A Structure?Activity Relationship Study of Novel Hydroxamic Acid Inhibitors around the S1 Subsite of Human Aminopeptidase N

Article abstract of DOI:10.1002/cmdc.202000527

Aminopeptidase N (APN/CD13) is a zinc-dependent ubiquitous transmembrane ectoenzyme that is widely present in different types of cells. APN is one of the most extensively studied metalloaminopeptidases as an anti-cancer target due to its significant role in the regulation of metastasis and angiogenesis. Previously, we identified a potent and selective APN inhibitor, N-(2-(Hydroxyamino)-2-oxo-1-(3′,4′,5′-trifluoro-[1,1′-biphenyl]-4-yl)ethyl)-4-(methylsulfonamido)benzamide (3). Herein, we report the further modifications performed to explore SAR around the S1 subsite of APN and to improve the physicochemical properties. A series of hydroxamic acid analogues were synthesised, and the pharmacological activities were evaluated in vitro. N-(1-(3′-Fluoro-[1,1′-biphenyl]-4-yl)-2-(hydroxyamino)-2-oxoethyl)-4-(methylsulfonamido)benzamide (6 f) was found to display an extremely potent inhibitory activity in the sub-nanomolar range.

Full text of DOI:10.1002/cmdc.202000527

Accepted Article
Title: A Structure–Activity Relationship Study of Novel Hydroxamic Acid
Inhibitors around the S1 Subsite of Human Aminopeptidase N
Authors: Peter J. Scammells, Jisook Lee, Nyssa Drinkwater, and
Sheena McGowan
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000527
Link to VoR: https://doi.org/10.1002/cmdc.202000527
01/2020

10.1002/cmdc.202000527
ChemMedChem
FULL
PAPER
A Structure–Activity Relationship Study of Novel
Hydroxamic Acid Inhibitors around the S1 Subsite of
Human Aminopeptidase N
Jisook Lee,[a] Nyssa Drinkwater,[b] Sheena McGowan,*[b] and Peter Scammells*[a]
[a]
Dr. J. Lee, Prof. P. J. Scammells
Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences
Monash University, Parkville Campus
Parkville, VIC 3052, Australia
E-mail: peter.scammells@monash.edu
[b]
Dr. N. Drinkwater, Assoc. Prof. S. McGowan
Department of Microbiology, Monash Biomedicine Discovery Institute
Monash University, Clayton Campus
Clayton, VIC 3800, Australia
E-mail: sheena.mcgowan@monash.edu
Abstract: Aminopeptidase N (APN/CD13) is a zinc-dependent
ubiquitous transmembrane ectoenzyme that is widely present in
different types of cells. APN is one of the most extensively studied
metalloaminopeptidases as an anti-cancer target due to its significant
role in the regulation of metastasis and angiogenesis. Previously we
identified a potent and selective APN inhibitor, N-(2-(hydroxyamino)-
2-oxo-1-(3',4',5'-trifluoro-[1,1'-biphenyl]-4-yl)ethyl)-4-
Aminopeptidase N (APN/CD13; EC 3.4.11.2) is one of the
most studied metalloaminopeptidases associated with cancer.[5]
A strong correlation between the level of APN expression of a cell
and its invasive characteristics has been established through
multiple studies.[6] Dysregulation of APN has been found to evolve
in almost all types of human malignancies.[7] Over-expression of
APN has been observed in various cancers including small cell
lung carcinoma,[8] thyroid carcinoma,[9] acute myeloid
leukaemia,[10] colon carcinoma[11] and prostate carcinoma.[12]
Selective expression of APN has also been found on the surface
of angiogenic blood vessels that are mostly absent in normal
blood vessels.[13] In addition, APN activity affects metastasis, a
complex biological process that contributes to more than 90% of
cancer related deaths, by promoting cell adhesion, cell motility,
angiogenesis and extracellular matrix (ECM) degradation.[14, 15]
(methylsulfonamido)benzamide (3). Herein, we report the further
modifications performed to explore SAR around the S1 subsite of APN
and to improve the physicochemical properties.
A series of
hydroxamic acid analogues were synthesised, and the
pharmacological activities were evaluated in vitro. N-(1-(3'-fluoro-
[1,1'-biphenyl]-4-yl)-2-(hydroxyamino)-2-oxoethyl)-4-
(methylsulfonamido)benzamide (6f) was found to display an
extremely potent inhibitory activity in the sub-nanomolar range.
As a member of M1 aminopeptidase enzyme superfamily,
APN contains a catalytic zinc ion in the active site and is
responsible for the cleavage of amino acids from the N-terminus
of polypeptide substrates.[5] APN displays an identical gene
sequence to the human lymphocyte surface cluster of
differentiation CD13 antigen; therefore, APN is also called as
CD13.[7, 16] APN possesses a HEXXHX18E zinc-binding motif and
GXMEN substrate-recognition motif that can be found across M1
aminopeptidases.[17] The enzyme is involved in various biological
responses with different roles, therefore it is often known as a
“moonlighting” enzyme.[18] Many peptides, including angiotensin
III and IV, neuropeptides and chemokines are processed by
APN.[19] Moreover, APN acts as a signalling molecule for cell
Introduction
Cancer is one of the most concerning public health problems
worldwide. According to the GLOBOCAN 2018 studies, more than
18 million new cancer cases and 9 million cancer related deaths
were estimated.[1] Although there has been a promising increase
in cancer survival rates and reduced rates of cancer mortality,
cancer still ranks the first or the second most common cause of
death in many countries.[1] Unfortunately, many cancer drugs are
associated with undesirable side effects, primarily due to their
non-selective activity towards non-cancerous cells.[2] Furthermore,
the chronic use of anti-cancer medications inevitably
accompanies drug resistance which reduces its efficacy.[3]
Therefore, the development of novel anti-cancer drugs with less
toxicity, improved efficacy, and higher selectivity continues to be
critically important. With the ever-growing interests in the
discovery of less toxic and more selective anti-cancer agents,
extensive research has been conducted to identify and
characterise therapeutic targets for cancer at the molecular
level.[4]
adhesion and endocytosis and is also
coronavirus.[18, 20]
a
receptor for
Amongst the APN inhibitors developed previously, bestatin (1)
is the most extensively investigated competitive APN inhibitor
(Figure 1).[7, 21] Bestatin is a natural dipeptide, originally isolated
from Streptomyces olivoreticuli as an immunomodulating
agent.[22] Later clinical trials proved the therapeutic activity of
1
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10.1002/cmdc.202000527
ChemMedChem
FULL PAPER
S1
bestatin against lung cancer, gastric cancer, and acute myeloid
leukaemia, and it is currently available in Japan as adjuvant
therapy for acute non-lymphatic leukaemia.[23] Another well-
known APN inhibitor is Tosedostat (2), which is an orally
bioavailable prodrug that is converted to a pharmacologically
active drug intracellularly.[24] Tosedostat exhibited anti-leukemic
activity in phase II clinical trials of acute myeloid leukaemia.[25]
− Halogen
− Heteroaryl
− Cycloalkyl
− Amino
R =
R
O
H
N
HO
N
O
O
S
H
O
N
H
O
OH
Figure 2. Structures of designed hydroxamic acid analogues
NH2
O
OH O
H
N
O
N
H
HO
N
The molecular sizes of pyridine and pyrimidine are
comparable to that of benzene. The designed analogues would
retain the biphenyl core scaffold so that the compounds could
engage in π-stacking hydrophobic interactions with the
phenylalanine residues of domain IV as described in earlier
studies.[26] Introducing additional polar atoms into the molecule
increases the hydrogen bonding capacity, which may also
enhance binding interactions with APN. Moreover, incorporating
H
OH
O
O
bestatin (1)
Tosedostat (2)
F
F
F
nitrogen-containing
heteroaromatics
may
change
the
physicochemical properties significantly due to lowered
LogP/LogD and increased polar surface area, which would also
assist in improving aqueous solubility and other aspects of the
pharmacokinetic profile.[27]
O
H
N
HO
N
H
O
O
O
S
N
3
H
Figure 1. Structures of APN inhibitors.
Apart from six-membered heteroaromatic, it was thought that
five-membered heteroaryl groups such as furan and thiophene
would be worthy of investigation. Furan bears an electronegative
oxygen atom which is capable of capturing additional hydrogen
bonding interactions with the nearby residues. Thiophene is one
of the most widely exploited sulfur-containing heteroaromatics in
medicinal chemistry due to its unique electropositive properties
that allow interactions with electronegative atoms.[28]
We previously synthesised a series of hydroxamic acid
analogues to optimise the binding interactions around and beyond
the S1′ subsite of APN. We discovered that the sulfonamide
compound (3) showed the most potent inhibition against APN
(Ki(app) = 4.8 nM), which was a 500-fold improved activity
compared to bestatin (Ki(app) = 2.4 µM).[26] Despite its potent
activity and promising pharmacokinetic properties, compound 3
possessed sub-optimal aqueous solubility (12.5 – 25 µg/mL in pH
2.0 and 6.0)[26] which could potentially lead to a limited absorption
and low efficacy in the cellular environment.
Earlier studies heavily focussed on the biaryl or 4-haloaryl
29]
units.[26,
Therefore, incorporating sp3-rich groups as
replacements of the 3,4,5-trifluorophenyl moiety may create new
chemical scaffolds to probe the activities around the S1 pocket of
APN. Moreover, the installation of sp3-groups has been used as
effective strategies to enhance solubility by changing the crystal
polymorphs.[30] Therefore, introducing cycloalkyl groups such as
cyclohexyl and cyclopropyl may remove the aromaticity and
symmetry of the biphenyl scaffold to reach an ideal balance
between solubility and hydrophobicity, which is essential for
optimal oral bioavailability.[30] Piperidine and piperazine
derivatives may have advantages in two-fold because they are
not only sp3-rich but also contain additional ionisable nitrogen
atom(s).
In this study, we proposed further modifications to compound
3 not only to improve aqueous solubility but also to maintain or
improve the potent inhibitory activity by replacing the hydrophobic
3,4,5-trifluorophenyl moiety that is positioned at the S1 binding
site of APN. The 3,4,5-trifluorophenyl group was derived from the
SAR studies and structure-guided optimisation of the parasitic
homologue PfA-M1.[29a] However, there was a limited exploration
of the S1 subsite of APN to probe binding interactions.
Results and Discussion
Chemistry
Synthesis. The synthesis of monoaryl, biaryl, and cycloalkyl
compounds 6a–s was achieved as described in Scheme 1.
Commercially available phenylglycine derivatives were protected
by acid-catalysed esterification, followed by EDCI/DMAP amide
coupling with (methylsulfonamido)benzoic acid to produce methyl
ester intermediates 5a-d successfully. The aryl bromide 5d was
used as the key intermediate to generate biaryl compounds 5e-n
through microwave-assisted Suzuki-Miyaura coupling reactions in
the presence of K3PO4 and Pd(PPh3)2Cl2 under anhydrous
conditions.
Compound Design. A set of target compounds were designed to
investigate the binding interactions at the S1 pocket of APN as
well as to achieve favourable aqueous solubility (Figure 2).
According to the previous molecular modelling studies, the 3,4,5-
trifluorophenyl ring formed various hydrophobic interactions,
creating a stable binding site for the inhibitors within the S1
subsite of APN.[26] Therefore, the complete removal of the
trifluorophenyl group would allow us to investigate if the biphenyl
system is essential for obtaining potency against APN. In addition,
replacing the hydrophobic 3,4,5-trifluorophenyl to various
isosteric heteroaromatic groups such as pyridyl and pyrimidyl
moieties may improve both the solubility and binding interactions.
2
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Scheme 1. Reagents and conditions: (a) conc. H2SO4, MeOH, reflux, overnight, 75–90%; (b) 4-(methylsulfonamido)benzoic acid, EDCI, DMAP, DCM, rt, overnight,
61–92%; (c) arylboronic acid, K3PO4, anhydrous DMF, Pd(PPh3)2Cl2, 100 °C, microwave, 1–20 h, 33–95%; (d) alkylboronic acid, K2CO3, anhydrous THF, Pd(dppf)Cl2,
reflux, overnight, 78–94%; (e) EtOH or MeOH, 10% Pd/C, NH4HCO2, reflux, 2–16 h, 80–83%; (f) NH2OH·HCl, 5 M KOH/MeOH, anhydrous MeOH, rt, overnight, 5–
78%.
The coupling reaction between 5d and cyclopropylboronic
acid under the same conditions only resulted in a minimal
conversion to the desired product. Cross-coupling reactions of
alkylboronic acids remain challenging because of several serious
issues such as decomposition of palladium-alkyl intermediate via
β-elimination, slow transmetallation, and formation of side
products.[31] Molander et al. reported successful coupling
reactions of alkylboronic acids with electron-deficient and
electron-excessive aryl bromides in the presence of Pd(dppf)Cl2,
K2CO3, and biphasic solvent THF/water.[32] It was also noted that
Pd(dppf)Cl2 was commonly used catalyst for coupling reactions
with alkylboronic acids which usually required for forcing
conditions.[33] Therefore, the modified reaction conditions
reported by Molander et al. were attempted in the absence of
water to avoid hydrolysis of methyl ester, which formed the
cyclopropyl 5o in an excellent yield of 94%.
cyclopentylboronic acid, a complete debromination of 5d was
observed. Therefore, cyclopentenyl and cyclohexenylboronic
acids were used as alternative substrates because sp2
vinylboronic acids are known to undergo Suzuki-Miyaura
couplings more readily than their sp3 alkylboronic acid pairs. The
reaction outcome was significantly improved and generated
cyclopentenyl and cyclohexenyl analogues 5p and 5q in yields of
87% and 78%, respectively. The C=C bonds present in 5p and 5q
were reduced by a transfer catalytic hydrogenation to afford fully
saturated cyclopentyl and cyclohexyl analogues 5r and 5s in good
yields (80% and 83%, respectively).
In order to synthesise analogues containing piperidine and
piperazine derivatives, a study was conducted to optimise
Buchwald-Hartwig amination reaction of 5d with 1-
methylpiperazine and 4-methylpiperidine. Different combinations
of commonly used catalyst Pd2(dba)3, ligands (XPhos and BINAP),
and bases (NaOtBu, K3PO4, and Cs2CO3) were assessed.
Despite substantial efforts to optimise the Buchwald-Hartwig
aminations, we were not able to generate the desired compounds
successfully. Debromination of the aryl bromide 5d was prevalent
and minimal conversion to the desired product was observed. As
such, an alternative synthetic pathway was established, which
involved the use of relatively simple molecule methyl 2-(4-
bromophenyl)-2-((tert-butoxycarbonyl)amino)acetate (7) as the
key aryl bromide intermediate (Scheme 2).
Unfortunately, the reaction conditions applied for the
cyclopropyl 5o failed to produce the cyclopentyl 5r and cyclohexyl
5s analogues. Minimal conversion to the desired product was
observed, and the starting material 5d was mostly recovered.
While cyclopropylboronic acid derivatives are widely explored as
a competent nucleophile in Suzuki-Miyaura coupling reactions[32-
33], there is limited evidence of other secondary alkylboronic acids
due to slow transmetallation rates.[33-34] A strategy applied to
accelerate the transmetallation step involved was the use of
silver(II) oxide (Ag2O) as an additive.[33, 35] However, when this
approach was applied to couple the aryl bromide 5d with
3
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FULL PAPER
R
Br
Br
(a)
(b)
O
O
O
O
O
N
O
NH2
N
H
O
H
O
O
O
4d
7
8a-i
(c)
R
a: N,N-diethylamino
b: Piperidyl
R
c: Morpholinyl
(e)
d: 4-Methylpiperidyl
e: 4-Fluoropiperidyl
f: 4,4-Difluoropiperidyl
g: 4-(Trifluoromethyl)piperidyl
h: 1-Methylpiperazinyl
i: Cbz-piperazinyl
O
O
H
O
N
N
O
S
HO
N
O
S
O
O
H
H
O
O
N
N
H
H
10a-h, j
j: Piperazinyl
9a-i
9i R = Cbz-piperazinyl
(d)
9j R = Piperazinyl
Scheme 2. Reagents and conditions: (a) 4d, Boc2O, sat. NaHCO3, water, THF, rt, overnight; 75%; (b) amines, Cs2CO3, Pd2(dba)3, XPhos, anhydrous toluene, reflux,
overnight, 50–99%; (c) (i) 20% TFA/DCM, rt, 3 h, quantitative; (ii) HCTU, DIPEA, 4-(methylsulfonamido)benzoic acid, rt, overnight, 41–70%; (d) MeOH, 10% Pd/C,
NH4HCO2, reflux, 4 h, 59%; (e) NH2OH·HCl, 5 M KOH/MeOH, anhydrous MeOH, rt, overnight, 35–73%.
