A scaffold replacement approach towards new sirtuin 2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.115231

Sirtuins (SIRT1–SIRT7) are an evolutionary conserved family of NAD⁺-dependent protein deacylases regulating the acylation state of ε-N-lysine residues of proteins thereby controlling key biological processes. Numerous studies have found association of the aberrant enzymatic activity of SIRTs with various diseases like diabetes, cancer and neurodegenerative disorders. Previously, we have shown that substituted 2-alkyl-chroman-4-one/chromone derivatives can serve as selective inhibitors of SIRT2 possessing an antiproliferative effect in two human cancer cell lines. In this study, we have explored the bioisosteric replacement of the chroman-4-one/chromone core structure with different less lipophilic bicyclic scaffolds to overcome problems associated to poor physiochemical properties due to a highly lipophilic substitution pattern required for achieve a good inhibitory effect. Various new derivatives based on the quinolin-4(1H)-one scaffold, bicyclic secondary sulfonamides or saccharins were synthesized and evaluated for their SIRT inhibitory effect. Among the evaluated scaffolds, the benzothiadiazine-1,1-dioxide-based compounds showed the highest SIRT2 inhibitory activity. Molecular modeling studies gave insight into the binding mode of the new scaffold-replacement analogues.

Full text of DOI:10.1016/j.bmc.2019.115231

Journal Pre-proofs
A scaffold replacement approach towards new sirtuin 2 inhibitors
Tina Seifert, Marcus Malo, Tarja Kokkola, E. Johanna L. Stéen, Kristian
Meinander, Erik A.A. Wallén, Elina M. Jarho, Kristina Luthman
PII:
S0968-0896(19)31698-0
https://doi.org/10.1016/j.bmc.2019.115231
BMC 115231
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
15 October 2019
20 November 2019
20 November 2019
Please cite this article as: T. Seifert, M. Malo, T. Kokkola, E.J.L. Stéen, K. Meinander, E.A.A. Wallén, E.M.
Jarho, K. Luthman, A scaffold replacement approach towards new sirtuin 2 inhibitors, Bioorganic & Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.115231
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A scaffold replacement approach towards new sirtuin 2 inhibitors
Tina Seifert,a,* Marcus Malo,a Tarja Kokkola,b E. Johanna L. Stéen,a Kristian Meinander,c Erik A. A.
Wallén,c Elina M. Jarho,b Kristina Luthmana
aDepartment of Chemistry and Molecular Biology, Medicinal Chemistry, University of Gothenburg, SE-412 96
Göteborg, Sweden
bSchool of Pharmacy, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland
cDrug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of
Helsinki, P.O. Box 56,FI- 00014 Helsinki, Finland
Abstract
Sirtuins (SIRT1–SIRT7) are an evolutionary conserved family of NAD+-dependent protein
deacylases regulating the acylation state of -N-lysine residues of proteins thereby controlling key
biological processes. Numerous studies have found association of the aberrant enzymatic activity of
SIRTs with various diseases like diabetes, cancer and neurodegenerative disorders. Previously, we
have shown that substituted 2-alkyl-chroman-4-one/chromone derivatives can serve as selective
inhibitors of SIRT2 possessing an antiproliferative effect in two human cancer cell lines. In this study,
we have explored the bioisosteric replacement of the chroman-4-one/chromone core structure with
different less lipophilic bicyclic scaffolds to overcome problems associated to poor physiochemical
properties due to a highly lipophilic substitution pattern required for achieve a good inhibitory effect.
Various new derivatives based on the quinolin-4(1H)-one scaffold, bicyclic secondary sulfonamides
or saccharins were synthesized and evaluated for their SIRT inhibitory effect. Among the evaluated
scaffolds, the benzothiadiazine-1,1-dioxide-based compounds showed the highest SIRT2 inhibitory
activity. Molecular modeling studies gave insight into the binding mode of the new scaffold-
replacement analogues.
KEYWORDS Scaffold; Sirtuin; SIRT2; Inhibitor; Benzothiazine-1,1-dioxide; Benzothiadiazine-1,1-
dioxides; Saccharin; Quinolin-4-one; Docking.
1

1
Introduction
Bioisosterism is a common strategy for optimization of lead compounds in medicinal chemistry
which involves structural modifications of bioactive compounds by replacement of functional groups
or scaffolds. We have used the chromone and chroman-4-one ring systems as scaffolds for the
development of potent and selective sirtuin 2 (SIRT2) enzyme inhibitors (Fig. 1).[1,2] Sirtuins (SIRTs)
catalyze the deacylation of lysine residues on numerous protein substrates requiring NAD+ as a co-
substrate.[3,4] The SIRT enzymes are considered to be important in various pathologies such as cancer,
neurodegeneration, diabetes, inflammation and cardiovascular diseases and therefore, the development
of SIRT inhibitor is of vast interest.[5-10] Despite the synthetic efforts to develop potent inhibitors the
majority of the reported inhibitors show IC50 values in the submicromolar to micromolar range with
only a handful of examples exibiting nanolar inhibitory activity.[9,11]
A drawback of the previously described chromone and chroman-4-one based SIRT2 inhibitors (e.g.
1, Fig. 1) [1] was their high lipophilicity which prevented their use in more advanced biological assays
as they precipitated at relevant test concentrations. Efforts to increase the hydrophilicity afforded
compounds containing heterofunctional groups such as pyridyl or oxadiazole moieties (3 and 5,
respectively, Fig. 1) that allowed them to be tested for their SIRT2-mediated antiproliferative effects
in cancer cells.[2]
O
O
O
O
Cl
Cl
Cl
Cl
O
O
O
O
X
O
O
O
N
Br
Br
Br
5,
IC50 = 12.2 M (77% inh.)
N
Br
2,
3,
X = CH, IC50 = 6.8 M
(81% inh.)
X = N, IC50 = 3.7 M
(86% inh.)
1
4,
, IC50 = 4.3 M (88% inh.)
-1
(S) , IC50 = 1.5 M (91% inh.)
-1
(R) , IC50 = 4.5 µM (70% inh.)
IC50 = 2.0 M (90% inh.)
Fig. 1. SIRT2 selective chroman-4-one based inhibitors. (%-Inhibition at 200 µM inhibitor conc.)
To further improve the physicochemical properties of potential SIRT2 inhibitors we decided to
investigate the effect of replacing the chroman-4-one scaffold with other heterocyclic frameworks
(Scheme 1).
In the present study, a set of compounds based on three types of bicyclic scaffolds has been
synthesized and evaluated for their activity as sirtuin inhibitors.
2

Hydrogen-bond interaction
O
Halogen-bond
interaction
Cl
O
O
Hydrophobic interaction
O
6
S
O
R'
N
R
R'
2
8
Hydrophobic
pocket
Br
N
H
R
O
2
, clogP =1.99
A
E
, cLogP = 2.5
, cLogP = 0.91
O
S
O
O
S
O
N
O
S
O
R'
NH
R'
N
H
R
NH
R
R'
B
, cLogP = 1.45
D
, cLogP = 0.16
N
H
R
C
, cLogP = 0.36
Scheme 1. Overview over the scaffolds envisioned to replace the chroman-4-one framework in the SIRT2 inhibitors. Apart
from Scaffold A, the new bicyclic ring systems show lower lipophilicity than the chroman-4-one scaffold as reflected by
the calculated logP values (logP values are calculated of the unsubstituted scaffolds (R=R’=H)). The key-binding
interactions of the chroman-4-one based inhibitors with SIRT2 have been highlighted in 2.[2]
Quinolin-4(1H)-ones (A) and bicyclic secondary sulfonamides such as benzothiazine-1,1-dioxides
(B) and benzothiadiazine-1,1-dioxides (C–D), or saccharins (E) are versatile scaffolds found in
bioactive compounds. The quinolin-4(1H)-one is structurally similar to the chromone and is a common
scaffold in antibacterial agents such as fluoroquinolones.[12] Benzothiadiazine-1,1-dioxide
derivatives have been used as diuretic drugs since the 1950s and this scaffold is a frequent motif also
in other biologically relevant substances.[13,14] Saccharin, widely used as an artificial sweetener, has
been successfully used as a core structure in e.g. inhibitors of carbonic anhydrases [15] and is also a
key element of repinotan, a highly selective 5-HT1A-receptor agonist.[16] The new scaffold analogues
contained comparable substitution patterns to the previously studied chroman-4-ones/chromones. The
observed structure-activity relationships (SAR) were in agreement with the findings from docking
studies conducted with a SIRT2 3D model previously published.[2]
2
Results and discussion
2.1 Design
The design of new scaffold analogues was guided by SAR studies and the suggested binding mode
achieved from molecular modelling trials performed around the parent chroman-4-one/chromone
scaffold.[1,2] The main focus was the replacement of the core structure with less lipophilic ring
systems in order to improve the physicochemical properties. To elucidate the influence of the scaffold
replacement on the inhibitory properties, we aimed to retain a characteristic substitution pattern which
has proven to be vital for inhibitory activity of derivatives based on the chroman-4-one/chromone
3

framework. Hence, the scaffolds should contain a hydrogen bond acceptor (HBA) group mimicking
the carbonyl group thereby retaining the hydrogen bond to a structural water molecule. In addition, the
introduction of functional groups in positions mimicking the 2- and 6-position of the chroman-4-
ones/chromones should be allowed (see Scheme 1 for numbering). The new compounds should
preferably be brominated at the site corresponding to the 6-position of the parent scaffold since
chroman-4-one derivatives holding a Br-substituent in this position showed higher inhibitory activity
than other substituents due to a halogen bond interaction with a backbone carbonyl group of SIRT2.
Introduction of an additional Br-group in the corresponding 8-position might be beneficial to increase
the inhibitory activity even further due to hydrophobic interactions with the target protein. The
substituent in the 2-position of the chroman-4-ones/chromones is located in a narrow hydrophobic
tunnel pointing towards the surrounding water environment. Consequently, the group representing this
substituent should be a linear aliphatic moiety which can be substituted with heterocycles and bulkier
groups when separated from the core structure with at least an ethylene spacer.[1,2]
The heterocyclic frameworks shown in Scheme 1 emerged as promising alternatives as they are
similar in size to the chroman-4-ones/chromones, readily accessible by synthesis and can be substituted
with functional groups in equivalent positions as the parent scaffold to maintain the key binding
properties of known, potent SIRT2-selective inhibitors (Fig. 1, 1–5). The calculated logP values of
scaffolds B–E are significantly lower than the one of the chroman-4-one scaffold, indicating a good
starting point for addressing the issues of the less favourable physicochemical properties exhibited by
the chroman-4-one series. In addition, the quinolin-4-(1H)-one framework (A) was also considered to
be of interest due to its structural similarities to the chromone scaffold despite its higher clogP-value.
2.2 Chemistry
The synthesis towards the quinolin-4-(1H)-one-based analogues is shown in Scheme 2. The scaffold
was synthesized from β-ketoesters and anilines employing the Conrad-Limpach reaction. The β-
ketoesters 8a–c were obtained in good yields by reacting monomethyl potassium malonate (6) with
the CDI-activated carboxylic acids 7a–c in anhydrous THF at room temperature.[16] 2-Bromo-4-
chloroaniline was then reacted with 8a–c under argon at 50 °C for 48 h to afford the intermediate
enaminoesters 9a–c. After removal of excess aniline and p-toluenesulfonic acid (p-TSA) the
cyclization was achieved by heating the enaminoesters to 250 °C in diphenyl ether for 45 min using
4

