Discovery of a Candidate Containing an (S)-3,3-Difluoro-1-(4-methylpiperazin-1-yl)-2,3-dihydro-1 H-inden Scaffold as a Highly Potent Pan-Inhibitor of the BCR-ABL Kinase including the T315I-Resistant Mutant for the Treatment of Chronic Myeloid Leukemia

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Discovery of a Candidate Containing an (S)‑3,3-Diﬂuoro-1-(4-

methylpiperazin-1-yl)-2,3-dihydro‑1H‑inden Scaﬀold as a Highly

Potent Pan-Inhibitor of the BCR-ABL Kinase Including the T315I-

Resistant Mutant for the Treatment of Chronic Myeloid Leukemia

Dongfeng Zhang,^∇Peng Li,^∇Yongxin Gao, Yaoyao Song, Yaqin Zhu, Hong Su, Beibei Yang, Li Li,

Gang Li, Ningbo Gong, Yang Lu, Huanjie Shao,* Chunrong Yu,* and Haihong Huang *

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ABSTRACT: BCR-ABL kinase inhibition is an eﬀective strategy

for the treatment of chronic myeloid leukemia (CML). Herein, we

report compound 3a-P1, bearing a diﬂuoro-indene scaﬀold, as a

novel potent pan-inhibitor against BCR-ABL mutants, including

the most refractory T315I mutant. As the privileged (S)-isomer

compared to its (R)-isomer 3a-P2, 3a-P1 exhibited potent

antiproliferative activities against K562 and Ku812 CML cells

and BCR-ABL and BCR-ABL^T315IBaF3 cells, with IC₅₀values of

0.4, 0.1, 2.1, and 4.7 nM, respectively. 3a-P1 displayed a good

safety proﬁle in a battery of assays, including single-dose toxicity,

hERG K⁺, and genotoxicity. It also showed favorable mice

pharmacokinetic properties with a good oral bioavailability (32%), a reasonable half-life (4.61 h), and a high exposure (1386 h·

ng/mL). Importantly, 3a-P1 demonstrated a higher potency than ponatinib in a mice xenograft model of BaF3 harboring BCR-

ABL^T315I. Overall, the results indicate that 3a-P1 is a promising drug candidate for the treatment of CML to overcome the imatinib-

resistant T315I BCR-ABL mutation.

to inhibit native ABL, BCR-ABL, BCR-ABL^T315I, and other

INTRODUCTION

■

clinically important ABL kinase domain mutants (i.e., Q252H,

Chronic myeloid leukemia (CML) results in a malignant

Y253H, M351T, and H396P). Additionally, ponatinib also

tumor and is characterized by the production of a large number

inhibits the sarcoma gene (SRC) and members of the vascular

of immature white blood cells, which accumulate in the bone

endothelial growth factor receptor (VEGFR), ﬁbroblast growth

marrow and inhibit normal hematopoietic function. Most

factor receptor (FGFR), and platelet-derived growth factor

CML cases are driven by the breakpoint cluster region-abelson

receptor (PDGFR) families of receptor tyrosine kinases.¹⁰

leukemia virus (BCR-ABL), which constitutes an activated

Although the Food and Drug Administration (FDA) issued a

fusion tyrosine kinase formed by the translocation of the

black-framed warning of the cardiovascular risk and liver

Philadelphia chromosome (Ph) between the ABL oncogene

toxicity of ponatinib as a multitarget tyrosine kinase inhibitor,

from the long arm of chromosome 9 and the BCR from the

ponatinib was recently proven to be a valuable alternative

long arm of chromosome 22.^1,2

treatment to allogeneic stem cell transplantation in patients

Imatinib (Figure 1) is a ﬁrst-generation BCR-ABL inhibitor

with T315I-positive advanced CML and Philadelphia-chromo-

and is highly eﬀective in approximately 60% of CML patients.³

some-positive (Ph⁺) acute lymphoblastic leukemia.¹¹

However, the acquired resistance to imatinib has become a

Among the more than 100 resistant mutants of BCR-ABL

major challenge for the clinical management of CML.⁴

that have been clinically identiﬁed, the “gatekeeper” T315I is

Signiﬁcant eﬀorts have been devoted to identiﬁng novel

the most common mutation, accounting for approximately

BCR-ABL inhibitors to overcome imatinib resistance.

Nilotinib, dasatinib, and bosutinib have been approved as

Received: January 17, 2021

Published: May 20, 2021

second-generation drugs to treat adult patients in all phases of

CML with an acquired resistance to imatinib.⁵⁻⁷However,

these second-generation inhibitors do not target the most

refractory mutant of BCR-ABL, T315I.⁸

To date, the only approved third-generation inhibitor for

CML is ponatinib⁹(Figure 1), which has been demonstrated

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Figure 1. Chemical structures of BCR-ABL inhibitors.

Figure 2. (A) Design strategy for ponatinib-based BCR-ABL kinase inhibitors. (B) Crystal structure of ponatinib in a complex with the BCR-

ABL^T315Imutant kinase (PDB 3IK3). Ponatinib is shown in cyan. (C) The predicted binding modes of 3a with the BCR-ABL^T315Imutant kinase

(PDB 3IK3). 3a is shown in pink, hydrogen bonds are shown as green dashed lines, and van der Waals interactions are shown as magenta dashed

lines.

15%−20% of all clinically acquired mutants.¹²The T315I

mutation stops the binding of ATP-competitive inhibitors to

the ABL catalytic domain by either direct steric hindrance or

stabilizing the active kinase conformation. The development of

new drugs to target the open and active conformation of the

T315I mutant is therefore a major challenge. Several small-

molecule inhibitors targeting BCR-ABL^T315Ihave been

reported (Figure 1), among which PF-114¹³and AT-

9283^14,15are in clinical phase II investigations and exhibit

potent inhibitory activities toward the BCR-ABL^T315Imutant.

HQP1351,¹⁶developed by Ascentage Pharma, has completed

its phase II clinical trial, and a New Drug Application (NDA)

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has been submitted to regulators in China. Therefore, the

development of structurally distinct BCR-ABL inhibitors,

particularly, those capable of inhibiting the mutant T315I,

would facilitate drug discovery for CML treatment.

the hERG inhibition by enhancing the conﬁrmation rigidity

and lowering the lipophilicity (ClogP 4.69) of 3a.

Subsequently, we selected compound 3a for a computational

study to investigate its potential binding mode with BCR-

ABL^T315I. The study predicted that the nitrogen atom in the

imidazo[1,2-b]pyridazine group of 3a would form a hydrogen

bond with the aminocarbonyl oxygen of Met318 in the ABL

kinase. A gatekeeper isoleucine residue was found at the

interface formed by the van der Waals contacts between

compound 3a and the T315I mutant of ABL (Figure 2C),

which indicated that favorable hydrophobic interactions were

occurred with the T315I mutant. Interestingly, the hydrogen in

the amide group of 3a formed a hydrogen bond with Met290,

which was diﬀerent from the interactions of ponatinib and

indicated that compound 3a may have better potency than

ponatinib.

The in vitro potencies of the designed compounds 1a, 1b,

2a−2k, and 3a−3e were ﬁrst assessed against K562, a CML

cell line harboring native BCR-ABL, with ponatinib included

for comparison. As shown in Table 1, compounds 1a and 1b

showed activities similar to that of ponatinib. The compounds

with a benzimidazole core (2a−2k), where R₁is either

imidazo[1,2-b]pyridazine (2a and 2g) or N-cyclopropylpyr-

imidin-2-amine (2e and 2k), were highly potent against K562.

The identity of R₂as a methyl or chloro group did not aﬀect

the potency (2a vs 2g and 2e vs 2k). The compounds with a

diﬂuoro-indene scaﬀold (3a−3e), except for 3c−3e, were

potent inhibitors of the proliferation of K562.

Herein, we report the design and evaluation of 3a-P1

bearing a novel diﬂuoro-indene scaﬀold with the privileged

(S)-conﬁguration, which is a new orally bioavailable pan-BCR-

ABL inhibitor with a high potency against T315I and several

other BCR-ABL mutants, including p-loop region mutants

(G250E, Q252H, Y253F, and E255K), an ATP-binding

domain hinge region mutant (F317L), a catalytic segment

mutant (M351T), and an activation loop region mutant

(H396P). The compound 3a-P1 is more potent than ponatinib

in mouse xenograft models of BCR-ABL^T315I-driven tumor

growth. Furthermore, 3a-P1 exhibits an acceptable safety

proﬁle as evaluated by preliminary safety assessments,

including hERG channel inhibition assay, the Ames test,

chromosome aberration, bone marrow micronucleus, and

single-dose toxicity assessments.

RESULTS AND DISCUSSION

■

It was evident that most of the reported BCR-ABL inhibitors

have an amide group between two aryl rings, a heterocycle tail,

and an aryl group head. Furthermore, it was established that

the arylacetylene (head) and N-methylpiperazine (tail) motifs

of ponatinib and its analogues are important for their activities

9,16

against the gatekeeper mutant BCR-ABL^T315I

.

On the basis

of this analysis, we intended to exploit structural modiﬁcations

of the A (left phenyl ring), B (amide group), and C (right

phenyl ring) regions of ponatinib to achieve the goal of

identifying novel chemical scaﬀolds with potent activities

toward the BCR-ABL^T315Imutant and improved drug-like

properties (Figure 2A).

To evaluate the cellular activity of these compounds toward

BCR-ABL^T315I, we employed BaF3 cells engineered to express

T315I-mutated BCR-ABL. As shown in Table 1, the designed

cyclic compounds 3a and 3b with the diﬂuoro-indene scaﬀold

demonstrated comparable potencies to that of ponatinib in

regard to inhibiting the proliferation of BaF3 expressing BCR-

ABL^T315I, whereas the other compounds (1a, 1b, and 2a−2k)

were much less potent. The results indicated that keeping the

diarylamide fragment had a very important eﬀect on the

biological activity. The potencies of the diﬂuoro-indene

scaﬀold compounds against BCR-ABL^T315Iwere greatly

reduced both when the methyl on the phenyl group in the A

region was changed to a hydrogen (3c and 3d) and when N-

methyl piperazine was translocated to the adjacent position of

the diﬂuoro group (3e). The structure−activity relationship

(SAR) studies revealed that the phenyl group of the A region is

essential for the potency. Interestingly, the eﬀect of the methyl

group on the phenyl ring on the activity is similar to that in the

ponatinib derivatives. In addition, the SAR results disclosed

that the amide of the B region is necessary for potent activity,

and the diﬂuoro-indene scaﬀold is a favored moiety for strong

in vitro potencies.

Based on both the racemic compounds 3a and 3b, which

displayed exceptionally high inhibitory activities, further eﬀorts

were focused on investigating the relationship between the

conﬁguration and activity due to the fact that chirality is of

great importance in modern drug development. First, we

sought a facile method to chemically resolve the compounds

3a and 3b and explored the biological properties of the pure

stereoisomers. We separated the enantiomers with chiral

supercritical ﬂuid chromatography (SFC) and then assessed

their cellular activity using BCR-ABL-positive CML cells

(K562 and Ku812), BCR-ABL-negative CML cells (U937 and

JURKAT), and BaF3 cells expressing either native or T315I

mutated BCR-ABL. Four FDA-approved BCR-ABL inhibitors

As disclosed in Figure 2B, the crystal structure of ponatinib

in a complex with the BCR-ABL^T315Imutant kinase (PDB

3IK3) revealed that the slim alkyne linker between the

diarylamide scaﬀold and the imidazo[1,2-b]pyridazine motif is

crucial to the success of ponatinib, as it avoids a steric clash

with a bulky isoleucine residue at the gatekeeper position.

Additionally, the ethynyl linkage and phenyl group in the A

region of ponatinib have favorable van der Waals interactions

with the Ile315 mutated residue. Moreover, the N-methyl-

piperazinyl group of ponatinib occupies the pocket formed by

the P-loop, the DFG motif, and the catalytic loop and thus

plays an important role in the key interactions (Figure 2B).

According to the analysis of the crystal structure and our

general optimization strategies, we ﬁrst used a heterocycle to

replace the phenyl group of ponatinib in the A region to

generate piperidine compound 1a with a ClogP of 3.62, which

was lower than that of ponatinib (ClogP 5.77) (Figure 2A). It

is likely that the van der Waals interactions of the heterocycle

derivative 1a with Ile315 were enhanced because of the

introduction of piperidine. Inspired by the unique structure of

AT-9283, which contains a benzimidazole fragment, we

replaced the amide group with an imidazole group, as

exempliﬁed by compound 2a containing the benzimidazole

scaﬀold, through a bioisosterism strategy. Considering the

potential cardiovascular risk displayed by ponatinib due to the

moderate inhibition of the hERG K⁺channel, the diﬂuoro-

indene moiety was introduced to the C region, therefore

keeping the key methylpiperazine group and taking advantage

of a cyclization strategy to generate 3a with the aim of reducing

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(Table 2). The (S)-enantiomers of 3a (3a-P1) and 3b (3b-P1)

were more potent against cells expressing native or T315I-

mutated BCR-ABL than the racemic mixtures, whereas the

(R)-enantiomers (3a-P2 and 3b-P2) displayed even weaker

activities than the racemic mixtures. In particular, compared

with 3b-P1 and even ponatinib, 3a-P1 demonstrated a higher

potency against the cells harboring native or T315I-mutated

BCR-ABL and a weaker activity toward BCR-ABL-negative

human leukemia cells. The results suggest that 3a-P1 possesses

not only an improved potency but also an improved selectivity

compared to those of ponatinib.

