Discovery of Novel Dual Histone Deacetylase and Mammalian Target of Rapamycin Target Inhibitors as a Promising Strategy for Cancer Therapy

Article abstract of DOI:10.1021/acs.jmedchem.8b01825

In the present study, a series of novel dual-target histone deacetylase (HDAC) and mammalian target of rapamycin (mTOR) inhibitors were designed and synthesized using pyrimidine-pyrazolyl pharmacophore to append HDAC recognition cap and hydroxamic acid as a zinc-binding motif. Among them, 12l was the optimal lead compound with potent inhibition activities against mTOR and HDAC1 with half-maximal inhibitory concentration of 1.2 and 0.19 nM, respectively. Western blot confirmed that 12l could upregulate acetylation of H3 and α-tubulin and downregulate mTOR-related downstream mediators. 12l could also stimulate cell cycle arrest in G₀/G₁ phase and induce tumor cell apoptosis. 12l showed comparable antitumor activity with the combination medication in MM1S xenograft model with a tumor growth inhibitory rate of 72.5%, without causing significant loss of body weight and toxicity. All of the results indicated that 12l could be a promising dual target inhibitor for treating hematologic malignancies.

Full text of DOI:10.1021/acs.jmedchem.8b01825

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Article
Discovery of a Novel Dual Histone Deacetylases (HDACs)
and Mammalian Target of Rapamycin (mTOR) Target
Inhibitor as a Promising Strategy for Cancer Therapy
Yong Chen, Xue Yuan, Wanhua Zhang, Minghai Tang, Li Zheng, Fang
Wang, Wei Yan, Shengyong Yang, Yuquan Wei, Jun He, and LiJuan Chen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01825 • Publication Date (Web): 10 Jan 2019
Downloaded from http://pubs.acs.org on January 11, 2019
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Discovery of a Novel Dual Histone Deacetylases (HDACs) and
Mammalian Target of Rapamycin (mTOR) Target Inhibitor as a
Promising Strategy for Cancer Therapy
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a,§
a,§
b,§
a
a
Yong Chen , Xue Yuan , Wanhua Zhang , Minghai Tang , Li Zheng , Fang
a
a
a
a
a,
a,
Wang , Wei Yan , Shengyong Yang , Yuquan Wei , Jun He *, Lijuan Chen *
a
State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy
and Cancer Center, West China Hospital of Sichuan University, Chengdu, 610041,
China.
b
Department of Hematology and Research Laboratory of Hematology, West China
Hospital of Sichuan University, Chengdu, 610041, China.
§
These authors contributed equally and should be considered as co-first author
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Abstract
In the present study, a series of novel dual target HDAC and mTOR inhibitors were
designed and synthesized, using pyrimidine-pyrazolyl pharmacophore to append
HDAC recognition cap and hydroxamic acid as a zinc-binding motif. Among them,
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2l was the optimal lead compound with potent inhibition activities against mTOR
and HDAC1 with IC of 1.2 nM and 0.19 nM. Western blot confirmed that 12l could
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upregulate acetylation of H3 and α-tubulin and downregulate mTOR related
downstream mediators. 12l could also stimulate cell cycle arrest in G /G phase and
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induce tumor cell apoptosis. 12l showed comparable anti-tumor activity with the
combination medication in MM1S xenograft model with TGI of 72.5%, without
causing significant loss of body weight and toxicity. All the results indicated that 12l
could be a promising dual target inhibitor for treating hematologic malignancies.
Introduction
The mammalian target of rapamycin (mTOR) regulates cell growth and survival by
integrating both extracellular and intracellular signals1, 2. These signaling functions
are distributed in distinct multiprotein complexes: mTOR complex 1 (mTORC1) and
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mTOR complex 2 (mTORC2) . mTOR is a downstream mediator of the PI3K/Akt
pathway that is over activated in numerous tumors. mTORC1 is sensitive to
rapamycin, while mTORC2 is resistant to rapamycin because of rapamycin triggering
a negative feedback mechanism by activating survival pathways involving Akt and
eIF4e, which may limit the anti-cancer efficacy1, 4-6. Several dual mTORC1 and
mTORC2 inhibitors, such as AZD80557, 8, Torkinib (PP242) , CC223 , not only
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inhibit multisite eIF4E-bing protein 1 (4E-BP1) phosphorylation but also block
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PI3K/Akt negative feedback . They belong to second-generation ATP competive
inhibitors and more effective than first-generation inhibitors. However, mTOR
resistance mutations, mainly including FRB mutations and kinase domain mutations,
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led to poor therapeutical effects and prognosis in clinical applications . Numerous
reports revealed that histone deacetylases (HDACs) are considered to be among the
most promising targets for cancer therapy, and four HDAC inhibitors have been
approved by FDA for treating hematologic malignancies12-17. Researchers tried to
adopt a combination treatment strategy of mTOR and HDAC inhibitors for treating
relapsed/refractory lymphoma in recent years18, 19. Emerging data indicated that
mTOR inhibitors were more effective when combined with HDAC inhibitors due to
synergy effects18, 20-24. One report demonstrated that panobinostat (LBH-589) and
everolimus (RAD001) combination resulted in enhanced anti-tumor efficacy mediated
by decreasing tumor growth concurrent with augmentation of p21 and p27 expression
and the attenuation of angiogenesis and tumor proliferation via androgen receptor,
c-Myc and HIF-1α signaling16, 22. Moreover, panobinostat could overcome
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rapamycin-mediated resistance by inhibiting Akt signaling through mTORC2 . Such
promising research drew our attention to develop novel and highly effective small
molecule inhibitors of dual mTOR and HDAC targets. Up to now, no dual mTOR and
HDAC target inhibitors are available in clinical or on market. Our research groups
decided to design a series of novel target molecules in order to develop a dual mTOR
and HDAC inhibitor. Many researchers utilized pyrimidine-pyrazolyl analogues as
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stem nucleus to develop several effective mTORC1/mTORC2 inhibitors, such as
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PP242, AZD8055 and Sapanisertib (INK128) . Hydroxamic acid is the most
common zinc binding group (ZBG) moiety in HDAC inhibitors owing to its ability
to reliably chelate active-site zinc ions by far24, 25, such as LBH-589, Vorinostat
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(
SAHA), Ricolinostat (ACY1215) . We thought of designing pyrimidine-pyrazolyl
pharmacophore to append HDAC recognition cap and hydroxamic acid as a zinc
binding motif (Figure 1). A series of compounds were synthesized and evaluated for
the cell inhibition activities and (or) enzymatic inhibition activities. As expected, 12l
potently inhibited mTOR and HDAC1 with IC50 values of 1.2 nM and 0.19 nM,
respectively. Western blot analysis reconfirmed dual inhibition on HDACs and mTOR
kinase in a concentration dependent manner. 12l could stimulate cell cycle arrest in
G /G phase and induce tumor cell apoptosis. And in vivo anti-tumor activity in
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xenograft models suggested that 12l could be a suitable drug candidate for treating
hematologic malignancies.
