, pyridinyl-H). ¹³C NMR (CDCl₃)

d 31.5, 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 (ESIþ) 344.0167, 346.0149

& 348.0118 [MþH]^þ; calcd. for C₁₃H₁₆BrClN₃O^þ344.0160, 346.0140

& 348.0110 [MþH]^þ.

observed). ¹³C NMR (DMSO‑d₆)

d 38.8, 110.7, 118.0, 121.1, 123.9,

125.0, 127.5, 128.3, 128.8, 128.9, 130.9, 133.0, 135.5, 136.3, 142.0,

149.0, 149.9, 165.2 (two carbon signals overlapping or obscured).

HRMS (ESIþ) 379.0959 [M(³⁵Cl)þH]^þ& 381.0932 [M(³⁷Cl)þH]^þ;

8-(3-Chloro-5-(4-(1-methyl-1H-pyrazol-4-yl)phenyl)pyridin-

4-yl)-2,8-diazaspiro[4.5]decan-1-one (CCT251545)

calcd. for

C

₂₀H₁₆ClN₄O^þ₂379.0957 [M(³⁵Cl)þH]^þ

&

381.0927

Bromide 11 (345 mg, 1.00 mmol) and boronate ester 6 (299 mg,

1.05 mmol) were reacted at 120 ^ꢁC using general synthetic pro-

cedure A. The residue was puriﬁed by Biotage® FlashMaster Per-

[M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method A: t_R16.13 min, purity >97%;

Method B: t_R9.60 min, purity >97%.

1-(3-Chloro-5-(4-(1-methyl-1H-pyrazol-4-yl)phenyl)pyridin-

4-yl)-1H-pyrrole-3-carboxamide (9)

sonal^þ

ﬂash

chromatography

(DCM

ramping

to

DCM:CH₃OH ¼ 96:4), and triturated with EtOAc (3 mL) to give

Carboxylic acid 8 (152 mg, 401 mmol) and NH₃(0.5 M in 1,4-

CCT251545 as a white solid (300 mg, 71%). R_F(DCM:CH₃OH ¼ 96:4)

dioxane, 4.00 mL, 2.00 mmol) were coupled using general syn-

thetic procedure B. The precipitate was collected by centrifugation,

puriﬁed by Biotage® FlashMaster Personal^þﬂash chromatography

(DCM ramping to DCM:CH₃OH ¼ 94:6), and triturated with n-

hexane (3 mL) to give 9 as a white solid (111 mg, 73%). R_F

(DCM:CH₃OH ¼ 9:1) 0.47. m.p. 245e246 ^ꢁC. ¹H NMR (DMSO‑d₆)

0.20. m.p. 216e217 ^ꢁC. ¹H NMR (CDCl₃)

d 1.35 (app d, 2H, J 13.0,

CH₂),1.94e1.99 (m, 2H, CH₂),1.97 (t, 2H, J 6.5, CH₂), 2.74 (t, 2H, J 11.0,

CH₂), 3.16 (dt, 2H, J 13.0 & 4.0, CH₂), 3.28 (t, 2H, J 6.5, CH₂), 3.96 (s,

3H, CH₃), 5.96 (br s,1H, CONH), 7.28 (d, 2H, J 8.0, 2 ꢂ phenyl-H), 7.56

(d, 2H, J 8.0, 2 ꢂ phenyl-H), 7.68 (s, 1H, pyrazolyl-H), 7.82 (s, 1H,

pyrazolyl-H), 8.21 (s, 1H, pyridinyl-H), 8.43 (s, 1H, pyridinyl-H). ¹³

C

d

3.84 (s, 3H, CH₃), 6.55 (dd, 1H, J 2.5 & 1.5, pyrrolyl-H), 6.74 (app t,

NMR (CDCl₃) d 31.7, 32.4, 38.7, 39.3, 41.8, 47.8, 122.7, 125.6, 127.2,

1H, J 2.5, pyrrolyl-H), 6.84 (br s, 1H, CONHH), 7.13 (d, 2H, J 8.0,

2 ꢂ phenyl-H), 7.32 (app t, 1H, J 1.5, pyrrolyl-H), 7.39 (br s, 1H,

CONHH), 7.52 (d, 2H, J 8.0, 2 ꢂ phenyl-H), 7.88 (s, 1H, pyrazolyl-H),

8.16 (s, 1H, pyrazolyl-H), 8.72 (s, 1H, pyridinyl-H), 8.85 (s, 1H,

128.3, 129.8, 132.3, 133.7, 135.7, 136.9, 149.6, 150.9, 152.9, 181.7 (four

carbon signals overlapping or obscured). HRMS (ESIþ) 422.1750

[M(³⁵Cl)þH]^þ& 424.1724 [M(³⁷Cl)þH]^þ; calcd. for C₂₃H₂₅ClN₅O^þ

422.1742 [M(³⁵Cl)þH]^þ& 424.1713 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC

Method A: t_R14.55 min, purity >99%; Method B: t_R8.41 min, pu-

rity >98%.

pyridinyl-H). ¹³C NMR (DMSO‑d₆)

d 38.8, 109.6, 121.1, 121.6, 123.2,

124.9, 125.0, 128.3, 128.7, 128.8, 130.9, 132.9, 135.4, 136.3, 142.1,

149.0, 150.0, 165.1 (two carbon signals overlapping or obscured).

HRMS (ESIþ) 378.1125 [M(³⁵Cl)þH]^þ& 380.1099 [M(³⁷Cl)þH]^þ;

8-(3-Chloro-5-((1-methyl-1H-indazol-5-yl)amino)pyridin-4-

yl)-2,8-diazaspiro[4.5]decan-1-one (12)

calcd. for

C

₂₀H₁₇ClN₅O^þ378.1117 [M(³⁵Cl)þH]^þ

&

380.1087

To a suspension of bromide 11 (345 mg, 1.00 mmol) and 1-

14

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

methyl-1H-indazol-5-amine (148 mg, 1.00 mmol) in 1,4-dioxane

118.4, 118.8, 125.9, 126.6, 126.8, 128.2, 129.7, 139.4, 141.1, 148.8,

150.3, 152.2, 180.1 (four carbon signals overlapping or obscured).

HRMS (ESIþ) 408.1586 [M(³⁵Cl)þH]^þ& 410.1558 [M(³⁷Cl)þH]^þ;

(10 mL) were added Pd₂(dba)₃(45.8 mg, 50.0

mmol), Xantphos

(28.9 mg, 50.1 mol) and Cs₂CO₃(652 mg, 2.00 mmol). The reaction

m

mixture was heated under microwave irradiation at 180 ^ꢁC for 1 h,

cooled down, and partitioned between distilled H₂O (40 mL) and

DCM (40 mL). The organic layer was separated, and the aqueous

layer extracted with DCM (2 ꢂ 40 mL). The organic layer and ex-

tracts were combined and concentrated under reduced pressure,

and the residue was puriﬁed by Biotage® FlashMaster Personal^þ

ﬂash chromatography (EtOAc ramping to EtOAc:CH₃OH ¼ 96:4),

and triturated with EtOAc/n-hexane (1 mL/2 mL) to give 12 as a

beige solid (103 mg, 25%). R_F(EtOAc:CH₃OH ¼ 95:5) 0.22. m.p.

calcd. for

C

₂₂H₂₃ClN₅O^þ408.1586 [M(³⁵Cl)þH]^þ

& 410.1556

[M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method A: t_R11.40 min, purity >97%;

Method B: t_R9.14 min, purity >99%.

8-(3-(1-Benzyl-1H-pyrazol-4-yl)-5-chloropyridin-4-yl)-2,8-

diazaspiro[4.5]decan-1-one (14)

Bromide 11 (173 mg, 502

mmol) and 1-benzyl-4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole

(157 mg,

552 m

mol) were reacted at 120 ^ꢁC using general synthetic proced-

ure A. The residue was puriﬁed by Biotage® FlashMaster Personal^þ

ﬂash chromatography (DCM ramping to DCM:CH₃OH ¼ 96:4),

triturated with n-hexane (3 mL), and washed with boiling EtOAc

203e204 ^ꢁC. ¹H NMR (CDCl₃)

d 1.60 (app d, 2H, J 13.0, CH₂), 2.09 (td,

2H, J 12.5 & 4.0, CH₂), 2.22 (t, 2H, J 6.5, CH₂), 3.04 (app br s, 2H, CH₂),

3.40 (t, 2H, J 7.0, CH₂), 3.51 (app br s, 2H, CH₂), 4.09 (s, 3H, CH₃), 5.87

(br s,1H), 6.84 (br s,1H) (total 2H, CONH & pyridinyl-NH-indazolyl),

7.24 (dd, 1H, J 9.0 & 2.0, indazolyl-H), 7.39 (d, 1H, J 8.5, indazolyl-H),

7.53 (s,1H, indazolyl-H), 7.92 (app s, 2H, indazolyl-H & pyridinyl-H),

(5 mL) to give 14 as

a beige solid (70 mg, 33%). R_F

(DCM:CH₃OH ¼ 95:5) 0.29. m.p. 167e168 ^ꢁC. ¹H NMR (DMSO‑d₆)

d

1.25 (app d, 2H, J 12.5, CH₂), 1.75 (td, 2H, J 12.0 & 4.0, CH₂), 1.82 (t,

2H, J 6.5, CH₂), 2.82 (t, 2H, J 10.5, CH₂), 2.94 (app d, 2H, J 12.5, CH₂),

3.14 (t, 2H, J 6.5, CH₂), 5.39 (s, 2H, phenyl-CH₂), 7.28e7.38 (m, 5H,

5 ꢂ phenyl-H), 7.57 (s, 1H, CONH), 7.70 (s, 1H, pyrazolyl-H), 8.07 (s,

1H, pyrazolyl-H), 8.30 (s, 1H, pyridinyl-H), 8.37 (s, 1H, pyridinyl-H).

8.20 (s, 1H, pyridinyl-H). ¹³C NMR (CDCl₃)

d 31.1, 33.3, 35.9, 38.9,

42.0, 45.5, 110.2, 113.8, 124.0, 124.7, 129.8, 132.4, 133.6, 133.9, 137.8,

140.4, 140.6, 141.3, 181.7 (two carbon signals overlapping or

obscured). HRMS (ESIþ) 411.1697 [M(³⁵Cl)þH]^þ

&

413.1680

¹³C NMR (DMSO‑d₆)

d 30.4, 31.8, 37.9, 41.3, 46.7, 55.0, 116.7, 126.5,

[M(³⁷Cl)þH]^þ; calcd. for C₂₁H₂₄ClN₆O^þ411.1695 [M(³⁵Cl)þH]^þ

&

127.8, 128.2, 128.6, 130.1, 137.4, 138.8, 148.3, 150.4, 152.2, 180.1 (ﬁve

carbon signals overlapping or obscured). HRMS (ESIþ) 422.1742

[M(³⁵Cl)þH]^þ& 424.1719 [M(³⁷Cl)þH]^þ; calcd. for C₂₃H₂₅ClN₅O^þ

422.1742 [M(³⁵Cl)þH]^þ& 424.1713 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC

Method A: t_R11.16 min, purity >95%; Method B: t_R8.69 min, pu-

rity >98%.

