Design, synthesis and biological evaluation of 4,5-dibromo-N-(thiazol-2-yl)-1H-pyrrole-2-carboxamide derivatives as novel DNA gyrase inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.10.038

Development of novel DNA gyrase B inhibitors is an important field of antibacterial drug discovery whose aim is to introduce a more effective representative of this mechanistic class into the clinic. In the present study, two new series of Escherichia coli DNA gyrase inhibitors bearing the 4,5-dibromopyrrolamide moiety have been designed and synthesized. 4,5,6,7-Tetrahydrobenzo[1,2-d]thiazole-2,6-diamine derivatives inhibited E. coli DNA gyrase in the submicromolar to low micromolar range (IC₅₀values between 0.891 and 10.4 μM). Their “ring-opened” analogues, based on the 2-(2-aminothiazol-4-yl)acetic acid scaffold, displayed weaker DNA gyrase inhibition with IC₅₀values between 15.9 and 169 μM. Molecular docking experiments were conducted to study the binding modes of inhibitors.

Full text of DOI:10.1016/j.bmc.2016.10.038

Accepted Manuscript
Design, synthesis and biological evaluation of 4,5-dibromo-N-(thiazol-2-
yl)-1H-pyrrole-2-carboxamide derivatives as novel DNA gyrase inhibitors
Tihomir Tomašič, Matic Mirt, Michaela Barančoková, Janez Ilaš, Nace Zidar,
Päivi Tammela, Danijel Kikelj
PII:
S0968-0896(16)31093-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.10.038
BMC 13365
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 September 2016
27 October 2016
29 October 2016
Please cite this article as: Tomašič, T., Mirt, M., Barančoková, M., Ilaš, J., Zidar, N., Tammela, P., Kikelj, D.,
Design, synthesis and biological evaluation of 4,5-dibromo-N-(thiazol-2-yl)-1H-pyrrole-2-carboxamide derivatives
as novel DNA gyrase inhibitors, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.
2016.10.038
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Design, synthesis and biological evaluation of 4,5-dibromo-N-
(thiazol-2-yl)-1H-pyrrole-2-carboxamide derivatives as novel DNA
gyrase inhibitors
Tihomir Tomašič,a,* Matic Mirt,a Michaela Barančoková,a Janez Ilaš,a Nace Zidar,a Päivi
Tammela,b Danijel Kikelja
aUniversity of Ljubljana, Faculty of Pharmacy, Aškerčeva cesta 7, 1000 Ljubljana, Slovenia
bUniversity of Helsinki, Faculty of Pharmacy, Division of Pharmaceutical Biosciences, P.O.
Box 56, FI-00014 Helsinki, Finland
Corresponding Author
*To whom correspondence should be addressed. Phone: +386-1-4769537; Fax: +386-1-
4258031; e-mail: tihomir.tomasic@ffa.uni-lj.si
1

ABSTRACT
Development of novel DNA gyrase B inhibitors is an important field of antibacterial drug
discovery whose aim is to introduce a more effective representative of this mechanistic class
into the clinic. In the present study, two new series of Escherichia coli DNA gyrase inhibitors
bearing the 4,5-dibromopyrrolamide moiety have been designed and synthesized. 4,5,6,7-
Tetrahydrobenzo[1,2-d]thiazole-2,6-diamine derivatives inhibited E. coli DNA gyrase in the
submicromolar to low micromolar range (IC50 values between 0.891 and 10.4 µM). Their
“ring-opened” analogues, based on the 2-(2-aminothiazol-4-yl)acetic acid scaffold, displayed
weaker DNA gyrase inhibition with IC50 values between 15.9 and 169 µM. Molecular
docking experiments were conducted to study the binding modes of inhibitors.
KEYWORDS antibacterial; DNA gyrase; docking; inhibitor; thiazole
2

1. Introduction
Antibacterial drug resistance, especially that of ‘ESKAPE’ organisms (Enterococcus
faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii and
Enterobacter spp.), is a growing threat to human health, not only in hospitals but in the
community in general [1]. Therefore, continuous discovery and development of new
antibacterial agents that avoid the existing resistance mechanisms is of great importance to
increase the number of drug candidates in the pipeline for treating these serious, life-
threatening infections.
DNA gyrase is a well-established and validated target for antibacterial drug discovery.
It offers opportunities for the development of drugs that could avoid some of the existing
resistance mechanisms. It catalyzes changes in DNA topology during replication by
introducing negative supercoils. It is a heterotetrameric enzyme consisting of two GyrA
subunits, which are involved in cleavage and reunion of the DNA substrate, and two GyrB
subunits that, by hydrolysis of ATP, provide energy for the supercoiling reaction of GyrA.
Topoisomerase IV, which is involved in decatenation of daughter DNA following DNA
replication, is structurally similar to DNA gyrase, being composed of two ParC and two ParE
subunits that are homologous to GyrA and GyrB, respectively [2].
The structural similarity of DNA gyrase and topoisomerase IV provides the
opportunity for dual targeting in most bacteria, which prolongs the onset of resistance, making
these two enzymes attractive targets for antibacterial drug discovery [3, 4]. The GyrA
subunits are targets of the clinically important class of antibacterial drugs, the
fluoroquinolones, which stabilize the complex between DNA gyrase and DNA [5]. The
increasing level of resistance to fluoroquinolones limits their therapeutic use [6] and
stimulates the search either for novel structural classes of inhibitors targeting GyrA or for
development of inhibitors of the GyrB ATPase activity [4, 7, 8]. Novobiocin, an
3

aminocoumarin antibiotic, remains the first and still the only, example of GyrB inhibitor that
has been used in therapy, primarily for infections caused by Gram positive bacterial strains.
Its use declined during the 1960s and early 1970s following the introduction of penicillinase-
stable penicillins and the first generation of cephalosporins, and was eventually withdrawn
from the market [4]. Despite extensive efforts to develop a new representative of this
mechanistic class, no GyrB inhibitor has progressed beyond phase 1 clinical trials [4]. Several
structural classes of GyrB and/or ParE inhibitors, such as cyclothialidines [9], ethylureas [10-
14], azaindoles [15], and pyrrolamides [16-18], were investigated in the past decades by the
pharmaceutical industry. The majority of these GyrB and/or ParE inhibitors possess potent
enzyme inhibitory activity and antibacterial activity, but mainly against Gram positive strains
[4]. In contrast, there are few examples of GyrB/ParE inhibitors displaying anti-Gram
negative activity, such as pyrrolopyrimidines [19] and pyrimidinoindoles [20]. DNA gyrase
inhibitors have been discovered by academic research groups, for example by virtual
screening [21-23] and by structure-based design of marine alkaloid analogues [24, 25].
Although many antibacterial drug discovery projects in the pharmaceutical industry were
recently terminated, the knowledge gathered over more than 50 years of research directed
towards ATPase inhibitors of DNA gyrase and topoisomerase IV provides an excellent
foundation for further research to finally introduce a new and effective representative of this
mechanistic class in antibacterial therapy [4].
We have recently designed several structural classes of analogues of marine alkaloids
based on clathrodin, hymenidin and oroidin, isolated from sponges of the genus Agelas, and
evaluated their voltage-gated sodium channel modulatory activity [26-29], inhibition of
biofilm formation [30], antimicrobial activity [31], and pro-apoptotic activity in HepG2 and
THP-1 cell lines [32]. The similarity of oroidin and its analogues to the known pyrrolamide-
based inhibitors [17, 18] stimulated us to evaluate selected oroidin analogues from our library
4

for E. coli DNA gyrase inhibition. Optimization of the hit compounds [24] and design of
novel 4,5-dibromopyrrolamide derivatives [25] resulted in potent DNA gyrase inhibitors
exhibiting IC50 values in the low nanomolar range, but possessing weak antibacterial activity.
The X-ray crystal structure of 4,5-dibromopyrrolamide-based inhibitor in complex with E.
coli DNA gyrase subunit B was solved, which confirmed the assumed binding mode of
inhibitor to the ATP-binding site of DNA gyrase [25]. The pyrrolamide moiety was found to
occupy the same binding pocket of the E. coli GyrB ATP-binding site as that observed for
other pyrrolamide-based DNA gyrase inhibitors, such as natural antibiotics clorobiocin [33]
and kibdelomycin [34], and some described synthetic inhibitors [17, 18] (Figure 1). We
describe here the design and synthesis of a novel series of 4,5-dibromopyrrolamides and the
evaluation of their E. coli DNA gyrase inhibition as well as their antibacterial activity against
selected Gram positive and Gram negative bacterial strains.
Figure 1. Pyrrolamide-based inhibitors of DNA gyrase.
2. Results and discussion
2.1. Design
X-Ray crystal structure of the 4,5-dibromopyrrolamide-based inhibitor I in the ATP-
binding site of E. coli DNA gyrase revealed three hydrogen bonds formed between the
5

