Discovery of DDO-2213 as a Potent and Orally Bioavailable Inhibitor of the WDR5-Mixed Lineage Leukemia 1 Protein-Protein Interaction for the Treatment of MLL Fusion Leukemia

Article abstract of DOI:10.1021/acs.jmedchem.1c00091

WD repeat-containing protein 5 (WDR5) is essential for the stability and methyltransferase activity of the mixed lineage leukemia 1 (MLL1) complex. Dysregulation of theMLL1gene is associated with human acute leukemias, and the direct disruption of the WDR5-MLL1 protein-protein interaction (PPI) is emerging as an alternative strategy for MLL-rearranged cancers. Here, we represent a new aniline pyrimidine scaffold for WDR5-MLL1 inhibitors. A comprehensive structure-activity analysis identified a potent inhibitor 63 (DDO-2213), with an IC₅₀of 29 nM in a competitive fluorescence polarization assay and aK_dvalue of 72.9 nM for the WDR5 protein. Compound 63 selectively inhibited MLL histone methyltransferase activity and the proliferation of MLL translocation-harboring cells. Furthermore, 63 displayed good pharmacokinetic properties and suppressed the growth of MV4-11 xenograft tumors in mice after oral administration, first verifying thein vivoefficacy of targeting the WDR5-MLL1 PPI by small molecules.

Full text of DOI:10.1021/acs.jmedchem.1c00091

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Article
Discovery of DDO-2213 as a Potent and Orally Bioavailable Inhibitor
of the WDR5−Mixed Lineage Leukemia 1 Protein−Protein
Interaction for the Treatment of MLL Fusion Leukemia
Published as part of the Journal of Medicinal Chemistry special issue “Epigenetics 2022”.
∇
∇
Weilin Chen, Xin Chen, Dongdong Li, Jianrui Zhou, Zhengyu Jiang,* Qidong You,*
and Xiaoke Guo*
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sı Supporting Information
ABSTRACT: WD repeat-containing protein 5 (WDR5) is essential for the stability and methyltransferase activity of the mixed
lineage leukemia 1 (MLL1) complex. Dysregulation of the MLL1 gene is associated with human acute leukemias, and the direct
disruption of the WDR5−MLL1 protein−protein interaction (PPI) is emerging as an alternative strategy for MLL-rearranged
cancers. Here, we represent a new aniline pyrimidine scaffold for WDR5−MLL1 inhibitors. A comprehensive structure−activity
analysis identified a potent inhibitor 63 (DDO-2213), with an IC of 29 nM in a competitive fluorescence polarization assay and a
5
0
Kd value of 72.9 nM for the WDR5 protein. Compound 63 selectively inhibited MLL histone methyltransferase activity and the
proliferation of MLL translocation-harboring cells. Furthermore, 63 displayed good pharmacokinetic properties and suppressed the
growth of MV4-11 xenograft tumors in mice after oral administration, first verifying the in vivo efficacy of targeting the WDR5−
MLL1 PPI by small molecules.
INTRODUCTION
weak when it is present in isolation, and the constitution of the
MLL core complex (MLL1, WDR5, ASH2L, RbBP5, and
DPY-30) dramatically enhances the enzymatic activity.
■
1
,2
WDR5, a member of the WD40 repeat protein family,
functions as a critical scaffolding component in numerous
protein complexes that relate epigenetic machinery and
transcriptional regulation. WDR5 is comprised of a circular
seven-bladed β-propeller fold where each blade interconnects
through four-stranded antiparallel β-sheets, which generally
mediate protein−protein interactions (PPIs).
two major binding sites: WDR5 binding motif (WBM) and
1
5
Among these interactions, the WDR5−MLL1 interaction is
necessary for the stability and H3K4 HMT activity of MLL1.
The chromosomal translocation of an allele in the 11q23
MLL1 gene to one of over 70 different partner genes causes a
unique group of leukemias with poor prognoses and treatment
3
1
−4
WDR5 has
16
outcomes. It has been reported that 70% of infant acute
5
−11
lymphoid leukemia (ALL) cases and 5−10% of adult acute
WDR5 interaction motif (WIN) sites.
The most well-
studied role of WDR5 is the formation of MLL/SET (MLL1−
12
Received: January 19, 2021
Published: June 9, 2021
4
, SET1A, and SET1B) complexes via the WIN site. In these
complexes, MLL1 and WDR5 PPI are uniquely required for
MLL1 histone methyltransferase activity. In addition, MLL1
regulates gene transcription by mediating the monomethyla-
tion, dimethylation, and trimethylation of histone 3 lysine 4
1
3,14
(
H3K4).
However, this enzymatic activity is relatively
©
2021 American Chemical Society
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Figure 1. Representative peptidomimetic and nonpeptide inhibitors of the WDR5 WIN-site.
myeloid leukemia (AML) cases are associated with MLL
none bicyclic core, showed a picomolar binding affinity and
32,33
rearrangements, such as MLL−AF4, MLL−AF9, and MLL−
inhibited the proliferation of MYC-driven cancer cells.
In
1
7−19
ENL.
However, the lack of a C-terminal catalytic SET
conclusion, these studies suggest that the WDR5 protein,
especially the WIN site, could be a promising anticancer target
and promote the further study of new inhibitors.
Here, we describe the design and synthesis of a series of new
aminopyrimidine-based WDR5−MLL1 PPI inhibitors and
discuss the structure−activity relationship (SAR). The
represented compound 63 showed tight binding to the
domain in MLL fusion proteins leads to the loss of their
histone methyltransferase activity. Interestingly, the synergy
between the remaining wild-type MLL allele and the MLL
14,19
fusion allele is necessary for leukemogenesis.
Therefore,
targeting the MLL1−WDR5 PPI is considered to be an
effective strategy for the treatment of leukemia containing
13−22
MLL fusion proteins (Figure S1).
WDR5 protein (K = 72.99 nM) in vitro, selectively inhibited
d
Because the interaction of WDR5−MLL1 is important for
the MLL1 complex, various WDR5−MLL1 PPI antagonists,
including peptides and small molecules, have been discovered,
and the four representative types of WDR5 WIN site inhibitors
that disrupt this interaction are shown in Figure 1.
Peptidomimetic compounds with sub-nanomolar affinities to
the WDR5 protein, low-nanomolar inhibitions of MLL1
enzyme catalytic activity, and antiproliferative activity in cells,
such as MM-401 (1) and MM-589 (2), were obtained by
the HMT activity of MLL1 both in vitro and in cells, effectively
reduced MLL fusion-protein-dependent gene expressions, and
selectively restrained the proliferation of leukemia cells
harboring MLL fusion proteins (MLL-FPs) with little toxicity
to non-MLL cells. Moreover, compound 63 showed favorable
drug-like properties and exhibited obvious anticancer effects in
the MLL fusion leukemia MV4-11 xenografts after oral
administration. This research not only reports a potent and
orally bioavailable WDR5−MLL1 inhibitor with a novel
aminopyrimidine scaffold but also is the first to show the
therapeutic potential of small-molecule WDR5−MLL1 inhib-
itors against MLL fusion leukemia in vivo.
23−26
imitating the MLL peptide residues.
The first nonpeptide
class of small-molecule antagonists (WDR5−0102, 3) was
identified by the high-throughput screening of a compound
2
7
library. According to the cocrystal structures of WDR5
inhibitors, several more potent small molecules were obtained
in the subsequent optimization, such as OICR-9429 (4) with a
high affinity to the WDR5 protein (IC50 = 64 nM) and the
favorable antiproliferative activity of human C/EBPα mutant
RESULTS AND DISCUSSION
■
Discovery of Initial Hit Compound 9 as a WDR5−
MLL1 Inhibitor. Targeting a protein−protein interaction is an
34−38
emerging strategy for drug design.
For the WDR5−MLL1
2
8,29
AML cells.
We previously reported that DDO-2117 (5)
PPI, the MLL1 binding site in the WDR5 domain is a well-
defined cavity. In an effort to discover a new scaffold for
WDR5−MLL1 inhibitors, we pursued a scaffold-hopping
approach utilizing the structural information on reported
inhibitor binding to the WDR5 protein to identify a novel hit.
We observed the cocrystal structure between WDR5 and
compound 3 (PDB 3SMR), the first WDR5 WIN site small-
disturbed the WDR5−MLL1 interaction (IC = 7.6 nM) in
5
0
3
0
vitro. Recently, the third class of representative small
molecules, 6e (6), was discovered by NMR-based fragment
screening, displaying a remarkable affinity to the WDR5
31
protein (K < 1 nM). The fourth class of representative small
i
molecules, compound 16 (7) containing a dihydroisoquinoli-
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Figure 2. (A) Cocrystal structure of compound 3 with WDR5 (PDB 3SMR). (B) Binding mode of compound 3 (yellow) with the key residues
green) in the WDR5 WIN site (PDB 3SMR). (C) The design of hit compound 9.
(
molecule inhibitor, and found that the O atom of the amide
and the Cl atom of the benzene ring were on the same side.
Simultaneously, considering the contributions of the O atom
to the binding activity via the formation of a water-mediated
hydrogen bond with the backbone nitrogen of Cys261 (Figure
amino substituent (17) at the 5-position gave about a fourfold
increase in inhibitory activity. In addition, the decreased
inhibitory activity of compound 18 again demonstrated that
the 2-position was not suitable for substitution. Compound 19,
which removed the chloro atom from compound 17, also
showed a dramatic loss of activity. Replacing the 4-chloro-
substituted pyrimidine ring with several heterocycles also failed
to produce active compounds (20−22). Thus, 17 contained
the most suitable group for the optimization of the pyrimidine
moiety due to its favorable inhibitory activity, indicating that
the 6-choro-5-aminopyrimidine group was essential for the
inhibitory activity.
To investigate the binding mode of this chemotype with the
WDR5 protein, a molecular docking study was performed
utilizing DS 3.0 for the most potent compound 17. The
binding mode showed that 17 can insert into the arginine-
binding pocket at the central cavity (Figure 3A). The N-
methylpiperazine moiety was also anchored at the bottom of
the pocket and formed a water-mediated hydrogen bond with
the backbone carbonyl of Cys261. The 6-chloro-5-amino-
pyrimidine group lay in a hydrophobic and shallow cavity
surrounded by Ala47, Ser49, Ala65, Ser91, Ile305, and Leu321.
The core phenyl ring formed a π−π stack interaction with
Phe133. As expected, the nitrogen atom at the 3-position of
the pyrimidine formed a water-mediated hydrogen bond with
the backbone nitrogen of Cys261, which was a similar
interaction to that observed with the amide group of the
benzamide compound 3. Further, the amino group of the
aniline formed a direct hydrogen bond with the side chain of
Ser91. Moreover, the newly introduced amino group on the
pyrimidine ring can form a direct hydrogen bond interaction
with Asp107, which may be the reason for the increased
potency of 17 compared to that of 9 (Figure 3A). Then, we
explored the SAR of 17 around three parts, including the N-
methylpiperazine substituents (exhibiting a potential space to
2
A, 2B), we supposed that the cyclization of the O atom with
the Cl moiety may result in a conformational restriction,
producing compound 8 (Figure 2C). Compound 8 showed
weak inhibitory activity against the WDR5−MLL1 PPI as
determined by a fluorescence polarization (FP) assay (35.8%
25
inhibition at 100 μM). We speculated that the volume of the
quinoline ring was too large to extend well into the
hydrophobic cavity, formed by Leu321, Ala65, Ser49, and
Ser91, resulting in the dramatically decreased activity.
Following a reduction in the molecular rigidity, compound 9
was obtained by opening the quinoline ring, which showed an
activity equivalent to that of 3 (as assessed by an FP assay, IC50
=
10.3 μM) (Figure 2C). This starting compound represents a
new scaffold for WDR5 inhibition and deserves further
optimization.
SAR of the Pyrimidine Ring. To improve the WDR5−
MLL1 inhibitory potency, we explored the effects of the
pyrimidine ring by including various substituents on the
pyrimidine ring in compound 9 and changing the pyrimidine
ring, as shown in Table 1. First, the chloro group was replaced
by different secondary amines, which resulted in target
compounds 10−12. These analogues gave the abolished
activity. Therefore, we decided to preserve the original chloro
group to synthesize the subsequent analogues. We then
introduced methyl, amino, and nitro groups at different
positions around the pyrimidine ring, and compounds 13−
18 were obtained. The inhibition activity of compounds with
substitutions at the 2-position (13 and 14) dramatically
decreased. Compounds with a methyl (15) or nitro (16)
substitution at the 5-position were also not ideal, while the
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Table 1. Effects of Pyrimidine Ring Modifications on WDR5−MLL1 Inhibition
a
IC50 values represent the mean ± SD of three independent experiments.
form polar and π−π stack interactions), the nitro substituent
which is surrounded by hydrophobic residues involving
Phe133, Phe149, Pro173, and Tyr191 and is closed to the
protein surface), and the core phenyl substituents (Figure 3B).
SAR of the N-Methylpiperazine Substituent. On the
one hand, the docking study showed the available space and
possibility of hydrogen bond formation with Asp92 in addition
to the π−π stack interaction formation with Phe263 at the
(
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Figure 3. (A) Proposed binding modes showing the structures of 17 docked into WDR5 (PDB 4QL1). The green dashed lines represent hydrogen
bonds, and the orange lines represent π−π stacking interactions. (B) The optimization of compound 17.
Table 2. Effects of N-Methylpiperazine Region Modifications on WDR5−MLL1 Inhibition
a
IC50 values represent the mean ± SD of three independent experiments.
bottom of the arginine-binding cleft (Figure 3). On the other
hand, the structural features of 17 may be different from those
of the benzamide compound 3. Therefore, we further tried to
investigate the N-methylpiperazine substituents of 17. Differ-
ent amines were introduced to this region to obtain the analogs
listed in Table 2. Unfortunately, all the compounds with N-
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Table 3. Effects of Nitro Substituents on WDR5−MLL1
a
IC50 values represent the mean ± SD of three independent experiments.
Figure 4. (A) Proposed binding modes showing the structures of 29 docked into WDR5 (PDB 4QL1). The green dashed lines represent the
hydrogen bonds, and the orange lines represent the π−π stacking interactions. (B) The optimization of compound 29 and design of 34−48.
methylpiperazine modifications had lower or no activities,
including demethylated compounds (23), compounds with a
morpholine replacement (24), and N-ethylation (25), N-
cyclopropanation (26), N-hydroxyethyl (27), and N-phenyl
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28) modifications of the piperazine ring. These results further
emphasized the importance of the conformational and steric
constraints of the methylpiperazine moiety. Therefore, we
opted to retain the N-methylpiperazine moiety and chose 17
for further optimization.
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(
Table 4. Effects of Side-Chain Phenyl Ring Modifications on
WDR5−MLL1 Inhibition
SAR of the Nitro Substituent. A subsequent SAR
optimization was conducted to the focus on the nitro group
of 17. Previous SAR studies at this position showed that the
introduction of an aromatic ring formed van der Waals
interactions with Phe133, Phe149, Pro173, and Tyr191 and led
29,30,39,40
to favorable improvements in potency.