413.1665 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method A: t_R14.97 min,

purity >98%; Method B: t_R8.58 min, purity >98%.

1-Phenyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-

1H-pyrazole

To

10.0

a

solution of 4-bromo-1-phenyl-1H-pyrazole (2.24 g,

mmol) and

4,4,4⁰,4⁰,5,5,5⁰,5⁰-octamethyl-2,2⁰-bi(1,3,2-

8-(3-Chloro-5-(1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-

yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (15)

dioxaborolane) (2.79 g, 11.0 mmol) in DMSO (25 mL) were added

AcOK (2.95 g, 30.1 mmol) and Pd(dppf)Cl₂$CH₂Cl₂(409 mg,

Bromide 11 (173 mg, 502

yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole

(167 mg, 600

mol) were reacted at 135 ^ꢁC using general synthetic

procedure A. The residue was puriﬁed by Biotage® FlashMaster

Personal^þ

ﬂash chromatography (DCM ramping to

mmol) and 1-(tetrahydro-2H-pyran-4-

501 m

mol). The reaction mixture was heated under N₂at 80 ^ꢁC for

20 h, cooled down, and partitioned between distilled H₂O (250 mL)

and EtOAc (250 mL). The mixture was ﬁltered through a pad of

Celite®, and the solids were washed with EtOAc (250 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,

washed with distilled H₂O (2 ꢂ 100 mL) and brine (100 mL), and

concentrated under reduced pressure. The residue was puriﬁed by

Biotage® FlashMaster Personal^þﬂash chromatography (DCM

ramping to DCM:CH₃OH ¼ 98:2) to give 1-phenyl-4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole as a white solid

(1.16 g, 43%). R_F(EtOAc:petroleum benzine ¼ 1:9) 0.28. ¹H NMR

m

DCM:EtOH ¼ 94:6) to give 15 as a beige solid (133 mg, 64%). R_F

(DCM:EtOH ¼ 94:6) 0.21. m.p. 215e216 ^ꢁC. ¹H NMR (DMSO‑d₆)

d

1.31 (app d, 2H, J 12.5, CH₂), 1.65e1.85 (m, 2H, CH₂), 1.94 (t, 2H, J

6.5, CH₂), 1.96e2.15 (m, 4H, 2 ꢂ CH₂), 2.96 (app br s, 4H, 2 ꢂ CH₂),

3.17 (t, 2H, J 6.5, CH₂), 3.49 (t, 2H, J 11.5, CH₂), 3.97 (d, 2H, J 10.5,

CH₂), 4.35e4.55 (m, 1H, tetrahydropyranyl-CH), 7.56 (s, 1H), 7.72 (s,

1H), 8.10 (s, 1H), 8.34 (s, 1H), 8.36 (s, 1H) (total 5H, CONH &

2 ꢂ pyrazolyl-H & 2 ꢂ pyridinyl-H). ¹³C NMR (DMSO‑d₆)

d 30.6, 31.8,

32.9, 37.9, 41.2, 46.6, 57.4, 65.9, 115.9, 127.0, 127.6, 128.3, 138.0,

148.3, 150.1, 152.1, 180.1 (four carbon signals overlapping or

(CDCl₃)

2H, J 8.0, 2 ꢂ phenyl-H), 7.70 (d, 2H, J 7.5, 2 ꢂ phenyl-H), 7.98 (s, 1H,

pyrazolyl-H), 8.24 (s, 1H, pyrazolyl-H). ¹³C NMR (CDCl₃)

25.0, 83.7,

d

1.35 (s, 12H, 4 ꢂ CH₃), 7.30 (t, 1H, J 7.5, phenyl-H), 7.45 (t,

obscured). HRMS (ESIþ) 416.1855 [M(³⁵Cl)þH]^þ

& 418.1813

d

[M(³⁷Cl)þH]^þ; calcd. for C₂₁H₂₇ClN₅O^þ₂416.1848 [M(³⁵Cl)þH]^þ

&

119.6, 126.9, 129.6, 133.8, 140.0, 146.9 (seven carbon signals over-

418.1818 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method C: t_R14.89 min,

purity >99%; Method D: t_R10.00 min, purity >99%.

Tert-butyl 4-(4-(5-chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-

8-yl)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate

(16)

lapping or obscured). HRMS (ESIþ) 271.1621 [MþH]^þ; calcd. for

C

₁₅H₂₀BN₂O^þ₂271.1613 [MþH]^þ.

8-(3-Chloro-5-(1-phenyl-1H-pyrazol-4-yl)pyridin-4-yl)-2,8-

diazaspiro[4.5]decan-1-one (13)

Bromide 11 (173 mg, 502

m

mol) and 1-phenyl-4-(4,4,5,5-

Bromide 11 (173 mg, 502

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)piperidine-

1-carboxylate (208 mg, 551

mol) were reacted at 120 ^ꢁC using

mmol) and tert-butyl 4-(4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole

552

(149 mg,

m

mol) were reacted at 120 ^ꢁC using general synthetic proced-

m

ure A. The residue was puriﬁed by Biotage® FlashMaster Personal^þ

ﬂash chromatography (DCM ramping to DCM:EtOH ¼ 96.5:3.5),

and washed with EtOAc (6 mL) to give 13 as a pinkish solid (90 mg,

44%). R_F(DCM:CH₃OH ¼ 95:5) 0.34. m.p. 225e226 ^ꢁC. ¹H NMR

general synthetic procedure A. The residue was puriﬁed by Bio-

tage® FlashMaster Personal^þﬂash chromatography (DCM ramping

to DCM:CH₃OH ¼ 96:4), and crystallised with DCM/n-hexane to

give 16 as a tan solid (80 mg, 31%). R_F(DCM:CH₃OH ¼ 95:5) 0.29.

(DMSO‑d₆)

d

1.35 (app d, 2H, J 12.5, CH₂), 1.75e1.88 (m, 2H, CH₂),

m.p. 179e180 ^ꢁC. ¹H NMR (DMSO‑d₆)

d 1.30 (app d, 2H, J 13.0, CH₂),

1.96 (t, 2H, J 7.0, CH₂), 2.95e3.12 (m, 4H, 2 ꢂ CH₂), 3.16 (t, 2H, J 7.0,

CH₂), 7.34 (t, 1H, J 7.5, phenyl-H), 7.53 (t, 2H, J 7.5, phenyl-H), 7.57 (s,

1H, CONH), 7.93 (d, 2H, J 8.0, phenyl-H), 8.01 (s, 1H, pyrazolyl-H),

8.42 (s, 1H), 8.44 (s, 1H), 8.80 (s, 1H) (total 3H, pyrazolyl-H &

1.41 (s, 9H, C(CH₃)₃), 1.66e1.88 (m, 4H, 2 ꢂ CH₂) 1.94 (t, 2H, J 6.5,

CH₂), 2.08 (app d, 2H, J 11.0, CH₂), 2.70e3.06 (m, 6H, 3 ꢂ CH₂), 3.17

(t, 2H, J 6.5, CH₂), 4.04 (br d, 2H, J 9.5, CH₂), 4.36e4.46 (m, 1H, CH),

7.56 (s, 1H, CONH), 7.70 (s, 1H, pyrazolyl-H), 8.08 (s, 1H, pyrazolyl-

H), 8.32 (s, 1H, pyridinyl-H), 8.36 (s, 1H, pyridinyl-H). ¹³C NMR

2 ꢂ pyridinyl-H). ¹³C NMR (DMSO‑d₆)

d 30.6, 31.8, 37.9, 41.3, 46.8,

15

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

(DMSO‑d₆)

d

28.1, 30.6, 31.8, 32.0, 38.0, 41.3, 42.0, 42.7, 46.6, 58.3,

41.3, 46.4, 100.0, 111.4, 120.4, 121.0, 126.1, 127.1, 127.2, 128.9, 132.2,

135.9, 148.6, 150.8, 153.1, 179.9 (two carbon signals overlapping or

79.0, 116.0, 127.0, 127.7, 128.3, 138.1, 148.3, 150.1, 152.2, 153.9, 180.1

(ﬁve carbon signals overlapping or obscured). HRMS (ESIþ)

obscured). HRMS (ESIþ) 381.1484 [M(³⁵Cl)þH]^þ

& 383.1442

515.2529 [M(³⁵Cl)þH]^þ

&

517.2504 [M(³⁷Cl)þH]^þ; calcd. for

[M(³⁷Cl)þH]^þ; calcd. for C₂₁H₂₂ClN₄O^þ381.1477 [M(³⁵Cl)þH]^þ

&

C

₂₆H₃₆ClN₆O₃^þ515.2532 [M(³⁵Cl)þH]^þ& 517.2502 [M(³⁷Cl)þH]^þ.

383.1447 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method C: t_R14.57 min,

purity >99%; Method D: t_R10.54 min, purity >99%.

8-(3-Chloro-5-(isoquinolin-4-yl)pyridin-4-yl)-2,8-diazaspiro

[4.5]decan-1-one (20)

Anal. RP-HPLC Method A: t_R11.58 min, purity >97%; Method B: t_R

9.31 min, purity >99%.

8-(3-Chloro-5-(1-methyl-1H-indazol-4-yl)pyridin-4-yl)-2,8-

diazaspiro[4.5]decan-1-one (17)

Bromide 11 (173 mg, 502

mmol) and isoquinolin-4-ylboronic

Bromide 11 (173 mg, 502

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole

554

m

mol) and 1-methyl-4-(4,4,5,5-

acid (95.5 mg, 552

m

mol) were reacted at 120 ^ꢁC using general

(143 mg,

synthetic procedure A. The residue was puriﬁed by Biotage®

FlashMaster Personal^þﬂash chromatography (DCM ramping to

DCM:EtOH ¼ 93:7), and crystallised with EtOAc/DCM to give 20 as a

m

mol) were reacted at 120 ^ꢁC using general synthetic proced-

ure A. The residue was puriﬁed by Biotage® FlashMaster Personal^þ

ﬂash chromatography (DCM ramping to DCM:CH₃OH ¼ 96.5:3.5),

triturated with n-hexane (5 mL), and washed with EtOAc (5 mL) to

give 17 as a pinkish solid (76 mg, 38%). R_F(DCM:CH₃OH ¼ 94:6)

tan solid (70 mg, 36%). R_F(DCM:EtOH

¼

96:4) 0.14. m.p.

