"a Sweet Combination": Developing Saccharin and Acesulfame K Structures for Selectively Targeting the Tumor-Associated Carbonic Anhydrases IX and XII

Article abstract of DOI:10.1021/acs.jmedchem.9b01669

The sweeteners saccharin (SAC) and acesulfame K (ACE) recently entered the topic of anticancer human carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, as they showed to selectively inhibit the tumor-associated CAs IX/XII over ubiquitous CAs. A drug design s

Full text of DOI:10.1021/acs.jmedchem.9b01669

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Article
“A Sweet Combination”: Developing saccharin and
acesulfame K structures for selectively targeting the
tumor-associated carbonic anhydrases IX and XII.
Silvia Bua, Carrie L. Lomelino, Akilah B. Murray, Sameh M. Osman, Zeid
Abdullah Alothman, Murat Bozdag, Hatem A. Abdel Aziz, Wagdy M.
Eldehna, Robert McKenna, Alessio Nocentini, and Claudiu Trandafir Supuran
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01669 • Publication Date (Web): 03 Dec 2019
Downloaded from pubs.acs.org on December 5, 2019
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“A Sweet Combination”: Developing saccharin and acesulfame K structures for selectively
targeting the tumor-associated carbonic anhydrases IX and XII.
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Silvia Buaa, Carrie Lomelinob, Akilah B. Murrayb, Sameh M. Osmanc, Zeid A. ALOthmanc, Murat
Bozdaga, Hatem A. Abdel-Azizd, Wagdy M. Eldehnae, Robert McKennab, Alessio Nocentinia,*,
Claudiu T. Supurana,*.
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aUniversity of Florence, Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, via Ugo Schiff 6,
50019, Sesto Fiorentino, Florence, Italy
bUniversity of Florida, Department of Biochemistry and Molecular Biology, 1200 Newell Dr, Gainesville, FL 32610, U.S.A.
cChemistry Department, College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia.
dDepartment of Applied Organic Chemistry, National Research Center, Dokki, Giza, P.O. Box 12622, Egypt
eDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh 33516, Egypt
ABSTRACT
The sweeteners saccharin (SAC) and acesulfame K (ACE) recently entered the topic of anticancer
human carbonic anhydrase (CA, EC 4.2.1.1) inhibitors as they showed to selectively inhibit the
tumor-associated CAs IX/XII over ubiquitous CAs. A drug design strategy is here reported which
took SAC and ACE as leads and produced a series of 2H-benzo[e][1,2,4]thiadiazin-3(4H)-one-1,1-
dioxides (BTD). Many derivatives showed greater potency (KIs-CA IX 19.1-408.5 nM) and
selectivity (II/IX SI 2-76) than the leads (KIs-CA IX 103, 2400 nM; II/IX-SI 56, >4) against CA
IX/XII over off-target isoforms. A thorough X-ray crystallographic study depicted their binding
mode to both CA II and IX-mimic. The most representative BTDs were characterized in vitro for
their antitumor activity against A549, PC-3 and HCT-116 cancer cell lines both in normoxia and
hypoxia. The two most effective compounds were assayed for their effect on several apoptosis
markers, identifying promising leads for the development of new anticancer drugs.
Keywords: tumor; metalloenzyme; sweetener; saccharin; acesulfame; optimization; inhibition.
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1. Introduction
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The tumor microenvironment considerably influences cancerogenesis.1-3 Hypoxic tumors
are often characterized by a disproportion between the demand of rapidly proliferating cancer cells
and the ability of the forming vascular system to provide an adequate oxygen supply.4,5 As a
response, cancer cells adapt their metabolism by undergoing a glycolytic shift, which decreases
cellular pH and changes gene expression patterns in order to survive in an environment unsuitable
for normal cell growth.4,5 Notably, an orchestra of membrane proteins, among which transporters
such as sodium-proton exchangers (NHE1), anion exchangers (AE2), sodium-bicarbonate
transporters (NBCe1), monocarboxylate transporters (MCT4) and carbonic anhydrases (CA, EC
4.2.1.1), is assembled to regulate the pH within and outside the cells, maintaining a slightly alkaline
intracellular pH and acidifying the extracellular environment.1-5 CA isoform IX (CA IX), which is
not significantly expressed in most normal tissues, has been shown to be associated with hypoxic
cancer phenotypes.4-8 CA XII also intervenes in the microenvironment acidification for a number of
tumor types.4,9 Therefore CA IX and XII are factors driving tumor growth, invasiveness,
proliferation, metastasis, and resistance to common radio- and chemotherapy, resulting in
unfavourable prognoses for cancer patients.3 As such, agents inhibiting CA IX and XII have shown
to provide inhibition of the primary tumor growth, invasion and metastasis, and reduction of cancer
stem cell population.10
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The main challenge encountered in the design of anticancer CA inhibitors (CAIs) has been
the lack of isoform-selectivity exhibited by the most classical inhibitory chemotypes, namely the
sulfonamides and their bioisosteres.11,12 A comparison of the 12 catalytically active human CAs
shows a high homology between the active sites that complicates the design of disease-associated
selective inhibitors.13 Nonetheless, a plethora of strategies have been developed to overcome this
issue, including the optimization of classical CAIs (i.e. the tail approach, which led to SLC-0111,
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the first-in-class CAI entering clinical trials for the treatment of hypoxic tumors)12 or the use of
alternative chemotypes adopting different mechanisms of enzymatic inhibition.11,12
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Figure 1: Active site view of the CA IX adduct with A) SAC (PDB 4RIV) and B) ACE (PDB 5WGP).
The cyclic sulfimide saccharin (SAC), the oldest artificial sweetener, has recently entered
this topic. SAC, which is 450 times sweeter than sucrose, was shown to selectively discriminate
among the diverse isoforms, distinctly from classical CAIs such as acetazolamide (AAZ).14 It is, in
particular, a selective nanomolar inhibitor of CA IX, showing weaker, micromolar inhibition of the
ubiquitous CA I and II (60-fold II/IX selectivity).14 Its cyclic acylsulfonamide, which is much more
acidic than primary sulfonamides, functions as a zinc binding group (ZBG) within the CA active
site. X-ray and neutron crystallography studies pointed out that alternative interactions with residue
Gln67 (leading to remodelling of H-bonds and water orientations) contributed to the CA IX
preferential binding of SAC compared to CA II (that reports an Asn in position 67).15,16 A SAC
derivative, called SGC (saccharin-glucose conjugate), was designed utilizing the tail approach with
a polar carbohydrate linked by a triazole as the tail moiety of the CA inhibitor.15 It showed even
further enhanced selectivity for CA IX over both CA I and CA II (>1000-fold), with the specificity
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of binding explained by evident differences in interacting active site residues between CA II and
CA IX.
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Another FDA approved food additive, acesulfame K (ACE, a cyclic acylsulfamate), was
recently kinetically and structurally characterized with CAs.17 Interestingly, ACE induced no
inhibition of CA I, II and XII while inhibiting CA IX in the low micromolar range, with its weak
inhibition being likely due to the absence of the aromatic core. Furthermore, it exhibits different
active site binding modes between isoforms, namely coordinating the Zn(II) ion via the aromatic
nitrogen in the case of CA IX, and anchoring to zinc-bound water molecule/hydroxide ion in CA II.
This behaviour is reflected by ACE demonstrating measurable inhibition toward CA IX but not to
other CA isoforms.17 This study showed the value of ACE as a lead compound for designing new
CA IX specific inhibitors.
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In this context, libraries of N- and/or O-substituted SAC and ACE derivatives have been
recently reported as potent and selective inhibitors of CA IX and XII.18-20 Nonetheless, this type of
functionalization decoupled these chemotypes away from a zinc-binder character (or water-
anchoring, vide supra) promoting likely alternative binding modes, which have not yet been
elucidated.
Here a new series of SAC- and ACE-based derivatives is reported by combining and
developing the lead chemotypes structures to produce cyclic ureidosulfonamides, namely 2H-
benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxides (BTD), which preserve a zinc-binding group and
include another foothold for ligand/target interactions and/or functionalization.
