Synthesis, biological evaluation and QSAR studies of new thieno[2,3-d]pyrimidin-4(3H)-one derivatives as antimicrobial and antifungal agents

Article abstract of DOI:10.1016/j.bioorg.2020.104509

A series of new thieno[2,3-d]pyrimidin-4(3H)-one derivatives were synthesized and evaluated for their activity against four gram-positive and four gram-negative bacterial and eight fungal species. The majority of the compounds exhibited excellent antimicr

Full text of DOI:10.1016/j.bioorg.2020.104509

Bioorganic Chemistry xxx (xxxx) xxx
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, biological evaluation and QSAR studies of new thieno[2,3-d]
pyrimidin-4(3H)-one derivatives as antimicrobial and antifungal agents
,
,
1
b
a
George E. Magoulasa 1, Lefkothea Kalopetridoua , Ana Ciric , Eftichia Kritsi ,
´
´
a
a
a
b
´
Paraskevi Kouka , Panagiotis Zoumpoulakis , Niki Chondrogianni , Marina Sokovic ,
Kyriakos C. Prousisa, Theodora Calogeropouloua
,
*
a National Hellenic Research Foundation, Institute of Chemical Biology, 48 Vassileos Constantinou Avenue, 11653 Athens, Greece
b
ˇ
´
University of Belgrade, Institute for Biological Research “Sinisa Stankovic” – National Institute of Republic of Serbia, Blvd. despot Stefan 142, 11000 Belgrade, Serbia
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of new thieno[2,3-d]pyrimidin-4(3H)-one derivatives were synthesized and evaluated for their activity
against four gram-positive and four gram-negative bacterial and eight fungal species. The majority of the
compounds exhibited excellent antimicrobial and antifungal activity, being more potent than the control com-
pounds. Compound 22, bearing a m-methoxyphenyl group and an ethylenediamine side chain anchored at C-2 of
the thienopyrimidinone core, is the most potent antibacterial compound with broad antimicrobial activity with
MIC values in the range of 0.05–0.13 mM, being 6 to 15 fold more potent than the controls, streptomycin and
ampicillin. Furthermore, compounds 14 and 15 which bear a p-chlorophenyl and m-methoxyphenyl group,
respectively, and share a 2-(2-mercaptoethoxy)ethan-1-ol side chain showed the best antifungal activity, being
10–15 times more potent than ketoconazole or bifonazole with MIC values 0.013–0.026 and 0.027 mM,
respectively. Especially in the case of compound 15 the low MIC values were accompanied by excellent MFC
values ranging from 0.056 to 0.058 mM. Evaluation of toxicity in vitro on HFL-1 human embryonic primary cells
and in vivo in the nematode C. elegans revealed no toxic effects for both compounds 15 and 22 tested at the MIC
concentrations. Ligand-based similarity search and molecular docking predicted that the antibacterial activity of
analogue 22 is related to inhibition of the topoisomerase II DNA gyrase enzyme and the antifungal activity of
compound 15 to CYP51 lanosterol demethylase enzyme. R-Group analysis as a means of computational structure
activity relationship tool, highlighted the compounds’ crucial pharmacophore features and their impact on the
antibacterial and antifungal activity. The presence of a N-methyl piperidine ring fused to the thienopyrimidinone
core plays an important role in both activities.
Thienopyrimidinones
Antimicrobial activity
Toxicity studies
Pharma RQSAR
1. Introduction
700,000 deaths annually, mainly in developing countries, with consid-
erable economic consequences [1]. Thus, there is still an unmet need for
Infectious diseases caused by bacteria and fungi emerged and still
evolve side by side with life itself. They have played a very important
role in the history of mankind, even changing the course of events in
terms of war, migration of population etc. From the empirical use of
plant extracts during ancient times to the impressive advance of modern
medicine, the ceaseless effort of humans to subdue infectious diseases is
evident. However, this is battle has not been completely won yet. The
abundance of new and effective antimicrobial drugs comes along with
their misuse or overuse leading eventually to antimicrobial resistance
(AMR). According to WHO, AMR is considered the cause of at least
new antimicrobial drugs with reduced toxicity and activity against
resistant strains.
Heterocycles and especially fused heterocyclic rings play a signifi-
cant role in medicinal chemistry as they can be found in many approved
drugs, exhibiting a wide range of biological activities [2,3].
Thienopyrimidinones represent a scaffold that has drawn much
attention as a source of bioactive compounds. A large number of de-
rivatives containing the thienopyrimidinone moiety were evaluated as
MCH-R1 [4] and mGluR1 antagonists [5], or as FGFR1 kinase [6], PARP-
1 [7], SIRT-2 [8] and phosphodiesterase 7 inhibitors [9]. Additionally,
* Corresponding author at: Institute of Chemical Biology, National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, 11635 Athens, Greece.
E-mail address: tcalog@eie.gr (T. Calogeropoulou).
1
Equally contributed.
https://doi.org/10.1016/j.bioorg.2020.104509
Received 12 September 2020; Received in revised form 11 November 2020; Accepted 19 November 2020
Available online 26 November 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: George E. Magoulas, Bioorganic Chemistry, https://doi.org/10.1016/j.bioorg.2020.104509

G.E. Magoulas et al.
Bioorganic Chemistry xxx (xxxx) xxx
this class of compounds has shown anti-malarial [10], anti-tubercular
[11], antiviral [12], anti-inflammatory and antimicrobial properties
[13,14].
Finally, the synthesis of the targeted compounds 31–33 was realized
using the synthetic methodology followed above for thioureas 7–9,
starting from the commercially available cyclohexanone (26) (Scheme
4). Condensation of 26 with ethyl cyanoacetate (2) led to 2-aminothio-
phene 27 in 52% yield. The latter was reacted with 3-methoxy benzene
isothiocyanate (5) to give thiourea 28 [17] in 95% yield which, in turn,
was converted to thiolate 29 in the presence of ethanolic KOH solution
and finally to the S-methylated compound 30 upon treatment with
iodomethane. Reaction of compound 30 with ethane-1,2-diamine, or
propane-1,3-diamine or 2-(piperazin-1-yl)ethane-1-amine under
solvent-free conditions at elevated temperatures, afforded compounds
31–33, respectively in 20–74% yield.
Inspired by the broad-spectrum activity of the thienopyrimidinone
compound class, we embarked upon the synthesis of twelve new de-
rivatives bearing structural modifications for SAR purposes concerning
their antibacterial and antifungal properties. More specifically: (a) a
phenyl ring substituted by electron-donating (-OMe) or electron-
withdrawing (-Cl) group, was introduced at the N-3 position of the
thieno[2,3-d]pyrimidin-4(3H)-one skeleton; (b) biocompatible aliphatic
amines or an ethoxyethanol group were anchored at the C-2 position
through a sulfur bridge or an amino group for enhanced hydrophilicity
and (c) a N-methylpiperidine or a cyclohexane moiety was fused to the
core. The changes that were applied to the thieno[2,3-d]pyrimidin-4
(3H)-one core are summarized in Fig. 1.
2.2. Antimicrobial evaluation
All new compounds (14–16, 18–20, 22–25 and 31–33) were eval-
uated in vitro for their antimicrobial and antifungal activity against eight
Gram-negative and Gram-positive bacterial species and eight fungi,
respectively.
2.2.1. Evaluation of in vitro antibacterial activity
The antibacterial activity of all synthesized compounds (including
side-product 23) was evaluated against four gram-positive (Bacillus.
cereus, Micrococcus flavus, Staphylococcus aureus, and Listeria mono-
cytogenes) and four gram-negative (Escherichia coli, Enterobacter cloacae,
Pseudomonas aeruginosa, and Salmonella typhimurium) bacterial strains,
using streptomycin and ampicillin as controls. The results are displayed
in Table 1 as minimal inhibitory concentrations (MICs), the lowest
concentration that inhibits the growth of the bacteria, and minimal
bactericidal concentrations (MBCs), the lowest concentration that kills
99.5% of the bacteria.
2. Results and discussion
2.1. Chemistry
The synthetic strategy followed for the preparation of compounds
14–16 is depicted in Scheme 1. A Gewald reaction between the
commercially available N-methyl pyrrolidone 1 and ethyl cyanoacetate
2, in the presence of sulfur and diethylamine in MeOH under microwave
irradiation, afforded the 2-aminothiophene derivative 3 in 69% yield
[15]. The latter reacted with the corresponding substituted aryl-
isothiocyanates 4–6 in a SN2 fashion in the presence of pyridine. The
reaction proceeded either by conventional heating or by using micro-
wave irradiation to give thioureas 7–9 in 45–80% yield. The final
compounds 14–16 were obtained, in 60–72% yield, in a one-pot reac-
tion which initially involved the cyclization of thioureas 7–9 to the
corresponding potassium thiolate salts 10–12, upon treatment with
ethanolic KOH solution under reflux, followed by the addition of mono-
tosylated diethylene glycol 13.
In detail, compounds 14 and 16 bearing a 2(2-thio)ethoxy)ethanol
side chain and a 4-chlorophenyl or 4-methoxyphenyl substituent at N-
3, respectively, showed very good antimicrobial activity. In particular,
analogue 16 was very potent against gram-negative bacteria, exhibiting
higher activity than streptomycin or ampicillin with MIC values ranging
between 0.02 and 0.22 mM while the MCB was 0.45 ± 0.00 mM. In
addition, compound 16 was also more potent than the controls against
the gram positive bacteria B. cereus and M. flavus exhibiting MIC and
MBC values of 0.11 ± 0.01 mM and 0.45 ± 0.05 mM, respectively.
Replacement of the C-2 2(2-thio)ethoxy)ethanol side chain in compound
14 by
ω-diamines or a 3-hydroxypropylamine (compounds 18–20)
resulted in a slight decrease in the activity. However, the 4-hydroxybu-
tylamine-substituted compound 19 was more potent than 14 against
M. flavus and P. aeruginosa with MIC = 0.06 ± 0.005 mM and MCB =
0.45 ± 0.00 mM. The C-2 amino-substituted congeners of compound 15,
derivatives 22, 24 and 25, possess good to excellent activities against all
bacterial strains tested. The most potent was compound 22 bearing an
ethylene diamine side chain, which exhibited an impressive broad-
spectrum antimicrobial activity with MIC (MBC) values ranging be-
tween 0.05 (0.06) and 0.13(0.26) mM, thus, being 6–15 fold more
potent than the controls, streptomycin and ampicillin. The elongation of
the C-2 side chain by one carbon in compound 24 reduces significantly
the antimicrobial activity which is even more pronounced in 25 which
bears a constrained piperazinyl-substituted side chain. Compound 23,
which was isolated as a side product towards the synthesis of 22,
possessed low activity, suggesting the importance of an aryl substitution
at the N-3 position of the thieno[2,3-d]pyrimidin-4(3H)-one core.
Since the 3-methoxyphenyl substituted series resulted in the most
potent derivative 22 we set out to explore the effect of the N-methyl
piperidine moiety on the antimicrobial activity, by the synthesis of the
cyclohexyl-substituted derivatives 31–33. As a first observation all the
new compounds 31–33 were less potent than their N-methyl piperidine
congeners 22, 24 and 25. The most potent was the C-2 (2-aminoethyl-
amine)-substituted derivative 31, being more active than streptomycin
or ampicillin in almost all the bacterial strains tested, with MIC values
ranging between 0.18 and 0.36 mM and MBC values ranging between
0.36 and 0.73 mM. However, compound 31 was less potent than its N-
methyl piperidine congener 22.
The synthesis of compounds 18–20 was accomplished in an analo-
gous manner as described above. Thus, treatment of thiourea derivative
7 with ethanolic KOH afforded the intermediate thiolate 10 which was
converted to the S-methylated compound 17, in 97% yield, upon addi-
tion of iodomethane. Subsequently, compound 17 was reacted under
solvent-free conditions with a large excess of propane-1,3-diamine or
butane-1,4-diamine or 3-aminopropan-1-ol to afford the desired com-
pounds 18–20, respectively in 46–55% yield (Scheme 2).
Compounds 22 and 24, 25 were obtained following the same
methodology as above for compound 17, using the previously synthe-
sized thiourea derivative 8 to obtain the S-methylated compound 21.
Reaction of 21 under solvent-free conditions with a large excess of
ethane-1,2-diamine, propane-1,3-diamine, or 2-(piperazin-1-yl)ethane-
◦
1-amine at elevated temperatures above 100 C, gave compounds 22
and 24, 25, respectively, although in very low yields 10–28%. In the
case of ethane-1,2-diamine, compound 23 [16] was also obtained in
39% yield along with compound 22 (see Scheme 3).
Fig. 1. General structure of the new thieno[2,3-d]pyrimidin-4(3H)-one de-
rivatives (14–16, 18–20, 22–25 and 31–33).
2

