Synthesis of GluN2A-selective NMDA receptor antagonists with an electron-rich aromatic B-ring

Article abstract of DOI:10.1016/j.ejmech.2020.112939

Glutamatergic N-Methyl-D-aspartate (NMDA) receptors are heterotetrameric ion channels that can be comprised of different subunits. GluN2A subunit-containing NMDA receptors are associated with diseases like anxiety, depression, and schizophrenia. However,

Full text of DOI:10.1016/j.ejmech.2020.112939

Journal Pre-proof
Synthesis of GluN2A-selective NMDA receptor antagonists with an electron-rich
aromatic B-ring
Remya Rajan, Dirk Schepmann, Julian A. Schreiber, Guiscard Seebohm, Bernhard
Wünsch
PII:
S0223-5234(20)30911-9
DOI:
https://doi.org/10.1016/j.ejmech.2020.112939
EJMECH 112939
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 August 2020
Revised Date: 9 October 2020
Accepted Date: 10 October 2020
Please cite this article as: R. Rajan, D. Schepmann, J.A. Schreiber, G. Seebohm, B. Wünsch, Synthesis
of GluN2A-selective NMDA receptor antagonists with an electron-rich aromatic B-ring, European Journal
of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112939.
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1
Synthesis of GluN2A-selective NMDA receptor antagonists with an electron-
rich aromatic B-ring
1,2
1
1,3
3
Remya Rajan,
Dirk Schepmann, Julian A. Schreiber,
Guiscard Seebohm,
1,2
Bernhard Wünsch*
1
Institut für Pharmazeutische und Medizinische Chemie der Westfälischen
Wilhelms-Universität Münster, Corrensstraße 48, D-48149 Münster, Germany.
Tel.: +49-251-8333311; Fax: +49-251-8332144; E-mail: wuensch@uni-muenster.de
2
Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM), Westfälische Wilhelms-
Universität Münster, D-48149 Münster, Germany
3
Cellular Electrophysiology and Molecular Biology, Institute for Genetics of Heart
Diseases (IfGH), Department of Cardiovascular Medicine, University Hospital
Münster, Robert-Koch-Str. 45, D-48149 Münster, Germany
*
Corresponding author:
Prof. Dr. Bernhard Wünsch, Institute of Pharmaceutical and Medicinal Chemistry,
University of Münster, Corrensstr. 48, 48149 Münster, Germany, Tel.: +49-251-83-
3
3311; Fax: +49-251-83-32144; Email: wuensch@uni-muenster.de
Abstract
Glutamatergic N-Methyl-D-aspartate (NMDA) receptors are heterotetrameric ion
channels that can be comprised of different subunits. GluN2A subunit-containing
NMDA receptors are associated with diseases like anxiety, depression, and
schizophrenia. However, the exact contribution of these NMDA receptor subtypes is

2
still unclear. To understand better the role of the GluN2A-containing receptors, novel
ligands were designed. In co-crystallization with the isolated binding site, TCN-201
(
1) and analogs adopt a U-shape conformation with parallel orientation of rings A
and B. In order to increase the π/π-interactions between these rings, ring B of TCN-
01 was replaced bioisosterically by different electron-rich thiazole, oxazole, and
2
isoxazole heterocycles. The inhibitory activity was measured by two-electrode
voltage clamp experiments with Xenopus laevis oocytes expressing GluN2A-
containing NMDA receptors. It was found that 21c, 31a, 37a, and 37b were able to
inhibit the ion channel. The isoxazole derivative 37b was the most potent negative
allosteric modulator displaying 40 % of the TCN-201 activity at a concentration of 10
µM.
Keywords
NMDA receptor; GluN2A selective antagonists; negative allosteric modulator; TCN-
2
01; bioisosteric replacement; electron rich heterocycles; two-electrode voltage
clamp; biological evaluation; electrophysiology.
1
. Introduction
The N-methyl-D-aspartate (NMDA) receptor belongs to the group of ionotropic
1
glutamate receptors and is mainly expressed in the central nervous system. This
ligand gated ion channel is one of the most intensively studied topics in medical
research due to its complexity, its diversity and its participation in a variety of
2
physiological processes and neurological disorders. The name has been derived
from N-methyl-D-aspartate (NMDA), which binds with high affinity and selectivity to
3
+
+
it. In addition to Na and K ions, the NMDA receptor is able to conduct high

3
2+
2+
amounts of Ca ions leading to elevated intracellular Ca concentrations that are
4
important for synaptic plasticity. Highly enhanced NMDA receptor activity can lead
2+
to Ca overload and subsequent excitotoxicity. Consequently, overactivation of
NMDA receptors is associated with neurodegenerative disorders including
5,6
Alzheimer’s, Huntington’s, and Parkinson’s disease.
Seven different subunits form different heterotetrameric NMDA receptors: GluN1,
GluN2A-D, and GluN3A-B subunits. A functional NMDA receptor is composed of two
7,8
GluN1 and typically two GluN2 subunits. While different genes encode for different
GluN2 and GluN3 subunits, only one gene encodes for GluN1, which exists due to
alternate splicing in 8 different variants GluN1a-h. Even though different genes
9,10
encode the subunits, they share a common structure.
Each subunit comprises of
an extracellular amino-terminal domain (ATD), the ligand-binding domain (LBD), a
transmembrane domain (TMD) with three transmembrane helices and an
intracellular C-terminal domain (CTD). The physiological gating properties of the
11
NMDA receptor depend manly on the receptor forming GluN2 subunit.
A particular feature of the NMDA receptor is the large number of binding sites for
endogenous molecules as well as for drugs. For activation, NMDA receptors require
the simultaneous binding of the two endogenous agonists (S)-glutamate and glycine
together. A slight depolarization of the plasmic membrane is required for the relief of
2+
the pore blocking Mg ion to allow for ion flux through the activated receptor. The
binding sites for the two agonists are located at the channel forming subunits GluN1
(glycine) and GluN2 ((S)-glutamate). Channel blockers such as phencyclidine (PCP
2+
12,13
binding site), Mg ions and ketamine are located within the channel pore.

4
Furthermore, several allosteric binding sites exist at the amino terminal domain
+
2+
14
including binding sites for H , spermidine and Zn ions.
GluN2A subunit-containing NMDA receptors are highly expressed in the
hippocampus and cerebral cortex and moderately expressed in the midbrain,
cerebellum, striatum, and brainstem. Beside the mentioned pathophysiological
processes, GluN2A-containing NMDA receptors are also involved in diseases like
epilepsy, cerebral ischemia and schizophrenia. However, the GluN2A subunit is not
only found in the brain but also in peripheral tissues. Makhro et al. reported the
expression of NMDA receptor subunits in rat hearts. The expression of the GluN2A
subunit was restricted to the atria. In the heart, activation of NMDA receptors is
15
associated with tachycardia, sinus arrhythmia and ischemia. Due to the lack of
appropriate ligands and tool compounds, the exact role of the GluN2A subunit
remains to be elucidated.
The first class of GluN2A-selective negative allosteric modulators (NAMs) was
16
reported by Bettini et al. in 2010. TCN-201 (1) represents the prototypical member
of this class. (Figure 1) It inhibits glycine binding to the GluN1 subunit of the
GluN1/GluN2A NMDA receptor (here GluN2A receptor) by binding at the interface
between GluN1 and GluN2A subunits. With an IC50 value of 109 nM TCN-201
inhibits the ion flux in HEK cells. TCN-201 does not inhibit GluN2B and GluN2D
receptors. However, the potency of TCN-201 to inhibit GluN2A receptors decreases
with increasing concentrations of extracellular glycine. At a concentration of 300 µM
of glycine, TCN-201 could no longer inhibit the ion flux. Also, TCN-201 has very low
17-19
19
solubility, which limits its use in biological studies.
In 2016, Volkmann et al.

