Synthesis and biological evaluation of diaryl urea derivatives as FLT3 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127525

As a class III receptor tyrosine kinase (RTK), FMS-like tyrosine kinase 3 (FLT3) is always overexpressed in many cases of acute leukemia. This paper studies the structure-based synthesis and biological evaluation of diaryl urea derivatives as FLT3 inhibitors. Encouragingly, compounds 15b, 16b, 24a, and 24c showed excellent biological activities in a low nanomolar range. In particular, compound 16b demonstrated significant inhibitory potency against FLT3-ITD (IC₅₀ = 5.60 nM) and better antiproliferative activity than quizartinib against MV4-11 cell line (IC₅₀ = 0.176 nM). It is indicated that compound 16b for the treatment of acute myeloid leukemia could be very promising.

Full text of DOI:10.1016/j.bmcl.2020.127525

Bioorganic&MedicinalChemistryLetters30(2020)127525
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of diaryl urea derivatives as FLT3
inhibitors
Qing Zhanga, Kuantao Zhaoa, Lixun Zhang, Xiaoyu Jiao, Yongjie Zhang, Chunlei Tang⁎
School of Pharmaceutical Science, Jiangnan University, Wuxi, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
As a class III receptor tyrosine kinase (RTK), FMS-like tyrosine kinase 3 (FLT3) is always overexpressed in many
cases of acute leukemia. This paper studies the structure-based synthesis and biological evaluation of diaryl urea
derivatives as FLT3 inhibitors. Encouragingly, compounds 15b, 16b, 24a, and 24c showed excellent biological
activities in a low nanomolar range. In particular, compound 16b demonstrated significant inhibitory potency
against FLT3-ITD (IC50 = 5.60 nM) and better antiproliferative activity than quizartinib against MV4-11 cell line
(IC50 = 0.176 nM). It is indicated that compound 16b for the treatment of acute myeloid leukemia could be very
promising.
FLT3 inhibitor
Internal tandem duplication
MV4-11
Biological evaluation
Acute myeloid leukemia (AML) is a malignant tumor of the bone
marrow and blood caused by immature bone marrow progenitor
cells.1,2 In the United States, about 12,000 new cases of AML are re-
ported each year. In China, the incidence of acute leukemia is sig-
nificantly higher than that of chronic leukemia, with a ratio of 5.5–1.3
Currently, conventional treatments include intensive chemotherapy
and allogeneic hematopoietic stem cell transplantation (HSCT), but the
efficacy is poor in elderly patients who are not able to withstand in-
tensive chemotherapy.4,5 Thus, there is a compelling medical need for
developing more efficient targeted agents.
FLT3 inhibitors based on their mechanism of action. Type I inhibitors
(e.g., midostaurin and gilteritinib) bind the FLT3 receptor in the active
conformation near the activation loop or near the ATP binding pocket,
type II (e.g., sorafenib and quizartinib (Fig. 1)) bind FLT3 receptor in
the inactive conformation in a region adjacent to the ATP binding do-
main.15,16 Diaryl ureas were identified as a scaffold that elicited potent
inhibition of FLT3,17 because it can form hydrogen bond interactions
with Asp 829 and Glu 661 of the FLT3 receptor, and can form π-π stacks
with Phe 691 and Phe 830, which has the effect of stabilizing the
conformation of the compound.18 These results indicate that diaryl
ureas play a pivot role in maintaining the activities of the compounds.
Based on the above analysis, our team retained the diaryl ureas and
designed a series of diaryl urea compounds with imidazobenzothiazole
as the basic skeleton. Here, the synthesis process and the relationship
between structure and activity are reported.
FMS-like tyrosine kinase 3 (FLT3) is an expression product of a
proto-oncogene located on chromosome 13q12, which plays an im-
portant role in the regulation of survival, proliferation, and differ-
entiation of hematopoietic progenitor cells.6–8 FLT3 mutations are de-
tected in approximately 30% of newly diagnosed AML cases and are
associated with a high leukemic burden and poor prognosis.9,10 The two
most common FLT3 mutations are internal tandem duplication (ITD) in
the juxtamembrane domain, which has been validated as the driving
mutation, and point mutations with in the tyrosine kinase domain
(TKD).11,12 Despite the different activation pathways, both the ITD
mutation and the TKD mutation could cause the abnormally activation
of ligand-independent FLT3, thereby inducing the survival and pro-
liferation of cytokine-independent AML cells.13 Therefore, FLT3 is re-
garded as the most potential target for the treatment of AML.
The synthetic methods of these compounds are shown in Schemes
1–3. The commercially available 4-nitrophenylacetic acid (1) was es-
terified and reduced with Pd/C catalyst under H2 to obtain compound
3. Phenyl chloroformate reacted with the corresponding heterocyclic
amine to produce 4a-4d. Then 4a-4d with compound 3 by amine
transesterification obtained the corresponding ureas, and the esters
were hydrolyzed with 0.5 mol/L NaOH aqueous solution to give the
corresponding acids 6a-6d. 6a-6d were submitted to the coupling with
commercially available 2-amino-6-methoxybenzothiazole (7) to afford
compounds 8a-8d (Scheme 1).
At present, many small molecule FLT3 inhibitors have been devel-
oped,14 and they are divided into type I FLT3 inhibitors and type II
Alkylation of 4-nitrophenol (9) with 4-(2-chloroethy)morpholine
⁎ Corresponding author.
E-mail address: tangcl@jiangnan.edu.cn (C. Tang).
a These authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2020.127525
Received 20 July 2020; Received in revised form 18 August 2020; Accepted 25 August 2020
Availableonline06September2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

