Optimization and Evaluation of 5-Styryl-Oxathiazol-2-one Mycobacterium tuberculosis Proteasome Inhibitors as Potential Antitubercular Agents

Article abstract of DOI:10.1002/open.201500001

This is the first report of 5-styryl-oxathiazol-2-ones as inhibitors of the Mycobacterium tuberculosis (Mtb) proteasome. As part of the study, the structure-activity relationship of oxathiazolones as Mtb proteasome inhibitors has been investigated. Furthermore, the prepared compounds displayed a good selectivity profile for Mtb compared to the human proteasome. The 5-styryl-oxathiazol-2-one inhibitors identified showed little activity against replicating Mtb, but were rapidly bactericidal against nonreplicating bacteria. (E)-5-(4-Chlorostyryl)-1,3,4-oxathiazol-2-one) was most effective, reducing the colony-forming units (CFU)/mL below the detection limit in only seven days at all concentrations tested. The results suggest that this new class of Mtb proteasome inhibitors has the potential to be further developed into novel antitubercular agents for synergistic combination therapies with existing drugs.

Full text of DOI:10.1002/open.201500001

DOI: 10.1002/open.201500001
Optimization and Evaluation of 5-Styryl-Oxathiazol-2-one
Mycobacterium tuberculosis Proteasome Inhibitors as
Potential Antitubercular Agents
Francesco Russo,[a] Johan Gising,[a] Linda Škerbladh,[a] Annette K. Roos,[b] Agata Naworyta,[c]
Sherry L. Mowbray,[b] Anders Sokolowski,[a] Ian Henderson,[d] Torey Alling,[e] Mai A. Bailey,[e]
Megan Files,[e] Tanya Parish,[e] Anders KarlØn,[a] and Mats Larhed*[f]
This is the first report of 5-styryl-oxathiazol-2-ones as inhibitors
of the Mycobacterium tuberculosis (Mtb) proteasome. As part of
the study, the structure–activity relationship of oxathiazolones
as Mtb proteasome inhibitors has been investigated. Further-
more, the prepared compounds displayed a good selectivity
profile for Mtb compared to the human proteasome. The
5-styryl-oxathiazol-2-one inhibitors identified showed little ac-
tivity against replicating Mtb, but were rapidly bactericidal
against nonreplicating bacteria. (E)-5-(4-Chlorostyryl)-1,3,4-oxa-
thiazol-2-one) was most effective, reducing the colony-forming
units (CFU)/mL below the detection limit in only seven days at
all concentrations tested. The results suggest that this new
class of Mtb proteasome inhibitors has the potential to be fur-
ther developed into novel antitubercular agents for synergistic
combination therapies with existing drugs.
Introduction
Tuberculosis (TB) has been declared a global health emergency
by the World Health Organization (WHO). It is estimated that
about one third of the world’s population is currently infected
with the responsible actinobacteria Mycobacterium tuberculosis
(Mtb).[1] Resistance of Mtb to available antibiotics has increased
drastically, and multidrug-resistant (MDR) strains of Mtb as well
as extensively drug-resistant (XDR) strains are becoming
a major health problem worldwide.[1] Furthermore, cases have
recently been reported where no available first- and second-
line drugs are effective, and this has led to the term totally
drug-resistant (TDR) TB.[2] Resistant strains of Mtb require
longer treatment, which may lead to interruption of therapy
and, in turn, lead to further drug resistance. The usual drug
regimen for TB at present consists of antibiotics discovered
more than 60 years ago, and includes isoniazid, rifampicin, pyr-
azinamide, and ethambutol.[3] These antibiotics act by disrup-
tion of cell wall synthesis[4] and inhibition of RNA synthesis.[4c,5]
It was quickly discovered that monotherapy led to resistance
in the bacteria, and therefore the recommended TB treatment
today utilizes a combination of the four first-line drugs for two
months, and thereafter a pharmacotherapy with rifampicin and
isoniazid for an additional four months.[3] Considering the long
treatment time, the adverse effects associated with the drugs,
and the problems with resistant strains, it is clear that there is
a great need for novel antibiotics that possess new antituber-
cular modes of action and that can be used in combination
with existing drugs.
[a] Dr. F. Russo, Dr. J. Gising, L. Škerbladh, Dr. A. Sokolowski, Prof. A. KarlØn
Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry
BMC, Uppsala University, Box 574, 751 23 Uppsala (Sweden)
[b] Dr. A. K. Roos, Prof. S. L. Mowbray
Department of Cell and Molecular Biology, Science for Life Laboratory
BMC, Uppsala University, Box 596, 751 24 Uppsala (Sweden)
[c] Dr. A. Naworyta
Department of Cell and Molecular Biology, BMC, Uppsala University
Box 596, 751 24 Uppsala (Sweden)
Proteasomes are responsible for degrading proteins, and so
help maintain intracellular protein homeostasis.[6] The human
proteasome consists of a cylindrical 26S particle composed of
a 20S core catalytic component capped at one or both ends
with a 19S regulatory subunit, which recognizes and binds the
substrate protein. In eukaryotic proteasomes, the 20S core par-
ticle is composed of four heptameric rings, of which the two
inner rings are made up of seven different b-subunits, with
only three of them responsible for the proteolytic activity (b1,
b2, b5, with peptidyl-glutamyl-peptide-hydrolyzing, trypsin-like
and chymotrypsin-like activities, respectively).[7] The Mtb pro-
teasome displays the same overall structure as the protea-
somes of eukaryotic systems.[8] However, prokaryotic 20S pro-
[d] Dr. I. Henderson
Medivir AB, PO Box 1086, 141 22 Huddinge (Sweden)
[e] T. Alling, M. A. Bailey, M. Files, Prof. T. Parish
TB Discovery Research, Infectious Disease Research Institute
Seattle, WA 98102 (USA)
[f] Prof. M. Larhed
Department of Medicinal Chemistry, Science for Life Laboratory
BMC, Uppsala University, Box 574, 751 23 Uppsala (Sweden)
E-mail: mats.larhed@orgfarm.uu.se
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are
made.
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teasomes, including those from Mtb, have been shown to have
open cylindrical ends, in contrast to the tighter ends of eukary-
otic proteasomes, and possess only one type of b-subunit, usu-
ally resembling the eukaryotic b5 chymotrypsin-like struc-
ture.[8–9] Proteolytic activity involves the b-hydroxyl group of
the N-terminal threonine, which is present in the active site of
proteasome subunits.[7a–d]
They demonstrated that the oxathiazol-2-one moiety acts as
an electrophilic warhead, such that the active-site nucleophile,
the N-terminal threonine residue, is effectively cyclocarbonylat-
ed by an irreversible covalent attack on the heterocycle.[7c,8b]
Moreover, oxathiazolones have been reported to have anti-
mycobacterial activity when they were exploited as carboxylic
acid bioisosteres in substituted pyridines and pyrazines.[17] In
an attempt to identify a new generation of species-selective
proteasome inhibitors, Gryder et al. investigated the effect of
replacing the boronate group on the dipeptide backbone of
Bortezomib with an oxathiazol-2-one moiety. Unfortunately,
the resulting compound was not active on the Mtb protea-
some and only slightly active on the human 20S protea-
some.[18] More recently, Yang et al. assessed the antitubercular
activity of a series of GL5-type oxathiazol-2-ones, together with
their dithiazol-3-one analogues, on the virulent Mtb H37Rv
strain, achieving a lowest minimum inhibitory concentration
(MIC) value of 4 mgmLÀ1 (15 mm) for HT1171.[16g] These com-
pounds also possessed mycobacterial cell wall permeation
properties, and were active against nonreplicating Mtb. They
have therefore been proposed as highly interesting antituber-
cular agents for the synergistic combination treatment of TB.[7c]
In the present study, we further investigate the structure–ac-
tivity relationships of 1,3,4-oxathiazol-2-one derivatives with re-
spect to both their potency as Mtb proteasome inhibitors and
their selectivity over the chymotrypsin-like catalytic activity of
the human proteasome as an early assessment of potential
toxicity. Furthermore, the compounds were optimized with
regard to solubility and stability. Finally, activity against repli-
cating and nonreplicating Mtb and cytotoxicity to mammalian
cells are reported for a collection of selective 5-styryl-1,3,4-oxa-
thiazol-2-one inhibitors.
In many diseases, such as cancer, auto-immune diseases,
and neurodegenerative diseases, cells accumulate various pro-
teins, and the human proteasome has thus emerged as a prom-
ising therapeutic target.[6a,7a] The first human proteasome in-
hibitor, Bortezomib (Velcade), was approved for clinical use in
the USA in 2003 and in Europe in 2004 for the treatment of
multiple myeloma (Figure 1). The X-ray structure of the yeast
Figure 1. Structures of the proteasome inhibitors Bortezomib and oxathia-
zol-2-one compounds HT1171 and GL5.
proteasome in complex with the compound was reported in
2006, giving valuable insights into its binding interactions.[10]
Serious toxicity associated with peptidyl boronates, such as
Bortezomib, is mainly due to the inhibition of the b5 subunit
of the human proteasome,[11] precluding the use for treatment
of, for example, infectious diseases. Therefore, the search for
alternative classes of inhibitors with greater selectivity towards
prokaryotic proteasomes is ongoing.
Results and Discussion
Proteasomes have been shown to be of great importance to
Mtb; they help the bacteria survive the nitrosative stress
caused by the host’s immune response[12] and increase their
ability to persist in infected mice.[13] Furthermore, the actino-
bacteria are unusual among prokaryotes in that they express
proteasomes, meaning that selective action against bacteria of
this phylum should be possible.[6b,14] It is particularly interesting
that proteasome inhibitors have been shown to be active on
nonreplicating Mtb. This is important since nonreplicating Mtb
(which to a large extent underlie the need for long treatment
times), display reduced protein synthesis, and are left unaffect-
ed by most antitranscription or antitranslation agents currently
used for treatment.[15] Only a few classes of compounds have
proven to be active on nonreplicating Mtb, for example, oxa-
thiazol-2-ones, quinolones, and allylic thiocyanates.[7c,16] Mtb
proteasome inhibitors displaying species selectivity, thus low
associated toxicity, are promising candidates for the develop-
ment of new antitubercular drugs.
In a first effort to investigate the chemical space, a wide variety
of oxathiazol-2-ones were prepared from the corresponding
commercially available amides by treatment with chlorocar-
bonyl sulfenyl chloride at 1008C for 15 min with microwave
(MW) heating (Method A) or at room temperature overnight
(Method B). Isolated products were thereafter evaluated for
proteasome inhibition based on the assay procedures de-
scribed by Lin et al.[7c] (Table 1, entries 1–23). Compounds were
tested for chymotrypsin-like peptidase activity against both
the Mtb 20S open-gate proteasome (in which the alpha subu-
nit has an 8-residue deletion at the N-terminus, as described
by Lin et al.[8a]) and the human 20S proteasome, tracked by
cleavage of the fluorogenic substrate suc-LLVY-7-amino-4-
methylcoumarin.
The 5-aryl-substituted 1,3,4-oxathiazol-2-ones in entries 1–4
(Table 1, 1–4) have previously been reported as inhibitors of
the Mtb proteasome.[19] To further develop the core structure,
we decided to find out if elongated compounds with different
spacers could have a positive impact on the activity. For this
purpose, various tethers between the aryl and the heterocyclic
warhead were introduced in the 5-position, for example,
alkene, alkyne, and methylene linkers. We found that an sp2-
Lin et al. have previously reported that the 1,3,4-oxathiazol-
2-one compounds HT1171 and GL5 (Figure 1) act as suicide-
substrate inhibitors of the Mtb proteasome displaying high se-
lectivity over the human proteasome and other proteases, in-
cluding trypsin, cathepsin B, and matrix metalloproteases.[7c]
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Table 1. Activity, solubility, and stability of proteasome inhibitors.
Entry
Structure
Cmpd
Method, Yield
Mtb proteasome
Human proteasome
IC50 [nM]
Solubility [mM]
63.8
Stability [%][a]
IC50 [nM]
1
2
1
2
A, 43%
1350
2300
6200
4500
97.4
3.6
B, 23%
35.1
3
4
3
4
B, 23%
B, 83%
665
630
15000
11 000
30.9
86.2
15.4
33.5
5
6
5
6
A, 60%
A, 27%
>100000
>100000
>100000
>100000
—
—
—
—
7
8
9
7
8
9
A, 56%
B, 24%
A, 43%
>100000
80500
>100000
1400
—
—
—
—
—
—
>100000
1900
10
11
10
11
A, 27%
A, 39%
>100000
>100000
>100000
>100000
—
—
—
—
12
12
A, 51%
>100000
>100000
—
—
13
14
15
13
14
15
B, 56%
B, 40%
B, 71%
>100000
>100000
>100000
8700
20000
6700
—
—
—
—
—
—
16
17
16
17
B, 58%[b]
B, 40%
>100000
5800
—
—
420
>100000
19.1
38.7
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Table 1. (Continued)
Entry
Structure
Cmpd
Method, Yield
B, 42%
Mtb proteasome
Human proteasome
IC50 [nM]
Solubility [mM]
Stability [%][a]
IC50 [nM]
18
18
640
>100000
0.1
n.d.
19
19
57%[c]
2250
2950
5.7
46.8
20
21
22
20
21
22
B, 24%
B, 8%
1035
27000
38000
4200
9100
24.9
—
89.6
—
B, 81%
>100000
44.1
79.9
23
24
23
24
B, 90%
>100000
14000
—
—
49%[d]
31500
>100000
7.6
8.5
25
26
25
26
51%[d,e]
54500
—
12.3
—
—
—
[f]
—
>100000
>100000
[f]
27
28
27
—
—
>100000
>100000
—
—
—
—
Bortezomib
—
135
(2.56)[g]
General method for the synthesis of 5-substituted oxathiazol-2-ones. Method A reagents and conditions: amide (1.0 equiv), chlorocarbonyl sulfenyl chloride
(1.5 equiv), 1,4-dioxane (4.0 mLmmolÀ1), 1008C MW, 15 min. Method B: amide (1.0 equiv), chlorocarbonyl sulfenyl chloride (2.0 equiv), THF (4.0 mLmmolÀ1),
rt, o/n.
[a] Chemical stability in PBS pH 7.4 at 258C as % remaining after 24 h. [b] The amide was prepared, and the crude sample was used without further purifi-
cation. [c] Reagents and conditions: 14 (1.0 mmol), Dess–Martin periodinane (1.1 mmol), CH2Cl2 (5 mL), 08C, 2 h. [d] General method for the synthesis of 3-
substituted dioxazolones. Reagents and conditions: starting hydroxamic acid (1.0 equiv), 1,1’-carbonyldiimidazole (1.1 equiv), CH3CN (4.0 mLmmolÀ1), 08C,
1 h. [e] The hydroxamic acid was prepared and used as starting material for the dioxazolone synthesis without further purification. [f] Commercially avail-
able compound. [g] Literature value.[18] n.d.=not determined.
hybridized carbon directly coupled to the oxathiazol-2-one
ring in the 5-postion was crucial for the compound’s activity.
Additionally, the introduction of the (E)-alkene linker (en-
tries 17–18) gave inhibitors equally or more potent (17 and 18;
IC50 =420 and 640 nm, respectively) toward the Mtb protea-
some compared to when the aryl was substituted directly onto
the oxathiazol-2-one (1–4). More importantly, this type of com-
pound was not active on the human proteasome (IC50 >
100000 nm). For compounds with an alkyne- or carbonyl-con-
taining spacer (19 and 20), the activities on the Mtb protea-
some were slightly lower compared to olefinic inhibitors 17
and 18. Moreover, these linkers provided less selective inhibi-
tors. When an sp3-hybridized carbon was used as a linker
atom, no activity was obtained against the Mtb proteasome,
and little or no inhibition was seen towards the human protea-
some (entries 9–16). Interestingly, when an ethyl bridge was in-
troduced instead of the double bond between the phenyl and
oxathiazol-2-one (compare moieties 15 and 17), no inhibition
was observed for the Mtb proteasome, but the saturated com-
pound instead gained some activity toward the human protea-
some. This suggests that the (E)-double bond is important
both for the mycobacterial inhibition and the selectivity over
the human proteasome. However, the allowed substitution
pattern of the (E)-alkene linker seems to be slightly constrain-
ed; when an additional phenyl group was introduced onto the
ethylene linker, no activity was detected against the Mtb pro-
teasome (entry 23). Instead, compound 23 became a more
potent inhibitor of the human proteasome.
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The aryl group of the styryl moiety also seems to be impor-
tant for the interaction with the Mtb protein. When a vinyl
group was substituted onto the oxathiazol-2-one, the activity
decreased from 420 nm for 17 to 27000 nm for 21. This pat-
tern was also observed when aliphatic substituents were intro-
duced (5–8). The effect is probably due to the lack of an aryl
group as well as the effect of the hybridization of the carbon
directly attached to the oxathiazol-2-one.
giving a poorer selectivity profile. However, based on the rela-
tive activities for nonstyryl containing 21 and 22 (Table 1, en-
tries 21–22), it is evident that the phenyl group is important
for inhibition of the Mtb proteasome.
Four of the compounds with the best inhibition and selectiv-
ity profiles (Table 2, 30–33) were chosen for further investiga-
tion. For these compounds, the corresponding ortho- and
meta-analogues were synthesized and evaluated in the
enzyme activity assay (Table 2, entries 14–21). The para-substi-
tuted analogues (Table 2, entries 3–6) were more active than
ortho- and meta-substituted analogues on the Mtb protea-
some, with IC50 values in the range of 475–1080 nm. Further-
more, no inhibition of the human proteasome was detected
for these compounds. When substituents were introduced in
the meta-position (Table 2, entries 14–17), inhibitory activity of
the Mtb proteasome decreased (IC50 1450–9900 nm) and
poorer selectivity was observed. The ortho-substituted ana-
logues (entries 18–21), despite the same mesomeric contribu-
tion as the para-analogues on the styryl moiety, showed
almost no inhibition of the Mtb proteasome (IC50 22000–
>100000 nm). The same trend was observed for chloro-func-
tionalized 39 and disubstituted 40 (Table 2, entries 12–13); the
para-substituted compound was more active on the Mtb pro-
teasome and had superior selectivity with regard to the
human proteasome.
