Human carbonic anhydrase inhibitory profile of mono- and bis-sulfonamides synthesized via a direct sulfochlorination of 3- and 4-(hetero)arylisoxazol-5-amine scaffolds

Article abstract of DOI:10.1016/j.bmc.2017.02.018

Three distinct series of isoxazole-based primary mono- and bis-sulfonamides have been synthesized via direct sulfochlorination, each of them delivering nanomolar inhibitors of human carbonic anhydrase. Certain pronounced SAR trends have been established a

Full text of DOI:10.1016/j.bmc.2017.02.018

Accepted Manuscript
Human carbonic anhydrase inhibitory profile of mono- and bis-sulfonamides
synthesized via a direct sulfochlorination of 3-and 4-(hetero)arylisoxazol-5-
amine scaffolds
Mikhail Krasavin, Mikhail Korsakov, Zhanna Zvonaryova, Evgenii
Semyonychev, Tiziano Tuccinardi, Stanislav Kalinin, Muhammet Tanç,
Claudiu T. Supuran
PII:
S0968-0896(17)30004-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.02.018
BMC 13546
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
3 January 2017
7 February 2017
Please cite this article as: Krasavin, M., Korsakov, M., Zvonaryova, Z., Semyonychev, E., Tuccinardi, T., Kalinin,
S., Tanç, M., Supuran, C.T., Human carbonic anhydrase inhibitory profile of mono- and bis-sulfonamides
synthesized via a direct sulfochlorination of 3-and 4-(hetero)arylisoxazol-5-amine scaffolds, Bioorganic &
Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.02.018
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Human carbonic anhydrase inhibitory profile of mono- and bis-
sulfonamides synthesized via a direct sulfochlorination of 3-and 4-
(hetero)arylisoxazol-5-amine scaffolds
Mikhail Krasavin,* Mikhail Korsakov, Zhanna Zvonaryova, Evgenii Semyonychev, Tiziano
Tuccinardi, Stanislav Kalinin, Muhammet Tanç, Claudiu T. Supuran*

Human carbonic anhydrase inhibitory profile of mono- and bis-
sulfonamides synthesized via a direct sulfochlorination of 3-and 4-
(hetero)arylisoxazol-5-amine scaffolds
Mikhail Krasavin,a,* Mikhail Korsakov,b Zhanna Zvonaryova,b Evgenii Semyonychev,b Tiziano
Tuccinardi,c Stanislav Kalinin,a Muhammet Tanç,d Claudiu T. Supurand *
a Institutes of Chemistry and Translational Biomedicine, Saint Petersburg State University, Saint
Petersburg, 199034 Russian Federation
b The Ushinsky Yaroslavl State Pedagogical University, Yaroslavl 150000, Russian Federation
c Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126, Pisa, Italy
d Neurofarba Dept., Universita degli Studi di Firenze, Florence, Italy
* Corresponding authors.
E-mail: m.krasavin@spbu.ru (M. Krasavin), claudiu.supuran@unifi.it (C. T. Supuran).
ABSTRACT
Three distinct series of isoxazole-based primary mono- and bis-sulfonamides have been
synthesized via direct sulfochlorination, each of them delivering nanomolar inhibitors of human
carbonic anhydrase. Certain pronounced SAR trends have been established and rationalized by in
silico docking. These findings expand the structure-activity knowledge base for heterocycle-
containing sulfonamide carbonic anhydrase inhibitors and further validate the power of direct
electrophilic sulfochlorination as a means of introducing the pharmacophoric primary
sulfonamide group into structurally diverse aromatic precursors.
Keywords: carbonic anhydrase inhibitors, isoform selectivity, zinc binding group, primary
sulfonamide, aromatic sulfochlorination, alternative binding mode.
1. Introduction
Primary aromatic and heteroaromatic sulfonamides represent a robust platform for the discovery
of novel, isoform-selective inhibitors of human carbonic anhydrase (hCA) as they ensure specific
binding of the enzyme’s active site zinc ion.1 Once anchored to the prosthetic metal ion, the
periphery groups in the primary sulfonamide template can be either serendipitously screened or
judiciously optimized to improve ligand’s affinity and ability to selectively inhibit any of the 16

known hCA isoforms.2 The availability of true isoform-selective inhibitors is essential from the
standpoint of ‘cleaner’ drug discovery strategies unencumbered by the need to tackle off-target
effects.3 Additionally, isoform-selective hCA inhibitors are of value as chemical biology probes
which can greatly aid in further deciphering the implications of the normal and aberrant
functioning of particular isoforms for the onset and treatment of disease.4 The majority of the
clinically used carbonic anhydrase inhibitors (CAIs), exemplified by primary sulfonamides
acetazolamide (1), methazolamide (2), dorzolamide (3) and brinzolamide (4) (Figure 1), are non-
selective ligands for several hCA isoforms.5 At the same time, the concept of isoform-selective
hCA targeting is substantiated by the ample pre-clinical data on numerous advanced leads6 and
by the recently completed Phase I clinical study involving selective hCA IX inhibitor SLC-
0111.7
Figure 1. Examples clinically used primary sulfonamide drugs – pan-CA inhibitors.
HN
HN
O
O
O
N
N
O
O
N
N
O
S NH2
O
S NH2
O
S NH2
O
S NH2
O
N
S
S
N
S
S
S
N
H
S
O O
O O
O
brinzolamide
methazolamide
acetazolamide
dorzolamide
Direct sulfochlorination of aromatic substrates, followed by a reaction of the respective sulfonyl
chloride with ammonia, offers a convenient and straightforward way of introducing the primary
sulfonamide zinc-binding group (ZBG) into a wealth of atomatic precursors.8 While often
chemoselective, electrophilic aromatic sulfochlorination can also take several alternative courses
and thus generate more chemical diversity in the initial, SAR-probing set of compounds.
However, this transformation has been relatively seldom reported in the literature as a direct link
between the chemical space of aromatic precursors and that of CA inhibitory sulfonamides
(compared to the more frequently reported synthesis of CAIs using reagents already containing
the SO2NH2 functionality).
Recently, we reported the synthesis and biological profiling of aromatic, 1,3-oxazol-5-yl-
substituted primary sulfonamides obtained via chemoselective sulfochlorination of the aromatic
and heteroaromatic sybstrates.9 The compounds were designed to systematically explore
opportunities to build additional contacts with the protein environment of the active site where
the presence of two distinct parts – hydrophobic and hydrophilic – had been confirmed earlier
from crystallography studies.10 As the result, selective picomolar inhibitors (exemplified by 4)
of hCA II were identified and their high potency was rationalized by the efficient interaction of
the carboxamide periphery of the molecule with the target’s hydrophobic half (Figure 2).

Figure 2. Selective picomolar inhibitor of hCA II 4 and its positioning in the enzyme’s active
site.
This success prompted us to consider applying the same strategy to other azolyl-substituted
aromatic substrates – in particular, those that offer an opportunity of introducing two primary
sulfonamides ZBGs in the same molecule. Herein, we disclose a series of primary mono- and
bis-sulfonamides based on 3- an 4-(hetero)arylisoxazol-5-amine scaffolds and the results of their
profiling as inhibitors of several hCA isoforms.
2. Results and discussion
Scheme 1. Synthesis of 5-acetylaminoisoxazole substrates 7 for subsequent sulfochlorination.
A set of isoxazol-5-amines 6a-g was prepared from readily available 2-cyanoketones 5 on
reaction with hydroxylamine – and the amino group in 6 was protected with an acetyl group, to
avoid unwanted reactions in the chlorosulfonylation step, to give 7a-g. The yields of 6 and 7
were good to excellent in all instances (Scheme 1). Direct chlorosulfonylation of 7 proceeded
smoothly at 60 ºC in excess chlorosulfonic acid containing thionyl chloride (10:1) and afforded
respective primary sulfamides 8a-f, as a single regioisomer, after the treatment of the respective
intermediate sulfonyl chlorides with 25% aqueous ammonia in acetone (Scheme 2). While

compounds 8a-f contain an acetylamino group, which makes them somewhat similar to
acetazolamide and methazolamide, we were curious to see if respective de-acetylated
compounds 9 could be obtained. Unfortunately, compounds 8 proved to be totally resistant to de-
acetylation, under basic or acidic conditions, whereupon destruction of the isoxazole nucleus was
observed on attempts to apply forcing temperature.
Scheme 2. Synthesis of primary mono-sulfonamides 8.
Reagents and conditions: (a) HSO3Cl (10 equiv.), SOCl2 (1.0 equiv.), 60 ºC, 2 h; (b) 25%
NH4OH (5 equiv.), acetone, 50 ºC, 1h.
Interestingly, doubling the quantity of the sulfochlorination reagents and increasing the reaction
temperature and time not only allowed achieving double sulfochlorination of substrates 7 but
also led to a spontaneous loss of the acetyl group to produce a set of bis-sulfamides 10a-g. This
lability of the acetyl function en route to 10 is in sharp contrast to that in compounds 8 (vide
supra). However, it is perhaps an unsurprising outcome as sulfochlorination of position 4 in
isoxazole essentially turns the compounds into vinylogous N-acetylsilfamides which would be
easy to de-acetylate. The intermediate sulfonyl chlorides 11 are also rather intriguing as they
contain two different SO2Cl moieties in combination with an unprotected amino group (Scheme
3). As they can be of interest as novel linkers for the design of dendritic structures, we also
isolated and characterized them as individual compounds.
Scheme 3. Synthesis of primary bis-sulfonamides 10 and intermediate bis-sulfonylchlorides 11.
a, b
O
O
O
a
N
N
N
NH2
NH2
NH
O
(Het)Ar
ClO2S
(Het)Ar
(Het)Ar
SO2Cl
SO2NH2
H2NO2S
11
10
7
Reagents and conditions: (a) HSO3Cl (20 equiv.), SOCl2 (2.0 equiv.), 80-110 ºC, 12-48 h; (b)
25% NH4OH (10 equiv.), acetone, 50 ºC, 1h.

