7-Substituted-sulfocoumarins are isoform-selective, potent carbonic anhydrase II inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.05.032

A series of 7-substituted sulfocoumarins and 3,4-dihydrosulfocoumarins was obtained by cyclization of the methanesulfonate of 2,4-dihydroxy- or 2-hydroxy-4-methoxybenzaldehyde, followed by derivatization reactions. The new compounds incorporate a range of substituents in position 7 of the heterocyclic ring (hydroxyl, methoxy, carboxylic and alkylsulfonate ester). The compounds were tested for the inhibition of the zinc enzyme human (h) carbonic anhydrase (hCA, EC 4.2.1.1). Unlike the 6-substituted sulfocoumarins which were potent hCA IX and XII inhibitors and ineffective hCA I and II inhibitors, compounds from this series showed low nanomolar hCA II inhibitory properties, and inhibited the mitochondrial isoform hCA VA with K_Is in the range of 91-9960 nM, but were ineffective as hCA I, IX and XII inhibitors. The structure activity relationship for this class of inhibitors was rather clear, with the nature of the 7-substituent strongly influencing hCA VA inhibition, whereas the nature of these groups were less relevant for hCA II inhibition (all reported compounds were highly effective hCA II inhibitors, with K_Is in the range of 1.5-8.4 nM). Since both hCA II and hCA VA are important drug targets (hCA II for antiglaucoma agents; hCA VA for antiobesity drugs), these isoform-selective inhibitors reported here may be considered of interest for various biomedical applications.

Full text of DOI:10.1016/j.bmc.2013.05.032

Accepted Manuscript
7-Substituted-sulfocoumarins are isoform-selective, potent carbonic anhydrase
II inhibitors
Muhammet Tanc, Fabrizio Carta, Murat Bozdag, Andrea Scozzafava, Claudiu
T. Supuran
PII:
S0968-0896(13)00481-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.05.032
BMC 10858
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
5 April 2013
14 May 2013
21 May 2013
Please cite this article as: Tanc, M., Carta, F., Bozdag, M., Scozzafava, A., Supuran, C.T., 7-Substituted-
sulfocoumarins are isoform-selective, potent carbonic anhydrase II inhibitors, Bioorganic & Medicinal Chemistry
(2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.05.032
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7-Substituted-sulfocoumarins are isoform-selective, potent carbonic anhydrase II inhibitors
Muhammet Tanc,a,b Fabrizio Cartab, Murat Bozdag,b Andrea Scozzafava,b and Claudiu T. Supuran*a,b
aUniversità degli Studi di Firenze, NEUROFARBA Department, Section of Pharmaceutical
Chemistry, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence); Italy.
b
Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm.
188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence); Italy.
Abstract: A series of 7-substituted sulfocoumarins and 3,4-dihydrosulfocoumarins was obtained by
cyclization of the methanesulfonate of 2,4-dihydroxy- or 2-hydroxy-4-methoxybenzaldehyde,
followed by derivatization reactions. The new compounds incorporate a range of substituents in
position 7 of the heterocyclic ring (hydroxyl, methoxy, carboxylic and alkylsulfonate ester). The
compounds were tsted for the inhibition of the zinc enzyme human (h) carbonic anhydrase (hCA,
EC 4.2.1.1). Unlike the 6-substituted sulfocoumarins which were potent hCA IX and XII inhibitors
and ineffective hCA I and II inhibitors, compounds from this series showed low nanomolar hCA II
inhibitory properties, and inhibited the mitochondrial isoform hCA VA with KIs in the range of 91 –
9960 nM, but were ineffective as hCA I, IX and XII inhibitors. The structure activity relationship
for this class of inhibitors was rather clear, with the nature of the 7-substituent strongly influencing
hCA VA inhibition, whereas the nature of these groups were less relevant for hCA II inhibition (all
reported compounds were highly effective hCA II inhibitors, with KIs in the range of 1.5 – 8.4 nM).
Since both hCA II and hCA VA are important drug targets (hCA II for antiglaucoma agents; hCA
VA for antiobesity drugs), these isoform-selective inhibitors reported here may be considered of
interest for various biomedical applications.
Keywords: carbonic anhydrase; sulfocoumarin; coumarin; sulfonamide; isoform-selective inhibitor
______
*Correspondence author. Phone: +39-055-4573005; Fax: +39-055-4573385; E-mail:
claudiu.supuran@unifi.it

1. Introduction
Sulfocoumarins (1,2-benzoxathiine 2,2-dioxides) were recently reported to act as efficient
inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) and to possess a novel
mechanism of inhibition.1 Similar to coumarins,2-4 which were used as lead compounds for their
design as CA inhibitors (CAIs) – Chart 1, the sulfocoumarins undergo a CA-mediated hydrolysis
leading to 2-hydroxyphenyl--ethenylsulfonic acids which thereafter bind to the zinc-coordinated
water molecule from the enzyme active site,1 as shown in Fig. 1. In this last step of the inhibition
mechanism, sulfocoumarins act diversely from the isostructural coumarins, which generate 2-
hydroxy-cinnamic acid derivatives, which neither coordinate to the zinc ion (as the sulfonamide
inhibitors),1 nor anchor to the water molecule coordinated to it, but occlude the entrance to the
enzyme active site.2-4
Fig. 1 and Chart 1 here
It should be noted that the sulfocoumarins represent just the last chemotype shown to
possess significant CA inhibitory properties, rationally designed by considering the coumarins as
lead molecules (Chart 1). Indeed, in the last several years a rather large number of new classes of
CAIs were reported starting from coumarins as leads, among which the thiocoumarins, 2-thioxo-
coumarins, coumarin oximes, 5-/6-membered /thio)lactones, etc.4-6 Many of these new classes of
CAIs showed a high degree of selectivity for inhibiting various mammalian CA isoforms, of the 16
presently known.7,8 Indeed, the main problem of the sulfonamide/sulfamate clinically used CAIs is
represented by the fact that most of them indiscriminately inhibit most of these isoforms, leading
thus to deleterious side effects. 7-9 The sulfonamide/sulfamates CAIs are used for the treatment of
glaucoma, obesity, epilepsy, and as diuretics. 7-9 Recently, significant antitumor and antimetastatic
effects were reported for many CAIs specifically targeting the tumor-associated isoforms CA IX
and XII.10,11 It should be stressed here that these very different pharmacological applications of the
CAIs are due to the fact that different isoforms are responsible for diverse biological functions: CA
II and XII are targeted by the antiglaucoma such drugs, CA II, IV and XIV for the diuretic ones, CA
VII and XIV for the antiepileptic ones, and as mentioned above, CA IX and XII for the
antitumor/antimetastatic effects.7-11 It is thus highly desirable to develop selective inhibitors for all
these isoforms, which has been a challenge for a long period, when only sulfonamides (and their
bioisosteres) were known as effective classes of CAIs. However, this situation changed dramatically
in the last several years, with the report of a rather large number of new chemotypes acting as
efficient and isoform-selective CAIs. In addition to the coumarins and the compounds developed

