Discovery of small molecule FLT3 inhibitors that are able to overcome drug-resistant mutations

Article abstract of DOI:10.1016/j.bmcl.2020.127532

Herein we report the discovery of 1-(5-(tert-butyl)isoxazol-3-yl)-3- (3-fluorophenyl)urea derivatives as new FLT3 inhibitors that are able to overcome the drug resistance mutations: the secondary D835Y and F691L mutations on the basis of the internal tandem duplications (ITD) mutation of FLT3 (FLT3-ITD/D835Y and FLT3-ITD/F691L, respectively). The most potent compound corresponds to 1-(5-(tert-butyl)isoxazol-3-yl)-3-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3- fluorophenyl)urea (4d), which showed IC₅₀s (half maximal inhibitory concentrations) of 0.072 nM, 5.86 nM and 3.48 nM against FLT3-ITD, FLT3-ITD/F691L and FLT3-ITD/D835Y, respectively. Compound 4d also showed good selectivity for FLT3 in a kinase profiling assay. Collectively, 4d could be a good lead compound and deserves further in-depth studies.

Full text of DOI:10.1016/j.bmcl.2020.127532

Bioorganic&MedicinalChemistryLetters30(2020)127532
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of small molecule FLT3 inhibitors that are able to overcome drug-
resistant mutations
Guo Zhanga,1, Wenqing Zhangc,1, Chenjian Shena, Jinshan Nana, Ming Chenb, Shusheng Laib,
⁎
Jiemin Zhongb, Bolin Lib, Tianqi Wanga, Yifei Wangc, Shengyong Yanga, Linli Lic,
a State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Sichuan 610041, China
b Guangxi Wuzhou Pharmaceutical Co. Ltd, Wuzhou, Guangxi 543000, China
c Key Laboratory of Drug Targeting and Drug Delivery System of Ministry of Education, West China School of Pharmacy, Sichuan University, Sichuan 610041, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Herein we report the discovery of 1-(5-(tert-butyl)isoxazol-3-yl)-3- (3-fluorophenyl)urea derivatives as new FLT3
inhibitors that are able to overcome the drug resistance mutations: the secondary D835Y and F691L mutations
on the basis of the internal tandem duplications (ITD) mutation of FLT3 (FLT3-ITD/D835Y and FLT3-ITD/F691L,
respectively). The most potent compound corresponds to 1-(5-(tert-butyl)isoxazol-3-yl)-3-(4-((6,7-dimethox-
yquinolin-4-yl)oxy)-3- fluorophenyl)urea (4d), which showed IC50s (half maximal inhibitory concentrations) of
0.072 nM, 5.86 nM and 3.48 nM against FLT3-ITD, FLT3-ITD/F691L and FLT3-ITD/D835Y, respectively.
Compound 4d also showed good selectivity for FLT3 in a kinase profiling assay. Collectively, 4d could be a good
lead compound and deserves further in-depth studies.
FLT3
Secondary point mutations
Small molecule inhibitor
Structure-activity relationship
Drug-resistant mutation
Acute myeloid leukemia (AML) is a hematological malignancy
caused by abnormal proliferation of myeloid hematopoietic progenitor
cells in the bone marrow. Clinical data have shown that approximately
30% of patients have activating mutations of FMS-like tyrosine kinase 3
(FLT3), and the most prevalent of which is internal tandem duplications
(ITD) mutation in the juxtamembrane domain of FLT3.1–5 Numerous
studies have indicated that FLT3 mutations are associated with a poor
prognosis for overall survival.6–9 FLT3 has thus been considered as a
valid target for the treatment of AML.10 Currently, a number of FLT3
inhibitors have been reported, and three of them, Midostaurin, Gilter-
itinib and Quizartinib (AC220), have been approved to clinical use for
the treatment of AML.11–19 Unfortunately, recent studies have shown
that resistance to currently known FLT3 inhibitors emerged due to
secondary point mutations in the kinase domain (KD) of FLT3. The most
common mutation sites are phenylalanine at position 691 (F691) and
aspartic acid at position 835 (D835).20–23 Drug discovery targeting the
secondary mutations of FLT3-ITD has thus been an urgent task.
D835Y and F691L of FLT3-ITD with IC50 values of 0.129 μM and
0.108 μM, respectively. Obviously, the potencies against these second
mutations of FLT3-ITD are not good and need further improvement. We
will in this investigation carry out a structural optimization to this
compound to improve its potency against the second mutations of
FLT3-ITD.
Our structural optimization will focus on the 1H-pyrazolo [3,4-d]
pyrimidine region (Figure 1 b) because 1-(5-(tert-butyl)isoxazol-3-yl)-
3-(3-fluoro-4-hydroxyphenyl)urea has been determined as an optimal
fragment in our previous study.24 We synthesized a total of 22 deri-
vatives (4a, 11a-o) of compound SKLB-677 with 1H-pyrazolo[3, 4-d]
pyrimidine substituted by different heteroaryl rings containing a pyr-
idine or pyrimidine ring.
In the first step, we replaced 1H-pyrazolo[3,4-d]pyrimidine with
various pyridine (or pyrimidine) -fused five (or six) membered ring
fragments and synthesized 7 compounds (4a-g). Synthetic routes for
compounds 4a-g are depicted in Scheme 1. Commercially available
reagents 1a-g reacted with 4-amino-2-fluorophenol through a nucleo-
philic substitution to give intermediate 2a-g. Condensation reaction
between 2a-g and 5-(tert-butyl)-3-isocyanatoisoxazole (3) produced
target compounds 4a-g.
In a previous study, we obtained a potent FLT3 inhibitor, 1-(4-((1H-
pyrazolo [3, 4-d]pyrimidin-4-yl)oxy)-3-fluorophenyl)-3-(5-(tert-butyl)
isoxazol-3-yl)urea (Figure 1a, SKLB-677). This compound showed an
excellent activity against FLT3-ITD mutant with an IC50 value of
1.3 nM.24 It also showed activity against the secondary mutations
Bioactivities of compounds 4a-g together with AC220 and SKLB-
⁎ Corresponding author.
E-mail address: lilinli@scu.edu.cn (L. Li).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2020.127532
Received 23 April 2020; Received in revised form 24 August 2020; Accepted 29 August 2020
Availableonline03September2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

