Synthesis, brain antihypoxic activity and cell neuroprotection of 1-substituted-3,7-dimethylxanthines

Article abstract of DOI:10.1016/S0223-5234(00)01172-7

Five newly synthesised original compounds were investigated for cute toxicity, influence on hexobarbital sleeping time, effect on the locomotor activity, and brain antihypoxic activity. Two of the compounds were tested in a model of glutamate induced neurotoxicity in the brain cell culture using a cell viability test. Our studies indicate that compounds 2a-c and 4 prolonged the survival time of mice in the model of anoxic hypoxia. Only compound 3 expressed antihypoxic activity in the model of circulatory hypoxia, evaluated with a statistical significant increase of the survival time. Compound 4 (1-[3-(2,3,dihydro-3-oxobenzisosulfonazol-2-yl)-propyl]-3,7-dimethylxanthine) in concentration range 0.3-3 μM statistically significantly antagonised the gutamate induced neurotoxicity. Compound 4 is important for further investigations on in vivo models of brain dementia. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)01172-7

Eur. J. Med. Chem. 35 (2000) 941–948
´
© 2000 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
Preliminary communication
PII: S0223-5234(00)01172-7/SCO
Synthesis, brain antihypoxic activity and cell neuroprotection of
1-substituted-3,7-dimethylxanthines
A. Zlatkova, P. Peikova, J. Rodriguez-Alvarezb*, N. Danchevc, I. Nikolovac, J. Mitkova
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, 2 Dunav Street, 1000 Sofia, Bulgaria
bDepartment of Biochemistry, Medical Faculty-UAB, Bellaterra, Barcelona, Spain
cDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, 2 Dunav Street, 1000 Sofia, Bulgaria
Received 30 September 1999; revised 4 May 2000; accepted 5 May 2000
Abstract – Five newly synthesised original compounds were investigated for acute toxicity, influence on hexobarbital sleeping time,
effect on the locomotor activity, and brain antihypoxic activity. Two of the compounds were tested in a model of glutamate induced
neurotoxicity in the brain cell culture using a cell viability test. Our studies indicate that compounds 2a–c and 4 prolonged the
survival time of mice in the model of anoxic hypoxia. Only compound 3 expressed antihypoxic activity in the model of circulatory
hypoxia, evaluated with a statistical significant increase of the survival time. Compound 4 (1-[3-(2,3-dihydro-3-oxobenzisosulfonazol-
2-yl)-propyl]-3,7-dimethylxanthine) in concentration range 0.3–3 mM statistically significantly antagonised the glutamate induced
´
neurotoxicity. Compound 4 is important for further investigations on in vivo models of brain dementia. © 2000 Editions
scientifiques et me´dicales Elsevier SAS
3,7-dimethylxanthines / acute toxicity / brain antihypoxic activity / glutamate induced neurotoxicity
1. Introduction
most important neurotoxic amino acid in the CNS [3,
4], we have also studied the effects of two 3,7-
dimethylxanthine derivatives on the glutamate in-
duced neurotoxicity in brain cell cultures.
Xanthine derivatives possess different pharmaco-
logical activity. Some of the asymmetrically substi-
tuted xanthines are useful for treating CNS diseases
such as Alzheimer’s type dementia [1]. For example, a
new xanthine derivative, Propentophylline, was re-
cently introduced as a drug with neuroprotective
properties for treatment of brain dementia [2], boost-
ing the search for new xantine derivatives with neuro-
protective actions.
We report here the preparation of five newly syn-
thesised 3,7-dimethylxanthine derivatives, and de-
scribe their pharmacological and toxicological
properties: acute toxicity, effect on the locomotor
activity, influence on hexobarbital sleeping time and
brain antihypoxic activity. Since glutamate is the
2. Results and discussion
2.1. Chemistry
The 1-substituted 3,7-dimethylxanthine derivatives
(2a–c, 3 and 4) indicated in figure 1 were prepared by
reaction between 1-(v-haloalkyl)-3,7-dimethylxanthines
(1a–c) and previously prepared sodium salts of imida-
zole derivatives and sodium 2,3-dihydro-3-oxobenziso-
sulfonazole. The 1-(v-haloalkyl)-3,7-dimethylxanthines
used were obtained by known methods [5, 6]. The
progress of the reaction was followed on TLC.