241e242 ^ꢁC. ¹H NMR (DMSO‑d₆)

d 1.03 (d, 2H, J 12.5, CH₂),1.40e1.50

(m, 2H, CH₂), 1.49 (t, 2H, J 6.5, CH₂), 2.24 (t, 1H, J 11.0, CHH), 2.47 (t,

1H, J 11.0, CHH), 2.97 (t, 2H, J 6.5, CH₂), 2.97e3.07 (m, 2H, CH₂), 7.42

(s, 1H, CONH), 7.57 (d, 1H, J 8.0, isoquinolinyl-H), 7.75 (t, 1H, J 8.0,

isoquinolinyl-H), 7.80 (t, 1H, J 8.0, isoquinolinyl-H), 8.23 (s, 1H), 8.24

(d, 1H, J 8.0), 8.44 (s, 1H), 8.59 (s, 1H), 9.40 (s, 1H) (total 5H,

0.37. m.p. 248e249 ^ꢁC. ¹H NMR (DMSO‑d₆)

d 1.12 (app d, 2H, J 12.5,

CH₂), 1.59 (app t, 2H, J 10.5, CH₂), 1.64 (t, 2H, J 6.5, CH₂), 2.46 (t, 2H, J

12.0, CH₂), 2.90e3.10 (m, 2H, CH₂), 3.04 (t, 2H, J 6.5, CH₂), 4.10 (s, 3H,

CH₃), 7.02 (d, 1H, J 6.5, indazolyl-H), 7.47 (s, 1H, CONH), 7.50 (t, 1H, J

7.5, indazolyl-H), 7.70 (d, 1H, J 8.5, indazolyl-H), 7.87 (s, 1H,

3 ꢂ isoquinolinyl-H & 2 ꢂ pyridinyl-H). ¹³C NMR (DMSO‑d₆)

d 30.2,

indazolyl-H), 8.23 (s, 1H, pyridinyl-H), 8.51 (s, 1H, pyridinyl-H). ¹³

C

31.6, 31.8, 37.7, 41.0, 46.5, 47.3, 124.3, 127.0, 127.8, 127.9, 128.0, 128.2,

128.4, 131.5, 134.1, 143.4, 150.1, 151.2, 152.8, 153.9, 179.7. HRMS

(ESIþ) 393.1476 [M(³⁵Cl)þH]^þ& 395.1446 [M(³⁷Cl)þH]^þ; calcd. for

NMR (DMSO‑d₆) d 30.3, 31.7, 35.6, 37.7, 41.2, 47.0, 109.5, 121.7, 123.1,

126.1, 127.1, 130.0, 130.6, 131.5, 139.7, 149.3, 150.6, 152.9, 179.8 (two

carbon signals overlapping or obscured). HRMS (ESIþ) 396.1590

[M(³⁵Cl)þH]^þ& 398.1560 [M(³⁷Cl)þH]^þ; calcd. for C₂₁H₂₃ClN₅O^þ

396.1586 [M(³⁵Cl)þH]^þ& 398.1556 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC

Method A: t_R10.51 min, purity >98%; Method B: t_R8.20 min, pu-

rity >99%.

C

₂₂H₂₂ClN₄O^þ393.1477 [M(³⁵Cl)þH]^þ& 395.1447 [M(³⁷Cl)þH]^þ.

Anal. RP-HPLC Method A: t_R10.76 min, purity >97%; Method B: t_R

8.16 min, purity >99%.

8-(3-Chloro-5-(quinolin-4-yl)pyridin-4-yl)-2,8-diazaspiro

[4.5]decan-1-one (21)

8-(3-Chloro-5-(2-methyl-2H-indazol-4-yl)pyridin-4-yl)-2,8-

diazaspiro[4.5]decan-1-one (18)

Bromide 11 (173 mg, 502

(95.5 mg, 552

mol) were reacted at 120 ^ꢁC using general synthetic

procedure A. The residue was puriﬁed by Biotage® FlashMaster

Personal^þ

ﬂash chromatography (DCM ramping to

DCM:CH₃OH ¼ 95:5), and washed with EtOAc (5 mL) and n-hexane

mmol) and quinolin-4-ylboronic acid

m

Bromide 11 (173 mg, 502

tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole

553

m

mol) and 2-methyl-4-(4,4,5,5-

(143 mg,

m

mol) were reacted at 120 ^ꢁC using general synthetic proced-

ure A. The residue was puriﬁed by Biotage® FlashMaster Personal^þ

ﬂash chromatography (DCM ramping to DCM:CH₃OH ¼ 96:4), and

triturated and washed with EtOAc (10 mL) to give 18 as a beige solid

(80 mg, 40%). R_F(DCM:CH₃OH ¼ 94:6) 0.36. m.p. 255e256 ^ꢁC. ¹H

(3 mL) to give 21 as a beige solid (136 mg, 69%). R_F

(DCM:CH₃OH ¼ 94:6) 0.39. m.p. 218e219 ^ꢁC. ¹H NMR (DMSO‑d₆)

d

1.04 (dd, 2H, J 16.0 & 14.5, CH₂), 1.45 (td, 2H, J 12.5 & 2.5, CH₂), 1.51

(t, 2H, J 6.5, CH₂), 2.23 (t, 1H, J 11.0, CHH), 2.53 (t, 1H, J 11.0, CHH),

2.98 (t, 2H, J 6.5, CH₂), 2.98e3.08 (m, 2H, CH₂), 7.42 (s, 1H, CONH),

7.50 (d, 1H, J 4.0, quinolinyl-H), 7.55e7.66 (m, 2H, 2 ꢂ quinolinyl-H),

7.77e7.87 (m, 1H, quinolinyl-H), 8.12 (d, 1H, J 8.5, quinolinyl-H),

8.21 (s, 1H, pyridinyl-H), 8.60 (s, 1H, pyridinyl-H), 8.99 (d, 1H, J

NMR (DMSO‑d₆) d 1.12 (app d, 2H, J 12.5, CH₂), 1.59 (app t, 2H, J 10.0,

CH₂), 1.66 (t, 2H, J 6.5, CH₂), 2.50e2.54 (m, 2H, CH₂), 2.90e3.10 (m,

2H, CH₂), 3.04 (t, 2H, J 6.5, CH₂), 4.14 (s, 3H, CH₃), 6.91 (d, 1H, J 7.0,

indazolyl-H), 7.34 (t, 1H, J 8.0, indazolyl-H), 7.47 (s, 1H, CONH), 7.63

(d, 1H, J 8.5, indazolyl-H), 8.24 (s, 1H), 8.25 (s, 1H) (total 2H,

indazolyl-H & pyridinyl-H), 8.49 (s, 1H, pyridinyl-H). ¹³C NMR

4.5, quinolinyl-H). ¹³C NMR (DMSO‑d₆)

d 30.4, 31.6, 31.8, 37.8, 41.1,

46.5, 47.3, 122.8, 125.6, 126.8, 127.0, 127.5, 128.7, 129.6, 130.0, 143.7,

147.9, 150.3, 150.4, 150.6, 153.4, 179.8. HRMS (ESIþ) 393.1476

[M(³⁵Cl)þH]^þ& 395.1451 [M(³⁷Cl)þH]^þ; calcd. for C₂₂H₂₂ClN₄O^þ

393.1477 [M(³⁵Cl)þH]^þ& 395.1447 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC

Method C: t_R17.90 min, purity >98%; Method D: t_R10.98 min,

purity >99%.

(DMSO‑d₆) d 30.4, 31.7, 37.8, 40.1, 41.2, 47.0, 116.6, 122.0, 122.1, 124.6,

125.4,127.0,129.6,131.3,148.1,149.2,150.4,152.8,179.9 (two carbon

signals overlapping or obscured). HRMS (ESIþ) 396.1587 [M(³⁵Cl)þ

H]^þ& 398.1559 [M(³⁷Cl)þH]^þ; calcd. for C₂₁H₂₃ClN₅O^þ396.1586

[M(³⁵Cl)þH]^þ& 398.1556 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method C:

t_R16.72 min, purity >98%; Method D: t_R10.30 min, purity >99%.

8-(3-Chloro-5-(1H-indol-4-yl)pyridin-4-yl)-2,8-diazaspiro

[4.5]decan-1-one (19)

8-(3-Chloro-5-(quinolin-5-yl)pyridin-4-yl)-2,8-diazaspiro

[4.5]decan-1-one (22)

Bromide 11 (173 mg, 502

(95.5 mg, 552

mol) were reacted at 120 ^ꢁC using general synthetic

procedure A. The residue was puriﬁed by Biotage® FlashMaster

Personal^þ

ﬂash chromatography (DCM ramping to

DCM:CH₃OH ¼ 94:6), and washed with EtOAc (5 mL) and n-hexane

mmol) and quinolin-5-ylboronic acid

Bromide 11 (173 mg, 502

mmol) and 4-(4,4,5,5-tetramethyl-

m

1,3,2-dioxaborolan-2-yl)-1H-indole (134 mg, 551

mmol) were

reacted at 120 ^ꢁC using general synthetic procedure A. The residue

was puriﬁed by Biotage® FlashMaster Personal^þﬂash chromatog-

raphy (DCM ramping to DCM:CH₃OH ¼ 95:5), and crystallised with

DCM/CH₃OH/EtOAc to give 19 as a pinkish solid (80 mg, 42%). R_F

(DCM:CH₃OH ¼ 94:6) 0.38. m.p. 296e297 ^ꢁC. ¹H NMR (DMSO‑d₆)

(3 mL) to give 22 as

a beige solid (100 mg, 51%). R_F

(DCM:CH₃OH ¼ 94:6) 0.30. m.p. 195e196 ^ꢁC. ¹H NMR (DMSO‑d₆)

d

1.01 (t, 2H, J 12.0, CH₂), 1.30e1.50 (m, 2H, CH₂), 1.50 (t, 2H, J 6.5,

d

1.10 (app d, 2H, J 13.0, CH₂), 1.50e1.70 (m, 4H, 2 ꢂ CH₂), 2.30e2.50

CH₂), 2.24 (t, 1H, J 11.0, CHH), 2.38 (t, 1H, J 12.0, CHH), 2.92 (d, 1H, J

12.5, CHH), 2.97 (t, 2H, J 6.5, CH₂), 3.02 (d, 1H, J 12.5, CHH), 7.40 (s,

1H, CONH), 7.52 (dd, 1H, J 9.0 & 4.5, quinolinyl-H), 7.54 (d, 1H, J 7.0,

quinolinyl-H), 7.87 (t, 1H, J 8.0, quinolinyl-H), 7.96 (d, 1H, J 8.5,

quinolinyl-H), 8.10 (d, 1H, J 8.5, quinolinyl-H), 8.18 (s, 1H, pyridinyl-

H), 8.56 (s, 1H, pyridinyl-H), 8.94 (dd, 1H, J 4.0 & 1.5, quinolinyl-H).