O
O
S
H-bond
acceptor
NH
Zinc
binding
function
O O
S
O
Saccharin
(SAC)
NH
R
N
O
(BTD)
O
O
R1
Interaction or
functionalization
point
K+
S
N
O
O
Acesulfame K
(ACE)
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Figure 2: Rational development of benzothiadiazin-3-one 1,1-dioxides (BTD), based on the structures and
inhibition profiles of SAC and ACE.
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2. Results and discussion
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2.1 Drug design and chemistry
To methodically explore the chemical space that induces cyclic acylsulfonamides or
sulfamates to yield specific inhibition of CA IX and XII, an approach to merge SAC and
ACE structures was developed and is here reported, producing a series of cyclic
ureidosulfonamides 2a-2t. The redesigned scaffold BTD preserves the zinc-binding group of SAC
and ACE and includes another interaction and/or functionalization point (i.e. the NH between the
carbonyl and the aromatic ring).
In order to identify new CA inhibitory chemotypes, a previous screening included the
unsubstituted 2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide 2a,21 further supporting the
production of such a series as this derivative was shown to hold selectivity for CA IX/XII over CAs
I and II. Here an extended SAR study was worked out by appending a wealth of substituents both in
R and R1 position (Figure 2) by exploiting a more versatile synthetic procedure than that reported
by Bozdag et al. for 2a.21 Instead of treating o-aminobenzenesulfonamide with phosgene, which
does not allow wide and accessible BTD functionalization, a forward strategy recently proposed by
Zhang and Kornahrens (for providing new classes of aldose reductase and irreversible serine
hydrolase inhibitors, respectively)22,23 is here applied and extended.
The adopted synthetic procedure is depicted in Scheme 1. Chlorosulfonyl isocyanate (CSI)
was treated at -40°C with commercially available variously substituted anilines (1a-n), N-
methylanilines (1o,p) and indoline (1q) using nitromethane or nitroethane as solvent to yield the
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intermediates chlorosulfamoylureas βa-q. Thus, aluminium trichloride (AlCl3) was added and
temperature risen to 110°C to promote a Friedel-Crafts-like core cyclization. The absence of a base
and a very low temperature are important to avoid reaction of the aniline with the sulfamoyl portion
of CSI. In contrast, the choice of nitromethane or nitroethane is driven by the necessity to use a
polar aprotic solvent which can span from markedly under-zero temperatures to over 100°C without
solidifying or boiling. Reaction attempts with markedly electron-poor anilines of the nitro-,
sulfonamido- or polyhalo-substituted types crashed with their low reactivity in both reaction steps.
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NH2
R
2a
: R=H
O
S
O
2b
2c
H
H
: R=5-CH3
: R=7-CH3
1a-n
AlCl3
N
N
Cl
NH
S
R
R
2d
2e
2f
: R=5,7-diCH3
: R=5,8-diCH3
: R=6,8-diCH3
O
O
110°C
45 min
O
CH3NO2 or CH3CH2NO2
-40°C, 15 min
N
H
O
a-n
2a-n
2g
2h
: R=5-F
: R=7-F
: R=5-Cl
: R=7-Cl
H
N
2i
2j
R
O
S
O
H
2k
2l
: R=5-Br
: R=7-Br
O
O
AlCl3
N
N
Cl
1o,p
S
NH
S
R
Cl
NCO
R
O
O
2m
: R=7-OCH3
: R=5,6-benzo
2o
O
110°C
45 min
CH3CH2NO2
-40°C, 15 min
N
O
2n
CSI
o,p
: R=H
2o,p
2p
: R=7-Cl
H
N
O
S
O
H
N
N
Cl
AlCl3
NH
1q
S
O
O
O
N
110°C
45 min
O
CH3CH2NO2
-40°C, 15 min
q
2q
Scheme 1: Synthesis of 2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxides 2a-q.
The 5- and 7-methyl derivatives 2b and 2c were also oxidized with potassium permanganate
to give benzoic acids 2r and 2s respectively, as illustrated in Scheme 2. The 7-methoxy-BTD
underwent demethylation by treatment with 1.0 M BBr3 in DCM to produce alcohol 2t (Scheme 2).
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Compounds 2a-2t were obtained in high yields and characterized by means of H-, C-, F-NMR
spectroscopy and HRMS. Compounds used in biological assays were >95% pure, as determined by
HPLC. Sodium salts thereof were in case produced by treating BTD derivatives with NaOH in
methanol.
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O
S
O
O
S
O
KMnO4
NaOHaq
O
7
7
NH
NH
H3C
H2O
HO
N
H
O
N
H
O
5
5
0°C
80°C
2b:
2c:
R = 5-CH3
R = 7-CH3
2r:
2s:
R = 5-COOH
R = 7-COOH
7
8
9
O
S
O
O
S
O
O
HO
BBr3
DCM
NH
NH
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N
H
O
N
H
O
r.t
0°C
2m
2t
Scheme 2: Synthesis of derivatives 2r-t.
2.2 Carbonic Anhydrases Inhibition
Compounds 2a-t were assayed in vitro for their inhibitory action against six physiologically
relevant human CA isoforms – i.e. the cytosolic I, II (ubiquitous isoforms) and VII (defined as a
CNS-associated isozyme) and the membrane associated IV (overexpressed in some CNS tumors),
IX and XII - by means of a Stopped-Flow CO2 hydrase assay using the clinically used drug AAZ as
a standard.24 The following structure−activity relationship (SAR) can be assembled from the data
reported in Table 1.
Table 1. Inhibition data of human CA isoforms I, II, IV, VII, IX, XII with compounds 2a-t and the standard
inhibitor acetazolamide (AAZ) by a Stopped Flow CO2 Hydrase Assay.24
O
S
O
8
7
6
NH
R
N
O
5
R1
KI (nM)a
CA IV
Cmpd
R
R1
CA I
866.0
968.7
1066
1274
2541
1920
546.7
1289
930.2
1864
2324
4403
3809
2071
3406
>10000
CA II
150.3
143.8
871.7
763.5
2192
2936
140.1
533.6
685.5
1141
526.9
2362
1922
483.6
853.1
2337
CA VII
465.8
975.3
956.0
690.9
1899
CA IX
78.7
63.9
45.8
25.8
367.6
408.5
58.4
19.1
28.2
56.9
73.3
31.0
35.8
60.2
139.5
188.9
CA XII
53.1
69.3
74.3
38.3
458.7
764.0
42.7
24.9
42.5
70.8
75.8
96.6
64.5
104.9
181.5
325.7
2ab
2b
2c
2d
2e
2f
2g
2h
2i
H
5-CH3
7-CH3
5,7-diCH3
5,8-diCH3
6,8-diCH3
5-F
H
H
H
H
H
H
H
H
H
675.3
1228
896.6
1637
1427
2692
461.0
708.6
1031
1748
853.6
1557
1945
2674
3459
5885
1790
125.5
435.5
389.3
1178
620.3
467.8
1364
3949
7507
3238
7-F
5-Cl
2j
7-Cl
H
2k
2l
5-Br
7-Br
H
H
2m
2n
2o
2p
7-OCH3
5,6-benzo
H
H
H
CH3
CH3
7-Cl
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2q
2r
2s
5-CH2-CH2-
5-COOH
7-COOH
7-OH
6857
5783
>10000
1894
>10000
>10000
>10000
5080
1060
2445
6775
3024
5950
>10000
>10000
960.0
12.5
3894
379.8
76.1
452.4
7920
n.d.
n.d.
286.0
74.0
1282
2796
4965
2565
10
n.d.
n.d.
8550
2.5
273.4
91.2
98.7
88.1
103
247.2
27.0
15.5
34.6
633
600
>10000
4.5
H
H
H
2t
SACc
SCGd
ACEe
SLC-0111
AAZ
-
-
-
-
-
50
2400
45.0
25.0
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250
5.7
a. Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5-10 % of the reported values); b.
data in agreement with ref. 21; c. data from ref. 14; d. data from ref. 15; e. data from ref. 17.