The intermediate 7 was synthesised by Boc protection of the
amino acid derivative 4d. A total of 10 alkyl amines were coupled
partitioning of a molecule between lipophilic and aqueous layers,
was calculated in silico by ChemAxon Instant JChem software
with 7 smoothly in the presence of Pd2(dba)3, XPhos, and Cs2CO3. 16.9.12.0. (Table 1). While there was no striking trend between
The installation of N,N-diethyl moiety resulted in modest yield
(42%) because of the higher volatility of N,N-diethylamine (b.p. =
56 °C) compared to other piperidine derivatives, which quickly
evaporated at a high reaction temperature of 120 °C. This
problem was easily rectified by increasing the amount of N,N-
diethylamine and by heating the reaction mixture in a closed
microwave vessel, which significantly improved the yield to 84%.
The Boc group of intermediates 8a–i was cleaved by acid
hydrolysis with 20% TFA in DCM to give the corresponding free
amines as TFA salts. The subsequent amide coupling reaction
was performed in the presence of excess DIPEA, HCTU, and 4-
(methylsulfonamido)benzoic acid to produce methyl ester
intermediates 9a-i. Synthesis of the piperazinyl analogue 9j
required additional deprotection of the mono-benzyl carbamate
(Cbz) group of compound 9i which was removed by a transfer
catalytic hydrogenation with ammonium formate and 10% Pd/C.
Finally, the methyl ester intermediates 9a-h and 9j were
converted to the corresponding hydroxamic acids 10a-h and 10j
via direct aminolysis.
the inhibitory activity and cLogP observed, all the hydroxamic acid
analogues displayed smaller cLogP values than the lead
compound 3 (cLogP = 2.10), suggesting that this series of
analogues could potentially have improved solubility than
compound 3.
Considering phenylglycine analogues 6a–d, it was noted that
complete removal of the 3,4,5-trifluorophenyl group resulted in a
significant loss in the activity from Ki(app) = 4.90 nM (3) to Ki(app)
=
116 nM (6a). However, inhibitory activity was regained once
halogen groups were introduced in the descending order of Cl >
Br > F, indicating dipole-induced interactions play a role in
improving potency. The result also suggested that biphenyl was
the preferred scaffold over phenylglycines; the phenyl analogue
6e (Ki(app) = 4.1 nM) was comparable to that of the lead compound
3 but more potent than the bromophenylglycine analogue 6d
(Ki(app) = 9.47 nM) which contained bromo group as a bioisostere
of phenyl.
The fluorine substituted phenyl compounds 6f–i were well
tolerated. For example, the 3-fluorophenyl analogue 6f was
determined to be the most potent inhibitor of the series with a
Ki(app) value of 0.66 nM. A decrease in potency was observed
when fluorine was substituted at the 4-position. For instance, the
4-fluorophenyl analogue 6g showed a reduced inhibitory activity
of Ki(app) = 9.54 nM. Similarly, the 3,4-difluorophenyl analogue 6h
possessed a Ki(app) = 6.15 nM, which was approximately 6-fold
less potent than the 3,5-difluorophenyl analogue 6i (Ki(app) = 1.19
nM). Based on these results, it was proposed that substituents at
the 3-position were preferred to 4-position and trifluoro group was
not essential for achieving the potent inhibitory activity.
Biology
A total of 28 novel hydroxamic acid analogues were successfully
synthesised (6a–s, 10a–h, and 10j). The inhibitory activities of
hydroxamic acid analogues were evaluated by a fluorescence-
based assay using recombinant human APN in the presence of
L-Leucine-7-amido-4-methylcoumarin
hydrochloride
as
a
competitive substrate (Table 1).[26] The measured inhibition
constants (Ki(app)) were compared between different analogues
comprehensively based on their structural variations to determine
which groups successfully replaced the 3,4,5-trifluorophenyl
group without sacrificing activity. In addition, as a measure of
solubility, partition coefficient cLogP, which reflects the
4
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Table 1. Inhibition activities (Ki(app)) of synthesised compounds against APN.
in electronic characteristics and basicity. Due to the electron-
withdrawing and inductive effect, protonation at the second
nitrogen atom is unlikely, making pyrimidine less basic than
pyridine. Therefore, it can be suggested that more basic group is
favoured at the S1 subsite of APN.
Cpd
3
R
3,4,5-Trifluorophenyl
H
Ki(app) ± SEM / nM[a]
4.90 ± 0.80
166 ± 15
cLogP
2.10
0.02
0.17
0.63
0.79
1.67
1.81
1.81
1.96
1.96
0.81
1.45
0.45
0.45
-0.25
0.81
1.43
1.87
1.70
2.14
0.85
0.98
-0.09
1.27
0.34
1.20
1.55
-0.02
-0.40
6a
According to our molecular modelling in the previous studies,
the biphenyl scaffold engaged in multiple hydrophobic
interactions with a flexible loop located in domain IV, and it was
proposed that these interactions might play an imperative role in
locking inhibitors within the active site of APN effectively.[26]
Therefore, it was hypothesised that analogues containing alkyl
groups as replacement of 3,4,5-trifluorophenyl (i.e. compounds
6o–s, 10a–h, and 10j) would display reduced potency. Indeed, a
decreased level of potency was observed from the majority of
alkyl analogues, except the cyclopropyl analogue 6o and
cyclohexyl analogue 6s which displayed tight-bindings to APN.
Despite the small size and less degree of planarity, the
cyclopropyl analogue 6o retained good activity (Ki(app) = 9.74 nM).
Interestingly, a trend of decreasing activities was observed from
cycloalkenyl analogues compared to its saturated cycloalkyl pairs.
Cyclopentyl 6r showed a Ki(app) value of 11.4 nM, but the
cyclopentenyl analogue 6p showed a decreased potency (Ki(app)
of 90.6 nM). Similarly, the cyclohexyl 6s (Ki(app) = 4.87 nM) which
was equipotent to the parent compound 3, was nearly 70-fold
more active than the cyclohexenyl 6q (Ki(app) = 343 nM). However,
there was an exceptional case as well, where the phenyl analogue
6e was equally potent to the cyclohexyl 6s.
6b
6c
F
48.6 ± 1.3
7.38 ± 0.17
9.47 ± 0.17
4.15 ± 0.24
0.66 ± 0.06
9.54 ± 0.97
6.15 ± 0.29
1.19 ± 0.13
34.6 ± 1.2
10.2 ± 1.0
9.77 ± 0.19
2.41 ± 0.30
49.0 ± 4.4
9.74 ± 0.62
90.6 ± 4.1
343 ± 5
Cl
6d
6e
Br
Phenyl
6f
3-Fluorophenyl
4-Fluorophenyl
3,4-Difluorophenyl
3,5-Difluorophenyl
3-Furan
6g
6h
6i
6j
6k
3-Thiophene
3-Pyridyl
6l
6m
6n
6o
6p
6q
6r
4-Pyridyl
Pyrimidyl
Cyclopropyl
1-Cyclopentenyl
1-Cyclohexenyl
Cyclopentyl
Cyclohexyl
The amine analogues 10a–h and 10j were not well tolerated
and showed significantly diminished inhibitory activities. N,N-
Diethyl analogue 10a showed a potency of Ki(app) = 54.8 nM and
the activity deteriorated even more when cyclised amino groups
were incorporated (10b–g). Additional fluorine(s) of 4-
fluoropiperidyl 10e (Ki(app) = 54.8 nM) and 4,4-difluoropiperidyl 6f
were able to enhance the potency (Ki(app) = 63.5 nM) compared to
the unsubstituted piperidyl analogue 10b (Ki(app) = 151 nM) or the
4-methylpiperidyl analogue 10d (Ki(app) = 212 nM). Piperazinyl
analogues were determined to be more potent than its
corresponding piperidyl compounds. For example, the piperazinyl
analogue 10j (Ki(app) = 27.0 nM) was nearly 6-fold more active than
the piperidyl analogue 10b (Ki(app) = 151 nM). A similar relationship
was observed between the 4-methylpiperidyl analogue 10d and
the 1-methylpiperazinyl 10h (Ki(app) = 42.4 nM). Two aspects
potentially contributed to the enhanced potency of piperazinyl
compounds compared to piperidyl compounds are suggested.
Firstly, piperazine derivatives possess an additional basic
aliphatic nitrogen atom that can be readily ionised in physiological
pH condition. However, the nitrogen atom present in piperidine
derivatives would have similar properties as arylamines, which
are significantly less basic than aliphatic amines. With the greater
capacity of ionisation, the piperazinyl analogues may form strong
ionic interactions with nearby residues. Secondly, the aliphatic
amine of piperazinyl is also able to engage in hydrogen bonding
interactions, acting as a hydrogen bond donor. These results were
also in line with the relationships between the pyridyl (6l and 6m)
and the pyrimidyl analogue (6n) where the compound bearing
more basic moiety was favoured.
11.4 ± 1.2
4.87 ± 0.34
54.8 ± 4.1
151 ± 13
6s
10a
10b
10c
10d
10e
10f
10g
10h
10j
N,N-Diethylamino
Piperidyl
Morpholine
224 ± 6
4-Methylpiperidyl
4-Fluoropiperidyl
4,4-Difluoropiperidyl
4-(Trifluoromethyl)piperidyl
1-Methylpiperazinyl
Piperazinyl
212 ± 9
54.8 ± 4.6
63.5 ± 6.2
158 ± 10
42.4 ± 1.8
27.0 ± 1.9
[a] SEM values of Ki(app) are obtained from three individual experiments
Most heteroaromatic analogues 6j–n displayed a decreased level
of potency against APN, except 4-pyridyl analogue 6m which
showed an increased inhibitory activity of Ki(app) = 2.41 nM. The
potent activity of compound 6m compared to phenyl analogue 6e
could be due to the pyridine moiety that is capable of participating
in hydrogen bonding interactions. Five-membered heteroaromatic
furan analogue 6j and thiophene analogue 6k displayed potency
of Ki(app) = 34.6 nM and 10.2 nM, respectively. In addition,
pyrimidyl analogue 6n exhibited a reduced inhibition activity
(Ki(app) = 49.0 nM) compared to 3-pyridyl analogue 6l and 4-pyridyl
analogue 6m by 5- and 10-fold. The preference towards pyridyl
compounds over pyrimidyl could be explained by the differences
5
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Conclusion
Chemistry: Chemicals and solvents were purchased from standard
suppliers and used without further purification unless otherwise indicated.
1H, 19F and 13C NMR spectra were recorded on a Bruker Avance Nanobay
III 400 MHz Ultrashield Plus spectrometer at 400.13, 376.46 and 100.61
MHz, respectively. Data acquisition and processing were managed using
Topspin software package version 3. Chemical shifts (δ) are recorded in
parts per million with reference to the chemical shift of the deuterated
solvent. Coupling constants (J) and carbon-fluorine coupling constants
(JCF) are recorded in hertz and multiplicities are described as singlet (s),
doublet (d), triplet (t), multiplet (m), doublet of doublets (dd), doublet of
triplets (dt), doublet of doublets of doublets (ddd), and broad (br).
Overlapped non-equivalent 13C peaks were identified by HSQC and HMBC
NMR.
Interests in the discovery of novel therapeutic agents targeting
metastasis and angiogenesis for the treatment of cancer are ever-
increasing. APN has been shown to involved in a number of
complex biological processes such as ECM degradation,
angiogenesis, cell adhesion, and cell motility which can all
accelerate metastasis. Therefore, APN has been extensively
studied as a therapeutic target for cancer. In this study, we have
performed further modifications of a previously discovered potent
APN inhibitor 3 by replacing the 3,4,5-trifluorophenyl moiety. A
small library of hydroxamic acid containing analogues was
designed to probe the binding interactions around the S1 pocket
of APN. A diverse range of substituents was introduced to
enhance the solubility of the lead compound. Comprehensive
SAR were established, and an extremely potent APN inhibitor 6f
(Ki(app) = 0.66 nM) was identified, which displayed 7-fold improved
inhibitory activity compared to the parent compound 3 with a
reduced cLogP value of 1.81. Further pharmacological
evaluations on compound 6f will be required to measure the
cellular activity, toxicity, and selectivity prior to pursuing as a
potential anti-cancer drug candidate.
Analytical thin-layer chromatography (TLC) was performed on Merck silica
gel 60 F254 aluminium-backed plates were visualised by fluorescence
quenching under a UV lamp at 254 nm or by Fe(III)Cl3 staining for
hydroxamic acid compounds. Flash chromatography was performed with
silica gel 60 (particle size 0.040–0.063 µm).
Analytical HPLC was performed using an Agilent 1260 Infinity Analytical
HPLC with a Zorbax Eclipse Plus C18 Rapid Resolution 4.6 × 100 m, 3.5
m column. Buffer A: 0.1% TFA in H2O and buffer B: 0.1 % TFA in MeCN
were used. Samples were run at a gradient of 5% buffer B/ buffer A (0–9
min) to 100% buffer B (9–10 min) at a flow rate of 1 mL/min. Unless
otherwise indicated, all compounds were > 95% by HPLC (254 nm and
214 nm) prior to biological evaluation.
Experimental Section
Biology
Preparative HPLC was performed using an Agilent 1260 Infinity instrument
coupled with a binary preparative pump and an Agilent 1260 FC-PS
fraction collector using Agilent OpenLAB CDS software (revision C.01.04)
and an Altima C8 22 × 250 mm, 5 μM column and a 1260 Infinity diode
array detector VL. The following buffers were used: buffer A: 0.1% TFA in
H2O and buffer B: 0.1% TFA in MeCN. The sample was run at a gradient
of 30% to 100% buffer B over 10 min at a flow rate of 20 mL/min.
Protein Expression and Purification: A soluble form of human APN
ectodomain was expressed and purified as reported previously.[26] Human
APN was expressed in a transfected HEK293S GnT1¯ cell line, which was
a kind gift from Professor James Rini from the University of Toronto,
Canada. The cell growth, cell passaging, and collection of the culture
supernatant were conducted by Monash Protein Production Unit. Cells
were grown in DMEM/F-12 supplemented with 3% FBS Invitrogen, 1 ×
penicillin-streptomycin (Invitrogen), 1 mg/liter of doxycycline (Sigma), and
1 mg/liter of aprotinin (Bioshop Canada). The APN–protein A fusion
protein was purified by IgG-Sepharose (GE Healthcare) affinity
chromatography. The protein A tag was removed by on-column tobacco
etch virus protease digestion, and the liberated APN was further purified
by size exclusion chromatography on a Superdex S200 10/300 column in
50 mM HEPES pH 8.0, 300 mM NaCl, 5% glycerol buffer. Biochemical
parameters Km (31 ± 5 μM) and kcat (456 ± 14 FU/sec-1) were determined
in the presence of L-Leucine-7-amido-4-methylcoumarin hydrochloride (H-
Leu-NHMec) (Sigma L2145).
LC-MS was performed using Agilent 6120 series Single Quadrupole
instrument coupled to an Agilent 1260 series HPLC instrument fitted with
a Poroshell 120 EC-C18 50 × 3.0 mm, 2.7 µm column. The following
buffers were used: buffer A, 0.1% formic acid in H2O; buffer B, 0.1% formic
acid in MeCN. Samples were run at a flow rate of 0.5 mL/min for 5 min:
5% buffer B/ buffer A (0–1 min), 100% buffer B (1–2.5 min) and held at this
composition until 3.8 min, 5% buffer B/ buffer A (3.8–4 min) and held until
5 min at this composition. Mass spectra were obtained in positive and
negative ion modes with a scan range of 100-1000 m/z. UV detection was
carried out at 214 and 254 nm.
APN Enzymatic Analysis: Aminopeptidase assays were based on the
previously published Lee et al.[26] The activity of APN was determined by
measuring the release of the fluorogenic leaving group, NH2Mec, from the
fluorogenic peptide H-Leu-NHMec (Sigma L2145). The reactions were
carried out in 384-well microtitre plates, 50 µL total volume at 37 ºC using
a spectrofluorimeter (BMG FLUOstar) with excitation at 355 nm and
emission at 460 nm. APN was pre-incubated in 100 mM Tris pH 8.0 at 37
ºC with the inhibitors for 10 min prior to the addition of substrate (25 μM).
Inhibitor concentrations were diluted 1:4 to assess an overall 1000-fold
concentration series. The fluorescence signal was monitored for 1 hour at
37 ºC. The only linear range of velocity was considered in data analysis.
Ki(app) values were then calculated by using the Morrison equation for
competitive and tight-binding inhibitors.[37] Analysis and graphical output
were performed in GraphPad Prism® 7.
HRMS was carried out using an Agilent 6224 TOF LC-MS mass
spectrometer coupled to an Agilent 1290 Infinity. All data were acquired
and referenced via dual-spray electrospray ionization (ESI) source.
Acquisition was performed using Agilent Mass Hunter Data Acquisition
software version B.05.00 Build 5.0.5042.2 and analysis was conducted
using Mass Hunter Qualitative Analysis version B.05.00 Build 5.0.519.13.
