Combining structure- and property-based optimization to identify selective FLT3-ITD inhibitors with good antitumor efficacy in AML cell inoculated mouse xenograft model

Article abstract of DOI:10.1016/j.ejmech.2019.05.021

FLT3 mutation is among the most common genetic mutations in acute myeloid leukemia (AML), which is also related with poor overall survival and refractory in AML patients. Recently, FLT3 inhibitors have been approved for AML therapy. Herein, a series of new compounds with pyrazole amine scaffold was discovered, which showed potent inhibitory activity against FLT3-ITD and significant selectivity against both FLT3-ITD and AML cells expressing FLT3-ITD. Compound 46, possessing the most promising cellular activity, blocked the autophosphorylation of FLT3 pathway in MV4-11 cell line. Furthermore, the apoptosis and downregulation of P-STAT5 were also observed in tumor cells extracted from the MV4-11 cell xenografts model upon compound 46 treatment. Compound 46 was also metabolically stable in vitro and suppressed tumor growth significantly in MV4-11 xenografts model in vivo. Compound 46 showed no toxicity to the viscera of mice and caused no decrease in body weight of mice. In conclusion, the results of this study could provide valuable insights into discovery of new FLT3 inhibitors, and compound 46 was worthy of further development as potential drug candidate to treat AML.

Full text of DOI:10.1016/j.ejmech.2019.05.021

Accepted Manuscript
Combining structure- and property-based optimization to identify selective FLT3-ITD
inhibitors with good antitumor efficacy in AML cell inoculated mouse xenograft model
Hao Heng, Zhijie Wang, Hongmei Li, Yatian Huang, Qingyuan Lan, Xiaoxing Guo,
Liang Zhang, Yanle Zhi, Jiongheng Cai, Tianren Qin, Li Xiang, Shuxian Wang,
Yadong Chen, Tao Lu, Shuai Lu
PII:
S0223-5234(19)30430-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.021
EJMECH 11331
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 March 2019
Revised Date: 3 May 2019
Accepted Date: 6 May 2019
Please cite this article as: H. Heng, Z. Wang, H. Li, Y. Huang, Q. Lan, X. Guo, L. Zhang, Y. Zhi, J.
Cai, T. Qin, L. Xiang, S. Wang, Y. Chen, T. Lu, S. Lu, Combining structure- and property-based
optimization to identify selective FLT3-ITD inhibitors with good antitumor efficacy in AML cell inoculated
mouse xenograft model, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.05.021.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Combining structure- and property-based optimization to identify selective
FLT3-ITD inhibitors with good antitumor efficacy in AML cell inoculated mouse
xenograft model
Hao Heng†, #, Zhijie Wang†, #, Hongmei Li†, #, Yatian Huang†, Qingyuan Lan†,
Xiaoxing Guo†, Liang Zhang†, Yanle Zhiǁ, Jiongheng Cai‡, Tianren Qin‡, Li Xiang‡,
Shuxian Wang‡, Yadong Chen§, *, Tao Lu¶, Shuai Lu†, *
† School of Science, China Pharmaceutical University, Nanjing 211198, P.R. China.
ǁ School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046, P.
R. China
‡ School of Pharmacy, China Pharmaceutical University, Nanjing 210009, P.R. China.
§
Laboratory of Molecular Design and Drug Discovery, China Pharmaceutical
University, Nanjing, 211198, P.R. China.
¶
State Key Laboratory of Natural Medicines, China Pharmaceutical University,
Nanjing 210009, P.R. China
Abstract
FLT3 mutation is among the most common genetic mutations in acute myeloid
leukemia (AML), which is also related with poor overall survival and refractory in
AML patients. Recently, FLT3 inhibitors have been approved for AML therapy.
Herein, a series of new compounds with pyrazole amine scaffold was discovered,
which showed potent inhibitory activity against FLT3-ITD and significant selectivity
against both FLT3-ITD and AML cells expressing FLT3-ITD. Compound 46,
possessing the most promising cellular activity, blocked the autophosphorylation of
FLT3 pathway in MV4-11 cell line. Furthermore, the apoptosis and downregulation of
P-STAT5 were also observed in tumor cells extracted from the MV4-11 cell
xenografts model upon compound 46 treatment. Compound 46 was also metabolically
stable in vitro and suppressed tumor growth significantly in MV4-11 xenografts
model in vivo. Compound 46 showed no toxicity to the viscera of mice and caused no
decrease in body weight of mice. In conclusion, the results of this study could provide
valuable insights into discovery of new FLT3 inhibitors, and compound 46 was
worthy of further development as potential drug candidate to treat AML.
Key words: FLT3-ITD; inhibitor; selectivity; AML
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Introduction
Acute myeloid leukemia (AML) is one severe type of tumor that occurs mostly in
children and adolescents [1], causing about ten thousand deaths in the United States in
2017, with a 5-year survival rate of 27%.[2]
It is reported that approximately 30%
of AML patients harbored the Fms-like tyrosine kinase 3 (FLT3) mutation.[3]
Normally, FLT3 is one member of type III receptor tyrosine kinase that is mainly
expressed in hematopoietic and lymphocytes cells, and regulates the growth,
differentiation and apoptosis of cells. [4] Pathologically, FLT3 mutations are often
capable of the autophosphorylation and excessive activation of its downstream
signaling pathways, including signal transducer and activator of transcription 5
(STAT5), Ras/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3
kinase (PI3K) / AKT pathway.[5] FLT3 mutations can be divided into two types, i.e.
internal-tandem duplication (ITD) in the juxtamembrane domain of kinase [6] and
tyrosine kinase domain (TKD)[7] point mutations. After standard chemotherapy, AML
patients with FLT3-ITD mutation had a worse prognosis than those with wild-type
FLT3.[8-10] Actually, both types of mutations not only correlate with negative
prognosis, but also contribute greatly to the drug resistance for current FLT3
inhibitors.[9, 11] Therefore, FLT3, especially mutant FLT3, has attracted numerous
interests to develop targeting inhibitors toward AML therapy.
According to their binding modes, FLT3 inhibitors can be categorized into type I
and type II.[12] Crenolanib, sorafenib, quizartinib, gilteritinib and midostaurin
(Figure 1) are the representative and advanced inhibitors. The combination of
midostaurin with standard chemotherapy can effectively prolong the overall survival
of AML patients with FLT3 mutations.[13] Quizartinib has an excellent effect on
FLT3-ITD mutation, and can effectively prevent fatal bone marrow suppression,
prolonging the survival and improving the life quality of AML patients.[14] Sorafenib
was reevaluated in several clinical trials for AML, and improved the outcomes for
patients with FLT3-ITD.[15-17] Despite of the reported FLT3 inhibitors,[18] novel
chemical entities that are effective and selective against either FLT3-ITD and
FLT3-TKD alone or concurrent mutations[19] are still urgently needed due to the fast
developing drug resistance. [20]
2

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O
O
O
N
N
Cl
O
O
N
N
N
H
N
F3C
N
N
H
H
NH2
Sorafenib
Crenolanib
H
N
O
S
N
N
N
N
O
N
O
O
O
H
N
N
H
N
H
O
O
O
N
Midostaurin
Quizartinib
H2N
O
H
N
N
H
O
N
N
N
H
N
N
N
N
NH
O
FN-1501
N
NH
N
N
HN
N
N
O
Gilteritinib
N
NH
O
S
N
H
NC
N
H
LT-180-0712
Figure 1. Represented FLT3 inhibitors and hit compound LT-180-0712 of this work.
In our previous efforts on FLT3 inhibitor research, several potent compounds
have been identified, among which FN-1501 is being under evaluation in phase I
clinical trial.[21, 22] However, FN-1501 was also potent CDKs inhibitors, making it
not a selective FLT3 inhibitor. To discover more selective FLT3-ITD inhibitors, a
virtual screening was performed based on our in-house molecule database, and a hit
compound (LT-180-0712) with good selectivity against FLT3 was identified by us.[23]
Herein, a series of new pyrazole amines with improved cellular activity was
discovered by hit to lead optimization, of which a representative compound showed
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high FLT3 selectivity from enzyme and cell-based assays as well as significant
anti-AML efficacy in vivo.
Chemistry
Scheme 1. Synthesis of compounds 8-18.
Reagents and conditions: (i) SOCl2, MeOH, reflux; (ii) CuCN, KI, DMF, reflux; (iii)
NH2NH2·H2O, EtOH, 45 °C; (iv) Amines, Et3N, DMF, r.t.; (v) Fe, NH4Cl, EtOH, reflux; (vi)
CSCl2, DCM, NaHCO3, H2O, 0 °C; (vii) Amines, HATU, DIPEA, DMF; (viii) Pyridine, EtOH,
reflux; (ix) Hg(OAc)2, EtOH, reflux; (x) NaOH, 30% H2O2, EtOH, 50 °C.
Compounds 8-18 were prepared by a general approach shown in Scheme 1.
4-Bromo-thiophene-2-carboxylic acid was used as the starting material to synthesize
compound 3 through a sequence of esterification, cyano substitution and
hydrazinolysis.[24] 4-Fluoronitrobenzene or 3-fluoronitrobenzene was used as the
starting material to synthesize compounds 4a-4b through a nucleophilic substitution
reaction with the appropriate amines. Reduction of compound 4a-4b gave compounds
5a-5b, followed by the reaction with thiophosgene to obtain intermediates 6a-6b.
Amides 4c-4g were synthesized by coupling 4-nitrobenzoic acid or 3-nitrobenzoic
4

