Development of oxathiino[6,5-b]pyridine 2,2-dioxide derivatives as selective inhibitors of tumor-related carbonic anhydrases IX and XII

Article abstract of DOI:10.1016/j.ejmech.2020.112300

Oxathiino[6,5-b]pyridine 2,2-dioxides are identified as a new class of isoform-selective nanomolar inhibitors of tumor associated human carbonic anhydrases (hCA) IX and XII. At the same time they do not inhibit or poorly inhibit cytosolic isoforms hCA I and II. Oxathiino[6,5-b]pyridine 2,2-dioxides exhibited good antiproliferative properties on tumor cell lines MCF-7 (Human breast adenocarcinoma), A549 (human lung (alveolar) adenocarcinoma) and HeLa (epithelioid cervix carcinoma).

Full text of DOI:10.1016/j.ejmech.2020.112300

Journal Pre-proof
Development of oxathiino[6,5-b]pyridine 2,2-dioxide derivatives as selective inhibitors
of tumor-related carbonic anhydrases IX and XII
Aiga Grandāne, Alessio Nocentini, Ilona Domračeva, Raivis Žalubovskis, Claudiu T.
Supuran
PII:
S0223-5234(20)30269-5
DOI:
https://doi.org/10.1016/j.ejmech.2020.112300
EJMECH 112300
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 March 2020
Revised Date: 1 April 2020
Accepted Date: 2 April 2020
Please cite this article as: A. Grandāne, A. Nocentini, I. Domračeva, R. Žalubovskis, C.T. Supuran,
Development of oxathiino[6,5-b]pyridine 2,2-dioxide derivatives as selective inhibitors of tumor-related
carbonic anhydrases IX and XII, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
j.ejmech.2020.112300.
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Graphical Abstract
Oxathiino[6,5-b]pyridine 2,2-dioxides and compound 17 in particular noticeably decreases viability
of human cancer cells (MCF-7, HeLa, A549), having IC values in the range from 4.8 to 20 µM.
5
0
Oxathiino[6,5-b]pyridine 2,2-dioxides very potent and selective inhibitors of cancer associated
carbonic anhydrases isoforms – CA IX and CA XII. At the same time these compounds have poor
or no inhibition of ubiquitous CA I and CA II.

Development of oxathiino[6,5-b]pyridine 2,2-dioxide derivatives as selective
inhibitors of tumor-related carbonic anhydrases IX and XII
a
b
a
a,c,
Aiga Grandāne , Alessio Nocentini , Ilona Domračeva , Raivis Žalubovskis *, Claudiu T.
b
Supuran
a
Latvian Institute of Organic Synthesis, Aizkraukles 21, LV-1006, Riga, Latvia
b
Università degli Studi di Firenze, NEUROFARBA Department, Section of Pharmaceutical
Chemistry, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy
c
Institute of Technology of Organic Chemistry, Faculty of Materials Science and Applied
Chemistry, Riga Technical University, 3/7 Paula Valdena Str., Riga LV-1048, Latvia
Abstract
Oxathiino[6,5-b]pyridine 2,2-dioxides are identified as a new class of isoform-selective nanomolar
inhibitors of tumor associated human carbonic anhydrases (hCA) IX and XII. At the same time they
do not inhibit or poorly inhibit cytosolic isoforms hCA I and II. Oxathiino[6,5-b]pyridine 2,2-
dioxides exhibited good antiproliferative properties on tumor cell lines MCF-7 (Human breast
adenocarcinoma), A549 (human lung (alveolar) adenocarcinoma) and HeLa (epithelioid cervix
carcinoma).
Keywords: Carbonic anhydrase; oxathiino[6,5-b]pyridine 2,2-dioxide; inhibitor; tumor.
*
Corresponding author. E-mail addresses: raivis@osi.lv (R. Žalubovskis)
1. Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes, which are present in
organisms all over the phylogenetic tree and being encoded by at least eight different genetic
families[1-10]. CAs catalyze a crucial physiologic reaction – reversible hydratation of CO to
2
produce a weak base (bicarbonate) and a strong acid (hydronium ions). CAs are involved in many
physiologic processes, starting with pH regulation and ending with metabolism[1-3, 7-10, 11-22].
These enzymes are also involved in different pathological processes and therefore are drug targets
for decades, and pharmacological applications of CA inhibitors (CAI) have found place in many
fields[1-3, 7-19]. The primary sulfonamides were discovered as CA inhibitors (CAIs) as early as in
the 40s. In the next decades numerous CAIs based on this class of compounds were approved for
clinical use as diuretics, antiepileptics, or antiglaucoma drugs[ 1-3, 7-19]. Although highly potent as
CAIs[1-3], the sulfonamides are generally non-selective inhibitors of most of α-CA isoforms

