Discovery of CJ-2360 as a Potent and Orally Active Inhibitor of Anaplastic Lymphoma Kinase Capable of Achieving Complete Tumor Regression

Article abstract of DOI:10.1021/acs.jmedchem.0c01550

We report herein the discovery of a class of potent small-molecule inhibitors of anaplastic lymphoma kinase (ALK) containing a fused indoloquinoline scaffold. The most promising compound CJ-2360 has an IC50 value of 2.2 nM against wild-type ALK and low-nanomolar potency against several clinically reported ALK mutants. This compound is capable of achieving complete tumor regression in the ALK-positive KARPAS-299 xenograft model with oral administration in mice. CJ-2360 represents a promising ALK inhibitor for advanced preclinical development.

Full text of DOI:10.1021/acs.jmedchem.0c01550

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Discovery of CJ-2360 as a Potent and Orally Active Inhibitor of
Anaplastic Lymphoma Kinase Capable of Achieving Complete
Tumor Regression
Jianyong Chen,¶ Yunlong Zhou,¶ Xuyuan Dong,¶ Liu Liu, Longchuan Bai, Donna McEachern,
Sally Przybranowski, Chao-Yie Yang, Jeanne Stuckey, Xiaoqin Li, Bo Wen, Ting Zhao, Siwei Sun,
Duxin Sun, Lingling Jiao, Yu Jing, Ming Guo, Dajun Yang, and Shaomeng Wang*
Cite This: https://dx.doi.org/10.1021/acs.jmedchem.0c01550
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ABSTRACT: We report herein the discovery of a class of potent small-molecule inhibitors of anaplastic lymphoma kinase (ALK)
containing a fused indoloquinoline scaffold. The most promising compound CJ-2360 has an IC50 value of 2.2 nM against wild-type
ALK and low-nanomolar potency against several clinically reported ALK mutants. This compound is capable of achieving complete
tumor regression in the ALK-positive KARPAS-299 xenograft model with oral administration in mice. CJ-2360 represents a
promising ALK inhibitor for advanced preclinical development.
Extensive efforts have been devoted in the last decade to the
discovery and development of ALK inhibitors.16−23 Crizotinib
(1)24,25 was the first ALK inhibitor approved by the U.S. FDA in
2011 as a first-line treatment for ALK-positive lung cancer
patients. Subsequently, ceritinib (2, LDK378),26 alectinib (3,
CH5424802),27,28 and brigatinib (4, AP26113)29 were
approved by the U.S. FDA for treatment of patients with
advanced (metastatic), ALK-positive non-small-cell lung cancer
(NSCLC), whose disease has deteriorated after treatment, or
who could not tolerate treatment with crizotinib (Figure 1).
Recently, lorlatinib30 (5, PF-6463922) received accelerated
approval by the U.S. FDA for patients with ALK-positive
NSCLC, whose metastatic disease has progressed on crizotinib
and at least one other ALK inhibitor or whose disease has
INTRODUCTION
■
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase
belonging to the insulin receptor (IR) kinase superfamily. ALK
is expressed primarily in adult brain tissue and plays an
important role in the development of the nervous system.1
Deregulation of ALK was originally discovered by the
identification of a t(2;5) chromosomal translocation in
anaplastic large cell non-Hodgkin’s lymphoma (ALCL).2 The
nucleophosmin (NPM)-ALK fusion protein produced by this
translocation results in constitutive activation of the ALK
kinase.3 Subsequently, fusion of ALK with other genes has been
identified in ALCL,4 inflammatory myofibroblastic tumor,5
diffuse large B-cell lymphoma,6 squamous cell carcinoma,7 and
non-small-cell lung cancer.8−10 In addition to chromosomal
translocations leading to fusion proteins, amplification of the
ALK gene and activating point mutations of the wild-type ALK
protein have been reported in neuroblastoma,11−13 ovarian
cancer,14 and patients with inflammatory breast cancer.15
Accordingly, ALK has been pursued as an attractive therapeutic
target for treatment of various blood and solid tumors
containing an ALK fusion (ALK-positive tumors).
Received: September 4, 2020
https://dx.doi.org/10.1021/acs.jmedchem.0c01550
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Figure 1. Chemical structures of ALK inhibitors approved or currently in clinical trials.
Table 1. Design of Indoloquinolones as Potential ALK Inhibitors, Chemical Structures, and Their In Vitro ALK Inhibitory
Activity
compound
IC50(nM) in inhibition of ALK enzymatic activity
7a
7b
7c
7d
>50,000
2503 545
28.6 12.6
21.3 7.4
progressed on alectinib or ceritinib as the first ALK inhibitor
therapy for metastatic disease. Additional ALK inhibitors,
including ASP3026 (6),31 are currently in clinical development.
In the present study, we report the discovery of a new class of
potent and selective ALK inhibitors, exemplified by CJ-2360
(11n). CJ-2360 (11n) potently inhibits wild-type ALK kinase
and several clinically reported ALK mutants and is capable of
achieving complete tumor regression in the ALK-positive
KARPAS-299 xenograft model with oral administration.
synthesized compounds 7a and 7b using the same soluble group
as in alectinib (Table 1). While 7a shows no inhibitory activity in
our in vitro ALK enzymatic assay at concentrations up to 50 μM,
7b displays an IC50 value of 2.5 μM (Table 1). Compound 7b
therefore represented a reasonable starting point for further
optimization.
In our modeled structure of 7b in complex with ALK, there is
additional room available around the N-methyl group in 7b
(Supporting Information). We explored this site by changing the
N-methyl group in 7b to N-isopropyl, yielding 7c (Table 1).
Compound 7c exhibits an IC50 value of 28.6 nM in ALK
inhibition (Table 1) and is nearly 100 times more potent than
7b.
RESULTS AND DISCUSSION
■
Among all the ALK inhibitors that have been approved or are
currently in clinical trials, alectinib (3) has a unique, fused
tetracyclic system and has been shown to be highly selective
against ALK over other kinases.27 We selected alectinib (3) as
the template for the design of a new class of ALK inhibitors.
We proposed that the indoloquinolone structure could mimic
the tetracyclic core in alectinib and, to test this idea, we
The 4-(piperidin-4-yl)morpholinyl soluble group in alectinib
was shown to be the primary metabolic site.32 Accordingly, we
replaced the 4-(piperidin-4-yl)morpholinyl group in 7c with 1-
methyl-piperazinyl and obtained 7d (Table 1). Compound 7d
has an IC50 value of 21.3 nM in ALK inhibition and is thus
B
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equipotent with 7c (Table 1). We employed 7c as our template
compound for further modifications.
We developed the binding model of 7d in complex with ALK
based on the cocrystal alectinib (CH5424802) complexed with
ALK (PDB ID: 3AOX,27 Figure 2A) to guide our further
Table 2. Structure−Activity Relationship Studies of Phenyl
Substitutions on ALK Enzymatic Inhibition and Cell Growth
Inhibitory Activity in the ALK-Positive KARPAS-299 Cell
Line
Figure 2. (A) Previously reported cocrystal structure of alectinib
(CH5424802) in complex with ALK (PDB ID: 3AOX). (B) Modeled
structure of compound 7d in complex with ALK. Four sites in 7d
identified for optimization are labeled with red arrows.
optimization efforts. The predicted binding model for 7d shows
that the tetracyclic core structure in 7d mimics the tetracyclic
core in alectinib, its cyano group interacts with a deep
hydrophobic pocket formed by the L1196, L1171, and V1180
residues in ALK, and the carbonyl group of 7d forms a hydrogen
bond with the backbone amide group of L1198. Furthermore,
the isopropyl group of 7d occupies the same space as the
dimethyl group in CH5424802.
Compound 7d lacks the corresponding ethyl group in
alectinib, which in the cocrystal structure (Figure 2A), has
hydrophobic interactions with L1198, A1200, and L1122
residues in ALK. We thus performed modifications on the
phenyl ring in 7d to explore the interactions with L1198, A1200,
and L1122 residues, obtaining the results summarized in Table
2.
Installation of an ethyl group on the phenyl ring in 7d yielded
compound 8b, which has an IC50 value of 2.1 nM in inhibition of
ALK and is therefore 10 times more potent than 7d. Changing
the ethyl group in 8b to n-propyl resulted in 8c, which has an
IC50 value of 1.0 nM in ALK inhibition and is thus 2 times more
potent than 8b. Replacing the ethyl group in 8b with a bromine
atom generated 8a, which is slightly more potent than 8b.
Installation of an isobutyl group at the same site resulted in 8d,
which has an IC50 value of 2.5 nM and is thus equipotent with
8b. Interestingly, replacing the isobutyl group in 8d with an
isopropyl group, which led to 8e, reduces the ALK inhibitory
activity by a factor of 16.
We next synthesized compounds 8f−8i by introducing a
series of alkoxy groups at this site. Compounds 8f with a
methoxyl group, 8g with a propoxyl group, 8h with a butoxyl
group, and 8i with an isopropoxyl group all have potent and
similar ALK inhibitory activity with IC50 values of 3.7−5.8 nM.
We evaluated these potent ALK inhibitors in Table 2 for their
cell growth inhibitory activity in the KARPAS-299 cell line
carrying an ALK fusion protein (ALK-positive), obtaining the
results in Table 2. Compound 7d has an IC50 value of 514.6 nM.
In comparison, compounds 8a−8d, which have an improved
ALK inhibitory activity over 7d, have IC50 values of 24.2−36.8
nM and are >10 times more potent than 7d. Although 8e is 2
times less potent than 7d in ALK inhibition, 8e is 4 times more
potent than 7d in inhibition of KARPAS-299 cell growth,
suggesting enhanced cell permeability.
Next, we installed one or two fluorine atoms onto the phenyl
ring in 7d to examine the effect on ALK inhibition and cell
growth inhibition in the KARPAS-299 cell line and obtained the
results summarized in Table 3. Installation of a single fluorine
atom onto the phenyl ring of 7d gave 9a, whose ALK inhibitory
activity is similar to that of 7d. However, the cellular activity of
9a in KARPAS-299 is improved by 10 times over that of 7d.
Compound 9b containing two fluorine substitutions in the
phenyl ring of 7d is 3 times more potent than 9a in ALK
inhibition but has a similar cell growth inhibitory activity in
KARPAS-299 when compared to 9a. Compound 9c, which was
obtained by introduction of a fluorine atom into 8f, is 2 times
less potent than 8f in ALK inhibition but is 10 times more potent
than 8f in cell growth inhibition in the KARPAS-299 cell line.
These data indicated that installation of one or two fluorine
atoms on the phenyl ring significantly improves the cellular
potency of the resulting compounds.
Next, we explored the effect of different substitutions on the
quinolone nitrogen in 8f and obtained the results summarized in
Table 4.
Replacement of the isopropyl group in 8f by an ethyl or n-
propyl group yielded 10a and 10b, respectively. While 10a and
10b are only 2−5 times less potent than 8f in ALK inhibition,
they are much less potent than 8f in cell growth inhibition in the
KARPAS-299 cell line. In fact, 10a with an ethyl group has a
minimal cell growth inhibitory activity at concentrations up to
100 μM. Replacement of the isopropyl group in 8f with an
isobutyl or isopentyl group yielded 10c and 10d, respectively.
Compounds 10c and 10d display very similar ALK inhibition
and cell growth inhibition activity compared to 8f. Replacing the
isopropyl group in 8f with a cyclopropyl, cyclobutyl, cyclopentyl,
or cyclohexyl group resulted in 10e, 10f, 10g, and 10h,
respectively. Compound 10e is 3 times less potent in ALK
inhibition than 8f but is >30 times less potent than 8f in
C
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Table 3. Effect of Fluorine Substitution on the Phenyl Ring
compound
IC50 (nM) (ALK inhibition)
IC50 (nM) (cell growth inhibition in KARPAS-299)
7d
9a
9b
8f
21.3 7.4
19.8 3.5
6.6 0.5
4.8 1.0
8.9 1.5
514.6
47.9
66.0
206.7
19.7
9c
potent in inhibition of ALK (IC50 = 5.8 nM) but has a much
weaker activity in inhibition of KARPAS-299 cell growth (IC50 =
275 nM) than ALK-68. Although the reason why CJ-2355 was
much less potent than ALK-68 in inhibition of cell growth
needed further investigation, the data nevertheless indicated the
critical importance of the solubilizing group at this site for
achieving potent cellular activity. Accordingly, we have
synthesized a series of compounds with diverse solubilizing
groups.
Table 4. Effect of Different Substitutions on the Quinolone
Nitrogen Atom
IC50 (nM) (ALK
Inhibition)
IC50 (nM) (cell growth inhibition in
KARPAS-299)
compound
Changing 1-methylpiperazine in ALK-68 with 1-methyl-
piperidine resulted in CJ-2212, which is more potent than ALK-
68 in inhibition of ALK activity (IC50 = 0.49 nM) and cell
growth in the KARPAS-299 cell line (IC50 = 9.7 nM).
8f
4.8 1.0
20.6 4.1
13.5 3.7
5.3 2.1
8.2 2.1
16.5
12.7 1.7
5.2 0.7
1.6 0.3
206.7
>100,000
4957
10a
10b
10c
10d
10e
10f
10g
10h
271.8
147.5
9884
138
129.8
151.6
Encouraged by the excellent potencies for CJ-2212, we
replaced the 1-methyl group in CJ-2212 with oxetane, isopropyl,
tetrahydro-2H-pyran, and ethan-1-ol, respectively, yielding CJ-
CJ-2224, CJ-2225, CJ-2226, and CJ-2227, respectively. These
four compounds all potently inhibit ALK activity with IC50
values of 0.62−5.5 nM, with CJ-2224 being the least potent
(IC50 = 5.5 nM) among them. They also displayed potent cell
growth inhibition in the KARPAS-299 cell line with IC50 values
of 17−96 nM, with CJ-2224 being the least potent (IC50 = 96
nM).
We next synthesized a total of eight compounds containing a
piperazine group substituted with one or more methyl group
(CJ-2349, CJ-2350, CJ-2351, CJ-2356, CJ-2357, CJ-2358, CJ-
2359, and CJ-2360). These eight compounds display potent and
similar inhibitory activities of ALK with IC50 values of 1.5−4.9
nM. While CJ-2349, CJ-2350, and CJ-2351 only display
moderate cell growth inhibition activity in the KARPAS-299
cell line with IC50 values of 116−162 nM, CJ-2356, CJ-2357, CJ-
2358, CJ-2359, and CJ-2360 achieve potent cell growth
inhibition with IC50 values of 1.8−29 nM. Among them, CJ-
2360 achieves an IC50 value of 1.8 nM in inhibition of cell growth
in the KARPAS-299 cell line. We further tested CJ-2360 for its
potency in cell growth inhibition in the H3122 non-small-cell
lung cell line carrying EML4-ALK and obtained an IC50 value of
3 nM. CJ-2360 is thus the most potent ALK inhibitor among all
inhibition of cell growth in the KARPAS-299 cell line.
Compound 10g has a similar ALK inhibitory activity as 8f,
whereas 10f is 2 times less potent than 8f in ALK inhibition, and
10h is 3 times more potent than 8f. However, these three
compounds show very similar cell growth inhibition activity in
the KARPAS-299 cell line when compared to 8f.
Among those ALK inhibitors shown in Tables 1−4,
compound 9c potently inhibits ALK enzymatic activity with
an IC50 value of 8.9 nM and is also the most potent compound in
inhibition of cell growth in the KARPAS-299 cell line with an
IC50 value of 19.7 nM. We performed further optimization of 9c
by focusing on modifications of its solubilizing group (Table 5).
We replaced the 1-methylpiperazine in 9c (ALK-68) with 4-
(piperidin-4-yl)morpholine, the same solubilizing group used in
3 (alectinib), yielding ALK-73. ALK-73 is similarly potent in
inhibition of both ALK enzymatic activity and cell growth
inhibition in the KARPAS-299 cell line, as compared to ALK-68.
We replaced 1-methylpiperazine in ALK-68 with N,N-
dimethylpiperidin-4-amine, which yielded CJ-2355. CJ-2355 is
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Table 5. Investigation of the Structure−Activity Relationships of the Solubilizing Group
IC50 (nM) (ALK
inhibition)
IC50 (nM) (cell growth inhibition in
KARPAS-299)
IC50 (nM) (ALK
inhibition)
IC50 (nM) (cell growth inhibition in
KARPAS-299)
compound
9c
compound
CJ-2349
8.9 1.5
19.7
4.9 1.4
162
(ALK-68)
ALK-73
CJ-2355
CJ-2212
CJ-2225
CJ-2226
CJ-2224
CJ-2227
4.0
5.8
0.49
2.5
1.3
40.5
275
9.7
17
30
96
CJ-2350
CJ-2351
CJ-2356
CJ-2357
CJ-2358
CJ-2359
CJ-2360
3.9 1.6
2.6 1.6
4.8 0.8
2.2 0.9
1.5 0.2
2.6 0.9
2.2 0.3
116
117
29
20
5
5.5
0.62
10
1.8
36
the ALK inhibitors we have synthesized and evaluated in this
study.
To understand the structural basis for its potent ALK
inhibitory activity, we modeled the binding model for CJ-2360
in complex with ALK and compared the predicted binding
model for CJ-2360 to the cocrystal structure of alectinib in
complex with ALK (Figure 3). Overall, CJ-2360 has a very
similar binding mode as compared to alectinib. However, there
are some subtle differences for alectinib and CJ-2360 on their
interactions with CJ-2360. For example, the ethyl group in
alectinib has hydrophobic interactions with the side chains of
Ala1200 and Leu1122, the corresponding methoxyl group in CJ-
2360 maintains those hydrophobic interactions with the side
chains of Ala1200 and Leu1122, and the oxygen atom in the
methoxyl group has close contacts with the guanidinium group
of Arg1120.
Evaluation of Pharmacokinetics of CJ-2360. Because
CJ-2360 is the most potent ALK inhibitor we have identified
based on its cell growth inhibitory activity in the KARPAS-299
cell line, we evaluated its pharmacokinetics of CJ-2360 in mice
and rats, obtaining the results summarized in Table 6.
The PK data for CJ-2360 in mice showed that it achieves a
reasonable oral exposure and an overall oral bioavailability of
38.2%. It has a modest clearance rate of 1.2 L/h/kg and a large
volume distribution of 7.5 L/kg, suggesting extensive tissue
distribution in mice.
CJ-2360 has a similar PK profile in rats as compared to that in
mice. It achieves an overall oral bioavailability of 32.0%. It has a
moderate clearance rate of 2.9 L/h/kg and a large volume
distribution of 8.4 L/kg, suggesting extensive tissue distribution
in rats.
Seeking an understanding on the ability of CJ-2360 to
penetrate into tumor tissues, we performed an analysis of the
drug concentrations in plasma and tumor tissue in mice bearing
KARPAS-299 xenograft tumors (Table 7). Our data showed that
CJ-2360 achieves good plasma exposure in mice following a
single dose of oral administration. Significantly, the drug
Figure 3. (A) Interaction residues in ALK with alectinib in previously
reported cocrystal structure (PDB ID: 3AOX). (B) Interaction residues
in ALK in our modeled structure of CJ2360 in complex with ALK. (C)
Detailed hydrophobic and hydrogen bonding interactions between
alectinib and ALK and between CJ-2360 and ALK, as analyzed by
LigPlot.
concentrations in the tumor tissue are >5 times higher than
those in the plasma at different time points, indicating drug
accumulation in the tumor.
We next tested CJ-2360 in KARPAS-299 xenograft tumors for
its ability to inhibit ALK activity with alectinib included as a
positive control in a pharmacodynamic (PD) experiment
(Figure 4).
