Class I/IIb-Selective HDAC Inhibitor Exhibits Oral Bioavailability and Therapeutic Efficacy in Acute Myeloid Leukemia

Article abstract of DOI:10.1021/acsmedchemlett.9b00471

The HDAC inhibitor 4-tert-butyl-N-(4-(hydroxycarbamoyl)phenyl)benzamide (AES-350, 51) was identified as a promising preclinical candidate for the treatment of acute myeloid leukemia (AML), an aggressive malignancy with a meagre 24% 5-year survival rate. Through screening of low-molecular-weight analogues derived from the previously discovered novel HDAC inhibitor, AES-135, compound 51 demonstrated greater HDAC isoform selectivity, higher cytotoxicity in MV4-11 cells, an improved therapeutic window, and more efficient absorption through cellular and lipid membranes. Compound 51 also demonstrated improved oral bioavailability compared to SAHA in mouse models. A broad spectrum of experiments, including FACS, ELISA, and Western blotting, were performed to support our hypothesis that 51 dose-dependently triggers apoptosis in AML cells through HDAC inhibition.

Full text of DOI:10.1021/acsmedchemlett.9b00471

Subscriber access provided by - Access paid by the | UCSB Libraries
Letter
Class I/IIb-Selective HDAC Inhibitor Exhibits Oral Bioavailability
and Therapeutic Efficacy in Acute Myeloid Leukemia
Andrew E. Shouksmith, Justyna M. Gawel, Nabanita Nawar, Diana Sina, Yasir S. Raouf,
Shazreh Bukhari, Liying He, Alexandra E. Johns, Pimyupa Manaswiyoungkul, Olasunkanmi O
Olaoye, Aaron D. Cabral, Abootaleb Sedighi, Elvin D de Araujo, and Patrick Thomas Gunning
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00471 • Publication Date (Web): 13 Dec 2019
Downloaded from pubs.acs.org on December 13, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 19
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
Class I/IIb-Selective HDAC Inhibitor Exhibits Oral Bioavailability and Therapeutic
Efficacy in Acute Myeloid Leukemia
7
8
9
Andrew E. Shouksmith†,#, Justyna M. Gawel†,#, Nabanita Nawar†, Diana Sina†, Yasir S. Raouf†,
Shazreh Bukhari†, Liying He†, Alexandra E. Johns†, Pimyupa Manaswiyoungkul†, Olasunkanmi
O. Olaoye†, Aaron D. Cabral†, Abootaleb Sedighi†, Elvin D. de Araujo†, Patrick T. Gunning†*
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
†Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359
Mississauga Road, Mississauga, Ontario, L5L 1C6, Canada
Abstract
The HDAC inhibitor 4-(tert-butyl)-N-(4-(hydroxycarbamoyl)phenyl)benzamide (AES-350, 51)
was identified as a promising pre-clinical candidate for the treatment of acute myeloid leukemia
(AML); an aggressive malignancy with a meagre 24% 5-year survival rate. Through screening of
low molecular-weight analogues derived from the previously discovered novel HDAC inhibitor,
AES-135, compound 51 demonstrated greater HDAC isoform selectivity, higher cytotoxicity in
MV4-11 cells, an improved therapeutic window, and more efficient absorption through cellular
and lipid membranes. Compound 51 also demonstrated improved oral bioavailability compared to
SAHA in mouse models. A broad spectrum of experiments, including FACS, ELISA, and western
blotting, were performed to support our hypothesis that 51 dose-dependently triggers apoptosis in
AML cells through HDAC inhibition.
Key Words
Histone deacetylases (HDACs), acute myeloid leukemia (AML), enzyme inhibition,
pharmacokinetics
Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are two protein classes with
antagonistic roles that regulate acetylation of histones as well as various cytoplasmic non-histone
proteins.1,2 HATs catalyze the transfer of acetyl groups to the ε-amino substituent of specific Lys
residues, whereas classical HDACs reverse this process using a metal co-factor to catalyze
hydrolysis of the acetyl group.1 There are 18 HDACs in the human proteome grouped into four
1
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 19
1
2
3
4
5
6
7
8
9
classes.2 Classes I, II, and IV are metal-dependent with corresponding HDAC inhibitors typically
coordinating to the metal co-factor rendering the protein inactive. Class III HDACs (sirtuins)
operate in conjunction with an NAD+ co-factor and are not assessed in this study. To date, four
small-molecule HDAC inhibitors have received FDA approval for cancer treatment: Vorinostat
(SAHA, 1),3 Belinostat (PXD101, 2),4 Panobinostat (LBH-589, 3)5 and Romidepsin (depsipeptide-
FK228, 4)6 (Figure 1). All compounds are approved specifically for hematological malignancies
although multiple clinical trials are ongoing with these compounds in combination studies against
various cancers, including gliomas, solid tumors, and AML. In contrast to the FDA approved
drugs, Ricolinostat and Citarinostat are the first HDAC6 selective inhibitors in clinical trials.7
HDAC6 is unique among the HDACs in that it is predominantly cytosolic and facilitates
microtubule deacetylation as well as regulation of PDL1 and other important targets related to
cancer immunotherapy. As such, HDAC6 has been implicated in oncogenesis and metastasis, with
the emergence of selective inhibitors as viable cancer therapeutics.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
Recently, we reported the discovery of AES-135 (5) (Figure 1); a novel nanomolar
inhibitor of HDAC3, 6, and 11 with in vitro cytotoxicity in low passage patient-derived pancreatic
cancer cells, even in the presence of cancer-associated fibroblasts (CAFs).10 Herein, we report
efforts to optimize the ligand efficiency of 5 via a focused SAR analysis. Several stripped-down
analogues of 5 exhibited enhanced HDAC6 inhibition and selectivity, with one inhibitor (51) also
demonstrating nanomolar cytotoxicity in MV4-11 (AML) cells.