Table 1. Inhibitory Activities of Compounds Against K562

a

and BaF3 (BCR-ABL^T315I) Cellular Proliferation

To understand the variation in the activity proﬁle of the pure

enantiomers 3a-P1 (S) and 3a-P2 (R), we performed a

docking study (Figure 3). The predicted binding models

showed that the conformations of 3a-P1 and 3a-P2 were

similar in the protein binding pocket except for the diﬂuoro-

indene and the N-methylpiperazine moieties (Figure 3B). The

imidazo[1,2-b]pyridazine core and the amide group of 3a-P1

and 3a-P2 could both form four hydrogen bond interactions

with Met318, Met290, Glu286, and Asp381 (Figure 3C and

3D). The phenyl and “ﬂag-methyl” moieties of 3a-P1 and 3a-

P2 could make favorable van der Waals interactions with the

gatekeeper Ile315. Although the diﬂuoro-indene moiety and

the N-methylpiperazine group of 3a-P1 and 3a-P2 could bind

into the hydrophobic pocket, the diﬂuoro-indene moiety of 3a-

P1 could form more van der Waals interactions with Ile293

and Leu354 (Figure 3D). The results of the predicted binding

modes suggested that 3a-P1 could bind more tightly to the

hydrophobic pocket, which is formed by the P-loop, the DFG

motif, and the catalytic loop.

We further investigated the eﬀects of 3a-P1 and ponatinib

on the cell death of K562 and BaF3/BCR-ABL^T315I(Figure 4).

The results showed that treatment with 3a-P1 and ponatinib

caused similar levels of cell death in K562 (IC₅₀∼ 10 nM).

Compared with ponatinib, 3a-P1 was more eﬀective at killing

BaF3/BCR-ABL^T315Icells.

To further conﬁrm the target inhibition in cells expressing

native or T315I-mutated BCR-ABL, we examined the eﬀect of

3a-P1 on the tyrosine phosphorylation status of BCR-ABL and

the direct BCR-ABL substrate CRKL using Western blotting

assays (Figure 5). The results showed that 3a-P1 and

ponatinib demonstrated similar activities against the phosphor-

ylation of BCR-ABL in K562 and BaF3 expressing native and

T315I-mutated BCR-ABL. Notably, 3a-P1 seemed to be more

potent at inhibiting CRKL phosphorylation than ponatinib.

As compound 3a-P1 demonstrated a potent antitumor

activity and the clear target inhibition on T315I-mutated BCR-

ABL, 3a-P1 was subsequently characterized using mouse

pharmacokinetic (PK) studies before the in vivo eﬃcacy

investigation was conducted (Table 3). When intravenously

administered at a dose of 5 mg/kg in mice, compound 3a-P1

displayed a slower clearance rate than that of ponatinib. When

orally administered at a dose of 15 mg/kg, the oral exposure of

3a-P1 was higher than that of ponatinib. The oral

bioavailability of compound 3a-P1 was determined to be

32%. Moreover, the elimination half-life of 3a-P1 was longer

than that of ponatinib. Taken together, its good oral

bioavailability and ideal half-life suggested that 3a-P1 may

have good eﬃcacy in vivo when orally administered and may be

more potent in vivo than ponatinib at the same dose.

a

b

The tested concentration of the compounds was 100 nM. The data

represent the mean values of three independent experiments.

(imatinib, nilotinib, dasatinib, and ponatinib) were included

for comparison. As expected, imatinib, nilotinib, and dasatinib

were eﬀective toward native BCR-ABL but not BCR-ABL^T315I

Based on the promising in vitro ﬁndings combined with the

excellent in vivo PK proﬁles of 3a-P1, we conducted

experiments to determine the in vivo antitumor potency of

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Table 2. Eﬀect of BCR-ABL Inhibitors on the Cell Viability of BCR-ABL-Positive and BCR-ABL-Negative Leukemia Cell Lines

a

and a Panel of BaF3 Cell Transfectants (IC₅₀as the Mean SEM (nM))

BaF3 cells

CML cells

non-CML cells

compounds

native BCR-ABL

>100

11.8 0.4

T315I BCR-ABL

K562

Ku812

U937

>1000

245.8 12.5

607.8 42.2

867.6 88.7

JURKAT

>1000

295.0 5.0

672.4 77.6

850.7 25.7

imatinib

nilotinib

dasatinib

ponatinib

3a

3a-P1

3a-P2

3b

>100

9.6 1.7

11.0 0.2

4.7 1.3

>100

40.0 9.6

12.4 0.4

>100

82.9 11.2

1.8 0.4

0.2 0.1

0.3 0.1

0.5 0.1

0.4 0.1

3.6 0.1

1.0 0.2

0.6 0.1

10.4 1.5

51.3 4.3

1.8 0.1

0.1 0.1

0.2 0.1

0.1 0.1

2.7 0.1

0.4 0.1

0.2 0.1

5.2 0.2

0.7 0.1

0.9 0.1

2.7 0.2

2.1 0.2

31.2 3.8

9.3 2.5

3.5 0.9

62.5 6.9

>1000

190.8 3.3

165.0 15.0

465.3 9.7

292.0 8.0

280.0 20.0

399.9 64.9

3b-P1

3b-P2

a

The data represent the mean values of three independent experiments.

Figure 3. (A) Structures of 3a-P1 and 3a-P2. (B) Overlays of the docked poses of 3a-P1 and 3a-P2 with the BCR-ABL^T315Imutant kinase (PDB

3IK3). (C) The predicted binding modes of 3a-P2 with the BCR-ABL^T315Imutant kinase (PDB 3IK3). (D) The predicted binding modes of 3a-P1

with the BCR-ABL^T315Imutant kinase (PDB 3IK3). 3a-P1 is shown in green, 3a-P2 is shown in pink, hydrogen bonds are shown as green dashed

lines, and van der Waals interactions are shown as magenta dashed lines.

3a-P1 in a subcutaneous xenograft model of BaF3 expressing

T315I-mutated BCR-ABL. Considering that ponatinib was

approved as its hydrochloride salt, we synthesized the

hydrochloride of 3a-P1 to evaluate its in vivo eﬃcacy. As

shown in Figure 6A, ponatinib in its hydrochloride salt

inhibited tumor growth, with a tumor growth inhibition (TGI)

of 53% based on daily oral dosing at 5 mg/kg. Compared with

the vehicle control, daily oral dosing of 2.5, 5, and 10 mg/kg of

3a-P1·HCl caused a reduction in mean tumor volumes at the

ﬁnal measurements, with TGI values of 69%, 87% and 107%,

respectively, showing an obvious dose−eﬀect relationship.

Notably, 3a-P1·HCl demonstrated a higher activity in terms of

suppressing tumor growth than that of ponatinib, as treatment

with 3a-P1·HCl at 2.5 mg/kg per day achieved a similar TGI

level to that of ponatinib at a higher dose (5 mg/kg). 3a-P1·

HCl likely could achieve a TGI of more than 100% at 10 mg/

kg per day. It is noteworthy that no obvious eﬀect on the

mouse body weight was observed for 15 days of treatment

(Figure 6B).

Given its highly potent in vitro and in vivo antiproliferative

activities, we further investigated the preliminary druggability

proﬁle of compound 3a-P1 (Table 4). In a hERG inhibition

assay, compound 3a-P1 showed hERG channel activity with an

IC₅₀value of 11.1 μM that was lower than that of ponatinib

(IC₅₀= 8 μM), which indicates that it had less potential for

cardiotoxicity. We also studied its mutagenicity using a

bacterial reverse mutation (Ames) test. Compound 3a-P1

was nonmutagenic at the tested concentrations (0.5−312.5 μg

per plate) with or without metabolic activation (using the ﬁve

Salmonella typhimurium test strains TA97a, TA98, TA100,

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Figure 4. Eﬀect of 3a-P1 and ponatinib on the cell death of K562 and BaF3/BCR-ABL^T315I

.

Figure 5. Eﬀect of 3a-P1 and ponatinib on the inhibition the phosphorylation of ABL and ABL^T315I. K562, BaF3/BCR-ABL, and BaF3/BCR-

ABL^T315Icells were treated with ponatinib and 3a-P1 at the indicated concentrations. After 4 h of treatment, cells were harvested, lysed, and

analyzed by immunoblots using an antibody for either phosphorylated BCR-ABL (Y245) or CRKL (Y207). eIF 4e was employed as an internal

control.

a

Table 3. Pharmacokinetic Parameters Following Intravenous and Oral Dosing in ICR Mice

T_max

(h)

C_max(ng/

mL)

AUC_0‑t(h·ng

AUC_0‑∞(h·ng

MRT

(h)

T_1/2

(h)

CL (L/h per

kilogram)

Vss (L/

kg)

F

(%)

compounds

route

/mL)

3a-P1

po (15 mg/kg)

iv (5 mg/kg)

5.0

0.08

1.5

129

1470

70.0

270

1386

1389

363

1443

1502

432

8.22

5.11

5.25

4.29

4.61

5.30

2.84

3.28

32

27

3.72

9.68

18.1

41.0

ponatinib po (15 mg/kg)

iv (5 mg/kg)

0.08

497

528

a

Oral (po) and intravenous (iv); n = 4 animals per study (po) and n = 3 animals per study (iv).

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Figure 6. Eﬃcacy of 3a-P1 and ponatinib in mouse subcutaneous xenograft models driven by BCR-ABL^T315I. (A) Mice bearing BaF3/BCR-

ABL^T315Ixenografts were orally treated once daily with either a vehicle or the indicated doses of 3a-P1·HCl and ponatinib. (B) Changes in mice

body weight after treatment with 3a-P1 and ponatinib. Results are expressed as the mean tumor volume SD (n = 10 for each group in panel A).

TGI stands for tumor growth inhibition. A TGI larger than 100% indicates that tumors treated with the drugs are smaller than their original sizes.

Table 4. Safety Proﬁle of Compound 3a-P1

d

hERG K⁺inhibition

IC₅₀(μM)

11.1

single-dose toxicity

ICR mice, MTD (mg/kg)

100

a

b

c

compound

Ames

negative

chromosome aberration

negative

mice bone marrow micronucleus

negative

3a-P1

a

Compound 3a-P1 was tested from 0.5 to 312.5 μg/plate with or without metabolic activation using the ﬁve Salmonella typhimurium test strains

b

TA97a, TA98, TA100, TA102, and TA1535; 3a-P1·HCl was used. Doses were as follows: 0.563, 1.125, 2.25, and 4.5 μg/mL; 3a-P1·HCl was

used. Doses were as follows: 25, 50, and 100 mg/kg; 3a-P1·HCl was used. Mice were observed for 14 d after a single-dose oral administration;

3a-P1·HCl was used.

c

d

TA102, and TA1535). In the chromosome aberration and

mouse bone marrow micronucleus assays, compound 3a-P1

was also found to be negative. A mice single-dose toxicity study

was also performed for compound 3a-P1, and the maximum

tolerated dose was determined to be 100 mg/kg.

4b. Second, compound 7 was coupled with compounds 6a and

6b under Sonogashira coupling conditions, followed by

deprotection to yield compounds 9a and 9b, respectively.

Finally, the target compounds 1a and 1b were obtained

through a condensation reaction between intermediates 9a or

9b, respectively, and 10. Compound 10 was prepared

according to the reported methods.⁹

The preparation of benzimidazole compounds 2a−e and

2g−k is illustrated in Scheme 2. The starting material 3,4-

dinitrobenzoic acid (11) reacted with N-methyl piperazine to

aﬀord compound 12, which was then reduced to give

intermediate 13. The reduction of the nitro group of

compound 13 by catalytic hydrogenation aﬀorded compound

14. The benzimidazole core structure was constructed by

reacting compound 14 with the iodide-substituted benzoic

acids (15a and 15b). Finally, compounds 16a and 16b were

coupled with aryl alkynyl compounds (17a−e) under

Sonogashira coupling conditions to deliver the ﬁnal products

2a−e and 2g−k.

Finally, to further understand its kinase selectivity, the

inhibitory activities of compound 3a-P1 against diﬀerent ABL

kinases and other kinases were evaluated. As depicted in Figure

7, both 3a-P1 and ponatinib exhibited strong and similar

inhibitory eﬀects on human ABL kinase and its mutants,

resulting in only 0.1%−6% of the kinase activity remaining

compared with the DMSO control group shown in the

enlarged segment. In particular, for the mutant T315I, the

inhibition of 3a-P1 and ponatinib both reached about 95% at

the tested concentration of 10 nM. Additionally, the IC₅₀

values of 3a-P1 for the c-Kit and TIE2/TEK kinases were

predicted to be about 10 nM. By comparison, the IC₅₀values

of 3a-P1 for c-Src, FGFR1, FLT3, KDR/VEGFR2, LYN, and

PDGFRa kinases were all less than 10 nM. These results

illustrated that compound 3a-P1 may be useful for the

treatment of CML as well as other solid tumors, such as triple-

negative breast cancers.

The synthetic procedure for compound 2f is brieﬂy

described in Scheme 3. The starting material 18 was coupled

with imidazole to aﬀord compound 19, which was converted to

the benzimidazole core structure by sequential reduction and

condensation reactions. Finally, the target compound 2f was

obtained by the Sonogashira coupling reaction of iodide

intermediate 22 and the aryl alkynyl compound 17a.