Instant JChem was used for data management and calculation of cLogP
values; Instant JChem 16.9.12.0, ChemAxon (http://www.chemaxon.com).
General Procedure A: Acid catalysed the esterification of substituted
2-amino-2-phenylacetic acids. To a round bottom flask was added
appropriate 2-amino-2-phenylacetic acid (1.0 eq.), MeOH (13 mLmmol-1)
and conc. H2SO4 (4.0 eq.) dropwise. The mixture was heated at reflux for
overnight. Upon completion, the mixture was concentrated in vacuo and
neutralised by sat. NaHCO3 to pH 7–8 and extracted with EtOAc (3 × 20
mL). The combined organic layers were dried over anhydrous Na2SO4 and
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000527
ChemMedChem
FULL PAPER
concentrated in vacuo. The crude product obtained was used for
subsequent reactions without further purifications.
General Procedure G: Direct aminolysis of methyl ester to the
hydroxamic acid. To a solution of methyl ester (1.0 eq.) in anhydrous
MeOH (3 mLmmol-1) was added NH2OH·HCl (8.0 eq.), followed by 5 M
KOH in anhydrous MeOH (10 eq.). The reaction was stirred at room
temperature for overnight. Upon completion, the suspension was
concentrated in vacuo followed by addition of a 10% citric acid (10 mL)
and extraction with EtOAc (3 × 10 mL). The combined organic layers were
dried over anhydrous Na2SO4 and concentration in vacuo. The crude
product was purified by column chromatography or preparative HPLC.
General Procedure B: Amide coupling using EDCI and DMAP. Methyl
2-amino-2-phenylacetate (1.0 eq.), carboxylic acid (1.2 eq.), EDCI (1.2
eq.) and DMAP (1.3 eq.) were dissolved in DCM/DMF (1:1) mixture (10
mLmmol-1) and stirred at room temperature for overnight. DCM was
removed in vacuo, and the resulting mixture was diluted with a 1 M HCl
(20 mL) and extracted with EtOAc (3 × 15 mL). The combined organic layer
were washed with water (2 × 20 mL) and brine (20 mL), which was then
dried over anhydrous Na2SO4 and concentrated in vacuo. The crude was
taken up into a minimum amount of DMF and added to ice-water dropwise.
The solid was filtered with minimal MeOH wash and dried under vacuum
overnight.
Methyl 2-amino-2-phenylacetate (4a). Compound 4a was synthesised
from 2-amino-2-phenylacetic (200 mg, 1.32 mmol) according to General
Procedure A. The crude product (164 mg, 75%) was obtained as a brown
oil. 1H NMR (CDCl3) δ 7.42–7.29 (m, 5H), 4.65 (s, 1H), 3.70 (s, 3H), 2.49
(br. s, 2H); 13C NMR (CDCl3) δ 170.6, 129.0, 128.3, 128.2, 127.0, 58.9,
52.6;
General Procedure C: Suzuki-coupling reaction between aryl
bromide 5d and arylboronic acids. To a N2 flushed 0.5–2 mL microwave
vial was added the aryl bromide 5d (1.0 eq.), boronic acid (1.2 eq.), K3PO4
(2.4 eq.) and anhydrous DMF (2 mL), which was stirred and degassed for
5 min before adding Pd(PPh3)2Cl2 (0.05 eq.). The mixture was heated at
100 °C for 1–20 h in a microwave reactor until completion. The reaction
mixture was diluted with a 1 M HCl (20 mL) and extracted with EtOAc (3 ×
15 mL). The combined organic layers were further washed with water (2 ×
20 mL) and brine (20 mL), which was then dried over anhydrous Na2SO4
followed by concentration in vacuo. The crude was taken up into a minimal
amount of DMF and added to ice-water dropwise. The solid was filtered
with minimal MeOH or Et2O wash and dried under vacuum overnight.
Methyl 2-amino-2-(4-fluorophenyl)acetate (4b). Compound 4b was
synthesised from 2-amino-2-(4-fluorophenyl)acetic acid (200 mg, 1.18
mmol) according to General Procedure A. The crude product (188 mg,
87%) was obtained as a brown oil. 1H NMR (CDCl3) δ 7.41–7.29 (m, 2H),
7.11–6.96 (m, 2H), 4.63 (s, 1H), 3.70 (s, 3H), 2.27 (br. s, 2H); 19F NMR
(CDCl3) δ -114.1; 13C NMR (CDCl3) δ 174.2, 162.6 (d, JCF = 246.7 Hz),
135.8 (d, JCF = 3.3 Hz), 128.7 (d, JCF = 8.2 Hz), 115.8 (d, JCF = 21.6 Hz),
58.1, 52.7.
Methyl 2-amino-2-(4-chlorophenyl)acetate (4c). Compound 4c was
synthesised from 2-amino-2-(4-chlorophenyl)acetic acid (200 mg, 1.08
mmol) according to General Procedure A. The crude product (181 mg,
84%) was obtained as a brown oil. 1H NMR (CDCl3) δ 7.33 (app. s, 4H),
4.61 (s, 1H), 3.70 (s, 3H), 2.13 (br. s, 2H); 13C NMR (CDCl3) δ 174.0, 138.5,
134.11, 129.1, 128.4, 58.6, 52.7.
General Procedure D: Suzuki-coupling reaction between aryl
bromide 5d and alkenylboronic acids. A dry round bottom flask was
charged with compound 5d (1.0 eq.), cycloalkenylboronic acid (1.6 eq.),
K2CO3 (4.5 eq.), and anhydrous THF (10 mLmmol-1). The mixture was
bubbled through N2 for 10 min prior to the addition of Pd(dppf)Cl2 (0.15 eq).
The mixture was refluxed for overnight. Upon completion, THF was
removed in vacuo, the residue was acidified with a 1 M HCl (10 mL) and
extracted with EtOAc (3 × 10 mL). The combined organic layers were dried
over anhydrous Na2SO4 and concentrated in vacuo. The crude product
was triturated with appropriate organic solvents or was purified by column
chromatography.
Methyl 2-amino-2-(4-bromophenyl)acetate (4d). Compound 4d was
synthesised from 2-amino-2-(4-bromophenyl)acetic acid (2.00 g, 8.69
mmol) according to General Procedure A. The crude product (1.90 g, 90%)
was obtained as a brown oil. 1H NMR (CDCl3) δ 7.48 (d, J = 7.4 Hz, 2H),
7.30 (d, J = 7.9 Hz, 2H), 4.70 (s, 1H), 3.70 (s, 3H), 3.42 (br. s, 2H).
Methyl 2-(4-(methylsulfonamido)benzamido)-2-phenylacetate (5a).
Compound 4a (164 mg, 0.993 mmol) was coupled to
(methylsulfonamido)benzoic acid (228 mg, 1.19 mmol) according to
General Procedure B. The desired product was obtained as a white solid
(220 mg, 61%). 1H NMR (DMSO-d6) δ 10.13 (s, 1H), 9.06 (d, J = 7.1 Hz,
1H), 8.01–7.79 (m, 2H), 7.47 (m, 2H), 7.44–7.33 (m, 3H), 7.30–7.09 (m,
2H), 5.65 (d, J = 7.1 Hz, 1H), 3.65 (s, 3H), 3.06 (s, 3H); 13C NMR (DMSO-
d6) δ 171.1, 165.8, 141.5, 136.2, 129.2, 128.5, 128.2, 128.2, 128.1, 117.8,
56.9, 52.2, 39.5; m/z MS C17H19N2O5S [MH]+ 362.8.
General Procedure E: Buchwald-Hartwig amination between aryl
bromide 7 and alkylamines. A mixture of aryl bromide 7 (1.0 eq.),
Pd2(dba)3 (0.03 eq.), XPhos (0.1 eq.), Cs2CO3 (2.5–5.0 eq.), and amine
(2.5 eq.) in a degassed anhydrous toluene (10 mL) was heated at reflux
for overnight. After cooling, the mixture was diluted with water (6 mL) and
extracted with EtOAc (3 × 20 mL). The combined organic layers were
washed with brine, dried over anhydrous Na2SO4, and concentrated in
vacuo. The crude was purified by column chromatography.
General Procedure F: Synthesis of aryl sulfonamide compounds 5t-
ab from Boc-protected intermediates 8a–i. A round bottom flask was
charged with Boc-protected intermediate (1.0 eq.) and 20% TFA in DCM
(10 mLmmol-1). The mixture was stirred at room temperature for 3 h. Upon
completion, excess TFA was removed in vacuo, and the residue was dried
under vacuum overnight. The residue was dissolved in DCM (5 mLmmol-
1) and DIPEA (3.0 eq.), which was stirred for 30 min (mixture 1). To a
Methyl 2-(4-fluorophenyl)-2-(4-(methylsulfonamido)benzamido)
acetate (5b). Compound 4b (188 mg, 1.03 mmol) was coupled to
(methylsulfonamido)benzoic acid (236 mg, 1.23 mmol) according to
General Procedure B. The desired product was obtained as a white solid
(300 mg, 77%). 1H NMR (DMSO-d6) δ 10.13 (s, 1H), 9.07 (d, J = 7.1 Hz,
1H), 7.89 (d, J = 8.8 Hz, 2H), 7.59–7.45 (m, 2H), 7.27–7.21 (m, 4H), 5.67
(d, J = 7.0 Hz, 1H), 3.65 (s, 3H), 3.06 (s, 3H); 19F NMR (DMSO-d6) δ -
114.1; 13C NMR (DMSO-d6) δ 171.0, 165.8, 161.9 (d, JCF = 244.4 Hz),
separate
dried
round
bottom
flask
was
added
4-
(methylsulfonamido)benzoic acid (1.1 eq.), HCTU (1.2 eq.), DIPEA (2.0
eq.), and DMF (5 mLmmol-1) and the mixture was stirred for 30 min
141.5, 132.5, 130.4 (d, JCF = 8.4 Hz), 129.2, 128.1, 117.8, 115.3 (d, JCF
21.5 Hz), 56.1, 52.3, 39.6; m/z MS C17H18FN2O5S [MH]+ 380.8.
=
(mixture 2). Mixture
1 and 2 were combined and stirred at room
temperature for overnight. Upon completion, the mixture was concentrated
in vacuo, diluted with a half sat. NaHCO3 (10 mL) and extracted with EtOAc
(3 × 15 mL). The combined organic layers were further washed with water
(3 × 20 mL) and brine (20 mL) followed by drying over anhydrous Na2SO4
and concentration in vacuo. The crude was purified by column
chromatography.
Methyl 2-(4-chlorophenyl)-2-(4-(methylsulfonamido)benzamido)
acetate (5c). Compound 4c (181 mg, 0.907 mmol) was coupled to
(methylsulfonamido)benzoic acid (209 mg, 1.09 mmol) according to
General Procedure B. The reaction produced the desired product (332 mg,
92%), which was used for the next reaction without further purification. 1H
NMR (DMSO-d6) δ 10.14 (s, 1H), 9.10 (d, J = 7.1 Hz, 1H), 7.89 (d, J = 8.8
Hz, 2H), 7.53–7.44 (m, 4H), 7.25 (d, J = 8.8 Hz, 2H), 5.69 (d, J = 7.1 Hz,
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000527
ChemMedChem
FULL PAPER
1H), 3.66 (s, 3H), 3.06 (s, 3H); 13C NMR (DMSO-d6) δ 171.3, 166.3, 142.1,
135.9, 133.4, 130.7, 129.7, 129.0, 128.5, 118.3, 56.6, 52.8, 40.2; m/z MS
C17H18ClN2O5S [MH]+ 396.8.
10.14 (s, 1H), 9.13 (d, J = 7.1 Hz, 1H), 7.92 (d, J = 8.8 Hz, 2H), 7.84–7.75
(m, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.61–7.50 (m, 4H), 7.26 (d, J = 8.8 Hz,
2H), 5.73 (d, J = 7.1 Hz, 1H), 3.68 (s, 3H), 3.07 (s, 3H); 19F NMR (DMSO-
d6) δ -138.1 (d, J = 22.5 Hz), -140.6 (d, J = 22.5 Hz); 13C NMR (DMSO-d6)
δ 171.0, 165.8, 149.8 (dd, JCF = 245.3/12.6 Hz), 149.2 (dd, JCF
=
Methyl 2-(4-bromophenyl)-2-(4-(methylsulfonamido)benzamido)
acetate (5d). 4-(Methylsulfonamido)benzoic acid (2.06 g, 9.59 mmol) was
coupled to compound 4d (1.95 g, 7.99 mmol) according to General
Procedure B. The desired product was obtained as a light yellow solid
(2.32 g, 66%). 1H NMR (DMSO-d6) δ 10.15 (s, 1H), 9.11 (d, J = 7.1 Hz,
1H), 7.97–7.81 (m, 2H), 7.64–7.56 (m, 2H), 7.48–7.39 (m, 2H), 7.31–7.20
(m, 2H), 5.67 (d, J = 7.1 Hz, 1H), 3.66 (s, 3H), 3.07 (s, 3H); 13C NMR
(DMSO-d6) δ 170.7, 165.8, 141.6, 135.8, 131.4, 130.5, 129.2, 128.0, 121.5,
117.8, 56.2, 52.4, 39.6; m/z MS C17H18BrN2O5S [MH]+ 441.3.
246.4/12.7 Hz), 141.6, 137.8, 137.3 (dd, JCF = 6.1/3.9 Hz), 136.1, 129.2,
128.9, 128.1, 126.9, 123.5 (dd, JCF = 6.6/3.3 Hz), 118.0 (d, JCF = 17.0 Hz),
117.8, 115.8 (d, JCF = 17.8 Hz), 56.5, 52.3, 39.64; m/z MS C23H21F2N2O5S
[MH]+ 474.8.
Methyl 2-(3',5'-difluoro-[1,1'-biphenyl]-4-yl)-2-(4-(methylsulfonamido)
benzamido)acetate (5i). (3,5-Difluorophenyl)boronic acid (51.5 mg, 0.326
mmol) was coupled to compound 5d (120 mg, 0.271 mmol) according to
General Procedure C. The reaction mixture was heated at 100 °C for 1.5
h to complete. The desired product was obtained as a yellow solid (69.0
mg, 53%). 1H NMR (DMSO-d6) δ 10.14 (s, 1H), 9.14 (d, J = 7.2 Hz, 1H),
7.92 (d, J = 8.8 Hz, 2H), 7.77 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.3 Hz, 2H),
7.47 (dd, J = 9.0/2.2 Hz, 2H), 7.32–7.18 (m, 3H), 5.74 (d, J = 7.1 Hz, 1H),
3.68 (s, 3H), 3.07 (s, 3H); 19F NMR (DMSO-d6) δ -109.4; 13C NMR (DMSO-
d6) δ 171.0, 165.8, 162.9 (dd, JCF = 245.6/13.7 Hz), 143.3 (dd, JCF = 9.8/9.8
Hz), 141.6, 137.4 (dd, JCF = 2.4/2.4 Hz), 136.9, 129.2, 128.9, 128.1, 127.1,
117.8, 111.2–109.1 (m), 102.9 (dd, JCF = 26.0/26.0 Hz), 56.5, 52.4, 39.6;
m/z MS C23H21F2N2O5S [MH]+ 474.8.
Methyl 2-([1,1'-biphenyl]-4-yl)-2-(4-(methylsulfonamido)benzamido)
acetate (5e). Phenylboronic acid (49.7 mg, 0.408 mmol) was coupled to
compound 5d (150 mg, 0.340 mmol) according to General Procedure C.
The reaction mixture was heated at 120 °C for 4 h to complete. The desired
product was obtained as a white solid (58.5 mg, 39%). 1H NMR (DMSO-
d6) δ 10.13 (s, 1H), 9.11 (d, J = 7.0 Hz, 1H), 7.92 (d, J = 8.6 Hz, 2H), 7.73–
7.62 (m, 4H), 7.56 (d, J = 8.2 Hz, 2H), 7.52–7.43 (m, 2H), 7.42–7.34 (m,
1H), 7.25 (d, J = 8.6 Hz, 2H), 5.71 (d, J = 7.0 Hz, 1H), 3.68 (s, 3H), 3.07
(s, 3H); 13C NMR (DMSO-d6) δ 171.1, 165.8, 141.5, 140.1, 139.7, 135.4,
129.2, 129.0, 128.9, 128.1, 127.6, 126.9, 126.7, 117.8, 56.6, 52.3, 39.6;
m/z MS C23H23N2O5S [MH]+ 438.8.
Methyl 2-(4-(furan-3-yl)phenyl)-2-(4-(methylsulfonamido)benzamido)
acetate (5j). Furan-3-ylboronic acid (36.5 mg, 0.326 mmol) was coupled
to compound 5d (120 mg, 0.271 mmol) according to General Procedure C.
