Thiazole analogues of the marine alkaloid nortopsentin as inhibitors of bacterial biofilm formation

Article abstract of DOI:10.3390/molecules26010081

Anti-virulence strategy is currently considered a promising approach to overcome the global threat of the antibiotic resistance. Among different bacterial virulence factors, the biofilm formation is recognized as one of the most relevant. Considering the high and growing percentage of multi-drug resistant infections that are biofilm-mediated, new therapeutic agents capable of counteracting the formation of biofilms are urgently required. In this scenario, a new series of 18 thiazole derivatives was efficiently synthesized and evaluated for its ability to inhibit biofilm formation against the Gram-positive bacterial reference strains Staphylococcus aureus ATCC 25923 and S. aureus ATCC 6538 and the Gram-negative strain Pseudomonas aeruginosa ATCC 15442. Most of the new compounds showed a marked selectivity against the Gram-positive strains. Remarkably, five compounds exhibited BIC₅₀ values against S. aureus ATCC 25923 ranging from 1.0 to 9.1 μM. The new compounds, affecting the biofilm formation without any interference on microbial growth, can be considered promising lead compounds for the development of a new class of anti-virulence agents.

Full text of DOI:10.3390/molecules26010081

molecules
Article
Thiazole Analogues of the Marine Alkaloid Nortopsentin as
Inhibitors of Bacterial Biofilm Formation
Anna Carbone †,‡, Stella Cascioferro † , Barbara Parrino, Daniela Carbone , Camilla Pecoraro,
Domenico Schillaci , Maria Grazia Cusimano, Girolamo Cirrincione and Patrizia Diana *
Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF),
Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy; anna.carbone@unipa.it (A.C.);
stellamaria.cascioferro@unipa.it (S.C.); barbara.parrino@unipa.it (B.P.); daniela.carbone@unipa.it (D.C.);
camilla.pecoraro@unipa.it (C.P.); domenico.schillaci@unipa.it (D.S.); mariagrazia.cusimano@unipa.it (M.G.C.);
girolamo.cirrincione@unipa.it (G.C.)
* Correspondence: patrizia.diana@unipa.it
† Present address: Dipartimento di Farmacia, Università degli Studi di Genova, Viale Benedetto XV 3,
16132 Genova, Italy.
‡ These authors contributed equally to this work.
Abstract: Anti-virulence strategy is currently considered a promising approach to overcome the
global threat of the antibiotic resistance. Among different bacterial virulence factors, the biofilm
formation is recognized as one of the most relevant. Considering the high and growing percentage
of multi-drug resistant infections that are biofilm-mediated, new therapeutic agents capable of
counteracting the formation of biofilms are urgently required. In this scenario, a new series of
18 thiazole derivatives was efficiently synthesized and evaluated for its ability to inhibit biofilm
formation against the Gram-positive bacterial reference strains Staphylococcus aureus ATCC 25923
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
and S. aureus ATCC 6538 and the Gram-negative strain Pseudomonas aeruginosa ATCC 15442. Most of
ꢀꢁꢂꢃꢄꢅꢆ
the new compounds showed a marked selectivity against the Gram-positive strains. Remarkably,
five compounds exhibited BIC50 values against S. aureus ATCC 25923 ranging from 1.0 to 9.1 µM.
The new compounds, affecting the biofilm formation without any interference on microbial growth,
can be considered promising lead compounds for the development of a new class of anti-virulence
agents.
Citation: Carbone, A.; Cascioferro, S.;
Parrino, B.; Carbone, D.; Pecoraro, C.;
Schillaci, D.; Cusimano, M.G.;
Cirrincione, G.; Diana, P. Thiazole
Analogues of the Marine Alkaloid
Nortopsentin as Inhibitors of
Keywords: antibiofilm agents; antibiotic resistance; thiazole derivatives; marine alkaloids ana-
logues; nortopsentin
Bacterial Biofilm Formation. Molecules
2021, 26, 81. https://dx.doi.org/
10.3390/molecules26010081
Academic Editor: Manuel Simões
Received: 30 October 2020
1. Introduction
Accepted: 23 December 2020
Published: 27 December 2020
The development of synthetic small molecules able to counteract antibiotic resistance
(AMR) mechanisms is urgently needed [1]. In fact, most antibiotics used to date to treat
the most common infections are becoming ineffective. Many bacteria, including the well-
known ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,
Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) evolved in highly
resistant forms through different mechanisms that include the inactivation of the antibiotic,
chemical modification of the antibiotic target, alteration of cell permeability, and biofilm
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional claims
in published maps and institutional
affiliations.
formation [
2]. In particular, bacterial biofilm is currently considered one of the most
relevant virulence factors, which is capable of making pathogens up to 1000 times more
Copyright: © 2020 by the authors. Li-
censee MDPI, Basel, Switzerland. This
article is an open access article distributed
under the terms and conditions of the
Creative Commons Attribution (CC BY)
license (https://creativecommons.org/
licenses/by/4.0/).
resistant than their planktonic form [
infections are caused by biofilm formation on indwelling medical devices or host tissues [
3
]. It was estimated that more than 80% of chronic
].
4
Biofilm is a complex multicellular structure in which bacterial cells are embedded in a
matrix constituted of extracellular polymeric substance (EPS), which is mainly formed by
polysaccharides, proteins, lipids, extracellular DNA (e-DNA), and molecules originating
from the host including mucus and DNA [5]. In the past decade, many efforts have
Molecules 2021, 26, 81. https://dx.doi.org/10.3390/molecules26010081
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 81
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been made for identifying new therapeutic strategy able to eradicate biofilm-associated
infections, [6] and, despite numerous compounds being described as potent anti-biofilm
agents, no new derivative has reached the clinic. The lack of approved anti-biofilm drugs
together with the increase in the spread of chronic biofilm-related nosocomial infections
make the research in this field particularly relevant.
Among the bioactive scaffolds recently described for their interesting anti-biofilm
properties, thiazole derivatives are considered among the most promising compounds [7].
Sulfur-containing heterocycles are often involved in attractive nonbonding interactions
that play an important role in the control of molecular conformation. In comparison with
other five-membered heterocycles, the thiazole nucleus has unique features due to the
presence of the low-lying C
on the sulfur atom, known as
−
S
σ
* orbitals. The small regions of low electron density present
σ
-holes, are often involved in drug–target interactions, thus
improving the affinity toward the biological receptor [8].
Many thiazole compounds were reported in the last decade as potent anti-biofilm
agents. The 4-(o-methoxyphenyl)-2-aminothiazoles 1a,b (Figure 1) were found to be able to
significantly inhibit P. aeruginosa biofilm formation at concentrations in the low micromolar
range, interfering with the quorum sensing (QS) system [9]. The thiazole derivatives 2a,b
(Figure 1) showed potent anti-biofilm activity against eight methicillin-resistant (MRSE)
and two reference (ATCC 12228, ATCC 35984) strains of Staphylococcus epidermidis eliciting
BIC50 values ranging from 0.35 to 7.32 µg/mL [10].
OCH3
R
HO
N
S
N
N
R
S
N
H
N
H
Cl
S
1a, R=5-Br
1b, R=2-NH
2a, R=Cl
2b, R=F
R
S
N
N
N
N
R2
R1
3
Figure 1. Chemical structures of the thiazole compounds 1a,b; 2a,b; and 3.
On the basis of the interesting anti-biofilm properties described for the thiazole scaf-
fold and continuing our search for new nortopsentin alkaloid analogues with promising
biological activity [11
of the new nortopsentin analogues of type
compound was replaced by the thiazole ring, and the indole moiety in position 4 was
replaced by 7-azaindole (Figure 1) [14]. The thiazole derivatives were tested against
–13], we recently reported the synthesis and the anti-biofilm activity
3, in which the imidazole nucleus of the natural
3
S. aureus ATCC 25923, S. aureus ATCC 6538, and P. aeuruginosa ATCC 15442 in order to
evaluate their ability to inhibit biofilm formation and microbial growth. Most of the new
thiazole nortopsentin analogues proved to be active as inhibitors of biofilm formation
exhibiting marked selectivity toward staphylococcal biofilms, showing BIC50 values in
the low micromolar range. Compounds of type 3 showed a typical anti-virulence profile,
fighting bacterial virulence factors, such as biofilm formation, without interfering with
the bacterial growth, thus imposing a low selective pressure for the onset of antibiotic
resistance mechanisms.
With the aim to obtain more potent anti-biofilm agents that could be effective in the
treatment of staphylococcal infections that are biofilm-mediated, herein, we report the
synthesis of a new series of thiazole derivatives, structurally related to the nortopsentin

Molecules 2021, 26, 81
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analogues
3
, in which the 7-azaindole nucleus in position 4 of the thiazole ring was replaced
by a thiophene (1a–q) or a pyridine ring (2a–q), and the aromatic bicyclic system in position
2 of the thiazole nucleus can be either an indole or a 7-azaindole moiety.
In fact, thiophene and pyridine moieties are recognized as valuable scaffolds in the
development of potent anti-biofilm derivatives [15,16]. Additionally, the thiophene ring
was recently discovered as a key nucleus in a series of compounds able to potently inhibit
the virulence of relevant Gram-negative pathogens interfering with bacterial Disulfide
bond enzyme A (DsbA) enzymes, which catalyzes disulfide bond formation in secreted
and outer membrane proteins with virulence functions [17]. Therefore, since the so-far
synthesized thiazole nortopsentin analogues have shown a strong selectivity toward the
Gram-positive pathogens, we investigated whether the introduction of the thiophene ring
could improve the anti-biofilm activity against the Gram-negative bacteria.
2. Results and Discussion
2.1. Chemistry
The new substituted thiazoles
1 and 2 (Scheme 1, Tables 1 and 2) were efficiently
synthesized by Hantzsch reaction between an appropriate thioamide of type 3–8, previously
obtained [18], and a suitable α-bromoacetyl derivative 11,12.
O
R2
S
R
9 R2=H
11 R2=Br
i
S
X
N
S
N
R1
1a-n
S
NH2
for
1a,d,g
1o-q
iii
R
iv
N
X
R
R1
3a-c R1 = Boc, X = CH
4a-d R1 = Me, X = CH
5a-d R1 = CH2CH2OMe, X = CH
S
N
X
O
N
R1
N
6d
7a
8a
R1 = H, X = CH
R1 = H, X = N
R1 = Me, X = N
R2
2a-n
for
N
a R = H, b R = OMe, c R = Br, d R = F
HBr
2a,d,g
2o-q
v
10 R2=H
12 R2=Br
ii
Scheme 1. Synthesis of new thiazole derivatives of type 1a–q and 2a–q. Reagents: (i) (a) Br2, DCM, r.t., 1 h; (b) aq. NaHCO3,
◦
◦
80%; (ii) (a) 48% HBr, r.t. then 80 C, Br2, 2 h; (b) acetone, 0 C, 90%; (iii) EtOH, reflux, 30 min, 74–98% for 1a–n and 73–99%
for 2a–n; (iv) (a) TFA, DCM, reflux, 24 h; (b) aq NaHCO3, 72–98%; (v) TFA, DCM, reflux, 24 h, 77–95%.
The key intermediates 11,12 were prepared from the corresponding ethanones 9,10
.
In particular, 1-thiophene-3-yl-ethanone
9 was converted into 2-bromo-1-thiophen-3-yl-
ethanone 11 (80%), using bromine in dichloromethane [19]; while 1-pyridin-3-yl-ethanone
10 was turned into 2-bromo-1-pyridin-3-yl-ethanone hydrobromide 12 (90%) by reaction
with 48% hydrobromic acid and bromine [20].
The reaction of thioamides 3a–c, 4a–d, 5a–d, 6d, 7a, and 8a with 2-bromo-1-heteroaryl-
3-yl-ethanones 11 and 12 provided the desired 3-[4-(thiophen-3-yl)-1,3-thiazol-2-yl]-1H-
indoles 1a–n and 3-[4-(pyridin-3-yl)-1,3-thiazol-2-yl]-1H-indole hydrobromides 2a–n in