When 1-(3-chloropropyl)-3,7-dimethylxanthine 1a was
used as starting material to obtain 2a,c, 3 and 4 their yield
was found to be remarkably lower and reaction time about
two times longer. This was probably due to low reac-
tivity of this compound in comparison with 1b. We also
* Correspondence and reprints:
E-mail address: jose.rodriguez@uab.es (J. Rodriguez-Al-
varez).

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A. Zlatkov et al. / European Journal of Medicinal Chemistry 35 (2000) 941–948
found that if there is no protection from the atmospheric
moisture whilst obtaining 2a,c, 3 and 4 from both of 1a
and 1b, the hydrolysis took place and the yield of the new
compounds decreased. The hydrolysis product is 1-(3-hy-
droxypropyl)-3,7-dimethylxanthine [5].
The second approach to obtain the title compounds was
illustrated with the synthesis of 2b as outlined in figure 2.
Starting from 1-(2-bromoethyl)-2-methyl-5-nitroimida-
zole (6) and 3,7-dimethylxanthine (5) via its sodium salt
(5a), 2b was prepared in a fairly good yield without the
presence of contaminating by-products.
Compounds 2a–c and 4 are partly soluble in water and
alcohols, but soluble in dimethylformamide and dimethyl-
sulfoxide. Compound 3 is soluble in water, dimethylfor-
mamide, dimethylsulfoxide and sparingly in alcohols.
The new compounds were characterised by microanaly-
ses, FTIR and NMR spectrometry and results were
consistent with the assigned structures.
1H- and 13C-NMR spectra of 3 are of basic interest.
The proton signals between 5 and 7 ppm are characteris-
tic for substituted urea, due to to N-(2,5-dioxo-4-imida-
zolidinyl)-ureido side chain. Thus, the signal at 5.82
ppm as singlet corresponds to amino group protons.
Each of the signals at 5.27 and 6.91 ppm, as doublets,
correspond to one proton – first of allantoine ring and
second from the ureido side chain. The signal corre-
sponding to NH-proton at position 3 of allantoine ring,
as singlet, appears at 8.33 ppm. In unsubstituted N-(2,5-
dioxo-4-imidazolidinyl)-urea there is one more signal at
about 10 ppm, which corresponds to NH-proton at
position 1 [7]. In the spectrum of 3 this signal is not
present. The rest of the signals correspond to the pro-
tons of the substituted xanthine ring and propyl side
chain. The values of the chemical shifts of the carbon
atoms registered by 13C-NMR spectra were compared
with simulated values [8]. These data confirm the struc-
Figure 1. Reaction pathway for the synthesis of 1-substituted-3,7-dimethylxanthines (2a–c, 3 and 4).

A. Zlatkov et al. / European Journal of Medicinal Chemistry 35 (2000) 941–948
943
All tested compounds, when administered in a dose
1/10 and 1/20 ratio of LD50, significantly (P50.05)
increased the hexobarbital sleeping time in comparison
with 3,7-dimethylxanthine, which at similar doses does
not affect the hexobarbital sleeping time (table II).
The locomotor activity investigations show that all
tested compounds, except 2b, decreased the spontaneous
locomotor activity in comparison with the control group
1
1
at doses /10 and /20 of LD50 (figures 3 and 4).
Accordingly with data from Ishii et al. [9] medial
temporal oxygen metabolism was markedly affected in
patients with mild-to-moderate Alzheimer’s disease. In
this respect, we wanted to analyse the effect of the
xantine derivatives in different models of provoked
brain hypoxia. The results showed that compounds 2a–
c and 4 prolonged the survival time of mice in the model
of anoxic hypoxia, whereas only compound 3 had an
antihypoxic activity in the model of circulatory hypoxia
(table III).
Table I. Acute toxicity (LD50) of compounds 2a–c, 3, 4 and
theobromine in mice after intraperitoneal administration.
Compound
LD50 (mg/kg)
b.w.
Range of values
Figure 2. Second synthetic approach for obtaining compound
2b.