F
F
O
O
O
O
H
N
OH
S
OH
N
N
N
F
F
H
H
H
O
O
F
O
O
O
HN
S
O
N
1
2
S
N
S
F
HN
HN
O
NH
O
OH
O
N
H
H
O
N
O
NH
OH
O
HN
3
5
4
Figure 1: Vorinostat (1), Belinostat (2), Panobinostat (3), Romidepsin (4) and AES-135 (5)
Given the molecular weight of 5 (693.7 g/mol), the relative importance of each substituent
towards in vitro HDAC potency was evaluated through the synthesis of truncated analogues (35–
40, 46, 47, 51, and 54). Analogues lacking one substituent were synthesized using Scheme 1.
2
ACS Paragon Plus Environment

Page 3 of 19
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Starting from tert-butyl glycine or sarcosine hydrochloride salts (6–7), sulfonylation was followed
either by acid-mediated removal of the tert-butyl protecting group, or by alkylation of the
sulfonamide and then tert-butyl deprotection to generate carboxylic acids 13–15. Microwave-
assisted coupling with various anilines generated a series of secondary and tertiary amides.
Secondary amides were alkylated prior to hydrogenation of the benzyloxy group, and tertiary
amides were hydrogenated directly. The resulting carboxylic acids 23–28 were coupled with O-
benzylhydroxylamine, and hydrogenation of the O-benzyl group yielded hydroxamic acids 35–40.
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 1. Synthesis of compounds 35–40
O
O
R1
N
O
R1
N
H
a
c
d
N
O
O
O
O
tBuO
R1
tBuO
S
R2
HO
S
R2
6 - 7
8 - 10
13 - 15
R1 = H
R1 = CH2C6F5
R1 = H
R2 = C6H5F
8 - 9
b
11 - 12
O
R1
O
R1
O
R1
N
R3
N
O
O
R3
N
O
O
R3
N
O
N
S
R2
N
S
R2
S
R2
O
f
g
O
OBn
16 - 21
O
OH
O
NH
OR
23 - 28
R3 = H
16
22
29 - 34
35 - 40
R = Bn
R = H
e
f
R3 = Me
i
(a) R2SO2Cl, Pr2Net, CH2Cl2, 16 h, RT, N2; (b) Pentafluorobenzyl bromide, Cs2CO3, MeCN, 16 h, RT; (c)
CF3CO2H/CHCl3 (1:3), 24 h, RT; (d) Appropriate aniline, PPh3Cl2, CHCl3, 90 min, 100 °C, MW, N2; (e) MeI, Cs2CO3,
MeCN, 20 h, RT; (f) H2, 10% Pd/C, THF/MeOH (2:1), 16 h, RT; (g) (i) (COCl)2, THF, DMF, 2 h, 0 °C; (ii) O-
benzylhydroxylamine, iPr2NEt, THF, 16 h, RT, N2. R1, R2, and R3 are variable depending on the molecule and shown
below in Table 1.
Fragment derivatives of 5 lacking the sulfonamide moiety were generated using Scheme
2. Reductive amination of benzyl 4-aminobenzoate (41) with 4-tert-butylbenzaldehyde and
protection of the resulting secondary aniline yielded carbamate 43. Hydrogenation of the
3
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 19
1
2
3
4
5
6
7
8
9
benzyloxy group, coupling with O-benzylhydroxylamine, and removal of the O-benzyl group
generated hydroxamic acid 46. Subsequent acid-mediated removal of the Boc group yielded
compound 47. Deprotection of the Boc group and acylation of the aniline prior to hydrogenation
of the O-benzyl group formed compound 54. Coupling of 10 with 4-tert-butylbenzoic acid,
followed by conversion of the benzyl ester to the hydroxamic acid, as described previously, gave
analogue 51 (AES-350).
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. Synthesis of compounds 46, 47, 51, and 54
O
O
NH2
O
N
OtBu
NH
N
OtBu
c
OtBu
a
b
d
e
NH
N
O
OBn
O
NH
OH
O
NH
OH
O
NH
O
OBn
O
OR
OBn
41
42
R=Bn 43
44
46
47
45
c
R=H
f
e
O
d
g
NH
O
NH
O
NH
N
O
NH
OBn
O
OR
O
NH
OR
O
NH
OR
R=Bn
R=Bn
R=H
48
49
50
51
R=Bn
R=H
53
54
52
c
c
c
R=H
(a) (i) 4-Tert-butylbenzaldehyde, THF/TFE (4:1), 16 h, RT; (ii) NaBH4, MeOH, 6 h, RT; (b) (Boc)2O, DMAP, MeCN,
2 h, 80 °C, MW; (c) H2, 10% Pd/C, THF/MeOH (2:1), 16 h, RT; (d) (i) (COCl)2, THF, DMF, 2 h, 0 °C; (ii) O-
benzylhydroxylamine, iPr2NEt, THF, 16 h, RT, N2; (e) CF3CO2H/CHCl3 (1:3), 18 h, RT; (f) 4-Tert-butylbenzoic acid,
PPh3Cl2, CHCl3, 90 min, 100 °C, MW, N2; (g) AcCl, CH2Cl2, 24 h, 0 °C – RT.