E
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Table 6. Pharmacokinetic Parameters of CJ-2360 in Mice and Rats with Intravenous and Oral Administration
dose (mg/kg)
C
max (ng/mL)
AUC0−t (ng·h/mL)
t1/2 (h)
CJ-2360
IV
PO
IV
PO
IV
PO
IV
PO
CL (IV) (L/h/kg)
Vss (IV) (L/kg)
F (PO)
mouse
rat
2
2
20
20
213
202
458
639
1783
2219
2.1
2.4
7.5
8.4
1.2
2.9
7.5
8.4
38.2%
32.0%
a
Table 7. Analysis of CJ-2360 Concentrations in Mouse Plasma and Tumor Tissue
PO (100 mg/kg)
time point (h)
concentration in plasma (ng/mL)
concentration in tumor (ng/g)
mouse 1
mouse 2
mean
right tumor
left tumor
right tumor
left tumor
mean
1
3
6
2780
2220
890
3540
7000
850
3160
4610
870
10,000
25,600
28,000
990
14,050
23,000
22,600
895
12,100
32,600
38,200
1335
12,850
37,000
38,400
790
12,250
29,550
31,800
1003
24
9.26
24.3
16.78
a
Mice bearing KARPAS-299 tumors were dosed with a single dose of CJ-2360 at 100 mg/kg via oral gavage and mice were sacrificed at indicated
time points, with two mice for each time point. Each mouse had two tumors.
time point in our PD experiment (Figure 4), we chose twice
daily dosing for testing its antitumor activity. Ceritinib was
included as a positive control in this efficacy experiment because
of its demonstrated excellent efficacy in the KARPAS-299
xenograft model.26
Our efficacy experiment showed that CJ-2360 is very
efficacious in the KARPAS-299 xenograft model (Figure 5A).
It achieves complete tumor regression in 100% of tumors (7 out
of 7) and all tumors did not return until day 53, 23 days after the
last dose. Hence, CJ-2360 is capable of achieving complete and
long-lasting tumor regression in the KARPAS-299 xenograft
tumor model. CJ-2360 is well tolerated in mice and did not cause
weight loss (Figure 5B) or other signs of toxicity during the
entire experiment. In our experiment, ceritinib is also very
efficacious, but it only achieved complete tumor regression in 4
out of 7 tumors with 50 mg/kg, daily administration, suggesting
that higher doses or a more frequent dosing schedule is needed
for ceritinib to achieve 100% tumor regression in mice.
Inhibitory Activity of CJ-2360 against Clinically
Relevant ALK Mutants. In clinic, patients with ALK-positive
tumors treated with crizotinib (1) developed resistance to the
drug due to generation of ALK mutations and a number of
clinical ALK mutants have been identified.27 We evaluated CJ-
Figure 4. Pharmacodynamic study to evaluate CJ-2360 for its ability to
inhibit ALK activity in KARPAS-299 tumors in mice. Mice were dosed
with a single dose of CJ-2360 (2360) at 100 mg/kg or alectinib (Ale) at
20 mg/kg via oral gavage.
Our PD data showed that a single dose of CJ-2360 is very
effective in inhibition of ALK phosphorylation, as well as ERK
and STAT3 phosphorylation in KARPAS-299 tumor tissue, with
the effect persisting for at least 24 h.
Evaluation of CJ-2360 for Its Antitumor Efficacy In
Vivo. Based on its promising in vivo PD data and excellent tumor
tissue exposure, we evaluated CJ-2360 for its antitumor efficacy
in the KARPAS-299 xenograft model in mice. Since CJ-2360
effectively inhibited the AKT activity at the 6 h time point but
had a slightly reduced inhibition of the AKT activity at the 24 h
Figure 5. Antitumor activity of CJ-2360 in the KARPAS-299 xenograft tumor model. CJ-2360 was orally administered twice daily at 100 mg/kg and
ceritinib was orally administered daily at 50 mg/kg for 22 days (from days 9 to 30).
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Table 8. Inhibitory Activity of Crizotinib, CJ-2360, Alectinib, and Ceritinib against Wild-Type ALK and Clinical ALK Mutants
IC50 (nM)
compound
ALK wild type
ALK F1197M
ALK G1269A
ALK L1196M
ALK S1206Y
crizotinib
CJ-2360
alectinib
ceritinib
34.5 5.0
2.2 0.3
0.59 0.09
0.61 0.02
416 43
4.0 1.4
1.0
1035 194
8.8 2.2
3.9
1540 270
6.3 2.2
1.5 0.3
1.1 0.1
1066 232
8.9 2.6
0.96 0.24
1.6 0.3
3.5
1.9
2360, together with crizotinib (1), alectinib, and ceritinib, for its
inhibitory activity against four clinically reported ALK mutants,
namely, F1197M, G1269A, L1196M, and S1206Y, and obtained
the results summarized in Table 8.
260 nM, respectively (Table 9). CJ-2360 displays >100-fold
selectivity for ALK over the insulin receptor kinase (INSR) and
shows no significant activity against insulin-like growth factor 1
receptor (IGF1R), two kinases for which ALK belongs to the
same subfamily of kinases. Hence, the KINOMEscan data
showed that CJ-2360 is a potent inhibitor against wild-type ALK
and clinically reported ALK mutants and displays potent
inhibitory activity against only a few other kinases (LTK,
MERTK, CLK1, DAPK1, and DAPK2) among the 468 kinases
evaluated. Mer tyrosine kinase and DAPK kinases are
considered as potential therapeutic targets for human cancer.
Therefore, CJ-2360 may serve as a promising lead compound for
these kinases.
Previously, alectinib was tested for its selectivity against 402
kinases at 10 and 1000 nM.27 Alectinib inhibited 29 other non-
ALK kinases with >95% inhibition at 1 μM among 402 kinases
tested.27 In our study, CJ-2360 only inhibited 11 other non-ALK
kinases with >50% inhibition at 100 nM. Alectinib and CJ-2360
also inhibited the same kinases with different potencies. For
example, alectinib inhibited GAK with 79% inhibition at 10 nM
(IC50 < 10 nM) and CJ-2360 inhibited GAK with 59% inhibition
at 100 nM. Taken together, our data showed that CJ-2360 is a
highly selective ALK inhibitor.
Our data showed that CJ-2360 demonstrates potent
inhibitory activity with IC50 values of 2.2, 4.0, 8.8, 6.3, and 8.9
nM against wild-type ALK and F1197M, G1269A, L1196M, and
S1206Y ALK mutants, respectively. Crizotinib has IC50 values of
34.5, 416, 1035, 1540, and 1066 nM against wild-type ALK and
F1197M, G1269A, L1196M, and S1206Y ALK mutants,
respectively. Alectinib has IC50 values of 0.59, 1.0, 3.9, 1.5, and
0.96 nM against wild-type ALK and F1197M, G1269A,
L1196M, and S1206Y ALK mutants, respectively, whereas
ceritinib achieves IC50 values of 0.61, 3.5, 1.9, 1.1, and 1.6 nM
against wild-type ALK and F1197M, G1269A, L1196M, and
S1206Y ALK mutants, respectively.
Hence, the fold of resistance for crizotinib ranges from 12 to
44 for these clinically relevant ALK mutants over wild-type ALK,
whereas the fold of resistance for CJ-2360 is 2−4 for these
clinically relevant ALK mutants over wild-type ALK. Alectinib
and ceritinib have 2−6-fold resistance for these clinically
relevant ALK mutants over wild-type ALK. CJ-2360 is thus
>100 times more effective than crizotinib against these four
clinically relevant ALK mutants.
CHEMISTRY
Selectivity of CJ-2360 against Other Human Kinases
by KINOMEscan. CJ-2360 was evaluated by KINOMEscan in
DiscoverX for its inhibitory activity in a panel of 468 kinases
(Table 9 and Table S1).
■
The synthesis of compounds 7a−7d is outlined in Scheme 1.
The intermediate methyl 2-(phenylamino)-1H-indole-3-car-
boxylates (13a−13c) were synthesized according to published
methods33 with minor modifications. Compounds 13a−13c are
obtained by chlorination of 12b with NCS in the presence of
DABCO, followed by addition of the aniline as its trichlor-
oacetate.
Table 9. Profiling the Selectivity of CJ-2360 against 468
Kinases by KINOMEscan
kinase
Kd (nM)
kinase
FAK
IGF1R
INSR
INSRR
LTK
Kd (nM)
Cyclization of 13a−13c in boiling diphenyl ether affords the
corresponding 5,6-dihydro-11H-indolo[2,3-b]quinolin-11-one
(14a−14c).33 Substitution with 4-morpholinopiperidine or 1-
methylpiperazine in DMSO in the presence of DIPEA affords
compounds 7a−7d.
ALK
5.6
1.2
4.8
31
>3000
>3000
950
1600
6.3
ALK (C1156Y)
ALK (L1196M)
CLK1
DAPK1
23
The synthesis of compounds 8a−8e is outlined in Scheme 2.
Methyl 2-((4-bromo-3-fluorophenyl)(isopropyl)amino)-6-
cyano-1H-indole-3-carboxylate (15) is obtained by chlorination
of 12b with NCS in the presence of DABCO, followed by
addition of the aniline as the trichloroacetate. Cyclization of 15
in boiling diphenyl ether affords 5,6-dihydro-11H-indolo[2,3-
b]quinolin-11-one (16). Reaction with 1-methylpiperazine in
DMSO in the presence of DIPEA afforded the designed
compound (8a). Compounds 8b−8e were obtained by Suzuki
coupling of 8a with appropriate 4,4,5,5-tetramethyl-1,3,2-
dioxaborolanes, followed by hydrogenation in THF.
The synthesis of compounds 8f−8i is outlined in Scheme 3.
Alkoxyl-substituted methyl 2-((4-alkoxyl-3-fluorophenyl)-
(isopropyl)amino)-6-cyano-1H-indole-3-carboxylates (17a−
17d) are obtained by chlorination of 12b with NCS in the
presence of DABCO, followed by addition of the aniline as its
trichloroacetate. Cyclization of 17a−17d in boiling diphenyl
DAPK2
DAPK3
22
260
MERTK
ROS1
11
>3000
Consistent with the ALK inhibition data obtained in the ALK
kinase assay developed in our laboratory, CJ-2360 achieves
potent inhibitory activity against wild-type ALK with IC50 = 5.6
nM. In addition, CJ-2360 is also potent against two clinically
reported ALK mutants (C1156Y and L1196M) included in the
468 kinase panel.
Outside of ALK, CJ-2360 only inhibited 11 other non-ALK
kinases at 100 nM with >50% inhibition (Table S1). CJ-2360
displays potent inhibitory activity against LTK (leukocyte
receptor tyrosine kinase), which belongs to the same subfamily
of the insulin receptor kinase superfamily (Table 9). CJ-2360
inhibits Mer tyrosine-protein kinase (MERTK), CLK1, DAPK1,
DAPK2, and DAPK3 with IC50 values of 6.3, 11, 31, 23, 22, and
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a
Scheme 1. Synthesis of 7a−7d
a
Reagents and conditions: (a) (i) NCS, DABCO, DCM, 0 °C, 2 h; (ii) trichloroacetic acid, DCM, RT, 2 h; (b) Ph2O, reflux, 1 h; (c) 4-
morpholinopiperidine or 1-methylpiperazine, DIPEA, DMSO, 120−140 °C, 3 days.
a
Scheme 2. Synthesis of 8a−8e
a
Reagents and conditions: (a) (i) NCS, DABCO, DCM, 0 °C, 2 h; (ii) 4-bromo-3-fluoro-N-isopropylaniline, trichloroacetic acid, DCM, RT, 2 h;
(b) Ph2O, reflux, 1 h; (c) 1-methylpiperazine, DIPEA, DMSO, 120−140 °C, 3 days; (d) (i) appropriate 4,4,5,5-tetramethyl-1,3,2-dioxaborolane,
Pd(PPh3)2Cl2, Na2,CO3, DME/H2O, 80 °C; (ii) 10% Pd-C, H2, THF.
ether affords the alkoxyl-substituted 5,6-dihydro-11H-indolo-
[2,3-b]quinolin-11-ones (18a−18d). Substitution with 1-
methylpiperazine in DMSO in the presence of DIPEA affords
the designed compounds (8f−8i).
The synthesis of compounds 9a−9c is outlined in Scheme 4.
Di- or trifluoro-substituted methyl 6-cyano-2-(isopropyl-
(phenyl)amino)-1H-indole-3-carboxylates (19a−19c) are ob-
tained by chlorination of 12b with NCS in the presence of
DABCO, followed by addition of the appropriate aniline as its
trichloroacetate. Cyclization of 19a−19c in boiling diphenyl
ether affords the di- or trifluoro-substituted 5,6-dihydro-11H-
indolo[2,3-b]quinolin-11-ones (20a−20c), and substitution
with 1-methylpiperazine in DMSO in the presence of DIPEA
affords the designed compounds (9a−9c). The synthesis of
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a
Scheme 3. Synthesis of 8f−8i
a
Reagents and conditions: (a) (i) NCS, DABCO, SCM, 0 °C, 2 h; (ii) trichloroacetic acid; appropriate anilines, RT, 2 h; (b) Ph2O, reflux, 1 h; (c)
1-methylpiperazine, DIPEA, DMSO, 120−140 °C, 3 days.
a
Scheme 4. Synthesis of 9a−9c
a
Reagents and conditions: (a) (i) NCS, DABCO, DCM, 0 °C, 2 h; (ii) trichloroacetic acid, appropriate anilines, RT, 2 h; (b) Ph2O, reflux, 1 h; (c)
1-methylpiperazine, DIPEA, DMSO, 120−140 °C, 3 days.
compounds 10a−10h is outlined in Scheme 5. Methyl 6-cyano-
2-(phenylamino)-1H-indole-3-carboxylates (23a−23h) are ob-
tained by chlorination of methyl 6-cyano-1H-indole-3-carbox-
ylate with NCS in the presence of DABCO, followed by addition
of the appropriate alkyl- or cycloalkyl-substituted aniline as the
trichloroacetate. Cyclization of 23a−23h in boiling diphenyl
ether affords alkyl- or cycloalkyl-substituted 5,6-dihydro-11H-
indolo[2,3-b]quinolin-11-ones (24a−24h). Substitution with 1-
methylpiperazine in DMSO in the presence of DIPEA affords
the designed compounds (10a−10h).
The synthesis of compounds ALK-73, CJ-2349-2351, and CJ-
2356-2360 is outlined in Scheme 6. Compound 20c was
substituted with 1-methylpiperazine in DMSO in the presence
of DIPEA at 120−140 °C for 3 days to give the designed
compounds.
The synthesis of compounds CJ-2212 and CJ-2224-2227 is
outlined in Scheme 7. Nucleophilic substitution of F in 25 by
sodium methoxide gave anisole 26. Aniline 28 was obtained by
Suzuki coupling of 26 followed by hydrogenation. Reductive
amination of 28 afforded isopropyl-substituted aniline 29.
Methyl 6-cyano-2-(phenylamino)-1H-indole-3-carboxylate 30
was obtained by chlorination of methyl 6-cyano-1H-indole-3-
carboxylate with NCS in the presence of DABCO, followed by
addition of substituted aniline 29 and trichloroacetate.
Deprotection of Boc in 30 followed by reductive amination
gave piperidine 31b−31f. Cyclization of 31b−31f in boiling
diphenyl ether affords target molecules.
CONCLUSIONS
■
We have designed, synthesized, and evaluated a new class of
ALK inhibitors containing a fused indoloquinoline scaffold. Our
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a
Scheme 5. Synthesis of 10a−10h
a
Reagents and conditions: (a) (i) methyl 6-cyano-1H-indole-3-carboxylate, NCS, DABCO, DCM, 0 °C, 2 h; (ii) trichloroacetic acid, appropriate
anilines, RT, 2 h; (b) Ph2O, reflux, 1 h; (c) 1-methylpiperazine, DIPEA, DMSO, 120−140 °C, 3 days.
a
Scheme 6. Synthesis of ALK-73, CJ-2349-2351, and CJ-2356-2360
a
Reagents and conditions: (a) appropriate amines, DIPEA, DMSO, 120−140 °C, 3 days.
efforts have resulted in the discovery of CJ-2360 as a potent,
orally bioavailable, and highly efficacious ALK inhibitor. CJ-
cell growth in the ALK-positive KARPAS-299 anaplastic large-
cell lymphoma cell line carrying NPM-ALK fusion and the
H3122 non-small-cell lung cell line carrying EML4-ALK with
IC50 values of 2 and 3 nM, respectively. CJ-2360 has good oral
pharmacokinetics in mice and rats. CJ-2360 effectively inhibits
ALK activity in the ALK-positive KARPAS-299 xenograft tumor
with a single administration in mice. CJ-2360 is capable of
2360 effectively inhibits wild-type ALK activity with an IC50
=
2.2 nM. Importantly, CJ-2360 displays potent inhibitory activity
against four clinically reported ALK mutants with IC50 values of
4.4−8.9 nM and is >100 times more potent than crizotinib
against these clinical ALK mutants. CJ-2360 potently inhibits
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a
Scheme 7. Synthesis of CJ-2212 and CJ-2224-2227
a
Reagents and conditions: (a) MeONa, MeOH, 0 °C to RT, 4 h, 55%; (b) tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-
dihydropyridline-1(2H)-carboxylate, Pd(dppf)Cl2, and K2CO3, 92%; (c) 10% Pd-C, ethanol, RT, overnight, 92%; (d) acetone, acetic acid,
NaBH(OAc)3, DCM, RT, 12 h, 94%; (e) (i) methyl 6-cyano-1H-indole-3-carboxylate, NCS, DABCO, DCM, 0 °C, 2 h; (ii) trichloroacetic acid,
RT, 2 h, 69%; (f) (i) TFA; (ii) appropriate aldehyde or ketone, NaBH(OAc)3, acetic acid, DCM; (g) Ph2O, reflux, 1 h.
was added dropwise and the reaction was stirred for 2 h at RT. The
reaction was quenched by adding H2O and the mixture was extracted
with DCM. Solvent was removed under vacuum and the residue was
purified by silica gel chromatography with hexane/EtOAc (4/1, v/v) to
achieving complete and long-lasting tumor regression in the
KARPAS-299 xenograft model in mice at a well-tolerated dose
schedule. Collectively, our data demonstrate that CJ-2360 is a
promising ALK inhibitor for advanced preclinical development.
1
afford the title compound as a colorless oil (220 mg, 68% yield). H
NMR (400 MHz, CDCl3) δ ppm 8.26 (s, 1H), 8.03 (d, J = 8.6 Hz, 1H),
7.46 (d, J = 1.8 Hz, 1H), 7.39 (dd, J = 8.6, 1.8 Hz, 1H), 7.21−7.18 (m,
1H), 6.66−6.49 (m, 3H), 3.85 (s, 3H), 3.43 (s, 3H).
EXPERIMENTAL SECTION
■
General Methods. Solvents and reagents were obtained
commercially and used without further purification. Reactions were
monitored by TLC on 250 μm E. Merck silica gel plates (60F-254)
using UV light for visualization. E. Merck silica gel (60; particle size,
15−40 μm) was used for flash column chromatography. The NMR
spectra were recorded on a Bruker Avance300 spectrometer (300
MHz) and Bruker Avance III spectrometer (400 MHz). Chemical shifts
are reported as δ values (ppm) downfield relative to TMS as an internal
standard, with multiplicities reported in the standard form. All final
compounds have purities >95%, as determined by HPLC (UV
detection at 254 nm).
Methyl 2-((3-Fluorophenyl)amino)-1H-indole-3-carboxylate
(13a). DABCO (62 mg, 0.55 mmol) was added to a solution of methyl
1H-indole-3-carboxylate (175 mg, 1 mmol) in DCM (20 mL) and the
mixture was cooled down to 0 °C. NCS (147 mg, 1.1 mmol) was then
added and the reaction was stirred at 0 °C for 2 h. A solution of 3-
fluoroaniline (111 mg, 1 mmol) and trichloroacetic acid (41 mg, 0.25
mmol) in DCM (5 mL) was added dropwise and the reaction was
stirred for 2 h at RT. The reaction was quenched by adding H2O and the
mixture was extracted with DCM. Solvent was removed under vacuum
and the residue was purified by silica gel chromatography with hexane/
EtOAc (4/1, v/v) to afford the title compound as a colorless oil (200
mg, 70% yield). 1H NMR (400 MHz, CDCl3) δ ppm 9.14 (s, 1H), 8.30
(s, 1H), 7.88−7.81 (m, 1H), 7.43−7.37 (m, 1H), 7.30−7.16 (m, 2H),
7.16−6.95 (m, 3H), 6.94−6.87 (m, 1H), 3.98 (s, 3H).
