Discovery of novel 4-azaaryl-N-phenylpyrimidin-2-amine derivatives as potent and selective FLT3 inhibitors for acute myeloid leukaemia with FLT3 mutations

European Journal of Medicinal Chemistry 213 (2021) 113215

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Discovery of novel 4-azaaryl-N-phenylpyrimidin-2-amine derivatives

as potent and selective FLT3 inhibitors for acute myeloid leukaemia

with FLT3 mutations

Yi Long, Mingfeng Yu, Aleksandra M. Ochnik, Jasmine D. Karanjia, Sunita KC. Basnet,

*

Alemwork A. Kebede, Lianmeng Kou, Shudong Wang

Drug Discovery and Development, Clinical and Health Sciences, University of South Australia, Adelaide, SA, 5001, Australia

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 6 December 2020

Received in revised form

Feline McDonough sarcoma (FMS)-like tyrosine kinase 3 (FLT3) is one of the most pursued targets in the

treatment of acute myeloid leukaemia (AML) as its gene ampliﬁcation and mutations, particularly in-

ternal tandem duplication (ITD), contribute to the pathogenesis of AML and the resistance to known FLT3

inhibitors. To conquer this challenge, there is a quest for structurally novel FLT3 inhibitors. Herein, we

report the discovery of a new series of 4-azaaryl-N-phenylpyrimidin-2-amine derivatives as potent and

selective FLT3 inhibitors. Compounds 12b and 12r were capable of suppressing a wide range of mutated

FLT3 kinases including ITD and D835Y mutants; the latter isoform is closely associated with acquired

drug resistance. In addition, both compounds displayed an anti-proliferative speciﬁcity for FLT3-ITD-

harbouring cell lines (i.e., MV4-11 and MOLM-13 cells) over those with expression of the wild-type ki-

nase or even without FLT3 expression. In mechanistic studies using MV4-11 cells, 12b was found to

diminish the phosphorylation of key downstream effectors of FLT3 and induce apoptosis, supporting an

FLT3-ITD-targeted mechanism of its anti-proliferative action.

15 January 2021

Accepted 15 January 2021

Available online 22 January 2021

Keywords:

AML

FLT3 inhibitor

FLT3-ITD

FLT3-D835Y

Anti-AML drug discovery

©

1

. Introduction

unphosphorylated monomer, and is stimulated upon binding to its

ligand and undergoes a conformational transformation that facili-

tates receptor-receptor dimerisation and activation of its intrinsic

tyrosine kinase activity [10,11]. Subsequently, activated FLT3 auto-

phosphorylates multiple sites in the intracellular domain, pro-

moting the recruitment of various proteins and giving rise to a

series of sophisticated protein-protein interactions [12]. The pro-

tein complex thus assembled initiates the cascades of phosphory-

lation reactions among diverse intracellular kinases and enables

the activation of their associated signalling pathways (e.g., RAS/

RAF/MEK/ERK, JAK/STAT5 and PI3K/AKT/mTOR), thereby regulating

differentiation, proliferation and survival of normal haematopoietic

cells [2,10,13e15].

However, genetic alterations of the FLT3 gene, mainly

comprising internal tandem duplications (ITDs) in the juxtamem-

brane domain and point mutations in the tyrosine kinase domain,

have been identiﬁed in approximately one third of AML patients,

and are among the most prevalent genetic abnormalities occurring

in AML [16,17]. These FLT3 mutations confer ligand-independent,

constitutive activation of downstream signalling pathways and

subsequently induce aberrant proliferation of AML cells, leading to

Acute myeloid leukaemia (AML) is an aggressive haematopoietic

malignancy originating from abnormal proliferation of myeloid

progenitor cells within the blood circulation and bone marrow, and

is associated with a poor prognosis and a sluggish response to

chemotherapy [1e3]. The clinical prognosis is contingent on patient

age, exposure to upfront chemotherapy, performance status, and

genetic proﬁle including the occurrence of mutations in the feline

McDonough sarcoma (FMS)-like tyrosine kinase 3 (FLT3) gene [4,5],

with survival rates being 60e70% in paediatric patients and lower

than half in adult patients [6].

FLT3 is a member of the class III subfamily of receptor tyrosine

kinases, and is primarily expressed in the haematopoietic system

(

e.g., immature myeloid and lymphoid progenitors), while its ligand

is present in a number of tissues such as liver, thymus, spleen,

placenta, heart, lung and so forth [2,7e9]. Inactive FLT3 exists as an

*

Corresponding author.

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

https://doi.org/10.1016/j.ejmech.2021.113215

0

Y. Long, M. Yu, A.M. Ochnik et al.

European Journal of Medicinal Chemistry 213 (2021) 113215

a higher relapse rate and an inferior overall survival in AML patients

positive patients last year. Nevertheless, the clinical success of other

FLT3 inhibitors under development is mostly hampered by partial

and transient clinical responses, adverse events as well as unfav-

ourable pharmacokinetics. These roadblocks might be overcome,

for example, by the discovery and development of FLT3 inhibitors

with new pharmacophores.

[

4,18,19]. The same holds true for the cases with overexpression of

wild-type (WT) FLT3 receptors yet in the absence of FLT3-ITD [20].

Therefore, FLT3 and its common mutant forms have been regarded

as legitimate molecular therapeutic targets for the treatment of

AML.

An arsenal of FLT3 inhibitors with various degrees of inhibitory

potency and selectivity have been developed (Fig. 1) [4,21]. The

initial wave of FLT3 inhibitors were co-opted from the molecules

originally designed to target alternative kinases, and therefore are

non-speciﬁc towards FLT3 [17,22,23]. Well-studied inhibitors of this

generation include sunitinib, sorafenib, midostaurin and lestaurti-

nib. Despite their high potency against FLT3 in vitro, most of these

inhibitors have toxicity issues and limited efﬁcacy, and thus

generally failed in early-phase clinical trials where they had been

used as single agents in relapsed or refractory AML patients

In our previous tremendous attempts to identify novel, potent

and selective inhibitors of individual CDKs [26e32], a few of the

compounds with little CDK inhibitory activity unexpectedly exerted

potent anti-proliferative effects on MV4-11 AML cells. This

intriguing phenomenon has urged us to explore their bona ﬁde

kinase targets, and one of such interesting compounds was 12b

with an N-phenylpyrimidin-2-amine scaffold (Fig. 2). Herein, we

report the identiﬁcation of 12b as an FLT3 inhibitor, molecular-

docking-guided structural modiﬁcation of this compound, and

cellular mechanistic investigation of 12b and its analogue 12r.

[

2,10,17,22,24,25]. Accordingly, newer-generation FLT3 inhibitors

with enhanced selectivity have emerged; typical examples are

quizartinib, crenolanib, gilteritinib and pexidartinib. These in-

hibitors were found generally well-tolerated in phase I/II clinical

trials [17,24], but their inhibition of multiple other kinases and

suboptimal pharmacokinetics remain severe disadvantages.

Among both generations of FLT3 inhibitors, midostaurin and gil-

teritinib were approved by the USFDA for the treatment of FLT3-

mutated AML in 2017 and 2018, respectively, whereas quizartinib

received Japanese regulatory approval for its use in FLT3-ITD-

2

. Results and discussion

2.1. Identiﬁcation of 12b as an FLT3 inhibitor

As brieﬂy aforementioned, compound 12b was inactive towards

CDKs (K

i

> 5

mM), but displayed potent anti-proliferative activity

against MV4-11 AML cells with a GI50 value of 0.074

identify its genuine kinase targets, this compound was further

assessed against a panel of 44 non-CDK kinases at a concentration

mM (Fig. 2). To

Fig. 1. Chemical structures of selected FLT3 inhibitors.

2

Y. Long, M. Yu, A.M. Ochnik et al.

European Journal of Medicinal Chemistry 213 (2021) 113215

Kinase inhibition, K

i

(μM)

FLT3-ITD

0.002

72 h growth inhibition, GI50 (μM)

CDKs 1-7 & 9

FLT3-WT

0.009

MV4-11

>

5

0.074 ± 0.010

Fig. 2. Chemical structure and biological activity of 12b.

þ

of 1

mM, revealing that: (i) 12b potently inhibited FLT3 and its ITD

(NH

3

) of Lys644 and the backbone NH of Asp829 from the DFG

mutant with inhibition being > 90%; and (ii) the majority of the

other kinases tested were not, or only slightly, suppressed (i.e., <

motif, while the bridging secondary amino and terminal acetyl

groups generate a total of two hydrogen bonds with the backbone

carbonyl of Asp829 and the backbone NH of the hinge residue

Cys694, respectively. The phenyl ring of 12b is sandwiched be-

5

0% inhibition) (Fig. 3). Later, K

i

values of 12b for FLT3-WT and -ITD

M, respectively (Fig. 2).

were determined to be 0.009 and 0.002

m

These enzymatic data indicate that 12b is a potent inhibitor of FLT3

with a reasonable degree of selectivity.

tween the gatekeeper residue Phe691 and DFG-Phe830, forming

p-

p

stacking that likely reinforces the stabilisation of 12b-FLT3

To probe the molecular basis for the potent FLT3 inhibitory ac-

tivity of 12b, this compound was docked into the crystal structure

of FLT3 (PDB ID: 4RT7) using Glide (Schr o€ dinger Inc., 2013) (Fig. 4A),

and the binding mode thus obtained was compared to that of the

quizartinib-FLT3 co-crystal structure (PDB ID: 4RT7) (Fig. 4B).

Compound 12b is predicted to bind to FLT3 through interactions of

different nature (Fig. 4A). Speciﬁcally, pyrimidinyl-N3 of 12b forms

a bifurcated hydrogen bond with both the ε-ammonium group

binding. In addition, the pyrrolo[2,3-b]pyridin-3-yl moiety extends

into the back cleft [33] of the ATP-binding pocket and hydro-

phobically interacts with multiple residues including Met664,

Met665, Leu802, Cys807 and Val808 (not shown in Fig. 4A). All of

these binding features are also seen in the co-crystal structure of

quizartinib in complex with FLT3 (Fig. 4B) despite the disparate

chemical structures of the two inhibitors, implying that: (i) 12b

might interact with the kinase in a similar manner to quizartinib,

Fig. 3. Kinase selectivity proﬁle of 12b over a panel of 52 kinases (8 CDKs and 44 non-CDK kinases). Assays were conducted in duplicate at a compound concentration of 1 mM by

Reaction Biology Corporation, and the average percentages of residual kinase activity are illustrated.

3

Y. Long, M. Yu, A.M. Ochnik et al.

European Journal of Medicinal Chemistry 213 (2021) 113215

Fig. 4. (A) Predicted 12b-FLT3 binding mode generated using Glide (Schr o€ dinger Inc., 2013), and (B) the co-crystal structure of quizartinib in complex with FLT3 (PDB ID: 4RT7).

Hydrogen bonds are drawn as black dotted lines and selected residues of FLT3 annotated. The ﬁgures were generated using PyMOL 1.7.

0

and (ii) it would be practical to derive novel FLT3 inhibitors from

the structural alteration of 12b with the aim of improving inhibi-

tory potency and speciﬁcity.

reacted with either DMF-DMA or 1-tert-butoxy-N,N,N ,N -tetra-

methylmethanediamine (Bredereck’s reagent) to afford a variety of

enaminones 8 in good to excellent yields. In parallel, the other

building blocksdguanidines 11dwere either commercially pur-

chased or modularly synthesised via high-yielding guanidinylation

of arylamines 10 with aqueous cyanamide, following quantitative

palladium-catalysed reduction of the corresponding aryl nitro

compounds 9 with gaseous hydrogen (Scheme 2). Ring closure of

an enaminone 8 with a guanidine 11 under microwave irradiation

eventually gave the desired 4-azaaryl-N-phenylpyrimidin-2-

amines 12a-12s in various yields.

2.2. Rational design of 12b analogues

Given its decent FLT3 inhibitory proﬁle, compound 12b was

considered as a starting point to develop a series of novel analogues

as more potent and selective FLT3 inhibitors. The 12b-FLT3 binding

pose generated from the above docking experiment suggested the

signiﬁcance of the N-phenylpyrimidin-2-amine scaffold for the

inhibition of this kinase (Fig. 4A). Hence, this scaffold was retained.

Comparison between the predicted 12b-FLT3 binding mode and

the co-crystal structure of the quizartinib-FLT3 complex revealed

that (i) the back cleft of the ATP-binding pocket might be able to

accommodate large-size aromatic groups, and (ii) the rigidity could

not be required for the hinge-binding moieties. As such, the

structural modiﬁcation of 12b focused on the diversiﬁcation of the

substituents on the pyrimidinyl-C4 position and the phenyl ring.

Speciﬁcally, a variety of fused azabiaryl ring systems, including 1H-

indol-3-yl, 1H-indazol-3-yl, 1H-pyrrolo[2,3-b]pyridin-3-yl and 1H-

pyrrolo[3,2-b]pyridin-3-yl, were introduced onto the C4 position of

the central pyrimidine ring on the one hand, and the position and

nature of aliphatic azacycles on the phenyl ring, i.e., p-(4-

acetylpiperazin-1-yl), m-(4-methylpiperazin-1-yl) and m-(4-

acetyl-1,4-diazepan-1-yl), were varied on the other hand. The

preparation and biological evaluation of these rationally designed

molecules are described below.

2.4. Structure-activity relationships

All compounds were evaluated for their activity against both

FLT3-WT and -ITD. Owing to the wide application of their common

scaffold, i.e., N-phenylpyrimidin-2-amine, in engineering CDK in-

hibitors, compounds 12a-12s were additionally evaluated for their

potency against CDKs 1, 2 and 7 to initially deﬁne some off-targets.

MV4-11 cells harbour a homozygous FLT3-ITD mutation, and their

proliferation is known to be FLT3-dependent [34e37]. As a result,

this AML cell line was chosen to measure the in vitro anti-

proliferative effects of the compounds. Clinical FLT3 inhibitor gil-

teritinib served as a positive control, and all the kinase inhibition as

well as cell viability data are summarised in Tables 1 and 2.

CH ) started with

3

unmasking the NH site of the 1H-pyrrolo[2,3-b]pyridin-3-yl moi-

1

Structural modiﬁcation of 12b (R

¼

1

ety; removal of the methyl group afforded 12a (R ¼ H) with

improved inhibitory activity against FLT3-WT (K

i

¼ 0.0008

mM),

2.3. Chemistry

FLT3-ITD (K

i

¼ 0.0001

mM) and MV4-11 cells (GI50 < 0.001 mM) but

with a diminished selectivity for FLT3 over CDKs (Table 1). The

1

The synthetic route deployed to prepare 4-azaaryl-N-phenyl-

same phenomenon was also observed for 12c (R ¼ H) and its

1

pyrimidin-2-amine derivatives 12a-12s is illustrated in Schemes 1

and 2. Acetylation of indoles 1 or azaindoles 3 at their respective

C3 positions was effected with cyanoacetic acid or acetyl chloride,

respectively, with the yields ranging from 77 to 92% (Scheme 1).

While 3-acetylindole 2c was masked with a toluenesulfonyl (Ts)

group to afford 2d in near-quantitative yield, 3-acetyl-7-azaindole

methyl counterpart 12d (R ¼ CH

3

), both of which contain a 4-

methylpiperazin-1-yl pendant at the meta position of the phenyl

4

ring [i.e., R ¼ m-(4-methylpiperazin-1-yl)], as well as for the pair of

1

compounds 12e (X ¼ N, R ¼ H) and 12f (X ¼ NCH

3

, no R sub-

stituent) with a larger terminal aliphatic azacycle, i.e., m-(4-acetyl-

1,4-diazepan-1-yl). Moving the nitrogen atom of 12a, 12c and 12e

from the X to Y position gave rise to 12g, 12h and 12i, respectively,

which were much less effective inhibitors of FLT3 kinases (K

0.0001e0.004 M vs. 0.004e0.217 M), CDKs (K : 0.029e0.531

vs. M) and MV4-11 cells (GI50 0.020

0.405e0.688 M). A close examination of the impact of the R

4

b

was further methylated with N,N-dimethylformamide

dimethyl acetal (DMF-DMA) to give 4c and 4d in a yield of 51% and

6%, respectively. On the other hand, starting from 1H-indazole-3-

i

:

2

m

i

mM

carboxylic acid 5, the corresponding carboxamide 6a and its Boc-

protected form 6b were prepared in 85% and 98% yields, and

were subsequently converted into their respective ketones 7a (95%)

and 7b (81%) using a metal-based reagent. All of ketones 2, 4 and 7

>

5

m

:

ꢀ

mM vs.

