Synthesis, pharmacological and antiviral activity of 1,3-thiazepine derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.08.013

The preparation of new fourteen thiourea and fourteen product of their condensation with 1,4-dibromobutane, viz. 1,3-thiazepine derivatives, of 10-isopropyl-8-methyl-4-aza-tricyclo[5.2.2.0^2,6]undec-8-ene-3,5-dione and 1-isopropyl-7-methyl-4-aza

Full text of DOI:10.1016/j.ejmech.2009.08.013

European Journal of Medicinal Chemistry 44 (2009) 4960–4969
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, pharmacological and antiviral activity of 1,3-thiazepine derivatives
,
a
b
c
c
Marta Struga a , Jerzy Kossakowski , Anna E. Koziol , Ewa Kedzierska , Sylwia Fidecka ,
**
*
Paolo La Colla d , Cristina Ibba , Gabriella Collu , Giuseppina Sanna , Barbara Secci , Roberta Loddo
,
d
d
d
d
d
a Department of Medical Chemistry, Medical University, 3 Oczki Str., 02-007 Warszawa, Poland
b Faculty of Chemistry, Maria Curie-Sklodowska University, 20-031 Lublin, Poland
c Department of Pharmacology and Pharmacodynamics, Medical University, 4 Staszica Str., 20-081 Lublin, Poland
d Department of Biomedical Science and Technology, University of Cagliari, 09042 Monserrato (CA), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The preparation of new fourteen thiourea and fourteen product of their condensation with 1,4-dibro-
mobutane, viz. 1,3-thiazepine derivatives, of 10-isopropyl-8-methyl-4-aza-tricyclo[5.2.2.02,6]undec-
8-ene-3,5-dione and 1-isopropyl-7-methyl-4-aza-tricyclo[5.2.2.02,6]undec-8-ene-3,5-dione is described.
Elemental analysis, MS and 1H NMR spectra confirmed the identity of the products. The molecular
structure of linear disubstituted thiourea derivative and its cyclization product was determined by an
X-ray crystal structure analysis. Two of new obtained compounds (6b0 and 7a0) were tested for their
pharmacological activity on animal central nervous system (CNS) in behavioral animal tests. With
relatively low acute toxicity (LD50 lower than 2000 mg kgꢀ1 i.p.) they exhibited significant influence on
spontaneous locomotor activity and body temperature. Additionally, compounds reduced number of the
‘‘head twitch’’ episodes after 5-hydroksytryptophan (5-HTP) administration.
Received 21 April 2009
Received in revised form
20 August 2009
Accepted 26 August 2009
Available online 6 September 2009
Keywords:
CNS activity
5-HT system connection
Cytotoxicity, antiviral activity Thiourea and
1,3-thiazepine derivatives of tricyclic imide
X-ray crystal structure analysis
New compounds were evaluated in vitro against representatives of different virus classes, such as
a HIV-1 (Retrovirus), a HBV (Hepadnavirus) and the single-stranded RNAþ viruses Yellow fever virus
(YFV) and Bovine viral diarrhea virus (BVDV), both belonging to Flaviridae. Three of new obtained
compounds showed a modest activity against HIV-1 wtIIIB, BVDV and YFV.
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
conformation [31]. This conformation is a part of structure of
NNRTIs (nucleoside reverse transcriptase inhibitors); anti-HIV
Thiourea and urea derivatives show a broad spectrum of
biological activities such as antibacterial, antiviral, anticancer,
anticonvulsion, analgesic and HDL-elevating properties [1–12].
Numerous compounds containing thiourea group are selective
ligands for 5-HT family receptors, including 5-HT2A, 5-HT2B and
5-HT2C [13–17].
The drug-elicited ‘‘head twitch’’ response (HTR) [18,19] is
a selective behavioral model for 5-HT2 agonist activity in the
rodent, and several previous studies have established that direct
and indirect 5-HT agonists induce this effect [20–27]. Additionally,
5-HT2 receptor antagonists selectively block HTR [27–29], and their
potency is highly correlated with the antagonist’s affinity for 5-HT2
receptors [20,30].
agents (Scheme 1) [32]. The main target in contemporary drug
discovery efforts against HIV-1 is RT (reverse transcriptase), a vital
enzyme that is responsible for the reverse transcription of retro-
viral RNA to proviral DNA [33]. Non-nucleoside reverse transcrip-
tase inhibitors (NNRTI’s) inhibit HIV RT by altering either the
conformation or mobility of RT by binding to a specific allosteric
site near the polymerase site, thereby resulting in non-competitive
inhibition of the enzyme [34,35].
Derivatives of 1,3-thiazepine (seven-membered cyclic thiourea
derivatives) are important compounds, because of their biological
activity as nitric oxide synthase inhibitors [36–39]. This is a rare
system in heterocyclic chemistry.
1,3-Thiazepine ring is present in Omipatrilat which is currently
in the phase IV of clinical trials. By inhibiting the activity of the
angiotensin converting enzyme (ACE), which causes blood vessels
to constrict, Omapatrilat lowers blood pressure. Another advantage
of this drug is inhibition of enzyme known as neutral endopepti-
dase (NEP), which causes blood vessels to relax [40,41].
Here we report the synthesis of compounds which are
composed of thiourea system and the products of their condensa-
tion, viz. 1,3-thiazepine system attached to polycyclic imide.
Furthermore, structural studies of active thiourea derivatives
have shown that these compounds contain a central hydrophilic
part and two hydrophobic moieties forming
a butterfly-like
* Corresponding author. Tel./fax: þ48 0226280679.
** Corresponding author. Tel.: þ39 070 6754147; fax: þ39 070 6754210.
E-mail addresses: marta.struga@wum.edu.pl (M. Struga), placolla@unica.it
(C. La Colla).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.08.013

M. Struga et al. / European Journal of Medicinal Chemistry 44 (2009) 4960–4969
4961
S
2.2. Pharmacology
HN
Cl
Two of the above compounds, 6b0 and 7a0, were tested for their
pharmacological activity.
N
CH3
They exhibited weak acute toxicity lower than 2000 mg kgꢀ1 i.p.
The progressive doses, calculated as a part of LD50 for compounds
tested, were used in behavioral experiments.
N
The significant depressive action on spontaneous locomotor
activity in mice was observed after administration of both inves-
tigated compounds at a dose of 0.1 LD50 (p < 0.05), but not at 0.05
LD50 (Fig. 3). Amphetamine hyperactivity was not changed by
either of the compounds (Fig. 4).
These compounds did not produce any protection in clonic
seizures and tonic convulsions evoked by pentetrazole and in
abdominal constriction induced by i.p. administration of the acetic
acid solution (Fig. 5).
TIBO
S
N
S
N
N
H
N
NH
Br
N
NH
H
S
The investigated substances did not affect motor coordination
(rota rod and chimney tests) (Figs. 6 and 7).
Phenethylthiazolylthiourea (PETT)
Trovirdine
Body temperature of normothermic mice was decreased
significantly (within 30–90 min, 7a0; 60–120 min, 6b0) by both
compounds (Fig. 8). 5-HT has been reported to play an important
role in central regulation of body temperature [48]. The MAO
(monoamine oxidase) type A inhibitors appear to be crucially
involved in hypothermia [49]. As a result of MAO-inhibition, 5-HT
levels in the body are increased and may precipitate a serotonin
syndrome. Hypothermia in rodents has been reported for MAO type
A enzyme inhibitors (antidepressant drugs), such as clorgyline [50]
Scheme 1. Structures representative of NNRTIs.
Method of synthesis of 1,3-thiazepine ring connected to Diels–
Alder product had been reported for authors [42].
In a previous study [43,44], we synthesized a series of urea and
thiourea derivatives of tricyclic imide. Reported urea and thiourea
derivatives exhibited activity on animal central nervous system
(CNS) and characterized by low toxicity. This study is a continuation
of our research.
and harman (1-methyl-b-carboline) [51].
The ‘‘head twitch’’ responses after 5-hydroxtryptophan (5-HTP)
administration were significantly decreased by 6b0 and markedly,
but not significantly, by compound 7a0 (Fig. 9). The result seems to
point out some connection with the 5-HT system. Since it appears
that headshakes induced by 5-HTP are mediated by 5-HT2 recep-
tors, these data suggest that 6b0 and 7a0 may interact with 5-HT2
receptors in the brain.
2. Results and discussion
2.1. Chemistry
Based on our previous research on central nervous system (CNS)
for urea [43] and thiourea [44] derivatives of tricyclic imide and
described in this paper for cyclic derivatives of thiourea (1,3-thia-
zepine system), we could conclude that this group of compounds
possesses significant influence on CNS of laboratory animals.
Comparison of urea and thiourea derivatives of tricyclic imide
shows that urea derivatives have influence on ‘‘head twitch
response’’ only. However thiourea and 1,3-thiazepine derivatives
were active in ‘‘head twitch’’ test similarly to urea derivatives and
have influence on spontaneous motor activity and decrease body
temperature of normothermic mice.
The preparation of new fourteen thiourea and fourteen product
of their condensation with 1,4-dibromobutane, viz. 1,3-thiazepine
derivatives, of 10-isopropyl-8-methyl-4-aza-tricyclo[5.2.2.02,6
]
undec-8-ene-3,5-dione and 1-isopropyl-7-methyl-4-aza-tricy-
clo[5.2.2.02,6]undec-8-ene-3,5-dione (Scheme 2) is described.
Anhydrides obtained in Diels–Alder reaction were used as
starting materials. Anhydride (a) was obtained in the reaction of
enantiomeric (R)-(ꢀ)-
a
-phellandrene with furan-2,5-dione [45].