Molecules 2021, 26, 81
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good to excellent yields (74–98% and 73–99%, respectively). The subsequent deprotec-
tion of N-tert-butylcarboxylate derivatives 1a,d,g and 2a,d,g using trifluoroacetic acid
in dichloromethane under reflux, after neutralization with aqueous hydrogen carbonate
solution, gave the thiazoles 1o–q (72–98%), whereas thiazoles 2o–q were directly isolated
without neutralization (72–95%).
Table 1. Substituted 3-[4-(thiophen-3-yl)-1,3-thiazol-2-yl]-1H-indoles 1a–q.
S
R
N
X
N
S
R1
Compd
R
R1
X
Compd
R
R1
X
1a
1b
1c
1d
1e
1f
1g
1h
1i
H
H
H
OMe
OMe
OMe
Br
Boc
Me
CH2CH2OMe
Boc
CH
CH
CH
CH
CH
CH
CH
CH
CH
1j
1k
1l
1m
1n
1o
1p
1q
F
F
F
H
H
H
OMe
Br
H
Me
CH
CH
CH
N
CH2CH2OMe
H
Me
H
H
H
Me
N
CH2CH2OMe
Boc
CH
CH
CH
Br
Br
Me
CH2CH2OMe
Table 2. Substituted 3-[4-(pyridin-3-yl)-1,3-thiazol-2-yl]-1H-indole hydrobromides 2a–q.
S
R
N
X
N
N
R1
Compd
R
R1
X
Compd
R
R1
X
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
H
H
OMe
OMe
OMe
Br
Boc
Me
CH2CH2OMe
Boc
CH
CH
CH
CH
CH
CH
CH
CH
CH
2j
2k
2l
2m
2n
2o
2p
2q
F
F
F
H
H
H
OMe
Br
H
Me
CH
CH
CH
N
CH2CH2OMe
H
Me
H
H
H
Me
N
CH2CH2OMe
Boc
CH
CH
CH
Br
Br
Me
CH2CH2OMe
2.2. Biological Studies
All the new compounds were first tested for evaluating the antibacterial activity
against the planktonic form of the Gram-positive S. aureus ATCC 25923, S. aureus ATCC
6538, and of the Gram-negative pathogen P. aeruginosa ATCC 15442. All the new thiazole
derivatives, analogously to the precursors 3, did not affect the microbial growth, showing
Minimum Inhibitory Concentration (MIC) values greater than 100
agreement with the desired anti-virulence profile.
µg/mL. This result is in
Inhibition of biofilm formation of the same bacterial strains was evaluated for all
the new derivatives 1a–q and 2a–q at sub-MIC concentrations, and BIC50 values (the
concentration of compound needed to inhibit biofilm formation by 50%) were determined
for the compounds that showed a percentage of biofilm inhibition greater than 20% at the
screening concentration of 10 µg/mL at least against one bacterial strain (Table 3).

Molecules 2021, 26, 81
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Table 3. Inhibition of biofilm formation, BIC50, µg/mL and µM.
S.aureus ATCC 25923 S. aureus ATCC 6538
Comp
P. aeruginosa ATCC 15442
µg/mL
n.s.
n.s.
µM
n.s.
n.s.
16.7
n.s.
n.s.
49.9
17.5
26.9
26.7
n.s.
n.s.
3.9
µg/mL
µM
37.9
n.s.
19.3
13.8
116.7
n.s.
27.5
n.s.
4.8
63.5
12.4
12.8
13.0
77.1
n.s.
45.4
34.6
34.5
11.9
37.9
20.1
24.4
7.6
µg/mL
5.7 ± 0.5
14.7 ± 1.2
n.s.
25.7 ± 1.7
11.2 ± 0.8
n.s.
µM
14.9
49.5
n.s.
62.2
34.3
n.s.
22.9
n.s.
n.s.
118.5
n.s.
n.s.
n.s.
44.6
43.5
22.4
n.s.
31.9
n.s.
n.s.
n.s.
n.s.
n.s.
28.7
5.5
1a
1b
1c
1d
1e
1f
1g
1h
1i
14.5 ± 0.9
n.s.
5.7 ± 0.7
6.6 ± 0.5
5.7 ± 0.3
38.1 ± 1.8
n.s.
n.s.
n.s.
18.5 ± 0.9
8.1 ± 0.4
10.1 ± 0.1
11.2 ± 0.8
n.s.
12.7 ± 0.9
10.6 ± 0.9
n.s.
n.s.
2.04 ± 0.07
19.1 ± 0.9
3.9 ± 0.1
4.6 ± 0.2
3.7 ± 0.08
21.8 ± 1.3
n.s.
16.4 ± 1.2
12.9 ± 0.9
14.4 ± 1.1
4.8 ± 0.2
17.1 ± 0.9
10.4 ± 0.8
9.2 ± 0.7
3.0 ± 0.01
n.s.
n.s.
1j
1k
1l
35.6 ± 1.3
n.s.
n.s.
1.4 ± 0.09
n.s.
n.s.
1m
1o
1p
1q
2b
2c
2e
2h
2i
2j
2k
2l
2m
2o
2p
n.s.
n.s.
n.s.
n.s.
n.s.
6.9
n.s.
n.s.
n.s.
12.6 ± 0.7
13.6 ± 1.1
8.1 ± 0.6
n.s.
2.6 ± 0.1
3.8 ± 0.2
n.s.
9.1
13.3 ± 0.5
n.s.
n.s.
1.0
14.8
8.1
15.8
n.s.
42.4
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
0.5 ± 0.008
5.6 ± 0.4
3.2 ± 0.2
6.9 ± 0.4
n.s.
n.s.
n.s.
19.2
25.2
12.5 ± 1.5
0.2 ± 0.007
n.s.
n.s.
15.2 ± 1.2
6.9 ± 0.5
n.s.
n.s.
n.s.
9.8 ± 0.3
n.s.
n.s.: not significant because lower than 20% of inhibition percentage at the screening concentration of 100
least three independent experiments are reported with standard deviation (SD).
µg/mL. The averages from at
All derivatives
previously observed for the nortopsentin analogues of type
the Gram-positive pathogens, in particular toward S. aureus ATCC 25923. Compounds
1l 2b 2c 2i, and 2k exhibited the highest potency with BIC50 values ranging from 1.0
to 9.1 M. The replacement of the indole ring with the 7-azaindole moiety, as well as
1
and most of the compounds
2
showed antibiofilm activity, eliciting, as
3, a marked selectivity against
,
,
,
µ
its substitution at position 5 with a halogen atom or a methoxy group, does not entail
advantages in terms of the biofilm inhibition. Instead, the presence of a methoxyethyl
group on the indole nitrogen generally led to an improvement of the antibiofilm activity
against the staphylococcal strains. Compounds 1l, 2b, 2c, 2i, and 2k were also tested by
using viable plate count, and the activity of inhibition of staphylococcal biofilm formation
was reported in terms of log reduction. By using such a method, compound 2i was the
most effective compound in interfering with biofilm formation, since it causes the greatest
log reduction ranging from 2.62 to 1.73 at concentrations between 10 and 0.1
Figure 2).
µg/mL (see
Most of the new thiazole derivatives 1 and 2 were inactive or weakly active against
the Gram-negative strain. Only compounds 1a and 2m showed a significant inhibition of
P. aeruginosa biofilm formation, eliciting BIC50 values of 14.9 and 5.5 µM, respectively.
Additionally, the most active compounds, for every bacterial strain, were selected and
tested at the screening concentration of 100 µg/mL, for evaluating their dispersal activity
against the 24 h preformed biofilm. No derivatives was able to interfere with the biofilm
architecture; only compound 2m showed weak dispersal activity eliciting a percentage of
inhibition of 36% against P. aeruginosa at the screening concentration. Biological results
highlighted the ability of the new compounds to interfere with the first stage of the biofilm
life cycle, which consists in the bacterial adhesion to surfaces [21]. Anti-adhesion agents

Molecules 2021, 26, 81
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represent a valuable alternative to antibiotics, since they deprive the bacterium of its
pathogenicity by preventing its adhesion to the host cells.
Figure 2. Inhibition of staphylococcal biofilm formation (viable plate count). Activity expressed in terms of log reduction.
Each value is the average (reported with the SD values and error bars) from at least two independent experiments, p < 0.05.
3. Materials and Methods
3.1. Chemistry
3.1.1. General
All melting points were taken on a Büchi-Tottoly capillary apparatus (Büchi, Cornaredo,
Italy) and are uncorrected. IR spectra were determined in bromoform with a Shimadzu
1
FT/IR 8400S spectrophotometer (Shimadzu Corporation, Milan, Italy). H and 13C NMR
spectra were measured at 200 and 50.0 MHz, respectively, in DMSO-d6 solution, using a
Bruker Avance II series 200 MHz spectrometer (Bruker, Milan, Italy). Column chromatog-
raphy was performed with Merk silica gel 230–400 mesh ASTM or with Büchi Sepacor
chromatography module (prepacked cartridge system). Elemental analyses (C, H, and
N) were within
±
0.4% of theoretical values. The purity of all the tested compounds was
greater than 95%, as determined by HPLC (Agilent 1100 Series).
3.1.2. General Procedures for the Synthesis of Thioamides 3a–c
,
4a–d
,
5a–d
,
6d
,
7a, and 8a
18]. Ana-
These compounds were prepared using procedures previously reported [17
,
lytical and spectroscopic data are in agreement with those previously reported.
3.1.3. General Procedures for the Synthesis of 3-bromoacetyl compounds 11 and 12
These compounds were prepared using known procedures (80–90%). Analytical and
spectroscopic data are compatible with those previously reported [19,20].
3.1.4. General Procedure for the Synthesis of Thiazole Derivatives (1a–n) and (2a–n)
A suspension of the proper thioamides 3a–c, 4a–d, 5a–d, 6d, 7a, 8a (2 mmol) and
bromoacetyl derivatives 11, 12 (2 mmol) in anhydrous ethanol (8 mL) was refluxed for
30 min. After cooling, the obtained precipitate was filtered off, dried, and recrystallized
from ethanol to give the desired thiazoles 1a–n and 2a–n.

