Discovery of benzoylsulfonohydrazides as potent inhibitors of the histone acetyltransferase KAT6A

Article abstract of DOI:10.1021/acs.jmedchem.9b00665

A high-throughput screen for inhibitors of the histone acetyltransferase, KAT6A, led to identification of an aryl sulfonohydrazide derivative (CTX-0124143) that inhibited KAT6A with an IC₅₀ of 1.0 μM. Elaboration of the structure-activity relationship and medicinal chemistry optimization led to the discovery of WM-8014 (97), a highly potent inhibitor of KAT6A (IC₅₀ = 0.008 μM). WM-8014 competes with acetyl-CoA (Ac-CoA), and X-ray crystallographic analysis demonstrated binding to the Ac-CoA binding site. Through inhibition of KAT6A activity, WM-8014 induces cellular senescence and represents a unique pharmacological tool.

Full text of DOI:10.1021/acs.jmedchem.9b00665

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Article
Discovery of benzoylsulfonohydrazides as potent
inhibitors of the histone acetyltransferase KAT6A
David Leaver, Benjamin Cleary, Nghi Tiger-Eye Nguyen, Daniel L. Priebbenow, H. Rachel Lagiakos, Julie
Sanchez, Lian Xue, Fei Huang, Yuxin Sun, Prashant Mujumdar, Ramesh Mudududdla, Swapna Varghese,
Silvia Teguh, Susan A. Charman, Karen White, Kasiram Katneni, Matthew Cuellar, Jessica Strasser, Jayme
Dahlin, Michael A. Walters, Ian Street, Brendon Monahan, Kate E. Jarman, Helene Jousset Sabroux,
Hendrik Falk, Matthew Chung, Stefan Hermans, Michael W. Parker, Tim Thomas, and Jonathan B. Baell
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00665 • Publication Date (Web): 29 Jun 2019
Downloaded from http://pubs.acs.org on June 30, 2019
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Parker, Michael; Saint Vincent's Institute of Medical Research, Biota
Structural Biology Laboratory
Thomas, Tim; Walter and Eliza Hall Institute
Baell, Jonathan; Monash Institute of Pharmaceutical Sciences, Medicinal
Chemistry
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Discovery of benzoylsulfonohydrazides as potent
inhibitors of the histone acetyltransferase KAT6A
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‡
‡
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‡
David J. Leaver, Benjamin Cleary, Nghi Nguyen, Daniel L. Priebbenow, H.
Rachel Lagiakos,‡,§ Julie Sanchez,, # Lian Xue, Fei Huang, Yuxin Sun,
†
†
‡
‡
∫
‡
‡
‡
Prashant Mujumdar, Ramesh Mudududdla, Swapna Varghese, Silvia Teguh,
£
£
£
Susan A. Charman, Karen L. White, 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, Michael W. Parker, Tim
¥
¥
¥
,#
†,‡,
Thomas and Jonathan B. Baell *
†School of Pharmaceutical Sciences, Nanjing Tech University, No. 30 South Puzhu Road,
Nanjing 211816, People’s Republic of China.
‡Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University
(Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052, Australia
Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria
3
052, 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 University (Parkville Campus), 381 Royal
Parade, Parkville, Victoria 3052, Australia
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∫Institute for Therapeutics Discovery and Development, University of Minnesota, 717
Delaware Street SE, Minneapolis, Minnesota, United States
∞Department of Pathology, Brigham and Women’s Hospital, Boston, 75 Francis Street,
Boston, Massachusetts 02115, United States
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¥ACRF Rational Drug Discovery Centre, St. Vincent’s Institute of Medical Research, Fitzroy,
Victoria 3065, Australia
*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
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Abstract
A high-throughput screen for inhibitors of the histone acetyltransferase, KAT6A, led to
identification of an aryl sulfonohydrazide derivative that inhibited KAT6A with an IC of
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.0 M. Elaboration of the structure-activity relationship (SAR) and medicinal chemistry
optimization led to discovery of WM-8014 (97), a highly potent inhibitor of KAT6A (IC =
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0
.008 M). WM-8014 competes with Ac-CoA and X-ray crystallographic analysis
demonstrated binding to the Ac-CoA binding site. Through inhibition of KAT6A activity,
WM-8014 induces cellular senescence and represents a unique pharmacological tool.
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■
INTRODUCTION
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Histone acetyltransferases (HATs) are enzymes that play a key role in a range of epigenetic
pathways through the acetylation of histones using acetyl-CoA (Ac-CoA) as a substrate.1-4
4
While acetyltransferase activity has been attributed to nearly 40 proteins, in only minority of
cases has acetyltransferase activity been confirmed in nuclear enzymes regulating transcription.
These HATs can be grouped into three main families based on the structure of the lysine
3
acetyltransferase domain: (i) p300/CBP; (ii) GNAT, and; (iii) MYST. The largest family of
HATs are the MYST proteins, responsible for a wide range of cellular events and comprising
approximately one third of HATs in the human genome.3,4 Proteins within the MYST family
of HATs have a conserved “MYST” domain responsible for the acetyl transferase activity.5,6
The MYST family governs the acetylation of histones (and other nuclear-bound proteins) and
7
are involved in DNA repair, replication and transcriptional activation. There are five MYST
HATs found in mammals; KAT6A (MOZ/MYST3), KAT6B (Morf/MYST4/Qkf), KAT8
6
,8
(Mof/MYST 1), KAT5 (Tip60), and KAT7 (Hbo1/MYST 2). Rearrangement of the KAT6A
gene leads to a particularly aggressive form of acute myeloid leukaemia (AML), with few
patients achieving sustained remission.9,10
Despite their potential roles in human disease, until recently there were limited
examples of on-target inhibitors with cellular activity, not just for any MYST HAT, but for the
whole field of lysine acetyltransferases (KATs). Of the many reported inhibitors, structural
flaws suggest they are predominantly pan assay interference compounds (PAINS - compounds
associated with non-progressable biological activity)11,12 or nuisance screening compounds.
