Biological evaluation of novel 1,4-dithiine derivatives as potential antimicrobial agents

Article abstract of DOI:10.1007/s00044-010-9513-0

The preparation of twelve aminoalkanol derivatives of 2,3-dihydro-5H-[1,4] dithiino[2,3-c]pyrrole- 5,7(6H)-dione was described. Newly obtained compounds, as well as their propyl and butyl analogues, were evaluated in vitro against selected viruses. Select

Full text of DOI:10.1007/s00044-010-9513-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:1411–1420
DOI 10.1007/s00044-010-9513-0
ORIGINAL RESEARCH
Biological evaluation of novel 1,4-dithiine derivatives as potential
antimicrobial agents
•
Anna Bielenica Jerzy Kossakowski Marta Struga
•
•
•
•
•
Izabela Dybała Paolo La Colla Elena Tamburini
Roberta Loddo
Received: 1 April 2010 / Accepted: 12 November 2010 / Published online: 28 November 2010
Ó Springer Science+Business Media, LLC 2010
Abstract The preparation of twelve aminoalkanol
derivatives of 2,3-dihydro-5H-[1,4]dithiino[2,3-c]pyrrole-
5,7(6H)-dione was described. Newly obtained compounds,
as well as their propyl and butyl analogues, were evaluated
in vitro against selected viruses. Selected derivatives were
tested for their antibacterial and antifungal activity. Com-
pounds 3h, 3j, 4b and 5a–d showed moderate to significant
protections against CVB-2, HSV-1 and YFV viruses. The
molecular structures of 4a, 5c and 5g were determined by
an X-ray analysis.
et al., 2009; Struga et al., 2009), antibacterial (Foroumadi
et al., 2005; Bozdag-Du¨ndar et al., 2007; Prasad and
´
Kishore, 2007; Stefanska et al., 2009; Tandon et al., 2009)
´
and antifungal (Stefanska et al., 2009) agents. Recently,
derivatives of other heterocycle, 1,4-dithiine, were found as
the new class of antimicrobial agents with activity against
broad spectrum of Gram-positive and Gram-negative
pathogens and fungal strains (Zentz et al., 2005). Sulphur
atom could be also incorporated in nonaromatic part of the
drug molecule, forming sulfanyl (Nawrozkij et al., 2008) or
thiol (Hadizadeh and Mehrparvar, 2004; Ahn et al., 2006)
groups. There is an increasing concern on thioureas as
some of them also have been reported as antiviral agents,
e.g. against HIV-1 (Venkatachalam et al., 2004; Ranise
et al., 2003; Ku¨c¸u¨kgu¨zel et al., 2008).
Keywords 1,4-Dithiine ꢀ X-ray analysis ꢀ
Antiviral activity ꢀ Conformations
Introduction
The anti-HIV-1 agent in a group of reverse transcriptase
inhibitors (RTIs), delavirdine, as well as protease inhibitor
nelfinavir, are sulphonoamido and phenylthio derivatives,
respectively. Their activity is also connected with the
presence of pyridinylpiperazine or 2-hydroxypropyl frag-
ments (Fig. 1). Literature survey revealed that modifica-
tions of their structures lead to new potential antiviral drugs
(Wang et al., 2006; Pinna et al., 2001; Di Santo et al.,
2002; Ragno et al., 2004).
Literature survey shows that sulphur play significant role in
enhancement of biological activity (Tandon et al., 2005,
2006). Sulphur-containing heterocyclic rings, such as thi-
azole, isothiazole, thiadiazole, thiazepine, tiophene, or
benzothiazole are nuclei of antiviral (Vicini et al., 2003;
Venkatachalam et al., 2004; Wang et al., 2006; Mercorelli
N-(4-Arylpiperazin-1-yl-alkyl) substituted derivatives of
cyclic imides are the largest group of ligands of seroto-
nergic 5-HT1A receptors. According to pharmacophore
model of 5-HT1A receptor proposed by Chilmonczyk et al.
(1997) and Bronowska’s group calculations (Bronowska
et al., 2006), a folded conformer of a ligand promotes high
affinity for the 5-HT1A receptor. In the solid state buspi-
rone, the free base as well as its hydrochloride, exist as
entended conformers (Chilmonczyk et al., 1995; Kozioł
et al., 2006). Theoretical calculations indicated that the
fully extended conformations are those of minimum energy
A. Bielenica (&) ꢀ J. Kossakowski ꢀ M. Struga
Department of Medical Chemistry, The Medical University
of Warsaw, 3 Oczki Street, 02-007 Warsaw, Poland
e-mail: anna.bielenica@wum.edu.pl
I. Dybała
Faculty of Chemistry, Maria Curie-Sklodowska University,
20-031 Lublin, Poland
P. La Colla ꢀ E. Tamburini ꢀ R. Loddo
Department of Biomedical Science and Technology,
University of Cagliari, Cittadella Universitaria,
09042 Monserrato, Cagliari, Italy
123

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Med Chem Res (2011) 20:1411–1420
Fig. 1 Chemical structures of
nelfinavir, delaviridine and
2,3-dihydro-5H-[1,4]dithiino
[2,3-c]pyrrole-5,7(6H)-dione
derivatives considered in this
study
OH
CH3
O
H3C
CH3
H3C
OH
S
O
NH
N
O
HN
N
HN
O
S
NH
N
H3C
CH3
N
N
H
CH3
O
DELAVIRDINE
NELFINAVIR
S
X = -CH2-
O
N
R
-CH2CH2-
-CH(OH)-
S
N
N
X
O
(Chilmonczyk et al., 1995). The conformationally flexible
n-butoxyl moiety of other neuroleptic aripiprazole adopts
either folded or extended molecular conformation. Both
structures are equally stable (Tessler and Goldberg, 2006).
second route of the synthesis was alkylation of imide 2
with 1,3-dibromopropane or 1,4-dibromobutane, in which
the respective bromoalkyl derivatives were obtained. Next,
they were condensed with appropriate arylpiperazines to
give derivatives 4a–4d and 5a–5g (Table 1). The structure
of all newly synthesized compounds have been established
´
´
In our previous studies (Stefanska et al., 2009; Stefanska
et al., 2010), we presented strongly hydrophobic aromatic
compounds with nitrogen-containing imide part that have
shown good antimicrobial activity. This study reports
biological activity of compounds purposely designed to
combine the heterocyclic sulphur containing ring of
1,4-dithiine with aminoalkanol or alkyl linker bearing
different substituents. Cytotoxicicty as well as activity of
selected compounds against virus, bacteria and fungi were
examined.