2a
\5000
740.08
5000
\2000
\3000
552.4
–
2b
588.66/930.44
–
–
–
ture of 3 and prove that 2,5-dioxo-4-ureidoimidazolidine
was bound to the propyl side chain at position 1, and
not at position 3.
2c
3
4
Theobromine
472.7/645.6
2.2. Pharmacology
Table II. Influence of compounds 2a–c, 3, 4 and theobromine
on hexobarbital sleeping time of mice.
Newly synthesised compounds 2a–c, 3, 4 were investi-
gated for acute toxicity, influence on Hexobarbital sleep-
ing time and locomotor activity of mice. Assessment of
the compounds’ effect in three different models of pro-
voked brain hypoxia is also reported. Two compounds
were studied for the antagonism of the glutamate in-
duced neurotoxicity.
Analysis of the obtained experimental data on the
acute toxicity (LD50) of 2a–c, 3, 4, as compared to
3,7-dimethylxanthine, showed that these compounds
caused an acute toxicity (LD50) which was significantly
(P50.05) lower than when 3,7-dimethylxanthine was
used (table I).
Compound
Doses (mg/kg)
b.w., i.p.
Sleeping time
(X/SD) min
Control (HB)
80
250
500
37
17.6/4.0
2a
25.0/16.5a
41.5/12.0a
20.0/9.9
2a
2b
2b
74
24.1/8.3b
57.9/4.4a
58.3/2.0a
24.4/2.2a
21.8/1.7a
26.5/2.5a
14.7/6.16
2c
250
500
100
150
300
55
2c
3
4
4
Theobromine
Compounds 2a and 2c (LD50\5000 mg/kg b.w.)
were practically nontoxic. This effect is probably due to
the influence of the substituent in the side chain of the
structures.
a P50.05 statistically significance in comparison with control
group.
b P50.1 statistically significance in comparison with control
group.

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A. Zlatkov et al. / European Journal of Medicinal Chemistry 35 (2000) 941–948
we have evaluated the effect of compounds 2c and 4 on
the brain hypocampal neuronal toxicity induced by glu-
tamate (see figure 5). The results indicated that com-
pound 4 significantly antagonises the glutamate induced
toxicity in concentration range 0.3–3 mM.
The fact that compound 4 antagonises the glutamate
induced toxicity in neuronal cultures together with its
lower toxicity suggest that compound 4 could be a
good candidate to be used in the treatment of neurode-
generative diseases. Obviously, further and extensive
research should be done using in vivo models of brain
dementia.
3. Experimental protocols
Figure 3. Effects of compounds 2a–c, 3, 4 and theobromine
1
(Theobr.) given i.p. in /10 ratio of LD50 on locomotor activity
of mice at 20 min intervals up to 120 min with P50.05
(indicated by asterisk) in comparison with control group.
3.1. Chemistry
Melting points were measured in °C and corrected
(Bu¨chi 535). FTIR spectra were recorded on a Shimadzu
1
FTIR 8101M. H- and 13C-NMR spectra were recorded
at ambient temperature on a Bruker-250 WM (250
MHz) spectrometer. Chemical shifts are given in parts
per million (l-ppm) downfield from tetramethylsilane as
internal standard. Splitting patterns are designed as
follows: s, singlet; d, doublet; t, triplet; and m, multiplet.
The following abbreviations are used: Im – imidazole
ring, Xanth – xanthine ring, Allan – alantoine ring, Ur
– ureido side chain and Ar – aromatic protons. Prepar-
ative thin-layer chromatography was performed on DC-
Alufolien Kieselgel 60 F254 (Merck) (0.20 mm) sheets
with solvents (vol. parts): system I: 25 % ammonium
hydroxide–chloroform–acetone–ethanol (1:3:3:4), sys-
tem II: (chloroform–ethanol (1:1), detected at UV 254
nm. Elemental analyses were performed by the microan-
alytical laboratory of Faculty of Pharmacy (Medical
University-Sofia). Microanalytical results were within9
0.4 % of the theoretical values. All solutions were dried
over anhydrous sodium sulfate and evaporated on a
Bu¨chi rotary evaporator at reduced pressure. Non-com-
mercially available intermediates required for the syn-
thesis of novel derivatives of 3,7-dimethylxanthine
were obtained as referenced: 1-(3-chloropropyl)-3,7-di-
methylxanthine (1a) [5]; 1-(3-iodoropropyl)-3,7-dimethyl-
xanthine (1b) [5]; 1-(2-chloroethyl)-3,7-dimethyl-
xanthine (1c) [6]; 1-(2-bromoethyl)-2-methyl-5-nitroimi-
dazole (6) [11].
