Rational Design and Evaluation of 6-(Pyrimidin-2-ylamino)-3,4-dihydroquinoxalin-2(1 H)-ones as Polypharmacological Inhibitors of BET and Kinases

Article abstract of DOI:10.1021/acs.jmedchem.0c00962

Cancer exhibits diverse heterogeneity with a complicated molecular basis that usually harbors genetic and epigenetic abnormality, which poses a big challenge for single-target agents. In the current work, we proposed a hybrid strategy by incorporating pharmacophores that bind to the acetylated lysine binding pocket of BET proteins with a typical kinase hinge binder to generate novel polypharmacological inhibitors of BET and kinases. Through elaborating the core structure of 6-(pyrimidin-2-ylamino)-3,4-dihydroquinoxalin-2(1H)-one, we demonstrated that this rational design can produce high potent inhibitors of CDK9 and BET proteins. In this series, compound 40 was identified as the potential lead compound with balanced activities of BRD4 (IC50 = 12.7 nM) and CDK9 (IC50 = 22.4 nM), as well as good antiproliferative activities on a small cancer cell panel. Together, the current study provided a new method for the discovery of bromodomain and kinase dual inhibitors rather than only being discovered by serendipity.

Full text of DOI:10.1021/acs.jmedchem.0c00962

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Article
Rational Design and Evaluation of 6-(Pyrimidin-2-
ylamino)-3,4-dihydroquinoxalin-2(1H)-ones as
Polypharmacological Inhibitors of BET and Kinases
Kaikai Lv, Weicong Chen, Danqi Chen, Jie Mou, Huijie Zhang, Tiantian Fan, Yanlian
Li, Danyan Cao, Xin Wang, lin Chen, Jingkang Shen, Dongsheng Pei, and Bing Xiong
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00962 • Publication Date (Web): 11 Aug 2020
Downloaded from pubs.acs.org on August 11, 2020
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Rational Design and Evaluation of
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6-(Pyrimidin-2-ylamino)-3,4-dihydroquinoxalin-2(1H)-ones as
Polypharmacological Inhibitors of BET and Kinases
Kaikai Lv1,2,#, Weicong Chen3,#, Danqi Chen1, Jie Mou4,*, Huijie Zhang1,2, Tiantian
Fan1,2, Yanlian Li1, Danyan Cao1, Xin Wang1, Lin Chen1, Jingkang Shen1, Dongsheng
Pei3,*, Bing Xiong1,*
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1Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zuchongzhi Road, Shanghai, 201203, China
2University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing
100049, China
3Department of Pathology, Xuzhou Medical University, 209 Tongshan Road, Xuzhou,
221006, China
4Jiangsu Key Laboratory of New drug and Clinical Pharmacy, School of Pharmacy,
Xuzhou Medical University, 209 Tongshan Road, Xuzhou, 221006, China
#these authors contributed equally
*corresponding authors: B.X.: bxiong@simm.ac.cn; D.P.: dspei@xzhmu.edu.cn;
J.M.: 100002009710@xzhmu.edu.cn
ABSTRACT
Cancer exhibits diverse heterogeneity with a complicated molecular basis that usually
harbors genetic and epigenetic abnormality, which poses a big challenge for
single-target agents. In the current work, we proposed a hybrid strategy by
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incorporating pharmacophores that bind to the acetylated lysine binding pocket of
BET proteins with typical kinases hinge binder to generate novel
polypharmacological inhibitors of BET and kinases. Through elaborating the core
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structure
of
6-(Pyrimidin-2-ylamino)-3,4-dihydroquinoxalin-2(1H)-one,
we
demonstrated that this rational design can produce high potent inhibitors of CDK9 and
BET proteins.In this series, compound 40 was identified as the potential lead
compound with balanced activities of BRD4 (IC50=12.7 nM) and CDK9 (IC50=22.4
nM), as well as good antiproliferative activities on a small cancer cell panel. Together,
current study provided a new method for the discovery of bromodomain and kinases
dual inhibitors rather than only discovered by serendipity.
KEYWORD: Polypharmacology; Bromodomain; Kinases; Antitumor;
Dihydroquinoxalin-2(1H)-one
INTRODUCTION
Complex diseases, such as cancer, can be attributed to the dysregulation of multiple
signal pathways and physiological processes, which are hardly cured by the specific
modulation of a single target. Polypharmacological inhibitor as an intriguing
therapeutic option is gaining a lot of attention in current anticancer drug development1,
2. It has several advantages with respect to single-target drugs and combination
therapies3, and the most important aspects can be summarized as follows: 1) a
multitarget drug could avoid the drug-drug interactions issues; 2) it may demonstrate
a more predictable pharmacokinetic profile compared with combination therapies; 3)
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it guarantees the simultaneous presence in the tissues to reduce the acute and delayed
toxicities in comparison with a combination therapy; 4) it usually has synergistic
effects to reduce the administration dosage and therefore may avoid the associated
toxicity issues of a single-target agent.
