Discovery of acylsulfonohydrazide-derived inhibitors of the lysine acetyltransferase, kat6a, as potent senescence-inducing anti-cancer agents

Article abstract of DOI:10.1021/acs.jmedchem.9b02071

A high-throughput screen designed to discover new inhibitors of histone acetyltransferase KAT6A uncovered CTX-0124143 (1), a unique aryl acylsulfonohydrazide with an IC₅₀ of 1.0 μM. Using this acylsulfonohydrazide as a template, we herein disclose the results of our extensive structure-activity relationship investigations, which resulted in the discovery of advanced compounds such as 55 and 80. These two compounds represent significant improvements on our recently reported prototypical lead WM-8014 (3) as they are not only equivalently potent as inhibitors of KAT6A but are less lipophilic and significantly more stable to microsomal degradation. Furthermore, during this process, we discovered a distinct structural subclass that contains key 2-fluorobenzenesulfonyl and phenylpyridine motifs, culminating in the discovery of WM-1119 (4). This compound is a highly potent KAT6A inhibitor (IC₅₀ = 6.3 nM; K_D = 0.002 μM), competes with Ac-CoA by binding to the Ac-CoA binding site, and has an oral bioavailability of 56% in rats.

Full text of DOI:10.1021/acs.jmedchem.9b02071

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Article
Discovery of Acylsulfonohydrazide-Derived Lysine Acetyltransferase
KAT6A) Inhibitors as Potent Senescence-Inducing Anti-Cancer Agents
(
Daniel L. Priebbenow, David Leaver, Nghi Nguyen, Benjamin Cleary, H. Rachel Lagiakos, Julie
Sanchez, Lian Xue, Fei Huang, Yuxin Sun, Prashant Mujumdar, Ramesh Mudududdla, Swapna
Varghese, Silvia C Teguh, Susan A. Charman, Karen L. White, David M. Shackleford, Kasiram Katneni,
Matthew E Cuellar, Jessica M. Strasser, Jayme L. Dahlin, Michael A. Walters, Ian P. Street, Brendon
J. Monahan, Kate E. Jarman, Helene Jousset Sabroux, Hendrik Falk, Matthew C. Chung, Stefan J.
Hermans, Natalie L. Downer, Michael W. Parker, Anne K. Voss, Tim Thomas, and Jonathan B. Baell
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02071 • Publication Date (Web): 02 Mar 2020
Downloaded from pubs.acs.org on March 3, 2020
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Jarman, Kate; The Walter and Eliza Hall Institute; University of
Melbourne
Jousset Sabroux, Helene; The Walter and Eliza Hall Institute; University
of Melbourne
Falk, Hendrik; Walter and Eliza Hall Institute,
Chung, Matthew; Monash Institute of Pharmaceutical Sciences, Medicinal
Chemistry
Hermans, Stefan; The University of Melbourne St Vincent's Department
of Medicine
Downer, Natalie; Walter and Eliza Hall Institute of Medical Research
Bioinformatics Division
Parker, Michael; Saint Vincent's Institute of Medical Research, Biota
Structural Biology Laboratory
Voss, Anne; The Walter and Eliza Hall Institute
Thomas, Tim; Walter and Eliza Hall Institute of Medical Research
Bioinformatics Division
Baell, Jonathan; Monash Institute of Pharmaceutical Sciences, Medicinal
Chemistry
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Discovery of Acylsulfonohydrazide-Derived Lysine Acetyltransferase (KAT6A)
Inhibitors as Potent Senescence-Inducing Anti-Cancer Agents
‡
,
‡,
‡
‡
§
Daniel L. Priebbenow, David J. Leaver, Nghi Nguyen, Benjamin Cleary, H. Rachel Lagiakos,
Julie Sanchez, Lian Xue, Fei Huang, Yuxin Sun, Prashant Mujumdar, Ramesh Mudududdla,
,#
†
†
‡
‡
‡
£
∫
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‡
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£
Swapna Varghese, Silvia Teguh, Susan A. Charman, Karen L. White, David M. Shackleford,
£
∫
∫
∞
Kasiram Katneni, Matthew Cuellar, Jessica M. Strasser, Jayme L. Dahlin, Michael A. Walters,
Ian P. Street, Brendon J. Monahan,,§,# Kate E. Jarman,,# Helene Jousset Sabroux,,# Hendrik
,§,#
Falk, Matthew C. Chung, Stefan J. Hermans, Natalie L. Downer, Michael W. Parker, Anne K.
,§,#
¥
¥

¥
Voss,,# Tim Thomas,# and Jonathan B. Baell†,‡,⸹ ,
*
†School of Pharmaceutical Sciences, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing
11816, People’s Republic of China.
2
‡Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville
Campus), Parkville, Victoria 3052, Australia
Walter and Eliza Hall Institute, 1G Royal Parade, Parkville, Victoria 3052, Australia.
§Cancer Therapeutics CRC, 343 Royal Parade, Parkville, Victoria 3052, Australia
#Department of Medical Biology, University of Melbourne, Parkville, Victoria 3050, Australia.
£Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash
University, 381 Royal Parade, Parkville, Victoria 3052, Australia
∫Institute for Therapeutics Discovery and Development, University of Minnesota, Minneapolis, MN,
USA
∞Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA
¥ACRF Rational Drug Discovery Centre, St. Vincent’s Institute of Medical Research, Fitzroy,
Victoria 3065, Australia
⸹
ARC Centre for Fragment-Based Design, Monash University, Parkville, VIC 3052, Australia

These two authors contributed equally
*Correspondence to:
Jonathan B. Baell
Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences,
Monash University,
Parkville, VIC 3052, Australia
Phone: +61 3 9903 9044
Email: jonathan.baell@monash.edu
■
KEYWORDS: MOZ, MYST3, KAT6A, histone acetyltransferase, HAT inhibitor, senescence
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■ ABSTRACT
A high throughput screen designed to discover new inhibitors of histone acetyltransferase KAT6A
uncovered CTX-124143 (1) a unique aryl acylsulfonohydrazide with an IC of 1.0 M. Using this
5
0
acylsulfonohydrazide as a template, we herein disclose the results of our extensive structure-activity
relationship (SAR) investigations, which resulted in the discovery of advanced compounds such as
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5 and 80. These two compounds represent significant improvements on our recently reported
prototypical lead WM-8014 (3), as they are not only equivalently potent as inhibitors of KAT6A but
are less lipophilic and significantly more stable to microsomal degradation. Furthermore, during this
process we discovered a distinct structural sub-class that contains key 2-fluorobenzenesulfonyl and
phenylpyridine motifs, culminating in the discovery of WM-1119 (4). This compound is a highly
potent KAT6A inhibitor (IC =6.3 nM; K =0.002 M), competes with AcCoA by binding to the
50
D
AcCoA binding site, and has an oral bioavailability of 56% in rats.