Molecules 2021, 26, 81
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Tert-Butyl-3-[4-(Thiophen-3-yl)-1,3-Thiazol-2-yl]-1H-Indole-1-Carboxylate (1a)
Yellow solid; yield: 79%; mp: 219–220 ◦C; IR cm−01: 1750 (CO); 1H NMR (200 MHz,
0
00
DMSO-d6)
δ
: 1.68 (s, 9H, 3 x CH3), 7.40–7.53 (m, 2H, H-5 and H-6 ), 7.66–7.74 (m, 2H, H-4
00
00
0
and H-5 ), 8.00 (s, 1H, H-5), 8.10 (dd, 1H, J = 2.8, 1.3 Hz, H-2 ), 8.16–8.20 (m, 1H, H-7 ), 8.33
(s, 1H, H-20), 8.47–8.52 (m, 1H, H-40); 13C (50 MHz, DMSO-d6)
δ
: 27.6 (3 x q), 84.8 (s), 112.2
(d), 114.9 (d), 121.5 (d), 122.5 (d), 123.8 (d), 125.5 (d), 125.6 (d), 126.3 (d), 126.6 (s), 126.8 (s),
127.1 (d), 135.0 (s), 136.2 (s), 148.6 (s), 150.9 (s), 160.2 (s). Anal. Calcd for C20H18N2O2S2: C,
62.80; H, 4.74; N, 7.32%. Found: C, 62.59; H, 4.56; N, 7.21%.
1-Methyl-3-[4-(Thiophen-3-yl)-1,3-Thiazol-2-yl]-1H-Indole (1b)
Yellow solid; yield: 98%; mp: 248–249 ◦C; 1H NMR (200 MHz, DMSO-d6)
δ
0 : 3.89 (s,
0
0
00
00
3H, CH3), 7.23–7.36 (m, 2H, H-5 and H-6 ), 7.55–7.72 (m, 3H, H-4 , H-5 and H-7 ), 7.79 (s,
00
0
0
1H, H-5), 8.03 (dd, 1H, J = 2.8, 1.3 Hz, H-2 ), 8.19 (s, 1H, H-2 ), 8.31–8.35 (m, 1H, H-4 ); 13
C
(50 MHz, DMSO-d6)
δ
: 32.9 (q), 109.1 (s), 110.0 (d), 110.6 (d), 120.6 (d), 121.1 (d), 122.2 (d),
122.5 (d), 124.5 (s), 126.3 (d), 127.0 (d), 130.8 (d), 136.2 (s), 137.0 (s), 149.8 (s), 162.5 (s). Anal.
Calcd for C16H12N2S2: C, 64.83; H, 4.08; N, 9.45%. Found: C, 65.11; H, 3.91; N, 9.59%.
1-(2-Methoxyethyl)-3-[4-(Thiophen-3-yl)-1,3-Thiazol-2-yl]-1H-Indole (1c)
Orange solid; yield: 80%; mp: 169–170 ◦C; 1H NMR (200 MHz, DMSO-d6)
δ: 3.24 (s,
3H, CH3), 3.72 (t, 2H, J = 5.1 Hz, CH2), 4.46 (t, 2H, J = 5.1 Hz, CH2), 7.23–7.33 (m, 2H, H-50
and H-60), 7.62–7.81 (m, 3H, H-400, H-500 and H-70), 7.81 (s, 1H, H-5), 8.04 (dd, 1H, J = 2.8,
1.4 Hz, H-200), 8.17 (s, 1H, H-20), 8.30–8.34 (m, 1H, H-40); 13C (50 MHz, DMSO-d6)
δ: 45.7
(t), 58.1 (q), 70.6 (t), 109.3 (s), 110.1 (d), 110.9 (d), 120.5 (d), 121.1 (d), 122.3 (d), 122.5 (d),
124.6 (s), 126.3 (d), 127.0 (d), 130.3 (d), 136.1 (s), 136.5 (s), 149.7 (s), 162.4 (s). Anal. Calcd for
C18H16N2OS2: C, 63.50; H, 4.74; N, 8.23%. Found: C, 63.26; H, 4.90; N, 8.02%.
Tert-Butyl 5-Methoxy-3-[4-(Thiophen-3-yl)-1,3-Thiazol-2-yl]-1H-Indole-1-Carboxylate (1d
)
◦
1
Pink solid; yield: 89%; mp: 185–186 C; IR cm−1: 1733 (CO); H NMR (200 MHz,
DMSO-d6)
δ
: 1.67 (s,090 H, 3 x C0H3), 3.90 (s, 3H, CH3), 7.09 (dd, 1H, J = 090.0, 2.6 Hz, H-60),
0
00
7.66–7.73 (m, 2H, H-4 and H-7 ), 7.98 (s, 1H, H-5), 8.04–8.07 (m, 3H, H-2 , H-4 and H-5 ),
8.29 (s, 1H, H-20); 13C (50 MHz, DMSO-d6)
δ: 27.6 (3 x q), 55.3 (q), 84.7 (s), 103.8 (d), 112.2
(d), 114.2 (d), 114.7 (s), 115.8 (d), 122.4 (d), 126.1 (d), 126.2 (d), 127.2 (d), 127.6 (s), 129.6 (s),
136.2 (s), 148.6 (s), 150.8 (s), 156.2 (s), 160.4 (s). Anal. Calcd for C21H20N2O3S2: C, 61.14; H,
4.89; N, 6.79%. Found: C, 60.89; H, 5.03; N, 6.56%.
5-Methoxy-1-Methyl-3-[4-(Thiophen-3-yl)-1,3-Thiazol-2-yl]-1H-Indole (1e)
Orange solid; yield: 98%; mp: 232–233 ◦C; 1H NMR (200 MHz, DMSO-d6)
δ
: 3.86 (s,
0
0
3H, CH3), 3.87 (s, 3H, CH3), 6.95 (dd, 1H, J = 8.9, 2.5 Hz, H-6 ), 7.48 (d, 1H, J = 8.9 Hz, H-7 ),
7.66–7.69 (m, 2H, H-400 and H-500), 7.77 (s, 1H, H-5), 7.83 (d, 1H, J = 2.5 Hz, H-40), 8.00 (dd,
1H, J = 2.7, 1.5 Hz, H-200), 8.13 (s, 1H, H-20); 13C (50 MHz, DMSO-d6)
δ: 33.1 (q), 55.3 (q),
102.3 (d), 108.5 (s), 109.8 (d), 111.5 (d), 112.4 (d), 122.2 (d), 125.1 (s), 126.2 (d), 127.1 (d), 131.2
(d), 132.2 (s), 136.0 (s), 149.5 (s), 155.0 (s), 162.7 (s). Anal. Calcd for C17H14N2OS2: C, 62.55;
H, 4.32; N, 8.58%. Found: C, 62.31; H, 4.15; N, 8.32%.
5-Methoxy-1-(2-Methoxyethyl)-3-[4-(Thiophen-3-yl)-1,3-Thiazol-2-yl]-1H-Indole (1f)
Yellow solid; yield: 91%; mp: 213–214 ◦C; 1H NMR (200 MHz, DMSO-d6)
δ: 3.24 (s,
3H, CH3), 3.69 (t, 2H, J = 5.1 Hz, CH2), 3.87 (s, 3H, CH3), 4.41 (t, 2H, J = 5.1 Hz, CH2), 6.93
(dd, 1H, J = 8.9, 2.5 Hz, H-60), 7.55 (d, 1H, J = 8.9 Hz, H-70), 7.64–7.71 (m, 2H, H-400 and
H-500), 7.78 (s, 1H, H-5), 7.83 (d, 1H, J = 2.5 Hz, H-40), 8.00 (dd, 1H, J = 2.7, 1.5 Hz, H-200),
8.10 (s, 1H, H-20); 13C (50 MHz, DMSO-d6)
δ: 45.9 (t), 55.3 (q), 58.1 (q), 70.7 (t), 102.3 (d),
108.8 (s), 109.8 (d), 111.8 (d), 112.3 (d), 121.2 (d), 125.1 (s), 126.2 (d), 127.1 (d), 130.6 (d), 131.6
(s), 136.1 (s), 149.5 (s), 154.9 (s), 162.6 (s). Anal. Calcd for C19H18N2O2S2: C, 61.60; H, 4.90;
N, 7.56%. Found: C, 61.45; H, 4.79; N, 7.39%.
Tert-Butyl 5-Bromo-3-[4-(Thiophen-3-yl)-1,3-Thiazol-2-yl]-1H-Indole-1-Carboxylate (1g)
◦
1
Pink solid; yield: 85%; mp: 174–175 C; IR cm
−
1: 1739 (CO); H NMR (200 MHz,
DMSO-d6)
δ
: 1.68 (s, 9H, 3 x CH3), 7.64 (dd, 1H, J = 8.9, 2.0 Hz, H-60), 7.70–7.71 (m, 2H,