present in humans and mammals[1-3] and CAs from the other genetic families (β-, γ-, δ-, ζ-, η-, θ-
and ι-CAs)[4-19], therefore novel, isoform selective CAI classes were searched. A multitude of
novel chemotypes and new CA inhibition mechanisms were reported in the last decade[1-3, 11-14,
23-30].
Highly enriched understanding of these enzymes allowed obtaining of isoform-selective CAIs that
target physiologically relevant isoforms[11-14, 23-27]. Among the new chemotypes, which also
exhibited the highest levels of isoform selectivity, were saccharin derivatives[31-33], the
coumarins[27], the sulfocoumarins[23-26, 34] and homosulfocoumarins (3H-1,2-benzoxathiepine
2,2-dioxides)[35-37].
Considering high interest in design of non-sulfonamide CAIs for various potential applications, we
report here a facile synthesis of a new class of compounds, namely oxathiino[6,5-b]pyridine 2,2-
dioxides, their inhibitory profiles against the major human (h) CA isoforms, hCA I, II, IX, and XII,
involved in many pathologies, including cancer, and in vitro cytotoxicity against cancer cell lines.
2. Results and discussion
2.1. Chemistry
The synthesis of oxathiino[6,5-b]pyridine 2,2-dioxides is presented in Schemes 1 and 2.
The synthesis of desired oxathiino[6,5-b]pyridine 2,2-dioxide (5) was started with the reduction of
commercially available 2-hydroxynicotinic acid (1) by LiAlH where intermediate 2 was isolated in
4
good yield (Scheme 1). Alcohol 2 was oxidized by MnO to corresponding aldehyde 3. Mesylation
2
of compound 3 provided compound 4, which in turn was submitted to intramolecular aldol
condensation affording 5 in acceptable yield.
Scheme 1. Reagents and conditions: (i) LiAlH , THF, relux, 2h, 80%. (ii) MnO , EtOAc, 50 °C,
4
2
7
0%. (iii) MsCl, NaH, THF, rt, 16 h, 30%. (iv) a) K CO , DMF rt, 1 h; b) MsCl, NEt , DCM, rt, 3
2 3 3
h, 40%.
Similar strategy was used for the synthesis of 11 – 20, where synthesis was started with reduction of
commercially available carboxylic acid 6 thus obtaining alcohol 7 in good yield (Scheme 2).
Oxidation of 7 by MnO provided aldehyde 8 also in good yield. Compound 8 was mesylated and
2

intermediate 9 formed was in situ submitted to intramolecular aldol condensation affording the key
intermediate 10. Bromide 10 was reacted with a series of organo-tin derivatives (prepared
separately) under Stille reaction conditions providing a series of the inhibitors 11-20 in 29% to 73%
yield.
Scheme 2. Reagents and conditions: (i) BH , THF, rt, 18 h, 53%. (ii) MnO , EtOAc, 60 °C, 18 h,
3
2
5
3%. (iii) a) MsCl, NaH, DMF, rt, 25 min; b) K CO , DMF, rt, 75 min, 58%. (iv) Ar-SnBu or
2 3 3
Ar-SnMe , Pd(OAc) , tri(2-furyl)phosphine, dioxane, 100 °C, 18 h.
3
2
2.2. Determination of inhibition of carbonic anhydrases in vitro
All synthesized oxathiino[6,5-b]pyridine 2,2-dioxides were studied for the inhibition of
physiologically relevant human CA isoforms - the cytosolic, widespread hCA I and hCA II (off-
targets in this case) and transmembrane tumor-associated hCA IX and hCA XII which have been
defined as anti-cancer drug targets[19, 30, 38-43].
From the collected and presented data in Table 1, following SAR can be drawn:
All compounds studies except 5, showed no inhibition of ubiquitous (off-target) CA I and CA II
with K values greater than 100 ꢀM (Table 1). Compound 5 exhibited moderate inhibition of CA I
I
and CA II with K values of 64.11 and 14.05 ꢀM, respectively.
I
All compounds showed very good inhibition of cancer associated CA IX and CA XII.
CA IX was inhibited with K values in range from 31.4 to 220.7 nM. Even though specific SAR
I
cannot be depicted, the inhibitors could be split into two groups. Most active ones having K s from
I
31.4 to 86.8 nM are compounds 11, 13, 14, 17 and 20. This compounds, except 17, have small
substituents at 4-posion of phenyl ring. The most active compound 13 from this group having