13
Indeed, we have recently demonstrated this to be the case. To date, only two HAT inhibitor
classes with mechanism-based cellular activity have been reported: (i) hydantoin-based
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inhibitors of p300/CPB developed at Abbvie, and (ii) acylsulfonohydrazide-based inhibitors
of KAT6A/B (Figure 1), with key compounds WM-8014 and WM-1119 recently reported by
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our research groups. As the medicinal chemistry-led discovery of these compounds has not
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been published in detail, we herein outline our research efforts that enabled the development
of WM-8014, the first known inhibitor of KAT6A with mechanism-based cellular activity.
F
N
O
CF3
N
F
N
O
O
O
O
O
H
H
H
O
S
N
S
N
S
N
N
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F
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O
H
O
H
H
O
F
O
F
O
HN
HN
O
A-485
p300 HAT SPA IC50 = 0.060 M
CTx-0124143
KAT6A HAT IC50 = 1.0 M
WM-8014
KAT6A HAT IC50 = 0.008 M KAT6A HAT IC50 = 0.0063 M
WM-1119
Figure 1: Structures of reported HAT inhibitors
■
RESULTS AND DISCUSSION
Chemistry. The majority of compounds reported herein were synthesized as outlined
in Scheme 1, by coupling an aryl sulfonohydrazide with an aryl carboxylic acid using N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI.HCl) and 1-hydroxy-7-
azabenzotriazole (HOAt) or N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium
hexafluorophosphate (HBTU).
Scheme 1. Synthesis of N'-aroylaryl-2-sulfonohydrazidesa
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R5
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O
O
O
R6
R4
R
S
R
S
NH2
Cl
O
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H
N
(b) or (c)
(a)
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H
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N
N
R3
H
R2
O
1
2
R = H
R = 3,4-naphthyl
3 R = H (58%)
4 R = 3,4-naphthyl (99%)
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CTX-0124143 R = 3,4-naphthyl, R = H (56%)
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9 R = 3,4-naphthyl, R = R = R = R = H, R = OMe (26%)
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4-6
2 R = 3,4-naphthyl, R = R = F, R = H (17%)
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4-6
3 R = 3,4-naphthyl, R = F, R = Cl, R = H (38%)
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35 R = 3,4-naphthyl, R = F, R = Et, R = H (17%)
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36 R = 3,4-naphthyl, R = F, R = CF , R = H (24%)
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7 R = 3,4-naphthyl, R = F, R = R = R = H, R = Me (25%)
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8 R = 4-CN, R = H (74%)
0 R1
-4,6
5
= H, R = Ph (13%)
1
,3-4,6
2
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2 R
= H,R = F, R = Ph (3%)
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64 R = H, R = [N] (68%)
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65 R = R = R = R = H, R = [N], R = Ph (56%)
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6 R = R = R = R = H, R = [N], R = Ph (29%)
1 R1-4,6 H, R = pyrimidin-5-yl (10%)
5
2 R1-4,6 H, R = pyridyl-3-yl (11%)
5
3 R1-4,6 H, R = thiophen-3-yl (7%)
5
84 R1-4,6 H, R = thiophen-2-yl (40%)
5
8
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5 R1-6 = H (83%)
6 R1,4-6 H, R = F, R = Cl (29%)
2
3
7 R1,4-6 H, R = F, R = Me (8%)
2
3
8 R1,4-6 H, R = F, R = Et (8%)
2
3
89 R1,4-6 H, R = F, R = OMe (31%)
2
3
90 R1,4-6 H, R = R = F (32%)
2
3
1 R1,4-6 H, R = F, R = CF (29%)
2
3
9
3
a
o
Reagents and conditions: (a) NH NH .H O, DCM, 0 C to rt, 3 h; (b) EDCI.HCl, HOAt, ACN,
2
2
2
o
aryl carboxylic acid 50 C o/n; (c) HBTU, DIPEA, MeCN, aryl carboxylic acid, reflux. Note
that [N] implies endocyclic nitrogen atom, such as in pyridyl.
For the more advanced compounds designed to investigate additive SAR, the synthesis
of the benzoic acid reagents was required, the process for which is outlined in Scheme 2, For
these examples, the substituted aryl bromide was subjected to Suzuki coupling with phenyl
boronic acid followed by ortho-lithiation and subsequent quenching with carbon dioxide
followed by aqueous work-up) to afford the requisite carboxylic acid.16,17
(
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Scheme 2. Synthesis of benzoic acid precursors (R=Me, Cl) a
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Br
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(b)
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R
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O
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F
9
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3 R = Cl (84%)
6 R = Me (33%)
5
6
R = Cl
R = Me
92 R = Cl (43%)
95 R = Me (100%)
a
Reagents and conditions: (a) Phenylboronic acid, K CO , PdCl (PPh ) , THF:water (1:1),
2
3
2
3 2
o
reflux o/n; (b) n-BuLi, CO THF, -78 C
2
,
SAR. Screening of our Stage 5 and Stage 6 HTS libraries of approximately 245,000
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structurally diverse “hit-like” compounds led to the identification of the primary screening
hit CTX-0124143 as a fully reversible and Ac-CoA-competitive inhibitor of KAT6A.19
Assessment of this compound against our internal criteria for progressable chemical matter –
our eight-point action plan (EPAP), was favorable. EPAP comprises (i) activity confirmed on
purified, resynthesized material, (ii) activity confirmed on orthogonal assay, (iii) counter-
screen assay clean, (iv) direct binding observed, (v) screening deck history clean, (vi) literature
history clean, (vii) library SAR clean, (viii) clear SAR with optimization demonstrated. CTX-
0
124143 passed all of the first seven criteria needing only SAR with optimization to
demonstrate its progressability and concomitantly yielding a potential tool compound.