G. Zhang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127532
Compounds 11a-o all showed potent activity against FLT3-ITD, ITD/
D835Y and ITD/F691L, except 11 g, which displaced moderate activity.
Nevertheless, their potency did not exceed that of compound 4d.
Overall, through the above structural optimization and SAR studies,
we obtained a number of new FLT3 inhibitors containing the scaffold 1-
(5-(tert-butyl)
isoxazol-3-yl)-3-(3-fluoro-4-(quinolin-4-yloxy)phenyl)
urea. Among them, 4d is the most potent one, which exhibited excellent
inhibitory activity against FLT3-ITD and drug-resistance mutations
FLT3-ITD/D835Y and FLT3-ITD/F691L (Figure 2). Further bioactivity
evaluations were then carried out on this compound.
Fig. 1. (a) The chemical structure of compound SKLB-677; (b) Schematic
showing the region being the focus of structural modification.
To examine the kinase selectivity of compound 4d, a kinase pro-
filing assay was performed through DiscoverX KINOMEscan kinase
profiling services. The results showed that 4d has a good kinase se-
lectivity with the calculated selectivity scores, S(1), S(5) and S(10),
being 0.082, 0.119 and 0.164, respectively (See Supporting Information
Table S1).
677 were then examined. Here cell-based bioactivity assays were
adopted, in which Ba/F3 cells expressing FLT3-ITD, FLT3-ITD/D835Y
and FLT3-ITD/F691L, as well as the parental Ba/F3 cells were used. The
measured bioactivities of these compounds are displayed in Table 1.
Compounds 4a, 4b, 4d and 4 g showed potent activity against both
FLT3-ITD mutation and drug resistance mutations FLT3-ITD/D835Y
and FLT3-ITD/F691L. Among them, 4d and 4 g, bearing quinoline and
quinazoline in region A, respectively, are the most active ones in terms
of the potency against drug resistance mutations FLT3-ITD/D835Y and
FLT3-ITD/F691L. All of these compounds showed weak or no activity
against the parental Ba/F3 cells, indicating that they indeed target the
mutated FLT3, and also have a low toxicity. Considering the poor water
solubility of 4 g, we chose 4d for further structural optimization.
In the second step, we tested the possible influence of 6 and 7-po-
sitions of quinoline on the bioactivity. To this end, a total of 15 com-
pounds (11a-o) with difference substituents at the 6 or 7-position of
quinoline were synthesized. Reaction routes for compounds 11a-o are
summarized in Scheme 2. Demethylation of 4-chloro-6,7-dimethox-
yquinoline (5) under different conditions gave intermediates4-chloro-7-
methoxyquinolin-6-ol (7) or 4-chloroquinoline-6, 7-diol (8). Commer-
cially available 4-chloro-6-methoxyquinolin-7-ol (6) and the synthe-
sized intermediates 7 and 8 reacted with various alkyl chloride to
provide 9a-o. Nucleophilic substitution reactions between 9a-o and 4-
amino-2-fluorophenol produced corresponding 10a-o. Reactions of in-
termediate 10a-o and 5-(tert-butyl)-3-isocyanatoisoxazole (3) offered
final product compounds 11a-o by condensation reaction.
Molecular docking was then used to predict the binding model of
the most active compound 4d in the active pocket of FLT3. The receptor
structure was taken from the crystal structure of FLT3 (PDB ID: 4RT7).
The preparation and preprocessing of the receptor and the ligand were
performed on the platform of Discovery Studio 3.1. The program GOLD
version 5.1 was adopted for molecular docking. The predicted inter-
action model between compound 4d and FLT3 is depicted in Figure 3.
4d suitably resides in the ATP binding pocket of FLT3. Four hydrogen
bonds are formed between 4d and FLT3: one is between quinoline (1-N)
and Cys694; one is between oxygen of urea and ASP829; and the other
two are between nitrogen of urea and Glu661. It is also obvious that the
benzene ring of 4d forms good π-π interactions with the benzene rings
of residues Phe691 and Phe830.
Our previous study has shown that SKLB-677 could block the Wnt/
β-catenin signaling pathway. To examine whether 4d has a similar ef-
fect, we used the same STF3a cell model as before,24 which stably ex-
press both the STF (Super Top Flash) luciferase reporter promoter and
the wnt3a gene. The results indicated that 4d was able to dose-de-
pendently inhibit the Wnt/β-catenin signaling with an IC50 value
of < 0.5 μM (Figure 4), which is very similar with SKLB-677. In the
same experiment, AC220 exhibited very weak effect (Figure 4).
In summary, we obtained a new 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-
Bioactivities of compounds 11a-o are displayed in Table 2.
Scheme 1. Synthetic routes for compounds 4a-g. Reagents and conditions: (a)t-BuOK, anhydrous DMF, 100 °C, overnight; (b) Et3N, EtOAc, 80 °C, overnight.
2

