Identification of novel inhibitors of Aurora A with a 3-(pyrrolopyridin-2-yl)indazole scaffold

Article abstract of DOI:10.1016/j.bmc.2015.02.004

A novel series of 3-(pyrrolopyridin-2-yl)indazole derivatives were synthesized and biologically evaluated for their anti-proliferative effects on five human cancer cell lines. As a result, all of them exhibited vigorous potency against HL60 cell line with IC₅₀ values ranging from singe digital nanomolar to micromolar level. Besides, a majority of them displayed modest to good antiproliferative activities against the other four cell lines, including KB, SMMC-7721, HCT116, and A549. Particularly, compound 2y, as the most distinguished one in this series, demonstrated IC₅₀ values of 8.3 nM and 1.3 nM against HL60 and HCT116 cell lines, respectively. Afterwards, for exploring the molecular target, compounds2d, 2g and 2y were further selected to evaluate the inhibitory activities against a panel of kinases. Finally, they were identified to be targeting Aurora A kinase with significant selectivity over other kinases, such as CHK1, CDK2, MEK1, GSK3β, BRAF, IKKβ and PKC.

Full text of DOI:10.1016/j.bmc.2015.02.004

Accepted Manuscript
Identification of novel inhibitors of Aurora A with a 3-(pyrrolopyridin-2-yl)in-
dazole scaffold
Pinrao Song, Ming Chen, Xiaodong Ma, Lei Xu, Tao Liu, Yubo Zhou,
Yongzhou Hu
PII:
S0968-0896(15)00088-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.02.004
BMC 12067
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 November 2014
3 February 2015
5 February 2015
Please cite this article as: Song, P., Chen, M., Ma, X., Xu, L., Liu, T., Zhou, Y., Hu, Y., Identification of novel
inhibitors of Aurora A with a 3-(pyrrolopyridin-2-yl)indazole scaffold, Bioorganic & Medicinal Chemistry (2015),
doi: http://dx.doi.org/10.1016/j.bmc.2015.02.004
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Identification of novel inhibitors of Aurora A with a 3-(pyrrolopyridin-2-yl)indazole
scaffold
Pinrao Songa, Ming Chena, Xiaodong Maa, Lei Xub, Tao Liua,*, Yubo Zhoub,*, Yongzhou Hua,*
a
ZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang Province Key Laboratory of
Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou
310058, China
b
National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
Abstract: A novel series of 3-(pyrrolopyridin-2-yl)indazole derivatives were synthesized and
biologically evaluated for their anti-proliferative effects on five human cancer cell lines. As a result, all
of them exhibited vigorous potency against HL60 cell line with IC50 values ranging from singe digital
nanomolar to micromolar level. Besides, a majority of them displayed modest to good antiproliferative
activities against the other four cell lines, including KB, SMMC-7721, HCT116, and A549. Particularly,
compound 2y, as the most distinguished one in this series, demonstrated IC50 values of 8.3 nM and 1.3
nM against HL60 and HCT116 cell lines, respectively. Afterwards, for exploring the molecular target,
compounds 2d, 2g and 2y were further selected to evaluate the inhibitory activities against a panel of
kinases. Finally, they were identified to be targeting Aurora A kinase with significant selectivity over
other kinases, such as CHK1, CDK2, MEK1, GSK3β, BRAF, IKKβ and PKC.
Keywords: Pyrrolopyridin-indazoles; Antiproliferative agents; Cell cycle profile; Aurora A inhibitors;
Selectivity
*Corresponding author:
Tao Liu, Tel: +86-571-88208458; Fax: +86-571-88981051; E-mail address: lt601@zju.edu.cn
Yubo Zhou, Tel: +86-21-5080-1313; Fax: +86-21-5080-1313; E-mail address: ybzhou@mail.shcnc.ac.
cn
Yongzhou Hu, Tel/Fax: +86-571-8820-8460; E-mail address: huyz@zju.edu.cn
1

1. Introduction
Cellular proliferation and growth, as a set of timely ordered events, are governed by a plethora of
protein kinases. Among them, checkpoint kinases (CHKs), cyclin-dependent kinases (CDKs), aurora
kinases (AURKs) and polo-like kinases (PLKs), play an essential role in the cell cycle progression.1
Phosphorylation or dephosphorylation of these functional proteins may be viewed as molecular on/off
switches involved in many distinct cellular processes, such as proliferation, differentiation,
transcription, translation, and apoptosis.2 On the other hand, the frequent mutation or overexpression of
these kinases has been observed in a broad range of human malignancies, thereby providing a unique
opportunity for anti-cancer intervention.3 As validated by ample experimental results, inhibition of
these functional proteins would result in cell accumulation at certain phases (G1, S, or G2/M) and
induce apoptosis of tumor cells.4 Currently, quite a number of small-molecule inhibitors targeting cell
cycle kinases have been developed and advanced into clinical trials.5 However, their further
development as chemotherapeutic drugs has been compromised due to lack of long term efficacy and
severe side effects caused by low selectivity over normal proliferating cells. Hence, there is presently
an urgent demand of more drug-like small-molecule inhibitors with novel and distinct chemotypes.
Our group has been continuously devoted to the synthesis and biological evaluations of small
molecule heterocyclic compounds and has established an in-house compound library. As a part of our
recent work, a novel structural series of 5-amide pyrrolopyridin-indazoles have been prepared. During
our speculation of their potential biological activities, we were attracted by their structural similarity to
a novel chemical entity 1 that targeting cell cycle. Discovered by Merck Research Laboratories, this
5-substituted indole-indazole derivative exhibited IC50 values of 30 nM and 11 nM against CHK1 and
CDK7, respectively (Fig. 1). The indole-indazole scaffold of 1 provided a hydrogen bond
donor-acceptor-donor motif, through which contacts to the hinge backbones of the ATP-binding sites of
numerous protein kinases could be captured.6 The pyrrolopyridin-indazole moiety in our compounds
could be viewed as a bioisostere of the indole-indazole core, which may represent a novel and distinct
template for the exploration of chemotherapeutic antitumor agents. Meanwhile, the
pyrrolopyridin-indazole core may mimic the indole-indazole as the hinge binder to develop kinase
inhibitors. With such a 5-amide pyrrolopyridin-indazole scaffold in hand, our medicinal chemistry
effort was geared towards variation of the substitutions on the C5-amide linker with a variety of
aromatic or alkyl groups to explore the antiproliferative effect. Besides, sulfamide and urea were also
investigated as surrogates for the amide linker. Of greater gratification was that derivative 2y bearing a
urea linker displayed antiproliferative activity in the low nanomolar range and induced G2/M cell cycle
arrest in the cell cycle profile (FACS) analysis. In a biochemical assay, the representative compounds
2d, 2g and 2y, showed great specificity for Aurora A inhibition and little inhibition against other
kinases being tested. Moreover, the binding modes of 2d and 2y bound to Aurora A kinase were
interpreted using in silico docking experiments.
< Insert Fig. 1>
2. Results and discussion
2.1. Chemistry
The general synthetic route of these 5-substituted pyrrolopyridin-indazole derivatives are
illustrated in Schemes 1-3. The intermediates indazoles 7a-c and 4-azaindole 14 were prepared
2

following the method described in the literature with minor modifications.5 Starting from the
commercial available 2-methylanilines 3a-c, the 1H-indazoles 4a-c were obtained via diazotization
using NaBF4/NaNO2 and followed by an in-situ intramolecular cyclization. After substitution of 4a-c
with I2 in the presence of KOH, the newly formed 5a-c were coped with ethylmagnesium bromide and
chlorotributyltin to afford 6a-c. Subsequently, N-Boc protection was carried out to afford intermediates
7a-c.
Meanwhile, the commercially available 8 was subjected to nitration with a mixture of
concentrated HNO3 and H2SO4, which furnished 9 as the product. Afterwards, its amine was masked
with Boc-protecting group and then methylated with methyl iodide to afford 11. Via condensation with
DMFDMA, the generated intermediate underwent a one pot catalytic hydrogenation and cyclization to
produce 12. At the next step, the iodide fragment 14 was achieved by treatment of Boc-protected
product 13 with I2 in the presence of n-butyllithium.
With 7a-c and 14 in hand, critical intermediates 15a-c were prepared from 7a-c and 14 through a
classical Stille coupling reaction. Subsequent removal of all the Boc-protecting groups under acidic
conditions gave the intermediates 16a-c. Finally, the secondary amines were converted into amides,
sulfonamides or ureas via reacting with corresponding acid chlorides, sulfonyl chlorides or isocyanates
to afford the target compounds 2a-n, 2o-x, and 2y-z, respectively.
< Insert Scheme 1>
< Insert Scheme 2>
< Insert Scheme 3>
2.2. Tumor cell growth inhibition studies
All the above compounds were tested for their antiproliferative activities in vitro against five
human cancer cell lines (HL60, KB, SMMC-7721, HCT116, and A549) with MTT assay via
introducing Taxol as a positive control.
As illustrated in Table 1 and 2, it is clear that most of the compounds bearing an amide or
sulfamide linkage were more effective against HL60 cell line than against the other four cell lines with
IC50 values at the submicromolar or low micromolar level (HL60: IC50 = 0.45-6.80 μM) except for the
compounds bearing a urea linkage (2y and 2z). Incorporation of halogen functionalities on the
4-position of the phenyl ring slightly enhanced the potency, as exemplified by compounds 2f-h, which
were over 3-fold more potent relative to the unsubstituted counterpart 2d. Among them, 2g bearing a
chlorine substituent was the most cytotoxic compound with IC50 values of 0.45 μM and 0.63 μM
against HL60 and KB cell lines, respectively. Introduction of electron-donating methoxyl group (2i and
3