Although HTS was undertaken in 2009 and screening hit validation reported in 2011,
it was only in 2018 that we published the screening hit structure along with its optimized
progeny, WM-8014.15,19 During the intervening nine years, assays were conducted under
several different conditions, and in particular SAR discussed here involves assays with two
differing Ac-CoA concentrations (0.4 and 15 µM) and MYST domain constructs (NusA and
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SGC constructs). For the purposes of SAR interpretation and the discussion herein, we
determined that there was sufficient correlation between assay protocols (supplementary
information) without having to retrieve historical compounds, resurrect the historical assays
conditions, and retest all compounds under both assay conditions, which in any case in now
not feasible.
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In practical terms, compounds competing with higher Ac-CoA concentration appear to
be less potent. SAR therefore needs to be interpreted in this context. We initially focused on
the right-hand side aryl ring system of CTX-0124143 (Figure 1 and Table 1). SAR by
commercial catalogues was relatively limited, so we supplemented commercially available
compounds with targeted analogues synthesized in-house. Since mono-substituted benzoic
acids were more readily accessible than substituted 2-fluorobenzoic acids, we based our initial
SAR on the des-fluoro compound 7 after ascertaining that while less potent, it still gave a
readout in our assay with an IC of 4.2 µM, deemed an acceptable starting point for a medicinal
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chemistry SAR investigation.
Table 1. Inhibitory potency of benzoylnaphthalene-2-sulfonohydrazide analogues
against KAT6A with variation of the right-hand sidea
R4
O
O
H
S
N
N
R3
H
R2
O
IC50 (µM)
ID
R2
F
R3
R4
-
0.4 µM [Ac-CoA]
15 µM [Ac-CoA]
CTX-0124143
-
-
-
-
1.0
4.2
-
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18
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8
9
-
-
Cl
OH
-
>125
>125
-
49
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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
1
1
1
1
0
1
2
3
Me
-
-
-
19
42
>125
-
OMe
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Cl
-
0.52
1.4
7.8
9.0
18
Me
i-Pr
F
-
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
14
15
16
17
18
19
-
45
-
2.1
33
-
OMe
-
18
CF
3
-
11.0
-
CN
Ac
-
-
24
>125
>125
-
-
2
2
2
2
2
2
2
2
2
2
3
3
0
1
2
3
4
5
6
7
8
9
0
1
OCF
3
-
2.8
NHBoc
-
-
71
NH
2
-
-
0.88
-
19
Ph
-
-
3.0
60
Cl
OBu
OH
Me
F
-
-
>125
30
-
-
-
-
30
-
-
-
-
-
120
120
>125
>125
-
OMe
CN
-
-
OCF
3
aDash denotes absence of substituent or, for biological data, the compound was not tested
under these conditions.
2
As shown in Table 1, a variety of ortho (R ) substituents including chloro (8), hydroxyl
(
9), methyl (10) and methoxy (11) all gave less potent or inactive compounds compared with
3
the unsubstituted parental compound 7. Substitution at the meta (R ) position was unfavorable
for certain groups, such as isopropyl (14), fluoro (15), trifluoromethyl (17), nitrile (18), acetyl
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9
(
19), butyloxycarbonylamino (21), but was tolerated or slightly favorable for chloro (12),
methyl (13), methoxy (16), trifluoromethoxy (20), amino (22), and phenyl (23) groups. At the
para position, substitution was less favorable and chloro (24), butyloxy (25), hydroxyl (26),
methyl (27), fluoro (28), methoxy (29), nitrile (30) and trifluoromethoxy (31) groups were all
relatively less potent.
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
We then undertook preliminary investigations to see if any additive activity was
2
observed. In particular, we focused on compounds with the favored R -fluoro substituent and
a small hydrophobic group in either of the two possible meta positions. As shown in Table 2,
2
3
some particularly potent analogues were identified with the R , R substitution pattern. For
2
3
2
3
2
3
example, R = F, R = F (32), R = F, R = Cl (33) and R = F, R = Me (34) analogues registered
IC values of 0.12 µM, 0.043 µM and 0.12 µM respectively. An ethyl or trifluoromethyl group
5
0
3
at the R -position was particularly highly favored with compounds 35 and 36 returning IC
5
0
values of 0.062 µM and 0.077 µM respectively. A methyl group at the alternative meta position
5
(R ) was also investigated and was tolerated but not improved, with 37 returning an IC value
5
0
of 0.91 µM.
Table 2. Inhibitory potency of second generation benzoylnaphthalene-2-
sulfonohydrazide analogues against KAT6A with variation of the right-hand sidea
R5
O
O
H
N
S
N
R3
H
O
R2
ID
R2
R3
R5
IC50 (µM)
0
.4 µM [Ac-CoA]
15 µM [Ac-CoA]
CTX-0124143
F
F
-
-
-
1.0
12
32
F
0.13
2.7
1
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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
3
3
3
3
3
4
5
6
F
F
F
F
F
Cl
Me
Et
-
-
0.043
0.12
0.60
1.3
-
0.062
0.077
0.91
1.0
CF
3
-
0.87
8.0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
37
-
Me
aDash denotes an absence of substituent.
Although 3-phenyl compound 23 was also a clear candidate for additive SAR studies,
we were concerned about the increasing bulk and aromatic ring accumulation. Therefore, at
this point, we undertook preliminary SAR around the left-hand aryl region (Table 3). First, we
2
replaced the naphthyl ring with a phenyl ring whilst retaining the R fluorine and were intrigued
to note that only a minor loss in potency was observed, with 38 returning an IC value of 1.8
5
0
µM. We reasoned that the simpler phenyl ring represented an advance in terms of drug-likeness
possessing a lower molecular weight whilst retaining a relatively high potency: LE and LLE)
(
and would also be more versatile than the naphthyl ring for further elaboration, and as such this
modification was retained. Compound 38 was thus employed as a convenient template around
which to explore the effect of different substituents. As shown in Table 3, all substituents
investigated at the ortho position led to lower activity, and this was the case with groups
comprising fluoro (39), trifluoromethyl (40), methyl (42), methoxy (43), and nitrile (44). On
the other hand, several groups were well tolerated at the meta position, including bromo (45),
chloro (46), methyl (47), fluoro (48), methoxy (49), and amino (51), with 45 and 51 being
noteworthy in their low IC values of 0.46 µM and 0.49 µM respectively. In the para position,
5
0
a bromo group (55) was well tolerated, fluoro (53) less so, and isopropyl (54), methyl (56),
methoxy (57), nitrile (58), and amino (59) groups all markedly less so.
