Synthesis and pharmacological activity of urea and thiourea derivatives of 4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione

Article abstract of DOI:10.1248/cpb.55.796

A series of nineteen new thiourea and urea derivatives of 10-isopropyl-8-methyl-4-azatricyclo[5.2.2.0^2,6]-undec-8-ene-3,5- dione, 1-isopropyl-7-methyl-4-azatricyclo[5.2.2.0^2,6]undec-8-ene-3,5- dione and 1,7,8,9,10-pentamethyl-4-azatricyclo[5.2.1.0^2,6]dec-8-ene- 3,5-dione have been prepared and studied by ¹H-NMR. The compound k1a (1-(1,7,8,9,10-pentamethyl-3,5-dioxo-4-aza-tricyclo[5.2.1.0^2,6]dec-8- en-4-yl)-3-phenyl-urea) was tested for pharmacological activity on animal central nervous system (CNS). The activities of synthesized compounds were evaluated for their cytotoxicity and anti-HIV-1 activity in MT-4 cells. Antimicrobial activity of the newly obtained derivatives was tested against some Gram-positive and Gram-negative bacteria and fungi of the Candida species.

Full text of DOI:10.1248/cpb.55.796

796
Notes
Chem. Pharm. Bull. 55(5) 796—799 (2007)
Vol. 55, No. 5
Synthesis and Pharmacological Activity of Urea and Thiourea Derivatives
of 4-Azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione
c
Marta STRUGA, ,a Jerzy KOSSAKOWSKI,a Ewa KE˛DZIERSKA,b Sylwia FIDECKA,b and Joanna STEFAN´SKA
*
a Department of Medical Chemistry, The Medical University; 3 Oczki Str., 02–007 Warsaw, Poland: b Department of
Pharmacology and Pharmacodynamics, Medical University School; Staszica 4, 20–081 Lublin, Poland: and c Department
of Pharmaceutical Microbiology, Medical University; 3 Oczki Str., 02–007 Warsaw, Poland.
Received October 12, 2006; accepted February 3, 2007
A series of nineteen new thiourea and urea derivatives of 10-isopropyl-8-methyl-4-azatricyclo[5.2.2.02,6]-
undec-8-ene-3,5-dione, 1-isopropyl-7-methyl-4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione and 1,7,8,9,10-penta-
1
methyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione have been prepared and studied by H-NMR. The compound
k1a (1-(1,7,8,9,10-pentamethyl-3,5-dioxo-4-aza-tricyclo[5.2.1.02,6]dec-8-en-4-yl)-3-phenyl-urea) was tested for
pharmacological activity on animal central nervous system (CNS). The activities of synthesized compounds were
evaluated for their cytotoxicity and anti-HIV-1 activity in MT-4 cells. Antimicrobial activity of the newly ob-
tained derivatives was tested against some Gram-positive and Gram-negative bacteria and fungi of the Candida
species.
Key words urea derivative; thiourea derivative; pharmacological activity; cytotoxicity; antimicrobial activity
Thiourea and urea derivatives show a broad spectrum of lower than 2000 mg/kg i.p. and therefore LD50ꢁ2000 mg/kg
biological activities as anti-HIV, antiviral, HDL-elevating, was accepted for the continuation of the studies.
antibacterial, analgesic properties.1—4)
Spontaneous motor activity, amphetamine-induced hyper-
Numerous compounds containing thiourea group are se- activity, motor coordination and body temperature, were not
lective ligands for 5-HT family receptors, including 5-HT2A, changed by k1a. This compound did not protect from clonic
5-HT2B and 5-HT2C.5—9) The drug-elicited head twitch re- seizures and tonic convulsions evoked by pentetrazole and
sponse (HTR)10,11) is a selective behavioral model for 5-HT2 from abdominal constriction induced by i.p. administration
agonist activity in rodents, and several previous studies have of the acetic acid solution.
established that direct and indirect 5-HT agonists induce this
Only the head-twitch responses to 5-HTP were signifi-
effect.12—19) Additionally, 5-HT2 receptor antagonists selec- cantly decreased by 77.88% (pꢂ0.05), from 14.13ꢃ4.24 to
tively block HTR,19—21) and their potency is highly correlated 3.125ꢃ1.007, by k1a (Fig. 1). The result seems to point out
with the antagonist’s affinity for 5-HT2 receptors.12,22)
some connection with 5-HT the system. Since it appears that
Here we report the synthesis of compounds h2, h2a, d2, head shakes induced by 5-HTP are mediated by 5-HT2 recep-
d2a, k2, k2a—h4, h4a, d4, d4a, k4, k4a which composed of
thiourea or urea system attached to policyclic imide.
Chemistry The preparation of new nineteen thiourea
and urea derivatives of 10-isopropyl-8-methyl-4-azatricy-
clo[5.2.2.02,6]undec-8-ene-3,5-dione, 1-isopropyl-7-methyl-4-
azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione and 1,7,8,9,10-
pentamethyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione
(Chart 1) is described.
