Design of Hydrazide-Bearing HDACIs Based on Panobinostat and Their p53 and FLT3-ITD Dependency in Antileukemia Activity

Article abstract of DOI:10.1021/acs.jmedchem.0c00442

Here, we present a new series of hydrazide-bearing class I selective HDAC inhibitors designed based on panobinostat. The cap, linker, and zinc-binding group were derivatized to improve HDAC affinity and antileukemia efficacy. Lead inhibitor 13a shows picomolar or low nanomolar IC₅₀ values against HDAC1 and HDAC3 and exhibits differential toxicity profiles toward multiple cancer cells with different FLT3 and p53 statuses. 13a indirectly inhibits the FLT3 signaling pathway and down-regulates master antiapoptotic proteins, resulting in the activation of pro-caspase3 in wt-p53 FLT3-ITD MV4-11 cells. While in the wt-FLT3 and p53-null cells, 13a is incapable of causing apoptosis at a therapeutic concentration. The MDM2 antagonist and the proteasome inhibitor promote 13a-triggered apoptosis by preventing p53 degradation. Furthermore, we demonstrate that apoptosis rather than autophagy is the key contributing factor for 13a-triggered cell death. When compared to panobinostat, 13a is not mutagenic and displays superior in vivo bioavailability and a higher AUC_0-inf value.

Full text of DOI:10.1021/acs.jmedchem.0c00442

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Article
Design of hydrazide-bearing HDACIs based on panobinostat
and their p53 and FLT3-ITD dependency in anti-leukemia activity
Xiaoyang Li, Yuqi Jiang, Yuri K. Peterson, Xu Tongqiang, Richard A. Himes, Luo Xin, Guilin Yin,
Elizabeth S. Inks, Nathan Dolloff, Stephanie Halene, Sherine S. L. Chan, and C. James Chou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00442 • Publication Date (Web): 22 Apr 2020
Downloaded from pubs.acs.org on April 22, 2020
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Design of hydrazide-bearing HDACIs based on
panobinostat and their p53 and FLT3-ITD dependency
in anti-leukemia activity
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Xiaoyang Li1,2,#,*, Yuqi Jiang , Yuri K. Peterson , Tongqiang Xu , Richard A. Himes , Xin Luo , Guilin
1,#
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Yin , Elizabeth S. Inks , Nathan Dolloff , Stephanie Halene , Sherine S.L. Chan , C. James Chou *
1Ocean University of China, School of Medicine and Pharmacy, Qingdao, Shandong, 266071, China
2Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, Medical University of
South Carolina, Charleston, South Carolina 29425, USA
3Department of Chemistry and Biochemistry, College of Charleston, 66 George Street, Charleston, South
Carolina 29424, USA
4Technology Center of Qingdao Customs, Qingdao, Shandong, 266002, China
5Department of Cellular and Molecular Pharmacology & Experimental Therapeutics, Medical University
of South Carolina, Charleston, SC29425, USA
6Section of Hematology, Department of Internal Medicine and Yale Cancer Center, Yale University
School of Medicine, New Haven, CT 06511, USA.
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Abstract
Here we present a new series of hydrazide-bearing class I selective HDAC inhibitors designed based
on panobinostat. The cap, linker, and zinc-binding group were derivatized to improve HDAC affinity and
anti-leukemia efficacy. Lead inhibitor 13a shows picomolar or low nanomolar IC against HDAC1 and
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HDAC3 and exhibits differential toxicity profiles toward multiple cancer cells with different FLT3 and
p53 status. 13a indirectly inhibits the FLT3 signaling pathway and down-regulates the master anti-
apoptotic proteins, resulting in the activation of pro-caspase3 in in wt-p53 FLT3-ITD MV4-11 cells. While
in the wt-FLT3 and p53-null cells, 13a is incapable of causing apoptosis at therapeutic concentration. The
MDM2 antagonist and the proteasome inhibitor promote 13a-triggered apoptosis by preventing p53
degradation. Furthermore, we demonstrate that apoptosis rather than autophagy is the key contributing
factor for 13a-triggered cell death. When compared to panobinostat, 13a is not mutagenic and displays
superior in vivo bioavailability and higher AUC0-inf
.
Introduction
Acute myelogenous leukemia (AML) is characterized by the uncontrolled proliferation and survival of
immature malignant myeloid cells in parallel with the concurrent loss of normal hematopoiesis.1-2 The
standard anti-AML therapies since 1973 are based on cytotoxic chemotherapy using antimetabolites such
as cytarabine (ara-C), and the DNA intercalating anthracyclines such as daunorubicin or idarubicin.3
Although a series of targeted drugs including FLT3 inhibitors, IDH2 inhibitors, and Bcl-2 inhibitors have
been approved for the treatment of AML, their uses limit specific patient population and undergo a high
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possibility of clonal resistance.4-5 Three-quarters of all AML patients are > 60 years of age, only less than
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0% of them achieve disease-free survival greater than 5 years. With an increase in life expectancy in the
U.S., AML cases are expected to become more prevalent, and there is a need for more effective and better-
tolerated therapies.6
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Unlike chronic myelogenous leukemia (CML), which is characterized by a more uniform genetic
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abnormality and a reciprocal translocation of the BCR and ABL genes, AML has various cytogenetic
abnormalities and mutations, such as FLT3, NPM1, c-kit tyrosine kinase and Ras mutations.8-15 These
constitutively active kinases initiate multiple pro-growth and pro-survival signaling through the mitogen-
activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), signal transducer and
activator of transcription 5 (STAT5), and PI3K/Akt kinase family mediated pathways and confer poor
prognosis in AML.8-9, 16-19 These genetic aberrations are not mutually exclusive and commonly coexist in
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AML cells. Thus, the biggest challenge is to develop pharmacologic agents that possess significant
specificity, yet are capable of attenuating multiple oncogenic signals in AML.