5
reported two analogs of TCN-201 by replacing the phenyl ring in the middle of TCN-
2
01 (ring B) by a pyrazine ring resulting in MPX-004 (2) and MPX-007 (3). (Figure 1)
Both pyrazine derivatives 2 and 3 show higher activity (IC50 = 79 nM and 27 nM),
better solubility, and less glycine dependence when compared to TCN-201.
Systematic modification of the benzenesulfonamide part of 1 was reported recently.
It was shown that a halogen atom in 3-position of the benzenesulfonamide part
resulted in high potency at GluN2A receptors. The 3-bromo derivative 4 was 2.5-fold
20
more potent than TCN-201 (1). Modifications of the terminal benzoyl moiety of 1
21
led to the thiophen-2-ylcarbonyl derivative 5 with also 2.5-fold increased activity.
Figure 1: GluN2A selective NMDA ligands

6
The X-ray crystal structure of the GluN1-GluN2A ligand binding domain together with
the lead compound 1 shows a hairpin-like or U-shaped conformation of 1 within the
binding pocket of the receptor. This U-shaped conformation of 1 is favored by π
stacking of the benzene rings A and B. However, both benzene rings are electron
deficient aromatic rings. Therefore, the idea came up to replace the benzene ring in
the middle (ring B) by an electron-rich five membered heterocycle, which should
increase the π stacking with ring A and at the end the interactions with the GluN2A
22
receptor. Herein, we wish to report the synthesis and pharmacological evaluation of
novel analogs of TCN-201 (1) containing electron rich five-membered heterocycles
thiazole, oxazole and isoxazole instead of benzene ring B, which should help to
O
O
Cl
S
N
O
A
H
B
H
N
F
N
H
C
O
1
R
S
O
R
N
n
N
O
H
R
O
O
H
N
N
HN R’
CH3
H
O
N
HN R’
N
HN R’
CH3
6
8
7
better understand the role of ring B in NAM activity. (Figure 2)
Figure 2: GluN2A selective NMDA receptor antagonists designed by replacement of
ring B of the lead compound 1 by thiazole (6), oxazole (7), and isoxazole (8) rings.

7
2
. Synthesis
Scheme 1: Synthesis of aminothiazole-based TCN-201 analogs 15a-d. Reagents
and reaction conditions: (a) 1. NBS, THF : H O 2 : 1, 0 °C to rt, 3 h; 2. thiourea, 80
2
°
C, 2 h, 84 %. (b) (Boc) O, Et N, DMAP, THF, rt, 16 h, 87 %. (c) 2 M KOH, CH OH :
2 3 3
THF 1 : 1, rt, 16 h, 75 %. (d) BnNH Cl, COMU, DIPEA, THF; rt, 16 h, 77 %. (e)
3
1
F CCO H, CH Cl , rt, 1 h, 85 %. (f) R SO Cl, NaH, THF, 0 °C to rt, overnight, 15a:
3
2
2
2
2
7
5 %; 15b: 67 %; 15c: 76 %; 15d: 84 %.
At first, TCN-201 analogs 15a-d with a thiazole moiety as ring B were envisaged.
The synthesis started with a two-step one-pot preparation of thiazolecarboxylate
23
1
0. At first ethyl acetoacetate (9) was converted with NBS into the corresponding
2
-bromo derivative, which reacted with thiourea affording the thiazolecarboxylate 10
in 84 % yield. After introduction of the Boc-protective group, ester 11 was saponified
by KOH to provide the acid 12. Acid 12 was coupled with benzylamine in the
presence of COMU leading to the N-benzylcarboxamide 13 in 85 % yield. Finally, the
24
Boc-protective group was removed by F CCO H to give the primary amine 14,
3
2

8
which allowed the introduction of various sulfonyl moieties at the very end of the
synthesis providing diverse sulfonamides 15a-d. (Scheme 1)
Scheme 2: Synthesis of (aminomethyl)thiazole-based TCN-201 analogs 21 and 22.
1
Reagents and reaction conditions: (a) R SO Cl, Et N, THF : H O 5 : 1, 0 °C to rt, 3 h,
2
3
2
o
1
1
7a: 68 %; 17b: 95 %; 17c: 67 %. (b) Lawesson’s reagent, dioxane, 70 C, 27 h,
8a: 95 %; 18b: 76 %; 18c: 86 %. (c) 1. NBS, THF : H O 2 : 1, 0 °C to rt, 3 h; 2. 18a-
2
c, 80 °C, 16 h, 19a: 42 %; 19b: 56 %; 19c: 71 %. (d) 2 M KOH, CH OH : THF 1 : 1,
3
rt, 16 h, 20a: 81 %; 20b: 75 %; 20c: 79 %. (e) PhC(=O)NHNH , COMU, DIPEA, THF;
2
rt, 16 h, 21a: 78 %; 21b: 69 %; 21c: 66 %. (f) Thiophen-2-C(=O)NHNH , COMU,
2
DIPEA, THF; rt, 16 h, 22a: 72 %; 22b: 66 %; 22c: 73 %.
The first step in the synthesis of (aminomethyl)thiazole analogs 21 and 22 was the
reaction of glycinamide (16) with different sulfonyl chlorides to obtain sulfonylated
glycinamides 17a-c, which were then converted into thioglycinamides 18a-c upon

9
treatment with Lawesson’s reagent. (Scheme 2) The one-pot reaction of ethyl
acetoacetate (9), N-bromosuccinimide, and the sulfonylated thiogycinamides 18a-c
in water/THF led to thiazole-5-carboxylates 19a-c. Saponification of ethyl esters 19a-
c with KOH provided acids 20a-c, which were then coupled with benzoylhydrazine
and thiophen-2-ylcarbonylhydrazine to afford the diacylhydrazine derivatives 21a-c
and 22a-c. (Scheme 2)
O
O
O
CbzHN
O
O
Cl
O
(
a)
(b)
(d)
RHN
OEt
OEt
CH3
OEt
CH3
O
N
O
CH3
25: R = Cbz
24
2
3
(
c)
26: R = H
O
O
O
O
O
O O
O
O
O
O
O
(
e)
(h)
S
S
N
S
R1
N
H
OEt
R1
OH
R1
N
NHBn
N
H
N
H
N
CH3
CH3
(g)
CH3
1a
2
7a,b
28a,b
3
(
f)
O
H
O
O O
O O
O
O
H
S
R1S N
N
N
R1S N
N
N
H
N
H
H
N
H
CH3
9a,b
O
CH3
O
2
30a,b
1
2
7-31a: R = 3-Br-C H
6
4
1
2
7-30b: R = 3 -Cl-4-F-C H
6
3
Scheme 3: Synthesis of (aminomethyl)oxazole-based TCN-201 analogs 29-31.
Reagents
and
reaction
conditions:
(a)
N-[(benzyloxy)carbonyl]glycine
(
CbzNHCH CO H), Et N, ethyl acetate, 0 °C to 70 °C, 16 h, 60 %. (b) CH CO NH ,
2
2
3
3
2
4
CH CO H, 135 °C, 16 h, 95 %. (c) H , Pd/C, THF : CH OH 1 : 1, rt, 10 h, 78 %. (d)
3
2
2
3
1
R SO Cl, Et N, THF, 0 °C to rt, 16 h; 27a: 57 %; 27b: 47 %. (e) 2 M KOH, THF :
2
3
CH OH 1 : 1, rt, 16 h; 28a: 86 %; 28b: 72 %. (f) PhC(=O)NHNH , COMU, DIPEA,
3
2
THF, rt, 16 h; 29a: 76 %; 29b: 71 %. (g) Thiophen-2-C(=O)NHNH , COMU, DIPEA,
2
THF; rt, 16 h, 30a: 70 %; 30b: 75 %. (h) BnNH , COMU, DIPEA, THF; rt, 16 h, 71 %.
2

1
0
In the next series of GluN2A ligands, the S-atom of the thiazoles 21 and 22 was
replaced by an O-atom (29-31). For the synthesis of oxazole derivatives 29-31 a
nucleophilic substitution of α-chloro-β-ketoester 23 with Cbz-protected glycinate was
performed providing the α-substituted β-ketoester 24, which reacted with ammonium
25
acetate to form oxazolecarboxylate 25. The primary amine 26 was obtained by
hydrogenolytic removal of the Cbz-protective group of 25 using Pd/C as catalyst.
Reaction of the primary amine 26 with two different sulfonyl chlorides provided
sulfonamides 27a and 27b. After hydrolysis of the esters 27a and 27b with KOH, the
free acids 28a and 28b were coupled with benzoylhydrazine, (thiophen-2-
ylcarbonyl)hydrazine and benzylamine to achieve the oxazole-based test
compounds 29-31. (Scheme 3)
Scheme 4: Synthesis of (aminomethyl)isoxazole-based TCN-201 analogs. Reagents
and reaction conditions: (a) Et N, THF : Et O 1 : 3, rt, 16 h, 69 %. (b) 2 M KOH,
3
2
CH OH : THF 1 : 1, rt, 16 h, 86 %. (c) benzoylhydrazine, COMU, DIPEA, THF; rt, 16
3
1
h, 75 %. (d) F CCO H, CH Cl , rt, 1h, 83 %. (e) R SO Cl, Et N, THF, 0 °C to rt, 16 h;
3
2
2
2
2
3
3
7a: 66 %; 37b: 67 %; 37c: 64 %.