K =31.4 nM is nearly as active as nonselective CA inhibitor acetazolamide (AAZ). The rest of the
I
compounds having bulkier substituents on phenyl ring, except compounds 15 and 19, have KI
values in range from 134.3 to 220.7 nM.
CA XII inhibited stronger than CA IX where K values are in the range from 3.8 to 63.8 nM. Also in
I
case of CA XII distinct SAR cannot be concluded, but all compounds can be split into two group. In
the first group compounds 11, 13, 14, 17, 19 and 20 with low nanomolar K s values ranging from
I
3.8 to 9.5 nM can be included. Compounds in this group, except 17, have small substituents at 4-
position of phenyl ring. Notably, compounds 17 and 20 have stronger inhibition of CA XII compare
to nonselective AAZ. The other compounds having bulkier substituents on phenyl ring or no phenyl
ring exhibited slightly lower inhibition of CA with K s values ranging from 18.4 to 63.8 nM.
I
It is interesting to note, that compare to previous results obtained on structurally similar compounds
-
sulfocoumarins all oxathiino[6,5-b]pyridine 2,2-dioxides except compound 5 also exhibited no
inhibition of ubiquitous CA I and CA II. At the same time CA IX inhibition is similar to one
observed for sulfocoumarins. However the oxathiino[6,5-b]pyridine 2,2-dioxide 17 exhibited better
inhibition of CA IX compare to sulfocoumarins where best inhibitor had K =4.3 nM.[23, 44-46]
I
Table 1: Inhibition data of human CA isoforms hCA I, II, IX and XII by compounds 5, 11-20 and
the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow CO hydrase assay[47].
2
CA inhibition, K (nM)*
I
Comp.
hCA I
64110
hCA II
14050
hCA IX
169.3
86.8
hCA XII
63.8
9.5
5
1
1
2
3
4
5
6
7
8
9
0
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
250
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
12
1
1
1
1
1
1
1
1
2
137.3
31.4
18.4
7.2
8.7
22.7
39.8
3.8
29.4
8.5
86.3
134.3
220.7
56.9
210.0
200.1
70.1
5.3
6
AAZ
25
*
Mean from 3 different assays, by a stopped flow technique (errors were in the range of ± 5-10 % of the
reported values). Data were collected after 6 h of incubation period of the enzyme and inhibitor solution.
It was observed, that after 15 min incubation of inhibitor and enzyme solution (data not shown) no
inhibition of any of four isoforms studied was observed, therefore we assume that similar to

sulfocoumarins oxatiine ring opening takes place in oxathiino[6,5-b]pyridine 2,2-dioxide, and
formed sulfonate binds in the active center of CA[23, 48].
2.3. Cell viability
Compounds 13, 17 and 20 having best CA inhibition profile were used for determination of their
antiproliferative properties in vitro on monolayer tumor cell lines MCF-7 (human breast
adenocarcinoma), A549 (human lung (alveolar) adenocarcinoma) and HeLa (epithelioid cervix
carcinoma), the results are summarized in Table 2.
Compounds 17 and 20 showed very good cytotoxic effect on MCF-7 cell line with IC values 20
5
0
and 75 ꢀM, respectively. All three compounds showed moderate to very good cytotoxic effect on
HeLa cell line with IC values from 171 to 4.8 ꢀM. Also moderate to very good cytotoxic effect on
5
0
A549 cell line was observed for 13, 17 and 20 with IC values ranging from 149 to 14 ꢀM. It is
5
0
interesting to note, that compound 17 exhibited very good cytotoxic effect on all three cancer cell
lines.
Table 2. In vitro cytotoxicity of compounds 5, 11-20 on monolayer tumor cell lines MCF-7 (human
breast adenocarcinoma), HeLa (epithelioid cervix carcinoma) and A549 (human lung (alveolar)
adenocarcinoma).
Cytotoxicity, IC (ꢀM)
5
0
Comp.
Formulas
MCF-7
HeLa
A549
>
500
171±11
149±27
1
1
3
7
20±1
75±7
4.8±0.03
42±9
14±2
77±6
2
0
3. Conclusions
In conclusion, we report here the facile synthesis of new class of compounds – oxathiino[6,5-
b]pyridine 2,2-dioxides. We have demonstrated that oxathiino[6,5-b]pyridine 2,2-dioxides are a
new class of tumor associated carbonic anhydrases IX and XII isoform-selective inhibitors. The title

compounds were investigated for the inhibition of four hCA isoforms with medicinal chemistry
applications, the cytosolic hCA I and II, and the transmembrane, tumor-associated hCA IX and XII.
Oxathiino[6,5-b]pyridine 2,2-dioxides generally do not inhibit the ubiquitous, off-target cytosolic
isoforms hCA I and II. However, these compounds showed selective and strong inhibition of the
two transmembrane hCAs, with K s ranging from 31.4 to 220.7 nM against hCA IX, and from 3.8 to
I
63.8 nM against hCA XII. The compounds 13, 17 and 20 having best CA inhibition profile were
studied on antiproliferative properties on tumor cell lines MCF-7 (human breast adenocarcinoma),
A549 (human lung (alveolar) adenocarcinoma) and HeLa (epithelioid cervix carcinoma), where
very good IC50 values were observed on all three cell line. Compound 17 showed excellent
activities on all three cell lines with IC ranging from 4.8 to 20 ꢀM. Thus we have demonstrated
5
0
that oxathiino[6,5-b]pyridine 2,2-dioxides is suitable class of compounds for further studies to
identify suitable drug candidates for preclinical and clinical studies.
4
4
. Experimental
.1. Chemistry
All reagents were purchased from commercial sources and used as received without further
purification. Thin layer chromatography was performed on Merck TLC-plates with fluorescence
indication (silica type 60, F ), spots were visualized using UV-light or KMnO stains. Flash
2
54
4
chromatography was performed using silica with a grain size of 40–63 µm from Macherey-Nagel.
Deuterated chloroform was purchased from Deutero. NMR spectra were recorded on Bruker 300
1
Fourier, Bruker AV 300 and Bruker AV 400 spectrometers. The chemical shifts (
δ
) for H in CDCl3
are given in parts per million (ppm) and referenced to 7.26, respectively. Coupling constants are
expressed in Hertz (Hz). The following abbreviations are used: s= singlet, d= doublet, t= triplet, q=
quadruplet, m= multiplet.
4.2. Synthesis of inhibitors
3-(Hydroxymethyl)pyridin-2-ol (2)
2-Hydroxynicotinic acid (1) (1.00 g, 7.19 mmol) was suspended in dry THF (100 mL). The
suspension was cooled to 0 °C and LiAlH (0.655 g, 17.3 mmol) was added. The mixture was
4
refluxed for 2 h, then cooled to room temperature and poured onto ice. To the mixture 10% HCl
was added (pH=1), then NaHCO was added (pH=8). The volatiles were evaporated. The crude
3