Imides obtained in Diels–Alder reaction were used
as starting material. 10-Isopropyl-8-methyl-4-azatricyclo-
[5.2.2.02,6]undec-8-ene-3,5-dione was obtained in reaction
of enantiomeric (R)-(ꢀ)-a-phellandrene with pyrrole-2,5-
Chart 1. Synthesis of the Obtained Compounds h2, h2a, d2, d2a,
k2, k2a—h4, h4a, d4, d4a, k4, k4a
Table 1. Structure of the Investigated Compounds h2, h2a, d2, d2a, k2,
dione,23) 1-isopropyl-7-methyl-4-azatricyclo[5.2.2.02,6]undec- k2a—h4, h4a, d4, d4a, k4, k4a
8-ene-3,5-dione in reaction of a-terpinene with pyrrole-2,5-
dione23) and 1,7,8,9,10-pentamethyl-4-azatricyclo[5.2.1.02,6]-
dec-8-ene-3,5-dione in reaction 1,2,3,4,5-pentamethylcyclo-
pentadiene with pyrrole-2,5-dione.24)
R
The obtained tricyclic imides reacted with hydrazine (80%
aqueous solution). Afterwards the compounds were subjected
to the reaction with phenyl, 4-methoxyphenyl, cyclohexyl
isocyanate or isothiocyanate in order to be transformed into
the corresponding urea or thiourea derivatives.
R1
h1,a) h1ab)
h2, h2a
d1, h1a
d2, d2a
d3, d3a
k1, k1a
k2, k2a
k3, k3a
All final compounds were characterized by 1H-NMR spec-
tra which corresponded with the proposed structures.
The general synthetic pathway is given in Chart 1.
h3, h3a
Pharmacology Acute toxicity of tested compound was
a) XꢁS; b) a for XꢁO.
∗ To whom correspondence should be addressed. e-mail: strugam@wp.pl
© 2007 Pharmaceutical Society of Japan

May 2007
797
1
Bruker DMX400 spectrometer, operating at 400.13 MHz for H or with a
1
Varian UNITYplus-200 spectrometer, operating at 199.97 MHz for H. The
chemical shift values, expressed in ppm, were referenced downfield to TMS
at ambient temperature. Microanalysis was performed at the Microanalysis
Laboratory of Warsaw Technical University and all values were within
ꢃ0.4% of the calculated compositions. Flash chromatography was per-
formed 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).
(1R,2S,6R,7R,10R) 4-Amino-10-isopropyl-8-methyl-4-aza-tricyclo-
[5.2.2.02,6]undec-8-ene-3,5-dione (h) This compound has been synthe-
sized as described previously.28)
(1S,2R,6S,7R) 4-Amino-1,7,8,9,10-pentamethyl-4-azatricyclo-
[5.2.1.02,6]dec-8-ene-3,5-dione (k) and (1S,2R,6S,7S) 4-Amino-1-iso-
propyl-7-methyl-4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione (d).
General Procedure A mixture of imide (0.01 mol) and hydrazine (80%
water solution) (50 ml) was refluxed for 5 h. After solvents were evaporated,
the residue was purified by a column chromatography (silica gel) to give
compound (d or k). The product was recrystallized from heptane.
(1S,2R,6S,7S) 4-Amino-1-isopropyl-7-methyl-4-azatricyclo-
Fig. 1. The Influence of k1a on the Head Twitch Responses Evoked by 5-
Hydroxytryptophan (5-HTP) (180 mg/kg i.p.)
The results are expressed as meanꢃS.E.M. (nꢁ8).
Table 2. Cytotoxicity and Anti-HIV Activity of Compounds (h, h2, h2a,
d, d2, d2a, k, k2, k2a—h4, h4a, d4, d4a, k4, k4a)
1
[5.2.2.02,6]undec-8-ene-3,5-dione (d) Yield 2.18 g (88%). mp 147 °C. H-
NMR (200 MHz, CDCl3) d: 0.98 (d, Jꢁ7.2 Hz, 3H, CH3), 1.11 (d, Jꢁ6.8 Hz,
3H, CH3), 1.23—1.36 (m, 2H, CH2), 1.4—1.46 (m, 2H, CH2), 1.48 (s, 3H,
CH3), 2.53—2.62 (m, 2H, CH), 2.57 (d, Jꢁ8 Hz, 1H, CH), 4.24 (s, 2H,
NH2), 5.88 (d, Jꢁ8.4 Hz, 1H, CHꢁ), 5.96 (d, Jꢁ8.4 Hz, 1H, CHꢁ).
C14H20O2N2 (248.32): Calcd C 67.71, H 8.12, N 11.28; Found C 67.64, H
8.08, N 11.36.
(1S,2R,6S,7R) 4-Amino-1,7,8,9,10-pentamethyl-4-azatricyclo-
[5.2.1.02,6]dec-8-ene-3,5-dione (k) Yield 2.23 g (90%). mp 171—172 °C.
1H-NMR (200 MHz, CDCl3) d: 0.61 (d, Jꢁ6.4 Hz, 3H, CH3); 1.34 (s, 6H,
CH3); 1.48 (s, 6H, CH3); 1.54 (q, 1H, CH); 2.87 (s, 2H, CH); 3.49 (s, 1H,
NH2). C14H20O2N2 (248.32): Calcd C 67.71, H 8.12, N 11.18; Found: C
67.58, H 8.03, N 11.31.
Urea or Tiourea Derivatives of (1R,2S,6R,7R,10R) 10-Isopropyl-
8-methyl-4-azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione (h1, h1a—h3,
h3a), (1S,2R,6S,7S) 1-Isopropyl-7-methyl-4-azatricyclo[5.2.2.02,6]undec-
8-ene-3,5-dione (d1, d1a—d3, d3a) and (1S,2R,6S,7R) 1,7,8,9,10-Pen-
tamethyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (k1, k1a—k3, k3a).