All compounds were screened against HDAC3, 6, 8, and 11 in an enzymatic activity assay
and fluorescence polarization binding assay. (Table S2, Supporting Information). Compounds 35–
40, bearing a central amide and lacking one substituent relative to 5, displayed increased potency
against HDAC6 (1.6–128-fold improvement) (Table 1). Ostensibly, variations in ligand efficiency
altered interactions with other HDAC proteins. Notably, when R3 is a benzyl substituent (35),
4
ACS Paragon Plus Environment

Page 5 of 19
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
general HDAC inhibition increases substantially over the larger tert-butylbenzyl or smaller methyl
group. When R1 was changed from a pentafluorobenzene (PFB) (5) to a methyl group (37) or
deleted (38), potency against HDACs 3 and 11 was unaffected, but there was a significant gain in
HDAC8 potency. Conversion of R2 from a 4-fluorobenzene (5) to a methyl group (39) increased
HDAC6 and 8 inhibition, but with a concomitant loss in HDAC11 inhibition.
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
Analogues of 5 with both the PFB and 4-fluorobenzenesulfonamide substituents removed
showed high variation in HDAC selectivity (Table 1). Boc-protected derivative 46 showed higher
potency and selectivity for HDAC6 than 5 or its deprotected derivative 47. The structurally more
rigid amide 51 was similarly potent against HDAC6, but also highly potent against HDAC3 and
8. Acetyl derivative 54 was one of the most potent HDAC8 inhibitors in this series (IC50 = 0.085
μM), with comparably high activity against HDAC6 (IC50 = 0.071 μM). None of the inhibitors
lacking two aromatic substituents from compound 5 showed significant potency against HDAC11,
highlighting the importance of steric bulk in the design of HDAC11-targeting compounds.
In conjunction with biochemical assays, inhibitors were assessed for cytotoxic activity in
several cancer cell lines including MV4-11 and MOLM-13 (AML), MDA-MB-231 and MDA-
MB-468 (breast cancer), as well as MRC-9 lung cells (non-cancerous) and compared to Vorinostat
(1) and 5 (Table 1). Deletion of different substituents resulted in complete abrogation of
cytotoxicity in breast cancer cells and reduction in potency in MOLM-13 cells (2–12 fold), with
the exception of 39. In MV4-11 cells, loss of activity was smaller (2–3 fold), with 39 again
demonstrating similar potency. While 35 showed impressive HDAC6-selectivity and potency in
the enzymatic assay, potency in MV4-11 cells was markedly weaker (>5 µM). Contrastingly, 51
was more potent than 5, with sub-micromolar activity (IC50 = 0.58 ± 0.13 μM) akin to Vorinostat
(IC50 = 0.31 ± 0.061 μM). At <50% the molecular weight of 5, compound 51 is more ligand
efficient, and exemplifies a large therapeutic index (IC50>30 µM in non-cancerous MRC-9 cells).
Compound 51 was also shown to be effective in AML-3 cell lines (IC50 = 0.73 ± 0.12 μM) similar
to SAHA (IC50 = 1.30 ± 0.85 μM) and Citarinostat (IC50 = 4.40 ± 0.99 μM). In combination with
the observed in vitro potency and selectivity of 51 (HDAC6 IC50 = 24 nM), it was selected for
further pharmacologic studies.
5
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 19
1
2
3
4
5
6
Table 1. IC50 values for compounds 35 – 40, 46, 47, 51, and 54 against HDACs 3, 6, 8, and 11
(EMSA, n = 1) and in MV4-11, MOLM-13, MDA-MB-231, MDA-MB-468, and MRC-9 cells (±
SD), and Ki values against zebra-fish HDAC6 catalytic domain 2.