1
on the basis of elemental analysis, H-NMR and MS. For
biochemical studies free bases were converted into their
hydrochlorides.
Molecular structure
The crystal and molecular structure of 4a, 5c and 5g have
been determined (Fig. 3). The torsion angle values
describing the alkyl chain conformation indicate that in the
solid state the n-alkyl linker of 4a and 5g adopts the folded
conformation, whereas the compound 5c exists as an
extended conformer. Rigid fragments of the molecules, i.e.
the imide, piperazine, and aryl groups have varying relative
orientations due to linker flexibility (Fig. 4). In the crystal
structures of 4a, 5c and 5g different intermolecular bonding
motifs are observed depending on the number of proton
donor/acceptor groups. The strongest intermolecular
interactions are formed in the crystal 5g where the hydro-
xyl groups participate as donors in the O–HꢀꢀꢀNpiperazine
hydrogen bonds. In the crystal of 5c, the molecules form
layers via intermolecular C–HꢀꢀꢀX (X = O, N, S) weak
hydrogen bonds. Molecules of 4a are connected by C–Hꢀꢀꢀp
interactions between piperazine and pyrimidine fragments
to give centrosymmetric dimers.
Results and discussion
Chemistry
Series of aminoalkanol and alkyl derivatives of 1,4-dithiine
was designed and synthesized, in order to check the impact
of different substituents at N-4 on antimicrobial activity.
The starting material for the synthesis was 2,3-dihydro-
5H-[1,4]dithiino[2,3-c]pyrrole-5,7(6H)-dione (2), prepared
from its anhydride. It was subjected to the reaction with
2-(chloromethyl)oxirane in anhydrous medium to give
oxirane 3. Next the semiproduct was reacted with short-
chain or cyclic amines, as well as with piperazine deriva-
tives giving amino alcohol derivatives 3a–3k (Fig. 2). The
123

Med Chem Res (2011) 20:1411–1420
1413
Fig. 2 Method of preparation
of compounds 1, 2, 3, 3a–3k
O
O
O
Cl
S
S
S
S
S
S
NH3 aq
O
NH
O
N
O
O
O
1
2
3
O
CH3OH
RH
O
S
R
N
HO
S
O
3a - 3k
CH
3
CH
CH
3
CH2
CH2
CH3
CH3
CH
3
N
N
N
NH
CH
N
CH
R:
3
NH
3
CH
3
CH
3
3
3a
3b
3c
3d
3e
3f
N
N
N
F
N
N
N
N
3g
3h
3i
F
H3CO
N
N
N
N
3j
3k
Antimicrobial activity
Gram-positive and Gram-negative bacteria (Staphylococ-
cus aureus, Pseudomonas aeruginosa), yeasts and moulds
(Candida albicans and Aspergillus niger).
AZT (30-azido-thymidine), NM 108 (20-ß-methyl-gua-
nosine), NM 176 (20-ethynyl-D-citidine), M 5255 (myco-
phenolic acid) and ACG (acycloGuanosine) were used as
reference inhibitors of ssRNA?, ssRNA- and DNA
viruses, respectively.
Twenty two compounds, derivatives of 2,3-dihydro-5H-
[1,4]dithiino[2,3-c]pyrrole-5,7(6H)-dione, were evaluated
in vitro against viruses, and 17 of them were tested against
bacteria and fungi.
Title compounds were evaluated for antiviral activity
against viruses representative of two of the three genera of
Flaviviridae family, that is, Flaviviruses (Yellow Fever
Virus, YFV) and Pestiviruses (Bovine Viral Diarrhoea
Virus, BVDV), as Hepaciviruses can hardly be used in
routine cell-based assays. Compounds were also tested
against representatives of other virus families. Among
ssRNA? were a retrovirus (human immunodeficiency virus
type 1, HIV-1) and two Picornaviruses (Coxsackie Virus
type B2, CVB-2 and Poliovirus type-1, Sabin strain, Sb-1);
among ssRNA- were a Rhabdoviridae (Vesicular Stoma-
titis Virus, VSV) representative. Among double-stranded
RNA (dsRNA) viruses was a Reoviridae representative
(Reo-1). Two representatives of DNA virus families were
also included: Herpes Simplex type-1, HSV-1 (Herpesvir-
idae), and Vaccinia Virus, VV (Poxviridae).
Seven of tested derivatives presented antiviral activity
(Tables 2, 3), which varied from moderate (compounds 3h,
3j, 5c, 5d) to significant (4b, 5a, 5b). The data may serve
as a good subject for discussion of the influence of
molecular structure of a compound on its biological
activity. It seems that antiviral activity depends on the type
of the substituent of the hydrocarbon branch and the linker
itself. The representative viruses tested were susceptible to
unsubstituted or para-substituted long-chain arylpiperazine
derivatives. The most active compounds were alkyl
derivatives of 1-(4-fluorophenyl)-piperazine (4b, 5b) and
1-phenylpiperazine (5a). Introduction of any substituent in
ortho-position as well as inserting of short-amine or cyclic
moiety to the molecule dramatically decreases antimicro-
bial activity of the compound. Derivatives 4b and 5b were
most potent against YFV, at a concentration that was at
In addition to the antiviral activity, compounds were
evaluated in vitro against representative strains of
123

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Med Chem Res (2011) 20:1411–1420
derivatives of 1,4-dithiine, only 3h and 3j revealed moderate
activity against YFV. The activity of presented compounds
decreased when three-unit propyl or 2-hydroxypropyl
branch is inserted, instead of the butyl one.
Table 1 Structures of (4-aryl-piperazin-1-ylalkyl) derivatives of 2,3-
dihydro-5H-[1,4]dithiino[2,3-c]pyrrole-5,7(6H)-dione
O
Gram-negative rods as well as gram-positive strains
and fungal organisms were resistant to all tested agents.
Their minimal inhibitory concentration (MIC) values for
all compounds were above 100 lM (Table 4). Moreover,
none of title compounds turned out to be active against
HIV-1, BVDV or representatives of ssRNA- and dsRNA
viruses.
