Design, synthesis, biological evaluation and molecular modeling of novel 1H-pyrrolo[2,3-b]pyridine derivatives as potential anti-tumor agents

Article abstract of DOI:10.1016/j.bioorg.2019.103474

A class of 3-substituted 1H-pyrrolo[2,3-b]pyridine derivatives were designed, synthesized and evaluated for their in vitro biological activities against maternal embryonic leucine zipper kinase (MELK). Among these derivatives, the optimized compound 16h e

Full text of DOI:10.1016/j.bioorg.2019.103474

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Design, synthesis, biological evaluation and molecular modeling of novel 1H-
pyrrolo[2,3-b]pyridine derivatives as potential anti-tumor agents
Ruifeng Wang, Yixuan Chen, Bowen Yang, Sijia Yu, Xiangxin Zhao, Cai
Zhang, Chenzhou Hao, Dongmei Zhao, Maosheng Cheng
PII:
S0045-2068(19)30892-2
DOI:
https://doi.org/10.1016/j.bioorg.2019.103474
YBIOO 103474
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
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19 November 2019
24 November 2019
Please cite this article as: R. Wang, Y. Chen, B. Yang, S. Yu, X. Zhao, C. Zhang, C. Hao, D. Zhao, M. Cheng,
Design, synthesis, biological evaluation and molecular modeling of novel 1H-pyrrolo[2,3-b]pyridine derivatives
as potential anti-tumor agents, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103474
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Design, synthesis, biological evaluation and molecular modeling of novel
1H-pyrrolo[2,3-b]pyridine derivatives as potential anti-tumor agents
Ruifeng Wanga, Yixuan Chena, Bowen Yanga, Sijia Yua, Xiangxin Zhaoa, Cai Zhangb, Chenzhou Haoa,
Dongmei Zhaoa,*, Maosheng Chenga
a
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang,
110016, China
b
The School of life Science and Biopharmaceutical, Shenyang Pharmaceutical University, 103 Wenhua Road,
Shenhe District, Shenyang, 110016, China
*Corresponding author
E-mail address: medchemzhao@163.com (D. Zhao).
Abstract
A class of 3-substituted 1H-pyrrolo[2,3-b]pyridine derivatives were designed, synthesized and evaluated for
their in vitro biological activities against maternal embryonic leucine zipper kinase (MELK). Among these
derivatives, the optimized compound 16h exhibited potent enzyme inhibition (IC50 = 32 nM) and excellent
anti-proliferative effect with IC50 values from 0.109 μM to 0.245 μM on A549, MDA-MB-231 and MCF-7 cell
lines. The results of flow cytometry indicated that 16h promoted apoptosis of A549 cells in a dose-dependent
manner and effectively arrested A549 cells in the G0/G1 phase. Further investigation indicated that compound 16h
potently suppressed the migration of A549 cells, had moderate stability in rat liver microsomes and showed
moderate inhibitory activity against various subtypes of human cytochrome P450. However, compound 16h is a
multi-target kinase inhibitor and recently several studies reported MELK expression is not required for cancer
growth, suggesting that compound 16h suppressed the proliferation and migration of cancer cells should through
an off-target mechanism. Collectively, compound 16h has the potential to serve as a new lead compound for
further anticancer drug discovery.
Keywords: MELK inhibitor; 1H-pyrrolo[2,3-b]pyridine; Structure-activity relationship; Biological evaluation.
1. Introduction
Maternal embryonic leucine zipper kinase (MELK) is a member of the snf1/AMPK family of serine-threonine
kinases that has been implicated in various cellular processes, including stem cell renewal, pre-mRNA splicing,
cell proliferation, cell cycle progression and cell migration[1-5]. More importantly, the overexpression of MELK
has been reported in various human cancers and is associated with more aggressive forms of astrocytoma[6],
melanoma[7,8], breast cancer[9], and glioblastoma[10]. Furthermore, increased MELK expression correlates with
the pathologic grade of brain tumors[11], and its expression levels are significantly correlated with poor prognosis
of prostate, breast, and glioblastoma cancer patients[12,13]. It has been demonstrated in numerous experimental
systems that si/shRNA-mediated knockdown of MELK leads to decreased viability of kidney, colon, breast and
pancreas cancer cell lines and inhibits colony formation and tumor growth in vivo[13,14]. Several correlative
studies have shown higher levels of MELK in human cancers than in normal tissue[6]. Additionally, normal adult
cells have a low expression of MELK, offering promise for selective treatment and rendering the assessment of the
role of MELK in tumor growth, metastasis and carcinogenesis even more urgent. In contrast to these results, there
is some controversy about the MELK because recently reported MELK expression correlates with tumor mitotic
activity but is not required for cancer growth. In these studies, neither pharmacological inhibition nor
1

CRISPR-Cas9–mediated knockout of the protein showed a growth-inhibiting phenotype[15].
Several potent and selective MELK inhibitors have been designed and developed, including type I (1-5) and
type II (6,7) inhibitors that by definition bind to the DFG-in and DFG-out conformations of kinases, respectively.
The only designated MELK inhibitor currently in clinical trials is OTS-167 (phase Ι). OTS-167 exhibits a strong
in vitro activity, conferring an IC50 of 0.41 nM at the enzyme level and an in vivo effect on various human cancer
xenograft models. Kinobeads profiling for this drug showed that it is a broad multikinase inhibitor[16]. Compound
2 retains biochemical potency against MELK at physiologically relevant high ATP concentration and demonstrates
selective antiproliferation and cell cycle effects in MELK-dependent cancer cells[17]. Astex reported compound
3[18] and compound 7[19] exhibit low nanomolar potencies in biochemical assays, and these compounds were
shown to be cell permeable; however, no MELK cellular activity or PK data were reported. Compound 4[20] and
compound 5[21] display nanomolar activity against MELK and compound 4 inhibits the expression of the
anti-apoptotic protein Mcl-1 and the proliferation of TNBC cells, exhibiting selectivity for cells expressing high
levels of MELK. Compound 6[22] is a highly selective inhibitor, and it affects only one off-target kinase with IC50
< 10 μM. To date, inhibitors that have not been developed with MELK as the primary target are FDA-approved. A
primary goal of this study was to identify different scaffolds from which to develop new inhibitors of MELK.
O
Me
Me
HN
N
Cl
N
Me
N
N
NH
N
OMe
H
N
HO
Cl
Me
O
HN
N
N
N
Me
N
NH
N
N
N
O
O
HN
O
OMe
NH
1,OTS167
IC50 = 0.41 nM PDB 4CQG
2,NVS-MELK8a
3,MELK-T1
4
N
IC50 = 2 nM PDB 5IH9
IC50 = 37 nM PDB 4D2V
O
IC50 = 3 nM
H
H
N
N
NH
O
N
H
N
S
Hinge binding
O
N
NH
MeO
S
O
CF3
CN
Lys40 region binding
Glu93 region binding
N
H2N
Br
N
O
H
Me
NH
5
IC50 = 98 nM
N
6
7,MELK-T2
IC50 = 19 nM
H
IC50 = 7 nM
Figure 1. Structures of potent and selective MELK inhibitors.
Pharmacophore is a set of structural features responsible for the biological activity of a molecule. It allows
compounds with diverse structures to find common chemical features by ligand pharmacophore mapping to extract
the important interaction features between the enzyme and its inhibitors[23-25]. Herein, ligand-based
pharmacophore models have been carried out on there known type I ATP competitive inhibitors
(compounds 1-3) of MELK (Supporting Information Table S1). Common features of these pharmacophore
models include: two hydrogen bond acceptors form interactions with the hinge and Lys40 region, positive
ionizable features form a salt bridge with Glu93 region and hydrophobic interactions with protein skeleton amino
acid residues. At the same time, the structural features of representative inhibitors are marked as shown in Figure
1.
Based on the binding features analysis above, in order to discover novel MELK inhibitors, our design process
involves the application of a scaffold hopping strategy to use 1H-pyrrolo[2,3-b]pyridine scaffold as the hinge
binding group due to its planar geometric conformation and critical hydrogen bond acceptors and donors to
interact with the amino acid residues of the kinases hinge region[26]. Then, by an accelerated knowledge-based
fragment growing approach[27], essential pharmacophores were added in reasonable positions. First, introducing
pyridine or pyrimidine-containing fragments to the 1H-pyrrole[2,3-b]pyridine yielded compounds 17 and 22,
which could form an hydrogen bond interaction with the Lys40 region and exhibit micromolar potency against
MELK(IC50 values of 2.48 μM and 1.03 μM, respectively). Next, the hydrophilic fragments were introduced as the
R2 and R3, which could form an interaction with the Glu93 region (see in Figure 2). On the basis of above strategy,
the synthesis and biological evaluation of two series of 3-substituted 1H-pyrrolo[2,3-b]pyridine derivatives as
2