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acid with the related amines. Similar steps were conducted to convert 4c-4g to 6c-6g
as used in the synthesis of 6a-6b. Reaction of compound 3 with intermediates 6a-6g
followed by cyclization gave title compounds 8-14. The hydrolysis of corresponding
compounds gave title compounds 15-18.[25, 26] Compounds 23-27 and 45-54 were
prepared by a general approach shown in Scheme 2. The pyrazolamines (20 and
21a-21f) were synthesized using similar procedures to those in Scheme 1. Coupling
reaction catalyzed by palladium gave intermediates 22a-22i. Then, title compounds
23-26 and 45-54 were obtained by the amination reactions of intermediates 20,
21a-21f and 22a-22i. Hydrolysis of hit compound LT-180-0712 gave compound 27.
Accordingly, compounds 28-34 and 38-44 (Scheme 3) were synthesized using similar
procedures to those shown in Scheme 1 and Scheme 2.
Scheme 2. Synthesis of compounds 23-27 and 45-54.
Reagents and conditions: (i) SOCl2, MeOH, reflux; (ii) CuCN, KI, DMF, reflux; (iii) NaH, THF,
CH3CN, reflux; (iv) NH2NH2·H2O, EtOH, reflux; (v) Amines, Pd(OAc)2, BINAP, Cs2CO3, Tol,
5

ACCEPTED MANUSCRIPT
90 °C; (vi) 20 or 21a-21f, Al(CH3)3 , Tol, 90 °C; (vii) KOH, EtOH, reflux.
Scheme 3. Synthesis of compounds 28-34 and 38-44.
Reagents and conditions: (i) Intermediates 35a-35g: acetic anhydride, HClO4, 0 °C;
Intermediates 35h-35k: corresponding carboxylic acids, H3PO4, TFAA, CH3CN, r.t.; (ii) CuCN,
KI, DMF, reflux; (iii) 1-Methylpiperazine, Et3N, DMF, r.t.; (iv) Fe, NH4Cl, EtOH, reflux; (v)
CSCl2, DCM, NaHCO3, H2O, 0 °C; (vi) Intermediate 6a, NaH, THF/DMF, 0 °C; (vii)
6

ACCEPTED MANUSCRIPT
NH2NH2·H2O, EtOH, reflux; (viii) KOH, EtOH, reflux.
Results and discussion
Structure and activity relationship (SAR). Previously, we found a potent and
selective FLT3-ITD inhibitor LT-180-0712 (IC50 = 0.9400 nM), but its cellular
activity against MV4-11 cell line was moderate (IC50 = 917 nM).[23] To discover new
selective FLT3-ITD inhibitors with improved cell efficacy, we firstly tried to increase
the cellular activity by structure-based optimization on LT-180-0712 and maintain the
kinase inhibitory activity. However, the initial oxadiazole bioisosteres of LT-180-0712
showed nearly eliminated FLT3-ITD inhibitory activity (Table S1), suggesting that
the pyrazole ring was indispensable for the enzymatic efficacy. To verify this
speculation, we docked compound 8 and LT-180-0712 to FLT3 in active form (since
no activated FLT3 crystal structure was revealed, the homology model [23] that we
constructed before was used). As shown in Figure 2, neither key hydrogen bonding
interaction nor π-cation interaction formed between compound 8 and FLT3.
Figure 2. Docking binding mode of LT-180-0712 (A) and compound 8 (B) in FLT3 homology
model. Yellow ribbon represents hinge region. Red dash line represents hydrogen bond; magenta
dash line π-cation interaction; black dash line represents the close contact of atoms. Carbon atoms
of the FLT3 residues and the ligand are shown in green and cyan color, respectively.
Hence, we focused on the study of pyrazole compounds. As shown in Table 1,
when keeping the cyano group unchanged, the introduction of chlorine, fluorine atom
or methyl group at position 3 of benzene ring (23-25) made FLT3-ITD inhibitory
potency decrease in different degrees. The sharp attenuation of kinase activity for
compounds 23 and 24 was probably due to unfavorable steric clash between the
chlorine atom or methyl group and Tyr693 of FLT3 and thus lack of key hydrogen
boning with hinge region (Figure 3A and 3B). Both replacing the benzene ring with
pyridine ring (26) and introducing hydrogen bonding donor on thiophene ring (27)
only slightly attenuated the FLT3-ITD inhibitory activity (IC50 = 8.963 and 3.120 nM,
respectively). However, cell potencies of compounds 26 and 27 against MV4-11 cells
were moderately improved (IC50 = 298 and 244 nM, respectively). This discrepancy
7

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on kinase and cell activities reminded us that weak cellular potency of this kind of
compounds was probably due to the unfavorable transmembrane capacity. Hence,
solubility and transmembrane properties of those compounds were predicted. The data
(Table 1) showed that compounds 23-27 and LT-180-0712 possessed weaker
predicted Caco-2 cell permeability and lower logP values than the clinical FLT3
inhibitors did, such as Crenolanib, Midostaurin, Sorafenib and Quzartinib. Therefore,
we turned to use the property-based optimization strategy.
Figure 3. Docking binding mode of compounds 23 (A), 24 (B), 29 (C), 30 (D) and 31 (E) in FLT3
homology model. Yellow ribbon represents hinge region. Red dash line represents hydrogen bond;
black dash line represents the close contact of atoms. Carbon atoms of the FLT3 residues and the
ligand are shown in green and cyan color, respectively.
Table 1. Structures and biological data of compounds 23-27.
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FLT3-ITD
MV4-11
Cpd.
R1
R2
X
PlogP d
PCaco e
IC50 (nM) a IC50 (nM) b
c
23
24
25
26
CN
CN
CN
CN
Cl
CH3
F
C
C
C
N
1198±102
398.7±10.4
39.61±2.3
8.963±1.1
-
2.929
2.869
2.709
2.44
43.612
53.13
42.4
-
-
H
298±11
29.013
27
H
C
3.120±0.8
244±16
917±27
2.171
2.532
18.308
44.735
0.9400±0.0
2
LT-180-0712
Crenolanib
Midostaurin
Sorafenib
4.041
5.394
4.11
359.609
2266.241
322.725
110.126
1.300±0.3
1.59±0.15
Quzartinib
4.553
a
In the presence of 10 µM ATP, the values are the mean from three independent experiments (±
c
d
SD); b the values are the mean from three independent experiments (± SD); not determined;
predicted logP; e predicted Caco-2 cell permeability in nm/second.
Considering that the amide linker between pyrazole and benzene ring may
contribute negatively to transmembrane capacity, a -NH- group was used as the new
linker (Table 2). Although enzymatic activities of compound 28 decreased by more
than 10 times, compounds 29, 30 and 31 possessed comparable FLT3-ITD inhibitory
potency with hit compound, and their binding modes in FLT3 (Figure 3C-3E) were
also similar with that of hit compound. Therefore, the -NH- linker was acceptable to
maintain kinase activity. More importantly, compounds 29 and 30 also displayed six
times higher cell efficacy (IC50 = 130 nM and 148 nM, respectively) than hit
compound did. Indeed, compounds 29 and 30 had obviously increased predicted logP
values and Caco-2 cell permeability (Table 2), which probably facilitated these two
compounds to transfer into MV4-11 cells and inhibited cell growth. Consistently,
compound 31 that had lower predicted logP values and Caco-2 cell permeability than
compounds 29 and 30, showed decreased cellular potency (IC50 = 756 nM).
Next, we continued to investigate various substituents with different volume or
electrostatic property on thiophene ring. When changing substitutions on position 5 of
thiophene ring (32, 33, 34, 38, 39), only compound 32 with 5-Br group showed
10-fold decreased kinase activity and the other compounds displayed maintained
kinase activities, perhaps due to too close distance between the bromine atom of
compound 32 and Phe691 of FLT3 (Figure 4A). Compounds 33 and 34 showed better
cellular activities (IC50 = 85 and 80 nM, respectively) than compounds 38 and 39,
which was consistent with their differences in the predicted logP and Caco-2 cell
9