We thought
that this moiety deserved to be broadly explored to capture the
aforementioned interactions with nearby residues. To this end,
we introduced benzene rings and different aromatic hetero-
cycles via Suzuki coupling reactions. The SAR data of these
compounds are summarized in Table 3. The introduction of
benzene rings (29 and 30) improved the potency relative to
that of 17, and 29 (with a 4-amino phenyl substituent) showed
a greater inhibition than 30 (with a 4-methoxy phenyl
substituent), suggesting that hydrophilic substituents at the
4
-position of the newly introduced phenyl ring may be
beneficial for the activity. The binding mode from the docking
study consistently predicted that the introduced phenyl ring of
29 can form a π−π stack interaction with Tyr191 and Phe133
in an appropriate manner close to the solvent area (Figure 4A).
A substituted 1,2,3-triazole was also introduced at this position,
resulting in compound 33 with about a threefold improvement
in activity. However, introducing pyrimidine and furan groups
(
31 and 32, respectively) diminished the potency (IC = 5.71
50
and 11.43 μM, respectively).
According to the SAR information above, further mod-
ifications to the side chain of the phenyl ring were performed
to optimize the binding activity and physicochemical proper-
ties, resulting in analogues 34−48 (Figure 4B). As shown in
Table 4, the binding affinity to WDR5 was significantly
improved in all cases except for aromatic substitution (38),
with IC50 values ranging from 0.110 to 0.490 μM. These
compounds maintained their potencies when the amide bond
at the 4-position was reversed (34 vs 42 and 35 vs 45).
Additionally, the terminal hydrophilic tag may be more
beneficial for inhibitory activity (36 vs 46 and 47). Finally,
the potency seemed to increase as the length of the linker
between the terminal group and the amide increased (39 < 40
<
41, 43 < 44 and 45). As expected, the side chains extended
into the solvent area, which is consistent with the prediction
(
Figure S2).
SAR of the Core Phenyl Ring. Since the exact binding
mode of this chemotype with WDR5 was not available, we
further tried to investigate the substituents on the core phenyl
ring. First, based on the previously obtained SAR, a small
electron-withdrawing F atom was added to the 6-position of
the core phenyl ring; this series of compounds (49−56)
resulted in slightly increased inhibitory activities compared to
that of compounds without the F substituent (Table 5),
indicating a tolerable modification when employing a small
substituent. However, introducing an electron-donating methyl
group at this position led to the great loss in activity, as seen in
5
7 (IC = 670 nM). Encouragingly, moving the F atom to the
50
4
-position of the core phenyl ring, significantly enhanced the
a
IC50 values represent the mean ± SD of three independent
experiments.
activity, as seen in 58−63. Moreover, compound 64
demonstrated that an electron-donating group was also not
suitable at this site. The SARs of the substituents on the upper
benzene ring were consistent with that described before. To
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Table 5. Effects of the Core Phenyl Ring Substituents on WDR5−MLL1 Inhibition
a
IC50 values represent the mean ± SD of three independent experiments.
understand the reason why a minor change in the structure led
to such a great increase in the potency, we compared the
docked pose of 52 (Figure 5A) to that of the more active 60
atom of 60 was capable of forming an additional potential
hydrogen bond with the sulfhydryl of Cys261. The result may
explain the ca. fourfold improvement of the in vitro binding
affinity of 60 compared to that of 52. Together, all these
(Figure 5B) in the WDR5 binding site. In contrast to 52, the F
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Figure 5. Proposed binding modes showing the structures of (A) 52 and (B) 60 docked into WDR5 (PDB 4QL1). Carbons are represented in
yellow, the green dashed lines represent the hydrogen bonds, and the orange lines represent the π−π stacking interactions.
Table 6. Inhibition of Cell Growth and Physicochemical Properties of Representative Compounds
a
antiproliferation activity IC50 (μM)
P at pH 7.4 (× 10− cm/s)
6
b
compound
MV4-11
MOLM-13
K562
HUVEC
CLogP
e
5
5
6
6
6
8
9
0
2
3
19.6 ± 2.10
27.7 ± 2.80
25.6 ± 2.98
15.7 ± 2.27
12.6 ± 1.91
16.5 ± 2.05
23.1 ± 3.47
17.3 ± 1.56
12.3 ± 1.09
13.1 ± 1.31
88.7 ± 9.80
>100
70.1 ± 7.40
81.7 ± 8.90
>100
>100
>100
>100
>100
>100
3.16
3.53
3.95
4.39
3.42
13.73 ± 0.87
10.51 ± 0.46
9.140 ± 0.58
16.87 ± 0.64
20.22 ± 1.43
a
b
IC50 values represent the mean ± SD of three independent experiments. P at pH 7.4 values represent the mean ± SD of three independent
experiments.
e
modifications led to a new series of compounds with a ∼350-
fold improvement on the in vitro binding affinity compared to
that of hit 9, such as compound 60 with an IC50 value of 21.0
nM.
(PAMPA) on a PAMPA Explore instrument (pION). As
shown in Table 6, all compounds showed reasonable lipid−
water distribution coefficients. In particular, compound 63
showed the best permeability at pH 7.4. These results may
explain, to some degree, why compound 63 displayed the best
antiproliferative activity.
Biophysical Characterization of Compound 63 Bind-
ing to WDR5. Compound 63 showed impressive inhibitory
activity in terms of the WDR5−MLL1 PPI, selective cellular
activity toward cell lines harboring MLL fusion proteins, and
favorable physicochemical properties. To validate the strong
WDR5 binding affinity and analyze the WDR5 binding
characteristics of 63, an isothermal titration calorimetry assay
was applied to assess the thermodynamics and affinity of the
Antiproliferative Activities and Physicochemical
Properties of Representative Compounds. On the basis
of the binding assays, representative compounds were next
evaluated for their antiproliferation activity against three
human acute leukemia cell lines, including cell lines harboring
MLL fusion proteins (MLL-FP, MV4-11, and MOLM-13), a
cell line without the MLL fusion protein (K562), and a
noncancer cell line (HUVEC, normal human umbilical vein
endothelial cells). The data are summarized in Table 6. All
compounds showed moderate potencies in terms of inhibiting
cell growth in the cell lines harboring MLL fusion proteins.
However, these compounds were much less active in the case
of the K562 cell line lacking the MLL translocation, showing
desirable selectivities between the leukemia cell lines with
MLL-FPs and the cell line without MLL-FPs. In addition,
these compounds displayed low toxicities against HUVECs,
with IC values >100 μM indicating their low toxicities against
ligand. The resulting ITC profile gave K = 72.99 nM (Figure
d
6). Notably, the thermodynamic analysis showed that the
binding of 63 had a strong enthalpic component (ΔH =
−12.25 ± 0 0.17 kcal/mol) and an acceptable entropy (TΔS=
−2.51 kcal/mol), which suggested that the binding of 63 to
WDR5 was an enthalpy-driven process. This binding assay
confirmed the direct and tight binding of 63 to the WDR5
protein.
5
0
normal cells.
To evaluate the drug-like properties of these analogues,
lipid−water distribution coefficients (CLogP) were computa-
tionally calculated using the ADMET Predictor 10.0 software.
Compound 63 Selectively Inhibited MLL Complex
HMT Activity In Vitro. As mentioned before, the WDR5−
MLL1 PPI plays a critical role in the integrity and HMT
activity of the MLL1 complex. The assessment of the
methylation-inhibitory activity of H3K4 served as a method
The permeability coefficients (P ) were determined by a
e
standard parallel artificial membrane permeability assay
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Figure 6. ITC profile of the titration of WDR5 with 63. The thermodynamic parameters of the interaction of WDR5 with 63 as determined by the
ITC assay are listed in the table. N is the stoichiometric coefficient; K is the binding constant; ΔH, ΔS, and ΔG refer to the changes in binding
d
enthalpy, entropy, and total Gibbs free energy, respectively; and ΔG was calculated according to the equation ΔG = ΔH − TΔS, where T is the
absolute temperature used for the ITC experiment.
Figure 7. Selectivity of compound 63 to the HMT activity of the reconstituted MLL1 core complex over other methyltransferases in vitro.
to evaluate the effects on MLL-mediated HMT activity. We
therefore evaluated MLL1 HMT activity in vitro. Compound
that 63 could selectively inhibit the H3K4 methyltransferase
activity of MLL1 in vitro.
6
3 efficiently inhibited MLL-mediated HMT activity (IC50
=
Compound 63 Effectively Inhibited MLL Complex
HMT Activity and Reduced Downstream Gene Ex-
pression in MV4-11 cells. Based on the inhibition of
MLL1 enzymatic activity in vitro, the methyltransferase activity
of compound 63 in cells was then examined. MV4-11 cells
were treated with different concentrations (1, 2.5, 5, and 10
μM) of compounds. As shown in Figure 8A, compound 63
induced the concentration-dependent downregulation of
0
.78 μM) (Figure 7). In addition to the MLL1 complex, 63
was assessed for its inhibitory activity against other protein
lysine methyltransferases, including G9a (a H3K9 PKMT),
DOT1L (a H3K79 PKMT), EZH2 (a H3K27 PKMT), SET8
(
a H4K20 PKMT), and PRMT5 (a protein arginine
methyltransferase), and was found to be inactive in these
assays (IC50 > 100 μM) (Figure 7). These results indicated
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Figure 8. (A) Western blot analyses for H3K4 methylation activity after the treatment of MV4-11 cells with DMSO and 1, 2.5, 5.0, and 10 μM
compound 63 for seven days. H3K4me1, H3K4me2, and H3K4me3 were determined using histone as loading control. (B) The inhibition of
Hoxa9 and Meis1 gene expressions after treatment with different concentrations (0, 1, 2.5, 5, and 10 μM) of compound 63 in MV4-11 cells for
seven days as assessed by RT-PCR. Each experiment was performed in triplicate (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 as calculated by
Student’s t-tests.
a
H3K4me1, H3K4me2, and H3K4me3, showing the inhibition
of the methyltransferase activity of MLL1 in cells.
Table 8. In Vivo Pharmacokinetic Profiles of 63
parameters
p.o. (10 mg/kg)
i.v. (2 mg/kg)
1.70 ± 0.63
MLL1 is essential for development, neurogenesis, and
hematopoiesis through the regulation of Hox genes and the
t1/2 (hr)
4.94 ± 0.04
2.00 ± 1.73
159 ± 60.0
1316 ± 683
1738 ± 255
37.1 ± 11.3
97.2 ± 14.0
5.42 ± 1.20
82.1%
41−45
tmax (hr)
expression of other transcription cofactors.
The dysregu-
Cmax (ng/mL)
lation of Hox genes and the cofactor Meis1 gene was observed
46
AUC0−t (ng·hr/mL)
AUC0−∞ (ng·hr/mL)
Vz_F (L/kg)
Cl_F (mL/min/kg)
MRT (hr)
411 ± 90.6
424 ± 98
12.2 ± 6.24
81.3 ± 16.7
1.18 ± 0.40
in leukemias associated with MLL1 genetic rearrangements.
Thus, we investigated the effects of 63 on the MLL1
downstream gene expression in MV4-11 cells. As shown in
Figure 8B, compound 63 decreased the mRNA level of Hoxa9
and Meis1 in MV4-11 cells in a concentration-dependant
manner. This result indicated that disturbing the WDR5−
MLL1 PPI with 63 efficiently regulated the expression of MLL
fusion protein-dependent genes.
Metabolic and Pharmacokinetic Profiles of 63. We
further examined the metabolic stability of 63 in vitro in human
and rat liver microsomes. As shown in Table 7, compound 63
possessed a suitable metabolic stability, with half-life values
bioavailability (%)
a
Cmax, maximum plasma concentration; tmax, time to reach the
maximum plasma concentration; AUC, area under the plasma
concentration−time curve; t1/2, half-life; MRT, mean residence
time; Vz, apparent volume of distribution; Cl_F, apparent clearance;
n = 3, mean ± SEM.
>
60 min in both human and rat liver microsomes.
compound 63 significantly inhibited the tumor growth. The
tumor volume growth inhibition (GI) values of the mice
treated with 40, 80, and 120 mg/kg doses of 63 were 39.40%,
Table 7. In Vitro Metabolic Stability in Human and Rat
Liver Microsomes of 63
5
3.59%, and 54.51%, respectively (Figure 9A). Simultaneously,
human liver microsomes
rat liver microsomes
treatment at the dosage of 40 mg/kg led to 36.43% tumor
weight growth inhibition (TGI) compared with the control
T
CL
T
CL
1
/2
1/2
(min)
compound
(min)
(μL/min/mg)
(μL/min/mg)
(
Figure 9B). Nevertheless, the antitumor effects did not
6
3
70.0
14.1
66.0
15.0
increase significantly at doses of 80 and 120 mg/kg, which may
need to be further explored. In addition, compound 63 was
well tolerated, and no significant loss of body weight was
observed in 63-treated mice during the treatment period
(Figure 9C). These results suggested that 63 had potential
efficacy against the growth of implanted MV4-11 cells in mice.
Chemistry. The target compounds 8−28 were synthesized
using routes summarized in Scheme 1. Commercially available
2-fluoro-5-nitroaniline 65 was reacted with 1-methyl piperazine
to give the intermediate 66, which was then coupled with the
corresponding chlorinated heterocyclic to give compounds 8,
19, and 22, respectively. The substitution reaction of 65 with
different pyrimidines afforded intermediates 67a−67i, which
were then reacted with 1-methyl piperazine to create
compounds 9, 13−18, 20, and 21, respetively. The chlorine
atom of 9 was substituted with different secondary amines to
obtain target compounds 10−12. The fluorine atom of
With the aim to explore the potentially use of 63 in acute
leukemia models in vivo and determine the method of
administration, we next undertook an assessment of
pharmacokinetic (PK) parameters in healthy rats (Table 8).
63 exhibited an acceptable half-life (T1/2) (4.94 ± 0.04 h) after
oral administration. Moreover, a good bioavailability was
observed (F = 82.1%), verifying the oral administration of this
compound for further research in vivo.
