Hydroxypyridinethione Inhibitors of Human Insulin-Degrading Enzyme

Article abstract of DOI:10.1002/cmdc.202100111

Insulin-degrading enzyme (IDE) is a human mononuclear Zn²⁺-dependent metalloenzyme that is widely regarded as the primary peptidase responsible for insulin degradation. Despite its name, IDE is also critically involved in the hydrolysis of several other disparate peptide hormones, including glucagon, amylin, and the amyloid β-protein. As such, the study of IDE inhibition is highly relevant to deciphering the role of IDE in conditions such as type-2 diabetes mellitus and Alzheimer disease. There have been few reported IDE inhibitors, and of these, inhibitors that directly target the active-site Zn²⁺ ion have yet to be fully explored. In an effort to discover new, zinc-targeting inhibitors of IDE, a library of ～350 metal-binding pharmacophores was screened against IDE, resulting in the identification of 1-hydroxypyridine-2-thione (1,2-HOPTO) as an effective Zn²⁺-binding scaffold. Screening a focused library of HOPTO compounds identified 3-sulfonamide derivatives of 1,2-HOPTO as inhibitors of IDE (K_i values of ～50 μM). Further structure-activity relationship studies yielded several thiophene-sulfonamide HOPTO derivatives with good, broad-spectrum activity against IDE that have the potential to be useful pharmacological tools for future studies of IDE.

Full text of DOI:10.1002/cmdc.202100111

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Hydroxypyridinethione Inhibitors of Human Insulin-
Degrading Enzyme
+ [a]
+ [b]
[a]
[b]
Rebecca N. Adamek , Caitlin N. Suire , Ryjul W. Stokes, Monica K. Brizuela,
[a]
[b]
Seth M. Cohen,* and Malcolm A. Leissring*
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Insulin-degrading enzyme (IDE) is a human mononuclear Zn
targeting inhibitors of IDE, a library of ~350 metal-binding
pharmacophores was screened against IDE, resulting in the
identification of 1-hydroxypyridine-2-thione (1,2-HOPTO) as an
effective Zn -binding scaffold. Screening a focused library of
HOPTO compounds identified 3-sulfonamide derivatives of 1,2-
-dependent metalloenzyme that is widely regarded as the
primary peptidase responsible for insulin degradation. Despite
its name, IDE is also critically involved in the hydrolysis of
several other disparate peptide hormones, including glucagon,
amylin, and the amyloid β-protein. As such, the study of IDE
inhibition is highly relevant to deciphering the role of IDE in
conditions such as type-2 diabetes mellitus and Alzheimer
disease. There have been few reported IDE inhibitors, and of
2
+
HOPTO as inhibitors of IDE (K values of ~50 μM). Further
i
structure-activity relationship studies yielded several thiophene-
sulfonamide HOPTO derivatives with good, broad-spectrum
activity against IDE that have the potential to be useful
pharmacological tools for future studies of IDE.
2
+
these, inhibitors that directly target the active-site Zn ion
have yet to be fully explored. In an effort to discover new, zinc-
Introduction
Insulin-degrading enzyme (IDE, EC 3.4.24.56), also known as
insulysin and insulinase, is found across nearly all domains of
life, and within mammals, is widely considered to be the
[1]
primary protease responsible for insulin degradation. IDE
belongs to the inverzincin family of metalloproteases, which
2
+
use an active-site Zn metal ion to catalyze the hydrolysis of
[1]
peptide bonds. The protease is also inhibited by thiol-
modifying compounds, and was at one point miscategorized as
a cysteine-dependent protease, though it is now known that
the cysteine residues within IDE are not involved directly in the
Figure 1. a) The complete structure of IDE, with the NTD in blue and green,
[1]
proteolytic activity of the enzyme.
the CTD in yellow and red, and the connecting hinge in white. b) Image of
2+
2
+
the buried catalytic Zn ion and metal-coordinating residues within the
Human IDE is a ~113-kDa Zn -metalloendopeptidase (Fig-
ure 1) that is classified as cryptidase-type protease, as it uses a
clamshell-like mechanism to encapsulate substrate peptides
2
+
active site (highlighted in black box in (a)), with the Zn ion shown as an
orange sphere, water as a red sphere, and coordination bonds as yellow
dashes. Images produced from PDB ID: 4NXO.
3
[2]
within its abnormally large, 13000-Å internal chamber. IDE is
comprised of two bowl-shaped N- and C-terminal domains
(
“
NTD, CTD) linked by a hinge that can adopt “open” and
closed” conformations. Notably, the active site is bipartite,
[2]
[2,3]
conformation. Because substrates must fit completely within
comprised of regions within both the NTD and CTD, and is fully
formed only when the protease is in the closed
the internal chamber to be hydrolyzed, this constrains the size
of the substrates that can be processed, rendering IDE a true
[
4–6]
peptidase and also resulting in exquisite substrate selectivity.
2
+
The catalytic Zn is coordinated by His108, His112, and Glu188,
with an axially bound water (which is also H-bonded to Glu111)
that completes the tetrahedral coordination geometry of the
+
[a] R. N. Adamek, R. W. Stokes, Prof. S. M. Cohen
Department of Chemistry and Biochemistry
University of California, San Diego
La Jolla, CA 92093 (USA)
[2]
metal center (Figure 1b). To aid in substrate binding, IDE uses
an exosite ~30 Å away from the catalytic site to anchor the N-
terminal domain of various substrates and facilitate substrate
E-mail: scohen@ucsd.edu
+
[
[
b] C. N. Suire, M. K. Brizuela, Prof. M. A. Leissring
Institute for Memory Impairments and Neurological Disorders
University of California, Irvine
Irvine, CA 92697 (USA)
E-mail: m.leissring@uci.edu
[7–8]
unfolding.
Mechanistically, IDE is believed to operate by first
enveloping its peptide substrates, with hydrolysis occurring
only upon complete encapsulation of the substrate.
[7–8]
+
In the
] These authors contributed equally to this work.
case of insulin, IDE is able to perform multiple peptide
cleavages without impacting the disulfide bonds that hold the
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202100111
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[
7]
hormone peptide together. Following substrate cleavage, IDE
re-opens to release the hydrolyzed products, and the cycle
Considering the relevance of IDE to various disease states,
there has been great interest in developing substrate-selective
inhibitors of IDE, with a particular emphasis on the develop-
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[7–8]
[18]
begins anew.
Homologues of IDE are found across all domains of life, but
within humans, misregulation of this mononuclear Zn metal-
lopeptidase has been implicated in both type-2 diabetes
mellitus (T2DM) and Alzheimer disease (AD).
biological roles of IDE are not yet fully understood, this
metalloenzyme is expressed throughout the body, and is found
in both the intra- and extra-cellular compartments.
functions to hydrolyze a broad range of peptide hormones, yet
paradoxically only hydrolyzes certain members of these dispa-
ment of tool compounds to disentangle the different biological
roles of IDE in relation to its various substrates. Among the
more sought-after goals related to IDE are compounds that act
as activators in respect to Aβ degradation, as a means to
2
+
[9–10]
While the
[16–17]
decrease levels of this neurotoxic peptide.
In addition,
there is particular interest in the discovery of compounds that
inhibit insulin degradation, while retaining IDE activity against
other substrates. Such a strategy would theoretically provide a
[11]
IDE
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novel pharmacological approach to boosting insulin signaling
[2]
[9,19–20]
rate peptide families. Chief among the known IDE substrates
and thereby treating T2DM.
Selective inhibitors of either
are insulin, glucagon, amylin, and the amyloid β-protein (Aβ;
glucagon or amylin would be of considerable value as
pharmacological tools to aid in evaluating the biological roles
of IDE. However, few substrate selective inhibitors of IDE are
available. Of the reported IDE inhibitors, most are peptidic and
[12–13]
Figure 2).
Of these, three are relevant to T2DM, as amylin
[14]
promotes satiety, and insulin and glucagon work in tandem
to down- and up-regulate blood glucose, respectively. Addi-
tionally, Aβ accumulates abnormally in AD.
[15]
[16–17]
2+
bind at the exosite distal to the catalytic Zn (Figure 3). The
current best-in-class inhibitor is a small molecule that is both
potent and selective for IDE-mediated insulin hydrolysis (Fig-
[18]
ure 3c); however, this compound is difficult to synthesize and
has yet to be examined in cellular studies. Finally, there remains
a great experimental need for broad-spectrum inhibitors of IDE
that are inexpensive to synthesize at scale.
Metal-binding inhibitors of IDE have the potential to
provide alternative avenues of IDE inhibition, but these
inhibitors have not been thoroughly explored. Only two
hydroxamic acid peptide compounds and 1,10-phenanthroline
have been reported as metal-binding inhibitors of IDE (Fig-
[21–22]
ure 3).
Hydroxamic acids may display potential issues with
selectivity, pharmacokinetics, and metabolism and 1,10-phenan-
throline is a non-selective, metal-stripping agent, leaving ample
room for the development of more selective, potent, and
druglike inhibitors of IDE.
Herein, a library of metal-binding pharmacophores (MBPs)
was screened against IDE. MBPs are small molecules (<
3
00 MW) containing one or more donor atoms suited for metal
[23]
binding. Metal binding (a.k.a., dative bonding, coordination
bonding) is a strong, but reversible interaction, making MBPs
ideal starting points for metalloenzyme drug discovery efforts.
The results of screening this library against IDE are described
here, revealing that 1,2-hydroxypyridinethione (1,2-HOPTO) is a
promising fragment lead against this important metalloenzyme.
Results and Discussion
Screening of the MBP Library
To identify MBPs with the potential for inhibitor development
[29–30]
against IDE, a ~350-component library
was screened
against IDE, testing for inhibition against the degradation of
several key biological IDE substrates, including insulin, gluca-
gon, amylin, and Aβ, as well as the synthetic fluorogenic
Figure 2. Binding of IDE to a broad range of substrates, as exemplified by: a)
[2]
insulin, b) glucagon, and c) Aβ. The full structure of IDE bound to each
substrate is shown on the left, with close-ups of the catalytic site shown on
the right. The zinc ion is depicted as an orange sphere in (a). Images
produced from PDB IDs 2G54, 2G49, and 2G47, respectively.
[31–32]
peptide, substrate V.
Compounds were initially screened
against all five substrates at fragment concentrations of 500
and 50 μM, then for compounds showing good inhibitory
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[
3,18,21,22,24–28]
Figure 3. Structures and inhibitory values of reported IDE inhibitors.
a) Cysteine-modifying IDE inhibitors. b) Metal-binding IDE inhibitors; inset:
2+ [22]
crystal structure of hydroxamate-based compound 2 coordinating to the active site Zn , PDB ID: 4NXO. c) Exosite-binding IDE inhibitors; inset: crystal
structure of 4 (green) bound to the exosite and co-crystallized with insulin (cyan) PDB ID: 6EDS.
[
18]
activity, K values were established via dose-response experi-
identified hits, 1,2-hydroxypyridinethione (1,2-HOPTO, 5, Fig-
i
ments. Approximately 14 compounds exhibited average Ki
values <100 μM across the five different substrates. These
initial hits showed varied degrees of substrate selectivity,
including three amylin-sparing compounds and another two
showing selectivity for glucagon (data not shown). In addition,
consistent with previous reports showing differential activities
ure 4), with an average K =86 μM against the evaluated
i
[33]
substrates, was identified as a fragment lead
against IDE
based on criteria of potency, synthetic tractability, and the
observation that it belonged to a larger family of hits that also
contained members exhibiting substrate-selective properties.