CHEMISTRY

■

Synthetic route of urea compounds 1a and 1b is schematically

illustrated in Scheme 1. First, the key intermediates 6a and 6b

were prepared from commercially available compounds 4a and

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Figure 7. Kinase inhibition proﬁles of compound 3a-P1 and ponatinib for native ABL, diﬀerent mutants, and selected kinases at 10 nM.

a

Scheme 1. Synthesis of Compounds 1a and 1b

a

Reagents and conditions are as follows: (a) pyridine·SO₃, DMSO, rt, 2 h, 69−70%; (b) (diazomethyl)phosphonic acid dimethyl ester, K₂CO₃,

MeOH, 0 °C to rt, overnight, 100−101%; (c) Pd(PPh₃)₂Cl₂, CuI, DIPEA, DMF, 80 °C, Ar₂, 6 h, 60−61%; (d) TFA, CH₂Cl₂, overnight, rt, 107−

108%; and (e) 4-nitrophenyl chloroformate, pyridine, dioxane, 60 °C, 11 h, 19−32%.

The designed diﬂuoro-indene compounds 3a−e were

prepared using a palladium-catalyzed Sonogashira coupling

reaction as the key step (Scheme 4). Brieﬂy, the key

intermediate 23 was synthesized according to our reported

method using commercially available 2,3-dihydro-1H-inden-1-

one as the starting material.¹⁷The following reduction of the

NO₂group aﬀorded the amine intermediate 24, which was

reacted with the substituted benzoic acids (15a and 15b) to

give the amide intermediates 25a and 25b, respectively.

Compounds 25a and 25b were converted to the target

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a

Scheme 2. Synthesis of Compounds 2a−e and 2g−k

a

Reagents and conditions are as follows: (a) (i) SOCl₂, reﬂux, 6 h, (ii) N-methylpiperazine, Et₃N, 5 °C to rt, CH₂Cl₂, overnight, 95%; (b) NaBH₄,

BF₃·OEt₂, 5 °C to rt, THF, 5 h, 80%; (c) 10% Pd/C, H₂, DMF/MeOH = 1:1, 12 h; (d) (i) EDCI, HOBt, DMF, rt, 24 h, (ii) AcOH, reﬂux, 3 h,

30−43%; and (e) Pd(PPh₃)₄, CuI, DIPEA, DMF, rt, Ar₂, 20 h, 9−78%.

a

Scheme 3. Synthesis of Compound 2f

a

Reagents and conditions are as follows: (a) K₂CO₃, CuI, 8-hydroxyquinoline, 4-methylimidazole, DMSO, sealed tube, 120 °C, Ar₂, 29 h, 57%; (b)

Raney Ni, H₂, rt, 7 h, 96%; (c) (i) 3-iodo-4-methylbenzoic acid, SOCl₂, reﬂux, 2 h, (ii) Et₃N, DMAP, rt, THF, 20 h, 37%; (d) AcOH, reﬂux, 8 h,

65%; and (e) Pd(PPh₃)₄, CuI, DIPEA, DMF, rt, 20 h, 63%.

compounds 3a−d via a Sonogashira coupling reaction.

Compound 3e was prepared from compound 26, which was

prepared according to our published method,¹⁷by a similar

procedure.

Enantiomerically pure 3a and 3b isomers were prepared via

supercritical ﬂuid chromatography resolution through a chiral

stationary phase (Scheme 5). Racemates 3a and 3b were

cleanly separated into the optically pure enantiomers 3a-P1

and 3a-P2 and 3b-P1 and 3b-P2, respectively.

(Scheme 6). The key chiral compound (S)-23 was prepared by

the chiral resolution of 23, and ^D-camphorsulfonic acid was

used as the chiral resolving agent. Compound 3a-P1 was

synthesized from compound (S)-23 by procedures similar to

those shown in Scheme 4.

The absolute conﬁguration of the synthesized compound

(S)-23, which was dissociated from the ^D-camphorsulfonic salt

of 29, was determined by comparing the experimental ECD

spectrum of compound (S)-23 and the corresponding

calculated ECD spectra of the optical isomers (S)-23 and

(R)-23. The ECD spectrum recorded for (S)-23 matched the

calculated ECD curve of (S)-23 well but was opposite to that

To determine the absolute conﬁguration of the above

prepared compounds by SFC, we synthesized the optical pure

chiral compound 3a from the chiral isomer of compound 23

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a

Scheme 4. Synthesis of Compounds 3a−e

a

Reagents and conditions are as follows: (a) 10% Pd/C, H₂, MeOH, 12 h; (b) (i) substituted benzoic acids, SOCl₂, reﬂux, 6 h, (ii) Et₃N, DMAP,

rt, THF, overnight, 51−92%; and (c) Pd(PPh₃)₄, CuI, DIPEA, DMF, rt, 20 h, 5−30%.

a

Scheme 5. Separation of the Optical Isomers of Compounds 3a and 3b

a

Reagents and conditions are as follows: (a) CHIRALCEL AS-H, CO₂/MeOH (0.1% diethyl amine) = 60:40 (v/v) and (b) CHIRALCEL OJ-H,

CO₂/EtOH (0.1% diethyl amine) = 75:25 (v/v).

of (R)-23 (Figure 8A). Moreover, the calculated UV spectrum

of (S)-23 displayed a curve similar to the experimental UV

spectrum of (S)-23 (Figure 8B). Therefore, the synthesized

compound (S)-23 was deduced to have the (S) absolute

conﬁguration. Furthermore, the single crystal of compound 29

was obtained and characterized by X-ray diﬀraction. The

crystal structure of compound 29 is depicted in Figure 9. Two

molecules of D-camphorsulfonic acid were bound with one

molecule of compound 29, and the absolute conﬁguration of

29 was (S). Thus, the absolute conﬁguration of (S)-23 was

conﬁrmed as the (S)-conﬁguration. The absolute conﬁgu-

rations of compounds 3a-P1 and 3a-P2, respectively, were

determined comparing the experimental ECD spectra. The

experimental ECD spectrum of 3a-P1 displayed a CD diagram

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a

Scheme 6. Synthesis of Compounds 3a-P1 and 3a-P1·HCl

a

Reagents and conditions are as follows: (a) D-camphorsulfonic acid, MeOH/iPrOH = 1:2, reﬂux to rt, 32%; (b) NaOH, H₂O, rt, 100%; (c) 10%

Pd/C, H₂, MeOH, 12 h, 95%; (d) (i) SOCl₂, reﬂux, 1 h; (ii) Et₃N, DMAP, rt, THF, overnight, 69%; (e) Pd(PPh₃)₄, CuI, DIPEA, DMF, rt, 20 h,

37%; and (f) acetone, 1 M HCl in EtOH, rt, 6 h, 102%.

Figure 8. (A) Calculated and experimental ECD spectra of compounds (R)-23 and (S)-23. (B) Calculated and experimental UV spectra of

compound (S)-23.

similar to the experimental spectrum of (S)-3a (Figure 10).

Therefore, compound 3a-P1 was elucidated to have the (S)

absolute conﬁguration, and 3a-P2 was elucidated to have (R)

absolute conﬁguration. The absolute conﬁgurations of 3b-P1

and 3b-P2 were consistent with those of 3a-P1 and 3a-P2,

respectively, according to the same rotation.

CONCLUSION

■

In summary, we have identiﬁed a novel diﬂuoro-indene

derivative 3a-P1 in the (S)-conﬁguration as a highly potent

BCR-ABL inhibitor that displays signiﬁcant activities against a

broad spectrum of BCR-ABL mutants, including the gate-

keeper T315I mutant, through a structure-based medicinal

chemistry optimization strategy. Additionally, 3a-P1 showed

potent inhibition against the kinases c-KIT, TIE2/TEK, c-Src,

FGFR1, FLT3, KDR/VEGFR2, LYN, and PDGFRa. The

results provide a strong rationale for the potential evaluation of

3a-P1 in CML and a range of other cancers driven by multiple

tyrosine kinases such as c-KIT. The PK proﬁle of compound

3a-P1 was also favorable, and its exposure value in ICR mice

was higher than that of ponatinib. In particular, 3a-P1

Figure 9. X-ray crystallographic structure of 29.

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mixture was evaporated in a vacuum. To the residue were added

EtOAc and water. The organic layer was separated, and the water

layer was extracted with EtOAc (100 mL × 3). The combined organic

layers were successively washed with water and brine, dried with

Na₂SO₄, and ﬁltered, and the ﬁltrate was evaporated in a vacuum to

1

give 1.44 g of the product as colorless oil (100%). H NMR (300

MHz, CDCl₃): δ 3.90 (1 H, brs), 3.75−3.70 (1 H, m), 3.02−2.95 (2

H, m), 2.47−2.40 (1 H, m), 2.05 (1 H, d, J = 2.1 Hz), 1.99−1.94 (1

H, m), 1.73−1.69 (1 H, m), 1.63−1.50 (2 H, m), 1.46 (9 H, s).

1-Boc-3-(2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl)piperidine

(8a). A solution of compound 6a (0.55 g, 2.6 mmol), 3-

bromoimidazo[1,2-b]pyridazine 7 (0.40 g, 2 mmol), Pd(PPh₃)₂Cl₂

(70 mg, 0.1 mmol), CuI (29 mg, 0.15 mmol), and DIPEA (0.39 g, 3

mmol) in DMF (20 mL) was stirred at 80 °C under Ar₂for 6 h. The

mixture was poured into 100 mL of water and extracted with EtOAc

(60 mL × 3). The organic layer was washed with brine, dried with

Na₂SO₄, and ﬁltered, and the ﬁltrate was evaporated in a vacuum. The

crude product was puriﬁed by chromatography on silica gel (PE/

EtOAc = 7:3 to 3:2) to give 0.39 g of the product as yellow oil (60%).

¹H NMR (300 MHz, CDCl₃): δ 8.50 (1 H, d, J = 4.2 Hz), 8.08−7.98

Figure 10. Experimental ECD spectra of compounds 3a-P1, 3a-P2,

and (S)-3a.

(2 H, m), 7.15 (1 H, dd, J = 4.5 and 8.7 Hz), 4.09 (1 H, brs), 3.82−

3.76 (1 H, m), 3.19 (1 H, brs), 3.09−3.02 (1 H, m), 2.88−2.81 (1 H,

m), 2.32 (1 H, brs), 2.15−2.11 (1 H, m), 1.81−1.67 (2 H, m), 1.46 (9

H, s). LC-MS: m/z [M + H]⁺327.2112.

demonstrated a higher potency than that of ponatinib in a mice

xenograft model of BaF3 expressing BCR-ABL^T315I. Further-

more, a panel of safety assessments, including a hERG K⁺

channel inhibition assay, an Ames test, chromosome aberration

and mice bone marrow micronucleus assays, and acute toxicity

studies, demonstrated that 3a-P1 displayed an acceptable

safety proﬁle for further characterization. Overall, these

pharmacological and safety data have laid a solid foundation

to develop 3a-P1 as a promising drug candidate.

3-(2-(Imidazo[1,2-b]pyridazin-3-yl)ethynyl)piperidine (9a). To a

solution of compound 8a (0.39 g, 1.2 mmol) in CH₂Cl₂(10 mL) was

added TFA (1.23 g, 10.8 mmol). The mixture was stirred at rt

overnight and evaporated in a vacuum. To the residue was added 10

mL of a 10% K₂CO₃solution, and the mixture was extracted with

EtOAc (20 mL × 3). The combined organic layers were washed with

brine, dried with Na₂SO₄, and ﬁltrated, and the ﬁltrate was evaporated

1

in a vacuum to give 0.33 g of the product as yellow oil (107%). H

NMR (300 MHz, CDCl₃): δ 8.43 (1 H, d, J = 4.5 Hz), 7.95 (1 H, dd,

J = 1.8 and 9.0 Hz), 7.91 (1 H, s), 7.07 (1 H, dd, J = 4.5 and 9.0 Hz),

2.98−2.82 (4 H, m), 2.11−2.08 (1 H, m), 1.84−1.75 (2 H, m), 1.58−

1.53 (2 H, m). LC-MS: m/z [M + H]⁺227.1325.

EXPERIMENTAL SECTION

Chemistry. H NMR spectra were obtained on a Varian mercury

■

1

spectrometer at 300 or 400 MHz in CDCl₃, DMSO-d₆, or CD₃OD.

Chemical shifts and coupling constants are recorded in units of parts

per million and hertz, respectively. Melting points were determined

on a Yanaco MP-J3 melting point apparatus. High-resolution mass

spectra were recorded on an Agilent 1100 series LC/MSD trap mass

spectrometer (ESI-TOF). All target compounds were puriﬁed by

chromatography and have purities of ≥95% as determined by HPLC/

MS analyses conducted on an Agilent 1100 system using a reversed-

phase C18 column with 75% CH₃CN in water (0.1% HCOOH) and a

ﬂow rate of 0.4 mL/min. All reagents and solvents were purchased

from commercial sources unless otherwise indicated. Thin layer

chromatography was carried out on silica gel plates (GF254), and

components were visualized by either UV light (254 nm) or exposure

to I₂. Column chromatography was carried out on silica gel (300−400

mesh).

Synthesis of Compounds 1a and 1b. 1-Boc-3-formylpiperidine

(5a). 1-Boc-3-hydroxymethylpiperidine 4a (2.15 g, 10 mmol) and

triethylamine (3.03 g, 30 mmol) were dissolved in 10 mL of DMSO.