solid was washed with EtOAc (5×100 mL). The combined organic layer was evaporated to give 2
1
(
0.720 g, 80%) as a white solid. H NMR (400 MHz, DMSO-d , δ): 4.28 (s, 2H), 4.99 (br s, 1H),
6
6.19 (t, J = 6.6 Hz, 1H), 7.22–7.27 (m, 1H), 7.38–7.43 (m, 1H), 11.4 (br s, 1H).
2-Hydroxynicotinaldehyde (3)
O
N
OH
3-(Hydroxymethyl)pyridin-2-ol (2) (1.50 g, 12.0 mmol) was suspended in EtOAc (150 mL). To the
suspension MnO (10.4 g, 120 mmol) was added. The resulting suspension was stirred at 50 °C for
2
18 h. The mixture was filtered through celite pad and celite was washed with EtOAc. The solvent
1
was evaporated to yield 3 (1.03 g, 70%) as a yellow solid. H NMR (400 MHz, DMSO-d , δ): 6.37
6
(t, J = 6.7 Hz, 1H), 7.82 (dd, J = 6.3, 2.4 Hz, 1H), 7.98 (dd, J = 7.1, 2.3 Hz, 1H), 10.09 (d, J = 0.7
Hz, 1H), 12.3 (br s, 1H).
3-Formylpyridin-2-yl methanesulfonate (4)
2-Hydroxynicotinaldehyde (3) (206 mg, 1.67 mmol) was suspended in dry THF (20 mL). The
suspension was cooled to 0 °C and 57-63% NaH on mineral oil (240 mg, 6.0 mmol) was added. The
mixture was stirred at 0 °C under argon for 15 min. To the mixture mesyl chloride (0.65 mL, 8.40
mmol) was added. The resulting mixture was stirred at room temperature under argon for 16 h. To
the mixture sat. NH Cl (15 mL) was added. The mixture was extracted with EtOAc (3×25 mL). The
4
organic layer was dried over Na SO and the volatiles were evaporated. The crude was purified by
2
4
reversed phase column chromatography (sorbent C-18 modified silica gel, eluent MeCN/H O
2
-1
gradient) to yield 4 (100 mg, 30%) as a yellow oil. IR (KBr, cm ) υmax: 1707 (C=O), 1328 (S=O),
1
1
7
4
2
153 (S=O). H NMR (400 MHz, DMSO-d , δ): 3.86 (s, 3H), 6.61 (t, J = 7.0 Hz, 1H), 8.14 (dd, J =
6
13
.0, 2.0 Hz, 1H), 8.26 (dd, J = 7.0, 2.0 Hz, 1H), 10.08 (s, 1H). C NMR (100 MHz, DMSO-d , δ):
6
+
1.5, 106.4, 126.5, 138.7, 145.1, 160.1, 188.1. HRMS-ESI (m/z) calcd for C H NO S [M] :
7
7
4
01.1960. Found: 201.1957.
[
1,2]Oxathiino[6,5-b]pyridine 2,2-dioxide (5)