from them shown in Chart 1, one can mention the polyamines,12 the phenols13 and the
dithiocarbamates14 as novel, interesting classes of CAIs. Many of these compounds possess
inhibition mechanisms distinct of those of the sulfonamides (or coumarins), as determined by X-ray
crystallography of enzyme-inhibitor adducts.2,7,12-14
In the previous work1 we have primarily explored sulfocoumarins possessing various
substituents in the 6 position of the heterocyclic ring. As for the coumarins,1-4 also for
sulfocoumarins the substitution pattern and especially the position of the substituent on the
heterocyclic ring system, are the main factors influencing CA inhibitory properties. In this paper we
report the synthesis and explore CA inhibition with a series of sulfocoumarins possessing various
substituents in the 7 position of the ring.
2. Results and Discussion
2.1. Chemistry. In a recent paper we investigated a rather large series of sulfocoumarins
incorporating simple functionalities in the 6 position of the ring, such as hydroxyl, methanesulfonyl,
benzyloxy, bromo, nitro, amino and azido, as well as the 5,6-benzo- and 6,8-dichloro-substituted
derivatives. By using click chemistry on 6-azido-sulfocoumarin which was reacted with various
alkynes, a large series of 6-substituted-1,2,3-triazolo-sulfocoumarins were also obtained.1 These
derivatives (most of which were 6-substituted-sulfocoumarins, except for the two compounds with a
diverse substitution pattern mentioned above)1 were generally ineffective as inhibitors of the
cytosolic isoforms hCA I and II, but some of them were highly potent inhibitors of the
transmembrane, tumnor-associated isoforms hCA IX and XII.1 The high resolution X-ray crystal
structure of a mutant hCA II (in which some residues present in the hCA IX active site were
introduced into hCA II) in complex with 6-bromosulfocoumarin also allowed us to understand the
inhibition mechanism by this new class of CAIs, which has been discussed in the Introduction (see
Fig. 1). Considering the very interesting inhibition profile of the four investigated CA isoforms (CA
I, II, IX and XII) with these 6-substituted sulfocoumarins, it appeared of interest to further explore
other substitution patterns on this ring system. In this paper we report two structural modifications
in the sulfocoumarin scaffold: (i) a series of compounds were prepared which incorporate various
moieties in the 7 position of the sulfocoumarin ring (as compounds possessing this substitution
pattern were not yet investigated as CAIs), and (ii) we have also explored what are the
consequences on the CA inhibitory effects if the double bond between the C3 and C4 atoms is

reduced in the sulfocoumarin scaffold (i.e., we investigated the CA inhibitory properties of 3,4-
dihydrosulfocoumarins substituted in the 7 position of the heterocyclic ring).
Schemes 1-3 here
The general strategy of Zalubovskis’s group1,15 for the preparation of 6-substituted sulfocoumarins
was applied by us for preparing 7-substituted such derivatives (Scheme 1-3). Reaction of 2,4-
dihydroxybenzaldehyde with one equivalent of benzyl bromide afforded the benzyl derivative 1
which was treated with methanesulfonyl chloride followed by cyclization of the sulfonate 2, in the
presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), with formation of the racemic 4-hydroxy-7-
benzyloxy-3,4-dihydrosulfocoumarin 3 (Scheme 1). The alcohol 3 was dehydrated in the presence
of mesityl chloride leading to 7-benzyloxy-sulfocoumarin 4, which by hydrogenation, depending on
the pressure of H2 used, was transformed in the benzyloxy-dihydrosulfocoumarin derivative 5 or the
debenzylated dihydrosulfocoumarin 6 which has been transformed to the corresponding
methanesulfonate 6a by reaction with the sulfonyl chloride (Scheme 1). The key intermediate 3 was
deprotected at the 7 position by hydrogenation, with formation of the 7-hydroxy derivative 7, which
by dehydration (in the same conditions as mentioned above for the transformation of 3 to 4) and
alkylsulfonylation of the phenol group led to 8. Hydrolysis of the methylsulfonate ester 8 in the
presence of tetraethylammonium hydroxide led to the formation of 7-hydroxysulfocoumarin 9
(Scheme 1). A similar strategy has been applied by using 2-hydroxy-4-methoxy benzaldehyde,
which led after several steps, to the formation of the 7-methoxy-sulfocoumarin 12 and the
corresponding dihydro derivative 13 (Scheme 2).
In order to create chemical diversity, the 7-hydroxy-3,4-dihydrosulfocoumarin 6 and 7-hydroxy-
sulfocoumarin 9 were acylated with alkyl- ar arylalkyl acyl chlorides, leading to the esters 14-16
and 17-19, respectively (Scheme 3).
Table 1 here
2.2. CA inhibition. Sulfocoumarins and their derivatives of types 3-19 reported here were assayed
as inhibitors of five physiologically relevant CA isoforms, the cytosolic hCA I and II (h = human
isoform), the mitochondrial hCA VA, and the transmembrane, tumor-associated hCA IX and XII
(Table 1). The sulfonamide in clinical use acetazolamide (AAZ) has been used as standard in these
measurements, for comparison reasons. The following should be noted on the CA inhibitory
properties of the compounds 3-19 reported here:

(i) Three of the investigated isoforms, i.e., hCA I, IX and XII, were not significantly inhibited by
compounds 3-19 up to 50 M concentration of inhibitor in the assay system. These data are not
very much intriguing regarding hCA I, which was also not inhibited by the 6-substituted
sulfocoumarins reported earlier.1 However, the 6-substututed sulfocoumarins were highly efficient
hCA IX and XII inhibitors,1 unlike the 7-substituted compounds 3-19 reported here, which are not
inhibiting these isozymes (Table 1). This is, we think, a highly interesting result, confirming the fact
that the position of the substituents in the sulfocoumarin ring (as in the case of the coumarins)2-4 is
the main factor influencing the CA inhibitory properties of these compounds. In fact, the nature of
these substituents seemed to be rather irrelevant, as all these derivatives were ineffective as hCA I,
IX and XII inhibitors, irrespective of the fact that they possesses small and compact (OH, OMe) or
bulkier (4-chlorophenylacetyl; 2-bromophenylacetyl, etc.) moieties in the 7 position of the ring.
(ii) Although the 6-substututed sulfocoumarins reported earlier were also ineffective as hCA II
inhibitors,1 the 7-substituted compounds 3-19 reported here were excellent, low nanomolar
inhibitors of this isoform, with inhibition constants in the range of 1.5 – 8.4 nM (Table 1) being thus
much more effective compared to acetazolamide, a clinically used sulfonamide CAI. It should be
mentioned that this type of behavior, i.e., important differences in the CA inhibitory properties
against various CA isoforms of regioisomers, has also been reported by us earlier for coumarin
CAIs. For example, disubstituted coumarins incorporating ether and acetyl/propionyl moieties in
positions 6,7- and 7,8- of the heterocyclic ring differed very much in their inhibition profiles against
hCA I, II, IX and XII.3a Whereas 6,7-disubstituted series showed ineffective inhibition for the
transmembrane tumor-associated isoforms CA IX and XII (and micromolar affinity for hCA I and
II), the corresponding isomeric 7,8-disubstituted coumarins showed nanomolar/subnanomolar
3a
inhibition of CA IX/XII (but were ineffective as hCA I and II inhibitors). Interestingly, the
structure-activity relationship (SAR) for the inhibition of hCA II with compounds 3-19 is very flat,
with all these derivatives possessing inhibition constants in a limited range (1.5 – 8.4 nM). Thus,
the nature of the substituent in position 7 has an influence (but not a very important one) on the
hCA II inhibitory properties. For example, The least “effective” hCA II inhibitors detected here,
compounds 6 and 9, possess a HO moiety in position 7 of the heterocyclic ring, and they showed
KIs of 7.6 – 8.4 nM (being extremely effective as hCA II inhibitors). Derivatives of 6 (such as 14-
16) or 9 (such as 17-19) possessing bulkier moieties at the 7 position (of the ester type) were more
effective hCA II inhibitors compared to the parent phenols 6 and 9, with KIs in the range of 1.5 –
3.4 nM. There were however not big differences of activity between the more compact acetyl esters
(14 and 17) and the bulkier phenyl-substituted acetates 15, 16 and 18, 19, respectively (Table 1).
The same may be observed for the methoxy derivatives 12 and 13 (Table 1), which do not differ

