Characterization of Conformationally Constrained Benzanilide Scaffolds for Potent and Selective HDAC8 Targeting

Article abstract of DOI:10.1021/acs.jmedchem.0c01025

Histone deacetylases (HDACs) are an attractive therapeutic target for a variety of human diseases. Currently, all four FDA-approved HDAC-targeting drugs are nonselective, pan-HDAC inhibitors, exhibiting adverse side effects at therapeutic doses. Although selective HDAC inhibition has been proposed to mitigate toxicity, the targeted catalytic domains are highly conserved. Herein, we describe a series of rationally designed, conformationally constrained, benzanilide foldamers which selectively bind the catalytic tunnel of HDAC8. The series includes benzanilides, MMH371, MMH409, and MMH410, which exhibit potent in vitro HDAC8 activity (IC50 = 66, 23, and 66 nM, respectively) and up to 410-fold selectivity for HDAC8 over the next targeted HDAC. Experimental and computational analyses of the benzanilide structure docked with human HDAC8 enzyme showed the adoption of a low-energy L-shaped conformer that favors HDAC8 selectivity. The conformationally constrained HDAC8 inhibitors present an alternative biological probe for further determining the clinical utility and safety of pharmacological knockdown of HDAC8 in diseased cells.

Full text of DOI:10.1021/acs.jmedchem.0c01025

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Article
Characterization of Conformationally Constrained Benzanilide
Scaffolds for Potent and Selective HDAC8 Targeting
Muhammad Murtaza Hassan, Johan Israelian, Nabanita Nawar, Giovanni Ganda, Pimyupa Manaswiyoungkul,
Yasir Raouf, David Armstrong, Abootaleb Sedighi, Olasunkanmi O Olaoye, Fettah Erdogan, Aaron
D. Cabral, Fabrizio Angeles, Rabia Altintas, Elvin D de Araujo, and Patrick Thomas Gunning
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c01025 • Publication Date (Web): 16 Jul 2020
Downloaded from pubs.acs.org on July 16, 2020
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Characterization of Conformationally Constrained Benzanilide
Scaffolds for Potent and Selective HDAC8 Targeting
Muhammad Murtaza Hassan†,‡, Johan Israelian†,‡, Nabanita Nawar†,‡, Giovanni Ganda†,‡, Pimyupa
Manaswiyoungkul†,‡, Yasir S. Raouf†,‡, David Armstrong†, Abootaleb Sedighi†,‡, Olasunkanmi O.
Olaoye†,‡, Fettah Erdogan†,‡, Aaron D. Cabral†,‡, Fabrizio Angeles†,‡, Rabia Altintas§,#, Elvin D. de
Araujo†, Patrick T. Gunning*†,‡
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†
Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road North,
Mississauga, ON L5L 1C6, Canada.
‡
Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON M5S 3H6, Canada.
§ Clinical Cooperation Unit Pediatric Oncology (G340), German Cancer Research Center (DKFZ), Im Neuenheimer Feld
280, Heidelberg 69120, Germany
#
Heidelberg Medical Faculty, University of Heidelberg, Heidelberg 691171, Germany
KEYWORDS HDAC8-selective, small molecule inhibitor, conformational constraint, neuroblastoma, immunofluorescence,
histone deacetylase 8 (HDAC8), benzanilide, L-shaped.
ABSTRACT: HDACs are an attractive therapeutic target for a variety of human diseases. Currently, all four FDA-approved HDAC-
targeting drugs are non-selective, pan-HDAC inhibitors, exhibiting adverse side-effects at therapeutic doses. While selective HDAC-
inhibition has been proposed to mitigate toxicity, the targeted catalytic domains are highly conserved. Herein, we describe a series of
rationally designed, conformationally constrained, benzanilide foldamers which selectively bind the catalytic tunnel of HDAC8. The
series includes benzanilides, MMH371, MMH409, and MMH410, which exhibit potent in vitro HDAC8 activity (IC50 = 66, 23, and
66 nM, respectively), and up to 410-fold selectivity for HDAC8 over the next targeted HDAC. Experimental and computational
analysis of the benzanilide structure docked with human HDAC8 enzyme showed the adoption of a low energy L-shape conformer
that favours HDAC8 selectivity. Conformationally constrained HDAC8-inhibitors present an alternative biological probe for further
determining the clinical utility and safety of pharmacological knockdown of HDAC8 in diseased cells.
INTRODUCTION
Due to their biological roles in the progression of multiple
cancers, including peripheral T-cell lymphoma (PTCL),
multiple myeloma, and neuroblastoma, there is considerable
interest in targeting HDACs. The United States Food and Drug
Administration (FDA) has approved four pan-HDAC inhibitors
to date: Vorinostat (SAHA), Romidepsin (FK228), Belinostat
(PXD101) and Panobinostat (LBH-589).10 Pan-HDAC
inhibition profiles are associated with adverse side-effects such
as diarrhea, thrombocytophenia, anemia and cardiac
toxicity.2,4,9,13,14 Mouse models have highlighted that selective
knockdown of HDAC enzymes can improve therapeutic
outcomes whereas the knockdown of other HDACs result in
lethal side-effects. For instance, knockdown studies of HDAC6
and HDAC8 have resulted in impediment of tumor growth,15,16
whereas embryonic disruption of specific HDACs (including 1,
2, 3, 7) in mouse models is associated with lethal outcomes.11
The toxic side-effects observed in clinical trials of approved
pan-HDAC inhibitors correlate with side effects observed in
certain HDAC knockouts (e.g. HDAC2, 3, 5, 9).11 Selective
HDAC inhibitors (HDACi) therefore stand to have wider
therapeutic windows. As a result, significant efforts towards the
development of small molecule HDAC-selective inhibitors
Histone Deacetylases (HDACs) constitute a family of eraser
proteins that are canonically known to remove acetyl groups
from specific Lys side-chains of histones in order to regulate
transcription. Deacetylation of Lys affords the positively
charged amino group (pKa ≈ 10.3), which strengthens the
electrostatic interactions between histones and the DNA
backbone, reducing access to DNA by transcription factors.
HDACs are known to be involved in gene transcription and
other cellular processes.1 Due to their role in gene transcription,
they have the ability to increase the expression of growth
promoting factors and decrease the expression of tumour
suppressors.2 Recently, HDACs have been found to have other
non-histone substrates.3–5 For instance, knockdown of over-
expressed HDAC2 in colorectal cells6 resulted in upregulation
of the cyclin-dependent kinase inhibitor, p21, independent of
p53, supporting the hypothesis that HDAC2 is involved in
carcinogenesis.7
Moreover, numerous studies have
demonstrated increased HDAC levels in several cancers
compared to normal tissues.6,8–12
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have been made in the past decade. One of the main challenges
between the L1 and L6 loops (Figure 1a).21 Quisinostat adopts
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with targeting HDACs selectively arises from the highly
conserved amino acid sequence in the catalytic domain.17–20
Although the catalytic tunnel is highly conserved, the surface
topology and tunnel shape vary between HDAC family
members, thus providing an opportunity for selective small
molecule intervention.
a linear conformation that does not interact with the L1-L6
HDAC8 selectivity filter, thereby exhibiting non-selective
HDAC inhibition. Furthermore, the lack of rigidity in clinically
approved HDAC inhibitors (Vorinostat, Panobinstat, Belinostat
etc.) afforded by their multiple rotatable centers, allows them to
adapt to the various HDAC tunnel shapes, and results in pan-
HDAC inhibition.
We were interested in the design of rigid L-shaped scaffolds
that interact favourably with the HDAC8 selectivity pocket and
are poorly accommodated by other HDACs such as HDAC6
(Figure 1b). The traditional pharmacophoric model of HDACi
consists of three groups – the cap group, the linker, and the zinc-
binding group (Figure 2a), with a majority of inhibitors
containing flexible motifs with rotatable centers. Herein,
conformationally constrained benzanilide foldamers, adopting
an L-shaped conformation, are shown to afford HDAC8 tunnel
selectivity (Figure 1b, 2c).
HDAC8 has been implicated in multiple neurological and T-
cell malignancies as well as Schistosomiasis, caused by the
human flat-worm Schistosoma mansoni. Previous studies
involving small molecule inhibition of HDAC8 have revealed
unique structural features that can be exploited for selective
HDAC8 ablation.21 More specifically, soaking experiments of
apo Schistosoma mansoni HDAC8 with HDAC8-selective
inhibitors PCI34051 (Figure 2b), NCC-149, as well as pan-
HDAC inhibitor Quisinostat, indicated that HDAC8 selective
inhibitors adopt an L-shape that allows them to interact with the
‘HDAC8-specific pocket’ formed by the catalytic tyrosine
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Figure 1. (a) Birds-eye view of hHDAC8 (PDB 1W22) compared to hHDAC6 (PDB 5EDU) surface topology. HDAC8 contains an L1-L6 basin
whereas the L1 and L6 loops of HDAC6 are locked creating a linear Lys tunnel. (b) A ‘tetris’-based model for determining HDAC8 selectivity. L-
shaped inhibitors are accommodated by the HDAC8 tunnel but not in other HDACs.
potency of 245 nM against HDAC8, it was hoped that a
medicinal chemistry program could improve HDAC8 activity
whilst reducing other HDAC potency. As part of the SAR, the
potency and selectivity of each small molecule was assessed in
an enzymatic-activity based Electrophoretic Mobility Shift
Assay (EMSA) against HDACs 3, 6, 8 and 11 (Nanosyn, USA).