1
1
The regioisomeric bioisosteric isoxazole derivatives 37 were prepared starting with a
,3-dipolar cycloaddition of ethyl 2-chloro-2-(hydroxyimino)acetate (32) with N-Boc
1
26
propargylamine. The resulting ethyl isoxazolecarboxylate 33 was hydrolyzed to
yield the acid 34, which was coupled with benzoylhydrazine to obtain die
diacylhydrazine derivative 35. Subsequent removal the Boc protective group with
CF CO H provided the primary amine 36, which was sulfonylated with three different
3
2
sulfonyl chlorides to yield the isoxazole-based TCN-201 analogous test compounds
7a-c. (Scheme 4)
3
Synthesis of (aminomethyl)thiazole and -oxazole TCN-201 analogs 21,22 and 29,30
involved the introduction of the acylhydrazine moiety as last step allowing facile
diversification of rind C. The last step in the synthesis of aminothiazoles 15 and
(aminomethyl)isoxazoles 37 was the sulfonylation of a primary amine allowing the
facile diversification of the arylsulfonyl part (ring A) of TCN-201 analogs. All the acid-
amine coupling reactions were performed in the presence of the coupling reagent
COMU giving high yields, respectively.
3
. Biological evaluation
The inhibitory activity of the synthesized test compounds was assessed
electrophysiologically by two-electrode voltage clamp (TEVC) technique in Xenopus
laevis oocytes expressing functional GluN1a-GluN2A NMDA receptors. In each
experiment, 10 µM (S)-glutamate and 10 µM glycine were used to activate the
NMDA receptor. The resulting cationic current was recorded and the changes of the
current upon addition of the test compounds (c = 10 µM) were measured. The
inhibition caused by the test compound (c = 10 µM) was normalized to the inhibition

1
2
generated by TCN-201 (1, c = 10 µM) and is summarized in Table 1 for each
compound.
Table 1: Normalized GluN2A receptor inhibition Inorm of the synthesized test
compounds.
normalized Inhibition Inorm
1
2
compd
(TCN-201)
R
R
±
SEM (%)
1
1
1
1
1
2
2
2
2
2
2
2
2
3
-
-
100 ± 0.06 %
8 ± 1.2 %
7 ± 0.2 %
6 ± 0.9 %
7 ± 1.3 %
7 ± 1.1 %
3 ± 0.7 %
37 ± 5 %
5a
5b
5c
5d
1a
1b
1c
2a
2b
2c
9a
9b
0a
2-NO -C H
CH C H
2 6
2
6
4
5
5
5
5
4-NO -C H
CH C H
2 6
2
6
4
4 CN-C H4
CH C H
2 6
6
CH -C H
CH C H
2 6
2
6
5
3-Br-C H4
NHC(=O)C H
6
6
5
5
5
3-Cl-4-F-C H4
NHC(=O)C H
6
6
4-CH -C H
NHC(=O)C H
3
6
4
6
#
#
#
3-Br-C H4
NHC(=O)thioph
NHC(=O)thioph
NHC(=O)thioph
NHC(=O)C H
2 ± 0.7 %
2 ± 0.9 %
2 ± 1.2 %
-5 ± 3.9 %
13 ± 1.3 %
2 ± 0.8 %
6
3-Cl-4-F-C H4
6
4-CH -C H
4
3
6
3-Br-C H4
6
6
5
5
3-Cl-4-F-C H4
NHC(=O)C H
6
6
#
3-Br-C H4
NHC(=O)thioph
6

1
3
#
3
3
3
3
3
0b
1a
7a
7b
3-Cl-4-F-C H4
NHC(=O)thioph
CH C H
14 ± 1.5 %
34 ± 1.9 %
31 ± 1.4 %
40 ± 1.9 %
12 ± 0.8 %
6
3-Br-C H4
6
2
6
5
3-Br-C H4
NHC(=O)C H
6
6
5
5
5
3-Cl-4-F-C H4
NHC(=O)C H
6
6
7c
4-CH -C H
NHC(=O)C H
3
6
4
6
#
thioph = thiophen-2-yl
Relative percent inhibition of ion current measured by TEVC at GluN1a-GluN2A-
expressing oocytes caused by test compounds (c = 10 µM) after activation by 10 µM
(S)-glutamate and 10 µM glycine. Inhibition was normalized to the inhibition of 10 µM
TCN-201 (1) (n = 3 in total) measured in the same experiment. Each compound was
measured with three independent oocytes (n = 3).
The aminothiazole-based TCN-201 analogs 15a-d displayed normalized inhibition of
the GluN2A receptor ion channel lower than 10 % of the lead compound TCN-201
(1). Among the (aminomethyl)thiazole TCN-201 analogs, the tosyl derivative 21c
showed the highest normalized inhibition of 38 % (p < 0.05). All the other thiazoles
revealed less than 7 % GluN2A receptor inhibition of TCN-201.
The aminomethyl oxazole and isoxazole derivatives 31a (34 %), 37a (29 %) and 37b
(40 %) showed the highest normalized GluN2A inhibition. The presence of a methyl
group at the 4-position of the phenylsulfonyl moiety led to a considerable decrease in
the normalized inhibition of isoxazole 37c (11 %, p < 0.05) when compared with
halogenated analogs 37a and 37b. This data synchronizes with the results published
20
by Müller et al. It can be concluded that halogen atoms at ring A increase the
GluN2A inhibitory activity.

1
4
In addition to the inhibition of GluN2A receptor associated ion channel, the affinity of
the thiazoles 15, 21, and 22, the oxazoles 29-31 and isoxazoles 37 towards the
30,31
32-34
ifenprodil binding site of GluN2B NMDA receptors,
σ und σ receptors
was
1
2
recorded in radioligand receptor binding assay. However, up to a concentration of 10
µM, the novel ligands did not interact with the ifenprodil binding site, σ und σ
1
2
receptors. The low affinity towards the ifenprodil binding site and both σ receptor
subtypes indicates high selectivity for the GluN2A receptor over these related
receptors.
4
. Conclusion
To analyze the effect of the bioisosteric replacement of ring B of the GluN2A inhibitor
TCN-201 (1) by electron-rich aromatic rings on the negative allosteric modulation of
GluN2A subunit-containing NMDA receptors, four different classes of compounds
were designed and synthesized. The GluN2A channel blocking activity of the
synthesized compounds was determined electrophysiologically by two-electrode
voltage clamp technique. The oxazole and isoxazole derivatives 31a, 37a and 37b
have GluN2A NMDA receptor inhibitory activity in the range of 29-40 % of the lead
compound TCN-201. However, none of the compounds reached the activity of TCN-
2
01. In particular, the most potent ligands display high selectivity for GluN2A over
GluN2B, σ und σ receptors.
1
2
5
5
. Experimental part
.1. Chemistry, general methods
Oxygen and moisture sensitive reactions were carried out under nitrogen, dried with
silica gel with moisture indicator (orange gel, VWR, Darmstadt, Germany) and in dry

1
5
glassware (Schlenk flask or Schlenk tube). Temperature was controlled with dry
ice/acetone (-78 °C), ice/water (0 °C), Cryostat (Julabo TC100E-F, Seelbach,
Germany), magnetic stirrer MR 3001 K (Heidolph, Schwalbach, Germany) or RCT
CL (IKA, Staufen, Germany), together with temperature controller EKT HeiCon
(Heidolph) or VT-5 (VWR) and PEG or silicone bath. All solvents were of analytical
or technical grade quality. Demineralized water was used. CH Cl was distilled from
2
2
CaH ; THF was distilled from sodium/benzophenone; MeOH was distilled from
2
magnesium methanolate. Thin layer chromatography (tlc): tlc silica gel 60 F254 on
aluminum sheets (VWR). Flash chromatography (fc): Silica gel 60, 40–63 µm
(VWR); parentheses include: diameter of the column (∅), length of the stationary
TM
phase (l), fraction size (v) and eluent. Automated flash chromatography: Isolera
®
Spektra One (Biotage ); parentheses include: cartridge size, flow rate, eluent,
fractions size was always 20 mL. Melting point: Melting point system MP50 (Mettler
Toledo, Gießen, Germany), open capillary, uncorrected. MS: MicroTOFQII mass
spectrometer (Bruker Daltonics, Bremen, Germany); deviations of the found exact
masses from the calculated exact masses were 5 ppm or less; the data were
®
analyzed with DataAnalysis (Bruker Daltonics). NMR: NMR spectra were recorded
in deuterated solvents on Agilent DD2 400 MHz and 600 MHz spectrometers
(Agilent, Santa Clara CA, USA); chemical shifts (δ) are reported in parts per million
(ppm) against the reference substance tetramethylsilane and calculated using the
solvent residual peak of the undeuterated solvent; coupling constants are given with
1
13
0
.5 Hz resolution; assignment of H and C NMR signals was supported by 2-D
®
NMR techniques where necessary.IR: FT/IR IR Affinity -1 spectrometer (Shimadzu,
Düsseldorf, Germany) using ATR technique.