To summarize the results depicted in Table 1, it appears that
an aryl group conjugated with the oxathiazolone scaffold is
highly beneficial for the inhibition of the Mtb proteasome (see
products 1–4 and 17–22), and in particular high selectivity
over the human proteasome is caused by the presence of the
(E)-alkene linker (17 and 18). This may also be coupled to the
loss of activity for the unconjugated 15.
To increase the scope of the investigation, we wished to
evaluate other heterocyclic warheads such as the dioxazolones
(Table 1, entries 24–25), but these compounds suffered from
poor stability due to rapid hydrolysis. The oxathiazol-2-one
was also replaced with a boronic acid functionality (Table 1, en-
tries 26–27) in an attempt to mimic the reversible covalently-
bonded warhead used in Bortezomib. However, the simple
boronic acid analogues were completely inactive on both the
Mtb and human proteasomes, suggesting that the oxathiazol-
2-one moiety is a more useful electrophile for the inactivation
of the target.
Overall, the styryl-analogues 28–48 in Table 2 displayed
good stability but poor solubility (0.1–7.1 mm). To hopefully im-
prove the solubility of the compounds, a small series of nitro-
gen-, oxygen- and sulfur-containing heterocyclic derivatives
were prepared (Table 3). The introduction of five-membered,
six-membered, and bicyclic heterocycles into the scaffold gen-
erally improved the solubility with retained chemical stability.
Furthermore, the heterocyclic analogues were active on the
Mtb proteasome, although slightly higher IC50 values were ob-
tained compared to the compounds in Table 2. However, for
the five-membered heterocycle-substituted oxathiazolones, the
compounds were not as selec-
A series of 5-styryl-oxathiazol-2-ones was synthesized to fur-
ther investigate the effect of the phenyl substitution on the
structure–activity relationship of proteasome inhibitors. For
this, we developed a synthetic route where aryl iodides or bro-
mides were used as starting material in a microwave-assisted
Mizoroki–Heck cross-coupling reaction with acrylamide to yield
the corresponding 3-substituted acrylamides.[20, 21] The final 5-
substituted-oxathiazolones were obtained by treating the cor-
responding amides with chlorocarbonyl sulfenyl chloride
(Scheme 1).
tive with regard to the human
proteasome (Table 3, entries 3–5
and 9).
Based on these promising re-
sults for both inhibition and se-
lectivity of the 5-styryl-oxathia-
zol-2-ones, a collection of syn-
Scheme 1. General synthetic route for the preparation of 5-styryl-oxathiazolones.
thesized compounds was select-
ed for evaluation of biological
activity. Active Mtb proteasome
A diverse set of para-substituted 5-styryl-oxathiazolones
were chosen to explore the structural requirements of the
active site (Table 2, entries 1–12). The majority of the com-
pounds displayed the same range of inhibiting activity against
the Mtb proteasome as 17 and 18 (IC50 values between 415–
1700 nm). The results showed no clear trend in activity for elec-
tron-rich and electron-poor aryl substituents, although elec-
tron-poor phenyl groups gave slightly better inhibition of the
Mtb proteasome compared to electron-rich phenyls. The inhibi-
tion of the human proteasome was unfortunately also affected,
inhibitors displaying full to moderate selectivity were tested
against a virulent strain of Mtb (H37Rv). Four 5-aryl-oxathiazol-
2-ones (Table 4, entries 1–4) were also tested for comparison,
as well as three known antitubercular agents as a reference
(entries 30-32). Cytotoxicity for mammalian cells was investigat-
ed using the Vero cell line (African green monkey kidney cells);
the results are reported in Table 4.
The oxathiazolones had little or no activity against replicat-
ing bacteria when compared to the known antitubercular
agents used as reference (entries 30-32 in Table 4). A few com-
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Table 2. Structure–activity relationship of 5-styryl-oxathiazolone inhibitors with the Mtb and human proteasome.
Structure
Mtb proteasome
Human proteasome
IC50 [nM]
Entry
Cmpd
Method, Yield
Solubility [mM]
Stability [%][a]
IC50 [nM]
1
2
28
29
C, 75%; B, 71%
C, 65%; B, 66%
1350
1700
8500
3.9
0.4
1.5
14500
99.8
3
4
30
31
C, 59%; B, 43%
C, 50%; B, 43%
1000
1080
>100000
>100000
1.1
0.1
96.0
98.2
5
6
32
33
D; B, 64%[b]
780
475
>100000
>100000
0.2
0.3
94.8
91.1
D, 67%; B, 69%
7
34
D, 63%; B, 80%
1190
87000
<2.5
96.1
8
35
36
37
D, 60%; B, 68%
D, 49%; B, 80%
D; B, 20%[b]
415
1400
495
27500
26000
41500
0.8
2.4
5.2
97.3
96.9
95.4
9
10
11
38
D; B, 15%[b]
695
32500
4.7
91.4
12
13
39
40
D, 68%; B, 73%
D; B, 11 %[b]
565
785
>100000
0.3
0.1
98.2
98.5
50000
14
15
16
41
42
43
D; A, 35%[b]
E; A, 17%[b]
D; A, 61%[b]
1600
1450
1800
39000
32500
39500
0.7
5
98.1
96.4
96.2
0.2
17
18
19
44
45
46
D; A, 43%[b]
D; A, 32%[b]
E; A, 19%[b]
9900
22000
68500
>100000
>100000
>100000
7.1
1.2
0.8
96
94
98
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Table 2. (Continued)
Structure
Mtb proteasome
Human proteasome
IC50 [nM]
Entry
Cmpd
Method, Yield
Solubility [mM]
Stability [%][a]
IC50 [nM]
20
21
47
48
D; A, 38%[b]
E; A, 32%[b]
>100000
>100000
>100000
>100000
0.3
0.4
95.7
97.7
General method for the synthesis of 2-substituted acrylamide. Method C reagents and conditions: starting aryl iodide (1.0 equiv), acrylamide (2.0 equiv),
Pd(OAc)2 (0.05 equiv), tri-tert-butylphosphonium tetrafluoroborate (0.10 equiv), Et3N (3.0 equiv), CH3CN (4.0 mLmmolÀ1), 1208C MW, 15 min. Method D re-
agents and conditions:. starting aryl iodide (1.0 equiv), acrylamide (2.0 equiv) and Pd(OAc)2 (0.05 equiv), Et3N (3.0 equiv), CH3CN (4.0 mLmmolÀ1), 1208C MW,
15 min. Method E reagents and conditions: starting aryl bromide (1.0 equiv), acrylamide (1.5 equiv) and trans-bis(acetato)bis[o-(di-o-tolylphosphino)benzyl]di-
palladium(II) (0.05 equiv), NaOAc (3.0 equiv), DMF (4.0 mLmmolÀ1), 1408C MW, 15 min.
[a] Chemical stability in PBS pH 7.4 at 258C; as % remaining after 24 h. [b] Isolated yield over two steps.
Table 3. Optimization of 5-styryl-oxathiazolone inhibitors with regard to solubility.
Structure
Mtb proteasome
IC50 [nM]
Human proteasome
IC50 [nM]
Entry
1
Cmpd
Method, Yield
D; B, 7%[b]
Solubility [mM]
Stability [%][a]
49
2750
77000
84.7
91.6
2
3
4
50
51
52
D; A, 39%[b]
15000
2100
1150
29500
18500
11 300
2.1
97.7
97.6
99.1
C, 46%; B, 45%
C, 75%; B, 53%
22.6
19.7
5
6
7
53
54
55
C; B, 5%[b]
E; A, 3%[b]
E; A, 15%[b]
735
3300
56.1
64.6
70.6
88.9
n.d.
n.d.
2350
1350
>10000[c]
>10000[c]
8
9
56
57
E; B, 4%[b]
2100
1200
>10000[c]
2.4
4.1
n.d.
n.d.
–, 31%;[d] B, 12%
5000
General method for the synthesis of 2-substituted acrylamides. Method C reagents and conditions: starting aryl iodide (1.0 equiv), acrylamide (2.0 equiv),
Pd(OAc)2 (0.05 equiv), tri-tert-butylphosphonium tetrafluoroborate (0.10 equiv), Et3N (3.0 equiv), CH3CN (4.0 mLmmolÀ1), 1208C MW, 15 min. Method D re-
agents and conditions:. starting aryl iodide (1.0 equiv), acrylamide (2.0 equiv) and Pd(OAc)2 (0.05 equiv), Et3N (3.0 equiv), CH3CN (4.0 mLmmolÀ1), 1208C MW,
15 min. Method E reagents and conditions: starting aryl bromide (1.0 equiv), acrylamide (1.5 equiv) and trans-bis(acetato)bis[o-(di-o-tolylphosphino)benzyl]di-
palladium(II) (0.05 equiv), NaOAc (3.0 equiv), DMF (4.0 mLmmolÀ1), 1408C MW, 15 min.
[a] Chemical stability in PBS pH 7.4 at 258C;as % remaining after 24 h. [b] Isolated yield over two steps. [c] The compound interferes with the assay at
10 mm, therefore it was not possible to measure human proteasome inhibition. [d] Reagents and conditions: 1) (2E)-3-(1H-Indolyl-3-yl)acrylic acid (2.0 mmol),
1,1’-carbonyldiimidazole (2.0 mmol), DMF (10 mL), rt, 30 min, 2) NH4HCO3 (4.0 mmol), rt, o/n.
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generally 1000/4000-fold less toxic than the approved human
proteasome inhibitor Bortezomib (TC50 less than 10 nm).[7c] The
observed cytotoxicity might be related to the inhibition of the
trypsin-like and caspase-like activity of the mammalian protea-
some.[22] Alternatively, it might be due to the non-proteasome-
related activity of the styryl oxathiazolone compounds towards
other cellular enzymes that catalyze bond cleavage via the
same mechanism as the Mtb-proteasome (i.e. nucleophilic
attack). On the other hand, a previous report from Lin et al.
demonstrated generally reduced inhibition potency for the
parent aryl-oxathiazolone inhibitors (e.g. compounds 1–4 in-
cluded in our study) toward b1 and b2 non-chymotrypsin-like
active sites of the proteasome.[7c] Additionally high selectivity
towards other human proteases, including trypsin, cathepsin B,
and matrix metalloproteases was reported for the same pro-
teasome inhibitors.[7c] However, recent research by Bassett
et al. has revealed cytotoxicity towards Vero cells for a number
of proteasome inhibitors reported as antitubercular agents.[23]
Previous work suggested that the proteasome plays a key
role during oxidative and nitrosative stress,[12] and that protea-
some inhibitors are bactericidal against nonreplicating Mtb
subjected to nitrosative stress.[7c] We therefore determined
whether the 5-styryl-oxathiazolone class of compounds had ac-
tivity against Mtb under nonreplicating conditions. For this we
used a starvation model, in which bacteria are nutrient-
deprived for 14 days,[24] to induce the nonreplicating state,
after which they are exposed to compounds over 21 days in
this state (Figure 2).
Table 4. Activity of compounds against actively-replicating Mtb.
Entry Cmpd
OD-
MIC
[mm]
OD-
RFU-MIC RFU-
Vero cell
cytotoxicity
inhib.
at 20 mm
[%]
[mm]
inhib.
at 20 mm TC50 [mm]
[%]
1
1
>20
33
>20
32
12
2
3
4
5
6
7
8
9
2
3
4
inactive
>20
> 20
>20
>20
inactive
inactive
>20
inactive
>20
>20
inactive
>20
>20
>20
>20
>20
>20
>20
inactive
>20
>20
inactive
>20
>20
>20
>20
inactive
inactive
>20
inactive
>20
>20
inactive
>20
>20
>20
>20
>20
>20
>20
inactive
>20
>20
18
11
21
22
41
19
21
n.d.
17
21
17
20
n.d.
16
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
27
22
22
28
30
24
27
n.d.
17
18
19
22
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Isoniazid[a]
33
30
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
41
41
43
46
45
83
33
48
36
32
36
45
85
30
45
35
32
36
35
26
24
27
32
24
27
39
>20
>20
>20
>20
inactive
inactive
inactive
inactive
0.3
inactive
inactive
inactive
inactive
0.3
All four 5-styryl-oxathiazol-2-ones tested (17, 33, 37, and 39)
were bactericidal against nonreplicating Mtb, displaying a very
rapid kill (>4 logs in 7–14 days). All compounds gave com-
plete sterilization of cultures within 14 days at the lowest con-
centration tested (20 mm). The 5-(4-chloro)styryl-oxathiazol-2-
one inhibitor (39) was most effective, reducing the colony-
forming units (CFU)/mL below the detection limit in only
seven days at all concentrations. These data confirm the selec-
tivity of proteasome inhibitors for nonreplicating over replicat-
ing bacteria.
Ethambutol[a] 6.5
Ofloxacin[a]
1.2
6.6
1.3
The minimum inhibitory concentration (MIC) was determined against aer-
obically-grown Mtb H37Rv using a 10-point serial dilution by optical den-
sity (OD) and relative fluorescence units (RFU) measurement. The highest
concentration of compound tested was 20 mm. The% growth was plot-
ted, and a curve was fitted using the Gompertz fit. The MIC was defined
as the minimum concentration at which growth was completely inhibited
and was calculated from the inflection point of the fitted curve to the
lower asymptote (zero growth). The % inhibition of growth at 20 mm is
given. Compounds defined as inactive gave% inhibition <20 at 20 mm.
Cytotoxicity was measured against the Vero cell line; the TC50 is the con-
centration required to inhibit growth by 50% over 48 h. n.d.=not deter-
mined. [a] Known antitubercular agents were used as reference com-
pounds.[3]
Conclusions
Our results demonstrate that 5-styryl-oxathiazol-2-ones provide
a promising scaffold for Mtb proteasome inhibitors. In the cur-
rent study we have further evaluated the structure–activity re-
lationship of oxathiazolones as proteasome inhibitors. We
found that conjugated 5-styryl-oxathiazolones are equally or
more potent inhibitors of the Mtb proteasome compared to
the previously reported 5-aryl-oxathiazolones. As part of this
investigation we prepared a series of novel 5-styryl-oxathiazo-
lones from the corresponding aryl iodides or bromides by a mi-
crowave-assisted Mizoroki–Heck coupling with acrylamide.
Subsequent cyclization using chlorocarbonyl sulfenyl chloride
yielded the oxathiazolones. Our enzyme activity results
showed that para-substituted 5-styryl-oxathiazol-2-ones were
superior to ortho- and meta-substitution with regard to Mtb
proteasome IC50 values, as well as their relative lack of effects
against the chymotrypsin-like peptidase activity of human pro-
pounds showed >20% inhibition of growth at the maximum
concentration tested (20 mm), but none had an MICꢀ20 mm.
There was no clear difference between the 5-styryl-oxathiazol-
2-ones (entries 5–29) and the previously reported 5-aryl-ana-
logues (entries 1–4) or with respect to the electronic nature
and/or position of the ring substitution. This finding is consis-
tent with the MIC values (15–299 mm) for a series of 5-aryl-oxa-
thiazol-2-ones recently tested on the same Mtb strain.[16g] Com-
pounds demonstrated some cytotoxicity against the Vero cell
line, with TC50 values (concentration required to inhibit growth
by 50% over 48 h) in the range of 11–41 mm (when tested).
However, these results indicate that our new inhibitors are
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Figure 2. 5-Styryl-oxathiazol-2-ones are bactericidal against nonreplicating Mycobacterium tuberculosis (Mtb). Mtb was grown aerobically, washed, and resus-
pended in PBS-tyloxapol for 14 d. Compounds were added with the indicated concentrations. Colony forming units (CFU) were determined by plating serial
dilutions. The lower limit of detection (LoD) is indicated.
teasomes. We also produced a small series of oxathiazolones in
which solubility-enhancing heterocyclic groups were intro-
duced. However, we gained solubility at the expense of selec-
tivity regarding the human proteasome. Finally, a selection of
the synthesized inhibitors showed rapid bactericidal activity
against nonreplicating Mtb, although they were not very active
against actively replicating cells.
with UV light. Flash column chromatography was performed on
columns prepacked with PHARMA-SILꢁ (5 g/10 g, hydrophilic high
1
surface activity silica, UCT, Inc., Bristol, USA). H and 13C NMR spec-
tra were recorded on Varian Mercury Plus instruments (Palo Alto,
USA); 1H NMR spectra at 399.9 MHz and 13C NMR spectra at
1
100.5 MHz. The chemical shifts for H NMR and 13C NMR were refer-
enced to tetramethylsilane (TMS) via residual solvent signals (1H,
CDCl3 at 7.26 ppm and [D6]-dimethylsulfoxide (DMSO) at 2.50 ppm;
13C, CDCl3 at 77.0 ppm and [D6]DMSO at 39.5 ppm), while chemical
shifts for 19F NMR were referenced to CFCl3 used as internal stan-
dard (0.0 ppm). The microwave reactions were performed in a Biot-
age Initiator (Uppsala, Sweden) producing controlled irradiation at
2450 MHz with a power of 0–300 W. The reaction temperature was
determined using the built-in on-line infrared (IR) sensor. All reac-
tions were performed in sealed microwave-transparent process
vials designed for 0.5–2, 2—5, or 10–20 mL reaction volumes. Gas
chromatography/electron ionization mass spectrometry (GC/EI-MS)
was performed on a Varian Saturn 3900/2100 system equipped
with a CP-Sil 8 CB capillary column (30 m”0.25 mm, 0.25 mm) oper-
ating at an EI potential of 70 eV. The oven temperature (GC) was
70–3008C. Analytical high-performance liquid chromatography/
The present results suggest that this new class of Mtb pro-
teasome inhibitors can be used as the basis for development
of novel antitubercular drugs that are effective when protein
synthesis of the mycobacteria is drastically reduced, such as in
the nonreplicative state or during antibiotic treatment. Further
development will include the improvement of the safety pro-
file of this class of Mtb proteasome inhibitors.[30]
Experimental Section
General Chemistry. Analytical thin-layer chromatography (TLC)
was performed on silica gel 60 F-254 plates (Merck) and visualized
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electrospray ionization mass spectrometry (HPLC/ESI-MS) was per-
formed on a Gilson HPLC system (Middleton, USA) with a Finnigan
AQA ESI quadropole mass spectrometer with electrospray ioniza-
tion (Thermo Fisher Scientific, Waltham, USA) and using an Onyx
Monolithic C18 column 4.6”50 mm (Phenomenex, Torrance, USA)
with CH3CN/H2O in 0.05% HCOOH as mobile phase at a flow rate
of 4 mLminÀ1 or on a Dionex UltiMate 3000 HPLC system (Sunny-
vale, USA) with a Bruker amaZon SL ion trap mass spectrometer
(Billerica, USA), using a Phenomenex Kinetex C18 column (50”
3.0 mm, 2.6 mm particle size, 100 Š pore size) with CH3CN/H2O in
0.05% HCOOH as mobile phase at a flow rate of 1.5 mLminÀ1. De-
tection was by UV (diode array detector) and MS (ESI+ mode).
