Synthesis of 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides and their evaluation as ligands for NMDA receptor glycine binding site

Article abstract of DOI:10.3109/14756366.2015.1057722

A series of 2-substituted 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides were synthesized and evaluated for their affinity to the glycine binding site of the N-methyl-d-aspartate (NMDA) receptor. The binding affinity was determined by the displacement of radioligand [³H]MDL-105,519 from rat cortical membrane preparations. The most attractive structures in the search for prospective NMDA receptor ligands were identified to be 2-arylcarbonylmethyl substituted 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides. It has been demonstrated for the first time that the replacement of NH group in the ligand by sp³ CH₂ is tolerated. This finding may pave the way for previously unexplored approaches for designing new ligands of the NMDA receptor.

Full text of DOI:10.3109/14756366.2015.1057722

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Synthesis of 3,4-dihydro-2H-1,2-benzothiazine-3-
carboxylic acid 1,1-dioxides and their evaluation as
ligands for NMDA receptor glycine binding site
Zanda Blukeab, Einars Paassb, Meik Sladekc, Ulrich Abelc & Valerjans Kaussb
a Riga Technical University, Riga, Latvia,
b Latvian Institute of Organic Synthesis, Riga, Latvia, and
c Merz Pharmaceuticals GmbH, Frankfurt am Main, Germany
Published online: 30 Jun 2015.
To cite this article: Zanda Bluke, Einars Paass, Meik Sladek, Ulrich Abel & Valerjans Kauss (2015): Synthesis of 3,4-
dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides and their evaluation as ligands for NMDA receptor glycine
binding site, Journal of Enzyme Inhibition and Medicinal Chemistry
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1057722
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–10
2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1057722
!
RESEARCH ARTICLE
Synthesis of 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid
1,1-dioxides and their evaluation as ligands for NMDA receptor
glycine binding site
Zanda Bluke1,2, Einars Paass2, Meik Sladek3, Ulrich Abel3, and Valerjans Kauss2
1Riga Technical University, Riga, Latvia, 2Latvian Institute of Organic Synthesis, Riga, Latvia, and 3Merz Pharmaceuticals GmbH, Frankfurt am Main,
Germany
Abstract
Keywords
A series of 2-substituted 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides were
synthesized and evaluated for their affinity to the glycine binding site of the N-methyl-
D-aspartate (NMDA) receptor. The binding affinity was determined by the displacement of
radioligand [3H]MDL-105,519 from rat cortical membrane preparations. The most attractive
structures in the search for prospective NMDA receptor ligands were identified to
be 2-arylcarbonylmethyl substituted 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid
1,1-dioxides. It has been demonstrated for the first time that the replacement of NH group
in the ligand by sp3 CH2 is tolerated. This finding may pave the way for previously unexplored
approaches for designing new ligands of the NMDA receptor.
3,4-Dihydro-2H-1,2-benzothiazine-3-
carboxylic acid 1,1-dioxide, glycine
binding site, N-methyl-D-aspartate
receptor, structure–activity relationship
History
Received 2 April 2015
Revised 8 April 2015
Accepted 9 April 2015
Published online 26 June 2015
Introduction
selective antagonist is MRZ 2/596 (V)4, demonstrating efficacy in
several pain models10. Successful optimisation of peripherally
restricted NMDAR glycine binding site antagonists, based on the
quinoxaline-2,3-dione structure VI, has already been reported11.
As it targets peripheral NMDA receptors, the glycine antagonist
pharmacophore model is much more promising for the optimisa-
tion of bioavailability of active compounds.
To search for new starting points in the design of effective
glycine B antagonists based on structure II12, we focused on
information from the early studies of 5,7-dichlorokynurenic acid
(I, DCKA)13. It has been shown that 4-O-substitution dramatic-
ally reduced the activity of the compounds. The carbonyl group in
structure I ensured the interaction with the receptor via water
molecule hydrogen bonding14. Alternatively, the activity was
improved by the introduction of acidic functional group. This
occurred in our series of naphthalenes II, where the most active
compounds contained a carboxyl group in the substituent R
(Figure 1).
Based on the speculations mentioned above, we expected that
the H-bond acceptor in the position 4 of naphthalene II would allow
to reduce the polarity of compounds by removing the carboxyl
group, while not affecting the affinity for the NMDA receptor. The
necessary H-bond acceptor can be included in structure II by using
carboxamide or sulfonamide group. In both cases, the structure may
be derivatised on nitrogen atom. However, compounds containing
carboxamide group in this position (4-oxo-quinazolidine-2-
carboxylic acids) were inactive in [3H]-Gly displacement at
100 mM concentration15. Surprisingly, sulfonamides were highly
active in corresponding benzo-1,2,4-thiadiazine-3-carboxylic acid
1,1-dioxide series14. The role of the sulfonamide group in this
The N-methyl-D-aspartate (NMDA) receptor is a member of the
glutamate receptor superfamily and plays an important role in
neurotransmission within the central nervous system (CNS), as
well as in the synaptic plasticity that underlies learning processes
and memory1,2. Dysfunction of this receptor is thought to be
involved in various disorders including epilepsy, ischemic brain
damage and neurodegenerative disorders, such as Parkinson’s and
Alzheimer’s diseases3,4. Overstimulation of the NMDA receptor
caused by excessive glutamate in the synaptic region is believed to
contribute to the symptomatology of these disorders. The NMDA
receptor, being organized as a ligand-gated ion channel, is
activated by glutamate in the presence of a co-agonist – glycine or
D-serine. The presence of a co-agonist, which binds to a specific
strychnine–insensitive glycine binding site (glycine B) of the
NMDA receptor, is necessary for ion channel activity5. Therefore,
the glycine binding site was perceived as a unique pharmaco-
logical target and several highly potent glycine binding site
antagonists have been discovered6–8. As, unsurprisingly, glycine B
antagonist pharmacophore9 does not look like a brain penetrant,
this was one of the reasons for low efficacy in vivo. The NMDA
receptors are present not only in the CNS but also in the periphery
and there is growing interest in the potential use of glycine
antagonist as treatment for different neuropathic pain conditions.
An example of peripheral activity of a brain impermeant glycine
Address for correspondence: Valerjans Kauss, Latvian Institute of
Organic Synthesis, Aizkraukles 21, Riga LV-1006, Latvia. Tel: +371
67014815. Fax: +371 67014848. E-mail: valerjan@osi.lv

2
Z. Bluke et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Cl
O
Cl
R
Cl
O
R
Cl
R
O
O
O
N
O
N
S
S
Cl
N
H
CO H
2
CO H
2
Cl
Cl
CO H
2
Cl
CO H
2
IDCKA
II
III
IV
O
O
S
HO
O
Me
N
H
N
Cl
Cl
N
O
O
NH
NH
Cl
N
H
O
V MRZ-2/596
VI
Figure 1. Structure of DCKA, naphthoic acid II, target compounds III and IV, MRZ-2/596 and quinoxaline VI.
particular class of compounds has never been explained. However, 4-Chloro-2-methylbenzene-1-sulfonyl chloride (2a)
combination of sulfonamide and carboxy functions has already
To a mixture of ClSO3H (26.0 mL, 395 mmol) and SOCl2
been known to be of great importance in this type of structures15,16
.
(2.3 mL, 31.6 mmol) stirred at 0 ꢁC was slowly added 1-chloro-
3-methylbenzene (1a) (18.7 mL, 158 mmol) and stirred for 1 h.
Then stirring was continued at room temperature for 19 h. The
black reaction mixture was poured into ice. The solid precipitated
was filtered, washed with water and dried to give the title
compound (30.67 g, 86%) as a white solid; mp441 ꢁC (decomp.).
TLC: Rf ¼ 0.24 (petroleum ether). 1H NMR (200 MHz, CDCl3), ꢀ
ppm: 2.77 (s, 3H), 7.37–7.42 (m, 2H), 7.99 (d, J ¼ 8.2 Hz, 1H).
13C NMR (100 MHz, CDCl3), ꢀ ppm: 20.2, 126.9, 130.2, 133.2,
140.0, 141.3, 141.7.
Both modifications described above have been designed based on a
common pharmacophore model of 5,7-dichlorokynurenic acid (I,
DCKA)13, including NH as a presumable H-donor and specific
substituents as in structure 1. The surprising loss of activity, when
the sulfonyl group was replaced by carbonyl15, prompted us to
evaluate carba-analogs III possessing the same sulfonamide
fragment and carboxyl group as crucial pharmacophores. The
efficiency of sp2 CH, rather than NH, for the development of new
GlyB ligands has already been proved12,17. Altogether, this
information provided a highly promising background to identify
novel potent and less polar ligands for the NMDA receptor glycine
binding site, using structure III and, possibly structure IV.
