New dual ATP-competitive inhibitors of bacterial DNA gyrase and topoisomerase IV active against ESKAPE pathogens

^cDrug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, P.O. Box 56 (Viikinkaari 5 E), 00014, Helsinki,

Finland

^dSARomics Biostructures, Medicon Village, Lund, Sweden

^eUniversity of Hradec Kralove, Faculty of Science, Department of Chemistry, Rokitanskeho 62, 500 03, Hradec Kralove, Czech Republic

a r t i c l e i n f o

a b s t r a c t

Article history:

The rise in multidrug-resistant bacteria deﬁnes the need for identiﬁcation of new antibacterial agents

that are less prone to resistance acquisition. Compounds that simultaneously inhibit multiple bacterial

targets are more likely to suppress the evolution of target-based resistance than monotargeting com-

pounds. The structurally similar ATP binding sites of DNA gyrase and topoisomerase Ⅳ offer an oppor-

tunity to accomplish this goal. Here we present the design and structure-activity relationship analysis of

balanced, low nanomolar inhibitors of bacterial DNA gyrase and topoisomerase IV that show potent

antibacterial activities against the ESKAPE pathogens. For inhibitor 31c, a crystal structure in complex

with Staphylococcus aureus DNA gyrase B was obtained that conﬁrms the mode of action of these

compounds. The best inhibitor, 31h, does not show any in vitro cytotoxicity and has excellent potency

Received 6 October 2020

Received in revised form

10 November 2020

Accepted 12 January 2021

Available online 22 January 2021

Keywords:

Dual inhibitor

DNA gyrase

Topoisomerase IV

Benzothiazole

Antibacterial

against Gram-positive (MICs: range, 0.0078e0.0625

mg/mL) and Gram-negative pathogens (MICs: range,

1e2 g/mL). Furthermore, 31h inhibits GyrB mutants that can develop resistance to other drugs. Based

m

on these data, we expect that structural derivatives of 31h will represent a step toward clinically efﬁ-

cacious multitargeting antimicrobials that are not impacted by existing antimicrobial resistance.

1. Introduction

use of each antibiotic was soon followed by the development of

resistance in the target bacterial species [1]. With inappropriate

The discovery of antibiotics led to tremendous improvements in

the success rates of treating bacterial infections, and the conse-

quent reduced mortality. This big step forward was also seen for

other medical areas, as safe organ transplants and cancer and heart

disease surgery then became possible [1,2]. Not surprisingly, the

use, and indeed major overuse mainly in treating livestock, the

problem of antibacterial resistance escalated [3]. One of the major

threats both in hospitals and in the community are the ‘ESKAPE’

pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella

pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa,

Enterobacter species) [2,4]. Most of these pathogens are included in

the World Health Organization priority list of pathogens for which

new therapies are urgently needed [5].

At the same time, the pharmaceutical industry has been with-

drawing from antibacterial research and development, due to the

high costs of the research needed and the low potential return as a

result of the development of bacterial resistance [6e8]. On a

** Corresponding author.

* Corresponding author.

ꢁ ꢁ

E-mail addresses: Lucija.PeterlinMasic@ffa.uni-lj.si (L.P. Masic), Tihomir.

ꢁ ꢁ

Tomasic@ffa.uni-lj.si (T. Tomasic).

1

Present address: Department of Genetics, Harvard Medical School, Boston, MA

02215, USA.

https://doi.org/10.1016/j.ejmech.2021.113200

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M. Durcik, A. Nyerges, Z. Skok et al.

European Journal of Medicinal Chemistry 213 (2021) 113200

Abbreviations

LDH

lactate dehydrogenase

MCF-7

MH

MRSA

NMM

NMP

ParC

breast cancer cell line

MuellereHinton

ATCC

DCM

DMF

DMSO

GHKL

GyrA

GyrB

American Type Culture Collection

dichloromethane

N,N-dimethylformamide

dimethylsulfoxide

gyrase Hsp90 histidine kinase and MutL

DNA gyrase subunit A

methicillin-resistant Staphylococcus aureus

N-methylmorpholine

N-methyl-2-pyrrolidone;

topoisomerase IV subunit C

topoisomerase IV subunit E

phenylalanine-arginine

triﬂuoroacetic acid;

tetrahydrofuran;

ParE

DNA gyrase subunit B

PA

b

N

b-naphthylamide

EDC

N-(3-Dimethylaminopropyl)-N⁰-ethylcarbodiimide

human hepatocellular carcinoma cell line

hydroxybenzotriazole

TFA

THF

Topo IV

VISA

HepG2

HOBt

hTopoII

topoisomerase IV

vancomycin-intermediate S. aureus

a

human DNA topoisomerase II

a

positive note, initiatives have been promoted to boost research and

development into new antibacterials, such as the Innovative Med-

icines Initiative of ‘New Drugs for Bad Bugs’ programme, the

‘Combating Antibiotic-Resistant Bacteria Biopharmaceutical

Accelerator’, the ‘Joint Programming Initiative on Antimicrobial

Resistance’, and others [9,10].

inhibitor of this series, 31h, showed potent low nanomolar dual

inhibition of E. coli and S. aureus DNA gyrase and TopoⅣ, selectivity

against human DNA topoisomerase IIa (hTopoIIa), broad spectrum

antibacterial activity including against GyrB mutants and clinical

isolates, and no in vitro cytotoxicity. In the in vitro metabolic study

using an S9 fraction, we also identiﬁed the main metabolites of 31h,

with no reactive metabolites detected.

The bacterial enzymes DNA gyrase and topoisomerase Ⅳ

(TopoⅣ) are validated targets for the development of new anti-

bacterial agents. Both are heterotetrameric enzymes that catalyze

changes in DNA topology during DNA replication, recombination,

and transcription [1 1]. DNA gyrase consists of two GyrA and two

GyrB subunits, while its paralog TopoⅣ consists of two ParC and

two ParE subunits. The GyrA and ParC subunits are responsible for

cleavage and reunion of the DNA and are the targets of the thera-

peutically used ﬂuoroquinolones [12]. However, resistance against

broad-spectrum ﬂuoroquinolones is rising rapidly and serious side-

effects have emerged that led to the EMA recommendation in 2018

to restrict, and even suspend, the use of some ﬂuoroquinolone

antibiotics [13,14]. The role of the GyrB and ParE subunits is to

provide the energy necessary for the DNA ligation process through

ATP hydrolysis [1 1,15]. The most well-known inhibitors of GyrB/

ParE are aminocoumarins, with their representative novobiocin,

which was used in the clinic primarily to treat penicillin-resistant

S. aureus infections, although it was later withdrawn from ther-

apy due to the development of target-based resistance and toxicity

[15].

In the last years, we have discovered and optimized several new

structural classes of pyrrolamide-based GyrB and ParE inhibitors

[16e22]. Recently, we also developed inhibitors I and II (Figs. 1 and

2) as balanced dual-targeting inhibitors of S. aureus GyrB and ParE.

These inhibitors interact with multiple evolutionarily conserved

amino acids in the ATP binding pockets of both enzymes. I and II are

highly potent against a broad panel of multidrug-resistant S. aureus

clinical strains and resistance mutations against these compounds

are particularly rare [23]. I and II bypass the existing and clinically

widespread resistance mechanisms, including those that reduce

the efﬁcacy of other DNA gyrase and TopoⅣ inhibitors. Addition-

ally, de novo resistance mutations against I and II are rare and have

a limited impact on resistance levels. Furthermore, I and II inhibit

Gram-negative pathogens in vitro, although at higher concentra-

tions than Gram-positive pathogens [23].

2. Results and discussion

2.1. Structure-based design

Recently, we carried out the rational design and evaluation of

two new benzothiazole-6-carboxylic acid-based inhibitors I and II

that showed low target-based resistance potential (Figs. 1 and 2)

[23]. The crystal structure of inhibitor II in complex with S. aureus

GyrB (Supplementary information, Fig. S1) revealed that the pyr-

rolamide moiety forms crucial hydrogen bonds with Asp81 of

S. aureus GyrB (Asp73 in E. coli) and the conserved water molecule,

along with additional hydrophobic interactions. The benzothiazole

core of II is important for cation-p interactions with Arg84 (Arg76

in E. coli). Hydrophobic interactions with Pro87 (Pro79 in E. coli) are

formed with the benzothiazole core and the benzyloxy group of II,

while the carboxyl group forms a salt bridge with Arg144 (Arg136

in E. coli) [23]. This crystal structure and the promising microbio-

logical data for inhibitors I and II served as the basis for hit-to-lead

optimization of the benzothiazole class of GyrB inhibitors, with the

aim to obtain new balanced dual GyrB and ParE inhibitors with

broad spectrum activities against the ESKAPE pathogens, and with

low target-based resistance potential. The latter was pursued by

structure-based design of dual GyrB/ParE inhibitors that form

potent interactions with amino-acid residues that are essential for

the enzymatic function (e.g., Asp73, Arg76, Pro79 in E. coli GyrB),

and therefore do not mutate and are conserved within the bacterial

strains [23,24]. The second aim of the present study was to deﬁne

the in vitro selectivity, cytotoxicity, genotoxicity, and metabolism of

the representative new inhibitor. This design of the optimized an-

alogs of I and II is presented in Figs. 1 and 2, respectively.

Based on compound I, we synthesized four new series of in-

hibitors with different substitution patterns around the benzo-

thiazole scaffold (Fig. 1, type 1e4 compounds). For the type 1

compounds, 4,5-dichloro-, 4,5-dibromo-, and 4-bromo-3-chloro-5-

methyl-1H-pyrrole were introduced into the molecules. However,

better results overall were obtained for inhibitor I than for type 1

compounds, therefore 3,4-dichloro-5-methyl-1H-pyrrole was used

as the pyrrole moiety in compounds of types 2e4. In the type 2

series, the carboxylic acid was replaced with its bioisosteres or with

hydrogen-bond-acceptor moieties, which can form hydrogen

In the present study, we present the structure-based design of I

and II analogs and a comprehensive structure-activity relationship

study, with the aim to develop a class of optimized ATP-competitive

dual-targeting inhibitors with broad-spectrum activities against

the emerging ESKAPE pathogens. The crystal structure of a repre-

sentative inhibitor 31c in complex with S. aureus GyrB showed the

detailed interaction pattern at the molecular level. The selected

2

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M. Durcik, A. Nyerges, Z. Skok et al.

European Journal of Medicinal Chemistry 213 (2021) 113200

Fig. 1. Design of type 1e4 compounds based on inhibitor I [23] and the predicted docking pose for type 3 and 4 compounds 17c and 25 in the ATP-binding site of E. coli DNA gyrase B

(gray cartoon; PDB code: 4DUH). For clarity, only selected amino-acid residues are shown as sticks. Hydrogen bonds are presented as black dashed lines. Conserved water molecule

is presented as a red sphere.

Fig. 2. Design of type 5 and 6 compounds based on inhibitor II [23] and the predicted docking pose for type 6 compound 44 in the ATP-binding site of E. coli DNA gyrase B (gray

cartoon; PDB code: 4DUH). For clarity, only selected amino-acid residues are shown as sticks. Hydrogen bonds are presented as black dashed lines. Conserved water molecule is

presented as a red sphere.

bonds or ionic interactions with Arg136 (E. coli numbering). The

aim of this modiﬁcation was to reduce the acidic character of

compound I, to improve bacterial cell-wall penetration. To make

inhibitors less planar than I, type 3 compounds were prepared that

were derivatized at the amide nitrogen with aliphatic groups (Fig. 1,

compound 17c). In the type 4 compounds, we attached ﬂuoro,

3

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M. Durcik, A. Nyerges, Z. Skok et al.

European Journal of Medicinal Chemistry 213 (2021) 113200

methoxy, or hydroxyl groups to positions 4 or 5 of the bicycle,

which can form additional interactions in the binding site accord-

ing to the predictions from the docking analysis (Fig. 1, compound

25).

For the optimization of compound II, we changed the part with

the phenyl substituent to gain additional interactions with the

target in the lipophilic ﬂoor of GyrB and ParE (Fig. 2, type 5, 6

compounds). In the type 5 compounds, the phenyl ring was

replaced with a variety of aromatic and aliphatic substituents with

hydrophobic, acidic, and basic properties. In the type 6 compounds,

we replaced the carboxylic group with its bioisotere tetrazole

(Fig. 2, compound 44), and with cyano or (substituted) carbox-

amide groups (Fig. 2).

and 6b was removed with triﬂuoroacetic acid, to obtain the ﬁnal

hydroxamic acids 7a and 7b, and the ethyl ester of 6c was hydro-

lyzed under alkaline conditions to obtain 7c.

Scheme 2 presents the synthesis of the type 3 compounds, with

substituents at the amide nitrogen. To prepare N-alkyl derivatives,

ﬁrst the carboxyl group of 2-aminobenzo[d]thiazole-6-carboxylic

acid (8) was protected as para-methoxybenzyl ester 9. The use of

this protecting group was crucial for the successful preparation of

17a-c, as alkaline hydrolysis of ethyl esters also resulted in cleavage

of the alkylated amide moiety. The amino group of 9 was replaced

with bromine using tert-butyl nitrite and copper (II) bromide, and

the bromide 10 obtained was substituted with either ethylamine

(for 15a) or cyclopropylamine (for 15b). In parallel, ethyl 2-

aminobenzo[d]thiazole-6-carboxylate (11) was also converted

into bromine derivative 12, which was substituted with iso-

propylamine to obtain compound 13. Ethyl ester of 13 was hydro-

lyzed to obtain 14, and replaced with a para-methoxybenzyl group

to obtain compound 15c. Intermediates 15a-c were reacted with

3,4-dichloro-5-methyl-1H-pyrrole-2-carbonyl chloride to obtain

compounds 16a-c. In the ﬁnal step, para-methoxybenzyl ester was

cleaved with acidolysis, to yield 17a-c.

Scheme 3 shows the synthesis of type 4 compounds 25, 28a, and

28b with hydroxy substituents on positions 4 or 5 of the benzo-

thiazole core. First, methyl ester 19 was formed from the 4-

aminosalicylic acid (18) using Fischer esteriﬁcation, after which

the amino group was protected as tert-butyl carbamate, to obtain

20. Then the hydroxyl group was protected with acetyl chloride, to

provide compound 21. The Boc protecting group was removed, to

get 22, and then cyclization was performed using Br₂and KSCN in

acetic acid, to obtain the benzothiazole intermediate 23. Compound

2.2. Chemistry

The synthesis of the type 1 and type 2 compounds is shown in

Scheme 1. First, a set of commercially available 2-aminobenzo [d]

thiazoles 1a-c were coupled to pyrrole derivatives, to obtain 2a-d.

Esters 2a-d were subjected to alkaline hydrolysis, to obtain ﬁnal

compounds 3a-d. Commercially available 2-aminobenzo[d]thia-

zoles 4a and 4b were coupled to 3,4-dichloro-5-methyl-1H-pyr-

role-2-carboxylic acid, to obtain ﬁnal compounds 5a and 5b, and

compound 5b was additionally cyclized to its tetrazole analog 5c

using sodium azide and ammonium chloride. Compound 5b was

converted also to its amide analog 5d using KOH in N-methyl-2-

pyrrolidone. Compounds 6a-d were prepared by coupling O-(tet-

rahydro-2H-pyran-2-yl)hydroxylamine (for 6a, 6b), b-alanine ethyl

ester (for 6c), or N-cyanomethylamine (for 6d) to the carboxylic

acids 3b-d. The tetrahydro-2H-pyran-2-yl protecting group of 6a

Scheme 1. Reagents and conditions: (a) 2,2,2-trichloro-1-(4,5-dichloro-1H-pyrrol-2-yl)ethan-1-one (for 2a) or 2,2,2-trichloro-1-(4,5-dibromo-1H-pyrrol-2-yl)ethan-1-one (for 2b),

Na₂CO₃, DMF, 80 ^ꢀC, 15 h; (b) i: 4-bromo-3-chloro-5-methyl-1H-pyrrole-2-carboxylic acid (for 2c) or 3,4-dichloro-5-methyl-1H-pyrrol-2-carboxylic acid (for 2d and 5a, 5b), (COCl)₂,

anhydrous DCM, 20 ^ꢀC, 15 h, ii: 1a-c, toluene, 130 ^ꢀC, 15 h; (c) 2 M NaOH, 1,4-dioxane or MeOH, 80 ^ꢀC (for 3a-d) or 20 ^ꢀC (for 7c), 15 h; (d) NaN₃, NH₄Cl, DMF, 100 ^ꢀC, 15 h; (e) KOH,

NMP, 115 ^ꢀC, 48 h; (f) O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (for 6a, 6b) or

DMF, 20 ^ꢀC, 15 h; (g) CF₃COOH, DCM, 20 ^ꢀC, 2 h (for 7a, 7b).

b-alanine ethyl ester (for 6c), or 2-aminoacetonitrile hydrochloride (for 6d), EDC, HOBt, NMM,

4

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M. Durcik, A. Nyerges, Z. Skok et al.

European Journal of Medicinal Chemistry 213 (2021) 113200

Scheme 2. Reagents and conditions: (a) 4-methoxybenzyl chloride, K₂CO₃, anhydrous DMF, 20 ^ꢀC, 15 h; (b) CuBr₂, tert-butyl nitrite, CH₃CN 0 ^ꢀCe20 ^ꢀC, 15 h; (c) ethylamine (for

15a), cyclopropylamine (for 15b), or isopropylamine (for 13), THF, 20 ^ꢀC, 15 h; (d) 2 M NaOH, 1,4-dioxane, 20 ^ꢀC, 15 h; (e) i: 3,4-dichloro-5-methyl-1H-pyrrol-2-carboxylic acid,

(COCl)₂, anhydrous DCM, 20 ^ꢀC, 15 h, ii: 15a-c, toluene, 130 ^ꢀC, 15 h; (f) 1 M HCl in CH₃COOH, CH₃COOH, 20 ^ꢀC, 15 h.

Scheme 3. Reagents and conditions: (a) H₂SO₄, MeOH, 65 ^ꢀC, 15 h; (b) di-tert-butyl dicarbonate, 70 ^ꢀC, 48 h; (c) acetic anhydride, pyridine, CH₃CN, 70 ^ꢀC, 48 h; (d) 2 M HCl in diethyl

ether, 20 ^ꢀC, 15 h; (e) KSCN, Br₂, CH₃COOH, 10 ^ꢀC, then 20 ^ꢀC, 15 h, saturated aq. NaHCO₃solution; (f) i: 3,4-dichloro-5-methyl-1H-pyrrol-2-carboxylic acid, (COCl)₂, anhydrous DCM,

20 ^ꢀC, 15 h, ii: 23 (for 24) or 26b (for 27b), toluene, 130 ^ꢀC, 15 h; (g) 4 M NaOH (for 25) or 1 M NaOH (for 28a, 28b), 1,4-dioxane or methanol, 80 ^ꢀC (for 25) or 40 ^ꢀC (for 28a, 28b); (h)

26a, 2,2,2-trichloro-1-(4,5-dibromo-1H-pyrrol-2-yl)ethan-1-one, Na₂CO₃, DMF, 80 ^ꢀC, 15 h (for 27a).

23 was then coupled to 3,4-dichloro-5-methyl-1H-pyrrole-2-

carbonyl chloride, and ﬁnally, compound 24 obtained was sub-

jected to alkaline hydrolysis, to provide target compound 25. To

synthesize 4-hydroxy substituted 28a and 28b, ﬁrst 26a and 26b

were prepared according to previously described procedures [25].

