Towards the inhibition of protein–protein interactions (PPIs) in STAT3: Insights into a new class of benzothiadiazole derivatives

Article abstract of DOI:10.3390/molecules25153509

Signal transducer and activator of transcription 3 (STAT3) is a validated anticancer target due to the relationship between its constitutive activation and malignant tumors. Through a virtual screening approach on the STAT3-SH2 domain, 5,6-dimethyl-1H,3H-2,1,3-benzothiadiazole-2,2-dioxide (1) was identified as a potential STAT3 inhibitor. Some benzothiadiazole derivatives were synthesized by employing a versatile methodology, and they were tested by an AlphaScreen-based assay. Among them, benzosulfamide 1 showed a significant activity with an IC₅₀ = 15.8 ± 0.6 μM as a direct STAT3 inhibitor. Notably, we discovered that compound 1 was also able to interact with cysteine residues located around the SH2 domain. By applying mass spectrometry, liquid chromatography, NMR, and UV spectroscopy, an in-depth investigation was carried out, shedding light on its intriguing and unexpected mechanism of interaction.

Full text of DOI:10.3390/molecules25153509

molecules
Article
Towards the Inhibition of Protein–Protein
Interactions (PPIs) in STAT3: Insights into a New
Class of Benzothiadiazole Derivatives
Matteo Mori 1 , Ettore Gilardoni 1 , Luca Regazzoni * , Alessandro Pedretti
1,
1
,
Diego Colombo 2 , Gary Parkinson 3 , Akira Asai , Fiorella Meneghetti
4
1
, Stefania Villa *
1,
and Arianna Gelain 1
1
Department of Pharmaceutical Sciences, University of Milan, via L. Mangiagalli 25, 20133 Milano, Italy;
matteo.mori@unimi.it (M.M.); ettore.gilardoni@unimi.it (E.G.); alessandro.pedretti@unimi.it (A.P.);
fiorella.meneghetti@unimi.it (F.M.); arianna.gelain@unimi.it (A.G.)
2
3
4
Department of Medical Biotechnology and Translational Medicine, University of Milan, via C. Saldini 50,
2
0133 Milano, Italy; diego.colombo@unimi.it
Department of Pharmaceutical and Biological Chemistry—UCL School of Pharmacy, University College
London, 29/39 Brunswick Square, London WC1N 1AX, UK; gary.parkinson@ucl.ac.uk
Center for Drug Discovery—Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada,
Suruga-ku, Shizuoka 422-8526, Japan; aasai@u-shizuoka-ken.ac.jp
*
Correspondence: luca.regazzoni@unimi.it (L.R.); stefania.villa@unimi.it (S.V.); Tel.: +39-02-50319368 (S.V.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 24 June 2020; Accepted: 29 July 2020; Published: 31 July 2020
Abstract: Signal transducer and activator of transcription 3 (STAT3) is a validated anticancer target
due to the relationship between its constitutive activation and malignant tumors. Through a virtual
screening approach on the STAT3-SH2 domain, 5,6-dimethyl-1H,3H-2,1,3-benzothiadiazole-2,2-
dioxide (
synthesized by employing a versatile methodology, and they were tested by an AlphaScreen-based
assay. Among them, benzosulfamide showed a significant activity with an IC50 = 15.8 0.6
as a direct STAT3 inhibitor. Notably, we discovered that compound was also able to interact
1) was identified as a potential STAT3 inhibitor. Some benzothiadiazole derivatives were
1
±
µM
1
with cysteine residues located around the SH2 domain. By applying mass spectrometry, liquid
chromatography, NMR, and UV spectroscopy, an in-depth investigation was carried out, shedding
light on its intriguing and unexpected mechanism of interaction.
Keywords: benzosulfamides; structure-based virtual screening; STAT3-SH2 domain; cysteine binder;
diversity-oriented synthesis
1
. Introduction
Since the discovery of the relationship between the constitutive activation of signal transducer
and activator of transcription 3 (STAT3) and malignant tumors, the validation of STAT3 as a target
has been supported by a large number of studies. Various compounds with STAT3 inhibitory activity
have been reported in recent years, including potent inhibitors such as Stattic, S3I-201, and erasin [
Nevertheless, these molecules are mostly at the experimental stage, and only few are in clinical trials [
1
–
5
4
].
].
Hence, the challenge is the discovery of new selective candidates with high potency and in vivo activity.
Since a direct inhibitory approach should be preferred, we addressed our efforts to the disruption of
STAT3 homodimerization, an essential step in its activation, targeting the protein–protein interactions
(PPIs). The large surface area of STAT3 and the chemistry of these interactions [6], which are very
different from those of better-known targets such as enzymes and G-protein-coupled receptors, could
represent significant hurdles, although a number of successful examples have demonstrated that it is
Molecules 2020, 25, 3509; doi:10.3390/molecules25153509
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Molecules 2020, 25, 3509
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possible to overcome them and develop PPI modulators [
7]. Since the binding of a ligand to a hot spot
should compete with the original protein partner of the PPI, thus resulting in disruption of its function,
the aim of our ongoing studies [ 13] is the identification of small molecules that are able to bind the Src
8
–
homology 2 (SH2) domain of STAT3, preventing STAT3 dimerization and thus its activation. With this
purpose, in the present work, some compound libraries were analyzed by means of a structure-based
virtual screening performed on the STAT3-SH2 domain (see the Molecular Modeling section). Among
the candidates that reached the best scores in the docking study, we decided to focus our attention on
the compounds that bear a sulfone moiety, which is a common feature in some STAT3-SH2 inhibitors
reported in the literature [1,2].
In order to support the docking results, the selected compounds were either purchased or
synthesized, and their ability to interact with the STAT3-SH2 domain was evaluated via an in vitro
binding test (AlphaScreen-based assay) at 30 µM; the most interesting results are reported in Table 1.
Hence, we identified the benzothiadiazole derivative
chemically accessible moiety for further development.
1 as a promising hit compound that carries a
Table 1. Inhibitory activities of compounds 1 and I–III.
Compound
Structure
% Inhibition
H
H C
3
N
1
I
SO2
96.5
79.0
N
H
H C
3
CH3
N
Cl
N
S
O
2
CH3
Cl
N
II
43.4
85.4
N
S
O
2
NH2
N
N
III
O
O S
2
N
N
H
The significant inhibitory activity exhibited by 5,6-dimethyl-1H,3H-2,1,3-benzothiadiazole-2,2-
dioxide ( ) prompted us to fully characterize it by means of an X-ray analysis. Moreover, we designed
and synthesized a small library of derivatives bearing different substituents on the aromatic and
thiadiazole rings (1a 2a ). Furthermore, imidazol-2-one ( ) and thiophene dioxide ( ) moieties
replacing the thiadiazole ring (Figure 1)) were assessed to develop a preliminary structure-activity
relationship (SAR) on this class of compounds.
For the synthesis of 1a , we employed an adaptable methodology [14] that also considered a
diversity-oriented approach using suitable monoprotected intermediates. For derivatives 2a,b , and
our synthetic strategy was planned to allow for the introduction of different substituents on compound
1
–
i
,
,
b,
3,
4
5
6
(
–
i
,
3
4,
(1) by the direct alkylation of the sulfamide moiety.
Herein, we describe the in silico-based rational design, synthesis, characterization, and interaction
with the STAT3-SH2 domain of new inhibitors bearing the benzosulfamide scaffold. Furthermore,
since several STAT3 inhibitors have been reported as irreversible cysteine binders (e.g., Stattic and
S3I-201) [15–18], we decided to evaluate the activity of compound 1 versus the cysteine residues located
around the STAT3-SH2 domain (Cys468, Cys542, Cys550, Cys687, and Cys712) and to shed light on its
mechanism of interaction by MS, UV, LC, and NMR studies.

Molecules 2020, 25, 3509
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R2
N
H
N
H C
3
R
SO2
SO2
N
N
H
R
1
R3
=
=
=
=
,
=
=
1
1
1
1
1
1
1
1
1
a R
b R CH3
H
2a R1 Br; R R3
H
2
,
2b R1 H; R2 R3 CH3
=
c R CF3
=
d R NO2
=
e R Cl
=
=
=
N
f
R
NHCOCH3
SO2
g2 R
CH2 CONH CH CH CH
3
2 2 2 2
( ) ( ) ( )
N
h R CH2 2CONHPh
(
Ph
)
=
i
R
O
3
N
H
N
H
N
SO2
O
SO
2
N
N
H
H
4
5
6
Figure 1. Library of 5,6-dimethyl-1H,3H-2,1,3-benzothiadiazole-2,2-dioxide (1) derivatives.
. Results and Discussion
2
2
.1. Molecular Modeling—Design and Chemical Space Exploration
In order to identify new direct STAT3 inhibitors, molecular modeling studies were carried out.
In particular, a virtual screening approach was applied to compound databases with the aim to
select a series of candidates endowed with the best binding scores. Therefore, we performed a
structure-based virtual screening, choosing STAT3 in complex with a DNA fragment (Protein Data
Bank (PDB) ID 1BG1) [19] as a model and employing a set of databases of small molecules, namely:
AKos (544391 molecules), ChemPDB (4009), ChemBank (2344), Kyoto Encyclopedia of Genes and
Genomes–Compound (KEGG, 10005), National Cancer Institute Anti-HIV( NCI’s Anti-HIV, 42689),
and National Cancer Institute (NCI, 15237).
Each molecule was ranked on the basis of the docking score (interaction energy of AutoDock
4) [20] of the best solution. In this way, a list of compounds was obtained in which the first and
the last molecules were, respectively, the best and the worst interacting ones. This list made the
next step of the analysis easier because only the first 500 compounds for each database were further
considered. Hence, the best molecules were classified on the basis of the most recurrent moieties: (e.g.,
sulfonic acids, sulfonamides, sulfamates, phosphates, and diketones; Tables S1–S5, Supplementary
Materials). This kind of clustering was performed by encoding the moieties into SMARTS patterns
and performing queries on the databases of the top-ranked poses, as implemented in the VEGA
ZZ program. These results were very interesting because the functional groups included negatively
charged atoms, such as oxygens, which can mimic the phosphate moiety of Tyr705 (pTyr-705), upon
interaction with positively charged amino acids. A further selection was performed based on the
commercial availability, synthetic accessibility, and size of the molecules. In particular, small molecules
were privileged because, in the future, they may be included in peptidic derivatives to improve their
selectivity. Therefore, drawing inspiration from compounds reported in the literature [1,2], we decided
to focus on the cyclic sulfonamide moiety, ultimately selecting compounds with a molecular weight
lower than 300 Da. The biological activity of several candidates belonging to the NCI collection was
evaluated by an AlphaScreen-based assay, and among the most interesting hits, compound
as a promising lead for further development (Table 1). The analysis of its docking pose (see Figure S1)
revealed that compound may interact with the SH2 domain through the insertion of its sulfonamide
moiety into the polar pocket lined by Arg-609 and Lys-591; moreover, it showed that the compound
1 emerged
1

Molecules 2020, 25, 3509
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occupies most of the pocket volume normally filled by pTyr-705 in the dimerized STAT3. Starting from
this qualitative analysis of the binding mode, it seems reasonable to hypothesize that this molecule
could act as a reversible inhibitor of STAT3 dimerization.
2
.2. Chemistry
Compounds
1
and 1a–d were synthesized from the corresponding commercially available
ortho-diamines through a direct cyclization in the presence of sulfamide and diglyme (Scheme 1) [21].
H
R1
NH2
NH2
R1
R
N
SO2
R
N
H
,
=
=
1
1
1
1
1
R R
CH3
H
,
,
,
1
,
a R R1
b R CH3 R1
c R CF3 R1
d R NO2 R1
=
=
=
=
=
=
H
H
H
Scheme 1. Reagents and conditions: NH SO NH , diglyme, reflux, 40 min (10 min for compound 1).
2
2
2
Compound
1
showed acidic properties (pKa = 6.88
±
0.03) [22] and was fully characterized by
X-ray diffraction. Its crystal structure is shown in Figure 2 as an ORTEP [23] drawing (Figure 2).
Figure 2. ORTEP [23] view of compound 1 and the relative arbitrary atom-numbering scheme (thermal
ellipsoids at 50% probability).
Products 1e and 2a were obtained starting from 2,4-dichloro-1-nitrobenzene and 1,5-dibromo-
2
-methyl-4-nitrobenzene, respectively. They were reacted with 2,4-dimethoxybenzylamine (DMB-NH2)
or 4-methoxybenzylamine (PMB-NH ) to give intermediates
7
and
and 10 through reduction with zinc and ammonium chloride.
and 10 underwent a cyclization reaction, thus affording intermediates
1 and 12. These benzothiadiazole derivatives were deprotected with trifluoroacetic acid (TFA) to yield
8, respectively, which were
2
converted to the corresponding amines
By treatment with sulfamide,
9
9
1
the final products 1e and 2a, respectively (Scheme 2).
Product 1f was obtained starting from compound 1d, which was protected by treatment with
PMB-Br and potassium carbonate to afford intermediate 13; its reduction with zinc and ammonium
chloride gave the amine 14. The latter, through a reaction with acetic anhydride, afforded intermediate
1
5, which was deprotected with TFA to give product 1f (Scheme 3).
Intermediate 19, the starting reagent for the synthesis of products 1g–i, was obtained as reported
in Scheme 4: the reaction of 2,5-dibromo-nitrobenzene with PMB-NH led to intermediate 16, which
2
was reduced to give the amine 17 in the presence of zinc and ammonium chloride. By treatment with
sulfamide, 17 was cyclized, thus affording the benzothiadiazole derivative 18, which was subsequently
protected with PMB-Br to yield compound 19 (Scheme 4).