1
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Table 3. Inhibitory potency of second generation benzoylsulfonohydrazide analogues
against KAT6A with variation of the left-hand sidea
Y2
O
O
H
N
Y3
S
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
H
O
F
Y4
ID
Y2
Y3
Y4
IC50 (µM)b
1.0
CTX-0124143
-
-
3,4-naphthyl
3
3
4
4
4
4
4
4
4
4
4
5
5
8
9
0
2
3
4
5
6
7
8
9
0
1
-
-
-
-
-
-
-
-
-
-
-
-
1.8
F
5.7
CF
3
4.4
Me
6.2
OMe
17
CN
-
7.3
Br
Cl
Me
F
-
0.46
1.7
-
-
-
-
3.5
-
-
1.3
-
OMe
CN
-
1.1
-
-
14
-
NH
2
-
CONH
F
0.49
12
52
53
54
55
56
57
-
-
-
-
-
-
-
-
-
2
-
4.1
-
CHMe
Br
2
19
-
1.0
-
Me
>12.5
>12.5
>12.5
>12.5
-
OMe
CN
5
5
8
9
-
-
NH
2
a
b
Dash denotes an absence of substituent. 0.4 µM [Ac-CoA]
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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
Having determined that the naphthyl ring was not vital for biochemical inhibition of
KAT6A, we returned to investigate right-hand side additive SAR with inclusion of a meta-
phenyl group, which had shown promise previously (23 in Table 1). As shown in Table 4, we
observed that the presence of the left-hand side naphthalene ring (23) was actually detrimental
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
for activity when a right-hand side phenyl ring was installed in the meta position: with IC of
50
0
.29 µM, 60 was several times more potent than 23. Reincorporation of the favored fluoro
substituent in the ortho position was readily undertaken by incorporating 4-fluoro-[1,1'-
biphenyl]-3-carboxylic acid (61) into the general synthetic procedure, leading to 62, which
exhibited an impressive IC of 0.017 µM. Just as there are two meta positions, so are there
5
0
6
two ortho positions and installation of the fluoro substituent (R ) in the alternative ortho
position (63) did not improve potency.
Table 4. Inhibitory potency of second-generation benzoylsulfonohydrazide analogues
against KAT6A with variation of the right-hand sidea
R6
O
O
H
Y
S
N
N
H
O
R2
ID
23
60
62
Y
R2
-
R6
-
IC50 (µM)b
0.88
3,4-naphthyl
-
-
-
-
-
0.29
F
-
0.017
0.27
6
3
-
F
a
b
Dash denotes an absence of substituent. 0.4 µM [Ac-CoA]
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Once again using 60 as a reference, we further probed right-hand side SAR. As shown in
Table 5, conversion of the benzoyl group to any sort of pyridyl group was disfavored and 64-
66 all lost substantial activity. Substitution of the meta-phenyl ring with 2-fluoro (67) was
tolerated, but chloro (68) or methoxy (69) here was not. Similarly, a 3-fluoro group (70) was
tolerated, but chloro (71), methoxy (72), and nitrile (73) groups were not. In the 4 position,
neither fluoro (74), chloro (75), methoxy (76), or nitrile (77) groups were tolerated.
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
We then investigated the replacement of the 5-phenyl motif with a variety of different
heterocyclic ring systems, including 1H-pyrazol-1-yl (78), piperazin-1-yl (80), pyrimidin-5-yl
(81), pyridin-3-yl (82), thiophen-3-yl (83), and thiophen-2-yl (84). All compounds except the
thiophene containing analogues, compounds 83 and 84, lost potency. Since the thiophene ring
of 83 was seen as an additional metabolic liability, and as none of the other investigated ring
5
systems delivered improvements over the unsubstituted R -phenyl system 60, this scaffold was
retained in further SAR exploration.
Table 5. Inhibitory potency of second generation benzoylsulfonohydrazide analogues
against KAT6A with variation of the right-hand sidea
R5
R6
O
R4
R3
O
O
H
S
N
N
H
R3
R4
R5
R6
IC50 (µM) b
ID
6
6
6
0
4
5
-
-
Ph
Ph
-
0.29
18
[N] c
-
-
-
[N] c
Ph
-
-
-
-
19
6
6
[N] c
-
-
-
Ph
22
6
7
-
-
Ph-2-F
Ph-2-Cl
0.62
8.2
68
1
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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
6
7
7
7
9
0
1
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Ph-2-OMe
Ph-3-F
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
11
0.79
9
Ph-3-Cl
Ph-3-OMe
Ph-3-CN
3.4
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
73
74
75
76
77
78
9.5
Ph-4-F
4.0
Ph-4-Cl
25.8
41
Ph-4-OMe
Ph-4-CN
>125d
7.4
1H-pyrazol-1-yl
piperazin-1-yl
pyrimidin-5-yl
pyridyl-3-yl
thiophen-3-yl
thiophen-2-yl
8
8
8
8
8
0
1
2
3
4
29
>125
13
0.16
0.47
a
b
c
Dash denotes an absence of substituent. 0.4 µM [Ac-CoA]. This signifies endocyclic nitrogen atom, in these
d
cases therefore pyridyl. 15 µM [Ac-CoA].
3
It was shown in Table 1 that 12, with a chloro group in the meta (R ) position, displayed
3
5
favorable inhibitory activity. Given that there are two possible meta positions (R and R ),
2
5
having determined that 62, with R = F and R = phenyl, was optimum so far, we wondered
whether additional activity might be discovered through installation of a small hydrophobic
3
group at the R position. In order to simplify this task, we chose 38 as a template. For the sake
of SAR completion, we also made and tested the des-fluoro compound 85, which returned an
IC of 6.7 M. In comparison, 38 was several times more potent attributable to favourable
5
0
effects conferred by the 2-fluoro substituent. As hoped, installation of an adjacent meta
substituent was highly favourable, in the order chloro (86), methyl (87), then ethyl (88) and
methoxy (89), followed by fluoro (90) and trifluoromethyl (91).