First, moving the tBu moiety on the cap-group of AES-350
from the para- to meta- position gave 1, and resulted in
increased HDAC8 activity, while retaining HDAC6 potency
RESULTS AND DISCUSSION
Structure Activity Relationship Studies.
The HDAC
inhibitor,
4-tert-butyl-N-[4-
(hydroxycarbamoyl)phenyl]benzamide, previously published
as AES-350 (HDAC8 IC50 = 245 nM, HDAC6 IC50 = 24.4
nM)22, was the starting point for our structure-activity
relationship (SAR). Whilst more selective for HDAC6, with
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(HDAC8 IC50 = 34 nM, HDAC6 IC50 = 71 nM). To improve The identification of 2 prompted a thorough and systematic
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8
selectivity, it was hypothesized that N-amide substitution might
hinder the hydroxamic acid moiety from entering the linear
HDAC6 tunnel and coordinating the Zn2+ atom at the base of
the tunnel. The hypothesis was explored first with an N-Et (2)
substituent, which exhibited HDAC8 activity (IC50 = 83.5 nM),
and >12-fold selectivity over HDACs 3, 6, and 11 (IC50’s > 1
µM). Exploration of N-alkylated benzamides for deriving
HDAC8 selective inhibitors was then conducted. Interestingly,
structural studies of benzanilide-scaffolds have shown the
adoption of either an E- or Z conformation (or cis and trans)-
depending on the selection of substitution at N. For example, a
proton substituent primarily favours a Z conformation, whereas
N-methylated homologs favour the E conformation.23 In the
case of 2, it was hypothesized that the Et group favoured an E-
(or ‘L-shaped’) conformation, which was proposed to be
complimentary to the topology of HDAC8’s catalytic pocket
(Figure 1).
SAR, guided by Hirano and Marek’s structural observations, to
identify an HDAC8 selective pharmacophore. First, to
investigate the sp2 amide bond, compound 3, containing a more
flexible sp3 sulfonylamide, was prepared. Compound 3
exhibited pan-HDAC activity against representative HDACs, 3,
6, and 11. This data supports the hypothesis that conformational
rigidity is important for HDAC8 selectivity with benzanilide
scaffolds. To better interact with hydrophobic Phe residues in
the tunnel, a second tBu substituent (3,5-tBu) was introduced to
the cap group (7). Despite its size, the potency and selectivity
of 7 were retained, with an IC50 = 80 nM against HDAC8 and a
> 12-fold selectivity for the nearest HDAC enzyme. Next, a 3-
CF3 substituted analog, 4, was prepared, which marginally
increased HDAC8 potency (IC50 = 62 nM) and selectivity (16-
fold). Replacing the amide group (4) with a sulfonamide (5)
resulted in a drop in potency (HDAC6; IC50 = 129 nM) and
selectivity (>12 to 5-fold). Transposed-amide homologues, 13,
14, and 23, which are unable to adopt the L-shaped conformer,
showed reduced HDAC8 potency and selectivity. Additionally,
an N-alkylated transposed amide analogue, 24, also showed
diminished HDAC8 potency and selectivity. This data
supported the hypothesis that positioning of the amide, in
addition to adopting the preferred L-shape, is critical to
maintaining HDAC8 potency and selectivity.
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Given the role of the N-substitution in promoting the KE/Z
(Figure 2c), we probed the importance of the side group (R1)
by preparing a series of bulky N-aryl homologues of 4 (8-10).
All benzanilides with bulky aryl side-groups, including those
that incorporated the 3,5-tBu benzyl (8) and 3-CF3 benzyl (9),
resulted in a loss of selectivity and potency against HDAC8.
Further side-group optimization efforts included incorporation
of an iPr (16) and methylene cPr on each of the HDAC8
selective leads, 2, 4, and 7. The inclusion of iPr (16) had
minimal effects on both potency and selectivity relative to the
N-Et analog (4), whereas the inclusion of the methylene cPr
group (MMH371, 15, and 17) resulted in a decrease in both
potency and selectivity with the exception of MMH371.
MMH371, which includes a 3-tBu cap group with a methylene
cPr R1 side group, showed an increase in both potency and
selectivity (HDAC8 IC50 = 66 nM and >15-fold selectivity).
MMH371 was also tested against the remaining class I HDAC
family members in the same EMSA assay, and showed no
activity against HDACs 1 and 2, IC50’s >10 M (table S2). Due
to the potency and selectivity profiles of N-iPr derivatives, the
N-iPr moiety was incorporated in a variety of subsequent
derivatives. It is known that iPr and cPr groups are less-likely
to be recognized and metabolized in biological systems.24
Figure 2. (a) Pharmacophoric model of HDACi. (b) Structure of PCI-
34051, an HDAC8 selective inhibitor. (c) Controlling E–Z equilibrium
via N-substitution of benzanilide based hydroxamic acids. N-H
benzanilides favor the Z conformation whereas, N-alkylation (R1
alkyl group) favors the E conformation.
=
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Table 1 –IC50 values for inhibitors against HDACs 3, 6, 8, and 11 in an activity-based Electrophoretic Mobility Shift Assay (EMSA) (treatment
range, 0.000244 - 1 M). Equilibrium dissociation constants (KD) and IC50 independently determined using Surface Plasmon Resonance (SPR) and
Fluorescence Polarization (FP) assays, respectively, against HDAC8.
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EMSA HDAC IC50 (M)
FP
IC50
(M)
Code
R1
X
R2
SI
SPR KD (M)
3
6
8
11
7
8
9
1
3-tBu
>1
0.066
0.0337
>1
1.96
–
–
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11
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2
3
4
3-tBu
3-tBu
3-CF3
>1
0.377
>1
>1
0.866
>1
0.0835
0.0553
0.0623
>1
0.802
>1
>12.0
6.82
0.0261
0.0239
0.0375
0.203
0.117
0.144
>16.1
5
6
3-CF3
0.661
>1
0.712
>1
0.129
0.308
0.695
>1
5.12
0.0459
0.0947
0.340
0.547
3,5-CF3
>3.25
7
3,5-tBu
>1
>2
>1
0.0798
>2
>1
>12.5
0.337
0.0951
0.108
3.030
8a
3-CF3
0.752
0.673
–
9a
3-CF3
>2
0.225
0.648
0.526
0.347
–
0.727
10a
11
12
13
14
3-CF3
3-CF3
2-CF3
3-CF3
3,5-CF3
>2
0.175
0.0596
0.821
0.118
>1
0.264
0.478
0.823
>1
>2
>1
>1
>1
>1
0.663
0.124
0.998
0.118
0.528
–
–
–
–
–
0.0596
0.266
0.368
–
0.254
0.904
0.312
>1
>1
–
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15
3-CF3
3-CF3
>1
>1
>1
>1
0.116
0.0651
0.0660
0.225
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
–
>8.62
>15.4
>15.2
>4.44
0.0996
410
0.00981
0.00770
0.0108
–
0.0328
0.0625
0.0587
1
2
3
4
5
6
7
8
16
MMH371b
3-tBu
>1
>1
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Below
limit
17
3,5-tBu
3,5-tBu
3,5-CF3
3-OCF3
>1
>1
18
0.0996
>1
0.19
>1
–
–
MMH409c
0.0234
0.0655
0.0885
0.119
0.107
0.682
>1
0.00350
0.00848
0.00840
0.00880
0.0260
–
0.113
0.0728
0.0418
MMH410b
>1
>1
>15.3
>11.3
>8.40
>9.35
0.137
0.345
2-F, 3-
CF3
19
20
>1
>1
3-CF3,
4-F
Below
limit
>1
>1
21
3,5-tBu
3,5-CF3
3-tBu
>1
>1
0.0119
–
22
0.672
0.345
0.112
0.414
23d
>1
>1
–
1.04
24d
3-tBu
–
>1
>1
>1
>1
0.732
1.37
120
–
0.358
0.134
0.0040
3
0.48
2
PCI34051c
–
–
0.00310
Note: All compounds are based on the Markush structure shown on the left except for 25 and 26 and PCI34051. PCI34051 is a known HDAC8
selective inhibitor. The Selectivity Index (SI) is calculated as a ratio between the lowest non-HDAC8 IC50 value and HDAC8 IC50 value. aCompounds
tested at a top concentration of 2 M. HDAC2, and 4 IC50 values for 8-10 were >2 M. bMMH371, and MMH410 were both additionally tested
c
against HDACs 1 and 2 yielding IC50 values >10 M. MMH409 and PCI34051 were also re-tested against HDACs (1-7, 9-11) at a top concentration
of 10 M to obtain a complete selectivity profile (SI = 410). IC50 values for HDAC 1, 2, 4, 5, 7, 9, and 10 were >10 M and the IC50 value against
HDAC11 was 9.6 M. dCompounds based on the Markush structure to the right. A colour gradient is applied for visualization of potency.
A 3,5-CF3 substitution on the N-iPr benzanilide structure 16,
resulted in a potent and most selective HDAC8 inhibitor,
MMH409 (IC50 = 23 nM). A complete HDAC inhibition panel
revealed a 410-fold selectivity for HDAC8 (Table 1, S2).