Methyl 6-Cyano-2-((3-fluorophenyl)(isopropyl)amino)-1H-
indole-3-carboxylate (13c). DABCO (62 mg, 0.55 mmol) was
added to a solution of methyl 6-cyano-1H-indole-3-carboxylate (200
mg, 1 mmol) in DCM (20 mL) and the mixture was cooled down to 0
°C. NCS (147 mg, 1.1 mmol) was then added and the reaction was
stirred at 0 °C for 2 h. A solution of 3-fluoro-N-isopropylaniline (153
mg, 1 mmol) and trichloroacetic acid (41 mg, 0.25 mmol) in DCM (5
mL) was added dropwise and the reaction was stirred for 2 h at RT. The
reaction was quenched by adding H2O and the mixture was extracted
with DCM. Solvent was removed under vacuum and the residue was
purified by silica gel chromatography with hexane/EtOAc (4/1, v/v) to
1
afford the title compound as a colorless oil (265 mg, 75% yield). H
NMR (400 MHz, CDCl3) δ ppm 8.39 (s, 1H), 8.23 (d, J = 8.4 Hz, 1H),
7.64−7.59 (m, 1H), 7.50 (dd, J = 8.4, 1.5 Hz, 1H), 7.24−7.22 (m, 1H),
6.74−6.64 (m, 1H), 6.60−6.56 (m, 2H), 4.56−4.49 (m, 1H), 3.81 (s,
3H), 1.32 (d, J = 6.6 Hz, 6H).
3-Fluoro-5,6-dihydro-11H-indolo[2,3-b]quinolin-11-one
(14a). Methyl 2-((3-fluoro-phenyl)amino)-1H-indole-3-carboxylate
(193 mg, 0.68 mmol) was dissolved in diphenyl ether (10 mL) and
the solution was refluxed for 1 h. The mixture was then cooled down to
RT. Hexane (20 mL) was added and the precipitate was collected by
filtration to afford the title compound as a pale yellow solid (82 mg, 48%
yield). 1H NMR (400 MHz, DMSO-d6) δ 12.41 (s, 1H), 11.80 (s, 1H),
8.32 (dd, J = 8.9, 6.6 Hz, 1H), 8.16 (d, J = 6.6 Hz, 1H), 7.48 (d, J = 7.7
Hz, 1H), 7.40 (dd, J = 10.5, 2.5 Hz, 1H), 7.30−7.10 (m, 3H).
3-Fluoro-5-methyl-11-oxo-6,11-dihydro-5H-indolo[2,3-b]-
quinoline-8-carbonitrile (14b). Methyl 6-cyano-2-((3-
fluorophenyl)(methyl)amino)-1H-indole-3-carboxylate (220 mg, 0.68
mmol) was dissolved in diphenyl ether (10 mL) and the solution was
refluxed for 1 h. The mixture was cooled down to RT. Hexane (20 mL)
was added and the precipitate was collected by filtration to afford the
Methyl 6-Cyano-2-((3-fluorophenyl)(methyl)amino)-1H-in-
dole-3-carboxylate (13b). DABCO (62 mg, 0.55 mmol) was
added to a solution of methyl 6-cyano-1H-indole-3-carboxylate (200
mg, 1 mmol) in DCM (20 mL) and the mixture was cooled down to 0
°C. NCS (147 mg, 1.1 mmol) was then added and the reaction was
stirred at 0 °C for 2 h. A solution of 3-fluoro-N-methylaniline (125 mg,
1 mmol) and trichloroacetic acid (41 mg, 0.25 mmol) in DCM (5 mL)
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title compound as a pale yellow solid (178 mg, 90% yield). This product
was used directly in the next step without further purification. MS: m/z
= 292 [M + H].
reaction was stirred at 0 °C for 2 h. A solution of 4-bromo-3-fluoro-N-
isopropylaniline (232 mg, 1 mmol) and trichloroacetic acid (41 mg,
0.25 mmol) in DCM (5 mL) was added dropwise and the reaction was
stirred for 2 h at RT. The reaction was quenched by adding H2O and the
mixture was extracted with DCM. Solvent was removed under vacuum
and the residue was purified by silica gel chromatography with hexane/
EtOAc (4/1, v/v) to afford the title compound as a colorless oil (245
mg, 57% yield). 1H NMR (400 MHz, CDCl3) δ ppm 8.44 (s, 1H), 8.27
(d, J = 8.3 Hz, 1H), 7.73−7.64 (m, 1H), 7.54 (dd, J = 8.3, 1.4 Hz, 1H),
7.34 (dd, J = 9.0, 7.9 Hz, 1H), 6.56 (dd, J = 11.3, 2.7 Hz, 1H), 6.41 (dd, J
= 9.0, 2.7 Hz, 1H), 4.45−4.41 (m, 1H), 3.81 (s, 3H), 1.31 (d, J = 6.6 Hz,
6H).
2-Bromo-3-fluoro-5-isopropyl-11-oxo-6,11-dihydro-5H-
indolo[2,3-b]quinoline-8-carbonitrile (16). Methyl 2-((4-bromo-
3-fluorophenyl)(isopropyl)amino)-6-cyano-1H-indole-3-carboxylate
(245 mg, 0.57 mmol) was dissolved in diphenyl ether (10 mL) and the
solution was refluxed for 1 h. The mixture was cooled down to RT.
Hexane (20 mL) was added and the precipitate was collected by
filtration. The precipitate was purified by silica gel chromatography with
hexane/EtOAc (2/1, v/v) to afford the title compound as a pale yellow
solid (110 mg, 49% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.30 (s,
1H), 8.54 (d, J = 8.3 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 11.9
Hz, 1H), 7.92 (d, J = 1.4 Hz, 1H), 7.63 (dd, J = 8.0, 1.4 Hz, 1H), 5.37−
5.28 (m, 1H), 1.74 (d, J = 7.0 Hz, 6H).
2-Bromo-5-isopropyl-3-(4-methylpiperazin-1-yl)-11-oxo-
6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (8a). 1-
Methylpiperazine (0.5 mL) and DIPEA (0.5 mL) were added to a
solution of 2-bromo-3-fluoro-5-isopropyl-11-oxo-6,11-dihydro-5H-
indolo[2,3-b]quinoline-8-carbonitrile (110 mg, 0.28 mmol) in
DMSO (2 mL) and the mixture was heated to 120−140 °C for 3
days. The reaction mixture was cooled down to RT and purified by
preparative HPLC to afford the title compound as a pale yellow solid
(29 mg, 22% yield). 1H NMR (300 MHz, CD3OD) δ ppm 8.58 (s, 1H),
8.29 (d, J = 7.9 Hz, 1H), 7.77 (s, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.49 (s,
1H), 5.44−5.30 (m, 1H), 3.90−3.70 (m, 4H), 3.60−3.30 (m, 4H), 3.08
(s, 3H), 1.89 (d, J = 7.1 Hz, 6H).
2-Ethyl-5-isopropyl-3-(4-methylpiperazin-1-yl)-11-oxo-
6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (8b). A
mixture of 2-bromo-5-isopropyl-3-(4-methylpiperazin-1-yl)-11-oxo-
6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (30 mg, 0.063
mmol), Na2CO3 (25 mg, 0.183 mmol), Pd(PPh3)2Cl2 (4.3 mg, 0.0061
mmol), and 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (14 mg,
0.091 mmol) in DME/H2O (4.8 mL, 5/1, v/v) was stirred under
nitrogen at 80 °C overnight. After the reaction was complete as
indicated by LC−MS, the mixture was purified by preparative HPLC to
afford 5-isopropyl-3-(4-methylpiperazin-1-yl)-11-oxo-2-vinyl-6,11-di-
hydro-5H-indolo[2,3-b]quinoline-8-carbonitrile, which was dissolved
in THF (5 mL). Pd/C (10%, 20 mg) was added to the THF solution
and the mixture underwent hydrogenation at RT overnight until LC−
MS indicated that the reaction was complete. The filtrate after filtration
was concentrated and the residue was purified by preparative HPLC to
afford the title compound as a pale yellow solid (14 mg, 52% yield for
two steps). 1H NMR (300 MHz, CD3OD) δ ppm 8.38 (s, 1H), 8.29 (d,
J = 8.0 Hz, 1H), 7.70 (s, 1H), 7.50 (s, 1H), 7.44 (d, J = 8.0 Hz, 1H),
5.40−5.30 (m, 1H), 3.75−3.65 (m, 2H), 3.63−3.25 (m, 6H), 3.06 (s,
3H), 2.90 (q, J = 7.4 Hz, 2H), 1.88 (d, J = 7.1 Hz, 6H), 1.40 (t, J = 7.4
Hz, 3H).
3-Fluoro-5-isopropyl-11-oxo-6,11-dihydro-5H-indolo[2,3-
b]quinoline-8-carbonitrile (14c). Methyl 6-cyano-2-((3-
fluorophenyl)(isopropyl)amino)-1H-indole-3-carboxylate (265 mg,
0.75 mmol) was dissolved in diphenyl ether (10 mL) and the solution
was refluxed for 1 h. The mixture was cooled down to RT. Hexane (20
mL) was added and the precipitate was collected by filtration. The
precipitate was purified by silica gel chromatography with hexane/
EtOAc (2/1, v/v) to afford the title compound as a pale yellow solid
(190 mg, 79% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 12.24 (s,
1H), 8.45 (dd, J = 8.8, 7.1 Hz, 1H), 8.32 (dd, J = 7.9, 2.7 Hz, 1H), 8.05−
7.84 (m, 2H), 7.66−7.64 (m, 1H), 7.34−7.25 (m, 1H), 5.35−5.32 (m,
1H), 1.75 (d, J = 7.1 Hz, 6H).
3-(4-Morpholinopiperidin-1-yl)-5,6-dihydro-11H-indolo-
[2,3-b]quinolin-11-one (7a). 4-Morpholinopiperidine (262 mg, 1.56
mmol) and DIPEA (0.5 mL) were added to a solution of 3-fluoro-5H-
indolo[2,3-b]quinolin-11(6H)-one (130 mg, 0.52 mmol) in DMSO (2
mL) and the mixture was heated to 120−140 °C for 3 days. The
reaction mixture was cooled down to RT and purified by preparative
HPLC to afford the title compound as a pale yellow solid (60 mg, 29%
yield). 1H NMR (400 MHz, DMSO-d6) δ 12.40 (s, 1H), 11.84 (s, 1H),
10.02 (s, 1H), 8.18−8.11 (m, 2H), 7.46 (d, J = 7.7 Hz, 1H), 7.28−7.14
(m, 2H), 7.09 (dd, J = 9.1, 2.3 Hz, 1H), 7.02 (d, J = 2.3 Hz, 1H), 4.00−
3.85 (m, 8H), 3.75−3.68 (m, 2H), 3.55−3.40 (m, 3H), 3.18−3.08 (m,
2H), 2.91 (t, J = 12.5 Hz, 2H), 2.23−2.14 (m, 2H), 1.78−1.64 (m, 2H).
5-Methyl-3-(4-morpholinopiperidin-1-yl)-11-oxo-6,11-dihy-
dro-5H-indolo[2,3-b]quinoline-8-carbonitrile (7b). 4-Morpholi-
nopiperidine (262 mg, 1.56 mmol) and DIPEA (0.5 mL) were added to
a solution of 3-fluoro-5-methyl-11-oxo-6,11-dihydro-5H-indolo[2,3-
b]quinoline-8-carbonitrile (150 mg, 0.52 mmol) in DMSO (2 mL) and
the mixture was heated to 120−140 °C for 3 days. The reaction mixture
was cooled down to RT and purified by preparative HPLC to afford the
title compound as a pale yellow solid (70 mg, 31% yield). 1H NMR (300
MHz, CD3OD) δ ppm 8.30 (d, J = 7.0 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H),
7.58 (s, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.12 (d, J = 7.0 Hz,1H), 6.84 (s,
1H), 4.37−4.10 (m, 4H), 4.00−3.72 (m, 2H), 3.89 (s, 3H), 3.70−3.20
(m, 5H), 3.18−3.00 (m, 2H), 2.40−2.26 (m, 2H), 1.97−1.86 (m, 2H).
5-Isopropyl-3-(4-morpholinopiperidin-1-yl)-11-oxo-6,11-di-
hydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (7c). 4-Morpho-
linopiperidine (262 mg, 1.56 mmol) and DIPEA (0.5 mL) were added
to a solution of 3-fluoro-5-isopropyl-11-oxo-6,11-dihydro-5H-indolo-
[2,3-b]quinoline-8-carbonitrile (190 mg, 0.59 mmol) in DMSO (2 mL)
and the mixture was heated to 120−140 °C for 3 days. The reaction
mixture was cooled down to RT and purified by preparative HPLC to
afford the title compound as a pale yellow solid (75 mg, 27% yield). 1H
NMR (300 MHz, CD3OD) δ ppm 8.39 (d, J = 8.0 Hz, 1H), 8.38 (d, J =
8.9 Hz, 1H), 7.82 (s, 1H), 7.56 (dd, J = 8.0, 1.3 Hz, 1H), 7.30−7.15 (m,
2H), 5.45−5.32 (m, 1H), 4.38−4.10 (m, 4H), 3.90−3.72 (m, 2H),
3.70−3.20 (m, 5H), 3.18−3.00 (m, 2H), 2.40−2.30 (m, 2H), 1.97−
1.80 (m, 2H), 1.87 (d, J = 7.1 Hz, 6H).
5-Isopropyl-3-(4-methylpiperazin-1-yl)-11-oxo-6,11-dihy-
dro-5H-indolo[2,3-b]Quinoline-8-Carbonitrile (7d). 1-Methylpi-
perazine (0.5 mL) and DIPEA (0.5 mL) were added to a solution of 3-
fluoro-5-isopropyl-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-
carbonitrile (190 mg, 0.59 mmol) in DMSO (2 mL) and the mixture
was heated to 120−140 °C for 3 days. The reaction mixture was cooled
down to RT and purified by preparative HPLC to afford the title
5-Isopropyl-3-(4-methylpiperazin-1-yl)-11-oxo-2-propyl-
6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (8c). A
mixture of 2-bromo-5-isopropyl-3-(4-methylpiperazin-1-yl)-11-oxo-
6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (30 mg, 0.061
mmol), Na2CO3 (25 mg, 0.183 mmol), Pd(PPh3)2Cl2 (4.3 mg, 0.0061
mmol), and 4,4,5,5-tetramethyl-2-(prop-1-en-1-yl)-1,3,2-dioxaboro-
lane (16 mg, 0.091 mmol) in DME/H2O (4.8 mL, 5/1, v/v) was
stirred under N2 at 80 °C overnight. After the reaction was complete as
indicated by LC−MS, the mixture was purified by preparative HPLC to
afford 5-isopropyl-3-(4-methylpiperazin-1-yl)-11-oxo-2-(prop-1-en-1-
yl)-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile, which was
dissolved in THF (5 mL). Pd/C (10%, 20 mg) was added to the THF
solution. The mixture underwent hydrogenation at RT overnight until
1
compound as a pale yellow solid (48 mg, 20% yield). H NMR (300
MHz, CD3OD) δ ppm 8.42 (d, J = 8.1 Hz, 1H), 8.39 (d, J = 9.2 Hz, 1H),
7.81 (d, J = 1.3 Hz, 1H), 7.55 (dd, J = 8.1, 1.3 Hz, 1H), 7.28 (d, J = 1.8
Hz, 1H), 7.21 (dd, J = 9.2, 1.8 Hz, 1H), 5.50−5.30 (m, 1H), 4.36−4.10
(m, 2H), 3.80−3.60 (m, 2H), 3.55−3.20 (m, 4H), 3.04 (s, 3H), 1.87 (d,
J = 7.1 Hz, 6H).
Methyl 2-((4-Bromo-3-fluorophenyl)(isopropyl)amino)-6-
cyano-1H-indole-3-carboxylate (15). DABCO (62 mg, 0.55
mmol) was added to a solution of methyl 6-cyano-1H-indole-3-
carboxylate (200 mg, 1 mmol) in DCM (20 mL) and the mixture was
cooled down to 0 °C. NCS (147 mg, 1.1 mmol) was then added and the
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LC−MS indicated that the reaction was complete. The filtrate was
concentrated and the residue was purified by preparative HPLC to
afford the title compound as a pale yellow solid (8 mg, 29% yield for two
steps). 1H NMR (300 MHz, CD3OD) δ ppm 8.37 (s, 1H), 8.32 (d, J =
8.1 Hz, 1H), 7.74 (d, J = 1.3 Hz, 1H), 7.56−7.39(m, 2H), 5.39−5.32
(m, 1H), 3.71−3.68 (m, 2H), 3.55−3.37 (m, 4H), 3.29−3.22(m, 2H),
3.05 (s, 3H), 2.90−2.70 (m, 2H), 1.87 (d, J = 7.1 Hz, 6H), 1.85−
1.73(m, 2H), 1.04 (t, J = 7.3 Hz, 3H).
4-propoxyaniline (211 mg, 1 mmol) and trichloroacetic acid (41 mg,
0.25 mmol) in DCM (5 mL) was added dropwise and the reaction was
stirred for 2 h at RT. The reaction was quenched by adding H2O and the
mixture was extracted with DCM. Solvent was removed under vacuum
and the residue was purified by silica gel chromatography with hexane/
EtOAc (4/1, v/v) to afford the title compound as a colorless oil (235
mg, 57% yield). 1H NMR (400 MHz, CDCl3) δ ppm 8.05 (d, J = 8.3 Hz,
1H), 7.95 (s, 1H), 7.41 (dd, J = 8.3, 1.4 Hz, 1H), 7.37 (d, J = 1.4 Hz,
1H), 7.01 (t, J = 8.9 Hz, 1H), 6.97 (dd, J = 12.0, 2.6 Hz, 1H), 6.90−6.85
(m, 1H), 4.91−4.82 (m, 1H), 4.03 (t, J = 6.6 Hz, 2H), 3.89 (s, 3H),
1.90−1.88 (m, 2H), 1.26 (d, J = 6.6 Hz, 6H), 1.09 (t, J = 7.4 Hz, 3H).
Methyl 2-((4-Butoxy-3-fluorophenyl)(isopropyl)amino)-6-
cyano-1H-indole-3-carboxylate (17c). DABCO (62 mg, 0.55
mmol) was added to a solution of methyl 6-cyano-1H-indole-3-
carboxylate (200 mg, 1 mmol) in DCM (20 mL) and the mixture was
cooled down to 0 °C. NCS (147 mg, 1.1 mmol) was then added and the
reaction was stirred at 0 °C for 2 h. A solution of 4-butoxy-3-fluoro-N-
isopropylaniline (225 mg, 1 mmol) and trichloroacetic acid (41 mg,
0.25 mmol) in DCM (5 mL) was added dropwise and the reaction was
stirred for 2 h at RT. The reaction was quenched by adding H2O and the
mixture was extracted with DCM. Solvent was removed under vacuum
and the residue was purified by silica gel chromatography with hexane/
EtOAc (4/1, v/v) to afford the title compound as a colorless oil (245
mg, 58% yield). 1H NMR (400 MHz, CDCl3) δ ppm 8.05 (d, J = 8.3 Hz,
1H), 7.86 (s, 1H), 7.41 (dd, J = 8.3, 1.4 Hz, 1H), 7.37 (d, J = 1.4 Hz,
1H), 7.01 (t, J = 8.9 Hz, 1H), 6.97 (dd, J = 12.1, 2.6 Hz, 1H), 6.91−6.88
(m, 1H), 4.91−4.83 (m, 1H), 4.08 (t, J = 6.5 Hz, 2H), 3.90 (s, 3H),
1.87−1.83 (m, 2H), 1.58−1.48 (m, 2H), 1.27 (d, J = 6.7 Hz, 6H), 1.02
(t, J = 7.4 Hz, 3H).