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mTOR ATP
domain binding
group
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merge key pharmacophores
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OH
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key HDAC
Zn binding group
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Vorinostat
Figure 1. Schematic showing plans for merged mTOR-HDAC pharmacophore taking
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advantage of ATP domain binding group and HDAC zinc binding group1.
Results and Discussion
Chemistry
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The general procedure to synthesize the target molecules 6a-i has been outlined in
Scheme 1. Commercially available 4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (1) as
the starting material reacted with morphline to give 2. Under N atmosphere
2
protection, 2 was treated with morphline/N-methylpiperazine to obtain 3/4 The
compound 3/4 was introduced an aliphatic chain group by reacting with bromoacetate
analogues, which got the corresponding compounds 5a-g and 5h-i (3 was treated with
methyl 3-(4-bromomethyl)cinnamate to get 5j). These compounds were directly
converted into targets molecules 6a-j by NH OH.
2
Scheme 1. Syntheses of compounds 6a-i.a
O
N
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Cl
a
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d
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H
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NH
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OH
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, R=morpholino;
, R=N-methylpiperazine
5a-g, R=morpholino, n=0-6;
5h-i, R=N-methylpiperazine, n=4, 5
6a-g, R=morpholino, n=0-6;
6h-i, R=N-methylpiperazine, n=4, 5
a
Reagents and Conditions: (a) morphline, CH OH, rt, 0.5 h; (b)
3
o
morphline/N-methylpiperazine, DIEA, KI, NMP, 90 C, 8 h; (c) BrCH (CH ) COOEt,
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2 n
o
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In Vitro Cell Growth Inhibitory Effects of Compounds 6a-j. We primarily began
our research by screening the inhibitory activities of these compounds in HCT116 and
Ramos cells. We first tested two concentrations in both cells including 5 μM and 0.5
μM, and the data was shown in Table 1. The inhibition rate below 50 % can be
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considered to be ineffective or inefficient. Obviously, compounds 6a-c showed no
inhibitory activities at two concentrations in HCT116 and Ramos cells. When the
chain length of the linker region became three carbon atoms, compound 6d exhibited
weak inhibitory activity at 5 μM. Similarly, compound 6e slightly improved inhibitory
activity and the inhibition rate was > 60 % at 0.5 μM. Not surprisingly, compound 6f
showed higher efficiency in two cells even at 0.5 μM. However, compound 6g which
contained seven carbon atoms in linker region sharply reduced inhibitory activities at
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.5 μM. Meantime, compounds 6h and 6i also showed equivalent inhibitory activities
in HCT116 cells, especially remarkable inhibitory efficacy in Ramos cells. Compound
j consisting of vinylbenzene instead of aliphatic chain also showed weak activity
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inferior to 6f. These results suggested that six carbon atoms chain of the linker region
was the optimal group to maintain the inhibitory activities in solid and hematological
cancer cells.
Table 1. Cell growth inhibition rate of compounds 6a-j in HCT116 and Ramos cells.
O
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a-g n=0-6
6h-i n=4, 5
6j
inhibition rate (%) a
HCT116
0.5 μM
Compd
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μM
5 μM
0.5 μM
6a
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The inhibition rate values are the means of at least two experiments.
To search the structure and activity relationship (SAR) of cap with the fixed linker
region, we also synthesized compounds 10a-j (Scheme 2). The synthetic route was
similar to Scheme 1, compound 2 reacted with ethyl 7-bromoheptanoate to give 7.
Then 7 was introduced kinds of aromatic nucleus by Suzuki coupling reaction to
obtain 9a-j. These compounds were also directly converted into target molecules
10a-j by NH OH.
2
Scheme 2. Syntheses of compounds 10a-j.a
O
N
O
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O
N
a
b
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H
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9a-j, n=5
n
7, n=5
10a-j, n=5
n
OH
O
O
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O
O
O
H
a
Reagents and Conditions ： (a) Br(CH ) COOEt, Cs CO , DMF, MW, 1 h; (b)
2
6
2
3
o
boronic acid derivatives, Dioxane/EtOH/water(v/v/v, 7/3/4), 80 C, 2 h; (c) NH OH,
2
NaOH, CH OH/CH Cl , 1 h.