7
8
9
F
F
F
F
R2
F
R3
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
F
F
F
F
O
F
O
O
O
H
O
S
O
S
O
S
O
S
N
O
N
O
N
O
N
O
N
N
N
N
R1
OH
F
OH
F
OH
F
OH
F
O
N
H
O
N
H
O
N
H
O
N
H
36
37
38
35
F
F
F
F
O
O
O
F
O
O
O
S
N
O
N
O
N
S
N
O
NH
O
NH
N
N
OH
F
OH
OH
OH
OH
OH
O
N
O
N
O
N
O
N
O
N
H
O
N
H
H
H
H
H
39
HDAC Activity IC50 (μM)a
40
46
47
51
Cytotoxicity (μM)
54
HDAC6 Ki (μM)
#
HDAC3
HDAC6
HDAC8
HDAC11
HDAC6
MV4-11
MOLM-13
MDA-MB-231
MDA-MB-468
MRC-9
1
5
0.00657
0.0058
0.497
>1
<0.035
0.310 ± 0.061c
1.88 ± 0.89d
0.345 ± 0.035b
-
-
>25b
19.2 ±
5.80c
0.654
0.190
>1
0.636
3.600
2.10 ± 0.22c
2.62 ± 0.56c
4.21 ± 1.76d
35
36
0.132
0.213
0.00149
0.0201
0.0906
0.224
0.0519
>1
1.860
0.573
5.80 ± 1.37b
3.24 ± 1.14b
8.05 ± 1.31b
14.1 ± 4.56b
-
-
-
>25b
>25b
>25b
11.8 ±
1.46b
37
38
0.492
0.372
0.0552
0.0528
0.0379
0.0280
0.697
0.665
0.768
0.293
3.41 ± 0.29b
3.33 ± 0.34b
5.46 ± 0.74b
5.11 ± 0.88b
>25b
>12.5b
>12.5b
10.8 ±
0.45b
-
39
40
0.213
0.708
0.0277
0.117
0.133
0.573
>1
>1
1.093
0.793
1.62 ± 0.29c
14.6 ± 1.44b
2.98 ± 0.36b
24.8 ± 1.30b
>12.5b
-
>25b
-
>25b
-
9.49 ±
0.57b
46
0.374
0.0282
0.635
0.837
0.201
3.12 ± 0.66b
10.3 ± 0.95b
>25b
>25b
6
ACS Paragon Plus Environment

Page 7 of 19
ACS Medicinal Chemistry Letters
1
2
3
4
16.1 ±
0.34b
47
0.503
0.110
>1
>1
0.451
3.49 ± 1.06b
9.47 ± 2.12b
>25b
>25b
5
6
7
8
33.2 ±
10.3b
51
54
0.187
0.276
0.0244
0.0713
0.245
>1
>1
0.035
0.199
0.576 ± 0.131e
4.24 ± 2.83b
6.00 ± 2.74c
10.6 ± 2.17b
>25b
>25b
>50b
>25b
0.0854
-
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
aCompounds evaluated to 1 μM; bn = 2; cn = 3; dn = 4; en = 8; R1, R2, R3 shown in blue, red, and green respectively.
The cytotoxicity data for 51 was corroborated by fluorescence-activated cell sorting (FACS)
(Figure 2). MV4-11 cells were cultured for 18 h with increasing concentrations of 51 or SAHA,
before treatment with apoptosis indicators Annexin V and propidium iodide. The findings revealed
a clear dose-dependent increase with the percentage of cells entering late-stage apoptosis, similar
to SAHA, further supporting the anticancer activity of 51 in this cell line. In vitro stability of 51
was evaluated in mouse hepatocytes, by comparing the rates of intrinsic clearance of Verapamil
(control) with 51. Compound 51 (t = 28.3 min) exhibited ~1.5-fold longer half life than Verapamil
½
(t = 18.0 min). Previous assays under the same conditions revealed a longer half-life for 5 (t =
½
½
38.5 min), although Verapamil also showed a 17.8% variation (t = 21.9 min).10 Normalizing the
½
data of 5 and 51 according to the control Verapamil, suggests that after removing >50% of the
total mass of 5, the hepatocyte half-life of 51 was reduced by only 10%. Similarly, the clearance
rate of 51 (49.1 μL/min per 106 cells) was lower than that of Verapamil (77.0 μL/min per 106 cells)
but faster than 5 (36.0 μL/min per 106 cells) (Table S5, Figures S12 – S15, Supporting
Information).
7
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 8 of 19
1
2
3
A.
4
5
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
8
ACS Paragon Plus Environment

Page 9 of 19
ACS Medicinal Chemistry Letters
B.
1
2
3
4
5
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
Figure 2: (A) Dot plots and (B) stacked bar graphs representing the distribution of MV4-11 cells
classed as healthy, early apoptosis, and late apoptosis 18 h post-dosing with varying concentrations
of SAHA and 51 (AES-350) using FACs.
Notably, 51 is markedly less bound to plasma proteins (95.3% protein bound) compared to 5
(99.6%), although both compounds were poorly recovered (~20%) suggesting a metabolic liability
in their composition. (Tables S6 – S8, Supporting Information).10
The significantly lower molecular weight of 51 (312.4 g/mol) suggests it would exhibit
superior membrane permeability in comparison to 5. Parallel artificial membrane permeability
assay (PAMPA) was used to approximate permeation of 51 through the blood-brain barrier, where
a permeability coefficient (-Log Pe) <6 is defined as having high permeability. Compound 51 was
found to have a -Log Pe of 5.14, indicating relatively good lipid bilayer permeability, and was
87.1% recovered in comparison to the parent 5 (-Log Pe = 7.73, 28.7 % recovery, Tables S9–S12,
Supporting Information). 51 was further analyzed in a small intestinal membrane model (Caco-2)
assay to gauge permeability through a monolayer of epithelial cells as well as compound efflux.
