Synthesis and biological evaluation of novel indole-derived thioureas

Article abstract of DOI:10.3390/molecules23102554

A series of 2-(1H-indol-3-yl)ethylthiourea derivatives were prepared by condensation of 2-(1H-indol-3-yl)ethanamine with appropriate aryl/alkylisothiocyanates in anhydrous media. The structures of the newly synthesized compounds were confirmed by spectroscopic analysis and the molecular structures of 8 and 28 were confirmed by X-ray crystallography. All obtained compounds were tested for antimicrobial activity against Gram-positive cocci, Gram-negative rods and for antifungal activity. Microbiological evaluation was carried out over 20 standard strains and 30 hospital strains. Compound 6 showed significant inhibition against Gram-positive cocci and had inhibitory effect on the S. aureus topoisomerase IV decatenation activity and S. aureus DNA gyrase supercoiling activity. Compounds were tested for cytotoxicity and antiviral activity against a large panel of DNA and RNA viruses, including HIV-1 and other several important human pathogens. Interestingly, derivative 8 showed potent activity against HIV-1 wild type and variants bearing clinically relevant mutations. Newly synthesized tryptamine derivatives showed also a wide spectrum activity, proving to be active against positive- And negative-sense RNA viruses.

Full text of DOI:10.3390/molecules23102554

molecules
Article
Synthesis and Biological Evaluation of Novel
Indole-Derived Thioureas
Giuseppina Sanna 1 , Silvia Madeddu 1, Gabriele Giliberti 1,*, Sandra Piras 2, Marta Struga 3,4
,
5
6
Małgorzata Wrzosek 4,5, Grazyna Kubiak-Tomaszewska , Anna E. Koziol ,
Oleksandra Savchenko 6, Tadeusz Lis 7, Joanna Stefanska 8, Piotr Tomaszewski 9,
Michał Skrzycki 9 and Daniel Szulczyk 3,4
˙
1
Department of Biomedical Science, University of Cagliari, 09042 Monserrato, Italy;
g.sanna@unica.it (G.S.); silvia.madeddu@unica.it (S.M.)
Department of Chemistry and Pharmacy, University of Sassari, 07100 Sassari, Italy; piras@uniss.it
Chair and Department of Biochemistry, First Faculty of Medicine, Medical University,
2
3
02-097 Warszawa, Poland; mstruga@wum.edu.pl (M.S.); daniel.szulczyk@wum.edu.pl (D.S.)
Laboratory of Centre for Preclinical Research, Medical University of Warsaw, 02-097 Warsaw, Poland;
4
malgorzata.wrzosek@wum.edu.pl
Department of Biochemistry and Pharmacogenomics, Faculty of Pharmacy, Medical University of Warsaw,
02-097 Warsaw, Poland; grazyna.kubiak-tomaszewska@wum.edu.pl
5
6
Faculty of Chemistry, Maria Curie-Sklodowska University, 20-031 Lublin, Poland;
anna.koziol@poczta.umcs.lublin.pl (A.E.K.); oleksandra.savchenko@poczta.umcs.lublin.pl (O.S.)
Faculty of Chemistry, Univeristy of Wrocław, 50-383 Wrocław, Poland; tadeusz.lis@chem.uni.wroc.pl
Department of Pharmaceutical Microbiology, Medical University, 02-007 Warszawa, Poland;
joanna.stefanska@wum.edu.pl
Department of Biochemistry, Second Faculty of Medicine, Medical University of Warsaw,
02-097 Warszawa, Poland; piotr.tomaszewski@wum.edu.pl (P.T.); mskrzycki@wum.edu.pl (M.S.)
Correspondence: ggiliberti@unica.it; Tel.: +39-070-6754-275
7
8
9
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 16 July 2018; Accepted: 26 September 2018; Published: 7 October 2018
Abstract: A series of 2-(1H-indol-3-yl)ethylthiourea derivatives were prepared by condensation
of 2-(1H-indol-3-yl)ethanamine with appropriate aryl/alkylisothiocyanates in anhydrous media.
The structures of the newly synthesized compounds were confirmed by spectroscopic analysis
and the molecular structures of
8 and 28 were confirmed by X-ray crystallography. All obtained
compounds were tested for antimicrobial activity against Gram-positive cocci, Gram-negative rods
and for antifungal activity. Microbiological evaluation was carried out over 20 standard strains
and 30 hospital strains. Compound
6 showed significant inhibition against Gram-positive cocci
and had inhibitory effect on the S. aureus topoisomerase IV decatenation activity and S. aureus
DNA gyrase supercoiling activity. Compounds were tested for cytotoxicity and antiviral activity
against a large panel of DNA and RNA viruses, including HIV-1 and other several important human
pathogens. Interestingly, derivative 8 showed potent activity against HIV-1 wild type and variants
bearing clinically relevant mutations. Newly synthesized tryptamine derivatives showed also a wide
spectrum activity, proving to be active against positive- and negative-sense RNA viruses.
Keywords: antibacterial activity; anti-HIV activity; antiviral activity; thiourea derivatives of indole;
topoisomerase
1. Introduction
Indole derivatives have attracted a great deal of attention due to their wide therapeutic applications
(Figure 1). Tryptamine-containing arylpiperazine derivatives (e.g., delavirdine, ateviridine) belong
Molecules 2018, 23, 2554; doi:10.3390/molecules23102554
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2554
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to the group of non-nucleoside reverse transcriptase inhibitors (NNRTIs), used in the treatment of
AIDS that represents the major health problem worldwide, with 1.0 million deaths and 36.7 million
people living with HIV in 2016 [1]. Anti-retroviral therapy, nowadays based on combinations of drugs
belonging to the NNRTI, nucleoside reverse transcriptase (NRTI), protease (PR) and, more recently,
integrase (IN) inhibitor classes, has improved the quality of life of AIDS patients. However, due to a
side effect and rapid emergence of drug-resistant viruses drug [2], a continuous development of new
anti-HIV compounds is needed to overcome these limitations.
Figure 1. Pharmacologically active indole ring-containing derivatives.
Indole-derived azonine compounds, such as pericine, are CNS-active alkaloids, ligands of opioid
receptors. Gliotoxin and its analogues act as antimicrobials and have antiproliferative activity in
lymphosarcoma cells. Indometacin is a non-steroidal anti-inflammatory agent that reduces fever,
pain or swelling. Current research on panobinostat is focused on its antiproliferative effects in various
cancer cell lines [3].
In order to search for potent inhibitors of hepatitis B virus (HBV) and hepatitis C virus (HCV),
a series of N-substituted tryptamine analogues were synthesized [
1,3,5-triazine derivatives of tryptamine show anti-HIV activity both by inhibiting cell-free virus
infection and cell-to-cell virus transmission [ ]. Furthermore, various families of indole-derived
compounds exhibit antimicrobial activity against Gram-positive [ 11] and Gram-negative bacteria [ 10],
as well as fungi [ 12]. The cytotoxic [13 16] and anticancer potency of substituted indole compounds
4,5]. What is more, series of
6
7–
7,9,
7
,
–
was also proven. In this field they act as inhibitors of serine/threonine kinase (AKT) [17], tyrosinase [18],
human sirtuins SIRT1-3 [19] or transcription factors NF-kB activation [20].
The thiourea moiety is an important synthon responsible for numerous biological activities,
such as antimicrobial [21–23] antiviral [24,25], antiproliferative [26] and cytotoxic effects [22,27].
The 1,3-disubstituted thiourea scaffold is a structure with a huge potential for multi-direction activity.
Through rationally planned modifications of thiourea derivative structures, a wide range of compounds
with high pharmacological activity and low side-effects can be obtained. The literature indicates
that including an electron acceptor group (e.g., a halogen atom) in a strategic part of a molecule
significantly influences its biological activity, leading to an increase in lipophilicity, and as a result in
the speed of absorption and transport of substances in vivo. The presence of a strongly electronegative
substituent also changes the properties of the adjacent functional groups, which influences the reactivity,