G.E. Magoulas et al.
Bioorganic Chemistry xxx (xxxx) xxx
Scheme 1. Synthesis of compounds 14–16. Reagents and conditions: (i) Sulfur, diethylamine, MeOH,
μ
W, Τ = 100 ◦C, t = 4 min, P = 120 W; (ii) Method А: pyridine,
45 ◦C, 16 h or method В: pyridine,
μ
W, Τ = 100 ◦C, t = 2 min, P = 150 W; (iii) (a) Ethanolic KOH, reflux; (b) EtOH, reflux, 18 h.
Scheme 2. Synthesis of compounds 18–20. Reagents and conditions:(i) (a) Ethanolic KOH, reflux; (b) Iodomethane, ethanol, rt. 1 h; (ii) Propane-1,3-diamine (30 eq),
100 ◦C, 16 h, or butane-1,4-diamine (30 eq) 140 ◦C, 16 h, or 3-aminopropan-1-ol, (30 eq), 150 ◦C, 16 h.
2.2.2. Evaluation of in vitro antifungal activity
ketoconazole and 17–23 fold higher activity than bifonazole. Interest-
ingly, compound 15 not only presents high potency against all the fungi
tested but in addition a very low MFC value of 0.056 ± 0.005 mM.
Compounds 14 and 16 were also more potent than the controls with MIC
values ranging from 0.013 to 0.027 mM, but the MFC values were higher
than those of 15. Replacement of the C-2 side chain of compound 14
All the new compounds were also tested for their antifungal activity
against eight fungal species namely, Aspergillus fumigatus, Aspergillus
versicolor, Aspergillus ochraceus, Aspergillus niger, Trichoderma viride,
Penicillium funiculosum, Penicillium ochrochloron, and Penicillium verru-
cosum var. cyclopium. Ketoconazole and bifonazole were used as con-
trols. The antifungal activity is presented in Table 2, as the minimal
minimal inhibitory concentration (MIC) and the minimal fungicidal
concentration (MFC).
with
ω-diamines (compounds 18 and 19) resulted in a slight improve-
ment in activity for the 3-aminopropylamino C-2-substituted derivative
18 (MIC (MFC) values 0.015 (0.233) mM). Furthermore, the 4-aminobu-
tylamino C-2-substituted derivative 19 possessed similar activity to 14
with MIC values ranging between 0.029 and 0.255 mM and MFC be-
tween 0.06 and 0.112 mM. Conversely, the C-2 substituted 3-
The most potent compound among derivatives 14–16 which bear a 2
(2-thio)ethoxy)ethanol group at C-2, is the N-3-(3-methoxyphenyl)-
substituted compound 15. It exhibits 10–100 fold higher activity than
3

G.E. Magoulas et al.
Bioorganic Chemistry xxx (xxxx) xxx
Scheme 3. Synthesis of compounds 22, 24 and 25. Reagents and conditions:(i) (a) Ethanolic KOH, reflux; (b) Iodomethane, ethanol, rt, 1 h; (ii) Ethane-1,2-diamine
(35 eq), 100 ◦C, 72 h, or propane-1,3-diamine (30 eq), 120 ◦C, 72 h, or 2-(piperazin-1-yl)ethane-1-amine (56 eq), 130 ◦C, 72 h.
Scheme 4. Synthesis of compounds 31–33. Reagents and conditions: (i) Sulfur, diethylamine, MeOH,
μ
W, Τ = 100 ◦C, t = 2 min, P = 150 W; (ii) 5, pyridine, 45 ◦C, 18
h; (iii) (a) Ethanolic KOH, reflux; (b) Iodomethane, ethanol, rt. 1 h; (iv) Ethane-1,2-diamine (69 eq), 100 ◦C, 48 h or propane-1,3-diamine (47 eq) 140 ◦C, 18 h, or 2-
(piperazin-1-yl)ethane-1-amine (52 eq), 140 ◦C, 18 h.
aminopropanol derivative 20 presented moderate activity against the
majority of the fungal strains tested. The C-2 amino-substituted conge-
ners of compound 15, derivatives 22, 24 and 25 presented slightly
reduced antifungal activity with respect to the parent compound.
However, derivative 22 was more potent than the controls against the
eight fungal strains tested. Surprisingly, compound 23, which lacks both
the N-3-aryl group and the C-2 side chain, showed significant inhibitory
and fungicidal potency with MIC value 0.046 ± 0.01 mM (0.041 ± 0.01
for A. niger) and MFC value 0.095 ± 0.03 mM against all the tested fungi.
Finally, the replacement of the N-methyl piperidine moiety by a
cyclohexane ring (compounds 31–33) led to significant loss of activity.
general toxicity, compounds 15 and 22 were further investigated for
their potential toxic effects in vitro on HFL-1 human embryonic primary
cells and in vivo in an established model for toxicity testing [18,19],
namely the nematode C. elegans. Both cells and nematodes were exposed
to the concentrations corresponding to the (MIC) /MFC values for
compound 15 and the MIC (0.05 mM) /highest MBC (0.26 mM) values
for compound 22.
Toxicity testing in human primary fibroblasts is more appropriate as
compared to cancer or immortalized cells since primary cells are the
ones that will be most probably affected by a potential antimicrobial or
antifungal treatment. Thus, concerning the in vitro toxicity, the number
of living cells was determined through crystal violet staining after 24
and 48 h of exposure to each compound, while DMSO was added in
control cultures. Both compounds were not toxic at the concentrations
tested since they did not induce any significant changes in the survival of
HFL-1 human primary fibroblasts (Fig. 2).
2.3. Evaluation of in vitro and in vivo toxicity
As discussed above the most potent antibacterial agent was com-
pound 22, presenting broad spectrum activity with MIC (MBC) values
ranging between 0.05 (0.06) and 0.06 (0.26) mM against all strains
tested. In addition, the most potent antifungal agent was compound 15
with MIC = 0.027 ± 0.005 mM and MFC = 0.056 ± 0.005 mM against all
fungal strains tested. To obtain a better understanding as to whether the
antimicrobial effect of these compounds was specific and not due to
Subsequently, the toxicity of compounds 15 and 22 was evaluated on
the multicellular organism C. elegans. Standardized toxicity tests using
this nematode have been introduced since 1990s [20] to define a variety
of endpoints for toxic effects [21,22]. Screening of drug and pollutants
toxicity has been shown to induce marked effects on various phenotypic
4

G.E. Magoulas et al.
Bioorganic Chemistry xxx (xxxx) xxx
Table 1
In vitro antibacterial activity of compounds 14–16, 18–20, 22–25 and 31–33.
Compounds
MIC/MBC (mM)
B.c.
0.11 ± 0.01b
M.f.
S.a.
L.m.
E.c.
En. cl.
P.a.
S.t.
14
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
0.44 ± 0.08d
0.88 ± 0.05bc
2.23 ± 0.25f
4.47 ± 0.28ef
0.11 ± 0.03b
0.45 ± 0.00ab
0.50 ± 0.00d
2.48 ± 0.30d
0.06 ± 0.005a
0.45 ± 0.00b
0.25 ± 0.00c
0.49 ± 0.03b
0.13 ± 0.01b
0.26 ± 0.02a
2.67 ± 0.16f
5.72 ± 0.33f
0.13 ± 0.03b
1.00 ± 0.00c
2.20 ± 0.43f
3.30 ± 0.06e
0.18 ± 0.01bc
0.36 ± 0.06a
1.77 ± 0.12e
3.54 ± 0.67e
2.27 ± 0.25f
2.84 ± 0.30d
0.35 ± 0.025 cd
0.53 ± 0.05b
0.86 ± 0.04de
1.14 ± 0.15c
0.11 ± 0.01b
0.22 ± 0.02b
0.56 ± 0.05d
4.47 ± 0.28de
0.22 ± 0.02c
0.45 ± 0.00bc
0.12 ± 0.04b
0.25 ± 0.00b
0.24 ± 0.02c
0.45 ± 0.00bc
0.49 ± 0.03d
0.99 ± 0.03c
0.05 ± 0.005a
0.06 ± 0.003a
0.76 ± 0.33de
5.72 ± 0.33e
0.50 ± 0.15d
1.00 ± 0.00c
2.20 ± 0.43f
4.40 ± 0.20de
0.18 ± 0.01bc
0.36 ± 0.06bc
1.77 ± 0.12e
3.54 ± 0.67d
4.55 ± 0.30 g
5.69 ± 1.15e
0.09 ± 0.03ab
0.18 ± 0.02b
0.86 ± 0.04de
1.14 ± 0.15c
0.44 ± 0.01b
1.77 ± 0.30c
4.47 ± 0.28e
8.94 ± 1.23f
0.45 ± 0.00b
0.89 ± 0.06b
0.50 ± 0.00b
2.48 ± 0.30d
0.45 ± 0.00b
0.90 ± 0.03b
0.49 ± 0.03b
0.99 ± 0.03b
0.06 ± 0.005a
0.26 ± 0.03a
5.72 ± 1.18f
11.44 ± 2.2 g
1.00 ± 0.00c
2.00 ± 0.15c
4.40 ± 0.60e
8.80 ± 1.15f
0.36 ± 0.06b
0.73 ± 0.15ab
3.54 ± 0.15c
6.20 ± 0.15c
4.55 ± 0.15d
5.69 ± 1.15e
0.35 ± 0.025b
0.53 ± 0.05ab
1.14 ± 0.15c
2.29 ± 0.25c
0.44 ± 0.01c
0.88 ± 0.05b
2.23 ± 0.25e
4.47 ± 0.28d
0.04 ± 0.002a
0.45 ± 0.00ab
1.24 ± 0.10de
2.48 ± 0.30c
0.22 ± 0.02b
0.45 ± 0.00ab
0.25 ± 0.00b
0.49 ± 0.03ab
0.06 ± 0.005a
0.13 ± 0.01a
2.67 ± 0.16e
5.72 ± 0.33e
0.06 ± 0.005a
0.50 ± 0.05ab
2.20 ± 0.43e
4.40 ± 0.20d
0.18 ± 0.01b
0.36 ± 0.06ab
2.66 ± 0.16e
3.54 ± 0.67 cd
2.27 ± 0.25e
2.84 ± 0.90c
0.35 ± 0.025 cd
0.53 ± 0.05ab
0.86 ± 0.04d
0.94 ± 0.03b
0.22 ± 0.02b
0.44 ± 0.02b
2.23 ± 0.25d
4.47 ± 0.28de
0.02 ± 0.01a
0.45 ± 0.00b
0.25 ± 0.00b
0.50 ± 0.00b
0.45 ± 0.00c
0.90 ± 0.03c
0.49 ± 0.03c
0.99 ± 0.03c
0.05 ± 0.001a
0.06 ± 0.003a
0.76 ± 0.33c
5.72 ± 0.33e
0.50 ± 0.05c
1.00 ± 0.00c
2.20 ± 0.05b
4.40 ± 0.05b
0.18 ± 0.01b
0.36 ± 0.06ab
1.77 ± 0.12d
3.54 ± 0.67d
4.55 ± 0.30e
5.69 ± 1.15e
0.53 ± 0.05c
0.89 ± 0.09bc
1.14 ± 0.15 cd
1.51 ± 0.06c
0.22 ± 0.02b
0.44 ± 0.02b
2.23 ± 0.25d
4.47 ± 0.28e
0.04 ± 0.02a
0.45 ± 0.00b
0.25 ± 0.00b
0.50 ± 0.00b
0.06 ± 0.005a
0.45 ± 0.00b
0.25 ± 0.00b
0.49 ± 0.03b
0.05 ± 0.001a
0.06 ± 0.003a
0.76 ± 0.33c
5.72 ± 0.33f
0.06 ± 0.005a
0.50 ± 0.05b
2.20 ± 0.05d
4.40 ± 0.05e
0.18 ± 0.01b
0.36 ± 0.05b
0.89 ± 0.09c
3.54 ± 0.67d
2.84 ± 0.30d
5.69 ± 1.15f
0.35 ± 0.025bc
0.53 ± 0.05b
2.29 ± 0.25d
2.35 ± 0.10c
0.22 ± 0.02b
0.44 ± 0.02b
2.23 ± 0.25e
4.47 ± 0.28e
0.22 ± 0.02b
0.45 ± 0.00b
0.25 ± 0.00b
0.50 ± 0.00b
0.22 ± 0.02b
0.45 ± 0.02b
0.49 ± 0.03c
0.99 ± 0.03c
0.05 ± 0.001a
0.06 ± 0.003a
1.52 ± 0.16d
5.72 ± 0.33e
0.50 ± 0.00c
1.00 ± 0.00c
4.40 ± 0.22f
8.80 ± 0.60f
0.18 ± 0.01b
0.36 ± 0.05b
1.77 ± 0.12d
3.54 ± 0.67d
2.84 ± 0.30e
5.69 ± 0.15e
0.35 ± 0.025bc
0.53 ± 0.05b
0.86 ± 0.04 cd
0.94 ± 0.03c
0.22 ± 0.02b
0.56 ± 0.05d
4.47 ± 1.12ef
0.11 ± 0.01b
0.45 ± 0.05c
0.12 ± 0.04b
0.25 ± 0.02b
0.11 ± 0.01b
0.45 ± 0.05c
0.25 ± 0.05c
0.49 ± 0.06c
0.05 ± 0.001a
0.06 ± 0.001a
2.67 ± 1.12f
5.72 ± 1.18f
0.25 ± 0.05c
1.00 ± 0.1d
15
16
18
19
20
22
23
24
25
1.65 ± 0.9e
3.30 ± 1.56e
0.18 ± 0.04bc
0.36 ± 0.06bc
1.77 ± 0.9e
31
32
3.54 ± 1.15e
2.84 ± 0.9f
33
5.69 ± 0.1.15f
0.18 ± 0.01bc
0.35 ± 0.01bc
0.86 ± 0.04d
1.14 ± 0.15d
Streptomycin
Ampicillin
B.c.: B. cereus, M.f.: M. flavus, S.a.: S. aureus, L.m.: L. monocytogenes, E.c.: E. coli, En.cl.: En. cloacae, P.a.: P. aeruginosa, S.t.: S. typhimurium.
Values are expressed as means ± SD. Different letters in each column indicate statistically significant differences between samples (p < 0.05).
characteristics of C. elegans such as pharyngeal pumping rate, defecation
rhythm, locomotion, development and reproduction [23–25]. Conse-
quently, three phenotypic characteristics of the wild type (wt) animals
were evaluated upon treatment with the tested compounds, namely the
pharyngeal pumping rate, the defecation rate and the developmental
timing which have been previously shown to respond to various toxic
treatments [18,26]. Day 1 is considered as short-term exposure while
day 3 as long-term exposure. Regarding the effects observed following
exposure to compounds 15 and 22 at the MIC concentrations all
phenotypic characteristics, including developmental timing, did not
show any significant changes at any time point with the exception of a
slight deceleration in the defecation rate at day 1 and a slight decrease in
the pharyngeal pumping rate at day 3 of the animals treated with 0.05
mM compound 22 (Table 3). Therefore, based on this model, both
compounds may be considered as non-toxic at MIC concentrations. With
regard to exposure to compound 15 at the MFC concentration, a sig-
nificant change was observed at day 1 for the pharyngeal pumping rate
(but no difference during the long exposure) and at day 3 for the defe-
cation rate (Table 3). Significant changes were recorded at all time
points for compound 22 after exposure to the MBC concentration
(Table 3). Finally, the developmental timing was found increased by 1.1
h at the MFC and MBC concentrations for compounds 15 and 22,
respectively (Table 3). Thus, our results suggest that compound 22 is
more toxic than compound 15 at high concentrations. The observed
differences are in accordance with previous studies examining the
toxicity of various agents. More specifically, pharyngeal pumping rate
and defecation rhythms have been shown to be highly responsive to
various toxic agents such as anti-obesity drugs (Reductil® and Xenical®;
reduction of pharyngeal pumping rate upon exposure to Reductil® and
increase of defecation rate upon exposure to Xenical®; [23]), biochars
(increased interval time between two defecations, no impact on the
pumping velocity; [24]), soils with various degrees of pollution
(significantly reduced pharyngeal pumping velocity; [25]).
Developmental delay has been also observed in nematodes after expo-
sure to arsenic, lead and mercury heavy metals [27]. In total, we
revealed alterations in our animal populations mainly following expo-
sure to the highest effective concentrations (MFC and MBC) but
importantly not to the MIC concentrations. Therefore, our results
advocate for rather low toxicity effects of both compounds.
2.4. Computational studies
2.4.1. Insights into the mechanisms of antimicrobial activity
In order to propose a putative mechanism for the antimicrobial ac-
tivity of the most potent compounds, a two-step computational process
was applied, using ligand-based similarity search and molecular dock-
ing, in order to predict the binding of the active candidates to the target
receptor of similar antibiotics [28,29].
In the case of antibacterial activity, the similarity metrics analysis
indicates that the most potent synthetic derivatives (compounds 14 and
22 - query molecule) present fingerprint similarity with the known FDA-
approved drug Levofloxacin [30], a broad-spectrum, third-generation
fluoroquinolone antibiotic. It is well known that Levofloxacin diffuses
through the bacterial cell wall and its activity is through inhibition of
bacterial topoisomerase II DNA gyrase, an enzyme required for bacterial
DNA replication and repair as well as RNA transcription. Overall, its
inhibition blocks the bacterial cell growth. Taking this observation into
account, the crystal structure of S. aureus gyrase co-crystallized with
ciprofloxacin was selected for further docking studies (PDB ID:2XCT)
[31].
Similarly, querying compound 15 (the most potent antifungal
analogue) revealed itraconazole, a first generation triazole antifungal
drug, among the structurally similar compounds. Itraconazole inhibits
the synthesis of fungal sterols by inhibiting the cytochrome P450-
dependentlanosterol 14α-demethylase (CYP51A) enzyme [32]. The
referred enzyme plays a crucial role in the biosynthesis of ergosterol
5