4

m

group on FLT3 inhibitory activity (12a vs. 12c and 12e; 12b vs. 12d;

12g vs. 12h and 12i) disclosed that para-substitution of the phenyl

4

Y. Long, M. Yu, A.M. Ochnik et al.

European Journal of Medicinal Chemistry 213 (2021) 113215

ꢁ

Scheme 1. Synthesis of enaminones 8a-8l. Reagents and conditions: (a) cyanoacetic acid, acetic anhydride, 85 C, 10 min, 2a: 90% and 2b: 92%; (b) 4-toluenesulfonyl chloride,

tetrabutylammonium hydrogen sulfate, 50% (w/v) aqueous NaOH, DCM, rt, o/n, 99%; (c) acetyl chloride, AlCl

3

, DCM, rt, o/n, 4a: 82% and 4b: 77%; (d) DMF-DMA, reﬂux, o/n, 4c: 51%

ꢁ

and 4d: 26%; (e) N,O-dimethylhydroxylamine hydrochloride, pyridine, EDC$HCl, THF, 0 C to rt, o/n, 85%; (f) Boc

2

O, DMAP, Et

3

N, DCM, 0 C to rt, o/n, 98%; (g) 6a e CH

3

ꢁ

MgBr,

ꢁ

THF, ꢂ78 C to rt, 3.5 h, 7a: 95%; 6b e CH

3

CN, LiN(iPr)

2

, THF, ꢂ78 C to rt, o/n, 7b: 81%; (h) DMF-DMA, (1) rt, 5e30 min, (2) reﬂux, 2e36 h, or (3) microwave 150e300 W, 180 C, 1 h,

0 0

7

0e98%; or 1-tert-butoxy-N,N,N ,N -tetramethylmethanediamine, reﬂux, 6 h, 69e90%.

Scheme 2. Synthesis of 4-azaaryl-N-phenylpyrimidin-2-amine derivatives 12a-12s. Reagents and conditions: (a) H

2

, 10% Pd/C, CH

3

OH, rt, o/n, 10a: 100% and 10b: 100%; (b) H

2

NCN,

ꢁ

(

CH

3

)

3

SiCl, CH

3

CN, 0 C to reﬂux, o/n, 11a: 85% and 11b: 95%; (c) appropriate enaminone 8, NaOH, 2-methoxyethanol or CH

3

CN, microwave 150e300 W, 160 C, 1 h, 13e64%.

5

Y. Long, M. Yu, A.M. Ochnik et al.

European Journal of Medicinal Chemistry 213 (2021) 113215

Table 1

Structure and biological activities of 12a-12i.

Compound

Structure

X

Kinase inhibition, K

i

(

m

M)^a

Growth inhibition, GI50

MV4-11

(m

M)^b

Y

R¹

R⁴

FLT3-WT

FLT3-ITD

CDK1B

CDK2A

CDK7H

1

2a

2b

2c

2d

2e

2f

2g

2h

2i

N

NCH

CH

N

H

CH

H

CH

H

p-(4-acetylpiperazin-1-yl)

0.0008

0.009

0.004

0.040

0.0004

0.001

0.032

0.217

0.150

0.010

0.0001

0.002

0.013

0.0001

0.0007

0.004

0.020

0.107

0.002

0.091

>5

0.531

>5

0.051

1.756

>5

0.049

>5

0.084

0.970

0.031

>5

1.485

>5

0.142

>5

0.029

>5

<0.001

3

0.074 ± 0.010

0.005 ± 0.004

0.024 ± 0.012

0.020 ± 0.003

0.024 ± 0.002

0.420 ± 0.035

0.405 ± 0.177

0.688 ± 0.041

0.002 ± 0.000

m-(4-methylpiperazin-1-yl)

m-(4-acetyl-1,4-diazepan-1-yl)

p-(4-acetylpiperazin-1-yl)

m-(4-methylpiperazin-1-yl)

m-(4-acetyl-1,4-diazepan-1-yl)

e

3

e

H

N

1

CH

gilteritinib

a

Apparent inhibition constants (K

i

) were determined either by Reaction Biology Corporation or using in-house kinase assays; K

i

values were calculated using the half

b

maximal inhibition (IC50) and the appropriate K

m

(ATP) of each kinase. Anti-proliferative activity was determined using 72 h resazurin assays, and the data given are mean

values derived from at least two replicates ± SD.

Table 2

Structure and biological activities of 12j-12s.

Compound

Structure

Z

Kinase inhibition, K

i

(

m

M)^a

Growth inhibition, GI50

MV4-11

(m

M)^b

R¹

R²

R³

FLT3-WT

FLT3-ITD

CDK1B

CDK2A

CDK7H

1

2j

2k

2l

2m

C

N

H

CH

Ts

H

CH

H

CH

H

CN

H

0.022

0.149

>5

0.004

0.037

0.119

>5

0.883

0.021

0.001

0.010

0.011

0.133

0.128

0.0004

0.007

0.002

0.207

0.274

0.0002

0.0006

0.002

>5

4.055

1.581

>5

1.654

0.727

>5

0.426

1.625

0.536

>5

0.033 ± 0.021

0.252 ± 0.103

1.559 ± 0.675

0.004 ± 0.001

0.030 ± 0.006

0.390 ± 0.496

0.346 ± 0.130

0.181 ± 0.054

0.017 ± 0.012

0.018 ± 0.002

0.002 ± 0.000

3

>5

1

2n

2o

2p

2q

3

1.120

2.170

>5

CH

e

3

e

H

CN

1

2r

2s

e

>5

1.485

gilteritinib

a

Apparent inhibition constants (K

i

) were determined either by Reaction Biology Corporation or using in-house kinase assays; K

i

values were calculated using the half

b

maximal inhibition (IC50) and the appropriate K

values derived from at least two replicates ± SD.

m

(ATP) of each kinase. Anti-proliferative activity was determined using 72 h resazurin assays, and the data given are mean

ring with 4-acetylpiperazin-1-yl offered the most potent FLT3 in-

hibitors, so this substituent remained unchanged during the further

variation of the fused azabiaryl ring system at the pyrimidinyl-C4

position, with the single exception of 12s (Table 2).

enzymatic and cellular potencies, with K

i

values for FLT3-WT and

-ITD being greater than 0.2 M (Tables 1 and 2). However, either

m

introduction of a cyanide group at the pyrimidinyl-C5 position [i.e.,

3

12r (R ¼ CN)] or substitution of m-(4-acetyl-1,4-diazepan-1-yl) for

Replacement of the 1H-pyrrolo[2,3-b]pyridin-3-yl headgroup of

p-(4-acetylpiperazin-1-yl) on the terminal phenyl ring (i.e., 12s)

1

2a and 12b (X ¼ N, Y ¼ Z ¼ CH) with the 1H-indazol-3-yl moiety

restored the inhibitory activity against FLT3 kinases

[

i.e., 12p and 12q (X ¼ Y¼ CH, Z ¼ N)] drastically decreased

(K

i

¼ 0.0002e0.021

mM) and MV4-11 cells (GI50 ¼ 0.017e0.018

mM)

6

Y. Long, M. Yu, A.M. Ochnik et al.

European Journal of Medicinal Chemistry 213 (2021) 113215

(

Table 2). The former tactic was also deployed to ameliorate the

inhibitory effect of the 1H-indol-3-yl-headed compounds

X ¼ Y ¼ Z ¼ CH) on both FLT3 kinases and MV4-11 cells. As ex-

statuses (Table 4). AML cell lines MV4-11 and MOLM-13 carry ITD

mutation; THP1 and U937, the other two AML cell lines tested,

together with NB4 acute promyelocytic leukaemia cells harbour

unmutated FLT3 gene; no FLT3 is expressed in Jurkat T-cell

leukaemia and K562 chronic myeloid leukaemia cell lines [37]. As

expected, FLT3-ITD-expressing cells, i.e., MV4-11 and MOLM-13,

(

1

3

pected, cyano compounds 12m (R ¼ H, R ¼ CN) and 12n

1

3

(

R

¼ CH

3

, R ¼ CN) more potently suppressed the activity of the

kinases and the proliferation of MV4-11 cells than did their

1

3

respective non-substituted counterparts 12j (R ¼ H, R ¼ H) and

were

(GI50 ¼ 0.0004e0.074

low or no expression of FLT3 (i.e., U937, Jurkat and K562) was not

affected by either inhibitor (GI50 > 10 M). The single exception of

the latter case was the moderate anti-proliferative activity of 12r

M), suggesting that the com-

very

sensitive

to

compounds

12b

and

12r

1

3

1

2k (R ¼ CH

3

, R ¼ H). Alternative elaborations of the 1H-indol-3-

mM), whereas the viability of the cells with

yl moiety included (1) masking of the NH site with a bulky tosyl

group [i.e.,12j (R¹¼ H) vs. 12l (R ¼ Ts)] and (2) relocation of the

methyl group from the indolyl-N1 to -C2 position in the presence of

1

m

1

2

3

1

a cyanide group [i.e.,12n (R ¼ CH

3

, R ¼ H, R ¼ CN) vs. 12o (R ¼ H,

towards K562 cells (GI50 ¼ 0.185

m

2

3

R

¼ CH

3

, R ¼ CN)]. Both strategies were found to be generally

pound might target alternative kinase(s) pivotal for the growth and

survival of this cell line. NB4 and THP1 cells with expression of WT

FLT3 were inhibited weakly by 12b or 12r. Taken together, these

results argue an FLT3-ITD-targeted mechanism of anti-proliferative

action within leukaemic cells, which is consistent with previous

observations [35,37].

detrimental to inhibition of both FLT3 kinases and MV4-11 cells,

with the effect being much more profound in the former structural

modiﬁcation.

To sum up, the majority of the compounds are not only single-

digit nanomolar or even sub-nanomolar FLT3 inhibitors with

decent degrees of selectivity over CDKs, but also effective anti-

proliferative agents of MV4-11 cells. Such potencies are compara-

ble, if not superior, to those of gilteritinib. While 1H-pyrrolo[2,3-b]

pyridin-3-yl is the most favourable headgroup in terms of FLT3

inhibitory activity and anti-proliferative effect on MV4-11 cells,

2.6. Cellular mechanistic studies

To decipher the plausible cellular mechanism of their anti-

proliferative action, compounds 12b and 12r were assessed

against MV4-11, a sensitive cell line with FLT3-ITD mutation, and

U937, a non-sensitive cell line with low expression of WT FLT3 only,

using annexin V/propidium iodide (PI) assays and western blotting

analysis.

installation of a cyanide group at the pyrimidinyl-C5 position (i.e.,

3

R

¼ CN) in the context of the 1H-ind(az)ol-3-yl-headed com-

pounds generally boosts both enzymatic and cellular potencies.

2.5. Further characterisation of 12b and 12r

Firstly, annexin V/PI double staining was carried out to examine

the apoptotic effects of each compound at concentrations of 1 ꢃ ,

In addition to FLT3-ITD, there are other mutations that occur in

5

11

ꢃ ,10 ꢃ and 20 ꢃ its 24 h GI50 value (that was determined in MV4-

AML patients and are highly correlated with a poor prognosis [19].

Consequently, a range of FLT3 mutants were selected to deﬁne the

scope of the inhibitory activity of the hit compound 12b and its

analogue 12r (one of the most potent and selective inhibitor of

FLT3-ITD). As presented in Table 3, 12r is far more active than 12b

against every single FLT3 mutant tested. While 12r is a sub-

nanomolar inhibitor of all eight FLT3-mutated isoforms (i.e.,

cells using the 24

h

resazurin assay, i.e., 12b: 24

h

GI50 ¼ 0.125 M and 12r: 24 h GI50 ¼ 0.085

m

M) after incubation

with either cell line (MV4-11 or U937) for a period of 24 or 48 h.

Compound 12b was capable of inducing apoptosis of MV4-11 cells

in a concentration-dependent manner, with the effect being more

profound at later time point (Fig. 5). For example, in comparison

with DMSO diluent, 12 b at the highest concentration (i.e.,

K

i

< 0.0003

However, the latter shows a degree of speciﬁcity for FLT3-ITD

M) over the other mutants (K M).

i

mM), K values of 12b range from 0.002 to 0.262 mM.

2

0 ꢃ GI50) triggered approximately 9% and 26% more apoptotic cells

after 24- and 48-h treatments, respectively (Fig. 5A and B); this

effect was almost wholly attributed to the late apoptosis (Fig. 5C).

Compound 12r exerted the apoptosis-inducing effect on MV4-

(

K

i

¼ 0.002

m

i

¼ 0.047e0.262

m

Notably, both 12b and 12r are potent inhibitors of FLT3-D835Yda

mutant implicated in acquired clinical resistance to known FLT3

inhibitors [22,38,39], and therefore hold promise to overcome this

challenge.

11 cells to a similar extent as 12b (Fig. S1). In contrast, neither

compound caused apparent apoptosis of U937 cells at all four

concentrations. Collectively, these data are in agreement with GI50

values of compounds 12r and 12b obtained from 72 h cell viability

assays using resazurin (Table 4), supporting that apoptosis con-

tributes to the anti-proliferative effects of the two compounds on

MV4-11 cells.

To understand the mechanism underlying the induction of

apoptosis described above as well as to investigate the cellular FLT3

inhibitory potency of 12b and 12r, MV4-11 and U937 cells were

As shown in Tables 1e3, both 12b and 12r effectively suppressed

the kinase activity of both WT FLT3 and its mutants, particularly,

FLT3-ITD, in non-cell-based biochemical assays. To investigate

whether such effects could be translated into the anti-proliferative

potencies in cell viability assays, the two compounds were screened

against a panel of seven leukaemic cell lines with different FLT3

Table 3

Inhibitory effect of 12b and 12r on a panel of FLT3-mutated isoforms.

Table 4

i

(m

M)^a

FLT3 mutant

Kinase inhibition, K

2b

Anti-proliferative activities of 12b and 12r against multiple leukaemic cell lines.

1

12r

_M₎a

(m

Human leukaemia cell line FLT3 status

Growth inhibition, GI50

2b 12r

0.074 ± 0.010 0.017 ± 0.012

FLT3 (ITD)

FLT3 (ITD)-NPOS

FLT3 (ITD)-W51

0.002

0.262

0.084

0.047

0.185

0.054

0.064

0.077

0.0002

<0.0003

1

MV4-11

MOLM-13

NB4

THP1

U937

FLT3-ITD

FLT3-WT

FLT3 (D835Y)

0.023 ± 0.001 0.0004 ± 0.0002

FLT3 (Y591-V592insVDFREYEYD)

FLT3 (F594_R595insR)

FLT3 (F594_R595insREY)

FLT3 (R595_E596insEY)

1.060 ± 0.080

7.720 ± 3.949

>10

0.126 ± 0.041

3.037 ± 3.351

>10

0.185 ± 0.055

FLT3-WT

FLT3-WT/low

FLT3-null

Jurkat

K562

>10

a

i

Apparent inhibition constants (K ) were determined by Reaction Biology Cor-

a

poration; K

appropriate K

i

values were calculated using the half maximal inhibition (IC50) and the

(ATP) of each FLT3 mutant.

GI50 values were determined using 72 h resazurin assays and are presented as

mean ± SD derived from at least two replicates.

m

7

Y. Long, M. Yu, A.M. Ochnik et al.

European Journal of Medicinal Chemistry 213 (2021) 113215

Fig. 5. Detection of apoptosis of MV4-11 or U937 cells after incubation with 12b for 24 (A) or 48 (B) hours using annexin V/PI double staining assays, followed by quantitative

analysis (C). GI50 in the ﬁgure refers to the 24 h value of MV4-11 cells (i.e., 0.125 M). Independent experiments were conducted in triplicate. Statistical analysis was performed using

m

GraphPad one-way ANOVA analysis with Tukey’s post-hoc test, and statistical signiﬁcance deﬁned as a p-value of <0.05 (*), <0.01 (**), <0.001 (***) or < 0.0001 (****) (GraphPad

Prism v.7, La Jolla, CA, USA).

individually incubated with each compound at concentrations of

[13,15]. To determine the effect of 12b on these signalling pathways,

several key pathway components were examined for their total and

phosphorylated levels. As shown in Figs. 6, 12b was capable of

signiﬁcantly reducing, if not abolishing, FLT3-driven phosphoryla-

tion of STAT5, ERK, AKT, S6RP and eIF4E (the last two are

1

ꢃ , 10 ꢃ and 20 ꢃ GI50(24 h, MV4-11) for 6 or 24 h, and analysed

using western blotting (Fig. 6 and Fig. S2). As mentioned previously,

autophosphorylated FLT3 lies upstream of three oncogenic path-

ways, namely JAK/STAT5, RAS/RAF/MEK/ERK and PI3K/AKT/mTOR

8

Y. Long, M. Yu, A.M. Ochnik et al.

European Journal of Medicinal Chemistry 213 (2021) 113215

Further proﬁling of 12b and 12r using a panel of seven additional

FLT3 mutants and a selection of six extra leukaemic cell lines with

different FLT3 statuses revealed their board FLT3 inhibitory spectra

and high anti-proliferative speciﬁcity for the cell lines with ITD

mutation, respectively. The hit compound 12b with a decent

selectivity for FLT3-ITD was capable of inducing apoptosis and

downregulating the phosphorylation of key downstream effectors

of FLT3 in MV4-11 cells, substantiating the FLT3-ITD-targeted

mechanism of its anti-proliferative action.