MELK inhibitors were reported in this article.
Hinge
Hinge
Hinge
H
H
H
N
N
Hinge
N
N
Hinge
N
N
Add
Pharmacophore
Fragments
Scaffod
Hopping
H
N
N
N
X=N/CH
R=R1/H
N
X
N
R1
O
R
N
N
N
R2
N
N
The scaffold
of OTS167
H2N
New scaffold
H
R3
H
Lys40
Lys40
Lys40
Glu93
Glu93
A series
Figure 2. Rational design of the target compounds.
B series
2. Results and discussion
2.1 Chemistry
Compounds 15a-e, 16f-j and 17 were synthesized as depicted in Scheme 1. N-tosyl protection starting from
the material 1H-pyrrolo[2,3-b] pyridine (8), followed by bromination at C-3, enabled efficient preparation of the
3-bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridine
(10).
Treatment
with
bis(pinacolato)diboron
and
(1,1'-Bis(diphenyl-phosphino)ferrocene)dichloropalladium provided the required Suzuki coupling partner,
intermediate 11. Intermediates 12a-d were obtained in a rapid and efficient way via Suzuki coupling of boronic
esters 11 with corresponding requisite aromatic halogen substitutes. Then, this step was followed by oxidation via
m-CPBA to provide the corresponding sulfones 13a-d. These intermediates allowed for SNAr displacement with
corresponding anilines by using triethylamine as acid binging agent to provide the desired analogues 14a-j. The
tosyl group at N-1 was subsequently removed under basic conditions (NaOH, MeOH/H2O) to give compounds 17,
15a-e and intermediates 15f-j. Finally, removal of the Boc group of intermediates 15f-j with 4N HCl/ethyl acetate
gave compounds 16f-j.
Compounds 20g-i, 21a-f and 22 were synthesized as depicted in Scheme 2. Intermediate 18 was synthesized
via Suzuki coupling of 11 with 4-bromopyridin-2-amine. Subsequently, the intermediate 18 was coupled with
corresponding acids using HATU and ethyldiisopropylamine in DMF to give 19a-j. Finally, target compounds
20f-j, 21a-e and 22 were prepared as previously described procedures and as shown in Scheme 1.
Me
S
Me
Me
Me
Me
N
Br
R1
a
b
c
d
N
O
12a:R1=Cl
12b:R1=H
12c:R1=Me
12d:R1=OMe
O
B
N
N
Ts
N
N
H
N
Ts
N
12
9
11
8
10
N
Ts
N
Ts
N
N
Me
O
R1
R1
S
S
H
H
N
N
Me
N
N
O
N
N
R1
Cl
R2
R2
N
N
N
N
g
e
f
g
12
17
13
15-16
14
N
Ts
N
H
N
N
N
Ts
N
N
N
H
15a:R1=Cl,R2=CH2CH2NH2 15b:R1=Cl,R2= CH2CH(CH3)2CH2NH2
15c:R1=Cl,R2= 3-aminocyclohexyl 15d:R1=Cl,R2= 4-hydroxycyclohexyl
15e:R1=Cl,R2= tetrahydro-2H-pyran-4ylmethyl
13a:R1=Cl
13b:R1=H
13c:R1=Me
14a:R1=Cl,R2=CH2CH2NH2
14b:R1=Cl,R2=CH2CH(CH3)2CH2NH2
14c:R1=Cl,R2= 3-aminocyclohexyl
15f:R1=Cl,R2=N-Boc-piperidin-4-yl
16f:R1=Cl,R2=N-piperidin-4-yl
h
13d:R1=OMe 14d:R1=Cl,R2= 4-hydroxycyclohexyl
14e:R1=Cl,R2= tetrahydro-2H-pyran-4ylmethyl
14f:R1=Cl,R2=N-Boc-piperidin-4-yl
14g:R1=Cl,R2=N-Boc-piperidin-4-ylmethyl
14h:R1=H,R2=N-Boc-piperidin-4-ylmethyl
14i:R1=Me,R2=N-Boc-piperidin-4-ylmethyl
14j:R1=OMe,R2=N-Boc-piperidin-4-ylmethyl
15g:R1=Cl,R2=N-Boc-piperidin-4-ylmethyl
16g:R1=Cl,R2=N-piperidin-4-ylmethyl
h
15h:R1=H,R2=N-Boc-piperidin-4-ylmethyl
16h:R1=H,R2=N-piperidin-4-ylmethyl
15i:R1=Me,R2=N-Boc-piperidin-4-ylmethyl
16i:R1=Me,R2=N-piperidin-4-ylmethyl
15j:R1=OMe,R2=N-Boc-piperidin-4-ylmethyl
16j:R1=OMe,R2=N-piperidin-4-ylmethyl
h
h
h
3

Scheme 1. Preparation of Compounds 15a-e, 16f-j and 17. Reagents and conditions: (a) Ts-Cl, NaH, anhydrous
THF, 0 ℃, 4 h. 93%. (b) NBS, anhydrous CH2Cl2, rt, 24 h. 89%. (c) Bis(pinacolato)diboron, Pd(dppf)Cl2, AcOK,
1,4-dioxane, 80 ℃, 3 h. 72%. (d) corresponding aromatic halogen substitutes, Pd(dppf)Cl2, K2CO3,
1,4-dioxane/H2O, 90 ℃, 5 h. 53-72%. (e) m-CPBA, DMF, rt, 63-80%. (f) R2-NH2, Et3N, 1,4-dioxane, 60 ℃-80 ℃,
4 h. 23%-66%. (g) NaOH, MeOH/H2O, 40 ℃, 5 h. 44%-67%. (h) 4N HCl-EA, rt, 6 h. 88%-95%.
19a:R3=CH2NHBoc
19b:R3=CH2CH2NBoc
H
R3
NH2
N
N
N
19c:R3=N-Boc-(S)-piperid-3-yl
19d:R3=N-Boc-(R)-piperid-3-yl
19e:R3=N-Boc-piperid-4-yl
19f: R3=N-Boc-piperidin-4-ylmethyl
19g:R3=tetrahydro-2H-pyran-4-yl
19h: R3=cyclohexaneyl
O
a
b
11
N
Ts
N
N
N
19i: R3=phenyl
Ts
18
19
c
c
20a:R3=CH2NHBoc
d
21a:R3=CH2NH2
H
R3
NH2
20b:R3=CH2CH2NHBoc
21b:R3=CH2CH2NH2
20c:R3=N-Boc-(S)-piperid-3-yl
N
N
d
N
O
d
21c:R3=N-(S)-piperid-4-yl
20d:R3=N-Boc-(R)-piperid-3-yl
21d:R3=N-(R)-piperid-4-yl
20e:R3=N-Boc-piperid-4-yl
21e:R3=N-piperid-4-yl
d
N
H
N
N
N
d
H
22
20-21
20f: R3=N-Boc-piperidin-4-ylmethyl
21f: R3=N-piperidin-4-ylmethyl
20g:R3=tetrahydro-2H-pyran-4-yl
20h: R3=cyclohexaneyl
d
20i: R3=phenyl
Scheme 2. Preparation of Compounds 20g-i, 21a-f and 22. Reagents and conditions: (a) 4-bromopyridin-2-amine,
Pd(dppf)Cl2, K2CO3, 1,4-dioxane/H2O, 90 ℃, 5 h. 74%. (b) R3-COOH, HATU, DIEA, anhydrous DMF, 30 ℃-50
℃, 12 h. 42%-67%. (c) NaOH, MeOH/H2O, 40 ℃, 5 h. 62%-78%. (d) HCl-EA, rt, 6 h. 86%-94%.
2.2 Biological activity
All newly synthesized compounds were evaluated for their activity against the MELK enzyme using the
homogeneous time-resolved fluorescence (HTRF) assays, as well as for their antiproliferative activity in
MDA-MB-231 (human breast cancer cell) and A549 (human lung cancer cell) cell lines, in which MELK has been
found to be overexpressed[14,28,29], using the MTT assay. Sunitinib and staurosporine were tested for
comparison.
The kinase-based test results indicated that most of these compounds exhibit moderate inhibitory activity
against the MELK enzyme within nanomolar concentrations, confirming the validity of rational designs. In the A
series of compounds, we first focused on the R2 moieties by fixing the R1 moieties. The inhibitory activities (IC50)
of these compounds against MELK are shown in Table 1. The results indicated that when keeping R1 as -Cl, and
varying the R3 group as different hydrophilic fragment-based derivatives (15a-e,16f-g) most compounds exhibited
moderate inhibitory activity against the MELK within nanomolar concentrations. In particular, the
N-piperidin-4-ylmethyl at R2 (16g) exhibited the best activity to MELK (IC50 = 16 nM). Keeping compound 16g's
R3 group and removing the chlorine group at R2 (16h) led to a 2-fold less potency against MELK (IC50 = 32 nM).
Replacement of the -Cl atom with a methyl group (16i) led to significant activity loss against MELK (IC50 = 129
nM). Additionally, switching R1 to a much larger group (methoxy) (16j) caused obvious activity loss. Clearly,
increasing the size of the substituent even moderately (16j) produced a significant reduction in activity,
presumably due to deleterious steric interactions with the protein skeletal amino acid residues. The activity of B
series compounds was generally weaker than that of the A series, as shown in Table 2, the introduction of
hydrophilic amino chain, morpholine or piperidine-containing fragments as the R3 yielded compounds 21a-f and
20g that retained moderate potency against MELK with IC50 values ranging between 122 and 558 nM.
Replacement of the piperidinyl group of 21e with cyclohexyl or benzyl groups provided 20h and 20i, with
4