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Started with imatinib, a Bcr-Abl inhibitor for cancer treatment, developing kinases
inhibitors for cancer therapies have stimulated tremendous efforts and resulted in
magnificent successes with more than 50 kinases inhibitors into the market. However,
application of these targeted kinases drugs are impeded by acquired resistance which
usually involves drug-induced mutations and epigenetic reprogramming. Based on the
recent advance of epigenetic drug development, especially the bromodomain
inhibitors, there are a lot of clinical trials are ongoing and the investigations indicated
that these epigenetic drugs can rewire the gene expression and regulation network to
execute its anticancer effects. Besides, there are many studies indicated that
combining bromodomain and extra-terminal domain (BET) inhibitors with kinases
inhibitors can enhance the antitumor effects through simultaneous targeting the
abnormal signaling kinases pathways and epigenetics-related cellular
reprogramming4-6. Moreover, the combined use of BET inhibitor could reduce the
probability of acquired resistance of kinases drugs. Giving the distinct benefits of
combining BET inhibitors with kinases inhibitors, considerable efforts are dedicated
to developing the multi-targeted molecules of BET and kinases7-9. However, to the
best of our knowledge, all of them are discovered by serendipity (compounds 1-3 of
Figure 1),10, 11 as exemplified by the PLK1 and BET dual inhibitor BI-253612, or
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optimized from these known inhibitors (4).
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To overcome this limitation, in this work, we presented a rational strategy by
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hybridising the acetylated lysine (KAc) mimic of BET inhibitors with the hinge
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binder motif extracted from typical kinases inhibitors. We demonstrated that this
design can provide a new opportunity to elegantly develop bromodomain-kinases
multi-targeted agents for antitumor treatment.
O
O
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O
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N
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O
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H
N
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H
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O
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O
1 (BI-2536)
BRD4 Ki=56 nM
PLK1 Ki=0.22 nM
2 (ERK5-IN-1)
BRD4IC50=217 nM
ERK5 IC50=162 nM
N
N
N
N
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O
S
NH
N
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H
O
S
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N
H
O
H
4
3 (TG101209)
BRD4 IC50=130 nM
JAK2 IC50=6 nM
BRD4 Kd=44 nM
ALK IC50=17 nM
Figure 1. The representative dual bromodomain-kinases inhibitors.
RESULTS AND DISCUSSION
Inhibitor Design Strategy
In the past two decades, more than 50 kinases inhibitors have been approved by FDA
to enter into drug market13 and most of them are ATP-competitive inhibitors with a
conserved hydrogen-bond acceptor motif, usually pyridine or pyrimidine, to interact
with the backbone of hinge segment in kinases catalytic domains14. Since the ATP
binding site of kinases is located at the cleft formed by the N-lobe and the C-lobe, it
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also has a large solvent-accessible are that can accommodate diverse and large
chemical groups. Based on the binding mode of kinases inhibitors and the biological
importance of drug combination of BET and kinases inhibitors, we assumed that
hybridize kinases essential hinge binder with the well-characterized acetylated
lysine mimic from bromodomain inhibitors may provide new polypharmacological
inhibitors with unique biological effects as anticancer drugs. Previously, the
dihydropteridinone in BI-2536 was identified as an essential component for
developing dual BET-kinase inhibitors. However, this moiety is both a kinase hinger
binder and BET protein’s pharmacophore, which makes the optimization highly
interlocked. Therefore, it limits the target scope and currently only PLK1 and ALK
were found to be the targets of this chemotype11, 12, 15. We have discovered a series of
dihydroquinoxalin-2(1H)-one derivatives as potent BET inhibitors, and found the
scaffold dihydroquinoxalin-2(1H)-one is a selective and potent acetylated lysine
mimic, which can form several direct and water-bridged hydrogen bonds with BRD4.
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Also, the 3,5-dimethylisoxazole motif first reported by Hewings et al.,16, 17
is
considered a general pharmacophore for the KAc binding pocket and has been
incorporated into many series of BET and non-BET inhibitors. We proposed to use
the hybridization strategy ,which will incorporate the acetylated lysine binding moiety
with a typical kinases hinge binder to discover novel dual BET-kinases inhibitors.
This strategy could enable us to select more biological relevant kinases as
BET-kinases ploypharmacological targets. To test the hypothesis of hybrid strategy,
we designed four prototype compounds (5-8 in Table 1) by combining
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pyrimidin-2-amine group with two acetylated lysine mimic initialized a docking study
to check the binding feasibility of them in the first bromodomain of BRD4
(BRD4-BD1). The docking result indicated that the compounds can bind to the
acetylated lysine binding pocket of BRD4 and maintain the essential hydrogen-bond
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interactions
between
the
residue
Asn140
and
the
selected
dihydroquinoxalin-2(1H)-one18-20 or 3,5-dimethylisoxazole motif21-25 (see the Figure
S1 for the calculated binding conformation). Based on this feasibility to maintain the
key interactions in the KAc binding site of BRD4-BD1 and the existence of large
solvent-accessible subpocket in kinase ATP binding site able to accommodate the
BET pharmacophore, we synthesized these four compounds to subject to the
biological tests of BRD4 and several kinasess to verify this approach.