Alternative di-substituted analogs assessed included 2-F, 3-CF3
(19), 3-CF3, 4-F (20), 3,5-tBu (7), and 3-OCF3 (MMH410). In
addition to the 3-CF3 substitution, fluorine substitution in the 2-
and 4-positions positions (19 and 20, respectively) both resulted
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in lower potency of 119 and 89 nM, respectively. A change in is known to be one of the most dynamic of HDAC proteins,
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the CF3 position from the 3-CF3-substituted analog (4) to 2-CF3
(12) also resulted in a loss of HDAC8 potency and selectivity.
(4; IC50 = 62 nM, 12; IC50 = 823 nM, 4; SI >16, 12; SI ~ 1). The
SAR strongly supports the hypothesis that either a mono- (3-)
or di-(3,5-) substituted cap-group is a favourable chemotype for
improved potency and selectivity against HDAC8. Notably,
previous studies have also demonstrated the use of meta-
substituted hydroxamic acid zinc-binding groups to target
HDAC8 selectively.25–27
which may permit binding to different HDAC8 conformations
and result in varying degrees of stabilization.29,30
The kinetic binding profile of the benzanilides was
investigated using surface plasmon resonance (SPR) with full-
length HDAC8 (Figure 3c). All of the sulfonamide derivatives
showed potent equilibrium dissociation constants (KD) of
between 10 and 100 nM. N-alkylated benzanilides, MMH371,
MMH409, MMH410, and 19 achieved a KD lower than 10 nM.
Notably, MMH409 demonstrated a KD of 3.5 nM, comparable
to PCI34051 (KD = 3.1 nM; Table 1, S3¸S4). The entire subset
of SAR derivatives displayed similar association rate constants
ka (~10-5 M-1s-1, Table S3). The SPR results suggested that
increased binding affinity was primarily due to the decrease in
the kinetic dissociation rate, kd. For instance, the N-Et
sulfonamide, 6, had a 30-fold lower kd than that of the N-iPr-
benzanilide, MMH409, despite both compounds containing the
same 3,5-CF3 cap-group (Table S3, S4). All N-iPr-substituted
derivatives demonstrated a lower kd when compared to their N-
Et counterparts; 21 and 16 had a 1.8-fold and 2.4-fold lower kd
compared to counterparts 2 and 4, respectively. The N-
methylene cPr derivative, MMH371 also had a marginally
lower kd (2-fold) compared to its ethylated counterpart, 2.
9
Next, to confirm our hypothesis that N-alkylation facilitates L-
shaped benzanilides and HDAC8 selectivity, we synthesized
the unsubstituted N-amide derivatives of HDAC8 selective
compounds 4, 7, and MMH409. All analogs lost HDAC8
potency (11 (~8-fold), 18 (~13-fold) and 22 (29-fold)), and
selectivity (11 (>133-fold), 18 (>127-fold) and 22 (2994-fold)).
All analogs showed increased potency against HDAC6,
suggesting that N-H benzanilides preferentially adopt a linear
conformation, more suitable for the HDAC6 tunnel.
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Collectively, the benzanilide scaffold achieves HDAC8
potency and selectivity by: 1) incorporation of either a mono-
(3-) or (3,5-) di-substituted (R2) cap group; 2)
a
conformationally rigid amide motif (i.e. sp2 carbonyl preferred
rather than sp3 sulfonyl, or methylene carbon); 3) restriction of
the size of N-alkyl group (R1) on the benzanilide.
Based on literature precedent, it can be assumed that the
difference in N-alkyl group bulk (Et, iPr, and methylene cPr) is
responsible for inducing a more favorable L-shape and
corresponding slower dissociation rate. This also implies that
the N-iPr and N-methylene cPr benzanilide scaffolds tend to
have longer drug-target residence times (MRT = 1/koff)
compared to their N-Et counterparts (Table S3).31,32 Another
explanation for the decrease in dissociation rates may be due to
a conformational change in the binary complex32 after inhibitor
binding, leading to a longer residence time. This is in
agreement with the statistical fitting of SPR response curves
using a two-state binding model33 of N-iPr benzanilides
MMH409, MMH410, and 21 with HDAC8 (Table S4, Chi²
(RU²) ~ 0.045-0.062).
In vitro Target Validation and Determining the Kinetic
Binding Profile. In order to corroborate the SAR trends in the
EMSA activity assay, we assessed the library with an HDAC8
fluorescence polarization (FP) assay using an FP-probe, FITC-
M344 (Table S5).28 HDAC8 potency in the FP assay was
consistent with the trends observed in the EMSA activity
assays, with MMH371, MMH409 and MMH410 all showing
nanomolar IC50 values comparable to PCI34051 (PCI34051,
FP; IC50 = 133 nM; MMH371, FP; IC50 = 59 nM; MMH409,
FP; IC50 = 110 nM; MMH410, FP; IC50 = 72 nM; Figure 3b,
Table 1, S5). In addition to the FP assessment, a Thermal Shift
Assay (TSA) was performed to corroborate HDAC8-target
engagement. A shift in HDAC8 melting temperature (Tm) was
observed upon dosing with most HDAC8-targeting compounds
(Table S6, Figure S4, S5). For example, 2 showed a 9 °C shift
in stabilization relative to apo-HDAC8 (Tm = 37 °C) while other
HDAC8 selective inhibitors only showed a 1 oC shift. HDAC8
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Figure 3. (a) Structures of MMH371, MMH409, and MMH410; (b) HDAC8 IC50 for PCI34051, MMH371, MMH409, and MMH410 using
FITC-M344 in a competition-based Fluorescence Polarization (FP) assay28; (c) Logarithmic isoaffinity plot generated from rates of association (ka)
and dissociation (kd) as determined by Surface Plasmon Resonance (SPR). The plot is divided into regions defined by their dissociation constant
(KD = kd/ka) (d) HDAC3, 6, 8, and 11 IC50 plots for the three compounds determined by Nanosyn using an activity-based Electrophoretic Mobility
Shift Assay (EMSA).
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equilibrium was much lower, while still predominantly preferring
Structural Studies of Benzanilide Scaffolds. Thus far, our
1
the E conformation (% E = 56 and 70 %, respectively). Some N-
aryl benzanilides however (9, 10), do not display HDAC8
potency despite their preference for the E conformation as
predicted by DFT (~96% and ~95% E, respectively, Table S11).
We presume that the greater steric bulk of 9 and 10 may prevent
entry into the HDAC8 tunnel, thereby reducing its activity.
Similarly, one of the N-alkylated transposed amides, 24, although
preferring the L-shape (log KE/Z = 1.74, % E = 98%), is not potent
against HDAC8 (IC50 = 732 nM). It is hypothesized that
transposition of the amide may result in a steric clash between the
N-alkyl group and the tunnel entrance residues, or, result in a loss
of favourable H-bonding interactions with the tunnel entrance
residues. X-ray and NMR studies of our benzanilide-based
hydroxamic acid pharmacophores confirmed previous
conformational studies conducted on the effect of N-alkylation on
conformation.23,34–39 While HDAC8 selectivity is proposed to be
due to the adoption of the L-shape, there are additional factors,
such as sterics and intermolecular bonding, that can also
contribute to HDAC8 selectivity, or lack thereof.
hypothesis was that incorporation of small alkyl groups such as
iPr (16, MMH409, MMH410, etc.) and methylene cPr (15, 17,
MMH371) impart HDAC8 selectivity via an E conformation
(Figure 1b, 2c). X-ray crystal structures of MMH371 and
MMH409, confirmed that both molecules had non-linear
conformations in the crystallized state (Figure 4b). The
structures revealed the close proximity of the aryl groups of the
cap- and linker moiety for both MMH371 and MMH409, with
the distance between the ortho-hydrogens of the cap-group and
linker, being 3.105 Å and 2.995 Å, respectively. The two phenyl
motifs of the benzanilide scaffold were approximately
perpendicular to each other. In MMH371, the distance between
the ortho-protons of one phenyl ring to the other (3.206 Å) was
larger than in MMH409, with the bulky tBu group oriented away
from the hydroxamic acid substituted phenyl ring. 2D-NOESY
experiments of N-alkylated HDAC8-selective inhibitors, 16, 17,
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20 and MMH409 all showed H-1H NOE correlations between
1
the protons from the two phenyl rings (Figure 4c, S10)
confirming the E conformation is retained in solution (deuterated
methanol). For instance, 17 (Figure 4c) showed a strong NOE
correlation consistent with the L-shaped configuration. As
expected, the N-H benzanilides, 11, 18 and 22, showed no NOE
correlations in solution (Figure S11). DFT analysis was
performed on the HDAC8 library to correlate the dependence of
In Silico Docking Studies. In order to validate the mode of
binding of HDAC8 selective inhibitors, in silico computational
studies were performed using Schrödinger Maestro 11.9.011
software with Glide within the Small-Molecule Drug Discovery
Suite 2019-1.40–42 The HDAC8 inhibitor library was docked with
hHDAC8 (PDB: 1W22) and docking scores were reported as a
free energy of binding (ΔGB) in the supplementary section (Table
S12). All of the N-H benzanilides (1, 18, and 22), with the
exception of 11, showed a higher ΔGB than their N-iPr and N-
methylene cPr HDAC8 selective counterparts, in agreement with
the activity assays. Compound 1 had a ΔGB of -8.097 kcal/mol as
compared to its N-methylene cPr HDAC8-selective analogue,
MMH371, which was calculated to have an ΔGB of -8.563
kcal/mol. Similarly, 18 and 22, had docking scores of -4.680
kcal/mol and -4.696 kcal/mol, whereas their N-iPr HDAC8
selective counterparts, 21 and MMH409 had a ΔGB of -4.725
kcal/mol and -7.873 kcal/mol, respectively. The three HDAC8
selective leads MMH371, MMH409 and MMH410 were chosen
for studying the docking poses in comparison with the N-H
benzanilides 1, 11, 18, and 22. All three inhibitors showed
binding to hHDAC8 in their L-shaped conformation, with the
cap-groups bound in the HDAC8 L1-L6 basin (Figure 5). This
was consistent with previous HDAC8-inhibitor structural studies
that indicated that HDAC8-selective inhibitors bind to a unique
HDAC8-selective pocket formed by a catalytic tyrosine and L1-
L6 loops.21 The N-H benzanilides were accommodated within the
lysine tunnel in a linear fashion as predicted by DFT analysis
without any interaction with the L1-L6 basin.