4,5-Dichloro-2-methylbenzene-1-sulfonyl chloride (2b)
Prepared according to the procedure for compound 2a using 1,2-
dichloro-3-methylbenzene (1b) (12 mL, 93.15 mmol) to give the
title compound (22.99 g, 95%) as a white solid; mp 78–81 ꢁC.
Materials and methods
Chemistry
1
TLC: Rf ¼ 0.24 (petroleum ether). H NMR (200 MHz, CDCl3),
Commercial grade reagents and anhydrous solvents were used as ꢀ ppm: 2.74 (s, 3H), 7.53 (s, 1H), 8.15 (s, 1H). 13C NMR
received. 1H NMR and 13C NMR spectra were recorded on a (100 MHz, CDCl3), ꢀ ppm: 19.7, 130.3, 131.2, 134.8, 137.5,
Varian Mercury BB 200 MHz and Varian Mercury plus 400 140.0, 141.7.
(400 MHz) (Ex Varian, Agilent Technologies, Santa Clara, CA)
spectrometer. Chemical shift values are given in parts-per-million N-tert-butyl-4-chloro-2-methylbenzenesulfonamide (3a)
(ppm) relative to tetramethylsilane (TMS) with the residual
To a solution of 4-chloro-2-methylbenzenesulfonyl chloride (2a)
solvent proton resonance as internal standard (CDCl3, 7.26 ppm;
(26.8 g, 119.1 mmol, 1 eq) in CH2Cl2 (250 mL) at 0 ꢁC were added
DMSO-d6, 2.50 ppm). LC–MS analyses were performed on
t-BuNH2 (14.94 mL, 142.9 mmol, 1.2 eq) and Et3N (33 mL,
Shimadzu CBM – 20A (Kyoto, Japan) with Applied Biosystems
238.2 mmol, 2 eq). The reaction mixture was stirred at room
API 2000 (Foster City, CA) mass detector. Elemental analyses
temperature for 22 h. Water was added (800 mL) and the mixture
were carried out on a Carlo Erba Instruments (Milan, Italy)
was extracted with CH2Cl2 (3 ꢀ 700 mL). The combined organic
EA1108 analyser. Thin layer chromatography was performed on
layers were dried over Na2SO4, filtered and evaporated. The
DC Alufolien, Kieselgel 60 F254 (Merck, Darmstadt, Germany)
residue was washed with Et2O and filtered to give the title
plates; UV detection. Flash column chromatography was carried
compound (22.39 g, 72%) as a white solid; mp 142–145 ꢁC. TLC:
out using silica gel (Merck silica gel 60). Melting points were
Rf ¼ 0.36 (Hex:EtOAc, 6:1). 1H NMR (200 MHz, CDCl3), ꢀ ppm:
determined on a Stanford Research System MPA100 (Sunnyvale,
1.21 (s, 9H), 2.63 (s, 3H), 7.27 (m, 2H), 7.95 (d, J ¼ 9.0 Hz, 1H).
CA) Automated Melting Point Apparatus. The purity of all
13C NMR (100 MHz, CDCl3), ꢀ ppm: 20.1, 30.1, 54.8, 126.2,
compounds was determined by HPLC on a Shimadzu CBM – 20A
130.5, 132.1, 138.3, 138.7, 139.6. MS m/z 260 [MꢂH]ꢂ.
(HPLC column: Phenomenex (Torrance, CA), Gemini C18,
See Supplementary Material for the respective compounds:
50 ꢀ 2 mm, 5 mm, solvent: MeCN; flow rate: 0.3 mL/min;
N-tert-butyl-4,5-dichloro-2-methylbenzenesulfonamide (3b).
column temperature: 25 ꢁC; mobile phase: 0.1% HCOOH in
N-tert-butyl-2,4-dichloro-6-methylbenzenesulfonamide (3c).
water/MeCN; detection: UV at 254 nm; injection volume: 3 mL).
Method I – gradient: from 10% MeCN/0.1% HCO2H to 90%
2-(Tert-butyl)-5-chlorobenzo[d]isothiazol-3(2H)-one 1,1-dioxide
MeCN/0.1% HCO2H in 12 min, hold 3 min 90% MeCN/0.1%
(4a)
HCO2H. Method II – gradient: from 40% MeCN/0.1% HCO2H to
90% MeCN/0.1% HCO2H in 12 min, hold 3 min 90% MeCN/0.1% A mixture of H5IO6 (155.96 g, 684.23 mmol, 8 eq) in MeCN
HCO2H. Method III – gradient: from 10% MeCN/0.1% HCO2H to (200 mL) was stirred vigorously at room temperature for 1 h, then
95% MeCN/0.1% HCO2H in 7 min, hold 3 min 95% MeCN/0.1% CrO3 (0.86 g, 8.55 mmol, 0.1 eq) was added followed by acetic
HCO2H.
anhydride (64.8 mL, 684.2 mmol, 8 eq). The mixture was cooled

DOI: 10.3109/14756366.2015.1057722
Synthesis of 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides
3
to ꢂ4 ꢁC then N-tert-butyl-4-chloro-2-methylbenzenesulfonamide concentrated under reduced pressure. The crude product was
(3a) (22.39 g, 85.53 mmol, 1 eq) was added in a small portions. washed with hexane to give the title compound (1.779 g, 95%) as a
1
After stirring at 0 ꢁC for 30 min, the reaction mixture was allowed white solid; mp 93–98 ꢁC. TLC: Rf ¼ 0.25, (Hex:EtOAc, 2:1). H
to reach room temperature and stirring was continued for 15 h. NMR (400 MHz, CDCl3), ꢀ ppm: 3.76 (s, 3H), 4.03 (d, J ¼ 6.0 Hz,
The solvent was removed under reduced pressure. Then water was 2H), 4.93 (s, 2H), 5.53 (t, J ¼ 6.0 Hz, 1H), 6.75 (d, J ¼ 8.4 Hz,
added (200 mL), and the precipitate was filtered and dried to 2H), 7.05 (d, J ¼ 9.2 Hz, 2H), 7.37 (dd, J ¼ 8.4, 2.0 Hz, 1H), 7.48
give the title compound (18.34 g, 78%) as a white solid; mp (d, J ¼ 2.4 Hz, 1H), 7.85 (d, J ¼ 8.8 Hz, 1H). 13C NMR (100 MHz,
138–141 ꢁC. TLC: Rf ¼ 0.38 (Hex:EtOAc, 6:1). 1H NMR CDCl3), ꢀ ppm: 47.0, 55.3, 63.1, 114.0, 127.7, 128.1, 129.3,
(200 MHz, CDCl3), ꢀ ppm: 1.76 (s, 9H), 7.77 (s, 2H), 7.95 131.0, 131.3, 136.4, 139.2, 140.1, 159.3. MS m/z 340 [MꢂH]ꢂ.
(s, 1H). 13C NMR (100 MHz, CDCl3), ꢀ ppm: 27.7, 61.7, 121.5,
See Supplementary Material for the respective compounds:
124.8, 129.2, 134.5, 136.0, 140.9, 158.7. MS m/z 216 [Mꢂ57]ꢂ 4,5-Dichloro-2-(hydroxymethyl)-N-(4-methoxybenzyl)-benzene-
(tert-Bu).
sulfonamide (7b).
2,4-Dichloro-6-(hydroxymethyl)-N-(4-methoxybenzyl)-benzene-
See Supplementary Material for the respective compounds:
2-(Tert-butyl)-5,6-dichlorobenzo[d]isothiazol-3(2H)-one 1,1-dioxide sulfonamide (7c).
(4b).
Ethyl 2-(4-chloro-2-(hydroxymethyl)-N-(4-methoxybenzyl)
phenylsulfonamido)acetate (8a)
2-(Tert-butyl)-5,7-dichlorobenzo[d]isothiazol-3(2H)-one 1,1-dioxide
(4c).