enzyme and the pyrrolamide moiety of inhibitor [25]. A hydrogen bond is seen between a
pyrrole NH group and the Asp73 side chain, while a second hydrogen bond is formed
between an adjacent carbonyl group and a structurally conserved water molecule that is in
contact with the Asp73 side chain (Figure 2) [25]. With this conserved binding motif of
adjacent hydrogen bond donor/acceptor groups, GyrB inhibitors mimic the binding of ATP
[8]. The NH of the amide group is additionally bridged by a water molecule to the side chain
of Thr365 (Figure 2). The formation of several hydrophobic interactions between the dibromo
substituted pyrrole moiety and the protein is crucial for achieving potent E. coli DNA gyrase
inhibition, since 4-bromopyrrole, 4,5-dichloropyrrole and indole derivatives displayed weaker
inhibition of the enzyme [24, 25]. In this new series, the 4,5-dibromopyrrolamide moiety has
thus been retained. To further explore the structure-activity relationship of the 4,5,6,7-
tetrahydrobenzo[1,2-d]thiazole-2,6-diamine derivatives we synthesized compounds in which
the 4,5-dibromopyrrole-2-carbonyl moiety is attached to the amino group at position 2,
instead of that at position 6, resulting in the “reversed” type III analogues (Figure 3) of the
parent compounds (compound II and its analogues [24], Figure 3). Additional substituents
designed to target the Arg76/Arg136 side chain were then introduced at the 6-amino group
(Figure 3). In addition, a series of 2-(2-aminothiazol-4-yl)acetic acid derivatives (type IV
analogues, Figure 3) were designed and synthesized. These so-called “ring-opened”
compounds are more flexible than type III compounds and possess a smaller central scaffold,
and thereby offering the opportunity for the introduction of larger substituents for potential
interaction with Arg76/Arg136. The design of all these compounds was supported by
molecular docking to the E. coli DNA gyrase ATP-binding site. The predicted binding modes
are presented in Figures 4 and 5.
6

Figure 2. Schematic representation of hydrogen bonds between inhibitor I and E. coli DNA gyrase ATP-binding
site residues (PDB entry: 4ZVI [25]).
a
b
Figure 3. a) Molecular docking pose of E. coli DNA gyrase inhibitor II in the ATP-binding site of E. coli DNA
gyrase [24]. b) Design of a novel series of 4,5-dibromopyrrolamide-based DNA gyrase inhibitors.
2.2. Chemistry
The designed 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole-2,6-diamine derivatives 5-8 were
prepared according to the synthetic procedure presented in Scheme 1. Enantiomerically pure
starting (S)- and (R)-4,5,6,7-tetrahydrobenzo[1,2-d]thiazole-2,6-diamines ((R)-1 and (S)-1)
were synthesized according to the reported procedure [24, 35]. In the first step, the amino
group at position 6 was protected as a tert-butylcarbamate using di-tert-butyl dicarbonate in
tetrahydrofuran to yield (R)-2 and (S)-2. The amino group at position 2 of (R)-2 and (S)-2 was
then acylated with 2,2,2-trichloro-1-(4,5-dibromo-1H-pyrrol-2-yl)ethanone in N,N-
7

dimethylformamide (DMF) at elevated temperature. The Boc protecting groups of the
compounds (R)-3 and (S)-3 obtained were removed with HClgas generated in situ by the slow
addition of acetyl chloride to methanol. This acidolysis yielded hydrochlorides (R)-4 and (S)-4
which were then acylated with ethyl oxalyl chloride ((R)-5 and (S)-5) or methyl malonyl
chloride ((S)-6) in DMF at room temperature. In the final step, esters (R)-5, (S)-5 and (S)-6
were hydrolyzed to their acid derivatives (R)-7, (S)-7 and (S)-8 using alkaline conditions.
Scheme 1. Reagents and conditions. a) Boc2O, THF, r.t., 24 h; b) 2,2,2-trichloro-1-(4,5-dibromo-1H-pyrrol-2-
yl)ethanone, Na2CO3, DMF, 80 °C, 18 h; c) acetyl chloride, MeOH, 0 °C, 1 h, then r.t., 18 h; d) ethyl oxalyl
chloride (for (R)-5 and (S)-5) or methyl malonyl chloride (for (S)-6), DBU, DMF, r.t., 24 h; e) 1 M NaOH,
MeOH/H2O, r.t., 24 h.
Synthesis of the 2-(2-aminothiazol-4-yl)acetic acid derivatives 11-20 is outlined in
Scheme 2. 2-(2-Aminothiazol-4-yl)acetic acid (9) was first esterified with methanol to give 10
and the amino group at position 2 of the thiazole moiety of 10 then acylated by 2,2,2-
trichloro-1-(4,5-dibromo-1H-pyrrol-2-yl)ethanone, as described above, to give compound 11.
Hydrolysis of the methyl ester 11 resulted in carboxylic acid 12, which was coupled with
either glycine methyl ester hydrochloride to give 13 or substituted aniline derivatives 15-18
using EDC/HOBt-promoted amide bond formation. Further, methyl esters 13, 17 and 18 were
converted to their carboxylic acid counterparts 14, 19 and 20 by alkaline hydrolysis.
8

Scheme 2. Reagents and conditions. a) SOCl2, MeOH, reflux, 1.5 h; b) 2,2,2-trichloro-1-(4,5-dibromo-1H-
pyrrol-2-yl)ethanone, Na2CO3, DMF, 80 °C, 18 h; c) 2 M NaOH, MeOH, r.t., 24 h; d) H-Gly-OMe HCl, EDC,
HOBt, NMM, DMF, r.t., 18 h; e) corresponding H2NC6H4R1R2, EDC, HOBt, NMM, DMF, r.t., 18 h.
2.3. Biological evaluation, structure-activity relationship and molecular modeling
The final compounds (R)-5, (S)-5, (S)-6, (R)-7, (S)-7, (S)-8, 13-20 were tested for E.
coli DNA gyrase inhibition using the DNA gyrase supercoiling assay (Tables 1 and 2).
Results are presented as residual activities (RA) of the enzyme at 100 μM of the tested
compound, or as IC50 values for the more active compounds (RA<50% at 100 μM). All
prepared final compounds were screened for their antibacterial activity at 50 μM against two
Gram positive (Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC
25923) and two Gram negative (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa
ATCC 27853) bacterial strains. Results of the antibacterial activity evaluation are presented in
Tables 1 and 2.
2.3.1. 4,5,6,7-Tetrahydrobenzo[1,2-d]thiazoles
The in vitro E. coli DNA gyrase inhibitory activities of 4,5,6,7-tetrahydrobenzo[1,2-
d]thiazoles (R)-5, (S)-5, (S)-6, (R)-7, (S)-7 and (S)-8 (Table 1) showed weaker DNA gyrase
inhibition than that of the parent compounds bearing a pyrrole moiety on the amino group at
9

position 6 (compound II and its analogues [24]). With an IC50 value of 4.47 μM the oxalic
acid ester (S)-5 was a 2-fold weaker inhibitor than the malonic acid ester (S)-6 with an IC50
value of 2.34 μM. Hydrolysis of these esters to their carboxylic acid counterparts resulted in
improved E. coli DNA gyrase inhibition. The oxalic acid derivative (S)-7 (IC50 = 1.12 μM)
was approximately 4-fold more potent than its parent ester (S)-5 (IC50 = 4.47 μM), while the
activity of the malonic acid derivative (S)-8 was improved to the submicromolar range (IC50 =
0.891 μM). These results highlight the importance of the chain length, since malonic acid
ester and acid (compounds (S)-6 and (S)-8), with an additional methylene group, displayed
more potent E. coli DNA gyrase inhibition than their oxalic acid-based analogues (S)-5 and
(S)-7. The inhibitory activity of the oxalic acid-based R-enantiomers (compounds (R)-5 and
(R)-7) was weaker (IC50 values of 10.4 μM and 3.58 μM, respectively) than that of their S-
enantiomers (S)-5 and (S)-7. A similar phenomenon was observed in our previous series of the
4,5,6,7-tetrahydrobenzo[1,2-d]thiazole-2,6-diamines with pyrrole moiety attached to the
amino group at position 6 [24].
Molecular docking experiments using FlexX [36, 37], as available in LeadIT
(BioSolveIT GmbH) [38], were conducted to study the binding modes of E. coli DNA gyrase
inhibitors (R)-5, (S)-5, (S)-6, (R)-7, (S)-7 and (S)-8. We used our already described and
validated docking protocol [24] using the high-resolution crystal structure of the 4,5’-
bithiazole inhibitor in complex with E. coli DNA gyrase (PDB entry: 4DUH) as the target
protein [22]. The obtained docking poses and scoring function scores cannot explain the
observed differences in binding affinities. All tested inhibitors are predicted to form two
hydrogen bonds in the pyrrolamide-binding pocket, one direct and one bridged by a conserved
water molecule to Asp73 side chain (Figure 4). Moreover, docking predicts formation of
additional hydrogen bonds with Arg76 and/or Arg136 side chains, but the binding modes
suggest that there is no interaction between a salt bridge made by Glu50-Arg76 residues and
10