Methyl 6-Cyano-2-((3-fluoro-4-isopropoxyphenyl)-
(isopropyl)amino)-1H-indole-3-carboxylate (17d). DABCO (62
mg, 0.55 mmol) was added to a solution of methyl 6-cyano-1H-indole-
3-carboxylate (200 mg, 1 mmol) in DCM (20 mL) and the mixture was
cooled down to 0 °C. NCS (147 mg, 1.1 mmol) was then added and the
reaction was stirred at 0 °C for 2 h. A solution of 3-fluoro-4-isopropoxy-
N-isopropylaniline (211 mg, 1 mmol) and trichloroacetic acid (41 mg,
0.25 mmol) in DCM (5 mL) was added dropwise and the reaction was
stirred for 2 h at RT. The reaction was quenched by adding H2O and the
mixture was extracted with DCM. Solvent was removed under vacuum
and the residue was purified by silica gel chromatography with hexane/
EtOAc (4/1, v/v) to afford the title compound as a colorless oil (220
mg, 54% yield). 1H NMR (300 MHz, CDCl3) δ ppm 8.05 (d, J = 8.3 Hz,
1H), 7.85 (s, 1H), 7.45−7.33 (m, 2H), 6.99 (t, J = 8.9 Hz, 1H), 6.91
(dd, J = 12.1, 2.7 Hz, 1H), 6.82−6.80 (m, 1H), 4.81 (m, 1H), 4.54 (m,
1H), 3.86 (s, 3H), 1.39 (d, J = 6.1 Hz, 6H), 1.31−1.21 (m, 6H).
3-Fluoro-5-isopropyl-2-methoxy-11-oxo-6,11-dihydro-5H-
indolo[2,3-b]quinoline-8-carbonitrile (18a). Methyl 6-cyano-2-
((3-fluoro-4-methoxyphenyl)(isopropyl)amino)-1H-indole-3-carbox-
ylate (250 mg, 0.66 mmol) was dissolved in diphenyl ether (10 mL) and
the solution was refluxed for 1 h. The mixture was cooled down to RT.
Hexane (20 mL) was added and the precipitate was collected by
filtration. The precipitate was purified by silica gel chromatography with
hexane/EtOAc (2/1, v/v) to afford the title compound as a pale yellow
solid (175 mg, 76% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.15 (s,
1H), 8.32 (d, J = 8.0 Hz, 1H), 8.04−7.94 (m, 2H), 7.90 (d, J = 1.3 Hz,
1H), 7.63 (dd, J = 8.0, 1.3 Hz, 1H), 5.35−5.27 (m, 1H), 3.98 (s, 3H),
1.73 (d, J = 6.9 Hz, 6H).
2-Isobutyl-5-isopropyl-3-(4-methylpiperazin-1-yl)-11-oxo-
6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (8d). A
mixture of 2-bromo-5-isopropyl-3-(4-methylpiperazin-1-yl)-11-oxo-
6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (30 mg, 0.061
mmol), Na2CO3 (25 mg, 0.183 mmol), Pd(PPh3)2Cl2 (4.3 mg, 0.0061
mmol), and 4,4,5,5-tetramethyl-2-(2-methylprop-1-en-1-yl)-1,3,2-di-
oxaborolane (16 mg, 0.091 mmol) in DME/H2O (4.8 mL, 5/1, v/v)
was stirred under nitrogen at 80 °C overnight. After the reaction was
complete as indicated by LC−MS, the mixture was purified by
preparative HPLC to afford 5-isopropyl-3-(4-methylpiperazin-1-yl)-2-
(2-methylprop-1-en-1-yl)-11-oxo-6,11-dihydro-5H-indolo[2,3-b]-
quinoline-8-carbonitrile, which was dissolved in THF (5 mL). Pd/C
(10%, 20 mg) was added to the THF solution. The mixture underwent
hydrogenation at RT overnight until LC−MS indicated that the
reaction was complete. The filtrate was concentrated and the residue
was purified by preparative HPLC to afford the title compound as a pale
1
yellow solid (10 mg, 35% yield for two steps). H NMR (300 MHz,
CD3OD) δ ppm 8.45 (s, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.62 (s, 1H),
7.39 (s, 1H), 7.18 (d, J = 8.0 Hz, 1H), 5.50−5.40 (m, 1H), 3.70−3.60
(m, 2H), 3.55−3.30 (m, 6H), 2.75 (d, J = 7.1 Hz, 2H), 2.53 (s, 3H),
2.25−2.15 (m, 1H), 1.90 (d, J = 7.1 Hz, 6H), 0.99 (d, J = 6.6 Hz, 6H).
2,5-Diisopropyl-3-(4-methylpiperazin-1-yl)-11-oxo-6,11-di-
hydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (8e). A mixture
of 2-bromo-5-isopropyl-3-(4-methylpiperazin-1-yl)-11-oxo-6,11-dihy-
dro-5H-indolo[2,3-b]quinoline-8-carbonitrile (30 mg, 0.061 mmol),
Na2CO3 (25 mg, 0.183 mmol), Pd(PPh3)2Cl2 (4.3 mg, 0.0061 mmol),
and 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborolane (16
mg, 0.091 mmol) in DME/H2O (4.8 mL, 5/1, v/v) was stirred under
nitrogen at 80 °C overnight. After the reaction was complete as
indicated by LC−MS, the mixture was purified by preparative HPLC to
afford 5-isopropyl-3-(4-methylpiperazin-1-yl)-11-oxo-2-(prop-1-en-2-
yl)-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile, which was
dissolved in THF (5 mL). Pd/C (10%; 20 mg) was added to the THF
solution. The resulting mixture underwent hydrogenation at RT
overnight until LC−MS indicated that the reaction was complete. The
filtrate after filtration was concentrated and the residue was purified by
preparative HPLC to afford the title compound as a pale yellow solid (9
1
mg, 32% yield for two steps). H NMR (300 MHz, CD3OD) δ ppm
8.53 (s, 1H), 8.42 (d, J = 8.0 Hz, 1H), 7.82 (m, 1H), 7.60−7.52 (m,
2H), 5.45−5.27 (m, 1H), 3.92−3.25 (m, 9H), 3.07 (s, 3H), 1.89 (d, J =
6.1 Hz, 6H), 1.38 (d, J = 6.8 Hz, 6H).
Methyl 6-Cyano-2-((3-fluoro-4-methoxyphenyl)(isopropyl)-
amino)-1H-indole-3-carboxylate (17a). DABCO (62 mg, 0.55
mmol) was added to a solution of methyl 6-cyano-1H-indole-3-
carboxylate (200 mg, 1 mmol) in DCM (20 mL) and the mixture was
cooled down to 0 °C. NCS (147 mg, 1.1 mmol) was then added and the
reaction was stirred at 0 °C for 2 h. A solution of 3-fluoro-N-isopropyl-
4-methoxyaniline (183 mg, 1 mmol) and trichloroacetic acid (41 mg,
0.25 mmol) in DCM (5 mL) was added dropwise and the reaction was
stirred for 2 h at RT. The reaction was quenched by adding H2O and the
mixture was extracted with DCM. The solvent was removed under
vacuum and the residue was purified by silica gel chromatography with
hexane/EtOAc (4/1, v/v) to afford the title compound as a colorless oil
(250 mg, 66% yield). 1H NMR (400 MHz, CDCl3) δ ppm 8.08−8.06
(m, 2H), 7.45−7.36 (m, 2H), 7.05−6.87 (m, 3H), 4.88−4.80 (m, 1H),
3.92 (s, 3H), 3.88 (s, 3H), 1.26 (d, J = 6.6 Hz, 6H).
Methyl 6-Cyano-2-((3-fluoro-4-propoxyphenyl)(isopropyl)-
amino)-1H-indole-3-carboxylate (17b). DABCO (62 mg, 0.55
mmol) was added to a solution of methyl 6-cyano-1H-indole-3-
carboxylate (200 mg, 1 mmol) in DCM (20 mL) and the mixture was
cooled down to 0 °C. NCS (147 mg, 1.1 mmol) was then added and the
reaction was stirred at 0 °C for 2 h. A solution of 3-fluoro-N-isopropyl-
3-Fluoro-5-isopropyl-11-oxo-2-propoxy-6,11-dihydro-5H-
indolo[2,3-b]quinoline-8-carbonitrile (18b). Methyl 6-cyano-2-
((3-fluoro-4-propoxyphenyl)(isopropyl)amino)-1H-indole-3-carboxy-
late (235 mg, 0.57 mmol) was dissolved in diphenyl ether (10 mL) and
the solution was refluxed for 1 h. The mixture was cooled down to RT.
Hexane (20 mL) was added and the precipitate was collected by
filtration to afford the title compound as a pale yellow solid (130 mg,
60% yield). This product was used directly in the next step without
further purification. MS: m/z = 378 [M + H].
2-Butoxy-3-fluoro-5-isopropyl-11-oxo-6,11-dihydro-5H-
indolo[2,3-b]quinoline-8-carbonitrile (18c). Methyl 2-((4-bu-
toxy-3-fluorophenyl)(isopropyl)amino)-6-cyano-1H-indole-3-carbox-
ylate (245 mg, 0.58 mmol) was dissolved in diphenyl ether (10 mL) and
M
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the solution was refluxed for 1 h. The mixture was cooled down to RT.
Hexane (20 mL) was added and the precipitate was collected by
filtration to afford the title compound as a pale yellow solid (165 mg,
73% yield). This product was used directly in the next step without
further purification. MS: m/z = 392 [M + H].
3-Fluoro-2-isopropoxy-5-isopropyl-11-oxo-6,11-dihydro-
5H-indolo[2,3-b]quinoline-8-carbonitrile (18d). Methyl 6-cyano-
2-((3-fluoro-4-isopropoxyphenyl)(isopropyl)amino)-1H-indole-3-car-
boxylate (220 mg, 0.54 mmol) was dissolved in diphenyl ether (10 mL)
and the solution was refluxed for 1 h. The mixture was cooled down to
RT. Hexane (20 mL) was added and the precipitate was collected by
filtration to afford the title compound as a pale yellow solid (130 mg,
70% yield). This product was used directly in the next step without
further purification. MS: m/z = 378 [M + H].
5-Isopropyl-2-methoxy-3-(4-methylpiperazin-1-yl)-11-oxo-
6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (8f). 1-
Methylpiperazine (0.5 mL) and DIPEA (0.5 mL) were added to a
solution of 3-fluoro-5-isopropyl-2-methoxy-11-oxo-6,11-dihydro-5H-
indolo[2,3-b]quinoline-8-carbonitrile (175 mg, 0.50 mmol) in DMSO
(2 mL) and the mixture was heated to 120−140 °C for 3 days. The
reaction mixture was cooled down to RT and purified by preparative
HPLC to afford the title compound as a pale yellow solid (56 mg, 26%
yield). 1H NMR (300 MHz, CD3OD) δ ppm 8.42 (d, J = 8.1 Hz, 1H),
8.00 (s, 1H), 7.82 (s, 1H), 7.57 (d, J = 8.1 Hz, 1H), 7.36 (s, 1H), 5.44−
5.35 (m, 1H), 4.05 (s, 3H), 4.02−3.92 (m, 2H), 3.70−3.63 (m, 2H),
3.50−3.18 (m, 4H), 3.05 (s, 3H), 1.88 (d, J = 7.1 Hz, 6H).
5-Isopropyl-3-(4-methylpiperazin-1-yl)-11-oxo-2-propoxy-
6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (8g). 1-
Methylpiperazine (0.5 mL) and DIPEA (0.5 mL) were added to a
solution of 3-fluoro-5-isopropyl-11-oxo-2-propoxy-6,11-dihydro-5H-
indolo[2,3-b]quinoline-8-carbonitrile (130 mg, 0.34 mmol) in DMSO
(2 mL) and the mixture was heated to 120−140 °C for 3 days. The
reaction mixture was cooled down to RT and purified by preparative
HPLC to afford the title compound as a pale yellow solid (28 mg, 18%
yield). 1H NMR (300 MHz, CD3OD) δ ppm 8.37 (d, J = 8.1 Hz, 1H),
7.93 (s, 1H), 7.78 (s, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.33 (s, 1H), 5.46−
5.25 (m, 1H), 4.18 (t, J = 6.4 Hz, 2H), 4.10−3.97 (m, 2H), 3.75−3.65
(m, 2H), 3.50−3.20 (m, 4H), 3.06 (s, 3H), 2.03−1.94 (m, 2H), 1.88 (d,
J = 7.1 Hz, 6H), 1.17 (t, J = 7.4 Hz, 3H).
2-Butoxy-5-isopropyl-3-(4-methylpiperazin-1-yl)-11-oxo-
6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (8h). 1-
Methylpiperazine (0.5 mL) and DIPEA (0.5 mL) were added to a
solution of 2-butoxy-3-fluoro-5-isopropyl-11-oxo-6,11-dihydro-5H-
indolo[2,3-b]quinoline-8-carbonitrile (165 mg, 0.42 mmol) in
DMSO (2 mL) and the mixture was heated to 120−140 °C for 3
days. The reaction mixture was cooled down to RT and purified by
preparative HPLC to afford the title compound as a pale yellow solid
(36 mg, 18% yield). 1H NMR (300 MHz, CD3OD) δ ppm 8.39 (d, J =
8.1 Hz, 1H), 7.95 (s, 1H), 7.79 (d, J = 1.4 Hz, 1H), 7.55 (dd, J = 8.1, 1.4
Hz, 1H), 7.32 (s, 1H), 5.40−5.32 (m, 1H), 4.21 (t, J = 6.4 Hz, 2H),
3.99−3.95 (m, 2H), 3.71−3.69 (m, 2H), 3.43−3.40 (m, 2H), 3.28−
3.12 (m, 2H), 3.04 (s, 3H), 1.96−1.89 (m, 2H), 1.87 (d, J = 7.1 Hz,
6H), 1.68−1.54 (m, 2H), 1.06 (t, J = 7.4 Hz, 3H).
2-Isopropoxy-5-isopropyl-3-(4-methylpiperazin-1-yl)-11-
oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile
(8i). 1-Methylpiperazine (0.5 mL) and DIPEA (0.5 mL) were added to
a solution of 3-fluoro-2-isopropoxy-5-isopropyl-11-oxo-6,11-dihydro-
5H-indolo[2,3-b]quinoline-8-carbonitrile (130 mg, 0.34 mmol) in
DMSO (2 mL) and the mixture was heated to 120−140 °C for 3 days.
The reaction mixture was cooled down to RT and purified by
preparative HPLC to afford the title compound as a pale yellow solid
(35 mg, 22% yield). 1H NMR (300 MHz, CD3OD) δ ppm 8.43 (d, J =
8.6 Hz, 1H), 8.01 (s, 1H), 7.84 (s, 1H), 7.59 (d, J = 8.6 Hz, 1H), 7.36 (s,
1H), 5.42−5.35 (m, 1H), 4.90−4.80 (m, 1H), 4.02−3.95 (m, 2H),
3.70−3.63 (m, 2H), 3.50−3.10 (m, 4H), 3.05 (s, 3H), 1.88 (d, J = 7.1
Hz, 6H), 1.47 (d, J = 6.0 Hz, 6H).
°C. NCS (147 mg, 1.1 mmol) was then added and the reaction was
stirred at 0 °C for 2 h. A solution of 2,3-difluoro-N-isopropylaniline
(171 mg, 1 mmol) and trichloroacetic acid (41 mg, 0.25 mmol) in
DCM (5 mL) was added dropwise and the reaction was stirred for 2 h at
RT. The reaction was quenched by adding H2O and the mixture was
extracted with DCM. Solvent was removed under vacuum and the
residue was purified by silica gel chromatography with hexane/EtOAc
(4/1, v/v) to afford the title compound as a colorless oil (200 mg, 54%
yield). 1H NMR (400 MHz, CDCl3) δ ppm 8.18 (s, 1H), 8.09 (d, J = 8.4
Hz, 1H), 7.51 (d, J = 1.4 Hz, 1H), 7.45 (dd, J = 8.4, 1.4 Hz, 1H), 7.17−
7.07 (m, 3H), 4.77−4.61 (m, 1H), 3.82 (s, 3H), 1.34 (d, J = 6.6 Hz,
6H).
Methyl 6-Cyano-2-(isopropyl(2,3,4-trifluorophenyl)amino)-
1H-indole-3-carboxylate (19b). DABCO (62 mg, 0.55 mmol) was
added to a solution of methyl 6-cyano-1H-indole-3-carboxylate (200
mg, 1 mmol) in DCM (20 mL) and the mixture was cooled down to 0
°C. NCS (147 mg, 1.1 mmol) was then added and the reaction was
stirred at 0 °C for 2 h. A solution of 2,3,4-trifluoro-N-isopropylaniline
(189 mg, 1 mmol) and trichloroacetic acid (41 mg, 0.25 mmol) in
DCM (5 mL) were added dropwise and the reaction was stirred for 2 h
at RT. The reaction was quenched by adding H2O and the mixture was
extracted with DCM. Solvent was removed under vacuum and the
residue was purified by silica gel chromatography with hexane/EtOAc
(4/1, v/v) to afford the title compound as a colorless oil (210 mg, 54%
yield). 1H NMR (400 MHz, CDCl3) δ ppm 8.36 (s, 1H), 8.07 (d, J = 8.3
Hz, 1H), 7.53 (d, J = 1.4 Hz, 1H), 7.44 (dd, J = 8.3, 1.4 Hz, 1H), 7.15−
7.10 (m, 1H), 7.05−7.00 (m, 1H), 4.65−4.60 (m, 1H), 3.82 (s, 3H),
1.33 (dd, J = 6.5, 0.9 Hz, 6H).
Methyl 6-Cyano-2-((2,3-difluoro-4-methoxyphenyl)-
(isopropyl)amino)-1H-indole-3-carboxylate (19c). DABCO (62
mg, 0.55 mmol) was added to a solution of methyl 6-cyano-1H-indole-
3-carboxylate (200 mg, 1 mmol) in DCM (20 mL) and the mixture was
cooled down to 0 °C. NCS (147 mg, 1.1 mmol) was then added and the
reaction was stirred at 0 °C for 2 h. A solution of 2,3-difluoro-N-
isopropyl-4-methoxyaniline (201 mg, 1 mmol) and trichloroacetic acid
(41 mg, 0.25 mmol) in DCM (5 mL) were added dropwise and the
reaction was stirred for 2 h at RT. The reaction was quenched by adding
H2O and the mixture was extracted with DCM. The solvent was
removed under vacuum and the residue was purified by silica gel
chromatography with hexane/EtOAc (4/1, v/v) to afford the title
compound as a colorless oil (225 mg, 56% yield). 1H NMR (400 MHz,
CDCl3) δ ppm 8.25 (s, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.45−7.36 (m,
2H), 7.13−7.06 (m, 1H), 6.846.80 (m, 1H), 4.85−4.79 (m, 1H), 3.95
(s, 3H), 3.85 (s, 3H), 1.27 (dd, J = 6.5, 1.1 Hz, 6H).
3,4-Difluoro-5-isopropyl-11-oxo-6,11-dihydro-5H-indolo-
[2,3-b]quinoline-8-carbonitrile (20a). Methyl 6-cyano-2-((2,3-
difluorophenyl)(isopropyl)amino)-1H-indole-3-carboxylate (200 mg,
0.54 mmol) was dissolved in diphenyl ether (10 mL) and the solution
was refluxed for 1 h. The mixture was cooled down to RT. Hexane (20
mL) was added and the precipitate was collected by filtration to afford
the title compound as a pale yellow solid (112 mg, 61% yield). This
product was used directly in the next step without further purification.
MS: m/z = 338 [M + H].
2,3,4-Trifluoro-5-isopropyl-11-oxo-6,11-dihydro-5H-indolo-
[2,3-b]quinoline-8-carbonitrile (20b). Methyl 6-cyano-2-
(isopropyl(2,3,4-trifluorophenyl)amino)-1H-indole-3-carboxylate
(210 mg, 0.54 mmol) was dissolved in diphenyl ether (10 mL) and the
solution was refluxed for 1 h. The mixture was cooled down to RT.
Hexane (20 mL) was added and the precipitate was collected by
filtration to afford the title compound as a pale yellow solid (100 mg,
52% yield). This product was used directly in the next step without
further purification. MS: m/z = 356 [M + H].