Compounds 26a and 26b were then coupled with the

corresponding pyrrole derivative, to obtain compounds 27a and

27b, which were subjected to alkaline hydrolysis to obtain 28a and

28b. In the same reaction step, the tert-butyldimethylsilyl pro-

tecting group was removed from 27b.

The combined synthetic steps for the preparation of type 4 and 5

compounds with substituents on position 4 of the benzothiazole

5

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M. Durcik, A. Nyerges, Z. Skok et al.

European Journal of Medicinal Chemistry 213 (2021) 113200

core are shown in Scheme 4. Various 4-substituted 2-aminobenzo

[d]thiazoles (29a-i) were synthesized according to described pro-

cedures [25]. Compounds 29a-i were coupled to 3,4-dichloro-5-

methyl-1H-pyrrole-2-carbonyl chloride, to obtain methyl esters

30a-i, which were then converted to the ﬁnal carboxylic acid an-

alogs (31a-i) under alkaline hydrolysis conditions. Finally, com-

pound 32 was prepared by Boc deprotection of 31d using acidolysis.

Scheme 5 presents the synthesis of type 6 compounds with the

carboxyl group at position 6 replaced. Compound 33 was synthe-

sized with EDC and HOBt-promoted coupling between 31c and 2-

aminoacetonitrile hydrochloride. Compound 37 was prepared by

ﬁrst hydrolyzing benzothiazole 34 to its carboxylic acid derivative

35, which was coupled to 2-aminoacetonitrile hydrochloride.

Compound 36 obtained was then reacted with 3,4-dichloro-5-

methyl-1H-pyrrole-2-carbonyl chloride to obtain ﬁnal product 37.

To obtain tetrazole (44) and amide (47) analogs of compound II,

methyl 3-(benzyloxy)-4-nitrobenzoate (38) was converted to

amide 39 using saturated NH₃solution in methanol. Compound 39

was then used in two different pathways. Reaction with triphe-

nylphosphine oxide, triethylamine, and oxalyl chloride afforded the

cyano analog 40. The nitro group of 40 was reduced to obtain amine

41 using tin (II) chloride, and then cyclization was performed with

Br₂and KSCN in acetic acid, to obtain the benzothiazole 42. Com-

pound 43 was prepared by coupling 42 with 3,4-dichloro-5-

methyl-1H-pyrrole-2-carbonyl chloride, and then the tetrazole

ring was formed from the cyano group using ammonium chloride

and sodium azide in DMF, to obtain ﬁnal compound 44. Also,

compound 39 was reduced to 45 with tin (II) chloride. Cyclization of

45 to the benzothiazole intermediate 46, and the subsequent

coupling of 46 with 3,4-dichloro-5-methyl-1H-pyrrole-2-carbonyl

chloride were performed, to obtain the ﬁnal amide analog 47.

enzymes. In particular, type 5 compounds 31c and 31e-i showed

potent inhibition of S. aureus and E. coli DNA gyrase and S. aureus

TopoⅣ (IC₅₀< 100 nM), but weaker E. coli TopoⅣ inhibition

(IC₅₀< 400 nM). Compounds with IC₅₀values < 500 nM against

DNA gyrase from E. coli were tested for antibacterial activity against

E. faecalis and the ESKAPE pathogens S. aureus, K. pneumoniae,

A. baumannii, P. aeruginosa, and E. coli (Table 3 and Supplementary

Information, Table S1).

Bacterial strains that were used: S. aureus ATCC 29213, S. aureus

(MRSA) ATCC 43300, E. faecalis ATCC 29212, A. baumannii ATCC

17978, P. aeruginosa ATCC 27853, K. pneumoniae ATCC 10031, and

E. coli ATCC 25922.

Measurements were performed according to the Clinical and

Laboratory Standards Institute guidelines, with three independent

measurements.

Type 1 compounds 3a-c with 4,5-dichloro-, 4,5-dibromo-, and

4-bromo-3-chloro-5-methylpyrroles, respectively, are potent in-

hibitors of E. coli DNA gyrase (Table 1, IC₅₀< 20 nM). Among these,

3c with 4-bromo-3-chloro-5-methylpyrrole displayed balanced

low nanomolar dual inhibition of S. aureus DNA gyrase and TopoIV

(Table 1, IC₅₀24 nM, 61 nM, respectively), as comparable to in-

hibitor I. This also resulted in broad-spectrum activity for 3c

(Table 3, MICs: 0.0625 mg/mL against E. faecalis; 2 mg/mL against

K. pneumoniae).

Moving the carboxyl group to position 5 of the benzothiazole

core (type 2 compound 3e) or replacing the carboxyl group on

position 6 with hydrogen bond acceptors (type 2 compounds 5a,

5b) led to loss of enzyme inhibition, and consequently of the

antibacterial activities. This appears to be because 5a and 5b cannot

form ionic interactions with Arg136, which are crucial for potent

activity. Compounds 7a and 7b with a hydroxamic acid group as a

less acidic bioisostere of the carboxyl group were weaker inhibitors

of DNA gyrase and TopoⅣ. On the other hand, compounds 5c, 5d,

6d, and 7c with the carboxylic acid bioisostere tetrazole or car-

boxamide, all retained potent activities against DNA gyrase from

E. coli and S. aureus (Table 1), which for compounds 5c and 7c

2.3. In vitro enzyme inhibition and antibacterial activity

The new compounds were examined for their inhibitory activ-

ities against DNA gyrase from E. coli and S. aureus in supercoiling

assays, and against TopoⅣ from E. coli and S. aureus in relaxation

assays (Table 1, types 1e4; Table 2, types 5, 6). Compounds showed

potent inhibition of E. coli DNA gyrase, with several with IC₅₀

values < 10 nM. Several of the new compounds showed potent dual

S. aureus DNA gyrase and TopoⅣ inhibition; e.g., 3c, 25, 31c, and

31e-i had IC₅₀values < 100 nM against both of these target

translated in activities against E. faecalis, with MICs of 1

g/mL, respectively.

Type 3 compounds with substituents at the amide nitrogen

mg/mL and

2

m

were prepared with the aim to reduce the high level of planarity in

the structure. Additionally, with substituents at this position, there

was the possibility to form additional interactions with the

Scheme 4. Reagents and conditions: (a) i: 3,4-dichloro-5-methyl-1H-pyrrol-2-carboxylic acid, (COCl)₂, anhydrous DCM, 20 ^ꢀC, 15 h, ii: 29a-i (for 30a-i), toluene, 130 ^ꢀC, 15 h; (b) 1 M

NaOH, 1,4-dioxane or MeOH, 40 ^ꢀC, 15e72 h; (c) 4 M HCl in 1,4-dioxane, 1,4-dioxane, 3 h, 20 ^ꢀC.

6

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European Journal of Medicinal Chemistry 213 (2021) 113200

Scheme 5. Reagents and conditions: (a) 2-aminoacetonitrile hydrochloride, EDC, HOBt, NMM, DMF, 20 ^ꢀC, 15 h; (b) 1 M NaOH, MeOH, 80 ^ꢀC, 15 h; (c) i: 3,4-dichloro-5-methyl-1H-

pyrrol-2-carboxylic acid, (COCl)₂, anhydrous DCM, 20 ^ꢀC, 15 h, ii: 36, 42 or 46, toluene, 130 ^ꢀC, 15 h; (d) saturated NH₃solution in MeOH, 65 ^ꢀC, 15 h; (e) PPh₃O, Et₃N, (COCl)₂,

anhydrous CH₃CN, 20 ^ꢀC, 30 min; (f) SnCl₂, MeOH/EtOAc, 55 ^ꢀC, 15 h; (g) KSCN, Br₂, CH₃COOH, 10 ^ꢀC, then 20 ^ꢀC, 15 h, 25% NH₃aq. solution; (h) NaN₃, NH₄Cl, DMF, 125 ^ꢀC, 15 h.

lipophilic ﬂoor of the enzyme. The difference in activities between

compounds 17a-c showed that increasing the size of the sub-

stituents from ethyl to cyclopropyl to isopropyl led to weaker ac-

tivities (Table 1). Compounds 17a and 17b showed potent

antibacterial activities against Gram-positive methicillin-resistant

complex with the 24 kDa fragment of S. aureus GyrB at a resolution

of 1.7 Å (Fig. 3, PDB code 6TTG). This conﬁrmed the predicted

binding mode of these inhibitors in the ATP binding site of S. aureus

DNA gyrase. One hydrogen bond was formed between the pyrrole

NH group and Asp81 (Asp73 in E. coli), and a second hydrogen bond

was formed between the amide carbonyl oxygen and the conserved

water molecule, which was coordinated by Asp81, Gly85, and

Thr173. The amide carbonyl oxygen also formed a direct hydrogen

bond with Thr173. In the lipophilic pocket of the enzyme, 3,4-

dichloro-5-methylpyrrole formed hydrophobic interactions with

Ile51, Val79, Gly85, Ile86, Pro87, Ile102, Leu103, Thr173, and Ile175.

S. aureus (MRSA) and E. faecalis, with MICs of 0.125

mg/mL to 8

m

g/

mL. For 17a, moderate activity was also observed against Gram-

negative A. baumannii and K. pneumoniae (Table 3, MICs of 16

m

g/

mL).

Type 4 and 5 inhibitors were the most potent compounds in this

class. Adding small substituents, such as hydroxyl, methoxy, or

ﬂuoro (i.e., type 4 compounds), or larger substituents (i.e., type 5

compounds) to position 4 resulted in strong inhibition of DNA

gyrase. Except for dibromopyrrole analog 28a, all of these com-

pounds showed IC₅₀< 10 nM for E. coli DNA gyrase and <61 nM for

S. aureus DNA gyrase (Tables 1 and 2).

The benzothiazole moiety formed a

p-cation interaction with the

Arg84 side chain (Arg76 in E. coli), while the aromatic carboxylate

formed two additional hydrogen bonds with Arg144 (Arg136 in

E. coli). As predicted, a 2-morpholinoethoxy substituent at position

4 of the benzothiazole core was oriented toward the lipophilic ﬂoor,

although due to the missing loop in the crystal structure (amino

A co-crystal structure was obtained for type 5 compound 31c in

7

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M. Durcik, A. Nyerges, Z. Skok et al.

European Journal of Medicinal Chemistry 213 (2021) 113200

Table 1

Inhibitory activities of type 1e4 compounds against E. coli and S. aureus DNA gyrase and topoisomerase Ⅳ (TopoⅣ).

Cpd.

R¹

R²

IC₅₀(nM)^a

E. coli

S. aureus

DNA gyrase

TopoⅣ

DNA gyrase

TopoⅣ

Type 1 inhibitors

3a

3b

3c

4,5-diCl

19

1

7

280 20

>1000

500 280

>1000

e

4,5-diBr

12

300 150

61 25

3-Cl-4-Br-5-Me

<10

220 90

24 13

Type 2 inhibitors

3d

5a

5b

5c

5d

6d

7a

7b

7c

3,4-diCl-5-Me

5-COOH

6-OMe

6-CN

6-(Tetrazol-5-yl)

6-CONH₂

6-CONHCH₂CN

6-CONHOH

6-CONHCH₂CH₂COOH

830 150

>1000

<10

>1000

660 180

490 80

>1000

25 12

59 28

310 100

>1000

720 310

71 12

>1000

270 140

320 160

>1000

3,4-diCl-5-Me

4,5-diBr

11

16

2

4

89 20

280 10

<10

3,4-diCl-5-Me

210 20

40 11

Type 3 inhibitors

17a

Ethyl

<10

>1000

32 15

>1000

230 80

>1000

e

17b

Cyclopropyl

Isopropyl

24

1

e

17c

320 170

>1000

e

Type 4 inhibitors

25

28a

28b

31a

31b

I

II

3,4-diCl-5-Me

4,5-diBr

3,4-diCl-5-Me

5-OH

4-OH

4-F

<10

46 33

<10

13

<10

170 20

95

4

61

170 70

22

17

44 12

72 20

4

46 10

180 40

130 10

200 10

160 20

500 280

350 50

11000 2000

240 110

120 60

240 40

390 60

66 22

6

0

4-OMe

0

16

34

5

7

68 31

27000 7000

Novobiocin

^aIC₅₀, concentration (mean SD of three independent experiments) that inhibits enzyme activity by 50%.

acids 105e127), no interactions with the enzyme were observed,

except from a hydrogen bond with a crystal water molecule.

Compound 31c therefore targets amino acids Asp81, Arg84, and

Pro87 in S. aureus GyrB, which have been shown to be essential for

the enzymatic activity of GyrB in E. coli [24], and to be fully

conserved across over 1000 phylogenetically diverse bacterial ge-

nomes [23].

bacterial cell walls, and consequently why they lack antibacterial

activities. On the other hand, replacing the phenyl ring of inhibitor

II with its bioisosteric replacement thiophene ring resulted in the

two best compounds of the series, 31g and 31h, which showed

exceptional activities against Gram-positive and Gram-negative

bacteria (Table 3).

Type 6 compounds with an aliphatic nitrile group in the side

chain on position 6 (33, 37) or carboxyl group bioisosteres on po-

sition 6 (44, 47), were potent inhibitors of E. coli DNA gyrase

(IC₅₀< 11 nM), while the aromatic nitrile 43 was devoid of activity,

which is in line with the observations for 5b. Tetrazole 44 also

showed potent S. aureus DNA gyrase inhibition and good TopoIV

inhibition (Table 2). The only notable antibacterial activity against

Gram-positive bacteria was seen for 37 and 44 (Table 3).

The structureeactivity relationship of the new series of in-

hibitors is summarized in Supplementary Information Fig. S2 while

the predicted docking pose of type 5 inhibitor 31h and the in-

teractions that it forms in the binding site are shown in Fig. 4. The

strongest activity of type 5 compounds can be attributed to the

substituents at position 4, which form additional interactions with

the lipophilic ﬂoor of the binding site, as observed for the co-crystal

structure of S. aureus GyrB in complex with 31c (Fig. 3) and by the

docking binding mode of 31h (Fig. 4). Furthermore, we super-

imposed the crystal structures of E. coli GyrB (PDB entry: 1KZN) and

ParE (PDB entry: 1S14) with those of S. aureus GyrB (PDB entry:

6TTG) and ParE (PDB entry: 4URN) (Fig. S4) to better understand

the difference in the observed inhibition of DNA gyrase and

Type 5 compounds with larger and mostly aromatic substituents

on position 4 of the benzothiazole core were the best balanced

dual-acting compounds, as seen for 31e, 31f, and 31i with IC₅₀

values of 13 nM, 12 nM, and <10 nM against S. aureus TopoIV

(Table 2). Type 4 compound 31a, with a ﬂuoro substituent on po-

sition 4, showed very good inhibition of Gram-positive bacteria and

Gram-negative K. pneumoniae (Table 3), although these activities

were not improved over those of the inhibitor I. The addition of

polar substituents to the type 4 and 5 compounds (i.e., hydroxyl:

25, 28a, 28b; morpholinoethoxy: 31c; aminoethoxy: 32) had

negative impacts on their antibacterial activities (Supplementary

Information, Table S1). Although the recently reported rules for

compound accumulation in Gram-negative bacteria, known as the

‘eNTRy rules’ [26], indicate that primary amines are important for

drug entry, compound 32 was devoid of antibacterial activity.

Similarly, other compounds with polar groups lacked antibacterial

activities despite their excellent on-target inhibition (i.e., 31e, 31f,

32, 33). These inhibitors have greater topological polar surface

areas in comparison with other compounds (Supplementary In-

formation, Table S2), which might be why they cannot penetrate

8

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M. Durcik, A. Nyerges, Z. Skok et al.

European Journal of Medicinal Chemistry 213 (2021) 113200

Table 2

Inhibitory activities of type 5 and 6 compounds against E. coli and S. aureus DNA gyrase and topoisomerase Ⅳ (TopoⅣ).

Cpd.

R¹

R²

IC₅₀(nM)^a

E. coli

S. aureus

DNA gyrase

TopoⅣ

DNA gyrase

TopoⅣ

Type 5 inhibitors

31c

<10

220 140

17

6

66

3

e

31e

31f

<10

170 10

260 70

<10

13

12

0

5

e

31g

31h

<10

300 80

340 70

<10

88 38

84 38

e

31i

32

<10

54 28

>1000

<10

e

48 25

160 60

520 370

e

Type 6 inhibitors

33

CONHCH₂CN

100 40

37

43

44

47

CONHCH₂CN

CN

<10

>1000

180 80

>1000

89 17

>1000

e

>1000

<10

>1000

Tetrazol-5-yl

CONH₂

680 250

>1000

26

7

11

2

0

>1000

I

II

13

<10

170 20

500 280

350 50

11000 2000

72 20

66 22

68 31

27000 7000

16

34

5

7

Novobiocin

topoisomerase IV. The most noticeable differences in binding sites

are observed in the pyrrole binding pocket (Fig. S4). However, the

unresolved loop in the lipophilic ﬂoor surrounding the substituent

at position 4 of the benzothiazole core may also contribute signif-

icantly to the observed difference in IC₅₀values against the four

enzymes.

E. faecalis and the compound was exceptionally active also against

the MRSA strain (Table 3, MIC 0.0625 g/mL). Additionally, 31h was

tested on the ESKAPE pathogens E. faecium (ATCC 700221) and

vancomycin-intermediate S. aureus (VISA; ATCC 700699), where it

m

showed MICs of 0.0078

mg/mL and 0.0313 mg/mL, respectively.

Therefore, 31h was further explored in terms of its microbiological

proﬁle. We examined how certain GyrB mutations (Arg144, Thr173

and Ile175) in MRSA and VISA strains inﬂuence the antibacterial

activities of 31h. As 31h also forms important interactions among

others also with these three amino acids of GyrB, their mutations

might lead to decreased susceptibility to inhibitor 31h. However,

31h also showed very potent activities here, with MICs against the

2.4. Microbiological proﬁling of compound 31h

Overall, 31h was selected as the best compound of this class of

inhibitors, as in addition to its potent inhibition of DNA gyrase and

TopoIV, it had the best antibacterial activity proﬁle against all of the

ESKAPE pathogens. The MICs of 31h against the Gram-negative

bacteria A. baumannii, P. aeruginosa, K. pneumoniae, and E. coli

MRSA mutants of 0.125e0.25

mg/mL, and against the VISA mutants

of 0.0625 g/mL (Table 4). These mutants thus confer very low

m

levels of resistance to 31h.

were 1, 2, 2, and 4

mg/mL, respectively. For the Gram-positive

To determine whether compound 31h undergoes efﬂux in

bacteria, the MICs were even better, as 0.0156

mg/mL for

9

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M. Durcik, A. Nyerges, Z. Skok et al.

European Journal of Medicinal Chemistry 213 (2021) 113200

Table 3

Minimum inhibitory concentrations (MICs) for the DNA gyrase and/or topoisomerase Ⅳ inhibitors with activities against the indicated Gram-positive and Gram-negative

bacterial strains.

Cpd.

MIC (m

g/mL) ^a

Gram positive

Gram-negative

S. aureus

S. aureus (MRSA)^b

E. faecalis

A. baumannii

P. aeruginosa

K. pneumoniae

E. coli

3c

5c

7b

7c

0.125

16

64

32

0.125

2

16

32

8

2

4

0.0625

0.5

0.0625

4

0.0625

1

0.5

4

8

2

>64

32

>64

16

64

8

4

>64

4

>64

16

>64

8

>64

64

4

2

64

16

>64

8

16

64

8

4

2

4

2

17a

17b

25

28b

31a

31b

31g

31h

31i

37

0.125

0.25

1

16

2

0.5

2

0.065

0.25

0.0625

4

1

0.5

0.0625

0.25

0.0163

0.0156

0.001

16

0.125

<0.0313

16

1

2

>64

1

>64

4

>64

8

44

I

II

4

0.125

0.0625

2

4

16

a

MIC, minimum inhibitory concentration.