the 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole ring of inhibitors (Figure 4). This could explain
why oxalic acid-based compound (S)-7 (IC50 = 1.12 μM) is less potent that its counterpart II
(Figure 3, IC50 = 0.058 μM, while its benzothiazole-based analogue, in which such interaction
is still possible, was shown to possess almost equipotent activity as compound II [39].
Compounds (R)-5, (S)-5, (S)-6, (R)-7, (S)-7 and (S)-8 were inactive or only weakly
active against selected Gram positive and Gram negative bacterial strains at 50 μM (i.e.
growth inhibition less than 50% for all compounds) (Table 1). There are several possible
explanations for the observed lack of correlation between in vitro enzyme inhibition and
antibacterial activity against selected bacterial strains. Firstly, the enzymatic activity could be
too low (IC50 values between 0.891 and 10.4 μM) to result in inhibition of bacterial growth of
Gram negative E. coli, since subnanomolar Ki values against cytoplasmic targets could be
required to generate sufficient antibacterial potency on Gram negative bacteria [20].
Pyrrolamide-based type II analogues (compound II and its analogues, Figure 3) that
displayed potent E. coli DNA gyrase inhibition in the low nanomolar range were also devoid
of antibacterial activity against Gram negative strains [24]. Secondly, type II compounds [24]
and pyrrolamides presented in Figure 1 were shown to possess more potent antibacterial
activity against the ΔtolC E. coli strain, which lacks the outer membrane tolC efflux pump,
than against the wild-type E. coli strain [16, 17]. Therefore, active efflux of the compounds
from bacterial cells could be the second reason for the inactivity of pyrrolamide-based DNA
gyrase inhibitors including compounds (R)-5, (S)-5, (S)-6, (R)-7, (S)-7 and (S)-8 in vitro.
We assume that the 4,5-dibromopyrrole moiety is too bulky to occupy the
hydrophobic pocket of S. aureus DNA gyrase [24], resulting in weak inhibition of S. aureus
DNA gyrase by 4,5-dibromopyrrolamide-based compounds and, hence, in the weak
antibacterial activity against Gram positive S. aureus [24, 25]. To evaluate this hypothesis,
compounds (S)-7 and (S)-8 were tested for S. aureus DNA gyrase inhibition and showed
11

weak inhibition with enzyme residual activities of 77% and 69% at 10 µM, respectively,
which is probably too weak inhibition to result in antibacterial activity.
Table 1. Inhibition of Escherichia coli DNA gyrase and antibacterial activity of 4,5,6,7-tetrahydrobenzo[1,2-
d]thiazoles (R)-5, (S)-5, (S)-6, (R)-7, (S)-7 and (S)-8.
E. coli DNA gyrase
IC50 [µM]
4.47 µM
% inhibitiona
E. faecalis E. coli
cpd
n
R
S. aureus
P. aeruginosa
(S)-5
(S)-6
(S)-7
(S)-8
(R)-5
(R)-7
0
1
0
1
0
0
Et
Me
H
0
0
26
48
4
7
0
0
8
0
3
4
6
2.34 µM
1.12 µM
11
1
5
H
0.891 µM
10.4 µM
0
0
Et
H
19
0
5
10
8
3.58 µM
3
a% growth inhibition at 50 µM of compound.
Figure 4. Molecular docking pose of inhibitor (S)-7 (magenta sticks) in the E. coli DNA gyrase ATP-binding
site (PDB entry: 4DUH [22]) in yellow cartoon representation. For clarity, only amino acid residues forming
hydrogen bonds with inhibitor are presented as yellow sticks. The water molecule is shown as a red sphere.
Figure was prepared by PyMOL [40].
2.3.2. 2-(2-Aminothiazol-4-yl)acetic acids
12

2-(2-Aminothiazol-4-yl)acetic acids 11-20 (Table 2) were designed as ring-opened
analogues of the 4,5,6,7-tetrahydrobenzo[1,2-d]thiazoles (Figure 3). In the first step,
compounds 13 and 14, which by their length mimic the structures of 5 and 7, were prepared.
Their E. coli DNA gyrase inhibitory activity, with IC50 values of 170 μM for glycine ester
derivative 13 and 16 μM for its carboxylic acid 14, are weaker than those for 5 and 7.
However, docking studies suggest similar binding modes for 14 (Figure 5) and (S)-7 (Figure
4), with formation of two hydrogen bonds with Arg136.
The lack of antibacterial activity of compounds (R)-5, (S)-5, (S)-6, (R)-7, (S)-7, (S)-8,
13 and 14, as well as of previously reported type II analogues (compound II and its analogues
[24]), which all possess flexible substituents addressing Arg136, suggested the introduction of
more lipophilic and rigid aromatic moieties decorated with substituents with hydrogen bond
acceptor properties and acidic character. Although sulfonamide 15, phenol 16 and methyl
esters 17 and 18 were all devoid of inhibitory activity, benzoic acid 19 and salicylic acid 20
derivatives inhibited E. coli DNA gyrase with IC50 values of 33 μM and 40 μM. Despite being
more lipophilic (ChemBioDraw clogP = 3.37 for 19 vs clogP = 1.42 for 14) and less acidic
than their aliphatic analogues, their antibacterial activity was not improved, because their
inhibitory activity is probably still too weak to result in potent antibacterial activity (Table 2).
Table 2. Inhibition of Escherichia coli DNA gyrase and antibacterial activity of 2-(2-aminothiazol-4-yl)acetic
acid derivatives 13-20.
E. coli DNA
gyrase IC50 [µM]
or RAa [%]
% inhibitionb
cpd
R1
R2
S. aureus
E. faecalis
E. coli
P. aeruginosa
Me
-
169 µM
13
10
7
0
13
13

H
SO2NH2
H
-
15.9 µM
82%
15
0
14
0
7
6
0
0
0
0
0
0
0
14
15
16
17
18
19
20
H
OH
143 µM
64%
0
0
3
H
COOMe
COOMe
COOH
COOH
8
11
4
25
7
OH
H
100%
12
6
32.8 µM
39.8 µM
2
8
OH
0
0
3
aResidual activity of the enzyme at 100 µM of compound.
b% growth inhibition at 50 µM of compound.
Figure 5. Molecular docking pose of inhibitor 14 (in magenta sticks) in the E. coli DNA gyrase ATP-binding
site (PDB entry: 4DUH [22]; in yellow cartoon representation). For clarity, only amino acid residues forming
hydrogen bonds with inhibitor are presented as yellow sticks. The water molecule is shown as a red sphere. The
figure was prepared by PyMOL [40].
3. Conclusions
Two series of novel E. coli DNA gyrase inhibitors possessing 4,5,6,7-
tetrahydrobenzo[1,2-d]thiazole (compounds (R)-5, (S)-5, (S)-6, (R)-7, (S)-7, (S)-8) or 2-(2-
aminothiazol-4-yl)acetic acid as a central core (compounds 13-20) were designed and
synthesized. Compounds were designed based on our recently reported series of potent DNA
gyrase inhibitors [24] in order to further explore the SAR and to improve their antibacterial
activity. The results of in vitro enzymatic activity measurements using the E. coli DNA
14

