Design, synthesis and biological evaluation of novel DNA gyrase inhibitors and their siderophore mimic conjugates

Article abstract of DOI:10.1016/j.bioorg.2019.103550

Bacterial DNA gyrase is an important target for the development of novel antibacterial drugs, which are urgently needed because of high level of antibiotic resistance worldwide. We designed and synthesized new 4,5,6,7-tetrahydrobenzo[d]thiazole-based DNA gyrase B inhibitors and their conjugates with siderophore mimics, which were introduced to increase the uptake of inhibitors into the bacterial cytoplasm. The most potent conjugate 34 had an IC₅₀ of 58 nM against Escherichia coli DNA gyrase and displayed MIC of 14 μg/mL against E. coli ΔtolC strain. Only minor improvements in the antibacterial activities against wild-type E. coli in low-iron conditions were seen for DNA gyrase inhibitor – siderophore mimic conjugates.

Full text of DOI:10.1016/j.bioorg.2019.103550

Journal Pre-proofs
Design, Synthesis and Biological Evaluation of Novel DNA Gyrase Inhibitors
and their Siderophore Mimic Conjugates
Andraž Lamut, Cristina D. Cruz, Žiga Skok, Michaela Barančoková, Nace
Zidar, Anamarija Zega, Lucija Peterlin Mašič, Janez Ilaš, Päivi Tammela,
Danijel Kikelj, Tihomir Tomašič
PII:
S0045-2068(19)31659-1
DOI:
https://doi.org/10.1016/j.bioorg.2019.103550
YBIOO 103550
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
3 October 2019
19 December 2019
21 December 2019
Please cite this article as: A. Lamut, C.D. Cruz, Z. Skok, M. Barančoková, N. Zidar, A. Zega, L. Peterlin Mašič,
J. Ilaš, P. Tammela, D. Kikelj, T. Tomašič, Design, Synthesis and Biological Evaluation of Novel DNA Gyrase
Inhibitors and their Siderophore Mimic Conjugates, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/
j.bioorg.2019.103550
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Design, Synthesis and Biological Evaluation of Novel DNA Gyrase Inhibitors and their
Siderophore Mimic Conjugates
Andraž Lamut,a Cristina D. Cruz,b Žiga Skok,a Michaela Barančoková,a Nace Zidar,a Anamarija
Zega,a Lucija Peterlin Mašič,a Janez Ilaš,a Päivi Tammela,b Danijel Kikelj,a and Tihomir
Tomašič,a,*
aUniversity of Ljubljana, Faculty of Pharmacy, Aškerčeva cesta 7, 1000 Ljubljana, Slovenia
bDrug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy,
University of Helsinki, P.O. Box 56 (Viikinkaari 5 E), FI-00014 Helsinki, Finland
*Corresponding author: Tihomir Tomašič
University of Ljubljana, Faculty of Pharmacy,
Aškerčeva cesta 7, 1000 Ljubljana, Slovenia
Tel: +386-1-4769556
E-mail: tihomir.tomasic@ffa.uni-lj.si
1

Abstract
Bacterial DNA gyrase is an important target for the development of novel antibacterial drugs,
which are urgently needed because of high level of antibiotic resistance worldwide. We
designed and synthesized new 4,5,6,7-tetrahydrobenzo[d]thiazole-based DNA gyrase B
inhibitors and their conjugates with siderophore mimics, which were introduced to increase the
uptake of inhibitors into the bacterial cytoplasm. The most potent conjugate 34 had an IC50 of
58 nM against Escherichia coli DNA gyrase and displayed MIC of 14 µg/mL against E. coli
ΔtolC strain. Only minor improvements in the antibacterial activities against wild-type E. coli
in low-iron conditions were seen for DNA gyrase inhibitor – siderophore mimic conjugates.
Graphical abstract
Keywords: antibiotics; catechol; DNA gyrase; inhibitors; siderophore mimic;
Abbreviations
EDC, 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide; GyrB, DNA gyrase subunit B;
HOBt, 1-hydroxybenzotriazole; MIC, minimum inhibitory concentration; OM, outer
membrane.
2

Highlights
ꢀ Siderophore mimics were conjugated with 4,5,6,7-tetrahydrobenzo[d]thiazole-based DNA
gyrase B inhibitors.
ꢀ The most potent conjugate had an IC50 of 58 nM against Escherichia coli DNA gyrase.
ꢀ Two conjugates displayed MICs of 14 µg/mL against E. coli ΔtolC strain.
ꢀ Most conjugates showed improved activities against wild-type Escherichia coli in iron-
depleted medium.
3

1. Introduction
In recent decades, infectious diseases have been recognized as one of the most important global
health issues, which is mainly attributed to the increasing problem of microbial resistance [1,
2]. According to the World Health Organization, the priority pathogens for which research and
development of new treatment options are most urgently needed include 12 families of bacteria.
Among these, Staphylococcus aureus, Pseudomonas aeruginosa and the Enterobacteriaceae
family (including Escherichia coli) have been described as critical high-priority pathogens that
are especially difficult to treat [3].
Unfortunately, the urgent need for development of new antibiotics is, however,
accompanied by a lack of interest of the pharmaceutical industry for economic reasons and
because of strict regulatory requirements [4]. Due to the problem of bacterial resistance,
antibiotics with new mechanisms of action are needed, as they would then not be expected to
be removed by the pre-existing resistance mechanisms, with the development of cross
resistance also less likely to occur. In the last 10 years, some successful new drugs have been
approved that have alternative mechanisms of action; e.g., antibiotics belonging to the class of
oxazolidinones and lipopeptides [5]. In addition to overexpression of efflux pumps, reduced
permeability of the outer membrane (OM) is recognized as a major mechanism that underlies
resistance of Gram-negative bacteria and that can cause the failure of existing antibacterial
therapies [6].
DNA gyrase is a validated target for antibacterial drug discovery [7, 8]. This enzyme
belongs to the class of bacterial type IIa topoisomerases, and it is responsible for the
introduction of negative supercoils during bacterial DNA replication. DNA gyrase is a
heterotetrameric protein that is composed of two GyrA subunits and two ATP-binding GyrB
subunits [7]. Many studies have already been carried out to develop clinically useful ATP-
4

competitive GyrB inhibitors; however, none are currently available for therapeutic use [8, 9].
In 2015 we discovered structurally novel GyrB inhibitors based on the 4,5,6,7-
tetrahydrobenzo[d]thiazole-2,6-diamine core (1-3, Figure 1) [10,11]. Compounds 1 and 2
showed low-nanomolar inhibition of E. coli DNA gyrase, but did not show activity against
wild-type Gram-negative strains of E. coli and P. aeruginosa, which was most likely the
consequence of cell-wall penetration issues and/or active efflux from the bacterial cells that is
mediated by their efflux pump machineries. This latter explanation was supported by the
findings that some of these compounds showed improved antibacterial activities when tested
against the E. coli JW5503 strain that has a defective efflux pump, with MICs as low as 31 μM
(e.g. 2, Figure 1) [10]. Similar observations were also made for other structural classes of GyrB
inhibitors, including benzo[d]thiazoles [12], N-phenylpyrrolamides [13] and ethyl ureas [14,
15].
Figure 1. Representative structures of previously reported DNA gyrase inhibitors with the 4,5,6,7-
tetrahydrobenzo[d]thiazole-2,6-diamine scaffold.
One strategy to overcome the problem of low bacterial cell-wall penetration is
conjugation of small molecular weight siderophore mimics to molecules that have antibacterial
properties [16, 17]. Siderophores are high-affinity iron-chelating compounds that are secreted
by microorganisms and are among the strongest known soluble iron-binding agents [18].
Bacteria use siderophores to exploit the very limited amounts of freely available iron in the host
environment during infection, and hence to survive the proliferation process [19]. Iron–
siderophore complexes are then transported across the bacterial membrane into the cytoplasm
5

via iron-uptake machineries. Attaching a siderophore or a siderophore mimic to an antibacterial
drug can therefore be used as a means to deliver antibiotics into bacteria [20, 21]. Siderophore
mimics are small fragments of natural siderophores that have the similar structural features that
are responsible for iron chelation. Commonly used siderophore mimics are shown in Figure 2,
and these include catechols, hydroxypyranones, hydroxypyridones and dihydroxypyridones.
Some antibiotic–siderophore mimic conjugates have recently been taken to clinical trials, such
as the monosulfactam BAL30072 [22] and cephalosporin cefiderocol [23] (Figure 2). In
November 2019 U.S. Food and Drug Administration approved cefiderocol for the treatment of
complicated urinary tract infections [24].
Figure 2. Structures of the commonly used siderophore mimics and the most developed antibiotic–siderophore
mimic conjugates BAL30072 and cefiderocol.
In the present study, we designed new DNA gyrase inhibitors starting from compounds
presented in Figure 1. Moreover, we attached the siderophore mimics to selected DNA gyrase
inhibitors to evaluate their impact on enzyme inhibition and antibacterial activities against
Gram-negative bacteria.
6

