Benzothiazolyl ureas are low micromolar and uncompetitive inhibitors of 17Β-HSD10 with implications to Alzheimer’s disease treatment

Article abstract of DOI:10.3390/ijms21062059

Human 17β-hydroxysteroid dehydrogenase type 10 is a multifunctional protein involved in many enzymatic and structural processes within mitochondria. This enzyme was suggested to be involved in several neurological diseases, e.g., mental retardation, Parkinson’s disease, or Alzheimer’s disease, in which it was shown to interact with the amyloid-beta peptide. We prepared approximately 60 new compounds based on a benzothiazolyl scaffold and evaluated their inhibitory ability and mechanism of action. The most potent inhibitors contained 3-chloro and 4-hydroxy substitution on the phenyl ring moiety, a small substituent at position 6 on the benzothiazole moiety, and the two moieties were connected via a urea linker (4at, 4bb, and 4bg). These compounds exhibited IC₅₀ values of 1–2 μM and showed an uncompetitive mechanism of action with respect to the substrate, acetoacetyl-CoA. These uncompetitive benzothiazolyl inhibitors of 17β-hydroxysteroid dehydrogenase type 10 are promising compounds for potential drugs for neurodegenerative diseases that warrant further research and development.

Full text of DOI:10.3390/ijms21062059

International Journal of
Molecular Sciences
Article
Benzothiazolyl Ureas are Low Micromolar and
Uncompetitive Inhibitors of 17β-HSD10 with
Implications to Alzheimer’s Disease Treatment
Monika Schmidt 1, ,† , Ondrej Benek 1,2,3, ,† , Lucie Vinklarova 1,2,†, Martina Hrabinova 2,4
* ,
*
Lucie Zemanova 1, Matej Chribek 5, Vendula Kralova 5, Lukas Hroch 2, Rafael Dolezal 1,2
Antonin Lycka 1, Lukas Prchal 2, Daniel Jun 4 , Laura Aitken 6 , Frank Gunn-Moore 6,
Kamil Kuca 1 and Kamil Musilek 1,2
,
1
University of Hradec Kralove, Faculty of Science, Department of Chemistry, Rokitanskeho 62,
500 03 Hradec Kralove, Czech Republic; lucie.vinklarova@uhk.cz (L.V.); lucie.zemanova@uhk.cz (L.Z.);
rafael.dolezal@gmail.com (R.D.); antonin.lycka@uhk.cz (A.L.); kamil.kuca@uhk.cz (K.K.);
kamil.musilek@uhk.cz (K.M.)
2
University Hospital Hradec Kralove, Biomedical Research Centre, Sokolska 581, 500 05 Hradec Kralove,
Czech Republic; martina.hrabinova@unob.cz (M.H.); lukashroch@gmail.com (L.H.); prchal.l@email.cz (L.P.)
National Institute of Mental Health, Topolova 748, 250 67 Klecany, Czech Republic
University of Defence, Faculty of Military Health Sciences, Department of Toxicology and Military Pharmacy,
Trebesska 1575, 500 01 Hradec Kralove, Czech Republic; daniel.jun@unob.cz
Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Department of Pharmaceutical
Chemistry and Drug Control, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic;
chribek.matej@email.cz (M.C.); vendula.kralova@gmail.com (V.K.)
University of St. Andrews, School of Biology, Medical and Biological Science Building, North Haugh,
St. Andrews KY16 9TF, UK; la49@st-andrews.ac.uk (L.A.); fjg1@st-andrews.ac.uk (F.G.-M.)
Correspondence: monika.schmidt@uhk.cz (M.S.); benek.ondrej@gmail.com (O.B.);
Tel.: +420-493-332-791 (M.S.); +420-493-332-783 (O.B.)
3
4
5
6
*
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 28 February 2020; Accepted: 12 March 2020; Published: 17 March 2020
Abstract: Human 17β-hydroxysteroid dehydrogenase type 10 is a multifunctional protein involved
in many enzymatic and structural processes within mitochondria. This enzyme was suggested to be
involved in several neurological diseases, e.g., mental retardation, Parkinson’s disease, or Alzheimer’s
disease, in which it was shown to interact with the amyloid-beta peptide. We prepared approximately
60 new compounds based on a benzothiazolyl scaffold and evaluated their inhibitory ability and
mechanism of action. The most potent inhibitors contained 3-chloro and 4-hydroxy substitution
on the phenyl ring moiety, a small substituent at position 6 on the benzothiazole moiety, and the
two moieties were connected via a urea linker (4at
IC50 values of 1–2 M and showed an uncompetitive mechanism of action with respect to the
substrate, acetoacetyl-CoA. These uncompetitive benzothiazolyl inhibitors of 17 -hydroxysteroid
, 4bb, and 4bg). These compounds exhibited
µ
β
dehydrogenase type 10 are promising compounds for potential drugs for neurodegenerative diseases
that warrant further research and development.
Keywords: neurodegeneration; Alzheimer’s disease; 17β-hydroxysteroid dehydrogenase type 10;
ABAD; inhibitor; benzothiazole
1. Introduction
Human 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) is an enzyme belonging to
the superfamily of short-chain dehydrogenases/reductases (SDR) (SDR5C1 in SDR nomenclature)
Int. J. Mol. Sci. 2020, 21, 2059; doi:10.3390/ijms21062059
www.mdpi.com/journal/ijms

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encoded by the HSD17B10 gene. The enzyme was discovered by a yeast two-hybrid screening
assay conducted to detect novel protein interaction partners of the amyloid-beta peptide (A ) and
was denoted as ERAB (endoplasmic reticulum-associated binding protein) or ABAD (amyloid-beta
binding alcohol dehydrogenase) [ ]. At around the same time, the enzyme was found to be
identical to L-3-hydroxyacyl-CoA dehydrogenase [ ], denoted as HADH2 or SCHAD (short chain to
L-3-hydroxyacyl-CoA dehydrogenase) and was identified as a novel 17 -hydroxysteroid dehydrogenase
localized in mitochondria and responsible for the inactivation of sex hormones [ ]. 17 -HSD10 is
a NAD+-dependent dehydrogenase with a very wide range of substrates and functions, including
the metabolism of branched-chain fatty acids, the catabolism of isoleucine [ ], and activation or
inactivation of potent sex hormones or steroid modulators [ ]. Besides the enzymatic activities of this
multifunctional enzyme, the non-enzymatic activities appear to be critical for either mitochondrial
health or neuronal cells development. 17 -HSD10 was found to be a component of mitochondrial
RNAse P complex [ ], and certain mutations in the HSD17B10 gene result in abnormal mitochondrial
RNA processing, leading to impaired mitochondrial respiration and energy failure [ ]. The protein
β
1,2
3
β
3,4
β
5
6
β
7
7,8
is expressed in a variety of tissues and the expression pattern in the human brain appears to be
region-specific [1,9,10].
Human 17
peptide, a key peptide involved in Alzheimer’s disease pathogenesis. The production and deposition
of the A into extracellular insoluble plaques is one of the morphological hallmarks of AD and the
pathological mechanism responsible for massive neuron deaths in the human brain [11]. A interacts
with 17 -HSD10 via several amino acids within the D-loop region, a unique sequence insertion
β-HSD10 enzyme is one of the known interaction partners of the amyloid beta (Aβ)
β
β
β
that is absent in all other members of the SDR family. This interaction was detected in the brains
of patients with AD and experimental transgenic mice overexpressing amyloid precursor protein
and 17
condensation and the partial loss of cristae structure [13].
The inhibition of 17 -HSD10 enzymatic activity or its interaction with the A
approaches for the protection of mitochondria against A -driven toxicity. Several inhibitors of
17 -HSD10 were developed; however, to date, none has reached the drug market. The first structure
known to inhibit the interaction complex of the 17 -HSD10 enzyme and A was the “decoy peptide”.
This peptide contains amino acids located in the D-loop of the 17 -HSD10 enzyme, a region responsible
for the 17 -HSD10-A interaction [12]. The decoy peptide inhibited the binding of 17 -HSD10 and A
(1–42) in vitro, with the half maximal inhibitory concentration (IC50) value of 1.7 M. The administration
of the decoy peptide to transgenic animals overexpressing amyloid beta precursor protein (APP)
and 17 -HSD10 enzyme protected mice neurons from amyloid-induced cytotoxicity [12], preserved
mitochondrial and neuronal functions, and improved spatial memory [14].
Shortly after the 17 -HSD10 decoy peptide was introduced, Xie et al. identified small-molecule
β-HSD10 [12]. Overexpression of the 17β-HSD10 enzyme resulted in mitochondrial matrix
β
β peptide are vital
β
β
β
β
β
β
β
β
β
µ
β
β
inhibitors that could block the enzyme-peptide interaction. Their screening revealed benzothiazole
derivatives thioflavin T and frentizole (Figure 1). Frentizole, structurally a 1-(6-methoxy-1,3-benzothiazol-
2-yl)-3-phenylurea, is known potent non-toxic immunosuppressive agent used in treatment of
rheumatoid arthritis and systemic lupus erythematosus [15]. Based on its structure, several series
of benzothiazolyl ureas and thioureas were synthesized, and the best hits showed IC50 values of
< 10
and synthesized benzothiazolyl phosphonate derivatives to identify novel inhibitors of the 17
enzyme-A complex [17]. Interestingly, these compounds were only tested for the ability to inhibit
enzymatic function of 17
µM [16]. By using the benzothiazolyl urea compounds as a vital scaffold, Valasani et al. designed
β-HSD10
β
β
-HSD10 instead of testing their ability to block 17
β
-HSD10-A interaction.
β
The best hit compound 4b (Figure 1), exhibited an IC50 value of 52.7
µM (Ki at 14.9 µM) against
17β-HSD10 enzyme activity [18].
Further compounds targeting 17
activity. A small molecule 17 -HSD10 nonsteroidal irreversible inhibitor was developed by
Kissinger et al. (Figure 1). The compound designated AG18051 ((1-azepan-1-yl-2-phenyl-2-(4-thioxo-1,4-
β-HSD10 are focused on the inhibition of the enzymatic
β

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dihydropyrazolo [3–d] pyrimidin-5-yl)-ethanone) was found to be a very potent inhibitor of
the 17 -HSD10 enzyme, with an IC50 value of 92 nM, that functions irreversibly [19]. Other
novel 17 -HSD10 enzyme inhibitors were developed with the aim to treating androgen- and
estrogen-dependent diseases. The best hit compound, a steroidal inhibitor designated as RM-532-46
(Figure 1), had an IC50 value of 0.55 in vitro using an HEK-293 cell line stably overexpressing
17 -HSD10, and this derivative was hypothesized to be a reversible inhibitor of the 17 -HSD10
β
β
µM
β
β
enzyme [20]. A subsequent study of the steroid inhibitor RM-532-46 and non-steroidal benzothiazole
phosphonate derivatives that used purified enzyme and stably transfected HEK-293 cells revealed that
the transformation of 17
this steroidal inhibitor was also active against the 17
likely to be a promising drug candidate for the treatment of prostate cancer [20]. Recently, several
publications came out focusing on development of 17 -HSD10 inhibitors that were structurally
derived from benzothiazolyl urea scaffold of frentizole (Figure 1), however, the mode of inhibition
has not been established in these publications, except of the study by Aitken et al. [22 26]. In 2016
Hroch et al. published a series of benzothiazolyl ureas with the best compounds showing 60% decrease
in 17 -HSD10 activity in 25 M screening (Figure 1; compounds 13 14) [22]. In 2017 Hroch et al.
published a series of indolyl urea compounds that were designed as dual inhibitors of 17 -HSD10
and monoamine oxidase enzymes. However, there was no improvement in 17 -HSD10 inhibition
β-estradiol to estrone was inhibited with an IC50 of 1.7
µM [21]. Moreover,
β-HSD3 enzyme; thus, this inhibitor is more
β
–
β
µ
,
β
β
compared to the previous publication [23]. In 2017 Benek et al. published series of frentizole analogues
with urea linker replaced with thiourea or guanidine. The most promising compound with guanidine
linker showed good inhibition activity with IC50 3.06 µM [24]. The latest publication from 2019
by Aitken et al. presents large structure–activity relationship (SAR) study addressing changes to
benzothiazole moiety, urea linker and phenyl moiety of frentizole and previously identified inhibitors
13 and 14 (Figure 1). The most promising compounds identified in this study showed IC50 values
around 1–2 µM with mixed type of inhibition mechanism [26].
Figure 1. Structures of known 17β-HSD10 inhibitors and frentizole.
In this work, we aimed to design, synthesize, and evaluate new compounds based on
benzothiazolyl urea scaffold with enhanced inhibitory ability against the human 17
As a starting point for the design of novel compounds, the most potent benzothiazolyl urea-based
17 -HSD10 inhibitors, published by Hroch et al. [22], were employed (Figure 1; compounds 13 and 14).
β-HSD10 enzyme.
β
The structural scaffold was divided into four separate parts and each part was modified to identify the
structure–activity relationship (SAR) for a particular molecular fragment (Figure 2).

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Figure 2. Design of novel 17β-HSD10 inhibitors. The original scaffold was divided into for parts
(depicted in violet, blue, green, and red) and each part was modified separately.
2. Results
2.1. Chemical Synthesis
Generally, the compounds were prepared in a two-step process. Firstly, the corresponding
benzothiazole-2-amine, benzoxazole-2-amine, or benzimidazole-2-amine ( ) was treated with
1,10-carbonyldiimidazole to form an imidazolyl intermediate (
), which was then treated with
the corresponding aniline derivative ( ) to yield the 1,3-disubstituted urea ( ) as a final product
1
2
3
4
(Scheme 1) [22,27]. The thiourea analogue (4ak) was prepared in a similar way by just using
1,10-thiocarbonyldiimidazole instead of 1,10-carbonyldiimidazole in the first reaction step.
Scheme 1. Synthesis of urea and thiourea final products. Reagents and conditions: (i) CDI, DMF/DCM,
reflux; (ii) SCDI, DMF/DCM, reflux; (iii) Et3N, DMF, RT.
An inverse reaction setup was used for the synthesis of the final products 4aj and 4bb, i.e., CDI
was first reacted with the corresponding aniline derivative, and the resulting intermediate was treated
with 6-hydroxybenzothiazole-2-amine (1f) to give the final product 4aj. O-Demethylation of compound
4aj then yielded the final product 4bb (Scheme 2).