supercoiling assay showed that active compounds (R)-5, (S)-5, (S)-6, (R)-7, (S)-7, (S)-8, 13,
14, 19 and 20 inhibit E. coli DNA gyrase in the sub-micromolar to low micromolar range,
however they display weak activity against Gram positive S. aureus and E. faecalis and Gram
negative E. coli and P. aeruginosa. Nevertheless, the described results for this novel structural
class of DNA gyrase inhibitors provides valuable information for the discovery of improved
DNA gyrase B inhibitors.
4. Experimental section
4.1. Chemistry
4.1.1. General procedures
Chemicals were obtained from Acros Organics (Geel, Belgium), Sigma-Aldrich (St.
Louis, MO, USA) and TCI Europe N.V. (Zwijndrecht, Belgium) and used without further
purification. Analytical TLC was performed on silica gel Merck 60 F254 plates (0.25 mm),
using visualization with UV light and spray reagents. Column chromatography was carried
out on silica gel 60 (particle size 240–400 mesh). HPLC analyses were performed on Agilent
Technologies 1100 instrument with G1365B UV-VIS detector, G1316A thermostat and
G1313A autosampler using Agilent Eclipse Plus C18 column (5 µm, 4.6 × 150 mm) using
Method A: mobile phase: 0.1% trifluoroacetic acid in water (A) and methanol (B); gradient:
90% A to 10% A in 20 min, then 5 min 10 % A; flow rate 1.0 mL/min; injection volume: 10
μL; Method B: mobile phase: 0.1% trifluoroacetic acid in water (A) and acetonitrile (B);
gradient: 2 min 95 % A, 90% A to 10% A in 12 min, 10% A to 5% A in 1 min, then 5 min 5%
A; flow rate 1.0 mL/min; injection volume: 10 μL. All tested compounds were ≥95% pure by
HPLC. Melting points were determined on a Reichert hot stage microscope and are
1
13
uncorrected. H and C NMR spectra were recorded at 400 and 100 MHz, respectively, on a
Bruker AVANCE III 400 spectrometer (Bruker Corporation, Billerica, MA, USA) in DMSO-
15

d6 or CDCl3 solutions, with TMS as the internal standard. IR spectra were recorded on a
Thermo Nicolet Nexus 470 ESP FT-IR spectrometer (Thermo Fisher Scientific, Waltham,
MA, USA). Mass spectra were obtained using a VG Analytical Autospec Q mass
spectrometer (Fisons, VG Analytical, Manchester, UK). Optical rotations were measured on a
Perkin-Elmer 241 MC polarimeter. The reported values for specific rotation are average
values of 5 successive measurements using an integration time of 5 s.
4.1.2. General procedure A. Synthesis of compounds (R)-2 and (S)-2
A solution of (S)- or (R)-4,5,6,7-tetrahydrobenzo[1,2-d]thiazole-2,6-diamine ((S)- or
(R)-1) (1 mmol) in tetrahydrofuran (THF) (5 mL) was cooled to 0 °C on an ice bath. Then a
solution of di-tert-butyl dicarbonate (Boc2O) (1.1 mmol) in THF (3 mL) was added dropwise
over 20 min. The reaction mixture was stirred at room temperature overnight. The solvent was
removed under reduced pressure and the crude product dissolved in ethyl acetate (10 mL).
Organic phase was successively washed with saturated aqueous NaHCO3 solution (10 mL)
and brine (10 mL), dried over Na2SO4, filtered and the solvent removed under reduced
pressure. Product was used without further purification.
4.1.2.1. tert-Butyl (S)-(2-amino-4,5,6,7-tetrahydrobenzo[1,2-d]thiazol-6-yl)carbamate ((S)-2)
Compound was prepared from (S)-1 (2.436 g, 14.40 mmol) and Boc2O (3.457 g, 15.84
mmol) according to the general procedure A. Yield: 3.892 g (100%); white solid; m.p. 147-
149 °C; [α]D -40.3 (c 0.26, MeOH); IR (ATR) ν 3363, 2976, 1684, 1634, 1514, 1445, 1365,
1307, 1282, 1249, 1229, 1167, 1090, 1050, 975, 880, 829, 783, 760, 742, 690, 609 cm-1; 1H
NMR (400 MHz, DMSO-d6): δ 1.38 (s, 9H, C(CH3)3), 1.54-1.65 (m, 1H, HA-7), 1.80-1.86 (m,
1H, HB-7), 2.31-2.47 (m, 3H, HA-4, H-5), 2.68 (dd, 1H, J1 = 14.8 Hz, J2 = 5.5 Hz, HB-4),
13
3.57-3.69 (m, 1H, CHNH), 6.64 (s, 2H, NH2), 6.94 (d, 1H, J = 7.9 Hz, NH) ppm; C NMR
16

(100 MHz, DMSO-d6): δ 24.9, 28.2 (3C), 28.87, 28.94, 46.8, 77.6, 112.4, 144.1, 154.9, 166.1
ppm.
4.1.2.2. tert-Butyl (R)-(2-amino-4,5,6,7-tetrahydrobenzo[1,2-d]thiazol-6-yl)carbamate ((R)-2)
Compound was prepared from (R)-1 (1.000 g, 5.92 mmol) and Boc2O (1.360 g, 6.21
mmol) according to the general procedure A. Yield: 1,59 g (100%); off-white solid; m.p. 153-
154 °C; [α]D +39.7 (c 0.24, MeOH); IR (ATR) ν 3361, 3122, 2976, 2934, 1684, 1514, 1443,
1367, 1307, 1251, 1228, 1171, 1090, 1047, 993, 975, 866, 827, 744 cm-1; 1H NMR (400 MHz,
DMSO-d6): δ 1.38 (s, 9H, C(CH3)3), 1.54-1.65 (m, 1H, HA-7), 1.80-1.86 (m, 1H, HB-7), 2.31-
2.47 (m, 3H, HA-4, H-5), 2.68 (dd, 1H, J1 = 14.8 Hz, J2 = 5.5 Hz, HB-4), 3.57-3.69 (m, 1H,
CHNH), 6.64 (s, 2H, NH2), 6.94 (d, 1H, J = 7.9 Hz, NH) ppm.
4.1.3. General procedure B. Synthesis of compounds (R)-3 and (S)-3
A solution of (S)- or (R)-2 (1 mmol) and Na2CO3 (1 mmol) in N,N-dimethylformamide
(5 mL) was stirred at room temperature for 15 min. Then 2,2,2-trichloro-1-(4,5-dibromo-1H-
pyrrol-2-yl)ethan-1-one (1.1 mmol) was added and the mixture stirred at 80 °C overnight.
Solvent was removed under reduced pressure and the crude product dissolved in ethyl acetate
(10 mL). Organic phase was successively washed with 10% citric acid (10 mL), saturated
aqueous NaHCO3 solution (10 mL) and brine (10 mL), dried over Na2SO4, filtered and the
solvent removed under reduced pressure. Product was purified by column chromatography
using dichloromethane/methanol (30:1) as eluent.
4.1.3.1.
tert-Butyl
(S)-(2-(4,5-dibromo-1H-pyrrole-2-carboxamido)-4,5,6,7-
tetrahydrobenzo[1,2-d]thiazol-6-yl)carbamate ((S)-3)
Compound was prepared from (S)-2 (0.800 g, 2.97 mmol) according to the general
procedure B. Yield: 0.610 g (39.5%); white crystals; m.p. 176-178 °C; [α]D -36.8 (c 0.21,
MeOH); IR (ATR) ν 3366, 3120, 2844, 1680, 1616, 1556, 1457, 1379, 1335, 1280, 1160,
17

1
1090, 972, 856, 779, 750, 708, 680 cm-1; H NMR (400 MHz, DMSO-d6): δ 1.41 (s, 9H,
C(CH3)3), 1.68-1.78 (m, 1H, HA-7), 1.91-1.99 (m, 1H, HB-7), 2.55-2.76 (m, 3H, HA-4, H-5),
2.91 (dd, 1H, J1 = 15.1 Hz, J2 = 4.5 Hz, HB-4), 3.69-3.79 (m, 1H, CHNH), 7.01 (d, J = 7.7 Hz,
13
CHNH), 7.40 (s, 1H, pyrrole-H), 12.18 (s, 1H, NH), 13.08 (s, 1H, NH) ppm; C NMR (100
MHz, DMSO-d6): δ 24.5, 28.2 (3C), 28.4, 28.8, 46.5, 77.7, 98.7, 107.6, 115.1, 119.7, 123.3,
125.97, 126.01, 154.7, 160.2 ppm; HRMS (ESI-) m/z for C17H19N4O3SBr2 ([M-H]-): calcd
516.9545, found 516.9541.
4.1.3.2.
tert-Butyl
(R)-(2-(4,5-dibromo-1H-pyrrole-2-carboxamido)-4,5,6,7-
tetrahydrobenzo[1,2-d]thiazol-6-yl)carbamate ((R)-3)
Compound was prepared from (R)-2 (1.000 g, 3.72 mmol) according to the general
procedure B. Yield: 0.645 g (33.4%); white crystals; m.p. 174-176 °C; [α]D +35.2 (c 0.23,
MeOH); IR (ATR) ν 3227, 2973, 1735, 1655, 1541, 1442, 1406, 1377, 1315, 1291, 1215,
1178, 1090, 1046, 1015, 992, 975, 858, 744 cm-1; 1H NMR (400 MHz, DMSO-d6): δ 1.40 (s,
9H, C(CH3)3), 1.67-1.77 (m, 1H, HA-7), 1.90-1.99 (m, 1H, HB-7), 2.55-2.76 (m, 3H, HA-4, H-
5), 2.91 (dd, 1H, J1 = 15.7 Hz, J2 = 4.4 Hz, HB-4), 3.69-3.79 (m, 1H, CHNH), 7.03 (d, J = 7.9
13
Hz, CHNH), 7.38 (s, 1H, pyrrole-H), 12.21 (s, 1H, NH), 13.11 (s, 1H, NH) ppm; C NMR
(100 MHz, DMSO-d6): δ 24.5, 28.2 (3C), 28.4, 28.8, 46.5, 77.7, 98.7, 107.6, 115.1, 119.7,
123.3, 125.97, 126.01, 154.7, 160.2 ppm; HRMS (ESI-) m/z for C17H19N4O3SBr2 ([M-H]-):
calcd 516.9545, found 516.9540.
4.1.4. General procedure C. Synthesis of compounds (R)-4 and (S)-4
Methanol (10 mL) was cooled on an ice bath and then acetyl chloride (10 mmol) was
added dropwise. The mixture was stirred at 0 °C for 30 min and then solution of (R)-3 or (S)-3
(1 mmol) in methanol (10 mL) was added. Reaction mixture was stirred at 0 °C for 1 h and
then at room temperature overnight.
18