Figure 4. Effects of compounds 2a–c, 3, 4 and theobromine
1
(Theobr.) given i.p. in /20 ratio of LD50 on locomotor activity
of mice at 20 min intervals up to 120 min with P50.05
(indicated by asterisk) in comparison with control group.
Several data in the literature suggest that excitotoxic-
ity plays an important role in the Alzheimer’s disease.
For example, Jansen et al. [10] observed reduction of
about 40–50 % of the NMDA receptor binding sites
(including glutamate) in different brain regions, assessed
post-mortem in patients with Alzheimer’s disease and
Malcolm et al. [3] have showed that transient exposure
to 100 mM glutamate results in neurotoxicity in primary
cultures of mouse cerebellar granule cells. In this respect
The given yields are those of analytically pure
product. No efforts were made to optimise the yields.

A. Zlatkov et al. / European Journal of Medicinal Chemistry 35 (2000) 941–948
945
Table III. Brain antihypoxic activity of compounds 2a–c, 3, 4 and theobromine on mice.
Compound
Doses (mg/kg),
b.w., i.p.
Antihypoxic activity
Anoxic hypoxia
(X9SD) min
Haemic hypoxia
(X9SD) min
Circulatory hypoxia
(X9SD) min
Control
–
14.690.8
21.196.4a
24.496.6a
15.992.9
18.392.2a
18.492.2b
21.193.0a
14.692.1
12.291.5a
9.591.3c
7.190.6c
10.091.8c
9.591.6c
7.991.9c
19.895.3
16.894.8
15.398.1
16.694.0
18.295.6
16.193.9
16.996.8
19.294.9
40.492.5a
2a
250
500
37
2a
2b
2b
74
2c
250
500
100
200
150
300
55
2c
3
3
14.4
25.4a
13.3
4
4
30.2a
11.4
13.292.6
19.991.3
Theobromine
17.591.8
39.9919.4a
a P50.05 statistically significance in comparison with control group.
b P50.1 statistically significance in comparison with control group.
c P50.001 statistically significance in comparison with control group.
3.1.1. 1-(3-Chloropropyl)-3,7-dimethyl-2,3,6,7-
tetrahydro-1H-2,6-purinedione (1a)
3.1.3. 1-(2-Chloroethyl)-3,7-dimethyl-2,3,6,7-
tetrahydro-1H-2,6-purinedione (1c)
Sodium methoxide 30% in absolute methanol (2.7 g,
0.05 mol) was added to a 3,7-dimethylxanthine (9 g, 0.05
mol) dissolved in 250 mL absolute methanol. The reac-
tion mixture was stirred under reflux for 1 h and 1-
bromo-3-chloropropane (47.2 g, 0.3 mol) was added.
The reaction mixture was stirred under reflux for 17 h.
The pH of the solution was maintained at 9 by addition
of sodium methoxide. The progress of the reaction was
followed on TLC (II). After removing the solvent, chlo-
roform was added and separated sodium bromide was
filtered. Chloroform was removed under reduced pres-
sure and the crude product was recrystallised from
absolute methanol. Yield: 78 %. M.p.: 142–144 [5].
Sodium methoxide 30% in absolute methanol (2.7 g,
0.05 mol) was added to a 3,7-dimethylxanthine (9 g, 0.05
mol) dissolved in 250 mL absolute methanol, and after
that the solvent was removed. The sodium salt of 3,7-
dimethylxanthine was dissolved in 70 mL water, and
1,2-dichloroethane (130 g, 1.5 mol) and Linopharm
3.1.2. 1-(3-Iodopropyl)-3,7-dimethyl-2,3,6,7-tetrahydro-
1H-2,6-purinedione (1b)
Sodium iodide (7.5 g, 0.05 mol) was added to a
solution of 1a (10.3 g, 0.04 mol) in 250 mL anhydrous
acetone. The reaction mixture was stirred under reflux
for 56 h. The progress of the reaction was followed on
TLC (II). After removing the solvent, chloroform was
added and separated sodium chloride was filtered. Chlo-
roform was removed under reduced pressure and the
crude product was recrystallised from absolute
methanol. Yield: 77 %. M.p.: 120–122 [5].