In Vivo Antitumor Efficacy of 63. With the potent
inhibitory activity and drug-like properties both in vitro and in
vivo established, compound 63 was further assessed in an
MV4-11 xenograft model to examine its in vivo antitumor
efficacy. In this study, the hydrochloride salt of compound 63
was administered daily to mice by oral administration at 40, 80,
and 120 mg/kg for three weeks. As shown in Figure 9,
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Figure 9. Compound 63 suppressed the tumor growth in vivo in the MV4-11 tumor xenografts nude model. (A) Changes in the tumor volume of
MV4-11 tumor-bearing mice after treatment for three weeks. (B) Tumor weights after treatment. (C) Body weights of the treated mice. (D)
Images of the excised tumors for each group.
a
Scheme 1. Synthetic Routes for Compounds 8−28
a
Reagents and conditions are as follows: (a) 1-methyl piperazine, DMF, DIPEA, 100 °C, 8 h, 53%; (b) the corresponding chlorinated heterocyclic,
dioxane, Pd (dba) , BINAP, Cs CO , 100 °C, 12 h, 37−64%; (c) pyrimidines, IPA (EtOH/H O), HCl, reflux, 18−24 h, 56−86%; (d) different
2
3
2
3
2
secondary amines, DMF, DIPEA, 50 °C, 2−5 h, 45−89%; (e) different amines, DMF, DIPEA, 120 °C, 4−6 h, 47−50%; (f) DCM/MeOH, TFA,
r.t., 2 h, 65%. The detailed R groups for 9−22 are shown in Table 1.
intermediate 67i was substituted with different piperazines or
morpholine to yield the target compounds 23−28.
The synthesis of compounds 29−32 and 34−38 are shown
in Scheme 2. The substitution reaction of 68 with 1-methyl
piperazine and the reduction of the nitro group with hydrogen
and Pd/C gave 69. Subsequent substitution and reduction
yielded 70. Then, 70 was reacted with different boronic acids
through the Suzuki coupling reaction in the presence of
8
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a
Scheme 2. Synthetic Routes for the Target Compounds 29−32 and 34−38
a
Reagents and conditions are as follows: (a) 1-methyl piperazine, DMF, DIPEA, 80 °C, 5 h; (b) Pd/C, H , r.t., 2 h, 86%; (c) 4,6-dichloro-5-
2
nitropyrimidine, THF, Et N, 0 °C, 6 h; (d) SnCl , EA, reflux, 4 h, 75%; (e) different boronic acids, Pd(PPh ) Cl , Cs CO , dioxane, reflux, 20 h,
4
shown in Table 4.
3
2
3
2
2
2
3
6−70%, (f) acids, BOP, Et N, DMF, r.t., 12 h, 64−80%. The detailed R groups for 29−32 are shown in Table 3, and R groups for 34−38 are
3
1
a
Scheme 3. Synthetic Routes for the Target Compound 33
a
Reagents and conditions are as follows: (a) 1-methylpiperazine, DIPEA, CH CN, reflux, 12 h, 95%; (b) (i) NaNO , 6 M/HCl, 0 °C, 0.5 h, (ii)
3
2
NaN , 0 °C, 0.5 h, then r.t., 2 h; (c) methyl propiolate, CuI, MeOH, DIPEA, reflux, 48 h, 63%; (d) H , Pd/C, MeOH, r.t., 6 h, 85%; (e) 4,6-
dichloro-5-nitropyrimidine, THF, Et N, 0 °C, 6 h; (f) SnCl , EA, reflux, 4 h, 66%.
3
2
3
2
a
Scheme 4. Synthetic Routes for the Target Compounds 39−48
a
Reagents and conditions are as follows: (a) (4-(methoxycarbonyl)phenyl)boronic acid, Pd(PPh ) Cl , Cs CO , dioxane, reflux, 20 h, 70%; (b) 1-
3
2
2
2
3
methylpiperazine, CH CN, DIPEA, 80 °C, 5 h; (c) Pd/C, H , r.t., 2 h, 85%; (d) 4,6-dichloro-5-nitropyrimidine, THF, Et N, 0 °C, 2−6 h; (e)
3
2
3
SnCl , EA, reflux, 4 h, 46%; (f) 1 M LiOH, MeOH, r.t., 2 h; (g) different amines, BOP, Et N, DMF, r.t., 12 h, 42−79%. The detailed R groups for
2
3
39−48 are shown in Table 4.
Cs CO to obtain 29−32. Compound 29 was condensed with
was hydrolyzed by LiOH in MeOH/H O, and the subsequent
condensation with different amines generated compounds 39−
2
3
2
corresponding acids to afford compounds 34−38.
The synthetic route of compound 33 is listed in Scheme 3.
Briefly, 4-fluoro-3-nitroaniline 71 was substituted with N-
methylpiperazine to produce intermediate 72. The key
intermediate 73 was prepared by diazotization to form the
azide and cyclized via click chemistry. Subsequent reduction,
substitution, and reduction reactions yielded the target
compound 33.
The compounds 39−48 were synthesized according to
Scheme 4. 68 was reacted with (4-(methoxycarbonyl)phenyl)-
boronic acid via coupling to create the intermediate 75. 76 was
synthesized similar to 69. After similar substitution and
reduction reactions, 77 was obtained. Then, the ester group
4
8.
The compounds 49−53 were synthesized (Scheme 5) from
commercially available 2,6-difluoronitrobenzene. 79 was
obtained by an NBS bromination reaction, and the target
compounds 49−53 were synthesized similar to 39.
The synthesis of compounds 54−56 are shown in Scheme 6.
The intermediate 84 was obtained by coupling and
substitution reactions from 79. To avoid the selective
condensation of amino groups, 84 was first acylated with
different acids, then nitro groups of the core phenyl were
reduced to afford 85a−85c. Subsequently, analogues 54−56,
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a
Scheme 5. Synthetic Routes for the Target Compounds 49−53
a
Reagents and conditions are as follows: (a) NBS, TFA, H SO , 0 °C, 18 h, 90%; (b) (4-(methoxycarbonyl)phenyl)boronic acid, Pd(PPh ) Cl ,
2
4
3
2
2
Cs CO , dioxane, reflux, 20 h, 65%; (c) 1-methyl piperazine, DMF, DIPEA, 80 °C, 5 h; (d) Pd/C, H , r.t., 2 h, 65%; (e) 4,6-dichloro-5-
2
3
2
nitropyrimidine, THF, Et N, 0 °C, 6 h; (f) SnCl , EA, reflux, 4 h, 56%; (g) 1 M LiOH, MeOH, r.t., 2 h; (h) different amines, BOP, Et N, DMF, r.t.,
3
2
3
1
2 h. The detailed R groups for 49−53 are shown in Table 5.
a
Scheme 6. Synthetic Routes for the Target Compounds 54−56
a
Reagents and conditions are as follows: (a) (4-aminophenyl)boronic acid, Pd(PPh ) Cl , Cs CO , dioxane, reflux, 20 h, 69%; (b) 1-
3
2
2
2
3
methylpiperazine, CH CN, DIPEA, 80 °C, 5 h, 88%; (c) different acids, HATU, Et N, DMF, r.t., 12 h; (d) Pd/C, H , r.t., 2 h, 69−88%; (e) 4,6-
3
3
2
dichloro-5-nitropyrimidine, THF, Et N, 0 °C, 2−6 h; (f) SnCl , EA, reflux, 4 h, 35−38%.
3
2
a
Scheme 7. Synthetic Routes for the Target Compound 57
a
Reagents and conditions are as follows: (a) NBS, TFA, H SO , 0 °C, 18 h 87%; (b) (4-(dimethylcarbamoyl)phenyl)boronic acid, Pd(PPh ) Cl ,
2
4
3
2
2
Cs CO , dioxane, reflux, 20 h, 77%; (c) 1-methylpiperazine, CH CN, DIPEA, 80 °C, 5 h; (d) Pd/C, H , r.t., 2 h, 65%; (e) 4,6-dichloro-5-
nitropyrimidine, THF, Et N, 0 °C, 6 h; (f) SnCl , EA, reflux, 4 h, 44%.
2
3
3
2
3
2
respectively, were synthesized according to a procedure similar
Compound 64 was synthesized (Scheme 9) starting from
commercially available 2-fluoro-6-methyl nitrobenzene accord-
ing to a procedure similar to that of 60.
to that of 33.
The compound 57 was synthesized (Scheme 7) from
commercially available 2-fluoro-6-methyl nitrobenzene. 87 was
CONCLUSIONS
■
obtained by an NBS bromination reaction, then the target
The dysregulation of MLL1 catalytic functions is associated
with MLL-rearranged cancers. Directly disrupting the WDR5−
MLL1 PPI has been increasingly recognized as a target and a
specific way to develop potential therapeutic agents. However,
the efficacy of small-molecule WDR5−MLL1 inhibitors in vivo
is still unclear. In this study, we initially identified compound 9
as an active hit with an aniline pyrimidine scaffold by utilizing a
compound 57 was synthesized similar to 54.
The synthesis of compounds 58−63 are shown in Scheme 8.
Analogues 58−63 were synthesized from commercially
available 1-bromo-2,4-difluoro-5-nitrobenzene according to a
procedure similar to that of 49.
8
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a
Scheme 8. Synthetic Routes for the Target Compounds 58−63
a
Reagents and conditions are as follows: (a) (4-(methoxycarbonyl)phenyl)boronic acid, Pd(PPh ) Cl , Cs CO , dioxane, reflux, 20 h, 68%; (b) 1-
3
2
2
2
3
methyl piperazine, CH CN, DIPEA, 80 °C, 5 h; (c) Pd/C, H , r.t., 2 h, 64%; (d) 4,6-dichloro-5-nitropyrimidine, THF, Et N, 0 °C, 6 h; (e) SnCl ,
3
2
3
2
EA, reflux, 4 h, 57%; (f) 1 M LiOH, MeOH, r.t., 2 h; (g) different amines, BOP, Et N, DMF, r.t., 12 h, 65−78%.
3
a
Scheme 9. Synthetic Routes for the Target Compound 64
a
Reagents and conditions are as follows: (a) (4-(methoxycarbonyl)phenyl)boronic acid, Pd(PPh ) Cl , Cs CO , dioxane, reflux, 20 h, 69%; (b) 1-
3
2
2
2
3
methylpiperazine, CH CN, DIPEA, 80 °C, 5 h; (c) Pd/C, H , r.t., 2 h, 65%; (d) 4,6-dichloro-5-nitropyrimidine, THF, Et N, 0 °C, 6 h; (e) SnCl ,
3
2
3
2
EA, reflux, 4 h, 46%; (f) 1 M LiOH, MeOH, r.t., 2 h; (g) different amines, BOP, Et N, DMF, r.t., 12 h, 78%.
3
scaffold-hopping strategy from benzamide inhibitor 3. Then,
further systematic SAR studies led to the discovery of inhibitor
using TLC silica gel plates (GF254, 0.25 mm) and visualized
1
13
under UV light. The H NMR and C NMR spectra were
recorded on a Bruker AV-300 instrument using deuterated
solvents with tetramethylsilane (TMS) as internal standard. EI-
MS were recorded on Shimadzu Model GCMS-2010 instru-
ments. ESI-MS and high-resolution mass spectra (HRMS)
were collected on a Waters Q-Tof micro mass spectrometer
63, which has the most efficient WDR5 inhibitory activity as
well as preferable physicochemical properties, making it a
valuable asset for further research. Compound 63 showed the
selective inhibition of the proliferation of MLL-FPs leukemia
cells over K562 cells without MLL-FPs and displayed little
toxicity to the noncancerous human cell line. In addition, we
described its inhibition activity of the MLL histone
methyltransferase in vitro and in cells, followed by the
validation of the downregulation of MLL fusion protein-
dependent genes. Moreover, 63 possessed an ideal metabolic
stability in both human and rat microsomes and an appropriate
pharmacokinetic profile for oral administration. Additionally,
(
all data were within 0.40% of the theoretical values). The
purity (≥95%) of the target compounds was verified by high-
performance liquid chromatography (HPLC) analysis (Agilent
C18 column, 4.6 mm × 150 mm, 0.5 mL/min).
N-(2-(4-Methylpiperazin-1-yl)-5-nitrophenyl)quinazolin-
4-amine (8). 66 (0.2 g, 0.846 mmol) was dissolved in the
solution of 1,4-dioxane (20 mL), Pd (dba) (0.15 g, 0.253
63 demonstrated potent in vivo activity in MV4-11 xenograft
2
3
mmol), and BINAP (0.12 g, 0.13 mmol). Then, 4-
chloroquinazoline (0.27 g, 1.69 mmol) and Cs CO were
tumors in mice. In conclusion, we discovered a new chemotype
small-molecule inhibitor of WDR5−MLL1 PPI that served as a
potential starting point toward MLL1-driven acute myeloid
leukemia carrying MLL translocation therapeutics. We hope
these results can provide useful implications for the develop-
ment of therapeutic agents to target WDR5−MLL1.
2
3
added into the mixture. The reaction was carried out in 100 °C
for 12 h under the protection of nitrogen. After cooling to the
room temperature, the reaction mixture was filtered to remove
the catalytic agent and Cs CO . Then, the solvent was
2
3
removed by a rotary evaporator and purified by column
chromatography on silica gel to afford compound 8 as light
yellow solid. Yield: 63%. mp >250 °C. H NMR (300 MHz,
EXPERIMENTAL SECTION
■
1
General Chemistry. The synthesis of the test compounds
is highlighted in Schemes 1−9. All reagents were purchased
from commercial sources. Organic solvents were concentrated
̈
via an evaporator (Buchi Rotavapor) below 60 °C under
reduced pressure. Melting points (mp) were detected by a
Melt-Temp II apparatus. All the reactions were monitored
DMSO-d ): δ 9.57 (s, 1H), 8.61 (s, 2H), 8.43 (s, 1H), 8.07 (s,
6
1H), 7.86 (d, J = 10.0 Hz, 2H), 7.69 (s, 1H), 7.30 (s, J = 8.8
Hz, 1H), 3.09 (s, 4H), 2.32 (s, 4H), 2.13 (s, 3H). HRMS
+
(ESI): calcd. for m/z C19H N O , [M + H] 365.1720, found
20 6 2
365.1722. HPLC (100% MeOH): t = 7.772 min, 99.16%.
R
8
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4
N -(2-(4-Methylpiperazin-1-yl)-5-nitrophenyl)pyrimidine-
found 363.1349. HPLC (100% MeOH): tR = 6.297 min,
95.67%.
4
,5-diamine (19). The same procedure as 8. Yellow solid,
1
yield: 44%. mp >250 °C. H NMR (300 MHz, DMSO-d ): δ
6-Chloro-N-(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)-5-
6
1
8
2
.25 (s, 1H), 7.82 (dd, J = 7.4, 1.9 Hz, 1H), 7.81−7.77 (m,
nitropyrimidin-4-amine (16). Yield: 53%. mp >250 °C. H
H), 7.67 (s, 1H), 7.04 (d, J = 7.5 Hz, 1H), 5.82 (s, 2H), 3.20
NMR (300 MHz, DMSO-d ): δ 10.20 (s, 1H), 8.85 (d, J = 6.5
6
(
t, J = 5.3 Hz, 4H), 2.98 (t, J = 5.3 Hz, 4H), 2.60 (s, 3H).