3,4-Difluoro-5-isopropyl-2-methoxy-11-oxo-6,11-dihydro-
5H-indolo[2,3-b]quinoline-8-carbonitrile (20c). Methyl 6-cyano-
2-((2,3-difluoro-4-methoxyphenyl)(isopropyl)amino)-1H-indole-3-
carboxylate (225 mg, 0.56 mmol) was dissolved in diphenyl ether (10
mL) and the solution was refluxed for 1 h. The mixture was cooled
down to RT. Hexane (20 mL) was added and the precipitate was
collected by filtration to afford the title compound as a pale yellow solid
Methyl 6-Cyano-2-((2,3-difluorophenyl)(isopropyl)amino)-
1H-indole-3-carboxylate (19a). DABCO (62 mg, 0.55 mmol) was
added to a solution of methyl 6-cyano-1H-indole-3-carboxylate (200
mg, 1 mmol) in DCM (20 mL) and the mixture was cooled down to 0
N
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(135 mg, 65% yield). This product was used directly in the next step
without further purification. MS: m/z = 368 [M + H].
reaction was stirred at 0 °C for 2 h. A solution of N-(sec-butyl)-3-fluoro-
4-methoxyaniline (197 mg, 1 mmol) and trichloroacetic acid (41 mg,
0.25 mmol) in DCM (5 mL) was added dropwise and the reaction was
stirred for 2 h at RT. The reaction was quenched by adding H2O and the
mixture was extracted with DCM. The solvent was removed under
vacuum and the residue was purified by silica gel chromatography with
hexane/EtOAc (4/1, v/v) to afford the title compound as a colorless oil
(210 mg, 53% yield). 1H NMR (400 MHz, CDCl3) δ ppm 8.05 (d, J =
8.3 Hz, 1H), 7.93 (s, 1H), 7.41 (dd, J = 8.3, 1.4 Hz, 1H), 7.37 (d, J = 1.4
Hz, 1H), 7.07−6.85 (m, 3H), 4.60−4.54 (m, 1H), 3.93 (s, 3H), 3.88 (s,
3H), 1.79−1.75 (m, 1H), 1.54−1.51 (m, 1H), 1.26 (d, J = 6.7 Hz, 3H),
0.98 (t, J = 7.4 Hz, 3H).
Methyl 6-Cyano-2-((3-fluoro-4-methoxyphenyl)(pentan-2-
yl)amino)-1H-indole-3-carboxylate (23d). DABCO (62 mg, 0.55
mmol) was added to a solution of methyl 6-cyano-1H-indole-3-
carboxylate (200 mg, 1 mmol) in DCM (20 mL) and the mixture was
cooled down to 0 °C. NCS (147 mg, 1.1 mmol) was then added and the
reaction was stirred at 0 °C for 2 h. A solution of 3-fluoro-4-methoxy-N-
(pentan-2-yl)aniline (211 mg, 1 mmol) and trichloroacetic acid (41 mg,
0.25 mmol) in DCM (5 mL) was added dropwise and the reaction was
stirred for 2 h at RT. The reaction was quenched by adding H2O and the
mixture was extracted with DCM. The solvent was removed under
vacuum and the residue was purified by silica gel chromatography with
hexane/EtOAc (4/1, v/v) to afford the title compound as a colorless oil
(196 mg, 48% yield). 1H NMR (400 MHz, CDCl3) δ ppm 8.05 (d, J =
8.3 Hz, 1H), 7.77 (br s, 1H), 7.41 (dd, J = 8.3, 1.5 Hz, 1H), 7.36 (d, J =
1.5 Hz, 1H), 7.07−6.90 (m, 3H), 4.78−4.61 (m, 1H), 3.95 (s, 3H), 3.89
(s, 3H), 1.72−1.65 (m, 1H), 1.49−1.41 (m, 3H), 1.26 (d, J = 6.8 Hz,
3H), 0.92 (t, J = 7.2 Hz, 3H).
M e t h y l 6 - C y a n o - 2 - ( c y c l o p r o p y l ( 3 - fl u o r o - 4 -
methoxyphenyl)amino)-1H-indole-3-carboxylate (23e).
DABCO (62 mg, 0.55 mmol) was added to a solution of methyl 6-
cyano-1H-indole-3-carboxylate (200 mg, 1 mmol) in DCM (20 mL)
and the mixture was cooled down to 0 °C. N-Chlorosuccinimide (147
mg, 1.1 mmol) was then added and the reaction was stirred at 0 °C for 2
h. A solution of N-cyclopropyl-3-fluoro-4-methoxyaniline (181 mg, 1
mmol) and trichloroacetic acid (41 mg, 0.25 mmol) in DCM (5 mL)
was added dropwise and the reaction was stirred for 2 h at room
temperature. The reaction was quenched by adding water and the
mixture was extracted with DCM. The solvent was removed under
vacuum and the residue was purified by silica gel chromatography
(Hex:EA = 4:1) to afford the title compound (180 mg, 47% yield). 1H
NMR (400 MHz, CDCl3) δ 8.94 (s, 1H), 8.17 (d, J = 8.4 Hz, 1H), 7.57
(d, J = 1.4 Hz, 1H), 7.48 (dd, J = 8.4, 1.4 Hz, 1H), 6.94−6.82 (m, 2H),
6.77−6.69 (m, 1H), 3.85 (s, 3H), 3.74 (s, 3H), 3.17−2.99 (m, 1H),
1.00−0.93 (m, 2H), 0.81−0.74 (m, 2H).
Methyl 6-Cyano-2-(cyclobutyl(3-fluoro-4-methoxyphenyl)-
amino)-1H-indole-3-carboxylate (23f). DABCO (62 mg, 0.55
mmol) was added to a solution of methyl 6-cyano-1H-indole-3-
carboxylate (200 mg, 1 mmol) in DCM (20 mL) and the mixture was
cooled down to 0 °C. NCS (147 mg, 1.1 mmol) was then added and the
reaction was stirred at 0 °C for 2 h. A solution of N-cyclobutyl-3-fluoro-
4-methoxyaniline (195 mg, 1 mmol) and trichloroacetic acid (41 mg,
0.25 mmol) in DCM (5 mL) was added dropwise and the reaction was
stirred for 2 h at RT. The reaction was quenched by adding H2O and the
mixture was extracted with DCM. The solvent was removed under
vacuum and the residue was purified by silica gel chromatography with
hexane/EtOAc (4/1, v/v) to afford the title compound as a colorless oil
(245 mg, 62% yield). 1H NMR (400 MHz, CDCl3) δ ppm 8.62 (s, 1H),
8.17 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 1.5 Hz, 1H), 7.47 (dd, J = 8.4, 1.5
Hz, 1H), 6.89 (t, J = 9.1 Hz, 1H), 6.64 (dd, J = 12.9, 2.8 Hz, 1H), 6.60−
6.51 (m, 1H), 4.55−4.43 (m, 1H), 3.86 (s, 3H), 3.81 (s, 3H), 2.40−
2.28 (m, 2H), 2.00−1.90 (m, 2H), 1.77−1.63 (m, 2H).
4-Fluoro-5-isopropyl-3-(4-methylpiperazin-1-yl)-11-oxo-
6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (9a). 1-
Methylpiperazine (0.5 mL) and DIPEA (0.5 mL) were added to a
solution of 3,4-difluoro-5-isopropyl-11-oxo-6,11-dihydro-5H-indolo-
[2,3-b]quinoline-8-carbonitrile (112 mg, 0.33 mmol) in DMSO (2
mL) and the mixture was heated to 120−140 °C for 3 days. The
reaction mixture was cooled down to RT and purified by preparative
HPLC to afford the title compound as a pale yellow solid (22 mg, 16%
yield). MS: m/z = 418 [M + H].
2,4-Difluoro-5-isopropyl-3-(4-methylpiperazin-1-yl)-11-
oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile
(9b). 1-Methylpiperazine (0.5 mL) and DIPEA (0.5 mL) were added to
a solution of 2,3,4-trifluoro-5-isopropyl-11-oxo-6,11-dihydro-5H-
indolo[2,3-b]quinoline-8-carbonitrile (100 mg, 0.28 mmol) in
DMSO (2 mL) and the mixture was heated to 120−140 °C for 3
days. The reaction was cooled down to RT and purified by preparative
HPLC to afford the title compound as a pale yellow solid (25 mg, 20%
1
yield). MS: m/z = 436 [M + H]. H NMR (400 MHz, DMSO-d6) δ
12.16 (s, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 1.3 Hz, 1H), 7.82
(dd, J = 11.8, 1.6 Hz, 1H), 7.61 (dd, J = 8.0, 1.3 Hz, 1H), 5.02 (hept, J =
7.0 Hz, 1H), 3.61−3.52 (m, 4H), 3.44−3.24 (m, 4H), 2.91 (s, 3H), 1.66
(dd, J = 7.0, 1.9 Hz, 6H).
4-Fluoro-5-isopropyl-2-methoxy-3-(4-methylpiperazin-1-
yl)-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carboni-
trile (9c). 1-Methylpiperazine (0.5 mL) and DIPEA (0.5 mL) were
added to a solution of 3,4-difluoro-5-isopropyl-2-methoxy-11-oxo-6,11-
dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (135 mg, 0.37
mmol) in DMSO (2 mL) and the mixture was heated to 120−140
°C for 3 days. The reaction mixture was cooled down to RT and purified
by preparative HPLC to afford the title compound as a pale yellow solid
1
(24 mg, 15% yield). H NMR (300 MHz, CD3OD + CDCl3) δ ppm
8.33 (d, J = 8.0 Hz, 1H), 7.77 (s, 1H), 7.72 (s, 1H), 7.48 (d, J = 8.0 Hz,
1H), 5.20−5.00 (m, 1H), 4.04 (s, 3H), 3.80−3.50 (m, 6H), 3.40−3.25
(m, 2H), 3.00 (s, 3H), 1.74 (dd, J = 5.6, 1.8 Hz, 6H).
Methyl 6-Cyano-2-(ethyl(3-fluoro-4-methoxyphenyl)-
amino)-1H-indole-3-carboxylate (23a). DABCO (62 mg, 0.55
mmol) was added to a solution of methyl 6-cyano-1H-indole-3-
carboxylate (200 mg, 1 mmol) in DCM (20 mL) and the mixture was
cooled down to 0 °C. NCS (147 mg, 1.1 mmol) was then added and the
reaction was stirred at 0 °C for 2 h. A solution of N-ethyl-3-fluoro-4-
methoxyaniline (169 mg, 1 mmol) and trichloroacetic acid (41 mg, 0.25
mmol) in DCM (5 mL) was added dropwise and the reaction was
stirred for 2 h at RT. The reaction was quenched by adding H2O and the
mixture was extracted with DCM. The solvent was removed under
vacuum and the residue was purified by silica gel chromatography with
hexane/EtOAc (4/1, v/v) to afford the title compound as a colorless oil
(160 mg, 44% yield). 1H NMR (400 MHz, CDCl3) δ ppm 8.31 (s, 1H),
8.09 (d, J = 8.7 Hz, 1H), 7.47−7.39 (m, 2H), 7.02−6.84 (m, 3H), 4.01
(q, J = 7.1 Hz, 2H), 3.89 (s, 3H), 3.87 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H).
Methyl 6-Cyano-2-((3-fluoro-4-methoxyphenyl)(propyl)-
amino)-1H-indole-3-carboxylate (23b). DABCO (62 mg, 0.55
mmol) was added to a solution of methyl 6-cyano-1H-indole-3-
carboxylate (200 mg, 1 mmol) in DCM (20 mL) and the mixture was
cooled down to 0 °C. NCS (147 mg, 1.1 mmol) was then added and the
reaction was stirred at 0 °C for 2 h. A solution of N-propyl-3-fluoro-4-
methoxyaniline (183 mg, 1 mmol) and trichloroacetic acid (41 mg, 0.25
mmol) in DCM (5 mL) was added dropwise and the reaction was
stirred for 2 h at RT. The reaction was quenched by adding H2O and the
mixture was extracted with DCM. The solvent was removed under
vacuum and the residue was purified by silica gel chromatography with
hexane/EtOAc (4/1, v/v) to afford the title compound as a colorless oil
(170 mg, 45% yield). 1H NMR (400 MHz, CDCl3) δ ppm δ 8.28 (s,
1H), 8.10 (d, J = 8.7 Hz, 1H), 7.47−7.41 (m, 2H), 7.02−6.79 (m, 3H),
4.00−3.79 (m, 8H), 1.80−1.65 (m, 2H), 0.96 (q, J = 7.3 Hz, 3H).
Methyl 2-(sec-Butyl(3-fluoro-4-methoxyphenyl)amino)-6-
cyano-1H-indole-3-carboxylate (23c). DABCO (62 mg, 0.55
mmol) was added to a solution of methyl 6-cyano-1H-indole-3-
carboxylate (200 mg, 1 mmol) in DCM (20 mL) and the mixture was
cooled down to 0 °C. NCS (147 mg, 1.1 mmol) was then added and the
Methyl 6-Cyano-2-(cyclopentyl(3-fluoro-4-methoxyphenyl)-
amino)-1H-indole-3-carboxylate (23g). DABCO (62 mg, 0.55
mmol) was added to a solution of methyl 6-cyano-1H-indole-3-
carboxylate (200 mg, 1 mmol) in DCM (20 mL) and the mixture was
cooled down to 0 °C. NCS (147 mg, 1.1 mmol) was then added and the
reaction was stirred at 0 °C for 2 h. A solution of N-cyclopentyl-3-
fluoro-4-methoxyaniline (209 mg, 1 mmol) and trichloroacetic acid (41
O
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mg, 0.25 mmol) in DCM (5 mL) was added dropwise and the reaction
was stirred for 2 h at RT. The reaction was quenched by adding H2O
and the mixture was extracted with DCM. Solvent was removed under
vacuum and the residue was purified by silica gel chromatography with
hexane/EtOAc (4/1, v/v) to afford the title compound as a colorless oil
(280 mg, 69% yield).1H NMR (400 MHz, CDCl3) δ ppm 8.55 (s, 1H),
8.13 (d, J = 8.3 Hz, 1H), 7.50 (d, J = 1.4 Hz, 1H), 7.44 (dd, J = 8.3, 1.4
Hz, 1H), 6.92 (t, J = 9.1 Hz, 1H), 6.79 (dd, J = 12.7, 2.7 Hz, 1H), 6.75−
6.71 (m, 1H), 4.61−4.52 (m, 1H), 3.87 (s, 3H), 3.83 (s, 3H), 2.09−
1.98 (m, 2H), 1.66−1.62 (m, 4H), 1.59−1.47 (m, 2H).
Methyl 6-Cyano-2-(cyclohexyl(3-fluoro-4-methoxyphenyl)-
amino)-1H-indole-3-carboxylate (23h). DABCO (62 mg, 0.55
mmol) was added to a solution of methyl 6-cyano-1H-indole-3-
carboxylate (200 mg, 1 mmol) in DCM (20 mL) and the mixture was
cooled down to 0 °C. NCS (147 mg, 1.1 mmol) was then added and the
reaction was stirred at 0 °C for 2 h. A solution of N-cyclohexyl-3-fluoro-
4-methoxyaniline (223 mg, 1 mmol) and trichloroacetic acid (41 mg,
0.25 mmol) in DCM (5 mL) was added dropwise and the reaction was
stirred for 2 h at RT. The reaction was quenched by adding H2O and the
mixture was extracted with DCM. The solvent was removed under
vacuum and the residue was purified by silica gel chromatography with
hexane/EtOAc (4/1, v/v) to afford the title compound as a colorless oil
(240 mg, 57% yield). 1H NMR (400 MHz, CDCl3) δ ppm 8.06 (d, J =
8.3 Hz, 1H), 8.02 (s, 1H), 7.45−7.37 (m, 2H), 7.04−6.82 (m, 3H),
4.37−4.26 (m, 1H), 3.92 (s, 3H), 3.88 (s, 3H), 2.10−2.06 (m, 2H),
1.85−1.62 (m, 2H), 1.67−1.63 (m, 2H), 1.47−1.35 (m, 2H), 1.30−
1.27 (m, 2H).
5-Ethyl-3-fluoro-2-methoxy-11-oxo-6,11-dihydro-5H-
indolo[2,3-b]quinoline-8-carbonitrile (24a). Methyl 6-cyano-2-
(ethyl(3-fluoro-4-methoxyphenyl)amino)-1H-indole-3-carboxylate
(160 mg, 0.44 mmol) was dissolved in diphenyl ether (10 mL) and the
solution was refluxed for 1 h and then cooled down to RT. Hexane (20
mL) was added and the precipitate was collected by filtration. The
precipitate was purified by silica gel chromatography with hexane/
EtOAc (2/1, v/v) to afford the title compound as a colorless oil (98 mg,
67% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 12.58 (s, 1H), 8.29
(d, J = 8.1 Hz, 1H), 7.98 (d, J = 9.7 Hz, 1H), 7.94−7.84 (m, 2H), 7.62
(dd, J = 8.1, 1.5 Hz, 1H), 4.52 (d, J = 7.4 Hz, 2H), 3.97 (s, 3H), 1.38 (t, J
= 7.4 Hz, 3H).
3-Fluoro-2-methoxy-11-oxo-5-propyl-6,11-dihydro-5H-
indolo[2,3-b]quinoline-8-carbonitrile (24b). Methyl 6-cyano-2-
(propyl(3-fluoro-4-methoxyphenyl)amino)-1H-indole-3-carboxylate
(150 mg, 0.39 mmol) was dissolved in diphenyl ether (10 mL) and the
solution was refluxed for 1 h and then cooled down to RT. Hexane (20
mL) was added and the precipitate was collected by filtration. The
precipitate was purified by silica gel chromatography with hexane/
EtOAc (2/1, v/v) to afford the title compound as a colorless oil (95 mg,
69% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 12.50 (s, 1H), 8.30
(d, J = 8.0 Hz, 1H), 7.98 (d, J = 9.7 Hz, 1H), 7.93−7.82 (m, 2H), 7.62
(dd, J = 8.0, 1.4 Hz, 1H), 4.41 (t, J = 7.8 Hz, 2H), 3.98 (s, 3H), 1.80 (q, J
= 7.8 Hz, 2H), 1.02 (t, J = 7.8 Hz, 3H).
3H), 4.00−3.82 (m, 2H), 3.75−3.60 (m, 2H), 3.50−3.10 (m, 4H), 3.05
(s, 3H), 2.50−2.10 (m, 2H), 1.95−1.75 (m, 3H), 1.50−1.15 (m, 2H),
0.91 (t, J = 7.3 Hz, 3H).
5-Cyclopropyl-3-fluoro-2-methoxy-11-oxo-6,11-dihydro-
5H-indolo[2,3-b]quinoline-8-carbonitrile (24e). Methyl 6-cyano-
2-(cyclopropyl(3-fluoro-4-methoxyphenyl)amino)-1H-indole-3-car-
boxylate (180 mg, 0.47 mmol) was dissolved in diphenyl ether (10 mL)
and the solution was refluxed for 1 h. The mixture was cooled down to
RT. Hexane (20 mL) was added and the precipitate was collected by
filtration. The precipitate was purified by silica gel chromatography
(Hex:EA = 2:1) to afford the title compound (102 mg, 62% yield). MS
m/z = 348 [M + H].
5-Cyclobutyl-3-fluoro-2-methoxy-11-oxo-6,11-dihydro-5H-
indolo[2,3-b]quinoline-8-carbonitrile (24f). Methyl 6-cyano-2-
(cyclobutyl(3-fluoro-4-methoxyphenyl)amino)-1H-indole-3-carboxy-
late (245 mg, 0.66 mmol) was dissolved in diphenyl ether (10 mL) and
the solution was refluxed for 1 h. The mixture was cooled down to RT.
Hexane (20 mL) was added and the precipitate was collected by
filtration. The precipitate (155 mg, 65% yield) was used directly in the
next step without further purification.
5-Cyclopentyl-3-fluoro-2-methoxy-11-oxo-6,11-dihydro-
5H-indolo[2,3-b]quinoline-8-carbonitrile (24g). Methyl 6-cyano-
2-(cyclopentyl(3-fluoro-4-methoxyphenyl)amino)-1H-indole-3-car-
boxylate (280 mg, 0.69 mmol) was dissolved in diphenyl ether (10 mL)
and the solution was refluxed for 1 h. The mixture was cooled down to
RT. Hexane (20 mL) was added and the precipitate was collected by
filtration. The precipitate (130 mg, 50% yield) was used directly in the
next step without further purification.