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Scheme 2. Synthesis of products 4aj and 4bb. Reagents and conditions: (i) CDI, DCM, reflux; (ii) DMF,
60 ◦C; (iii) AlCl3, DCM, reflux.
Guanidine 4al was prepared from thiourea 4ak by reaction with mercury oxide and ammonia
(Scheme 3) [28].
Scheme 3. Synthesis of guanidine analogue. Reagents and conditions: (i) HgO, NH3/MeOH, RT.
Compound 4bg was prepared from the corresponding 6-nitro substituted product (4bf) by
reduction with iron powder in acidic conditions (Scheme 4).
Scheme 4. Synthesis of compound 4bg. Reagents and conditions: (i) Fe, NH4Cl, THF/MeOH/water,
50 ◦C.
In several cases, the required aromatic precursors
thus, they were synthesized as described. 5- and 7-Chlorobenzothiazole-2-amines (1a
prepared from corresponding 2-aminobenzenethiols by cyclization with cyanogen bromide [29] or
di(1H-imidazol-1-yl)methanimine [30 31]. 2-Amino-6-chlorobenzenethiol ( ) was prepared by heating
7-chlorobenzothiazole-2-thiol with hydrazine hydrate [32] (Scheme 5).
1
and
3
were not commercially available;
,
1b) were
,
5
Benzoxazole-2-amine (1c), 6-chlorobenzoxazole-2-amine (1d), and 6-chlorobenzimidazole-2-amine
1e) were prepared from corresponding 2-aminophenols resp. 4-chlorobenzene-1,2-diamine in reaction
(
with cyanogen bromide (Scheme 5) [29]. 6-Hydroxybenzothiazole-2-amine (1f) was synthesized from
6-methoxybenzothiazole-2-amine by O-demethylation (Scheme 6).

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Scheme 5. Synthesis of intermediates 1a–
1e. Reagents and conditions: (i) hydrazine hydrate, 110 ◦C;
(ii for 1a,1e) BrCN, water/MeOH, RT; (iii for 1b) di(1H-imidazol-1-yl)methanimine, dioxane, reflux;
(iv for 1c,1d) BrCN, THF, RT.
Scheme 6. Synthesis of 6-hydroxybenzothiazole-2-amine. Reagents and conditions: (i) AlCl3,
toluene, reflux.
Benzothiazole-2-amines substituted in position 6 with acetyl, methylcarboxylate, carbonitrile
or nitro groups were prepared from corresponding para-substituted anilines by reaction with
potassium thiocyanate and bromine. In the case of 6-iodobenzothiazol-2-amine (1g) synthesis,
benzyltrimethylammonium dichloroiodate was used as an oxidizer instead of bromine, because it
offered improved reaction selectivity (Scheme 7) [22,33].
Scheme 7. Synthesis of 6-substituted benzothiazole-2-amines. Reagents and conditions: (i = 1g
)
KSCN, benzyltrimethylammonium dichloroiodate, DMSO/H2O, RT–70 ◦C; (ii) KSCN, Br2, acetic acid,
10 ◦C–RT.
Aniline derivatives 3a
catalyzed with palladium on activated carbon [34]. In several cases (3c
–
3j were prepared from corresponding nitrobenzenes by hydrogenation
3e 3f), this reaction did not
,
,
give sufficient yield or purity; thus, reduction with iron powder in acidic conditions was used instead
(Scheme 8) [35].
Scheme 8. Reduction of nitrobenzenes to anilines. Reagents and conditions: (i) H2, Pd/C, EtOH, RT;
(ii = 3c, 3e, 3f) NH4Cl, Fe, THF/MeOH/H2O, 50 ◦C.

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For the preparation of substituted anilines 3g and 3h, the corresponding nitrobenzene derivative
(8
) had to be first synthesized by nitration of 3-chloro-2-methoxybenzoic acid [36]. In the case of
aniline 3g synthesis, the obtained 3-chloro-2-methoxy-5-nitrobenzoic acid (6) was O-demethylated
using aluminum trichloride to obtain intermediate 7 [37] prior to the reduction step (Scheme 9).
Scheme 9. Synthesis of aniline intermediates 3g and 3h. Reagents and conditions: (i) HNO3, H2SO4,
0 ◦C–RT; (ii) AlCl3, DCM, reflux; (iii) H2, Pd/C, EtOH, RT.
All synthesized final products (4a–
4bg) were sufficiently characterized by 1H NMR, 13C NMR
spectroscopy and high-resolution mass spectrometry (HRMS) analyses. Intermediate products were
generally characterized only by 1H NMR (see Supplementary Materials). In case of fluorinated final
products (4a–4u and 4az)
19F NMR spectra were recorded. To remove potential aluminum residues
(demethylation using AlCl3) or mercury residues (guanidine formation using HgO) corresponding
final products and/or their intermediates were purified by suitable procedures, commonly used
for this purpose, e.g., extraction followed by column chromatography or filtration through Celite
followed by precipitation and filtration of the product (detailed synthetic procedures can be found in
Supplementary Materials).
2.2. Biological Evaluation
2.2.1. Recombinant Enzyme Production
The human recombinant 17β-HSD10 enzyme was expressed and purified using standard molecular
biological and chromatographic techniques. Briefly, the human 17β-HSD10 enzyme was expressed
as polyhistidine-tagged fusion protein using bacterial expression system and autoinduction culture
medium (detailed procedures are presented in the Material and Methods Section). Immobilized metal
affinity chromatography was used for polyhistidine-tagged recombinant 17β-HSD10 purification.
The results of purification procedures were confirmed using SDS-PAGE analysis, followed by Western
blotting analysis, resulting in a monomeric molecular mass of approximately 27 kDa (Figure 3).
The typical yield obtained after a single purification procedure, starting from 50 mL of overexpressed
bacterial culture prepared in autoinduction culture medium, was 1.5 mg, and the purity was over 95%.

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Figure 3. SDS-PAGE and western blotting analysis of recombinant human 17β-HSD10. Lanes: 1,
purified recombinant human 17β-HSD10 stained by Coomassie Brilliant Blue; 2, purified recombinant
17 -HSD10 transferred onto PVDF membrane and detected by the monoclonal anti-17
β
β-HSD10
antibody, showing monomeric and also dimeric form (54 kDa).
2.2.2. 17β-HSD10 Reductase Assay and Enzyme Kinetics
The enzyme activity of the human recombinant 17
β-HSD10 was determined from previously
published methods using acetoacetyl-CoA (AAC) as a substrate [
4
,19,38,39]. The method was based
on the decrease in nicotinamide adenine dinucleotide (NADH) cofactor absorbance at 340 nm during
its utilization by the enzyme. To establish the reaction properties correctly, the enzyme kinetic
parameters were determined. The Michaelis constant (Km) obtained for acetoacetyl-CoA as a substrate,
79.2 ± 15.0 µM at pH 7.4, corresponded to the previously reported values of 117
89 ± 5.4 µM [3] for the same substrate. The maximum reaction rate, Vmax, for the enzyme, was
estimated to be 27.7 1.8
min−1. Based on known Km values, the substrate concentration for
±
28 µM [38] and
±
µM
·
compound screening and IC50 evaluation was chosen as 320 µM (4 × the Km value).
2.2.3. Screening Inhibitory Effects of Novel Compounds
Initially, all newly synthesized compounds were screened to determine inhibitory potency against
17β-HSD10 enzyme at 10 µM. Previously identified benzothiazolyl urea inhibitors 13 and 14 [22],
together with the irreversible inhibitor AG18051 [19], and the template structure frentizole [16], were
assayed as the standards (Table 1).
The 13 most potent compounds (residual activity < 55%) showed similar or even stronger inhibitory
effects on 17
All these compounds were used for the determination of IC50 values, which ranged from ~1 to 7
(Table 1). The irreversible 17 -HSD10 inhibitor AG18051 was used as a control showing more than
96% of enzyme inhibition at 10 M and the IC50 value for this compound was determined as 97 nM,
which is consistent with the published data (92 nM; [19]).
The most potent inhibitors 4at 4bb, and 4bg (IC50 < 2
β-HSD10 compared to the previously published compounds 13 and 14 (Figure 1) [22].
µM
β
µ
,
µM) were further used for kinetic experiments
to determine the type of inhibition. The acquired data were linearized by using Lineweaver-Burk
and Hanes-Wolf plots (Figure 4A,B). Km and Vmax values in presence of inhibitors were assessed with
respect to the uninhibited 17
both the maximum reaction velocity and the Michaelis constant relative to the uninhibited enzyme
(Figure 4C). The performed kinetic experiments indicate that the selected inhibitors (4at 4bb, and
β-HSD10 enzymatic reaction (Figure 4C). All three inhibitors decreased
,
4bg) act via an uncompetitive mode of action with respect to AAC as a substrate of the reaction. Such
inhibitors are only able to bind the enzyme-substrate complex and the substrate concentration enhances
the inhibitory ability (in contrast to competitive inhibition).

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Table 1. List of synthesized compounds and their in vitro inhibitory potency against the human
17β-HSD10 enzyme.
4
5
N
R1
6
NH
Y
X
NH
2
4
7
R2
3
5
Percentage of Residual
Compound
R1
X
Y
R2
IC50 (µM)
Activity at 10 µM
AG18051
frentizole
13
3.4 ± 0.01
85.6 ± 4.8
60.3 ± 2.9
55.7 ± 9.7
0.097 ± 0.013
6-OMe
6-F
6-Cl
S
S
S
O
O
O
-
-
3-Cl, 4-OH
3-Cl, 4-OH
16.8 ± 1.0
14
-
4a
4b
4c
4d
4e
4f
6-F
6-Cl
6-OMe
6-F
6-Cl
6-OMe
S
S
S
S
S
S
O
O
O
O
O
O
2-F, 4-OH
2-F, 4-OH
2-F, 4-OH
3-F, 4-OH
3-F, 4-OH
3-F, 4-OH
3-F, 4-OH,
5-F
3-F, 4-OH,
5-F
3-F, 4-OH,
5-F
75.5 ± 5.2
73.8 ± 5.6
74.9 ± 6.5
85.0 ± 2.1
80.2 ± 4.2
84.5 ± 4.8
62.5 ± 3.7
58.3 ± 2.5
56.9 ± 4.4
-
-
-
-
-
-
4g
4h
4i
6-F
6-Cl
S
S
S
O
O
O
-
-
-
6-OMe
4j
4k
4l
6-F
6-Cl
6-OMe
S
S
S
O
O
O
2-OH, 4-F
2-OH, 4-F
2-OH, 4-F
2-OH, 3-F,
4-F
2-OH, 3-F,
4-F
2-OH, 3-F,
4-F
85.6 ± 3.2
91.8 ± 5.7
92.1 ± 2.5
80.5 ± 5.7
68.7 ± 7.6
69.8 ± 5.7
-
-
-
4m
4n
4o
6-F
6-Cl
S
S
S
O
O
O
-
-
-
6-OMe
4p
4q
4r
4s
4t
6-F
6-Cl
6-OMe
6-F
6-Cl
6-OMe
S
S
S
S
S
S
O
O
O
O
O
O
2-OMe, 3-F
2-OMe, 3-F
2-OMe, 3-F
2-OMe, 4-F
2-OMe, 4-F
2-OMe, 4-F
82.8 ± 4.1
69.3 ± 4.1
76.6 ± 3.2
80.5 ± 2.9
72.4 ± 4.8
71.0 ± 1.7
-
-
-
-
-
-
4u
4v
4w
4x
4y
4z
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
S
S
S
S
S
O
O
O
O
O
3-Cl
4-Cl
3-Cl, 4-Cl
2-Cl, 4-OH
3-Cl, 4-OMe
3-Cl,
4-COOH
3-Cl, 4-OH,
5-Cl
3-Cl, 4-OMe,
5-Cl
3-Cl, 4-OH,
5-COOH
3-Cl, 4-OMe,
5-COOH
2-OH, 4-OH
3-OH, 4-OH
3-OMe,
100.2 ± 10.4
100.8 ± 0.8
100.8 ± 7.8
94.0 ± 4.8
95.2 ± 7.8
90.4 ± 9.3
65.9 ± 2.8
91.2 ± 5.5
64.2 ± 5.5
83.9 ± 9.0
-
-
-
-
-
4aa
4ab
4ac
4ad
4ae
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
S
S
S
S
S
O
O
O
O
O
-
-
-
-
-
4af
4ag
6-Cl
6-Cl
S
S
O
O
75.5 ± 5.2
-
-
84.5 ± 1.7
4ah
6-Cl
S
O
76.0 ± 2.4
-
4-OH
3-OH,
4-OMe
3-Cl, 4-OMe
4ai
4aj
6-Cl
S
S
O
O
80.5 ± 4.2
78.3 ± 6.4
-
-
6-OH