2. Design
New (S)-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl-1H-pyrrole-2-carboxamide derivatives were
designed using co-crystal structures that we reported previously, where the inhibitors bind to
the 43-kDa N-terminal fragment of E. coli GyrB (PDB codes: 4ZVI, 5L3J) [11, 25]. In these
structures, the pyrrole-2-carboxamide moiety is bound deep in the hydrophobic pocket and
forms hydrogen bonds with Asp73 of E. coli GyrB and a conserved water molecule. In addition,
substituents on the 2-amino group of the central (S)-4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-
diamine scaffold can form additional interactions with Arg136 and adjacent amino acid residues
of E. coli GyrB, as was predicted by molecular docking (Figure 3A) [10].
In the present designed series of DNA gyrase inhibitors and inhibitor–siderophore
mimic conjugates, two different pyrrole moieties were used to achieve favorable interactions in
the hydrophobic pocket of E. coli GyrB, namely dibromopyrrole and dichloromethylpyrrole, as
halogen atoms have been shown to be important for binding [10, 26]. Further, based on the
described orientation of these inhibitors in the GyrB ATP-binding site, the 2-amino group was
identified as a suitable position on which to attach additional substituents or siderophore mimic
moieties (Figure 3).
Based on molecular docking calculations (Figure 3A), substituents directly attached to
the 2-amino group (Figure 3B and 3C, type I) can form additional interactions with the binding
site residues. In addition, to explore the impact of siderophore mimics on DNA gyrase
inhibition, we also designed analogs that had the siderophore mimic attached via different
linkers to the 2-amino group of the central scaffold (Figure 3B and 3D, type II). Three different
siderophore mimics were used: hydroxypyranone, hydroxypyridone and catechol. Different
amino acid linkers were explored for the possibility of hydrophobic interactions of their
lipophilic side chains with hydrophobic amino-acid residues in the binding site, or cation-π
7

interactions with the Arg136 for compounds with phenylalanine and tyrosine linkers. The
length of the linkers was also examined, with this extended by one carbon atom using β-alanine.
The catechol siderophore mimic was attached via two different substitution patterns, to obtain
either 3,4-dihydroxyphenyl-based or 2,3-dihydroxyphenyl-based conjugates. To compare the
effects of the siderophore moiety on DNA gyrase inhibition, the compounds were also prepared
with free amino (11, 12, 29, 30) or acetamido (13, 14, 18-23) groups.
a
b
c
d
Figure 3. a) Molecular docking calculated binding mode of amine 11 (in cyan sticks) in the E. coli DNA gyrase
ATP-binding site (PDB entry: 4DUH, in grey). b) Design of the DNA gyrase inhibitor–siderophore mimic
conjugates, based on the binding mode of 11. c, d) Docking binding modes of compounds 17 (in orange sticks)
and 34 (in yellow sticks) in the E. coli DNA gyrase ATP-binding site (in grey). For clarity, only selected amino
acid residues are presented as sticks. Conserved water molecule is presented as a red sphere. Hydrogen bonds are
presented as black dashed lines.
8

3. Chemistry
The synthesis of the siderophore mimics was performed starting from kojic acid (Scheme 1, 4).
First, the hydroxyl group directly attached to the ring was protected with either benzyl or para-
methoxybenzyl functionality using benzyl bromide or 4-methoxybenzyl chloride under alkaline
conditions, to obtain compounds 5 and 6. Subsequent oxidation of the aliphatic hydroxyl group
with Jones reagent (chromium trioxide in sulfuric acid) yielded compounds 7 and 8. The
removal of the O-benzyl group of 7 with catalytic hydrogenation using palladium on activated
charcoal as catalyst resulted in siderophore mimic 9, and treating 8 with 25% aqueous ammonia
gave compound 10.
Scheme 1. Reagents and conditions. (a) For 5: benzyl bromide, 10 M NaOH, MeOH, 80 °C, overnight; for 6: 4-
methoxybenzyl chloride, K2CO3, DMF, 50 °C, 4 h. (b) CrO3 / H2SO4, acetone, 30 min at -5 °C, then 3 h at 20 °C.
(c) H2, Pd/C, abs. EtOH, 20 °C, 60 min. (d) 25% NH3 (aq), 80 °C, 4 h.
All of these final compounds were synthesized from the parent (S)-4,5,6,7-
tetrahydrobenzo[d]thiazol-6-yl-1H-pyrrole-2-carboxamides 11 and 12 (Scheme 2), which were
obtained according to previously reported procedures [10]. Also, acetamides 13 and 14 (Table
1) were obtained as reported previously [10]. Hydroxypyranone siderophore mimics in the form
9

of carboxylic acids and either O-benzyl protected or not (7, 9) were coupled to the 2-amino
group of 11 and 12 by 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide (EDC)/ 1-
hydroxybenzotriazole (HOBt)-promoted amide bond formation, to yield compounds 15-17. In
the same manner, compounds 18-23 were prepared using N-acetylated amino acids (Gly, L-Ala,
L-Val, L-Phe, L-Tyr) and compounds 24-28, using N-Boc-protected amino acids (Gly, L-Ala, L-
Val, β-Ala). Removal of the Boc protecting group was performed with 4 M HCl in 1,4-dioxane
or acetyl chloride in methanol, to obtain compounds 29-33, which were used in another EDC/
HOBt coupling to prepare analogs 34-43. Finally, removal of the para-methoxybenzyl
protection group from compounds 40-43 with 1 M HCl in acetic acid resulted in compounds
44-47.
10

Scheme 2. Reagents and conditions. (a) For 15-17: hydroxypyranone acid 7 or 9; for 18-23 corresponding N-
acetylated amino acid derivative; for 24-28 corresponding N-Boc-protected amino acid derivative; for 34: 3,4-
dihydroxybenzoic acid; for 35: 2,3-dihydroxybenzoic acid; for 36-43: corresponding hydroxypyranone acid 7 or
9 or hydroxypyridone acid 10; EDC, HOBt, NMM, DMF, 0 °C, then 20 °C, overnight, except 60 h for 15 and 25,
96 h for 17 and 72 h for 40; additional heating 5 h at 60 °C for 16. (b) 24, acetyl chloride, MeOH, 90 min, 0 °C;
then 20 °C, 18 h. (c) Corresponding N-Boc-protected compounds 25-28, 4 M HCl in 1,4-dioxane, 20 °C, 16 h. (d)
Corresponding 4-methoxybenzyl-protected compounds 40-43, 1 M HCl in acetic acid, 20 °C, 16 h.
11