HDAC8-selectivity on L-shaped conformation.
The E-Z
equilibrium constant KE/Z (Figure 2c, 4a) was predicted for the
complete SAR using Gaussian at the ωB97XD/6-31++G(d,p)
level of theory with Polarizable Continuum Model (PCM) water
solvent correction by determining the Gibbs free energy of the E
(GE) and Z (GZ) foldamers (Table S11). The E-Z equilibrium
constant (KE/Z) was calculated by using ΔGE/Z (Equation 1).
퐺퐸 ― 퐺푍 = ― 푅푇푙푛(퐾퐸 )
(1)
푍
A plot of log KE/Z versus the selectivity of HDAC8 molecules
(Figure 5d) was used to computationally determine the
dependence of HDAC8 selectivity on the conformational bias of
the HDAC8 SAR library. A log KE/Z > 1 represents an E
conformer population of > 90%. Interestingly, all of the HDAC8
selective molecules with an SI > 8 displayed a log KE/Z > 1
indicating a high-preference for the E (L-shaped) conformation.
The N-H benzanilides (1, 11, 22) had >90% Z population with the
exception of 20, a 3,5-tBu cap group benzanilide (~72% Z, Table
S11).
Although, some of the sulfonamides (3, 5) displayed some
selectivity due to their flexible sp3 sulfur atom, their E-Z
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Figure 4. N-alkylation of benzanilide scaffolds facilitate adoption of an E or L-shaped conformation. (a) The E-Z equilibrium of the benzanilide
MMH410. MMH410 is 93% E as predicted by DFT analysis. The E-Z equilibrium of MMH410 depends on R1 composition; (b) X-ray crystal
structures of MMH371 and MMH409; (c) Two-dimensional NOESY experiment on 17, a potent and selective HDAC8 inhibitor, showing NOE
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correlations consistent with the L-shape; (d) A log KE/Z versus HDAC8 selectivity plot, showing the dependence of HDAC8 selectivity on the E-Z
benzanilide equilibrium. G of E-Z equilibrium was calculated using Density Functional Theory (DFT) analysis of the HDAC8 SAR and used to
determine Log KE/Z. All values for Selectivity Index (SI), were plotted as the maximum reported value and are likely underestimates of the true
HDAC8 selectivity. For example, 17 displayed SI >4.44, and the value 4.44 was used for the plot.
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Immunofluorescence imaging in neuroblastoma cells (BE(2)-
C) was employed to determine the acetylation levels of the
HDAC8 substrate, Structural Maintenance of Chromosomes
protein 3 (SMC3), after being dosed with MMH410.3,26,44,46,47
Inhibition of HDAC enzymes typically leads to increased
acetylation of their substrates. Immunofluorescence imaging
revealed a dose-dependent increase in acetylation levels similar
to that of PCI34051 (Figure 6c, S15). The neuroblastoma cells
were found to form visible clusters upon addition of MMH410,
which may be indicative of a stress response.48 Furthermore,
Western blot analysis of Ac-SMC3 and Ac-tubulin (HDAC6
substrate) demonstrated in cellulo HDAC8 selectivity over
HDAC6 for MMH410 whereas MMH371 did not. Ac-SMC3
Determining Biological Applicability. Lead compounds,
MMH371, MMH409, and MMH410, were evaluated for
cellular permeability by conducting a Parallel Artificial
Membrane Permeability Assay (PAMPA) (Table 2).43 In the
PAMPA assay, methotrexate and testosterone were used as
negative and positive controls, respectively. A permeability
coefficient (−Log Pe) lower than 6 generally defines a
compound as having a high permeability profile. All
compounds tested displayed a −Log Pe < 6 predicting a high
permeability profile (Table 2, S7). To gauge structural
stability, compounds were assessed for stability in human
plasma. MMH371 and MMH-410 demonstrated good
stability in human plasma (t1/2 > 4 h) with MMH409 being
less stable (~1.5 h, Table 2).
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showed
a dose-dependent increase for MMH371 and
MMH410, whereas Ac-tubulin remained constant even at a
concentration of 10 M for MMH410 but showed a dose-
dependent increase for MMH371 (Figure 6d). MMH410
displayed a greater selectivity for HDAC8 in cells than
MMH371.
Table 2 – Properties of top three HDAC8 selective molecules. In
vitro permeability (-Log Pe and % recovery) and stability profiles
(t1/2) in human plasma.
PAMPA
MMH371 and MMH410 were selected for further stability
profiling to determine their application as in vivo probes. CD-1
mice were given HDAC8 inhibitors interperitoneally (IP), at a
single dose (100 mg/kg), and blood was collected at multiple
time points over a 12 h period post-administration. MMH371
exhibited a modest increase in t1/2 as compared to PCI34051
(4.1 h vs. 3.3 h, respectively; Figure 6b). While the maximal
concentration, Cmax, (2893 ng/mL, 7.88 M) was significantly
lower than that of PCI34051 (Cmax = 16967 ng/mL, 57.6 M),
Plasma Half-life
Compound
t1//2 (min)a
-Log Pe
Recovery%
ID
MMH371
MMH409
MMH410
5.70
5.42
5.80
102.5
95.4
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93
101.8
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aHalf-life determined from an average of three runs.
their overall exposures were comparable (MMH371, AUClast
=
5851 h*ng/mL; PCI34051, AUClast = 6946 h*ng/mL; Table S9,
S10). MMH410 was shown to exhibit a t1/2 comparable to
PCI34051, but exhibited a significantly lower maximal
concentration and exposure (t1/2 = 2.7 h; Cmax = 440 ng/mL, 1.14
M; AUClast = 1653 h*ng/mL; Figure 6b, table S8). MMH371
and MMH410 both demonstrated a greater in vivo mean
residence time (MRT) over PCI34051 (MMH371, MRT = 2.77
h; MMH410, MRT = 3.00 h; PCI23051, MRT = 0.45 h; Table
S8, S9, S10).
While not fully understood, the disease relevance of
aberrant HDAC8 activity has been implicated in advanced-
stage neuroblastoma.3,9,44 Neuroblastoma is
extracranial solid-tumour associated with
a
a
childhood
lack of
differentiation and maturation of neural crest cells, and <50%
long-term survival probability of high-risk patients.3
Additionally, patients that undergo chemotherapy suffer from
immediate and long-term toxicities, which makes newer
therapies with minimal toxicities, such as those targeting cell
maturation, highly desirable.3,45 The three lead HDAC8
inhibitors were assessed in a neuroblastoma cell (BE(2)-C
colony assay and the potency was determined. It was found that
MMH371 (IC50 = 2.1 µM) was essentially equipotent with
known HDAC8 inhibitor, PCI34051 (IC50 = 3.1 M) (Figure
6a).
Conclusion and Future Remarks. HDAC selective inhibitors
are hypothesized to prevent or lower acute therapy related
toxicities associated with pan-HDAC inhibitors. One approach
to achieving selectivity has been to exploit the topological
differences between various HDACs.
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Figure 5. Computational docking studies to determine mode of action of N-alkylated versus N-H benzanilide scaffolds against hHDAC8 (PDB
1W22). (a) Three HDAC8 selective inhibitors (MMH371, MMH409, and MMH410) overlaid and docked onto hHDAC8. The N-alkylated
benzanilide scaffolds adopt an L-shape where the cap-groups dock in the L1-L6 basin. (b) The N-H benzanilide scaffolds (1, 11, 18, 22)
preferentially adopt a linear conformation that is unable to interact with the L1-L6 basin.
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Figure 6. (a) Colony assay of neuroblastoma (BE(2)-C) cells with three HDAC8 selective inhibitors (MMH371, MMH409, MMH410). (b) In
vivo half-life of MMH371 (t1/2 = 4.1 h), MMH410 (t1/2 = 2.7 h) and PCI34051 (t1/2 = 3.3 h) in CD-1 mice dosed at 100 mg/kg, measured as a
function of time (h) versus concentration (ng/mL) of MMH410 in blood. (c) Immunofluorescence images of DAPI (blue) nuclear staining
and Ac-SMC3 (red) showing a dose dependent increase of Ac-SMC3 upon addition of MMH410 and PCI34051. (d) Western blot analyses of
Ac-SMC3 and Ac-tubulin in BE(2)-C cells treated with MMH410 and MMH371 (DMSO, 2.5, 5, 7.5, and 10 M).