4.1.4.1.
(S)-2-(4,5-dibromo-1H-pyrrole-2-carboxamido)-4,5,6,7-tetrahydrobenzo[1,2-
d]thiazol-6-aminium chloride ((S)-4)
Compound was prepared from (S)-3 (0.505 g, 0.97 mmol) according to the general
procedure C. The precipitate was filtered off and dried. Additional amount of (S)-4 was
obtained from mother liquor, which was concentrated in vacuo and dried. Yield: 0.443 g
(100%); off-white solid; m.p. >300 °C; [α]D -60.7 (c 0.20, MeOH); IR (ATR) ν 3394, 2828,
1
1692, 1631, 1567, 1519, 1382, 1338, 1199, 1183, 1073, 970, 904, 845, 733, 616 cm-1; H
NMR (400 MHz, DMSO-d6): δ 1.87-1.97 (m, 1H, HA-7), 2.11-2.19 (m, 1H, HB-7), 2.71-2.80
(m, 3H, HA-4, H-5), 3.10 (dd, 1H, J1 = 16.0 Hz, J2 = 5.1 Hz, HB-4), 3.51-3.54 (m, 1H,
+
CHNH), 7.40 (d, 1H, J = 2.7 Hz, pyrrole-H), 8.28 (d, 3H, J = 4.4 Hz, NH3 ), 12.30 (s, 1H,
NH), 13.14 (s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 23.5, 26.4, 26.6, 46.4, 98.8,
107.7, 115.4, 117.8, 124.3, 125.9, 143.2, 156.2 ppm; HRMS (ESI-) m/z for C12H11N4OSBr2
([M-H]-): calcd 416.9020, found 416.9030.
4.1.4.2.
(R)-2-(4,5-dibromo-1H-pyrrole-2-carboxamido)-4,5,6,7-tetrahydrobenzo[1,2-
d]thiazol-6-aminium chloride ((R)-4)
Compound was prepared from (R)-3 (0.600 g, 1.15 mmol) according to the general
procedure C. The precipitate was filtered off and dried. Additional amount of (R)-4 was
obtained from mother liquor, which was concentrated in vacuo and dried. Yield: 0.527 g
(100%); off-white solid; m.p. >300 °C; [α]D +58.7 (c 0.21, MeOH); IR (ATR) ν 3355, 3312,
3226, 3127, 2980, 1697, 1651, 1547, 1508, 1436, 1409, 1375, 1292, 1219, 1175, 1115, 1089,
1010, 979, 855, 825, 781, 736, 704 cm-1; 1H NMR (400 MHz, DMSO-d6): δ 1.86-1.96 (m, 1H,
HA-7), 2.09-2.18 (m, 1H, HB-7), 2.71-2.80 (m, 3H, HA-4, H-5), 3.10 (dd, 1H, J1 = 15.7 Hz, J2
+
= 5.0 Hz, HB-4), 3.51-3.54 (m, 1H, CHNH), 7.41 (s, 1H, pyrrole-H), 8.23 (s, 3H, NH3 ), 12.29
(s, 1H, NH), 13.13 (s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 23.5, 26.4, 26.6,
19

46.4, 98.8, 107.7, 115.4, 117.8, 124.3, 125.9, 143.2, 156.2 ppm; HRMS (ESI-) m/z for
C12H11N4OSBr2 ([M-H]-): calcd 416.9020, found 416.9030.
4.1.5. General procedure D. Synthesis of compounds (S)-5, (R)-5 and (S)-6
To a solution of amine (1 mmol) in N,N-dimethylformamide (DMF) (5 mL) cooled to
0 °C, 1,8-diazabicyclo[5.4.0]undec-7-en (DBU) (3 mmol) and ethyl 2-chloro-2-oxoacetate or
methyl 3-chloro-3-oxopropanoate (1.3 mmol) were added dropwise. Reaction mixture was
stirred at room temperature overnight. The solvent was evaporated under reduced pressure
and the oily residue dissolved in ethyl acetate (20 mL). Organic phase was successively
washed with 10% citric acid (2 × 10 mL), saturated aqueous NaHCO3 solution (2 × 10 mL)
and brine (10 mL), dried over Na2SO4, filtered and the solvent removed under reduced
pressure.
4.1.5.1.
Ethyl
(S)-2-((2-(4,5-dibromo-1H-pyrrole-2-carboxamido)-4,5,6,7-
tetrahydrobenzo[1,2-d]thiazol-6-yl)amino)-2-oxoacetate ((S)-5)
Compound was prepared from (S)-4 (0.115 g, 0.25 mmol) according to the general
procedure D. Yield: 0.080 g (61.1%); off-white solid; m.p. 160-162 °C; [α]D -19.8 (c 0.11,
MeOH); IR (ATR) ν 3228, 2937, 1734, 1668, 1535, 1443, 1405, 1378, 1318, 1284, 1262,
1211, 1110, 1017, 973, 933, 859, 745, 699, 639, 620 cm-1; 1H NMR (400 MHz, DMSO-d6): δ
1.28 (t, 3H, J = 7.1 Hz, CH2CH3), 1.87-1.98 (m, 2H, H-7), 2.69-2.76 (m, 3H, HA-4, H-5), 2.93
(dd, 1H, J1 = 15.8 Hz, J2 = 5.5 Hz, HB-4), 4.07-4.14 (m, 1H, CHNH), 4.25 (q, 2H, J = 7.1 Hz,
CH2CH3), 7.41 (s, 1H, pyrrole-H), 9.04 (d, 1H, J = 8.1 Hz, CONHCH), 12.23 (s, 1H, NH),
13.12 (s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 13.8, 24.5, 27.5, 28.0, 45.7, 62.0,
98.8, 107.7, 115.2, 119.5, 130.8, 131.5, 141.0, 155.8, 156.9, 160.9 ppm; HRMS (ESI-) m/z for
C16H15N4O4SBr2 ([M-H]-): calcd 516.9181, found 516.9186. HPLC: method B, tr 12.91 min
(95.1% at 254 nm).
20