Class I HDACs play a crucial role in the transformation and survival of myeloid and lymphoid
malignancies.21-23 Inhibition or co-depletion of HDACs 1 and 2 elicits pro-apoptotic responses in
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leukemia. HDAC3 activity is required for the initiation of leukemogenesis in acute leukemia. Relevant
to cancer therapy, HDAC3 depletion or inhibition significantly reduces proliferation and promotes
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differentiation in leukemia. In our previous study, we demonstrated that our HDAC1, 2, and 3 selective
inhibitors cause apoptosis in the AML cell line MV4-11, and displayed low nano-molar EC , which
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suggests that class I HDACs 1, 2, and 3 are potential molecular targets for the treatment of AML.25-26
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The mechanisms of HDACIs lethality against leukemia and other cancer types can be elucidated as
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follows: 1) HDACIs activate the endogenous cyclin-dependent kinase (CDK) inhibitor p21 and disrupt
cell cycle (especially mitotic spindle assembly) checkpoints;28-29 2) HDACIs activate both the intrinsic
(mitochondrial) and extrinsic (death receptor-mediated) pathways of apoptosis by down-regulating the
anti-apoptotic proteins such as X-linked inhibitor of apoptosis (XIAP) and cellular FLICE-like inhibitory
protein (c-FLIP),30-32 while up-regulating the pro-apoptotic proteins (Bim, Bmf and Noxa) through
acetylation of p5333-34 and inducing Bid cleavage; 3) induction of autophagy by HDACIs through
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acetylation of the autophagy signaling component including Atg3 and regulation of mammalian target
of rapamycin (mTOR) pathway.37
The actions of HDACIs in cancer cells reveal that in addition to epigenetic modifications, HDACs
also control cell proliferation, differentiation, migration, and death by modification of non-histone
proteins.38 The tumor suppressor p53 is the first characterized example of non-histone protein
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acetylation. It plays an important role in cellular signaling and stress responses and can either positively
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or negatively regulate apoptosis, cell cycle arrest, and autophagy. P53 regulates apoptosis through
control of transcription of pro-apoptotic members of the Bcl-2 family, including Bax, Puma, Noxa, and
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Bid. P53 transcriptionally activates the endogenous CDK inhibitor p21, which can in turn inhibit cyclin
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E(A)/CDK2 and preserve the association of the tumor suppressor retinoblastoma protein. Additionally,
damage-regulated autophagy modulator (DRAM) that modulates autophagosome formation is also
activated by p53.37, 43
Four HDACIs have been approved by the FDA: vorinostat,44 romidepsin,45 belinostat46 and
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panobinostat, among these, panobinostat is the most potent HDACI in vitro and in vivo. Structural
regions and pharmacophore models of HDACIs contain three critical parts: a cap group (interacts with the
surface of the enzyme), a linker (occupies the long hydrophobic tunnel), and a zinc-binding group (ZBG,
functioning within the catalytic site).48-49 Although hydroxamic acid is the most commonly used ZBG, it
has disadvantages in its structural instability, lack of isoform selectivity, and the hydroxamic acid ZBG is
mutagenic. Hydrazide-based ZBG-HDACIs have emerged in recent years in exploring new ZBGs of
HDACIs that confirms to the physiological substrate geometric.25-26, 50-51 As a new motif for HDACIs, its
potency, structural stability, and off-target toxicity all need to be addressed. In our previous studies, we
have demonstrated HDAC inhibitory activity and superior stability of the hydrazide ZBG.25-26 However,
the potential genotoxicity is a remains an important question. In fact, the hydrazine/hydrazide motif was
extensively used in the past in anti-depressants as well as first line antituberculosis agents today best
characterized by isocarboxazid and isoniazid for chronic diseases.52-54 Therefore, the hydrazide motif is
expected to be better tolerated in vivo. Here we will describe and discuss the discovery of hydrazide-
bearing HDACIs based on the structure of panobinostat with the comparative chemical structural studies,
detailed p53 and FLT3 dependent anti-tumor mechanisms of action studies in AML cells, and preliminary
mutagenic and pharmacokinetic studies.