5-Cyclohexyl-3-fluoro-2-methoxy-11-oxo-6,11-dihydro-5H-
indolo[2,3-b]quinoline-8-carbonitrile (24h). Methyl 6-cyano-2-
(cyclohexyl(3-fluoro-4-methoxyphenyl)amino)-1H-indole-3-carboxy-
late (240 mg, 0.57 mmol) was dissolved in diphenyl ether (10 mL) and
the solution was refluxed for 1 h. The mixture was cooled down to RT.
Hexane (20 mL) was added and the precipitate was collected by
filtration. The precipitate (160 mg, 72% yield) was used directly in the
next step without further purification.
5-Ethyl-2-methoxy-3-(4-methylpiperazin-1-yl)-11-oxo-6,11-
dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (10a). 1-
Methylpiperazine (0.5 mL) and DIPEA (0.5 mL) were added to a
solution of 5-ethyl-3-fluoro-2-methoxy-11-oxo-6,11-dihydro-5H-
indolo[2,3-b]quinoline-8-carbonitrile (98 mg, 0.29 mmol) in DMSO
(2 mL) and the mixture was heated to 120−140 °C for 3 days. The
reaction mixture was cooled down to RT and purified by preparative
HPLC to afford the title compound as a pale yellow solid (25 mg, 21%
yield). 1H NMR (300 MHz, CD3OD) δ ppm 8.31 (d, J = 7.6 Hz, 1H),
7.91 (s, 1H), 7.59 (d, J = 0.7 Hz, 1H), 7.50 (dd, J = 7.6, 0.7 Hz, 1H),
7.06 (s, 1H), 4.50 (q, J = 7.3 Hz, 2H), 4.06 (s, 3H), 4.04−3.86 (m, 2H),
3.75−3.63 (m, 2H), 3.50−3.37 (m, 2H), 3.26−3.17 (m, 2H), 3.05 (s,
3H), 1.53 (t, J = 7.3 Hz, 3H).
5-Propyl-2-methoxy-3-(4-methylpiperazin-1-yl)-11-oxo-
6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (10b).
1-Methylpiperazine (0.5 mL) and DIPEA (0.5 mL) were added to a
solution of 5-propyl-3-fluoro-2-methoxy-11-oxo-6,11-dihydro-5H-
indolo[2,3-b]quinoline-8-carbonitrile (90 mg, 0.26 mmol) in DMSO
(2 mL) and the mixture was heated to 120−140 °C for 3 days. The
reaction mixture was cooled down to RT and purified by preparative
HPLC to afford the title compound as a pale yellow solid (22 mg, 20%
yield). 1H NMR (400 MHz, DMSO-d6) δ 12.50 (s, 1H), 9.97 (s, 1H),
8.29 (d, J = 8.1 Hz, 1H), 7.92−7.74 (m, 2H), 7.60 (dd, J = 8.1, 1.5 Hz,
1H), 7.09 (s, 1H), 4.49 (t, J = 7.6 Hz, 2H), 3.94 (s, 3H), 3.84 (d, J = 11.8
Hz, 2H), 3.59 (d, J = 11.8 Hz, 2H), 3.33−3.25 (m, 2H), 3.10 (m, 2H),
2.92 (s, 3H), 1.86 (m, 2H), 1.01 (t, J = 7.5 Hz, 3H).
5-(sec-Butyl)-3-fluoro-2-methoxy-11-oxo-6,11-dihydro-5H-
indolo[2,3-b]quinoline-8-carbonitrile (24c). Methyl 2-(sec-butyl-
(3-fluoro-4-methoxyphenyl)amino)-6-cyano-1H-indole-3-carboxylate
(210 mg, 0.53 mmol) was dissolved in diphenyl ether (10 mL) and the
solution was refluxed for 1 h. The mixture was cooled down to RT.
Hexane (20 mL) was added and the precipitate was collected by
filtration. The precipitate (150 mg, 78% yield) was used directly in the
next step without further purification.
3-Fluoro-2-methoxy-11-oxo-5-(pentan-2-yl)-6,11-dihydro-
5H-indolo[2,3-b]quinoline-8-carbonitrile (24d). Methyl 6-cyano-
2-((3-fluoro-4-methoxyphenyl)(pentan-2-yl)amino)-1H-indole-3-car-
boxylate (196 mg, 0.48 mmol) was dissolved in diphenyl ether (10 mL)
and the solution was refluxed for 1 h. The mixture was cooled down to
RT. Hexane (20 mL) was added and the precipitate was collected by
filtration. The precipitate was purified by silica gel chromatography with
hexane/EtOAc (2/1, v/v) to afford the title compound as a pale yellow
5-(sec-Butyl)-2-methoxy-3-(4-methylpiperazin-1-yl)-11-oxo-
6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (10c).
1-Methylpiperazine (0.5 mL) and DIPEA (0.5 mL) were added to a
solution of 5-(sec-butyl)-3-fluoro-2-methoxy-11-oxo-6,11-dihydro-5H-
indolo[2,3-b]quinoline-8-carbonitrile (150 mg, 0.41 mmol) in DMSO
(2 mL) and the mixture was heated to 120−140 °C for 3 days. The
reaction mixture was cooled down to RT and purified by preparative
HPLC to afford the title compound as a pale yellow solid (35 mg, 19%
1
solid (148 mg, 82% yield). MS m/z = 378 [M + H]. H NMR (300
MHz, CD3OD) δ ppm 8.43 (d, J = 8.0 Hz, 1H), 8.00 (s, 1H), 7.82 (s,
1H), 7.58 (d, J = 8.0 Hz, 1H), 7.36 (s, 1H), 5.05−4.85 (m, 1H), 4.05 (s,
P
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Article
yield). 1H NMR (300 MHz, CD3OD) δ ppm 8.46 (d, J = 8.1 Hz, 1H),
8.02 (s, 1H), 7.84 (s, 1H), 7.59 (d, J = 8.1, Hz, 1H), 7.36 (s, 1H), 5.05−
4.80 (m, 1H), 4.05 (s, 3H), 4.02−3.79 (m, 2H), 3.77−3.61 (m, 2H),
3.51−3.32 (m, 2H), 3.28−3.12 (m, 2H), 3.03 (s, 3H), 2.48−2.40 (m,
1H), 2.25−2.18 (m, 1H), 1.88 (d, J = 6.8 Hz, 3H), 0.90 (t, J = 7.3 Hz,
3H).
2H), 3.69−3.63 (m, 2H), 3.50−3.37(m, 2H), 3.21−3.11(m, 2H), 3.04
(s, 3H), 2.70−2.49 (m, 2H), 2.11−1.40 (m, 8H).
(S)-4-Fluoro-5-isopropyl-2-methoxy-3-(3-methylpiperazin-
1-yl)-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-car-
bonitrile (CJ-2349). (S)-2-Methylpiperazine (0.5 mL) and DIPEA
(0.5 mL) were added to a solution of 3,4-difluoro-5-isopropyl-2-
methoxy-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carboni-
trile (367 mg, 1.0 mmol) in DMSO (3 mL) and the mixture was heated
to 120−140 °C for 3 days. The reaction mixture was cooled down to RT
and purified by preparative HPLC to afford the title compound (150
mg, 33% yield). 1H NMR (400 MHz, CD3OD) δ 8.36 (d, J = 8.1 Hz,
1H), 7.82−7.76 (m, 2H), 7.55 (d, J = 8.1 Hz, 1H), 5.15−5.08 (m, 1H),
4.04 (s, 3H), 3.65−3.55 (m, 5H), 3.50−3.45 (m, 1H), 3.44−3.35 (m,
1H), 1.80−1.72 (m, 6H), 1.41 (d, J = 6.4 Hz, 3H).
2-Methoxy-3-(4-methylpiperazin-1-yl)-11-oxo-5-(pentan-2-
yl)-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile
(10d). 1-Methylpiperazine (0.5 mL) and DIPEA (0.5 mL) were added
to a solution of 3-fluoro-2-methoxy-11-oxo-5-(pentan-2-yl)-6,11-
dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (148 mg, 0.39
mmol) in DMSO (2 mL) and the mixture was heated to 120−140
°C for 3 days. The reaction was cooled down to RT and purified by
preparative HPLC to afford the title compound as a pale yellow solid
1
(R)-4-Fluoro-5-isopropyl-2-methoxy-3-(3-methylpiperazin-
1-yl)-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-car-
bonitrile (CJ-2350). (R)-2-Methylpiperazine (0.5 mL) and DIPEA
(0.5 mL) were added to a solution of 3,4-difluoro-5-isopropyl-2-
methoxy-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carboni-
trile (367 mg, 1.0 mmol) in DMSO (3 mL) and the mixture was heated
to 120−140 °C for 3 days. The reaction mixture was cooled down to RT
and purified by preparative HPLC to afford the title compound (132
mg, 30% yield). 1H NMR (400 MHz, CD3OD) δ 8.36 (d, J = 8.1 Hz,
1H), 7.82−7.76 (m, 2H), 7.55 (d, J = 8.1 Hz, 1H), 5.15−5.08 (m, 1H),
4.04 (s, 3H), 3.65−3.55 (m, 5H), 3.50−3.45 (m, 1H), 3.44−3.35 (m,
1H), 1.80−1.72 (m, 6H), 1.41 (d, J = 6.4 Hz, 3H).
3-(3,3-Dimethylpiperazin-1-yl)-4-fluoro-5-isopropyl-2-me-
thoxy-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-car-
bonitrile (CJ-2351). 2,2-Dimethylpiperazine (0.5 mL) and DIPEA
(0.5 mL) were added to a solution of 3,4-difluoro-5-isopropyl-2-
methoxy-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carboni-
trile (367 mg, 1.0 mmol) in DMSO (3 mL) and the mixture was heated
to 120−140 °C for 3 days. The reaction mixture was cooled down to RT
and purified by preparative HPLC to afford the title compound (200
mg, 43% yield). 1H NMR (300 MHz, CD3OD) δ 8.34 (d, J = 7.8 Hz,
1H), 7.80−7.76 (m, 2H), 7.53 (d, J = 7.8 Hz, 1H), 5.15−5.05 (m, 1H),
4.05 (s, 3H), 3.61−3.36 (m, 6H), 1.75 (d, J = 6.7 Hz, 6H), 1.56 (s, 6H).
(S)-3-(3,4-Dimethylpiperazin-1-yl)-4-fluoro-5-isopropyl-2-
methoxy-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-
carbonitrile (CJ-2356). Formaldehyde solution (37%; 33 mg, 0.40
mmol), acetic acid (12 mg, 0.20 mmol), and sodium triacetoxybor-
ohydride (43 mg, 0.20 mmol) were added to a solution of (S)-4-fluoro-
5-isopropyl-2-methoxy-3-(3-methylpiperazin-1-yl)-11-oxo-6,11-dihy-
dro-5H-indolo[2,3-b]quinoline-8-carbonitrile (60 mg, 0.134 mmol) in
DCM (10 mL) and the mixture was stirred at RT for 12 h. Water was
added to quench the reaction and the mixture was extracted with DCM.
The solvent was removed under vacuum and the residue was purified by
preparative HPLC to afford the title compound (40 mg, 65% yield). 1H
NMR (400 MHz, CD3OD) δ 8.32 (d, J = 8.0 Hz, 1H), 7.84−7.72 (m,
2H), 7.50 (d, J = 8.0 Hz, 1H), 5.20−5.01 (m, 1H), 4.04 (s, 3H), 3.71−
3.59 (m, 5H), 3.50−3.44 (m, 1H), 3.40−3.34 (m, 1H), 3.05 (s, 3H),
1.75 (d, J = 6.8 Hz, 6H), 1.47 (d, J = 6.2 Hz, 3H).
(45 mg, 23% yield). MS: m/z = 458 [M + H]. H NMR (400 MHz,
DMSO-d6) δ 9.98 (s, 1H), 8.32 (d, J = 8.0 Hz, 1H), 7.91−7.87 (m, 2H),
7.61 (dd, J = 8.0, 1.4 Hz, 1H), 7.23 (s, 1H), 5.11−5.05 (m, 1H), 3.95 (s,
3H), 3.86−3.77 (m, 2H), 3.59 (d, J = 11.9 Hz, 2H), 3.35−3.23 (m,
2H), 3.12−3.02 (m, 2H), 2.92 (s, 3H), 2.35−2.20 (m, 1H), 2.05−2.01
(m, 1H), 1.76 (d, J = 6.9 Hz, 3H), 1.32−1.25 (m, 1H), 1.11−1.05 (m,
1H), 0.83 (t, J = 7.3 Hz, 3H).
5-Cyclopropyl-2-methoxy-3-(4-methylpiperazin-1-yl)-11-
oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile
(10e). 1-Methylpiperazine (0.5 mL) and DIPEA (0.5 mL) were added
to a solution of 5-cyclopropyl-3-fluoro-2-methoxy-11-oxo-6,11-dihy-
dro-5H-indolo[2,3-b]quinoline-8-carbonitrile (160 mg, 0.41 mmol) in
DMSO (2 mL) and the mixture was heated to 120 to 140 °C for 3 days.
The reaction was cooled down to RT and purified by preparative HPLC
to afford the title compound (36 mg, 18% yield). 1H NMR (300 MHz,
CD3OD) δ ppm 8.27 (d, J = 7.8 Hz, 1H), 7.84 (s, 1H), 7.70 (s, 1H),
7.54 (s, 1H), 7.47 (d, J = 7.8 Hz, 1H), 4.04 (s, 3H), 4.03−3.90 (m, 2H),
3.72−3.63 (m, 2H), 3.60−3.37 (m, 3H), 3.25−3.12 (m, 2H), 3.05 (s,
3H), 1.66−1.57 (m, 2H), 1.30−1.18 (m, 2H).
5-Cyclobutyl-2-methoxy-3-(4-methylpiperazin-1-yl)-11-
oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile
(10f). 1-Methylpiperazine (0.5 mL) and DIPEA (0.5 mL) were added
to a solution of 5-cyclobutyl-3-fluoro-2-methoxy-11-oxo-6,11-dihydro-
5H-indolo[2,3-b]quinoline-8-carbonitrile (155 mg, 0.50 mmol) in
DMSO (2 mL) and the mixture was heated to 120−140 °C for 3 days.
The reaction mixture was cooled down to RT and purified by
preparative HPLC to afford the title compound as a pale yellow solid
(43 mg, 20% yield). 1H NMR (300 MHz, CD3OD) δ ppm 8.39 (d, J =
8.1 Hz, 1H), 7.94 (s, 1H), 7.79 (s, 1H), 7.55 (d, J = 8.1 Hz, 1H), 7.14 (s,
1H), 5.31−5.25 (m, 1H), 4.03 (s, 3H), 3.95−3.91 (m, 2H), 3.69−3.65
(m, 2H), 3.45−3.37 (m, 2H), 3.19−3.14 (m, 2H), 3.07−2.99 (m, 5H),
2.68−2.53 (m, 2H), 2.04−2.02 (m, 2H).
5-Cyclopentyl-2-methoxy-3-(4-methylpiperazin-1-yl)-11-
oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile
(10g). 1-Methylpiperazine (0.5 mL) and DIPEA (0.5 mL) were added
to a solution of 5-cyclopentyl-3-fluoro-2-methoxy-11-oxo-6,11-dihy-
dro-5H-indolo[2,3-b]quinoline-8-carbonitrile (130 mg, 0.35 mmol) in
DMSO (2 mL) and the mixture was heated to 120 to 140 °C for 3 days.
The reaction mixture was cooled down to RT and purified by
preparative HPLC to afford the title compound as a pale yellow solid
(51 mg, 32% yield). 1H NMR (300 MHz, CD3OD) δ ppm 8.29 (d, J =
8.1 Hz, 1H), 7.94 (s, 1H), 7.65 (d, J = 1.3 Hz, 1H), 7.45 (dd, J = 8.1, 1.3
Hz, 1H), 7.08 (s, 1H), 5.40−5.28 (m, 1H), 4.03 (s, 3H), 3.96−3.94 (m,
2H), 3.71−3.67 (m, 2H), 3.51−3.18 (m, 4H), 3.03 (s, 3H), 2.54−2.47
(m, 2H), 2.41−2.13 (m, 4H), 2.10−1.89 (m, 2H).
(R)-3-(3,4-Dimethylpiperazin-1-yl)-4-fluoro-5-isopropyl-2-
methoxy-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-
carbonitrile (CJ-2357). Formaldehyde solution (37%; 33 mg, 0.40
mmol), acetic acid (12 mg, 0.20 mmol), and sodium triacetoxybor-
ohydride (43 mg, 0.20 mmol) were added to a solution of (R)-4-fluoro-
5-isopropyl-2-methoxy-3-(3-methylpiperazin-1-yl)-11-oxo-6,11-dihy-
dro-5H-indolo[2,3-b]quinoline-8-carbonitrile (60 mg, 0.134 mmol) in
DCM (10 mL) and the mixture was stirred at RT for 12 h. Water was
added to quench the reaction and the mixture was extracted with DCM.
The solvent was removed under vacuum and the residue was purified by
preparative HPLC to afford the title compound (47 mg, 76% yield). 1H
NMR (400 MHz, CD3OD) δ 8.32 (d, J = 8.0 Hz, 1H), 7.84−7.72 (m,
2H), 7.50 (d, J = 8.0 Hz, 1H), 5.20−5.01 (m, 1H), 4.04 (s, 3H), 3.71−
3.59 (m, 5H), 3.50−3.44 (m, 1H), 3.40−3.34 (m, 1H), 3.05 (s, 3H),
1.75 (d, J = 6.8 Hz, 6H), 1.47 (d, J = 6.2 Hz, 3H).
5-Cyclohexyl-2-methoxy-3-(4-methylpiperazin-1-yl)-11-
oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile
(10h). 1-Methylpiperazine (0.5 mL) and DIPEA (0.5 mL) were added
to a solution of 5-cyclohexyl-3-fluoro-2-methoxy-11-oxo-6,11-dihydro-
5H-indolo[2,3-b]quinoline-8-carbonitrile (160 mg, 0.41 mmol) in
DMSO (2 mL) and the mixture was heated to 120−140 °C for 3 days.
The reaction mixture was cooled down to RT and purified by
preparative HPLC to afford the title compound as a pale yellow solid
(53 mg, 27% yield). 1H NMR (300 MHz, CD3OD) δ ppm 8.42 (d, J =
8.1 Hz, 1H), 7.98 (s, 1H), 7.82 (d, J = 1.4 Hz, 1H), 7.57 (dd, J = 8.1, 1.4
Hz, 1H), 7.42 (s, 1H), 4.90−4.78 (m, 1H),4.03 (s, 3H), 4.00−3.82 (m,
4-Fluoro-5-isopropyl-2-methoxy-11-oxo-3-(3,3,4-trimethyl-
piperazin-1-yl)-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-car-
bonitrile (CJ-2358). Formaldehyde solution (37%; 175 mg, 2.17
mmol), acetic acid (39 mg, 0.65 mmol), and sodium triacetoxybor-
Q
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ohydride (138 mg, 0.65 mmol) were added to a solution of 3-(3,3-
dimethylpiperazin-1-yl)-4-fluoro-5-isopropyl-2-methoxy-11-oxo-6,11-
dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (200 mg, 0.43
mmol) in DCM (10 mL) and the mixture was stirred at RT for 12 h.
Water was added to quench the reaction and the mixture was extracted
with DCM. The solvent was removed under vacuum and the residue
was purified by preparative HPLC to afford the title compound (150
mg, 73% yield). 1H NMR (300 MHz, CD3OD + CDCl3) δ 8.39 (d, J =
8.0 Hz, 1H), 7.81−7.75 (m, 2H), 7.53 (d, J = 8.0 Hz, 1H), 5.19−4.99
(m, 1H), 4.04 (s, 3H), 3.66−3.35 (m, 6H), 2.88 (s, 3H), 1.82−1.70 (m,
6H), 1.63 (s, 3H), 1.50 (s, 3H).