The reaction of
a-terpinene with furan-2,5-dione gave compound
b [46].
Obtained tricyclic anhydrides were subjected to the reaction of
hydrazine (80% aqueous solution), described previously [43,47].
In order to obtain corresponding thiourea derivatives of above
compounds they were subjected to the reaction with appropriate
isothiocyanates. Products were transformed into 1,3-thiazepine
derivatives by condensation with 1,4-dibromobutane. The general
synthesis pathway is given in Scheme 2.
Based on performed research we could conclude that urea
derivatives of polycyclic imides were less active than thiourea and
cyclic thiourea (1,3-thiazepine system). Comparison of the activity
on CNS of thiourea and 1,3-thiazepine derivatives could lead to the
conclusion that there are no differences between free thiourea
system and the system inbuilt in 1,3-thiazepine ring.
Obtained compounds were purified by flash chromatography.
Elemental analysis, MS, 1H NMR and 13C NMR spectra confirmed the
identity of the products. The molecular structure of linear disub-
stituted thiourea derivative 7a and its cyclization product 7a0 was
determined by an X-ray crystal structure analysis (Figs. 1 and 2).
The crystal structure of 7a is formed by of two crystallographi-
cally independent molecules, A and B, adopting different confor-
mations of the thiourea fragment; the orientation of the N–H bonds
is trans and cis in the molecules A and B, respectively (Fig. 1). These
conformers show substantially different bond distances (Table 1).
The disubstituted 1,3-thiazepine ring in 7a0 adopts slightly
distorted chair conformation (Fig. 2).
2.3. Cytotoxicity and antiviral activity
Title compounds were evaluated in cell-based assays for cyto-
toxicity and antiviral activity against viruses representative of two
of the three genera of the Flaviviridae family, i.e. Flaviviruses
(Yellow Fever Virus, YFV) and Pestiviruses (Bovine Viral Diarrhoea
Virus, BVDV), as Hepaciviruses can hardly be used in routine cell-
based assays. Title compounds were also tested against represen-
tatives of other virus families. Among ssRNAþ were a retrovirus
(Human Immunodeficiency Virus Type 1, HIV-1) and two Picorna-
viruses (Coxsackie Virus Type B2, CVB-2 and Poliovirus Type-1,
Sabin strain, Sb-1); among ssRNAꢀ were
a Paramixoviridae

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M. Struga et al. / European Journal of Medicinal Chemistry 44 (2009) 4960–4969
S
H
N
R2
R1
N
H
R1 NH2
R2NCS
Br
+
1a - 7a, 1b - 7b
S
H
N
N
+
R1
S
R2
O
R1
N
H
Br
N
R2
1a' - 7a', 1b' - 7b'
O
N
N
R1 =
O
O
a
b
R2 = allyl (1a,b; 1a',b'), benzyl (2a,b; 2a',b'), 4-fluoroPhe ( 3a,b; 3a',3b'),
4-chloroPhe ( 4a,b; 4a',b' ), 4-iodoPhe (5a,b; 5a',b'), 3,4-dichloroPhe ( 6a,b; 6a',b'),
4-methylPhe (7a,b; 7a',b')
Scheme 2. Synthesis of studied compounds.
(Respiratory Syncytial Virus, RSV) and a Rhabdoviridae (Vesicular
Stomatitis 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
Symplex Type-1, HSV-1 (Herpesviridae) and Vaccinia Virus, VV
(Poxviridae).
Unfortunately, none of the compounds showed any activity
against any viruses tested; with the exception of compounds 2a, 1b
and 2b (Tables 2–4), that showed a modest activity against HIV-1
wtIIIB, BVDV and YFV (EC50 ¼ 30, 52 and 41
mM, respectively).
3. Experimental protocol
AZT (30-azido-thymidine), NM 108 (20-
b-methyl-guanosine),
NM 176 (20-ethynyl-D-citidine), NM 299 (6-azauridine), M 5255
(Mycophenolic Acid) and ACG (acycloGuanosine) were used as
reference inhibitors of ssRNAþ, ssRNAꢀ and DNA viruses,
respectively.
3.1. Chemistry
Melting points were determined in a Kofler’s apparatus and are
uncorrected. The NMR spectra were recorded on a Bruker AVANCE
DMX400 spectrometer, operating at 300 MHz (1H NMR) and
75 MHz (13C NMR). The chemical shift values are expressed in ppm
relative to TMS as an internal standard. Elemental analyses were
recorded with a CHN model 2400 Perkin–Elmer. Mass spectral ESI
Fig. 2. Perspective view of molecule 7a0. Bond distances of 1,3-thiazepine ring are: S1–
C15 1.768(4), S1–C16 1.828(7), C16–C17 1.503(9), C17–C18 1.528(9), C18–C19 1.509(9),
C19–N2 1.457(6), N2–C15 1.388(6) Å.
Fig. 1. Perspective view of two symmetry independent molecules 7a.

M. Struga et al. / European Journal of Medicinal Chemistry 44 (2009) 4960–4969
4963
Table 1
Bond lengths (Å) within the thiourea fragment of two conformers of 7a.
S]C15
C15–N3
C15–N2
N2–N1
N3–C1 p
mol. A – trans
mol. B – cis
1.689(5)
1.631(6)
1.321(7)
1.362(7)
1.346(7)
1.373(6)
1.395(6)
1.364(6)
1.423(7)
1.422(7)
measurements were carried out on Waters ZQ Micro-mass instru-
ments with quadrupol mass analyzer. The spectra were performed
in the positive ion mode at a declustering potential of 40–60 V. The
sample was previously separated on a UPLC column (C18) using
UPLC ACQUITYÔ system by Waters connected with DPA detector.
Flash chromatography was performed on Merck silica gel 60
(200–400 mesh) using chloroform/methanol (19:1 vol) mixture as
eluent. Analytical TLC was carried out on silica gel F254 (Merck)
plates (0.25 mm thickness).
C]O); 4.1–4.16 (m, 1H, CH2); 4.26–4.31 (m, 1H, CH2); 5.17–5.26 (m,
2H, CH2]); 5.69 (d, 1H, CH, J ¼ 8 Hz); 5.78–5.91 (m, 1H, CH]); 6.33
(s, 1H, NH); 8.07 (s, 1H, NH). 13C NMR (CDCl3)
d: 20.61, 21.16, 21.24,
30.76, 33.43, 35.51, 37.98, 42.53, 44.56, 45.54, 49.09, 117.40, 132.78,
134.25, 135.64, 175.43, 175.80, 181.63. Anal. Calcd for C18H25N3O2S:
C 62.22, H 7.25, N 12.09. Found: C 62.00, H 7.26, N 12.08. ESI MS:
m/z ¼ 370.2 [M þ Na]þ (100%).
3.2.2. 1-Benzyl-3-(10-isopropyl-8-methyl-3,5-dioxo-4-
X-ray crystallography: intensity measurements were carried out
azatricyclo[5.2.2.02,6] undec-8-en-4-yl)-thiourea (2a)
at 295 K with
chromated CuK
a
KM4 diffractometer, using graphite mono-
Yield 67%. Mp. 165 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.78 (d, 3H, CH3,
a
radiation ( /2 scan mode.
l
¼ 1.54178 Å) and
u
q
J ¼ 8.8 Hz); 0.86 (d, 3H, CH3, J ¼ 8.8 Hz); 0.96–1.04 (m, 2H, CH2);
1.28–1.36 (m,1H, CH); 1.46 (d, 3H, CH3, J ¼ 1.2 Hz); 1.69–1.82 (m,1H,
CH); 2.84–2.94 (m, 3H, CH, CH–C]O); 3.09–3.12 (m, 1H, CH–C]O);
4.61–4.89 (m, 1H, CH2); 5.29 (d, 1H, CH, J ¼ 7.6 Hz); 6.37 (s, 1H, NH);
Structures were solved by the SHELXS-97 program and refined by
full-matrix least squares on F2 using the SHELXL-97 program [52].
Non-hydrogen atoms were refined anisotropically. Hydrogen atoms
were positioned geometrically and C–H bond ‘riding’ model was
used in the refinement. The experimental details and final atomic
parameters for 7a and 7a0 have been deposited with the Cambridge
Crystallographic Data Centre as supplementary material; No CCDC
721055 and CCDC 721056, respectively. Copies of the data can be
obtained free of charge on request to The Director, CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK (www: http://www.ccdc.cam.ac.uk).
7.3–7.37 (m, 5H, CHarom,); 7.96 (s, 1H, NH). 13C NMR (CDCl3)
d: 20.62,
21.16, 21.28, 30.78, 33.42, 35.52, 37.96, 42, 53, 40.56, 45.50, 49.83,
128.42 (2C), 128.68 (2C), 129.12 (2C), 136.63, 137.37, 173.15, 173.72,
181.70. Anal. Calcd for C18H25N3O2S: C 62.22, H 7.25, N 12.09.
Found: C 62.00, H 7.26, N 12.08. ESI MS: m/z ¼ 370.2 [M þ Na]þ
(100%).