Preparative HPLC purification was performed by UV-triggered
(254 nm) fraction collection with a Dionex UltiMate 3000 HPLC
system, using an Agilent PrepHT Zorbax SB-C8 column (21.2”
150 mm, 5 mm particle size) (Santa Clara, USA) with CH3CN/H2O in
0.05% trifluoroacetic acid (TFA) as mobile phase. High-resolution
mass spectrometry (HRMS) was performed on a Micromass Q-Tof2
mass spectrometer (Waters, Milford, USA) equipped with an elec-
trospray ion source. HRMS data are provided for all novel com-
pounds, unless low ionization level, intrinsic to the physicochemi-
cal properties of the specific molecules, did not permit the mea-
surement of the corresponding molecular mass data. However, in
some cases, it was possible to determine the low resolution molec-
ular mass by the Varian Saturn 3900/2100 GC/MS system and/or by
the Bruker amaZon SL ion trap mass spectrometer during the
LC/MS analysis. All starting materials and reagents are commercial-
ly available and were used as received.
144.3, 140.2 ppm; ESI-MS m/z: 182 [M+H]+; HRMS m/z calcd for
C6H3N3O2S [M+H]+ 182.0024, found 182.0031.
5-(3-Methoxyphenyl)-1,3,4-oxathiazol-2-one (3).[7c] According to
Method B, 3-methoxybenzamide (151 mg, 1.0 mmol) was reacted
with chlorocarbonyl sulfenyl chloride to give, after purification by
precipitation from EtOAc/pentane, 80 mg (38% isolated yield) of
the title compound as a white powder. LC purity (254 nm): 97%;
1H NMR (400 MHz, [D6]DMSO): d=7.53–7.49 (m, 2H), 7.41–7.39 (m,
1H), 7.27–7.20 (m, 1H), 3.84 ppm (s, 3H); 13C NMR (100 MHz,
[D6]DMSO): d=173.7, 159.5, 156.6, 130.6, 126.8, 119.5, 118.9, 111.7,
55.5 ppm; ESI-MS m/z: 210 [M+H]+; HRMS m/z for C9H7NO3S as
[M+H]+ adduct not found due to low ionization level.
5-(3-Fluorophenyl)-1,3,4-oxathiazol-2-one (4).[7c] According to
Method B, 3-fluorobenzamide (139 mg, 1.0 mmol) was reacted with
chlorocarbonyl sulfenyl chloride to give, after silica column purifi-
cation (pentane/EtOAc 100:0 ! 100:1), 163 mg (83% isolated
yield) of the title compound as a white solid. TLC: Rf =0.79 (pen-
tane/ EtOAc 100:3); LC purity (254 nm): 97%; 1H NMR (400 MHz,
[D6]DMSO): d=7.78 (dt, J=7.6, 1.2 Hz, 1H), 7.70 (ddd, J=9.2, 2.8,
1.6 Hz, 1H), 7.65 (td, J=8.0, 5.9 Hz, 1H), 7.53 ppm (tdd, J=8.8, 2.8,
0.8 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=173.5, 162.1 (d, J=
244.0 Hz), 155.6 (d, J=3.1 Hz), 131.7 (d, J=8.4 Hz), 127.6 (d, J=
9.2 Hz), 123.4 (d, J=3.0 Hz), 119.7 (d, J=21.4 Hz), 113.8 ppm (d, J=
23.7 Hz); 19F NMR (376 MHz, [D6]DMSO): d=À111.4 ppm (td, J=9.3,
5.3 Hz); EI-MS m/z: 197 [M]+; HRMS m/z for C8H4FNO2S as [M+H]+
adduct not found due to low ionization level.
5-(2-Chloroethyl)-1,3,4-oxathiazol-2-one (5). According to Meth-
od A, 3-chloropropionamide (81 mg, 0.75 mmol) was used to give,
after silica column purification (pentane/EtOAc 100:2), 75 mg (60%
isolated yield) of the title compound as a colorless oil. TLC: Rf =
0.31 (isohexane/EtOAc 10:1); LC purity (254 nm):99%; 1H NMR
(400 MHz, CDCl3): d=3.84 (t, J=6.4 Hz, 2H), 3.11 ppm (t, J=6.4 Hz,
2H); 13C NMR (100 MHz, CDCl3): d=173.5, 157.9, 38.4, 33.7 ppm; EI-
MS m/z: 166/168 [M+1]+; ESI-MS m/z: 166/168 [M+H]+; HRMS m/
z for C4H4ClNO2S as [M+H]+ adduct not found due to low ioniza-
tion level.
General method for the synthesis of 5-substituted oxathiazol-2-
one.
Method A: A mixture of starting amide (1.0 equiv) and chlorocar-
bonyl sulfenyl chloride (1.5 equiv) in 1,4-dioxane (4.0 mLmmolÀ1
)
was irradiated with microwaves at 1008C for 15 min. The mixture
was allowed to cool down to rt, and the solvent evaporated at re-
duced pressure. The crude material was purified by silica gel
column chromatography and/or preparative HPLC to give the final
compound with purity>95% by HPLC analysis (l=254 nm).
Method B: A mixture of starting amide (1.0 equiv) and chlorocar-
bonyl sulfenyl chloride (2.0 equiv) in freshly distilled THF
(4.0 mLmmolÀ1) was stirred o/n at rt. The solvent was evaporated
at reduced pressure, and the crude material was purified by silica
gel column chromatography and/or preparative HPLC to give the
final compound with purity>95% by HPLC analysis (l=254 nm).
5-Cyclopropyl-1,3,4-oxathiazol-2-one (6). According to Method A,
cyclopropanecarboxamide (85 mg, 1.0 mmol) was reacted to give,
after silica column purification (pentane/EtOAc 100:1), 38 mg (27%
isolated yield) of the title compound as a colorless oil. TLC: Rf =
0.51 (isohexane/EtOAc 10:1); LC purity (254 nm) 99%; 1H NMR
(400 MHz, CDCl3): d=1.98–1.90 (m, 1H), 1.18–1.12 (m, 2H), 1.11–
1.04 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=174.2, 162.7, 11.2,
8.4 ppm; EI-MS m/z: 143 [M]+; HRMS m/z for C5H5NO2S as [M+H]+
adduct not found due to low ionization level.
5-Phenyl-1,3,4-oxathiazol-2-one (1).[7c] According to Method A,
benzamide (121 mg, 1.0 mmol) was used to give, after preparative
HPLC purification, 77 mg (43% isolated yield) of the title com-
pound as a white powder. TLC: Rf =0.53 (isohexane/EtOAc 10:1);
LC purity (254 nm): 99%; 1H NMR (400 MHz, CDCl3): d=7.99–7.96
(m, 2H), 7.60–7.55 (m, 1H), 7.52–7.47 (m, 2H); 13C NMR (100 MHz,
CDCl3): d=173.8, 157.4, 132.6, 129.0, 127.4, 125.8; HRMS m/z for
C8H5NO2S as [M+H]+ adduct not found due to low ionization
level.
5-Pentyl-1,3,4-oxathiazol-2-one (7). According to Method A, hexa-
namide (86 mg, 0.75 mmol) was used to give, after silica column
purification (pentane/EtOAc 100:1), 73 mg (56% isolated yield) of
the title compound as colorless oil. TLC: Rf =0.65 (isohexane/EtOAc
1
10:1); LC purity (254 nm): 99%; H NMR (400 MHz, CDCl3): d=2.61
(t, J=7.0 Hz, 2H), 1.73 (quin, J=7.0 Hz, 2H), 1.40–1.30 (m, 4H),
0.91 ppm (t, J=7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): d=174.4,
162.0, 30.9, 30.4, 25.0, 22.1, 13.8 ppm; EI-MS m/z: 174 [M+1]+; ESI-
MS m/z: 174 [M+H]+; HRMS m/z for C7H11NO2S as [M+H]+ adduct
not found due to low ionization level.
5-(Pyrazin-2-yl)-1,3,4-oxathiazol-2-one (2).[17] According to Meth-
od B, pyrazinamide (123 mg, 1.0 mmol) was reacted with chlorocar-
bonyl sulfenyl chloride to give, after preparative HPLC purification,
42 mg (23% isolated yield) of the title compound as white crystals.
TLC: Rf =0.29 (pentane/EtOAc 1:1); LC purity (254 nm): 99%;
1H NMR (400 MHz, CDCl3): d=9.29 (s, 1H), 8.79 (s, 1H), 8.75 ppm (s,
1H); 13C NMR (100 MHz, CDCl3): d=172.2, 154.6, 147.2, 144.7,
5-(Fluoromethyl)-1,3,4-oxathiazol-2-one (8). According to Meth-
od B, fluoroacetamide (154 mg, 2.0 mmol) was reacted with chloro-
carbonyl sulfenyl chloride to give, after silica column purification
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(pentane/EtOAc 100:0 ! 100:2), 64 mg (24% isolated yield) of the
title compound as a colorless oil. TLC: Rf =0.55 (pentane/EtOAc
10:1); LC purity (254 nm)=96%; H NMR (400 MHz, [D6]DMSO): d=
HRMS m/z calcd for C10H6F3NO2S [M+H]+ 262.0150, found
262.0154.
1
5.39 ppm (d, J=45.6 Hz, 2H); 13C NMR (100 MHz, [D6]DMSO): d=
173.4, 155.7 (d, J=21.5 Hz), 76.9 ppm (d, J=171.0 Hz); 19F NMR
(376 MHz, [D6]DMSO): d=0.96 ppm (t, J=45.5 Hz); EI-MS m/z: 135
[M]+, 196; HRMS m/z for C3H2FNO2S as [M+H]+ adduct not found
due to low ionization level.
5-(Hydroxy(phenyl)methyl)-1,3,4-oxathiazol-2-one (14). Accord-
ing to Method B, 2-hydroxy-2-phenylacetamide (151 mg, 1.0 mmol)
was reacted with chlorocarbonyl sulfenyl chloride to give, after
silica column (pentane/EtOAc 100:0 ! 1:1) and preparative HPLC
purification, 84 mg (40% isolated yield) of the title compound as
a pale yellow oil. TLC: Rf =0.17 (pentane/EtOAc 5:1); LC purity
(254 nm): 98%; 1H NMR (400 MHz, [D6]DMSO): d=7.48–7.32 (m,
5H), 6.73 (d, J=5.5 Hz, 1H), 5.65 ppm (d, J=5.5 Hz, 1H); 13C NMR
(100 MHz, [D6]DMSO): d=174.0, 161.9, 138.3, 128.5, 128.4, 126.7,
69.9 ppm; ESI-MS m/z: 232 [M+Na]+; HRMS m/z for C9H7NO3S as
[M+H]+ adduct not found due to low ionization level.
5-Benzyl-1,3,4-oxathiazol-2-one (9).[25] According to Method A, 2-
phenylacetamide (135 mg, 1.0 mmol) was reacted to yield, after
preparative HPLC purification, the title compound as a white
powder (83 mg, 43%). TLC: Rf =0.39 (isohexane/EtOAc 10:1); LC
1
purity (254 nm): 97%; H NMR (400 MHz, CDCl3): d=7.40–7.28 (m,
5H), 3.93 ppm (s, 2H); 13C NMR (100 MHz, CDCl3): d=174.0, 160.0,
132.3, 129,1, 129.0, 127.9, 37.0 ppm; HRMS m/z for C9H7NO2S as
[M+H]+ adduct not found due to low ionization level.
5-Phenethyl-1,3,4-oxathiazol-2-one (15). 3-Phenylpropionic acid
(150 mg, 1.0 mmol) and 1,1’-carbonyldiimidazole (357 mg,
2.2 mmol) were dissolved in DMF (5 mL), and the mixture was
stirred at rt. After 30 min, NH4HCO3 (316 mg, 4.0 mmol) was added,
and the resulting suspension was stirred at rt for 3 d. EtOAc
(50 mL) was added, and the organic phase was washed with H2O
(2”20 mL) and brine (20 mL). Evaporation of solvent gave 171 mg
of the crude corresponding amide, purified by precipitation from
EtOAc/pentane to afford 92 mg (62% isolated yield) of 3-phenyl-
propionamide as a white solid. TLC: Rf =0.28 (pentane/EtOAc 1:1);
GC/MS purity: 99%; 1H NMR (400 MHz, [D6]DMSO): d=7.35–6.98
(m, 6H), 6.76 (bs, 1H), 2.81 (t, J=8.0 Hz, 2H), 2.36 ppm (t, J=
8.0 Hz, 2H); 13C NMR (100 MHz, [D6]DMSO): d=173.4, 141.5, 128.2,
128.1, 125.8, 36.7, 30.9 ppm; EI-MS m/z: 149 [M]+. According to
Method B, 3-phenylpropionamide (150 mg, 1.0 mmol) was reacted
with chlorocarbonyl sulfenyl chloride to give, after silica column
purification (pentane/EtOAc 100:0 ! 100:1), 147 mg (71% isolated
yield) of the title compound as a colorless oil. TLC: Rf =0.79 (pen-
tane/EtOAc 10:1); LC purity (254 nm): 98%; 1H NMR (400 MHz,
[D6]DMSO): d=7.35–7.20 (m, 5H), 2.98 ppm (s, 4H); 13C NMR
(100 MHz, [D6]DMSO): d=174.3, 161.2, 139.6, 128.4, 128.3, 126.4,
31.2, 30.4 ppm; ESI-MS m/z: 208 [M+H]+; HRMS m/z for C10H9NO2S
as [M+H]+ adduct not found due to low ionization level.
5-(3-Methoxybenzyl)-1,3,4-oxathiazol-2-one (10). According to
Method A, 4-methoxyphenylacetamide (83 mg, 0.50 mmol) was
used to yield, after preparative HPLC purification, 30 mg (27% iso-
lated yield) of the title compound as a white powder. TLC: Rf =0.35
(isohexane/EtOAc 10:1); LC purity (254 nm): 97%; 1H NMR
(400 MHz, CDCl3): d=7.28 (t, J=7.8 Hz, 1H), 6.90–6.82 (m, 3H),
3.90 (s, 2H), 3.81 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=174.0,
160.0, 133.7, 130.0, 121.3, 114.9, 113.2, 55.3, 37.0 ppm; EI-MS m/z:
184, 147; ESI-MS m/z: 224 [M+H]+; HRMS m/z calcd for C10H9NO3S
[M+H]+ 224.0381, found 224.0380.
4-((2-Oxo-1,3,4-oxathiazol-5-yl)methoxy)benzaldehyde (11). Ac-
cording to Method A, 2-(4-Formylphenoxy)acetamide (90 mg,
0.50 mmol) was used to yield, after preparative HPLC purification,
47 mg (39% isolated yield) of the title compound as white powder.
TLC: Rf =0.10 (isohexane/EtOAc 10:1); LC purity (254 nm): 99%;
1H NMR (400 MHz, CDCl3): d=9.92 (s, 1H), 7.89 (d, J=8.8 Hz, 2H),
7.09 (d, J=8.8 Hz, 2H), 5.05 ppm (s, 2H); 13C NMR (100 MHz,
CDCl3): d=190.5, 172.6, 161.9, 155.8, 132.1, 131.3, 115.0, 63.2 ppm;
EI-MS m/z: 238 [M+1]+; ESI-MS m/z: 238 [M+H]+; HRMS m/z
calcd for C10H7NO4S [M+H]+ 238.0174, found 238.0175.
(R)-5-(Methoxy(phenyl)methyl)-1,3,4-oxathiazol-2-one (16). (R)-
(À)-2-Methoxyphenylacetic acid (332 mg, 2.0 mmol) and 1,1’-car-
bonyldiimidazole (649 mg, 4.0 mmol) were dissolved in DMF
(10 mL), and the mixture was stirred at rt. After 30 min, NH4HCO3
(632 mg, 8.0 mmol) was added and the resulting suspension was
stirred at rt for 3 d. EtOAc (50 mL) was added, and the organic
phase was washed with H2O (2”30 mL) and brine (30 mL). Evapo-
ration of solvent gave 180 mg (55% yield) of the crude (R)-2-me-
thoxy-2-phenylacetamide as a white powder, used for the next
step without further purification. GC/MS purity: 99%; EI-MS m/z:
166 [M+1]+. According to Method B, (R)-2-methoxy-2-phenylace-
tamide (180 mg, 1.1 mmol) was reacted with chlorocarbonyl sulfe-
nyl chloride to give, after silica column purification (pentane/EtOAc
100:0 ! 100:1), 140 mg (58% isolated yield) of the title compound
as a colorless oil. TLC: Rf =0.64 (pentane/EtOAc 10:1); LC purity
(254 nm): 95%; 1H NMR (400 MHz, [D6]DMSO): d=7.48–7.38 (m,
5H), 5.44 (s, 1H), 3.43 ppm (s, 3H); 13C NMR (100 MHz, [D6]DMSO):
d=173.7, 159.4, 135.4, 129.0, 128.7, 127.2, 78.8, 57.3 ppm; ESI-MS
m/z: 164; HRMS m/z calcd for C10H9NO3S [M+H]+ 224.0381, found
224.0379.