1
6
5
.2. HPLC method for the determination of the purity
Equipment 1: Pump: L-7100, degasser: L-7614, autosampler: L-7200, UV detector:
L-7400, interface: D-7000, data transfer: D-line, data acquisition: HSM-Software (all
from Merck Hitachi, Darmstadt, Germany); Equipment 2: Pump: LPG-3400SD,
degasser: DG-1210, autosampler: ACC-3000T, UV-detector: VWD-3400RS,
interface: DIONEX UltiMate 3000, data acquisition: Chromeleon 7 (equipment and
software from Thermo Fisher Scientific, Lauenstadt, Germany); column:
®
®
LiChrospher 60 RP-select B (5 µm), LiChroCART 250-4 mm cartridge; flow rate:
.0 mL/min; injection volume: 5.0 µL; detection at λ = 210 nm; solvents: A:
demineralized water with 0.05 % (V/V) trifluoroacetic acid, B: CH CN with 0.05 %
1
3
(
V/V) trifluoroacetic acid; gradient elution (% A): 0 - 4 min: 90 %; 4 - 29 min: gradient
from 90 % to 0 %; 29 - 31 min: 0 %; 31 - 31.5min: gradient from 0 % to 90 %; 31.5 -
0 min: 90 %. Unless otherwise mentioned, the purity of all test compounds is
greater than 95 %.
4
5
.3. Synthetic procedures
23, 24
The synthesis of the intermediates 10 – 14 is reported in references.
The
synthesis of the intermediates 24 and 25 is reported in reference. The 1,3-dipolar
25
26
cycloaddition to obtain 33 is described in referende. Without any information about
synthesis and properties compounds 17a and 18a can be found in databases
https://pubchem.ncbi.nlm.nih.gov/compound/2556165
and
https://pubchem.ncbi.nlm.nih.gov/compound/61127496, respectively. The synthesis
32
of 17c has already been mentioned in a patent, but herein the procedure was
33
optimized. For the synthesis of 18c, Lawesson’s reagent was used instead of H S,
2
herein.

1
7
5
.3.1. N-Benzyl-4-methyl-2-[(2-nitrophenyl)sulfonamido]thiazole-5-carboxamide
15a)
Primary amine 14
(
23,24
(0.3 g, 1.2 mmol, 1.0 equiv.) was dissolved in THF (15 mL)
o
and the solution was cooled to 0 C. NaH (87 mg, 3.6 mmol, 3.0 equiv.) and a
solution of 2-nitrobenzenesulfonyl chloride (0.27 g, 1.2 mmol, 1.0 equiv.) in THF (5
mL) were added. The reaction mixture was stirred overnight at room temperature.
After completion of the transformation, the solvent was evaporated in vacuo and
ethyl acetate (30 mL) and H O (30 mL) were added. The organic layer was
2
separated, and the aqueous layer was extracted with ethyl acetate (3 x 30 mL). The
organic layer was dried (Na SO ) and concentrated in vacuo. The residue was
2
4
purified by FCC (Ø = 2 cm, l = 20 cm, V = 45 mL, CH Cl :CH OH 95:5). R = 0.45
2
2
3
f
o
(
CH Cl :CH OH 95:5). Pale yellow solid, mp 192 C, yield 0.39 g (75 %). Purity
2 2 3
1
(
HPLC): 96.9 % (t = 18.7 min). C H N O S (432.5 g/mol). H NMR (400 MHz,
R
18 16
4
5 2
CD OD): δ (ppm) = 2.42 (s, 3H, CH -B), 4.45 (s, 2H, CONHCH ), 7.20 – 7.25 (m,
3
3
2
1
H, 4-H ), 7.28 – 7.32 (m, 4H, 2-H , 3-H , 5-H , 6-H ), 7.70 – 7.75 (m, 3H, 4-H , 5-
C C C C C A
13
H , 6-H ), 8.12 (dd, J = 7.3 / 2.3 Hz, 1H, 3-H ). C NMR (101 MHz, CD OD): δ
A
A
A
3
(
ppm) = 11.9 (CH -B), 43.0 (CONHCH ), 112.5 (C-5 ), 123.9 (C-5 ), 126.8 (C-4 ),
3 2 B A C
1
27.1 (2C, C-2 , C-6 ), 128.1 (2C, C-3 , C-5 ), 129.2 (C-3 ), 131.5 (C-4 ), 133.2
C C C C A A
(
2C, C-1 , C-6 ), 134.4 (C-2 ), 138.4 (C-1 ), 139.1 (C-4 ), 161.4 (C-2 ), 167.1
A
A
A
C
B
B
(CONHCH2).
5
.3.2. N-Benzyl-4-methyl-2-[(4-nitrophenyl)sulfonamido]thiazole-5-carboxamide
(15b)

1
8
23,24
Primary amine 14
(0.3 g, 1.2 mmol, 1.0 equiv.) was dissolved in THF (15 mL)
o
and the solution was cooled to 0 C. NaH (87 mg, 3.6 mmol, 3.0 equiv.) and a
solution of 4-nitrobenzenesulfonyl chloride (0.27 g, 1.2 mmol, 1.0 equiv.) in THF (5
mL) were added. The reaction mixture was stirred overnight at room temperature.
After completion of the transformation, the solvent was evaporated in vacuo and
ethyl acetate (30 mL) and H O (30 mL) were added. The organic layer was
2
separated and the aqueous layer was extracted with ethyl acetate (3 x 30 mL). The
organic layer was dried (Na SO ) and concentrated in vacuo. The residue was
2
4
purified by FCC (Ø = 2 cm, l = 20 cm, V = 40 mL, CH Cl :CH OH 95:5). R = 0.45
2
2
3
f
o
(
CH Cl :CH OH 95:5). Light yellow solid, mp 197 C, yield 0.35 g (67 %).
2 2 3
1
C H N O S (432.5 g/mol). H NMR (600 MHz, DMSO-D ): δ (ppm) = 2.34 (s, 3H,
18
16
4
5
2
6
CH -B), 4.34 (d, J = 6.0 Hz, 2H, CONHCH ), 7.21 (t, J = 7.2 Hz, 1H, 4-H ), 7.25 (d, J
3
2
C
=
2
6.6 Hz, 2H, 2-H , 6-H ), 7.30 (t, J = 7.3 Hz, 2H, 3-H , 5-H ), 8.03 (d, J = 8.9 Hz,
C C C C
H, 2-H , 6-H ), 8.34 (d, J = 8.9 Hz, 2H, 3-H , 5-H ), 8.64 (t, J = 5.9 Hz, 1H,
A
A
A
A
13
CONHCH ), 13.18 (s, 1H, SO NHthiaz ). C NMR (151 MHz, DMSO-D ): δ (ppm) =
2
2
B
6
1
1
1
6.3 (CH -B), 45.8 (CONHCH ), 115.3 (C-5 ) 127.7 (2C, C-3 , C-5 ), 129.9 (C-4 ),
3 2 B A A C
30.3 (2C, C-2 , C-6 ), 130.4 (2C, C-2 , C-6 ), 131.4 (2C, C-3 , C-5 ), 142.2 (C-1 ),
A
A
C
C
C
C
C
42.3 (C-4 ), 150.4 (C-1 ), 152.6 (C-4 ), 163.1 (C-2 ), 169.5 (CONHCH ).
B
A
A
B
2
5
.3.3.
N-Benzyl-2-[(4-cyanophenyl)sulfonamido]-4-methylthiazole-5-
carboxamide (15c)
23,24
Primary amine 14
(0.1 g, 0.4 mmol, 1.0 equiv.) was dissolved in THF (10 mL)
o
and the solution was cooled to 0 C. NaH (29 mg, 1.2 mmol, 3.0 equiv.) and a
solution of 4-cyanobenzenesulfonyl chloride (80 mg, 0.4 mmol, 1.0 equiv.) in THF (5
mL) were added. The reaction mixture was stirred overnight at room temperature.