3
2
2
In Vitro Cell Growth Inhibitory Effects of Compounds 10a-j. On the basis of the
former researches and discussions, compounds with C-2 position modification were
chosen to further evaluate cell growth inhibitory effects in different cancer cells,
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including HCT116, Raji, MCF-7 and MM1S cells. In view of the inhibition rates of 6f
in HCT116 and Ramos cells, we directly tested the IC values in the four cancer
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cells. Though compound 6f showed high efficacy at 0.5 μM, the highest inhibition
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activity (IC ) in HCT116 was only 89.9 nM and IC values were >100 nM in other
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cancer cells. Satisfactorily acceptably, compound 10a with anilino group at C-2
position exhibited remarkable inhibition activities in four cancer cells. Dramatically,
the IC values toward HCT116 and Raji were 7.0 nM and 7.1 nM, respectively.
5
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However, 10c exhibited equivalent effects in hematological tumors but worse
activities toward solid tumor cells when pyridyl replaced phenyl. When phenyl was
replaced by pyrimidinyl (10b), the inhibition activities sharply declined in tested
tumor cells. And 10f almost lost inhibition activities in comparison with 10a. We can
draw conclusion that phenyl group contributed to increasing inhibition activities
toward these cancer cells. Predictably, 10e showed better inhibitory activities than
10d but inferior to 10a. In addition, amino group played an important role in
maintaining potent inhibition activities statistically. When five-membered heterocycle
groups were introduced at C-2 position (10h, 10i and 10j), the inhibition activities in
hematological tumors still remained at nM level but inferior to 10a. 10a with aniline
group at C-2 position was preferred pilot structure with excellent cell inhibitory
effects in solid and hematological tumor cells. Moreover, the research outcome was in
accordance with our previous conclusion.28
Table 2. IC values in several cancer cells of compounds 10a-j, 6f and SAHA.
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0a-j, n=5
n
OH
N
O
H
a
IC ± SEM , nM
5
0
Compd
R
HCT116
89.9 ± 14.5
7.1 ± 1.8
Raji
MCF-7
MM1S
6
f
158.6 ± 30.6 371.9 ± 14.3 192.3 ± 32.9
1
0a
0b
0c
0d
0e
0f
7.1 ± 2.6
31.7 ± 2.3
7.1 ± 0.2
63.7 ± 6.7
221 ± 5.4
163 ± 6
18.9 ± 1.5
32.6 ± 1.6
8.4 ± 1.9
> 500
NH2
NH2
H2N
N
1
163 ± 10
61.3 ± 8.1
66.9 ± 7.8
38.0 ± 2.0
296.3 ± 62.8
N
1
N
1
44.9 ± 10.4 372.4 ± 52.6
O
O
N
1
29.3 ± 9.7
> 500
70.6 ± 17.5 353.5 ± 83.5
1
> 500
> 500
> 500
N
1
0g
O
S
76.5 ± 7.0
111.4 ± 5.4
168.1 ± 4.3
O
10h
10i
10j
34.9 ± 0.6
106.9 ± 40.6
72.8 ± 2.7
7.7 ± 0.2
15.3 ± 2.1
14.6 ± 0.4
196 ± 7
156 ± 3.6
15.6 ± 1.3
52.3 ± 3.0
35.6 ± 0.4
573 ± 9
O
1
80.4 ± 57.3
NH
S
202 ± 5
354 ± 2
SAHA
882.5 ± 66.5
a
IC = compound concentration required to inhibit tumor cell proliferation by 50%;
5
0
data are expressed as the mean ± SEM from the dose-response curves of at least three
independent experiments.
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In Vitro Enzymatic Inhibitory Effects for PI3Kα and mTOR. Owing to most
HDAC inhibitors acting on hematological cancers and in view of several compounds
showing remarkable inhibitory activities in hematological cancer cells, we decided to
test enzymatic inhibitory effects for mTOR first. Moreover, PI3K kinases are the
upstream signal mediators of mTOR. PI3Kα and mTOR kinases targets were chosen
to test these compounds and data was shown in Table 3. Obviously, all these
compounds hardly inhibited PI3Kα kinase. However, several compounds could
partially inhibit mTOR, such as 10a whose inhibition rate was 95% and 10i with 85%
inhibition rate at 500 nM. Theoretically, 10a may be an efficient mTOR inhibitor
which needed subsequent specific data.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Enzymatic inhibition rate for PI3Kα and mTOR.
inhibition rate (%) at 500 nM a
Compd
PI3Kα
5.9 ± 2.7
11 ± 1.7
17 ± 1.6
15 ± 3.7
1.0 ± 0.1
4.5 ± 2.1
-1.2 ± 0.7
1.5 ± 0.8
-6.2 ± 0.8
-3.0 ± 1.3
16 ± 1.6
1.2 ± 0.7
1.9 ± 0.8
mTOR
-12 ± 2.1
-5.0 ± 0.5
21 ± 0.5
-5.4 ± 4.5
-4.2 ± 0.4
2.2 ± 2.3
4.4 ± 0.7
-1.7 ± 1.6
-0.4 ± 1.4
-5.4 ± 1.7
95 ± 0.2
40 ± 1.1
35 ± 0.9
6
a
6b
6c
6
d
6
e
6
f
6
g
6
h
6
i
6
j
10a
10b
1
0c
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5
5
5
5
5
6
1
0d
0e
0f
0g
0h
0i
10j
3.3 ± 5.7
-1.0 ± 1.5
-0.1 ± 0.3
31 ± 4.6
58 ± 1.0
38 ± 2.3
17 ± 7.4
64 ± 1.5
35 ± 2.9
85 ± 0.0
55 ± 3.2
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1
0
1
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4
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6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8.4 ± 1.1
-3.8 ± 1.0
9.7 ± 5.6
1
a
The inhibition rate values are the means of at least two experiments.