1
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Table 6. Inhibitory potency of second generation benzoylsulfonohydrazide analogues
against KAT6A with variation of the right-hand sidea
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
O
O
H
S
N
R3
N
H
O
R2
ID
R2
R3
-
IC50 (µM)b
6.7
8
3
8
8
8
8
9
9
5
8
6
7
8
9
0
1
-
F
F
F
F
F
F
F
-
1.8
Cl
Me
Et
0.13
0.23
0.58
OMe
F
0.49
0.74
CF
3
0.39
a
b
A dash means an absence of substituent. 0.4 µM [Ac-CoA]
Comparing the inhibitory potency of 86 with 33 (respective KAT6A IC values of 0.13 and
50
0
.043 M, and 87 with 34 (respective KAT6A IC values of 0.23 and 0.12 M), it can be seen
5
0
that the replacement of a naphthyl ring with phenyl ring results in only a relatively minor loss
of potency. This parallels the observations that were made for 38 and CTX-0124143.
With the significant boosts in potency resulting from the addition of small hydrophobic
3
5
groups at the R position and the R phenyl substitution, we sought to further increase potency
2
3
of these analogues by incorporating all the favorable substitutions (R fluoro, R small
5
hydrophobic, and R phenyl) onto the phenyl sulfonyl core system. The two analogues designed
to explore this hypothesis were 94 and 97 containing either a chloro or methyl group
respectively in the meta position. As can be seen in Table 7, the respective compounds were
highly potent KAT6A inhibitors with IC values of 10 and 8 nM respectively. In addition, the
5
0
1
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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
5
importance of the R -phenyl was demonstrated by the relatively great loss of activity of the
des-phenyl compounds 86 and 87 (Table 6).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 7. Inhibitory potency of third-generation benzoylsulfonohydrazide analogues
against KAT6Aa
O
O
H
S
N
N
R3
H
O
R2
ID
60
62
94
R2
-
R3
-
IC50 (µM)b
0.29
F
F
F
-
0.017
Cl
0.010
9
7
Me
0.008
a
b
Dash denotes an absence of substituent. 0.4 µM [Ac-CoA]
Selectivity and cell-based activity. In our recent publication in the journal, Nature, we
have shown that 97 (WM-8014) induces cell cycle arrest in embryonic day (E)14.5 mouse
1
5
embryonic fibroblasts (MEFs). Cells treated with WM-8014 failed to proliferate after ten
days of treatment (IC 2.4 μM). Higher doses of WM-8014 (up to 40 μM) did not accelerate
5
0
growth arrest, which after eight days of treatment was irreversible. Cell cycle analysis showed
an increase in the proportion of cells in G0/G1 phase after six days of treatment and a
corresponding reduction in cells in G2/M- and S-phases. This report also describes how 97 is
highly potent towards the highly similar enzyme, KAT6B (IC 0.028M), but does not bind
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appreciably to any of the diverse panel of around 160 pharmacological targets tested.15
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ALARM NMR. ALARM NMR is an industry-developed HMQC counter-screen to detect
nonspecific thiol-reactive compounds using a protein-based probe. Although not precluding
development, it is generally preferable for drug and chemical probe candidates to not exhibit
nonspecific protein reactivity. As shown in Figure 2, 97 did not perturb the La antigen
conformation in the presence or absence of excess DTT, suggesting it may not react
indiscriminately with protein cysteine thiol residues.20,21
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Figure 2. Compound 97 does not perturb the La antigen conformation by ALARM
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NMR counter-screen for nonspecific reactivity with protein thiols. Shown are H- C
13
HMQC spectra of key C-labeled methyl groups of the La antigen after incubation with
either DMSO or 97, 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.
ADMET parameters for 94 and 97. With the development of two highly potent
inhibitors of KAT6A, we sought to further explore their physicochemical properties in order
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to evaluate their potential as drug–like molecules. Both 94 and 97 were attractively small,
possessing molecular weights of 404.84 g/moland 384.42 g/molrespectively. Both compounds
had calculated polar surface areas of 72.5 Å, a relatively low value conducive to passive cellular
permeability.22 Indeed, permeability studies undertaken using differentiated Caco-2 cell
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monolayers, found that both 94 and 97 display high permeability, with A-B Papp (10 cm/s) of
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7.5 ± 12.5 and 35.9 ± 2.8 respectively, and thus are unlikely to be subject to permeability-
limited absorption. Calculated partition values for both compounds were high (cLogD >5) and
aqueous solubility was limited, under both acidic and neutral pH, being in the range of 3.1-6.3
g/mL. The plasma protein binding (PPB) of 94 was very high, being greater than 99.9%.
Table 8. Key physicochemical parameters and permeability properties of selected
compounds
cLogDa
PSAa PPBb Solubility (µg/mL)c
(Å2)
A-B Papp (10-6
cm/s)
ID
MW
(pH 7.4)
(%)
pH 2.0
pH 6.5
9
9
4
7
384.42
404.84
5.1
72.5 >99.9 3.1 - 6.3 3.1 - 6.3
72.5 -- 3.1 - 6.3 3.1 - 6.3
77.5 ± 12.5
35.9 ± 2.8
5.1
a
b
Calculated using ChemAxon JChem for Excel. Determined in mouse plasma using
c
d
ultracentrifugation. Kinetic solubility range. Caco-2 permeability assessed in the apical-to-
basolateral (A-B) direction
Metabolism was assessed by incubating compounds 94 and 97 with mouse and human
liver microsomes in the presence and absence of an NADPH-regenerating system. In the
presence of NADPH, both compounds were metabolized relatively rapidly, with degradation
half-lives of ≤ 8 min and ≤ 32 min in mouse and human liver microsomes, respectively.