microwave heating. The quinolin-4-(1H)-ones 10a–c were finally isolated by crystallization from
hexane.
O
O
O
O
O
Cl
Cl
a
O
O
c
O
b
+
O
O-
K+
R2
O
R2
N
H
R2
HO
7a-c
N
H
R2
6
8a-c
Br
Br
9a c
-
10a-c
2 = (CH2)4CH3
R
2 = CH2Ph
2 = (CH2)4CO2CH3
a:
b:
c:
R
R
Scheme 2. Synthesis of quinolin-4-(1H)-one derivative 10a–c. Reagents and conditions: (a) CDI, MgCl2, THF, room temp,
16 h, 74–78%; (b) 2-bromo-4-chloroaniline, p-TSA, neat, 50 °C, 48 h; (c) i. cyclohexane, reflux, 30 min; ii. Ph2O, 250 °C,
45 min, MW, 24–26% over two steps from 8a–c.
Four analogues with the benzothiazine-1,1-dioxide scaffold were synthesized as outlined in Scheme
3. 2-Aminobenzenesulfonamide (11) was reacted in a Sandmeyer-type reaction with NaNO2 at 0 °C
followed by the addition of KI. Introduction of the bromide in the 5-position was pursued via the
reaction of 12 with N-bromosuccinimide (NBS). Aryl iodides 12 and 13 were successfully used in a
Sonogashira reaction yielding the precursor for the subsequent 6-endo-dig cyclization to afford the
desired products 15a−d.
O
O
O
O
O
O
O O
S
R5
S
I
R5
S
R7
S
a
c
d
NH2
NH2
NH2
NH
R3
NH2
11
R3
5 = H, R3 = (CH2)3CH3
5 = H, R3 = CH2CH2Ph
5 = Br, R3 = (CH2)3CH3
5 = Br, R3 = CH2CH2Ph
12:
R
R
5 = H
5 = Br
b
14a:
14b:
14c:
14d:
15a:
15b:
15c:
15d:
R
R
7 = H, R3 = (CH2)3CH3
7 = H, R3 = CH2CH2Ph
7 = Br, R3 = (CH2)3CH3
R
7 = Br, R3 = CH2CH2Ph
13:
R
R
R
R
R
Scheme 3. Synthesis of 2H-benzo[e][1,2]thiazine-1,1-dioxide 15a–d. Reagents and conditions: (a) i. NaNO2, H2O:HCl
(6:4), 0 °C, 40 min; ii. KI, 90 °C, 5 h, 63%; (b) NBS, conc. H2SO4, 60 °C, 24 h; 87%; (c) 12 or 13, Pd(PPh3)2Cl2, CuI,
Et3N, appropriate alkyne, DMF, room temp., overnight, 64–77%; (d) Pd(PPh3)2(OAc)2, KOH, DMF, 60 °C, 4.5–5 h, 25–
46%.
The synthesis of derivatives based on the benzothiadiazine-1,1-dioxide scaffolds is shown in
Scheme 4. 2-Amino-3,5-dibromobenzenesulfonamide 17 was obtained by reacting 2-aminobenzene-
sulfonamide with Br2 in DMF at room temperature. Sulfonamide 17 was then either reacted with
various aldehydes or carboxylic acids which defined the substituent in the 3-position and the degree
of saturation of the scaffold. Aldehydes 18a and 18b employed in the aforementioned reaction were
commercially available. Whereas, compound 18c was obtained via a reduction-oxidation protocol
from 3-(3-bromophenyl)propionic acid (Supporting Information) and 18d, used to introduce the
5

quinolin-2(1H)-one moiety in the 3-position of 19d, was synthesized in three steps as previously
reported by our group.[2] The acid-catalyzed reaction of 17 with aldehydes 18a–d was carried out
under microwave heating at 120 °C in dioxane to afford 19a–d in 34–53% yield.
O
O
Br
S
b
19a:
19b:
19c:
19d:
R
3 = (CH2)4CH3
3 = CH2CH2Ph
3 = CH2CH2(m-Br)Ph
3 = CH2CH2(6-quinolin-2(1H)-one-yl)
NH
R3
R
O
N
H
R
R3
18a-d
Br
R
19a-d
O
S
O
O
O
O
O
O
O
3 = CH2CH2Ph
3 = CH2CH2(m-Br)Ph
3 = CH2CH2(p-Br)Ph
Br
S
Br
S
Br
22a:
22b:
R3 22c:
R
a
c
d
S
NH2
NH2
N
NH2
R
O
N
NH2
O
N
H
R
NH2
R3
H
Br
Br
17
R3
Br
HO
20a-c
16
21a-c
22a-c
Scheme 4. Synthesis of the saturated and unsaturated benzothiadiazine-1,1-dioxides 19a–d and 22a–c. Reagents and
conditions: (a) Br2, DMF, 10 °C→room temp, 22 h, 99% crude yield; (b) appropriate aldehyde, 4 M HCl in dioxane,
120 °C, MW, 1–2.5 h, 34–53%; (c) i. appropriate carboxylic acid, CDI, CH2Cl2, room temp, 1.5 h; ii. 17, CH2Cl2, DMF,
reflux, 21 h; (d) Cs2CO3, EtOH, 120 °C, 1.5 h, MW, 21–37% over two steps.
As outlined in Scheme 4, the unsaturated benzothiadiazine-1,1-dioxides 22a–c were synthesized
via a reaction of CDI-activated carboxylic acids with 17 leading to amide intermediates 21a–c which
were cyclized under microwave heating to afford 22a–c.[18]
The synthesis of saccharin derivatives substituted in the 2-position is shown in Scheme 5. As earlier
mentioned the SAR study of the chroman-4-ones revealed that halogen atoms in the 6- and
8-positions are essential for activity. Thus, dibrominated saccharins were considered promising
derivatives. However, bromination of 2-methylbenzenesulfonamide by the previously used method
(Br2 in DMF) was unsuccessful. Attempts to obtain a dihalogenated precursor for saccharin synthesis
via chlorosulfonation of 4-bromo-2-chlorotoluene yielded only the non-desired isomer, with the
sulfonyl chloride moiety in the meta-position to the methyl group.
Instead, the mono-brominated derivative 24 was synthesized by chlorosulfonation of 4-
bromotoluene (23). The desired isomer 24 was obtained together with small amounts of the meta-
substituted regioisomer (14–21%). The isomeric mixture was used in the following reaction converting
24 to the corresponding sulfonamide 25 with aqueous ammonia. A subsequent oxidation reaction, as
reported by Xu et al. using HI5O6 and sub-stoichiometric amounts of CrO3 furnished 6-bromosaccharin
(29) in 46% yield.[19] Alternatively, 29 was obtained in 62% overall yield via oxidative cyclization
under similar conditions of N-tert-butyl sulfonamide 26 followed by removal of the tert-butyl group
6

in refluxing trifluoroacetic acid (TFA). However, the final deprotection step of 27 proceeds very
slowly on larger scale and is therefore time consuming.
O
O
H
O
S
Br
N
S
e
Br
N
O
d
f
O
26
27
O
O
O
O
O
S
O
S
R6
R6
Cl
NH2
g
Br
S
S
a
b
c
Na
Br
Br
N
NH
O
O
O
O
23
24
25
28:
29:
30:
31:
R
R
6 = H
6 = Br
R
R
6 = H
6 = Br
h
2 = (CH2)4CH3
2 = CH2CH2Ph
R
2 = (CH2)4CH3
2 = CH2CH2Ph
2 = CH2CH2(p-OH)Ph
2 = CH2CH2(m-Br)Ph
2 = CH2CH2(m-NO2)Ph
2 = CH2CH2(1-succinimidyl)
32a:
32b:
32c:
32d:
33a:
33b:
33c:
33d:
33e:
33f:
R
O
R
R
O
R6
S
2
(
R
R
=
CH2)3CO2Et
R
N
R2
2 = (CH2)3(3-pyridyl
R
R
O
R
6 = H
6 = Br
32a-d:
33a-f:
R
R
Scheme 5. Synthesis of saccharin derivatives. Reagents and conditions: (a) ClSO3H, CH2Cl2, 0 °C→room temp, o.n., 81%;
(b) NH4OH, Et2O, 0 °C→reflux→room temp, 48 h, 74%; (c) HI5O6, CrO3, MeCN, reflux, 20 h, 38%; (d) tert-BuNH2, Et3N,
Et2O, 0 °C→room temp, 33 h, 81%; (e) HI5O6, CrO3, Ac2O, MeCN, 0 °C→room temp, 20 h; (f) TFA, reflux→room temp,
4 d, 51% over three steps (d–f); (g) NaOMe, MeOH, 6 h, room temp, >99%; (h) Alkyl halide, DMF, MW, 145 °C, 15 min.
or 1 h, 12–79%.
Finally, commercially available saccharin 28 and 6-bromosaccharin 29 were converted to their
corresponding sodium salts using NaOMe in MeOH and were further subjected to an alkylation with
a variety of alkyl halides containing different terminal heterofunctionalities and aryl groups (32a–d,
33a–f).
2.3 Physicochemical evaluation
In this study we attained scaffold analogues with an improved physicochemical profile (Supporting
Information, Table S1). The computed physicochemical properties (molecular weight, clogP, PSA,
HBD, HBA) of the new derivatives conformed with Lipinski’s rule of five. Even though we maintained
the highly lipophilic substitution pattern required to assure a minor loss in inhibitory activity [1,2] a
clear improvement in polarity can be seen in the increase in PSA and the number of HBD and HBA.
Derivatives based on the bicyclic sulfonamides (B–D) and saccharins (E) showed at a least twofold
increase in PSA and a decrease by one unit in clogP compared to active chroman-4-one based
derivatives (e.g. 1–3).
7