1
9
After completion of the transformation, the solvent was evaporated in vacuo and
ethyl acetate (30 mL) and H O (30 mL) were added. The organic layer was
2
separated, and the aqueous layer was extracted with ethyl acetate (3 x 30 mL). The
organic layer was dried (Na SO ) and concentrated in vacuo. The residue was
2
4
purified by FCC (Ø = 1 cm, l = 15 cm, V = 20 mL, CH Cl :CH OH 95:5). R = 0.48
2
2
3
f
o
(
CH Cl :CH OH 95:5). Colorless solid, mp 200 C, yield 0.13 g (76 %).
2 2 3
1
C H N O S (432.5 g/mol). H NMR (600 MHz, DMSO-D ): δ (ppm) = 2.34 (s, 3H,
19
16
4
3
2
6
CH -B), 4.33 (d, J = 5.9 Hz, 2H, CONHCH ), 7.21 (t, J = 7.2 Hz, 1H, 4-H ), 7.25 (d, J
3
2
C
=
2
8.4 Hz, 2H, 2-H , 6-H ), 7.30 (t, J = 8.1 Hz, 2H, 3-H , 5-H ), 7.94 (d, J = 8.5 Hz,
C C C C
H, 3-H , 5-H ), 8.01 (d, J = 8.6 Hz, 2H, 2-H , 6-H ), 8.63 (t, J = 6.0 Hz, 1H,
A
A
A
A
13
CONHCH ), 13.15 (s, 1H, SO2NHthiaz ). C NMR (151 MHz, DMSO-D ): δ (ppm) =
2
B
6
1
6.3 (CH -B), 45.8 (CONHCH ), 115.2 (C-5 ), 117.8 (C≡N), 120.9 (C-1 ), 129.6 (2C,
3 2 B A
C-3 , C-5 ), 130.0 (C-4 ), 130.4 (2C, C-2 , C-6 ), 131.4 (2C, ,C-3 , C-5 ), 136.5
A
A
C
C
C
C
C
(
2C, C-2 , C-6 ) 142.3 (C-4 , C-1 ), 149.0 (C-4 ), 163.1 (C-2 ), 169.5 (CONHCH ).
A A B C A B 2
5
.3.4. N-Benzyl-4-methyl-2-[(phenylmethyl)sulfonamido]thiazole-5-carboxamide
(15d)
23,24
Primary amine 14
(10 mg, 0.04 mmol, 1.0 equiv.) was dissolved in THF (5 mL)
o
and the solution was cooled to 0 C. NaH (0.2 mg, 0.1 mmol, 3.0 equiv.) and a
solution of phenylmethanesulfonyl chloride (7 mg, 0.4 mmol, 1.0 equiv.) in THF (5
mL) were added. The reaction mixture was stirred overnight at room temperature.
After completion of the transformation, the solvent was evaporated in vacuo and
ethyl acetate (15 mL) and H O (15 mL) were added. The organic layer was
2
separated, and the aqueous layer was extracted with ethyl acetate (3 x 15 mL). The
organic layer was dried (Na SO ) and concentrated in vacuo. The residue was
2
4

2
0
purified by FCC (Ø = 1 cm, l = 10 cm, V = 15 mL, CH Cl :CH OH 95:5). R = 0.45
2
2
3
f
o
(
(
(
CH Cl :CH OH 95:5). Colorless solid, mp 194 C yield 14 mg (84 %). C H N O S
2 2 3 19 19 3 3 2
1
432.5 g/mol). H NMR (600 MHz, DMSO-D ): δ (ppm) = 2.34 (s, 3H, CH -B), 4.30
6
3
d, J = 6.5 Hz, 2H, CONHCH ), 4.31 (s, 2H, CH SO NH), 7.21 (t, J = 7.1 Hz, 1H, 4-
2
2
2
H ), 7.24 (d, J = 7.1 Hz, 2H, 2-H , 6-H ), 7.27 – 7.31 (m, 5H, 3-H , 5-H , 3-H , 4-H ,
C
C
C
C
C
A
A
5
1
-H ), 7.33 (dd, J = 7.9, 1.3 Hz, 2H, 2-H , 6-H ), 8.51 (t, J = 5.9 Hz, 1H, CONHCH ),
A A A 2
13
2.78 (s, 1H, CH SO NH). C NMR (151 MHz, DMSO-D ): δ (ppm) = 16.23 (CH -
2
2
6
3
B), 45.7 (CONHCH ), 61.4 (CH SO NH), 113.9 (C-5 ), 129.9 (C-4 ), 130.3 (2C, C-
2
2
2
B
C
2C, C-6 ), 130.9 (C-4 ), 131.2 (2C, C-3 , C-5 ) 131.4 (2C, C-3 , C-5 ), 133.4 (2C, C-
C
A
C
C
A
A
1A, C-1 ), 134.0 (2C, C-2 , C-6 ), 142.4 (C-4 ), 163.3 (C-2 ), 169.4 (CONHCH ).
C
A
A
B
B
2
5
.3.5. 2-[(3-Bromophenyl)sulfonamido]acetamide (17a)
o
At 0 C, Et N (0.25 mL, 1.8 mmol, 2.0 equiv.) was added to a solution of 16 (0.10 g,
3
0
.9 mmol, 1.0 equiv.) in DMF (25 mL). After 10 min, 3-bromobenzenesulfonyl
chloride (0.13 g, 0.9 mmol, 1.0 equiv.) was added and the reaction mixture was
stirred overnight at rt. After completion of the transformation, the solvent was
evaporated in vacuo and CH Cl (25 mL) and H O (25 mL) were added. The organic
2
2
2
layer was separated, and the aqueous layer was extracted with CH Cl (3 x 25 mL).
2
2
The organic layer was dried (Na SO ) and concentrated in vacuo. The residue was
2
4
purified by FCC (Ø = 2 cm, l = 20 cm, V = 20 mL, CH Cl /CH OH/Et N 95:5:0.1). R =
2
2
3
3
f
o
0
.5 (CH Cl /CH OH 9:1). Colorless solid, mp 186 C, yield 0.18 g (68 %).
2 2 3
1
C H BrN O S (293.1 g/mol). H NMR (400 MHz, DMSO-D ): δ (ppm) = 3.41 (s, 2H,
8
9
2
3
6
SO NHCH ), 7.06 (s, 1H, N-H), 7.28 (s, 1H, N-H), 7.53 (t, J = 7.9 Hz, 1H, 5-Harom),
2
2
7
.77 (ddd, J = 7.9 / 1.8 / 1.0 Hz, 1H, 6-Harom), 7.83 (ddd, J = 8.0 / 2.1 / 1.0 Hz, 1H, 4-
Harom), 7.91 – 7.97 (m, 1H, 2-Harom), 8.02 (s, 1H, SO NHCH ).
2
2

2
1
5
.3.6. 2-[(3-Chloro-4-fluorophenyl)sulfonamido]acetamide (17b)
o
At 0 C, Et N (0.25 mL, 1.8 mmol, 2.0 equiv.) was added to a solution of 16 (0.10 g,
3
0
.9 mmol, 1.0 equiv.) in DMF (25 mL). After 10 min, 3-chloro-4-
fluorobenzenesulfonyl chloride (0.13 g, 0.9 mmol, 1.0 equiv.) was added and the
reaction mixture was stirred overnight at rt. After completion of the transformation,
the solvent was evaporated in vacuo and CH Cl (20 mL) and H O (20 mL) were
2
2
2
added. The organic layer was separated, and the aqueous layer was extracted with
CH Cl (3 x 25 mL). The organic layer was dried (Na SO ) and concentrated in
2
2
2
4
vacuo. The residue was purified by FCC (Ø = 2 cm, l = 15 cm, V = 25 mL,
CH Cl /CH OH/Et N 95:5:0.1). R = 0.45 (CH Cl /CH OH 9:1). Colorless solid, mp
2
2
3
3
f
2
2
3
o
1
1
84 C, yield 0.16 g (95 %). C H ClFN O S (266.7 g/mol). H NMR (400 MHz,
8 8 2 3
DMSO-D ): δ (ppm) = 3.44 (s, 2H, SO NHCH ), 7.06 (s, 1H, N-H), 7.29 (s, 1H, N-H),
6
2
2
7
.62 (t, J = 8.9 Hz, 1H, 5-Harom), 7.79 (ddd, J = 8.7 / 4.5 / 2.3 Hz, 1H, 6-Harom), 7.98
(
dd, J = 6.9 / 2.3 Hz, 1H, 2-Harom), 8.05 (s, 1H, SO NHCH ).
2
2
32
5
.3.7. 2-[(4-Methylphenyl)sulfonamido]acetamide (17c)
A solution of glycinamide 16 (0.10 g, 0.9 mmol, 1.0 equiv.) in THF:H O (5:1, 30 mL)
2
and Et N (0.25 mL, 1.8 mmol, 2.0 equiv.) was cooled to 0 °C. After 10 min, p-TsCl
3
(0.17 g, 0.9 mmol, 1.0 equiv.) was added and the reaction mixture was stirred
overnight at rt. After completion of the transformation, the solvent was evaporated in
vacuo and CH Cl (25 mL) and H O (25 mL) were added. The organic layer was
2
2
2
separated, and the aqueous layer was extracted with CH Cl (3 x 25 mL). The
2
2
organic layer was dried (Na SO ) and concentrated in vacuo. The residue was
2
4
purified by FCC (Ø = 2 cm, l = 10 cm, V = 20 mL, CH Cl /CH OH/Et N 95:5:0.1). R =
2
2
3
3
f