General Procedure A solution of 4-aminoimide (h) or (d) or (k)
(0.01 mol) in acetonitrile (6 cm3) was treated with phenyl, 4-metoxyphenyl,
cyclohexylisocyanate or isothiocyanate (0.011 mol) and the mixture was re-
fluxed for 6 h. The precipitate was filtered and then washed with ether to
give compounds (h1, h1a—h3, h3a), (d1, d1a—d3, d3a), (k1, k1a—k3,
k3a). The product was recrystallized from ethanol.
a)
b)
CC50
EC50
Compound
MT-4
HIV-1
h
h1a
h1
h2a
h2
h3a
h3
d
d1a
d1
d2a
d2
d3a
d3
ꢄ100
ꢄ100
48
ꢄ100
62
ꢄ48
ꢄ46
ꢄ52
ꢄ63
ꢄ47
ꢄ100
ꢄ100
ꢄ60
ꢄ57
ꢄ76
ꢄ72
ꢄ53
ꢄ100
ꢄ42
ꢄ55
ꢄ12
ꢄ48
46
52
63
47
ꢄ100
ꢄ100
60
57
76
72
53
k
k1a
k2
k3a
k3
ꢄ100
ꢄ42
55
12
48
(1R,2S,6R,7R,10R) 1-(10-Isopropyl-8-methyl-3,5-dioxo-4-aza-tricyclo-
[5.2.2.02,6]undec-8-en-4-yl)-3-phenyl-thio-urea (h1) Yield 2.93 g (67%).
EFV
35
0.003
1
mp 188 °C. H-NMR (400 MHz, CDCl3) d: 0.803 (d, Jꢁ6.2 Hz, 3H, CH3),
0.9 (d, Jꢁ6.4 Hz, 3H, CH3), 0.94—1.45 (m, 2H, CH), 1.34—1.41 (m, 1H,
CH), 1.66 (s, 3H, CH3), 1.78—1.88 (m, 1H, CH–CH3), 2.75 (m, 1H, CH),
2.91—2.98 (m, 3H, CH2, CH), 5.61 (d, Jꢁ4.4 Hz, 1H, CH), 7.14—7.36 (m,
5H, Carom.), 9.88 (s, 1H, NH), 10.22 (s, 1H, NH). C21H25O3N3S (387.53):
Calcd C 65.08, H 7.54, N 10.84; Found C 65.0, H 7.65, N 10.76.
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 cy-
topathogeneticy, as determined by the MTT method.
(1R,2S,6R,7R,10R) 1-(10-Isopropyl-8-methyl-3,5-dioxo-4-aza-tricyclo-
[5.2.2.02,6]undec-8-en-4-yl)-3-phenyl-urea (h1a) Yield 2.89 g (78%). mp
143 °C. H-NMR (400 MHz, CDCl3) d: 0.81 (d, Jꢁ6.4 Hz, 3H, CH3), 0.89
(d, Jꢁ6.2 Hz, 3H, CH3), 1.1—1.12 (m, 2H, CH), 1.31—1.33 (m, 1H, CH),
1.71 (s, 3H, CH3), 1.76—1.82 (m, 1H, CH), 2.76—2.84 (m, 2H, CH2), 2.92
(s, 1H, CH), 3.17 (s, 1H, CH), 4.92 (s, 1H, NH), 5.63 (d, Jꢁ5.4 Hz, 1H,
CH), 7.14—7.37 (m, 5H, Carom.), 8.66 (s, 1H, NH). C21H25O3N3 (367.44):
Calcd C 68.64, H 6.86, N 11.44; Found C 68.68, H 7.65, N 11.53.
tors, these data suggest that k1a significantly interact with 5-
HT2 receptors in the brain.
Antiviral Activities The synthesized compounds were
evaluated for their cytotoxicity and anti-HIV-1 activity in
MT-4 cells. None of the synthesized compounds showed ac-
tivity against HIV-1.
Microbiology All newly synthesized compounds were
tested for their antibacterial and antifungal activities. Gram-
negative and Gram-positive bacterial strains and Candida al-
bicans used in this study have common application in the an-
timicrobial activity tests for many substances like antibiotics,
disinfectants and antiseptic drugs or in research on new an-
timicrobial agents.25—27) All synthesized compounds were
completely inactive against tested microorganisms.
1
(1R,2S,6R,7R,10R) 1-(10-Isopropyl-8-methyl-3,5-dioxo-4-aza-tricyclo-
[5.2.2.02,6]undec-8-en-4-yl)-3-(4-methoxy-phenyl)-thiourea (h2) Yield
1
3.34 g (81%). mp 186 °C. H-NMR (400 MHz, CDCl3) d: 0.81 (d, Jꢁ4 Hz,
3H, CH3), 0.90 (d, Jꢁ4 Hz, 3H, CH3), 1.07—1.08 (m, 2H, CH), 1.34—1.45
(m, 1H, CH), 1.71 (s, 3H, CH3), 1.78—1.83 (m, 1H, CH), 2.85—2.95 (m,
3H, CH, CH2), 3.19 (s, 1H, CH), 3.78 (s, 3H, O-CH3), 5.67 (s, 1H, CH),
6.87—6.89 (m, 2H, Carom.), 7.28—7.3 (m, 2H, Carom.), 7.94 (s, 1H, NH), 8.32
(s, 1H, NH). C22H27O3N3S (413.53): Calcd C 63.90, H 6.58, N 10.16; Found
C 63.82, H 6.30, N 10.23.