4. Experimental

4.1. Docking studies

The potential modes of the binding of 12b to FLT3 were inves-

tigated with Schr o€ dinger suite docking program, Glide, under the

Maestro graphical user interface. Other associated programs

included LigPrep and Protein Preparation Wizard. Preparing 3D

conformers using LigPrep: the 3D conformers of 12b were generated

by LigPrep with default settings. Docking using Glide: the ﬁrst step

of performing structure-based docking was to generate the recep-

tor grid for FLT3. The required atomic coordinates of FLT3 were

obtained from the Protein Data Bank (PDB ID: 4RT7) and loaded

onto the Maestro interface. Any missing atoms in the structures

were added using Protein Preparation Wizard. Whilst retaining the

default settings, the docking receptor was deﬁned and generated

with the centre at the ATP-binding pocket. Subsequently, the pre-

pared conformers of 12b were docked into the ATP-binding site of

FLT3 using Glide. During the docking process, the extra precision

mode was selected, and the number of energy minimisation steps

was maximised to 400 to ensure convergence. The results were

ranked according to the Glide-XP docking score, and the best

scoring pose was analysed in Maestro and viewed with PyMOL.

Fig. 6. Western blot analysis of MV4-11 and U937 cells treated with 12b for a period of

6

h. GI50 in the ﬁgure refers to the 24 h value of MV4-11 cells (i.e., 0.125

mM). DMSO

diluent was used as control in each experiment, and -actin as a loading control. The

b

blots present the results of at least two independent experiments.

4.2. Chemistry

downstream effectors of mTOR) [40e43] at no expense of their

respective total levels after 6-h incubation with MV4-11 cells,

indicating that FLT3-mediated signalling pathways were greatly

dampened or even blocked by this compound. These suppressive

effects were also found in U937 cells treated with 12b, but in a

much weaker manner at each of the same compound concentra-

tions as used in MV4-11 cells. In addition, 12b increased the

cleavages of poly(ADP-ribose)polymerase (PARP) and caspase-3 in

MV4-11 cells, but these cleavages were nearly not detectable in

U937 cells. However, the level of anti-apoptotic protein myeloid cell

leukaemia 1 (Mcl-1) was downregulated in both cell lines after 24-

h incubation with either 12b or 12r, with the reducing effect being

far more marked in MV4-11 cells than that in U937 cells (Fig. S2).

These results correlate well with the observations obtained from

cell viability and apoptosis assays, suggesting the anti-proliferative

effects of 12b and 12r on MV4-11 cells could originate from their

inhibition of FLT3-ITD.

Chemical reagents and solvents including anhydrous solvents

were purchased from commercial sources, and used as received

unless speciﬁed otherwise. All reactions except microwave-assisted

syntheses were carried out with continuous magnetic stirring in

ordinary glassware; microwave-assisted syntheses were performed

in a 10 mL Asynt DrySyn insert (Isleham, UK) using a CEM Discover

SP and Explorer 48/72/96 microwave system (Matthews, NC, USA)

controlled by Synergy™ software (Firmware version DSCA02.17).

Heating of reactions was conducted with a DrySyn® single- or

multi-position heating block (Isleham, UK); cooling of reactions

was achieved using an ice bath. Analytical TLC was performed on

Merck silica gel 60 F₂₅₄pre-coated aluminium plates (0.2 mm) and

visualised under UV light (254 nm). Flash column chromatography

was carried out using a fritted solid loader packed with GRACE

Davison DAVISIL® silica gel 60 Å (40e63

m

m) on a Biotage Flash-

þ

Master Personal ﬂash chromatography system or using a con-

1

13

ventional glass column. H and C NMR spectra were recorded at

3

. Conclusions

298 K (unless otherwise speciﬁed) on a Bruker AVANCE III HD 500

1

13

spectrometer ( H at 500.20 MHz and C at 125.79 MHz; Fae-

llanden, Switzerland), and were analysed using Bruker Topspin 3.2

Capitalising on the serendipitous discovery of 12b as an FLT3

1

13

1

inhibitor, the utility and versatility of its core scaffold, i.e., N-phe-

nylpyrimidin-2-amine, for the fabrication of novel, potent and se-

lective FLT3 inhibitors were explored using an analogue-based

design approach. A series of 4-azaaryl-N-phenylpyrimidin-2-amine

derivatives was synthesised, and their structure-activity relation-

ships were analysed. All of these compounds displayed nanomolar

potencies against FLT3-ITD with one third of them being sub-

nanomolar inhibitors; these potencies were well reﬂected in their

anti-proliferative effects on FLT3-ITD-harbouring MV4-11 cells.

software. H and C NMR spectra are referenced to H signals of

13

residual nondeuterated solvents and C signals of deuterated sol-

vents, respectively. H NMR signals are reported with chemical shift

1

values

d

(ppm), multiplicity (s ¼ singlet, d ¼ doublet, t ¼ triplet,

dd ¼ doublet of doublets, td ¼ triplet of doublets, ddd ¼ doublet of

doublet of doublets, m ¼ multiplet and br ¼ broad), relative inte-

gral, coupling constants J (Hz) and assignments. High resolution

mass spectra were recorded on an AB SCIEX TripleTOF 5600 mass

spectrometer (Concord, ON, Canada), and ionisation of all samples

9

Y. Long, M. Yu, A.M. Ochnik et al.

European Journal of Medicinal Chemistry 213 (2021) 113215

was carried out using ESI. Melting points were determined using an

open capillary on a Stuart SMP10 or Mettler Toledo MP50 melting

point apparatus, and are uncorrected. The purity of compounds

used for biological evaluation was determined by analytic RP-HPLC

which was carried out on a Shimadzu Prominence UltraFast Liquid

Chromatograph (UFLC) system (Kyoto, Japan) equipped with a

CBM-20A communications bus module, a DGU-20A5R degassing

unit, an LC-20AD liquid chromatograph pump, an SIL-20AHT auto-

sampler, an SPD-M20A photo diode array detector, a CTO-20A

General synthetic procedure C: reduction of nitro compounds to

amines

To a suspension of a nitro compound (1.0 equiv.) in CH

(50 mM in the nitro compound) was added 10% Pd/C (0.01 equiv.).

The reaction mixture was bubbled with H gas at room temperature

for 30 min, stirred under H overnight, and ﬁltered through a pad of

Celite®. The solids were washed with CH OH (300 mL). The ﬁltrate

3

OH

2

3

and washing were combined and concentrated under reduced

pressure to give the desired amine.

column oven and

50 mm ꢃ 4.60 mm column. Method A (gradient 5%e95% CH

containing 0.1% formic acid (FA) over 20 min at a ﬂow rate of 1 mL/

min, followed by 95% CH OH containing 0.1% FA over 5 min), and

method B (gradient 5%e95% CH CN containing 0.1% FA over

0 min at a ﬂow rate of 1 mL/min, followed by 95% CH CN con-

a

Phenomenex Kinetex 5u C18 100A

General synthetic procedure D: guanidinylation of amines

To a solution of an amine (1.0 equiv.) and cyanamide (2.0 equiv.)

2

3

OH

3

in CH CN (100 mL) on an ice bath was added TMSCl (2.0 equiv.)

3

dropwise. The reaction mixture was heated at reﬂux overnight and

3

ﬁltered while hot. The precipitate was washed with Et

2

O or EtOAc

2

3

(100e150 mL) to give the desired guanidine.

taining 0.1% FA over 5 min) were used for analytic RP-HPLC. Data

acquired from analytic RP-HPLC were processed using LabSolutions

Analysis Data System.

General synthetic procedure E: formation of pyrimidines

To a solution of a guanidine (2.0 equiv.) in CH CN or 2-

3

methoxyethanol (4 mL) were added an enaminone (1.0 equiv.)

ꢁ

General synthetic procedure A: acetylation of (aza)indoles.

and NaOH (2.0 equiv.). The reaction mixture was heated at 160 C

under microwave irradiation for 1 h, cooled down to room tem-

perature and concentrated under reduced pressure. The residue

was puriﬁed by FlashMaster Personal chromatography or glass

(

i) To a solution of an indole (1.00 equiv.) in acetic anhydride

þ

(

1 M in indole) was added cyanoacetic acid (1.18 equiv.), and

ꢁ

the reaction mixture was heated at 85 C for 10 min, cooled

down to room temperature. The crystals thus formed were

column (silica gel) and washed with ice-cold CH

(3 ꢃ 5 mL) to give the desired pyrimidine.

3-(1H-indol-3-yl)-3-oxopropanenitrile (2a)

3

OH or EtOAc

ﬁltered, washed with CH

reduced pressure to give the desired 3-acetylindole.

ii) To a suspension of AlCl (5.0 equiv.) in anhydrous DCM

100 mM in azaindole) under N was added an azaindole (1.0

3

OH (50 mL) and dried under

Indole (1a, 5.86 g, 50.0 mmol) and cyanoacetic acid (5.00 g,

(

3

58.8 mmol) were reacted according to general synthetic procedure

1

(

2

A(i) to give 2a as an off-white solid (8.29 g, 90%). H NMR

equiv.). The reaction mixture was stirred at room tempera-

ture for 1 h, and acetyl chloride (5.0 equiv.) was added

dropwise. The reaction mixture was stirred at room tem-

perature overnight, cooled down on an ice bath for 15 min,

6 2

(DMSO‑d ) d 4.49 (s, 2H, CH ), 7.22e7.27 (m, 2H, indolyl-H),

7.50e7.51 (d, 1H, J 7.0, indolyl-H), 8.14 (d, 1H, J 7.0, indolyl-H),

8.37 (s, 1H, indolyl-H), 12.18 (br s, 1H, indolyl-NH). HRMS (ESI-

þ

TOF) m/z 185.0713 [MþH] ; calcd. for

C

11

H

9

N

2

O

185.0709

þ

quenched by slow addition of CH

room temperature for 1 h, and concentrated under reduced

pressure. The residue was dissolved in H O (50 mL), basiﬁed

to pH 12 with 2 M NaOH, and extracted with EtOAc

3

OH (150 mL), stirred at

[MþH] .

3-(2-Methyl-1H-indol-3-yl)-3-oxopropanenitrile (2b)

2

2-Methylindole (1b, 6.56 g, 50.0 mmol) and cyanoacetic acid

(5.00 g, 58.8 mmol) were reacted according to general synthetic

1

(

3 ꢃ 200 mL) and DCM (3 ꢃ 200 mL). The organic extracts

procedure A(i) to give 2B as an off-white solid (9.12 g, 92%). H NMR

were combined and concentrated under reduced pressure,

6 3 2

(DMSO‑d ) d 2.68 (s, 3H, CH ), 4.52 (s, 2H, CH ), 7.15e7.19 (m, 2H,

and the residue was puriﬁed by ﬂash column chromatog-

indolyl-H), 7.39 (d, 1H, J 7.0, indolyl-H), 7.97 (d, 1H, J 7.0, indolyl-H),

þ

raphy (silica gel, DCM ramping to DCM:CH

give the desired 3-acetylazaindole.

3

OH ¼ 92:8) to

12.09 (br s,1H, indolyl-NH). HRMS (ESI-TOF) m/z 199.0867 [MþH] ;

þ

calcd. for C12

11

H N

2

O

199.0866 [MþH] .

1

-(1-Tosyl-1H-indol-3-yl)ethan-1-one (2d)

General synthetic procedure B: formation of enaminones.

To a solution of 3-acetylindole (2c, 15.9 g, 100 mmol) and tet-

rabutylammonium hydrogen sulfate (3.40 g, 10.0 mmol) in DCM

was added 50% (w/v) NaOH (60 mL, 750 mmol) slowly and stirred

vigorously at room temperature for 15 min. 4-Toluenesulfonyl

chloride (28.9 g, 150 mmol) was added slowly in portions and

stirred vigorously at room temperature overnight. The reaction

(

i) To DMF-DMA (1.20 equiv.) was added a ketone (1.00 equiv.).

The reaction mixture was stirred at room temperature for

5

min, ﬁltered and crystallised with EtOH to afford the

desired enaminone.

(

ii) To DMF-DMA (5.00e10.0 equiv.) was added a ketone (1.00

equiv.). The reaction mixture was heated at reﬂux for 2e36 h,

concentrated under reduced pressure, and either crystallised

mixture was poured into H

(3 ꢃ 100 mL). The organic extracts were combined, dried over

Na SO and concentrated under reduced pressure. The resulting oil

2

O (100 mL), and extracted with DCM

2

4

with EtOH or washed with Et

enaminone.

iii) To DMF-DMA (3.00e5.00 equiv.) was added a ketone (1.00

2

O to afford the desired

was crystallised upon standing to yield 2d as an off-white solid

(31.1 g, 99%) which was used in the next step without further pu-

1

(

riﬁcation. H NMR (DMSO‑d

6

)

d

2.31 (s, 3H, tosyl-CH

3

), 2.60 (s, 3H,

equiv.). The reaction mixture was heated under microwave at

3

acetyl-CH ), 7.35 (td, 1H, J 8.0 & 1.0, indolyl-H), 7.38 (td, 1H, J 8.0 &

ꢁ

180 C for 1 h, concentrated under reduced pressure, and

1.0, indolyl-H), 7.41 (d, 2H, J 8.0, tosyl-H), 7.94 (d, 1H, J 8.0, indolyl-

H), 8.03 (d, 2H, J 8.0, tosyl-H), 8.19 (d, 1H, J 8.0, indolyl-H), 8.79 (s,

puriﬁed by ﬂash column chromatography (silica gel, DCM

þ

ramping to DCM/CH

enaminone.

3

OH

¼

9:1) to give the desired

1H, indolyl-H). HRMS (ESI-TOF) m/z 314.0849 [MþH] ; calcd. for

þ

C

17

H

16NO

3

S

314.0845 [MþH] .

0

(

iv) To

1-tert-butoxy-N,N,N ,N -tetramethylmethanediamine

1-(1H-Pyrrolo[3,2-b]pyridin-3-yl)ethan-1-one (4a)

(2.00 equiv.) was added a ketone (1.00 equiv.). The reaction

4-Azaindole (3a, 2.36 g, 20.0 mmol) and acetyl chloride (7.10 mL,

mixture was heated at reﬂux for 6 h, and concentrated via

forced evaporation under a stream of air and in vacuo. The

residue was washed with DCM (3 ꢃ 6 mL) to give the desired

enaminone.

99.9 mmol) were reacted according to general synthetic procedure

1

A(ii) to give 4a as a white solid (2.64 g, 82%). H NMR (DMSO‑d

2.72 (s, 3H, CH ), 7.23 (dd, 1H, J 8.0 & 4.5, azaindolyl-H), 7.88 (dd,

1H, J 8.0 & 1.5, azaindolyl-H), 8.29 (s,1H, azaindolyl-H), 8.50 (dd,1H,

6

)

d

3

10

Y. Long, M. Yu, A.M. Ochnik et al.

European Journal of Medicinal Chemistry 213 (2021) 113215

J 4.5 & 1.5, azaindolyl-H) (one proton signal (azaindolyl-NH) not

A solution of 6a (8.21 g, 40.0 mmol) in anhydrous THF (150 mL)

under N was cooled down on an acetone-dry ice bath for 30 min,

2

and methyl magnesium bromide (3.0 M in diethyl ether, 33.4 mL,

100 mmol) was added. The reaction mixture was stirred on the

acetone-dry ice bath for 2.5 h and at room temperature for 1 h,

þ

observed). HRMS (ESI-TOF) m/z 161.0709 [MþH] ; calcd. for

þ

C

9

H

1

9

N

2

O

161.0709 [MþH] .