Hz, 1H), 8.16 (d, J = 6.0 Hz, 2H), 7.62 (t, J = 9.5 Hz, 1H),
+
HRMS (ESI): calcd. for m/z C H N O , [M + H]
3
7
3.52 (br s, 4H), 2.42 (br s, 4H), 2.22 (s, 3H). HRMS (ESI):
1
5
19
7
2
+
30.1673, found 330.1675. HPLC (100% MeOH): tR
=
calcd. for m/z C H ClN O , [M + H] 394.0671, found
15
16
7
4
.673 min, 97.05%.
394.0674. HPLC (100% MeOH): t = 6.083 min, 97.01%.
R
2
4
N -(2-(4-Methylpiperazin-1-yl)-5-nitrophenyl)pyridine-
6-Chloro-N -(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)-
2
,3-diamine (22). The same procedure as 8. Yellow solid,
pyrimidine-4,5-diamine (17). Yield: 85%. mp 185−187 °C.
1
1
yield: 37%. mp 210−212 °C. H NMR (300 MHz, DMSO-d ):
δ 9.06 (d, J = 2.8 Hz, 1H), 7.77 (dd, J = 8.8, 2.8 Hz, 1H),
H NMR (300 MHz, DMSO-d ): δ 8.65 (d, J = 2.6 Hz, 1H),
6
6
8.22 (s, 1H), 7.98−7.91 (m, 2H), 7.27 (d, J = 8.9 Hz, 1H),
7
1
.68−7.59 (m, 2H), 7.28 (d, J = 8.8 Hz, 1H), 7.77 (dd, J = 7.6,
5.41 (s, 2H), 2.91 (br s, 4H), 2.47 (br s, 4H), 2.20 (s, 3H).
+
.7 Hz, 1H), 6.75 (dd, J = 7.6, 1.7 Hz, 1H), 4.94 (s, 2H), 2.90
HRMS (ESI): calcd. for m/z C H ClN O , [M + H]
1
5
18
7
2
(
t, J = 4.8 Hz, 4H), 2.54 (s, 4H), 2.25 (s, 3H). HRMS (ESI):
364.1283, found 364.1286. HPLC (100% MeOH): tR =
+
calcd. for m/z C H N O , [M + H] 329.1720, found
6.581 min, 98.22%.
15
20
6
2
4
3
29.1722. HPLC (100% MeOH): t = 4.151 min, 95.29%.
6-Chloro-2-methyl-N -(2-(4-methylpiperazin-1-yl)-5-
R
General Procedure for the Synthesis of the Com-
nitrophenyl)pyrimidine-4,5-diamine (18). Yield: 68%. mp
1
pounds 9, 13−18, and 20−21. The intermediates 67a−67i
1 mmol) were dissolved in the solution of DMF (5 mL) and
DIPEA (3 mmol) before the addition of 1-methyl piperazine
3 mmol). The reaction was carried out at 50 °C for 2−5 h.
248−250 °C. H NMR (300 MHz, DMSO-d ): δ 8.87 (d, J =
6
(
2.8 Hz, 1H), 8.22 (s, 1H), 7.94 (dd, J = 8.8, 2.8 Hz, 1H), 7.28
(d, J = 8.9 Hz, 1H), 5.12 (s, 2H), 2.98 (t, J = 4.8 Hz, 4H), 2.46
(s, 4H), 2.32 (s, 3H), 2.21 (s, 3H). HRMS (ESI): calcd. for m/
(
+
The reaction mixture was subsequently diluted with dichloro-
methane (20 mL) and washed with water (3 × 10 mL) and a
saturated sodium chloride solution (10 mL). The organic
phase was then separated, dried with sodium sulfate (Na SO ),
z C H ClN O , [M + H] 378.1440, found 378.1440. HPLC
1
6
20
7
2
(100% MeOH): t = 6.251 min, 97.99%.
R
N-(2-(4-Methylpiperazin-1-yl)-5-nitrophenyl)pyrimidin-2-
1
2
4
amine (20). Yield: 57%. mp 195−197 °C. H NMR (300
filtered, and concentrated to provide crude products. The
crude products were washed with CH CN (5 mL) to obtain 9,
1
MHz, CDCl ): δ 9.47 (s, 1H), 8.54 (d, J = 3.0 Hz, 2H), 8.03
3
3
(s, 1H), 7.88 (d, J = 3.0 Hz, 1H), 7.22 (d, J = 9.0 Hz, 1H),
6.85 (t, J = 9.0 Hz, 1H), 3.04 (t, J = 5.6 Hz, 4H), 2.70 (br s,
4H), 2.42 (s, 3H). HRMS (ESI): calcd. for m/z
3−18, 20, and 21 as yellow solids.
6
-Chloro-N-(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)-
1
+
pyrimidin-4-amine (9). Yield: 66%. mp 169−171 °C. H
NMR (300 MHz, DMSO-d ): δ 9.36 (s, 1H), 8.48 (s, 1H),
C H ClN O , [M + H] 315.1564, found 315.1566. HPLC
1
5
18
7
2
6
(100% MeOH): t = 16.340 min, 96.09%.
R
2
8
.42 (d, J = 2.7 Hz, 1H), 8.02 (dd, J = 8.9, 2.8 Hz, 1H), 7.23
N -(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)pyrimidine-
2,4-diamine (21). Yield: 46%. mp 210−212 °C. H NMR (300
MHz, DMSO-d ): δ 9.30 (s, 1H), 7.94 (d, J = 6.0 Hz, 1H),
1
(
(
d, J = 9.0 Hz, 1H), 6.81 (s, 1H), 3.04 (t, J = 4.6 Hz, 4H), 2.35
t, J = 4.7 Hz, 4H), 2.16 (s, 3H). C NMR (75 MHz, DMSO-
13
6
d6): δ 161.34, 158.74, 158.69, 151.70, 141.57, 130.46, 129.94,
21.67, 121.11, 119.89, 105.90, 54.60, 49.90, 46.16. HRMS
7.80 (dd, J = 8.7 Hz,1H), 7.63 (s, 1H), 7.32 (d, J = 8.8
Hz,1H), 6.81 (br s, 2H), 6.03 (d, J = 5.8 Hz,1H), 2.93 (t, J =
4.7 Hz, 4H), 2.54 (br s, 4H), 2.26 (s, 3H). HRMS (ESI):
1
+
(
ESI): calcd. for m/z C H ClN O , [M + H] 349.1174,
15 17 6 2
+
found 349.1177. HPLC (100% MeOH): tR = 6.097 min,
6.84%.
-Chloro-2-methyl-N-(2-(4-methylpiperazin-1-yl)-5-
calcd. for m/z C H N O , [M + H] 330.1673, found
15
19
7
2
9
330.1679. HPLC (100% MeOH): t = 6.354 min, 96.85%.
R
6
General Procedure for the Synthesis of 10−12.
Compound 9 (0.35 mmol) was dissolved in the solution of
DMF (5 mL) and DIPEA (3 mmol) before the addition of
different amines (1 mmol). The reaction was carried out at 120
°C for 4 h. The reaction mixture was subsequently diluted with
EA (20 mL) and washed with water (10 mL × 3) and a
saturated sodium chloride solution (10 mL). The organic layer
was then separated, dried with sodium sulfate (Na SO ),
nitrophenyl)pyrimidin-4-amine (13). Yield: 45%. mp 196−
1
1
1
6
98 °C. H NMR (300 MHz, CDCl ): δ 9.09 (d, J = 2.6 Hz,
H), 7.97 (dd, J = 8.8, 2.7 Hz, 1H), 7.55 (s, 1H), 7.23 (s, 1H),
.59 (s, 1H), 3.02 (t, J = 4.8 Hz, 4H), 2.65−2.61 (m, 7H), 2.40
3
(
s, 3H). HRMS (ESI): calcd. for m/z C H ClN O , [M +
16 19 6 2
+
H] 363.1331, found 363.1335. HPLC (100% MeOH): t =
R
8
.574 min, 97.99%.
2
4
4
6
-Chloro-N -(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)-
filtered, and concentrated to provide crude products. The
purified products 10−12 were obtained by column chroma-
tography (DCM/MeOH = 50:1) to afford yellow solids.
pyrimidine-2,4-diamine (14). Yield: 78%. mp 248−251 °C.
1
H NMR (300 MHz, CDCl ): δ 9.13 (s, 1H), 7.94 (dd, J = 8.7,
3
4
4
6
2
2
.6 Hz, 1H), 7.41 (s, 1H), 7.20 (d, J = 2.6 Hz, 1H), 5.16 (s,
H), 3.00 (t, J = 4.8 Hz, 4H), 2.64 (s, 4H), 2.41 (s, 3H).
N ,N -Dimethyl-N -(2-(4-methylpiperazin-1-yl)-5-
nitrophenyl)pyrimidine-4,6-diamine (10). Yield: 49%. mp
+
1
HRMS (ESI): calcd. for m/z C H ClN O , [M + H]
152−154 °C. H NMR (300 MHz, CDCl ): δ 8.69 (d, J = 2.7
15
18
7
2
3
3
5
64.1283, found 364.1289. HPLC (100% MeOH): tR
.850 min, 97.14%.
=
Hz, 1H), 8.28 (s, 1H), 7.80 (dd, J = 8.7, 2.7 Hz, 1H), 7.06 (d, J
= 8.8 Hz, 1H), 6.94 (s, 1H), 5.82 (s, 1H), 3.03 (s, 6H), 2.94 (t,
J = 4.8 Hz, 4H), 2.65−2.48 (m, 4H), 2.30 (s, 3H). HRMS
6
-Chloro-5-methyl-N-(2-(4-methylpiperazin-1-yl)-5-
+
nitrophenyl)pyrimidin-4-amine (15). Yield: 60%. mp 259−
(ESI): calcd. for m/z C H N O , [M + H] 358.1986, found
1
7
23
7
2
1
2
1
3
61 °C. H NMR (300 MHz, CDCl ): δ 9.55 (s, 1H), 8.57 (s,
358.1992. HPLC (100% MeOH): t = 4.759 min, 99.20%.
3
R
H), 8.18 (s, 1H), 7.95 (dd, J = 8.7, 2.7 Hz, 1H), 7.27 (s, 1H),
6-(Azetidin-1-yl)-N-(2-(4-methylpiperazin-1-yl)-5-
.02 (t, J = 4.8 Hz, 4H), 2.66 (s, 4H), 2.42 (s, 6H). HRMS
nitrophenyl)pyrimidin-4-amine (11). Yield: 51%. mp 209−
+
1
(ESI): calcd. for m/z C H ClN O , [M + H] 363.1331,
211 °C. H NMR (300 MHz, CDCl ): δ 8.64 (d, J = 2.7 Hz,
16
19
6
2
3
8
236
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Journal of Medicinal Chemistry
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Article
1
8
4
H), 8.26 (s, 1H), 7.81 (dd, J = 8.8, 2.6 Hz, 1H), 7.06 (d, J =
.8 Hz, 1H), 6.95 (s, 1H), 5.54 (s, 1H), 4.03 (t, J = 7.5 Hz,
H), 2.93 (t, J = 4.9 Hz, 4H), 2.55 (d, J = 5.2 Hz, 4H), 2.43 (q,
390.1440, found 390.1446. HPLC (100% MeOH): t = 4.811
min, 99.31%.
R
2-(4-(2-((5-Amino-6-chloropyrimidin-4-yl)amino)-4-
nitrophenyl)p-iperazin-1-yl)ethan-1-ol (27). The same pro-
J = 7.5 Hz, 2H), 2.30 (s, 3H). HRMS (ESI): calcd. for m/z
C H N O , [M + H] 370.1986, found 370.1993. HPLC
+
1
cedure as 23. Yellow solid, yield: 76%. mp 202−204 °C. H
1
8
23
7
2
(
100% MeOH): t = 5.293 min, 96.43%.
NMR (300 MHz, CDCl ): δ 9.33 (s, 1H), 8.32 (s, 1H), 8.15
3
R
6
-Cyclopentyl-N-(2-(4-methylpiperazin-1-yl)-5-
(s, 1H), 7.94 (d, J = 8.7 Hz, 1H), 7.27 (s, 1H), 3.70 (s, 2H),
3.64−2.46 (m, 2H), 3.02 (s, 4H), 2.78 (s, 4H), 2.68 (s, 2H),
nitrophenyl)pyrimidin-4-amine (12). Yield: 47%. mp 223−
25 °C. H NMR (300 MHz, CDCl ): δ 8.74 (d, J = 2.6 Hz,
H), 8.36 (s, 1H), 7.89 (dd, J = 8.8, 2.7 Hz, 1H), 7.14 (d, J =
.8 Hz, 1H), 7.00 (s, 1H), 5.80 (s, 1H), 3.49 (s, 4H), 3.03 (t, J
4.8 Hz, 4H), 2.64 (s, 4H), 2.39 (s, 3H), 2.03 (s, 4H). HRMS
ESI): calcd. for m/z C H N O , [M + H] 384.2142, found
84.2154. HPLC (100% MeOH): t = 9.383 min, 99.41%.
R
6
pyrimidine-4,5-diamine (23). 67f (0.35 mmol) was dissolved
in the solution of DMF (5 mL) and DIPEA (3 mmol). Then,
tert-butyl-piperazine-1-carboxylate (1 mmol) was added. The
reaction was carried out at 50 °C for 5 h. The reaction mixture
was subsequently diluted with EA (20 mL), then washed with
water (10 mL × 3) and a saturated sodium chloride solution
1
2
1
8
=
(
2.57 (s, 1H). HRMS (ESI): calcd. for m/z C16H20ClN O , [M
7 3
3
+
+
H] 394.1389, found 394.1387. HPLC (100% MeOH): t =
R
7.347 min, 96.12%.
4
6
-Chloro-N -(5-nitro-2-(4-phenylpiperazin-1-yl)phenyl)-
+
pyramidi-ne-4,5-diamine (28). The same procedure as 23.
19 26 7 2
1
Yellow solid: yield 76%. mp >250 °C. H NMR (300 MHz,
3
4
−
CDCl ): δ 9.39 (d, J = 2.7 Hz, 1H), 8.35 (s, 1H), 8.25 (s, 1H),
-Chloro-N (5-nitro-2-(piperazin-1-yl)phenyl)-
3
7
.97 (dd, J = 8.9, 2.6 Hz, 1H), 7.39−7.28 (m, 3H), 7.06−6.90
m, 3H), 3.54 (s, 2H), 3.44 (t, J = 4.9 Hz, 4H), 3.16 (t, J = 4.9
Hz, 4H). HRMS (ESI): calcd. for m/z C H ClN O , [M +
(
20 20
7
2
+
H] 426.1440, found 426.1449. HPLC (100% MeOH): t =
R
4
.905 min, 95.52%.
General Procedure for the Synthesis of Compounds
2
9−32. 70 (1.0 g, 2.5 mmol) was dissolved in the solution of
(
10 mL). The organic layer was then separated, dried by
sodium sulfate (Na SO ), filtered, and concentrated to provide
dioxane (30 mL), and different boronic acids (1.2 equiv) were
2
4
added to the solution. Then, Cs CO (1.6 g, 5 mmol) and a
2
3
the crude product. The obtained intermediate was dissolved in
the solution of DCM/MeOH (20 mL, 2:1). Then, TFA (2
mL) was dropped. The mixture was stirred at room
temperature for 2 h. The mixture was neutralized with a
saturated sodium bicarbonate solution and extracted with EA
catalytic amount of Pd(PPh ) Cl were added to the mixture.