3
-Formylpyridin-2-yl methanesulfonate (4) (147 mg, 0.731 mmol) and K CO (131 mg, 0.950
2 3
mmol) were suspended in dry DMF (5 mL). The suspension was stirred at room temperature under
argon for 1 h. To the mixture 10% HCl was added (pH=1). The resulting mixture was extracted
with EtOAc (3×20 mL). The organic layer was washed with H O (3×20 mL) and brine (1×20 mL),
2
dried over Na SO . The volatiles were evaporated. The crude was dissolved in dry CH Cl (5 mL).
2
4
2
2
The solution was cooled to 0 °C. To the solution mesyl chloride (0.08 mL, 1.03 mmol) and NEt3
0.14 mL, 1.01 mmol) were added. The resulting mixture was stirred at room temperature under
(
argon for 3 h. The volatiles were evaporated. The crude was purified by reversed phase column
chromatography (sorbent C-18 modified silica gel, eluent MeCN/H O gradient) to yield 5 (54 mg,
2
-1
1
4
0%) as a light pink solid. Mp 103–105 °C. IR (KBr, cm ) υ : 1378 (S=O), 1169 (S=O). H NMR
max
(
400 MHz, DMSO-d , δ): 7.57 (dd, J = 7.6, 4.9 Hz, 1H), 7.68 (d, J = 10.3 Hz, 1H), 7.81 (d, J = 10.3
6
1
3
Hz, 1H), 8.26 (dd, J = 7.6, 1.9 Hz, 1H), 8.47 (dd, J = 4.9, 1.9 Hz, 1H). C NMR (100 MHz,
DMSO-d , δ): 114.3, 123.3, 123.5, 134.9, 140.2, 150.1, 155.5. Anal. Calcd for C H NO S: C,
6
7
5
3
45.90; H, 2.75; N, 7.65. Found: C, 45.99; H, 2.84; N, 7.55.
5-Bromo-3-(hydroxymethyl)pyridin-2-ol (7)
5-Bromo-2-hydroxynicotinic acid (6) (2.00 g, 9.17 mmol) was suspended in dry THF (100 mL).
The suspension was cooled to 0 °C and 1M BH /THF (36.7 mL, 36.7 mmol) was added. The
3
mixture was stirred at room temperature under argon for 18 h. To the mixture 10% HCl was added
(pH=1) and the resulting mixture was refluxed for 1 h. The volatiles were evaporated. The crude
was purified by reversed phase column chromatography (sorbent C-18 modified silica gel, eluent
1
MeCN/H O gradient) to yield 7 (0.993 g, 53%) as a white solid. H NMR (300 MHz, DMSO-d , δ):
2
6
4.26-4.29 (m, 2H), 7.41–7.44 (m, 1H), 7.52–7.55 (m, 1H), 11.82 (br s, 1H).
5-Bromo-2-hydroxynicotinaldehyde (8)
5-Bromo-3-(hydroxymethyl)pyridin-2-ol (7) (0.820 g, 4.02 mmol) was suspended in EtOAc (100
mL). To the suspension MnO (3.49 g, 40.2 mmol) was added. The suspension was stirred at 60 °C
2
for 18 h. The resulting mixture was filtered through celite pad and celite was washed with EtOAc.
1
The solvent was evaporated to yield 8 (0.432 g, 53%) as a yellow solid. H NMR (300 MHz,
DMSO-d , δ): 7.97 (d, J = 3.0 Hz, 1H), 8.12 (d, J = 3.0 Hz, 1H), 10.01 (s, 1H), 12.7 (br s, 1H).
6

6-Bromo-[1,2]oxathiino[6,5-b]pyridine 2,2-dioxide (10)
5-Bromo-2-hydroxynicotinaldehyde (8) (1.39 g, 6.88 mmol) was dissolved in dry DMF (54 mL).
The solution was cooled to 0 ºC and 57–63% NaH on mineral oil (0.339 g, 8.26 mmol) was added.
The mixture was stirred under argon at 0 ºC for 10 min. To the mixture mesyl chloride (0.64 mL,
8.26 mmol) was added. The resulting mixture was stirred at room temperature for 25 min. To the
mixture K CO (1.23 g, 8.90 mmol) was added. The resulting suspension was stirred at room
2
3
temperature under argon for 75 min. The mixture was poured into 10% HCl (pH=1) and extracted
with EtOAc (3×50 mL). The organic layer was washed with H O (2×40 mL) and sat. NaCl (1×40
2
mL) and dried over Na SO . The volatiles were evaporated. The crude was dissolved in dry DCM
2
4
(
15 mL). The solution was cooled to 0 ºC and NEt (1.44 mL, 10.3 mmol) and mesyl chloride (0.80
3
mL, 10.3 mmol) were added. The resulting mixture was stirred at room temperature under argon for
h. To the mixture H O (10 mL) was added and the resulting mixture was extracted with EtOAc
3
2
(
3×20 mL). The organic layer was dried over Na SO and the volatiles were evaporated. The crude
2 4
was purified by reversed phase column chromatography (sorbent C-18 modified silica gel, eluent
-1
MeCN/H O gradient) to yield 10 (1.04 g, 58%) as a yellow solid. Mp 164–166 °C. IR (neat, cm )
2
1
υmax: 1366 (S=O), 1177 (S=O), 1114 (S=O). H NMR (400 MHz, DMSO-d , δ): 7.74 (d, J = 10.3
6
1
3
Hz, 1H), 7.78 (d, J = 10.3 Hz, 1H), 8.56 (d, J = 2.5 Hz, 1H), 8.62 (d, J = 2.5 Hz, 1H). C NMR
100 MHz, DMSO-d , δ): 115.8, 117.3, 124.4, 133.9, 142.0, 150.4, 154.4. Anal. Calcd for
(
6
C H BrNO S: C, 32.08; H, 1.54; N, 5.34. Found: C, 32.58; H, 1.80; N, 5.41.
7
4
3
General procedure for the synthesis of 6-aryl-[1,2]oxathiino[6,5-b]pyridine 2,2-dioxides
0 (200 mg, 0.763 mmol), appropriate organotin derivative (1.3 equiv.), tri(2-furyl)phosphine (71
1
mg, 0.305 mmol) and Pd(OAc) (34 mg, 0.151 mmol) were suspended in dry 1,4-dioxane (0.7 mL).
2
The suspension was stirred under argon at 100 ºC for 18 h. To the mixture EtOAc (5 mL) was
added and the resulting mixture was filtrated through celite pad and the volatiles were evaporated.
The crude was purified by reversed phase column chromatography (sorbent C-18 modified silica
gel, eluent MeCN/H O gradient).
2
6-(4-Methoxyphenyl)-[1,2]oxathiino[6,5-b]pyridine 2,2-dioxide (11)