To the mixture was added 15 mL of a DMSO solution of Py·SO₃

(4.77g, 30 mmol) dropwise, and the result mixture was stirred at rt for

2 h. The mixture was poured into 100 mL of ice−water and extracted

with EtOAc (100 mL × 3). The organic layer was washed with brine,

dried with Na₂SO₄, and ﬁltered, and the ﬁltrate was evaporated in a

vacuum. The crude product was puriﬁed by chromatography on silica

gel (PE/EtOAc 5:1) to give 1.46 g of the product as colorless oil

(69%).¹H NMR (300 MHz, CDCl₃): δ 9.70 (1 H, s), 3.93−3.91 (1

H, m), 3.67−3.62 (1 H, m), 3.36−3.29 (1 H, m), 3.13−3.05 (1 H,

m), 2.45−2.40 (1 H, m), 1.97−1.95 (1 H, m), 1.72−1.63 (2 H, m),

1.59−1.49 (1 H, m), 1.46 (9 H, s).

N-(3-(Triﬂuoromethyl)-4-((4-methylpiperazin-1-yl)methyl)-

phenyl)-3-(2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl)piperidine-1-

carboxamide (1a). To a solution of compound 10 (0.1 g, 0.37 mmol)

in 15 mL of anhydrous dioxane was added pyridine (0.036 mL, 0.45

mmol) and 4-nitrophenyl chloroformate (90 mg, 0.45 mmol). The

mixture was stirred at 60 °C for 2 h and then cooled to rt, and

compound 9a (0.1 g, 0.44 mmol) was added to the above mixture.

The mixture was stirred at 60 °C for 9 h and evaporated in a vacuum.

The residue was puriﬁed by chromatography on silica gel (CH₂Cl₂/

CH₃OH = 25:1) to give 140 mg of the crude product, and continued

puriﬁcation via preparation TLC (CH₂Cl₂/CH₃OH = 120:15) gave

60 mg of the product as a pale yellow solid (32%). mp: 68−70 °C. ¹H

NMR (300 MHz, CDCl₃): δ 8.38 (1 H, s), 7.97 (1 H, d, J = 9.0 Hz),

7.88 (1 H, s), 7.61−7.58 (2 H, m), 7.43 (1 H, d, J = 7.8 Hz), 7.10−

7.06 (1 H, m), 7.03 (1 H, s), 3.99−3.95 (2 H, m), 3.65−3.49 (5 H,

m), 3.02 (4 H, brs), 2.79 (4 H, s), 2.69 (3 H, s), 2.13 (1 H, brs), 1.93

(2 H, brs), 1.63 (1 H, brs). HRMS (ESI-TOF⁺): m/z [M + H]⁺calcd

for C₂₇H₃₁F₃N₇O 526.2537, found 526.2536.

1-Boc-3-formylpyrrolidine (5b). In a similar manner to 5a,

compound 5b was prepared from 4b (1.40 g, colorless oil, 70%).

¹H NMR (300 MHz, CDCl₃): δ 9.68 (1 H, s), 3.72−3.68 (1 H, m),

3.51−3.48 (1 H, m), 3.37 (2 H, brs), 3.03−2.99 (1 H, m), 2.24−2.05

(2 H, m), 1.45 (9 H, s).

1-Boc-3-ethylnylpyrrolidine (6b). In a similar manner to 6a,

compound 6b was prepared from 5b and (diazomethyl)phosphonic

acid dimethyl ester (1.39 g, colorless oil, 101%). ¹H NMR (300 MHz,

CDCl₃): δ 3.60−3.47 (2 H, m), 3.30 (2 H, brs), 2.95−2.90 (1 H, m),

2.18−2.12 (1 H, m), 2.10 (1 H, d, J = 1.8 Hz), 1.96−1.90(1 H, m),

1.45 (9 H, s).

1-Boc-3-(2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl)pyrrolidine

(8b). In a similar manner to 8a, compound 8b was prepared from 6b

and 3-bromoimidazo[1,2-b]pyridazine 7 (0.38 g, yellow oil, 61%). ¹H

NMR (300 MHz, CDCl₃): δ 8.43 (1 H, d, J = 3.6 Hz), 7.99−7.92 (2

1-Boc-3-ethynylpiperidine (6a). A solution of compound 5a (1.46

g, 6.85 mmol) and (diazomethyl) phosphonic acid dimethyl ester

(1.79 g, 11.94 mmol) in 50 mL of methanol was stirred at 0 °C for 10

min. To the mixture was added K₂CO₃(1.96 g, 14.2 mmol). The

mixture was stirred at 0 °C for 2 h, then stirred at rt overnight. The

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H, m), 7.09 (1 H, dd, J = 4.2 and 9.0 Hz), 3.77−3.75 (1 H, m), 3.60−

3.45 (4 H, m), 2.32−2.12 (2 H, m), 1.47 (9 H, s).

2-(4-Chloro-3-iodophenyl)-6-((4-methylpiperazin-1-yl)methyl)-

1H-benzo[d]imidazole (16b). In a similar manner to 16a, compound

16b was prepared from 14 and 15b (0.9 g, a pale yellow solid,

43%).¹H NMR (300 MHz, CDCl₃): δ 8.52 (1 H, s), 7.97 (1 H, d, J =

8.1 Hz), 7.46−7.36 (2 H, m), 7.24−7.21 (2 H, m), 3.59 (2 H, s), 2.51

(8 H, brs), 2.29 (3 H, s).

3-(2-(Imidazo[1,2-b]pyridazin-3-yl)ethynyl)pyrrolidine (9b). In a

similar manner to 9a, compound 9b was prepared from 8b (0.28 g,

1

yellow oil, 108%). H NMR (300 MHz, CDCl₃): δ 8.43 (1 H, d, J =

4.2 Hz), 7.95 (1 H, d, J = 9.0 Hz), 7.91 (1 H, s), 7.08 (1 H, dd, J = 4.2

and 9.0 Hz), 3.35−3.10 (5 H, m), 2.31−2.24 (1 H, m), 2.09−2.05 (1

H, m). LC-MS: m/z [M + H]⁺213.1192.

3-(2-(2-Methyl-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo-

[d]imidazol-2-yl)phenyl)ethynyl)imidazo[1,2-b]pyridazine (2a).

Compound 17a (29 mg, 0.2 mmol), compound 16a (110 mg, 0.2

mmol), Pd(PPh₃)₄(17 mg, 0.015 mmol), and CuI (2 mg, 0.01 mmol)

were placed in a two-neck ﬂask with a rubber plug. The mixture

underwent three cycles of vacuuming and ﬁlling with Ar₂. Then, a

solution of DIPEA (58 mg, 0.45 mmol) and DMF (2 mL) was

injected into the ﬂask. The mixture was stirred at rt for 20 h, then

poured into 25 mL of water and extracted with CH₂Cl₂(20 mL × 3).

The organic layer was washed with brine, dried over Na₂SO₄, and

ﬁltered, and the ﬁltrate was evaporated in vacuo. The residue was

puriﬁed by chromatography on silica gel (CH₂Cl₂/CH₃OH = 97:3 to

97:6) to give 0.1 g of the crude product, and continued puriﬁcation

via preparation TLC (CH₂Cl₂/CH₃OH = 120:8) gave 15 mg of the

N-(3-(Triﬂuoromethyl)-4-((4-methylpiperazin-1-yl)methyl)-

phenyl)-3-(2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl)pyrrolidine-1-

carboxamide (1b). In a similar manner to 1a, compound 1b was

prepared from 9b and 10 (46 mg, pale yellow solid, 19%). mp: 83−85

1

°C. H NMR (300 MHz, CDCl₃): δ 8.44 (1 H, s), 7.98−7.92 (2 H,

m), 7.64−7.58 (3 H, m), 7.10 (1 H, dd, J = 4.5 and 9.0 Hz), 6.37 (1

H, s), 3.93−3.88 (1 H, m), 3.76 (1 H, m), 3.70−3.49 (4 H, m), 2.62

(8 H, brs), 2.44 (4 H, brs), 2.31−2.29 (2 H, m). HR-MS (ESI-

TOF⁺): m/z [M + H]⁺calcd for C₂₆H₂₉F₃N₇O 512.2380, found

512.2375.

Synthesis of Compounds 2a−k. (3,4-Dinitrophenyl)-(4-methyl-

piperazin-1-yl)-methanone (12). A mixture of 3,4-dinitrobenzoic

acid 11 (10.6 g, 50 mmol) and SOCl₂(50 mL) was heated at reﬂux

for 6 h. Then, the mixture was evaporated to dryness in vacuo. The

residue was dissolved in CH₂Cl₂(50 mL) and cooled to 5 °C. To this

solution were added N-methylpiperazine (5.5 g, 55 mmol) and Et₃N

(5.5 g, 55 mmol) dropwise as a solution in CH₂Cl₂(50 mL). After

stirring overnight at rt, the combined organic phase was washed with

water (100 mL), dried over Na₂SO₄, ﬁltered, and concentrated. The

resulting residue was puriﬁed by silica gel chromatography (eluent of

5% MeOH in CH₂Cl₂) to give a yellow solid (14.0 g, 95%). ¹H NMR

(300 MHz, DMSO-d₆): δ 8.29 (1 H, s), 8.26 (1 H, d, J = 9.0 Hz),

7.96 (1 H, d, J = 9.0 Hz), 3.62 (2 H, brs), 3.28 (2 H, brs), 2.38 (2 H,

brs), 2.26 (2 H, brs), 2.19 (3 H, s).

1-(3,4-Dinitrobenzyl)-4-methylpiperazine (13). To a cooled

solution (5 °C) of compound 12 (5.88 g, 20 mmol) in THF was

added powdered NaBH₄(1.89 g, 50 mmol), followed by the dropwise

addition of BF₃·Et₂O (6.4 mL, 50 mmol) while keeping the

temperature below 5 °C. The mixture was allowed to come to rt

over 2 h and then stirred for a further 3 h at rt. MeOH was then

added cautiously to the mixture. Stirring was continued for 10 min,

then the mixture was concentrated. The residue was partitioned

between EtOAc (150 mL) and saturated aqueous NaHCO₃(150

mL). The organic layer was washed with water (100 mL) and brine

(100 mL), then dried over Na₂SO₄, ﬁltered, and concentrated. The

residue was puriﬁed by silica gel chromatography (eluent of 2%

MeOH in CH₂Cl₂) to give a yellow solid (4.5 g, 80%). ¹H NMR (400

MHz, CDCl₃): δ 7.92−7.90 (2 H, m), 7.70 (1 H, d, J = 8.4 Hz), 3.73

(2 H, s), 3.10−3.07 (2 H, m), 2.99−2.94 (2 H, m), 2.81−2.76 (2 H,

m), 2.67 (3 H, s), 2.56−2.53 (2 H, m).

1-(3,4-Diaminobenzyl)-4-methylpiperazine (14). Compound 13

(2.8 g, 10 mmol) was dissolved in DMF/MeOH (1:1, 20 mL) and

agitated with 10% Pd/C (280 mg) under an atmosphere of H₂for 12

h. The reaction was monitored by TLC. The mixture was then ﬁltered

and evaporated to give a dark solid, which was used immediately

without any further puriﬁcation.

2-(3-Iodo-4-methylphenyl)-6-((4-methylpiperazin-1-yl)methyl)-

1H-benzo[d]imidazole (16a). A mixture of compound 14 (10

mmol), 3-iodo-4-methylbenzoic acid 15a (2.6 g, 10 mmol), EDCI (11

mmol), and HOBt (11 mmol) in dry DMF (25 mL) was stirred at

ambient temperature for 24 h. The mixture was then evaporated in

vacuo, and the crude residue was dissolved in CH₂Cl₂(100 mL),

washed with water (100 mL) and brine (100 mL), dried over Na₂SO₄,

ﬁltered, and concentrated. The residue obtained was dissolved in

AcOH (30 mL), and the mixture was heated at reﬂux for 3 h. The

reaction mixture was evaporated in vacuo, and the residue was puriﬁed

by silica gel chromatography (eluent of 10% MeOH in CH₂Cl₂,

MeOH was added to 0.5% Et₃N) to give a yellow solid (1.35 g, 30%).

¹H NMR (300 MHz, CDCl₃): δ 8.50 (1 H, s), 7.96 (1 H, d, J = 9.0

1

product as a pale yellow solid (16%). mp: 114−115 °C. H NMR

(300 MHz, CDCl₃): δ 8.47 (1 H, d, J = 3.6 Hz), 8.25 (1 H, s), 8.15 (1

H, s), 8.11 (1 H, d, J = 7.5 Hz), 7.98 (1 H, d, J = 9.0 Hz), 7.59 (2 H,

brs), 7.36 (1 H, d, J = 7.8 Hz), 7.22 (1 H, s), 7.13 (1 H, dd, J = 9.3

and 4.2 Hz), 3.64 (2 H, s), 2.60 (11 H, brs), 2.34 (3 H, s). ¹³C NMR

(150 MHz, DMSO-d₆): δ 151.01, 145.53, 141.52, 140.12, 138.71,

131.00, 129.26, 128.71, 127.38, 126.89, 126.59, 124.55, 122.75,

119.53, 119.39, 118.93, 112.25, 110.38, 97.04, 81.40, 62.31, 54.19,

51.51, 44.69, 20.74. HR-MS (ESI-TOF⁺): m/z [M + H]⁺calcd for

C₂₈H₂₈N₇462.2401, found 462.2413.