In this study, we have developed a series of potent and selective benzanilides as potential chemotherapeutics and biological
HDAC8 inhibitors based on the conformational restrictions probes. The current FDA approved HDAC inhibitors have pan-
afforded by N-alkylated benzanilides. We have shown the inhibition profiles due to their predominantly linear conformation
following: (1) A thorough SAR demonstrating the structural and/or conformational degrees of freedom which permit
determinants required for HDAC8 selectivity; (2) N-alkylation interaction with a variety of HDAC catalytic Lys tunnels.
of benzanilides affords an L-shaped conformation required for Utilizing conformationally rigid scaffolds for exploiting tunnel
HDAC8 selectivity; (3) the biological utility of L-shaped shapes and specific surface topologies is a promising strategy for
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attaining HDAC protein-selectivity. The conformationally Column Chromatography was conducted using a gradient of
constrained benzanilide foldamer-based hydroxamic acid ethyl acetate and hexane on manually packed SNAP 10, 25, 50,
pharmacophore presents an interesting pharmacophore for 100 g cartridges with 40-60 µm silica gel on a Biotage
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8
achieving selective targeting of HDAC8.
system. Compounds were wet-loaded by dissolving in minimal
DCM or dry-loaded by adsorbing onto silica gel. Flow rates 12,
25, and 50 mL/min were used for cartridges SNAP 10, 25, 50 and
100 respectively. Compounds were detected by measuring
absorbance at 254 nm and 280 nm. Preparatory High-
Performance Liquid Chromatography (PrepHPLC) was carried
out on all final compounds to ensure a final purity >95%. The
PrepHPLC was equipped with an XSelect CSH Phenyl-Hexyl
C18 column (30 x 250mm), a Waters 2489 Dual Absorbance UV-
Visible Detector, a Waters 2545 Quaternary Gradient Module,
and a Waters 2707 Autosampler. All compounds were detected
using 254/280 nm UV-light and purified using a linear gradient
of a mixture of H2O and 0.1 % formic acid (A), and acetonitrile
and 0.1% formic acid (B) using two different methods as
indicated. The methods are illustrated below as the linear
gradient of B depicted by its % (v/v) composition and its time
duration at a flow rate of 50 mL/min.
EXPERIMENTAL DETAILS
General Methods. All solvents and chemicals were purchased
from Sigma-Aldrich, Combi-Blocks, Alfa-Aesar, Caledon
Laboratory Chemicals, and Oakwood Chemical. Anhydrous
solvents such as methanol, acetonitrile, and DMF were used from
anhydrous-grade Sure-Seal bottles without further drying
treatments. THF, DCM, DCE, and chloroform were dispensed
using the MBraun-Solvent Dispensary System (MB-SPS). All
reactions under non-aqueous conditions were conducted in oven-
dried glassware. Reaction progress was monitored by Thin Layer
Chromatography (TLC) and visualized by Ultraviolet light (UV,
254/365 nm). Microwave irradiation was carried out in an
Initiator Sixty Biotage apparatus at a high-absorption setting at
2.54 GHz.
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A 400 MHz Bruker, 600 MHz Agilent DD2, and 700 MHz
Agilent DD2 NMR spectrometer equipped with an HCN
cryoprobe, were used to collect 1H, 13C, and 19F NMR
spectra. Two dimensional 1H-1H Nuclear Overhauser
Spectroscopy (NOESY) studies were conducted on the 700 MHz
Varian at room temperature in deuterated methanol. All
deuterated solvents for NMR were purchased from either Sigma-
Aldrich or Cambridge Isotope Laboratories. Chemical shifts (δ)
are reported in parts per million after calibration to the residual
solvent peak from the deuterated solvent. Coupling constants (J)
are reported in Hertz (Hz) and multiplicities are reported as
follows: singlet (s), doublet (d), triplet (t), quartet (q), pentet (p),
sextet (sex), septet (sep), multiplet (m), broad (br), and the
combination of these is represented as a combination of their
abbreviations (eg. doublet of triplet (dt)). Low-resolution mass
spectrometry (LRMS) using Waters LC-MS equipped with a
Micromass ZQ MS in both positive and negative ESI modes was
used to confirm the success of the reaction along with NMR.
High-resolution mass spectrometry was performed for all final
compounds at the Advanced Instrumentation for Molecular
Structure (AIMS) at the University of Toronto using the Agilent
6538 Ultra High Definition (UHD) Accurate-Mass Q-TOF
LC/MS System.
Method 1: 2% → 100% over 50 minutes then 100% for 10
minutes
Method 2: 5% → 100% over 50 minutes then 100% for 10
minutes
Analytical HPLC was used to determine the purity of final
compounds after PrepHPLC. It was carried out using a
Phenomenex Luna 5.0 µm C18 4.6 mm x 150 mm column fitted
on a Hewlett Packard Series 1100 analytical HPLC at a flow rate
of 1.2 mL/min using method 1 and 2 as indicated. Compound
purity is reported as the relative area of the compound peak to all
other peaks on the chromatogram. The retention time (tR) for each
peak is reported in minutes. The purity of all compounds reported
is >95% as determined by the analytical HPLC.
X-ray diffraction data were collected on a Bruker Kappa APEX-
DUO diffractometer using a Copper ImuS (microsource) tube
with multi-layer optics and were measured using a combination
of f scans and w scans. The data were processed using APEX3 3
(Bruker, 2018). Absorption corrections were carried out using
SADABS.49 The structures were solved using SHELXT50 and
refined using SHELXL-201851 for full-matrix least-squares
refinement that was based on F2.
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Synthesis of 2-9, 11 (skipped STAB step), 12, 15-17, 19-21, MMH371, MMH409, and MMH410
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Synthesis of 10
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Synthesis of 13, and 14
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Synthesis of 23 and 24
Note: First step was skipped for 23.
Esterification of 4-aminobenzoic acid
were added concurrently. The reaction was stirred for 4 hours
and concentrated in vacuo and adsorbed onto silica gel. The
crude mixture with silica was dry-loaded for column
chromatography which afforded the desired intermediates.
To a solution of 0.1 mmol/mL solution of 4-aminobenzoic acid in
ethanol was added sulfuric acid to make a sulfuric acid:ethanol
ratio of 1:20. The reaction was heated to 90 C overnight then
diluted with ethyl acetate and extracted with 0.5 M NaOH Acylation/sulfonylation of aryl amines
followed by a brine wash. The solution was concentrated in
vacuo to give a peachy-flaky solid.
o
To a solution of the respective sulfonyl or acyl chloride (1.5 to 2
eq.) in dichloromethane (0.1 mmol of aryl amine/mL) was added
diisopropyl amine (DIPEA, 4 eq.) which produced a smoke
suspended above the solution. Then aryl amine was added, and
the solution was left to stir overnight. The solution was dried in
vacuo with silica gel for dry-loading on a column for column
chromatography which yielded the desired intermediates.
Reductive amination of aryl amines
Aryl amines from schemes 1,4 and 5 were dissolved in DCE to
make a 0.1 mmol/mL solution. Subsequently, the respective
aldehyde (1.5 eq.), acetic acid (1.5 eq.), sodium
triacetoxyborohydride (STAB, 3 eq.) and one scoop of MgSO4
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Deprotection of methyl/ethyl esters
131.2, 128.0, 127.2, 126.8, 126.2, 125.4, 125.2, 124.6, 119.5,
116.7, 34.1, 30.1. HRMS (ESI+) m/z calcd for [C18H20N2O3]+:
312.37, found: 312.20. HPLC tR = 11.586 min (97.6% purity).
1
2
3
To a solution of THF, methanol and MilliQ H2O in a ratio of 3:1:1
containing the methyl/ethyl ester (0.1 mmol/mL) was added
lithium hydroxide (3 eq.). The mixture was left to stir overnight. 3-(tert-butyl)-N-ethyl-N-(4-
The reaction was complete in approximately 16 hours as indicated (hydroxycarbamoyl)phenyl)benzamide (2).1H NMR (700
by TLC and LRMS. The solution was diluted with ethyl acetate MHz, Methanol-d4) δ 7.64 (d, J = 8.5 Hz, 2H), 7.31 (dt, J = 7.5,
and washed twice with 1 M HCl followed by a brine wash and 1.8 Hz, 1H), 7.22 – 7.14 (m, 5H), 4.00 (q, J = 7.1 Hz, 2H), 1.20
drying with magnesium sulfate. Drying in vacuo yielded relative (t, J = 7.1 Hz, 3H), 1.10 (s, 9H). 13C NMR (176 MHz, Methanol-
pure intermediates that were used directly for the subsequent step d4) δ 174.1, 168.1, 153.2, 148.7, 137.9, 133.1, 130.5, 130.3,
4
5
6
7
8
9
without further purification.
130.3, 129.3, 128.3, 128.2, 47.7, 36.7, 32.7, 14.3. HRMS (ESI+)
m/z calcd for [C20H25N2O3]+: 341.18652, found: 341.18632,
found: 341.18632. HPLC tR = 19.045 min (>99% purity).