G.E. Magoulas et al.
Bioorganic Chemistry xxx (xxxx) xxx
Table 2
In vitro antifungal evaluation of compounds 14–16, 18–20, 22–25 and 31–33.
Compounds
MIC/MFC (mМ)
A. fum.
A.v.
A.o.
A.n.
T.v.
P. f.
P.o.
P.v.c.
14
15
MIC
MFC
MIC
MFC
0.013 ± 0.03a
0.013 ± 0.03a
0.208 ± 0.005c
0.027 ± 0.005b
0.056 ± 0.005a
0.026 ± 0.004b
0.208 ± 0.005c
0.027 ± 0.005b
0.058 ± 0.005a
0.026 ± 0.004c
0.208 ± 0.005c
0.027 ±
0.026 ± 0.004b
0.208 ± 0.005c
0.027 ±
0.026 ± 0.004b
0.208 ± 0.005c
0.027 ±
0.026 ± 0.004b
0.208 ± 0.005c
0.027 ±
0.013 ± 0.03a
0.208 ± 0.005c
0.027 ± 0.005b
0.056 ± 0.005a
0.416 ± 0.03c
0.027 ± 0.005a
0.056 ± 0.005a
0.005b
0.005b
0.005b
0.005b
0.056 ± 0.005a
0.105 ± 0.02d
0.21 ± 0.02c
0.015 ± 0.008a
0.233 ± 0.05c
0.029 ± 0.005b
0.05 ± 0.015a
0.464 ± 0.02e
0.926 ± 0.14e
0.065 ± 0.03c
0.129 ± 0.02b
0.056 ± 0.005a
0.027 ± 0.01b
0.056 ± 0.03a
0.015 ± 0.008a
0.233 ± 0.05c
0.029 ± 0.005b
0.06 ± 0.015a
0.059 ± 0.01c
0.464 ± 0.02d
0.034 ± 0.01b
0.065 ± 0.03a
0.056 ± 0.005a
0.027 ± 0.01b
0.056 ± 0.03a
0.015 ± 0.008a
0.233 ± 0.05c
0.029 ± 0.005b
0.06 ± 0.015a
0.059 ± 0.01c
0.464 ± 0.02d
0.065 ± 0.03c
0.13 ± 0.02b
0.056 ± 0.005a
0.027 ± 0.01b
0.056 ± 0.03a
0.015 ± 0.008a
0.233 ± 0.05c
0.029 ± 0.005b
0.06 ± 0.015a
0.059 ± 0.01c
0.464 ± 0.02d
0.034 ± 0.01bc
0.065 ± 0.03a
16
18
19
20
22
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
0.210 ± 0.03d
0.420 ± 0.06c
0.233 ± 0.03d
0.465 ± 0.04d
0.057 ± 0.005b
0.112 ± 0.02b
0.926 ± 0.14c
1.852 ± 0.20e
0.065 ± 0.03b
0.259 ± 0.06bc
0.045 ± 0.03c
0.056 ± 0.03a
0.012 ± 0.01a
0.233 ± 0.08c
0.029 ± 0.003b
0.059 ± 0.003a
0.464 ± 0.02 g
0.926 ± 0.14f
0.034 ±
0.027 ± 0.01b
0.056 ± 0.03a
0.015 ± 0.008a
0.233 ± 0.05c
0.029 ± 0.005b
0.06 ± 0.015a
0.232 ± 0.02d
0.464 ± 0.02d
0.034 ± 0.01b
0.13 ± 0.03b
0.045 ± 0.03c
0.056 ± 0.03a
0.015 ± 0.008a
0.232 ± 0.05c
0.225 ± 0.025e
0.45 ± 0.05d
0.464 ± 0.02f
0.926 ± 0.14e
0.065 ± 0.03d
0.259 ± 0.03c
0.002bc
0.259 ± 0.04c
0.046 ± 0.06c
0.095 ± 0.02b
23
24
MIC
MFC
MIC
MFC
0.046 ± 0.01a
0.095 ± 0.03b
0.063 ± 0.003b
2.00 ± 0.40e
0.046 ± 0.01bc
0.041 ± 0.01bc
0.095 ± 0.03b
0.125 ± 0.003d
0.501 ± 0.05d
0.046 ± 0.01bc
0.046 ± 0.01bc
0.046 ± 0.01c
0.046 ± 0.01c
0.095 ± 0.03b
0.125 ±
0.095 ± 0.03ab
0.095 ± 0.03b
0.095 ± 0.03b
0.095 ± 0.03b
NA
NA
NA
NA
NA
NA
NA
NA
NA
0.003de
NA
1.001 ± 0.05 e
1.98 ± 0.10 h
3.96 ± 0.30 g
0.171 ± 0.06e
0.342 ± 0.02 cd
0.443 ± 0.03f
3.188 ± 0.90 g
25
31
32
MIC
MFC
MIC
MFC
MIC
MFC
1.980 ± 0.67f
3.960 ± 1.12f
0.171 ± 0.06 cd
1.363 ± 0.30d
0.433 ± 0.03e
3.188 ± 0.90f
0.98 ± 0.067d
1.98 ± 0.10 g
0.044 ± 0.02c
0.171 ± 0.06bc
0.035 ±
0.99 ± 0.08c
1.98 ± 0.10f
0.109 ± 0.08c
0.171 ± 0.06b
0.021 ± 0.01ab
0.044 ± 0.04a
1.32 ± 0.30f
0.55 ± 0.00d
0.55 ± 0.00c
0.55 ± 0.00c
3.96 ± 0.30 h
0.044 ± 0.02c
0.342 ± 0.02d
0.443 ± 0.03d
0.797 ± 0.03e
1.98 ± 0.10f
1.98 ± 0.10f
3.96 ± 0.30 g
0.044 ± 0.02bc
0.342 ± 0.02 cd
0.443 ± 0.03e
0.797 ± 0.03e
0.171 ± 0.06d
0.342 ± 0.02 cd
0.443 ± 0.03e
0.797 ± 0.03de
0.044 ± 0.02bc
0.171 ± 0.06bc
0.035 ± 0.005b
0.044 ± 0.02a
0.005bc
0.044 ± 0.04a
0.227 ± 0.03e
0.455 ± 0.05d
0.376 ± 0.004f
0.941 ± 0.03f
33
MIC
MFC
MIC
MFC
0.227 ± 0.03d
1.820 ± 0.50e
0.376 ±
0.227 ± 0.03d
0.455 ± 0.05d
0.282 ± 0.04d
0.376 ± 0.004 cd
0.455 ± 0.05e
0.91 ± 0.01e
0.376 ± 0.004e
0.941 ± 0.03e
0.057 ± 0.005c
0.455 ± 0.05d
1.882 ± 0.07e
2.822 ± 0.50 g
0.057 ± 0.005c
0.901 ± 0.05f
0.376 ± 0.004d
0.941 ± 0.03f
0.455 ± 0.05d
1.82 ± 0.025f
1.882 ± 0.07f
2.882 ± 0.50 g
0.455 ± 0.05f
1.82 ± 0.025f
2.882 ± 0.50 h
3.763 ± 0.60 g
Ketoconazole
0.004de
0.941 ± 0.03d
0.483 ± 0.01e
0.644 ± 0.07 cd
Bifonazole
MIC
0.332 ± 0.04f
0.644 ± 0.07e
0.483 ± 0.01e
0.644 ± 0.07e
0.483 ± 0.01e
0.644 ± 0.07de
0.483 ± 0.01d
0.644 ± 0.07e
0.644 ± 0.07f
0.805 ± 0.06e
0.644 ± 0.07e
0.805 ± 0.07e
0.644 ± 0.07 g
0.966 ± 0.08e
MFC
NA: not active.
A.fum.: A. fumigatus, A.v.: A. versicolor, A.o.: A. ochraceus, A.n.: A. niger, T.v.: T. viride, P.f.: P. funiculosum, P.o.: P. ochrochloron, P.v.c.: P. verrucosum var. cyclopium.
Values are expressed as means ± SD. Different letters in each column indicate statistically significant differences between samples (p < 0.05).
Fig. 2. Absence of toxic effects of compounds 15 and 22 in HFL-1 human primary cells. Survival ratio following crystal violet staining of HFL-1 human embryonic
primary fibroblasts treated with (A) 0.027 and 0.058 mM compound 15 and B) 0.05 and 0.26 mM compound 22 or the appropriate amount of DMSO (control) for 24
and 48 h. The dye absorbance exhibited by the control cells was set to 100%. All values are reported as mean of three independent experiments. Error bars denote ±
SEM. NS (not significant).
catalyzing the demethylation step of lanosterol to ergosterol. Due to the
absence of crystal structures of CYP51A enzyme of the examined fungi, a
previously published homology model of A. fumigatus was used for
docking studies [33].
acid Arg1122 through a π-cation interaction. (Fig. 3B, Table 4). Finally,
the amine group of compound 22 is located in close proximity with the
Mn cation although metal chelation interactions were not conserved in
all docked poses.
The docking results of compounds 14 and 22 on S. aureus topo-
isomerase II DNA gyrase present Glide scores (ꢀ 7.2 and ꢀ 7.1 kcal⋅molꢀ 1
respectively), in the range of previously reported active antibacterials
[34] as well as interactions with enzyme residues and DNA bases at the
active site of the enzyme. Specifically, compound 22 interacts similarly
to ciprofloxacin and levofloxacin [30] with the crucial residue Ser1084
through H bond formation and with DG8 and DG9 DNA chains through
The binding poses of compound 14 are also located in the active site
of the enzyme interacting through π-π interactions and hydrogen bonds
with the DG8 and DG9 DNA chains, but lacks the interaction with the
amino acid Ser1084. Interestingly, it seems to interact with Asp437
through halogen bond, a residue also found in contact with the bound
pose of levofloxacin in previous studies [30] (Fig. 3A, Table 4).
The docking results of compounds 14 and 15 present Glide scores
ꢀ 8.6 ⋅and ꢀ 8.1 kcal molꢀ 1 respectively with both compounds being
π-π interactions. Additionally, it’s binding pose interacts with the amino
6