S
N
(CH2)n
N
R
N
S
O
Compound
n
R
N
4a
3
3
3
3
4
4
4
4
4
4
4
F
4b
4c
4d
5a
5b
5c
5d
5e
5f
Experimental
H3CO
Chemistry
All chemicals and solvents were purchased from Aldrich
(Vienna, Austria). Melting points were determined on
Electrothermal Digital Melting Point Apparatus (Essex,
UK) and are uncorrected. The 1H-NMR spectra were
recorded on a Bruker (Rheinstetten, Germany) spectrom-
eter, operating at 400 or 300 MHz. The chemical shift
values are expressed in ppm relative to TMS as an internal
standard. Elemental analyses were recorded on a CHN
model 2400 Perkin-Elmer (Hitachi, Tokyo, Japan). Mass
spectra were performed on MARINER PE Biosystems
instrument (Foster City, USA) with TOF detector. Metha-
nol was used as a solvent. The spectra were performed in
the positive ion mode with a declustering potential
140–300 V. TLC was carried out using silica gel 60 F254,
layer thickness 0.25 mm (E. Merck, Darmstadt, Germany)
and the results were visualized using UV lamp at 254 nm.
Column chromatography was carried out using silica gel 60
(200–400 mesh, Merck).
HO
F
N
H3CO
N
N
HO
X-ray crystallography
5g
Diffraction data for 4a, 5g and 5c were measured at 293 K
on a KM4 diffractometer using graphite monochromated
˚
CuKa radiation (k = 1.54178 A) and variable scan speed
least 4.8-fold lower than the cytotoxic concentration. The
50% cytotoxic concentration (CC50) of 5a and 5d in Vero-
76 cells was higher than 100 lM, resulting in a selectivity
index (SI = CC50/EC50) of above 5 and 3.5, respectively.
The compound 5b was found as the most selective and
potent anti-CVB-2 agent. Compounds 4b and 5a expressed
the broadest antiviral activity, viz against Flaviviruses,
DNA and ssRNA viruses. For 1-(4-fluorophenyl)-pipera-
zine derivatives 4b and 5b the observed EC50 value in
anti-CVB tests was lower than for the standard 20-ethenyl-
D-cytidine (NM 176). In a group of aminoalkanol
in the x - 2h scan mode. The structures were solved by
direct methods using the SHELXS-97 (Sheldrick, 1997)
and refinement was performed by the full-matrix least-
squares method on F2 using the SHELXL-97 (Sheldrick,
1997) program. The non-hydrogen atoms were refined with
anisotropic displacement parameters. H-atom positions
were calculated from the geometry and were given iso-
tropic factors of 1.2 Ueq of the bonded non-H atoms.
Compound 4a crystallizes as monoclinic, in the space
˚
group P21/n, a = 12.512(3) A, b = 6.181(1) A, c =
˚
3
˚
˚
24.210(5) A, b = 94.25(3)°, V = 1867.2(7) A , Z = 4,
123

Med Chem Res (2011) 20:1411–1420
1415
Fig. 3 Perspective view of
molecules 4a, 5g and 5c with
atom numbering scheme and
values of torsion angles [°]
within the N-alkyl linker
4a
N4-C1-C2-C3 -167.5(6)
C1-C2-C3-N1 56.3(5)
5g
N4-C1-C2-C3 158.7(5)
C1-C2-C3-C4 58.9(7)
C2-C3-C4-N1 -163.9(4)
5c
N4-C1-C2-C3 -175.7(4)
C1-C2-C3-C4 178.8(4)
C2-C3-C4-N1 -177.4(4)
253 parameters refined the final discrepancy factors are:
R1 = 0.0389, wR2 = 0.0973 for reflections with I [ 2r(I)
and R1 = 0.1968, wR2 = 0.1373 for all data.
Compound 5c crystallizes in the monoclinic system,
˚
space group P21/n, a = 14.295(3) A, b = 9.013(2) A,
˚
3
˚
˚
c = 16.325(3) A, b = 107.81(3), V = 2002.5(7) A , Z = 4,
dcalc = 1.342, F(000) = 856, l(CuKa) = 2.589 cm-1. In the
h range 4.93–72.14°, 3,248 reflections were collected and
3,051 unique reflections were used in the refinement. For
245 parameters refined the final discrepancy factors are:
R1 = 0.0455, wR2 = 0.124 for reflections with I [ 2r(I) and
R1 = 0.1485, wR2 = 0.1604 for all data. Extinction coeffi-
cient equals 0.0014(3).
Fig. 4 Fit of molecules 4a, 5c and 5g through the atoms of imide ring
Crystallographic data haves been deposited with the
Cambridge Crystallographic Data Center as CCDC Nos
758466, 758465 and 758464 for compounds 4a, 5g, and 5c,
respectively. Copies of the data can be obtained on appli-
cation to the Director, CCDC Union Road, Cambridge CB2
1EZ U.K. (Fax: ?44-1223-336-033; e-mail: deposite@
ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk).
dcalc = 1.393 g cm-3, F(000) = 824, l(CuKa) = 2.775
cm-1. In the h range 6.76–72.11°, 3,575 reflections were
collected and 1,883 unique reflections were used in the
refinement. For 231 parameters refined the final discrep-
ancy factors are: R1 = 0.032, wR2 = 0.043 for reflections
with I [ 2r(I) and R1 = 0.1932, wR2 = 0.0602 for all
data. Extinction coefficient equals 0.0043(1).
2,3-Dihydro-5H-[1,4]dithiino[2,3-c]pyrrole-5,7(6H)-
dione (2) was obtained according to the method descri-
bed previously (Schweizer, 1969) from commercially
available anhydride 1.
Compound 5g crystallizes in the orthorhombic system,
˚
˚
space group P 212121, a = 7.440(1) A, b = 9.429(2) A,
3
˚
˚
c = 28.552(6) A, V = 2003.0(7) A , Z = 4, dcalc
=
1.391 g cm-3, F(000) = 888, l(CuKa) = 2.633 cm-1. In
the (range 4.94–72.22°, 4,163 reflections were collected and
3,876 unique reflections were used in the refinement. For
6-(Oxiran-2-ylmethyl)-2,3-dihydro-5H-[1,4]dithiino[2,
3-c]pyrrole-5,7(6H)-dione (3) was prepared by dissolving
of 2 g (0.011 mol) of the starting imide 2 in 29 cm3 of
123

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Med Chem Res (2011) 20:1411–1420
Table 2 Cytotoxicity and antiviral activity of compounds 3h, 3j, 4b and 5a–5d
Compds. MT-4a HIV- MDBKc BVDVd BHK- YFVf SI1
Reo-1f Vero- HSV- SI2
76g 1h
EC50 CC50 EC50
VVh VSV CVB- SI3
Sb-1h
1b
21e
2h
EC50 (lM) EC50
(lM)
CC50 EC50 CC50
(lM) (lM) (lM)
EC50
(lM)
CC50 EC50
(lM) (lM)
EC50
(lM)
(lM) (lM) (lM)
3h
3j
[100 [100 [100
[100 [100 [100
[100 [100 [100
[100 [100 [100
[100 [100 [100
[100 [100 [100
[100 [100 [100
[100
[100
[100
[100
[100
[100
[100
[100 80
[1.25 [100 [100 [100
[1.33 [100 [100 [100
–
–
–
[100 [100 [100
[100 [100 [100
[96 [96 20
–
–
[100
[100
[100 75
4b
5a
5b
5c
5d
[100 52
[1.92 [100 96
[96
[4.8 [96
[1.51 [100
[4.76 [100
[3.12 [100
[100 88
[1.14 [100 [100 19
[5.26 [100 [100 66
[100 [100
[100 [100
[100 [100
–
–
–
[100 [100 [100
[100 [100 [100
[100 [100 29
–
–
[100 [100 21
[100 [100 32
[3.45 [100 [100 [100
–
[100
Ref. compds.