3
2
2
The reaction bottle was protected with N , and the mixture
2
was refluxed for 20 h. The solution was filtered to remove the
catalytic agent and Cs CO , then purified by column
2
3
chromatography on silica gel using a common 1−5%
MeOH/DCM gradient to afford compounds 29−32 as light
yellow solids.
(
10 mL× 3). The organic layer was collected to be
concentrated by a rotary evaporator and then washed with
CH CN to afford the crude product as a yellow solid, yield:
3
4
N -(4′-Amino-4-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-
1
6
(
8
(
6%. mp >250 °C. H NMR (300 MHz, DMSO-d ): δ 9.09
6
3
2
8
-yl)-6-chloropyrimid-in-e-4,5-diamine (29). Yield: 67%. mp
br s, 1H), 8.60 (d, J = 2.7 Hz, 1H), 8.33 (s, 1H), 7.89 (dd, J =
.9, 2.7 Hz, 1H), 7.83 (s, 1H), 7.23 (d, J = 8.9 Hz, 1H), 5.55
s, 2H), 3.21 (s, 4H), 3.42 (t, J = 6.0 Hz, 4H). HRMS (ESI):
calcd. for m/z C H ClN O , [M + H] 350.1127, found
50.1131. HPLC (100% MeOH): t = 4.069 min, 96.17%.
1
12−214 °C. H NMR (300 MHz, DMSO-d ): δ 8.16 (d, J =
6
.1 Hz, 2H), 7.90 (s, 1H), 7.65 (d, J = 8.2 Hz, 2H), 7.55 (d, J
=
8.4 Hz, 2H), 7.37 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 8.2 Hz,
+
14
17
7
2
1
2
H), 5.43 (s, 2H), 5.15 (s, 2H), 3.17 (s, 4H), 3.06 (s, 4H),
.75 (s, 3H). m/z (EI-MS): 410.2 [M] . HPLC (100%
3
+
R
4
6
-Chloro-N -(2-morpholino-5-nitrophenyl)pyrimidine-
methanol): t = 7.489 min, 95.67%.
R
4
,5-diamine (24). The same procedure as 23. Yellow solid,
yield: 71%. mp >250 °C. H NMR (300 MHz, DMSO-d ): δ
.70 (s, 1H), 8.32 (s, 1H), 8.08−7.86 (m, 2H), 7.29 (d, J = 9.0
Hz, 1H), 5.46 (s, 2H), 3.73 (s, 4H), 2.99 (s, 4H). HRMS
4
6
-Chloro-N -(4′-methoxy-4-(4-methylpiperazin-1-yl)-
1
6
[
6
8
1,1′-biphenyl]-3-yl)pyram-idine-4,5-diamine (30). Yield:
8
1
0%. mp 211−213 °C. H NMR (300 MHz, DMSO-d ): δ
6
.24 (s, 2H), 7.90 (s, 1H), 7.57 (d, J = 7.9 Hz, 2H), 7.40−7.35
+
(
ESI): calcd. for m/z C H ClN O , [M + H] 351.0967,
14 16 6 3
(m, 3H), 7.24 (d, J = 8.3 Hz, 1H), 5.37 (s, 2H), 5.22 (s, 1H),
found 351.0967. HPLC (100% MeOH): tR = 7.041 min,
8.84%.
-Chloro-N -(2-(4-ethylpiperazin-1-yl)-5-nitrophenyl)-
4.52 (s, 2H), 2.85 (t, J = 4.5 Hz, 4H), 2.53 (s, 4H), 2.24 (s,
9
+
3
H). (EI-MS): m/z 425.2 [M] . HPLC (80% methanol in
4
6
water): t = 4.590 min, 99.44%.
R
pyrimidine-4,5-diamine (25). The same procedure as 23.
Yellow solid, yield: 47%. mp 185−187 °C. H NMR (300
MHz, CDCl ): δ 9.34 (s, 1H), 8.32 (s, 1H), 8.18 (s, 1H), 7.93
4
6
-Chloro-N -(2-(4-methylpiperazin-1-yl)-5-(pyrimidin-5-
1
yl)phenyl)pyrimidine-4,5-diamine (31). Yield: 46%. mp 189−
1
3
191 °C. H NMR (300 MHz, DMSO-d ): δ 9.17 (s, 1H), 9.10
6
(
3
2
dt, J = 8.8, 2.7 Hz, 1H), 7.27−7.20 (m, 1H), 3.60 (s, 2H),
(
s, 2H), 8.57 (s, 1H), 7.80 (s, 1H), 7.10 (dd, J = 7.5, 2.0 Hz,
.08−2.93 (m, 4H), 2.70 (s, 4H), 2.53 (qd, J = 7.2, 2.7 Hz,
1
H), 6.99 (d, J = 7.4 Hz, 1H), 6.82 (d, J = 2.0 Hz, 1H), 5.32
H), 1.16 (td, J = 7.3, 2.7 Hz, 3H). HRMS (ESI): calcd. for m/
(
s, 2H), 2.85 (s, 4H), 2.54 (s, 4H), 2.34 (s, 3H). HRMS (ESI):
calcd. for m/z C H ClN , [M + H] 397.1651, found
+
+
z C H ClN O , [M + H] 378.1440, found 348.1449. HPLC
1
6
21
7
2
19 22 8
(
100% MeOH): t = 4.542 min, 97.65%.
6
nitrophenyl)pyr-imidine-4,5-diamine (26). The same proce-
dure as 23. Yellow solid, yield: 59%. mp 223−225 °C. H
NMR (300 MHz, CDCl ): δ 9.35 (d, J = 2.7 Hz, 1H), 8.32 (s,
397.1653. HPLC (80% methanol in water): t = 3.973 min,
R
R
4
-Chloro-N -(2-(4-cyclopropylpiperazin-1-yl)-5-
97.50%.
4
6-Chloro-N -(5-(furan-3-yl)-2-(4-methylpiperazin-1-yl)-
1
phenyl)pyrimidine-4,5-diamine (32). Yield: 70%. mp 164−
1
3
166 °C. H NMR (300 MHz, DMSO-d ): δ 8.23 (s, 1H), 7.90
6
1
8
4
2
H), 8.21 (s, 1H), 7.92 (dd, J = 8.7, 2.7 Hz, 1H), 7.22 (d, J =
.8 Hz, 1H), 3.62 (s, 2H), 2.96 (t, J = 4.7 Hz, 4H), 2.87 (s,
H), 1.78−1.71 (m, 1H), 0.56−0.51 (m, 2H), 0.49−0.44 (m,
(s, 1H), 7.54 (d, J = 7.5 Hz, 1H), 7.43 (s, 1H), 7.07 (d, J = 7.7
Hz, 1H), 6.91 (d, J = 7.4 Hz, 1H), 6.82 (s, 1H), 6.53 (d, J =
8.0 Hz, 1H), 5.36 (s, 2H), 2.85 (s, 4H), 2.61 (s, 4H), 2.25 (s,
+
+
H). HRMS (ESI): calcd. for m/z C H ClN O , [M + H]
3H). HRMS (ESI): calcd. for m/z C H ClN O, [M + H]
17
21
7
2
19 22
6
8
237
https://doi.org/10.1021/acs.jmedchem.1c00091
J. Med. Chem. 2021, 64, 8221−8245

Journal of Medicinal Chemistry
85.1538, found 385.1538. HPLC (90% methanol in water): tR
pubs.acs.org/jmc
Article
3
δ 10.00 (s, 1H), 8.21−8.16 (m, 2H), 7.90 (d, J = 6.0 Hz, 1H),
7.71−7.69 (m, 2H), 7.59−7.54 (m, 2H), 7.38 (d, J = 7.8 Hz,
1H), 7.27 (d, J = 8.2 Hz, 1H), 5.44 (s, 2H), 3.51−3.46 (m,
4H), 3.27−3.16 (m, 4H), 2.88 (s, 3H), 2.32−2.27 (m, 2H),
=
6.859 min, 96.26%.
General Procedure for the Synthesis of Compounds
4−38. To a solution of different acids (0.35 mmol) in DMF
3
(
1
3
8 mL) at room temperature was added the BOP reagent (0.26
1.68−1.58 (m, 2H), 0.96−0.89 (m, 3H). C NMR (75 MHz,
g, 0.59 mmol), followed by Et N (0.1 mL, 0.88 mmol). The
DMSO-d ): δ 166.01, 151.06, 146.61, 143.61, 140.32, 138.84,
3
6
mixture was stirred for 0.5 h before compound 29 (0.12 g, 0.29
mmol) was added. Then, the mixture was stirred at room
temperature for 12 h. The aqueous solution was poured into
135.73, 135.52, 135.42, 133.91, 132.05, 128.87, 128.15, 126.97,
125.53, 122.22, 121.30, 121.19, 120.73, 55.53, 51.51, 46.32.
+
HRMS (ESI): calcd. for m/z C H ClN O, [M + H]
2
8
28
7
5
0 mL of water and extracted with EA (3 × 50 mL), and the
514.2117, found 514.2115. HPLC (90% methanol in water):
tR = 3.889 min, 95.09%.
organic phase was dried over Na SO , filtered, and
2
4
concentrated to obtain the crude product, which was then
purified by column chromatography on silica gel using a
common 1−5% MeOH/DCM gradient to afford compounds
Methyl 1-(3-((5-Amino-6-chloropyrimidin-4-yl)amino)-4-
(4-methylpiperazin-1-yl) phenyl)-1H-1,2,3-triazole-4-car-
boxylate (33). 33 was prepared from 74 and 4,6-dichloro-5-
34−38 as light yellow solids.
nitropyrimidine according to the procedure of 70. Yield: 66%.
1
N-(3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-4′-(4-
mp 231−233 °C. H NMR (300 MHz, DMSO-d ): δ 9.46 (s,
6
methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)tetrahydro-2H-
1H), 8.62 (s, 1H), 8.34 (s, 1H), 7.95 (s, 1H), 7.64 (d, J = 8.6
Hz, 1H), 7.37 (d, J = 8.7 Hz, 1H), 5.44 (s, 2H), 3.89 (s, 3H),
2.88 (s, 4H), 2.51 (s, 4H), 2.23 (s, 3H). HRMS (ESI): calcd.
1
pyran-4-carboxamide (34). Yield: 78%. mp 207−209 °C. H
NMR (300 MHz, DMSO-d ): δ 9.99 (s, 1H), 8.21 (s, 1H),
6
+
7
2
3
4
.90 (s, 1H), 7.68 (dd, J = 8.4, 5.9 Hz, 3H), 7.57−7.54 (m,
H), 7.38−7.35 (m, 1H), 7.24−7.22 (m, 1H), 5.38 (s, 2H),
.94−3.90 (m, 3H), 3.37−3.35 (m, 2H), 2.92 (s, 4H), 2.76 (s,
for m/z C H ClFN O , [M + H] 444.1657, found 444.1660.
1
9
22
9
2
HPLC (90% methanol in water): t = 3.775 min, 97.63%.
R
Methyl 4′-Fluoro-3′-nitro-[1,1′-biphenyl]-4-carboxylate
(75). 4-Bromo-1-fluoro-2-nitrobenzene (5.0 g, 22.8 mmol)
was dissolved in the solution of dioxane (150 mL) before the
addition of boronic acid (27.3 mmol). Then, Cs CO (14.8 g,
13
H), 2.43 (s, 3H), 1.72−1.69 (m, 4H). C NMR (75 MHz,
DMSO-d ): δ 173.52, 150.72, 146.29, 142.40, 140.15, 139.15,
6
136.23, 134.86, 134.03, 127.06, 125.57, 122.34, 121.86, 121.07,
2
3
1
20.00, 66.88, 53.87, 48.83, 42.21, 29.36. HRMS (ESI): calcd.
45.6 mmol) and a catalytic amount of Pd(PPh ) Cl were
3 2 2
+
for m/z C H ClN O , [M + H] 522.2379, found 522.2377.
HPLC (80% methanol in water): t = 4.280 min, 95.50%.
added. The mixture was protected with N and then refluxed
2
7
32
7
2
2
for 20 h. The solution was filtered to remove the catalytic agent
and Cs CO , then purified by column chromatography on
R
N-(3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-4′-(4-
2
3
methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)-1-methylpiperi-
silica gel using a common 1−5% MeOH/DCM gradient to
1
1
dine-4-carboxamide (35). Yield: 64%. mp 157−160 °C. H
afford intermediate 75. Yield: 70%. mp 145−147 °C. H NMR
NMR (300 MHz, DMSO-d ): δ 10.19 (s, 1H), 8.22−8.19 (m,
(300 MHz, CDCl ): δ 8.31−8.13 (m, 3H), 7.88−7.59 (m,
6
3
2
H), 7.95 (s, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.7
1H), 8.28 (m, 3H), 7.42−7.36 (m, 1H), 3.94 (s, 3H). (EI-
+
Hz, 2H), 7.36 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 8.1 Hz, 1H),
MS): m/z 276.2 [M] .
5
2
.42 (s, 2H), 2.99−2.90 (m, 5H), 2.85 (s, 4H), 2.73 (s, 3H),
Methyl 3′-Amino-4′-(4-methylpiperazin-1-yl)-[1,1′-bi-
.53 (s, 4H), 2.49 (s, 3H), 2.03−1.99 (m, 4H). HRMS (ESI):
phenyl]-4-carboxylate (76). 76 was prepared from 75 similar
+
1
calcd. for m/z C H ClN O, [M + H] 535.2695, found
to intermediate 69. Yield: 90%. mp 229−231 °C. H NMR
28
36
8
5
35.2710. HPLC (80% methanol in water): t = 4.518 min,
(300 MHz, CDCl ): δ 8.07 (d, J = 8.5 Hz, 2H), 7.61 (dd, J =
R
3
9
8.25%.
6.7, 1.8 Hz, 2H), 7.09 (d, J = 8.5 Hz, 1H), 7.03 (m, 2H), 4.06
(s, 2H), 3.93 (s, 3H), 3.01 (t, J = 4.4 Hz, 4H), 2.61 (s, 4H),
N-(3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-4′-(4-
+
m e t h y l p i p e r a z i n - 1 - y l ) - [ 1 , 1 ′ - b i p h e n y l ] - 4 - y l ) -
2.38 (s, 3H). (EI-MS): m/z 326.2 [M] .
cyclohexanecarboxamide (36). Yield: 80%. mp 256−258 °C.
Methyl 3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-4′-
1
H NMR (300 MHz, DMSO-d ): δ 9.91 (s, 1H), 8.18 (d, J =
(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylate (77).