3.2.3. 1-(4-Fluorophenyl)-3-(10-isopropyl-8-methyl-3,5-dioxo-
4-aza-tricyclo [5.2.2.02,6]undec-8-en-4-yl)-thiourea (3a)
3.2. Thiourea derivatives of 10-isopropyl-8-methyl-4-
Yield 82%. Mp. 166.7 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.82 (d, 3H,
azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione (1a–7a)
CH3, J ¼ 6.8 Hz); 0.91 (d, 3H, CH3, J ¼ 6.4 Hz); 1.06–1.13 (m, 2H,
CH2); 1.34–1.37 (m, 1H, CH); 1,69 (s, 3H, CH3); 1.71–1.85 (m, 1H,
CH); 2.92–3.0 (m, 3H, CH, CH–C]O); 3.2 (d, 1H, CH–C]O, J ¼ 6 Hz);
5.7 (d, 1H, CH], J ¼ 5.6 Hz); 7.0–7.08 (m, 2H, CHarom,); 7.35–7.38 (m,
and 1-isopropyl-7-methyl-4-azatricyclo[5.2.2.02,6
]
undec-8-ene-3,5-dione (1b–7b)
General procedure:
A solution of 4-amino-10-isopropyl-8-
2H, CHarom,); 8.15 (s, 1H, NH). 13C NMR (CDCl3)
d: 20.62, 21.16, 21.28,
methyl-4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione or 4-amino-
1-isopropyl-7-methyl-4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione
(0.0025 mol, 0.62 g) in acetonitrile (10 mL) was treated with appro-
priate isothiocyanate (0.003 mol) and the mixture was refluxed for
6 h. Then solvent was removed on rotary evaporator. The residue was
purified by column chromatography (chloroform:methanol; 9.5:0.5
vol.). The compound was crystallized from ethanol.
30.78, 33.42, 35.52, 37.96, 40.56, 42.53, 45.50, 116.29, 116.59, 127.31
(2C), 132.93, 136.24, 137.02, 163.02, 174.06, 174.56, 181.89. Anal.
Calcd for C21H24FN3O2S: C 62.82, H 6.03, N 10.47. Found: C 62.92, H
6.09, N 10.49. ESI MS: m/z ¼ 418.1 [M þ H]þ (100%).
3.2.4. 1-(4-Chlorophenyl)-3-(10-isopropyl-8-methyl-3,5-dioxo-4-
aza-tricyclo [5.2.2.02,6]undec-8-en-4-yl)-thiourea (4a)
Yield 82%. Mp. 179.5 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.82 (d, 3H,
3.2.1. 1-Allyl-3-(10-isopropyl-8-methyl-3,5-dioxo-4-
CH3, J ¼ 6.4 Hz); 0.91 (d, 3H, CH3, J ¼ 6.4 Hz); 1.06–1.13 (m, 2H,
CH2); 1.35–1.36 (m, 1H, CH); 1,69 (s, 3H, CH3); 1.75–1.84 (m, 2H, CH,
NH); 2.92–2.98 (m, 3H, CH, CH–C]O); 3.2 (d, 1H, CH–C]O,
J ¼ 5.6 Hz); 5.69 (d,1H, CH], J ¼ 5.2 Hz); 7.29–7.36 (m, 4H, CHarom,);
azatricyclo[5.2.2.02,6] undec-8-en-4-yl)-thiourea (1a)
Yield 67%. Mp. 165 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.73 (d, 3H, CH3,
J ¼ 8.8 Hz); 0.90 (d, 3H, CH3, J ¼ 8.8 Hz); 1.04–1.15 (m, 2H, CH2);
1.34–1.422 (m, 1H, CH); 1.75 (d, 3H, CH3, J ¼ 2.4 Hz); 1.8–1.88 (m,
1H, CH); 2.91–3.01 (m, 3H, CH, CH–C]O); 3.18–3.21 (n, 1H, CH–
8.38 (s, 1H, NH). 13C NMR (CDCl3)
d: 20.61, 21.17, 21.22, 30.73, 33.42,
400
2500
2000
Control
Amphetamine
5 mg/kg
Control
300
0.1 LD50
*
1500
0.1 LD50
0.05 LD50
p < 0.05
vs control
200
100
*
p < 0.05
vs control
*
1000
500
0
*
*
0
6b'
7a'
6b'
7a'
Fig. 3. The influence of 6b0and 7a0 on the spontaneous motor activity.
Fig. 4. The influence 6b0 and 7a0 on amphetamine-induced hyperactivity.

4964
M. Struga et al. / European Journal of Medicinal Chemistry 44 (2009) 4960–4969
40
30
20
10
0
15
Control
0.1 LD50
Control
0.1 LD50
10
5
0
6b'
7a'
6b'
7a'
Fig. 7. The influence of 6b0 and 7a0 on motor coordination evaluated in chimney test.
Fig. 5. The influence of 6b0 and 7a0 on nociceptive reactions studied in the acetic acid
(0.6%) induced writhing test.
3.2.7. 1-(4-Methylphenyl)-3-(10-isopropyl-8-methyl-3,5-dioxo-4-
azatricyclo [5.2.2.02,6]undec-8-en-4-yl)-thiourea (7a)
Yield 82%. Mp. 195 ꢁC. 1H NMR 300 MHz (CDCl3)
d (ppm): 0.76
35.51, 37.98, 42.55, 44.55, 45.54, 122.50 (2C), 126.53 (2C), 129.66,
132.79, 135.64, 142.59, 175.45, 175.80, 181.62. Anal. Calcd for
C21H24ClN3O2S: C 60.35, H 5.79, N 10.05. Found: C 60.32, H 5.92, N
10.09. ESI MS: m/z ¼ 424.1 [M þ Na]þ (100%).
(d, 3H, CH3, J ¼ 6.3 Hz); 0.84 (d, 3H, CH3, J ¼ 6.3 Hz); 0.98–1.07 (m,
2H, CH2); 1.25–1.29 (m, 1H, CH); 1,58 (s, 3H, CH3); 1.62–1.79 (m, 2H,
CH, NH); 2.28 (s, 3H, CH3); 2.81–2.92 (m, 3H, CH, CH–C]O); 3.15 (d,
1H, CH–C]O, J ¼ 6.3 Hz); 5.63 (d, 1H, CH], J ¼ 6.3 Hz); 7.12–7.22
(m, 4H, CHarom,); 8.05 (s, 1H, NH). 13C NMR (CDCl3)
d: 20.61, 21.17,
3.2.5. 1-(4-Iodophenyl)-3-(10-isopropyl-8-methyl-3,5-dioxo-4-
aza-tricyclo [5.2.2.02,6]undec-8-en-4-yl)-thiourea (5a)
21.22, 22.73, 30.73, 33.42, 35.51, 37.98, 42.55, 44.55, 45.54, 125.41
(2C), 130.55 (2C), 133.76, 136.23, 137.04, 137.82, 173.66, 174.10,
181.75. Anal. Calcd for C22H27N3O2S: C 66.47, H 6.85, N 10.57. Found:
C 66.56, H 6.91, N 10.67. ESI MS: m/z ¼ 398.28 [M þ H]þ (100%).
Crystal data: crystal system monoclinic, space group P21, unit
cell dimensions a ¼ 14.056(3), b ¼ 10.933(2), c ¼ 15.955(3) Å,
Yield 82%. Mp. 181 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.82 (d, 3H, CH3,
J ¼ 6.4 Hz); 0.91 (d, 3H, CH3, J ¼ 6.4 Hz); 1.06–1.14 (m, 2H, CH2);
1.35–1.36 (m, 1H, CH); 1,69 (s, 3H, CH3); 1.72–1.85 (m, 2H, CH, NH);
2.92–2.98 (m, 3H, CH, CH–C]O); 3.2 (d, 1H, CH–C]O, J ¼ 5.6 Hz);
5.69 (d, 1H, CH], J ¼ 5.2 Hz); 7.1 (d, 2H, CHarom, J ¼ 8.4 Hz); 7.6 (d,
2H, CHarom, J ¼ 8.4 Hz) 8.4 (s, 1H, NH). 13C NMR (CDCl3)
d: 20.64,
b
¼111.06(3)ꢁ, V ¼ 2288.1(8) Å3; Z ¼ 4, dc ¼ 1.154 g cmꢀ3
,
m
¼
1.415 mmꢀ1, F(000) ¼ 848. A crystal of dimensions 0.5 ꢂ 0.32 ꢂ
21.17, 21.18, 30.76, 33.38, 35.53, 37.93, 42.51, 44.53, 45.54, 91.08,
126.23 (2C), 135.76, 136.49, 137.22, 138.38 (2C), 174.14, 174.68,
181.22. Anal. Calcd for C21H24IN3O2S: C 49.51, H 4.75, N 8.25. Found:
C 49.58, H 4.77, N 8.29. ESI MS: m/z ¼ 532.1 [M þ Na]þ (100%).
0.21 mm was used for intensity measurements. Within the
q range
2.97–75.14ꢁ [ꢀ16 ꢃ h ꢃ 16, ꢀ13 ꢃ k ꢃ 13, 0 ꢃ l ꢃ 19] 9034 reflections
were collected. The 8898 unique reflections [R(int) ¼ 0.0476] were
used for the refinement of 513 parameters, including extinction
coefficient [x ¼ 0.0022(3)]. Final R indices on F2 for 3456 observed
3.2.6. 1-(3,4-Dichlorophenyl)-3-(10-isopropyl-8-methyl-3,5-dioxo-
4-aza-tricyclo [5.2.2.02,6]undec-8-en-4-yl)-thiourea (6a)
reflections [I > 2
s
(I)] were: R1 ¼0.0544, wR2 ¼ 0.1454, goodness-
Yield 82%. Mp. 154 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.83 (d, 3H, CH3,
of-fit 0.964, and largest difference peak/hole 0.29/ꢀ0.23 e Åꢀ3
.