AZTi 50
0.01
NM
108j
1.8
2.5
NM
176k
23
18
M
5255l
1.8
ACGm
3
Data represent mean values for three independent determinations. Variation among duplicate samples was less than 15%
Antiviral activity is given as EC50 (median effective concentration—the concentration of a drug (lM) required to induce a 50% effect), and
cytotoxicity is given as CC50 (cytotoxic concentration—the amount of a drug (lM) at which 50% of cells become dead)
SI (selectivity index) was determined as the ratio between CC50 and EC50 for YFV (SI1), HSV-1 (SI2) and CVB-2 (SI3)
a
Compd. concn. (lL) required to reduce the viability of mock-infected MT-4 (CD4? Human T-cells containing an integrated HTLV-1 genome)
cells by 50%, as determined by the colorimetric MTT method
b
Compd. concentration (lL) required to achieve 50% protection of MT-4 cells from the HIV-1-induced cytopathogenicity, as determined by
the MTT method
c
Compd. concn. (lL) required to reduce the viability of mock-infected MDBK (Bovine normal kidney) cells by 50%, as determined by the
MTT method
d
Compd. concn. (lL) required to achieve 50% protection of MDBK cells from the BVDV (Bovine Viral Diarrhoea Virus)-induced cyto-
pathogenicity, as determined by the MTT method
e
Compd. concn. (lL) required to reduce the viability of mock-infected BHK (Hamster normal kidney fibroblast) monolayers by 50%, as
determined by the MTT method
f
Compd. concentration (lL) required to achieve 50% protection of BHK (Kidney fibroblast) cells from the YFV (Yellow Fever Virus) and Reo
(Reovirus 1)-induced cytopathogenicity, as determined by the MTT method
g
Compd. concn. (lM) required to reduce the viability of mock-infected VERO-76 (Monkey normal kidney) monolayers by 50%, as determined
by the MTT method
h
Compd. concn. (lM) required to reduce the plaque number of HSV-1 (Herpesvirus 1), VV (Vaccinia virus), VSV (Vesicular Stomatitis virus),
CVB-2 (Coxsackievirus B2), Sb-1 (Poliovirus 1) and RSV (Respiratory Syncytial Virus) by 50% in VERO-76 monolayers
i
Azido-thymidine
20-b-methyl-guanosine
20-Ethynyl-D-citidine
j
k
l
Mycophenolic acid
m
AcycloGuanosine
1
2-(chloromethyl)oxirane, to which 2 g (0.014 mol) of
anhydrous potassium carbonate was added. The mixture
was stirred under reflux condenser at 40°C for 24 h. The
crude product was filtered off, the solvent was evaporated
and the oily residue was purified by column chromatog-
raphy using chloroform as eluent.
Yield 87%, oil, H-NMR (400 MHz, CDCl3) d (ppm):
4.08 (2H, m, N–CH2), 3.33 (4H, s, (CH2)2–S), 3.15 (1H, m,
OCH), 2.79 (1H, dd, J1 = 4.4 Hz, J2 = 8.4 Hz, CH2–O),
2.63 (1H, m, CH2–O); ESI MS for C9H9NO3S2: m/z
[%]: 265.9 [M ? Na]? 100, 243.9 [M ? H]? 39, 509.0
[2 M ? Na]? 23.
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Med Chem Res (2011) 20:1411–1420
1417
Table 3 Cytotoxicity and antiviral activity of 2,3-dihydro-5H-[1,4]dithiino[2,3-c]pyrrole-5,7(6H)-dione derivatives
Compds.
MT-4a
CC50
(lM)
HIV-1b
EC50
(lM)
MDBKc
CC50
(lM)
BVDVd
EC50
(lM)
BHK-21e YFVf Reo-1f Vero-76g HSV-1h VVh VSVh CVB-2h hSb-1
CC50
(lM)
EC50 (lM)
CC50
(lM)
EC50 (lM)
3a, 3b, 3d, [100
3e, 3f, 3g,
3i, 4a, 5
2, 4c, 4d, 5e, [100
5f, 5g
ND
ND not determined
Table 4 Antibacterial and antifungal activities of 2,3-dihydro-5H-[1,4]dithiino[2,3-c]pyrrole-5,7(6H)-dione derivatives
MICa (lM)
Compdsb
S.aureus
DSM 2569
P. aeruginosa
DSM 1117
C. albicans
DSM 1386
A. niger
DSM 1988
1, 2, 3a, 3b, 3d, 3e, 3f, 3g, 3h, 3i, 3j, 4a, 4b, 5a, 5b, 5c, 5d
[100
[100
[100
[100
Ref. compds.
Ciprofloxacin
Miconazole
4
–
0.8
–
–
–
0.8
20
Miconazole was solubilized in DMSO (0.1 M solution) and stored at 4° C overnight
Reference Compounds were diluted from 100 to 0.0013 lM
a
The antimicrobial activity is given as minimum inhibitory concentration (MIC) corresponding to the lowest concentration of an antimicrobial
compound that showed complete growth inhibition
b
Ciprofloxacin was solubilized in water (0.1 M solution) according to the British Society for Antimicrobial Chemotherapy (BSAC) protocol and
stored at 4°C overnight
General procedure for preparation of 6-(3-amino-2-
hydroxypropyl)-2,3-dihydro-5H-[1,4]dithiino
[2,3-c]pyrrole-5,7(6H)-diones (3a–3k)
J2 = 14.4 Hz, N-CH2), 3.33 (4H, s, (CH2)2–S), 2.88
(6H, m, CH2–N–(CH2)2), 1.29 (6H, m, N(CH3)2). C13H20
N2O3S2: Calcd. C 49.34, H 6.37, N 8.85. Found C 49.29, H
6.29, N 8.79.