6
8
=
1
2
.2 Hz, 2H), 7.90 (s, 1H), 7.69 (d, J = 8.3 Hz, 2H), 7.55 (d, J
8.4 Hz, 2H), 7.37 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 8.2 Hz,
H), 5.43 (s, 2H), 3.17 (s,4H), 3.06 (s, 4H), 2.75 (s, 3H),
The same procedure as that for 33 was used. Yield: 46%. mp
1
209−210 °C. H NMR (300 MHz, DMSO-d ): δ 8.53−8.42
6
(m, 1H), 8.23 (s, 1H), 7.96−7.90 (m, 3H), 7.70 (d, J = 8.4 Hz,
2H), 7.46−7.42 (m, 1H), 7.26 (d, J = 8.3 Hz, 1H), 5.36 (s,
2H), 3.93 (s, 3H), 3.57 (t, J = 4.6 Hz, 4H), 2.87 (t, J = 4.7 Hz,
.34−2.30 (m, 1H), 1.76 (t, J = 12.5 Hz, 4H), 1.42 (t, J = 11.6
Hz, 2H), 1.33−1.09 (m, 4H). HRMS (ESI): calcd. for m/z
C H ClN O, [M + H] 520.2586, found 520.2584. HPLC
+
+
4H), 2.24 (s, 3H). (EI-MS): m/z 453.2 [M] .
2
8
34
7
(
90% methanol in water): t = 4.003 min, 97.87%.
N-(3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-4′-(4-
General Procedure for Preparation of Compounds
R
39−48. The ester group was hydrolyzed by LiOH in MeOH/
methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)butyramide (37).
H O to give the acid intermediate. Then, the BOP reagent
2
1
Yield: 80%. mp 196−198 °C. H NMR (300 MHz, DMSO-
(0.22 g, 0.49 mmol) was added to a solution of acid (0.1 g,
0.26 mmol) in DMF (10 mL) at room temperature, followed
by TEA (0.12 mL, 0.88 mmol). Then, different amines (0.39
mmol) were added to reaction mixture, and the mixture was
stirred for 30 min. Then, the resultant solution was stirred at
room temperature for 12 h. The mixture was poured into 50
mL of water and extracted with EA (3 × 50 mL). The organic
layer was dried by Na SO , filtered, and concentrated by a
d ): δ 10.00 (s, 1H), 8.21−8.16 (m, 2H), 7.90 (d, J = 6.0 Hz,
6
1
H), 7.71−7.69 (m, 2H), 7.59−7.54 (m, 2H), 7.38 (d, J = 7.8
Hz, 1H), 7.27 (d, J = 8.2 Hz, 1H), 5.44 (s, 2H), 3.51−3.46 (m,
4
1
H), 3.27−3.16 (m, 4H), 2.88 (s, 3H), 2.32−2.27 (m, 2H),
.68−1.58 (m, 2H), 0.96−0.89 (m, 3H). HRMS (ESI): calcd.
+
for m/z C H ClN O, [M + H] 480.2273, found 480.2274.
HPLC (80% methanol in water): t = 4.696 min, 96.97%.
2
5
31
7
R
2
4
N-(3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-4′-(4-
rotary evaporator below 50 °C under reduced pressure to
obtain the crude products. The final products were purified by
column chromatography on silica gel using a common 1−10%
methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)benzamide (38).
1
Yield: 78%. mp >250 °C. H NMR (300 MHz, DMSO-d ):
6
8
238
https://doi.org/10.1021/acs.jmedchem.1c00091
J. Med. Chem. 2021, 64, 8221−8245

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
+
MeOH/DCM gradient to afford compounds 39−48 as white
solids.
[M + H] 521.2937, found 521.2937. HPLC (80% methanol
in water): t = 4.094 min, 98.10%.
R
(
3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-4′-(4-meth-
3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-4′-(4-meth-
ylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)(morpholino)-
ylpiperazin-1-yl)-N-(1-methylpiperidin-4-yl)-[1,1′-biphenyl]-
1
1
methanone (39). Yield: 68%. mp 225−227 °C. H NMR (300
4-carboxamide (45). Yield: 47%. mp >250 °C. H NMR (300
MHz, DMSO-d ): δ 8.45 (d, J = 13.6 Hz, 2H), 7.90 (s, 1H),
MHz, DMSO-d ): δ 8.48 (s, 1H), 8.22 (s, 1H), 8.14 (s, 1H),
6
6
7
8
4
3
5
.69 (d, J = 7.9 Hz, 2H), 7.49 (d, J = 7.7 Hz, 2H), 7.42 (d, J =
.4 Hz, 1H), 7.27 (d, J = 8.2 Hz, 1H), 5.39 (s, 2H), 3.61 (s,
H), 3.35 (s, 4H), 2.98−2.90 (s, 4H), 2.64 (s, 4H), 2.34 (s,
7.87 (d, J = 7.9 Hz, 2H), 7.77 (s, 1H), 7.61 (d, J = 8.0 Hz,
2H), 7.36 (d, J = 8.1 Hz, 1H), 7.16 (d, J = 7.5 Hz, 1H), 5.40
(s, 2H), 3.92 (s, 1H), 3.22 (s, 4H), 2.87(s, 4H), 2.64 (s, 2H),
2.41 (s, 6H), 2.30 (s, 2H), 1.86 (s, 4H). HRMS (ESI): calcd.
+
H). HRMS (ESI): calcd. for m/z C H ClN O , [M + H]
26
30
7
2
+
08.2222, found 508.2223. HPLC (80% methanol in water): tR
3.507 min, 98.81%.
for m/z C H ClN O, [M + H] 535.2695, found 535.2700.
28
35
8
=
HPLC (80% methanol in water): t = 4.512 min, 97.75%.
R
3
′-((5-Amino-6-chloropyrimidin-4-yl)amino)-4′-(4-meth-
(3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-4′-(4-meth-
ylpiperazin-1-yl)-N-(2-morpholinoethyl)-[1,1′-biphenyl]-4-
ylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)(4-hydroxycyclohexyl)-
1
1
carboxamide (40). Yield: 56%. mp 230−232 °C. H NMR
methanone (46). Yield: 42%. mp 190−192 °C. H NMR (300
(
7
300 MHz, DMSO-d ): δ 8.45 (s, 1H), 8.27−8.22 (m, 2H),
MHz, DMSO-d ): δ 8.24 (d, J = 8.4 Hz, 2H), 7.89 (s, 1H),
6
6
.92−7.90 (m, 3H), 7.71 (d, J = 8.0 Hz, 2H), 7.45 (d, J = 8.2
7.67 (d, J = 7.9 Hz, 2H), 7.50−7.37 (m, 3H), 7.26 (d, J = 8.3
Hz, 1H), 5.41 (s, 2H), 4.82 (s, 1H), 3.74 (m, 1H), 3.25−3.16
(m, 4H), 2.92 (m, 4H), 2.68 (s, 4H), 2.36 (s, 3H), 1.75 (s,
Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H), 5.39 (s, 2H), 3.58 (s, 4H),
3
2
+
.40−3.34 (m, 4H), 2.90 (s, 4H), 2.61 (s, 4H), 2.44 (s, 4H),
.31 (s, 3H). HRMS (ESI): calcd. for m/z C H ClN O , [M
H] 551.2644, found 551.2639. HPLC (80% methanol in
water): t = 3.196 min, 98.91%.
1
3
2H), 1.35 (s, 2H). C NMR (75 MHz, DMSO-d ): δ 169.15,
28
35
8
2
6
+
150.72, 146.37, 143.10, 141.25, 140.25, 135.48, 133.96, 127.93,
126.81, 125.58, 122.83, 122.12, 121.07, 65.96, 54.46, 49.62.
R
+
3
′-((5-Amino-6-chloropyrimidin-4-yl)amino)-4′-(4-meth-
HRMS (ESI): calcd. for m/z C H ClN O , [M + H]
2
7
32
7
2
ylpiperazin-1-yl)-N-(3-morpholinopropyl)-[1,1′-biphenyl]-4-
522.2378, found 522.2379. HPLC (90% methanol in water):
tR = 3.965 min, 96.50%
1
carboxamide (41). Yield: 49%. mp 210−212 °C. H NMR
(
300 MHz, DMSO-d ): δ 8.53−8.42 (m, 1H), 8.28 (d, J = 2.8
(3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-4′-(4-meth-
6
Hz, 1H), 8.23 (s, 1H), 7.96−7.90 (m, 3H), 7.70 (d, J = 8.4 Hz,
ylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)(4-hydroxy-4-methylpi-
2
2
4
3
H), 7.46−7.42 (m, 1H), 7.26 (d, J = 8.3 Hz, 1H), 5.36 (s,
H), 3.57 (t, J = 4.6 Hz, 4H), 3.32−3.30 (m, 2H), 2.87 (t, J =
.7 Hz, 4H), 2.51−2.49 (m, 4H), 2.37−2.32 (m, 6H), 2.24 (s,
peridin-1-yl)methanone (47). Yield: 45%. mp 185−187 °C.
1
H NMR (300 MHz, DMSO-d ): δ 8.16 (s, 2H), 7.80 (s, 1H),
6
7.57 (d, J = 7.9 Hz, 2H), 7.37−7.31 (m, 3H), 7.17 (d, J = 8.3
Hz, 1H), 5.34 (s, 2H), 4.37 (s, 1H), 3.23−3.03 (m, 4H), 2.83
(s, 4H), 2.60 (s, 4H), 2.27 (s, 3H), 1.50−1.37 (m, 4H), 1.06
(s, 3H). HRMS (ESI): calcd. for m/z C H ClN O , [M +
H), 1.74−1.70 (m, 2H). HRMS (ESI): calcd. for m/z
+
C H ClN O , [M + H] 565.2801, found 565.2801. HPLC
2
9
37
8
2
(
80% methanol in water): t = 3.947 min, 96.00%.
3
R
28 34
7
2
+
′-((5-Amino-6-chloropyrimidin-4-yl)amino)-4′-(4-meth-
H] 536.2535, found 536.2533. HPLC (80% methanol in
ylpiperazin-1-yl)-N-(tetrahydro-2H-pyran-4-yl)-[1,1′-biphen-
water): t = 4.567 min, 95.52%.
R
1
yl]-4-carboxamide (42). Yield: 76%. mp 218−220 °C. H
3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-N-isopropyl-
NMR (300 MHz, DMSO-d ): δ 8.35 (d, J = 7.7 Hz, 1H), 8.26
4′-(4-methylpipera-zin-1-yl)-[1,1′-biphenyl]-4-carboxamide
6
1
(
2
(
4
1
1
1
3
5
s, 1H), 8.20 (s, 1H), 8.01−7.82 (m, 3H), 7.71 (d, J = 8.2 Hz,
H), 7.46 (d, J = 8.1 Hz, 1H), 7.28 (d, J = 8.4 Hz, 1H), 5.44
s, 2H), 4.01 (s, 1H), 3.88 (d, J = 10.5 Hz, 2H), 3.42−3.34 (m,
(48). Yield: 64%. mp 184−186 °C. H NMR (300 MHz,
DMSO-d ): δ 8.27−8.22 (m, 2H), 7.91 (d, J = 8.1 Hz, 2H),
6
7.71 (s, 1H), 7.59 (d, J = 8.0 Hz, 2H), 7.43−7.30 (m, 2H),
7.04 (d, J = 8.2 Hz, 1H), 5.54 (s, 2H), 4.12 (s, 1H), 3.33 (s,
4H), 3.07 (s, 4H), 2.82 (s, 3H), 1.18 (s, 6H). HRMS (ESI):
H), 3.00 (s, 6H), 2.51 (s, 3H), 1.90−1.78 (m, 2H), 1.60−
1
3
.56 (m, 2H). C NMR (75 MHz, DMSO-d ): δ 165.68,
6
+
58.03, 150.68, 146.29, 142.80, 140.19, 133.91, 133.69, 128.48,
26.54, 125.59, 122.98, 122.34, 121.04, 66.66, 54.46, 46.22,
2.92. HRMS (ESI): calcd. for m/z C H ClN O , [M + H]
22.2379, found 522.2379. HPLC (80% methanol in water): tR
4.383 min, 95.49%.
3
calcd. for m/z C H ClN O, [M + H] 480.2279, found
2
5
30
7
480.2276. HPLC (80% methanol in water): t = 3.601 min,
R
+
96.77%.
27
32
7
2
3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-
N,N-dimethyl-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-
carboxamide (49). The same procedure as that for 39. White
=
′-((5-Amino-6-chloropyrimidin-4-yl)amino)-N,N-dimeth-
1
yl-4′-(4methylpipera-zin-1-yl)-[1,1′-biphenyl]-4-carboxa-
solid, yield 77%. mp 158−160 °C. H NMR (300 MHz,
1
mide (43). Yield: 79%. mp 142−145 °C. H NMR (300 MHz,
DMSO-d ): δ 8.03 (s, 1H), 7.71 (s, 1H), 7.60−7.46 (m, 4H),
6
DMSO-d ): δ 8.24 (s, 1H), 7.70 (s, 2H), 7.56−7.41 (m, 5H),
7.40 (t, J = 8.5 Hz, 1H), 7.00 (d, J = 8.6 Hz, 1H), 5.49 (s, 2H),
2.99−2.98 (m, 6H), 2.87 (s, 4H), 2.38 (s, 4H), 2.18 (s, 3H).
6
7
2
+
.02 (s, 1H), 5.57 (s, 2H), 3.19−2.97 (m, 10H), 2.75 (s, 4H),
+
.43 (s, 3H). HRMS (ESI): calcd. for m/z C H ClN O, [M
HRMS (ESI): calcd. for m/z C H ClFN O, [M + H]
24
28
7
24 27
7
+
H] 466.2117, found 466.2121. HPLC (80% methanol in
water): t = 2.489 min, 95.56%.
484.2202, found 484.2203. HPLC (80% methanol in water):
tR = 3.448 min, 95.30%.