To a solution of compound 7a (4.37 g, 12.8 mmol) in THF
(30 mL) was added NaH (60% in mineral oil, 0.57 g, 14.2 mmol)
and heated at 50 ꢁC for 30 min. Then ethyl bromoacetate
(1.56 mL, 14.08 mmol) was added and heating was continued at
the same temperature for 24 h. The solvent was removed under
reduced pressure. To the residue water was added (30 mL) and
extracted with EtOAc (3 ꢀ 30 mL). The combined organic
extracts were dried over Na2SO4, filtered and concentrated
under reduced pressure. Purified by flash chromatography
(petroleum ether:EtOAc, 4:1) to give the title compound
(3.541 g, 64%) as a colorless oil. TLC: Rf ¼ 0.33 (Hex:EtOAc,
4:1). 1H NMR (200 MHz, CDCl3), ꢀ ppm: 1.17 (t, J ¼ 6.8 Hz, 3H),
3.16 (t, J ¼ 7 Hz, 1H), 3.80 (s, 3H), 3.88 (s, 2H), 4.05 (q,
J ¼ 6.8 Hz, 2H), 4.52 (s, 2H), 4.92 (d, J ¼ 7.6 Hz, 2H), 6.85 (d,
J ¼ 8.6 Hz, 2H), 7.16 (d, J ¼ 8.8 Hz, 2H), 7.39 (dd, J ¼ 8.2,
2.4 Hz, 1H), 7.69 (d, J ¼ 2.4 Hz, 1H), 7.98 (d, J ¼ 8.6 Hz, 1H). 13C
NMR (100 MHz, CDCl3), ꢀ ppm: 14.0, 45.9, 50.8, 55.3, 61.7,
61.8, 114.2, 126.1, 127.7, 130.2, 131.0, 131.3, 135.5, 139.8,
142.4, 159.7, 168.9. MS m/z low ionization. Purity (HPLC,
254 nm, Method I, tR ¼ 9.47 min) 98%.
5-Chlorobenzo[d]isothiazol-3(2H)-one 1,1-dioxide (5a)
A solution of compound 4a (18.3 g, 66.85 mmol) in TFA (70 mL)
was heated at reflux for 70 h (TLC control). Then TFA was
removed under reduced pressure. The residue was treated with
dry Et2O and filtered to give the title compound (12.8 g, 88%)
as a white solid; mp4140 ꢁC (decomp.). TLC: Rf ¼ 0.23
(EtOAc:AcOH, 1:0.01). 1H NMR (200 MHz, DMSO-d6),
ꢀ ppm: 5.02 (s, 1H), 7.95–8.00 (m, 2H), 8.12 (d, J ¼ 8.8 Hz,
1H). 13C NMR (100 MHz, CDCl3), ꢀ ppm: 122.9, 124.5, 130.3,
135.0, 138.2, 139.3, 160.0. MS m/z 216 [MꢂH]ꢂ.
See Supplementary Material for the respective compounds:
5,6-Dichlorobenzo[d]isothiazol-3(2H)-one 1,1-dioxide (5b).
5,7-Dichlorobenzo[d]isothiazol-3(2H)-one 1,1-dioxide (5c).
5-Chloro-2-(4-methoxybenzyl)benzo[d]isothiazol-3(2H)-one
1,1-dioxide (6a)
To a solution of compound 5a (1.6 g, 7.35 mmol) in DMF (5 mL)
was added NaH (60% in mineral oil, 0.29 g, 7.35 mmol) and
heated at 80 ꢁC for 30 min. Then 4-methoxybenzyl chloride
(1.19 mL, 8.82 mmol) was added and heated at 80 ꢁC for 16 h. The
reaction mixture was allowed to reach room temperature, then
water was added (100 mL) and extracted with EtOAc (3 ꢀ 50 mL).
The combined organic extracts were dried over Na2SO4, filtered
and evaporated. Purified by flash chromatography (petroleum
ether:EtOAc, 4:1) to give the title compound (1.9 g, 75%) as a
white solid; mp4108 ꢁC (decomp.). TLC: Rf ¼ 0.33 (Hex:EtOAc,
4:1). 1H NMR (200 MHz, CDCl3), ꢀ ppm: 3.79 (s, 3H), 4.84
(s, 2H), 6.87 (d, J ¼ 8.8 Hz, 2H), 7.43 (d, J ¼ 8.8 Hz, 2H), 7.77–
7.88 (m, 2H), 7.99 (s, 1H). 13C NMR (100 MHz, CDCl3), ꢀ ppm:
42.5, 55.2, 114.0, 122.2, 125.3, 126.1, 129.1, 130.4, 134.8, 135.8,
141.2, 157.6, 159.6;. MS m/z 216 [Mꢂ121]ꢂ (elimination of
p-metoxybenzyl (p-MeOBn)).
See Supplementary Material for the respective compounds:
Ethyl 2-(4,5-dichloro-2-(hydroxymethyl)-N-(4-methoxybenzyl)
phenylsulfonamido)acetate (8b).
Ethyl 2-(2,4-dichloro-6-(hydroxymethyl)-N-(4-methoxybenzyl)
phenylsulfonamido)acetate (8c).
Ethyl 2-(2,4-dichloro-6-formyl-N-(4-methoxybenzyl)
phenylsulfonamido)acetate (9)
To a solution of compound 8c (0.48 g, 1.04 mmol, 1 equiv.) in
dry THF (6 mL) was added MnO2 (0.36 g, 4.14 mmol, 4 equiv.)
and stirred at room temperature for 16 h. Reaction was not
complete, therefore 8 equiv. of MnO2 were added and stirring
was continued for 22 h. The reaction mixture was filtered
through celite, washed with EtOAc and the filtrate concentrated
under reduced pressure to give the title compound (0.15 g, 31%)
as a white solid, mp: 117–121 ꢁC. Rf ¼ 0.30 (Hex/EtOAc 6:1).
1H NMR (200 MHz, CDCl3), ꢀ ppm: 1.21 (t, J ¼ 7.2 Hz, 3H);
3.78 (s, 3H); 4.11 (q, J ¼ 7.2 Hz, 2H); 4.54 (s, 2H); 6.79
(d, J ¼ 8.8 Hz, 2H); 7.06 (d, J ¼ 8.8 Hz, 2H); 7.45 (d, J ¼ 2.0 Hz,
1H); 7.66 (d, J ¼ 2.0 Hz, 1H); 10.52 (s, 1H). 13C NMR
(100 MHz, CDCl3): ꢀ ¼ 14.0; 47.7; 51.9; 55.3; 61.6; 114.2;
125.8; 127.6; 130.1; 134.4; 134.6; 135.8; 139.5; 142.0; 159.7;
168.3; 189.5. MS: m/z 438]Mꢂ121]ꢂ (p-MeOBn).
See Supplementary Material for the respective compounds:
5,6-Dichloro-2-(4-methoxybenzyl)benzo[d]isothiazol-3(2H)-one
1,1-dioxide (6b).
5,7-Dichloro-2-(4-methoxybenzyl)benzo[d]isothiazol-3(2H)-one
1,1-dioxide (6c).
4-Chloro-2-(hydroxymethyl)-N-(4-methoxybenzyl)-benzenesulfo-
namide (7a)
To a solution of compound 6a (1.84 g, 5.44 mmol) in THF
(20 mL) was added solution of NaBH4 (0.68 g, 19.95 mmol) in
water (20 mL) and stirred at room temperature for 17 h. The
solvent was removed under reduced pressure and the aqueous
Ethyl 6,8-dichloro-2-(4-methoxybenzyl)-2H-1,2-benzothiazine-
3-carboxylate 1,1-dioxide (10)
solution was extracted with EtOAc (3 ꢀ 20 mL). The combined To a solution of compound 9 (0.14 g, 0.3 mmol, 1 equiv.) in dry
organic extracts were dried over Na2SO4, filtered and THF (0.5 mL) was added NaH (60% in mineral oil, 3 mg,

4
Z. Bluke et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
0.08 mmol, 0.25 equiv.) and stirred at room temperature for 1 h. 6,8-Dichloro-2-(4-methoxybenzyl)-2H-1,2-benzothiazine-
The solvent was removed under reduced pressure. To the residue 3-carboxylic acid 1,1-dioxide (13a)
was added water (20 mL) and it was extracted with EtOAc
To a solution of compound 10 (20 mg, 0.05 mmol) in THF (1 mL)
(3 ꢀ 20 mL). The combined organic extracts were dried over
was added a solution of NaOH (2 mg, 0.05 mmol) in water (1 mL)
Na2SO4, filtred and concentrated under reduced pressure. The
and stirred at room temperature for 6 h. THF was removed under
residue was triturated with hexane and filtered to give the title
reduced pressure and the aqueous residue was acidified till pHꢃ2
compound (0.12 g, 87%) as a white solid, mp: 91–95 ꢁC. Rf ¼ 0.21
by addition of 1 N HCl. The precipitate was filtered, washed with
(petroleum ether/EtOAc 6:1). 1H NMR (200 MHz, CDCl3), ꢀ:
water and dried to give the title compound (12 mg, 53%) as a light
1.39 (t, J ¼ 7.0 Hz, 3H); 3.68 (s, 3H); 4.38 (q, J ¼ 7.0 Hz, 2H);
1
brown solid, mp: 184–187 ꢁC. H NMR (200 MHz, CDCl3): ꢀ:
5.02 (s, 2H); 6.59 (d, J ¼ 9.0 Hz, 2H); 6.90 (d, J ¼ 9.0 Hz, 2H);
3.69 (s, 3H); 5.05 (s, 2H); 6.60 (d, J ¼ 8.0 Hz, 2H); 6.94 (d,
7.19 (d, J ¼ 1.6 Hz, 1H); 7.34 (s, 1H); 7.49 ppm (d, J ¼ 1.4 Hz,
J ¼ 8.0 Hz, 2H); 7.19 (s, 1H), 7.48 (s, 1H); 7.53 ppm (s, 1H).