2
2
o
0
.45 (CH Cl /CH OH 9:1). Colorless solid, mp 180 C, yield 0.14 g (67 %).
2 2 3
1
C H N O S (228.3 g/mol). H NMR (400 MHz, DMSO-D ): δ (ppm) = 2.36 (s, 3H,
9
12
2
3
6
CH ), 3.31 (s, 2H, SO NHCH ), 7.07 (s, 1H, N-H), 7.22 (s, 1H, N-H), 7.36 (d, J = 7.7
3
2
2
Hz, 2H, 3-Harom, 5-Harom), 7.67 (d, J = 8.3 Hz, 2H, 2-Harom, 6-Harom), 7.72 (s, 1H,
SO NHCH ).
2
2
5
.3.8. 2-[(3-Bromophenyl)sulfonamido]thioacetamide (18a)
Compound 17a (0.2 g, 0.6 mmol, 1.0 equiv.) and Lawesson’s reagent (0.16 g, 0.3
mmol, 0.6 equiv.) were dissolved in dioxane (20 mL) and the mixture was heated to
o
7
0 C for 27 h. After completion of transformation, the solvent was evaporated in
vacuo. The residue was dissolved in saturated NaHCO solution (20 mL) and the
3
mixture was stirred for 30 min. The precipitated solid was filtered through a sintered
o
funnel, washed with distilled H O and dried. Colorless solid, mp 189 C, yield 0.20 g
2
1
(
95 %). C H BrN O S (309.2 g/mol). H NMR (400 MHz, DMSO-D ): δ (ppm) = 3.70
8
9
2
2
2
6
(
d, J = 6.2 Hz, 2H, SO NHCH ), 7.54 (t, J = 7.9 Hz, 1H, 5-Harom), 7.79 (ddd, J = 7.9 /
2 2
1
.8 / 1.0 Hz, 1H, 6-Harom), 7.85 (ddd, J = 8.0 / 2.1 / 1.0 Hz, 1H, 4-Harom), 7.93 – 7.95
(
m, 1H, 2-Harom), 8.20 (t, J = 6.2 Hz, 1H, SO NHCH ), 9.07 (s, 1H, N-H), 9.78 (s, 1H,
2 2
N-H).
5
.3.9. 2-[(3-Chloro-4-fluorophenyl)sulfonamido]thioacetamide (18b)
Compound 17b (0.16 g, 0.6 mmol, 1.0 equiv.) and Lawesson’s reagent (0.15 g, 0.3
o
mmol, 0.6 equiv.) were dissolved in dioxane (15 mL) and heated to 70 C for 27 h.
After completion of the transformation, the solvent was evaporated in vacuo. The
residue was dissolved in saturated NaHCO solution (20 mL) and the mixture was
3
stirred for 30 min. The precipitated solid was filtered through a sintered funnel,

2
3
o
washed with distilled H O and dried. Colorless solid, mp 187 C, yield 0.13 g (76 %).
2
1
C H ClFN O S (282.7 g/mol). H NMR (400 MHz, DMSO-D ): δ (ppm) = 3.71 (s,
8
8
2
2
2
6
2
1
H, SO NHCH ), 7.61 (t, J = 8.9 Hz, 1H, 5-Harom), 7.78 (ddd, J = 8.7 / 4.6 / 2.3 Hz,
2 2
H, 6-Harom), 7.96 (dd, J = 6.9 / 2.3 Hz, 1H, 2-Harom). The signals for the N-H protons
are not seen in the spectrum.
33
5
.3.10. 2-[(4-Methylphenyl)sulfonamido]thioacetamide (18c)
Compound 17c (0.12 g, 0.5 mmol, 1.0 equiv.) and Lawesson’s reagent (0.12 g, 0.3
mmol, 0.6 equiv.) were dissolved in dioxane (20 mL) and the mixture was heated at
o
7
0 C for 27 h. After completion of the transformation, the solvent was evaporated in
vacuo. The residue was dissolved in saturated NaHCO solution (20 mL) and the
3
mixture was stirred for 30 min at rt. The precipitated solid was filtered through a
o
sintered funnel, washed with distilled H O and dried. Light yellow solid, mp 189 C,
2
1
yield 0.11 g (86 %). C H N O S (244.3 g/mol). H NMR (400 MHz, DMSO-D ): δ
9
12
2
2
2
6
(
ppm) = 2.37 (s, 3H, CH ), 3.61 (s, 2H, SO NHCH ), 7.38 (d, J = 7.8 Hz, 2H, 3-Harom,
3 2 2
5
-Harom), 7.66 (d, J = 8.3 Hz, 2H, 2-Harom, 6-Harom). The signals for the N-H protons
are not seen in the spectrum.
5
.3.11.
Ethyl
2-[(3-bromophenyl)sulfonamidomethyl]-4-methylthiazole-5-
carboxylate (19a)
N-Bromosuccinimide (1.77 g, 9.9 mmol, 1.2equiv.) was added to a mixture of 9 (1.05
o
mL, 8.3 mmol, 1.0 equiv.) in THF/H O (2:1, 60 mL) at 0 C,. The mixture was stirred
2
at rt for 3 h. 18a (2.57 g, 8.3 mmol, 1.0 equiv.) was added and the mixture was
o
heated to 80 C for 16 h. THF was removed in vacuo and ethyl acetate (50 mL) was
added. The organic layer was separated, and the aqueous layer was extracted with

2
4
ethyl acetate (3 x 50 mL). The organic layer was dried (Na SO ) and concentrated
2
4
under reduced pressure. The residue was purified by FCC (Ø = 6 cm, l = 22 cm, V
=
100 mL, cyclohexane:ethyl acetate 7:3). R = 0.68 (cyclohexane:ethyl acetate 1:1).
f
1
Light brown oil, yield 1.5 g (42 %). C H BrN O S (419.3 g/mol). H NMR (400
14
15
2
4 2
MHz, DMSO-D ): δ (ppm) = 1.27 (t, J = 7.1 Hz, 3H, CO CH CH ), 2.51 (s, 3H, CH -
6
2
2
3
3
B), 4.23 (q, J = 7.1 Hz, 2H, CO CH CH ), 4.35 (d, J = 6.3 Hz, 2H, SO NHCH ), 7.51
2
2
3
2
2
(
t, J = 7.9 Hz, 1H, 5-H ), 7.77 (ddd, J = 7.8 / 1.8 / 1.0 Hz, 1H, 6-H ), 7.82 (ddd, J =
A
A
8
.0 / 2.0 / 1.0 Hz, 1H, 4-H ), 7.86 (t, J = 1.8 Hz, 1H, 2-H ), 8.85 (t, J = 6.3 Hz, 1H,
A A
13
SO NHCH ). C NMR (151 MHz, DMSO-D ): δ (ppm) = 17.2 (CO CH CH ), 19.9
2
2
6
2
2
3
(
(
(
CH -B), 47.1 (SO NHCH ), 64.2 (CO CH CH ), 124.8 (C-5 ), 125.2 (C-3 ), 128.7
3 2 2 2 2 3 B A
C-6 ), 131.9 (C-2 ), 134.5 (C-5 ), 138.5 (C-4 ), 145.5 (C-1 ), 162.2 (C-4 ), 164.4
A
A
A
A
A
B
C=O), 174.4 (C-2B).
5
5
.3.12. Ethyl 2-[(3-chloro-4-fluorophenyl)sulfonamidomethyl]-4-methylthiazole-
-carboxylate (19b)
N-Bromosuccinimide (0.10 g, 0.48 mmol, 1.2equiv.) was added to a mixture of 9
o
(
0.06 mL, 0.4 mmol, 1.0 equiv.) in THF/H O (2:1, 5mL) at 0 C. The mixture was
2
stirred at rt for 3 h and TLC showed the disappearance of ethyl acetoacetate. 18b
o
(
0.13 g, 0.4 mmol, 1.0 equiv.) was added and the mixture was heated at 80 C for 16
h. THF was removed in vacuo and ethyl acetate (10mL) was added. The organic
layer was separated, and the aqueous layer was extracted with ethyl acetate (3 x 10
ml). The organic layer was dried (Na SO ) and concentrated under reduced
2
4
pressure. The residue was purified by FCC (Ø = 2 cm, l = 22 cm, V = 30 mL,
cyclohexane:ethylacetate 7:3). R = 0.67 (cyclohexane:ethylacetate 1:1). Yellow oil,
f
1
yield 0.10 g (56 %). C H ClFN O S (392.8 g/mol). H NMR (400 MHz, DMSO-D ):
14
14
2
4
2
6