Most compounds among 2a-t inhibited the ubiquitous off-target CA I in the micromolar range with
inhibition constants (KI) spanning between 1066 and over 10000 nM, whereas the unsubstituted
(2a), 5-CH3 (2b), 5-F (2g) and 5-Cl (2i) derivatives were high nanomolar inhibitors (KIs in the
range 546.7-968.7 nM).
The physiologically dominant CA II was shown to be slightly more inhibited by BTDs than
CA I as a minor number of compounds acted in the micromolar range, from 1060 to 6775 nM (2j,
2l, 2m, 2p-t) and most displayed medium to high nanomolar KIs (in the range 140.1-871.7 nM).
The unsubstituted (2a), 5-CH3 (2b) and 5-F (2g) derivatives resulted to be the most potent CA II
inhibitors of the series with KIs of 150.3, 143.8 and 140.1 nM), showing that the CA II active site
efficiently tolerates solely small substituents appended at the 5-position of the scaffold.
CA IV was inhibited in a comparable range with CA I by BTDs 2a-2t with most compounds
functioning with KIs between 1031 and 5885 nM, and a small subset (2a, 2c, 2g, 2h, 2k) acting in
the high nanomolar range (461.0-896.6 nM). Of note is the case of derivatives bearing hydrophilic
groups on the aromatic ring, that are 2r-t, which showed enhanced CA IV inhibitory properties such
as the 76.1 nM KI of the 7-carboxy-BTD 2s. The presence of a markedly hydrophilic pocket is a
unique hallmark of CA IV active site structure that might account for the inhibition data shown by
2r-t.25
The other cytosolic isoform CA VII showed, in contrast, an inhibition profile that resembled that of
CA II, according significant similarities existing between their active sites.26 Nonetheless, the 5-F-
BTD 2g solely displayed a KI below 200 nM (125.5 nM) and the halogen swapping with a chlorine
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atom produced the second best CA VII inhibitor with a KI of 389.3 nM. Also the unsubstituted (2a),
7-F (2h) and 7-Br (2l) derivatives reported KIs minor than 500 nM (435.5-467.8 nM), whereas the
5-CH3-derivative 2b showed a worsening of CA VII inhibition when compared to CA II (from
143.8 to 975.3 nM).
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In agreement with the planned drug-design, the inhibitory profiles measured against CA IX
for BTDs 2a-2t deviate from those of the other assayed isoforms. The compounds KI values
spanned between 19.1 and 408.5 nM. A rather flat inhibition trend can be seen from data in Table 1
for derivatives acting on CA IX below 100 nM, as KIs lie in the range 31.0-98.7 nM, except for the
5,7-diCH3 (2d), 7-F (2h) and 5-Cl (2i) derivatives which resulted to be the best CA IX inhibitor of
the study with KIs of 25.8, 19.1 and 28.2 nM, respectively. A subset of compounds, among which
the 5,8-diCH3 (2e) and 6,8-diCH3 (2f) derivatives and the N-substituted 2o-q reported, in contrast,
over 100 nM KIs which spanned from 139.5 and 408.5 nM.
Again, structural similarities between CA IX and XII active sites yielded analogies in the
inhibitory profiles of the two isoforms with BTDs 2a-2t.26 CA XII was, in fact, efficiently inhibited
by most derivatives with KIs settling in the range 15.5-764.0 nM. While the 7-F derivative 2h
retained one of the most efficient activities with a KI of 24.9 nM, a worsening can be observed for
2d and 2i (KI of 38.3 and 42.5 nM). In contrast, an inhibition increase with respect to CA IX was
shown by the carboxy and hydroxy derivatives 2r-t (KIs in the range 15.5-34.6 nM), with the 7-
carboxy 2r resulting the best CA XII inhibitor of the series. It should be stressed that one of the
dissimilarities in the active site architectures of the tumor-associated CAs is the presence of a set of
Thr and Ser residues uniquely in CA XII, which might produce H-bond networks accounting for the
2r-t inhibition potency. Again, the 5,8-diCH3 (2e) and 6,8-diCH3 (2f) derivatives and the N-
substituted 2o-q reported the weakest CA XII inhibition.
Overall, most compounds of the BTD series showed significantly improved CA IX and XII
inhibitory action than the lead SAC (KIs of 103 and 633 nM) and ACE (KIs of 2400 and >10000
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nM), but also reported an increased binding to CA I, II and IV with respect to the leads. A marked
drop of efficacy was instead displayed by 2a-t against CA VII compared to SAC (KIs of 10 nM).
Comparable inhibition profiles were detected for most BTDs with SLC-0111. In fact, this
ureidobenzenesulfonamide, currently in Phase II/b clinical trials as antitumor CAI, inhibits CA I
and VII in the low micromolar range and CA II with a KI of 960 nM. The target CA IX is inhibited
with a KI of 45.0 nM. In contrast, it displays a more marked CA IV inhibition (KI of 286.0 nM) than
BTD compounds as well as a more intense inhibition of the target CA XII (KI of 4.5 nM).
As for the selectivity of action of the newly reported CAIs, the selectivity index (SI) for CA IX and
XII over the remaining isoforms are reported in Table S1, Supporting Information. It is noteworthy
that all derivatives are selective inhibitors of the tumor-related CAs over off-target ones, with the
unique exception of compound 2s in the IV/IX ratio (SI = 0.8). Almost all substitution patterns on
the BTD scaffold induced better II/IX and II/XII SI than the unsubstituted 2a (1.9 and 2.8,
respectively), ranging from 2.3 to 68.6 and 2.1 to 437.1, respectively. While SI calculated for CA
VII settle in a comparable range with CA II, those reported for CA I, II and IV are markedly
greater, reaching a value major than 645.2 for 2s in the I/XII ratio.
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While SI calculated for the clinically used AAZ are easily exceeded by BTDs 2a-2t, it is reasonable
to comment on their comparison with SI measured for SAC, SCG, ACE and SLC-0111. In spite of
a more efficient inhibition of off-target CAs by the BTDs than the leads, comparable or even more
favourable SI could be extrapolated for many compounds of the series from the inhibition data of
Table 1. In fact, depending on the substitution pattern on the main scaffold some derivatives, such
as 2l, 2m and 2s in the II/IX ratio and many more in the II/XII ratio, showed increased selectivity
than the lead SAC, with the SI of ACE SI being difficult to comment because of its weak CA
inhibition. A subset of derivatives might even compete with SCG in terms of I/XII and II/XII
specificity of action. Finally, most BTDs show higher II/IX SI than SLC-0111 (21.3). However, the
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latter reports a rather high II/XII SI (213.3), which is overcome uniquely by 2s (437.1) within the
BTD series.
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2.2 X-ray Crystallography
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Crystal structures of CA II and CA IX-mimic in complex with cyclic ureidosulfonamide
inhibitors 2a, 2d, 2h, 2i, and 2o were determined in order to analyze compound binding and
rationalize the observed inhibition profiles. The crystallographic data are reported in Tables S2 and
S3, Supporting Information. Overall, these compounds bind similarly to the lead compounds SAC
and ACE (bound in CA IX) (Figure 3).
Figure 3. A) Overlay of ureidosulfonamide compounds 2a (orange, PDB 6U4Q), 2d (green, PDB 6UGN),
2h (purple, PDB 6UGR), 2i (blue, PDB 6UGP), 2o (yellow, PDB 6UH0) and lead compound SAC (pink,
PDB 2Q1B) in CA II. B) Overlay of ureidosulfonamide compounds 2a (orange, PDB 6U4T), 2d (green,
PDB 6UGO), 2h (purple, PDB 6UGZ), 2i (blue, PDB 6UGQ) and lead compounds SAC (pink, PDB 4RIV)
and ACE (salmon, PDB 5WGP) in CA IX-mimic.