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Results and discussion
Chemistry
Scheme 1 showed the synthesis of intermediates 6a-6c, 10a, and 10b. Phenylhydrazine (1) and 5-
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chloropentan-2-one (2) afford 2-(2-methyl-1H-indol-3-yl)ethanamine (3). 3 was mixed with aldehyde 4a-
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c, NaBH CN and CH COOH in methanol to give 5a-5c, respectively. Boc-protection of 5a-5c got 6a-
3 3
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c. Similarly, compound 3, 8a, and 8b were reacted with benzaldehyde, then NaBH CN was added to
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obtain 9a and 9b, respectively, which also converted to Boc-protected product 10a and 10b.
Scheme 1. Synthesis of 3, 6a-6c, 10a and 10b.
H
O
N
NH2
a
NH2
Cl
+
HN
3
1
2
Boc
N
H
O
NH2
+
N
R1
c
R1
b
b
R1
H
HN
HN
HN
3
4a-4c
5a-5c
6a-6c
O
O
R2
O
R2
O
R2
N
c
N
H
H N
2
H
+
O
8a, 8b
O
Boc O
0a
7
9a, 9b
1
O
O
OH
O
O
4
a-6a R1:
4b-6b R1:
4c-6c R1:
O
8a, 9a, 10a R2:
8b, 9b R2:
Reagents and conditions: (a) Ethanol, NaOH, reflux, 47%; (b) NaBH CN, CH COOH, methanol, 70-80%
3
3
yield; (c) Boc O, TEA, DCM, 80-85% yield; (d) Propionaldehyde or benzaldehyde, NaBH CN, HCl,
2
3
Ethanol, 60-70%.
Synthesis of product 13a-13d was shown in Scheme 2. 6a connected with hydrazine monohydrate by
TBTU-mediated amide formation afforded 11. 11 was reacted with propionaldehyde or N-Boc-2-
aminoacetaldehyde to get Schiff base, which was suffered by NaBH CN gave reductant product 12a or
3
1
2b, respectively. 12c and 12d were achieved by reaction of 11 with acrylonitrile or methyl acrylate in
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ethanol. Boc de-protection of 12a-12d leading end products 13a-13d, respectively.
Scheme 2. Synthesis of 13a-13d.
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OH
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HN
b,c or d
O
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H
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H
R3
H
N
H
e
13a: R =
13c: R = CN
12a: R =
12c: R = CN
3
3
3
3
O
O
1
3b: R = NH2
13d: R =
12b: R =
N
H
Boc
12d: R3 =
3
3
3
O
O
Reagents and conditions: (a) hydrazine monohydrate, TBTU, TEA, DMF, yield 50%; (b) propionaldehyde
or N-Boc-2-aminoacetaldehyde, MgSO , ethanol, yield 60-70%; (c) NaBH CN, HCl, methanol, H O,
4
3
2
methyl orange, yield 60-70%; (d) acrylonitrile or methyl acrylate, ethanol, refluxed, yield 40-50%; (e)
TFA, DCM, yield 60%.
6
b, 6c, and 10a were refluxed with hydrazine monohydrate to get 14a-14c, which was reacted with
propionaldehyde in the presence of magnesium sulfate followed by a reduction with sodium
cyanoborohydride in acidified methanol to give the desired 15a-15c (Scheme 3). Boc de-protection by
TFA afforded 16a-16c.
Scheme 3. Synthesis of 16a-16c.
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TFA
H
H
H
R5
O
R5
N
R5
N
N
R5
R4
N
a
R4
N
NH2
b, c
R4
N
N
d
R4
N
H
N
H
H
Boc O
Boc O
Boc O
15a-15c
O
6
b, 6c, 10a
14a-14c
16a-16c
6
b, 14a-16a R4:
R :
6c, 14b-16b R4:
R :
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HN
HN
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0a, 14c-16c R4:
R :
5
Reagents and conditions: (a) hydrazine monohydrate, methanol, reflux, yield 90-95%; (b)
propionaldehyde, MgSO , ethanol, yield 60-70%; (c) NaBH CN, HCl, methanol, H O, methyl orange,
4
3
2
yield 60-70%; (d) TFA, DCM, yield 60-70%.
Scheme 4 described the synthesis of 24a-24c. 17 connected with 18 got amide 19, 20 connected with
1 by Mitsunobu reaction afforded ether 22. 23a, 23b, and 23c, the hydrazine product of 9b, 19 and 22
2
respectively, were treated with propionaldehyde followed by NaBH CN to get end product 24a, 24b, and
3
2
4c.
Scheme 4. Synthesis of 24a-24c.
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O
O
O
O
O
O
O
a
Cl
O
O
N
H
+
H N
2
19
17
18
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O
OH
b
O
+
HO
20
21
22
R6
R6
R6
c
d, e
H
H
O
N
N
NH2
N
H
O
O
O
24a, 24b, 24c
9
b, 19, 22
23a, 23b, 23c
O
N
9
b, 23a, 24a R6:
N
19, 23b, 24b R6:
22, 23c, 24c R6:
H
O
H
Reagents and conditions: (a) DCM, TEA, DMF, yield 80%; (b) PPh , DEAD, anhydrous THF, yield 53%;
3
(
c) hydrazine monohydrate, methanol, reflux, yield 95%; (d) propionaldehyde, MgSO , ethanol, yield 60-
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7
0%; (e) NaBH CN, HCl, methanol, H O, methyl orange, yield 60-70%.