J ¼ 6.4 Hz); 0.91 (d, 3H, CH3, J ¼ 6.4 Hz); 1.07–1.14 (m, 2H, CH2);
1.35–1.37 (m, 1H, CH); 1,7 (s, 3H, CH3); 1.74–1.85 (m, 2H, CH, NH);
2.93–3.01 (m, 3H, CH, CH–C]O); 3.2 (d, 1H, CH–C]O, J ¼ 5.6 Hz);
5.69 (d, 1H, CH], J ¼ 5.6 Hz); 7.24–7.36 (m, 3H, CHarom,); 7.6 (s, 1H,
3.2.8. 1-(Allyl)-3-(1-isopropyl-7-methyl-3,5-dioxo-4-
azatricyclo[5.2.2.02,6] undec-8-en-4-yl)-thiourea (1b)
Yield 84%. Mp. 135 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.99 (d, 3H, CH3,
CHarom,) 8.56 (s, 1H, NH). 13C NMR (CDCl3)
d: 20.64, 21.17, 21.18,
J ¼ 6.9 Hz); 1.04 (d, 3H, CH3, J ¼ 6.9 Hz); 1.25–1.37 (m, 2H, CH2); 1.48
(s, 3H, CH3); 1.52–1.58 (m, 2H, CH2); 2.48–2.57 (m, 1H, CH); 2.65 (d,
1H, CH–C]O, J ¼ 8.1 Hz); 3.08 (d, 1H, CH–C]O, J ¼ 8.1 Hz); 4.19 (s,
2H, CH2); 5.79–5.86 (m, 1H, CH]); 6.03 (dd, 2H, CH], J ¼ 11.3 Hz);
30.76, 33.38, 35.53, 37.93, 42.51, 44.53, 45.54, 123.52, 125.88, 130.19,
130.69, 132.93, 136.28, 136.96, 137.04, 174.46, 175.00, 181.18. Anal.
Calcd for C21H23Cl2N3O2S: C 55.75, H 5.12, N 9.29. Found: C 55.80, H
5.21, N 9.31. ESI MS: m/z ¼ 473.7 [M þ Na]þ (100%).
6.29 (s, 1H, NH); 7.7 (s, 1H, NH). 13C NMR (CDCl3)
d
: 17.01, 18.57,
C
0.0
60
7a'
Control
0.1 LD50
6b'
-0.5
control
40
-1.0
-1.5
-2.0
** p < 0.01
**
**
**
**
20
0
**
**
[min]
0
30
60
90
120 150 180
7a'
6b'
Fig. 8. The influence of 6b0 and 7a0 on the body temperature of mice. Note: each point
represents the mean for a group of 10 mice.
Fig. 6. The influence of 6b0 and 7a0 on motor coordination evaluated in rota-rod test.

M. Struga et al. / European Journal of Medicinal Chemistry 44 (2009) 4960–4969
4965
Table 3
8
6
4
2
0
Cytotoxicity and anti-HIV-1 activity of compounds (1b–7b, 1b0–7b0).
Cmpds
MT-4
HIV-1
Cmpds
MT-4
HIV-1
Control
a
b
a
b
CC50
EC50
CC50
EC50
0.1 LD50
1b
2b
3b
4b
5b
6b
7b
EFV
>100
58
nt
nt
>100
59
>100
>58
nt
1b0
2b0
3b0
4b0
5b0
6b0
7b0
EFV
58
>100
86
32
59
>100
nt
35
>58
p < 0.05
vs control
*
>100
>86
>32
>59
>100
nt
*
nt
>100
>59
nt
nt
35
0.003
0.003
a
Compound concentration (mM) required to reduce the viability of mock-infected
6b'
7a'
MT-4 cells by 50%, as determined by the MTT method.
b
Compound concentration (mM) required to achieve 50% protection of MT-4 cells
Fig. 9. The influence of 6b0 and 7a0 on the head twitch response evoked by
5–hydroxytryptophan (5-HTP). The results are expressed as mean ꢄS.E.M. (n ¼ 10).
from the HIV-1 induced cytopathogenicity, as determined by the MTT method.
22.69, 23.15, 29.81, 34.31, 37.29, 40.61, 44.12, 45.42, 49.09, 117.4,
134.25, 136.54, 137.27, 174.02, 174.55, 182.23. Anal. Calcd for C18H25
N3O2S: C 62.22, H 7.25, N 10.09. Found: C 62.0, H 7.26, N 12.08. ESI
MS: m/z ¼ 358.2 [M þ Na]þ (100%).
3.2.11. 1-(4-Chlorophenyl)-3-(1-isopropyl-7-methyl-3,5-dioxo-
4-aza-tricyclo [5.2.2.02,6]undec-8-en-4-yl)-thiourea (4b)
Yield 84%. Mp. 142 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.92 (d, 3H, CH3,
J ¼ 6.8 Hz); 1.1 (d, 3H, CH3, J ¼ 6.8 Hz); 1.26–1.41 (m, 2H, CH2); 1.47
(s, 3H, CH3); 1.49–1.63 (m, 2H, CH2); 2.49–2.54 (m,1H, CH2); 2.76 (d,
1H, CH–C]O, J ¼ 8 Hz); 3.12 (d, 1H, CH–C]O, J ¼ 8 Hz); 6.01 (dd,
2H, CH], J ¼ 12.8 Hz); 7.27–7.28 (m, 2H, CHarom,); 7.36–7.42 (m, 2H,
3.2.9. 1-(4-Benzylphenyl)-3-(1-isopropyl-7-methyl-3,5-dioxo-
4-aza-tricyclo [5.2.2.02,6]undec-8-en-4-yl)-thiourea (2b)
Yield 84%. Mp. 208 ꢁC. 1H NMR (CDCl3) 300 MHz
d (ppm): 0.74
CHarom,); 8.66 (s, 1H, NH). 13C NMR (CDCl3)
d: 17.04, 18.59, 22.73,
(d, 3H, CH3, J ¼ 5.1 Hz); 1.1 (d, 3H, CH3, J ¼ 5.1 Hz); 1.12–1.26 (m, 2H,
CH2); 1.28 (s, 3H, CH3); 1.38–1.45 (m, 2H, CH2); 2.26–2.33 (m, 1H,
CH2); 2.51 (d, 1H, CH–C]O, J ¼ 5.7 Hz); 2.58 (d, 1H, CH–C]O,
J ¼ 5.7 Hz); 3.14 (s, 1H, NH); 4.5–4.69 (m, 2H, CH2); 5.89 (dd, 2H,
23.20, 29.90, 34.35, 37.21, 44.06, 45.54, 49.25, 126.07 (2C), 130.03
(2C), 132.60, 135.61, 136.29, 137.05, 174.03, 174.35, 181.50. Anal.
Calcd for C21H24 ClN3O2S: C 60.35, H 5.79, N 10.05. Found: C 60.47, H
5.82, N 10.08. ESI MS: m/z ¼ 418.1 [M þ H]þ (100%).
CH], J ¼ 13.1 Hz); 7.19–7.29 (m, 4H, CHarom,); 7.68 (s, 1H, NH). 13
C
NMR (CDCl3) d: 16.99, 18.49, 22.55, 23.01, 29.66, 34.08, 37.27, 44.11,
3.2.12. 1-(4-Iodophenyl)-3-(1-isopropyl-7-methyl-3,5-dioxo-
4-aza-tricyclo [5.2.2.02,6]undec-8-en-4-yl)-thiourea (5b)
45.24, 49.00, 49.83, 128.42 (2C), 128.68 (2C), 129.12 (2C), 136.63,
137.37, 173.15, 173.72, 181.70. Anal. Calcd for C22H27N3O2S: C 66.47,
H 6.85, N 10.57. Found: C 66.52, H 6.89, N 10.67. ESI MS: m/z ¼ 420.2
[M þ Na]þ (100%).
Yield 84%. Mp. 140.8 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.89 (d, 3H,
CH3, J ¼ 6.8 Hz); 1.1 (d, 3H, CH3, J ¼ 6.8 Hz); 1.25–1.36 (m, 2H, CH2);
1.47 (s, 3H, CH3); 1.4–1.53 (m, 2H, CH2); 2.5–2.59 (m, 3H, CH2, CH–
C]O); 2.95 (d, 1H, CH–C]O, J ¼ 7.6 Hz); 5.93 (dd, 2H, CH],
J ¼ 13.4 Hz); 7.34–7.36 (m, 2H, CHarom,); 7.59–7.61 (m, 2H, CHarom,);
3.2.10. 1-(4-Fluorophenyl)-3-(1-isopropyl-7-methyl-3,5-dioxo-
4-aza-tricyclo [5.2.2.02,6]undec-8-en-4-yl)-thiourea (3b)
9.11 (s, 1H, NH). 13C NMR (CDCl3)
d: 17.04, 18.59, 22.74, 23.06, 29.87,
Yield 84%. Mp. 142 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.98 (d, 3H, CH3,
34.35, 37.18, 44.03, 45.53, 49.23, 91.08, 126.23 (2C), 135.76, 136.49,
137.22, 138.38 (2C), 174.14, 174.68, 181.22. Anal. Calcd for C21H24
IN3O2S: C 49.51, H 4.75, N 8.25. Found: C 49.56, H 4.76, N 8.25. ESI
MS: m/z ¼ 508.2 [M ꢀ H]ꢀ (100%).