2j), electron-withdrawing nitro group (2k) or replacement of the phenyl ring with a furan moiety (2l),
despite retention of cytotoxicity against HL60 cell line, led to a decrease in efficacy against the other
four cell lines. Besides, a slight loss in antiproliferative activities was observed when replacing the
benzene ring with alkyl groups (2a-c versus 2d). The chlorine atom at the 6-position of the indazole
ring was tolerable since the analog 2m displayed little differentiation in cytotoxic activity compared to
2d, whereas introduction of a cyano group (2n) caused a dramatic loss of potency against KB,
SMMC-7721, HCT116, and A549, except for HL60. Similarly, derivatives 2o and 2q-s with a common
tertiary sulfamide linker exhibited comparable activities to their amide counterparts 2d and 2g-i. It is
noteworthy that compounds 2y and 2z with a urea linker stood out as the most potent compounds with
nanomolar IC50 values against HL60 and HCT116 cell lines, respectively. Particularly, derivative 2y
with an inhibitory IC50 value of 1.3 nM against HCT116 cell line, was found to be 3-fold more efficient
than the well-known cytotoxic agent Taxol.
< Insert Table 1>
< Insert Table 2>
2.3. Cell cycle profile (FACS) analysis
By virtue of its distinguished antiproliferative activities, compound 2y was further evaluated for
its effects on the cell cycle by flow cytometry using HCT116 cells (Fig. 2). Our results demonstrated
that treatment of HCT116 cells with 2y at 50, 100 and 200 nM for 24 h induced G2/M cell cycle arrest
in a concentration-dependent manner. At concentration up to 100 nM, compound 2y caused significant
alteration of cell distribution relative to the DMSO control. Treatment with 2y at a higher concentration
of 200 nM led to a remarkable arrest at G2/M phase and apoptosis as illustrated by 25.4 % of the total
cells accumulating in the apoptotic sub-G1 phase．The flow cytometric data clearly indicated that
compound 2y possessed the capacity of dysregulation of cell cycle progression and induction of
apoptosis in HCT116 cells.
< Insert Fig. 2>
2.4. Kinase inhibitory assays
4

To elucidate the molecular target of this new class of compounds, profiling of representative
compounds, including 2d, 2g and 2y, was carried out over a panel of eight kinases. As a result, they
were identified to be Aurora A inhibitors with respective IC50 values of 32 nM, 46 nM, and 519 nM.
Aurora A kinase, a conserved serine/threonine kinase locates mainly to the centrosomes, is
responsible for mitotic entry and exit, centrosome maturation, separation, and bipolar spindle
assembly.7 There are accumulating experimental results illustrating the intimate roles played by
overexpression of Aurora A in genomic instability and tumorigenesis.8 In contrast, inhibition of Aurora
A kinase would induce G2/M tumor cell cycle delay and subsequent apoptotic cell death, which make it
a viable anticancer target for solid tumors and hematological malignancies, including primary colon,
breast cancers, and leukemias.9 Until now, a variety of small-molecule inhibitors of Aurora A, as
exemplified by VX-680 (MK-0457), PHA-739358, SNS-314, CYC-116, AZD1152, MLN8054 and
MLN8237, have been advanced into clinical evaluation stage. Hence, the discovery of 2d, 2g and 2y as
potential Aurora A kinase inhibitors is meaningful and elicits our further research in this area.
As shown in Table 3, derivatives 2d and 2g which only differed in a chlorine substituent on the
phenyl ring displayed comparable inhibitory activities against Aurora A and were over 10-fold more
effective relative to the most cytotoxic compound 2y. The factors attributing to such incompatible
results between the cytotoxicity and enzymatic assay might be multifarious, such as impaired cellular
uptake, metabolism, and/or off target activities unrelated to cancer cell growth and apoptosis.
Interestingly, we did found some discrepancy in cell permeability of these compounds that could to
some extent explained the consequence (Table S2). Clearly, replacement of the hydrophilic morpholine
of compound 1 by more lipophilic phenyl rings in our compounds 2d, 2g and 2y abolished CHK1
inhibitory potency, which was consistent with the fact that simultaneous inhibition of CHK1 and other
cell cycle associated kinases may result in counterproductive effects in the cell cycle control.10 As
expected, these compounds also demonstrated significant selectivity over other kinases namely CDK2,
MEK1, GSK3β, BRAF, IKKβ, and PKC.
< Insert Table 3>
2.5. Molecular docking study
To gain better understanding of the interactions of 2d and 2y with Aurora A protein, we conducted
a molecular docking study utilizing C-DOCKER program within Discovery Studio 2.5. The binding
modes of compounds 2d and 2y bound to Aurora A kinase were depicted in Fig. 3A and Fig. 3B,
respectively. Both compounds 2d and 2y occupied the ATP binding site of Aurora A kinase via
substantial hydrophobic contacts with a number of neighboring hydrophobic residues, and the binding
was stabilized by the hydrogen bonding network with the hinge backbone Ala213 and Glu211 as
expected. The C5 attachments in 2d and 2y with corresponding amide and urea linkages allowed the
phenyl ring extending into the solvent front. Besides, an additional contact between the amide carbonyl
oxygen and Arg220 was observed in 2d. As for 2y, the urea carbonyl oxygen located too far to generate
hydrogen bond contact with the Arg220 residue as 2d did, which suggested that the 5-amide linkage
was beneficial for adopting an appropriate orientation so as to confer additional interactions with the
kinase and increase binding affinity.
< Insert Fig. 3A >
< Insert Fig. 3B >
5

3. Conclusions
A series of novel 3-(pyrrolopyridin-2-yl)indazoles was synthesized and evaluated for their
anti-proliferative activities against five human cancer cell lines. All of them exhibited vigorous potency
against HL60 cell line with IC50 values ranging from singe digital nanomolar to micromolar level.
Besides, a majority of them displayed modest to good antiproliferative activities against the other four
cell lines, including KB, SMMC-7721, HCT116, and A549. In particular, compound 2y, as the most
distinguished one (IC50 values of 8.3 nM and 1.3 nM against HL60 and HCT116, respectively) in this
series, induced cell cycle arrest at G2/M phase and apoptosis in a cell cycle profile (FACS) analysis. To
elucidate the molecular target underlying this result, in vitro biochemical assays were conducted for the
representative compounds 2d, 2g and 2y. Of the eight kinases being tested, good inhibitory activities
were obtained against Aurora A at the nanomolar level and almost no inhibitory efficacy has been
observed against seven other kinase targets. Furthermore, the binding modes of derivatives 2d and 2y
bound to Aurora A were explored using in silico docking. These findings will facilitate the development
of potent pyrrolopyridin-indazoles as a novel class of Aurora A inhibitors. And further structure
optimization work such as addressing the solubility issue to improve the physical chemical properties
will be reported in the near future.
4. Experimental
4.1. Chemistry
Melting points were determined with a B-540 Büchi melting-point apparatus and are uncorrected.
1H NMR spectra were recorded on a 500 MHz, 13C NMR were recorded on a 125 MHz spectrometer at
room temperature (chemical shifts are given in ppm (δ) relative to TMS as internal standard, coupling
constants (J) are in hertz (Hz), and signals are designated as follows: s, singlet; d, doublet; t, triplet; m,
multiplet; br, broad singlet, etc.). Mass spectra (MS), ESI (positive) were recorded on an
Esquire-LC-00075 spectrometer. Thin layer chromatography was carried out using plate silica gel F254
Merck. Reagents and solvents were purchased from common commercial suppliers and some
unhydrous solvents were further purified before usage. All yields are unoptimized and generally
represent the result of a single experiment.
4.1.1. General procedure for the synthesis of indazole intermediates (4a-c).
NaNO2 aqueous solution (13.80 g, 0.2 mol, 32.20 mL) was added dropwise to a mixture of 3a
o
(21.40 g, 0.2 mol), H2O (190 mL), and 12 N HCl aqueous solution (50 mL, 0.6 mol) at 0 C. The
o
mixture was stirred at 0 C for 30 min and then was filtered. The precooled NaBF4 (24.20 g, 0.22 mol)
o
dissolved in H2O (90 mL) was added to the filtrate and kept stirring at 0 C for an additional 40 min.
The precipitate was collected by filtration, washed with cold ethanol (50 mL×3), cold ethyl ether (50
mL×3), and concentrated in vacuo to deliver diazonium salt (19.19 g) as a yellow solid. To a solution
of diazonium salt (19.19 g, 0.093 mol) in CHCl3 (231 mL), KOAc (15.15 g, 0.155 mol) was added. The
mixture was stirred at room temperature for 4 h. Afterwards, the reaction was quenched with water
(200 mL) and extracted with CH2Cl2 (70 mL×3). The organic phase was washed with brine (50 mL×3),
dried over anhydrous Na2SO4, and concentrated under reduced pressure. The obtained crude residue
was purified by recrystallization to give the title compound 7.51 g.
o
1
4.1.1.1. 1H-Indazole (4a). Brown solid (75%), mp: 148-150 C. H NMR (500 MHz, DMSO-d6) δ
13.05 (s, 1H), 8.06 (s, 1H), 7.70 (d, 1H, J = 8.0 Hz), 7.53 (d, 1H, J = 8.0 Hz), 7.34 (t, 1H, J = 7.6 Hz),
7.10 (t, 1H, J = 7.6 Hz). ESI-MS: m/z = 119 [M+1]+.
6