Obtained according to the general procedure from tributyl(4-methoxyphenyl)stannane (394 mg,
0.992 mmol). The crude was recrystallized from EtOH to yield 11 (133 mg, 60%) as a yellow solid.
1
Mp 165–166 °C. H NMR (400 MHz, DMSO-d , δ): 3.82 (s, 3H), 7.07–7.12 (m, 2H), 7.69–7.74 (m,
6
1
3
3
H), 7.81 (d, J = 10.3 Hz, 1H), 8.52 (d, J = 2.5 Hz, 1H), 8.72 (d, J = 2.5 Hz, 1H). C NMR (100
MHz, DMSO-d , δ): 55.3, 114.1, 114.7, 123.8, 127.4, 128.3, 134.9, 135.0, 137.3, 147.2, 154.3,
6
159.9. Anal. Calcd for C H NO S: C, 58.12; H, 3.83; N, 4.84. Found: C, 58.15; H, 3.81; N, 4.70.
14 11 4
6-(3-Methoxyphenyl)-[1,2]oxathiino[6,5-b]pyridine 2,2-dioxide (12)
Obtained according to the general procedure from tributyl(3-methoxyphenyl)stannane (394 mg,
0.992 mmol). The crude was recrystallized from EtOH to yield 12 (150 mg, 68%) as a yellow solid.
1
Mp 151–152 °C. H NMR (400 MHz, DMSO-d , δ): 3.84 (s, 3H), 7.02–7.06 (m, 1H), 7.31–7.35 (m,
6
2
1
1
5
H), 7.42–7.48 (m, 1H), 7.73 (d, J = 10.3 Hz, 1H), 7.82 (d, J = 10.3 Hz, 1H), 8.59 (d, J = 2.5 Hz,
1
3
H), 8.79 (d, J = 2.5 Hz, 1H). C NMR (100 MHz, DMSO-d , δ): 55.3, 112.5, 114.0, 114.4, 119.2,
6
23.9, 130.4, 134.9, 135.0, 136.5, 138.1, 147.9, 154.8, 159.9. Anal. Calcd for C H NO S: C,
1
4
11
4
8.12; H, 3.83; N, 4.84. Found: C, 58.12; H, 3.79; N, 4.72.
6-(4-(tert-Butyl)phenyl)-[1,2]oxathiino[6,5-b]pyridine 2,2-dioxide (13)
Obtained according to the general procedure from tributyl(4-(tert-butyl)phenyl)stannane (420 mg,
1
0
.992 mmol) as yellow needles (144 mg, 60%). Mp 212–214 °C. H NMR (400 MHz, DMSO-d ,
6
δ): 1.32 (s, 9H), 7.53–7.58 (m, 2H), 7.67–7.72 (m, 2H), 7.73 (d, J = 10.3 Hz, 1H), 7.84 (d, J = 10.3
1
3
Hz, 1H), 8.56 (d, J = 2.5 Hz, 1H), 8.76 (d, J = 2.5 Hz, 1H). C NMR (100 MHz, DMSO-d , δ):
6
31.0, 34.4, 114.1, 123.9, 126.1, 126.7, 132.3, 135.0, 135.1, 137.7, 147.6, 151.3, 154.6. Anal. Calcd
for C H NO S: C, 64.74; H, 5.43; N, 4.44. Found: C, 64.54; H, 5.73; N, 4.43.
1
7
17
3