2.4 Biological evaluation
The synthesized compounds were evaluated in a fluorescence-based assay for their inhibitory
activity against SIRT2 at a compound concentration of 200 µM.[2,20] The most active derivatives
from each scaffold class were also tested for their inhibitory effect on SIRT1 and SIRT3 to investigate
their selectivity towards the SIRT2-isoform. The results are summarized in Tables 1−3.
The quinolone-4-(1H)-one series (Table 1) resembles chromone in which the oxygen in the 1-
position is replaced with a hydrogen bond donating NH-group. The evaluation of the inhibitory effect
towards SIRT2 showed that similar biological effects cannot be achieved in this series despite its
structural similarity to the chromone scaffold. (Table 1).
Table 1
SIRT2 inhibitory activities of quinolin-4-(1H)-one derivatives 10a–c. For selected analogues inhibition of SIRT1 and
SIRT3 was investigated as well.a,b,c
O
O
Cl
Cl
O
R2
N
H
R2
Br
34-35
Br
10a-c
Inhibition (%)a,b,c
SIRT2
No.
R2
SIRT1
SIRT3
34
35
9.8 ± 2.8
n.d.
82 ± 0.4
75 ± 2.6
26  1.4
58  2.1
53  1.1
4.5 ± 1.6
n.d.
n.d.
10a
10b
10c
n.d.
7.6  2.2
n.d.
41  3.0
n.d.
O
O
aSD, standard deviation (n = 3). bInhibition at 200 µM inhibitor concentration.
cn.d. = not determined.
Further, as summarized in Table 2, we tested various analogues based on bicyclic sulfonamide
scaffolds B–D substituted with aliphatic groups (butyl, pentyl, phenethyl) in the 3-position as well as
the bulkier 2-quinolinone group (19d). The mono-brominated bicyclic sulfonamides 15c and 15d
based on scaffold B (Table 2) displayed only poor inhibitory activity with the phenethyl derivative
15d being the most active (35% inhibition at 200 µM concentration). Compared to non-brominated
15a and b, introduction of the Br-group had only a marginal effect on the inhibitory activity as
compared to the corresponding effect among the chroman-4-ones.[1]
8

Table 2
SIRT2 inhibitory activities of benzothiazine-1,1-dioxide 15a–d and benzothiadiazine-1,1-dioxides 19a–d and 22a–c. For
selected analogues inhibition of SIRT1 and SIRT3 was also investigated.a,b,c
O
O
O
O
Br
S
Br
S
O
O
O
O
NH
R3
N
S
Br
S
NH
R3
NH
R3
N
H
N
H
R3
Br
Br
15a-b
15c-d
19a-d
22a-c
Inhibition (%)a,b,c
SIRT2
No.
Scaffold
R3
SIRT1
n.d.
SIRT3
n.d.
15a
15b
15c
15d
19a
19b
19c
B
B
B
B
C
C
C
12  2.3
15  6.5
29  4.1
35  1.7
32  2.2
47  1.9
59  0.4
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
4.4  2.8
4.6  4.2
9.7  3.9
5.8  3.4
54  9.2
9.9  1.5
Br
19d
C
25  1.6
74  1.3
28  1.5
N
H
O
22a
22b
22c
D
D
D
n.d.
0  3.7
n.d.
n.d.
3.2  2.0
n.d.
15  3.1
24  0.8
10  1.5
Br
Br
aSD, standard deviation (n = 3). bInhibition at 200 µM inhibitor concentration. cn.d. = not
determined.
The di-brominated derivatives 19a–d based on the saturated bicyclic sulfonamide core structure C
showed increased activity compared to 15c–d. Compound 19d was the most potent compound of the
benzothiadiazine-1,1-dioxides with 74% inhibition of SIRT2. The unsaturated analogues 22a–c were
considerably less potent (10–24% inhibition) with inhibitory activities in the range with the non-
halogenated analogues based on scaffold B. Moreover, 22a–c showed very low solubility in most
solvents and were therefore not considered for future optimization.
The last scaffold evaluated in the study was the saccharin core, i.e. compounds 32a–d and 33a–f.
As shown in Table 3, derivatives based on this core structure showed only a minimal inhibitory effect,
9

32a and 32b as most active showed 34% at 200 µM concentration. For this series, the introduction of
a bromide in the aromatic ring (33a–f) did not improve the activity.
Table 3
SIRT2 inhibitory activities of saccharin derivatives 32a–d and 33a–f. For selected analogues inhibition of SIRT1 and
SIRT3 was also investigated.a,b,c
O
O
S
R6
N
R2
O
32a-d, 33a-f
Inhibition (%)a,b,c
SIRT2
No.
R6
R2
SIRT1
n.d.
SIRT3
n.d.
32a
32b
32c
32d
33a
33b
33c
33d
33e
H
H
34  2.5
34  2.8
13  5.0
12  4.9
6.9  2.6
4.7  3.5
12  4.8
15  1.9
6.2  4.2
14  6.3
8.7  3.4
17  4.3
n.d.
14  2.8
7.2  1.9
14  2.4
n.d.
O
H
O
H
N
Br
Br
Br
Br
Br
3.8  4.3
5.2  4.0
n.d.
1.6  2.2
15  5.6
n.d.
OH
Br
NO2
n.d.
n.d.
O
N
33f
Br
n.d.
n.d.
13  4.4
O
aSD, standard deviation (n = 3). bInhibition at 200 µM inhibitor concentration.
cn.d. = not determined.
2.5 Structure-activity relationship and molecular modeling
In order to investigate and get a better understanding of the observed SAR among these new series
of compounds molecular modeling and docking studies were performed. For the docking trials we
used our 3D model of SIRT2 which was previously used for docking studies of the corresponding
chromones and chroman-4-ones.[2] The docking was performed using Glide with the OPLS3 force
field [21] as implemented in the Schrödinger software.[22] OPLS3 is superior in predicting protein-
ligand binding energy to commonly used force fields (e.g. OPLS_2005 and MMFF) [23,24] and it is
10

also parametrized to predict the geometry and energy contribution of aryl halogen bonds more
accurately.[25] In the docking procedure the SIRT2 3D-structure was kept rigid while the ligands were
flexible. A representative compound from each scaffold series was included together with a set of
relevant previously published structurally analogous chroman-4-ones as reference ligands. Docking
scores obtained from representative compounds of each compound class were compared (Supporting
Information, Table S2). The rank of the docking scores was in good agreement with the decreasing
inhibitory activity from the most potent chroman-4-one (3) to the saccharins comprising the least active
series of the new analogues.
As illustrated in Fig. 2, docking studies of quinolone-4-(1H)-one 10b revealed a similar binding
mode as observed for the chroman-4-ones/chromones with the presence of the key enzyme/inhibitor
interactions (see Fig. 1) such as the halogen bond and the hydrogen bond-interaction as well as the π-
π interactions. However, in the suggested binding mode of this scaffold the polar NH-group of the
quinolone-4-(1H)-ones is unfavourably positioned in a hydrophobic region close to Leu103 and 138
(Fig. 2). This in turn, might explain the decrease in inhibitory activity.
Leu103
Leu138
Pro94
Phe96
Ile93
Asp170
Asn168
W1
His187
Gln167
Fig. 2. Quinolone-4-(1H)-one analogue 10b docked in the C-pocket of SIRT2. The key enzyme/inhibitor interactions
are present, i.e. the halogen bond to His187, the hydrogen bond to W1, and the π-π interaction to Phe96. The NH moiety
in 10b is positioned in a hydrophobic region, close to two leucine residues (Leu103 and Leu138). The hydrophilic (purple)
and hydrophobic (green) regions are highlighted on the surface of the binding site.
The docking trial of the bicyclic sulfonamide scaffolds B–D revealed that they adopt similar binding
poses as that observed for the parent scaffold. When looking at the binding pose of 15d and 19d as
representative structures (Fig. 3A and B) in the SIRT2 homology model, the Br-group in the 7-position
(corresponding to 6-position of the chroman-4-ones) forms the expected halogen-bond with a
backbone carbonyl group of SIRT2. The other Br-group of the dibrominated derivatives (Fig. 3B) is
buried in the hydrophobic pocket. The SO2NH-group is placed in close proximity of a hydrogen
11

bonding network comprising Ser88, His149, Thr166, Asn168, Asp170, Glu173 and a structural water
molecule (W2) (see Fig. 3A), this network is highly conserved throughout the sirtuin family. The
sulfonyl interacts with the protein via a hydrogen bond of one of the sulfonyl oxygens with W1, while
the second oxygen is positioned in close proximity to Asp170 (3.9 Å) as shown in Fig. 3A and 3B.
This results in an electrostatic repulsion as the side-chain conformation of the aspartate is rotationally
restricted due to its participation in the conserved hydrogen bonding network. The distance between
the sulfonyl oxygen atom and Asp170 (3.9 Å) is also considerably shorter as compared to that between
the carbonyl oxygen of 2 and Asp170 (5.5 Å, Fig. 3C). The electrostatic repulsion could be the main
contributor to the overall decreased inhibitory activity observed for scaffolds B–D as compared to the
chroman-4-ones/chromones. In addition, a certain degree of flexibility in the scaffold might be
required for reasonable binding to SIRT2 since derivatives 22a–c that are based on the more rigid
scaffold D only showed negligible activity.
Phe119
C
A
B
Leu103
Phe119
Phe119
Leu103
Gln142
Gln142
Leu103
Gln142
Leu138
Leu138
Pro94
Leu138
Ile235
Ile235
3.9 Å
Asp170
Ser88
His187
5.5 Å
Asn168
W1
Ile93
Asp170
His187
3.9 Å
His187
W1
Asn168
Asp170
W1
W2
His149
Gln167
Asn168
Gln167
Glu173
Gln167
Fig. 3. (A) Binding pose of 15d and the observed conserved hydrogen-bond network formed by Ser88, His149, Asp170,
Asn168, Glu173 and a structural water molecule (W2). (B) Docking solution of 19d. (C) The docking pose of the chroman-
4-one analogue 2. The carbonyl oxygen is interacting with the structural water molecule W1 and the distance to Asp170 is
5.5 Å. The hydrophilic (purple) and hydrophobic (green) regions are highlighted in on the molecular surface of the binding
site .
In general, derivatives based on the saccharin scaffold compose a series of only weak SIRT2
inhibitors with 34% inhibitory activity as the best (32a–b). Docking studies of this compound class
revealed that they can adopt two different binding poses with either the sulfonyl group (Fig. 4A) or the
carbonyl moiety (Fig. 4B) interacting with the structural water (W1) thereby mimicking the carbonyl
group of the chroman-4-ones. Depending on the nature of the hydrogen bond interacting group, the
mono-brominated compounds can additionally, either form the halogen bond (Fig. 4A) or the π-π-
interaction with Phe96 (Fig. 4B).
12