Structure-Activity Relationship
As shown in Table 1, two compounds (5 and 6) containing 3,5-dimethylisoxazole did
not show more than 50% inhibition ratio at the concentration of 1 μM in the
BRD4-BD1 fluorescence polarization (FP) assay. However, the compounds (7 and 8)
with dihydroquinoxalin-2(1H)-one scaffold showed encouraging binding activities
towards BRD4, with the IC50 values below 200 nM. Based on this initial results, we
prepared two derivatives (9 and 10) to check the possibility of optimization. Clearly,
they demonstrated that the modification at the benzene group could remain a good
binding activity to BRD4, especially at the meta-position of the benzene group (10
with IC50=160.5 nM). Previous studies have identified the synergistic effect of BRD4
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with kinases such as CDKs26, PI3Ks27and Aurora kinases28, 29. Therefore, we tested
the inhibition effects of compounds 7-10 on these selected kinases. As listed in Table
1, compounds 7-10 showed differential activities to four selected kinases. In detail,
compounds 7-9 have good activities towards CDK9 but with no activity on CDK6,
indicating they have the potential to be developed into selective inhibitors. Besides
the promising activity on CDK9, compound 8 also showed good activity on PI3Kα
and compound 9 showed good activity on Aurora kinase A. For the comparison, we
also synthesized the compounds 11-14 by substituting the pyrimidine core with
benzene motif, which eliminate the hinge region binding capability and therefore
should not inhibit the kinases activity. As expected, compounds 11-14 did not
show any activity on these four kinases, further demonstrating that the pyrimidine, a
common hinge binder acting as hydrogen-bond acceptor, is very important for the
binding to kinases. Contrarily, since the pyrimidine in compounds 7-10 is not
essential for binding to BRD4, the substituents (11-14) still have good binding
activities on BRD4, as indicated by the similar IC50 values from BRD4-BD1 FP assay
relative to the corresponding compounds 7-10.
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Table 1. The proof-of-concept of molecular designa
R1
N
N
O
X
X
N
H
9
10
11
12
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Inhibition Values(%)
CDK6 PI3Kα
BRD4
BD1
CDK9
100nM
Aurora A
1μM 100nM
Compd.
X
R1
IC50(nM)
1μM
1μM 100nM 1μM 100nM
N
5
6
2.2%@1μM
N
N
H
O
N
/
N
N
37.7%@1μM
N
N
H
O
N
*
7
N
N
N
N
C
C
C
C
193.5±49.5
83.0±13.0
101.9
98.0
103.5
71.8
1.3
83.4
94.5
67.9
25.2
-6.1
2.2
4.8
7.7
8.1
2.8
0.9
0.7
-0.1
-3.3
-0.8
0.2
2.3
-0.8
1.8
2.7
-1.4
2.3
22.9
82.4
0.1
0.0
37.5
-2.1
14.1
-4.8
-4.1
-2.6
-0.1
67.4
10.6
4.0
*
N
8
43.7
86.8
54.4
-1.5
14.2
-0.8
-1.8
*
9
477.0±61.0
160.5±15.5
357.5±21.5
77.3±2.3
37.4
8.7
O
*
10
11
12
13
14
63.1
6.1
CN
*
-3.4
-6.1
1.1
*
N
0.7
-1.0
3.0
*
820.5±138.5
666.5±56.5
3.8
-3.0
2.0
O
*
-4.1
5.2
-3.0
CN
aAll BRD4-BD1 IC50 values and Kinases inhibition values are mean values from at
least two independent experiments.
To assist the further optimization, we solved the cocrystal structure of compound 7
bound to the BD1 domain of BRD4. As shown in Figure 2A, the
dihydroquinoxalin-2(1H)-one scaffold situated at the same position in the acetylated
lysine binding site with respect to our previously reported crystal structure and makes
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essential hydrogen-bond interactions with the conserved residue Asn140. The binding
conformation of compound 7 in the solved structure adopted an U shape by rotating
the 4-phenylpyrimidin-2-amine group to the residue Asp145. Comparison with our
previous solved crystal structure, the residue Asp145 slightly receded to eliminate the
steric clash with the ligand (Figure 2B).
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A)
B)
C)
D)
Figure 2. The cocrystal structure of compound 7 (cyans) and compound 40 (blue) in
complex with BRD4-BD1 (PDB entry number 6LIH and 6LIM, respectively). A)
binding mode of compound 7 in the KAc binding pocket. Asn140,Tyr97,Trp81 and
Asp145 are represented as stick model and hydrogen bonds are represented by dashed
lines. B) Superimposing the structure of compound 7 to our previous crystal structure
(PDB entry number：6JI5) (green). C) Superimposing the structure of compound 7
and compound 40. D) the interactions between compound 40 and WPF pocket that is
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represented as surface diagram.