In addition to mono- (8a-f) and bis-sulfonamides (10a-g) we were interested to explore
sulfonamides of different topology where the pharmacophoric ZBG would be positioned, also
via direct sulfochlorination, within 4-aryl substituent of respective 5-acetylamino isoxazole
substrates. The latter was obtained analogously to 7 from 2-aryl-2-cyanoacetones 12 and
sulfochlorinated under the same set of conditions. While sulfochlorination (followed by aqueous
ammonia treatment) proved smooth and reasonably yielding, the respective 5-acetylamino
compounds 15a-e were also resistant to all our efforts to remove the acetyl group (Scheme 4).
Scheme 4. Synthesis of primary sulfonamides 15.
The relatively low yield of compound 15e was due to the fact that it was accompanied by an
intriguing cyclic by-product 16 formed in roughly the same amount. Its formation can be
justified by the deprotonation (in basic aqueous ammonia medium) of the nearby acetylamino
group and its intramolecular interaction with the sulfonyl chloride moiety prior to its conversion
to primary sulfamide. This view was supported by 16 being the sole product (isolated in 46%
yield) of treatment of 17 with trimethylamine (Scheme 5).
Scheme 5. Formation of tricyclic sultam 16.

The inhibitory profile of compounds 8a-f against hCA I, II, IV and VII isoforms was assessed in
a stopped-flow kinetics assay as reported earlier.9 Examination of the data presented in Table 1
reveals a strong dependence of the inhibition activity on the topology of the ZBG relative to the
periphery of the molecule. In particular, compound 8a (having a roughly 120º relationship
between the sulfonamide and the isoxazole moiety) displays a remarkable (~100-fold) selectivity
toward hCA II while compound 8b is completely inactive. Small variations in the substitution
pattern in the thiophene-linked compounds 8d-f appear to have a strong effect on the inhibition
profile. A sharp change in geometry drastically increases (8f) the overall potency profile of
relatively weak pan-inhibitor 8d. Compound 8e displayed a pronounced (>100-fold) selectivity
toward hCA II while not inhibiting the other three isoforms. Particularly intriguing if the
detrimental effect of methoxy group in 8b on the hCA II and VII potency of its close, methyl-
substituted analog 8a.
Table 1. CA inhibitory profile of sulfonamides 8a-f.
Ki, nM
Yield
(%)
Compound (Het)ArSO2NH2
hCA I
hCA II
hCAIV
hCAVII
61a
8a
>10,000
390.4
>10,000
84.5
79
8b
>10,000
>10,000
>10,000
>10,000

65
8c
>10,000
>10,000
>10,000
>10,000
56
72
63
8d
8e
8f
615.8
272.4
89.9
6148.0
281.6
>10,000
>10,000
>10,000
13.6
250
2.4
12
94.1
74
6.7
2.5
Acetazolamide
a Sulfochlorination reaction time – 8 h.
Figure 3. Docking of compounds 8a (A) and 8b (B) into hCA II.

In order to understand the observed difference in hCA II potency between compounds 8a and 8b,
we docked both of them into the crystal structure of hCA II12 (PDB13 code 2AW1) using GOLD
software (Figure 3).14 As expected, the sulfonamide portion of the molecule acts as a zinc
binding group, also showing two additional H-bond interactions with the protein backbone and
the hydroxyl group of T198. The 4-methylphenyl ring shows lipophilic interactions with V121,
V142 and L197, the isoxazole ring forms two H-bonds with the side chains of Q92 and N67
whereas the acetamide portion does not show any important interactions and is directed towards
the solvent-exposed entrance of the binding site cavity. Compound 8b is not able to display the
same binding mode of 8a because of the steric clash effects between the methyl group of the
methoxy substituent and the sidechain of V142. For this reason, as shown in Figure 3B
compound 8b displays an unusual distorted trigonal bipyramidal coordination of the zinc ion
with both the nitrogen of the sulfonamide and oxygen of the methoxy group interacting with the
zinc ion. Therefore, the impossibility for compound 8b to display the common sulfonamide
coordination of the zinc ion could explain the sharp inhibition activity decrease observed in 8b
relative to 8a. Moreover, this finding does not contradict similar ‘detrimental methoxy’ SAR
trend observed earlier for o-methoxy (vs. o-methyl) substituted arenesulfonamides as hCA II
inhibitors.9, 15
Table 2. CA inhibitory profile of bis-sulfonamides 10a-g.
Yield Yield
Ki, nM
Compound (Het)ArSO2NH2
10
(%)
11
(%)
hCA I
hCA II
hCAIV
hCAVII
10(11)aa
10(11)bb
10(11)cc
31
54
61
40
77
82
3,649
376.3
6.0
1,752
553.5
3,661
8.9
9.3
3.8
>10,000 >10,000
>10,000

10(11)dc
37
49
>10,000 >10,000 >10,000 >10,000
10(11)ed
10(11)fd
10(11)gd
49
48
41
57
59
53
802.5
792.3
20.3
67.6
530.5
533.4
8.5
78.1
66.3
250
24.6
12
184.5
74
10,1
2.5
Acetazolamide
Conditions for the bis-sulfochlorination step:
a 110 ºC over 48 h.
b 90 ºC over 24 h.
c 80 ºC over 24 h.
d 90 ºC over 12 h.
Some SAR trends observed for bis-sulfonamide CAIs 10a-g (Table 2) appear to parallel those of
mono-sulfonamides 8a-f. The ‘detrimental methoxy’ trend seems to be maintained for hCA I, II
and VII inhibition (cf. 10b and 10c). Likewise, o-isoxazolyl-substituted sulfonamide 10d was
similarly inactive. These instances suggest that the second, isoxazole-bound primary
sulfonamide group probably does not act as a ZBG and is rather an element of the isoxazole
periphery. However, certain inhibitory properties that were not observed for mono-sulfonamides
appear to be restored in bis-sulfonamides, thus strongly suggesting the existence of an alternative
zinc anchoring mode for the bis-sulfonamide compounds. The most notable instances are: (i) the
restoration of hCA VII potency of compound 10b (in comparison to its N-acetyl mono-
sulfonamide counterpart 8a); (ii) the low nanomolar potency of compound 10c toward hCA IV
(compared to the inactivity of 8b toward this isoform) and the pronounced selectivity (>1,000-
fold) vs. the other three isoforms; (iii) the restoration of inhibitory potency (in particular, against
hCA I, VI and VII) for compound 10f in comparison to its mono-sulfonamide congener 8e.
These observations suggest that introduction of a second primary sulfonamide ZBG16 into
isoxazole nucleus can carry strong advantages for selective targeting of certain CA isoforms, in
particular hCA IV and VII.
Analysis of the structure-activity relationships for the isoxazol-4-yl arenesulfonamides 15a-e
(Table 3) also reveals several notable trends. Firstly, the ‘detrimental methoxy’ phenomenon
(manifesting itself in compound 15c) seems to persist in this series of CAIs, in particular, when it
comes to ablating the nanomolar inhibitory activity of 15b against hCA II, IV and VII. The lack

of inhibitory activity for the o-isoxazolyl benzene sulfonamide 15e vs. all four isoforms parallels
that of compounds 8c and 10d (vide supra). The effect of the methyl group in 15d on hCA VII
activity in comparison to the unsubstituted phenyl-linked compound 15a is quite pronounced.
The latter compound is rather exemplary of the strong hCA VII inhibition (in the low nanomolar
range) observed for selected compounds in all three series (cf. 8f, 10b, 10e).
Table 3. CA inhibitory profile of sulfonamides 15.
Ki, nM
Yield
(%)
Compound
15a
ArSO2NH2
hCA I
hCA II
hCAIV
hCAVII
56
52
96.1
5.4
207.2
4.5
15b
7,488
14.9
7.5
9.2
61
47
15c
15d
>10,000 >10,000 >10,000 >10,000
872.9
197.1
>10,000
707.1
27
15e
>10,000 >10,000 >10,000 >10,000
250
12
74
2.5
Acetazolamide
3. Conclusion
In summary, we have described synthesis and inhibitory activity against four isoforms of human
carbonic anhydrase of three distinct series of primary mono- and bis-sulfonamides. Nanomolar
levels of inhibition were achieved in all three series. Certain SAR trends have been established,
in particular, the ablation of hCA inhibitory potency by replacing an o-methyl substituent with o-

methoxy (the ‘detrimental methoxy phenomenon’) as well as a possibility of an alternative
binding mode for bis-sulfonamides and the suitability of the latter for selective targeting of hCA
IV and VII isoforms.
4. Experimental section
4.1. General experimental
All reactions were carried out in oven-dried glassware in atmosphere of nitrogen. Melting points
were measured with a Buchi В-520 melting point apparatus and are uncorrected. Thin-layer
chromatography was carried out on Silufol UV-254 silica gel plates using an appropriate mixture
1
of ethyl acetate and hexane. Compounds were visualized with short-wavelength UV light. H
NMR and 13C NMR spectra were recorded on Bruker MSL-300 spectrometers in DMSO-d6
using TMS as an internal standard. Elemental analyses were obtained at Research Institute for
Chemical Crop Protection (Moscow, Russia) using Carlo ErbaStrumentazione 1106 analyzer.
Mass spectra were recorded using Shimadzu LCMS-2020 system with electron impact (EI)
ionization. All and reagents and solvents were obtained from commercial sources and used
without purification.
4.2. Synthetic organic chemistry
4.2.1. General procedure for the preparation of 5-aminoisoxazoles 6a-g.
Ketonitrile 5 (100 mmol) was added to 15% aqueous NaOH solution (100 mL) followed by
hydroxylamine (200 mmol). The resulting mixture was heated at reflux for 14 h, cooled down to
r. t., the resulting precipitate was isolated by filtration and crystallized from isopropyl alcohol.
4.2.1.1. 3-(Thiophen-2-yl)-1,2-oxazol-5-amine (6a)
Yield 12,450 mg (75%). White solid, m.p. 96-98 °С (i-PrOH). 1H NMR (300 MHz, DMSO-d6) δ
ppm 7.60 (dd, J3-4=5.0 Hz, J3-5=1.0 Hz, 1H, 3-Hthiophene), 7.51 (dd, J4-5=3.6 Hz, J3-5=1.0 Hz, 1H,
5-Hthiophene), 7.13 (dd, J3-4=5.0 Hz, J 4-5=3.6 Hz, 1H, 4-Hthiophene), 6.81 (s, 2H, NH2), 5.36 (s, 1H,
13
Hisoxazole). C NMR (75 MHz, DMSO-d6) δ ppm 171.3, 158.4, 131.9, 128.1, 128.0, 127.8, 75.6.
LC MS (ESI): m/z [M+H]+ 167. Anal. calcd for C7H6N2OS (166.20): C, 50.59; H, 3.64; N,
16.86; found: C, 50.51; H, 3.64; N, 16.83.
4.2.1.2.
3-(5-Methylthiophen-2-yl)-1,2-oxazol-5-amine (6b)
1
Yield 12,780 mg (71%). White solid, m.p. 134-136 °С (i-PrOH). H NMR (300 MHz, DMSO-
d6) δ ppm 7.29 (d, J=3.30 Hz, 1H, Hthiophene), 6.80 (d, J=3.30 Hz, 1H, Hthiophene), 5.29 (s, 2H,