dramatically decreased enzyme activities (IC50 values of 2771 and > 5000 nM, respectively), which confirms the
importance of the R3 as a hydrophilic fragment for achieving activity.
As shown in Table 1-2, in cell-based assays, a large portion of these compounds displayed moderate
inhibitory potency against these cancer cells at drug concentrations lower than 10 μM. Five of the compounds
(15b, 15c, 15d, 16f and 16h) displayed favorable antiproliferative activity against A549 and MDA-MB-231 cell
lines with IC50 values on the submicromolar scale. Among these compounds, compound 16h, which had an IC50
value of 0.109 μM against A549, and 0.149 μM against MDA-MB-231 cells, was the strongest inhibitor against
these two cancer cell lines. Furthermore, the results in Figure 3 revealed that the cell viability was significantly
decreased in both A549 and MDA-MB-231 cells treated with inhibitor 16h in a time and concentration-dependent
manner.
Table 1 Biological activity of the compounds 15a-e and 16f-j.
R1
HN
N
N
N
NH
R2
A series
antiproliferative activity IC50 (μM)b
Compd.
15a
R1
Cl
Cl
R2
MELK, IC50 (nM)a
A549
MDA-MB-231
NH2
255
494
9.228 ± 0.219
7.768 ± 0.167



NH2
15b
0.940 ± 0.0448
0.529 ± 0.040
0.566 ± 0.030
0.172 ± 0.0171
NH2
15c
15d
15e
16f
16g
16h
16i
Cl
Cl
245
102
156
116
16

0.861 ± 0.044
13.029 ± 0.798
0.693 ± 0.016
2.479 ± 0.155
0.109 ± 0.009
14.410 ± 0.404
12.71 ± 1.006
0.579 ± 0.047
13.36 ± 0.774
0.196 ± 0.014
1.770 ± 0.125
0.149 ± 0.003
7.894 ± 0.382
8.863 ± 0.461
OH
O

Cl

Cl
NH




Cl
NH
NH
NH
NH
H
32
Me
OMe
129
240
16j
5

sunitinib
26
23
2.024 ± 0.189
1.817 ± 0.121
-
c
staurosporine
-
a The IC50 values are shown as the mean (nM) values from two separate experiments.
b Concentration that inhibits the proliferation of cancer cells by 50%. The cell proliferation was determined by
MTT assay after incubation with the compounds for 72h. The mean values of three independent experiments ± SE
are reported.
c Untested
Table 2 Biological activity of the compounds 20g-i and 21a-f.
HN
N
N
NH
O
R3
B series
antiproliferative activity IC50 (μM)b
Compd.
21a
R3
MELK, IC50 (nM)a
A549
MDA-MB-231
122
324
0.323 ± 0.011
6.965 ± 0.279
2.319 ± 0.189


NH2
NH2
21b
11.160 ± 0.702
1.913 ± 0.123

21c
21d
168
329
1.163 ± 0.045
2.941 ± 0.252
N
H

1.513 ± 0.092
N
H


21e
21f
20g
139
551
558
9.532 ± 0.532
23.42 ± 0.253
3.042 ± 0.114
6.608 ± 0.359
5.674 ± 0.291
5.750 ± 0.341
NH
NH

O


c
20h
20i
2771
-
-
>5000
26
-
-
sunitinib
2.024 ± 0.189
1.817 ± 0.121
6

staurosporine
23
-
-
a The IC50 values are shown as the mean (nM) values from two separate experiments.
b Concentration that inhibits the proliferation of cancer cells by 50%. The cell proliferation was determined by
MTT assay after incubation with the compounds for 72h. The mean values of three independent experiments ± SE
are reported.
c Untested
Figure 3. The effects of treatment time and concentration of 16h on A549 and MDA-MB-231 cell viability.
2.3 Cellular selectivity assay
We evaluated the effects of several compounds on cell growth in a MELK-independent cell line MCF-7[17],
in which MELK has been found to be low in expression, using the MTT assay. Most of the compounds possessed
significant anticancer activities with IC50 values ranging from 0.245 to 6.292 μM. Noteworthy, compound 16h
potently inhibited the proliferation of MCF-7 cells (IC50 = 0.245 μM). These data suggest that these compounds
exhibit non-selective activity targeting MELK, leading to equal effective proliferation inhibition in MELK low
expressing cancer cells compared to MELK high expressing cells.
Table 3 Antiproliferative activity of several compounds.
Compd.
15b
MCF-7 (μM)a
1.132 ± 0.014
1.190 ± 0.093
1.156 ± 0.105
0.868 ± 0.010
5.517 ± 0.324
Compd.
16h
MCF-7 (μM)a
0.245 ± 0.012
2.153 ± 0.115
4.800 ± 0.084
6.292 ± 0.489
15c
21a
15d
21c
16f
21d
sunitinib
a Concentration that inhibits the proliferation of cancer cells by 50%. Cell proliferation was determined by MTT
assay after incubation with compounds for 72 h. The mean values of three independent experiments ± SE are
reported.
2.4 Kinase selectivity profile of compound 16h
The kinase selectivity of 16h was profiled against a panel of 50 kinases covering the major tumor
progression, metastasis, angiogenesis, oncogenic activation and mitogenic stimulation kinases of the human
protein kinome at a concentration of 1.0 μM, and the percent inhibition values are reported in Table 4. Compound
16h is a multi-target kinase inhibitor, with six kinases (CDK2, CLK1, FLT3, MELK, PKA and ROCK1)
producing greater than 80% inhibition. Five kinases (AMPK, CSF1R, JAK3, MINK1 and TYK2) were inhibited
by 16h to an extent of 40% - 80%. Given that all major human cancers seem to harbor not a single but several
concomitant dysregulation of kinase pathways, suggesting that compound 16h suppressed the proliferation of
7

cancer cells should through the combined effect of multiple targets.
Table 4 Kinase selectivity profile of compound 16h.a
%inhibition
%inhibition
%inhibition
%inhibition
Kinase
Kinase
Kinase
Kinase
at 1 μM
9
at 1 μM
7
at 1 μM
24
7
at 1 μM
84
18
8
ABL1
AKT1
ALK
ERK1
ERK2
FGFR1
FGFR4
FLT1
FLT3
FYN
KDR
KIT
PKA
PTK2
RET
17
18
43
32
8
7
19
26
8
LCK
23
99
-9
AMPK
Aurora A
BMX
MELK
MET
ROCK1
SRC
99
7
86
9
MINK1
MKNK1
NEK2
NTRK1
PAK4
PDGFRA
PIM1
72
15
1
SYK
10
10
15
45
12
7
BRAF
BTK
22
15
91
5
TBK1
TEK
HER2
IGF1R
IRAK4
ITK
-18
9
CDK2
CHEK1
CLK1
CSF1R
EGFR
29
12
8
TYK2
YES1
ZAP70
14
30
14
73
95
63
8
JAK1
JAK3
27
6
PLK1
a Selectivity profile of compound 16h measured at a concentration of 1 μM in a panel of 50 kinases generated with
the SelectScreen® Profiling Service from Life Technologies. The results represent the mean of three independent
experiments performed in triplicate.
2.5 Effects of compound 16h on cell apoptosis and cell cycle progression
To further investigate the antiproliferative mechanism of 16h on A549 cells, the effects of 16h on apoptosis
in A549 cells was tested by flow cytometry (Figure 4A). Compound 16h strongly induced apoptosis of A549 cells
in a concentration-dependent manner, with apoptotic rates of 9.20%, 15.22%, and 35.02% at concentrations of 50,
100, and 200 nM, respectively.
The effect of 16h on cell cycle progression in A549 cells was analyzed using flow cytometry analysis (Figure
4B and 4C). Compared with the control group, the percentages of cells in the G0/G1 phase increased from 50.76%
to 61.62%, whereas cells in the G2/M phase decreased from 14.02% to 4.39% after treatment with 16h at
concentrations of 50, 100, and 200 nM for 72 h. The percentages of cells in the S phase showed only minor
changes. Evidently, 16h effectively arrested A549 cells in the G0/G1 phase.
8