Metoprolol and Digoxin were used as controls with low and high efflux ratios respectively. In
agreement with PAMPA, 51 demonstrated a high permeability (apparent permeability coefficient,
Papp A-B = 3.45 × 10-6 cm/s), approximately 13-times the rate of 5 (Papp A-B = 0.27 × 10-6 cm/s),
Additionally, efflux ratios for Metoprolol, Digoxin and 5 were all higher (1.00, 72.99 and 3.83,
respectively) in comparison to 51 (0.64) indicating that 51 would likely demonstrate intestinal
absorption if ingested orally (Tables S13 – S17, Supporting Information).11
9
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 10 of 19
1
2
3
4
5
6
7
8
9
To gain a comprehensive HDAC selectivity profile of 51, the compound was screened in
vitro against all 11 metal-dependent HDACs (Table 2). Known pan-HDAC inhibitors Trichostatin
A (TSA), Quisinostat (JNJ-26481585, Phase II clinical trials in CTCL and ovarian cancer), and
Group I-selective inhibitor Entinostat (MS-275, Phase III clinical trials in breast cancer / Phase II
trials in renal cell carcinoma) were used as positive controls and showed IC50 values/selectivity
profiles consistent with the literature.12–14 51 was found to be selective for HDAC6 with no
inhibition observed for the remaining HDACs at >1 μM, in contrast to both Quisinostat and
Entinostat which displayed low nM IC50 values against almost all HDACs (Tables S2–S4, Figures
S1–S11, Supporting Information). Indeed, 51 could be considered equally to marginally more
selective for HDAC6 than Ricolinostat and Citarinostat under these assay conditions. Although
both compounds are more potent than 51 against HDAC6, they are also more potent against
HDAC3, which reduces their selectivity.
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
N
N
O
O
HN
NH
N
O
N
O
N
HN
O
H2N
O
NH
OH
NH
OH
Quisinostat
(JNJ-26481585)
Entinostat
(MS-275)
Trichostatin A
(TSA)
Table 2. IC50 Values for 51 Against 11 Classical HDACs (EMSA, n = 1)
HDAC IC50 (μM)a
Cpd
51
1
2
3
4
>1
5
6
7
8
9
>1
10
11
>1
>1
>1
0.605
>1
0.187
>1
0.0244
0.000954
0.0399
>1
0.245
0.207
0.0024
0.899
TSA
0.000681
0.000617
0.00274
0.00216
0.000404
0.000478
>1
0.776
0.00595
0.482
0.00404
>1
0.00161
0.00196
JNJ-26481585
0.00461
0.0067
10
ACS Paragon Plus Environment

Page 11 of 19
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
MS-275
0.118
0.247
0.379
0.318
0.223
0.0143
0.0125
>1
>1
>1
>1
-
>1
>1
-
>1
>1
-
>1
-
>1
>1
>1
Ricolinostat
Citarinostat
-
-
0.00259
0.00221
0.245
0.171
-
-
-
-
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
To explain the observed selectivity of 51, the compound was modelled in the catalytic
domain 2 (CD2) of HDAC6 and HDAC8 using Maestro v11.9.01 (Schrödinger, LLC, New York,
NY 2019).15–17 (Figure 3A–D) The hydroxamic acid of 51 formed a H-bond between the carbonyl
oxygen and hydroxyl group of Tyr745 (HDAC6) or Tyr306 (HDAC8), and a salt bridge
interaction between the deprotonated acid and protonated His573 (HDAC6) or His142 (HDAC8).
Crucially, the benzene ring of 51 formed π-stacking interactions with Phe583 and Phe643 of
HDAC6 not observed in HDAC8. This twin π-stacking phenomenon is responsible for selectivity
of this structural motif for HDAC6 over other isoforms.18,19 Additionally, the amide NH in 51
formed a hydrogen bond with the hydroxyl of Ser531, giving an average free energy of binding
(ΔGB) of -8.5 kcal/mol. Neither of these interactions were observed with HDAC8 (ΔGB was -7.8
kcal/mol, Figure S16, Supporting Information).
11
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 12 of 19
1
2
3
4
5
6
7
8
9
Figure 3: A. Docking of 51 with zebra fish HDAC6 CD2 (light blue) (PDB: 6CSR), B. Ligand
interaction diagram depicting the key interactions of 51 in HDAC6 CD2. C. Docking of 51 in the
human HDAC8 catalytic domain (green) (PDB: 6HSK). D. Ligand interaction diagram depicting
the key interactions of 51 in the HDAC8 catalytic domain. E. Overlap of 51 (grey) with Quisinostat
(red) in the human HDAC8 catalytic domain (PDB: 6HSK). In A, C, and E, 51 is illustrated by
grey (C), white (H), blue (N), red (O), yellow (Zn2+). Other interactions are shown as follows: π-
stacking (green dashed lines), H-bonds and salt bridges (yellow dashed lines) and Zn2+ chelation
(yellow/red dots).
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
To validate the proposed cellular HDAC inhibition, an enzyme-linked immunosorbent-
based assay (ELISA) was performed using HeLa cervical cancer cell lysates. HeLa cells highly
express HDAC6, and were sensitive to 51 (IC50 = 2.53 ± 0.57 μM) (Table S1, Supporting
Information). Correspondingly, ELISA-assays depicted a dose-dependent increase in HDAC6
inhibition (IC50 = 0.58 μM), and supported 51-induced cell death via HDAC6 inhibition (Figure
4A). ELISA-based data were also supported by western blot analysis of 51-treated MV4-11 cells
(Figure 4B) with a dose-dependent increase in acetylated alpha-tubulin (Ac-α-tubulin), a substrate
of HDAC6.20 Collectively, ELISA and western blot analysis suggest 51 elicits HDAC6 inhibition
in vitro to dose-dependently facilitate apoptosis. It is important to note that despite the observed
HDAC6 selectivity in vitro there is also a significant reduction of the biomarker of acetylated
histone (Ac-Histone H3, which is a biomarker of pan-HDAC activity),21 and acetylated tubulin in
cellulo which suggests there may be additional Class I activity of the compound.9 As such, the in
vitro selectivity of 51 may not be observed in cellulo to same extent which may be the result of
alternative inhibition mechanisms.