G. Zhang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127532
Table 1
Inhibitory activities of compounds 4a-g against Ba/F3 cells expressing FLT3-ITD, FLT3-ITD/D835Y and FLT3-ITD/F691L, as well as the parental Ba/F3 cells.
Cpd
Region A
FLT3-ITD(IC50, nM)
FLT3-ITD/D835Y(IC50, nM)
FLT3-ITD/F691L(IC50, nM)
Ba/F3(IC50, nM)
AC220
SKLB-677
4a
–
–
3.01
0.79
520
130
34.3
27.8
14.6
7.2
625
108
35.0
31.2
17.3
2.7
9312.2
3156.3
2623.2
16.0
17.1
10.0
0.038
0.292
0.001
0.054
4b
4c
0.015
140.9
0.004
7.3
12.4
5.7
18.6
2.4
2084.0
13.0
741.1
35.6
1023.7
9.8
> 10000
4d
0.072
0.02
23.9
3.48
0.59
5.86
0.62
1212.4
10.9
12.0
4e
4f
1258.0
1.20
> 10000
> 10000
1021.7
> 10000
3035.4
0.24
64.2
7.93
11.7
6.8
4 g
1.51
0.32
1.29
13.8
2.8
> 1988.1
13.5
aAll IC50 values were obtained by triplet testing.
3

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Bioorganic&MedicinalChemistryLetters30(2020)127532
Scheme 2. Synthetic routes for compounds 11a-o. Reagents and conditions: (a) L-Methionine, Methanesulfonic acid, 120 °C, 12 h; (b) BBr3, DCM, 0 °C-25 °C, 6 h; (c)
K2CO3, DMF, 150 °C, 4 h; (d) t-BuOK, anhydrous DMF, 100 °C, overnight; (e) Et3N, EtOAc, 80 °C, overnight.
fluorophenyl)urea derivative (4d), which showed potent activity
against both FLT3-ITD mutation and drug resistance mutations FLT3-
ITD/D835Y and FLT3-ITD/F691L. This compound has a good kinase
selectivity, and is able to inhibit the Wnt/β-catenin signaling pathway.
Nevertheless, we have to mention that more studies, including in vivo
anti-AML activity, and pharmacokinetic properties, as well as me-
chanism of actions, are still needed to evaluate its druggability.
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.
4