J ¼ 6.8 Hz); 1.1 (d, 3H, CH3, J ¼ 6.8 Hz); 1.27–1.41 (m, 2H, CH2); 1.47
(s, 3H, CH3); 1.49–1.56 (m, 2H, CH2); 1.89 (s, 1H, NH); 2.48–2.55 (m,
1H, CH2); 2.76 (d, 1H, CH–C]O, J ¼ 8 Hz); 3.12 (d, 1H, CH–C]O,
J ¼ 8 Hz); 5.99 (dd, 2H, CH], J ¼ 12.8 Hz); 7.0–7.09 (m, 2H, CHarom,);
7.36–7.39 (m, 2H, CHarom,); 8.04 (s, 1H, NH). 13C NMR (CDCl3)
d:
3.2.13. 1-(3,4-Dichlorophenyl)-3-(1-isopropyl-7-methyl-3,5-dioxo-
4-aza-tricyclo [5.2.2.02,6]undec-8-en-4-yl)-thiourea (6b)
17.02, 18.57, 22.79, 23.18, 29.88, 34.33, 37.16, 44.02, 45.52, 49.23,
116.29, 116.59, 127.31 (2C), 132.93, 136.24, 137.02, 163.02, 174.06,
174.56, 181.89. Anal. Calcd for C21H24 FN3O2S: C 62.82, H 6.03, N
10.47. Found: C 62.98, H 6.11, N 10.52. ESI MS: m/z ¼ 424.1
[M þ Na]þ (100%).
Yield 84%. Mp. 143 ꢁC. 1H NMR (CDCl3)
d (ppm): 1.0 (d, 3H,
CH3, J ¼ 7.2 Hz); 1.13 (d, 3H, CH3, J ¼ 6.8 Hz); 1.27–1.42 (m, 2H,
Table 4
Cytotoxicity, anti-HIV-1, anti-BVDV and anti-YFV activity of compounds (1b and 2b).
Table 2
Compds
MT-4
HIV-1
BVDV
YFV
Cytotoxicity and anti-HIV-1 activity of compounds (1a–7a, 1a0–7a0).
a
b
c
d
CC50
EC50
EC50
EC50
Cmpds
MT-4
HIV-1
Cmpds
MT-4
HIV-1
1b
2b
>100
58
>100
>58
52
>100
>100
41
a
b
a
b
CC50
EC50
CC50
EC50
1a0
2a0
3a0
4a0
5a0
6a0
7a0
EFV
59
47
49
30
22
100
nt
>59
>47
>49
>30
>22
>100
nt
EFV
NM–108
35
0.003
1a
2a
3a
4a
5a
6a
7a
EFV
>100
54
69
45
45
41
Nt
35
>100
30
1.8
2.5
>69
>45
>45
>41
nt
a
Compound concentration (
mM) required to reduce the viability of mock-infected
MT-4 cells by 50%, as determined by the MTT method.
b
Compound concentration (
mM) required to achieve 50% protection of MT-4 cells
from the HIV-1 induced cytopathogenicity, as determined by the MTT method.
c
Compound concentration (mM) required to achieve 50% protection of MDBK
0.003
35
0.003
cells from the BVDV (Bovine Viral Diarrhea Virus) – induced cytopathogenicity, as
a
determined by the MTT method.
Compound concentration (mM) required to reduce the viability of mock-infected
d
Compound concentrarion (mM) required to achieve 50% protection of BHK
MT-4 cells by 50%, as determined by the MTT method.
b
(Kidney fibroblast) cells from the YFV (Yellow Fever Virus) – induced cytopatho-
genicity, as determined by the MTT method.
Compound concentration (
mM) required to achieve 50% protection of MT-4 cells
from the HIV-1 induced cytopathogenicity, as determined by the MTT method.

4966
M. Struga et al. / European Journal of Medicinal Chemistry 44 (2009) 4960–4969
CH2); 1.48 (s, 3H, CH3); 1.49–1.57 (m, 2H, CH2); 1.96 (s, 1H, NH);
2.47–2.59 (m, 1H, CH2); 2.78 (d, 1H, CH–C]O, J ¼ 8.4 Hz); 3.14 (d,
1H, CH–C]O, J ¼ 8 Hz); 6.02 (dd, 2H, CH], J ¼ 13 Hz); 7.28–7.44
3.3.3. 10-Isopropyl-8-methyl-4-{2-[(Z)-4-fluorophenylimino]-
1,3-thiazepan-3-yl}-4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-
dione (3a0)
(m, 4H, CHarom,); 8.22 (s, 1H, NH). 13C NMR (CDCl3)
d
: 17.05, 18.59,
Yield 45%. Mp. 119.6 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.80 (d, 3H,
22.74, 23.31, 29.93, 34.33, 37.24, 44.12, 45.6, 49.31, 123.52, 125.88,
130.19, 130.69, 132.93, 136.28, 136.96, 137.04, 174.46, 175.00,
181.18. Anal. Calcd for C21H23 Cl2N3O2S: C 55.75, H 5.12, N 9.29.
Found: C 55.78, H 5.18, N 9.31. ESI MS: m/z ¼ 474.2 [M þ Na]þ
(100%).
CH3, J ¼ 6.8 Hz); 0.9 (d, 3H, CH3, J ¼ 6.8 Hz); 1.01–1.14 (m, 2H,
CH2); 1.25–1.33 (m, 1H, CH); 1.36–1.38 (m, 1H, CH); 1.62–1.86 (m,
2H, CH2); 1,77 (s, 3H, CH3); 1.9–2.0 (m, 2H, CH2); 2.72 (d, 1H,
CH–C]O, J ¼ 2.4 Hz); 2.74 (d, 1H, CH–C]O, J ¼ 2.8 Hz); 2.81–2.6
(m, 2H, CH); 3.08–3.19 (m, 2H, CH2); 3.83–3.92 (m, 2H, CH2); 5.73
(d, 1H, CH], J ¼ 5.6 Hz); 6.57–6.64 (m, 2H, CHarom,); 6.88–7.0
3.2.14. 1-(4-Methylphenyl)-3-(1-isopropyl-7-methyl-3,5-dioxo-
(m, 2H, CHarom,). 13C NMR (CDCl3)
d: 20.62, 21.16, 21.28, 27.53,
4-aza-tricyclo [5.2.2.02,6]undec-8-en-4-yl)-thiourea (7b)
28.14, 30.79, 33.42, 34.54, 35.52, 37.96, 40.56, 42.56, 44.53, 45.50,
54.45, 115.21 (2C), 122, 84 (2C), 135.92, 136.75, 161.80, 162.82,
174.05, 174.39. Anal. Calcd for C25H30FN3O2S: C 65.91, H 6.64, N
9.22. Found: C 66.02, H 6.68, N 9.22. ESI MS: m/z ¼ 456.3
[M þ H]þ (100%).
Yield 84%. Mp. 142 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.96 (d, 3H, CH3,
J ¼ 7.2 Hz); 1.08 (d, 3H, CH3, J ¼ 7.2 Hz); 1.2–1.37 (m, 2H, CH2); 1.46
(s, 3H, CH3); 1.399–1.54 (m, 2H, CH2); 2.32 (s, 3H, CH3); 2.47–2.57
(m, 1H, CH); 2.72 (d, 1H, CH–C]O, J ¼ 8.1 Hz); 3.08 (d, 1H, CH–C]O,
J ¼ 8.4 Hz); 5.94 (dd, 2H, CH], J ¼ 10.5 Hz); 7.17 (d, 2H, CHarom,
J ¼ 8.1 Hz); 7.25 (d, 2H, CHarom, J ¼ 8.7 Hz); 7.59 (s, 1H, NH); 8.26 (s,
3.3.4. 10-Isopropyl-8-methyl-4-{2-[(Z)-4-chlorophenylimino]-1,
3-thiazepan-3-yl}-4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-
dione (4a0)
1H, NH). 13C NMR (CDCl3)
d: 17.03, 18.59, 21.27, 22.73, 23.19, 29.85,
34.38, 37.29, 43.97, 45.49, 49.20, 125.41 (2C), 130.55 (2C), 133.76,
136.23, 137.04, 137.82, 173.66, 174.10, 181.75. Anal. Calcd for C23H28
N3O2S: C 66.47, H 6.85, N 10.57. Found: C 66.67, H 6.91, N 10.62. ESI
MS: m/z ¼ 398.16 [M þ Na]þ (100%).
Yield 45%. Mp. 164 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.82 (d, 3H,
CH3, J ¼ 6.4 Hz); 0.91 (d, 3H, CH3, J ¼ 6.4 Hz); 1.05–1.15 (m, 2H,
CH2); 1.25–1.28 (m, 1H, CH); 1.36–1.38 (m, 1H, CH); 1.73–1.86 (m,
2H, CH2); 1,77 (s, 3H, CH3); 2.03–2.04 (m, 2H, CH2); 2.73 (d, 1H,
CH–C]O, J ¼ 2.4 Hz); 2.76 (d, 1H, CH–C]O, J ¼ 2.8 Hz); 2.81–2.86
(m, 2H, CH); 3.09–3.19 (m, 2H, CH2); 3.83–3.92 (m, 2H, CH2); 5.73
(d, 1H, CH], J ¼ 5.6 Hz); 6.57–6.81 (m, 2H, CHarom,); 7.23–7.32 (m,
3.3. 1,3-Thiazepine derivatives (1a0–7a0, 1b0–7b0)
General procedure: Sodium hydride dispersion (60%) in mineral
oil (0.44 g, w10 mmol) was added in a single portion to a stirred
solution of thiourea derivative (10 mmol) in anhydrous N,N-dime-
thylformamide at room temperature. After hydrogen evolution
ceased, 1,4-dibromobutane (15 mmol) was added to the reaction
mixture, after 5 and 15 min., respectively. The mixture was stirred
for 6 h. Evaporation in vacuum gave a residue which was then
purified by column chromatography (chloroform was used as
eluent). The compound was crystallized from methanol.
2H, CHarom,). 13C NMR (CDCl3)
d: 20.61, 21.17, 21.22, 27.45, 28.17,
30.73, 33.42, 34.56, 35.51, 37.98, 42.55, 44.55, 45.54, 49.05, 54.12,
124.19 (2C), 128.64, 135.51, 135.95, 136.75, 148.35, 153.68, 173.54,
174.32. Anal. Calcd for C25H30ClN3O2S: C 63.61, H 6.41, N 8.9.
Found: C 63.72, H 6.45, N 8.93. ESI MS: m/z ¼ 471.8 [M þ H]þ
(100%).