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H-400 and H-500), 8.01 (s, 1H, H-5), 8.04 (dd, 1H, J = 2.1, 1.3 Hz, H-200), 8.11 (d, 1H, J = 8.9
Hz, H-70), 8.38 (s, 1H, H-20), 8.61 (d, 1H, J = 2.0 Hz, H-40); 13C (50 MHz, DMSO-d6)
δ: 27.6
(3 x q), 85.4 (s), 112.6 (d), 116.4 (s), 116.9 (d), 119.3 (d), 122.4 (d), 123.8 (d), 126.2 (d), 127.0
(d), 127.3 (s), 128.1 (d), 128.4 (s), 133.9 (s), 136.1 (s), 148.3 (s), 150.9 (s), 159.8 (s). Anal. Calcd
for C20H17BrN2O2S2: C, 52.06; H, 3.71; N, 6.07%. Found: C, 51.85; H, 3.94; N, 6.22%.
5-Bromo-1-Methyl-3-[4-(Thiophen-3-yl)-1,3-Thiazol-2-yl]-1H-Indole (1h)
Orange solid; yield: 90%; mp: 250–251 ◦C; 1H NMR (200 MHz, DMSO-d6)
δ
: 3.89 (s,
0
0
3H, CH3), 7.44 (dd, 1H, J = 8.7, 1.9 Hz, H-6 ), 7.58 (d, 1H, J = 8.7 Hz, H-7 ), 7.67–7.68 (m, 2H,
00
00
00
0
H-4 and H-5 ), 7.81 (s, 1H, H-5), 7.99 (dd, 1H, J = 2.1, 1.3 Hz, H-2 ), 8.24 (s, 1H, H-2 ), 8.47
(d, 1H, J = 1.9 Hz, H-40); 13C (50 MHz, DMSO-d6)
δ
: 33.1 (q), 108.8 (s), 110.3 (d), 112.8 (d),
113.8 (s), 122.1 (d), 122.8 (d), 125.0 (d), 126.1 (s), 126.2 (d), 127.1 (d), 132.0 (d), 135.8 (s), 136.4
(s), 150.3 (s), 161.8 (s). Anal. Calcd for C16H11BrN2S2: C, 51.20; H, 2.95; N, 7.46%. Found: C,
50.93; H, 2.90; N, 7.18%.
5-Bromo-1-(2-Methoxyethyl)-3-[4-(Thiophen-3-yl)-1,3-Thiazol-2-yl]-1H-Indole (1i)
Yellow solid; yield: 92%; mp: 226–227 ◦C; 1H NMR (200 MHz, DMSO-d6)
δ: 3.24 (s,
3H, CH3), 3.72 (t, 2H, J = 5.0 Hz, CH2), 4.46 (t, 2H, J = 5.0 Hz, CH2), 7.42 (dd, 1H, J = 8.8,
2.0 Hz, H-60), 7.65 (d, 1H, J = 8.8 Hz, H-70), 7.63–7.68 (m, 2H, H-400 and H-500), 7.81 (s, 1H,
H-5), 7.99 (dd, 1H, J = 2.1, 1.3 Hz, H-200), 8.22 (s, 1H, H-20), 8.46 (d, 1H, J = 2.0 Hz, H-40);
13C (50 MHz, DMSO-d6)
δ: 46.0 (t), 58.1 (q), 70.6 (t), 109.0 (s), 110.4 (d), 113.1 (d), 113.8 (s),
122.1 (d), 122.7 (d), 125.0 (d), 126.1 (s), 126.2 (d), 127.1 (d), 131.5 (d), 135.4 (s), 136.3 (s), 150.2
(s), 161.8 (s). Anal. Calcd for C18H15BrN2OS2: C, 51.55; H, 3.61; N, 6.68%. Found: C, 51.27;
H, 3.77; N, 6.81%.
5-Fluoro-3-[4-(Thiophen-3-yl)-1,3-Thiazol-2-yl]-1H-Indole (1j)
Yellow solid; yield: 74%; mp: 209–210 ◦C; IR cm−1: 3270 (NH); 1H NMR (200 MHz,
DMSO-d6)
7.64–7.71 (m, 2H, H-400 and H-500), 7.79 (s, 1H, H-5), 8.01–8.08 (m, 2H, H-200 and H-40),
8.23 (d, 1H, J = 2.9 Hz, H-20); 11.90 (bs, 1H, NH); 13C (50 MHz, DMSO-d6)
: 105.3 (d,
δ
: 7.10 (td, 1H, J = 11.6, 9.2, 2.6 Hz, H-60), 7.51 (dd, 1H, J = 11.6, 4.6 Hz, H-70),
δ
JC60-F = 24.4 Hz), 110.0 (d), 110.4 (d, JC70a-F = 4.6 Hz), 110.2 (d, JC40F = 26.0 Hz), 113.4 (d, JC70-F
= 9.6 Hz), 122.2 (d), 124.5 (d, JC30a-F = 10.8 Hz), 126.2 (d), 127.0 (d), 128.7 (d), 133.2 (s), 134.3
(s), 136.4 (s), 150.1 (s), 157.9 (d, JC50-F = 235 Hz). Anal. Calcd for C15H9FN2S2: C, 59.98; H,
3.02; N, 9.33%. Found: C, 59.67; H, 2.93; N, 9.18%.
5-Fluoro-1-Methyl-3-[4-(Thiophen-3-yl)-1,3-Thiazol-2-yl]-1H-Indole (1k)
◦
Yellow solid; yield: 95%; mp: 231–232 C; 1H NMR (200 MHz, DMSO-d6)
δ: 3.90 (s, 3H,
CH3), 7.18 (td, 1H, J = 11.7, 9.2, 2.6 Hz, H-60), 7.57–7.71 (m, 3H, H-400, H-500 and H-70), 7.78
(s, 1H, H-5), 8.02–8.08 (m, 2H, H-200 and H-40), 8.25 (s, 1H, H-20); 13C (50 MHz, DMSO-d6)
δ: 33.2 (q), 105.5 (d, JC60-F = 24.4 Hz), 109.3 (d, JC70a-F = 4.6 Hz), 110.0 (d), 110.7 (d, JC40F =
26.3 Hz), 113.5 (d, JC70-F = 9.7 Hz), 122.6 (d), 124.8 (d, JC30a-F = 10.8 Hz), 126.2 (d), 127.0 (d),
132.4 (d), 133.8 (s), 136.3 (s), 150.2 (s), 157.2 (d, JC50-F = 234 Hz), 162.1 (s). Anal. Calcd for
C16H11FN2S2: C, 61.12; H, 3.53; N, 8.91%. Found: C, 60.88; H, 3.47; N, 8.74%.
5-Fluoro-1-(2-Methoxyethyl)-3-[4-(Thiophen-3-yl)-1,3-Thiazol-2-yl]-1H-Indole (1l)
Yellow solid; yield: 88%; mp: 200–201 ◦C; 1H NMR (200 MHz, DMSO-d6)
δ: 3.25 (s,
3H, CH3), 3.72 (t, 2H, J = 5.0 Hz, CH2), 4.46 (t, 2H, J = 5.0 Hz, CH2), 7.16 (td, 1H, J = 11.8,
9.2, 2.6 Hz, H-60), 7.64–7.71 (m, 3H, H-400, H-500 and H-70), 7.80 (s, 1H, H-5), 8.01–8.07 (m,
2H, H-200 and H-40), 8.23 (s, 1H, H-20); 13C (50 MHz, DMSO-d6)
δ: 46.0 (t), 58.1 (q), 70.7 (t),
105.5 (d, JC60-F = 25.0 Hz), 109.5 (d, JC70a-F = 4.6 Hz), 110.1 (d), 110.7 (d, JC40F = 26.1 Hz), 117.3
(d, JC70-F = 9.8 Hz), 122.3 (d), 124.9 (d, JC30a-F = 10.7 Hz), 126.2 (d), 127.0 (d), 131.8 (d), 133.3
(s), 136.3 (s), 150.2 (s), 158.1 (d, JC50-F = 234 Hz), 162.0 (s). Anal. Calcd for C18H15FN2OS2:
C, 60.31; H, 4.22; N, 7.82%. Found: C, 60.03; H, 4.05; N, 7.64%.
3-[4-(Thiophen-3-yl)-1,3-Thiazol-2-yl]-1H-Pyrrolo[2,3-b]Pyridine (1m)
Yellow solid; yield: 93%; mp: 268–269 ◦C; IR cm−1: 3273 (NH); 1H NMR (200 MHz,
DMSO-d6) δ: 7.37 (dd, 1H, J = 7.9, 4.9 Hz, H-50), 7.64–7.73 (m, 2H, H-400 and H-500), 7.84 (s,
1H, H-5), 8.09 (dd, 1H, J = 2.8, 1.3 Hz, H-200), 8.34 (d, 1H, J = 2.0 Hz, H-20), 8.41 (dd, 1H,