[32]
against short peptide substrates, five compounds were in fact
activators of the degradation of substrate V. From among the
Figure 4. HOPTO derivatives tested for IDE inhibition.
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Screening of the elaborated HOPTO sublibrary
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HOPTOs are a known class of ligands particularly suited for Zn
[
34,35]
binding based on hard-soft acid-base theory.
They have
been established to affect bidentate binding through a set of
oxygen and sulfur donor atoms, where the sulfur has been
demonstrated to exist predominately as the thione isomer. In
an effort to find HOPTO derivatives as warheads for metal-
loenzyme inhibition, an expanded library of HOPTO derivatives
[36]
2
+
Figure 5. Model of 8 bound to the Zn within the active site of IDE. a)
Superposition of 8 and 2, which was used to create b) a model of 8 bound
(Figure 4) was prepared and demonstrated to have druglike
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to the Zn in the IDE active site. c) A 2-D representation of the model of 8
binding, with Zn in orange, coordination bonds as pink dashes,
interactions as green arrows, and solvent exposed regions as blue spheres.
[37]
qualities while maintaining the core metal-binding capacities.
2+
As 1,2-HOPTO was identified as a broad-spectrum scaffold for
IDE inhibition, it was decided to employ this elaborated HOPTO
sublibrary against IDE to better optimize the MBP warhead
before pursuing further inhibitor elaboration. Screening of this
focused HOPTO sublibrary revealed both 3-sulfonamide-1,2-
HOPTO (8) and isoquinoline-1,2-HOPTO (10) to have improved
Ph,Me
both the model [(Tp
structures.
)Zn(8)] and prior hydroxamic crystal
inhibition activity, with K values of ~50 μM for both com-
i
pounds. Compound 8 was selected for further development
against IDE.
Sulfonamide HOPTO derivative synthesis and inhibitory
activity
Modeling of 8in the IDE active site
To facilitate the use of these sulfonamide derivatized HOPTOs
as tool compounds, a readily adaptable and simple 3-step
synthetic route amenable towards rapid derivatization was
developed, as detailed in Scheme 1. Combining a sulfonyl
chloride with a suitable amine heterocycle starting material in a
microwave reactor yielded the desired sulfonamide product. It
was found that using excess amine relative to sulfonyl chloride
aided in decreasing the amount of undesired disulfonamide
side products generated. This synthetic step is highly adaptable
to a broad scope of both amine and sulfonyl chloride partners,
as evidenced by the scope of sulfonamide HOPTOs prepared in
this study (Figure 6). The subsequent step of oxidation using
mCPBA achieves selective oxidation at the pyridine nitrogen.
The final step of thionation was achieved by heating the
oxidized product in a solution of freshly prepared saturated
NaSH in the presence of KI. This reaction was quenched with
aqueous HCl, and subsequent work-up and chromatography
followed by recrystallization from iPrOH with varying amounts
In the absence of a co-crystal structure, 8 was modeled in the
active site of IDE to gain a rudimentary idea of how the
compound could potentially bind. The lead fragment 8 has
been previously crystallized in
a
model hydrotris(3,5-
)Zn(8)] complex, demon-
strating a preferred Zn metal-coordination through donor
Ph,Me
phenylmethylpyrazolyl)borate [(Tp
2
+
[37]
atoms of an axial thione and equatorial hydroxy group.
Additionally, there have been previously reported crystal
structures of hydroxamic acid-based inhibitors of IDE, showing
2
+
that these compounds bind the active-site Zn through axial
[22]
carbonyl and equatorial hydroxy groups (Figure 3b). Consid-
ering the 1,2-HOPTO MBP core as a cyclized thione analog of a
hydroxamic acid, the structure of hydroxamic acid 2 bound to
IDE was used to model the binding of 8 in the IDE active site.
To prepare the in silico model, the Molecular Operating
Environment (MOE, Chemical Computing Group) software suite
was employed, using the structure of 8 from the crystal
of H O yielded final HOPTO products. In the case of compounds
2
Ph,Me
structure of [(Tp
)Zn(8)] and superposing it over the hydrox-
47 and 48 (which are designed as negative controls because
they lack one donor group), thionation was instead achieved by
heating the halogenated sulfonamide to reflux at 150°C in DMF
amate of 2 bound in IDE by aligning the thione of 8 with the
carbonyl of 2, and the hydroxy group of 8 with the hydroxy
group of 2. This resulted in the model displayed in Figure 5,
with compound 8 being predicted to bind IDE through the
thione and hydroxy donor atoms in a manner consistent with
in the presence of excess thiourea. Finally, it should be noted
that some HOPTO products were isolated as mixtures with the
oxidized dimer (through the sulfur atom). This dimer is easily
Scheme 1. Synthesis of select sulfonamide HOPTO derivatives reported in this study. a) sulfonyl chloride, pyridine, microwave irradiation, 120°C, 15 min, 25–
7
1%; b) mCPBA, CH
2
Cl
2
, MeOH, 25–35°C, 16–72 h, 28–64%; c) sat. aq. NaSH, KI, MeOH, 50–100°C, 4–16 h, 6–58%; d) thiourea, DMF, 150°C, followed by 6 M
HCl, 110°C, 2 h, 22–40%.than the amide derivatives.
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Figure 6. Sulfonamide HOPTO compounds prepared and tested against IDE. Compounds 30 and 31 are amide derivatives, and 47 and 48 are controls for
metal binding by lacking the hydroxy moiety that should be essential for metal coordination.
reverted back to the active thione monomer in the presence of
DTT as a reductant. Considering IDE requires DTT in the assay
buffer to ensure reduction of its 13 Cys residues, DTT in the
biochemical assays served the dual function of also ensuring
the HOPTO species were in the active thione form.
active than 29. On the whole, these data suggest that the
sulfonamide compounds are more active.
Sulfonamide HOPTO SAR analysis
To further elaborate the lead 8, it was decided to investigate
the structure-activity relationship (SAR) of the sulfonamide
moiety to explore other potential interactions within the active
site. A preliminary test of phenyl (27) and benzyl (28)
derivatives showed some improvement relative to parent 8,
In an effort to further improve the activity of the lead 29,
compounds 32–46 were synthesized (Figure 6). The methyl
derivatives 32–35, were prepared to probe which positions
would be amenable to further elaboration. These methyl
derivatives exhibited a flat SAR, revealing little difference in
activity against IDE. Among a set of derivatives with alkyl
sulfonamide substituents, cyclopropane 36 had a comparatively
with 27 having an average K value (over all tested substrates)
i
of 22 μM, and 28 having an average K value of 35 μM. Utilizing
i
an aromatic isostere replacement of the phenyl ring to a
thiophene substituent yielded 29 with a further improved
average Ki value of 9 μM (Figure S1 in the Supporting
Information).
In addition, to validate the contribution of the sulfonamide
to the overall activity of 29, amide linked compounds 30 and
high average K value of 45 μM, whereas the bulky camphor 37
i
was slightly more on par with the thiophene, with an average Ki
value of 16 μM. Pyrazole derivatives 38 and 39 were prepared
as analogues of the five-member thiophene ring, and again
showed somewhat flat SAR with average K values of 16 and
i
3
3
1 were prepared as analogs of 27 and 29. As shown in Table 1,
23 μM, respectively. To explore alternate variations of the
position of the thiophene, the 3-thiophene 40 and alternate
position 2-thiophene compounds 41 and 42 were prepared. Of
these, 42 was the most interesting, although it had a higher
0 had an average K value of 57 μM (37 μM not including
i
Substrate V) rendering 30 approximately 2.5 times less active
than 27, and 31 had average K values of 55 μM (38 μM not
i
including substrate V) making 31 approximately six times less
average K value of 43 μM, it was the most selective in this study
i
towards sparing amylin degradation (Table 2, Figure S1), mak-
ing 42 of potential interest as a tool compound in this regard.
To examine more lipophilic substituents, compounds 43–46
were synthesized. The halogenated derivatives 43 and 44
showed improvement relative to the parent thiophene, with
Table 1. Inhibitory activity of HOPTO compounds against IDE substrates.
i
K [μM]
Cmpd
Insulin
Gluc.
Amylin
Aβ
V
Ave.
Avg. w/o V
respective average K values of 11 and 7 μM (Figure S1).
i
Between the halogenated derivatives, the brominated
thiophene 44 displayed good broad-spectrum activity, inhibit-
ing the degradation of all four physiological substrates (i.e.,
excluding substrate V) with relatively similar, low micromolar
activity (Table 2). Intriguingly, the chlorinated thiophene 43
exhibited a relatively insulin-sparing inhibition profile, for
5
8
2
39
24
26
57
9
23
27
217
114
10
14
3
26
26
41
31
26
40
10
63
62
108
61
13
19
6
33
36
25
19
36
47
15
140
126
86
50
22
35
9
57
55
101
58
19
33
7
37
38
7
28
2
3
3
9
0
1
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biochemical tools and potential starting points for future
Table 2. Inhibitory activity of thiophene sulfonamide derivatives against
IDE substrates.
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therapeutic development. IDE relies on an active-site Zn ion
for catalytic activity, and controls the levels of several bio-
logically important peptide substrates, making IDE inhibition of
high importance towards conditions such as T2DM as well as
AD. Screening an MBP library of ~350 unique compounds led
to the identification of the HOPTO scaffold as a novel zinc-
binding moiety having good activity against IDE. Subsequent
testing of a HOPTO sublibrary resulted in the identification of 3-
sulfonamide-1,2-HOPTO as a novel IDE inhibitor, with an
i
K [μM]
Insulin
Cmpd
Gluc.
Amylin
Aβ
V
Ave.
Ave. w/o V
3
3
3
2
3
4
11
23
18
21
51
7
15
21
16
8
27
30
9
26
55
>200
>200
6
5
6
5
20
7
6
7
13
12
13
14
35
19
15
19
14
27
64
7
8
6
7
7
22
11
8
13
9
13
20
3
27
22
18
27
95
46
36
54
26
29
84
13
14
31
10
61
77
13
14
12
15
45
18
16
23
15
17
43
11
7
10
11
11
12
32
11
11
15
12
15
33
11
5
35
3
3
3
6
7
8
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
39
average K value of 50 μM against the tested IDE substrates, and
i
4
4
4
0
1
2
8
11
20
2
2
5
2
172
62
derivatization of the sulfonamide resulted in thiophene sulfona-
mide 29, with an average K of 9 μM against the IDE substrates.
i
43
Further SAR studies yielded halogen derivatives 43 and 44, of
which the bromine 44 had broad-spectrum activity, with
average Ki value of 5 μM against the physiological IDE
substrates. In addition, compounds 43 and 46 showed a
promising degree of selectivity between insulin and other
diabetes-related IDE substrates, glucagon and amylin. Com-
pounds 29, 43, 44 and 46 possess many features not present in
most existing IDE inhibitors, including a small size (e.g., MW
288-367) and other druglike properties (Table S3), suggesting
they may represent useful pharmacophores for future drug
development. Overall, these compounds represent a new class
of metal-binding IDE inhibitors that, due to their facile
syntheses, can be readily made available to serve as useful tools
for investigating the role of IDE and the various substrates it
regulates.
4
4
4
47
4
4
5
6
7
3
11
28
>200
84
10
13
>200
102
16
23
n.a.
n.a.
13
25
n.a.
n.a.