■
INTRODUCTION
A requirement for cancer progression is that tumor cells can avoid the normal cellular regulation
1
of the cell cycle. One defense against uncontrolled cell cycle progression is permanent cell cycle
exit: cellular senescence. In therapeutic settings, there exists considerable potential for the application
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of senescence-inducing drugs in combination with senotherapeutics. Indeed, a recent study readily
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identified multiple different cancers with a patient/market need in this specific context. Melanoma
tumors, for example, have proven to be very responsive to treatment with the CDK4/6 inhibitor
palbociclib, which induces senescence.4
The acetylation of histones by lysine acetyltransferases (KATs) plays a critical role in the
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epigenetic regulation of gene expression. Among the genes encoding for the MYST family of KATs
(KAT5–KAT8) are the oncogenes KAT6A (MOZ/MYST3) and KAT6B (MORF/MYST4/Qkf).
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KAT6A is essential for the formation and maintenance of normal haematopoietic stem cells9-11 and is
the target of recurrent chromosomal translocations which result in acute myeloid leukaemia.12
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Furthermore, heterozygous loss of KAT6A inhibits lymphoma progression. KAT6A is known to
suppress cellular senescence through the regulation of suppressors of the CDKN2A locus, a function
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that requires its KAT activity. Thus, the opportunity to deliver therapeutic benefits particularly in
cancer, but also in a range of other disorders would be realized through discovery of specific inhibitors
of this class of lysine acetylases.
To this end, the overarching aim of our research program has been to discover novel potent
inhibitors of KAT6A. Initially, screening of HTS libraries of 243,000 structurally diverse “hit-like”
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compounds identified CTX-0124143 (1, Figure 1A) as a reversible AcCoA-competitive inhibitor
of KAT6A.15,17,18 While the original HTS and hit validation were conducted around ten years ago,
after a thorough optimization campaign we only recently reported the discovery of compounds 2 and
3
(Figure 1B) as potent inhibitors of KAT6A that bind reversibly in the AcCoA binding site as
determined by X-ray crystallography and SPR.15,18
Figure 1. Overview of SAR efforts to progress from the initial screening hit to highly potent HAT
inhibitors
The SAR summary of this preliminary work is shown in Figure 1B. Compound 3 (WM-8014) was
observed to induce cell cycle exit and cellular senescence (INK4A/ARF-dependent) without
damaging DNA and enhanced oncogene-induced senescence in a zebrafish model of hepatocellular
1
5
carcinoma. With regard to their physicochemical properties, 2 and 3 are both relatively hydrophobic,
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rapidly metabolized by human and mouse liver microsomes and highly bound to plasma protein. As
more lipophilic compounds tend to be more strongly bound to plasma proteins and rapidly
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metabolised, we set out to investigate whether we could emulate the potency of the lead compounds
whilst reducing lipophilicity. Using 2 and 3 as templates, we herein disclose our investigations to
further explore the SAR of acylsulfonohydrazides aiming to improve the physicochemical properties
to afford improved tool compounds to further probe KAT6A pharmacology.
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We also recently described the cellular effects of acylsulfonohydrazide 4 (WM-1119, Figure 1C),
a highly potent and selective inhibitor of KAT6A and KAT6B which possessed the ability to arrest
the progression of lymphoma in mice.15 Biochemical and structural studies demonstrated that
acylsulfonohydrazide 4 and related analogues were reversible competitors of acetyl coenzyme A that
inhibit MYST-catalyzed histone acetylation. Both compounds 3 and 4 induced cell cycle exit and
cellular senescence yet did not damage DNA, properties which suggest such compounds could be
further developed as anti-cancer agents. Limited testing against KAT6B suggests these compounds
are inherently dual KAT6A/6B inhibitors, but our focus has been on KAT6A. As the comprehensive
medicinal chemistry efforts that led to the discovery of 4 and related analogues has not yet been
disclosed in detail, we herein describe our efforts that culminated in the development of a series of
highly potent KAT6A inhibitors.
■
RESULTS AND DISCUSSION
Chemistry: Target compounds were synthesized as summarized in Schemes 1 and 2 by coupling
either (i) an aryl carboxylic acid (e.g. 7, 8, or 9) with an arylsulfonylhydrazide 11 (prepared from
sulfonyl chloride 10); or (ii) an aryl sulfonyl chloride 10 with a benzoyl hydrazide 12 (prepared from
the corresponding carboxylic acid). If not commercially available, carboxylic acid derivatives 7/8/9
were prepared from the bromo-benzoic acids using the strategies summarized in Scheme 1.18,20 To
prepare the requisite ether-substituted carboxylic acids, bromobenzoic acid 5 was converted to the
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corresponding phenol 6 utilizing an Ullman type coupling reaction (Scheme 1). The next step
involved alkylation of 6 with an alkyl halide followed by hydrolysis to reveal the free carboxylic acid
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which was subsequently coupled to benzene sulfonohydrazide to yield the benzoylsulfonohydrazide
derivatives (Scheme 2). The incorporation of 5- or 6-membered heterocyclic rings at the meta-
position was achieved by converting the substituted bromobenzoic acids 5 to the corresponding
substituted heterocyclic carboxylic acids via an Ullman-type or Suzuki reaction (Scheme 1). The
substituted carboxylic acids derivatives were then coupled with benzoylsulfonohydrazide 12 to afford
the corresponding benzoyl sulfonohydrazide derivatives (Scheme 2).
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Scheme 1. Synthesis of benzoic acid intermediates: Reagents and conditions: (a) Na CO , CuBr ,
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trans-N,N′-dimethyl-1,2-cyclohexanediamine, H O, reflux o/n; (b) alkyl halide, K CO , DMF reflux
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h; (c) NaOH, EtOH 50°C o/n; (d) 1,4-dioxane, KOAc, PdCl (dppf), bis(pinacolato)diboron, reflux
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o/n, then DMF/H O (10:1), heteroaryl halide, Cs CO , Pd(PPh ) , 100°C, o/n; (e) 1,4-dioxane/H O
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(9:1), K CO , heteroaryl boronic acid, PdCl (PPh ) , 80 C, μW, 30 min. (f) K CO , CuI, DMF, trans-
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N,N′-dimethyl-1,2-cyclohexanediamine, N- or O-heterocycle, 110°C o/n.
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Scheme 2. Synthesis of N'-aroylaryl-2-sulfonohydrazides: Reagents and conditions: (a)
o
NH NH H O, CH Cl , 0 C to rt, 3 h; (b) (i) HBTU, DIPEA, MeCN, aryl carboxylic acid, reflux o/n;
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
or (ii) EDCIHCl, HOAt, MeCN, aryl carboxylic acid, 40 C o/n; (c) MeOH, cat. H SO , reflux o/n
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(d) NH NH H O, EtOH, reflux o/n; (e) arylsulfonyl chloride, pyridine, rt 2 h.
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The synthesized compounds were then evaluated for their ability to inhibit KAT6A. Over the
course of our program we employed both lower (0.4 M) and higher (15 M) Ac-CoA concentrations
in our competition assay. The net effect was that compounds appeared about ten-fold weaker when
competing with higher concentrations of Ac-CoA but the correlation between the two was otherwise
excellent.18
SAR Discussion
The first section (Tables 1-5) of the SAR discussion describes our efforts to develop analogues of
3
containing the key 2-fluorobenzoyl motif (RHS of the molecule as drawn in Figure 1A and B), that
possessed improved physicochemical properties. At the same time, a parallel investigation into
acylsulfonohydrazide derived KAT6A inhibitors containing a 2-fluorobenzenesulfonyl group (LHS
of the molecule as drawn in Figure 1C) was conducted, and these efforts are described in the second
section (Tables 6-13) of the SAR discussion. Following the SAR discussion, the outcomes of
structural biology and physicochemical evaluation of compounds from both SAR streams are
described and discussed.