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Table 1. Cont.
Percentage of Residual
Compound
R1
X
Y
R2
IC50 (µM)
Activity at 10 µM
4ak
4al
6-Cl
6-Cl
S
S
S
NH
3-Cl, 4-OH
3-Cl, 4-OH
89.5 ± 1.7
-
-
72.6 ± 8.4
4am
4an
4ao
4-Cl
5-Cl
7-Cl
S
S
S
O
O
O
3-Cl, 4-OH
3-Cl, 4-OH
3-Cl, 4-OH
58.6 ± 1.8
52.9 ± 5.6
38.0 ± 5.9
41.7 ± 4.2
57.1 ± 5.4
50.7 ± 5.5
36.0 ± 5.7
30.4 ± 4.8
42.2 ± 2.8
54.1 ± 1.4
69.8 ± 2.9
65.9 ± 8.4
43.9 ± 3.7
49.0 ± 7.3
38.3 ± 3.2
25.9 ± 2.1
61.4 ± 3.2
72.6 ± 2.4
72.1 ± 3.5
55.7 ± 7.2
38.0 ± 0.8
-
6.7 ± 1.6
2.5 ± 0.3
4ap
4aq
4ar
4as
4at
-
O
O
O
NH
NH
O
O
O
O
O
3-Cl, 4-OH
3-Cl, 4-OH
3-Cl, 4-OH
3-Cl, 4-OH
3-Cl, 4-OH
4.7 ± 2.3
-
5-Cl
6-Cl
-
2.4 ± 0.6
2.3 ± 0.5
1.9 ± 0.4
6-Cl
4au
4av
4aw
4ax
4ay
4az
4ba
4bb
4bc
4bd
4be
4bf
4bg
-
S
S
S
S
S
S
S
S
S
S
S
S
S
O
O
O
O
O
O
O
O
O
O
O
O
O
3-Cl, 4-OH
3-Cl, 4-OH
3-Cl, 4-OH
3-Cl, 4-OH
3-Cl, 4-OH
3-Cl, 4-OH
3-Cl, 4-OH
3-Cl, 4-OH
3-Cl, 4-OH
3-Cl, 4-OH
3-Cl, 4-OH
3-Cl, 4-OH
3-Cl, 4-OH
2.2 ± 0.5
5-Br
6-Br
6-I
6-Me
6-CF3
6-OMe
6-OH
6-COMe
6-COOMe
6-CN
-
-
-
5.5 ± 1.4
3.4 ± 1.1
2.4 ± 0.6
1.2 ± 0.2
-
-
-
6-NO2
6-NH2
5.0 ± 1.2
1.6 ± 0.3
Figure 4. Determination of the mechanism of action for compounds 4at
compounds, the uninhibited enzymatic reaction was compared with enzymatic reaction at two
concentrations of each particular inhibitor. ( ) Lineweaver-Burk plot of compound 4at (plots for
4bb, and 4bg in Supplementary Materials); ( ) Hanes-Wolf plot of compound 4at (plots for 4bb, and
) comparison of kinetic parameters Km and Vmax between uninhibited and
, 4bb, and 4bg. For all
A
B
4bg in supplementary); (
C
inhibited reactions.
3. Discussion
In our previous work, compounds harboring the 3-chloro-4-hydroxy substitution on the phenyl
ring alongside the 6-substitution (fluorine, chlorine, trifluoromethyl) on the benzothiazole moiety

Int. J. Mol. Sci. 2020, 21, 2059
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(Figure 2; compounds 13 and 14) showed good inhibitory activities towards 17β-HSD10, resulting in
less than 40% residual enzymatic activity at 25
been determined [22]. Within this study we made different modification into the structure of previous
hits (compounds 13 and 14) in order to establish the SAR and find more potent 17 -HSD10 inhibitors,
and further we performed kinetic experiments to establish the mode of inhibition.
µM, however, their mechanism of inhibition has not
β
Firstly, a series was prepared that combined different patterns of fluorine, hydroxy, or methoxy
substitutions of the phenyl ring, together with fluorine, chlorine, or methoxy substituents in position
6 of the benzothiazole moiety (4a–4u). Fluorine substitution of the phenyl ring was used instead
of the original chlorine to improve physico-chemical properties, and methoxy substitution of the
benzothiazole moiety instead of the original halogen substituent was introduced for the same reason.
Most compounds within this series were found to be less active thanthe parental compounds 13 and 14
Any deviation from the original 3-chloro, 4-hydroxy substitution of the phenyl ring resulted in decrease
in inhibitory activity except for 3,5-fluoro, 4-hydroxy substitution pattern (4g 4i), which resulted in
.
–
inhibition comparable to the parental molecules 13 and 14. There was no significant difference in
activity between compounds substituted with fluorine, chlorine of methoxy group in the position 6 of
benzothiazole moiety.
Secondly, we explored the SAR of the phenyl ring by changing the position and amount of
hydroxy and chlorine substituents and, further, by introducing methoxy and carboxy groups, while
the original 6-chlorobenzothiazole moiety was fixed along with the urea linker (4v–4aj). However,
none of modification led to stronger inhibitory ability compared to the parental compound 14.
We also focused on the urea linker, which was replaced with thiourea or guanidine. These
substitutions (4ak, 4al) did not lead to enhanced inhibitory ability and resulted in more than 70% of
residual enzyme activity.
In the next series, the original 6-chlorine substitution of the benzothiazole moiety was moved
to alternative positions 4, 5, and 7 (4am, 4an, and 4ao). Derivatives with chlorine in position 5 or 6
showed inhibition similar to parental compound 14 (~55% residual activity). While the substitution in
position 7 (4ao) increased the inhibition (38% residual activity).
Further, the benzothiazole core was replaced with benzoxazole (4ap, 4aq, and 4ar) or benzimidazole
(4as, 4at) while relocating or omitting the original 6-chlorine substitution. 5- and 6-chlorobenzoxazole
derivatives were of similar potency compared to the parental compound 14, however, the unsubstituted
benzoxazole derivative was found slightly better (41% residual activity). 5- and 6-chlorobenzimidazoles
showed improved potency resulting in 30% and 36% residual enzymatic activity respectively.
Finally, different substituents in position 6 of the benzothiazole moiety, along with fixed
3-chloro-4-hydroxy pattern on the phenyl ring and urea linker (4au to 4bg), were evaluated.
The preferred substituents for inhibition were hydroxy (4bb) > amine (4bg) ~ methoxy (4ba) >
hydrogen (4au) ~ methyl (4ay) > trifluoromethyl (4az) moiety, resulting in 26% to 49% residual
enzymatic activity. In contrast, substitution with bromine, iodine, acetyl, carboxymethylate, nitrile or
nitro group led to similar or decreased inhibition ability compared to original 6-chloro substitution.
In the next step, we have selected 13 compounds (4an
–
4ap, 4ar–4au, 4ay–4bb, 4bf, and 4bg),
which showed similar or even stronger inhibitory effects on 17
β-HSD10 than the previously published
compounds 13 and 14, to determine their IC50 values. All these compounds shared a common phenyl
part, with 3-chlorine, 4-hydroxy substitutions, together with the urea linker, but they differed in the
substitution of the benzothiazole, benzoxazole, or benzimidazole moiety. The obtained IC50 values
ranged from 1 to 7 µM (Table 1).
The three most potent inhibitors 4at, 4bb, and 4bg with IC50 < 2 µM were further used for
kinetic experiments to determine the type of inhibition. From this point of view, all the selected
inhibitors were found to act via an uncompetitive mode of action with respect to acetoacetyl-CoA
as a substrate of the reaction. This is in contrast to the mixed inhibition mode described for similar
type of inhibitors by Aitken et al. [26]. However, when looking closely at the previously published
data, the mode of inhibition seems to be identical. This is because the uncompetitive inhibition can be

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considered as a subtype of mixed inhibition and deeper analysis of the previous kinetic experiment
would probably yield the same conclusion, i.e., uncompetitive mode of inhibition. In drug research
and development, uncompetitive inhibitors are relatively rare, although they can provide unique
physiological consequences. The inhibitors can bind the target only if the substrate is present and this
mechanism of action should be favorable for enzymes with cellular substrate concentration higher than
the enzyme Km value [40]. Several drugs act in an uncompetitive manner towards dehydrogenases or
reductases with a NAD+/NADH pair as their enzyme cofactor: e.g., mycophenolic acid, reversible
inhibitor of inosine 50-monophosphate dehydrogenase [41
,42]; ononetin, an inhibitor of dihydrofolate
reductase [43]; or epristeride, an inhibitor of human steroid 5α-reductase [44].
Development of uncompetitive inhibitors is a very valuable strategy in drug discovery in
terms of specificity to the target when such inhibitors can only recognize the enzyme-substrate
complex and therefore should not inhibit other enzymes with a similar structure. For this reason,
the presented uncompetitive inhibitors of 17
research and development.
β-HSD10 appear to be promising molecules for further
4. Materials and Methods
4.1. Chemistry
4.1.1. General Chemistry
Solvents and reagents were purchased from Fluka and Sigma-Aldrich (Prague, Czech Republic)
and used without further purification. Reactions were monitored by thin layer chromatography
performed on aluminum sheets pre-coated with silica gel 60 F254 (Merck, Prague, Czech Republic) and
detected under 254 nm UV light. Column chromatography was performed on a silica gel 60 column
(230 mesh). Melting points were measured by using a Stuart SMP30 melting point apparatus (Bibby
Scientific Limited, Staffordshire, UK) and were uncorrected.
1H and 13C NMR spectra were recorded at Varian Gemini 300 (1H 300 MHz, 13C 75 MHz, Palo
Alto CA, USA) or Varian S500 (1H 500 MHz, 13C 126 MHz, Palo Alto CA, USA). In all cases, the
chemical shift values for 1H spectra were reported in ppm (
or CDCl3 (
dimethylsulfoxide-d6 (
δ
) relative to residual CHD2SO2CD3 (
7.27); shift values for 13C spectra are reported in ppm (
) relative to the solvent peak for
39.52) or CDCl3 (
77.2). Proton decoupled 19F NMR spectra were recorded
δ 2.50)
δ
δ
δ
δ
on a Bruker AVANCE III HD 500 spectrometer (Billerica, MA, USA) operating at 470.55 MHz for
fluorine using 5 mm broadband tunable probe. Samples were dissolved in dimethylsulfoxide-d6 and
19F chemical shifts were referred to internal CFCl3.
For HRMS determination, a Dionex UltiMate 3000 analytical LC-MS system coupled with a Q
Exactive Plus hybrid quadrupole-orbitrap spectrometer (both produced by ThermoFisher Scientific,
Bremen, Germany) was used. The LC-MS system consisted of a binary pump HPG-3400RS connected to
a vacuum degasser, a heated column compartment TCC-3000, an autosampler WTS-3000 equipped with
a 25
column was used as the stationary phase. The analytical column was protected against mechanical
particles by an in-line filter (Vici Jour) with a frit with 0.5 m pores. Water (MFA) and acetonitrile
µL loop, and a VWD-3000 ultraviolet detector. A Waters Atlantis dC18 100Å (2.1
×
100 mm/ 3 µm)
µ
(MFB) used in the analyses were acidified with 0.1% (v/v) formic acid. Ions for mass spectrometry
were generated by heated electro-spray ionization source (HESI) working in positive mode, with the
following settings: sheath gas flow rate 40, aux gas flow rate 10, sweep gas flow rate 2, spray voltage
3.2 kV, capillary temperature 350 ◦C, aux gas temperature 300 ◦C, S-lens RF level 50, microscans 1,
maximal injection time 35 ms, resolution 140,000. The full-scan MS analyses monitored ions within m/z
range 100–1500. The studied compounds were dissolved in methanol, and 1 µL of the solution was
injected into the LC-MS system. For elution, following ramp-gradient program was used: 0–1 min: 10%
MFB, 1–4 min: 10–100% MFB, 4–5 min: 100% MFB, 5–7.5 min: 10% MFB. The flow-rate in the gradient
elution was set to 0.4 mL/min. To increase the accuracy of HRMS, internal lock-mass calibration was
employed using polysiloxane traces of m/z = 445.12003 ([M+H]+, [C2H6SiO]6) present in the mobile