(1R,2S,6R,7R,10R) 1-(10-Isopropyl-8-methyl-3,5-dioxo-4-aza-tricyclo-
[5.2.2.02,6]undec-8-en-4-yl)-3-(4-methoxy-phenyl)-urea (h2a) Yield
1
3.21 g (81%). mp 108 °C. H-NMR (400 MHz, CDCl3) d: 0.8 (d, Jꢁ4.4 Hz,
Experimental
3H, CH3), 0.91 (d, Jꢁ8 Hz, 3H, CH3), 1.04—1.05 (m, 2H, CH), 1.34—1.45
Chemistry Melting points were determined in a capillary on Kofler’s
apparatus and are uncorrected. The 1H-NMR spectra were recorded with
(m, 1H, CH), 1.72 (s, 3H, CH3), 1.79—1.84 (m, 1H, CH), 2.77—2.95 (m,

798
Vol. 55, No. 5
[5.2.1.02,6]dec-8-en-4-yl)-3-phenyl-thiourea (k1) Yield 3.18 g (83%). mp
191 °C. H-NMR (400 MHz, CDCl3) d: 0.62 (d, Jꢁ8 Hz, 3H, CH3), 1.35 (s,
3H, CH, CH2), 3.2 (s, 1H, CH), 3.79 (s, 3H, O-CH3), 5.7 (d, Jꢁ5.2 Hz, 1H,
CH), 6.81—6.88 (m, 3H, NH, Carom.), 7.22—7.24 (m, 2H, Carom.).
C22H27O4N3 (397.46): Calcd C 66.48, H 6.85, N 10.57; Found C 66.40, H
6.86, N 10.44.
1
6H, CH3), 1.48 (s, 6H, CH3), 1.61 (q, 1H, CH), 3.0 (s, 2H, CH), 7.27—7.38
(m, 5H, CHarom.), 7.71 (s, 1H, NH), 8.10 (s, 1H, NH). C21H25O2N3S
(383.50): Calcd C 65.77, H 6.57, N 10.96; Found C 65.74, H 6.5, N 10.84.
(1S,2R,6S,7R) 1-(1,7,8,9,10-Pentamethyl-3,5-dioxo-4-aza-tricyclo-
[5.2.1.02,6]dec-8-en-4-yl)-3-phenyl-urea (k1a) Yield 3.9 g (82%). mp
(1R,2S,6R,7R,10R) 1-Cyclohexyl-3-(10-isopropyl-8-methyl-3,5-dioxo-
4-aza-tricyclo[5.2.2.02,6]undec-8-en-4-yl)-thiourea (h3) Yield 2.8 g
(72%). mp 201 °C. 1H-NMR (400 MHz, CDCl3) d: 0.81 (d, Jꢁ6 Hz, 3H,
CH3), 0.92 (d, Jꢁ7.2 Hz, 3H, CH3), 1.07—1.15 (m, 6H, CH, CHcyclohexyl),
1.35—1.38 (m, 1H, CH), 1.62—1.86 (m, 4H, Ccyclohexyl), 1.78 (s, 3H, CH3),
1.98—12.05 (m, 1H, CH), 2.9—3.01 (m, 3H, CH, CH2), 3.2 (s, 1H, CH),
4.12 (s, 1H, NH), 5.72 (d, Jꢁ4 Hz, 1H, CH), 6.22 (s, 1H, NH). C21H31O2N3S
(389.55): Calcd C 64.75, H 7.02, N 10.79; Found C 65.02, H 7.07, N 10.95.
(1R,2S,6R,7R,10R) 1-Cyclohexyl-3-(10-isopropyl-8-methyl-3,5-dioxo-
4-aza-tricyclo[5.2.2.02,6]undec-8-en-4-yl)-urea (h3a) Yield 2.46 g (66%).
1
240 °C. H-NMR (400 MHz, CDCl3) d: 0.62 (d, Jꢁ6.4 Hz, 3H, CH3), 1.35
(s, 6H, CH3), 1.55 (s, 6H, CH3), 1.57—1.65 (m, 1H, CH), 3.07 (s, 2H, CH),
7.51—7.84 (m, 5H, CHarom.), 9.25 (s, 1H, NH), 11.82 (s, 1H, NH).
C21H25O3N3 (367.44): Calcd C 68.64, H 6.86, N 11.44; Found C 68.9, H
6.75, N 11.46.
(1S,2R,6S,7R) 1-(4-Methoxy-phenyl)-3-(1,7,8,9,10-pentamethyl-3,5-
dioxo-4-aza-tricyclo [5.2.1.02,6]dec-8-en-4-yl)-thiourea (k2) Yield 2.84 g
1
1
(69%). mp 127 °C. H-NMR (400 MHz, CDCl3) d: 0.61 (d, Jꢁ12 Hz, 3H,
mp 107 °C. H-NMR (400 MHz, CDCl3) d: 0.83 (d, Jꢁ6.8 Hz, 3H, CH3),
CH3), 1.34 (s, 6H, CH3), 1.53 (s, 6H, CH3), 1.6 (q, 1H, CH), 3.03 (s, 2H,
CH), 3.79 (s, 3H, O-CH3), 6.89 (d, Jꢁ12 Hz, 2H, CHarom.), 7.27 (d, Jꢁ12 Hz,
2H, CHarom.), 7.9 (s, 1H, NH), 8.12 (s, 1H, NH). C22H27O3N3S (413.53):
Calcd C 62.9, H 6.58, N 10.16; Found C 62.78, H 6.29, N 10.07.