Figure 4. Flow cytometry was used to investigate the effect of compound 16h on A549 cells apoptosis (A) and
cell cycle progression (B). (C). Quantitative analysis of cell cycle.
2.6 Compound 16h inhibited A549 cells migration
In order to investigate the effect of 16h on the migration of A549 cells, transwell assays were carried out.
A549 cells were incubated with DMSO and 16h (50, 100 and 200 nM) for 72 h. Under these conditions, as
displayed in Figure 5A and 5B, the number of cells that permeated the membrane were reduced by 11%, 25% and
36%, respectively. These results demonstrated that 16h had a significant anti-metastatic ability against A549 cells.
Figure 5. (A). The effect of compound 16h on the migration of A549 cells were investigated by transwell assays.
(B). Quantitative analysis of migration.
2.7 Molecular modeling analysis
To elucidate the interaction mode of compound 16h, a docking study of this compound in the ATP-binding
site of MELK (PDB entry 4CQG) was performed. The best predicted binding mode is shown in Figure 6 and has a
calculated binding energy of -9.0 kcal/mol. As shown in Figure 6, the 1H-pyrrolo[2,3-b]pyridine scaffold forms
two hydrogen bonds with the NH of Cys89 and the backbone carbonyl oxygen of Glu87 in the hinge region. The
pyrimidine nitrogen forms a hydrogen bond with Lys40, and further stabilization may be achieved through the
hydrophobic interactions of the pyrimidine ring with the hydrophobic side chains of Ile148, Leu86 and Leu61.
Furthermore, the piperidine moiety appeared to point toward the solvent region by forming a salt bridge with
Glu93 located at the edge of the active pocket.
9

Figure 6. Molecular docking mode of compound 16h into the MELK active site. (A) Detailed interactions with the
protein residues. Each of the dashed red and yellow lines represent hydrogen bonds and salt bridges. (B) The
two-dimensional interaction diagram showing the interaction of compound 16h with key amino acid residues in
MELK active site.
2.8 In vitro metabolic stability and CYP450 inhibition
We investigated the in vitro metabolic stability of compound 16h in rat liver microsomes and evaluated its
ability to inhibit human cytochrome P450 in vitro. As shown in Table 5 and Table 6, compound 16h possessed
moderate microsomal metabolic stability, and it had weak inhibitory effect on CYP2C9, CYP2D6 and CYP3A4
metabolism and had moderate inhibitory effect on CYP1A2 and CYP2C19 metabolism.
Table 5 Liver microsomal stability
Remaining (%)
T = 1 h
Remaininga (%)
T = 1 h
CLint(mic)
(μL/min/mg)
17.5
CLint(liver)
(μL/min/mg)
31.5
parameters
in rat
T1/2 (min)
79.2
59.0
87.6
a
The NADPH regeneration system (replaced with buffer) was not added to the sample during the one hour
incubation.
Table 6 Cytochrome P450 inhibitiona
isozyme
%inhibition at 10 μM
CYP1A2
26.7
CYP2C9
4.9
CYP2C19
37.8
CYP2D6
5.8
CYP3A4
1.3
a Assays were performed in pooled human liver microsomes.
2.9 Physicochemical and in vitro ADME properties of 16h
In recent years, in order to increase the success rate of drug discovery and promote high-quality drug
candidates into clinical research, the importance of optimizing the absorption, distribution, metabolism, and
excretion (ADME) properties of compounds has been extensively discussed[30]. To further characterize the
drug-likeness of the most promising compound 16h, the physiochemical parameters were calculated using the
Qikprop, and the results obtained are shown in Table 7.
According to recent literature[31,32], 28 small molecule kinase inhibitors approved by the US Food and Drug
Administration (FDA) have the optimal range of physicochemical characteristics as shown in Table 7. The
drug-like properties of compound 16h all fall within the optimum ranges. In addition, to assess the contribution of
structural and lipophilicity to in vitro potency, ligand efficiency (LE)[33], ligand lipophilicity efficiency
(LLE)[34], and ligand-efficiency-dependent lipophilicity (LELP)[35] were calculated using the in vitro MELK
inhibition data. Compound 16h displayed excellent LE, LLE, and LELP values within the preferred range for good
hits and leads.
Table 7 Drug-likeness of compound 16ha
b
c
d
e
f
g
h
i
j
MV (g.mol-1)
clogP
2-5
tPSA (Å2)
HDBs
≤4
HBAs
≤9
NRBs
3-9
4
LE
LLE
LELP
optimum range
300-600
308.17
60-140
71.09
16h
2.31
3
5
0.46
5.18
5.02
a Properties calculated using QikProp, version 4.3, Schrödinger. b MV: molecular weight; c clog P: calculated log P;
d tPSA: topological polar surface area; e HBDs: hydrogen-bond donors; f HBAs: hydrogen-bond acceptors; g NRBs:
i
the number of rotatable bonds; h LE = -1.4(log[IC50 (MELK, mol/L)/number of heavy atoms]); LLE = - log[IC50
(MELK, mol/L)] - clogP; j LELP = clogP/LE.
10

3. Conclusions
In conclusion, based on a scaffold hopping strategy and fragment growing approach, a class of MELK
inhibitors bearing a 3-substituted 1H-pyrrolo[2,3-b]pyridine scaffold were designed and synthesized. The
structure-activity relationships of these compounds were discussed from the perspective of enzymatic and cellular
activities. Compounds 16g and 16h exhibited the most potent inhibitory activity against MELK (IC50 = 16 nM and
32 nM, respectively). Compound 16h exhibited excellent anti-proliferative effect with IC50 values from 0.109 μM
to 0.245 μM on A549, MDA-MB-231 and MCF-7 cell lines with high or low expression of MELK. Furthermore,
compound 16h effectively induced apoptosis and G0/G1 phase arrest in A549 cells and suppressed the migration
of A549 cells. Further investigation demonstrated that compound 16h had moderate stability in rat liver
microsomes (T1/2 = 79.2 min) and showed moderate inhibitory activity against various subtypes of human
cytochrome P450. Finally, molecular docking demonstrated the binding pose between compound 16h and MELK
and explained its anti-MELK activity. Drug-likeness predictions indicate that compound 16h had better
pharmacological properties and drug development capabilities. However, Compound 16h was a multi-target kinase
inhibitor and exhibited potent potency against cancer cell lines with high or low expression of MELK. Meanwhile,
recently several studies reported MELK expression correlates with tumor mitotic activity but is not required for
cancer growth. These data indicated that compound 16h suppressed the proliferation and migration of cancer cells
should through an off-target mechanism. Taken together, these results provided a practical basis for the further
structural optimization of 3-substituted 1H-pyrrolo[2,3-b]pyridine derivatives as MELK inhibitors and
demonstrated that compound 16h may serve as a new lead compound for further discovery of anticancer drugs.
4. Experimental section
4.1 Chemistry
Starting materials, reagents and solvents were obtained from commercial suppliers and used without further
purification unless otherwise indicated. Anhydrous solvents were dried and stored according to standard
procedures. All reactions were monitored by thin layer chromatography (TLC) on silica gel plates with
fluorescence F-254 and visualized with UV light. Column chromatography was carried out on silica gel (200-300
1
mesh). H NMR and 13C NMR spectral data were recorded in DMSO-d6, MeOD or CDCl3 on Bruker ARX-600
NMR or Bruker ARX-400 NMR spectrometers with TMS as an internal standard. High-resolution accurate mass
spectrometry (HRMS) determinations for all final target compounds were obtained on a Bruker micromass time of
flight mass spectrometer equipped with an electrospray ionization (ESI) detector. All melting points were obtained
on a Büchi melting point B-540 apparatus and are uncorrected.
4.1.1 Preparation of 1-tosyl-1H-pyrrolo[2,3-b]pyridine(9)
1-tosyl-1H-pyrrolo[2,3-b]pyridine(9) was synthesized according to previously reported methods[36].
4.1.2 Preparation of 3-bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (10)
A solution of 9 (1.84 mmol) in CH2Cl2 (30 mL) was slowly added to NBS (2.02 mmol) at 0°C, The mixture
was stirred at 25°C for 24 h. The mixture was diluted with CH2Cl2, and the organic layer was washed with brine,
dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by column chromatography to
afford compound 10 as a white solid, yield: 89%. 1H NMR (600 MHz, DMSO-d6) δ 8.45 (dd, J = 4.8, 1.4 Hz, 1H),
8.21 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.96–7.94 (m, 1H), 7.44–7.40 (m, 3H), 2.33 (s, 3H).
4.1.3 Preparation of 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (11)
11