Int. J. Mol. Sci. 2020, 21, 2059
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phases. The chromatograms and mass spectra were processed in Chromeleon 6.80 and Xcalibur 3.0.63
software, respectively (both produced by ThermoFisher Scientific, Bremen, Germany).
Novelty of prepared final products was checked using Reaxys database (www.reaxys.com). Three
final products were found not to be novel structures (4v, 4w and 4af). Two of those compounds, 4w [45]
and 4af 16], were previously mentioned in scientific articles and compound 4v is indexed within
[
Pubchem database (https://pubchem.ncbi.nlm.nih.gov) and can be supplied by commercial vendors.
However, none of those compounds has ever been tested for inhibition of 17β-HSD10 enzyme.
4.1.2. Chemical Synthesis
Detailed description of chemical synthesis and characterization of intermediate products can be
found in Supplementary Materials.
4.1.3. Final Products and their Characterization
1-(2-fluoro-4-hydroxyphenyl)-3-(6-fluorobenzo[d]thiazol-2-yl)urea (4a)
Yield 66%; mp: 270 ◦C decomp.; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 10.91 (s, 1H), 9.75 (s,
1H), 8.70 (s, 1H), 7.83 (dd, J = 8.6, 2.3 Hz, 1H), 7.71–7.62 (m, 2H), 7.23 (td, J = 9.1, 2.6 Hz, 1H), 6.67
(dd, J = 12.5, 2.3 Hz, 1H), 6.61 (d, J = 8.8 Hz, 1H); 13C NMR (126 MHz, DMSO-d6):
(ppm) 159.13,
δ
158.30 (d, J = 239.2 Hz), 154.87 (d, J = 11.0 Hz), 154.21 (d, J = 242.5 Hz), 151.65, 145.76, 132.71 (d,
J = 7.8 Hz), 124.16, 120.90, 116.87 (d, J = 11.4 Hz), 113.80 (d, J = 24.3 Hz), 111.11 (d, J = 2.8 Hz), 108.04 (d,
J = 27.0 Hz), 102.74 (d, J = 21.6 Hz); 19F NMR (471 MHz, DMSO-d6):
δ
(ppm)
−
118.9, 124.8; ESI-HRMS:
−
m/z 322.0454 [M+H]+ (calc. for C14H10F2N3O2S: 322.0456 [M+H]+).
1-(6-chlorobenzo[d]thiazol-2-yl)-3-(2-fluoro-4-hydroxyphenyl)urea (4b)
Yield 94%; mp: 261–262 ◦C decomp.; 1H NMR (500 MHz, DMSO-d6):
(ppm) 10.98 (s, 1H), 9.76 (s,
δ
1H), 8.71 (s, 1H), 8.05 (d, J = 2.0 Hz, 1H), 7.68 (d, J = 9.1 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.39 (dd, J = 8.6,
2.2 Hz, 1H), 6.67 (dd, J = 12.5, 2.6 Hz, 1H), 6.61 (dd, J = 8.8, 2.4 Hz, 1H); 13C NMR (126 MHz, DMSO-d6):
δ
(ppm) 160.02, 154.91 (d, J = 10.9 Hz), 154.23 (d, J = 243.1 Hz), 151.62, 147.92, 133.21, 126.91, 126.17,
124.17, 121.19, 121.06, 116.82 (d, J = 11.6 Hz), 111.11 (d, J = 2.8 Hz), 102.74 (d, J = 21.6 Hz); 19F NMR
(471 MHz, DMSO-d6):
338.0161 [M+H]+).
δ
(ppm)
124.7; ESI-HRMS: m/z 338.0157 [M+H]+ (calc. for C14H10ClFN3O2S:
−
1-(2-fluoro-4-hydroxyphenyl)-3-(6-methoxybenzo[d]thiazol-2-yl)urea (4c)
Yield 97%; mp: 241 ◦C decomp.; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 10.75 (s, 1H), 9.73 (s,
1H), 8.72 (s, 1H), 7.69 (t, J = 9.1 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.51 (d, J = 2.6 Hz, 1H), 6.98 (dd,
J = 8.8, 2.6 Hz, 1H), 6.67 (dd, J = 12.5, 2.6 Hz, 1H), 6.64–6.58 (m, 1H), 3.79 (s, 3H); 13C NMR (126 MHz,
DMSO-d6):
δ (ppm) 157.20, 155.70, 154.72 (d, J = 10.9 Hz), 154.12 (d, J = 242.3 Hz), 151.62, 143.11, 132.66,
124.04, 120.46, 117.05 (d, J = 11.7 Hz), 114.38, 111.09 (d, J = 2.8 Hz), 104.88, 102.72 (d, J = 21.6 Hz),
55.60; 19F NMR (471 MHz, DMSO-d6):
C15H13FN3O3S: 334.0656 [M+H]+).
δ
(ppm) -125.0; ESI-HRMS: m/z 334.0663 [M+H]+ (calc. for
1-(3-fluoro-4-hydroxyphenyl)-3-(6-fluorobenzo[d]thiazol-2-yl)urea (4d)
Yield 72%; mp: 243–244 ◦C; 1H NMR (300 MHz, DMSO-d6):
(ppm) 10.84 (br s, 1H), 9.62 (br s,
1H), 9.04 (s, 1H), 7.82 (dd, J = 8.7, 2.6 Hz, 1H), 7.64 (dd, J = 8.8, 4.8 Hz, 1H), 7.43 (dd, J = 13.2, 2.4 Hz,
1H), 7.22 (td, J = 9.1, 2.7 Hz, 1H), 7.07–6.97 (m, 1H), 6.96–6.85 (m, 1H); 13C NMR (75 MHz, DMSO-d6):
δ
δ
(ppm) 159.55, 158.30 (d, J = 239.4 Hz), 150.48 (d, J = 239.5 Hz), 145.11, 140.59 (d, J = 12.2 Hz), 132.49 (d,
J = 10.6 Hz), 130.26 (d, J = 9.2 Hz), 120.49 (d, J = 11.6 Hz), 117.76 (d, J = 4.0 Hz), 113.80 (d, J = 24.4 Hz),
108.22 (d, J = 11.8 Hz), 107.89 (d, J = 7.7 Hz); 19F NMR (471 MHz, DMSO-d6):
δ (ppm) -119.0, -134.1;
ESI-HRMS: m/z 322.0455 [M+H]+ (calc. for C14H10F2N3O2S: 322.0456 [M+H]+).
1-(6-chlorobenzo[d]thiazol-2-yl)-3-(3-fluoro-4-hydroxyphenyl)urea (4e)
Yield 29%; mp: 281–282 ◦C decomp.; 1H NMR (500 MHz, DMSO-d6):
δ (ppm) 10.89 (br s, 1H),
9.61 (br s, 1H), 9.04 (s, 1H), 8.04 (d, J = 2.2 Hz, 1H), 7.63 (d, J = 8.6 Hz, 1H), 7.43 (dd, J = 13.2, 2.6 Hz,
1H), 7.39 (dd, J = 8.6, 2.2 Hz, 1H), 7.06–6.99 (m, 1H), 6.91 (dd, J = 9.8, 8.7 Hz, 1H); 13C NMR (126 MHz,
DMSO-d6):
δ (ppm) 160.23, 150.46 (d, J = 239.2 Hz), 140.62 (d, J = 12.0 Hz), 133.01, 130.16, 126.87, 126.20,

Int. J. Mol. Sci. 2020, 21, 2059
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121.23, 120.68, 117.74 (d, J = 3.9 Hz), 115.62, 107.99 (d, J = 22.5 Hz); 19F NMR (471 MHz, DMSO-d6): δ
(ppm) -134.2; ESI-HRMS: m/z 338.0158 [M+H]+ (calc. for C14H10ClFN3O2S: 338.0161 [M+H]+).
1-(3-fluoro-4-hydroxyphenyl)-3-(6-methoxybenzo[d]thiazol-2-yl)urea (4f)
Yield 30%; mp: 250 ◦C decomp.; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 10.68 (br s, 1H), 9.57 (s,
1H), 9.05 (s, 1H), 7.54 (d, J = 8.6 Hz, 1H), 7.50 (s, 1H), 7.43 (d, J = 13.0 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H),
6.98 (d, J = 8.5 Hz, 1H), 6.91 (t, J = 9.2 Hz, 1H), 3.79 (s, 3H); 13C NMR (126 MHz, DMSO-d6):
(ppm)
δ
157.49, 155.67, 151.91, 150.47 (d, J = 239.0 Hz), 140.43 (d, J = 12.0 Hz), 132.39, 130.39, 120.06, 117.73,
115.43, 114.35, 107.82 (d, J = 22.8 Hz), 104.96, 55.60; 19F NMR (471 MHz, DMSO-d6):
ESI-HRMS: m/z 334.0653 [M+H]+ (calc. for C15H13FN3O3S: 334.0656 [M+H]+).
1-(3,5-difluoro-4-hydroxyphenyl)-3-(6-fluorobenzo[d]thiazol-2-yl)urea (4g)
δ
(ppm) 134.2;
−
Yield 62%; mp: 317–319 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 10.94 (br s, 1H), 9.83 (br s, 1H),
9.25 (s, 1H), 7.88–7.79 (m, 1H), 7.65 (s, 1H), 7.28–7.19 (m, 3H); 13C NMR (126 MHz, DMSO-d6):
(ppm)
159.68, 158.31 (d, J = 239.2 Hz), 152.45, 152.16 (dd, J = 239.6, 8.7 Hz), 144.41, 132.22, 129.88 (t, J = 11.2 Hz),
129.03 (t, J = 16.3 Hz), 120.14, 113.85 (d, J = 24.2 Hz), 108.17 (d, J = 26.9 Hz), 103.34–102.69 (m); 19
NMR (471 MHz, DMSO-d6): (ppm) -118.8 (s, 1F),
131.0 (s, 2F); ESI-HRMS: m/z 340.0371 [M+H]+
(calc. for C14H9F3N3O2S: 340.0362 [M+H]+).
1-(6-chlorobenzo[d]thiazol-2-yl)-3-(3,5-difluoro-4-hydroxyphenyl)urea (4h)
Yield 90%; mp: 304 ◦C; 1H NMR (500 MHz, DMSO-d6):
(ppm) 9.82 (br s, 1H), 9.34 (s, 1H), 8.04
(d, J = 2.2 Hz, 1H), 7.62 (d, J = 8.6 Hz, 1H), 7.40 (dd, J = 8.6, 2.2 Hz, 1H), 7.26–7.18 (m, 2H); 13C NMR
(126 MHz, DMSO-d6): (ppm) 160.52, 152.52, 152.15 (dd, J = 239.7, 8.7 Hz), 146.42, 132.68, 129.84 (t,
J = 12.6 Hz), 129.07 (t, J = 16.3 Hz), 126.95, 126.26, 121.30, 120.19, 103.24–102.85 (m); 19F NMR (471 MHz,
DMSO-d6): (ppm)
131.0 (s, 2F); ESI-HRMS: m/z 356.0077 [M+H]+ (calc. for C14H9ClF2N3O2S:
356.0067 [M+H]+).
1-(3,5-difluoro-4-hydroxyphenyl)-3-(6-methoxybenzo[d]thiazol-2-yl)urea (4i)
Yield 93%; mp: 161–162 ◦C; 1H NMR (500 MHz, DMSO-d6):
(ppm) 9.80 (s, 1H), 9.28 (s, 1H),
7.54 (d, J = 8.9 Hz, 1H), 7.51 (d, J = 2.6 Hz, 1H), 7.27–7.17 (m, 2H), 6.98 (dd, J = 8.8, 2.6 Hz, 1H), 3.79
(s, 3H); 13C NMR (126 MHz, DMSO-d6):
(ppm) 155.94, 155.73, 152.17 (dd, J = 239.5, 8.8 Hz), 132.01,
130.27–129.85 (m), 128.89 (t, J = 17.3 Hz), 119.74, 114.68, 114.43, 105.22, 105.08, 103.22–102.56 (m), 55.63;
19F NMR (471 MHz, DMSO-d6): 131.0 (s, 2F); ESI-HRMS: m/z 352.0558 [M+H]+ (calc. for
(ppm)
C15H12F2N3O3S: 352.0562 [M+H]+).
1-(4-fluoro-2-hydroxyphenyl)-3-(6-fluorobenzo[d]thiazol-2-yl)urea (4j)
Yield 78%; mp: 226–228 ◦C decomp.; 1H NMR (500 MHz, DMSO-d6):
δ
F
δ
−
δ
δ
δ
−
δ
δ
δ
−
δ
(ppm) 11.16 (s, 1H), 10.63
(s, 1H), 8.76 (s, 1H), 7.99 (dd, J = 9.0, 6.3 Hz, 1H), 7.83 (dd, J = 8.7, 2.7 Hz, 1H), 7.66 (dd, J = 8.8, 4.8 Hz,
1H), 7.22 (td, J = 9.1, 2.8 Hz, 1H), 6.69 (dd, J = 10.0, 2.9 Hz, 1H), 6.63 (td, J = 8.8, 2.9 Hz, 1H); 13C NMR
(126 MHz, DMSO-d6):
δ (ppm) 159.10, 158.28 (d, J = 238.9 Hz), 157.91 (d, J = 239.3 Hz), 151.48, 147.73
(d, J = 10.9 Hz), 145.77, 132.71 (d, J = 10.9 Hz), 122.98 (d, J = 2.8 Hz), 120.83 (d, J = 8.9 Hz), 120.18 (d,
J = 9.6 Hz), 113.76 (d, J = 24.2 Hz), 107.99 (d, J = 26.9 Hz), 105.04 (d, J = 21.8 Hz), 102.11 (d, J = 25.1 Hz);
19F NMR (471 MHz, DMSO-d6):
C14H10F2N3O2S: 322.0456 [M+H]+).
δ
(ppm)
−
118.1,
118.9; ESI-HRMS: m/z 322.0453 [M+H]+ (calc. for
−
1-(6-chlorobenzo[d]thiazol-2-yl)-3-(4-fluoro-2-hydroxyphenyl)urea (4k)
Yield 94%; mp: 216–218 ◦C decomp.; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 10.74 (br s, 1H),
8.85 (s, 1H), 8.05 (d, J = 2.2 Hz, 1H), 7.96 (dd, J = 9.0, 6.3 Hz, 1H), 7.64 (d, J = 8.6 Hz, 1H), 7.39 (dd,
J = 8.6, 2.2 Hz, 1H), 6.74 (dd, J = 10.1, 2.9 Hz, 1H), 6.62 (td, J = 8.7, 2.9 Hz, 1H); 13C NMR (126 MHz,
DMSO-d6):
δ (ppm) 160.03, 157.99 (d, J = 239.2 Hz), 151.55, 148.00 (d, J = 11.1 Hz), 147.84, 133.22, 126.87,
126.18, 122.90 (d, J = 2.7 Hz), 121.17, 120.97, 120.31 (d, J = 9.7 Hz), 104.95 (d, J = 21.6 Hz), 102.20 (d,
J = 25.2 Hz); 19F NMR (471 MHz, DMSO-d6):
for C14H10ClFN3O2S: 338.0161 [M+H]+).
δ
(ppm)
118.0; ESI-HRMS: m/z 338.0157 [M+H]+ (calc.
−
1-(4-fluoro-2-hydroxyphenyl)-3-(6-methoxybenzo[d]thiazol-2-yl)urea (4l)
Yield 99%; mp: 207–208 ◦C decomp.; 1H NMR (500 MHz, DMSO-d6):
(ppm) 10.72 (br s, 1H),
8.88 (s, 1H), 7.96 (dd, J = 9.0, 6.3 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 2.6 Hz, 1H), 6.99 (dd,
δ