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permeability values. Unexpectedly, compound 40, with no substitution at thiophene
ring, also demonstrated potent enzyme inhibitory activity (IC50 = 0.327 nM),
suggesting that simple substituents on thiophene were not necessary to intermolecular
binding (Figure 4B). And higher predicted logP and Caco-2 cell permeability values
and the improved cell efficacy (IC50 = 74 nM) were concurrent for compound 40.
However, substitution on pyrazole ring (41-44) led to eliminated potency against
FLT3-ITD and MV4-11 cells (Table S2), though these compounds had high predicted
Caco-2 cell permeability. Molecular docking results indicated that the
cyclopentylmethyl group (41) formed unfavorable steric clashes with Leu616 and
Val624 of FLT3, which probably caused the intermolecular binding unstable (Figure
S1), suggesting unsubstitution on the pyrazole ring was optimal.
Table 2. Structures and biological data of compounds 28-34 and 38-40.
Cpd.
R1
4-CN
4-Br
FLT3-ITD IC50 (nM) a
30.12±1.6
MV4-11 IC50 (nM) b PlogP PCaco
c
28
29
30
31
32
33
34
38
-
1.711±0.42
0.7991±0.09
1.960±0.1
130±11
148±12
756±31
-
4.361 643.97
4.283 642.941
3.782 488.549
4-Cl
4-OCH3
5-Br
48.31±1.3
5-Cl
1.503±0.17
1.711±0.2
85±6
4.275 637.811
4.026 454.706
2.918 92.801
5-OCH3
5-CN
80±4
2.006±0.59
261±18
39
3.212±0.88
418±24
1.988 37.645
40
H
0.3270±0.05
1.300±0.3
74±3
3.78 639.025
4.11 322.725
Sorafenib
1.59±0.15
a
In the presence of 10 µM ATP, the values are the mean from three independent experiments (±
SD); b The values are the mean from three independent experiments (± SD); c Not determined.
Figure 4. Overlapped docking binding mode of compounds 32 and 33 (A) and binding mode of
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compound 40 (B) in FLT3 homology model. Yellow ribbon represents hinge region. Red dash line
represents hydrogen bond; green dash line represents π-π interaction; black dash line represents
the close contact of atoms. Carbon atoms of the FLT3 residues and the ligand are shown in green
and cyan color, respectively.
Since the simple modification on hit compound only enhanced the cellular
potency by 12 folds (40), we wondered whether other moieties could replace
thiophene ring and further improve the cellular potency. Hence, the scaffold hopping
was adopted on the thiophene ring (Table 3) because substitutions on this ring were
generally tolerated in kinase activity. Initially, fusing a benzene ring with thiophene
led to the obvious decrease in enzymatic and cellular activity (45). Further, the
thiophene was replaced with benzodioxol and 2,3-dihydrobenzodioxin ring (46 and
47), respectively. Compound 46 showed ~3 times more potent cellular efficacy (IC50
= 27 nM) than compound 40 did. However, compound 47 with the expanded
fused-ring system, possessed 10 times weaker cell efficacy than compound 46 did.
Subsequently, we investigated the analogues of compound 46. The addition of two
fluorine atoms at the benzodioxol moiety (48) caused a sharp decline in cell potency
and enzyme activity. And substituting an oxygen atom with CH2 group (49) in
benzodioxol ring also had a negative consequence on cell potency (IC50 = 339 nM).
When the oxygen containing ring was open (50), both enzyme and cell inhibitory
potencies decreased dramatically. The data implied that structural modification on the
benzodioxol moiety was detrimental. Therefore, the benzodioxol ring was reserved as
the optimal substituent.
Table 3. Structures and biological data of compounds 45-50.
FLT3-ITD IC50
(nM) a
MV4-11 IC50
(nM) b
Compound
R1
PlogP
4.731
3.439
3.276
3.808
4.024
PCaco
221.618
219.834
225.442
224.695
227.32
45
46
47
48
49
10.51±1.2
1.336±0.3
12.50±2.1
123.9±10.3
25.88±1.6
335±23
27±2
236±9
1175±106
339±3
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50
63.65±1.9
1.300±0.3
335±12
3.573
4.11
224.836
322.725
Sorafenib
1.59±0.15
a
In the presence of 10 µM ATP, the values are the mean from three independent experiments (±
SD); b The values are the mean from three independent experiments (± SD).
In order to investigate the effect of hydrophilic amines on the activity, compounds
51-54 with varying cyclic amines were synthesized. The data (Table 4) showed that
piperazine and substituted piperazine (51-53) were tolerable to kinase activity but
made cell efficacy decrease moderately. For morpholine (54), it made an obvious
reduction in the kinase and cell activities. Hence, we kept N-methyl piperidine as the
current optimal group in hydrophilic region of FLT3. The SAR was collectively
illustrated in Figure 5.
Table 4. Structures and biological data of compounds 51-54.
Compound
R1
FLT3-ITD IC50 (nM) a
1.518±0.12
MV4-11 IC50 (nM) b
62±1
51
4.434±0.6
2.773±0.1
54±4
64±7
52
53
8.422±0.25
1.300±0.3
96±4
54
1.59±0.15
Sorafenib
a
In the presence of 10 µM ATP, the values are the mean from three independent experiments (±
SD); b The values are the mean from three independent experiments (± SD).
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Property directed optimization improved cellular potency by 12 folds
Optimal scaffold:
Pyrazole ring
was favorable
Hydrophilic
group
Enhanced cellular activity
Varying R2
improved
cellular
activity
moderately
Structure-based scaffold hopping improved
cellular potency by ~3 folds
Benzene ring
was better
than pyridine
Substitution here was unfavorable
Figure 5. Summarized SAR.
Inhibitory activity of compound 46 against other kinases. We tested
FLT3-ITD selectivity of compound 46 over a panel of 48 kinases at the concentration
of 0.1 µM. As shown in Table S3, only six kinases had an inhibitory rate greater than
60%, which includes FMS and c-kit that are very closely homologous to FLT3.
Furthermore, at least 100-fold selectivity of FLT3-ITD over those 6 kinases was
observed (Table 5). In addition, the high selectivity of compound 46 over c-kit
(461-fold) was thought to be beneficial for avoiding the potential synthetic lethal
myelosuppreion.[27-30] Moreover, compound 46 almost showed no inhibitory
activity against CDKs that were among the targets of FN-1501.
As reported,[31, 32] some FLT3 inhibitors were subjected to drug resistance due
to FLT3 mutation. Therefore, we tested inhibitory activities of compound 46 against a
variety of commonly occurred FLT3 mutants. As shown in Table 6, compound 46
suppressed those FLT3-ITD or FLT3-TKD mutants with IC50 values lower than 25
nM. Notably, compound 46 inhibited FLT3-D835Y (IC50 = 18.71 nM) or
FLT3-D835V (Kd = 7.3 nM) potently, which could cause drug resistance to type II
28
FLT3 inhibitors. And it bound two concurrent FLT3-ITD-TKD mutants with Kd
values around 100 nM. To further validate potency of compound 46 against FLT3
mutants, its antiproliferative activities were investigated in BaF3 cells transformed
with FLT3 mutants. The data showed that compound 46 indeed inhibited growth of
BaF3 cells harboring FLT3 mutations; while it did not obviously affect the growth of
parental BaF3 cells (Table 7).
Table 5. IC50 of compound 46 against 6 kinases.
Kinase IC50 (nM) a
ABL1 1340±102
BTK
161±8
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c-Kit
FMS
616±13
247±11
860±25
432±19
LCK
TRKA
a Mean value of results from triplicate runs (± SD).
Table 6. Inhibitory efficacy of compound 46 against wild-type FLT3 and several FLT3 mutants.
Kinase
Activity
FLT3
66.75±1.32 a
18.71±1.62 a
11.22±1.31 a
10.26±0.39 a
24.06±1.02 a
18.23±0.88 a
6.867±0.92 a
FLT3 (D835Y)
FLT3 (F594_R595insR)
FLT3 (F594_R595insREY)
FLT3 (ITD)-NPOS
FLT3 (ITD)-W51
FLT3 (R595_E596insEY)
FLT3 (Y591-V592insVDFREYEYD) 17.72±1.8 a
FLT3 (D835V)
7.3±1.2 b
160±8 b
90±6 b
FLT3 (ITD)-D835V
FLT3 (ITD)-F691L
a IC50 (nM) value in the presence of 10 µM ATP, n = 3 (± SD); b Kd value (nM), n = 3 (± SD).
Table 7. Antiproliferative effects of compound 46 against a panel of FLT3 mutants transformed
BaF3 cells a.
IC50 (µM)
Cell line
Cpd. 46
sorafenib
BaF3
9.197±0.293 7.997±0.221
0.107±0.006 0.006±0.001
BaF3-FLT3 (ITD)
BaF3-FLT3 (ITD-D835V) 0.146±0.009 1.454±0.028
BaF3-FLT3 (ITD-F691L) 0.532±0.021 1.348±0.021
BaF3-FLT3 (D835V)
0.211±0.004 1.112±0.032
a All IC50 values were obtained by triple testing (± SD).
Binding mode simulation of compound 46. We simulated the binding modes of
compound 46 with FLT3, FMS and c-kit using induce-fit docking module, and the
corresponding results were analyzed using pose view module in Schrödinger software.
As presented in Figure 6A, two hydrogen bonds formed between the amino moiety of
14