4.1.5.2.
Ethyl
(R)-2-((2-(4,5-dibromo-1H-pyrrole-2-carboxamido)-4,5,6,7-
tetrahydrobenzo[1,2-d]thiazol-6-yl)amino)-2-oxoacetate ((R)-5)
Compound was prepared from (R)-4 (0.150 g, 0.33 mmol) according to the general
procedure D. Crude product was purified by column chromatography using
dichloromethane/methanol (40:1) as eluent. Yield: 0.021 g (11.5%); yellow solid; m.p. 161-
164°C; [α]D +20.1 (c 0.12, MeOH); IR (ATR) ν 3356, 3226, 2936, 1735, 1696, 1651, 1538,
1
1442, 1407, 1376, 1315, 1290, 1214, 1177, 1091, 1014, 992, 976, 858, 826, 742 cm-1; H
NMR (400 MHz, DMSO-d6): δ 1.28 (t, 3H, J = 7.1 Hz, CH2CH3), 1.85-1.99 (m, 2H, H-7),
2.67-2.76 (m, 3H, HA-4, H-5), 2.93 (dd, 1H, J1 = 15.6 Hz, J2 = 4.9 Hz, HB-4), 4.04-4.14 (m,
1H, CHNH), 4.25 (q, 2H, J = 7.1 Hz, CH2CH3), 7.41 (s, 1H, pyrrole-H), 9.04 (d, 1H, J = 8.1
Hz, CONHCH), 12.23 (s, 1H, NH), 13.12 (s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6):
δ 13.8, 24.5, 27.5, 28.0, 45.7, 62.0, 98.8, 107.7, 115.2, 119.5, 130.8, 131.5, 141.0, 155.8,
156.9, 160.9 ppm; HRMS (ESI-) m/z for C16H15N4O4SBr2 ([M-H]-): calcd 516.9181, found
516.9186. HPLC: method B, tr 12.90 min (95.3% at 254 nm).
4.1.5.3.
Methyl
(S)-3-((2-(4,5-dibromo-1H-pyrrole-2-carboxamido)-4,5,6,7-
tetrahydrobenzo[1,2-d]thiazol-6-yl)amino)-3-oxopropanoate ((S)-6)
Compound was prepared from (S)-4 (0.100 g, 0.22 mmol) according to the general
procedure D. Yield: 0.062 g (46.3%); white solid; m.p. 205-207 °C; [α]D -17.5 (c 0.13, MeOH);
IR (ATR) ν 3261, 3124, 2952, 1721, 1641, 1538, 1438, 1384, 1324, 1352, 1281, 1226, 1177,
1122, 1018, 991, 975, 902, 856, 832, 773, 740, 696 cm-1; 1H NMR (400 MHz, DMSO-d6): δ
1.74-1.84 (m, 1H, HA-7), 1.87-1.95 (m, 1H, HB-7), 2.51-2.57 (m, 2H, HA-4, HA-5), 2.67-2.73
(m, 1H, HB-5), 2.94 (dd, 1H, J1 = 15.8 Hz, J2 = 5.0 Hz, HB-4), 3.24 (d, 2H, J = 1.9 Hz,
COCH2CO), 3.59 (s, 3H, CH3), 4.02-4.10 (m, 1H, CHNH), 7.38 (s, 1H, pyrrole-H), 8.23 (d,
1H, J = 7.6 Hz, CONHCH), 12.21 (s, 1H, NH), 13.08 (s, 1H, NH) ppm; 13C NMR (100 MHz,
DMSO-d6): δ 23.6, 27.7, 28.2, 42.2, 44.8, 51.8, 98.7, 107.7, 115.2, 119.4, 126.0, 132.5, 143.6,
21

156.6, 164.7, 168.4 ppm; HRMS (ESI-) m/z for C16H15N4O4SBr2 ([M-H]-): calcd 516.9181,
found 516.9188. HPLC: method B, tr 12.26 min (95.5% at 254 nm).
4.1.6. General procedure E. Synthesis of compounds (S)-7, (R)-7 and (S)-8
To a solution of ester (1 mmol) in methanol (5 mL), 1 M NaOH (5 mmol) was added
and the reaction mixture stirred at room temperature overnight. Methanol was evaporated
under reduced pressure and reaction mixture extracted with ethyl acetate (10 mL). Water
phase was acidified with 1 M HCl to pH ~ 2, precipitate was filtered off and purified by flash
column chromatography using dichloromethane/methanol (1:1) as eluent.
4.1.6.1.
(S)-2-((2-(4,5-Dibromo-1H-pyrrole-2-carboxamido)-4,5,6,7-tetrahydrobenzo[1,2-
d]thiazol-6-yl)amino)-2-oxoacetic acid ((S)-7)
Compound was prepared from (S)-5 (0.050 g, 0.097 mmol) according to the general
procedure E. Yield: 0.032 g (67.7%); off-white solid; m.p. 236-238 °C; [α]D -48.6 (c 0.18,
MeOH); IR (ATR) ν 2941, 1657, 1555, 1408, 1383, 1325, 1223, 1171, 1079, 973, 864, 829,
742, 662, 618 cm-1; 1H NMR (400 MHz, DMSO-d6): δ 1.89-1.97 (m, 2H, H-7), 2.45-2.47 (m,
1H, H-4/H-5), 2.70-2.77 (m, 2H, H-4/H-5), 2.92 (dd, 1H, J1 = 15.6 Hz, J2 = 5.1 Hz, HB-4),
4.03-4.12 (m, 1H, CHNH), 7.40 (s, 1H, pyrrole-H), 8.95 (d, 1H, J = 8.4 Hz, CONHCH),
13
12.23 (s, 1H, NH), 13.11 (s, 1H, NH) ppm; C NMR (100 MHz, DMSO-d6): δ 24.5, 27.6,
28.0, 45.7, 98.8, 107.6, 115.2, 119.4, 126.0, 137.1, 143.2, 156.5, 158.0, 162.3 ppm; HRMS
(ESI-) m/z for C14H11N4O4SBr2 ([M-H]-): calcd 488.8868, found 488.8879. HPLC: method B,
tr 11.74 min (95.0% at 254 nm).
4.1.6.2.
(R)-2-((2-(4,5-Dibromo-1H-pyrrole-2-carboxamido)-4,5,6,7-tetrahydrobenzo[1,2-
d]thiazol-6-yl)amino)-2-oxoacetic acid ((R)-7)
22

Compound was prepared from (R)-5 (0.010 g, 0.020 mmol) according to the general
procedure E. Yield: 0.008 g (84.2%); off-white solid; m.p. 240-242°C; [α]D +47.7 (c 0.20,
1
MeOH); H NMR (400 MHz, DMSO-d6): δ 1.89-1.97 (m, 2H, H-7), 2.44-2.47 (m, 1H, H-
4/H-5), 2.70-2.78 (m, 2H, H-4/H-5), 2.92 (dd, 1H, J1 = 15.4 Hz, J2 = 5.2 Hz, HB-4), 4.03-4.12
(m, 1H, CHNH), 7.40 (s, 1H, pyrrole-H), 8.94 (d, 1H, J = 8.2 Hz, CONHCH), 12.24 (s, 1H,
NH), 13.10 (s, 1H, NH) ppm; HRMS (ESI-) m/z for C14H11N4O4SBr2 ([M-H]-): calcd
488.8868, found 488.8872. HPLC: method B, tr 11.744 min (100% at 254 nm).
4.1.6.3.
(S)-3-((2-(4,5-Dibromo-1H-pyrrole-2-carboxamido)-4,5,6,7-tetrahydrobenzo[1,2-
d]thiazol-6-yl)amino)-3-oxopropanoic acid ((S)-8)
Compound was prepared from (S)-6 (0.040 g, 0.0077 mmol) according to the general
procedure E. Yield: 0.020 g (51.3%); off-white solid; m.p. 123-125 °C; [α]D -15.6 (c 0.12,
MeOH); IR (ATR) ν 3287, 2924, 1711, 1656, 1558, 1383, 1328, 1304, 1260, 1213, 1182,
1099, 1014, 977, 921, 863, 836, 802, 740, 673, 632 cm-1; 1H NMR (400 MHz, DMSO-d6): δ
1.77-1.87 (m, 1H, HA-7), 1.90-2.00 (m, 1H, HB-7), 2.64-2.76 (m, 3H, HA-4, H-5), 2.97 (dd,
1H, J1 = 15.5 Hz, J2 = 4.7 Hz, HB-4), 3.15 (d, 2H, J = 2.6 Hz, COCH2CO), 4.05-4.17 (m, 1H,
CHNH), 7.40 (s, 1H, pyrrole-H), 8.21 (d, 1H, J = 7.5 Hz, CONHCH), 12.37 (s, 1H, NH),
13.11 (s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 23.7, 27.8, 28.9, 42.6, 44.7, 98.7,
107.6, 115.1, 121.0, 126.0, 133.4, 142.5, 156.5, 157.7, 169.5 ppm; HRMS (ESI-) m/z for
C15H13N4O4SBr2 ([M-H]-): calcd 502.9024, found 502.9010. HPLC: method B, tr 11.70 min
(96.2% at 254 nm).
4.1.7. Methyl 2-(2-aminothiazol-4-yl)acetate (10)
Methanol (30 mL) was cooled to 0 °C and thionyl chloride (3.45 mL, 47.6 mmol) was
added dropwise. Then 2-(2-aminothiazol-4-yl)acetic acid (9) (3.00 g, 19.0 mmol) was added
and reaction mixture stirred under reflux for 1.5 h. The solvent was evaporated and the oily
23