To a solution of 10 (18.51 mmol), bis(pinacolato)diboron (22.21 mmol), and AcOK (55.52 mmol) in
1,4-dioxane (50 mL) was added Pd(dppf)Cl2 (1.48 mmol) under a nitrogen atmosphere. The mixture was purged
with nitrogen for 5 min and then heated at 80 °C until the completion of the reaction. The mixture was diluted with
ethyl acetate, and the organic layer was washed with brine, dried over anhydrous Na2SO4 and filtered. The filtrate
was concentrated and purified by chromatograph to give the coupling intermediate as light white solid, yield: 72%.
1H NMR (600 MHz, CDCl3) δ 8.41 (dd, J = 4.8, 1.6 Hz, 1H), 8.19 (dd, J = 7.8, 1.6 Hz, 1H), 8.14 (s, 1H), 8.09 (d, J
= 8.4 Hz, 2H), 7.28–7.25 (m, 2H), 7.18 (dd, J = 7.8, 4.8 Hz, 1H), 2.36 (d, J = 9.1 Hz, 3H), 1.35 (s, 12H).
4.1.4 Compounds 12a-d were prepared following the synthetic procedure of 12a.
4.1.4.1 3-(6-chloro-2-(methylthio)pyrimidin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (12a)
To a solution of 11 (0.25 mmol), 4,6-dichloro-2-(methylthio)pyrimidine (0.25 mmol), and K2CO3 (0.75 mmol)
in 1,4-dioxane:H2O(4:1)(4 mL) was added Pd(dppf)Cl2 (0.013 mmol) under a nitrogen atmosphere. The mixture
was purged with nitrogen for 5 min, and then heated at 90 °C until the completion of the reaction. The mixture was
diluted with ethyl acetate, and the organic layer was washed with brine, dried over anhydrous Na2SO4 and filtered.
The filtrate was concentrated and purified by column chromatography to give the coupling intermediate as white
solid, yield: 64%.1H NMR (600 MHz, DMSO-d6) δ 9.13 (s, 1H), 8.78 (dd, J = 8.0, 1.6 Hz, 1H), 8.46 (dd, J = 4.7,
1.5 Hz, 1H), 8.19 (s, 1H), 8.07 (d, J = 8.4 Hz, 2H), 7.48–7.44 (m, 3H), 2.63 (s, 3H), 2.35 (s, 3H).
4.1.5 Compounds 13a-d were prepared following the synthetic procedure of 13a.
4.1.5.1 3-(6-chloro-2-(methylsulfonyl)pyrimidin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (13a)
m-CPBA (6 mmol) was added to a DMF solution of 12a (2 mmol) at 0 °C. The mixture was stirred at room
temperature for 20 h. The reaction mixture was diluted with ethyl acetate, washed with a saturated solution of
sodium sulfite (2×50 mL) and then with a saturated solution of sodium hydrocarbonate (2×50 mL). The organic
phases were dried over anhydrous Na2SO4. The organic phase was evaporated, and the residue was purified by
1
column chromatography to provide the title compound 13a as white solid, yield: 78%. H NMR (600 MHz,
DMSO-d6) δ 9.34 (s, 1H), 8.84 (dd, J = 8.0, 1.6 Hz, 1H), 8.81 (s, 1H), 8.50 (dd, J = 4.7, 1.6 Hz, 1H), 8.08 (d, J =
8.5 Hz, 2H), 7.52 (dd, J = 8.0, 4.7 Hz, 1H), 7.47 (d, J = 8.2 Hz, 2H), 3.51 (s, 3H), 2.36 (s, 3H).
4.1.6 Compounds 14a-j were prepared following the synthetic procedure of 14g.
4.1.6.1 tert-butyl 4-(((4-chloro-6-(1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-yl)amino)methyl)piperidine-
1-carboxylate (14g)
Tert-butyl 4-(aminomethyl) piperidine-1-carboxylate (1.1 mmol) and Et3N (3 mmol) were added to a solution
of 13a (1 mmol) in dioxane (5 mL). The mixture was heated to 70 °C and stirred for 4 h. Water was added, and the
mixture was extracted with CH2Cl2. The combined organic extracts were washed with brine, dried over anhydrous
Na2SO4, filtered, and concentrated. The residue was purified by column chromatography to afford compound 14g
1
as a yellow solid, yield: 35%. H NMR (600 MHz, CDCl3) δ 8.63 (d, J = 6.8 Hz, 1H), 8.48 (d, J = 3.7 Hz, 1H),
8.33 (s, 1H), 8.13 (d, J = 8.2 Hz, 2H), 7.29 (d, J = 8.2 Hz, 2H), 7.28–7.26 (m, 1H), 6.92 (s, 1H), 5.47 (s, 1H), 4.12
(s, 2H), 3.42 (s, 2H), 2.68 (s, 2H), 2.38 (s, 3H), 1.83–1.74 (m, 3H), 1.44 (s, 9H), 1.23–1.17 (m, 2H).
4.1.7 Compounds 15a-j, 17 were prepared following the synthetic procedure of 15a.
4.1.7.1 N1-(4-chloro-6-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-yl)ethane-1,2-diamine (15a)
14a (0.2 mmol) was dissolved in 5 mL methanol. 2 N sodium hydroxide solution (1.0 mL) was added to the
mixture. The mixture was heated to 40 °C and stirred for 5 h. The majority of the solvent was removed under
vacuum, and the residue was partitioned between ethyl acetate and water. The organic layer was dried over
12