Molecules 2020, 25, 3509
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H
N
R1
NO2
R
R1
R
NO2
R1
R
NH2
R1
R
a
b
c
SO2
R
NH
R2
NH
R2
N
R
2
=
=
,
,
=
=
,
,
,
R
R
Cl R
H
7 8
9 10
11 12
1
Br R CH3
1
H
N
, ,
=
=
=
=
,
,
=
=
=
,
=
R1
R
7 9 11
R
Cl R
1
H R
2
DMB
d
,
,
,
=
8
1
2
10 12 R Br R
CH3 R2 PMB
H
R1 CH3
SO2
1
,
e
a
N
H
R
R
Cl R
1
r,
=
B
,
1
e 2a
Scheme 2. Reagents and conditions: ) or
(
a
) 2,4-dimethoxybenzylamine (DMB-NH ) (for
7
2
4
-methoxybenzylamine (PMB-NH ) (for 8), isopropanol, reflux, 12 h; (b) Zn, NH Cl, MeOH/H O (9:1),
2
4
2
◦
◦
reflux, 3 h; (
.5 h.
c
) NH SO NH , dry diglyme, 160 C, 1.5 h; and (
d) trifluoroacetic acid (TFA), DCM, 50 C,
2
2
2
1
PMB
N
SO2
PMB
H
N
N
a
b
c
SO2
SO2
N
H
N
N
O N
O N
H N
2
2
2
PMB
PMB
1
d
13
14
PMB
N
SO2
H
N
O
O
d
SO2
N
H
N
H C
N
H
H C
N
3
3
H
PMB
15
1f
◦
Scheme 3. Reagents and conditions: (a) PMB-Br, dry K CO , dry DMF, 80 C, 2 h; (b) Zn, NH Cl,
2 3 4
MeOH/H O (9:1), reflux, 3 h; (c) (CH COO) O, (CH ) CO, r.t., 3 h; and (d) TFA, DCM, r.t., 8 h.
2
3
2
3 2
PMB
NH
PMB
NH
Br
a
b
c
Br
NO2
Br
NO2
Br
NH2
1
6
17
PMB
N
SO2
PMB
N
SO2
d
B
r
N
Br
N
H
PMB
1
8
19
Scheme 4. Reagents and conditions: (
a) PMB-NH , reflux, 8 h; (b) Zn, NH Cl, MeOH/H O (9:1), reflux,
2 4 2
◦
◦
4
h; (c) NH SO NH , dry diglyme, 160 C, 3 h; and (d) PMB-Br, dry K CO , dry DMF, 80 C, 2 h.
2
2
2
2
3
Afterwards, intermediate 19 was reacted with ethyl acrylate in the presence of tri-o-tolylphosphine
and tris(dibenzylideneacetone)dipalladium(0) to afford the ester 20. The latter was hydrolyzed under
basic conditions to the corresponding acid 21, which was converted to the amides 22 and 23 using
isopentylamine, HATU, and N,N-diisopropylethylamine or aniline, TBTU, and N-methylmorpholine,
respectively. By catalytic hydrogenation with 10% Pd/C, they were reduced and deprotected to give
products 1g and 1h (Scheme 5).

Molecules 2020, 25, 3509
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PMB
N
SO2
PMB
PMB
N
SO
N
N
SO
N
a
b
2
2
H C
O
HO
3
B
r
N
PMB
O
d
PMB
O
PMB
1
9
20
21
PMB
H
N
N
,
,
=
=
c
22 1g R isopentyl
H
SO
H
SO2
2
N
23 1h R Ph
N
N
H
N
R
R
PMB
O
O
,
,
2
2 23
1g 1h
◦
Scheme 5. Reagents and conditions: (a) ethyl acrylate, P(o-tol) , Pd (dba) , TEA, dry DMF, 80 C, 20 h;
3 2 3
◦
(b) 3 M NaOH, MeOH, 80 C, 3 h; (c) isopentylamine, HATU, DIPEA, DCM, r.t., 20 h (for 22) or aniline,
TBTU, N-methylmorpholine, DCM, r.t., 24 h (for 23); and (d) H (3 atm), 10% Pd/C, EtOH, r.t., 48 h.
2
Moreover, compound 19 was arylated through a Suzuki reaction with phenylboronic acid in
the presence of tetrakis(triphenylphosphine)palladium(0) to give intermediate 24, which was then
deprotected by treatment with TFA, thus affording product 1i (Scheme 6).
PMB
N
SO2
PMB
N
SO2
H
N
a
b
SO2
N
H
B
r
N
N
PMB
PMB
1
9
24
1i
Scheme 6. Reagents and conditions: (
a) PhB(OH) , Pd(PPh ) , Na CO , 1,4-dioxane, reflux, 20 h; and
2
3 4
2
3
(
b) TFA, DCM, r.t., 3 h.
Products 2b and 3 were obtained from compounds 1b and 1a, respectively, through alkylation
with iodomethane or benzyl bromide in the presence of potassium carbonate (Scheme 7).
R1
H
N
N
a
SO2
SO
2
N
H
R
N
R
R2
=
=
=
,
,
,
=
=
1
1
a R
b R CH3
H
3
R
H R R
Bn
1
2
,
2b R R R
CH3
1
2
◦
Scheme 7. Reagents and conditions: CH I (for 2b) or BnBr (for
3), dry K CO , and dry DMF, 80 C, 2 h.
3
2
3
Product
4 was prepared through a reaction of 1-chloro-2-nitrobenzene with DMB-NH to obtain
2
intermediate 25, which was reduced using zinc and ammonium chloride to give the amine 26. Its
cyclization with sulfamide led to intermediate 27, which was firstly esterified with ethyl bromoacetate
to give intermediate 28 and then hydrolyzed under basic conditions to the corresponding acid
2
9
. This intermediate was condensed with 2-phenylethylamine to afford the amide 30, which was
deprotected with TFA, thus leading to the product 4 (Scheme 8).

Molecules 2020, 25, 3509
7 of 29
H C
O
3
O
H
N
NO2
Cl
NO2
NH2
N
N
c
d
a
b
SO2
SO
2
N
NH
NH
DMB
DMB
DMB
DMB
25
26
27
28
OH
O
O
O
N
H
N
H
e
N
N
N
N
N
f
g
SO2
SO2
SO2
N
H
DMB
DMB
2
9
30
4
Scheme 8. Reagents and conditions: (
9:1), reflux; ( ) NH SO NH , dry diglyme, 160 C, 1.5 h; (d) BrCH COOCH CH , dry K CO , dry
2 2 3 2 3
a) DMB-NH , EtOH, reflux, 12 h; (b) Zn, NH Cl, MeOH/H O
2 4 2
◦
(
c
2
2
2
◦
◦
DMF, 80 C, 2 h; (e) K CO , MeOH, 50 C, 2 h; (f) Ph(CH ) NH , HATU, DIPEA, DCM, r.t., 12 h; and
2
3
2 2
2
◦
(
g) TFA, DCM, 45 C, 45 min.
Product
5 was achieved through the cyclization of benzene-1,2-diamine in the presence of
carbonyldiimidazole (CDI) (Scheme 9).
H
N
NH2
O
N
H
NH2
5
Scheme 9. Reagents and conditions: CDI, dry THF, reflux, 12 h.
Finally, compound 6 was purchased from commercial sources.
2
.3. Biological Evaluation
AlphaScreen is an in vitro competitive binding test used to identify compounds that are able to
inhibit the binding of proteins containing an SH2 domain to their corresponding phosphopeptides
5-carboxyfluorescein (FITC)-GpYLPQTV for STAT3 and FITC-GpYDKPHVL for STAT1) (Table 2).
(
Compounds characterized by an interesting STAT3 affinity were further investigated for their selectivity
versus STAT1, which shows a high degree of sequence similarity to STAT3 but an opposite physiological
role [24,25], and Grb2, as a model for other SH2 domain-containing proteins.
The significant activity of compound
1, revealed at 30 µM (Table 2), prompted us to test this
compound at different concentrations. Therefore, we calculated its IC50 values against STAT3 and
STAT1 to be 15.8
concentration).
±
0.6 and >50 µM, respectively, while the inhibition versus Grb2 was 23% (at a 30 µM
To identify a trend in terms of structure–activity relationship, we firstly explored the role of the
substituents on the benzene ring (1a ). In this respect, we found that lipophilicity does not play
an important role in modulating STAT3 inhibitory activity. Bulky lipophilic substituents at position
(derivatives 1g and 1h) induced a significant drop in the inhibition properties (Table 2). Among
–
i
5
these derivatives, only 1d, bearing a NO group at position 5, exhibited a noticeable activity against
2
STAT3. For this compound, we calculated the IC50 values against STAT3 and STAT1, which were
2
5.1 ± 2.6 µM and 15.9
introduced different groups on the nitrogen atoms of the thiadiazole ring (2a
to explore asymmetrical substitutions: The presence of the N-substituent did not improve activity.
±
2.0
µ
M, respectively, revealing no selectivity for STAT3 inhibition. Then, we
,b,
3
, and ), in order
4

Molecules 2020, 25, 3509
8 of 29
The enhanced bulkiness and hydrophobicity of these substituents compared to compound
lead to a better interaction.
1
did not
Table 2. Inhibitory activities of compounds
1,
1a–
i
,
2a–
b, and
3–
6. STAT3: signal transducer and
activator of transcription 3.
R2
H
R
N
N
SO2
O
SO2
N
R3
,
N
H
R1
,
-
,
-
,
1
1a 1h 2a b 3 4
5
6
Compound
1
Substituents
R, R = CH ; R , R = H
STAT3 % Inhibition (30 µM)
[
a]
1
3
2
3
96.5 ± 1.1
16.4 ± 0.8
14.5 ± 0.6
[
b]
b]
1
a
R, R , R , R = H
1 2 3
[
1
b
R = H; R = CH ; R , R = H
1 3 2 3
[
b]
1
c
R = H; R = CF ; R , R = H
8.2 ± 1.6
56.9 ± 3.6
28.2 ± 1.4
18.9 ± 2.3
1
3
2
3
[
c]
b]
b]
1
d
R = H; R = NO ; R , R = H
1 2 2 3
[
[
1
e
R = H; R = Cl; R , R = H
1 2 3
1
f
R = H; R = NHCOCH ; R , R = H
1 3 2 3
[
b]
1
g
R = H; R = (CH ) CONH(CH ) CH ; R , R = H
11 ± 2.1
1
2 2
2 2
3
2
3
1
h
R = H; R = (CH ) CONHPh; R , R = H
n.a.
1 2 2 2 3
[
b]
1
i
R = H; R = Ph; R , R = H
6.1 ± 0.9
25.6 ± 1.2
8.9 ± 1.1
n.a.
1
2
3
[
b]
2
a
R = CH ; R = Br; R , R = H
3 1 2 3
[
b]
2
b
R = H; R , R , R = CH
1 2 3 3
3
R, R = H; R , R = CH Ph
1 2 3 2
[
b]
4
5
6
R, R , R = H; R = CH CONH(CH ) Ph
17.3 ± 1.3
3.5 ± 5.6
11.5 ± 0.8
1
3
2
2
2 2
[
b]
-
-
[
b]
[
[
a]
c]
M. [b] STAT3 inhibition: IC50 > 30
STAT3 inhibition: IC50 = 15.8
STAT3 inhibition: IC50 = 25.1 ± 2.6 µM; STAT1 inhibition: IC50 = 15.9 ± 2.0 µM. n.a.: not active.
±
0.6
µM; STAT1 inhibition: IC50 > 50
µ
µ
M.
Finally, we investigated the importance of the thiadiazole ring (5 and 6) through the exploration
of the function of the SO group and of the nitrogen atoms: The results of the AlphaScreen-based assay
2
indicated that these moieties play a key role in the interaction with the STAT3-SH2 domain.
Overall, our data seem to indicate that this class of benzothiadiazole derivatives is characterized
by an extremely “tight” SAR: Most of the modifications applied to the scaffold of compound
1 resulted
in a considerable loss of activity, with the sole exception of 1d, which retained a partial inhibitory effect.
Since from the literature it is known that some benzothiadiazoles could covalently bind to
proteins [26], an in silico prediction to evaluate whether the proposed derivatives were pan-assay
interference compounds (PAINS) was performed by submitting their structures to five on-line services
(
FAF-Drugs4 [27], PAINS remove [28], SmartsFilter, (http://pasilla.health.unm.edu/tomcat/biocomp/
smartsfilter), SwissADME [29], and Zinc Patterns (http://zinc15.docking.org/patterns/home/)). Three of
them (SmartsFilter, SwissADME, and Zinc Patterns) identified compounds
PAINS (see Table S6).
1, 1a–i, and 2a as potential
This result was experimentally confuted, at least for compound
selective STAT3 inhibitor with an IC50 of 15.8 0.6 M, which was significantly lower than the IC50
shown versus STAT1 ( >50 M). This selectivity was remarkable considering the high similarity and
identity of both STAT1 and STAT3, for the SH2 domain in particular (73.5% sequence similarity and
1, because it was found to act as a
±
µ
µ
5
6.1% sequence identity, calculated by EMBOSS Needle [30]).
Furthermore, to gain insight into the activity of compound
1
, its interaction with the cysteine
residues located in the vicinities of the SH2 domain (Figure 3) was investigated and evaluated by an
AlphaScreen-based assay using different mutants (Table 3).