(1S,2R,6S,7R) 1-(4-Methoxy-phenyl)-3-(1,7,8,9,10-pentamethyl-3,5-
dioxo-4-aza-tricyclo[5.2.1.02,6]dec-8-en-4-yl)-urea (k2a) Yield 3.13 g
(79%). mp 205 °C. 1H-NMR (400 MHz, CDCl3) d: 0.62 (d, Jꢁ8 Hz, 3H,
CH3), 1.33 (s, 6H, CH3), 1.54 (s, 6H, CH3), 1.65 (s, 1H, CH), 2.95 (s, 2H,
CH), 3.74 (s, 3H, O-CH3), 4.81 (s, 1H, NH), 6.75 (d, 2H, CHarom.), 7.17 (s,
1H, NH), 7.17—7.23 (m, 2H, CHarom.). C22H27O4N3 (397.46): Calcd C
66.48, H 6.85, N 10.57; Found C 66.52, H 6.47, N 10.47.
(1S,2R,6S,7R) 1-Cyclohexyl-3-(1,7,8,9,10-pentamethyl-3,5-dioxo-4-
aza-tricyclo[5.2.1.02,6]dec-8-en-4-yl)-thiourea (k3) Yield 3.07 g (72%).
mp 127 °C. 1H-NMR (400 MHz, CDCl3) d: 0.61 (d, Jꢁ8 Hz, 3H, CH3),
1.15—1.27 (m, 6H, CHcyclohexyl), 1.37 (s, 6H, CH3), 1.53 (s, 6H, CH3),
1.58 (q, 1H, CH), 1.70—1.73 (m, 2H, CHcyclohexyl), 2.03—12.06 (m, 2H,
CHcyclohexyl), 3.02 (s, 2H, CH), 4.13 (s, 1H, NH), 6.13 (s, 1H, NH).
C21H31O2N3S (389.55): Calcd C 64.75, H 8.02, N 10.79; Found C 64.96;
H 8.02, N 10.72.
0.93 (d, Jꢁ8 Hz, 3H, CH3), 1.08—1.14 (m, 6H, CH, CHcyclohexyl), 1.26—1.34
(m, 5H, CH, CHcyclohexyl), 1.84 (s, 3H, CH3), 1.86—1.92 (m, 1H, CH), 2.95
(m, 1H, CH), 3.02 (s, 2H, CH2), 3.23 (s, 1H, CH), 4.93 (s, 1H, NH), 5.77 (d,
Jꢁ4 Hz, 1H, CH), 8.66 (s, 1H, NH). C21H31O3N3 (373.48): Calcd C 67.53, H
8.37, N 11.25; Found C 67.94, H 8.39, N 10.88.
(1S,2R,6S,7S) 1-(1-Isopropyl-7-methyl-3,5-dioxo-4-aza-tricyclo-
[5.2.2.02,6]undec-8-en-4-yl)-3-phenyl-thiourea (d1) Yield 2.87 g (75%).
1
mp 108 °C. H-NMR (400 MHz, CDCl3) d: 0.98 (d, Jꢁ6.8 Hz, 3H, CH3),
1.12 (d, Jꢁ6.4 Hz, 3H, CH3), 1.26—1.37 (m, 2H, CH2), 1.49 (s, 3H, CH3),
1.51—1.57 (m, 2H, CH2), 2.53—2.58 (m, 1H, CH), 2.76 (d, Jꢁ8 Hz, 1H,
CH), 3.12 (d, Jꢁ8.4 Hz, 1H, CH), 5.95 (d, Jꢁ8.4 Hz, 1H, CHꢁ), 6.03 (d,
Jꢁ8.4 Hz, 1H, CHꢁ), 7.28—7.43 (m, 5H, CHarom.), 7.53 (s, 1H, NH), 8.17
(s, 1H, NH). C21H25O2N3S (383.50): Calcd C 65.77, H 6.57, N 10.96; Found
C 65.93, H 6.55, N 10.97.
(1S,2R,6S,7S) 1-(1-Isopropyl-7-methyl-3,5-dioxo-4-aza-tricyclo-
[5.2.2.02,6]undec-8-en-4-yl)-3-phenyl-urea (d1a) Yield 2.71 g (74%). mp
1
171 °C. H-NMR (400 MHz, CDCl3) d: 1.03 (d, Jꢁ6.8 Hz. 3H, CH3), 1.14
(d, Jꢁ6.8 Hz, 3H, CH3), 1.36—1.46 (m, 2H, CH2), 1.54 (s, 3H, CH3),
1.56—1.69 (m, 2H, CH2), 2.52—2.59 (m, 1H, CH), 2.89 (d, Jꢁ8 Hz, 1H,
CH), 3.28 (d, Jꢁ8,4 Hz, 1H, CH), 6.21 (d, Jꢁ8.4 Hz, 1H, CHꢁ), 6.29 (d,
Jꢁ8.4 Hz, 1H, CHꢁ), 7.05—7.47 (m, 7H, CHarom., NH). C21H25O3N3
(367.44): Calcd C 68.64, H 6.86, N 11.44; Found C 68.50, H 6.71, N 11.37.
(1S,2R,6S,7S) 1-(1-Isopropyl-7-methyl-3,5-dioxo-4-aza-tricyclo-
[5.2.2.02,6]undec-8-en-4-yl)-3-(4-methoxy-phenyl)-thiourea (d2) Yield
2.56 g (62%). mp 126 °C. 1H-NMR (400 MHz, CDCl3) d: 0.98 (d, Jꢁ3.6 Hz,
3H, CH3), 1.11 (d, Jꢁ3.6 Hz, 3H, CH3), 1.27—1.40 (m, 2H, CH2), 1.49 (s,
3H, CH3), 1.58—1.53 (m, 2H, CH2), 2.55—2.57 (m, 1H, CH), 2.75 (d,
Jꢁ6 Hz, 1H, CH), 3.11 (d, Jꢁ6 Hz, 1H, CH), 3.81 (s, 3H, O-CH3), 5.93 (d,
1H, CHꢁ), 5.99 (d, 1H, CHꢁ), 6.91—6.93 (m, 2H, CHarom.), 7.30—7.36 (m,
3H, CHarom., NH), 8.0 (s, 1H, NH). C22H27O3N3S (413.53): Calcd C 63.9, H
6.58, N 10.16; Found C 64.26, H 6.64, N 10.14.