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Bioorganic&MedicinalChemistryLetters30(2020)127532
Table 2
Inhibitory activities of compounds 11a-o against Ba/F3 cells expressing FLT3-ITD, FLT3-ITD/D835Y and FLT3-ITD/F691L, as well as parental Ba/F3 cells.
Cpd
R1
R2
FLT3-ITD (IC50, nM)
FLT3-ITD/D835Y (IC50, nM)
FLT3-ITD/F691L (IC50, nM)
Ba/F3 (IC50, nM)
4d
0.072
5.20
19.2
0.020
3.48
5.96
14.9
0.59
0.57
3.2
5.86
8.31
54.3
0.62
1.33
5.9
1212.4
1645.6
4935.0
10.9
11a
11b
0.68
9.4
7.2
15.0
11c
3.22
0.35
5.09
1.18
9.93
2.75
1097.1
17.7
11d
11e
4.73
66.5
0.24
9.5
5.35
104
1.20
16.0
11.9
189
1.6
1125.2
5556.1
13.0
12.4
25.1
11f
36.5
4.5
62.4
11.3
127
13.4
4817.0
9203.9
7.2
11g
2649.0
11.1
3540.8
21.3
5270.6
15.5
28.5
11h
11i
11j
7.25
76.1
5.77
0.21
9.4
10.9
160
2.9
29.9
296
6.1
19.3
3.4
2877.7
7774.1
1840.0
10.3
20.0
13.8
12.9
0.82
0.30
6.28
16.8
11k
11l
4.91
6.88
0.53
0.74
10.1
10.6
3.1
4.0
20.8
23.1
4.5
3.3
3000.2
2034.8
11.4
15.6
11m
11n
11o
3.08
5.17
37.2
1.16
0.55
6.3
15.5
11.0
27.9
2.5
3.7
7.4
32.6
16.0
38.9
1.4
4.4
9.1
3137.5
1996.3
6500.1
28.6
17.6
19.4
aAll IC50 values were obtained by triplet testing.
5