significantly in their hCA II inhibitory properties. The other structural element differentiating
compounds 3-19 reported here, was the presence or the absence of the double bond between
carbons 3 and 4 of the heterocyclic ring. It may be observed that among the least effective hCA II
inhibitors (6 and 9), one compound is saturated (6) whereas the second one has the double bond (9).
Comparing the saturated (14-16) and aromatic (17-19) esters one may again see irrelevant
differences of activity between the two series of compounds. We may thus conclude that the
presence or the absence of the C3-C4 double bond does not significantly influence the hCA II
inhibitory properties of these compounds. Probably both the sulfocoumarins as well as their dihydro
derivatives possess a similar mechanism of hCA II inhibition, as depicted in Fig. 1.
(iii) The mitochondrial isoform CA VA, involved in biosynthetic reactions, and thus a target for
anti-obesity agents,17 was also inhibited by compounds 3-19 reported here, with KIs in the range of
91 nM – 9.96 M (Table 1). The best hCA VA inhibitors were compounds 13, 15 and 16 (KIs in the
range of 91 – 206 nM). They all incorporate the 3,4-dihydrosulfocoumarin scaffold, as well as a
methoxy, 4-chlorophenylacetyl and 2-bromophenylacetayl moiety in position 7 of the heterocycle.
It is interesting to note that the corresponding aromatic compounds, 12, 18 and 19, are much weaker
hCA VA inhibitors (KIs in the range of 3910 – 9960 nM, Table 1). Thus, a very simple feature vin
the scaffold of these compounds drastically influences affinity for the mitochondrial isoform. The
remaining compounds investigated here were medium potency – weak hCA VA inhibitors, with
inhibition constants in the range of 327 – 5090 nM (Table 1). Compound 13, the best hCA VA
inhibitor detected here, showed an inhibition profile against this isoform similar to that of
acetazolamide AAZ.
3. Conclusions
We report here a series of 7-substituted sulfocoumarins and 3,4-dihydrosulfocoumarins, obtained by
cyclization of the methanesulfonate of 2,4-dihydroxy- or 2-hydroxy-4-methoxybenzaldehyde,
possessing a range of substituents in position 7 of the heterocyclic ring. Unlike the 6-substituted
sulfocoumarins investigated earlier, which were potent hCA IX and XII inhibitors and ineffective
hCA I and II inhibitors, compounds from this series showed low nanomolar hCA II inhibitory
properties, inhibited the mitochondrial isoform hCA VA with KIs in the range of 91 – 9960 nM, and
were ineffective as hCA I, IX and XII inhibitors. The structure activity relationship for this class of
CAIs was rather well defined (at least for hCA II and VA), with the nature of the 7-substituent

strongly influencing hCA VA inhibition, whereas the nature of these groups were less relevant for
hCA II inhibition (all the reported compounds were highly effective hCA II inhibitors, with KIs in
the range of 1.5 – 8.4 nM). Since both hCA II and hCA VA are important drug targets (hCA II for
obtaining antiglaucoma agents; hCA VA for antiobesity drugs), these isoform-selective CAIs
reported here may be considered of interest for various biomedical applications.
4. Experimental
4.1. Chemistry
Anhydrous solvents and all reagents were purchased from Sigma-Aldrich, Alfa Aesar and TCI. All
reactions involving air- or moisture-sensitive compounds were performed under a nitrogen
atmosphere using dried glassware and syringes techniques to transfer solutions. Nuclear magnetic
resonance (1H-NMR, 13C-NMR, DEPT-135, DEPT-90, HSQC, HMBC) spectra were recorded
using a Bruker Advance III 400 MHz spectrometer in DMSO-d6. Chemical shifts are reported in
parts per million (ppm) and the coupling constants (J) are expressed in Hertz (Hz). Splitting patterns
are designated as follows: s, singlet; d, doublet; sept, septet; t, triplet; q, quadruplet; m, multiplet;
brs, broad singlet; dd, double of doublet,appt, aparent triplet, appq, aparent quartet, brt broad triplet.
The assignment of exchangeable protons (OH and NH) was confirmed by the addition of D2O.
Analytical thin-layer chromatography (TLC) was carried out on Merck silica gel F-254 plates. Flash
chromatography purifications were performed on Merck Silica gel 60 (230-400 mesh ASTM) as the
stationary phase and ethylacetate/n-hexane were used as eluents. Melting points (m.p.) were carried
out in open capillary tubes and are uncorrected.
Synthesis of 4-benzyloxy-2-hydroxy-benzaldehyde 1
O
O
BnBr, NaHCO
ACN; quant.
3
HO
OH
BnO
OH
1
2,4-Dihydroxybenzaldehyde (3.0 g, 1.0 eq) was dissolved in acetonitrile (220 ml) and treated with
NaHCO3 (2.2 eq) for 45 min at r.t. followed by the addition of benzylbromide (1.1 eq). The reaction