MRSA, methicillin-resistant S. aureus.

b

Fig. 3. Co-crystal structure of S. aureus DNA gyrase B (gray cartoon; PDB entry: 6TTG)

in complex with inhibitor 31c (yellow sticks). For clarity, only amino-acid residues that

interact with 31c are shown as sticks. Water molecules are presented as red spheres

and hydrogen bonds are shown as dashed black lines.

Fig. 4. The predicted docking pose of type 5 inhibitor 31h (yellow sticks) that shows

the interactions that are important for activity against the E. coli DNA gyrase B (gray

cartoon; PDB code 4DUH) ATP-binding site. For clarity, only selected amino-acid res-

idues are shown as sticks. Hydrogen bonds are presented as black dashed lines. The

detailed interaction pattern is shown in Supplementary Information Fig. S3.

Gram-negative bacteria, it was tested against the highly resistant

E. coli MG1655 strain without and with the efﬂux pump substrate

Additionally, 31h was tested against Gram-negative clinical

isolates, where it showed good activity against P. aeruginosa, E. coli,

and ciproﬂoxacin resistant A. baumannii and E. coli clinical strains,

phenylalanine-arginine

31h showed no activity, whereas with PA

E. coli strain with a very good MIC of 0.125

b

-naphthylamide (PA

N, 31h inhibited this

g/mL (Table 4). This

bN). Without PAbN,

b

m

with MICs from 0.5 mg/mL to 16 mg/mL (Table 5).

conﬁrmed that compound 31h is a substrate for the efﬂux pumps in

the E. coli MG1655 strain. To determine the importance of the

interaction between the terminal carboxylate of 31h and Arg136 in

E. coli, inhibitor 31h was tested on the E. coli MG1655 strain with

2.5. In vitro selectivity and toxicity evaluation of compound 31h

the R136C mutation in the absence and presence of PAbN and the

activity of 31h was only 8-fold weaker (Table 4). Although inter-

action with Arg136 is important for bacterial inhibition, a very good

MIC of 2 mg/mL was obtained against the mutated strain, which

Human DNA TopoII catalyzes the introduction of topological

changes into the DNA molecule. Its ATP-binding domain belongs to

the GHKL ATPase family, and it is similar to those of DNA gyrase and

TopoIV [27]. To determine the selectivity of 31h for these bacterial

showed that the interactions formed between 31h and the rest of

the GyrB active site are strong enough to provide good antibacterial

activity. These promising data are in line with our design strategy,

where compounds form strong interactions with Asp81, Arg84, and

Pro87 (S. aureus GyrB numbering), which are essential for the

enzymatic function of GyrB.

enzymes, we evaluated its inhibition of hTopoII

relaxation assay. Compound 31h had an IC₅₀of 45.0

hTopoII (Supplementary Information, Fig. S5). 31h is thus over

450-fold selective for DNA gyrase from E. coli or S. aureus, and 13-

fold and 54-fold selective for TopoIV from E. coli and S. aureus,

respectively.

a

in the DNA

mM against

a

10

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M. Durcik, A. Nyerges, Z. Skok et al.

European Journal of Medicinal Chemistry 213 (2021) 113200

Table 4

Minimum inhibitory concentrations (MICs) for 31h against wild-type methicillin-resistant S. aureus (MRSA), vancomycin-intermediate S. aureus (VISA), and E. coli, and their

GyrB mutants.

Bacteria

Strain

MIC (

m

g/mL)^a

Fold-change (mutant vs. wild type)

S. aureus (MRSA)

Wild-type

GyrB R144I

GyrB T173A

Wild-type

GyrB R144I

GyrB I175T

Wild-type

0.0625

0.25

-

4-fold

2-fold

-

2-fold

0.125

0.0313

0.0625

>64

S. aureus (VISA)

E. coli MG1655

e

GyrB R136C

Wild type

>64

0.125

2

e

(þ50

m

g/mL PA

b

N^b)

GyrB R136C

8-fold

a

MIC, minimum inhibitory concentration.

b

PAbN, phenylalanine-arginine b-naphthylamide (efﬂux pump substrate).

Table 5

MICs for 31h against Gram-negative clinical isolates.

concentrations up to 16

m

M, and no formation of reactive metab-

olites. Further microbiological evaluation against mutated strains

revealed promising results. Thus, analogs of this class represent

new and much-needed scaffolds for the further development of

new antibacterials for treatment of resistant Gram-positive and

Gram-negative infections.

Bacteria

Strain

MIC (m

g/mL)^a

A. baumannii CIPr^b

E. coli

ATCC BAA-1605

20204

0.5

1

2

E. coli CIPr

31995

E. coli CIPr

31859

2

P. aeruginosa

19488

16

a

MIC, minimum inhibitory concentration.

CIPr, ciproﬂoxacin-resistant clinical isolate.

4. Experimental section

b

4.1. General chemistry information

Compound 31h was tested for in vitro cytotoxicity using the

lactate dehydrogenase assay in liver cancer HepG2 cells and breast

cancer MCF-7 cells. Here, 31h showed no cytotoxicity up to 100

(Supplementary Information, Fig. S6). The genetic toxicity of 31h

was also evaluated in a standard micronucleus test on Chinese

hamster ovary K1 cells, without and with a rat liver S9 fraction

(Supplementary Information, Table S3). In the absence of the S9

Chemicals were obtained from Acros Organics (Geel, Belgium),

Sigma-Aldrich (St. Louis, MO, USA), and Apollo Scientiﬁc (Stockport,

UK), and were used without further puriﬁcation. Analytical TLC was

performed on silica gel Merck 60 F₂₅₄plates (0.25 mm), using

visualization with UV light and spray reagents. Column chroma-

tography was carried out on silica gel 60 (particle size, 240e400

mesh). Analytical reversed-phase HPLC analyses were performed

on a 1260 Inﬁnity II LC system (Agilent Technologies Inc., Santa

Clara, CA, USA) for method A or a Dionex Ultimate 3000 binary

rapid separation LC system (Thermo Scientiﬁc, Thermo Fisher Sci-

entiﬁc, Waltham, MA, USA) for methods B and C. Method A: A

Waters XBridge C18 column was used (3.5

with ﬂow rate of 1.5 mL/min and sample injection volume of 10

The mobile phase consisted of acetonitrile (solvent A) and 0.1%

formic acid in 1% acetonitrile in ultrapure water (solvent B). The

gradient (deﬁned for solvent A) was: 0e1.0 min, 25%; 1.0e6.0 min,

25%e98%; 6.0e6.5 min, 98%; 6.5e7.5 min, 98%e25%; 7.5e10.5 min,

m

M

fraction, 31h showed no genetic toxicity up to 16 mM, which was

495-fold, 123-fold, and 247-fold higher than the MICs of 31h

against E. faecalis, MRSA, and VISA, respectively. Conversely, in the

presence of the S9 fraction, compound 31h showed no genotoxicity

up to 62 mM, which was 1917-fold, 478-fold, and 957-fold higher

than the MICs against E. faecalis, MRSA, and VISA. To identify the

main in vitro metabolites of 31h, in vitro metabolic transformation

studies were performed using the rat liver S9 fraction. Here, we

identiﬁed three main metabolites shown in Scheme 6: a metabolite

hydroxylated on the methylpyrrole moiety; a metabolite hydrox-

ylated on the benzothiazole ring; and a direct glucuronide conju-

gate (as either an acylglucuronide or an N-glucuronide)

(Supplementary Information, Figs. S7eS10).

m

m, 4.6 mm ꢁ 150 mm),

mL.

25%. Method B: An Agilent Extend-C18 column was used (3.5

4.6 ꢁ 150 mm), with ﬂow rate of 1.0 mL/min and sample injection

volume of 10 L. The mobile phase consisted of acetonitrile (solvent

mm,

m

3. Conclusions

A) and 0.1% triﬂuoroacetic acid (TFA) in ultrapure water (solvent B).

The gradient (deﬁned for solvent A) was: 0e16 min, 30e90%;

16e20 min, 90%; 20e21 min, 90-30%. Method C: A Waters Acquity

In conclusion, we have designed and synthesized new multi-

targeting benzothiazole-based DNA gyrase and TopoⅣ inhibitors.

The crystal structure of inhibitor 31c in complex with S. aureus DNA

gyrase resolved to a resolution of 1.7 Å conﬁrmed the binding mode

of these types of inhibitors in the ATP binding site. Compound 31h

showed potent low nanomolar dual inhibition of GyrB and ParE

from S. aureus and E. coli, and excellent broad-spectrum antibac-

terial activity against resistant pathogens belonging to ESKAPE

group, including MRSA, VISA, K. pneumoniae, A. baumannii, and

P. aeruginosa. Compound 31h also showed potency against clinical

A. baumannii, E. coli, and P. aeruginosa isolates. Furthermore, 31h

showed good selectivity for DNA gyrase and TopoⅣ over hTopoII,

no cytotoxicity in MCF-7 and HepG2 cells, no in vitro genotoxicity at

UPLC CSH C18 column was used (1.7

rate of 0.4 mL/min and sample injection volume of 1e4

m

m, 2.1 ꢁ 50 mm), with ﬂow

m

L. The

mobile phase consisted of acetonitrile (solvent A) and 0.1% tri-

ﬂuoroacetic acid (TFA) in ultrapure water (solvent B). The gradient

(deﬁned for solvent A) was: 0e8 min, 30e90%; 8e10 min, 90%;

10e11 min, 90-30%. ¹H and ¹³C NMR spectra were recorded at 400

and 100 MHz, respectively, on a Bruker AVANCE III 400 spectrom-

eter (Bruker Corporation, Billerica, MA, USA) in DMSO‑d₆or CDCl₃

solutions, with TMS as the internal standard. Mass spectra were

obtained using Exactive Plus Orbitrap mass spectrometer (Thermo

Fisher Scientiﬁc, Waltham, MA, USA) or an ADVION expression

CMS^Lmass spectrometer (Advion Inc., Ithaca, USA).

11

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M. Durcik, A. Nyerges, Z. Skok et al.

European Journal of Medicinal Chemistry 213 (2021) 113200

Scheme 6. Identiﬁed metabolic transformation products of 31h.

4.2. Synthetic procedures and analytical data

dichloromethane (10 mL) oxalyl chloride (0.181 mL, 2.11 mmol) was

added dropwise and the solution stirred at room temperature un-

der an argon atmosphere overnight. The solvent was evaporated

under reduced pressure, ethyl 2-aminobenzo [d]thiazole-6-

carboxylate (1b, 94 mg, 0.42 mmol) and toluene (20 mL) were

added, and the suspension was stirred at 130 ^ꢀC overnight. The

precipitate in the reaction mixture was ﬁltered off, resuspended in

1 M HCl (100 mL), sonicated and ﬁltered off. The crude product was

dispersed in methanol (100 mL), heated, ﬁltered off and dried, to

obtain 2c (121 mg) as a gray solid.

General procedure A. Synthesis of compounds 2a, 2b, and 27a

(with 2a as an example). To a solution of methyl 2-aminobenzo[d]

thiazole-6-carboxylate (1a, 201 mg, 0.964 mmol) in N,N-dime-

thylformamide, Na₂CO₃(102 mg, 0.964 mmol) and 2,2,2-trichloro-

1-(4,5-dichloro-1H-pyrrole-2-yl)-ethan-1-one

(295

mg,

1.06 mmol) were added, and the reaction mixture was stirred at

80 ^ꢀC overnight. The reaction mixture was cooled to room tem-

perature, 10% citric acid aqueous solution was added, and the

mixture was cooled in an ice bath. The precipitate that formed was

ﬁltered off and dried to give 2a (240 mg) as an off-white solid.

Methyl 2-(4,5-dichloro-1H-pyrrole-2-carboxamido)benzo[d]

thiazole-6-carboxylate (2a). Yield: 240 mg (70.0%); off-white

Ethyl

2-(4-bromo-3-chloro-5-methyl-1H-pyrrole-2-

carboxamido)benzo[d]thiazole-6-carboxylate (2c). Synthesized

according to general procedure B using ethyl 2-aminobenzo [d]

thiazole-6-carboxylate (1b, 0.094 mg, 0.42 mmol). Yield: 121 mg

solid. ¹H NMR (400 MHz, DMSO‑d₆)

d

3.89 (s, 3H), 7.54 (s, 1H),

(64.6%); gray solid. ¹H NMR (400 MHz, DMSO‑d₆)

d

1.35 (t, J ¼ 7.1 Hz,

7.84 (d, J ¼ 8.5 Hz, 1H), 8.04 (dd, J ¼ 8.5, 1.7 Hz, 1H), 8.68 (d,

3H), 2.28 (s, 3H), 4.35 (q, J ¼ 7.1 Hz, 2H), 7.83 (s,1H), 8.03 (dd, J ¼ 8.5,

1.8 Hz, 1H), 8.66 (s, 1H), 11.95 (s, 1H), 12.43 (s, 1H). HRMS (ESI^þ) m/z

for C₁₄H₉Cl₂N₄O₂S ([MþH]^þ): calculated 441.9622, found 441.9619.

J ¼ 1.7 Hz, 1H), 12.87 (s, 1H), 13.37 (s, 1H). HRMS (ESI^ꢂ) m/z for

C

₁₄H₈Cl₂N₃O₃S ([M ꢂ H]^-): calculated 367.9660, found 367.9663.

Methyl 2-(4,5-dibromo-1H-pyrrole-2-carboxamido)benzo[d]

Methyl

2-(3,4-dichloro-5-methyl-1H-pyrrole-2-

thiazole-6-carboxylate (2b). Synthesized according to general

carboxamido)benzo[d]thiazole-5-carboxylate (2d). Synthesized

according to general procedure B using methyl 2-aminobenzo [d]

thiazole-5-carboxylate (1c, 250 mg, 1.20 mmol). The crude product

was dispersed in acetone (100 mL), sonicated, ﬁltered off and dried.

Yield: 210 mg (45.5%); off-white solid. ¹H NMR (400 MHz,

procedure A. Yield: 91.6% (1.01 g); light brown solid. ¹H NMR

(400 MHz, DMSO‑d₆)

d

3.89 (s, 3H), 7.55 (d, J ¼ 8.5 Hz, 1H), 7.81 (s,

1H), 8.04 (dd, J ¼ 8.5, 1.8 Hz, 1H), 8.68 (d, J ¼ 1.8 Hz, 1H), 12.84 (s,

1H), 13.30 (s, 1H). HRMS (ESI^ꢂ) m/z for C₁₄H₈Br₂N₃O₃S ([M ꢂ H]^-):

calculated 455.8653, found 455.8663.

DMSO‑d₆)

(dd, J ¼ 8.9, 2.5 Hz, 1H), 8.54 (d, J ¼ 2.5 Hz, 1H), 9.92 (s, 1H), 12.20 (s,

1H). ¹³C NMR (101 MHz, DMSO‑d₆)

10.92, 53.26, 108.85, 111.83,

d

2.24 (s, 3H), 3.93 (s, 3H), 7.81 (d, J ¼ 8.8 Hz, 1H), 8.09

General procedure B. Synthesis of compounds 2c, 2d, 5a, 5b,

16a-c, 24, 27b, 30a-i, 37, 43, and 47 (with 2c as an example). To a

d

suspension

of

4-bromo-3-chloro-5-methyl-1H-pyrrole-2-

112.32, 119.13, 122.35, 122.45, 125.79, 126.38, 128.34, 128.61, 138.86,

carboxylic acid (101 mg, 0.42 mmol) in anhydrous

157.52, 165.94. HRMS (ESI^ꢂ) m/z for C₁₅H₁₀Cl₂N₃O₃S ([M ꢂ H]^-):

12

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M. Durcik, A. Nyerges, Z. Skok et al.

European Journal of Medicinal Chemistry 213 (2021) 113200

calculated 381.9825, found 381.9830.

General procedure C. Synthesis of compounds 3a-d, 7c, 14, 25,

28a, 28b, 31a-f, and 35 (with 3b as an example). Methyl 2-(4,5-

methanol, sonicated, ﬁltered off, washed with methanol and dried.

Yield: 74 mg (40%); brown solid. ¹H NMR (400 MHz, DMSO‑d₆)

d

2.26 (s, 3H), 3.82 (s, 3H), 7.05 (dd, J ¼ 2.6, 8.8 Hz, 1H), 7.58 (d,

dibromo-1H-pyrrole-2-carboxamido)benzo

[d]thiazole-6-

J ¼ 2.6 Hz, 1H), 7.64 (d, J ¼ 8.8 Hz, 1H), 11.57 (s, 1H), 12.30 (s, 1H). ¹³

C

carboxylate (2b, 0.800 g, 1.74 mmol) was suspended in 1,4-

dioxane (30 mL), 2 M NaOH (4.35 mL, 8.70 mmol) was added,

and the reaction mixture was stirred at 80 ^ꢀC overnight. The solvent

was removed under reduced pressure, the residue was acidiﬁed

with 1 M HCl to pH 1, and the precipitate obtained was ﬁltered off

and dried to give 3b (565 mg) as a brown solid.

NMR (101 MHz, DMSO‑d₆) d 10.89, 55.55, 104.80, 109.53, 114.44,

114.89, 117.49, 120.32, 128.11, 128.80, 129.55, 132.38, 141.39, 156.11.

HRMS (ESI^þ) m/z for C₁₄H₁₂Cl₂N₃O₂S ([MþH]^þ): calculated

356.0022, found 356.0020. HPLC: t_r7.19 min (98.0% at 254 nm),

method A.

3,4-Dichloro-N-(6-cyanobenzo[d]thiazol-2-yl)-5-methyl-1H-

pyrrole-2-carboxamide (5b). Synthesized according to general

procedure B using 2-aminobenzo [d]thiazole-6-carbonitrile (4b,

400 mg, 2.28 mmol). The crude product was dispersed in acetoni-

trile, sonicated, ﬁltered off, and dried. Yield: 310 mg (82%); gray

2-(4,5-Dichloro-1H-pyrrole-2-carboxamido)benzo [d]thia-

zole-6-carboxylic acid (3a). Synthesized according to general

procedure

C

using methyl 2-(4,5-dichloro-1H-pyrrole-2-

carboxamido)benzo [d]thiazole-6-carboxylate (2a, 240 mg,

0.65 mmol). Yield: 205 mg (88.7%); brown solid. ¹H NMR (400 MHz,

solid. ¹H NMR (400 MHz, DMSO‑d₆)

d 2.27 (s, 3H), 7.78e8.00 (m,

DMSO‑d₆)

J ¼ 8.5, 1.7 Hz, 1H), 8.64 (d, J ¼ 1.7 Hz, 1H), 12.86 (s, 1H), 13.37 (s, 1H).

¹³C NMR (101 MHz, DMSO‑d₆)

109.71, 114.13, 119.05, 120.34,

122.80, 124.27, 126.28, 127.84, 132.12, 152.17, 158.06, 161.97, 167.51.

HRMS (ESI^ꢂ) m/z for C₁₃H₆Cl₂N₃O₃S ([M H]^-): calculated

d

7.53 (d, J ¼ 2.1 Hz, 1H), 7.82 (d, J ¼ 8.4 Hz, 1H), 8.02 (dd,

2H), 8.59 (s, 1H), 12.06 (s, 1H), 12.39 (s, 1H). HRMS (ESI^þ) m/z for

C

₁₄H₉Cl₂N₄OS ([MþH]^þ): calculated 350.9869, found 350.9859.

d

HPLC: t_r7.06 min (98.0% at 254 nm), method A.