5-(Naphthalen-1-ylmethyl)-1,3,4-oxathiazol-2-one (12). According
to Method A, 1-naphthylacetamide (93 mg, 0.50 mmol) was used
to yield, after preparative HPLC purification, 62 mg (51% isolated
yield) of the title compound as a white powder. TLC: Rf =0.48 (iso-
hexane/EtOAc 10:1); LC purity (254 nm): 99%; 1H NMR (400 MHz,
CDCl3): d=8.03 (d, J=7.8 Hz, 1H), 7.92–7.83 (m, 2H), 7.61–7.51 (m,
2H), 7.50–7.45 (m, 2H), 4.38 ppm (s, 2H); 13C NMR (100 MHz,
CDCl3): d=173.9, 159.9, 133.9, 131.6, 129.0, 128.5, 128.3, 126.9,
126.1, 125.5, 123.2, 34.6 ppm; EI-MS m/z: 228, 167; ESI-MS m/z: 244
[M+H]+; HRMS m/z calcd for C13H9NO2S [M+H]+ 244.0432, found
244.0427.
5-(2-(Trifluoromethyl)benzyl)-1,3,4-oxathiazol-2-one (13). Accord-
ing to Method B, 2-(trifluoromethyl)phenylacetamide (152 mg,
0.75 mmol) was reacted with chlorocarbonyl sulfenyl chloride to
give, after two silica column purification (1st: pentane/EtOAc 100:0
! 10:1, 2nd: pentane/EtOAc 100:0 ! 100:2), 110 mg (56% isolated
yield) of the title compound as a colorless oil. TLC: Rf =0.53 (pen-
tane/EtOAc 10:1); LC purity (254 nm): 99%; 1H NMR (400 MHz,
CDCl3): d=7.72 (d, J=7.8 Hz, 1H), 7.58 (t, J=7.8 Hz, 1H), 7.49–
7.41 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=173.7, 158.9,
132.3, 132.0, 130.5 (q, J=1.5 Hz), 129.1 (q, J=30.7 Hz), 128.3, 126.6
(q, J=5.4 Hz), 124.0 (q, J=273.7 Hz), 33.8 ppm (q, J=2.3 Hz);
19F NMR (376 MHz, CDCl3) d: À59.8 ppm; ESI-MS m/z: 262 [M+H]+;
(E)-5-Styryl-1,3,4-oxathiazol-2-one (17).[26] According to Method B,
trans-cinnamamide (147 mg, 1.0 mmol) was reacted with chlorocar-
bonyl sulfenyl chloride to give, after silica column purification (pen-
tane/EtOAc 100:0 ! 10:1) and trituration in pentane/EtOAc/MeOH
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1
10:1:1 drop, 82 mg (40% isolated yield) of the title compound as
(pentane/EtOAc 9 :1); LC purity (254 nm): 98%; H NMR (400 MHz,
a pale yellow powder. TLC: Rf =0.76 (pentane/EtOAc 10:1); LC
purity (254 nm): 99%; H NMR (400 MHz, CDCl3): d=7.56–7.49 (m,
[D6]DMSO): d=7.75–7.71 (m, 2H), 7.64–7.58 (m, 1H), 7.56–
7.50 ppm (m, 2H); 13C NMR (100 MHz, [D6]DMSO): d=172.3, 141.5,
132.6, 131.6, 129.2, 118.1, 92.3, 75.7 ppm; ESI-MS m/z: 204 [M+H]+
; HRMS m/z for C10H5NO2S as [M+H]+ adduct not found due to
low ionization level.
1
2H), 7.50 (d, J=16.4 Hz, 1H), 7.44–7.39 (m, 3H), 6.64 ppm (d, J=
16.4 Hz, 1H); 13C NMR (100 MHz, CDCl3): d=173.4, 157.6, 142.0,
134.1, 130.6, 129.1, 127.8, 112.8 ppm; EI-MS m/z: 191, 161, 129; ESI-
MS m/z: 206 [M+H]+; HRMS m/z calcd for C10H7NO2S [M+H]+
262.0276, found 262.0273.
5-Vinyl-1,3,4-oxathiazol-2-one (21).[27] According to Method B, ac-
rylamide (107 mg, 1.5 mmol) was reacted with chlorocarbonyl sul-
fenyl chloride to give, after two silica column purification (1 st: pen-
tane/EtOAc 100:0 ! 10:1, 2nd: pentane/EtOAc 100:0 ! 100:2),
15 mg (8% isolated yield) of the title compound as a colorless oil.
TLC: Rf =0.73 (pentane/EtOAc 10:1); LC purity (254 nm): 98%;
1H NMR (400 MHz, CDCl3): d=6.32 (dd, J=17.6, 10.9 Hz, 1H), 6.23
(d, J=17.6 Hz, 1H), 5.92 ppm (d, J=10.5 Hz, 1H); 13C NMR
(100 MHz, CDCl3): d=173.1, 157.0, 127.9, 122.9 ppm; EI-MS m/z:
129 [M]+; HRMS m/z for C4H3NO2S as [M+H]+ adduct not found
due to low ionization level.
(E)-5-(4-(Trifluoromethyl)styryl)-1,3,4-oxathiazol-2-one (18). Ac-
cording to Method B, 4-(trifluoromethyl)cinnamamide (183 mg,
0.85 mmol) was reacted with chlorocarbonyl sulfenyl chloride to
give, after purification by precipitation from EtOAc/pentane, 97 mg
(42% isolated yield) of the title compound as a white powder. TLC:
Rf =0.57 (pentane/EtOAc 10:1); LC purity (254 nm): 99%; 1H NMR
(400 MHz, [D6]DMSO): d=8.01 (d, J=8.2 Hz, 2H), 7.79 (d, J=8.2 Hz,
2H), 7.63 (d, J=16.4 Hz, 1H), 7.17 ppm (d, J=16.4 Hz, 1H);
13C NMR (100 MHz, [D6]DMSO): d=173.3, 157.0, 139.5, 138.2 ppm
(q, J=1.5 Hz), 129.9 (q, J=32.2 Hz), 128.8, 125.7 (q, J=3.8 Hz),
124.0 (q, J=272.2 Hz), 116.0; 19F NMR (376 MHz, [D6]DMSO): d=
À61.3 ppm; ESI-MS m/z: 274 [M+H]+; HRMS m/z for C11H6F3NO2S
as [M+H]+ adduct not found due to low ionization level.
(E)-5-(3,3,3-Trifluoroprop-1-en-1-yl)-1,3,4-oxathiazol-2-one (22).
According to Method B, 4,4,4-trifluorocrotonamide (306 mg,
2.2 mmol) was reacted with chlorocarbonyl sulfenyl chloride to
give, after silica column purification (pentane/EtOAc 100:0 !
100:2), 350 mg (81% isolated yield) of the title compound as a col-
orless oil. TLC: Rf =0.69 (pentane/EtOAc 10:1); LC purity (254 nm):
99%; 1H NMR (400 MHz, [D6]DMSO): d=7.12–7.01 ppm (m, 2H);
13C NMR (100 MHz, [D6]DMSO): d=172.7, 154.2, 127.1 (q, J=
34.3 Hz), 124.0 (q, J=6.9 Hz), 122.5 ppm (q, J=268.4 Hz); 19F NMR
(376 MHz, [D6]DMSO): d=À63.5 ppm (d, J=5.6 Hz); HRMS m/z for
C5H2F3NO2S as [M+H]+ adduct not found due to low ionization
level.
5-Benzoyl-1,3,4-oxathiazol-2-one (19).[25] In a round-bottom flask,
Dess–Martin periodinane (467 mg, 1.1 mmol) was added to a pre-
cooled solution (T=08C) of 5-(hydroxy(phenyl)methyl)-1,3,4-oxa-
thiazol-2-one (14) (209 mg, 1.0 mmol) in anhydrous CH2Cl2 (5 mL).
The resulting reaction mixture was stirred 2 h at 08C and then fil-
tered to remove the formed solid. The mother liquor was washed
with H2O (10 mL) and dried on anhydrous Na2SO4. Evaporation of
the solvent under reduced pressure gave 347 mg of the crude ma-
terial that was triturated with EtOAc for 30 min at rt. Filtration of
the formed solid and evaporation of the solvent gave 117 mg of
a colorless oil that was purified by silica column purification (pen-
tane/EtOAc 100:0 ! 10:1) to give 118 mg (57% isolated yield) of
the title compound as a colorless oil. TLC: Rf =0.48 (pentane/EtOAc
(E)-5-(1,2-Diphenylvinyl)-1,3,4-oxathiazol-2-one (23). 2-Phenyl-
trans-cinnamic acid (224 mg, 1.0 mmol) and 1,1’-carbonyldiimida-
zole (357 mg, 2.2 mmol) were dissolved in DMF (5 mL) and the
mixture was stirred at rt. After 30 min, NH4HCO3 (316 mg,
4.0 mmol) was added and the resulting suspension was stirred at rt
for 3 d. EtOAc (50 mL) was added, and the organic phase was
washed with H2O (2”20 mL) and brine (20 mL). Evaporation of sol-
vent gave 212 mg of the crude corresponding amide, purified by
silica column chromatography (pentane/EtOAc 1:1 ! 3:1) to afford
150 mg (67% isolated yield) of 2-phenyl-trans-cinnamamide as
white crystals. TLC: Rf =0.63 (pentane/EtOAc=1:1); GC/MS purity:
1
10:1); LC purity (254 nm): 99%; H NMR (400 MHz, [D6]DMSO): d=
8.18–8.13 (m, 2H), 7.78–7.72 (m, 1H), 7.63–7.57 ppm (m, 2H);
13C NMR (100 MHz, [D6]DMSO): d=177.4, 172.3, 153.3, 134.5, 133.5,
130.6, 128.6 ppm; EI-MS m/z: 208 [M+1]+; ESI-MS m/z: 208 [M+
H]+; HRMS m/z calcd for C9H5NO3S [M+H]+ 208.0068, found
208.0070.
1
5-(Phenylethynyl)-1,3,4-oxathiazol-2-one (20). In a round-bottom
flask, SOCl2 (714 mg, 6.0 mmol) was added to a solution of phenyl-
propiolic acid (730 mg, 5.0 mmol) in CHCl3 (10 mL) at rt After addi-
tion of DMF (1 drop), the reaction mixture was stirred 1 h at rt To
the resulting solution was then added 25% aq NH4OH (2.5 mL) at
À108C. The reaction mixture was allowed to return to rt and
stirred 30 min at this temperature. CHCl3 (10 mL) and H2O (10 mL)
where added, the aqueous layer was extracted with CHCl3 (2”
20 mL) and the combined organic phases were dried over Na2SO4.
Evaporation of the solvent under reduced pressure gave 377 mg
(52% isolated yield) of the crude 3-phenylpropiolamide as a white
solid, which was used for the next step without further purification.
99%; H NMR (400 MHz, [D6]DMSO): d=7.46–7.35 (m, 4H), 7.30 (bs,
1H), 7.24–7.15 (m, 5H), 7.03–6.97 (m, 2H), 6.90 ppm (bs, 1H);
13C NMR (100 MHz, [D6]DMSO): d=170.0, 136.7, 136.6, 135.0, 133.9,
129.7, 129.3, 128.8, 128.2, 128.1, 127.8 ppm; EI-MS m/z: 223 [M]+.
According to MethodB, 2-phenyl-trans-cinnamamide (256 mg,
1.1 mmol) was reacted with chlorocarbonyl sulfenyl chloride to
give, after silica column purification (pentane/EtOAc 100:0 !
100:1), 290 mg (90% isolated yield) of the title compound as
a white solid. TLC: Rf =0.77 (pentane/EtOAc 10:1); LC purity
(254 nm): 95%; 1H NMR (400 MHz, [D6]DMSO): d=7.65 (s, 1H),
7.50–7.43 (m, 3H), 7.35–7.29 (m, 2H), 7.28–7.20 (m, 3H), 7.13–
7.08 ppm (m, 2H); 13C NMR (100 MHz, [D6]DMSO): d=173.6, 158.5,
137.6, 134.1, 134.0, 130.3, 129.9, 129.4, 128.9, 128.6, 128.4,
127.8 ppm; ESI-MS m/z: 282 [M+H]+; HRMS m/z calcd for
C16H11NO2S [M+H]+ 282.0589, found 282.0587.
1
TLC: Rf =0.72 (EtOAc); LC purity (254 nm): 95%; H NMR (400 MHz,
[D6]DMSO): d=8.14 (bs, 1H), 7.66 (bs, 1H), 7.61–7.52 (m, 2H), 7.51–
7.42 ppm (m, 3H); 13C NMR (100 MHz, [D6]DMSO): d=153.9, 132.0,
130.2, 128.9, 119.9, 84.2, 82.9 ppm; ESI-MS m/z: 146 [M+H]+, 291
[2M+H]+. According to Method B, 3-phenylpropiolamide (150 mg,
1.03 mmol) was reacted with chlorocarbonyl sulfenyl chloride to
give, after purification by silica column (pentane/EtOAc 100:1 !
10:1) followed by preparative HPLC purification, 51 mg (24% isolat-
ed yield) of the title compound as a white powder. TLC: Rf =0.80
3-Phenyl-1,4,2-dioxazol-5-one (24). Benzhydroxamic acid (137 mg,
1.0 mmol) was reacted with 1,1’-carbonyldiimidazole to give, after
silica column purification (pentane/EtOAc 100:0 ! 100:5), 86 mg
of the crude dioxazolone which was purified by precipitation from
EtOAc/pentane to give 80 mg (49% isolated yield) of the title com-
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ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

pound as a white solid. TLC: Rf =0.87 (pentane/EtOAc=9:1); LC
purity (254 nm): 98%; H NMR (400 MHz, [D6]DMSO): d=7.87–7.83
(m, 2H), 7.76–7.71 (m, 1H), 7.67–7.61 ppm (m, 2H); 13C NMR
(100 MHz, [D6]DMSO): d=163.0, 153.9, 133.7, 129.6, 126.4,
120.3 ppm; HRMS m/z for C8H5NO3 as [M+H]+ adduct not found
due to low ionization level.
waves at 1408C for 15 min. The mixture was allowed to cool down
to rt, and the collected crude material was purified by silica gel
column chromatography to give the desired 3-substituted acrylam-
ide.
1
(E)-5-(4-Methoxystyryl)-1,3,4-oxathiazol-2-one (28). According to
Method C, 4-iodoanisole (234 mg, 1.0 mmol) was used as starting
iodide in the Mizoroki–Heck reaction. After microwave irradiation,
the reaction mixture was cooled down to 08C and the collected
precipitate was washed with cold CH3CN (3 mL) and dried by
vacuum to give 139 mg of the crude corresponding acrylate, that
was purified by silica column chromatography (EtOAc/MeOH 100:5
! 100:10) to afford 133 mg (75% isolated yield) of (E)-3-(4-me-
thoxyphenyl)acrylamide as a white powder. TLC: Rf =0.34 (EtOAc);
LC purity (254 nm): 95%; 1H NMR (400 MHz, [D6]DMSO): d=7.52
(dm, J=8.6 Hz, 2H), 7.45 (bs, 1H), 7.38 (d, J=16.0 Hz, 1H), 7.03–
6.97 (m, 3H), 6.47 (d, J=15.6 Hz, 1H), 3.80 ppm (s, 3H); 13C NMR
(100 MHz, [D6]DMSO): d=166.9, 160.3, 138.8, 129.1, 127.4, 119.8,
114.4, 55.2 ppm; ESI-MS m/z: 178 [M+H]+. According to Method B,
(E)-3-(4-methoxyphenyl)acrylamide (256 mg, 1.4 mmol) was reacted
with chlorocarbonyl sulfenyl chloride to give, after purification by
precipitation from EtOAc/pentane, 240 mg (71% isolated yield) of
the title compound as a pale yellow powder. LC purity (254 nm):
98%; 1H NMR (400 MHz, [D6]DMSO): d=7.73 (dm, J=8.6 Hz, 2H),
7.49 (d, J=16.0 Hz, 1H), 6.99 (dm, J=8.6 Hz, 2H), 6.83 (d, J=
16.4 Hz, 1H), 3.81 ppm (s, 3H); 13C NMR (100 MHz, [D6]DMSO): d=
173.6, 161.1, 157.7, 141.2, 130.0, 126.8, 114.4, 110.6, 55.4 ppm; ESI-
MS m/z: 236 [M+H]+; HRMS m/z calcd for C11H9NO3S [M+H]+
236.0381, found 236.0383.
(E)-3-Styryl-1,4,2-dioxazol-5-one
(25).
trans-Cinnamic
acid
(296 mg, 2.0 mmol) and 1,1’-carbonyldiimidazole (486 mg,
3.0 mmol) were dissolved in THF (8 mL), and the mixture was
stirred at rt. After 1 h, hydroxylamine hydrochloride (278 mg,
4.0 mmol) was added, and the resulting suspension was stirred at
rt for 2 d. 5% aq KHSO4 (30 mL) was added, and the mixture was
extracted with EtOAc (2”30 mL). The collected organic phases
were washed with brine (30 mL) and dried over Na2SO4. Evapora-
tion of solvent gave 280 mg (86% isolated yield) of the corre-
sponding N-hydroxycinnamamide as a pale pink solid, used in the
next step without further purification. LC purity (254 nm)=95%;
ESI-MS: m/z 164 [M+H]+. The obtained N-hydroxycinnamamide
(140 mg, 0.86 mmol) was reacted with 1,1’-carbonyldiimidazole to
give, after silica column purification (CH3CN) and evaporation of
the solvent by freeze-drying, 123 mg of the crude dioxazolone
which was purified by preparative HPLC to give 83 mg (51% isolat-
ed yield) of the title compound as a white solid. TLC: Rf =0.64
1
(pentane/EtOAc 10:1); LC purity (254 nm): 97%; H NMR (400 MHz,
[D6]DMSO): d=7.84–7.78 (m, 2H), 7.56 (d, J=16.4 Hz, 1H), 7.49–
7.43 (m, 3H), 7.15 ppm (d, J=16.4 Hz, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=163.2, 153.6, 142.2, 133.8, 130.8, 129.0, 128.4,
106.9 ppm; ESI-MS m/z: 190 [M+H]+; HRMS m/z for C10H7NO3 as
[M+H]+ adduct not found due to low ionization level.