1H). MS: m/z 440 [MꢂH]ꢂ, 320 [Mꢂ121]ꢂ (p-MeOBn). Anal.
HPLC: (Method I) tR ¼ 10.38 min, purity 98%. MS m/z 412
Calcd. for C19H17Cl2NO5SxH2O: C 49.58, H 4.16, N 3.04, found:
[MꢂH]ꢂ.
C 49.21, H 3.78, N 3.05.
See Supplementary Material for the respective compounds:
2-(3-Bromobenzyl)-6,8-dichloro-2H-1,2-benzothiazine-3-carboxylic
acid 1,1-dioxide (13b).
6,8-Dichloro-2-(3-chlorobenzyl)-2H-1,2-benzothiazine-3-carboxylic
acid 1,1-dioxide (13c).
Ethyl 6,8-dichloro-2H-1,2-benzothiazine-3-carboxylate
1,1-dioxide (11)
A solution of compound 10 (1.64 g, 3.7 mmol) in TFA (8 mL) was
stirred at room temperature for 6 h then it was concentrated under
Ethyl 2-(2-(bromomethyl)-4-chloro-N-(4-methoxybenzyl)
phenylsulfonamido)acetate (14a)
reduced pressure. Purification by flash chromatography gave the
title product 11 (0.85 g, 71%) as a light yellow solid, mp: 173–
177 ꢁC. Rf ¼ 0.35 (petroleum ether/EtOAc/AcOH 4:1:0.01). 1H
NMR (200 MHz, CDCl3): ꢀ ¼ 1.41 (t, J ¼ 7.2 Hz, 3H); 4.42 (q,
J ¼ 7.2 Hz, 2H); 7.05 (s, 1H); 7.42 (d, J ¼ 2.2 Hz, 1H); 7.54 (d,
J ¼ 2.2 Hz, 1H); 7.86 ppm (s, 1H). 13C NMR (100 MHz, CDCl3):
ꢀ ¼ 14.1; 63.3; 111.2; 127.5; 129.3; 130.2; 131.2; 131.5; 135.3;
138.3; 161.1. MS: m/z 320 [MꢂH]ꢂ. Anal. Calcd. for
C11H9Cl2NO4Sx0.2C6H14: C 43.23, H 3.39, N 4.13, found: C
43.39, H 3.07, N 3.92.
To a solution of compound 8a (2.68 g, 6.26 mmol) in dry Et2O
(20 mL) was added PBr3 (0.21 mL, 2.19 mmol) and stirred at
room temperature for 4 h. The solvent was removed under reduced
pressure. Purified by flash chromatography (petroleum
ether:EtOAc, 4:1) to give the title compound (2.09 g, 68%) as a
white solid; mp 112–115 ꢁC. TLC: Rf ¼ 0.29 (Hex:EtOAc, 4:1).
1H NMR (200 MHz, CDCl3), ꢀ ppm: 1.18 (t, J ¼ 7.2 Hz, 3H), 3.79
(s, 3H), 3.95 (s, 2H), 4.07 (q, J ¼ 7.2 Hz, 2H), 4.49 (s, 2H), 4.91
(s, 2H), 6.80 (d, J ¼ 8.8 Hz, 2H), 7.13 (d, J ¼ 8.6 Hz, 2H), 7.38
(dd, J ¼ 8.2, 2.4 Hz, 1H); 7.65 (d, J ¼ 2.2 Hz, 1H), 7.98 (9 Hz,
1H). 13C NMR (100 MHz, CDCl3), ꢀ ppm: 14.0, 28.4, 46.4, 51.0,
55.3, 61.4, 114.1, 126.0, 128.6, 130.3, 131.3, 133.1, 136.6, 138.3,
139.3, 159.6, 168.6.
Ethyl 2-(3-bromobenzyl)-6,8-dichloro-2H-1,2-benzothiazine-
3-carboxylate 1,1-dioxide (12b)
To a solution of compound 11 (70 mg, 0.22 mmol, 1 equiv.) in
DMF (1.5 mL) was added DIEA (0.06 mL, 0.33 mmol) and
3-bromobenzyl bromide (70 mg, 0.24 mmol) and the mixture was
stirred at room temperature for 12 h. Then water (10 mL) was
added and the mixture was extracted with EtOAc (3 ꢀ 10 mL).
The combined organic extracts were dried over Na2SO4, filtered
and concentrated. Purification by column chromatography gave
the title compound (100 mg, 95%) as a white solid, mp: 111–
114 ꢁC. Rf ¼ 0.29 (petroleum ether:EtOAc, 4:1). 1H NMR
(400 MHz, CDCl3): ꢀ ¼ 1.38 (t, J ¼ 7.2 Hz, 3H); 4.37 (q,
J ¼ 7.2 Hz, 2H); 5.03 (s, 2H); 6.94 (d, J ¼ 7.6 Hz, 1H); 7.00 (t,
J ¼ 7.6 Hz, 1H); 7.19 (s, 1H); 7.24 (d, J ¼ 1.6 Hz, 1H); 7.27–7.28
(m, 1H); 7.39 (s, 1H); 7.54 ppm (d, J ¼ 2.0 Hz, 1H). 13C NMR
(100 MHz, CDCl3): ꢀ ¼ 14.2; 52.8; 62.6; 122.3; 123.8; 126.9;
127.8; 129.8; 130.9. 131.2; 131.2; 131.3; 132.5; 132.9; 134.2;
136.5; 137.9; 161.6. MS: low ionization. Anal. Calcd. for
C18H14BrCl2NO4S: C 44.02, H 2.87, N 2.85, found: C 44.09,
H 2.95, N 2.81.
See Supplementary Material for the respective compounds:
Ethyl 2-(2-(bromomethyl)-4,5-dichloro-N-(4-methoxybenzyl)
phenylsulfonamido)acetate (14b).
Ethyl 2-(2-(bromomethyl)-4,6-dichloro-N-(4-methoxybenzyl)
phenylsulfonamido)acetate (14c).
Ethyl 6-chloro-2-(4-methoxybenzyl)-3,4-dihydro-2H-1,2-
benzothiazine-3-carboxylate 1,1-dioxide (15a)
To a solution of compound 14a (2.08 g, 4.24 mmol) in THF
(20 mL) was added NaH (60% in mineral oil, 0.17 g, 4.25 mmol)
and stirred at room temperature for 6 h. The solvent was removed
under reduced pressure. To the residue water (30 mL) was added
and the mixture was extracted with EtOAc (3 ꢀ 30 mL). The
combined organic extracts were dried over Na2SO4, filtered and
concentrated under reduced pressure. The residue was separated
by column chromatography (petroleum ether:EtOAc, 4:1) to give
the title compound (1.38 g, 79%) as a white solid; mp 103–107 ꢁC.
TLC: Rf ¼ 0.27 (Hex:EtOAc, 4:1). 1H NMR (200 MHz, CDCl3), ꢀ
ppm: 1.22 (t, J ¼ 7.2 Hz, 2H), 3.09 (dd, J ¼ 16.6, 7.0 Hz, 1H), 3.44
(dd, J ¼ 17.0, 9.4 Hz, 1H), 3.78 (s, 3H), 4.02–4.26 (m, 3H), 4.30
Ethyl 6,8-dichloro-2-(3-chlorobenzyl)-2H-1,2-benzothiazine-
3-carboxylate 1,1-dioxide (12c)
Prepared according to procedure for compound 12b using (d, J ¼ 15.0 Hz, 1H), 2.43 (d, J ¼ 15.0 Hz, 1H), 6.77 (d,
3-chlorobenzyl chloride. Yield 82%. White solid, mp: 113– J ¼ 8.8 Hz, 2H), 7.13 (d, J ¼ 8.8 Hz, 2H), 7.26 (s, 1H), 7.39 (dd,
116 ꢁC. Rf ¼ 0.39 (Hex/EtOAc 6:1). 1H NMR (200 MHz, CDCl3), J ¼ 8.0, 2 Hz, 1H), 7.79 (d, J ¼ 8.0 Hz, 1H). 13C NMR (100 MHz,
ꢀ ppm: 1.37 (t, J ¼ 7.2 Hz, 3H); 4.37 (q, J ¼ 7.2 Hz, 2H); 5.03 (s, CDCl3), ꢀ ppm: 14.0, 27.6, 51.6, 55.2, 57.2, 62.0, 113.8, 125.0,
2H); 6.88–6.92 (m, 1H); 7.01–7.14 (m, 3H); 7.25 (d, J ¼ 2.0 Hz, 126.7, 127.7, 129.1, 130.2, 136.1, 136.5, 138.3, 159.4, 169.5. MS
1H); 7.39 (s, 1H); 7.54 ppm (d, J ¼ 2.2 Hz, 1H). 13C NMR low ionization.