Substantial non-NADPH dependent metabolism of both compounds was also observed in the
absence of NADPH, particularly in mouse liver microsomes. This suggests optimization of
metabolic stability would be useful for progression of these compounds as candidates for in
vivo investigations.
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Structural Biology. The crystal structures of Ac-CoA and six acylsulfonohydrazides
(CTX-124143, 34, 60, 62, 63 and 97) were solved with a modified MYST histone
acetyltransferase domain (MYSTCryst) as shown in Figure 4. The positioning of the main-chain
atoms of the six acylsulfonohydrazides bound to MYSTCryst (Figure 4h) were almost identical
with r.m.s.d. values ranging from 0.1- 0.4 Å. Key hydrogen bonds formed by the
acylsulfonohydrazide core involves the side chain of Ser690 and the main chain atoms of
Gly657, Gly659, Arg655, and Arg660. The biphenyl group of 60, 62, 63 and 97 (Figure 4d-g)
further extends into the acetyl-CoA binding pocket enabling van der Waal interactions with
Leu601, Leu686, Ile649 and Ser684 on top of the van der Waal interactions with Ile649, Ile663,
and Ile647 shared with all acylsulfonohydrazides. The naphthalene group of 34 and CTX-
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24143 (Figure 4b-c) picks up additional van der Waal interactions with Tyr694 and Trp697
on top of the van der Waal interactions with Arg660 and Ser693 shared with all
acylsulfonohydrazides. CTX-124143, 34, 62 and 97 each formed a pseudo six-membered ring
that was stabilized by an intramolecular hydrogen bond between the ortho fluorine atom and
the hydrogen atom attached to the terminal hydrazide nitrogen. The incorporation of the meta
methyl group increased affinity to KAT6A an order of magnitude for both 34 and 97 in
comparison to the nonmethylated derivatives CTX-124143 and 62, respectively. The fluorine
atom in 97 forms an electrostatic interaction with Ser690, while the meta methyl of the biphenyl
group in 97 forms van der Waal interactions with Ile647, Ile649, Ile663, and Ser690. For 63,
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the fluorine atom in the R position and the R phenyl was associated with the loss of one order
of magnitude of biological activity relative to its regioisomer 62, which is probably due to the
loss of the electrostatic interaction between Ser690 and the disruption of the pseudo six-
membered ring.
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a)
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Figure 4. Crystal structures of Ac-CoA and derivatized acylsulfonohydrazides bound to the
MYST lysine acetyltransferase domain (MYSTCryst). a) Ribbon diagram showing Ac-CoA
bound to MYSTCryst (PDB ID: 6BA4); b) Ribbon diagram showing CTX-124143 bound to
MYSTCryst (PDB ID: 6OIN); c) Ribbon diagram showing 34 bound to MYSTCryst (PDB ID:
6
OIP); d) Ribbon diagram showing 60 bound to MYSTCryst (PDB ID: 6OIO); e) Ribbon
diagram showing 62 bound to MYSTCryst (PDB ID: 6OIR); f) Ribbon diagram showing 63
bound to MYSTCryst (PDB ID: 6OIQ); g) Ribbon diagram showing WM-8014 bound to
MYSTCryst (PDB ID: 6BA2); h) Ribbon diagram showing the superimposition of CTX-
124143, 34, 60, 62, 63, and 97 bound to MYSTCryst. Hydrogen bonds are shown as dashed
lines.
■
CONCLUSION
Elaboration of HTS hit CTX-0124143 culminated in a series of potent inhibitors of the histone
acetyltransferase KAT6A, accompanied by extensive and readily interpretable SAR. Leading
compounds 94 and 97 are single-digit nanomolar inhibitors of KAT6A and compete with Ac-
CoA, binding in the Ac-CoA-binding site as clearly demonstrated through X-ray
crystallographic analysis. In this manner, the acyl sulfonohydrazide core mimics the
pyrophosphate group of Ac-CoA. Both 94 and 97 are highly cell-permeable to Caco2
monolayers and thus present the acylsulfonohydrazide group as a cell-permeable
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pyrophosphate mimetic that could be of wider utility in this context. Imparting membrane
permeability is the most challenging task in phosphate group isostere discovery23,24 and
although a report on a related core appears to be able to emulate pyrophosphate group binding
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interactions, specific identification of cellular permeability as an associated and beneficial
property of acylsulfonohydrazides is new and herein reported for the first time.
Properties for improvement comprise significant protein binding and relatively rapid
microsomal metabolism. Until the discovery by us of this class of small-molecule inhibitors
of KAT6A, and by workers at Abbvie of p300/CBP inhibitors, HATs were beginning to be
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viewed as an undruggable class of enzymes. Of the three major HAT classes – p300/CBP,
GNAT and MYST, the MYST HATS comprise about one-third of the HATs in the human
genome and yet have remained entirely undrugged. Compound 97 binds reversibly to KAT6A
at the Ac-CoA binding site, potently inhibiting KAT6A in biochemical assays, inactivating its
catalytic machinery, inducing cellular senescence as a consequence, and represents a unique
pharmacological tool to probe the cellular sequalae of KAT6A inhibition.
■
EXPERIMENTAL SECTION
Chemistry. General Experimental Methods. TLC was performed on silica gel 60F254
pre-coated aluminum sheets (0.25 mm, Merck) and was visualized with UV light at 254nm
and/or a potassium permanganate stain. Flash column chromatography was carried out using
Merck silica gel 60, 0.63 – 0.20 mm (70-230 mesh). Melting points were recorded using a
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Mettler Toledo MP50 melting point system. H and C nuclear magnetic resonance spectra
were recorded at 400 Hz and 100 Hz respectively, on an Avance III Nanobay 400 MHz Bruker
spectrometer coupled to the BACS 60 automatic sample changer or a Bruker Avance DRX 300
1
spectrometer ( H at 300 MHz). Results are recorded as follows: chemical shifts (δ) in ppm
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acquired in either CDCl (7.26 ppm for H and 77.16 ppm for C), acetone-d ((2.05 ppm for
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H and 29.84 ppm for C) or DMSO-d (2.50 ppm for H and 39.52 ppm for C) as a solvent
6
and reference. Solvents used for NMR studies are from Cambridge Isotope Laboratories. Each
proton resonance was assigned according to the following convention: chemical shift (δ),
multiplicity, coupling constant (J, Hz) and number of protons. In reporting of the spectral data,
the following abbreviations are utilized: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet.