R
(
3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-4′-(4-meth-
(3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-
4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)-
(morpholino)methanone (50). The same procedure as that
ylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)(4-methylpiperazin-1-
1
yl)methanone (44). Yield: 57%. mp >250 °C. H NMR (300
1
MHz, DMSO-d ): δ 8.54 (d, J = 13.6 Hz, 2H), 7.90 (s, 1H),
for 39. White solid, yield 85%. mp 145−148 °C. H NMR
6
7
8
4
3
.68 (d, J = 7.9 Hz, 2H), 7.45 (d, J = 7.7 Hz, 2H), 7.39 (d, J =
.4 Hz, 1H), 7.27 (d, J = 8.1 Hz, 1H), 5.35 (s, 2H), 3.61 (s,
H), 3.35 (s, 4H), 2.98−2.90 (s, 4H), 2.64 (s, 4H), 2.34 (s,
H), 2.30 (s, 3H). HRMS (ESI): calcd. for m/z C H ClN O,
(300 MHz, DMSO-d ): δ 8.05 (s, 1H), 7.72 (s, 1H), 7.54 (dd,
6
J = 8.5, 5.4 Hz, 4H), 7.41 (t, J = 8.5 Hz, 1H), 7.03 (d, J = 8.6
Hz, 1H), 5.48 (s, 2H), 3.61 (s, 4H), 3.34 (s, 4H), 2.98 (s, 4H),
1
3
2.65 (s, 4H), 2.38 (s, 3H). C NMR (75 MHz, DMSO-d ): δ
27
33
8
6
8
239
https://doi.org/10.1021/acs.jmedchem.1c00091
J. Med. Chem. 2021, 64, 8221−8245

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1
1
69.20, 156.80, 150.45, 145.58, 138.73, 137.73, 136.78, 134.93,
3.01−2.96 (m, 6H), 2.63−2.60 (m, 4H), 2.37 (s, 3H), 2.21−
2.18 (m, 4H), 2.07−2.02 (m, 4H). HRMS (ESI): calcd. for m/
28.97, 128.95, 127.93, 127.82, 125.43, 121.23, 121.08, 115.70.
+
+
HRMS (ESI): calcd. for m/z C H ClFN O , [M + H]
z C H ClFN O, [M + H] 553.2607, found 553.2606. HPLC
26
29
7
2
28 34
8
5
=
26.2128, found 526.2122. HPLC (80% methanol in water): tR
3.560 min, 98.66%.
′-((5-Amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4′-
4-methylpiperazin-1-yl)-N-(2-morpholinoethyl)-[1,1′-bi-
phenyl]-4-carboxamide (51). The same procedure as that for
(80% methanol in water): t = 3.168 min, 97.33%.
R
N-(3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-
4′-(4-methylpipera-zin-1-yl)-[1,1′-biphenyl]-4-yl)-2-(tetrahy-
dro-2H-pyran-4-yl)acetamide (56). The same procedure as
3
(
1
that for 33. White solid, yield: 38.6%. mp >250 °C. H NMR
1
3
9. White solid, yield: 75%. mp 199−202 °C. H NMR (300
(300 MHz, DMSO-d ): δ 8.24 (s, 1H), 8.11 (s, 1H), 7.80 (s,
6
MHz, DMSO-d ): δ 8.48 (t, J = 5.5 Hz, 1H), 8.09 (s, 1H), 7.90
1H), 7.40 (s, 4H), 7.15−7.12 (m, 1H), 6.73 (d, J = 7.5 Hz,
1H), 5.42 (s, 2H), 3.55−3.50 (m, 4H), 3.53 (t, J = 4.6 Hz,
4H), 2.48 (s, 4H), 2.26 (s, 3H), 2.15 (d, J = 7.0 Hz, 2H),
2.03−2.01 (m, 1H), 1.70−1.64 (m, 4H). HRMS (ESI): calcd.
6
(
d, J = 8.1 Hz, 2H), 7.71 (s, 1H), 7.59 (d, J = 7.9 Hz, 2H),
7
3
2
1
1
5
.41 (t, J = 8.5 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 5.50 (s, 2H),
.57 (s, 4H), 3.41−3.38 (m, 2H), 2.91 (s, 4H), 2.49 (s, 4H),
.42 (s, 6H), 2.21 (s, 3H). C NMR (75 MHz, DMSO-d ): δ
67.95, 166.21, 150.46, 149.96, 145.53, 138.60, 138.27, 133.78,
28.80, 127.90, 125.45, 116.36, 115.49, 99.99, 66.63, 65.69,
7.81, 54.94, 53.74, 50.43, 37.00. HRMS (ESI): calcd. for m/z
13
+
for m/z C H ClFN O , [M + H] 554.2441, found 554.2440.
6
28 33
7
2
HPLC (80% methanol in water): t = 3.524 min, 99.10%.
R
3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-N,N, 2′-tri-
methyl-4′-(4-methylpi-perazin-1-yl)-[1,1′-biphenyl]-4-car-
boxamide (57). The same procedure as that for 54. White
+
C H ClFN O , [M + H] 569.2550, found 569.2548. HPLC
2
8
34
8
2
1
(
80% methanol in water): t = 3.634 min, 98.88%.
solid, yield: 44%. mp 165−167 °C. H NMR (300 MHz,
R
3
′-((5-Amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4′-
4-methylpiperazin-1-yl)-N-(3-morpholinopropyl)-[1,1′-bi-
phenyl]-4-carboxamide (52). The same procedure as that for
DMSO-d ): δ 8.09 (s, 1H), 7.87−7.85 (m, 2H), 7.80 (s, 1H),
6
(
7.58 (d, J = 7.4 Hz, 2H), 7.10 (d, J = 7.5 Hz, 1H), 6.86 (d, J =
7.5 Hz, 1H), 5.49 (s, 2H), 3.01−2.98 (m, 6H), 2.87 (s, 4H),
1
3
9. White solid, yield: 65%. mp 202−205 °C. H NMR (300
2.38 (s, 4H), 2.18 (s, 3H), 2.09 (s, 3H). (EI-MS): m/z 480.2
+
MHz, DMSO-d ): δ 8.46 (t, J = 5.7 Hz, 1H), 7.96 (s, 1H), 7.81
[M ] . HPLC (80% methanol in water): t = 3.543 min,
6
R
(
d, J = 8.2 Hz, 2H), 7.31 (t, J = 8.6 Hz, 2H), 6.90 (d, J = 8.7
Hz, 1H), 5.40 (s, 2H), 3.47 (t, J = 5.4 Hz, 4H), 3.21−3.19 (m,
H), 2.80 (s, 4H), 2.28−2.22 (m, 10H), 2.09 (s, 3H), 1.60 (t, J
7.1 Hz, 2H). 13C NMR (75 MHz, DMSO-d ): δ 166.21,
54.80, 150.45, 149.97, 145.55, 138.63, 138.21, 133.89, 128.77,
27.88, 125.44, 124.46, 124.21, 122.23, 122.13, 121.16, 121.05,
96.37%.
(5′-((5-Amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-
4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)-
(morpholino)methanone (58). The same procedure as that
for 49. White solid, yield: 78%. mp 136−138 °C. H NMR
(300 MHz, DMSO-d ): δ 8.09 (s, 1H), 7.93 (d, J = 8.7 Hz,
2
=
6
1
1
1
1
6
15.46, 66.59, 56.48, 54.99, 53.76, 50.51, 46.02, 38.19, 26.38.
1H), 7.82 (s, 1H), 7.59 (s, 2H), 7.51 (d, J = 7.8 Hz, 2H), 7.12
(d, J = 8.6 Hz, 1H), 5.40 (s, 2H), 3.61 (s, 4H), 2.97 (s, 4H),
2.72 (s, 4H), 2.54−2.51 (m, 4H), 2.41 (s, 3H). HRMS (ESI):
+
HRMS (ESI): calcd. for m/z C H ClFN O , [M + H]
29
36
8
2
5
=
83.2706, found 583.2707. HPLC (80% methanol in water): tR
3.601 min, 97.46%.
3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-
′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)(4-ethylpi-
perazin-1-yl)methanone (53). The same procedure as that for
+
calcd. for m/z C H ClFN O , [M + H] 526.2128, found
2
6
29
7
2
(
526.2130. HPLC (80% methanol in water): t = 4.046 min,
96.85%.
5′-((5-Amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4′-
(4-methylpiperazin-1-yl)-N-(2-morpholinoethyl)-[1,1′-bi-
phenyl]-4-carboxamide (59). The same procedure as that for
R
4
1
3
9. White solid, yield: 65%. mp 229−232 °C. H NMR (300
MHz, DMSO-d ): δ 8.05 (s, 1H), 7.71 (s, 1H), 7.57 (d, J = 8.2
Hz, 2H), 7.47 (d, J = 8.1 Hz, 2H), 7.39 (t, J = 8.5 Hz, 2H),
7
4
2
C H ClFN O, [M + H] 553.2587, found 553.2596. HPLC
(
6
1
49. White solid, yield: 78%. mp 199−201 °C. H NMR (300
.00 (d, J = 8.7 Hz, 1H), 5.49 (s, 2H), 3.61 (s, 2H), 2.91 (s,
MHz, DMSO-d ): δ 8.47 (s, 1H), 8.09 (s, 1H), 7.95−7.92 (m,
6
H), 2.38−2.34 (m, 10H), 2.20 (s, 3H), 1.17 (t, J = 7.1 Hz,
3H), 7.81 (s, 1H), 7.62 (d, J = 7.9 Hz, 2H), 7.11 (d, J = 12.4
Hz, 1H), 5.39 (s, 2H), 3.58 (t, J = 4.6 Hz, 4H), 3.41−3.40 (m,
4H), 2.92 (s, 4H), 2.56 (s, 4H), 2.45 (s, 4H), 2.29 (s, 3H).
H), 1.01 (t, J = 7.1 Hz, 2H). HRMS (ESI): calcd. for m/z
+
2
8
34
8
+
80% methanol in water): t = 3.794 min, 97.08%.
HRMS (ESI): calcd. for m/z C H ClFN O , [M + H]
R
28 34
8
2
N-(3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-
569.2550, found 569.2548. HPLC (80% methanol in water): tR
= 4.049 min, 98.64%.
4
2
′-(4-methylpipera-zin-1-yl)-[1,1′-biphenyl]-4-yl)tetrahydro-
H-pyran-4-carboxamide (54). The same procedure as that
5′-((5-Amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4′-
(4-methylpiperazin-1-yl)-N-(3-morpholinopropyl)-[1,1′-bi-
phenyl]-4-carboxamide (60). The same procedure as that for
1
for 33. White solid, yield: 37%. mp >250 °C. H NMR (300
MHz, DMSO-d ): δ 9.56 (s, 1H), 8.25 (s, 1H), 7.80−7.77 (m,
6
1
3
(
=
2
H), 7.41−7.39 (m, 2H), 7.07 (dd, J = 7.5, 5.7 Hz, 1H), 6.68
49. White solid, yield: 78%. mp 204−206 °C. H NMR (300
d, J = 7.3 Hz, 1H), 5.43 (s, 2H), 3.52−3.49 (m, 4H), 3.34 (t, J
4.6 Hz, 4H), 2.70−2.67 (m, 1H), 2.44 (s, 4H), 2.22 (s, 3H),
.06−1.99 (m, 2H), 1.85−1.78 (m, 2H). HRMS (ESI): calcd.
MHz, DMSO-d ): δ 8.55 (s, 1H), 8.09 (s, 1H), 7.93−7.90 (m,
6
3H), 7.81 (s, 1H), 7.63−7.60 (m, 2H), 7.11 (d, J = 12.3 Hz,
1H), 5.39 (s, 2H), 3.59 (s, 4H), 3.32 (s, 4H), 2.92 (s, 4H),
2.58 (s, 4H), 2.41 (s, 4H), 2.31 (s, 3H), 1.72 (s, 2H). HRMS
+
for m/z C H ClFN O , [M + H] 540.2285, found 540.2276.
2
7
31
7
2
+
HPLC (90% methanol in water): t = 3.656 min, 98.94%.
(ESI): calcd. for m/z C H ClFN O , [M + H] 583.2693,
R
29 36
8
2
N-(3′-((5-Amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-
found 583.2699. HPLC (80% methanol in water): t = 3.885
min, 99.38%.
R
4
′-(4-methylpipera-zin-1-yl)-[1,1′-biphenyl]-4-yl)-1-methyl-
piperidine-4-carboxamide (55). The same procedure as that
5′-((5-Amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-N-
(4-hydroxycyclohexy-l)-4′-(4-methylpiperazin-1-yl)-[1,1′-bi-
phenyl]-4-carboxamide (61). The same procedure as that for
1
for 33. White solid, yield: 35%. mp >250 °C. H NMR (300
MHz, DMSO-d ): δ 9.66 (s, 1H), 8.18 (s, 1H), 7.80 (s, 1H),
6
1
7
6
.63 (d, J = 7.5, 2H), 7.40 (d, J = 7.6, 2H), 7.08−7.05 (m, 1H),
.61 (d, J = 7.5 Hz, 1H), 5.30 (s, 2H), 3.20 (t, J = 4.9 Hz, 4H),
49. White solid, yield: 65%. mp 200−203 °C. H NMR (300
MHz, DMSO-d ): δ 9.67 (s, 1H), 8.06 (s, 1H), 7.92 (d, J = 8.4
6
8
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Article
Hz, 1H), 7.82 (s, 1H), 7.58 (d, J = 7.7 Hz, 2H), 7.47 (d, J =
FAM-based probe, and 20 μL of the tested compounds). The
FP assay (λex = 485 ± 25 nm; λem = 535 ± 25 nm) was
recorded using a SpectraMax GeminiXS (Molecular Devices,
Sunnyvale, CA) instrument. The data were analyzed using
GraphPad Prism ver. 6.0 software. IC50 values represent three
independent replicate determinations ± the standard deviation.
ITC Assay. ITC (MicroCal iTC200) was used to determine
the binding affinities between WDR5 and 63. Proteins with a
concentration of 10 μM were prepared in the assay buffer (PBS
buffer, pH 7.4) and subsequently injected into titration cells,
and compounds with a concentration of 100 μM in the same
assay buffer were in syringes. The whole injection procedure
was set with intervals of 180 s and a stirring speed of 750 rpm
and involved 19 injections in total. To prevent interference and
ensure the accuracy, the first titration of the compound
solution was 0.5 μL. Finally, Origin ver. 7.0 software was used
to analyze the data obtained to determine the binding
parameters, including the enthalpy value (ΔH), the entropy
value (ΔS), and the association constant (K = 1/K ).
7
1
1
.9 Hz, 2H), 7.21 (d, J = 12.2 Hz, 1H), 5.44 (s, 2H), 4.82 (s,
H), 4.01 (s, 2H), 3.52 (s, 2H), 3.16 (s, 8H), 2.78 (s, 3H),
.76 (s, 2H), 1.36 (s, 2H).HRMS (ESI): calcd. for m/z
+
C H ClFN O , [M + H] 554.2441, found 554.2442. HPLC
2
7
31
7
2
(
80% methanol in water): t = 4.057 min, 98.48%.
R
5
′-((5-Amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4′-
(
4-methylpiperazin-1-yl)-N-((tetrahydro-2H-pyran-4-yl)-
methyl)-[1,1′-biphenyl]-4-carboxamide (62). The same
procedure as that for 49. White solid, yield: 75%. mp 189−
1
1
(
91 °C. H NMR (300 MHz, DMSO-d ): δ 8.54 (s, 1H), 8.10
6
s, 1H), 7.94−7.91 (m, 3H), 7.61 (d, J = 8.0 Hz, 2H), 7.11 (d,
J = 12.2 Hz, 1H), 5.40 (s, 2H), 3.87−3.83 (m, 2H), 3.17 (s,
4
1
[
H), 2.92 (s, 4H), 2.57 (s, 4H), 2.30 (s, 3H), 1.81 (s, 1H),
.23 (s, 4H). HRMS (ESI): calcd. for m/z C H ClFN O ,
28
33
7
2
+
M + H] 540.2285, found 540.2284. HPLC (100%
methanol): t = 7.989 min, 99.16%.