N-(6-(1H-Tetrazol-5-yl)benzo[d]thiazol-2-yl)-3,4-dichloro-5-

methyl-1H-pyrrole-2-carboxamide (5c). A solution of 3,4-

ꢂ

353.9498, found 353.9507. HPLC: t_r5.80 min (92.9% at 254 nm),

method A.

dichloro-N-(6-cyanobenzo [d]thiazol-2-yl)-5-methyl-1H-pyrrole-

2-carboxamide (5b, 100 mg, 0.285 mmol), ammonium chloride

(152 mg, 2.85 mmol), and sodium azide (185 mg, 2.85 mmol) in

DMF (5 mL) was heated to 100 ^ꢀC for 15 h. The mixture was cooled

down to room temperature, water (7 mL) and ethyl acetate (7 mL)

were added, and the mixture was acidiﬁed with 1 M HCl to pH 3e4.

The precipitate was ﬁltered off, washed with ethyl acetate, and

dried. The crude product was dispersed in acetone (5 mL), soni-

cated, ﬁltered off, and dried. Yield: 67 mg (59.7%); brown solid. ¹H

2-(4,5-Dibromo-1H-pyrrole-2-carboxamido)benzo[d]thia-

zole-6-carboxylic acid (3b). Yield: 565 mg (68.5%); brown solid. ¹H

NMR (400 MHz, DMSO‑d₆)

J ¼ 8.5 Hz, 1H), 8.02 (dd, J ¼ 8.5, 1.7 Hz, 1H), 8.64 (d, J ¼ 1.4 Hz, 1H),

12.83 (s, 1H), 13.31 (s, 1H). ¹³C NMR (101 MHz, DMSO‑d₆)

99.10,

d

7.55 (d, J ¼ 2.7 Hz, 1H), 7.82 (d,

d

110.11, 116.74, 120.14, 124.11, 126.45, 127.03, 127.77, 132.13, 152.26,

158.46, 162.21, 167.70. HRMS (ESI^ꢂ) m/z for C₁₃H₆Br₂N₃O₃S

([M ꢂ H]^-): calculated 441.8497, found 441.8501. HPLC: t_r5.89 min

(96.1% at 254 nm), method A.

2-(4-Bromo-3-chloro-5-methyl-1H-pyrrole-2-carboxamido)

benzo[d]thiazole-6-carboxylic acid (3c). Synthesized according to

general procedure C using ethyl 2-(4-bromo-3-chloro-5-methyl-

1H-pyrrole-2-carboxamido)benzo [d]thiazole-6-carboxylate (2c,

100 mg, 0.23 mmol). Yield: 80 mg (85.4%). ¹H NMR (400 MHz,

NMR (400 MHz, DMSO‑d₆) d 2.29 (s, 3H), 7.95 (s, 1H), 8.08e8.18 (m,

1H), 8.70 (s, 1H), 12.00 (s, 1H), 12.41 (s, 1H), the signal for the tet-

razole NH not seen. HRMS (ESI^þ) m/z for C₁₄H₁₀Cl₂N₇OS ([MþH]^þ):

calculated 394.0039, found 394.0039. HPLC: t_r6.13 min (95.3% at

254 nm), method A.

2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo

[d]thiazole-6-carboxamide (5d). 3,4-dichloro-N-(6-cyanobenzo

[d]thiazol-2-yl)-5-methyl-1H-pyrrole-2-carboxamide (5b, 100 mg,

0.28 mmol) was dissolved in N-methyl-2-pyrrolidone (10 mL),

pulverized KOH (320 mg, 5.96 mmol) was added, and the reaction

mixture was stirred at 100 ^ꢀC overnight. Additional 10 equivalents

of KOH (160 mg, 2.8 mmol) were added and the reaction mixture

was stirred at 115 ^ꢀC overnight. The reaction mixture was cooled to

room temperature, diluted with water, and acidiﬁed with 2 M HCl.

The resulting precipitate was ﬁltered off and puriﬁed with ﬂash

column chromatography using hexane/ethyl acetate/THF (1:1:1) /

ethyl acetate/THF (1:1) as eluent. Yield: 10 mg, (10.4%); white solid.

DMSO‑d₆)

d

2.28 (d, J ¼ 11.0 Hz, 3H), 7.74 (d, J ¼ 8.2 Hz, 1H), 7.99 (d,

J ¼ 8.6 Hz, 1H), 8.56 (s, 1H), 12.53 (s, 2H). ¹³C NMR (101 MHz,

DMSO‑d₆)

d 12.60, 98.29, 117.47, 119.30, 119.53, 124.17, 126.62,

127.87, 131.76, 132.08, 151.17, 158.65, 162.81, 167.86. HRMS (ESI^þ) m/

z

for

C

₁₄H₁₀BrClN₃O₃S ([MþH]^þ): calculated 413.9309, found

413.9321. HPLC: t_r6.44 min (95.5% at 254 nm), method A.

2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo

[d]thiazole-5-carboxylic acid (3d). Synthesized according to gen-

eral procedure C using methyl 2-(3,4-dichloro-5-methyl-1H-pyr-

role-2-carboxamido)benzo [d]thiazole-5-carboxylate (2e, 100 mg,

0.260 mmol) at room temperature. The product was isolated by

cooling the reaction mixture to room temperature, then it was

acidiﬁed using ion exchange resin (Amberlite IR120; pH˂5) and

stirred at room temperature for 5 min. DMF was added to dissolve

the precipitate that formed. The resin was ﬁltered off and the

ﬁltrate was evaporated to dryness. The residue was dispersed in

acetone, sonicated and ﬁltered off. The product was washed with

acetonitrile and diethyl ether and dried. Yield: 64 mg (66.4%); off-

¹H NMR (400 MHz, DMSO‑d₆)

d 2.27 (s, 3H), 7.40 (s, 1H), 7.76 (d,

J ¼ 8.5 Hz, 1H), 7.96 (d, J ¼ 8.1 Hz, 1H), 8.04 (s, 1H), 8.49 (s, 1H), 11.99

(s, 1H), 12.52 (s, 1H). HRMS (ESI^ꢂ) m/z for C₁₄H₉Cl₂N₄O₂S ([M ꢂ H]^-):

calculated 366.9829, found 366.9822. HPLC: t_r5.70 min (95.3% at

254 nm), method A.

General procedure D. Synthesis of compounds 6a-d, 33, and

36 (with 6a as an example). A solution of 2-(4,5-dibromo-1H-

pyrrole-2-carboxamido)benzo [d]thiazole-6-carboxylic acid (3b,

100 mg, 0.225 mmol) in N,N-dimethylformamide (3 mL) was cooled

to 0 ^ꢀC and then EDC (42 mg, 0.27 mmol) and HOBt (40 mg,

0.295 mmol) were added. The pH was adjusted to 8 with N-

methylmorpholine, and the reaction mixture was stirred for

20 min at 0 ^ꢀC. Then O-(tetrahydro-2H-pyran-2-yl)hydroxylamine

(26 mg, 0.225 mmol) was added, and reaction mixture was stirred

overnight at room temperature. The solvent was evaporated in

vacuo and the residue dissolved in ethyl acetate (30 mL), and

washed successively with 1% citric acid (15 mL), saturated aqueous

NaHCO₃solution (15 mL), and brine (30 mL). The organic phase was

dried over Na₂SO₄and ﬁltered, and the solvent evaporated off

white solid. ¹H NMR (400 MHz, DMSO‑d₆)

J ¼ 8.8 Hz, 1H), 8.09 (dd, J ¼ 8.8, 2.3 Hz, 1H), 8.50 (d, J ¼ 2.2 Hz, 1H),

9.88 (s, 1H), 12.21 (s, 1H). ¹³C NMR (101 MHz, DMSO‑d₆)

11.30,

109.23, 112.62, 112.71, 119.61, 123.04, 125.79, 127.87, 128.26, 128.93,

139.11, 157.90, 167.94. HRMS (ESI^ꢂ) m/z for C₁₄H₈Cl₂N₃O₃S

([M ꢂ H]^-): calculated 367.9669, found 367.9661. HPLC: t_r6.61 min

(96.1% at 254 nm), method A.

d 2.25 (s, 3H), 7.79 (d,

d

3,4-Dichloro-N-(6-methoxybenzo[d]thiazol-2-yl)-5-methyl-

1H-pyrrole-2-carboxamide (5a). Synthesized according to general

procedure

B using 6-methoxybenzo [d]thiazol-2-amine (4a,

110 mg, 0.515 mmol). The crude product was dispersed in

13

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European Journal of Medicinal Chemistry 213 (2021) 113200

under reduced pressure.

7.78 (d, J ¼ 8.6 Hz, 1H), 7.85 (dd, J ¼ 8.4, 1.7 Hz, 1H), 8.39 (d,

J ¼ 1.6 Hz, 1H), 11.27 (s, 1H), 11.92 (s, 1H), 12.40 (s, 1H). HRMS (ESI^ꢂ)

m/z for C₁₄H₉Cl₂N₄O₃S ([M ꢂ H]^-): calculated 382.97669, found

382.97842. HPLC: t_r5.46 min (97.4% at 254 nm), method A.

3-(2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)

benzo[d]thiazole-6-carboxamido)propanoic acid (7c). Synthe-

sized according to general procedure C using ethyl 3-(2-(3,4-

dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo [d]thiazole-

6-carboxamido)propanoate (6c, 33 mg, 0.070 mmol) and 1 M

NaOH, and the reaction mixture was stirred at room temperature,

not at 80 ^ꢀC. Yield: 30 mg (95.7%); brown solid. ¹H NMR (400 MHz,

2-(4,5-Dibromo-1H-pyrrole-2-carboxamido)-N-((tetrahydro-

2H-pyran-2-yl)oxy)benzo[d]thiazole-6-carboxamide (6a). Crude

product was puriﬁed using ﬂash column chromatography using

dichloromethane/methanol (20:1) as eluent. Yield: 8.7 mg (7.0%);

beige solid. ¹H NMR (400 MHz, DMSO‑d₆)

d

1.57 (s, 3H), 1.74 (s, 3H),

3.50e3.59 (m,1H), 4.01e4.15 (m,1H), 5.03 (s,1H), 7.55 (d, J ¼ 2.7 Hz,

1H), 7.79e7.89 (m, 2H), 8.42 (d, J ¼ 1.6 Hz,1H), 11.69 (s, 1H), 12.79 (s,

1H), 13.28 (s, 1H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 18.78, 25.22,

28.38, 61.81, 97.68, 101.41, 116.00, 119.85, 121.45, 123.41, 124.67,

125.61, 127.14, 132.14, 151.79, 159.66, 161.96, 164.71. HRMS (ESI^þ) m/

z

for

C

₁₈H₁₇Br₂N₄O₄S ([MþH]^þ): calculated 542.9332, found

DMSO‑d₆)

d

2.28 (s, 3H), 2.55 (t, J ¼ 7.5 Hz, 2H), 3.49 (q, J ¼ 7.0, Hz,

542.9334.

2H), 7.78 (d, J ¼ 8.4 Hz, 1H), 7.93 (dd, J ¼ 8.5, 1.8 Hz, 1H), 8.47 (d,

2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-N-

((tetrahydro-2H-pyran-2-yl)oxy)benzo[d]thiazole-6-

J ¼ 1.2 Hz, 1H), 8.61 (t, J ¼ 5.5 Hz, 1H), 12.43 (s, 1H). HRMS (ESI^ꢂ) m/z

for

C₁₇H₁₃Cl₂N₄O₄S ([M

ꢂ

H]^-): calculated 439.0040, found

carboxamide (6b). Synthesized according to general procedure D

using 2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo

[d]thiazole-6-carboxylic acid (3d, 0.150 g, 0.41 mmol). Yield: 70 mg

439.0043. HPLC: t_r5.72 min (90.7% at 254 nm), method A.

General procedure F. Synthesis of compounds 9 and 15c (with

9 as an example). To a solution of 2-aminobenzo [d]thiazole-6-

carboxylic acid (1.50 g, 7.7 mmol) in dry DMF (10 mL), potassium

carbonate (1.60 g, 11.58 mmol) and 4-methoxybenzyl chloride

(1.45 mL, 9.24 mmol) were added, and the mixture was stirred at

room temperature for 14 h. The solvent was removed under

reduced pressure, and to the residue, ethyl acetate (30 mL) and

water (30 mL) were added. The organic phase was dried over

Na₂SO₄and ﬁltered, and the solvent evaporated under reduced

pressure, to give 9 (219 mg) as a white solid.

(36.8%); off-white solid. ¹H NMR (400 MHz, DMSO‑d₆)

d 1.56 (s, 3H),

1.74 (s, 3H), 2.24 (d, J ¼ 2.2 Hz, 3H), 3.50e3.58 (m, 1H), 4.03e4.13

(m, 1H), 5.01 (s, 1H), 7.63 (d, J ¼ 9.5 Hz, 1H), 7.72e7.80 (m, 1H), 8.26

(d, J ¼ 8.6 Hz, 1H), 11.61 (s, 1H). HRMS (ESI^þ) m/z for C₁₄H₉Cl₂N₄O₂S

([MþH]^þ): calculated 469.0499, found 469.0495.

Ethyl

3-(2-(3,4-dichloro-5-methyl-1H-pyrrole-2-

carboxamido)benzo[d]thiazole-6-carboxamido)propanoate

(6c). Synthesized according to general procedure D using 3,4-

dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo [d]thiazole-

6-carboxylic acid (3d, 80 mg, 0.22 mmol). Yield: 47 mg (45.5%);

4-Methoxybenzyl

(9). Yield: 219 mg (9.0%); white solid. ¹H NMR (400 MHz,

DMSO‑d₆) 3.76 (s, 3H), 5.26 (s, 2H), 6.93e6.99 (m, 2H), 7.40e7.48

(m, 3H), 7.88 (dd, J ¼ 8.5, 1.8 Hz, 1H), 8.37 (d, J ¼ 1.8 Hz, 1H), 8.53 (s,

2H). ¹³C NMR (101 MHz, DMSO‑d₆)

55.59, 66.19, 114.35, 117.58,

2-aminobenzo[d]thiazole-6-carboxylate

light brown solid. ¹H NMR (400 MHz, DMSO‑d₆)

d

1.19 (t, J ¼ 7.0 Hz,

d

3H), 2.28 (s, 3H), 2.61 (t, J ¼ 6.9 Hz, 2H), 3.53 (q, J ¼ 5.7 Hz, 2H), 4.09

(q, J ¼ 7.2 Hz, 2H), 7.79 (s, 1H), 7.93 (d, J ¼ 8.5 Hz, 1H), 8.46 (s, 1H),

8.63 (t, J ¼ 4.6 Hz, 1H), 11.92 (s, 1H), 12.34 (s, 1H). HRMS (ESI^þ) m/z

d

122.20, 123.07, 127.62, 128.74, 130.42, 131.67, 157.47, 159.65, 166.01,

for

469.0497.

C

₁₉H₁₉Cl₂N₄O₄S ([MþH]^þ): calculated 469.0499, found

170.32. MS (ESI) m/z ¼ 314.5 ([MþH]^þ).

General procedure G. Synthesis of compounds 10 and 12

(with 12 as an example). To a solution of ethyl 2-aminobenzo [d]

thiazole-6-carboxylate (11, 8.0 g, 36.0 mmol) and CuBr₂(16.07 g,

72.0 mmol) in acetonitrile (200 mL), tert-butyl nitrite (7.42 mL,

72.0 mmol) was added in an ice bath. The reaction mixture was

stirred at room temperature for 14 h. The solvent was removed

under reduced pressure, and to the residue, ethyl acetate (200 mL)

and NH₄Cl solution (200 mL) were added. The organic phase was

washed with brine (100 mL) and NH₄Cl solution (100 mL), dried

over Na₂SO₄, and ﬁltered, and the solvent was evaporated under

reduced pressure. Compound 12 (8.0 g) was obtained as a pale

brown solid.

N-(Cyanomethyl)-2-(3,4-dichloro-5-methyl-1H-pyrrole-2-

carboxamido)benzo[d]thiazole-6-carboxamide (6d). Synthe-

sized according to general procedure D using 3,4-dichloro-5-

methyl-1H-pyrrole-2-carboxamido)benzo [d]thiazole-6-carboxylic

acid (3d, 60 mg, 0.162 mmol). Yield: 45 mg (68.0%); gray solid. ¹H

NMR (400 MHz, DMSO‑d₆)

d

2.28 (s, 3H), 4.35 (d, J ¼ 5.4 Hz, 2H),

7.83 (s, 1H), 7.95 (dd, J ¼ 8.5, 1.8 Hz, 1H), 8.52 (s, 1H), 9.27 (t,

J ¼ 5.5 Hz, 1H), 11.97 (s, 1H), 12.34 (s, 1H). HRMS (ESI^þ) m/z for

C

₁₆H₁₂Cl₂N₅O₂S ([MþH]^þ): calculated 408.0083, found 408.0082.

HPLC: t_r6.14 min (95.2% at 254 nm), method A.

General procedure E. Synthesis of compounds 7a, 7b (with 7a

as an example).

carboxamido)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzo

A

solution of 2-(4,5-dibromo-1H-pyrrole-2-

[d]thia-

4-Methoxybenzyl 2-bromobenzo[d]thiazole-6-carboxylate

(10). Synthesized according to general procedure G using 4-

methoxybenzyl 2-aminobenzo [d]thiazole-6-carboxylate (9,

0.300 g, 0.95 mmol). The reaction mixture was stirred at room

temperature for only 2 h. Yield: 0.354 g (98.1%); light brown solid.

zole-6-carboxamide (6a, 8.7 mg, 0.016 mmol) in dichloromethane

(5 mL) and CF₃COOH (0.012 mL, 0.161 mmol) was stirred at room

temperature for 2 h. The precipitate was ﬁltered off, washed with

dichloromethane, and dried to give 7a (1.7 mg) as a white solid.

2-(4,5-Dibromo-1H-pyrrole-2-carboxamido)-N-hydrox-

¹H NMR (400 MHz, DMSO‑d₆)

d

3.77 (s, 3H), 5.32 (s, 2H), 6.98 (d,

J ¼ 8.7 Hz, 2H), 7.45 (d, J ¼ 8.7 Hz, 2H), 88.07e8.08 (m, 2H), 8.80 (s,

1H). ¹³C NMR (101 MHz, DMSO‑d₆)

55.61, 66.97, 114.39, 122.74,

ybenzo[d]thiazole-6-carboxamide (7a). Yield: 1.7 mg (23%);

d

white solid. ¹H NMR (400 MHz, DMSO‑d₆)

d

7.55 (s, 1H), 7.76e7.90

(m, 2H), 8.40 (s, 1H), 11.27 (s, 1H), 12.78 (s, 1H), 13.28 (s, 1H). ¹³C

NMR (101 MHz, DMSO‑d₆) 99.62, 109.34, 116.80, 120.38, 121.37,

124.68, 127.31, 127.96, 128.27, 130.62, 137.72, 144.76, 155.21, 159.79,

165.53. MS (ESI) m/z ¼ 398.8 ([M þ Nae2H]^-).

d

Ethyl 2-bromobenzo[d]thiazole-6-carboxylate (12) [28].