ACCEPTED MANUSCRIPT
compound 46 and residue Cys694 of FLT3, and the pyrazole moiety and Glu692,
respectively. And the π-cation and π-π interactions formed between compound 46 and
Lys614 and Phe691, respectively. An unfavored close contact formed between the
pyrazole moiety and backbone NH of Cys694. For FMS and c-kit (Figure 6B and 6C),
compound 46 bound to the proteins more deeply in ATP site, perhaps due to the
smaller size of their gatekeeper residues Thr663 and Thr623, respectively, compared
with Phe691 of FLT3. Hydrogen bonds also formed between compound 46 and the
residues in the hinge region of those two kinases; instead, the π-cation and π-π
interactions did not form for compound 46. Furthermore, more unfavorable close
contacts emerged when compound 46 interacted with FMS and c-kit. For example, the
1-methylpiperidine moiety may clashed with Lys586 of FMS and Lys593 of c-kit,
respectively. It was speculated that the larger amount of the close contacts and the
lack of π-π interactions may contributed to the weaker inhibitory activities of
compound 46 against FMS and c-kit.
Figure 6. Proposed binding mode of compound 46 with FLT3 homology model (A), FMS (B) and
c-kit (C) from molecular docking simulations. Red dash line represents hydrogen bond; green dash
line represents π-π interaction or π-cation interaction; black dash line represents the close contact
of atoms. Carbon atoms of the residues and the ligand are shown in grey and cyan color,
respectively. PDB code for FMS and c-kit crystals: 2I1M and 3G0E.
Cell growth inhibition of compound 46 against other cell lines. Proliferative
inhibitory efficacy of compound 46 against other leukemia cell lines and
histologically different cell lines was showed in Table 8. Only leukemia cell line
Molm-13 was sensitive to this compound (IC50 = 33.2 nM), which also expressed
FLT3-ITD. The other leukemia and solid tumor cell lines, which either expressed
wild-type FLT3 or lack expression of FLT3, were resistant to compound 46. This
indicated that anti-growth effect of compound 46 against MV4-11 and Molm-13 cells
were dependent on the expression of FLT3-ITD in cells. We also investigated in vitro
antitumor of 46 against more tumor cell lines by performing the NCI-60 panel screen.
As data shown in Table S4, most screened tumor cell lines exhibited weak sensitivity
to compound 46 even at a high concentration of 10 µM, further suggesting its
15

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selectivity of FLT3-ITD expressing cells.
Table 8. Antiproliferative activity of compound 46 against a variety of cell lines a.
Cancer type
Cell line
MCF-7
HT-29
FLT3 status Inhibition % (1 µM) / IC50 (nM)
Breast Cancer
Colorectal
unknown
unknown
unknown
unknown
unknown
unknown
unknown
unknown
unknown
unknown
9.2±1.4 / - b
-13.5±1.9 / -
4.3±1.3 / -
Liver Cancer
Kidney Cancer
Lung Cancer
Ovarian Cancer
Hep G2
ACHN
A-549
-8.3±1.1 / -
-2.9±0.9 / -
SK-OV-3
18.8±1.7 / -
2.6±0.5 / -
Pancreatic Cancer BxPC-3
Prostate Cancer
Stomach Cancer
Glioblastoma
Esophageal
Leukemia
PC-3
1.5±0.2 / -
MKN-45
U251
24.1±1.4 / -
1.3±0.2 / -
KYSE-520 unknown
-0.2±0.1 / -
K-562
FLT3-null
WT/low
ITD
-14.9±1.1 / -
1.6±0.2 / -
Leukemia
HL-60
Leukemia
Molm-13
90.3±2.9 / 33.2±4.6
46.0±1.9 / 3795±304
28.7±2.1 / -
Myeloma
RPMI 8226 unknown
OCI-LY19 unknown
lymphoma
a Data from triple testing (± SD); b Not determined.
Cellular mode of action of compound 46. We analyzed the phosphorylation
levels of FLT3, STAT5, ERK and AKT in MV4-11 cells after treated with compound
46 at various concentrations. As shown in Figure 7, compound 46 decreased the
phosphorylation of FLT3 and its downstream signaling proteins in a dose-dependent
manner, suggesting compound 46 could interfere the transduction of FLT3 signal
pathway in MV4-11 cells.
Figure 7. Western blotting analysis of compound 46. (A) MV4-11 cells were treated with
16

ACCEPTED MANUSCRIPT
compound 46 or sorafenib for 24 h; (B) Immunoblotting quantification of P-FLT3, P-STAT5,
P-AKT, and P-ERK, respectively. All bands were quantified and normalized by GAPDH.
In vitro and in vivo PK evaluation. The metabolic stability of compound 40 and
46 was firstly evaluated by human and rat liver microsomes assay in vitro. As the data
shown (Table 9), compound 40 was metabolically stable in both human and rat liver
microsomes. In comparison, compound 46 was more stable in human liver
microsomes than in that of rat. Then, PK properties of compound 46 in rats following
intravenous and oral administration were studied (Table 10). Upon oral
administration, the bioavailability was only 22.8%. Upon intravenous administration,
a higher Cmax was observed, which may be beneficial to achieve the antitumor
efficacy in vivo. Hence, the intravenous administration was selected to evaluate in
vivo antitumor effect of compound 46, and the dosing frequency was set as twice per
day to offset its shorter T1/2. To assist the determination of safe dosage of compound
46 in vivo, we investigated its maximum tolerated dose in healthy SPFICR rats. The
result showed that the rats were tolerable to dosage up to 200 mg/kg followed the
intravenous administration.
Table 9. Pharmacokinetics of compound 40 and 46 in human and rat liver microsomes.
CLint b
Compound Liver microsomes T1/2 a (min)
(µL/min/mg protein)
human
rat
59.8±7.8
41.3±4.2
117±8
23.2±1.1
33.6±0.9
11. 8±0.6
168±5
40
46
human
rat
8.27±1.3
a T1/2 is half life; b CLint is the intrinsic clearance (± SD).
Table 10. Pharmacokinetics of compound 46 in SD rats. a
Parameter
T1/2 (h)
iv (1 mg/kg)
1.96 ± 0.71
po (5 mg/kg)
3.07 ± 2.58
27.00 ± 5.36
2.83 ±2.02
Cmax (ng/mL)
Tmax (h)
205.37 ± 11.91
AUC(0-t) (h*ng/mL) 152.11 ± 38.96 173.18 ± 29.52
AUC(0-∞) (h*ng/mL) 160.58 ± 46.35 193.66 ± 40.06
MRT(0-t) (h)
Vz (L/kg)
2.75 ± 2.13
18.19 ± 6.58
6.63 ± 2.09
5.90 ± 1.96
105.05 ± 81.04
26.68 ± 6.27
Clz (L/h/kg)
17