mixture was stirred under reflux O.N. and then quenched with H2O (100 ml) and extracted with
ethyl acetate (3 x 25 ml). The combined organic layers were washed with brine (2 x 15 ml), dried
over Na2SO4, filtered and the solvent was eliminated under vacuo to give the title compound 12 as a
white solid that was used as it is.
4-Benzyloxy-2-hydroxy-benzaldehyde 1: quantitative; silica gel TLC Rf 0.65 (Ethyl acetate/n-
hexane 20 % v/v); δH (400 MHz, DMSO-d6): 5.21 (s, 2H), 6.60 (d, J 2.2, 1H), 6.68 (dd, J 2.2, 8.6,
1H), 7.44 (m, 5H), 7.67 (d, J 8.6, 1H), 10.5 (s, 1H); δC (100 MHz, DMSO-d6) 70.6, 102.6, 108.9,
117.2, 128.7, 129.0, 129.4, 133.2, 137.1, 163.9, 165.9, 192.0. Experimental data in agreement with
reported data.18
Synthesis of methanesulfonic acid 5-benzyloxy-2-formyl-phenyl ester 2
ET N MsCl
O
O
3
DCM; quant.
BnO
OH
BnO
O
O S O
2
1
4-Benzyloxy-2-hydroxy-benzaldehyde 1 (5.61 g, 1.0 eq) was dissolved in dry DCM (250 ml)
followed by addition of triethylamine (1.2 eq) and methanesulfonylchloride (1.6 eq). The reaction
was stirred at r.t. until starting material was consumed (TLC monitoring) and then quenched with a
1.0 M hydrochloric acid aqueous solution (100 ml) and extracted with ethyl acetate (3 x 25 ml). The
combined organic layers were washed with brine (2 x 15 ml), dried over Na2SO4, filtered and the
solvent was eliminated under vacuo to give the title compound 2 as a white solid that was used as it
is.
Methanesulfonic acid 5-benzyloxy-2-formyl-phenyl ester 2: quantitative yield; silica gel TLC Rf
0.35 (Ethyl acetate/n-hexane 30 % v/v); δH (400 MHz, DMSO-d6): 3.61 (s, 3H), 5.30 (s, 2H), 7.22
(d, J 2.4, 1H), 7.25 (dd, J 2.4, 8.6, 1H), 7.45 (m, 5H), 7.92 (d, J 8.6, 1H), 10.11 (s, 1H); δC (100
MHz, DMSO-d6) 30.8, 71.3, 110.7, 115.5, 123.6, 129.0, 129.2, 129.5, 131.5, 136.7, 152.5, 165.0,
188.0. Experimental data in agreement with reported data.15
Synthesis of 7-benzyloxy-2,2-dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiin-4-ol 3

OH
O
DBU
BnO
O
S
DCM; 68% Yield
S
O
O
O
O
BnO
O
3
2
Methanesulfonic acid 5-benzyloxy-2-formyl-phenyl ester 2 (7.82 g, 1.0 eq) was dissolved in DCM
(320 ml) and then the solution was cooled down to 0°C and DBU (1.8 eq) was added drop-wise.
The reaction was stirred at r.t. until consumption of the starting material (TLC monitoring) and then
quenched with a 1.0 M hydrochloric acid aqueous solution (100 ml) and extracted with ethyl acetate
(3 x 25 ml). The combined organic layers were washed with brine (2 x 15 ml), dried over Na2SO4,
filtered and the solvent was eliminated under vacuo to give a residue that was purified by silica gel
column chromatography eluting with 30 % EtOAc in n-hexane to afford the title compound 3 as a
white solid.
7-Benzyloxy-2,2-dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiin-4-ol 3: 68% yield; m.p. 105-
107°C; silica gel TLC Rf 0.35 (Ethyl acetate/n-hexane 30 % v/v); δH (400 MHz, DMSO-d6): 3.65
(dd, J 9.1, 13.6, 1H), 4.26 (dd, J 6.2, 13.6, 1H), 5.09 (m, 1H), 5.19 (s, 2H), 6.30 (d, J 7.0, 1H), 6.86
(d, J 2.5, 1H), 7.01 (dd, J 2.5, 8.7, 1H), 7.44 (m, 6H), 9.70 (s, 1H); δC (100 MHz, DMSO-d6) 52.3,
65.2, 70.5, 104.7, 114.0, 119.1, 128.7, 128.9, 129.4, 130.7, 137.5, 150.3, 160.1; Elemental analysis:
calc: C 58.81, H 4.61, S 10.47; found: C 58.16, H 4.79, S 11.44. Experimental data in agreement
with reported data.15
Synthesis of 7-benzyloxy-benzo[e][1,2]oxathiine 2,2-dioxide 4
OH
ET N MsCl
3
S
O
S
O
DCM; 97% yield
BnO
O
BnO
O
O
O
3
4
7-Benzyloxy-2,2-dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiin-4-ol 3 (1.2 g, 1.0 eq) was
dissolved in dry DCM (50 ml) and treated with triethylamine (2.0 eq) then the solution was cooled
down to 0°C and methanesulfonyl chloride (1.6 eq) was added drop-wise. The reaction was stirred
at r.t. until complete consumption of starting material (TLC monitoring) and then quenched with a
1.0 M hydrochloric acid aqueous solution (15 ml) and extracted with ethyl acetate (3 x 10 ml). The
combined organic layers were washed with brine (2 x 10 ml), dried over Na2SO4, filtered and the
solvent was eliminated under vacuo to give the title compound 4 as a white solid that was used as it
is.

7-Benzyloxy-benzo[e][1,2]oxathiine 2,2-dioxide 4: 97% yield; m.p. 118-119°C; silica gel TLC Rf
0.39 (Ethyl acetate/n-hexane 30 % v/v); δH (400 MHz, DMSO-d6): 5.25 (s, 2H), 7.09 (dd, J 2.5 8.6,
1H), 7.19 (d, J 2.5, 1H), 7.34 (d, J 10.3, 1H), 7.45 (m, 5H), 7.65 (d, J 2.5, 1H), 7.67 (m, 1H); δC
(100 MHz, DMSO-d6) 71.0, 105.6, 113.0, 114.4, 120.1, 128.9, 129.1, 129.5, 132.1, 137.0, 137.4,
153.2, 162.3; Elemental analysis: calc: C 62.49, H 4.20, S 11.12; found: C 61.52, H 4.18, S 11.75.
Synthesis of 7-benzyloxy-3,4-dihydro-benzo[e][1,2]oxathiine 2,2-dioxide 5
10 % Pd/C H
2
S O
O
S O
O
EtOAc; 87% Yield
BnO
O
BnO
O
4
5
A solution of 7-benzyloxy-benzo[e][1,2]oxathiine 2,2-dioxide 4 (0.15 g, 1.0 eq) in 20 ml of ethyl
acetate was hydrogenated for 1 h at 20 Psi using 10 % Pd/C (0,05g) as catalyst. The mixture filtered
through Celite®, the solvent was eliminated under vacuo to give a residue that was purified by
silica gel column chromatography eluting with 50 % EtOAc in n-hexane to afford the title
compound 5 as a white solid.
7-Benzyloxy-3,4-dihydro-benzo[e][1,2]oxathiine 2,2-dioxide 5: 87% yield; m.p. 120-121°C; silica
gel TLC Rf 0.52 (Ethyl acetate/n-hexane 30 % v/v); δH (400 MHz, DMSO-d6): 3.28 (t, J 6.8, 2H),
3.87 (t, J 6.8, 2H), 5.15 (s, 2H), 6.82 (d, J 2.5, 1H), 6.93 (dd, J 2.5, 8.6, 1H), 7.29 (d, J 8.6, 1H),
7.43 (m, 5H); δC (100 MHz, DMSO-d6) 26.3, 44.8, 70.5, 105.2, 113.7, 113.7, 128.6, 128.8, 129.4,
131.4, 137.5, 152.6, 159.0; Elemental analysis: calc: C 62.05, H 4.86, S 11.04; found: C 61.80, H
4.11, S 11.20. Experimental data in agreement with reported data.18
Synthesis of 2,2-dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiin-7-ol 6
10 % Pd/C H
2
S
O
O
S
O
O
EtOAc; 87% Yield
BnO
O
HO
O
4
6
A solution of 7-benzyloxy-benzo[e][1,2]oxathiine 2,2-dioxide 4 (0.10 g, 1.0 eq) in 20 ml of ethyl
acetate was hydrogenated O.N. at 75 Psi using 10 % Pd/C (0,05g) as catalyst. The mixture was
filtered through Celite®, the solvent was eliminated under vacuo to give the title compound 6 as a
white solid and was used as it is.