G.E. Magoulas et al.
Bioorganic Chemistry xxx (xxxx) xxx
Table 3
Phenotypic analysis of C. elegans N2 wt strain treated with compounds 15 and 22 or DMSO (control) at day 1 and 3 of adulthood.
DAY 1
DAY 3
Pharyngeal pumping a
Concentration (mM)
Pharyngeal pumping a
Defecation b
Defecationb
Developmental timing c
DMSO (15)
225.3 ± 4.5
222.4 ± 3.0
182.2 ± 3.6***
213.6 ± 1.7
214.7 ± 4.5
176.7 ± 2.1***
47.4 ± 1.1
47.2 ± 0.7
46.3 ± 0.5
43.8 ± 0.4
45.6 ± 0.8*
47.4 ± 0.4***
199.3 ± 3.4
195.3 ± 2.4
204.4 ± 1.8
212.7 ± 1.3
200.4 ± 2.2***
203.2 ± 1.9***
51.2 ± 0.7
51.6 ± 0.6
52.4 ± 0.36
53.4 ± 0.22
Compound 15
0.027
0.058
44.8 ± 0.7***
52.6 ± 0.5
53.5 ± 0.20*
52.8 ± 0.18
53.6 ± 0.22
53.9 ± 0.22**
DMSO (22)
Compound 22
0.05
0.26
51.2 ± 0.8
57.9 ± 0.5***
All assays were performed at 20 ◦C. The results are presented as mean (n = 3) ± SEM.
***p < 0.001,**p < 0.005, *p < 0.05 as compared to DMSO.
a
b
Number of Pumps in 1 min at day 1 and 3 of adulthood.
Duration of defecation cycle in seconds at day 1 and 3 of adulthood.
c
Duration of post-embryonic development (hours from egg hatching to L4 moult).
Fig. 3. 2D protein- ligand interaction diagrams of (A) compound 14 (docking score = ꢀ 7.2 kcal⋅molꢀ 1) and (B) compound 22 (docking score = ꢀ 7.1 kcal⋅molꢀ 1) into
the active site of S. aureus topoisomerase II DNA gyrase (PDB ID:2XCT) and (C) compound 14 (docking score = ꢀ 8.6 kcal⋅molꢀ 1) and (D) 15 (docking score = ꢀ 8.1
kcal⋅molꢀ 1) into the active site of A. fumigatus CYP51A enzyme [33].
7

G.E. Magoulas et al.
Bioorganic Chemistry xxx (xxxx) xxx
Table 4
Docking interactions with S. aureus topoisomerase II DNA gyrase enzyme.
Protein Residues / DNA
Ciprofloxacin
Compound
14
Compound 22
bases
Mn2+
Chelation
–
Contact/
chelation
H-bond
Ser1084
Arg1122
Asp437 / Ser438
DG8
H-bond
–
–
–
–
π
–
π
π
-cation
Halogen bond
π
-
π
π
&
&
π
-cation
π
π
-
-
π
π
& H-bond
& H-bond
-
-
π
π
DG9
π-
π-cation
Fig. 4. (A) The pyrimidinone ring as a common scaffold of the synthesized
compounds, and (B) the selected core scaffold including a thienopyrimidinone
ring substituted with a phenyl and a N-methylpiperidine ring. The attachment
positions are designated as Rs.
DC13
H-bond
Contact
Contact
positioned into the A. fumigatus CYP51A active site. Docking poses
reveal a binding mode similar to R-econazole, with formation of
π-π
affect M. flavus, L. monocytogenes, E. coli and S. typhimurium (i.e. com-
pound 25). Comparing compounds 22, 24 and 25 which all bear HB-D or
HB-A at R4, the ethylene diamine side chain of compound 22 signifi-
cantly reinforces its antibacterial activity in all tested strains compared
to compounds 24 and 25, indicating as optimal the two-carbon atom
chain. In contrast, the addition of one more carbon in the side chain
induces a reduction in the activity while the replacement by a piperazine
ring leads to a complete loss of antibacterial activity. Finally, the effect
of R3 substitutions could not be evaluated since all tested compounds
bear a methyl group.
interactions with Tyr121, a consistent amino acid interaction found in
azole family of CYP51A inhibitors [33]. Furthermore, the hydroxyl
group of compound 15 forms an H-bond with Ser363, a residue also
noted to contribute in binding [33]. Finally, the ether oxygen atom of
both compounds 14 and 15, is in proximity with the heme iron atom
(Fig. 3C and D, respectively and Table 5).
2.4.2. Computational model for structure activity relationship analysis
In order to obtain a better understanding about the structure activity
relationships a Pharma QSAR model was developed based on scaffold
decomposition and R-group analysis (Materials and methods section).
Specifically, the 13 studied compounds were subjected to scaffold
decomposition to identify common structural features (core structural
motifs). Although the pyrimidinone ring (Fig. 4A) was found as a com-
mon scaffold among all the synthesized compounds, it cannot provide
satisfactory SAR results since it is a generic core with many and different
substitutions. At a step further, a core scaffold consisting of a thieno-
pyrimidinone ring substituted with a phenyl and a N-methylpiperidine
ring was selected (Fig. 4B) which is encountered in 10 out of 13 com-
pounds. Subsequently, R-group analysis was performed to identify the
attachment points on the core scaffold, (R-groups), and their correlation
with the molecular properties.
Similar models were generated to correlate the antifungal activity
with the pharmacophore features of the new compounds. Statistical
models were generated focusing on the previous core scaffold, offering a
putative explanation of compounds’ antifungal activity against
A. fumigatus, A. niger and P. verrucosum var. cyclopium (Table 7A, Fig. S3,
Supplementary Material). Since compound 24 was completely inactive
against A. versicolor, A. ochraceus, T. viride, P. funiculosum and
P. ochrochloron, it was excluded from the dataset and models were re-
generated (Table 7B).
The Pharma RQSAR models indicate that the presence of a hydrogen
bond acceptor (HB-A) or a hydrophobic (H) group at R1 position has a
negative effect on activity against A. niger (i.e. compound 24), while this
is not observed for the cases of A. fumigatus and P. verrucosum var.
cyclopium. Regarding the R2 position, a hydrophobic (H) substituent
results into a positive effect on activity against A. niger (i.e. compounds
18 and 19) while again A. fumigatus and P. verrucosum var. cyclopium are
not affected by substitutions at R2 (similarly to R1). Regarding the R4
position, a hydrogen bond acceptor (HB-A) leads to increase of activity
against A. niger, A. fumigatus and P. verrucosum var. cyclopium species.
Substitution by an HB-D increases the antifungal activity against A. niger
and P. verrucosum var. cyclopium species. (i.e. compounds 14 and 15).
Specifically, a comparison between compounds 14 and 16, sharing a
common substitution at R4, indicated the crucial role of chlorine (-Cl) at
R2 position which is reflected by the strongest antifungal activity of
compound 14. Comparison of compounds 18, 19 and 20, bearing a
common chlorine (-Cl) atom at R2 position, showed a stronger antifungal
activity for compounds 18 and 19 compared to 20 which may be
explained by the replacement of the terminal amino group of the
aliphatic amines by a hydroxyl group at R4 position.
The correlation of the R-groups (based on their pharmacophore
features) with the in vitro antibacterial activity (MIC values) for each
bacterial species is presented in Table 6 and Fig. S2 (Supplementary
Material). Some general observations can be drawn from the results. The
presence of a hydrogen bond acceptor (HB-A) or a hydrophobic group
(H) at R1 position have a negative impact in almost all bacterial strains
with the exception of L. monocytogenes and E. coli (i.e. compounds 22
and 24). Furthermore, a hydrophobic substituent is preferred at R2 po-
sition except for the cases of L. monocytogenes and E. coli while, a
hydrogen bond acceptor (HB-A) at R2 is beneficial for M. flavus E. coli
and En. cloacae (i.e. compounds 16 and 19). Thus, among compounds
14, 15 and 16, the methoxy (-OMe) substitution at meta position (R1) of
compound 15 caused a significant decrease in activity compared to
compound 14 bearing the same (-OMe) group at the para position (R2) of
the phenyl ring (Table 6). Substitution of the methoxy group with a
chlorine (-Cl) atom (as in the case of compound 16) exhibited one-fold
increase of the activity against E. coli. With respect to R4 position, a
hydrogen bond donor (HB-D) is clearly preferred for most species, while,
the HB-A at R4 positively affects the species of B. cereus, S. aureus, En.
cloacae and S. typhimurium. Finally, hydrophobic groups at R4 negatively
According to the second model for A. versicolor, A. ochraceus, T. vir-
ide, P. funiculosum and P. ochrochloron (Fig. S4, Supplementary Mate-
rial), common SAR patterns are observed for all species (Table 7).
Specifically, a substitution at R1 position with an HB-A or a hydrophobic
(H) group (compound 15) negatively affects the activity while hydro-
phobic (H) groups at R2 position (compounds 14, 18 and 19) induce a
positive effect. Moreover, a hydrogen bond acceptor (HB-A) at R4 po-
sition positively affects antifungal activity (compounds 18, 19 and 22).
Indicatively, comparison between compounds 22 and 25 proved that in
the case of compound 22 the ethylenediamine side chain at R4 position
contributes positively to antifungal activity while its replacement by the
piperazine moiety (compound 25) leads to loss of activity.
Table 5
Docking interactions with A. fumigatus CYP51A enzyme.
Protein Residues
Econazole
Compound 14
Compound 15
Fe3+
Chelation
Contact
Contact
Tyr107
Tyr121
Ser363
π
π
–
-
-
π
π
–
–
π
-
π
π
-π
–
H-bond
8