Molecules 2018, 23, 2554
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metabolic stability and chemical nature of the molecule [28
–30]. High stability, in turn, increases the
bioavailability of the compound, which improves its pharmacokinetic parameters. For these reasons,
halogen derivatives constitute ca. 20% of the total number of medical drugs currently available
on the world market [29]. Encouraged by the above observations and considering the interesting
pharmacological profile of indole derivatives, we have synthesized new tryptamines incorporating the
1,3-disubstituted thiourea scaffold as promising antimicrobial and antiviral agents.
2. Results and Discussion
2.1. Chemistry
The synthesis of 28 thiourea derivatives is described. Sixteen of them had not been described
previously in the literature. To prepare the thiourea derivatives 2-(1H-indol-3-yl)ethanamine was
condensed with the corresponding isothiocyanate (Scheme 1 and Table 1).
Scheme 1. Synthesis of thiourea derivatives of 2-(1H-indol-3-yl)ethanamine.
Table 1. Structure and molar weight of the investigated compounds 1–28.
S
NH
R
NH
N
H
Number
R
Mol. Weight (g/mol)
1
2
3
4
5
6
7
8
Phenyl
4-Methoxyphenyl
4-Methylphenyl
4-Chlorophenyl
Benzoyl
3,4-Dichlorophenyl
3-Bromophenyl
4-Bromophenyl
Cyclohexyl
295.40
325.43
309.43
329.85
323.41
364.29
374.30
374.30
301.45
309.43
313.39
313.39
329.85
329.85
374.30
323.46
313.39
259.37
273.40
291.37
421.30
247.36
343.87
363.40
233.33
343.87
365.53
347.84
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Benzyl
2-Fluorophenyl
3-Fluorophenyl
2-Chlorophenyl
3-Chlorophenyl
2-Bromophenyl
Phenethyl
4-Fluorophenyl
Allyl
Methallyl
Ethoxycarbonyl
4-Iodophenyl
Ethyl
3-Chloro-4-methylphenyl
3-Trifluoromethylphenyl
Methyl
3-Chloro-6-methylphenyl
4-Butyl-2-methylphenyl
3-Chloro-4-fluorophenyl

Molecules 2018, 23, 2554
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Obtained compounds were purified by column chromatography and/or crystallized from a
1
nonpolar solvent. MS, H-NMR and 13C-NMR spectra confirmed the identity of the products.
The molecular structures of
8 and 28 (Supplementary Material Figure S1) were also determined
by an X-ray crystal structure analysis.
2.2. Microbiology
Twenty eight thiourea derivatives of tryptamine were examined in vitro against Gram-positive
cocci, Gram-negative rods and Candida albicans. The microorganisms used in this study have universal
utility in the antimicrobial tests in the research for new active agents [21]. All tested compounds were
screened by disc diffusion method [31], next these showing significant activity, were examined for their
minimal inhibitory concentration (MIC) [32]. The results of antimicrobial activity are summarized in
Table 2. In Table 2 only the results for active derivatives are included.
Table 2. Activities against Gram-positive bacteria—minimal inhibitory concentrations (MIC,
and diameter of growth inhibitory zone (GIZ, mm, applied 400 µg per disc).
µg/mL)
1
Strains
6
14
15
19
Ref.
S. aureus NCTC 4163
S. aureus ATCC 25923
S. aureus ATCC 6538
S. aureus ATCC 29213
S. epidermidis ATCC 12228
B. subtilis ATCC 6633
B. cereus ATCC 11778
M. luteus ATCC 9341
6.25 (10)
6.25 (15)
12.5 (16)
6.25 (15)
12.5 (16)
12.5 (15)
6.25 (15)
12.5 (15)
6.25 (23)
50 (13)
50 (14)
50 (13)
50 (15)
50 (12)
50 (12)
25 (15)
200 (12)
12.5 (20)
50 (11)
100 (11)
50 (12)
50 (11)
-
400 (12)
400 (12)
200 (11)
-
0.25 (26)
0.5 (26)
0.25 (28)
0.5 (22)
-
0.25 (30)
<0.12 (38)
0.25 (26)
0.25 (26)
1 (25)
50 (15)
50 (14)
200 (13)
50 (14)
400 (13)
200 (13)
200 (13)
50 (14)
M. luteus ATCC 10240
1
Ref.—ciprofloxacin (GIZ—5 µg/9 mm disc). Lack of the growth inhibition area.
Preliminary tests by disc-diffusion method indicated antimicrobial activity against standard
Gram-positive cocci. The next step was estimation of MIC values for not only standard but also
hospital strains. The study was carried out over 20 standard, 20 hospital of Staphylococcus aureus and 10
hospital of Staphylococcus epidermidis strains used for routine antimicrobial media susceptibility testing.
Hospital strains were isolated from different biological materials of the patients hospitalized in the
Warsaw Medical University hospitals. MIC values for the standard Gram-positive strains were in the
range 400–6.25
µg/mL (Table 3). For the hospital Staphylococcus epidermidis (MSSA) rods the MIC value
ranged from 400 to 6.25 µg/mL and the average value was 25 µg/mL.

Molecules 2018, 23, 2554
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Table 3. Activity of compounds against hospital methicillin-susceptible strains of S. aureus (MSSA),
methicillin-resistant strains of S. aureus (MRSA) and S. epidermidis (MRSE)—minimal inhibitory
concentrations (MIC, µg/mL).
S. aureus MSSA
440/11 441/11 442/11 443/11 444/11
445/11
446/11
447/11 448/11 449/11
6
14
6.25
25
0.4
6.25
25
0.4
6.25
25
0.4
6.25
25
0.4
6.25
12,5
0.8
6.25
25
0.4
6.25
25
0.4
6.25
25
0.8
6.25
25
0.8
6.25
25
0.4
Ciprofloxacin
S. aureus MRSA
389/10 390/10 391/10 392/10 393/10
394/10
399/10
450/11 451/11 452/11
6
14
6.25
25
25
6.25
25
100
6.25
25
100
6.25
25
100
6.25
25
50
6.25
25
25
6.25
25
200
6.25
25
100
6.25
25
100
6.25
25
200
Ciprofloxacin
S. epidermidis MRSE
430/11 431/11 432/11 433/11 434/11
435/11
436/11
437/11 438/11 439/11
6
6.25
25
400
12.5
6.25
6.25
50
6.25
25
50
6.25
25
50
12.5
25
50
6.25
25
100
50
6.25
50
100
100
6.25
25
100
200
6.25
50
200
100
6.25
25
50
14
15
19
100
12.5
50
12.5
50
Ciprofloxacin
0.8
6.25
50
50
50
0.4
0.4
0.8
0.4
50
On the other hand, the MIC values obtained for the hospital Staphylococcus aureus (MSSA) for the
derivatives 6 and 14 varied in the range 25–6.25 µg/mL. MRSA strains were also susceptible to these
compounds, and the MIC value range changed from 25 to 6.25 µg/mL. The comparison between the
chemical structure and the biological activity led to the conclusion that thiourea-derived tryptamines
with halogen substituents at the ring are more potent antimicrobials than alkylthioureas. Analysis of a
position and a type of an electronegative element at a benzene ring showed that chlorine at the position
C-3 promotes antimicrobial activity (derivatives
second chlorine atom in the 4-position increases antimicrobial activity (compound
of another substituent in the 4-position (fluorine, methyl) causes the disappearance of antimicrobial
activity (compounds 23 28). Other halogens (F and Br) in position 3 do not affect antimicrobial
6
and 14). Substitution of the benzene ring with a
6). The introduction
,
activity. On the other hand, the substitution of the benzene ring with bromine at position 2 implies
the moderate antimicrobial activity, as in the previous derivatives, only for Gram-positive strains
(compound 15). The methallyl indole derivative 19 expressed a moderate antibacterial activity. These
results are in the general agreement with our previous research concerning the activity of heterocyclic
thiourea compounds [33]. To assess the mechanism of antimicrobial activity, S. aureus topoisomerase
IV decatenation assay and S. aureus DNA gyrase supercoiling assay were performed. Ciprofloxacin
(CFX) was used as a positive control. The decatenation activity of S. aureus topoisomerase IV was
almost completely inhibited by CFX at 32
S. aureus topoisomerase with IC50 = 28.9
supercoiling activity of S. aureus gyrase but weaker than that of S. aureus topoisomerase, and the IC50
for inhibition of DNA gyrase supercoiling activity for compound 6 was 72.6 ± 1.2 µg/mL (Table 4).
µ
g/mL and compound
6
has an inhibitory activity against
shows inhibition of the
±
0.3 g/mL (Table 4). Compound 6
µ
Table 4. Inhibitory activities against DNA topoisomerase IV and DNA gyrase, expressed as IC50
(µg/mL).
±
SEM
Compounds
S. aureus Topoisomerase IV
S. aureus DNA Gyrase
6
28.90 ± 0.30
72.60 ± 1.2
Ciprofloxacin
1.70 ± 0.15
3.55 ± 0.13
IC50—half of the maximal inhibitory concentration.
On the contrary, compound 14 was not significantly effective against DNA topoisomerase IV
(Figure 2) and DNA gyrase (data not shown).