As such, the aromatic N interacts directly with the zinc, displacing the zinc-bound water that is
essential for catalytic activity, in addition to DW and W1 of the proton wire, which facilitate the
transfer of a proton to bulk solvent during catalysis. Inhibitor binding was determined to be
stabilized by the formation of hydrogen bonds between the compound carbonyl and T199 side chain
hydroxyl in addition to inhibitor sulfonamide and T199 backbone amide. Binding is further
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supported by VDW interactions with active site residues such as V121, F131 (CA II), V135, L141,
V143, L198, T200, P202, and W209 (Figures 4 and S1, Supporting Information).
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Figure 4. Binding of BTD compounds displayed in the active sites of CA II (gray) and CA IX-mimic (pale
cyan). Hydrophobic residues are shaded orange and hydrophilic residues purple. The 2a (panel A and B,
PDBs 6U4Q and 6U4T), 2d (panel C and D, PDBs 6UGN and 6UGO), 2h (panel E and F, PDBs 6UGR and
6UGZ), 2i (panel G and H, PDBs 6UGP and 6UGQ), and 2o (panel J, PDB 6UH0) inhibitors are shown as
orange, green, purple, blue, and yellow sticks, respectively.
These five compounds all exhibit a greater affinity for CA IX over the off-target, ubiquitous
CA II. Interestingly, an overlay of each inhibitor bound in CA II and CA IX demonstrates that these
inhibitors bind in a similar orientation in both isoforms (Figure 5) (excluding 2o which was only
determined in CA II), which has also been previously observed for SAC binding. Therefore, the
observed specificity for CA IX is likely attributed to the feasibility of inhibitor movement and entry
into the active site. CA IX has a larger hydrophobic pocket, along which substrate and many
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inhibitors travel, due to residue V131 in place of F131 in CA II. The decrease in steric hindrance of
this residue at the opening of the active site facilitates the binding of bulkier, aromatic compounds
such as these ureidosulfonamides.
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Figure 5. Overlay of inhibitors bound in CA II and CA IX-mimic. Inhibitors are displayed in the active site
of CA IX-mimic as sticks. A) 2a (orange, PDBs 6U4Q and 6U4T), B) 2d (green, PDBs 6UGN and 6UGO),
C) 2h (purple, PDBs 6UGR and 6UGZ), and D) 2i (blue, PDBs 6UGP and 6UGQ).
Compounds 2d, 2h, 2i have similar affinities for CA IX that are approximately 3- and 7-fold
higher than those of 2a and 2o, respectively. Generally, the affinity of the inhibitor is observed to
increase with the hydrophobicity of substitutions. Substitution of two positions or the addition of a
halogen increases the number and/or strength of VDW and hydrophobic interactions with
surrounding active site residues. Although the structure of 2o was not determined in complex CA
IX-mimic, an overlay of the five compounds in CA II shows that 2o is shifted, reflecting the
weakest affinity observed for both CA II and CA IX. This observation can be attributed to steric
hindrance of the addition methyl group on the N with the side chain of T200, likely forcing the
compound to bind in a less energetically favorable orientation.
2.3 Anticancer Activity
2.3.1. Anti-proliferative activity
The in vitro anti-proliferative action of the BTDs 2a, 2d, 2h, 2i, 2o was assessed against three
human cancer cell lines overexpressing CA IX, namely A549 (lung), PC-3 (prostate) and HCT-116
(colon),27-29 using an MTT reduction assay as described by T. Mosmann.30 Staurosporine was used
as reference anticancer drug. The five BTDs were evaluated under both normoxic and hypoxic
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conditions, where the cobalt (II) chloride hexahydrate was adopted as the chemical inducer of HIF-
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1α to establish the hypoxic conditions.31 The results are expressed as IC50 values and listed in Table
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Table 2. In vitro anti-proliferative activity of compounds 2a, 2d, 2h, 2i, 2o against lung A549, prostate PC-3
and colon HCT116 cancer cell lines under normoxic and hypoxic conditions.
IC50 (μM)a
Compound
A549
PC-3
HCT116
Normoxia
3.53±0.16
4.77±0.22
18.32±1.3
10.88±0.73
19.86±0.97
7.47±0.39
Hypoxia
2.68±0.31
5.74±0.28
Normoxia
Hypoxia
12.25±1.1
0.82±0.06
6.53±0.43
3.52±0.24
16.93±0.99
5.92±0.24
Normoxia
Hypoxia
1.50±0.53
4.18±0.27
2.73±0.14
7.57±0.51
7.13±0.38
16.15±0.57
2a
13.95±0.75
6.31±0.41
11.98±1.2
16.23±0.92
23.76±1.92
4.91±0.17
1.30±0.06
3.85±0.22
3.86±0.19
5.26±0.36
10.88±0.41
8.86±0.33
2d
2h
15.01±0.92
7.35±0.36
16.34±2.1
13.22±0.82
2i
2o
Staurosporine
a. IC50 values are the mean ± S.D. of three separate experiments.
The IC50 values in Table 2 revealed that 2a, 2d, 2h, 2i and 2o exhibited moderate to excellent
growth inhibition activity against the tested cancer cell lines. In particular, the assayed BTDs were
more effective against colon HCT-116 cells than A549 and PC-3 cells, except 2d and 2i which
displayed an enhanced growth inhibitory activity against PC-3 cells under the hypoxic conditions.
As for the activity against HCT-116, the data displayed in Table 2 ascribed to all the examined
BTDs excellent efficacy in inhibiting the growth of the cells under both normoxic (IC50 in the range
1.30 – 10.88 µM) and hypoxic (IC50 in the range 1.50 – 7.57 µM) conditions. In particular,
compounds 2a and 2h were found to be the most potent of the series against HCT-116 cells under
hypoxic conditions with IC50 values of 1.50±0.53 and 2.73±0.14 μM, respectively. In contrast,
compounds 2a and 2d emerged as the most potent derivatives against A549 cells under both
normoxic (IC50 = 3.53±0.16 and 4.77±0.22 µM, respectively) and hypoxic (IC50 = 2.68±0.31 and
5.74±0.28 µM, respectively) conditions. Compound 2d, instead, showed the best growth inhibitory
activity against PC-3 cells with IC50 values of 6.31±0.41 and 0.82±0.06 μM under normoxic and
hypoxic conditions, respectively.
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The SAR outcomes illustrate that the substituents on the BTD scaffold such as in 2d, 2h and 2i
increase the growth inhibition action against prostate PC-3 cells compared to the unsubstituted 2a.
On the other hand, N-4 methylation (2o), produced a worsening of effectiveness against the three
tested cancer cell lines in comparison to N-4 unsubstituted analogue 2a. Notably, such methylation
decreased the CA IX and XII inhibitory activities as well.
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Additionally, compounds 2a, 2d, 2h, 2i and 2o were examined for their cytotoxic effect toward the
non-tumorigenic lung fibroblast WI-38 cell line to investigate their safety profile (Table 3). The
examined BTDs displayed significantly minor cytotoxic impact against the non-tumorigenic cell
line when compared to HCT116 cells with IC50 in the range 23.5 ± 0.8 – 124.6±3.6 μM. Derivatives
2a and 2h stood out among the others for a good safety profile with SI of 30.7 and 32.3,
respectively (Table 3).
Table 3. In vitro cytotoxic activity of BTDs 2a, 2d, 2h, 2i and 2o against non-tumorigenic WI-38
cells, and WI-38/HCT116 SI.
Compound
IC50 WI-38 (μM)a
SI WI-38/HCT116
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2d
2h
2i
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23.5 ± 0.8
124.6 ± 3.6
36.6 ± 1.8
40.89 ± 3.2
30.7
6.1
32.3
7.0
2o
3.8
a. IC50 values are the mean ± S.D. of three separate experiments.
2.3.1. Effects on the levels of apoptotic markers
The ability of 2a and 2h to provoke apoptosis in colon HCT-116 cells was assessed by
measuring the expression levels of apoptosis hallmark parameters upon treatment with the
compounds at their IC50 concentrations (1.30 and 3.86 µM, respectively) (Figure 6, Tables S3 and
S4). The exposure of HCT-116 cells to 2a and 2h induced a significant down-regulation of the
expression levels of the anti-apoptotic protein Bcl-2 by approximately 59% and 42%,
respectively, with a concurrent 8.12- and 4.73-fold increase, respectively, in the expression of the
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pro-apoptotic protein Bax, compared to the control. Accordingly, the Bax/Bcl-2 ratio was
calculated to be boosted by 21.4- and 8.6-fold, respectively, in comparison to the control (Figure 6
and Table S4, Supporting Information).