A.
B.
12
ACS Paragon Plus Environment

Page 13 of 19
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
Figure 4: A. HDAC6 inhibition profile with varying concentrations of 51 in HeLa cell lysates
(IC50 = 0.58 ± 0.13 μM, n = 2). B. Western blots probing for Ac-α-tubulin, Ac-Histone H3, and
HSC70 from MV4-11 after 6 h with varying concentrations of 51 and SAHA.
7
8
9
To evaluate pharmacokinetic (PK) properties of 51, CD-1 mice were dosed (20 mg/Kg,
PO, and 5 mg/Kg IV; Figure 5, Table S18 – S22, Supporting Information). The single dose oral
bioavailability (F) of 51 was 19.8%. In comparison, the reported F value for SAHA in mice is
significantly lower (8%).22 The rapid clearance rate of 51 would require regular dosing, alternative
administration routes, or larger dosages. In previous studies, 5 achieved an average Cmax of 10.74
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
μM in NSG mice that was sustained for 8 h (t = 5.0 h).10 However, it should be noted that 5 could
½
only be dosed by IP, due to poor absorption profiles.
Figure 5. Mean Plasma Concentrations of 51 in CD-1 Mice Following PO (20 mg/Kg, n = 3) and
IV administration (5 mg/Kg, n = 3)
Systematic modifications of novel HDAC inhibitor, 5, led to the discovery of 51, a
comparatively ligand-efficient compound. Encouragingly, 51, exhibited an in vitro HDAC6
selectivity profile comparable to that of Ricolinostat and Citarinostat. This translated into cellular
target engagement, with dose-dependent HDAC6 inhibition in HeLa and MV4-11 cells. Although
51 did not display the same extent of HDAC6 selectivity in cellulo as observed in the in vitro
studies, the compound demonstrated superior cytotoxicity compared to its predecessor in MV4-
11, which was corroborated with FACS experiments. The membrane permeability of 51 was also
significantly improved compared to 5 based on PAMPA and Caco-2 assays, which translated into
13
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 14 of 19
1
2
3
4
5
6
7
8
9
an orally absorbed therapeutic in CD-1 mice. While initial pharmacological data have been
promising, ultimately, 51 was not as potent as its FDA-approved competitor SAHA in AML cells.
Moreover, although 51 was discovered during our SAR studies, we subsequently discovered it had
been previously documented in patent literature.23–25 Despite these shortcomings, this investigation
represents the first disclosure of the mechanism of action for 51 in cancer cells with potency for
AML, potentially enabling close analogues to reach pre-clinical investigation for AML treatment.
Overall, 51 represents an improved orally bioavailable analogue of 5, and a promising candidate
for further medicinal chemistry efforts to build upon the HDAC selectivity/potency and
pharmacological properties.
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
Supporting Information
 Details of compound synthesis, characterization, in vitro, and in cellulo assays
 Molecular formula strings for final compounds
Author Information:
Author Addresses:
Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359
Mississauga Rd N., Mississauga, Ontario L5L 1C6, Canada
*E-mail: patrick.gunning@utoronto.ca. Telephone: (905) 828-5354.
Author Contributions:
#A.E.S and J.M.G contributed equally to this work. A.E.S., J.M.G., L.H., A.E.J., A.D.C
synthesized/characterized the compounds. J.M.G., N.N., D.S., S.B., P.M., O.O.O., E.D.A., A.S.
carried out biological and biophysical evaluation of the compounds. Y.S.R. performed in silico
experiments. A.E.S., J.M.G., E.D.A., and P.T.G. wrote the manuscript with input from all authors.
Acknowledgements:
P.T.G is supported by research grants from NSERC (RGPIN-2014-05767), CIHR (MOP-130424,
MOP-137036), Canada Research Chair (950-232042), Canadian Cancer Society (703963),
Canadian Breast Cancer Foundation (705456), Leukemia and Lymphoma Society of Canada and
14
ACS Paragon Plus Environment

Page 15 of 19
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
infrastructure grants from CFI (33536) and the Ontario Research Fund (34876). A.E.S. is
supported by a Mitacs Accelerate Grant (IT05960).
7
8
9
Abbreviations used:
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
HAT, histone acetyltransferase; HDAC, histone deacetylase; SAHA, suberanilohydroxamic acid;
CTCL, cutaneous T-cell lymphoma; PTCL, peripheral T-cell lymphoma; MM, multiple myeloma;
AML, acute myeloid leukemia; CAF, cancer-associated fibroblast; IP, intraperitoneal; PO, per os;
Boc, tert-butoxycarbonyl; PFB, pentafluorobenzyl; FB, fluorobenzene; EMSA, electrophoretic
mobility shift assay; FACS, fluorescence-activated cell sorting; ELISA, enzyme-linked
immunosorbent assay; PAMPA, parallel artificial membrane permeability assay; TSA, trichostatin
A;
References:
(1)
(2)
(3)
Khan, O.; La Thangue, N. B. HDAC Inhibitors in Cancer Biology: Emerging Mechanisms
and Clinical Applications. Immunology and Cell Biology. 2012, pp 85–94.
https://doi.org/10.1038/icb.2011.100.