4.1.1.2. 6-Chloro-1H-indazole (4b). Yellow solid (65%), mp: 172-174 oC (lit. 175-176 oC).
o
1
4.1.1.3. 1H-Indazole-6-carbonitrile (4c). Yellow solid (53%), mp: 127-129 C. H NMR (500 MHz,
DMSO-d6) δ 13.64 (s, 1H), 8.28 (s, 1H), 8.17 (s, 1H), 7.99 (d, 1H, J = 8.0 Hz), 7.45 (d, 1H, J = 8.0 Hz).
ESI-MS: m/z = 144 [M+1]+.
4.1.2. General procedure for the synthesis of 3-iodo-1H-indazoles (5a-c).
o
KOH (31.10 g, 0.555 mol) was added in portions at 0 C to a solution of 4a (15.80 g, 0.134 mol)
and I2 (85.40 g, 0.336 mol) in DMF (263.40 mL). The mixture was stirred for 4 h at room temperature,
quenched with saturated Na2S2O3 solution (50 mL), diluted with H2O (150 mL), and extracted with
EtOAc (70 mL×3). The organic phase was washed with brine (50 mL×3), dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The obtained residue was purified by silica gel
column chromatography (petroleum ether/ethyl acetate = 15:1, v/v) to give an orange solid (16.78 g).
4.1.2.1. 3-Iodo-1H-indazole (5a). Orange solid (52%), mp: 142-143 oC. 1H NMR (500 MHz,
DMSO-d6) δ 13.50 (s, 1H), 7.55 (d, 1H, J = 8.4 Hz), 7.43 (d, 1H, J = 8.4 Hz), 7.43 (t, 1H, J = 7.2 Hz),
7.20 (t, 1H, J = 7.2 Hz). ESI-MS: m/z = 245 [M+1]+.
4.1.2.2. 6-Chloro-3-iodo-1H-indazole (5b). Orange solid (51%), mp: 162-165 oC. 1H NMR (500 MHz,
DMSO-d6) δ 13.39 (s, 1H), 7.70 (s, 1H), 7.56 (d, 1H, J = 8.5 Hz), 7.45 (d, 1H, J = 8.5 Hz). ESI-MS:
m/z = 279 [M+1]+.
4.1.2.3. 3-Iodo-1H-indazole-6-carbonitrile (5c). Yellow solid (47%), mp: 259 oC (dec). 1H NMR (500
MHz, DMSO-d6) δ 14.06 (s, 1H), 8.20 (s, 1H), 7.62 (d, 1H, J = 8.5 Hz), 7.49 (d, 1H, J = 8.5 Hz).
ESI-MS: m/z = 270 [M+1]+.
4.1.3. General procedure for the synthesis of 3-(tributylstannyl)-1H-indazoles (6a-c).
Bromoethane (9 mL, 121 mmol) was added slowly to a solution of magnesium (2.70 g, 112.5
o
mmol) and I2 (catalytic amount) in dry ethyl ether (114 mL) at 0 C under N2 atmosphere. Until the
magnesium being completely consumed, 3-iodo-indazole (7.59 g, 31.1 mmol) dissolved in THF (190
mL) was added dropwise to the solution. The mixture was stirred at 0 oC for 30 min. Then a solution of
tributylstannanylium chloride (41.80 g, 128 mmol) in THF (10 mL) was added dropwise. The mixture
was stirred at 0 oC for an additional 40 min, quenched with saturated NH4Cl solution, diluted with H2O
(70 mL), and extracted with EtOAc (30 mL×3). The organic phase was washed with brine (20 mL×3),
dried over anhydrous Na2SO4, and concentrated under reduced pressure. The obtained mixture was
purified by silica gel column chromatography (petroleum ether/ethyl acetate = 10:1, v/v) to give the
title compound as a yellow liquid (7.73 g).
1
4.1.3.1. 3-(Tributylstannyl)-1H-indazole (6a). Yellow oil (61%). H NMR (500 MHz, DMSO-d6) δ
11.05 (s, 1H), 7.76 (d, 1H, J = 8.4 Hz), 7.55 (d, 1H, J = 8.4 Hz), 7.37 (t, 1H, J = 7.6 Hz), 7.15 (t, 1H, J
= 7.6 Hz), 1.56 (m, 6H), 1.33 (m, 6H), 1.27 (t, 6H, J = 8.8 Hz), 0.90 (t, 9H, J = 7.2 Hz). ESI-MS: m/z =
409 [M+1]+.
4.1.3.2. 6-Chloro-3-(tributylstannyl)-1H-indazole (6b). Yellow oil (53%). 1H NMR (500 MHz,
DMSO-d6) δ 13.39 (s, 1H), 7.62 (d, 2H, J = 8.5 Hz), 7.04 (d, 1H, J = 8.5 Hz), 1.48 (m, 6H), 1.25 (q, 6H,
7

J = 7.5 Hz), 1.12 (m, 6H), 0.79 (t, 9H, J = 7.5 Hz). ESI-MS: m/z = 443 [M+1]+.
1
4.1.3.3. 3-(Tributylstannyl)-1H-indazole-6-carbonitrile (6c). Yellow oil (45%). H NMR (500 MHz,
DMSO-d6) δ 13.39 (s, 1H), 8.12 (d, 2H, J = 8.5 Hz), 7.64 (d, 1H, J = 8.5 Hz), 1.58 (m, 6H), 1.35 (q, 6H,
J = 7.5 Hz), 1.22 (m, 6H), 0.79 (t, 9H, J = 7.5 Hz). ESI-MS: m/z = 434 [M+1]+.
4.1.4. General procedure for the synthesis of Boc-protected indazoles (7a-c).
(Boc)2O (4.91 g, 22.50 mmol) was added to a solution of 6a (7.73 g, 18.90 mmol) and DMAP
(46.30 mg, 0.38 mmol) in THF (88.80 mL). The mixture was stirred for 30 min at room temperature,
concentrated under reduced pressure and subjected to silica gel column chromatography (petroleum
ether/ethyl acetate = 25:1, v/v) to yield a yellow liquid (6.48 g).
1
4.1.4.1. Tert-butyl 3-(tributylstannyl)-1H-indazole-1-carboxylate (7a). Yellow oil (68%). H NMR
(500 MHz, CDCl3) δ 8.05 (d, 1H, J = 8.0 Hz), 7.71 (d, 1H, J = 8.0 Hz), 7.52 (t, 1H, J = 7.5 Hz), 7.31 (t,
1H, J = 7.5 Hz), 1.63 (s, 9H), 1.55 (m, 6H), 1.26 (q, 6H, J = 7.5 Hz), 1.19 (t, 6H, J = 7.5 Hz), 0.81 (t,
9H, J = 7.5 Hz). ESI-MS: m/z = 509 [M+1]+.
4.1.4.2. Tert-butyl 6-chloro-3-(tributylstannyl)-1H-indazole-1-carboxylate (7b). Yellow oil (63%).
1H NMR (500 MHz, CDCl3) δ 8.15 (s, 1H), 7.55 (d, 1H, J = 9.0 Hz), 7.24 (d, 1H, J = 9.0 Hz), 1.72 (s,
9H), 1.57 (m, 6H), 1.32 (q, 6H, J = 7.0 Hz), 1.24 (t, 6H, J = 8.0 Hz), 0.87 (t, 9H, J = 7.0 Hz). ESI-MS:
m/z = 543 [M+1]+.
4.1.4.3. Tert-butyl 6-cyano-3-(tributylstannyl)-1H-indazole-1-carboxylate (7c). Yellow oil (58%).
1H NMR (500 MHz, CDCl3) δ 8.65 (s, 1H), 8.15 (d, 1H, J = 9.0 Hz), 7.34 (d, 1H, J = 9.0 Hz), 1.72 (s,
9H), 1.67 (m, 6H), 1.42 (q, 6H, J = 7.0 Hz), 1.34 (t, 6H, J = 8.0 Hz), 0.87 (t, 9H, J = 7.0 Hz). ESI-MS:
m/z = 534 [M+1]+.
4.1.5. Synthesis of indazole 6-methyl-5-nitropyridin-2-amine (9).
The 6-methylpyridin-2-amine (20 g, 185mmol) was dissolved in 98% H2SO4 (91 mL). A mixture
of fuming HNO3 (9 mL) and 98% H2SO4 (9 mL) was added dropwise to the vigorously stirring solution
over 30 min at -6 oC. The mixture was stirred for 2 h at 0 oC and slowly warmed to 10 oC over 1 h and
kept stirring for an additional hour. Afterwards, the solution was warmed to 20 °C over 1 h and stirred
for 2 h. The reaction mixture was poured onto ice and basified with ammonium hydroxide (pH ~ 9).
The precipitated solid was collected by filtration and purified by steam distillation to afford compound
9 (9.99 g). Yellow solid (36%), mp: 189-191 oC. 1H NMR (500 MHz, DMSO-d6) δ 7.77 (d, 1H, J = 9.2
Hz), 6.09 (d, 1H, J = 9.2 Hz), 6.11 (s, 2H), 2.36 (s, 3H). ESI-MS: m/z = 154 [M+1]+.
4.1.6. Synthesis of tert-butyl (6-methyl-5-nitropyridin-2-yl) carbamate (10).
(Boc)2O (16.94 g, 77.62 mmol) was added to a solution of 9 (9.99 g, 65.2 mmol) and DMAP
(160.2 mg, 1.31 mmol) in THF (306 mL). After stirring at room temperature for 30 min, THF was
removed under reduced pressure. The obtained crude product was purified by chromatography
(petroleum ether/ethyl acetate = 25:1, v/v) to afford compound 10 (14.3 g). White solid (87%), mp:
127-129 oC. 1H NMR (500 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.41 (d, 1H, J = 8.8 Hz), 7.84 (d, 1H, J =
8.8 Hz), 2.67 (s, 3H), 1.46 (s, 9H). ESI-MS: m/z = 254 [M+1]+.
8