8
example, inhibiting the degradation of glucagon ~15 times
more potently than insulin (Table 2, Figure S1). As a preliminary
test of inhibitor selectivity, compounds 43 and 44 were
screened at a concentration of 100 μM against human carbonic
anhydrase II (hCAII); no inhibition of the Zn-dependent hCAII
metalloenzyme was observed. The thiadiazole 45 and 2-phenyl
thiophene 46 showed no improvement relative to 29 in terms
of average K values, but notably 46 inhibited the degradation
i
of glucagon 27 and 14 times more potently than insulin and
amylin, respectively (Table 2, Figure S1).
Experimental Section
Validation of the metal-binding mechanism
Synthesis
Unless otherwise noted, all reagents and solvents were purchased
from commercial suppliers and used with no additional purification.
Microwave reactions were performed using a CEM Discover series
S-class microwave reactor in pressure-sealed vessels. Silica gel
To validate the mechanism of action as metal binding at the
2
+
active site Zn ion, a set of non-metal-binding controls were
prepared (Figure 6). Control compounds 47 and 48 were
prepared as respective methyl and thiophene sulfonamide
derivatives lacking the oxygen donor atom of the HOPTO. These
controls represent a deletion of a crucial portion of the donor
atoms required for the bidentate metal coordination of the 1,2-
HOPTO ligand, and neither should be capable of strong metal
coordination. As expected, 47 displayed essentially no inhib-
itory activity (except against the short, fluorogenic peptide,
Substrate V, Table 2), while compound 48 displayed greatly
reduced activity across all IDE substrates (Table 2). The residual
activity for compound 48 is likely due to the strong interactions
of the thiophene ring and some metal-binding ability by the
remaining thione group from the MBP; however, overall these
data are supporting of a metal-binding mode of action by the
lead compounds in this study.
+
column chromatography was performed using a CombiFlash Rf
Teledyne ISCO system, using hexane, ethyl acetate, CH Cl or MeOH
2
2
+
as eluents. Separations were monitored via a Teledyne ISCO RF
1
PurIon ESI-MS detector with 1 Da resolution. H NMR spectra were
obtained using Varian 400-MHz and 500-MHz spectrometers at the
1
Department of Chemistry and Biochemistry at UC San Diego. H
NMR data is reported in parts per million relative to the residual
non-deuterated solvent signals, and spin multiplicities are given as
s (singlet), br s (broad singlet), d (doublet), dd (doublet of doublets),
t (triplet), dt (doublet of triplets), q (quartet), and m (multiplet).
When available, coupling constants (J) are reported in hertz (Hz)
(
Figure S2–S22). Standard resolution mass spectrometry was per-
formed at either the UC San Diego Molecular Mass Spectrometry
Facility or on the previously described Teledyne ISCO RF PurIon
+
ESI-MS detector. High resolution mass spectrometry (HRMS) was
measured with an Agilent 6230 time-of-flight (TOF) mass spectrom-
eter using a jet stream electrospray ionization source (ESI).
[37]
The synthesis of 5–28 was previously reported in Adamek et al.
While 29 was previously reported in Adamek et al., this key
compound was prepared again for this work. The purity of
compounds 29–48 was evaluated either by analytical HPLC or
elemental (combustion) analysis (Table S1); all assayed compounds
were >90% purity, with the exception of compounds 33 and 34,
Conclusion
The goal of this work was to develop novel, easy-to-synthesize,
metal-binding inhibitors of IDE that could be useful as
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1
13
which were >84% pure by HPLC. Experimental H and C NMR for
N-(2-Bromopyridin-3-yl)benzamide (30a): To a 0°C solution of 2-
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
final compounds are reported in Figures S2–S22).
bromopyridin-3-amine (0.600 g, 3.47 mmol) and pyridine (0.56 mL,
6
.9 mmol) in 10 mL CH Cl , a mixture of benzoyl chloride (269 μL,
2 2
2.31 mmol) in 5 mL CH Cl was added dropwise. The reaction
2
2
Generation of sulfonyl chloride
mixture was allowed to slowly warm to 25°C for 16 h, and was then
diluted with an additional 15 mL CH Cl , and was washed with
2
2
5
-Phenylthiophene-2-sulfonyl chloride (49): To a solution of DMF
2
0 mL 4 M HCl. The aqueous was back-extracted with 10 mL CH Cl ,
2 2
(0.87 mL, 11.2 mmol) cooled to 0°C while under a nitrogen
atmosphere, sulfuryl chloride (913 μL, 11.2 mmol) was added
dropwise, and the reaction was stirred for 15 min, until the mixture
formed a white solid. Then 2-phenylthiophene (1.50 g, 9.36 mmol)
was added in one portion, and the solids were heated as a melt at
and the combined organic phase was washed with brine, dried
over magnesium sulfate, and the solids were filtered off and
discarded. The filtrate was concentrated under reduced pressure,
and the resulting crude was purified by column chromatography,
running gradient from 100% hexanes to 100% ethyl acetate. The
desired product eluted in 50% ethyl acetate in hexanes. Like
fractions were combined and concentrated under reduced pressure
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
1
00°C for 45 min, noting that upon initial addition of the white
thiophene, the solid mixture turned yellow, and upon heating, melt
was observed to occur at 55°C. Upon reaching 100°C, the reaction
mixture had turned from clear yellow to clear green. After heating
at 100°C for 45 min, upon reaction completion, the melt was
cooled to 25°C, diluted with ethyl acetate, and ice water was
added. With a chilled separatory funnel, the product was extracted
into organic using 2×15 mL ethyl acetate, and the combined
organic was washed with brine and dried over magnesium sulfate.
The solids were filtered off and discarded, and the resulting filtrate
was concentrated under reduced pressure to yield a blue liquid.
The product was then purified by column chromatography, running
an isocratic in gradient 100% hexanes. Like fractions of the desired
product were combined and concentrated under reduced pressure,
until there was a remaining volume of ~5 mL hexanes, containing a
solid precipitate of the desired product. The precipitate was
collected via vacuum filtration and washed with hexanes to obtain
to obtain 30a (0.549 g, 1.98 mmol, 86%) as a clear oil that solidified
1
upon standing. H NMR (400 MHz, [D ]DMSO): δ 10.10 (br s, 1H),
6
8.30 (dd, J =4.4, J =1.2, 1H), 8.01 (d, J=7.2, 3H), 7.65–7.47 (m, 4H);
1
2
+
ESI-MS(+): m/z 277.12 [M+H] .
N-(2-Bromopyridin-3-yl)thiophene-2-carboxamide (31a): Follow-
ing the same protocol used in 30a, from 2-bromopyridin-3-amine
(0.600 g, 3.47 mmol), pyridine (0.56 mL, 6.9 mmol), and thiophene-
2-carbonyl chloride (247 μL, 2.31 mmol), the desired 31a (0.536 g,
1.89 mmol, 82%) was obtained as a clear oil that solidified upon
1
standing. H NMR (400 MHz, [D ]DMSO): δ 10.23 (br s, 1H), 8.30 (d,
6
J=4.8, 1H), 8.02 (d, J=3.6, 1H), 7.96 (d, J=7.6, 1H), 7.90 (d, J=5.2,
1H), 7.51 (dd, J =7.6, J =0.8, 1H), 7.25 (t, J=5.2, 1H); ESI-MS(+): m/
1
2
+
z 283.11 [M+H] .
N-(2-Bromopyridin-3-yl)-5-methylthiophene-2-sulfonamide (32a):
Following the general method for sulfonamide coupling, from 2-
bromopyridin-3-amine (0.600 g, 3.47 mmol) and 5-meth-
ylthiophene-2-sulfonyl chloride (608 μL, 4.51 mmol), the desired
product 32a (0.380 g, 1.14 mmol, 33%) was obtained as a light
4
9 (1.25 g, 4.81 mmol, 51%) as a powdery solid that was pale
1
yellow with a greenish tint in color. H NMR (400 MHz, (CD ) SO):
3
2
7.60 (d, J=7.6, 2H), 7.39 (t, J=7.6, 2H), 7.31–7.27 (m, 2H), 7.11 (d,
J=2.8, 1H).
1
yellow oil that solidified upon standing. H NMR (400 MHz, [D6]
DMSO): δ 10.31 (br s, 1H), 8.24 (dd, J =4.8, J =1.6, 1H), 7.68 (dd,
Sulfonamide coupling
1
2
J =7.6, J =1.6, 1H), 7.45 (dd, J =8.0, J =4.8, 1H), 7.30 (d, J=3.6,
1
2
1
2
General sulfonamide coupling protocol: Unless otherwise noted,
the following protocol was used for sulfonamide coupling. To a
heat-gun dried microwave vessel equipped with stir bar and
charged with a solution of starting amine (1.3 equiv) in 2 mL dry
pyridine, sulfonyl chloride (1.0 eq) was added in one portion. The
mixture was placed in a microwave reactor and irradiated at 120°C
1H), 6.87 (dd, J =4.0, J =1.2, 1H), 2.48 (s, 3H); ESI-MS(+): m/z
1
2
+
333.10 [M+H] .
N-(2-Bromopyridin-3-yl)-4-methylthiophene-2-sulfonamide (33a):
Following the general method for sulfonamide coupling, from 2-
bromopyridin-3-amine (0.484 g, 2.80 mmol) and 4-meth-
ylthiophene-2-sulfonyl chloride (0.500 g, 2.54 mmol), the desired
product 33a (0.560 g, 1.68 mmol, 66%) was obtained as a slightly
for 15 min. Upon cooling, the reaction mixture was diluted with
15 mL ethyl acetate, and washed with 2×15 mL 4 M HCl. The
aqueous was back-extracted with an additional 15 mL ethyl acetate,
and the combined organic was washed with brine, dried over
magnesium sulfate, and the solids were filtered off and discarded.
The filtrate was concentrated under reduced pressure, and the
resulting crude was purified by column chromatography, running
gradient from 100% hexanes to 100% ethyl acetate. The desired
sulfonamide product typically eluted in 50% ethyl acetate in
hexanes. Like fractions were combined and concentrated under
reduced pressure to obtain the desired sulfonamide product that
was used directly in the subsequent step. As previously noted,
some coupling reactions resulted in an inseparable mix of
sulfonamide and disulfonamide products. These mixtures were
carried over directly into the next step with no additional
purification.
impure white solid that was used directly in the next step without
1
any additional purification. H NMR (400 MHz, [D ]DMSO): δ 10.39
6
(
1
br s, 1H), 8.24 (dd, J =4.4, J =1.2, 1H), 7.67 (dd, J =8.0, J =1.2,
1
2
1
2
H), 7.55–7.54 (m, 2H), 7.45 (dd, J =7.6, J =4.4, 1H), 7.35 (s, 1H),
1 2
À
2.19 (s, 3H); ESI-MS(À ): m/z 333.15 [MÀ H] .
N-(2-Bromo-5-methylpyridin-3-yl)thiophene-2-sulfonamide (34a):
Following the general method for sulfonamide coupling, from 2-
bromo-5-methylpyridin-3-amine
(0.500 g,
2.67 mmol)
and
thiophene-2-sulfonyl chloride (0.635 g, 3.48 mmol), the desired
product 34a (0.513 g, 1.20 mmol, 43%) was obtained as a white
solid contaminated with approximately 25% disulfonamide prod-
uct. The product was used directly in the next step without any
1
additional purification. H NMR (400 MHz, [D ]DMSO): δ 10.31 (br s,
6
1
H), 8.10 (s, 1H), 7.96 (d, J=5.2, 1H), 7.52 (s, 1H), 7.48 (d, J=3.6, 1H),
À
7.16 (t, J=4.8, 1H), 2.25 (s, 3H); ESI-MS(À ): m/z 331.29 [MÀ H] .