Molecules 2020, 25, 3509
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Table 3. Sensitivity of single-mutated STAT3 proteins (Cys468, Cys542, Cys550, Cys687, and Cys712) to
compound . SH2 binding to the phosphotyrosine peptide was assessed by the AlphaScreen assay.
1
The means ± SD were calculated from triplicate experiments.
Compound 1 vs. STAT3
Mutated Cys
IC50 (µM)
Mutated Cys
IC50 (µM)
WT
Cys468A
Cys542A
15.8 ± 0.6
Cys550A
Cys687A
Cys712A
>30
>30
>30
>30
>30
Figure 3. Ribbon diagram of STAT3 (blue), with the SH2 domain in green and the mutated cysteine
residues in yellow (Protein Data Bank (PDB) ID 1BG1).
Compound
1 was inactivated by the addition of an excess amount of cysteine into the assay
medium (see Table S7). Moreover, the point mutations of Cys468, Cys542, Cys550, Cys687, or Cys712
in STAT3 conferred resistance to the compound (see Table S8), suggesting that each cysteine residue
partially contributes to the inhibition mediated by the molecule.
Though STAT3 contains twelve cysteine residues, the assays were performed only on clones
bearing mutations on the five cysteines located near the STAT3-SH2 domain. Therefore, we cannot
exclude the possibility that other cysteines may be involved in the interaction with compound 1.
Considering the affinity of compound
of interaction with cysteines by means of analytical studies.
In order to analyze the binding mode of compound
1 for STAT3 wild-type, we decided to explore its mechanism
1
to the pockets containing the mutated
cysteine residues, a molecular docking study was carried out. In particular, we applied the same
protocol used for the virtual screening, but, in this case, the grid maps were calculated by selecting the
atoms included in a sphere of 12 Å radius that were centered on each cysteine residue. The so-obtained
complexes were optimized with the same minimization protocol employed for the preparation of the
STAT3 crystal structure (30,000 steps of conjugate gradients minimization). For a better analysis of
the poses, the distances between the cysteine sulfur and the two hypothetical reactive centers (the
methyl in 5 and the aromatic carbon in 4) of compound
1 were evaluated. The results in Table S9 show
that only the complex with Cys712 seems to have the suitable prerequisites in terms of distances to

Molecules 2020, 25, 3509
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the reactive centers (4.70 and 4.95 Å for CH –S and CH–S, respectively; see Figure S72) and docking
3
energy (
it was calculated by molecular mechanics. The complex with Cys550 also seemed to have the right
distances to react with compound (3.64 and 5.13 Å for CH –S and CH–S, respectively); however,
the interaction energy was found to be significantly worse than that of the complex with Cys712
3.52 versus 5.93 Kcal/mol). Unfortunately, this kind of behavior could not be confirmed by the
−
5.93 Kcal/mol), even if the latter parameter must be taken into account with caution because
1
3
(−
−
experimental data; this was probably due to the intrinsic problem afflicting all docking calculations
that could only evaluate the physicochemical and steric complementarity of two interacting partners
and not the reactivity of the atoms.
2
.4. Analytical Studies
2
.4.1. MS, UV, and LC Studies
MS studies revealed that compound
1 reacts with glutathione (GSH, used as a model for
cysteine-containing peptides) to generate stable covalent adducts. Since no adducts were detectable
at the incubation starting point, such reactions did not take place during sample transfer into the
MS analyzer. Similar approaches have already been used to identify reactive intermediates by MS
studies [31]. The reaction was pH-dependent and required a neutral or basic pH to take place, whereas
no reactivity was observed at an acidic pH (Figure 4).
Figure 4. MS spectra of glutathione (GSH) incubated with compound
starting point ( ), at incubation endpoint in a pH 2 buffer ( ), at incubation endpoint in a pH 7.4
buffer (C), and at incubation endpoint in a pH 10 buffer (D).
1 as recorded at incubation
A
B
The absence of reactivity at the acidic pH can be explained on one hand by the pH-dependent
nucleophilicity of GSH [32] and on the other hand by the deprotonation of compound occurring at a
neutral/basic pH (pKa = 6.88 0.03) [22], which can lead to structure rearrangements that possibly
contribute to an increased reactivity towards GSH. However, the methyl groups seem fundamental for
1
±
the covalent reaction, since no adducts were detectable upon incubation of GSH with compound 1d
which was the second most active compound according to the results reported in Table 2.
At a neutral pH (Figure 4C), a single adduct was detectable at the reaction endpoint (504.12121 m/z),
whereas at a basic pH (Figure 4D), three signals at 502.10569, 504.12120, and 520.11607 m/z were
detected along with the complete oxidation of GSH to its dimer GSSG (i.e., the conversion of the singly
charged GSH ion at 308.09106 m/z to the doubly charged GSSG ion at 307.08332 m/z).
,

Molecules 2020, 25, 3509
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The signals at 502.10569, 504.12120, and 520.11607 m/z were all within 1 ppm from the theoretical
m/z values expected for singly charged ions of molecules C H N O S , C H N O S , and
18
25
5
8
2
18 23
5
8 2
C H N O S , respectively.
18
25
5
9 2
Interestingly, at pH 10, the adducts were also detected at the incubation starting point if compound
was added before GSH into the buffer (Figure 5A), but not if GSH (Figure 5B) or Na S O (Figure 5C)
1
2
2
5
were added into the buffer before compound 1.
Figure 5. MS spectra at the reaction starting point for a 1:1 GSH:compound
1
mixture incubated at pH
) or GSH (spectrum
) and endpoint (spectrum
D) for a 1:1 GSH:compound 1 mixture incubated at pH 10 with Na S O in the buffer.
1
0 (spectra A and B). Samples were prepared by adding compound
1 (spectrum A
B
) as the first reagent. MS spectra at the incubation starting point (spectrum
C
2
2
5
Since GSH is an antioxidant, we speculated on the involvement of oxidation steps for the formation
of adducts. To address such a hypothesis, the experiments were repeated in a buffer containing Na S O ,
2
2
5
which had similar effect of GSH at the incubation starting point (Figure 5C) and prevented the formation
of adducts at 502.10569 and 520.11607 m/z at the incubation endpoint (Figure 5D), suggesting that such
adducts are generated only in oxidizing conditions.
On the contrary, the adduct at 504.12130 m/z was detectable at the incubation endpoint, despite
Na S O addition (Figure 5D).
2
2
5
Consistently with MS data, LC-UV experiments detected a single adduct when compound
1 was
incubated with GSH and Na S O (i.e., chromatographic peak at RT = 8.7 min, Figure 6B), whereas
2
2
5
multiple peaks were generated if no antioxidants were added into the buffer (Figure 6C).
Na S O also prevented the complete consumption of compound (Figure 5B), thus confirming
1
2
2
5
that oxidizing conditions trigger a faster reaction rate.
Moreover, despite MS experiments detecting an adduct at 504.12125
and without Na S O , the corresponding chromatograms had no common peaks. This means that
±
0.00006 m/z, both with
2
2
5
oxidizing and non-oxidizing conditions lead to the formation of adducts with different structures but
the same elemental composition.
This hypothesis was also supported by the UV spectra collected for the corresponding reaction
batches. As reported in Figure 6, the UV spectra at the reaction endpoint look alike for reactions
buffered at pH 7.4 or pH 10 with Na S O , with no shifts compared to the starting point (data not
2
2
5
shown). On the contrary, the incubation at pH 10 without Na S O was only found to have a similar
2
2
5
shape at the starting point, while a significant shift was observed at the reaction endpoint (Figure 7).

Molecules 2020, 25, 3509
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Figure 6. Chromatograms of GSH incubated with compound
), at incubation endpoint in a pH 10 buffer containing Na S O (B
5
1
as recorded at incubation starting point
(
A
), and at incubation endpoint in a
2
2
Na S O -free pH 10 buffer (C).
2
2
5
Figure 7. UV spectra at the endpoint of the reaction between GSH and compound
1. Reaction was
buffered at pH 7.4 (blue line), pH 10 (green line), or pH 10 with Na S O (orange line).
2
2
5
The reaction mechanism in Figure 8 accounts for all the observations described above.
Specifically, the overall mechanism requires a neutral-to-basic pH for compound deprotonation to
generate adducts. All included oxidation steps are dependent on the basic pH activation of compound
, like with catechol [33], and provide the generation of reactive intermediates similar to quinones and
quinone-methides that are able to covalently react with GSH [34 36]. The final products VIb and VIIa
are generated only after the oxidative rearrangement of compound and have molecular formulas in
1
1
–
1
agreement with the MS signals at 502 and 520 m/z, found only in reaction batches without antioxidants.
The products IVa and IIb were found to have the same molecular formula, which was in agreement
with the signal at 504 m/z that was found in reaction batches with or without antioxidants. However,
consistently with LC experiments, the products IVa and IIb were found to have different structures
and formation mechanisms: IVa was found to be generated after compound
formation of IIb was not found to require the oxidation of compound . Interestingly, such mechanism
is not specific for the class of compounds since it was only observed for compound . The removal of
1 oxidation, while the
1
1

Molecules 2020, 25, 3509
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the methyl groups led to compounds that were unable to covalently react with glutathione, despite
some of them still being capable of inhibiting STAT3 activity (e.g., compound 1d; see Table 2).
H
H C
H C
3
H C
H C
3
N
O
N
N
3
N
O
b
ase
3
N
O
ox
1
O
S
S
S
S
O
O
O
O
N
H
N
H
N
H
H C
H C
3
H C
H C
2
3
3
GSH
I
a
III
a
GSH
IIIb
Compound 1
H
H C
H C
3
N
O
H C
3
N
O
3
N
O
S
S
S
S
S
O
O
N
H
O
N
H
H C
N
H
3
H C
3
SG
SG
SG
Ib
IVa 504 m/z
IVb
(
)
base
base
H
H C
H C
3
N
N
O
3
N
N
O
H C
3
N
N
O
S
S
S
O
O
OH
H
C
3
H C
3
GSH
SG
Ic
Va
Vb
ox1
ox
2
H
N
H C
H C
H
3
C
N
N
3
N
O
2
O
O
S
O
O
O
N
H
N
H C
3
H C
3
SG
+
SG
VIa
SG
VIb 502 m/z
IIa
(
)
H+
+ H O
2
OH
SG
H
N
H C
H
N
3
O
O
S
S
O
N
H
O
H C
N
3
H
SG
IIb 504 m/z
VIIa 520 m/z
( )
(
)
Figure 8. Proposed reaction mechanisms for the generation of compound 1—glutathione adducts.
Group SG indicates covalently attached glutathione via thioether linkage.
1
2
.4.2. H-NMR Analysis
With the aim of confirming the stated hypotheses, NMR studies were performed in the experimental
conditions previously considered for the MS analysis in the absence or in the presence of GSH. Actually,
few NMR studies concerning covalent interactions between GSH/STAT proteins and small molecules
have been reported. In one case [37], the authors exploited the presence of fluorine atoms on the
studied compounds and used 19F-NMR spectroscopy, which has the advantage of simple spectra with
a wide spread of resonances.
1
In our case, we used H-NMR spectroscopy even though it is known that GSH, like other small
peptides, presents a great conformational variability, also depending on the pH, that influences its
1
ionization state [38]; consequently, its H-NMR spectra could appear quite complex.
Therefore, to study the interaction of compound 1 with the model peptide GSH, we decided to
perform a comparison, at different times, of the spectra of compound
spectrum of a mixture of them at pH 10 and 37 C. These conditions were chosen because they proved
to be optimal for the interaction between the two compounds in the MS investigations. Moreover,
1 and of GSH alone with the
◦
in order to make the NMR study easier, we decided to add Na S O to the mixture to reduce the
2
2
5
number of adducts that could potentially derive from the reactions between compound 1 and GSH.
+
The use of this reducing agent made the MS spectra less complex, mainly showing a 504 [M + H]
ion (Figure 5D) corresponding to few hypothetical structures (IIb and IVa; Figure 8); in the same
conditions, the LC chromatogram revealed essentially one peak at 8.7 min, even if this analysis showed
only a reduced transformation of the starting compound
1 at the incubation endpoint (Figure 6B).

Molecules 2020, 25, 3509
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Moreover, the addition of Na S O should have prevented the presence of interfering NMR resonances
2
2
5
due to the formation of oxidized GSSG.
Though this study did not demonstrate the formation of the expected compounds (see discussion
in Supplementary Materials and Figures S72–S74), we believe that the NMR outcomes cannot disprove
the results obtained by MS spectroscopy.
3
. Experimental Section
3
.1. Computational Methods
The structural coordinates of STAT3, co-crystallized with a DNA fragment, were downloaded
from the Protein Data Bank (PDB ID 1BG1) [19]. The protein was dimerized by applying the
transformation matrix, as shown in the PDB file, and the model was completed by the addition
of the hydrogens in two steps: (1) to STAT3, applying the algorithm for proteins, and (2) to DNA,
applying the algorithm for nucleic acids. In both cases, we used the features included in the VEGA
ZZ package [39]. Atom charges (Gasteiger–Marsili method) [40] and potentials (CHARMM 22 for
proteins and nucleic acids) [41,42] were assigned to the obtained structure. Finally, the model was
optimized by a conjugate gradient minimization (30,000 steps) to reduce the high-energy steric
interactions. In order to preserve the experimental data, atom constraints were applied to protein and
DNA backbones. This step was carried out by NAMD 2.13 [43], which was integrated in the VEGA
ZZ graphic environment. To perform virtual screening calculations, the following databases were
chosen: AKos (AKos Consulting & Solutions GmbH, Steinen, Germany; 544,391 molecules), ChemPDB
(4009), ChemBank (Broad Institute, Cambridge, MA, USA; 2344), KEGG (Kyoto Encyclopedia of
Genes and Genomes, Kanehisa Laboratories, Kyoto, Japan; 10005), NCI’s Anti-HIV (National Cancer
Institute, National Institute of Health, USA; 42689), and NCI (National Cancer Institute, National
Institute of Health, USA; 15237). These databases are available for free at the mirror site of Ligand.info
(http://biophysics.med.jhmi.edu/~{}yliu120/dockingdatabase.html; the original database site is no
longer available) [44], which includes collections of compounds for virtual screening. These structures
are already converted to 3D and fully optimized by molecular mechanics. Nevertheless, all molecules
were pre-processed setting their ionization state at a physiological pH, and the resulting structures were
optimized and refined by PM7 semi-empirical method, as implemented in MOPAC 2016. To perform
this calculation, the WarpEngine technology implemented in VEGA ZZ [45] was used, reducing the
computational time to few tens of minutes for the whole dataset through the distribution of the MOPAC
calculation on a network of PCs. In more detail: Three blade servers equipped with two CPUs, each of
Intel Xeon E5 class, were used for a total of 52 physical cores and 104 threads. The virtual screening
was carried out by the GriDock software, which was especially designed to run on high performance
computing systems (HPC) and distributed computer resources (GRID) as front-end to the well-known
AutoDock 4 package [20]. Specifically, GriDock is able to extract each molecule from a database
and perform a docking calculation through AutoDock 4. Though it is currently possible to perform
screenings with faster and more efficient programs, we preferred to spend more time on the calculation
because we had already obtained good results with AutoDock in previous studies on STAT3. Before
running GriDock, a single STAT3 monomer was considered, and the grid maps, required to evaluate
docking scores, were calculated by selecting the atoms included in a sphere of 12 Å radius centered on
the phosphorylated Tyr-705 (PTR-705 in the PDB file). This pocket represented a possible hot spot of
interaction with STAT3 for ligands, since it is known to play a pivotal role in STAT3 dimerization and
activation. This process was carried out by AutoGrid 4 interfaced to VEGA ZZ. The compounds of
the considered databases were docked by GriDock/AutoDock using the genetic-algorithm search and
generating ten possible solutions.