anhydrous Na2SO4 and concentrated. The residue was purified by column chromatography to afford compound
15a as a yellow solid. Yield: 62%; mp:287.5–288.6 ℃; 1H NMR (400 MHz, MeOD) δ 8.85 (s, 1H), 8.29–8.27 (m,
1H), 8.26 (s, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.26–7.19 (m, 2H), 7.16 (s, 1H), 3.77 (s, 2H), 3.26 (t, J = 5.4 Hz, 2H),
2.34(s, 2H). 13C NMR (150 MHz, MeOD) δ 161.85, 150.16, 144.54, 143.46, 141.72, 130.38, 130.36, 129.82,
126.95, 118.40, 105.86, 40.21, 21.29. HRMS calcd for C13H13ClN6, [M+H]+, 289.0963; found 289.0962.
4.1.7.2 N1-(4-chloro-6-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-yl)-2,2-dimethyl-propane-1,3-diamine (15b)
White solid, yield: 56%; mp: 222.4–223.1 ℃; 1H NMR (400 MHz, MeOD) δ 8.94 (s, 1H), 8.33–8.28 (m, 2H),
7.27 (dd, J = 7.8, 4.8 Hz, 1H), 7.15 (s, 1H), 3.38 (s, 2H), 2.81 (s, 2H), 1.12 (s, 6H). HRMS calcd for C16H19ClN6,
[M+H]+, 331.1432; found 331.1429.
4.1.7.3 N1-(4-chloro-6-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-yl)cyclohexane-1,3-diamine (15c)
White solid, yield: 45%; mp: 229.6–230.4 ℃; 1H NMR (400 MHz, MeOD) δ 8.88 (s, 1H), 8.27 (dd, J = 4.8,
1.5 Hz, 1H), 8.20 (s, 1H), 7.24 (dd, J = 8.0, 4.8 Hz, 1H), 7.03 (s, 1H), 3.98 (s, 1H), 3.00–2.93 (m, 1H), 2.39–2.34
(m, 1H), 2.11 (s, 1H), 2.00–1.88 (m, 2H), 1.58–1.48 (m, 1H), 1.33–1.17 (m, 3H). 13C NMR (150 MHz, MeOD) δ
165.50, 163.19, 161.74, 150.11, 144.44, 129.80, 126.96, 120.08, 118.26, 114.02, 104.54, 53.56, 50.62, 41.42,
34.46, 32.80, 24.13. HRMS calcd for C17H19ClN6, [M+H]+, 343.1432; found 343.1440.
4.1.7.4 4-((4-chloro-6-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-yl)amino)cyclo-hexan-1-ol (15d)
Yellow solid, yield: 48%; mp: 296.3–298.6 ℃; 1H NMR (400 MHz, DMSO-d6) δ 12.32 (s, 1H), 8.97 (s, 0.5H),
8.75 (s, 0.5H), 8.50 (s, 1H), 8.30 (d, J = 3.6 Hz, 1H), 7.42 (s, 1H), 7.25–7.13 (m, 2H), 4.59 (s, 1H), 3.83–3.61 (m,
1H), 3.44 (s, 1H), 2.03–1.88 (m, 4H), 1.36–1.28 (m, 4H). 13C NMR (150 MHz, DMSO-d6) δ 163.89, 161.56,
159.55, 149.23, 143.71, 129.89, 129.66, 117.81, 117.02, 111.86, 102.40, 68.40, 49.83, 34.19 (2C), 30.07 (2C).
HRMS calcd for C17H18ClN5O, [M-H]-, 342.1127; found 342.1159.
4.1.7.5 4-chloro-6-(1H-pyrrolo[2,3-b]pyridin-3-yl)-N-((tetrahydro-2H-pyran-4-yl) methyl)pyrimidin-2-amine(15e)
White solid, yield: 67%; mp: 235.2–235.9 ℃; 1H NMR (400 MHz, DMSO-d6) δ 12.33 (s, 1H), 8.97 (s, 0.5H),
8.77 (s, 0.5H), 8.50 (s, 1H), 8.30 (d, J = 4.6 Hz, 1H), 7.63 (s, 1H), 7.26–7.15 (m, 2H), 3.85 (d, J = 10.9 Hz, 2H),
3.28–3.10 (m, 4H), 2.02–1.81 (m, 1H), 1.67 (d, J = 12.4 Hz, 2H), 1.28–1.22 (m, 2H). 13C NMR (150 MHz,
DMSO-d6) δ 163.83, 162.38, 159.57, 149.23, 143.71, 131.06, 129.93, 117.78, 117.08, 111.85, 102.59, 66.83 (2C),
46.95, 34.59 (2C), 30.69. HRMS calcd for C17H18ClN5O, [M-H]-, 342.1127; found 342.1129.
4.1.7.6 3-(6-chloro-2-(methylthio)pyrimidin-4-yl)-1H-pyrrolo[2,3-b]pyridine (17)
White solid, yield: 57%; 1H NMR (400 MHz, DMSO-d6) δ 12.42 (s, 1H), 8.71 (dd, J = 7.9, 1.4 Hz, 1H), 8.49
(s, 1H), 8.29 (dd, J = 4.6, 1.4 Hz, 1H), 7.21 (dd, J = 7.9, 4.7 Hz, 1H), 7.05 (s, 1H), 2.62 (s, 3H).
4.1.8 Compounds 16f-j were prepared following the synthetic procedure of 16f.
4.1.8.1 4-chloro-N-(piperidin-4-yl)-6-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine Hydrochloride (16f)
4N HCl in ethyl acetate (4 mL) was added to a solution of 15f (0.35 mmol). The mixture was stirred for 10 h
at room temperature, and then, the resulting solid was filtered and dried to give the title compound as a white solid.
Yield: 92%; mp: 351.4–352.3 ℃; 1H NMR (400 MHz, MeOD) δ 9.64 (s, 1H), 8.57–8.53 (m, 2H), 7.73 (t, J = 8.0
Hz, 1H), 7.24 (s, 1H), 4.27–4.20 (m, 1H), 3.50 (d, J = 13.0 Hz, 2H),3.28–3.21 (m, 2H), 2.32 (d, J = 12.4 Hz, 2H),
1.89 (td, J = 14.1, 3.7 Hz, 2H). 13C NMR (150 MHz, MeOD) δ 163.42, 163.07, 162.74, 141.93, 141.47, 136.02,
132.70, 125.05, 118.48, 115.89, 105.91, 46.95, 44.13 (2C), 29.66 (2C). HRMS calcd for C16H17ClN6, [M+H]+,
13

329.1276; found 329.1274.
4.1.8.2 4-chloro-N-(piperidin-4-ylmethyl)-6-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine Hydrochloride
(16g)
Yellow solid, yield: 88%; mp: 335.5–336.6 ℃; 1H NMR (400 MHz, MeOD) δ 9.58 (d, J = 6.2 Hz, 1H), 8.58
(s, 1H), 8.56 (d, J = 5.7 Hz, 1H), 7.78–7.73 (m, 1H), 7.22 (s, 1H), 3.49–3.41 (m, 4H), 3.01 (t, J = 11.8 Hz, 2H),
2.08 (d, J = 11.5 Hz, 3H), 1.61–1.51 (m, 2H). HRMS calcd for C17H19ClN6, [M+H]+, 343.1432; found 343.1429.
4.1.8.3 N-(piperidin-4-ylmethyl)-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine Hydrochloride (16h)
1
White solid, yield: 95%; mp: 299.7–301.4 ℃; H NMR (400 MHz, MeOD) δ 8.84 (dd, J = 7.9, 1.6 Hz, 1H),
8.20 (s, 1H), 8.19 (d, J = 1.6 Hz, 1H), 8.04 (d, J = 5.5 Hz, 1H), 7.13 (dd, J = 7.9, 4.8 Hz, 1H), 6.95 (d, J = 5.5 Hz,
1H), 3.33–3.29 (m, 2H), 3.02 (d, J = 12.3 Hz, 2H), 2.53 (td, J = 12.3, 2.3 Hz, 2H), 1.90–1.79 (m, 3H), 1.28–1.19
(m, 2H). 13C NMR (100 MHz, MeOD) δ 164.74, 163.81, 157.12, 153.08 , 143.22, 133.75, 131.91, 121.00, 117.17,
113.99, 105.86, 48.36, 46.87 (2C), 37.49, 31.85 (2C). HRMS calcd for C17H20N6, [M+H]+, 309.1822; found
309.1846.
4.1.8.4 methyl-N-(piperidin-4-ylmethyl)-6-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine Hydrochloride (16i)
Yellow solid, yield: 93%; mp: 220.6–221.5 ℃; 1H NMR (600 MHz, DMSO-d6) δ 13.12 (s, 1H), 9.21 (d, J =
7.7 Hz, 1H), 9.02 (d, J = 9.8 Hz, 1H), 8.86 (s, 1H), 8.73 (d, J = 7.6 Hz, 1H), 8.52 (s, 1H), 8.41 (d, J = 4.2 Hz, 1H),
7.47 (s, 1H), 7.44–7.38 (m, 1H), 3.56 (s, 2H), 3.28 (d, J = 12.1 Hz, 2H), 2.83 (dd, J = 23.1, 11.8 Hz, 2H), 2.45 (s,
3H), 2.05–1.98(m, 1H), 1.92 (d, J = 11.0 Hz, 2H), 1.59–1.49 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 167.08,
155.48, 154.69, 149.04, 143.98, 134.95, 130.73, 118.47, 117.98, 111.46, 104.25, 45.64, 42.58 (2C), 33.35, 26.09
(2C), 18.75. HRMS calcd for C18H22N6, [M+H]+, 323.1979; found 323.1983.
4.1.8.5 4-methoxy-N-(piperidin-4-ylmethyl)-6-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine Hydrochloride
(16j)
1
White solid, yield: 94%; mp: 234.0–235.7 ℃; H NMR (600 MHz, DMSO-d6) δ 13.03 (s, 1H), 9.22 (d, J =
8.9 Hz, 1H), 9.08 (s, 1H), 9.04–8.86 (m, 2H), 8.58 (d, J = 7.7 Hz, 1H), 8.41 (d, J = 4.4 Hz, 1H), 7.34–7.29 (m,
1H), 6.79 (s, 1H), 4.06 (s, 3H), 3.45 (s, 2H), 3.26 (d, J = 11.9 Hz, 2H), 2.85 (dd, J = 22.8, 11.4 Hz, 2H), 1.93–1.86
(m, 3H), 1.49 (dd, J = 23.3, 11.0 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) δ 172.18, 156.02, 150.58, 148.46,
144.16, 131.25, 129.53, 117.94, 116.60, 104.88, 92.03, 55.29, 45.34, 42.63 (2C), 33.50, 26.03 (2C). HRMS calcd
for C18H22N6O, [M+H]+, 339.1928; found 339.1930.
4.1.9 Preparation of 4-(1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2-amine (18).
Compound 18 was prepared in a similar manner as compound 12. 1H NMR (600 MHz, CDCl3) δ 8.48 (d, J =
4.3 Hz, 1H), 8.12 (d, J = 8.0 Hz, 4H), 7.99 (s, 1H), 7.29 (d, J = 8.2 Hz, 2H), 7.24 (dd, J = 7.7, 4.8 Hz, 1H), 6.87 (d,
J = 4.9 Hz, 1H), 6.73 (s, 1H), 4.71 (s, 2H), 2.37 (s, 3H).
4.1.10 General procedure for the synthesis of compounds 19a-i.
To a solution of 18 (0.2 mmol) and an appropriate acid (0.26 mmol) in CH2Cl2 (3 mL) at 0 °C, HATU (0.26
mmol) and DIEA(0.4 mmol) were added. The resulting mixture was warmed to 30 ℃–50 ℃ and stirred until the
completion of the reaction. The mixture was diluted with EtOAc and washed successively with water and brine.
The organic layer was dried over Na2SO4, filtered, and evaporated. The resulting intermediate was purified by
chromatograph.
14