Then IC values of 10a for HDACs and mTOR were evaluated. The results were
5
0
shown in Table 4. As expected, 10a showed excellent inhibition activities toward
HDACs. The IC values were 0.21 nM, 1.6 nM, 4.21 nM for HDAC 1, 2, 3 and 2.43
5
0
nM, 1.43 nM for HDAC 6, 10, respectively. Moreover, the IC values for HDAC 4,
50
5
, 7, 9, and 11 were > 1 μM. The result indicated 10a was a class I and class IIb
selective HDAC inhibitor. 10a also exhibited potent inhibition effect for mTOR
kinase with IC of 40 nM. By far, 10a was selected out to be an acceptable dual
5
0
HDAC and mTOR inhibitor. Improvement of inhibition efficacy for mTOR kinase
need further structure modification and optimization based on lead compound 10a.
Table 4. IC values for HDACs and mTOR of compound 10a.
5
0
Class I
HDAC1 HDAC2 HDAC3 HDAC8 HDAC6 HDAC10 mTOR
0.21 1.60 4.21 35.67 2.43 1.43 40
IC values for enzymatic inhibition of HDACs and mTOR kinase. The IC values
Class IIb
IC ,nM
5
0
a
5
0
50
are the means of at least two experiments, with intra- and inter- assay variations of <
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10%.
As reported, introduction of urea linkage to aniline could improve inhibition efficacy
1
for mTOR kinase . We adopted the similar strategy and 12a-p were designed and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
synthesized. 9a was acylated by triphosgene and then RNH or RNH was added to
2
give urea intermediates 11a-p. These compounds were also directly converted into
target molecules 12a-p by NH OH.
2
Scheme 3. Syntheses of compounds 12a-p.a
O
O
N
O
N
N
N
N
N
a
N
b
N
N
N
N
O
N
N
N
O
N
R
n
n
R
n
N
N
H2N
N
N
H
H
12a-p, n=5
NH
OH
9
a, n=5
O
H
H
11a-p, n=5
O
O
O
O
a
Reagents and Conditions: (a) triphosgene, NEt , CH Cl ; RNH or RNH ; (b)
3
2
2
2
NH OH, NaOH, CH OH/CH Cl , 1 h.
2
3
2
2
To validate our design strategy of improving cell inhibition activities, compounds
12a-p were first evaluated IC values in HCT116, Raji, and MM1S cell lines. The
5
0
data was outlined in Table 5. 12a promoted inhibition efficacy in hematological tumor
cells with IC values of 3.5 nM, 13.4 nM for Raji and MM1S, respectively. When
5
0
ethyl was introduced (12b), the inhibition effect was inferior to 12a and 10a.
However, the inhibition activity of 12c for tested cells with propyl substituent
decreased sharply. The potency performed differently when various short aliphatic
chains were introduced but all inferior to 12a. Additionally, aromatic rings (12g, 12h,
12i, and 12j) were inserted instead of aliphatic chains. 12g and 12h exhibited
equivalent inhibition activities both in solid and hematological tumor cells with
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2
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2
2
2
2
2
2
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6
average IC value of 20 nM. Trialkylamine as one domain of urea was introduced to
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obtain compounds 12k-p. Morpholine substituent derivatives (12k-l) all exhibited
potent anti-tumor activities. However, piperazine substituent derivatives (12n-p)
reduced inhibitory effects than 12k-l.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. IC values against several cancer cells of compounds 12a-p, and SAHA as
5
0
positive control.
O
N
N
N
N
O
N
R
n
OH
N
H
N
H
N
12a-p, n=5
O
H
IC50 a ± SEM, nM
Raji
Compd
R
HCT116
51.6 ± 30.9
61.9 ± 2.8
103.9 ± 1.9
33.9 ± 0.6
MM1S
1
2a
12b
2c
2d
2e
2f
12g
Me
Et
3.5 ± 0.2
13.4 ± 0.3
11.8 ± 0.4
83.4 ± 1.5
15.3 ± 0.4
19.7 ± 0.2
42.5 ± 1.7
19.1 ± 0.4
1
Pro
1
n-butyl
HO
1
440.7 ± 25.4 152.6 ± 24.9 305.4 ± 21.4
HO
1
57.2 ± 24.9
26.1 ± 0.8
24.6 ± 1.3
143.5 ± 14.5
> 500
61.08 ± 5.9
22.1 ± 1.2
19.0 ± 1.4
60.8 ± 1.3
60.3 ± 9.5
24.7 ± 0.9
20.2 ± 1.9
64.3 ± 4.8
1
2h
N
N
Br
N
1
2i
1
2j
247.3 ± 95.4 464.3 ± 20.5
O
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O
O
12k
100.1 ± 3.1
17.2 ± 0.3
14.6 ± 0.7
1.9 ± 0.4
27.8 ± 0.8
7.3 ± 0.2
N
1
2l
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
S
HN
N
1
2m
2n
68.0 ± 2.0
147.5 ± 3.2
127.3 ± 3.3
70.4 ± 1.8
34.8 ± 1.4
196 ± 7
39.2 ± 1.4
126.4 ± 3.6
62.2 ± 3.1
52.1 ± 2.1
573 ± 9
1
110.9 ± 1.9
N
1
2o
83.5 ± 2.1
42.9 ± 1.2
882.5 ± 66
N
N
1
2p
N
SAHA
a
IC = compound concentration required to inhibit tumor cell proliferation by 50%;
5
0
data are expressed as the mean ± SEM from the dose-response curves of at least three
independent experiments.