6-(4-Chlorophenyl)-[1,2]oxathiino[6,5-b]pyridine 2,2-dioxide (14)
Obtained according to the general procedure from tributyl(4-chlorophenyl)stannane (398 mg, 0.992
mmol). The crude was recrystallized from EtOH to yield 14 (115 mg, 51%) as an orange solid. Mp
1
2
03–205 °C. H NMR (400 MHz, DMSO-d , δ): 7.58–7.64 (m, 2H), 7.74 (d, J = 10.2 Hz, 1H),
6
1
3
7
.79–7.84 (m, 3H), 8.60 (d, J = 2.5 Hz, 1H), 8.79 (d, J = 2.5 Hz, 1H). C NMR (100 MHz, DMSO-
d6, δ): 114.1, 124.0, 128.8, 129.2, 133.7, 133.9, 134.0, 134.9, 138.1, 147.8, 154.9. Anal. Calcd for
C H ClNO S: C, 53.16; H, 2.75; N, 4.77. Found: C, 53.06; H, 2.80; N, 4.64.
13
8
3
6-Phenyl-[1,2]oxathiino[6,5-b]pyridine 2,2-dioxide (15)
Obtained according to the general procedure from tributyl(phenyl)stannane (364 mg, 0.992 mmol).
The crude was recrystallized from EtOH to yield 15 (145 mg, 73%) as a yellow solid. Mp 187–188
1
°
C. H NMR (400 MHz, DMSO-d , δ): 7.44–7.50 (m, 1H), 7.51–7.57 (m, 2H), 7.74 (d, J = 10.3 Hz,
6
1
1
1
3
H), 7.76–7.79 (m, 2H), 7.84 (d, J = 10.3 Hz, 1H), 8.59 (d, J = 2.5 Hz, 1H), 8.78 (d, J = 2.5 Hz,
1
3
H). C NMR (100 MHz, DMSO-d , δ): 114.1, 123.9, 127.0, 128.7, 129.3, 134.9, 135.1, 135.2,
6
38.0, 147.8, 154.8. Anal. Calcd for C H NO S: C, 60.22; H, 3.50; N, 5.40. Found: C, 60.02; H,
1
3
9
3
.43; N, 5.27.
6-(Naphthalen-1-yl)-[1,2]oxathiino[6,5-b]pyridine 2,2-dioxide (16)
Obtained according to the general procedure from tributyl(naphthalen-1-yl)stannane (414 mg, 0.992
mmol). The crude was crystallized from Et O to yield 16 (96 mg, 41%) as a yellow solid. Mp 148–
2
1
1
50 °C. H NMR (400 MHz, DMSO-d , δ): 7.53–7.67 (m, 4H), 7.75–7.81 (m, 2H), 7.87 (d, J = 10.3
6
13
Hz, 1H), 8.04–8.09 (m, 2H), 8.45 (d, J = 2.4 Hz, 1H), 8.58 (d, J = 2.4 Hz, 1H). C NMR (100
MHz, DMSO-d , δ): 113.8, 123.8, 124.7, 125.6, 126.4, 127.1, 127.9, 128.5, 129.0, 130.7, 133.4,
6

1
33.8, 134.9, 135.0, 141.0, 150.0, 154.8. Anal. Calcd for C H NO S: C, 66.01; H, 3.58; N, 4.53.
17 11 3
Found: C, 65.79; H, 3.90; N, 4.51.
6-(3,5-Dichlorophenyl)-[1,2]oxathiino[6,5-b]pyridine 2,2-dioxide (17)
Obtained according to the general procedure from (3,5-dichlorophenyl)trimethylstannane (307 mg,
0.992 mmol). The crude was recrystallized from EtOH to yield 17 (116 mg, 46%) as a yellow solid.
1
Mp 220–222 °C. H NMR (400 MHz, DMSO-d , δ): 7.71 (t, J = 1.9 Hz, 1H), 7.75 (d, J = 10.3 Hz,
6
1H), 7.78 (d, J = 10.3 Hz, 1H), 7.90 (d, J = 1.9 Hz, 2H), 8.68 (d, J = 2.5 Hz, 1H), 8.86 (d, J = 2.5
1
3
Hz, 1H). C NMR (100 MHz, DMSO-d , δ): 114.0, 124.1, 125.8, 128.1, 132.3, 134.7, 134.9, 138.6,
6
1
2
38.7, 148.3, 155.3. Anal. Calcd for C H Cl NO S: C, 47.58; H, 2.15; N, 4.27. Found: C, 47.45; H,
13 7 2 3
.02; N, 4.29.
6-([1,1'-Biphenyl]-4-yl)-[1,2]oxathiino[6,5-b]pyridine 2,2-dioxide (18)
Obtained according to the general procedure from [1,1'-biphenyl]-4-yltrimethylstannane (314 mg,
0.992 mmol). The crude was recrystallized from EtOH to yield 18 (112 mg, 44%) as a brown solid.
1
Mp 239–240 °C. H NMR (400 MHz, DMSO-d , δ): 7.37–7.43 (m, 1H), 7.47–7.53 (m, 2H), 7.72–
6
1
3
7
.78 (m, 3H), 7.82–7.91 (m, 5H), 8.65 (d, J = 2.5 Hz, 1H), 8.84 (d, J = 2.5 Hz, 1H). C NMR (100
MHz, DMSO-d , δ): 114.1, 123.9, 126.7, 127.4, 127.5, 127.8, 129.0, 134.1, 134.6, 134.9, 137.9,
6
139.2, 140.4, 147.6, 154.8. Anal. Calcd for C H NO S: C, 68.05; H, 3.91; N, 4.18. Found: C,
19 13 3
67.84; H, 4.20; N, 4.16.
6-(4-(Trifluoromethoxy)phenyl)-[1,2]oxathiino[6,5-b]pyridine 2,2-dioxide (19)