The reaction mixture was heated at 100 °C for 1 h to complete. The desired
product was obtained as a pale yellow solid (96 mg, 82%). 1H NMR
(DMSO-d6) δ 10.13 (s, 1H), 9.06 (d, J = 7.0 Hz, 1H), 8.26–8.08 (m, 1H),
7.91 (d, J = 8.8 Hz, 2H), 7.78–7.68 (m, 1H), 7.63 (d, J = 8.3 Hz, 2H), 7.48
(d, J = 8.3 Hz, 2H), 7.30–7.20 (m, 2H), 6.97 (dd, J = 1.9/0.8 Hz, 1H), 5.65
(d, J = 7.0 Hz, 1H), 3.66 (s, 3H), 3.06 (s, 3H); 13C NMR (DMSO-d6) δ 170.9,
165.5, 141.5, 134.8, 131.9, 129.2, 128.7, 128.1, 125.6, 125.4, 117.8, 108.6,
52.3, 39.6 *CH signals of furan around 144.0 and 139.0 ppm were very
small to be detected but these signals were easily found in the
corresponding hydroxamic acid analogue 6j.; m/z MS C21H21N2O6S [MH]+
428.8.
Methyl 2-(3'-fluoro-[1,1'-biphenyl]-4-yl)-2-(4-(methylsulfonamido)
benzamido)acetate (5f). (3-Fluorophenyl)boronic acid (57.1 mg, 0.406
mmol) was coupled to compound 5d (150 mg, 0.340 mmol) according to
General Procedure C. The reaction mixture was heated at 100 °C for 1.5
h to complete. The desired product was obtained as a light pink solid (114
mg, 74%). 1H NMR (DMSO-d6) δ 10.14 (s, 1H), 9.13 (d, J = 7.1 Hz, 1H),
8.00–7.84 (m, 2H), 7.78–7.70 (m, 2H), 7.58 (d, J = 8.3 Hz, 2H), 7.56–7.48
(m, 3H), 7.29–7.24 (m, 2H), 7.23–7.18 (m, 1H), 5.73 (d, J = 7.1 Hz, 1H),
3.68 (s, 3H), 3.07 (s, 3H); 19F NMR (DMSO-d6) δ -112.7; 13C NMR (DMSO-
d6) δ 171.0, 165.8, 162.7 (d, JCF = 243.3 Hz), 142.1 (d, JCF = 7.8 Hz), 141.6,
138.6 (d, JCF = 2.2 Hz), 136.1, 130.9 (d, JCF = 8.6 Hz), 129.2, 128.9, 128.1,
127.0, 122.8, 117.8, 114.3 (d, JCF = 21.0 Hz), 113.4 (d, JCF = 22.1 Hz),
56.5, 52.3, 39.6; m/z MS C23H22FN2O5S [MH]+ 456.7.
Methyl 2-(4-(methylsulfonamido)benzamido)-2-(4-(thiophen-3-yl)
phenyl)acetate (5k). Thiophen-3-ylboronic acid (41.8 mg, 0.326 mmol)
was coupled to compound 5d (120 mg, 0.271 mmol) according to General
Procedure C. The reaction mixture was heated at 100 °C for 1 h to
complete. The crude obtained (116 mg, 95%) was used for the subsequent
reaction without further purification. 1H NMR (DMSO-d6) δ 10.13 (s, 1H),
9.08 (d, J = 7.1 Hz, 1H), 7.94–7.90 (m, 2H), 7.90–7.85 (m, 1H), 7.76–7.71
(m, 2H), 7.67–7.64 (m, 1H), 7.57–7.54 (m, 2H), 7.52–7.49 (m, 1H), 7.25
Methyl 2-(4'-fluoro-[1,1'-biphenyl]-4-yl)-2-(4-(methylsulfonamido)
benzamido)acetate (5g). A nitrogen flushed 5 mL microwave vial was
charged with compound 5d (150 mg, 0.340 mmol), (4-fluorophenyl)boronic
acid (57.1 mg, 0.408 mmol), 1 M Na2CO3 (1 mL) and degassed DMF (3
mL). The mixture was bubble through N2 while stirring for 5 min before
adding Pd(PPh3)2Cl2 (11.9 mg, 0.017 mmol). The mixture was heated at
100 °C for 15 min. After cooling, the mixture was diluted with a 1 M HCl
(10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic layer
was washed with water (2 × 20 mL) and brine (20 mL) and then dried over
anhydrous Na2SO4 before concentration in vacuo. The crude product was
purified by column chromatography using DCM/MeOH (95:5 to 90:10) to
afford the desired product (118 mg, 77%) as a pale yellow solid. 1H NMR
(DMSO-d6) δ 10.14 (s, 1H), 9.12 (d, J = 7.1 Hz, 1H), 8.00–7.87 (m, 2H),
7.75–7.69 (m, 2H), 7.68–7.64 (m, 2H), 7.56 (d, J = 8.3 Hz, 2H), 7.36–7.21
(m, 4H), 5.72 (d, J = 7.0 Hz, 1H), 3.68 (s, 3H), 3.07 (s, 3H); 19F NMR
(d, J = 8.8 Hz, 2H), 5.67 (d, J = 7.0 Hz, 1H), 3.67 (s, 3H), 3.06 (s, 3H); 13
C
NMR (DMSO-d6) δ 169.5, 158.2, 141.5, 140.9, 135.1, 134.9, 129.2, 128.9,
128.8, 128.7, 126.2, 126.1, 121.3, 117.8, 56.6, 52.3, 39.6; m/z MS
C21H19N2O5S2 [M-H]- 442.8.
Methyl 2-(4-(methylsulfonamido)benzamido)-2-(4-(pyridin-3-yl)
phenyl)acetate (5l). Pyridin-3-ylboronic acid (40.4 mg, 0.326 mmol) was
coupled to compound 5d (120 mg, 0.272 mmol) according to General
Procedure C. Only 70% of conversion to the desired product was observed
based on LC-MS after heating at 100 °C for 20 h. After cooling, the solvent
was removed by concentrating in vacuo. 1 M HCl was added dropwise to
the residue to adjust pH to 8–9 and extracted with EtOAc (3 × 10 mL). The
combined organic layers were washed with water (2 × 20 mL) and brine
(20 mL) and then dried over anhydrous Na2SO4 before concentration in
vacuo. The crude was dissolved in a minimum amount of DMF and added
into ice-water mixture dropwise. The precipitate was filtered and washed
with minimal MeOH to afford the desired product (40.0 mg, 33%) as an off-
white solid. 1H NMR (DMSO-d6) δ 10.14 (s, 1H), 9.13 (d, J = 7.1 Hz, 1H),
8.96–8.86 (m, 1H), 8.64–8.53 (m, 1H), 8.14–8.04 (m, 1H), 7.92 (d, J = 8.8
Hz, 2H), 7.79–7.73 (m, 2H), 7.64–7.58 (m, 3H), 7.25 (d, J = 8.8 Hz, 2H),
5.74 (d, J = 7.1 Hz, 1H), 3.68 (s, 3H), 3.07 (s, 3H); 13C NMR (DMSO-d6) δ
(DMSO-d6) δ -115.2; 13C NMR (DMSO-d6) δ 171.1, 165.9, 162.0 (d, JCF
=
244.5 Hz), 141.6, 139.1, 136.2 (d, JCF = 3.0 Hz), 135.4, 129.2, 128.9, 128.7
(d, JCF = 8.2 Hz), 128.2, 126.8, 117.8, 115.8 (d, JCF = 21.4 Hz), 56.6, 52.3,
39.6; m/z MS C23H22FN2O5S [MH]+ 456.8.
Methyl 2-(3',4'-difluoro-[1,1'-biphenyl]-4-yl)-2-(4-(methylsulfonamido)
benzamido)acetate (5h). (3,4-Difluorophenyl)boronic acid (51.5 mg,
0.326 mmol) was coupled to compound 5d (120 mg, 0.271 mmol)
according to General Procedure C. The reaction mixture was heated at
100 °C for 1.5 h to complete. The crude was dissolved in minimum amount
of DMF and added into ice-water mixture dropwise. The desired product
was obtained as a light beige solid (95.0 mg, 74%). 1H NMR (DMSO-d6) δ
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000527
ChemMedChem
FULL PAPER
171.0, 165.8, 148.7, 147.7, 141.6, 137.0, 136.3, 135.1, 134.2, 129.3, 129.1,
128.1, 127.1, 124.0, 117.8, 56.5, 52.3, 39.6; m/z MS C22H22N3O5S [MH]+
439.8.
1.67–1.47 (m, 2H); 13C NMR (DMSO-d6) δ 171.1, 165.8, 141.8, 141.5,
135.6, 134.4, 129.2, 128.2, 128.2, 124.8, 124.7, 117.8, 56.6, 52.2, 39.6,
26.7, 25.3, 22.5, 21.6; m/z MS C23H27N2O5S [MH]+ 443.0.
Methyl 2-(4-(methylsulfonamido)benzamido)-2-(4-(pyridin-4-yl)
phenyl)acetate (5m). Pyridin-4-ylboronic acid (40.1 mg, 0.326 mmol) was
coupled to compound 5d (120 mg, 0.271 mmol) according to General
Procedure C. The reaction mixture was heated at 100 °C for 1.5 h to
complete. The desired product was obtained as a yellow solid (67.4 mg,
56%). 1H NMR (DMSO-d6) δ 10.14 (s, 1H), 9.15 (d, J = 7.1 Hz, 1H), 8.65
(d, J = 6.1 Hz, 2H), 7.92 (d, J = 8.8 Hz, 2H), 7.86–7.81 (m, 2H), 7.75–7.69
(m, 2H), 7.65–7.59 (m, 2H), 7.25 (d, J = 8.8 Hz, 2H), 5.75 (d, J = 7.1 Hz,
1H), 3.68 (s, 3H), 3.07 (s, 3H); 13C NMR (DMSO-d6) δ 170.9, 165.8, 150.3,
147.2, 141.4, 137.4, 137.0, 129.2, 129.1, 128.2, 127.0, 121.2, 117.8, 56.5,
52.4, 39.6; m/z MS C22H22N3O5S [MH]+ 439.8.
Methyl 2-(4-cyclopentylphenyl)-2-(4-(methylsulfonamido)benzamido)
acetate (5r). A 50 mL round bottom flask was charged with compound 5p
(174 mg, 406 µmol), ammonium formate (256 mg, 4.06 mmol) and EtOH
(10 mL). The mixture was stirred and purged with N2 thoroughly before
adding 10% Pd/C (20 mg), which was refluxed for 2 h. Upon completion,
the reaction mixture was cooled to room temperature and filtered through
Celite. The filtrate was concentrated in vacuo and extracted with EtOAc (3
× 10 mL) from water (10 mL). The combined organic layers were dried over
anhydrous Na2SO4 and concentrated in vacuo. The crude was obtained
(144 mg, 83%) as a white solid and was used for subsequent reaction
without further purification. 1H NMR (DMSO-d6) δ 10.12 (s, 1H), 8.99 (d, J
= 6.9 Hz, 1H), 7.94–7.87 (m, 2H), 7.37 (d, J = 8.2 Hz, 2H), 7.31–7.20 (m,
4H), 5.58 (d, J = 6.9 Hz, 1H), 3.64 (s, 3H), 3.06 (s, 3H), 3.00–2.90 (m, 1H),
2.06–1.89 (m, 2H), 1.82–1.71 (m, 2H), 1.71–1.59 (m, 2H), 1.59–1.45 (m,
2H); 13C NMR (DMSO-d6) δ 171.2, 165.8, 146.0, 141.5, 133.5, 129.2,
128.2, 128.1, 127.2, 117.8, 56.7, 52.2, 45.1, 39.6, 34.2, 25.0; m/z MS
C22H27N2O5S [MH]+ 431.0.
Methyl 2-(4-(methylsulfonamido)benzamido)-2-(4-(pyrimidin-5-yl)
phenyl)acetate (5n). Pyrimidin-5-ylboronic acid (40.4 mg, 0.326 mmol)
was coupled to compound 5d (120 mg, 0.272 mmol) according to General
Procedure C. The reaction mixture was heated at 100 °C for 6 h to
complete. After cooling, the solvent was removed by concentrating in
vacuo. 1 M HCl was added dropwise to the concentrated residue and
sonicated for 2 min. The precipitate formed was filtered and washed with
minimal Et2O to afford the desired product (70 mg, 58%) as a white solid.
1H NMR (DMSO-d6) δ 10.14 (s, 1H), 9.20 (s, 1H), 9.18–9.14 (m, 3H), 7.91
(d, J = 8.8 Hz, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.3 Hz, 2H), 7.25
Methyl 2-(4-cyclohexylphenyl)-2-(4-(methylsulfonamido)benzamido)
acetate (5s). A 50 mL round bottom flask was charged with compound 5q
(263 mg, 594 µmol), ammonium formate (375 mg, 5.94 mmol) and MeOH
(10 mL). The mixture was stirred and purged with N2 thoroughly before
adding 10% Pd/C (20 mg), which was refluxed for overnight. Upon
completion, the reaction mixture turned turbid and DMF was added until
the mixture becomes a homogeneous black solution to dissolve the solid
formed. Pd was filtered through Celite and the filtrate was concentrated in
vacuo and extracted with EtOAc (3 × 20 mL) from water (30 mL). The
combined organic layers were further washed with water (3 × 50 mL) and
brine (50 mL), dried over anhydrous Na2SO4 and concentrated in vacuo to
afford the desired product (210 mg, 80%) as a beige solid. 1H NMR
(DMSO-d6) δ 10.10 (s, 1H), 9.00 (d, J = 6.9 Hz, 1H), 7.97–7.76 (m, 2H),
7.36 (d, J = 8.2 Hz, 2H), 7.28–7.21 (m, 4H), 5.58 (d, J = 6.9 Hz, 1H), 3.64
(s, 3H), 3.06 (s, 3H), 1.87–1.49 (m, 6H), 1.49–1.09 (m, 5H); 13C NMR
(DMSO-d6) δ 171.2, 165.8, 147.6, 141.5, 133.5, 129.2, 128.2, 128.1, 126.9,
117.8, 56.7, 52.2, 43.5, 39.6, 33.9, 33.8, 26.3, 25.6; m/z MS C23H29N2O5S
[MH]+ 445.0.
(d, J = 8.8 Hz, 2H), 5.76 (d, J = 7.2 Hz, 1H), 3.68 (s, 3H), 3.07 (s, 3H); 13
C
NMR (DMSO-d6) δ 170.9, 165.8, 154.8, 141.6, 137.2, 133.6, 132.8, , 129.2,
128.1, 127.1, 126.9, 117.8, 56.4, 52.4, 39.6; m/z MS C21H21N4O5S [MH]+
440.8.
Methyl 2-(4-cyclopropylphenyl)-2-(4-(methylsulfonamido)
benzamido)acetate (5o). Cyclopropylboronic acid (21.4 mg, 0.245 mmol)
was coupled to compound 5d (100 mg, 0.227 mmol) according to General
Procedure D. The crude obtained (85.9 mg, 94%) was used for the
subsequent reaction without further purification. 1H NMR (DMSO-d6) δ
10.13 (s, 1H), 8.98 (d, J = 7.0 Hz, 1H), 7.89 (d, J = 8.7 Hz, 2H), 7.33 (d, J
= 8.2 Hz, 2H), 7.24 (d, J = 8.7 Hz, 2H), 7.09 (d, J = 8.2 Hz, 2H), 5.57 (d, J
= 6.9 Hz, 1H), 3.64 (s, 3H), 3.06 (s, 3H), 1.95–1.79 (m, 1H), 1.06–0.83 (m,
2H), 0.77–0.53 (m, 2H); 13C NMR (DMSO-d6) δ 171.2, 165.8, 143.9, 141.5,
133.0, 129.2, 128.2, 125.5, 117.8, 56.6, 52.2, 39.6, 14.8, 9.5, 9.4 *one
quaternary carbon around 128.0 was extremely small to be detected.
However, this signal was easily observed in the corresponding hydroxamic
acid compound 6o; m/z MS C20H23N2O5S [MH]+ 402.8.
N-(2-(Hydroxyamino)-2-oxo-1-phenylethyl)-4-(methylsulfonamido)
benzamide (6a). Compound 5a (120 mg, 0.331 mmol) was converted to
the corresponding hydroxamic acid according to General Procedure G.
The crude was purified by column chromatography using DCM/MeOH
(100:0 to 90:10) to afford the desired product (27 mg, 23%) as a white solid.
1H NMR (DMSO-d6) δ 11.02 (s, 1H), 10.12 (s, 1H), 9.02 (s, 1H), 8.75 (d, J
= 8.2 Hz, 1H), 7.91 (d, J = 8.7 Hz, 2H), 7.55–7.46 (m, 2H), 7.41–7.32 (m,
2H), 7.32–7.27 (m, 1H), 7.27–7.20 (m, 2H), 5.60 (d, J = 8.1 Hz, 1H), 3.06
(s, 3H); 13C NMR (DMSO-d6) δ 166.7, 165.5, 141.4, 138.5, 129.2, 128.5,
128.3, 127.6, 127.4, 117.8, 54.6, 39.6; m/z HRMS (TOF ES+) C16H18N3O5S
[MH]+ calcd 364.0962, found 364.0950; HPLC 98%.