(100 MHz, CDCl3): ꢀ ppm: 14.1; 52.8; 62.6; 123.6; 126.4; 127.8;
See Supplementary Material for the respective compounds:
128.3; 128.4; 129.5; 131.1; 132.4; 132.9; 134.1; 136.3; 138.0; Ethyl 6,7-dichloro-2-(4-methoxybenzyl)-3,4-dihydro-2H-1,2-ben-
161.6. MS m/z 447 [M+H]+ (low ionization).
zothiazine-3-carboxylate 1,1-dioxide (15b).

DOI: 10.3109/14756366.2015.1057722
Synthesis of 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides
5
Ethyl 6,8-dichloro-2-(4-methoxybenzyl)-3,4-dihydro-2H-1,2-ben- Ethyl 6-chloro-2-[(2-pyridinyl)-methyl]-3,4-dihydro-2H-1,2-ben-
zothiazine-3-carboxylate 1,1-dioxide (15c).
zothiazine-3-carboxylate 1,1-dioxide (17f).
Ethyl 6-chloro-2-[(3-pyridinyl)-methyl]-3,4-dihydro-2H-1,2-ben-
zothiazine-3-carboxylate 1,1-dioxide (17g).
Ethyl 6-chloro-3,4-dihydro-2H-1,2-benzothiazine-3-carboxylate
1,1-dioxide (16a)
Ethyl
6-chloro-2-[(2-methyl-1,3-thiazolyl-4)-methyl]-3,4-dihy-
dro-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide (17h).
Ethyl 2-(tert-butoxycarbonylmethyl)-6-chloro-3,4-dihydro-2H-
1,2-benzothiazine-3-carboxylate 1,1-dioxide (17i).
Ethyl 6-chloro-2-(2-phenethyl)-3,4-dihydro-2H-1,2-benzothia-
zine-3-carboxylate 1,1-dioxide (17j).
Ethyl 6-chloro-2-[2-(3-methoxyphenyl)-ethyl]-3,4-dihydro-2H-
1,2-benzothiazine-3-carboxylate 1,1-dioxide (17k).
Ethyl 6-chloro-2-[2-(2-methoxyphenyl)-ethyl]-3,4-dihydro-2H-
1,2-benzothiazine-3-carboxylate 1,1-dioxide (17l).
Ethyl 6-chloro-2-[2-(2-fluorophenyl)-ethyl]-3,4-dihydro-2H-1,2-
benzothiazine-3-carboxylate 1,1-dioxide (17m).
Ethyl 6-chloro-2-[2-(3-methoxyphenyl)-2-oxoethyl]-3,4-dihydro-
2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide (17n).
Ethyl 6-chloro-2-[2-(2-methoxyphenyl)-2-oxoethyl]-3,4-dihydro-
2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide (17o).
To a solution of compound 15a (0.47 g, 1.15 mmol) in CH2Cl2
(1 mL) was added TFA (1 mL) and stirred at room temperature for
5 h. The solvent was removed under reduced pressure. Purified by
column chromatography (petroleum ether:EtOAc, 2:1) to give the
title compound (0.25 g, 75%) as a white solid; mp 135–138 ꢁC.
TLC: Rf ¼ 0.44 (Hex:EtOAc, 2:1). 1H NMR (400 MHz, CDCl3), ꢀ
ppm: 1.33 (t, J ¼ 7.2 Hz, 3H), 3.16 (dd, J ¼ 16.8, 8.4 Hz, 1H), 3.43
(dd, J ¼ 16.8, 6.0 Hz, 1H), 4.29 (q, J ¼ 7.2 Hz, 2H), 4.62 (dd,
J ¼ 13.8, 7.6 Hz, 1H), 7.27 (d, J ¼ 2 Hz, 1H), 7.41 (dd, J ¼ 8.4,
1.6 Hz, 1H), 7.78 (d, J ¼ 8.4 Hz, 1H). 13C NMR (100 MHz,
CDCl3), ꢀ ppm: 14.1, 30.5, 54.4, 62.8, 125.2, 128.4, 129.2, 135.0,
136.5, 138.6, 169.4. MS m/z 290 [M+H]+.
Ethyl 6,7-dichloro-3,4-dihydro-2H-1,2-benzothiazine-3-carboxy-
late 1,1-dioxide (16b)
Ethyl
6-chloro-2-[2-(3-fluorophenyl)-2-oxoethyl]-3,4-dihydro-
Prepared in 77% yield from compound 15b according to 2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide (17p).
procedure for compound 16a. A light pink solid; mp 154– Ethyl 6-chloro-2-[2-(4-chlorophenyl)-2-oxoethyl]-3,4-dihydro-
1
158 ꢁC. TLC: Rf ¼ 0.19 (Hex:EtOAc, 4:1). H NMR (200 MHz, 2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide (17q).
CDCl3), ꢀ ppm: 1.33 (t, J ¼ 7.0 Hz, 3H); 3.13 (dd, J ¼ 17.0, Ethyl 6,7-dichloro-2-(3-chlorobenzyl)-3,4-dihydro-2H-1,2-ben-
8.0 Hz, 1H), 3.41 (dd, J ¼ 17.0, 5.8 Hz, 1H), 4.29 (q, J ¼ 7.0 Hz, zothiazine-3-carboxylate 1,1-dioxide (17r).
2H); 4.61 (dd, J ¼ 8.0, 5.8 Hz, 1H), 7.39 (s, 1H), 7.93 (s, 1H). 13C Ethyl
6,7-dichloro-2-(2-phenylethyl)-3,4-dihydro-2H-1,2-ben-
NMR (100 MHz, CDCl3), ꢀ ppm: 14.1, 29.8, 54.3, 63.0, 125.7, zothiazine-3-carboxylate 1,1-dioxide (17s).
131.0, 132.8, 137.0, 137.4, 169.3. MS m/z 324 [M+H]+. Anal.
Calcd. for C11H11Cl2NO4S: C, 40.76; H, 3.42; N, 4.32. Found: C, Hydrolysis of ethyl 3,4-dihydro-2H-1,2-benzothiazine-3-carbox-
40.89; H, 3.47; N, 4.23.
ylates 15, 16 and 17 (general procedure B).
To a solution of ethyl 3,4-dihydro-2H-1,2-benzothiazine-3-carb-
oxylate 1,1-dioxide (1 mmol) in THF (2 mL) was added a solution
of LiOHꢄH2O or NaOH (1 mmol) in H2O (2 mL), then it was
Synthesis of 2-substituted ethyl 3,4-dihydro-2H-1,2-benzothia-
zine-3-carboxylates 17 (general procedure A)
To a solution of ethyl 3,4-dihydro-2H-1,2-benzothiazine-3-carb- stirred at room temperature for 2–8 h (TLC control). The reaction
oxylate 1,1-dioxide 16 (1 mmol) in DMF (2 mL) was added NaH mixture was acidified by 1 N HCl (ꢃpH 2) and extracted
(60% in mineral oil, 1 mmol) and it was stirred at room with CH2Cl2 (3 ꢀ 15 mL). The combined organic layers were
temperature for 30 min. Then the alkyl halogenide (1.1 mmol) dried over Na2SO4, filtered and concentrated. The residue was
was added and stirred at room temperature for 4–15 h (TLC triturated with Et2O or hexane and washed with small amount of
control). To the reaction mixture water (10 mL) was added and it Et2O, CH2Cl2 or CHCl3. Dried in vacuo over P2O5 (room
was extracted with EtOAc (3 ꢀ 10 mL). The combined organic temperature).
extracts were dried over Na2SO4, filtered and concentrated under
reduced pressure. The product was purified by column chroma- 6-Chloro-2-(4-methoxybenzyl)-3,4-dihydro-2H-1,2-benzothiazine-
tography (petroleum ether:EtOAc).