Molecules 2020, 25, 3509
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3
.2. Chemistry
3
.2.1. Materials and Methods
All starting materials, chemicals, and solvents were purchased from commercial suppliers
Sigma-Aldrich-Merck, St. Louis, MI, USA; FluoroChem, Hadfield, UK) and used as received.
Anhydrous solvents were utilized without further drying. Aluminum-backed silica gel 60 plates
0.2 mm; Merck, Darmstadt, Germany) were used for analytical thin-layer chromatography (TLC)
to follow the course of the reactions. Silica gel 60 (40–63 m; Merck) was used for the purification
(
(
µ
of intermediates and final compounds through flash column chromatography. Melting points were
determined in open capillary tubes with a Stuart SMP30 Melting Point Apparatus (Cole-Parmer Stuart,
1
13
Stone, UK) and are uncorrected. H and C-NMR spectra were acquired at ambient temperature with
1
a Varian Oxford 300 MHz instrument (Varian, Palo Alto, CA, USA),operating at 300 MHz for H and
7
5 MHz for 13C. Chemical shifts are expressed in ppm (
δ) from tetramethylsilane (TMS) resonance
in the indicated solvent (TMS:
δ = 0.0 ppm), while J-couplings are given in hertz. All the spectra
are reported in the Supplementary Materials. The purity of the tested compounds was assessed by
means of elemental analysis using a EuroVector EA 3000 CHNS-O analyzer (EuroVector, Pavia, Italy).
All experimental values are within ±0.40% of the theoretical predictions, indicating a ≥95% purity.
3
.2.2. Synthetic Procedures
5
,6-Dimethyl-1H,3H-2,1,3-benzothiadiazole-2,2-dioxide, 1
Procedure A: To a solution of 4,5-dimethyl-1,2-phenylenediamine (100 mg, 0.73 mmol) in dry
diglyme (0.75 mL) under reflux, sulfamide (86 mg, 0.89 mmol) dissolved in dry diglyme (0.73 mL) was
◦
slowly added. The mixture was stirred for 10 min at 160 C. After cooling, the dark brown mixture
was diluted with ice-H O and extracted with EtOAc (3
×
5 mL). The organic phase was washed
2
repeatedly with cold H O to remove the diglyme. Then, it was treated with Na SO , filtered, and
2
2
4
evaporated under reduced pressure. The crude product was purified by flash column chromatography
(
0
cyclohexane/EtOAc 8:2) to obtain
1
as a brownish solid. Yield: 50%. TLC (cyclohexane/EtOAc 8:2): Rf:
.36. Mp: 148 C. H-NMR (300 MHz, acetone-d ): 9.25 (s, 2H, NH), 6.72 (s, 2H, ArH), 2.18 (s, 6H,
134.6, 127.0, 111.1, 18.4 ppm. Anal. calcd (%) for C H N O S:
◦
1
δ
6
1
3
CH ) ppm. C-NMR (75 MHz, D O):
δ
3
2
8
10
2
2
C, 48.47; H, 5.08; N, 14.13; S, 16.17; found: C, 48.21; H, 5.03; N, 14.67; S, 16.34.
1
H,3H-2,1,3-Benzothiadiazole-2,2-dioxide, 1a
Benzene-1,2-diamine was reacted for 1.5 h using Procedure A; the purification was performed by
flash column chromatography (cyclohexane/EtOAc 6:4) to afford the compound as a dark yellow solid.
◦
1
Yield: 88%. TLC (cyclohexane/EtOAc 6:4): R : 0.23. Mp: 171–173 C. H-NMR (300 MHz, CD OD):
δ
f
3
13
6.93–6.91 (m, 2H, ArH), 6.84–6.81 (m, 2H, ArH) ppm. C-NMR (75 MHz, CD OD):
δ
129.9, 121.5,
3
110.2 ppm. Anal. calcd (%) for C H N O S: C, 42.34; H, 3.55; N, 16.46; S, 18.84; found: C, 41.98; H,
6 6 2 2
3
.59; N, 16.35; S, 18.75.
5
-Methyl-1H,3H-2,1,3-benzothiadiazole-2,2-dioxide, 1b
-methylbenzene-1,2-diamine was reacted for 1.5 h using Procedure A; the crude residue was
purified by flash column chromatography (DCM/MeOH 98:2) to give a dark red solid. Yield: 55%.
4
◦
1
TLC (DCM/MeOH 9:1): R : 0.54. Mp: 157 C. H-NMR (300 MHz, CD OD):
2
δ
6.73–6.66 (m, 3H, ArH),
131.6, 130.1, 127.5, 121.8, 110.8, 110.2, 19.9 ppm.
Anal. calcd (%) for C H N O S: C, 45.64; H, 4.38; N, 15.21; S, 17.41; found: C, 45.11; H, 4.51; N, 14.96;
f
3
13
.28 (s, 3H, CH ) ppm. C-NMR (75 MHz, CD OD): δ
3
3
7
8
2
2
S, 17.25.

Molecules 2020, 25, 3509
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5
-(Trifluoromethyl)-1H,3H-2,1,3-benzothiadiazole-2,2-dioxide, 1c
-(trifluoromethyl)benzene-1,2-diamine was reacted for 1.5 h using Procedure A; the purification
was performed by flash column chromatography (DCM/MeOH 9:1) to give a white solid. Yield: 66%.
4
◦
1
TLC (DCM/MeOH 9:1): R : 0.19. Mp: 143 C. H-NMR (300 MHz, CD OD):
1
δ
7.23 (dd, J = 8.2, 2.0 Hz,
f
3
13
H, ArH), 7.06 (d, J = 2.0 Hz, 1H, ArH), 6.94 (d, J = 8.2 Hz, 1H, ArH) ppm. C-NMR (75 MHz, CD OD):
3
δ
132.61, 129.65, 123.38, 123.06, 123.00, 118.66 (q, J = 4.2 Hz), 109.34, 106.41 (q, J = 4.2 Hz) ppm. Anal.
calcd (%) for C H F N O S: C, 35.30; H, 2.12; N, 11.76; O, 13.43; S, 13.46; found: C, 35.61; H, 2.17; N,
7
5
3
2
2
1
1.94; S, 13.78.
5
-Nitro-1H,3H-2,1,3-benzothiadiazole-2,2-dioxide, 1d
-nitrobenzene-1,2-diamine was reacted for 1.5 h using Procedure A; the residue was purified by
flash column chromatography (DCM/MeOH 9:1) to give a red solid. Yield: 50%. TLC (DCM/MeOH
4
◦
:1): R : 0.15. Mp: 190 C. H-NMR (300 MHz, CD OD): δ 7.66 (dd, J = 8.7, 2.5 Hz, 1H, ArH), 7.38 (d,
f 3
1
9
13
J = 2.5 Hz, 1H, ArH), 6.53 (d, J = 8.7 Hz, 1H, ArH) ppm. C-NMR (75 MHz, CD OD):
δ
144.2, 140.1,
3
1
1
33.4, 119.6, 108.9, 104.3 ppm. Anal. calcd (%) for C H N O S: C, 33.49; H, 2.34; N, 19.53; O, 29.74; S,
4.90; found: C, 33.74; H, 2.38; N, 19.41; S, 14.51.
6
5
3
4
5
-Chloro-N-[(2,4-dimethoxyphenyl)methyl]-2-nitroaniline, 7
Procedure B: A mixture of 2,4-dichloro-1-nitrobenzene (191 mg, 1 mmol), 2,4-dimethoxybenzylamine
334 mg, 2 mmol), and isopropanol (2 mL) was heated at reflux for 16 h. After cooling, the reaction
was diluted with H O (4 mL) and extracted with EtOAc (2 4 mL). The collected organic layers were
5 mL) and then dried over anhydrous Na SO , filtered, and concentrated
(
×
2
washed with 0.5 M HCl (1
×
2
4
in vacuo. The crude residue was purified by recrystallization from EtOH to obtain intermediate
orange solid. Yield: 60%. TLC (hexane/EtOAc 85:15): R : 0.36. Mp: 107–109 C. H-NMR (300 MHz,
7
as an
◦
1
f
CDCl3):
δ 8.44 (br s, 1H, NH), 8.13 (d, J = 9.1 Hz, 1H, ArH), 7.18 (d, J = 8.2 Hz, 1H, ArH), 6.96 (d,
J = 2.2 Hz, 1H, ArH), 6.60 (dd, J = 9.1, 2.2 Hz, 1H, ArH), 6.53 (d, J = 2.4, 1H, ArH), 6.49 (dd, J = 8.2,
13
2
.4 Hz, 1H, ArH), 4.44 (d, J = 5.7 Hz, 2H, CH ), 3.89 (s, 3H, CH ), 3.84 (s, 3H, CH ) ppm. C-NMR
2 3 3
(
75 MHz, CDCl3): δ 160.9, 158.4, 145.8, 142.7, 130.7, 129.6, 128.2, 117.1, 115.7, 113.6, 104.3, 98.8, 55.42,
5
5.41, 42.2 ppm.
5
-Bromo-N-[(4-methoxyphenyl)methyl]-4-methyl-2-nitroaniline, 8
,5-dibromo-2-methyl-4-nitrobenzene and PMB-NH were reacted according to Procedure B to
1
2
◦
8 as an orange solid. Yield: 95%. TLC (hexane/EtOAc 9:1): R : 0.28. Mp: 115–116 C. H-NMR
f
1
yield
300 MHz, CD OD):
(
δ
8.37 (s, 1H, ArH), 8.27 (br s, 1H, NH), 7.28 (d, J = 8.7 Hz, 2H, ArH), 6.93 (d,
3
J = 8.7 Hz, 2H, ArH), 6.75 (s, 1H, ArH), 4.46 (d, J = 5.4 Hz, 2H, CH ), 3.84 (s, 3H, CH ), 2.37 (s, 3H,
2
3
13
CH ) ppm. C-NMR (75 MHz, CD OD):
δ
159.3, 147.1, 144.1, 132.0, 129.6, 128.9, 128.4, 115.4, 114.4,
3
3
1
10.1, 55.3, 46.7, 23.8 ppm.
5
-Chloro-N1-[(2,4-dimethoxyphenyl)methyl]benzene-1,2-diamine, 9
Procedure C: Zn dust (350 mg, 5.36 mmol) and NH Cl (46.6 mg, 0.87 mmol) were added in a
4
single portion to a stirred suspension of intermediate
The reaction was stirred at reflux for 3 h. After completion, the mixture was filtered through celite, and
the filtrate was evaporated under vacuum. The residue was diluted with H O (6 mL) and extracted
7 (216 mg, 0.67 mmol) in MeOH/H O 9:1 (10 mL).
2
2
with EtOAc (3
in vacuo. The crude residue was purified by flash column chromatography (hexane/EtOAc from 7:3 to
:4) to obtain the desired compound as a brown solid. Yield: 71%. TLC (hexane/EtOAc 7:3): R : 0.28.
×
5 mL). The collected organic layers were dried over Na SO , filtered, and concentrated
2 4
6
f
◦
1
Mp: 135–138 C. H-NMR (300 MHz, CDCl ):
δ
7.21 (d, J = 8.2 Hz, 1H, ArH), 6.71 (d, J = 2.0 Hz, 1H,
ArH), 6.65–6.62 (m, 2H, ArH), 6.52 (d, J = 2.4 Hz, 1H, ArH), 6.48 (dd, J = 8.2, 2.4 Hz, 1H, ArH), 4.22 (s,
H, CH ), 3.86 (s, 3H, CH ), 3.83 (s, 3H, CH ), 3.48 (br s, 2H, NH ) ppm.
3
2
2
3
3
2