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Based on the cocrystal structure, we found that adding a small group at the 2- or
3-position of the benzene group could extend the ligand into the WPF subpocket,
which may enhance the binding affinity with BRD4. Since compounds 7-10 all
showed activities on CDK9, as a proof-of-concept project, we decided to
simultaneously optimize the compounds as dual inhibitors of BET and CDK9. To
probe the binding mode of 7 with CDK9, we docked the compound into the ATP
binding site of CDK9 by utilizing the crystal structure 6GZH as the template30. From
the docking model (Figure 3A), compound 7 adopted similar binding interactions to
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the
ligand
A86
([4-[[4-[5-(cyclopropylmethyl)-1-methyl-pyrazol-4-yl]-5-fluoranyl-pyrimidin-2-yl]am
ino]cyclohexyl]azanium) in the original crystal structure, forming the essential
hydrogen-bond interactions with the hinge residue Cys106 and stretching the benzene
group upwards(Figure 3B). The dihydroquinoxalin-2(1H)-one ring extends to the
solvent-accessible area with the isopropyl group reverting to the vicinity of the
benzene group. According this predicted binding mode, substitutions on benzene
group also are feasible to enhance the binding activity on CDK9, which is consistent
with the idea proposed for the BRD4 optimization.
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A)
B)
C)
D)
Figure 3. The binding conformation of compounds 7, 21 and 40 in CDK9 ATP
binding site from the docking study. The protein was shown in cartoon mode and the
residues of binding site were shown in stick mode. A) predicted conformation of
compound 7 (cyans) bound to the ATP binding site of CDK9. It can form two
hydrogen bonds with hinge residue Cys106. B) superimposition of compound 7 with
original ligand A86 (green) in the crystal structure 6GZH. C) predicted conformation
of compound 21 (orange) bound to the ATP binding site of CDK9. D) predicted
conformation of compound 40 (blue) bound to the ATP binding site of CDK9.
As shown in Table 2, various small functional groups were added to the different
position of the benzene group. According to the analysis of the cocrystal structure of
compound 7 with BRD4-BD1, the 4-position is at the vicinity of the protein residue
Asp145. Therefore, substitutions at the 4-position of the benzene may cause a steric
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crash to reduce the binding activities to BRD4, which is consistent with the low
activities of compounds 16, 19 and 22, especially of compound 19 (IC50=5850.0 nM).
And compounds with substitutions at 3-position of benzene group also showed
decreased activities except compound 21 with a NH2 group that may form
electrostatic interaction with the nearby negative-charge residue Asp145. Since the
2-position faces the WPF subpocket, the substitutions at 2-position generally have less
detriment effect on the binding with BRD4. Compounds 15-24 were tested in CDK9
enzymatic inhibition assay and all of them except 17 showed moderate to high
inhibitory activities. Among them, compound 21 showed very good activity towards
CDK9, with an IC50 value of 5.9 nM. We docked compound 21 into the ATP binding
site of CDK9 (Figure 3C), and found that its binding conformation is very similar to
the previous predicted binding mode of compound 7, only the amino group entering
into a hydrophilic subpocket formed by residues Lys48, Glu66 and Asp167, which
may create favorable electrostatic interactions.
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Considering the promising inhibitory activities of compound 21 on BRD4 and
CDK9,we further explore the structure-activity relationship (SAR) around the
3-position of the benzene group in order to obtain potent dual-inhibitors.
Table 2. The exploration of different substituents on benzene groupa
4
3
R2
2
N
N
O
N
N
N
H
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BRD4
CDK9
Compd.
R2
BD1
IC50(nM)
6
IC50(nM)
7
8
9
15
16
3-OCH3
4-OCH3
965.0±135.0
534.5±9.5
126.4 ± 20.1
31.7 ± 0.9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1.0%@1μM
17
18
19
20
21
22
23
24
2-CH3
3-CH3
271.0±12.0
759.5±42.5
5850.0±150.0
251.5±73.5
119.5±30.5
480.0±48.0
496.5±82.5
156.2±34.3
1.5%@100nM
195.8 ± 5.2
488.5± 34.9
39.2 ± 2.8
5.9±0.5
4-CH3
2-NH2
3-NH2
4-NH2
48.9 ±8.7
46.9 ± 6.2
578.2 ±97.9
2-OCH3-4-F
2-CH3-4-F
aAll BRD4-BD1 IC50 values and CDK9 IC50 values are mean values from at least two
independent experiments. The BET selective inhibitor JQ-1 and CDK9 selective
inhibitor Dinaciclib were used as the positive control in our enzymatic assay,
respectively exhibiting an IC50 value of 41.5 nM and 3.0 nM.
On the basis of compound 21, we synthesized compounds 25-30 by substituted
various groups on the 3-NH2 of benzene group. Compound 25 with the added methyl
group maintained the binding activity on BRD4 relative to 21. Compounds with
slightly large groups such as ethyl or isopropyl (26 and 27) decreased the potency
about two folds. the substituents larger than isopropyl (28-30) are not tolerable at this
position as they showed a dramatic loss in BRD4 binding activities. Since compounds
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with 2-methyl group on the benzene (17 and 24) also showed good binding activities
on BRD4, we prepared compounds 31 and compound 32 to further explore the
binding motif around the WPF subpocket. Comparison of compounds 31 and 32, it
was found the methyl at the ortho-position of amino group in compound 31 may
extend into WPF pocket and make favorable Van Der Waals (VDW) interactions with
the residues lining this subpocket, which slightly increased the binding affinity with
BRD4-BD1. Compound 33 with the tetrahydroquinoline motif at this part exhibited
high potency to BRD4 (IC50=27.0 nM), indicating this added piperidine ring indeed
entered into the WPF subpocket. We also investigated the activity of these
compounds on CDK9. The result showed that adding the methyl group on the
amine ,the potency of compound 25 on CDK9 decreased about 4-fold relative to
compound 21. Substituted with larger groups further reduced the inhibitory activity on
CDK9, which generally follows the similar trends in the BRD4 binding assay.