4. Results and Discussion
4.1 In vitro enzyme inhibition
In total, 23 compounds (15-23, 34-47) were synthesized and tested in the DNA gyrase
supercoiling assay to determine their in vitro inhibitory activities against E. coli DNA gyrase.
These data are presented in Figures 4 and 5, and Tables S1 and S2 as IC50 values.
Overall, 12 of the compounds showed E. coli DNA gyrase inhibitory activities with IC50
<1 μM, with seven of these at <0.2 μM. Six of these seven were more potent inhibitors than the
positive control novobiocin (IC50, 0.17 μM): 19, 20, 21, 34, 35 and 39. For the synthesized
GyrB inhibitors bearing a siderophore mimic, six of 17 showed IC50 against E. coli DNA gyrase
<0.5 μM, with the most potent compound 34 with an IC50 of 0.058 μM (Figure 5, Table S2).
Moreover, this IC50 for 34 is comparable to that of the oxalyl acid 1 (Figure 2), which is one of
the most potent 4,5,6,7-tetrahydrobenzo[d]thiazole-based DNA gyrase inhibitors described to
date [10].
The E. coli DNA gyrase inhibition was improved by acylation of the 2-amino group of
the central scaffold (Figure 4, Table S1), as the acetamides 13 and 14 (IC50, 0.15 μM, 0.48 μM,
respectively) were more potent than their free amino counterparts 11 and 12 (IC50, >10 μM).
The same was seen for the acetamides 18 and 19 and the amines 29 and 30 with glycine as the
linker (Figure 4, Table S1). Amino acids with small side chains as linkers (Gly, L-Ala, L-Val,
β-Ala) were well tolerated (IC50, 0.10-0.52 μM for 18-21), while bulkier side chains in the
linkers (L-Phe, L-Tyr) led to a loss of activity (IC50, 7.0 μM, 2.8 μM for 22, 23, respectively).
Therefore, only DNA gyrase inhibitors with non-aromatic amino acids as linkers were
considered for the synthesis of the conjugates.
In the type I compounds (Figure 5, Table S2), the dibromopyrrole analog 16 (IC50, 0.18
μM) was significantly more potent than its dichloromethylpyrrole counterpart 17 (IC50, 2.1
12

μM). The attached hydroxypyranone siderophore mimic did not affect the binding, as starting
compound 13 (IC50, 0.15 μM) and its conjugate analog 16 had comparable IC50 values.
Furthermore, the additional benzyl group in 15 (IC50, 3.4 μM) largely reduced the affinity for
E. coli DNA gyrase, which was also observed in the type II compounds 36 and 38 (Figure 5,
Table S2, IC50, 2.0 μM, 2.1 μM, respectively). Catechol-based conjugates 34 (IC50, 0.058 μM)
and 35 (IC50, 0.11 μM) with a glycine linker were more potent E. coli DNA gyrase inhibitors
than the hydroxypyranone 37 (IC50, 0.69 μM) and the hydroxypyridinone 44 (IC50, 1.6 μM) and
had comparable activities to the hydroxypyranone conjugate 39 (IC50, 0.090 μM). Increasing
the linker length of 44 in the β-Ala analog 47 (IC50, 0.35 μM) improved the DNA gyrase
inhibition. Moreover, introduction of a methyl (45; IC50, 0.26 μM) or i-Pr (46; IC50, 0.64 μM)
group into the linker also increased the DNA gyrase inhibitory activities.
Figure 4. Enzyme inhibition data of the (S)-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl-1H-pyrrole-2-carboxamide-
based compounds against Escherichia coli DNA gyrase.
E. coli DNA Gyrase inhibition
20.0
12.5
5.0
5.0
3.0
1.0
1.0
0.5
0.0
1
3
4
8
9
0
1
2
3
9
0
3
B
1
1
1
1
1
2
2
2
2
2
N
Compounds
13

Figure 5. Enzyme inhibition data of DNA gyrase inhibitor – siderophore mimic conjugates against Escherichia
coli DNA gyrase.
E. coli DNA Gyrase inhibition
4.5
2.5
0.5
0.4
0.2
0.0
5
6
7
4
5
6
7
8
9
4
5
6
7
4
B
1
1
1
3
3
3
3
3
3
4
4
4
N
Compounds
4.2 Antibacterial activities
All of these compounds were evaluated for their in vitro antibacterial activities against two
Gram-positive (Enterococcus faecalis ATCC 29212, S. aureus ATCC 25923 or 29213) and two
Gram-negative (E. coli ATCC 25922, P. aeruginosa ATCC 27853) bacterial strains. In
addition, they were tested against two E. coli mutant strains: JD17464, as an lpxC deletion
mutant with an impaired outer membrane; and JW5503, as a tolC deletion mutant with a
defective efflux pump. Preliminary tests were performed at 50 µM, and growth inhibition was
measured after 24 h incubation. These data are presented in Figures S1 and S2 in the
Supplementary Information. MICs were determined in dose-response assays for compounds
that showed >90% growth in the preliminary assay (Table S3) [27]. The compounds containing
the siderophore mimics were evaluated also against E. coli (ATCC 25922), P. aeruginosa
(ATCC 27853) and A. baumannii (ATCC 19606) in cation-adjusted Mueller-Hinton broth
(CAMHB) as iron-depleted (ID-CAMHB) or when supplemented with iron, to determine the
effects of the attached siderophore mimics on the antibacterial activities in iron depleted
conditions. These data are presented in Table S4 in the Supplementary Information as
14

percentages of bacterial growth inhibition at 50 µM of the tested compounds and in Figure 6.
Data of growth inhibition for non-conjugated compounds 13, 14, 18, 20, 21 and 29 against E.
coli ATCC 25922, in CAMHB and ID-CAMHB, are also shown in Table S4. Since there is no
difference in activity, any possible direct effect due to different culture medium composition
can be excluded.
These antibacterial activity assays revealed activity of compounds 30 and 35 against the
Gram-positive bacterial strain of E. faecalis with MICs of 11 µg/mL and 14 µg/mL,
respectively. On the other hand, tested compounds were generally inactive against the wild-
type Gram-negative bacterial strains in iron-supplemented medium. However, weak
correlations between in vitro inhibitory activities and antibacterial properties have also been
reported previously for other structural classes of DNA gyrase B inhibitors [8]. In addition, the
inhibitory activities here in the iron-depleted medium were generally also weak against all of
the bacterial strains tested, with only two compounds (45, 46) showing >50% growth inhibition
against A. baumannii (Figure 6). For compounds 35, 44, 45 and 47 we observed an opposite
effect on bacterial growth of P. aeruginosa, since they showed a drop in growth inhibition in
iron-supplemented compared to iron-depleted medium. Several groups have investigated the
effect of introducing siderophores mimics into the structure of fluoroquinolones, well-known
DNA gyrase inhibitors, but the activities of most of these conjugates were also not greater
compared to those of the parent antibiotics, or in many cases activities were even lost [28].
However, increases in the inhibitory effects were seen for iron-depleted versus iron-
supplemented media for almost all of the compounds tested against wild-type E. coli,
presumably as a consequence of the attached siderophore mimics. Moreover, inhibition against
the ΔtolC E. coli JW5503 strain was seen (Supplementary Figures S1, S2 and Table S3). For
this E. coli mutant with a defective efflux pump, the most potent bacterial growth inhibition
was for compound 19 with an MIC of 1 µg/mL. Among the siderophore mimic conjugates, the
15

most promising activities were shown by the catechol derivatives 34 and 35, with MICs of 14
µg/mL against the ΔtolC E. coli JW5503 strain, which is more effective than for the best
compounds from the first generation 4,5,6,7-tetrahydrobenzo[d]thiazoles [10]. In contrast, none
of the tested compounds showed significantly improved antibacterial activity against ΔlpxC E.
coli JD17464 compared to wild-type strain. These data suggest that efflux problems, but not
cell wall penetration, remain the issue in terms of the weak antibacterial activities of the 4,5,6,7-
tetrahydrobenzo[d]thiazole derivatives.
Figure 6. Antibacterial activity of DNA gyrase B inhibitor–siderophore mimic conjugates against E. coli, P.
aeruginosa and A. baumannii in iron-depleted (ID-CAMHB) and iron-supplemented CAMHB media at 50 μM
concentration. Cefiderocol MICs in CAMHB were 0.25, 1.0, and 2.0 µg/mL, and in ID-CAMHB 0.25, 0.5 and
0.25 µg/ml against E. coli, P. aeruginosa and A. baumannii, respectively (complying with approved CLSI values)
[29]. Ceftazidime was used as a negative control and MICs were 0.25, 2, and > 4 µg/mL both in CAMHB and ID-
CAMHB against E. coli, P. aeruginosa and A. baumannii, respectively.
Recent studies revealed that not only siderophore-mimic moieties, but also other groups
present in the molecule could contribute to iron chelation [30, 31]. For example, BAL30072
(Figure 1) has been found to form compact complex in solution comprising two BAL30072
molecules and one Fe3+ ion. In this complex sulfate group of BAL30072 participates as a
chelating functionality in addition to the dihydroxypyridone moiety [30]. In another study with
(pre)acinetobactin, a siderophore secreted by A. baumannii, the same ligand to metal
stoichiometry in the solution was found to be favorable in low iron conditions and such complex
16