To further evaluate the inhibitory activities of compounds 12b, 12d, and 12k-p, acute
myeloid leukemia cells (MV4-11, OCI-AML2, and OCI-AML3) were cultured with
these compounds8, 29. All these compounds exhibited potent inhibitory activities in
these tested cells. Morpholine substituent derivatives (12k-l) showed more potent in
leukemia cells than other tumor cells with IC values below 10 nM, equivalent with
50
piperazine substituent derivatives (12n-p). However, RAPA (rapamycin) hardly
inhibited the three cancer cells and SAHA only showed slight inhibitory effects
(
IC > 200 nM).
50
Table 6. IC values against leukemia cells of optimal compounds, rapamycin and
5
0
SAHA as positives.
IC50 a ± SEM, nM
Compd
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1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
MV4-11
OCI-AML2
OCI-AML3
1
2b
12d
2k
2l
2m
2n
12o
2p
10.38 ± 1.56
18.32 ± 2.56
12.64 ± 2.56
4.05 ± 0.41
13.87 ± 0.60
26.53 ± 1.55
7.01 ± 1.42
5.86 ± 0.80
220 ± 8
18.41 ± 0.04
9.71 ± 0.38
28.96 ± 1.92
9.01 ± 0.53
16.95 ± 0.03
32.78 ± 2.28
8.55 ± 0.46
3.97 ± 0.27
390 ± 11
16.32 ± 1.21
8.79 ± 0.50
30.11 ± 2.11
9.98 ± 0.62
14.98 ± 1.01
40.11 ± 2.83
35.33 ± 2.22
8.01 ± 0.43
300 ± 10
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
1
1
1
SAHA
RAPA
> 500
> 500
> 500
a
IC = compound concentration required to inhibit tumor cell proliferation by 50%;
5
0
data are expressed as the mean ± SEM from the dose-response curves of at least three
independent experiments.
Based on potent inhibition effects in cancer cells, the inhibition effects on mTOR
were inevitable to be evaluated to obtain dual target inhibitors. Meanwhile, the
morpholinopyrimidine function group was frequently merged in PI3K inhibitors. Thus
the selected compounds were evaluated efficacy on mTOR and PI3Kα together.
Surprisingly, all the three compounds showed excellent inhibitory activities on mTOR
kinase, especially 12l with IC of 1.2 nM with improved efficacy > 30 - fold (10a, 40
5
0
nM). Furthermore, 12l showed more than 500 - fold selectivity on mTOR inhibition
activity vs PI3Kα. 12o and 12p showed slightly decreased selectivity on mTOR
inhibition activity to PI3Kα.
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Table 7. The IC values on mTOR kinase and inhibition rates on PI3Kα of selected
5
0
compounds.
Enzyme
types
IC50 a, nM
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
12l
12o
12p
mTOR
1.2
3.4
3.1
PI3Kα,
1000 nM
00 nM
57.3 ± 13.7
28.3 ± 6.7
98.1 ± 29.6
52.7 ± 1.8
83.4 ± 8.1
69.3 ± 7.4
inhibition rate
%)
1
(
a
The IC values and inhibition rates are the means of at least two experiments, with
5
0
intra- and inter- assay variations of < 10%.
Up to now, 12l was the optimal potent mTOR inhibitor without PI3Kα inhibition
activity, which proved structure modification strategy correctness. The inhibitory
activity on HDACs was further evaluated to confirm dual targets on HDACs and
mTOR kinase. As shown in Table 4, 7 and 8, 12l could maintain inhibitory activities
on HDACs with improving potency on mTOR. 12l showed potent inhibitory activities
on class I and IIb isoforms with nanomolar or sub-nanomolar IC values, much more
50
3
0
potent than SAHA . Additionally, the IC values were all > 1000 nM for class IIa
5
0
and IV isoforms. We can draw conclusion that 12l indeed was a potent HDAC and
mTOR dual target inhibitor.
Table 8. The IC values on HDACs of compound 12l, and SAHA as positive control.
5
0
a
HDACs IC , nM
5
0
Class I
Class IIb
6 10
Class IIa and IV
4, 5, 7, 9, 11
Compd
1
2
3
8
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1
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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5
5
5
5
5
6
1
2l
0.19
11
0.61
35
1.47
30
1.28
172
1.8
15
0.58
170
> 1000
> 1000
SAHA
a
The IC values and inhibition rate are the means of at least two experiments, with
50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
intra- and inter- assay variations of < 10%.
Kinase Selectivity Profiling. In order to further understand compound 12l’s
selectivity, we then examined its kinome wide selectivity profile with KinaseProfileTM
technology by Eurofins Discovery Pharma Services. A panel of 99 related kinases
were tested at 1 μM concentration (Table S1). Not surprisingly, 12l displayed strong
binding affinities against mTOR and mTOR/FKBP12. As for the other kinases,
especially for PI3K related kinases, 12l hardly showed inhibitory activities. In
conclusion, 12l didn’t inhibit kinases activity except for mTOR kinase.
Figure 2. Kinome wide selectivity profiling of compound 12l. Measurements were
performed at a concentration of 1 μM of the inhibitor in duplicate. The % percent
control means remaining active kinase percentage. The affinity was defined with
respect to a DMSO control.
Molecular Docking Study. To better understand the different activities of 12l on
HDAC1 and mTOR, the molecular docking was performed. The result was shown in
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Figure 3A. The hydroxamic tail of 12l was buried within the narrow active-site
channel with the carbonyl oxygen chelating with Zn ion in a similar manner with
other class I HDAC inhibitors (for example SAHA bound to HDAC2, PDB code:
4LXZ). The scaffold of pyrazolopyrimidine located at the rim of the HDAC1 active
site. In addition, the linking benzene ring formed the π-π stacking with the residues of
Phe150 and His28. When aligned the docking pose of 12l and SAHA in HDAC2
crystal structure, the benzene rings in both structures located at the same position and
formed the same contacts with the corresponding residues of Phe and His. The
molecular docking with 12l in mTOR was also performed, and the result was shown
in Figure 3B. The docking pose of 12l could well overlap with the bound PI-103 in
mTOR crystal structure. The morpholine ring of 12l linking to pyrazolopyrimidine
made hydrogen bond with Val804 in the hinge region, whereas
1
1
1
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2
2
2
2
2
2
3
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3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
-(N-phenylcarbamoyl)-2-methylmorpholine binded to the inner pocket and made
hydrogen bond with the residue of Asp759. Moreover, the hydroxamic tail formed
three hydrogen bonds with the residues of Thr809, Ala812 and Ser906.