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Development of WM-8014 analogues with improved microsomal stability
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During our initial investigations, we identified that large substitutions at the meta-position on the
RHS of the hydrazide core (e.g. phenyl) governed the orientation and substitution pattern which
enhanced the potency of these compounds. Subsequently in the medicinal chemistry program, a retest
of collections of our compounds revealed the meta-methoxy derivative 14 to be unexpectedly potent
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(
Table 1), returning an IC value of 0.69 M. In prior assays using higher AcCoA concentrations
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such aberrantly potent activity had not been observed, for reasons that remain unclear. We proposed
that the methoxy substitution in compound 14 was adopting the same orientation as the meta-phenyl
group in 2 and 3. We were therefore interested in exploring the SAR of this position further to
determine whether less hydrophobic groups than the phenyl ring in 2 or 3 could reduce lipophilicity
and plasma protein binding, while enhancing metabolic stability and maintaining potency.
It was speculated that if the methoxy group of derivative (14) sub-optimally occupied the large
hydrophobic pocket where the meta-phenyl of 3 binds tightly, that KAT6A affinity would increase if
the alkoxy group was made longer and bulkier. When compared with the unsubstituted parent
compound 13 (IC =4.2 µM) the methoxy (14), ethoxy (16) and n-propoxy (18) naphthyl derivatives
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returned respective IC values of 0.69, 0.57 and 0.12 µM, which supported our prediction that this
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group occupied the hydrophobic 5-phenyl-binding region, as opposed to the other meta-position,
18
which favored small hydrophobic groups in 2 and 3. In our previous study , we found that
simplifying the naphthyl group to a phenyl group led to only a minor loss of activity, and that potent
compounds were obtained following installation of a 2-fluoro, and/or 3-methyl or 3-chloro
substituents on the RHS of the molecule. First, we investigated the importance of the naphthyl group
1
when combined with the aforementioned C3-alkoxy substitutions (R , Table 1).
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Table 1. Inhibitory potency of alkoxy benzoylnaphthalene-2-sulfonohydrazide analogues and
alkoxy benzoylsulfonohydrazide analogues against KAT6A.
IC (µM)
.4 µM [AcCoA]
R1
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Entry
Compound
LHS
0
1
2
3
4
5
6
7
13
14
15
16
17
18
19
Naphthalene
Naphthalene
Phenyl
Naphthalene
Phenyl
H
4.2
0.69
2.3
0.57
0.37
0.12
0.38
OMe
OMe
OEt
OEt
n
Naphthalene
Phenyl
O Pr
n
O Pr
It was apparent from the activity of the methoxy (15), ethoxy (17) and n-propoxy (19) derivatives
that the naphthyl group in the corresponding analogues 14, 16 and 18 did contribute to potency by
severalfold. Nevertheless, we chose to continue on with the phenyl substituted analogues over the
naphthalene system as a key focus to reduce the overall lipophilicity of these compounds. Earlier
investigations revealed that multiple substitutions on the RHS phenyl ring were incompatible with
the bulky naphthalene ring on the LHS, and as such we decided to focus our efforts on exploration of
the benzenesulfonyl rather than naphthalenesulfonyl analogues to allow for a broader scope of
exploration. We then sought to expand the SAR exploration at the C3-meta-position of the RHS
phenyl ring of the benzenesulfonyl motif by introducing a number of alkoxy and alkyl modifications
at this site (Table 2).
Table 2. Inhibitory potency of alkoxy and alkyl benzoylsulfonohydrazide analogues against
KAT6A.
IC (µM)
.4 µM [AcCoA]
R1
50
Entry
Compound
0
1
2
3
15
17
19
OMe
OEt
O Pr
2.3
0.37
0.38
n
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
i
4
5
6
7
8
20
21
22
23
24
25
26
27
28
29
30
31
O Pr
0.33
3.0
0.24
0.44
4.7
10
2.3
3.6
3.3
6.2
7.2
11
n
O Bu
i
O Bu
Ocyclopentyl
OPh
OCF3
Me
Et
nPr
iPr
9
10
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
1
2
13
1
1
4
5
F
CF3
From this investigation, an increase in potency was observed for 17 and 19 from 15, as we
progressed from methoxy (IC = 2.3 µM), ethoxy (IC = 0.37 µM), n-propoxy (IC = 0.38 µM), iso-
5
0
50
50
propoxy (IC = 0.33 µM) and iso-butoxy (IC = 0.24 µM). Interestingly, there was a sharp decrease
50
50
in activity observed for the n-butoxy analogue 21 (IC = 3.0 µM) suggesting a size limit for optimal
5
0
binding being a 3-4 atom alkoxy motif (ethyl or propyl ethers). Additional substitution in the form of
the isopropyl (20) and isobutyl (22) substitutions afforded no observable advantage in potency. Also
evaluated were analogues containing alkyl, fluoro and fluoroalkyl substituents at the R1-position
(
Table 2, entries 10-15), which led to a significant loss in potency. This indicated that the presence
of the oxygen heteroatom in this SAR exploration was important for the activity of this class of
compounds however the phenoxy (24) and trifluoromethoxy (25) derivatives were inactive.
We then set out to further elaborate the SAR around the RHS ether-substituted benzoyl ring. In
order to ascertain whether a C2-fluoro substituent increased activity in the same manner as observed
during previous studies (i.e. 2 and 3), we synthesised and evaluated the series of compounds outlined
2
in Table 3. From the results outlined in Table 3, a fluorine atom at the R position (entries 1-5) had a
remarkable effect on activity, as seen through the comparison of compounds 32, 33 and 34 with their
2
respective des-fluoro counterparts 17, 19 and 20 (Table 2). In each case, introduction of R fluoro
resulted in a significant increase in inhibitory activity ranging from two- to five-fold. Also
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3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
investigated with this substitution pattern were analogues containing terminal alkene (35) and alkyne
(
36) functional groups (Table 3, entries 4 and 5) of which compound 35 possessed impressive potency
IC = 0.070 M).
(
50
Table 3. Inhibitory potency of second generation alkoxy benzoylsulfonohydrazide analogues
against KAT6A.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
IC (µM)
.4 µM [AcCoA]
0.19
Entry
Compound
R1
R2
R3
50
0
1
2
3
4
5
6
7
8
9
32
33
34
35
36
37
38
39
40
41
42
43
44
45
OEt
F
F
F
F
F
n
O Pr
0.081
0.062
0.070
0.12
0.40
0.11
0.062
0.063
0.052
0.054
0.061
0.017
0.029
i
O Pr
O-allyl
O-propargyl
n
O Pr
F
F
OCH cyclopropyl
2
OEt
F
F
F
F
F
F
F
Me
Me
Me
Me
Me
Me
Me
i
O Pr
n
1
1
0
1
O Pr
O-allyl
OCH cyclopropyl
12
2
1
1
3
4
O-2-MeAllyl
CH OEt
2
The logical next step was to attempt to engage the small hydrophobic pocket in the region of the
3
R -substituent by introduction of either only a fluoro substituent (Table 3, entries 6 and 7) or a methyl
3
2
substituent at the R -position with retention of the R -fluorine substitution (Table 3, entries 8-14).