NH2), 2.45 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) δ ppm 171.1, 158.5, 141.4, 129.6,
128.0, 126.4, 75.3, 15.4. LC MS (ESI): m/z [M+H]+ 181. Anal. calcd for C8H8N2OS (180.23): C,
53.31; H, 4.47; N, 15.54; found: C, 53.28; H, 4.48; N, 15.50.
4.2.1.3.
3-(Thiophen-3-yl)-1,2-oxazol-5-amine (6c)
Yield 12,284 mg (74%). White solid, m.p. 177-178 °С (i-PrOH).1H NMR (300 MHz, DMSO-d6)
δ ppm 7.94 (dd, J2-5=3.0 Hz, J2-4=1.3 Hz, 1H, 2-Hthiophene), 7.61 (dd, J4-5=5.0 Hz, J2-5=3.0 Hz, 1H,
4-Hthiophene), 7.41 (dd, J4-5=5.0 Hz, J2-4=1.3 Hz, 1H, 4-Hthiophene), 6.73 (s, 2H, NH2), 5.34 (s, 1H,
13
Hisoxazole). C NMR (75 MHz, DMSO-d6) δ ppm 171.1, 159.2, 131.8, 127.6, 126.0, 125.1, 75.9.
LC MS (ESI): m/z [M+H]+ 167. Anal. calcd for C7H6N2OS (166.20): C, 50.59; H, 3.64; N,
16.86; found: C, 50.56; H, 3.64; N, 16.90.
4.2.1.4.
3-Phenyl-1,2-oxazol-5-amine (6d)
1
Yield 12,000 mg (75%). White solid, m.p. 109-112 °С (i-PrOH). H NMR (400 MHz, DMSO-
13
d6) δ ppm 7.72 (m, 2H, HAr), 7.44 (m, 3H, HAr), 6.76 (s, 2H, NH2), 5.39 (s, 1H, Hisoxazole). C
NMR (75 MHz, DMSO-d6) δ ppm 171.5, 163.0, 130.5, 129.9, 129.2, 126.7, 75.7. LC MS (ESI):
m/z [M+H]+ 161. Anal. calcd for C9H8N2O (160.18): C, 67.49; H, 5.03; N, 17.49; found: C,
67.44; H, 5.03; N, 17.50.
4.2.1.5.
3-(4-Methylphenyl)-1,2-oxazol-5-amine (6e)
1
Yield 12,702 mg (73%). White solid, m.p. 155-156 °С (i-PrOH). H NMR (400 MHz, DMSO-
d6) δ ppm 7.60 (d, J=8.0 Hz, 2H, HAr), 7.24 (d, J=8.0 Hz, 2H, HAr), 6.73 (s, 2H, NH2), 5.35 (s,
1H, Hisoxazole), 2.33 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) δ ppm 171.3, 162.9, 139.5,
129.8, 127.7, 126.6, 75.5, 21.3. LC MS (ESI): m/z [M+H]+ 175. Anal. calcd for C10H10N2O
(174.20): C, 68.95; H, 5.79; N, 16.08; found: C, 68.91; H, 5.79; N, 16.06.
4.2.1.6.
3-(4-Methoxyphenyl)-1,2-oxazol-5-amine (6f)
1
Yield 14,060 mg (74%). White solid, m.p. 132-134 °С (i-PrOH). H NMR (400 MHz, DMSO-
d6) δ ppm 7.65 (m, 2H, HAr), 6.98 (m, 2H, HAr), 6.68 (s, 2H, NH2), 5.32 (s, 1H, Hisoxazol), 3.79 (s,
3H, OCH3). 13C NMR (75 MHz, DMSO-d6) δ ppm 171.2, 162.6, 160.6, 128.1, 122.8, 114.6,
75.4, 55.6. LC MS (ESI): m/z [M+H]+ 191. Anal. calcd for C10H10N2O2 (190.20): C, 63.15; H,
5.30; N, 14.73; found: C, 63.10; H, 5.30; N, 14.73.
4.2.1.7.
3-(3-Methoxyphenyl)-1,2-oxazol-5-amine (6g)

Yield 13,680 mg (72%). White solid, m.p. 83-85 °С (i-PrOH). 1H NMR (300 MHz, DMSO-d6) δ
ppm 7.39 (m, 3H, HAr), 7.07 (m, 1H, HAr), 6.68 (s, 2H, NH2), 5.34 (s, 1H, Hisoxazol), 3.80 (s, 3H).
13C NMR (75 MHz, DMSO-d6) δ ppm 171.3, 162.8, 159.9, 130.3, 119.2, 116.5, 115.4, 111.5,
75.3, 55.6. LC MS (ESI): m/z [M+H]+ 191. Anal. calcd for C10H10N2O2 (190.20): C, 63.15; H,
5.30; N, 14.73; found: C, 63.07; H, 5.30; N, 14.76.
4.2.2. General procedure for the preparation of 5-acetylaminoisoxazoles 7a-g.
A solution of 5-aminoisoxazole 6 (50 mmol) and pyridine (100 mmol) in acetonitrile (800 mL)
was cooled to 0 ºC and treated with acetyl chloride (50 mmol). The resulting mixture was heated
at reflux for 3 h, cooled to r. t., concentrated in vacuo and the residue was treated with water
(800 mL). The resulting precipitate was isolated by filtration and crystallized from ethanol.
4.2.2.1. N-[3-(Thiophen-2-yl)-1,2-oxazol-5-yl]acetamide (7a)
Yield 8,778 mg (84%). White solid, m.p. 132-134 °С (EtOH). 1H NMR (400 MHz, DMSO-d6) δ
ppm 11.62 (br.s., 1H, AcNH), 7.73 (dd, J4-5=3.6 Hz, J3-5=1.0 Hz, 1H, 5-Hthiophene), 7.71 (dd, J3-
4=5.0 Hz, J3-5=1.0 Hz, 1H, 3-Hthiophene), 7.19 (dd, J3-4=5.0 Hz, J 4-5=3.6 Hz, 1H, 4-Hthiophene), 6.67
13
(s, 1H, Hisoxazole), 2.12 (s, 3H, CH3). C NMR (75 MHz, DMSO-d6) δ ppm 167.4, 162.5, 158.7,
132.5, 128.4, 128.2, 127.9, 86.6, 15.5. LC MS (ESI): m/z [M+H]+ 209. Anal. calcd for
C9H8N2O2S (208.24): C, 51.91; H, 3.87; N, 13.45; found: C, 51.89; H, 3.88; N, 13.44.
4.2.2.2.
N-[3-(5-Methylthiophen-2-yl)-1,2-oxazol-5-yl]acetamide (7b)
Yield 9,324 mg (84%). White solid, m.p. 193-195 °С (EtOH). 1H NMR (300 MHz, DMSO-d6) δ
ppm 11.66 (s, 1H, AcNH), 7.50 (d, J=3.3 Hz, 1H, Hthiophene), 6.86 (d, J=3.3 Hz, 1H, Hthiophene),
6.61 (s, 1H, Hisoxazole), 2.47 (s, 3H, Th-CH3), 2.12 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) δ
ppm 167.4, 162.3, 158.6, 142.5, 129.4, 128.2, 126.8, 85.8, 23.5, 15.5. LC MS (ESI): m/z [M+H]+
223. Anal. calcd for C10H10N2O2S (222.27): C, 54.04; H, 4.54; N, 12.60; found: C, 53.99; H,
4.54; N, 12.63.
4.2.2.3.
N-[3-(Thiophen-3-yl)-1,2-oxazol-5-yl]acetamide (7c)
Yield 8,632 mg (83%). White solid, m.p. 197-199 °С (EtOH). 1H NMR (400 MHz, DMSO-d6) δ
ppm 11.65 (br s, 1H), 8.19 (dd, J2-5=2.8 Hz, J2-4=1.0 Hz, 1H, 2-Hthiophene), 7.68 (dd, J4-5=5.0, J2-
5=2.8 Hz, 1H, 5-Hthiophene), 7.52 (dd, J4-5=5.0 Hz, J2-4=1.0 Hz, 1H, 4-Hthiophene), 6.66 (s, 1H,
13
Hisoxazole), 2.12 (s, 3H, CH3). C NMR (75 MHz, DMSO-d6) δ ppm 167.4, 162.0, 159.4, 131.9,
127.2, 125.5, 125.3, 86.5, 21.5. LC MS (ESI): m/z [M+H]+ 209. Anal. calcd for C9H8N2O2S
(208.24): C, 51.91; H, 3.87; N, 13.45; found: C, 51.82; H, 3.87; N, 13.40.