3-carboxylic acid 1,1-dioxide (18a)
Prepared according to general procedure B using compound 15a
and LiOHꢄH2O. Yield 62%. white solid; mp 179–181 ꢁC. 1H NMR
(400 MHz, CDCl3), ꢀ ppm: 3.21 (dd, J ¼ 16.2, 7.8 Hz, 1H), 3.43
Ethyl 6-chloro-2-(3-methoxybenzyl)-3,4-dihydro-2H-1,2-ben-
zothiazine-3-carboxylate 1,1-dioxide (17a)
Prepared according to general procedure A using compound 16a (dd, J ¼ 15.4, 9.6 Hz, 1H), 3.77 (s, 3H), 4.11 (dd, J ¼ 9.4, 7.2 Hz,
and 3-methoxybenzyl bromide to give product 17a in 62% yield 1H), 4.21 (d, J ¼ 14.6 Hz, 1H), 4.43 (d, J ¼ 14.4 Hz, 1H), 6.77 (d,
1
as a colorless oil; TLC: Rf ¼ 0.33 (Hex:EtOAc, 4:1). H NMR J ¼ 8.8 Hz, 2H), 7.09 (d, J ¼ 7.8 Hz, 2H), 7.29 (s, 1H), 7.43 (dd,
(400 MHz, CDCl3), ꢀ ppm: 1.22 (t, J ¼ 7.2 Hz, 3H), 3.14 (dd, J ¼ 7.8, 1.6 Hz, 1H), 7.81 (d, J ¼ 8.4 Hz, 1H). MS m/z 380
J ¼ 16.8, 6.8 Hz, 1H), 3.46 (dd, J ¼ 16.2, 9.8 Hz, 1H), 3.72 (s, [MꢂH]ꢂ. Purity (HPLC, 254 nm, Method I, tR ¼ 8.74 min) 99%.
3H), 4.05–4.30 (m, 3H), 4.35 (d, J ¼ 15.2 Hz, 1H), 4.48 (d, Anal. Calcd. for C17H16ClNO5S: C, 53.48; H, 4.22; N, 3.67.
J ¼ 15.2 Hz, 1H), 6.77–6.80 (m, 3H), 7.16 (t, J ¼ 8.0 Hz, 1H), 7.28 Found: C, 53.56; H, 3.98; N, 3.67.
(s, 1H), 7.40 (dd, J ¼ 8.8, 2.0 Hz, 1H), 7.80 (d, J ¼ 8.8 Hz, 1H).
See Supplementary Material for the respective compounds 18:
6,8-Dichloro-2-(4-methoxybenzyl)-3,4-dihydro-2H-1,2-benzothiazine-
MS m/z 410 [M+H]+.
See Supplementary Material for the respective compounds 17: 3-carboxylic acid 1,1-dioxide (18b).
Ethyl 6-chloro-2-(4-chlorobenzyl)-3,4-dihydro-2H-1,2-benzothia- 6-Chloro-2-(3-methoxybenzyl)-3,4-dihydro-2H-1,2-benzothiazine-3-
zine-3-carboxylate 1,1-dioxide (17b).
Ethyl 6-chloro-2-(3-chlorobenzyl)-3,4-dihydro-2H-1,2-benzothia- 6-Chloro-2-(4-chlorobenzyl)-3,4-dihydro-2H-1,2-benzothiazine-3-
zine-3-carboxylate 1,1-dioxide (17c). carboxylic acid 1,1-dioxide (18d).
Ethyl 2-(3-bromobenzyl)-6-chloro-3,4-dihydro-2H-1,2-benzothia- 6-Chloro-2-(3-chlorobenzyl)-3,4-dihydro-2H-1,2-benzothiazine-3-
zine-3-carboxylate 1,1-dioxide (17d). carboxylic acid 1,1-dioxide (18e).
Ethyl 2-benzyl-6-chloro-3,4-dihydro-2H-1,2-benzothiazine-3- 2-(3-Bromobenzyl)-6-chloro-3,4-dihydro-2H-1,2-benzothiazine-3-
carboxylate 1,1-dioxide (17e). carboxylic acid 1,1-dioxide (18f).
carboxylic acid 1,1-dioxide (18c).

6
Z. Bluke et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
2-Benzyl-6-chloro-3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic thawed and washed four times by re-suspension in 50 mM Tris-
acid 1,1-dioxide (18g). HCl, pH 7.5 and centrifugation at 48 000g for 20 min. The final
6,7-Dichloro-2-(3-chlorobenzyl)-3,4-dihydro-2H-1,2-benzothiazine- pellet was suspended in assay buffer. The amount of protein in the
3-carboxylic acid 1,1-dioxide (18h). final membrane preparation was determined according to the
6-Chloro-2-(2-phenylethyl)-3,4-dihydro-2H-1,2-benzothiazine-3- method of Lowry et al.20 with some modifications21. The final
carboxylic acid 1,1-dioxide (18i).
protein concentration used for our studies was 400 mg/mL.
6-Chloro-2-[2-(3-methoxyphenyl)-ethyl]-3,4-dihydro-2H-1,2-
benzothiazine-3-carboxylic acid 1,1-dioxide (18j).
6-Chloro-2-[2-(2-methoxyphenyl)-ethyl]-3,4-dihydro-2H-1,2-
benzothiazine-3-carboxylic acid 1,1-dioxide (18k).
6-Chloro-2-[2-(2-fluorophenyl)-ethyl]-3,4-dihydro-2H-1,2-ben-
zothiazine-3-carboxylic acid 1,1-dioxide (18l).
[3H]MDL-105,519 displacement studies
A robotic system designed for binding assays (Tecan Deutschland
GmbH, Crailsheim, Germany) was loaded with the membrane
solution, solutions for bound control (buffer/DMSO 20%),
unlabeled glycine (1 mM) for evaluation of non-specific binding,
all compounds to be tested (at 20-fold concentrations), radi-
oligand and respective 96-well plates. Before performing dis-
placement studies, saturation experiments were performed to
determine the equilibrium dissociation constant (Kd) of [3H]-
MDL-105,519, which is a parameter for the affinity of the
radioligand to the binding site. The protein/receptor concentration
was held constant, whereas the amount of specific bound
radioligand was determined using increasing concentrations of
ligand. On the basis of the saturation experiments, a final [3H]-
MDL-105,519 concentration of 2 nM was selected. First, the assay
plates were loaded with membrane solution and were shaken at
4 ꢁC. The mother plates were then prepared by pipetting the
compounds into assay buffer/20% DMSO to obtain the desired
final concentrations (dose response curve with five different
concentrations, e.g. 10, 3, 1, 0.3 and 0.1 mM). After transferring
radioligand into the assay plates, the compounds were added
(including the bound and the non-specific binding control). The
final DMSO concentration was 1%. The assay plates were
incubated and shaken at 4 ꢁC for 1 h, before the mixture was
exhausted as rapidly as possible via a vacuum manifold using the
Multiscreen HTS glass fibre (type B) filter plates (Millipore,
6,7-Dichloro-2-(2-phenylethyl)-3,4-dihydro-2H-1,2-benzothiazine-
3-carboxylic acid 1,1-dioxide (18m).
6-Chloro-2-[2-(3-methoxyphenyl)-2-oxoethyl]-3,4-dihydro-2H-
1,2-benzothiazine-3-carboxylic acid 1,1-dioxide (18n).
6-Chloro-2-[2-(2-methoxyphenyl)-2-oxoethyl]-3,4-dihydro-2H-
1,2-benzothiazine-3-carboxylic acid 1,1-dioxide (18o).
6-Chloro-2-[2-(3-fluorophenyl)-2-oxoethyl]-3,4-dihydro-2H-1,2-
benzothiazine-3-carboxylic acid 1,1-dioxide (18p).
6-Chloro-2-[2-(4-chlorophenyl)-2-oxoethyl]-3,4-dihydro-2H-1,2-
benzothiazine-3-carboxylic acid 1,1-dioxide (18q).
6-Chloro-2-[(2-pyridinyl)-methyl]-3,4-dihydro-2H-1,2-benzothia-
zine-3-carboxylic acid 1,1-dioxide (18r).
6-Chloro-2-[(3-pyridinyl)-methyl]-3,4-dihydro-2H-1,2-benzothia-
zine-3-carboxylic acid 1,1-dioxide (18s).
6-Chloro-2-[(2-methyl-1,3-thiazolyl-4)-methyl]-3,4-dihydro-2H-
1,2-benzothiazine-3-carboxylic acid 1,1-dioxide (18t).
2-(Tert-butoxycarbonylmethyl)-6-chloro-3,4-dihydro-2H-1,2-ben-
zothiazine-3-carboxylic acid 1,1-dioxide (18u).