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J = 8.8, 2.7 Hz, 1H), 6.75 (dd, J = 10.1, 2.9 Hz, 1H), 6.61 (td, J = 8.8, 3.0 Hz, 1H), 3.79 (s, 3H); 13C NMR
(126 MHz, DMSO-d6): (ppm) 157.91 (d, J = 239.0 Hz), 157.51, 155.76, 151.54, 147.97 (d, J = 11.0 Hz),
δ
142.36, 132.42, 123.03 (d, J = 2.8 Hz), 120.28 (d, J = 10.1 Hz), 120.17, 114.48, 104.9, 104.90 (d, J = 21.6 Hz),
102.18 (d, J = 25.0 Hz), 55.64; 19F NMR (471 MHz, DMSO-d6):
[M+H]+ (calc. for C15H13FN3O3S: 334.0656 [M+H]+).
δ
(ppm) 118.2; ESI-HRMS: m/z 334.0652
−
1-(3,4-difluoro-2-hydroxyphenyl)-3-(6-fluorobenzo[d]thiazol-2-yl)urea (4m)
Yield 96%; mp: 209–210 ◦C; 1H NMR (500 MHz, DMSO-d6):
(ppm) 11.23 (s, 1H), 10.88 (s, 1H),
8.91 (s, 1H), 7.84 (dd, J = 8.7, 2.7 Hz, 1H), 7.83–7.78 (m, 1H), 7.67 (dd, J = 8.8, 4.7 Hz, 1H), 7.24 (td,
J = 9.1, 2.7 Hz, 1H), 6.91–6.82 (m, 1H); 13C NMR (75 MHz, DMSO-d6):
(ppm) 159.03 (d, J = 2.3 Hz),
δ
δ
158.32 (d, J = 239.0 Hz), 151.73, 146.29 (dd, J = 240.5, 10.7 Hz), 145.65, 140.11 (dd, J = 238.6, 15.0 Hz),
136.27 (dd, J = 13.5, 2.0 Hz), 132.70 (d, J = 11.1 Hz), 125.39–125.25 (m), 120.87 (d, J = 9.0 Hz), 114.15
(dd, J = 8.1, 3.6 Hz), 113.84 (d, J = 24.3 Hz), 108.05 (d, J = 27.0 Hz), 106.17 (d, J = 17.7 Hz); 19F NMR
(471 MHz, DMSO-d6):
δ
(ppm) -118.8, -143.9 (3J(19F, 19F) = 22.6 Hz), 158.4 (3J(19F, 19F) = 22.6 Hz);
−
ESI-HRMS: m/z 340.0361 [M+H]+ (calc. for C14H9F3N3O2S: 340.0362 [M+H]+).
1-(6-chlorobenzo[d]thiazol-2-yl)-3-(3,4-difluoro-2-hydroxyphenyl)urea (4n)
Yield 75%; mp: 212–213 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 11.29 (s, 1H), 10.89 (s, 1H),
8.92 (s, 1H), 8.07 (d, J = 2.2 Hz, 1H), 7.84–7.77 (m, 1H), 7.66 (d, J = 8.6 Hz, 1H), 7.41 (dd, J = 8.6, 2.2 Hz,
1H), 6.93–6.82 (m, 1H); 13C NMR (126 MHz, DMSO-d6):
(ppm) 159.87, 151.53, 147.85, 146.26 (dd,
δ
J = 240.8, 10.7 Hz), 140.07 (dd, J = 238.4, 15.1 Hz), 136.10 (d, J = 13.3 Hz), 133.20, 126.94, 126.20, 125.26 (t,
J = 3.0 Hz), 121.18, 121.04, 113.96 (d, J = 6.4 Hz), 106.19 (d, J = 17.6 Hz); 19F NMR (471 MHz, DMSO-d6):
δ
(ppm) -143.9 (3J(19F, 19F) = 22.5 Hz), 158.5 (3J(19F, 19F) = 22.5 Hz); ESI-HRMS: m/z 356.0065 [M+H]+
−
(calc. for C14H9ClF2N3O2S: 356.0067 [M+H]+).
1-(3,4-difluoro-2-hydroxy◦phenyl)-3-(6-methoxybenzo[d]thiazol-2-yl)urea (4o)
1
Yield 99%; mp: 198–200 C; H NMR (500 MHz, DMSO-d6):
δ
(ppm) 10.73 (br s, 2H), 9.05 (s,
1H), 7.83–7.75 (m, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 2.6 Hz, 1H), 6.99 (dd, J = 8.9, 2.6 Hz, 1H),
6.90–6.80 (m, 1H), 3.79 (s, 3H); 13C NMR (126 MHz, DMSO-d6):
δ
(ppm) 157.27, 155.77, 151.68, 146.22
(dd, J = 240.9, 11.1 Hz), 142.61, 140.11 (dd, J = 238.3, 14.9 Hz), 136.18 (d, J = 13.6 Hz), 132.52, 125.46 (d,
J = 3.1 Hz), 120.32, 114.47, 114.07 (dd, J = 7.5, 3.0 Hz), 106.15 (d, J = 17.4 Hz), 104.92, 55.62; 19F NMR
(471 MHz, DMSO-d6):
δ
(ppm) -144.1 (3J(19F, 19F) = 22.6 Hz), 158.4 (3J(19F, 19F) = 22.6 Hz); ESI-HRMS:
−
m/z 352.0560 [M+H]+ (calc. for C15H12F2N3O3S: 352.0562 [M+H]+).
1-(3-fluoro-2-methoxyphenyl)-3-(6-fluorobenzo[d]thiazol-2-yl)urea (4p)
Yield 85%; mp: 366–368 ◦C decomp.; 1H NMR (500 MHz, DMSO-d6):
δ (ppm) 11.34 (s, 1H), 9.16
(br s, 1H), 8.01 (d, J = 8.4 Hz, 1H), 7.84 (dd, J = 8.7, 2.5 Hz, 1H), 7.69 (dd, J = 8.8, 4.7 Hz, 1H), 7.24 (td,
J = 9.0, 2.5 Hz, 1H), 7.09 (td, J = 8.3, 6.3 Hz, 1H), 6.99–6.92 (m, 1H), 3.92 (s, 3H); 13C NMR (126 MHz,
DMSO-d6):
δ (ppm) 158.96, 158.36 (d, J = 239.3 Hz), 154.79 (d, J = 243.4 Hz), 151.24, 145.75, 136.19 (d,
J = 13.5 Hz), 133.02 (d, J = 4.6 Hz), 132.63 (d, J = 10.9 Hz), 124.09 (d, J = 8.9 Hz), 121.00 (d, J = 8.1 Hz),
114.59 (d, J = 2.5 Hz), 113.88 (d, J = 24.3 Hz), 110.50 (d, J = 18.5 Hz), 108.07 (d, J = 27.0 Hz), 61.53 (d,
J = 4.8 Hz); 19F NMR (471 MHz, DMSO-d6):
(calc. for C15H12F2N3O2S: 336.0613 [M+H]+).
1-(6-chlorobenzo[d]thiazol-2-yl)-3-(3-fluoro-2-methoxyphenyl)urea (4q)
Yield 71%; mp: 341–343 ◦C decomp.; 1H NMR (500 MHz, DMSO-d6):
(ppm) 11.41 (s, 1H), 9.16
(br s, 1H), 8.06 (s, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.40 (dd, J = 8.7, 1.7 Hz, 1H),
7.13–7.05 (m, 1H), 6.99–6.92 (m, 1H), 3.92 (s, 3H); 13C NMR (126 MHz, DMSO-d6):
(ppm) 159.79,
δ
(ppm)
−
119.7,
−
130.5; ESI-HRMS: m/z 336.0607 [M+H]+
δ
δ
154.78 (d, J = 243.3 Hz), 151.17, 147.94, 136.19 (d, J = 13.5 Hz), 133.15, 132.96 (d, J = 4.5 Hz), 127.04,
126.23, 124.08 (d, J = 8.9 Hz), 121.21, 121.16, 114.58 (d, J = 3.2 Hz), 110.54 (d, J = 18.6 Hz), 61.53 (d,
J = 4.9 Hz); 19F NMR (471 MHz, DMSO-d6):
for C15H12ClFN3O2S: 352.0317 [M+H]+).
δ
(ppm)
130.5; ESI-HRMS: m/z 352.0316 [M+H]+ (calc.
−
1-(3-fluoro-2-methoxyphenyl)-3-(6-methoxybenzo[d]thiazol-2-yl)urea (4r)
Yield 54%; mp: 329–331 ◦C decomp.; 1H NMR (500 MHz, DMSO-d6):
(ppm) 11.20 (s, 1H), 9.22
(br s, 1H), 8.02 (d, J = 8.2 Hz, 1H), 7.59 (d, J = 8.7 Hz, 1H), 7.53 (s, 1H), 7.14–7.04 (m, 1H), 7.00 (d,
δ

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J = 8.6 Hz, 1H), 6.97–6.91 (m, 1H), 3.92 (s, 3H), 3.80 (s, 3H); 13C NMR (126 MHz, DMSO-d6):
δ
(ppm)
157.04, 155.79, 154.82 (d, J = 244.9 Hz), 151.20, 143.14, 136.13 (d, J = 13.5 Hz), 133.18 (d, J = 4.5 Hz),
132.60, 124.08 (d, J = 9.0 Hz), 120.59, 114.56, 114.47, 110.35 (d, J = 18.6 Hz), 104.90, 61.54, 55.60; 19F NMR
(471 MHz, DMSO-d6):
348.0813 [M+H]+).
δ
(ppm)
130.5; ESI-HRMS: m/z 348.0806 [M+H]+ (calc. for C16H15FN3O3S:
−
1-(4-fluoro-2-methoxyphenyl)-3-(6-fluorobenzo[d]thiazol-2-yl)urea (4s)
Yield 97%; mp: 364–366 ◦C decomp.; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 11.30 (br s, 1H),
8.96 (br s, 1H), 8.05 (dd, J = 8.9, 6.3 Hz, 1H), 7.83 (dd, J = 8.7, 2.6 Hz, 1H), 7.71–7.62 (m, 1H), 7.23 (td,
J = 9.1, 2.6 Hz, 1H), 7.01 (dd, J = 10.6, 2.6 Hz, 1H), 6.77 (td, J = 8.7, 2.7 Hz, 1H), 3.90 (s, 3H); 13C NMR
(126 MHz, DMSO-d6):
δ (ppm) 159.13, 158.32 (d, J = 239.6 Hz), 158.30 (d, J = 238.9 Hz), 151.52, 149.63
(d, J = 10.3 Hz), 145.67, 132.64 (d, J = 11.1 Hz), 123.66 (d, J = 3.0 Hz), 120.85 (d, J = 9.0 Hz), 119.91 (d,
J = 9.4 Hz), 113.81 (d, J = 24.3 Hz), 108.02 (d, J = 27.0 Hz), 106.16 (d, J = 21.8 Hz), 99.72 (d, J = 27.2 Hz),
56.38; 19F NMR (471 MHz, DMSO-d6):
for C15H12F2N3O2S: 336.0613 [M+H]+).
1-(6-chlorobenzo[d]thiazol-2-yl)-3-(4-fluoro-2-methoxyphenyl)urea (4t)
Yield 94%; mp: 356–358 ◦C decomp.; 1H NMR (300 MHz, DMSO-d6):
(ppm) 11.28 (s, 1H), 8.89 (s,
1H), 8.11–8.01 (m, 2H), 7.66 (d, J = 8.6 Hz, 1H), 7.40 (dd, J = 8.6, 2.2 Hz, 1H), 7.02 (dd, J = 10.7, 2.7 Hz,
1H), 6.78 (td, J = 8.7, 2.8 Hz, 1H), 3.91 (s, 3H); 13C NMR (75 MHz, DMSO-d6):
(ppm) 159.92, 158.33 (d,
δ
(ppm) -116.9,
118.8; ESI-HRMS: m/z 336.0610 [M+H]+ (calc.
−
δ
δ
J = 239.8 Hz), 151.35, 149.52 (d, J = 10.2 Hz), 147.99, 133.21, 126.95, 126.22, 123.61 (d, J = 3.1 Hz), 121.21,
121.12, 119.76 (d, J = 9.5 Hz), 106.21 (d, J = 21.9 Hz), 99.73 (d, J = 27.2 Hz), 56.42; 19F NMR (471 MHz,
DMSO-d6):
[M+H]+).
δ
(ppm)
116.8; ESI-HRMS: m/z 352.0316 [M+H]+ (calc. for C15H12ClFN3O2S: 352.0317
−
1-(4-fluoro-2-methoxyphenyl)-3-(6-methoxybenzo[d]thiazol-2-yl)urea (4u)
Yield 88%; mp: 324 ◦C decomp.; 1H NMR (500 MHz, DMSO-d6):
(ppm) 11.07 (s, 1H), 8.94 (br s,
1H), 8.08 (dd, J = 8.9, 6.3 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 2.6 Hz, 1H), 7.01 (dd, J = 10.7,
2.8 Hz, 1H), 6.98 (dd, J = 8.8, 2.6 Hz, 1H), 6.77 (td, J = 8.7, 2.8 Hz, 1H), 3.91 (s, 3H), 3.79 (s, 3H); 13
NMR (126 MHz, DMSO-d6): (ppm) 158.19 (d, J = 239.3 Hz), 157.16, 155.71, 149.45 (d, J = 10.3 Hz),
δ
C
δ
143.16, 132.61, 123.82 (d, J = 3.0 Hz), 120.49, 119.68 (d, J = 9.5 Hz), 114.40, 106.15 (d, J = 21.8 Hz), 104.86,
99.67 (d, J = 27.2 Hz), 56.38, 55.59; 19F NMR (471 MHz, DMSO-d6):
348.0812 [M+H]+ (calc. for C16H15FN3O3S: 348.0813 [M+H]+).
1-(6-chlorobenzo[d]thiazol-2-yl)-3-(3-chlorophenyl)urea (4v)
δ
(ppm) 117.1; ESI-HRMS: m/z
−
Yield 83%; mp: 351–353 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 11.07 (br s, 1H), 9.36 (br
s, 1H), 8.04 (s, 1H), 7.73 (s, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.47–7.27 (m, 3H), 7.10 (d, J = 7.3 Hz, 1H);
13C NMR (126 MHz, DMSO-d6):
δ (ppm) 160.42, 152.17, 146.76, 139.99, 133.28, 132.77, 130.54, 127.04,
126.30, 122.69, 121.32, 120.54, 118.29, 117.40; ESI-HRMS: m/z 337.9914 [M+H+] (calc. for C14H9Cl2N3OS:
337.9916 [M+H]+).
1-(6-chlorobenzo[d]thiazol-2-yl)-3-(4-chlorophenyl)urea (4w)
Yield 86%; mp: 335–337 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 10.98 (br s, 1H), 9.29 (br s,
1H), 8.04 (s, 1H), 7.63 (d, J = 8.3 Hz, 1H), 7.55 (d, J = 8.6 Hz, 2H), 7.47–7.27 (m, 3H); 13C NMR (126 MHz,
DMSO-d6):
δ (ppm) 160.24, 152.01, 147.33, 137.40, 132.83, 128.79, 126.99, 126.68, 126.26, 121.28, 120.48;
ESI-HRMS: m/z 337.9914 [M+H+] (calc. for C14H9Cl2N3OS: 337.9916 [M+H]+).
1-(6-chlorobenzo[d]thiazol-2-yl)-3-(3,4-dichlorophenyl)urea (4x)
Yield 85%; mp: 334–336 ◦C; 1H NMR (300 MHz, DMSO-d6):
s, 1H), 8.03 (s, 1H), 7.90 (s, 1H), 7.77–7.19 (m, 4H); 13C NMR (75 MHz, DMSO-d6):
δ
(ppm) 11.25 (br s, 1H), 9.47 (br
δ
(ppm) 160.70,
152.89, 145.92, 138.79, 132.47, 131.13, 130.66, 127.05, 126.31, 124.36, 121.35, 120.01, 119.01; ESI-HRMS:
m/z 371.9526 [M+H+] (calc. for C14H8Cl3N3OS: 371.9526 [M+H]+).
1-(2-chloro-4-hydroxyphenyl)-3-(6-chlorobenzo[d]thiazol-2-yl)urea (4y)
Yield 71%; mp: 283–285 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ (ppm) 11.29 (br s, 1H), 9.77 (br
s, 1H), 8.74 (br s, 1H), 8.05 (d, J = 2.4 Hz, 1H), 7.73 (d, J = 8.9 Hz, 1H), 7.65 (d, J = 8.6 Hz, 1H), 7.40
(dd, J = 8.6, 2.4 Hz, 1H), 6.90 (d, J = 2.6 Hz, 1H), 6.77 (dd, J = 8.9, 2.5 Hz, 1H); 13C NMR (126 MHz,