3-(cis-3,5-Dimethylpiperazin-1-yl)-4-fluoro-5-isopropyl-2-
methoxy-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-
carbonitrile (CJ-2359). cis-2,6-Dimethylpiperazine (0.5 mL) and
DIPEA (0.5 mL) were added to a solution of 3,4-difluoro-5-isopropyl-
2-methoxy-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbon-
itrile (300 mg, 0.817 mmol) in DMSO (3 mL) and the mixture was
heated to 120−140 °C for 3 days. The reaction mixture was cooled
down to RT and purified by preparative HPLC to afford the title
compound (162 mg, 43% yield). 1H NMR (300 MHz, CD3OD) δ 8.39
(d, J = 8.1 Hz, 1H), 7.88−7.82 (m, 2H), 7.59 (d, J = 8.1 Hz, 1H), 5.15−
5.01 (m, 1H), 4.04 (s, 3H), 3.70−3.50 (m, 4H), 3.40−3.32 (m, 2H),
1.76 (d, J = 6.5 Hz, 6H), 1.39 (d, J = 6.3 Hz, 6H).
4-Fluoro-5-isopropyl-2-methoxy-11-oxo-3-(cis-3,4,5-trime-
thylpiperazin-1-yl)-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-
carbonitrile (CJ-2360). Formaldehyde solution (37%; 175 mg, 2.17
mmol), acetic acid (39 mg, 0.65 mmol), and sodium triacetoxybor-
ohydride (138 mg, 0.65 mmol) were added to a solution of 3-(cis-3,5-
dimethylpiperazin-1-yl)-4-fluoro-5-isopropyl-2-methoxy-11-oxo-6,11-
dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (200 mg, 0.43
mmol) in DCM (10 mL) and methanol (2 mL) and the mixture was
stirred at RT for 12 h. Water was added to quench the reaction and the
mixture was extracted with DCM. The solvent was removed under
vacuum and the residue was purified by preparative HPLC to afford the
title compound (135 mg, 66% yield). Purity by HPLC >99%. 1H NMR
(400 MHz, CD3OD) δ 8.33 (d, J = 8.1 Hz, 1H), 7.87−7.70 (m, 2H),
7.52 (d, J = 8.1 Hz, 1H), 5.25−5.10 (m, 1H), 4.05 (s, 3H), 3.66−3.40
(m, 6H), 3.06 (s, 3H), 1.72 (d, J = 7.1 Hz, 6H), 1.49 (d, J = 6.0 Hz, 6H).
13C NMR (101 MHz, CD3OD) δ 172.14, 151.29 (d, JC−F = 5 Hz),
(20 mL) and the mixture underwent hydrogenation at RT overnight.
The Pd-C was filtered off. Evaporation of ethanol under reduced
pressure afforded the title compound (1.1 g, 92% yield). 1H NMR (300
MHz, CDCl3) δ ppm 6.65−6.45 (m, 2H), 4.30−4.10 (m, 2H), 3.74 (s,
3H), 3.43 (s, 2H), 3.30−3.20 (m, 1H), 2.80−2.70 (m, 2H), 2.20−2.00
(m, 2H), 1.70−1.55 (m, 2H), 1.49 (s, 9H).
t er t -B u tyl 4 - (2 - Fl u oro -3 - (i so pro pyl ami no )- 6-
methoxyphenyl)piperidine-1-carboxylate (29). Acetone (1.74
g, 30 mmol), acetic acid (0.27 g, 4.5 mmol), and sodium
triacetoxyborohydride (0.98 g, 4.5 mmol) were added to a solution
of tert-butyl 4-(3-amino-2-fluoro-6-methoxyphenyl)piperidine-1-car-
boxylate (1.0 g, 3 mmol) in DCM (20 mL) and the mixture was stirred
at RT for 12 h. Water was added to quench the reaction and the mixture
was extracted with DCM. The solvent was removed under and the
residue was purified by silica gel chromatography to afford the title
compound (1.06 g, 94% yield). 1H NMR (300 MHz, CDCl3) δ ppm
6.58−6.44 (m, 2H), 4.30−4.10 (m, 2H), 3.74 (s, 3H), 3.60−3.50 (m,
1H), 3.74 (s, 1H), 3.30−3.17 (m, 1H), 2.83−2.70 (m, 2H), 2.40−2.00
(m, 2H), 1.63−1.55 (m, 2H), 1.49 (s, 9H), 1.21 (d, J = 6.2 Hz, 6H).
Methyl 2-((3-(1-(tert-Butoxycarbonyl)piperidin-4-yl)-2-fluo-
ro-4-methoxyphenyl)(isopropyl)amino)-6-cyano-1H-indole-3-
carboxylate (30). DABCO (84 mg, 0.75 mmol) was added to a
solution of methyl 6-cyano-1H-indole-3-carboxylate (273 mg, 1.366
mmol) in DCM (20 mL) and the mixture was cooled down to 0 °C. N-
Chlorosuccinimide (200 mg, 1.5 mmol) was then added and the
reaction was stirred at 0 °C for 2 h. A solution of tert-butyl 4-(2-fluoro-
3-(isopropylamino)-6-methoxyphenyl)piperidine-1-carboxylate (500
mg, 1.366 mmol) and trichloroacetic acid (56 mg, 0.342 mmol) in
DCM (10 mL) was added dropwise and the reaction was stirred for 2 h
at room temperature. The reaction was quenched by adding water and
the mixture was extracted with DCM. The solvent was removed under
vacuum and the residue was purified by silica gel chromatography
(Hex:EA = 4:1) to afford the title compound (530 mg, 69% yield). 1H
NMR (300 MHz, CDCl3) δ ppm 8.34 (s, 1H), 7.96 (d, J = 8.2 Hz, 1H),
7.40−7.30 (m, 2H), 7.18 (t, J = 8.7 Hz, 1H), 6.72 (dd, J = 8.9, 1.1 Hz,
1H), 4.90−4.75 (m, 1H), 4.25−4.10 (m, 2H), 3.86 (s, 3H), 3.84 (s,
3H), 3.30−3.20 (m, 1H), 2.80−2.65 (m, 2H), 2.20−2.00 (m, 2H),
1.65−1.50 (m, 2H), 1.46 (s, 9H), 1.20 (d, J = 6.5 Hz, 6H).
Methyl 6-Cyano-2-((2-fluoro-4-methoxy-3-(1-methylpiperi-
din-4-yl)phenyl)(isopropyl)amino)-1H-indole-3-carboxylate
(31b). TFA (1 mL) was added to a solution of methyl 2-((3-(1-(tert-
butoxycarbonyl)piperidin-4-yl)-2-fluoro-4-methoxyphenyl)-
(isopropyl)amino)-6-cyano-1H-indole-3-carboxylate (240 mg, 0.425
mmol) in DCM (5 mL) and the mixture was stirred at RT for 12 h. The
solvent was removed under reduced pressure and the residue was
neutralized with NaHCO3 and extracted with EtOAc. EtOAc was
removed under reduced pressure and the residue was dissolved in DCM
(5 mL). HCHO (37%; 100 mg, 1.28 mmol), acetic acid (38 mg, 0.64
mmol), and NaBH(OAc)3 (135 mg, 0.64 mmol) were then added and
the reaction mixture was stirred at RT for 6 h. The solvent was removed
under reduced pressure and the product was extracted with EtOAc.
EtOAc was evaporated under reduced pressure and the residue was
purified by silica gel chromatography (EA:MeOH = 80:20) to afford
the title compound (180 mg, 89% yield). 1H NMR (300 MHz, CDCl3)
δ ppm 8.85 (s, 1H), 7.94 (d, J = 8.3 Hz, 1H), 7.38 (s, 1H), 7.32 (d, J =
8.3 Hz, 1H), 7.15 (t, J = 8.7 Hz, 1H), 6.69 (d, J = 8.7 Hz, 1H), 4.95−
4.80 (m, 1H), 3.83 (s, 3H), 3.81 (s, 3H), 3.20−3.00 (m, 3H), 2.45−
2.25 (m, 2H), 2.35 (s, 3H), 2.20−2.02 (m, 2H), 1.73−1.60 (m, 2H),
1.19 (d, J = 6.6 Hz, 6H).
Methyl 6-Cyano-2-((2-fluoro-3-(1-isopropylpiperidin-4-yl)-
4-methoxyphenyl)(isopropyl)amino)-1H-indole-3-carboxylate
(31c). TFA (1 mL) was added to a solution of methyl 2-((3-(1-(tert-
butoxycarbonyl)piperidin-4-yl)-2-fluoro-4-methoxyphenyl)-
(isopropyl)amino)-6-cyano-1H-indole-3-carboxylate (365 mg, 0.647
mmol) in DCM (5 mL) and the mixture was stirred at RT for 12 h. The
solvent was removed under reduced pressure and the residue was
neutralized with NaHCO3 and extracted with EtOAc. EtOAc was
removed under reduced pressure and the residue was dissolved in DCM
(5 mL). Acetone (113 mg, 1.94 mmol), acetic acid (58 mg, 0.97 mmol),
and NaBH(OAc)3 (206 mg, 0.97 mmol) were then added and the
148.37, 148.16 (d, JC−F = 245 Hz), 134.22, 130.56 (d, JC−F = 13 Hz),
126.29, 125.68 (d, JC−F = 8 Hz), 124.56, 122.86, 120.47, 119.58, 114.18,
104.95, 103.67, 101.40, 61.81, 56.42, 56.31, 55.26, 54.89, 36.01, 19.43,
19.39, 13.59. MS m/z = 476 [M + H].
2-Bromo-3-fluoro-1-methoxy-4-nitrobenzene (26). Sodium
methoxide (25%; 0.9 g, 4.2 mmol) was added dropwise to a solution
of 2-bromo-1,3-difluoro-4-nitrobenzene (1 g, 4.2 mmol) in MeOH (5
mL) and the mixture was stirred at 0 °C for 1 h and RT for 4 h. The
reaction was quenched by addition of water and the product was
extracted with EtOAc. Solvent was removed under reduced pressure
and the residue was purified by silica gel chromatography (Hex:EA =
10:1) to afford the title compound (0.58 g, 55% yield). 1H NMR (300
MHz, CDCl3) δ ppm 8.14 (dd, J = 9.3, 8.3 Hz, 1H), 6.80 (dd, J = 9.3, 1.7
Hz, 1H), 4.03 (s, 3H).
tert-Butyl 4-(2-Fluoro-6-methoxy-3-nitrophenyl)-5,6-dihy-
dropyridine-1(2H)-carboxylate (27). tert-Butyl 4-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxy-
late (1.854 g, 6 mmol), Pd(dppf)Cl2 (117 mg, 0.16 mmol), and K2CO3
(1.656 g, 12 mmol) were added to a solution of 1-bromo-2-iodo-4-
nitrobenzene (1 g, 4 mmol) in DME-H2O (22 mL, 10:1 mixture). The
mixture was stirred at 80 °C for 12 h under nitrogen. The reaction was
cooled down to RT and the product was extracted with EtOAc. The
solvent was removed under reduced pressure and the residue was
purified by silica gel chromatography (Hex:EA = 3:1) to afford the title
compound (1.3 g, 92% yield). 1H NMR (300 MHz, CDCl3) δ ppm 8.06
(m, 1H), 6.74 (d, J = 9.3 Hz, 1H), 5.72 (s, 1H), 4.15−4.03 (m, 2H),
3.82 (s, 3H), 3.70−3.60 (m, 2H), 2.40−2.30 (m, 2H), 1.50 (s, 9H).
tert-Butyl 4-(3-Amino-2-fluoro-6-methoxyphenyl)-
piperidine-1-carboxylate (28). Pd-C (10%; 140 mg) was added to
a solution of tert-butyl 4-(2-fluoro-6-methoxy-3-nitrophenyl)-5,6-
dihydropyridine-1(2H)-carboxylate (1.3 g, 3.69 mmol) in ethanol
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reaction mixture was stirred at RT for 12 h. The solvent was removed
under reduced pressure and the product was extracted with EtOAc.
EtOAc was evaporated under reduced pressure and the residue was
purified by silica gel chromatography (EA:MeOH = 80:20) to afford
the title compound (150 mg, 46% yield). 1H NMR (300 MHz, CDCl3)
δ ppm 7.98−7.90 (m, 2H), 7.35 (dd, J = 8.3, 1.4 Hz, 1H), 7.31 (d, J =
1.4 Hz, 1H), 7.16 (t, J = 8.8 Hz, 1H), 6.72 (d, J = 8.8 Hz, 1H), 4.95−
4.80 (m, 1H), 3.87 (s, 6H), 3.20−2.75 (m, 4H), 2.40−2.20 (m, 4H),
1.75−1.55 (m, 2H), 1.19 (d, J = 6.4 Hz, 6H), 1.08 (d, J = 6.5 Hz, 6H).
Methyl 6-Cyano-2-((2-fluoro-4-methoxy-3-(1-(tetrahydro-
2H-pyran-4-yl)piperidin-4-yl)phenyl)(isopropyl)amino)-1H-in-
dole-3-carboxylate (31d). TFA (1 mL) was added to a solution of
methyl 2-((3-(1-(tert-butoxycarbonyl)piperidin-4-yl)-2-fluoro-4-
methoxyphenyl)(isopropyl)amino)-6-cyano-1H-indole-3-carboxylate
(365 mg, 0.647 mmol) in DCM (5 mL) and the mixture was stirred at
RT for 12 h. The solvent was removed under reduced pressure and the
residue was neutralized with NaHCO3 and extracted with EtOAc.
EtOAc was removed under reduced pressure and the residue was
dissolved in DCM (5 mL). Dihydro-2H-pyran-4(3H)-one (194 mg,
1.94 mmol), acetic acid (58 mg, 0.97 mmol), and NaBH(OAc)3 (206
mg, 0.97 mmol) were then added and the reaction mixture was stirred at
RT for 12 h. The solvent was removed under reduced pressure and the
product was extracted with EtOAc. EtOAc was evaporated under
reduced pressure and the residue was purified by silica gel
chromatography (EA:MeOH = 80:20) to afford the title compound
(140 mg, 39% yield). 1H NMR (300 MHz, CDCl3) δ ppm 8.55 (s, 1H),
7.95 (d, J = 8.3 Hz, 1H), 7.40−7.27 (m, 2H), 7.16 (t, J = 8.7 Hz, 1H),
6.70 (d, J = 8.3 Hz, 1H), 4.95−4.75 (m, 1H), 4.12−4.00 (m, 2H), 3.84
(s, 3H), 3.82 (s, 3H), 3.45−3.25 (m, 2H), 3.15−3.02 (m, 3H), 2.75−
2.27 (m, 5H), 1.80−1.60 (m, 6H), 1.20 (d, J = 6.5 Hz, 6H).
Methyl 6-Cyano-2-((2-fluoro-4-methoxy-3-(1-(oxetan-3-yl)-
piperidin-4-yl)phenyl)(isopropyl)amino)-1H-indole-3-carbox-
ylate (31e). TFA (1 mL) was added to a solution of methyl 2-((3-(1-
(tert-butoxycarbonyl)piperidin-4-yl)-2-fluoro-4-methoxyphenyl)-
(isopropyl)amino)-6-cyano-1H-indole-3-carboxylate (365 mg, 0.647
mmol) in DCM (5 mL) and the mixture was stirred at RT for 12 h. The
solvent was removed under reduced pressure and the residue was
neutralized with NaHCO3 and extracted with EtOAc. EtOAc was
removed under reduced pressure and the residue was dissolved in DCM
(5 mL). Cyclobutanone (140 mg, 1.94 mmol), acetic acid (58 mg, 0.97
mmol), and NaBH(OAc)3 (206 mg, 0.97 mmol) were then added and
the reaction mixture was stirred at RT for 6 h. The solvent was removed
under reduced pressure and the product was extracted with EtOAc.
EtOAc was evaporated under reduced pressure and the residue was
purified by silica gel chromatography (EA:MeOH = 80:20) to afford
the title compound (190 mg, 58% yield). 1H NMR (300 MHz, CDCl3)
δ ppm 8.20 (s, 1H), 7.96 (d, J = 8.2 Hz, 1H), 7.48−7.30 (m, 2H), 7.17
(t, J = 8.7 Hz, 1H), 6.73 (d, J = 8.9 Hz, 1H), 4.95−4.80 (m, 1H), 4.64
(d, J = 6.7 Hz, 4H), 3.88 (s, 3H), 3.85 (s, 3H), 3.51−3.49 (m, 1H),
3.25−3.05 (m, 1H), 2.95−2.75 (m, 2H), 2.45−2.25 (m, 2H), 2.00−
1.80 (m, 2H), 1.73−1.60 (m, 2H), 1.20 (d, J = 6.5 Hz, 6H).
(m, 8H), 3.20−3.05 (m, 3H), 2.62 (t, J = 6.3 Hz, 2H), 2.45−2.20 (m,
4H), 1.70−1.55 (m, 2H), 1.20 (d, J = 6.5 Hz, 6H), 0.89 (s, 9H), 0.06 (s,
6H).
4-Fluoro-5-isopropyl-2-methoxy-3-(4-morpholinopiperidin-
1-yl)-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-car-
bonitrile (ALK-73). 4-Morpholinopiperidine (0.5 g) and DIPEA (0.5
mL) were added to a solution of 3,4-difluoro-5-isopropyl-2-methoxy-
11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carbonitrile (135
mg, 0.37 mmol) in DMSO (2 mL) and the mixture was heated to
120 to 140 °C for 3 days. The reaction mixture was cooled down to RT
and purified by preparative HPLC to afford the title compound (24 mg,
15% yield). 1H NMR (300 MHz, CD3OD) δ ppm 8.21 (d, J = 8.0 Hz,
1H), 7.64 (s, 1H), 7.60 (s, 1H), 7.39 (d, J = 8.0 Hz, 1H), 5.10−4.90 (m,
1H), 4.20−4.10 (m, 2H), 4.00 (s, 3H), 3.90−3.70 (m, 2H), 3.65−3.50
(m, 4H), 3.45−3.20 (m, 5H), 2.33−2.20 (m, 2H), 2.10−1.85 (m, 2H),
1.70 (dd, J = 6.9, 1.9 Hz, 6H).
4-Fluoro-5-isopropyl-2-methoxy-3-(1-methylpiperidin-4-
yl)-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-carboni-
trile (CJ-2212). Methyl 6-cyano-2-((2-fluoro-4-methoxy-3-(1-meth-
ylpiperidin-4-yl)phenyl)(isopropyl)amino)-1H-indole-3-carboxylate
(180 mg, 0.38 mmol) was dissolved in diphenyl ether (10 mL) and the
solution was refluxed for 1 h. The mixture was cooled down to RT.
Hexane (20 mL) was added and the precipitate was collected by
filtration. The precipitate was purified by preparative HPLC to afford
the title compound (100 mg, 59% yield). 1H NMR (300 MHz,
CD3OD) δ ppm 8.25 (d, J = 8.0 Hz, 1H), 7.76 (s, 1H), 7.67 (s, 1H),
7.44 (d, J = 8.0 Hz, 1H), 5.10−5.00 (m, 1H), 4.03 (s, 3H), 3.80−3.62
(m, 3H), 3.30−3.12 (m, 2H), 2.96 (s, 3H), 2.80−2.60 (m, 2H), 2.18−
2.00 (m, 2H), 1.73 (dd, J = 7.0, 2.0 Hz, 6H).
4-Fluoro-5-isopropyl-3-(1-isopropylpiperidin-4-yl)-2-me-
thoxy-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-car-
bonitrile (CJ-2225). Methyl 6-cyano-2-((2-fluoro-3-(1-isopropylpi-
peridin-4-yl)-4-methoxyphenyl)(isopropyl)amino)-1H-indole-3-car-
boxylate (150 mg, 0.296 mmol) was dissolved in diphenyl ether (10
mL) and the solution was refluxed for 1 h. The mixture was cooled
down to RT. Hexane (20 mL) was added and the precipitate was
collected by filtration. The precipitate was purified by preparative
HPLC to afford the title compound (35 mg, 25% yield). 1H NMR (300
MHz, CD3OD) δ ppm 8.26 (d, J = 8.0 Hz, 1H), 7.76 (s, 1H), 7.68 (s,
1H), 7.44 (d, J = 8.0 Hz, 1H), 5.10−5.00 (m, 1H), 4.03 (s, 3H), 3.80−
3.50 (m, 4H), 3.30−3.20 (m, 2H), 2.80−2.60 (m, 2H), 2.15−2.03 (m,
2H), 1.73 (dd, J = 7.0, 2.0 Hz, 6H), 1.45 (d, J = 6.7 Hz, 6H).