3c. Yield 25%, oil, 1H-NMR (300 MHz, CDCl3) d
(ppm): 4.33 (1H, m, CH–OH), 3.70 (2H, m, NCH2), 3.32
(4H, s, (CH2)2S), 3.15 (1H, m, CH(CH3)2), 3.07 (1H, dd,
J1 = 2.4 Hz, J2 = 12.3 Hz, CH2N), 2.87 (1H, dd, J1 =
9.9 Hz, J2 = 12.0 Hz, CH2N), 1.40 (6H, dd, J1 = 3.6 Hz,
J2 = 6.6 Hz, (CH3)2); ESI MS for C12H18N2O3S2: m/z
[%]: 303,1 [M ? H]? 100.
To 0.3 g (0.0012 mol) of 6-(oxiran-2-ylmethyl)-2,3-dihy-
dro-5H-[1,4]dithiino[2,3-c]pyrrole-5,7(6H)-dione (3) dis-
solved in 35 cm3 of methanol:water mixture (33.9:1.1 vol),
appropriate chain or cyclic amine (0.0024 mol) was added.
The mixture was stirred at 60°C under reflux condenser.
The reaction was monitored by TLC (silica gel, developing
system: chloroform:methanol, 4.5:0.5). The liquid was
distilled off and the oily residue was purified by column
chromatography (chloroform:methanol, 9:1).
1
3d. Yield 50%, m.p. 156–158°C, H-NMR (400 MHz,
CDCl3) d (ppm): 4.00 (1H, m, CH–OH), 3.63 (2H, m, N–
CH2), 3.32 (4H, s, (CH2)2–S), 2.86 (1H, dd, J1 = 2.4 Hz,
J2 = 12.4 Hz, CH2–N), 2.60 (1H, dd, J1 = 8.0 Hz,
J2 = 12.0 Hz, CH2–N), 1.23 (9H, s, (CH3)3).
C13H20N2O3S2ꢀH2O: Calcd. C 46.68, H 6.63, N 8.38.
Found C 47.07, H 6.35, N 8.18.
1
3a. Yield 65%, m.p. 240–242°C, H-NMR (400 MHz,
CDCl3) d (ppm): 4.25 (1H, m, CH–OH), 3.69 (1H, dd,
J1 = 6.4 Hz, J2 = 14.4 Hz, N–CH2), 3.44 (1H, dd,
J1 = 4.8 Hz, J2 = 14.0, N–CH2), 3.33 (4H, s, (CH2)2–S),
2.90 (2H, m, CH2–N), 2.77 (6H, s, N(CH3)2).
C11H17ClN2O3S2: Calcd. C 40.67, H 5.27, N, 8.63; Found
C 40.86, H 5.43, N, 8.63.
3b. Yield 60%, m.p. 91–93°C, 1H-NMR (400 MHz,
CDCl3) d (ppm): 4.30 (1H, m, CH–OH), 3.69 (1H, dd, J1 =
6.4 Hz, J2 = 14.0 Hz, N–CH2), 3.57 (1H, dd, J1 = 5.2 Hz,
1
3e. Yield 60%, m.p. 112–113°C, H-NMR (400 MHz,
CDCl3) d (ppm): 4.19 (1H, m, CH–OH), 3.66 (1H, dd,
J1 = 6.4 Hz, J2 = 14.0 Hz, N-CH2), 3.55 (1H, dd,
J1 = 5.2 Hz, J2 = 14.4 Hz, N–CH2), 3.32 (4H, s, (CH2)2–
S), 2.64 (6H, m, CH2–N, Hapiper), 1.79 (4H, m, Hbpiper),
123

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Med Chem Res (2011) 20:1411–1420
1.41 (2H, m, Hcpiper). C14H20N2O3S2: Calcd. C 51.19, H
6.14, N 8.53. Found C 50.68, H 6.18, N 8.05.
Microbiological assays
1
3f. Yield 65%, m.p. 173–175°C, H-NMR (400 MHz,
Compounds
CDCl3) d (ppm): 4.40 (1H, m, CH–OH), 3.69 (1H, dd,
J1 = 6.0 Hz, J2 = 14.0 Hz, N–CH2), 3.58 (1H, dd,
J1 = 5.6 Hz, J2 = 14.4 Hz, N–CH2), 3.33 (4H, s, (CH2)2–
S), 2.87 (2H, m, CH2–N), 2.59 (2H, m, Hapiper), 1.81 (4H,
br. s, Hbpiper), 1.60 (2H, br. s, Hbpiper), 1.02 (3H, d,
J = 6.2 Hz, CH3), 0.94 (1H, m, Hcpiper). C15H23ClN2O3S2:
Calcd. C 47.54, H 6.12, N 7.39. Found C 47.56, H 6.10, N
7.29.
Compounds were dissolved in DMSO at 100 mM and then
diluted in culture medium.
Cells and viruses
Cell lines were purchased from American Type Culture
Collection (ATCC). The absence of mycoplasma contam-
ination was checked periodically by the Hoechst staining
method. Cell lines supporting the multiplication of RNA
viruses were the following: CD4? human T-cells contain-
ing an integrated HTLV-1 genome (MT-4); Madin Darby
Bovine Kidney (MDBK); Baby Hamster Kidney (BHK-21)
and Monkey kidney (Vero 76) cells.
1
3g. Yield 62%, m.p. 168–170°C, H-NMR (400 MHz,
CDCl3) d (ppm): 6.88 (4H, m, Harom), 4.15 (1H, m, CH–
OH), 3.62 (2H, m, N–CH2), 3.31 (4H, s, (CH2)2–S), 3.18
(4H, m, N(CH2)2piperazine–Ph), 2.98 (2H, m, CH2–
N(CH2)piperazine), 2.59 (2H, m, CH2-N(CH2) piperazine), 2.44
(2H, m, CH2–N). C19H24ClN3O3S2ꢀ2H2O: Calcd. C 47.74,
H 5.90, N 8.79. Found C 47.74, H 5.54, N 8.69.