residue triturated with diethyl ether. White precipitate (3.564 g, 89.9%) was filtered off and
dried. 1H NMR (400 MHz, DMSO-d6) δ 3.66 (s, 3H, CH3), 3.77 (s, 2H, CH2), 6.71 (s, 1H, Ar-
H), 9.30 (s, 2H, NH2) ppm; 13C NMR (100 MHz, DMSO-d6): δ 32.7, 52.1, 105.6, 132.4,
169.0, 169.5 ppm.
4.1.8. Methyl 2-(2-(4,5-dibromo-1H-pyrrole-2-carboxamido)thiazol-4-yl)acetate (11)
A solution of 10 (1.044 g, 5.00 mmol) and Na2CO3 (0.530 g, 5.00 mmol) in DMF (20
mL) was stirred at room temperature for 15 min. 2,2,2-Trichloro-1-(4,5-dibromo-1H-pyrrol-2-
yl)ethan-1-one (1.852 g, 5.00 mmol) was added and mixture was stirred at 80 °C overnight.
Solvent was removed under reduced pressure, residue was suspended in ethyl acetate (60 mL)
and successively washed with 10% citric acid (2 × 30 mL), saturated aqueous NaHCO3
solution (2 × 30 mL) and brine (30 mL), dried over Na2SO4, filtered and the solvent removed
under reduced pressure. The crude product was recrystallized from methanol. Yield: 1.630 g
(77.0%); white crystals; m.p. 200-202 °C; IR (ATR) ν 3352, 3232, 3129, 2982, 1698, 1650,
1543, 1505, 1442, 1410, 1368, 1274, 1218, 1172, 1116, 1085, 1010, 980, 886, 854, 823, 782,
729, 687 cm-1; 1H NMR (400 MHz, DMSO-d6): δ 3.63 (s, 3H, CH3), 3.75 (s, 2H, CH2), 7.04
(s, 1H, thiazole-H), 7.45 (d, 1H, J = 2.6 Hz, pyrrole-H), 12.42 (s, 1H, NH), 13.12 (s, 1H, NH)
13
ppm; C NMR (100 MHz, DMSO-d6): δ 36.4, 51.7, 98.8, 107.8, 110.8, 115.4, 125.8, 143.7,
156.6, 157.8, 170.5 ppm; HRMS (ESI-) m/z for C11H8N3O3SBr2 ([M-H]-):calcd 419.8653,
found 419.8650; HPLC: method A, tr 13.09 min (95.4% at 254 nm).
4.1.9. 2-(2-(4,5-Dibromo-1H-pyrrole-2-carboxamido)thiazol-4-yl)acetic acid (12)
To a solution of 11 (0.702 g, 1.66 mmol) in methanol (25 mL) 2 M NaOH (1.66 mL,
3.32 mmol) was added and the reaction mixture stirred at room temperature overnight.
Methanol was evaporated under reduced pressure. Water phase was acidified with 2 M HCl to
pH ~ 2, white precipitate filtered and dried. Yield: 0.504 g (74.2%); white crystals; m.p. 240-
24

242 °C; IR (ATR) ν 3353, 3230, 3129, 2983, 1699, 1652, 1545, 1505, 1442, 1408, 1367,
1292, 1217, 1174, 1115, 1087, 1010, 989, 898, 856, 823, 782, 740 cm-1; 1H NMR (400 MHz,
DMSO-d6): δ 3.64 (s, 2H, CH2), 7.00 (s, 1H, thiazole-H), 7.44 (d, 1H, J = 2.7 Hz, pyrrole-H),
13
12.41 (s, 1H, NH), 13.13 (s, 1H, NH) ppm; C NMR (100 MHz, DMSO-d6): δ 97.5, 109.9,
110.0, 115.3, 127.8, 130.5, 139.9, 157.9, 160.3, 164.7 ppm, signal for CH2 overlapped with
DMSO-d6; HRMS (ESI-) m/z for C10H6N3O3SBr2 ([M-H]-): calcd 405.8497, found 405.8488.
4.1.10. Methyl (2-(2-(4,5-dibromo-1H-pyrrole-2-carboxamido)thiazol-4-yl)acetyl)glycinate
(13)
A solution of 12 (0.200 g, 0.49 mmol) in N,N-dimethylformamide (5 mL) was cooled
to 0 °C and then EDC (0.113 g, 0.51 mmol) and HOBt (0.072 g, 0.51 mmol) were added. pH
was adjusted to 8 with N-methylmorpholine and the reaction mixture stirred for 20 min at 0
°C. Then glycine methyl ester hydrochloride (0.061 g, 0.49 mmol) was added and reaction
mixture stirred overnight at room temperature. The solvent was evaporated in vacuo and the
oily residue dissolved in ethyl acetate (30 mL) and washed successively with 10% citric acid
(2 × 30 mL), saturated aqueous NaHCO3 solution (2 × 30 mL) and brine (30 mL). The
organic phase was dried over Na2SO4, filtered and the solvent evaporated under reduced
pressure. Yield: 0.159 g (69.4%); white crystals; m.p. 231-233 °C; IR (ATR) ν 3354, 3225,
3129, 2980, 1698, 1650, 1543, 1508, 1441, 1409, 1369, 1290, 1216, 1172, 1116, 1086, 1010,
981, 856, 824, 782, 739 cm-1; 1H NMR (400 MHz, DMSO-d6): δ 3.58 (s, 2H, CH2CO), 3.64
(s, 3H, CH3), 3.87 (d, 2H, J = 5.8 Hz, NHCH2CO), 6.97 (s, 1H, thiazole-H), 7.45 (d, 1H, J =
2.5 Hz, pyrrole-H), 8.40 (t, 1H, J = 5.8 Hz), NHCH2CO), 12.40 (s, 1H, NH), 13.11 (s, 1H,
NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 38.0, 40.7, 51.7, 98.8, 107.8, 110.2, 115.3,
125.9, 145.2, 156.5, 157.6, 169.5, 170.4 ppm; HRMS (ESI-) m/z for C13H11N4O4SBr2 ([M-H]-
): calcd 476.8868, found 476.8867; HPLC: method B, tr 11.98 min (95.0% at 254 nm).
4.1.11. (2-(2-(4,5-dibromo-1H-pyrrole-2-carboxamido)thiazol-4-yl)acetyl)glycine (14)
25

To a solution of 13 (0.122 g, 0.25 mmol) in methanol (5 mL) 1 M NaOH (0.42 mL,
0.42 mmol) was added and the reaction mixture stirred at room temperature overnight.
Methanol was evaporated under reduced pressure. Water phase was acidified with 2 M HCl to
pH ~ 2, white precipitate filtered and dried. Yield: 0.050 g (42.4%); white crystals; m.p. >300
°C; IR (ATR) ν 3355, 3228, 3128, 2982, 1699, 1651, 1543, 1512, 1441, 1408, 1376, 1294,
1220, 1175, 1090, 1011, 981, 857, 826, 738 cm-1; 1H NMR (400 MHz, DMSO-d6): δ 3.56 (s,
2H, CH2CO), 3.77 (d, 2H, J = 5.6 Hz, NHCH2CO), 6.96 (s, 1H, thiazole-H), 7.43 (s, 1H,
pyrrole-H), 8.28 (t, 1H, J = 5.6 Hz), NHCH2CO), 12.41 (s, 1H, NH), 13.11 (s, 1H, NH) ppm,
13
signal for COOH is not seen in the spectrum; C NMR (100 MHz, DMSO-d6): δ 38.0, 40.7,
98.8, 107.7, 110.1, 115.3, 125.9, 145.2, 156.6, 157.6, 169.3, 171.3 ppm; HRMS (ESI-) m/z for
C12H9N4O4SBr2 ([M-H]-): calcd 462.8711, found 462.8721; HPLC: method B, tr 11.24 min
(95.2% at 220 nm).
4.1.12.
pyrrole-2-carboxamide (15)
A solution of 12 (0.207 g, 0.51 mmol) in N,N-dimethylformamide (5 mL) was cooled
4,5-Dibromo-N-(4-(2-oxo-2-((4-sulfamoylphenyl)amino)ethyl)thiazol-2-yl)-1H-
to 0 °C and then EDC (0.120 g, 0.61 mmol) and HOBt (0.082 g, 0.61 mmol) were added. pH
was adjusted to 8 with N-methylmorpholine and the reaction mixture stirred for 20 min at 0
°C. Then 4-aminobenzenesulfonamide (0.087 g, 0.51 mmol) was added and reaction mixture
stirred overnight at room temperature. The solvent was evaporated in vacuo and the oily
residue dissolved in ethyl acetate (30 mL) and washed successively with 10% citric acid (2 ×
30 mL), saturated aqueous NaHCO3 solution (2 × 30 mL) and brine (30 mL). The organic
phase was dried over Na2SO4, filtered and the solvent evaporated under reduced pressure.
Crude product was recrystallized from methanol. Yield: 0.044 g (15.4%); white crystals; m.p.
>300 °C; IR (ATR) ν 3244, 3130, 2981, 1698, 1651, 1544, 1513, 1443, 1409, 1368, 1292,
1218, 1172, 1116, 1086, 1010, 982, 886, 855, 823, 782, 738, 660 cm-1; 1H NMR (400 MHz,
26