Yield: 8.0 g (78%); pale brown solid. ¹H NMR (400 MHz, DMSO‑d₆):

125.53, 126.05, 128.51, 132.03, 151.07, 157.72, 160.81, 164.57. HRMS

(ESI^þ) m/z for C₁₃H₉Br₂N₄O₃S ([MþH]^þ): calculated 458.87566,

found 458.87604. HPLC: t_r5.12 min (96.1% at 254 nm), method A.

2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-N-

hydroxybenzo[d]thiazole-6-carboxamide (7b). Synthesized ac-

cording to general procedure E using 2-(3,4-dichloro-5-methyl-1H-

pyrrole-2-carboxamido)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzo

[d]thiazole-6-carboxamide (6b, 0.070 g, 0.15 mmol). Yield: 23 mg

d

1.36 (t, J ¼ 7.1 Hz, 3H), 4.37 (q, J ¼ 7.1 Hz, 2H), 8.10 (s, 2H), 8.82 (s,

1H).

General procedure H. Synthesis of compounds 13, 15a, and

15b (with 13 as an example). A solution of ethyl 2-bromobenzo [d]

thiazole-6-carboxylate (12, 1.00 g, 3.49 mmol) and isopropylamine

(2.86 mL, 34.9 mmol) in THF (70 mL) was stirred at room temper-

ature for 14 h. The solvent was removed under reduced pressure.

The crude product was dissolved in ethyl acetate (100 mL) and

(40.0%); brown solid. ¹H NMR (400 MHz, DMSO‑d₆)

d 2.28 (s, 3H),

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European Journal of Medicinal Chemistry 213 (2021) 113200

washed successively with 1% citric acid (2 ꢁ 50 mL), saturated

NaHCO₃solution (50 mL), and brine (50 mL). The organic phase was

dried over Na₂SO₄and ﬁltered, and the solvent was removed under

reduced pressure, to give 13 (0.915 g) as a yellow oil.

methoxybenzyl

carboxylate (15b, 0.201 g, 0.57 mmol). Yield: 0.092 g (30.6%);

white solid. ¹H NMR (400 MHz, DMSO‑d₆)

0.59e0.66 (m, 2H), 1.04

2-(cyclopropylamino)benzo

[d]thiazole-6-

d

(q, J ¼ 6.9 Hz, 2H), 2.26 (s, 3H), 3.68 (tt, J ¼ 7.3, 3.9 Hz, 1H), 3.77 (s,

3H), 5.31 (s, 2H), 6.95e7.01 (m, 2H), 7.43e7.47 (m, 2H), 7.87e7.93

(m, 1H), 8.02 (dt, J ¼ 8.6, 2.1 Hz, 1H), 8.66e8.69 (m, 1H), 12.31 (s,

1H). MS (ESI) m/z ¼ 530.0 ([MþH]^þ).

Ethyl

(13). Yield: 0.915 g (99.1%), yellow oil. ¹H NMR (400 MHz,

DMSO‑d₆):

1.22 (d, J ¼ 6.5 Hz, 6H),1.32 (t, J ¼ 7.1 Hz, 3H), 3.96e4.11

2-(isopropylamino)benzo[d]thiazole-6-carboxylate

d

(m, 1H), 4.28 (q, J ¼ 7.1 Hz, 2H), 7.41 (d, J ¼ 8.4 Hz, 1H), 7.81 (dd,

4-Methoxybenzyl 2-(3,4-dichloro-N-isopropyl-5-methyl-1H-

pyrrole-2-carboxamido)benzo[d]thiazole-6-carboxylate (16c).

J ¼ 8.4,1.8 Hz,1H), 8.28 (d, J ¼ 1.6 Hz,1H), 8.35 (d, J ¼ 7.3 Hz,1H). ¹³

C

NMR (101 MHz, DMSO‑d₆)

d

14.72, 22.72, 46.57, 60.81, 117.69,

Synthesized according to general procedure

methoxybenzyl 2-(isopropylamino)benzo

B

using 4-

122.34, 122.90, 127.57, 130.88, 157.27, 166.09, 168.54. MS (ESI) m/

[d]thiazole-6-

z ¼ 265.0 ([MþH]^þ).

carboxylate (15c, 0.150 g, 0.42 mmol). The crude product was pu-

riﬁed by preparative TLC using dichloromethane/methanol (20:1)

as eluent. Yield: 0.031 g (13.8%); white solid. ¹H NMR (400 MHz,

2-(Isopropylamino)benzo[d]thiazole-6-carboxylic acid (14).

Synthesized according to general procedure C using ethyl 2-(iso-

propylamino)benzo [d]thiazole-6-carboxylate (13, 0.940 g,

3.56 mmol), with pH was adjusted to 3. Yield: 0.749 g (89.1%); gray

CDCl₃)

d

1.56 (s, 6H), 2.30 (s, 3H), 3.82 (s, 3H), 5.03 (p, J ¼ 6.8 Hz,1H),

5.31 (s, 2H), 6.90e6.95 (m, 2H), 7.37e7.43 (m, 2H), 7.81 (d,

J ¼ 8.5 Hz, 1H), 8.10 (dd, J ¼ 8.6, 1.7 Hz, 1H), 8.40 (d, J ¼ 1.6 Hz, 1H),

9.09 (s, 1H).

solid. ¹H NMR (400 MHz, DMSO‑d₆):

d

1.22 (d, J ¼ 6.6 Hz, 6H), 4.02

(q, J ¼ 6.6 Hz, 1H), 7.39 (d, J ¼ 8.1 Hz, 1H), 7.79 (d, J ¼ 8.3 Hz, 1H),

8.13e8.45 (m, 2H), 12.62 (s, 1H). ¹³C NMR (101 MHz, DMSO‑d₆)

General procedure I. Synthesis of compounds 17a-c (with 17a

as an example). To a suspension of 4-methoxybenzyl 2-(3,4-

dichloro-N-ethyl-5-methyl-1H-pyrrole-2-carboxamido)benzo [d]

thiazole-6-carboxylate (63 mg, 0.12 mmol) in glacial acetic acid

(5 mL), 1 M HCl in acetic acid (1.2 mmol) was added, and the re-

action mixture was stirred at room temperature overnight. The

precipitate was ﬁltered off, washed with diethyl ether, and dried in

vacuo to obtain 17a (13 mg) as a white solid.

d

22.73, 46.53, 117.62, 123.10, 123.33, 127.80, 130.71, 156.98, 167.67,

168.30. MS (ESI) m/z ¼ 237.1 ([MþH]^þ).

4-Methoxybenzyl

2-(ethylamino)benzo[d]thiazole-6-

carboxylate (15a). Synthesized according to general procedure H

using 4-methoxybenzyl 2-bromobenzo [d]thiazole-6-carboxylate

(11, 0.230 g, 0.61 mmol). Yield: 0.203 g (97.5%); white solid. ¹H

NMR (400 MHz, DMSO‑d₆)

d

1.21 (t, J ¼ 7.0 Hz, 3H), 3.41 (q,

J ¼ 7.0 Hz, 2H), 3.76 (s, 3H), 5.25 (s, 2H), 6.92e7.01 (m, 2H), 7.42 (dt,

J ¼ 8.5, 2.3 Hz, 3H), 7.82 (dd, J ¼ 8.5, 1.8 Hz, 1H), 8.30 (d, J ¼ 1.8 Hz,

1H), 8.42 (t, J ¼ 5.3 Hz, 1H). MS (ESI) m/z ¼ 343.1 ([MþH]^þ).

4-Methoxybenzyl 2-(cyclopropylamino)benzo[d]thiazole-6-

carboxylate (15b). Synthesized according to general procedure H

using 4-methoxybenzyl 2-bromobenzo [d]thiazole-6-carboxylate

(11, 0.279 g, 0.74 mmol). Yield: 0.211 g (80.7%); white solid. ¹H

2-(3,4-Dichloro-N-ethyl-5-methyl-1H-pyrrole-2-

carboxamido)benzo[d]thiazole-6-carboxylic acid (17a). Yield:

13 mg (26.9%); white solid. ¹H NMR (400 MHz, DMSO‑d₆)

d 1.23 (t,

J ¼ 6.9 Hz, 3H), 2.26 (s, 3H), 4.41 (q, J ¼ 6.9 Hz, 2H), 7.90 (d,

J ¼ 8.5 Hz, 1H), 8.02 (dd, J ¼ 8.5, 1.6 Hz, 1H), 8.63 (d, J ¼ 1.5 Hz, 1H),

12.52 (s, 1H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 10.86, 13.96, 45.20,

108.46, 112.44, 117.65, 120.67, 123.68, 126.26, 127.26, 129.69, 132.13,

151.42, 161.70, 162.66, 166.88. HRMS (ESI^þ) m/z for C₁₆H₁₄Cl₂N₃O₃S

([MþH]^þ): calculated 398.0127, found 398.0125. HPLC: t_r3.813 min

(97.4% at 254 nm), method C.

2-(3,4-Dichloro-N-cyclopropyl-5-methyl-1H-pyrrole-2-

carboxamido)benzo[d]thiazole-6-carboxylic acid (17b). Synthe-

sized according to general procedure I using 4-methoxybenzyl 2-

(3,4-dichloro-N-cyclopropyl-5-methyl-1H-pyrrole-2-

NMR (400 MHz, DMSO‑d₆)

d

0.55e0.64 (m, 2H), 0.79 (td, J ¼ 6.9,

4.7 Hz, 2H), 2.73 (s, 1H), 3.76 (s, 3H), 5.26 (s, 2H), 6.93e7.00 (m, 2H),

7.39e7.51 (m, 3H), 7.82e7.88 (m, 1H), 8.38 (d, J ¼ 1.8 Hz,1H), 8.77 (s,

1H). ¹³C NMR (101 MHz, DMSO‑d₆)

d

7.32, 26.71, 55.59, 66.22,

114.36, 118.03, 122.16, 123.28, 127.70, 128.73, 130.43, 131.12, 133.51,

157.27, 159.66, 166.02. MS (ESI) m/z ¼ 355.0 ([MþH]^þ).

4-Methoxybenzyl

2-(isopropylamino)benzo[d]thiazole-6-

carboxylate (15c). Synthesized according to general procedure F

using 2-(isopropylamino)benzo [d]thiazole-6-carboxylic acid (14,

0.568 g, 2.40 mmol). The crude product was recrystallized from

ethyl acetate. Yield: 0.806 g (94.1%), white solid. ¹H NMR (400 MHz,

carboxamido)benzo

0.094 mmol). Yield: 10 mg (25.9%); white solid. ¹H NMR (400 MHz,

DMSO‑d₆)

0.63 (s, 2H), 1.04 (d, J ¼ 6.9 Hz, 2H), 2.27 (s, 3H), 7.89 (d,

J ¼ 8.5 Hz, 1H), 8.00 (dd, J ¼ 8.6, 1.8 Hz, 1H), 8.62 (d, J ¼ 1.8 Hz, 1H),

12.32 (s, 1H). ¹³C NMR (101 MHz, DMSO- d₆)

10.95, 11.44, 32.53,

109.50, 114.65, 119.20, 121.17, 124.14, 126.52, 127.71, 130.32, 132.64,

152.49, 162.34, 165.03, 167.51. HRMS (ESI^þ) m/z for C₁₇H₁₄Cl₂N₃O₃S

([MþH]^þ): calculated 410.0127, found 410.0124. HPLC: t_r3.820 min

(92.5% at 254 nm), method C.

[d]thiazole-6-carboxylate

(50

mg,

d

DMSO‑d₆)

d

1.22 (d, J ¼ 6.5 Hz, 6H), 3.76 (s, 3H), 4.02 (q, J ¼ 6.7 Hz,

d

1H), 5.25 (s, 2H), 6.92e7.02 (m, 2H), 7.31e7.50 (m, 3H), 7.82 (dd,

J ¼ 8.4,1.8 Hz, 1H), 8.29 (d, J ¼ 1.8 Hz, 1H), 8.36 (d, J ¼ 7.3 Hz, 1H). ¹³

C

NMR (101 MHz, DMSO‑d₆)

d 22.67, 46.73, 55.57, 66.21, 114.34,

117.55, 122.31, 123.11, 127.77, 128.71, 128.96, 130.41, 156.60, 159.65,

165.95, 168.56. MS (ESI) m/z ¼ 355.1 ([M ꢂ H]^-).

2-(3,4-Dichloro-N-isopropyl-5-methyl-1H-pyrrole-2-

4-Methoxybenzyl 2-(3,4-dichloro-N-ethyl-5-methyl-1H-pyr-

role-2-carboxamido)benzo[d]thiazole-6-carboxylate (16a). Syn-

thesized according to general procedure B using 4-methoxybenzyl

2-(ethylamino)benzo [d]thiazole-6-carboxylate (15a, 0.180 g,

0.53 mmol). Yield: 0.078 g (28.6%); white solid. ¹H NMR (400 MHz,

carboxamido) benzo[d]thiazole-6-carboxylic acid (17c). Synthe-

sized according to general procedure I using 4-methoxybenzyl 2-

(3,4-dichloro-N-isopropyl-5-methyl-1H-pyrrole-2-carboxamido)

benzo [d]thiazole-6-carboxylate (29 mg, 0.055 mmol). Yield: 18 mg

(80.2%); white solid. ¹H NMR (400 MHz, DMSO‑d₆)

d 1.51 (d,

DMSO‑d₆)

d

1.22 (t, J ¼ 7.0 Hz, 3H), 2.25 (s, 3H), 3.76 (s, 3H), 4.40 (q,

J ¼ 6.2 Hz, 6H), 2.23 (s, 3H), 4.96 (s, 1H), 7.83 (d, J ¼ 8.2 Hz, 1H), 7.97

J ¼ 7.0Hz, 2H), 5.30 (s, 2H), 6.97e7.02 (m, 2H), 7.45 (d, J ¼ 8.7 Hz,

2H), 7.88e7.95 (m, 1H), 8.03 (dd, J ¼ 8.5, 1.8 Hz, 1H), 8.68 (dd, J ¼ 1.7,

(d, J ¼ 7.0 Hz, 1H), 8.53 (s, 1H), 12.53 (s, 1H). HRMS (ESI^þ) m/z for

C

₁₇H₁₆Cl₂N₃O₃S ([MþH]^þ): calculated 412.0284, found 412.0279.

0.4 Hz, 1H), 12.50 (s, 1H). ¹³C NMR (101 MHz, DMSO‑d₆)

d

11.43,

HPLC: t_r3.873 min (98.0% at 254 nm), method C.

14.52, 45.79, 55.61, 66.62, 109.09, 113.10, 114.38, 118.17, 121.42,

124.30, 125.67, 127.67, 128.51, 130.33, 130.50, 132.90, 152.32, 159.72,

162.29, 163.66, 165.81. MS (ESI) m/z ¼ 516.0 ([M ꢂ H]^-).

Methyl 4-amino-2-hydroxybenzoate (19) [29]. To a solution of

4-aminosalicylic acid (18, 7.50 g, 49.0 mmol) in methanol (70 mL),

H₂SO₄(4 mL, 75 mmol) was added, and the solution was stirred at

65 ^ꢀC for 24 h. The solvent was removed in vacuo, and the residue

dissolved in ethyl acetate (100 mL) and neutralized with saturated

aqueous NaHCO₃solution (100 mL). The phases were separated, the

4-Methoxybenzyl 2-(3,4-dichloro-N-cyclopropyl-5-methyl-

1H-pyrrole-2-carboxamido)benzo[d]thiazole-6-carboxylate

(16b). Synthesized according to general procedure B using 4-

15

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M. Durcik, A. Nyerges, Z. Skok et al.

European Journal of Medicinal Chemistry 213 (2021) 113200

water phase was extracted with ethyl acetate (3 ꢁ 75 mL), and the

combined organic phases were washed with brine (3 ꢁ 75 mL),

dried over Na₂SO₄, and ﬁltered, and the solvent removed in vacuo.

Yield: 7.26 g (88.6%); brown solid; mp: 99e104 ^ꢀC. ¹H NMR

reﬂux, and the undissolved solid was ﬁltered off. Methanol was

removed under reduced pressure, to obtain 23 (205 mg) as a yellow

solid.

Methyl 5-acetoxy-2-aminobenzo[d]thiazole-6-carboxylate

(400 MHz, CDCl₃) d 3.90 (s, 3H), 4.12 (s, 2H), 6.12e6.20 (m, 2H), 7.64

(23). Yield: 205 mg (23%); yellow solid.¹H NMR (400 MHz,

(dd, J ¼ 8.0, 0.9 Hz, 1H), 10.97 (s, 1H).

DMSO‑d₆) d 2.28 (s, 3H), 3.78 (s, 3H), 7.09 (s, 1H), 8.08 (s, 2H), 8.29

Methyl 4-((tert-butoxycarbonyl)amino)-2-hydroxybenzoate

(20) [30]. To a solution of methyl 4-amino-2-hydroxybenzoate

(9.57 g, 57.3 mmol), di-tert-butyl dicarbonate (13.8 g, 63.0 mmol)

was added, and the mixture was stirred at 70 ^ꢀC for 48 h. The

solvent was removed under reduced pressure, to the residue ethyl

acetate (100 mL) and water were added, and the phases were

separated. The organic phase was washed with 1 M HCl (3 ꢁ 40 mL)

and brine (3 ꢁ 40 mL), dried over Na₂SO₄, and ﬁltered, and the

solvent was removed in vacuo. The crude product was puriﬁed with

ﬂash column chromatography using ethyl acetate/hexane (1:7) as

eluent. Yield: 6.52 g (43%); white crystals. ¹H NMR (400 MHz,

(s, 1H). MS (ESI) m/z ¼ 289.0 ([MþNa]^þ).

Methyl

5-acetoxy-2-(3,4-dichloro-5-methyl-1H-pyrrole-2-

carboxamido)benzo[d]thiazole-6-carboxylate (24). Synthesized

according to general procedure B using methyl 5-acetoxy-2-

aminobenzo [d]thiazole-6-carboxylate (23, 100 mg, 0.376 mmol).

The crude product was dispersed in diethyl ether (20 mL), soni-

cated, ﬁltered off, and dried. Yield: 56 mg (33%); brown solid. ¹H

NMR (400 MHz, DMSO‑d₆)

d 2.28 (s, 3H), 2.32 (s, 3H), 3.84 (s, 3H),

7.60 (s, 1H), 8.67 (s, 1H), 12.06 (s, 1H), 12.36 (s, 1H).

2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-5-

hydroxybenzo[d]thiazole-6-carboxylic acid (25). Synthesized

according to general procedure C using methyl 5-acetoxy-2-(3,4-

dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo [d]thiazole-

6-carboxylate (24, 39 mg, 0.088 mmol) and 4 M NaOH (0.221 mL,

0.88 mmol). The crude product was dispersed in diethyl ether

(10 mL), sonicated, ﬁltered off, and dried. Yield: 22.3 mg (66%);

CDCl₃)

d

1.54 (s, 9H), 3.94 (s, 3H), 6.62 (s, 1H), 6.95 (dd, J ¼ 8.7,

2.2 Hz, 1H), 7.01 (d, J ¼ 2.1 Hz, 1H), 7.76 (d, J ¼ 8.7 Hz, 1H), 10,86 (s,

1H).