Q. Zhang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127525
Fig. 1. Structures of some FLT3 inhibitors.
Scheme 1. Synthesis of compounds 8a-8d. Reagents and conditions: (i) SOCl2, EtOH, r.t., 2 h, 89.3%; (ii) 10% Pd/C, H2, MeOH, r.t., 12 h, 92.5%; (iii) 4a-4d, DMAP,
TEA, CHCl3, 50 °C, 12 h, 55.7–83.5%; (iv) 0.5 mol/L NaOH-wt, THF, 40 °C, overnight, 76.2–95.8%; (v) 2-amino-6-methoxybenzothiazole, HATU, DIPEA, DMF, r.t.,
20 h, 15.2–48.3%.
hydrochloride followed by reduction of the nitro group with Pd/C
catalyst under H2 gave compound 11. 11 and ammonium thiocyanate
gave rise to compound 12 under the action of bromine and acetic acid.
Then compound 12 or 2-amino-6-methoxybenzothiazole (7) was con-
densed with 2-bromo-4′-nitroacetophenone in refluxing isopropanol to
yield 13a-13b. Reduction of the nitro group using iron and ammonium
chloride gave the corresponding amines 14a-14b, which were reacted
with the corresponding compound to yield the ureas 15a-16c. 14b
reacted with the corresponding carboxylic acid to provide amides 17a-
17d and 19a-19d (Scheme 2).
were all less than 10%. This may be due to the change in the connection
between the basic skeleton and the phenyl ring of the diaryl urea
structure, resulting in it not being between the two phenylalanine re-
sidues of Phe 691 in the gatekeeper residue and Phe 830 in the DFG
motif. It can only form a π-π interaction with Phe 691 in the gatekeeper
residue, and cannot achieve the effect of stabilizing the active con-
formation of the compound.
Based on the above analysis, we fixed the imidazobenzothiazole ring
as the basic skeleton and explored the effect of R2 group as methyl or 2-
morpholinoethyl on inhibitory activities of compounds. 2-morpholi-
noethyl substituted compounds 16a-16c showed higher FLT3 kinase
inhibition rates compared to 15a-15c. Overall, when the R2 group was
2-morpholinoethyl substituted, the kinase activities of the compounds
were better than that of the methyl substituted compounds.
Subsequently, we introduced p-substituted amide between the diaryl
urea structure and the basic skeleton to obtain compounds 17a-17d and
19a-19d. As is showed in the results, this amide bonding pattern sig-
nificantly reduced the kinase inhibitory activities of the compounds.
Compared with compound 16b (inhibition rate of 99.6%), the inhibi-
tion rates against FLT3-ITD of compounds 17c and 19c lowered to
38–41%. According to the above activity data, we try to exchange the
urea structure and 2-morpholinoethoxy position in the structure of
compounds 16a-16c to obtain compounds 24a-24d. We hypothesized
that the position of urea structure and 2-morpholinoethoxy are ex-
changed, the benzene ring of the basic skeleton can play the same role
as the benzene ring on the diaryl urea structure. So the benzene ring
The synthetic route of the compounds 24a-24d is shown in Scheme
3. Compound 21 was obtained by cyclization of 2-amino-6-ni-
trobenzothiazole (20) and 2-bromo-4′-hydroxyacetophenone. Mitsu-
nobu reaction of 21 with 4-(2-chloroethy)morpholine hydrochloride
gave compound 22 in basic conditions. After reduction of the nitro
group, compound 23 and 4a-4d reacted to generate 24a-24d.
In order to have a preliminary screening, all synthesized compounds
were tested for their inhibitory activities against FLT3-ITD at 500 nM
with quizartinib as a positive control (Tables 1 and 2). Remarkably,
many compounds showed good inhibitory activities against FLT3-ITD at
500 nM. We analyzed the preliminary structure–activity relationships
(SARs) based on the results in Tables 1 and 2. Firstly, we tried to syn-
thesize compounds 8a-8d by ring-opening of the imidazole ring in the
basic skeleton. Compared with compounds 15a-15c (inhibition rates of
52.6–97.2%), ring-opening compounds 8a-8d led to a significantly
decreased inhibition rates against FLT3-ITD, and the inhibition rates
2