-(1H-Pyrrolo[2,3-b]pyridin-3-yl)ethan-1-one (4b)

7

-Azaindole (3b, 5.91 g, 50.0 mmol) and acetyl chloride (17.8 mL,

2

50 mmol) were reacted according to general synthetic procedure

4

quenched with saturated aqueous NH Cl solution (50 mL), extrac-

1

A(ii) to give 4b as a beige solid (6.15 g, 77%). H NMR (DMSO‑d

d

6

)

ted with EtOAc (2 ꢃ 100 mL). The organic extracts were combined

and concentrated under reduced pressure, and the residue was

puriﬁed by ﬂash column chromatography (silica gel, DCM ramping

2.46 (s, 3H, CH

3

), 7.23 (dd, 1H, J 7.5 & 5.0, azaindolyl-H), 8.31 (dd,

1

H, J 5.0 & 1.5, azaindolyl-H), 8.45 (s,1H, azaindolyl-H), 8.46 (dd,1H,

1

J 7.5 & 1.5, azaindolyl-H), 12.42 (br s, 1H, azaindolyl-NH). HRMS

to DCM:MeOH ¼ 98:2) to give 7a as a white solid (6.09 g, 95%). H

þ

(

[

ESI-TOF) m/z 161.0700 [MþH] ; calcd. for C

H

9 9

N

2

O

161.0709

3 3

NMR (CDCl ) d 2.76 (s, 3H, CH ), 7.35 (ddd, 1H, J 8.0 & 7.0 & 1.0,

þ

MþH] .

indazolyl-H), 7.46 (ddd, 1H, J 8.0 & 7.0 & 1.0, indazolyl-H), 7.55 (d,

1H, J 8.5, indazolyl-H), 8.40 (dt, 1H, J 8.0 & 1.0, indazolyl-H), 10.49

1

-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)ethan-1-one (4c) &

þ

1

-(7-methyl-7H-pyrrolo[2,3-b]pyridin-3-yl)ethan-1-one (4d)

To DMF-DMA (4.00 mL, 30.1 mmol) was added 4b (1.61 g,

(br s, 1H, indazolyl-NH). HRMS (ESI-TOF) m/z 161.0709 [MþH] ;

þ

calcd. for C

9

H

9

N

2

O

161.0709 [MþH] .

10.0 mmol). The reaction mixture was heated at reﬂux overnight

3-(1H-indazol-3-yl)-3-oxopropanenitrile (7b)

and concentrated under reduced pressure. The residue was puriﬁed

by FlashMaster Personal chromatography (silica gel, DCM ramping

To a solution of 6b (2.75 g, 9.01 mmol) in anhydrous THF (50 mL)

under N was added anhydrous CH CN (706 mL, 13.5 mmol). The

2 3

þ

to DCM:CH

3

OH ¼ 95:5) to give 4c as a beige solid (891 mg, 51%) and

reaction mixture was cooled down on an acetone-dry ice bath for

30 min, and lithium diisopropylamide (2.0 M in THF/heptane/eth-

ylbenzene, 6.75 mL, 13.5 mmol) was added dropwise over 15 min.

The reaction mixture was stirred on the acetone-dry ice bath for 2 h

4

d as a beige solid (457 mg, 26%).

1

4

c: H NMR (DMSO‑d 2.44 (s, 3H, COCH ), 3.87 (s, 3H,

6

)

d

3

azaindolyl-NCH ), 7.28 (dd, 1H, J 7.5 & 4.5, azaindolyl-H), 8.37 (dd,

H, J 4.5 & 1.5, azaindolyl-H), 8.46 (dd, 1H, J 7.5 & 1.5, azaindolyl-H),

3

1

8

and at room temperature overnight, quenched with H

acidiﬁed with 2.0 M HCl to pH 1, extracted with CHCl

The organic extracts were combined and concentrated under the

2

O (10 mL),

(3 ꢃ 100 mL).

þ

.52 (s, 1H, azaindolyl-H). HRMS (ESI-TOF) m/z 175.0878 [MþH] ;

3

þ

calcd. for C10

H

11

N

2

O

175.0866 [MþH] .

1

4

d: H NMR (DMSO‑d

6

)

d

2.43 (s, 3H, COCH

3

), 4.29 (s, 3H,

reduced pressure, the residue was puriﬁed by FlashMaster Per-

þ

azaindolyl-NCH

3

), 7.26 (dd, 1H, J 7.5 & 6.5, azaindolyl-H), 8.28 (d,

H, J 6.0, azaindolyl-H), 8.44 (s, 1H, azaindolyl-H), 8.69 (dd, 1H, J 7.5

sonal

chromatography (silica gel, DCM ramping to

1

DCM:EtOAc ¼ 9:1) to give 7b as a beige solid (1.36 g, 81%). H NMR

þ

&

for C10

1.0, azaindolyl-H). HRMS (ESI-TOF) m/z 175.0841 [MþH] ; calcd.

(CDCl 4.31 (s, 2H, CH ), 7.41 (ddd, 1H, J 8.0 & 7.0 & 1.0, indazolyl-

3

)

d

2

þ

H

11

N

2

O

175.0866 [MþH] .

H), 7.52 (ddd, 1H, J 8.0 & 7.0 &1.0, indazolyl-H), 7.59 (d, 1H, J 8.5,

indazolyl-H), 8.34 (d, 1H, J 8.5, indazolyl-H), 10.50 (br s, 1H,

N-Methoxy-N-methyl-1H-indazole-3-carboxamide (6a)

To a solution of 1H-indazole-3-carboxylic acid (5, 4.87 g,

0.0 mmol) in THF (250 mL) was added N,O-dimethylhydroxyl-

þ

indazolyl-NH). HRMS (ESI-TOF) m/z 186.0667 [MþH] ; calcd. for

þ

3

C

10

8

H N

3

O

186.0662 [MþH] .

amine hydrochloride (3.22 g, 33.0 mmol) at room temperature. The

reaction mixture was cooled on an ice-salt bath for 30 min, and

pyridine (5.34 mL, 66.0 mmol) was added. The resultant reaction

mixture was stirred on the ice-salt bath for 2 h and at room tem-

perature for 1 h. Pyridine (5.34 mL, 66.0 mmol) and EDC$HCl (11.5 g,

3-(Dimethylamino)-2-(1H-indole-3-carbonyl)acrylonitrile (8a)

2a (1.85 g, 10.0 mmol) and DMF-DMA (1.50 mL, 11.3 mmol) were

reacted according to general synthetic procedure B(i) to afford 8a as

1

a yellow solid (2.31 g, 96%). H NMR (DMSO‑d

6 3

) d 3.27 (s, 3H, NCH ),

3.37 (s, 3H, NCH ), 7.12e7.20 (m, 2H, indolyl-H), 7.47 (d, 1H, J 8.0,

3

6

0.0 mmol) were added. The reaction mixture was stirred at room

temperature overnight and concentrated under reduced pressure,

and the residue was triturated with H O (500 mL). The solids were

ﬁltered and washed with H O (400 mL) to give 6a as a white solid

5.26 g, 85%). ¹H NMR (CDCl

3.57 (s, 3H, NCH ), 3.81 (s, 3H,

), 7.24e7.28 (m, 1H, indazolyl-H), 7.39e7.43 (m, 1H, indazolyl-

indolyl-H), 8.00 (s,1H, NCH]C(CO)CN), 8.13 (d,1H, J 8.0, indolyl-H),

8.27 (s, 1H, indolyl-H), 11.76 (br s, 1H, indolyl-NH). HRMS (ESI-TOF)

þ

2

240.1130 ([MþH] ); calcd. for C14

H

14

N

3

O

([MþH] ) 240.1131.

2

3-(Dimethylamino)-2-(1-methyl-1H-indole-3-carbonyl)acrylo-

(

3

)

d

3

nitrile (8b)

OCH

3

2a (478 mg, 3.00 mmol) and DMF-DMA (4.30 mL, 32.4 mmol)

were reacted according to general synthetic procedure B(ii) in the

presence of DMF (6 mL). The residue was crystallised with EtOH

H), 7.55 (d, 1H, J 8.5, indazolyl-H), 8.21 (d, 1H, J 8.0, indazolyl-H),

11.24 (br s, 1H, indazolyl-NH). HRMS (ESI-TOF) m/z 206.0927

þ

1

[

MþH] ; calcd. for C10

Tert-butyl

carboxylate (6b)

H

12

N

3

O

2

206.0924 [MþH] .

(5 mL) to afford 8b as a yellow solid (545 mg, 81%). H NMR

3-(methoxy(methyl)carbamoyl)-1H-indazole-1-

(DMSO‑d

6

)

d

3 3 3 3

3.27 (s, 3H, CH NCH ), 3.36 (s, 3H, CH NCH ), 3.86 (s,

3H, indolyl-NCH

3

), 7.18e7.28 (m, 2H, indolyl-H), 7.52 (d, 1H, J 8.0,

To a solution of 6a (4.10 g, 20.0 mmol) in DCM (250 mL) on an

ice-salt bath were added DMAP (489 mg, 4.00 mmol), triethylamine

indolyl-H), 7.99 (s, 1H, NCH]C(CO)CN), 8.14 (d, 1H, J 7.5, indolyl-H),

þ

8.24 (s, 1H, indolyl-H). HRMS (ESI-TOF) 254.1311 ([MþH] ); calcd.

þ

(

4

5.58 mL, 40.0 mmol) and di-tert-butyl dicarbonate (8.73 g,

0.0 mmol). The reaction mixture was stirred on the ice-salt bath

for 1 h and at room temperature overnight, washed with 0.5 M HCl

100 mL), H O (100 mL) and brine (100 mL), dried over Na SO , and

for C15

H

16

N

3

O

([MþH] ) 254.1288.

3-(Dimethylamino)-2-(2-methyl-1H-indole-3-carbonyl)acrylo-

nitrile (8c)

(

2

4

2b (1.98 g, 10.0 mmol) and DMF-DMA (1.50 mL, 11.3 mmol) were

concentrated under the reduced pressure. The residue was puriﬁed

by ﬂash column chromatography (silica gel, petroleum benzine

reacted according to general synthetic procedure B(i) to afford 8c as

1

a yellow solid (2.48 g, 98%). H NMR (DMSO‑d

6

)

d

2.50 (s, 3H,

ramping to petroleum benzine:EtOAc ¼ 7:3) to give 6b as a white

indolyl-CH

3

), 3.20 (s, 3H, NCH

3

), 3.35 (s, 3H, NCH

3

), 7.02e7.09 (m,

1

solid (5.98 g, 98%). H NMR (CDCl

3

)

d

1.67 (s, 9H, C(CH

3

)

3

), 3.43 (br s,

2H, indolyl-H), 7.31 (d,1H, J 8.0, indolyl-H), 7.56 (d,1H, J 8.0, indolyl-

H), 7.74 (s, 1H, NCH]C(CO)CN), 11.56 (br s, 1H, indolyl-NH). HRMS

3

H, NCH

3 3

), 3.86 (s, 3H, OCH ), 7.32 (app t, 1H, J 8.0, indazolyl-H),

þ

7.49 (ddd, 1H, J 8.0 & 7.0 &1.0, indazolyl-H), 8.08 (app br s, 1H,

(ESI-TOF) 254.1269 ([MþH] ); calcd. for C15

H

16

N

3

O

([MþH] )

indazolyl-H), 8.11 (d, 1H, J 8.5, indazolyl-H). HRMS (ESI-TOF) m/z

254.1288.

þ

8

6

33.2660 [2MþNa] ; calcd. for

C

H

30 38

N

6

NaO

633.2643

3-(Dimethylamino)-1-(1-methyl-1H-indol-3-yl)prop-2-en-1-

one (8d)

3-Acetylindole (2c, 5.00 g, 31.4 mmol) and DMF-DMA (45.0 mL,

þ

[2MþNa] .

1

-(1H-indazol-3-yl)ethan-1-one (7a)

11

Y. Long, M. Yu, A.M. Ochnik et al.

European Journal of Medicinal Chemistry 213 (2021) 113215

3

39 mmol) were reacted according to general synthetic procedure

(E)-3-(Dimethylamino)-1-(1H-indazol-3-yl)prop-2-en-1-one

B(ii) in the presence of DMF (63 mL). The residue was crystallised

with EtOH (20 mL) to afford 8d as a yellow solid (5.45 g, 76%). H

(8j)

1

0

7a (4.80 g, 30.0 mmol) and 1-tert-butoxy-N,N,N ,N -tetrame-

NMR (DMSO‑d

5

1

6

)

d

2.96 (s, 6H, CH

.72 (d, 1H, J 12.5, CH]CHCO), 7.14 (t, 1H, J 7.5, indolyl-H), 7.20 (t,

H, J 7.5, indolyl-H), 7.46 (d, 1H, J 8.0, indolyl-H),7.54 (d, 1H, J 12.5,

3

NCH

3

), 3.82 (s, 3H, indolyl-NCH

3

),

thylmethanediamine (12.4 mL, 60.0 mmol) were reacted according

to general synthetic procedure B(iv) to give 8j as a beige solid

1

(5.82 g, 90%). H NMR (DMSO‑d

6

)

d

2.89 (s, 3H, CH

3

), 3.14 (s, 3H,

CH]CHCO), 8.16 (s, 1H, indolyl-H), 8.30 (d, 1H, J 8.0, indolyl-H).

3

CH ), 6.09 (d, 1H, J 12.5, CH]CHCO), 7.19e7.23 (m, 1H, indazole-H),

þ

HRMS (ESI-TOF) 229.1339 ([MþH] ); calcd. for

C

14

H

17

N

2

O

7.37 (ddd, 1H, J 8.0 & 7.0 & 1.0, indazole-H), 7.58 (d, 1H, J 8.5,

indazole-H), 7.74 (d, 1H, J 12.5, CH]CHCO), 8.28 (d, 1H, J 8.0,

indazole-H), 13.42 (br s, 1H, indazole-NH). HRMS (ESI-TOF) m/z

þ

(

[MþH] ) 229.1335.

3

-(Dimethylamino)-1-(1-tosyl-1H-indol-3-yl)prop-2-en-1-one

þ

(

8e)

216.1136 [MþH] ; calcd. for C12

H

14

N

3

O

216.1131 [MþH] .

2

d (3.13 g, 10.0 mmol) and DMF-DMA (3.0 mL, 22.6 mmol) were

(E)-3-(Dimethylamino)-1-(1-methyl-1H-indazol-3-yl)prop-2-

en-1-one (8k)

reacted according to general synthetic procedure B(ii). The solid

thus formed was ﬁltered and washed with Et O to afford 8e as a

2.32 (s, 3H, tosyl-

), 5.93 (d, 1H, J 12.5, CH]CHCO), 7.11 (d,

2

d

7a (1.61 g, 10.0 mmol) and DMF-DMA (13.3 mL, 100 mmol) were

1

yellow solid (2.87 g, 78%). H NMR (DMSO‑d

CH ), 3.10 (s, 6H, CH NCH

6

)

reacted according to general synthetic procedure B(iii) to give 8k as

1

3

a yellow solid (1.60 g, 70%). H NMR (CDCl

3

)

d

2.87 (br s, 3H,

2

8

H, J 8.0, tosyl-H), 7.29 (td, 1H, J 8.0 & 1.0, indolyl-H), 7.35 (td, 1H, J

.0 & 1.0, indolyl-H), 7.48 (d, 1H, J 8.0, indolyl-H), 7.64 (d, 1H, J 12.5,

CH

3

NCH

3

), 3.04 (br s, 3H, CH

3

NCH

3

), 4.03 (s, 3H, indazolyl-CH ),

3

6.12 (d, 1H, J 12.5, CH]CHCO), 7.21 (ddd, 1H, J 8.0 & 6.0 & 1.5,

indazolyl-H), 7.29e7.35 (m, 1H, indazolyl-H), 7.32 (d, 1H, J 6.5,

indazolyl-H), 7.80 (d, 1H, J 12.5, CH]CHCO), 8.43 (d, 1H, J 8.5,

CH]CHCO), 7.95 (d, 2H, J 8.0, tosyl-H), 8.30 (d, 1H, J 8.0, indolyl-H),

þ

8

.61 (s, 1H, indolyl-H). HRMS (ESI-TOF) 369.1274 ([MþH] ); calcd.

þ

for C20

H

21

N

2

O

3

S

([MþH] ) 369.1267.

indazolyl-H). HRMS (ESI-TOF) m/z 230.1291 [MþH] ; calcd. for

þ

(

E)-3-(Dimethylamino)-1-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-

C

13

16

H N

3

O

230.1288 [MþH] .