(E)-5-(4-Isopropylstyryl)-1,3,4-oxathiazol-2-one (29). According to
Method C, 1-iodo-4-isopropylbenzene (246 mg, 1.0 mmol) was
used as starting iodide in the Mizoroki–Heck reaction. After micro-
wave irradiation, the reaction mixture was cooled down to 08C,
and the collected precipitate was washed with cold CH3CN (3 mL)
and dried by vacuum to give 150 mg of the crude corresponding
acrylate, which was purified by silica column chromatography
(EtOAc/MeOH 100:0 ! 100:10) to afford 118 mg (62% isolated
yield) of (E)-3-(4-isopropylphenyl)acrylamide as an off-white
powder. TLC: Rf =0.42 (EtOAc); LC purity (254 nm): 98%; 1H NMR
(400 MHz, [D6]DMSO): d=7.49 (bs, 1H), 7.47 (d, J=8.0 Hz, 2H), 7.37
(d, J=16.0 Hz, 1H), 7.28 (d, J=8.0 Hz, 2H), 7.05 (bs, 1H), 6.55 (d,
J=16.0 Hz, 1H), 2.90 (sep, J=7.0 Hz, 1H), 1.20 ppm (d, J=7.0 Hz,
6H); 13C NMR (100 MHz, [D6]DMSO): d=166.8, 150.0, 139.1, 132.5,
127.6, 126.9, 121.3, 33.3, 23.7 ppm; ESI-MS m/z: 190 [M+H]+, 231
[M+CH3CN+H]+, 379 [2M+H] +. According to Method B, (E)-3-
(4-isopropylphenyl)acrylamide (118 mg, 0.62 mmol) was reacted
with chlorocarbonyl sulfenyl chloride to give, after purification by
silica column chromatography (pentane/EtOAc 100:0 ! 100:2)
and preparative HPLC purification, 102 mg (66% isolated yield) of
the title compound as a white solid. TLC: Rf =0.60 (pentane/EtOAc
General method for the synthesis of 2-substituted acrylamide.
Method C (Mizoroki–Heck reaction, electron-rich iodide) A suitable
microwave vial was loaded with solid reactants and reagents: start-
ing iodide (1.0 equiv), acrylamide (2.0 equiv), palladium(II) acetate
(0.05 equiv), and tri-tert-butylphosphonium tetrafluoroborate
(0.10 equiv). CH3CN (4.0 mLmmolÀ1) was added, followed by liquid
reactants: starting iodide (1.0 equiv, in case). Oxygen was removed
by bubbling gaseous nitrogen into the resulting mixture for
15 min, then triethylamine (3.0 equiv) was added, the vial was
capped, and the reaction mixture was irradiated with microwaves
at 1208C for 15 min. The mixture was allowed to cool down to rt,
and the collected crude material was purified by silica gel column
chromatography to give the desired 3-substituted acrylamide.
Method D (Mizoroki–Heck reaction, electron-poor iodide) A suitable
microwave vial was loaded with solid reactants and reagents: start-
ing iodide (1.0 equiv), acrylamide (2.0 equiv), and palladium(II) ace-
tate (0.05 equiv). CH3CN (4.0 mLmmolÀ1) was added, followed by
liquid reactants: starting iodide (1.0 equiv, in case). Oxygen was re-
moved by bubbling gaseous nitrogen into the resulting mixture
for 15 min, then triethylamine (3.0 equiv) was added, the vial was
capped, and the reaction mixture was irradiated with microwaves
at 1208C for 15 min. The mixture was allowed to cool down to rt,
and the collected crude material was purified by silica gel column
chromatography to give the desired 3-substituted acrylamide.
1
10 :1); LC purity (254 nm): 99%; H NMR (400 MHz, [D6]DMSO): d=
7.69 (d, J=8.1 Hz, 2H), 7.50 (d, J=16.3 Hz, 1H), 7.31 (d, J=8.1 Hz,
2H), 6.93 (d, J=16.3 Hz, 1H), 2.92 (sep, J=6.8 Hz, 1H), 1.21 ppm
(d, J=6.8 Hz, 6H); 13C NMR (100 MHz, [D6]DMSO): d=173.5, 157.5,
151.2, 141.4, 131.9, 128.3, 126.9, 112.2, 33.4, 23.6 ppm; ESI-MS m/z:
248 [M+H]+, 289 [M+CH3CN+H]+; HRMS m/z for C13H13NO2S as
[M+H]+ adduct not found due to low ionization level.
Method E (Mizoroki–Heck reaction, electron-poor bromide) A suitable
microwave vial was loaded with solid reactants and reagents: start-
ing bromide (1.0 equiv), acrylamide (1.5 equiv), trans-bis(acetato)-
bis[o-(di-o-tolylphosphino)benzyl]dipalladium(II) (0.05 equiv), and
NaOAc (3.0 equiv). Dry DMF (4.0 mLmmolÀ1) was added, followed
by liquid reactants: starting bromide (1.0 equiv, in case). The vial
was capped, and the reaction mixture was irradiated with micro-
(E)-5-(4-Methylstyryl)-1,3,4-oxathiazol-2-one (30). According to
Method C, 1-iodo-4-methylbenzene (218 mg, 1.0 mmol) was used
as starting iodide in the Mizoroki–Heck reaction. After microwave
irradiation, the reaction mixture was cooled down to 08C, and the
collected precipitate was washed with cold CH3CN (3 mL) and
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dried by vacuum to give 126 mg of the crude corresponding acry-
late, which was purified by silica column chromatography (EtOAc/
MeOH 100:0 ! 100:10) to afford 95 mg (59% isolated yield) of (E)-
3-(p-tolyl)acrylamide as a white powder. TLC: Rf =0.36 (EtOAc); LC
purity (254 nm): 99%; 1H NMR (400 MHz, [D6]DMSO): d=7.48 (bs,
1H), 7.44 (d, J=8.0 Hz, 2H), 7.37 (d, J=16.0 Hz, 1H), 7.22 (d, J=
8.0 Hz, 2H), 7.04 (bs, 1H), 6.55 (d, J=16.0 Hz, 1H), 2.32 ppm (s,
3H); 13C NMR (100 MHz, [D6]DMSO): d=166.8, 150.8, 139.1, 132.1,
129.5, 127.5, 121.3, 20.9 ppm; ESI-MS m/z: 162 [M+H]+, 203 [M+
CH3CN+H] +, 323 [2M+H]+. According to Method B, (E)-3-(p-toly-
l)acrylamide (95 mg, 0.59 mmol) was reacted with chlorocarbonyl
sulfenyl chloride to give, after purification by precipitation from
EtOAc/pentane, 41 mg (32% isolated yield) of the title compound
as white crystals. LC purity (254 nm): 99%; 1H NMR (400 MHz,
[D6]DMSO): d=7.66 (d, J=8.1 Hz, 2H), 7.49 (d, J=16.4 Hz, 1H),
7.25 (d, J=8.1 Hz, 2H), 6.92 (d, J=16.4 Hz, 1H), 2.34 ppm (s, 3H);
13C NMR (100 MHz, [D6]DMSO): d=173.5, 157.5, 141.4, 140.5, 131.4,
129.6, 128.2, 112.2, 21.0 ppm; ESI-MS m/z: 220 [M+H]+, 261 [M+
CH3CN+H]+; HRMS m/z for C11H9NO2S as [M+H]+ adduct not
found due to low ionization level.
as a white powder. TLC: Rf =0.55 (pentane/EtOAc 10:1); LC purity
(254 nm): 94%; 1H NMR (400 MHz, [D6]DMSO): d=7.93 (dm, J=
9.0 Hz, 2H), 7.58 (d, J=16.4 Hz, 1H), 7.43 (dm, J=8.0 Hz, 2H),
7.05 ppm (d, J=16.4 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=
173.4, 157.2, 149.4, 139.7, 133.5, 130.2, 121.3, 120.0 (q, J=256.9 Hz),
114.4 ppm; ESI-MS m/z: 290 [M+H]+; HRMS m/z for C11H6F3NO3S as
[M+H]+ adduct not found due to low ionization level.
(E)-Ethyl 4-(2-(2-oxo-1,3,4-oxathiazol-5-yl)vinyl)benzoate (33).
According to Method D, ethyl 4-iodobenzoate (276 mg, 1.0 mmol)
was used as starting iodide in the Mizoroki–Heck reaction. After
microwave irradiation, the reaction mixture was cooled down to
08C, and the collected precipitate was washed with cold CH3CN
(3 mL) and dried by vacuum. The crude corresponding acrylate
was purified by silica column chromatography (EtOAc/MeOH 1:0
! 10:1) to afford 147 mg (67% isolated yield) of (E)-ethyl 4-(3-
amino-3-oxoprop-1-en-1-yl)benzoate as an off-white powder. TLC:
Rf =0.53 (EtOAc); LC purity (254 nm): 95%; 1H NMR (400 MHz,
[D6]DMSO): d=7.98 (dm, J=8.3 Hz, 2H), 7.69 (dm, J=8.3 Hz, 2H),
7.61 (bs, 1H), 7.46 (d, J=16.0 Hz, 1H), 7.20 (bs, 1H), 6.73 (d, J=
16.0 Hz, 1H), 4.31 (q, J=6.9 Hz, 2H), 1.33 ppm (t, J=6.9 Hz, 3H);
13C NMR (100 MHz, [D6]DMSO): d=166.2, 165.3, 139.4, 137.8, 130.2,
129.7, 127.7, 124.9, 60.8, 14.1 ppm; ESI-MS m/z: 220 [M+H]+, 439
[2M+H]+. According to Method B, (E)-ethyl 4-(3-amino-3-oxoprop-
1-en-1-yl)benzoate (147 mg, 0.67 mmol) was reacted with chloro-
carbonyl sulfenyl chloride to give, after purification by precipitation
from EtOAc/pentane, 129 mg (69% isolated yield) of the title com-
pound as a white powder. TLC: Rf =0.39 (pentane/EtOAc 10:1); LC
purity (254 nm): 99%; 1H NMR (400 MHz, [D6]DMSO): d=7.98 (bd,
J=8.6 Hz, 2H), 7.92 (bd, J=8.6 Hz, 2H), 7.59 (d, J=16.4 Hz, 1H),
7.13 (d, J=16.4 Hz, 1H), 4.33 (q, J=7.1 Hz, 2H), 1.33 ppm (t, J=
7.1 Hz, 3H); 13C NMR (100 MHz, [D6]DMSO): d=173.3, 165.2, 157.0,
139.9, 138.6, 130.9, 129.5, 128.4, 115.7, 60.9, 14.1 ppm; ESI-MS m/z:
278 [M+H]+; HRMS m/z calcd for C13H11NO4S [M+H]+ 278.0487,
found 278.0482.
(E)-5-(4-Ethylstyryl)-1,3,4-oxathiazol-2-one (31). According to
Method C, 1-ethyl-4-iodobenzene (232 mg, 1.0 mmol) was used as
starting iodide in the Mizoroki–Heck reaction. After microwave irra-
diation, the reaction mixture was cooled down to 08C, and the col-
lected precipitate was washed with cold CH3CN (3 mL) and dried
by vacuum to give 170 mg of the crude corresponding acrylate,
which was purified by silica column chromatography (EtOAc/MeOH
100:0 ! 100:10) to afford 88 mg (50% isolated yield) of (E)-3-(4-
ethylphenyl)acrylamide as a white powder. TLC: Rf =0.40 (EtOAc);
LC purity (254 nm): 99%; 1H NMR (400 MHz, [D6]DMSO): d=7.49
(bs, 1H), 7.47 (d, J=8.0 Hz, 2H), 7.38 (d, J=16.0 Hz, 1H), 7.25 (d,
J=8.0 Hz, 2H), 7.04 (bs, 1H), 6.55 (d, J=16.0 Hz, 1H), 2.62 (q, J=
7.6 Hz, 2H), 1.18 ppm (t, J=7.6 Hz, 3H); 13C NMR (100 MHz,
[D6]DMSO): d=166.8, 145.4, 139.1, 132.4, 128.3, 127.6, 121.3, 28.1,
15.4 ppm; ESI-MS m/z: 176 [M+H]+, 217 [M+CH3CN+H] +, 351
[2M+H]+. According to Method B, (E)-3-(4-ethylphenyl)acrylamide
(88 mg, 0.50 mmol) was reacted with chlorocarbonyl sulfenyl chlo-
ride to give, after purification by precipitation from EtOAc/pentane,
50 mg (43% isolated yield) of the title compound as yellow crys-
tals. LC purity (254 nm): 98%; 1H NMR (400 MHz, [D6]DMSO): d=
7.68 (d, J=8.1 Hz, 2H), 7.50 (d, J=16.4 Hz, 1H), 7.28 (d, J=8.1 Hz,
2H), 6.93 (d, J=16.4 Hz, 1H), 2.63 (q, J=7.7 Hz, 2H), 1.18 ppm (t,
J=7.7 Hz, 3H); 13C NMR (100 MHz, [D6]DMSO): d=173.5, 157.5,
146.7, 141.4, 131.7, 128.4, 128.3, 112.2, 28.1, 15.3 ppm; ESI-MS m/z:
234 [M+H]+, 275 [M+CH3CN+H] +; HRMS m/z for C12H11NO2S as
[M+H]+ adduct not found due to low ionization level.
(E)-5-(4-Benzoylstyryl)-1,3,4-oxathiazol-2-one (34). According to
Method D, 4-iodobenzophenone (308 mg, 1.0 mmol) was used as
starting iodide in the Mizoroki–Heck reaction. After microwave irra-
diation, the reaction mixture was cooled down to 08C, and the col-
lected precipitate was washed with cold CH3CN (3 mL) and dried
by vacuum. The crude corresponding acrylate was purified by silica
column chromatography (EtOAc/MeOH 1:0 ! 10:1) to afford
158 mg (63% isolated yield) of (E)-3-(4-benzoylphenyl)acrylamide
as an off-white powder. TLC: Rf =0.47 (EtOAc); LC purity (254 nm):
1
99%; H NMR (400 MHz, [D6]DMSO): d=7.80–7.66 (m, 7H), 7.63 (bs,
1H), 7.61–7.55 (m, 2H), 7.50 (d, J=15.8 Hz, 1H), 7.21 (bs, 1H),
6.75 ppm (d, J=15.8 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=
156.0, 127.1, 99.8, 98.8, 98.1, 97.8, 93.6, 91.1, 90.4, 89.5, 88.5,
85.8 ppm; ESI-MS m/z: 252 [M+H]+, 503 [2M+H]+. According to
Method B, (E)-3-(4-benzoylphenyl)acrylamide (158 mg, 0.63 mmol)
was reacted with chlorocarbonyl sulfenyl chloride to give, after pu-
rification by precipitation from EtOAc/pentane, 157 mg (80% iso-
lated yield) of the title compound as a white powder. TLC: Rf =0.26
(E)-5-(4-(Trifluoromethoxy)styryl)-1,3,4-oxathiazol-2-one (32). Ac-
cording to Method D, 1-iodo-4-trifluoromethoxy-benzene (288 mg,
1.0 mmol) was used as starting iodide in the Mizoroki–Heck reac-
tion. After microwave irradiation, the reaction mixture was cooled
down to rt, and the formed Pd black was removed by filtration
through Celite. Evaporation of the solvent gave the crude corre-
sponding acrylate, which was purified by silica column chromatog-
raphy (double purification: 1st EtOAc; 2nd pentane/EtOAc 1:1 ! 0:1)
to afford 221 mg of a 1:1 mixture of (E)-3-(4-(trifluoromethoxy)phe-
nyl)acrylamide and starting acrylamide as a white powder. TLC: Miz-
oroki–Heck product Rf =0.48, acrylamide Rf =0.31 (EtOAc); ESI-MS
m/z: 232 [M+H]+. According to Method B, the obtained Mizoroki–
Heck reaction mixture (221 mg) was reacted with an excess of
chlorocarbonyl sulfenyl chloride (0.25 mL) to give, after purification
by silica column chromatography (pentane/EtOAc 100:0 ! 100:2),
185 mg (64% isolated yield after two steps) of the title compound
1
(pentane/EtOAc 10:1); LC purity (254 nm): 95%; H NMR (400 MHz,
[D6]DMSO): d=7.96 (dm, J=8.8 Hz, 2H), 7.80–7.74 (m, 4H), 7.73–
7.67 (m, 1H), 7.64 (d, J=16.4 Hz, 1H), 7.61–7.55 (m, 2H), 7.16 ppm
(d, J=16.4 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=195.1, 173.4,
157.1, 140.0, 138.1, 138.0, 136.8, 132.9, 130.1, 129.6, 128.6, 128.2,
115.6 ppm; ESI-MS m/z: 310 [M+H]+; HRMS m/z calcd for
C17H11NO3S [M+H]+ 310.0538, found 310.0535.
(E)-Methyl 4-(2-(2-oxo-1,3,4-oxathiazol-5-yl)vinyl)benzoate (35).
According to Method D, methyl 4-iodobenzoate (262 mg,
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1.0 mmol) was used as starting iodide in the Mizoroki–Heck reac-
tion. After microwave irradiation, the reaction mixture was cooled
down to 08C, and the collected precipitate was washed with cold
CH3CN (3 mL) and dried by vacuum. The crude corresponding acry-
late was purified by silica column chromatography (EtOAc/MeOH
1:0 ! 10:1) to afford 124 mg (60% isolated yield) of (E)-methyl 4-
(3-amino-3-oxoprop-1-en-1-yl)benzoate as an off-white powder.
from EtOAc/pentane, 47 mg (20% isolated yield after two steps) of
the title compound as an off-white powder. TLC: Rf =0.21 (pen-
tane/EtOAc 10:1); LC purity (254 nm): 99%; 1H NMR (400 MHz,
[D6]DMSO): d=7.98 (bd, J=8.4 Hz, 2H), 7.90 (bd, J=8.4 Hz, 2H),
7.61 (d, J=16.4 Hz, 1H), 7.20 ppm (d, J=16.4 Hz, 1H); 13C NMR
(100 MHz, [D6]DMSO): d=173.3, 156.9, 139.3, 138.8, 132.8, 128.8,
118.6, 116.6, 112.2 ppm; ESI-MS m/z: 231 [M+H]+; HRMS m/z calcd
for C11H6N2O2S [M+H]+ 231.0228, found 231.0226.