Ceccacci, E.; Minucci, S. Inhibition of Histone Deacetylases in Cancer Therapy: Lessons
from Leukaemia. British Journal of Cancer 2016, 114 (6), 605–611.
https://doi.org/10.1038/bjc.2016.36.
Kumagai, T.; Wakimoto, N.; Yin, D.; Gery, S.; Kawamata, N.; Takai, N.; Komatsu, N.;
Chumakov, A.; Imai, Y.; Koeffler, H. P. Histone Deacetylase Inhibitor, Suberoylanilide
Hydroxamic Acid (Vorinostat, SAHA) Profoundly Inhibits the Growth of Human
Pancreatic Cancer Cells. International Journal of Cancer 2007, 121 (3), 656–665.
https://doi.org/10.1002/ijc.22558.
(4)
(5)
Qian, X.; Ara, G.; Mills, E.; LaRochelle, W. J.; Lichenstein, H. S.; Jeffers, M. Activity of
the Histone Deacetylase Inhibitor Belinostat (PXD101) in Preclinical Models of Prostate
Cancer. International Journal of Cancer 2008, 122 (6), 1400–1410.
https://doi.org/10.1002/ijc.23243.
Ellis, L.; Pan, Y.; Smyth, G. K.; George, D. J.; McCormack, C.; Williams-Truax, R.; Mita,
15
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 16 of 19
1
2
3
4
5
6
7
8
9
M.; Beck, J.; Burris, H.; Ryan, G.; et al. Histone Deacetylase Inhibitor Panobinostat
Induces Clinical Responses with Associated Alterations in Gene Expression Profiles in
Cutaneous T-Cell Lymphoma. Clinical Cancer Research 2008, 14 (14), 4500–4510.
https://doi.org/10.1158/1078-0432.CCR-07-4262.
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
(6)
(7)
Piekarz, R. L.; Frye, R.; Turner, M.; Wright, J. J.; Allen, S. L.; Kirschbaum, M. H.; Zain,
J.; Prince, H. M.; Leonard, J. P.; Geskin, L. J.; et al. Phase II Multi-Institutional Trial of
the Histone Deacetylase Inhibitor Romidepsin as Monotherapy for Patients with
Cutaneous T-Cell Lymphoma. Journal of Clinical Oncology 2009, 27 (32), 5410–5417.
https://doi.org/10.1200/JCO.2008.21.6150.
Dasmahapatra, G.; Patel, H.; Friedberg, J.; Quayle, S. N.; Jones, S. S.; Grant, S. In Vitro
and in Vivo Interactions between the HDAC6 Inhibitor Ricolinostat (ACY1215) and the
Irreversible Proteasome Inhibitor Carfilzomib in Non-Hodgkin Lymphoma Cells.
Molecular Cancer Therapeutics 2014, 13 (12), 2886–2897. https://doi.org/10.1158/1535-
7163.MCT-14-0220.
(8)
(9)
Li, T.; Zhang, C.; Hassan, S.; Liu, X.; Song, F.; Chen, K.; Zhang, W.; Yang, J. Histone
Deacetylase 6 in Cancer. Journal of Hematology and Oncology. 2018.
https://doi.org/10.1186/s13045-018-0654-9.
Vogl, D. T.; Raje, N.; Jagannath, S.; Richardson, P.; Hari, P.; Orlowski, R.; Supko, J. G.;
Tamang, D.; Yang, M.; Jones, S. S.; et al. Ricolinostat, the First Selective Histone
Deacetylase 6 Inhibitor, in Combination with Bortezomib and Dexamethasone for
Relapsed or Refractory Multiple Myeloma. Clinical Cancer Research 2017, 23 (13),
3307–3315. https://doi.org/10.1158/1078-0432.CCR-16-2526.
(10) Shouksmith, A. E.; Shah, F.; Grimard, M. L.; Gawel, J. M.; Raouf, Y. S.; Geletu, M.;
Berger-Becvar, A.; De Araujo, E. D.; Luchman, H. A.; Heaton, W. L.; et al. Identification
and Characterization of AES-135, a Hydroxamic Acid-Based HDAC Inhibitor That
Prolongs Survival in an Orthotopic Mouse Model of Pancreatic Cancer. Journal of
Medicinal Chemistry 2019, 62 (5), 2651–2665.
https://doi.org/10.1021/acs.jmedchem.8b01957.
(11) Sambuy, Y.; De Angelis, I.; Ranaldi, G.; Scarino, M. L.; Stammati, A.; Zucco, F. The
16
ACS Paragon Plus Environment

Page 17 of 19
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
Caco-2 Cell Line as a Model of the Intestinal Barrier: Influence of Cell and Culture-
Related Factors on Caco-2 Cell Functional Characteristics. Cell Biology and Toxicology.
2005, pp 1–26. https://doi.org/10.1007/s10565-005-0085-6.
7
8
9
(12) Tong, W. G.; Wei, Y.; Stevenson, W.; Kuang, S. Q.; Fang, Z.; Zhang, M.; Arts, J.; Garcia-
Manero, G. Preclinical Antileukemia Activity of JNJ-26481585, a Potent Second-
Generation Histone Deacetylase Inhibitor. Leukemia Research 2010, 34 (2), 221–228.
https://doi.org/10.1016/j.leukres.2009.07.024.