Molecules 2018, 23, 2554
6 of 17
Figure 2. Effect of the tested compounds on S. aureus topoisomerase IV activity. Decreasing amounts of
compounds 14 and were incubated with 200 ng kinetoplast DNA and run on agarose gel. Lane 1:
6
incubation mixture without enzyme (negative control). Lane 2: S. aureus topoisomerase IV assay
with dilution buffer (control). Lane 3: S. aureus topoisomerase IV with DMSO (control). Lane: 4:
Ciprofloxacin at concentration 32
µg/mL. Lane: 5–11: compound 14 at concentrations 64, 32, 16,
8, 4, 2, 0.5 g/mL, respectively. Lane: 12–18: compound
µ
6
at concentrations 64, 32, 16, 8, 4, 2,
0.5 µg/mL, respectively.
2.3. Antiviral and Antiproliferative Evaluation
All twenty-eight thiourea derivatives were tested in cell-based assays against the Human
Immunodeficiency Virus type-1 (HIV-1) and against representative of several RNA and DNA virus
families. The results are reported in the Tables 5 and 6.
Table 5. Antiviral activity of thiourea derivatives against HIV-1 and ssRNA+ (BVDV, YFV, CV-B5, Sb-1)
viruses and cytotoxicity against the cell lines used in the assays.
MT-4 a
CC50
MDBK c
CC50
BHK e
CC50
Vero-76 g
CC50
HIV-1 b
EC50
BVDV d
EC50
YFV f
EC50
CV-B5 h
EC50
Sb-1 i
EC50
Compounds
1
2
3
4
5
6
7
8
31
32
33
45
38
45
45
45
49
78
48
47
47
42
46
49
44
>31
>32
>33
>45
>38
>100
>100
>100
>100
67
27
55
52
55
>100
69
49
49
32
48
>100
53
>100
>100
>100
>100
>100
25
>100
>100
≥100
>100
20 ± 1.5
>27
48
44
16
47
34
10
10
10
13
52
34
16
11
6.0
28
18
30
>100
35
>100
11
>100
10
9.0
>100
12
9.5
30
>48
>44
>16
>47
>34
>10
>10
>10
>13
>52
>34
>16
>11
>6.0
>28
>18
>30
>100
>35
>100
>11
>100
>10
>9.0
>100
>12
>9.5
>30
64
56
75
55
29
13
12
13
14
>100
73
60
53
32
46
>100
53
>100
89
>100
11
>64
>56
>75
>55
>29
>13
>12
>13
>14
>64
>56
>75
>55
>29
>13
>12
>13
>14
>100
>73
>60
>53
>32
>46
>100
>53
>100
>89
>100
>11
>100
>17
>16
>100
>17
>12
>32
>45
>45
20 ± 2.0
8.7 ± 0.4
>52
9
>49
>55
>100
>69
>49
>49
>32
>48
46
>53
>100
≥100
35 ± 2.8
>100
>100
>25
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
>78
>48
>47
>47
>42
>46
>49
>44
>100
>100
>100
>2.4
>100
>44
>44
>100
>45
30 ± 2.4
>73
>60
12 ± 0.9
9.0 ± 1.2
>46
18 ± 1.7
>53
>100
>100
>100
2.4 ± 0.2
>100
44
44
>100
45
>100
>89
>100
>11
>100
>17
>16
>100
>17
>12
20 ± 2.1
>100
17
16
>100
17
12
23
>100
42
11
37
>23
>100
15 ± 1.2
>11
13 ± 1.2
45
45
>45
>45
32
Efavirenz
40
0.002 ± 0.0002
0
2 -C-methyl-guanosine
2 -C-methyl-cytidine
>10
1.1 ± 0.1
>10
1.9 ± 0.1
0
>100
18 ± 0.9
7.3 ± 0.4
Data represent mean values for three independent determinations. Standard deviations are reported for the more
a
active compounds. Also for the others values the variation was less than 15%. Compound concentration (
µ
M)
b
required to reduce the proliferation of mock-infected MT-4 cells by 50%. Compound concentration (
µM) required
c
to achieve 50% protection of MT-4 cells from HIV-1 induced cytopathogenicity. Compound concentration (
µ
M)
d
required to reduce the viability of mock-infected MDBK cells by 50%. Compound concentration (
µM) required to
e
achieve 50% protection of MDBK cells from BVDV-induced cytopathogenicity. Compound concentration (
µ
M)
f
required to reduce the viability of mock-infected BHK cells by 50%. Compound concentration (
µ
M) required
g
to achieve 50% protection of BHK cells from YFV-induced cytopathogenicity. Compound concentration (
µ
M)
h,i
required to reduce the viability of mock-infected VERO-76 cells by 50%. Compound concentration (
µM) required
to reduce the plaque number of CV-B5 h, Sb-1 i by 50% in VERO-76 monolayers.

Molecules 2018, 23, 2554
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Table 6. Antiviral activity of thiourea derivatives against ssRNA-(RSV, VSV), dsRNA (Reo-1) and
dsDNA (VV, HSV-1) viruses and cytotoxicity against the cell lines used in the assays.
BHK a
CC50
Vero-76 c
CC50
VSV e
EC50
HSV-1 g
EC50
Reo-1 b
EC50
RSV d
EC50
VV f
EC50
Compounds
1
2
3
4
5
6
7
8
48
44
16
47
34
10
10
10
13
52
34
16
11
6.0
28
18
30
>100
35
>100
11
>100
10
9.0
>100
12
9.5
30
>48
>44
>16
>47
>34
>10
>10
>10
>13
>52
>34
>16
>11
>6.0
>28
>18
>30
61
>35
>100
>11
>100
>10
>9.0
>100
>12
>9.5
>30
64
56
75
55
29
13
12
13
14
>100
73
60
53
32
46
>100
53
>100
89
>100
11
>64
>56
>64
>56
>75
>55
>29
>13
>12
>13
>14
>100
>73
>60
>53
>32
>46
>100
>53
>100
>89
>100
>11
>100
>17
>16
>100
>17
>12
>32
>64
>56
>75
>55
>29
>13
>12
>13
>14
>100
>73
>60
>53
>32
>46
>100
>53
>100
>89
>100
>11
>100
>17
>16
>100
>17
>12
>32
>64
>56
>75
>55
>29
>13
>12
>13
>14
>100
>73
>60
>53
>32
>46
>100
>53
>100
>89
>100
>11
>100
>17
>16
>100
>17
>12
>32
20 ± 3.0
>55
>29
>13
>12
>13
>14
>100
>73
>60
>53
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
>32
20 ± 2.5
30 ± 2.6
>53
>100
>89
>100
>11
>100
>17
>16
>100
>17
>12
>32
>100
17
16
>100
17
12
32
0
2 -C-methyl-cytidine>100
6-Aza-uridine
10 ± 1.2
12
1.4 ± 0.06
1.5 ±
Mycophenolic acid
Acycloguanosine
13
0.2
>100
3.0 ± 0.2
Data represent mean values for three independent determinations. Standard deviations are reported for the more
a
active compounds. Also for the others values the variation was less than 15%. Compound concentration (
µ
M)
b
required to reduce the proliferation of mock-infected BHK cells by 50%. Compound concentration (
µ
M) required
M)
µM) required
c
to achieve 50% protection of BHK cells from Reo-1-induced cytopathogenicity. Compound concentration (
µ
required to reduce the viability of mock-infected VERO-76 cells by 50%. d–g Compound concentration (
to reduce the plaque number of RSV d, VSV e, VV f, HSV-1 g by 50% in VERO-76 monolayers.
Interestingly, fourteen derivatives exhibited an antiviral activity, although in some cases not
very potent, against one or more viruses. None of them turned out to be active against YFV, Sb-1,
VSV, VV and HSV-1; also the activity against Reo-1 of derivative 18 was not particularly significant.
In general, tryptamine derivatives showed different degree of cytotoxicity for the cell monolayers
used to support the multiplication of different viruses (MDBK, BHK, Vero-76) in stationary growth;
in particular, sixteen compounds turned out to be cytotoxic against all cell lines used, four compounds
turned out to be not cytotoxic.
Only derivative
moderate cytotoxicity (CC50 = 45
16 20 26 28) showed activity against BVDV, a member of Pestiviruses that have a serious
impact on livestock [34], associated in two cases (16 20) with lack of cytotoxicity for MDBK cell lines.
Five compounds (10 13 14 16, 28) exhibited activity (EC50 from 9.0 up to 30 M) against CV-B5
virus, a member of Enterovirus genus that are important human pathogens that cause both acute and
8
showed interesting activity (EC50 = 8.7
µM) against HIV-1, associated with a
µM). As far as the antiviral activity is concerned, six compounds
(5
,
7
,
,
,
,
,
,
,
,
µ