4.2.2.4.
N-(3-Phenyl-1,2-oxazol-5-yl)acetamide (7d)
1
Yield 7,272 mg (72%). White solid, m.p. 152-155 °С (EtOH). H NMR (400 MHz, DMSO-d6)
δ ppm 11.68 (br s, 1H, AcNH), 7.84 (m, 2H, HAr), 7.49 (m, 3H, HAr), 6.69 (s, 1H, Hisoxazole), 2.13
13
(s, 3H, CH3). C NMR (75 MHz, DMSO-d6) δ ppm 167.4, 162.9, 162.7, 130.6, 129.5, 129.3,
126.9, 86.1, 23.6. LC MS (ESI): m/z [M+H]+ 203. Anal. calcd for C11H10N2O2 (202.21): C,
65.34; H, 4.98; N, 13.85; found: C, 65.25; H, 4.99; N, 13.80.
4.2.2.5.
N-[3-(4-Methylphenyl)-1,2-oxazol-5-yl]acetamide (7e)
Yield 8,856 mg (82%). White solid, m.p. 197-199 °С (EtOH). 1H NMR (300 MHz, DMSO-d6) δ
ppm 11.64 (br s, 1H, AcNH), 7.72 (d, J=7.9 Hz, 2H, HAr), 7.29 (d, J=7.9 Hz, 2H, HAr), 6.65 (s,
13
1H, Hisoxazole), 2.35 (s, 3H, ArCH3), 2.13 (s, 3H, CH3). C NMR (75 MHz, DMSO-d6) δ ppm
167.4, 162.9, 162.5, 140.3, 126.8, 126.5, 86.0, 23.5, 21.4. LC MS (ESI): m/z [M+H]+ 217. Anal.
calcd for C12H12N2O2 (216.24): C, 66.65; H, 5.59; N, 12.96; found: C, 66.61; H, 5.60; N, 13.00.
4.2.2.6.
N-[3-(4-Methoxyphenyl)-1,2-oxazol-5-yl]acetamide (7f)
Yield 9,396 mg (81%). White solid, m.p. 166-168 °С (EtOH). 1H NMR (400 MHz, DMSO-d6) δ
ppm 11.62 (br s, 1H, AcNH), 7.77 (m, 2H, HAr), 7.03 (m, 2H, HAr), 6.62 (s, 1H, Hisoxazole), 3.80
13
(s, 3H, OCH3), 2.12 (s, 3H, CH3). C NMR (75 MHz, DMSO-d6) δ ppm 167.5, 162.6, 162.3,
159.7, 129.8, 122.1, 114.7, 86.5, 55.7, 20.5. LC MS (ESI): m/z [M+H]+ 233. Anal. calcd for
C12H12N2O3 (232.24): C, 62.06; H, 5.21; N, 12.06; found: C, 61.98; H, 5.21; N, 12.11.
4.2.2.7.
N-[3-(3-Methoxyphenyl)-1,2-oxazol-5-yl]acetamide (7g)
1
Yield 8,120 mg (70%). White solid, m.p. 153-156 °С (EtOH). H NMR (300 MHz, DMSO-d6)
δ ppm 11.68 (br s, 1H), 7.40 (m, 3H, HAr), 7.05 (m, 1H, HAr), 6.71 (s, 1H, Hisoxazol), 3.82 (s, 3H),
2.10 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ ppm 167.4, 162.9, 162.6, 160.1, 130.6, 119.3,
116.6, 111.8, 86.3, 55.7, 23.5. LC MS (ESI): m/z [M+H]+ 233. Anal. calcd for C12H12N2O3
(232.24): C, 62.06; H, 5.21; N, 12.06; found: C, 62.00; H, 5.21; N, 12.00.
4.2.3. N-[3-(4-methyl-3-sulfamoylphenyl)-1,2-oxazol-5-yl]acetamide (8a).
Compound 7d (10 mmol) was added portionwise to a stirred and cooled mixture of
chlorosulfonic acid (100 mmol) and thionyl chloride (10 mmol). The resulting mixture was
heated at 60 °С over 8 h, cooled to r. t. and poured over crushed ice. The resulting precipitate
was isolated by filtration and dissolved in dichloromethane (100 mL). The solution was washed
with 5% aqueous K2CO3 (50 mL), dried over anhydrous CaCl2, filtered and concentrated in

vacuo. The residue was briefly fractionated on silica gel using 25% ethyl acetate in hexanes and
the fractions containing the intermediate sulfonyl chloride were pooled and concentrated in
vacuo. The residue was dissolved in acetone (40 mL) and the solution was treated with 25%
aqueous ammonia (25 mmol). The resulting mixture was heated at 50 °С over 1 h, cooled and the
volatiles were removed in vacuo. The residue was treated with ice-cold water (40 mL) and the
mixture was stirred until a thick precipitate formed. The latter was filtered off and crystallized
from isopropyl alcohol to provide the title compound.
1
Yield 1,805 mg (61%). White solid, m.p. 237-239 °С (i-PrOH). H NMR (300 MHz, DMSO-d6)
δ ppm 11.70 (s, 1H, AcNH), 8.30 (d, J2-6=1.3 Hz, 1H), 7.94 (dd, J5-6=7.9 Hz, J2-6=1.3 Hz, 1H, 6-
HAr), 7.50 (m, 3H, 5-HAr, NH2), 6.72 (s, 1H, Hisoxazol), 2.64 (s, 3H, Ar-CH3), 2.14 (s, 3H, CH3).
13C NMR (75 MHz, DMSO-d6) δ ppm 167.5, 162.9, 162.0, 143.4, 138.4, 133.5, 129.9, 127.2,
125.2, 86.0, 23.6, 20.2. LC MS (ESI): m/z [M+H]+ 296. Anal. calcd for C12H13N3O4S (295.32):
C, 48.81; H, 4.44; N, 14.23; found: C, 48.79; H, 4.44; N, 14.21.
4.2.4.
General procedure for the synthesis of sulfonamides 8b-f.
The procedure was identical to that for the preparation of compound 8a but the heating in the
first step continued for 2 h.
4.2.4.1. N-[3-(4-Methoxy-3-sulfamoylphenyl)-1,2-oxazol-5-yl]acetamide (8b)
1
Yield 2,457 mg (79%). White solid, m.p. 278-280 °С (i-PrOH). H NMR (300 MHz, DMSO-d6)
δ ppm 11.66 (s, 1H, AcNH), 8.17 (d, J2-6=1.6 Hz, 1H, 2-HAr), 8.04 (dd, J5-6=8.6 Hz, J2-6=1.6 Hz,
1H, 6-HAr), 7.32 (d, J5-6=8.6 Hz, 1H, 5-HAr), 7.19 (s, 2H, SO2NH2), 6.67 (s, 1H, Hisoxazol), 3.97 (s,
3H, OCH3), 2.14 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) δ ppm 167.5, 162.4, 158.2, 135.9,
129.5, 126.9, 120.1, 112.8, 85.6, 56.1, 23.6. LC MS (ESI): m/z [M+H]+ 312. Anal. calcd for
C12H13N3O5S (311.32): C, 46.30; H, 4.21; N, 13.50; found: C, 46.24; H, 4.21; N, 13.55.
4.2.4.2.
N-[3-(5-Methoxy-2-sulfamoylphenyl)-1,2-oxazol-5-yl]acetamide (8c)
1
Yield 2,022 mg (65%). White solid, m.p. 207-209 °С (i-PrOH). H NMR (300 MHz, DMSO-d6)
δ ppm 11.71 (s, 1H, AcNH), 7.98 (d, J3-4=9.0 Hz, 1H, 3-HAr), 7.21 (dd, J3-4=9.0 Hz, J4-6=2.3 Hz,
1H, 4-HAr), (s, 2H, NH2), 7.09 (d, J4-6=2.3 Hz, 1H, 6-HAr), 6.57 (s, 1H, Hisoxazol), 3.87 (s, 3H,
13
OCH3), 2.13 (s, 3H, CH3). C NMR (75 MHz, DMSO-d6) δ ppm 168.4, 167.4, 163.1, 161.7,
135.1, 130.4, 129.5, 117.4, 115.2, 90.3, 66.8, 23.5. LC MS (ESI): m/z [M+H]+ 312. Anal. calcd
for C12H13N3O5S (311.32): C, 46.30; H, 4.21; N, 13.50; found: C, 46.18; H, 4.21; N, 13.44.

4.2.4.3.
N-[3-(5-Sulfamoylthiophen-2-yl)-1,2-oxazol-5-yl]acetamide (8d)
Yield 1,607 mg (56%). Brown solid, m.p. 201-203 °С (i-PrOH). 1H NMR (300 MHz, DMSO-d6)
δ ppm 13.40 (br s, 2H, SO2NH2), 11.67 (s, 1H, AcNH), 7.48 (d, J=3.63 Hz, 1H, Hthiophene), 7.12
(d, J=3.63 Hz, 1H, Hthiophene), 6.62 (s, 1H, Hisoxazole), 2.12 (s, 3H, CH3). 13C NMR (75 MHz,
DMSO-d6) δ ppm 167.5, 162.5, 158.5, 153.6, 130.3, 128.3, 126.5, 86.1, 23.5. LC MS (ESI): m/z
[M+H]+ 288. Anal. calcd for C9H9N3O4S2 (287.32): C, 37.62; H, 3.16; N, 14.63; found: C, 37.60;
H, 3.16; N, 14.60.
4.2.4.4.
N-[3-(5-Methyl-4-sulfamoylthiophen-2-yl)-1,2-oxazol-5-yl]acetamide (8e)
Yield 2,167 mg (72%). Brown solid, m.p. 161-165 °С (i-PrOH). 1H NMR (300 MHz, DMSO-d6)
δ ppm 12.75 (br s, 2H, SO2NH2), 11.68 (br s, 1H, AcNH), 7.47 (s, 1H, Hthiophene), 6.54 (s, 1H,
Hisoxazole), 2.55 (s, 3H, Th-CH3), 2.11 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) δ ppm 167.5,
162.5, 158.1, 145.8, 139.5, 129.6, 124.6, 85.7, 23.5, 14.5. LC MS (ESI): m/z [M+H]+ 302. Anal.
calcd for C10H11N3O4S2 (301.35): C, 39.86; H, 3.68; N, 13.94; found: C, 39.82; H, 3.68; N,
13.98.
4.2.4.5.
N-[3-(5-Sulfamoylthiophen-3-yl)-1,2-oxazol-5-yl]acetamide (8f)
1
Yield 1,808 mg (63%). White solid, m.p. 226-228 °С (i-PrOH). H NMR (400 MHz, DMSO-d6)
δ ppm 11.70 (br s, 1H, AcNH), 8.45 (s, 1H, Hthiophene), 7.91 (s, 1H, Hthiophene), 7.79 (s, 2H,
SO2NH2), 6.72 (s, 1H, Hisoxazole), 2.13 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) δ ppm 167.5,
162.7, 158.5, 147.7, 130.7, 130.2, 127.9, 86.4, 23.6. LC MS (ESI): m/z [M+H]+ 288. Anal. calcd
for C9H9N3O4S2 (287.32): C, 37.62; H, 3.16; N, 14.63; found: C, 37.59; H, 3.16; N, 14.61.
4.2.5. 5-Amino-3-(3-sulfamoylphenyl)-1,2-oxazole-4-sulfonamide (10a).
Compound 7d (10 mmol) was added portionwise to a stirred and cooled mixture of
chlorosulfonic acid (200 mmol) and thionyl chloride (20 mmol). The resulting mixture was
heated at 60 °С over 1 h, then at 110 ºC for 48 h, cooled to r. t. and poured over crushed ice. The
mixture was extracted with dichloromethane (150 mL). The solution was washed with 5%
aqueous K2CO3 (50 mL), dried over anhydrous CaCl2, filtered and concentrated in vacuo. The
residue was dissolved in acetone (40 mL) and the solution was treated with 25% aqueous
ammonia (50 mmol). The resulting mixture was heated at 50 °С over 1 h, cooled and the
volatiles were removed in vacuo. The residue was treated with ice-cold water (40 mL) and the
mixture was extracted with ethyl acetate (50 mL). The extract was dried over anhydrous CaCl2,