is readily recognized and transported by the outer membrane (OM) transporter into the
periplasm of A. baumannii. Interestingly, only one oxygen from a catechol moiety of
preacinetobactin participates in complex formation, together with three other atoms in the
scaffold [31]. Thus, more focused investigation of the interactions of our DNA gyrase inhibitor–
siderophore mimic conjugates with Fe3+ is required in the future to completely elucidate
stoichiometry of these complexes as well as to identify functional groups responsible for iron
chelation. Moreover, since the interactions with the OM transporters also play a key role in
circumventing poor permeation in Gram-negative bacteria, further studies on affinity and
binding mode of our conjugates in OM transporters are needed, such as recently published study
on enterobactin-iron complex with P. aeruginosa OM transporter PfeA [32]. Inappropriate
shape and size of our conjugates and consequentially their poor affinity toward OM transporters
might be a reason for only slight improvements in antibacterial activities in iron-depleted versus
iron-supplemented media against tested wild-type Gram-negative bacterial strains. Moreover,
the solving of the efflux problems should be considered as equally important to achieve
improved antibacterial activities against Gram-negative bacterial strains.
5. Conclusion
These new 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives conjugated with three types of
siderophore mimics were designed, synthesized and evaluated in biological assays. In vitro
enzyme inhibition of E. coli DNA gyrase revealed that compounds 34 and 35, which include a
catechol siderophore mimic moiety, are more potent inhibitors than the positive control
novobiocin, with IC50 in the low nanomolar range (<0.2 µM). The most potent among all of the
compounds tested was 34, with IC50 of 0.058 μM, which is comparable to the best 4,5,6,7-
tetrahydrobenzo[d]thiazole-based DNA gyrase inhibitors described to date [10]. This shows
17

that it is possible to design potent DNA gyrase inhibitor–siderophore mimic conjugates with
non-cleavable linkers. In vitro antibacterial assays showed no significant activities against wild-
type Gram-negative bacterial strains. For the efflux pump mutant strain, both of these potent
catechol mimic conjugates 34 and 35 showed a MIC of 14 µg/mL against E. coli JW5503,
demonstrating that active efflux together with permeability remains the issue for these
compounds. Both 34 and 35 also showed minor improvements in their antibacterial activities
against wild-type E. coli in iron-depleted medium. Therefore, the use of catechols as
siderophore mimics appears to be a promising approach for further investigations into DNA
gyrase inhibitor–siderophore mimic conjugates with sufficient antibacterial activity against
wild-type Gram-negative bacterial strains.
6. Experimental Section
6.1 Materials and Methods
The chemicals were obtained from Acros Organics (Geel, Belgium), Sigma-Aldrich (St. Louis,
MO, USA), TCI Europe N.V. (Zwijndrecht, Belgium) and Apollo Scientific (Stockport, UK),
and were used without further purification. Analytical TLC was performed on silica gel Merck
60 F254 plates (0.25 mm), with 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: (i) an Agilent Technologies 1100 instrument with a UV-VIS detector
(G1365B), a thermostat (G1316A), an autosampler (G1313A) and a C18 column (Eclipse Plus;
5 µm, 4.6 × 150 mm; Agilent); (ii) a Thermo Scientific Dionex Ultimate 3000 Binary Rapid
Separation LC System (Thermo Fisher Scientific, Waltham, MA, USA) with an autosampler, a
binary pump system, a photodiode array detector, a thermostated column compartment and a
C18 column (Zorbax Extend; 3.5 µm, 4.6 × 150 mm; Agilent). The following gradient elution
18

was used with mobile phases A (0.1% trifluoroacetic acid in water) and B (acetonitrile): 0-16
min, 95%-5% A; 16-21 min, 5% A. The flow rate was 1.0 mL/min, and the injection volume
was 20 μL. All of the tested compounds were ≥95% pure by HPLC. Melting points were
determined on a Reichert hot-stage microscope, and are uncorrected. 1H and 13C NMR spectra
were recorded at 400 MHz and 100 MHz, respectively, on a spectrometer (AVANCE III 400;
Bruker Corporation, Billerica, MA, USA) in DMSO-d6 or CDCl3 solutions, with TMS as the
internal standard. Mass spectra were obtained using Advion expression CMS mass
spectrometer (Advion Inc., Ithaca, USA) and high resolution mass spectra were obtained using
a Q-TOF Premier mass spectrometer (Micromass, Waters, Manchester, UK) or an Exactive
Plus Orbitrap mass spectrometer (Thermo Fischer Scientific Inc., Waltham, MA, USA). Optical
rotations were measured on a polarimeter (241 MC; Perkin-Elmer). The reported values for
specific rotation were the means of five successive measurements, using an integration time of
5 s.
6.2 Synthesis
6.2.1 Synthesis of siderophore mimics
Siderophore mimics were synthesized according to the reported procedures [33, 34].
6.2.1.1 General procedure A. Chromium trioxide (2.8 mmol) was suspended in water (1 mL)
and concentrated sulfuric acid (0.3 mL) was added dropwise on an ice bath. The mixture was
quantitatively introduced into a solution of corresponding alcohol (1 mmol), which was
previously dissolved in acetone (10 mL) with additional heating and then cooled on an ice bath.
Reaction mixture was stirred for 30 min at 0 °C and then for 3 h at room temperature. The
precipitate was filtered off through a layer of Celite and mother liquid was evaporated under
reduced pressure. Water (2 mL) was added to the residue and precipitate formed was filtered
off and dried at 40 °C.
19

6.2.1.1.1 5-(Benzyloxy)-2-(hydroxymethyl)-4H-pyran-4-one (5). Kojic acid (10.0 g, 70.4 mmol)
was suspended in methanol (80 mL) and 10 M NaOH (7.74 mL, 77.4 mmol) was added. Then
benzyl bromide (9.61 mL, 80.9 mmol) was added dropwise and reaction mixture was stirred at
80 °C overnight. Reaction mixture was allowed to cool to room temperature and solvent was
evaporated under reduced pressure. Acetone (12 mL) and water (120 mL) were added to the
crude residue and precipitate was formed, which was filtered off and dried at 40 °C. Yield
16.845 g (99.0 %); yellow solid; m. p. 123-125 °C; 1H NMR (400 MHz, CDCl3) δ 4.48 (s, 2H,
CH2), 5.08 (s, 2H, CH2), 6.57 (s, 1H, pyran-H), 7.31-7.42 (m, 5H, Ar-H), 7.56 (s, 1H, pyran-H)
ppm, signal for OH not seen in the spectrum.
6.2.1.1.2 2-(Hydroxymethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one (6). Kojic acid (5.0 g,
35.2 mmol) and potassium carbonate (10.7 g, 77.4 mmol) were suspended in dry N,N-
dimethylformamide (DMF) (80 mL). The round-bottom flask was filled with argon and 4-
methoxybenzyl chloride (5.23 mL, 38.7 mmol) was added dropwise. Reaction mixture was
stirred at 50 °C for 4 h. Then DMF was evaporated under reduced pressure and water (100 mL)
was added. A precipitate was formed, which was filtered off and dried at 60 °C. Yield 8.145 g
(88 %); yellow solid; m. p. 114-116 °C; 1H NMR (400 MHz, DMSO-d6) δ 3.77 (s, 3H, CH3),
4.30 (d, 2H, J = 4.0 Hz, CH2OH), 4.86 (s, 2H, CH2), 5.75 (br s, 1H, OH), 6.32 (t, 1H, J = 0.9
Hz, pyran-H), 6.92-6.99 (m, 2H, Ar-H), 7.31-7.39 (m, 2H, Ar-H), 8.16 (s, 1H, pyran-H) ppm;
HRMS ESI+ m/z for C14H15O5 ([M+H]+): calcd 263.0919, found 263.0921.
6.2.1.1.3 5-(Benzyloxy)-4-oxo-4H-pyran-2-carboxylic acid (7). Prepared from 5 (5.0 g, 21.5
mmol) and chromium trioxide (6.02 g, 60.2 mmol) according to the general procedure A. Yield
1
3.437 g (65 %); yellow solid; m. p. 162 - 164 °C; H NMR (400 MHz, DMSO-d6) δ 4.99 (s,
2H, CH2), 6.93 (s, 1H, pyran-H), 7.34-7.47 (m, 5H, Ar-H), 8.37 (s, 1H, pyran-H) ppm, signal
for COOH not seen in the spectrum; HRMS ESI- m/z for C13H9O5 ([M-H]-): calcd 245.0450,
found 245.0451.
20