2

-en-1-one (8f)

a (2.56 g, 16.0 mmol) and 1-tert-butoxy-N,N,N ,N -tetrame-

(E)-3-(Dimethylamino)-2-(1H-indazole-3-carbonyl)acryloni-

0

4

trile (8l)

To DMF-DMA (2.00 mL, 15.0 mmol) was added to 7b (352 mg,

thylmethanediamine (6.53 mL, 32.0 mmol) were reacted according

to general synthetic procedure B(iv) to give 8f as a dark brown solid

1.90 mmol). The suspension was stirred at room temperature for

1

(

2.38 g, 69%). H NMR (DMSO‑d

6

)

d

2.96 (br s, 3H, CH

3

), 3.07 (br s,

30 min, and concentrated via forced evaporation under a stream of

þ

3

H, CH

azaindolyl-H), 7.69 (d, 1H, J 12.5, CH]CHCO), 7.83 (dd, 1H, J 8.5 &

.5, azaindolyl-H), 8.12 (s, 1H, azaindolyl-H), 8.46 (dd, 1H, J 4.5 & 1.5,

3

), 6.83 (d, 1H, J 12.5, CH]CHCO), 7.16 (dd, 1H, J 8.5 & 4.5,

N

2

and in vacuo. The residue was puriﬁed by FlashMaster Personal

chromatography (silica gel, DCM ramping to DCM:CH

3

OH ¼ 97:3)

to give 8l as a yellow solid (330 mg, 72%). H NMR (DMSO‑d 3.28

(s, 3H, CH ), 3.39 (s, 3H, CH ), 7.26 (app t, 1H, J 7.5, indazolyl-H), 7.42

1

6

) d

azaindolyl-H) (one proton signal (azaindolyl-NH) not observed).

3

þ

HRMS (ESI-TOF) m/z 216.1134 [MþH] ; calcd. for C12

H

14

N

3

O

(app t, 1H, J 7.5, indazolyl-H), 7.62 (d, 1H, J 8.5, indazolyl-H), 8.09 (d,

1H, J 8.0, indazolyl-H), 8.51 (s, 1H, CH]C(CN)CO), 13.69 (br s, 1H,

þ

2

16.1131 [MþH] .

þ

(

E)-3-(Dimethylamino)-1-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-

-en-1-one (8g)

b (1.61 g, 10.0 mmol) and 1-tert-butoxy-N,N,N ,N -tetrame-

indazolyl-NH). HRMS (ESI-TOF) m/z 503.1917 [2MþNa] ; calcd. for

þ

2

C

H N

26 24 8

NaO

2

503.1914 [2MþNa] .

0

4

1-(4-(4-Aminophenyl)piperazin-1-yl)ethan-1-one (10a)

thylmethanediamine (4.13 mL, 20.0 mmol) were reacted according

1-(4-(4-Nitrophenyl)piperazin-1-yl)ethan-1-one (9a, 2.50 g,

to general synthetic procedure B(iv) to give 8g as a beige solid

10.0 mmol) was reduced according to general synthetic procedure

1

(

1.83 g, 85%). H NMR (DMSO‑d

6

)

d

2.98 (br s, 6H, CH

3

NCH

3

), 5.80 (d,

C to give 10a as a grey solid (2.19 g, 100%). H NMR (CDCl

3H, CH ), 3.55e3.60 (m, 2H, CH

), 2.92e2.94 (m, 4H, 2 ꢃ CH

3.73e3.78 (m, 2H, CH ), 3.45 (br s, 2H, NH ), 6.64 (d, 2H, J 9.0,

3

):

d

2.09 (s,

1

(

8

H, J 12.5, CH]CHCO), 7.15 (dd, 1H, J 7.5 & 4.5, azaindolyl-H), 7.56

d, 1H, J 12.5, CH]CHCO), 8.24 (dd, 1H, J 4.5 & 1.5, azaindolyl-H),

.29 (s, 1H, azaindolyl-H), 8.54 (dd, 1H, J 7.5 & 1.5, azaindolyl-H),

3

2

),

2

2 ꢃ Ph-H), 6.81 (d, 2H, J 9.0, 2 ꢃ Ph-H). HRMS (ESI-TOF) m/z

þ

1

2.11 (br s, 1H, azaindolyl-NH). HRMS (ESI-TOF) m/z 216.1137

220.1452 [MþH] ; calcd. For C12

H

18

N

3

O

220.1444 [MþH] .

þ

[MþH] ; calcd. for C12

H

14

N

3

O

216.1131 [MþH] .

3-(4-Methylpiperazin-1-yl)aniline (10b)

(

E)-3-(Dimethylamino)-1-(1-methyl-1H-pyrrolo[2,3-b]pyridin-

1-Methyl-4-(3-nitrophenyl)piperazine (9b, 5.00 g, 22.6 mmol)

3

-yl)prop-2-en-1-one (8h)

c (732 mg, 4.20 mmol) and DMF-DMA (3.00 mL, 22.6 mmol)

were reacted according to general synthetic procedure B(iii) to give

was reduced according to general synthetic procedure C to give 10b

1

4

as an orange solid (4.32 g, 100%). H NMR (CDCl

3

):

d

2.34 (s, 3H,

), 3.60 (br s,

), 6.21 (d, 1H, J 8.0, Ph-H), 6.25 (s, 1H, Ph-H), 6.37 (d, 1H, J

CH

2H, NH

3

), 2.55 (t, 4H, J 5.0, 2 ꢃ CH

2

), 3.18 (t, 4H, J 5.0, 2 ꢃ CH

2

1

8

h as an orange solid (819 mg, 85%). H NMR (CDCl

3

)

d

2.73 (br s,

2

6

H, CH NCH ), 3.68 (s, 3H, azaindolyl-CH ), 5.36 (d, 1H, J 12.5, CH]

3

8.0, Ph-H), 7.06 (t, 1H, J 8.0, Ph-H). HRMS (ESI-TOF) m/z 192.1483

þ

CHCO), 6.99 (dd, 1H, J 8.0 & 5.0, azaindolyl-H), 7.53 (d, 1H, J 12.5,

CH]CHCO), 7.60 (s, 1H, azaindolyl-H), 8.16 (dd, 1H, J 4.5 & 1.5,

azaindolyl-H), 8.49 (dd, 1H, J 8.0 & 1.5, azaindolyl-H). HRMS (ESI-

[MþH] ; calcd. for C11

H

18

N

3

192.1495 [MþH] .

1-(4-(4-Acetylpiperazin-1-yl)phenyl)guanidine hydrochloride

(11a)

þ

230.1288

TOF) m/z 230.1285 [MþH] ; calcd. for C13

H

16

N

3

O

Amine 10a (1.50 g, 6.84 mmol) and cyanamide (580 mg,

þ

[MþH] .

13.7 mmol) were reacted using general synthetic procedure D to

1

(

E)-3-(Dimethylamino)-1-(7-methyl-7H-pyrrolo[2,3-b]pyridin-

-yl)prop-2-en-1-one (8i)

d (435 mg, 2.50 mmol) and DMF-DMA (1.00 mL, 7.53 mmol)

were reacted according to general synthetic procedure B(iii) to give

give 11a as a grey solid (1.73 g, 85%). H NMR (D

2

O)

d 2.17 (s, 3H,

3

CH

4H, 2 ꢃ CH

(ﬁve proton signals (NH

3

), 3.20 (t, 2H, J 5.0, CH

2

), 3.26 (t, 2H, J 5.0, CH

2

), 3.70e3.75 (m,

4

2

), 7.01 (d, 2H, J 9.0, 2 ꢃ Ph-H), 7.63 (d, 2H, J 9.0, 2 ꢃ Ph-H)

2

& 2 ꢃ NH & HCl) not observed due to H/D

1

þ

8

i as a brown solid (486 mg, 85%). H NMR (CDCl

3

)

d

2.77 (br s, 6H,

exchange). HRMS (ESI-TOF) m/z 262.1675 [M-HClþH] ; calcd. For

þ

CH NCH ), 4.04 (s, 3H, azaindolyl-CH ), 5.54 (d, 1H, J 12.0, CH]

3

C

13

20

H N

5

O

262.1662 [M-HClþH] .

CHCO), 6.74 (app t, 1H, J 6.5, azaindolyl-H), 7.41 (d, 1H, J 6.0,

azaindolyl-H), 7.49 (d,1H, J 12.5, CH]CHCO), 8.18 (s,1H, azaindolyl-

H), 8.70 (d, 1H, J 7.0, azaindolyl-H). HRMS (ESI-TOF) m/z 230.1285

1-(3-(4-Methylpiperazin-1-yl)phenyl)guanidine

dihydro-

chloride (11b)

Amine 10b (4.00 g, 20.9 mmol) and cyanamide (1.77 g,

41.8 mmol) were reacted using general synthetic procedure D to

þ

[MþH] ; calcd. for C13

H

16

N

3

O

230.1288 [MþH] .

12

Y. Long, M. Yu, A.M. Ochnik et al.

European Journal of Medicinal Chemistry 213 (2021) 113215

1

give 11b as a grey solid (5.35 g, 95%). H NMR (DMSO‑d

H, CH ), 3.13 (t, 4H, J 4.5, 2 ꢃ CH

), 2.42 (t, 4H, J 4.5, 2 ꢃ CH

d,1H, J 8.0, Ph-H), 6.68 (s,1H, Ph-H), 6.84 (d,1H, J 8.5, Ph-H), 7.01 (t,

H, J 8.0, Ph-H) (six proton signals (NH

& 2 ꢃ NH & 2 ꢃ HCl) not

6

)

d

2.19 (s,

157.1, 161.9, 167.4 (two carbon signals overlapping or obscured).

þ

3

(

1

3

2

), 6.58

HRMS (ESI-TOF) m/z 386.2081 [MþH] ; calcd. for C22

H

24

N

7

þ

386.2088 [MþH] . Anal. RP-HPLC: t

R

¼ 13.42 min, purity >95%

2

(method A); t

R

¼ 9.82 min, purity > 95% (method B).

observed due to H/D exchange). HRMS (ESI-TOF) m/z 234.1710 [M-

4-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-N-(3-(4-

þ

2

HClþH] ; calcd. For C12

20

H N

5

234.1713 [M-2HClþH] .

methylpiperazin-1-yl)phenyl)pyrimidin -2-amine (12d)

1

-(4-(4-((4-(1H-Pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-yl)

Guanidine 11b (233 mg, 1.00 mmol) and enaminone 8h (115 mg,

500 mol) were coupled using general synthetic procedure E. The

residue was puriﬁed by glass column (silica gel, DCM ramping to

DCM:CH

OH ¼ 96:4) and washed with EtOAc (10 mL) to give 12d as

a beige solid (63 mg, 32%). m.p. 192e194 C (decomposed). H NMR

(DMSO‑d 2.22 (s, 3H, piperazinyl-CH

), 2.44 (t, 4H, J ¼ 5.0,

CH N(CH N(Ph)CH ), 3.91 (s, 3H,

), 3.12 (t, 4H, J ¼ 5.0, CH

azaindolyl-CH

amino)phenyl)piperazin-1-yl)ethan-1-one (12a)

Guanidine 11a (522 mg, 2.00 mmol) and enaminone 8g (216 mg,

.00 mmol) were coupled using general synthetic procedure E. The

m

1

3

þ

ꢁ

1

residue was puriﬁed by FlashMaster Personal chromatography

silica gel, DCM ramping to DCM:CH

OH ¼ 92:8) and washed with

CH OH (15 mL) to give 12a as a golden solid (230 mg, 56%). m.p.

48e150 C (decomposed). H NMR (DMSO‑d

.04 (t, 2H, J 5.0, CH N(Ph)CH ), 3.11 (t, 2H, J 5.0, CH

t, 2H, J 6.0, CH N(COCH )CH ), 3.61 (t, 2H, J 6.0, CH

.98 (d, 2H, J 9.0, 2 ꢃ Ph-H), 7.25 (dd, 1H, J 8.0 & 5.0, azaindolyl-H),

(

3

6

)

d

3

2

3

)CH

2

ꢁ

1

3

(

6

)

d

2.05 (s, 3H, CH

N(Ph)CH ), 3.59

N(COCH )CH ),

3

),

3

), 6.58 (d, 1H, J ¼ 8.0, Ph-H), 7.14 (t, 1H, J ¼ 8.0, Ph-H),

2

7.21 (d, 1H, J ¼ 5.0, pyrimidinyl-H), 7.24 (dd, 1H, J ¼ 8.0 & 4.5,

azaindolyl-H), 7.29 (d, 1H, J ¼ 8.0, Ph-H), 7.40 (s, 1H, Ph-H),

8.35e8.38 (m, 2H, azaindolyl-H & pyrimidinyl-H), 8.50 (s, 1H,

azaindolyl-H), 8.94 (d, 1H, J ¼ 7.5, azaindolyl-H), 9.28 (s, 1H, pyr-

2

3

2

3

2

6

7

2

.44 (d, 2H, J 9.0, 2 ꢃ Ph-H), 7.62e7.66 (m, 3H, pyrimidinyl-H &

ꢃ azaindolyl-H), 8.46 (d, 1H, J 5.0), 8.67 (d, 1H, J 5.0) (total 2H,

pyrimidinyl-H & azaindolyl-H), 9.42 (s, 1H, pyrimidinyleNHePh),

3.63 (s, 1H, azaindolyl-NH). C NMR (DMSO‑d

8.6, 49.3, 49.7, 106.9, 110.6, 116.6, 121.1, 121.8, 123.2, 126.6, 133.1,

13

6

imidinyleNHePh). C NMR (DMSO‑d ) d 31.4, 45.7, 48.2, 54.6,

106.8, 109.0, 110.4, 111.1, 117.1, 117.9, 128.8, 130.9, 132.6, 141.4, 143.5,

148.2, 151.4, 157.2, 160.2, 161.5, 167.0 (two carbon signals over-

13

1

6

) d 21.3, 40.8, 45.6,

þ

4

lapping or obscured). HRMS (ESI-TOF) m/z 400.2243 [MþH] ; calcd.

þ

1

41.3, 141.5, 146.2, 158.1, 160.4, 160.7, 168.3 (two carbon signals

for C23H

26

N

7

400.2244 [MþH] . Anal. RP-HPLC: t

R

¼ 14.01 min,

þ

overlapping or obscured). HRMS (ESI-TOF) m/z 414.2041 [MþH] ;

purity >96% (method A); t

1-(4-(3-((4-(1H-Pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-yl)

amino)phenyl)-1,4-diazepan-1-yl)ethan-1-one (12e)

R

¼ 10.33 min, purity > 96% (method B).

þ

calcd. for

C

H N

23 24 7

O

414.2037 [MþH] . Anal. RP-HPLC:

t

R

¼ 18.32 min, purity >98% (method A); t ¼ 13.34 min, pu-

R

rity > 98% (method B).