Interestingly, although compound 33 coupled a large bicyclic group on the
pyrimidine, it still has a good activity to CDK9 (IC50=67.0 nM), prompting us that the
bicycle motifs could be used to further probe the dual inhibitiors.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. The exploration of 3-NH2 on benzene groupa
H
N
R3
N
N
O
N
N
N
H
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9
BRD4
CDK9
Compd.
R3
BD1
IC50 (nM)
IC50 (nM)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
25
26
27
Me
Et
173.4±11.8
319.2±21.2
268.1±37.1
22.0±1.0
37.8 ± 2.0
50.9 ± 1.5
iPr
*
28
29
30
11%@10μM
121.9± 25.2
168.8± 11.3
*
13%@10μM
47.8%@1μM
Ph
/
8.8%@100nM
NH2
N
N
O
31
32
33
115.0±14.0
236.0±98.0
27.0±5.3
182.9 ± 4.4
151.2 ± 0.8
67.0±3.7
N
N
N
H
NH2
N
O
N
N
N
N
H
H
N
N
N
O
N
N
N
H
aAll BRD4-BD1 IC50 values and CDK9 IC50 values are mean values from at least two
independent experiments.
From the above-mentioned SAR study, compounds 34-40 with bicycles coupled on
the pyrimidine group were synthesized. From the BRD4 binding assay, it was
revealed that compounds containing [6,6]-bicyclic ring system (37-40) have better
activities than [6,5]-bicycle compounds (34-36), especially compounds 38 and 40
with IC50 values lower than 20 nM. We checked the CDK9 enzymatic inhibition of
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8
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these compounds, and found that compound 35 with NH group at 3-position of
benzene group has superior inhibitory activity on CDK9 (IC50=13.8 nM). Fortunately,
the BRD4-potent compound 40 also showed high activity to CDK9, with an IC50
value of 81.6 nM. Comparing compounds 38 with 40, clearly the nitrogen atom in
bicycle played an important role for the binding interactions. We utilized the
molecular docking method to examine the possible binding mechanism. Based on the
docking model(Figure 3D), the nitrogen atom in isoquinoline ring of compound 40
located at the same position as the 2-nitrogen atom in parazole of the original ligand,
which is at the vicinity of the residue Lys48 that participates in the salt bridge
interaction with DFG motif. It was noticed that, as comparison with the predicted
binding conformation of compounds 7 and 21, the dihydroquinoxalin-2(1H)-one ring
in compound 40 adopted a different orientation to alleviate the possible clash with the
larger isoquinoline group.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Since the R4 substitutions on pyrimidine are usually utilized to enhance the selectivity
on CDK9 (as exemplified in crystal structure 6GZH, the ligand has a fluorine atom at
this position, Figure 3B), then we prepared compounds 41-43 and examined them in
this two assays. From the Table 4, we can find that although 41 and 42 retained the
activity on CDK9, they showed about 5-fold reduction in BRD4 binding activities.
While compound 43 remained similar high binding activity on BRD4-BD1 as
compound 40, it did not maintain the inhibitory activity on CDK9. Taking together,
compound 40 showed high potency on both BRD4-BD1 and CDK9 proteins, and
were selected as a promising dual inhibitor for further profiling the binding mode,
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selectivity, and cellular activity.
6
7
8
9
Table 4. The exploration of bicycles on the WPF subpocketa
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
R5
R4
N
N
O
N
N
N
H
BRD4
BD1
CDK9
Compd.
34
R4
R5
IC50(nM)
IC50(nM)
H
N
58.6%@1μM
H
329.1±40.9
288.0±26.0
7.6%@100nM
*
H
N
35
H
13.8±0.9
*
N
N
36
37
38
39
40
41
42
H
H
H
H
H
F
167.8±35.5
75.9±13.2
15.2±2.6
110.0±7.0
79.8±11.2
81.6±4.5
19.8±1.5
22.4±2.3
23.9±2.5
23.0±1.0
*
N
*
N
*
N
25.3±3.9
*
*
N
12.7±4.6
N
81.3±15.7
58.1±14.9
*
N
Cl
*
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N
43
Me
19.1±1.3
100.0±4.0
*
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aAll BRD4-BD1 IC50 values and CDK9 IC50 values are mean values from at least two
independent experiments.