Synthesis of THP-protected hydroxamic acids
10
11
12
13
14
15
16
17
18
19
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60
The aryl carboxylic acid intermediates were converted to their
acyl chloride intermediates by the use of the Vilsmeier-Haack
reagent generated in situ. The aryl carboxylic acid intermediate
was added to a solution of THF (0.1 mmol/mL), followed by the
addition of no more than two drops of DMF, and oxalyl chloride
(5 eq.) which caused immediate effervescence. The solution was
purged with nitrogen gas until the effervescence halted. The
reaction was then stirred for 1 hour under inert atmosphere. The
solution containing the acyl chloride was dried immediately in
vacuo (15 min rotary evaporator followed by 15 min high-
4-((3-(tert-butyl)-N-ethylphenyl)sulfonamido)-N-
hydroxybenzamide (3). H NMR (400 MHz, Acetonitrile-d3) δ
1
7.75 – 7.70 (m, 3H), 7.54 – 7.47 (m, 2H), 7.41 (s, 1H), 7.15 (d, J
= 8.3 Hz, 2H), 3.63 (q, J = 7.1 Hz, 2H), 1.26 (s, 9H), 1.03 (t, J =
7.1 Hz, 3H). 13C NMR (101 MHz, Acetonitrile-d3) δ 164.6, 152.5,
142.1, 137.4, 130.2, 129.0, 128.5, 127.6, 124.5, 124.2, 117.4,
45.0, 34.6, 30.2, 13.3. HRMS (ESI+) m/z calcd for
[C19H25N2O4S]+: 377.1530, found: 377.1534. HPLC tR = 21.260
min (99.4%).
vacuum) with minimal exposure to air and moisture. The crude N-ethyl-N-(4-(hydroxycarbamoyl)phenyl)-3-
mixture was re-dissolved in THF, and DIPEA (4 eq.) was added, (trifluoromethyl)benzamide (4). 1H NMR (400 MHz,
resulting in a layer of smoke above the solution. Subsequently, Acetonitrile-d3) δ 7.67 – 7.58 (m, 4H), 7.53 (t, J = 7.4 Hz, 1H),
O-(Tetrahydro-2H-pyran-2-yl)hydroxylamine (2 eq.) was added 7.45 – 7.38 (m, 1H), 7.23, (d, J = 8.5 Hz, 2H), 4.77 (br, 2H), 3.97
and the mixture was left to stir overnight. Drying in vacuo (q, J = 7.1 Hz, 2H), 1.19 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz,
followed by column chromatography yielded the desired THP- Acetonitrile-d3) δ 168.2, 145.8, 137.5, 132.0, 128.9, 128.3, 128.2,
protected hydroxamic acid.
127.9, 126.8, 126.1, 125.3, 117.3, 45.0, 12.2. 19F NMR (376
MHz, Acetonitrile-d3) δ -63.42 (s, 3F). HRMS (ESI+) m/z calcd
for [C17H16F3N2O3]+: 353.1108, found: 353.1109. HPLC tR =
18.184 min (>99% purity).
Deprotection of the THP-protected hydroxamic acids
The THP-protected hydroxamic acid was dissolved in 4 M
hydrochloric acid in dioxanes (0.1 mmol/mL dioxane) and left to
stir for 4 hours. The reaction was monitored by TLC and LRMS
which indicated a complete conversion. The solution was dried
in vacuo and the crude mixture was purified by Preparative
HPLC.
4-((N-ethyl-3-(trifluoromethyl)phenyl)sulfonamido)-N-
hydroxybenzamide (5). 1H NMR (400 MHz, Acetone-d6) δ
10.87 (br, 1H), 8.07 (d, J = 7.2 Hz, 1H), 7.94 – 7.81 (m, 5H), 7.26
(d, J = 8.6 Hz, 2H), 3.76 (q, J = 7.0 Hz, 2H), 1.09 (t, J = 7.1 Hz,
3H). 13C NMR (101 MHz, Acetone-d6) δ 205.3, 141.4, 139.6,
131.8, 131.3, 131.0, 130.7, 129.6, 128.6, 127.8, 126.1, 124.1,
45.3, 13.4. 19F NMR (376 MHz, Acetone-d6) δ -63.43 (s, 3F).
HRMS (ESI+) m/z calcd for [C16H16F3N2O4S]+: 389.0775, found:
389.0770. HPLC tR = 20.241 min (>99% purity).
Benzyl protection of 4-aminobenzoic acid
4-aminobenzoic acid was dissolved in DMF (2 mmol/mL) and
stirred with Cs2CO3 (1.1 eq.) for 20 minutes. Benzyl bromide (1
eq.) was added subsequently, and the solution was stirred
overnight. The mixture was diluted with ethyl acetate and
washed thrice with water followed by a brine wash. Drying by
MgSO4 and concentrating in vacuo, followed by column
chromatography afforded two products: a peachy mono-O-
benzylated product, and another di-N,O-benzylated product (used
for scheme 3).
4-((N-ethyl-3,5-bis(trifluoromethyl)phenyl)sulfonamido)-N-
hydroxybenzamide (6). 1H NMR (400 MHz, Acetone-d6) δ
10.90 (br, 1H), 8.43 (s, 1H), 8.14 – 8.09 (m, 2H), 7.90 (d, J = 8.6,
2H), 7.32 (d, J = 8.5, 2H), 3.81 (q, J = 7.1 Hz, 2H), 1.11 (t, J =
7.1 Hz, 3H). 13C NMR (101 MHz, Acetone-d6) δ 205.3, 141.0,
132.3, 128.9, 128.8, 127.9, 126.7, 126.3, 124.1, 121.4, 45.7, 13.3.
19F NMR (376 MHz, Acetone-d6) δ -63.52 (s, 6F). HRMS (ESI+)
m/z calcd for [C17H15F6N2O4S]+: 457.0649, found: 457.0644.
HPLC tR = 22.030 min (>99% purity).
Benzyl deprotection of benzyl ester intermediates
To a solution of benzyl ester in THF (0.1mmol/mL) was added
methanol to make a THF:methanol ratio of 2:1. The solution was
purged with nitrogen for 15 minutes before the addition of 10%
palladium on carbon (0.1 eq.) followed by another 5 minute
nitrogen purge. Hydrogen gas was bubbled through the solution
for 1 hour using a long-needle submerged in the solution. TLC
indicated the reaction was complete and the reaction was diluted
with ethyl acetate and filtered through celite.
3,5-di-tert-butyl-N-ethyl-N-(4-
(hydroxycarbamoyl)phenyl)benzamide (7). 1H NMR (400
MHz, Acetone-d6) δ 10.56 (s, 1H), 7.70 (d, J = 8.5 Hz, 2H), 7.37
(t, J = 1.9 Hz, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.18 (d, J = 1.8 Hz,
2H), 3.99 (q, J = 7.1 Hz, 2H), 1.22 – 1.14 (m, 21H). 13C NMR
(101 MHz, Acetone-d6) δ 205.9, 170.0, 150.1, 146.9, 135.5,
127.9, 127.6, 123.4, 123.2, 117.0, 44.7, 34.4, 30.5, 12.4. HRMS
(ESI+) m/z calcd for [C24H33N2O3]+: 397.2486, found: 397.2487.
HPLC tR = 22.814 min (>99% purity).
3-(tert-butyl)-N-(4-(hydroxycarbamoyl)phenyl)benzamide
1
(1). H NMR (400 MHz, DMSO-d6) δ 10.77 – 10.71 (br, 1H),
N-(3,5-di-tert-butylbenzyl)-N-(4-
(hydroxycarbamoyl)phenyl)-3-(trifluoromethyl)benzamide
8.70 – 8.61 (m, 1H), 7.51 – 7.19 (m, 6H), 6.65 (d, J = 8.4 Hz, 2H),
1.19 (s, 9H). 13C NMR (101 MHz, Methanol-d4) δ 151.2, 144.1,
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(8). 1H NMR (400 MHz, Acetone-d6) δ 7.76 – 7.58 (m, 5H), 7.52 4-(N-(3,5-bis(trifluoromethyl)benzyl)acetamido)-N-
1
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2
3
4
5
6
7
8
– 7.43 (m, 1H), 7.37 (s, 1H), 7.22 – 7.20, 7.12 (m, 4H), 5.24 (s, hydroxybenzamide (14). H NMR (700 MHz, Methanol-d4) δ
2H), 1.28 (s, 18H). 13C NMR (101 MHz, Acetone-d6) δ 205.3, 7.84 (s, 1H), 7.84 – 7.77 (m, 4H), 7.26 (d, J = 8.5 Hz, 2H), 5.09
168.3, 150.7, 145.9, 137.5, 136.4, 132.3, 130.2, 129.9, 129.5, (s, 2H), 1.94 (s, 3H). 13C NMR (176 MHz, Methanol-d4) δ 174.2,
129.0, 127.9, 127.7, 126.3, 125.4, 122.4, 121.0, 53.5, 34.4, 30.8. 168.1, 147.4, 143.1 134.9, 134.1, 131.4, 131.1, 130.6, 125.9,
19F NMR (376 MHz, Acetone-d6) δ -63.35 (s, 3F). HRMS (ESI+) 123.6, 54.1, 23.8. 19F NMR (658 MHz, Methanol-d4) δ -64.44 (s,
m/z calcd for [C30H34F3N2O3]+: 527.2516, found: 527.2518. 6F). HRMS (ESI+) m/z calcd for [C18H15F6N2O3]+: 421.0979,
HPLC tR = 28.483 min (>99% purity).
found: 421.0978. HPLC tR = 20.187 min (98.6% purity).