(1S,2R,6S,7R) 1-Cyclohexyl-3-(1,7,8,9,10-pentamethyl-3,5-dioxo-4-
aza-tricyclo[5.2.1.02,6]dec-8-en-4-yl)-urea (k3a) Yield 2.72 g (73%). mp
1
256 °C. H-NMR (400 MHz, CDCl3) d: 0.62 (d, Jꢁ8 Hz, 3H, CH3), 1.09—
1.14 (m, 6H, CHcyclohexyl), 1.33 (s, 6H, CH3), 1.51 (s, 6H, CH3), 1.60 (q, 1H,
CH), 1.67—1.7 (m, 2H, CHcyclohexyl), 1.90—1.93 (m, 2H, CHcyclohexyl), 2.96
(s, 2H, CH), 4.77 (s, 1H, NH), 6.38 (s, 1H, NH). C21H31O3N3 (373.48):
Calcd C 67.53, H 8.37, N 11.25; Found C 67.62, H 8.17, N 11.23.
Pharmacology The experiments were performed on male Albino Swiss
mice (18—30 g). 8—10 animals were kept in a cage, at room temp. of
20ꢃ1 °C, on a 12 : 12 h dark–light cycle. Standard food (Bacutil, Motycz)
and water were available ad libitum. The investigated substance, marked as
k1a, was administered i.p. in the volume of 10 ml/kgꢀ1 as suspensions in
aqueosus solution of 0.5% methylcellulose (tylose). The compounds were
injected 60 min before the test. Controls received the equivalent volume of
the solvent.
(1S,2R,6S,7S) 1-(1-Isopropyl-7-methyl-3,5-dioxo-4-aza-tricyclo-
[5.2.2.02,6]undec-8-en-4-yl)-3-(4-methoxy-phenyl)-urea (d2a) Yield
1
3.13 g. mp 145 °C. H-NMR (400 MHz, CDCl3) d: 1.02 (d, Jꢁ6.8 Hz, 3H,
All performed tests, suggested by Vogel and Vogel29) are generally ac-
cepted as basic in investigation of the central activity by behavioral methods.
The acute toxicity of the compound was assessed in mice according to
Litchfield and Wilcoxon method,30) as the LD50 calculated from mortality
within 48 h. In addition, the activity of compounds was assessed in the fol-
lowing test:
• Locomotor activity was measured in photoresistor actometers for single
mice for 30 min as a) spontaneous activity and b) amphetamine-induced hy-
peractivity: mice received subcutaneusly (s.c.) 5 mg/kg of amphetamine
30 min before the test.
CH3), 1.14 (d, Jꢁ6.8 Hz, 3H, CH3), 1.34—1.45 (m, 2H, CH2), 1.53 (s, 3H,
CH3), 1.59—1.65 (m, 2H, CH2), 2.52—2.57 (m, 1H, CH), 2.87 (d,
Jꢁ8.4 Hz, 1H, CH), 3.27 (d, Jꢁ8.8 Hz, 1H, CH), 3.81 (s, 3H, O-CH3), 5.95
(d, Jꢁ8.4 Hz, 1H, CHꢁ), 6.25 (d, Jꢁ8.4 Hz, 1H, CHꢁ), 6.81—6.92 (m, 3H,
CHarom., NH), 7.20—7.37 (m, 2H, CHarom.), 10.65 (s, 1H, NH). C22H27O4N3
(397.46): Calcd C 66.48, H 6.85, N 10.57; Found C 66.76, H 6.58, N 10.47.
(1S,2R,6S,7S) 1-Cyclohexyl-3-(1-isopropyl-7-methyl-3,5-dioxo-4-aza-
tricyclo[5.2.2.02,6]undec-8-en-4-yl)-thiourea (d3) Yield 2.56 g (66%). mp
1
112 °C. H-NMR (200 MHz, DMSO) d: 1.01 (d, Jꢁ6.8 Hz, 3H, CH3), 1.11
(d, Jꢁ6.4 Hz, 3H, CH3), 1.15—1.43 (m, 10H, CH2 cyclohexyl), 1.49 (s, 3H,
CH3), 1.52—1.57 (m, 2H, CH2), 1.7—1.74 (m, 2H, CHcyclohexyl), 2.50—2.55
(m, 1H, CH), 2.71 (d, Jꢁ8.4 Hz, 1H, CH), 3.08 (d, Jꢁ4 Hz, 1H, CH), 4.0 (s,
1H, NH), 5.98 (d, Jꢁ8 Hz, 1H, CHꢁ), 6.08 (d, Jꢁ8 Hz, 1H, CHꢁ), 7.32 (s,
1H, NH). C21H31O2N3S (389.55): Calcd C 67.75, H 8.02, N 10.79; Found C
67.69, H 7.80, N 11.24.
• Nociceptive reactions were studied in 0.6% acetic acid-induced
writhing test.31) The number of writhing episodes was measured for 10 min
starting 5 min after i.p. administration of acid solution.
• Motor coordination was evaluated in rota rod test32) and chimney test.33)
• Body temperatue in normothermic mice was measured in the rectum by
thermistor thermometer.