N-(2-Bromopyridin-3-yl)thiophene-2-sulfonamide (29a): Following
the general method for sulfonamide coupling, from 2-bromopyr-
idin-3-amine (1.50 g, 8.67 mmol) and thiophene-2-sulfonyl chloride
N-(2-Bromo-6-methylpyridin-3-yl)thiophene-2-sulfonamide (35a):
Following the general method for sulfonamide coupling, from 2-
(1.27 g, 6.94 mmol), the desired product 29a (1.53 g, 4.80 mmol,
bromo-6-methylpyridin-3-amine
(0.500 g,
2.67 mmol)
and
1
55%) was obtained as a peach colored solid. H NMR (400 MHz, [D ]
6
thiophene-2-sulfonyl chloride (0.635 g, 3.48 mmol), the desired
product 35a (0.746 g, 1.90 mmol, 71%) was obtained as a white
solid contaminated with approximately 15% disulfonamide prod-
DMSO): δ 10.41 (br s, 1H), 8.25 (d, J=4.4, 1H), 7.97 (d, J=5.2, 1H),
7
.70 (d, J=7.6, 1H), 7.49–7.44 (m, 2H), 7.16 (t, J=4.4, 1H); ESI-MS(À ):
À
m/z 317.18 [MÀ H] .
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uct. The product was used directly in the next step without any
N-(6-Bromopyridin-3-yl)thiophene-2-sulfonamide (42a): Following
the general method for sulfonamide coupling, from 6-bromopyr-
idin-3-amine (0.750 g, 4.34 mmol) and thiophene-2-sulfonyl
chloride (1.19 g, 6.50 mmol), the desired product 42a (1.38 g,
4.29 mmol) was obtained in theoretical yield as a pale yellow solid
contaminated with approximately 50% disulfonamide product. The
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
1
additional purification. H NMR (400 MHz, [D ]DMSO): δ 10.25 (br s,
6
1
H), 7.96 (d, J=4.8, 1H), 7.53 (d, J=8.0, 1H), 7.45 (d, J=3.6, 1H),
7.30 (d, J=7.6, 1H), 7.15 (t, J=4.8, 1H), 2.41 (s, 3H); ESI-MS(À ): m/z
À
331.26 [MÀ H] .
N-(2-Bromopyridin-3-yl)cyclopropanesulfonamide (36a): Follow-
ing the general method for sulfonamide coupling, from 2-
bromopyridin-3-amine (0.750 g, 4.34 mmol) and cyclopropanesul-
product was used directly in the next step without any additional
1
purification. H NMR (400 MHz, [D ]DMSO): δ 10.87 (br s, 1H), 8.26
6
(
dd, J =4.8, J =1.2, 1H), 8.12 (d, J=2.8, 1H), 7.95 (dd, J =5.2, J =
1
2
1
2
fonyl chloride (662 μL, 6.50 mmol), the desired product 36a
1.2, 1H), 7.79 (dd, J =4.0, J =1.6, 1H), 7.62–7.57 (m, 2H), 7.30 (t, J=
5.2, 1H); ESI-MS(+): m/z 321.15 [M+H] .
1
1
2
(
0.850 g, 3.07 mmol, 71%) was obtained as a light yellow solid. H
+
NMR (400 MHz, [D ]DMSO): δ 9.70 (br s, 1H), 8.26 (dt, J =4.4, J =
6
1
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
1
.6, 1H), 7.84 (dt, J =8.0, J =1.6, 1H), 7.46 (qd, J =4.4, J =1.6, 1H),
N-(2-Bromopyridin-3-yl)-5-chlorothiophene-2-sulfonamide (43a):
Following the general method for sulfonamide coupling, from 2-
bromopyridin-3-amine (3.00 g, 17.3 mmol) and 5-chlorothiophene-
2-sulfonyl chloride (1.86 mL, 13.9 mmol), the desired product 43a
1
2
1
2
2.77–2.70 (m, 1H), 1.01–0.96 (m, 2H), 0.90–0.86 (m, 2H); ESI-MS(+):
+
m/z 279.12 [M+H] .
N-(2-Chloropyridin-3-yl)-1-((1R,4S)-7,7-dimethyl-2-oxobicyclo
(
2.27 g, 6.41 mmol, 37%) was obtained as a light tan solid
[
2.2.1]heptan-1-yl)methanesulfonamide (37a): Following the gen-
contaminated with approximately 10% disulfonamide product. The
eral method for sulfonamide coupling, from 2-chloropyridin-3-
amine (0.350 g, 2.02 mmol) and ((1R,4S)-7,7-dimethyl-2-oxobicyclo
product was used directly in the next step without any additional
1
purification. H NMR (400 MHz, [D ]DMSO): δ 10.65 (br s, 1H), 8.28
6
[
2.2.1]heptan-1-yl)methanesulfonyl chloride (0.650 g, 2.59 mmol),
(
J
dd, J =4.4, J =1.2, 1H), 7.71 (dd, J =8.0, J =1.6, 1H), 7.48 (dd,
1
2
1
2
the desired product 37a (0.382 g, 1.11 mmol, 43%) was obtained as
a pale yellow solid. H NMR (400 MHz, [D ]DMSO): δ 9.79 (br s, 1H),
=8.0, J
=4.8, 1H), 7.37 (d, J=4.0, 1H), 7.25 (d, J=4.0, 1H); ESI-
1
1
2
6
À
MS(À ): m/z 353.09 [MÀ H] .
8
1
.25 (d, J=4.8, 1H), 7.95 (d, J=8.0, 1H), 7.47 (dd, J =8.0, J =4.8,
H), 3.50 (d, J=14.8, 1H), 3.15 (d, J=14.8, 1H), 2.38–2.29 (m, 2H),
1 2
5-Bromo-N-(2-bromopyridin-3-yl)thiophene-2-sulfonamide (44a):
Following the general method for sulfonamide coupling, from 2-
bromopyridin-3-amine (1.00 g, 5.78 mmol) and 5-bromothiophene-
2.06 (t, J=4.4, 1H), 1.97–1.91 (m, 2H), 1.59–1.52 (m, 1H), 1.43–1.37
+
(m, 1H), 1.01 (s, 3H), 0.79 (s, 3H); ESI-MS(+): m/z 343.31 [M+H] .
2
-sulfonyl chloride (1.21 g, 4.62 mmol), the desired product 44a
N-(2-Bromopyridin-3-yl)-1-methyl-1H-pyrazole-3-sulfonamide
(38a): Following the general method for sulfonamide coupling,
from 2-bromopyridin-3-amine (0.500 g, 2.89 mmol) and 1-methyl-
(1.41 g, 3.54 mmol, 61%) was obtained as a pale yellow solid
contaminated with approximately 20% disulfonamide product. The
product was used directly in the next step without any additional
1
H-pyrazole-3-sulfonyl chloride (0.475 g, 2.63 mmol), the desired
1
purification. H NMR (400 MHz, [D ]DMSO): δ 10.64 (br s, 1H), 8.28
6
product 38a (0.354 g, 1.12 mmol, 43%) was obtained as a pale tan
oil that solidified upon standing. H NMR (400 MHz, [D ]DMSO): δ
(d, J=4.4, 1H), 7.72–7.68 (m, 2H), 7.51–7.46 (m, 2H); ESI-MS(À ): m/z
1
6
À
3
97.03 [MÀ H] .
1
0.18 (br s, 1H), 8.21 (dd, J =4.8, J =1.6, 1H), 7.88 (d, J=2.0, 1H),
1 2
7
.72 (dd, J =8.0, J =1.6, 1H), 7.42 (dd, J =8.0, J =4.8, 1H), 6.57 (d,
N-(2-bromopyridin-3-yl)benzo[c][1,2,5]thiadiazole-4-sulfonamide
(45a): Following the general method for sulfonamide coupling,
from 2-bromopyridin-3-amine (0.350 g, 2.02 mmol) and benzo
1
2
1
2
+
J=2.4, 1H), 3.91 (s, 3H); ESI-MS(+): m/z 317.25 [M+H] .
N-(2-bromopyridin-3-yl)-1-methyl-1H-pyrazole-4-sulfonamide
[
c][1,2,5]thiadiazole-4-sulfonyl chloride (0.380 g, 1.62 mmol), the
(
39a): Following the general method for sulfonamide coupling,
desired product 45a (0.382 g, 1.03 mmol, 64%) was obtained as a
cream colored solid. H NMR (400 MHz, [D ]DMSO): δ 10.49 (br s,
from 2-bromopyridin-3-amine (0.500 g, 2.89 mmol) and 1-methyl-
1H-pyrazole-4-sulfonyl chloride (0.626 g, 3.47 mmol), the desired
product 39a (0.611 g, 1.93 mmol, 67%) was obtained as a clear oil
1
6
1
6
H), 8.41 (d, J=8.8, 1H), 8.21 (dd, J =4.8, J =1.6, 1H), 8.13 (d, J=
1 2
.8, 1H), 7.84–7.76 (m, 2H), 7.43 (dd, J =8.0, J =4.8, 1H); ESI-MS(+):
1
1
2
that solidified upon standing to a white solid. H NMR (400 MHz,
+
m/z 373.16 [M+H] .
[
1
3
D ]DMSO): δ 9.97 (br s, 1H), 8.23 (s, 1H), 8.21 (dd, J =4.8, J =1.6,
6
1
2
H), 7.69 (s, 1H), 7.67 (d, J=1.6, 1H), 7.42 (dd, J =8.0, J =4.8, 1H),
N-(2-bromopyridin-3-yl)-5-phenylthiophene-2-sulfonamide (46a):
Following the general method for sulfonamide coupling, from 2-
bromopyridin-3-amine (1.08 g, 6.23 mmol) and 49 (1.24 g,
4.79 mmol), the desired product 46a (1.13 g, 2.85 mmol, 60%) was
1
2
À
.85 (s, 3H); ESI-MS(À ): m/z 315.20 [MÀ H] .
N-(2-Bromopyridin-3-yl)thiophene-3-sulfonamide (40a): Following
the general method for sulfonamide coupling, from 2-bromopyr-
idin-3-amine (0.600 g, 3.47 mmol) and thiophene-3-sulfonyl
chloride (823 mg, 4.51 mmol), the desired product 40a (0.583 g,
1
obtained as a fluffy bright white solid. H NMR (400 MHz, [D6]
DMSO): δ 10.53 (br s, 1H), 8.28–8.26 (m, 1H), 7.75–7.70 (m, 3H), 7.56
À
(
d, J=2.4, 1H), 7.48–7.39 (m, 5H); ESI-MS(À ): m/z 393.23 [MÀ H] .
1.83 mmol, 53%) was obtained as a light brown oil that solidified
1
upon standing. H NMR (400 MHz, [D ]DMSO): δ 10.17 (br s, 1H),
N-(2-bromopyridin-3-yl)methanesulfonamide (47a): Following the
general method for sulfonamide coupling, from 2-bromopyridin-3-
amine (1.08 g, 6.23 mmol) and methanesulfonyl chloride (645 μL,
8.33 mmol), the desired product 47a (0.510 g, 2.05 mmol, 25%) was
obtained as a pale yellow liquid contaminated with approximately
50% disulfonamide product. The product was used directly in the
6
8
.22–8.21 (m, 1H), 8.12–8.11 (m, 1H), 7.76–7.74 (m, 1H), 7.64–7.61
(
m, 1H), 7.44–7.40 (m, 1H), 7.28–7.26 (m, 1H); ESI-MS(À ): m/z 317.19
À
[
MÀ H] .