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F (%)
22.77 ± 3.88
aAll testing data were obtained from three independent mice (± SD).
In vivo effects of compound 46 in MV4-11 tumor xenografts. The in vivo
antitumor efficacy of compound 46 was evaluated in MV4-11 cells inoculated
xenograft mouse model, in which a FLT3 inhibitor, sorafenib, was used as the positive
control. In addition, compound 40 was tested simultaneously to compare with
compound 46. As data shown in Figure 8, in vivo tumor inhibition of sorafenib in
MV4-11 xenograft mouse model [33] was reproduced, suggesting the results of this
experiment was reliable. The 14-day administration of compound 46 and 40 caused
almost no body weight loss compared with sorafenib (Figure 8A), indicating good
tolerance of mice to both compounds. The injections of compound 46 at dosage of 20
and 40 mg/kg twice per day inhibited tumor progression significantly, and its
antitumor activity was dose dependent (Figure 8B). The antitumor efficacy of 20
mg/kg 46 was comparable to that of sorafenib in terms of inhibitory rate (IR) and rate
of relative tumor growth (T/C%) (Figure 8B and 8D), which indicated that 46 showed
similar performance with sorafenib in the same total daily dose. The 40 mg/kg 46
produced a T/C% of 8.01% (Figure 8B) and a significant IR of 94.65% in tumor
weight (Figure 8D), which were moderately better than those values of sorafenib.
However, the increment of antitumor efficacy for 46 was moderate when its daily
dose was doubled. For compound 40, the IR (81.14%) was weaker than compound 46
at the same dosage, which was consistent with its weaker inhibitory activity against
MV4-11 cells in vitro.
800.00
25.00
20.00
15.00
10.00
Vehicle, iv, BID
GI %
119.86
95.25
T/C %
10.10
28.51
8.01
A
B
Vehicle, iv, BID
Sorafenib, 40, ig, QD
Cpd. 40, 40, iv, BID
Cpd. 46, 40, iv, BID
Cpd. 46, 20, iv, BID
Sorafenib, 40, ig, QD
Cpd. 40, 40, iv, BID
Cpd. 46, 40, iv, BID
Cpd. 46, 20, iv, BID
122.77
120.66
600.00
400.00
200.00
0.00
9.64
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
10
12
14
16
18
20
22
24
10
12
14
16
18
20
22
24
Days Post Implantation
Days Post Implantation
18

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1.0
D
C
No. 1
No. 2
No. 3
No. 4
No. 5
No. 6
No. 7
No. 8
0.8
0.6
0.4
0.2
0.0
Vehicle
Sorafenib, 40,
ig, QD
Cpd. 40, 40,
iv, BID
Cpd. 46, 40,
iv, BID
IR = 93.19%
IR = 81.14%
**
IR = 94.65%
IR = 93.64%
Cpd. 46, 20,
iv, BID
**
**
**
Vehicle, iv, BID Sorafenib, 40, Cpd. 40, 40, iv, Cpd. 46, 40, iv, Cpd. 46, 20, iv,
ig, QD BID BID BID
Figure 8. (A) Means of body weights for compound 40, 46 and sorafenib mice groups and vehicle
group in an in vivo MV4-11 xenograft model (n = 8). (B) Antitumor efficacy of compound 40, 46
and sorafenib in an MV4-11 xenograft model (n = 8). (C) Comparison of the final means tumor
volume in each group after 14-day treatment period with compound 40, 46 and sorafenib. (D) The
means of tumor weight and inhibition rate for compound 40, 46 and sorafenib mice groups in an
MV4-11 xenograft model (n = 8). ** P < 0.01.
To further characterize in vivo biological effects of compound 46, the
immunohistochemistry stain assays were performed in the tumor tissues after test
compound treatment. Figure 9A showed that compound 46 induced apoptosis of
tumor cells and inhibited cell proliferation at both dosages, respectively; while tumor
cells from vehicle-treated animals had a high proliferative index. Besides,
phosphorylation of STAT5 was blocked intensely by compound 46 (Figure 9A),
which demonstrated the on-target effect of compound 46 in vivo. Nevertheless,
hematoxylin and eosin (HE) staining results of the viscera of mice indicated that
compound 46 were not toxic to normal cells from heart, liver, spleen, lung and kidney
as compared with the HE staining results of vehicle groups (Figure 9B).
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Figure 9. (A) Representative micrographs of hematoxylin and eosin (HE), KI-67, P-STAT5 and
TUNEL staining of tumor tissues of 46 treated groups in comparison with the vehicle and
sorafenib group. (B) Representative micrographs of hematoxylin and eosin (HE) staining of
normal tissues of 46 treated groups in comparison with the vehicle and sorafenib group.
Conclusion
Initiated from our hit compound from virtual screening, a series of FLT3-ITD
inhibitors were investigated to improve efficacy against MV4-11 cells that harbored
FLT3-ITD mutation, from which we discovered new FLT3 inhibitors with stronger
20

ACCEPTED MANUSCRIPT
selectivity than FN-1501. The representative compound (46) showed potent cellular
efficacy and specificity against MV4-11 cells, consistently with its high selectivity
against FLT3-ITD over the structurally similar c-kit and FMS kinases. Meanwhile,
this compound also displayed inhibitory activities against some concurrent
FLT3-ITD-TKD mutants biochemically and in transformed BaF3 cells. Further, the
suppression of FLT3 signal pathway by compound 46 was confirmed in MV4-11 cells
both in vitro and in vivo. Compound 46 was well tolerated to mice at dosage up to 200
mg/kg followed the intravenous administration. Furthermore, significant antitumor
efficacy was observed for compound 46 in MV4-11-xenograft model with IR of
94.7% and T/C% of 8% at dosage of 40 mg/kg; meanwhile it caused little decrease in
mice body weight and showed no obvious toxicity to the viscera of treated mice,
indicating its good safety profile. In all, compound 46 represented a potential drug
candidate for AML driven by FLT3-ITD mutation.
EXPERMENTAL SECTION
Reagents and General Methods. Unless otherwise specified, reagents were purchased from
commercial suppliers and used without further purification. Melting points were determined by
X-4 digital display micro-melting point apparatus (Beijing Tech Instrument Co., Ltd.); NMR
1
spectra were recorded on Bruker AVANCE AV-600 spectrometer (600 MHz for H, 151 MHz for
13C) or AVANCE AV-500 spectrometer (500 MHz for 1H, 126 MHz for 13C) or AVANCE AV-400
spectrometer (400 MHz for 1H, 101 MHz for 13C) or Bruker AVANCE AV-300 spectrometer (300
1
MHz for H, 75 MHz for 13C); Mass spectra were obtained on the Agilent 1100 LC / MSD mass
spectrometer (Agilent, USA) or Q-tofmicro MS (micromass company). All reactions were
monitored by TLC (Merck Kieselgel GF254) and spots were visualized with UV light or iodine.
The purity of biologically evaluated compounds was >95% as determined by HPLC (Agilent
Technologies). Ten µL of the solution was injected on a column (150 mm × 4.6 mm; 5 µm;
Symmetry C18, ZORBAX SB-C18 PN883975-902). The column was kept in a thermostat at 25
°C. Water and methanol were used as the mobile phase at flow rate of 1 mL·min−1.
Procedure for the synthesis of compound 1. To
a
stirring solution of
4-bromo-2-thiophenecarboxylic acid (3 g, 14.5 mmol) in methanol (25 mL) was added thionyl
chloride (1.74 g, 14.5 mmol). Reaction mixture was heated to reflux and stirred for 6 h. Upon
completion, the residue was taken up in ice water (60 mL). The pH was adjusted to 9-10 with
saturated sodium carbonate solution. The aqueous phase was extracted with ethyl acetate (40 mL
× 3). The combined organic phase was washed twice with saturated brine (20 mL × 2) and dried
over anhydrous MgSO4. After filtering out MgSO4, the solvent of filtrate was removed in vacuo to
21