-(4-(4-((4-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyr-

Guanidine 11c (300 mg, 1.08 mmol) and enaminone 8g (110 mg,

1

510

m

mol) were coupled using general synthetic procedure E. The

þ

imidin-2-yl)amino)phenyl)-piperazin-1-yl)ethan-1-one (12b)

residue was puriﬁed by FlashMaster Personal chromatography

Guanidine 11a (522 mg, 2.00 mmol) and enaminone 8h

(silica gel, DCM ramping to DCM:CH

O)) and washed with CH OH (10 mL) to give 12e as a beige solid

(65 mg, 30%). m.p. 150e152 C. H NMR (DMSO‑d

two rotamers in approximately 1:1 ratio.) 1.79e1.83 (m, 1H,

CH CH CH of one rotamer), 1.87 (s, 1.5H, CH of one rotamer),

1.89e1.93 (m, 1H, CH CH CH of the other rotamer), 1.99 (s, 1.5H,

CH of the other rotamer), 3.29 (t, 2H, J 6.0), 3.50 (t, 2H, J 6.0), 3.53

(t, 1H, J 6.0), 3.59e3.63 (m, 2H), 3.64 (t, 1H, J 5.0) (total 8H,

CH N(COCH )CH & CH N(Ph)CH ), 6.39 (d, 1H, J 8.0, Ph-H), 7.09 (t,

3

OH ¼ 91:9 þ 0.2% NH

3

(32% in

(

230 mg, 1.01 mmol) were coupled using general synthetic pro-

H

2

3

þ

ꢁ

1

cedure E. The residue was puriﬁed by FlashMaster Personal

chromatography (silica gel, DCM ramping to DCM:CH

and washed with CH OH (15 mL) to give 12b as a beige solid

245 mg, 57%). m.p. 218e220 C. H NMR (DMSO‑d

COCH ), 3.03 (t, 2H, J 5.0, CH N(Ph)CH ), 3.10 (t, 2H, J 5.0, CH

CH ), 3.59 (t, 2H, J 6.0, CH N(COCH )CH ), 3.60 (t, 2H, J 6.0,

CH N(COCH )CH ), 3.90 (s, 3H, azaindolyl-CH ), 6.96 (d, 2H, J 9.0,

6

) (12e exists as

3

OH ¼ 96:4)

d

3

2

3

ꢁ

1

(

6

)

d

2.05 (s, 3H,

2

3

2

N(Ph)

3

2

3

2

3

2

3

2

3

2

ꢃ Ph-H), 7.16 (d, 1H, J 5.5, pyrimidinyl-H), 7.24 (dd, 1H, J 7.5 & 4.5,

1H, J 8.0, Ph-H), 7.15 (d, 1H, J 8.0, Ph-H), 7.18e7.27 (m, 2H, Ph-H &

azaindolyl-H), 7.29 (d, 1H, J 5.5, pyrimidinyl-H), 8.31 (dd, 1H, J 4.5 &

1.5, azaindolyl-H), 8.34 (d, 1H, J 5.5, pyrimidinyl-H), 8.45 (s, 1H,

azaindolyl-H), 7.63 (d, 2H, J 9.0, 2 ꢃ Ph-H), 8.31 (d, 1H, J 5.5,

pyrimidinyl-H), 8.36 (dd, 1H, J 4.5 & 1.0, azaindolyl-H), 8.48 (s, 1H,

azaindolyl-H), 8.90 (apparent d, 1H, J 6.5, azaindolyl-H), 9.22 (s, 1H,

azaindolyl-H), 8.97 (d, 1H, J 7.5, azaindolyl-H), 9.21 (s, 1H, pyr-

pyrimidinyleNHePh). ¹³C NMR (DMSO‑d

d

21.2, 31.4, 40.8, 45.7,

imidinyleNHePh), 12.30 (s, 1H, azaindolyl-NH).

(DMSO‑d

13

C

NMR

6

)

4

9.4, 49.8, 106.3, 111.2, 116.6, 117.1, 118.0, 120.8, 130.9, 132.5, 133.4,

6

)

d

21.0, 21.3 (one carbon), 24.0, 25.9 (one carbon), 43.9,

1

43.4, 146.0, 148.2, 157.2, 160.3, 161.5, 168.3 (two carbon signals

44.5, 46.9, 47.0, 47.1, 48.2, 49.0, 49.7 (four carbons),102.5,102.7 (one

carbon), 105.3, 105.4 (one carbon), 106.9, 107.4, 112.4, 116.9, 117.7,

129.1, 129.2 (one carbon), 130.7, 141.9, 143.6, 147.3, 147.5, 149.3,

157.0, 160.2, 161.9, 169.0, 169.3 (one carbon). HRMS (ESI-TOF) m/z

þ

overlapping or obscured). HRMS (ESI-TOF) m/z 428.2198 [MþH] ;

þ

calcd. for

C

H

24 26

N

7

O

428.2193 [MþH] . Anal. RP-HPLC:

t

R

¼ 16.32 min, purity >95% (method A); t ¼ 11.57 min, pu-

R

þ

rity > 95% (method B).

N-(3-(4-methylpiperazin-1-yl)phenyl)-4-(1H-pyrrolo[2,3-b]

pyridin-3-yl)pyrimidin-2-amine (12c)

428.2194 [MþH] ; calcd. for C24

H

26

N

7

O

428.2193 [MþH] . Anal.

RP-HPLC: t

R

¼ 16.74 min, purity >96% (method A); t ¼ 11.76 min,

R

purity > 95% (method B).

Guanidine 11b (233 mg, 1.00 mmol) and enaminone 8g (108 mg,

1-(4-(3-((4-(7-Methyl-7H-pyrrolo[2,3-b]pyridin-3-yl)pyr-

imidin-2-yl)amino)phenyl)-1,4-diazepan-1-yl)ethan-1-one (12f)

Guanidine 11c (300 mg, 1.08 mmol) and enaminone 8i (115 mg,

5

00

residue was puriﬁed by glass column (silica gel, DCM ramping to

DCM:CH

OH ¼ 95:5) and washed with EtOAc (10 mL) to give 12c as

a beige solid (33 mg, 17%). m.p. 228e230 C (decomposed). H NMR

DMSO‑d 2.25 (s, 3H, CH N(Ph)CH ),

.58 (d, 1H, J ¼ 8.0, Ph-H), 7.14 (t, 1H, J ¼ 8.0, Ph-H), 7.19 (dd, 1H, J ¼

.0 & 4.5, azaindolyl-H), 7.28e7.31 (m, 2H, pyrimidinyl-H & Ph-H),

mmol) were coupled using general synthetic procedure E. The

3

500

m

mol) were coupled using general synthetic procedure E. The

ꢁ

1

þ

residue was puriﬁed by FlashMaster Personal chromatography

(

6

8

7

1

6

)

d

3

), 3.13 (t, 4H, J ¼ 4.5, CH

2

(silica gel, DCM ramping to DCM:CH

O)) and washed with Et O (10 mL) to give 12f as a yellow solid

(56 mg, 25%). m.p. 144e146 C. H NMR (DMSO‑d

two rotamers in approximately 1:1 ratio.) 1.79e1.83 (m, 1H,

CH CH CH of one rotamer), 1.87 (s, 1.5H, CH of one rotamer),

1.89e1.93 (m, 1H, CH CH CH of the other rotamer), 1.99 (s, 1.5H,

CH of the other rotamer), 3.30 (t, 2H, J 6.0), 3.50 (t, 2H, J 5.5), 3.53

(t, 1H, J 5.5), 3.60e3.64 (m, 2H), 3.65 (t, 1H, J 5.0) (total 8H,

CH N(COCH )CH & CH N(Ph)CH ), 4.31 (s, 3H, azaindolyl-CH ),

3

OH ¼ 97:3 þ 0.2% NH

3

(32% in

H

2

ꢁ

1

6

) (12f exists as

.41 (s, 1H, Ph-H), 8.31 (dd, 1H, J ¼ 4.5 & 1.5, azaindolyl-H), 8.35 (d,

H, J ¼ 5.5, pyrimidinyl-H), 8.45 (d, 1H, J ¼ 3.0, azaindolyl-H), 8.94

s, 1H, azaindolyl-H), 9.27 (s, 1H, pyrimidinyleNHePh), 12.31 (s, 1H,

azaindolyl-NH) (four proton signals (CH N(CH CH not

observed). ¹³C NMR (DMSO‑d

45.7, 48.2, 54.6, 106.5, 107.0, 109.0,

10.4, 112.4, 116.9, 117.6, 128.8, 129.1, 130.7, 141.5, 143.6, 149.3, 151.4,

d

2

3

(

2

3

)

2

)

3

6

) d

1

2

3

2

3

13

Y. Long, M. Yu, A.M. Ochnik et al.

European Journal of Medicinal Chemistry 213 (2021) 113215

6

.38 (d, 1H, J 8.0, Ph-H), 7.08 (t, 1H, J 8.0, Ph-H), 7.14e7.23 (m, 3H,

Ph-H & azaindolyl-H & pyrimidinyl-H), 7.24 (d, 1H, J 8.0, Ph-H), 8.25

d, 1H, J 5.5, pyrimidinyl-H), 8.26 (d, 1H, J 5.5, azaindolyl-H), 8.54 (s,

1.91e1.95 (m, 1H, CH

CH of the other rotamer), 3.29 (t, 2H, J 5.5), 3.52 (t, 2H, J 5.5), 3.56

(t, 1H, J 5.5), 3.60e3.64 (m, 2H), 3.66 (t, 1H, J 5.0) (total 8H,

CH N(COCH )CH & CH N(Ph)CH ), 6.37 (d, 1H, J 8.0, Ph-H), 7.09 (t,

2 2 2

CH CH of the other rotamer), 2.00 (s, 1.5H,

3

(

1

H, azaindolyl-H), 9.09 (s, 1H, pyrimidinyleNHepyridinyl), 9.19 (d,

H, J 7.5, azaindolyl-H). C NMR (DMSO‑d

2

3

2

13

6

)

d

21.0, 21.3 (one car-

1H, J 8.0, Ph-H), 7.21e7.23 (m, 1H, Ph-H), 7.24 (dd, 1H, J 8.0 & 4.5,

azaindolyl-H), 7.30e7.32 (m, 1H, Ph-H), 7.92 (dd, 1H, J 8.0 & 1.5,

azaindolyl-H), 8.18 (d, 1H, J 5.0, pyrimidinyl-H), 8.38 (dd, 1H, J 4.5 &

1.5, azaindolyl-H), 8.45 (s, 1H, azaindolyl-H), 8.51 (d, 1H, J 5.0,

pyrimidinyl-H), 9.15 (s, 1H, pyrimidinyleNHePh), 11.98 (s, 1H,

bon), 24.0, 25.9 (one carbon), 43.9, 44.5, 46.9, 47.0, 47.1, 48.2, 49.1,

9.7 (four carbons), 102.4, 102.6 (one carbon), 105.0, 105.1 (one

carbon), 106.4, 107.3, 111.6, 113.5, 126.4, 129.1, 133.1, 134.3, 142.1,

47.2, 147.4, 147.5 (one carbon), 150.7, 156.5, 160.2, 162.2, 169.0,

69.3 (one carbon) (one carbon signal overlapping or obscured).

4

1

13

6

azaindolyl-NH). C NMR (DMSO‑d ) d 21.0, 21.3 (one carbon), 24.1,

þ

HRMS (ESI-TOF) m/z 442.2353 [MþH] ; calcd. for C25

H

28

N

7

O

26.0 (one carbon), 44.0, 44.6, 46.9, 47.0, 47.1, 48.3, 48.9, 49.7 (four

carbons), 102.0, 102.1 (one carbon), 104.9, 105.0 (one carbon), 107.0,

108.7, 113.3, 117.2, 119.8, 129.3, 129.9, 130.5, 142.1, 143.6, 147.2, 147.6,

158.0, 159.9, 160.0, 160.1 (one carbon), 169.1, 169.3 (one carbon).

þ

4

42.2350 [MþH] . Anal. RP-HPLC: t

R

¼ 13.68 min, purity >98%

(

method A); t

R

¼ 10.18 min, purity > 98% (method B).

1

-(4-(4-((4-(1H-Pyrrolo[3,2-b]pyridin-3-yl)pyrimidin-2-yl)

þ

26 7

H N O

amino)phenyl)piperazin-1-yl)ethan-1-one (12g)

HRMS (ESI-TOF) m/z 428.2206 [MþH] ; calcd. for C24

þ

Guanidine 11a (523 mg, 2.01 mmol) and enaminone 8f (220 mg,

428.2193 [MþH] . Anal. RP-HPLC: t

R

¼ 15.21 min, purity >95%

1.02 mmol) were coupled using general synthetic procedure. The

(method A); t

R

¼ 10.93 min, purity > 95% (method B).

þ

residue was puriﬁed by FlashMaster Personal chromatography

silica gel, DCM ramping to DCM:CH

OH ¼ 95:5) and washed with

CH OH (10 mL) to give 12g as a yellow solid (213 mg, 51%). m.p.

1-(4-(4-((4-(1H-Indol-3-yl)pyrimidin-2-yl)amino)phenyl)

piperazin-1-yl)ethan-1-one (12j) & 1-(4-(4-((4-(1-Tosyl-1H-indol-

3-yl)pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)ethan-1-one

(12l)

(

3

ꢁ

1

293e295 C (decomposed). H NMR (DMSO‑d

3.01 (t, 2H, J 5.0, CH N(Ph)CH ), 3.07 (t, 2H, J 5.0, CH

3.57 (t, 2H, J 6.0, CH N(COCH )CH ), 3.59 (t, 2H, J 6.0, CH

6

)

d

2.04 (s, 3H, CH

3

2

),

2

N(Ph)CH

Guanidine 11a (522 mg, 2.00 mmol) and enaminone 8e (368 mg,

1.00 mmol) were coupled using general synthetic procedure E. The

residue was puriﬁed by glass column chromatography (silica gel,

2

3

2

N(COCH

3

)

CH

2

), 6.96 (d, 2H, J 9.0, 2 ꢃ Ph-H), 7.24 (dd, 1H, J 8.0 & 4.5,

azaindolyl-H), 7.73 (d, 2H, J 9.0, 2 ꢃ Ph-H), 7.92 (dd, 1H, J 8.0 & 1.5,

azaindolyl-H), 8.15 (d, 1H, J 5.0, pyrimidinyl-H), 8.39 (s, 1H,

azaindolyl-H), 8.41 (d, 1H, J 5.0, pyrimidinyl-H), 8.51 (dd, 1H, J 4.5 &

DCM ramping to DCM:CH

DCM:CH

give 12l as a beige solid (78 mg, 14%) and 12j (185 mg, 45%) as a

3

OH ¼ 99:1 for 12l, and ramping to

3

OH ¼ 96:4 for 12j) and washed with CH

3

OH (15 mL) to

1

.5, azaindolyl-H), 9.19 (s, 1H, pyrimidinyleNHePh), 11.93 (s, 1H,

white solid.

13

ꢁ

1

azaindolyl-NH). C NMR (DMSO‑d

6

)

d

21.2, 40.8, 45.7, 49.4, 49.9,

12j: m.p. 250e252 C (decomposed). H NMR (DMSO‑d

(s, 3H, CH N(Ph)CH ), 3.09 (t, 2H, J ¼ 5.0,

), 3.03 (t, 2H, J ¼ 5.0, CH

CH N(Ph)CH ), 3.57e3.61 (m, 4H, CH N(COCH )CH ), 6.95 (d, 2H,

6

) d 2.05

108.4, 113.3, 116.8, 117.2, 119.8, 119.9, 130.0, 130.7, 133.9, 143.6, 143.7,

3

2

145.5, 158.0, 159.9, 160.1, 168.3 (two carbon signals overlapping or

2

3

2

þ

obscured). HRMS (ESI-TOF) m/z 414.2049 [MþH] ; calcd. for

J ¼ 9.0, 2 ꢃ Ph-H), 7.13 (t, 1H, J ¼ 7.5, indolyl-H), 7.17e7.21 (m, 2H,

indolyl-H & pyrimidinyl-H), 7.46 (d, 1H, J ¼ 8.0, indolyl-H), 7.67 (d,

2H, J ¼ 9.0, 2 ꢃ Ph-H), 8.26e8.29 (m, 2H, pyrimidinyl-H & indolyl-

H), 8.58 (br s,1H, pyrimidinyleNHePh), 9.16 (s,1H, indolyl-H),11.75

þ

C

23

24

H N

7

O

414.2037 [MþH] . Anal. RP-HPLC: t

R

¼ 13.57 min,

purity >98% (method A); t

N-(3-(4-methylpiperazin-1-yl)phenyl)-4-(1H-pyrrolo[3,2-b]

pyridin-3-yl)pyrimidin-2-amine (12h)

R

¼ 9.78 min, purity > 98% (method B).

13

6

(s, 1H, indolyl-NH). C NMR (DMSO‑d ) d 21.2, 40.1, 40.8, 45.6, 49.4,

Guanidine 11b (233 mg, 1.00 mmol) and enaminone 8f (108 mg,

49.8, 106.8, 111.9, 113.7, 116.6, 120.5, 120.6, 122.1, 122.4, 125.2, 128.8,

133.7, 137.1, 145.8, 156.7, 160.3, 162.5, 168.2 (one carbon signal

5

00

residue was puriﬁed by glass column (silica gel, DCM ramping to

DCM:CH

OH ¼ 94:6) and washed with EtOAc (10 mL) to give 12h as

a beige solid (74 mg, 38%). m.p. 222e224 C (decomposed). H NMR

DMSO‑d 2.25 (s, 3H, CH N(Ph)CH ),

.54 (d, 1H, J ¼ 8.0, Ph-H), 7.13 (t, 1H, J ¼ 8.0, Ph-H), 7.25 (dd, 1H, J ¼

mmol) were coupled using general synthetic procedure E. The

þ

overlapping or obscured). HRMS (ESI-TOF) m/z 413.2099 [MþH] ;

þ

3

calcd. for

C

24

H

25

N

6

O

413.2084 [MþH] . Anal. RP-HPLC:

ꢁ

1

t

R

¼ 15.81 min, purity >96% (method A); t ¼ 11.82 min, pu-

R

(

6

8

6

)

d

3

), 3.16 (t, 4H, J ¼ 5.0, CH

2

rity > 95% (method B).