(1S,2R,6S,7S) 1-Cyclohexyl-3-(1-isopropyl-7-methyl-3,5-dioxo-4-aza-
tricyclo[5.2.2.02,6]undec-8-en-4-yl)-urea (d3a) Yield 2.31 g (62%). mp
• Pentylenetetrazole (110 mg/kg, s.c.)-induced convulsions were evalu-
ated as the number of mice with clonic seizures, tonic convulsions and dead
animals.
• “Head twitch” responses after 5-hydroxytryptophan (5-HTP), according
to Corne et al.34) Mice received 5-HTP (180 mg/kg, 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).
1
203 °C. H-NMR (400 MHz, CDCl3) d: 0.98 (d, Jꢁ6.8 Hz, 3H, CH3), 1.10
(d, Jꢁ6.4 Hz, 3H, CH3), 1.06—1.34 (m, 4H, CH2 CHcyclohexyl), 1.28—1.38
(m, 6H, CH2 CHcyclohexyl), 1.35 (s, 3H, CH3), 1.59—1.7 (m, 2H, CH2), 1.91—
1.94 (m, 2H, Ccyclohexyl), 2.52—2.59 (m, 1H, CH), 2.67 (d, Jꢁ8 Hz, 1H, CH),
3.05 (d, Jꢁ8 Hz, 1H, CH), 4.80 (s, 1H, NH), 5.97 (d, Jꢁ8.4 Hz, 1H, CHꢁ),
6.05 (d, Jꢁ8.4 Hz, 1H, CHꢁ), 6.35 (s, 1H, NH). C21H31O3N3 (373.48):
Calcd C 67.53, H 8.37, N 11.25; Found C 67.67, H 8.32, N 11.22.
The compound was injected in doses equivalent to 0.1 LD50 (200 mg/kg).
Statistics Obtained data were calculated by Student’s t-test and c2 test
(1S,2R,6S,7R) 1-(1,7,8,9,10-Pentamethyl-3,5-dioxo-4-aza-tricyclo-

May 2007
799
with Yates correction (pentylenetetrazole-induced seizures).
Antiviral Assay Procedures The antiviral investigations of the com-
pounds were performed in Dipartamento di Scienze e Tecnologie Bio-
mediche, Universita di Cagliari, Monserato, Italy.
Compounds Compounds were solubilized in DMSO at 200 mM and
then diluted into a culture medium.
Buerki H. R., J. Med. Chem., 38, 28—33 (1995).
7) Forbes I. T., Jones G. E., Murphy O. E., Holland V., Barter G. S., J.
Med. Chem., 38, 855—857 (1995).
8) Fludzinski P., Wittenauer L. A., Schenck K. W., Cohen M. L., J. Med.
Chem., 29, 2415—2418 (1986).
9) Audia J. E., Evrard D. A., Murdoch G. R., Droste J. J., Nissen J. S.,
Schenck K. W., Fludzinski P., Lucaites V. L., Nelson D. L., Cohen M.
L., J. Med. Chem., 39, 2773—2780 (1996).
Cells and Viruses MT-4 cells were grown at 37 °C in a 5% CO2 atmo-
sphere in RPMI 1640 medium, supplemented with 10% fetal calf serum
(FCS), 100 IU/ml penicillin G, and 100 mg/ml streptomycin. Cell cultures 10) Corne S. J., Pickering R. W., Warner B. T., Brit. J. Pharmacol, 20,
were checked periodically for the absence of mycoplasma contamination 106—120 (1963).
wuth a Myco Tect Kit (Gibco). Human immunodeficiency viruses HIV-1, 11) Corne S. J., Pickering R. W., Psychopharmacologia, 11, 65—78
IIIB strain were obtained from supernatants of persistently infected H9/IIIB (1967).
cells. HIV-1 stock solutions had titers of 4.5ꢅ106 and 1.4ꢅ105 50% cell cul- 12) Peroutka S. J., Lebovitz R. M., Snyder S. H., Science, 212, 827—829
ture infectious dose (CCID50)/ml, respectively. (1981).
Anti-HIV Assays Activity of the compound against HIV-1 multiplica- 13) Colpaert F. C., Janssen P. A. J., Neuropharmacology, 22, 993—1000
tion in acutely infected cells was based on the inhibition of virus-induced (1983).
cytopathogenicity in MT-4 cells. Briefly, 50 ml of culture medium containing 14) Green A. R., O’Shaughnessy K., Hammond M., Schachter M., Gra-
1ꢅ104 cells was added to each well of flat-bottom microtiter plates contain-
hame-Smith D. G., Neuropharmacology, 22, 573—578 (1983).
ing 50 ml of culture medium with or without various concentrations of com- 15) Goodwin G. M., Green A. R., Brit. J. Pharmacol., 84, 743—755
pounds. Then 20 ml of an HIV suspensions (containing the appropriate (1985).
amount of CCID50 to cause complete cytopathogenicity at day 4) was added. 16) Darmani N. A., Martin B. R., Glennon R. A., Eur. J. Pharmacol., 186,
After incubation at 37 °C, the number of viable cells was determined by 115—118 (1990).
the 3-(4,5-dimethylthiazol-1-yl)-2,5-diphenyltetrazolium bromide (MTT) 17) Darmani N. A., Martin B. R., Pandy U., Glennon R. A., Pharmacol.
method.35) Cytotoxicity of the compounds was evaluated in parallel with
Biochem. Behav., 36, 901—906 (1990).
their antiviral activity. It was based on the viability of mock-infected cells, 18) Darmani N. A., Martin B. R., Glennon R. A., J. Pharmacol. Exp.
as monitored by MTT method. Ther., 262, 692—698 (1992).