N-Cyclobutyl-6-(2-(2-methyl-5-(6-((4-methylpiperazin-1-yl)-

methyl)-1H-benzo[d]imidazol-2-yl)phenyl)ethynyl)pyridazin-3-

amine (2b). In a similar manner to 2a, compound 2b was prepared

from 16a and 17b (87 mg, a pale yellow solid, 59%). mp: 148−150

°C. ¹H NMR (300 MHz, CDCl₃): δ 8.08 (1 H, d, J = 8.1 Hz), 7.90 (1

H, s), 7.66−7.63 (2 H, m), 7.24−7.15 (3 H, m), 6.62 (1 H, d, J = 9.0

Hz), 5.63 (1 H, d, J = 6.6 Hz), 4.33−4.26 (1 H, m), 3.62 (2 H, s),

2.58−2.45 (10 H, m), 2.35 (3 H, s), 2.31 (3 H, s), 2.03−1.94 (2 H,

m), 1.85−1.82 (2 H, m). ¹³C NMR (150 MHz, CDCl₃): δ 157.13,

151.77, 143.32, 142.12, 138.96, 136.96, 131.22, 130.22, 130.12,

129.99, 127.93, 127.54, 126.47, 124.20, 122.43, 112.20, 101.35, 90.23,

89.89, 63.00, 54.63, 52.08, 47.12, 45.38, 31.01, 20.51, 15.29. HR-MS

(ESI-TOF⁺): m/z [M + H]⁺calcd for C₃₀H₃₄N₇492.2870, found

492.2856.

N-Cyclopropyl-6-(2-(2-methyl-5-(6-((4-methylpiperazin-1-yl)-

methyl)-1H-benzo[d]imidazol-2-yl)phenyl)ethynyl)pyridazin-3-

amine (2c). In a similar manner to 2a, compound 2c was prepared

from 16a and 17c (30 mg, a khaki solid, 28%). mp: 145−146 °C. ¹H

NMR (300 MHz, CDCl₃): δ 8.07 (1 H, d, J = 8.7 Hz), 7.99 (1 H, s),

7.64−7.61 (2 H, m), 7.39 (1 H, d, J = 9.3 Hz), 7.22−7.18 (2 H, m),

7.02 (1 H, d, J = 9.3 Hz), 5.98 (1 H, s), 3.61 (2 H, s), 2.55−2.30 (9

H, m), 2.58 (1 H, m), 2.28 (3 H, s), 2.26 (3 H, s), 0.87−0.84 (2 H,

m), 0.64 (2 H, brs). ¹³C NMR (150 MHz, DMSO-d₆): δ 159.65,

150.84, 144.25, 143.45, 141.77, 138.30, 131.14, 130.93, 129.71,

128.73, 127.30, 123.52, 122.77, 119.47, 118.82, 112.31, 111.76, 92.29,

88.42, 62.92, 55.26, 52.99, 46.21, 23.99, 20.70, 7.22. HR-MS (ESI-

TOF⁺): m/z [M + H]⁺calcd for C₂₉H₃₂N₇478.2714, found 478.2721.

N-Isopropyl-6-(2-(2-methyl-5-(6-((4-methylpiperazin-1-yl)-

methyl)-1H-benzo[d]imidazol-2-yl)phenyl)ethynyl)pyridazin-3-

amine (2d). In a similar manner to 2a, compound 2d was prepared

from 16a and 17d (40 mg, a pale yellow solid, 37%). mp: 129−130

°C. ¹H NMR (300 MHz, CDCl₃): δ 8.07 (1 H, d, J = 8.1 Hz), 7.87 (1

H, s), 7.65 (2 H, brs), 7.27 (1 H, s), 7.25−7.19 (2 H, m), 6.62 (1 H,

d, J = 9.0 Hz), 5.02 (1 H, d, J = 7.5 Hz), 4.15−4.11 (1 H, m), 3.61 (2

H, s), 2.43 (8 H, brs), 2.32 (3 H, s), 2.27 (3 H, s), 1.28 (6 H, d, J =

6.0 Hz). ¹³C NMR (150 MHz, CDCl₃): δ 157.13, 151.63, 142.08,

138.83, 131.05, 130.10, 130.08, 129.76, 128.88, 128.36, 127.94,

127.44, 124.14, 122.45, 121.09, 112.68, 90.25, 89.77, 63.38, 55.07,

52.95, 45.94, 43.38, 22.76, 20.50. HR-MS (ESI-TOF⁺): m/z [M +

H]⁺calcd for C₂₉H₃₄N₇480.2870, found 480.2847.

Hz), 7.55 (1 H, d, J = 9.0 Hz), 7.54 (1 H, d, J = 9.0 Hz), 7.30 (1 H,

s), 7.22 (1 H, d, J = 9.0 Hz), 3.60 (2 H, s), 2.54 (8 H, brs), 2.45 (3 H,

s), 2.32 (3 H, s).

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N-Cyclopropyl-5-(2-(2-methyl-5-(6-((4-methylpiperazin-1-yl)-

methyl)-1H-benzo[d]imidazol-2-yl)phenyl)ethynyl)pyrimidin-2-

amine (2e). In a similar manner to 2a, compound 2e was prepared

from 16a and 17e (30 mg, a brown solid, 23%). mp: 136−137 °C. ¹H

NMR (300 MHz, CDCl₃): δ 8.40 (2 H, s), 8.21 (1 H, s), 7.98 (1 H,

d, J = 8.1 Hz), 7.55−7.48 (2 H, m), 7.23−7.15 (2 H, m), 5.81 (1 H,

s), 5.28 (1 H, s), 3.57 (2 H, s), 2.79−2.77 (1 H, m), 2.55 (8 H, brs),

2.46 (3 H, s), 2.32 (3 H, s), 0.87−0.83 (2 H, m), 0.56 (2 H, brs). ¹³C

NMR (150 MHz, CDCl₃): δ 161.58, 160.20, 151.64, 141.87, 131.17,

130.90, 130.29, 129.87, 128.89, 128.81, 127.61, 126.43, 125.04,

124.32, 123.69, 108.36, 90.78, 88.66, 62.55, 54.04, 51.12, 44.73, 23.99,

20.75, 7.44. HR-MS (ESI-TOF⁺): m/z [M + H]⁺calcd for C₂₉H₃₂N₇

478.2714, found 478.2718.

H, d, J = 9.3 Hz), 7.54−7.46 (3 H, m), 7.19 (1 H, d, J = 7.8 Hz), 7.11

(1 H, d, J = 4.5 Hz), 3.60 (2 H, s), 2.61 (8 H, brs), 2.31 (3 H, s). ¹³

NMR (150 MHz, CDCl₃): δ 150.51, 144.10, 139.92, 139.86, 138.81,

138.72, 137.06, 131.16, 130.45, 130.11, 128.93, 128.07, 125.76,

123.17, 123.11, 118.25, 118.09, 117.68, 112.90, 94.94, 81.51, 62.76,

54.36, 51.64, 45.08. HR-MS (ESI-TOF⁺): m/z [M + H]⁺calcd for

C₂₇H₂₅ClN₇482.1854, found 482.1841.

C

6-(2-(2-Chloro-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo-

[d]imidazol-2-yl)phenyl)ethynyl)-N-cyclobutylpyridazin-3-amine

(2h). In a similar manner to 2a, compound 2g was prepared from 17b

1

and 16b (70 mg, a pale yellow solid, 64%). mp: 161−163 °C. H

NMR (300 MHz, CDCl₃): δ 8.14 (1 H, d, J = 7.2 Hz), 7.95 (1 H, s),

7.65−7.62 (2 H, m), 7.38 (1 H, d, J = 8.7 Hz), 7.31 (1 H, d, J = 9.0

Hz), 7.19 (1 H, d, J = 8.1 Hz), 6.65 (1 H, d, J = 9.3 Hz), 5.80 (1 H, d,

J = 5.7 Hz), 4.30−4.28 (1 H, m), 3.63 (2 H, s), 2.63−2.40 (13 H, m),

2.02−1.96 (2 H, m), 1.81−1.79 (2 H, m). ¹³C NMR (100 MHz,

CDCl₃): δ 157.22, 150.53, 138.33, 136.91, 132.49, 131.47, 131.26,

130.01, 129.87, 129.56, 129.19, 129.06, 128.51, 128.02, 124.56,

122.41, 112.27, 91.11, 87.86, 63.23, 54.85, 52.63, 47.08, 45.73, 30.94,

15.29. HR-MS (ESI-TOF⁺): m/z [M + H]⁺calcd for C₂₉H₃₁ClN₇

512.2324, found 512.2303.

4-(4-Methyl-1H-imidazol-1-yl)-2-nitroaniline (19). A suspension

of 4-bromo-2-nitroaniline 18 (4.34 g, 20 mmol), 4-methylimidazole

(1.97 g, 24 mmol), K₂CO₃(3.04 g, 22 mmol), CuI (0.57 g, 3 mmol)

and 8-hydroxyquinoline (0.44 g, 3 mmol) in 20 mL of DMSO was

stirred at 120 °C in a sealed tube under Ar₂for 29 h. The mixture was

cooled to rt, and 28% aqueous ammonia (10 mL) was added. To the

mixture were added H₂O and EtOAc. The aqueous layer was

extracted with EtOAc (80 mL × 3), and the organic layers were

washed with brine, dried over Na₂SO₄, and ﬁltered. The ﬁltrate was

evaporated in a vacuum, and the residue was washed with PE/EtOAc

6-(2-(2-Chloro-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo-

[d]imidazol-2-yl)phenyl)ethynyl)-N-cyclopropylpyridazin-3-amine

(2i). In a similar manner to 2a, compound 2i was prepared from 17c

1

to give 2.47 g of the product as a red solid (57%). H NMR (300

1

and 16b (30 mg, a khaki solid, 28%). mp: 130−131 °C. H NMR

MHz, DMSO-d₆): δ 8.06−8.02 (2 H, m), 7.68 (1 H, dd, J = 2.7 and

9.0 Hz), 7.56 (2 H, s), 7.36 (1 H, s), 7.15 (1 H, d, J = 9.3 Hz), 2.15 (3

H, s). LC-MS: m/z [M + H]⁺219.0895.

(300 MHz, CDCl₃): δ 8.12 (1 H, d, J = 8.1 Hz), 7.86 (1 H, s), 7.64−

7.55 (2 H, m), 7.45−7.39 (2 H, m), 7.22 (1 H, s), 7.02 (1 H, d, J =

9.3 Hz), 6.00 (1 H, s), 3.61 (2 H, s), 2.60−2.44 (9 H, m), 2.27 (3 H,

s), 0.89−0.87 (2 H, m), 0.65 (2 H, brs). ¹³C NMR (150 MHz,

DMSO-d₆): δ 159.75, 149.77, 144.31, 143.38, 137.73, 135.89, 131.25,

130.68, 130.01, 128.60, 124.79, 123.74, 122.66, 119.60, 119.01,

112.18, 111.55, 93.32, 86.35, 62.88, 55.24, 52.98, 46.19, 23.99, 7.20.

HR-MS (ESI-TOF⁺): m/z [M + H]⁺calcd for C₂₈H₂₉ClN₇498.2167,

found 498.2150.

2-Amino-4-(4-methyl-1H-imidazol-1-yl)aniline (20). Compound

19 (0.22 g, 1 mmol) was suspended in 20 mL of anhydrous methanol.

The mixture was hydrogenated with 0.11 g of Raney Ni at 40 psi for 7

h. Then, the Raney Ni was removed by ﬁltration. The ﬁltrate was

evaporated to give 0.18 g of the title compound as a yellow solid

(96%), which was used immediately without any further puriﬁcation.

N-(2-Amino-4-(4-methyl-1H-imidazol-1-yl)phenyl)-3-iodo-4-

methylbenzamide (21). A solution of 3-iodo-4-methylbenzoic acid

(0.26 g, 1 mmol) in SOCl₂(5 mL) was reﬂuxed for 2 h and then

evaporated in a vacuum to remove the residual SOCl₂. The residue

was dissolved in 5 mL of anhydrous THF and added dropwise to a

solution of triethylamine (0.12 g, 1.2 mmol), compound 20 (0.18 g, 1

mmol), and DMAP (24 mg) in 5 mL of anhydrous THF. The result

mixture was stirred at rt for 20 h and then evaporated in a vacuum.

The residue was puriﬁed by chromatography on silica gel (CH₂Cl₂/

CH₃OH = 97:3) to give 0.16 g of the product as a pale yellow solid

(37%). LC-MS: m/z [M + H]⁺433.0520.

2-(3-Iodo-4-methylphenyl)-6-(4-methyl-1H-imidazol-1-yl)-1H-

benzo[d]imidazole (22). A solution of compound 21 (0.16 g, 0.37

mmol) in 5 mL of glacial acetic acid was reﬂuxed for 8 h. Then, the

mixture was evaporated in a vacuum, and the residue was puriﬁed by

chromatography on silica gel (CH₂Cl₂/CH₃OH = 97:3 to 94:6) to

give 0.1 g of the product as a pale yellow solid (65%). ¹H NMR (300

MHz, DMSO-d₆): δ 8.65 (1 H, s), 8.13−8.11 (2 H, m), 7.71 (1 H, d,

J = 8.4 Hz), 7.55−7.51 (2 H, m), 7.45−7.39 (3 H, m), 2.45 (3 H, s),

2.19 (3 H, s). LC-MS: m/z [M + H]⁺415.0409.