Each carbon resonance was assigned according to the following convention: chemical shift (δ),
multiplicity (where no multiplicity is assigned a singlet peak was observed), coupling constants
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(J, Hz) for carbon-fluorine coupling. In reporting of the spectral data, the following
abbreviations are utilized: s, singlet; d, doublet. In the case where multiple splitting of a single
peak was observed (e.g. splitting due to coupling of the carbon atom to multiple fluorine atoms)
the peak was assigned as m, multiplet. Microwave syntheses were performed with a CEM
Focused Microwave™ synthesis system, Discover® SP. Low resolution mass spectrometry
analyses were performed on an Agilent 6100 Series single quad LC/MS coupled with an
Agilent 1200 Series HPLC, 1200 Series G1311A quaternary pump, 1200 series G1329A
thermostatted autosampler and 1200 series G1314B variable wavelength detector. The
conditions for mass spectrometry were: quadrupole ion source; ion mode, multimode-ES;
drying gas temp, 300 °C; vaporizer temperature, 200 °C; capillary voltage, 2000 V (positive),
4
000 V (negative); scan range, 100-1000 m/z; step size, 0.1 s; acquisition time, 5 min. Low-
resolution mass spectra were also performed on a Finnigan LCQ Advantage MAX
spectrometer with a Phenomenex, Gemini 5 m, C-18, 110Å, 50 x 2.0 mm column. Low
resolution mass spectrometry analyses were also performed on an Agilent 6120 Series single
quad UHPLC/MS coupled with a 1260 Infinity G1312B binary pump, 1260 Infinity G1367E
1260 HiP ALS and a 1290 Infinity G4212A 1290 DAD variable wavelength detector. The
conditions for mass spectrometry were: quadrupole ion source; ion mode, API-ES; drying gas
temp, 350 °C; capillary voltage, 3000 V (positive), 3000 V (negative); scan range, 100-1000
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m/z; step size, 0.1 s; acquisition time, 5 min. Unless otherwise noted, all tested compounds
were found to be greater than 95% pure by UV absorption at 254 nm. All HRMS analyses were
done on an Agilent 6224 TOF LC/MS mass spectrometer coupled to an Agilent 1290 Infinity
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(Agilent, Palo Alto, CA). All data were acquired and reference mass corrected via a dual‐spray
electrospray ionisation (ESI) source. Each scan or data point on the total ion chromatogram
(TIC) is an average of 13,700 transients, producing a spectrum every second. Mass spectra
were created by averaging the scans across each peak and background subtracted against the
first 10 seconds of the TIC. Acquisition was performed using the Agilent Mass Hunter data
acquisition software version B.05.00 Build 5.0.5042.2 and analysis was performed using Mass
Hunter qualitative analysis version B.05.00 Build 5.0.519.13 mass spectrometer with the
following conditions: ESI mode; desolvation gas flow, L/min; nebulizer, 45 psi; drying gas
temperature, 325 °C; capillary voltage, 4000 V; fragmentor, 160 V; skimmer, 65 V; OCT RFV,
750 V; scan range acquired, 100-1500 m/z; internal reference ions: positive ion mode m/z =
1
21.050873 and 922.009798.
Purity. The purity of all compounds submitted for biological testing was greater than
95%, as determined using methods described (vide supra).
General procedure A. A solution of sulfonyl chloride (1.0 eq.) in dichloromethane
o
(
DCM, 1.0 M) was cooled to 0 C and to this a solution of hydrazine monohydrate (20.0 eq.)
in DCM was added. The reaction mixture was stirred for 3 h at room temperature. The reaction
mixture was then sequentially washed with water and brine and the organic phases collected,
dried over MgSO , filtered and concentrated in vacuo to yield the desired compound.
4
General procedure B. The benzoic acid (1.0 eq.), sulfonyl hydrazide (1.25 eq.), HOAt
(1.25 eq.) and EDCI.HCl (1.25 eq.) were all dissolved in CH CN (0.8 M), under an atmosphere
3
of nitrogen. The solution was heated to 40 °C and allowed to stir for 17 h, after which time the
reaction was cooled and concentrated in vacuo. The resulting residue was partitioned between
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water and EtOAc and the desired organics were extracted from the aqueous layer with EtOAc.
The combined organic fractions were dried over Na SO , filtered and then evacuated in vacuo
2
4
with silica gel. The crude material was purified by flash chromatography using silica gel and
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5% EtOAc/petroleum benzine to yield the compound of interest.
General Procedure C. To a solution of desired benzoic acid (1.0 eq.) in CH CN (5.0
3
o
mL per 0.5 mmol) was added HBTU (1.25 eq). The reaction mixture was then cooled to 0 C
and DIPEA (1.25 eq.) was added and the reaction stirred until all carboxylic acid had been
consumed according to TLC. The sulfonohydrazide (1.5 eq.) was then added and the reaction
was refluxed for 18 h. The crude material was then evaporated in vacuo with silica gel and was
purified by flash chromatography using silica gel and a gradient of 10-20% EtOAc/petroleum
benzine. The compound of interest was then recrystallized from petroleum benzine/toluene.
General Procedure D. To a reaction mixture of the benzoic acid (1 eq.) in CH OH
3
(excess) a few drops of H SO were added and the reaction mixture refluxed for 16 h. The
2
4
mixture was then cooled to room temperature and the volatiles removed in vacuo. To the
resulting residue was added EtOAc and the organic layer was subsequently washed with water
and NaHCO (sat.). The organic layer was dried over MgSO filtered and concentrated in
3
4,
vacuo. The resulting oil was dissolved in EtOH to which hydrazine monohydrate (10 eq.) was
added and the reaction mixture refluxed for 16 h. The mixture was then cooled to room
temperature and concentrated in vacuo. To this mixture, water was added and the resulting
precipitate was filtered, washed with water and dried under vacuum to afford the compound of
interest.