Molecules 2020, 25, 3509
17 of 29
5
-Bromo-N1-[(4-methoxyphenyl)methyl]-4-methylbenzene-1,2-diamine, 10
The compound was obtained from following Procedure C. The crude residue was purified by
flash column chromatography (hexane/EtOAc from 8:2 to 7:3) to afford a yellow solid. Yield: 71%. TLC
8
◦
1
(hexane/EtOAc 8:2): R : 0.31. Mp: 147–149 C. H-NMR (300 MHz, CDCl ):
δ
7.32 (d, J = 8.8 Hz, 2H,
f
3
ArH), 6.92 (d, J = 8.8 Hz, 2H, ArH), 6.91 (s, 1H, ArH), 6.56 (s, 1H, ArH), 4.22 (s, 2H, CH ), 3.84 (s, 3H,
2
13
CH ), 3.30 (br s, 2H, NH ), 2.30 (s, 3H, CH ) ppm. C-NMR (75 MHz, CDCl3):
δ 159.0, 137.3, 133.1,
3
2
3
1
31.1, 129.2, 129.1, 119.9, 114.1, 113.9, 112.4, 55.3, 48.1, 22.3 ppm.
6
-Chloro-1-[(2,4-dimethoxyphenyl)methyl]-1H,3H-2,1,3-benzothiadiazole-2,2-dioxide, 11
Intermediate was reacted for 1.5 h following Procedure A. The crude residue was purified by flash
9
column chromatography (hexane/EtOAc 6:4) to afford a brown oil. Yield: 55%. TLC (hexane/EtOAc
1
6
:4): R : 0.21. H-NMR (300 MHz, CDCl ):
δ
7.33 (d, J = 8.3 Hz, 1H, ArH), 6.86 (dd, J = 8.3, 2.0 Hz, 1H,
f
3
ArH), 6.80–6.73 (m, 2H, ArH), 6.53–6.44 (m, 2H, ArH), 4.82 (s, 2H, CH ), 3.91 (s, 3H, CH ), 3.82 (s, 3H,
2
3
CH ) ppm.
3
6
-Bromo-1-[(4-methoxyphenyl)methyl]-5-methyl-1H,3H-2,1,3-benzothiadiazole-2,2-dioxide, 12
Intermediate 10 was reacted for 1.5 h following Procedure A. The purification was performed by
flash column chromatography (hexane/EtOAc 6:4) to give the desired compound as a yellow oil. Yield:
1
5
7
2
6%. TLC (hexane/EtOAc 6:4): R : 0.27. H-NMR (300 MHz, CDCl ):
δ
7.39 (d, J = 8.8 Hz, 2H, ArH),
f
3
.02 (s, 1H, ArH), 6.93 (d, J = 8.8 Hz, 2H, ArH), 6.42 (s, 1H, ArH), 4.79 (s, 2H, CH ), 3.83 (s, 3H, CH ),
.25 (s, 3H, CH ) ppm. C-NMR (75 MHz, CDCl3):
2
3
13
δ 159.6, 132.6, 130.7, 128.9, 126.0, 125.6, 115.8, 115.4,
3
1
14.4, 110.9, 55.3, 46.1, 23.0 ppm.
5
-Chloro-1H,3H-2,1,3-benzothiadiazole-2,2-dioxide, 1e
Procedure D: Intermediate 11 (40 mg, 0.113 mmol) was dissolved in DCM (1 mL), and TFA (1 mL)
◦
was added dropwise. The reaction mixture was stirred for 45 min at 40 C. After the evaporation of
TFA under vacuum, MeOH was added and the white precipitate was filtered off. The filtrate was
concentrated in vacuo and purified by flash column chromatography (hexane/EtOAc 6:4) to give
◦
1
product 1e as a pale pink solid. Yield: 82%. TLC (hexane/EtOAc 6:4): R : 0.48. Mp: 183 C. H-NMR
f
(
1
300 MHz, CD OD):
δ
6.91 (dd, J = 8.4, 2.1 Hz, 1H, ArH), 6.83 (d, J = 2.1 Hz, 1H, ArH), 6.78 (d, J = 8.4 Hz
,
3
13
H, ArH) ppm. C-NMR (75 MHz, CD OD): 130.8, 128.4, 126.4, 121.0, 111.0, 110.1 ppm. Anal. calcd
δ
3
(
%) for C H ClN O S: C, 35.22; H, 2.46; N, 13.69; S, 15.67; found: C, 34.98; H, 2.49; N, 13.42; S, 15.48.
6 5 2 2
5
-Bromo-6-methyl-1H,3H-2,1,3-benzothiadiazole-2,2-dioxide, 2a
The product was obtained starting from intermediate 12 following Procedure D. The crude residue
was purified by flash column chromatography (DCM/MeOH 95:5) to yield the desired compound as a
◦
1
white solid. Yield: 85%. TLC (DCM/MeOH 95:5): R : 0.19. Mp: 185 C. H-NMR (300 MHz, CD OD):
f
3
13
δ
6.99 (s, 1H, ArH), 6.77 (s, 1H, ArH), 2.32 (s, 3H, CH ) ppm. C-NMR (75 MHz, CD OD):
δ
130.5,
3
3
1
29.5, 129.0, 115.2, 113.6, 112.0, 21.3 ppm. Anal. calcd (%) for C H BrN O S: C, 31.95; H, 2.68; N, 10.65;
7 7 2 2
S, 12.19; found: C, 32.08; H, 2.75; N, 10.29; S, 12.31.
1
,3-Bis[(4-methoxyphenyl)methyl]-5-nitro-2,1,3-benzothiadiazole-2,2-dioxide, 13
Procedure E: To a stirred solution of 1d (140 mg, 0.65 mmol) in dry DMF (2 mL), dried K CO
2
3
(
8
179.7 mg, 1.3 mmol) and PMB-Br (313.6 mg, 1.56 mmol) were added. The mixture was stirred at
◦
0 C for 2 h and then cooled to room temperature, diluted with H O (6 mL), and extracted with
2
EtOAc (2
×
5 mL). The collected organic layers were dried over anhydrous Na SO , filtered, and
2 4
evaporated in vacuo. The crude residue was purified by flash chromatography (hexane/EtOAc 8:2) to
◦
give intermediate 13 as a yellow solid. Yield: 90%. TLC (hexane/EtOAc 8:2): R : 0.28. Mp: 116 C.
f
1
H-NMR (300 MHz, CDCl3):
δ 7.71 (dd, J = 8.8, 2.2 Hz, 1H, ArH), 7.38–7.33 (m, 3H, ArH), 7.30 (d,

Molecules 2020, 25, 3509
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J = 8.8 Hz, 2H, ArH), 6.85 (d, J = 8.8 Hz, 2H, ArH), 6.83 (d, J = 8.8 Hz, 2H, ArH), 6.50 (d, J = 8.8 Hz,
13
1
H, ArH), 4.86 (s, 4H, CH ), 3.73 (s, 3H, CH ), 3.72 (s, 3H, CH ) ppm. C-NMR (75 MHz, CDCl3): δ
2 3 3
159.88, 159.86, 142.1, 133.7, 129.3, 129.1, 128.7, 124.96, 124.93, 118.4, 114.59, 114.56, 107.3, 103.7, 55.3,
4
6.5, 46.2 ppm.
5
-Amino-1,3-bis[(4-methoxyphenyl)methyl]-2,1,3-benzothiadiazole-2,2-dioxide, 14
Intermediate 13 was reacted for 4 h following Procedure C. The crude residue was purified by
flash column chromatography (cyclohexane/EtOAc from 1:1 to 4:6) to give the desired product as a
1
yellow oil. Yield: 46%. TLC (cyclohexane/EtOAc 6:4): R : 0.23. H-NMR (300 MHz, CDCl ):
δ 7.32–7.25
f
3
(
5
m, 4H, ArH), 6.84–6.77 (m, 4H, ArH), 6.25 (d, J = 8.3 Hz, 1H, ArH), 6.01 (dd, J = 8.3, 2.3 Hz, 1H, ArH),
.85 (d, J = 2.3 Hz, 1H, ArH), 4.68 (s, 2H, CH ), 4.67 (s, 2H, CH ), 3.71 (s, 6H, CH ), 3.31 (br s, 2H, NH )
2
2
3
2
13
ppm. C-NMR (75 MHz, CDCl3):
δ 159.40, 159.39, 141.7, 130.8, 129.0, 128.8, 127.0, 126.7, 121.9, 114.3,
1
14.2, 110.2, 107.5, 97.3, 55.29, 55.28, 47.2, 46.1 ppm.
N-{1,3-Bis[(4-methoxyphenyl)methyl]-2,2-dioxo-2,1,3-benzothiadiazol-5-yl}acetamide, 15
Intermediate 14 (114.9 mg, 0.27 mmol) was dissolved in acetone (2 mL), and the mixture was
◦
cooled to 0 C in an ice bath. Acetic anhydride (41.3 mg, 0.41 mmol) was added dropwise, and the
solution was stirred at room temperature for 2 h. After completion, the solvent was removed in vacuo
and the residue was diluted with 1 M NaOH and extracted with EtOAc (2
×
3 mL). The collected
organic layers were dried over anhydrous Na SO , filtered, and concentrated in vacuo. The crude
2
4
residue was purified by flash column chromatography (cyclohexane/EtOAc from 5:5 to 4:6) to obtain
◦
intermediate 15 as a dark green solid. Yield: 98%. TLC (cyclohexane/EtOAc 4:6): R : 0.22. Mp: 153 C.
H-NMR (300 MHz, CDCl3):
f
1
δ 7.43–7.37 (m, 5H, ArH and NH), 7.11 (d, J = 2.1 Hz, 1H, ArH), 6.94–6.85
(
m, 4H, ArH), 6.73 (dd, J = 8.4, 2.1 Hz, 1H, ArH), 6.44 (d, J = 8.4 Hz, 1H, ArH), 4.82 (s, 2H, CH ), 4.81 (s,
2
13
2
δ
4
H, CH ), 3.81 (s, 3H, OCH ), 3.78 (s, 3H, OCH ), 2.06 (s, 3H, CH ) ppm. C-NMR (75 MHz, CDCl3):
2 3 3 3
168.4, 159.49, 159.47, 132.4, 129.5, 129.2, 129.0, 126.4, 126.3, 125.7, 114.3, 112.8, 108.7, 101.8, 55.29, 55.25,
6.4, 46.1, 24.3 ppm.
N-(2,2-Dioxo-1H,3H-2,1,3-benzothiadiazol-5-yl)acetamide, 1f
Intermediate 15 was reacted for 8 h at room temperature following Procedure D. The crude residue
was purified by flash column chromatography (DCM/MeOH from 95:5 to 9:1) to afford a white foam.
1
Yield: 52%. TLC (DCM/MeOH 85:15): R : 0.37. H-NMR (300 MHz, CD OD):
δ 7.14 (d, J = 2.1 Hz,
f
3
1
H, ArH), 6.83 (dd, J = 8.4, 2.1 Hz, 1H, ArH), 6.64 (d, J = 8.4 Hz, 1H, ArH), 1.99 (s, 3H, CH ) ppm.
3
1
3
C-NMR (75 MHz, CD OD):
δ
170.0, 133.0, 130.3, 126.2, 113.1, 110.3, 103.3, 22.2 ppm. Anal. calcd (%)
3
for C H N O S: C, 42.28; H, 3.99; N, 18.49; O, 21.12; S, 14.11; found: C, 42.37; H, 4.02; N, 18.65; S, 14.32.
8
9
3
3
4
-Bromo-N-[(4-methoxyphenyl)methyl]-2-nitroaniline, 16
A mixture of 2,5-dibromo-nitrobenzene (200 mg, 0.71 mmol) and PMB-NH (293 mg, 2.14 mmol)
2
◦
was heated at 160 C for 8 h. After cooling, the reaction was diluted with H O (4 mL) and extracted
2
with EtOAc (2
×
4 mL). The collected organic layers were washed with 0.5 M HCl (1
×
8 mL) and then
dried over anhydrous Na SO , filtered, and concentrated in vacuo. The crude residue was purified by
2
4
flash chromatography (cyclohexane/EtOAc 8:2) to afford the desired compound as an orange solid.
◦
1
Yield: 95%. TLC (cyclohexane/EtOAc 9:1): R : 0.35. Mp: 121 C. H-NMR (300 MHz, CDCl ):
(
δ
8.25
br s, 1H, NH), 8.23 (d, J = 2.4 Hz, 1H, ArH), 7.35 (dd, J = 9.2, 2.4 Hz, 1H, ArH), 7.16 (d, J = 8.8 Hz, 2H,
ArH), 6.81 (d, J = 8.8 Hz, 2H, ArH), 6.65 (d, J = 9.2 Hz, 1H, ArH), 4.37 (d, J = 5.5 Hz, 2H, CH ), 3.72 (s,
f
3
2
13
3
H, CH ) ppm. C-NMR (75 MHz, CDCl3): δ 159.3, 144.1, 138.9, 132.5, 128.9, 128.7, 128.4, 116.0, 114.4,
3
1
06.8, 55.3, 46.7 ppm.