N-(4-(hydroxycarbamoyl)phenyl)-3-(trifluoromethyl)-N-(3-
N-(cyclopropylmethyl)-N-(4-(hydroxycarbamoyl)phenyl)-3-
1
(trifluoromethyl)benzyl)benzamide (9). H NMR (700 MHz, (trifluoromethyl)benzamide (15). 1H NMR (700 MHz,
Methanol-d4) δ 7.66 (s, 1H), 7.60 – 7.56 (m, 6H), 7.54 (d, J = 7.7 Methanol-d4) δ 7.67 (d, J = 8.5 Hz, 2H), 7.61 (s, 1H), 7.56 (d, J =
Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 7.14 7.9 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.39 (t, J = 7.8 Hz, 1H),
(d, J = 8.6 Hz, 2H), 5.28 (s, 2H).13C NMR (176 MHz, Methanol- 7.29 (d, J = 8.5 Hz, 2H), 3.84 (d, J = 7.2 Hz, 2H), 1.11 – 1.04 (m,
d4) δ 172.3, 168.0, 147.7, 140.6, 139.0, 134.6, 133.7, 132.9, 1H), 0.48 – 0.41 (m, 2H), 0.18 – 0.12 (m, 2H). 13C NMR (176
131.8, 131.4, 130.6, 130.5, 129.1, 129.0, 127.9, 127.6, 127.0, MHz, Methanol-d4) δ 172.1, 168.2, 148.5, 139.6, 134.4, 133.6,
126.8, 126.0, 125.5, 55.3. 19F NMR (658 MHz, Methanol-d4) δ - 132.7, 131.3, 131.0, 130.5, 128.7, 127.7, 126.3, 57.1, 11.9, 5.5.
64.24 (s, 3F), -64.50 (s, 3F). HRMS (ESI+) m/z calcd for 19F NMR (658 MHz, Methanol-d4) δ -64.38 (s, 3F). HRMS
[C23H17F6N2O3]+: 483.1147, found: 483.1144. HPLC tR = 23.546 (ESI+) m/z calcd for [C19H18F3N2O3]+: 379.1262, found:
9
10
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13
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17
18
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42
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53
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59
60
min (>99% purity).
379.1259. HPLC tR = 21.253 min (>99% purity).
N-benzyl-N-(4-(hydroxycarbamoyl)phenyl)-3-
N-(4-(hydroxycarbamoyl)phenyl)-N-isopropyl-3-
(trifluoromethyl)benzamide (10). 1H NMR (700 MHz, (trifluoromethyl)benzamide (16). 1H NMR (700 MHz,
Methanol-d4) δ 7.66 (s, 1H), 7.60 – 7.50 (m, 4H), 7.36 (t, J = 7.8 Methanol-d4) δ 7.66 (d, J = 8.3 Hz, 2H), 7.57 (s, 1H), 7.50 (t, J =
Hz, 1H), 7.28 (d, J = 7.3 Hz, 2H), 7.24 (t, J = 7.3 Hz, 2H), 7.20 7.7 Hz, 2H), 7.36 (t, J = 7.9 Hz, 1H), 7.27 (d, J = 8.3 Hz, 2H),
(t, J = 7.3 Hz, 1H), 7.10 (d, J = 8.4 Hz, 2H), 5.18 (s, 2H). 13C 4.99 (br, 1H), 1.23 (d, J = 6.9 Hz, 6H). 13C NMR (176 MHz,
NMR (176 MHz, Methanol-d4) δ 172.2, 168.1, 147.9, 139.3, Methanol-d4) δ 172.3, 168.1, 144.5, 140.5, 134.4, 133.9, 133.4,
139.2, 134.6, 133.5, 132.7, 131.4, 130.9, 130.8, 130.7, 130.4, 132.6, 131.2, 130.1, 128.3, 127.2, 126.4, 51.3, 22.4. 19F NMR
130.0, 129.0, 127.9, 126.3, 55.8. 19F NMR (658 MHz, Methanol- (658 MHz, Methanol-d4) δ -64.37 (s, 3F). HRMS (ESI+) m/z
d4)
δ -64.22 (s, 3F). HRMS (ESI+) m/z calcd for calcd for [C18H18F3N2O3]+: 367.1264, found: 367.1263. HPLC tR
[C22H18F3N2O3]+: 415.1273, found: 415.1275. HPLC tR = 22.033 = 20.680 min (>99% purity).
min (>99% purity).
3-(tert-butyl)-N-(cyclopropylmethyl)-N-(4-
(hydroxycarbamoyl)phenyl)benzamide (MMH371). H NMR
1
N-(4-(hydroxycarbamoyl)phenyl)-3-
(trifluoromethyl)benzamide (11). 1H NMR (700 MHz, (700 MHz, Methanol-d4) δ 7.64 (d, J = 8.6 Hz, 2H), 7.30 (dt, J =
Methanol-d4) δ 8.24 (s, 1H), 8.19 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 7.6, 1.7 Hz, 1H), 7.23 (d, J = 8.6 Hz, 2H), 7.22 – 7.19 (m, 2H),
7.7 Hz, 1H), 7.84 (d, J = 8.7 Hz, 2H), 7.76 (d, J = 8.7 Hz, 2H), 7.17 (t, J = 7.6 Hz, 1H), 3.84 (d, J = 7.1 Hz, 2H), 1.11 (s, 9H),
7.71 (t, J = 7.8 Hz, 1H). 13C NMR (176 MHz, Methanol-d4) δ 1.09 – 1.05 (m, 1H), 0.46 – 0.42 (m, 2H), 0.14 (q, J = 5.2 Hz, 2H).
168.9, 168.4, 144.3, 138.3, 133.6, 133.3, 131.9, 130.7, 130.4, 13C NMR (176 MHz, Methanol-d4) δ 174.3, 168.1, 153.2, 149.3,
130.2, 126.9, 126.6, 122.8. 19F NMR (658 MHz, Methanol-d4) δ 137.9, 133.1, 130.8, 130.28, 130.27, 129.2, 128.3, 128.2, 57.0,
-64.22 (s, 3F). HRMS (ESI+) m/z calcd for [C15H12F3N2O3]+: 36.7, 32.7, 12.0, 5.4. HRMS (ESI+) m/z calcd for [C22H27N2O3]+:
325.0795, found: 325.0794. HPLC tR = 10.522 min (>99% 367.1943, found: 367.2016. HPLC tR = 27.958 min (>99%
purity).
purity).
N-ethyl-N-(4-(hydroxycarbamoyl)phenyl)-2-
3,5-di-tert-butyl-N-(cyclopropylmethyl)-N-(4-
(trifluoromethyl)benzamide (12). 1H NMR (700 MHz, (hydroxycarbamoyl)phenyl)benzamide (17). 1H NMR (700
Methanol-d4) δ 7.61 (d, J = 8.47 Hz, 2H), 7.59 (d, J = 7.7 Hz, 1H), MHz, Methanol-d4) δ 7.66 (d, J = 8.3 Hz, 2H), 7.33 (s, 1H), 7.22
7.40 – 7.33 (m, 2H), 7.26 – 7.24 (m, 3H), 3.99 (br, 2H), 1.22 (t, J (d, J = 8.1 Hz, 2H), 7.14 (s, 2H), 3.85 (d, J = 7.2 Hz, 2H), 1.15 (s,
= 7.2 Hz, 3H). 13C NMR (176 MHz, Methanol-d4) δ 171.0, 168.1, 18H), 0.43 (q, J = 7.9 Hz, 2H), 0.14 (q, J = 5.2 Hz, 2H). 13C NMR
147.1, 137.1, 134.1, 134.0, 131.9, 131.4, 130.9, 130.5, 129.7, (176 MHz, Methanol-d4) δ 174.6, 168.0, 153.0, 149.6, 137.3,
129.0, 126.6, 47.1, 14.1. 19F NMR (658 MHz, Methanol-d4) δ - 132.9, 130.7, 130.3, 126.1, 125.8, 57.0, 36.9, 32.9, 12.0, 5.5.
59.94 (s, 3F). HRMS (ESI+) m/z calcd for [C17H16F3N2O3]+: HRMS (ESI+) m/z calcd for [C26H35N2O3]+: 423.2642, found:
353.1108, found: 353.1107. HPLC tR = 9.442 min (>99% purity). 423.2636. HPLC tR = 34.486 min (>99% purity).
N-hydroxy-4-(N-(3-
3,5-di-tert-butyl-N-(cyclopropylmethyl)-N-(4-
1
(trifluoromethyl)benzyl)acetamido)benzamide (13). H NMR (hydroxycarbamoyl)phenyl)benzamide (18). 1H NMR (700
(700 MHz, Methanol-d4) δ 7.76 (d, J = 8.4 Hz, 2H), 7.53 (d, J = MHz, Methanol-d4) δ 7.85-7.80 (m, 2H), 7.79 – 7.73 (m, 4H),
7.2 Hz, 1H), 7.49 (s, 1H), 7.48 – 7.43 (m, 2H), 7.23 (d, J = 8.4 7.69 – 7.65 (m, 1H), 1.37 (s, 18H). 13C NMR (176 MHz,
Hz, 2H), 5.01 (s, 2H), 1.92 (s, 3H). 13C NMR (176 MHz, Methanol-d4) δ 171.3, 169.0, 153.8, 144.6, 136.8, 130.1, 130.1,
Methanol-d4) δ 174.0, 168.2, 147.6, 141.0, 134.7, 134.6, 133.1, 128.6, 124.2, 122.9, 37.2, 33.0. HRMS (ESI+) m/z calcd for
131.7, 130.9, 130.7, 127.5, 126.6, 126.0, 54.4, 23.9. 19F NMR [C22H29N2O3]+: 369.2173, found: 369.2172. HPLC tR = 35.032
(658 MHz, Methanol-d4) δ -64.21 (s, 3F). HRMS (ESI+) m/z min (>99% purity).
calcd for [C17H16F3N2O3]+: 353.1108, found: 353.1108. HPLC tR
= 18.616 min (98.3% purity).