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Figure 6. Increase in Bax/Bcl-2 ratio and expression levels of Bax, caspase-3, caspase-9, and p53 in
HCT116 cancer cells upon treatment with 2a and 2h in comparison to the control.
Thereafter, the expression levels of active caspase-3, caspase-9 and p53 tumor suppressor
protein were evaluated. Treatment of HCT-116 cells with 2a and 2h led to a significant increase
in the expression levels of the pro-apoptotic caspase-3 (by 21.7- and 16.3-fold, respectively),
caspase-9 (by 11.7- and 8.2-fold, respectively), and p53 (by 25.5- and 15.3-fold, respectively),
compared to control (Figure 6 and Table S5, Supporting Information).
3. Conclusions
After their kinetical and structural characterization as selective inhibitors of the tumor-
associated CAs IX and XII, the sweeteners SAC and ACE have entered the topic of anticancer
CAIs. Here, a drug design strategy over the structures of SAC and ACE was reported, which
produced a new series of 2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxides (BTD). Many such
derivatives showed enhanced potency (CA IX KIs in the range 19.1-408.5 nM; CA XII KIs in the
range 15.5-764.0 nM) and in some cases selectivity (II/IX selectivity index between 2 and 76; II/XII
selectivity index between 2 and 440) when compared to the leads against the target CA IX and XII
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over off-target isoforms. A thorough X-ray crystallographic study conducted on 5 derivatives with
both CA II and IX-mimic enabled the speculation that the larger hydrophobic pocket in CA IX
compared to CA II, along which substrate and many inhibitors travel, facilitates the binding of
bulkier, aromatic compounds such as these ureidosulfonamides. A subset of compounds selected for
the in vitro evaluation of their anticancer properties (2a, 2d, 2h, 2i and 2o) exhibited a greater
efficacy against colon HCT-116 than A549 and PC-3 cells, except 2d and 2i which displayed an
enhanced growth inhibition against PC-3 cells under the hypoxic conditions. In particular, BTDs 2a
and 2h (IC50 against HCT-116 cells of 1.30±0.06 and 3.86±0.19 μM in normoxia, and 1.50±0.53
and 2.73±0.14 μM in hypoxia, respectively) were selected to further assess their effect on apoptosis
markers in HCT-116 cells. Treatment with 2a and 2h led to a significant increase in the expression
levels of the pro-apoptotic proteins Bax, caspase-3, caspase-9, and p53 and a down-regulation of the
anti-apoptotic protein Bcl-2. These outcomes indicate that SAC and ACE, widely used as
sweeteners might be used for designing promising leads for the development of new anticancer
drugs.
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4. Experimental Section
4.1 Chemistry
Anhydrous solvents and all reagents were purchased from Sigma-Aldrich (Milan, Italy), Alfa Aesar
(Milan, Italy) and TCI (Milan, Italy). All reactions involving air- or moisture-sensitive compounds
were performed under a nitrogen atmosphere using dried glassware and syringes techniques to
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transfer solutions. Nuclear magnetic resonance spectra (1H-NMR: 400 MHz; C-NMR: 100 MHz;
19F-NMR: 376 MHz) were recorded in DMSO-d6 using an Avance III 400 MHz spectrometer
(Bruker, Milan, Italy). 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; t, triplet; q, quadruplet; m, multiplet; brs, broad singlet; dd, double of doublets. The
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assignment of exchangeable protons (OH and NH) was confirmed by the addition of D2O.
Analytical thin-layer chromatography (TLC) was carried out on silica gel F-254 plates (Merck,
Milan, Italy). Flash chromatography purifications were performed on Merck Silica gel 60 (230-400
mesh ASTM) as the stationary phase and ethyl acetate/n-hexane were used as eluents. Melting
points (m.p.) were carried out in open capillary tubes and are uncorrected.
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The HPLC analysis was performed by using an Agilent 1200 Series equipped by autosampler,
binary pump system and diode array detector (DAD). The column used was a Luna PFP 30 mm
length, 2 mm internal diameter and 3 μm particle size (Phenomenex, Bologna, Italy) at constant
flow of 0.25 mL min–1, employing a binary mobile phase elution gradient. The eluents used were 10
mM formic acid and 5 mM ammonium formate in mQ water solution (solvent A) and 10 mM
formic acid and 5 mM ammonium formate in methanol (solvent B) according to the elution gradient
as follows: initial at 90% solvent A, which was then decreased to 10% in 8 min, kept for 3 min,
returned to initial conditions in 0.1 min and maintained for 3 min for reconditioning, to a total run
time of 14 min. The stock solution of each analyte was prepared in methanol at 1.0 mg mL-1 and
stored at 4 °C. The sample solution of the analyte was freshly prepared by diluting its stock solution
up to a concentration of 10 μg mL-1 in mixture of mQ water:methanol 50:50 (v/v) and 5 μL were
injected in the HPLC system. All compounds reported were >95% HPLC pure. The solvents used in
HPLC measures were methanol (Chromasolv grade), purchased from Sigma-Aldrich (Milan - Italy),
and mQ water 18 MΩ cm, obtained from Millipore's Simplicity system (Milan-Italy).
High resolution mass spectrometry (HR-MS) analysis was performed with a Thermo Finnigan LTQ
Orbitrap mass spectrometer equipped with an electrospray ionization source (ESI). Analysis were
carried out in negative ion mode monitoring the [M-H]- species, and a proper dwell time acquisition
was used to achieve 60,000 units of resolution at Full Width at Half Maximum (FWHM). Elemental
composition of compounds were calculated on the basis of their measured accurate masses,
accepting only results with an attribution error less than 5 ppm and a not integer RDB (double
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bond/ring equivalents) value, in order to consider only the deprotonated species.32 None of the
screened derivatives reported PAINS alerts determined by SwissADME server
(www.swissadme.ch).
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General procedure for the synthesis of compounds 2a-q.
A solution of chlorosulfonyl isocyanate (0.48 mL, 1.1 eq.) in the proper solvent (nitromethane or
nitroethane) was treated with a solution of the appropriate aromatic amine (1.0 eq.) in the same
solvent (5 mL) at –40 °C. The reaction mixture was stirred for 15 min before AlCl3 (1.0 eq.) was
added and then it was warmed at 110 °C and stirred for 45 min. The resulting mixture was cooled
and poured onto ice. The formed precipitate was collected by filtration and dried to obtain a powder
that was purified by silica gel column chromatography eluting with appropriate mixture of solvents
or alternatively by dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment with
charcoal, filtration over Celite and re-acidification to pH 1. The precipitate was filtered in vacuo to
afford the titled compounds 2a-q.
2H-Benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (2a)
Compound 2a was obtained according to the general procedure earlier reported adding dropwise a
solution of aniline (0.46 mL, 1.0 eq.) in nitromethane (6.0 mL) to a solution of chlorosulfonyl
isocyanate (0.48 mL, 1.12 eq.) in nitromethane (6.0 mL). The crude compound was purified by
dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment with charcoal, filtration
over Celite and re-acidification to pH 1. The precipitate was filtered in vacuo to afford the title
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compound 2a. 31% yield; m.p. >300°C; TLC: Rf = 0.30 (methanol/dichloromethane 20% v/v); H-
NMR (DMSO-d6, 400 MHz): δ 7.27 (2H, m, Ar-H), 7.64 (1H, t, J = 8.0, Ar-H), 7.78 (1H, d, J =
13
7.6, Ar-H), 11.09 (1H, s, exchange with D2O, CONH); C-NMR (DMSO-d6, 100 MHz): δ 117.7,
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122.9, 123.5, 124.1, 134.6, 136.2, 151.9; ESI-HRMS (m/z) [M-H]-: calculated for C7H5N2O3S
197.0021; found 197.0028.