Molecules 2020, 25, 3509
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4
-Bromo-N1-[(4-methoxyphenyl)methyl]benzene-1,2-diamine, 17
Compound 16 was reacted according to Procedure C; the resulting crude residue was purified by
flash column chromatography (cyclohexane/EtOAc from 7.5:2.5 to 7:3) to afford the desired product
◦
1
as a pale yellow solid. Yield: 54%. TLC (cyclohexane/EtOAc 7:3): R : 0.36. Mp: 116 C. H-NMR
f
(
300 MHz, CDCl3):
δ 7.32 (d, J = 8.8 Hz, 2H, ArH), 6.96–6.88 (m, 3H, ArH), 6.86 (d, J = 2.4 Hz, 1H, ArH),
6
.55 (d, J = 8.4 Hz, 1H, ArH), 4.23 (s, 2H, CH ), 3.84 (s, 3H, CH ), 3.57 (br s, 1H, NH), 3.40 (br s, 2H,
2
3
13
NH ) ppm. C-NMR (75 MHz, CDCl3):
δ 159.0, 136.6, 135.8, 131.0, 129.0, 123.0, 118.9, 114.1, 113.3,
2
1
10.7, 55.3, 48.1 ppm.
5
-Bromo-1-[(4-methoxyphenyl)methyl]-3H-2,1,3-benzothiadiazole-2,2-dioxide, 18
Intermediate 17 was reacted for 2 h following Procedure A. The crude residue was purified by
flash column chromatography (cyclohexane/EtOAc from 6:4 to 1:1), to afford compound 18 as a red
◦
1
solid. Yield: 60%. TLC (cyclohexane/EtOAc 6:4): R : 0.19. Mp: 166 C. H-NMR (300 MHz, CDCl ):
δ
f
3
7
.26 (d, J = 8.8 Hz, 2H, ArH), 6.87–6.82 (m, 2H, ArH), 6.80 (d, J = 8.8 Hz, 2H, ArH), 6.28 (d, J = 8.4 Hz,
13
1
1
H, ArH), 4.69 (s, 2H, CH ), 3.70 (s, 3H, CH ) ppm. C-NMR (75 MHz, CDCl3):
27.8, 125.8, 125.5, 114.4, 114.3, 113.7, 110.1, 55.3, 46.0 ppm.
δ
159.6, 129.7, 129.0,
2
3
5
-Bromo-1,3-bis[(4-methoxyphenyl)methyl]-2,1,3-benzothiadiazole-2,2-dioxide, 19
The desired product was obtained starting from 18 following Procedure E. The crude residue was
purified by flash column chromatography (cyclohexane/EtOAc 8:2) to afford a pale yellow solid. Yield:
◦
1
9
4
4
8%. TLC (cyclohexane/EtOAc 7:3): R : 0.59. Mp: 134 C. H-NMR (300 MHz, CDCl ):
δ
7.48–7.36 (m,
f
3
H, ArH), 6.98–6.84 (m, 5H, ArH), 6.70 (d, J = 1.9 Hz, 1H, ArH), 6.42 (d, J = 8.4 Hz, 1H, ArH), 4.86 (s,
1
3
H, CH ), 3.84 (s, 3H, CH ), 3.83 (s, 3H, CH ) ppm. C-NMR (75 MHz, CDCl3):
δ 159.7, 159.6, 130.2,
2
3
3
1
29.0, 128.1, 125.9, 125.8, 124.1, 114.5, 114.4, 113.8, 111.5, 109.7, 55.3, 46.3 ppm.
Ethyl (2E)-3-{1,3-bis[(4-methoxyphenyl)methyl]-2,2-dioxo-2,1,3-benzothiadiazol-5-yl} prop-2-enoate, 20
To a solution of intermediate 19 (200 mg, 0.41 mmol) in DMF (2.0 mL), triethylamine (0.28 mL,
2.05 mmol), ethyl acrylate (0.17 mL, 1.64 mmol), tri-o-tolylphosphine (74.8 mg, 0.25 mmol), and
tris(dibenzylideneacetone)dipalladium(0) (75.1 mg, 0.08 mmol) were added, and the mixture was
◦
heated to 80 C for 20 h. The reaction was then cooled to ambient temperature, and DMF was
evaporated. The residue was diluted with H O (3 mL) and extracted with EtOAc (3
×
3 mL).
2
The collected organic layers were dried over Na SO , filtered, concentrated in vacuo, and purified by
2
4
flash column chromatography (cyclohexane/EtOAc 8:2) to afford the desired product as a yellow foam.
1
Yield: 50%. TLC (cyclohexane/EtOAc 7:3): R : 0.31. H-NMR (300 MHz, CDCl ):
δ 7.39 (d, J = 16.0 Hz,
f
3
1
H, CH), 7.35–7.30 (m, 4H, ArH), 6.90 (dd, J = 8.2, 1.6 Hz, 1H, ArH), 6.87–6.80 (m, 4H, ArH), 6.62 (d,
J = 1.6 Hz, 1H, ArH), 6.46 (d, J = 8.2 Hz, 1H, ArH), 6.04 (d, J = 16.0 Hz, 1H, CH), 4.80 (s, 2H, CH2),
4
3
.79 (s, 2H, CH ), 4.14 (q, J = 7.1 Hz, 2H, OCH ) 3.74 (s, 3H, CH ), 3.72 (s, 3H, CH ), 1.23 (t, J = 7.1 Hz,
2 2 3 3
H, CH3). 1 C-NMR (75 MHz, CDCl3):
3
δ
167.0, 159.63, 159,62, 144.1, 130.7, 129.5, 128.99, 128.95, 128.2,
1
25.95, 125.93, 122.8, 116.5, 114.5, 114.4, 108.4, 106.9, 60.5, 55.3, 46.3, 46.2, 14.3 ppm.
(
2E)-3-{1,3-Bis[(4-methoxyphenyl)methyl]-2,2-dioxo-2,1,3-benzothiadiazol-5-yl} prop-2-enoic acid, 21
To a solution of intermediate 20 (53.4 mg, 0.105 mmol) in MeOH (2 mL), 3 M NaOH (1 mL) was
◦
added, and the mixture was stirred at 80 C for 3 h. After cooling, MeOH was evaporated under
vacuum and 2 M HCl (1.5 mL) was added until pH 2. Extraction with EtOAc (3
×
3 mL) was carried
out, and the collected organic layers were dried over Na SO , filtered, and evaporated in vacuo to
2
4
afford the acid 21 as a pale yellow solid, which was used in the next step for further purification. Yield:
◦
1
quantitative. TLC (DCM/MeOH 95:5): R : 0.26. Mp: 125–127 C. H-NMR (300 MHz, CDCl ):
(
δ
7.55
d, J = 16 Hz, 1H, CH), 7.47–7.34 (m, 4H, ArH), 7.00 (dd, J = 8.1, 1.6 Hz, 1H, ArH), 6.97–6.84 (m, 4H,
ArH), 6.70 (d, J = 1.6 Hz, 1H, ArH), 6.54 (d, J = 8.1 Hz, 1H, ArH), 6.11 (d, J = 16 Hz, 1H, CH), 4.89
f
3

Molecules 2020, 25, 3509
20 of 29
s, 4H, CH ), 3.82 (s, 3H, CH ), 3.80 (s, 3H, CH ) ppm. 3C-NMR (75 MHz, CDCl3):
1
δ
172.35, 159.85,
(
2
3
3
1
46.87, 131.29, 129.71, 129.21, 127.5, 126.04, 123.58, 115.33, 114.66, 108.64, 107.23, 55.45, 46.40 ppm.
(
2E)-3-{1,3-Bis[(4-methoxyphenyl)methyl]-2,2-dioxo-2,1,3-benzothiadiazol-5-yl}-N-(3-methylbutyl)prop-
2
-enamide, 22
Procedure F: To a mixture of 21 (50.5 mg, 0.105 mmol) in DCM (1 mL), isopentylamine (13.7 mg, 0.15
mmol), N,N-diisopropylethylamine (DIPEA, 27.1 mg, 0.21 mmol), and HATU (47.9 mg, 0.126 mmol
were added. The reaction mixture was stirred at room temperature for 16 h; after completion, the
solvent was evaporated and the residue was diluted with H O (3 mL) and extracted with EtOAc
)
2
(
3 × 3 mL). The organic layers were washed with 1 M HCl (1
×
3 mL) and then dried over Na SO ,
2 4
filtered, and concentrated in vacuo. Purification by flash column chromatography (DCM/MeOH 95:5)
afforded compound 22 as a yellow oil. Yield: 62%. TLC (DCM/EtOAc 9:1): R : 0.55. H-NMR (300
1
f
MHz, CDCl3):
δ
7.45–7.36 (m, 5H, ArH), 7.00–6.87 (m, 5H, ArH, CH), 6.67 (d, J = 1.6 Hz, 1H, ArH), 6.53
(
d, J = 8.2 Hz, 1H, ArH), 6.09 (d, J = 15.5 Hz, 1H, CH), 5.62 (br t, J = 5.7 Hz, 1H, NH), 4.87 (s, 4H, CH2),
3
2
.82 (s, 3H, OCH ), 3.81 (s, 3H, OCH ), 3.40–3.35 (m, 2H, CH ), 1.74–1.56 (m, 1H, CH), 1.47–1.41 (m,
3 3 2
13
H, CH ), 0.91 (d, J = 6.6 Hz, 6H, CH ) ppm. C-NMR (75 MHz, CDCl3):
δ 165.8, 159.58, 159.57, 140.3,
2
3
130.1, 129.5, 128.99, 128.92, 128.7, 128.6, 126.1, 126.0, 122.0, 119.3, 114.43, 114.37, 114.0, 108.5, 107.1, 55.3,
4
6.23, 46.17, 38.6, 38.0, 25.9, 22.4 ppm.
(
2E)-3-{1,3-Bis[(4-methoxyphenyl)methyl]-2,2-dioxo-2,1,3-benzothiadiazol-5-yl}-N-phenylprop-2-
enamide, 23
To a stirred solution of 21 (50 mg, 0.104 mmol) in DCM (2.5 mL), aniline (0.03 mL, 0.32 mmol)
,
TBTU (46 mg, 0.142 mmol), and N-methylmorpholine (up to pH 7) were added. The reaction mixture
was stirred at room temperature for 24 h. After completion, the product was extracted with DCM
(
3
×
3 mL), and the organic layer was washed with 1 M HCl (1
solution of NaHCO (1 3 mL) and finally with brine (1 3 mL). The organic phase was dried over
anhydrous Na SO , filtered, and concentrated in vacuo. Purification by flash column chromatography
×
3 mL) and then with a saturated
×
×
3
2
4
(
6
cyclohexane/EtOAc 7:3) yielded intermediate 23 as a white solid. Yield: 69%. TLC (cyclohexane/EtOAc
◦
:4): R : 0.47. Mp: 180–182 C. H-NMR (300 MHz, CDCl ): δ 7.57 (br s, 1H, NH), 7.52 (d, J = 15.4 Hz,
f 3
1
1
H, CH), 7.43–7.27 (m, 8H, ArH), 7.11 (t, J = 7.4, 1H, ArH), 6.98 (dd, J = 8.1, 1.6 Hz, 1H, ArH), 6.96–6.87
(
m, 4H, ArH), 6.67 (d, J = 1.6 Hz, 1H, ArH), 6.53 (d, J = 8.1 Hz, 1H, ArH), 6.21 (d, J = 15.4 Hz, 1H, CH),
4
.87 (s, 4H, CH ), 3.81 (s, 3H, CH ), 3.80 (s, 3H, CH ) ppm.
2 3 3
3
-(2,2-Dioxo-1H,3H-2,1,3-benzothiadiazol-5-yl)-N-(3-methylbutyl)propanamide, 1g
Procedure G: Intermediate 22 (100 mg, 0.182 mmol) was dissolved in EtOH (10 mL), and the
solution was degassed by bubbling Ar for 10 min. Pd/C (10% w/w) was then added, and the mixture
was stirred at room temperature under an H atmosphere (3 atm) for 48 h. After completion, the
2
reaction mixture was filtered through celite, and the filtrate was evaporated under vacuum. The crude
residue was purified by flash column chromatography (DCM/MeOH 95:5 to 9:1) to obtain product
1
1
g
as a pale brown oil. Yield: 3%. TLC (DCM/MeOH 9:1): R : 0.41. H-NMR (300 MHz, CD OD):
δ
f
3
6
.78–6.55 (m, 3H, ArH), 3.12 (t, J = 7.5 Hz, 2H, CH ), 2.82 (t, J = 7.5 Hz, 2H, CH ), 2.41 (t, J = 7.5 Hz, 2H,
2 2
13
CH ), 1.55–1.37 (m, 1H, CH), 1.35–1.18 (m, 2H, CH ), 0.86 (d, J = 6.6 Hz, 6H, CH ) ppm. C-NMR
2
2
3
(
(
75 MHz, CDCl3): δ 173.5, 134.6, 130.2, 128.2, 121.2, 110.2, 37.8, 37.1, 31.4, 25.2, 21.3 ppm. Anal. calcd
%) for C H N O S: C, 54.00; H, 6.80; N, 13.49; S, 10.30; found: C, 54.08; H, 6.95; N, 13.27; S, 10.41.
14
21
3
3
3
-(2,2-Dioxo-1H,3H-2,1,3-benzothiadiazol-5-yl)-N-phenylpropanamide, 1h
◦
The compound was obtained by reacting 23 at 50 C for 5 h following Procedure G. The crude
residue was purified by flash column chromatography (DCM/MeOH 9:1) to afford a white solid. Yield:
◦
0%. TLC (DCM/MeOH 9:1): R : 0.40. Mp: 192–197 C. H-NMR (300 MHz, CD OD): δ 7.51–7.45 (m,
f 3
1
1
2
H, ArH), 7.33–7.22 (m, 2H, ArH), 7.13–6.99 (m, 1H, ArH), 6.80 (dd, J = 8.1, 1.7 Hz, 1H, ArH), 6.76–6.70