G.E. Magoulas et al.
Bioorganic Chemistry xxx (xxxx) xxx
Table 6
Effect of substituents on antibacterial activity as derived from the Pharma RQSAR models.
Attachment Positions – Activity Effect
R1
R2
R4
Bacterial strains
HB-A
H
HB-A
H
HB-D
HB-A
H
B. cereus
Negative
Negative
Negative
Weak
Negative
Negative
Negative
Weak
Weak
Positive
Positive
Positive
Weak
Positive
Positive
Positive
Positive
Positive
Weak
Positive
Weak
Weak
M. flavus
Positive
Weak
Negative
Weak
S. aureus
Positive
Weak
L. monocytogenes
E. coli
Weak
Negative
Negative
Weak
Weak
Weak
Positive
Positive
Weak
Weak
Weak
En. cloacae
P. aeruginosa
S. typhimurium
Negative
Negative
Negative
Negative
Negative
Negative
Positive
Positive
Positive
Positive
Weak
Positive
Positive
Weak
Weak
Positive
Negative
Attachment positions: R1 (meta) - R2 (ortho) substituted phenyl ring is attached at the N-3 position of the thieno[2,3-d]pyrimidin-4(3H) scaffold, R4 aliphatic amines
or alcohols are anchored at the C-2 position through a sulfur or amino bridge. Pharmacophore features representation: Hydrogen bond acceptor (HB-A), Hydrogen
bond donor (HB-D), Hydrophobic region (H).
Table 7
Effect of substituents on the antifungal activity as derived from the Pharma RQSAR models.
Attachment Positions – Activity Effect
Fungal strains
R1
R2
R4
1st model (Table 7A)
HB-A
H
HB-A
H
HB-D
HB-A
H
A. fumigatus
A. niger
Weak
Weak
Weak
Weak
Weak
Weak
Weak
Positive
Positive
Positive
Positive
Weak
Negative
Weak
Negative
Weak
Positive
Weak
Positive
Positive
P. cycl.
Weak
2nd model (Table 7B)
A. ochraceus
A. versicolor
P. funiculosum
P. ochrochloron
T. viride
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Weak
Weak
Weak
Weak
Weak
Positive
Positive
Positive
Positive
Positive
Weak
Weak
Weak
Weak
Weak
Positive
Positive
Positive
Positive
Positive
Weak
Positive
Weak
Weak
Weak
Attachment positions: R1 (meta) - R2 (ortho) substituted phenyl ring is attached at the N-3 position of the thieno[2,3-d]pyrimidin-4(3H) scaffold, R4 aliphatic amines
or alcohols are anchored at the C-2 position through a sulfur or amino bridge. Pharmacophore features representation: Hydrogen bond acceptor (HB-A), Hydrogen
bond donor (HB-D), Hydrophobic region (H).
Finally, the molecular properties of the synthesized compounds were
calculated indicating that they are within the ranges of the recom-
mended values for drug likeliness (Table S3, Supplementary Material).
embryonic primary cells and in vivo on the nematode C. elegans, revealed
absence of in vitro toxicity at MIC/MFC and MIC/MBC concentrations,
and of in vivo toxicity at MIC concentrations for both compounds.
Furthermore, R-Group analysis as a means of structure activity re-
lationships computational tool, highlighted the effect of specific chem-
ical features (as substituents on a core scaffold) on the antibacterial and
antifungal activity.
3. Conclusions
A series of new thieno[2,3-d]pyrimidin-4(3H)-one derivatives were
synthesized and evaluated for their activity against four gram-positive
and four gram-negative bacterial strains and eight fungal species. The
structural variations included substitution at N-3 position by a phenyl
ring bearing an electron-donating (-OMe) or electron-withdrawing (-Cl)
group, biocompatible aliphatic amines or a hydroxyalkoxy group
anchored at the C-2 position through a sulfur or amino bridge and a N-
methylpiperidine or a cyclohexane moiety fused to the thieno[2,3-d]
pyrimidin-4(3H)-one core.
Finally, a putative mechanism of antibacterial activity is related to
inhibition of the topoisomerase II DNA gyrase enzyme as shown for the
case of S. aureus and the active analogue 22, while inhibition of CYP51
lanosterol demethylase enzyme may be a putative mechanism for anti-
fungal activity as presented for the case of A. fumigatus and compound
15.
In conclusion, compounds 15 and 22 represent promising leads for
the further development of potent antimicrobial and antifungal agents
and future studies are required to optimize their efficacy against addi-
tional strains including resistant ones and to delineate the mechanism(s)
of their activity.
The majority of the compounds exhibited excellent antimicrobial
and/or antifungal activity, being more potent than the control
compounds.
The most potent antibacterial agent was compound 22, bearing at N-
3 a m-methoxyphenyl group and an ethylenediamine side chain at C-2,
presenting broad spectrum activity with MIC = 0.027 ± 0.005 mM and
MFC = 0.056 ± 0.005 mM against all strains tested. The most potent
antifungal agent was 15 substituted by a m-methoxyphenyl group at N-3
and a 2-(2-mercaptoethoxy)ethan-1-ol side chain at C-2 with MIC =
0.027 ± 0.005 mM and MFC = 0.056 ± 0.005 mM against all fungal
strains tested. Furthermore, compound 22 also showed high antifungal
potency with MIC values ranging between 0.034 and 0.065 mM and
MFC ranging between 0.065 and 0.259 mM. Evaluation of the potential
toxic effects of compounds 15 and 22 in vitro on HFL-1 human
4. Experimental
4.1. Chemistry
Melting points were determined with a Buchi 510 apparatus and are
1
uncorrected. H NMR spectra were recorded on Varian spectrometers
operating at 300 MHz or 600 MHz and 13C NMR spectra were recorded
at 75 MHz or 150 MHz respectively. Chemical shifts are reported in δ
units, parts per million (ppm) downfield from TMS. IR spectra were
recorded by using the attenuated total reflection (ATR) method on a
9

G.E. Magoulas et al.
Bioorganic Chemistry xxx (xxxx) xxx
FTIR Bruker Tensor 27. Electro-spray ionization (ESI) mass spectra were
recorded on a LC-MSn Fleet, Thermo spectrometer using MeOH as sol-
vent. HRMS spectra were recorded, in the ESI mode, on UPLC-MSn
Orbitrap Velos-Thermo. Reactions under microwave irradiation were
performed in a CEM Discover Lab Mate reactor. Flash column chroma-
tography (FCC) was performed on Merck silica gel 60 (230–400 mesh)
and TLC on Merck 60 F254 films (0.2 mm) precoated glass plates. Spots
were visualized with UV light at 254 nm and ninhydrin or PMA stain. All
solvents were dried and/or purified according to standard procedures
prior to use. All reagents employed in the present work were purchased
from commercial suppliers and used without further purification. Re-
actions were run in flame-dried glassware under an atmosphere of
argon.
4.1.2.3. Ethyl 2-(3-(4-methoxyphenyl)thioureido)-6-methyl-4,5,6,7-tetra-
hydrothieno [2,3-c]pyridine-3-carboxylate (9). Using the general pro-
cedure above, compound 3 (0.3 g, 1.25 mmol), 4-methoxyphenyl
isothiocyanate (9) (0.21 mL, 1.56 mmol) the title compound was ob-
tained as a yellow solid (0.23 g, 45%) after FCC (EtOAc/MeOH, 97:3); Rf
= 0.62 (DCM/MeOH 9:1); 1H NMR (300 MHz, CDCl3): δ 11.96 (s, 1H),
7.70 (s, 1H), 7.24 (d, J = 8.8 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 4.12 (q, J
= 7.1 Hz, 2H), 3.85 (s, 3H), 3.50 (s, 2H), 2.85 (t, J = 5.9 Hz, 2H), 2.66 (t,
J = 5.8 Hz, 2H), 2.46 (s, 3H), 1.24 (t, J = 7.1 Hz, 3H).
4.1.3. 2-(2-hydroxyethoxy)ethyl 4-methylbenzenesulfonate (13)
To a solution of diethylene glycol (7.1 mL, 74.8 mmol) in THF (2.2
mL) a solution of NaOH (0.43 g, 10.85 mmol) in H2O (2 mL) was added.
The mixture was cooled at 0 ◦C and a solution of p-toluenesulfonyl
chloride (1.38 g, 7.2 mmol) in THF (2.0 mL) was added dropwise. After
stirring for 2 h at rt, the reaction mixture was partitioned between cold
water (20 mL) and DCM (30 mL). The organic layer was separated and
the aqueous layer was then extracted with DCM (2 × 30 mL). The
combined organic layers were washed with brine, dried over Na2SO4
and evaporated under reduced pressure to afford the crude title com-
pound 13 which was dried over P2O5 for 3 h under high vacuum and
used to the next step without further purification. Colourless oil (1.19 g,
63%). Rf = 0.27 (petroleum ether/EtOAc 1:1); 1H NMR (300 MHz,
CDCl3): δ 7.78 (d, J = 7.9 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H), 4.19 (t, J =
4.5 Hz, 2H), 3.74–3.59 (m, 4H), 3.52 (t, J = 4.4 Hz, 2H), 2.44 (s, 3H),
1.96 (s, 1H).
4.1.1. Ethyl 2-amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxylate (3)
To an ice-cold rb-flask containing N-methyl-4-piperidone (1) (2.87
mL, 25.0 mmol), sulfur (0.8 g, 25.0 mmol) and ethyl cyanoacetate (2)
(2.66 mL, 25.0 mmol), a solution of diethylamine (2.57 mL, 25.0 mmol)
in MeOH (2 mL) was added. Then, a condenser was installed and irra-
◦
diated for 4 min at 120 W and 100 C in a CEM-Discover microwave
reactor. The reaction mixture was cool to room temperature and then
diluted with DCM (50 mL). The organic layer was washed with brine (30
mL), dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The residue was recrystallized using MeOH/diethyl ether/
hexane to afford compound 3 as a brownish solid (4.2 g, 69%); Rf = 0.30
(DCM/MeOH 95:5); m.p: 82–84 ◦C; 1H NMR (300 MHz, CDCl3): δ 4.26
(q, J = 7.1 Hz, 2H), 3.38 (s, 2H), 2.84 (t, J = 5.9 Hz, 2H), 2.66 (t, J = 5.9
Hz, 2H), 2.44 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H); 13CNMR (75 MHz, CDCl3):
δ 165.9, 162.0, 130.6, 114.5, 105.3, 59.5, 53.3, 52.4, 45.4, 27.3, 14.4;
ESI-MS: m/z 241 [M+H]+.
4.1.4. General procedure for the preparation of compounds 14–16
To a solution of compound 7, 8 or 9 (0.46 mmol), an ethanolic KOH
solution (2 mL, 0.21 М) was added and the reaction mixture heated to
reflux. The progress of the reaction was monitored by TLC. Upon
completion, the reaction mixture was filtered and to the filtrate con-
taining the corresponding thiolate 10–12, 2-(2-hydroxyethoxy)ethyl 4-
methylbenzenesulfonate (13) (0.143 g, 0.55 mmol) was added and the
reaction was further heated to reflux for 18 h. The solvent was evapo-
rated under reduced pressure and the projected compounds were iso-
lated in pure form after FCC.
4.1.2. General procedure for the preparation of compounds 7–9
In a 10 mL microwave reaction tube, compound 3 (0.3 g, 1.25 mmol)
and the corresponding aryl isothiocyanate 4, 5 or 6 were mixed (3 mL of
dry pyridine was also added in the case of compound 4). The tube was
sealed and irradiated in a CEM Discover microwave reactor at 120 W,
100 ◦C, over 2 min. After cooling at ambient temperature the residues
were subjected to FCC to afford the desired compounds in pure form. In
the case of 4 pyridine was evaporated under reduced pressure first and
the residue was subjected to FCC to afford the desired compound in pure
form.
4.1.4.1. 3-(4-Chlorophenyl)-2-[2-(2-hydroxyethoxy)ethylsulfanyl]-7-
methyl-5,6,7,8-tetrahydro-3H-pyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4-
one (14). Using the general procedure above from compound 7 (0.209
g, 0.46 mmol) the title compound 14 was obtained as a yellow solid
(0.13 g, 63%) after FCC (EtOAc/MeOH, 95:5). Rf = 0.53 (DCM/MeOH,
9:1); m.p: 180 – 182 ◦C; IR: 3371, 2939, 1676, 1510 cmꢀ 1; 1H NMR (600
MHz, CDCl3): δ 7.50 (d, J = 8.7 Hz, 2H), 7.19 (d, J = 8.7 Hz, 2H), 3.73 (t,
J = 6.2 Hz, 2H), 3.70 (dd, J = 4.8 and 4.2 Hz, 2H), 3.63 (bs, 2H), 3.57
(dd, J = 4.8 and 4.2 Hz, 2H), 3.34 (t, J = 6.2 Hz, 2H), 3.06 (t, J = 5.3 Hz,
2H), 2.75 (t, J = 5.3 Hz, 2H), 2.50 (s, 3H); 13C NMR (150 MHz, CDCl3): δ
162.7, 158.4, 156.9, 136.2, 133.8, 130.3, 130.0, 129.9, 128.7, 118.6,
71.9, 68.8, 61.7, 53.5, 51.8, 45.3, 31.6, 25.7; ESI-MS: m/z 452.1 and
454.2 (3:1) [M+H]+; ESI-HRMS: m/z [M+H]+ calcd for
4.1.2.1. Ethyl 2-(3-(4-chlorophenyl)thioureido)-6-methyl-4,5,6,7-tetrahy-
drothieno [2,3-c]pyridine-3-carboxylate (7). Using the general procedure
above compound 3 (0.3 g, 1.25 mmol), 4-chlorophenyl isothiocyanate
(7) (0.32 g, 1.88 mmol) and pyridine (3 mL), the title compound was
obtained as a yellow solid (0.256 g, 50%) after FCC (EtOAc/ MeOH,
97:3); Rf = 0.24 (DCM/MeOH 95:5); m.p. 204–206 ◦C (Lit. [35] –
206–208 ◦C); 1H NMR (300 MHz, CDCl3): δ 12.21 (s, 1H), 7.84 (s, 1H),
7.43 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 4.19 (q, J = 7.1 Hz,
2H), 3.51 (s, 2H), 2.87 (t, J = 5.8 Hz, 2H), 2.68 (t, J = 5.8 Hz, 2H), 2.47
(s, 3H), 1.29 (t, J = 7.1 Hz, 3H).
C20
Н
23N3O335ClS2 452.0864, found 452.0860.
4.1.4.2. 2-[2-(2-Hydroxyethoxy)ethylsulfanyl]-3-(3-methoxyphenyl)-7-
methyl-5,6,7,8-tetrahydro-3H-pyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4-
one (15). Using the general procedure above from compound 8 (0.187
g, 0.46 mmol) the title compound 15 was obtained as a yellow gummy
solid (0.148 g, 71.5%) after FCC (EtOAc/MeOH, 9:1); Rf = 0.36 (EtOAc/
MeOH 9:1); IR: 3394, 2919, 1677 cmꢀ 1; 1H NMR (300 MHz, CDCl3): δ
7.43 (t, J = 8.1 Hz, 1H), 7.05 (ddd, J = 8.1, 2.1 and 0.8 Hz, 1H), 6.85
(ddd, J = 8.1, 2.1 and 0.8 Hz, 1H), 6.79 (t, J = 2.1 Hz, 1H), 3.83 (s, 3H),
3.73 (t, J = 6.3 Hz, 2H), 3.70 (m, 4H), 3.57 (m, 2H), 3.33 (t, J = 6.3 Hz,
4.1.2.2. Ethyl 2-(3-(3-methoxyphenyl)thioureido)-6-methyl-4,5,6,7-tetra-
hydrothieno [2,3-c]pyridine-3-carboxylate (8). Using the general pro-
cedure above, compound 3 (0.3 g, 1.25 mmol) and 3-methoxyphenyl
isothiocyanate (8) (0.22 mL, 1.56 mmol), the title compound was ob-
tained as a yellow solid (0.41 g, 80%) after FCC (EtOAc/MeOH, 97:3); Rf
= 0.42 (EtOAc/MeOH 9:1); 1H NMR (600 MHz, CDCl3): δ 12.19 (s, 1H),
7.94 (s, 1H), 7.34 (t, J = 8.4 Hz, 1H), 6.91–6.85 (m, 3H), 4.14 (q, J = 7.1
Hz, 2H), 3.81 (s, 3H), 3.51 (s, 2H), 2.86 (t, J = 5.7 Hz, 2H), 2.68 (t, J =
5.8 Hz, 2H), 2.47 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H); 13C NMR (150 MHz,
CDCl3): δ 175.9, 166.0, 160.7, 150.5, 136.7, 130.7, 128.9, 123.4 117.2,
113.9, 112.5, 110.5, 60.5, 55.4, 53.0, 52.3, 45.3, 26.6, 14.1.
2H), 3.09 (t, J = 6.0 Hz, 2H), 2.80 (t, J = 5.8 Hz, 2H), 2.53 (s, 3H); 13
C
NMR (75 MHz, CDCl3): δ 162.9, 160.7, 158.6, 157.6, 136.6, 130.6,
129.7, 128.2 121.2, 118.8, 116.1, 114.6, 72.2, 69.1, 61.8, 55.6, 53.6,
10