6-Chloro-3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid
1,1-dioxide (19)
Prepared according to general procedure B using compound 16a Schwalbach, Germany) under a constant vacuum of 450 mbar.
and LiOHꢄH2O. White solid; mp 177–181 ꢁC. 1H NMR The membranes were washed four times with cold assay buffer
(400 MHz, DMSO-d6), ꢀ ppm: 3.08 (dd, J ¼ 17.2, 12.0 Hz, 1H), (100 mL).
A 50 mL of Ultima Gold scintillation cocktail
3.25 (dd, J ¼ 17.6, 4.8 Hz, 1H), 4.42 (td, J ¼ 12.0, 4.8 Hz, 1H), (PerkinElmer, Rodgau-Ju¨gesheim, Germany) was added to the
7.52 (dd, J ¼ 8.0, 2.0 Hz, 1H), 7.60 (d, J ¼ 1.6 Hz, 1H), 7.74 (d, wet filter plates and incubated at room temperature overnight
J ¼ 8.0 Hz, 1H), 7.86 (d, J ¼ 10.8 Hz, 1H). LC/MS m/z 260 before counting the disintegration per minutes using a liquid
[MꢂH]ꢂ. Purity (HPLC, 254 nm, Method II, tR ¼ 7.35) 97%.
scintillation counter (MicroBeta, PerkinElmer, Rodgau-
Ju¨gesheim, Germany).
Binding experiments
Analysis of data
For the evaluation of binding affinity of the test compounds on the
glycine binding pocket of the NMDA receptor, [3H]-MDL-
105,519 (GE Healthcare, Freiburg, Germany) displacement
studies were performed using a 96-well plate robotic platform.
MDL-105,519 is a selective, high affinity antagonist at the
NMDA receptor glycine binding site.
For the evaluation of binding affinity of the test compound to the
glycine B binding site and its potency to displace [3H]-MDL-
105,519, the measured radioactivity of the radioligand alone is set
as 100% bound control and the non-specific binding of the
radioligand (which could not be displaced by glycine, 1 mM)
represented the 0% control. The residual radioactivity after
displacement of the test compound (n ¼ 5) is then corrected
with respect to the set controls.
Preparation of cortical membranes
Tissue preparation was performed according to Foster & Wong18
with some modifications19. For isolation of the cell membranes,
rat cortices were homogenized in 20 volumes of ice-cold 0.32 M
sucrose (Sigma-Aldrich, Taufkirchen, Germany) using a glass-
Teflon homogenizer. The homogenate was centrifuged at 1000g
for 10 min, the pellet was discarded and the supernatant
centrifuged at 20 000g for 20 min. The resulting pellet was re-
suspended in 20 volumes of distilled water and centrifuged for
20 min at 8000g. The supernatant and the buffy coat were then
centrifuged three times (48 000g for 20 min) in the presence of
50 mM Tris-HCl, pH 8.0 (assay buffer). All centrifugation steps
were carried out at 4 ꢁC. After re-suspension in 5 volumes of
50 mM Tris-HCl, pH 7.5, the membrane suspension was frozen
rapidly at ꢂ80 ꢁC. On the day of assay, the membranes were
Results and discussion
Chemistry
There are two main approaches for the synthesis of 2H-1,2-
benzothiazine-3-carboxylates 10–12. One of them is based on the
diazotation of an appropriate aniline and subsequent Meerwein
arylation of acrylonitrile22. Then sulfonation with chlorosulfonic
acid gives the corresponding benzenesulfonyl chloride, which is
cyclised to a derivative of 3,4-dihydro-2H-1,2-benzothiazine.
First, we attempted the synthesis according to the procedure
described above. Unfortunately, the Meerwein arylation of
acrylonitrile with 3,5-dichlorobenzenediazonium chloride did
not work well in our hands. All attempts to optimise the reaction

DOI: 10.3109/14756366.2015.1057722
Synthesis of 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides
7
1
R
1
1
R
O O
S
R
O O
S
2
R
2
3
2
3
R
R
b
a
c
Cl
N
H
3
R
R
R
1a, b
2a, b
3a, b, c
1
1
R
R
1
O
S
R
O
N
O
O
O
S
2
O
2
3
R
R
2
d
S
e
R
f
NH
N
PMB
3
R
R
3
R
O
O
O
4a, b, c
5a, b, c
6a, b, c
1
1
R
R
O
O
O
O
2
1
2
3
2
3
PMB
R
R
S
PMB
1-8 a R = R = H, R = Cl;
R
S
g
N
N
1
2
3
b R = H, R = R = Cl;
H
1
3
2
3
c R = R = Cl, R = H
R
CO Et
2
OH
OH
7a, b, c
8a, b, c
Scheme 1. Synthesis route to compounds 8a, b, c. Reagents and conditions: (a) SOCl2, ClSO3H, 0 ꢁC – r.t.; (b) t-BuNH2, Et3N, CH2Cl2; (c) H5IO6,
CrO3, (CH3CO)2O, MeCN, 0 ꢁC – r.t.; (d) TFA; (e) PMBCl, NaH, DMF, 80 ꢁC; (f) NaBH4, THF/H2O; (g) NaH, ethyl bromoacetate, THF, 50 ꢁC.
conditions resulted in only a 9% isolated yield of corresponding carboxylic acids. Thus, alkylation of benzothiazine 11 with 3-
3-aryl-2-chloropropionitrile. Therefore, we focused on the second bromobenzylbromide gave the expected ester 12b in 95% yield.
approach comprising a six-step synthesis from saccharin23. This Finally, alkaline hydrolysis of esters 10 and 12 by NaOH or LiOH
involved chemical transformation of saccharin derivatives to was used to afford the desired carboxylic acids 13.
sulfonamides 8 is shown in Scheme 1. Further on, the hydroxyl
Compounds 18 were synthesized according to the previously
group of compound 8 was transformed to bromide which, in turn, described approach22 shown in Scheme 3. Benzyl alcohols 8 were
was cyclised in the presence of a base to the corresponding 3,4- treated with PBr3 to give bromides 14, which were then cyclised
dihydro-2H-1,2-benzothiazine. Finally, oxidation with NBS gave to benzothiazines 15, using NaH for deprotonation. Cleavage of
2H-1,2-benzothiazine analogues of structure 10. Synthesis of N-(p-methoxybenzyl) group with TFA gave 1,2-benzothiazines
necessary benzisothiazolones 5 was attempted in accordance with 16, which were used in N-alkylation reactions with different aryl-
the previously described procedure24. As phenylsulfonyl chlorides and heteroaryl-methyl halides. Then alkaline hydrolysis of ethyl
2a and 2b were not commercially available, they were prepared esters 17 gave the desired carboxylic acids 18 in variable yields.
by chlorosulfonation of toluene derivatives 1a and 1b. In the next The N-(p-methoxybenzyl) group containing compounds 18 was
step, sulfonyl chlorides 2 were treated with t-butyl amine to prepared by hydrolysis of the corresponding esters 15.
generate corresponding sulfonamides 3. The N-tert-butyl group is
compatible with the oxidative cyclisation of compounds 3 with
H5IO6 and CrO3 in acetonitrile to obtain benzisothiazoles 4 in
Binding results
high yield24. The cleavage of N-tert-butyl group and
The affinity of benzothiazine compounds 13a, b, c to the glycine
binding site of the NMDA receptor was determined by the
displacement of radioligand [3H]MDL-105,51925–27 from rat
cortical membrane preparations. Surprisingly, all 2H-benzothia-
zine-3-carboxylic acids 13a, b, c were found completely inactive
at a 10 mM concentration. Only compound 13b displayed weak
affinity (Ki ¼ 90 mM) for the glycine binding site.
subsequent N-protection with p-methoxybenzyl group gave
benzisothiazoles 6.
The next step was crucial since the authors of this synthetic
pathway for the reductive opening of the isothiazolone ring used
lithium aluminum hydride, which gave the product only with 41%
yield24. It appeared that the formation of byproducts could be
overcome by using sodium borohydride in THF/water (1:1).
Thus, benzenesulfonamides 7 were obtained in 94–98% yield.
Alkylation of sulfonamides 7 with ethyl bromoacetate in the
presence of base gave compounds 8. The reported three-step
transformation of 2-(hydroxymethyl)-benzenesulfonamide to 2H-
1,2-benzothiazines included the hydroxide conversion to bromide,
ring closure, and finally the oxidation of the ring system23. To
prepare the crucial intermediate 10 from compound 8c we used a
two-step procedure as shown in Scheme 2. Oxidation of benzylic
hydroxyl group with MnO2 gave aldehyde 9, which was easily
cyclised to benzothiazine 10 in the presence of base. The N-(p-
methoxybenzyl) group of compound 17 was cleaved with
trifluoroacetic acid to give benzothiazine 11 in good yield.