Figure 5. Effects of compounds 2c, and 4 on glutamate
induced neurotoxicity.

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A. Zlatkov et al. / European Journal of Medicinal Chemistry 35 (2000) 941–948
(0.35 g, 5 % of water amount) were added. The reaction
mixture was stirred under reflux for 70 h. The progress
of the reaction was followed on TLC (I). After cooling,
the two layers were separated by filtration. After drying
with anhydrous sodium sulfate and filtration, the or-
ganic layer was distilled. The residual solid was recrys-
tallised from ethanol. The product was dried at
90–95 °C. Yield: 85 %. M.p.: 149–151 [6].
3.1.7. 3,7-dimethyl-1-[2-(2-methyl-5-nitro-1H-
imidazol-1-yl)-ethyl]-2,3,6,7-tetrahydro-1H-2,6-
purinedione (2b)
3.1.7.1. Method A
Compound 2b was synthesised in the same manner in
methanol from 2-methyl-5-nitroimidazole sodium salt
and 1c for 20 h in yield 1.95 g (59 %).
3.1.7.2. Method B
3.1.4. General procedure for the preparation of sodium
salts of imidazole deri6ati6es
Sodium methoxide (0.012 mol, 1.94 g 33 % methano-
lic solution in 10 mL absolute methanol) was added to
3,7-dimethylxanthine (0.01 mol, 1.8 g) dissolved in 140
mL absolute methanol and the mixture was refluxed for
3 h. Then the volume of the solution was reduced to 60
mL and 1-(2-bromoethyl)-2-methyl-5-nitroimidazole (6)
(0.01 mol, 2.34 g) dissolved in 20 mL methanol was
added. The reaction mixture was heated under reflux for
15 h. The isolation of 2b was carried out as described in
General procedure. Yield: 2.25 g (68%). Rf=0.47 (I).
M.p.: \300 °C. IR (cm−1) Nujol: 1694 (wCꢀO), 1668
(wCꢀO); 1605, 1592, 1545, (wCꢀC, wCꢀN), 1345 (wNO2);
1H-NMR (DMSO-d6): 3.35 (s, 3H, CH3); 3.57 (s, 3H,
CH3); 4.11 (t, 2H, J=4.2 Hz, CH2); 4.35 (t, 2H, J=5.4
Hz, CH2); 7.95 (s, 1H, C88ꢁH), 8.20 (s, 1H, CꢁHꢁIm).
Anal. C13H15N7O40 (N).
Sodium methoxide (0.012 mol, 2.17 g 30 % methano-
lic solution in 10 mL absolute methanol) was added to
the corresponding imidazole derivative (0.01 mol) dis-
solved in 30 mL absolute methanol. The mixture was
heated under reflux for 1.5 h. Then this solution was
used for obtaining the novel compounds. If necessary
the solvent was removed and imidazole derivative
sodium salt was dissolved in an appropriate solvent to
prepare the novel products.
3.1.5. General procedure for the preparation of
compounds 2a–c and 3
To a solution of fresh prepared imidazole derivative
sodium salt in appropriate anhydrous solvent was added
1b (3.48 g, 0.01 mol) or 1c (2.42 g, 0.01 mol) dissolved
in 20 mL of the same solvent. The reaction mixture was
stirred under reflux. The reaction time was established
by TLC monitoring. Separated sodium halide was
filtered when hot, the filtrate was kept for 12 h at 4 °C.
The crude product was filtered and washed three fold
with methanol to give corresponding compound. If nec-
essary the product was recrystallised from methanol or
ethanol.