Molecules 2018, 23, 2554
8 of 17
chronic diseases in infants, young children and immunocompromised individuals [35]. Finally, three
derivatives ( 15 16) resulted moderately active against RSV, the most common respiratory pathogen
3
,
,
in infants and young children worldwide [36].
Since a critical issue in the long-term clinical management of HIV disease is the development
of drug resistance, compound
confer selective resistance to NRTI and NNRTI inhibitors, and that often appear during HAART
therapy, reducing its effectiveness [37
38]. Interestingly, the activity against A17, AZTR and MDR
8 was further tested against a set of viruses possessing mutations that
,
strains (Table 7) is comparable with those of HIV-1 wild-type, demonstrating its activity against
variants carrying clinically relevant NRTI and NNRTI mutations.
Table 7. Cytotoxicity and antiviral activity of tryptamine derivative
NNRTI-resistant mutants.
8 against HIV-1 and its NRTI- and
a
b
CC50
MT
45
EC50
Compounds
HIV-1
N119
>45
A17
EFVR
>45
AZTR
MDR
9.0 ± 0.8
NA
8
8.7 ± 0.8
12 ± 1.5
9.0 ± 1.0
Efavirenz
AZT
40
0.002 ± 0.0003
0.022 ± 0.004
0.080 ± 0.005
0.018 ± 0.002
0.025 ± 0.004
6.3 ± 0.05
0.075 ± 0.009
0.01 ± 0.001
80
7.0 ± 0.8
0.001 ± 0.0002
0.35 ± 0.04
0.078 ± 0.008
45
0.02 ± 0.001
0.08 ± 0.006
5.0 ± 0.6
M) required to
µM) required to achieve
Nevirapine
>100
100
a
Data represent mean values for three independent determinations. Compound concentration (
reduce the proliferation of mock-infected MT-4 cells by 50%. Compound concentration (
50% protection of MT-4 cells from HIV-1 induced cytopathogenicity.
µ
b
Generally, no correlation could be found between the type of substituent in position 3 of the
thiourea and antiviral activity. It can only be seen that aliphatic substituents (cyclohexyl, ethyl, methyl)
block both antimicrobial and antiviral activity (compounds 9, 22, 25). Other derivatives, those with
an aromatic ring or a double bond (allyl, methallyl) are characterized by activity against various
viruses. Many of the tested compounds showed a moderate cytotoxicity against the MT4 cells, with
the compound 21 interestingly turning out to be cytotoxic at a very low value (CC50 = 2.4
µM).
Its antiproliferative properties were further evaluated against different cell lines derived from human
haematological tumours (Table 8), showing potent activities at micromolar level (CC50 = 5.0–12
and confirming its promising potential.
µM)
Table 8. Cytotoxicity of tryptamine derivative 21 against human leukaemia/lymphoma cell lines.
a
CC50
Compounds
WIL-2NS c
CCRF-CEM b
5.0 ± 0.8
CCRF-SB d
12 ± 1.6
21
5.8 ± 1.0
Doxorubicin
0.02 ± 0.002
0.02 ± 0.003
0.03 ± 0.003
a
Data represent mean values for three independent determinations. Compound concentration (
µM) required to
reduce cell proliferation by 50%, under conditions allowing untreated controls to undergo at least three consecutive
b
c
rounds of multiplication. CD4+ human acute T-lymphoblastic leukaemia. Human splenic B-lymphoblastoid
d
cells. Human acute B-lymphoblastic leukemia.
Biological data suggested that tryptamine derivatives proved to be active against different
viruses. Their wide spectrum of activity against different RNA viruses and their different degree of
cytotoxicity offer interesting indications for SAR studies, with the aim to design and develop more
potent derivatives. Of particular interest are the derivative 8, strongly active against HIV-1 and variants
carrying clinically relevant NRTI and NNRTI mutations, and the derivative 16 that showed activity,
although moderate, against three different RNA viruses, belonging to both ssRNA+ (BVDV, CV-B5)
and ssRNA-(RSV), with lack of cytotoxicity (CC50 > 100 µM) for MDBK and Vero-76 cell lines.

Molecules 2018, 23, 2554
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3. Materials and Methods
3.1. General Information
1H-NMR and 13C-NMR spectra were recorded on a model Avance DMX 300 spectrometer (Bruker,
Billerica, MA, USA, 1H at 300 MHz and 13C at 75, MHz respectively). The chemical shift values are
expressed in ppm relative to TMS as an internal standard. Mass spectral ESI measurements were
carried out on a ZQ Micromass instrument (Waters, Milford, MA, USA) equipped with a quadrupole
mass analyzer. The spectra were recorded in the positive ion mode at a declustering potential of
40–60 V. The samples were previously separated on a UPLC column (C18) using an ACQUITY UPLC
system by Waters connected with a DPA detector. Flash chromatography was performed on silica gel
60 (200–400 mesh, Merck, Kenilworth, NJ, USA) using chloroform/methanol (19:1 vol) mixture as
eluent. Analytical TLC was carried out on silica gel F254 (Merck) plates (0.25 mm thickness).
The diffraction data for the X-ray crystal structure analysis were collected for
8 and 28 at 200 K and
150 K, respectively, with an Xcalibur CCD diffractometer (Oxford Diffraction Ltd, Abingdon, UK), using
graphite monochromated MoKα radiation. Crystal structures were solved by direct methods using
the SHELXS-97 program and refined by full-matrix least squares method on F2 using the SHELXL-97
program [39]. All ordered non-hydrogen atoms were refined with anisotropic displacement parameters.
Hydrogen atoms were positioned geometrically and allowed to ride on their parent atoms, with
Uiso(H) = 1.2 Ueq(C). The experimental details and final atomic parameters for 8 and 28 have been
deposited with the Cambridge Crystallographic Data Centre as supplementary material (CCDC ID:
992,428 and 992,429). Copies of the data can be obtained free of charge on request by e-mailing
data_request@ccdccam.ac.uk or via www.ccdc.cam.ac.uk/data_request/cif.
3.2. Chemistry
General Procedure for the Synthesis of Thiourea Derivatives of 2-(1H-indol-3-yl)ethanamine
A solution of 2-(1H-indol-3-yl)ethanamine (0.0038 mol, 0.61 g) in anhydrous acetonitrile (25 mL)
was treated with an appropriate isothiocyanate (0.0042 mol) and the mixture was refluxed for 8 h.
Then solvent was removed on rotary evaporator. The residue was purified by column chromatography
(chloroform/methanol; 9.8:0.2 vol.). The compound was crystallized from acetonitrile or another
appropriate solvent.
1-(2-(1H-Indol-3-yl)ethyl)-3-phenylthiourea (
1-(2-(1H-Indol-3-yl)ethyl)-3-(4-methoxyphenyl)thiourea (
(DMSO-d6) (ppm): 3.07 (t, 2H, CH2, J = 6.6 Hz); 3.77 (s, 3H, CH3); 3.89 (t, 2H, CH2, J = 6.3 Hz); 6.42 (s,
1H, NH); 6.71 (d, 1H,CHarom., J = 8.7 Hz); 6.83 (d, 2H, CHarom., J = 9.3 Hz); 7.02 (bs, 1H, NH); 7.35–7.41
(m, 3H, CHarom.); 7.55–7.67 (m, 3H, CHarom.); 7.99 (bs, 1H, NH). 13C-NMR (DMSO-d6)
(ppm): 24.6
1). This compound was synthesized as described previously [40].
◦
1
2
). Yield 87%. m.p. 146–148 C. H-NMR
δ
δ
(CH2), 44.6 (CH2), 55.2 (CH3), 111.3 (C), 111.6 (CH), 113.9 (CH, CH), 118.2 (CH), 118.5 (CH), 120.9 (CH),
122.7 (CH), 125.8 (CH), 127.3 (C), 128.6 (C), 131.5 (C), 136.3 (C), 156.5 (CH), 180.4 (C). HRMS (ESI) calc.
for C18H19N3OS [M − H]−: 324.4279, found: 324.4298.
1-(2-(1H-Indol-3-yl)ethyl)-3-(4-methylphenyl)thiourea (3). This compound was synthesized as described
previously [41].
1-(2-(1H-Indol-3-yl)ethyl)-3-(4-chlorophenyl)thiourea (4). This compound was synthesized as described
previously [39].
1-(2-(1H-Indol-3-yl)ethyl)-3-benzoylthiourea (5). This compound was synthesized as described
previously [42].
◦
1
1-(2-(1H-indol-3-yl)ethyl)-3-(3,4-dichlorophenyl)thiourea (
(DMSO-d6) (ppm): 2.99 (t, 2H, CH2, J = 7.2 Hz); 3.76 (q, 2H, CH2, J = 5.4 Hz); 6.98 (t, 1H, CHarom.,
J = 7.8 Hz); 7.08 (t, 1H,CHarom., J = 7.2 Hz); 7.18 (d, 1H, CHarom., J = 2.4 Hz); 7.30–7.36 (m, 2H, CHarom.);
6). Yield 81%. m.p. 168–169 C. H-NMR
δ