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J = 4.9, 1.5 Hz, H-60), 8.80 (dd, 1H, J = 7.9, 1.5 Hz, H-40), 12.50 (bs, 1H, NH); 13C (50 MHz,
DMSO-d6) δ: 111.0 (s), 112.1 (d), 118.2 (d), 120.2 (s), 123.4 (d), 127.2 (d), 128.0 (d), 129.2 (d),
134.4 (d), 137.2 (s), 141.0 (d), 145.2 (s), 151.4 (s), 162.1 (s). Anal. Calcd for C14H9N3S2: C,
59.34; H, 3.20; N, 14.83%. Found: C, 59.03; H, 3.44; N, 15.06%.
1-Methyl-3-[4-(Thiophen-3-yl)-1,3-Thiazol-2-yl]-1H-Pyrrolo[2,3-b]Pyridine (1n)
Yellow solid; yield: 96%; mp: 254–255 ◦C; 1H NMR (200 MHz, DMSO-d6)
δ: 3.92 (s,
0
00
00
3H, CH3), 7.34 (dd, 1H, J = 7.9, 4.7 Hz, H-5 ), 7.64–7.73 (m, 2H, H-4 and H-5 ), 7.83 (s, 1H,
H-50), 8.07 (dd, 1H, J = 2.8, 1.3 Hz, H-200), 8.38 (s, 1H, H-20), 8.42 (dd, 1H, J = 4.7, 1.6 Hz,
H-6 ), 8.72 (dd, 1H, J = 7.9, 1.6 Hz, H-4 ); 13C (50 MHz, DMSO-d6)
δ: 31.7 (q), 108.3 (s), 110.7
0
(d), 117.3 (d), 118.0 (s), 122.4 (d), 126.3 (d), 127.0 (d), 131.1 (2 x d), 136.3 (s), 142.2 (d), 146.0
(s), 150.4 (s), 161.3 (s). Anal. Calcd for C15H11N3S2: C, 60.58; H, 3.73; N, 14.13%. Found: C,
C, 60.33; H, 3.59; N, 13.89%.
Tert-Butyl 3-[4-(Pyridin-3-yl)-1,3-Thiazol-2-yl]-1H-Indole-1-Carboxylate Hydrobromide (2a
)
Pink solid; yield: 90%; mp: 171–172 ◦C; IR cm−1: 1737 (CO), 3420 (NH+); 1H NMR
(200 MHz, DMSO-d6) 00
δ
: 1.69 (s, 9H, 3 x CH3), 07.43–7.54 (m, 2H,0H-50 and H-60), 8.09 (dd,
0
1H, J = 8.1, 5.4 Hz, H-5 ), 8.14–8.23 (m, 1H, H-7 ), 8.44 (s, 1H, H-2 ), 8.49–8.53 (m, 1H, H-4 ),
8.62 (s, 1H, H-5), 8.88 (d, 1H, J = 5.4 Hz, H-600), 9.08 (d, 1H, J = 8.1 Hz, H-400), 9.53 (s, 1H,
H-200); 13C (50 MHz, DMSO-d6)
δ: 27.6 (3xq), 85.0 (s), 114.3 (s), 115.0 (d), 118.1 (d), 121.5
(d), 124.0 (d), 125.6 (d), 126.3 (d), 126.5 (s), 127.2 (d), 132.4 (s), 134.9 (s), 140.1 (d), 141.3 (d),
141.6 (d), 148.4 (s), 148.5 (s), 161.8 (s). Anal. Calcd for C21H20BrN3O2S: C, 55.03; H, 4.40; N,
9.17%. Found: C, 54.78; H, 4.57; N, 8.98%.
1-Methyl-3-[4-(Pyridin-3-yl)-1,3-Thiazol-2-yl]-1H-Indole Hydrobromide (2b)
Orange solid; yield: 79%; mp: 266–267 ◦C; IR cm−1 3423 (NH+); 1H NMR (200 MHz,
DMSO-d6) δ
: 3.91 (s, 3H, CH3), 7.25–7.38 (m, 2H, H-50 and H-60), 7.57–7.61 (m, 1H, H-70),
8.21 (dd, 1H, J = 8.2, 5.7 Hz, H-500), 8.29 (s, 1H, H-20), 8.35–8.40 (m, 1H, H-40), 8.52 (s, 1H,
H-5), 8.94 (d, 1H, J = 5.7 Hz, H-600), 9.21 (d, 1H, J = 8.2 Hz, H-400), 9.55 (d, 1H, J = 1.7 Hz,
H-200); 13C (50 MHz, DMSO-d6)
δ: 32.9 (q); 108.8 (s), 110.7 (d), 116.2 (d), 120.6 (d), 121.3 (d),
122.7 (d), 124.4 (s), 127.5 (d), 131.4 (d), 133.1 (s), 137.0 (s), 139.0 (d), 140.3 (d), 142.1 (d), 147.5
(s), 163.9 (s). Anal. Calcd for: C17H14BrN3S: C, 54.85; H, 3.79; N, 11.29%. Found: C, 54.64;
H, 4.00; N, 11.47%.
1-(2-Methoxyethyl)-3-[4-(Pyridin-3-yl)-1,3-Thiazol-2-yl]-1H-Indole Hydrobromide (2c)
Orange solid; yield: 73%; mp: 190–191 ◦C; IR cm−1 3425 (NH+); 1H NMR (200 MHz,
DMSO-d6)
δ
: 3.24 (s,03H, CH3), 3.73 (t, 2H, J = 5.0 Hz, CH2), 4.47 (t, 2H, J = 5.0 Hz, CH2),
0
0
00
7.25–7.35 (m, 2H, H-5 and H-6 ), 7.64–7.68 (m, 1H, H-7 ), 8.23 (dd, 1H, J = 8.2, 5.8 Hz, H-5 ),
8.27 (s, 1H, H-20), 8.33–8.3070 (m, 1H, H-40), 8.56 (s, 1H, H-5), 8.82 (d, 1H, J = 5.8 Hz, H-600),
9.24 (d, 1H, J = 8.2 Hz, H-4 ), 9.58 (d, 1H, J = 1.5 Hz, H-2 ); 13C (50 MHz, DMSO-d6)
δ: 45.8
00
(t), 50.1 (q), 70.6 (t), 109.1 (s), 111.0 (d), 116.3 (d), 120.6 (d), 121.3 (d), 122.7 (d), 124.5 (s),
127.6 (d), 131.0 (d), 133.1 (s), 136.6 (s), 139.1 (d), 140.4 (d), 142.3 (d), 147.5 (s), 163.9 (s). Anal.
Calcd for: C19H18BrN3OS: C, 54.81; H, 4.36; N, 10.09%. Found: C, 54.60; H, 4.21; N, 9.92%.
Tert-Butyl 5-Methoxy-3-[4-(Pyridin-3-yl)-1,3-Thiazol-2-yl]-1H-Indole-1-Carboxylate Hydro-
bromide (2d)
Orange solid; yield: 88%; mp: 154–155 ◦C; IR cm−1: 1734 (CO), 3422 (NH+); 1H NMR
(200 MHz, DMSO-d6) δ: 1.68 (s, 9H, 3 x CH3), 3.91 (s, 3H, CH3), 7.11 (dd, 1H, J = 9.1, 2.6 Hz,
H-60), 7.82 0(0d, 1H, J = 2.6 Hz0, H-40), 8.05 (d, 1H, J = 9.1 Hz, H-70), 8.13 (dd, 1H, J = 8.2,
00
5.6 Hz, H-5 ), 8.38 (s, 1H, H-2 ), 8.63 (s, 1H, H-5), 8.91 (d, 1H, J = 5.6 Hz, H-6 ), 9.07 (d, 1H,
00
00
J = 8.2 Hz, H-4 ), 9.50 (d, 1H, J = 2.0 Hz, H-2 ); 13C (50 MHz, DMSO-d6)
δ
: 27.6 (3 x q), 55.4
(q), 84.9 (s), 103.8 (d), 114.0 (d), 115.8 (d), 118.0 (d), 126.9 (d), 127.2 (d), 129.5 (s), 132.4 (s),
140.0 (d), 141.2 (d), 141.3 (s), 141.5 (d), 148.2 (s), 148.5 (s), 149.7 (s), 156.2 (s), 161.9 (s). Anal.
Calcd for: C22H22BrN3O3S: C, 54.10; H, 4.54; N, 8.60%. Found: C, 54.35; H, 4.30; N, 8.46%.
5-Methoxy-1-Methyl-3-[4-(Pyridin-3-yl)-1,3-Thiazol-2-yl]-1H-Indole Hydrobromide (2e)
Orange solid; yield: 96%; mp: 210–211 ◦C; IR cm−1 3429 (NH+); 1H NMR (200 MHz,
0
DMSO-d6)
δ
: 3.87 (s, 3H, CH3), 3.90 (s, 3H, CH3), 6.99 (dd, 1H, J = 8.9, 2.4 Hz, H-6 ), 7.63 (d,