3.1.8. 3,7-dimethyl-1-[3-(4(5)-nitro-1H-imidazol-1-yl)-
propyl]-2,3,6,7-tetrahydro-1H-2,6-purinedione (2c)
2c was obtained as described in general method from
4(5)-nitroimidazole sodium salt and 1b for 21 h in
absolute methanol to give 2.0 g (56%). Rf=0.68 (I).
M.p.: 214–217, IR (cm−1) Nujol: 1698 (wCꢀO), 1685
(wCꢀO); 1600, 1590, 1543, (wCꢀC, wCꢀN), 1350 (wNO2);
1H-NMR (DMSO-d6): 2.04–2.15 (m, 2H, CH2); 3.33 (s,
3H, CH3); 3.41 (s, 3H, CH3); 3.91 (t, 2H, J=6.2 Hz,
CH2); 4.11 (t, 2H, J=7 Hz, CH2); 8.01 (s, 1H, C8ꢁH);
8.30 (s, 1H, CꢁH, Im); 8.43 (s, 1H, CꢁH, Im). Anal.
C13H15N7O4 (N).
3.1.6. 3,7-dimethyl-1-[3-(2-methyl-5-nitro-1H-
imidazol-1-yl)-propyl]-2,3,6,7-tetrahydro-
1H-2,6-purinedione (2a)
Compound 2a was prepared from 2-methyl-5-nitroim-
idazole sodium salt and 1b in anhydrous 2-
ethoxyethanol as described above for 6 h. Yield: 2.6 g
(75%). Rf=0.73 (I). M.p.: 230–233 (ethanol). IR
(cm−1) Nujol: 1710 (wCꢀO), 1695 (wCꢀO), 1651, 1610,
3.1.9. N-[1-[3-(3,7-dimethyl-2,6-dioxo-2,3,6,7-
tetrahydro-1H-1-purinyl)propyl]-2,5-dioxo-4-
imidazolidinyl]-urea (3)
Compound 3 was obtained as described above from
N-(2,5-dioxo-4-imidazolidinyl)-urea (allantoine) sodium
salt and 1b for 36 h in absolute methanol. After concen-
tration of the filtrate to minimal volume the separated
solid was filtered and the combined precipitate was
washed with cold methanol. Yield: 1.5 g (40%); Rf=
0.22 (I). M.p.: 194–196 (methanol). IR (cm−1) Nujol:
1
1550 (wCꢀC, wCꢀN), 1344 (wNO2); H-NMR (DMSO-
d6): 2.38 (s, 3H, ꢁCH3); 2.46–2.52 (m, 2H, CH2); 3.21 (s,
3H, CH3); 3.37 (s, 3H, CH3); 4.14 (t, 2H, J=6.5 Hz,
CH2); 4.62 (t, 2H, J=7 Hz, CH2); 7.59 (s, 1H, C8ꢁH);
8.29 (s, 1H, CꢁH, Im). Anal. C14H16N7O4 (N).

A. Zlatkov et al. / European Journal of Medicinal Chemistry 35 (2000) 941–948
947
3565–3370 (wNꢁH), 1774–1651 (wC=O); 1617, 1553
under study. Sleeping time was measured in minutes by
observing the righting reflex recovery.
1
(wCꢀC, wCꢀN). H-NMR (DMSO-d6): 1.79 (t, 2H, J=
6.4 Hz, CH2); 3.33–3.42 (m, 5H, CH3, CH2); 3.82–3.86
(m, 5H, CH3, CH2); 5.27 (d, 1H, J=8 Hz, Im); 5.82 (s,
2H, NH2); 6.91 (d, 1H, J=8 Hz, Im); 7.97 (s, 1H,
C8ꢁH); 8.33 (s, 1H, NH). 13C-NMR (DMSO-d6): 172
(CO-5-Allan), 157.5 (CO-Ur), 156.4 (CO-2-Allan), 154.4
(CO-6-Xanth), 150.9 (CO-2-Xanth), 148.4 (C-4-Xanth),
143 (C-8-Xanth), 106.7 (C-5-Xanth), 61.5 (C-4-Allan),
38.4 (−CH2-N-1-Xanth), 35.9 (CH3-N-7-Xanth), 33.3
(CH2-N-1-Allan), 29.5 (CH3-N-3-Xanth), 26.5 (CH2).