Molecules 2018, 23, 2554
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7.52 (d, 1H,CHarom., J = 8.7 Hz); 7.62 (d, 1H, CHarom., J = 7.8 Hz); 7.85 (s, 1H, CHarom.); 7.99 (bs, 1H,
NH); 9.72 (s, 1H, NH); 10.84 (s, 1H, NH). 13C-NMR (DMSO-d6)
(ppm): 24.3 (CH2), 44.5 (CH2), 111.4
δ
(C), 111.4 (CH), 118.3 (CH), 118.4 (CH), 120.9 (CH), 122.5 (CH), 122.8 (CH), 123.7 (CH), 125.3 (C), 127.2
−
(C), 130.2 (C), 130.5 (C), 136.3 (C), 139.7 (CH), 180.2 (C). HRMS (ESI) calc. for C17H15 Cl2N3S [M
363.2921, found: 363.2943.
−
H] :
1-(2-(1H-Indol-3-yl)ethyl)-3-(3-bromophenyl)thiourea (
(DMSO-d6) (ppm): 3.09 (t, 2H, CH2, J = 6.6 Hz); 3.97 (q, 2H, CH2, J = 5.7 Hz); 6.06 (s, 1H, NH); 6.81 (d,
7
). Yield 79%. m.p. 152–154 ◦C. 1H-NMR
δ
1H, CHarom., J = 7.2 Hz); 7.00 (s, 1H, CHarom.); 7.05 (d, 1H, CHarom., J = 8.1 Hz); 7.08 (d, 1H, CHarom.,
J = 9.0 Hz); 7.13 (s, 1H, CHarom.); 7.20 (t, 1H, CHarom., J = 7.2 Hz); 7.29 (d, 1H,CHarom., J = 8.1 Hz); 7.36
(d, 1H, CHarom., J = 8.1 Hz); 7.40 (bs, 1H, NH); 7.57–7.60 (d, 1H, CHarom., J = 8.7 Hz); 8.03 (s, 1H, NH).
13C-NMR (DMSO-d6)
δ (ppm): 24.3 (CH2), 44.5 (CH2), 111.4 (C), 111.5 (CH), 118.3 (CH), 118.5 (CH),
121.0 (CH), 121.1 (CH), 121.3 (CH), 122.8 (CH), 124.9 (CH), 126.3 (C), 127.2 (C), 130.4 (C), 136.3 (C),
141.2 (CH), 180.1 (C). HRMS (ESI) calc. for C17H16BrN3S [M − H]−: 373.2980, found: 373.2978.
1-(2-(1H-Indol-3-yl)ethyl)-3-(4-bromophenyl)thiourea (
(DMSO-d6) (ppm): 3.09 (t, 2H, CH2, J = 6.6 Hz); 3.95 (t, 2H, CH2, J = 6.6 Hz); 6.24 (bs, 1H, NH);
8
). Yield 81%. m.p. 156–158 ◦C. 1H-NMR
δ
6.71 (d, 1H, CHarom., J = 8.7 Hz); 6.97 (s, 1H, CHarom.); 7.11 (t, 1H, CHarom., J = 7.8 Hz); 7.17–7.29
(m, 3H, CHarom.); 7.39 (d, 1H,CHarom., J = 8.1 Hz); 7.56 (d, 1H, CHarom., J = 8.1 Hz); 7.62 (bs, 1H,
NH); 8.01 (bs, 1H, NH). 13C-NMR (DMSO-d6)
δ
(ppm): 24.4 (CH2), 44.5 (CH2), 111.4 (C), 111.5 (CH),
115.8 (CH), 118.2 (CH, CH), 118.5 (CH), 121.0 (CH), 122.8(CH), 124.7 (CH), 127.2 (CH), 131.3(C, C),
136.3 (C), 138.7, 180.1 (C). HRMS (ESI) calc. for C17H16BrN3S [M
H]−: 373.2980, found: 373.2978.
Crystal data: crystal system monoclinic, space group Cc, unit cell dimensions a = 35.17(2), b = 10.863(4),
c = 8.798(3) Å,
= 100.44(5) ◦, V = 3306(3) Å3; Z = 8, dc = 1.504 g/cm3, = 2.613 mm−1, F(000) = 1520.
0.04 mm was used for intensity measurements. Within the
13, 10), 9510 reflections were collected. The 4871
−
β
µ
A crystal of dimensions 0.20
×
0.15
×
θ
◦
range 2.98–26.31 (
−
43
≤
h
≤
33, 13
−
≤
k
≤
−
7
≤ ≤
l
unique reflections [R(int) = 0.0699] were used for the refinement of 397 parameters. Final R indices
on F2 for 2599 observed reflections [I > 2
σ(I)] were: R1 = 0.0615, wR2 = 0.0825, goodness-of-fit = 0.941,
∆$max/min = 0.57/−0.44 e Å−3
.
1-(2-(1H-Indol-3-yl)ethyl)-3-cyclohexylthiourea (9). This compound was synthesized as described
previously [40].
1-(2-(1H-Indol-3-yl)ethyl)-3-benzylthiourea (10). This compound was synthesized as described
previously [43].
1
1-(2-(1H-Indol-3-yl)ethyl)-3-(2-fluorophenyl)thiourea (11). Yield 90%. m.p. 167–168 ◦C. H-NMR
(DMSO-d6)
6.88–6.98 (m, 3H, CHarom.); 7.04–7.17 (m, 3H, CHarom.); 7.35 (d, 2H,CHarom., J = 8.1 Hz); 7.58 (d, 1H,
CHarom., J = 7.5 Hz); 7.62 (bs, 1H, NH); 7.99 (s, 1H, NH). 13C-NMR (DMSO-d6)
(ppm): 24.5 (CH2),
δ (ppm): 3.09 (t, 2H, CH2, J = 6.6 Hz); 3.98(t, 2H, CH2, J = 6.3 Hz); 6.06 (bs, 1H, NH);
δ
44.7 (CH2), 111.3 (C), 111.5 (CH), 115.6 (CH), 115.9 (d, J = 31.2 Hz, CH), 118.2 (CH), 118.5 (CH), 121.0
(CH), 122.8 (CH), 124.0 (d, J = 3.7 Hz, CH), 126.6 (d, J = 11.8 Hz, CH), 127.2 (C), 127.9 (d, J = 7.8 Hz, C),
136.3 (C), 157.5 (d, J = 245.3Hz, CH), 181.1 (C). HRMS (ESI) calc. for C17H16FN3S [M
found: 312.3921.
−
H]−: 312.3924,
1
1-(2-(1H-Indol-3-yl)ethyl)-3-(3-fluorophenyl)thiourea (12). Yield 88%. m.p. 162–164 ◦C. H-NMR
(DMSO-d6) δ (ppm): 3.10 (t, 2H, CH2, J = 6.6 Hz); 3.98 (q, 2H, CH2, J = 5.7 Hz); 6.13 (s, 1H, NH); 6.65
(d, 2H, CHarom., J = 6.9 Hz); 6.87 (d, 1H, CHarom., J = 6.9 Hz); 6.98 (s, 1H, CHarom.); 7.05–7.23 (m, 3H,
CHarom.); 7.37 (d, 2H, CHarom., J = 8.1 Hz); 7.49 (s, 1H, NH); 7.58–7.60 (d, 2H, CHarom., J = 7.8 Hz); 8.01
(s, 1H, NH). 13C-NMR (DMSO-d6)
δ (ppm): 24.4 (CH2), 44.5 (CH2), 109.2 (C), 110.3 (d, J = 24,9 Hz, CH),
111.4 (d, J = 21,3 Hz, CH), 111.5 (CH), 118.3 (CH), 118.5 (q, J = 3.9 Hz, CH), 121.0 (CH), 122.8 (CH),
127.2 (CH), 130.1 (q, J = 32 Hz, CH), 136.3 (C), 141.3 (d, J = 10.8 Hz, C), 160.3 (d, J = 242 Hz, C), 163.5
(CH), 180.1 (C). HRMS (ESI) calc. for C17H16FN3S [M − H]−: 312.3924, found: 312.3921.