6-(2-(2-Chloro-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo-

[d]imidazol-2-yl)phenyl)ethynyl)-N-isopropylpyridazin-3-amine

(2j). In a similar manner to 2a, compound 2j was prepared from 17d

1

and 16b (70 mg, a khaki solid, 65%). mp: 127−128 °C. H NMR

(300 MHz, CDCl₃): δ 8.12 (1 H, d, J = 7.5 Hz), 7.82 (1 H, s), 7.64−

7.53 (3 H, m), 7.36 (1 H, d, J = 8.4 Hz), 7.30 (1 H, d, J = 9.3 Hz),

6.68 (1 H, d, J = 9.3 Hz), 5.22 (1 H, d, J = 6.6 Hz), 4.14−4.12 (1 H,

m), 3.62 (2 H, s), 2.51 (8 H, brs), 2.31 (3 H, s), 1.30 (6 H, d, J = 6.0

Hz). ¹³C NMR (100 MHz, CDCl₃): δ 162.61, 157.33, 150.63, 138.26,

136.97, 131.33, 131.24, 129.82, 129.58, 129.23, 129.15, 128.56,

128.03, 124.53, 123.48, 122.37, 112.98, 91.14, 87.85, 63.31, 55.03,

52.84, 45.89, 43.45, 22.73. HR-MS (ESI-TOF⁺): m/z [M + H]⁺calcd

for C₂₈H₃₁ClN₇500.2324, found 500.2313.

5-(2-(2-Chloro-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo-

[d]imidazol-2-yl)phenyl)ethynyl)-N-cyclopropylpyrimidin-2-amine

(2k). In a similar manner to 2a, compound 2k was prepared from 17e

1

and 16b (10 mg, a brown solid, 9%). mp: 160−162 °C. H NMR

(300 MHz, CDCl₃): δ 8.44 (2 H, d, J = 4.5 Hz), 8.13 (1 H, d, J = 7.8

Hz), 7.59 (1 H, d, J = 7.5 Hz), 7.52 (1 H, s), 7.46 (1 H, d, J = 8.7

Hz), 7.39 (1 H, d, J = 8.4 Hz), 7.16 (1 H, d, J = 7.5 Hz), 5.80 (1 H,

s), 3.60 (2 H, s), 3.12−2.81 (9 H, m), 2.59 (3 H, s), 0.88−0.82 (2 H,

m), 0.61−0.58 (2 H, m). ¹³C NMR (150 MHz, DMSO-d₆): δ 162.27,

160.69, 150.22, 135.87, 135.60, 134.96, 130.98, 130.71, 130.56,

129.84, 129.50, 128.87, 128.69, 128.11, 123.31, 105.93, 91.36, 88.80,

63.27, 52.90, 49.61, 45.79, 24.41, 6.78. HR-MS (ESI-TOF⁺): m/z [M

+ H]⁺calcd for C₂₈H₂₉ClN₇498.2167, found 498.2163.

3-((2-Methyl-5-(5-(4-methyl-1H-imidazol-1-yl)-1H-benzo[d]-

imidazol-2-yl)phenyl)ethynyl)imidazo[1,2-b]pyridazine (2f). In a

similar manner to 2a, compound 2f was prepared from 17a and 22

1

(80 mg, a pale yellow solid, 63%). mp: 182−184 °C. H NMR (400

MHz, CD₃OD): δ 8.57 (1 H, d, J = 3.6 Hz), 8.36 (1 H, s), 8.24 (1 H,

s), 8.05−7.98 (3 H, m), 7.70 (1 H, s), 7.67 (1 H, d, J = 9.0 Hz),

7.47−7.40 (3 H, m), 7.30 (1 H, dd, J = 4.2 and 9.2 Hz), 2.59 (3 H, s),

2.28 (3 H, s). ¹³C NMR (150 MHz, CD₃OD): δ 153.42, 144.66,

142.71, 139.83, 136.69, 134.16, 134.04, 131.52, 130.27, 129.41,

126.93, 126.81, 124.99, 123.15, 119.10, 117.19, 116.93, 112.97, 96.26,

80.08, 19.47, 9.94. HR-MS (ESI-TOF⁺): m/z [M + H]⁺calcd for

C₂₆H₂₀N₇430.1775, found 430.1778.

Synthesis of Compounds 3a−e. 3,3-Diﬂuoro-1-(4-methylpiper-

azin-1-yl)-2,3-dihydro-1H-inden-5-amine (24). Compound 23 (0.15

g, 0.5 mmol) was dissolved in MeOH (20 mL) and agitated with 10%

Pd/C (15 mg) under an atmosphere of H₂for 12 h. The reaction was

monitored by TLC. The mixture was then ﬁltered and evaporated to

give a gray solid, which was used immediately without any further

puriﬁcation.

N-(3,3-Diﬂuoro-1-(4-methylpiperazin-1-yl)-2,3-dihydro-1H-

inden-5-yl)-3-iodo-4-methylbenzamide (25a). 3-Iodo-4-methylben-

zoyl chloride, which was prepared from the reaction of 3-iodo-4-

methylbenzoic acid (79 mg, 0.3 mmol) and SOCl₂, was added to a

3-(2-(2-Chloro-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo-

[d]imidazol-2-yl)phenyl)ethynyl)imidazo[1,2-b]pyridazine (2g). In a

similar manner to 2a, compound 2g was prepared from 17a and 16b

(80 mg, a khaki solid, 78%). mp: 156−157 °C. ¹H NMR (300 MHz,

CDCl₃): δ 8.43 (1 H, s), 8.36 (1 H, s), 8.14−8.11 (2 H, m), 7.93 (1

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solution of compound 24 (78 mg, 0.3 mmol), Et₃N (37 mg 0.36

mmol), and a catalytic amount of DMAP in THF (5 mL). After

stirring at rt overnight, the reaction was quenched with water,

extracted with CH₂Cl₂(20 mL × 3), dried over Na₂SO₄, and puriﬁed

by chromatography on sililca gel (CH₂Cl₂/MeOH = 95:5) to aﬀord

140 mg of the product as a yellow solid in a 92% yield. LC-MS: m/z

[M + H]⁺512.1049.

N-(3,3-Diﬂuoro-1-(4-methylpiperazin-1-yl)-2,3-dihydro-1H-

inden-5-yl)-3-iodobenzamide (25b). In a similar manner to 25a,

compound 25b was prepared from compound 24 and 3-iodo-benzoyl

chloride (100 mg, yellow solid, 51%). LC-MS: m/z [M + H]⁺

498.0801.

H, s), 7.95 (1 H, s), 7.85 (2 H, d, J = 8.0 Hz), 7.80 (1 H, s), 7.68 (1

H, d, J = 7.6 Hz), 7.52−7.47 (2 H, m), 5.55 (1 H, s), 4.51−4.49 (1 H,

m), 2.82 (1 H, m), 2.66−2.52 (10 H, m), 2.36 (3 H, s), 0.91−0.86 (2

H, m), 0.59 (2 H, brs). ¹³C NMR (150 MHz, CDCl₃): δ 164.90,

161.63, 160.40, 139.30, 138.66, 137.99 (t, J = 26.1 Hz), 134.91,

134.62, 129.75, 129.11, 127.77 (t, J = 243.0 Hz), 126.96, 126.30,

124.07, 123.93, 114.34, 107.87, 91.41, 85.53, 64.31, 54.96, 45.33,

35.65 (t, J = 22.8 Hz), 29.71, 23.98, 7.44. HR-MS (ESI-TOF⁺): m/z

[M + H]⁺calcd for C₃₁H₃₄F₂N₆O 529.2522, found 529.2506.

3,3-Diﬂuoro-2-(4-methylpiperazin-1-yl)-2,3-dihydro-1H-inden-5-

amine (27). Compound 26 (1.06 g, 3.56 mmol) was dissolved in

MeOH (20 mL) and agitated with 10% Pd/C (15 mg) under an

atmosphere of H₂for 12 h. The reaction was indicated by TLC. The

mixture was then ﬁltered and evaporated to give a gray solid, which

was used immediately without any further puriﬁcation.

N-(3,3-Diﬂuoro-2-(4-methylpiperazin-1-yl)-2,3-dihydro-1H-

inden-5-yl)-3-iodo-4-methylbenzamide (28). In a similar manner to

25a, compound 28 was prepared from compound 27 and 3-iodo-4-

methylbenzoyl chloride (220 mg, a colorless solid, 86%). LC-MS: m/z

[M + H]⁺512.0918.

N-(3,3-Diﬂuoro-2-(4-methylpiperazin-1-yl)-2,3-dihydro-1H-

inden-5-yl)-3-(imidazo[1,2-b]pyridazin-3-ylethynyl)-4-methylben-

zamide (3e). In a similar manner to 3a, compound 3e was prepared

from 17a and 28 (5 mg, a pale yellow solid, 5%). mp: 250−252 °C.

¹H NMR (400 MHz, CDCl₃): δ 8.48 (1 H, d, J = 3.2 Hz), 8.08−8.05

(3 H, m), 8.00 (1 H, d, J = 9.2 Hz), 7.83−7.78 (3 H, m), 7.40 (1 H, d,

J = 7.6 Hz), 7.29 (1 H, s), 7.13 (1 H, dd, J = 8.8 and 4.0 Hz), 4.50−

4.52 (1 H, m), 3.27−2.96 (6 H, m), 2.74−2.64 (7 H, m), 2.36 (3 H,

s).). ¹³C NMR (150 MHz, CDCl₃): δ 164.78, 144.53, 143.92, 139.77,

138.43, 137.67, 136.58 (t, J = 21.5 Hz), 135.65, 132.25, 130.26,

130.03, 127.71, 126.76 (t, J = 234.0 Hz), 125.96, 125.91, 124.07,

122.99, 117.78, 114.86, 113.07, 96.65, 80.87, 69.76 (t, J = 23.7 Hz),

54.54, 32.54, 29.70, 20.96. HR-MS (ESI-TOF⁺): m/z [M + H]⁺calcd

for C₃₀H₂₉F₂N₆O: 527.2365, found 527.2337.

Separation of the Optical Isomers of Compound 3a. The

racemate product 3a (0.2 g) was subjected to a separation by the SFC

method using a chiral column (CHIRALCEL AS-H, 0.46 cm I.D. ×25

cm·L). Mobile phase: CO₂/MeOH (0.1% DEA) = 60/40 (v/v). Flow

rate: 2.0 mL/min. Wavelength: UV 254 nm. The products were

respectively collected at peak 1 with a retention time of 19.492 min

(3a-P1, 89 mg, [α]²_D⁰= +15.96 (c 1.0, CHCl₃)) and peak 2 with a

retention time of 24.422 min (3a-P2, 90 mg, [α]²_D⁰= −16.19 (c 1.0,

CHCl₃)).

Separation of the Optical Isomers of Compound 3b. The

racemate product 3b (0.16 g) was subjected to separation by the SFC

method using a chiral column (CHIRALCEL OJ-H, 0.46 cm I.D. ×25

cm·L). Mobile phase: CO₂/EtOH (0.1% DEA) = 75/25 (v/v). Flow

rate: 2.0 mL/min. Wavelength: UV 280 nm. The products were

respectively collected at peak 1 with a retention time of 7.093 min

(3b-P1, 57 mg, [α]²_D⁰= +14.79 (c 1.0, CHCl₃)) and peak 2 with a

retention time of 8.287 min (3b-P2, 69 mg, [α]²_D⁰= −12.69 (c 1.0,

CHCl₃)).

Synthesis of Compound 3a-P1. (S)-1-(3,3-Diﬂuoro-5-nitro-2,3-

dihydro-1H-inden-1-yl)-4-methylpiperazine ((S)-23). To a solution

of compound 23 (5 g, 16.8 mmol) in 50 mL of methanol was added

D-camphorsulfonic acid (8.99 g, 38.7 mmol) at room temperature.

Then, 100 mL of isopropanol was added. The mixture was reﬂuxed

and then cooled to room temperature. The solution was stirred at rt

overnight, and the resultant solid was collected by ﬁltration (5 g). The

solid was recrystallized in a solution of methanol/isopropanol = 1:3 to

give a colorless solid (4.1 g, 32%). The solid was dissolved in 20 mL

of H₂O, then a 15 mL of a 1 mol/L NaOH solution was added in

dropwise. The mixture was stirred for 0.5 h, then the aqueous layer

was separated, which was further extracted with CH₂Cl₂(3 × 20 mL).

The combined organic layers were washed with brine, dried over

Na₂SO₄, and evaporated to give 1.6 g of the product as yellow oil

(100%). ¹H NMR (400 MHz, CDCl₃): δ 8.47−8.36 (2 H, m), 7.66 (1

H, d, J = 8.4 Hz), 4.58 (1 H, brs), 2.74−2.55 (10 H, m), 2.36 (3 H, s).

[α]²_D⁰= +18.51 (c 1.1, CHCl₃).

N-(3,3-Diﬂuoro-1-(4-methylpiperazin-1-yl)-2,3-dihydro-1H-

inden-5-yl)-3-(imidazo[1,2-b]pyridazin-3-ylethynyl)-4-methylben-

zamide (3a). Compound 17a (27 mg, 0.19 mmol), compound 25a

(80 mg, 0.16 mmol), Pd(PPh₃)₄(9 mg, 0.008 mmol), and CuI (2.2

mg, 0.012 mmol) were placed in a two-neck ﬂask with a rubber plug.

The mixture underwent three cycles of vacuuming and ﬁlling with Ar₂,

then a solution of DIPEA (30 mg, 0.24 mmol) and DMF (2 mL) was

injected into the ﬂask. The mixture was stirred at rt for 20 h and then

poured into 25 mL of water and extracted with CH₂Cl₂(20 mL × 3).