The propyl ether analogues were observed to have slight increases over the des-methyl analogues
with the ethoxy analogue 39 exhibiting a three-fold increase in activity over its des-methyl
2
3
counterpart 32. In general, the combination of the R = F and R = Me appeared to be the most
favorable substitution pattern for this class of compounds, affording highly active analogues.
To further probe the SAR of these compounds, we next set out to explore the incorporation of
1
heteroaryl substituents at the C5-position (Table 4, R ) of the benzoyl ring, in place of the previously
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3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
explored alkoxy motifs. During this study, a variety of substituents at the C2- and C3-positions of the
benzoyl ring were concurrently explored.
Table 4. Inhibitory potency of heteroaryl benzoylsulfonohydrazide analogues against KAT6A
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
IC (µM)
.4 µM [AcCoA]
0.12
Entry
Compound
R1
R2
R3
50
0
1
2
3
4
5
6
8
9
10
11
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
furan-2-yl
furan-2-yl
furan-2-yl
furan-2-yl
furan-2-yl
furan-2-yl
thiazol-2-yl
thiazol-4-yl
triazol-2-yl
F
Me
Cl
F
OMe
Me
0.13
0.11
0.14
0.48
0.080
0.16
0.27
0.34
0.038
0.61
0.14
0.44
1.2
0.078
0.16
0.43
0.065
0.22
F
F
F
triazol-2-yl
Me
1
1
1
1
1
2
3
4
5
6
1H-pyrazol-1-yl
1H-pyrazol-1-yl
1H-pyrazol-1-yl
1H-pyrazol-1-yl
1H-pyrazol-1-yl
3-methyl-1H-pyrazol-1-yl
3-methyl-1H-pyrazol-1-yl
3-methyl-1H-pyrazol-1-yl
4-methyl-1H-pyrazol-1-yl
4-methyl-1H-pyrazol-1-yl
4-methyl-1H-pyrazol-1-yl
4-fluoro-1H-pyrazol-1-yl
4-fluoro-1H-pyrazol-1-yl
5-methyl-1H-pyrazol-1-yl
pyridin-2-yl
F
F
OMe
Me
F
F
17
1
1
2
2
2
8
9
0
1
2
F
Me
F
F
F
0.80
F
Me
Me
Me
Me
0.084
0.60
23
24
a
F
F
0.052
2
2
2
2
2
5
6
7
8
9
0.087
0.14
0.09
0.10
0.39
0.073
0.63
0.14
0.23
0.45
0.67
0.067
>125
>125
pyridin-2-yl
pyridin-2-yl
pyridin-2-yl
pyridin-2-yl
pyrimidin-2-yl
pyrimidin-2-yl
pyrimidin-2-yl
pyrimidin-2-yl
pyrimidin-2-yl
pyrimidin-2-yl
pyrimidin-5-yl
pyridazin-4-yl
F
F
OMe
Me
30
31
F
F
3
3
3
3
3
2
3
4
5
6
Me
F
OMe
Me
F
F
37
3
8
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5
As shown in Table 4, preliminary investigations with the unsubstituted thiazole, pyrazole, pyridine
and pyrimidine all proved promising, with compounds 56, 70 and 75 recording IC values below 1.0
5
0
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
µM. On this basis, we undertook the synthesis of the more elaborated analogues with a fluoro
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
substituent in the C2-position (R ), a strategy which had been previously shown to significantly
improve the activity of the alkoxy analogues (see Table 3). As shown in Table 4, this trend was again
observed for the heterocycle analogues, in particular, the pyrazole (57) and pyridin-2-yl (71)
analogues returning increased potency when compared with the corresponding C2-des-fluoro
compounds. Also tested with this substitution pattern were the furan-2-yl (46), 3-methyl-1H-pyrazol-
1
yl (61) and 4-methyl-1H-pyrazol-1yl (64) analogues, all of which possessed reasonable potency.
We continued to probe the SAR of these heterocyclic analogues by introducing a substituent at the
3
3
R position. As shown in Table 4, the small hydrophobic substituents (R = F, Me or Cl) afforded
3
more potent compounds than the larger, more polar substitutions (R = methoxy). This is exemplified
by the furan-2-yl (Table 4, entries 2-5) and pyrimidin-2-yl (Table 4, entries 33-35) series whereby
3
the general trend observed for R substituents with regard to potency was Me>F>OMe.
1
To further explore the value of heterocyclic substitution at the C5-position (R ) we next assembled
2
3
a series of analogues containing both R fluoro and R methyl groups. From this investigation, we
identified a number of highly potent KAT6A inhibitors with all analogues in this class returning sub-
1
00 nM IC values. In particular the effect of introducing the methyl substituted analogues can be
50
seen through the comparison of the triazole analogues 54 and 55 which returned a nine-fold increase
3
in activity following the inclusion of an R methyl substituent. In addition, the decorated 3-methyl,
2
3
4
-methyl and 5-methyl pyrazolyl analogues 63, 66 and 69 containing the R -fluoro, R -methyl
substitution pattern were also identified as very potent analogues. Overall, there were no significant
differences in potency when comparing the oxygen-based heterocycles with the nitrogen
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
heterocycles, however, the pyrimidin-5-yl (81) and pyridazin-4-yl (82) analogues were completely
inactive against KAT6A.
Finally, to complete our investigations we selected two compounds and reintroduced the
naphthalene sulfonyl ring in place of the benzenesulfonyl group (Table 5, entries 2 and 4). As shown
in Table 5, compounds 83 and 84 were significantly less potent than their phenyl counterparts 39 and
55, even though earlier studies (see Table 1) indicated that the naphthalene ring appeared to be
important for activity. This confirmed our previously established theory that a highly decorated RHS
was incompatible with a bulky LHS naphthalene.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. Comparison of inhibitory potency of benzoylnaphthalene-2-sulfonohydrazide
analogues and benzoylsulfonohydrazide analogues against KAT6A.
IC (µM)
0.4 µM [AcCoA]
0.064
R1
50
Entry
Compound
LHS
1
2
3
4
5
3
Phenyl
Naphthyl
Phenyl
Naphthyl
Phenyl
Ph
OEt
OEt
83
39
84
55
0.082
0.062
0.096
0.038
triazol-2-yl
triazol-2-yl
Of note, and as shown in Table 5, reference compound 3 returned an IC value of 0.064 µM under
5
0
the assay conditions used throughout this section of the program, showcasing the utility of replacing
the pendant phenyl ring with the smaller, less hydrophobic heterocycles of triazol-2-yl for 55 or
pyrimidin-2-yl for 80 (Table 4). With potent new compounds in hand, analyses to determine if the
structural modifications provided increased microsomal stability were conducted and the outcomes
of this investigation are discussed towards the end of the manuscript.