The cocrystal structure of compound 40 bound to BRD4-BD1 was solved with X-ray
crystallography. By superimposing on the cocrystal structure of compound 7, we can
realize that the bicycle ring on the pyrimidine extended into the WPF subpocket,
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making favorable VDW interactions with this hydrophobic area as expected. The
essential dihydroquinoxalin-2(1H)-one scaffold established the same hydrogen bond
interactions with the residues of acetylated lysine binding pocket: one hydrogen bond
between the oxygen atom of amide of compound 40 and the side chain of Asn140,
one water-bridged hydrogen bond between the same oxygen and the phenol group of
residue Tyr97. The notable difference between the binding mode of compound 40 and
of compound 7 lies in the pyrimidine ring, which is slightly backward to eliminate the
steric clash with the helix around the residue Asp145 and Ile146, and therefore make
a new hydrogen bond with the residue Lys91.
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13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Selectivity profile of compound 40
Although compound 40 was optimized as a dual inhibitor of BRD4 and CDK9, we
examined its selectivity in a diverse kinases panel provided by Eurofins Scientific and
in-house
purified
bromodomains.
From
the
result
of
DiscoveRx
diversity-kinases-panel assay that contains 54 kinases, at 1 μM concentration
compound 40 showed the highest percent inhibition of CDK9, although 5 others also
displayed more than 80% inhibition (Figure 4). Two other CDK kinases (CDK1 and
CDK2) were also strongly inhibited by compound 40 with the inhibition ratio around
90% at 1 μM concentration(Table S1). So we obtained the detailed IC50 value(Table
S2) of CDK2 (168.9 nM), and showed that compound 40 has about 8-fold selectivity
towards CDK9 over CDK2. While for CDK6, compound 40 only showed about 22%
inhibition at tested concentration. We also tested the compound on seven in-house
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prepared bromodomains, including BRD4-BD1, BRD4-BD2, BRD2-BD1,
BRD2-BD2 and non-BET bromomain proteins EP300, CECR2 and BRD931. As
demonstrated in Figure 4 and Table S4, it is a potent BET inhibitor and showed less
than 12% inhibition activity to three other bromodomains at 1 μM concentration.
Overall, the selectivity measurement of compound 40 revealed that the design strategy
by hybridising acetylated lysine mimic with hinge binder of the kinases is feasible to
provide new options for developing polypharmacological inhibitors of bromodomain
and kinases. With the detailed analysis of cocrystal structure of 40, we could find that
although the benzene motif of the bicycle ring extends into the WPF pocket, the other
side of bicycle points to the solvent-accessible part. Therefore, we think that it is
possible to obtain more selective inhibitors by modifying the bicycle ring motif, as it
is the determinant for kinases selectivity.
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. the selectivity profile of compound 40 on selected bromodomains and
DiscoveRx diversity-kinases-panel. All inhibition values are mean values from at
least two independent experiments at the concentration of 1μM. And for kinases
selectivity assay, only kinases with inhibition ratio of more than 50% were depicted in
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the figure 4 and whole data were listed in supporting material(Table S1).
6
7
8
9
Cellular Activity and Western Blot
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. Proliferation inhibition of 22 cancer cell lines by Compound 40.The cutoff
line (red dotted-line) represents IC50 values were larger than 500 nM. Precise value
were listed in SI Table S5.
To further examine the cellular activity of compound 40, total 22 cancer cell lines
were screened to identify the sensitive cancer cells. As shown in Figure 5 and Table
S5, compound 40 showed differential effects on the tested cell lines. After 96 hours of
treating compound 40, several cancer cell lines were remarkably sensitive to
compound 40, but others were not. This discrepancy may be due to the heterogeneity
of tumor cells. Lars Tögel et al.32 detected molecular subgroups of colon cancer,
which included microsatellite instability (MSI)/ microsatellite stable (MSS) and CpG
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island methylator phenotype (CIMP), mutation status of commonly altered oncogenes
and tumor suppressor genes in colorectal cell lines; and they found that colorectal cell
lines with MSI were significantly more sensitive to JQ1 compared to MSS cell lines.
However, no association between JQ1 response and cell lines wild type or mutant for
RAS/BRAF, PIK3CA/PTEN or TP53 was observed in their study, nor was there a
correlation between JQ1 response and CIMP. The positive control compounds of
BRD4 inhibitor JQ-1 and CDK9 inhibitor Atuveciclib were applied to these cell lines,
and the results (SI Table S5) indicated the dual inhibitor 40 could enhance the
antiproliferative activities, suggesting a synergy effect in the cellular context. Hence,
the relatively sensitive solid tumor cell line HCT-116 was selected to detect the
cellular activity of drug targets.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. HCT-116 cells were treated with the indicated concentration of JQ1,
Atuveciclib or compound 40 and examined BRD4, c-Myc, CDK9, p-RNA II CTD,
P-AR and GAPDH protein levels by Western Blotting. The molecular weight of
different isoform of BRD4, c-Myc and CDK9 were shown at the right of the bands.