Methyl 2-(4-(cyclopent-1-en-1-yl)phenyl)-2-(4-(methylsulfonamido)
benzamido)acetate (5p). 1-Cyclopentenylboronic acid (162 mg, 1.45
mmol) was coupled to compound 5d (400 mg, 906 µmol) according to
General Procedure D. The crude product was triturated with Et2O and the
filtered solid was washed with minimum amount of 1:2 MeOH/Et2O to
obtain the desired product (337 mg, 87%) as a dark brown solid. 1H NMR
(DMSO-d6) δ 10.13 (s, 1H), 9.04 (d, J = 7.1 Hz, 1H), 7.93–7.85 (m, 2H),
7.47 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.29–7.17 (m, 2H), 6.35–
6.18 (m, 1H), 5.63 (d, J = 7.0 Hz, 1H), 3.65 (s, 3H), 3.06 (s, 3H), 2.73–2.58
(m, 2H), 2.49–2.44 (m, 2H), 2.04–1.84 (m, 2H); 13C NMR (DMSO-d6) δ
171.1, 165.8, 141.5 (2C), 136.1, 134.7, 129.2, 128.3, 128.2, 126.6, 125.6,
117.8, 56.6, 52.2, 39.6, 32.9, 32.7, 22.7; m/z MS C22H25N2O5S [MH]+ 429.0.
N-(1-(4-Fluorophenyl)-2-(hydroxyamino)-2-oxoethyl)-4-
(methylsulfonamido)benzamide (6b). Compound 5b (100 mg, 0.263
mmol) was converted to the corresponding hydroxamic acid according to
General Procedure G. The crude was purified by column chromatography
using DCM/MeOH (100:0 to 90:10) to afford the desired product (78 mg,
78%) as a white solid. 1H NMR (DMSO-d6) δ 11.03 (s, 1H), 10.12 (s, 1H),
9.04 (s, 1H), 8.79 (d, J = 8.1 Hz, 1H), 7.91 (d, J = 8.7 Hz, 2H), 7.58–7.50
(m, 2H), 7.25 (d, J = 8.7 Hz, 2H), 7.23–7.15 (m, 2H), 5.60 (d, J = 8.1 Hz,
1H), 3.06 (s, 3H); 19F NMR (DMSO-d6) δ -114.9; 13C NMR (DMSO-d6) δ
166.7, 165.6, 161.7 (d, JCF = 243.7 Hz), 141.4, 134.8 (d, JCF = 3.0 Hz),
129.5 (d, JCF = 8.3 Hz), 129.3, 128.5, 117.8, 115.1 (d, JCF = 21.4 Hz), 53.9,
39.5; m/z HRMS (TOF ES+) C16H17FN3O5S [MH]+ calcd 382.0867, found
382.0861; HPLC 98%.
Methyl 2-(4-(methylsulfonamido)benzamido)-2-(2',3',4',5'-tetrahydro-
[1,1'-biphenyl]-4-yl)acetate (5q). 1-Cyclohexenylboronic acid (185 mg,
1.46 mmol) was coupled to compound 5d (404 mg, 915 µmol) according
to General Procedure D. The crude product was purified by column
chromatography using PE/EtOAc (70:30 to 0:100) to afford the desired
product (315 mg, 78%) as a brown solid. 1H NMR (DMSO-d6) δ 10.13 (s,
1H), 9.03 (d, J = 7.0 Hz, 1H), 8.03–7.79 (m, 2H), 7.40 (app. s, 4H), 7.29–
7.19 (m, 2H), 6.23–6.09 (m, 1H), 5.62 (d, J = 7.0 Hz, 1H), 3.65 (s, 3H),
3.06 (s, 3H), 2.41–2.27 (m, 2H), 2.26–2.05 (m, 2H), 1.82–1.68 (m, 2H),
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000527
ChemMedChem
FULL PAPER
N-(1-(4-Chlorophenyl)-2-(hydroxyamino)-2-oxoethyl)-4-
mg, 0.179 mmol) was converted to the corresponding hydroxamic acid
according to General Procedure G. The crude was purified by column
chromatography using DCM/MeOH (100:0 to 90:10) to afford the desired
product (38 mg, 45%) as a white solid. 1H NMR (DMSO-d6) δ 11.06 (s, 1H),
10.12 (s, 1H), 9.05 (s, 1H), 8.82 (d, J = 8.1 Hz, 1H), 7.96–7.87 (m, 2H),
7.82–7.73 (m, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.55–
(methylsulfonamido)benzamide (6c). Compound 5c (152 mg, 0.383
mmol) was converted to the corresponding hydroxamic acid according to
General Procedure G. The crude was purified by column chromatography
using DCM/MeOH (100:0 to 90:10) to afford the desired product (80 mg,
53%) as a white solid. 1H NMR (DMSO-d6) δ 11.05 (s, 1H), 10.13 (s, 1H),
9.06 (s, 1H), 8.82 (d, J = 8.1 Hz, 1H), 7.91 (d, J = 8.8 Hz, 2H), 7.52 (d, J =
8.5 Hz, 2H), 7.46–7.38 (m, 2H), 7.24 (d, J = 8.8 Hz, 2H), 5.60 (d, J = 8.1
Hz, 1H), 3.06 (s, 3H); 13C NMR (DMSO-d6) δ 166.4, 165.6, 141.5, 137.5,
132.3, 129.3, 129.2, 128.4, 128.3, 117.8, 54.0, 40.0; m/z HRMS (TOF ES+)
C16H17ClN3O5S [MH]+ calcd 398.0572, found 398.0569; HPLC 98%.
7.45 (m, 2H), 7.31–7.19 (m, 2H), 5.65 (d, J = 8.1 Hz, 1H), 3.07 (s, 3H); 19
F
NMR (DMSO-d6) δ -138.2 (d, J = 22.5 Hz), -140.8 (d, J = 22.5 Hz); 13C
NMR (DMSO-d6) δ 166.6, 165.7, 149.8 (dd, JCF = 245.2/12.8 Hz), 149.2
(dd, JCF = 246.2/12.6 Hz), 141.5, 138.4, 137.5 (dd, JCF = 6.1/3.8 Hz), 137.4,
129.3, 128.5, 128.1, 126.8, 123.5 (dd, JCF = 6.4/3.3 Hz), 118.0 (d, JCF
=
17.0 Hz), 117.8, 115.8 (d, JCF = 17.7 Hz), 54.3, 39.7; m/z HRMS (TOF ES+)
C22H20F2N3O5S [MH]+ calcd 476.1086, found 476.1078; HPLC 95%.
N-(1-(4-Bromophenyl)-2-(hydroxyamino)-2-oxoethyl)-4-
(methylsulfonamido)benzamide (6d). Compound 5d (600 mg, 1.36
mmol) was converted to the corresponding hydroxamic acid according to
General Procedure G. The crude was purified by column chromatography
using DCM/MeOH (100:0 to 90:10) to afford the desired product (109 mg,
18%) as a white solid. 1H NMR (DMSO-d6) δ 11.04 (app. d, J = 1.0 Hz,
1H), 10.12 (s, 1H), 9.05 (app. d, J = 1.3 Hz, 1H), 8.81 (d, J = 8.1 Hz, 1H),
8.01–7.85 (m, 2H), 7.62–7.54 (m, 2H), 7.50–7.42 (m, 2H), 7.26–7.19 (m,
2H), 5.57 (d, J = 8.1 Hz, 1H), 3.06 (s, 3H); 13C NMR (DMSO-d6) δ 166.3,
165.6, 141.4, 137.9, 131.2, 129.6, 129.2, 128.4, 120.8, 117.7, 54.0, 39.6;
m/z HRMS (TOF ES+) C16H17BrN3O5S [MH]+ calcd 442.0067, found
442.0073; HPLC 95%.
N-(1-(3',5'-Difluoro-[1,1'-biphenyl]-4-yl)-2-(hydroxyamino)-2-
oxoethyl)-4-(methylsulfonamido)benzamide (6i). Compound 5i (59.0
mg, 0.124 mmol) was converted to the corresponding hydroxamic acid
according to General Procedure G. The crude was purified by column
chromatography using DCM/MeOH/AcOH (100:0:1 to 89:9:1) and
preparative HPLC to afford the desired product (13.3 mg, 23%) as a white
solid. 1H NMR (DMSO-d6) δ 11.06 (s, 1H), 10.13 (s, 1H), 9.05 (s, 1H), 8.83
(d, J = 8.1 Hz, 1H), 7.92 (d, J = 8.7 Hz, 2H), 7.74 (d, J = 8.3 Hz, 2H), 7.60
(d, J = 8.3 Hz, 2H), 7.45 (dd, J = 8.9/2.0 Hz, 2H), 7.34–7.03 (m, 3H), 5.65
(d, J = 8.1 Hz, 1H), 3.07 (s, 3H); 19F NMR (DMSO-d6) δ -109.4; 13C NMR
(DMSO-d6) δ 166.5, 165.6, 162.9 (dd, JCF = 245.5/13.7 Hz), 143.4 (d, JCF
= 9.8 Hz), 141.4, 139.1, 136.9, 129.2, 128.4, 128.0, 126.8, 117.8, 110.1–
109.6 (m), 102.6 (d, JCF = 26.2 Hz), 54.23, 39.6; m/z HRMS (TOF ES+)
C22H20F2N3O5S [MH]+ calcd 476.1086, found 476.1085; HPLC 98%
N-(1-([1,1'-Biphenyl]-4-yl)-2-(hydroxyamino)-2-oxoethyl)-4-
(methylsulfonamido)benzamide (6e). Compound 5e (126 mg, 0.287
mmol) was converted to the corresponding hydroxamic acid according to
General Procedure G. The crude was purified preparative HPLC to afford
the desired product (31 mg, 14%) as a white solid. 1H NMR (DMSO-d6) δ
11.04 (s, 1H), 10.12 (s, 1H), 9.03 (s, 1H), 8.79 (d, J = 8.1 Hz, 1H), 7.92 (d,
J = 8.7 Hz, 2H), 7.71–7.63 (m, 4H), 7.58 (d, J = 8.3 Hz, 2H), 7.54–7.42 (m,
2H), 7.39–7.33 (m, 1H), 7.24 (d, J = 8.8 Hz, 2H), 5.64 (d, J = 8.1 Hz, 1H),
3.06 (s, 3H); 13C NMR (DMSO-d6) δ 166.6, 165.5, 141.4, 139.8, 139.6,
137.7, 129.2, 128.9, 128.5, 128.0, 127.5, 126.7, 126.6, 117.8, 54.3, 39.6;
m/z HRMS (TOF ES+) C22H22N3O5S [MH]+ calcd 440.1275, found
440.1276; HPLC 95%.
N-(1-(4-(Furan-3-yl)phenyl)-2-(hydroxyamino)-2-oxoethyl)-4-
(methylsulfonamido)benzamide (6j). Compound 5j (96.0 mg, 0.224
mmol) was converted to the corresponding hydroxamic acid according to
General Procedure G. The crude was purified by column chromatography
using DCM/MeOH (100:0 to 90:10) to afford the desired product (59.5 mg,
62%) as a white solid. 1H NMR (DMSO-d6) δ 11.03 (s, 1H), 10.13 (s, 1H),
8.77 (d, J = 8.1 Hz, 1H), 8.26–8.13 (m, 1H), 7.92 (d, J = 8.7 Hz, 2H), 7.77–
7.70 (m, 1H), 7.60 (d, J = 8.3 Hz, 2H), 7.51 (d, J = 8.3 Hz, 2H), 7.25 (d, J
= 8.8 Hz, 2H), 6.96 (dd, J = 1.8/0.7 Hz, 1H), 5.60 (d, J = 8.1 Hz, 1H), 3.07
(s, 3H) *one exchangeable proton of hydroxamic acid was not observed;
13C NMR (DMSO-d6) δ 166.7, 165.6, 144.3, 141.4, 139.4, 137.1, 131.4,
129.2, 128.6, 127.9, 125.5, 125.4, 117.8, 108.8, 54.4, 39.7; m/z HRMS
(TOF ES+) C20H20N3O6S [MH]+ calcd 430.1067, found 430.1072; HPLC
98 %.
N-(1-(3'-Fluoro-[1,1'-biphenyl]-4-yl)-2-(hydroxyamino)-2-oxoethyl)-4-
(methylsulfonamido)benzamide (6f). Compound 5f (114 mg, 0.250
mmol) was converted to the corresponding hydroxamic acid according to
General Procedure G. The crude was purified by column chromatography
using DCM/MeOH/AcOH (99:0:1 to 84:15:1) and preparative HPLC to
afford the desired product (4.2 mg, 4%) as a white solid. 1H NMR (DMSO-
d6) δ 11.05 (s, 1H), 8.81 (d, J = 8.1 Hz, 1H), 7.92 (d, J = 8.8 Hz, 2H), 7.70
(d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.3 Hz, 2H), 7.56–7.42 (m, 3H), 7.24 (d, J
= 8.7 Hz, 2H), 7.23–7.10 (m, 1H), 5.64 (d, J = 8.1 Hz, 1H), 3.06 (s, 3H)
*exchangeable protons of hydroxamic acid and sulfonamide were not
N-(2-(Hydroxyamino)-2-oxo-1-(4-(thiophen-3-yl)phenyl)ethyl)-4-
(methylsulfonamido)benzamide (6k). Compound 5k (116.0 mg, 0.260
mmol) was converted to the corresponding hydroxamic acid according to
General Procedure G. The crude was purified by column chromatography
using DCM/MeOH (100:0 to 90:10) to afford the desired product (64.0 mg,
55%) as a white solid. 1H NMR (DMSO-d6) δ 11.05 (s, 1H), 10.13 (s, 1H),
9.05 (s, 1H), 8.79 (d, J = 8.1 Hz, 1H), 7.93 (d, J = 8.7 Hz, 2H), 7.89–7.85
(m, 1H), 7.71 (d, J = 8.3 Hz, 2H), 7.66–7.61 (m, 1H), 7.60–7.49 (m, 3H),
7.26 (d, J = 8.7 Hz, 2H), 5.62 (d, J = 8.1 Hz, 1H), 3.07 (s, 3H); 13C NMR
(DMSO-d6) δ 166.7, 165.6, 141.4, 141.1, 137.3, 134.6, 129.2, 128.5, 127.9,
127.1, 126.2, 126.0, 121.0, 117.8, 54.4, 39.7; m/z HRMS (TOF ES+)
C20H20N3O5S2 [MH]+ calcd 446.0839, found 446.0837; HPLC 95%
observed; 19F NMR (DMSO-d6)
δ
-112.8; m/z HRMS (TOF ES+)
C22H21FN3O5S [MH]+ calcd 458.1180, found 458.1180; HPLC 95%.
N-(1-(4'-Fluoro-[1,1'-biphenyl]-4-yl)-2-(hydroxyamino)-2-oxoethyl)-4-
(methylsulfonamido)benzamide (6g). Compound 5g was converted to
the corresponding hydroxamic acid according to General Procedure G.
The crude was purified by column chromatography using
DCM/MeOH/AcOH (99:0:1 to 89:9:1) to afford the desired product (60 mg,
52%) as a white solid. 1H NMR (DMSO-d6) δ 11.06 (s, 1H), 10.15 (s, 1H),
9.06 (s, 1H), 8.81 (d, J = 8.1 Hz, 1H), 7.93 (d, J = 8.8 Hz, 2H), 7.75–7.67
(m, 2H), 7.64 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.33–7.20 (m,
4H), 5.65 (d, J = 8.1 Hz, 1H), 3.07 (s, 3H); 19F NMR (DMSO-d6) δ -115.4;
13C NMR (DMSO-d6) δ 166.7, 165.6, 161.9 (d, JCF = 244.3 Hz), 141.5,
138.6, 137.7, 136.3 (d, JCF = 3.1 Hz), 129.2, 128.7 (d, JCF = 8.2 Hz), 128.5,
128.0, 126.6, 117.8, 115.8 (d, JCF = 21.3 Hz), 54.3, 39.7; m/z HRMS (TOF
ES+) C22H21FN3O5S [MH]+ calcd 458.1180, found 458.1185; HPLC 98%.