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Bioorganic&MedicinalChemistryLetters30(2020)127532
Fig. 2. Dose-response curves of the inhibitory activities of SKLB-677, AC220, and 4d against Ba/F3-FLT3-ITD (A), Ba/F3-FLT3-ITD/D835Y (B) and Ba/F3-FLT3-ITD/
F691L (C).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127532.
References
1. Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Blood.
2002;100:1532–1542.
2. Kelly LM, Yu J-C, Boulton CL, et al. CT53518, a novel selective FLT3 antagonist for
the treatment of acute myelogenous leukemia (AML). Cancer Cell. 2002;1:421–432.
3. Metzelder S, Wang Y, Wollmer E, et al. A Compassionate use of sorafenib in FLT3-
ITD–positive acute myeloid leukemia: sustained regression before and after allo-
geneic stem cell transplantation. Blood. 2009;113:6567–6571.
4. Cao Z, Liu J, Zheng R, et al. SKLB1028, a novel oral multikinase inhibitor of EGFR,
FLT3 and Abl, displays exceptional activity in models of FLT3-driven AML and
considerable potency in models of CML harboring Abl mutants. Leukemia.
2012;26:1892–1895.
5. Yang J, Wang L-J, Liu J-J, et al. Structural Optimization and Structure-Activity
Relationships of N2-(4-(4-Methylpiperazin-1-yl)phenyl)-N8-phenyl-9H-purine-2,8-
diamine Derivatives, a New Class of Reversible Kinase Inhibitors Targeting both
EGFR-Activating and Resistance Mutations. J Med Chem. 2012;55:10685–10699.
6. Kuchenbauer F, Kern W, Schoch C, et al. Detailed analysis of FLT3 expression levels
in acute myeloid leukemia. Haematologica. 2005;90:1617–1625.
Fig. 3. Prediction binding model of compound 4d in the active pocket of
FLT3(Protein Data Bank: 4RT7). Compound 4d is color coded with carbon
atoms by brown, nitrogen atoms by blue, and oxygen atoms by red. Hydrogen
bonds are shown in purple dashed line. The π-π interactions are shown in da-
shed red line. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)
7. Levis M. FLT3/ITD AML and the law of unintended consequences. Blood.
2011;117:6987–6990.
8. Wang W, Wang X, Xu X, Lin G. Prevalence and prognostic significance of FLT3 gene
mutations in patients with acute leukaemia: analysis of patients from the Shanghai
Leukaemia Co-operative Group. J Int Med Res. 2010;38:432–442.
9. Whitman SP, Ruppert AS, Radmacher MD, et al. FLT3 D835/I836 mutations are as-
sociated with poor disease-free survival and a distinct gene-expression signature
among younger adults with de novo cytogenetically normal acute myeloid leukemia
lacking FLT3 internal tandem duplications. Blood. 2008;111:1552–1559.
10. Smith CC, Wang Q, Chin C-S, et al. Validation of ITD mutations in FLT3 as a ther-
apeutic target in human acute myeloid leukaemia. Nature. 2012;485:260–263.
11. Zarrinkar PP, Gunawardane RN, Cramer MD, et al. AC220 is a uniquely potent and
selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML). Blood.
2009;114:2984–2992.
12. Pauwels D, Sweron B, Cools J. The N676D and G697R mutations in the kinase do-
main of FLT3 confer resistance to the inhibitor AC220. Haematologica.
2012;97:1773–1774.
13. Smith CC, Lasater EA, Zhu X, et al. Activity of ponatinib against clinically-relevant
AC220-resistant kinase domain mutants of FLT3-ITD. Blood. 2013;121:3165–3171.
14. Li C, Liu L, Liang L, et al. AMG 925 is a dual FLT3/CDK4 inhibitor with the potential
to overcome FLT3 inhibitor resistance in acute myeloid leukemia. Mol cancer ther.
2015;14:375–383.
Fig. 4. Inhibitory activities of compounds 4d, SKLB-677 and AC220 against
Wnt/β-catenin pathway were determined using STF3a cells treated with various
concentrations of compounds for 24 h before luciferase activity measurements.
All data points are means of triplicates
SD. ns, not statistically significant,
15. Smith CC, Zhang C, Lin KC, et al. Characterizing and overriding the structural me-
chanism of the Quizartinib-Resistant FLT3 “Gatekeeper” F691L mutation with
PLX3397. Cancer Discov. 2015;5:668–679.
**P < 0.01, ***P < 0.005 among groups.
16. Lee HK, Kim HW, Lee IY, et al. G-749, a novel FLT3 kinase inhibitor, can overcome
drug resistance for the treatment of acute myeloid leukemia. Blood.
2014;123:2209–2219.
Acknowledgments
17. Smith CC, Lasater E, Mccreery M, et al. Crenolanib (CP-868,596) is a potent and
selective type I FLT3 inhibitor that retains activity against AC220 resistance-causing
FLT3 kinase domain mutants. Blood. 2012;120 141-141.
This work was supported by the National Natural Science
Foundation of China (81573349, 81773633, 21772130, and
81930125), National Science and Technology Major Project
18. Kesarwani M, Huber E, Azam M. Overcoming AC220 resistance of FLT3-ITD by
SAR302503. Blood Cancer J. 2013;3:e138.
(2018ZX09711002-014-002,
2018ZX09711002-011-019,
2018ZX
19. Li G-B, Ma S, Yang L-L, et al. Drug Discovery against Psoriasis: Identification of a New
Potent FMS-like Tyrosine Kinase 3 (FLT3) Inhibitor, 1-(4-((1 H-Pyrazolo [3, 4-d]
pyrimidin-4-yl) oxy)-3-fluorophenyl)-3-(5-(tert-butyl) isoxazol-3-yl) urea, That
Showed Potent Activity in a Psoriatic Animal Model. J Med Chem.
2016;59:8293–8305.
09201018 and 2018ZX09711003-003-006), and 1.3.5 project for dis-
ciplines of excellence, West China Hospital, Sichuan University.
6

G. Zhang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127532
20. Xu B, Zhao Y, Wang X, Gong P, Ge W. MZH29 is a novel potent inhibitor that
overcomes drug resistance FLT3 mutations in acute myeloid leukemia. Leukemia.
2017;31:913–921.
Kinase 3. ChemMedChem. 2010;5:513–516.
23. Li W-W, Wang X-Y, Zheng R-L, et al. Discovery of the novel potent and selective FLT3
inhibitor 1-{5-[7-(3-morpholinopropoxy) quinazolin-4-ylthio]-[1, 3, 4] thiadiazol-2-
yl}-3-p-tolylurea and its anti-acute myeloid leukemia (AML) activities in vitro and in
vivo. J Med Chem. 2012;55:3852–3866.
21. Wedge SR, Kendrew J, Hennequin LF, et al. AZD2171: a highly potent, orally bioa-
vailable, vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor for
the treatment of cancer. Cancer Res. 2005;65:4389–4400.
24. Ma S, Yang L-L, Niu T, et al. SKLB-677, an FLT3 and Wnt/β-catenin signaling in-
hibitor, displays potent activity in models of FLT3-driven AML. Sci Rep. 2015;5
15646-15646.
22. Li WW, Chen JJ, Zheng RL, et al. Taking Quinazoline as a General Support-Nog to
Design Potent and Selective Kinase Inhibitors: Application to FMS-like Tyrosine
7