R
5
′-((5-Amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-
N,N-dimethyl-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-
a
d
carboxamide (63). The same procedure as that for 49. White
In Vitro MLL HMT Functional Assay. The MLL1
1
solid, yield: 75%. mp 157−159 °C. H NMR (300 MHz,
DMSO-d ): δ 8.01 (s, 1H), 7.83 (dd, J = 8.7, 3.7 Hz, 1H), 7.72
enzymatic reactions were conducted at room temperature for
6
60 min in a 50 μL mixture containing the methyltransferase
(
d, J = 3.6 Hz, 1H), 7.49 (d, J = 7.1 Hz, 2H), 7.45−7.36 (m,
assay buffer (50 mM HEPES, 100 mM NaCl, 1.0 mM EDTA,
and 5% glycerol, pH 7.8), 1 μM (S)-adenosylmethionine
2
2
1
1
1
H), 7.14−7.01 (m, 1H), 5.35 (s, 2H), 3.27−2.87 (m,14H),
1
3
.40 (s, 3H). C NMR (75 MHz, DMSO-d ): δ 170.20,
6
(
SAM), the MLL protein complex (MLL1/WDR5/ASH2/
57.23, 155.28, 150.61, 146.08, 145.70, 144.62, 139.76, 136.28,
36.08, 129.63, 128.86, 128.83, 127.87, 126.85, 125.39, 122.45,
RbBP5/DPY30 = 1:1:1:1:1), and the test compound. These 50
μL reactions were carried out in the wells of a Histone
substrate-precoated plate. Blocking buffer was added to stop
the methylation reactions for 10 min. Then, 100 μL of the
diluted primary antibody was added, and the mixture was
slowly shaken for 60 min at room temperature. As before, the
plate was emptied, washed three times, and shaken with
blocking buffer for 10 min at room temperature. After
discarding the blocking buffer, 100 μL of the diluted secondary
antibody was added. The plate was then slowly shaken for 30
min at room temperature. As before, the plate was emptied,
washed three times, and shaken with blocking buffer for 10
min at room temperature. The blocking buffer was discarded,
and a mixture of the HRP chemiluminescent substrates was
freshly prepared; 100 μL of this mixture was added to each
empty well. Immediately, the luminescence of the samples was
measured in a BioTek SynergyTM 2 microplate reader. The
IC50 values were calculated using nonlinear regression with a
normalized dose−response fit using GraphPad Prism ver. 6.0
08.70, 108.51, 54.04, 48.91, 35.26, 29.29, 22.57. HRMS
+
(
ESI): calcd. for m/z C H ClFN O, [M + H] 484.2022,
24
27
7
found 484.2022. HPLC (80% methanol in water): t = 4.751
R
min, 95.34%.
5
′-((5-Amino-6-chloropyrimidin-4-yl)amino)-2′-methyl-
4
′-(4-methylpiperazin-1-yl)-N-(3-morpholinopropyl)-[1,1′-
biphenyl]-4-carboxamide (64). The same procedure as that
1
for 60, yield: 78%. mp 204−206 °C. H NMR (300 MHz,
DMSO-d ): δ 8.65 (s, 1H), 8.10 (s, 1H), 7.93−7.90 (m, 3H),
6
7
5
4
.80 (s, 1H), 7.66−7.62 (m, 2H), 7.11 (d, J = 12.3 Hz, 1H),
.39 (s, 2H), 3.59 (s, 4H), 3.32 (s, 4H), 2.92 (s, 4H), 2.58 (s,
H), 2.41 (s, 4H), 2.31 (s, 6H), 1.72 (s, 2H). HRMS (ESI):
calcd. for m/z C H ClN O , [M + H] 579.2944, found
+
20
39
8
2
5
9
79.2950. HPLC (80% methanol in water): t = 3.830 min,
8.82%.
R
Molecular Docking. The crystal structure of WDR5 (PDB
SMR and 4QL1) was downloaded from the Protein Data
3
Bank (PDB). All the compounds and protein structures were
imported to Discovery Studio (DS) ver. 3.0, and the
conformations were generated with the protocols “Prepare
Protein” and “Prepare Ligands”, respectively. Molecular
docking was performed using the CDOCKER tool, and the
protein residues around the identified critical residues for
WDR5-OICR-9429 were defined as the binding sites. The
docking process was conducted with the default parameters
unless otherwise mentioned. The high fitness score model was
selected to analyze the binding model.
software. IC values represent three independent replicate
50
determinations ± the standard deviation.
AlphaScreen Assay. To verify whether compound 63 had
a good MLL1 HMT selectivity, the AlphaScreen assay was
used to evaluated the compound’s activity on other
methyltransferases (G9a, Dot1L, EZH2, PRMT5, and
SET8). The AlphaScreen IgG kit provides the necessary
reagents for the test. The test was performed in a 384-well
white plate (PerkinElmer). The buffer solution contained 10
mM PBS, compound 63, SAM, and proteins, and the solution
was mixed at room temperature for 30 min. After the
enzymatic reaction, 5 μL of receptor beads and 5 μL of the
substrate antibodies were added to the reaction system, and
the system was incubated for 30 min at room temperature.
Then, 10 μL of AphaScreen streptavidin-coupled donor beads
were added. The 384-well white plate was investigated in an
AlphaScreen microplate reader (EnSpire Alpha 2390 Multi-
label Reader, PerkinElmer).
Fluorescence Polarization Competition Assay. The
binding affinities of the synthesized compounds were tested
using a fluorescence polarization (FP)-based competitive
3
0,40,47
binding assay, which was described earlier.
The
experimental buffer contained 100 mM KH PO , 25 mM
2
4
KCl, and 0.01% Triton X-100 at pH 6.5. 10mer-Thr-FAM was
used as a probe, and the reactions were performed in 384
black-well plates (no. 3575, Corning) with a total reaction
volume of 60 μL (20 μL of the WDR5 protein, 20 μL pf the
8
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Article
In Vitro Antiproliferative Assay. Antiproliferative
activities of all the compounds against different cell lines
were determined using the CCK8 assay kit. Cells were seeded
as 3000−6000 cells per well in 96-well plates and treated with
an inhibitor for 72 h at different concentrations in the culture
medium. Cell viability was determined by the CCK8 kit
In Vivo Pharmacokinetic Assay. Animal experiments
were conducted according to protocols approved by the
Institutional Animal Care and Use Committee of China
Pharmaceutical University. Six healthy male SD rats, which
were 6−8 weeks old and weighed 220 ± 30 g, were supplied by
Joinn Laboratories. The rats (three for p.o. and three for i.v.)
were administered with 63. For p.o. administration, the test
compound (10 mg/kg) was administered orally; for i.v.
administration, the test compound (2 mg/kg) was adminis-
tered intravenously. Blood samples were collected at times of
(
Beyotime, Jiangsu, China) according to the manufacturer’s
instructions.
Western Blot Analysis. Anti-H3K4me1 (ab8895), anti-
H3K4me2 (ab32356), anti-H3K4me3 (ab8580), and anti-H3
0
, 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after administration
for the i.v. group and at times of 0, 0.25, 0.5, 1, 2, 4, 6, 8, and
4 h after administration for the p.o. group. The resulting
(
ab1791) were purchased from Abcam (Abcam, UK). The
isolation of cell fractions and Western blotting were performed
28,35
2
as detailed previously.
Briefly, the extracts were separated
plasma samples were stored at −80 °C and analyzed by LC-
MS/MS. Pharmacokinetic parameters shown are the mean ±
SEM.
by SDS-PAGE and then electrotransferred to PVDF
membranes (PerkinElmer, Northwalk, CT). Membranes were
blocked with 1% BSA for 1 h, followed by incubation with a
primary antibody at 4 °C overnight. Then, the membranes
were washed and treated with a DyLight 800-labeled secondary
antibody at 37 °C for 1 h. The membranes were screened
through the odyssey infrared imaging system (LI-COR,
Lincoln, NE).
In Vivo Antitumor Activity. Animal experiments were
conducted according to protocols approved by the Institutional
Animal Care and Use Committee of China Pharmaceutical
University. six to eight week old female BALB/c nude mice
were purchased from Shanghai Laboratory Animal Center,
China Academy of Sciences, raised in air-conditioned rooms
with 12 h of light per day, and fed with standard laboratory
RNA Extraction and qRT-PCR Analysis. The exper-
imental procedure for quantitative real-time RT-PCR analysis
6
35,47
food and water. Approximately 5 × 10 MV4-11 cells
was previously reported.
Briefly, the total RNA of MV4-11
suspended in PBS (0.2 mL) were injected into the flanks of
cells was extracted from the treated cells using the TRIzol
reagent (Invitrogen). Then, the RNA was converted to cDNA
by reverse transcriptase (PrimeScript RT reagent kit)
according to the manufacturer’s instructions. Quantitative
real-time RT-PCR analyses of Hoxa9 and Mesi1 were
performed by using the StepOne System Fast real-time PCR
system (Applied Biosystems). The mRNA expression of all
genes was normalized against the GAPDH expression.
3
nude mice. After the tumors grew to 100−150 mm , all the
mice were randomized into four groups (six mice for each
group) and treated with the vehicle (0.9% saline solution) or
compound 63 (40, 80 , or 120 mg/kg) by oral administration
daily for 21 days. Tumor volume and body weight were
determined every other day by measuring the two
perpendicular diameters of the tumors and using the formula
2
V = length (mm) × width (mm) /2. After 21 days of
Cell Culture. MV4-11, MOLM-13, K562, and HUVEC cell
lines were purchased from the Cell Resource Center of
Shanghai Institute for Biological Sciences, Chinese Academy of
Sciences. The MV4-11 and K562 cells were cultured in a
modified IMDM medium with 10% fetal bovine serum (FBS)
and penicillin/streptomycin in a 37 °C incubator with 5%
treatment, the mice were sacrificed, and the tumors were
dissected and weighed. The drug efficacy was assessed by
calculating the GI% = [1 − (TV − TV )/(CV − CT )] ×
t
0
t
0
1
00%, where TV and CV are the tumor volume of treatments
t t
group and the control group measured at each time point,
respectively, and TV and CV are the tumor volume of
0
0
CO . MOLM-13 cells were cultured in an RPMI-1640 medium
2
treatment group and the control group monitored at the
beginning, respectively. The drug efficacy was also assessed by
calculating TGI% = (mean Wcontrol − mean Wtumor)/mean
Wcontrol × 100%.
supplemented with 10% (v/v) FBS. HUVEC cells were
maintained in a modified DMEM medium that was
supplemented with 10% FBS.
Physicochemical Properties Experiments. The distri-
bution coefficients (CLogP) were computationally calculated
using the ADMET Predictor 10.0 software. The permeability
coefficients were determined via double-sink PAMPA on a
PAMPA Explorer instrument (pION). The test compounds
were diluted with the donor buffer at pH 7.4, placed in the
donor side, and allowed to permeate to the acceptor side
through the artificial membrane over 4 h of incubation at 25
ASSOCIATED CONTENT
■
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00091.
Molecular formula strings (CSV)
°
C. After incubation, the “sandwich” plate was separated.
Schematic diagram for targeting the WDR5−MLL1 PPI
Then, the acceptor and donor solutions were each measured
with a UV plate reader. Lastly, the permeability value was
calculated by pION software.
for the treatment of leukemia; docking study for
1
13
compound 40; and representative H NMR, C NMR,
HRMS and HPLC spectra for the target compounds
(PDF)
In Vitro Microsomal Stability Assay. The metabolic
stability of 63 was assessed using human and rat liver
microsomes. 63 was preincubated with human and rat
microsomes (0.7 mg/mL) at 1 μM for 5 min at 37 °C in
PBS buffer (100 mM, pH 7.4) before 1 mM NADPH was
added to initiate the reaction. Then, cold acetonitrile was
utilized to precipitate the protein. Lastly, the samples were
centrifuged for further analysis by LC-MS/MS.
Dock study of compound 40 (PDB )
Dock study of compound 52 and 60 (PDB)
Dock study of compound 17 (PDB)
Dock study of compound 29 (PDB)
8
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Article
AUTHOR INFORMATION
Corresponding Authors
Xiaoke Guo − Jiangsu Key Laboratory of Drug Design and
Optimization and State Key Laboratory of Natural
Medicines and Department of Medicinal Chemistry, School of
Pharmacy, China Pharmaceutical University, Nanjing
Academic Program Development of Jiangsu Higher Education
Institutions, and the Natural Science Research Project of
Jiangsu Higher Education (20KJB350001).
■
ABBREVIATIONS USED
■
ALL, acute lymphoid leukemia; AML, acute myeloid leukemia;
FP, fluorescence polarization; GI, growth inhibition; HMT,
histone methyltransferase; H3K4, histone 3 lysine 4; ITC,
isothermal titration calorimetry; MLL1, mixed lineage
leukemia 1; PPI, protein−protein interaction; TGI, tumor
weight growth inhibition; WBM, WDR5 binding motif;
WDR5, WD repeat-containing protein 5; WIN, WDR5
interaction motif
2
10009, China; orcid.org/0000-0002-7558-5504;
Email: kexin95@126.com
Qidong You − Jiangsu Key Laboratory of Drug Design and
Optimization and State Key Laboratory of Natural
Medicines and Department of Medicinal Chemistry, School of
Pharmacy, China Pharmaceutical University, Nanjing
2
10009, China; orcid.org/0000-0002-8587-0122;
Email: youqd@163.com
Zhengyu Jiang − Jiangsu Key Laboratory of Drug Design and
Optimization and State Key Laboratory of Natural
Medicines and Department of Medicinal Chemistry, School of
Pharmacy, China Pharmaceutical University, Nanjing
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Author Contributions
These authors contributed equally to this work.
∇
(
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Sun, Q.; Olejniczak, E. T.; Clark, T.; Dey, S.; Lorey, S.; Alicie, B.;
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
4
(
40−452.
■
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We are thankful for the financial support of the National
Natural Science Foundation of China (nos. 81872799,
J. G.; Howes, J. E.; Sai, J.; Yuh, J. P.; Camper, D.; Alicie, B. M.;
Alvarado, J.; Nikhar, S.; Payne, W.; Aho, E. R.; Bauer, J. A.; Zhao, B.;
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of Jiangsu Province of China (no. BK 20191321), Funda-
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2
632018ZD14), the National Science & Technology Major
Project “Key New Drug Creation and Manufacturing Program”
of China (2018ZX09711002), the Double First Class
Innovation Team of China Pharmaceutical University
2
(
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NOTE ADDED AFTER ASAP PUBLICATION
Published ASAP on June 9, 2021; Table 5 revised on June 14,
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