1
TLC: Rf =0.47 (EtOAc); LC purity (254 nm): 99%; H NMR (400 MHz,
[D6]DMSO): d=7.98 (bd, J=8.3 Hz, 2H), 7.70 (bd, J=8.3 Hz, 2H),
7.61 (bs, 1H), 7.46 (d, J=15.9 Hz, 1H), 7.20 (bs, 1H), 6.73 (d, J=
15.9 Hz, 1H), 3.86 ppm (s, 3H); 13C NMR (100 MHz, [D6]DMSO): d=
166.2, 165.8, 139.5, 137.8, 129.9, 129.7, 127.8, 125.0, 52.2 ppm; ESI-
MS m/z: 206 [M+H]+, 411 [2M+H]+. According to Method B, (E)-
(E)-4-(2-(2-Oxo-1,3,4-oxathiazol-5-yl)vinyl)benzaldehyde (38). Ac-
cording to Method D, 4-iodobenzaldehyde (232 mg, 1.0 mmol) was
used as starting iodide in the Mizoroki–Heck reaction. After micro-
wave irradiation, the reaction mixture was cooled down to rt, and
the formed Pd black was removed by filtration through Celite.
Evaporation of the solvent gave the crude corresponding acrylate,
which was purified by silica column chromatography (double pu-
rification: 1st EtOAc, 2nd pentane/EtOAc 1:1 ! 0:1) to afford
100 mg of a 2:3 mixture of (E)-3-(4-formylphenyl)acrylamide and
starting acrylamide as a white powder. TLC: Mizoroki–Heck product
Rf =0.37, acrylamide Rf =0.31 (EtOAc); ESI-MS m/z: 176 [M+H]+.
According to Method B, the obtained Mizoroki–Heck reaction mix-
ture (100 mg) was reacted with an excess of chlorocarbonyl sulfe-
nyl chloride (0.10 mL) to give, after purification by precipitation
from EtOAc/pentane, 36 mg (15% isolated yield after two steps) of
the title compound as a pale yellow powder. TLC: Rf =0.18 (pen-
tane/EtOAc 10:1); LC purity (254 nm): 98%; 1H NMR (400 MHz,
[D6]DMSO): d=10.03 (s, 1H), 8.01 (bd, J=8.4 Hz, 2H), 7.95 (bd, J=
8.4 Hz, 2H), 7.62 (d, J=16.4 Hz, 1H), 7.19 ppm (d, J=16.4 Hz, 1H);
13C NMR (100 MHz, [D6]DMSO): d=192.6, 173.3, 157.0, 139.9, 139.8,
136.8, 129.9, 128.8, 116.2 ppm; ESI-MS m/z: 234 [M+H]+; HRMS
m/z for C11H7NO3S as [M+H]+ adduct not found due to low ioniza-
tion level.
methyl
4-(3-amino-3-oxoprop-1-en-1-yl)benzoate
(124 mg,
0.60 mmol) was reacted with chlorocarbonyl sulfenyl chloride to
give, after purification by precipitation from EtOAc/pentane,
108 mg (68% isolated yield) of the title compound as white
powder. TLC: Rf =0.32 (pentane/EtOAc 10:1); LC purity (254 nm):
99%; 1H NMR (400 MHz, [D6]DMSO): d=7.98 (bd, J=8.6 Hz, 2H),
7.92 (bd, J=8.6 Hz, 2H), 7.60 (d, J=16.4 Hz, 1H), 7.13 (d, J=
16.4 Hz, 1H), 3.87 ppm (s, 3H); 13C NMR (100 MHz, [D6]DMSO): d=
173.3, 165.7, 157.0, 139.8, 138.7, 130.6, 129.6, 128.4, 115.7,
52.3 ppm; ESI-MS m/z: 264 [M+H]+; HRMS m/z calcd for
C12H9NO4S [M+H]+ 264.0331, found 264.0336.
(E)-5-(4-Nitrostyryl)-1,3,4-oxathiazol-2-one (36). According to
Method D, 1-iodo-4-nitrobenzene (249 mg, 1.0 mmol) was used as
starting iodide in the Mizoroki–Heck reaction. After microwave irra-
diation, the reaction mixture was cooled down to 08C, and the col-
lected precipitate was washed with cold CH3CN (3 mL) and dried
by vacuum. The crude corresponding acrylate was purified by silica
column chromatography (EtOAc/MeOH 1:0 ! 10:1) to afford
95 mg (49% isolated yield) of (E)-3-(4-nitrophenyl)acrylamide as
a yellow powder. TLC: Rf =0.53 (EtOAc); LC purity (254 nm): 99%;
1H NMR (400 MHz, [D6]DMSO): d=8.26 (bd, J=9.0 Hz, 2H), 7.83
(bd, J=9.0 Hz, 2H), 7.66 (bs, 1H), 7.52 (d, J=15.9 Hz, 1H), 7.27 (bs,
1H), 6.80 ppm (d, J=15.9 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO):
d=166.9, 147.5, 141.5, 136.8, 128.6, 126.7, 124.1 ppm; ESI-MS m/z:
193 [M+H]+. According to Method B, (E)-3-(4-nitrophenyl)acryla-
mide (95 mg, 0.49 mmol) was reacted with chlorocarbonyl sulfenyl
chloride to give, after purification by precipitation from EtOAc/pen-
tane, 99 mg (80% isolated yield) of the title compound as a pale
yellow powder. TLC: Rf =0.24 (pentane/EtOAc 10:1); LC purity
(254 nm): 99%; 1H NMR (400 MHz, [D6]DMSO): d=8.26 (bd, J=
8.9 Hz, 2H), 8.07 (bd, J=8.9 Hz, 2H), 7.67 (d, J=16.4 Hz, 1H),
7.24 ppm (d, J=16.4 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=
173.3, 156.8, 147.9, 140.7, 138.7, 129.2, 124.0, 117.3 ppm; ESI-MS
m/z: 251 [M+H]+; HRMS m/z for C10H6N2O4S as [M+H]+ adduct
not found due to low ionization level.
(E)-5-(4-Chlorostyryl)-1,3,4-oxathiazol-2-one (39). According to
Method D, 1-chloro-4-iodobenzene (238 mg, 1.0 mmol) was used as
starting iodide in the Mizoroki–Heck reaction. After microwave irra-
diation, the reaction mixture was cooled down to 08C, and the col-
lected precipitate was washed with cold CH3CN (3 mL) and dried
by vacuum. The crude corresponding acrylate was purified by silica
column chromatography (EtOAc/MeOH 1:0 ! 10:1) to afford
123 mg (68% isolated yield) of (E)-3-(4-chlorophenyl)acrylamide as
a yellow powder. TLC: Rf =0.60 (EtOAc); LC purity (254 nm): 99%;
1H NMR (400 MHz, [D6]DMSO): d=7.58 (dm, J=8.4 Hz, 2H), 7.54
(bs, 1H), 7.47 (dm, J=8.4 Hz, 2H), 7.40 (d, J=15.9 Hz, 1H), 7.13 (bs,
1H), 6.61 ppm (d, J=15.9 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO):
d=166.4, 137.8, 133.9, 133.8, 129.2, 128.9, 123.2 ppm; ESI-MS m/z:
182/184 [M+H]+, 363/365 [2M+H]+. According to Method B, (E)-
3-(4-chlorophenyl)acrylamide (123 mg, 0.68 mmol) was reacted
with chlorocarbonyl sulfenyl chloride to give, after purification by
precipitation from EtOAc/pentane, 118 mg (73% isolated yield) of
the title compound as a white powder. TLC: Rf =0.68 (pentane/
EtOAc 10:1); LC purity (254 nm): 99%; 1H NMR (400 MHz,
[D6]DMSO): d=7.81 (dm, J=8.4 Hz, 2H), 7.54 (d, J=16.3 Hz, 1H),
7.50 (dm, J=8.4 Hz, 2H), 7.03 ppm (d, J=16.3 Hz, 1H); 13C NMR
(100 MHz, [D6]DMSO): d=173.4, 157.2, 140.0, 134.9, 133.2, 129.9,
129.0, 114.0 ppm; ESI-MS m/z: 240/242 [M+H]+; HRMS m/z for
C10H6ClNO2S as [M+H]+ adduct not found due to low ionization
level.
(E)-4-(2-(2-Oxo-1,3,4-oxathiazol-5-yl)vinyl)benzonitrile (37). Ac-
cording to Method D, 4-iodobenzonitrile (229 mg, 1.0 mmol) was
used as starting iodide in the Mizoroki–Heck reaction. After micro-
wave irradiation, the reaction mixture was cooled down to rt, and
the formed Pd black was removed by filtration through Celite.
Evaporation of the solvent gave the crude corresponding acrylate,
which was purified by silica column chromatography (double pu-
rification: 1st EtOAc, 2nd pentane/EtOAc 1:1 ! 0:1) to afford
123 mg of a 1:2 mixture of (E)-3-(4-cyanophenyl)acrylamide and
starting acrylamide as white a powder. TLC: Mizoroki–Heck product
Rf =0.37, acrylamide Rf =0.31 (EtOAc); ESI-MS: m/z 173 [M+H]+.
According to Method B, the obtained Mizoroki–Heck reaction mix-
ture (123 mg) was reacted with an excess of chlorocarbonyl sulfe-
nyl chloride (0.21 mL) to give, after purification by precipitation
(E)-5-(2,5-Dichlorostyryl)-1,3,4-oxathiazol-2-one (40). According
to Method D, 1,4-dichloro-2-iodobenzene (273 mg, 1.0 mmol) was
used as starting iodide in the Mizoroki–Heck reaction. After micro-
wave irradiation, the reaction mixture was cooled down to rt, and
the formed Pd black was removed by filtration through Celite.
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Evaporation of the solvent gave the crude corresponding acrylate,
which was purified by silica column chromatography (double pu-
rification: 1st EtOAc; 2nd pentane/EtOAc 1:1 ! 0:1) to afford
184 mg of a 1:1 mixture of (E)-3-(2,5-dichlorophenyl)acrylamide
and starting acrylamide as a white powder. TLC: Mizoroki–Heck
product Rf =0.69, acrylamide Rf =0.31 (EtOAc); ESI-MS m/z: 216/
217 [M+H]+. According to Method B, the obtained Mizoroki–Heck
reaction mixture (184 mg) was reacted with an excess of chlorocar-
bonyl sulfenyl chloride (0.24 mL) to give, after purification by pre-
cipitation from EtOAc/pentane, 31 mg (11% isolated yield after
two steps) of the title compound as an off-white powder. TLC: Rf =
0.79 (pentane/EtOAc 10:1); LC purity (254 nm): 99%; 1H NMR
(400 MHz, [D6]DMSO): d=8.18 (d, J=2.5 Hz, 1H), 7.63 (d, J=
16.2 Hz, 1H), 7.61 (d, J=8.7 Hz, 1H), 7.53 (dd, J=8.7, 2.5 Hz, 1H),
7.25 ppm (d, J=16.2 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=
173.2, 156.6, 134.4, 133.5, 132.7, 132.1, 131.6, 131.4, 127.5,
117.6 ppm; ESI-MS m/z: 274/276 [M+H]+; HRMS m/z for
C10H5Cl2NO2S as [M+H]+ adduct not found due to low ionization
level.
through Celite. Evaporation of the solvent gave the crude corre-
sponding acrylate, which was purified by silica column chromatog-
raphy (EtOAc/isohexane 50:50 ! 75:25) to (E)-3-(3-(trifluorome-
thoxy)phenyl)acrylamide as a pale yellow solid. TLC: Rf =0.47
(EtOAc:iso-hexane, 75:25); ESI-MS m/z: 232 [M+H]+. According to
Method A, the crude 3-substituted acrylamide was reacted with an
excess of chlorocarbonyl sulfenyl chloride (0.22 mL) to give, after
silica column chromatography (etylacetate/isohexane 1:100),
176 mg (61% isolated yield after two steps) as a white solid. ESI-
1
MS m/z: 299 [M+H]+; LC purity (254 nm): 98%; H NMR (400 MHz,
[D6]DMSO): d=7.81 (m, 1H), 7.78, (dm, J=8.0 Hz, 1H), 7.55 (d, J=
16.4 Hz, 1H), 7.54 (m, 1H), 7.38 (m, 1H), 7.01 ppm (d, J=16.4 Hz,
1H); 13C NMR (100 MHz, [D6]DMSO): d=173.8, 157.5, 149.3, 140.0,
137.1, 131.3, 127.7, 122.9, 121.0, 120.5 (q, J=257 Hz), 115.6 ppm;
HRMS m/z calcd for C11H6F3NO3S [M+H]+ 299.0099, found
299.0103.
(E)-Ethyl 3-(2-(2-Oxo-1,3,4-oxathiazol-5-yl)vinyl)benzoate (44).
According to Method D, ethyl-3-iodobenzoate (276 mg, 1.0 mmol)
was used as starting iodide in the Mizoroki–Heck reaction. After
microwave irradiation, the reaction mixture was cooled down to rt,
and the formed Pd black was removed by filtration through Celite.
Evaporation of the solvent gave the crude corresponding acrylate,
which was purified by silica column chromatography (EtOAc/iso-
hexane 50:50 ! 75:25) to afford 206 mg of (E)-3-(3- ethoxycarbo-
nyl)acrylamide as pale yellow solid. TLC: Rf =0.58, (EtOAc); ESI-MS
m/z: 220 [M+H]+. According to Method A, the crude 3-substituted
acrylamide (206 mg) was reacted with an excess of chlorocarbonyl
sulfenyl chloride (0.17 mL) to give, after recrystallization in ethyla-
cetate/isohexane, 118 mg (43% isolated yield after two steps) as
a white solid, 43% yield. ESI-MS m/z: 278 [M+H]+; LC purity
(254 nm): 99%; 1H NMR (400 MHz, [D6]DMSO): d=8.24 (m, 1H),
8.05, (dm, J=7.7 Hz, 1H), 7.97 (dm, J=7.8 Hz, 1H), 7.60 (d, J=
16.4 Hz, 1H), 7.58 (dd, J=7.7, 7.8 Hz, 1H), 7.05 (d, J=16.4 Hz, 1H),
4.32 ppm (q, J=7.2 Hz, 2H), 1.32 (t, J=7.2 Hz, 3H); 13C NMR
(100 MHz, [D6]DMSO): d=173.9, 165.8, 157.6, 140.7, 135.2, 132.6,
131.2, 131.1, 129.9, 129.4, 114.9, 61.5, 14.6 ppm; HRMS m/z calcd
for C13H11NO4S [M+H]+ 278.0487, found 278.0486.
(E)-5-(3-Methylstyryl)-1,3,4-oxathiazol-2-one (41). According to
Method D, 1-iodo-3-methylbenzene (218 mg, 1.0 mmol) was used
as starting iodide in the Mizoroki–Heck reaction. After microwave
irradiation, the reaction mixture was cooled down to rt, and the
formed Pd black was removed by filtration through Celite. Evapo-
ration of the solvent gave the crude to (E)-3-(m-tolyl)acrylamide,
ESI-MS m/z: 162 [M+H]+. According to Method A, the crude 3-
substituted acrylamide was reacted with an excess of chlorocar-
bonyl sulfenyl chloride (0.34 mL) to give, after silica column chro-
matography (CH2Cl2/isohexane 1:9) and recrystallization in CH3CN,
76 mg (35% isolated yield after step two) as a white solid. ESI-MS
m/z: 220 [M+H]+; LC purity (254 nm): 99%; 1H NMR (400 MHz,
CDCl3): d=7.47 (d, J=16.2 Hz, 1H), 7.34–7.20 (m, 4H), 6.63 (d, J=
16.2 Hz, 1H), 2.39 ppm (m, 3H); 13C NMR (100 MHz, CDCl3): d=
173.4, 157.7, 142.1, 138.8, 134.0, 131.4, 129.0, 128.5, 125.0, 112.6,
21.3 ppm; HRMS m/z calcd for C11H9NO2S [M+H]+ 220.0432, found
220.0437.
(E)-5-(3-Ethylstyryl)-1,3,4-oxathiazol-2-one (42). According to
Method E, 1-bromo-3-ethylbenzene (370 mg, 2.0 mmol) was used
as starting bromide in the Mizoroki–Heck reaction. After microwave
irradiation, the reaction mixture was cooled down to rt, filtered
through a plug of cotton, and concentrated under reduced pres-
sure. The residue was dissolved in EtOAc (25 mL) and washed with
H2O (25 mL), and the organic phase was dried over MgSO4 and
evaporated to yield the crude (E)-3-(3-ethylphenyl)acrylamide as
a pale yellow solid. ESI-MS m/z: 176 [M+H]+. According to Meth-
od A, the crude 3-substituted acrylamide was reacted with an
excess of chlorocarbonyl sulfenyl chloride (0.67 mL) to give, after
silica column chromatography (EtOAc/isohexane 2:100 ! 5:100)
and recrystallization in CH3CN, 77 mg (17% isolated yield after two
steps) as a white solid. ESI-MS m/z: 234 [M+H]+; LC purity
(254 nm): 99%; 1H NMR (400 MHz, CDCl3): d=7.48 (d, J=16.2 Hz,
1H), 7.36–7.23 (m, 4H), 6.63 (d, J=16.2 Hz, 1H), 2.68 (q, J=7.6 Hz,
2H), 1.27 ppm (t, J=7.6 Hz, 3H); 13C NMR (100 MHz, CDCl3): d=
173.4, 157.8, 145.1, 142.2, 134.1, 130.3, 129.1, 127.4, 125.3, 112.6,
28.7, 15.5 ppm; HRMS m/z calcd for C12H11NO2S [M+H]+ 234.0589,
found 234.0591.