4.1.7. Synthesis of tert-butyl methyl (6-methyl-5-nitropyridin-2-yl) carbamate (11).
NaH (3.11 g, 130 mmol) was added in portions to a solution of 10 (14.3 g, 56.50 mmol) in dry
o
DMF (172 mL) at -5 C. After stirring for 40 min, iodomethane (4.17 mL, 66.98 mmol) was added
dropwise over 30 min and stirred for 1 h at room temperature. H2O (200 mL) was added and the
resulting solution was extracted with ethyl acetate (70 mL×3), washed with brine (50 mL×3), dried
over anhydrous Na2SO4, and concentrated under reduced pressure. The obtained residue was purified
by chromatography (petroleum ether/ethyl acetate = 25:1, v/v) to afford 11 (9.52 g). Yellow solid (58%),
1
mp: 141-143 oC. H NMR (500 MHz, DMSO-d6) δ 8.39 (d, 1H, J = 9.2 Hz), 7.84 (d, 1H, J = 9.2 Hz),
3.39 (s, 3H), 2.73 (s, 3H), 1.50 (s, 9H). ESI-MS: m/z = 268 [M+1]+.
4.1.8. Synthesis of tert-butyl methyl (1H-pyrrolo[3,2-b]pyridin-5-yl) carbamate (12).
DMFDMA (20.38 g, 171 mmol) was added to a solution of 11 (21.85 g, 81.80 mmol) in DMF
o
(278 mL) and stirred at 95 C for 5 h. Then the solvent was removed under reduced pressure. The
obtained crude product was subjected to chromatography (petroleum ether/ethyl acetate = 10:1, v/v).
The obtained intermediate was dissolved in MeOH (450 mL) and was added 10% Pd/C (0.29 g). The
mixture was stirred at room temperature overnight under H2 atmosphere. After filtration through
Celite® and concentration under vacuo, the obtained crude product was recrystallized from EtOH to
give 12 (13.91 g). White solid (69%), mp: 157-159 oC. 1H NMR (500 MHz, DMSO-d6) δ 11.30 (s, 1H),
7.73 (d, 1H, J = 8.8 Hz), 7.60 (d, 1H, J = 3.2 Hz), 7.17 (d, 1H, J = 8.8 Hz), 6.45 (d, 1H, J = 3.2 Hz),
3.27 (s, 3H), 1.42 (s, 9H). ESI-MS: m/z = 248 [M+1]+.
4.1.9.
Synthesis of tert-butyl 5-((tert-butoxycarbonyl)(methyl)amino)-1H-pyrrolo[3,2-b]-
pyridine-1-carboxylate (13).
(Boc)2O (14.6 g, 66.90 mmol) was added to a solution of 12 (13.91 g, 56.2 mmol) and DMAP
(138.0 mg, 1.13 mmol) in THF (264 mL). After stirring at room temperature for 30 min, THF was
removed under reduced pressure. The obtained crude product was purified by chromatography
(petroleum ether/ethyl acetate = 20:1, v/v) to afford compound 13 (14.3 g). White solid (77%), mp:
157-159 oC. 1H NMR (500 MHz, CDCl3) δ 8.26 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 3.6 Hz, 1H), 7.44 (d,
1H, J = 8.8 Hz), 6.67 (d, 1H, J = 3.6 Hz), 3.41 (s, 3H), 1.65 (s, 9H), 1.48 (s, 9H). ESI-MS: m/z = 348
[M+1]+.
4.1.10.
Synthesis of tert-butyl 5-((tert-butoxycarbonyl)(methyl)amino)-2-iodo-1H-pyrrolo-
[3,2-b]pyridine-1-carboxylate (14).
N-butyllithium (2.5 mol/L, 9.2 mL) was added dropwise to a solution of 13 (6.6 g, 19.0 mmol) in
dry THF (60 mL) at -78 oC over 30 min under N2 atmosphere. The mixture was allowed to warm to -60
oC and stirred for 1 h. Then a solution of I2 (5.3 g) dissolved in dry THF (40 mL) was slowly added at
o
-72 °C. The reaction was stirred at -72 C for 2 h and stirred at room temperature for 1 h, quenched
with saturated Na2S2O3 (50 mL), diluted with H2O (100 mL), extracted with ethyl acetate (50 mL×3),
and washed with brine (30 mL×3). The solvent was dried over anhydrous Na2SO4, and concentrated
under reduced pressure. The obtained residue was purified by chromatography (petroleum
1
ether/acetone = 15:1, v/v) to afford 14 (5.42 g). Yellow oil (61%). H NMR (500 MHz, DMSO-d6) δ
8.19 (d, 1H, J = 8.8 Hz), 7.42 (d, 1H, J = 8.8 Hz), 6.35 (s, 1H), 3.28 (s, 3H), 1.66 (s, 9H), 1.43 (s, 9H).
ESI-MS: m/z = 474 [M+1]+.
9

4.1.11. General procedure for the synthesis of 15a-c.
Compound 7a (3.14 g, 6.18 mmol) in toluene (10 mL) was added dropwise to a mixture of 14
(2.75 g, 5.81 mmol), Pd(PPh3)4 (0.67 g, 0.581 mmol), CuI (0.15 g, 0.79 mmol) in toluene (110 mL)
under N2 atmosphere and stirred at 95 oC for 24 h. The reaction mixture was cooled to room
temperature and filtered. The filtrate was concentrated under reduced pressure and followed by
purification through chromatography (petroleum ether/acetone = 15:1, v/v) to afford 15a (3.35 g).
4.1.11.1.
Tert-butyl 3-(1-(tert-butoxycarbonyl)-5-((tert-butoxycarbonyl)(methyl)amino)-
1
1H-pyrrolo[3,2-b]pyridin-2-yl)-1H-indazole-1-carboxylate (15a). Yellow oil (63%). H NMR (500
MHz, DMSO-d6) δ 8.42 (d, 1H, J = 7.2 Hz), 8.18 (d, 1H, J = 6.4 Hz), 7.78 (d, 1H, J = 6.4 Hz), 7.67 (t,
1H, J = 6.4 Hz), 7.64 (d, 1H, J = 7.2 Hz), 7.43 (t, 1H, J = 6.4 Hz), 7.20 (s, 1H), 3.35 (s, 3H), 1.66 (s,
9H), 1.47 (s, 9H), 1.14 (s, 9H). ESI-MS: m/z = 564 [M+1]+.
4.1.11.2.
Tert-butyl 3-(1-(tert-butoxycarbonyl)-5-((tert-butoxycarbonyl)(methyl)amino)-1H-
1
pyrrolo[3,2-b]pyridin-2-yl)-6-chloro-1H-indazole-1-carboxylate (15b). Yellow oil (56%). H NMR
(500 MHz, DMSO-d6) δ 8.44 (d, 1H, J = 9.5 Hz), 8.29 (s, 1H), 7.59 (d, 1H, J = 9.5 Hz), 7.54 (d, 1H, J
= 8.5 Hz), 7.32 (d, 1H, J = 8.5 Hz), 7.04 (s, 1H), 3.46 (s, 3H), 1.73 (s, 9H), 1.52 (s, 9H), 1.24 (s, 9H).
ESI-MS: m/z = 598 [M+1]+.
4.1.11.3.
Tert-butyl 3-(1-(tert-butoxycarbonyl)-5-((tert-butoxycarbonyl)(methyl)amino)-
1H-pyrrolo[3,2-b]pyridin-2-yl)-6-cyano-1H-indazole-1-carboxylate (15c). Yellow oil (35%). 1H
NMR (500 MHz, DMSO-d6) δ 8.43 (d, 1H, J = 9.0 Hz), 8.32 (s, 1H), 7.94 (d, 1H, 8.5 Hz), 7.62 (d, 1H,
J = 9.0 Hz), 7.55 (d, 1H, J = 8.5 Hz), 7.10 (s, 1H), 3.38 (s, 3H), 1.71 (s, 9H), 1.51 (s, 9H), 1.17 (s, 9H).
ESI-MS: m/z = 589 [M+1]+.
4.1.12. General procedure for the synthesis of 16a-c.
12 N HCl aqueous solution (37 mL) was added dropwise to a solution of 15a (1.55 g, 2.75 mmol)
in EtOH (37 mL). The mixture was stirred at room temperature for 5 h. Then 10% NaOH aqueous
solution was added to the solution to basify the mixture to neutral. The mixture was extracted with
ethyl acetate (50 mL×3), washed with brine (30 mL×3), dried over anhydrous Na2SO4, and
concentrated under reduced pressure. The obtained residue was purified by chromatography (petroleum
ether/ethanol = 3:1, v/v) to afford 16a (0.3 g).
4.1.12.1.
2-(1H-Indazol-3-yl)-N-methyl-1H-pyrrolo[3,2-b]pyridin-5-amine (16a).
o
1
White solid (42%), mp: 244-246 C. H NMR (500 MHz, DMSO-d6) δ 13.27 (s, 1H), 11.25 (s, 1H),
8.18 (d, 1H, J = 8.4 Hz), 7.59 (d, 1H, J = 8.0 Hz), 7.46 (d, 1H, J = 8.0 Hz), 7.42 (t, 1H, J = 7.2 Hz),
7.23 (t, 1H, J = 7.2 Hz), 6.92 (s, 1H), 6.35 (d, 1H, J = 8.4 Hz), 6.04 (q, 1H, 4.8 Hz), 2.81 (d, 3H, J =
4.8 Hz). ESI-MS: m/z = 264 [M+1]+.
4.1.12.2.
2-(6-Chloro-1H-indazol-3-yl)-N-methyl-1H-pyrrolo[3,2-b]pyridin-5-amine (16b).
1
Yellow solid (39%), mp: 250 oC (dec). H NMR (500 MHz, DMSO-d6) δ 13.37 (s, 1H), 11.26 (s, 1H),
8.19 (d, 1H, J = 8.5 Hz), 7.65 (s, 1H), 7.44 (d, 1H, J = 9.0 Hz), 7.20 (d, 1H, J = 9.0 Hz), 6.93 (s, 1H),
6.34 (d, 1H, J = 8.5 Hz), 6.02 (q, 1H, J = 4.5 Hz), 2.81 (d, 3H, J = 4.5 Hz). ESI-MS: m/z = 298
[M+1]+.
4.1.12.3.
3-(5-(Methylamino)-1H-pyrrolo[3,2-b]pyridin-2-yl)-1H-indazole-6-carbonitrile
10