6.2.1.1.4 5-((4-Methoxybenzyl)oxy)-4-oxo-4H-pyran-2-carboxylic acid (8). Prepared from 6
(2.05 g, 7.82 mmol) and chromium trioxide (2.19 g, 21.9 mmol) according to the general
procedure A. Precipitate was additionally washed with hot MeOH (2 × 30 mL). Yield 0.980 g
(45 %); yellow solid; m. p. 165 - 167 °C; 1H NMR (400 MHz, DMSO-d6) δ 3.77 (s, 3H, CH3),
4.90 (s, 2H, CH2), 6.93 (s, 1H, pyran-H); 6.94-7.00 (m, 2H, Ar-H), 7.34-7.40 (m, 2H, Ar-H),
8.36 (s, 1H, pyran-H) ppm; HRMS ESI- m/z for C14H11O6 ([M-H]-): calcd 275. 0556, found
275.0562.
6.2.1.1.5 5-Hydroxy-4-oxo-4H-pyran-2-carboxylic acid (9). Compound 7 (0.400 g, 1.22 mmol)
was dissolved in dry ethanol (80 mL) and put under argon. Then 5 % m/m of Pd/C (0.020 g)
was added and reaction mixture was stirred for 1 h at room temperature under hydrogen
atmosphere. Then Pd/C was filtered off and the solvent evaporated under reduced pressure.
1
Yield 0.236 g (93 %); yellow amorphous powder; H NMR (400 MHz, DMSO-d6) δ 6.96 (s,
1H, pyran-H); 8.21 (s, 1H, pyran-H), 9.67 (br s, 1H, OH) ppm; HRMS ESI- m/z for C6H3O5
([M-H]-): calcd 154.9986, found 154.9981.
6.2.1.1.6 5-((4-Methoxybenzyl)oxy)-4-oxo-1,4-dihydropyridine-2-carboxylic acid (10).
Compound 8 (0.390 g, 1.41 mmol) was dissolved in 25% aqueous ammonia (10 mL) and
refluxed for 4 h at 80 °C. Then reaction mixture was concentrated to about 5 mL and slowly
acidified with concentrated hydrochloric acid to pH = 2. Precipitate formed was filtered off and
dried under reduced pressure (co-evaporation with 2 × 5 mL diethyl ether). Yield 0.325 g (84
%); white amorphous solid; 1H NMR (400 MHz, DMSO-d6) δ 3.76 (s, 3H, CH3), 5.12 (s, 2H,
CH2), 6.96 (m, 2H, Ar-H), 7.24 (br s, 1H, pyridine-H), 7.39 (m, 2H, Ar-H), 7.93 (br s, 1H,
pyridine-H) ppm, signals for NH and COOH not seen in the spectrum; HRMS ESI- m/z for
C14H12NO5 ([M-H]-): calcd 274.0715, found 274.0723.
6.2.2 Synthesis of compounds
21

6.2.2.1 General procedure B. A solution of carboxylic acid (1 mmol) in N,N-
dimethylformamide (10 mL) was cooled to 0 °C and then EDC (1.2 mmol) and HOBt (1.3
mmol) were added. pH was adjusted to 8 with N-methylmorpholine and the reaction mixture
stirred for 20 min at 0 °C. Then amine (1 mmol) was added and reaction mixture 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 (2 × 30 mL), saturated
aqueous NaHCO3 solution (2 × 30mL), and brine (30 mL). The organic phase was dried over
Na2SO4, filtered and solvent evaporated under reduced pressure.
6.2.2.1.1
(S)-N-(2-(5-(Benzyloxy)-4-oxo-4H-pyran-2-carboxamido)-4,5,6,7-
tetrahydrobenzo[d]thiazol-6-yl)-4,5-dibromo-1H-pyrrole-2-carboxamide (15). Prepared from
11 (0.300 g, 0.714 mmol) and 7 (0.176 g, 0.714 mmol) according to the general procedure B.
Product was purified by flash column chromatography using dichloromethane/methanol (15:1)
as eluent. Yield 0.094 g (20 %); yellow solid; m. p. 168 - 170 °C; [α]D +32.8 (c 0.19, DMF); 1H
NMR (400 MHz, DMSO-d6) δ 1.83-1.94 (m, 1H, HA-7), 1.95-2.08 (m, 1H, HB-7), 2.57-2.66
(m, 1H, HA-5), 2.70-2.78 (m, 2H, HB-5, HA-4), 2.94-3.06 (m, 1H, HB-4), 4.16-4.31 (m, 1H,
CHNH), 5.02 (s, 2H, OCH2), 7.01 (d, 1H, J = 2.6 Hz, pyrrole-H), 7.10 (s, 1H, pyran-H), 7.35-
7.49 (m, 5H, Ar-H), 8.12 (d, 1H, J = 7.8 Hz, CHNH), 8.33 (s, 1H, pyran-H), 12.72 (d, 1H, J =
2,5 Hz, pyrrole-NH) ppm; 13C NMR (100 MHz, DMSO-d6) δ 27.8, 70.1, 97.2, 104.0, 112.4,
114.7, 127.4, 127.7, 127.9, 135.3, 140.3, 147.5, 157.8 ppm; HRMS ESI- m/z for
C25H19Br2N4O5S ([M-H]-): calcd 644.9443, found 644.9439; HPLC: tr 13.15 min (95 % at 254
nm).
6.2.2.1.2
(S)-4,5-Dibromo-N-(2-(5-hydroxy-4-oxo-4H-pyran-2-carboxamido)-4,5,6,7-
tetrahydrobenzo[d]thiazol-6-yl)-1H-pyrrole-2-carboxamide (16). Prepared from 11 (0.300 g,
0.714 mmol) and 9 (0.111 g, 0.714 mmol) according to the general procedure B. After addition
of 10 % citric acid and ethyl acetate, precipitate was formed, which was filtered off and dried.
22