In this series we observed an opposite trend in the inhibitory effect where the lack of the halogen
substituents slightly increased the activity. The non-halogenated saccharin derivatives can bind closer
to His187 thereby increasing the distance between the sulfonyl oxygen to the acidic Asp170 (4.8 Å).
At the same time the carbonyl group is positioned further away from the lipophilic leucine residues
Leu103 and Leu138 which could result in a more favourable binding mode which could explain the
slight increase in inhibitory activity.
C
A
B
Leu103
Leu103
Leu103
Leu138
Leu138
Leu138
Pro94
Pro94
Pro94
Ile93
Ile93
Phe96
Ile93
Asp170
Asp170
Asp170
Phe96
Phe96
Asn168
Asn168
Asn168
W1
W1
W1
His187
His187
His187
Gln167
Gln167
Gln167
Fig. 4. (A). Binding pose of 33b which is similar to that of the sulfonamides in where the sulfonyl group interacts with
W1 and also forms a halogen bond with the backbone carbonyl moiety of His187. (B) The alternative pose of 33b rotated
180° in which the hydrogen bond interaction with W1 is maintained via the carbonyl oxygen. However, this binding
prevents any potential halogen bonding with His187. (C) Binding pose of the non-halogenated saccharin analogue 32b
which interacts with the structural water W1 and places the sulfonyl and carbonyl group further away from the acidic
Asp170 (4.8 Å) and Leu103 and Leu138. However, the distance between the aromatic ring in the ligand and Phe96 is
longer which weakens the π-π-interaction. The hydrophilic (purple) and hydrophobic (green) regions are highlighted in on
the molecular surface of the binding site
3
Conclusion
We have shown that bicyclic sulfonamides can act as scaffolds for SIRT inhibitors. The new
scaffold analogues comprised a set of promising SIRT2 inhibitors (50–75% inhibition at 200 µM) with
19d, based on the sulfonamide scaffold C substituted with a 2-quinolinone containing alkyl group
(Table 2), being the most potent inhibitor identified in our study. Further optimization guided by
molecular modeling will be performed around the benzothiadiazine-1,1-dioxides, to identify
compounds that can be expected to be more potent.
Interestingly, two compounds (10b and 19b) show higher activity against SIRT3 than what we have
previously seen for the chroman-4-ones. This could be a potential starting point for the development
of either more general SIRT inhibitors, so called pan inhibitors, or of more SIRT3 selective
compounds.
13

4. Experimental
3.1 General information
All reactions were carried out using magnetic stirring under ambient atmosphere if not otherwise
stated. Room temperature corresponds to a temperature interval from 20 to 22 ºC. All starting materials
and reagents were obtained from commercial producers and were used without prior purification.
Solvents were generally used as supplied by the manufacturer. Microwave reactions were carried out
using a Biotage Initiator™ with fixed hold time modus in 0.5–2 mL, 2–5 mL or 10–20 mL capped
microwave vials. All reactions were monitored by thin-layer chromatography (TLC) on silica plated
aluminum sheets (Silica gel 60 F254, E. Merck). Spots were detected by UV light (254 or 365 nm).
Purification by flash column chromatography was performed using an automatic Biotage SP4 Flash+®
instrument. Prefabricated columns of different cartridge sizes (surface area 500 m2/g, porosity 60 Å,
particle size 40–63μm) were used. The NMR spectra were measured on a Varian 400-MR spectrometer
1
13
or a 300 MHz instrument. H and C NMR spectra were measured at 400 MHz and 100 MHz,
respectively. Chemical shifts are reported in ppm with the solvent residual peak as internal standard
[CHCl3 δH 7.26, CDCl3 δC 77.16; CD2HOD H 3.31, CD3OD C 49.00; acetone-d6 δH 2.05, δC 29.84;
DMSO-d6 δH 2.50, δC 39.52]. All NMR experiments were measured at ambient temperature. If not
otherwise stated the NMR experiments were run in CDCl3 at 400 MHz. Combustion analyses for CHN
were measured on a Thermo Quest CE Instruments EA 1110 CHNS-O elemental analyzer or Perkin
Elmer 2400 Series II CHNS/O system elemental analyzer. High-resolution mass spectrometry
(HRMS) analysis was obtained from Recipharm OT Chemistry AB, Uppsala (Sweden).
3.2 General procedure for the synthesis of β-ketoesters 8a–c
The carboxylic acid (1 equiv) and CDI (1.1 equiv) were dissolved in anhydrous THF (0.5 M) and
stirred at room temperature for 1 h. Monomethyl potassium malonate 6 (1.2 equiv) and anhydrous
MgCl2 (1.5 equiv) were added and the reaction was stirred at room temperature overnight. The reaction
was treated with 1 M HCl (aq). The phases were separated and the aqueous phase was extracted with
EtOAc. The combined organic phases were washed with water and brine, dried over Na2SO4, filtered
and concentrated under reduced pressure. Purification by flash column chromatography (2→22%
EtOAc in pentane) afforded β-ketoesters 8a–c. All 1H-NMR spectra also showed trace amounts of the
enol tautomer of the product.
14

Methyl 3-oxooctanoate (8a). The compound was synthesized according to the general procedure
1
from 7a (1.39 g, 12.0 mmol) to afford 8a (1.53 g, 74%) as a colourless oil. H NMR (300 MHz)
δ 3.73 (s, 3H), 3.45 (s, 2H), 2.53 (t, J = 7.4 Hz, 2H), 1.66–1.54 (m, 2H), 1.38–1.22 (m, 4H), 0.89 (t,
J = 6.8 Hz, 3H). 13C NMR (75 MHz) δ 202.8, 167.7, 52.3, 49.0, 43.0, 31.2, 23.2, 22.4, 13.9.
Methyl 3-oxo-4-phenylbutanoate (8b). The compound was synthesized according to the general
1
procedure from 7b (1.04 g, 7.63 mmol) to afford 8b as a colourless oil (1.30 g, 88%). H NMR
13
(300 MHz) δ 7.37–7.13 (m, 5H), 3.80 (s, 2H), 3.68 (s, 3H), 3.44 (s, 2H). C NMR (75 MHz)
δ 200.3, 167.5, 133.3, 129.6, 128.9, 127.4, 52.3, 50.0, 48.0.
Methyl 7-methoxycarbonyl-3-oxooctanoate (8c). The compound was synthesized according to the
general procedure from 7c (1.46 g, 13.8 mmol) to afford 8c as a colourless oil (1.51 g, 77%).
1H NMR (300 MHz) δ 3.74 (s, 3H), 3.67 (s, 3H), 3.45 (s, 2H), 2.57 (t, J = 6.9 Hz, 2H), 2.33 (t,
13
J = 7.1 Hz, 2H), 1.69–1.58 (m, 4H). C NMR (75 MHz) δ 202.2, 173.8, 167.6, 52.4, 51.6, 49.0, 42.6,
33.8, 24.3, 22.9.
3.3 General procedure for the synthesis of 4-quinolones 10a–c
β-Ketoester (1 equiv), 2-bromo-4-chloroaniline (1.2 equiv) and p-TSA (0.1 equiv) were stirred in a
sealed reaction vessel at 50 °C for approximately 48 h. The mixture was transferred to a flask and
refluxed briefly in cyclohexane. The solid was filtered off and the solvent was evaporated. The residue
was dissolved in diphenyl ether (5 mL) and the solution was heated in a microwave reactor at 250 °C
for 45 min. Excess hexane was added and the product was collected the next day by filtration. The
compounds were recrystallized from chloroform/hexane to remove traces of diphenyl ether.
8-Bromo-6-chloro-2-pentylquinolin-4(1H)-one (10a). The compound was synthesized according
to the general procedure from 8a (544 mg, 3.87 mmol) to afford 10a (255 mg, 25%) as a brown solid.
1H NMR (300 MHz) δ 8.28 (d, J = 2.3 Hz, 1H), 8.26 (br s, 1H), 7.78 (d, J = 2.3 Hz, 1H), 6.18 (s, 1H),
2.67 (t, J = 7.7 Hz, 2H), 1.85–1.66 (m, 2H), 1.46–1.32 (m, 4H), 0.99-0.83 (m, 3H). 13C NMR (75 MHz)
δ 177.2, 152.9, 135.8, 134.7, 129.5, 127.0, 125.7, 111.7, 109.7, 34.6, 31.2, 27.9, 22.5, 14.0. Anal. Calcd
for C14H15BrClNO: C, 51.17; H, 4.60; N, 4.26. Found: C, 51.67; H, 4.80; N, 4.21.
2-Benzyl-8-bromo-6-chloroquinolin-4(1H)-one (10b). The compound was synthesized according
to the general procedure from 8b (322 mg, 1.68 mmol) to afford 10b (143 mg, 25%) as a brown solid.
1H NMR (300 MHz) δ 8.26 (d, J = 2.3 Hz, 1H), 8.20 (s, 1H), 7.72 (d, J = 2.3 Hz, 1H),
7.49–7.27 (m, 5H), 6.24 (s, 1H), 4.04 (s, 2H). 13C NMR (75 MHz) δ 177.2, 150.8, 135.7, 134.7, 134.3,
15