Molecules 2021, 26, 81
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0
0
0
00
1H, J = 8.9 Hz, H-7 ), 7.83 (d, 1H, J = 2.4 Hz, H-4 ), 8.19–8.25 (m, 2H, H-2 and H-5 ), 8.50 (s,
1H, H-5), 8.94 (d, 1H, J = 5.3 Hz, H-600), 9.20 (d, 1H, J = 8.3 Hz, H-400), 9.51 (s, 1H, H-200);
13C (50 MHz, DMSO-d6)
δ: 33.1 (q), 55.4 (q), 102.5 (d), 108.4 (s), 111.6 (d), 112.3 (d), 115.9
(d), 125.0 (s), 127.5 (d), 131.7 (d), 132.2 (s), 133.1 (s), 139.1 (d), 140.4 (d), 141.9 (d), 147.4 (s),
155.1 (s), 164.2 (s). Anal. Calcd for: C18H16BrN3OS: C, 53.74; H, 4.01; N, 10.44%. Found: C,
53.50; H, 3.83; N, 10.70%.
5-Methoxy-1-(2-Methoxyethyl)-3-[4-(Pyridin-3-yl)-1,3-Thiazol-2-yl]-1H-Indole
Hydrobromide (2f)
Orange solid; yield: 89%; mp: 122–123 ◦C; IR cm−1 3426 (NH+); 1H NMR (200 MHz,
DMSO-d6)
J = 5.0 Hz, CH2), 6.95 (dd, 1H,
1H,
= 2.5 Hz, H-40), 8.18–8.22 (m, 2H, H-20 and H-500), 8.50 (s, 1H, H-5), 8.93 (d, 1H,
5.2 Hz, H-600), 9.15 (d, 1H, = 8.4 Hz, H-400), 9.51 (d, 1H, = 1.3 Hz, H-200); 13C (50 MHz,
DMSO-d6) : 46.0 (t), 55.4 (q), 58.1 (q), 70.6 (t), 102.5 (d), 108.7 (s), 111.9 (d), 112.2 (d), 115.8
δ
: 3.24 (s, 3H, CH3), 3.71 (t, 2H,
J
= 5.0 Hz, CH2), 3.89 (s, 3H, CH3), 4.42 (t, 2H,
J
= 8.9, 2.5 Hz, H-60), 7.56 (d, 1H,
J
= 8.9 Hz, H-70), 7.81 (d,
J
J =
J
J
δ
(d), 125.1 (s), 127.3 (d), 131.1 (d), 131.7 (s), 133.0 (s), 139.5 (d), 140.8 (d), 141.6 (d), 147.6 (s),
155.0 (s), 164.1 (s). Anal. Calcd for: C20H20BrN3O2S: C, 53.82; H, 4.52; N, 9.41%. Found: C,
53.65; H, 4.47; N, 9.66%.
Tert-Butyl 5-Bromo-3-[4-(Pyridin-3-yl)-1,3-Thiazol-2-yl]-1H-Indole-1-Carboxylate Hydro-
bromide (2g)
Yellow solid; yield: 78%; mp: 185–186 ◦C; IR cm−1: 1749 (CO), 3421 (NH+); 1H NMR
(200 MHz, DMSO-d6)
2H, H-500 and H-70), 8.45 (s, 1H, 20), 8.57 (d, 1H, J = 2.0 Hz, H-40), 8.60 (s, 1H, H-5), 8.89
(d, 1H, J = 5.5 Hz, H-600), 8.99 (d, 1H, J = 8.3 Hz, H-400), 9.46 (d, 1H, J = 1.8 Hz, H-200); 13
(50 MHz, DMSO-d6) : 27.6 (3 x q), 85.5 (s), 113.4 (s), 116.5 (d), 116.8 (s), 118.2 (d), 123.5
δ
: 1.68 (s, 9H, CH3); 7.63 (dd, 1H, J = 8.9, 2.0 Hz, H-60), 8.01–8.13 (m,
C
δ
(d), 127.1 (d), 127.6 (d), 128.0 (s), 128.1 (d), 132.2 (s), 133.7 (s), 140.3 (d), 140.9 (d), 141.9 (d),
148.1 (s), 148.5 (s), 161.3 (s). Anal. Calcd for: C21H19Br2N3O2S: C, 46.95; H, 3.56; N, 7.82%.
Found: C, 47.18; H, 3.41; N, 7.56%.
5-Bromo-1-Methyl-3-[4-(Pyridin-3-yl)-1,3-Thiazol-2-yl]-1H-Indole Hydrobromide (2h)
Orange solid; yield: 94%; mp: 279–280 ◦C; IR cm−1: 3420 (NH+); 1H NMR (200 MHz,
DMSO-d6) δ
: 3.89 (s, 3H, CH3), 7.41 (dd, 1H, J = 8.7, 1.9 Hz, H-60), 7.56 (d, 1H, J = 8.7 Hz,
H-70), 8.18 (dd, 1H, J = 8.2, 5.6 Hz, H-500), 8.31 (s, 1H, H-20), 8.43 (d, 1H, J = 1.9 Hz, H-40),
8.49 (s, 1H, H-5), 8.92 (d, 1H, J = 5.6 Hz, H-600), 9.09 (d, 1H, J = 8.2 Hz, H-400), 9.47 (d, 1H,
00
J = 1.8 Hz, H-2 ); 13C (50 MHz, DMSO-d6)
δ: 33.2 (q), 108.4 (s), 112.9 (d), 114.1 (s), 116.2 (d),
122.7 (d), 125.2 (d), 125.9 (s), 127.3 (d), 132.7 (d), 132.8 (s), 135.8 (s), 139.6 (d), 141.0 (d), 141.4
(d), 147.8 (s), 163.4 (s). Anal. Calcd for: C17H13Br2N3S: C, 45.26; H, 2.90; N, 9.31%. Found:
C, 45.44; H, 2.65; N, 9.47%.
5-Bromo-1-(2-Methoxyethyl)-3-[4-(Pyridin-3-yl)-1,3-Thiazol-2-yl]-1H-Indole
Hydrobromide (2i)
Red solid; yield: 87%; mp: 196–197 C; IR cm−1: 3427 (NH+); H NMR (200 MHz,
◦
1
DMSO-d6)
δ
: 3.23 (s, 3H, CH3),03.72 (t, 2H, J = 5.0 Hz, CH2), 4.46 (t, 2H, J = 5.0 Hz, CH2),
0
7.42 (dd, 1H, J = 8.8, 2.0 Hz, H-6 ), 7.65 (d, 1H, J = 8.8 Hz, H-7 ), 8.18 (dd, 1H, J = 8.2, 5.6 Hz,
H-500), 8.32 (s, 1H, H-20), 8.44 (d, 1H, J = 2.0 Hz, H-40), 8.51 (s, 1H, H-5), 8.92 (d, 1H, J =
5.6 Hz, H-600), 9.10 (d, 1H, J = 8.2 Hz, H-400), 9.49 (d, 1H, J = 1.7 Hz, H-200); 13C (50 MHz,
DMSO-d6) δ: 46.0 (t), 58.1 (q), 70.6 (t), 108.7 (s), 113.2 (d), 114.0 (s), 116.3 (d), 122.7 (d), 125.2
(d), 126.0 (s), 127.2 (d), 132.2 (d), 132.7 (s), 135.5 (s), 139.8 (d), 141.2 (d), 141.3 (d), 148.0 (s),
163.3 (s). Anal. Calcd for: C19H17Br2N3OS: C, 46.08; H, 3.46; N, 8.48%. Found: C, 46.33; H,
3.60; N, 8.66%.
5-Fluoro-3-[4-(Pyridin-3-yl)-1,3-Thiazol-2-yl]-1H-Indole Hydrobromide (2j)
Yellow solid; yield: 90%; mp: 284–285 ◦C; IR cm−1: 3159 (NH), 3428 (NH+); 1H NMR
0
(200 MHz, DMSO-d6)
δ
: 7.13 (td, 1H, J = 11.6, 9.3, 2.5 Hz, H-6 ), 7.54 (dd, 1H, J = 9.3, 4.6 Hz,
0
0
00
0
H-7 ), 8.06–9.19 (m, 2H, H-4 and H-5 ), 8.34 (d, 1H, J = 2.9 Hz, H-2 ), 8.48 (s, 1H, H-5), 8.90
(d, 1H, J = 4.8 Hz, H-600), 9.14 (d, 1H, J = 8.4 Hz, H-400), 9.54 (d, 1H, J = 1.7 Hz, H-200), 12.02

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(bs, 1H, NH); 13C (50 MHz, DMSO-d6): 105.4 (d, JC60-F = 24.2 Hz), 110.0 (d, JC’7a-F = 4.6 Hz),
110.9 (d, JC40-F = 26.2 Hz), 113.5 (d, JC70-F = 10.2 Hz), 116.2 (d), 124.4 (d, JC30a-F = 10.9 Hz),
127.4 (d), 129.4 (d), 133.0 (s), 133.2 (s), 139.4 (d), 140.6 (d), 141.9 (d), 147.6 (s), 158.1 (d, JC50-F
= 234 Hz), 164.1 (s). Anal. Calcd for: C16H11BrFN3S: C, 51.08; H, 2.95; N, 11.17%. Found: C,
51.36; H, 3.09; N, 11.41%.
5-Fluoro-1-Methyl-3-[4-(Pyridin-3-yl)-1,3-Thiazol-2-yl]-1H-Indole Hydrobromide (2k)
Orange solid; yield: 92%; mp: 277–278 ◦C; IR cm−1: 3420 (NH+0); 1H NMR (200 MHz,
DMSO-d6)
δ
0
: 3.89 (s, 3H, CH3), 7.13 (td, 1H, J = 11.7, 9.2, 2.6 Hz, H-6 ), 7.60 (dd, 1H, J = 9.2,
0
00
4.4 Hz, H-7 ), 8.05 (dd, 1H, J = 11.7, 2.6 Hz, H-4 ), 8.16 (dd, 1H, J = 8.2, 5.6 Hz, H-5 ), 8.32 (s,
0
00
00
1H, H-2 ), 8.48 (s, 1H, H-5), 8.91 (d, 1H, J = 5.6 Hz, H-6 ), 9.19 (d, 1H, J = 8.2 Hz, H-4 ), 9.52
(d, 1H, J = 1.6 Hz, H-2 ); 13C (50 MHz, DMSO-d6): 33.2 (q), 105.6 (d, JC60-F = 24.9 Hz), 110.0
00
(d, JC’7a-F = 4.6 Hz), 110.9 (d, JC40-F = 25.7 Hz), 112.1 (d, JC70-F = 10.3 Hz), 115.9 (d), 124.7 (d,
JC30a-F = 11.0 Hz), 127.2 (d), 132.8 (s), 133.0 (d), 133.8 (s), 139.7 (d), 141.0 (d), 141.5 (d), 147.8
(s), 158.3 (d, JC50-F = 236 Hz), 163.6 (s). Anal. Calcd for: C17H13BrFN3S: C, 52.32; H, 3.36; N,
10.77%. Found: C, 52.14; H, 3.48; N, 10.53%.
5-Fluoro-1-(2-Methoxyethyl)-3-[4-(Pyridin-3-yl)-1,3-Thiazol-2-yl]-1H-Indole
Hydrobromide (2l)
Orange solid; yield: 88%; mp: 202 C; IR cm−1: 3421 (NH+); H NMR (200 MHz,
◦
1
DMSO-d6) : 3.24 (s, 3H, CH3), 3.72 (t, 2H, J = 5.1 Hz, CH2), 4.46 (t, 2H, J = 5.1 Hz, CH2),
δ
7.17 (td, 1H, J = 11.7, 9.2, 2.6 Hz, H-60), 7.69 (dd, 1H, J = 9.2, 4.5 Hz, H-70), 8.07 (dd, 1H,
J = 11.7, 2.6 Hz, H-40), 8.16 (dd, 1H, J = 8.2, 5.6 Hz, H-500), 8.33 (s, 1H, H-20), 8.50 (s, 1H,
H-5), 8.91 (d, 1H, J = 5.6 Hz, H-600), 9.14 (d, 1H, J = 8.2 Hz, H-400), 9.54 (d, 1H, J = 1.7 Hz,
H-200); 13C (50 MHz, DMSO-d6): 46.1 (t), 58.1 (q), 70.6 (t), 105.6 (d, JC60-F = 25.0 Hz), 109.2
(d, JC’7a-F = 4.7 Hz), 110.9 (d, JC40-F = 26.3 Hz), 112.4 (d, JC70-F = 9.8 Hz), 116.0 (d), 124.8 (d,
JC30a-F = 10.8 Hz), 127.2 (d), 132.5 (d), 132.8 (s), 133.4 (s), 139.9 (d), 141.2 (d), 141.4 (d), 147.9
(s), 162.1 (d, JC50-F = 234 Hz), 163.6 (s). Anal. Calcd for: C19H17BrFN3OS: C, 52.54; H, 3.95;
N, 9.67. Found: C, 52.23; H, 3.77; N, 9.81.
3-[4-(Pyridin-3-yl)-1,3-Thiazol-2-yl]-1H-Pyrrolo[2,3-b]Pyridine Hydrobromide (2m)
Yellow solid; yield: 95%; mp: 324–325 ◦C; IR cm−1: 3227 (NH), 3423 (NH+); 1H NMR
(200 MHz, DMSO-d6)
δ
: 7.406 (dd, 1H, J = 7.9, 5.1 Hz, H-50), 8.23 (dd, J = 8.2,05.7 Hz, 10H0 ,
00
0
H-5 ), 8.47–8.49 (m, 2H, H-2 and H-6 ), 8.63 (s, 1H, H-5), 8.94–8.95 (m, 2H, H-4 and H-6 ),
9.27 (d, 1H, J = 8.2 Hz, H-4 ), 9.63 (d, 1H, J = 1.4 Hz, H-2 ), 12.50 (bs, 1H, NH); 13C (50 MHz,
DMSO-d6): 109.3 (s), 117.3 (2 x d), 118.4 (s), 127.5 (d), 128.8 (d), 132.3 (d), 132.9 (s), 139.2 (d),
140.5 (d), 141.3 (d), 142.4 (d), 145.4 (s), 147.7 (s), 165.0 (s). Anal. Calcd for: C15H11BrN4S: C,
50.15; H, 3.09; N, 15.60%. Found: C, 50.42; H, 3.20; N, 15.83%.
00
00
1-Methyl-3-[4-(Pyridin-3-yl)-1,3-Thiazol-2-yl]-1H-Pyrrolo[2,3-b]Pyridine
Hydrobromide (2n)
Yellow solid; yield: 99%; mp: 280–282 ◦C; IR cm−1: 3425 (NH+); 1H NMR (200 MHz,
DMSO-d6) δ
: 3.92 (s, 3H, CH3), 7.37 (dd, 1H, J = 7.9, 4.8 Hz, H-50), 8.21 (dd, J = 8.2, 5.7 Hz,
1H, H-500), 8.44 (dd, 1H, J = 4.8, 1.5 Hz, H-60), 8.49 (s, 1H, H-20), 8.58 (s, 1H, H-5), 8.78 (dd,
1H, J = 7.9, 1.5 Hz, H-40), 8.95 (d, 1H, J = 5.7 Hz, H-600), 9.23 (d, 1H, J = 8.2 Hz, H-400), 9.59
(d, 1H, J = 1.7 Hz, H-2”); 13C (50 MHz, DMSO-d6): 31.6 (q), 107.6 (s), 116.9 (d), 117.3 (s),
117.5 (d), 127.5 (d), 130.2 (d), 131.6 (d), 132.9 (s), 139.1 (d), 140.4 (d), 142.3 (d), 143.2 (d),
146.8 (s), 147.6 (s), and 163.2 (s). Anal. Calcd for: C16H13BrN4S: C, 51.48; H, 3.51; N, 15.01%.
Found: C, 51.76; H, 3.72; N, 14.92%.
3.1.5. General Procedure for the Synthesis of Thiazole Compounds (1o–q)
To a suspension of appropriate thiazole 1a,d,g (0.38 mL) in DCM (5 mL) trifluoroacetic
acid (7.0 mmol, 0.54 mL) was added, and the mixture was heated under reflux for 24 h
.
After cooling, the mixture was neutralized with saturated aqueous sodium hydrogen
carbonate solution and extracted with dichloromethane (3 x 20 mL). The organic phases
were dried (Na2SO4), evaporated under reduced pressure, and the obtained residue was
recrystallized with ethanol to afford the desidered thiazoles 1o–q.