(m, 2H, CH₂), 2.70e3.15 (m, 2H, CH₂), 3.03 (t, 2H, J 6.5, CH₂), 6.20

(app s, 1H, indolyl-H), 6.86 (d, 1H, J 7.0, indolyl-H), 7.19 (t, 1H, J 7.5,

indolyl-H), 7.38 (app s, 1H, indolyl-H), 7.45 (d, 1H, J 7.0, indolyl-H),

7.46 (s, 1H, CONH), 8.19 (s, 1H, pyridinyl-H), 8.46 (s, 1H, pyridinyl-

H), 11.28 (s, 1H, indolyl-NH). ¹³C NMR (DMSO‑d₆)

d 30.2, 31.7, 37.7,

16

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

¹³C NMR (DMSO‑d₆)

d

30.4, 31.7, 31.9, 37.9, 41.2, 46.5, 47.3, 122.3,

(d,1H, J 8.0, quinolinyl-H), 8.49 (s,1H, pyridinyl-H), 8.87 (d,1H, J 2.5,

126.9,127.2,128.7,129.5,129.6,130.3,134.1,135.4,147.8,150.0,151.0,

quinolinyl-H). ¹³C NMR (DMSO‑d₆)

d 30.1, 31.5, 31.8, 37.6, 41.1, 46.0,

151.2, 153.9, 180.0. HRMS (ESIþ) 393.1478 [M(³⁵Cl)þH]^þ& 395.1454

47.0, 121.8, 126.4, 126.8, 127.9, 128.8, 130.8, 131.0, 136.5, 136.6, 146.1,

148.8, 150.6, 151.3, 153.9, 179.7. HRMS (ESIþ) 393.1477 [M(³⁵Cl)þ

H]^þ& 395.1450 [M(³⁷Cl)þH]^þ; calcd. for C₂₂H₂₂ClN₄O^þ393.1477

[M(³⁵Cl)þH]^þ& 395.1447 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method A:

t_R9.82 min, purity >97%; Method B: t_R7.89 min, purity >99%.

8-(3-Chloro-5-(naphthalen-1-yl)pyridin-4-yl)-2,8-diazaspiro

[4.5]decan-1-one (26)

[M(³⁷Cl)þH]^þ; calcd. for C₂₂H₂₂ClN₄O^þ393.1477 [M(³⁵Cl)þH]^þ

&

395.1447 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method C: t_R17.08 min,

purity >99%; Method D: t_R10.09 min, purity >99%.

8-(3-Chloro-5-(isoquinolin-5-yl)pyridin-4-yl)-2,8-diazaspiro

[4.5]decan-1-one (23)

Bromide 11 (173 mg, 502

acid (95.5 mg, 552

mmol) and isoquinolin-5-ylboronic

m

mol) were reacted at 120 ^ꢁC using general

Bromide 11 (173 mg, 502

mmol) and naphthalen-1-ylboronic

synthetic procedure A. The residue was puriﬁed by Biotage®

FlashMaster Personal^þﬂash chromatography (DCM ramping to

DCM:CH₃OH ¼ 94:6), and washed with EtOAc (5 mL) and n-hexane

acid (95.0 mg, 552

m

mol) were reacted at 120 ^ꢁC using general

synthetic procedure A. The residue was puriﬁed by Biotage®

FlashMaster Personal^þﬂash chromatography (DCM ramping to

DCM:EtOH ¼ 97:3), triturated with n-hexane (5 mL), and washed

with EtOAc (3 mL) to give 26 as a white solid (120 mg, 61%). R_F

(DCM:CH₃OH ¼ 96:4) 0.33. m.p. 203e204 ^ꢁC. ¹H NMR (DMSO‑d₆)

(3 mL) to give 23 as

a beige solid (45 mg, 23%). R_F

(DCM:CH₃OH ¼ 94:6) 0.30. m.p. 205e206 ^ꢁC. ¹H NMR (DMSO‑d₆)

d

1.02 (d, 2H, J 13.0, CH₂), 1.44 (t, 2H, J 12.0, CH₂), 1.50 (t, 2H, J 6.5,

CH₂), 2.25 (t, 1H, J 12.0, CHH), 2.44 (t, 1H, J 12.0, CHH), 2.94 (d, 1H, J

13.0, CHH), 2.98 (t, 2H, J 6.5, CH₂), 3.02 (d, 1H, J 13.0, CHH), 7.40 (d,

1H, J 6.5, isoquinolinyl-H), 7.41 (s, 1H, CONH), 7.73 (d, 1H, J 7.0,

isoquinolinyl-H), 7.80 (t, 1H, J 7.5, isoquinolinyl-H), 8.19 (s, 1H,

pyridinyl-H), 8.23 (d, 1H, J 8.0, isoquinolinyl-H), 8.48 (d, 1H, J 6.0,

isoquinolinyl-H), 8.58 (s,1H, pyridinyl-H), 9.40 (s,1H, isoquinolinyl-

d 0.96e1.08 (m, 2H, CH₂), 1.46 (t, 2H, J 7.0, CH₂), 1.46e1.54 (m, 2H,

CH₂), 2.17 (t, 1H, J 11.5, CHH), 2.40 (t, 1H, J 11.5, CHH), 2.85e3.05 (m,

2H, CH₂), 2.97 (t, 2H, J 7.0, CH₂), 7.35e7.47 (m, 2H), 7.47e7.54 (m,

2H), 7.54e7.59 (m, 1H) (total 5H, CONH & 4 ꢂ naphthalenyl-H), 7.62

(t, 1H, J 7.5, naphthalenyl-H), 8.02 (d, 2H, J 8.0, 2 ꢂ naphthalenyl-H),

8.18 (s, 1H, pyridinyl-H), 8.56 (s, 1H, pyridinyl-H). ¹³C NMR

H). ¹³C NMR (DMSO‑d₆)

d

30.3, 31.6, 31.8, 37.7, 41.0, 46.4, 47.3, 118.1,

(DMSO‑d₆) d 30.1, 31.6, 31.7, 37.6, 41.0, 46.1, 47.2, 125.3, 125.6, 126.3,

127.1, 127.4, 128.3, 130.0, 132.4, 133.9, 134.1, 143.7, 149.9, 151.1, 152.9,

153.8, 179.8 (one carbon signal overlapping or obscured). HRMS

(ESIþ) 393.1478 [M(³⁵Cl)þH]^þ& 395.1450 [M(³⁷Cl)þH]^þ; calcd. for

126.8, 127.0, 128.1, 128.4, 128.5, 131.2, 131.6, 133.1, 134.6, 149.4, 151.1,

153.7, 179.7. HRMS (ESIþ) 392.1527 [M(³⁵Cl)þH]^þ& 394.1501

[M(³⁷Cl)þH]^þ; calcd. for C₂₃H₂₃ClN₃O^þ392.1524 [M(³⁵Cl)þH]^þ

&

C

₂₂H₂₂ClN₄O^þ393.1477 [M(³⁵Cl)þH]^þ& 395.1447 [M(³⁷Cl)þH]^þ.

394.1495 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method A: t_R9.28 min,

purity >98%; Method B: t_R11.84 min, purity >97%.

8-(3-Chloro-5-(4-methylnaphthalen-1-yl)pyridin-4-yl)-2,8-

diazaspiro[4.5]decan-1-one (27)

Anal. RP-HPLC Method C: t_R15.94 min, purity >97%; Method D: t_R

9.25 min, purity >98%.

8-(3-Chloro-5-(isoquinolin-8-yl)pyridin-4-yl)-2,8-diazaspiro

[4.5]decan-1-one (24)

Bromide 11 (173 mg, 502

boronic acid (103 mg, 554 m

m

mol) and (4-methylnaphthalen-1-yl)

Bromide 11 (173 mg, 502

m

mol) and isoquinolin-8-ylboronic

mol) were reacted at 120 ^ꢁC using

acid (95.5 mg, 552

m

mol) were reacted at 120 ^ꢁC using general

general synthetic procedure A. The residue was puriﬁed by Bio-

tage® FlashMaster Personal^þﬂash chromatography (DCM ramping

to DCM:CH₃OH ¼ 97.5:2.5), and triturated and washed with n-

hexane (10 mL) to give 27 as a white solid (110 mg, 54%). R_F

(DCM:CH₃OH ¼ 94:6) 0.33. m.p. 195e196 ^ꢁC. ¹H NMR (DMSO‑d₆)

synthetic procedure A. The residue was puriﬁed by Biotage®

FlashMaster Personal^þﬂash chromatography (DCM ramping to

DCM:CH₃OH ¼ 94:6), and washed with EtOAc (5 mL) and n-hexane

(3 mL) to give 24 as

a beige solid (110 mg, 56%). R_F

(DCM:CH₃OH ¼ 94:6) 0.34. m.p. 198e199 ^ꢁC. ¹H NMR (DMSO‑d₆)

d 1.04 (app d, 2H, J 13.0, CH₂), 1.47 (t, 2H, J 6.5, CH₂), 1.50e1.60 (m,

d

1.00 (t, 2H, J 8.5, CH₂), 1.32e1.46 (m, 2H, CH₂), 1.48 (t, 2H, J 6.5,

2H, CH₂), 2.18 (t, 1H, J 12.0, CHH), 2.42 (t, 1H, J 12.0, CHH), 2.72 (s,

3H, CH₃), 2.92 (app d, 1H, J 13.0, CHH), 2.97 (t, 2H, J 6.5, CH₂), 3.02

(app d, 1H, J 12.5, CHH), 7.31 (d, 1H, J 7.0, naphthalenyl-H), 7.42 (s,

1H, CONH), 7.47 (d, 1H, J 7.0, naphthalenyl-H), 7.50e7.57 (m, 2H,

2 ꢂ naphthalenyl-H), 7.57e7.66 (m, 1H, naphthalenyl-H), 8.11 (d,

1H, J 8.5, naphthalenyl-H), 8.12 (s, 1H, pyridinyl-H), 8.54 (s, 1H,

CH₂), 2.23 (t, 1H, J 11.0, CHH), 2.42 (t, 1H, J 11.0, CHH), 2.97 (t, 2H, J

6.5, CH₂), 2.97e3.15 (m, 2H, CH₂), 7.40 (s, 1H, CONH), 7.59 (d, 1H, J

6.5, isoquinolinyl-H), 7.88 (t, 1H, J 7.5, isoquinolinyl-H), 7.93 (d, 1H, J

5.5, isoquinolinyl-H), 8.07 (d, 1H, J 8.0, isoquinolinyl-H), 8.26 (s, 1H,

pyridinyl-H), 8.56 (d, 1H, J 5.5, isoquinolinyl-H), 8.59 (s, 1H,

pyridinyl-H), 8.89 (s, 1H, isoquinolinyl-H). ¹³C NMR (DMSO‑d₆)

pyridinyl-H). ¹³C NMR (DMSO‑d₆)

d 19.3, 30.3, 31.7, 31.8, 37.7, 41.2,

d

30.3, 31.5, 31.8, 37.7, 41.0, 46.5, 47.2, 120.8,126.5,127.1,127.3,129.6,

46.2, 47.3, 124.8, 126.0, 126.3, 126.4, 126.5, 127.1, 127.9, 131.4, 131.8,

132.2, 133.0, 134.7, 149.3, 151.4, 153.8, 179.8. HRMS (ESIþ) 406.1679

[M(³⁵Cl)þH]^þ& 408.1655 [M(³⁷Cl)þH]^þ; calcd. for C₂₄H₂₅ClN₃O^þ

406.1681 [M(³⁵Cl)þH]^þ& 408.1651 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC

Method C: t_R21.36 min, purity >99%; Method D: t_R14.02 min,

purity >97%.