(16c). Yellow solid (43%), mp: 250 oC (dec). 1H NMR (500 MHz, DMSO-d6) δ 13.45 (s, 1H), 11.40 (s,
1H), 8.41 (d, 1H, J = 8.0 Hz), 8.26 (s, 1H), 7.53 (d, 1H, J = 9.0 Hz), 7.49 (d, 1H, J = 9.0 Hz), 7.01 (s,
1H), 6.39 (d, 1H, J = 8.5 Hz), 6.10 (q, 1H, J = 5.0 Hz), 2.85 (d, 3H, J = 5.0 Hz). ESI-MS: m/z = 289
[M+1]+.
4.1.13. General procedure for the synthesis of 2a-x.
A mixture of 16a (26.30 mg, 0.10 mmol), acetyl chloride (7.80 mg, 0.10 mmol), and pyridine (2.0
mL) was stirred at 60 oC and monitored by TLC. Pyridine was removed under reduced pressure. Then
the mixture was basified by 1 N NaOH aqueous solution (pH ~ 9), extracted with CH2Cl2 (20 mL×3),
washed with brine (5 mL×3), dried over anhydrous Na2SO4, and concentrated under reduced pressure.
The obtained residue was purified by chromatography (petroleum ether/ethanol = 8:1, v/v) to afford 2a
(0.3 g).
4.1.13.1. N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-N-methylacetamide (2a). Yellow
solid (43%), mp: 275 oC (dec). 1H NMR (500 MHz, DMSO-d6) δ 13.66 (s, 1H), 12.02 (s, 1H), 8.23 (d,
1H, J = 8.4 Hz), 7.84 (d, 1H, J = 8.4 Hz), 7.63 (d, 1H, J = 8.4 Hz), 7.44 (t, 1H, J = 7.2 Hz), 7.26 (t, 1H,
J = 7.2 Hz), 7.24 (s, 1H), 7.13 (d, 1H, J = 8.0 Hz), 3.25 (s, 3H), 1.89 (s, 3H). ESI-MS: m/z = 306
[M+1]+.
4.1.13.2. N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-N-methylpropionamide (2b).
o
1
Yellow solid (51%), mp: 219-221 C. H NMR (500 MHz, DMSO-d6) δ 13.55 (s, 1H), 12.02 (s, 1H),
8.27 (d, 1H, J = 8.0 Hz), 7.89 (d, 1H, J = 8.5 Hz), 7.67 (d, 1H, J = 8.5 Hz), 7.49 (t, 1H, J = 7.5 Hz),
7.31 (t, 1H, J = 8.0 Hz), 7.29 (s, 1H), 7.16 (d, 1H, J = 8.0 Hz), 3.83 (s, 3H), 2.19 (q, 2H, J = 7.5 Hz),
0.98 (t, 3H, J = 7.5 Hz). ESI-MS: m/z = 320 [M+1]+.
4.1.13.3.
N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-N-methylpivalamide (2c).
o
1
Yellow solid (77%), mp: 260 C (dec). H NMR (500 MHz, DMSO-d6) δ 13.67 (s, 1H), 12.03 (s, 1H),
8.23 (d, 1H, J = 8.4 Hz), 7.84 (d, 1H, J = 8.0 Hz), 7.63 (d, 1H, J = 8.4 Hz), 7.44 (t, 1H, J = 7.6 Hz),
7.25 (t, 1H, J = 7.6 Hz), 7.23 (s, 1H), 7.11 (d, 1H, J = 8.0 Hz), 3.16 (s, 3H), 0.96 (s, 9H). ESI-MS: m/z
= 348 [M+1]+.
4.1.13.4.
N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-4-chloro-N-methylbenzamide
o
1
(2d). White solid (67%), mp: 178-180 C. H NMR (500 MHz, DMSO-d6) δ 13.51 (s, 1H), 11.90 (s,
1H), 8.22 (d, 1H, J = 8.0 Hz), 7.62 (d, 1H, J = 8.4 Hz), 7.57 (d, 1H, J = 8.8 Hz), 7.44 (t, 1H, J = 7.2
Hz), 7.23 (m, 4H), 7.17 (m, 3H), 6.75 (d, 1H, J = 8.4 Hz), 3.48 (s, 3H). ESI-MS: m/z = 368 [M+1]+.
4.1.13.5.
N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-N-methyl-3-phenylpropana-
mide (2e). Yellow solid (49%), mp: 86-88 oC. 1H NMR (500 MHz, DMSO-d6) δ 11.98 (br, 1H), 11.52
(s, 1H), 8.22 (d, 1H, J = 6.5 Hz), 7.82 (d, 1H, J = 8.5 Hz), 7.63 (d, 1H, J = 8.5 Hz), 7.43 (t, 1H, J = 7.5
Hz), 7.25 (t, 1H, J = 7.5 Hz), 7.22 (s, 1H), 7.16 (m, 2H), 7.03 (m, 4H), 3.25 (s, 3H), 2.79 (t, 2H, J = 8.0
Hz), 2.40 (t, 2H, J = 8.0 Hz). ESI-MS: m/z = 396 [M+1]+.
4.1.13.6. N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-4-fluoro-N-methylbenzamide (2f).
o
1
Yellow solid (19%), mp: 162-164 C. H NMR (500 MHz, DMSO-d6) δ 10.45 (br, 1H), 9.27 (s, 1H),
8.16 (d, 1H, J = 8.0 Hz), 7.60 (d, 1H, J = 8.5 Hz), 7.53 (t, 1H, J = 8.5 Hz), 7.51 (d, 1H, J = 8.5 Hz),
11