129.72, 129.70, 129.5, 128.4, 126.8, 125.7, 111.9, 110.3, 40.4. Anal. (C16H11BrClNO)
C, H, N.
Methyl 5-(8-bromo-6-chloro-quinoline-4(1H)-one-2-yl)pentanoate (10c). The compound was
synthesized according to the general procedure from 8c (248 mg, 1.15 mmol) to afford 10c (129 mg,
1
30%) as a light brown solid. H NMR (300 MHz) δ 8.41 (s, 1H), 8.27 (d, J = 2.3 Hz, 1H), 7.79 (d,
J = 2.3 Hz, 1H), 6.18 (s, 1H), 3.69 (s, 3H), 2.70 (t, J = 7.2 Hz, 2H), 2.40 (t, J = 6.8 Hz, 2H),
1.90–1.68 (m, 4H).13C NMR (75 MHz) δ 177.2, 173.7, 152.4, 135.8, 134.8, 129.7, 127.0, 125.7, 111.8,
109.8, 51.8, 34.2, 33.6, 27.8, 24.1. Anal. (C15H15BrClNO3) C, H, N.
3.4 2-Iodobenzenesulfonamide (12)
To a suspension of 2-aminobenzenesulfonamide 11 (1.5 g, 8.71 mmol) in H2O/HCl (16:11 mL)
cooled to 0 °C was added a solution of NaNO2 (0.91 g, 13.2 mmol) in H2O (16 mL). The mixture was
stirred until a clear yellow solution was formed. A solution of KI (4.34 g, 26.1 mmol) in H2O (16 mL)
was added and the red coloured mixture was heated to 90 °C for 4 h. The mixture was allowed to cool
to room temperature and the formed solid was filtered off, washed with water and pentane and dried
under vacuum to afford 12 (1.65 g, 63%) as a light brown solid. 1H NMR (DMSO-d6) δ 8.11 (dd, J =
7.8, 1.2 Hz, 1H), 8.01 (dd, J = 7.9, 1.5 Hz, 1H), 7.58 (ddd, J = 7.9, 7.4, 1.2 Hz, 1H), 7.49 (s, 2H), 7.27
(ddd, J = 7.4, 1.6 Hz, 1H). 13C NMR (DMSO-d6) δ 145.9, 142.1, 132.8, 128.3, 92.4.
3.5 5-Bromo-2-iodobenzenesulfonamide (13)
2-Iodobenzenesulfonamide 12 (846 mg, 2.99 mmol) was suspended in conc. H2SO4 (20 mL) and
warmed to 60 °C. N-Bromosuccinimde (638 mg, 3.59 mmol) was added in one portion and the mixture
was stirred at 60 °C for 24 h. The mixture was poured on ice and the formed precipitate was filtered
off, washed with water and pentane and dried in vacuo to afford 13 (941 mg, 87 %) as off-white solid.
1H NMR (DMSO-d6) δ 8.08 (d, J = 2.3 Hz, 1H), 8.03 (d, J = 8.3 Hz, 1H), 7.67 (br s, 2H), 7.49 (dd, J
= 8.3, 2.3 Hz, 1H). 13C NMR (DMSO-d6) δ 147.8, 144.0, 135.5, 130.6, 121.4, 91.3
3.6 General procedure for synthesis of alkyne derivatives 14a–c
To a vial with the appropriate 2-iodobenzenesulfonamide (1 equiv), Pd(PPh3)2Cl2 (0.1 equiv) and
CuI (0.1 equiv) was added DMF (0.2 M) and Et3N (10 equiv) and the mixture was purged with N2 gas.
The appropriate alkyne (1.5 equiv) was added and the reaction mixture was stirred at room temperature
overnight. The mixture was diluted with EtOAc and filtered through CeliteTM. The organic phase was
washed with 0.1 M HCl, water (3×) and brine (2×), dried over MgSO4, filtered and concentrated under
16

reduced pressure. The crude product was purified by automated flash column chromatography
(15%→20% EtOAc/pentane) to afford the desired products.
2-(Hex-1-yn-1-yl)benzenesulfonamide (14a) The compound was synthesized according to the
general procedure from 12 (400 mg, 1.40 mmol), Purification by automated flash column
1
chromatography (0→45% EtOAc/pentane) to afford 14a (224 mg, 67%) as a white solid. H NMR
δ 8.07 (d, J = 2.0 Hz, 1H), 7.58 (dd, J = 8.2, 2.1 Hz, 1H), 7.40 (d, J = 8.2 Hz, 1H), 5.36 (s, 3H), 2.50
13
(t, J = 7.2 Hz, 2H), 1.69–1.56 (m, 2H), 1.54–1.40 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H). C NMR
δ 142.7, 134.3, 131.9, 127.6, 126.8, 121.1, 100.2, 77.4, 30.3, 22.0, 19.3, 13.5.
2-(4-Phenylbut-1-yn-1-yl)benzenesulfonamide (14b) The compound was synthesized according to
the general procedure from 12 (400 mg, 1.40 mmol), Purification by automated flash column
chromatography (0→45% EtOAc/pentane) to afford 14b (309 mg, 77%) as a yellow solid. 1H NMR
δ 7.92 (dd, J = 7.9, 1.3 Hz, 1H), 7.51 (dd, J = 7.6, 1.4 Hz, 1H), 7.45 (td, J = 7.5, 1.4 Hz, 1H),
7.39–7.19 (m, 6H), 4.77 (br s, 1H), 2.96 (td, J = 7.0, 1.3 Hz, 2H), 2.86 (td, J = 6.8, 1.3 Hz, 2H).
13C NMR δ 143.2, 140.1, 134.5, 132.1, 128.8, 128.7, 128.0, 127.2, 126.8, 121.0, 99.0, 78.4, 34.3, 21.7.
5-Bromo-2-(hex-1-yn-1-yl)benzenesulfonamide (14c) The compound was synthesized according
to the general procedure from 13 (108 mg, 0.30 mmol), Purification by automated flash column
chromatography (0→30% EtOAc/pentane) to afford 14c (60 mg, 64%) as an off-white solid. 1H NMR
δ 8.11 (d, J = 2.0 Hz, 1H), 7.60 (dd, J = 8.2, 2.1 Hz, 1H), 7.41 (d, J = 8.2 Hz, 1H), 5.27 (s, 2H), 2.51
(t, J = 7.2 Hz, 2H), 1.69–1.58 (m, 2H), 1.54–1.41 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H). 13C NMR δ 144.3,
135.7, 135.2, 130.2, 121.8, 120.2, 101.7, 76.9, 30.4, 22.2, 19.6, 13.7.
5-Bromo-2-(4-phenylbut-1-yn-1-yl)benzenesulfonamide (14d) The compound was synthesized
according to the general procedure from 13 (105 mg, 0.29 mmol), Purification by automated flash
column chromatography (0→40% EtOAc/pentane) to afford 14d (68 mg, 65%) as an off-white solid.
1H NMR δ 8.03 (d, J = 2.0 Hz, 1H), 7.55 (dd, J = 8.2, 2.1 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H),
7.34–7.19 (m, 5H), 4.79 (s, 3H), 2.95 (dd, J = 7.3, 5.8 Hz, 2H), 2.85 (td, J = 6.7, 1.2 Hz, 2H).
13C NMR δ 144.4, 139.9, 135.7, 135.1, 130.1, 128.8, 128.6, 126.9, 121.9, 119.9, 100.2, 77.7, 34.1,
21.7.
3.7 General procedure for synthesis of benzothiazine-1,1-dioxides 15a–d
A solution of the alkyne (1 equiv) in DMF (0.1 M) was added to a oven-dried MW vial containing
KOH (4 equiv) and Pd(PPh3)2OAc2 (0.05 equiv). The mixture was heated to 60 °C for 4.5–5 h. The
mixture was diluted with EtOAc and washed with 1 M HCl (aq.), water and brine, dried over MgSO4,
17

filtered and concentrated to afford the desire products after purification by automated flash column
chromatography.
3-Butyl-2H-benzo[e][1,2]thiazine 1,1-dioxide (15a) The compound was synthesized according to
the general procedure from 14a (224 mg, 0.94 mmol). Purification by automated flash column
chromatography (0→20% EtOAc/pentane) to afford 15a (95 mg, 42%) as a colourless oil that
1
crystalizes over time. H NMR δ 7.93–7.79 (m, 1H), 7.54 (br s, 1H, NH), 7.53 (ddd, J=7.9, 7.4, 1.3
Hz, 1H), 7.38 (td, J=7.7, 1.2 Hz, 1H), 7.32–7.28 (m, 1H), 6.03 (s, 1H), 2.48–2.33 (m, 2H), 1.62 (ddt,
J=8.7, 7.5, 6.4 Hz, 2H), 1.53–1.28 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H). 13C NMR δ 141.3, 133.8, 132.2,
130.4, 126.7, 126.6, 121.3, 104.8, 34.4, 29.2, 22.0, 13.9. Anal. (C12H15NO2S) C, H, N.
3-Phenethyl-2H-benzo[e][1,2]thiazine 1,1-dioxide (15b) The compound was synthesized
according to the general procedure from 14b (309 mg, 1.08 mmol). Purification by automated flash
column chromatography (0→20% EtOAc/pentane) to afford 15b (110 mg, 36%) as a yellow oil.
1H NMR δ 7.85 (dd, J = 8.0, 1.2 Hz, 1H), 7.80 (s, 1H), 7.50 (td, J = 7.6, 1.3 Hz, 1H), 7.36 (td,
J = 7.7, 1.2 Hz, 1H), 7.32–7.15 (m, 6H), 5.97 (s, 1H), 2.95 (dd, J = 8.9, 6.7 Hz, 2H), 2.66 (dd,
J = 8.8, 6.8 Hz, 2H). 13C NMR δ 140.3, 140.2, 133.6, 132.2, 130.4, 128.6, 128.6 126.8, 126.7, 126.4,
121.2, 105.3, 36.7, 33.6. Anal. (C16H15NO2S·0.6 H2O) C, H, N.
7-Bromo-3-butyl-2H-benzo[e][1,2]thiazine 1,1-dioxide (15c) The compound was synthesized
according to the general procedure from 14c (60 mg, 0.19 mmol). Purification by automated flash
column chromatography (0→20% EtOAc/pentane) to afford 15c (15 mg, 25%) as a colourless oil that
1
crystalizes over time. H NMR δ 7.97 (d, J = 2.1 Hz, 1H), 7.64 (dd, J = 8.4, 2.0 Hz, 1H), 7.19 (d,
J = 8.4 Hz, 1H), 7.13 (br s, 1H), 6.03 (s, 1H), 2.39 (t, J = 7.6 Hz, 2H), 1.63 (dddd, J = 8.6, 7.5, 7.0, 5.8
Hz, 2H), 1.47–1.33 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H). 13C NMR δ 141.6, 135.3, 132.6, 131.8, 128.3,
124.2, 119.7, 104.8, 34.6, 29.2, 22.1, 13.9. HRMS (ESI) m/z calcd for C12H15BrNO2S [M+H]+
316.0007, found 316.0009.
7-Bromo-3-phenethyl-2H-benzo[e][1,2]thiazine 1,1-dioxide (15d) The compound was synthesized
according to the general procedure from 14c (68 mg, 0.19 mmol). Purification by automated flash
column chromatography (0→20% EtOAc/pentane) to afford 15c (31 mg, 46%) as a colourless oil that
crystalizes over time. 1H NMR δ 7.98 (d, J = 2.1 Hz, 1H), 7.64 (dd, J =8.4, 2.0 Hz, 1H), 7.38–7.19 (m,
5H), 7.16 (d, J = 8.4 Hz, 1H), 6.79 (br s, 1H), 5.99 (s, 1H), 2.97 (t, J = 7.6 Hz, 1H), 2.69 (t, J = 7.6 Hz,
13
2H). C NMR δ 140.5, 139.8, 135.4, 132.3, 132.0, 128.9, 128.6, 128.5, 126.8, 124.3, 120.1, 105.6,
36.9, 33.7. HRMS (ESI) m/z calcd for C16H15BrNO2S [M+H]+ 364.0007, found 364.0011.
18