N-(2-Bromopyridin-4-yl)thiophene-2-sulfonamide (41a): Following
the general method for sulfonamide coupling, from 2-bromopyr-
idin-4-amine (0.750 g, 4.34 mmol) and thiophene-2-sulfonyl
chloride (1.19 g, 6.50 mmol), the desired product 41a (1.37 g,
1
next step without any additional purification. H NMR (400 MHz,
[
7
D ]DMSO): δ 9.66 (br s, 1H), 8.23 (d, J=8.0, 1H), 7.83 (d, J=7.6, 1H),
.48–7.45 (m, 1H), 3.11 (s, 3H); ESI-MS(À ): m/z 249 [MÀ H] .
6
À
4.29 mmol, 55%) was obtained as a pale yellow oil that solidified
1
upon standing to a white solid. H NMR (400 MHz, [D
]DMSO): δ
6
1
7
1.57 (br s, 1H), 8.19 (d, J=6.0, 1H), 8.01 (dd, J =4.8, J =1.2, 1H),
1 2
Oxidation
.79 (dd, J =3.6, J =1.2, 1H), 7.24 (d, J=2.0, 1H), 7.20–7.16 (m, 2H);
1
2
+
ESI-MS(+): m/z 321.13 [M+H] .
General oxidation coupling protocol: Unless otherwise noted, the
following protocol was used. To a solution of sulfonamide in 20 mL
1
:1 CH Cl /MeOH, a solution of mCPBA (5 equiv) in 10 mL 1:1
2 2
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1
CH Cl /MeOH was added in one portion. The reaction mixture was
was obtained as a white solid. H NMR (400 MHz, [D ]DMSO): δ
2
2
6
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
then heated at 35°C for 72 h. Upon completion, the reaction
mixture was concentrated under reduced pressure and the
resulting crude was purified by column chromatography, running
gradient first from 100% hexanes to 100% ethyl acetate, and then
from 100% CH Cl to 15% MeOH in CH Cl . The desired products
10.50 (br s, 1H), 7.98 (d, J=4.8, 1H), 7.52 (d, J=3.6, 1H), 7.46 (d, J=
8.4, 1H), 7.16 (t, J=8.8, 1H), 7.11 (d, J=8.4, 1H), 2.37 (s, 3H); ESI-
MS(+): m/z 349.14 [M+H] .
+
2
-Bromo-3-(cyclopropanesulfonamido)pyridine 1-oxide (36b):
2
2
2
2
Following the general method for oxidation, from 36a (0.890 g,
typically eluted in 8–10% MeOH in CH Cl . Like fractions were
2
2
3.13 mmol) and mCPBA (3.60 g, 77.0% Wt, 16.1 mmol), at 30°C for
combined and concentrated under reduced pressure to obtain the
desired oxidized product that was used directly in the subsequent
step
16 h, the desired product 36b (0.470 g, 0.160 mmol, 50%) was
1
obtained as a white solid. H NMR (400 MHz, [D ]DMSO): δ 9.87 (br
6
s, 1H), 8.36 (dd, J =5.2, J =2.4, 1H), 7.42–7.37 (m, 2H), 2.81–2.75
1
2
2
-Bromo-3-(thiophene-2-sulfonamido)pyridine 1-oxide (29b): Fol-
(m, 1H), 1.03–0.98 (m, 2H), 0.97–0.91 (m, 2H); ESI-MS(+): m/z 295.14
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
+
lowing the general method for oxidation, from 29a (1.50 g,
[M+H] .
4
.70 mmol) and mCPBA (6.32 g, 77.0% Wt, 28.2 mmol), at 25°C for
2
-Chloro-3-((((1R,4S)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)
16 h, the desired product 29b (0.742 g, 2.21 mmol, 55%) was
obtained as a light yellow solid. H NMR (400 MHz, [D ]DMSO): δ
1
methyl)sulfonamido)pyridine 1-oxide (37b): Following the general
method for oxidation, from 37a (0.380 g, 1.11 mmol) and mCPBA
6
1
3
3
0.61 (br s, 1H), 8.35 (d, J=6.8, 1H), 7.99 (d, J=4.8, 1H), 7.55 (d, J=
(
1.24 g, 77.0% Wt, 5.54 mmol), at 40°C for 4 d, the desired product
37b (0.241 g, 0.672 mmol, 61%) was obtained as a yellow oil that
1
.6, 1H), 7.39 (t, J=6.8, 1H), 7.21–7.16 (m, 2H); ESI-MS(À ): m/z
À
33.11 [MÀ H] .
solidified upon standing. H NMR (400 MHz, [D ]DMSO): δ 7.98 (d,
6
3
-Benzamido-2-bromopyridine 1-oxide (30b): Following the gen-
J=6.4, 1H), 7.42 (d, J=8.4, 1H), 7.19 (t, J=8.0, 1H), 3.37 (d, J=5.6,
1H), 2.95 (d, J=14.8, 1H), 2.52–2.46 (m, 1H), 2.33–2.27 (m, 1H), 2.01
(t, J=4.0, 1H), 1.90–1.86 (m, 2H), 1.53–1.46 (m, 1H), 1.39–1.33 (m,
eral method for oxidation, from 30a (0.540 g, 1.95 mmol) and
mCPBA (2.18 g, 77.0% Wt, 9.74 mmol), at 35°C for 16 h, the desired
À
product 30b (0.169 g, 0.577 mmol, 30%) was obtained as an off-
1H), 1.00 (s, 3H), 0.76 (s, 3H); ESI-MS(À ): m/z 357.31 [MÀ H] .
1
white solid. H NMR (400 MHz, [D ]DMSO): δ 10.34 (br s, 1H), 8.42 (d,
6
2
-Bromo-3-((1-methyl-1H-pyrazole)-3-sulfonamido)pyridine
oxide (38b): Following the general method for oxidation, from 38a
0.350 g, 1.10 mmol) and mCPBA (1.24 g, 77.0% Wt, 5.52 mmol), at
1-
J=6.4, 1H), 7.98 (d, J=7.2, 1H), 7.64 (t, J=7.2, 1H), 7.58–7.52 (m,
+
2H), 7.46 (t, J=6.4, 1H); ESI-MS(+): m/z 293.05 [M+H] .
(
2
-Bromo-3-(thiophene-2-carboxamido)pyridine 1-oxide (31b):
30°C for 72 h, the desired product 38b (0.166 g, 0.498 mmol, 45%)
1
Following the general method for oxidation, from 31a (0.530 g,
was obtained as a yellow solid. H NMR (400 MHz, [D ]DMSO): δ
6
1.87 mmol) and mCPBA (2.10 g, 77.0% Wt, 9.36 mmol), at 35°C for
10.40 (br s, 1H), 8.33 (d, J=6.4, 1H), 7.90 (s, 1H), 7.36 (d, J=1.2, 1H),
7.25 (d, J=8.4, 1H), 6.61 (s, 1H), 3.91 (s, 3H); ESI-MS(+): m/z 333.14
1
6 h, the desired product 31b (0.358 g, 1.20 mmol, 64%) was
1
+
obtained as a yellow oil that solidified upon standing. H NMR
[M+H] .
(
1
400 MHz, [D ]DMSO): δ 10.38 (br s, 1H), 8.42 (dd, J =6.0, J =1.6,
H), 8.01 (dd, J =2.8, J =0.8, 1H), 7.92 (dd, J =4.8, J =0.8, 1H),
1 2 1 2
6
1
2
2-Bromo-3-((1-methyl-1H-pyrazole)-3-sulfonamido)pyridine
1-
oxide (39b): Following the general method for oxidation, from 39a
(0.600 g, 1.89 mmol) and mCPBA (2.12 g, 77.0% Wt, 9.46 mmol), at
7
.51–7.46 (m, 2H), 7.25 (dt, J =3.6, J =1.2, 1H); ESI-MS(+): m/z
1
2
+
299.02 [M+H] .
3
0°C for 16 h, the desired product 39b (0.212 g, 0.636 mmol, 34%)
1
2
-Bromo-3-((5-methylthiophene)-2-sulfonamido)pyridine 1-oxide
was obtained as an off-white solid. H NMR (400 MHz, [D ]DMSO): δ
10.18 (br s, 1H), 8.31 (dd, J =6.4, J =1.2, 1H), 8.28 (s, 1H), 7.74 (s,
6
(
32b): Following the general method for oxidation, from 32a
1
2
(0.380 g, 1.28 mmol) and mCPBA (1.28 g, 77.0% Wt, 5.70 mmol), at
1H), 7.36 (t, J=6.8, 1H), 7.20 (dd, J =8.4, J =0.8, 1H), 3.85 (s, 3H);
1
2
+
25°C for 67 h, the desired product 32b (0.202 g, 0.578 mmol, 51%)
ESI-MS(+): m/z 331.17 [M+H] .
1
was obtained as an off-white solid. H NMR (400 MHz, [D ]DMSO): δ
6
2
-Bromo-3-(thiophene-3-sulfonamido)pyridine 1-oxide (40b): Fol-
0.52 (br s, 1H), 8.35 (d, J=6.4, 1H), 7.40–7.37 (m, 2H), 7.21 (d, J=
lowing the general method for oxidation, from 40a (0.580 g,
À
1.82 mmol) and mCPBA (2.04 g, 77.0% Wt, 9.09 mmol), at 25°C for
6
7 h, the desired product 40b (0.256 g, 0.763 mmol, 42%) was
1
2
(
(
-Bromo-3-((4-methylthiophene)-2-sulfonamido)pyridine 1-oxide
33b): Following the general method for oxidation, from 33a
0.560 g, 1.68 mmol) and mCPBA (1.88 g, 77.0% Wt, 8.40 mmol), at
obtained as an off-white solid. H NMR (400 MHz, [D ]DMSO): δ
6
10.61 (br s, 1H), 8.35 (d, J=6.8, 1H), 7.99 (d, J=4.8, 1H), 7.55 (d, J=
3.6, 1H), 7.39 (t, J=6.8, 1H), 7.21–7.16 (m, 2H); ESI-MS(À ): m/z
À
2
5°C for 72 h, the desired product 33b (0.192 g, 0.550 mmol, 33%)
333.22 [MÀ H] .
1
was obtained as a yellow solid. H NMR (400 MHz, [D ]DMSO): δ
10.56 (br s, 1H), 8.35 (d, J=6.4, 1H), 7.58 (s, 1H), 7.42 (s, 1H), 7.38 (t,
6
2
-Bromo-4-(thiophene-2-sulfonamido)pyridine 1-oxide (41b): Fol-
lowing the general method for oxidation, from 41a (1.00 g,
J=6.4, 1H), 7.19 (d, J=8.4, 1H), 2.20 (s, 3H); ESI-MS(À ): m/z 349.10
À
3.13 mmol) and mCPBA (2.11 g, 77.0% Wt, 9.40 mmol), at 35°C for
[
MÀ H] .