DMSO-d6): δ 3.78 (s, 2H, CH2), 7.03 (s, 1H, thiazole-H), 7.07 (t, 1H, J = 8.1 Hz, Ar-H), 7.27
(s, 2H, SO2NH2), 7.42 (s, 1H, pyrrole-H), 7.77 (s, 4H, 4 × Ar-H), 10.51 (s, 1H, CH2CONH),
12.38 (br s, 1H, NH), 13.12 (br s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 98.7,
107.7, 110.5, 115.3, 118.6, 126.7, 138.3, 142.0, 144.8, 149.0, 156.7, 157.8, 168.5 ppm, signal
for CH2 overlapped with DMSO-d6; HRMS (ESI-) m/z for C16H12N5O4S2Br2 ([M-H]-): calcd
559.8697, found 559.8687; HPLC: method B, tr 12.31 min (95.2% at 254 nm).
4.1.13. 4,5-Dibromo-N-(4-(2-((3-hydroxyphenyl)amino)-2-oxoethyl)thiazol-2-yl)-1H-pyrrole-
2-carboxamide (16)
A solution of 12 (0.200 g, 0.49 mmol) in N,N-dimethylformamide (5 mL) was cooled
to 0 °C and then EDC (0.113 g, 0.59 mmol) and HOBt (0.079 g, 0.59 mmol) were added. pH
was adjusted to 8 with N-methylmorpholine and the reaction mixture stirred for 20 min at 0
°C. Then 3-aminophenol (0.053 g, 0.49 mmol) was added and reaction mixture stirred
overnight at room temperature. The solvent was evaporated in vacuo and the oily residue
dissolved in ethyl acetate (30 mL) and washed successively with 10% citric acid (2 × 30 mL),
saturated aqueous NaHCO3 solution (2 × 30 mL) and brine (30 mL). The organic phase was
dried over Na2SO4, filtered and the solvent evaporated under reduced pressure. Crude product
was recrystallized from methanol. Yield: 0.110 g (45.3%); white crystals; m.p. 288-290 °C;
IR (ATR) ν 3259, 1655, 1604, 1530, 1489, 1443, 1411, 1377, 1323, 1280, 1225, 1170, 1044,
980, 860, 779, 732, 686 cm-1; 1H NMR (400 MHz, DMSO-d6): δ 3.71 (s, 2H, CH2), 6.45 (ddd,
1H, J1 = 7.9 Hz, J2 = 2.3 Hz, J3 = 0.9 Hz, Ar-H), 6.98 (ddd, 1H, J1 = 8.1 Hz, J2 = 1.8 Hz, J3 =
0.9 Hz, Ar-H), 7.00 (s, 1H, thiazole-H), 7.07 (t, 1H, J = 8.1 Hz, Ar-H), 7.20 (t, 1H, J = 2.1
Hz, Ar-H), 7.44 (d, 1H, J = 2.6 Hz, Ar-H), 9.39 (s, 1H, OH), 10.02 (s, 1H, CH2CONH), 12.38
(br s, 1H, NH), 13.12 (s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 98.8, 106.1,
107.8, 109.8, 110.3, 115.3, 125.8, 129.4, 140.2, 145.3, 156.5, 157.6, 157.7, 167.7 ppm, signal
27

for CH2 overlapped with DMSO-d6; HRMS (ESI-) m/z for C16H11N4O3SBr2 ([M-H]-): calcd
496.8919, found 496.8908; HPLC: method B, tr 12.61 min (100% at 254 nm).
4.1.14.
Methyl
3-(2-(2-(4,5-dibromo-1H-pyrrole-2-carboxamido)thiazol-4-
yl)acetamido)benzoate (17)
A solution of 12 (0.155 g, 0.38 mmol) in N,N-dimethylformamide (5 mL) was cooled
to 0 °C and then EDC (0.087 g, 0.45 mmol) and HOBt (0.061 g, 0.45 mmol) were added. pH
was adjusted to 8 with N-methylmorpholine and the reaction mixture stirred for 20 min at 0
°C. Then methyl 3-aminobenzoate (0.057 g, 0.38 mmol) was added and reaction mixture
stirred overnight at room temperature. The solvent was evaporated in vacuo and the oily
residue dissolved in ethyl acetate (30 mL) and washed successively with 10% citric acid (2 ×
30 mL), saturated aqueous NaHCO3 solution (2 × 30 mL) and brine (30 mL). The organic
phase was dried over Na2SO4, filtered and the solvent evaporated under reduced pressure.
Compound 17 was obtained as a white solidified oil. Yield: 0.158 g (77.1%); white solidified
1
oil; H NMR (400 MHz, DMSO-d6): δ 3.79 (s, 2H, CH2), 3.86 (s, 3H, CH3), 7.03 (s, 1H,
thiazole-H), 7.43 (s, 1H, pyrrole-H), 7.48 (t, 1H, J = 8.0 Hz, Ar-H-3), 7.65 (d, 1H, J = 8.0 Hz,
Ar-H-2/4), 7.87 (d, 1H, J = 8.0 Hz, Ar-H-2/4), 8.30 (s, 1H, Ar-H-6), 10.40 (s, 1H,
13
CH2CONH), 12.38 (s, 1H, NH), 13.12 (s, 1H, NH) ppm; C NMR (100 MHz, DMSO-d6): δ
52.2, 98.8, 107.8, 110.5, 115.4, 119.5, 123.5, 123.8, 125.8, 129.3, 130.1, 139.5, 145.0, 156.6,
157.7, 166.0, 168.3 ppm, signal for CH2 overlapped with DMSO-d6; HRMS (ESI-) m/z for
C18H13N4O4SBr2 ([M-H]-): calcd 538.9024, found 538.9033; HPLC: method A, tr 21.45 min
(100% at 254 nm).
4.1.15. Methyl 5-(2-(2-(4,5-dibromo-1H-pyrrole-2-carboxamido)thiazol-4-yl)acetamido)-2-
hydroxybenzoate (18)
28

A solution of 12 (0.209 g, 0.51 mmol) in N,N-dimethylformamide (5 mL) was cooled
to 0 °C and then EDC (0.117 g, 0.61 mmol) and HOBt (0.083 g, 0.61 mmol) were added. pH
was adjusted to 8 with N-methylmorpholine and the reaction mixture stirred for 20 min at 0
°C. Then methyl 5-amino-2-hydroxybenzoate (0.085 g, 0.51 mmol) was added and reaction
mixture stirred overnight at room temperature. The solvent was evaporated in vacuo and the
oily residue dissolved in ethyl acetate (30 mL) and washed successively with 10% citric acid
(2 × 30 mL), saturated aqueous NaHCO3 solution (2 × 30 mL) and brine (30 mL). The
organic phase was dried over Na2SO4, filtered and the solvent evaporated under reduced
pressure. Crude product was recrystallized from methanol. Yield: 0.100 g (35.2%); white
crystals; m.p. 257-259 °C; IR (ATR) ν 3235, 3129, 2981, 1697, 1651, 1543, 1505, 1441,
1409, 1367, 1288, 1216, 1172, 1116, 1085, 1009, 981, 899, 856, 824, 782, 737, 692 cm-1; 1H
NMR (400 MHz, DMSO-d6): δ 3.71 (s, 2H, CH2), 3.90 (s, 3H, CH3), 6.93-7.01 (m, 2H,
thiazole-H, Ar-H-3), 7.44 (s, 1H, pyrrole-H), 7.67 (dd, 1H, J1 = 9.2 Hz, J2 = 2.6 Hz, Ar-H-4),
8.17 (d, 1H, J = 2.6 Hz, Ar-H-6), 10.17 (s, 1H, CH2CONH or OH), 10.28 (s, 1H, CH2CONH
13
or OH), 12.37 (s, 1H, NH), 13.12 (s, 1H, NH) ppm; C NMR (100 MHz, DMSO-d6): δ 52.5,
98.8, 107.8, 110.4, 112.4, 115.4, 117.6, 120.0, 125.9, 127.2, 131.1, 145.1, 145.2, 155.9, 156.6,
167.7, 169.0 ppm, signal for CH2 overlapped with DMSO-d6; HRMS (ESI-) m/z for
C18H13N4O5SBr2 ([M-H]-): calcd 554.8973, found 554.8979; HPLC: method B, tr 10.46 min
(95.8% at 254 nm).
4.1.16. 3-(2-(2-(4,5-Dibromo-1H-pyrrole-2-carboxamido)thiazol-4-yl)acetamido)benzoic acid
(19)
To a solution of 17 (0.113 g, 0.21 mmol) in ethanol (5 mL) 2 M NaOH (0.42 mL, 0.42
mmol) was added and the reaction mixture stirred at room temperature for 8 h. Ethanol was
evaporated under reduced pressure. Water phase was acidified with 2 M HCl to pH ~ 2, white
precipitate filtered and dried. Yield: 0.103 g (93.6%); white crystals; m.p. >300 °C; IR (ATR)
29