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5-Methyl-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (2b)
Compound 2b was obtained according to the general procedure earlier reported adding dropwise a
solution of o-toluidine (1.0 g., 1.0 eq.) in nitroethane (8.0 mL) to a solution of chlorosulfonyl
isocyanate (0.89 mL, 1.1 eq.) in nitroethane (7.0 mL). The crude compound was purified by
dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment with charcoal, filtration
over Celite and re-acidification to pH 1. The precipitate was filtered in vacuo to afford 2b. 26%
yield; m.p. 292-293°C; TLC: Rf = 0.13 (methanol/dichloromethane 15% v/v); 1H-NMR (DMSO-d6,
400 MHz): δ 2.40 (3H, s, CH3), 7.25 (1H, t, J = 7.6, Ar-H), 7.55 (1H, d, J = 7.6, Ar-H), 7.66 (1H, d,
J = 7.6, Ar-H), 10.42 (1H, s, exchange with D2O, CONH); 13C-NMR (DMSO-d6, 100 MHz): δ 18.4,
120.4, 124.1, 124.4, 127.3, 134.1, 136.1, 151.6; ESI-HRMS (m/z) [M-H]-: calculated for
C8H7N2O3S 211.0177; found 211.0181.
7-Methyl-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (2c)
Compound 2c was obtained according to the general procedure earlier reported adding dropwise a
solution of p-toluidine (1.0 g., 1.0 eq.) in nitromethane (9.7 mL) to a solution of chlorosulfonyl
isocyanate (0.60 mL, 1.2 eq.) in nitromethane (6.4 mL). The crude compound was purified by
dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment with charcoal, filtration
over Celite and re-acidification to pH 1. The precipitate was filtered in vacuo to afford 2c as a white
1
solid. 40% yield; m.p. 287-288°C; TLC: Rf = 0.30 (methanol/dichloromethane 30% v/v); H-NMR
(DMSO-d6, 400 MHz): δ 2.38 (3H, s, CH3), 7.17 (1H, d, J = 8.4, Ar-H), 7.49 (1H, dd, J = 8.0, 1.2,
13
Ar-H), 7.63 (1H, s, Ar-H), 11.18 (1H, s, exchange with D2O, CONH); C-NMR (DMSO-d6, 100
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MHz): δ 21.0, 117.8, 122.4, 123.3, 133.6, 134.0, 135.6, 151.5; ESI-HRMS (m/z) [M-H]-: calculated
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5
6
for C8H7N2O3S 211.0177; found 211.0169.
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5,7-Dimethyl-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (2d)
Compound 2d was obtained according to the general procedure earlier reported adding dropwise a
solution of 2,4-dimenthylaniline (1.02 mL, 1.0 eq.) in nitromethane (5.0 mL) to a solution of
chlorosulfonyl isocyanate (0.93 mL, 1.3 eq.) in nitromethane (5.0 mL). The crude compound was
purified by dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment with
charcoal, filtration over Celite and re-acidification to pH 1. The precipitate was filtered in vacuo to
afford 2d as a white solid. 60% yield; m.p. 290-291°C; TLC: Rf = 0.23 (methanol/dichloromethane
1
20% v/v); H-NMR (DMSO-d6, 400 MHz): δ 2.35 (6H, d, J = 5.6, 2 x CH3), 7.37 (1H, s, Ar-H),
7.48 (1H, s, Ar-H), 10.35 (1H, s, exchange with D2O, CONH); 13C-NMR (DMSO-d6, 100 MHz): δ
18.3, 20.7, 119.9, 124.3, 127.1, 131.7, 133.7, 136.9, 151.5; ESI-HRMS (m/z) [M-H]-: calculated for
C9H9N2O3S 225.0334; found 225.0329.
5,8-Dimethyl-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (2e)
Compound 2e was obtained according to the general procedure earlier reported adding dropwise a
solution of 2,5-dimenthylaniline (1.03 mL, 1.0 eq.) in nitromethane (8.0 mL) to a solution of
chlorosulfonyl isocyanate (0.93 mL, 1.3 eq.) in nitromethane (7.0 mL). The crude compound was
purified by dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment with
charcoal, filtration over Celite and re-acidification to pH 1. The precipitate was filtered in vacuo to
afford 2e as a white solid. 66% yield; m.p. 297-298°C; TLC: Rf = 0.16 (methanol/dichloromethane
20% v/v); 1H-NMR (DMSO-d6, 400 MHz): δ 2.35 (3H, s, 5-CH3), 2.57 (3H, s, 8-CH3), 7.04 (1H, d,
13
J = 7.6, Ar-H), 7.38 (1H, d, J = 8.0, Ar-H), 10.17 (1H, s, exchange with D2O, CONH); C-NMR
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(DMSO-d6, 100 MHz): δ 18.4, 20.0, 123.2, 124.6, 126.7, 132.9, 134.4, 135.1, 151.2; ESI-HRMS
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6
(m/z) [M-H]-: calculated for C9H9N2O3S 225.0334; found 225.0338.
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6,8-Dimethyl-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (2f)
Compound 2f was obtained according to the general procedure earlier reported adding dropwise a
solution of 3,5-dimethylaniline (1.03 mL, 1.0 eq.) in nitromethane (5.0 mL) to a solution of
chlorosulfonyl isocyanate (0.93 mL, 1.3 eq.) in nitromethane (5.0 mL). The crude compound was
purified by dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment with
charcoal, filtration over Celite and re-acidification to pH 1. The precipitate was filtered in vacuo to
afford 2f as a solid. 32% yield; m.p. 282-283°C; TLC: Rf = 0.11 (methanol/dichloromethane 20%
v/v); 1H-NMR (DMSO-d6, 400 MHz): δ 2.32 (3H, s, 6-CH3), 2.54 (3H, s, overlap with DMSO peak,
13
8-CH3), 6.89 (1H, s, Ar-H), 6.95 (1H, s, Ar-H), 11.17 (1H, s, exchange with D2O, CONH); C-
NMR (DMSO-d6, 100 MHz): δ 20.0, 21.9, 115.6, 119.7, 127.9, 135.5, 136.4, 144.2, 151.1; ESI-
HRMS (m/z) [M-H]-: calculated for C9H9N2O3S 225.0334; found 225.0341.
5-Fluoro-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (2g)
Compound 2g was obtained according to the general procedure earlier reported adding dropwise a
solution of 2-fluoroaniline (0.87 mL, 1.0 eq.) in nitroethane (5.0 mL) to a solution of chlorosulfonyl
isocyanate (0.94 mL, 1.2 eq.) in nitroethane (5.0 mL). The crude compound was purified by
dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment with charcoal, filtration
over Celite and re-acidification to pH 1. The precipitate was filtered in vacuo to afford 2g. 10%
yield; m.p. 277-278°C; TLC: Rf = 0.48 (ethyl acetate/n-hexane 70% v/v); 1H-NMR (DMSO-d6, 400
MHz): δ 7.34 (1H, m, Ar-H), 7.64 (2H, m, Ar-H), 11.31 (1H, s, exchange with D2O, CONH); 13C-
NMR (DMSO-d6, 100 MHz): δ 118.7 (J3 = 4 Hz), 120.5 (J2 = 18), 124.9 (J3 = 6), 125.2
C-F
C-F
C-F
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(J2 = 15), 125.8, 150.2 (J1 = 249), 151.5; F-NMR (DMSO-d6, 376 MHz) −125.93 (1F, s);
C-F
C-F
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ESI-HRMS (m/z) [M-H]-: calculated for C7H4FN2O3S 214.9927; found 214.9935.
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7-Fluoro-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (2h)
Compound 2h was obtained according to the general procedure earlier reported adding dropwise a
solution of 4-fluoroaniline (1.0 g., 1.0 eq.) in nitromethane (5.0 mL) to a solution of chlorosulfonyl
isocyanate (1.02 mL, 1.3 eq.) in nitromethane (5.0 mL). The crude compound was purified by
dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment with charcoal, filtration
over Celite and re-acidification to pH 1. The precipitate was filtered in vacuo to afford 2h as a solid.