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3-[4-(Thiophen-3-yl)-1,3-THIAZOL-2-yl]-1H-indole (1o)
Yellow solid; yield: 95%; mp: 181–182 ◦C; IR cm−1: 3216 (NH); 1H NMR (200 MHz,
DMSO-d6)
δ
: 070.22–7.27 (m, 2H, H-50 and H-60), 7.50–7.54 (m, 1H, H-70), 7.64–7.72 (m, 2H,
00
00
H-4 and H-5 ), 7.81 (s, 1H, H-5), 8.06 (dd, 1H, J = 2.8, 1.4 Hz, H-2 ), 8.19 (d, 1H, J = 2.9 Hz,
H-20), 8.29–8.33 (-1H, H-40); 11.86 (bs, 1H, NH); 13C (50 MHz, DMSO-d6)
δ: 109.8 (s), 110.1
(d), 112.3 (d), 120.3 (d), 121.0 (d), 122.4 (d), 122.5 (d), 124.2 (s), 126.3 (d), 127.1 (d), 127.2
(d), 135.9 (s), 136.5 (s), 149.4 (s), 163.0 (s). Anal. Calcd for C15H10N2S2: C, 63.80; H, 3.57; N,
9.92%. Found: C, 63.57; H, 3.45; N, 10.22%.
5-Methoxy-3-[4-(Thiophen-3-yl)-1,3-THIAZOL-2-yl]-1H-indole (1p)
Orange solid; yield: 98%; mp: 183–184 ◦C; IR cm−1: 3264 (NH); 1H NMR (200 MHz,
DMSO-d6)
δ
: 3.87 (s, 3H, CH3), 6.89 (dd, 1H, J = 8.8, 2.5 Hz, H-60), 7.40 (d, 1H, J = 8.8 Hz,
H-70), 7.63–7.75 (m, 2H, H-400 and H-500), 7.75 (s, 1H, H-5), 7.84 (d, 1H, J = 2.5 Hz, H-40),
8.00 (dd, 1H, J = 2.8, 1.4 Hz, H-2 ), 8.07 (d, 1H, J = 2.9 Hz, H-2 ), 11.63 (bs, 1H, NH); 13C (50
00
0
MHz, DMSO-d6) : 55.2 (q), 102.1 (d), 109.6 (d), 110.1 (s), 112.4 (d), 112.9 (d), 122.0 (d), 124.8
δ
(s), 126.2 (d), 127.0 (d), 127.2 (d), 131.5 (s), 135.5 (s), 150.0 (s), 154.6 (s), 163.0 (s). Anal. Calcd
for C16H12N2OS2: C, 61.51; H, 3.87; N, 8.97%. Found: C, 61.30; H, 3.68; N, 9.15%.
5-Bromo-3-[4-(Thiophen-3-yl)-1,3-Thiazol-2-yl]-1H-Indole (1q)
Yellow solid; yield: 72%; mp: 164–165 ◦C; IR cm−1: 3269 (NH); 1H NMR (200 MHz,
DMSO-d6)
2H, H-400 and H-500), 7.80 (s, 1H, H-5), 8.00 (t, 1H, J = 2.1 Hz, H-200), 8.22 (d, 1H, J = 2.8 Hz,
H-20), 8.61 (d, 1H, J = 1.9 Hz, H-40), 11.99 (bs, 1H, NH); 13C (50 MHz, DMSO-d6)
: 110.0
δ
: 7.37 (dd, 1H, J = 8.9, 1.9 Hz, H-60), 7.49 (d, 1H, J = 8.9 Hz, H-70), 7.68–7.69 (m,
δ
(s), 110.2 (d), 113.4 (s), 114.2 (d), 122.0 (d), 122.7 (d), 125.0 (d), 126.0 (s), 126.2 (d), 127.1 (d),
128.2 (d), 135.3 (s), 136.5 (s), 150.3 (s), 162.2 (s). Anal. Calcd for C15H9BrN2S2: C, 49.87; H,
2.51; N, 7.75%. Found: C, 49.66; H, 2.42; N, 7.57%.
3.1.6. General Procedure for the Synthesis of Thiazole Derivatives (2o–q)
To a suspension of appropriate thiazole 2a,d,g (0.38 mL) in DCM (5 mL), trifluoroacetic
acid (7.0 mmol, 0.54 mL) was added, and the mixture was heated under reflux for 24 h.
After cooling, the solvent was evaporated under reduced pressure, and the obtained residue
was recrystallized with ethanol to afford the desidered thiazoles 2-q.
3-[4-(Pyridin-3-yl)-1,3-Thiazol-2-yl]-1H-Indole Hydrobromide (2o)
Yellow solid; yield: 72%; mp: 204–205 ◦C0; IR cm−1: 3558 (NH), 3426 (NH+); 1H NMR
0 0
(200 MHz, DMSO-d6) δ: 7.24–7.31 (m, 2H, H-5 and H-6 ), 7.49–7.57 (m, 1H, H-7 ), 8.05 (dd,
1H, J = 8.1, 5.4 Hz, H-500), 8.25 (d, 1H, J = 2.9 Hz, H-20), 8.35–8.39 (m, 1H, H-40), 8.41 (s, 1H,
H-5), 8.85 (dd, 1H, J = 5.4, 1.8 Hz, H-600), 8.99–9.05 (m, 1H, H-400), 9.49 (d, 1H, J = 1.8 Hz,
H-200), 11.90 (bs, 1H, NH); 13C (50 MHz, DMSO-d6)
δ: 109.9 (s), 112.3 (d), 115.8 (d), 120.5
(d), 121.0 (d), 122.6 (d), 124.1 (s), 127.2 (d), 127.6 (d), 132.9 (s), 136.6 (s), 139.9 (d), 141.1 (d),
141.4 (d), 147.8 (s), 164.4 (s). Anal. Calcd for: C16H12BrN3S: C, 53.64; H, 3.38; N, 11.73%.
Found: C, 53.43; H, 3.24; N, 11.96%.
5-Methoxy-3-[4-(Pyridin-3-yl)-1,3-Thiazol-2-yl]-1H-Indole Hydrobromide (2p)
◦
Orange solid; yield: 95%; mp: 228–229 C; IR cm−1: 3313 (NH), 3421 (NH+); 1H NMR
(200 MHz, DMSO-d6)
: 3.89 (s, 3H, CH3), 6.72 (dd, 1H, J = 8.8, 2.5 Hz, H-60), 7.43 (d, 1H,
J = 8.8 Hz, H-70), 7.84 (d, 1H, J = 2.5 Hz, H-40), 8.09 (dd, 1H, J = 8.1, 5.5 Hz, H-500), 8.19 (d,
δ
0
00
1H, J = 2.9 Hz, H-2 ), 8.42 (s, 1H, H-5), 8.87 (d, 1H, J = 5.5 Hz, H-6 ), 9.06 (d, 1H, J = 8.1 Hz,
00
00
H-4 ), 9.50 (d, 1H, J = 2.0 Hz, H-2 ), 11.79 (d, 1H, J = 2.9 Hz, NH); 13C (50 MHz, DMSO-d6)
δ: 55.3 (q), 102.3 (d), 109.7 (s), 112.4 (d), 113.0 (d), 115.1 (d), 124.7 (s), 126.9 (d), 127.9 (d),
131.6 (s), 132.6 (s), 140.4 (d), 140.6 (d), 141.9 (d), 148.0 (s), 154.8 (s), 164.5 (s). Anal. Calcd for:
C17H14BrN3OS: C, 52.59; H, 3.63; N, 10.82%. Found: C, 52.35; H, 3.51; N, 11.08%.
5-Bromo-3-[4-(Pyridin-3-yl)-1,3-Thiazol-2-yl]-1H-Indole Hydrobromide (2q)
Yellow solid; yield: 86%; mp: 245–246 ◦C; IR cm−1: 3193 (NH), 3422 (NH+); 1H NMR
(200MHz, DMSO-d6)
δ
: 7.40 (dd, 1H, J = 8.6, 2.0 Hz, H-60), 7.51 (d, 1H, J = 8.6 Hz, H-70),
8.00 (dd, 1H, J = 8.1, 5.4 Hz, H-500), 8.31 (d, 1H, J = 2.9 Hz, H-20), 8.39 (s, 1H, H-5), 8.49 (d,