1
6 h, the desired product 41b (0.298 g, 0.889 mmol, 28%) was
1
2-Bromo-5-methyl-3-(thiophene-2-sulfonamido)pyridine 1-oxide
34b): Following the general method for oxidation, from 34a
0.500 g, 1.13 mmol) and mCPBA (1.68 g, 77.0% Wt, 7.50 mmol), at
obtained as a white solid. H NMR (400 MHz, [D ]DMSO): δ 11.30 (br
6
(
s, 1H), 8.31 (d, J=7.2, 1H), 7.98 (d, J=4.8, 1H), 7.70 (d, J=3.6, 1H),
7.44 (d, J=2.8, 1H), 7.19–7.14 (m, 2H); ESI-MS(+): m/z 335.10 [M+
(
3
+
H] .
0°C for 16 h, the desired product 34b (0.101 g, 0.289 mmol, 26%)
1
was obtained as a pale yellow solid. H NMR (400 MHz, [D ]DMSO):
δ 10.53 (br s, 1H), 8.29 (s, 1H), 7.98 (d, J=5.2, 1H), 7.55 (d, J=4.0,
1H), 7.16 (t, J=4.0, 1H), 7.01 (s, 1H), 2.20 (s, 3H); ESI-MS(+): m/z
6
2-Bromo-5-(thiophene-2-sulfonamido)pyridine 1-oxide (42b): Fol-
lowing the general method for oxidation, from 42a (1.00 g,
3.13 mmol) and mCPBA (2.11 g, 77.0% Wt, 9.40 mmol), at 35°C for
+
349.11 [M+H] .
1
6 h, the desired product 42b (0.323 g, 0.964 mmol, 31%) was
1
2
-Bromo-6-methyl-3-(thiophene-2-sulfonamido)pyridine 1-oxide
35b): Following the general method for oxidation, from 35a
(0.730 g, 1.86 mmol) and mCPBA (5.87 g, 77.0% Wt, 2.45 mmol), at
obtained as a white solid. H NMR (400 MHz, [D ]DMSO): δ 11.09 (br
6
(
s, 1H), 8.13 (d, J=2.0, 1H), 7.99 (dd, J =4.8, J =1.6, 1H), 7.81 (d, J=
1
2
8.8, 1H), 7.68 (dd, J =4.0, J =1.2, 1H), 7.17 (dd, J =5.2, J =4.0, 1H),
1
2
1
2
+
3
0°C for 20 h, the desired product 35b (0.216 g, 0.289 mmol, 33%)
7.04 (dd, J =8.8, J =2.0, 1H); ESI-MS(+): m/z 335.08 [M+H] .
1 2
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(
-Bromo-3-((5-chlorothiophene)-2-sulfonamido)pyridine 1-oxide
43b): Following the general method for oxidation, from 43a
1.80 g, 1.86 mmol) and mCPBA (6.84 g, 77.0% Wt, 5.09 mmol), at
J=4.8, 1H), 7.72 (d, J=4.0, 1H), 7.59 (d, J=8.0, 1H), 7.14 (t, J=4.8,
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
13
1H), 6.88 (d, J=7.6, 1H); C NMR (126 MHz, [D ]DMSO): δ 164.3,
6
(
138.6, 137.3, 135.4, 134.3, 134.0, 128.4, 119.4, 112.8; HRMS (ESI-TOF):
À
3 3
À
35°C for 48 h, the desired product 43b (0.876 g, 2.37 mmol, 47%)
m/z calcd for [C H N O S ] : 286.9624 [MÀ H] ; found: 286.9626.
9
7
2
1
was obtained as a yellow-tan solid. H NMR (400 MHz, [D
]DMSO): δ
6
N-(1-Hydroxy-2-thioxo-1,2-dihydropyridin-3-yl)benzamide
(30):
1
7
1
0.85 (br s, 1H), 8.39 (dd, J =6.4, J =0.8, 1H), 7.44 (d, J=4.0, 1H),
.40 (t, J=6.4, 1H), 7.25 (d, J=4.0, 1H), 7.22 (dd, J =8.4, J =0.8,
H); ESI-MS(À ): m/z 369.12 [MÀ H] .
1 2
Following the above general thionation protocol, from 30b
(0.160 g, 0.546 mmol) with KI (0.136 g, 0.819 mmol) in 5 mL
1
2
À
saturated NaSH solution (excess) at 40
0.260 mmol, 48%) was obtained as an off white solid. H NMR
°
C for 16 h, 30 (0.065 g,
1
2
-Bromo-3-((5-chlorothiophene)-2-sulfonamido)pyridine 1-oxide
(
44b): Following the general method for oxidation, from 44a
(400 MHz, [D ]DMSO): δ 10.55 (br s, 1H), 8.55 (d, J=7.6, 1H), 8.21 (d,
6
(
3
1.40 g, 3.52 mmol) and mCPBA (3.94 g, 77.0% Wt, 17.6 mmol), at
5°C for 6 d, the desired product 44b (0.497 g, 1.20 mmol, 34%)
J=7.2, 1H), 7.96 (d, J=7.6, 2H), 7.69–7.60 (m, 3H), 6.98 (t, J=7.6,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
13
1H); C NMR (126 MHz, [D ]DMSO): δ 165.2, 163.4, 138.7, 134.1,
6
1
was obtained as a tan solid. H NMR (400 MHz, [D ]DMSO): δ 10.81
(br s, 1H), 8.37 (d, J=6.4, 1H), 7.42–7.39 (m, 2H), 7.34 (d, J=4.0, 1H),
133.1, 132.6, 129.7, 127.5, 119.1, 113.4; HRMS (ESI-TOF): m/z calcd
6
À
À
for [C H N O S] : 245.0390 [MÀ H] ; found: 245.0391.
1
2
9
2
2
À
7.21 (d, J=8.4, 1H); ESI-MS(À ): m/z 413.02 [MÀ H] .
N-(1-Hydroxy-2-thioxo-1,2-dihydropyridin-3-yl)thiophene-2-car-
boxamide (31): Following the above general thionation protocol,
from 31b (0.350 g, 1.17 mmol) with KI (0.291 g, 1.76 mmol) in 5 mL
saturated NaSH solution (excess) at 40°C for 16 h, 31 (0.130 g,
3
-(Benzo[c][1,2,5]thiadiazole-4-sulfonamido)-2-bromopyridine 1-
oxide (45b): Following the general method for oxidation, from 45a
(0.350 g, 0.943 mmol) and mCPBA (1.06 g, 77.0% Wt, 4.71 mmol), at
1
3
0°C for 5 d, the desired product 45b (0.147 g, 0.380 mmol, 40%)
0.515 mmol, 44%) was obtained as a gray solid. H NMR (400 MHz,
1
was obtained as a tan solid. H NMR (400 MHz, [D ]DMSO): δ 8.26
[D ]DMSO): δ 10.42 (br s, 1H), 8.41 (d, J=7.6, 1H), 8.21 (d, J=6.8,
6
6
(d, J=8.8, 1H), 8.08 (d, J=7.2, 1H), 7.94–7.92 (m, 1H), 7.60 (t, J=6.8,
1H), 7.96 (d, J=4.8, 1H), 7.82 (d, J=3.2, 1H), 7.27 (t, J=3.6, 1H), 6.96
13
1H), 7.17 (d, J=8.4, 1H), 7.10–7.06 (m, 1H); ESI-MS(+): m/z 389.11
(t, J=6.8, 1H); C NMR (126 MHz, [D ]DMSO): δ 163.3, 160.0, 138.7,
6
+
[M+H] .
138.4, 133.5, 132.6, 130.0, 129.2, 119.3, 113.3; HRMS (ESI-TOF): m/z
À
2 2
À
calcd for [C H N O S ] : 250.9954 [MÀ H] ; found: 250.9957.
1
0
7
2
2
-Bromo-3-((5-phenylthiophene)-2-sulfonamido)pyridine 1-oxide
(
46b): Following the general method for oxidation, from 46a
N-(1-Hydroxy-2-thioxo-1,2-dihydropyridin-3-yl)-5-meth-
(
1.05 g, 2.66 mmol) and mCPBA (2.98 g, 77.0% Wt, 13.3 mmol), at
ylthiophene-2-sulfonamide (32): Following the above general
thionation protocol, from 32b (0.200 g, 0.573 mmol) with KI
(0.143 g, 0.859 mmol) in 5 mL saturated NaSH solution (excess) at
35°C for 16 h, the desired product 46b (0.590 g, 1.43 mmol, 54%)
1
was obtained as a pale yellow solid. H NMR (400 MHz, [D
]DMSO):
6
δ 10.73 (br s, 1H), 8.36 (d, J=6.4, 1H), 7.71 (d, J=7.6, 2H), 7.57–7.54
100°C for 8 h, 32 (0.058 g, 0.18 mmol, 33%), was obtained as a pale
1
(
4
m, 2H), 7.47–7.39 (m, 4H), 7.25 (d, J=8.4, 1H); ESI-MS(À ): m/z
yellow solid. H NMR (400 MHz, [D ]DMSO): δ 9.44 (br s, 1H), 8.17 (d,
6
À
13
09.17 [MÀ H] .
J=6.8, 1H), 7.58–7.56 (m, 2H), 6.90–6.87 (m, 2H), 2.45 (s, 3H);
C
NMR (126 MHz, [D ]DMSO): δ 164.0, 149.7, 137.4, 135.3, 134.8, 133.9,
6
1
27.0, 118.7, 112.9, 15.6; HRMS (ESI-TOF): m/z calcd for
À
3 3
À
Thionation
[C H N O S ] : 300.9781 [MÀ H] ; found: 300.9780.
1
0
9
2
General thionation protocol: The starting material and 1.5
equivalents of KI were dissolved in 5 mL DI water with a minimal
amount of MeOH to dissolve as needed. Then 5 mL of saturated
freshly prepared sodium hydrogen sulfide solution (excess) was
added to the solution containing the starting material. The reaction
mixture was then heated at 50–100°C for 3–16 h. Upon reaction
completion as indicated by TLC, the reaction mixture was cooled to
N-(1-Hydroxy-2-thioxo-1,2-dihydropyridin-3-yl)-4-meth-
ylthiophene-2-sulfonamide (33): Following the above general
thionation protocol, from 33b (0.190 g, 0.544 mmol) with KI
(0.135 g, 0.816 mmol) in 5 mL saturated NaSH solution (excess) at
100°C for 5 h, 33 (0.070 g, 0.230 mmol, 43%) was obtained as a
1
pale yellow solid. H NMR (400 MHz, [D ]DMSO): δ 9.47 (br s, 1H),
6
8.17 (dd, J =6.8, J =1.2, 1H), 7.58–7.56 (m, 3H), 6.89 (t, J=8.0, 1H),
1
2
13
0
°C, and slowly quenched with 6 M HCl. CAUTION! The neutraliza-
2.16 (s, 3H); C NMR (126 MHz, CD OD): δ 161.6, 138.4, 138.3, 137.2,
3
tion of NaSH using acid generates H S gas, which is both highly
flammable and highly toxic by inhalation; only perform neutraliza-
tion in a well-vented fume hood. After waiting ~5 min to allow the
resultant H S gas to evolve and disperse, the resulting mixture was
extracted into organic using 3×15 mL ethyl acetate. The combined
organic was washed with aqueous 1 M Na S O , until the aqueous
went from cloudy to clear. Then the organic was washed with brine,
dried over magnesium sulfate, and the solids were filtered off and
discarded. The filtrate was concentrated under reduced pressure,
and the remaining residue was then purified by column chroma-
tography, using a gradient of 100% hexanes to 100% CH Cl , and
then to 15% MeOH in CH Cl . The desired product typically eluted
around 80% CH Cl2 in hexanes. When necessary, the collected
product was recrystallized from 7:3 IPA to water. The crystals were
collected via vacuum filtration and rinsed with a small amount of
cold IPA to obtain the desired final products.