N-(2-(Hydroxyamino)-2-oxo-1-(4-(pyridin-3-yl)phenyl)ethyl)-4-
(methylsulfonamido)benzamide TFA salt (6l). Compound 5l (40.0 mg,
0.910 mmol) was converted to the corresponding hydroxamic acid
according to General Procedure G. The crude was purified by preparative
HPLC to afford the desired product (12.5 mg, 31%) as a white solid. 1H
NMR (DMSO-d6) δ 11.09 (s, 1H), 10.14 (s, 1H), 9.05 (br. s, 1H), 8.86 (d, J
= 8.1 Hz, 1H), 8.72 (br. s, 1H), 8.40 (br. d, J = 8.2 Hz, 1H), 7.92 (d, J = 8.7
Hz, 2H), 7.82–7.72 (m, 3H), 7.66 (d, J = 8.3 Hz, 2H), 7.25 (d, J = 8.8 Hz,
2H), 5.67 (d, J = 8.1 Hz, 1H), 3.07 (s, 3H); 19F NMR (DMSO-d6) δ -74.5;
13C NMR (DMSO-d6) δ 166.3, 165.5, 145.3, 144.6, 141.3, 139.2, 137.6,
136.4, 135.0, 129.1, 128.3, 128.1, 126.9, 125.1, 117.7, 54.2, 39.5 *one
N-(1-(3',4'-Difluoro-[1,1'-biphenyl]-4-yl)-2-(hydroxyamino)-2-
oxoethyl)-4-(methylsulfonamido)benzamide (6h). Compound 5h (85.0
10
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000527
ChemMedChem
FULL PAPER
exchangeable proton was not observed. Due to the extensive proton
exchange between the pyridinium and water present in DMSO the protons
of pyridinium were broadened. The carbon signals of pyridinium were
substantially small compared to the other aromatic CH signals; m/z HRMS
(TOF ES+) C21H21N4O5S [MH]+ calcd 441.1227, found 441.1233; HPLC
95%
N-(2-(Hydroxyamino)-2-oxo-1-(2',3',4',5'-tetrahydro-[1,1'-biphenyl]-4-
yl)ethyl)-4-(methylsulfonamido)benzamide (6q). Compound 5q (120
mg, 271 µmol) was converted to the corresponding hydroxamic acid
according to General Procedure G. The crude was purified by preparative
HPLC to afford the desired product (58 mg, 48%) as an apricot solid. 1
H
NMR (DMSO-d6) δ 10.99 (s, 1H), 10.11 (s, 1H), 9.01 (s, 1H), 8.71 (d, J =
8.1 Hz, 1H), 7.91 (d, J = 8.7 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.37 (d, J =
8.4 Hz, 2H), 7.24 (d, J = 8.7 Hz, 2H), 6.13 (m, 1H), 5.57 (d, J = 8.1 Hz, 1H),
3.06 (s, 3H), 2.40–2.23 (m, 2H), 2.26–2.04 (m, 2H), 1.79–1.66 (m, 2H),
1.66–1.48 (m, 2H); 13C NMR (DMSO-d6) δ 166.8, 165.5, 141.4, 141.3,
136.8, 135.7, 129.2, 128.5, 127.3, 124.6, 124.4, 117.8, 54.3, 39.6, 26.7,
25.3, 22.6, 21.7; m/z HRMS (TOF ES+) C22H26N3O5S [MH]+ calcd
444.1588, found 444.1600; 95%.
N-(2-(Hydroxyamino)-2-oxo-1-(4-(pyridin-4-yl)phenyl)ethyl)-4-
(methylsulfonamido)benzamide TFA salt (6m). Compound 5m (62.0
mg, 0.141 mmol) was converted to the corresponding hydroxamic acid
according to General Procedure G. The crude was purified by preparative
HPLC to afford the desired product (22.4 mg, 36%) as a white solid. 1H
NMR (DMSO-d6) δ 11.11 (s, 1H), 10.14 (s, 1H), 8.90 (d, J = 8.1 Hz, 1H),
8.83 (d, J = 6.3 Hz, 2H), 8.12 (d, J = 6.5 Hz, 2H), 8.04–7.86 (m, 4H), 7.70
(d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.8 Hz, 2H), 5.70 (d, J = 8.1 Hz, 1H), 3.07
(s, 3H) *one exchangeable proton was not observed. Due to extensive
proton exchange between the pyridinium and water present in DMSO, the
salt signal was observed as an extremely broadened signal at 4.64 ppm;
19F NMR (DMSO-d6) δ -74.3; 13C NMR (DMSO-d6) δ 166.4, 165.6, 149.8,
147.1, 141.4, 139.8, 136.4, 129.2, 128.4, 128.2, 126.8, 121.4, 117.8, 54.3,
39.6; m/z HRMS (TOF ES+) C21H21N4O5S [MH]+ calcd 441.1227, found
441.1223; HPLC 95 %
N-(1-(4-Cyclopentylphenyl)-2-(hydroxyamino)-2-oxoethyl)-4-
(methylsulfonamido)benzamide (6r). Compound 5r (134 mg, 311 µmol)
was converted to the corresponding hydroxamic acid according to General
Procedure G. The crude was purified by preparative HPLC to afford the
desired product (53 mg, 40%) as a white solid. 1H NMR (DMSO-d6) δ 10.96
(s, 1H), 10.11 (s, 1H), 8.97 (s, 1H), 8.67 (d, J = 8.1 Hz, 1H), 7.90 (d, J =
8.7 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.38–7.07 (m, 4H), 5.53 (d, J = 8.0
Hz, 1H), 3.06 (s, 3H), 2.99–2.86 (m, 1H), 2.07–1.92 (m, 2H), 1.83–1.70 (m,
2H), 1.69–1.57 (m, 2H), 1.58–1.42 (m, 2H); 13C NMR (DMSO-d6) δ 166.9,
165.5, 145.4, 141.4, 135.9, 129.2, 128.6, 127.3, 126.8, 117.8, 54.4, 45.0,
39.7, 34.3, 25.0; m/z HRMS (TOF ES+) C21H26N3O5S [MH]+ calcd
432.1588, found 432.1588; HPLC 98%.
N-(2-(Hydroxyamino)-2-oxo-1-(4-(pyrimidin-5-yl)phenyl)ethyl)-4-
(methylsulfonamido)benzamide TFA salt (6n). Compound 5n (87.0 mg,
0.198 mmol) was converted to the corresponding hydroxamic acid
according to General Procedure G. The crude was purified by preparative
HPLC to afford the desired product (12 mg, 14%) as a white solid. 1H NMR
(DMSO-d6) δ 11.07 (s, 1H), 10.13 (s, 1H), 9.19 (s, 1H), 9.14 (s, 2H), 8.85
(d, J = 8.1 Hz, 1H), 7.92 (d, J = 8.7 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.66
(d, J = 8.3 Hz, 2H), 7.25 (d, J = 8.7 Hz, 2H), 5.66 (d, J = 8.1 Hz, 1H), 3.07
(s, 3H) *one exchangeable proton of hydroxamic acid was not observed.
Due to extensive proton exchange between the pyridinium and water
present in DMSO, the salt signal was observed as an extremely broadened
signal at 5.20 ppm; 19F NMR (DMSO-d6) δ -74.5; 13C NMR (DMSO-d6) δ
166.5, 165.6, 157.3, 154.7, 141.4, 139.4, 133.1, 133.0, 129.2, 128.5, 128.3,
126.9, 117.8, 54.4, 39.6; m/z HRMS (TOF ES+) C20H20N5O5S [MH]+ calcd
442.1180, found 442.1166; HPLC 95%
N-(1-(4-Cyclohexylphenyl)-2-(hydroxyamino)-2-oxoethyl)-4-
(methylsulfonamido)benzamide (6s). Compound 5s (200 mg, 450 µmol)
was converted to the corresponding hydroxamic acid according to General
Procedure G. The crude product was purified by preparative HPLC to
afford the desired product (73 mg, 36%) as a light pink solid. 1H NMR
(DMSO-d6) δ 10.96 (s, 1H), 10.11 (s, 1H), 8.98 (s, 1H), 8.67 (d, J = 8.0 Hz,
1H), 7.90 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.28–7.14 (m, 4H),
5.54 (d, J = 8.0 Hz, 1H), 3.06 (s, 3H), 2.49–2.41 (m, 1H), 1.84–1.62 (m,
5H), 1.51–1.13 (m, 5H); 13C NMR (DMSO-d6) δ 166.9, 165.5, 147.0, 141.4,
135.9, 129.2, 128.5, 127.4, 126.5, 117.8, 54.4, 43.5, 39.6, 33.9, 26.4, 25.6;
m/z HRMS (TOF ES+) C22H28N3O5S [MH]+ calcd 446.1744, found
446.1744; HPLC 97%.
N-(1-(4-Cyclopropylphenyl)-2-(hydroxyamino)-2-oxoethyl)-4-
(methylsulfonamido)benzamide (6o). Compound 5o (86.0 mg, 0.214
mmol) was converted to the corresponding hydroxamic acid according to
General Procedure G. The crude was purified by column chromatography
using DCM/MeOH (100:0 to 90:10) and preparative HPLC to afford the
desired product (31.8 mg, 37%) as a sticky solid. 1H NMR (DMSO-d6) δ
10.94 (s, 1H), 10.10 (s, 1H), 8.97 (s, 1H), 8.66 (d, J = 8.1 Hz, 1H), 7.89 (d,
J = 8.7 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 8.6 Hz, 2H), 7.05 (d,
J = 8.2 Hz, 2H), 5.53 (d, J = 8.1 Hz, 1H), 3.06 (s, 3H), 2.03–1.69 (m, 1H),
1.10–0.86 (m, 2H), 0.76–0.57 (m, 2H); 13C NMR (DMSO-d6) δ 166.9, 165.4,
143.2, 141.5, 135.4, 129.2, 128.4, 127.3, 125.2, 117.8, 54.3, 39.7, 14.8,
9.4, 9.3; m/z HRMS (TOF ES+) C19H22N3O5S [MH]+ calcd 404.1275, found
404.1275; HPLC 95%
Methyl 2-(4-bromophenyl)-2-((tert-butoxycarbonyl)amino)acetate (7).
In a round bottom flask was added compound 4d (3.10 g, 12.7 mmol), sat.
NaHCO3 (30 mL) and water (30 mL). The mixture was stirred at room
temperature for 30 min after adding Boc2O (2.77 g, 12.7 mmol). Additional
Boc2O (1.39 g, 6.35 mmol) and THF (90 mL) were added. The reaction
mixture was stirred at room temperature for overnight. Upon completion,
THF was removed under reduced pressure. 1 M HCl was added to the
residue until pH 3 and the mixture was extracted with EtOAc (3 × 50 mL).
The combined organic layers were dried over anhydrous Na2SO4 and
concentrated in vacuo. The crude was purified by column chromatography
using PE/EtOAc (100:0 to 40:60) to afford the desired product (3.28 g,
75%) as a pale-yellow foam. 1H NMR (DMSO-d6) δ 7.84 (d, J = 8.1 Hz,
1H), 7.55 (d, J = 8.4 Hz, 2H), 7.43–7.12 (m, 2H), 5.23 (d, J = 8.1 Hz, 1H),
3.61 (s, 3H), 1.38 (s, 9H); 13C NMR (DMSO-d6) δ 171.0, 155.1, 136.1,
131.4, 130.1, 121.3, 78.7, 56.9, 52.3, 28.1.
N-(1-(4-(Cyclopent-1-en-1-yl)phenyl)-2-(hydroxyamino)-2-oxoethyl)-
4-(methylsulfonamido)benzamide (6p). Compound 5p (150 mg, 350
µmol) was converted to the corresponding hydroxamic acid according to
General Procedure G. The crude was purified by a preparative HPLC to
afford the desired product (30 mg, 20%) as a light apricot solid. 1H NMR
(DMSO-d6) δ 11.00 (s, 1H), 10.11 (s, 1H), 9.01 (s, 1H), 8.73 (d, J = 8.1 Hz,
1H), 7.90 (d, J = 8.7 Hz, 2H), 7.44 (app. s, 4H), 7.24 (d, J = 8.7 Hz, 2H),
6.32–6.21 (m, 1H), 5.57 (d, J = 8.1 Hz, 1H), 3.06 (s, 3H), 2.71–2.59 (m,
2H), 2.50–2.43 (m, 2H), 2.03–1.87 (m, 2H); 13C NMR (DMSO-d6) δ 166.7,
165.5, 141.6, 141.4, 137.1, 135.5, 129.2, 128.5, 127.4, 126.1, 125.4, 117.8,
54.4, 39.6, 32.9, 32.7, 22.7; m/z HRMS (TOF ES+) C21H24N3O5S [MH]+
calcd 430.1431, found 430.1436; HPLC 98%.
Methyl 2-((tert-butoxycarbonyl)amino)-2-(4-(diethylamino)phenyl)
acetate (8a). To a dried 20 mL microwave vessel was added 7 (250 mg,
726 µmol), Pd2(dba)3 (20.0 mg, 21.8 µmol), XPhos (34.6 mg, 72.6 µmol),
Cs2CO3 (592 mg, 1.82 mmol), diethylamine (376 µL, 3.63 mmol), and a
degassed anhydrous toluene (10 mL). The mixture was heated at reflux
for overnight. Upon completion, the mixture was diluted with water (6 mL)
and extracted with EtOAc (3 × 20 mL). The combined organic layers were
washed with brine (50 mL), dried over anhydrous Na2SO4 and
concentrated in vacuo. The crude was purified by column chromatography
using PE/EtOAc (100:0 to 70:30) to afford the desired product (204 mg,
83%) as a dark brown solid. 1H NMR (DMSO-d6) δ 7.51 (d, J = 7.7 Hz, 1H),
7.13 (d, J = 8.8 Hz, 2H), 6.58 (d, J = 8.8 Hz, 2H), 5.00 (d, J = 7.8 Hz, 1H),
11
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000527
ChemMedChem
FULL PAPER
3.58 (s, 3H), 3.34–3.12 (m, 4H), 1.38 (s, 9H), 1.06 (t, J = 7.0 Hz, 6H); 13
NMR (DMSO-d6) δ 172.1, 155.2, 147.1, 128.8, 122.3, 111.1, 78.3, 57.1,
C
= 8.8 Hz, 2H), 6.86 (d, J = 8.8 Hz, 2H), 5.04 (d, J = 7.9 Hz, 1H), 3.74–3.62
(m, 2H), 3.59 (s, 3H), 2.63 (td, J = 12.3/2.3 Hz, 2H), 1.75–1.58 (m, 2H),
1.57–1.44 (m, 1H), 1.38 (s, 9H), 1.25–1.15 (m, 2H), 0.92 (d, J = 6.5 Hz,
3H); 13C NMR (DMSO-d6) δ 171.9, 155.2, 150.9, 128.5, 125.7, 115.3, 78.4,
57.0, 52.0, 48.5, 33.4, 30.2, 28.2, 21.8; m/z MS C20H31N2O4 [MH]+ 363.1.
51.9, 43.6, 28.2, 12.4.
Methyl 2-(4-(diethylamino)phenyl)-2-(4-(methylsulfonamido)
benzamido)acetate (9a). Compound 8a (292 mg, 868 µmol) was
converted to the desired compound according to General Procedure F.
The crude was purified by column chromatography PE/EtOAc (100:0 to
40:60) to afford the desired product (264 mg, 70%) as an apricot solid. 1H
NMR (DMSO-d6) δ 10.12 (s, 1H), 8.82 (d, J = 6.6 Hz, 1H), 7.97–7.82 (m,
2H), 7.30–7.13 (m, 4H), 6.64 (d, J = 8.9 Hz, 2H), 5.41 (d, J = 6.6 Hz, 1H),
3.62 (s, 3H), 3.33 (q, J = 7.1 Hz, 4H), 3.06 (s, 3H), 1.08 (t, J = 7.0 Hz, 6H);
13C NMR (DMSO-d6) δ 171.7, 165.8, 147.3, 141.4, 129.3, 129.2, 128.3,
121.7, 117.7, 111.2, 56.6, 51.9, 43.6, 39.6, 12.4; m/z MS C21H28N3O5S
[MH]+ 434.0.
Methyl 2-(4-(4-methylpiperidin-1-yl)phenyl)-2-(4-(methylsulfonamido)
benzamido)acetate (9d). Compound 8d (137 mg, 378 µmol) was
converted to the titled compound according to General Procedure F. The
crude was purified by column chromatography using PE/EtOAc (70:30 to
20:80) to afford the desired product (99 mg, 62%) as a beige solid. 1H NMR
(CDCl3) δ 7.93 (s, 1H), 7.74 (d, J = 8.7 Hz, 2H), 7.29 (d, J = 8.7 Hz, 2H),
7.23 (d, J = 8.7 Hz, 2H), 7.12 (d, J = 6.8 Hz, 1H), 6.94 (d, J = 8.4 Hz, 2H),
5.64 (d, J = 6.8 Hz, 1H), 3.73 (s, 3H), 3.66–3.51 (m, 2H), 2.97 (s, 3H),
2.76–2.64 (m, 2H), 1.71 (app. d, J = 11.4 Hz, 2H), 1.60–1.45 (m, 1H), 1.44–
1.28 (m, 2H), 0.95 (d, J = 6.4 Hz, 3H); 13C NMR (CDCl3) δ 172.0, 166.2,
150.7, 140.9, 129.4, 129.0, 128.5, 119.1, 117.0 (br.), 60.6, 56.6, 53.0, 50.3,
39.7, 33.6, 30.5, 21.8; m/z MS C23H30N3O5S [MH]+ 460.0.