To test the effects of the drug on activities of CDK9 and BRD4, HCT-116 cell line
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was pretreated with compound JQ1, Atuveciclib or compound 40 (0 nM, 50 nM, 100
nM, 250 nM or 500 nM for 24 h respectively) and then immunoblotted. JQ1 is the
positive control of BRD4 and Atuveciclib is the positive control of CDK9. CDK9 and
BRD4 facilitate the transition from abortive to productive elongation by
phosphorylating the CTD (C-terminal domain) of the large subunit of RNA
polymerase II33. Regulation of c-Myc gene transcription appears to be the one
function of BRD4 targeted by identified cancer therapeutic compounds activates
related downstream target34, 35. CDK9 also specifically phosphorylates the AR on
Ser81 and regulates AR transcriptional activity33. Western blot analysis showed that
compounds JQ1 and compound 40 caused a dose-dependent reduction in c-Myc
protein levels in HCT-116 cell line. Meanwhile, Atuveciclib caused a dose-dependent
decrease in p-AR and p-RNAP II CTD but there was no significant trending effect on
other proteins (The normalized quantified WB data was shown in SI Figure S4). As
shown in Figure 6, compound 40 demonstrated good inhibitory activities on CDK9
and BRD4 similar to JQ1 in vitro and large doses could block CDK9 and BRD4
activities for a short period of time.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 5. Pharmacokinetic parameters of compound 40 in ICR mice a
Kel
(h-1)
T1/2
(h)
Tmax
(h)
Cmax
(ng·mL-1)
AUC0-t
(h·ng·mL-1)
AUC0-inf
(h·ng·mL-1)
route
IP
dose
3 mg/kg
1.45
0.480
0.194
1366
757
759
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a Mice (n = 3); Parameters were calculated from composite mean plasma concentration-time data.
5
6
7
8
9
To complete the valuation of the lead compound 40, we also investigated its in vivo
pharmacokinetics (PK) parameters in the mice model. From the in vivo PK test,
compound 40 dosed at 3 mg/kg showed a moderate plasma exposure with the AUC0-t
value of 757 h·ng·mL-1, indicating that the compound could be considered as a
potential lead structure for further optimization.
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21
22
23
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25
26
27
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29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Chemistry
Scheme 1. The Synthesis of Compound 7a
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6
H
N
NO2
NH
NO2
F
O
a
c
b
Br
Br
Br
N
H
OH
7
8
9
O
44
46
45
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
O
N
O
N
O
O
e
d
O
N
N
Br
N
Br
N
H
47
49
48
O
N
O
R
N
O
g
N
f
h
H2N
N
N
N
H
N
50
51a, 51b, 51c, 51d
OH
Cl
N
R
N
N
O
R
N
O
N
N
i
j
N
N
H
N
N
H
52a, 52b, 52c, 52d
53a, 53b, 53c, 53d
51a, 52a, 53a R = H
51b, 52b, 53b R = F
51c, 52c, 53c R = Cl
51d, 52d, 53d R = Me
N
O
N
N
N
N
H
7
aReagents and conditions: (a) D-alanine, K2CO3, EtOH: H2O = 3:1, 80 °C, 8 h; (b)
K2CO3, Na2S2O4, H2O, 60 °C, overnight; (c) acetone, phenylsilane,
dibutyltindichloride, THF, rt, 10 h; (d) NaH, 0 °C
- rt, 30 min and then
iodomethane, DMF，rt, 2 h; (e) tert-Butyl carbamate，CS2CO3 , Pa(OAc)2, Xphos,
1.4-dioxane, 85 °C, 2 d;（f）HCl in 1.4-dioxane, rt, 24 h; (g) AcOH, 1.4-dioxane,
0
100 °C, 8 h; (h) HCl in 1.4-dioxane ,100 °C, 8 h; (i) POCl3, 60 C, 8 h; (j) K2CO3,
Pd(dppf)2Cl2, 1.4-dioxane : H2O = 4：1, 80 °C,4 h;
Compound 7 is synthesized according to Scheme 118. 45 is obtained from nucleophilic
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substitution of 4-bromo-2-fluoro-1-nitrobenzene with D-alanine, followed by
cyclization to afford 46. 48 was prepared by reductive amination with acetone and
then alkylation with iodomethane. Then, palladium catalyzed C-N coupling reaction
was utilized to obtain 49. After removing the Boc protecting group ,a nucleophilic
substitution reaction was adopted to give the intermediate 51a-51d. Sequential acid
hydrolysis and chlorination produced the compounds 53a-53d. A palladium catalyzed
Suzuki coupling reaction between aryl chloride (53a) and phenylboronic acid yieldes
end-product 7.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2. The Synthesis of compounds 11-14a
R
Br
R
55a, 11 R=Ph
N
N
O
a
b
55b, 12 R=4-Pyridyl
55c, 13 R=2-OMe-Phenyl
55d, 14 R=3-cyano-Phenyl
N
H
NH2
NH2
55a, 55b, 55c, 55d
54
11, 12, 13, 14
aReagents and conditions:(a) phenylboronic acid, K2CO3, Pd(dppf)2Cl2, 1.4-dioxane :
H2O = 4:1, 80 °C, 8 h; (b) 48, t-BuONa, Pa2(dba)3, Xphos, 1.4-dioxane, 100 °C,
overnight;
Compounds 11-14, as depicted in Scheme 2, were obtained throught palladium
catalyzed Suzuki and Buchwald-Hartwig coupling reactions.