Crude residue was suspended in a small volume of ethyl acetate, heated until boiling and filtered
off. Product was dried in vacuo. Yield 0.044 g (11 %); brown amorphous solid; [α]D +41.5 (c
0.20, DMF); 1H NMR (400 MHz, DMSO-d6) δ 1.81-1.96 (m, 1H, HA-7), 1.96-2.07 (m, 1H, HB-
7), 2.60-2.80 (m, 3H, H-5, HA-4), 3.00 (m, 1H, HB-4), 4.16-4.29 (m, 1H, CHNH), 7.01 (d, 1H,
J = 2.4 Hz, pyrrole-H), 7.12 (s, 1H, pyran-H), 8.12 (d, 1H, J = 7.6 Hz, CHNH), 8.18 (s, 1H,
pyran-H), 9.62 (s, 1H, OH), 12.72 (d, 1H, J = 2.4 Hz, pyrrole-NH) ppm; 13C NMR (100 MHz,
DMSO-d6) δ 27.9, 28.4, 44.9, 97.8, 104.6, 113.0, 114.1, 128.0, 139.7, 147.5, 158.4, 173.7 ppm;
HRMS ESI- m/z for C18H13Br2N4O5S ([M-H]-): calcd 554.8968, found 554.8984; HPLC: tr
10.77 min (95 % at 254 nm).
6.2.2.1.3
(S)-3,4-Dichloro-N-(2-(5-hydroxy-4-oxo-4H-pyran-2-carboxamido)-4,5,6,7-
tetrahydrobenzo[d]thiazol-6-yl)-5-methyl-1H-pyrrole-2-carboxamide (17). Prepared from 12
(0.126 g, 0.365 mmol) and 9 (0.057 g, 0.365 mmol) according to the general procedure B. After
addition of 10% citric acid, precipitate was formed, which was filtered off and washed with
ethyl acetate (3 × 10 mL) and hot methanol (3 × 1 mL). Yield 0.030 g (17 %); brown amorphous
solid; [α]D −17.7 (c 0.19, DMSO); 1H NMR (400 MHz, DMSO-d6) δ 1.90-2.11 (m, 2H, H-7),
2.19 (s, 3H, CH3), 2.65-2.84 (m, 3H, H-5, HA-4), 3.02 (d, 1H, J1 = 14.1 Hz, HB-4), 4.20-4.35
(m, 1H, CHNH), 7.12 (s, 1H, pyran-H), 7.39 (d, 1H, J = 7.9 Hz, CHNH), 8.18 (s, 1H, pyran-
H), 9.63 (s, 1H, OH), 12.02 (s, 1H, pyrrole-NH) ppm; 13C NMR (100 MHz, DMSO-d6) δ 10.7,
27.5, 28.3, 44.9, 108.0, 110.0, 114.1, 119.2, 127.3, 139.7, 147.5, 158.3, 173.7 ppm; HRMS ESI+
m/z for C19H17N4O5SCl2 ([M+H]+): calcd 483.0291, found 483.0282; HPLC: tr 11.42 min (95
% at 254 nm).
6.2.2.1.4
(S)-N-(2-(2-Acetamidoacetamido)-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)-4,5-
dibromo-1H-pyrrole-2-carboxamide (18). Prepared from 11 (0.200 g, 0.476 mmol) and N-
acetylglycine (0.058 g, 0.476 mmol) according to the general procedure B. Product was purified
by flash column chromatography using ethyl acetate to ethyl acetate/methanol (5:2) as eluent.
23

Yield 0.086 g (35 %); yellow solid; m. p. 196-198 °C; [α]D −7.4 (c 0.30, MeOH); 1H NMR (400
MHz, DMSO-d6) δ 1.83-1.91 (m, 4H, HA-7, CH3), 1.96-2.03 (m, 1H, HB-7), 2.58-2.77 (m, 3H,
H-5, HA-4), 2.99 (dd, 1H, J1 = 15.5 Hz, J2 = 5.2 Hz, HB-4), 3.94 (d, 2H, J = 5.8 Hz, CH2NH),
4.13-4.24 (m, 1H, CHNH), 7.01 (d, 1H, J = 2.8 Hz, pyrrole-H), 8.11 (d, 1H, J = 7.7 Hz, CHNH),
8.26 (t, 1H, J = 5.9 Hz, CH2NH), 11.98 (s, 1H, NHCO), 12.71 (d, 1H, J = 2.6 Hz, pyrrole-NH)
ppm; 13C NMR (100 MHz, DMSO-d6) δ 22.3, 24.6, 28.4, 28.6, 41.9, 45.3, 97.8, 104.5, 112.9,
119.3, 128.0, 143.5, 155.3, 158.4, 167.9, 169.8 ppm; HRMS ESI- m/z for C16H16Br2N5O3S ([M-
H]-): calcd 515.9341, found 515.9346; HPLC: tr 11.48 min (99 % at 254 nm).
6.2.2.1.5
(S)-N-(2-(2-Acetamidoacetamido)-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)-3,4-
dichloro-5-methyl-1H-pyrrole-2-carboxamide (19). Prepared from 12 (0.150 g, 0.435 mmol)
and N-acetylglycine (0.051 g, 0.435 mmol) according to the general procedure B. Crude product
was crystalized from methanol. Yield 0.078 g (40 %); yellow solid; m. p. 168 – 171 °C; [α]D
−9.6 (c 0.13, DMF); 1H NMR (400 MHz, DMSO-d6) δ 1.88 (s, 3H, CH3), 1.91-2.07 (m, 2H, H-
7), 2.19 (s, 3H, pyrrole-CH3), 2.65-2.78 (m, 3H, H-5, HA-4), 3.01 (m, 1H, HB-4), 3.94 (d, 2H,
J = 5.8 Hz, CH2NH), 4.20-4.31 (m, 1H, CHNH), 7.32 (d, 1H, J = 7.8 Hz, CHNH), 8.27 (t, 1H,
J = 5.8 Hz, CH2NH), 11.99 (s, 1H, NHCO), 12.02 (s, 1H, pyrrole-NH) ppm; 13C NMR (100
MHz, DMSO-d6) δ 11.1, 22.8, 24.7, 28.5, 28.8, 42.4, 45.6, 108.5, 110.5, 112.6, 119.7, 127.8,
143.8, 155.8, 158.7, 168.4, 170.3 ppm; HRMS ESI- m/z for C17H18N5O3SCl2 ([M-H]-): calcd
442.0507, found 442.0496; HPLC: tr 10.55 min (97 % at 254 nm).
6.2.2.1.6 N-((S)-2-((S)-2-Acetamidopropanamido)-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)-
4,5-dibromo-1H-pyrrole-2-carboxamide (20). Prepared from 11 (0.160 g, 0.381 mmol) and N-
acetyl-L-alanine (0.050 g, 0.381 mmol) according to the general procedure B. Product was
purified by flash column chromatography using ethyl acetate to ethyl acetate/methanol (5:2) as
1
eluent. Yield 0.081 g (40 %); yellow solid; m. p. 160-162 °C; [α]D +10.3 (c 0.18, DMF); H
NMR (400 MHz, DMSO-d6) δ 1.27 (dd, 3H, J1 = 7.2 Hz, J2 = 1.6 Hz, CHCH3), 1.81-1.90 (m,
24

4H, HA-7, COCH3), 1.94-2.03 (m, 1H, HB-7), 2.59-2.75 (m, 3H, H-5, HA-4), 2.98 (m, 1H, HB-
4), 4.14-4.24 (m, 1H, CHNH), 4.40-4.48 (m, 1H, CHCH3), 7.00 (d, 1H, J = 2.5 Hz, pyrrole-H),
8.09 (t, 1H, J = 7.2 Hz, CHNH), 8.25 (d, 1H, J = 6.8 Hz, CHNH), 11.98 (d, 1H, J = 1.9 Hz,
pyrrole-NH), 12.70 (s, 1H, NHCO) ppm; 13C NMR (100 MHz, DMSO-d6) δ 18.1, 22.8, 25.1,
28.8, 29.1, 45.7, 48.8, 98.3, 105.0, 113.4, 119.9, 128.6, 144.0, 155.9, 158.9, 169.8, 171.9 ppm;
HRMS ESI- m/z for C17H18Br2N5O3S ([M-H]-): calcd 529.9497, found 529.9496; HPLC: tr
11.77 min (97.6 % at 254 nm).
6.2.2.1.7 N-((S)-2-((S)-2-Acetamido-3-methylbutanamido)-4,5,6,7-tetrahydrobenzo[d]thiazol-
6-yl)-4,5-dibromo-1H-pyrrole-2-carboxamide (21). Prepared from 11 (0.180 g, 0.428 mmol)
and N-acetyl-L-valine (0.068 g, 0.428 mmol) according to the general procedure B. Product
was purified by flash column chromatography using dichloromethane/methanol (20:1) as
eluent. Yield 0.047 g (20 %); white solid; m. p. 180-182 °C; 1H NMR (400 MHz, DMSO-d6) δ
0.88 (t, 6H, J = 7.5 Hz, 2 × CH3), 1.80-1.92 (m, 4H, HA-7, COCH3), 1.94-2.06 (m, 2H, HB-7,
CH(CH3)2), 2.59-2.74 (m, 3H, H-5, HA-4), 2.98 (dd, 1H, J1 = 14.4 Hz, J2 = 5.4 Hz, HB-4), 4.16-
4.23 (m, 1H, CHNH), 4.32-4.39 (m, 1H, CHCH), 7.00 (d, 1H, J = 2.5 Hz, pyrrole-H), 8.08-8.13
(m, 2H, 2 × CHNH), 12.04 (s, 1H, NHCO), 12.70 (d, 1H, J = 2.6 Hz, pyrrole-NH) ppm; HRMS
ESI- m/z for C19H22Br2N5O3S ([M-H]-): calcd 557.9810, found 557.9823; HPLC: tr 12.38 min
(100 % at 254 nm).
6.2.2.1.8
N-((S)-2-((S)-2-Acetamido-3-phenylpropanamido)-4,5,6,7-
tetrahydrobenzo[d]thiazol-6-yl)-4,5-dibromo-1H-pyrrole-2-carboxamide (22). Prepared from
11 (0.180 g, 0.428 mmol) and N-acetyl-L-phenylalanine (0.089 g, 0.428 mmol) according to
the general procedure B. Product was purified by flash column chromatography using
dichloromethane/methanol (20:1) as eluent. Yield 0.023 g (9 %); white solid; m. p. 143-145 °C;
[α]D +10.3 (c 0.18, DMF); 1H NMR (400 MHz, DMSO-d6) δ 1.77-1.91 (m, 4H, HA-7, COCH3),
1.95-2.04 (m, 1H, HB-7), 2.60-2.74 (m, 3H, H-5, HA-4), 2.84 (dd, 1H, J1 = 13.1 Hz, J2 = 3.2
25