Discovery of WM-1119 (4)
As discussed above, we had identified that the naphthyl group present in the initial hit CTX-
0
124143 (1) could be readily replaced with a phenyl group without significant loss of activity.18
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3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Whilst exploring this phenomenon, we identified that a 2-fluorobenzenesulfonyl moiety in place of
the naphthylsulfonyl group on the LHS of the molecule was particularly effective, leading to
increased potency in most cases. As such, in parallel to the work described above, the following
section describes the design, synthesis and evaluation of 2-fluorobenzene sulfonyl analogues,
exploring various additional substitution patterns on both the benzoyl ring (right-hand side of the
molecule as drawn in Figure 1) and the 2-fluorobenzensulfonyl motif (left-hand side).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In order to obtain potent biochemical activity against KAT6A, it was previously deemed important
1
8
to have a fluorine atom in the 2-position of the benzoyl ring. X-ray crystallographic analysis of the
complex between such compounds (for example 3) and KAT6A readily showed why, since the
fluorine atom formed a hydrogen bond with the benzamide proton, locking this moiety into a quasi-
planar conformation and aiding the orientation of the RHS ring allowing the other substituents to bind
in optimal locations.21 Unsurprisingly, addition of a fluorine atom in the 2-position of the
phenylsulfonyl ring had no such beneficial effect, and indeed proved detrimental to inhibitory potency
by several fold.18
During our investigation, we purchased the simple 2-fluorobenzenesulfonylhydrazide analogue
(
85), which when evaluated returned an IC value of 0.80 M. Given that the unsubstituted parent
5
0
compound exhibited an IC50 of 6.7 M, it became clear that a 2-fluoro substituent in the
phenylsulfonyl ring could be beneficial when an opposing 2-fluoro substituent on the benzoyl ring
was absent, an entirely unexpected result based on previous SAR investigations. Given the pseudo-
symmetrical nature of this system, we hypothesized that the core might have flipped 180 degrees
horizontally within the KAT6A binding site or adopted an unrelated binding mode. We thus embarked
on a new SAR investigation focused on 2-fluorobenzenesulfonohydrazides. This was influenced by
prior SAR knowledge gained when the 2-fluoro substituent was on the opposite ring, to gauge
whether parallel SAR might be observed.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
Initially, the unsubstituted benzoyl analogue where R = H (85) exhibited good activity at IC =
5
0
0
.8 M (Table 6, entry 1), with enhanced potency observed for the naphthoyl derivative 86 (entry 2,
1
IC = 0.23 M). As shown in Table 6, a variety of meta (R ) substituents afforded equipotent or more
5
0
potent compounds than the unsubstituted derivative. For example, introducing a methyl substituent
at the meta-position (87) led to a two-fold increase in potency, and the presence of an ethyl group at
the same position (88) further increased potency (IC = 0.22 M, Table 6 entry 4), matching the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
activity observed for the naphthoyl compound 86. Interestingly, the incorporation of an iso-propyl
group at the meta-position (89) led to a loss of activity whereas an n-propyl group (90) increased
activity (IC =0.094 M).
5
0
Table 6. Inhibitory potency of 2-fluorobenzenesulfonohydrazide analogues against KAT6A
with variation of the right-hand side meta-position
IC (µM)
5
0
Entry
Compound
R1
15 µM [AcCoA]
0.4 µM [AcCoA]
1
2
3
4
5
6
7
8
9
85
86
87
88
89
90
91
92
93
H
2-naphthyl
Me
7.0
3.5
2.2
3.5
13
0.85
8.9
0.56
1.8
0.80
0.23
0.38
0.22
0.97
0.094
0.14
0.060
0.18
Et
i
Pr
Pr
n
Ph
I
Br
When compared to the unsubstituted template molecule 85 (Table 6, entry 1), incorporation of a
bulky phenyl group (compound 91, Table 6, entry 7) led to a slight increase in potency. Further
elaboration at this site to incorporate a halogen (in the form of a bromine or iodine atom, Table 6,
entries 8 and 9) led to increased activity with the 3-iodobenzoyl analogue 92 proving to be the most
active of the two returning an IC = 0.060 M. These results indicated that the binding site could
5
0
tolerate the inclusion of larger lipophilic substituents at the meta-position of the benzoyl ring.
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4
Taking the relatively active meta-phenyl derivative 91 as a new template, we then set out to explore
the incorporation of additional functionality on both the left-hand 2-fluorobenzenesulfonyl (at the 3-
and 4-positions) and right-hand benzoyl ring (at the 2-, 5- and 6-positions, Table 7).
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 7. Inhibitory potency of 2-fluorobenzenesulfonohydrazide analogues against KAT6A
with variation of the left- and right-hand side
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
IC (µM)
15 µM [AcCoA] 0.4 µM [AcCoA]
5
0
Entry
Compound
R1 R2 R3 R4
R5
1
2
3
4
5
94
95
96
97
98
F
Cl
Me
F
F
F
0.017
0.048
F
OH
0.19
0.061
0.13
OH
OCH3
For this investigation, the additional functionality on the RHS benzoyl ring (in the form of small
lipophilic substituents such as F, Cl or CH , Table 7, entries 1-2) significantly increased activity with
3
the 2-fluoro-3-phenyl-5-chloro analogue (94) proving to be most active (Table 7, entry 1, IC = 0.017
5
0
M). Keeping the fluoro substituent constant at the 6-position of the RHS 3-phenylbenzoyl ring, the
incorporation of more polar hydroxy or methoxy groups on the LHS benzenesulfonyl ring (entries 3-
5
) also increased activity relative to the parent biphenyl (91), with 2-fluoro-4-hydroxybenzene
sulfonyl derivative 97 exhibiting the best activity of these analogues with an IC of 0.061 M against
5
0
KAT6A in the presence of 15 M of AcCoA.
To further interrogate substitution at the 3-position of the right-hand side benzoyl ring, heteroatom
derivatives in the form of alkyl ethers (Tables 8 and 9) and amines (predominantly N-heterocycles –
Tables 10 and 11) were explored whilst retaining the ortho-fluoro substituent on the left-hand
benzenesulfonyl ring.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 8. Inhibitory potency of C3-ether substituted 2-fluorobenzenesulfonohydrazide
analogues against KAT6A
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
IC (µM)
5
0
Entry
Compound
99
R1
Et
15 µM [AcCoA]
0.4 µM [AcCoA]
0.069
1
2
3
4
5
6
7
8
9
1.1
0.82
0.85
1.1
i
100
101
102
103
104
105
106
107
Pr
Pr
0.059
0.094
0.080
0.23
n
i
Bu
Bu
n
3.5
cyclopentyl
3.5
0.23
-CH -cyclopropyl
0.12
2
allyl
CF3
0.030
0.56
7.6
The ethyl (99), iso-propyl (100), n-propyl (101) and iso-butyl (102) ether derivatives all
demonstrated good inhibitory activity against KAT6A in the range of IC = 0.059 – 0.094 M (Table
5
0
8
, entries 1-4). The n-butyl (103), cyclopentyl (104), methylcyclopropyl (105) and trifluoromethoxy
107) ether derivatives however did not deliver the same increase in activity as observed for smaller
substituents (returning IC values in the range of 0.12 – 0.56 M). The most active compound
(
5
0
identified from this series turned out to be the O-allyl substituted benzoyl derivative 106 (Table 8,
entry 8) which returned an excellent IC of 0.030 M.