(E)-5-(2-Methylstyryl)-1,3,4-oxathiazol-2-one (45). According to
Method D, 1-iodo-2-methylbenzene (218 mg, 1.0 mmol) was used
as starting iodide in the Mizoroki–Heck reaction. After microwave
irradiation, the reaction mixture was cooled down to rt, and the
formed Pd black was removed by filtration through Celite. Evapo-
ration of the solvent gave the crude corresponding acrylate, which
was purified by silica column chromatography (EtOAc/isohexane
50:50 ! 75:25) to (E)-3-(o-tolyl)acrylamide as a pale yellow solid,
149 mg. TLC: Rf =0.48 (EtOAc); ESI-MS m/z: 162 [M+H]+. Accord-
ing to Method A, the crude 3-substituted acrylamide was reacted
with an excess of chlorocarbonyl sulfenyl chloride (0.26 mL) to
give, after silica column chromatography (EtOAc/isohexane 1:100),
70 mg (32% isolated yield after two steps) as a white solid. ESI-MS
m/z: 220 [M+H]+; LC purity (254 nm): 95%; 1H NMR (400 MHz,
CDCl3): d=7.69 (d, J=16.2 Hz, 1H), 7.48 (dm, J=7.8 Hz, 1H), 7.25–
7.13 (m, 3H), 6.49 (d, J=16.2 Hz, 1H), 2.37 ppm (m, 3H); 13C NMR
(100 MHz, CDCl3): d=173.5, 157.8, 139.5, 137.6, 133.0, 131.0, 130.3,
126.5, 125.9, 113.8, 19.8 ppm; HRMS m/z calcd for C11H9NO2S
[M+H]+ 220.0432, found 220.0436.
(E)-5-(3-(Trifluoromethoxy)styryl)-1,3,4-oxathiazol-2-one (43). Ac-
cording to Method D, 1-iodo-3-(trifluoromethoxy)benzene (288 mg,
1.0 mmol) was used as starting iodide in the Mizoroki–Heck reac-
tion. After microwave irradiation, the reaction mixture was cooled
down to rt, and the formed Pd black was removed by filtration
(E)-5-(2-Ethylstyryl)-1,3,4-oxathiazol-2-one (46). According to
Method E, 1-bromo-2-ethylbenzene (218 mg, 1.0 mmol) was used
as starting bromide in the Mizoroki–Heck reaction. After microwave
irradiation, the reaction mixture was cooled down to rt, and fil-
tered through a plug of cotton. The reaction mixture was then ex-
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tracted between EtOAc (25 mL) and H2O (25 mL), and the organic
phase was dried over MgSO4 and evaporated to yield the crude
(E)-3-(2-ethylphenyl)acrylamide as a pale yellow solid, 117 mg. ESI-
MS m/z: 176 [M+H]+. According to Method A, the crude 3-substi-
tuted acrylamide was reacted with an excess of chlorocarbonyl sul-
fenyl chloride (0.23 mL) to give, after silica column chromatogra-
phy (EtOAc/isohexane 2:100), 90 mg (19% isolated yield after two
steps) as a white solid. ESI-MS m/z: 234 [M+H]+; LC purity
(254 nm): 98%; 1H NMR (400 MHz, CDCl3): d=7.80 (d, J=16.2 Hz,
1H), 7.57 (m, 1H), 7.35 (m, 1H), 7.28–7.22 (m, 2H), 6.58 (d, J=
16.2 Hz, 1H), 2.79 (q, J=7.5 Hz, 2H), 1.24 ppm (t, J=7.5 Hz, 3H);
13C NMR (100 MHz, CDCl3): d=173.5, 157.8, 143.8, 139.3, 132.3,
130.6, 129.4, 126.5, 126.1, 113.9, 26.4, 15.9 ppm; HRMS m/z calcd
for C12H11NO2S [M+H]+ 234.0589, found 234.0584.
a pale yellow solid. TLC: Mizoroki–Heck product Rf =0.25, acrylam-
ide Rf =0.72 (EtOAc/MeOH 9:1); ESI-MS m/z: 149 [M+H]+. Accord-
ing to Method B, the obtained Mizoroki–Heck reaction mixture
(120 mg) was reacted with an excess of chlorocarbonyl sulfenyl
chloride (0.34 mL) to give, after purification by preparative HPLC,
15 mg (7% isolated yield after two steps) of the title compound as
an off-white powder. TLC: Rf =0.32 (pentane/EtOAc=1:1); LC
1
purity (254 nm): 95%; H NMR (400 MHz, [D6]DMSO): d=8.66–8.63
(m, 2H), 7.75–7.72 (m, 2H), 7.53 (d, J=16.3 Hz, 1H), 7.27 ppm (d,
J=16.3 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=173.2, 156.8,
150.4, 141.3, 138.7, 121.9, 117.6 ppm; ESI-MS m/z: 207 [M+H]+;
HRMS m/z calcd for C9H6N2O2S [M+H]+ 207.0228, found 207.0255.
(E)-5-(2-(2-Methoxypyridin-3-yl)vinyl)-1,3,4-oxathiazol-2-one (50).
According to Method D, 3-iodo-2-methoxy-pyridine (235 mg,
1.0 mmol) was used as starting iodide in the Mizoroki–Heck reac-
tion. After microwave irradiation, the reaction mixture was cooled
down to rt, and the formed Pd black was removed by filtration
through silica gel (CH3CN). Evaporation of the solvent gave the
crude corresponding acrylate, which was purified by silica column
chromatography (pentane/EtOAc 1:1 ! 0:1, then EtOAc/MeOH
9:1) to afford 184 mg of a 1:1 mixture of (E)-3-(2-methoxypyridin-3-
yl)acrylamide and starting acrylamide as a pale yellow oil. TLC: Miz-
oroki–Heck product Rf =0.68, acrylamide Rf =0.60 (EtOAc/MeOH
9:1); ESI-MS m/z: 179 [M+H]+. According to Method A, the ob-
tained Mizoroki–Heck reaction mixture (184 mg) was reacted with
an excess of chlorocarbonyl sulfenyl chloride (0.34 mL) to give,
after purification by trituration with Et2O followed by precipitation
from CH3CN/H2O, 91 mg (39% isolated yield after two steps) of the
title compound as a pale yellow solid. TLC: Rf =0.80 (EtOAc); LC
purity (254 nm): 95%; 1H NMR (400 MHz, [D6]DMSO): d=8.23 (dd,
J=4.9, 1.8 Hz, 1H), 8.17 (dd, J=7.4, 1.8 Hz, 1H), 7.56 (d, J=16.5 Hz,
1H), 7.09 (dd, J=7.4, 4.9 Hz, 1H), 7.06 (d, J=16.5 Hz, 1H),
3.98 ppm (s, 3H); 13C NMR (100 MHz, [D6]DMSO): d=173.6, 161.1,
157.4, 148.7, 138.2, 135.1, 117.8, 117.1, 115.4, 53.9 ppm; ESI-MS
m/z: 237 [M+H]+; HRMS m/z calcd for C10H8N2O3S [M+H]+
237.0334, found 237.0338.
(E)-5-(2-(Trifluoromethoxy)styryl)-1,3,4-oxathiazol-2-one (47). Ac-
cording to Method D, 1-iodo-2-(trifluoromethoxy)benzene (288 mg,
1.0 mmol) was used as starting iodide in the Mizoroki–Heck reac-
tion. After microwave irradiation, the reaction mixture was cooled
down to rt, and the formed Pd black was removed by filtration
through Celite. Evaporation of the solvent gave the crude corre-
sponding acrylate, which was purified by silica column chromatog-
raphy (EtOAc/isohexane 50:50 ! 75:25) to (E)-3-(2-(trifluorome-
thoxy)phenyl)acrylamide as a pale yellow solid, 229 mg. TLC: Rf =
0.49 (EtOAc/iso-hexane 75:25); ESI-MS m/z: 232 [M+H]+. Accord-
ing to Method A, the crude 3-substituted acrylamide was reacted
with an excess of chlorocarbonyl sulfenyl chloride (0.20 mL) to
give, after precipitation from EtOAc/isohexane, 110 mg (38% isolat-
ed yield after two steps) as a white solid. ESI-MS m/z: 299 [M+H]+;
1
LC purity (254 nm): 99%; H NMR (400 MHz, [D6]DMSO): d=8.04 (d,
J=7.8 Hz, 1H), 7.54 (m, 1H), 7.47 (d, J=16.4 Hz, 1H), 7.45–7.38 (m,
2H), 7.08 ppm (d, J=16.4 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO):
d=173.7, 157.1, 146.7, 132.9, 132.6, 128.7, 128.6, 127.5, 122.1, 120.6
(q, J=258 Hz), 110.0 ppm; HRMS m/z calcd for C11H6F3NO3S [M+
H]+ 299.0099, found 299.0096.
(E)-Ethyl 2-(2-(2-Oxo-1,3,4-oxathiazol-5-yl)vinyl)benzoate (48).
According to Method E, ethyl 2-bromobenzoate (506 mg,
2.2 mmol) was used as starting bromide in the Mizoroki–Heck reac-
tion. After microwave irradiation, the product was filtered and
washed with EtOAc (20 mL) give (E)-ethyl 2-(3-amino-3-oxoprop-1-
en-1-yl)benzoate as a pale white solid, 391 mg. According to Meth-
od A, the crude 3-substituted acrylamide was reacted with an
excess of chlorocarbonyl sulfenyl chloride (0.60 mL) to give, after
filtration and washing with EtOAc (10 mL), 198 mg (32% isolated
yield after two steps) as a white solid. ESI-MS m/z: 278 [M+H]+;
(E)-5-(2-(Thiophen-2-yl)vinyl)-1,3,4-oxathiazol-2-one (51). Accord-
ing to Method C, 2-iodothiophene (210 mg, 1.0 mmol) was used as
starting iodide in the Mizoroki–Heck reaction. After 15 min of mi-
crowave irradiation (T=1208C), the reaction mixture was irradiated
for additional 30 min at the same temperature to afford full con-
version. The resulting reaction mixture was cooled down to rt, and
the formed Pd black was removed by filtration through silica gel
(CH3CN!EtOAc!EtOAc/MeOH 10:1). Evaporation of the solvent
gave the crude corresponding acrylate, which was purified by pre-
cipitation from CH3OH/H2O to afford 70 mg (46% isolated yield) of
(E)-3-(thiophen-2-yl)acrylamide as an off-white solid. TLC: Rf =0.52
(EtOAc); LC purity (254 nm): 95%; 1H NMR (400 MHz, CDCl3): d=
7.71 (d, J=15.3 Hz, 1H), 7.28 (bd, J=5.1 Hz, 1H), 7.17 (bd, J=
3.5 Hz, 1H), 6.98 (dd, J=5.1, 3.5 Hz, 1H), 6.19 (d, J=15.3 Hz, 1H),
5.36 ppm (bs, 2H); ESI-MS m/z: 154 [M+H]+. According to Meth-
od B, (E)-3-(thiophen-2-yl)acrylamide (70 mg, 0,46 mmol) was react-
ed with an excess of chlorocarbonyl sulfenyl chloride (0.40 mL) to
give, after purification by silica column (pentane/EtOAc 100:0 !
1:1) followed by preparative HPLC purification, 46 mg (45% isolat-
ed yield) of the title compound as a white powder. TLC: Rf =0.63
1
LC purity (254 nm): 99%; H NMR (400 MHz, [D6]DMSO): d=8.18 (d,
J=16.2 Hz, 1H), 7.95 (dm, J=8.0 Hz, 1H), 7.90 (dd, J=7.8, 1.6 Hz,
1H), 7.65 (ddd, J=8.0, 7.6, 1.6 Hz, 1H), 7.54 (ddm, J=7.8, 7.6 Hz,
1H), 6.92 (d, J=16.2 Hz, 1H), 4.33 (q, J=7.2 Hz, 2H), 1.33 ppm (t,
J=7.2 Hz, 3H); 13C NMR (100 MHz, [D6]DMSO): d=173.8, 166.7,
157.6, 139.7, 125.1, 133.0, 130.9, 130.5, 130.3, 128.1, 116.0, 61.7,
14.5 ppm; HRMS m/z calcd for C13H11NO4S [M+H]+ 278.0487,
found 278.0485.
(E)-5-(2-(Pyridin-4-yl)vinyl)-1,3,4-oxathiazol-2-one (49). According
to Method D, 4-iodopyridine (205 mg, 1.0 mmol) was used as start-
ing iodide in the Mizoroki–Heck reaction. After microwave irradia-
tion, the reaction mixture was cooled down to rt, and the formed
Pd black was removed by filtration through silica gel (CH3CN).
Evaporation of the solvent gave the crude corresponding acrylate,
which was purified by silica column chromatography (pentane/
EtOAc 1:1 ! 0:1, then EtOAc/MeOH 9:1) to afford 120 mg of a 1:1
mixture of (E)-3-(pyridin-4-yl)acrylamide and starting acrylamide as
1
(pentane/EtOAc 20:1); LC purity (254 nm): 95%; H NMR (400 MHz,
[D6]DMSO): d=7.76 (bd, J=5.1 Hz, 1H), 7.72 (d, J=16.1 Hz, 1H),
7.59 (bd, J=3.7 Hz, 1H), 7.17 (dd, J=5.1, 3.7 Hz, 1H), 6.61 ppm (d,
J=16.1 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=173.4, 157.0,
138.9, 134.3, 131.8, 130.2, 128.7, 111.3 ppm; ESI-MS m/z: 212
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ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

[M+H]+; HRMS m/z calcd for C8H5NO2S2 [M+H]+ 211.9840, found
211.9837.
for additional 60 min at 1608C to afford full conversion. The result-
ing reaction mixture was cooled down to rt, and the formed Pd
black was removed by filtration through Celite (EtOAc/MeOH 1:1).
Evaporation of the solvent under reduced pressure gave a crude
mixture containing DMF, which was taken in EtOAc (20 mL). The or-
ganic phase was washed with H2O (2”40 mL), the H2O phases
were extracted with EtOAc (2”100 mL), and the collected organic
phases were dried over Na2SO4. Evaporation of solvent gave
126 mg of a mixture containing the desired (E)-3-(pyridin-2-yl)acry-
lamide as a yellow oil. TLC: Mizoroki–Heck compound Rf =0.07
(EtOAc/MeOH 10:1); LC purity (254 nm)=70%; ESI-MS m/z: 149
[M+H]+. According to Method A, the obtained Mizoroki–Heck re-
action mixture (126 mg) was reacted with an excess of chlorocar-
bonyl sulfenyl chloride (0.50 mL) to give, after purification by prep-
arative HPLC, 8 mg (3% isolated yield after two steps) of the title
(E)-5-(2-(Thiophen-3-yl)vinyl)-1,3,4-oxathiazol-2-one (52). Accord-
ing to Method C, 3-iodothiophene (210 mg, 1.0 mmol) was used as
starting iodide in the Mizoroki–Heck reaction. After 15 min of mi-
crowave irradiation (T=1208C), the reaction mixture was irradiated
for additional 30 min at the same temperature to afford full con-
version. The resulting reaction mixture was cooled down to rt, and
the formed Pd black was removed by filtration through silica gel
(CH3CN!EtOAc!EtOAc/MeOH 10:1). Evaporation of the solvent
gave the crude corresponding acrylate, that was purified by precip-
itation from EtOAc/pentane followed by silica column purification
(pentane/EtOAc 1:1 ! 0:1) to afford 115 mg (75% isolated yield)
of (E)-3-(thiophen-3-yl)acrylamide as a white solid. TLC: Rf =0.46
(EtOAc); LC purity (254 nm): 95%; 1H NMR (400 MHz, CDCl3): d=
7.64 (d, J=15.4 Hz, 1H), 7.47 (bd, J=2.9 Hz, 1H), 7.33 (dd, J=5.1,
2.9 Hz, 1H), 7.28 (bd, J=5.1 Hz, 1H), 6.30 ppm (d, J=15.4 Hz, 1H),
5.53 (bs, 2H); ESI-MS m/z: 154 [M+H]+. According to Method B,
(E)-3-(thiophen-3-yl)acrylamide (115 mg, 0,75 mmol) was reacted
with an excess of chlorocarbonyl sulfenyl chloride (0.40 mL) to
give, after purification by silica column (pentane/EtOAc 100:0 !
1:1) followed by preparative HPLC purification, 84 mg (53% isolat-
ed yield) of the title compound as a white powder. TLC: Rf =0.55
(pentane/EtOAc=20:1); LC purity (254 nm): 95%; 1H NMR
(400 MHz, [D6]DMSO): d=8.02–8.00 (m, 1H), 7.66–7.61 (m, 2H),
7.55 (d, J=16.2 Hz, 1H), 6.82 ppm (d, J=16.2 Hz, 1H); 13C NMR
(100 MHz, [D6]DMSO): d=173.5, 157.6, 137.5, 135.4, 129.6, 127.9,
125.7, 112.6 ppm; ESI-MS: m/z 212 [M+H]+; HRMS m/z calcd for
C8H5NO2S2 [M+H]+ 211.9840, found 211.9841.
1
compound as a brown powder. LC purity (254 nm): 92%; H NMR
(400 MHz, [D6]DMSO): d=8.69–8.65 (m, 1H), 7.89 (td, J=7.8,
2.0 Hz, 1H), 7.78 (dm, J=8.0 Hz, 1H), 7.58 (d, J=16.0 Hz, 1H), 7.43
(ddd, J=7.8, 4.7, 1.2 Hz, 1H), 7.20 ppm (d, J=16.0 Hz, 1H);
13C NMR (100 MHz, [D6]DMSO): d=173.3, 156.9, 152.0, 150.1, 140.3,
137.4, 125.0, 124.8, 116.0 ppm; ESI-MS m/z: 207 [M+H]+; HRMS
m/z calcd for C9H6N2O2S [M+H]+ 207.0228, found 207.0234.