Hz, HB-4), 2.95-3.06 (m, 2H, PhCH2), 4.14-4.24 (m, 1H, CHNH), 4.68-4.74 (m, 1H, CHCH2),
7.01 (d, 1H, J = 2.6 Hz, pyrrole-H), 7.18-7.31 (m, 5H, Ar-H), 8.07-8.13 (m, 1H, CHNH), 8.33
(d, 1H, J = 7.8 Hz, CHNH), 12.18 (s, 1H, NHCO), 12.71 (d, 1H, J = 1.9 Hz, pyrrole-NH) ppm;
HRMS ESI+ m/z for C23H24Br2N5O3S ([M+H]+): calcd 607.9961, found 607.9959; HPLC: tr
13.08 min (100 % at 254 nm).
6.2.2.1.9
N-((S)-2-((S)-2-Acetamido-3-(4-hydroxyphenyl)propanamido)-4,5,6,7-
tetrahydrobenzo[d]thiazol-6-yl)-4,5-dibromo-1H-pyrrole-2-carboxamide (23). Prepared from
11 (0.100 g, 0.238 mmol) and N-acetyl-L-tyrosine (0.053 g, 0.238 mmol) according to the
general procedure B. Product was purified by flash column chromatography using
dichloromethane/methanol (20:1) as eluent. Yield 0.037 g (25 %); white solid; m. p. 188-190
1
°C; [α]D +13.2 (c 0.08, DMF); H NMR (400 MHz, DMSO-d6) δ 1.80 (s, 3H, COCH3), 1.83-
1.91 (m, 1H, HA-7), 1.95-2.04 (m, 1H, HB-7), 2.59-2.74 (m, 4H, H-5, HA-4, PhCH2-HA), 2.87-
2.92 (m, 1H, PhCH2-HB), 2.99 (dd, 1H, J1 = 15.8 Hz, J2 = 5.1 Hz, HB-4), 4.13-4.24 (m, 1H,
CHNH), 4.57-4.67 (m, 1H, CHCH2), 6.64 (d, 2H, J = 8.5 Hz, Ar-H), 7.01 (dd, 1H, J1 = 2.6 Hz,
J2 = 1.1 Hz, pyrrole-H), 7.07 (d, 2H, J = 8.5 Hz, Ar-H), 8.10 (dd, 1H, J1 = 7.4 Hz, J2 = 2.1 Hz,
CHNH), 8.25 (d, 1H, J = 7.8 Hz, CHNH), 9.21 (d, 1H, J = 1.1 Hz, OH), 12.11 (s, 1H, NHCO),
12.70 (d, 1H, J = 2.7 Hz, pyrrole-NH) ppm; 13C NMR (100 MHz, DMSO-d6) δ 22.8, 25.1, 28.9,
37.1, 45.7, 55.0, 98.3, 105.0, 113.4, 115.4, 119.9, 127.8, 128.5, 130.6, 144.0, 155.7, 156.4,
158.9, 169.9, 170.9 ppm; HRMS ESI- m/z for C23H22Br2N5O4S ([M-H]-): calcd 621.9759, found
621.9741; HPLC: tr 12.19 min (97.6 % at 254 nm).
6.2.2.1.10
tert-Butyl
(S)-(2-((6-(4,5-dibromo-1H-pyrrole-2-carboxamido)-4,5,6,7-
tetrahydrobenzo[d]thiazol-2-yl)amino)-2-oxoethyl)carbamate (24). Prepared from 11 (0.918 g,
2.19 mmol) and N-Boc-glycine (0.383 g, 2.19 mmol) according to the general procedure B.
Product was purified by flash column chromatography using dichloromethane/methanol (20:1)
as eluent. Yield 0.672 g (53 %); yellow solid; m. p. 156-158 °C; [α]D −2.96 (c 0.15, MeOH);
26

1H NMR (400 MHz, DMSO-d6) δ 1.35 (s, 9H, 3 × CH3), 1.79-1.93 (m, 1H, HA-7), 1.95-2.04
(m, 1H, HB-7), 2.57-2.76 (m, 3H, H-5, HA-4), 2.98 (dd, 1H, J1 = 15.6 Hz, J2 = 4.9 Hz, HB-4),
3.81 (d, 2H, J = 6.2 Hz, CH2NH), 4.12-4.26 (m, 1H, CHNH), 7.00 (d, 1H, J = 1.9 Hz, pyrrole-
H), 7.14 (t, 1H, J = 6.1 Hz, CH2NH), 8.10 (d, 1H, J = 7.7 Hz, CHNH), 11.94 (s, 1H, NHCO),
12.70 (s, 1H, pyrrole-NH) ppm; 13C NMR (100 MHz, DMSO-d6) δ 24.6, 28.1, 28.4, 28.6, 42.9,
45.2, 78.2, 97.8, 104.5, 112.9, 119.2, 128.0, 143.5, 155.3, 155.8, 158.4, 168.2 ppm; HRMS ESI-
m/z for C19H22Br2N5O4S ([M-H]-): calcd 573.9759, found 573.9752; HPLC: tr 13.23 min (96 %
at 254 nm).
6.2.2.1.11 tert-Butyl (S)-(2-((6-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4,5,6,7-
tetrahydrobenzo[d]thiazol-2-yl)amino)-2-oxoethyl)carbamate (25). Prepared from 12 (0.220 g,
0.637 mmol) and N-Boc-glycine (0.112 g, 0.637 mmol) according to the general procedure B.
Yield 0.128 g (40 %); yellow amorphous solid; [α]D +3.3 (c 0.43, DMF); 1H NMR (400 MHz,
DMSO-d6) δ 1.30 and 1.39 (2 × s, 9H, 3 × CH3), 1.89-2.07 (m, 2H, H-7), 2.18 (s, 3H, pyrrole-
CH3), 2.66-2.77 (m, 3H, H-5, HA-4), 3.02 (dd, 1H, J1 = 15.9 Hz, J2 = 5.0 Hz, HB-4), 3.81 (d,
2H, J = 6.1 Hz, CH2NH), 4.21-4.32 (m, 1H, CHNH), 7.14 (t, 1H, J = 6.1 Hz, CH2NH), 7.32 (d,
1H, J = 7.8 Hz, CHNH), 11.95 (s, 1H, NHCO), 12.02 (s, 1H, pyrrole-NH) ppm; 13C NMR (100
MHz, DMSO-d6) δ 10.6, 24.1, 27.9, 28.1, 28.2, 42.9, 45.1, 78.1, 107.9, 109.8, 119.0, 119.1,
127.3, 143.3, 155.3, 155.8, 158.1, 168.1 ppm; HRMS ESI+ m/z for C20H26N5O4SCl2 ([M+H]+):
calcd 502.1077, found 502.1074; HPLC: tr 11.09 min (97.7 % at 254 nm).
6.2.2.1.12
tert-Butyl
((S)-1-(((S)-6-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-
4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)amino)-1-oxopropan-2-yl)carbamate (26). Prepared
from 12 (0.301 g, 0.872 mmol) and N-Boc-L-alanine (0.165 g, 0.872 mmol) according to the
general procedure B. Product was purified by flash column chromatography using
dichloromethane/methanol (20:1) as eluent. Yield 0.216 g (48 %); brown solid; m. p. 142-144
°C; [α]D −23.5 (c 0.19, DMF); 1H NMR (400 MHz, DMSO-d6) δ 1.18-1.30 (m, 4H, CH3), 1.38
27