G.E. Magoulas et al.
Bioorganic Chemistry xxx (xxxx) xxx
52.0, 45.4, 32.3, 25.6; ESI-MS: m/z 448.1 [M+H]+; ESI-HRMS: m/z
[M+H]+ calcd for C21H26O4N3S2 448.1359, found 448.1348.
128.6, 118.1, 113.4, 52.5, 52.0, 43.0, 41.7, 40.2, 26.9, 25.5, 24.1; ESI-
MS: m/z 418.2 [M+H]+; ESI-HRMS: m/z [M+H]+ calcd for
C20H25ON5ClS [M+Н]+, 418.1463, found 418.1451.
4.1.4.3. 2-[2-(2-Hydroxyethoxy)ethylsulfanyl]-3-(4-methoxyphenyl)-7-
methyl-5,6,7,8-tetrahydro-3H-pyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4-
one (16). Using the general procedure above from compound 9 (0.187
g, 0.46 mmol) the title compound 16 was obtained as a light yellow solid
(0.124 g, 60%) after FCC (EtOAc/MeOH, 9:1); Rf = 0.46 (DCM/MeOH,
9:1); m.p: 124 – 126 ◦C; IR: 3517, 2918, 1676, 1502 cmꢀ 1; 1H NMR (600
MHz, CDCl3): δ 7.17 (d, J = 8.9 Hz, 2H), 7.01 (d, J = 8.9 Hz, 2H), 3.85 (s,
3H), 3.72 (t, J = 6.3 Hz, 2H), 3.71–3.69 (m, 2H), 3.63 (bs, 2H), 3.57 (t, J
= 5.1 Hz, 2H), 3.31 (t, J = 6.3 Hz, 2H), 3.06 (t, J = 5.7 Hz, 2H), 2.75 (t, J
= 5.7 Hz, 2H), 2.49 (s, 3H); 13C NMR (75 MHz, CDCl3): δ 163.3, 160.7,
159.2, 158.0, 130.2, 129.8, 128.6, 128.1, 118.6, 115.2, 72.1, 69.1, 61.9,
55.7, 53.8, 52.1, 45.6, 32.4, 25.8; ESI-MS: m/z 448.1 [M+H]+; ESI-
HRMS: m/z [M+H]+ calcd for C21H26O4N3S2 448.1359, found
448.1353.
4.1.7. 3-(4-chlorophenyl)-2-((3-hydroxypropyl)amino)-7-methyl-5,6,7,8-
tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (20)
To a rb-flask compound 17 (0.102 g, 0.27 mmol) and 3-aminopro-
pan-1-ol (1.0 mL, 13 mmol) were added and the reaction mixture was
stirred for 16 h at 150 ◦C. Then, the mixture was allowed to cool down to
rt and was partitioned between brine and DCM. The aqueous layer was
further extracted twice with DCM and the combined organic layers were
dried over Na2SO4 and evaporated to dryness. Compound 20 was ob-
tained after FCC (DCM/MeOH 95:5) as a light-yellow sticky solid (0.050
g, 46%). Rf = 0.35 (DCM/MeOH, 9:1); IR: 3384, 2939, 1674, 1544 cmꢀ 1
;
1H NMR (600 MHz, CDCl3): δ 7.53 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 8.0
Hz, 2H), 4.80 (t, J = 5.4 Hz, 1H), 3.62 (t, J = 5.3 Hz, 2H), 3.56 (bs, 2H),
3.54 – 3.51 (m, 2H), 3.45 (s, 1H), 3.00 (t, J = 5.7 Hz, 2H), 2.73 (t, J =
5.7 Hz, 2H), 2.48 (s, 3H), 1.69 (dt, J = 11.1 and 5.4 Hz, 2H); 13C NMR
(150 MHz, CDCl3): δ 165.6, 158.6, 151.2, 136.1, 133.0, 131.0, 130.6,
129.3, 124.1, 114.3, 60.3, 53.6, 52.1, 45.5, 39.9, 31.8, 25.7; ESI-MS: m/
4.1.5. 3-(4-chlorophenyl)-7-methyl-2-(methylthio)-5,6,7,8-
tetrahydropyrido [4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (17)
To a rb-flask containing compound 7 (0.16 g, 0.42 mmol), an etha-
nolic KOH solution (2 mL, 0.21 М) was added and the reaction mixture
was heated to reflux until completion of the reaction (monitored by
TLC). The reaction mixture was then filtered and a solution of methyl
iodide (0.026 mL, 0.42 mmol) in EtOH (1 mL) was added dropwise to the
filtrate. The solution was stirred for 90 min and then H2O (1 mL) was
added. The resulting orange precipitate was filtered, washed with H2O,
dried under high vacuum and used in the next step without further
purification (0.154 g, 97%). Rf = 0.69 (DCM/MeOH, 9:1); 1H NMR (300
MHz, CDCl3): δ 7.50 (d, J = 8.6 Hz, 2H), 7.23 (d, J = 8.6 Hz, 2H), 3.84
(bs, 2H), 3.06 (t, J = 6.0 Hz, 2H), 2.76 (t, J = 6.0 Hz, 2H), 2.50 (s, 3H),
2.49 (s, 3H).
z 405.2 [M+H]+; ESI-HRMS: m/z [M+H]+ calcd for C19
[M+Н]+, 405.1147, found 405.1130.
Н
22N4O2ClS
4.1.8. 3-(3-methoxyphenyl)-7-methyl-2-(methylthio)-5,6,7,8-
tetrahydropyrido [4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (21)
Compound 21 was synthesised according to the procedure described
above for the preparation of compound 17. Starting from 8 (0.2 g, 0.49
mmol) the title compound 21 was obtained as an orange solid (0.122
mg, 67%); Rf = 0.49 (DCM/MeOH, 9:1); ESI-MS: m/z 374.2 [M+H]+,
388.2 [M+Na]+; 1H NMR (300 MHz, CDCl3): δ 7.43 (t, J = 8.1 Hz, 1H),
7.05 (m, 1H), 6.88–6.85 (m, 1H), 6.82–6.80 (m, 1H), 3.84 (s, 3H), 3.65
(bs, 2H), 3.08 (t, J = 5.7 Hz, 2H), 2.76 (t, J = 5.7 Hz, 2H), 2.50 (s, 3H),
2.49 (s, 3H).
4.1.6. General procedure for the preparation of compounds 18 and 19
To a rb-flask, compound 17 (0.102 g, 0.27 mmol) and the appro-
priate diamine were added and the reaction mixture was stirred at
120 ◦C for 18 h. Then, the mixture was cooled to rt and passed through a
column of neutral aluminium oxide (DCM/MeOH, 8:2). A second puri-
fication by FCC on silica gel (DCM/MeOH/25% NH4OH 6:4:0.5) affor-
ded the desired compounds.
4.1.9. General procedure for the synthesis of compounds 22, 24 and 25
To a rb-flask compound 21 (0.102 g, 0.27 mmol) and the appropriate
amine were added and the reaction mixture was stirred for 72 h at the
indicated temperature. Then, the mixture was left to cool down to rt and
it was partitioned between brine and THF. The aqueous layer was
further extracted twice with THF. The combined organic layers were
dried over Na2SO4 and evaporated to dryness. The desired compounds
were obtained in pure form after FCC purification (DCM/MeOH 2:8 to
MeOH/25% NH4OH 10:1).
4.1.6.1. 2-((3-aminopropyl)amino)-3-(4-chlorophenyl)-7-methyl-5,6,7,8-
tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (18). Using
the general procedure above from compound 17 (0.102 g, 0.27 mmol)
and propane-1,3-diamine (0.67 mL, 8.1 mmol) the title compound was
obtained as a light-yellow sticky solid (0.054 g, 50%). Rf = 0.15 (DCM/
4.1.9.1. 2-((2-aminoethyl)amino)-3-(3-methoxyphenyl)-7-methyl-
5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one
(22). Using the general procedure above compound 21 (0.102 g, 0.27
mmol) and ethane-1,2-diamine (0.65 mL, 9.7 mmol) compound 22
along with by-product 23 were obtained.
MeOH, 8:2); IR: 3407, 2933, 1664, 1546 cmꢀ 1
;
1H NMR (600 MHz,
CD3OD): δ 7.64 (d, J = 8.6 Hz, 2H), 7.36 (d, J = 8.6 Hz, 2H), 4.43 (bs,
2H), 3.58 (bs, 2H), 3.47 (t, J = 6.6 Hz, 2H), 3.24 (t, J = 5.6 Hz, 2H), 3.05
(s, 3H), 2.97 (t, J = 6.2 Hz, 2H), 1.93 (m, 2H); 13C NMR (75 MHz,
CD3OD): δ 167.5, 159.0, 152.0, 135.6, 132.8, 130.62, 130.57, 127.6,
117.4, 112.7, 51.7, 51.1, 42.2, 38.1, 37.1, 27.1, 23.1; ESI-MS: m/z
Compound 22. yellow sticky solid (0.022 g, 20%), Rf = 0.56 (MeOH/
25% NH4OH 99:1); IR: 3344, 3033, 1656, 1539 cmꢀ 1
;
1H NMR (600
MHz, CD3OD): δ 7.53 (t, J = 8.1 Hz, 1H), 7.14 (dd, J = 8.3 and 1.9 Hz,
1H), 6.94 (s, 1H), 6.91 (d, J = 8.3 Hz, 1H), 3.85 (s, 3H), 3.64–3.59 (m,
4H), 3.08 (t, J = 5.8 Hz, 2H), 2.99 (t, J = 5.4 Hz, 2H), 2.80 (t, J = 5.8 Hz,
2H), 2.50 (s, 3H);13C NMR (75 MHz, CD3OD): δ 167.7, 162.8, 160.5,
153.2, 136.6, 132.3, 129.9, 124.8, 121.9, 116.9, 115.7, 115.2, 56.1,
54.1, 52.9, 45.4, 41.1, 41.1, 26.7; ESI-MS: m/z 386.2 [M+H]+; ESI-
HRMS: m/z [M+H]+ calcd for C19H24O2N5S [M+Н]+ 386.1645, found
386.1638.
404.07 [M+H]+; ESI-HRMS: m/z [M+H]+ calcd for C19
[M+Н]+ 404.1306, found 404.1294.
Н23N5OClS
4.1.6.2. 2-((4-aminobutyl)amino)-3-(4-chlorophenyl)-7-methyl-5,6,7,8-
tetrahydro pyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (19).
Using the general procedure above from compound 17 (0.102 g, 0.27
mmol) and butane-1,4-diamine (0.8 mL, 8.1 mmol) the title compound
was obtained as a light-yellow sticky solid (0.062 g, 55%). Rf = 0.20
4.1.9.2. 8-methyl-2,3,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[4′,3′:4,5]
thieno[2,3-d]pyrimidin-5(1H)-one (23). Isolated as light yellow sticky
solid (0.028 g, 40%), Rf = 0.31 (MeOH 100%); IR: 3147, 2927, 1664,
1610 cmꢀ 1; 1H NMR (600 MHz, CD3OD): δ 4.17 (t, J = 8.6 Hz, 2H), 3.77
(t, J = 8.6 Hz, 2H), 3.54 (s, 2H), 3.02 (t, J = 5.9 Hz, 2H), 2.77 (t, J = 5.9
(DCM/MeOH, 8:2); IR: 3348, 2939, 1672, 1542 cmꢀ 1
;
1HNMR (600
MHz, CD3OD): δ 7.63 (d, J = 8.7 Hz, 2H), 7.32 (d, J = 8.7 Hz, 2H), 4.39
(bs, 2H), 3.55 (t, J = 5.6 Hz, 2H), 3.39 (t, J = 6.1 Hz, 2H), 3.23 (t, J = 6.0
Hz, 2H), 3.03 (s, 3H), 2.94 (t, J = 7.1 Hz, 2H), 1.67 – 1.61 (m, 4H); 13
C
NMR (75 MHz, CD3OD): δ 169.1, 160.1, 153.0, 136.7, 134.0, 131.6,
11