3.8 2-Amino-3,5-dibromobenzenesulfonamide (17)
Br2 (0.75 mL, 15 mmol) was added dropwise to a solution of 2-aminobenzenesulfonamide 16 (1.01
g, 5.85 mmol) in DMF (4 mL) at <10 °C. The dark brown solution was stirred at 10–15 °C for 15 min
and then continued to stir at room temperature for 23 h. Additional Br2 (0.15 mL, 2.9 mmol) was added
and the stirring was continued at room temperature for 22 h. The mixture was diluted with EtOAc and
treated with 10% Na2S2O3 (aq.). After 10 min of stirring water was added, the phases were separated
and the aqueous phase was extracted with EtOAc. The combined organic phases were washed with
sat. Na2CO3 (aq), water and brine, dried over MgSO4, filtered and concentrated under reduced pressure
to afford 17 (1.96 g, >99%) as a light-brown solid, which was directly used in the next step. 1H NMR
(DMSO-d6) δ 7.85 (d, J = 2.3 Hz, 1H), 7.71 (d, J = 2.4 Hz, 1H), 7.65 (s, 2H), 6.02 (s, 2H). 13C NMR
(DMSO-d6) δ 141.5, 137.6, 129.7, 127.0, 110.6, 105.1.
3.9 General procedure for synthesis of saturated benzothiadiazine derivatives 19a–d
Compound 17 (1.1–1.2 equiv) and the appropriate aldehyde 18a–d (1 equiv) were dissolved in
4 M HCl in dioxane (0.04–0.06 M). The mixture was heated in a microwave reactor at 120 °C for
1.5 h.
Work-up procedure A. The solvent was removed and the residue was diluted with EtOAc. The
organic phase was washed with 2 M NaOH (aq), water, 0.1 M HCl (aq), water and brine, dried over
MgSO4, filtered and concentrated under reduced pressure.
Work-up procedure B. The product was isolated by filtration and washed with EtOAc.
5,7-Dibromo-3-pentyl-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine-1,1-dioxide
(19a).
The
compound was synthesized according to the general procedure from 17 (198 mg, 0.60 mmol), hexanal
(18a, 0.05 mL, 0.50 mmol) and 4 M HCl in dioxane (10 mL). Work-up procedure A was used.
Purification by flash column chromatography (10→20% EtOAc/pentane) and trituration with MeOH
afforded 19a (106 mg, 51%) as a white solid. 1H NMR (acetone-d6) δ 7.81 (d, J = 2.2 Hz, 1H), 7.69
(d, J = 2.2 Hz, 1H), 6.62 (d, J = 12.2 Hz, 1H), 5.79 (s, 1H), 5.02–4.88 (m, 1H), 2.16–2.06 (m, 1H),
2.02–1.89 (m, 1H), 1.70–1.48 (m, 2H), 1.43–1.31 (m, 4H), 0.97–0.79 (m, 3H). 13C NMR (acetone-d6)
δ 141.0, 138.7, 126.9, 126.0, 111.1, 108.6, 67.5, 34.6, 32.0, 25.0, 23.1, 14.2. Anal. (C12H16Br2N2O2S)
C, H, N.
5,7-Dibromo-3-phenethyl-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine-1,1-dioxide (19b). The
compound was synthesized according to the general procedure from 17 (200 mg, 0.61 mmol) and 18b
(0.08 mL, 0.50 mmol). Work-up procedure A was used. Purification by flash column chromatography
19

(10→20% EtOAc/pentane) and trituration with MeOH afforded 19b (117 mg, 53%) as an white solid.
1H NMR (DMSO-d6) δ 8.02 (d, J = 11.4 Hz, 1H), 7.90 (d, J = 2.3 Hz, 1H), 7.67 (dd, J = 2.2, 0.6 Hz,
1H), 7.35–7.13 (m, 5H), 6.38 (s, 1H), 4.76–4.62 (m, 1H), 2.86–2.62 (m, 2H), 2.44–2.28 (m, 1H), 2.12–
1.96 (m, 1H).13C NMR (DMSO-d6) δ 140.8, 140.2, 138.0, 128.4, 128.4, 126.0, 125.6, 124.3, 110.3,
107.3, 65.6, 34.5, 30.2. Anal. (C15H14Br2N2O2S) C, H, N.
5,7-Dibromo-3-(3-bromophenethyl)-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine-1,1-dioxide
(19c). The compound was synthesized according to the general procedure from 17 (89 mg,
0.27 mmol) and 18c (63 mg, 0.29 mmol) in a microwave reactor at 120 °C for 1.5 h. Work-up
procedure A was used. Purification by flash column chromatography (20→100% EtOAc/pentane) and
recrystallization from MeCN afforded 19c (52 mg, 34%) as an off-white solid. 1H NMR (DMSO-d6)
δ 8.02 (d, J = 11.5 Hz, 1H), 7.91 (d, J = 2.2 Hz, 1H), 7.69 (dd, J = 2.3, 0.6 Hz, 1H), 7.51–7.46 (m,
1H), 7.43–7.36 (m, 1H), 7.31–7.23 (m, 2H), 6.40 (s, 1H), 4.77–4.64 (m, 1H),
13
2.90–2.60 (m, 2H), 2.45–2.27 (m, 1H), 2.13–1.95 (m, 1H). C NMR (DMSO-d6) δ 143.7, 140.2,
138.0, 131.1, 130.5, 129.0, 127.6, 125.6, 124.3, 121.7, 110.3, 107.3, 65.5, 34.2, 29.7. Anal.
(C15H13Br3N2O2S) C, H, N.
5,7-Dibromo-3-(2-(quinolin-2(1H)-one-6-yl)ethyl)-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine-
1,1-dioxide (19d). The compound was synthesized according to the general procedure from 17
(75 mg, 0.23 mmol) and 18d (42 mg, 0.21 mmol). Work-up procedure B was used and 19d
1
(39 mg, 37%) was afforded as a light-brown solid. H NMR (DMSO-d6) δ 11.69 (s, 1H), 8.04 (d,
J = 11.5 Hz, 1H), 7.91 (d, J = 2.2 Hz, 1H), 7.85 (d, J = 9.5 Hz, 1H), 7.69 (d, J = 2.2 Hz, 1H), 7.51 (d,
J = 1.9 Hz, 1H), 7.40 (dd, J = 8.4, 1.9 Hz, 1H), 7.25 (d, J = 8.4 Hz, 1H), 6.48 (d, J = 9.5 Hz, 1H), 6.37
(s, 1H), 4.77–4.66 (m, 1H), 2.92–2.68 (m, 2H), 2.46–2.32 (m, 1H), 2.16–2.01 (m, 1H).
13C NMR (DMSO-d6) δ 161.8, 140.2, 140.0, 137.9, 137.3, 134.1, 130.9, 127.0, 125.5, 124.3, 122.0,
119.1, 115.2, 110.3, 107.3, 65.5, 34.5, 29.5. HRMS (ESI) m/z calcd for C18H16Br2N3O3S [M+H]+
511.9279, found 511.9273.
3.10 General procedure for synthesis of unsaturated benzothiadiazine derivatives 22a–c
A solution of carboxylic acid (1.3 equiv) and CDI (1.2–1.8 equiv) in dry CH2Cl2 (0.4–0.7 M) was
stirred at room temperature for 1.5–4 h. Sulfonamide 17 (1 equiv) dissolved in dry CH2Cl2
(0.4–0.5 M) and 15–50 vol% DMF was added and the solution was heated to reflux for 21 h. The
solvent was removed and the residue was dissolved in a large quantity of EtOAc and washed with 0.1
M HCl (aq), sat. Na2CO3 (aq) and brine, dried over MgSO4, filtered and concentrated under reduced
20

pressure. The crude product was dissolved in EtOH, Cs2CO3 (2 equiv) was added and the mixture was
heated in a microwave reactor at 120 ºC for 1.5 h The solvent was removed and 1 M HCl (aq) was
added. The formed precipitation was filtered off and washed with EtOAc.
5,7-Dibromo-3-phenethyl-4H-benzo[e][1,2,4]thiadiazine-1,1-dioxide (22a). The compound was
synthesized according to the general procedure from 17 (373 mg, 1.13 mmol). Purification by
recrystallization from MeCN afforded 22a (120 mg, 21 % over two steps) as an off-white solid.
1H NMR (DMSO-d6) δ 7.98 (d, J = 2.4 Hz, 1H), 7.74 (d, J = 2.3 Hz, 1H), 7.31–7.20 (m, 4H), 7.14 (tt,
13
J = 5.9, 3.0 Hz, 1H), 2.96 (m, 2H), 2.76 (t, J = 7.9 Hz, 2H). C NMR (DMSO-d6) δ 163.0, 162.8,
141.0, 137.4, 128.4, 128.3, 126.0, 124.8, 124.4, 115.4, 115.1, 39.5, 32.2. HRMS (ESI) m/z calcd for
C15H13Br2N2O2S [M+H]+ 442.9064, found 442.9053.
5,7-Dibromo-3-(3-bromophenethyl)-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine-1,1-dioxide
(22b). The compound was synthesized according to the general procedure from 17 (251 mg,
0.76 mmol). Purification by recrystallization from MeCN afforded 22b (97 mg, 24% over two steps)
1
as an off-white solid. H NMR (DMSO-d6) δ 10.89 (s, 1H), 8.30 (d, J = 2.1 Hz, 1H), 8.01 (d,
J = 2.1 Hz, 1H), 7.52 (dd, J = 2.2, 1.5 Hz, 1H), 7.40 (ddd, J = 7.5, 2.1, 1.5 Hz, 1H), 7.32–7.23 (m,
2H), 3.15–3.06 (m, 2H), 2.98–2.92 (m, 2H). 13C NMR (DMSO-d6) δ 161.1, 142.9, 138.9, 132.7, 131.2,
130.5, 129.1, 127.6, 125.4, 123.7, 121.6, 117.7, 111.6, 36.3, 31.2. Anal. (C15H11Br3N2O2S)
C, H, N.
5,7-Dibromo-3-(4-bromophenethyl)-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine-1,1-dioxide
(22c). The compound was synthesized according to the general procedure from 17 (253 mg,
0.77 mmol). Purification by recrystallization from MeCN afforded 22c (146 mg, 37% over two steps)
1
as an off-white solid. H NMR (DMSO-d6) δ 10.89 (s, 1H), 8.29 (d, J = 2.1 Hz, 1H), 8.01 (d,
J = 2.1 Hz, 1H), 7.48 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H), 3.14–3.05 (m, 2H), 2.99–2.90 (m,
13
2H). C NMR (DMSO-d6) δ 161.1, 139.5, 138.9, 132.7, 131.2, 130.7, 125.4, 123.8, 119.3, 117.7,
111.6, 36.3, 31.0. Anal. (C15H11Br3N2O2S) C, H, N.
3.11 5-Bromo-2-methylbenzenesulfonyl chloride (24)
A solution of ClSO3H (7.00 mL, 103 mmol) in CH2Cl2 (12 mL) was added dropwise to an ice-cold
solution of 4-bromotoluene (2.61 g, 15 mmol) in CH2Cl2 (25 mL). The mixture was stirred in an ice-
bath overnight, while the temperature increased to 10 °C. The solvent was removed and the residue
was added dropwise to ice-water. The formed solid was filtered off and washed with water to afford a
1
mixture (3.85 g, 94%, 86:14 ratio according to H NMR) of 24 (major isomer, 81%) and
21