Molecules 2020, 25, 3509
21 of 29
(
m, 2H, ArH) 2.93 (t, J = 7.6 Hz, 2H, CH ), 2.61 (t, J = 7.6 Hz, 2H, CH ) ppm. 13C-NMR (75 MHz,
2
2
CD OD):
δ
172.1, 138.3, 134.8, 130.1, 128.3, 128.1, 123.8, 121.3, 120.0, 110.3, 38.7, 31.2 ppm. Anal. calcd
3
(
%) for C H N O S: C, 56.77; H, 4.76; N, 13.24; S, 10.10; found: C, 56.89; H, 4.81; N, 13.16; S, 10.14.
15
15
3
3
1
,3-Bis[(4-methoxyphenyl)methyl]-5-phenyl-2,1,3-benzothiadiazole-2,2-dioxide, 24
A flask was charged with 19 (70 mg, 0.14 mmol), Na CO (30 mg, 0.28 mmol), phenylboronic acid
2
3
(
18 mg, 0.15 mmol), and tetrakis(triphenylphosphine)palladium(0) (32 mg, 0.028 mmol) under an Ar
atmosphere. Then, 1,4-dioxane (3 mL) and a drop of H O were added. The resulting mixture was
2
heated to reflux for 5 h. After completion, the reaction was cooled to room temperature, H O was
2
added (3 mL), and the mixture was extracted with EtOAc (3
×
2 mL). The collected organic layers
were dried over Na SO , filtered, and evaporated in vacuo. The residue was purified by flash column
2
4
chromatography (cyclohexane/EtOAc 7:3) to afford the desired product as a pale-yellow oil. Yield:
1
6
8%. TLC (cyclohexane/EtOAc 7:3): R : 0.50. H-NMR (300 MHz, CDCl ):
δ
7.38–7.32 (m, 4H, ArH),
f
3
7.30–7.23 (m, 4H, ArH, partially hidden by solvent peak), 7.22–7.17 (m, 1H, ArH), 6.93 (dd, J = 8.1,
1
4
.8 Hz, 1H, ArH), 6.87–6.80 (m, 4H, ArH), 6.70 (d, J = 1.8 Hz, 1H, ArH), 6.52 (d, J = 8.1 Hz, 1H, ArH),
13
.83 (s, 2H, CH ), 4.80 (s, 2H, CH ), 3.72 (s, 3H, CH ), 3.71 (s, 3H, CH ) ppm. C-NMR (75 MHz,
2 2 3 3
CDCl3):
δ 159.5, 140.7, 135.2, 129.7, 129.04, 129.02, 128.97, 128.8, 128.6, 127.1, 126.8, 126.4, 120.4, 114.37,
1
14.35, 114.31, 108.8, 107.4, 55.3, 46.33, 46.29 ppm.
5
-Phenyl-1H,3H-2,1,3-benzothiadiazole-2,2-dioxide, 1i
Intermediate 24 was reacted for 3 h at room temperature following Procedure D. The crude residue
was purified by flash column chromatography (cyclohexane/EtOAc 6:4) to afford a white solid. Yield:
◦
0%. TLC (cyclohexane/EtOAc 6:4): R : 0.42. Mp: 180–185 C. H-NMR (300 MHz, CD OD): δ 7.47–7.38
f 3
1
2
(
m, 2H, ArH), 7.33–7.26 (m, 2H, ArH), 7.24–7.14 (m, 1H, ArH), 7.07 (dd, J = 8.1, 1.8 Hz, 1H, ArH), 6.96
(
d, J = 1.8 Hz, 1H, ArH), 6.78 (d, J = 8.1 Hz, 1H, ArH) ppm. Anal. calcd (%) for C H N O S: C, 58.52;
12
10
2
2
H, 4.09; N, 11.37; S, 13.02; found: C, 58.48; H, 4.11; N, 11.26; S, 13.18.
1
,3-Dibenzyl-2,1,3-benzothiadiazole-2,2-dioxide, 3
The compound was obtained starting from 1a and benzyl bromide following Procedure E.
The crude residue was purified by flash column chromatography (hexane/EtOAc 9:1) to afford the
◦
1
desired product as a white solid. Yield: 90%. TLC (hexane/EtOAc 7:3): R : 0.81. Mp: 134 C. H-NMR
f
(
(
300 MHz, CDCl3):
δ 7.56–7.48 (m, 4H, ArH), 7.45–7.39 (m, 4H, ArH), 7.38–7.32 (m, 2H, ArH), 6.86–6.81
13
m, 2H, ArH), 6.59–6.54 (m, 2H, ArH), 4.96 (s, 4H, CH ) ppm. C-NMR (75 MHz, CDCl3):
δ
134.6,
2
1
7
29.3, 128.9, 128.1, 127.5, 121.6, 108.5, 46.7 ppm. Anal. calcd (%) for C H N O S: C, 68.55; H, 5.18; N,
.99; S, 9.15; found: C, 68.63; H, 5.17; N, 8.13; S, 9.17.
20
18
2
2
1
,3,5-Trimethyl-2,1,3-benzothiadiazole-2,2-dioxide, 2b
In a flame-dried flask, 1b (50 mg, 0.27 mmol) was solubilized in dry DMF under an Ar atmosphere;
oven-dried K CO (111.9 mg, 0.81 mmol) was then added, and the mixture was stirred for 30 min.
2
3
◦
After cooling to 0 C on an ice bath, CH I (0.050 mL, 0.81 mmol) was added dropwise, and the
reaction was heated at 80 C for 2 h. After completion, DMF was removed under reduced pressure
3
◦
and the remaining mixture was diluted with 1 M HCl (3 mL), and then the product was extracted
with EtOAc (3 × 3 mL). The organic fractions were combined, dried over anhydrous Na SO , filtered,
2
4
and evaporated under vacuum. The crude residue was purified by flash column chromatography
(
0
6
hexane/EtOAc 8:2) to afford product 2b as a white foam. Yield: 87%. TLC (hexane/EtOAc 6:4): Rf:
1
.78. H-NMR (300 MHz, CDCl ):
δ
6.81 (dd, J = 8.0, 1.5 Hz, 1H, ArH), 6.64 (d, J = 8.0 Hz, 1H, ArH),
3
13
.58 (d, J = 1.5 Hz, 1H, ArH), 3.28 (s, 3H, NCH ), 3.27 (s, 3H, NCH ), 2.36 (s, 3H, CH ) ppm. C-NMR
3
3
3
(75 MHz, CDCl3):
δ 131.7, 130.4, 128.2, 122.0, 108.7, 107.7, 28.2, 27.9, 21.4 ppm. Anal. calcd (%) for
C H N O S: C, 50.92; H, 5.70; N, 13.20; S, 15.11; found: C, 50.78; H, 5.63; N, 13.28; S, 15.17.
9
12
2
2

Molecules 2020, 25, 3509
22 of 29
N-[(2,4-Dimethoxyphenyl)methyl]-2-nitroaniline, 25
In a two-neck flask, a solution of 1-chloro-2-nitrobenzene (200 mg, 1.27 mmol) and DMB-NH
637 mg, 3.8 mmol) in EtOH was heated overnight at 100 C. After completion, EtOH was evaporated
2
◦
(
under vacuum, and the residue was dissolved in EtOAc and washed with 1 M HCl (1
×
3 mL).
The organic layer was dried over anhydrous Na SO , filtered, and concentrated in vacuo. The crude
2
4
residue was purified by flash column chromatography (hexane/EtOAc 8:2) to give the desired compound
◦
1
as an orange solid. Yield: 80%. TLC (hexane/EtOAc 8:2): R : 0.32. Mp: 85–87 C. H-NMR (300 MHz,
f
CDCl3):
δ 8.43 (br s, 1H, NH), 8.20 (dd, J = 8.7, 1.7 Hz, 1H, ArH), 7.43–7.39 (m, 1H, ArH), 7.17 (d,
J = 8.4 Hz, 1H, ArH), 6.91 (dd, J = 8.7, 1.7 Hz, 1H, ArH), 6.67–6.62 (m, 1H, ArH), 6.52 (d, J = 2.4, 1H,
ArH), 6.46 (dd, J = 8.4, 2.4 Hz, 1H, ArH), 4.48 (d, J = 5.6 Hz, 2H, CH ), 3.88 (s, 3H, CH ), 3.83 (s, 3H,
2
3
CH ) ppm.
3
N1-[(2,4-Dimethoxyphenyl)methyl]benzene-1,2-diamine, 26
Intermediate 25 was reacted for 1 h at reflux following Procedure C. The crude residue was
purified by flash column chromatography (cyclohexane/EtOAc from 7:3 to 6:4) to afford a yellow solid.
◦
1
Yield: 73%. TLC (cyclohexane/EtOAc 6:4): R : 0.37. Mp: 107–112 C. H-NMR (300 MHz, CDCl ):
δ
f
3
7
.27 (d, J = 8.2 Hz, 1H, ArH), 6.92–6.82 (m, 1H, ArH), 6.78 (d, J = 8.4 Hz, 1H, ArH), 6.77–6.73 (m, 2H,
ArH), 6.56 (d, J = 2.4 Hz, 1H, ArH), 6.51 (dd, J = 8.2, 2.4 Hz, 1H, ArH), 4.30 (s, 2H, CH ), 3.88 (s, 3H,
2
13
CH ), 3.86 (s, 3H, CH ), 3.52 (br s, 2H, NH ) ppm. C-NMR (75 MHz, CDCl3):
δ 160.3, 158.6, 137.9,
3
3
2
1
34.8, 130.0, 120.5, 120.0, 118.8, 116.3, 112.7, 104.1, 98.7, 55.42, 55.40, 43.7 ppm.
1
-[(2,4-Dimethoxyphenyl)methyl]-3H-2,1,3-benzothiadiazole-2,2-dioxide, 27
◦
The product was obtained by reacting 26 for 2 h at 160 C following Procedure A. The crude
residue was purified by flash column chromatography (cyclohexane/EtOAc 6:4) to give the desired
1
compound as a sticky yellow solid. Yield: 78%. TLC (cyclohexane/EtOAc 1:1): R : 0.31. H-NMR
f
(
300 MHz, CDCl3):
δ 7.33 (d, J = 8.4 Hz, 1H, ArH), 7.03–6.84 (m, 3H, ArH), 6.72 (dd, J = 7.3, 1.1 Hz, 1H,
ArH), 6.52 (d, J = 2.4 Hz, 1H, ArH), 6.48 (dd, J = 8.4, 2.4 Hz, 1H, ArH), 4.86 (s, 2H, CH ), 3.90 (s, 3H,
2
CH ), 3.82 (s, 3H, CH ) ppm.
3
3
Ethyl 2-{3-[(2,4-dimethoxyphenyl)methyl]-2,2-dioxo-2,1,3-benzothiadiazol-1-yl}acetate, 28
Intermediate 27 (100 mg, 0.31 mmol), K CO (55.74 mg, 0.403 mmol), and ethyl bromoacetate
2
3
(
62.8 mg, 0.38 mmol) were reacted according to Procedure E. The crude residue was purified by flash
column chromatography (hexane/EtOAc 7:3) to afford the desired compound as a yellow solid. Yield:
◦
1
9
0%. TLC (hexane/EtOAc 6:4): R : 0.52. Mp: 128–132 C. H-NMR (300 MHz, CDCl ):
δ
7.34 (d,
f
3
J = 8.4 Hz, 1H, ArH), 6.97–6.90 (m, 2H, ArH), 6.75–6.67 (m, 2H, ArH), 6.51 (d, J = 2.4 Hz, 1H, ArH),
6.48 (dd, J = 8.4, 2.4 Hz, 1H, ArH), 4.90 (s, 2H, CH ), 4.47 (s, 2H, CH ), 4.29 (q, J = 7.1 Hz, 2H, OCH ),
2 2 2
3
.90 (s, 3H, OCH ), 3.82 (s, 3H, OCH ), 1.30 (t, J = 7.1 Hz, 3H, CH ) ppm.
3
3
3
2
-{3-[(2,4-Dimethoxyphenyl)methyl]-2,2-dioxo-2,1,3-benzothiadiazol-1-yl} acetic acid, 29
To a solution of intermediate 28 (63 mg, 0.16mmol) in MeOH (2 mL), 2% aqueous K CO (1 mL)
2
3
◦
was added, and the mixture was stirred at 50 C for 2 h. After cooling, MeOH was evaporated under
vacuum, and 2 M HCl (1.5 mL) was added until it reached pH 4. Extraction with EtOAc (3 3 mL)
was carried out, and the collected organic layers were dried over anhydrous Na SO , filtered, and
×
2
4
evaporated in vacuo to obtain the acid 29 as a yellow oil, which was used in the next step without any
1
further purification. Yield: quantitative. TLC (DCM/MeOH 9:1): R : 0.10. H-NMR (300 MHz, CDCl ):
f
3
δ
6
3
9.91 (br s, 1H, COOH), 7.32 (d, J = 8.4 Hz, 1H, ArH), 6.99–6.91 (m, 2H, ArH), 6.79–6.68 (m, 2H, ArH),
.51 (d, J = 2.4 Hz, 1H, ArH), 6.47 (dd, J = 8.4, 2.4 Hz, 1H, ArH), 4.90 (s, 2H, CH ), 4.52 (s, 2H, CH ),
2
2
.89 (s, 3H, CH ), 3.81 (s, 3H, CH ) ppm.
3
3

Molecules 2020, 25, 3509
23 of 29
2
-{3-[(2,4-Dimethoxyphenyl)methyl]-2,2-dioxo-2,1,3-benzothiadiazol-1-yl}-N-phenethylacetamide, 30
Intermediate 29 and 2-phenylethanamine were reacted for 24 h following Procedure F. The crude
residue was purified by flash column chromatography (hexane/EtOAc 6:4) to afford the desired
1
compound as a yellow oil. Yield: 47%. TLC (DCM/MeOH 95:5): R : 0.83. H-NMR (300 MHz, CDCl ):
f
3
δ
7.30 (d, J = 8.4 Hz, 1H, ArH), 7.20–7.16 (m, 3H, ArH), 7.10–7.03 (m, 2H, ArH), 7.01–6.96 (m, 2H, ArH),
6.85–6.78 (m, 1H, ArH), 6.70–6.64 (m, 1H, ArH), 6.63 (br t, J = 5.8 Hz, 1H, NH), 6.53 (d, J = 2.4 Hz, 1H,
ArH), 6.50 (dd, J = 8.4, 2.4 Hz, 1H, ArH), 4.88 (s, 2H, CH ), 4.34 (s, 2H, CH ), 3.92 (s, 3H, CH ), 3.83 (s,
2
2
3
3
H, CH ), 3.56 (td, J = 7.0, 5.8 Hz, 2H, NHCH ), 2.78 (t, J = 7.0 Hz, 2H, CH CH ) ppm.
3 2 2 2
2
-(2,2-Dioxo-3H-2,1,3-benzothiadiazol-1-yl)-N-(2-phenylethyl)acetamide, 4
Intermediate 30 was reacted according to Procedure D. The crude residue was purified by flash
column chromatography (DCM/MeOH 95:5) to give the product as a white solid. Yield: 88%. TLC
◦
1
(
DCM/MeOH 9:1): R : 0.41. Mp: 172 C. H-NMR (300 MHz, CD OD):
δ
8.06 (t, J = 5.9 Hz, 1H,
f
3
NH), 7.29–7.23 (m, 2H, ArH), 7.22–7.16 (m, 3H, ArH), 7.00–6.94 (m, 2H, ArH), 6.92–6.84 (m, 1H, ArH),
6
.70–6.58 (m, 1H, ArH), 4.27 (s, 2H, CH ), 3.49 (td, J = 7.2, 5.9 Hz, 2H, NHCH ), 2.81 (t, J = 7.2 Hz, 2H,
2
2
13
CH CH ) ppm. C-NMR (75 MHz, CD OD):
δ
167.6, 138.8, 131.3, 128.5, 128.1, 126.0, 121.9, 121.7,
2
2
3
1
10.5, 108.2, 44.2, 40.6, 35.0 ppm. Anal. calcd (%) for C H N O S: C, 57.99; H, 5.17; N, 12.68; S, 9.68;
16 17 3 3
found: C, 58.05; H, 5.21; N, 12.49; S, 9.72.
2
,3-Dihydro-1H-1,3-benzodiazol-2-one, 5
In a two-neck flamed-dried flask, a solution of o-phenylenediamine (30 mg, 0.28 mmol) and
carbonyldiimidazole (48 mg, 0.30 mmol) in dry THF was heated at reflux overnight under an Ar
atmosphere. After completion, the reaction was cooled to room temperature, and the solvent was
evaporated under vacuum. The residue was acidified to pH 2 with 2 M HCl, and the precipitate was
filtered off. The filtrate was dried in vacuo, and a white solid was obtained. The crude residue was
purified by flash column chromatography (DCM/MeOH 95:5) to obtain product
5
. Yield: 78%. TLC
7.06–7.04 (m, 4H, ArH)
δ 156.6, 129.4, 121.1, 108.9 ppm. Anal. calcd (%) for C H N O: C,
◦
DCM/MeOH 9:1): R : 0.22. Mp: 305–308 C. H-NMR (300 MHz, CD OD): δ
f 3
1
(
ppm. 13C-NMR (75 MHz, CDCl3):
7
6
2
6
2.68; H, 4.51; N, 20.88; found: C, 62.72; H, 4.58; N, 20.87.
3
.3. Single Crystal X-ray Analysis
Diffraction data for compound
1 were collected by means of a Bruker AXS CCD-based
three-circle diffractometer (Bruker AXS, Karlsruhe, Germany), working at ambient temperature
with graphite-monochromatized Mo-Kα X-radiation (λ = 0.71073 Å).
◦
X-ray diffraction data in the
θ
range of 2–30 were collected by acquiring 4 sets of 600 bidimensional
◦
CCD frames with the following operative conditions: omega rotation axis, scan width 0.3 , acquisition
time 30 s, sample-to-detector distance 50 mm, phi angle fixed at four different values (0 , 90 , 180 , and
◦
◦
◦
◦
2
70 ) for the four different sets.
Omega-rotation frames were processed with the SAINT software (Bruker) for data reduction
(including intensity integration, background, Lorentz, and polarization corrections) and for
determination of accurate unit-cell dimensions, obtained by the least-squares refinement of the
◦
positions of 6048 independent reflections with I > 10
σ
(I) in the
θ
range 2–25 . Absorption effects were
empirically evaluated by the SADABS software [46], and absorption correction was applied to the data
(0.847 and 0.973 min and max transmission factors, respectively).
The structures were solved by direct methods (SIR-2014) [47] and completed by iterative cycles
2
of full-matrix least-squares refinement on Fo and
∆
F synthesis using the SHELXL-17 [48] program
(WinGX suite) [49]. The positions of hydrogen atoms were introduced at calculated positions in their
described geometries and allowed to ride on the attached carbon atom with fixed isotropic thermal
parameters (1.2 Ueq of the parent carbon atom).