ACCEPTED MANUSCRIPT
afford crude product of title compound as a pale-yellow oil which was used for next step directly.
1
Yield: 98%; H NMR (300 MHz, CDCl3) δ: 7.69 (s, 1H), 7.45 (s, 1H), 3.90 (s, 3H). MS (m/z):
[M+H]+ 221.0, 223.0.
Procedure for the synthesis of compound 2. In a sealed tube, compound 1 (5.00 g, 22.6 mmol)
was dissolved in anhydrous DMF (5 mL) followed by the addition of cuprous cyanide (6.08 g,
67.9 mmol) and potassium iodide (0.01 g, 0.06 mmol) under an argon atmosphere. Reaction
mixture was heated at 160 °C for 10 h. After this period, the reaction was quenched with a mixed
solution of ammonia and ice water (v / v = 1 : 3, 50 mL). The precipitate was filtered out and
rinsed with ethyl acetate. The filtrate was` extracted with ethyl acetate (40 mL × 3). The combined
organic phase was washed twice with saturated brine (30 mL × 2) and dried over anhydrous
MgSO4. The solvent was removed in vacuo and the residue was purified by silica gel column
chromatography (PE : EA = 20 : 1) to give title compound as a white solid. Yield: 79%; mp:
107-109 °C. 1H NMR (300 MHz, CDCl3) δ: 8.09 (s, 1H), 7.93 (s, 1H), 3.93 (s, 3H). MS (m/z):
[M+H]+ 168.1.
Procedure for the synthesis of compound 3. To a stirring solution of compound 2 (1.67 g, 10
mmol) in anhydrous ethanol (25mL) was added hydrazine hydrate (1.19 g, 10 mmol). Reaction
was stirred at 45 °C for 10 h. The solvent was removed in vacuo to afford the pale-yellow solid,
which was recrystallized with 95% ethanol to give title compound as a white solid. Yield: 87%;
mp: 162-164 °C; 1H NMR (300 MHz, DMSO-d6) δ: 10.00 (s, 1H), 8.73 (d, J = 1.3 Hz, 1H), 7.99
(d, J = 1.3 Hz, 1H), 4.58 (s, 2H). MS (m/z): [M+H]+ 168.21, MS (m/z): [M-H]- 166.30.
General procedure for the synthesis of compound 4a-4b. To a stirring solution of
4-fluoronitrobenzene (1.41 g, 10.00 mmol) in anhydrous DMF (25 mL) were added
1-methylpiperazine (1.01 g, 10.00 mmol) and triethylamine (1.01 g, 10.00 mmol). Reaction was
stirred at r.t. for 24 h. Upon completion, the residue was taken up in ice water (60 mL). The pH
was adjusted to 8 with saturated sodium carbonate solution, and the aqueous phase was extracted
with ethyl acetate (40 mL × 3). The combined organic phase was washed twice with saturated
brine (30 mL × 2) and dried over anhydrous MgSO4. After filtering out MgSO4, the solvent of
filtrate was removed in vacuo and the residue was purified by silica gel column chromatography
(PE : EA = 1 : 1) to afford compound 4a as an orange solid. Yield: 85%. mp: 35-38 °C; 1H NMR
(300 MHz, DMSO-d6) δ: 8.05 (d, J = 9.5 Hz, 2H), 7.03 (d, J = 9.5 Hz, 2H), 3.54−3.37 (m, 4H),
2.47−2.38 (m, 4H), 2.22 (s, 3H). MS (m/z): [M+H]+ 222.3.
1-Methyl-4-(3-nitrophenyl)piperazine (4b)
Yellow solid was afforded as the crude product which was used in next step without further
purification. MS (m/z): [M+H]+ 222.2.
22

ACCEPTED MANUSCRIPT
General procedure for the synthesis of compound 4c-4g. To a stirring solution of
4-nitrobenzoic acid (1.67 g, 10 mmol) in anhydrous DMF (30 mL) were added
3-morpholinylpropylamine (1.44 g, 10 mmol), HATU (7.6 g, 20 mmol) and N,
N-diisopropylethylamine (3 mL, 4 mmol). Reaction mixture was stirred at 35 °C for 4 h. Upon
completion, the residue was taken up in ice water (60 mL). The aqueous phase was extracted with
ethyl acetate (50 mL × 3). The combined organic phase was washed twice with saturated brine (20
mL × 2) and dried over anhydrous MgSO4. After filtration, the solvent of filtrate was removed in
vacuo, and the residue was purified by silica gel column chromatography (DCM : MeOH : Et3N =
300 : 1 : 1) to afford compound 4d as a white solid; Yield: 70%; mp: 182-184 °C; 1H NMR (400
MHz, DMSO-d6) δ: 8.89 (s, 1H), 8.44−8.21 (m, 2H), 8.17−7.90 (m, 2H), 3.67 (s, 4H), 2.75 (s, 4H),
1.81 (s, 2H). MS (m/z): [M+H]+ 294.2.
(4-Methylpiperazin-1-yl)(4-nitrophenyl)methanone (4c)
White solid was afforded as the crude product which was used in next step without further
purification. MS (m/z): [M+H]+ 250.2.
N-(3-(diethylamino)propyl)-4-nitrobenzamide (4e)
White solid was afforded as the crude product which was used in next step without further
purification. MS (m/z): [M+H]+ 280.1.
(4-Methylpiperazin-1-yl)(3-nitrophenyl)methanone (4f)
White solid was afforded as the crude product which was used in next step without further
purification. MS (m/z): [M+H]+ 250.3.
N-(1-methylpiperidin-4-yl)-4-nitrobenzamide (4g)
White solid was afforded as the crude product which was used in next step without further
purification. MS (m/z): [M+H]+ 264.3.
General procedure for the synthesis of compound 5a-5g. To a stirring solution of compound 4a
(1.2 g, 4.77 mmol) in 70% ethanol (20 mL) were added ammonium chloride (1.28 g, 23.85 mmol)
and reduced iron powder (0.8 g, 14.31 mmol). The reaction was refluxed for 5 h. Upon completion,
the mixture was filtered while hot and rinsed with ethyl acetate. The solvent was removed in
vacuo, and the residue was purified by silica gel column chromatography (PE : EA = 1 : 2) to
1
afford compound 5a as an orange solid. Yield: 97%; mp: 108-110 °C; H NMR (300 MHz,
DMSO-d6) δ 6.67 (d, J = 8.8 Hz, 2H), 6.48 (d, J = 8.8 Hz, 2H), 4.53 (s, 2H), 2.92−2.84 (m, 4H),
2.46−2.36 (m, 4H), 2.19 (s, 3H). MS (m/z): [M+H]+ 192.3.
3-(4-Methylpiperazin-1-yl)aniline (5b)
Brown oil was afforded as the crude product which was used in next step without further
purification. MS (m/z): [M+H]+ 192.1.
(4-Aminophenyl)(4-methylpiperazin-1-yl)methanone (5c)
23

ACCEPTED MANUSCRIPT
White solid was afforded as the crude product which was used in next step without further
purification. MS (m/z): [M+H]+ 220.3.
4-Amino-N-(3-morpholinopropyl)benzamide (5d)
White solid was afforded as the crude product which was used in next step without further
purification. MS (m/z): [M+H]+ 264.2.
4-Amino-N-(3-(diethylamino)propyl)benzamide (5e)
White solid was afforded as the crude product which was used in next step without further
purification. MS (m/z): [M+H]+ 250.2.
(3-Aminophenyl)(4-methylpiperazin-1-yl)methanone (5f)
Light yellow solid was afforded as the crude product which was used in next step without further
purification. MS (m/z): [M+H]+ 220.3.
4-Amino-N-(1-methylpiperidin-4-yl)benzamide (5g)
Light yellow solid was afforded as the crude product which was used in next step without further
purification. MS (m/z): [M+H]+ 234.3.
General procedure for the synthesis of compound 6a-6g. An aqueous solution of sodium
bicarbonate (0.57 g, 6.78 mmol) (20 mL) was stirred at r.t. for 10 min, and then compound 5a
(0.50 g, 2.26 mmol) in dichloromethane (10 mL) was added under ice-cooling. Thiophosgene
(0.39 g, 3.39 mmol) in dichloromethane solution (5 mL) was added dropwise for 0.5 h. Reaction
was stirred under ice-cooling for 6 h. The aqueous phase was extracted with dichloromethane (20
mL × 3). The combined organic phase was washed twice with saturated brine (20 mL × 2) and
dried over anhydrous MgSO4. After filtration, the solvent of filtrate was removed in vacuo, and
the residue was purified by silica gel column chromatography (DCM : MeOH = 100 : 1) to afford
compound 6a as a pale-yellow solid; Yield: 63%; mp: 94-96 °C; 1H NMR (300 MHz, DMSO-d6)
δ: 7.27 (d, J = 9.0 Hz, 2H), 6.95 (d, J = 9.1 Hz, 2H), 3.24−3.15 (m, 4H), 2.49−2.39 (m, 4H), 2.23
(s, 3H). MS (m/z): [M+H]+ 234.1.
1-(3-Isothiocyanatophenyl)-4-methylpiperazine (6b)
Brown oil was afforded as the crude product which was used in next step without further
purification. MS (m/z): [M+H]+ 234.1, MS (m/z): [M-H]- 232.1.
(4-Isothiocyanatophenyl)(4-methylpiperazin-1-yl)methanone (6c)
White solid was afforded as the crude product which was used in next step without further
purification. MS (m/z): [M+H]+ 262.1, MS (m/z): [M-H]- 260.1.
4-Isothiocyanato-N-(3-morpholinopropyl)benzamide (6d)
White solid was afforded as the crude product which was used in next step without further
purification. MS (m/z): [M+H]+ 306.1.
N-(3-(diethylamino)propyl)-4-isothiocyanatobenzamide (6e)
24