3 2
6
a connected with 1,1-dimethylhydrazine, pyrrolidin-1-amine, piperidin-1-amine or pyrazolidine by
TBTU-mediated amide formation to get intermediates 25a-25c, respectively, which suffered TFA to get
rid of the Boc group achieved 26a-26c (Scheme 5). 12a reacted with ethanal, propanal, n-butanal, n-
pentanal or n-hexanal, followed by the addition of NaBH CN provided tertiary amine 27a-27e. The end
3
products 28a-28e were achieved by Boc deprotection (Scheme 5).
Scheme 5. Synthesis of 26a-26c, 28a-28e.
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5
5
5
5
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6
O
O
O
Boc
N
OH
a
Boc
N
R9
HN
6a
HN
25a-25c
b
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
0
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0
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2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HN
N
HN
N
2
5a, 26a R =
25b, 26b R =
R9
9
9
H
N
H
N N
2
5c, 26c R =
9
HN
TFA
6a-26c
2
O
O
R10
N
H
N
Boc
N
N
H
Boc
N
N
H
c
HN
12a
HN
27a-27e
b
O
R10
2
7a, 28a R10
=
27b, 28b R10
27d, 28d R10
=
=
N
N
H
H
N
2
7c, 28c R10
=
2
7e, 28e R10
=
HN
TFA
28a-28e
Reagents and conditions: (a) 1,1-dimethylhydrazine, pyrrolidin-1-amine, piperidin-1-amine or
pyrazolidine, TBTU, TEA, DMF, yield 50-60%; (b) TFA, DCM, yield 60-65%. (c) ethanal, propanal, n-
butanal, n-pentanal, n-hexanal, NaBH CN, CH COOH, methanol, yield 50-60%.
3
3
Intermediate 11 reacted with 3-Bromo-1-propanol to get the propanol di-substituent product 29,
which was treated with TFA to afford 30 (Scheme 6).
Scheme 6. Synthesis of 30.
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OH
OH
O
O
O
OH
OH
NH2
N
N
Boc
N
N
Boc
N
N
H
H
a
1
1
29
HN
HN
HN
b
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
N
H
H
N
TFA
30
Reagents and conditions: (a) 3-Bromo-1-propanol, K CO , ethanol, reflux, yield 70%；(b) TFA, DCM,
2
3
yield 60%.
Compounds design
Panobinostat is a highly potent hydroxamic acid-bearing pan-HDACI among all of the clinical
HDACIs with low nano-molar IC s against HDACs and cancer cells but with limited selectivity.
5
0
Although hydroxamic acid is the most commonly used ZBG for HDACIs, it shows limitations in isoform-
selectivity, which is hypothesized to cause greater side effects than more selective HDACIs. Further,
hydroxamic acid also suffers from reduction, hydrolysis, and glucuronidation in vivo, leading to the rapid
5
5
inactivation of HDACIs. This structural instability is the main reason for the poor pharmacokinetic
profiles (short t and low C and bioavailability) of clinical hydroxamate HDACIs.56-59 Therefore, the
1
/2
max
exploration of new ZBG motifs for HDACIs holds great interest. Initially, Wang and colleagues
51
demonstrated a novel hydrazide-based HDACI with class I selectivity. We have shown further that the
hydrazide motif as a potential ZBG displayed allosteric inhibition kinetics and was impervious to
glucuronidation,25-26 indicating that hydrazide could be a preferable ZBG fragment with improved
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6
selectivity and in vivo stability. Most recently, Son et al. also demonstrated hydrazide with long chain can
be used for the design of HDAC11 selective inhibitor.50
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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8
9
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Therefore, we modify the hydroxamic acid group of panobinostat using hydrazide motifs. It should
be noted that among all of the hydrazide-based HDAC inhibitor studies, only one single N-substituent at
the hydrazide tail was explored.25-26, 50-51 Here in this study, we first design compounds with di-N-
substituent at the hydrazide tail (Figure 1, R group). As modifications in the cap and linker groups can
4
also generate changes in enzymatic activity and selectivity, we also incorporate different fragments in the
cap and linker (Figure 1). These newly designed compounds are expected to have the following
improvements: 1) selectivity against class I HDACs; 2) similar potency toward class I HDACs and anti-
leukemia activity compared to the parent molecule panobinostat; 3) less toxicity and improved in vivo PK
profiles.
R2
O
H
O
O
R4
R6
N
OH
N
N
N
H
R1
N
H
H
N
R2
N
N
H
R5
HN
R1
R3
R4
HN
N
HN N
HN
Cap
Modification
HN
O
R3
Linker
Panobinostat
ZBG
(LBH589)
Figure 1. Design of hydrazide-bearing HDACIs based on the structure of panobinostat.