Compound 11 was expected to be a versatile synthon for the
parallel synthesis of N-substituted 2H-1,2-benzothiazine-3-
The in vitro activity data of compounds 18 and 19 are shown in
Table 1. Inhibition of radioligand binding at 10 mM concentration
of substrate (I, %) is given for less potent compounds in cases
when a correct dose-response curve has not been obtained.
Roughly, figures close to 50% of radioligand displacement
correspond to IC50 ꢃ 10 mM in Table 1. Our first synthesized
3,4-dihydrobenzothiazine 18b showed a considerable activity at
10 mM concentration, replacing ca 50% of radioligand. It should
be noted that the reference compound of kynurenic acid with two
halogen atoms in positions 6 and 8 (DCKA), showed higher
activity than its 6-monochloro analogue13. In our case, 6,8-
dichlorobenzothiazine 18b and monochloro derivative 18a dis-
played comparable activities. Therefore, the study was continued
mainly with 6-chlorosubstituted benzothiazines. An argument in
favour of this decision was based on the dramatic solubility

8
Z. Bluke et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Cl
Cl
O
O
Cl
O
O
O
O
PMB
PMB
S
S
S
PMB
b
a
N
N
c
N
Cl
Cl
CO Et
CO Et
2
2
Cl
CO Et
2
OH
O
O
9
10
8c
f
R
R
Cl
O
Cl
Cl
O
O
O
O
e
d
S
S
S
NH
CO Et
N
N
Cl
2
CO H
2
Cl
CO Et
Cl
2
11
13a R= 4-MeO,
13b R= 3-Br,
13c R= 3-Cl
12b R= 3-Br,
12c R= 3-Cl
Scheme 2. Synthesis route to compounds 13a, b, c. Reagents and conditions: (a) MnO2, THF; (b) NaH, THF; (c) TFA; (d) DIEA, DMF, 3-
ClC6H4CH2Cl or 3-BrC6H4CH2Br; (e) LiOH, THF/H2O; (f) NaOH, THF/H2O.
1
1
R
R
1
O
O
R
O
O
O
O
2
3
2
3
R
PMB
R
S
PMB
S
2
3
R
S
PMB
b
c
N
N
a
N
R
CO Et
R
CO Et
2
2
R
CO Et
2
Br
OH
8a, b, c
14a, b, c
15a, b, c
(15a, c)
e
4
4
R
1
O
O
R
R
O
O
O
O
2
2
R
S
R
2
S
R
S
NH
CO Et
e
d
N
N
3
3
R
R
CO Et
3
2
2
R
CO H
2
17
16a, b
18
e
2
3
4
2
3
4
k
l
a
17
17
R = H, R = Cl, R = 3-MeOPh;
R = H, R = Cl, R = 3-MeOPh;
2
3
4
2
3
4
R = H, R = Cl, R = 2-MeOBN;
b
c
d
e
f
g
h
i
R = R = Cl, R = 4-ClPh;
O
O
2
3
4
2
3
4
m R = H, R = Cl, R = 2-FBn;
R = H, R = Cl, R = 3-ClPh;
S
HN
CO H
2
3
4
2
3
4
n
o
p
q
r
R = H, R = Cl, R = 3-MeOPhCO;
R = H, R = Cl, R = 3-BrPh;
2
3
4
2
3
4
R = H, R = Cl, R = 2-MeOPhCO;
R = H, R = Cl, R = Ph;
Cl
2
2
3
4
2
3
4
R = H, R = Cl, R = 3-FPhCO;
R = H, R = Cl, R = 2-Py;
19
2
3
4
2
3
4
R = H, R = Cl, R = 4-CIPhCO;
R = H, R = Cl, R = 3-Py;
2
3
4
2
3
4
a
R = R = Cl, R = 3-CIPh;
R = H, R = Cl, R = 4-(2-MeTh)
8, 14-16
R1 = R2 = H, R3 = Cl;
b R1 = H, R2 = R3 = Cl;
c R1 = R3 = Cl, R2 = H
2
3
4
2
3
4
s
R = R = Cl, R = Bn;
R = H, R = Cl, R = CO tBu;
2
2
3
4
j
R = H, R = Cl, R = Bn;
Scheme 3. Synthesis of target compounds 18 and 19. Reagents and conditions: (a) PBr3, Et2O; (b) NaH, THF; (c) TFA, CH2Cl2; (d) R4CH2Hal, NaH,
DMF; (e) LiOH.H2O or NaOH, THF/H2O.
diminishing effect after the introduction of the second halogen in electronic properties of the substituent. Replacement of N-benzyl
position 8. Surprisingly, 6,7-dichlorobenzothiazines were better with N-phenethyl group resulted in lowered Gly B activity (18i,
soluble, allowing the screening to be performed at concentration R4 ¼ 3-MeOPh; Ki ¼ 2.5 mM and 18j, R4 ¼ 3-MeOBn; displacing
of at least 10 mM. We also tested 6,7-dichloro-1,2-benzothiazine- only ꢃ40% of radioligand at 10 mM). However, in the case of
3-carboxylic acids 18h and 18m and both showed significantly unsubstituted N-benzyl and N-phenethyl derivatives (18g and 18i,
lower activity, displacing only 45% and 20% of radioligand, respectively), this effect is not marked because of the reduced
respectively at 10 mM concentration. It is important to note that activity of both compounds. Actually, compound 18i is more
even 2-unsubstituted benzothiazine 19 displays moderate affinity active than 18g (62% and 41% displacement, respectively). The
for Gly B with Ki ꢃ 10 mM. N-benzyl derivatives displayed higher group of acetophenones (18n–18q) possessing additional hydro-
affinity if the substituents in the phenyl ring were in the meta gen bond acceptor displayed significantly higher activity than
position (MeO, 18c; Cl, 18e and Br, 18f), regardless of the N-phenethyl-benzothiazines. It is possible that the oxo group is

DOI: 10.3109/14756366.2015.1057722
Synthesis of 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides
9
Table 1. Activity of compounds 18 and 19 in binding experiments.
1
4
R
R
O
O
2
3
R
S
N
CO H
R
2
18
Nr.
R1
R2
R3
R4
I (%)*
IC50 (mM)
Ki (mM)y
18a
18b
18c
18d
18e
18f
18g
18h
18i
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
4-MeOPh
4-MeOPh
3-MeOPh
4-ClPh
3-ClPh
3-BrPh
Ph
3-ClPh
Bn
3-MeOBn
2-MeOBn
2-FBn
46
48
3.6
8.9
2.5
2.0
2.5
6.2
1.8
1.4
59
55
38
61
66
40
80
18j
18k
18l
18m
18n
18o
18p
18q
18r
18s
18t
18u
19
Bn
3-MeOPhCO
2-MeOPhCO
3-FPhCO
4-ClPhCO
2-Py
1.5
2.1
7.2
4.5
1.1
1.7
5.0
3.2
80
68
88
82
3-Py
4-(2-MeTh)z
CO2t-Bu
–ô
13.2
9.2
*Remaining radioactivity of [3H]MDL-105,519 at 10 mM concentration of substrate.
yAffinity for Gly binding site; displacement of [3H]MDL-105,519 from rat cortical membrane preparations.
z2-Methyl-1,3-thiazol-4-yl.
ôUnsubstituted at N(2).
involved in additional hydrogen bonding with the receptor. In this previously unexplored approaches for designing new ligands of
series of compounds the highest Gly B affinity was observed for the NMDA receptor.
compounds with methoxy group in meta and ortho positions of
the N-benzyl group (18n and 18o, Ki ¼ 1.1 mM and Ki ¼ 1.7 mM,
Acknowledgements
respectively). Considering the above-mentioned higher activity of
the phenone group containing benzothiazines, one could assume
that N-substitution with heteroaryl group, which may act as a
hydrogen bond acceptor, should also result in potentially active
compounds.
Nonetheless, N-heteroarylmethyl benzothiazines 18r, 18s and
18t were less active. Low activity of 2-pyridinyl derivative 18r
may point to different hydrogen bonding vectors in comparison to
the phenone group, if such an interaction occurs. To investigate if
there is any functional importance of phenyl group, ester 18u was
screened. Compound 18u was inactive, even though it possessed
an oxygen functionality at the same distance from the benzothia-
zine core as corresponding acetophenone 18o.
The authors thank Tanja Bauer and Sabine Denk for their expert technical
assistance in performing the in vitro experiments.
Declaration of interest
The authors declare no conflicts of interest.
This work was financially supported by Merz Pharmaceuticals GmbH.
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Supplementary material available online
Supplementary Information