Q. Zhang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127525
Scheme 2. Synthesis of compounds 15a-17d and 19a-19d. Reagents and conditions: (i) 4-(2-chloroethy)morpholine hydrochloride, Cs2CO3, DMF, 100 °C, 2.5 h,
91.1%; (ii) 10% Pd/C, H2, MeOH, reflux, 3.5 h, 94.0%; (iii) NH4SCN, Br2, AcOH, r.t., 4 h, 88.5%;(iv) 2-bromo-4′-nitroacetophenone, NaHCO3, i-PrOH, reflux, 3 h,
67.4–89.3%; (v) Fe, NH4Cl, EtOH, H2O, reflux, 2.5 h, 84.7–90.8%; (vi) 4b-4d, DMAP, TEA, CHCl3, 50 °C, overnight, 50.2–85.4%. (vii) HATU, DIPEA, DMF, r.t, 20 h,
54.3–79.8%.
Scheme 3. Synthesis of compounds 24a-24d. Reagents and conditions: (i) 2-bromo-4′-hydroxyacetophenone, NaHCO3, n-BuOH, reflux, 3 h, 32.4%; (ii) 4-(2-
chloroethy)morpholine hydrochloride, Cs2CO3, DMF, 120 °C, 3 h, 93.4%; (iii) Fe, NH4Cl, EtOH, H2O, reflux, 2.5 h, 84.2%; (iv) 4a-4d, DMAP, TEA, CHCl3, 50 °C,
overnight, 25.5–48.0%.
connected to the imidazole ring can be removed or replaced by other
groups, which is significant for reducing the molecular weight and
improving the physical and chemical properties of the compound.
Encouragingly, compounds 24a-24d displayed similar inhibition rates
against FLT3-ITD to 16a-16c.
Meanwhile, the inhibition rates of compounds 15b, 16b, 24a, and
24c at 500 nM were all more than 97.2%. Interestingly, we found that
the R1 groups of these compounds were all tert-butyl substituted
3

Q. Zhang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127525
Table 1
Inhibition rates against FLT3-ITD at 500 nM by compounds 8a-8d, 15a-15c, 16a-16c, 17a-17d, and 19a-19d.
Compd.
L
–
R1
R2
–
Inhibit rate a (at 500 nM) against FLT3-ITD
8a
2.5%
0.4
0.9
8b
8c
–
–
–
–
−2.0%
6.4%
–
3.3
0.2
8d
15a
–
4.5%
-CH3
67.3%
0.4
15b
15c
16a
16b
16c
17a
-CH3
-CH3
97.2%
52.6%
79.9%
99.6%
66.7%
36.0%
1.1
0.4
0.9
0.4
0.9
2.9
17b
17c
17d
19a
35.8%
38.5%
45.9%
35.0%
2.2
1.1
2.6
6.9
19b
19c
19d
29.0%
41.9%
28.1%
99.2%
3.6
0.7
3.3
0.9
Quizartinib b
a
–
–
–
Each experiment was run in duplicate and the values shown are the average of the two.
Used as a positive control.
b
isoxazole. The methyl-substituted isoxazole ring showed weaker in-
their IC50 values against FLT3-ITD and anti-proliferative activities
against human AML cell line MV4-11 (Table 3). The results suggested
hibition activities. The results revealed that the inhibition effect against
FLT3-ITD after methyl substitution was markedly reduced, compared
with the tert-butyl substituted compounds.
that compound 16b (FLT3-ITD IC50
=
5.60 nM, MV4-11
IC50 = 0.176 nM) showed better activity than quizartinib (FLT3-ITD
IC50 = 6.51 nM, MV4-11 IC50 = 0.836 nM). In particular, the IC50
value against the MV4-11 cell line of compound 16b was about 4.75-
On the basis of the above inhibition rate results, several potent
compounds (15b, 16b, 16c, and 24a-24d) were selected to evaluate for
4

Q. Zhang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127525
Table 2
exhibited excellent biological activity against FLT3-ITD kinase
(IC50 = 5.60–8.25 nM). Overall, the above statistics suggest the optimal
compound 16b with great promise for the treatment of AML.
Inhibition rates against FLT3-ITD at 500 nM by compounds 24a-24d.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Compd.
R1
Inhibit rate a (at 500 nM) against FLT3-ITD
Acknowledgements
24a
98.8%
82.5%
99.3%
76.9%
99.2%
2.2
0.7
1.8
1.5
0.9
This work was supported by the National First-class Discipline
Program of Food Science and Technology (JUFSTR20180101) and the
National Natural Science Foundation of China (Grant Nos. 21305051).
24b
24c
24d
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127525.
Quizartinib b
a
–
Each experiment was run in duplicate and the values shown are the average
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