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1H, J = 2.0 Hz, H-40), 8.82 (d, 1H, J = 5.4 Hz, H-600), 8.91 (d, 1H, J = 8.1 Hz, H-400), 9.44 (d,
1H, J = 1.6 Hz, H-200), 12.09 (bs, 1H, NH); 13C (50 MHz, DMSO-d6)
δ: 109.5 (s), 113.6 (s),
114.4 (d), 115.6 (d), 122.6 (d), 125.2 (d), 125.8 (s), 126.7 (d), 129.0 (d), 132.4 (s), 135.3 (s), 140.1
(d), 140.9 (d), 142.3 (d), 148.4 (s), 163.7 (s). Anal. Calcd for: C16H11Br2N3S: C, 43.96; H, 2.54;
N, 9.61%. Found: C, 43.68; H, 2.68; N, 9.42%.
3.2. Biology
3.2.1. Minimum Inhibitory Concentrations (MICs) Determination
MICs were determined by using a microdilution method as recommended by CLSI
for bacteria that grow aerobically (CLSI) (Clinical and Laboratory Standards Institute.
Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically;
Approved Standard—Ninth Edition. CLSI document M07-A9 (ISBN 1-56238-783-9 [Print];
ISBN 1-56238-784-7 [Electronic])) and Tryptic Soy Broth (TSB) (VWR International, Leuven,
Belgium) as medium. It starts with the preparation of the bacterial suspension by diluting
◦
the bacteria grown at 37 C for 24 h on Tryptic Soy Agar (TSA) (VWR International,
LLC, Leuven, Belgium ) in 5 mL of 0.9% NaCl to obtain a culture suspension whose
optical density (O.D.) at 570 nm is of about 0.190 (corresponding to 106 colony-forming
units CFU/mL); a further dilution 1:20 is always carried out in physiological solution.
Serial dilutions 1:2 of the substance to be tested are performed, starting from the highest
concentration, in culture medium (TSB); then, 10 µL of the prepared inoculum was added
to each well containing substance solution. A positive growth control (test bacterial
strain in the medium without inhibitor), a substance control (only the substance solution
without inoculum), and a negative control (only the medium without inoculum), to check
respectively the bacterial growth, the absorbance of substance at the highest concentration,
and the sterility of medium, were also included in the assay. Thus, the plate prepared is
◦
incubated at 37 C for 24 h, MICs were read by a microplate spectrophotometer (GloMax®-
Multi Detection System, Promega Italia s.r.l, Milan, Italy) as the lowest concentration of
sample whose O.D. at 570 nm is comparable to O.D. values of negative control wells.
3.2.2. Inhibition of Biofilm Formation (Crystal Violet Method)
Bacterial strains were incubated in test tubes with TSB (5 mL) containing 2% w/v
glucose at 37 ◦C for 24 h. After that, the bacterial suspensions were diluted to achieve a
turbidity equivalent to a 0.5 McFarland standard. The diluted suspension (2.5
added to each well of a single cell culture polystyrene sterile, flat-bottom 96-well plate filled
with TSB (200 L) with 2% w/v glucose. Sub-MIC concentration values of all compounds
were directly added to the wells to reach concentrations ranging from 100 to 0.1 M to
µL) was
µ
µ
assess BIC50 values—that is, the concentration at which the percentage of inhibition of
biofilm formation is equal to 50%. Plates were incubated at 37 ◦C for 24 h. After biofilm
growth, the content of each well was removed, wells were washed twice with st◦erile NaCl
0.9% and stained with 200 µL of 0.1% w/v crystal violet solution for 15 min at 37 C. Excess
solution was removed, and the plate was washed twice, using tap water. A volume of
200 µL of ethanol was added to each stained well to solubilize the dye. Optical density
(O.D.) was read at 600 nm using a microplate reader (GloMax®-Multi Detection System,
Promega Italia s.r.l, Milan, Italy).
The experiments were run at least in triplicates, and three independent experiments
were performed.
The percentage of inhibition was calculated through the formula:
% Inhibition = (OD growth − OD sample/OD growth control) × 100.
(1)
3.2.3. Antibiofilm Activity Against Pre-Formed Biofilm
A suspension of bacteria (0.5 McFarland standard) was obtained using the procedure
described above for the inhibition of biofilm formation test. First, 2.5 mL of suspension was
added to each well of a 96-well plate containing TSB (200 µL) with 2% w/v glucose. After

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the growth of a biofilm (24 h old), the content of each well was removed; then, wells were
washed up twice with sterile PBS (Phosphate Buffered Saline) and filled with fresh TSB
medium (200 µL). After that, different concentrations of compounds were added starting
from a concentration equal or greater than the MIC obtained against the planktonic form
of tested strains using TSB as the medium. The microtiter plate was sealed and incubated
at 37 ◦C for further 24 h. The content of each well was removed, wells were washed twice
with sterile PBS (100 mL to each well) and the 96-well plate was placed at 37 ◦C for 1 h
before staining with a 0.1% w/v crystal violet solution. After 30 min, plates were washed
with tap water to remove any excess stain.
Biofilm formation was determined by solubilizing crystal violet as above described,
and the absorbance was read at 540 nm using a microplate reader (Glomax Multidetection
System Promega, Promega Italia s.r.l, Milan, Italy). The percentages of inhibition were
calculated with the above-reported. Each assay was performed in triplicate and repeated
at least twice.
3.2.4. Inhibition of Biofilm Formation (Viable Plate Count)
Compounds 1l, 2b, 2c, 2i, and 2k, which exhibited the highest potency in inhibiting
S.aureus ATCC 25923 biofilm formation by the crystal violet method, were tested against
the same strain by using a viable plate counts method [22]. Briefly, a suspension of the
tested strain was obtained as described in Section 3.2.2. Polystyrene flat-bottom 24-well
plates were filled with 2 mL of TSB with 2% w/v glucose; then, we added 25
µL of bacterial
suspension and sub-MIC concentrations (10; 5; 1; 0.1
µg/mL) of the above-mentioned
compounds and incubated them for 24 h at 37 ◦C. After that time, the wells were washed
3 times with 1 mL of sterile NaCl (0.9% v/v solution), and the surface of each well was
scraped 3 times. The inocula were put in test tubes with 10 mL of NaCl (0.9% v/v solution)
and sonicated (ultrasonic nominal power equal to 215 kHz) for 2 min. Eight serial dilutions
1:10 were prepared and 100 µL aliquots of each dilution were plated onto tryptic soy agar
(TSA). Then, petri dishes were incubated at 37 ◦C and CFU/mL were counted after 24 h.
Each assay was performed in triplicate and repeated at least twice. Activity was expressed
as log reduction with respect to the not treated growth control.
3.2.5. Statistical Analysis
Mean values, standard deviation (SD), and significance testing (p-value) were calcu-
lated on a PC with the computer program, Microsoft Excel 2019 (Microsoft Corporation,
Redmond, WA, USA).
4. Conclusions
With the aim to identify novel therapeutic approaches targeting antibiotic resistance
mechanisms, two new series of 3-[4-(thiophen-3-yl)-1,3-thiazol-2-yl]-1H-indoles 1a–n and
3-[4-(pyridin-3-yl)-1,3-thiazol-2-yl]-1H-indole hydrobromides 2a-n were efficiently syn-
thesized and evaluated for their anti-biofilm activity. For almost all the new compounds,
a marked selectivity toward the staphylococcal strains has been observed. In particular,
compounds 1l and 2i showed the highest potency against S. aureus ATCC 25923, eliciting
BIC50 values of 3.9 and 1.0
µM, respectively. Whereas, the 3-[4-(thiophen-3-yl)-1,3-thiazol-
2-yl]-1H-indole 1a and 3-[4-(pyridin-3-yl)-1,3-thiazol-2-yl]-1H-indole 2m proved to be
good inhibitors of P. aeruginosa biofilm formation with BIC50 values of 14.9 and 5.5 µM,
respectively.
Among the novel approaches evaluated in response to the emergence of the antibiotic
resistance, the anti-virulence strategy is considered one of the most encouraging [23].
Disarming the bacteria from their pathogenicity tools, as the biofilm formation, was found
to be more beneficial than interfering with their growth. In this scenario, the new thiazole
derivatives 1l
, 2b, 2c, 2i, and 2m, which proved to be able to interfere with the biofilm
formation, without affecting the microbial vital processes, can be considered promising

Molecules 2021, 26, 81
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lead compounds for the development of new anti-virulence agents usable for the treatment
of biofilm-associated infections or for the prophylaxis of implant surgery.
Author Contributions: A.C., S.C., B.P., D.C., and C.P. performed chemical research and analyzed the
data; D.S. and M.G.C. performed biological research and analyzed the data; P.D. and G.C. participated
in the design of the research and the writing of the manuscript. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by European Union 2014–2020 PON Ricerca e Innovazione grant
from the Italian Ministry of Education, University and Research, entitled “PROGEMA—Processi
Green per l’Estrazione di Principi Attivi e la Depurazione di Matrici di Scarto e Non” (ARS01_00432)
to P.D.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
Sample Availability: Samples of the compounds are available from the authors.
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