1
2-bromo-5-methylbenzenesulfonyl chloride as a colourless oil. H NMR (24) δ 8.20 (d, J = 2.1 Hz,
13
1H), 7.72 (dd, J = 8.2, 2.0 Hz, 1H), 7.31 (dd, J = 8.2, 0.8 Hz, 1H), 2.74 (s, 3H). C NMR (24)
δ 144.1, 138.2, 137.0, 135.0, 131.4, 120.1, 20.0.
3.12 5-Bromo-2-methylbenzenesulfonamide (25)
To an ice cold solution of 24 (5.83 g, 21.6 mmol) in Et2O (100 mL) was added aqueous ammonia
(25%, 20 mL). The mixture was refluxed for 2 h and finally stirred at room temperature for 48 h. The
solvent was removed under reduced pressure and the formed solid was filtered off, extensively washed
with water and dried in vacuo to afford a mixture (5.29 g, 98%, 83:17 ratio according to
1H NMR signal of CH3-group) of 25 (major isomer, 81%) and 2-bromo-5-methylbenzenesulfonamide
(minor isomer, 17%) as
a
white solid. 1H NMR (CD3OD, 25)
δ
8.07 (d,
13
J = 2.1 Hz, 1H), 7.61 (dd, J= 8.2, 2.2 Hz, 1H), 7.28 (d, J = 8.1 Hz, 1H), 2.61 (s, 3H). C NMR
(CD3OD, 25) δ 144.0, 135.3, 134.4, 134.4, 129.3, 118.4, 19.3.
3.13 6-Bromosaccharin (29) synthesized from 25
To a mixture of H5IO6 (27.7 g, 122 mmol) and CrO3 (304 mg, 3.0 mmol) in MeCN (150 mL) was
added 25 (3.80 g, 15.2 mmol, isomer ratio 82:18) and the mixture was heated to reflux for 20 h.
Isopropanol (15 mL) was added and the mixture was heated to reflux for additional 10 min. The
mixture was allowed to cool to room temperature and the formed solid was filtered off, rinsed with
acetone and the filtrate was concentrated under reduced pressure. The green coloured crude solid was
triturated with 1M H2SO4 (aq, 15 mL) and the solid material was filtered off, washed with water and
pentane and dried in vacuo to afford 29 (1.60 g, 38% based on 25) as a white solid. 1H NMR (DMSO-
d6) δ 8.51 (d, J = 1.7 Hz, 1H), 8.09 (dd, J = 8.2, 1.7 Hz, 1H), 7.87 (d, J = 8.1 Hz, 1H).
13C NMR (DMSO-d6) δ 160.9, 141.5, 137.4, 128.7, 127.5, 126.4, 124.1.
3.14 6-Bromosaccharin (29) synthesized from 24
A solution of 24 (7.06 g, 26.2 mmol) in dry CH2Cl2 (60 mL) was dropwise added to a solution of
tert-butylamine (2.88 mL, 27.5 mmol) and Et3N (3.83 mL, 27.5 mmol) in CH2Cl2 (120 mL) at 0 °C.
The mixture was stirred at 0 °C for 1.5 h, was then allowed to warm to room temperature and stirred
at this temperature for 24 h. The mixture was washed with 0.1 M HCl (aq.) and sat. NaHCO3 (aq.),
dried over MgSO4, filtered and concentrated under reduced pressure. The crude product was purified
by flash column chromatography (17% EtOAc/pentane) to afford 26 (5.65 g, 70% yield, 23% of the
other regioisomer). H5IO6 (15.5 g, 68.0 mmol) was vigorously stirred in MeCN (85 mL) for 1 h. CrO3
22

(254 mg, 2.55 mmol) and acetic anhydride (6.4 mL, 68.0 mmol) were added and the suspension was
stirred for 10 min at room temperature. The mixture was cooled in an ice-bath and 26 (2.60 g,
1
8.49 mmol, ratio 77:23 according to H NMR spectra) was added in one portion. The mixture was
stirred at 0 °C for 15 min and then allowed to warm to room temperature and stirred for 13 h. The
solvent was removed and the remaining slurry was stirred in EtOAc (100 mL) for 10 min. The solid
was filtered off, washed with EtOAc and acetone and the filtrate was concentrated under reduced
pressure. Water was added and the aqueous phase was extracted with EtOAc. The combined organic
phases were washed with sat. NaHCO3 (aq), sat. Na2S2O3 (aq) and brine, dried over MgSO4, filtered
and concentrated under reduced pressure. The crude product was dissolved in TFA (20 mL) and heated
to reflux for 48 h and thereafter stirred at room temperature for 4 d. The TFA was removed from the
formed precipitate and concentrated under reduced pressure. The precipitate and the remaining solid
after evaporation were treated with CH2Cl2, and dried in vacuo to afford 29 (1.14 g, 62% overall yield
based on 26). 1H NMR (CD3OD) δ 8.25 (d, J = 1.6 Hz, 1H), 8.07 (dd, J = 8.1, 1.6 Hz, 1H), 7.91 (d, J
= 8.2 Hz, 1H). 13C NMR (DMSO-d6) δ 160.9, 141.5, 137.4, 128.7, 127.59, 126.4, 124.1.
3.15 General procedure for the synthesis of the sodium salt of saccharins (30 and 31)
To a 0.2 M solution of saccharin (1 equiv) in dry MeOH, NaOMe (1.01 equiv) was added and the
mixture was stirred for 6 h at room temperature. The solvent was removed and the residue was dried
in vacuo.
Sodium saccharin (30). The compound was synthesized according to the general procedure from
saccharin 28 (8.0 g, 44 mmol) to afford 30 (8.8 g, 98%) as a white solid which was directly used in the
next step.
Sodium 6-bromosaccharin (31). The compound was synthesized according to the general
procedure from 29 (824 mg, 2.97 mmol) to afford 31 (896 mg, >99%) as a white solid which was
directly used in the next step.
3.16 General procedure for the alkyl substituted saccharins 32a–d and 33a–f
The sodium salts of saccharin 30 or 31 (1 equiv) was dissolved in DMF (1 M) and the appropriate
halide (1.05 equiv) was added. The mixture was heated in a microwave reactor at 145 °C for 1 h if not
otherwise stated. The mixture was poured on ice-water and the precipitate was filtered off, washed
with water and pentane and dried in vacuo. If no precipitate was formed, the aqueous phase was
23

extracted with EtOAc and the combined organic phases were washed with brine, dried over MgSO4,
filtered and concentrated under reduced pressure.
2-Pentylbenzo[d]isothiazol-3(2H)-one-1,1-dioxide (32a). The compound was synthesized
according to the general procedure from 30 (150 mg, 0.731 mmol). Purification by flash column
1
chromatography (40% EtOAc/pentane) afforded 32a (143 mg, 77%) as a colourless oil. H NMR
δ 8.08–8.02 (m, 1H), 7.93–7.89 (m, 1H), 7.89−7.78 (m, 2H), 3.76 (t, J = 7.6 Hz, 2H), 1.92–1.78 (m,
2H), 1.44–1.32 (m, 4H), 0.91 (t, J = 6.9 Hz, 3H). 13C NMR δ 159.1, 137.9, 134.8, 134.4, 127.6, 125.2,
121.0, 39.6, 29.0, 28.2, 22.3, 14.0. Anal. (C12H15NO3S) C, H, N.
2-Phenethylbenzo[d]isothiazol-3(2H)-one-1,1-dioxide (32b). The compound was synthesized
according to the general procedure from 30 (200 mg, 0.97 mmol). Purification by flash column
chromatography (30% EtOAc/pentane) afforded 32b (197 mg, 67%) as a white solid. 1H NMR DMSO-
d6) δ 8.30 (ddd, J = 7.6, 0.9, 0.9 Hz, 1H), 8.09–7.94 (m, 3H), 7.34–7.15 (m, 5H), 3.99–3.88 (m, 2H),
13
3.09–2.97 (m, 2H). C NMR (DMSO-d6) δ 158.2, 137.6, 136.7, 135.8, 135.3, 128.7, 128.4, 126.6,
126.2, 125.0, 121.5, 39.8, 33.9. Anal. (C15H13NO3S) C, H, N.
Ethyl 4-(1,1-dioxo-3-oxobenzo[d]isothiazol-2(3H)-yl)butanoate (32c). The compound was
synthesized according to the general procedure from 30 (100 mg, 0.49 mmol) in a microwave reactor
at 145 °C for 15 min. Purification by flash chromatography (25% EtOAc/pentane) afforded 32c
(111 mg, 73%) as a colourless oil. 1H NMR δ 8.08–8.04 (m, 1H), 7.95–7.90 (m, 1H), 7.90–7.80 (m,
2H), 4.16 (q, J = 7.1 Hz, 2H), 3.86 (t, J = 7.0 Hz, 2H), 2.45 (t, J = 7.3 Hz, 2H), 2.18 (p, J = 7.2 Hz,
2H), 1.26 (t, J = 7.1 Hz, 3H). 13C NMR δ 172.5, 159.2, 137.8, 134.9, 134.5, 127.5, 125.3, 121.1, 60.7,
38.7, 31.4, 23.8, 14.4. Anal. (C13H15NO5S) C, H, N.
2-(3-(Pyridin-3-yl)propyl)benzo[d]isothiazol-3(2H)-one-1,1-dioxide (32d). The compound was
synthesized according to the general procedure from 30 (88 mg, 0.43 mmol). Purification by flash
column chromatography (30→80% EtOAc/pentane) afforded 32d (15 mg, 12%) as a yellow oil.
1H NMR δ 8.49 (d, J = 1.6 Hz, 1H), 8.45 (dd, J = 4.8, 1.6 Hz, 1H), 8.10–8.03 (m, 1H), 7.95–7.91 (m,
1H), 7.90–7.80 (m, 2H), 7.55 (ddd, J = 7.8, 2.3, 1.6 Hz, 1H), 7.22 (ddd, J = 7.8, 4.8, 0.9 Hz, 1H), 3.84
13
(t, J = 7.2 Hz, 2H), 2.83–2.68 (m, 2H), 2.24−2.13 (m, 2H). C NMR δ 159.1, 150.1, 147.9, 137.8,
136.1, 135.9, 135.0, 134.5, 127.4, 125.3, 123.5, 121.1, 39.0, 30.3, 29.8. HRMS (ESI) m/z calcd for
C15H15N2O3S [M+H]+ 303.0803, found 303.0776.
6-Bromo-2-pentylbenzo[d]isothiazol-3(2H)-one-1,1-dioxide (33a). The compound was
synthesized according to the general procedure from 31 (100 mg, 0.33 mmol) in a microwave reactor
at 145 °C for 30 min. to afford 33a (67 mg, 58%) as an off-white solid. 1H NMR δ 8.05 (dd, J = 1.6,
24