Scheme 3. The Synthesis of compounds 5 and 6a
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7
O
N
N
N
O
N
O
Br
I
HN
Br
8
a
b
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
56
57
58
OH
Cl
N
N
N
N
c
N
O
d
N
e
O
HN
HN
59
60
R
N
HN
N
N
5 R=Ph
6 R=4-Pyridyl
O
5, 6
aReagents and conditions: (a) NaHCO3, Pa(PPh3)4, 1.4-dioxane : H2O = 4：1, 70 °C, 2
h; (b) 2-Amino-4-methoxypyrimidine ， t-BuONa, Pa2(dba)3, Xphos, 1.4-dioxane,
0
100 °C, 8 h; (c) HCl in 1.4-dioxane, 100 °C, 8 h; (d) POCl3, 60 C, 8 h; (e)
K2CO3, Pd(dppf)2Cl2, 1.4-dioxane : H2O = 4：1, 80 °C, 4 h;
The synthetic routes of 3,5-dimethyl-isoxazole derivatives 5 and 6 are outlined in
Scheme 3. Like the preparation of compounds 11-14, Compound 58 was obtained
throught two successive palladium catalyzed
coupling reactions. Acid
hydrolysis ,which could remove the OMe group, and chlorination afford intermediate
60.As mentioned above,the final step also was Suzuki couploing rection between 60
and the corresponding boronic acid derivatives.
CONCLUSIONS
Over the past decades, many kinases inhibitors have entered into the market for the
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2
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5
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7
8
9
treatment of cancers. However, cancer is notorious for its high heterogeneity and
usually harboring genetic and epigenetic abnormality, therefore there is an urgent
need of polypharmacological agents to simultaneously targeting multiple
dysfunctional pathways. BET inhibitors have entered into clinical trials and
demonstrated its antitumor effects and promising synergistic effects by combining
with current kinases inhibitors. Although there are several dual inhibitors of BET and
kinases have been discovered by serendipity, it remains a challenge for the
researchers to rational design dual inhibitors of certain kinases and BET. In the
present work, we adopted the hybrid strategy by incorporating typical kinases hinge
binder with pharmacophores that bind to the acetylated lysine pocket of BET
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
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41
42
43
44
45
46
47
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49
50
51
52
53
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55
56
57
58
59
60
proteinsand
successfully
obtained
a
series
of
6-(Pyrimidin-2-ylamino)-3,4-dihydroquinoxalin-2(1H)-ones. Through structure-based
drug design, we investigated the structure-activity relationship and demonstrated that
it is feasible to discover potent inhibitors of dual BET and kinases, providing not only
a lead compound of CDK9 and BET, but also the promising approach for the
community to design specific kinase and BET dual inhibitors.
EXPERIMENTAL SECTION
General Chemistry
1H NMR spectra were recorded by using a Varian Mercury-400 high performance
digital FT-NMR spectrometer with tetramethylsilane (TMS) as an internal standard.
Low-resolution mass spectra were obtained with a Finnigan LCQ Deca XP mass
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spectrometer using a CAPCELL PAK C18 (50 mm × 2.0 mm, 5 μm) or an Agilent
ZORBAX Eclipse XDB C18 (50 mm × 2.1 mm, 5 μm) in positive or negative
electrospray mode. The purity of compounds was determined by high-performance
liquid chromatography (HPLC) and confirmed to be more than 95%, monitored by
UV absorption at 214 and 254 nm. TLC analysis was carried out with glass precoated
silica gel GF254 plates. TLC spots were visualized under UV light. All solvents and
reagents were used directly as obtained commercially unless otherwise noted.
10
11
12
13
14
15
16
17
18
19
20
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22
23
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56
57
58
59
60
N-(3-(3,5-dimethylisoxazol-4-yl)phenyl)-4-phenylpyrimidin-2-amine (5). Step
1: 4-(3-bromophenyl)-3,5-dimethylisoxazole (57). To a solution of compound 56 (4 g,
14.2 mmol) and 3,5-Dimethylisoxazole-4-boronic acid (1 g，7.1 mmol) in 1.4-dioxane
and H2O (4:1 50 mL) was added NaHCO3 (1.2 g ， 14.2 mmol), the mixture was
bubbled with N2 for 5 min, then Pa(PPh3)4 (820 mg, 0.71 mmol) was added, the
mixture was further bubbled with N2 for 5 min, the mixture was heated to 70 °C for 2
h. The reaction was monitored by TLC. Upon completion, the reaction mixture was
diluted with water and extracted with DCM (3 × 100 mL), The combined organic
layers were washed with brine, dried over Na2SO4 and concentrated by evaporation
under reduced pressure. Purification by silica gel column chromatography (gradient
elution, gradient 0 to 10% EtOAc/60-90 °C petroleum ether) to afford compound 57
1
(1.237 g, 4.906 mmol, 69.1% yield) as a white solid. H NMR (400 MHz,
Chloroform-d ) δ 7.52 (ddt, J = 8.4, 3.6, 1.5 Hz, 1H), 7.43 (dt, J = 3.5, 1.8 Hz, 1H),
7.34 (td, J = 7.8, 3.1 Hz, 1H), 7.24 – 7.15 (m, 1H), 2.43 (d, J = 3.1 Hz, 3H), 2.29 (d, J
= 3.1 Hz, 3H);
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