4.1.11 Preparation of compounds 20g-i and 22.
Compounds 20g-i and 22 were prepared in a similar manner as compounds 15a-j.
4.1.11.1 N-(4-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2-yl)tetrahydro-2H-pyran-4-carboxamide (20g)
White solid, yield: 67%; mp: 265.0–265.4 ℃; 1H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 10.45 (s, 1H),
8.59 (s, 1H), 8.36 (d, J = 8.0 Hz, 1H), 8.33 (dd, J = 4.6, 1.3 Hz, 1H), 8.28 (d, J = 5.3 Hz, 1H), 8.17 (d, J = 2.7 Hz,
1H), 7.47 (dd, J = 5.3, 1.6 Hz, 1H), 7.27 (dd, J = 8.0, 4.7 Hz, 1H), 3.95–3.89 (m, 2H), 3.39–3.32 (m, 2H), 2.85–
2.75 (m, 1H), 1.77–1.63 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 174.14, 152.80, 149.28, 148.09, 144.21,
143.38, 127.41, 126.23, 116.99, 116.69, 116.02, 111.70, 109.48, 66.41 (2C), 41.39, 28.81 (2C). HRMS calcd for
C18H18N4O2, [M+H]+, 323.1503; found 323.1504, [M+Na]+, 345.1322; found 345.1323.
4.1.11.2 N-(4-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2-yl)cyclohexanecarboxamide (20h)
White solid, yield: 74%; mp:256.5–257.0 ℃; 1H NMR (600 MHz, DMSO-d6) δ 12.22 (s, 1H), 10.36 (s, 1H),
8.59 (s, 1H), 8.35 (d, J = 7.9 Hz, 1H), 8.32 (d, J = 4.4 Hz, 1H), 8.26 (d, J = 5.2 Hz, 1H), 8.16 (d, J = 2.5 Hz, 1H),
7.45 (d, J = 5.4 Hz, 1H), 7.26 (dd, J = 7.9, 4.7 Hz, 1H), 2.58–2.52 (m, 1H), 1.82 (d, J = 12.1 Hz, 2H), 1.75 (d, J =
12.8 Hz, 2H), 1.65 (d, J = 12.2 Hz, 1H), 1.47–1.40 (m, 2H), 1.31–1.26 (m, 3H). 13C NMR (150 MHz, DMSO-d6) δ
175.55, 152.93, 149.30, 148.08, 144.16, 143.40, 127.45, 126.26, 117.02, 116.72, 115.90, 111.74, 109.45, 44.40,
29.12 (2C), 25.43, 25.24 (2C). HRMS calcd for C19H20N4O, [M+H]+, 321.1710; found 321.1737.
4.1.11.4 N-(4-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2-yl)benzamide (20i)
White solid, yield: 69%; mp:253.3–253.7 ℃; 1H NMR (600 MHz, DMSO-d6) δ 12.26 (s, 1H), 10.80 (s, 1H),
8.67 (s, 1H), 8.43 (d, J = 7.9 Hz, 1H), 8.36 (d, J = 5.2 Hz, 1H), 8.34 (d, J = 4.0 Hz, 1H), 8.22 (d, J = 2.3 Hz, 1H),
8.08 (d, J = 7.5 Hz, 2H), 7.61 (t, J = 7.3 Hz, 1H), 7.58–7.52 (m, 3H), 7.28 (dd, J = 7.9, 4.6 Hz, 1H). 13C NMR (100
MHz, DMSO-d6) δ 166.16, 152.78, 149.30, 148.11, 144.25, 143.43, 134.19, 131.90, 128.35 (2C), 128.00 (2C),
127.47, 126.32, 117.03, 116.71, 116.61, 111.71, 110.82. HRMS calcd for C19H14N4O, [M+H]+, 315.1240; found
315.1271, [M+Na]+, 337.1060; found 337.1024.
4.1.11.3 4-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2-amine (22)
White solid, yield: 78%; 1H NMR (600 MHz, DMSO-d6) δ 12.09 (s, 1H), 8.33 (d, J = 8.0 Hz, 1H), 8.29 (dd, J
= 4.6, 1.3 Hz, 1H), 8.02 (d, J = 2.6 Hz, 1H), 7.89 (d, J = 5.4 Hz, 1H), 7.20 (dd, J = 7.9, 4.6 Hz, 1H), 6.91 (s, 1H),
6.89 (dd, J = 5.4, 1.4 Hz, 1H), 5.94 (s, 2H).
4.1.12 Preparation of compounds 21a-f.
Compounds 21a-f were prepared in a similar manner as compounds 16f-j.
4.1.12.1 N-(4-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2-yl)-2-aminoacetamide Hydrochloride (21a)
White solid, yield: 91%; mp: 390.9–392.3 ℃; 1H NMR (600 MHz, DMSO-d6) δ 12.87 (s, 1H), 12.76 (s, 1H),
8.62–8.59 (m, 1H), 8.56–8.52 (m, 3H), 8.46 (s, 1H), 8.41 (dd, J = 4.7, 1.0 Hz, 1H), 8.31 (d, J = 6.2 Hz, 1H), 7.84
(d, J = 5.7 Hz, 1H), 7.35 (dd, J = 8.0, 4.8 Hz, 1H), 3.99 (d, J = 5.4 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) δ
167.27, 148.79, 148.71, 148.53, 143.12 , 142.15, 130.23, 128.81, 117.48, 117.44, 116.42, 110.57, 109.01, 41.34.
HRMS calcd for C14H13N5O, [M+H]+, 268.1193; found 268.1202.
4.1.12.2 N-(4-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2-yl)-3-aminopropanamide Hydrochloride (21b)
15