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DMSO-d6):
δ (ppm) 160.16, 154.72, 151.80, 147.81, 133.14, 126.94, 126.19, 125.94, 125.42, 125.04, 121.19,
121.00, 115.61, 114.65; ESI-HRMS: m/z 353.9864 [M+H+] (calc. for C14H9Cl2N3O2S: 353.9865 [M+H]+).
1-(3-chloro-4-methoxyphenyl)-3-(6-chlorobenzo[d]thiazol-2-yl)urea (4z)
Yield 93%; mp: 307–309 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 11.00 (br s, 1H), 9.12 (br s,
1H), 8.03 (d, J = 2.2 Hz, 1H), 7.68 (d, J = 2.4 Hz, 1H), 7.62 (d, J = 8.5 Hz, 1H), 7.39 (dd, J = 8.6, 2.2 Hz, 1H),
7.36 (dd, J = 8.9, 2.3 Hz, 1H), 7.11 (d, J = 9.0 Hz, 1H), 3.83 (s, 3H); 13C NMR (126 MHz, DMSO-d6):
δ
(ppm) 160.39, 152.14, 150.50, 147.12, 132.84, 131.96, 126.90, 126.20, 121.23, 120.86, 120.82, 119.16, 113.04,
56.20; ESI-HRMS: m/z 368.0021 [M+H+] (calc. for C15H11Cl2N3O2S: 368.0022 [M+H]+).
2-chloro-4-(3-(6-chlorobenzo[d]thiazol-2-yl)ureido)benzoic acid (4aa)
Yield 83%; mp: 324–326 ◦C; 1H NMR (300 MHz, DMSO-d6):
δ
(ppm) 12.27 (br s, 1H), 9.69 (br s,
1H), 8.05 (d, J = 1.9 Hz, 1H), 7.92–7.77 (m, 2H), 7.62 (d, J = 8.6 Hz, 1H), 7.50 (dd, J = 8.6, 1.6 Hz, 1H), 7.41
(dd, J = 8.6, 2.0 Hz, 1H); 13C NMR (75 MHz, DMSO-d6):
(ppm) 166.00, 160.99, 152.98, 145.64, 142.54,
δ
133.30, 132.53, 132.40, 127.13, 126.40, 124.12, 121.43, 119.90, 119.74, 116.74; ESI-HRMS: m/z 381.9815
[M+H+] (calc. for C15H9Cl2N3O3S: 381.9814 [M+H]+).
1-(6-chlorobenzo[d]thiazol-2-yl)-3-(3,5-dichloro-4-hydroxyphenyl)urea (4ab)
Yield 69%; mp: 300 ◦C decomp.; 1H NMR (300 MHz, DMSO-d6):
δ
(ppm) 10.61 (br s, 1H), 9.19 (br
s, 1H), 8.03 (d, J = 2.1 Hz, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.55 (s, 2H), 7.39 (dd, J = 8.6, 2.2 Hz, 1H); 13
C
NMR (75 MHz, DMSO-d6):
δ (ppm) 160.83, 152.82, 146.07, 144.82, 132.58, 131.55, 126.97, 126.28, 122.42,
121.33, 120.06, 119.41; ESI-HRMS: m/z 387.9476 [M+H+] (calc. for C14H8Cl3N3O2S: 387.9476 [M+H]+).
1-(6-chlorobenzo[d]thiazol-2-yl)-3-(3,5-dichloro-4-methoxyphenyl)urea (4ac)
Yield 90%; mp: 290 ◦C decomp.; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 11.26 (br s, 1H), 9.39 (br
s, 1H), 8.02 (d, J = 2.0 Hz, 1H), 7.65 (s, 2H), 7.60 (d, J = 8.6 Hz, 1H), 7.40 (dd, J = 8.7, 2.0 Hz, 1H), 3.79
(s, 3H); 13C NMR (126 MHz, DMSO-d6):
δ (ppm) 146.74, 135.97, 132.25, 128.14, 127.03, 126.31, 121.36,
119.07, 60.64; ESI-HRMS: m/z 401.9632 [M+H+] (calc. for C15H10Cl3N3O2S: 401.9632 [M+H]+).
3-chloro-5-(3-(6-chlorobenzo[d]thiazol-2-yl)ureido)-2-hydroxybenzoic acid (4ad)
Yield 68%; mp: 268–270 ◦C; 1H NMR (300 MHz, DMSO-d6):
δ
(ppm) 11.34 (br s, 1H), 9.23 (br s,
1H), 8.03 (d, J = 2.1 Hz, 1H), 7.93 (d, J = 2.6 Hz, 1H), 7.87 (d, J = 2.7 Hz, 1H), 7.61 (d, J = 8.6 Hz, 1H),
7.39 (dd, J = 8.6, 2.2 Hz, 1H); 13C NMR (75 MHz, DMSO-d6):
δ (ppm) 171.37, 160.76, 152.64, 146.45,
132.67, 130.24, 126.97, 126.73, 126.28, 121.30, 120.47, 119.53, 114.20; ESI-HRMS: m/z 397.9764 [M+H+]
(calc. for C15H9Cl2N3O4S: 397.9764 [M+H]+).
3-chloro-5-(3-(6-chlorobenzo[d]thiazol-2-yl)ureido)-2-methoxybenzoic acid (4ae)
Yield 60%; mp: 263.5–265 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 12.10 (br s, 1H), 9.41 (br s,
1H), 8.04 (d, J = 2.1 Hz, 1H), 7.89 (d, J = 2.7 Hz, 1H), 7.79 (d, J = 2.7 Hz, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.40
(dd, J = 8.6, 2.2 Hz, 1H), 3.80 (s, 3H); 13C NMR (126 MHz, DMSO-d6):
(ppm) 166.11, 160.84, 152.95,
δ
149.82, 145.83, 135.06, 132.50, 128.18, 128.02, 127.04, 126.31, 123.23, 121.34, 119.79, 61.67; ESI-HRMS: m/z
411.9919 [M+H+] (calc. for C16H11Cl2N3O4S: 411.9920 [M+H]+).
1-(6-chlorobenzo[d]thiazol-2-yl)-3-(2,4-dihydroxyphenyl)urea (4af)
◦
1
Yield 81%; mp: 241–243 C; H NMR (500 MHz, DMSO-d6):
δ (ppm) 8.60 (br s, 1H), 8.03 (d,
J = 2.1 Hz, 1H), 7.72–7.53 (m, 2H), 7.38 (dd, J = 8.6, 2.1 Hz, 1H), 6.42 (d, J = 2.6 Hz, 1H), 6.21 (dd, J = 8.7,
2.5 Hz, 1H); 13C NMR (126 MHz, DMSO-d6):
δ (ppm) 160.33, 153.99, 151.53, 148.23, 147.76, 133.19,
126.78, 126.16, 121.37, 121.15, 120.81, 117.75, 105.63, 102.57; ESI-HRMS: m/z 336.0202 [M+H+] (calc. for
C14H10ClN3O3S: 336.0204 [M+H]+).
1-(6-chlorobenzo[d]thiazol-2-yl)-3-(3,4-dihydroxyphenyl)urea (4ag)
Yield 28%; mp: 257–259 ◦C; 1H NMR (500 MHz DMSO-d6):
δ
(ppm) 9.18 (s, 1H), 8.03 (d, J = 2.2 Hz,
1H), 7.63 (d, J = 8.6 Hz, 1H), 7.38 (ddd, J = 8.6, 2.2, 0.5 Hz, 1H), 7.03 (d, J = 2.1 Hz, 1H), 6.73–6.64 (m, 2H);
13C NMR (126 MHz, DMSO-d6):
δ (ppm) 160.17, 151.60, 147.56, 145.29, 141.39, 133.14, 130.10, 126.75,
126.13, 121.13, 120.70, 115.58, 110.12, 107.69; ESI-HRMS: m/z 336.0202 [M+H]+ (calc. for C14H10ClN3O3S:
336.0204 [M+H]+).
1-(6-chlorobenzo[d]thiazol-2-yl)-3-(4-hydroxy-3-methoxyphenyl)urea (4ah)

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Yield 98%; mp: 257–259 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 9.42 (s, 1H), 8.03 (d, J = 2.2 Hz,
1H), 7.63 (d, J = 8.6 Hz, 1H), 7.39 (dd, J = 8.6, 2.2 Hz, 1H), 7.17 (d, J = 2.4 Hz, 1H), 6.82 (dd, J = 8.5,
2.4 Hz, 1H), 6.73 (d, J = 8.4 Hz, 1H), 3.77 (s, 3H); 13C NMR (126 MHz, DMSO-d6):
δ
(ppm) 160.19,
151.85, 147.51, 147.47, 142.61, 133.11, 130.21, 126.78, 126.16, 121.15, 120.68, 115.45, 111.74, 104.76, 55.61;
ESI-HRMS: m/z 350.0357 [M+H]+ (calc. for C15H12ClN3O3S: 350.0361 [M+H]+).
1-(6-chlorobenzo[d]thiazol-2-yl)-3-(3-hydroxy-4-methoxyphenyl)urea (4ai)
Yield 79%; mp: 281–283 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 10.77 (s, 1H), 9.12 (s, 1H), 8.88
(s, 1H), 8.04 (d, J = 2.1 Hz, 1H), 7.63 (d, J = 8.7 Hz, 1H), 7.39 (dd, J = 8.7, 2.2 Hz, 1H), 7.07 (d, J = 2.4 Hz,
1H), 6.86 (d, J = 8.8 Hz, 1H), 6.82 (dd, J = 8.7, 2.4 Hz, 1H), 3.73 (s, 3H); 13C NMR (126 MHz, DMSO-d6):
δ
(ppm) 160.23, 151.67, 147.38, 146.73, 143.85, 133.06, 131.68, 126.81, 126.15, 121.16, 120.65, 112.81, 109.67,
107.56, 55.96; ESI-HRMS: m/z 350.0359 [M+H]+ (calc. for C15H12ClN3O3S: 350.0361 [M+H]+).
1-(3-chloro-4-methoxyphenyl)-3-(6-hydroxybenzo[d]thiazol-2-yl)urea (4aj)
Yield 31%; mp: 290–292 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ (ppm) 10.68 (s, 1H), 9.43 (s, 1H),
9.09 (s, 1H), 7.69 (d, J = 2.6 Hz, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.35 (dd, J = 8.9, 2.5 Hz, 1H), 7.22 (d,
J = 2.4 Hz, 1H), 7.11 (d, J = 9.0 Hz, 1H), 6.84 (dd, J = 8.6, 2.4 Hz, 1H), 3.83 (s, 3H); 13C NMR (126 MHz,
DMSO-d6):
δ (ppm) 157.00, 153.67, 152.21, 150.31, 141.07, 132.26, 120.82, 120.67, 119.86, 118.95, 114.74,
113.09, 106.68, 56.20; ESI-HRMS: m/z 350.0354 [M+H]+ (calc. for C15H12ClN3O3S: 350.0361 [M+H]+).
1-(3-chloro-4-hydroxyphenyl)-3-(6-chlorobenzo[d]thiazol-2-yl)thiourea (4ak)
Yield 80%; mp: 232.5–234 ◦C; 1H NMR (300 MHz, DMSO-d6):
δ
(ppm) 12.60 (s, 1H), 10.67 (s, 1H),
10.15 (s, 1H), 8.00 (s, 1H), 7.75–7.27 (m, 4H), 6.95 (d, J = 8.7 Hz, 1H); 13C NMR (75 MHz, DMSO-d6):
δ
(ppm) 150.68, 131.10, 127.32, 126.75, 125.22, 124.04, 121.93, 118.86, 116.10, 114.96; ESI-HRMS: m/z
369.9637 [M+H]+ (calc. for C14H9Cl2N3OS2: 369.9637 [M+H]+).
1-(3-chloro-4-hydroxyphenyl)-3-(6-chlorobenzo[d]thiazol-2-yl)guanidine (4al)
Yield 38%; mp: 274–275 ◦C; 1H NMR (300 MHz, DMSO-d6):
δ
(ppm) 10.58 (s, 1H), 8.68 (s, 1H),
8.09 (d, J = 2.0 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.52–7.40 (m, 2H), 7.18 (dd, J = 8.7, 2.5 Hz, 1H), 7.11 (d,
J = 8.7 Hz, 1H); 13C NMR (75 MHz, DMSO-d6):
δ (ppm) 164.14, 155.06, 152.54, 130.55, 128.14, 127.02,
126.42, 125.50, 121.91, 119.91, 117.09; ESI-HRMS: m/z 353.0021 [M+H]+ (calc. for C14H10Cl2N4OS:
353.0025 [M+H]+).
1-(3-chloro-4-hydroxyphenyl)-3-(4-chlorobenzo[d]thiazol-2-yl)urea (4am)
Yield 54%; mp: 237–239 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 11.32 (br s, 1H), 9.97 (br
s, 1H), 8.83 (s, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.57 (d, J = 2.5 Hz, 1H), 7.47 (d, J = 7.2 Hz, 1H), 7.22 (t,
J = 7.9 Hz, 1H), 7.17 (dd, J = 8.7, 2.6 Hz, 1H), 6.93 (d, J = 8.7 Hz, 1H); 13C NMR (126 MHz, DMSO-d6):
δ
(ppm) 160.35, 151.59, 149.20, 145.83, 133.10, 130.33, 125.98, 123.70, 123.65, 121.18, 120.54, 119.83, 119.35,
116.66; ESI-HRMS: m/z 353.9861 [M+H]+ (calc. for C14H9Cl2N3O2S: 353.9865 [M+H]+).
1-(3-chloro-4-hydroxyphenyl)-3-(5-chlorobenzo[d]thiazol-2-yl)urea (4an)
Yield 49%; mp: 317.5–319 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 10.97 (s, 1H), 9.98 (s, 1H),
9.01 (s, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.70 (s, 1H), 7.59 (d, J = 2.5 Hz, 1H), 7.27 (dd, J = 8.4, 2.0 Hz,
1H), 7.18 (dd, J = 8.7, 2.4 Hz, 1H), 6.93 (d, J = 8.7 Hz, 1H); 13C NMR (126 MHz, DMSO-d6):
(ppm)
δ
161.45, 151.87, 149.93, 149.15, 130.68, 130.53, 130.17, 123.03, 122.84, 121.03, 119.70, 119.40, 119.09, 116.71;
ESI-HRMS: m/z 353.9858 [M+H]+ (calc. for C14H9Cl2N3O2S: 353.9865 [M+H]+).
1-(3-chloro-4-hydroxyphenyl)-3-(7-chlorobenzo[d]thiazol-2-yl)urea (4ao)
Yield 52%; mp: 272–274 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 9.54 (s, 1H), 7.69–7.53 (m, 2H),
7.41 (t, J = 7.9 Hz, 1H), 7.32 (d, J = 7.8 Hz, 1H), 7.19 (dd, J = 8.7, 2.2 Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H); 13
C
NMR (126 MHz, DMSO-d6):
δ (ppm) 159.38, 152.20, 149.29, 149.05, 130.63, 130.58, 127.39, 125.23, 122.50,
120.65, 119.38, 119.30, 118.20, 116.72; ESI-HRMS: m/z 353.9857 [M+H]+ (calc. for C14H9Cl2N3O2S:
353.9865 [M+H]+).
1-(benzo[d]oxazol-2-yl)-3-(3-chloro-4-hydroxyphenyl)urea (4ap)
Yield 59%; mp: 190.5–191.5 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 11.33 (s, 1H), 10.22 (s,
1H), 9.94 (s, 1H), 7.70 (d, J = 1.5 Hz, 1H), 7.60–7.47 (m, 2H), 7.33–7.18 (m, 3H), 6.95 (d, J = 8.7 Hz, 1H);
13C NMR (126 MHz, DMSO-d6):
(ppm) 156.78, 149.75, 149.39, 147.01, 140.03, 130.11, 124.62, 123.09,
δ