G.E. Magoulas et al.
Bioorganic Chemistry xxx (xxxx) xxx
Hz, 2H), 2.45 (s, 3H); 13C NMR (75 MHz, CD3OD): δ 168.5, 159.5, 157.5,
129.5, 124.6, 54.1, 52.9, 45.4, 43.6, 41.6, 26.7; ESI-MS: m/z 263.1
[M+H]+; ESI-HRMS: m/z [M+H]+ calcd for C12H15ON4S [M+Н]+
263.0957, found 263.0957.
4.1.12. 3-(3-methoxyphenyl)-2-(methylthio)-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (30)
Compound 30 was synthesised according to the method described
above for the preparation of compound 17, using compound 28 (0.16 g,
0.42 mmol) and iodomethane (0.26 mL, 0.42 mmol), as a white solid
which was used to the next step without further purification. Rf = 0.26
(Petroleum ether/EtOAc, 9:1); ESI-MS: m/z 359.2[M+H]+.
4.1.9.3. 2-((3-aminopropyl)amino)-3-(3-methoxyphenyl)-7-methyl-
5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one
(24). Using the general procedure above compound 21 (0.102 g, 0.27
mmol) and propane-1,3-diamine (0.82 mL, 9.8 mmol) compound 24 was
obtained as a yellow sticky solid (0.011 g, 10%); Rf = 0.3 (MeOH/25%
4.1.13. 2-((2-aminoethyl)amino)-3-(3-methoxyphenyl)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (31)
1
NH4OH 95:5); IR: 3342, 2970, 1642, 1542 cmꢀ 1; H NMR (600 MHz,
Compound 31 was synthesized according to the method described
above for the synthesis of 22 from compound 30 (0.150 g, 0.42 mmol)
and 1,2-diamino ethane (2.0 mL, 29 mmol) after FCC purification
(EtOAc/MeOH 9:1), as a light-yellow oil (0.096 g, 62%); Rf = 0.12
(EtOAc/MeOH 9:1). Compound 31 was then dissolved in MeOH (0.5
mL), and an equimolar amount of oxalic acid was added and the cor-
responding salt was precipitated as a white solid by the addition of
CD3OD): δ 7.51 (t, J = 8.1 Hz, 1H), 7.12 (dd, J = 8.3, 1.9 Hz, 1H), 6.89 (t,
J = 2.1 Hz, 1H), 6.86 (dd, J = 8.3 and 1.9, 1H), 3.85 (s, 3H), 3.60 (bs,
2H), 3.45–3.37 (m, 2H), 2.99 (t, J = 5.3 Hz, 2H), 2.79 (t, J = 5.7 Hz, 2H),
2.67 (t, J = 6.8 Hz, 2H), 2.48 (s, 3H), 1.76–1.67 (m, 2H);13C NMR (75
MHz, CD3OD): δ 168.3, 162.9, 160.7, 153.0, 136.9, 132.3, 129.8, 124.3,
121.9, 116.9, 115.6, 114.5, 56.1, 54.1, 52.9, 45.4, 40.1, 39.5, 32.4, 26.8;
ESI-MS: m/z 400.2 [M+H]+; ESI-HRMS: m/z [M+H]+ calcd for
◦
diethyl ether, filtered and dried (0.107 g, 90%); m.p: 148–151 C; IR:
3352, 2937, 1737, 1654, 1542 cmꢀ 1; 1H NMR (600 MHz, CD3OD): δ 7.46
(t, J = 8.1 Hz, 1H), 7.03 (d, J = 8.1 Hz, 1H), 6.87 (d, J = 8.1 Hz, 1H),
6.81 (s, 1H), 4.76 (t, J = 5.1 Hz, 1H), 3.82 (s, 3H), 3.48–3.40 (m, 2H),
2.90–2.83 (m, 4H), 2.68 (t, J = 5.2 Hz, 2H), 1.88–1.75 (m, 4H); 13C NMR
(75 MHz, CD3OD): δ 165.2, 161.4, 158.9, 150.8, 135.9, 131.42, 131.38,
127.2, 120.9, 115.9, 115.0, 114.5, 55.7, 43.5, 40.7, 25.6, 25.1, 23.4,
22.6; ESI-MS: m/z 371.2 [M+H]+; ESI-HRMS: m/z [M+H]+ calcd for
C
20H26O2N5S [M+Н]+ 400.1802, found 400.1792.
4.1.9.4. 3-(3-methoxyphenyl)-7-methyl-2-((2-(piperazin-1-yl)ethyl)
amino)-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-
one (25). Using the general procedure above compound 21 (0.102 g,
0.27 mmol) and 3-(piperazin-1-yl)propan-1-amine (2.0 mL, 15.2 mmol)
compound 25 was obtained. as a yellow sticky solid (0.042 g, 34%); Rf
= 0.56 (MeOH/25% NH4OH 5:1.2); IR: 3326, 2941, 2833, 1672, 1544
cmꢀ 1; 1H NMR (600 MHz, CD3OD): δ 7.53 (t, J = 8.1 Hz, 1H), 7.14 (dd, J
= 8.4 and 2.3 Hz, 1H), 6.92 (t, J = 2.1 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H),
3.84 (s, 3H), 3.58 (s, 2H), 3.41 (t, J = 6.2 Hz, 2H), 2.97 (t, J = 5.7 Hz,
2H), 2.79–2.76 (m, 6H), 2.49 (t, J = 6.0 Hz, 2H), 2.48 (s, 3H), 2.43 (bs,
4H); 13C NMR (75 MHz, CD3OD):δ 168.1, 162.9, 160.5, 152.6, 137.0,
132.4, 129.9, 124.4, 122.0, 116.7, 115.8, 114.8, 57.0, 56.2, 54.1, 53.0,
52.9, 45.8, 45.4, 39.0, 26.8; ESI-MS: m/z 455.1 [M+H]+; ESI-HRMS: m/
z [M+H]+ calcd for C23H31O2N6S [M+Н]+ 455.2224, found 455.2211.
C
19H23O2N4S [M+Н]+ 371.1536, found 371.1528.
4.1.14. 2-((3-aminopropyl)amino)-3-(3-methoxyphenyl)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (32)
Compound 32 was synthesized according to the method described
above for the synthesis of 22, from compound 30 (0.184 g, 0.51 mmol)
and 1,3-diaminopropane (2 mL, 23.9 mmol) after FCC purification
(EtOAc/MeOH 9:1), as a light-yellow oil (0.147 g, 74%); Rf = 0.12
(DCM/MeOH 8:2). Compound 32 was then dissolved in MeOH (0.5 mL)
and an equimolar amount of oxalic acid was added and the corre-
sponding salt was precipitated as a white solid by the addition of diethyl
ether, filtered and dried (0.198 g, 92%); m.p: 79–82 ◦C; IR: 3359, 2929,
1740, 1654, 1542 cmꢀ 1; 1H NMR (600 MHz, CD3OD): δ 7.52 (t, J = 8.1
Hz, 1H), 7.13 (dd, J = 8.5 and 2.5 Hz, 1H), 6.91 (t, J = 2.4 Hz, 1H), 6.87
(dd, J = 8.5 and 2.5, 1H), 3.84 (s, 3H), 3.44 (dd, J = 14.5 and 6.6 Hz,
2H), 2.93 (t, J = 7.3 Hz, 2H), 2.81 (t, J = 6.0 Hz, 2H), 2.68 (t, J = 6.0 Hz,
2H), 1.91–1.85 (m, 4H), 1.81–1.78 (m, 2H);13C NMR (150 MHz,
CD3OD):δ 167.0, 162.8, 160.6, 152.7, 136.9, 132.4, 131.9, 128.3, 121.9,
116.8, 115.7, 115.5, 56.1, 39.3, 38.4, 29.0, 26.6, 25.7, 24.3, 23.5; ESI-
MS: m/z 385.1 [M+H]+; ESI-HRMS: m/z [M+H]+ calcd for
C20H25O2N4S [M+Н]+, 385.1693, found 385.1682.
4.1.10. Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate
(27)
Compound 27 was synthesised according to the method for the
synthesis of compound 3, using cyclohexanone 26 (2.59 g, 25.0 mmol),
sulfur (0.8 g, 25.0 mmol), ethyl cyanoacetate 2 (2.66 mL, 25.0 mmol).
The title compound 27 was obtained as a yellow solid (2.89 g, 52%)
following recrystallisation from methanol; Rf = 0.37 (petroleum ether/
1
◦
◦
EtOAc 9:1); m.p 102–106 C (lit.[36] – 107 C); H NMR (600 MHz,
CDCl3):δ 4.24 (q, J = 7.1 Hz, 2H), 2.68 (t, J = 5.3 Hz, 2H), 2.47 (t, J =
5.3 Hz, 2H), 1.79–1.69 (m, 4H), 1.32 (t, J = 7.1 Hz, 3H); 13C NMR (75
MHz, CDCl3):δ 166.1, 161.6, 132.5, 117.7, 105.8, 59.4, 26.9, 24.5, 23.2,
22.8, 14.5; ESI-MS: m/z 226.0 [M+Н]+.
4.1.15. 3-(3-methoxyphenyl)-2-((2-(piperazin-1-yl)ethyl)amino)-5,6,7,8
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (33)
4.1.11. Ethyl 2-(3-(3-methoxyphenyl)thioureido)-4,5,6,7-tetrahydrobenzo
[b]thiophene-3-carboxylate (28)
Compound 33 was synthesized according to the method described
above for the synthesis of 22 from compound 30 (0.106 g, 0.29 mmol)
and 1-(2-aminoethyl)piperazine (2 mL, 15.2 mmol) after FCC purifica-
tion (MeOH/25% NH4OH 9:1) as a light-yellow sticky solid (0.024 g,
To a solution of 27 (0.1 g, 0.44 mmol) in anhydrous pyridine (1 mL),
3-methoxyphenyl isothiocyanate (5) (0.08 mL, 0.58 mmol) was added
dropwise. The reaction mixture was stirred at 45 ◦C for 16 h. Pyridine
was evaporated, and the residue was subjected to FCC (Petroleum ether/
EtOAc 95:5 to 85:15) to give the title compound as a light-yellow solid
(0.165 g, 95%). Rf = 0.56 (Petroleum ether/EtOAc 8:2); 1HNMR (600
MHz, CDCl3): δ 12.21 (s, 1H), 8.13 (s, 1H), 7.32 (t, J = 8.3 Hz, 1H),
6.88–6.85 (m, 3H), 4.12 (q, J = 7.1 Hz, 2H), 3.80 (s, 3H), 2.70 (t, J = 5.8
Hz, 2H), 2.61 (t, J = 5.7 Hz, 2H), 1.77–1.72 (m, 4H), 1.23 (t, J = 7.1 Hz,
3H); 13CNMR (75 MHz, CDCl3): δ 175.8, 166.3, 160.7, 149.8, 136.9,
130.8, 130.6, 126.9, 117.2, 113.8, 113.1, 110.5, 60.4, 55.4, 26.3, 24.3,
23.0, 22.8, 14.2; ESI-MS: m/z 391.0 [M+Н]+.
20%); Rf = 0.37 (DCM/MeOH/8:2); IR: 3336, 2941, 1676, 1546 cmꢀ 1
;
1H NMR (600 MHz, CD3OD): δ 7.54 (t, J = 8.1 Hz, 1H), 7.15 (dd, J = 8.5
and 1.7 Hz, 1H), 6.92 (t, J = 1.8 Hz, 1H), 6.89 (dd, J = 8.5 and 1.7 Hz,
1H), 3.85 (s, 3H), 3.40 (t, J = 6.2 Hz, 2H), 2.82 (t, J = 6.0 Hz, 2H), 2.68
(bs, 6H), 2.46 (t, J = 6.2 Hz, 2H), 2.36 (bs, 4H), 1.90–1.85 (m, 2H),
1.81–1.77 (m, 2H); 13C NMR (75 MHz, CD3OD):δ 167.5, 162.9, 160.6,
152.3, 137.2, 132.4, 131.8, 128.1, 122.0, 116.6, 115.8, 115.3, 57.2,
56.2, 53.9, 46.1, 39.0, 26.7, 25.7, 24.3, 23.6; ESI-MS: m/z 440.2
[M+H]+; ESI-HRMS: m/z [M+H]+ calcd for C23H30O2N5S [M+Н]+,
440.2115, found 440.2104.
12