129.9, 130.5, 135.2, 135.6, 143.2, 150.1, 150.2, 151.0, 153.7, 179.7.

HRMS (ESIþ) 393.1476 [M(³⁵Cl)þH]^þ& 395.1450 [M(³⁷Cl)þH]^þ;

calcd. for

C

₂₂H₂₂ClN₄O^þ393.1477 [M(³⁵Cl)þH]^þ

& 395.1447

[M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method C: t_R15.97 min, purity >99%;

Method D: t_R9.53 min, purity >98%.

8-(3-Chloro-5-(quinolin-8-yl)pyridin-4-yl)-2,8-diazaspiro

[4.5]decan-1-one (25)

8-(3-Chloro-5-(4-methoxynaphthalen-1-yl)pyridin-4-yl)-2,8-

diazaspiro[4.5]decan-1-one (28)

Bromide 11 (173 mg, 502

(95.5 mg, 552

mol) were reacted at 120 ^ꢁC using general synthetic

procedure A. The residue was puriﬁed by Biotage® FlashMaster

Personal^þ

ﬂash chromatography (DCM ramping to

m

mol) and quinolin-8-ylboronic acid

Bromide 11 (173 mg, 502

1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (157 mg, 552

m

mol) and 2-(4-methoxynaphthalen-

m

mmol)

were reacted at 120 ^ꢁC using general synthetic procedure A. The

residue was puriﬁed by Biotage® FlashMaster Personal^þﬂash

chromatography (DCM ramping to DCM:CH₃OH ¼ 97.5:2.5), and

washed with EtOAc (3 mL) and n-hexane (3 mL) to give 28 as a

white solid (120 mg, 57%). R_F(DCM:CH₃OH ¼ 94:6) 0.41. m.p.

DCM:EtOH ¼ 95.5:4.5), and crystallised with EtOAc/DCM to give 25

as a tan solid (60 mg, 30%). R_F(DCM:EtOH ¼ 96:4) 0.30. m.p.

203e204 ^ꢁC. ¹H NMR (DMSO‑d₆)

d 1.00 (d, 1H, J 12.5, CHH), 1.05 (d,

1H, J 12.5, CHH), 1.35e1.52 (m, 3H, CHH & CH₂), 1.53 (t, 1H, J 9.5,

CHH), 2.02 (t, 1H, J 10.0, CHH), 2.50e2.58 (m, 1H, CHH), 2.90 (d, 1H, J

12.0, CHH), 2.92e3.02 (m, 3H, CHH & CH₂), 7.42 (s, 1H, CONH), 7.59

(dd, 1H, J 8.0 & 4.5, quinolinyl-H), 7.72 (d, 2H, J 5.0, 2 ꢂ quinolinyl-

H), 8.08 (t, 1H, J 4.5, 2 ꢂ quinolinyl-H), 8.16 (s, 1H, pyridinyl-H), 8.46

255e256 ^ꢁC. ¹H NMR (DMSO‑d₆)

d 1.04 (t, 2H, J 11.5, CH₂),1.48 (t, 2H,

J 6.5, CH₂), 1.50e1.62 (m, 2H, CH₂), 2.16 (t, 1H, J 11.5, CHH), 2.46 (t,

1H, J 11.5, CHH), 2.90e3.20 (m, 2H, CH₂), 2.98 (t, 2H, J 6.0, CH₂), 4.02

(s, 3H, CH₃), 7.08 (d, 1H, J 8.0, naphthalenyl-H), 7.35 (d, 1H, J 8.0,

naphthalenyl-H), 7.42 (s,1H, CONH), 7.45 (d,1H, J 8.0, naphthalenyl-

17

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

H), 7.48e7.62 (m, 2H, 2 ꢂ naphthalenyl-H), 8.13 (s, 1H, pyridinyl-H),

8.24 (d, 1H, J 8.0, naphthalenyl-H), 8.52 (s, 1H, pyridinyl-H). ¹³C

H]^þ& 383.1440 [M(³⁷Cl)þH]^þ; calcd. for C₂₁H₂₂ClN₄O^þ381.1477

[M(³⁵Cl)þH]^þ& 383.1447 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method C:

t_R17.88 min, purity >98%; Method D: t_R12.72 min, purity >98%.

8-(3-Chloro-5-(1-methyl-1H-indol-2-yl)pyridin-4-yl)-2,8-

diazaspiro[4.5]decan-1-one (32)

NMR (DMSO‑d₆)

d 30.2, 31.6, 31.8, 37.8, 41.2, 46.2, 47.2, 55.8, 104.2,

122.0, 124.8, 125.4, 125.7, 126.7, 127.0, 127.2, 128.5, 131.2, 132.6,

149.2, 151.6, 154.0, 155.0, 179.9. HRMS (ESIþ) 422.1630 [M(³⁵Cl)þ

H]^þ& 424.1608 [M(³⁷Cl)þH]^þ; calcd. for C₂₄H₂₅ClN₃O^þ₂422.1630

[M(³⁵Cl)þH]^þ& 424.1600 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method C:

t_R20.85 min, purity >99%; Method D: t_R13.43 min, purity >99%.

8-(3-Chloro-5-(dibenzo[b,d]furan-4-yl)pyridin-4-yl)-2,8-

diazaspiro[4.5]decan-1-one (29)

Bromide 11 (173 mg, 502

mmol) and 1-methyl-2-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole

(194 mg,

754 m

mol) were reacted at 140 ^ꢁC using general synthetic procedure

A. The residue was puriﬁed by Biotage® FlashMaster Personal^þ

ﬂash chromatography (DCM ramping to DCM:CH₃OH ¼ 97.5:2.5) to

give 32 as a beige solid (25 mg, 13%). R_F(DCM:CH₃OH ¼ 96:4) 0.38.

Bromide 11 (173 mg, 502

mmol) and dibenzo[b,d]furan-4-

ylboronic acid (117 mg, 552

m

mol) were reacted at 120 ^ꢁC using

m.p. 129e130 ^ꢁC. ¹H NMR (DMSO‑d₆)

d 1.22 (app d, 2H, J 12.5, CH₂),

general synthetic procedure A. The residue was puriﬁed by Bio-

tage® FlashMaster Personal^þﬂash chromatography (DCM ramping

to DCM:CH₃OH ¼ 97.5:2.5) to give 29 as a beige solid (125 mg, 58%).

R_F(DCM:CH₃OH ¼ 94:6) 0.40. m.p. 215e216 ^ꢁC. ¹H NMR (DMSO‑d₆)

1.65 (t, 2H, J 11.5, CH₂), 1.77 (t, 2H, J 6.5, CH₂), 2.67 (t, 2H, J 12.0, CH₂),

3.06 (t, 2H, J 6.5, CH₂), 3.16 (app br s, 2H, CH₂), 3.60 (s, 3H, CH₃), 6.50

(s, 1H, indolyl-H), 7.09 (t, 1H, J 7.5, indolyl-H), 7.20 (t, 1H, J 7.5,

indolyl-H), 7.49 (s, 1H, CONH), 7.50 (d, 1H, J 7.5, indolyl-H), 7.59 (d,

1H, J 7.5, indolyl-H), 8.25 (s, 1H, pyridinyl-H), 8.53 (s, 1H, pyridinyl-

d

1.11 (app d, 2H, J 12.5, CH₂), 1.48 (t, 2H, J 6.5, CH₂), 1.59 (t, 2H, J 9.5,

CH₂), 2.50e2.72 (m, 2H, CH₂), 2.96 (t, 2H, J 6.5, CH₂), 3.07 (d, 2H, J

11.5, CH₂), 7.40e7.47 (m, 2H), 7.47e7.57 (m, 3H) (total 5H, CONH &

4 ꢂ dibenzofuranyl-H), 7.68 (d, 1H, J 8.5, dibenzofuranyl-H), 8.22 (t,

2H, J 7.0, 2 ꢂ dibenzofuranyl-H), 8.34 (s, 1H, pyridinyl-H), 8.55 (s,

H). ¹³C NMR (DMSO‑d₆)

d 30.7, 30.8, 31.9, 37.8, 41.1, 46.5,103.2,110.3,

119.7, 120.3,121.7, 123.2, 126.4, 127.4, 135.4, 137.3, 150.2, 151.9, 153.8,

179.8 (two carbon signals overlapping or obscured). HRMS (ESIþ)

395.1634 [M(³⁵Cl)þH]^þ

&

397.1611 [M(³⁷Cl)þH]^þ; calcd. for

1H, pyridinyl-H). ¹³C NMR (DMSO‑d₆)

d

30.2, 31.8, 37.6, 41.0, 46.7,

C

₂₂H₂₄ClN₄O^þ395.1633 [M(³⁵Cl)þH]^þ& 397.1604 [M(³⁷Cl)þH]^þ.

111.7, 121.2, 121.5, 121.7, 123.4, 123.5, 123.6, 123.7, 126.9, 127.2, 128.0,

128.5, 149.6, 151.2, 152.9, 153.2, 155.5, 179.7 (two carbon signals

Anal. RP-HPLC Method C: t_R19.28 min, purity >95%; Method D: t_R

13.81 min, purity >97%.

overlapping or obscured). HRMS (ESIþ) 432.1469 [M(³⁵Cl)þH]^þ

&

8-(3-(1-Benzyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-5-

chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (33)

434.1452 [M(³⁷Cl)þH]^þ; calcd. for C₂₅H₂₃ClN₃O^þ₂432.1473

[M(³⁵Cl)þH]^þ& 434.1444 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method C:

t_R20.74 min, purity >99%; Method D: t_R15.14 min, purity >99%.

8-(3-Chloro-5-(dibenzo[b,d]thiophen-4-yl)pyridin-4-yl)-2,8-

diazaspiro[4.5]decan-1-one (30)

Bromide 11 (173 mg, 502

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine

(190 mg, 568

mol) were reacted at 120 ^ꢁC using general synthetic

procedure A. The residue was puriﬁed by Biotage® FlashMaster Per-

sonal^þ

ﬂash chromatography (DCM ramping to

mmol) and 1-benzyl-3-(4,4,5,5-

m

Bromide 11 (173 mg, 502

ylboronic acid (126 mg, 552

m

mol) and dibenzo[b,d]thiophen-4-

mol) were reacted at 120 ^ꢁC using

DCM:CH₃OH ¼ 96.5:3.5), and washed with EtOAc (6 mL) to give 33 as

general synthetic procedure A. The residue was puriﬁed by Bio-

tage® FlashMaster Personal^þﬂash chromatography (DCM ramping

to DCM:CH₃OH ¼ 97.5:2.5) to give 30 as a white solid (100 mg, 44%).