Methyl 2-acetoxy-4-((tert-butoxycarbonyl)amino)benzoate

(21) [31]. To a solution of methyl 4-((tert-butoxycarbonyl)amino)-

2-hydroxybenzoate (1.28 g, 4.8 mmol) in acetonitrile (15 mL),

pyridine (0.694 mL, 8.6 mmol) and acetic anhydride (0.869 mL,

8.6 mmol) were added, and the mixture was stirred at 70 ^ꢀC for

48 h. The solvent was removed under reduced pressure, and the

residue was dissolved in ethyl acetate (40 mL). The organic phase

was washed with 1 M HCl (3 ꢁ 20 mL) and brine (3 ꢁ 20 mL), dried

over Na₂SO₄, and ﬁltered, and the solvent was removed in vacuo. To

the crude product, hexane was added, with the resulting suspen-

sion sonicated, and the solid was ﬁltered off, washed with hexane,

and dried. Yield: 1.24 g (84%); white solid. ¹H NMR (400 MHz,

brown solid. ¹H NMR (400 MHz, DMSO‑d₆)

d 2.27 (s, 3H), 6.94 (s,

1H), 8.22 (s, 1H), 12.00 (s, 1H), 12.31 (s, 1H). HRMS (ESI^ꢂ) m/z for

C

₁₄H₉Cl₂N₃O₄S ([M ꢂ H]^-): calculated 383.9618, found 383.9621.

HPLC: t_r8.890 min (99.1% at 254 nm), method B.

Methyl

2-(4,5-dibromo-1H-pyrrole-2-carboxamido)-4-

hydroxybenzo[d]thiazole-6-carboxylate (27a). Synthesized ac-

cording to general procedure

hydroxybenzo [d]thiazole-6-carboxylate

0.67 mmol). Yield: 167 mg (31.8%); off-brown solid. ¹H NMR

(400 MHz, DMSO‑d₆)

3.86 (s, 3H), 7.47 (d, J ¼ 1.6 Hz, 1H), 7.56 (d,

J ¼ 2.5 Hz, 1H), 8.10 (d, J ¼ 1.5 Hz, 1H), 10.32 (s, 1H), 12.90 (s, 1H),

13.26 (s, 1H). ¹³C NMR (101 MHz, DMSO‑d₆)

52.58, 99.59, 109.23,

A

using methyl 2-amino-4-

(26a, 248 mg,

d

CDCl₃)

d 1.54 (s, 9H), 2.36 (s, 3H), 3.86 (s, 3H), 6.74 (s, 1H) 7.14 (dd,

J ¼ 8.7, 1.7 Hz, 1H), 7.40 (d, J ¼ 1.7 Hz, 1H), 7.97 (d, J ¼ 8.7 Hz, 1H).

d

Methyl 2-acetoxy-4-aminobenzoate (22) [31]. To methyl 2-

111.98, 114.59, 116.73, 125.91, 126.08, 133.78, 150.03, 157.67, 159.74,

166.58, 171.75. HRMS (ESI^þ) m/z for C₁₄H₁₀Br₂N₃O₄S ([MþH]^þ):

calculated 473.8753, found 473.8749.

acetoxy-4-((tert-butoxycarbonyl)amino)benzoate

(1.24

g,

4.01 mmol), 2 M HCl in diethyl ether (35 mL) was added, and the

mixture was stirred at room temperature overnight. The product

precipitated as a white solid and was ﬁltered off, washed with

ether, and dried. The ﬁltrate contained the remaining starting

material, so the solvent was removed under reduced pressure, to

the residue, 2 M HCl in diethyl ether (5 mL) was added, and the

mixture was stirred at room temperature overnight. The white

precipitate was ﬁltered off, washed with ether, and dried. The

combined product in the form of a hydrochloride salt (0.911 g) was

dissolved in ethyl acetate (100 mL), and the organic phase was

washed with saturated aqueous NaHCO₃solution (50 mL) and brine

(50 mL), dried over Na₂SO₄, and ﬁltered, and the solvent was

removed in vacuo. Yield: 0.760 g (86%); light brown solid. ¹H NMR

Methyl

4-((tert-butyldimethylsilyl)oxy)-2-(3,4-dichloro-5-

methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6-

carboxylate (27b). Synthesized according to general procedure B

using methyl 2-amino-4-((tert-butyldimethylsilyl)oxy)benzo [d]

thiazole-6-carboxylate (26b, 87 mg, 0.26 mmol). Yield: 70 mg

(52.9%); white solid. ¹H NMR (400 MHz, DMSO‑d₆)

d 2.10 (s, 3H),

3.87 (s, 3H), 7.41 (s, 1H), 8.26 (s, 1H), 11.84 (s, 1H), 12.46 (s, 1H).

HRMS (ESI^þ) m/z for C₂₁H₂₆Cl₂N₃O₄SSi ([MþH]^þ): calculated

514.0785, found 514.0783.

2-(4,5-Dibromo-1H-pyrrole-2-carboxamido)-4-

hydroxybenzo[d]thiazole-6-carboxylic acid (28a). Synthesized

according to general procedure C using methyl 2-(4,5-dibromo-1H-

(400 MHz, DMSO‑d₆)

d

2.23 (s, 3H), 3.68 (s, 3H), 6.20 (br s, 2H), 6.22

pyrrole-2-carboxamido)-4-hydroxybenzo

carboxylate (100 mg, 0.21 mmol). Yield: 83 mg (85.5%); black

solid. ¹H NMR (400 MHz, DMSO‑d₆)

J ¼ 2.8 Hz, 1H), 8.04 (d, J ¼ 1.5 Hz, 1H), 10.31 (s, 1H), 12.87 (s, 1H),

13.27 (d, J ¼ 2.8 Hz, 1H). HRMS (ESI^ꢂ) m/z for C₁₃H₆Br₂N₃O₄S

([M ꢂ H]^-): calculated 457.8451, found 457.8458. HPLC: t_r5.173 min

(98.8% at 254 nm), method B.

2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-

hydroxybenzo[d]thiazole-6-carboxylic acid (28b). Synthesized

according to general procedure C using methyl 4-((tert-butyldi-

methylsilyl)oxy)-2-(3,4-dichloro-5-methyl-1H-pyrrole-2-

carboxamido)benzo [d]thiazole-6-carboxylate (27b, 70 mg,

[d]thiazole-6-

(d, J ¼ 2.2 Hz, 1H), 6.46 (dd, J ¼ 8.7, 2.2 Hz, 1H), 7.65 (d, J ¼ 8.7 Hz,

1H).

d

7.48 (d, J ¼ 1.6 Hz,1H), 7.56 (d,

General procedure J. Synthesis of compounds 23, 42, and 46

(with 23 as an example). A solution of methyl 2-acetoxy-4-

aminobenzoate (0.716 g, 3.43 mmol) and KSCN (1.33 g,

13.7 mmol) in acetic acid (12 mL) was stirred at room temperature

for 20 min. It was then cooled to 10 ^ꢀC, bromine (0.351 mL,

6.85 mmol) in acetic acid (3 mL) was added, and the mixture was

stirred at room temperature overnight. The suspension was cooled

in an ice bath, and neutralized with saturated aqueous NaHCO₃

solution (500 mL), with the resulting precipitate ﬁltered off and

dried at 60 ^ꢀC for 1.5 h. The solid was puriﬁed by adding diethyl

ether (50 mL), with the suspension obtained sonicated, and the

solid ﬁltered off and washed with diethyl ether. To the crude

product, methanol (100 mL) was added, the mixture was heated to

0.21 mmol). An additional 500

reaction mixture was stirred at 80 ^ꢀC for a further 15 h. Yield: 7.8 mg

(14.8%); brown solid. ¹H NMR (400 MHz, DMSO‑d₆)

2.27 (s, 3H),

7.44 (d, J ¼ 1.6 Hz, 1H), 8.05 (d, J ¼ 1.5 Hz, 1H), 10.25 (s, 1H), 11.93 (s,

mL of 2 M NaOH was added, and the

d

16

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M. Durcik, A. Nyerges, Z. Skok et al.

European Journal of Medicinal Chemistry 213 (2021) 113200

1H), 12.36 (s, 1H), 12.80 (s, 1H). HRMS (ESI^ꢂ) m/z for C₁₄H₈Cl₂N₃O₄S

([M ꢂ H]^-): calculated 383.9618, found 383.9622. HPLC: t_r6.310 min

(96.2% at 254 nm), method B.

Methyl

2-(3,4-dichloro-5-methyl-1H-pyrrole-2-

carboxamido)-4-(thiophen-2-ylmethoxy)benzo[d]thiazole-6-

carboxylate (30g). Synthesized according to general procedure B

using methyl 2-amino-4-(thiophen-2-ylmethoxy)benzo [d]thia-

zole-6-carboxylate (29g, 120 mg, 0.375 mmol). Yield: 122 mg

Methyl

2-(3,4-dichloro-5-methyl-1H-pyrrole-2-

(30a).

carboxamido)-4-ﬂuorobenzo[d]thiazole-6-carboxylate

Synthesized according to general procedure B using methyl 2-

amino-4-ﬂuorobenzo [d]thiazole-6-carboxylate (29a, 350 mg,

1.55 mmol). Yield: 242 mg (38.9%); brown solid. ¹H NMR (400 MHz,

(65.6%); light gray solid. ¹H NMR (400 MHz, DMSO‑d₆)

d 2.26 (s, 3H),

3.89 (s, 3H), 5.53 (s, 2H), 7.08 (dd, J ¼ 5.1, 3.4 Hz, 1H), 7.28 (dd,

J ¼ 3.5, 1.2 Hz, 1H), 7.60 (dd, J ¼ 5.1, 1.3 Hz, 1H), 7.65 (d, J ¼ 1.5 Hz,

1H), 8.31 (d, J ¼ 1.4 Hz, 1H), 12.21e12.23 (m, 2H). HRMS (ESI^þ) m/z

DMSO‑d₆)

d

2.28 (s, 3H), 3.90 (s, 3H), 7.78 (dd, J ¼ 11.2, 1.5 Hz, 1H),

8.55 (d, J ¼ 1.5 Hz, 1H), 12.30 (s, 1H), 12.37 (s, 1H). HRMS (ESI^þ) m/z

for

C

₂₀H₁₆Cl₂N₃O₄S ([MþH]^þ): calculated 495.9954, found

for

C

₁₅H₁₁FCl₂N₃O₃S ([MþH]^þ): calculated 401.9877, found

495.9946.

401.9853.

Methyl

2-(3,4-dichloro-5-methyl-1H-pyrrole-2-

Methyl

2-(3,4-dichloro-5-methyl-1H-pyrrole-2-

carboxamido)-4-(thiophen-3-ylmethoxy)benzo[d]thiazole-6-

carboxylate (30h). Synthesized according to general procedure B

using methyl 2-amino-4-(thiophen-3-ylmethoxy)benzo [d]thia-

zole-6-carboxylate (29h, 93 mg, 0.290 mmol). Yield: 63 mg (43.7%);

carboxamido)-4-methoxybenzo[d]thiazole-6-carboxylate (30b).

Synthesized according to general procedure B using methyl 2-

amino-4-methoxybenzo [d]thiazole-6-carboxylate (29b, 368 mg,

1.55 mmol). Yield: 345 mg (53.9%); brown solid. ¹H NMR (400 MHz,

light gray solid. ¹H NMR (400 MHz, DMSO‑d₆)

d 2.26 (s, 3H), 3.89 (s,

DMSO‑d₆)

d

2.28 (s, 3H), 3.90 (s, 3H), 4.00 (s, 3H), 7.50 (d, J ¼ 1.4 Hz,

3H), 5.32 (s, 2H), 7.26 (dd, J ¼ 4.9, 1.3 Hz, 1H), 7.58e7.64 (m, 2H),

7.64e7.70 (m, 1H), 8.30 (d, J ¼ 1.4 Hz, 1H), 12.20 (s, 1H), 12.22 (s, 1H).

HRMS (ESI^ꢂ) m/z for C₂₀H₁₄Cl₂N₃O₄S ([M ꢂ H]^-): calculated

493.9808, found 493.9810.

1H), 8.29 (d, J ¼ 1.2 Hz, 1H), 12.15 (s, 1H), 12.29 (s, 1H). HRMS (ESI^þ)

m/z for C₁₆H₁₄Cl₂N₃O₄S ([MþH]^þ): calculated 414.0077, found

414.0073.

4-(2-((2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-

Methyl

2-(3,4-dichloro-5-methyl-1H-pyrrole-2-

carboxamido)-6-(methoxycarbonyl)benzo[d]thiazol-4-yl)oxy)

ethyl)morpholin-4-ium chloride (30c). Synthesized according to

carboxamido)-4-((3-ﬂuorobenzyl)oxy)benzo[d]thiazole-6-

carboxylate (30i). Synthesized according to general procedure B

using methyl 2-amino-4-((3-ﬂuorobenzyl)oxy)benzo [d]thiazole-

6-carboxylate (29i, 110 mg, 0.331 mmol). Yield: 132 mg (71.7%);

general

procedure

B

using

methyl

2-amino-4-(2-

morpholinoethoxy)benzo [d]thiazole-6-carboxylate (29c, 261 mg,

0.773 mmol). Yield: 70 mg (16.5%); beige solid. ¹H NMR (400 MHz,

light gray solid. ¹H NMR (400 MHz, DMSO‑d₆)

d 2.27 (s, 3H), 3.89 (s,

DMSO‑d₆)

d

2.28 (s, 3H), 3.20e3.30 (m, 2H), 3.65 (s, 4H), 3.79 (t, 2H),

3H), 5.36 (s, 2H), 7.17e7.27 (m, 1H), 7.39e7.41 (m, 2H), 7.45e7.53

(m, 1H), 7.61 (s, 1H), 8.31 (s, 1H), 12.19 (s, 1H), 12.26 (s, 1H). HRMS

(ESI^þ) m/z for C₂₂H₁₇FCl₂N₃O₄S ([MþH]^þ): calculated 508.0295,

found 508.0293.

2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-

ﬂuorobenzo[d]thiazole-6-carboxylic acid (31a). Synthesized ac-

cording to general procedure C using methyl 2-(3,4-dichloro-5-

methyl-1H-pyrrole-2-carboxamido)-4-ﬂuorobenzo [d]thiazole-6-

carboxylate (30a, 100 mg, 0.249 mmol). The crude product was

dispersed in acetonitrile, sonicated, and heated, and the precipitate

was ﬁltered off and dried. Yield: 43 mg (44.5%); brown solid. ¹H

3.90 (s, 3H), 4.04 (d, J ¼ 12.5 Hz, 2H), 4.69 (s, 2H), 7.60 (d, J ¼ 1.4 Hz,

1H), 8.36 (s, 1H), 10.68 (s, 1H), 12.12 (s, 1H), 12.68 (s, 1H). HRMS

(ESI^þ) m/z for C₂₁H₂₃Cl₂N₄O₅S ([MþH]^þ): calculated 513.0761,

found 513.0751.

Methyl

4-(2-((tert-butoxycarbonyl)amino)ethoxy)-2-(3,4-

dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo[d]thia-

zole-6-carboxylate (30d). Synthesized according to general pro-

cedure B using methyl 2-amino-4-(2-((tert-butoxycarbonyl)amino)

ethoxy)benzo

0.544 mmol). Yield: 38 mg (12.8%); beige solid. ¹H NMR (400 MHz,

DMSO‑d₆) 1.40 (s, 9H), 2.28 (s, 3H), 3.41 (m, 2H, signal is over-

lapping with the signal for water),3.89 (s, 3H), 4.22 (t, J ¼ 5.8 Hz,

2H), 7.06 (t, J ¼ 4.8 Hz, 1H), 7.50 (d, J ¼ 1.4 Hz, 1H), 8.29 (s, 1H), 12.26

(s, 2H). HRMS (ESI^þ) m/z for C₂₂H₂₄Cl₂N₄O₆SNa ([MþNa]^þ): calcu-

lated 565.0686, found 565.0682.

[d]thiazole-6-carboxylate

(29d,

200

mg,

d

NMR (400 MHz, DMSO‑d₆)

1H), 8.52 (d, J ¼ 1.4 Hz, 1H), 12.26 (s, 1H), 12.34 (s, 1H), 13.23 (br s,

1H). ¹³C NMR (101 MHz, DMSO‑d₆)

11.04, 110.04, 112.37, 112.57,

d

2.29 (s, 3H), 7.75 (dd, J ¼ 11.2, 1.5 Hz,

d

115.79, 116.67, 120.08, 120.11, 126.81, 130.35, 134.56, 156.67, 161.63,

166.20. HRMS (ESI^ꢂ) m/z for C₁₄H₇Cl₂FN₃O₃S ([M ꢂ H]^-): calculated

385.9580, found 385.9564. HPLC: t_r8.983 min (95.1% at 254 nm),

method B.

Methyl

2-(3,4-dichloro-5-methyl-1H-pyrrole-2-

carboxamido)-4-((3-(methoxycarbonyl)benzyl)oxy)benzo[d]

thiazole-6-carboxylate (30e). Synthesized according to general

procedure

2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-

methoxybenzo[d]thiazole-6-carboxylic acid (31b). Synthesized

according to general procedure C using methyl 2-(3,4-dichloro-5-

methyl-1H-pyrrole-2-carboxamido)-4-methoxybenzo [d]thiazole-

6-carboxylate (30b, 162 mg, 0.391 mmol). Yield: 120 mg (76.7%);

B using methyl 2-amino-4-((3-(methoxycarbonyl)

benzyl)oxy)benzo [d]thiazole-6-carboxylate (29e, 77 mg,

0.207 mmol). Yield: 73 mg (64.4%); light gray solid. ¹H NMR

(400 MHz, DMSO‑d₆) d 2.27 (s, 3H), 3.88 (s, 3H), 3.89 (s, 3H), 5.43 (s,

2H), 7.61 (s, 1H), 7.63e7.65 (m, 1H), 7.83 (s, 1H), 7.98 (dt, J ¼ 7.7,

1.5 Hz, 1H), 8.14e8.17 (m, 1H), 8.32 (d, J ¼ 0.9 Hz, 1H), 12.21 (s, 1H),

12.25 (s, 1H). HRMS (ESI^ꢂ) m/z for C₂₄H₁₈Cl₂N₃O₆S ([M ꢂ H]^-):

calculated 546.0299, found 546.0300.

brown solid. ¹H NMR (400 MHz, DMSO‑d₆)

d

2.28 (s, 3H), 3.98 (s,

3H), 7.50 (d, J ¼ 1.4 Hz, 1H), 8.24 (d, J ¼ 1.4 Hz, 1H), 12.02 (br s, 1H),

12.41 (s, 1H), 12.66 (br s, 1H). ¹³C NMR (101 MHz, DMSO‑d₆)

11.01,

d

55.77, 107.78, 109.87, 115.41, 116.07, 116.91, 126.79, 130.03, 132.65,

Methyl

2-(3,4-dichloro-5-methyl-1H-pyrrole-2-

141.53, 151.03, 156.69, 159.52, 167.07. HRMS (ESI^ꢂ) m/z for

carboxamido)-4-((4-(methoxycarbonyl)benzyl)oxy)benzo[d]

thiazole-6-carboxylate (30f). Synthesized according to general

C

₁₅H₁₀Cl₂N₃O₄S ([M ꢂ H]^-): calculated 397.9764, found 397.9779.