Molecules 2018, 23, 2554
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1
1-(2-(1H-Indol-3-yl)ethyl)-3-(2-chlorophenyl)thiourea (13). Yield 79%. m.p. 170–172 ◦C. H-NMR
(DMSO-d6) (ppm): 2.97 (t, 2H, CH2, J = 7.5 Hz); 3.75 (q, 2H, CH2, J = 5.7 Hz); 6.98 (t, 1H,CHarom.,
δ
J = 7.5 Hz); 7.07 (t, 1H, CHarom., J = 6.9 Hz); 7.16–7.35 (m, 4H, CHarom.); 7.47 (d, 1H,CHarom., J = 7.8 Hz);
7.63 (d, 2H, CHarom., J = 6.9 Hz); 7.95 (bs, 1H, NH); 9.16 (s, 1H, NH); 10.83 (s, 1H, NH). 13C-NMR
(DMSO-d6)
δ (ppm): 24.5 (CH2), 44.7 (CH2), 111.3 (C), 111.5 (CH), 118.2 (CH), 118.5 (CH), 120.9 (CH),
122.7 (CH), 126.9 (CH), 127.1 (CH), 127.2 (CH), 128.9 (C), 129.1 (C), 129.4(C), 136.0(C), 136.2 (CH), 181.1
(C). HRMS (ESI) calc. for C17H16ClN3S [M − H]−: 328.8470, found: 328.8467.
1
1-(2-(1H-Indol-3-yl)ethyl)-3-(3-chlorophenyl)thiourea (14). Yield 85%. m.p. 169–171 ◦C. H-NMR
(DMSO-d6)
δ (ppm): 2.98 (t, 2H, CH2, J = 7.5 Hz); 3.77 (q, 2H, CH2, J = 5.7 Hz); 6.98 (t, 1H, CHarom.,
J = 8.1 Hz); 7.04–7.14 (m, 2H, CHarom.); 7.18 (d, 1H, CHarom., J = 2.4 Hz); 7.23–7.36 (m, 3H, CHarom.);
7.63 (d, 2H, CHarom., J = 8.4 Hz); 7.91 (bs, 1H, NH); 9.65 (s, 1H, NH); 10.84 (s, 1H, NH). 13C-NMR
(DMSO-d6)
δ (ppm): 24.4 (CH2), 44.5 (CH2), 111.4 (C), 111.5 (CH), 118.2 (CH, CH), 118.5 (CH), 120.96
(CH), 122.0 (CH), 122.8 (CH), 123.4 (CH), 127.2 (C), 130.1 (C), 132.6 (C), 136.3 (C), 140.9 (CH), 180.1 (C).
HRMS (ESI) calc. for C17H16ClN3S [M − H]−: 328.8470, found: 328.8468.
1-(2-(1H-Indol-3-yl)ethyl)-3-(2-bromophenyl)thiourea (15). Yield 83%.m.p. 150–152 ◦C. 1H-NMR
(DMSO-d6)
δ (ppm): 2.98 (t, 2H, CH2, J = 7.2 Hz); 3.75 (q, 2H, CH2, J = 5.4 Hz); 6.98 (t, 1H,CHarom.,
J = 7.8 Hz); 7.07 (t, 1H, CHarom., J = 6.6 Hz); 7.12–7.17 (m, 2H, CHarom.); 7.32–7.38 (m, 2H, CHarom.); 7.57
(d, 1H, CHarom., J = 7.2 Hz); 7.62–7.66 (m, 2H, CHarom.); 7.91 (bs, 1H, NH); 9.11 (s, 1H, NH); 10.83 (s,
1H, NH). 13C-NMR (DMSO-d6)
δ (ppm): 24.5 (CH2), 44.7 (CH2), 111.3 (C), 111.5 (CH), 118.2 (CH), 118.5
(CH), 120.2 (CH), 120.9 (CH), 122.8 (CH), 127.2 (CH), 127.4 (CH), 127.7 (C), 129.7 (C), 132.5 (C), 136.2
(C), 137.4 (CH), 181.1 (C). HRMS (ESI) calc. for C17H16BrN3S [M − H]−: 373.2980, found: 373.2978.
1-(2-(1H-Indol-3-yl)ethyl)-3-phenethylthiourea (16). This compound was synthesized as described
previously [44].
1-(2-(1H-Indol-3-yl)ethyl)-3-(4-fluorophenyl)thiourea (17). This compound was synthesized as described
previously [42].
1-(2-(1H-Indol-3-yl)ethyl)-3-allylthiourea (18).
This compound was synthesized as described
previously [40].
◦
1-(2-(1H-Indol-3-yl)ethyl)-3-(2-methylallyl)thiourea (19). Yield 76%. m.p. 141–143 C. 1H-NMR (DMSO-d6)
δ
(ppm): 1.67 (s, 3H, CH3); 2.90 (t, 2H, CH2, J = 7.2 Hz); 3.67 (bs, 2H, CH2); 3.98 (bs, 2H, CH2); 4.77
(s, 2H, =CH2); 6.98 (t, 1H, CHarom., J = 7.8 Hz); 7.08 (t, 1H, CHarom., J = 6.9 Hz); 7.14 (d, 1H, CHarom.,
J = 2.4 Hz); 7.33 (d, 1H, CHarom., J = 8.1 Hz); 7.42 (bs, 1H, NH); 7.56 (bs, 1H, NH); 7.59 (d, 1H, CHarom.,
J = 7.8 Hz); 10.82 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ (ppm): 20.3 (CH3), 24.9 (CH2), 44.4 (C), 48.7
(CH2), 109.9 (CH2), 111.3 (CH2), 111.6 (C), 118.2 (CH), 118.46 (CH), 120.9−(CH), 122.7 (CH), 127.3 (C),
136.2 (C), 142.4 (CH), 182.5 (C). HRMS (ESI) calc. for C15H19N3S [M
−
H] : 272.3964, found: 272.3486.
1-(2-(1H-Indol-3-yl)ethyl)-3-ethoxycarbonylthiourea (20). This compound was synthesized as described
previously [43].
◦
1-(2-(1H-Indol-3-yl)ethyl)-3-(4-iodophenyl)thiourea (21). Yield 88%. m.p. 162–165 C. 1H-NMR (DMSO-d6)
(ppm): 2.97 (t, 2H, CH2, J = 7.5 Hz); 3.75 (q, 2H, CH2, J = 6.0 Hz); 6.98 (t, 1H, CHarom., J = 6.9 Hz);
7.07 (t, 1H,CHarom., J = 6.9 Hz); 7.20 (m, 3H, CHarom.); 7.34 (d, 1H, CHarom., J = 8.1 Hz); 7.61 (m, 3H,
CHarom.); 7.83 (bs, 1H, NH); 9.57 (s, 1H, NH); 10.84 (s, 1H, NH). 13C-NMR (DMSO-d6)
(ppm): 24.39
δ
δ
(CH2), 44.53 (CH2), 111.34 (C), 111.48 (CH), 118.22 (CH, CH), 118.46 (CH), 120.95 (CH, CH), 122.81
(CH), 124.92 (CH), 127.21 (CH), 136.26 (C), 137.13 (C, C), 139.18 (CH), 180.03 (C). HRMS (ESI) calc. for
C17H16IN3S [M − H]−: 420.2985, found: 420.2987.
1-(2-(1H-Indol-3-yl)ethyl)-3-ethylthiourea (22). This compound was synthesized as described previously [45].
◦
1-(2-(1H-Indol-3-yl)ethyl)-3-(3-chloro-4-methylphenyl)thiourea (23). Yield 83%. m.p. 161–163 C. 1H-NMR
(DMSO-d6) (ppm): 2.28 (s, 3H, CH3); 2.98 (t, 2H, CH2, J = 7.5 Hz); 3.76 (q, 2H, CH2, J = 5.7 Hz); 6.98
δ