R_F(DCM:CH₃OH ¼ 94:6) 0.41. m.p. 245e246 ^ꢁC. ¹H NMR (DMSO‑d₆)

a beige solid (120 mg, 51%). R_F(DCM:CH₃OH ¼ 94:6) 0.38. m.p.

252e253 ^ꢁC.¹H NMR (DMSO‑d₆)

d 1.12 (app d, 2H, J 12.5, CH₂), 1.53 (t,

2H, J 6.5, CH₂), 1.64 (td, 2H, J 12.0 & 3.0, CH₂), 2.58 (t, 2H, J 12.0, CH₂),

2.98 (app d, 2H, J 12.5, CH₂), 3.03 (t, 2H, J 6.5, CH₂), 5.56 (s, 2H, phenyl-

CH₂), 7.18 (dd, 1H, J 7.5 & 4.5, pyrrolopyridinyl-H), 7.22e7.28 (m, 1H,

phenyl-H), 7.31e7.33(m, 4H, 4ꢂ phenyl-H), 7.50(s,1H, CONH), 7.72(s,

1H, pyrrolopyridinyl-H), 7.94 (d,1H, J 7.5, pyrrolopyridinyl-H), 8.29 (s,

1H, pyridinyl-H), 8.35 (d, 1H, J 4.0, pyrrolopyridinyl-H), 8.44 (s, 1H,

d

1.13 (t, 2H, J 14.0, CH₂), 1.40e1.75 (m, 4H, 2 ꢂ CH₂), 2.50e2.65 (m,

2H, CH₂), 2.76e2.92 (m, 1H, CHH), 3.00 (t, 2H, J 6.5, CH₂), 3.10e3.40

(m, 1H, CHH), 7.37e7.49 (m, 2H), 7.49e7.60 (m, 2H) (total 4H, CONH

& 3 ꢂ dibenzofuranyl-H), 7.66 (t, 1H, J 7.5, dibenzofuranyl-H), 8.00

(d, 1H, J 7.0, dibenzofuranyl-H), 8.31 (s, 1H, pyridinyl-H), 8.43 (t, 2H,

pyridinyl-H).¹³CNMR(MSO-d₆)

d30.2, 31.7, 37.8, 41.3, 46.9, 47.3,109.3,

J

8.0, dibenzofuranyl-H), 8.57 (s, 1H, pyridinyl-H). ¹³C NMR

116.6, 119.3, 126.1, 127.4, 127.5, 127.6, 128.6, 138.2, 143.4, 147.0, 148.4,

151.3,153.4,179.9 (six carbon signals overlapping or obscured). HRMS

(ESIþ) 472.1896 [M(³⁵Cl)þH]^þ& 474.1864 [M(³⁷Cl)þH]^þ; calcd. for

C₂₇H₂₇ClN₅O^þ472.1899 [M(³⁵Cl)þH]^þ& 474.1869 [M(³⁷Cl)þH]^þ.

Anal. RP-HPLC Method A: t_R11.77 min, purity >98%; Method B: t_R

9.22 min, purity >98%.

(DMSO‑d₆) d 30.4, 31.6, 31.8, 37.7, 41.1, 46.4, 47.6, 121.9, 122.5, 123.2,

125.1, 125.3, 127.0, 127.5, 128.8, 131.1, 132.1, 135.3, 135.6, 138.4, 139.1,

150.0, 150.2, 153.1, 179.7. HRMS (ESIþ) 448.1246 [M(³⁵Cl)þH]^þ

&

450.1206 [M(³⁷Cl)þH]^þ; calcd. for C₂₅H₂₃ClN₃OS^þ448.1245

[M(³⁵Cl)þH]^þ& 450.1215 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method C:

t_R21.48 min, purity >99%; Method D: t_R16.22 min, purity >99%.

8-(3-Chloro-5-(1H-indol-2-yl)pyridin-4-yl)-2,8-diazaspiro

[4.5]decan-1-one (31)

8-(3-Chloro-5-(1-(phenylsulfonyl)-1H-indol-3-yl)pyridin-4-

yl)-2,8-diazaspiro[4.5]decan-1-one (34)

Bromide 11 (173 mg, 502

mmol) and 1-(phenylsulfonyl)-3-

Bromide 11 (173 mg, 502

1,3,2-dioxaborolan-2-yl)-1H-indole (134 mg, 551

m

mol) and 2-(4,4,5,5-tetramethyl-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole (230 mg,

mmol) were

600 m

mol) were reacted at 135 ^ꢁC using general synthetic proced-

reacted at 120 ^ꢁC using general synthetic procedure A. The residue

was puriﬁed by Biotage® FlashMaster Personal^þﬂash chromatog-

raphy (DCM ramping to DCM:CH₃OH ¼ 97:3) to give 31 as a beige

solid (85 mg, 45%). R_F(DCM:CH₃OH ¼ 94:6) 0.38. m.p. 169e170 ^ꢁC.

ure A. The residue was puriﬁed by Biotage® FlashMaster Personal^þ

ﬂash chromatography (DCM ramping to DCM:CH₃OH ¼ 97.5:2.5) to

give 34 as a beige solid (128 mg, 49%). R_F(DCM:CH₃OH ¼ 94:6) 0.46.

m.p. 248e249 ^ꢁC. ¹H NMR (DMSO‑d₆)

d 0.96 (app d, 2H, J 12.5, CH₂),

¹H NMR (DMSO‑d₆)

d

1.26 (app d, 2H, J 12.0, CH₂), 1.75 (td, 2H, J 12.5

1.34 (t, 2H, J 6.0, CH₂),1.52 (t, 2H, J 10.0, CH₂), 2.33 (t, 2H, J 13.0, CH₂),

2.98 (t, 2H, J 6.5, CH₂), 3.05 (app d, 2H, J 12.5, CH₂), 7.29 (t, 1H, J 7.5,

indolyl-H), 7.33 (d, 1H, J 8.0, indolyl-H), 7.44 (t, 1H, J 7.5, indolyl-H),

7.48 (s, 1H, CONH), 7.58 (t, 2H, J 7.5, 2 ꢂ phenyl-H), 7.69 (t, 1H, J 7.5,

phenyl-H), 7.96 (s, 1H, indolyl-H), 8.04 (d, 1H, J 6.5, indolyl-H), 8.06

(d, 2H, J 7.0, 2 ꢂ phenyl-H), 8.24 (s, 1H, pyridinyl-H), 8.50 (s, 1H,

& 3.5, CH₂), 1.83 (t, 2H, J 6.5, CH₂), 2.81 (t, 2H, J 12.0, CH₂), 3.00e3.20

(m, 4H, 2 ꢂ CH₂), 6.54 (s, 1H, indolyl-H), 7.03 (t, 1H, J 7.0, indolyl-H),

7.13 (t, 1H, J 7.0, indolyl-H), 7.42 (d, 1H, J 8.0, indolyl-H), 7.52 (s, 1H,

CONH), 7.58 (d,1H, J 8.0, indolyl-H), 8.38 (s,1H, pyridinyl-H), 8.47 (s,

1H, pyridinyl-H), 11.35 (s, 1H, indolyl-NH). ¹³C NMR (DMSO‑d₆)

d

30.5, 31.8, 37.9, 41.4, 46.6, 102.7, 111.5, 119.5, 120.2, 121.7, 125.4,

pyridinyl-H). ¹³C NMR (DMSO‑d₆)

d 30.2, 31.7, 37.7, 41.0, 46.2, 113.6,

127.2, 128.2, 133.4, 136.7, 149.3, 151.0, 152.9, 180.0 (two carbon

120.0, 120.2, 123.5, 124.1, 125.5, 125.6, 126.9, 129.8, 130.8, 134.2,

134.7, 137.0, 149.7, 151.1, 153.5, 179.7 (ﬁve carbon signals

signals overlapping or obscured). HRMS (ESIþ) 381.1483 [M(³⁵Cl)þ

18

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

overlapping or obscured). HRMS (ESIþ) 521.1398 [M(³⁵Cl)þH]^þ

&

[M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method C: t_R15.86 min, purity >98%;

Method D: t_R9.38 min, purity >98%.

8-(3-(Benzofuran-2-yl)-5-chloropyridin-4-yl)-2,8-diazaspiro

[4.5]decan-1-one (38)

523.1371 [M(³⁷Cl)þH]^þ; calcd. for C₂₇H₂₆ClN₄O₃S^þ521.1409

[M(³⁵Cl)þH]^þ& 523.1379 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method C:

t_R20.26 min, purity >99%; Method D: t_R15.30 min, purity >98%.

8-(3-Chloro-5-(1H-indol-3-yl)pyridin-4-yl)-2,8-diazaspiro

[4.5]decan-1-one (35)

Bromide 11 (173 mg, 502

mmol) and 2-(benzofuran-2-yl)-

4,4,5,5-tetramethyl-1,3,2-dioxaborolane (129 mg, 528

mmol) were

Compound 34 (261 mg, 501

m

mol) was deprotected using gen-

reacted at 130 ^ꢁC using general synthetic procedure A. The residue

was puriﬁed by Biotage® FlashMaster Personal^þﬂash chromatog-

raphy (DCM ramping to DCM:CH₃OH ¼ 97:3) to give 38 as a white

solid (65 mg, 34%). R_F(DCM:CH₃OH ¼ 95:5) 0.33. m.p. 177e178 ^ꢁC.

eral synthetic procedure C. The residue was puriﬁed by Biotage®

FlashMaster Personal^þﬂash chromatography (DCM ramping to

DCM:CH₃OH ¼ 95:5) to give 35 as a white solid (152 mg, 80%). R_F

(DCM:CH₃OH ¼ 92:8) 0.46. m.p. 245e246 ^ꢁC. ¹H NMR (DMSO‑d₆)

¹H NMR (DMSO‑d₆)

d 1.32 (app d, 2H, J 12.5, CH₂), 1.79 (td, 2H, J 12.5

d

1.16 (app d, 2H, J 12.5, CH₂),1.55e1.75 (m, 4H, 2 ꢂ CH₂), 2.66 (t, 2H,

& 4.0, CH₂), 1.89 (t, 2H, J 6.5, CH₂), 2.84 (t, 2H, J 11.5, CH₂), 3.06e3.22

(m, 2H, CH₂), 3.13 (t, 2H, J 6.5, CH₂), 7.23 (s, 1H, benzofuranyl-H),

7.30 (td, 1H, J 7.0 & 0.5, benzofuranyl-H), 7.37 (td, 1H, J 7.0 & 1.0,

J 12.0, CH₂), 2.96e3.12 (m, 4H, 2 ꢂ CH₂), 7.05 (t, 1H, J 7.5, indolyl-H),

7.16 (t, 1H, J 7.5, indolyl-H), 7.41 (d, 1H, J 8.5, indolyl-H), 7.43 (s, 1H,

indolyl-H or CONH), 7.46 (d, 1H, J 8.5, indolyl-H), 7.48 (s, 1H, CONH

or indolyl-H), 8.24 (s, 1H, pyridinyl-H), 8.41 (s, 1H, pyridinyl-H),

benzofuranyl-H), 7.55 (br s, 1H, CONH), 7.66 (d, 1H,

J 8.0,

benzofuranyl-H), 7.72 (d, 1H, J 7.5, benzofuranyl-H), 8.55 (s, 2H,

11.40 (s, 1H, indolyl-NH). ¹³C NMR (DMSO‑d₆)

d

30.3, 31.8, 37.8,

2 ꢂ pyridinyl-H). ¹³C NMR (DMSO‑d₆)

d 30.4, 31.8, 37.9, 41.4, 46.1,

41.4, 46.5, 111.1, 111.9, 118.5, 119.7, 121.7, 125.1, 127.0, 127.1, 127.3,

136.0, 148.1, 151.6, 153.5, 178.0 (two carbon signals overlapping or

106.0, 111.4, 121.6, 122.9, 123.4, 125.1, 128.1, 128.6, 150.3, 150.8,151.8,

153.0, 154.6, 180.0 (two carbon signals overlapping or obscured).