2,2-Dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiin-7-ol 6: 87% yield; m.p. 149-151°C; silica gel
TLC Rf 0.14 (Ethyl acetate/n-hexane 50 % v/v); δH (400 MHz, DMSO-d6): 3.28 (t, J 6.8, 2H), 3.83
(t, J 6.8, 2H), 6.47 (d, J 2.5, 1H), 6.67 (dd, J 2.5, 8.4, 1H), 7.29 (d, J 8.4, 1H), 9.87 (s, 1H); δC (100
MHz, DMSO-d6) 26.3, 45.0, 105.5, 111.7, 113.9, 131.4, 152.6, 158.3; Elemental analysis: calc: C
47.99, H 4.03, S 16.02; found: C 47.54, H 4.14, S 16.41. Experimental data in agreement with
reported data.18
Synthesis of 2,2-dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiine-4,7-diol 7
OH
OH
10 % Pd/C H
2
S
O
O
EtOAc; 92 % yield
S
O
O
HO
O
BnO
O
3
7
A solution of 7-benzyloxy-2,2-dioxo-3,4-dihydro-2H-22λ6-benzo[e][1,2]oxathiin-4-ol 3 (0.20 g, 1.0
eq) in 20 ml of ethyl acetate was hydrogenated O.N. at 60 Psi using 10 % Pd/C (0,05g) as catalyst.
The mixture was filtered through Celite®, the solvent was eliminated under vacuo to to give a
residue that was purified by silica gel column chromatography eluting with 50 % EtOAc in n-
hexane to afford the title compound 7 as a white solid.
2,2-Dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiine-4,7-diol 7: 92 % yield; m.p. 142-143°C;
silica gel TLC Rf 0.26 (Ethyl acetate/n-hexane 50 % v/v); δH (400 MHz, DMSO-d6): 3.60 (dd, J 9.1,
13.7 , 1H), 4.22 (dd, J 6.1, 13.7, 1H), 5.05 (m, 1H), 6.21 (d, J 7.1, 1H), 6.50 (d, J 2.4, 1H), 6.76 (dd,
J 2.4, 8.6, 1H), 7.42 (d, J 8.6, 1H), 10.05 (s, 1H); δC (100 MHz, DMSO-d6) 52.4, 65.2, 105.0,
114.1, 117.3, 130.7, 150.3, 159.4; Elemental analysis: calc: C 44.44, H 3.73, S 14.83; found: C
44.30, H 3.66, S 14.62. Experimental data in agreement with reported data.18
Synthesis of methanesulfonic acid 2,2-dioxo-2H-2λ6-benzo[e][1,2]oxathiin-7-yl ester 8
OH
O
MsCl, ET N
THF; quant.
3
O
S
S
O
O
S O
O
O
O
HO
O
7
8
2,2-Dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiine-4,7-diol 7 (0.1 g, 1.0 eq) was dissolved in
THF (5.0 ml), then the solution was cooled down to 0°C and treated drop-wise with

methanesulfonylchloride (1.6 eq). The reaction was stirred at r.t until consumption of the starting
material (TLC monitoring) and then quenched with a 1.0 M hydrochloric acid aqueous solution (15
ml) and extracted with ethyl acetate (3 x 10 ml). The combined organic layers were washed with
brine (2 x 10 ml) dried over Na2SO4, filtered and the solvent was eliminated under vacuo to give the
title compound 8 as a white solid and was used as it is.
2,2-dioxo-2H-2λ6-benzo[e][1,2]oxathiin-7-yl ester 8: quantitative yield; m.p. 127-128°C; silica gel
TLC Rf 0.35 (Ethyl acetate/n-hexane 40 % v/v); δH (400 MHz, DMSO-d6): 3.54 (s, 3H), 7.47 (dd, J
2.4, 8.6, 1H), 7.61 (d, J 2.4, 1H), 7.63 (d, J 10.4, 1H), 7.80 (d, J 10.4 Hz, 1H), 7.90 (d, J 8.6, 1H);
δC (100 MHz, DMSO-d6) 39.0, 114.1, 119.0, 121.5, 123.6, 132.7, 136.8, 151.8, 152.3; Elemental
analysis: calc: C 39.12, H 2.92, S 23.21; found: C 39.15, H 3.02, S 26.41. Experimental data in
agreement with reported data.15
Synthesis of 2,2-dioxo-2H-2λ6-benzo[e][1,2]oxathiin-7-ol 9
Et NOH
O
4
O
S
O
S
S
1,4-Dioxane; 85% Yield
O
O
HO
O
O
O
O
8
9
2,2-Dioxo-2H-2λ6-benzo[e][1,2]oxathiin-7-yl ester 8 (0.13 g, 1.0 eq) was dissolved in 1,4-dioxane
(5.0 ml) and treated with a 20 % aqueous solution of tetraethylamonium hydroxide (2.0 eq). The
solution was stirred at r.t. until consumption of the starting material (TLC monitoring), then
quenched with a 1.0 M hydrochloric acid aqueous solution (15 ml) and extracted with ethyl acetate
(3 x 10 ml). The combined organic layers were washed with brine (2 x 10 ml), dried over Na2SO4,
filtered and the solvent was eliminated under vacuo to give the title compound 9 as a white solid
and was used as it is.
2,2-Dioxo-2H-2λ6-benzo[e][1,2]oxathiin-7-ol 9: 85% yield; m.p. 157-159°C; silica gel TLC Rf 0.43
(Ethyl acetate/n-hexane 40 % v/v); δH (400 MHz, DMSO-d6): 6.77 (d, J 2.4, 1H), 6.83 (dd, J 2.4,
8.5, 1H), 7.23 (d, J 10.3, 1H), 7.54 (d, J 8.5, 1H), 7.60 (d, J 10.3, 1H), 10.79 (s, 1H); δC (100 MHz,
DMSO-d6) 105.9, 111.5, 114.6, 119.0, 132.3, 137.7, 153.3, 162.1; Elemental analysis: calc: C
48.48, H 3.05, S 16.18; found: C 48.60, H 3.16, S 17.00. Experimental data in agreement with
reported data.19