4-Fluoro-5-isopropyl-2-methoxy-11-oxo-3-(1-(tetrahydro-
2H-pyran-4-yl)piperidin-4-yl)-6,11-dihydro-5H-indolo[2,3-b]-
quinoline-8-carbonitrile (CJ-2226). Methyl 6-cyano-2-((2-fluoro-
4-methoxy-3-(1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl)phenyl)-
(isopropyl)amino)-1H-indole-3-carboxylate (140 mg, 0.255 mmol)
was dissolved in diphenyl ether (10 mL) and the solution was refluxed
for 1 h. The mixture was cooled down to RT. Hexane (20 mL) was
added and the precipitate was collected by filtration. The precipitate
was purified by preparative HPLC to afford the title compound (70 mg,
53% yield). 1H NMR (300 MHz, CD3OD) δ ppm 8.25 (d, J = 8.0 Hz,
1H), 7.75 (s, 1H), 7.66 (s, 1H), 7.43 (d, J = 8.0 Hz, 1H), 5.15−4.95 (m,
1H), 4.20−4.07 (m, 2H), 4.03 (s, 3H), 3.82−3.70 (m, 3H), 3.60−3.40
(m, 3H), 3.30−3.20 (m, 2H), 2.80−2.60 (m, 2H), 2.20−1.80 (m, 6H),
1.73 (dd, J = 6.9, 1.8 Hz, 6H).
4-Fluoro-5-isopropyl-2-methoxy-3-(1-(oxetan-3-yl)-
piperidin-4-yl)-11-oxo-6,11-dihydro-5H-indolo[2,3-b]-
quinoline-8-carbonitrile (CJ-2224). Methyl 6-cyano-2-((2-fluoro-
4-methoxy-3-(1-(oxetan-3-yl)piperidin-4-yl)phenyl)(isopropyl)-
amino)-1H-indole-3-carboxylate (190 mg, 0.37 mmol) was dissolved in
diphenyl ether (10 mL) and the solution was refluxed for 1 h. The
mixture was cooled down to RT. Hexane (20 mL) was added and the
precipitate was collected by filtration. The precipitate was purified by
preparative HPLC to afford the title compound (90 mg, 51% yield). 1H
NMR (300 MHz, CD3OD) δ ppm 8.28 (d, J = 8.0 Hz, 1H), 7.78 (d, J =
1.1 Hz, 1H), 7.69 (d, J = 0.7 Hz, 1H), 7.46 (dd, J = 8.0, 1.1 Hz, 1H),
5.18−5.02 (m, 1H), 4.95−4.80 (m, 4H), 4.55−4.40 (m, 1H), 4.04 (s,
3H), 3.80−3.60 (m, 3H), 3.20−3.10 (m, 2H), 2.80−2.60 (m, 2H),
2.18−2.02 (m, 2H), 1.74 (dd, J = 7.0, 2.1 Hz, 6H).
Methyl 2-((3-(1-(2-((tert-Butyldimethylsilyl)oxy)ethyl)-
piperidin-4-yl)-2-fluoro-4-methoxyphenyl)(isopropyl)amino)-
6-cyano-1H-indole-3-carboxylate (31f). TFA (1 mL) was added to
a solution of methyl 2-((3-(1-(tert-butoxycarbonyl)piperidin-4-yl)-2-
fluoro-4-methoxyphenyl)(isopropyl)amino)-6-cyano-1H-indole-3-car-
boxylate (365 mg, 0.647 mmol) in DCM (5 mL) and the mixture was
stirred at RT for 12 h. The solvent was removed under reduced pressure
and the residue was neutralized with NaHCO3 and extracted with
EtOAc. EtOAc was removed under reduced pressure and the residue
was dissolved in DCM (5 mL). 2-((tert-Butyldimethylsilyl)oxy)-
acetaldehyde (164 mg, 0.97 mmol), acetic acid (58 mg, 0.97 mmol),
and NaBH(OAc)3 (206 mg, 0.97 mmol) were then added and the
reaction mixture was stirred at RT for 6 h. The solvent was removed
under reduced pressure and the product was extracted with EtOAc.
EtOAc was evaporated under reduced pressure and the residue was
purified by silica gel chromatography (EtOAc) to afford the title
compound (180 mg, 45% yield). 1H NMR (300 MHz, CDCl3) δ ppm
8.61 (s, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.40−7.30 (m, 2H), 7.15 (t, J =
8.7 Hz, 1H), 6.69 (d, J = 8.7 Hz, 1H), 4.95−4.78 (m, 1H), 3.90−3.75
S
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4-Fluoro-3-(1-(2-hydroxyethyl)piperidin-4-yl)-5-isopropyl-
2-methoxy-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-8-
carbonitrile (CJ-2227). Methyl 2-((3-(1-(2-((tert-
butyldimethylsilyl)oxy)ethyl)piperidin-4-yl)-2-fluoro-4-
methoxyphenyl)(isopropyl)amino)-6-cyano-1H-indole-3-carboxylate
(180 mg, 0.29 mmol) was dissolved in diphenyl ether (10 mL) and the
solution was refluxed for 1 h. The mixture was cooled down to RT.
Hexane (20 mL) was added and the precipitate was collected by
filtration. The precipitate was dissolved in MeOH (10 mL) and the
solution was treated with KHSO4 (197 mg, 1.45 mmol). The mixture
was stirred at RT for 12 h. The solvent was removed under reduced
pressure and the residue was purified by preparative HPLC to afford the
title compound (80 mg, 58% yield). 1H NMR (300 MHz, CD3OD) δ
ppm 8.26 (d, J = 8.0 Hz, 1H), 7.75 (s, 1H), 7.68 (s, 1H), 7.44 (d, J = 8.0
Hz, 1H), 5.20−5.02 (m, 1H), 4.03 (s, 3H), 4.00−3.84 (m, 2H), 3.80−
3.60 (m, 3H), 3.35−3.20 (m, 4H), 2.80−2.60 (m, 2H), 2.15−2.00 (m,
2H), 1.73 (dd, J = 6.9, 1.6 Hz, 6H).
affinity ligand) and incubated at RT with shaking for 30 min. The kinase
concentration in the eluates was measured by qPCR.
Cell Viability Assay. The effect of compounds on cell viability was
determined in a 4 day proliferation assay. Cells were maintained in
RPMI1640 culture medium with 10% FBS at 37 °C and an atmosphere
of 5% CO2. All the cell lines were used within 3 months of thawing fresh
vials. Cells were seeded in 96-well plates at a density of 3000−10,000
cells/well in 75 μL of culture medium. Compounds were serially diluted
in the appropriate medium, and 75 μL of the diluted compounds was
added to the appropriate wells of the cell plate. After the addition of
compounds, the cells were incubated at 37 °C in an atmosphere of 5%
CO2 for 4 days. Cell viability was determined using the CellTiter-Glo
Luminescent Cell Viability Assay Kit (Promega, Madison, WI) (for
KARPAS-299 cells) or WST (2-(2-methoxy-4-nitrophenyl)-3-(4-
nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazolium, monosodium
salt) Cell Counting-8 Kit (Dojindo Molecular Technologies, Inc.,
Rockville, MD) (for H3122 cells) according to the manufacturer’s
instructions.
Enzymatic Inhibition Assay. The cytoplasmic domain (amino
acid 1058−1620) of wild-type human ALK protein expressed as N-
terminal GST-fusion protein was purchased from Carna Biosciences,
Inc. (Japan). Mutated ALK proteins were expressed in SF9 insect cells
with N-terminal tags cleaved after purification. Kinase activities of all
enzymes were assessed using a Lance TR-FRET assay kit from
PerkinElmer Life Sciences (Waltham, MA). Compound solution (2.5
μL) and 5 μL of protein solution were added into a black low volume
384-well microtiter plate, which was incubated for 30 min with gentle
shaking at RT, followed by addition of 2.5 μL of fluorescently labeled
peptide substrate (Ulight-CKKSRGDYMTMQIG) and ATP mixture
solution. The kinase reaction was performed in 50 mM HEPES (pH
7.5) with 1 mM EGTA, 1 mM MgCl2, and 2 mM DTT, and 0.01%
Tween 20 was added immediately prior to the assay. Final
concentrations of ATP, substrates, and DMSO were 100 μM, 20 nM,
and 0.5%, respectively. Concentrations of different ALK proteins were
adjusted accordingly to achieve comparable enzymatic activities for
both wild-type and all mutated ALK proteins. The final ALK
concentrations were 1, 1, 1, 128, 2, and 4 nM for wild type, F1174L,
L1196M, S1206Y, G1269A, and G1202R, respectively. The reaction
was allowed to proceed for 90 min in the dark with gentle shaking at RT
after which 10 μL of 20 mM EDTA and 2 nM Eu-W1024 anti-
phosphotyrosine antibody (PT66) mixture solution in the detection
buffer from the manufacturer was added to terminate the reaction and
detect the phosphorylation of the peptide substrate. The final mixture
was incubated in the dark for 1 h before the plate was read on a Tecan
Infinite M−1000 multimode plate reader (Tecan, Durham, NC) with an
excitation wavelength of 320 nm. Emission intensities were measured at
both 620 and 665 nm with the intensity ratio between 665 and 620 nm
corresponding to the peptide substrate phosphorylation. IC50 values of
inhibitors were obtained by fitting the ratio of 665/620 nm vs inhibitor
concentrations in a sigmoidal dose−response curve (variable slope)
with a nonlinear regression.
Kinase Selectivity Enzymatic Assays. The experiments were
conducted at DiscoveRx Corporation. For most assays, kinase-tagged
T7 phage strains were prepared in an Escherichia coli host derived from
the BL21 strain. E. coli were grown to log phase, infected with T7 phage,
and incubated with shaking at 32 °C until lysis. The lysates were
centrifuged and filtered to remove cell debris. The remaining kinases
were produced in HEK-293 cells and subsequently tagged with DNA
for qPCR detection. Streptavidin-coated magnetic beads were treated
with biotinylated small-molecule ligands for 30 min at RT to generate
affinity resins for kinase assays. The liganded beads were blocked with
excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1%
BSA, 0.05% Tween 20, and 1 mM DTT) to remove unbound ligand and
to reduce nonspecific binding. Binding reactions were assembled by
combining kinases, liganded affinity beads, and test compounds in 1×
binding buffer (20% SeaBlock, 0.17× PBS, 0.05% Tween 20, and 6 mM
DTT). All reactions were performed in polystyrene 96-well plates in a
final volume of 0.135 mL. The assay plates were incubated at RT with
shaking for 1 h and the affinity beads were washed with wash buffer (1×
PBS and 0.05% Tween 20). The beads were then resuspended in
elution buffer (1× PBS, 0.05% Tween 20, and 0.5 μM nonbiotinylated
For WST assays, the WST-8 reagent was added to each well at a final
concentration of 10% (v/v), and then the plates were incubated at 37
°C for 1−2 h for color development. The absorbance was measured at
450 nm using a SPECTRAmax PLUS plate reader (Molecular Devices,
Sunnyvale, CA). The readings were normalized to the DMSO-treated
cells and the half-maximal inhibitory concentration (IC50) was
calculated by nonlinear regression analysis using GraphPad Prism 5
software (GraphPad Software, La Jolla, CA).
For CellTiter-Glo assay, 100 μL of CellTiter-Glo Reagent was added
to each well, and then the plates were incubated at RT for 10−20 min.
The luminescent signal was measured using a Tecan Infinite M1000
multimode microplate reader (Tecan, Morrisville, NC). The readings
were normalized to the DMSO-treated cells and the IC50 values were
calculated by nonlinear regression analysis using GraphPad Prism 5
software.
Computational Docking Simulations. All the docking simu-
lations were performed using GOLD (version 4.0.11).34,35 The ALK
structural coordinates were extracted from the cocrystal structure of
ALK in complex with CH5424802 (PDBID: 3AOX3)27,35 and used for
the docking calculations. ALK protein and the designed compounds
were prepared using the MOE program.36 The N- and C-termini of
ALK were capped with a methyl group and acetyl group, respectively,
and the missing hydrogen atoms were added by setting the pH at 7.0.
The binding site of ALK was centered at Glu1132 with a radius of 20 Å.
In each genetic algorithm (GA) run, a maximum number of 200,000
operations were set on a population of 5 islands of 100 individuals.
Operator weights for crossover, mutation, and migration were set to 95,
95, and 10, respectively. Twenty docking runs were used for each
compound. The PLP scoring function in the GOLD program35 was
used as the fitness function to assess the docked conformations. The
highest ranked docked conformation of compounds 7d and CJ-2360
were selected as their predicted binding poses, as shown in Figures 2
and 3, respectively.
In Vivo Pharmacodynamic and Efficacy Studies. Human
anaplastic large cell lymphoma (ALCL) KARPAS-299 cells were
purchased from Sigma and maintained at 37 °C, 95% air, and 5% carbon
dioxide in RPMI-1640 medium (with 2.05 mM L-glutamine; 0.1 μM,
sterile filtered) supplemented with 10% fetal bovine serum, 100 units/
mL penicillin, and 100 units/mL streptomycin (GIBCO, Invitrogen
Corp.) and passaged twice weekly.
Tumor cells for xenografts were passaged into fresh medium the day
before injection. For injection, a cell sample was mixed 1:1 with Trypan
Blue (GIBCO, Invitrogen Corp.) and counted on a hemocytometer to
determine the number of live/dead cells. Cells were washed twice with
1× PBS (GIBCO, Invitrogen Corp.) and resuspended in an ice-cold
mixture of 1:1 PBS and Matrigel (BD Biosciences, Invitrogen Corp.) for
a final Matrigel protein concentration of 5 mg/mL. All tumors were
inoculated subcutaneously (s.c.) into female SCID mice (strain: 236
C.B-17 SCID, Charles River) with 5 × 106 KARPAS-299 cells in 0.1 mL
with Matrigel in each mouse. The size of tumors growing in the mice
was measured in two dimensions using calipers. Tumor volume (mm3)
T
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= (A × B2)/2, where A and B are the tumor length and width (in mm),
respectively.
States; Ascentage Pharma (Jiangsu), Taizhou, Jiangsu
Province 225300, China
For pharmacodynamic (PD) experiments, tumors were grown to
200−600 mm3 and mice were randomly assigned for a single oral dose
of CJ-2360 or alectinib. The control group mice were treated with
vehicle alone. Tumor samples were collected at indicated time points
post dose. Control mice were given vehicle only and collected at the 6 h
time point. The mice were euthanized with CO2 and 0.3−0.5 mL of
blood was drawn from a cardiac puncture using a 25g needle and 1 mL
syringe, which was pretreated with heparin. Tumors were removed and
samples were placed in cassettes in 10% neutral-buffered formalin for 24
h and then transferred to 70% ethanol. Tumor sample was immediately
frozen in liquid nitrogen, ground into fine powder, and stored at −80
°C.
For western blot analysis, tumor tissues were lysed with radio-
immunoprecipitation assay buffer (RIPA buffer). The protein
concentration of tissue lysate was determined with Bio-Rad Protein
Assay Dye Reagent Concentrate (#5000006). The levels of
phosphorylated STAT3 protein (pTyr705 rabbit, Cell Signaling
Technology, Beverly, MA), phosphorylated AKT protein (pSer473,
rabbit, Cell Signaling Technology, Beverly, MA), and phosphorylated
ERK protein (pThr202/Tyr204, rabbit, Cell Signaling Technology,
Beverly, MA) in tissue lysate were examined by western blotting
analysis. GAPDH (sc-25,778, Santa Cruz Biotechnology) was used as a
loading control.
Yunlong Zhou − Ascentage Pharma (Jiangsu), Taizhou,
Jiangsu Province 225300, China
Xuyuan Dong − Rogel Cancer Center and Departments of
Internal Medicine, Pharmacology, and Medicinal Chemistry,
University of Michigan, Ann Arbor, Michigan 48109, United
States
Liu Liu − Rogel Cancer Center and Departments of Internal
Medicine, Pharmacology, and Medicinal Chemistry, University
of Michigan, Ann Arbor, Michigan 48109, United States
Longchuan Bai − Rogel Cancer Center and Departments of
Internal Medicine, Pharmacology, and Medicinal Chemistry,
University of Michigan, Ann Arbor, Michigan 48109, United
States
Donna McEachern − Rogel Cancer Center and Departments of
Internal Medicine, Pharmacology, and Medicinal Chemistry,
University of Michigan, Ann Arbor, Michigan 48109, United
States
Sally Przybranowski − Rogel Cancer Center and Departments
of Internal Medicine, Pharmacology, and Medicinal Chemistry,
University of Michigan, Ann Arbor, Michigan 48109, United
States
For the efficacy study, the size of tumors growing in the mice was
measured in two dimensions using calipers. Tumor volume (mm3) = (A
× B2)/2, where A and B are the tumor length and width (in mm),
respectively. During treatment, tumor volume and body weight were
measured three times a week. After the treatment was stopped, tumor
volume and body weight were measured at least once a week. Before
treatment began, tumors were allowed to grow to an average of 150
mm3 (70−270 mm3) in volume. Mice with tumors within the
acceptable size range were randomized into treatment groups of
seven to eight mice for each group. CJ-2360 or alectinib was
administered via oral gavage, twice or once daily for 3 weeks. Mice in
the control group received vehicle alone once daily for 3 weeks.
All animal experiments were performed under the guidelines of the
University of Michigan Committee for Use and Care of Animals and
using an approved animal protocol (PI, Shaomeng Wang).
Chao-Yie Yang − Rogel Cancer Center and Departments of
Internal Medicine, Pharmacology, and Medicinal Chemistry,
University of Michigan, Ann Arbor, Michigan 48109, United
States; orcid.org/0000-0002-5445-0109
Jeanne Stuckey − Life Sciences Institute, University of Michigan,
Ann Arbor, Michigan 48109, United States
Xiaoqin Li − Department of Pharmaceutical Sciences, College of
Pharmacy, University of Michigan, Ann Arbor, Michigan
48109, United States
Bo Wen − Department of Pharmaceutical Sciences, College of
Pharmacy, University of Michigan, Ann Arbor, Michigan
48109, United States
Ting Zhao − Department of Pharmaceutical Sciences, College of
Pharmacy, University of Michigan, Ann Arbor, Michigan
48109, United States
Siwei Sun − Department of Pharmaceutical Sciences, College of
Pharmacy, University of Michigan, Ann Arbor, Michigan
48109, United States
Duxin Sun − Department of Pharmaceutical Sciences, College of
Pharmacy, University of Michigan, Ann Arbor, Michigan
48109, United States
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01550.
■
sı
Summary of KINOMEscan screening data for CJ-2360
against 468 kinases; HPLC trace of CJ-2360 (PDF)
Modeled structure for compound 7d in complex with
Lingling Jiao − Ascentage Pharma (Jiangsu), Taizhou, Jiangsu
Province 225300, China
Yu Jing − Ascentage Pharma (Jiangsu), Taizhou, Jiangsu
Province 225300, China
ALK (PDB)
Modeled structure for CJ-2369 in complex with ALK
(PDB)
Molecular string file for all the final target compounds
(CSV)
Ming Guo − Ascentage Pharma (Jiangsu), Taizhou, Jiangsu
Province 225300, China
Dajun Yang − Ascentage Pharma (Jiangsu), Taizhou, Jiangsu
Province 225300, China
AUTHOR INFORMATION
Corresponding Author
Shaomeng Wang − Rogel Cancer Center and Departments of
Internal Medicine, Pharmacology, and Medicinal Chemistry,
University of Michigan, Ann Arbor, Michigan 48109, United
States; orcid.org/0000-0002-8782-6950; Phone: 1-734-
615-0362; Email: shaomeng@umich.edu
■
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01550
Author Contributions
¶J.C., Y.Z., and X.D. contributed equally to this work.
Funding
This work is supported by Ascentage Pharma Group (to S.W.).
Authors
Notes
Jianyong Chen − Rogel Cancer Center and Departments of
Internal Medicine, Pharmacology, and Medicinal Chemistry,
University of Michigan, Ann Arbor, Michigan 48109, United
The authors declare the following competing financial
interest(s): Ascentage has filed a patent application on this
U
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Ascentage. The University of Michigan also owns stock in
Ascentage.
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