HRMS (ESIþ) 382.1322 [M(³⁵Cl)þH]^þ& 384.1297 [M(³⁷Cl)þH]^þ;

obscured). HRMS (ESIþ) 381.1482 [M(³⁵Cl)þH]^þ

& 383.1439

[M(³⁷Cl)þH]^þ; calcd. for C₂₁H₂₂ClN₄O^þ381.1477 [M(³⁵Cl)þH]^þ

&

calcd. for

C

₂₁H₂₁ClN₃O^þ₂382.1317 [M(³⁵Cl)þH]^þ

& 384.1287

383.1447 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method C: t_R15.16 min,

[M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method A: t_R12.25 min, purity >95%;

purity >98%; Method D: t_R10.88 min, purity >98%.

Method B: t_R10.34 min, purity >95%.

8-(3-Chloro-5-(1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-

3-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (36)

8-(3-(Benzo[b]thiophen-2-yl)-5-chloropyridin-4-yl)-2,8-

diazaspiro[4.5]decan-1-one (39)

Bromide 11 (173 mg, 502

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]

pyridine (212 mg, 552

mol) were reacted at 120 ^ꢁC using general

m

mol) and 1-(phenylsulfonyl)-3-

Bromide 11 (173 mg, 502

mmol) and benzo[b]thiophen-2-

ylboronic acid (94.0 mg, 528

m

mol) were reacted at 130 ^ꢁC using

m

general synthetic procedure A. The residue was puriﬁed by Bio-

tage® FlashMaster Personal^þﬂash chromatography (DCM ramping

to DCM:CH₃OH ¼ 97:3) to give 39 as a white solid (70 mg, 35%). R_F

(DCM:CH₃OH ¼ 95:5) 0.33. m.p. 230e232 ^ꢁC. ¹H NMR (DMSO‑d₆)

synthetic procedure A. The residue was puriﬁed by Biotage®

FlashMaster Personal^þﬂash chromatography (DCM ramping to

DCM:CH₃OH ¼ 96.5:3.5), and triturated and washed with EtOAc

(10 mL) to give 36 as

a

white solid (45 mg, 17%). R_F

d 1.32 (app d, 2H, J 12.5, CH₂), 1.85 (td, 2H, J 12.0 & 3.0, CH₂), 1.90 (t,

(DCM:CH₃OH ¼ 96:4) 0.31. m.p. 252e253 ^ꢁC. ¹H NMR (DMSO‑d₆)

2H, J 7.0, CH₂), 2.98 (t, 2H, J 11.0, CH₂), 3.05e3.22 (m, 2H, CH₂), 3.12

(t, 2H, J 6.5, CH₂), 7.40 (td, 1H, J 7.0 & 1.5, benzothiophenyl-H), 7.43

(td, 1H, J 7.0 & 1.5, benzothiophenyl-H), 7.54 (s, 1H, CONH), 7.65 (s,

1H, benzothiophenyl-H), 7.91 (d, 1H, J 7.0, benzothiophenyl-H), 8.01

(d, 1H, J 7.0, benzothiophenyl-H), 8.51 (s, 1H, pyridinyl-H), 8.53 (s,

d

1.06 (app d, 2H, J 12.5, CH₂), 1.45e1.62 (m, 4H, CH₂), 2.50e2.58 (m,

2H, CH₂), 3.04 (t, 2H, J 6.5, CH₂), 3.11 (d, 2H, J 12.5, CH₂), 7.33 (dd,1H,

J 7.0 & 5.5, pyrrolopyridinyl-H), 7.50 (s, 1H, CONH), 7.61 (t, 2H, J 7.5,

2 ꢂ phenyl-H), 7.71 (t, 1H, J 7.5, phenyl-H), 7.89 (d, 1H, J 7.5,

pyrrolopyridinyl-H), 8.05 (s, 1H, pyrrolopyridinyl-H), 8.17 (d, 2H, J

7.5, 2 ꢂ phenyl-H), 8.31 (s, 1H, pyridinyl-H), 8.45 (d, 1H, J 4.5,

pyrrolopyridinyl-H), 8.50 (s, 1H, pyridinyl-H). ¹³C NMR (DMSO‑d₆)

1H, pyridinyl-H). ¹³C NMR (DMSO‑d₆)

d 30.4, 31.5, 37.9, 41.3, 46.6,

122.4, 124.0, 124.5, 124.9, 125.0, 127.5, 128.1, 137.8, 139.4, 140.2,

150.0, 150.7, 152.7, 180.1 (two carbon signals overlapping or

d

30.5, 31.8, 37.8, 41.0, 46.6, 116.2, 119.9, 122.6, 123.8, 125.4, 127.1,

obscured). HRMS (ESIþ) 398.1086 [M(³⁵Cl)þH]^þ

& 400.1068

127.6, 129.3, 129.7, 134.8, 137.4, 145.5, 146.6, 149.9, 151.1, 153.5, 179.8

[M(³⁷Cl)þH]^þ; calcd. for C₂₁H₂₁ClN₃OS^þ398.1088 [M(³⁵Cl)þH]^þ

&

(four carbon signals overlapping or obscured). HRMS (ESIþ)

400.1059 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method A: t_R12.42 min,

purity >97%; Method B: t_R10.56 min, purity >99%.

8-(3-(Benzofuran-3-yl)-5-chloropyridin-4-yl)-2,8-diazaspiro

[4.5]decan-1-one (40)

522.1347 [M(³⁵Cl)þH]^þ

&

524.1318 [M(³⁷Cl)þH]^þ; calcd. for

C₂₆H₂₅ClN₅O₃S^þ522.1361 [M(³⁵Cl)þH]^þ& 524.1332 [M(³⁷Cl)þH]^þ.

Anal. RP-HPLC Method C: t_R19.28 min, purity >98%; Method D: t_R

13.80 min, purity >98%.

Bromide 11 (173 mg, 502

mmol) and 2-(benzofuran-3-yl)-4,4,5,5-

8-(3-Chloro-5-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-4-yl)-

2,8-diazaspiro[4.5]decan-1-one (37)

tetramethyl-1,3,2-dioxaborolane (135 mg, 553

m

mol) were reacted at

120 ^ꢁC using general synthetic procedure A. The residue was puriﬁed

by Biotage® FlashMaster Personal^þﬂash chromatography (DCM

ramping to DCM:CH₃OH ¼ 97.5:2.5), and crystallised with EtOAc/n-

Compound 36 (157 mg, 301 mmol) was deprotected using gen-

eral synthetic procedure C. The residue was puriﬁed by Biotage®

FlashMaster Personal^þﬂash chromatography (DCM ramping to

DCM:CH₃OH ¼ 92:8) to give 37 as a white solid (86 mg, 75%). R_F

(DCM:CH₃OH ¼ 9:1) 0.44. m.p. 297e298 ^ꢁC. ¹H NMR (DMSO‑d₆)

hexane to give 40 as

a beige solid (67 mg, 35%). R_F

(DCM:CH₃OH ¼ 94:6) 0.40. m.p. 185e186 ^ꢁC. ¹H NMR (DMSO‑d₆)

d

1.18 (app d, 2H, J 12.5, CH₂), 1.64 (td, 2H, J 12.0 & 2.5, CH₂), 1.67 (t,

d

1.16 (app d, 2H, J 12.5, CH₂), 1.63 (td, 2H, J 12.0 & 2.5, CH₂), 1.67 (t,

2H, J 6.5, CH₂), 2.74 (t, 2H, J 12.0, CH₂), 3.05 (t, 2H, J 6.5, CH₂), 3.16 (d,

2H, J 12.5, CH₂), 7.31 (t, 1H, J 7.5, benzofuranyl-H), 7.40 (t, 1H, J 7.5,

benzofuranyl-H), 7.48 (s, 1H, CONH), 7.50 (d, 1H, J 8.0, benzofuranyl-

H), 7.68 (d, 1H, J 8.5, benzofuranyl-H), 8.15 (s, 1H, benzofuranyl-H),

8.27 (s, 1H, pyridinyl-H), 8.50 (s, 1H, pyridinyl-H). ¹³C NMR

2H, J 6.5, CH₂), 2.66 (t, 2H, J 12.0, CH₂), 3.05 (t, 2H, J 6.5, CH₂),

3.05e3.14 (m, 2H, CH₂), 7.11 (dd, 1H, J 7.5 & 4.5, pyrrolopyridinyl-H),

7.48 (s, 1H, CONH), 7.57 (s, 1H, pyrrolopyridinyl-H), 7.85 (d, 1H, J 8.0,

pyrrolopyridinyl-H), 8.24 (s, 1H, pyridinyl-H), 8.28 (d, 1H, J 4.5,

pyrrolopyridinyl-H), 8.42 (s, 1H, pyridinyl-H), 11.95 (s, 1H,

(DMSO‑d₆) d 30.4, 31.8, 37.8, 41.3, 46.7, 111.8, 117.4, 120.0, 122.7, 123.5,

pyrrolopyridinyl-NH). ¹³C NMR (DMSO‑d₆)

d

30.4, 31.8, 37.9, 41.4,

125.1, 127.0, 127.7, 144.1, 149.8, 151.1, 153.6, 154.5, 179.9 (two carbon

signals overlapping or obscured). HRMS (ESIþ) 382.1319 [M(³⁵Cl)þ

H]^þ& 384.1293 [M(³⁷Cl)þH]^þ; calcd. for C₂₁H₂₁ClN₃O^þ₂382.1317

[M(³⁵Cl)þH]^þ& 384.1287 [M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method C: t_R

19.76 min, purity >99%; Method D: t_R12.90 min, purity >99%.