ꢁ

1

12l: m.p. 229e231 C (decomposed). H NMR (DMSO‑d

6

)

d

2.04

N(Ph)

), 3.55e3.61 (m, 4H,

.0 & 4.5, azaindolyl-H), 7.30 (d, 1H, J ¼ 8.0, Ph-H), 7.60 (s, 1H, Ph-

(s, 3H, COCH

CH

), 3.09 (t, 2H, J ¼ 5.0, CH

CH N(COCH )CH

3

), 2.32 (s, 3H, tosyl-CH

3

), 3.02 (t, 2H, J ¼ 5.0, CH

2

H), 7.93 (dd, 1H, J ¼ 4.5 & 1.5, azaindolyl-H), 8.19 (d, 1H, J ¼ 5.0,

pyrimidinyl-H), 8.38 (s, 1H, azaindolyl-H), 8.46 (d, 1H, J ¼ 5.0,

pyrimidinyl-H), 8.52 (dd, 1H, J ¼ 4.5 & 1.5, azaindolyl-H), 9.24 (s, 1H,

pyrimidinyleNHePh), 12.02 (s, 1H, azaindolyl-NH) (four proton

2

N(Ph)CH

2

3

2

), 6.94 (d, 2H, J ¼ 9.0, 2 ꢃ Ph-H), 7.32 (t, 1H, J ¼ 7.5,

indolyl-H), 7.39e7.46 (m, 4H, pyrimidinyl-H, indolyl-H & 2 ꢃ tosyl-

H), 7.60 (d, 2H, J ¼ 9.0, 2 ꢃ Ph-H), 7.95e8.01 (m, 3H, indolyl-H &

2 ꢃ tosyl-H), 8.42 (d, 1H, J ¼ 5.0, pyrimidinyl-H), 8.63 (br s, 1H,

pyrimidinyleNHePh), 8.72 (s,1H, indolyl-H), 9.37 (s,1H, indolyl-H).

13

signals (CH

8.4, 54.7, 105.9, 108.7, 109.8, 113.3, 117.3, 119.8, 128.9, 130.0, 130.5,

41.8, 143.6, 143.7, 151.4, 158.1, 159.9, 160.1, 167.5 (two carbon sig-

2 3 2 6

N(CH ) CH ) not observed). C NMR (DMSO‑d ) d 45.8,

4

1

13

6

C NMR (DMSO‑d ) d 21.2, 21.3, 40.2, 40.8, 45.6, 49.2, 49.7, 108.1,

nals overlapping or obscured). HRMS (ESI-TOF) m/z 386.2072

113.1, 116.5,120.0,121.0, 123.6,124.0, 125.4,127.0, 127.7,128.5,130.4,

133.0, 133.8, 134.8, 146.0, 146.2, 158.0, 160.1, 160.3, 168.2 (three

carbon signals overlapping or obscured). HRMS (ESI-TOF) m/z

þ

[

t

(

MþH] ; calcd. for C22

H

24

N

7

386.2088 [MþH] . Anal. RP-HPLC:

R

¼ 10.96 min, purity >95% (method A); t ¼ 8.81 min, purity > 95%

R

þ

method B).

-(4-(3-((4-(1H-Pyrrolo[3,2-b]pyridin-3-yl)pyrimidin-2-yl)

567.2177 [MþH] ; calcd. for C31

H

31

N

6

O

3

S

567.2173 [MþH] . Anal.

1

RP-HPLC: t

R

¼ 22.53 min, purity >97% (method A); t ¼ 18.86 min,

R

amino)phenyl)-1,4-diazepan-1-yl)ethan-1-one (12i)

purity > 98% (method B).

Guanidine 11c (300 mg, 1.08 mmol) and enaminone 8f (110 mg,

1-(4-(4-((4-(1-Methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)

phenyl)piperazin-1-yl)ethan-1-one (12k)

Guanidine 11a (522 mg, 2.00 mmol) and enaminone 8d

(228 mg, 1.00 mmol) were coupled using general synthetic pro-

cedure E. The residue was puriﬁed by glass column chromatog-

0

.510 mmol) were coupled using general synthetic procedure E. The

þ

residue was puriﬁed by FlashMaster Personal chromatography

(

H

(

silica gel, DCM ramping to DCM:CH

3

OH ¼ 95:5 þ 0.2% NH

3

(32% in

2

O)) and washed with CH OH (10 mL) to give 12i as a beige solid

3

ꢁ

1

71 mg, 33%). m.p. 155e157 C. H NMR (DMSO‑d ) (12i exists as

6

raphy (silica gel, DCM ramping to DCM:CH

3

OH ¼ 97:3) and washed

two rotamers in approximately 1:1 ratio.) 1.82e1.86 (m, 1H,

d

with EtOAc (20 mL) to give 12k as a beige solid (273 mg, 64%). m.p.

ꢁ

1

CH CH CH of one rotamer), 1.88 (s, 1.5H, CH of one rotamer),

2

3

228e230 C. H NMR (DMSO‑d

6

)

d

2.05 (s, 3H, COCH ), 3.03 (t, 2H,

3

14

Y. Long, M. Yu, A.M. Ochnik et al.

European Journal of Medicinal Chemistry 213 (2021) 113215

J ¼ 5.0, CH

2

N(Ph)CH

N(COCH

2

), 3.09 (t, 2H, J ¼ 5.0, CH

2

N(Ph)CH

2

), 3.57e3.61

3H, indolyl-H & 2 ꢃ Ph-H), 8.82 (s, 1H, pyrimidinyl-H), 10.13 (s, 1H,

13

(

m, 4H, CH

2

3

)CH ), 3.88 (s, 3H, indolyl-CH

2

3

), 6.95 (d, 2H, J ¼

6

pyrimidinyleNHePh),11.74 (s,1H, indolyl-NH). C NMR (DMSO‑d )

9

.0, 2 ꢃ Ph-H), 7.12 (d, 1H, J ¼ 5.5, pyrimidinyl-H), 7.18 (t, 1H, J ¼ 7.5,

d 13.8, 21.3, 40.3, 40.8, 45.6, 48.9, 49.3, 111.2, 114.7, 116.3, 118.3,

indolyl-H), 7.26 (t,1H, J ¼ 7.5, indolyl-H), 7.52 (d,1H, J ¼ 8.0, indolyl-

120.2, 121.8, 126.4, 131.5, 135.3, 147.1, 159.9, 165.0, 165.9, 168.5 (ﬁve

carbon signals overlapping or obscured). HRMS (ESI-TOF) m/z

H), 7.67 (d, 2H, J ¼ 9.0, 2 ꢃ Ph-H), 8.27e8.28 (m, 2H, pyrimidinyl-H

þ

&

indolyl-H), 8.60 (br s, 1H, pyrimidinyleNHePh), 9.17 (s, 1H,

452.2185 [MþH] ; calcd. for C26

26

H N

7

O

452.2193 [MþH] . Anal.

13

indolyl-H). C NMR (DMSO‑d

6

)

d

21.2, 33.0, 40.8, 45.6, 49.4, 49.8,

RP-HPLC: t

R

¼ 19.24 min, purity >95% (method A); t ¼ 15.64 min,

R

106.6, 110.3, 112.6, 116.6, 120.6, 120.7, 122.2, 122.5, 125.6, 132.7,

purity > 96% (method B).

1

33.6, 137.6, 145.8, 156.8, 160.3, 162.0, 168.2 (two carbon signals

1-(4-(4-((4-(1H-Indazol-3-yl)pyrimidin-2-yl)amino)phenyl)

piperazin-1-yl)ethan-1-one (12p)

Guanidine 11a (522 mg, 2.00 mmol) and enaminone 8j (215 mg,

1.00 mmol) were coupled using general synthetic procedure E. The

residue was puriﬁed by glass column chromatography (silica gel,

þ

overlapping or obscured). HRMS (ESI-TOF) m/z 427.2232 [MþH] ;

þ

calcd. for

C

25

H

27

N

6

O

427.2241 [MþH] . Anal. RP-HPLC:

t

R

¼ 16.93 min, purity >98% (method A); t ¼ 12.58 min, pu-

R

rity > 99% (method B).

-((4-(4-Acetylpiperazin-1-yl)phenyl)amino)-4-(1H-indol-3-yl)

pyrimidine-5-carbonitrile (12m)

2

DCM ramping to DCM:CH

(20 mL) and CH OH (5 mL) to give 12p as a yellow solid (248 mg,

60%). m.p. 275e277 C (decomposed). H NMR (DMSO‑d

3H, CH N(Ph)CH ), 3.09 (t, 2H, J ¼ 5.0,

), 3.02 (t, 2H, J ¼ 5.0, CH

CH N(Ph)CH ), 3.58e3.59 (m, 4H, CH N(COCH )CH ), 6.95 (d, 2H,

3

OH ¼ 97:3) and washed with EtOAc

3

ꢁ

1

Guanidine 11a (261 mg, 1.00 mmol) and enaminone 8a (120 mg,

6

) d 2.04 (s,

5

00

mmol) were coupled using general synthetic procedure E. The

3

2

residue was puriﬁed by glass column chromatography (silica gel,

2

3

2

DCM ramping to DCM:CH

3

OH ¼ 98:2) and washed with CH

3

OH

J ¼ 9.0, 2 ꢃ Ph-H), 7.43 (d, 1H, J ¼ 5.0, pyrimidinyl-H), 7.49 (t, 1H, J ¼

7.5, indazolyl-H), 7.67 (d, 2H, J ¼ 9.0, 2 ꢃ Ph-H), 7.72 (d, 1H, J ¼ 8.0,

indazolyl-H), 7.85 (d, 1H, J ¼ 7.5, indazolyl-H), 8.52 (d, 1H, J ¼ 5.0,

pyrimidinyl-H), 8.85 (br s, 1H, pyrimidinyleNHePh), 9.49 (s, 1H,

ꢁ

(

15 mL) to give 12m as a beige solid (62 mg, 28%). m.p. 278e280 C

1

(

decomposed). H NMR (DMSO‑d

N(Ph)CH

), 3.14 (t, 2H, J ¼ 5.0, CH

N(COCH )CH

), 6.99 (d, 2H, J ¼ 9.0, 2 ꢃ Ph-H), 7.23e7.25

m, 2H, 2 ꢃ indolyl-H), 7.53 (d, 1H, J ¼ 8.5, indolyl-H), 7.57e7.59 (m,

H,

ꢃ Ph-H), 8.51e8.53 (m, 1H, indolyl-H), 8.73 (s, 1H,

pyrimidinyl-H), 10.01 (s, 1H, indolyl-H), 12.03 (s, 1H, indolyl-NH)

6

)

d

2.05 (s, 3H, CH

3

), 3.08 (t, 2H,

J ¼ 5.0, CH

2

N(Ph)CH

2

), 3.58e3.60

13

(

m, 4H, CH

2

3

2

6

indazolyl-H),13.26 (s,1H, indazolyl-NH). C NMR (DMSO‑d ) d 21.2,

(

2

40.8, 45.6, 49.2, 49.7, 108.4, 112.9, 116.6, 120.2, 120.4, 120.7, 125.8,

130.0,133.2,135.0,140.8,146.1,158.6,160.5,164.1,168.2 (two carbon

signals overlapping or obscured). HRMS (ESI-TOF) m/z 414.2041

2

13

þ

(

1

one proton signal (pyrimidinyleNHePh) not observed). C NMR

DMSO‑d 21.2, 40.2, 40.7, 45.5, 48.9, 49.3, 108.2, 112.1, 116.2,

20.0, 121.2, 122.9, 123.1, 123.5, 130.5, 136.4, 140.8, 163.1, 168.3 (six

[MþH] ; calcd. for C23

24

H N

7

O

414.2037 [MþH] . Anal. RP-HPLC:

6

)

d

t

R

¼ 18.51 min, purity >96% (method A); t ¼ 13.44 min, pu-

R

rity > 97% (method B).

carbon signals overlapping or obscured). HRMS (ESI-TOF) m/z

1-(4-(4-((4-(1-Methyl-1H-indazol-3-yl)pyrimidin-2-yl)amino)

phenyl)piperazin-1-yl)ethan-1-one (12q)

Guanidine 11a (522 mg, 2.00 mmol) and enaminone 8k

(230 mg, 1.00 mmol) were coupled using general synthetic pro-

cedure E. The residue was puriﬁed by glass column chromatog-

þ

4

38.2031 [MþH] ; calcd. for C25

H

24

N

7

O

438.2037 [MþH] . Anal.

RP-HPLC: t

R

¼ 19.80 min, purity >95% (method A); t

R

¼ 15.81 min,

purity > 95% (method B).

-((4-(4-Acetylpiperazin-1-yl)phenyl)amino)-4-(1-methyl-1H-

2

indol-3-yl)pyrimidine-5-carbonitrile (12n)

raphy (silica gel, DCM ramping to DCM:CH

with CH OH (15 mL) to give 12q as a yellow solid (154 mg, 36%).

m.p. 268e270 C (decomposed). H NMR (DMSO‑d

COCH N(Ph)CH ), 3.11 (t, 2H, J ¼ 5.0,

), 3.04 (t, 2H, J ¼ 5.0, CH

CH N(Ph)CH ), 3.58e3.61 (m, 4H, CH N(COCH )CH ), 4.17 (s, 3H,

indazolyl-CH

3

OH ¼ 98:2) and washed

Guanidine 11a (261 mg, 1.00 mmol) and enaminone 8b (127 mg,

3

ꢁ

1

5

00

residue was puriﬁed by glass column (silica gel, DCM ramping to

DCM:CH OH (15 mL) to give 12n

OH ¼ 99:1) and washed with CH

as a white solid (60 mg, 27%). m.p. 213e215 C (decomposed). H

NMR (DMSO‑d 2.05 (s, 3H, COCH N(Ph)

), 3.08 (t, 2H, J ¼ 5.0, CH

CH N(Ph)CH ), 3.59e3.60 (m, 4H,

), 3.14 (t, 2H, J ¼ 5.0, CH

CH N(COCH )CH ), 3.94 (s, 3H, indolyl-CH

), 6.99 (d, 2H, J ¼ 9.0,

m

mol) were coupled using general synthetic procedure E. The

6

) d

2.05 (s, 3H,

3

2

3

2

3

2

ꢁ

1

3

), 6.95 (d, 2H, J ¼ 9.0, 2 ꢃ Ph-H), 7.28 (t, 1H, J ¼ 7.5,

6

)

d

3

2

indazolyl-H), 7.39 (d, 1H, J ¼ 5.0, pyrimidinyl-H), 7.49 (t, 1H, J ¼ 7.5,

indazolyl-H), 7.65 (d, 2H, J ¼ 9.0, 2 ꢃ Ph-H), 7.74 (d, 1H, J ¼ 8.5,

indazolyl-H), 8.45 (d, 1H, J ¼ 5.0, pyrimidinyl-H), 8.67 (br s, 1H,

2

3

2

3

13

2

ꢃ Ph-H), 7.30e7.32 (m, 2H, 2 ꢃ indolyl-H), 7.56e7.58 (m, 3H,

6

pyrimidinyleNHePh), 9.41 (s,1H, indazolyl-H). C NMR (DMSO‑d )

indolyl-H & 2 ꢃ Ph-H), 8.47e8.49 (m, 1H, indolyl-H), 8.72 (s, 1H,

d 21.2, 36.0, 40.8, 45.6, 49.3, 49.7, 54.9, 106.7, 110.3, 116.5, 121.0,

121.7,122.0,123.3,126.6,133.1,139.8,141.3, 146.2,158.1,160.2,160.4,

pyrimidinyl-H), 10.01 (s, 1H, indolyl-H) (one proton signal (pyr-

imidinyleNHePh) not observed). ¹³C NMR (DMSO‑d

)

d

21.2, 33.4,

168.2 (one carbon signal overlapping or obscured). HRMS (ESI-TOF)

6

þ

4

0.1, 40.7, 45.5, 48.9, 49.3, 110.5, 116.2, 119.4, 121.5, 123.0, 126.0,

m/z 428.2198 [MþH] ; calcd. for C24

H

26

N

7

O

428.2193 [MþH] .

132.5, 134.0, 137.1, 160.2, 161.1, 163.3, 168.3 (six carbon signals

Anal. RP-HPLC: t

R

¼ 20.10 min, purity >99% (method A);

þ

overlapping or obscured). HRMS (ESI-TOF) m/z 452.2179 [MþH] ;

t

R

¼ 15.17 min, purity > 99% (method B).