1
38% yield; m.p. 288-289°C; TLC: Rf = 0.28 (methanol/dichloromethane 30% v/v); H-NMR
(DMSO-d6, 400 MHz): δ 7.32 (1H, m, Ar-H), 7.59 (1H, m, Ar-H), 7.72 (1H, m, Ar-H), 11.41 (1H,
s, exchange with D2O, CONH); 13C-NMR (DMSO-d6, 100 MHz): δ 109.6 (J2C-F = 26), 120.3 (J2
C-F
= 8), 122.5 (J3 = 23), 124.1 (J2 = 7), 132.7 (J4 = 2), 152.1, 158.3 (J1 = 242); F-NMR
19
C-F
C-F
C-F
C-F
(DMSO-d6, 376 MHz) −117.29 (1F, s); ESI-HRMS (m/z) [M-H]-: calculated for C7H4FN2O3S
214.9927; found 214.9921.
5-Chloro-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (2i)
Compound 2i was obtained according to the general procedure earlier reported adding dropwise a
solution of 2-chloraniline (0.82 mL, 1.0 eq.) in nitroethane (6.0 mL) to a solution of chlorosulfonyl
isocyanate (0.89 mL, 1.3 eq.) in nitroethane (6.0 mL). The crude compound was purified by
dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment with charcoal, filtration
over Celite and re-acidification to pH 1. The precipitate was filtered in vacuo to afford 2i as a white
1
solid. 34% yield; m.p. 246-247°C; TLC: Rf = 0.08 (methanol/dichloromethane 10% v/v); H-NMR
(DMSO-d6, 400 MHz): δ 7.36 (1H, t, J = 8.0, Ar-H), 7.85 (2H, m, Ar-H), 10.71 (1H, s, exchange
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with D2O, CONH); C-NMR (DMSO-d6, 100 MHz): δ 121.5, 122.0, 125.4, 126.1, 133.1, 135.1,
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6
151.4; ESI-HRMS (m/z) [M-H]-: calculated for C7H4ClN2O3S 230.9631; found 230.9626.
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7-Chloro-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (2j)
Compound 2j was obtained according to the general procedure earlier reported adding dropwise a
solution of 4-chloroaniline (1.0 g, 1.0 eq.) in nitroethane (6.0 mL) to a solution of chlorosulfonyl
isocyanate (0.82 mL, 1.2 eq.) in nitroethane (6.0 mL). The crude compound was purified by
dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment with charcoal, filtration
over Celite and re-acidification to pH 1. The precipitate was filtered in vacuo to afford 2j as a solid.
1
48% yield; m.p. 285-286°C; TLC: Rf = 0.11 (methanol/dichloromethane 15% v/v); H-NMR
(DMSO-d6, 400 MHz): δ 7.30 (1H, d, J = 8.8, Ar-H), 7.74 (1H, t, J = 8.0, 2.4, Ar-H), 7.86 (1H, d, J
= 2.0, Ar-H), 11.47 (1H, s, exchange with D2O, CONH); 13C-NMR (DMSO-d6, 100 MHz): δ 120.1,
122.6, 124.5, 128.1, 134.7, 135.0, 152.0; ESI-HRMS (m/z) [M-H]-: calculated for C7H4ClN2O3S
230.9631; found 230.9636.
5-Bromo-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (2k)
Compound 2k was obtained according to the general procedure earlier reported adding dropwise a
solution of 2-bromoaniline (1.0 g, 1.0 eq.) in nitroethane (5.0 mL) to a solution of chlorosulfonyl
isocyanate (0.61 mL, 1.2 eq.) in nitroethane (5.0 mL). The crude compound was purified by
dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment with charcoal, filtration
over Celite and re-acidification to pH 1. The precipitate was filtered in vacuo to afford 2k as a solid.
1
25% yield; m.p. 250-251°C; TLC: Rf = 0.19 (methanol/dichloromethane 15% v/v); H-NMR
(DMSO-d6, 400 MHz): δ 7.29 (1H, t, J = 8.0, Ar-H), 7.86 (1H, d, J = 7.2 Ar-H), 8.00 (1H, dd, J =
13
8.4, 1.2, Ar-H), 10.28 (1H, s, exchange with D2O, CONH); C-NMR (DMSO-d6, 100 MHz): δ
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111.1, 122.6, 125.8, 126.2, 134.2, 138.4, 151.4; ESI-HRMS (m/z) [M-H]-: calculated for
C7H4BrN2O3S 274.9126; found 274.9120.
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7-Bromo-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (2l)
Compound 2l was obtained according to the general procedure earlier reported adding dropwise a
solution of 4-bromoaniline (1.0 g., 1.0 eq.) in nitromethane (5.0 mL) to a solution of chlorosulfonyl
isocyanate (0.60 mL, 1.2 eq.) in nitromethane (5.0 mL). The crude compound was purified by
dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment with charcoal, filtration
over Celite, re-acidification to pH 1. The precipitate was filtered in vacuo to afford 2l as a white
1
solid. 30% yield; m.p. >300°C; TLC: Rf = 0.11 (methanol/dichloromethane 20% v/v); H-NMR
(DMSO-d6, 400 MHz): δ 7.24 (1H, d, J = 8.8, Ar-H), 7.85 (1H, dd, J =8.8, 2.2, Ar-H), 7.95 (1H, d,
J = 2.0, Ar-H), 11.46 (1H, s, exchange with D2O, CONH); 13C-NMR (DMSO-d6, 100 MHz): 115.5,
120.3, 124.9, 125.2, 135.4, 137.4, 152.0; ESI-HRMS (m/z) [M-H]-: calculated for C7H4BrN2O3S
274.9126; found 274.9130.
7-Methoxy-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (2m)
Compound 2m was obtained according to the general procedure earlier reported adding dropwise a
solution of p-anisidine (1.0 g, 1.0 eq.) in nitromethane (4.0 mL) to a solution of chlorosulfonyl
isocyanate (0.78 mL, 1.1 eq.) in nitromethane (5.7 mL). The crude compound was purified by silica
gel column chromatography eluting with from 1 to 15% v/v MeOH/DCM to give a residue which
was further purified by dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment
with charcoal, filtration over Celite, re-acidification to pH 1. The precipitate was filtered in vacuo to
afford 2m as a solid. 17% yield; m.p. 284-285°C; TLC: Rf = 0.15 (methanol/dichloromethane 5%
1
v/v); H-NMR (DMSO-d6, 400 MHz): δ 3.85 (3H, s, CH3), 7.26 (3H, m, Ar-H), 11.15 (1H, s,
13
exchange with D2O, CONH); C-NMR (DMSO-d6, 100 MHz): δ 56.9, 105.5, 119.7, 122.7, 123.9,
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129.5, 151.8, 156.1; ESI-HRMS (m/z) [M-H]-: calculated for C8H7N2O4S 227.0127; found
227.0122.
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1H-Naphtho[1,2-e][1,2,4]thiadiazin-2(3H)-one 4,4-dioxide (2n)
Compound 2n was obtained according to the general procedure earlier reported adding dropwise a
solution of 1-naphthylamine (1.0 g, 1.0 eq.) in nitroethane (5.0 mL) to a solution of chlorosulfonyl
isocyanate (0.67 mL, 1.1 eq.) in nitroethane (5.0 mL). The crude compound was purified by
dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment with charcoal, filtration
over Celite and re-acidification to pH 1. The precipitate was filtered in vacuo to afford a green
semisolid, that was purified by silica gel column chromatography eluting with 60 to 100%
EtOAc/n-hexane to give 2n. 3% yield; m.p. 293-294°C; TLC: Rf = 0.15 (methanol/dichloromethane
15% v/v); 1H-NMR (DMSO-d6, 400 MHz): δ 7.76 (3H, m, Ar-H), 7.86 (1H, d, J = 8.8, Ar-H), 8.08
(1H, d, J = 7.6, Ar-H), 8.72 (1H, d, J = 8.0, Ar-H), 11.34 (1H, s, exchange with D2O, CONH); 13C-
NMR (DMSO-d6, 100 MHz): δ 118.4, 118.8, 122.6, 123.7, 124.7, 128.5, 129.7, 129.9, 133.1, 135.9,
152.2; ESI-HRMS (m/z) [M-H]-: calculated for C11H7N2O3S 247.0177; found 247.0183.