1
3h. Yield 68%, m.p. 225–227°C, H-NMR (400 MHz,
CDCl3) d (ppm): 6.91 (4H, m, Harom), 3.98 (1H, m, CH–
OH), 3.63 (2H, m, N–CH2), 3.32 (4H, s, (CH2)2–S), 3.11
(4H, m, N(CH2)2piperazine–Ph), 2.79 (2H, m, CH2–
N(CH2)piperazine), 2.59 (2H, m, CH2–N(CH2)piperazine), 2.47
(2H, m, CH2–N). C19H24Cl2FN3O3S2: Calcd. C 45.97, H
4.87, N 8.47. Found C 45.59, H 4.99, N 8.25.
Bacterial strains
The antibacterial activity of compounds was tested against
collection strains representative of Gram-positive bacteria
(S. aureus DSM 2569) and Gram-negative bacteria (P.
aeruginosa DSM 1117). Antifungal activity was tested
against collection strains representative of yeasts (C. albi-
cans DSM 1386) and moulds (A. niger DSM 1988).
1
3i. Yield 65%, m.p. 178–180°C, H-NMR (400 MHz,
CDCl3) d (ppm): 8.20 (1H, m, Haarom), 7.53 (m, 1H,
Hcarom), 6.69 (2H, m, Hbarom), 4.32 (1H, m, CH–OH), 3.82
(4H, m, N(CH2)2piperazine–Ph), 3.65 (2H, m, N–CH2), 3.32
(4H, s, (CH2)2–S), 3.00 (4H, m, CH2–N(CH2)2piperazine),
2.79 (2H, m, CH2–N). C18H24Cl2N4O3S2ꢀ2H2O: Calcd.
C 41.94, H 5.48, N, 10.87. Found C 42.06, H 5.58,
N 10.54.
Cytotoxicity assays
For cytotoxicity tests, run in parallel with antiviral assays,
MDBK, BHK and Vero 76 cells were resuspended in
96-multiwell plates at an initial density of 6 9 105,
1 9 106 and 5 9 105 cells/ml, respectively, in mainte-
nance medium, without or with serial dilutions of tested
compounds. Cell viability was determined after 48–120 h
at 37°C in a humidified CO2 (5%) atmosphere by the MTT
method. The cell number of Vero 76 monolayers was
determined by staining with the crystal violet dye.
1
3j. Yield 60%, m.p. 185–187°C, H-NMR (400 MHz,
CDCl3) d (ppm): 6.93 (4H, m, Harom), 3.97 (1H, m, CH–OH),
3.86 (3H, s, OCH3), 3.64 (2H, m, N–CH2), 3.32 (4H, s,
(CH2)2–S), 3.07 (4H, m, N(CH2)2piperazine–Ph), 2.82 (2H, m,
CH2–N(CH2)piperazine), 2.63 (2H, m, CH2–N(CH2)piperazine),
2.49 (1H, m, CH2–N), 2.41 (1H, m, CH2–N).
C20H27Cl2N3O4S2ꢀ1‘H2O: Calcd. C 44.86, H 5.65, N 7.85.
Found C 45.33, H 5.80, N 7.32.
For cytotoxicity evaluations, exponentially growing
cells derived from human haematological tumours [CD4?
human T-cells containing an integrated HTLV-1 genome
(MT-4)] were seeded at an initial density of 1 9 105 cells/
ml in 96 well plates in RPMI-1640 medium, supplemented
with 10% foetal calf serum (FCS), 100 units/ml penicillin
G and 100 lg/ml streptomycin. Cell cultures were then
incubated at 37°C in a humidified, 5% CO2 atmosphere in
the absence or presence of serial dilutions of test com-
pounds. Cell viability was determined after 96 h at 37°C by
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) method (Pauwels et al., 1988).
1
3k. Yield 65%, m.p. 142–144°C, H-NMR (300 MHz,
CDCl3) d (ppm): 6,98 (4H, m, Harom), 4,01 (1H, m, CH–OH),
3,62 (2H, m, NCH2), 3,32 (4H, s, (CH2)2S), 3,11 (4H, m,
N(CH2)2piperazine-Ph), 2,84 (2H, m, CH2–N(CH2)2piperazine),
2,66 (2H, m, CH2–N(CH2)2piperazine), 2,49 (2H, m, CH2N);
ESI MS for C19H22FN3O3S2: m/z [%]: 425,11 [M ? 2H]?
81,72.
(4-aryl/heteroaryl)piperazin-1-yl)alkyl]-2,3-dihydro-5H-
[1,4]dithiino[2,3-c]-pyrrole-5,7- (6H)-diones (4a–4d, 5a–
5g) were obtained according to the method published
previously (Kossakowski et al., 2007).
123

Med Chem Res (2011) 20:1411–1420
Antiviral assay
1419
Antibacterial and antifungal assays
Activity of compounds against Human Immunodeficiency
virus type-1 (HIV-1) was based on inhibition of virus-
induced cytopathogenicity in MT-4 cells acutely infected
with a multiplicity of infection (m.o.i.) of 0.01. Briefly,
50 ll of RPMI containing 1 3 104 MT-4 were added to
each well of flat-bottom microtitre trays containing 50 ll
of RPMI, without or with serial dilutions of test com-
pounds. Then, 20 ll of an HIV-1 suspension containing
100 CCID50 were added. After a 4-day incubation, cell
viability was determined by the MTT method.
The antibacterial and antifungal activities were evaluated
by determining the Minimum inhibitory concentration
(MIC) by the broth microdilution procedure.
Bacterial strains were grown on Tryptic soy agar at 37°C
for 1 day. Cell suspensions of these recent cultures were
prepared in sterile 0.85% saline solution by 4–5 colonies.
The turbidity of the suspensions was adjusted to the
McFarland 0.5 standard. Suspensions were diluted in cat-
ion-supplemented Mueller–Hinton broth. For each micro-
organism, 100 ll of the fivefold serial dilutions of the
compounds in cation-supplemented Mueller–Hinton broth
and 100 ll of inoculum were added to each well of a
microdilution plate (final titre 5 9 105 CFU/ml). The
inoculated plates were incubated at 37°C in non-CO2
incubator and humid atmosphere. The MICs were deter-
mined after 16–20 h (EUCAST, 2003).
Activity of compounds against Yellow Fever Virus
(YFV) and Reo virus type-1 (Reo-1) was based on inhi-
bition of virus-induced cytopathogenicity in acutely
infected BHK-21 cells. Activities against Bovine Viral
Diarrhoea Virus (BVDV), in infected MDBK cells, were
also based on inhibition of virus-induced cytopath-
ogenicity.
Fungal strains were grown on Sabouraud’s dextrose agar
at 35°C for 1–5 days. Suspensions of these recent cultures
were prepared in sterile saline solution (NaCl 0.85%).