46.6, 110.0, 116.2, 119.4, 125.5, 126.4, 127.1, 127.2, 143.3, 148.4, 148.5,

151.5, 153.5, 180.0 (two carbon signals overlapping or obscured).

HRMS (ESIþ) 382.1429 [M(³⁵Cl)þH]^þ& 384.1403 [M(³⁷Cl)þH]^þ;

calcd. for

C

₂₀H₂₁ClN₅O^þ382.1429 [M(³⁵Cl)þH]^þ

& 384.1400

19

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

8-(3-(Benzo[b]thiophen-3-yl)-5-chloropyridin-4-yl)-2,8-

diazaspiro[4.5]decan-1-one (41)

Cell Culture. All the cell lines were obtained from the cell bank

at Drug Discovery 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 [63] was detected in cell culture.

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 reported previously [41,50]. Compound con-

centrations required to inhibit 50% of cell growth (GI₅₀) were

calculated using nonlinear regression analysis.

Bromide 11 (173 mg, 502

mmol) and benzo[b]thiophen-3-

ylboronic acid (94.0 mg, 528

m

mol) were reacted at 120 ^ꢁC using

general synthetic procedure A. The residue was puriﬁed by Bio-

tage® FlashMaster Personal^þﬂash chromatography (DCM ramping

to DCM:CH₃OH ¼ 97:3), and crystallised with DCM/EtOAc to give 41

as a grey solid (110 mg, 55%). R_F(DCM:CH₃OH ¼ 95:5) 0.30. m.p.

156e157 ^ꢁC. ¹H NMR (DMSO‑d₆)

d 1.11 (app d, 2H, J 13.0, CH₂),

1.50e1.65 (m, 2H, CH₂), 1.58 (t, 2H, J 7.0, CH₂), 2.50e2.60 (m, 2H,

CH₂), 3.02 (t, 2H, J 7.0, CH₂), 3.07 (app d, 2H, J 12.5, CH₂), 7.41 (td, 1H,

7.0 & 1.0, benzothiophenyl-H), 7.45 (td, 1H, J 7.0 & 1.0,

J

benzothiophenyl-H), 7.47 (s, 1H, CONH), 7.50 (dd, 1H, J 7.0 & 1.0,

benzothiophenyl-H), 7.79 (s, 1H, benzothiophenyl-H), 8.08 (d, 1H, J

7.0, benzothiophenyl-H), 8.22 (s, 1H, pyridinyl-H), 8.53 (s, 1H,

pyridinyl-H). ¹³C NMR (DMSO‑d₆)

d

30.3, 31.7, 37.7, 41.2, 46.5, 122.4,

Western Blot Analysis. Western blotting was performed as

described previously [64]. Primary antibodies included STAT1,

123.2, 124.8, 124.9, 126.6, 126.7, 127.0, 132.3, 138.8, 139.2, 149.6,

150.9, 153.7, 179.7 (two carbon signals overlapping or obscured).

HRMS (ESIþ) 398.1090 [M(³⁵Cl)þH]^þ& 400.1059 [M(³⁷Cl)þH]^þ;

STAT1 with phosphorylated Ser727 (p-STAT1^S727) and

b-actin (Cell

Signaling Technology, Danvers, MA, USA). The anti-rabbit immu-

noglobulin G (IgG) horseradish peroxidase-conjugated antibody

(Cell Signaling Technology) was used as a secondary antibody.

Enhanced Chemiluminescence reagents (GE Healthcare Life Sci-

ences, Rydalmere, NSW, Australia) were used for western blot

detection.

calcd. for

C

₂₁H₂₁ClN₃OS^þ398.1088 [M(³⁵Cl)þH]^þ

& 400.1059

[M(³⁷Cl)þH]^þ. Anal. RP-HPLC Method A: t_R11.82 min, purity >96%;

Method B: t_R9.39 min, purity >97%.

4.2. Biology

Proﬁling of In Vitro Drug-like Properties. All of the assays

were performed with either 38 or 39 at Cyprotex Discovery Ltd.

(Macclesﬁeld, UK). Log D_7.4was assessed using the shake ﬂask assay

with a buffer at pH 7.4, and pKa acquired with a fast UV spectro-

metric titration. Aqueous solubility was estimated by turbidimetry.

Bidirectional permeability was measured using Caco-2 cell line.

Inhibition of CYP450 enzymes and the hERG potassium channel

was evaluated using human liver microsomes and CHO-hERG cells,

respectively. All the experimental details can be found at https://

www.cyprotex.com/services.

Kinase Assays. A radiometric protein kinase assayd³³PanQinase®

Activity Assaydwas used to measure the residual kinase activity of

CDK8/cyclin C. The assay was performed in a 96-well FlashPlate™

from PerkinElmer (Boston, MA, USA)in a reactionvolumeof 50

Beckman Coulter/SAGIAN™ Core System. A reaction cocktail was

formed bypipettingthefollowingfoursolutions inanorderof[i] 20

of assay buffer (standard buffer), [ii] 5 L of ATP solution (in H₂O), [iii]

L of a test compound (in 10% DMSO) [iv] 10 L of enzyme (i.e.,

CDK8), solution & 10 L of substrate (i.e., RBER-IRStide) solution

(premixed). Each well contained 70 mM HEPES-NaOH pH 7.5, 3 mM

MgCl₂, 3 mM MnCl₂, 3 M sodium orthovanadate, 1.2 mM dithio-

threitol (DTT), 50 g/mL polyethylene glycol (PEG) 20000,1.0 M ATP

mL ona

mL

m

5

m

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 3e4 per group for rats, and n ¼ 10 per group for

mice. Rodents were administered an IV injection of 38 or

CCT251545 via the tail vein (5 mg/kg for rats, and 2 mg/kg for mice

for 38 only), or a single oral dose by gavage (20 mg/kg for rats, and

10 mg/kg for mice for 38 only; 2-h prior fasting was fulﬁlled). IV

formulations consisted of 38 or CCT251545 dissolved in a mixture

of 0.1 M sodium acetate buffer (pH 4.5):PEG 400:N-methyl-2-

pyrrolidone (5:4:1, v/v/v), and oral formulations were comprised

of 38 or CCT251545 dispersed in 1% (w/v) CMC 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 analysis. Concentrations

of 38 or CCT251545 in plasma samples were quantiﬁed using a

validated LC/MS/MS method with the AB SCIEX TripleTOF 5600

mass spectrometer. Brieﬂy, plasma samples were supplemented

with an internal standard (IS) and extracted with ethyl acetate, and

organic phases separated and dried in a Genevac HT-4X HCL cen-

trifugal vacuum evaporator (SP Scientiﬁc, Ipswich, UK). The resi-

dues were reconstituted in acetonitrile/water (50:50), injected into

a Shimadzu Nexera HPLC system, and resolved on a Phenomenex

m

(the concentration corresponded to the apparent K_m(ATP) value of

CDK8), [

g

-

³³P]-ATP (4e9 ꢂ 10⁵cpm per well), 1 ng/

mL CDK8 (ProQi-

nase lot: 004), and 20 ng/mL RBER-IRStide (ProQinase lot: 019 or 023).

The reaction cocktails were incubated at 30 ^ꢁC for 60 min. The re-

actions were stopped with addition of 50 L of 2% (v/v) H₃PO₄, and

plates aspirated and washed twice with 200 L of 0.9% (w/v) NaCl.

m

Incorporation of ³³P_iwas determined with a microplate scintillation

counter (Microbeta, Wallace). A residual kinase activity (%) at each

compound concentration and an IC₅₀value of a test compound were

calculated using Quattro Workﬂow V3.1.1 (Quattro Research GmbH,

Münich, Germany; www.quattro-research.com). The ﬁtting model

used for the IC₅₀determination was sigmoidal dose-response (vari-

ableslope)with the parametersof TOPand BOTTOMﬁxed at 100% and

0%, respectively. The method of least squares was used for regression

analysis. Residual kinase activities (%) of the kinases except CDK8 in

the presence of 1

against GSK-3

m

M 38 or 39 and IC₅₀values of the two compounds

a

/

b

and PCK- were determined using Euroﬁns Scien-

q

tiﬁc’s KinaseProﬁler™ kinase screening and proﬁling service (key

details of the service can be found at https://www.

euro ﬁ nsdiscoveryservices.com/services/in-vitro-assays/kinases/

kinase-pro ﬁ ler/) or by HotSpot™ radioisotope-based kinase assays at

Reaction Biology Corporation (the assay protocol is available at

https://www.reactionbiology.com/services/target-speci ﬁ c-assays/

kinase-assays/kinase-screening). All of the IC₅₀values determined by

three contract research organisations were converted to the K_ivalues

using the Cheng-Prusoff equation: K_i¼ IC₅₀/(1þ([ATP]/K_m(ATP))),

where [ATP] is the concentration of ATP used for the IC₅₀determina-

tion and K_m(ATP) for each kinase is determined experimentally [62].

Kinetex Phenyl-Hexyl column (2.6

m

m,100 Å, 50 mm ꢂ 2.1 mm) at a

mobile phase ﬂow rate of 0.6 mL/min. Mobile phase A was 5%

methanol and 0.1% FA in water, and mobile phase B (MPB) 95%

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

20

M. Yu, T. Teo, Y. Yang et al.

European Journal of Medicinal Chemistry 214 (2021) 113248

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 the unknown samples. The limit of

quantiﬁcation 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 experi-

ments were carried out under the approval of the University of

South Australia Animal Ethics Committee (Animal Ethics Number:

U21-16).

Assessment of In Vivo Toxicity. Female BALB/c nude mice were

allocated into three treatment groups of two each, and each group

was orally administered 38 at 70 mg/kg, CCT251545 at 70 mg/kg, or

vehicle (1% CMC aqueous solution) at 10 mL/kg once daily for seven

consecutive days, and observed for another seven days. One mouse

suffered a major, unrelated welfare issue after the ﬁrst dose of

CCT251545, and therefore was humanely killed. Individual body

weights were recorded daily (except day 13). On day 15, mice were

anaesthetised by inhalation of isoﬂurane gas, and killed by cervical

dislocation without regaining consciousness. Long bones (left

femora), lungs, hearts, livers, kidneys, intestines, uteri, ovaries, and

brains were collected and ﬁxed in 10% buffered formalin for his-

topathological examination, and spleens and bone narrows stored

in RPMI-1640 containing 10% FBS for FACS analysis. These experi-

ments were carried out under the approval of the University of

South Australia Animal Ethics Committee (Animal Ethics Number:

26/13).

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The authors declare that they have no known competing

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22

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Potent and orally bioavailable CDK8 inhibitors: Design, synthesis, structure-activity relationship analysis and biological evaluation

DOI: 10.1016/j.ejmech.2021.113248

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