The organic layer was washed with brine, dried with Na₂SO₄, and

ﬁltered, and the ﬁltrate was evaporated in a vacuum. The residue was

puriﬁed by chromatography on silica gel (CH₂Cl₂/CH₃OH = 97:3 to

97:6) to give 50 mg of the crude product, and continued puriﬁcation

via preparation TLC (CH₂Cl₂/CH₃OH = 150:15) gave 25 mg of the

1

product as a pale yellow solid (30%). mp: 111−113 °C. H NMR

(300 MHz, CDCl₃): δ 8.48 (1 H, d, J = 4.2 Hz), 8.18 (1 H, s), 8.08 (1

H, s), 8.06 (1 H, d, J = 1.8 Hz), 7.98 (1 H, dd, J = 9.6 and 1.5 Hz),

7.88−7.80 (3 H, m), 7.48 (1 H, d, J = 8.7 Hz), 7.39 (1 H, d, J = 8.7

Hz), 7.13 (1 H, dd, J = 9.0 and 4.2 Hz), 4.52−4.50 (1 H, m), 2.67−

2.58 (7 H, m), 2.49 (6 H, brs), 2.31 (3 H, s). ¹³C NMR (150 MHz,

CDCl₃): δ 164.90, 144.54, 143.95, 139.77, 139.51, 138.76, 138.40,

137.91 (t, J = 26.0 Hz), 132.29, 130.26, 130.05, 127.94 (t, J = 241.2

Hz), 127.82, 126.35, 125.94, 124.02, 122.91, 117.82, 114.28, 113.10,

96.65, 80.83, 64.35, 55.21, 45.86, 35.34 (t, J = 23.3 Hz), 20.95. HR-

+

MS (ESI-TOF⁺): m/z [M + H] calcd for C₃₀H₂₉F₂N₆O 527.2365,

found 527.2331.

3-((2-(Cyclopropylamino)pyrimidin-5-yl)ethynyl)-N-(3,3-di-

ﬂuoro-1-(4-methylpiperazin-1-yl)-2,3-dihydro-1H-inden-5-yl)-4-

methylbenzamide (3b). In a similar manner to 3a, compound 3b was

prepared from 17e and 25a (10 mg, a pale yellow solid, 23%). mp:

95−97 °C. ¹H NMR (300 MHz, CDCl₃): δ 8.50 (2 H, s), 7.95 (1 H,

s), 7.91 (1 H, s), 7.85 (1 H, d, J = 8.1 Hz), 7.79−7.74 (2 H, m), 7.48

(1 H, d, J = 7.8 Hz), 7.36 (1 H, d, J = 8.1 Hz), 4.51−4.49 (1 H, m),

2.82 (1 H, brs), 2.67−2.47 (13 H, m), 2.30 (3 H, s), 0.94−0.85 (2 H,

m), 0.62−0.60 (2 H, m). ¹³C NMR (150 MHz, CDCl₃): δ 164.86,

161.63, 160.24, 144.15, 139.42, 138.65, 137.90 (t, J = 26.1 Hz),

132.20, 130.15, 130.03, 127.87 (t, J = 243.3 Hz), 127.02, 126.31,

123.98, 123.55, 114.23, 108.11, 90.40, 89.14, 64.33, 55.12, 45.71,

35.53 (t, J = 23.0 Hz), 29.70, 23.98, 20.89, 7.43. HR-MS (ESI-TOF⁺):

m/z [M + H]⁺calcd for C₃₁H₃₃F₂N₆O 543.2684, found 543.2654.

N-(3,3-Diﬂuoro-1-(4-methylpiperazin-1-yl)-2,3-dihydro-1H-

inden-5-yl)-3-(imidazo[1,2-b]pyridazin-3-ylethynyl)benzamide

(3c). In a similar manner to 3a, compound 3c was prepared from 17a

1

and 25b (15 mg, a pale yellow solid, 29%). mp: 136−138 °C. H

NMR (400 MHz, CDCl₃): δ 8.49 (1 H, d, J = 3.2 Hz), 8.19 (1 H, s),

8.09 (2 H, s), 7.98 (1 H, d, J = 9.2 Hz), 7.92−7.85 (3 H, m), 7.78 (1

H, d, J = 7.6 Hz), 7.54−7.48 (2 H, m), 7.13 (1 H, dd, J = 9.2 and 4.0

Hz), 4.52−4.50 (1 H, m), 2.67−2.48 (10 H, m), 2.31 (3 H, s). ¹³C

NMR (150 MHz, CDCl₃): δ 164.81, 143.95, 140.76, 139.76, 139.53,

138.78, 138.58, 137.98 (t, J = 25.9 Hz), 135.00, 134.80, 129.81,

129.17, 127.89 (t, J = 249.2 Hz), 127.68, 126.37, 126.02, 124.01,

123.27, 117.85, 114.31, 112.88, 97.57, 64.34, 55.11, 45.69, 35.55 (t, J

= 23.1 Hz), 29.70. HR-MS (ESI-TOF⁺): m/z [M + H]⁺calcd for

C₂₉H₂₇F₂N₆O 513.2209, found 513.2200.

3-((2-(Cyclopropylamino)pyrimidin-5-yl)ethynyl)-N-(3,3-di-

ﬂuoro-1-(4-methylpiperazin-1-yl)-2,3-dihydro-1H-inden-5-yl)-

benzamide (3d). In a similar manner to 3a, compound 3d was

prepared from 17e and 25b (10 mg, a pale yellow solid, 19%). mp:

105−107 °C. ¹H NMR (400 MHz, CDCl₃): δ 8.50 (1 H, s), 7.98 (1

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(S)-N-(3,3-Diﬂuoro-1-(4-methylpiperazin-1-yl)-2,3-dihydro-1H-

inden-5-yl)-3-(imidazo[1,2-b]pyridazin-3-ylethynyl)-4-methylben-

zamide (3a-P1). In a similar manner to 3a, compound 3a-P1 was

prepared from compound (S)-23 (25 mg, a pale yellow solid, 37%).

mp: 125−127 °C. [α]¹_D⁷= +13.5 (c 1.0, CHCl₃). ¹H NMR (400 MHz,

CDCl₃): δ 8.49 (1 H, d, J = 3.6 Hz), 8.08−8.06 (2 H, m), 7.99 (1 H,

d, J = 9.2 Hz), 7.87 (1 H, d, J = 8.4 Hz), 7.83−7.81 (2 H, m), 7.48 (1

H, d, J = 8.4 Hz), 7.40 (1 H, d, J = 8.0 Hz), 7.15 (1 H, dd, J = 8.8 and

4.0 Hz), 4.51 (1 H, brs), 2.65−2.52 (13 H, m), 2.35 (3 H, s). ¹³C

NMR (150 MHz, CDCl₃): δ 164.98, 144.48, 143.94, 139.75, 139.55,

138.81, 138.36, 137.87 (t, J = 26.3 Hz), 132.31, 130.23, 130.06,

127.97 (t, J = 240.8 Hz), 127.91, 126.34, 125.90, 124.05, 122.82,

117.83, 114.31, 113.12, 96.66, 80.77, 64.36, 55.57, 45.97, 35.48 (t, J =

21.8 Hz), 20.93. HR-MS (ESI-TOF⁺): m/z [M + H]⁺calcd for

C₃₀H₂₉F₂N₆O 527.2365, found 527.2355.

to SDS-PAGE, followed by a transfer to the PVDF membranes

(BioRad, Hercules, CA). Membranes were immunoblotted with the

antibodies against phospho-c-ABL (Y245), phospho-Crkl (Y207), and

eIF 4E, which were purchased from Cell Signaling Technology

(Danvers, MA). Binding was detected with the Pierce ECL Western

blotting substrate (Thermo Scientiﬁc).

Pharmacokinetic Studies. An animal care and use application

for the study was approved by the Institutional Animal Care and Use

Committee of Shanghai Institute of Materia Medica, Chinese

Academy of Sciences. The tested compounds were subjected to PK

in ICR mice (male) weighing 20 to 25 g, with four mice in the oral

administration group and three mice in the intravenous injection

group. The tested compounds were formulated at a concentration of

10 mg/mL for a 15 mg/kg dose given orally and at a concentration of

1 mg/mL for a 5 mg/kg dose given intravenously. The tested

compound was formulated in a 25 mM sodium citrate buﬀer (pH

2.75) for p.o. administration and with 5% DMSO/5% Tween 80/90%

physiological saline for i.v. administration. Blood samples were

collected at 0.25, 0.5, 1, 2, 3, 5, 7, 9, and 24 h after oral dosing and 5

min and 0.25, 0.5, 1, 2, 3, 5, 7, 9, and 24 h after i.v. administration.

Plasma was harvested and stored at −20 °C until analyzed. Plasma

samples were extracted with acetonitrile that contained propranolol as

an internal standard using a 3:1 extractant-to-plasma ratio. The

concentrations of the tested compound were determined by high-

pressure liquid chromatography/tandem mass spectrometry (LC/

MS/MS). Chromatographic separation was performed on a Zobax

SB-C18 column (100 mm × 2.1 mm, 3.5 μm) with an isocratic mobile

phase of acetonitrile/water (70:30, v/v) containing 0.1% formic acid

at a ﬂow rate of 0.2 mL/min. The oral bioavailability was calculated as

the ratio between the area under the curve (AUC) following the

(S)-N-(3,3-Diﬂuoro-1-(4-methylpiperazin-1-yl)-2,3-dihydro-1H-

inden-5-yl)-3-(imidazo[1,2-b]pyridazin-3-ylethynyl)-4-methylben-

zamide Hydrochloride (3a-P1·HCl). To a solution of 3a-P1 (3.93 g,

7.5 mmol) in acetone (110 mL) was added 1 M HCl in an EtOH

solution (6.7 mL, 6.7 mmol). The mixture was stirred at rt for 6 h and

then ﬁltered. The resultant solid was washed with acetone and dried

to give the product as a pale yellow solid (3.84 g, 102%). mp: 240−

1

242 °C. H NMR (400 MHz, DMSO-d₆): δ 10.99 (1 H, brs), 10.63

(1H, s), 8.71 (1 H, dd, J = 4.4 and 1.2 Hz), 8.25 (1 H, dd, J = 9.2 and

1.6 Hz), 8.22 (1 H, s), 8.20 (1 H, d, J = 1.6 Hz), 8.13 (1 H, s), 8.02 (1

H, d, J = 8.4 Hz), 7.97 (1 H, dd, J = 8.0 and 1.6 Hz), 7.54 (1 H, d, J =

8.4 Hz), 7.46 (1 H, d, J = 8.4 Hz), 7.38 (1 H, dd, J = 8.8 and 4.4 Hz),

4.63 (1 H, brs), 3.39−3.30 (2 H, m), 3.05−2.95 (3 H, m), 2.77−2.48

(11 H, m). ¹³C NMR (100 MHz, DMSO-d₆): δ 164.59, 145.03,

143.39, 140.21, 139.61, 138.18, 136.68 (J = 26.3 Hz), 132.29, 130.22,

129.99, 128.50, 128.43 (J = 239.7 Hz), 126.06, 125.97, 124.36,

121.73, 119.05, 113.65, 111.68, 96.40, 81.08, 63.18, 52.57, 46.45,

42.75, 41.88, 35.44 (J = 24.4 Hz), 20.36. HR-MS (ESI-TOF⁺): m/z

[M-HCl+H]⁺calcd for C₃₀H₂₉F₂N₆O 527.2365, found 527.2361.

Cell Lines and Reagents. The cell lines K562, Ku812, U937, and

JURKAT were obtained from the American Type Culture Collection

(ATCC) (Manassas, VA). Native or T315I-mutated BCR-ABL

cDNAs were cloned into the expression lentiviral vector pCDH-

CMV-MCS-EF1-Puro (System Biosciences, Mountain View, CA).

Recombinant lentivirus was produced in 293TN cells with lentiviral

vectors and packaging plasmids using lipofectamine 2000 (Thermo

Fisher Scientiﬁc, Waltham, MA). Two days after the lentiviral

infection, the stably transduced BaF3 cells were established by

selection with 2 μg/mL puromycin for 2 weeks. All the cells were

cultured in RPMI 1640 supplemented with 10% FBS (Atlanta

Biological, Atlanta, GA) and 1% penicillin/streptomycin (Thermo

Fisher Scientiﬁc).

intravenous administration corrected for the dose (F = (AUC_p.o.

dose _i.v.)/(AUC_i.v.× dose_p.o.) × 100%).

×

Mice Xenograft Tumor Models. The experimental protocols

were approved by the Institutional Animal Care and Use Committee

of Shaanxi Normal University. Fifty female BALB/c-nu mice (SPF

(Beijing) Biotechnology Co., Ltd., Beijing, China) were implanted

subcutaneously in the right ﬂank with 5 × 10⁶cells of BaF3/BCR-

ABL^T315Icells. Tumor volumes were measured using a digital caliper

and calculated using the following formula: V = 0.5 × width²× length,

where V stands for tumor volume. When the tumor sizes approached

100−150 mm³, the mice were divided randomly into groups to be

treated once daily by oral gavage with either the vehicle or the BCR-

ABL inhibitor (10 mice per group). When the dosing period was

ﬁnished, the TGI was determined with the formula: TGI = {1 − [(V_te

− V_to/(V_ce− V_co)]} × 100%, where V_teis the mean tumor volume of

the treated group at the ﬁnal measurement, V_tois the mean tumor

volume of the treated group at the ﬁrst measurement, V_ceis the mean

tumor volume of control group at the ﬁnal measurement, and V_cois

the mean tumor volume of control group at the ﬁrst measurement.