3.3.5. 10-Isopropyl-8-methyl-4-{2-[(Z)-4-iodophenylimino]-1,3-
thiazepan-3-yl}-4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione (5a0)
Yield 43%. Mp. 163.1 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.82 (d, 3H,
3.3.1. 10-Isopropyl-8-methyl-4-{2-[(Z)-allylimino]-1,3-thiazepan-
3-yl}-4-aza-tricyclo [5.2.2.02,6]undec-8-ene-3,5-dione (1a0)
CH3, J ¼ 6.4 Hz); 0.91 (d, 3H, CH3, J ¼ 6.9 Hz); 1.05–1.15 (m, 2H, CH2);
1.3–1.42 (m, 1H, CH); 1.62–1.65 (m, 1H, CH); 1.73–1.85 (m, 2H, CH2);
1,79 (s, 3H, CH3); 2.0–2.02 (m, 2H, CH2); 2.81 (d, 1H, CH–C]O,
J ¼ 2.4 Hz); 2.84 (d, 1H, CH–C]O, J ¼ 2.8 Hz); 2.92–2.98 (m, 2H,
CH); 3.04–3.2 (m, 2H, CH2); 3.7–3.78 (m, 2H, CH2); 5.73 (d, 1H,
CH], J]6 Hz); 6.49–6.55 (m, 2H, CHarom,); 7.5–7.62 (m, 2H,
Yield 66%. Mp. 112 ꢁC. 0.82 (d, 3H, CH3, J ¼ 4.4 Hz); 0.84 (d, 3H,
CH3, J ¼ 4.4 Hz); 1.27–1.33 (m, 2H, CH2); 1.44–1.49 (m, 3H, CH2,
CH); 1.68 (s, 3H, CH3); 1.68–1.7 (m, 2H, CH2); 2.3–2.16 (m, 2H, CH2);
2.53–2.59 (m, 2H, CH2); 2.8–2.99 (m, 2H, CH–C]O); 3.569–3.87 (m,
2H, CH2); 3.92–4.05 (m, 2H, CH2); 4.94–5.03 (m, 1H, CH2]); 5.16–
5.25 (m, 1H, CH2]); 5.75–5.82 (m, 1H, CH]); 5.95 (dd, 2H, CH],
CHarom,). 13C NMR (CDCl3)
d: 20.64, 21.17, 21.18, 27.56, 28.17, 30.76,
33.38, 34.54, 35.53, 37.93, 42.51, 44.53, 45.54, 49.29, 54.23, 86.56,
124.70 (2C), 135.52, 135.89, 137.58 (2C), 153.61, 173.69, 174.11. Anal.
Calcd for C25H30IN3O2S: C 53.29, H 5.37, N 7.46. Found: C 53.35, H
5.43, N 7.46. ESI MS: m/z ¼ 564.2 [M þ H]þ (100%).
J1 ¼ J2 ¼ 8.4 Hz). 13C NMR (CDCl3)
d: 20.61, 21.16, 21.24, 27.54, 28.13,
30.76, 33.43, 34.54, 35.51, 37.98, 42.53, 44.56, 45.54, 49.09, 54.22,
117.4, 134.25, 136.54, 137.81, 163.01, 174.02, 174.45. Anal. Calcd for
C22H31N3O2S: C 65.8, H 7.78, N 10.46. Found: C 65.6, H 7.8, N 10.32.
ESI MS: m/z ¼ 402.2 [M þ H]þ (100%).
3.3.6. 10-Isopropyl-8-methyl-4-{2-[(Z)-3,4-dichlorophenylimino]-
1,3-thiazepan-3-yl}-4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-
dione (6a0)
3.3.2. 10-Isopropyl-8-methyl-4-{2-[(Z)-benzylimino]-1,3-thiazepan-
3-yl}-4-aza-tricyclo[5.2.2.02,6]undec-8-ene-3,5-dione (2a0)
Yield 45%. Mp. 95.3 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.81 (d, 3H,
Yield 38%. Mp. 107.1 ꢁC. 1H NMR (CDCl3)
d
(ppm): 0.82 (d, 3H,
CH3, J ¼ 6.4 Hz); 0.9 (d, 3H, CH3, J ¼ 6.4 Hz); 1.05–1.11 (m, 2H, CH2);
1.25–1.28 (m, 1H, CH); 1.47–1.49 (m, 1H, CH); 1.73–1.85 (m, 2H,
CH2); 1,77 (s, 3H, CH3); 2.04–2.08 (m, 2H, CH2); 2.76 (d, 1H, CH–
C]O, J ¼ 2.4 Hz); 2.79 (d, 1H, CH–C]O, J ¼ 2.8 Hz); 2.85–2.89 (m,
2H, CH); 3.08–3.12 (m, 2H, CH2); 3.84–3.86 (m, 2H, CH2); 5.74 (d,
1H, CH], J ¼ 5.6 Hz); 6.76–6.78 (m, 1H, CHarom,); 7.01–7.02 (m, 2H,
CH3, J ¼ 6.6 Hz); 0.91 (d, 3H, CH3, J ¼ 6.6 Hz); 1.03–1.17 (m, 2H, CH2);
1.25–1.28 (m, 1H, CH); 1.32–1.44 (m, 1H, CH); 1.72–1.87 (m, 2H,
CH2); 1,75 (s, 3H, CH3); 2.04–2.12 (m, 2H, CH2); 2.74 (d, 1H, CH–
C]O, J ¼ 3.6 Hz); 2.74 (d, 1H, CH–C]O, J ¼ 3.6 Hz); 2.82–2.89 (m,
2H, CH); 3.14–3.22 (m, 2H, CH2); 3.49–3.82 (m, 2H, CH2); 4.78–4.92
(m, 2H, CH2); 5.70 (d,1H, CH], J ¼ 6 Hz); 7.27–7.42 (m, 5H, CHarom,).
CHarom,); 7.27–7.29 (m, 2H, CHarom,). 13C NMR (CDCl3)
d: 20.64, 21.17,
13C NMR (CDCl3)
d
: 20.62, 21.16, 21.28, 27.56, 28.13, 30.78, 33.42,
21.18, 27.48, 28.12, 30.76, 33.38, 34.52, 35.53, 37.93, 42.51, 44.53,
45.54, 49.02, 54.24, 122.59, 124.76, 130.25, 132.29, 135.94, 136.74,
149.41, 154.68, 173.48, 173.98. Anal. Calcd for C25H29Cl2N3O2S: C
59.28, H 5.77, N 8.3. Found: C 59.29, H 5.77, N 8.35. ESI MS:
m/z ¼ 506.03 [M]þ (100%).
34.54, 35.52, 37.96, 42.53, 40.56, 45.50, 49.83, 54.23, 123.11, 128.42,
128.67 (2C), 129.12 (2C), 136.63, 137.37, 162.8, 173.15, 173.71. Anal.
Calcd for C26H33N3O2S: C 69.15, H 7.36, N 9.3. Found: C 69.18, H 7.43,
N 9.31. ESI MS: m/z ¼ 452.3 [M þ H]þ (100%).

M. Struga et al. / European Journal of Medicinal Chemistry 44 (2009) 4960–4969
4967
3.3.7. 10-Isopropyl-8-methyl-4-{2-[(Z)-4-methylphenylimino]-1,3-
CHarom,), 6.84–6.88 (m, 2H, CHarom,). 13C NMR (CDCl3)
d: 17.03, 18.61,
thiazepan-3-yl}-4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione (7a0)
22.89, 23.26, 27.28, 28.28, 29.55, 29.97, 33.43, 34.56, 37.18, 44.04,
45.55, 49.27, 54.45, 115.21 (2C), 122, 84 (2C), 135.92, 136.75, 161.80,
162.82, 174.05, 174.39. Anal. Calcd for C25H30 FN3O2S: C 65.91, H
6.64, N 9.22. Found: C 66.03, H 6.72, N 9.33. ESI MS: m/z ¼ 456.62
[M þ H]þ (100%).
Yield 45%. Mp. 148 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.82 (d, 3H, CH3,
J ¼ 6.4 Hz); 0.84 (d, 3H, CH3, J ¼ 6.4 Hz); 0.95–1.08 (m, 2H, CH2);
1.15–1.28 (m, 1H, CH); 1.34–1.5 (m, 1H, CH); 1.64–1.67 (m, 2H, CH2);
1,7 (s, 3H, CH3); 2.22 (s, 3H, CH3); 2.73 (d, 1H, CH–C]O, J ¼ 2.4 Hz);
2.76 (d, 1H, CH–C]O, J ¼ 2.8 Hz); 2.77–2.83 (m, 2H CH2); 2.89–3.03
(m, 2H, CH); 3.06–3.12 (m, 2H, CH2); 3.58–3.72 (m, 2H, CH2); 5.66
(d, 1H, CH], J ¼ 5.6 Hz); 6.47–6.55 (m, 2H, CHarom,); 6.95–6.97 (m,
3.3.11. 1-Isopropyl-7-methyl-4-{2-[(Z)-4-chlorophenylimino]-1,3-
thiazepan-3-yl}-4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione (4b0)
2H, CHarom,). 13C NMR (CDCl3)
d
: 20.61, 21.17, 21.22, 22.73, 27.54,
Yield 82%. Mp. 138 ꢁC. 1H NMR (CDCl3)
d (ppm): 1.0 (d, 3H, CH3,
28.17, 30.73, 33.42, 34.54, 35.51, 37.98, 42.55, 44.55, 45.54, 49.28,
54.15, 122.05, 139.25 (2C), 132.42, 135.91, 136.70, 147.26, 152.74,
173.66, 174.10. Anal. Calcd for C26H33N3O2S: C 69.15, H 7.36, N 9.3.