50
Building on the promising activity observed for ethereal substituents at the meta-position of the
benzoyl ring, we then set out to explore the incorporation of further functionality at varying points on
both aryl rings, retaining both the ether substituent on the RHS and the 2-fluorobenzenesulfonyl motif
on the LHS (Table 9).
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Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 9. Inhibitory potency of ether substituted 2-fluorobenzenesulfonohydrazide analogues
against KAT6A with variation on left- and right-hand sides
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
IC (µM)
5
0
Entry Compound
R1
R2 R3 R4 R5
R6
R7
0.4 µM
AcCoA]
3.6
[
1
2
3
4
5
6
7
8
9
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
-OEt
-OEt
-OEt
-OEt
-OEt
-OEt
-OEt
-OEt
-OEt
-OEt
-OEt
-OEt
-OEt
-OEt
-OEt
Br
Br
OMe
0.53
0.27
5.0
0.016
0.11
0.16
0.074
0.17
0.16
0.13
OMe
Me
F
OMe
Me
Me
Me
Me
Me
Me
Me
Me
Me
F
OMe
Me
F
Me
F
F
F
Me
10
1
1
1
1
1
1
2
3
4
5
F
Me
0.18
0.17
0.12
0.13
F
Me
F
i
16
17
-O Pr
0.015
0.061
0.11
0.090
0.091
0.016
0.020
0.072
0.027
0.024
i
-O Pr
i
1
1
2
2
2
8
9
0
1
2
-O Pr
-OCH -cyclopropyl
-OCH -cyclopropyl
2
2
-Oallyl
-Oallyl
n
23
-O Pr
F
Me
Me
2
2
4
5
-O-cyclopropyl
-CH OEt
2
1
Initially, for compounds containing the ethyl ether at R , bromo or methoxy substituents were
4
6
7
introduced at the R , R and R positions on the left-hand sulfonyl ring however potency was not
increased when compared to the parent compound 99 (Table 9, entries 1-4). Addition of a methyl
2
group on the RHS ring at the other meta-position (R =CH , Table 9, entry 5) led to a slight increase
3
1
in activity for the R ethyl ether (IC =0.016 M), however, interestingly, fluoro or methoxy groups
5
0
2
at this same R position resulted in decreased potency (Table 9, entries 6 and 7).
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
Keeping this R = CH substituent constant, additional functionality was then introduced at varying
3
positions on both LHS and RHS aryl rings. The 2-fluoro-3-methyl derivative 115 (Table 9, entry 8)
demonstrated good activity with an IC = 0.074 M, however for the most part these efforts did not
50
afford any significant increase in potency (Table 9, entries 9-15). Using a similar approach, methyl,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
fluoro or methoxy substitution at the R position for the iso-propoxy (Table 9, entries 16-18),
cyclopropylmethoxy (entries 19-20), allyloxy (entries 21-22), n-propoxy (entry 23), cyclopropoxy
1
i
2
(entry 24), and ethoxymethyl (entry 25) was explored, with compounds 123 (R = O Pr, R = CH ;
3
1
2
IC = 0.015 M) and 128 (R = Oallyl, R = CH ; IC = 0.016 M) identified as the most potent from
5
0
3
50
this investigation.
The incorporation of relatively small substituents around both aryl rings was tolerated but did not
offer any significant advantages with regard to potency when compared to the undecorated parent
molecules. Substitutions at the 3- and 5-positions of the benzoyl ring proved to be most favorable,
however the introduction of various substituents at any other position across the left- or right-hand
side aryl rings typically did not enhance potency.
Having explored the incorporation of a variety of relatively flexible aliphatic O-linked
substitutions at the meta-position, our focus then shifted to the incorporation of more rigid aromatic
N-linked heterocyclic substituents at this same position (Tables 10 and 11). In the first instance,
pyrazole derivatives at the meta-position of the RHS benzoyl ring were explored (Table 10) whilst
incorporating additional functionality in the form of fluoro, methyl or methoxy substituents at the
1
18
other meta-position (Table 10, R ) which was previously found to enhance potency.
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1
Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 10. Inhibitory potency of first-generation pyrazolyl N’-benzoyl-2-sulfonohydrazide
analogues against KAT6A with variation on left- and right-hand sides
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
IC (µM)
R1
R2
R3
R4
R5
R6
50
Entry
Compound
0
.4 µM [AcCoA]
0.043
0.013
0.023
0.27
1
2
3
4
5
6
7
8
9
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
OMe
F
F
F
F
OMe
OMe
0.35
0.026
0.1
0.027
0.062
0.23
0.39
0.53
0.18
0.12
OH
OMe
F
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
F
1
1
1
1
0
1
2
3
F
Me
OH
F
Me
14
1
1
1
1
1
5
6
7
8
9
Me
OH
OMe
0.096
0.23
0.073
0.18
F
OMe
0.11
The parent compound 133 (Table 10, entry 1) returned an excellent IC50 of 0.043 M.
Incorporation of additional substituents on both phenyl rings such as fluorine, methoxy, hydroxy and
methyl groups in varying combinations was then explored (Table 10, entries 2-21). In general, the
introduction of multiple substituents led to decreased potency, except in the case of compounds 134
1
1
2
6
(
R = OMe, IC = 0.013 M), 135 (R = F, IC = 0.023 M), 138 (R = F, R = OH, IC = 0.026
5
0
50
50
1
M), and 140 (R = Me, IC = 0.027 M) which ranged from two-fold more to two-fold less potent
5
0
than parent compound 133.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Variations in the substitution patterns on the pyrazole motif were then explored through
incorporation of fluoro, methyl and dimethyl pyrazoles (Table 11). Again, for this series, various
2
substituents at the other meta-position (Table 11, R ) was also explored.
Table 11. Inhibitory potency of second-generation pyrazolyl and triazolyl N’-benzoyl-2-
sulfonohydrazide analogues against KAT6A
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
IC (µM)
.4 µM [AcCoA]
0.34
Entry
Compound
R1
R2
50
0
1
2
3
4
5
6
7
8
9
152
153
154
155
156
157
158
159
160
161
162
163
164
165
4-fluoro-1H-pyrazol-1-yl
4-fluoro-1H-pyrazol-1-yl
3,5-dimethyl-1H-pyrazol-1-yl
3-methyl-1H-pyrazol-1-yl
3-methyl-1H-pyrazol-1-yl
3-methyl-1H-pyrazol-1-yl
3-methyl-1H-pyrazol-1-yl
4-methyl-1H-pyrazol-1-yl
4-methyl-1H-pyrazol-1-yl
4-methyl-1H-pyrazol-1-yl
5-methyl-1H-pyrazol-1-yl
5-methyl-1H-pyrazol-1-yl
2-triazolyl
Me
0.029
0.18
0.018
0.023
0.007
0.203
0.30
0.032
0.014
0.031
0.013
0.018
0.009
F
Me
OMe
F
Me
F
Me
Me
F
1
1
1
1
1
0
1
2
3
4
2-triazolyl
From this investigation, the 3-methylpyrazole (157), 4-methylpyrazole (161) and 5-
2
methylpyrazole (163) derivatives all possessing an R = Me group proved to be the most active
returning exceptional IC values of 0.007 M, 0.014 M and 0.013 M, respectively. In general, the
50
2
2
R = Me derivatives were more potent than the corresponding R = F derivatives, and interestingly,
inclusion of a methoxy substituent in this position (entry 7) led to a significant loss of activity. Finally,
2
two potent triazole derivatives were prepared (Table 11, entries 13 and 14), and in this case the R =
2
F analogue 165 (IC = 0.009 M) showed enhanced potency when compared to the R = Me analogue
5
0
1
64 (IC = 0.018 M).