2
5
δ (ppm) = 1.26 (t, J = 7.1 Hz, 3H, CO CH CH ), 2.51 (s, 3H, CH -B), 4.17 (d, J = 6.0
2
2
3
3
Hz, 0.6H, SO NHCH *), 4.24 (q, J = 7.1 Hz, 2H, CO CH CH ), 4.37 (d, J = 6.3 Hz,
2
2
2
2
3
2
H, SO NHCH ), 7.52 (s, 0.18H, N-H*), 7.62 (t, J = 8.9 Hz, 1H, 5-H ), 7.63 (t, J = 8.0
2 2 A
Hz, 0.07H, 5-H *), 7.69 (t, J = 8.9 Hz, 0.3H, 5-H *), 7.78 (ddd, J = 8.7 / 4.5 / 2.3 Hz,
A
A
1
2
0
H, 6-H ), 7.81 (ddd, J = 8.7 / 4.1 / 2.3 Hz, 0.07H, 6-H *), 7.85 (ddd, J = 8.7 / 4.5 /
A A
.3 Hz, 0.3H, 6-H *), 7.91 (dd, J = 6.8 / 2.3 Hz, 1H, 2-H ), 7.98 (dd, J = 6.9 / 2.3 Hz,
A
A
.09H, 2-H *), 8.03 (dd, J = 6.8 / 2.3 Hz, 0.3H, 2-H *), 8.71 (t, J = 6.0 Hz, 0.3H,
A
A
SO NHCH *), 8.86 (t, J = 6.3 Hz, 1H, SO NHCH ). The signals of 18b were also
2
2
2
2
13
seen in the spectra. The signals of 18b are marked with asterisks. C NMR (101
MHz, DMSO-D ): δ (ppm) = 14.5 (CO CH CH ), 17.3 (CH -B), 30.8 (0.3C,
6
2
2
3
3
SO NHCH *), 44.4 (SO NHCH ), 61.5 (CO CH CH ), 118.2 (d, J = 22.7 Hz, C-5 ),
2
2
2
2
2
2
3
A
1
1
21.2 (d, J = 18.0 Hz, C-3 ), 122.2 (C-5 ), 128.5 (d, J = 8.8 Hz, C-6 ), 129.6 (C-2 ),
A B A A
30.0 (0.3C, C-2 *), 138.5 (d, J = 3.8 Hz, C-1 ), 158.6 (d, J = 252.3 Hz, C-4 ), 159.5
A
A
A
(
C-4 ), 161.7 (CO CH CH ), 171.6 (C-2 ). The signals of 18b were also seen in the
B 2 2 3 B
spectra. The signals of 18b are marked with asterisks.
5
.3.13.
Ethyl
4-methyl-2-{[(4-methylphenyl)sulfonamido]methyl}thiazole-5-
carboxylate (19c)
N-Bromosuccinimide (89 mg, 0.5 mmol, 1.2equiv.) was added to a mixture of 9 (0.05
o
mL, 0.4 mmol, 1.0 equiv.) in THF/H O (2:1, 5mL) at 0 C. The mixture was stirred at
2
rt for 3 h and TLC showed the disappearance of ethyl acetoacetate. 18c (0.10 g, 0.4
o
mmol, 1.0 equiv.) was added and the mixture was heated to 80 C for 16 h. THF was
removed in vacuo and ethyl acetate (10 mL) was added. The organic layer was
separated, and the aqueous layer was extracted with ethyl acetate (3 x 10 mL). The
organic layer was dried (Na SO ) and concentrated under reduced pressure. The
2
4

2
6
residue was purified by FCC (Ø = 1 cm, l = 20 cm, V = 25 mL,
cyclohexane:ethylacetate 7:3). R = 0.75 (cyclohexane:ethyl acetate 1:1). Yellow
f
1
resin, yield 0.10 g (71 %). C H N O S (354.4 g/mol). H NMR (400 MHz, DMSO-
15
18
2
4 2
D ): δ (ppm) = 1.27 (t, J = 7.1 Hz, 3H, OCH CH ), 2.36 (s, 3H, CH -A), 2.53 (s, 3H,
6
2
3
3
CH -B), 4.24 (d, J = 6.2 Hz, 2H,SO NHCH ), 4.24 (q, J = 7.1 Hz, 2H, OCH CH ),
3
2
2
2
3
7
6
.37 (d, J = 7.8 Hz, 2H, 3-H , 5-H ), 7.67 (d, J = 8.3 Hz, 2H, 2-H , 6-H ), 8.61 (t, J =
A A A A
13
.3 Hz, 1H, SO NHCH ). C NMR (151 MHz, DMSO-D ): δ (ppm) = 17.2
2
2
6
(
OCH CH ), 20.1 (CH -B), 24.1 (CH -A), 47.2 (SO NHCH ), 64.2 (OCH CH ), 124.7
2 3 3 3 2 2 2 3
(
C-5 ), 129.7 (2C, C-2 , C-6 ), 132.8 (2C, C-3 , C-5 ) 140.4 (C-4 ), 146.2 (C-1 ),
B A A A A A A
1
62.3 (C-4 ), 164.5 (C=O), 175.5 (C-2 )
B B
5
.3.14. 2-[(3-Bromophenyl)sulfonamidomethyl]-4-methylthiazole-5-carboxylic
acid (20a)
Ester 19a (1.0 g, 2.3 mmol, 1.0 equiv.) was dissolved in a mixture of CH OH and
3
THF (1:1, 50 mL). Then, 2 M KOH (1.19 mL, 2.3 mmol, 1.0 equiv.) was added and
the reaction mixture was stirred for 16 h at rt. After completion of the conversion, the
solvent was evaporated in vacuo. 1 M HCl (4.0 mL) was added dropwise to the
residue untill the pH was <7 and the precipitated solid was collected by filtration. R =
f
o
0
.32 (CH Cl :CH OH 2:1). Colorless solid, mp 208 C, yield 0.74 g (81 %).
2
2
3
1
C H BrN O S (389.9 g/mol). H NMR (400 MHz, DMSO-d): δ (ppm) = 2.49 (s, 3H,
12
11
2
4 2
CH ), 4.33 (d, J = 6.3 Hz, 2H, SO NHCH ), 7.51 (t, J = 7.9 Hz, 1H, 5-H ), 7.76 (d, J =
3
2
2
A
8
.1 Hz, 1H, 6-H ), 7.82 (d, J = 8.1 Hz, 1H, 4-H ), 7.86 (s, 1H, 2-H ), 8.82 (t, J = 6.3
A A A
1
3
Hz, 1H, SO NHCH ), 13.24 (s, 1H, CO H). C NMR (101 MHz, DMSO-d ): δ (ppm)
2
2
2
6
=
17.2 (CH -B), 44.4 (SO NHCH ), 122.5 (C-3 ), 123.7 (C-5 ), 126.0 (C-6 ), 129.4
3 2 2 A B A