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121.47, 120.07, 119.34, 117.45, 116.63, 109.90; ESI-HRMS: m/z 304.0481 [M+H]+ (calc. for C14H10ClN3O3:
304.0484 [M+H]+).
1-(3-chloro-4-hydroxyphenyl)-3-(5-chlorobenzo[d]oxazol-2-yl)urea (4aq)
◦
1
Yield 89%; mp: 176–178 C; H NMR (500 MHz, DMSO-d6):
δ (ppm) 10.17 (s, 1H), 7.65 (d,
J = 2.5 Hz, 1H), 7.62–7.57 (m, 2H), 7.27–7.23 (m, 2H), 6.97 (d, J = 8.7 Hz, 1H); 13C NMR (126 MHz,
DMSO-d6):
δ (ppm) 158.08, 151.04, 149.25, 145.39, 140.22, 130.54, 128.71, 122.81, 121.11, 119.67, 119.35,
116.68, 116.33, 111.18; ESI-HRMS: m/z 338.0090 [M+H]+ (calc. for C14H9Cl2N3O3: 338.0094 [M+H]+).
1-(3-chloro-4-hydroxyphenyl)-3-(6-chlorobenzo[d]oxazol-2-yl)urea (4ar)
Yield 16%; mp: 188.5–190.5 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 11.46 (s, 1H), 10.21 (s, 1H),
10.00 (s, 1H), 7.77 (d, J = 1.7 Hz, 1H), 7.67 (d, J = 2.4 Hz, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.34 (dd, J = 8.4,
2.0 Hz, 1H), 7.26 (dd, J = 8.8, 2.6 Hz, 1H), 6.94 (d, J = 8.7 Hz, 1H); 13C NMR (126 MHz, DMSO-d6):
δ
(ppm) 157.70, 152.28, 149.23, 146.84, 138.05, 130.53, 126.96, 124.83, 121.26, 119.88, 119.34, 116.65, 116.62,
110.61; ESI-HRMS: m/z 338.0091 [M+H]+ (calc. for C14H9Cl2N3O3: 338.0094 [M+H]+).
1-(1H-benzo[d]imidazol-2-yl)-3-(3-chloro-4-hydroxyphenyl)urea (4as)
◦
1
Yield 90%; mp: 264–266 C; H NMR (500 MHz, DMSO-d6):
δ (ppm) 10.78 (br s, 3H), 9.43 (s,
1H), 7.73 (d, J = 2.6 Hz, 1H), 7.39–7.33 (m, 2H), 7.21 (dd, J = 8.7, 2.6 Hz, 1H), 7.08–7.02 (m, 2H), 6.91
(d, J = 8.7 Hz, 1H); 13C NMR (126 MHz, DMSO-d6):
δ (ppm) δ 154.49, 149.23, 148.33, 134.62, 131.84,
120.96, 120.38, 119.27, 118.93, 116.61, 112.69; ESI-HRMS: m/z 303.0645 [M+H]+ (calc. for C14H11ClN4O2:
303.0643 [M+H]+).
1-(5-chloro-1H-benzo[d]imidazol-2-yl)-3-(3-chloro-4-hydroxyphenyl)urea (4at)
Yield 37%; mp: 256–258 ◦C; 1H NMR (300 MHz, DMSO-d6):
δ
(ppm) 10.98 (s, 1H), 9.94 (s, 1H),
9.34 (s, 1H), 7.69 (d, J = 2.5 Hz, 1H), 7.40 (d, J = 1.9 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.18 (dd, J = 8.8,
2.5 Hz, 1H), 7.07 (dd, J = 8.4, 2.0 Hz, 1H), 6.93 (d, J = 8.7 Hz, 1H); 13C NMR (75 MHz, DMSO-d6):
δ
(ppm) 152.91, 149.27, 148.69, 136.85, 134.35, 131.21, 125.03, 120.78, 120.64, 119.32, 119.19, 116.66, 114.15,
113.01; ESI-HRMS: m/z 337.0251 [M+H]+ (calc. for C14H10Cl2N4O2: 337.0254 [M+H]+).
1-(benzo[d]thiazol-2-yl)-3-(3-chloro-4-hydroxyphenyl)urea (4au)
Yield 55%; mp: 115–117 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ (ppm) 10.90 (br s, 1H), 9.92 (s, 1H),
9.03 (s, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 2.4 Hz, 1H), 7.38 (t, J = 7.6 Hz,
1H), 7.23 (t, J = 7.6 Hz, 1H), 7.20 (dd, J = 8.7, 2.3 Hz, 1H), 6.94 (d, J = 8.7 Hz, 1H); 13C NMR (126 MHz,
DMSO-d6):
δ (ppm) 159.87, 152.35, 148.93, 148.17, 130.78, 125.95, 122.84, 121.52, 120.80, 119.46, 119.33,
116.65; ESI-HRMS: m/z 320.0252 [M+H]+ (calc. for C14H10ClN3O2S: 320.0255 [M+H]+).
1-(5-bromobenzo[d]thiazol-2-yl)-3-(3-chloro-4-hydroxyphenyl)urea (4av)
Yield 50%; mp: 319–321 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 11.18 (br s, 1H), 9.98 (br s,
1H), 9.03 (s, 1H), 7.88 (d, J = 8.5 Hz, 1H), 7.84 (d, J = 1.2 Hz, 1H), 7.59 (d, J = 2.4 Hz, 1H), 7.39 (dd, J = 8.5,
1.2 Hz, 1H), 7.19 (dd, J = 8.9, 2.4 Hz, 1H), 6.94 (d, J = 8.9 Hz, 1H); 13C NMR (126 MHz, DMSO-d6):
δ
(ppm) 161.26, 152.03, 149.78, 149.10, 130.49, 125.41, 123.32, 121.80, 120.98, 119.65, 119.34, 118.65, 116.65;
ESI-HRMS: m/z 397.9359 [M+H]+ (calc. for C14H9BrClN3O2S: 397.9360 [M+H]+).
1-(6-bromobenzo[d]thiazol-2-yl)-3-(3-chloro-4-hydroxyphenyl)urea (4aw)
Yield 38%; mp: 290–292 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 11.02 (br s, 1H), 9.94 (br s,
1H), 9.00 (s, 1H), 8.17 (s, 1H), 7.55 (dd, J = 30.6, 9.7 Hz, 3H), 7.18 (d, J = 8.2 Hz, 1H), 6.93 (d, J = 8.5 Hz,
1H); 13C NMR (126 MHz, DMSO-d6):
δ (ppm) 160.40, 152.20, 149.06, 133.39, 130.54, 128.88, 124.02,
120.92, 119.58, 119.33, 116.65, 114.66; ESI-HRMS: m/z 397.9360 [M+H]+ (calc. for C14H9BrClN3O2S:
397.9360 [M+H]+).
1-(3-chloro-4-hydroxyphenyl)-3-(6-iodobenzo[d]thiazol-2-yl)urea (4ax)
Yield 58%; mp: 271–273 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 10.86 (br s, 1H), 9.94 (s, 1H),
9.01 (s, 1H), 8.30 (s, 1H), 7.66 (dd, J = 8.4, 1.6 Hz, 1H), 7.59 (d, J = 2.2 Hz, 1H), 7.44 (d, J = 8.2 Hz,
1H), 7.18 (dd, J = 8.6, 1.8 Hz, 1H), 6.93 (d, J = 8.7 Hz, 1H); 13C NMR (126 MHz, DMSO-d6):
(ppm)
δ
160.16, 151.89, 149.04, 134.50, 133.90, 130.59, 129.67, 120.90, 119.56, 119.33, 116.65, 86.22; ESI-HRMS: m/z
445.9215 [M+H]+ (calc. for C14H9ClIN3O2S: 445.9221 [M+H]+).
1-(3-chloro-4-hydroxyphenyl)-3-(6-methylbenzo[d]thiazol-2-yl)urea (4ay)