5
0
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Journal of Medicinal Chemistry
1
2
3
4
1
We next set out to explore the suitability of C-linked heterocycles at the meta-position (R ) of the
right-hand side benzoyl ring (Table 12). To accomplish this, a series of analogues containing a furan,
thiophene, or oxadiazole motif was synthesized. Again, incorporation of additional functionality at
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
the other meta-position (R ) on the RHS benzoyl ring was concurrently explored.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 12. Inhibitory potency of C-linked 5-membered heteroaryl N’-benzoyl-2-
sulfonohydrazide analogues against KAT6A
IC (µM)
0.4 µM [AcCoA]
0.020
R1
R2
50
Entry
Compound
1
2
3
4
5
6
7
8
9
166
167
168
169
170
171
172
173
174
175
furan-2-yl
furan-2-yl
furan-2-yl
furan-2-yl
furan-2-yl
Cl
Me
OMe
F
0.0055
0.011
0.11
0.0094
0.025
0.024
0.014
0.49
4.63
thiophen-2-yl
thiophen-2-yl
thiophen-3-yl
3-(1,2,4-oxadiazol)yl
2-(1,3,4-oxadiazol)yl
Cl
1
0
From this investigation, it was observed that furan and thiophene substitution at the 3-position of
the RHS benzoyl ring was favorable (Table 12, entries 1-8) with these analogues predominantly
returning IC values of well under 0.1 M, whereas installation of an oxadiazole motif in this position
5
0
2
resulted in loss of activity (entries 9 and 10). Again, substitution at the other meta-position (R ) was
well tolerated, with 3-(furan-2-yl)-5-chloro benzoyl derivative 167, 3-(furan-2-yl)-5-methyl benzoyl
derivative 168 and 3-(furan-2-yl)-5-fluoro benzoyl derivative 170 exhibiting exceptional activity
against KAT6A with IC values of 0.0055, 0.011 and 0.0094 M, respectively when screened against
50
0
.4 M AcCoA concentration.
To further explore the chemical space, installation of C-linked 6-membered heterocycles (e.g.
pyridine, pyrimidine and pyridazines) at the 3-position of the benzoyl ring was explored (Table 13).
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
Again, incorporation of additional functionality at the other meta-position (R ) on the RHS benzoyl
3
4
ring was concurrently studied, in addition to the R and R -substitutions also highlighted in Table 13.
Table 13. Inhibitory potency of C-linked 6-membered heteroaryl N’-benzoyl-2-
sulfonohydrazide analogues against KAT6A
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
IC (µM)
Entry Compound
R1
R2
R3
R4
50
0
.4 µM [AcCoA]
0.006
0.008
0.005
0.0063
0.044
2.4
1
2
3
4
5
6
7
8
9
176
177
178
4
179
180
181
182
183
184
185
186
pyridin-2-yl
pyridin-2-yl
pyridin-2-yl
pyridin-2-yl
pyridin-2-yl
Me
OMe
F
Me
F
F
pyridin-2-yl (N-oxide)
pyridin-2-yl
F
OH
0.27
pyrimidin-2-yl
pyrimidin-2-yl
pyrimidin-2-yl
pyrimidin-2-yl
pyridazin-4-yl
0.054
0.009
0.014
0.024
1.9
Me
F
OMe
1
0
11
1
2
From this study, the pyridine analogues were observed to be most active, with methoxy (178) and
2
fluoro (4) substituents at the other meta-position (R ) affording the most potent analogues, exhibiting
IC values of 0.005 M and 0.0063 M respectively against KAT6A. Interestingly for this case, the
5
0
3
-aryl-5-fluoro analogue 4 was more active than the 3-aryl-5-methyl derivative 177 which reversed
the trend previously observed. The pyrimidinyl derivatives (Table 13, entries 8-11) were also very
2
active (with the R = Me derivative returning an IC = 0.009 M), however the pyridazine derivative
5
0
1
86 proved to be comparatively inactive. Subsequent efforts focused on incorporating a nitrogen atom
into the ring of the RHS benzoyl group (Figure 2), however these analogues were all relatively
inactive, and this strategy was not further pursued.
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Inhibitory potency of pyridinyl analogues screened against KAT6A
With multiple new highly potent KAT6A inhibitors identified from the two parallel SAR
investigations described above, additional studies were conducted which involved evaluating cell-
based activity, structural biology and physicochemical parameters of these compounds with the
results described in the following sections.
Cell-based activity.
We previously described a 7-day cell-based assay involving the culturing of mouse embryonic
fibroblasts (MEFs) under low oxygen conditions;15 both 3 and 4 inhibit proliferation at sub-
1
5
micromolar concentrations resulting in cells entering a senescent state. In the work reported herein,
seven new compounds were selected for testing in this assay. As shown in Table 14, the most potent
compounds 55, 80, 99 and 133 were similar to positive control and prototypical leading compound 3
1
5
that has an IC value of 0.551 M in the MEF assay as recently reported. In contrast, negative
5
0
control WM-2474, which is structurally and physicochemically similar to the active compounds
described herein does not exhibit biochemical activity against KAT6A and was inactive in this cell-
based assay.
Table 14. Inhibition of MEFs with selected 2-fluorobenzenesulfonohydrazidesa
MEF 168 h assay
IC [µM]
Entry
Compound ID
R2
50
1
2
3
4
5
6
55
80
99
112
133
135
0.61
0.63
0.65
1.17
0.48
0.99
0.941
0.958
0.984
0.870
0.995
0.985
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
8
164
WM-2474
1.26
>10
0.706
-
a
2
Early passage +/+ MEFs maintained at 3% O and cultured at low density (1000 cells/cm ). Cells
were not split over time course of experiment and media (1μL DMSO/mL (0.001%) and 10% FCS)
changed every 48h with fresh inhibitor. Each condition performed in technical triplicate.
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Alarm NMR.
ALARM NMR aims to detect the presence of any nonspecific thiol reactivity in the context of a
protein host. As shown in Figure 3, compounds 112 and 4 did not perturb the La antigen conformation
in the presence or absence of DTT, implying that these compounds should not react indiscriminately
with protein cysteine thiol residues.22,23
112
4
Figure 3. Compounds 112 and 4 did not perturb the La antigen conformation by ALARM NMR
counter-screen for nonspecific compound thiol reactivity. Shown are H- C HMQC spectra of key
C-labeled methyl groups of the La antigen after incubation with either DMSO, 112 or 4 which
was incubated with the La antigen probe in the presence (blue spectra) and absence (red spectra)
of excess DTT. Data are normalized to DMSO vehicle control. ALARM NMR-positive compounds
perturb the La antigen, as evidenced by pronounced significant peak shifts and/or peak signal
attenuations in the absence of DTT. CPM = positive reactive control compound; fluconazole =
negative reactive control compound.