Suspensions were then diluted in Sabouraud’s dextrose
broth. 100 ll of the fivefold serial dilutions of the com-
pounds in Sabouraud’s dextrose broth and 100 ll of inoc-
ulum were added to each well of a microdilution plate (C.
albicans 1 9 104 cell/ml; A. niger OD600 0.05). The
inoculated plates were incubated at 35°C in non-CO2
incubator and humid atmosphere. The MICs were deter-
mined after 24 and 48 h.
BHK and MDBK cells were seeded in 96-well plates at
a density of 5 9 104 and 3 9 104 cells/well, respectively,
and were allowed to form confluent monolayers by incu-
bating overnight in growth medium at 37°C in a humidified
CO2 (5%) atmosphere. Cell monolayers were then infected
with 50 ll of a proper virus dilution (in serum-free med-
ium) to give an m.o.i = 0.01. 1 h later, 50 ll of MEM
Earle’s medium, supplemented with inactivated foetal calf
serum (FCS), 1% final concentration, without or with serial
dilutions of test compounds, were added. After 3–4 days of
incubation at 37°C, cell viability was determined by the
MTT method.
The concentration of each inoculum was confirmed by
viable counts on agar plates by plating the appropriate
dilution of the growth control well, immediately after
inoculation, and incubating until visible growth. MIC
corresponded to the lowest concentration of an antimicro-
bial compound that showed complete growth inhibition.
Activity of compounds against Coxsackie virus, B-2
strain (CVB-2), Polio virus type-1 (Polio-1), Sabin strain,
Vesicular Stomatitis Virus (VSV), Vaccinia Virus (VV) and
Herpes Simplex Virus type-1 (HSV-1), in infected Vero 76
cells, was determined by plaque reduction assays in Vero
76 cell monolayers. To this end, Vero 76 cells were seeded
in 24-well plates at a density of 2 9 105 cells/well and
were allowed to form confluent monolayers by incubating
overnight in growth medium at 37°C in a humidified CO2
(5%) atmosphere. Then, monolayers were infected with
250 ll of proper virus dilutions to give 50–100 PFU/well.
Following removal of unadsorbed virus, 500 ll of Dul-
becco’s modified Eagle’s medium, supplemented with 1%
inactivated FCS and 0.75% methyl cellulose, without or
with serial dilutions of test compounds, were added. Cul-
tures were incubated at 37°C for 2 (Sb-1 and VSV) or 3
(CVB-2, VV and HSV-1) and then fixed with PBS con-
taining 50% ethanol and 0.8% crystal violet, washed and
air-dried. Plaques were then counted. 50% effective con-
centrations (EC50) were calculated by linear regression
technique.
Linear regression analysis
Viral and cell growth at each drug concentration was
expressed as percentage of untreated controls and the
concentrations resulting in 50% (EC50, CC50) growth
inhibition were determined by linear regression analysis.
References
Ahn S-H, Jeon H, Choi J-H, Baek D, Seo SH, Cho J-H, Oh Ch-H
(2006) Synthesis and antibacterial activity of 1b-methyl-2-[5-
(1,2-disubstitutedethyl)pyrrolidin-3-ylthio]-carbapenem deriva-
tives. Arch Pharm Life Sci 339:67–73
¨
¨
Bozdag-Du¨ndar O, Ozgen O, Mentes¸e A, Altanlar N, Ath O, Kendi E,
Ertan R (2007) Synthesis and antimicrobial activity of some new
123

1420
Med Chem Res (2011) 20:1411–1420
thiazolyl thiazolidine-2,4-dione derivatives. Bioorg Med Chem
15:6012–6017
2,3,4-trihydropyrimido[1,2-c]9,10,11,12-tetrahydrobenzo[b]thie-
no[3, 2-e]pyrimidines. Chem Pharm Bull 55:776–779
´
Bronowska A, Les A, Chilmonczyk Z, Filipek S, Edvardsen Ø,
Østensen R (2006) Molecular dynamics of buspirone analogues
interacting with the 5-HT1A and 5-HT2A serotonin receptors.
Bioorg Med Chem 9:881–895
Ragno R, Mai A, Sbardella G, Artico M, Massa S, Musiu Ch, Mura
M, Marturana F, Cadeddu A, La Colla P (2004) Computer-
aided design, synthesis, and anti-HIV-1 activity in vitro of 2-
alkylamino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkyl-
pyrimidin-4(3H)-ones as novel potent non-nucleoside reverse
transcriptase inhibitors, also active against the Y181C variant.
J Med Chem 47:928–934
Ranise A, Spallarossa A, Schenone S, Bruno O, Bondavalli F, Vargiu
L, Marceddu T, Mura M, La Colla P, Pani A (2003) Design,
synthesis, SAR, and molecular modeling studies of acylthiocar-
bamates: a novel series of potent non-nucleoside HIV-1 reverse
transcriptase inhibitors structurally related to phenethylthiazo-
lylthiourea derivatives. J Med Chem 46:768–781
´
Chilmonczyk Z, Les A, Wozniakowska A, Cybulski J, Kozioł AE,
Gdaniec M (1995) Buspirone analogues as ligands of the 5-HT1A
receptor. 1. The molecular structure of buspirone and its two
analogues. J Med Chem 38:1701–1710
´
Chilmonczyk Z, Szelejewska-Wozniakowska A, Cybulski J, Cybulski
M, Kozioł AE, Gdaniec M (1997) Conformational flexibility of
serotonin1A receptor ligands from crystallographic data. Updated
model of the receptor pharmacophore. Arch Pharm (Weinheim)
330:146–160
Di Santo R, Costi R, Artico M, Massa S, Ragno R, Marshall GR, La
Colla P (2002) Design, synthesis and QSAR studies on N-aryl
heteroarylisopropanolamines, a new class of non-peptidic HIV-1
protease inhibitors. Bioorg Med Chem 10:2511–2526
European Committee on Antimicrobial Susceptibility Testing (2003)
Determination of minimum inhibitory concentrations (MICs) of
antibacterial agents by broth microdilution. EUCAST Discussion
Document E Def 51. Clin Microbiol Infect 9:1–10
Schweizer HR (1969) Derivate des 5,6-dihydro-p-dithiin-2,3-dicar-
¨
bonsaure-anhydrids, I: Imide. Helv Chim Acta 52:2221–2235
Sheldrick GM (1997) SHELXS-97 and SHELXL-97. University of
¨
Gottingen, Germany
´
Stefanska J, Bielenica A, Struga M, Tyski S, Kossakowski J, Loddo
R, Ibba C, Collu D, Marongiu E, La Colla P (2009) Biological
evaluation of 10-(diphenylmethylene)-4-azatricyclo[5.2.1.02,
6]dec-8-ene-3,5-dione derivatives. Cent Eur J Biol 4:362–368
´
Foroumadi A, Emami S, Hassanzadeh A, Rajaee M, Sokhanvar K,
Moshafi MH, Shafiee A (2005) Synthesis and antibacterial
activity of N-(5-benzylthio-1,3,4-thiadiazol-2-yl) and N-(5-ben-
zylsulfonyl-1,3,4-thiadiazol-2-yl)piperazinyl quinolone deriva-
tives. Bioorg Med Chem Lett 15:4488–4492
Hadizadeh F, Mehrparvar A (2004) Synthesis of some new 1-[2-
(alkylthio-1-benzyl-5-imidazolyl)carbonyl]-4-[3-(isopropyl amino)-
2-pydridyl] piperazines as anti-HIV. J Sci Isl Rep Iran 15:
131–134
Stefanska J, Bielenica A, Struga M, Tyski S, Kossakowski J, La Colla
P, Tamburini E, Loddo R (2010) Antimicrobial activity of
10-(diphenylmethylene)-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-
dione derivatives. Ann Microbiol 60:151–155
Struga M, Kossakowski J, Kozioł AE, Ke˛dzierska E, Fidecka S, La
Colla P, Ibba C, Collu G, Sanna G, Secci B, Loddo R (2009)
Synthesis, pharmacological and antiviral activity of 1,3-thiaze-
pine derivatives. Eur J Med Chem 44:4960–4969
Tandon VK, Yadav DB, Singh RV, Vaish M, Chaturvedi AK, Shukla
PK (2005) Synthesis and biological evaluation of novel 1,4-
naphthoquinone derivatives as antibacterial and antiviral agents.