(E)-5-(2-(Pyridin-3-yl)vinyl)-1,3,4-oxathiazol-2-one (55). According
to Method E, 3-bromopyridine (237 mg, 1.5 mmol) was used as
starting bromide in the Mizoroki–Heck reaction. After 15 min of mi-
crowave irradiation (T=1408C), the reaction mixture was cooled
down to rt, and the formed Pd black was removed by filtration
through Celite (EtOAc/MeOH 1:1). Evaporation of the solvent under
reduced pressure gave a crude mixture containing DMF, which was
taken in EtOAc (20 mL). The organic phase was washed with H2O
(2”40 mL), the H2O phases were extracted with EtOAc (2”
100 mL), and the collected organic phases were dried over Na2SO4.
Evaporation of solvent gave 102 mg of a mixture containing the
desired (E)-3-(pyridin-3-yl)acrylamide as a white solid. TLC: Mizoro-
ki–Heck compound Rf =0.26 (EtOAc/MeOH 10:1); LC purity
(254 nm): 90%; ESI-MS m/z: 149 [M+H]+. According to Method A,
the obtained Mizoroki–Heck reaction mixture (102 mg) was reacted
with an excess of chlorocarbonyl sulfenyl chloride (0.50 mL) by mi-
crowave irradiation, followed by thermal heating at 708C for 2 d to
give, after purification by preparative HPLC, 45 mg (15% isolated
yield after two steps) of the title compound as a white powder. LC
purity (254 nm): 99%; 1H NMR (400 MHz, [D6]DMSO): d=8.92 (bd,
J=2.1 Hz, 1H), 8.59 (dd, J=4.9, 1.8 Hz, 1H), 8.24 (dm, J=8.0 Hz,
1H), 7.58 (d, J=16.4 Hz, 1H), 7.47 (bdd, J=7.5, 4.3 Hz, 1H),
7.16 ppm (d, J=16.4 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=
173.4, 157.0, 150.9, 149.8, 138.1, 134.4, 130.1, 123.9, 115.1 ppm; ESI-
MS m/z: 207 [M+H]+; HRMS m/z calcd for C9H6N2O2S [M+H]+
207.0228, found 207.0229.
(E)-5-(2-(2-Oxo-1,3,4-oxathiazol-5-yl)vinyl)furan-2-carbaldehyde
(53). According to Method C, 5-iodo-2-furaldehyde (222 mg,
1.0 mmol) was used as starting iodide in the Mizoroki–Heck reac-
tion. After 15 min of microwave irradiation (T=1208C), the reaction
mixture was irradiated for additional 30 min at the same tempera-
ture to afford full conversion. The resulting reaction mixture was
cooled down to rt, and the formed Pd black was removed by filtra-
tion through silica gel (CH3CN!EtOAc!EtOAc/MeOH 10:1). Evap-
oration of the solvent gave a crude mixture that was taken in
CH3OH/H2O and then filtered to remove the precipitated homo-
coupling byproduct. Evaporation of the solvent gave 262 mg of
a crude mixture, which was purified by flash silica column (pen-
tane/EtOAc 1:1 ! 0:1) to give 88 mg of a mixture containing the
desired (E)-3-(5-formylfuran-2-yl)acrylamide as a yellow solid. TLC:
Mizoroki–Heck compound Rf =0.44 (EtOAc); LC purity (254 nm):
30%; ESI-MS m/z: 166 [M+H]+. According to Method B, the ob-
tained Mizoroki–Heck reaction mixture (88 mg) was reacted with
an excess of chlorocarbonyl sulfenyl chloride (0.40 mL) to give,
after purification by silica column (pentane/EtOAc 100:0 ! 1:1) fol-
lowed by preparative HPLC purification, 10 mg (5% isolated yield
after two steps) of the title compound as a white powder. TLC:
(E)-5-(2-(2-Oxo-2H-[1,2,4]thiadiazolo[2,3-a]pyridin-8-yl)vinyl)-
1,3,4-oxathiazol-2-one (56). According to Method E, 2-amino-3-
bromopyridine (260 mg, 1.5 mmol) was used as starting bromide
in the Mizoroki–Heck reaction. After 15 min of microwave irradia-
tion (T=1408C), the reaction mixture was irradiated for additional
15 min at 1608C to afford full conversion. The resulting reaction
mixture was cooled down to rt, and the formed Pd black was re-
moved by filtration through Celite (EtOAc/MeOH 1:1). Evaporation
of the solvent under reduced pressure gave a crude mixture con-
taining DMF, which was taken in EtOAc (20 mL). The organic phase
was washed with H2O (2”40 mL), the H2O phases were extracted
with EtOAc (2”100 mL), and the collected organic phases were
dried over Na2SO4. Evaporation of solvent gave 94 mg of a mixture
containing the desired (E)-3-(2-aminopyridin-3-yl)acrylamide as
1
Rf =0.70 (pentane/EtOAc=20 :1); LC purity (254 nm): 90%; H NMR
(400 MHz, [D6]DMSO): d=9.66 (s, 1H), 7.63 (d, J=3.7 Hz, 1H), 7.47
(d, J=16.2 Hz, 1H), 7.24 (d, J=3.7 Hz, 1H), 6.83 ppm (d, J=
16.2 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=178.7, 173.1, 156.4,
154.5, 152.8, 127.1, 124.3, 116.8, 115.0 ppm; ESI-MS m/z: 224
[M+H]+; HRMS m/z calcd for C9H5NO4S [M+H]+ 224.0018, found
224.0015.
(E)-5-(2-(Pyridin-2-yl)vinyl)-1,3,4-oxathiazol-2-one (54). According
to Method E, 2-bromopyridine (237 mg, 1.5 mmol) was used as
starting bromide in the Mizoroki–Heck reaction. After 15 min of mi-
crowave irradiation (T=1408C), the reaction mixture was irradiated
ChemistryOpen 2015, 4, 342 – 362
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ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

a yellow solid. TLC: Mizoroki–Heck compound Rf =0.22 (EtOAc/
MeOH 10:1); LC purity (254 nm)=90%; ESI-MS m/z: 164 [M+H]+.
According to Method B, the obtained Mizoroki–Heck reaction mix-
ture (94 mg) was reacted with an excess of chlorocarbonyl sulfenyl
chloride (0.50 mL), to give, after purification by trituration in
CH3CN/H2O, 16 mg (4% isolated yield after two steps) of the title
many), and cloned in E. coli Top10 cells (Invitrogen, Carlsbad,
USA).
Expression and purification were performed as previously de-
scribed.[8a] Briefly, 6 L cultures of E. coli BL21(DE3) cells in Luria Ber-
tani broth with 30 mg/mL kanamycin were grown for 20–22 h at
378C after induction with 0.4 mm isopropyl beta-D1 thiogalacto-
pyranoside. The cells were harvested by centrifugation, resus-
pended in lysis buffer (50 mm Na3PO4, pH 8, 300 mm NaCl, 10 mm
imidazole with 0.25 mgmLÀ1 lysozyme, 0.02 mgmLÀ1 RNase, and
0.01 mgmLÀ1 DNase), incubated 30 min at rt and lysed by passage
through a Constant Cell Disruption System (Constant Systems Ltd,
Daventry, UK) at 2 kBar. Full proteasome molecules were isolated
from the cleared lysate with 1 mL Ni-Sepharose (GE-Healthcare, Up-
psala, Sweden), and eluted with 250 mm imidazole. The buffer was
exchanged on an Econo-Pac 10DG desalting column (Bio-Rad, Her-
cules, USA) to 10 mm Tris pH 7.5, 100 mm NaCl before a final purifi-
cation step by size-exclusion chromatography on a HiLoad 16/60
Superdex 200 column (GE Healthcare, Little Chalfront, UK)
equilibrated with the same buffer. The fractions containing the pro-
teasome as analyzed by SDS-PAGE were pooled and concen-
trated to 1 mgmLÀ1 (measured by A280, extinction coefficient
33810mÀ1 cmÀ1) in a Vivaspin 20, molecular weight cut-off 100 kDa
(Sartorius Stedin Biotech, Goettingen, Germany).
1
compound as a pale yellow solid. LC purity (254 nm): 97%; H NMR
(400 MHz, [D6]DMSO): d=8.02 (dd, J=7.0, 1.3 Hz, 1H), 7.95 (dm,
J=7.0 Hz, 1H), 7.72 (d, J=16.0 Hz, 1H), 7.62 (dd, J=16.2, 0.6 Hz,
1H), 6.89 ppm (t, J=7.0 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO):
d=174.3, 173.3, 157.1, 148.0, 137.1, 134.8, 126.1, 125.0, 117.4,
111.6 ppm; ESI-MS m/z: 280 [M+H]+; HRMS m/z calcd for
C10H5N3O3S2 [M+H]+ 279.9851, found 279.9843. gHSQC and
gHMQC experiments showed no correlations between proton HE
and carbon C10, suggesting the following structure (Figure 3) as
the most probable one.
Proteasome assay. The protein production and assay were based
on that of Lin et al.[8b] Both Mtb and human proteasomes were as-
sayed in 20 mm Hepes, 0.5 mm EDTA pH 7.5, with 25 mm suc-LLVY-
AMC (Bachem AG, Switzerland) as substrate, in a final volume of
100 mL on a white 96-well assay plate (Cliniplate, Thermo Fisher
Scientific OY, Finland). The increase in fluorescence caused by
cleavage of AMC (7-amino-4-methylcoumarin) was measured using
a Fluoroskan Ascent fluorescent plate reader (Thermo Fisher Scien-
tific OY, Finland) equipped with a 390 nm filter for excitation and
460 nm filter for emission. Human 20S proteasome (Boston Bio-
chemicals, USA) was assayed at 1 nm concentration in the presence
of a tenfold excess of human PA28 activator (Boston Biochemicals),
as recommended by the manufacturer. The Mtb proteasome was
assayed at a concentration of 2 nm. The assay was performed as
follows. Threefold dilutions of inhibitor (10 mm stock solution in
DMSO) from an initial concentration of 100 mm were performed on
the assay plate in buffer containing the above concentrations of
enzyme; the final volume in the well after dilution was 96 mL. The
plate was sealed and preincubated at rt for 3 h for Mtb proteasome
or 1 h for human proteasome. Next, substrate (5 mL of 500 mm in
a buffer made from a 10 mm stock solution in DMSO) was added,
and the fluorescence read in the plate reader immediately (t=0),
then after 3 h for the Mtb proteasome and after 1 h for the human
proteasome. The t=0 value was subtracted from the final value,
and plotted against the logarithm of the inhibitor concentration;
the data were fitted to the standard IC50 equation using GraphPad
Prism 5 (GraphPad Software Inc., USA).
Figure 3. gHSQC and gHMQC experiment results of compound 56.
5-Phenethyl-1,3,4-oxathiazol-2-one (57). (2E)-3-(1H-Indol-3-yl)-
acrylic acid (378 mg, 2.0 mmol) and 1,1’-carbonyldiimidazole
(656 mg, 2.0 mmol) were dissolved in anhydrous DMF (10 mL), and
the mixture was stirred at rt. After 30 min, NH4HCO3 (639 mg,
4.0 mmol) was added, and the resulting suspension was stirred at
rt o/n. EtOAc (100 mL) was added, and the organic phase was
washed with H2O (2”50 mL) and brine (50 mL). Evaporation of sol-
vent gave 397 mg of the crude corresponding amide, purified by
silica column chromatography (pentane/EtOAc 1:1 ! 0:1) to afford
116 mg (31% isolated yield) of (E)-3-(1H-indol-3-yl)acrylamide as
a yellow sticky oil. TLC: Rf =0.24 (EtOAc); LC purity (254 nm): 95%;
ESI-MS m/z: 187 [M+H]+. According to Method B, (E)-3-(1H-indol-
3-yl)acrylamide (58 mg, 0.31 mmol) was reacted with chlorocarbon-
yl sulfenyl chloride to give, after preparative HPLC purification,
9 mg (12% isolated yield) of the title compound as a pale yellow
powder. LC purity (254 nm): 97%; 1H NMR (400 MHz, [D6]DMSO):
d=11.84 (bs, 1H), 8.00 (s, 1H), 7.94 (dm, J=7.2 Hz, 1H), 7.75 (d,
J=16.2 Hz, 1H), 7.48 (dm, J=8.1 Hz, 1H), 7.26–7.15 (m, 2H),
6.66 ppm (d, J=16.2 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=
174.7, 159.3, 138.1, 136.7, 132.4, 125.4, 123.4, 121.8, 120.5, 113.1,
112.5, 107.0 ppm; ESI-MS m/z: 245 [M+H]+; HRMS m/z calcd for
C12H8N2O2S [M+H]+ 245.0385, found 245.0384.
Minimum inhibitory concentration (MIC). The MIC were deter-
mined as previously described[29] in Middlebrook 7H9 medium con-
taining 10% v/v OADC (oleic acid, bovine serum albumin, d-glu-
cose, catalase; Becton Dickinson, USA) and 0.05% w/v Tween 80.
Briefly, compounds were tested in 10-point, two-fold serial dilu-
tions with the highest concentration of 20 mm against Mtb H37Rv
expressing a fluorescent protein (DsRed). Growth was measured
using OD590 and RFU after 5 d. Curves were fitted using the Gom-
pertz function, with MIC defined as the minimum concentration re-
quired to inhibit growth. Data are reported for optical density at
590 nm (OD590) and relative fluorescence units (RFU).
Proteasome cloning, expression, and purification. The sequences
for the open-gate alpha[8a] (Rv2109c residues 9–240) and a slightly
truncated beta (Rv2110c) (residues 1–285) subunits of the M. tu-
berculosis proteasome were amplified by PCR from total DNA of
Mtb strain H37Rv[28] using the polymerase Pfu Ultra (Agilent Tech-
nologies, La Jolla, USA). The C-terminus of the beta subunit was
modified to incorporate a 6-histidine tag after the terminal glycine
residue.[8a] The genes were inserted into the vector pRSF Duet-1,
which is a vector for coexpressing two proteins, both controlled
by T7 promoters, from Novagen (MerckMillipore, Darmstadt, Ger-
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Kill kinetics against nonreplicating bacteria. Mtb was grown aer-
obically in Middlebrook 7H9 medium containing 10% v/v OADC
and 0.05% w/v Tween 80, washed, and resuspended in PBS with
0.05% w/v tyloxapol for 14 d. Compounds were added, and viabili-
ty was determined by plating serial dilutions to determine colony
forming units (CFU) on Middlebrook 7H10 agar plus 10% v/v
OADC. Colonies were counted after four weeks.
Acknowledgements
The authors would like to acknowledge VINNOVA–the Swedish
Governmental Agency for Innovation Systems for financial sup-
port and Medivir AB for technical support. The research was also
supported in part by funding from the Bill and Melinda Gates
Foundation, under grant OPP1024038, and by National Institute
of Allergy and Infectious Diseases (NIAID) of the National Insti-
tutes of Health under award numbers R56AI095652 and
R01AI095652. The content is solely the responsibility of the au-
thors and does not necessarily represent the official views of the
National Institutes of Health. The authors also thank Stephanie
Florio, Juliane Ollinger, and Julie Early at the Infectious Disease
Research Institute (Seattle, USA) for technical assistance and
helpful discussion.
Cytotoxicity assay. Cytotoxicity was monitored against Vero Afri-
can green monkey kidney epithelial cells (ATCC CCL-81) grown in
DMEM, High Glucose, GlutaMAXꢂ (Invitrogen), supplemented with
10% FBS, and penicillin–streptomycin solution (Gibco). The assay
was run in 96-well plates using a 10-point, serial dilution curve.
Cell viability was measured after 48 h using Cell Titer Glo (Prome-
ga), and growth inhibition curves were fitted using the Levenberg–
Marquardt algorithm. The TC50 was defined as the compound con-
centration that gave 50% inhibition of growth.
Determination of solubility in PBS (pH 7.4). HPLC analyses were
performed using a Shimadzu 10 A system with a SCL-10Avp con-
troller, 2 LC-10AD pumps with a 0.5 mL gradient mixer at the high
pressure side, a SIL-10 A (rebuilt to also run 96-well plates), a CMA
260 Degasser, and a Waters PDA 996 Photodiode Array Detector. A
Zorbax Eclipse XDB-C8, 5 mm, 2.1”50 mm column was utilized
from Dalco Chromtech AB (Sollentuna, Sweden) with CH3CN/H2O in
0.05% TFA as mobile phase and 1 mLminÀ1. The chromatograms
were registered at the wavelengths 230, 254, and 280 nm. All solu-
tions containing the sample compound were prepared from
10 mm DMSO stock solutions stored in glass vials at ambient tem-
perature. High standards (HS) were made by diluting the DMSO
stock solution 200 times with CH3CN+PBS pH 7.4 (1+1), obtaining
a 50 mm solution. Low standards (LS) were prepared by diluting HS
20 times with CH3CN+PBS pH 7.4 (1+1), obtaining a 2.5 mm solu-
tion. The HS solution was chromatographed a second time after
24 h to determine the chemical stability. It was very important to
exchange the septa after the first injection to avoid evaporation of
the CH3CN. The solubility solutions were prepared by diluting the
DMSO stock solution 100 times with PBS pH 7.4, obtaining a nomi-
nal concentration of 100 mm. The solutions were left for 18 h at
ambient temperature before filtration. When filtering the sample
solution, the first 0.1 mL was discarded, and the rest was collected.
The solubility was determined using a two-point calibration curve
forced through the origin. To evaluate the quality of the standard
curve, the quotient of the area of HS to the LS was also calculated.
The optimal quotient should equal the value of the concentration
ratio, that is, 20.0. When evaluating the solubility, the wavelength
used was that which gives the area quotient closest to 20. The sta-
bility (given in percent remaining) was obtained by multiplying the
area after the 24 h run by 100 and dividing it by the area of the
first HS injection.
Keywords: antitubercular agents · 5-styryl-oxathiazolones ·
Mtb proteasome inhibitor
· Mycobacterium tuberculosis ·
nonreplicating Mtb · rapid bactericidal activity
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