filtered and concentrated in vacuo. Chromatography on silica gel using 75% ethyl acetate in
hexanes as eluent afforded the title compound.
Yield 986 mg (31%). White solid, m.p. 153-155 °С (AcOEt). 1H NMR (300 MHz, DMSO-d6) δ
ppm 8.11 (s, 1H, 2-HAr), 8.02 (d, J=7.9 Hz, 1H, HAr), 7.95 (d, J=7.9 Hz, 1H, HAr), 7.67 (t, J=7.9
13
Hz, 1H, 4-HAr), 7.65 (s, 2H, NH2), 7.47 (s, 2H, NH2), 7.28 (s, 2H, NH2). C NMR (75 MHz,
DMSO-d6) δ ppm 169.6, 159.4, 144.7, 132.9, 129.6, 129.4, 127.4, 126.0, 94.5. LC MS (ESI): m/z
[M+H]+ 319. Anal. calcd for C9H10N4O5S2 (318.33): C, 33.96; H, 3.17; N, 17.60; found: C,
33.93; H, 3.17; N, 17.63.
4.2.6. 5-Amino-3-(4-methyl-3-sulfamoylphenyl)-1,2-oxazole-4-sulfonamide (10b).
Compound 7d (10 mmol) was added portionwise to a stirred and cooled mixture of
chlorosulfonic acid (200 mmol) and thionyl chloride (20 mmol). The resulting mixture was
heated at 60 °С over 1 h, then at 90 ºC for 24 h, cooled to r. t. and poured over crushed ice. The
mixture was extracted with dichloromethane (150 mL). The solution was washed with 5%
aqueous K2CO3 (50 mL), dried over anhydrous CaCl2, filtered and concentrated in vacuo. The
residue was briefly fractionated on silica gel using 25% ethyl acetate in hexanes and the fractions
containing the intermediate sulfonyl chloride were pooled and concentrated in vacuo. The
residue was dissolved in acetone (40 mL) and the solution was treated with 25% aqueous
ammonia (50 mmol). The resulting mixture was heated at 50 °С over 1 h, cooled and the
volatiles were removed in vacuo. The residue was treated with ice-cold water (40 mL) and the
mixture was and the mixture was extracted with ethyl acetate (50 mL). The extract was dried
over anhydrous CaCl2, filtered and concentrated in vacuo. Chromatography on silica gel using
75% ethyl acetate in hexanes as eluent afforded the title compound.
1
Yield 1,793 mg (54%). Beige solid, m.p. 200-202 °С (AcOEt). H NMR (400 MHz, DMSO-d6)
δ ppm 8.14 (d, J2-6=1.5 Hz, 1H, 2-HAr), 7.90 (dd, J5-6=7.8 Hz, J2-6=1.5 Hz, 1H, 6-HAr), 7.59 (s,
2H, NH2), 7.49 (m, 3H, NH2, 5-HAr), 7.24 (s, 2H, NH2), 2.64 (s, 3H, OCH3). 13C NMR (75 MHz,
DMSO-d6) δ ppm 169.6, 159.4, 142. 7, 138.2, 132.7, 132.7, 127.4, 126.6, 94.4, 20.2. LC MS
(ESI): m/z [M+H]+ 333. Anal. calcd for C10H12N4O5S2 (332.36): C, 36.14; H, 3.64; N, 16.86;
found: C, 36.14; H, 3.64; N, 16.85.
4.2.7. General procedure for the synthesis of sulfonamides 10c-d.
The procedure is identical to that for the synthesis of compound 10b but the heating in the first
step continued for 24 h at 80 ºC.

4.2.7.1. 5-Amino-3-(4-methoxy-3-sulfamoylphenyl)-1,2-oxazole-4-sulfonamide (10c)
Yield 2,123 mg (61%). Beige solid, m.p. 211-213 °С (i-PrOH). 1H NMR (300 MHz, DMSO-d6)
δ ppm 8.03 (m, 2H, 2-HAr, 6-HAr), 7.56 (s, 2H, NH2), 7.30 (d, J5-6=8.6 Hz, 1H, 5-HAr), 7.16 (s,
13
2H, NH2), 7.21 (s, 2H, NH2), 3.96 (s, 3H, CH3). C NMR (75 MHz, DMSO-d6) δ ppm 169.6,
159.2, 157.6, 134.9, 131.6, 128.3, 120.2, 113.0, 94.2, 56.8. LC MS (ESI): m/z [M+H]+ 349. Anal.
calcd for C10H12N4O6S2 (348.36): C, 34.48; H, 3.47; N, 16.08; found: C, 34.42; H, 3.47; N,
16.10.
4.2.7.2. N-[3-(5-Methoxy-2-sulfamoylphenyl)-4-sulfamoyl-1,2-oxazol-5-yl]acetamide (10d)
Yield 1,288 mg (37%). Beige solid, m.p. 125-129 °С (i-PrOH). 1H NMR (300 MHz, DMSO-d6)
δ ppm 7.92 (d, J3-4=8.9 Hz, 1H, 3-HAr), 7.57 (s, 2H, NH2), 7.21 (dd, J3-4=8.9 Hz, J4-6=2.8 Hz, 1H,
4-HAr), 7.07 (d, J4-6=2.8 Hz, 1H, 6-HAr), 6.94 (s, 2H, NH2), 6.89 (s, 2H, NH2), 3.85 (s, 3H, CH3).
13C NMR (75 MHz, DMSO-d6) δ ppm 168.5, 161.3, 159.5, 135.2, 130.1, 128.1, 118.1, 115.4,
95.86, 56.3. LC MS (ESI): m/z [M+H]+ 349. Anal. calcd for C10H12N4O6S2 (348.36): C, 34.48;
H, 3.47; N, 16.08; found: C, 34.45; H, 3.47; N, 16.11.
4.2.8. General procedure for the synthesis of sulfonamides 10e-g.
The procedure is identical to that for the synthesis of compound 10b but the heating in the first
step continued for 12 h at 90º C.
4.2.8.1. 5-Amino-3-(5-sulfamoylthiophen-2-yl)-1,2-oxazole-4-sulfonamide (10e)
1
Yield 1,588 mg (49%). Yellow solid, m.p. 137-139 °С (i-PrOH). H NMR (300 MHz, DMSO-
d6) δ ppm 7.35 (d, J=3.63 Hz, 1H, Hthiophene), 7.27 (br s, 2H, NH2), 7.17 (d, J=3.63 Hz, 1H,
13
Hthiophene), 7.10 ((br s, 2H, NH2), 6.93 ((br s, 2H, NH2). C NMR (75 MHz, DMSO-d6) δ ppm
172.6, 159.3, 155.3, 131.6, 127.8, 126.1, 94.7. LC MS (ESI): m/z [M+H]+ 325. Anal. calcd for
C7H8N4O5S3 (324.36): C, 25.92; H, 2.49; N, 17.27; found: C, 25.89; H, 2.49; N, 17.29.
4.2.8.2. 5-Amino-3-(5-methyl-4-sulfamoylthiophen-2-yl)-1,2-oxazole-4-sulfonamide (10f)
Yield 1,622 mg (48%). Beige solid, m.p. 229-231 °С (i-PrOH). 1H NMR (300 MHz, DMSO-d6)
δ ppm 7.99 (s, 1H, Hthiophene), 7.63 (s, 2H, NH2), 7.34 (s, 2H, NH2), 7.40 (s, 2H, NH2), 2.65 (s,
13
3H, CH3). C NMR (75 MHz, DMSO-d6) δ ppm 169.9, 153.4, 144.5, 140.4, 130.7, 124.6, 93.1,
14.4. LC MS (ESI): m/z [M+H]+ 339. Anal. calcd for C8H10N4O5S3 (338.39): C, 28.40; H, 2.98;
N, 16.56; found: C, 28.38; H, 2.98; N, 16.58.

4.2.8.3. 5-Amino-3-(5-sulfamoylthiophen-3-yl)-1,2-oxazole-4-sulfonamide (10g)
Yield 1,328 mg (41%). Beige solid, m.p. 215-217 °С (i-PrOH). 1H NMR (300 MHz, DMSO-d6)
δ ppm 8.42 (d, J=1.3 Hz, 1H, Hthiophene), 7.88 (d, J=1.3 Hz, 1H, Hthiophene), 7.77 (s, 2H, NH2), 7.59
(s, 2H, NH2), 7.35 (s, 2H, NH2) 13C NMR (75 MHz, DMSO-d6) δ ppm 169.7, 154.5, 146.5,
132.6, 129.7, 128.0, 93.5. LC MS (ESI): m/z [M+H]+ 325. Anal. calcd for C7H8N4O5S3 (324.36):
C, 25.92; H, 2.49; N, 17.27; found: C, 25.91; H, 2.49; N, 17.27.
4.2.9. 5-Amino-3-[3-(chlorosulfonyl)phenyl]-1,2-oxazole-4-sulfonyl chloride (11a).
Compound 7d (10 mmol) was added portionwise to a stirred and cooled mixture of
chlorosulfonic acid (200 mmol) and thionyl chloride (20 mmol). The resulting mixture was
heated at 60 °С over 1 h, then at 110 ºC for 48 h, cooled to r. t. and poured over crushed ice. The
mixture was extracted with dichloromethane (150 mL). The solution was washed with 5%
aqueous K2CO3 (75 mL), dried over anhydrous CaCl2, filtered and concentrated in vacuo. The
residue was briefly fractionated on silica gel using 25% ethyl acetate in hexanes and the fractions
containing the bis-sulfonyl chloride were pooled and concentrated in vacuo. The residue was
crystallized from diethyl ether to provide the title compound.
1
Yield 1,428 mg (40%). Beige solid, m.p. 121-124 °С (diethyl ether). H NMR (400 MHz,
CDCl3) δ ppm 8.51 (s, 1H, 2-HAr), 8.23 (d, J=8.0 Hz, 1H, HAr), 8.23 (d, J=8.0 Hz, 1H, HAr) 7.81
(t, J=8.0 Hz, 1H, 5-HAr), 6.28 (br s, 2H, NH2). 13C NMR (75 MHz, CDCl3) δ ppm 169.1, 157.6,
144.9, 135.6, 130.2, 129.1, 127.9, 127.8, 99.0. Anal. calcd for C9H6Cl2N2O5S2 (357.19): C,
30.26; H, 1.69; N, 7.84; found: C, 30.36; H, 1.73; N, 7.79.
5-Amino-3-[3-(chlorosulfonyl)-4-methylphenyl]-1,2-oxazole-4-sulfonyl chloride (11b).
The procedure is analogous to that for the preparation of compound 10a but the heating in the
first step continued at 90 ºC at 24 h.
1
Yield 2,857 mg (77%). Beige solid, m.p. 136-138 °С (diethyl ether). H NMR (300 MHz,
CDCl3) δ ppm 8.54 (s, 1H, 2-HAr), 8.07 (d, J=7.9 Hz, 1H, HAr), 7.60 (d, J=7.9 Hz, 1H, HAr), 6.29
(br s, 2H, NH2), 2.89 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3) δ ppm 169.0, 157.7, 143.4,
141.1, 135.3, 133.8, 129.5, 124.9, 99.0, 20.4. Anal. calcd for C10H8Cl2N2O5S2 (371.22): C,
32.35; H, 2.17; N, 7.55; found: C, 32.42; H, 2.12; N, 7.59.
4.2.11. General procedure for the synthesis of bis-sulfochlorides 11c-d.