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Figure 3. (A) Align the docking pose of 12l (green stick), the bound peptide
inhibitors in HDAC1 (magentas stick, PDB code: 5ICN), and SAHA in HDAC2
(yellow stick, PDB code: 4LXZ); (B) Align the docking pose of 12l (green stick) and
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the bound PI-103 in mTOR (yellow stick, PDB code: 4JT6).
Western Blot Confirmed Dual Targets for HDACs and mTOR Kinase. To
elucidate signaling pathway inhibition by 12l, mTOR kinase related upstream and
downstream signal factors were detected. As illuminated in Figure 4 (A and C),
p-p70S6K (T389) and p-4EBP1 (S65) decreased sharply or even vanished at 0.1 μM,
and RAPA showed similar effects only at 1 μM or higher concentration. RAPA
induced a rapid and sustained increase in Akt phosphorylation in several types of
cancer cells28, 29, but prolonged RAPA treatment could inhibit mTORC2 assembly and
3
1
Akt , which was consistent with our results. 12l could blockade phosphorylation of
Akt at S473 site at 0.1 μM and higher doses, which resulted from synergistic effects
2
5
of dual inhibition on HDACs and mTOR kinase . MTORC1 inhibition led to the
activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase
3
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(ERK/MAPK) cascade in many patients with metastatic cancer . Moreover, RAPA
increased p-ERK activity at low concentration while inhibited p-ERK expression at
high concentration. 12l could downregulate p-ERK in a dose-dependent manner from
0.1 to 10 μM, however RAPA decreased p-ERK only at 10 μM. The effects of 12l on
the acetylation level of histone H3 (a known substrate for HDAC 1, 2, and 3) and
α-tubulin (a known substrate for HDAC 6), the biomarkers of HDAC inhibition in
MV4-11 cells treated with different concentrations were shown in Figure 4 (B). 12l
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induced a concentration-dependent increase of the Ac-H3 from 0.01 μM to 1 μM,
however, SAHA only slightly upregulated the Ac-H3 at 1 μM. Furthermore, 12l
concentration-dependently upregulated acetylated α-tubulin, but SAHA induced
acetylated α-tubulin increase at slightly higher concentration. Western blot analysis of
HDACs indicated that 12l inhibited HDAC1 more potent than HDAC6 isoform,
which was consistent with previous HDACs enzyme assay. These results showed that
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2l was a potent HDACs and mTOR dual target inhibitor.
Figure 4. (A, C): MV4-11 cells were treated with 12l and RAPA for 24 h at 0.01, 0.1,
, 10 μM, respectively. The cells were harvested for preparation of whole-cell protein
1
lysates and subsequent western blot analysis with the indicated antibodies. (B):
Western blot analysis of acetylated α-tubulin, acetylated histone H3 in MV4-11 cells
after 6 h treatment with compound 12l, at 0.01, 0.1, 1 μM and SAHA at 0.1, 1 μM.
GAPDH was used as a loading control.
Cell Cycle Analysis and Apoptosis Assays by Flow Cytometry. mTOR is a
coordinator of cell fundamental biological processes, which regulates both cell growth
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and cell cycle progression . Compared to the vehicle, MV4-11 and MM1S cells
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treated with 12l demonstrated a loss of S-phase cells and an increase in the percentage
of cells in G /G phase (Figure 5A and Figure 6A). We next wanted to confirm that
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the phenotypic observation of cell death induced by 12l was specifically due to
apoptosis. As shown in Figure 5B and Figure 6B, RAPA (1 μM) alone hardly induced
cell apoptosis, and SAHA (1 μM) could induce cell apoptosis. When MV4-11 cells
were treated with 12l at 20 nM, the percentage of the early stage and the later stage of
apoptosis was 76.6%, approximatively with SAHA group. Furthermore, the apoptosis
effect was consistent as the dose increased. For MM1S cells, the apoptosis effect was
stronger as the dose increased. SAHA+RAPA (3:1) group didn’t induce stronger
apoptosis than SAHA group, indicating inapparent synergistic effects in MV4-11
cells. However, SAHA+RAPA (3:1) group could induce stronger apoptosis than
SAHA group, indicating apparent synergistic effects in MM1S cells. It was evident
that 12l blocked the tumor cells in G1-phase progression, which resulted in decreased
S-phase populations, and induced tumor cells apoptosis.
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Figure 5. (A): MV4-11 cells were cultured with 12l from 1.25 to 10 nM for 24 h, the
cell cycle distribution of these cells was analyzed. (B): MV4-11 cells were cultured
with 12l from 10 to 1000 nM for 48 h, and SAHA at 1 μM, RAPA at 1 μM, SAHA (1
μM)+RAPA (0.3 μM) were positive controls.
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Figure 6. (A): MM1S cells were cultured with 12l from 1.25 to 10 nM for 24 h, the
cell cycle distribution of these cells was analyzed. (B): MM1S cells were cultured
with 12l from 10 to 1000 nM for 48 h, and SAHA at 1 μM, RAPA at 1 μM, SAHA (1
μM)+RAPA (0.3 μM) were positive controls.