Inhibition Evaluation of the hERG K⁺Channel. HEK293 cells

were stably transfected with the human ether-a-go-go-related gene

(hERG) channel. The voltage-gated hERG potassium channel current

was recorded at room temperature (25 °C) from randomly selected

transfected cells using a whole-cell manual patch clamp system

equipped with a EPC10 USB (HEKA) or Multiclamp 700B ampliﬁer

(Molecular Devices), while electrical data were digitalized by a

Digidata1440A system with a sampling frequency at 10 kHz using a

Patchmaster instrument or pClamp10, respectively. The hERG

current inhibition was tested in the presence of ﬁve concentrations

of the inhibitor (30, 10, 3.0, 1.0, and 0.3 μM) for the IC₅₀

determination. Dofetilide was also included as a positive control to

ensure the accuracy and sensitivity of the test system. All experiments

were performed in duplicate for the IC₅₀determination.

Cell Cytotoxicity Assays. K562 and BaF3 (BCR-ABL^T315I) cells

were cultured in RPMI 1640 with 10% FBS and 100 units/mL

penicillin/streptomycin. The cell cytotoxicity assay was performed

with the trypan blue assay. K562 and BaF3 (BCR-ABL^T315I) cells were

seeded in 24-well plates with 1.5 × 10⁵cells/mL per well, then the

cells were treated with ponatinib and test compounds dosed as 100

nM for 48 h. Then, the cells were harvested at 4000 rpm × 4 min and

resuspended in 150 μL of PBS. A total of 10 μL of the resuspended

cells was stained with an equal volume of a 0.4% trypan blue solution.

The stained cell samples were added to the slide, and the number of

positive and negative trypan blue cells was counted by an automatic

cell counter TC20 (Bio-Rad, Richmond, CA).

Cell Proliferation Assays. Cells were seeded in 24-well plates

(0.5 × 10⁵cells/well) and incubated with increasing concentrations of

BCR-ABL inhibitors for 72 h. The inhibitor concentrations tested

ranged from 0 to 200 nM for cells expressing BCR-ABL and 0 to

10 000 nM for BCR-ABL-negative cells. Proliferation was assessed

using the Vybrant MTT cell proliferation assay kit (Thermo Fisher

Scientiﬁc).

Ames Assay. Five strains of Salmonella typhimurium bacteria

(TA97a, TA98, TA100, TA102, and TA1535) were used in the study,

which were originally obtained from Bioplus Biotech (Shanghai, CN).

DMSO was used as the solvent and the vehicle control, and the

highest concentration tested was 312.5 μg/plate. Bacteria, the tested

compound, or the vehicle or positive control formulation and a 10%

S9 mixture or PBS buﬀer were added to molten agar at 37 °C, mixed

Western Blot Analysis. A total of 1 × 10⁶cells were treated in 6-

well plates for 6 h with the indicated compounds. Cell lysates were

prepared in a RIPA buﬀer supplemented with a protease/phosphatase

inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) and subjected

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rapidly, and poured onto a 6-well plate containing minimal agar

medium. After the agar was solidiﬁed, the plates were inverted and

incubated at 37 °C for 48−72 h. Revertant colonies were counted

manually, and the background lawn was inspected for signs of

cytotoxicity. The results were considered to be positive for mutagenic

potential if the increase in the mean revertants at the peak of the

dose−response was greater than two- or threefold of the mean solvent

or vehicle control value and the increase was dose related.

kinase substrate was added into the reaction buﬀer, then the kinase

was added. Compounds at the indicated concentrations were then

delivered to the reaction using an echo liquid handler, and the mixture

was incubated for 30 min at room temperature. Then, a ³³P-ATP

solution (the concentration of ATP was 0.01 mCi/mL) was added to

initiate the reaction, and the mixture was further incubated for 2 h at

room temperature. The resulting reaction was ﬁltered through an ion-

exchange ﬁlter P81 (Whatman) and washed with 0.75% phosphoric

acid three times to remove the ³³P-ATP without adding the substrate

polypeptide. Finally, the ³³P-ATP samples on the ﬁlter paper were

quantiﬁed with a liquid scintillator. The data shown are the mean

value of three experiments.

Chromosome Aberration Test. Chinese hamster lung ﬁbroblasts

were used in the test. Cells were treated with a 0.25% EDTA-trypsin

solution to prepare the cell suspension (5.0 × 10⁵cells/mL) and

cultured for 24 h in a humidiﬁed atmosphere of 5% CO₂in air at 37

°C. Cultures treated with the metabolic activation system were re-fed

with the serum-free S9 mix just before treatment. Compounds were

dissolved in DMSO and added to the cultures, and the cultures were

incubated at 37 °C. Treatments were for either 4 or 20 h, and

aberrations were scored in cells harvested 20 h after the beginning of

the treatment. At the termination, the cultures were washed twice

with Hank’s balanced salt solution and re-fed with the culture

medium. Cultures were incubated for a further 18−20 h. Colcemid

(0.1 mL) was added 4 h before the monolayers were harvested by

trypsinization. A cell sample was counted using a Coulter counter to

determine the cell number as an indicator of the cytotoxicity. The

viable cells were veriﬁed by checking samples for trypan blue dye

exclusion. The cells were treated with hypotonic KCl (75 mM) for 25

min at room temperature, washed twice with a ﬁxative (methanol/

glacial acetic acid = 3:1, v/v), dropped onto slides, air-dried, and

stained with a Giemsa stain. A total of 200 metaphase cells from each

concentration were examined and scored for chromosome aberra-

tions. Compound 3a-P1·HCl was tested at 0.563, 1.125, 2.25, and 4.5

μg/mL for the chromosome analysis. A statistical analysis of the

chromosome aberration data was performed using TOXSTAT2006

software.

Mice Bone Marrow Micronucleus Assay. An animal care and

use application for the study was approved by the Institutional Animal

Care and Use Committee of Shandong XinBo Pharmaceutical R&D

Ltd. Compound 3a-P1·HCl was subjected to the assay in ICR mice

(male, n = 60) weighing 23.5 to 30.9 g. The mice were randomly

divided into ﬁve groups (solvent control, positive control, and 3a-P1·

HCl at dose levels of 25, 50, and 100 mg/kg) with 12 mice in per

group. Mice were dosed with the vehicle control and the tested article

via oral gavage. At 24 and 48 h after dosing, animals were sacriﬁced

for the bone marrow harvest. The bone marrow from each animal was

examined microscopically to determine the micronucleus frequency

and the proportion of polychromatic erythrocyte to total erythrocytes.

A statistical analysis was performed using TOXSTAT2006 software.

Single-Dose Toxicity Studies. An animal care and use

application for the study was approved by the Institutional Animal

Care and Use Committee of Shandong XinBo Pharmaceutical R&D

Ltd. ICR mice (40♀ and 40♂) were housed in standard SPF facilities.

After at least 10 days of acclimation, mice were randomly divided into

four groups (solvent control and the tested compound 3a-P1·HCl at

dose levels of 100, 200, and 300 mg/kg) with 20 mice in per group.

Diﬀerent doses of the tested compound were administered by oral

gavage to the animals. Body weight, health, mortality, and clinical

signs were monitored for a continuous period of 21 days during the

study. After the recovery period, all animals were euthanized and

dissected. Histopathological examinations of tissue samples were

conducted.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00082.

■

sı

Synthesis of compounds 17a−e, crystal data for

compound 29, SFC report for compounds 3a and 3b,

and the determination of the absolute conﬁguration of

compound (S)-23 (PDF)

Molecular formula strings (CSV)

X-ray crystal structure data for compound 29 (CIF)

AUTHOR INFORMATION

Corresponding Authors

■

Huanjie Shao − College of Life Sciences, Shaanxi Normal

University, Xi’an 710119, P. R. China; Email: hshao@

snnu.edu.cn

Chunrong Yu − Taizhou Astar BioTechnology Co. Ltd,

Taizhou 225300, P. R. China; Email: yuc804@gmail.com

Haihong Huang − Beijing Key Laboratory of Active Substance

Discovery and Druggability Evaluation, Institute of Materia

Medica, Peking Union Medical College and Chinese Academy

of Medical Sciences, Beijing 100050, P. R. China;

Email: joyce@imm.ac.cn

Authors

Dongfeng Zhang − Beijing Key Laboratory of Active

Substance Discovery and Druggability Evaluation, Institute of

Materia Medica, Peking Union Medical College and Chinese

Academy of Medical Sciences, Beijing 100050, P. R. China;

orcid.org/0000-0003-0870-3782

Peng Li − Beijing Key Laboratory of Active Substance

Discovery and Druggability Evaluation, Institute of Materia

Medica, Peking Union Medical College and Chinese Academy

of Medical Sciences, Beijing 100050, P. R. China

Yongxin Gao − Beijing Key Laboratory of Active Substance

Discovery and Druggability Evaluation, Institute of Materia

Medica, Peking Union Medical College and Chinese Academy

of Medical Sciences, Beijing 100050, P. R. China

Yaoyao Song − College of Life Sciences, Shaanxi Normal

University, Xi’an 710119, P. R. China

Yaqin Zhu − College of Life Sciences, Shaanxi Normal

University, Xi’an 710119, P. R. China

Hong Su − College of Life Sciences, Shaanxi Normal

University, Xi’an 710119, P. R. China

Beibei Yang − Beijing Key Laboratory of Active Substance

Discovery and Druggability Evaluation, Institute of Materia

Medica, Peking Union Medical College and Chinese Academy

of Medical Sciences, Beijing 100050, P. R. China

Li Li − Beijing Key Laboratory of Active Substance Discovery

and Druggability Evaluation, Institute of Materia Medica,

Peking Union Medical College and Chinese Academy of

Kinase Inhibitory Activity Assays. The kinases assayed were

ABL1, ABL (E255K), ABL (F317I), ABL (G250E), ABL (H396P),

ABL (M351T), ABL (Q252H), ABL (T315I), ABL (Y253F), c-Kit, c-

Src, FGFR1, FLT3, KDR/VEGFR2, LYN, PDGFR, and TIE2/TEK.

The substrate for ABL1 and its mutants and FLT3 was

EAIYAAPFAKKK at 20 mM; the substrate for the kinases c-Kit, c-

Src, KDR/VEGFR2, LYN, PDGFRa, and TIE2/TEK was poly-

[Glu:Tyr](4:1) at 0.2 mg/mL; the substrate for FGFR1 was

KKKSPGEYVNIEFG at 20 mM. The reaction buﬀer was 20 mM

HEPES (pH 7.5), 10 mM MgCl₂, 1 mM EGTA, 0.02% Brij35, 0.02

mg/mL BSA, 0.1 mM Na₃VO₄, 2 mM DTT, and 1% DMSO. The

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Medical Sciences, Beijing 100050, P. R. China; orcid.org/

0000-0002-9496-2280

Gang Li − Beijing Key Laboratory of Active Substance

Discovery and Druggability Evaluation, Institute of Materia

Medica, Peking Union Medical College and Chinese Academy

of Medical Sciences, Beijing 100050, P. R. China;

orcid.org/0000-0003-0924-7950

Ningbo Gong − Beijing Key Laboratory of Polymorphic

Drugs, Institute of Materia Medica, Peking Union Medical

College and Chinese Academy of Medical Sciences, Beijing

100050, P. R. China

Yang Lu − Beijing Key Laboratory of Polymorphic Drugs,

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Author Contributions

^∇These authors contributed equally.

Notes

The authors declare no competing ﬁnancial interest.

ABBREVIATIONS USED

■

Ames, bacterial reverse mutation; BCR-ABL, breakpoint

cluster region-abelson leukemia virus; CML, chronic myeloid

leukemia; c-KIT, proto-oncogene that encodes a type III

transmembrane tyrosine kinase; DMSO, dimethyl sulfoxide;

DMF, N,N-dimethylforamide; DIPEA,, diisopropyl ethyl-

amine; DMAP, 4-dimethylaminopyridine; DEA, diethylamine;

EDCI, 1-(3-(dimethylamino)-propyl)-3-ethylcarbodiimide hy-

drochloride; EtOAc, ethyl acetate; ECD, electronic circular

dichroism; FGFR, ﬁbroblast growth factor receptor; FLT3,

FMS-like tyrosine kinase-3; HOBt, 1-hydroxybenzotriazole;

hERG, human ether-a-go-go-related gene; LYN, Lck/Yes-

related novel protein tyrosine kinase; PDGFR, platelet-derived

growth factor receptor; PE, petroleum ether; PK, pharmaco-

kinetic; Src, sarcoma; SAR, structure−activity relationship;

SFC, supercritical ﬂuid chromatography; TIE2/TEK, tyrosine

kinase with immunoglobulin and EGF-like domains; TGI,

tumor growth inhibition; TFA, triﬂuoroacetic acid; THF,

tetrahydrofuran; VEGFR, vascular endothelial growth factor

receptor

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Article Doi

Discovery of a Candidate Containing an (S)-3,3-Difluoro-1-(4-methylpiperazin-1-yl)-2,3-dihydro-1 H-inden Scaffold as a Highly Potent Pan-Inhibitor of the BCR-ABL Kinase including the T315I-Resistant Mutant for the Treatment of Chronic Myeloid Leukemia

DOI: 10.1021/acs.jmedchem.1c00082

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