Molecules 2018, 23, 2554
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(t, 1H, CHarom., J = 8.1 Hz); 7.08 (t, 1H, CHarom., J = 6.9 Hz); 7.13–7.18 (m, 2H, CHarom.); 7.25 (d, 1H,
CHarom., J = 8.4 Hz); 7.34 (d, 1H,CHarom., J = 7.8 Hz); 7.57 (d, 1H,CHarom., J = 2.1 Hz); 7.62 (d, 1H,
CHarom., J = 7.5 Hz); 7.81 (bs, 1H, NH); 9.55 (s, 1H, NH); 10.84 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ
(ppm): 18.9 (CH3), 24.4 (CH2), 44.5 (CH2), 111.3 (C), 111.5 (CH), 118.2 (CH), 118.5 (CH), 120.9 (CH),
121.7 (CH), 122.8 (C), 123.0 (CH), 127.2 (CH), 130.7 (C), 130.9 (C), 132.6 (C), 136.3 (C), 138.4 (CH), 180.2
(C). HRMS (ESI) calc. for C18H18ClN3S [M − H]−: 342.8736, found: 342.8769.
◦
1-(2-(1H-Indol-3-yl)ethyl)-3-(3-(trifluoromethyl)phenyl)thiourea (24). Yield 92%. m.p. 169–171 C. 1H-NMR
(DMSO-d6) (ppm): 2.99 (t, 2H, CH2, J = 7.2 Hz); 3.79 (q, 2H, CH2, J = 5.7 Hz); 6.98 (t, 1H, CHarom.,
δ
J = 7.8 Hz); 7.08 (t, 1H, CHarom., J = 7.8 Hz); 7.18 (d, 1H,CHarom., J = 2.1 Hz); 7.35 (d, 1H, CHarom.,
J = 8.1 Hz); 7.40 (d, 1H, CHarom., J = 7.8 Hz); 7.50 (d, 1H, CHarom., J = 7.8 Hz); 7.64 (d, 1H, CHarom.,
J = 7.8 Hz); 7.98 (s, 1H, CHarom.); 7.98 (bs, 1H, NH); 9.79 (s, 1H, NH); 10.85 (s, 1H, NH). 13C-NMR
(DMSO-d6)
δ (ppm): 24.4 (CH2), 44.4 (CH2), 79.2 (C), 111.4 (C), 111.4 (CH), 118.2 (CH), 118.4 (CH),
118.6 (CH), 121.0 (q, 3.8 Hz, CH), 122.8 (q, J = 3.8 Hz, CH), 125.8 (q, J = 272 Hz, C), 126.1 (CH), 127.2
(CH), 128.8 (C), 129.5 (q, J = 33.6 Hz, C), 136.3 (C), 140.4, 180.4 (C). HRMS (ESI) calc. for C18H16F3N3S
[M − H]−: 362.3999, found: 362.3940.
1-(2-(1H-Indol-3-yl)ethyl)-3-methylthiourea (25). This compound was synthesized as described
previously [46].
◦
1-(2-(1H-Indol-3-yl)ethyl)-3-(3-chloro-6-methylphenyl)thiourea (26). Yield 87%. m.p. 163–165 C. 1H-NMR
(DMSO-d6) (ppm): 2.13 (s, 3H, CH3); 2.96 (t, 2H, CH2, J = 7.2 Hz); 3.73 (q, 2H, CH2, J = 5.1 Hz); 6.98
δ
(t, 1H,CHarom., J = 6.3 Hz); 7.07 (t, 1H, CHarom., J = 6.9 Hz); 7.15–7.26 (m, 3H, CHarom.); 7.34 (d, 1H,
CHarom., J = 8.1 Hz); 7.37 (s, 1H, CHarom.); 7.63 (d, 1H, CHarom., J = 7.8 Hz); 7.71 (bs, 1H, NH); 9.11 (s,
1H, NH); 10.82 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ
(ppm): 17.2 (CH3), 24.6 (CH2), 44.8 (CH2), 111.3
(C), 111.6 (CH), 118.2 (CH), 118.5 (CH), 120.9 (CH), 122.7 (CH), 125.8 (C), 127.0 (CH), 127.3 (CH), 129.7
(C), 131.8 (C), 133.1 (C), 136.2 (C), 138.6 (CH), 180.9 (C). HRMS (ESI) calc. for C18H18ClN3S [M
342.8736, found: 342.8772.
−
H]−:
1-(2-(1H-Indol-3-yl)ethyl)-3-(4-butyl-2-methylphenyl)thiourea (27). Yield 77%. m.p. 172–174 ◦C. 1H-NMR
(DMSO-d6) (ppm): 0.8 (t, 3H, CH3, J = 7.5 Hz); 1.25–1.37 (m, 2H, CH2, J = 7.5 Hz); 1.49–1.59 (m, 2H,
δ
CH2, J = 7.2 Hz); 2.11 (s, 3H, CH3); 2.49 (s, 2H, CH2); 2.93 (t, 2H, CH2, J = 7.5 Hz); 3.69 (q, 2H, CH2,
J = 6.0 Hz); 6.94–7.11 (m, 6H, CHarom.); 7.25 (bs, 1H, NH); 7.33 (d, 1H, CHarom., J = 8.1 Hz); 7.63 (d, 1H,
CHarom., J = 7.8 Hz); 9.01 (s, 1H, NH); 10.79 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ (ppm): 13.8 (CH3),
17.6 (CH2), 21.8 (CH2), 24.8 (CH3), 33.0 (CH2), 34.3 (CH2), 40.3 (CH2), 111.3 (C), 111.6 (CH), 118.1 (CH),
118.5 (CH), 120.9 (CH, CH), 122.6 (C), 126.2 (C), 127.3 (CH), 127.6 (CH), 130.4 (C), 134.5 (C), 136.2 (C),
140.6, 180.8 (C). HRMS (ESI) calc. for C22H27N3S [M − H]−: 364.5348, found: 364.5360.
1-(2-(1H-Indol-3-yl)ethyl)-3-(3-chloro-4-fluorophenyl)thiourea (28). Yield 91%. m.p. 162–164 ◦C. 1H-NMR
(DMSO-d6)
δ (ppm): 2.98 (t, 2H, CH2, J = 7.2 Hz); 3.76 (q, 2H, CH2, J = 5.7 Hz); 6.98 (t, 1H, CHarom.,
J = 8.1 Hz); 7.08 (t, 1H,CHarom., J = 7.2 Hz); 7.18 (d, 1H, CHarom., J = 2.1 Hz); 7.24–7.30 (m, 1H, CHarom.);
7.34 (d, 2H,CHarom., J = 8.7 Hz); 7.62 (d, 1H,CHarom., J = 7.8 Hz); 7.72 (d, 1H, CHarom., J = 6.6 Hz);
7.87 (bs, 1H, NH); 9.60 (s, 1H, NH); 10.84 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ (ppm): 24.4 (CH2), 44.5
(CH2), 111.4 (C), 111.5 (CH), 116.6 (d, J = 22 Hz, CH), 118.2 (CH), 118.4 (d, J = 18.6 Hz, CH), 121.0 (CH),
122.8 (CH), 123.6 (CH), 124.9 (d, J = 7.2 Hz, C), 127.2 (C), 136.3 (C), 136.5 (d, J = 3.3 Hz, C), 152.2 (C),
155.5 (d, J = 242.6 Hz, CH), 180.4 (C). HRMS (ESI) calc. for C17H15ClFN3S [M
346.8378. Crystal data: crystal system triclinic, space group P-1, unit cell dimensions a = 8.592(2) Å,
b = 12.4914(3) Å, c = 15.309(3) Å,
= 85.32(2)◦, = 81.17(2)◦, = 88.87(2)◦, V = 1678.1(6) Å3; Z = 4,
dc = 1.428 g/cm3, = 0.377 mm−1, F(000) = 720. A crystal of dimensions 0.40
was used for intensity measurements. Within the 11
range 2.04–28.54◦ [
20
−
H]−: 346.8375, found:
α
β
γ
µ
×
0.20
×
0.03 mm
16,
θ
−
≤
h
≤
11,
−
16
≤ ≤
k
−
≤
l
≤
20], reflections collected 22058. The 5859 unique reflections [R(int) = 0.0360] were used for
the refinement of 427 parameters. Final R indices on F2 for 5698 observed reflections [I > 2
σ(I)] were:
R1 = 0.0483, wR2 = 0.1019, goodness-of-fit 1.018, ∆$max/min = 0.76/−0.80 e Å−3
.

Molecules 2018, 23, 2554
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3.3. Biology
3.3.1. In Vitro Evaluation of Antimicrobial Activity
The antibacterial activity of compounds was tested against a series of Gram-positive bacteria:
Staphylococcus aureus ATCC 4163, Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC
29213, Staphylococcus aureus ATCC 6538,Staphylococcus epidermidis ATCC 12228, Bacillus subtilis ATCC
6633, Bacillus cereus ATCC 11778, Enterococcus hirae ATCC 10541, Micrococcus luteus ATCC 9341,
Micrococcus luteus ATCC 10240 and Gram-negative rods: Escherichia coli ATCC 10538, Escherichia
coli ATCC 25922, Escherichia coli NCTC 8196, Proteus vulgaris NCTC 4635, Pseudomonas aeruginosa ATCC
15442, Pseudomonas aeruginosa NCTC 6749, Pseudomonas aeruginosa ATCC 27853, Bordetella bronchiseptica
ATCC 4617. Antifungal activity was tested against yeasts: Candida albicans ATCC 10231, Candida albicans
ATCC 90028, Candida parapsilosis ATCC 220191. Microorganisms used in this study were obtained
from the collection of the Department of Pharmaceutical Microbiology, Medical University of
Warsaw, Poland.
3.3.2. Media, Growth Conditions and Antimicrobial Activity Assays
Antimicrobial activity was examined by the disc diffusion and Minimal Inhibitory Concentration
(MIC) method under standard conditions, using Mueller–Hinton II agar medium (Becton Dickinson,
Franklin Lakes, NJ, USA) for bacteria and RPMI agar with 2% glucose (Sigma, St. Louis, MO, USA)
for yeasts, according to CLSI (previously NCCLS) guidelines [31]. Solutions containing the tested
agents were prepared in methanol or DMSO. For the disc diffusion method, sterile paper discs (9 mm
diameter, Whatman No. 3 chromatography filter paper) were dripped with the compound solutions
tested to obtain 400 µg of substance per disc. Dry discs were placed on the surface of an appropriate
agar medium. The results (diameter of the growth inhibition zone) were read after 18 h of incubation
◦
at 35 C. MIC’s were examined by the twofold serial agar dilution technique [32]. Concentrations
of the tested compounds in solid medium ranged from 3.125 to 400 µg/mL. The final inoculum of
studied organisms was 104 CFU/mL (colony forming units per mL), except the final inoculum for
E. hirae ATCC 10541, which was 105 CFU/mL. Minimal inhibitory concentrations were read off after
18 h of incubation at 35 ◦C.
3.3.3. Inhibitory Activities against DNA Topoisomerase IV and DNA Gyrase
S. aureus topoisomerase IV decatenation assay was performed with 200 ng of kinetoplast DNA
(Inspiralis Limited, Norwich, UK) as a substrate. S. aureus DNA gyrase supercoiling assay was
performed with 0.5
detected by incubation for 30 min at 37 ◦C in a total reaction volume of 30
different concentrations of tested compounds and the reference ciprofloxacin at a concentration of
32 g/mL. The reactions were terminated by adding an equal volume of STEB buffer (40% sucrose,
100 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mg/mL bromophenol blue), followed by extraction with
1 volume of chloroform/isoamyl alcohol (24:1). Then, 20 L of the aqueous phase of each sample was
µg of relaxed pBR322 DNA (Inspiralis) as a substrate. Enzymes activity was
µL and in the presence of
µ
µ
loaded onto a 1% agarose gel. Following electrophoresis, gels were stained with ethidium bromide,
visualized under UV light in a transilluminator (ChemiDoc MP, BioRad, Hercules, CA, USA) and
analyzed by the Image Lab 6.0 software (Image Lab’s, Cleveland, OH, USA).
3.3.4. Cells and Viruses
Cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).
Cell lines supporting the multiplication of RNA and DNA viruses were the following: CD4+ human
T-cells containing an integrated HTLV-1 genome (MT-4); Madin Darby Bovine Kidney (MDBK) [ATCC
CCL 22 (NBL-1) Bos taurus]; baby hamster kidney (BHK-21) [ATCC CCL 10 (C-13) Mesocricetus auratus]
and monkey kidney (Vero-76) [ATCC CRL 1587 Cercopithecus aethiops]. Human Immunodeficiency
Virus type-1 (HIV-1) IIIB laboratory strain was obtained from the supernatant of the persistently