134.8, 129.4, 128.8, 118.9, 111.9, 13.9; HRMS (ESI-TOF): m/z calcd for
2
À
3 3
À
[C H N O S ] : 300.9781 [MÀ H] ; found: 300.9778.
1
0
9
2
N-(1-Hydroxy-5-methyl-2-thioxo-1,2-dihydropyridin-3-yl)
2
thiophene-2-sulfonamide (34): Following the above general thio-
nation protocol, from 34b (0.100 g, 0.286 mmol) with KI (0.071 g,
0.430 mmol) in 3 mL saturated NaSH solution (excess) at 75°C for
2
2
3
3
h, 34 (0.0057 g, 0.019 mmol, 7%) was obtained as a very pale
1
yellow solid. H NMR (400 MHz, [D ]acetone): δ 8.65 (br s, 1H), 8.17
6
(
s, 1H), 7.92 (d, J=4.8, 1H), 7.77 (d, J=4.0, 1H), 7.63 (s, 1H), 7.16 (t,
13
J=4.0, 1H), 2.34 (s, 3H); C NMR (126 MHz, [D ]DMSO): δ 165.8,
6
2
2
1
57.3, 134.7, 128.0, 116.6, 110.2, 67.3, 53.3, 39.8, 20.3. HRMS (ESI-
2
2
À
3 3
À
TOF): m/z calcd for [C H N O S ] : 300.9781 [MÀ H] ; found:
1
0
9
2
2
300.9779.
N-(1-Hydroxy-6-methyl-2-thioxo-1,2-dihydropyridin-3-yl)
thiophene-2-sulfonamide (35): Following the above general thio-
nation protocol, from 35b (0.210 g, 0.601 mmol) with KI (0.150 g,
N-(1-Hydroxy-2-thioxo-1,2-dihydropyridin-3-yl)thiophene-2-sulfo-
namide (29): Following the above general thionation protocol,
from 29b (0.500 g, 1.49 mmol) with KI (0.371 g, 2.24 mmol) in 5 mL
0
3
.902 mmol) in 3 mL saturated NaSH solution (excess) at 75°C for
h, 35 (0.011 g, 0.036 mmol, 6%) was obtained as a pale yellow
1
solid. H NMR (400 MHz, [D ]DMSO): δ 12.49 (br s, 1H), 9.35 (br s,
saturated NaSH solution (excess) at 100°C for 8 h, 29 (0.160 g,
6
1
1H), 7.95 (d, J=4.8, 1H), 7.68 (d, J=4.0, 1H), 7.55 (d, J=8.0, 1H),
0
.555 mmol, 37%) was obtained as a pale yellow solid. H NMR
(400 MHz, [D ]DMSO): δ 9.50 (br s, 1H), 8.17 (d, J=6.8, 1H), 7.98 (d,
6
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ChemMedChem
7
.12 (t, J=4.0, 1H), 6.85 (d, J=8.0, 1H), 2.41 (s, 3H); HRMS (ESI-TOF):
143.0, 139.6, 133.5, 133.4, 133.2, 127.3, 116.7, 105.1; HRMS (ESI-TOF):
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
À
À
À
3 3
À
m/z calcd for [C H N O S ] : 300.9781 [MÀ H] ; found: 300.9778.
N-(1-Hydroxy-2-thioxo-1,2-dihydropyridin-3-yl)
cyclopropanesulfonamide (36): Following the above general
thionation protocol, from 36b (0.460 g, 0.157 mmol) with KI
m/z calcd for [C H N O S ] : 286.9624 [MÀ H] ; found: 286.9627.
10
9
2
3
3
9
7
2
N-(1-Hydroxy-6-thioxo-1,6-dihydropyridin-3-yl)thiophene-2-sulfo-
namide (42): Following the above general thionation protocol,
from 42b (0.310 g, 0.925 mmol) with KI (0.230 g, 2.39 mmol) in
5 mL saturated NaSH solution (excess) at 100°C for 8 h, 42 (0.028 g,
(0.391 g, 2.35 mmol) in 5 mL saturated NaSH solution (excess) at
1
100°C for 8 h, 36 (0.132 g, 0.534 mmol, 34%) was obtained as a
0.097 mmol, 10%) was obtained as a pale yellow solid. H NMR
1
pale yellow solid. H NMR (400 MHz, [D
]DMSO): δ 8.94 (br s, 1H),
6
(400 MHz, [D ]acetone): δ 8.31 (dd, J =2.4, J =0.4, 1H), 7.93 (dd,
6
1
2
8
.22 (dd, J =6.8, J =1.2, 1H), 7.58 (dd, J =7.6, J =0.8, 1H), 6.90 (t,
J =4.8, J =1.2, 1H), 7.64 (dd, J =3.6, J =1.2, 1H), 7.58 (dd, J =9.2,
1 2 1 2 1
1
2
1
2
1
3
J=7.6, 1H), 2.87–2.81 (m, 1H), 1.05–0.95 (m, 4H); C NMR (126 MHz,
CD OD): δ 161.5, 138.1, 128.9, 119.6, 112.1, 29.8, 4.9; HRMS (ESI-
J =0.4, 1H), 7.33 (dd, J =9.2, J =2.4, 1H), 7.19 (dd, J =5.2, J =4.0,
2
1
2
1
2
1
3
1H); C NMR (126 MHz, CD OD): δ 164.8, 139.0, 133.1, 132.8, 131.7,
3
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
À
3 2
À
TOF): m/z calcd for [C H N O S ] : 245.0060 [MÀ H] ; found:
128.6, 127.4, 126.99, 126.2; HRMS (ESI-TOF): m/z calcd for
8
9
2
À
3 3
À
2
45.0061.
[C H N O S ] : 286.9624 [MÀ H] ; found: 286.9626.
9
7
2
1
-((1R,4S)-7,7-Dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)-N-(1-
5-Chloro-N-(1-hydroxy-2-thioxo-1,2-dihydropyridin-3-yl)
hydroxy-2-thioxo-1,2-dihydropyridin-3-yl)methanesulfonamide (37):
Following the above general thionation protocol, from 37b
(0.240 g, 0.669 mmol) with KI (0.167 g, 1.00 mmol) in 5 mL saturated
thiophene-2-sulfonamide (43): Following the above general thio-
nation protocol, from 43b (0.300 g, 0.812 mmol) with KI (0.202 g,
1.22 mmol) in 5 mL saturated NaSH solution (excess) at 65°C for
NaSH solution (excess) at 80°C for 6 h, 37 (0.011 g, 0.031 mmol,
16 h, 43 (0.152 g, 0.471 mmol, 58%) was obtained as a pale yellow
1
1
5
%) was obtained as a dark gray solid. H NMR (400 MHz, [D ]
solid. H NMR (400 MHz, [D ]DMSO): δ 9.64 (br s, 1H), 8.23 (dd, J =
6
6
1
DMSO): δ 11.63 (br s, 1H), 8.45 (br s, 1H), 8.31 (d, J=6.4, 1H), 7.81
6.8, J =0.8, 1H), 7.62–7.57 (m, 2H), 7.23 (d, J=4.0, 1H), 6.89 (t, J=
2
1
3
(d, J=7.6, 1H), 7.11 (t, J=7.2, 1H), 3.66 (d, J=15.2, 1H), 3.30 (d, J=
7.2, 1H); C NMR (126 MHz, CD OD): δ 162.4, 137.5, 137.4, 136.8,
3
1
0.8, 1H), 2.46–2.34 (m, 2H), 2.15–2.13 (m, 1H), 1.94 (d, J=18.4, 2H),
132.9, 130.1, 127.1, 120.6, 111.7; HRMS (ESI-TOF): m/z calcd for
À
3 3
À
1
.82–1.75 (m, 1H), 1.53–1.47 (m, 1H), 1.09 (s, 3H), 0.87 (s, 3H); HRMS
[C H ClN O S ] : 320.9235 [MÀ H] ; found: 320.9234.
9
6
2
À
4 2
À
(ESI-TOF): m/z calcd for [C H N O S ] : 355.0792 [MÀ H] ; found:
15
19
2
5-Bromo-N-(1-hydroxy-2-thioxo-1,2-dihydropyridin-3-yl)
355.0789.
thiophene-2-sulfonamide (44): Following the above general thio-
nation protocol, from 44b (0.300 g, 0.724 mmol) with KI (0.180 g,
1.09 mmol) in 5 mL saturated NaSH solution (excess) at 75°C for
N-(1-Hydroxy-2-thioxo-1,2-dihydropyridin-3-yl)-1-methyl-1H-
pyrazole-3-sulfonamide (38): Following the above general thiona-
tion protocol, from 38b (0.166 g, 0.498 mmol) with KI (0.124 g,
17 h, 44 (0.050 g, 0.14 mmol, 19%) was obtained as a pale yellow
1
0
.747 mmol) in 5 mL saturated NaSH solution (excess) at 100°C for
solid. H NMR (400 MHz, [D ]DMSO): δ 9.62 (br s, 1H), 8.22 (d, J=6.8,
6
1
3
5
h, 38 (0.047 g, 0.17 mmol, 33%) was obtained as a light gray solid.
1H), 7.59–7.56 (m, 2H), 7.32 (d, J=3.6, 1H), 6.89 (t, J=7.2, 1H); C
1
H NMR (400 MHz, [D ]DMSO): δ 9.35 (br s, 1H), 8.14 (d, J=6.8, 1H),
NMR (126 MHz, [D ]DMSO): δ 164.6, 139.7, 137.0, 134.9, 134.4, 132.1,
6
6
À
7
.88 (d, J=1.6, 1H), 7.54 (d, J=7.6, 1H), 6.87 (t, J=7.6, 1H), 6.78 (d,
121.0, 120.8, 112.8; HRMS (ESI-TOF): m/z calcd for [C H BrN O S ] :
364.8729 [MÀ H] ; found: 364.8728.
9
6
2
3 3
13
À
J=1.6, 1H), 3.87 (s, 3H); C NMR (126 MHz, CD OD): δ 160.8, 148.6,
3
137.3, 132.7, 128.7, 117.9, 111.9, 107.1, 38.5; HRMS (ESI-TOF): m/z
calcd for [C
À
À
N-(1-Hydroxy-2-thioxo-1,2-dihydropyridin-3-yl)benzo[c][1,2,5]
H
9
N
4
O
3
S
] : 285.0122 [MÀ H] ; found: 285.0122.
9
2
thiadiazole-4-sulfonamide (45): Following the above general
thionation protocol, from 45b (0.150 g, 0.387 mmol) with KI
(0.0965 g, 0.581 mmol) in 5 mL saturated NaSH solution (excess) at
N-(1-Hydroxy-2-thioxo-1,2-dihydropyridin-3-yl)-1-methyl-1H-
pyrazole-4-sulfonamide (39): Following the above general thiona-
tion protocol, from 39b (0.210 g, 0.630 mmol) with KI (0.157 g,
75°C for 17 h, 45 (0.018 g, 0.053 mmol, 14%) was obtained as a
1
0
.945 mmol) in 5 mL saturated NaSH solution (excess) at 100°C for
deep yellow solid. H NMR (400 MHz, [D ]DMSO): δ 9.73 (br s, 1H),
6
5
h, 39 (0.057 g, 0.20 mmol, 32%) was obtained as a dark gray solid.