Synthesis of methanesulfonic acid 2-formyl-5-methoxy-phenyl ester 10
O
O
Et N , MsCl
3
O
O
S
DCM, r.t.; 86% Yield
O
OH
O
O
10
2-Hydroxy-4-methoxybenzaldehyde (1.0 g, 1.0 eq) and triethylamine (1.2 eq) were dissolved in dry
DCM (40.0 ml) followed by addition of methanesulfonyl chloride (1.6 eq) at 0°C. The reaction was
stirred at r.t. under a nitrogen atmosphere until starting material was consumed (TLC monitoring)
then quenched with H2O (30 ml) and extracted with ethyl acetate (3 x 15 ml). The combined
organic layers were washed with brine (2 x 15 ml), dried over Na2SO4, filtered and the solvent was
eliminated under vacuo to give a residue that was purified silica gel column chromatography eluting
with 20 % EtOAc in n-hexane to afford the title compound 10 as a white solid.
Methanesulfonic acid 2-formyl-5-methoxy-phenyl ester 10: 86 % yield; m.p. 120-122°C silica gel
TLC Rf 0.05 (Ethyl acetate/n-hexane 20 % v/v); δH (400 MHz, DMSO-d6): 3.62 (s, 3H), 3.94 (s,
3H), 7.12 (d, J 2.4, 1H), 7.17 (dd, J 2.4, 8.6, 1H), 10.12 (s, 1H); δC (100 MHz, DMSO-d6) 38.8,
57.2, 109.9, 114.8, 123.4, 131.5, 152.5, 165.9, 188.1; Elemental analysis: calc: C 46.95, H 4.38, S
13.93; found: C 47.53, H 4.21, S 12.04.
Synthesis of 7-methoxy-benzo[e][1,2]oxathiine 2,2-dioxide 12
OH
O
POCl
3
DBU
O
S
O
O
DCM; 90 % Yield
O
S
O
O
Pyr, 30 % yield
over two steps
O
S
O
O
O
O
O
12
10
11
Methanesulfonic acid 2-formyl-5-methoxy-phenyl ester 10 (1.24 g, 1.0 eq) was dissolved in dry
DCM (25 ml) and then DBU (1.0 eq) was added. The solution was cooled down to 0°C and
mesylchloride (1.6 eq) was added drop-wise. The reaction was stirred at r.t. under a nitrogen
atmosphere for 2 hrs and then quenched with a 10% aqueous hydrochloric acid solution (30 ml) and
extracted with ethyl acetate (3 x 15 ml). The combined organic layers were washed with brine (2 x
15 ml), dried over Na2SO4, filtered and the solvent was eliminated under vacuo to give the
intermediate 11 which was dissolved in pyridine (20 ml) and the solution was treated at 0°C with
phosphorus oxychloride (1.5 eq). The reaction mixture was stirred at r.t. for 3 hrs at r.t. and then

quenched with slush (30 g). The precipitate formed was collected by filtration, dried under vacuo
and recrystallized from EtOH to afford the title compound 12 as a light brown solid.
7-Methoxy-benzo[e][1,2]oxathiine 2,2-dioxide 12: 30 % yield over 2 steps; m.p. 111-112°C; silica
gel TLC Rf 0.16 (Ethyl acetate/n-hexane 40 % v/v); δH (400 MHz, DMSO-d6): 3.89 (s, 3H), 6.84
(dd, J 2.4, 8.7, 1H), 7.29 (d, J 2.4, 1H), 7.33 (d, J 10.2, 1H), 7.66 (m, 2H); δC (100 MHz, DMSO-d6)
57.0, 104.7, 112.8, 113.8, 120.0, 132.0, 137.4, 153.3, 163.3; Elemental analysis: calc: C 50.94, H
3.80, S 15.11; found: C 50.61, H 3.69, S 16.99.
Synthesis of 7-methoxy-3,4-dihydro-benzo[e][1,2]oxathiine 2,2-dioxide 13
10 % Pd/C
O
S
O
O
S
O
O
O
O
O
EtOAc; 93 % Yield
12
13
A solution of 7-methoxy-benzo[e][1,2]oxathiine 2,2-dioxide 12 (0.10 g, 1.0 eq) in 20 ml of ethyl
acetate was hydrogenated for 2 hrs at 40 Psi using 10 % Pd/C (0,05g) as catalyst. The mixture was
filtered through Celite®, the solvent was eliminated under vacuo to give the title compound 13 as
white solid and was used as it is.
7-Methoxy-3,4-dihydro-benzo[e][1,2]oxathiine 2,2-dioxide 13: 93 %yield; m.p. 160-161°C; silica
gel TLC Rf 0.54 (Ethyl acetate/n-hexane 40 % v/v); δH (400 MHz, DMSO-d6): 3.32 (t, J 6.8, 2H),
3.79 (s, 3H), 3.87 (t, J 6.8, 2H), 6.74 (d, J 2.6, 1H), 6.84 (dd, J 2.6, 8.5, 1H), 7.29 (d, J 8.6, 1H); δC
(100 MHz, DMSO-d6) 26.4, 44.9, 56.5, 104.3, 113.0, 113.5, 131.4, 152.7, 160.1; Elemental
analysis: calc: C 50.46, H 4.70, S 14.97; found: C 49.45, H 4.64, S 15.75.
Synthesis of methanesulfonic acid 2,2-dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiin-7-yl ester
6a
O
Et3N , MsCl
O
S
O
S
S O
O
HO
O
THF, r.t.; 86% Yield
O
O
O
6a
6
2,2-Dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiin-7-ol
6
or
2,2-dioxo-2H-2λ6-benzo
[e][1,2]oxathiin-7-ol 9 (0.1 g, 1.0 eq) was dissolved in THF (10.0 ml) and treated with triethylamine
(1.5 eq). The solution was cooled down to 0°C and treated with methanesulfonyl chloride (1.6 eq).
The reaction was stirred at r.t. until starting material was consumed (TLC monitoring) then