1
13
1
3
Physicochemical parameters and predictive ADMET
With a number of potent compounds identified for the inhibition of KAT6A, several of the lead
compounds were evaluated for their physicochemical, metabolism and permeability properties. Key
physicochemical properties for selected compounds are shown in Table 15. The distribution
coefficients (cLogD) were favorable across all compounds, in the range of 1.3-4.1 at physiological
2
pH. All compounds have a topological polar surface area (tPSA) of between around 80-100 Å , well
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
within the range considered appropriate for passive absorption. Aqueous solubility varied from a
relatively low 6.3-12.5 µg/mL for 168 to a more acceptable value of up to 100 g/mL or greater for
3
5, 80, 100, 133, 176 and 177. In general, solubilities were similar at both pH 2.0 and pH 6.5 except
for 176 and 177 that were more soluble at pH 2.0, suggesting that for these two compounds the pyridyl
ring was basic enough to become protonated at the lower pH whereas neutrality would be the case
for all other compounds. Plasma protein binding (PPB) was measured for a selection of compounds
and varied from 98.0% for 35, 98.5% for 4, up to 99.6% for 112 and 99.9% for 74. We also assessed
the extent of binding in the cell culture medium, which for 135, 112, 133 and 4 was 67.2, 73.6, 36.2
and 74.9% respectively. Although more data would be needed to strengthen correlations, the
relatively better cell-based activity of 133 (cell IC 0.48 µM, KAT6A IC 0.050 µM) compared with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
50
1
35 (cell IC 0.99 µM, KAT6A IC 0.050 µM) supports an important role of free drug concentration
50 50
in governing cell-based potency.
Table 15. Physicochemical parameters of selected acylsulfonohydrazide derivatives
cLogDa
(pH 7.4)
3.5
PSA
a
PPBb
(%)
98.0
99.8
99.4
99.2
99.9
99.4
Solubility (µg/mL)c
Entry
Compound
2
(Å )
pH 2.0
50-100
12.5-25
25-50
pH 6.5
50-100
12.5-25
25-50
1
2
3
4
5
6
7
8
9
35
39
55
60
74
81.7
81.7
103.2
90.3
85.4
98.3
81.7
81.7
81.7
90.3
90.3
90.3
85.6
85.4
85.4
85.4
2.3
1.3
2.3
3.0
2.5
3.5
3.6
3.4
3.0
2.7
3.5
4.1
3.7
4.2
2.7
6.3-12.5
50-100
50-100
50-100
12.5-25
12.5-25
50-100
25-50
6.3-12.5
6.3-12.5
50-100
50-100
12.5-25
12.5-25
50-100
25-50
80
100
112
123
133
135
140
168
176
177
4
-
99.6/73.6
98.9
96.0/36.2
1
1
1
0
1
2
98.9/67.2
-
-
-
-
12.5-25
6.3-12.5
50-100
>100
12.5-25
6.3-12.5
12.5-25
12.5-25
25-50
13
14
1
1
5
6
98.5/74.9
25-50
a
b
Calculated using ChemAxon JChem for Excel. Determined in mouse plasma using
ultracentrifugation. Where a second value is listed, this is for protein binding in DMEM media
containing 10% FCS, for which values were also obtained for 164, 101, 153, and 129 were
c
respectively 68.3, 71.0, 83.2, and 80.6%. Kinetic solubility range.
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3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
6
Metabolic liabilities of key compounds were also investigated. This involved incubation of
compounds with mouse (MLM) and human (HLM) liver microsomes with the addition of an
NADPH-regenerating system. As can be seen in Table 16, the in vitro intrinsic clearance (CL ) in
int
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HLM was lowest for 133, 135 and 177 while for all compounds, there was significantly more rapid
degradation in MLM compared to HLM. A number of the compounds appeared to be subject to amide
hydrolysis which was particularly evident in MLM. Indeed, MLM half-lives were only a few minutes,
in contrast to HLM stability which was more moderate, the standout being compounds 80 and 133
(with a degradation half-life close to 3 h). This compares favorably with prototypical lead 2 with a
degradation half-life value of 24 min in HLM and represents a significant advance in terms of the
quality of these new compounds as improved biochemical probes.
Table 16. In Vitro Metabolic Stability of Selected Compounds
In Vitro CLint
(µL/min/mg protein)
Degradation half-life (min)
Entry
Compound ID
Mouse
3
nd*
<2
2
Human
25
Mouse
695
nd*
>866
865
567
Human
1
2
3
4
5
6
7
8
9
2
39
42
55
60
74
80
112
123
129
133
135
153
164
4
69
42
73
28
23
27
11
181
58
138*
12
41
24
61
75
65
163
9
30
13
141
96
12
22
56
3
4
6
424
279
<2
<2
2
10
5
4
<2
13
>866*
>866*
803*
178*
334*
464*
>866*
131
1
1
1
1
0
1
2
3
18
143
80
14
1
5
31
a
In vitro intrinsic clearance determined in human and mouse liver microsomes. * = significant non-
NADPH mediated degradation detected
Finally, unidirectional permeability studies were undertaken to determine the apparent
permeability of some of the lead compounds across differentiated Caco-2 cell monolayers. Table 17
shows the results of this investigation which revealed that all compounds have a high permeability,
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1
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and on this basis, there is no reason to expect that these compounds would suffer from permeability-
limited absorption in the gastrointestinal tract.
Table 17. Permeability of Selected Compounds
-
6
a
Entry
Compound ID
A-B Papp (10 cm/s)
65±9
Mass Balance (%)
118± 1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
39
55
60
74
80
53±13
111± 9
100 ± 8
103 ± 3
116 ± 6
90  6.3
107.8  6.2
92  2
87  6
54  5
96  11
48±4
63±9
77±10
92
60.4  17.7
77.3  7.7
47.0  3.5
55.3  6.6
27.8  9.9
60  6.1
101
112
166
172
4
1
1
0
1
a
Caco-2 permeability assessed in the apical-to-basolateral (A-B) direction
Structural Biology
The crystal structures of Ac-CoA, and nine benzoylsulfonohydrazide compounds 39, 40, 41, 42,
55, 60, 74, 80, and 83 were solved with a modified MYST histone acetyltransferase domain
MYSTCryst) as shown in Figures 4 and 5. The positioning of the main-chain atoms of MYSTCryst
(
bound to the nine benzoylsulfonohydrazide inhibitors were almost identical with r.m.s.d. values
ranging between 0.08 – 0.26 Å. Key hydrogen bonds formed by the acylsulfonohydrazide core are
entirely conserved across all structures and involve the side chain of Ser690 and the main chain atoms
of Arg655, Gly657, Gly659, and Arg660 (Figure 4I).
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