Found: C 69.23, H 7.42, N 8.99. ESI MS: m/z ¼ 452.18 [M þ H]þ
(100%).
J ¼ 6.8 Hz); 1.1 (d, 3H, CH3, J ¼ 6.8 Hz); 1.25–1.41 (m, 2H, CH2); 1.47–
1.55 (m, 3H, CH2, CH); 1.47 (s, 3H, CH3); 1.7–1.77 (m, 2H, CH2); 1.99–
2.02 (m, 2H, CH2); 2.55–2.63 (m, 2H, CH2); 3.02 (d, 1H, CH–C]O,
J ¼ 14.4 Hz); 3.06 (d, 1H, CH–C]O, J ¼ 14.4 Hz); 3.78–3.82 (m, 2H,
CH2); 5.95 (dd, 2H, CH], J1 ¼8.4 Hz, J2 ¼ 8.8 Hz); 6.59–6.81 (d, 2H,
Crystal data: crystal system orthorhombic, space group P212121,
unit cell dimensions a ¼ 8.203(2), b ¼ 12.925(3), c ¼ 23.349(5) Å,
CHarom,), 7.19–7.23 (m, 2H, CHarom,). 13C NMR (CDCl3)
d: 17.05, 18.60,
22.77, 23.25, 27.12, 28.13, 29.42, 29.97, 33.47, 34.54, 37.18, 44.04,
45.55, 49.05, 124.19 (2C), 128.64 (2C), 135.51, 135.95, 136.75, 148.35,
153.68,173.54,174.32. Anal. Calcd for C25H30 ClN3O2S: C 63.61, H 6.41,
N 8.9. Found:C 63.72, H 6.44, N 8.92. ESIMS: m/z ¼ 472.18 [M] (100%).
V ¼ 2475.5(10) Å3; Z ¼ 4, dc ¼ 1.198 g cmꢀ3
,
m
¼ 1.366 mmꢀ1
,
F(000) ¼ 948. A crystal of dimensions 0.4 ꢂ 0.38 ꢂ 0.1 mm was used
for intensity measurements. Within the
q
range 3.91–80.27ꢁ
[0 ꢃ h ꢃ 10, 0 ꢃ k ꢃ 16, 0 ꢃ l ꢃ 29] 3033 reflections were collected.
The 3022 unique reflections [R(int) ¼ 0.10] were used for the
refinement of 290 parameters, including extinction coefficient
[x ¼ 0.0053(7)]. Final R indices on F2 for 1609 observed reflections
3.3.12. 1-Isopropyl-7-methyl-4-{2-[(Z)-4-iodophenylimino]-1,3-
thiazepan-3-yl}-4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione (5b0)
Yield 78%. Mp. 188 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.93 (d, 3H, CH3,
[I > 2
s
(I)] were: R1 ¼0.0432, wR2 ¼ 0.1254, goodness-of-fit 1.038,
J ¼ 6.8 Hz); 1.04 (d, 3H, CH3, J ¼ 6.8 Hz); 1.16–1.28 (m, 2H, CH2);
1.36–1.48 (m, 3H, CH2, CH); 1.43 (s, 3H, CH3); 1.59–1.7 (m, 2H, CH2);
1.9–1.95 (m, 2H, CH2); 2.46–2.56 (m, 2H, CH2); 2.93 (d, 1H, CH–
C]O, J ¼ 7.6 Hz); 2.95 (d, 1H, CH–C]O, J ¼ 7.6 Hz); 3.71–3.76 (m,
2H, CH2); 5.96 (dd, 2H, CH], J1 ¼8.8 Hz, J2 ¼ 8.8 Hz); 6.35–6.45 (d,
and largest difference peak/hole 0.27/ꢀ0.25 e Åꢀ3
.
3.3.8. 1-Isopropyl-7-methyl-4-{2-[(Z)-4-allylimino]-1,3-thiazepan-
3-yl}-4-aza-tricyclo [5.2.2.02,6]undec-8-ene-3,5-dione (1b0)
Yield 62%. Mp. 123 ꢁC. 1H NMR (CDCl3)
d
(ppm): 0.98 (d, 3H, CH3,
2H, CHarom,), 7.45–7.47 (m, 2H, CHarom,). 13C NMR (CDCl3)
d: 17.04,
J ¼ 6.8 Hz); 1.09 (d, 3H, CH3, J ¼ 6.8 Hz); 1.27–1.33 (m, 2H, CH2);
1.44–1.49 (m, 3H, CH2, CH); 1.48 (s, 3H, CH3); 1.68–1.7 (m, 2H, CH2);
2.3–2.16 (m, 2H, CH2); 2.53–2.59 (m, 2H, CH2); 2.8–2.99 (m, 2H,
CH–C]O); 3.569–3.87 (m, 2H, CH2); 3.92–4.05 (m, 2H, CH2); 4.94–
5.03 (m, 1H, CH2]); 5.16–5.25 (m, 1H, CH2]); 5.75–5.82 (m, 1H,
18.62, 22.79, 23.15, 23.27, 27.54, 28.13, 29.65, 29.95, 33.23, 34.54,
37.18, 44.03, 45.56, 49.29, 86.56, 124.70 (2C), 135.52, 135.89, 137.58
(2C), 153.61, 173.69, 174.11. Anal. Calcd for C25H30IN3O2S: C 53.29, H
5.37, N 7.46. Found: C 53.33, H 5.42, N 7.52. ESI MS: m/z ¼ 563.85
[M] (100%).
CH]); 5.95 (dd, 2H, CH], J1 ¼ J2 ¼ 8.4 Hz). 13C NMR (CDCl3)
d:
18.27, 19.91, 22.69, 23.15, 23.53, 24.50, 29.52, 29.81, 34.31, 37.29,
40.61, 44.12, 45.42, 49.09, 117.4, 134.25, 136.54, 137.81, 163.01,
174.02, 174.45. Anal. Calcd for C22H31N3O2S: C 65.8, H 7.78, N 10.46.
Found: C 65.88, H 7.82, N 10.52. ESI MS: m/z ¼ 402.22 [M þH]þ
(100%).
3.3.13. 1-Isopropyl-7-methyl-4-{2-[(Z)-3,4-dichlorophenylimino]-1,3-
thiazepan-3-yl}-4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione (6b0)
Yield 80%. Mp. 144 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.93 (d, 3H, CH3,
J ¼ 5.1 Hz); 1.03 (d, 3H, CH3, J ¼ 5.1 Hz); 1.19–1.3 (m, 2H, CH2); 1.36–
1.49 (m, 3H, CH2, CH); 1.44 (s, 3H, CH3); 1.63–1.7 (m, 2H, CH2); 1.92–
1.96 (m, 2H, CH2); 2.45–2.56 (m, 2H, CH2); 2.94 (d, 1H, CH–C]O,
J ¼ 6.3 Hz); 2.97 (d, 1H, CH–C]O, J ¼ 6.3 Hz); 3.67–3.71 (m, 2H,
CH2); 2.95 (dd, 2H, CH], J1 ¼ J2 ¼ 6.3 Hz); 6.54 (d, 1H, CHarom,
J ¼ 1.5 Hz), 6.8 (s, 1H, CHarom,); 7.1–7.23 (m, 2H, CHarom.). 13C NMR
3.3.9. 1-Isopropyl-7-methyl-4-{2-[(Z)-4-benzylimino]-1,3-
thiazepan-3-yl}-4-aza-tricyclo[5.2.2.02,6]undec-8-ene-3,5-dione(2b0)
Yield 62%. Mp. 179 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.98 (d, 3H, CH3,
J ¼ 6.4 Hz); 1.06 (d, 3H, CH3, J ¼ 6.8 Hz); 1.23–1.33 (m, 2H, CH2);
1.44–1.49 (m, 3H, CH2, CH); 1.5 (s, 3H, CH3); 1.73–1.76 (m, 2H, CH2);
1.86–1.89 (m, 2H, CH2); 2.57–2.62 (m, 2H, CH2); 2.77 (d, 1H, CH–
C]O, J ¼ 6.4 Hz); 2.98 (d, 1H, CH–C]O, J ¼ 6.4 Hz); 3.51–3.54 (m,
2H, CH2); 4.74–4.96 (m, 2H, CH2); 6.0 (dd, 2H, CH],
(CDCl3) d: 17.01, 18.62, 22.81, 23.17, 27.15, 28.00, 29.42, 29.99, 33.48,
34.54, 37.21, 44.07, 45.26, 49.02, 122.59, 124.76, 130.21, 130.25,
132.29, 135.94, 136.74, 149.41, 154.68, 173.48, 173.98. Anal. Calcd for
C25H29 Cl2N3O2S: C 59.28, H 5.77, N 8.3. Found: C 59.29, H 5.82, N
8.31. ESI MS: m/z ¼ 507.25 [M þ H]þ
J1 ¼ J2 ¼ 7.2 Hz); 7.32–7.38 (m, 5H, CHarom,). 13C NMR (CDCl3)
d:
16.99, 18.45, 22.54, 23.01, 27.12, 28.13, 29.65, 29.97, 34.08, 34.54,
37.20, 44.11, 45.23, 49.00, 49.85, 123.11, 128.42, 128.67 (2C), 129.12
(2C), 136.63, 137.37, 162.8, 173.15, 173.71. Anal. Calcd for
C26H33N3O2S: C 69.15, H 7.36, N 9.3. Found: C 69.18, H 7.42, N 9.36.