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
(13) Arts, J.; King, P.; Mariën, A.; Floren, W.; Beliën, A.; Janssen, L.; Pilatte, I.; Roux, B.;
Decrane, L.; Gilissen, R.; et al. JNJ-26481585, a Novel “Second-Generation” Oral
Histone Deacetylase Inhibitor, Shows Broad-Spectrum Preclinical Antitumoral Activity.
Clinical Cancer Research 2009, 15 (22), 6841–6851. https://doi.org/10.1158/1078-
0432.CCR-09-0547.
(14) Tatamiya, T.; Saito, A.; Sugawara, T.; Nakanichi, O. Isozyme-Selective Activity of the
HDAC. In Proc Amer Assoc Cancer Res; 2004; Vol. 2004, pp 5–6.
(15) Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.; Halgren,
T. A.; Sanschagrin, P. C.; Mainz, D. T. Extra Precision Glide: Docking and Scoring
Incorporating a Model of Hydrophobic Enclosure for Protein-Ligand Complexes. Journal
of Medicinal Chemistry 2006, 49 (21), 6177–6196. https://doi.org/10.1021/jm051256o.
(16) Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W. T.;
Banks, J. L. Glide: A New Approach for Rapid, Accurate Docking and Scoring. 2.
Enrichment Factors in Database Screening. Journal of Medicinal Chemistry 2004, 47 (7),
1750–1759. https://doi.org/10.1021/jm030644s.
(17) Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.;
Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; et al. Glide: A New Approach for
Rapid, Accurate Docking and Scoring. 1. Method and Assessment of Docking Accuracy.
Journal of Medicinal Chemistry 2004, 47 (7), 1739–1749.
https://doi.org/10.1021/jm0306430.
(18) Porter, N. J.; Mahendran, A.; Breslow, R.; Christianson, D. W. Unusual Zinc-Binding
17
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 18 of 19
1
2
3
4
5
6
Mode of HDAC6-Selective Hydroxamate Inhibitors. Proceedings of the National
Academy of Sciences of the United States of America 2017, 114 (51), 13459–13464.
https://doi.org/10.1073/pnas.1718823114.
7
8
9
(19) Porter, N. J.; Wagner, F. F.; Christianson, D. W. Entropy as a Driver of Selectivity for
Inhibitor Binding to Histone Deacetylase 6. Biochemistry 2018, 57 (26), 3916–3924.
https://doi.org/10.1021/acs.biochem.8b00367.
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
(20) Hubbert, C.; Guardiola, A.; Shao, R.; Kawaguchi, Y.; Ito, A.; Nixon, A.; Yoshida, M.;
Wang, X. F.; Yao, T. P. HDAC6 Is a Microtubule-Associated Deacetylase. Nature 2002,
417 (6887), 455–458. https://doi.org/10.1038/417455a.
(21) Seto, E.; Yoshida, M. Erasers of Histone Acetylation: The Histone Deacetylase Enzymes.
Cold Spring Harbor Perspectives in Biology 2014, 6 (4).
https://doi.org/10.1101/cshperspect.a018713.
(22) Venkatesh, P. R.; Goh, E.; Zeng, P.; New, L. S.; Xin, L.; Pasha, M. K.; Sangthongpitag,
K.; Yeo, P.; Kantharaj, E. In Vitro Phase I Cytochrome P450 Metabolism, Permeability
and Pharmacokinetics of SB639, a Novel Histone Deacetylase Inhibitor in Preclinical
Species. Biological and Pharmaceutical Bulletin 2007, 30 (5), 1021–1024.
https://doi.org/10.1248/bpb.30.1021.
(23) Rho, H. S.; Baek, H. S.; Lee, J. A.; Park, C. M.; Kim, D. H.; Chang, I. S.; Lee, O. S.
Hydroxamic Acid Derivatives and the Preparation Method Thereof, 2009.
https://doi.org/WO2005/19162 A1.
(24) Lu, Q.; Yang, Y. T.; Chen, C. S.; Davis, M.; Byrd, J. C.; Etherton, M. R.; Umar, A.; Chen,
C. S. Zn2+-Chelating Motif-Tethered Short-Chain Fatty Acids as a Novel Class of
Histone Deacetylase Inhibitors, 2004. https://doi.org/10.1021/jm0303655.
(25) London, C. A.; Lin, T.-Y.; Kulp, S. Methods and Compositions for Inhibiting and
Preventing the Growth of Malignant Mast Cells, 2011. https://doi.org/WO2011/103563
A1.
18
ACS Paragon Plus Environment

Page 19 of 19
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
Class I/IIb-Selective HDAC Inhibitor Exhibits Oral Bioavailability and Therapeutic
Efficacy in Acute Myeloid Leukemia
7
8
9
Andrew E. Shouksmith†,#, Justyna M. Gawel†,#, Nabanita Nawar†, Diana Sina†, Yasir S. Raouf†,
Shazreh Bukhari†, Liying He†, Alexandra E. Johns†, Pimyupa Manaswiyoungkul†, Olasunkanmi
O. Olaoye†, Aaron D. Cabral†, Abootaleb Sedighi†, Elvin D. de Araujo†, Patrick T. Gunning†*
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
ACS Paragon Plus Environment