G.E. Magoulas et al.
Bioorganic Chemistry xxx (xxxx) xxx
4.2. In vitro evaluation of antimicrobial and antifungal activity
course of the experiment, provided by the CGC, which is funded by NIH
Office of Research Infrastructure Programs (P40 OD010440).
Microbial culture treated isolates: The antibacterial and antifungal
activity of all investigated samples was tested on strains obtained from
the Mycological laboratory, Department of Plant Physiology, Institute
4.4.2. Compound treatment
Nematodes were exposed to the following final designated concen-
trations per NGM plate: 0.027 and 0.058 mM compound 15 and 0.05
and 0.26 mM compound 22, corresponding to MIC/MFC (for compound
15) and MIC/MBC (for compound 22), respectively. Stock solutions of
the tested compounds were prepared after suspension in DMSO and
were kept at ꢀ 20 ◦C. The appropriate amount of the tested compounds
or DMSO (<1%) (control cultures) [41] were added on UV-irradiated
OP50 bacteria lawn. UV-killed bacteria were used to avoid the side ef-
fects of living bacteria metabolism.
ˇ
´
for Biological Research “Sinisa Stankovic” National Institute of Republic
of Serbia, Belgrade, Serbia. The following Gram (+) bacteria (Bacillus
cereus clinical isolate, Micrococcus flavus ATCC 10240, Staphylococcus
aureus ATCC 6538 and Listeria monocytogenes NCTC 7973), Gram (-)
bacteria (Escherichia coli ATCC 35210, Enterobacter cloacae human
isolate, Pseudomonas aeruginosa ATCC 27853 and Salmonella Typhimu-
rium ATCC 13311) and micromycetes Aspergillus fumigatus (ATCC 1022),
Aspergillus versicolor (ATCC 11730), Aspergillus ochraceus (ATCC 12066),
Aspergillus niger (ATCC 6275), Trichoderma viride (IAM 5061), Penicillium
funiculosum (ATCC 36839), Penicillium ochrochloron (ATCC 9112) and
P. verrucosum var. cyclopium (food isolates) were tested. Bacterial strains
4.4.3. Toxicity test in the nematode Caenorhabditis elegans though
phenotypic analysis
◦
were grown in Tryptic Soy Broth (TSB) at 37 C for 24 h. After har-
For all assays, N2 adult nematodes were set to lay eggs for 30 min on
NGM plates containing the tested compounds or the appropriate amount
of DMSO (control cultures). For the pharyngeal pumping and defecation
assays the nematodes were transferred to plates containing the tested
compounds or DMSO at the L4 stage. Standard procedures were fol-
lowed as described before [42]. More specifically:
vesting, microbial cells were suspended in sterile saline solution a con-
centration of approximately 1.0 × 106 CFU mlꢀ 1 and immediately used.
Fungi were maintained in solid Malt Agar (MA) medium at 120 h at
◦
28 C. The fungal spores were washed from the surface of agar plates
with sterile 0.85% saline containing 0.1% Tween 80 (v/v). The spore
suspension was adjusted with sterile saline to a concentration of
approximately 1.0 × 105 in a final volume of 100
μ
L per well. The cul-
4.4.3.1. Developmental timing. The progeny were frequently observed to
record the necessary time to reach the L4 larval stage from egg hatching.
The experiment was repeated five times.
◦
tures were subcultured once a month and stored at +4 C for further
usage [37].
Micro-well dilution assay: For the determination of the antimicrobial
activity of tested compounds, a modified microdilution method was
used [38–40]. The assay was performed by using sterile 96-well mi-
crotiter plates. Compounds were dissolved in 5% DMSO (1 mg/ml) and
the corresponding media – TSB and MB for bacteria and fungi, respec-
tively. The microplates were incubated for 24 h at 37 ◦C for bacterial and
for 72 h at 28 ◦C for fungal strains, respectively. The results were pre-
sented as minimum inhibitory concentrations (MIC), minimum bacte-
ricidal concentrations (MBC) and minimum fungicidal concentrations
(MFC). DMSO was used as a negative control, and commercial antibi-
otics streptomycin and ampicillin, and commercial fungicides, bifona-
zole and ketoconazole, were used as positive controls. All experiments
were performed in duplicate and repeated three times.
4.4.3.2. Pharyngeal pumping. At days 1 and 3 of adulthood, the
pharyngeal pumping rate was assessed for 15 s and expressed as number
of pumps/min. At least 30 animals per condition were evaluated.
4.4.3.3. Defecation assay. At days 1 and 3 of adulthood, the period in
seconds needed from defecation to defecation (defecation rate) was
recorded. At least 30 animals per condition were evaluated.
4.4.4. Statistical analysis
One-way ANOVA followed by Tukey’s and Dunnett’s tests were
applied to compare the means between the groups. The statistical sig-
nificance level was set at p < 0.05. The results are expressed as means ±
standard error of the means (SEM) of three independent experiments.
Asterisks denote p-values as follows: *p < 0.05, **p < 0.01, ***p <
0.001. Statistical analyses were performed using the SPSS software
(version 20.0; IBM Corp., Armonk, NY, USA).
4.3. Cell culture and cell viability assay
HFL-1 human embryonic primary fibroblasts were obtained from the
European Collection of Cell Cultures and were maintained in a humid-
ified incubator (37 ◦C and 5% CO2, 95% humidity) in Dulbecco’s
Modified Eagle’s Medium (DMEM; Invitrogen, Carlsbad, CA, USA)
supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA,
USA), 2 mM glutamine, 1% non-essential amino acids and 1% pen-
icillin–streptomycin (complete medium). Early passage cells (CPD < 37)
were maintained for 24 and 48 h in complete medium containing 0.027
and 0.058 mM compound 15 and 0.05 and 0.26 mM compound 22 or
dimethyl sulfoxide (DMSO; control). The percentage of cell survival was
determined through crystal violet staining. HFL-1 cells treated as
described above were fixed in 4% paraformaldehyde for 20 min at 37 ◦C
and then stained with 0.2% crystal violet in distilled water. The cells
were then washed with ddH2O and the dye was eluted with 30% acetic
acid. Viability was monitored via measurement of the dye absorbance at
595 nm using the Safire2 Multi-detection Microplate Reader (Tecan,
4.5. Similarity search and molecular docking studies
Structural similarity search was applied using the online platform
ChemMine Tools (http://chemmine.ucr.edu) [43]. Compounds 22 and
15, presenting the higher antibacterial and antifungal activity respec-
tively, were imported as the reference molecules for similarity search.
PubChem fingerprint algorithm was selected with a similarity cutoff
equal to 0.5.
S. aureus topoisomerase II DNA gyrase, co-crystallized with cipro-
floxacin and DNA (PDB:2XCT) was selected for molecular docking of
compounds 14 and 22 while a homology model of A. fumigatus CYP51A
was used for docking compounds 14 and 15.
¨
Molecular simulations were performed using Schrodinger Suite
¨
Grodig, Austria). Each sample was seeded in five replicates in 3 inde-
Release 2020–3. In an initial step, the examined targets and compounds
were prepared using the Protein Preparation Wizard [44] and LigPrep
[45], respectively. Molecular docking simulations were performed,
applying the Extra Precision (XP) mode and Standard Presision (SP)
mode of Glide and a grid box with dimensions 10 × 10 × 10 Å was
generated for both enzymes [46]. The number of poses were set equal to
10 and were visually inspected [47].
pendent experiments.
4.4. Evaluation of in vivo toxicity
4.4.1. Strains and nematode cultures
◦
We followed standard procedures for strain maintenance at 20 C.
C. elegans strain N2 (wild type Bristol isolate; wt) was used during the
For validation of docking parameters, the co-crystallized ligand
13

G.E. Magoulas et al.
Bioorganic Chemistry xxx (xxxx) xxx
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Declaration of Competing Interest
[13] A.A. Bekhit, A.M. Farghaly, R.M. Shafik, M.M.A. Elsemary, A.E.A. Bekhit, A.
A. Guemei, M.S. El-Shoukrofy, T.M. Ibrahim, Synthesis, biological evaluation and
molecular modeling of novel thienopyrimidinone and triazolothienopyrimidinone
derivatives as dual anti-inflammatory antimicrobial agents, Bioorg. Chem. 77
(2018) 38–46, https://doi.org/10.1016/j.bioorg.2017.12.028.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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Acknowledgments
We acknowledge support of this work by the project “OPENSCREEN-
GR: An Open-Access Research Infrastructure of Chemical Biology and
Target-Based Screening Technologies for Human and Animal Health
Agriculture and the Environment” “(2018–2020)” (MIS) 5002691 which
is implemented under the Action “Reinforcement of the Research and
Innovation Infrastructure”, funded by the Operational Programme
“Competitiveness, Entrepreneurship and Innovation” (NSRF 2014-
2020) and co-financed by Greece and the European Union (European
Regional Development Fund).
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