In Vivo Xenograft Model Experiments. MV4-11 xenograft NOD/SCID mouse
model was primarily established to investigate the efficacy of 12l in vivo. The
administration, dosing schedules, and results were presented in Table 9. 12l displayed
potent anti-tumor effect without significant toxicity: slight body weight change and no
death of mice were observed during the treatment period. The TGI (tumor growth
inhibitory rate) was up to 53.1%. In order to validate the potent anti-tumor effect due
to dual targets for HDACs and mTOR, MM1S model was established subsequently.
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SAHA and RAPA were chosen to be double positives. The SAHA group showed a
slight anti-tumor efficacy with TGI of 32.5%. As expected, the combination group
sharply inhibited tumor growth and TGI was up to 73.8%, which suggested
synergistic anti-tumor effects of two target inhibitors, which was consistent with
apoptosis effects. The low dosage group of 12l with 10 mg/kg iv treatment exhibited
potent anti-tumor effect and the TGI was 48.1%. The TGI increased to 72.5% after
treatment with 20 mg/kg iv of 12l, which was similar to the combination medication.
Conclusion might be drawn that 12l could exert synergistic effects of dual HDAC and
mTOR target inhibitor.
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Table 9. Summary of tumor growth inhibition of compound 12l.
Administration
Tumor
model
Survivors
(day)
Tumor mass
change(%)
Compd
Dose
Schedulea
Route
(
mg/kg)
MV4-11
12l
12l
Q2D×6
Q2D×5
Q2D×5
QD×10
QD×10
10
10
iv
iv
6/6
6/6
6/6
6/6
6/6
53.1
48.1
72.5
73.8
32.5
1
2l
20
iv
MM1S
SAHA+RAPA
SAHA
50+25
50
po
po
a
QD, every day; Q2D, every two days.
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Figure 7. (A): Anti-tumor activity of 12l in the MV4-11 xenograft model; (B):
Anti-tumor activity of 12l in MM1S xenograft model.
Conclusion
Based on the progress of clinical research of combination of mTOR inhibitors and
HDAC inhibitors, our groups decided to design and synthesize dual target inhibitors
for mTOR and HDACs. We designed pyrimidine-pyrazolyl pharmacophore to append
HDAC recognition cap and hydroxamic acid as a zinc binding motif. Among them,
12l was the optimal lead compound with potent inhibition for mTOR and HDAC1
with IC of 1.2 nM and 0.19 nM. Kinome profile confirmed that 12l hardly hit other
5
0
kinases except for mTOR. 12l also showed potent inhibition activities in hematologic
malignancies cells, such as MV4-11 and MM1S. Furthermore, western blot confirmed
the anti-tumor activities were indeed on-target. 12l could also stimulate cell cycle
arrest in G /G phase and induce tumor cell apoptosis. Most importantly, in MM1S
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xenograft model, 12l could inhibit tumor growth and the TGI was up to 72.5% at 20
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mg/kg iv. In combination medication group, SAHA and RAPA exerted synergistic
effects on inhibiting tumor growth. We speculated that the potent anti-tumor activities
of 12l were due to dual target effects. However, the poor bioavailability limited its
convenient administration and higher dosage in treating cancers. Thus an optimal
formulation may overcome this disadvantage. To sum up, 12l was a promising dual
target inhibitor for treating cancers, especially for hematologic malignancies.
Experimental Section
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Chemistry. All the chemical solvents and reagents, which were analytically pure
without further purification, were commercially available. TLC was performed on
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.20 mm Silica Gel 60 F254 plates (Qingdao Haiyang Chemical, China). H NMR and
13C NMR spectra were recorded on a Bruker Avance 400 spectrometer (Bruker
Company, Germany) or Varian spectrometer (Varian, Palo Alto, CA), using TMS as
an internal standard. Chemical shifts were given in ppm (parts per million). Mass
spectra were recorded on Q-TOF Priemier mass spectrometer (Micromass,
Manchester, UK). The purity of each compound (> 95%) was determined on an
Waters e2695 series LC system (column, Xtimate C18, 4.6 mm ×150 mm, 5 μm;
mobile phase, methanol (60%)/H O (40%); low rate, 1.0 mL/min; UV wavelength,
2
o
2
54 - 400 nM; temperature, 25 C; injection volume, 10 μL).
General Procedures of Method A for the Syntheses of compounds 6a-j.
-(6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)morpholine (2). To the mixture of 1
4
(9.45 g, 50 mmol, 1 equiv) in methanol (250 ml) was added morpholine (6.5 ml, 75
mmol, 1.5 equiv) slowly under ice-bath. The mixture was stirred for 10 min, and
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stirred at room temperature for another 2 h. Upon the reaction completed, large
amounts of precipitates formed. The precipitates were collected by filtration to get the
1
crude production. Yield, 95%, light yellow power solid. H NMR (400 MHz, CDCl )
3
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δ: 10.48 (s, 1H), 7.72 (s, 1H), 3.85 – 3.76 (m, 4H), 3.68 – 3.52 (m, 4H).
4
,4'-(1H-pyrazolo[3,4-d]pyrimidine-4,6-diyl)dimorpholine (3). To a solution of 2
(5 g, 17.2 mmol, 1 equiv) in N-methyl pyrrolidone (NMP) (100 ml) was added
morphline (7.5 ml, 86 mmol, 5 equiv), N,N-diisopropylethylamine (DIEA) (5.7 ml,
3
4.4 mmol, 2 equiv) and KI (catalytic amount). The vial was sealed under N2
o
production and heated with stirring at 90 C for 8 h. The solution was quenched with
water and extracted with ethyl acetate. The solvent was removed under reduced
1
pressure to afford the crude 3 without further purification. Yield, 85%. H NMR (400
MHz, CDCl ) δ: 10.48 (s, 1H), 7.72 (s, 1H), 3.84 – 3.78 (m, 4H), 3.75 – 3.66 (m, 8H),
3
3
.66 – 3.54 (m, 4H).