HPLC: t_r7.867 min (96.7% at 254 nm), method B.

procedure

B

using methyl 2-amino-4-((4-(methoxycarbonyl)

4-(2-((6-Carboxy-2-(3,4-dichloro-5-methyl-1H-pyrrole-2-

carboxamido)benzo[d]thiazol-4-yl)oxy)ethyl)morpholin-4-ium

chloride (31c). Synthesized according to general procedure C using

4-(2-((2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-6-

(methoxycarbonyl)benzo [d]thiazol-4-yl)oxy)ethyl)morpholin-4-

ium chloride (30c, 70 mg, 0.127 mmol). Yield: 54 mg (78.7%);

benzyl)oxy)benzo [d]thiazole-6-carboxylate (29f, 68 mg,

0.185 mmol). Yield: 72 mg (70.9%); light gray solid. ¹H NMR

(400 MHz, DMSO‑d₆) d 2.26 (s, 3H), 3.87 (s, 3H), 3.88 (s, 3H), 5.44 (s,

2H), 7.61 (d, J ¼ 1.3 Hz, 1H), 7.69 (d, J ¼ 8.2 Hz, 2H), 8.03 (d,

J ¼ 8.3 Hz, 2H), 8.31 (d, J ¼ 0.9 Hz, 1H), 12.19 (s, 1H), 12.27 (s, 1H).

HRMS (ESI^ꢂ) m/z for C₂₄H₁₈Cl₂N₃O₆S ([M ꢂ H]^-): calculated

546.0299, found 546.0302.

brownish-red solid. ¹H NMR (400 MHz, DMSO‑d₆)

3.32 (m, 2H), 3.66 (m, 4H), 3.84 (t, J ¼ 11.9 Hz, 2H), 4.06 (m, 2H), 4.71

d 2.29 (s, 3H),

17

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European Journal of Medicinal Chemistry 213 (2021) 113200

(s, 2H), 7.58 (d, J ¼ 1.4 Hz, 1H), 8.30 (d, J ¼ 1.3 Hz, 1H), 11.13 (s, 1H),

12.20 (br s, 1H), 12.86 (s, 1H). HRMS (ESI^ꢂ) m/z for C₂₀H₁₉Cl₂N₄O₅S

([M ꢂ H]^-): calculated 497.0448, found 497.0458. HPLC: t_r5.030 min

(98.0% at 254 nm), method B.

2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-

(thiophen-2-ylmethoxy)benzo[d]thiazole-6-carboxylic

acid

(31g). Synthesized according to general procedure K using methyl

2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(thio-

phen-2-ylmethoxy)benzo [d]thiazole-6-carboxylate (30g, 110 mg,

0.344 mmol). The crude product was puriﬁed with ﬂash column

chromatography using dichloromethane/methanol (9:1) as eluent.

Yield: 50 mg (46.7%); beige solid. ¹H NMR (400 MHz, DMSO‑d₆)

4-(2-((tert-Butoxycarbonyl)amino)ethoxy)-2-(3,4-dichloro-

5-methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6-

carboxylic acid (31d). Synthesized according to general procedure

C using methyl 4-(2-((tert-butoxycarbonyl)amino)ethoxy)-2-(3,4-

dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo [d]thiazole-

6-carboxylate (30d, 35 mg, 0.064 mmol). Yield: 7.6 mg (22.3);

d

2.26 (s, 3H), 5.52 (s, 2H), 7.08 (dd, J ¼ 5.1, 3.4 Hz, 1H), 7.28 (dd,

J ¼ 3.4, 1.2 Hz, 1H), 7.60 (dd, J ¼ 5.1, 1.2 Hz, 1H), 7.64 (d, J ¼ 1.5 Hz,

1H), 8.26 (d, J ¼ 1.4 Hz, 1H), 12.17 (s, 1H), 12.23 (s, 1H), 12.98 (s, 1H).

HRMS (ESI^þ) m/z for C₁₉H₁₄Cl₂N₃O₄S₂([MþH]^þ): calculated

481.9797, found 481.9792. HPLC: t_r4.503 min (95.4% at 254 nm),

method C.

brownish-red solid. ¹H NMR (400 MHz, DMSO‑d₆)

d 1.39 (s, 9H),

2.28 (s, 3H), 3.39 (m, 2H, signal is overlapping with the signal for

water), 4.10 (t, J ¼ 6.0 Hz, 2H), 6.96e7.05 (m, 1H), 7.34 (d, J ¼ 1.4 Hz,

1H), 7.92 (d, J ¼ 1.5 Hz, 1H), 12.70 (s, 1H), 12.80 (s, 1H), 12.97 (s, 1H).

HRMS (ESI^ꢂ) m/z for C₂₁H₂₁Cl₂N₄O₆S ([M ꢂ H]^-): calculated

527.0564, found 527.0565.

4-((3-Carboxybenzyl)oxy)-2-(3,4-dichloro-5-methyl-1H-pyr-

role-2-carboxamido)benzo[d]thiazole-6-carboxylic acid (31e).

To a suspension of methyl 2-(3,4-dichloro-5-methyl-1H-pyrrole-2-

carboxamido)-4-((3-(methoxycarbonyl)benzyl)oxy)benzo [d]thia-

zole-6-carboxylate (60 mg, 0.109 mmol) in 1,4-dioxane, 2 M NaOH

2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-

(thiophen-3-ylmethoxy)benzo[d]thiazole-6-carboxylic

(31h). Yield: 30 mg (68.3%); light gray solid. ¹H NMR (400 MHz,

DMSO‑d₆)

acid

d

2.26 (s, 3H), 5.30 (s, 2H), 7.25 (dd, J ¼ 4.9, 1.3 Hz, 1H),

7.57e7.63 (m, 2H), 7.63e7.68 (m, 1H), 8.25 (d, J ¼ 1.4 Hz, 1H), 12.17

(s, 1H), 12.22 (s, 1H), 13.00 (s, 1H). ¹³C NMR (101 MHz, DMSO‑d₆)

d

11.53, 65.84, 109.50, 110.41, 116.06, 116.76, 117.37, 125.36, 127.13,

(274

m

L, 0.547 mmol) was added, and the suspension stirred at

127.23, 128.62, 130.47, 133.28, 137.94, 142.24, 150.52, 157.08, 160.07,

167.54. HRMS (ESI^ꢂ) m/z for C₁₉H₁₄Cl₂N₃O₄S₂([MþH]^þ): calculated

481.9797, found 481.9794. HPLC: t_r4.527 min (95.1% at 254 nm),

method C.

2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-((3-

ﬂuorobenzyl)oxy)benzo[d]thiazole-6-carboxylic acid (31i). Syn-

thesized according to general procedure K using methyl 2-(3,4-

dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-((3-

60 ^ꢀC overnight. Then, 5 equivalents 2 M NaOH were added, and the

reaction mixture was stirred at 75 ^ꢀC for 7 days. The reaction was

followed using HPLC-MS, and 5 equivalents of fresh 2 M NaOH were

added, and the reaction mixture was stirred at 75 ^ꢀC for 7 days.

After the reaction was ﬁnished, the solvent was evaporated under

reduced pressure, the residue was acidiﬁed with 1 M HCl to pH 1,

and the precipitate obtained was ﬁltered off. The crude product was

suspended in methanol, heated, and ﬁltered off. Yield: 33 mg

ﬂuorobenzyl)oxy)benzo [d]thiazole-6-carboxylate (30i, 58 mg,

0.114 mmol) in methanol (15 mL), with the reaction mixture ﬁrst

(58.5%); brown solid. ¹H NMR (400 MHz, DMSO‑d₆)

d 2.26 (s, 3H),

5.41 (s, 2H), 7.58 (t, J ¼ 7.7 Hz, 1H), 7.63 (d, J ¼ 1.4 Hz, 1H), 7.80 (dt,

J ¼ 7.8,1.4 Hz,1H), 7.96 (dt, J ¼ 7.8,1.4 Hz,1H), 8.12 (d, J ¼ 1.8 Hz,1H),

8.27 (d, J ¼ 1.4 Hz, 1H), 12.19 (br s, 1H), 12.25 (s, 1H), 13.06 (br s, 2H).

stirred for 4 days. An additional 2 equivalents of 1 M NaOH (228 mL,

0.228 mmol) were added, and the reaction mixture was stirred for a

further 1 day. The crude product was puriﬁed by suspension in

methanol, heating, and ﬁltering off. Yield: 36 mg (64.3%); brown

¹³C NMR (101 MHz, DMSO‑d₆)

d 11.00, 69.49, 109.05, 109.93, 115.59,

116.38, 116.81, 126.69, 128.83, 128.86, 129.00, 130.03, 130.92, 132.58,

132.82, 137.17, 141.65, 149.92, 156.65, 159.72, 167.04, 167.17. HRMS

(ESI^þ) m/z for C₂₂H₁₆Cl₂N₃O₆S ([MþH]^þ): calculated 520.0131,

found 520.0146. HPLC: t_r8.323 min (95.8% at 254 nm), method B.

4-((4-Carboxybenzyl)oxy)-2-(3,4-dichloro-5-methyl-1H-pyr-

role-2-carboxamido)benzo[d]thiazole-6-carboxylic acid (31f).

Synthesized according to general procedure C using methyl 2-(3,4-

dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-((4-(methox-

ycarbonyl)benzyl)oxy)benzo [d]thiazole-6-carboxylate (30f, 63 mg,

0.115 mmol). Yield: 57 mg (95.5%); brown crystals. ¹H NMR

crystals. ¹H NMR (400 MHz, DMSO‑d₆)

d 2.27 (s, 3H), 5.35 (s, 2H),

7.18e7.26 (m, 1H), 7.38e7.40 (m, 2H), 7.46e7.53 (m, 1H), 7.61 (d,

J ¼ 1.2 Hz, 1H), 8.27 (d, J ¼ 1.1 Hz, 1H), 12.25 (s, 1H), 12.16 (s, 1H),

13.01 (br s, 1H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 11.02, 69.15, 109.04,

109.93, 114.59, 114.71, 114.81, 114.92, 115.60, 116.44, 116.85, 124.03,

124.05, 126.68, 129.97, 130.45, 130.53, 132.83, 139.48, 139.55,

149.84, 156.65, 159.75, 160.90, 163.32, 167.01. HRMS (ESI^ꢂ) m/z for

C

₂₁H₁₃Cl₂FN₃O₄S ([M ꢂ H]^-): calculated 491.9993, found 491.9997.

HPLC: t_r11.797 min (99.0% at 254 nm), method B.

2-((6-Carboxy-2-(3,4-dichloro-5-methyl-1H-pyrrole-2-

(400 MHz, DMSO‑d₆)

d

2.27 (s, 3H), 5.43 (s, 2H), 7.61 (d, J ¼ 1.4 Hz,

carboxamido)benzo[d]thiazol-4-yl)oxy)ethan-1-aminium chlo-

ride (32). To a suspension of 4-(2-((tert-butoxycarbonyl)amino)

ethoxy)-2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)

benzo [d]thiazole-6-carboxylic acid (31d, 7.6 mg, 0.014 mmol) in

1,4-dioxane (1 mL), 4 M HCl in 1,4-dioxane (1 mL) was added, and

the reaction mixture was stirred at room temperature for 5 h. The

precipitate in the reaction mixture was ﬁltered off, washed with

methanol, and dried. Yield: 3 mg (44.9%); brownish-red solid. ¹H

1H), 7.66 (d, J ¼ 8.4 Hz, 2H), 8.01 (d, J ¼ 8.4 Hz, 2H), 8.27 (d,

J ¼ 1.3 Hz,1H),12.17 (br s,1H),12.28 (s,1H),13.02 (br s, 2H). ¹³C NMR

(101 MHz, DMSO‑d₆)

d 11.03, 69.44, 109.19, 109.94, 115.63, 116.47,

116.84, 126.70, 127.45, 127.84, 129.48, 130.00, 130.34, 132.87, 141.63,

149.86, 156.68, 159.75, 167.01, 167.09. HRMS (ESI^ꢂ) m/z for

C

₂₂H₁₄Cl₂N₃O₆S ([M ꢂ H]^-): calculated 517.9986, found 517.9989.

HPLC: t_r8.380 min (95.4% at 254 nm), method B.

General procedure K. Synthesis of compounds 31g-i (with

31h as an example). To a suspension of methyl 2-(3,4-dichloro-5-

methyl-1H-pyrrole-2-carboxamido)-4-(thiophen-3-ylmethoxy)

benzo [d]thiazole-6-carboxylate (30h, 45 mg, 0.091 mmol) in

methanol (15 mL), 1 M NaOH (0.455 mL, 0.455 mmol) was added,

and the reaction mixture was stirred at 40 ^ꢀC overnight. An addi-

tional 0.455 mL 1 M NaOH was added, and the reaction mixture was

stirred at 40 ^ꢀC for 3 days. The solvent was evaporated in vacuo, the

residue was acidiﬁed with 1 M HCl to pH 1, and the precipitate

formed was ﬁltered off. The crude product was suspended in

methanol, heated, and ﬁltered to get 31h (30 mg) as a light gray

solid.

NMR (400 MHz, DMSO‑d₆) d 2.28 (s, 3H), 3.30 (m, 2H, signal is

overlapping with the signal for water), 4.45 (t, J ¼ 5.0 Hz, 2H), 7.59

(d, J ¼ 1.1 Hz, 1H), 8.10 (s, 3H), 8.31 (s, 1H), 12.07 (br s, 1H), 12.50 (s,

1H), 13.08 (br s, 1H). HRMS (ESI^þ) m/z for C₁₆H₁₅Cl₂N₄O₄S

([MþH]^þ): calculated 429.0186, found 429.0187. HPLC: t_r3.733 min

(95.1% at 254 nm), method B.

4-(2-((6-((Cyanomethyl)carbamoyl)-2-(3,4-dichloro-5-

methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazol-4-yl)oxy)

ethyl)morpholin-4-ium chloride (33). Synthesized according to

general procedure D using 4-(2-((6-carboxy-2-(3,4-dichloro-5-

methyl-1H-pyrrole-2-carboxamido)benzo

[d]thiazol-4-yl)oxy)

ethyl)morpholin-4-ium chloride (31c, 19 mg, 0.035 mmol). Here,

18

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European Journal of Medicinal Chemistry 213 (2021) 113200

1 M HCl was used in the isolation process instead of 1% citric acid.

After addition of organic and water phases, the precipitate formed

was ﬁltered off and dried. Yield: 14 mg (68.0%); beige solid. ¹H NMR

126.15, 128.12, 128.84, 129.08, 135.75, 142.96, 150.93. MS (ESI) m/

z ¼ 253.1 ([M ꢂ H]^-).

General procedure L. Synthesis of compounds 41 and 45 (with

41 as an example). 3-(Benzyloxy)-4-nitrobenzonitrile (643 mg,

2.53 mmol) was dissolved in methanol/ethyl acetate (1:3, 40 mL),

tin (II) chloride (1.7 g, 8.86 mmol) was added, and the reaction

mixture was stirred at 55 ^ꢀC overnight. The solvent was evaporated

in vacuo, the residue was neutralized with saturated aqueous

NaHCO₃solution and extracted with ethyl acetate (3 ꢁ 100 mL). The

combined organic phases were washed with brine (100 mL) and

ﬁltered, and the solvent was evaporated under reduced pressure, to

obtain 41 (321 mg) as a white solid.

(400 MHz, DMSO‑d₆)

d 2.29 (s, 3H), 3.25e3.34 (m, 2H), 3.62e3.73

(m, 4H), 3.76e3.88 (m, 2H), 4.01e4.10 (m, 2H), 4.36 (d, J ¼ 5.4 Hz,

2H), 4.71 (t, 2H), 7.62 (d, J ¼ 1.5 Hz, 1H), 8.21 (d, J ¼ 0.5 Hz, 1H), 9.40

(t, J ¼ 5.5 Hz, 1H), 10.93 (s, 1H), 12.20 (s, 1H), 12.78 (s, 1H). HRMS

(ESI^þ) m/z for C₂₂H₂₃Cl₂N₆O₄S ([MþH]^þ): calculated 537.0873,

found 537.0861. HPLC: t_r4.313 min (96.9% at 254 nm), method B.

2-Amino-4-(benzyloxy)benzo[d]thiazole-6-carboxylic acid

(35). Synthesized according to general procedure C using methyl 2-

amino-4-(benzyloxy)benzo [d]thiazole-6-carboxylate (34, 400 mg,

1.27 mmol). After addition of 1 M HCl, the water phase was cooled

to 0 ^ꢀC for 3 h, and the white precipitate formed was ﬁltered off.

Yield: 340 mg (89.0%); white solid. ¹H NMR (400 MHz, DMSO‑d₆)

4-Amino-3-(benzyloxy)benzonitrile (41) [32]. Yield: 321 mg

(56.6%); white solid. ¹H NMR (400 MHz, DMSO‑d₆)

d 5.16 (s, 2H),

5.82 (s, 2H), 6.69 (d, J ¼ 8.2 Hz), 7.12 (dd, J ¼ 8.2, 1.8 Hz, 1H), 7.22 (d,

J ¼ 1.7 Hz, 1H), 7.31e7.36 (m, 1H), 7.37e7.43 (m, 2H), 7.47e7.53 (m,

2H).

d

5.23 (s, 2H), 7.31e7.43 (m, 3H), 7.45 (d, J ¼ 1.5 Hz, 1H), 7.47e7.51

(m, 2H), 7.88 (s, 2H), 7.93 (d, J ¼ 1.5 Hz, 1H), 12.68 (s, 1H). ¹³C NMR

(101 MHz, DMSO‑d₆)

d

70.43, 110.73, 116.43, 124.05, 128.30, 128.33,

2-Amino-4-(benzyloxy)benzo[d]thiazole-6-carbonitrile (42).

Synthesized according to general procedure J using 4-amino-3-

(benzyloxy)benzonitrile (41, 315 mg, 1.31 mmol). The reaction

mixture was neutralized with 25% aqueous NH₃solution instead of

NaHCO₃, and the product was extracted with ethyl acetate

(2 ꢁ 50 mL). The combined organic phases were washed with brine

(20 mL), dried over Na₂SO₄, and ﬁltered, and the solvent was

evaporated under reduced pressure. The crude product was sus-

pended in methanol and ﬁltered off. Yield: 126 mg (34.0%); yellow

128.83, 132.03, 137.54, 146.53, 148.37, 167.64, 168.93. MS (ESI) m/

z ¼ 299.1 ([M ꢂ H]^-).

2-Amino-4-(benzyloxy)-N-(cyanomethyl)benzo[d]thiazole-

6-carboxamide (36). Synthesized according to general procedure D

using 2-amino-4-(benzyloxy)benzo [d]thiazole-6-carboxylic acid

(35, 330 mg, 1.10 mmol). Yield: 93 mg (27%); white solid. ¹H NMR

(400 MHz, DMSO‑d₆)

d

4.32 (d, J ¼ 5.4 Hz, 2H), 5.24 (s, 2H),

7.33e7.38 (m, 1H), 7.39e7.45 (m, 2H), 7.47 (d, J ¼ 1.6 Hz, 1H),

7.48e7.52 (m, 2H), 7.83 (s, 2H), 7.86 (d, J ¼ 1.6 Hz, 1H), 9.08 (t,

solid. ¹H NMR (400 MHz, DMSO‑d₆)

2H), 7.38e7.44 (m, 2H), 7.45e7.50 (m, 2H), 7.84 (d, J ¼ 1.4 Hz, 1H),

8.02 (s, 2H). ¹³C NMR (101 MHz, DMSO‑d₆)

70.69, 102.78, 112.77,

d 5.22 (s, 2H), 7.32e7.38 (m,

J ¼ 5.5 Hz, 1H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 28.26, 70.63, 109.30,

114.15, 118.28, 126.27, 128.38, 128.51, 128.85, 132.26, 137.48, 145.97,

d

148.54, 166.75, 168.29. MS (ESI) m/z ¼ 337.1 ([M ꢂ H]^-).