ACCEPTED MANUSCRIPT
White solid was afforded as the crude product which was used in next step without further
purification. MS (m/z): [M+H]+ 292.4.
(3-Isothiocyanatophenyl)(4-methylpiperazin-1-yl)methanone (6f)
White solid was afforded as the crude product which was used in next step without further
purification. MS (m/z): [M+H]+ 262.1.
4-Isothiocyanato-N-(1-methylpiperidin-4-yl)benzamide (6g)
White solid was afforded as the crude product which was used in next step without further
purification. MS (m/z): [M+H]+ 276.4.
General procedure for the synthesis of compound 7a-7g. To a stirring solution of compound 3
(0.17 g, 1 mmol) in anhydrous ethanol (10 mL) were added compound 6a (0.26 g, 1.1 mmol) and
pyridine (0.79 mg, 0.01 mmol). Reaction mixture was refluxed for 1 h. Upon completion, the
solvent was removed in vacuo, and the residue was Recrystallized with 95% ethanol to afford
compound 7a as a pale-yellow solid which was used in next step directly. Yield: 52%; mp:
1
192-194 °C; H NMR (300 MHz, DMSO-d6) δ: 12.52 (s, 1H), 10.60 (s, 1H), 10.39 (s, 1H), 8.28
(s, 1H), 8.07 (s, 1H), 6.66−6.64 (m, 2H), 6.30−6.28 (m, 2H), 3.31−2.43 (m, 8H), 2.21 (s, 3H). MS
(m/z): [M+H]+ 401.1, MS (m/z): [M-H]- 399.1.
General procedure for the synthesis of compound 8-14.
5-(5-((4-(4-Methylpiperazin-1-yl)phenyl)amino)-1,3,4-oxadiazol-2-yl)thiophene-3-carbonitril
e (8). To a stirring solution of compound 7a (0.4 g, 1 mmol) in anhydrous ethanol (15 mL) was
added mercury acetate (0.35 g, 1.1 mmol). Reaction mixture was heated to reflux and stirred for 3
h. Upon completion, the mixture was filtered and rinsed with methanol. The solvent was removed
in vacuo, and the residue was purified by silica gel column chromatography (DCM : MeOH : TEA
= 40 : 2 : 1) to afford title compound as a white solid. Yield: 26%; mp: 255-257 °C; 1H NMR (300
MHz, DMSO-d6) δ: 10.50 (s, 1H), 8.77−8.76 (m, 1H), 7.97−7.96 (m, 1H), 7.46−7.43 (m, 2H),
6.97−6.94 (m, 2H), 3.09−3.06 (m, 4H), 2.51−2.46 (m, 4H), 2.24 (s, 3H). 13C NMR (151 MHz,
DMSO-d6) δ: 60.53 (s), 153.04 (s), 146.70 (s), 140.66 (s), 131.08 (s), 128.90 (s), 127.79 (s),
118.87 (s), 116.95 (s), 115.01 (s), 110.94 (s), 63.00 (s), 61.18 (s), 54.51 (s), 52.45 (s), 29.66 (s),
7.65 (s). HRMS-EI m/z [M+H]+ calcd for C18H19N6OS: 367.1341, found: 367.1319.
5-(5-((3-(4-Methylpiperazin-1-yl)phenyl)amino)-1,3,4-oxadiazol-2-yl)thiophene-3-carbonitril
e (9). The preparation of compound 9 was similar with that of compound 8 to afford title
1
compound as a yellow solid. Yield: 42%; mp: 250-251 °C; H NMR (300 MHz, DMSO-d6) δ:
10.64 (s, 1H), 8.79−8.78 (m, 1H), 7.97−7.96 (m, 1H), 7.28−6.59 (m, 4H), 3.15-3.12 (m, 4H),
2.50−2.45 (m, 4H), 2.23 (s, 3H). HRMS-EI m/z [M+H]+ calcd for C18H19N6OS: 367.1341, found:
367.1311.
5-(5-((4-(4-Methylpiperazine-1-carbonyl)phenyl)amino)-1,3,4-oxadiazol-2-yl)thiophene-3-car
25

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bonitrile (10). The preparation of compound 10 was similar with that of compound 8 to afford
title compound as a yellow solid. Yield: 42%; mp: 292-293 °C; 1H NMR (300 MHz, DMSO-d6) δ:
11.26 (s, 1H), 8.83 (s, 1H), 8.04 (s, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 3.56
(m, 4H), 2.33 (m, 2H), 2.20 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ: 169.26 (s), 160.06 (s),
153.55 (s), 141.07 (s), 139.98 (s), 129.40 (s), 128.94 (s), 127.52 (s), 117.18 (s), 114.97 (s), 110.96
(s), 45.97 (s), 0.58 (s). HRMS-EI m/z [M+H]+ calcd for C19H19N6O2S: 395.1290, found: 395.1276.
5-(5-((3-(4-Methylpiperazine-1-carbonyl)phenyl)amino)-1,3,4-oxadiazol-2-yl)thiophene-3-car
bonitrile (11). The preparation of compound 11 was similar with that of compound 8 to afford
title compound as a white solid. Yield: 52%; mp: 261-263 °C; 1H NMR (300 MHz, DMSO-d6) δ:
10.99 (s, 1H), 8.80−8.79 (m, 1H), 8.02−8.01 (m, 1H), 7.66−7.01(m, 4H), 3.60−3.42 (m, 4H),
3.32−3.20 (m, 2H), 2.20 (s, 3H). HRMS-EI m/z [M+H]+ calcd for C19H19N6O2S: 395.1290, found:
395.1268.
4-((5-(4-Cyanothiophen-2-yl)-1,3,4-oxadiazol-2-yl)amino)-N-(1-methylpiperidin-4-yl)benzam
ide (12). The preparation of compound 12 was similar with that of compound 8 to afford title
compound as a white solid. Yield: 47%; mp: 266-268 °C; 1H NMR (300 MHz, DMSO-d6) δ: 11.10
(s, 1H), 8.81−8.80 (m, 1H), 8.12−8.09 (m, 1H), 8.03−8.01 (m, 1H), 7.89−7.86 (m, 2H), 7.66−7.63
(m, 2H), 3.75−3.67 (m, 1H), 2.79−2.75 (m, 2H), 2.17 (s, 3H), 1.98−1.91 (m, 2H), 1.77−1.74 (m,
2H), 1.64−1.52 (m, 2H). 13C NMR (151 MHz, DMSO-d6) δ: 172.61 (s), 165.60 (s), 159.96 (s),
153.59 (s), 141.22 (s), 141.00 (s), 129.37 (s), 129.07 (s), 128.46 (s), 127.51 (s), 116.79 (s), 114.95
(s), 111.00 (s), 54.24 (s), 52.46 (s), 46.10 (s), 45.29 (s), 31.00 (s), 21.66 (s), 7.66 (s). HRMS-EI
m/z [M+H]+ calcd for C20H21N6O2S: 409.1447, found: 409.1449.
4-((5-(4-Cyanothiophen-2-yl)-1,3,4-oxadiazol-2-yl)amino)-N-(3-morpholinopropyl)benzamid
e (13). The preparation of compound 13 was similar with that of compound 8 to afford title
compound as a white solid. Yield: 51%; mp: 279-281 °C; 1H NMR (300 MHz, DMSO-d6) δ: 11.11
(s, 1H), 8.81−8.80 (m, 1H), 8.37−8.34 (m, 1H), 8.03−8.01 (m, 1H), 7.88−7.85 (m, 2H), 7.66−7.63
(m, 2H), 3.58−3.55 (m, 4H), 3.31−3.25 (m, 2H), 2.34−2.29 (m, 6H), 1.72 (m, 2H). 13C NMR (151
MHz, DMSO-d6) δ: 165.99 (s), 159.99 (s), 153.60 (s), 141.18 (s), 141.04 (s), 129.41 (s), 128.86
(s), 128.56 (s), 127.52 (s), 116.88 (s), 114.96 (s), 110.99 (s), 56.59 (s), 55.39 (s), 53.81 (s), 40.47
(s), 38.17 (s), 29.65 (s), 26.50 (s). HRMS-EI m/z [M+H]+ calcd for C20H21N6O2S: 439.1552,
found: 439.1519.
4-((5-(4-Cyanothiophen-2-yl)-1,3,4-oxadiazol-2-yl)amino)-N-(3-(diethylamino)propyl)benza
mide (14). The preparation of compound 14 was similar with that of compound 8 to afford title
1
compound as a white solid. Yield: 52%; mp: 254-255 °C; H NMR (300 MHz, DMSO-d6) δ:
8.81−8.80 (m, 1H), 8.41−8.37 (m, 1H), 8.02−8.79 (m, 1H), 7.87−7.83 (m, 2H), 7.65−7.62 (m,
2H), 3.30−3.24 (m, 2H), 2.44−2.40 (m, 6H), 1.66−1.59 (m, 2H), 0.97−0.92 (m, 6H). 13C NMR
26