Bioorg Med Chem Lett 15:3463–3466
Kossakowski J, Wojciechowska A, Dybała I (2007) Derivatives of 2,3-
dihydro-5H-[1,4]dithiino[2,3-c]pyrrole-5,7(6H)-dione as poten-
tial anxiolytics. Ann Pol Chem Soc 1:249–252
Kozioł AE, Chilmonczyk Z, Cybulski J (2006) Buspiron free base.
Acta Crystallogr E62:o5616–o5618
Tandon VK, Maurya HK, Yadav DB, Tripathi A, Kumar M, Shukla
PK (2006) Naphtho[2,3-b][1,4]-thiazine-5,10-diones and 3-
substituted-1,4-dioxo-1,4-dihydronaphthalen-2-yl-thioalkanoate
derivatives: synthesis and biological evaluation as potential
antibacterial and antifungal agents. Bioorg Med Chem Lett
16:5883–5887
Tandon VK, Maurya HK, Mishra NN, Shukla PK (2009) Design,
synthesis and biological evaluation of novel nitrogen and sulphur
containing hetero-1,4-naphtoquinones as potent antifungal and
antibacterial agents. Eur J Med Chem 44:3130–3137
Tessler L, Goldberg I (2006) Crystal structure of arypiprazole, a new
anti-psychotic drug, and of its inclusion compounds with
methanol, ethanol and water. J Incl Phenom Macro Chem 55:
255–261
Venkatachalam TK, Mao C, Uckun FM (2004) Effect of stereochem-
istry on the anti-HIV activity of chiral thiourea compounds.
Bioorg Med Chem 12:4275–4284
Vicini P, Geronikaki A, Incerti M, Busonera B, Poni G, Cabras CA,
La Colla P (2003) Synthesis and biological evaluation of
benzo[d]isothiazole, benzothiazole and thiazole Schiff base.
Bioorg Med Chem 11:4785–4789
Wang Z, Wu B, Kuhen KL, Bursulaya B, Nguyen TN, Nguyen DG,
He Y (2006) Synthesis and biological evaluations of sulfanyl-
triazoles as novel HIV-1 non-nucleoside reverse transcriptase
inhibitors. Bioorg Med Chem Lett 16:4174–4177
Ku¨c¸u¨kgu¨zel I, Tatar E, Ku¨c¸u¨kgu¨zel SG, Rollas S, De Clercq E (2008)
Synthesis of some novel tiourea derivatives obtained from 5-[(4-
aminophenoxy)methyl]-4-alkyl/aryl-2,4-dihydro-3H–1,2,4-tria-
zole-3-thiones and evaluation as antiviral/anti-HIV and
anti-tuberculosis agents. Eur J Med Chem 43:381–392
Mercorelli B, Muratore G, Sinigalia E, Tabarrini O, Biasolo MA,
Cecchetti V, Palu G, Loregian A (2009) A 6-aminoquinolone
compound, WC5, with potent and selective anti-human cyto-
megalovirus activity. Antimicrob Agents Chemother 53:312–315
Nawrozkij MB, Rotili D, Tarantino D, Botta G, Eremiychuk AS,
Musmuca I, Ragno R, Samuele A, Zanoli S, Armand-Ugon M,
Clotet-Codina I, Novakov IA, Orlinson BS, Maga G, Este JA,
Artico M, Mai A (2008) 5-Alkyl-6-benzyl-2-(2-oxo-2-pheny-
lethylsulfanyl)-pyrimidyn-4(3H)-ones, a series of anti-HIV-1
agents of the dihydro-alkoxy-benzyl-oxopyrimidine family with
peculiar structure-acivity relationship profile. J Med Chem
51:4641–4652
Pauwels R, Balzarini J, Baba M, Snoeck R, Schols D, Hardewijn P,
Desmyster J, De Clercq
E (1988) Rapid and automated
tetrazolium-based colorimetric assay for the detection of anti-
HIV compounds. J Virol Methods 20:309–321
Pinna GA, Loriga G, Murineddu G, Grella G, Mura M, Vargiu L,
Murgioni Ch, La Colla P (2001) Synthesis and anti-HIV-1
activity of new delavirdine analogues carrying arylpyrrole
moieties. Chem Pharm Bull 49:1406–1411
Prasad MR, Kishore DP (2007) Multistep, microwave assisted,
solvent free synthesis and antibacterial activity of 6-substituted-
Zentz F, Labia R, Sirot D, Faure O, Grillot R, Valla A (2005)
Syntheses, in vitro antibacterial and antifungal activities of a
series of N-alkyl,1,4-dithiines. Farmaco 60:944–947
123