In our previous studies, hydrazide compounds with a 3 carbon on the hydrazide motif exhibited
excellent HDAC inhibitory activity.25-26 Therefore, we initiated this study by designing compounds in
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6
series 1 (Table 1) with N'-propylacetohydrazide as their ZBG and with modifications of panobinostat in
the cap and linker groups. In detail, direct modification of hydroximic acid of panobinostat with N'-
propylacetohydrazide affords 13a. While keeping the cap group and ZBG unchanged, substituting the
linker group of 13a with 1-ethyl-4-methylbenzene and 1-ethyl-3-methylbenzene results in 16a and 16b,
respectively; modifying both cap and linker of 13a affords 16c, 24a, 24b, and 24c. Compounds 12a, 15a,
15b, and 15c are the Boc-containing structures of 13a, 16a, 16b, and 16c, respectively. Recombinant
HDACs 1, 2, 3, and 6 are used to screen the newly designed compounds. IC of 13a for HDAC1, 2, and
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
5
0
3
are 5.17, 49.5 and 0.28 nM, respectively, while they do not show inhibition against HDAC6 up to
100,000 nM. This indicates that modification of panobinostat by replacing hydroxamic acid with N'-
propylacetohydrazide maintains its inhibitory activity toward class I HDACs, as well as acquired
selectivity between class I HDACs and HDAC6. The IC of 13a is much lower than that of 16a and 16b;
5
0
therefore, (E)-1-ethyl-4-(prop-1-en-1-yl)benzene of 13a is the optimized linker. Compound 16a with 2-
methyl-3-propyl-1H-indole as cap group is much more potent than 16c with an ethylbenzene cap.
Compounds 24a, 24b, and 24c with modifications in both cap and linker groups all show weaker activity
than 13a. Compounds 12a, 15a, 15b, and 15c with a Boc group exhibit reduced (12a, 15b, 15c) or
equivalent (15a) activity when compared with their relative compounds; therefore, a branched aliphatic
cap group may not improve the HDAC inhibitory activity of compounds.
Table 1. Structure IC of compounds in series 1 against HDAC1, 2, 3 and 6.
5
0
O
H
N
R1
N
H
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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
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4
5
5
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5
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5
5
5
5
5
6
a
Compound
No.
IC (nM)
50
R1
HDAC1
180.9
5.17
HDAC2
1298
HDAC3
HDAC6
>50,000
>50,000
>50,000
>50,000
>50,000
O
O
N
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
12a
16.76
0.28
HN
H
N
1
1
3a
5a
49.5
TFA
HN
O
O
N
28.84
29.43
2524
278.6
107.5
> 5000
12.59
19.94
> 5000
HN
H
N
16a
TFA
O
HN
O
1
1
5b
6b
N
HN
H
N
1674
216.5
87.98
15.8
4942
579.3
441.3
67.48
474.1
174.6
13.2
673.9
78.14
96.71
6.12
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
10.5
TFA
HN
O
O
1
1
5c
6c
N
H
N
TFA
H
N
2
4a
24b
4c
H
N
93.05
56.32
2.5
48.75
24.82
2.1
O
O
2
Panobinostat
SAHA
43.76
89.23
144.7
78.3
a
Values are average of three independent determinations, the SD values are < 20% of the mean.
As HDACs 1, 2, and 3 also have significant differences in surface charges and anchor interactions
with the binding site, we further modified the hydrazide anchor (R ) group of 13a with motifs of varying
3
electronegativity (13c and 13d) and ionization 13b (Table 2). HDAC inhibitory activity shows that the
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2
2
2
2
2
2
2
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3
3
3
3
3
3
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3
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newly designed 13b-13d all display less potency and selectivity when compare with 13a. We then
designed compounds with di-N-substituted and cyclic hydrazide groups (26a-26c, 28a-28e, 30) based on
the ZBG space size difference between HDACs 1, 2, and 3 which are expected to achieve improved
activity and selectivity these compounds. IC s of compounds with cyclic hydrazide (26b-26c) are all >
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
5
0
2
,000 nM, which indicates cyclized hydrazide is a weak ZBG. Although some of the di-N-substituted
compounds (28b and 28e) display selectivity for HDAC3 to some extent, their activity is at least 100 times
lower than N'-propylacetohydrazide 13a. Therefore, HDACIs with di-N-substituted and cyclized
hydrazide groups all have much lower potency than those with a 3-carbon linker chain mono-substitution
hydrazide. To the best of our knowledge, it is the first time we discuss the structure-activity relationship
(SAR) study of di-N-substituted hydrazide based HDACIs. These conclusions are important in the design
of hydrazide HDACIs.
Table 2. Inhibition of HDAC1, 2, 3, and 6 for Compounds in series 2.
O
R2
N
R3
TFA
HN
a
IC (nM)
5
0
R2
R3
HDAC1 HDAC2 HDAC3 HDAC6
H
N
1
3a
H
H
H
H
N
H
5.17
9842
58.12
1089
49.5
>10000
172
0.28
5241
34.82
309.7
>50,000
>50,000
>50,000
>50,000
H
N
1
3b
N
H
NH2
H
N
N
H
CN
O
1
3c
H
N
N
H
1
3d
4214
O
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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
N
2
6a
6b
6c
8a
28b
8c
8d
H
H
H
H
H
H
H
H
H
N
H
11000
8599
12930
996.4
3091
>50,000
>50,000
HN
N
2
>10000
H
N
2
N
>10000 >10000 >10000 >50,000
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
HN
N
2
2925
412.8
209.7
381.5
2880
8071
1366
3222
7316
9835
550.1
25.19
229.4
321.1
143.2
>50,000
>50,000
>50,000
>50,000
>50,000
HN
N
HN
N
2
2
HN N
2
8e
HN N
OH
OH
HN
N
3
0
>10000 >10000 >10000 >50,000
a
Values are average of three independent determinations, the SD values are < 20% of the mean.