0.6 Hz, 1H), 7.95 (dd, J = 8.1, 1.6 Hz, 1H), 7.90 (dd, J = 8.2, 0.6 Hz, 1H), 3.79–3.71 (m, 2H), 1.84 (p,
J = 7.5 Hz, 2H), 1.58–1.47 (m, 2H), 1.44–1.34 (m, 4H), 1.00–0.84 (m, 3H). 13C NMR δ 158.3, 139.2,
137.7, 129.8, 126.5, 126.3, 124.3, 39.8, 29.0, 28.2, 22.3, 14.0. HRMS (ESI) m/z calcd for
C12H14BrNO3S [M]+ 330.9878, found 330.9877.
6-Bromo-2-phenethylbenzo[d]isothiazol-3(2H)-one-1,1-dioxide (33b). The compound was
synthesized according to the general procedure from 31 (150 mg, 0.50 mmol). Purification by
1
recrystallization from EtOAc/pentane afforded 33b (148 mg, 77%) as a white solid. H NMR δ
8.08–8.04 (m, 1H), 7.95–7.90 (m, 1H), 7.90–7.80 (m, 2H), 4.16 (q, J = 7.1 Hz, 2H), 3.86 (t, J = 7.0
13
Hz, 2H), 2.45 (t, J = 7.3 Hz, 2H), 2.18 (p, J = 7.2 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H). C NMR
δ 158.1, 139.2, 137.8, 137.4, 129.9, 129.0, 128.8, 127.1, 126.6, 126.2, 124.3, 40.8, 34.8. Anal
(C15H12BrNO3S) C, H, N.
6-Bromo-2-(4-hydroxyphenethyl)benzo[d]isothiazol-3(2H)-one-1,1-dioxide
(33c).
The
compound was synthesized according to the general procedure from 31 (124 mg, 0.41 mmol).
Purification by recrystallization from EtOAc/pentane afforded 33c (108 mg, 66%) as a white solid. 1H
NMR (DMSO-d6) δ 9.22 (s, 1H), 8.74 (d, J = 1.6 Hz, 1H), 8.17 (dd, J = 8.2, 1.7 Hz, 1H), 7.96 (d, J =
8.2 Hz, 1H), 7.06 (d, J = 8.4 Hz, 2H), 6.67 (d, J = 8.4 Hz, 2H), 3.86 (dd, J = 8.4, 6.8 Hz, 2H), 2.91
13
(dd, J = 8.5, 6.7 Hz, 2H). C NMR (DMSO-d6) δ 157.6, 156.0, 138.3, 138.2, 129.7, 129.4, 127.6,
126.8, 125.4, 124.7, 115.3, 40.3, 33.1. Anal. (C15H12BrNO4S) C, H, N.
6-Bromo-2-(3-bromophenethyl)benzo[d]isothiazol-3(2H)-one-1,1-dioxide (33d). The compound
was synthesized according to the general procedure from 31 (114 mg, 0.38 mmol) to afford 33d
(133 mg, 76%) as a white solid. 1H NMR δ 8.06 (dd, J = 1.6, 0.5 Hz, 1H), 7.96 (dd, J = 8.2, 1.6 Hz,
1H), 7.89 (dd, J = 8.2, 0.5 Hz, 1H), 7.47–7.43 (m, 1H), 7.39 (dt, J = 7.5, 1.7 Hz, 1H), 7.25–7.16 (m,
13
2H), 4.01–3.93 (m, 1H), 3.14–3.06 (m, 2H). C NMR δ 158.1, 139.6, 139.1, 137.9, 132.1, 130.4,
130.3, 130.1, 127.7, 126.7, 126.1, 124.4, 122.8, 40.4, 34.4. Anal. (C15H11Br2NO3S) C, H, N.
6-Bromo-2-(3-nitrophenethyl)benzo[d]isothiazol-3(2H)-one-1,1-dioxide (33e). The compound
was synthesized according to the general procedure from 31 (150 mg, 0.53 mmol) to afford 33e
1
(166 mg, 76%) as an off-white solid. H NMR (DMSO-d6) δ 8.74 (dd, J = 1.7, 0.5 Hz, 1H),
8.20–8.15 (m, 2H), 8.08 (ddd, J = 8.2, 2.4, 1.0 Hz, 1H), 7.95 (dd, J = 8.2, 0.5 Hz, 1H), 7.74 (ddd,
J = 7.6, 1.7, 1.0 Hz, 1H), 7.58 (ddd, J = 8.1, 7.6, 0.4 Hz, 1H), 4.05 (t, J = 7.0 Hz, 1H), 3.19 (t, J = 6.9
25

Hz, 1H). 13C NMR (DMSO-d6) δ 157.8, 147.8, 140.1, 138.3, 138.1, 135.9, 129.9, 129.5, 126.8, 125.3,
1
124.8, 123.7, 121.7, 39.4 , 33.2. Anal. (C15H11BrN2O5S) C, H, N.
6-Bromo-2-(2-(1-succinimidyl)ethyl)benzo[d]isothiazol-3(2H)-one-1,1-dioxide
(33f).
The
compound was synthesized according to the general procedure from 31 (90 mg, 0.3 mmol).
Purification by recrystallization from EtOAc/pentane afforded 33f (71 mg, 59%) as a white solid.
1H NMR (DMSO-d6) δ 8.74 (dd, J = 1.7, 0.5 Hz, 1H), 8.20 (dd, J = 8.2, 1.7 Hz, 1H), 8.02 (dd,
13
J = 8.2, 0.5 Hz, 1H), 3.91–3.80 (m, 2H), 3.80–3.70 (m, 2H), 2.55 (s, 4H). C NMR (DMSO-d6)
δ 177.8, 157.9, 138.4, 137.9, 129.6, 126.8, 125.3, 125.0, 37.0, 35.6, 28.0. Anal. (C13H11BrN2O5S)
C, H, N.
3.17 SIRT1−3 in Vitro Assay
The Fluor de Lys fluorescence assays were based on the method described in the BioMol product
sheet (Enzo Life Sciences) using the BioMol KI177 substrate for SIRT1 and the KI179 substrate for
SIRT2 and SIRT3. The determined Km value of SIRT1 for KI177 was 58 μM, and the Km of SIRT2
for KI179 was 198 μM.[26] The Km of SIRT3 for KI179 was reported by Enzo Life Sciences to be 32
μM. The Km values of SIRT1, SIRT2 and SIRT3 for NAD+ were reported by BioMol to be
558 μM, 547 μM and 2 mM, respectively.
Briefly, assays were carried out using the Fluor de Lys acetylated peptide substrate at 0.7 Km and
NAD+ (Sigma N6522 or BioMol KI282) at 0.9 Km, recombinant GST-SIRT1/2-enzyme or
recombinant His-SIRT3 and SIRT assay buffer (KI286). GST-SIRT1 and GST-SIRT2 were produced
as described previously.[27,28] His-SIRT3 (BML-SE270) was purchased from Enzo Life Sciences.
The buffer, Fluor de Lys acetylated peptide substrate, NAD+ and DMSO/compounds in DMSO (2.5
μL in 50 μL total reaction volume; DMSO from Sigma, D2650) were preincubated for
5 min at room temperature. The reaction was started by adding the enzyme. The reaction mixture was
incubated for one hour at 37 °C. After that, Fluor de Lys developer (KI176) and 2 mM nicotinamide
(KI283) in SIRT assay buffer (total volume 50 μL) were added, and the incubation was continued for
45 min at 37 °C. Fluorescence readings were obtained using EnVision 2104 Multilabel Reader
(PerkinElmer) with excitation wavelength 370 nm and emission 460 nm.
1 Detected by HSQC.
26

3.18 Molecular Modeling
3.18.1 Docking
The previously published 3D-structure of SIRT2 [2] was prepared using Protein Preparation Wizard
[29] implemented in the Schrödinger Suite. Ensembles of conformations for each ligand to be docked
were generated in order to prevent the starting conformation bias the final docking solutions. In case
of a chiral compound, both enantiomers were docked. The docking was conducted using Glide XP
(extra precision) into the proposed binding site for the chroman-4-one inhibitors.[2] In the docking
trial the protein structure was held rigid while the ligands were flexible. The best scored docking
solutions that fulfilled the inhibitor-enzyme key interactions were reported. Single point energies for
each docked ligand conformation were calculated, to be able to determine their relative energiesError!
Reference source not found.. All calculations have been performed using OPLS3 force field [21] in
a water solvation model with tools implemented in Schrödinger Suite.[22]
Corresponding Author
*(T.S.) Tel.: +46 31 786 9115; fax: +46 31 772 3840; e-mail: tina.seifert@chem.gu.se.
Notes
The authors declare no competing financial interest.
Abbreviations
CDI, 1,1'-Carbonyldiimidazole; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; HBA,
hydrogen bond acceptor; HBD, hydrogen bond donator; HRMS, high resolution mass spectrometry;
MMFF, Merck Molecular Force Field; MW, microwave; NAD, Nicotinamide adenine dinucleotide,
NBS, N-bromosuccinimide , NMR, nuclear magnetic resonance; OPLS, Optimized Potentials for
Liquid Simulations; PSA, polar surface area; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC,
thin layer chromatography, TSA, toluenesulfonic acid.
+
Appendix A. Supplementary data.
27

Electronic Supplementary Information (ESI) available. Synthetic procedures for compounds 18c,
1
13
18d and 3-(3-bromopropyl)pyridine, H NMR and C NMR spectra of all compounds, elemental
analysis, HRMS and purity data of all biologically tested compounds, as well as the calculated
physicochemical properties.
Acknowledgments
We thank the Swedish Research Council (Projects #2013-4749 and 2017-3984), the Academy of
Finland (grants no 127062 and 132780) and the Department of Chemistry and Molecular Biology,
University of Gothenburg, for financial support and Biocenter Kuopio for providing facilities. We also
thank Sari Ukkonen for assistance with the sirtuin assays, and Dr. Maria Fridén-Saxin from
AstraZeneca R&D Mölndal for calculation of the physicochemical properties.
28

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