Molecules 2020, 25, 3509
24 of 29
These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or
from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB21EZ, UK;
Fax: +44-1223-336-033; or deposit@ccdc.cam.ac.uk). CCDC Number 1,982,708 number contains
the supplementary crystallographic data for this paper.
16 20 8 2 1
r
Crystal data for compound 1: C H N O S , M = 396.48 g/mol, Tetragonal, Space group I41/a,
3
3
a = 14.1731 (7) Å, b = 14.1731 (7) Å, c = 18.0847 (9) Å, V = 3632.8 (5) Å , Z = 8, D = 1.397 Mg/m ,
calc
R = 0.049 (2655 reflections), wR2 = 0.133, T = 293 (2) K, GOF = 1.029. The reflections were collected in
◦
◦
the range 1.78 ≤ θ ≤ 29.98 employing a 0.41 × 0.13 × 0.013 mm crystal.
3
.4. Biological Evaluation
AlphaScreen is a bead-based non-radioactive assay system for the detection of biomolecular
interactions in a microtiter plate format. The binding of biological partners brings donor and acceptor
beads into close proximity and, as a result, a fluorescent signal between 520 and 620 nm was produced.
The AlphaScreen-based assays [50,51] were performed in a final reaction volume of 25 µL of the
assay buffer containing 10 mM HEPES–NaOH (pH 7.4), 50 mM NaCl, 1 mM EDTA (pH 8.0), 0.1%
◦
NP-40, and 10 ng/
µ
L bovine serum albumin (BSA) in a 96-well microtiter plate at 25 C. The pTyr
peptide probes used in this study were 5-carboxyfluorescein (FITC)-GpYLPQTV for STAT3 and
FITC)-GpYDKPHVL for STAT1. Plasmid construction and protein expression were performed as
(
previously described [52]; the experimental method is briefly summarized as follows. Considering
that the expression of the full-length protein is troublesome, a truncated form of STAT3 (136-705)
was constructed and chromatographically purified (AKTA FPLC system; GE Healthcare Bio-Sciences,
Piscataway, NJ, USA). The soluble STAT3 fraction was loaded on a HisTrap
Bio-Sciences) and eluted with an imidazole gradient (50 mM Tris–HCl pH 7.4, 300 mM NaCl, 0.2 mM
EDTA, 0.1% (w/v) NP-40, 5 mM -mercaptoethanol, 10% (v/v) glycerol, and 10–300 mM imidazole).
™ column (GE Healthcare
β
An SDS-PAGE stained with Coomassie Brilliant Blue (CBB) was performed to check the purity of the
protein (see Figure S71). Subsequently, the protein was dialyzed (10 mM HEPES-NaOH pH 7.4, 50 mM
NaCl, 10 mM β-mercaptoethanol, 0.1 mM EGTA, 0.02% (w/v) NP-40, 0.2 mM phenylmethylsulphonyl
fluoride (PMSF), and 10% (v/v) glycerol).
Firstly, 75 nM of each SH2-containing protein were incubated with the test compound (at the
following concentrations: 1, 3, 10, and 30 µM) for 15 min. Each protein sample was then incubated
for 90 min with 50 nM of its corresponding FITC-pTyr peptide and simultaneously mixed with
streptavidin-coated donor beads and anti-FITC acceptor beads before detection at 570 nm using
EnVison Xcite (PerkinElmer, Waltham, MA, USA).
The same procedure was used to evaluate compound
residue mutants (Cys468A, Cys542A, Cys550A, Cys687A, and Cys712A). Briefly, 3
in DMSO (at 0.3, 1, 3, 10 and 30 M concentration) were premixed with 1 L of L-Cys in PBS and 16
1 activity versus STAT3 single-cysteine-
µL of compound
1
µ
µ
µL
of an AlphaScreen buffer. Then, the mixture was immediately (<5 min) mixed with the STAT3 mutant
solution before adding the FITC-peptide.
3
.5. Analytical Studies
3
.5.1. MS, UV, and LC Analyses
Sample Preparation
A compound
prepared at the same concentration in deionized water.
An aqueous buffer (10 mM) for experiments at pH 2 was prepared with 10 mM H PO /NaH PO ,
1
stock solution (1 mg/mL) was prepared in MeOH. The stock solution for GSH was
3
4
2
4
whereas 10 mM NaH PO /Na HPO and 10 mM NaHCO /Na CO were used for the physiological
2
4
2
4
3
2
3
buffer (pH 7.4) and the basic buffer (pH 10), respectively. For experiments under non-oxidizing
conditions, 5 mM Na S O was directly added into the buffers.
2
2
5

Molecules 2020, 25, 3509
25 of 29
Reaction batches were prepared by spiking into the desired buffer aliquots of stock solutions of
◦
compound
1
and GSH down to 0.5 and 1 mM, respectively. All reactions were kept at 37 C under
gentle stirring for up to 24 h.
Before MS experiments, all samples were diluted 1:10 with an aqueous solution containing 30%
acetonitrile and 0.1% formic acid to improve ionization.
Before LC injection, samples were diluted 1:5 with 24 mM HCl. No additional dilution/sample
treatment was required for UV spectroscopy.
MS Method
MS analyses were performed on an LTQ-Orbitrap
™ XL mass spectrometer (Thermo Fisher
Scientific, Waltham, MA, USA) equipped with a Finnigan Ion Max 2 electro-spray source (ESI) endowed
with a metal capillary that was 140 mm long and had a 160 µm inner diameter.
Samples were taken with a Hamilton glass syringe. The solution was injected by a pre-installed
infuser at a flow rate of 5
voltage of 4 kV, 10 units of sheath gas at a capillary temperature of 275 C, and an offset tube lens of
µ
L/min. Nebulization was carried out in positive ion mode with a spray
◦
7
5
4 V. Orbitrap
™
analyzer acquired mass spectra with a scan range from 200 to 1000 m/z, recording
5
× 10 ion per scan with a resolution of 100,000 (full-width at half-maximum at a 400 m/z).
UV Method
UV spectra were performed with a Shimadzu UV-1900 (Shimadzu, Kyoto, Japan) by using UV
PROBE 2.7 software (Shimadzu). Analyses were done with 1.5 mL quartz cuvettes (Hellma, Müllheim,
Germany), acquiring the spectra in a scan range from 200 to 600 nm.
LC Method
LC analysis was carried out with an HPLC Surveyor LC system (Thermo Finnigan) equipped
with a quaternary pump, diode array (PDA) detector, autosampler, online degasser, and thermostat.
Chromatographic separation was carried out with a Phenomenex® Kinetex C18 reverse-phase column
(
Phenomenex, Torrance, CA, USA; 25 mm
×
2.10 mm, 2.6 µm particle diameter with 100 Å pores).
®
An OPTI-SOLV Mini Filter (0.5 mm; Sigma-Aldrich-Merck) was installed before the column to protect
it from particulates. A gradient program reported in Table 4 was used at 200 µL/min flow rate; as a
mobile phase, 1 mM HCl (pH 3) and acetonitrile were employed. The PDA detector worked in scan
mode, acquiring UV spectra in a range from 200 to 600 nm.
Table 4. LC-UV gradient program.
Time (min)
1 mM HCl, pH 3 (A)
ACN (B)
0
1
.00
.00
95
95
30
30
95
95
5
5
70
70
5
1
1
1
2
6.00
6.50
6.51
2.00
5
1
3
.5.2. H-NMR Analysis
1H-NMR analysis was performed at 500 MHz with a Bruker FT-NMR AVANCE
™
DRX500
spectrometer (Bruker, Billerica, MA, USA) using a 5 mm z-PFG (pulsed field gradient) broadband
reverse probe at 298 K. The experimental procedure was designed as follows: a 10 mM pH 10 carbonate
buffer solution in D O was used as the solvent for NMR analysis. Na S O (20 mg, 0.1 mmol) was
2
2
2
5
dissolved in 1 mL of the solvent, and (1) compound
1 (5 mg, 0.025 mmol), (2) GSH (16 mg, 0.05 mmol),
or (3) compound
1 (5 mg, 0.025 mmol) and GSH (16 mg, 0.05 mmol) were dissolved in 0.7 mL of such a
solution. Notably, when Na S O was added, the pH of the solution was slightly lowered below the
2
2
5

Molecules 2020, 25, 3509
26 of 29
value of 10 (pH
≤
10). Each sample was immediately put into the NMR tube and analyzed (time 0)
◦
1
at 298 K. Then, they were heated at 37 C in a thermostatic bath, and their H-NMR spectra were
collected after different times at 298 K.
4
. Conclusions
Benzothiadiazole derivative
screening performed on the SH2 domain. The AlphaScreen-based assay confirmed, albeit indirectly,
that compound was able to inhibit STAT3 dimerization, exhibiting a good activity (STAT3 IC50
5.8 0.6 M versus STAT1 IC50 > 50 M). Therefore, a small set of derivatives was designed and
1
was identified as a novel STAT3 inhibitor by structure-based virtual
1
=
1
±
µ
µ
synthesized through a flexible diversity-oriented synthetic approach when required. The compounds
were tested to define the main structural features for the lead development. Among them, only
compound 1d exhibited a significant, albeit reduced, activity, thus suggesting a considerably “tight”
SAR for this class of derivatives. Moreover, because compound 1, like other known STAT3 inhibitors,
probably binds to cysteine residues, MS, UV, LC, and NMR studies were performed to understand
its mechanism of interaction. Mimicking the biological conditions, we conducted an MS analysis of
compound
1 in the presence of GSH: We discovered that this inhibitor can covalently bind to cysteine
residues through a Michael-like reaction, leading to the formation of some alkylation adducts.
In an attempt to confirm the results of the MS study, an NMR investigation was carried out
to further characterize the adducts formed by the interaction of compound
1 with GSH. However,
despite the same reaction conditions (pH, temperature, and use of an antioxidant) being employed,
the intrinsic differences between the two techniques prevented us from obtaining additional evidence
to support the MS conclusions. Notably, NMR spectroscopy is seldom used for this kind of study
in the literature [16,18], which suggests that its inherent limitations make it unsuitable for such an
application. Therefore, it would seem reasonable to claim that the NMR results neither supported nor
disproved our hypotheses.
The research of STAT3 inhibitors has led to several potent anti-tumor candidates over the years
(e.g., Stattic, S3I-201, and erasin). In this context, we are confident that our ongoing studies will
allow us to develop and improve our lead compound as a new PPI inhibitor endowed with a novel
1
mechanism of interaction and characterized by a pH-dependent activation.
Supplementary Materials: The following are available online: Tables S1–S5: Top-ranked compounds from
computational studies; Figure S1: Main SH2 domain residues involved in the interaction with compound 1;
Table S6: Pan-assay interference compound (PAINS) in silico prediction test, employing five on-line services
1
13
(
SmartsFilter, SwissADME, Zinc Patterns, FAF-Drugs4, and PAINS remover); Figures S2–S70: H and C-NMR
spectra of synthesized compounds; Table S7: Effect of cysteine on the inhibitory activity of compound versus
STAT3; Table S8: Effect of the mutation of cysteine residues located in the vicinities of the SH2 domain on
the inhibitory activity of compound versus STAT3; Figure S71: CBB staining of the STAT3(136–705) and
STAT5b(136–703) proteins. The purified soluble proteins (0.5 g) were analyzed by SDS-PAGE; Table S9:
into the pockets containing the
1
1
µ
Comparison of the best STAT3 complexes obtained by docking compound
1
mutated cysteines, considered in the mutagenesis study; Figure S72: Main interactions of compound
pocket lined by Cys712; Page S52: NMR studies; Figure S73–S75: H-NMR full spectra and expansions related to
1
into the
1
the NMR analysis of compound 1/GSH.
Author Contributions: Conceptualization, A.G. and S.V.; computational studies, A.P.; synthesis and
characterization, M.M., A.G., and S.V.; crystallographic studies, F.M., M.M., and G.P.; biological evaluation, A.A.;
1
M.S., U.V., and L.C. analyses, L.R. and E.G.; H-NMR analysis, D.C.; writing—original draft preparation, A.G. and
S.V.; writing—review and editing, A.G., S.V., and M.M.; supervision, A.G., S.V., and M.M. All authors have read
and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: The authors gratefully thank Rachele Iannarone, Carlo Baschieri, Luca Oliva, Daniela
Masciocchi and Daniela Barlocco for their helpful support.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2020, 25, 3509
27 of 29
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©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(