þ

calcd. for

C

26

H

26

N

7

O

452.2193 [MþH] . Anal. RP-HPLC:

2-((4-(4-Acetylpiperazin-1-yl)phenyl)amino)-4-(1H-indazol-3-

t

R

¼ 20.97 min, purity >99% (method A); t

R

¼ 17.13 min, pu-

yl)pyrimidine-5-carbonitrile (12r)

rity > 98% (method B).

-((4-(4-Acetylpiperazin-1-yl)phenyl)amino)-4-(2-methyl-1H-

Guanidine 11a (522 mg, 2.00 mmol) and enaminone 8l (240 mg,

1.00 mmol) were coupled using general synthetic procedure E. The

residue was puriﬁed by glass column (silica gel, DCM ramping to

2

indol-3-yl)pyrimidine-5-carbonitrile (12o)

Guanidine 11a (261 mg, 1.00 mmol) and enaminone 8c (127 mg,

.50 mmol) were coupled using general synthetic procedure E. The

residue was puriﬁed by glass column chromatography (silica gel,

DCM:CH

as a yellow solid (136 mg, 31%). m.p. > 300 C. H NMR (DMSO‑d

2.05 (s, 3H, CH N(Ph)CH ), 3.16 (t, 2H, J ¼

), 3.09 (t, 2H, J ¼ 5.0, CH

5.0, CH N(Ph)CH ), 3.58e3.60 (m, 4H, CH N(COCH )CH ), 7.01 (d,

3

OH ¼ 98:2) and washed with CH

3

OH (15 mL) to give 12r

ꢁ

1

0

6

)

d

3

2

DCM ramping to DCM:CH

3

OH ¼ 98:2) and washed with CH

3

OH

2

3

2

ꢁ

(

3

5

6

1

10 mL) to give 12o as a yellow solid (30 mg, 13%). m.p. 208e210 C

2H, J ¼ 8.5, 2 ꢃ Ph-H), 7.18e7.33 (m, 1H, indazolyl-H), 7.46e7.60 (m,

3H, indazolyl-H & 2 ꢃ Ph-H), 7.68 (d, 1H, J ¼ 8.5, indazolyl-H), 8.84

(s, 1H, pyrimidinyl-H), 10.22 (s, 1H, indazolyl-H), 13.99 (s, 1H,

indazolyl-NH) (one proton signal (pyrimidinyleNHePh) not

1

decomposed). H NMR (DMSO‑d

6

)

d

2.03 (s, 3H, COCH

), 3.02 (t, 2H, J ¼ 5.0, CH N(Ph)CH ), 3.09 (t, 2H, J ¼

N(Ph)CH ), 3.55e3.56 (m, 4H, CH N(COCH )CH ),

3

), 2.55 (s,

H, indolyl-CH

3

2

.0, CH

2

3

2

13

.91e6.93 (m, 2H, 2 ꢃ Ph-H), 7.08 (t, 1H, J ¼ 7.5, indolyl-H), 7.13 (t,

H, J ¼ 7.5, indolyl-H), 7.39 (d, 1H, J ¼ 8.0, indolyl-H), 7.62e7.64 (m,

observed). C NMR (DMSO‑d

6

) d 21.3, 40.7, 45.5, 48.6, 48.9, 49.3,

110.9, 116.3, 117.9, 121.6, 122.0, 122.6, 123.6, 127.0, 140.1, 141.2, 159.9,

15

Y. Long, M. Yu, A.M. Ochnik et al.

European Journal of Medicinal Chemistry 213 (2021) 113215

1

60.9, 164.1, 168.3 (four carbon signals overlapping or obscured).

binding white polystyrene plates. Compounds were added either at

þ

HRMS (ESI-TOF) m/z 439.1980 [MþH] ; calcd. for C24

H

23

N

8

O

single concentration of 1

centrations (1:3 serial dilutions from 3

in a total assay volume of 4 L with a ﬁnal DMSO concentration of

mM for initial evaluation or at 8e10 con-

þ

4

39.1989 [MþH] . Anal. RP-HPLC: t

R

¼ 19.40 min, purity >95%

mM) for IC50 determination

(

method A); t

-(4-(3-((4-(1H-Indazol-3-yl)pyrimidin-2-yl)amino)phenyl)-

,4-diazepan-1-yl)ethan-1-one (12s)

Guanidine 11c (300 mg, 1.08 mmol) and enaminone 8j (110 mg,

R

¼ 15.21 min, purity > 95% (method B).

m

1

0.5% in all wells. Optimised concentration of each kinase (CDK1/

cyclin B: 1 nM, CDK2/cyclin E1: 10 nM, and CDK7/cyclin H/MAT1:

40 nM) was then incubated with tested compounds, their sub-

strates (histone 1 protein for CDK1/cyclin B and CDK2/cyclin E1 and

a peptide based upon the c-terminal tail of RNA polymerase II, H-

YSPTSPSYSPTSPSYSPTSPSKKKK-OH for CDK7/cyclin H/MAT1) and

1

5

10

m

mol) were coupled using general synthetic procedure E. The

þ

residue was puriﬁed by FlashMaster Personal chromatography

(

silica gel, DCM ramping to DCM:CH

3

OH ¼ 96:4 þ 0.2% NH

3

(32% in

H

2

O)) and washed with CH OH (10 mL) to give 12s as a white solid

3

K

m

(ATP) for each kinase. The reaction was performed in kinase

ꢁ

1

(

42 mg, 19%). m.p. 189e191 C. H NMR (DMSO‑d ) (12s exists as

6

reaction buffer; 40 mM Tris base (pH 7.5), 20 mM MgCl , 50 M DTT

2

m

two rotamers in approximately 1:1 ratio.) 1.79e1.83 (m, 1H,

CH CH CH of one rotamer), 1.87 (s, 1.5H, CH of one rotamer),

.88e1.92 (m, 1H, CH CH CH of the other rotamer), 1.99 (s, 1.5H,

CH of the other rotamer), 3.31 (t, 2H, J 5.5), 3.50 (t, 2H, J 5.5), 3.54

t, 1H, J 5.5), 3.58e3.63 (m, 2H), 3.66 (t, 1H, J 5.0) (total 8H,

CH N(COCH )CH & CH N(Ph)CH ), 6.42 (d, 1H, J 8.0, Ph-H), 7.11 (t,

d

and 0.1 mg/mL BSA. Positive and negative controls were performed

in 0.5% DMSO in the presence and absence of each kinase. The assay

plate was then incubated at room temperature for an optimised

time period for each kinase, generally 60e120 min. Following in-

2

3

1

2

3

(

cubation, 4

m

L of ADP-Glo reagent was added to each well and

L of

2

3

2

incubated for 40 min in the dark at room temperature. Then 8 m

1

H, J 8.0, Ph-H), 7.15 (s, 1H, Ph-H), 7.25 (d, 1H, J 8.0, Ph-H), 7.28 (ddd,

H, J 7.5 & 6.5 & 0.5, indazolyl-H), 7.44 (t,1H, J 7.5, indazolyl-H), 7.48

detection reagent was added to all the wells and then further

incubated for 40 min in the dark. Luminescence was measured with

an EnVision Multilabel plate reader (PerkinElmer, Buck-

inghamshire, UK) with an integration time of 1 s per well. The

luminescence thus generated correlates with kinase activity. The

IC50 values were derived by ﬁtting a sigmoidal dose-response curve

using non-linear regression 4-parameter logistic ﬁts (variable

slope) by GraphPad Prism software 8.3.0 (La Jolla, CA, USA). For FLT3

kinase assays, compounds were used with 1:3 serial dilutions for 10

(

d, 1H, J 5.0, pyrimidinyl-H), 7.63 (d, 1H, J 8.5, indazolyl-H), 8.50 (d,

1

H, J 5.5, pyrimidinyl-H), 8.73 (d, 1H, J 7.5, indazolyl-H), 9.40 (s, 1H,

13

pyrimidinyleNHePh), 11.98 (s, 1H, indazolyl-NH).

C NMR

(

DMSO‑d

6

)

d

21.0, 21.3 (one carbon), 24.0, 25.8 (one carbon), 43.9,

4

4.5, 46.9, 47.0, 47.2, 48.2, 49.1, 49.7 (four carbons), 89.2, 102.8,

1

02.9 (one carbon), 105.6, 105.7 (one carbon), 107.3, 107.6 (one

carbon), 110.6, 121.1, 121.8, 123.2, 126.6, 129.2, 141.2, 141.5, 141.6,

41.7 (one carbon), 147.3, 147.6 (one carbon), 158.0, 160.3, 160.7,

1

concentrations (from 10 to 0.0005

performed with the kinase reaction buffer (40 nM Tris base pH 7.5,

20 mM MgCl , 50 M DTT, 0.1 mg/mL BSA), the poly(Glu, Tyr) (the

ratio of Glu/Tyr ¼ 4:1) peptide substrate, K ATP (FLT3-WT:

200 M; FLT3-ITD: 100 M) and an FLT3 kinase (FLT3-WT: 20 nM;

FLT3-ITD: 20 nM) in a total assay volume of 5 L. The kinase re-

actions were run for an optimised time period (FLT3-WT: 120 min;

FLT3-ITD: 80 min) at room temperature and stopped by adding 5

of ADP Glo reagent. After incubation at room temperature in the

dark for 40 min, 10 L of kinase detection reagent was added to

mM). The kinase reactions were

þ

1

69.0, 169.3 (one carbon). HRMS (ESI-TOF) m/z 428.2201 [MþH] ;

þ

calcd. for

C

H

24 26

N

7

O

428.2193 [MþH] . Anal. RP-HPLC:

2

m

t

R

¼ 19.61 min, purity >99% (method A); t

R

¼ 14.77 min, pu-

m

rity > 99% (method B).

.3. Kinase assays

The percentages of residual kinase activity of 12b in the panel

m

4

mL

screen and the half maximal inhibitory concentration (IC50) values

of the reported compounds against each kinase were mostly

determined by Reaction Biology Corporation (Malvern, PA, USA,

www.reactionbiology.com ). In brief, speciﬁc kinase-substrate pairs

together with the required cofactors were prepared in freshly-

m

each well and incubated for 30e40 min. Luminescence was

measured using the EnVision multilabel plate reader with an

integration time of 1 s per well. Positive and negative controls were

performed in 0.5% DMSO in the presence and absence of each FLT3

kinase, respectively.

made base reaction buffer (20 mM HEPES pH 7.5, 10 mM MgCl

2

,

1

mM EGTA, 0.02% Brij 35, 0.02 mg/mL BSA, 0.1 mM Na VO , 2 mM

3

4

DTT, 1% DMSO). Compounds in DMSO were delivered into the re-

action mixture by Acoustic technology (Echo550; nanoliter range).

The reaction mixtures were incubated for 20 min at room tem-

4.4. Cell culture

MV4-11 cells were kindly provided by Professor Richard D’An-

drea from University of South Australia. All other cell lines were

purchased from the American Tissue Culture Collection (ATCC)

(Manassas, VA, USA). The cell lines were maintained following

ATCC recommendation in Roswell Park Memorial Institute (RPMI)-

1640, Dulbecco’s Modiﬁed Eagle’s Medium (DMEM) or Minimum

Essential Medium (MEM) with 10% foetal bovine serum (FBS)

(Sigma-Aldrich, Castle Hill, NSW, Australia). All the cell lines were

perature and ³³P-ATP with a speciﬁc activity of 10

mCi/mL was added

to initiate the reaction. Reactions were carried out at room tem-

perature for 2 h, followed by spotting of the reactions onto P81 ion

exchange ﬁlter papers. Phosphoric acid (0.75%) was used to wash

unbound phosphate from the ﬁlters. The percentages of remaining

kinase activity in tested samples were measured by comparison to

vehicle (DMSO) reactions after subtraction of the background

derived from the control reactions containing inactive kinase. IC50

values and curve ﬁts were obtained using Prism (GraphPad Soft-

ꢁ

cultured at 37 C in a humidiﬁed incubator in the presence of 5%

2

CO atmosphere.

ware, La Jolla, California, USA). K

Cheng-Prusoff equation: K

¼ IC50/(1 þ ([ATP]/K

ATP] is K (ATP) according to the Reaction Biology Corporation

i

values were calculated using the

i

m

(ATP))), where

4.5. Cell viability assays

[

m

binning structure.

Resazurin assays were performed with each of leukaemic cell

lines (i.e., MV4-11, MOLM-13, NB4, THP1, U937, Jurkat and K562)

according to manufacturers’ instructions. Cells were seeded at

Some of the CDK and FLT3 kinase assays were performed in-

house. The CDK kinase activity assays were validated and per-

formed using an ADP-Glo™ assay kit (Promega Corporation, Mad-

ison, WI, USA). Compounds were prepared as 2 mM stocks in 100%

3

5 ꢃ 10 cells/well onto 96-well cell culture plates and incubated at

ꢁ

37 C, 5% CO

2

for 24 h. Tested compounds were diluted from a

ꢁ

DMSO and stored at ꢂ20 C. For determination of % inhibition and

10 mM stock solution to prepare a ﬁve-fold dilution series in 100 mL

ꢁ

of cell medium, added to cells (in triplicate), and incubated at 37 C,

IC50, the assays were conducted using 384-well, low volume, non-

16

Y. Long, M. Yu, A.M. Ochnik et al.

European Journal of Medicinal Chemistry 213 (2021) 113215

ꢁ

5

% CO

2

for 24 or 72 h. Resazurin (Sigma-Aldrich, Castle Hill, NSW,

plus 1 ꢃ TBST and further probed with primary antibodies at 4 C to

Australia) was made up as a stock of 0.1 mg/mL in cell medium, and

the solution was ﬁlter-sterilised. Resazurin solution was added at

detect the steady-state protein levels of phosphorylated and/or

total STAT5, AKT, ERK, S6RP, eIF4E, full-length and cleaved PARP and

caspase-3, Mcl-1 and b-actin according to manufacturers’ speciﬁ-

ꢁ

2

0

mL/well and incubated in the dark at 37 C, 5% CO

2

for 4 h. The

plate was left at room temperature for 10e15 min, and the ﬂuo-

rescent product quantiﬁed using the EnVision multi-label plate

reader with a 570 nm (excitation)/585 nm (emission) ﬁlter set. The

drug-treated samples were normalised to a vehicle control (100%

DMSO). Compound concentrations required to inhibit 50% of cell

growth (i.e., GI50 values) were calculated using non-linear regres-

sion analysis (GraphPad Prism v.7, La Jolla, CA, USA).

cations. The secondary anti-rabbit HRP antibody was further used

to probe the membranes for 1 h at room temperature. Subse-

quently, the ECL-reagent was added to the membranes and image

analysis using a Bio-Rad ChemiDoc™ MP imager was performed for

band detection and image production.

Declaration of competing interest

4

.6. Detection of apoptosis

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

5

MV4-11 and U937 cells were plated at a cell density of 3 ꢃ 10

and cultured in 3 mL of medium per well in a six-well plate for 24 h.

The cells were subsequently treated with the vehicle control (100%

DMSO) or each compound at concentrations of 1 ꢃ , 5 ꢃ , 10 ꢃ and

Acknowledgements

2

0 ꢃ GI50 (GI50 refers to the 24 h value of MV4-11 cells for each

This work was supported by The Financial Markets Foundation

for Children (Project 2019-112) to S.W. and Channel 7 Children’s

Research Foundation of South Australia (project ID 19/10674521) to

M.Y.

compound) for 24 and/or 48 h. The media containing the cells were

transferred to FACS tubes, centrifuged for 5 min at 300 g and re-

suspended in 1 mL of warm 1 ꢃ PBS to wash cells. The cells were

counted using an automated cell counter (Bio-Rad) using 10 mL of

5

the cell suspension, and subsequently diluted to 2 ꢃ 10 cells/mL

with 1 mL PBS in a new FACS tube. Lastly, the cells were centrifuged

for an additional 5 min at 300 g, re-suspended in 1 mL of ice-cold

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.113215.

1

ꢃ PBS, re-centrifuged at 300 g for 5 min, re-suspended in a so-

lution of 1 ꢃ binding buffer (100 L), annexin V (3 L) and PI (3 L),

and incubated at room temperature for 15 min. A ﬁnal 200 L of

m

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with annexin V (no PI), (3) cells stained with PI (no annexin V) and

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18

Article Doi

Discovery of novel 4-azaaryl-N-phenylpyrimidin-2-amine derivatives as potent and selective FLT3 inhibitors for acute myeloid leukaemia with FLT3 mutations

DOI: 10.1016/j.ejmech.2021.113215

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