MV4-11 is widely used for the biological evaluation of drugs targeting AML.60-62 What’s more, in
26
our previous study, MV4-11 cells showed high sensitivity to our hydrazide HDACIs. Therefore, we
chose the MV4-11 cell line to screen the antiproliferative activity of representative compounds 13a, 16a,
1
6b, and 16c with different linker- and cap-structures, plus the HDAC3 selective compound 28b (Table
3). Class I selective inhibitors entinostat, as well as pan-inhibitors vorinostat and panobinostat, were
chosen as positive controls. Results show that compounds 13a, 16a, 16b, and 16c have similar selective
profiles. 13a has the strongest enzyme inhibitory activity and antiproliferative activity with an EC of
5
0
15.35 nM, which is much lower than that of vorinostat and entinostat and comparable to that of
panobinostat. HDACIs 26a, 26b, 26c, and 30, with little HDAC inhibitory activity, also do not affect cell
proliferation at 50 μM.
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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
Table 3. EC of representative compounds.
50
EC50a (nM) of
Cpd No.
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
MV4-11
15.35±2.22
81.64±5.29
7696±1002
172.6±38.75
1054±240.6
418.1±72.27
806.2±68.72
5.2±1.87
13a
1
6a
1
6b
1
6c
2
8b
Vorinostat
Entinostat
Panobinostat
a
EC values were shown as the mean of three experiments ± the standard error of the mean.
50
1
3a is slow-on/slow-off class I inhibitor
Enzymatic and cell-based assays indicate that 13a is the most potent HDACI among all of the
compounds we designed. Thus, we chose 13a for further biological study. To assess the HDAC isozymes
2+
selectivity of 13a, we determined its IC for the other Zn -dependent HDACs (HDAC4, 5, 7, 8, and 9),
5
0
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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
and compared it with pan-HDAC inhibitor panobinostat, as well as class I selective inhibitor entinostat
(Table 4). As there are no appropriate substrates for HDAC10 and HDAC11, inhibitory activity toward
63
these two isotypes was not determined. IC s of 13a for class I HDAC1, 2, and 3 are 4.69, 46.0, and 0.28
5
0
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
nM, respectively, which is comparable to those of panobinostat. Compound 13a displays micro-molar
IC (1.75 μM) for HDAC8 and does not inhibit (up to 10 μM) HDAC4, 5, 6, 7, or 9; therefore, 13a is an
5
0
HDAC1, 2, and 3 selective inhibitor. Although panobinostat shows nanomolar IC for all types of HDACs,
5
0
its EC for MV4-11 cells (5.2 nM) is close to that of class I HDAC selective HDACI 13a (15.35 nM).
5
0
This indicates that 13a achieves HDAC isoform selectivity while maintaining potent anti-leukemia
activity. We also find that 13a does not show any inhibition to two metalloproteases, APN, and MMP
(supporting information Figure S4), and trypsin used for the HDAC assay. Therefore, the anti-tumor
activity of 13a comes from its inhibition toward HDACs rather than non-specific metalloprotease
inhibition.
Enzyme kinetic studies were also performed for HDACs 1 and 3 (Figure 2A and B). It is interesting
to note that 13a displays a non-competitive enzyme inhibition for HDAC1 and a mixed mode of inhibition
for HDAC3. To better understand the potential binding modes and patterns between HDAC1 and HDAC3,
we tested 13a in four conditions each by probing the competitive site (CS) and the allosteric site (AS) of
HDAC1 and HDAC3 using computational docking. As shown using the free-energy calculation in the
docking simulations (Figure 2C), there is very large discrimination between the high affinity AS for 13a
and the very low-affinity CS of 13a in HDAC1, in silico binding mode also shows 13a fails to chelate due
to the geometric constraint of the indole cap group preventing ZBG chelation with the Zn metal ion at the
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2
2
2
2
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3
3
3
3
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4
4
4
4
4
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4
5
5
5
5
5
5
5
5
5
5
6
CS (supporting information Figure S6). This might be the reason that 13a exhibits the non-competitive
inhibition against HDAC1. For HDAC3, 13a has a high binding affinity for both AS and CS sites based
on free-energy calculation (Figure 2C). Potential binding mode of 13a with HDAC3 shows in the catalytic
site, the top five docking poses all display a reasonable distance between ZBG and Zn metal ion (Figure
0
1
2
3
4
5
6
7
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9
0
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9
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0
2
D). These results are consistent with the kinetic studies that 13a inhibits HDAC1 non-competitively, and
13a has a mixed inhibition mode due to its interaction with both AS and CS of HDAC3.