7.42 (t, 2H, J = 5.5 Hz), 7.39 (t, 1H, J = 7.5 Hz), 7.43 (s, 1H), 6.86 (t, 2H, J = 8.5 Hz), 6.65 (d, 1H, J =
8.0 Hz), 3.71 (s, 3H). ESI-MS: m/z = 386 [M+1]+.
4.1.13.7.
N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-4-chloro-N-methylbenzamide
o
1
(2g). White solid (27%), mp: 260-262 C. H NMR (500 MHz, DMSO-d6) δ 13.60 (s, 1H), 11.94 (s,
1H), 8.22 (d, 1H, J = 7.6 Hz), 7.62 (dd, 2H, J = 2.8 Hz, 8.0 Hz), 7.43 (t, 1H, J = 7.2 Hz), 7.25 (m, 5H),
7.19 (s, 1H), 6.79 (d, 1H, J = 8.4 Hz), 3.47 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 168.57, 150.11,
145.39, 141.35, 136.62, 135.97, 135.51, 134.19, 130.00, 127.89, 127.65, 126.59, 121.51, 120.56,
120.02, 115.21, 110.69, 99.66, 36.39. ESI-MS: m/z = 402 [M+1]+.
4.1.13.8.
N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-4-bromo-N-methylbenzamide
o
(2h). Yellow solid (38%), mp: 272 C (dec). 1H NMR (500 MHz, DMSO-d6) δ 13.61 (s, 1H), 11.94 (s,
1H), 8.22 (d, 1H, J = 8.0 Hz), 7.63 (d, 2H, J = 8.5 Hz), 7.46 (t, 1H, J = 7.0 Hz), 7.41 (dd, 2H, J = 1.5
Hz, 6.5 Hz), 7.26 (t, 1H, J = 7.0 Hz), 7.21 (dd, 2H, J = 2.0 Hz, J = 7.0 Hz), 7.19 (s, 1H), 6.80 (d, 1H, J
= 10.5 Hz), 3.47 (s, 1H). ESI-MS: m/z = 446 [M+1]+.
4.1.13.9.
N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-4-methoxy-N-methylbenza-
o
1
mide (2i). Yellow solid (44%), mp: 265 C (dec). H NMR (500 MHz, DMSO-d6) δ 13.60 (s, 1H),
11.92 (s, 1H), 8.22 (d, 1H, J = 8.4 Hz), 7.63 (d, 1H, J = 8.4 Hz), 7.60 (d, 1H, J = 8.8 Hz), 7.43 (t, 1H,
J = 7.2 Hz), 7.25 (t, 1H, J = 7.2 Hz), 7.24 (s, 1H), 7.21 (d, 2H, J = 2.8 Hz), 6.71 (m, 3H), 3.66 (s, 3H),
3.45 (s, 3H). ESI-MS: m/z = 398 [M+1]+.
4.1.13.10.
N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-3-methoxy-N-methylbenza-
mide (2j). Yellow solid (45%), mp: 152-154 oC. 1H NMR (500 MHz, DMSO-d6) δ 13.57 (s, 1H), 11.90
(s, 1H), 7.23 (d, 1H, J = 8.0 Hz), 7.63 (d, 1H, J = 8.5 Hz), 7.60 (d, 1H, J = 8.5 Hz), 7.45 (t, 1H, J = 7.5
Hz), 7.26 (t, 1H, J = 7.5 Hz), 7.21 (s, 1H), 7.09 (t, 1H, J = 8.0 Hz), 6.85 (t, 1H, J = 2.0 Hz), 6.81 (m,
2H), 6.79 (d, 1H, J = 8.5 Hz), 3.58 (s, 3H), 3.47 (s, 3H). ESI-MS: m/z = 398 [M+1]+.
4.1.13.11. N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-N-methyl-4-nitrobenzamide (2k).
o
1
Yellow solid (26%), mp: 231 C (dec). H NMR (500 MHz, DMSO-d6) δ 13.59 (s, 1H), 11.94 (s, 1H),
8.20 (d, 1H, J = 8.5 Hz), 8.05 (d, 1H, J = 8.5 Hz), 7.63 (t, 2H, J = 8.0 Hz), 7.52 (d, 2H, J = 9.0 Hz),
7.44 (t, 1H, J = 7.0 Hz), 7.16 (m, 3H), 6.91 (d, 1H, J = 8.0 Hz), 3.51 (s, 3H). ESI-MS: m/z = 413
[M+1]+.
4.1.13.12.
N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-N-methylfuran-2-carbox-
o
1
amide (2l). Yellow solid (45%), mp: 110-112 C. H NMR (500 MHz, DMSO-d6) δ 13.59 (s, 1H),
12.00 (s, 1H), 8.22 (d, 1H, J = 8.0 Hz), 7.79 (d, 1H, J = 8.5 Hz), 7.63 (d, 1H, J = 8.5 Hz), 7.57 (d, 1H, J
= 1.0 Hz), 7.44 (t, 1H, J = 7.5 Hz), 7.25 (t, 1H, J = 7.5 Hz), 7.23 (s, 1H), 7.00 (d, 1H, J = 8.0 Hz), 6.33
(dd, 1H, J =1.5 Hz, 3.5 Hz), 6.01 (d, 1H, J = 3.5 Hz), 3.39 (s, 3H). ESI-MS: m/z = 358 [M+1]+.
4.1.13.13. N-(2-(6-Chloro-1H-indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-N-methylbenzamide
(2m). Yellow solid (27%), mp: 275 oC (dec). 1H NMR (500 MHz, DMSO-d6) δ 13.91 (s, 1H), 12.01 (s,
1H), 7.99 (d, 1H, J = 8.5 Hz), 7.81 (s, 1H), 7.53 (s, 1H), 7.42 (d, 1H, J = 8.5 Hz), 7.29 (dd, 2H, J = 1.5
Hz, 9.0 Hz), 7.22 (d, 2H, J = 7.5 Hz), 7.14 (t, 2H, J = 8.0 Hz), 6.64 (d, 1H, J = 8.5 Hz), 3.67 (s, 3H).
12

ESI-MS: m/z = 402 [M+1]+.
4.1.13.14.
N-(2-(6-Cyano-1H-indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-N-methylbenzamide
o
(2n). Yellow solid (19%), mp: 250 C (dec). 1H NMR (500 MHz, DMSO-d6) δ 14.11 (s, 1H), 12.04 (s,
1H), 8.49 (d, 1H, J = 8.5 Hz), 8.31 (s, 1H), 7.61 (td, 2H, J = 9.0 Hz, 1.0 Hz), 7.28 (m, 4H), 7.22 (m,
2H), 6.63 (d, 1H, J = 8.0 Hz), 3.51 (s, 3H). ESI-MS: m/z = 393 [M+1]+.
4.1.13.15. N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-N-methylbenzenesulfonamide
(2o). Yellow solid (40%), mp: 78-80 oC. 1H NMR (500 MHz, DMSO-d6) δ 13.46 (s, 1H), 11.93 (s, 1H),
8.18 (d, 1H, J = 8.4 Hz), 7.79 (d, 1H, J = 8.4 Hz), 7.61 (m, 1H), 7.58 (d, 1H, J = 8.4 Hz), 7.49 (m, 4H),
7.39 (t, 1H, J = 8.0 Hz), 7.29 (d, 1H, J = 8.8 Hz), 7.19 (t, 1H, J = 8.0 Hz), 7.13 (d, 1H, J = 1.6 Hz),
3.20 (s, 3H). ESI-MS: m/z = 404 [M+1]+.
4.1.13.16.
N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-N-4-dimethylbenzenesul-
o
1
fonamide (2p). Yellow solid (25%), mp: 91-93 C. H NMR (500 MHz, DMSO-d6) δ 13.39 (s, 1H),
11.88 (s, 1H), 8.17 (d, 1H, J = 8.0 Hz), 7.78 (d, 1H, J = 8.4 Hz), 7.58 (d, 1H, J = 8.4 Hz), 7.39 (m, 3H),
7.29 (d, 3H, J = 8.0 Hz), 7.19 (t, 1H, J = 7.6 Hz), 7.14 (d, 1H, J = 0.8 Hz), 3.18 (s, 3H), 2.76 (s, 3H).
ESI-MS: m/z = 418 [M+1]+.
4.1.13.17.
N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-4-chloro-N-methylbenzenes-
o
1
ulfonamide (2q). Yellow solid (40%), mp: 90-92 C. H NMR (500 MHz, DMSO-d6) δ 13.50 (s, 1H),
11.98 (s, 1H), 8.22 (d, 1H, J = 8.4 Hz), 7.83 (d, 1H, J = 8.8 Hz), 7.61 (m, 3H), 7.58 (s, 1H), 7.56 (d,
1H, J = 8.4 Hz), 7.43 (t, 1H, J = 8.0 Hz), 7.29 (d, 1H, J = 8.8 Hz), 7.23 (t, 1H, J = 8.0 Hz), 7.18 (d, 1H,
J = 1.2 Hz), 3.23 (s, 3H). ESI-MS: m/z = 438 [M+1]+.
4.1.13.18.
N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-4-bromo-N-methylbenzenes-
o
1
ulfonamide (2r). Yellow solid (42%), mp: 80-82 C. H NMR (500 MHz, DMSO-d6) δ 13.58 (s, 1H),
12.03 (s, 1H), 8.22 (d, 1H, J = 8.0 Hz), 7.84 (d, 1H, J = 8.8 Hz), 7.76 (d, 2H, J = 8.8 Hz), 7.62 (d, 1H,
J = 8.4 Hz), 7.48 (d, 2H, J = 8.8 Hz), 7.43 (t, 1H, J = 8.0 Hz), 7.28 (d, 1H, J = 8.4 Hz), 7.23 (t, 1H, J
= 8.0 Hz), 7.19 (s, 1H), 3.24 (s, 3H). ESI-MS: m/z = 482 [M+1]+.
4.1.13.19.
N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-4-methoxy-N-methylbenzene-
o
1
sulfonamide (2s). Yellow solid (43%), mp: 125-127 C. H NMR (500 MHz, DMSO-d6) δ 13.49 (s,
1H), 11.94 (s, 1H), 8.21 (d, 1H, J = 8.8 Hz), 7.81 (d, 1H, J = 8.0 Hz), 7.61 (d, 2H, J = 8.4 Hz), 7.46 (d,
2H, J = 8.4 Hz), 7.42 (t, 1H, J = 8.0 Hz), 7.32 (d, 1H, J = 8.4 Hz), 7.22 (t, 1H, J = 8.0 Hz), 7.17 (d, 1H,
J = 1.2 Hz), 7.03 (d, 1H, J = 9.2 Hz), 3.80 (s, 3H), 3.20 (s, 3H). ESI-MS: m/z = 434 [M+1]+.
4.1.13.20.
N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-3,4-dimethoxy-N-methylben-
o
1
zenesulfonamide (2t). Yellow solid (40%), mp: 88-90 C. H NMR (500 MHz, DMSO-d6) δ 13.50 (s,
1H), 11.96 (s, 1H), 8.21 (d, 1H, J = 8.0 Hz), 7.83 (d, 1H, J = 8.4 Hz), 7.61 (d, 1H, J = 8.4 Hz), 7.43 (t,
1H, J = 7.6 Hz), 7.33 (d, 1H, J = 8.0 Hz), 7.23 (t, 1H, J = 7.6 Hz), 7.19 (dd, 1H, J = 2.4 Hz, 8.8 Hz),
7.18 (d, 1H, J = 1.2 Hz), 7.07 (d, 1H, J = 8.8 Hz), 6.85 (d, 1H, J = 1.2 Hz), 3.81 (s, 3H), 3.55 (s, 3H),
3.19 (s, 3H). ESI-MS: m/z = 464 [M+1]+.
13