2
7
(
C-2 ), 131.8 (C-5 ), 135.8 (C-4 ), 142.9 (C-1 ), 158.8 (C-4 ), 163.2 (C=O), 170.9
A
A
A
A
B
(C-2B).
5
.3.15.
2-[(3-Chloro-4-fluorophenyl)sulfonamidomethyl]-4-methylthiazole-5-
carboxylic acid (20b)
Ester 19b (1.0 g, 2.5 mmol, 1.0 equiv.) was dissolved in a mixture of CH OH and
3
THF (1:1, 25 mL). Then, 2 M KOH (1.25 mL, 2.5 mmol, 1.0 equiv.) was added and
the reaction mixture was stirred for 16 h at rt. After completion of the conversion, the
solvent was evaporated in vacuo. 1 M HCl (4.0 mL) was added dropwise to the
residue untill the pH was <7 and the precipitated solid was collected by filtration. R =
f
o
0
.30 (CH Cl :CH OH 2:1). Colorless solid, mp 206 C, yield 0.69 g (75 %).
2
2
3
1
C H ClFN O S (364.8 g/mol). H NMR (400 MHz, DMSO-d ): δ (ppm) = 2.48 (s,
12
10
2
4
2
6
3
H, CH ), 4.35 (d, J = 6.3 Hz, 2H, SO NHCH ), 7.61 (t, J = 8.9 Hz, 1H, 5-H ), 7.77
3 2 2 A
(
ddd, J = 8.7 / 4.5 / 2.3 Hz, 1H, 6-H ), 7.90 (dd, J = 6.8 / 2.3 Hz, 1H, 2-H ), 8.83 (t, J
A
A
13
=
6.3 Hz, 1H, SO NHCH ), 13.25 (s, 1H, CO H). C NMR (151 MHz, DMSO-d ): δ
2 2 2 6
(
ppm) = 19.8 (CH -B), 47.1 (SO NHCH ), 120.9 (d, J = 22.6 Hz, C-5 ), 123.6 (d, J =
3 2 2 A
1
3
8.5 Hz, C-3 ), 126.3 (C-5 ), 131.2 (d, J = 8.9 Hz, C-6 ), 132.3 (C-2 ), 141.1 (d, J =
A B A A
.5 Hz, C-1 ), 161.4 (C-4 ), 161.7 (d, J = 252.7 Hz, C-4 ), 165.9 (C=O), 173.4(C-2 ).
A
B
A
B
5
.3.16.
4-Methyl-2-[(4-methylphenyl)sulfonamidomethyl]thiazole-5-carboxylic
acid (20c)
Ester 19c (1.0 g, 2.8 mmol, 1.0 equiv.) was dissolved in a mixture of CH OH and
3
THF (1:1, 30 mL). Then, 2 M KOH (1.41 mL, 2.7 mmol, 1.0 equiv.) was added and
the reaction mixture was stirred for 16 h at rt. After completion of the transformation,
the solvent was evaporated in vacuo. 1 M HCl (3.0 mL) was added dropwise to the

2
8
residue untill the pH was <7 and the precipitated solid was collected by filtration. R =
f
o
0
.30 (CH Cl /CH OH 2:1). Colorless solid, mp 199 C, yield 0.73 g (79 %).
2
2
3
1
C H N O S (326.4 g/mol). H NMR (400 MHz, DMSO-D ): δ (ppm) = 2.36 (s, 3H,
13
14
2
4
2
6
CH -A), 2.51 (s, 3H, CH -B), 4.21 (d, J = 6.3 Hz, 2H, SO NHCH ), 7.37 (d, 8.6 Hz,
3
3
2
2
2
H, 3-H , 5-H ), 7.67 (d, J = 8.3 Hz, 2H, 2-H , 6-H ), 8.58 (t, J = 6.3 Hz, 1H,
A A A A
13
SO NHCH ), 13.23 (s, 1H, CO H). C NMR (151 MHz, DMSO-d ): δ (ppm) = 19.9
2
2
2
6
(
CH -B), 24.1 (CH -A), 47.2 (SO NHCH ), 126.2 (C-5 ), 129.7 (2C, C-2 , C-6 ),
3 3 2 2 B A A
1
32.8 (2C, C-3 ,C-5 ), 140.4 (C-1 ), 146.1 (C-4 ), 161.5 (C-4 ), 165.9 (CO H), 174.6
A A A A B 2
(C-2B).
5
.3.17. N-({5[(2-Benzoylhydrazin-1-yl)carbonyl]-4-methylthiazol-2-yl}methyl)-3-
bromobenzenesulfonamide (21a)
Carboxylic acid 20a (530 mg, 1.36 mmol, 1.0 equiv.), benzoylhydrazine (184 mg,
1
.36 mmol, 1.0 equiv.) and COMU (640 mg, 1.49 mmol, 1.1 equiv.) were suspended
in THF (15 mL). After addition of DIPEA (472 µL, 2.71 mmol, 2.0 equiv.) the resulting
yellow solution was stirred at rt overnight. The solvent was removed under reduced
pressure and water (30 mL) was added to the residue. The resulting yellow solution
was stirred for 30 min at rt and ethyl acetate (30 mL) was added. The organic phase
was separated and the aqueous layer was extracted with ethyl acetate (3 x 30 mL).
The organic layer was dried (Na SO ) and concentrated in vacuo. The residue was
2
4
purified by FCC (Ø = 2 cm, l = 25 cm, V = 35 mL, CH Cl :CH OH 97:3). R = 0.66
2
2
3
f
o
(
CH Cl :CH OH 9:1). Colorless solid, mp 210 C, yield 530 mg (78 %).
2 2 3
1
C H BrN O S (509.4 g/mol). H NMR (400 MHz, DMSO-D ): δ (ppm) = 2.52 (s,
19
17
4
4
2
6
3
7
H, CH -B), 4.36 (s, 2H, SO NHCH ), 7.50 (t, J = 7.3 Hz, 2H, 3-H , 5-H ), 7.52 –
3 2 2 C C
.63 (m, 2H, 5-H , 4-H ), 7.81 (d, J = 8.6 Hz, 1H, 6-H ), 7.85 (d, J = 8.0 Hz, 1H, 4-
A
C
A

2
9
H ), 7.90 (d, J = 7.0 Hz, 2H, 2-H , 6-H ), 7.94 (t, J = 1.9 Hz, 1H, 2-H ), 8.87 (s, 1H,
A
C
C
A
13
SO NHCH ), 10.25 (s, 1H, NHNHCOaryl), 10.51 (s, 1H, NHNHCOaryl). C NMR
2
2
(
151 MHz, DMSO-D ): δ (ppm) = 20.0 (CH -B), 47.0 (SO NHCH ), 125.3 (C-3 ),
6 3 2 2 A
1
26.6 (C-5 ), 128.7 (C-6 ), 130.5 (2C, C-2 , C-6 ), 131.6 (2C, C-3 , C-5 ), 132.1 (C-
B A C C C C
2A), 134.6 (C-5 ), 135.0 (C-4 ), 135.5 (C-1 ), 138.6 (C-4 ), 145.4 (C-1 ), 158.8 (C-
A
C
A
A
C
4B), 164.0 (NHC(=O)Thiaz ), 168.8 (NHC(=O)Aryl), 171.9 (C-2 ).
B
B
5
.3.18. N-({5-[(2-Benzoylhydrazin-1-yl)carbonyl]-4-methylthiazol-2-yl}methyl)-3-
chloro-4-fluorobenzenesulfonamide (21b)
Carboxylic acid 20b (0.65 g, 1.8 mmol, 1.0 equiv.), benzoylhydrazine (0.25 g, 1.8
mmol, 1.0 equiv.) and COMU (0.8 g, 1.9 mmol, 1.1 equiv.) were suspended in THF
(25 mL). After addition of DIPEA (0.63 mL, 3.6 mmol, 2.0 equiv.). The resulting
yellow solution was stirred at rt overnight. The solvent was removed under reduced
pressure and water (30 mL) was added to the residue. The resulting yellow solution
was stirred for 30 min at rt and ethyl acetate (30 mL) was added. The organic phase
was separated, and the aqueous layer was extracted with ethyl acetate (3 x 30 mL).
The organic layer was dried (Na SO ) and concentrated in vacuo. The residue was
2
4
purified by FCC (Ø = 2 cm, l = 15 cm, V = 20 mL, CH Cl :CH OH 97:3). R = 0.7
2
2
3
f
o
(
CH Cl :CH OH 9:1). Pale yellow solid, mp 198 C, yield 0.6 g (69 %).
2 2 3
1
C H ClFN O S (482.9 g/mol). H NMR (400 MHz, DMSO-D ): δ (ppm) = 2.51 (s,
19
16
4
4
2
6
3
H, CH ), 4.37 (s, 2H, SO NHCH ), 7.50 (t, J = 7.8 Hz, 2H, 3-H , 5-H ), 7.56 – 7.61
3 2 2 C C
(
m, 1H, 4-H ), 7.63 (t, J = 8.9 Hz, 1H, 5-H ), 7.82 (ddd, J = 8.7 / 4.5 / 2.3 Hz, 1H, 6-
C A
H ), 7.89 (d, J = 7.1 Hz, 2H, 2-H , 6-H ), 7.98 (dd, J = 6.8 / 2.3 Hz, 1H, 2H ), 8.87
A
C
C
A
13
(
s, 1H, SO NHCH ), 10.25 (s, 1H, NHNHCOAryl), 10.51 (s, 1H, NHNHCOAryl). C
2 2
NMR (151 MHz, DMSO-D ): δ (ppm) = 20.0 (CH -B), 47.0 (SO NHCH ), 121.0 (d, J
6
3
2
2