119.27, 120.14, 128.15, 128.49, 128.88, 132.78, 137.12, 147.03, 148.77,

4-(Benzyloxy)-N-(cyanomethyl)-2-(3,4-dichloro-5-methyl-

1H-pyrrole-2-carboxamido)benzo[d]thiazole-6-carboxamide

(37). Synthesized according to general procedure B using 2-amino-

4-(benzyloxy)-N-(cyanomethyl)benzo [d]thiazole-6-carboxamide

(36, 77 mg, 0.228 mmol). Yield: 45 mg (38.5%); brown solid. ¹H

169.44. MS (ESI) m/z ¼ 280.1 ([M ꢂ H]^-).

N-(4-(Benzyloxy)-6-cyanobenzo[d]thiazol-2-yl)-3,4-

dichloro-5-methyl-1H-pyrrole-2-carboxamide (43). Synthesized

according to general procedure B using 2-amino-4-(benzyloxy)

benzo [d]thiazole-6-carbonitrile (42, 120 mg, 0.427 mmol). Yield:

NMR (400 MHz, DMSO‑d₆)

d

2.26 (s, 3H), 4.37 (d, J ¼ 5.4 Hz, 2H),

126 mg (64.6%); gray solid. ¹H NMR (400 MHz, DMSO‑d₆)

d 2.26 (s,

5.32 (s, 2H), 7.36e7.48 (m, 3H), 7.51e7.59 (m, 2H), 7.63 (d, J ¼ 1.3 Hz,

3H), 5.32 (s, 2H), 7.37e7.49 (m, 3H), 7.53e7.54 (m, 2H), 7.57 (d,

J ¼ 1.1 Hz, 1H), 8.21 (d, J ¼ 1.0 Hz, 1H), 12.25 (s, 1H), 12.29 (br s, 1H).

HRMS (ESI^ꢂ) m/z for C₂₁H₁₃Cl₂N₄O₂S ([M ꢂ H]^-): calculated

455.0142, found 455.0147. HPLC: t_r14.237 min (97.6% at 254 nm),

method B.

N-(4-(Benzyloxy)-6-(1H-tetrazol-5-yl)benzo[d]thiazol-2-yl)-

3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamide (44). To a so-

lution of N-(4-(benzyloxy)-6-cyanobenzo [d]thiazol-2-yl)-3,4-

1H), 8.15 (d, J ¼ 1.3 Hz, 1H), 9.28 (t, J ¼ 5.5 Hz, 1H), 12.18 (br s, 1H),

12.22 (s, 1H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 11.55, 28.35, 70.71,

108.17, 110.43, 114.35, 116.17, 117.25, 118.20, 128.67, 128.89, 128.96,

129.50, 130.44, 133.33, 136.98, 141.56, 150.81, 156.92, 159.38, 166.72.

HRMS (ESI^ꢂ) m/z for C₂₃H₁₆Cl₂N₅O₃S ([M ꢂ H]^-): calculated

512.0356, found 512.0361. HPLC: t_r11.253 min (95.2% at 254 nm),

method B.

3-(Benzyloxy)-4-nitrobenzamide (39). A solution of methyl 3-

(benzyloxy)-4-nitrobenzoate (1.19 g, 4.15 mmol) in saturated

methanol solution of ammonia was stirred in a pressure tube at

65 ^ꢀC overnight. The precipitate formed was ﬁltered off. The mother

liquid was evaporated under reduced pressure, the residue crys-

tallized from acetonitrile, and the crystals combined with the

ﬁltered precipitate. Yield: 1.01 g (89.4%); pale yellow crystals. ¹H

dichloro-5-methyl-1H-pyrrole-2-carboxamide

(100

mg,

0.219 mmol) in N,N-dimethylformamide (5 mL), ammonium chlo-

ride (117 mg, 2.19 mmol) and sodium azide (142 mg, 2.19 mmol)

were added, and the reaction mixture was stirred at 125 ^ꢀC over-

night. Ethyl acetate (10 mL) and 1 M HCl (5 mL) were added, and the

precipitate formed (the starting compound) was ﬁltered off. The

phases of the mother liquid were separated, the organic phase was

washed with brine (5 mL), dried over Na₂SO₄, and ﬁltered, and the

solvent was removed under reduced pressure. The crude product

was puriﬁed by ﬂash column chromatography using dichloro-

methane/methanol/acetic acid (30:1:0.1) as eluent. Yield: 4 mg

NMR (400 MHz, DMSO‑d₆)

d 5.37 (s, 2H), 7.33e7.49 (m, 5H), 7.59

(dd, J ¼ 8.4, 1.6 Hz, 1H), 7.73 (s, 1H), 7.87 (d, J ¼ 1.5 Hz, 1H), 7.98 (d,

J ¼ 8.4 Hz, 1H), 8.24 (s, 1H). MS (ESI) m/z ¼ 272.9 ([MþH]^þ).

3-(Benzyloxy)-4-nitrobenzonitrile (40). To a solution of 3-

(benzyloxy)-4-nitrobenzamide (700 mg, 2.57 mmol) in dry aceto-

nitrile (10 mL), triphenylphosphine oxide (7.15 mg, 0.026 mmol),

(3.7%); beige solid. ¹H NMR (400 MHz, DMSO‑d₆)

d 2.26 (s, 3H), 5.36

(s, 2H), 7.37e7.48 (m, 3H), 7.57e7.59 (m, 2H), 7.78 (d, J ¼ 1.5 Hz,1H),

8.29 (d, J ¼ 1.4 Hz, 1H), 12.18 (br s, 1H), 12.22 (s, 1H), signal for

tetrazole NH is not seen. HRMS (ESI^ꢂ) m/z for C₂₁H₁₄Cl₂N₇O₂S

triethylamine (1.08 mL, 7.71 mmol), and oxalyl chloride (441 mL,

5.14 mmol) were added, and the reaction mixture stirred at room

temperature for 30 min. The triethylammonium chloride that was

formed was ﬁltered off, and the mother liquid was evaporated. The

crude product was crystallized from methanol. Yield: 650 mg

([M

ꢂ

H]^-): calculated 498.0312, found 498.0319. HPLC: t_r

11.120 min (95.1% at 254 nm), method B.

4-Amino-3-(benzyloxy)benzamide (45). Synthesized accord-

ing to general procedure L using 3-(benzyloxy)-4-nitrobenzamide

(300 mg, 1.102 mmol). Yield: 260 mg (97.4%); yellow oil. ¹H NMR

(99.4%); off-white solid. ¹H NMR (400 MHz, DMSO‑d₆)

d

5.38 (s, 2H),

7.35e7.47 (m, 5H), 7.66 (dd, J ¼ 8.3, 1.6 Hz, 1H), 8.07e8.11 (m, 2H).

¹³C NMR (101 MHz, DMSO‑d₆)

71.66, 116.44, 117.74, 120.10, 125.52,

d

(400 MHz, DMSO‑d₆)

d

5.14 (s, 2H), 5.29 (s, 2H), 6.63 (d, J ¼ 8.2 Hz,

19

ꢀ

ꢁ

M. Durcik, A. Nyerges, Z. Skok et al.

European Journal of Medicinal Chemistry 213 (2021) 113200

1H), 6.92 (s, 1H), 7.28e7.36 (m, 3H), 7.36e7.44 (m, 2H), 7.45 (d,

J ¼ 1.8 Hz, 1H), 7.48e7.55 (m, 2H), 7.60 (s, 1H). MS (ESI) m/z ¼ 243.0

([MþH]^þ).

calculate the IC₅₀values. The data are reported as the means of

three independent measurements. Etoposide was used as the

positive control (IC₅₀71 mM).

2-Amino-4-(benzyloxy)benzo[d]thiazole-6-carboxamide

(46). Synthesized according to general procedure J using 4-amino-

3-(benzyloxy)benzamide (45, 255 mg, 1.053 mmol). The reaction

mixture was neutralized with 25% aqueous NH₃solution instead of

NaHCO₃. The crude product was puriﬁed only by resuspension in

methanol, heating to reﬂux, and ﬁltering off the undissolved solid.

The methanol was evaporated, to obtain the product 46. Yield:

4.5. Determination of antibacterial activity

Clinical microbiology control strains of A. baumannii (ATCC

17978, ATCC19606), ciproﬂoxacin-resistant A. baumannii (ATCC

BAA-1605), E. faecalis (ATCC 29212), E. faecium (ATCC 700221),

E. coli (ATCC 25922), K. pneumoniae (ATCC 700603, ATCC 10031),

P. aeruginosa (ATCC 27853), S. aureus (ATCC 25913), methicillin-

resistant S. aureus (ATCC 43300) and vancomycin-intermediate

S. aureus (ATCC 700699) were obtained from American Type Cul-

ture Collection (ATCC) via Microbiologics Inc. (St. Cloud, MN, USA).

E. coli MG1655 originated from the laboratory collection of Dr.

Csaba Pal. E. coli (20204), ciproﬂoxacin-resistant E. coli (31995,

31859), and P. aeruginosa (19488) clinical strains were obtained

from the strain collection of the University of Szeged (Hungary).

The GyrB mutant strains of methicillin-resistant S. aureus (ATCC

43300) and vancomycin-intermediate S. aureus (ATCC 700699)

were generated according to the published protocol [23]. The GyrB

mutant strain of E. coli MG1655 was constructed using pORTMAGE

[34] recombineering (Addgene plasmid # 120418; http://n2t.net/

addgene:120418; RRID:Addgene_120418) according to the pub-

lished protocol [35].

Mueller Hinton Agar or cation-adjusted Mueller Hinton II broth

(MHBII) were used for growth of the bacteria under standard lab-

oratory conditions, for antimicrobial susceptibility tests, and for

selection of resistant variants. To prepare the MHBII broth, 22 g

MHBII powder (containing 3 g beef extract, 17.5 g acid hydrolysate

of casein, 1.5 g starch; Becton Dickinson and Co.) was dissolved in

1 L water. MHBII agar was prepared by the addition of 14 g agar

(Bacto; Molar Chemicals) to 1 L broth.

307 mg (97.0%); orange solid. ¹H NMR (400 MHz, DMSO‑d₆)

d 5.22

(s, 2H), 7.23 (s, 1H), 7.32e7.37 (m, 1H), 7.38e7.44 (m, 2H), 7.45e7.51

(m, 3H), 7.74 (s, 2H), 7.83 (d, J ¼ 1.5 Hz, 1H), 7.86 (s, 1H). ¹³C NMR

(101 MHz, DMSO‑d₆)

d 70.52, 109.68, 113.99, 127.92, 128.01, 128.33,

128.83, 131.98, 137.64, 145.34, 148.43, 167.81, 167.99. MS (ESI) m/

z ¼ 300.1 ([MþH]^þ).

4-(Benzyloxy)-2-(3,4-dichloro-5-methyl-1H-pyrrole-2-

carboxamido)benzo[d]thiazole-6-carboxamide (47). Synthesized

according to general procedure B using 2-amino-4-(benzyloxy)

benzo [d]thiazole-6-carboxamide (46, 190 mg, 0.635 mmol). The

crude product was puriﬁed with ﬂash column chromatography

using dichloromethane/methanol (20:1) as eluent. Yield: 20 mg

(6.6%); light gray solid. ¹H NMR (400 MHz, DMSO‑d₆)

d 2.26 (s, 3H),

5.30 (s, 2H), 7.36e7.47 (m, 4H), 7.51e7.57 (m, 2H), 7.64 (d, J ¼ 1.3 Hz,

1H), 8.04 (s, 1H), 8.13 (s, 1H), 12.12 (s, 1H), 12.22 (s, 1H). HRMS (ESI^ꢂ)

m/z for C₂₁H₁₅Cl₂N₄O₃S ([M ꢂ H]^-): calculated 473.0247, found

473.0255. HPLC: t_r9.740 min (95.0% at 254 nm), method B.

4.3. Determination of inhibitory activities on E. coli and S. aureus

DNA gyrase and topoisomerase IV

The assay for the determination of the IC₅₀values was per-

formed according to previously reported procedures [33]. IC₅₀

values were determined using seven concentrations of the in-

hibitors, with GraphPad Prism 6.0 software used to calculate the

values. IC₅₀values were determined in three independent mea-

surements, and their means are given as the ﬁnal result.

Minimum inhibitory concentrations (MICs) were determined

using a standard serial broth microdilution technique, according to

the Clinical and Laboratory Standards Institute guidelines [36]. For

details on MIC determination see Supplementary Information.

4.4. Determination of inhibitory activities on human DNA

topoisomerase II

a

4.6. Molecular docking

Inhibitory activities were determined in an assay from Inspiralis

using streptavidin-coated 96-well microtiter plates (Thermo Sci-

entiﬁc Pierce). First, the plates were rehydrated with buffer (20 mM

Tris/HCl, 0.01% [w/v] bovine serum albumin, 0.05% [v/v] Tween 20,

137 mM NaCl, pH 7.6), and then the biotinylated oligonucleotide

was immobilized. After washing off the unbound oligonucleotide,

Three-dimensional models of the designed DNA gyrase B and

topoisomerase IV inhibitors were built in Chem3D 18.0 (Perki-

nElmer Inc., Massachusetts, USA). The geometries and charges of

the ligands were optimized using the MM2 force ﬁeld, and partial

atomic charges were assigned. The energy was minimized until the

gradient was <0.001 kcal/(mol ꢁ Å). Molecular docking calculations

the enzyme assays were performed. The reaction volume was 30

in buffer (50 mM Tris/HCl, 10 mM MgCl₂, 125 mM NaCl, 5 mM

dithiothreitol, 0.1 g/mL albumin, 1 mM ATP, pH 7.5) that contained

1.5 U human DNA topoisomerase II, 0.75 g supercoiled pNO1

plasmid, and 3 L inhibitor solution in 10% dimethylsulfoxide

mL

€

were performed using Schrodinger Release 2019e1 (Schrodinger,

LLC, New York, NY, USA, 2019). The crystal structure of E. coli DNA

gyrase B in complex with bithiazole inhibitor (PDB entry: 4DUH)

was retrieved from the Protein Data Bank. The protein was then

prepared using Protein Preparation Wizard with the default set-

tings. The receptor grid was calculated for the ligand-binding site,

and the designed compounds were docked using the Glide XP

m

(DMSO) containing 0.008% Tween 20. Reaction solutions were

incubated at 37 ^ꢀC for 30 min. TF buffer (50 mM NaOAc, 50 mM

NaCl, 50 mM MgCl₂, pH 5.0) was then added to terminate the

enzymatic reaction. After additional incubation for 30 min at room

temperature, during which the biotineoligonucleotideeplasmid

triplex was formed, the unbound plasmid was washed off using TF

buffer, and Diamond Dye in T10 buffer (10 mM Tris/HCl, 1 mm

EDTA, pH 8.0) was added. The ﬂuorescence was measured using a

microplate reader (Synergy™ 4 Hybrid; BioTek, VT, USA; excitation:

485 nm; emission: 537 nm). The initial screening was done at

€

protocol, as implemented in Schrodinger Release 2019e1 (Glide,

€

Schrodinger, LLC, New York, NY, USA, 2019).

4.7. Crystallography

Protein production, crystallization, preparation of the

inhibitorꢂprotein complexes, data collection and processing, and

structure modeling and reﬁnement were performed by SARomics

Biostructures, Medicon Village, Lund, Sweden. For details on crys-

tallography see Supplementary Information.

100 mM or 10mM concentrations of inhibitors. For the most active

inhibitors, the IC₅₀was determined using seven concentrations of

each compound. The GraphPad Prism 6.0 software was used to

20

ꢀ

ꢁ

M. Durcik, A. Nyerges, Z. Skok et al.

European Journal of Medicinal Chemistry 213 (2021) 113200

4.8. In vitro cytotoxicity and genotoxicity measurements

Declaration of Competing Interest

The cytotoxicity of compound 31h in HepG2 and MCF-7 cells

was determined using the lactate dehydrogenase (LDH) assay

(Thermo Fisher Scientiﬁc, Waltham, MA, USA). Brieﬂy, the HepG2

and MCF-7 cells were cultured in Eagle’s minimum essential me-

dium (Gibco, Thermo Fisher Scientiﬁc, Waltham, MA, USA) sup-

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

Acknowledgements

plemented with 2 mM

USA), 100 U/mL penicillin (Sigma-Aldrich, St. Louis, MO, USA),

100 g/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and

L-glutamine (Sigma-Aldrich, St. Louis, MO,

The study was funded by the Slovenian Research Agency (Grant

No. P1-0208, J1-9192 and BI-HU/19-20-008) and supported by the

following research grants: European Research Council H2020-ERC-

m

10% fetal bovine serum (Gibco, Thermo Fisher Scientiﬁc, Waltham,

MA, USA), at 37 ^ꢀC and under 5% CO₂. The LDH assays for compound

31h were performed according to manufacturer instructions using

cytotoxicity assay kits (CyQUANT™ LDH; Thermo Fisher Scientiﬁc,

Waltham, MA, USA). The genetic toxicity analysis of compound 31h

was performed in an in vitro micronucleus test, according to the

published protocol [37], at Euroﬁns Panlabs (St. Charles, MO, US).

For details on cytotoxicity and genotoxicity assays see Supple-

mentary Information.

ꢀ

2014-CoG 648364 e Resistance Evolution (C.P.); ‘Celzott Lendület’

Programme of the Hungarian Academy of Sciences LP-2017e10/

ꢀ

2017(C.P.);

‘Elvonal’

KKP

(EVOMER,

126506

to

(C.P.);

C.P.),

GINOP-

2.3.2e15e2016e00014

2.3.2e15e2016e00020 (MolMedEx TUMORDNS), and GINOP-

2.3.3e15e2016e00001; EFOP 3.6.3-VEKOP-16-406 2017e00009

(P.Sz, T.R.); UNKP-19-3 New National Excellence Program of the

Ministry for Innovation and Technology (P.Sz.); and a PhD Fellow-

ship from the Boehringer Ingelheim Funds (A.N.). The authors

thank Chris Berrie for editing the manuscript.

4.9. In vitro metabolism assay

Appendix A. Supplementary data

The in vitro metabolism study was performed using the rat he-

patic S9 fraction in phosphate buffer, pH 7.4, supplemented with

MgCl₂, NADPH, GSH, UDPGA, PAPS and alamethicin as co-factors/

additives. The following were purchased from Sigma-Aldrich (St.

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.113200.

Louis, MO, USA): pooled S9 from liver;

dinucleotide phosphate (NADPH, reduced tetra (cyclo-

hexylammonium salt; CAS 100929-71-3); -glutathione (GSH, 98%;

CAS 70-18-8); uridine diphosphate glucuronic acid (UDPGA;

ammonium salt; 98%e100%; CAS 43195-60-4); adenosine 3⁰-

phosphate 5⁰-phosphosulfate (PAPS, lithium salt hydrate; CAS

109434-21-1); alamethicin (CAS 27061-78-5); sodium phosphate

monobasic, anhydrous (>98%; CAS 7558-80-7); and magnesium

chloride (98%; CAS 7786-30-3). Details on metabolism assay can be

found in Supplementary Information.

b-nicotinamide adenine

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L

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Author contributions

L.P.M. and T.T. conceived the study. M.D. and O.B. carried out the

synthesis. T.T. planned and carried out docking experiments. Z.S.

ꢁ

carried out enzyme activity testing. A.N., C.D.C., P.T., T.R., G.D., P.S.,

L.D., and C.P. planned and carried out microbiology testing. M.W.,

Y.R.K., and D.F. carried out crystallography. M.D. and D.G.S. carried

out cytotoxicity testing. M.D., D.G.S., and J.T. carried out metabolism

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22

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New dual ATP-competitive inhibitors of bacterial DNA gyrase and topoisomerase IV active against ESKAPE pathogens

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