4.1.13.21.
N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-2,5-dimethoxy-N-methylbe-
nzenesulfonamide (2u). Yellow solid (30%), mp: 90-92 oC. 1H NMR (500 MHz, DMSO-d6) δ 13.47 (s,
1H), 11.86 (s, 1H), 8.22 (d, 1H, J = 8.0 Hz), 7.73 (d, 1H, J = 8.4 Hz), 7.61 (d, 1H, J = 8.0 Hz), 7.42 (t,
1H, J =7.2 Hz), 7.32 (d, 1H, J = 2.8 Hz), 7.23 (t, 1H, J = 7.2 Hz), 7.22 (d, 1H, J = 8.8 Hz), 7.17 (s,
1H), 7.15 (d, 1H, J = 2.8 Hz), 7.07 (d, 1H, J = 9.2 Hz), 3.74 (s, 3H), 3.48 (s, 3H), 3.37 (s, 3H). ESI-MS:
m/z = 464 [M+1]+.
4.1.13.22.
N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-2,4-dimethoxy-N-methylbe-
nzenesulfonamide (2v). Yellow solid (60%), mp: 81-83 oC. 1H NMR (500 MHz, DMSO-d6) δ 13.46 (s,
1H), 11.82 (s, 1H), 8.22 (d, 1H, J = 8.4 Hz), 7.69 (t, 2H, J = 8.4 Hz), 7.61 (d, 1H, J = 8.4 Hz), 7.42 (t,
1H, J = 7.2 Hz), 7.23 (m, 2H), 7.17 (d, 1H, J = 1.2 Hz), 6.58 (m, 2H), 3.80 (s, 3H), 3.47 (s, 3H), 3.32
(s, 3H). ESI-MS: m/z = 464 [M+1]+.
4.1.13.23.
N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-N-methyl-3-nitrobenzenesu-
o
1
lfonamide (2w). Yellow solid (37%), mp: 92-94 C. H NMR (500 MHz, DMSO-d6) δ 13.52 (s, 1H),
12.03 (s, 1H), 8.51 (dd, 1H, J = 2.4 Hz, 8.4 Hz), 8.34 (s, 1H), 8.18 (d, 1H, J = 8.0 Hz), 8.01 (d, 1H, J =
7.2 Hz), 7.86 (t, 2H, J = 8.0 Hz), 7.62 (d, 1H, J = 8.4 Hz), 7.43 (t, 1H, J = 8.4 Hz), 7.28 (d, 1H, J = 8.8
Hz), 7.24 (t, 1H, J = 8.4 Hz), 7.13 (s, 1H), 3.26 (s, 3H). ESI-MS: m/z = 449 [M+1]+.
4.1.13.24. N-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-N-methyl-4-(trifluoromethoxy)-
o
1
benzenesulfonamide (2x). Yellow solid (45%), mp: 107-109 C. H NMR (500 MHz, DMSO-d6) δ
10.73 (s, 1H), 9.54 (s, 1H), 8.05 (d, 1H, J = 8.5 Hz), 7.67 (m, 3H), 7.53 (d, 1H, J = 8.5 Hz), 7.46 (t, 1H,
J = 7.0 Hz), 7.44 (d, 1H, J = 8.5 Hz), 7.29 (t, 1H, J = 7.0 Hz), 7.25 (d, 2H, J = 8.5 Hz), 7.16 (d, 1H, J
= 1.0 Hz), 3.36 (s, 3H). ESI-MS: m/z = 488 [M+1]+.
4.1.14. General procedure for the synthesis of 2y and 2z.
Phenyl isocyanate (143 mg, 1.20 mmol) in dry CH2Cl2 (3 mL) was added dropwise to 16a (26.30
mg, 1 mmol) in dry CH2Cl2 (5 mL) at room temperature and stirred for 4 h. The reaction was quenched
with H2O (100 mL), extracted with CH2Cl2 (10 mL×3) and washed with brine (20 mL×3). The organic
layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The obtained residue
was purified by chromatography (petroleum ether/ethanol = 5:1, v/v) to afford 2y (15 mg).
4.1.14.1. 1-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-1-methyl-3-phenylurea (2y).
o
1
White solid (30%), mp: 217-219 C. H NMR (500 MHz, DMSO-d6) δ 13.52 (s, 1H), 12.21 (s, 1H),
11.94 (s, 1H), 8.34 (d, 1H, J = 10.5 Hz), 7.87 (d, 1H, J = 11.0 Hz), 7.64 (m, 3H), 7.46 (t, 1H, J = 9.0
Hz), 7.38 (d, 1H, J = 1.5 Hz), 7.29 (q, 3H, J = 8.0 Hz), 7.11 (d, 1H, J = 11.0 Hz), 7.02 (t, 1H, J = 9.5
Hz), 3.44 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 153.73, 150.19, 143.00, 141.38, 139.73, 136.85,
135.59, 128.73, 126.63, 126.15, 122.24, 121.61, 121.43, 120.79, 120.05, 119.36, 110.67, 108.32, 99.13,
33.71. ESI-MS: m/z = 383 [M+1]+.
4.1.14.2.
1-(2-(1H-Indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-3-(4-methoxyphenyl)-1-methyl-
urea (2z). White solid (34%), mp: 213-215 oC. 1H NMR (500 MHz, DMSO-d6) δ 11.98 (s, 1H), 11.88
(s, 1H), 10.08 (s, 1H), 8.52 (d, 1H, J = 8.0 Hz), 8.41 (d, 1H, J = 8.5 Hz), 8.04 (d, 1H, J = 9.0 Hz), 7.57
(m, 3H), 7.25 (d, 1H, J = 9.0 Hz), 7.05 (d, 2H, J = 9.0 Hz), 6.93 (d, 2H, J = 9.0 Hz), 3.80 (s, 3H), 3.75
(s, 3H). ESI-MS: m/z = 413 [M+1]+.
14

4.2. Cell proliferation assay
The antiproliferative activity in vitro was measured using the MTT assay. HL60, KB,
SMMC-7721, HCT116, and A549 cell lines were cultured in RPMI-1640 (Invitrogen Corp., Carlsbad,
CA) medium with heat-inactivated 10% fetal bovine serum, penicillin (100 units/mL) and streptomycin
o
(100 μg/mL) and incubated in normoxic atmosphere with 20% O2, 5% CO2 at 37 C. All tested
compounds were dissolved in DMSO at concentrations of 10.0 mg/mL and diluted to appropriate
concentrations. Cells were plated in 96-well plates for 24 h and subsequently treated with different
concentrations of all tested compounds for 72 h. Viable cells were determined using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay kit (MTT, Sigma) according to
operation instructions provided by the manufacturer. The concentration of drug causing 50% inhibition
in absorbance compared with control cells (IC50) was calculated using the software of dose-effect
analysis with microcomputers.
4.3. Cell cycle profile (FACS) analysis
HCT116 cells were incubated for 24 h in the presence of compound 2y (0, 50, 100, 200 nM). Cells
were harvested, fixed with 70% ethanol, washed twice with PBS, and stained by 50 μg/mL propidium
iodide (PI) solution containing 0.1% Triton X-100, 0.1 mM EDTA, and 50 μg/mL RNase A. Cell cycle
was detected by flow cytometry. The cellular DNA content was analyzed by measuring the
fluorescence with a FACS Calibur Flow Cytometer (Becton Dickinson Immunocytometry Systems,
San Jose, CA).
4.4. Kinase inhibitory assays
For the kinase inhibitory assays, the recombinant Aurora A, GSK3β, and MEK1 proteins were
expressed in Escherichia coli system. CDK2/cyclin A was purchased from Carna company. Aurora A,
GSK-3β and PKC kinase assays were carried out by using the Z'-LYTETM Kinase Assay kits
(Invitrogen). CDK2/cyclin A, IKKβ kinase assays were carried out by using the HTRF Kinase Assay
kits (Cisbio). CHK1 inhibitory activities were determined using the luminescent ADP-Glo assay kit
(Promega). Compounds were initially tested at a fixed concentration, and those displaying more than
50% inhibition were further tested for dose-response IC50 values. And the results represented an
average of at least three experiments.
4.5. Molecular docking
Docking analysis was carried out by using C-DOCKER module (Discovery Studio, version 2.5;
Accelrys, San Diego, CA, USA, 2008) to compare the binding modes between compound 2d bound to
Aurora A and 2y bound to Aurora A. The X-ray crystal structure of Aurora A (PDB ID: 4JAJ) was used
for the docking calculation. After removing the ligand and water molecules, the CHARMm-force field
was applied to the protein. And the ATP binding pocket was chosen as the active site with a radius set
as 8 Å. The ligands were generated random conformations using CHARMm-based molecular dynamics
(1000 steps), and then docked into the defined Aurora A binding site. The other parameters were set as
default. The final binding conformation of 2d and 2y was determined based on the calculated
CDOCKING ENERAGE. The most stable binding modes among the top 10 docking poses of 2d and
2y were presented in Fig. 3A and 3B, respectively.
15