Anal. C14H18N8O5 (N).
3.2.2. Influence on locomotor acti6ity
A group of six animals was put on in actometer
(Activity Cage, Ugo Basile, Italy) and the locomotor
activity in arbitrary units was determined at 20 min
intervals for 120 min. The tested compounds at a dose
1
/
10
of LD50 were administered to the animals and they
were tested in the apparatus for 120 min under analogi-
cal conditions, and the results were compared with those
in the control vehicle-treated group.
3.1.10. 2-[3-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-
1H-1-purinyl)propyl]-2,3-dihydro-1H-1u6-benzo[d]-
isothiazole-1,1,3-trione (4)
3.2.3. Antihypoxic acti6ity
1
The tested compounds were given in doses /20 and
1
/
of LD50. Three models of provoked brain hypoxia
10
A
solution of sodium 2,3-dihydro-1H-1l6-ben-
were used:
zo[d]isothiazole-1,1,3-trione (saccharin, 0.01 mol, 2.05 g)
and added 1b (3.48 g, 0.01 mol) in 40 mL DMF was
heated under reflux for 25 h. After cooling to room
temperature 160 mL water were added. A waxy product
was separated and after 24 h it became a yellow solid.
The crude product was recrystallised from abs. ethanol
to give 3.2 g (80%). Rf=0.84 (I). M.p.: 191–193; IR
(cm−1) Nujol: 1732 (wCꢀO), 1703 (wC=O), 1659, 1551
Anoxic hypoxia — 30 min after the i.p. administra-
tion of the tested compounds to the mice (8 per group),
they were placed into hermetic bottles of 200 mL vol-
ume. The survival time (in min) was determined and
compared with control vehicle treated group [13].
Haemic hypoxia — the animals (8 per group) were
injected with sodium nitrite in dose 360 mg/kg b.w. i.p.
30 min after the i.p. administration of compounds. The
survival time (in min) for each animal is defined as time,
measured from the induction of the hypoxia (caused by
the introduction of the hypoxant) until death [14].
Circulatory hypoxia was induced 30 min after the i.p.
administration of compounds by sodium fluoride in
dose 150 mg/kg b.w. i.p. to the mice (10 per group). The
survival time (in min) for each animal is defined as time,
measured from the induction of the hypoxia by the
hypoxic agent until death [15].
Excitotoxicity studies. The effect on the brain neu-
ronal toxicity was studied according the method of
Malcolm [3] using the neuronal cell culture vialibility
method. The studied compounds were added prelimi-
nary in the cell culture in concentrations 0.3, 3 and 30
mM and preincubated for 2 h.
All the pharmacological and biochemical results un-
derwent statistical processing by the Student–Fischer
t-test at P50.05.
1
(wCꢀC, wCꢀN), 1326 (was SO2), 1182 (ws SO2). H-NMR
(DMSO-d6): 2.11 (t, 2H, J=7 Hz, CH2), 3.46 (s, 3H,
CH3), 3.76–3.81 (m, 5H, CH2, CH3), 3.86–3.96 (m, 2H,
CH2), 8.00 (s, 1H, C8ꢁH), 8.07–8.24 (m, 4H, Ar). Anal.
C17H17N5O5S (N).
3.2. Biological e6aluation
The experiments were conducted on eight male white
rats (8-days-old) of the Wistar line, and 410 male white
mice, weighing 18/22 g.
Acute toxicity (LD50) of the studied compounds was
assessed by dissolving in saline (0.9% NaCl) with 1–2
drops of Tween 80, and administering to mice via
intraperitoneal (i.p.) route. LD50 was evaluated at four
or five different doses, each on the six animals and
calculated by the method of Litchfield–Wilcoxon [12].
3.2.1. Influence on hexobarbital sleeping time (HBST)
The studied compounds were administered to male
mice i.p. at doses 1/10 of LD50. The same volume 0.1/10 g
b.w., of solvent (0.9% NaCl) was administered to the
controls. The solution of Hexobarbital sodium at a dose
of 80 mg/kg b.w. was administered i.p. to the animals 30
min after administration of the compounds’ solutions
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