Methyl 2-((tert-butoxycarbonyl)amino)-2-(4-(piperidin-1-yl)phenyl)
acetate (8b). Compound 7 (363 mg, 1.05 mmol) was coupled to piperidine
(260 µL, 2.64 mmol) according to General Procedure E. The crude was
purified by column chromatography using PE/EtOAc (100:0 to 70:30) to
afford the desired product (186 mg, 50%) as a brown oil. 1H NMR (CDCl3)
δ 7.18 (d, J = 8.7 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 5.46 (d, J = 6.8 Hz, 1H),
5.19 (d, J = 7.3 Hz, 1H), 3.67 (s, 3H), 3.17–3.04 (m, 4H), 1.83–1.59 (m,
4H), 1.61–1.48 (m, 2H), 1.40 (s, 9H); 13C NMR (CDCl3) δ 172.0, 154.9,
151.7, 128.0, 127.0, 116.5, 79.9, 57.1, 52.5, 50.4, 28.3, 25.6, 24.2; m/z
MS C19H29N2O4 [MH]+ 349.1.
Methyl 2-((tert-butoxycarbonyl)amino)-2-(4-(4-fluoropiperidin-1-yl)
phenyl)acetate (8e). Compound 7 (250 mg, 726 µmol) was coupled to 4-
fluoropiperidine hydrochloride (253 mg, 1.82 mmol) according to General
Procedure E. The crude was purified by column chromatography using
PE/EtOAc (100:0 to 60:40) to afford the desired product (264 mg, 99%) as
a sticky yellow solid. 1H NMR (DMSO-d6) δ 7.61 (d, J = 7.9 Hz, 1H), 7.20
(d, J = 8.7 Hz, 2H), 6.98–6.82 (m, 2H), 5.06 (d, J = 7.9 Hz, 1H), 4.95–4.65
(m, 1H), 3.59 (s, 3H), 3.45–3.31 (m, 2H), 3.21–3.05 (m, 2H), 2.07–1.87 (m,
2H), 1.82–1.68 (m, 2H), 1.38 (s, 9H); 19F NMR (DMSO-d6) δ -177.2; 13C
NMR (DMSO-d6) δ 171.8, 155.2, 150.2, 128.6, 126.2, 115.5, 88.5 (d, JCF
= 169.4 Hz), 78.4, 57.0, 52.0, 44.8 (d, JCF = 6.8 Hz), 30.5 (d, J = 19.0 Hz),
28.2; m/z MS C19H28FN2O4 [MH]+ 367.0.
Methyl 2-(4-(methylsulfonamido)benzamido)-2-(4-(piperidin-1-yl)
phenyl)acetate (9b). Compound 8b (186 mg, 534 µmol) was converted to
the titled product according to General Procedure F. The crude was
purified by column chromatography using PE/EtOAc (70:30 to 10:90) to
afford the desired product (149 mg, 58%) as a brown solid. 1H NMR
(DMSO-d6) δ 10.12 (s, 1H), 8.90 (d, J = 6.7 Hz, 1H), 8.06–7.78 (m, 2H),
7.44–7.14 (m, 4H), 6.92 (br. d, J = 6.8 Hz, 2H), 5.48 (d, J = 6.7 Hz, 1H),
3.63 (s, 3H), 3.22–3.10 (m, 4H), 3.06 (s, 3H), 1.67–1.43 (m, 6H); 13C NMR
(DMSO-d6) δ 171.5, 165.8, 151.4, 141.4, 129.2, 129.0, 128.3 (2C), 117.8,
115.8 (br.), 56.5, 52.0, 49.3, 39.6, 25.1, 23.9; m/z MS C22H28N3O5S [MH]+
446.0.
Methyl 2-(4-(4-fluoropiperidin-1-yl)phenyl)-2-(4-(methylsulfonamido)
benzamido)acetate (9e). Compound 8e (226 mg, 617 µmol) was
converted to the titled compound according to General Procedure F. The
crude was taken up into 5 mL of MeOH and filtered to afford the desired
product (132 mg, 46%) as a light beige solid. 1H NMR (DMSO-d6) δ 10.06
(s, 1H), 8.91 (d, J = 6.8 Hz, 1H), 8.01–7.78 (m, 2H), 7.29 (d, J = 8.8 Hz,
2H), 7.26–7.18 (m, 2H), 6.96 (d, J = 8.8 Hz, 2H), 5.49 (d, J = 6.8 Hz, 1H),
5.01–4.65 (m, 1H), 3.63 (s, 3H), 3.44–3.33 (m, 2H), 3.21–3.09 (m, 2H),
3.06 (s, 3H), 2.04–1.86 (m, 2H), 1.86–1.69 (m, 2H); 19F NMR (DMSO-d6)
δ -177.2; 13C NMR (DMSO-d6) δ 171.5, 165.8, 150.4, 141.5, 129.2, 129.0,
128.2, 125.7, 117.7, 115.6, 88.5 (d, JCF = 169.4 Hz), 56.5, 52.1, 44.8 (d,
JCF = 6.9 Hz), 39.6, 30.5 (d, JCF = 19.0 Hz); m/z MS C22H27FN3O5S [MH]+
464.0.
Methyl 2-((tert-butoxycarbonyl)amino)-2-(4-morpholinophenyl)
acetate (8c). Compound 7 (250 mg, 726 µmol) was coupled to morpholine
(158 mg, 1.82 mmol) according to General Procedure E. The crude was
purified by column chromatography using PE/EtOAc (100:0 to 50:50) to
afford the desired product (233 mg, 91%) as a yellow oil. 1H NMR (DMSO-
d6) δ 7.62 (d, J = 7.9 Hz, 1H), 7.22 (d, J = 8.7 Hz, 2H), 6.89 (d, J = 8.8 Hz,
2H), 5.07 (d, J = 7.9 Hz, 1H), 3.80–3.64 (m, 4H), 3.59 (s, 3H), 3.19–2.97
(m, 4H), 1.38 (s, 9H); 13C NMR (DMSO-d6) δ 171.8, 155.2, 150.8, 128.5,
126.7, 114.8, 78.4, 66.1, 57.0, 52.0, 48.2, 28.2; m/z MS C18H27N2O5 [MH]+
351.0.
Methyl 2-((tert-butoxycarbonyl)amino)-2-(4-(4,4-difluoropiperidin-1-
yl)phenyl)acetate (8f). Compound 7 (270 mg, 784 µmol) was coupled to
4,4-difluoropiperidine hydrochloride (309 mg, 1.96 mmol) according to
General Procedure E. The crude was purified by column chromatography
using PE/EtOAc (100:0 to 70:30) to afford the desired product (245 mg,
81%) as an orange oil. 1H NMR (DMSO-d6) δ 7.63 (d, J = 7.9 Hz, 1H), 7.22
(d, J = 8.7 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 5.08 (d, J = 7.9 Hz, 1H), 3.59
(s, 3H), 3.39–3.28 (m, 4H), 2.02 (ddd, J = 19.8/14.1/6.0 Hz, 4H), 1.38 (s,
9H); 19F NMR (DMSO-d6) δ -95.2; 13C NMR (DMSO-d6) δ 171.8, 155.2,
149.3, 128.7, 126.8, 122.8 (t, JCF = 240.7/240.7 Hz), 115.8, 78.4, 57.0,
52.0, 45.5 (t, JCF = 5.0/5.0 Hz), 32.8 (t, JCF = 22.4/22.4 Hz), 28.2; m/z MS
C19H27F2N2O4 [MH]+ 385.0.
Methyl 2-(4-(methylsulfonamido)benzamido)-2-(4-morpholinophenyl)
acetate (9c). Compound 8c (218 mg, 622 µmol) was converted to the titled
compound according to General Procedure F. The crude was triturated
with MeOH (10 mL) and filtered to afford the desired product (114 mg,
41%) as a white solid. 1H NMR (DMSO-d6) δ 10.12 (s, 1H), 8.91 (d, J = 6.8
Hz, 1H), 7.89 (d, J = 8.8 Hz, 2H), 7.31 (d, J = 8.7 Hz, 2H), 7.23 (d, J = 8.7
Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 5.50 (d, J = 6.8 Hz, 1H), 3.78–3.69 (m,
4H), 3.63 (s, 3H), 3.16–3.08 (m, 4H), 3.06 (s, 3H); 13C NMR (DMSO-d6) δ
171.5, 165.8, 151.0, 141.4, 129.2., 129.0, 128.2, 126.3, 117.8, 114.9, 66.0,
56.5, 52.1, 48.3, 39.6; m/z MS C21H26N3O6S [MH]+ 447.9.
Methyl 2-(4-(4,4-difluoropiperidin-1-yl)phenyl)-2-(4-
(methylsulfonamido)benzamido)acetate (9f). Compound 8f (225 mg,
585 µmol) was converted to the titled compound according to General
Procedure F. The crude was purified by triturating with MeOH (10 mL) to
afford the desired product (123 mg, 44%) as an off-white solid. 1H NMR
(DMSO-d6) δ 10.12 (s, 1H), 8.92 (d, J = 6.9 Hz, 1H), 8.06–7.77 (m, 2H),
7.31 (d, J = 8.8 Hz, 2H), 7.26–7.19 (m, 2H), 7.01 (d, J = 8.8 Hz, 2H), 5.51
(d, J = 6.8 Hz, 1H), 3.63 (s, 3H), 3.40–3.33 (m, 4H), 3.06 (s, 3H), 2.03 (ddd,
Methyl 2-((tert-butoxycarbonyl)amino)-2-(4-(4-methylpiperidin-1-yl)
phenyl)acetate (8d). Compound 7 (200 mg, 582 µmol) was coupled to 4-
methylpiperidine (172 µL, 1.45 mmol) according to General Procedure E.
The crude was purified by column chromatography using PE/EtOAc (100:0
to 70:30) to afford the desired product (137 mg, 65%) as an orange
crystalline solid. 1H NMR (DMSO-d6) δ 7.59 (d, J = 7.9 Hz, 1H), 7.17 (d, J
12
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000527
ChemMedChem
FULL PAPER
J = 19.8/14.0/5.6 Hz, 4H); 19F NMR (DMSO-d6) δ -95.2; 13C NMR (DMSO-
d6) δ 171.5, 165.8, 149.5, 141.5, 129.2, 129.1, 128.2, 126.3, 122.8 (t, JCF
= 240.7/240.7 Hz), 117.8, 115.9, 56.4, 52.1, 45.5 (t, JCF = 5.2/5.2 Hz), 39.6,
32.8 (t, JCF = 22.4/22.4 Hz); m/z MS C22H26F2N3O5S [MH]+ 482.0.
3.15–3.09 (m, 4H), 1.38 (s, 9H); 13C NMR (DMSO-d6) δ 171.6, 154.4,
150.5, 136.8, 128.6, 128.4, 127.9, 127.6, 127.0, 115.6, 78.4, 66.3, 57.0,
52.0, 48.1, 43.2, 28.2 *one quaternary carbon at ~155.0 was difficult to
observe. However, this signal was easily detected from the corresponding
aryl sulfonamide analogue 5ab; m/z MS C26H34N3O6 [MH]+ 484.1.
Methyl 2-((tert-butoxycarbonyl)amino)-2-(4-(4-(trifluoromethyl)
piperidin-1-yl)phenyl)acetate (8g). Compound 7 (250 mg, 726 µmol) was
coupled to 4-(trifluoromethyl)piperidine hydrochloride (344 mg, 1.82 mmol)
according to General Procedure E. The crude was purified by column
chromatography using PE/EtOAc (100:0 to 70:30) to afford the desired
product (278 mg, 92%) as orange flakes. 1H NMR (DMSO-d6) δ 7.62 (d, J
= 7.8 Hz, 1H), 7.25–7.14 (m, 2H), 6.91 (d, J = 8.8 Hz, 2H), 5.06 (d, J = 7.9
Hz, 1H), 3.91–3.72 (m, 2H), 3.59 (s, 3H), 2.79–2.62 (m, 2H), 2.49–2.42 (m,
1H), 1.98–1.77 (m, 2H), 1.66–1.42 (m, 2H), 1.38 (s, 9H); 19F NMR (DMSO-
d6) δ -72.5; 13C NMR (DMSO-d6) δ 171.8, 155.2, 150.3, 129.2, 128.6,
126.4, 115.6, 78.4, 57.0, 52.0, 47.2 (d, JCF = 3.4 Hz), 38.9 (d, JCF = 26.5
Hz), 28.7, 23.7 *J coupling of CF3 signal was difficult to observe; m/z MS
C20H28F3N2O4 [MH]+ 417.0.
Benzyl 4-(4-(2-methoxy-1-(4-(methylsulfonamido)benzamido)-2-
oxoethyl)phenyl)piperazine-1-carboxylate (9i). Compound 8i (493 mg,
937 µmol) was converted to the titled compound according to General
Procedure F. The crude product was triturated with Et2O to afford the
desired product (271 mg, 50%) as an off-white solid. 1H NMR (DMSO-d6)
δ 10.12 (s, 1H), 8.92 (d, J = 6.8 Hz, 1H), 8.02–7.72 (m, 2H), 7.43–7.28 (m,
7H), 7.28–7.18 (m, 2H), 6.96 (d, J = 8.8 Hz, 2H), 5.51 (d, J = 6.8 Hz, 1H),
5.11 (s, 2H), 3.63 (s, 3H), 3.54 (br. s, 4H), 3.21–3.08 (m, 4H), 3.06 (s, 3H);
13C NMR (DMSO-d6) δ 171.4, 165.8, 154.4, 150.7, 141.4, 136.8, 129.2,
129.0, 128.4, 128.2, 127.9, 127.6, 126.6, 117.8, 115.8, 66.3, 56.4, 52.1,
48.1, 43.2, 39.6; m/z MS C29H33N4O7S [MH]+ 581.0.
Methyl 2-(4-(methylsulfonamido)benzamido)-2-(4-(piperazine-1-yl)
phenyl)acetate (9j). A 50 mL round bottom flask was charged with 9i (261
mg, 450 µmol), ammonium formate (283 mg, 4.49 mmol) and MeOH (10
mL). The mixture was stirred and purged with N2 thoroughly before adding
10 % Pd/C (20 mg), which was refluxed for 4 h. Upon completion, the
reaction mixture was cooled to room temperature and filtered through
Celite. The filtrate was concentrated in vacuo and extracted with EtOAc (3
× 10 mL) from water (10 mL). The combined organic layers were dried over
anhydrous Na2SO4 and concentrated in vacuo to give 14 mg of a clear oil.
LC-MS of the aqueous layer indicated the presence of the product which
was recovered by lyophilisation to afford the desired product (118 mg,
59%) as a white solid. 1H NMR (DMSO-d6) δ 8.91 (d, J = 6.9 Hz, 1H), 8.32
(s, 1H), 8.03–7.74 (m, 2H), 7.30 (d, J = 8.8 Hz, 2H), 7.26–7.16 (m, 2H),
6.95 (d, J = 8.8 Hz, 2H), 5.50 (d, J = 6.8 Hz, 1H), 3.63 (s, 3H), 3.28–3.14
(m, 4H), 3.05 (s, 3H), 3.02–2.90 (m, 4H) *NH of sulfonamide was not
observed; 13C NMR (DMSO-d6) δ 171.5, 165.8, 150.9, 141.7, 129.2, 129.0,
128.1, 126.4, 117.8, 115.4, 56.4, 52.1, 47.6, 44.1, 39.6; m/z MS
C21H27N4O5S [MH]+ 447.0.
Methyl 2-(4-(methylsulfonamido)benzamido)-2-(4-(4-(trifluoromethyl)
piperidin-1-yl)phenyl)acetate (9g). Compound 8g (258 mg, 620 µmol)
was converted to the titled compound according to General Procedure F.
The crude was triturated with MeOH to afford the desired product (134 mg,
56%) as a light beige solid. 1H NMR (DMSO-d6) δ 8.91 (d, J = 6.8 Hz, 1H),
7.95–7.79 (m, 2H), 7.29 (d, J = 8.8 Hz, 2H), 7.26–7.18 (m, 2H), 6.96 (d, J
= 8.9 Hz, 2H), 5.50 (d, J = 6.8 Hz, 1H), 3.92–3.72 (m, 2H), 3.63 (s, 3H),
3.06 (s, 3H), 2.81–2.62 (m, 3H), 1.92–1.79 (m, 2H), 1.68–1.40 (m, 2H) *
NH of sulfonamide was not observed; 19F NMR (DMSO-d6) δ -72.5; 13C
NMR (DMSO-d6) δ 171.5, 165.8, 150.5, 141.5, 129.2, 129.1, 128.2, 125.9,
117.8, 115.7, 56.5, 52.1, 47.2, 47.1, 39.6, 38.9–38.1 (m), 23.6; m/z MS
C23H27F3N3O5S [MH]+ 513.9.