Microbiology Microorganisms used in this study were as follows: 19) Fantegrossi W. E., Kiessel C. L., Leach P. T., Van Martin C.,
Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, Staphy-
lococcus aureus ATCC 29213, Staphylococcus aureus ATCC 6538P, Staphy-
Karabenick R. L., Chen X., Ohizumi Y., Ullrich T., Rice K. C., Woods
J. H., Psychopharmacology, 173, 270—277 (2004).
lococcus aureus NCTC 4163, Escherichia coli ATCC 25922, Escherichia 20) Lucki I., Nobler M. S., Frazer A., J. Pharmacol. Exp. Ther., 228,
coli ATCC 10538, Enterococcus hirae ATCC 10541, Pseudomonas aerugi- 133—139 (1984).
nosa ATCC 15442, Pseudomonas aeruginosa NCTC 6749, Bordetella bron- 21) Handley S. L., Singh L., Pharmacol. Sci., 7, 324—328 (1986).
chiseptica ATCC 4617, Candida albicans ATCC 10231. Other microorgan- 22) Ortmann R., Biscoff S., Radeke E., Bueche O., Delini-Stula A.,
isms used were obtained from the collection of the Department of Pharma-
ceutical Microbiology, Medical University of Warsaw, Poland.
Media, Growth Conditions and Antimicrobial Activity Assays An-
Naunyn Schmiedeberg’s Arch. Pharmacol., 321, 265—270 (1982).
23) Zawadowski T., Skowron A. A., Suski S., Rump S., Jakowicz J., Acta
Pol. Pharm., 52, 129—132 (1995).
tibacterial activity was examined by the disc-diffusion method under stan- 24) Kossakowski J., Kus´mierczyk J., Pharmazie, 55, 5—8 (2000).
dard conditions using Mueller-Hinton II agar medium (Becton Dickinson)
according to the guidelines established by the NCCLS (CLSI).36) Antifungal
activities were assessed using YNB-agar medium.
For the disc-diffusion assay, the solution of tested agents was prepared in
ethanol or dimethylsulfoxide. Sterile filter paper discs (9 mm diameter,
25) Orzeszko A., Kamien´ska B., Orzeszko G., Staros´ciak B. J., Farmaco,
55, 619—623 (2000).
26) Furmanowa M., Nosow A. M., Oreshnikow A. V., Kluski A. G., Kotin
´
M., Statros´ciak B. J., Sliwin´ska A., Guzewska J., Bloch R., Pharmazie,
57, 424—426 (2002).
Whatman No 3 chromatography paper) were dripped with test compound 27) Orzeszko A., Kamien´ska B., Staros´ciak B. J., Farmaco, 57, 619—624
solution to load 400 mg of a given compound per disc. (2002).
The results were read following 24—48 h incubation at 30 °C for fungi 28) Struga M., Miroslaw B., Wawrzycka-Gorczyca I., Kossakowski J.,
and 18 h incubation at 35 °C for bacterial.
Koziol A. E., Polish J. Chem., 81, 51—59 (2007).
29) Vogel G. H., Vogel W. H., “Drug Discovery and Evaluation. Pharma-
cological Assays,” Springer-Verlag, Berlin, 1997.
References
1) Venkatachalam T. K., Mao C., Uckun F. M., Bioorg. Med. Chem., 12, 30) Litchfield L. T., Wilcoxon F., J. Pharmacol. Exp. Ther., 96, 99—113
4275—4284 (2004). (1949).
2) Bloom J. D., Dushin R. G., Curran K. J., Donahue F., Norton E. B., 31) Koster R., Anderson M., DeBeer E. J., Fed. Proc., 18, 412—418
Terefenko E., Jonas T. R., Ross A. A., Feld B., Lang S. A., DiGrandi
M. J., Bioorg. Med. Chem., 14, 3401—3406 (2004).
(1959).
32) Gross F., Tripod J., Meir R., Doriden. Schweiz. Med. Wochschr., 85,
3) Seth P. P., Ranken R., Robinson D. E., Osgood S. A., Risen L. M.,
305—309 (1995).
Rodgers E. L., Migawa M. T., Jefferson E. A., Swayze E. E., Bioorg. 33) Boissier J. R., Tardy J., Diverres J. C., Med. Exp. (Basel), 3, 81—84
Med. Chem., 14, 5569—5572 (2004). (1960).
4) Lee J., Lee J., Kang M., Shin M., Kim J. M., Kang S. U., Lim J. O., 34) Corne S. J., Pickering R. W., Werner B. T., Brit. J. Pharmacol., 20,
Choi H. K., Suh Y. G., Park H. G., Oh U., Kim H. D., Park Y. H., Ha 106—120 (1963).
H. J., Kim Y. H., Toth A., Tran R., Pearce L. V., Lundberg D. J., Blum- 35) Pauwels P., Balzarini J., Baba M., Snoeck R., Schols R. D., Herdewijn
berg P. M., J. Med. Chem., 46, 3116—3126 (2003). P., Desmyster J., De Clercq E., J. Virol. Methods, 20, 309—321 (1988).
5) Forbes I. T., Ham P., Booth D. H., Martin R. T., Thompson M., Baxter 36) Clinical and Laboratory Standards Institute, Performance Standards
G. T., Blackburn T. P., Glen A., Kennet G. A., Wood M. D., J. Med.
Chem., 25, 2524—2530 (1995).
for Antimicrobial Disk Susceptibility Tests; Approved Standard–
Eighth Edition, Villanova, PA, U.S.A. 2003.
6) Nozulak J., Walkman H. O., Floerscheim P., Hoyer D., Schoeffer P.,