White solid, yield: 92%; mp: 338.5–339.7 ℃; 1H NMR (600 MHz, DMSO-d6) δ 13.07 (s, 1H), 12.94 (s, 1H),
8.70 (dd, J = 8.1, 1.2 Hz, 1H), 8.66 (s, 1H), 8.42 (d, J = 1.4 Hz, 1H), 8.41 (dd, J = 4.7, 1.3 Hz, 1H), 8.27 (d, J = 6.6
Hz, 1H), 8.21 (s, 2H), 7.94 (dd, J = 6.6, 1.6 Hz, 1H), 7.35 (dd, J = 8.0, 4.7 Hz, 1H), 3.20–3.13 (m, 2H), 3.03 (t, J =
7.1 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) δ 171.52, 150.52, 149.25, 148.08, 143.88, 138.76, 131.32, 128.69,
117.78, 117.04, 115.83, 109.95, 108.58, 34.22, 33.84. HRMS calcd for C15H15N5O, [M+H]+, 282.1349; found
282.1347.
4.1.12.3 (S)-N-(4-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2-yl)piperidine-3-carboxamide Hydrochloride (21c)
White solid, yield: 88%; mp: 253.5–254.6 ℃; 1H NMR (600 MHz, DMSO-d6) δ 13.18 (s, 1H), 12.98 (s, 1H),
9.51 (d, J = 9.9 Hz, 1H), 9.25 (d, J = 10.2 Hz, 1H), 8.72 (d, J = 7.8 Hz, 1H), 8.68 (s, 1H), 8.47–8.39 (m, 2H), 8.26
(d, J = 6.4 Hz, 1H), 7.96 (d, J = 5.5 Hz, 1H), 7.36 (dd, J = 8.0, 4.7 Hz, 1H), 3.47 (d, J = 11.5 Hz, 1H), 3.24–3.20
(m, 2H), 3.08 (dd, J = 21.9, 10.8 Hz, 1H), 2.89 (dd, J = 22.5, 11.9 Hz, 1H), 2.27 (d, J = 10.2 Hz, 1H), 1.92–1.85
(m, 1H), 1.84–1.75 (m, 1H), 1.68 (ddd, J = 15.7, 12.4, 3.6 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 173.84,
150.96, 148.93, 147.77, 143.62, 137.91, 131.76, 129.03, 117.85, 117.17, 115.92, 109.86, 108.79, 43.22, 42.67,
25.75, 21.09. HRMS calcd for C18H19N5O, [M+H]+, 322.1662; found 322.1672.
4.1.12.4 (R)-N-(4-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2-yl)piperidine-3-carboxamide Hydrochloride (21d)
Yellow solid, yield: 86%; mp:248.2–249.9 ℃; 1H NMR (600 MHz, DMSO-d6) δ 13.17 (s, 1H), 12.98 (s, 1H),
9.51 (d, J = 8.4 Hz, 1H), 9.25 (d, J = 9.7 Hz, 1H), 8.72 (d, J = 7.9 Hz, 1H), 8.68 (s, 1H), 8.45 (s, 1H), 8.41 (d, J =
4.4 Hz, 1H), 8.26 (d, J = 6.5 Hz, 1H), 7.96 (d, J = 6.4 Hz, 1H), 7.36 (dd, J = 7.9, 4.7 Hz, 1H), 3.48 (d, J = 10.9 Hz,
1H), 3.26–3.17 (m, 2H), 3.08 (dd, J = 21.7, 10.9 Hz, 1H), 2.89 (d, J = 11.0 Hz, 1H), 2.27 (d, J = 11.0 Hz, 1H),
1.93–1.86 (m, 1H), 1.84–1.76 (m, 1H), 1.74–1.65 (m, 1H). 13C NMR (150 MHz, DMSO-d6) δ 173.81, 150.84,
149.16, 147.91, 143.83, 138.19, 131.63, 128.84, 117.84, 117.07, 115.91, 109.87, 108.76, 43.26, 42.69, 25.80,
21.12. HRMS calcd for C18H19N5O, [M+H]+, 322.1662; found 322.1670.
4.1.12.5 N-(4-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2-yl)piperidine-4-carboxamide Hydrochloride (21e)
White solid, yield: 94%; mp: 275.1–276.7 ℃; 1H NMR (600 MHz, DMSO-d6) δ 12.95 (s, 1H), 12.89 (s, 1H),
9.09 (s, 1H), 9.00 (s, 1H), 8.69 (d, J = 8.0 Hz, 1H), 8.64 (s, 1H), 8.42 (s, 1H), 8.41 (dd, J = 4.6, 1.1 Hz, 1H), 8.25
(d, J = 6.6 Hz, 1H), 7.92 (d, J = 5.4 Hz, 1H), 7.35 (dd, J = 8.0, 4.7 Hz, 1H), 3.34 (d, J = 12.6 Hz, 2H), 3.03–2.94
(m, 3H), 2.15 (d, J = 11.7 Hz, 2H), 1.92 (td, J = 14.7, 3.7 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 175.41,
150.91, 149.21, 147.99, 143.86, 137.88, 131.56, 128.80, 117.82, 117.00, 115.75, 109.83, 108.65, 41.99 (2C), 24.39
(2C). HRMS calcd for C18H19N5O, [M+H]+, 322.1662; found 322.1665.
4.1.12.6 N-(4-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2-yl)-2-(piperidin-4-yl)acetamide Hydrochloride (21f)
White solid, yield: 89%; mp: 282.3–284.2 ℃; 1H NMR (400 MHz, DMSO-d6) δ 13.34 (s, 1H), 13.02 (s, 1H),
9.28 (d, J = 9.6 Hz, 1H), 8.96 (d, J = 9.9 Hz, 1H), 8.77 (dd, J = 8.2, 1.3 Hz, 1H), 8.69 (s, 1H), 8.45 (d, J = 1.6 Hz,
1H), 8.41 (dd, J = 4.7, 1.3 Hz, 1H), 8.24 (d, J = 6.7 Hz, 1H), 7.97 (dd, J = 6.8, 1.8 Hz, 1H), 7.35 (dd, J = 8.1, 4.8
Hz, 1H), 3.24 (d, J = 12.4 Hz, 2H), 2.87 (q, J = 12.0 Hz, 2H), 2.61 (d, J = 7.0 Hz, 2H), 2.21–2.08 (m, 1H), 1.88 (d,
J = 12.5 Hz, 2H), 1.53 (td, J = 15.1, 3.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 173.49, 151.07, 148.96,
147.73, 143.64, 137.45, 131.81, 129.10, 117.85, 117.14, 115.70, 109.81, 108.47, 42.72 (2C), 42.29, 30.51, 27.90
(2C). HRMS calcd for C19H21N5O, [M+H]+, 336.1819; found 336.1816.
4.2 Pharmacological assay
4.2.1 MELK HTRF assay
16

The MELK kinase assay was performed using the HTRF® KinEASE™-STK kit (Cisbio Bioassays, France)
in white 384 well small volume plate with a total working volume of 20 μL[37,38]. Purified MELK enzyme was
purchased from Carna Biosciences (Japan). Compounds were diluted step by step from a concentrated stock of 8
mM in 100% DMSO and with serial kinase reaction buffer dilutions. The IC50 measurements were performed in
replicates. For each assay, 4 μL dispensed compounds, 4 μL of mix 1 (ATP: 39.14 μM+substrate S1) and 2 μL
kinase (0.111 ng/µL) were added in the assay wells. The assay plate were incubated at 30°C for 50 min and
terminated by adding 10 µL of mix 2 (Sa-XL665+STK-Antibody-Cryptate). After a final incubation (60 min at
room temperature), HTRF signal was obtained by reading the plate in Infinite® F500 microplate reader (Tecan,
Switzerland). The fluorescence was measured at 620 nM (Cryptate) and 665 nM (XL665). A ratio was calculated
(665/620) for each well. For IC50 measurements, values were normalized and fitted with Prism (GraphPad
software).
4.2.2 Cell proliferation assay
A549 and MDA-MB-231 cells were cultured in a 96-well plate at a density of 4000–5000 cells/well and were
maintained at 37 °C in a 5% CO2 incubator for one day. The cells were then incubated with different
concentrations of compounds over the course of 72 h, then fresh MTT solution was added and incubated for 4 h.
After discarding the supernatant and adding 150 μL DMSO. Observation of each test well was performed at λ490nm
by a Thermo MULTISKAN GO reader.
4.2.3 Cell migration assay by transwell assays; cell apoptosis and cell cycle analysis by flow cytometry
The measurements were performed as previously described[38].
4.2.4 Molecular docking study
All calculations were performed using the AutoDock4.2. The X-ray cocrystal structure of MELK (PDB code:
4CQG) was obtained from the Protein Data Bank. The 3D structure of the compound 16h was generated using the
Corina server (www.mn-am.com/online_demos/corina_demo). The AutoDock 4.2 program equipped with ADT
was used to perform the automated molecular docking[39]. The top 10 docking poses were visually inspected and
the most favorable pose of 16h was displayed. In addition, the figure was prepared using PyMOL.
4.2.5 Cytochrome P450 inhibition assay
Cytochrome P450 inhibition effect was tested in human liver microsomes (0.253 mg/mL) using five special
probe substrates (CYP3A4, midazolam; CYP2C19, S-mephenytoin; CYP2C9, diclofenac; CYP1A2, phenacetin;
CYP2D6, dextromethorphan) in the presence of compound 16h (10 μM). After pre-incubation for 10 min at 37 ℃,
an NADPH regenerating system was added. After the mixed system was incubated for 15 min at 37 ℃, the
reaction was stopped by adding of 400 μL of cold stop solution (200 ng/mL labetalol and 200 ng/mL tolbutamide
in acetonitrile). Then, the incubation mixtures were were centrifuged, and the supernatants were analyzed by
LC/MS/MS.
4.2.6 Liver microsomal stability assay.
The liver microsomal stability assay was performed by the incubation of rat liver microsomes (0.5 mg/mL,
Biopredic, Lot No. MIC254034) at 37 °C with compound 16h at a final concentration of 1 μM in potassium
phosphate buffer (pH 7.4, 100 mM with 10 mM MgCl2). Incubation was initiated by the addition of prewarmed
cofactors (1 mmol NADPH). After incubation at 37 °C for different times (0, 5, 10, 20, 30, and 60 min), the
protein was precipitated by the addition of cold acetonitrile. Then, the precipitated proteins were removed by
17

centrifugation , and the supernatants were injected onto an LC-MS/MS system.
Acknowledgments
We gratefully acknowledge the financial support from the Program for Innovative Research Team of the
Ministry of Education and Program for Liaoning Innovative Research Team in University.
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Declaration of interests
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.
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Graphical abstract
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A library of 1H-pyrrolo[2,3-b]pyridine derivatives were designed and synthesized.
16h exhibited potent enzyme inhibition against MELK (IC50 = 32 nM).
16h potently inhibited the proliferation of A549, MDA-MB-231 and MCF-7 cell lines.
16h is a multi-target kinase inhibitor when tested on a panel of 50 kinases.
16h induced apoptosis and suppressed the migration of A549 cells.
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