N-(4-(hydroxycarbamoyl)phenyl)-N-isopropyl-3,5-
bis(trifluoromethyl)benzamide (MMH409). 1H NMR (700
MHz, Methanol-d4) δ 7.85 (s, 2H), 7.80 (s, 1H), 7.70 (d, J = 8.2
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Journal of Medicinal Chemistry
Hz, 2H), 7.33 (d, J = 8.2 Hz, 2H), 5.02 (br, 1H), 1.24 (d, J = 7.1 33.0. HRMS (ESI+) m/z calcd for [C18H21N2O3]+: 313.1547,
Hz, 6H). 13C NMR (176 MHz, Methanol-d4) δ 170.3, 167.9, found: 313.1545. HPLC tR = 27.776 min (>99% purity).
1
2
3
4
5
6
7
8
9
144.2, 141.9, 134.7, 133.6, 133.5, 131.0, 130.3, 125.5, 125.1,
51.4, 22.3. 19F NMR (658 MHz, Methanol-d4) δ -64.52 (s, 6F).
HRMS (ESI+) m/z calcd for [C19H17F6N2O3]+: 435.1138, found:
435.1131. HPLC tR = 28.875 min (>99% purity).
N1-(3-(tert-butyl)phenyl)-N1-ethyl-N4-
hydroxyterephthalamide (24). H NMR (700 MHz, Methanol-
d4) δ 7.55 (d, J = 7.7 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.23 –
7.16 (m, 2H), 7.05 – 6.93 (m, 2H), 4.00 – 3.96 (br, 2H), 1.21 (t, J
= 7.2 Hz, 3H), 1.12 (s, 9H). 13C NMR (176 MHz, Methanol-d4) δ
1
N-(4-(hydroxycarbamoyl)phenyl)-N-isopropyl-3-
(trifluoromethoxy)benzamide (MMH410). 1H NMR (700 172.9, 168.2, 155.1, 144.6, 142.3, 135.5, 131.4, 130.8, 128.9,
MHz, Methanol-d4) δ 7.66 (d, J = 8.4 Hz, 2H), 7.31 – 7.22 (m, 128.7, 126.9, 126.4, 47.4, 36.6, 32.7, 14.3. HRMS (ESI+) m/z
4H), 7.15 (s, 1H), 7.12 (d, J = 7.7 Hz, 1H), 4.97 (br, 1H), 1.22 (d, calcd for [C20H25N2O3]+: 341.1860, found: 341.1856. HPLC tR =
J = 6.9 Hz, 6H). 13C NMR (176 MHz, Methanol-d4) δ 172.1, 27.544 min (>99% purity).
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11
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13
14
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16
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19
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59
60
168.1, 151.0, 144.5, 141.6, 134.4, 133.3, 132.2, 130.1, 129.1,
124.3, 123.0, 122.9, 51.3, 22.4. 19F NMR (658 MHz, Methanol-
ASSOCIATED CONTENT
Supporting Information
d4)
δ -59.61 (s, 3F). HRMS (ESI+) m/z calcd for
[C18H18F3N2O4]+: 383.1211, found: 383.1209. HPLC tR = 18.415
min (>99% purity).
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
2-fluoro-N-(4-(hydroxycarbamoyl)phenyl)-N-isopropyl-3-
(trifluoromethyl)benzamide (19). 1H NMR (700 MHz,
Methanol-d4) δ 7.64 (d, J = 8.1 Hz, 2H), 7.52 (dt, J = 28.4, 7.1
Hz, 2H), 7.27 (d, J = 8.1 Hz, 2H), 7.17 (t, J = 7.8 Hz, 1H), 5.04
(h, J = 6.7 Hz, 1H), 1.22 (d, J = 7.0 Hz, 6H). 13C NMR (176 MHz,
Methanol-d4) δ 168.0, 167.7, 157.9, 156.4, 143.2, 135.7, 135.0,
133.0, 130.2, 130.0, 126.9, 124.9, 120.5, 50.8, 22.3. 19F NMR
(658 MHz, Methanol-d4) δ -63.05 (d, J = 12.6 Hz, 3F), -118.30 –
-118.44 (m, 1F). HRMS (ESI+) m/z calcd for [C18H17F4N2O3]+:
385.1170, found: 385.1171. HPLC tR = 18.324 min (>99%
purity).
Additional figures and tables illustrating the HDAC binding and
inhibition profiles using FP, EMSA, SPR, and TSA; additional
tables summarizing PAMPA, and in vivo pharmacokinetic (PK)
results of selected compounds from the SAR; additional figures
and tables illustrating the L-shape of inhibitors using two-
dimensional NOESY experiments, DFT, and docking
experiments; additional figures indicating the existence of a
conformational equilibrium using variable-temperature one-
dimensional NMR experiments; additional figures demonstrating
the
cellular
HDAC8-target
engagement
using
4-fluoro-N-(4-(hydroxycarbamoyl)phenyl)-N-isopropyl-3-
(trifluoromethyl)benzamide (20). 1H NMR (700 MHz,
Methanol-d4) δ 7.68 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 6.3 Hz, 1H),
7.56 – 7.52 (br, 1H), 7.27 (d, J = 8.4 Hz, 2H), 7.13 (t, J = 9.5 Hz,
1H), 4.97 (br, 1H), 1.23 (d, J = 6.9 Hz, 6H). 13C NMR (176 MHz,
Methanol-d4) δ 171.2, 168.1, 144.6, 136.9, 136.3, 134.5, 133.3,
130.2, 129.8, 124.8, 120.0, 119.1, 119.0, 51.5, 22.3. 19F NMR
(658 MHz, Methanol-d4) δ -63.10 (d, J = 12.7 Hz, 3F), -114.56 –
-114.67 (m, 1F). HRMS (ESI+) m/z calcd for [C18H17F4N2O3]+:
385.1167, found: 385.1166. HPLC tR = 18.703 min (>99%
purity).
immunofluorescence and western blotting; compound
characterization data (PDF)
X-ray crystallographic data (CIF)
AUTHOR INFORMATION
Corresponding Author
*patrick.gunning@utoronto.ca
Notes
3,5-di-tert-butyl-N-(4-(hydroxycarbamoyl)phenyl)-N-
1
isopropylbenzamide (21). H NMR (700 MHz, Methanol-d4) δ
All animal experiments performed in the manuscript were in
compliance with institutional guidelines.
7.65 (d, J = 8.4 Hz, 2H), 7.28 (s, 1H), 7.20 (d, J = 8.4 Hz, 2H),
7.08 (s, 2H), 4.99 (br, 1H), 1.24 (d, J = 6.8 Hz, 6H), 1.16 (s, 18H).
13C NMR (176 MHz, Methanol-d4) δ 174.8, 168.0, 152.9, 145.6,
138.3, 133.8, 133.2, 129.9, 125.6, 125.1, 51.2, 36.9, 32.9, 22.5.
HRMS (ESI+) m/z calcd for [C25H35N2O3]+: 411.2642, found:
411.2642. HPLC tR = 23.806 min (>99% purity).
ACKNOWLEDGMENT
P.T.G. is thankful for the financial support from research grants
such as NSERC (RGPIN-2014-05767), CIHR (MOP-130424,
MOP-137036), Canada Research Chair (950-232042), Canadian
Cancer Society (703963), Canadian Breast Cancer Foundation
(705456), infrastructure grants from CFI (33536), and the
Ontario Research Fund (34876).
N-(4-(hydroxycarbamoyl)phenyl)-3,5-
bis(trifluoromethyl)benzamide (22). 1H NMR (700 MHz,
Methanol-d4) δ 8.55 (s, 2H), 8.20 (s, 1H), 7.86 (d, J = 8.7 Hz, 2H),
7.82 – 7.75 (d, J = 8.7 Hz, 2H). 13C NMR (176 MHz, Methanol-
d4) δ 168.9, 166.6, 144.1, 139.8, 134.4, 130.7, 130.2, 127.5,
125.8, 122.8. 19F NMR (564 MHz, Methanol-d4) δ -64.38 (s, 6F).
HRMS (ESI+) m/z calcd for [C16H11F6N2O3]+: 393.0677, found:
393.0675. HPLC tR = 20.396 min (98.2% purity).
ABBREVIATIONS
HDAC, histone deacetylase; HDACi, HDAC inhibitor; PAMPA,
parallel artificial membrane permeability assay; FP, fluorescence
polarization; SPR, surface plasmon resonance; TLC, thin layer
chromatography; LRMS, low-resolution mass spectrometry;
HRMS, high-resolution mass spectrometry; NMR, nuclear
magnetic resonance; NOE, nuclear Overhauser effect; NOESY,
nuclear Overhauser effect spectroscopy; PK, pharmacokinetic;
DFT, density functional theory; Ac, acetylated; SMC3, Structural
N1-(3-(tert-butyl)phenyl)-N4-hydroxyterephthalamide (23).
1H NMR (700 MHz, Methanol-d4) δ 7.99 (d, J = 8.3 Hz, 2H), 7.85
(d, J = 8.3 Hz, 2H), 7.74 (s, 1H), 7.52 (d, J = 7.0 Hz, 1H), 7.27 (t,
J = 7.9 Hz, 1H), 7.20 (ddd, J = 7.8, 1.9, 1.0 Hz, 1H), 1.32 (s, 9H).
13C NMR (176 MHz, Methanol-d4) δ 169.1, 168.4, 154.4, 140.6,
140.5, 137.7, 130.7, 130.1, 129.6, 124.1, 120.79, 120.77, 36.9,
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