Figure 2A and B: Lineweaver-Burke plots of enzyme kinetics data in the presence of inhibitors. Y-axes
−
1
−1
units: (pmoles acetylated substrate cleaved/min) , Xaxes units: (μmoles) . Compound 13a for HDAC1,
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and 3, respectively. Intersection on Xaxes are indicative of non-competative inhibition (HDAC1, 3A),
while intersections in 2nd quadrant are indicative of mixed and competitive inhibition (HDAC3, 3B).
Representative plots of n ≥ 3 experiments. 2C. Molecular docking probe of the catalytic and allosteric
binding pockets of HDAC1 (PDB: 5ICN) and HDAC3 (PDB: 4A69). Data is represented as whisker plot
using the top 5 docking poses for each condition. The catalytic site was defined as the catalytic pocket
that incorporated the Zn metal iron, and the allosteric site was defined as the allosteric pocket created by
the interface between the HDAC heterodimer. 2D. Potential binding mode of 13a with catalytic site and
allosteric site of HDAC3 in silico. Top 5 docking poses for each site are displayed.
0
1
2
3
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9
0
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9
0
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1
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0
1
2
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4
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8
9
0
Inhibition of HDACs by hydrazide compound 13a is time-dependent (slow-on) as concentration-
response curves shift and IC values change with enzyme-inhibitor pre-incubation time (Figure 3A). For
5
0
example, the IC of 13a for HDAC3 following 90 min pre-incubation is 0.17 nM, which is 3 times lower
5
0
than IC measured after 10 min pre-incubation. However, panobinostat reaches a steady-state within 10
5
0
min pre-incubation, indicating classical fast-on binding (Figure 3A). We also studied the release profile
of 13a and panobinostat by western blot. MV4-11 cells were treated with 13a or panobinostat for 3 h. The
drugs were then washed out and the cells cultured for 30 min, 1 h, 3 h, and 6 h without drugs. The t = 0
min time point was collected immediately after 3 h incubation with drugs. Histone acetylation caused by
1
3a does not decrease within 6 h (Figure 3B). Acetylation of histone H3 caused by panobinostat begins
to decrease after 1 h, but persisted through 6 h (Figure 3B). Therefore, 13a and panobinostat both display
slow-off release kinetics for class I HDACs. Interestingly, the acetylation of tubulin caused by
panobinostat completely disappears after 30 min (Figure 3B). Inhibition of HDAC6 leads to tubulin
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acetylation, so panobinostat exhibits fast-off release kinetics against HDAC6, which is different from the
class I HDACs. 13a does not increase the acetylation of tubulin (Figure 3B), which is in agreement with
its isoform selectivity. Although our HDAC inhibitors display slow-off and likely irreversible inhibition,
the potent inhibition of HDACs are likely required because non-substituted hydrazine and hydrazides with
bulky, branched, and long-chain acyl-groups are significantly less effective at inhibiting HDACs and
cancer cell proliferation. 25-26, 64
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 4. IC of 13a against HDAC1-9.
5
0
IC of
IC50 a of
5
0
HDAC
Classes
HDAC
IC50 a of 13a
Panobinostat Entinostat
isotypes
(nM)
(
nM) 65
(nM)
HDAC1
HDAC2
HDAC3
HDAC8
HDAC4
HDAC5
4.69±1.28
46.0±5.39
0.28±0.12
1755±110.2
>10,000
2.5
53.89±5.29
108.2±8.28
77.18±6.30
>10,000
13.2
2.1
Class I
277
203
7.8
>10,000
Class IIa
>10,000
>10,000
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6
HDAC7
>10,000
>10,000
>10,000
531
>10,000
>10,000
>10,000
HDAC9
HDAC6
5.7
0
1
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0
Class IIb
10.5
a
IC values were shown as the mean of at least three experiments ± the standard error of the mean.
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6
Figure 3A: IC curves of 13a and panobinostat for HDACs 1, 2, and 3 after pre-incubation of 10 min, 30
50
min, 60 min, and 90 min, respectively. 13a shows time-dependent inhibition toward HDAC1 and 3, which
indicates slow-on inhibition; while panobinostat reaches a steady-state with 10 min pre-incubation,
indicating fast-on kinetics. Dose response curves for 13a and panobinostat performed in triplicates were
generated using GraphPad Prism software. 3B: Histone acetylation caused by 13a does not reduce within
6 h of drug removal, while panobinostat reduces acetylation within 30 min after removal of drug. MV4-
0
1
2
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9
0
1
1 cells are treated with 13a or panobinostat (Pano) for 3 h, control cells are collected for the 0 min point,
the drugs are washed out and the cells are cultured for various lengths of time.