4-(6-(4-methylpiperazin-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)morpholine (4).
1
Compound 4 could be gotten in similar method with 3. H NMR (400 MHz, CDCl )
3
δ: 10.50 (s, 1H), 7.79 (s, 1H), 3.95 – 3.79 (m, 12H), 2.61 – 2.48 (s, 4H), 2.39 (s, 3H).
Ethyl-3-(4,6-dimorpholino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acidate (5a-i). To a
mixture of 3 (4) in DMF was added Cs CO , followed by bromoacetate analogues.
2
3
o
The mixture was reacted using microwave (MW) at 120 C for 1 h. The mixture was
quenched with water and extracted with ethyl acetate. The organic layer was separated
and washed with saturated brine. After dried over Na SO , the solvent was removed
2
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under reduced pressure to afford the crude 5a-g and 5h-i. The crude was purified by
flash chromatograph as light yellow solid.
(E)-methyl-3-(4-((4,6-dimorpholino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)ph
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-enyl)acrylate (5j). The intermediate 5j was gotten in similar synthesis route with
1
methyl 3-(4-bromomethyl)cinnamate instead of bromoacetate analogues. H NMR
(400 MHz, CDCl ) δ: 7.74 (s, 1H), 7.64 (d, J = 16.0 Hz, 1H), 7.45 (d, J = 8.2 Hz, 2H),
3
7
3
.31 (d, J = 8.2 Hz, 2H), 6.39 (d, J = 16.0 Hz, 1H), 5.44 (s, 2H), 3.89 - 3.85 (m, 4H),
.85 – 3.80 (m, 8H), 3.79 (s, 3H), 3.78 – 3.74 (m, 4H).
General Procedures of Method A for the Syntheses of Hydroxamic Acid
Derivatives. The ester intermediate (1 mmol, 1 equiv) was dissolved in
o
CH Cl /CH OH (10 ml, v:v, 2:1). The resulting solution was cooled to 0 C, then
2
2
3
hydroxamic (50 wt % in water, 1 ml, 30 mmol, 30 equiv) and NaOH (400 mg, 10
mmol, 10 equiv) were added. The reaction was stirred for 1 h. The solvent was then
removed under reduced pressure, and the obtained solid was dissolved in water, which
was adjusted to pH 7-8 by acetic acid and the crude formed in large amounts. After
filtration, the crude obtained to yield a solid production which recrystallized with
EtOH to give the title compound.
N-Hydroxy-2-(4,6-dimorpholino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetamide
1
(6a). 6a was obtained from compound 5a as described for method A. H NMR (400
MHz, DMSO-d ) δ: 10.77 (s, 1H), 8.98 (s, 1H), 8.03 (s, 1H), 4.68 (s, 2H), 3.84 – 3.78
6
+
(m, 4H), 3.75 – 3.66 (m, 8H), 3.64 (m, 4H). HRMS (ESI), [M+H ] m/z: 364.1746.
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N-Hydroxy-3-(4,6-dimorpholino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propanamide
1
(
6b). 6b was obtained from compound 5b as described for method A. H NMR (400
MHz, DMSO-d ) δ: 10.47 (s, 1H), 8.76 (s, 1H), 8.00 (s, 1H), 4.33 (t, J = 7.4 Hz, 2H),
6
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3.83 – 3.77 (m, 4H), 3.70 (m, 8H), 3.65 (m, 4H), 2.50 – 2.49 (m, 2H). HRMS (ESI),
+
[
M+H ] m/z: 378.1914.
N-Hydroxy-4-(4,6-dimorpholino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butanamide
1
(
6c). 6c was obtained from compound 5c as described for method A. H NMR (400
MHz, DMSO-d ) δ: 10.32 (s, 1H), 8.67 (s, 1H), 8.01 (s, 1H), 4.14 (t, J = 6.4 Hz, 2H),
6
3.84 – 3.77 (m, 4H), 3.74 – 3.66 (m, 8H), 3.65 (m, 4H), 1.94 (m, 4H). HRMS (ESI),
+
[
M+H ] m/z: 392.2043.
N-Hydroxy-5-(4,6-dimorpholino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pentanamide
1
(
6d). 6d was obtained from compound 5d as described for method A. H NMR (400
MHz, DMSO-d ) δ: 10.31 (s, 1H), 8.64 (s, 1H), 8.00 (s, 1H), 4.13 (t, J = 6.7 Hz, 2H),
6
3.84 – 3.78 (m, 4H), 3.74 – 3.67 (m, 8H), 3.64 (m, 4H), 1.97 (t, J = 7.3 Hz, 2H), 1.81
+
–
1.67 (m, 2H), 1.46 – 1.35 (m, 2H). HRMS (ESI), [M+H ] m/z: 406.2186.
N-Hydroxy-6-(4,6-dimorpholino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)hexanamide
1
(
6e). 6e was obtained from compound 5e as described for method A. H NMR (400
MHz, DMSO-d ) δ: 10.29 (s, 1H), 8.63 (s, 1H), 8.00 (s, 1H), 4.12 (t, J = 6.8 Hz, 2H),
6
3.84 – 3.78 (m, 4H), 3.74 – 3.66 (m, 8H), 3.64 (m , 4H), 1.90 (t, J = 7.4 Hz, 2H), 1.80
+
–
1.69 (m, 2H), 1.57 – 1.46 (m, 2H), 1.17 (m, 2H). HRMS (ESI), [M+H ] m/z:
420.2356.
ACS Paragon Plus Environment