Obtained according to the general procedure from tributyl(4-(trifluoromethoxy)phenyl)stannane
(448 mg, 0.992 mmol). The crude was recrystallized from EtOH to yield 19 (131 mg, 50%) as a
1
yellow solid. Mp 143–144 °C. H NMR (400 MHz, DMSO-d , δ): 7.52–7.57 (m, 2H), 7.75 (d, J =
6
1
2
1
0.3 Hz, 1H), 7.83 (d, J = 10.3 Hz, 1H), 7.89–7.93 (m, 2H), 8.61 (d, J = 2.5 Hz, 1H), 8.80 (d, J =
1
3
.5 Hz, 1H). C NMR (100 MHz, DMSO-d , δ): 114.1, 120.0 (q, J = 256.6 Hz), 121.7, 124.0,
6
29.1, 133.8, 134.5, 134.8, 138.3, 148.0, 148.7 (q, J = 1.9 Hz), 155.0. Anal. Calcd for
C H F NO S: C, 48.99; H, 2.35; N, 4.08. Found: C, 48.78; H, 2.32; N, 3.92.
14
8
3
4
6-(4-(Trifluoromethyl)phenyl)-[1,2]oxathiino[6,5-b]pyridine 2,2-dioxide (20)
Obtained according to the general procedure from tributyl(4-(trifluoromethyl)phenyl)stannane (432
mg, 0.992 mmol). The crude was recrystallized from EtOH to yield 20 (72 mg, 29%) as a yellow
1
solid. Mp 185–187 °C. H NMR (400 MHz, DMSO-d , δ): 7.76 (d, J = 10.3 Hz, 1H), 7.85 (d, J =
6
10.3 Hz, 1H), 7.89–7.93 (m, 2H), 7.99–8.03 (m, 2H), 8.68 (d, J = 2.5 Hz, 1H), 8.86 (d, J = 2.5 Hz,
1
3
1
H). C NMR (100 MHz, DMSO-d , δ): 114.2, 124.0, 124.2 (q, J = 272.0 Hz), 126.1 (q, J = 3.9
6
Hz), 127.9, 129.0 (q, J = 32.0 Hz), 133.6, 134.8, 138.6, 139.3 (q, J = 1.2 Hz), 148.3, 155.3. Anal.
Calcd for C H F NO S: C, 51.38; H, 2.46; N, 4.28. Found: C, 51.07; H, 2.74; N, 4.25.
1
4
8
3
3
4.3. CA inhibition
An Applied Photophysics stoppedflow instrument has been used for assaying the CA catalysed CO2
hydration activity. 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 Hepes (pH 7.5) as buffer, and 20 mM Na SO4
2
(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 CO concentrations ranged from 1.7 to 17 mM for
2
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
0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were
preincubated together for 6 h at room temperature prior to assay, in order to allow for the formation
of the E–I complex. Data from Table 1 were obtained after 6 h incubation of enzyme and inhibitor,
as for coumarins reported earlier[2, 3, 27, 28, 30, 49-52]. The inhibition constants were obtained by

non-linear leastsquares methods using PRISM 3, as reported earlier[53-56], and represent the mean
from at least three different determinations. All CA isofoms were recombinant ones obtained in-
house as reported earlier[2, 3, 27, 28, 30, 49-52, 57-59].
4.4. In vitro viability
Cells were seeded on a 96-well plate in DMEM medium supplemented with 10% fetal bovine
serum and 2 mM glutamine and growth 24 h. Then the tested compound was added and cultivated
in thermostat 48 hours. Cell viability was measured by colouring with MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). After incubation with tested compound the
medium was replaced with new one containing 0.2 mg/ml MTT. After incubated 3 hours in
thermostat at 37 °C with 5% CO , removal of medium, the dye was extracted with 0.2 ml DMSO.
2
Optical density (OD) determine using multichannel spectrophotometer (Tecan multiplate reader
Infinite100) at 540 nm. Then calculate IC – the concentration of tested compound providing the
5
0
death of 50% of cells by the usage of program Graph Pad Prism® 3.0.
Supplementary data
Can be found on...
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the European Regional Development Fund (ERDF, project no.
1.1.1.2/VIAA/1/16/235).
Author Information
ORCID
Aiga Grandane: 0000-0002-9563-131X
Alessio Nocentini: 0000-0003-3342-702X
Ilona Domračeva: 0000-0003-3622-6178
Raivis Zalubovskis: 0000-0002-9471-1342
Claudiu T. Supuran: 0000-0003-4262-0323

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Highlights
•
•
•
The facile synthesis of oxathiino[6,5-b]pyridine 2,2-dioxides (OPDs) was developed.
OPDs are selectively and effectively inhibitors of CA IX and CA IX.
Compound 17 have good antiproliferative properties on MCF-7, A549 and HeLa cells.

Declaration of interest statement
Dear Editor,
the authors have declared no conflicts of interest.
Prof. Raivis Žalubovskis