Anti-cancer activity of 13a is p53 and FLT3 status-dependent
In our previous study, we concluded hydrazide-based HDACI 13e displayed different mechanisms in
2
6
MV4-11 and PC-3 cells depending on their p53 status. However, only two cell lines were used and we
2
6
only showed a simple mechanism with the phenomenon of apoptosis and cell cycle arrest. Therefore, in
this study, we further verify this hypothesis by conducting experiments to determine 13a’s GI (average
5
0
growth inhibition concentration) and LC (average lethal concentration) in the NCI60 tumor cell lines via
5
0
the National Cancer Institute therapeutics program (Table 5). GI s of 13a in 60 cell lines range from 17.3-
5
0
338 nM; however, 13a does not induce cell death at these concentrations. 13a only induces lethality in 10
of the 60 cell lines at concentrations under 10 μM, and LC values of 13a towards 7 of the 10 lethality-
5
0
sensitive cell lines are below 1 μM. Unlike 13a, panobinostat induces death in 18 of the 60 cell lines under
the concentration of 10 μM (Figure 4A), which indicates that 13a has a more selective profile in the NCI
6
0 cell screen. Deeper analysis of the NCI 60 cell line screen revealed that 4 (NCI-H226, HCT116, LOX
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IMVI, and UACC-62) of the 7 most sensitive cell lines to 13a are p53 wild type (wt-p53); the other three
sensitive cell lines are p53 mutants. Notably, none of the p53-null cell lines display sensitivity to 13a
induced lethality. This result coincides with our previous hypothesis that the response of cancer cell lines
to hydrazide inhibitor is related to the p53 status.
0
1
2
3
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9
0
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In AML, p53 mutations occur in only ∼8% of de novo AML.66-67 Interestingly, over-expression of
wt-p53 is associated with FLT3-ITD mutations suggesting a potential therapeutic niche.68 FLT3 is
important for the development of the hematopoietic and activating mutations of FLT3, which is now
recognized as the most common molecular abnormality in AML and other hematologic malignancies as
well. Patients with this activating mutation were found to have an increased incidence of leukocytosis and
6
9
a decreased overall survival (OS). Therefore, we also checked the dependency of 13a with FLT3 status
in addition to p53.
We chose 6 different leukemia and multiple myeloma cell lines with different p53 and FLT3 status,
including MV4-11 and Molm-14 cell lines with wt-p53 and FLT3-ITD, the RS4;11 cell line with wt-p53
and wt-FLT3, as well as U937 and HL60 cell lines with p53-null and wt-FLT3 to test our compounds
using the NCI-60 screening methodology. Results show that the wt-p53 leukemia cell lines are more
sensitive to 13a than p53-null cell lines. Furthermore, 13a can cause cell death in wt-p53 MV4-11, Molm-
1
4, and RS4;11, but only inhibits proliferation of p53-null HL-60 and U937 (Figure 4B). Within the wt-
p53 cell lines, FLT3-ITD MV4-11 and Molm-14 cell lines are highly sensitive to HDACI treatment with
significantly lower LC than wt-FLT3 cell line RS4;11. Therefore, wt-p53 and FLT3-ITD cell lines have
5
0
the highest sensitivity of the 6 cell lines tested. We also tested the effect of 13a in primary wt-p53 AML
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cells (Figure 4C). 13a is toxic in all three patients’ cells with < 1 μM EC .
50
Table 5. National Cancer Institute Developmental Therapeutics Program In Vitro Testing Results of 13a.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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9
0
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0
Cancer Types
Cell Line
CCRF-CEM
HL-60
GI (nM)
LC (nM)
5
0
50
40.6
34.2
35.2
47.7
26.8
94.7
176
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
460
K-562
Leukemia
MOLT-4
RPMI-8226
SR
A549/ATCC
EKVX
285
Non-Small Cell
Lung Cancer
HOP-62
HOP-92
NCI-H226
102
23.0
52.3
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2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
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NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
182
173
163
96.9
29.8
143
22.9
313
32.4
116
53.0
198
131
98.9
>10,000
>10,000
>10,000
>10,000
660
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
1760
879
Colon Cancer
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
HT29
KM12
SW-620
SF-268
CNS Cancer
SF-295
SF-539
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SNB-19
SNB-75
231
312
>10,000
>10,000
4280
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
U251
80.1
118
LOX IMVI
MALME-3M
M14
537
48.2
72.5
97.5
73.9
68.7
192
>10,000
>10,000
>10,000
>10,000
893
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
Melanoma
>10,000
>10,000
589
79.6
72.6
115
>10,000
>10,000
Ovarian Cancer
OVCAR-3
135
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OVCAR-4
OVCAR-5
105
31.6
41.3
516
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
4300
0
1
2
3
4
5
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7
8
9
0
1
2
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9
0
1
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9
0
1
2
3
4
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9
0
1
2
3
4
5
6
7
8
9
0
OVCAR-8
NCI/ADR-RES
SK-OV-3
98.3
161
7
86-0
ACHN
CAKI-1
RXF 393
SN12C
TK-10
85.5
101
Renal Cancer
23.4
158
666
>10,000
>10,000
>10,000
>10,000
>10,000
17.3
173
UO-31
PC-3
54.2
90.8
Prostate Cancer
DU-145
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MCF7
MDA-MB-231/ATCC
HS 578T
106
203
121
338
23.9
50.4
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
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
Breast Cancer
BT-549
T-47D
MDA-MB-468
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