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(151 MHz, DMSO-d6) δ: 165.96 (s), 159.98 (s), 153.60 (s), 141.16 (s), 141.01 (s), 129.40 (s),
128.81 (s), 128.59 (s), 127.52 (s), 116.89 (s), 114.96 (s), 111.02 (s), 55.38 (s), 50.53 (s), 46.72 (s),
38.33 (s), 26.76 (s), 11.83 (s). HRMS-EI m/z [M+H]+ calcd for C21H25N6O2S: 425.1760, found:
425.1745.
General procedure for the synthesis of compound 15-18.
5-(5-((3-(4-Methylpiperazin-1-yl)phenyl)amino)-1,3,4-oxadiazol-2-yl)thiophene-3-carboxami
de (15). To a stirring solution of compound 9 (0.125 g, 0.342 mmol) in anhydrous ethanol (4 mL)
were added 30% H2O2 (0.1 mL) and 6N aqueous NaOH (0.15 mL). Reaction was stirred at 50 °C
for 6 h. Upon completion, the mixture was filtered and rinsed with methanol. The solvent was
removed in vacuo, and the residue was purified by silica gel column chromatography (DCM :
MeOH : TEA = 40 : 2 : 1) to afford title compound as a white solid. Yield: 51%; mp: 255-256 °C;
1H NMR (300 MHz, DMSO-d6) δ: 10.95 (s, 1H), 8.35−8.34 (m, 1H), 8.03 (s, 1H), 7.93−7.92 (m,
1H), 7.43−7.42 (s, 1H), 7.27−6.60 (m, 4H), 3.15−3.12 (m, 4H), 2.50−2.45 (m, 4H), 2.23 (s, 3H).
HRMS-EI m/z [M+H]+ calcd for C18H21N6O2S: 385.1447, found: 385.1418.
5-(5-((4-(4-Methylpiperazine-1-carbonyl)phenyl)amino)-1,3,4-oxadiazol-2-yl)thiophene-3-car
boxamide (16). The preparation of compound 16 was similar with that of compound 15 to afford
title compound as a white solid. Yield: 52%; mp: 272-274 °C; 1H NMR (300 MHz, DMSO-d6) δ:
10.96 (s, 1H), 8.36 (d, 1H), 8.00 (s, 1H), 7.99−7.98 (m, 1H), 7.43 (s, 1H), 7.27−6.60 (m, 4H),
3.48−3.35 (m, 4H), 2.31−2.30 (m, 4H), 1.97 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ: 169.25
(s), 163.15 (s), 159.73 (s), 154.39 (s), 140.05 (s), 139.15 (s), 132.90 (s), 129.53 (s), 128.97 (s),
127.98 (s), 125.74 (s), 117.06 (s), 55.38 (s), 54.97 (s), 46.08 (s). HRMS-EI m/z [M+H]+ calcd for
C19H21N6O3S: 413.1396, found: 413.1407.
5-(5-((4-((3-Morpholinopropyl)carbamoyl)phenyl)amino)-1,3,4-oxadiazol-2-yl)thiophene-3-c
arboxamide (17). The preparation of compound 17 was similar with that of compound 15 to
1
afford title compound as a white solid. Yield: 44%; mp: 278-280 °C; H NMR (300 MHz,
DMSO-d6) δ: 11.02 (s, 1H), 8.81−8.79 (m, 2H), 8.02−7.98 (m, 2H), 7.88−7.86 (m, 2H), 7.69−7.67
(m, 2H), 7.44 (s, 1H), 3.59-3.56 (m, 4H), 3.33-3.26 (m, 2H), 2.35-2.31 (m, 6H), 1.72-1.64 (m,
2H). HRMS-EI m/z [M+H]+ calcd for C21H25N6O4S: 457.1658, found: 457.1637.
5-(5-((4-((3-(Diethylamino)propyl)carbamoyl)phenyl)amino)-1,3,4-oxadiazol-2-yl)thiophene-
3-carboxamide (18). The preparation of compound 18 was similar with that of compound 15 to
1
afford title compound as a white solid. Yield: 42%; mp: 272-274 °C; H NMR (300 MHz,
DMSO-d6) δ: 11.00 (s, 1H), 8.41-8.36 (m, 2H), 8.00−7.98 (m, 2H), 7.86−7.83 (m, 2H), 7.66−7.63
(m, 2H), 7.43 (s, 1H), 3.26−3.24 (m, 2H), 2.47−2.41 (m, 6H), 1.68−1.59 (m, 2H), 0.97−0.92 (m,
6H). 13C NMR (151 MHz, DMSO-d6) δ:166.05 (s), 163.15 (s), 159.67 (s), 154.45 (s), 141.33 (s),
139.16 (s), 132.94 (s), 128.83 (s), 128.39 (s), 128.05 (s), 125.70 (s), 116.83 (s), 52.42 (s), 50.28
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(s), 46.69 (s) , 38.10 (s), 26.33 (s), 11.31 (s), 7.61 (s), 0.56 (s). HRMS-EI m/z [M+H]+ calcd for
C21H27N6O3S: 443.1865, found: 443.1838.
General procedure for the synthesis of compound 19a-19g. A mixture of 60% NaH (0.12 g, 5
mmol) was added in anhydrous THF (10 mL), followed by the addition of anhydrous CH3CN
(0.21 g, 5 mmol) and compound 2 (0.42 g, 2.5 mmol) in the ice bath. Reaction was heated to
reflux and stirred for 6 h. Upon completion, the reaction mixture was taken up in ice water (30 mL)
and the pH was adjusted to 5. After filtration, the filtrate was extracted with ethyl acetate (20 mL ×
3). The combined organic phase was washed twice with saturated brine (20 mL × 2) and dried
over anhydrous MgSO4. Then, MgSO4 was filtered out, and the filtrate was concentrated in vacuo,
Followed by recrystallization of the residue to afford compound 19a as a pale-yellow solid. Yield：
52%. mp: 151-153 °C; 1H NMR (600 MHz, DMSO-d6) δ: 9.01(s, 1H), 8.43 (s, 1H), 4.72 (s, 2H).
MS (m/z): [M+H]+ 176. 1.
3-(Benzo[b]thiophen-2-yl)-3-oxopropanenitrile (19b)
1
Light yellow solid. Yield: 73%. mp: 120-121 °C; H NMR (300 MHz, DMSO-d6) δ: 13.51 (s,
0.8H), 8.42−8.11 (m, 1H), 8.11−7.95 (m, 2H), 7.61−7.41 (m, 2H), 4.86 (s, 0.4H). MS (m/z):
[M-H]- 199.9.
3-(Benzo[d][1,3]dioxol-5-yl)-3-oxopropanenitrile (19c)
Yellow solid. Yield: 71%. mp: 131-133 °C; 1H NMR (300 MHz, CDCl3) δ: 7.49 (dd, J = 8.2, 1.8
Hz, 1H), 7.40 (d, J = 1.8 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.10 (s, 2H), 4.01 (s, 2H). MS (m/z):
[M+H]+ 190.2.
3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-oxopropanenitrile (19d)
White solid. Yield: 92%. mp: 168-170 °C; 1H NMR (300 MHz, DMSO-d6) δ: 7.52−7.47 (m, 1H),
7.44−7.43 (m, 1H), 7.03−7.00 (m, 1H), 4.67 (s, 2H), 4.35−4.33 (m, 2H), 4.31−4.29 (m, 2H). MS
(m/z): [M+Na]+ 226.0.
3-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-3-oxopropanenitrile (19e)
Light yellow solid. Yield: 93%. mp: 69-70 °C; 1H NMR (300 MHz, DMSO-d6) δ: 11.70 (s, 0.09H)
7.97 (d, J = 1.7 Hz, 1H), 7.89 (dd, J = 8.5, 1.8 Hz, 1H), 7.62 (d, J = 8.5 Hz, 1H), 4.76 (s, 1.82H).
MS (m/z): [M-H]- 224.0.
3-(2,3-Dihydrobenzofuran-5-yl)-3-oxopropanenitrile (19f)
1
Yellow solid. Yield: 95%. mp: 123-124 °C; H NMR (300 MHz, CDCl3) δ: 7.83 (q, J = 1.5 Hz,
1H), 7.73 (dd, J = 8.5, 2.0 Hz, 1H), 6.85 (d, J = 8.5 Hz, 1H), 4.69 (d, J = 8.8 Hz, 2H), 3.99 (s, 2H),
3.27 (q, J = 8.8 Hz, 2H). MS (m/z): [M-H]- 185.9.
3-(3,4-Dimethoxyphenyl)-3-oxopropanenitrile (19g)
1
Light yellow solid. Yield: 96%. mp: 139-141 °C; H NMR (300 MHz, CDCl3) δ: 7.54−7.47 (m,
2H), 6.92 (d, J = 8.9 Hz, 1H), 4.05 (s, 2H), 3.98 (s, 3H), 3.95 (s, 3H). MS (m/z): [M+H]+ 206.1.
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