General Procedure E. To a degassed solution of 1,4-dioxane:H O (9:1, 0.2 M) was
2
added the benzoic acid (1.0 eq.), boronic acid (3.0 eq.), K CO (1.5 eq.) and the palladium
2
3
catalyst (0.05 eq.) under an atmosphere of nitrogen gas. The reaction mixture was irradiated in
a CEM microwave at 80 °C for 30 min after which time the reaction mixture was cooled to
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®
room temperature and passed through a pad of Celite . The Celite was washed with EtOAc
and the resulting filtrate acidified using 2 M HCl (1.0 mL). The organic solvents were removed
in vacuo and the solid precipitate collected via filtration to afford the compound of interest.
General procedure F. The acid chloride (1.02 eq.) was added dropwise to a suspension
of 2‐naphthalene sulfonohydrazide (1.0 eq.) in anhydrous THF (0.2 M) at 65 °C under an
atmosphere of nitrogen gas. After stirring at 65 °C for 24 h the reaction mixture was partitioned
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0
1
2
3
4
5
6
7
8
9
0
between EtOAc and sat. aq. NaCl solution. The organic layer was dried over MgSO , filtered
4
and concentrated in vacuo. The product was purified by flash chromatography using silica gel
and 30% EtOAc/cyclohexane.
General procedure G. To a solution of the benzoic acid (1.0 eq.) in anhydrous THF
(
5 mL) was added dicyclohexylcarbodiimide (1.1 eq.) under an atmosphere of nitrogen gas.
The resulting suspension was stirred for 5 min at room temperature, after which time,
2‐naphthalene sulfonohydrazide (1.0 eq) was added and the reaction mixture stirred at 65 °C
for 24 h. The mixture was then cooled to room temperature and the resulting suspension was
filtered to remove the dicyclohexylurea precipitate. The solution was concentrated in vacuo
and the product purified by flash chromatography using silica gel and 30% EtOAc/cyclohexane.
o
General procedure H. To a hydrazide solution (1.0 eq.) in pyridine (0.3 M) at 0 C
was added the sulfonyl chloride. The resulting reaction mixture was stirred for 2 h at room
temperature, after which time, the reaction mixture was poured onto water. The desired
organics were extracted with EtOAc and the combined organic layers washed with 1 M HCl,
water and then dried over MgSO . The solution was then concentrated in vacuo and the product
4
was purified by flash chromatography using silica gel and 30% EtOAc/cyclohexane.
General procedure I. The aryl bromide (1.0 eq), arylboronic acid (1.1 eq), potassium
carbonate (1.5 eq), and PdCl (PPh ) (5 mol%) were suspended with a 1:1 THF:H O mixture
2
3 2
2
(
4 mL per mmol) and the reaction mixture was refluxed overnight. The reaction mixture was
2
7
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Page 29 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
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then cooled to room temperature and separated between EtOAc and water. The organic layer
was washed with water, brine, then dried over Na SO , filtered, dry loaded onto silica and
2
4
purified by flash chromatography using a mixture of petroleum benzine/EtOAc as the eluent.
Commercial Compounds. Compounds 7-9, 12, 13, 24-26, 38-40, 45, 53-55, 67-77
were sourced from commercial suppliers with the purity of each compound being greater than
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
9
5% as determined using HPLC and assessed by UV absorption at 254 nm.
Benzenesulfonohydrazide (3). Prepared according to general procedure A (58%). H
NMR (400 MHz, [D ]DMSO) δ 8.39 (s, 1H), 7.81 (t, J = 1.4 Hz, 1H), 7.80 (t, J = 1.8 Hz, 1H),
1
6
7
.69 – 7.63 (m, 1H), 7.63 – 7.57 (m, 2H), 4.10 (d, J = 3.2 Hz, 2H).
Naphthalene-2-sulfonohydrazide (4). Prepared according to general procedure A
1
(
99%). H NMR (400 MHz, [D ]DMSO): δ 8.48 (s, 1H), 8.45 (d, J = 1.4 Hz, 1H), 8.16 (d, J =
6
7
.9 Hz, 1H), 8.13 (d, J = 8.8 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.82 (dd, J = 8.6, 1.8 Hz, 1H),
7.75 – 7.64 (m, 2H), 4.15 (d, J = 3.0 Hz, 2H).
N'-(2-Fluorobenzoyl)naphthalene-2-sulfonohydrazide (CTX-0124143). Prepared
1
according to general procedure B (56%). H NMR (400 MHz, [D ]DMSO): δ 10.63 (s, 1H),
6
1
7
7
0.25 (s, 1H), 8.52 (d, J = 1.3 Hz, 1H), 8.21 – 7.99 (m, 3H), 7.91 (dd, J = 8.7, 1.8 Hz, 1H),
.67 (dtd, J = 14.8, 6.9, 1.3 Hz, 2H), 7.55 – 7.47 (m, 1H), 7.38 (ddd, J = 7.4, 5.9, 1.7 Hz, 1H),
.29 – 7.19 (m, 2H). LRMS m/z 345.1 [M+H]+.
N’‐(2‐Methylbenzoyl)naphthalene‐2‐sulfonohydrazide (10). Prepared according to
1
general procedure F (3%). H NMR (400 MHz, [D ]acetone): δ 8.57 (s, 1H), 8.14 – 7.99 (m,
6
4
3
H), 7.69 – 7.64 (m, 2H), 7.36 – 7.26 (m, 2H), 7.18 – 7.12 (m, 2H), 2.76 (s, 3H). LRMS m/z
41.2 [M + H]+.
N’‐(2‐Methoxybenzoyl)naphthalene‐2‐sulfonohydrazide (11). Prepared according
1
to general procedure G (8%). H NMR (400 MHz, [D ]acetone): δ 8.52 (s, 1H), 8.07 – 7.95
6
2
8
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