Methyl 2-((tert-butoxycarbonyl)amino)-2-(4-(4-methylpiperazin-1-yl)
phenyl)acetate (8h). Compound 7 (250 mg, 726 µmol) was coupled to 1-
methylpiperazine (201 µL, 1.82 mmol) according to General Procedure E.
Upon completion, the mixture was cooled to room temperature and
concentrated in vacuo. The residue was extracted with 2 M HCl (3 × 20
mL) from Et2O (15 mL). The aqueous layer was basified with sat. NaHCO3
to pH 8 and extracted with EtOAc (3 × 100 mL). The combined organic
layer was washed with brine (200 mL), dried over anhydrous Na2SO4 and
concentrated in vacuo to afford the desired product (195 mg, 73%) as a
yellow oil. 1H NMR (DMSO-d6) δ 7.61 (d, J = 7.9 Hz, 1H), 7.19 (d, J = 8.8
Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 5.06 (d, J = 7.8 Hz, 1H), 3.59 (s, 3H),
3.16–3.02 (m, 4H), 2.46–2.35 (m, 4H), 2.21 (s, 3H), 1.38 (s, 9H); 13C NMR
(DMSO-d6) δ 171.8, 155.2, 150.7, 128.5, 126.3, 115.0, 78.4, 57.0, 54.5,
52.0, 47.8, 45.7, 28.2; m/z MS C19H30N3O4 [MH]+ 364.1.
N-(1-(4-(Diethylamino)phenyl)-2-(hydroxyamino)-2-oxoethyl)-4-
(methylsulfonamido)benzamide TFA salt (10a). Compound 9a (150 mg,
346 µmol) was converted to the corresponding hydroxamic acid according
to General Procedure G. The crude was purified by column
chromatography using DCM/MeOH (100:0 to 90:10) followed by
preparative HPLC to afford the desired product (88 mg, 59%) as a white
solid. 1H NMR (DMSO-d6) δ 11.01 (s, 1H), 10.13 (s, 1H), 8.72 (d, J = 7.5
Hz, 1H), 7.90 (d, J = 8.7 Hz, 2H), 7.51 (br. d, J = 6.2 Hz, 2H), 7.31–6.71
(m, 4H), 5.56 (d, J = 7.9 Hz, 1H), 3.48 (br. d, J = 6.9 Hz, 4H), 3.06 (s, 3H),
1.02 (t, J = 7.1 Hz, 6H) *one exchangeable proton of hydroxamic acid was
not observed. Due to extensive proton exchange between the ammonium
and water present in DMSO, the CH2 signals were observed as a
broadened peak, and the salt signal was observed as a broadened singlet
around 3.80 ppm; 19F NMR (DMSO-d6) δ -74.5; 13C NMR (DMSO-d6) δ
166.7, 165.5, 141.4, 129.2, 128.9, 128.5, 117.8 (2C), 54.1, 39.6, 11.2
*Quaternary carbon atoms of the arylamine group and ethylene carbon
atom were difficult to observe; m/z HRMS (TOF ES+) C20H27N4O5S [M]+
calcd 435.1697, found 435.1704; HPLC 95%
Methyl 2-(4-(4-methylpiperazin-1-yl)phenyl)-2-(4-
(methylsulfonamido)benzamido)acetate (9h). Compound 8h (195 mg,
537 µmol) was converted to the titled compound according to General
Procedure F. The crude was obtained (131 mg, 83%) as a brown oil and
was used for subsequent reaction without further purification. 1H NMR
(DMSO-d6) δ 10.20 (s, 1H), 8.90 (d, J = 6.8 Hz, 1H), 7.97–7.79 (m, 2H),
7.29 (d, J = 8.8 Hz, 2H), 7.26–7.20 (m, 2H), 6.93 (d, J = 8.9 Hz, 2H), 5.49
(d, J = 6.8 Hz, 1H), 3.63 (s, 3H), 3.21–3.08 (m, 4H), 3.06 (s, 3H), 2.47–
2.33 (m, 4H), 2.22 (s, 3H); 13C NMR (DMSO-d6) δ 171.5, 165.8, 150.9,
141.5, 129.1, 129.0, 128.2, 125.8, 117.8, 115.1, 56.5, 54.5, 52.1, 47.9,
45.7, 39.6; m/z MS C22H29N4O5S [MH]+ 461.0.
N-(2-(Hydroxyamino)-2-oxo-1-(4-(piperidin-1-yl)phenyl)ethyl)-4-
(methylsulfonamido)benzamide (10b). Compound 9b (122 mg, 274
µmol) was converted to the corresponding hydroxamic acid according to
General Procedure G. The crude was purified by column chromatography
using DCM/MeOH (100:0 to 90:10) to afford the desired product (58 mg,
48%) as a light pink solid. 1H NMR (DMSO-d6) δ 10.90 (s, 1H), 10.10 (s,
1H), 8.95 (s, 1H), 8.58 (d, J = 8.1 Hz, 1H), 7.90 (d, J = 8.8 Hz, 2H), 7.31
(d, J = 8.8 Hz, 2H), 7.24 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 5.47
(d, J = 8.0 Hz, 1H), 3.16–3.08 (m, 4H), 3.06 (s, 3H), 1.68–1.46 (m, 6H);
13C NMR (DMSO-d6) δ 167.3, 165.4, 151.2, 141.3, 129.2, 128.7, 128.1,
128.0, 117.8, 115.5, 54.1, 49.6, 39.6, 25.1, 24.0; m/z HRMS (TOF ES+)
C21H27N4O5S [MH]+ calcd 447.1697, found 447.1706; HPLC 95%
Benzyl 4-(4-(1-((tert-butoxycarbonyl)amino)-2-methoxy-2-oxoethyl)
phenyl)piperazine-1-carboxylate (8i). Compound 7 (350 mg, 1.02 mmol)
was coupled to benzyl piperazine-1-carboxylate (560 mg, 2.54 mmol)
according to General Procedure E. The crude was purified by column
chromatography using PE/EtOAc (100:0 to 50:50) to afford the desired
product (463 mg, 94%) as a yellow oil. 1H NMR (DMSO-d6) δ 7.63 (d, J =
8.0 Hz, 1H), 7.41–7.29 (m, 5H), 7.22 (d, J = 8.8 Hz, 2H), 7.01–6.84 (m,
2H), 5.10 (s, 2H), 5.07 (d, J = 7.8 Hz, 1H), 3.59 (s, 3H), 3.53 (br. s, 4H),
13
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000527
ChemMedChem
FULL PAPER
N-(2-(Hydroxyamino)-1-(4-morpholinophenyl)-2-oxoethyl)-4-
(methylsulfonamido)benzamide (10c). Compound 9c (104 mg, 232
µmol) was converted to the corresponding hydroxamic acid according to
General Procedure G. The crude was purified by column chromatography
using DCM/MeOH (100:0 to 90:10) to afford the desired product (36 mg,
35%) as an off-white solid. 1H NMR (DMSO-d6) δ 10.92 (s, 1H), 10.17 (s,
1H), 8.96 (s, 1H), 8.60 (d, J = 8.1 Hz, 1H), 7.90 (d, J = 8.8 Hz, 2H), 7.35
(d, J = 8.8 Hz, 2H), 7.29–7.12 (m, 2H), 6.92 (d, J = 8.8 Hz, 2H), 5.48 (d, J
= 8.0 Hz, 1H), 3.87–3.51 (m, 4H), 3.10–2.91 (m, 7H); 13C NMR (DMSO-
d6) δ 167.2, 165.4, 150.7, 141.4, 129.2, 129.0, 128.7, 128.2, 117.8, 114.9,
66.1, 54.1, 48.6, 39.6; m/z HRMS (TOF ES+) C20H25N4O6S [MH]+ calcd
449.1489, found 449.1466; HPLC 99%
2H), 1.63–1.43 (m, 2H) *CH of trifluoromethylpiperidine was hidden under
DMSO solvent peak; 19F NMR (DMSO-d6) δ -72.5; 13C NMR (DMSO-d6) δ
167.2, 165.4, 150.2, 141.3, 129.1, 128.7, 128.6, 128.2, 126.4, 117.8, 115.7,
54.0, 47.5, 39.6, 38.8–38.1 (m), 23.6; m/z HRMS (TOF ES+)
C22H26F3N4O5S [MH]+ calcd 515.1571, found 515.1558; HPLC 95%.
N-(2-(Hydroxyamino)-1-(4-(4-methylpiperazin-1-yl)phenyl)-2-
oxoethyl)-4-(methylsulfonamido)benzamide
TFA
salt
(10h).
Compound 9h (169 mg, 367 µmol) was converted to the corresponding
hydroxamic acid according to General Procedure G. Upon completion, the
reaction mixture was concentrated in vacuo. The residue was purified by
preparative HPLC to afford the desired product (63 mg, 37%) as a yellow
fluffy solid. 1H NMR (DMSO-d6) δ 10.95 (s, 1H), 10.13 (s, 1H), 9.88 (s, 1H),
8.98 (s, 1H), 8.64 (d, J = 8.2 Hz, 1H), 7.88 (d, J = 8.7 Hz, 2H), 7.39 (d, J =
8.8 Hz, 2H), 7.23 (d, J = 8.8 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 5.50 (d, J =
8.1 Hz, 1H), 3.83 (br. s, 2H), 3.51 (br. s, 2H), 3.15 (br. s, 2H), 3.06 (s, 3H),
2.94 (br. s, 2H), 2.85 (s, 3H); 19F NMR (DMSO-d6) δ -73.6; 13C NMR
(DMSO-d6) δ 166.9, 165.3, 148.9, 141.3, 129.9, 129.0, 128.5, 128.2, 117.7,
115.6, 53.8, 52.1, 45.6, 42.0, 39.5; m/z HRMS (TOF ES+) C21H28N5O5S
[M]+ calcd 462.1806, found 462.1815; HPLC 95%.
N-(2-(Hydroxyamino)-1-(4-(4-methylpiperidin-1-yl)phenyl)-2-
oxoethyl)-4-(methylsulfonamido)benzamide (10d). Compound 9d (96
mg, 209 µmol) was converted to the corresponding hydroxamic acid
according to General Procedure G. The crude was purified by column
chromatography using DCM/MeOH (100:0 to 90:10) to afford the desired
product (60 mg, 63%) as a white solid. 1H NMR (DMSO-d6) δ 10.90 (s, 1H),
10.11 (s, 1H), 8.95 (s, 1H), 8.58 (d, J = 8.1 Hz, 1H), 8.01–7.80 (m, 2H),
7.31 (d, J = 8.8 Hz, 2H), 7.29–7.15 (m, 2H), 6.89 (d, J = 8.9 Hz, 2H), 5.47
(d, J = 8.0 Hz, 1H), 3.79–3.53 (m, 2H), 3.06 (s, 3H), 2.62 (td, J = 12.3/2.4
Hz, 2H), 1.84–1.58 (m, 2H), 1.48 (ddd, J = 11.1/9.0/5.4 Hz, 1H), 1.19 (m,
2H), 0.92 (d, J = 6.5 Hz, 3H); 13C NMR (DMSO-d6) δ 167.3, 165.4, 150.8,
141.3, 129.2, 128.7, 128.2, 128.0, 117.8, 115.3, 54.1, 48.9, 39.6, 33.4,
30.3, 21.8; m/z HRMS (TOF ES+) C22H29N4O5S [MH]+ calcd 461.1853,
found 461.1864; HPLC 95%.
N-(2-(Hydroxyamino)-2-oxo-1-(4-(piperazin-1-yl)phenyl)ethyl)-4-
(methylsulfonamido)benzamide TFA salt (10j). Compound 9j (108 mg,
242 µmol) was converted to the corresponding hydroxamic acid according
to General Procedure G. Upon completion, the reaction mixture was
concentrated in vacuo and purified by preparative HPLC to afford the
desired product (67 mg, 62%) as a yellow solid. 1H NMR (DMSO-d6) δ
10.96 (s, 1H), 10.13 (s, 1H), 8.93 (s, 2H), 8.64 (d, J = 8.0 Hz, 1H), 7.89 (d,
J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.5 Hz, 2H), 6.97 (d,
J = 8.5 Hz, 2H), 5.50 (d, J = 8.0 Hz, 1H), 3.33 (br. s, 4H), 3.23 (br. s, 4H),
3.06 (s, 3H) *one exchangeable proton of hydroxamic acid was not
observed; 19F NMR (DMSO-d6) δ -73.9; 13C NMR (DMSO-d6) δ 167.0,
165.4, 149.6, 141.4, 123.0, 129.2, 128.6, 128.3, 117.8, 115.8, 54.0, 45.7,
42.6, 39.6; m/z HRMS (TOF ES+) C20H26N5O5S [M]+ calcd 448.1649, found
448.1657; HPLC 95%.
N-(1-(4-(4-Fluoropiperidin-1-yl)phenyl)-2-(hydroxyamino)-2-
oxoethyl)-4-(methylsulfonamido)benzamide (10e). Compound 9e (122
mg, 263 µmol) was converted to the corresponding hydroxamic acid
according to General Procedure G. The crude was purified by column
chromatography using DCM/MeOH (100:0 to 90:10) to afford the desired
product (88 mg, 72%) as an off-white solid. 1H NMR (DMSO-d6) δ 10.92
(s, 1H), 10.12 (s, 1H), 8.96 (s, 1H), 8.60 (d, J = 8.1 Hz, 1H), 8.13–7.77 (m,
2H), 7.33 (d, J = 8.8 Hz, 2H), 7.29–7.13 (m, 2H), 6.94 (d, J = 8.8 Hz, 2H),
5.48 (d, J = 8.0 Hz, 1H), 5.10–4.60 (m, 1H), 3.49–3.26 (m, 2H), 3.17–3.08
(m, 2H), 3.06 (s, 3H), 2.13–1.81 (m, 2H), 1.86–1.60 (m, 2H); 19F NMR
(DMSO-d6) δ -177.1; 13C NMR (DMSO-d6) δ 167.2, 165.4, 150.1, 141.4,
129.2, 128.7, 128.5, 128.3, 117.8, 115.5, 88.6 (d, JCF = 169.3 Hz), 54.1,
45.1 (d, JCF = 6.8 Hz), 39.6, 30.5 (d, JCF = 18.9 Hz); m/z HRMS (TOF ES+)
C21H26FN4O5S [MH]+ calcd 465.1602, found 465.1592; HPLC 99%
Acknowledgements
The authors thank Prof. James Rini at the University of Toronto,
Canada, for the kind donation of the recombinant human APN
expressing cell line. The authors also thank Monash University,
Sir James McNeil Foundation, and the Cancer Therapeutics CRC
CTx for scholarship support to J.L.
N-(1-(4-(4,4-Difluoropiperidin-1-yl)phenyl)-2-(hydroxyamino)-2-
oxoethyl)-4-(methylsulfonamido)benzamide (10f). Compound 9f (108
mg, 224 µmol) was converted to the corresponding hydroxamic acid
according to General Procedure G. The crude was purified by column
chromatography using DCM/MeOH (100:0 to 90:10) to afford the desired
product (76.5 mg, 71%) as an off-white solid. 1H NMR (DMSO-d6) δ 10.93
(s, 1H), 10.08 (s, 1H), 8.98 (s, 1H), 8.63 (d, J = 8.1 Hz, 1H), 7.91 (d, J =
8.8 Hz, 2H), 7.36 (d, J = 8.8 Hz, 2H), 7.24 (d, J = 8.8 Hz, 2H), 6.98 (d, J =
8.8 Hz, 2H), 5.50 (d, J = 8.1 Hz, 1H), 3.36–3.28 (m, 4H), 3.06 (s, 3H), 2.02
(ddd, J = 19.8/14.0/5.5 Hz, 4H); 19F NMR (DMSO-d6) δ -95.1; 13C NMR
(DMSO-d6) δ 167.2, 165.4, 149.2, 141.4, 129.2, 129.0, 128.7, 128.3, 122.8
(t, JCF = 240.7/240.7 Hz), 117.8, 115.8, 54.1, 45.8 (t, JCF = 5.1/5.1 Hz),
39.6, 32.8 (t, JCF = 22.4/22.4 Hz); m/z HRMS (TOF ES+) C21H25F2N4O5S
[MH]+ calcd 483.1508, found 483.1517; HPLC 98%
Keywords: Aminopeptidase N • cancer • hydroxamic acids •
metallopeptidases • zinc binding group
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Entry for the Table of Contents
Hydroxamic acid bearing small molecules were synthesised as potent inhibitors of Aminopeptidase N (APN) for the treatment of cancer.
The 3,4,5-trifluorophenyl group of the lead compound 3 was replaced with various substituents to improve both potency and solubility.
The SAR results indicated that 3-fluorophenyl analogue 6f was the most potent APN inhibitor that showed inhibitory activity in the sub-
nanomolar range.
16
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