quenched with a 1.0 M hydrochloric acid aqueous solution (15 ml) extracted with ethyl acetate (3 x
10 ml). The combined organic layers were washed with brine (2 x 10 ml) dried over Na2SO4,
filtered and the solvent was eliminated under vacuo to give the title compound 6a as a white solid
and was used as it is.
Methanesulfonic acid 2,2-dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiin-7-yl ester 6a: 86 %
yield; m.p. 106-107°C; silica gel TLC Rf 0.19 (Ethyl acetate/n-hexane 50 % v/v); δH (400 MHz,
DMSO-d6): 3.44 (t, J 7.0, 2H), 3.46 (s, 3H), 3.98 (t, J 7.0, 2H), 7.25 (d, J 2.4, 1H), 7.27 (dd, J 2.4,
8.2, 1H), 7.52 (d, J 8.2, 1H); δC (100 MHz, DMSO-d6) 26.8, 38.5, 44.5, 113.6, 120.8, 121.4, 132.2,
149.1, 152.4; Elemental analysis: calc: C 38.84, H 3.62, S 23.04; found: C 38.73, H 3.80, S 24.41.
Synthesis of 2,2-dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiin-7-acyl (14-16) and 2,2-dioxo-2H-
2λ6-benzo[e][1,2]oxathiin-7-acyl (17-19) derivatives.
2,2-Dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiin-7-ol
6
or
2,2-dioxo-2H-2λ6-benzo
[e][1,2]oxathiin-7-ol 9 (0.1 g, 1.0 eq) was dissolved in THF (10.0 ml) and treated with triethylamine
(1.5 eq). The solution was cooled down to 0°C and treated with the corresponding acyl halide (1.0
eq). The reaction was stirred at r.t. until starting material was consumed (TLC monitoring) then
quenched with a 1.0 M hydrochloric acid aqueous solution (15 ml) extracted with ethyl acetate (3 x
10 ml). The combined organic layers were washed with brine (2 x 10 ml), dried over Na2SO4,
filtered and the solvent was eliminated under vacuo to give a residue that was purified by silica gel
column chromatography eluting with the appropriate concentration of EtOAc in n-hexane to afford
the title compounds
Synthesis of acetic acid 2,2-dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiin-7-yl ester 14
O
O
S
O
O
O
14
The title compound 14 was obtained according to the procedure described above and used as it is.
Acetic acid 2,2-dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiin-7-yl ester 14: quantitative; m.p.
99-101°C; silica gel TLC Rf 0.55 (Ethyl acetate/n-hexane 50 % v/v); δH (400 MHz, DMSO-d6):

2.30 (s, 3H), 3.41 (t, J 6.6, 2H), 3.94 (t, J 6.6, 2H), 7.02 (d, J 2.0, 1H), 7.05 (dd, J 2.3, 8.4, 1H),
7.43 (d, J 8.4, 1H); δC (100 MHz, DMSO-d6) 21.7, 26.8, 44.6, 113.2, 119.5, 120.0, 131.5, 150.8,
152.2, 169.9; Elemental analysis: calc: C 49.58, H 4.16, S 13.24; found: C 49.32, H 4.15, S 13.72.
Synthesis of (4-chloro-phenyl)-acetic acid 2,2-dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiin-7-
yl ester 15
Cl
O
O
S
O
O
O
15
The title compound 15 was obtained according to the procedure described above and used as it is.
(4-Chloro-phenyl)-acetic acid 2,2-dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiin-7-yl ester 15:
quantitative; m.p. 97-99°C; silica gel TLC Rf 0.59 (Ethyl acetate/n-hexane 50 % v/v); δH (400 MHz,
DMSO-d6): 3.40 (t, J 6.8, 2H), 3.95 (t, J 6.8, 2H), 4.04 (s, 2H), 7.04 (s, 1H), 7.05 (dd, J 2.3, 8.4,
1H), 7.45 (m, 5H); δC (100 MHz, DMSO-d6) 26.9, 44.7, 113.2, 119.7 119.8, 120.0, 129.1, 129.4,
131.7, 132.6, 133.8, 150.9, 152.3, 170.8; Elemental analysis: calc: C 54.47, H 3.71, S 9.09; found:
C 54.25, H 3.67, S 8.92.
Synthesis of (2-bromo-phenyl)-acetic acid 2,2-dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiin-7-
yl ester 16
O
O
S
O
O
O
Br
16
The title compound 16 was obtained according to the procedure described above after purification
by silica gel column chromatography eluting with 50% EtOAc in n-hexane.
(2-Bromo-phenyl)-acetic acid 2,2-dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiin-7-yl ester 16: 67
% yield; m.p. 114-116°C; silica gel TLC Rf 0.53 (Ethyl acetate/n-hexane 50 % v/v); δH (400 MHz,
DMSO-d6): 3.40 (t, J 6.8, 2H), 3.94 (t, J 6.8, 2H), 4.15 (s, 2H), 7.01 (d, J 2.3, 1H), 7.06 (dd, J 2.3,
8.5, 1H), 7.31 (m, 1H), 7.44 (m, 2H), 7.55 (dd, J 1.6, 8.0, 1H), 7.70 (dd, J 1.6, 8.0, 1H); δC (100
MHz, DMSO-d6) 26.8, 41.8, 44.6, 112.9, 119.8, 125.6, 128.9, 129.2, 130.5, 131.7, 133.2, 133.35,

133.39, 150.7, 152.2, 169.8; Elemental analysis: calc: C 48.38, H 3.30, S 8.07; found: C 47.49, H
3.26, S 7.60.
Synthesis of acetic acid 2,2-dioxo-2H-2λ6-benzo[e][1,2]oxathiin-7-yl ester 17
The title compound 17 was obtained according to the procedure described above and used as it is.
Acetic acid 2,2-dioxo-2H-2λ6-benzo[e][1,2]oxathiin-7-yl ester 17: 62 % yield; m.p. 57-59°C; silica
gel TLC Rf 0.50 (Ethyl acetate/n-hexane 50 % v/v); δH (400 MHz, DMSO-d6): 2.34 (s, 3H), 7.27
(dd, J 2.2, 8.4, 1H), 7.41 (d, J 2.2, 1H), 7.56 (d, J 10.4, 1H), 7.76 (d, J 10.4, 1H), 7.80 (d, J 8.4,
1H); δC (100 MHz, DMSO-d6) 21.9, 113.8, 117.7, 121.3, 123.0, 132.1, 137.2, 152.2, 153.8, 169.8;
Elemental analysis: calc: C 50.00, H 3.36, S 13.35; found: C 50.91, H 4.60, S 11.43.
Sinthesis of (4-chloro-phenyl)-acetic acid 2,2-dioxo-2H-2λ6-benzo[e][1,2]oxathiin-7-yl ester 18
The title compound 18 was obtained according to the procedure described above after purification
by silica gel column chromatography eluting with 50% EtOAc in n-hexane.
(4-Chloro-phenyl)-acetic acid 2,2-dioxo-2H-2λ6-benzo[e][1,2]oxathiin-7-yl ester 18: 53 % yield;
m.p. 106-108°C; silica gel TLC Rf 0.61 (Ethyl acetate/n-hexane 50 % v/v); δH (400 MHz, DMSO-
d6): 4.07 (s, 2H), 7.28 (dd, J 2.2, 8.4, 1H), 7.44 (d, J 2.2, 1H), 7.47 (s, 4H), 7.57 (d, J 10.4, 1H),
7.78 (d, J 10.4, 1H), 7.81 (d, J 8.4, 1H); δC (100 MHz, DMSO-d6) 39.6, 113.6, 117.7, 121.0, 122.9,
129.3, 132.0, 132.5, 132. 8, 133.5, 137.0, 152.1, 153.6, 170.4; Elemental analysis: calc: C 54.78, H
3.16, S 9.14; found: C 53.75, H 3.29, S 8.18.
Synthesis of (2-bromo-phenyl)-acetic acid 2,2-dioxo-2H-2λ6-benzo[e][1,2]oxathiin-7-yl ester 19
The title compound 19 was obtained according to the procedure described above after purification
by silica gel column chromatography eluting with 40% EtOAc in n-hexane.
(2-Bromo-phenyl)-acetic acid 2,2-dioxo-2H-2λ6-benzo[e][1,2]oxathiin-7-yl ester 19: 50 % yield;
m.p. 132-135°C; silica gel TLC Rf 0.73 (Ethyl acetate/n-hexane 40 % v/v); δH (400 MHz, DMSO-
d6): 4.20 (s, 2H), 7.28 (dd, J 2.3, 8.4, 1H), 7.32 (m, 1H), 7.43 (m, 2H), 7.57 (d, J 10.4, 2H), 7.71
(m, 1H), 7.77 (d, J 10.4, 1H), 7.82 (d, J 8.4, 1H); δC (100 MHz, DMSO-d6) 41.8, 113.4, 117.8,