The procedure is analogous to that for the preparation of compound 10a but the heating in the
first step continued at 80 ºC at 24 h.
4.2.11.1. 5-Amino-3-[3-(chlorosulfonyl)-4-methoxyphenyl]-1,2-oxazole-4-sulfonyl chloride
(11c)
1
Yield 3,173 mg (82%). Beige solid, m.p. 148-151 °С (diethyl ether). H NMR (300 MHz,
CDCl3) δ ppm 8.46 (d, J2-6=2.3 Hz, 1H, 2-HAr), 8.18 (dd, J5-6=8.6 Hz, J2-6=2.3 Hz, 12 H), 7.29
(d, J5-6=8.6 Hz, 1H, 5-HAr), 6.27 (br s, 2H, NH2), 4.17 (s, 3H, OCH3). 13C NMR (75 MHz,
CDCl3) δ ppm 169.1, 159.0, 157.5, 137.7, 132.2, 130.8, 118.2, 113.5, 98.9, 57.0. Anal. calcd for
C10H8Cl2N2O6S2 (387.22): C, 31.02; H, 2.08; N, 7.23; found: C, C, 30.97; H, 2.16; N, 7.13.
4.2.11.2. 5-Amino-3-[2-(chlorosulfonyl)-5-methoxyphenyl]-1,2-oxazole-4-sulfonyl chloride
(11d)
1
Yield 1.896 mg (49%). Beige solid, m.p. 124-126 °С (diethyl ether). H NMR (300 MHz,
CDCl3) δ ppm 8.19 (d, J3-4=8.6 Hz, 1H, 3-HAr), 7.14 (m, 2H, 4-HAr, 6-HAr), 6.27 (br s, 2H, NH2),
3.94 (s, 3H, OCH3). 13C NMR (75 MHz, CDCl3) δ ppm 169.1, 161.5, 160.4, 134.8, 132.1, 131.3,
118.2, 115.3, 99.9, 56.2. Anal. calcd for C10H8Cl2N2O6S2 (387.22): C, 31.02; H, 2.08; N, 7.23;
found: C, C, 31.07; H, 2.10; N, 7.27.
4.2.12. General procedure for the synthesis of bis-sulfochlorides 11e-g.
The procedure is analogous to that for the preparation of compound 10a but the heating in the
first step continued at 90 ºC at 12 h.
4.2.12.1. 5-Amino-3-[5-(chlorosulfonyl)thiophen-2-yl]-1,2-oxazole-4-sulfonyl chloride (11e)
1
Yield 2,069 mg (57%). Beige solid, m.p. 116-118 °С (diethyl ether). H NMR (300 MHz,
CDCl3) δ ppm 7.99 (d, J=4.3 Hz, 1H, Hthiophene), 7.92 (d, J=4.3 Hz, 1H, Hthiophene), 6.40 (br s, 2H,
13
NH2). C NMR (75 MHz, CDCl3) δ ppm 169.2, 152.2, 146.4, 135.5, 134.6, 131.0, 98.2. Anal.
calcd for C7H4Cl2N2O5S3 (363.22): C, 23.15; H, 1.11; N, 7.71; found: C, 23.08; H, 1.17; N, 7.66.
5-Amino-3-[4-(chlorosulfonyl)-5-methylthiophen-2-yl]-1,2-oxazole-4-sulfonyl chloride (11f)
1
Yield 2,224 mg (59%). Beige solid, m.p. 113-116 °С (diethyl ether). H NMR (300 MHz,
CDCl3) δ ppm 8.23 (s, 1H, Hthiophene), 6.37 (br s, 2H, NH2), 2.88 (s, 3H, CH3). 13C NMR (75
MHz, CDCl3) δ ppm 169.1, 152.1, 152.0, 139.1, 130.4, 123.6, 97.9, 14.8. Anal. calcd for
C8H6Cl2N2O5S3 (377.24): C, 25.47; H, 1.60; N, 7.43; found: C, 25.42; H, 1.55; N, 7.47.

5-Amino-3-[5-(chlorosulfonyl)thiophen-3-yl]-1,2-oxazole-4-sulfonyl chloride (11g)
Yield 1,924 mg (53%). Beige solid, m.p. 97-99 °С (diethyl ether). 1H NMR (300 MHz, CDCl3) δ
ppm 8.63 (d, J=1.6 Hz, 1H, Hthiophene), 8.37 (d, J=1.6 Hz, 1H, Hthiophene), 6.32 (br s, 2H, NH2). 13C
NMR (75 MHz, CDCl3) δ ppm 169.0, 152.8, 145.2, 137.0, 134.2, 126.6, 98.3. Anal. calcd for
C7H4Cl2N2O5S3 (363.22): C, 23.15; H, 1.11; N, 7.71; found: C, 23.13; H, 1.09; N, 7.75.
4.2.13. General procedure for preparation of 3-methyl-4-aryl-1,2-oxazol-5-amines 13a-e.
To a solution of ketonitrile 12 (100 mmol) in ethanol (250 mL) hydroxylamine (100 mL) and
10% aqueous Na2CO3 (110 mL) were added. The mixture was stirred at r. t. until the full
dissolution of the solids and then heated at reflux for 3 h. It was then cooled to r. t. and the
volatiles were removed in vacuo. Water (10 mL) was added and the mixture was extracted with
ethyl acetate (250 mL). The extract was dried over anhydrous Na2SO4, filtered and concentrated
in vacuo to provide the title compounds.
4.2.13.1. 3-Methyl-4-phenyl-1,2-oxazol-5-amine (13a)
1
Yield 16,182 mg (93%). Yellow oil. H NMR (300 MHz, DMSO-d6) δ ppm 7.35 (m, 4H, HAr),
13
7.22 (m, 1H, HAr), 6.70 (s, 2H, NH2), 2.12 (s, 3H, CH3). C NMR (75 MHz, DMSO-d6) δ ppm
166.6, 158.9, 131.8, 129.1, 128.3, 126.0, 92.3, 11.6. LC MS (ESI): m/z [M+H]+ 175. Anal. calcd
for C10H10N2O (174.20): C, 68.95; H, 5.79; N, 16.08; found: C, 68.95; H, 5.79; N, 16.07.
4.2.13.2. 3-Methyl-4-(4-methylphenyl)-1,2-oxazol-5-amine (13b)
1
Yield 17,484 mg (91%). Yellow oil. H NMR (300 MHz, DMSO-d6) δ ppm 7.20 (m, 4H, HAr),
13
6.60 (s, 2H, NH2), 2.31 (s, 3H, CH3), 2.10 (s, 3H, CH3). C NMR (75 MHz, DMSO-d6) δ ppm
166.5, 158.9, 135.2, 129.7, 128.8, 128.3, 92.3, 21.2, 11.6. LC MS (ESI): m/z [M+H]+ 189. Anal.
calcd for C11H12N2O (188.23): C, 70.19; H, 6.43; N, 14.88; found: C, 69.99; H, 6.43; N, 14.82.
4.2.13.3. 4-(4-Methoxyphenyl)-3-methyl-1,2-oxazol-5-amine (13c)
1
Yield 17,425 mg (85%). Yellow solid, m.p.76 - 78 °С (AcOEt). H NMR (300 MHz, DMSO-d6)
δ ppm 7.22 (m, 2H, HAr), 6.95 (m, 2H, HAr), 6.53 (s, 2H), 3.75 (m, 3H), 2.06 (m, 3H). 13C NMR
(75 MHz, DMSO-d6) δ ppm 166.4, 159.0, 157.8, 129.7, 123.9, 114.6, 92.1, 55.5, 11.5. LC MS
(ESI): m/z [M+H]+ 205. Anal. calcd for C11H12N2O2 (204.23): C, 64.69; H, 5.92; N, 13.72;
found: C, 64.66; H, 5.93; N, 13.70.
4.2.13.4. 3-Methyl-4-(3-methylphenyl)-1,2-oxazol-5-amine (13d)