(s, 8H, CH3), 1.89-2.07 (m, 2H, H-7), 2.19 (s, 3H, pyrrole-CH3), 2.66-2.77 (m, 3H, H-5, HA-4),
3.01 (dd, 1H, J1 = 15.9 Hz, J2 = 5.1 Hz, HB-4), 4.15-4.32 (m, 2H, 2 × CHNH), 7.22 (d, 1H, J =
7.1 Hz, COCHNH), 7.34 (d, 1H, J = 7.7 Hz, CHNH), 11.96 (s, 1H, NHCO), 12.01 (s, 1H,
13
pyrrole-NH) ppm; C NMR (100 MHz, DMSO-d6) δ 11.1, 18.1, 24.7, 28.5, 28.7, 28.8, 45.6,
50.1, 78.6, 108.5, 110.4, 119.7, 127.8, 143.9, 155.6, 156.0, 158.7, 172.3 ppm; HRMS ESI+ m/z
for C21H28N5O4SCl2 ([M+H]+): calcd 516.12336, found 516.12366 ; HPLC: tr 13.82 min (96 %
at 254 nm).
6.2.2.1.13
tert-Butyl
((S)-1-(((S)-6-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-
(27).
4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate
Prepared from 12 (0.305 g, 0.883 mmol) and N-Boc-L-valine (0. 192 g, 0.883 mmol) according
to the general procedure B. Product was purified by flash column chromatography using
dichloromethane/methanol (20:1) as eluent. Yield 0.158 g (33 %); brown solid; m. p. 144-146
°C; [α]D −10.2 (c 0.20, DMF); 1H NMR (400 MHz, DMSO-d6) δ 0.80-0.95 (m, 6H, 2 × CHCH3),
1.28 and 1.38 (2 × s, 9H, 3 × CH3), 1.89-2.06 (m, 3H, H-7, CHCH3), 2.19 (s, 3H, pyrrole-CH3),
2.66-2.77 (m, 3H, H-5, HA-4), 2.96-3.06 (m, 1H, HB-4), 4.01 (t, 1H, J = 8.0 Hz, COCHNH),
4.21-4.32 (m, 1H, CHNH), 7.07 (d, 1H, J = 8.1 Hz, COCHNH), 7.35 (d, 1H, J = 7.5 Hz,
CHNH), 11.98 (s, 1H, NHCO), 12.01 (s, 1H, pyrrole-NH) ppm; 13C NMR (100 MHz, DMSO-
d6) δ 11.1, 19.0, 19.5, 24.7, 28.5, 28.6, 28.8, 30.5, 45.6, 60.3, 78.7, 108.5, 110.4, 119.7, 127.8,
143.9, 156.0, 158.7, 171.2 ppm; HRMS ESI+ m/z for C23H32N5O4SCl2 ([M+H]+): calcd
544.15466, found 544.15472; HPLC: tr 15.10 min (96 % at 254 nm).
6.2.2.1.14 tert-Butyl (S)-(3-((6-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4,5,6,7-
tetrahydrobenzo[d]thiazol-2-yl)amino)-3-oxopropyl)carbamate (28). Prepared from 12 (0.339
g, 0.982 mmol) and N-Boc-β-alanine (0.186 g, 0.982 mmol) according to the general procedure
1
B. Yield 0.361 g (71 %); brown solid; m. p. 167-169 °C; [α]D −6.8 (c 0.32, DMF); H NMR
(400 MHz, DMSO-d6) δ 1.37 (s, 9H, 3 × CH3), 1.87-2.07 (m, 2H, H-7), 2.19 (s, 3H, pyrrole-
28

CH3), 2.52-2.58 (m, 2H, COCH2), 2.65-2.73 (m, 3H, H-5, HA-4), 2.94-3.06 (m, 1H, HB-4), 3.22
(dd, 2H, J1 = 12.9 Hz, J2 = 6.8 Hz, CH2NH), 4.18-4.32 (m, 1H, CHNH), 6.89 (t, 1H, J = 5.5
Hz, CH2NH), 7.33 (d, 1H, J = 7.7 Hz, CHNH), 11.91 (s, 1H, NHCO), 12.03 (s, 1H, pyrrole-
NH) ppm; 13C NMR (100 MHz, DMSO-d6) δ 11.1, 24.7, 28.6, 28.7, 28.8, 35.9, 36.6, 45.7, 78.1,
108.4, 110.5, 119.4, 119.7, 127.8, 143.7, 155.9, 156.0, 158.7, 169.7 ppm; HRMS ESI+ m/z for
C21H28N5O4SCl2 ([M+H]+): calcd 516.12336, found 516.12366; HPLC: tr 13.44 min (95 % at
254 nm).
6.2.2.1.15
(S)-2-((6-(4,5-Dibromo-1H-pyrrole-2-carboxamido)-4,5,6,7-
tetrahydrobenzo[d]thiazol-2-yl)amino)-2-oxoethan-1-aminium chloride (29). To an ice-cold
anhydrous methanol (70 mL) acetyl chloride (1.2 mL, 12.0 mmol) was added dropwise.
Reaction mixture was stirred for 30 min at 0 °C. Then a solution of 24 (0.691 g, 1.20 mmol) in
anhydrous methanol (40 mL) was added and reaction mixture was stirred for 1 h at 0 °C and
then for 18 h at room temperature. Solvent was evaporated under reduced pressure. Yield 0.493
1
g (80 %); white solid; m. p. 197-199 °C; [α]D −4.42 (c 0.24, MeOH); H NMR (400 MHz,
DMSO-d6) δ 1.81-1.93 (m, 1H, HA-7), 1.96-2.04 (m, 1H, HB-7), 2.63-2.74 (m, 3H, H-5, HA-4),
+
2.98 (dd, 1H, J1 = 15.5 Hz, J2 = 5.4 Hz, HB-4), 3.86 (dd, 2H, J1 = 8.6 Hz, J2 = 3.3 Hz, CH2NH3 ),
4.14-4.24 (m, 1H, CHNH), 7.02 (d, 1H, J = 2.8 Hz, pyrrole-H), 8.23 (d, 1H, J = 7.8 Hz, CHNH),
8.31 (br s, 3H, NH3 ), 12.77 (d, 1H, J = 2.7 Hz, pyrrole-NH) ppm; 13C NMR (100 MHz, DMSO-
+
d6) δ 24.6, 28.4, 28.6, 40.6, 45.2, 97.8, 104.5, 113.1, 120.0, 128.0, 142.0, 157.3, 157.7, 158.3
ppm; HRMS ESI- m/z for C14H14Br2N5O2S ([M-H]-): calcd 473.9235, found 473.9230; HPLC:
tr 10.80 min (100 % at 254 nm).
6.2.2.2. General procedure C. Starting compound (1 mmol) was dissolved in anhydrous 1,4-
dioxane (10 mL) under argon atmosphere and cooled on an ice bath. Then a solution of 4 M
HCl in 1,4-dioxane (2.5 mL, 10 mmol) was added dropwise and the mixture was stirred
29