120.9, 123.0, 125.5, 128.9, 130.5, 132.1, 133.3, 133.4, 134.6, 137.0, 152.1, 153.5, 169.5; Elemental
analysis: calc: C 48.62, H 2.81, S 8.11; found: C 47.51, H 2.90, S 7.47.
4.2. CA inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed
CO2 hydration activity.16 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
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 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were
preincubated together for 15 min – 6 h at room temperature (15 min) 0r 4 °C (6h) prior to assay, in
order to allow for the formation of the E-I complex. Data from table 1 were obtained after 6 hours
incubation of enzyme and inhibitor, as for the sulfocoumarins and coumarins reported earlier.1-3 The
inhibition constants were obtained by non-linear least-squares methods using PRISM 3, as reported
earlier,2 and represent the mean from at least three different determinations. All CA isofoms were
recombinant ones obtained in-house as reported earlier.20,21
Acknowledgments: This work was financed by two FP7 EU grant (METOXIA and DYNANO).

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Table 1. hCA I, II, VA, IX and XII inhibition data with sulfocoumarins and their derivatives of
types 3-19 synthesized in this work, by a stopped-flow CO2 hydrase assay.16 The sulfonamide
inhibitor acetazolamide (AAZ, 5-acetamido-1,3,4-thiadiazole-2-sulfonamide) was used as standard.
Compound
Ki (nM)*
hCA VA
hCA I
hCA II
hCA IX
hCA XII
3
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
250
2.3
2.5
2.0
7.6
1.8
1.6
2.1
8.4
1.5
2.4
1.5
2.2
3.4
2.2
2.6
1.9
12
591
835
2150
327
517
5100
5090
788
9960
91
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
25
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
5.7
4
5
6
6a
7
8
9
12
13
14
15
16
17
18
19
AAZ
1675
206
141
716
3910
7060
64
* Errors in the range of  10 of the reported values, from 3 different assays.

R
R
O
O
S
O
Lactones
(6-membered)
R
Thiocoumarin
R
O
S
2-Thioxocoumarin
R
O
S
Thioactones
(6-membered)
R
S
S
O
O
R
COUMARIN
Dithiocoumarin
O
NOH
Lactone oximes
R
R
O
O
O
N OH
Lactones
(5-membered)
Coumarin-oximes
R
O
S
O
O
"Sulfocoumarin"
Chart 1: The various classes of CAIs developed by using the coumarins as lead molecules.

O
ET3N MsCl
O
O
BnBr, NaHCO3
ACN; quant.
HO
OH
DCM; quant.
BnO
OH
BnO
O
S
O
O
2
1
DBU
DCM
68 % Yield
OH
10 % Pd/C H2
ET3N MsCl
S
O
O
S
O
O
EtOAc; 87% Yield
75 Psi ON
S O
O
BnO
O
DCM;
97% Yield
HO
O
BnO
O
4
3
6
10 % Pd/C, H2
Et3N , MsCl
THF, r.t.;
86% Yield
10 % Pd/C H2
EtOAc; quant.
20 Psi 1 hr.
EtOAc; quant.
60 Psi ON
OH
O
Me
S
S O
O
S
O
O
O
O
BnO
O
S O
O
O
HO
O
6a
5
7
MsCl, ET3N
THF; quant.
Et4NOH
1,4-Dioxane; 85% Yield
O
O
S
S
S
O
O
O
O
HO
O
O
O
9
8
Scheme 1: Synthesis of sulfocoumarins 3-9.

OH
O
O
Et N , MsCl
3
DBU
O
S
O
DCM; 86 % Yield
O
O
S
DCM, r.t.,
86 % yield
O
O
O
OH
O
O
11
10
POCl
3
Pyr, 30 % yield
10 % Pd/C
O
S
O
S
EtOAc; 93 % Yield
O
O
O
O
O
O
13
12
Scheme 2: Synthesis of sulfocoumarins 12 and 13.
O
Et3N
R = Me; 14
R = 4-chlorobenzyl; 15
R = 2-bromobenzyl; 16
O
O
R
Cl
O
O
O
O
S
O
S
O
HO
HO
O
R
R
O
O
O
THF, r.t.
6
O
Et3N
R = Me; 17
R = 4-chlorobenzyl; 18
R = 2-bromobenzyl; 19
R
Cl
S
O
S
O
O
O
THF, r.t.
9
Scheme 3: Synthesis of 2,2-dioxo-3,4-dihydro-2H-2λ6-benzo[e][1,2]oxathiin-7-acyl (14-16) and
2,2-dioxo-2H-2λ6-benzo[e][1,2]oxathiin-7-acyl (17-19) derivatives.

H2O
SO3H
R
R
O
S
R
SO3H
OH
O
O
OH
Reaction intermediated
(not isolated)
Sulfocoumarin
Hydrolyzed sulfocoumarin
A
B
Fig. 1: CA inhibition mechanism by sulfocoumarins. A. The sulfocoumarin undergoes an enzyme-
mediated hydrolysis with formation of the trans-2-hydroxy-phenyl--ethenylsulfonic acid. B. The
sulfonic acid binds to a mutant CA II active site, by anchoring of the sulfonic acid group to the zinc-
coordinated water molecule. The Zn(II) ion (central larger sphere), its three His ligands (His94, 96
and 119), water molecule coordinated to the zinc (small sphere) as well as active site residues
Thr200 and Pro201 involved in the binding of the hydrolyzed sulfocoumarin are shown, as
determined by X-ray crystallography (PDB file 4bcw).1

7-Substituted-sulfocoumarins are isoform-selective, potent carbonic anhydrase II inhibitors
Muhammet Tanc, Fabrizio Carta, Murat Bozdag, Andrea Scozzafava, and Claudiu T. Supuran*b
H2O
SO3H
R
R
O
S
R
SO3H
OH
O
O
OH
Reaction intermediated
(not isolated)
Sulfocoumarin
Hydrolyzed sulfocoumarin
KIs (hCA II ) = 1.5 – 8.4 nM
KIs (hCA I, hCA IX, hCA XII ) > 10 M