4-Methyl-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (2o)
Compound 2o was obtained according to the general procedure earlier reported adding dropwise a
solution of N-methylaniline (1.01 mL, 1.0 eq.) in nitroethane (5.0 mL) to a solution of
chlorosulfonyl isocyanate (0.90 mL, 1.1 eq.) in nitroethane (5.0 mL). The crude compound was
purified by dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment with
charcoal, filtration over Celite and re-acidification to pH 1. The precipitate was filtered in vacuo to
afford 2o as a solid. 27% yield; m.p. 257-258°C; TLC: Rf = 0.20 (methanol/dichloromethane 15%
v/v); 1H-NMR (DMSO-d6, 400 MHz): δ 3.47 (3H, s, CH3), 7.42 (1H, t, J = 7.4, Ar-H), 7.56 (1H, d,
13
J = 8.4, Ar-H), 7.81 (1H, m, Ar-H), 7.89 (1H, dd, J = 8.0, 1.2, Ar-H); C-NMR (DMSO-d6, 100
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MHz): δ 32.5, 117.9, 122.7, 124.4, 126.1, 135.2, 137.9, 151.2; ESI-HRMS (m/z) [M-H]-: calculated
4
5
6
for C8H7N2O3S 211.0177; found 211.0182.
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7-Chloro-4-methyl-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (2p)
Compound 2p was obtained according to the general procedure earlier reported adding dropwise a
solution of 4-chloro-N-methylaniline (0.86 mL, 1.0 eq.) in nitroethane (6.0 mL) to a solution of
chlorosulfonyl isocyanate (0.74 mL, 1.2 eq.) in nitroethane (6.0 mL). The crude compound was
purified by dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment with
charcoal, filtration over Celite and re-acidification to pH 1. The precipitate was filtered in vacuo to
afford 2p as a white solid. 29% yield; m.p. 254-255°C; TLC: Rf = 0.20 (methanol/dichloromethane
15% v/v); 1H-NMR (DMSO-d6, 400 MHz): δ 3.33 (3H, m, overlap with water peak, CH3), 7.57 (1H,
d, J = 9.2, Ar-H), 7.85 (1H, dd, J = 6.4, 2.4, Ar-H), 7.94 (1H, d, J = 2.4); 13C-NMR (DMSO-d6, 100
MHz): δ 32.8, 120.1, 122.2, 127.4, 128.4, 134.8, 137.1, 151.2; ESI-HRMS (m/z) [M-H]-: calculated
for C8H6ClN2O3S 244.9788; found 244.9782.
5,6-Dihydro-[1,2,4]thiadiazino[6,5,4-hi]indol-3(2H)-one 1,1-dioxide (2q)
Compound 2q was obtained according to the general procedure earlier reported adding dropwise a
solution of indoline (0.94 mL, 1.0 eq.) in nitroethane (6.0 mL) to a solution of chlorosulfonyl
isocyanate (0.95 mL, 1.3 eq.) in nitroethane (6.0 mL). The crude compound was purified by
dissolution in a saturated solution of NaHCO3 in 1:1 H2O/MeOH, treatment with charcoal, filtration
over Celite and re-acidification to pH 1. The precipitate was filtered in vacuo to afford 2q as a solid.
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68% yield; m.p. 252-253°C; TLC: Rf = 0.15 (methanol/dichloromethane 15% v/v); H-NMR
(DMSO-d6, 400 MHz): δ 3.34 (2H, t, J = 8.6, CH2), 4.17 (2H, t, J = 8.4, CH2), 7.26 (1H, t, J = 7.6,
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Ar-H), 7.62 (2H, t, J = 8.6, Ar-H); C-NMR (DMSO-d6, 100 MHz): δ 28.0, 47.8, 119.6, 120.9,
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124.9, 130.7, 133.4, 139.4, 149.2; ESI-HRMS (m/z) [M-H]-: calculated for C9H7N2O3S 223.0177;
found 223.0173.
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3-Oxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine-5-carboxylic acid 1,1-dioxide (2r)
Compound 2b (0.5 g, 1.0 eq.) was first suspended in H2O (30 mL) and then dissolved during the
addition of saturated aqueous NaOH (0.56 g). KMnO4 (1.42 g, 3.8 eq.) was added portionwise at
0°C, and the reaction mixture was stirred at 80°C for 12 h. The insoluble material was removed by
filtration, and the filtrate was acidified to pH 1 with concentrated HCl. The precipitate was collected
by filtration, washed with Et2O, and dried to yield the product 2r as a white solid. 33% yield; m.p.
285-286°C; TLC: Rf = 0.14 (methanol/dichloromethane 20% v/v); 1H-NMR (DMSO-d6, 400 MHz):
δ 7.26 (1H, t, J = 7.8, Ar-H), 7.97 (1H, d, J = 7.6, Ar-H), 8.18 (1H, dd, J = 8.0, 0.8, Ar-H), 10.58
(1H, s, exchange with D2O, CONH), 14.01 (1H, s, exchange with D2O, COOH); 13C-NMR (DMSO-
d6, 100 MHz): δ 114.7, 122.1, 125.1, 128.8, 135.5, 139.0, 152.5, 169.3; ESI-HRMS (m/z) [M-H]-:
calculated for C8H5N2O5S 240.9919; found 240.9911.
3-Oxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine-7-carboxylic acid 1,1-dioxide (2s)
Compound 2c (0.2 g, 1.0 eq.) was first suspended in H2O (12 mL) and then dissolved during the
addition of saturated aqueous NaOH (0.23 g). KMnO4 (0.49 g, 3.3 eq.) was added portionwise at
0°C, and the reaction mixture was stirred at 80°C for 7 h. The insoluble material was removed by
filtration, and the filtrate was acidified to pH 1 with concentrated HCl. The precipitate was collected
by filtration, washed with acid water and Et2O, and dried to yield the product 2s as a white solid.
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21% yield; m.p. >300°C; TLC: Rf = 0.17 (methanol/dichloromethane 20% v/v); H-NMR (DMSO-
d6, 400 MHz): δ 7.37 (1H, d, J = 8.4, Ar-H), 8.19 (1H, m, Ar-H), 8.23 (1H, s, Ar-H), 11.64 (1H, s,
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MHz): δ 118.3, 123.2, 124.4, 126.6, 135.4, 139.5, 151.8, 166.4; ESI-HRMS (m/z) [M-H]-:
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calculated for C8H5N2O5S 240.9919; found 240.9923.
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7-Hydroxy-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (2t)
A solution of 2m (0.2 g, 1.0 eq.) in dichloromethane (15 mL) was treated with BBr3 (1.84 mL, 3.0
eq.) at –10 °C. The reaction mixture was stirred for 12 h at r.t. After completion of the reaction, the
mixture was quenched with ice and the dichloromethane was evaporated under vacuum. The
suspended solid was filtered in vacuo to afford 2t as a solid. 42% yield; m.p. 284-285°C; TLC: Rf =
0.56 (methanol/dichloromethane 30% v/v); 1H-NMR (DMSO-d6, 400 MHz): δ 7.12 (3H, m, Ar-H),
10.03 (1H, s, exchange with D2O, OH), 11.04 (1H, s, exchange with D2O, CONH), 12.67 (1H, s,
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exchange with D2O, SO2NH); C-NMR (DMSO-d6, 100 MHz): 107.3, 119.6, 122.8, 124.0, 127.9,
151.7, 154.2; ESI-HRMS (m/z) [M-H]-: calculated for C7H5N2O4S 212.9970; found 212.9974.
4.2. Carbonic anhydrases inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed
CO2 hydration activity.23 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-catalysed
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 1 h at room temperature prior to assay, in order to allow for the formation
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