1
Yield 17,296 mg (92%).Yellow solid, m.p. 93 - 95 °С (AcOEt). H NMR (300 MHz, DMSO-d6)
δ ppm 7.27 (t, J=7.60 Hz, 2H, 5-HAr), 7.11 (m, 2H, HAr), 7.03 (d, J=7.60 Hz, 1H, HAr), 6.65 (s,
13
2H, NH2), 2.32 (s, 3H, CH3), 2.11 (s, 3H, CH3). C NMR (75 MHz, DMSO-d6) δ ppm 166.6,
158.9, 138.2, 131.7, 128.9, 126.7, 125.3, 92.3, 21.5, 11.6. LC MS (ESI): m/z [M+H]+ 189. Anal.
calcd for C11H12N2O (188.23): C, 70.19; H, 6.43; N, 14.88; found: C, 70.17; H, 6.43; N, 14.89.
4.2.13.5. 4-(3-Methoxyphenyl)-3-methyl-1,2-oxazol-5-amine (13e)
Yield 18,156 mg (89%). Yellow solid, m.p. 94 - 96 °С (AcOEt). 1H NMR (300 MHz, DMSO-d6)
δ ppm 7.29 (t, J5-6=7.6 Hz, J4-5=8.3 Hz, 1H, 5-HAr), 6.89 (d, J5-6=7.6 Hz, 1H, 6-HAr), 6.85 (d, J2-
4=2.31 Hz, 1H, 2-HAr), 6.79 (dd, J4-5=8.3 Hz, J2-4=2.31 Hz, 1H, 4-HAr), 6.70 (s, 2H, NH2), 3.79
13
(m, 3H, CH3), 2.12 (s, 3H, CH3). C NMR (75 MHz, DMSO-d6) δ ppm 166.7, 159.9, 158.9,
133.2, 130.1, 120.5, 113.6, 111.8, 92.2, 55.4, 11.7. LC MS (ESI): m/z [M+H]+ 205. Anal. calcd
for C11H12N2O2 (204.23): C, 64.69; H, 5.92; N, 13.72; found: C, 64.65; H, 5.92; N, 13.75.
4.2.14. General procedure for the synthesis of N-(3-methyl-4-aryl-1,2-oxazol-5-
yl)acetamides 14a-e.
To a solution of amine 13 (20 mmol) and trimethylamine (4.7 mL, 30 mmol) in acetonitrile (25
mL) acetyl chloride (2 mL, 30 mmol) was added dropwise. The mixture was stirred at r. t. for 12
h and then water (50 mL) was added. The mixture was extracted with ethyl acetate (50 mL), the
extract was washed with water (50 mL), dried over anhydrous sodium sulfate, filtered and
concentrated. The residue was crystallized from isopropyl alcohol to provide the title compound.
4.2.14.1.
N-(3-Methyl-4-phenyl-1,2-oxazol-5-yl)acetamide (14a)
1
Yield 4,210 mg (97%). Yellow oil. H NMR (300 MHz, DMSO-d6) δ ppm 10.46 (br s, 1H,
AcNH), 7.39 (m, 5H, HAr), 2.26 (s, 3H, CH3), 2.01 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6)
δ ppm 169.7, 160.1, 157.7, 129.5, 129.2, 128.8, 128.0, 110.4, 23.1, 11.8. LC MS (ESI): m/z
[M+H]+ 217. Anal. calcd for C12H12N2O2 (216.24): C, 66.65; H, 5.59; N, 12.96; found: C, 66.62;
H, 5.60; N, 12.97.
4.2.14.2.
N-[3-Methyl-4-(4-methylphenyl)-1,2-oxazol-5-yl]acetamide (14b)
1
Yield 3,588 mg (78%). White solid, m.p. 115 - 118 °С (i-PrOH). H NMR (300 MHz, DMSO-
d6) δ ppm 10.42 (br s, 1H, AcNH), 7.26 (m, 4H, HAr), 2.33 (s, 3H, Ar-CH3), 2.25 (s, 3H, CH3),
13
2.00 (s, 3H, COCH3). C NMR (75 MHz, DMSO-d6) δ ppm 169.7, 160.2, 157.5, 137.4, 129.7,

128.7, 126.5, 110.6, 23.1, 21.2, 11.7. LC MS (ESI): m/z [M+H]+ 231. Anal. calcd for
C13H14N2O2 (230.27): C, 67.81; H, 6.13; N, 12.17; found: C, 67.80; H, 6.13; N, 12.19.
4.2.14.3.
N-[4-(4-Methoxyphenyl)-3-methyl-1,2-oxazol-5-yl]acetamide (14c)
Yield 4,379 mg (89%). Yellow solid, m.p. 101 - 103 °С (i-PrOH). 1H NMR (300 MHz, DMSO-
d6) δ ppm 10.38 (br s, 1H, AcNH), 7.31 (d, J=8.8 Hz, 2H, HAr), 7.01 (d, J=8.8 Hz, 2H, HAr), 3.78
13
(s, 3H, OCH3), 2.24 (s, 3H, CH3), 2.00 (s, 3H, COCH3). C NMR (75 MHz, DMSO-d6) δ ppm
169.7, 160.2, 159.2, 157.2, 130.1, 121.5, 114.6, 110.3, 55.5, 23.0, 11.7. LC MS (ESI): m/z
[M+H]+ 247. Anal. calcd for C13H14N2O3 (246.27): C, 63.40; H, 5.73; N, 11.38; found: C, 63.38;
H, 5.73; N, 11.40.
4.2.14.4.
N-[3-Methyl-4-(3-methylphenyl)-1,2-oxazol-5-yl]acetamide (14d)
1
Yield 3,772 mg (82%). Yellow solid, m.p. 85 - 87 °С (i-PrOH). H NMR (300 MHz, DMSO-d6)
δ ppm 10.43 (br s, 1H, AcNH), 7.33 (m, 1H, 5-HAr), 7.18 (m, 3H, HAr), 2.34 (s, 3H, Ar-CH3),
13
2.26 (s, 3H, CH3), 2.01 (s, 3H, COCH3). C NMR (75 MHz, DMSO-d6) δ ppm 169.7, 160.1,
157.6, 138.4, 129.4, 129.1, 128.7, 125.9, 110.5, 23.07, 21.5, 11.7. LC MS (ESI): m/z [M+H]+
231. Anal. calcd for C13H14N2O2 (230.27): C, 67.81; H, 6.13; N, 12.17; found: C, 67.77; H, 6.13;
N, 12.16.
4.2.14.5.
N-[4-(3-Methoxyphenyl)-3-methyl-1,2-oxazol-5-yl]acetamide (14e)
1
Yield 4,133 mg (84%). White solid, m.p. 122 - 124 °С (i-PrOH). H NMR (300 MHz, DMSO-
d6) δ ppm 10.46 (br s, 1H, AcNH), 7.35 (m, 1H, 5-HAr), 6.95 (m, 3H, HAr), 3.77 (s, 3H, OCH3),
13
2.27 (s, 3H, CH3), 2.01 (s, 3H, COCH3). C NMR (75 MHz, DMSO-d6) δ ppm 169.7, 160.1,
159.8, 157.7, 130.7, 130.2, 121.1, 114.4, 113.7, 110.4, 55.6, 23.1, 11.8. LC MS (ESI): m/z
[M+H]+ 247. Anal. calcd for C13H14N2O3 (246.27): C, 63.40; H, 5.73; N, 11.38; found: C, 63.32;
H, 5.74; N, 11.41.
4.2.15. General procedure for the preparation of sulfonamides 15a-d.
Compound 10 (10 mmol) was added portionwise to a stirred and cooled mixture of
chlorosulfonic acid (100 mmol) and thionyl chloride (10 mmol). The resulting mixture was
heated at 60 °С over 4 h, cooled to r. t. and poured over crushed ice. The resulting mixture was
extracted with ethyl acetate precipitate (75 mL). The extract was washed with 5% aqueous
K2CO3 (75 mL), dried over anhydrous CaCl2, filtered and concentrated in vacuo. The residue
was briefly fractionated on silica gel using 25% ethyl acetate in hexanes and the fractions

containing the intermediate sulfonyl chloride were pooled and concentrated in vacuo. The
residue was dissolved in acetone (40 mL) and the solution was treated with 25% aqueous
ammonia (25 mmol). The resulting mixture was heated at 50 °С over 1 h, cooled and the
volatiles were removed in vacuo. The residue was treated with ice-cold water (40 mL) and the
mixture was stirred until a thick precipitate formed. The latter was filtered off and crystallized
from acetone to provide the title compound.
4.2.15.1.
N-[3-Methyl-4-(4-sulfamoylphenyl)-1,2-oxazol-5-yl]acetamide (15a)
1
Yield 1,652 mg (56%). White solid, m.p. 212-214 °С (acetone). H NMR (300 MHz, DMSO-d6)
δ ppm 10.68 (br s, 1H, AcNH), 7.86 (d, J=8.3 Hz, 2H, HAr), 7.56 (d, J=8.3 Hz, 2H, HAr), 7.41 (s,
13
2H, SO2NH2), 2.28 (s, 3H, CH3), 2.02 (s, 3H, COCH3). C NMR (75 MHz, DMSO-d6) δ ppm
160.1, 158.3, 143.3, 133.3, 129.2, 126.4, 108.4, 23.2, 11.7. LC MS (ESI): m/z [M+H]+ 296. Anal.
calcd for C12H13N3O4S (295.32): C, 48.81; H, 4.44; N, 14.23; found: C, 48.78; H, 4.44; N, 14.21.
4.2.15.2.
N-[3-Methyl-4-(4-methyl-3-sulfamoylphenyl)-1,2-oxazol-5-yl]acetamide (15b)
Yield 1,607 mg (52%). White solid, m.p. 173-175 °С (acetone). 1H NMR (300 MHz, DMSO-d6)
δ ppm 10.62 (br s, 1H, AcNH), 7.84 (s, 1H, 2-HAr), 7.47 (m, 4H, 5-HAr, 6-HAr, SO2NH2), 2.63 (s,
13
3H, Ar-CH3), 2.28 (s, 3H, CH3), 2.06 (m, 3H, COCH3). C NMR (75 MHz, DMSO-d6) δ ppm
169.3, 160.0, 158.1, 142.9, 135.5, 133.1 131.8, 127.6, 127.1, 108.7, 23.1, 20.0, 11.7. LC MS
(ESI): m/z [M+H]+ 310. Anal. calcd for C13H15N3O4S (309.35): C, 50.47; H, 4.89; N, 13.58;
found: C, 50.44; H, 4.89; N, 13.56.
4.2.15.3. N-[4-(4-Methoxy-3-sulfamoylphenyl)-3-methyl-1,2-oxazol-5-yl]acetamide (15c)
1
Yield 1,987 mg (61%). White solid, m.p. 176-178 °С (acetone). H NMR (300 MHz, DMSO-d6)
δ ppm 10.50 (br s, 1H, AcNH), 7.70 (d, J2-6=2.0 Hz, 1H, 2-HAr), 7.56 (dd, J2-6=8.6 Hz, J5-6=2.31
Hz, 1H, 6-HAr), 7.28 (d, J5-6=8.6 Hz, 1H, 5-HAr), 7.10 (s, 2H, SO2NH2), 3.94 (s, 3H, OCH3), 2.25
13
(s, 3H, CH3), 2.00 (s, 3H, COCH3). C NMR (75 MHz, DMSO-d6) δ ppm 169.5, 160.1, 157.8,
155.8, 134.0, 131.9, 127.8, 121.2, 113.5, 109.0, 56.7, 23.1, 11.7. LC MS (ESI): m/z [M+H]+ 326.
Anal. calcd for C13H15N3O5S (325.35): C, 47.99; H, 4.65; N, 12.92; found: C, 47.94; H, 4.65; N,
12.92.
4.2.15.4. N-[3-Methyl-4-(3-methyl-4-sulfamoylphenyl)-1,2-oxazol-5-yl]acetamide (15d)
Yield 1,452 mg (47%). Gray solid, m.p. 215-217 °С (acetone). 1H NMR (300 MHz, DMSO-d6) δ
ppm 10.59 (br s, 1H, AcNH), 7.88 (d, J5-6=7.93 Hz, 1H, 5-HAr), 7.33 (m, 4H, 2-HAr, 6-HAr,