Molecules 2018, 23, 2554
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infected H9/IIIB cells (NIH 1983). Viruses representative of ssRNA+ were: (i) Flaviviridae: yellow
fever virus (YFV) [strain 17-D vaccine (Stamaril Pasteur J07B01)], bovine viral diarrhoea virus (BVDV)
[strain NADL (ATCC VR-534)]; (ii) Picornaviridae: enterovirus B [coxsackievirus B5 (CV-B5), strain
Ohio-1 (ATCC VR-29)], and enterovirus C [poliovirus type-1 (Sb-1), Sabin strain Chat (ATCC VR-1562)].
Viruses representative of ssRNA- were: (iii) Pneumoviridae: human respiratory syncytial virus (RSV)
[strain A2 (ATCC VR-1540)]; (iv) Rhabdoviridae: vesicular stomatitis virus (VSV) [lab strain Indiana
(ATCC VR 1540)]. The virus representative of dsRNA was: (v) Reoviridae: reovirus type-1 (Reo-1)
[simian virus 12, strain 3651 (ATCC VR-214)]. DNA virus representatives were: v) Poxviridae: vaccinia
virus (VV) [vaccine strain Elstree-Lister (ATCC VR-1549)]; (vi) Herpesviridae: human herpes 1 (HSV-1)
[strain KOS (ATCC VR-1493)]. Mutants carrying NNRTI mutations used: Y181C mutant (NIH-N119) of
HIV-1 derives from an AZT-sensitive clinical isolate passaged initially in CEM and then in MT-4 cells,
in the presence of nevirapine (up to 10
strain passaged in H9 cells in the presence of BI-RG 587 (up to 1
(EFVR) derives from an IIIB strain passaged in MT-4 cells in the presence of efavirenz (up to 2
µ
M); K103N + Y181C mutant (NIH A17) derives from an IIIB
M); K103R + V179D + P225H mutant
M).
107 CCID50/mL,
µ
µ
N119, A17 and EFVR stock solutions had titres of 1.2
×
108, 2.1
×
107, and 4.0
×
respectively. Mutants carrying NRTI mutations used: AZTR strain (67N, 70R, 215F, 219Q); MDR strain
(74V, 41L, 106A, 215Y).
3.3.5. Cytotoxicity Assays
Exponentially growing MT-4 cells were seeded at an initial density of 4
plates in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS), 100 units/mLpenicillin
G and 100 g/mL streptomycin. MDBK and BHK cells were seeded in 96-well plates at an initial
density of 6
105 and 1 106 cells/mL, respectively, in minimum essential medium with Earle’s salts
×
105 cells/mL in 96-well
µ
×
×
(MEM-E), L-glutamine, 1 mM sodium pyruvate and 25 mg/L kanamycin, supplemented with 10%
horse serum (MDBK) or 10% foetal bovine serum (FBS) (BHK). Vero-76 cells were seeded in 96-well
plates at an initial density of 4
×
105 cells/mL, in Dulbecco’s Modified Eagle Medium (D-MEM) with
L-glutamine and 25 mg/L kanamycin, supplemented with 10% FBS. Cell cultures were then incubated
at 37 ◦C in a humidified, 5% CO2 atmosphere, in the absence or presence of serial dilutions of test
compounds. Cell viability was determined after 48–96 h at 37 ◦C by MTT method for MT-4, Vero-76,
MDBK and BHK [47].
Cell lines derived from human haematological tumours [CD4+ human acute T-lymphoblastic
leukaemia (CCRF-CEM), human splenic B-lymphoblastoid cells (WIL-2NS), human acute
B-lymphoblastic leukaemia (CCRF-SB)] were seeded at an initial density of 1
×
105 cells/mL in
96 well plates in RPMI-1640 medium supplemented with 10% foetal calf serum (FCS), 100 units/mL
penicillin G and 100 µ
g/mL streptomycin. Cell viability was determined after 96 h at 37 ◦C by the
MTT method. All cell cultures were then incubated at 37 ◦C in a humidified, 5% CO2 atmosphere,
in the absence or presence of serial dilutions of test compounds in culture medium. Before dilutions,
compounds were dissolved in DMSO at 100 mM.
3.3.6. Antiviral Assays
Activity against HIV-1 wt and mutant strains (N119, A17, EFVR, AZTR, MDR) was based on
inhibition of virus-induced cytopathogenicity in exponentially growing MT-4 cell acutely infected
with a multiplicity of infection (m.o.i.) of 0.01. Briefly, 50
were added to each well of flat-bottom microtitre trays, containing 50
serial dilutions of extracts or fractions. Then, 20 L of a HIV-1 suspension containing 100 CCID50 were
µ
L of RPMI containing 1
×
104 MT-4 cells
µL of RPMI without or with
µ
added. After 4-days incubation at 37 ◦C, cell viability was determined by the MTT method.
Compound’s activity against YFV and Reo-1 was based on inhibition of virus-induced cytopathogenicity
in BHK-21 cells acutely infected with a m.o.i. of 0.01. Compound’s activity against BVDV was based
on inhibition of virus-induced cytopathogenicity in MDBK cells acutely infected at a m.o.i. of 0.01.
◦
After a 3 or 4-days incubation at 37 C, cell viability was determined by the MTT method, as described

Molecules 2018, 23, 2554
15 of 17
earlier [47]. Compound’s activity against CV-B5, Sb-1, VSV, VV, HSV-1 and RSV was determined by
plaque reduction assays in infected Vero-76 cell monolayers, as described earlier [48 49]. Concentrations
,
resulting in 50% inhibition (CC50 or EC50) were determined by linear regression analysis. Efavirenz,
20-C-methylguanosine, 20-C-methylcytidine, 6-azauridine, mycophenolic acid and acycloguanosine
were used as reference inhibitors.
Supplementary Materials: The following are available online.
Author Contributions: Conceptualization, D.S. and M.S. (Marta Struga); Formal Analysis, S.M.; J.S., G.K.-T.;
Investigation, G.S., S.M., G.G., M.W., A.E.K., O.S., T.L. and J.S.; Resources, G.K.-T.; Data Curation, G.S., S.P.,
D.S.; Writing—Original Draft Preparation, G.S.; Writing—Review & Editing, G.G.; Supervision, D.S.; Project
Administration, M.S. (Marta Struga); Funding Acquisition, M.S. (Marta Struga), P.T., M.S. (Michał Skrzycki). All
authors approved the final version of the manuscript.
Funding: This work was supported by the Medical University of Warsaw and carried out with the use of
CePT infrastructure financed by the European Union—the European Regional Development Fund within the
Operational Programme Innovative Economy for 2007–2013.
Acknowledgments: We gratefully acknowledge the Sardinia Regional Government for the financial support of
Silvia Madeddu through his scholarship (P.O.R. Sardegna F.S.E. Operational Programme of the Autonomous
Region of Sardinia, European Social Fund 2007–2013).
Conflicts of Interest: The authors declare no conflict of interest.
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,
Sample Availability: Samples of the compounds are available from the authors.
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