Acknowledgments
The authors are grateful to the support of the National Natural Science Foundation of China
(81172929) and the support of the Program for Zhejiang Leading Team of S&T Innovation
(2011R50014). The authors also thank Jianyang Pan (Institute of Pharmaceutical Informatics, Zhejiang
University, China) for performing NMR spectrometry and Mass spectrometry for structure elucidation.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at
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Pitts, T. M.; Davis, S. L.; Eckhardt, S. G.; Bradshaw-Pierce, E. L., Targeting nuclear kinases in cancer:
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Maugeri-Sacca, M.; Bartucci, M.; De Maria, R., Checkpoint kinase 1 inhibitors for potentiating
systemic anticancer therapy. Cancer Treat. Rev. 2013, 39 (5), 525-533.
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Y.; Hartman, G. D.; Stirdivant, S. M.; Drakas, B. A.; Rickert, K.; Walsh, E. S.; Hamilton, K.; Buser, C.
A.; Hardwick, J.; Tao, W.; Beck, S. C.; Mao, X.; Lobell, R. B.; Sepp-Lorenzino, L.; Yan, Y.; Ikuta, M.;
Munshi, S. K.; Kuo, L. C.; Kreatsoulas, C., 3-(Indol-2-yl)indazoles as Chek1 kinase inhibitors:
Optimization of potency and selectivity via substitution at C6. Bioorg. Med. Chem. Lett. 2006, 16 (23),
6049-6053.
7. Dar, A. A.; Goff, L. W.; Majid, S.; Berlin, J.; El-Rifai, W., Aurora Kinase Inhibitors - Rising Stars
in Cancer Therapeutics? Mol. Cancer Ther. 2010, 9 (2), 268-278.
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9. (a) Moore, A. S.; Blagg, J.; Linardopoulos, S.; Pearson, A. D. J., Aurora kinase inhibitors: novel
small molecules with promising activity in acute myeloid and Philadelphia-positive leukemias.
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new target for anticancer drug. Recent Pat. Anti-Canc. 2008, 3 (2), 114-122.
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Fischmann, T. O.; Duca, J. S.; Madison, V.; Parry, D.; Davis, N.; Seghezzi, W.; Wiswell, D.; Guzi, T. J.,
Discovery of pyrazolo 1,5-a pyrimidine-based CHK1 inhibitors: A template-based approach-Part 1.
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16

Figure captions:
Fig. 1 Design of 5-substituted pyrrolopyridin-indazoles as anticancer agents.
Fig. 2 Cell cycle distribution of HCT116 cells after compound 2y treatment for 24 h. The 2N (diploid)
and 4N (tetraploid) DNA content represented the G1 and G2/M phases of the cell cycle, respectively.
Fig. 3 Docking mode comparison between compounds 2d and 2y bound to Aurora A. (A) Molecular
docking analysis of 2d with Aurora A. (B) Molecular docking analysis of 2y with Aurora A. Red
dashed lines indicate hydrogen bonds, prepared using PyMOL, PDB ID: 4JAJ.
Scheme 1. Synthesis of substituted indazole intermediates (7a-c). Reagents and conditions: (a) NaNO2,
NaBF4, KOAc, CHCl3, 53-75%; (b) I2, KOH, DMF, 47-52%; (c) EtMgBr, Et2O, (n-Bu)3SnCl, 45-61%;
(d) (Boc)2O, DMAP, THF, 58-68%.
Scheme 2. Synthesis of 4-azaindole fragment (14). Reagents and conditions: (a) HNO3, H2SO4, 36%; (b)
(Boc)2O, DMAP, THF, 87%; (c) NaH, CH3I, DMF, 58%; (d) DMFDMA, DMF; Pd/C, H2, MeOH, 69%;
(e) (Boc)2O, DMAP, THF, 77%; (f) n-BuLi, I2, THF, 61%.
Scheme 3. Synthesis of pyrrolopyridin-indazoles (2a-z). Reagents and conditions: (a) Pd(PPh3)4, CuI,
toluene, 35-63%; (b) 12 N HCl, EtOH, 39-43%; (c) R1COCl, Pyridine, 19-77%; (d) R1SO2Cl, Pyridine,
25-60%; (e) R1NCO, CH2Cl2, 30-34%.
17

Table 1. In vitro antiproliferative activities of 2a-n against five cancer cell lines.
Cytotoxicity IC50a (μM)
Compd
R1
R2
HL60
KB
SMMC-7721
HCT116
A549
2a
2b
2c
2d
2e
2f
CH3-
CH3CH2-
(CH3)3C-
Ph-
5.11
6.13
4.99
4.85
4.73
1.21
0.45
1.08
3.32
1.42
6.80
4.54
5.62
6.43
10.33
9.30
23.38
33.45
22.16
12.53
20.93
9.15
14.79
10.75
17.98
10.60
11.23
9.27
>50
15.70
24.35
13.75
18.09
9.65
8.39
8.58
Ph-CH2-CH2-
4-F-Ph-
7.38
H
7.22
2g
2h
2i
4-Cl-Ph-
4-Br-Ph-
4-CH3O-Ph-
3-CH3O-Ph-
4-NO2-Ph-
furan-2-yl
Ph-
0.63
2.42
3.00
2.22
1.78
3.46
6.11
4.25
12.90
9.01
23.68
22.00
18.42
28.16
11.27
>50
29.47
14.24
19.90
29.99
13.14
24.04
14.56
14.87
34.47
15.49
9.59
2j
2k
2l
20.15
19.43
5.59
2m
Cl
2n
Ph-
CN
20.93
32.83
a
Values are means of three experiments.

Table 2. In vitro antiproliferative acitivities of 2o-z against five cancer cell lines.
Cytotoxicity IC50a (μM)
Compd
X
R1
HL60
KB
SMMC-7721
HCT116
A549
2o
2p
2q
2r
2s
2t
Ph-
3.70
3.03
0.77
6.24
4.34
6.32
2.40
9.50
12.00
16.34
12.68
11.53
12.95
26.55
6.74
13.33
5.98
14.01
27.13
18.20
19.70
24.25
>50
4-CH3-Ph-
4-Cl-Ph-
4-Br-Ph-
4-CH3O-Ph-
3,4-di
8.35
7.15
9.85
-SO2-
22.12
10.05
22.40
16.71
CH3O-Ph-
2,5-di
2u
2v
0.84
5.18
4.38
4.28
8.40
19.40
25.88
CH3O-Ph-
2,4-di
10.17
10.77
21.83
CH3O-Ph-
3-NO2-Ph-
4-CF3O-Ph-
Ph-
2w
2x
2y
2z
3.66
1.17
5.87
1.33
5.07
7.16
0.28
NTb
0.12
13.58
6.04
20.07
8.00
-NH-CO-
0.0083
0.0079
0.0017
0.032
NTb
0.0013
0.063
0.0049
1.43
NTb
4-CH3O-Ph-
Taxol
0.015
0.058
a
Values are means of three experiments.
NT, not tested.
b

Table 3. Kinase inhibitory profiles of compounds 2d, 2g and 2y.
%Inhibition (1 µg/mL)
Kinases
2d
2g
2y
CHK1
CDK2
MEK1
GSK3β
BRAF
IKKβ
8.3%
1.0%
20.5%
28.23%
3.85%
18.57%
11.66%
8.06%
5.40%
35.47%
1.98%
13.77%
6.64%
19.26%
16.37%
0.55%
0.62%
27.75%
14.37%
0.94%
PKC
10.60%
95.8%
(0.032 μM) a
95.0%
(0.046 μM) a
75.4%
(0.519 μM) a
Aurora A
a
Compounds were initially tested at a fixed concentration, those displaying more than 50% inhibition
were further tested for dose-response IC50 values and the results represented means of at least three
experiments.

Scheme 1

Scheme 2

Scheme 3

Fig. 1

Fig. 2

Fig. 3A

Fig. 3B

Graphical abstract