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Yield 61%; mp: 281–283 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 10.87 (br s, 1H), 9.91 (br s,
1H), 9.03 (s, 1H), 7.67 (s, 1H), 7.61 (d, J = 2.1 Hz, 1H), 7.51 (d, J = 8.3 Hz, 1H), 7.19 (d, J = 8.8 Hz, 2H),
6.93 (d, J = 8.9 Hz, 1H), 2.38 (s, 3H); 13C NMR (126 MHz, DMSO-d6):
δ
(ppm) 159.13, 152.44, 148.89,
145.54, 132.20, 131.02, 130.83, 127.14, 121.22, 120.74, 119.39, 119.33, 118.70, 116.65, 20.88; ESI-HRMS: m/z
334.0407 [M+H]+ (calc. for C15H12ClN3O2S: 334.0412 [M+H]+).
1-(3-chloro-4-hydroxyphenyl)-3-(6-(trifluoromethyl)benzo[d]thiazol-2-yl)urea (4az)
Yield 75%; mp: 223–225 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 9.78 (s, 1H), 8.40 (s, 1H), 7.80
(d, J = 8.5 Hz, 1H), 7.68 (dd, J = 8.5, 1.7 Hz, 1H), 7.59 (d, J = 2.5 Hz, 1H), 7.19 (dd, J = 8.6, 2.4 Hz, 1H),
6.96 (d, J = 8.7 Hz, 1H); 13C NMR (126 MHz, DMSO-d6):
(ppm) 162.41, 152.08, 151.18, 149.08, 131.92,
δ
130.63, 124.67 (q, J = 271.8 Hz), 123.05 (q, J = 31.9 Hz), 122.83 (q, J = 3.5 Hz), 120.59, 119.73, 119.51
(q, J = 4.0 Hz), 119.40, 119.25, 116.77; 19F NMR (471 MHz, DMSO-d6):
388.0126 [M+H]+ (calc. for C15H9ClF3N3O2S: 388.0129 [M+H]+).
δ (ppm) -58.8; ESI-HRMS: m/z
1-(3-chloro-4-hydroxyphenyl)-3-(6-methoxybenzo[d]thiazol-2-yl)urea (4ba)
Yield 63%; mp: 281–283 ◦C; 1H NMR (500 MHz, DMSO-d6):
(ppm) 9.33 (s, 1H), 7.69–7.40 (m,
3H), 7.17 (d, J = 7.5 Hz, 1H), 7.06–6.86 (m, 2H), 3.79 (s, 3H); 13C NMR (126 MHz, DMSO-d6):
(ppm)
δ
δ
157.70, 155.70, 152.14, 148.88, 142.05, 132.34, 130.85, 120.57, 119.92, 119.35, 119.26, 116.71, 114.42, 104.95,
55.63; ESI-HRMS: m/z 350.0357 [M+H]+ (calc. for C15H12ClN3O3S: 350.0361 [M+H]+).
1-(3-chloro-4-hydroxyphenyl)-3-(6-hydroxybenzo[d]thiazol-2-yl)urea (4bb)
Yield 85%; mp: 249–250 ◦C; 1H NMR (300 MHz, DMSO-d6):
δ (ppm) 10.57 (s, 1H), 9.91 (s, 1H),
9.44 (s, 1H), 8.98 (s, 1H), 7.59 (d, J = 2.5 Hz, 1H), 7.44 (d, J = 8.6 Hz, 1H), 7.22 (d, J = 2.3 Hz, 1H), 7.17
(dd, J = 8.8, 2.5 Hz, 1H), 6.92 (d, J = 8.7 Hz, 1H), 6.83 (dd, J = 8.7, 2.5 Hz, 1H); 13C NMR (75 MHz,
DMSO-d6):
δ (ppm) 156.96, 153.68, 152.29, 148.86, 141.49, 132.31, 130.88, 120.71, 120.02, 119.38, 119.33,
116.67, 114.75, 106.68; ESI-HRMS: m/z 336.0199 [M+H]+ (calc. for C14H10ClN3O3S: 336.0204 [M+H]+).
1-(6-acetylbenzo[d]thiazol-2-yl)-3-(3-chloro-4-hydroxyphenyl)urea (4bc)
Yield 56%; mp: 242–244 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ (ppm) 11.20 (br s, 1H), 9.96 (br s,
1H), 9.06 (s, 1H), 8.57 (d, J = 1.4 Hz, 1H), 7.96 (dd, J = 8.5, 1.6 Hz, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.61 (d,
J = 2.5 Hz, 1H), 7.20 (dd, J = 8.7, 2.5 Hz, 1H), 6.94 (d, J = 8.7 Hz, 1H), 2.61 (s, 3H); 13C NMR (126 MHz,
DMSO-d6):
δ (ppm) 196.69, 163.07, 152.33, 151.37, 149.12, 131.71, 131.28, 130.57, 126.24, 122.96, 120.98,
119.64, 119.38, 118.71, 116.68, 26.72; ESI-HRMS: m/z 362.0356 [M+H]+ (calc. for C16H12ClN3O3S:
362.0361 [M+H]+).
methyl 2-(3-(3-chloro-4-hydroxyphenyl)ureido)benzo[d]thiazole-6-carboxylate (4bd)
Yield 84%; mp: 278–280 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 11.14 (br s, 1H), 9.95 (s, 1H),
9.04 (s, 1H), 8.54 (s, 1H), 7.96 (dd, J = 8.5, 1.7 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 2.5 Hz, 1H),
7.20 (dd, J = 8.7, 2.4 Hz, 1H), 6.94 (d, J = 8.7 Hz, 1H), 3.86 (s, 3H); 13C NMR (126 MHz, DMSO-d6):
δ
(ppm) 166.00, 163.02, 152.18, 149.09, 131.35, 130.54, 127.11, 123.84, 123.49, 120.92, 119.58, 119.34, 116.65,
52.07; ESI-HRMS: m/z 378.0306 [M+H]+ (calc. for C16H12ClN3O4S: 378.0309 [M+H]+).
1-(3-chloro-4-hydroxyphenyl)-3-(6-cyanobenzo[d]thiazol-2-yl)urea (4be)
Yield 79%; mp: 309–311 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 10.98 br (s, 1H), 9.74 (br s,
1H), 8.99 (s, 1H), 8.32 (s, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.68–7.64 (m, 1H), 7.55 (d, J = 2.5 Hz, 1H), 7.13 (dd,
J = 8.7, 2.5 Hz, 1H), 6.90 (d, J = 8.7 Hz, 1H); 13C NMR (126 MHz, DMSO-d6):
(ppm) 162.89, 151.58,
δ
149.07, 132.11, 130.10, 129.10, 125.95, 120.83, 119.83, 119.41, 119.18, 118.89, 116.37, 104.59; ESI-HRMS:
m/z 345.0204 [M+H]+ (calc. for C15H9ClN4O2S: 345.0208 [M+H]+).
1-(3-chloro-4-hydroxyphenyl)-3-(6-nitrobenzo[d]thiazol-2-yl)urea (4bf)
Yield 52%; mp: 277–279 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 11.53 (br s, 1H), 9.99 (br s,
1H), 9.15 (s, 1H), 8.95 (d, J = 2.2 Hz, 1H), 8.22 (dd, J = 8.9, 2.3 Hz, 1H), 7.77 (d, J = 8.9 Hz, 1H), 7.59 (d,
J = 2.4 Hz, 1H), 7.20 (dd, J = 8.7, 2.5 Hz, 1H), 6.94 (d, J = 8.7 Hz, 1H); 13C NMR (126 MHz, DMSO-d6):
δ
(ppm) 164.86, 153.25, 152.02, 149.25, 142.44, 132.01, 130.32, 121.78, 121.05, 119.71, 119.37, 119.22, 118.64,
116.67; ESI-HRMS: m/z 365.0106 [M+H]+ (calc. for C14H9ClN4O4S: 365.0106 [M+H]+).
1-(6-aminobenzo[d]thiazol-2-yl)-3-(3-chloro-4-hydroxyphenyl)urea (4bg)

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Yield 77%; mp: 223–224 ◦C; 1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 10.48 (s, 1H), 9.88 (s, 1H),
8.98 (s, 1H), 7.60 (d, J = 2.6 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.16 (dd, J = 8.7, 2.6 Hz, 1H), 6.97 (d,
J = 2.2 Hz, 1H), 6.92 (d, J = 8.7 Hz, 1H), 6.66 (dd, J = 8.5, 2.2 Hz, 1H), 5.07 (br s, 2H); 13C NMR (126 MHz,
DMSO-d6):
δ (ppm) 155.24, 152.15, 148.75, 145.19, 139.03, 132.30, 130.98, 120.62, 119.61, 119.33, 119.28,
116.66, 114.00, 104.50; ESI-HRMS: m/z 335.0363 [M+H]+ (calc. for C14H11ClN4O2S: 335.0364 [M+H]+).
4.2. Biological Evaluation
4.2.1. Recombinant Production of Human 17β-HSD10 Enzyme
The recombinant 17β-HSD10 enzyme was produced in Escherichia coli (E. coli) BL21 (DE3) strain
and purified to homogeneity. Briefly, the DNA encoding the HSD17B10 sequence was obtained from
UniProtKB server (accession number: Q99714), codon optimized for E. coli as host cells, and produced
in GeneArt Synthesis service (Thermo Fisher Scientific). The enzyme coding sequence was amplified
by PCR using a pair of specific primers introducing restriction endonuclease recognition sites to both
ends, and the sequence was verified by Sanger sequencing (ABI Prism 3130xl). The PCR product was
subcloned into expression vector pET-28b(+) (Novagen) in frame with N-terminal hexa-histidine tag
coding sequence. The Overnight ExpressTM TB medium was used for enzyme autoinduction and
overexpression at 25 ◦C for 18 h. The bacterial cells were harvested using centrifugation (10,000
×
g,
10 min) and the bacterial pellet was lysed by sonication in combination with 1 mg/mL lysozyme in
50 mM sodium phosphate buffer, pH 8.0, 500 mM NaCl, 30 mM imidazole buffer containing 150 U/mL
benzonase, and Complete EDTA-free protease inhibitor cocktail (Sigma). The cleared crude lysate
was loaded onto Ni-NTA agarose (Qiagen, Hilden, Germany) for affinity purification under native
conditions using a hexahistidine tag. After several washing steps with low-concentration imidazole
buffer (50 mM sodium phosphate buffer, pH 8.0, 150 mM NaCl, 30 mM imidazole), elution with a high
concentration of imidazole (250 mM imidazole) was performed. The high concentration of imidazole
in elution buffer was removed by Amicon Ultra-15 units with a 10 kDa cut-off by using buffer exchange
protocol, and the protein was stored immersed in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 30%
glycerol at –80 ◦C for long-term storage.
4.2.2. Recombinant Protein Analyses
For the confirmation of the presence and purity of the recombinant protein, the final protein
fractions were analyzed by using SDS-PAGE followed by semi-dry western blotting. The purified
recombinant protein was loaded onto a 12% SDS-PAGE gel, electrophoresed, and transferred onto a
polyvinylidene fluoride membrane. The membrane was blocked with 3% non-fat dry milk (Bio-Rad,
Hercules, CA, USA) and probed with mouse monoclonal antibody specific to 17β-HSD10 protein
(dilution 1:500, Santa Cruz Biotechnology, Dallas, TX, USA, sc-393693) followed by the incubation
with anti-mouse binding protein conjugated with horseradish peroxidase (dilution 1:5000, SantaCruz
Biotechnology, sc-516102). Protein bands were visualized by the application of ECL Prime Western
blotting detection reagent (GE Healthcare, Chicago, IL, USA), and the images were captured using
Azure c400 imaging system (Azure Biosystem, Dublin, CA, USA). The protein concentration was
determined by using a MicroBCA kit (ThermoFisher Scientific).
4.2.3. AAC Reductase Assay and Enzyme Kinetics
The enzymatic activity of the recombinant protein was determined by using AAC as a substrate for
enzyme reduction. To determine the Michaelis constant Km and maximal rate of enzyme reaction Vmax
,
different substrate concentrations ranging from 0 to 500 M were incubated with a fixed concentration
µ
of cofactor and enzyme. The reaction readout was absorbance at 340 nm for 5 min in 20 s intervals at
37 ◦C. The data were analyzed by GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, USA)
and Vmax and Km were calculated by non-linear regression analysis using the reaction velocity and

Int. J. Mol. Sci. 2020, 21, 2059
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substrate concentration data. All reactions were measured in triplicate, and the values were expressed
as means ± SD.
The general reaction mixture for the reductase activity measurement consisted of 320
µ
M AAC,
g of recombinant enzyme in assay buffer (10 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, 0.001% Tween 20 and 0.01% bovine serum
albumin) in 100 L of reaction buffer. The process was as follows: the reaction mixture of enzyme and
320 µM NADH as the enzyme cofactor and 0.15 µ
µ
cofactor in assay buffer was preincubated for 5 min at 37 ◦C prior to substrate addition. The reaction
was performed in 96-well polystyrene plates at 37 ◦C (Tecan Spark 10M, Männedorf, Switzerland), and
enzyme activity was recorded as the change in absorbance at 340 nm over 1 min and calculated using
the molar extinction coefficient of NADH (ε = 6220 M−1·cm−1).
4.2.4. 17β-HSD10 Enzyme Inhibition Screening and IC50 Determination
The novel compounds were screened at 10
µM or 1 µM to determine their ability to inhibit
17
β-HSD10 enzyme. The tested compounds were dissolved in dimethyl sulfoxide (DMSO) to a stock
concentration of 10 mM and then diluted into deionized water to get the working concentration.
The inhibitory screening was based on AAC reductase assay. The remaining enzyme activity was
measured at 340 nm at 20 s intervals for 5 min and determined as the difference in absorbance between
inhibited and non-inhibited enzymatic reaction. The DMSO was used as well as the inhibitor as the
vehicle control. The potent known 17
enzymatic reaction.
β-HSD10 inhibitor AG18051 [19] was used as a control for the
For compounds with at least 50% 17
β
-HSD10 inhibition in the 10 M screening assay, the IC50 was
µ
determined. For this purpose, the AAC reductase assay was used and the remaining enzyme activity
was measured as dose-response inhibition with at least six different inhibitor concentrations (ranging
from 0 to 3 µM) at fixed substrate, enzyme, and cofactor concentrations. The data were analyzed by
using GraphPad Prism 7 software in non-linear regression, and IC50 values were calculated for each
inhibitor from at least three independent measurements, all in triplicate.
4.2.5. Determination of Inhibition Type
The determination of the inhibition type for three most potent compounds was made by using
an AAC reductase activity assay. The inhibitors were used at different concentrations (0–3
µM) in
combination with different substrate concentrations (25–400 M) and saturated NADH concentration
µ
to determine the cofactor-based absorbance change measured at 340 nm. The uninhibited enzymatic
reaction contained the same DMSO concentration as the inhibited one and was used as the vehicle control.
For data evaluation, the linearization of Lineweaver-Burk and Hanes-Wolf was used. To determine the
binding mechanism more precisely, the Km and Vmax values of inhibited reactions were compared
with uninhibited reactions.
5. Conclusions
In conclusion, we have prepared and in vitro evaluated over 50 novel compounds based on a
benzothiazolyl urea scaffold acting as 17β-HSD10 inhibitors. SAR study revealed crucial structural
aspects required for good inhibitory potency, namely 3-chlorine and 4-hydroxy substitution on the
phenyl ring, urea linker and small substituent at position 6 of the benzothiazole moiety. The most
potent inhibitors were able to inhibit 17
being ten times more potent compared to parental inhibitors. The novel compounds were found to be
uncompetitive inhibitors of 17 -HSD10 with selectivity towards the enzyme-substrate complex, which
β-HSD10 activity with IC50 at low micromolar level, thus
β
makes them interesting for further research and development as potential drugs.
Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/6/2059/s1.

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Author Contributions: Conceptualization, M.S., O.B. and K.M.; Data curation, M.S. and L.V.; Funding acquisition,
K.K. and K.M.; Methodology, M.S., O.B., L.V., M.H., M.C., V.K., L.H., R.D., A.L., L.P. and D.J.; Project administration,
K.K. and K.M.; Supervision, F.G.-M., K.K. and K.M.; Writing—original draft, M.S., O.B., L.V., L.Z. and K.M.;
Writing—review and editing, M.S., O.B., L.V., L.Z., L.A. and K.M. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by Ministry of Health of the Czech Republic (no. NV19-09-00578), Ministry of
Education, Youth and Sports of the Czech Republic (project ESF no. CZ.02.1.01/0.0/0.0/18_069/0010054), University
of Hradec Kralove (Faculty of Science, no. VT2019-2021, SV2115-2018, and Postdoctoral job positions at UHK),
University Hospital Hradec Kralove (UHHK, 00179906), National Institute of Mental Health (NIMH-CZ) project
no. LO1611 for financial support from MEYS under the program of NPU I, Wellcome Trust (204821/Z/16/Z),
The Rosetrees Trust, and the RS MacDonald Charitable Trust.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
17β-HSD10
SDR
Aβ
17β-hydroxysteroid dehydrogenase type 10
short-chain dehydrogenases/reductases
amyloid-beta peptide
ERAB
ABAD
HADH2
SCHAD
APP
endoplasmic reticulum-associated binding protein
amyloid-beta binding alcohol dehydrogenase
L-3-hydroxyacyl-CoA dehydrogenase
short chain to L-3-hydroxyacyl-CoA dehydrogenase
amyloid-beta precursor protein
half maximal inhibitory concentration
structure–activity relationship
IC50
SAR
AAC
acetoacetyl-CoA
NADH
Km
nicotinamide adenine dinucleotide
Michaelis constant
Vmax
maximum reaction rate
DMSO
dimethyl sulfoxide
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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