8.40 (d, J=8.8, 1H), 8.32 (d, J=7.2, 1H), 8.06 (d, J=6.8, 1H), 7.84 (t,
1
13
H NMR (400 MHz, [D ]DMSO): δ 9.24 (br s, 1H), 8.14 (d, J=6.8, 1H),
J=6.8, 1H), 7.56 (d, J=8.0, 1H), 6.78 (t, J=6.8, 1H); C NMR
6
8
.44 (s, 1H), 8.14 (d, J=6.8, 1H), 7.85 (s, 1H), 7.53 (d, J=8.0, 1H), 6.86
(126 MHz, CD OD): δ 156.7, 153.4, 146.8, 135.0, 129.8, 127.8, 125.8,
3
13
(t, J=7.6, 1H), 3.82 (s, 3H); C NMR (126 MHz, CD OD): δ 161.3,
125.4, 124.3, 115.7, 110.3; HRMS (ESI-TOF): m/z calcd for
3
À
3 3
À
138.4, 137.4, 133.1, 129.1, 120.6, 118.7, 112.0, 38.2; HRMS (ESI-TOF):
m/z calcd for [C
[C H N O S ] : 338.9686 [MÀ H] ; found: 338.9683.
1
1
7
4
À
À
H
9
N
4
O S
3
] : 285.0122 [MÀ H] ; found: 285.0121.
9
2
N-(1-Hydroxy-2-thioxo-1,2-dihydropyridin-3-yl)-5-phenylthio-
phene-2-sulfonamide (46): Following the above general thionation
protocol, from 46b (0.400 g, 0.973 mmol) with KI (0.242 g,
1.46 mmol) in 5 mL saturated NaSH solution (excess) at 60°C for
N-(1-Hydroxy-2-thioxo-1,2-dihydropyridin-3-yl)thiophene-3-sulfo-
namide (40): Following the above general thionation protocol,
from 40b (0.250 g, 0.746 mmol) with KI (0.186 g, 1.12 mmol) in
5
0
mL saturated NaSH solution (excess) at 100°C for 8 h, 40 (0.053 g,
16 h, 46 (0.153 g, 0.420 mmol, 43%) was obtained as a light yellow
1
1
.18 mmol, 25%) was obtained as a pale yellow solid. H NMR
solid. H NMR (400 MHz, [D ]DMSO): δ 9.58 (br s, 1H), 8.19 (d, J=7.2,
6
(400 MHz, [D ]DMSO): δ 9.36 (br s, 1H), 8.40–8.39 (m, 1H), 8.14 (d,
1H), 7.74 (d, J=3.6, 1H), 7.70–7.66 (m, 2H), 7.62 (d, J=8.0, 1H), 7.55
6
13
J=6.8, 1H), 7.73–7.71 (m, 1H), 7.54 (d, J=8.0, 1H), 7.35 (d, J=5.2,
(d, J=4.0, 1H), 7.51–7.39 (m, 4H), 6.90 (t, J=7.6, 1H); C NMR
À
1
H), 6.84 (t, J=8.0, 1H); HRMS (ESI-TOF): m/z calcd for [C H N O S ] :
(126 MHz, CD OD): δ 161.9, 152.1, 137.1, 137.1, 134.2, 132.3, 129.9,
9
7
2
3
3
3
À
286.9624 [MÀ H] ; found: 286.9623.
129.0, 128.9, 125.8, 123.0, 119.2, 111.9; HRMS (ESI-TOF): m/z calcd
À
À
for [C15
H
N
2
O
3
S
3
] : 362.9937 [MÀ H] ; found: 362.9935.
11
N-(1-Hydroxy-2-thioxo-1,2-dihydropyridin-4-yl)thiophene-2-sulfo-
namide (41): Following the above general thionation protocol,
from 41b (0.290 g, 0.865 mmol) with KI (0.215 g, 2.30 mmol) in
N-(2-Thioxo-1,2-dihydropyridin-3-yl)methanesulfonamide (47): To
a solution of 47a (0.500 g, 50%Wt, 0.996 mmol) in 15 mL DMF,
thiourea (1.82 g, 23.9 mmol) was added in one portion, and the
reaction was placed under argon and heated to reflux at 150°C for
5 mL saturated NaSH solution (excess) at 100°C for 8 h, 41 (0.083 g,
1
0
.288 mmol, 33%) was obtained as a very pale yellow solid. H NMR
(400 MHz, [D ]DMSO): δ 11.95 (br s, 1H), 11.44 (br s, 1H), 8.25 (dd,
12 h, after which the reaction mixture had changed color to dark
6
J =7.2, J =3.6, 1H), 8.04–8.02 (m, 1H), 7.76–7.74 (m, 1H), 7.22–7.18
brown. Then the reaction was cooled to 110°C, and 6 M HCl
1
2
13
(m, 2H), 6.64–6.62 (m, 1H); C NMR (126 MHz, CD OD): δ: 167.9,
(15 mL, 90 mmol) was added in one portion; the reaction was
3
heated at 110°C for an additional 2 h. Then the reaction mixture
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was cooled to 25°C, diluted with 50 mL brine, and extracted into
organic using 3×15 mL ethyl acetate. The combined organic was
washed with an additional 15 mL brine, dried over magnesium
sulfate, and the solids were filtered off and discarded. The filtrate
was concentrated under reduced pressure, and the resulting crude
was purified by column chromatography, running gradient from
Molecular modeling
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
The in silico model of 8 bound to the IDE active site was prepared
by using the superpose function within the Molecular Operating
Environment (MOE) software suite to align the structure of Zn-
Ph,Me
[37]
bound 8 from the structure of [(Tp
)Zn(8)] with the hydroxa-
mic acid of 2 from PDB ID: 4NXO. The atoms of the thione in 8 were
aligned with the carbonyl atoms of the hydroxamate group in 2,
and likewise, the hydroxy atoms of 8 were aligned with the hydroxy
atoms of 2, resulting in a model of 8 bound to the Zn within the
IDE active site. Upon modeling 8 in the IDE active site, the image
was visually rendered using PyMOL.
1
00% hexanes to 100% ethyl acetate, and then from 100% CH Cl
2 2
to 15% MeOH in CH Cl . The desired product eluted in 85% ethyl
2
2
acetate in hexanes. Like fractions of the desired product were
combined and concentrated under reduced pressure. The resulting
solid was recrystallized from 3:1 ethyl acetate to hexanes, and the
solid was collected via vacuum filtration and washed with hexanes
2
+
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
1
to obtain 47 (0.081 g, 0.40 mmol, 40%) as a pale yellow solid. H
NMR (400 MHz, [D ]DMSO): δ 8.71 (br s, 1H), 7.61–7.58 (m, 2H), 6.90
6
(
t, J=6.8, 1H), 3.16 (s, 3H); HRMS (ESI-TOF): m/z calcd for Acknowledgements
+
+
C H N O S : 205.0100 [M+H] ; found: 205.0103.
6 9 2 2 2
N-(2-Thioxo-1,2-dihydropyridin-3-yl)thiophene-2-sulfonamide
48): Following the same protocol used in 47, from 29a (0.500 g,
.57 mmol) and thiourea (1.43 g, 18.8 mmol) the desired 48
The authors would like to thank Dr. Yongxuan Su for mass
spectrometry sample analysis at the U.C. San Diego Chemistry and
Biochemistry Mass Spectrometry Facility. We also thank Dr. Cy V.
Credille for helpful discussions. This work was supported by
National Institutes of Health grant R01GM115617, and the
Chemical Biology Interfaces at U.C. San Diego training grant
T32GM112584-01. R.W.S. was supported, in part, by the Graduate
Research Fellowship Program (GRFP) from the National Science
Foundation (DGE-1650112).
(
1
1
(
0.094 g, 0.35 mmol, 22%) was obtained as a deep yellow solid. H
NMR (400 MHz, [D ]DMSO): δ 9.27 (br s, 1H), 7.98 (d, J=4.8, 1H),
.73 (d, J=0.8, 1H), 7.65 (d, J=7.6, 1H), 7.57 (d, J=6.0, 1H), 7.16–
6
7
7
1
1
3
.15 (m, 1H), 6.88 (d, J=7.6, 1H); C NMR (126 MHz, [D ]DMSO): δ
6
68.3, 138.6, 136.4, 135.3, 134.3, 133.8, 128.4, 122.6, 114.2; HRMS
À
2 3
À
(
2
ESI-TOF): m/z calcd for C H N O S : 270.9675 [MÀ H] ; found:
9
7
2
70.9675.
Activity assays
Conflict of Interests
The degradation of Aβ, glucagon, amylin and insulin was quantified
by fluorescence polarization-based assays using fluorescein-labeled
[
38–39]
S.M.C. is a cofounder of and has an equity interest in Cleave
Therapeutics and Forge Therapeutics, companies that might
potentially benefit from the research results. S.M.C. also serves
on the Scientific Advisory Board for Forge Therapeutics. The
terms of this arrangement have been reviewed and approved
by the University of California, San Diego in accordance with its
conflict of interests policies.
and biotinylated peptides, as described.
For substrate V,
fluorescence dequenching was monitored continuously (λ =
ex
3
85 nm; λ =425 nm). Bacterially expressed, recombinant human
em
[40]
IDE, was used in all assays. Reactions were performed in Buffer A
(
PBS supplemented with 0.05% BSA and 0.5 mM DTT) and, for
discontinuous assays, terminated by addition of 10-fold excess
avidin relative to the substrate concentration and 2 mM 1,10-
phenanthroline. A peptidic inhibitor of IDE, P12-3 A was used as
a positive control. All MBP fragments were tested in at least 3
independent experiments, with results normalized to DMSO-only
and no-enzyme controls. All activity assays were conducted in 384-
[
41]
Keywords: fragment-based drug discovery · high-throughput
screening · insulin-degrading enzymes · medicinal chemistry ·
metalloenzymes
e
well format on a SpectraMAX M5 multilable plate reader (Molecular
Devices). IC50 values were obtained from normalized activity data
by curve fitting within Prism v8.1 (GraphPad Software Inc.) and
converted to Ki values using the Cheng-Prusoff equation and
[
2,38–39]
published K values for each substrate.
M
hCAII assay. The plasmid for recombinant expression of hCAII with
a T7 RNA polymerase promoter and ampicillin resistance gene
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1420.
land). The protein for this activity assays was expressed in BL21
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9
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50 mM HEPES pH 8.0. Compounds were added from a 50 mM
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p-Nitrophenyl acetate was used as the substrate (500 μM final
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20 min at 1 min intervals. Percent inhibition was determined by
comparing the activity of wells containing inhibitors to the activity
of those without.
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FULL PAPERS
Metal-binding inhibitors of the
human metalloenzyme insulin-
R. N. Adamek, C. N. Suire, R. W. Stokes,
M. K. Brizuela, Prof. S. M. Cohen*,
Prof. M. A. Leissring*
degrading enzyme (IDE) were discov-
ered by screening a focused library
that revealed 3-sulfonamide-1-hy-
droxypyridine-2-thione as a novel
inhibitor of insulin degrading
enzyme (IDE). Structure-activity rela-
tionship (SAR) studies led to several
thiophene-sulfonamide-hydroxypyri-
dinethione compounds with broad-
spectrum activity against several IDE
substrates, thus demonstrating the
potential of these compounds to be
used as pharmacological tools for
probing IDE's biological functions.
1
– 14
Hydroxypyridinethione Inhibitors of
Human Insulin-Degrading Enzyme
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