ESI MS: m/z ¼ 474.3 [M þ Na]þ (100%).
3.3.14. 1-Isopropyl-7-methyl-4-{2-[(Z)-4-methylphenylimino]-1,3-
thiazepan-3-yl}-4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione (7b0)
Yield 85%. Mp. 142.9 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.91 (d,
3H, CH3, J ¼ 6.8 Hz); 1.04 (d, 3H, CH3, J ¼ 6.8 Hz); 1.18–1.33 (m,
2H, CH2); 1.4–1.49 (m, 3H, CH2, CH); 1.44 (s, 3H, CH3); 1.64–1.69 (m,
2H, CH2); 1.9–1.95 (m, 2H, CH2); 2.22 (s, 3H, CH3), 2.48–2.57 (m, 2H,
CH2); 2.92 (d, 1H, CH–C]O, J ¼ 6 Hz); 2.94 (d, 1H, CH–C]O,
J ¼ 6 Hz); 3.71–3.76 (m, 2H, CH2); 5.91 (dd, 2H, CH], J1 ¼6.3 Hz,
J2 ¼ 6.6 Hz); 6.49–6.55 (d, 2H, CHarom.), 6.98–7.05 (m, 2H, CHarom.).
3.3.10. 1-Isopropyl-7-methyl-4-{2-[(Z)-4-fluorophenylimino]-1,3-
thiazepan-3-yl}-4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione (3b0)
Yield 62%. Mp. 138 ꢁC. 1H NMR (CDCl3)
d (ppm): 0.93 (d, 3H, CH3,
J ¼ 6.8 Hz); 1.06 (d, 3H, CH3, J ¼ 6.8 Hz); 1.18–1.3 (m, 4H, CH2); 1.36–
1.43 (m, 3H, CH2, CH); 1.44 (s, 3H, CH3); 1.64–1.69 (m, 2H, CH2);
1.89–1.96 (m, 2H, CH2); 2.46–2.56 (m, 2H, CH2); 2.93 (d, 1H, CH–
C]O, J ¼ 8.4 Hz); 2.96 (d, 1H, CH–C]O, J ¼ 8.4 Hz); 3.71–3.76 (m,
2H, CH2); 5.96 (dd, 2H, CH], J1 ¼ J2 ¼ 8.8 Hz); 6.52–6.61 (m, 2H,
13C NMR (CDCl3)
d: 17.05, 18.62, 21.10, 22.88, 23.27, 27.79, 28.33,
29.82, 29.95, 33.17, 34.57, 37.15, 43.99, 45.56, 49.28, 54.15, 122.05,
139.25 (2C), 132.42, 135.91, 136.70, 147.26, 152.74, 173.66, 174.10.
Anal. Calcd for C26H33N3O2S: C 69.15, H 7.36, N 9.3. Found: C 69.23,
H 7.39, N 9.32. ESI MS: m/z ¼ 552.18 [M þ H]þ (100%).

4968
M. Struga et al. / European Journal of Medicinal Chemistry 44 (2009) 4960–4969
3.4. Pharmacology
plates at an initial density of 6 ꢂ 105, 1 ꢂ106 and 5 ꢂ105 cells mLꢀ1
,
respectively, in maintenance medium, with or without serial
dilutions of test compounds. Cell viability was determined after 48–
120 h at 37 ꢁC in a humidified CO2 (5%) atmosphere by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)
method [58]. The cell number of Vero 76 monolayers was deter-
mined by staining with the crystal violet dye.
The experiments were performed on male Albino Swiss mice
(18–30 g). The animals were kept 8–10 to a cage, at room temper-
ature of 20 ꢄ 1 ꢁC, on a 12:12 h dark–light cycle. Standard food
(LSM, Motycz, Poland) and water were available ad libitum. The
investigated compounds 6b0 and 7a0, were administered intra-
peritoneally (ip.) at a volume of 10 mL kgꢀ1, as suspensions in
aqueous solution of 0.5% methylcellulose (tylose). The compounds
were injected 60 min before the test. The controls received the
equivalent volume of the solvent.
All tests performed, suggested by Vogel and Vogel [53] are
generally accepted as basic in investigation of the central activity by
behavioral methods.
The acute toxicity of the compound was assessed in mice acc. to
Litchfield and Wilcoxon method [54], as the LD50 calculated on
mortality within 48 h. The compound was injected in doses
equivalent to 0.1 LD50 (200 mg kgꢀ1). In addition, the activity of
compounds was assessed in the following test:
For cytotoxicity evaluations, exponentially growing cells derived
from human haematological tumors [CD4þ human T-cells con-
taining an integrated HTLV-1 genome (MT-4)] were seeded at an
initial density of 1 ꢂ105 cells mLꢀ1 in 96 well plates in RPMI-1640
medium, supplemented with 10% fetal calf serum (FCS), 100 uni-
ts mLꢀ1 penicillin G and 100 g mLꢀ1 streptomycin. Cell cultures
m
were then incubated at 37 ꢁC in a humidified, 5% CO2 atmosphere in
the absence or presence of serial dilutions of test compounds. Cell
viability was determined after 96 h at 37 ꢁC by the MTT method.
3.5.4. Antiviral assay
Activity of compounds against Human Immunodeficiency Virus
type-1 (HIV-1) was based on inhibition of virus-induced cyto-
pathogenicity in MT-4 cells acutely infected with a multiplicity of
- locomotor activity was measured in photoresistor actometers
for single mice for 30 min as
a) spontaneous activity
infection (m.o.i.) of 0.01. Briefly, 50
MT-4 were added to each well of flat-bottom microtitre trays
m
L of RPMI containing 1 ꢂ104
b) amphetamine-induced hyperactivity: mice received subcuta-
neously (s.c.) 5 mg kgꢀ1 of amphetamine 30 min before the
test;
- nociceptive reactions were studied in the acetic acid (0.6%)
induced writhing test [55]. The number of writhing episodes
was measured for 10 min starting 5 min after i.p. administra-
tion of acid solution;
containing 50
compounds. Then, 20
CCID50 were added. After a 4-day incubation, cell viability was
determined by the MTT method.
Activity of compounds against Yellow Fever Virus (YFV) and Reo
Virus type-1 (Reo-1) was based on inhibition 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 cytopathogenicity.
BHK and MDBK cells were seeded in 96-well plates at a density
of 5 ꢂ104 and 3 ꢂ 104 cells per well, respectively, and were allowed
to form confluent monolayers by incubating overnight in growth
medium at 37 ꢁC in a humidified CO2 (5%) atmosphere. Cell
m
L of RPMI, with or without serial dilutions of test
m
L of an HIV-1 suspension containing 100
- motor coordination was evaluated in rota rod test [56] and
chimney test [57];
- body temperature in normothermic mice was measured in the
rectum by thermistor thermometer;
- pentylenetetrazole (110 mg kgꢀ1, s.c.)-induced convulsions were
evaluated as the number of mice with clonic seizures, tonic
convulsions and dead animals;
monolayers were then infected with 50 mL of a proper virus dilution
- ‘‘head twitch’’ responses after 5-hydroxytryptophan (5-HTP),
acc. to Corne et al. [18]. Mice received 5-HTP (180 mg kgꢀ1, i.p.)
and the number of head twitches was recorded in 6 two-minute
intervals (4–6, 14–16, 24–26, 34–36, 44–46, 54–56 min).
(in serum-free medium) to give an m.o.i. ¼ 0.01. One hr later, 50
mL
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 incubation at 37 ꢁC,
cell viability was determined by the MTT method.
Statistics. Obtained data were calculated by c2 test with Yates
correction (pentylenetetrazole-induced seizures), Dunnett’s test,
followed by one-way ANOVA (body temperature) and Students t-
test (other tests).
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), Herpes Virus 1 (HSV-1)
and against Respiratory Syncytial Virus (RSV), A-2 strain, 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 ꢂ 105 cells per 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
3.5. Cytotoxicity and antiviral activity assays
3.5.1. Compounds
Compounds were dissolved in DMSO at 100 mM and then
diluted in culture medium.
infected with 250
mL of proper virus dilutions to give 50–100 PFU/
well. Following removal of unadsorbed virus, 500
mL of Dulbecco’s
3.5.2. Cells and viruses
modified Eagle’s medium, supplemented with 1% inactivated FCS
and 0.75% methyl cellulose, with or without serial dilutions of test
compounds, were added. Cultures were incubated at 37 ꢁC for 2 (Sb-
1 and VSV), 3 (CVB-2, VV and HSV-1) or 5 days (RSV) and then fixed
with PBS containing 50% ethanol and 0.8% crystal violet, washed and
air-dried. Plaques were then counted. 50% effective concentrations
(EC50) were calculated by linear regression technique.
Cell lines were purchased from American Type Culture Collec-
tion (ATCC). The absence of mycoplasma contamination was
checked periodically by the Hoechst staining method. Cell lines
supporting the multiplication of RNA viruses were the following:
CD4þ human T-cells containing an integrated HTLV-1 genome (MT-
4), Madin Darby Bovine Kidney (MDBK), Baby Hamster Kidney
(BHK-21) and Monkey kidney (Vero 76) cells.
AZT (30-azido-thymidine), NM 108 (20-
b-methyl-guanosine),
NM 176 (20-ethynyl-D-citidine), NM 299 (6-azauridine), M 5255
(mycophenolic acid) and ACG (acycloGuanosine) were used as
reference inhibitors of ssRNAþ, ssRNAꢀ and DNA viruses,
respectively.
3.5.3. Cytotoxicity assays
For cytotoxicity tests, run in parallel with antiviral assays,
MDBK, BHK and Vero 76 cells were resuspended in 96 multiwell

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