13
SO2NH2), 2.62 (s, 3H, Ar-CH3), 2.28 (s, 3H, CH3), 2.02 (s, 3H, COCH3). C NMR (75 MHz,
DMSO-d6) δ ppm 169.2, 160.1, 158.3, 141.5, 136.7, 133.3, 132.3, 127.9, 126.3, 108.4, 23.2,
20.3, 11.7. LC MS (ESI): m/z [M+H]+ 310. Anal. calcd for C13H15N3O4S (309.35): C, 50.47; H,
4.89; N, 13.58; found: C, 50.45; H, 4.89; N, 13.59.
4.2.16. N-[4-(5-Methoxy-2-sulfamoylphenyl)-3-methyl-1,2-oxazol-5-yl]acetamide 15e and 1-
(8-methoxy-1-methyl-5,5-dioxido-4H-[1,2]oxazolo[5,4-c][1,2]benzothiazin-4-yl)ethanone 16.
Compound 14e (10 mmol) was added portionwise to a stirred and cooled mixture of
chlorosulfonic acid (100 mmol) and thionyl chloride (10 mmol). The resulting mixture was
heated at 60 °С over 4 h, cooled to r. t. and poured over crushed ice. The resulting mixture was
extracted with ethyl acetate (100 mL). The extract was washed with 5% aqueous K2CO3 (75
mL), dried over anhydrous CaCl2, filtered and concentrated in vacuo. The residue was briefly
fractionated on silica gel using 25% ethyl acetate in hexanes and the fractions containing the
intermediate sulfonyl chloride were pooled and concentrated in vacuo. The residue was dissolved
in acetone (40 mL) and the solution was treated with 25% aqueous ammonia (25 mmol). The
resulting mixture was heated at 50 °С over 1 h, cooled and the volatiles were removed in vacuo.
The residue was treated with ice-cold water (40 mL) and the mixture was stirred until a thick
precipitate formed. The latter was filtered off and chromatographed on silica gel using 85% ethyl
acetate un hexanes as eluent to provide 15e and 16.
4.2.16.1. N-[4-(5-Methoxy-2-sulfamoylphenyl)-3-methyl-1,2-oxazol-5-yl]acetamide (15e)
Yield 880 mg (27%). White solid, m.p. 162-164 °С (AcOEt). 1H NMR (300 MHz, DMSO-d6) δ
ppm 10.37 (br s, 1H, AcNH), 7.90 (d, J3-4=8.8 Hz, 1H, 3-HAr), 7.12 (dd, J3-4=8.8 Hz, J4-6=2.0 Hz,
1H, 4-HAr), 6.98 (br s, 2H, SO2NH2), 6.83 (d, J4-6=2.0 Hz, 1H, 6-HAr), 3.83 (s, 3H, OCH3), 1.97
(s, 3H, CH3), 1.92 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) δ ppm 168.8, 161.4, 161.3,
157.7, 136.2, 129.9, 129.5, 119.5, 113.7, 106.7, 56.1, 23.1, 11.3. LC MS (ESI): m/z [M+H]+ 326.
Anal. calcd for C13H15N3O5S (325.35): C, 47.99; H, 4.65; N, 12.92; found: C, 47.96; H, 4.65; N,
12.95.
4.2.16.2.
1-(8-Methoxy-1-methyl-5,5-dioxido-4H-[1,2]oxazolo[5,4-c][1,2]benzothiazin-4-
yl)ethanone (16)
1
Yield 739 mg (24%). White solid, m.p. 155-157 °С (AcOEt). H NMR (300 MHz, DMSO-d6)
δ ppm 7.58 (d, J6-7=8.6 Hz, 1H, 6-HAr), 6.90 (d, J7-9=2.0 Hz, 1H, 9-HAr), 6.78 (dd, J6-7=8.6, J7-
13
9=2.0 Hz, 1H, 7-HAr), 3.83 (s, 3H, OCH3), 2.40 (s, 3H, CH3), 1.91 (s, 3H, CH3). C NMR (75
MHz, DMSO-d6) δ ppm 172.4, 171.0, 160.7, 155.8, 131.6, 125.6, 121.6, 110.5, 104.3, 87.6, 55.6,

21.5, 13.3. LC MS (ESI): m/z [M+H]+ 309. Anal. calcd for C13H12N2O5S (308.31): C, 47.99; H,
3.72; N, 8.61; found: C, 47.98; H, 3.72; N, 8.64.
4.2.17. 2-[5-(Acetylamino)-3-methyl-1,2-oxazol-4-yl]-4-methoxybenzenesulfonyl chloride
(17).
Compound 14e (10 mmol) was added portionwise to a stirred and cooled mixture of
chlorosulfonic acid (100 mmol) and thionyl chloride (10 mmol). The resulting mixture was
heated at 60 °С over 4 h, cooled to r. t. and poured over crushed ice. The resulting mixture was
extracted with ethyl acetate (100 mL). The extract was washed with 5% aqueous K2CO3 (75
mL), dried over anhydrous CaCl2, filtered and concentrated in vacuo. The residue was purified
by chromatography on silica gel using 25% ethyl acetate in hexanes to provide the title
compound.
1
Yield 3,068 mg (89%). Brown solid, m.p. 155-157 °С (acetone). H NMR (300 MHz, CDCl3) δ
ppm 8.14 (d, J5-6=9.0 Hz, 1H, 6-HAr), 7.95 (br s, 1H, AcNH), 7.08 (dd, J5-6=9.0 Hz, J3-5=2.3 Hz,
1H, 5-HAr), 6.94 (d, J3-5=2.3 Hz, 1H, 3-HAr), 3.95 (s, 3H, OCH3), 2.13 (s, 3H, CH3), 2.12 (s, 3H,
13
CH3). C NMR (75 MHz, DMSO-d6) δ ppm 164.4, 160.8, 157.6, 135.9, 131.7, 120.0, 113.900,
102.9, 56.1, 23.1, 11.1. Anal. calcd for C13H13ClN2O5S (344.78): C, 45.29; H, 3.80; N, 8.13;
found: C, 45.25; H, 3.80; N, 8.14.
4.2.18.
1-(8-Methoxy-1-methyl-5,5-dioxido-4H-[1,2]oxazolo[5,4-c][1,2]benzothiazin-4-
yl)ethanone (16).
Compound 17 (5 mmol) was dissolved in acetone (20 mL) and trimethylamine (7 mmol) was
added. The mixture was stirred at r. t. for 24 h and concentrated in vacuo. Water (25 mL) was
added and the precipitate formed was filtered off, washed with water and air-dried to provide the
title compound in 46% yield, with analytical data identical to those presented above (4.2.16.2).
4.3 Carbonic anhydrase inhibition assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed
CO2 hydration activity.17 Phenol red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM Tris (pH 8.3) as buffer, and 20
mM Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the CA-
catalyzed CO2 hydration reaction for a period of 10-100 s. The CO2 concentrations ranged from
1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each

inhibitor at least six traces of the initial 5-10% of the reaction have been used for determining the
initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from
the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-
deionized water and dilutions up to 0.005 nM were done thereafter with the assay buffer.
Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior
to assay, in order to allow for the formation of the E-I complex. The inhibition constants were
obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as
reported earlier, and represent the mean from at least three different determinations. All CA
isoforms were recombinant ones obtained in-house.18-21
4.4 In Silico docking
The crystal structure of hCA II (pdb code 2AW112) was taken from the Protein Data Bank.13
After adding hydrogen atoms and removing the complexed ligand, the protein was minimized
using Amber 11 software and parm03 force field at 300 K. The protein was placed in a
rectangular parallelepiped water box, an explicit solvent model for water, TIP3P, was used and
the complex was solvated with a 20 Å water cap. Sodium ions were added as counter ions to
neutralize the system. Two steps of minimization were then carried out; in the first stage, we
kept the protein fixed with a position restraint of 500 kcal/mol•Å2 and we solely minimized the
positions of the water molecules. In the second stage, we minimized the entire system through
5000 steps of steepest descent followed by conjugate gradient until a convergence of 0.05
kcal/Å•mol. The region of interest used by the docking program GOLD version 5.1 was defined
in order to contain the residues within 15 Å from the original position of the ligand in the X-ray
structure. Metal coordination in GOLD is modeled as ‘pseudohydrogen bonding’ in which
metals can be considered to bind to H-bond acceptors and the metal will compete with H-bond
donors for interaction. The ‘allow early termination’ option was deactivated, while the
possibility for the ligand to flip ring corners was activated. The remaining GOLD default
parameters were used, and the ligands were submitted to 30 genetic algorithm runs. The docking
analysis was carried out using the ChemScore fitness function imposing the formation of an H
bond between the ligands and the hydroxy group of T198.14, 23 Cluster analysis was performed on
the results using an rmsd tolerance of 2.0 Å and the best docked conformation was taken into
account.
Acknowledgment
This research was supported by the Russian Scientific Fund (Project Grant 14-50-00069).

Supplementary data
Supplementary data associated with this article can be found, in the online version, at
http://dx.doi.org/xxx.
References and notes
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