Preparation of novel derivatives of pyridothiazine-1,1-dioxide and their CNS and antioxidant properties.

Article abstract of DOI:10.1016/S0014-827X(02)01267-3

Starting from isothiazolopyridine-1,1-dioxide (1), corresponding derivatives of 3-aryl-4-hydroxypyrido[3,2-e]-1,2-thiazine-1,1-dioxide (6) possessing the 3-[4-(substituted-phenyl)piperazinyl]propyl or 3-(4-substituted-piperidinyl)propyl side chain by the

Full text of DOI:10.1016/S0014-827X(02)01267-3

Il Farmaco 57 (2002) 737ꢀ746
/
www.elsevier.com/locate/farmac
Preparation of novel derivatives of pyridothiazine-1,1-dioxide and
their CNS and antioxidant properties
W. Malinka a,*, M. Kaczmarz a, B. Filipek b, J. Sapa b, B. Glod c
a Department of Chemistry of Drugs, Wroc law Medical University, ul. Tamka 1, 50-137 Wroc law, Poland
b Laboratory of Pharmacological Screening, Faculty of Pharmacy, Jagiellonian University Medical College, ul. Medyczna 9, 30-688 Krako´w, Poland
c Laboratory of Experimental Pharmacology, Polish Academy of Sciences, Medical Research Centre, ul. Pawinskiego 5, 02-106 Warsaw, Poland
Received 16 February 2002; received in revised form 18 March 2002; accepted 29 March 2002
Abstract
Starting from isothiazolopyridine-1,1-dioxide (1), corresponding derivatives of 3-aryl-4-hydroxypyrido[3,2-e]-1,2-thiazine-1,1-
dioxide (6) possessing the 3-[4-(substituted-phenyl)piperazinyl]propyl or 3-(4-substituted-piperidinyl)propyl side chain by the
nitrogen atom of the thiazine ring were prepared. Under pharmacological central nervous system (CNS) screening in animal models
(mice), all of the six pyridothiazines 6 tested exhibited analgesic action as the predominant profile of their activity (‘writhing’ test
12.5ꢀ50 mg/kg). Moreover, the radical scavenging activity against peroxyl radicals of the representative pyridothiazines 6 was
/
´
evaluated in vitro in water environment and some of them proved to be moderate antioxidants. # 2002 Editions scientifiques et
me´dicales Elsevier SAS. All rights reserved.
Keywords: Pyrido[3,2-e]-1,2-thiazine-1,1-dioxides; Analgesic activity; Antioxidant properties
1. Introduction
antiamphetamine (1/80 LD50) and hypothermic (1/80
LD50) effects (neuroleptic action).
In our earlier papers, we described synthesis, anti-
inflammatory and CNS (central nervous system) activ-
ities of some derivatives of the new biheterocylic system
pyrido[3,2-e]-1,2-thiazine of the general structure I (Fig.
Introduction of a substituent to the aromatic ring of
4-phenylpiperazinyl alkyl side chain of derivatives of
heterocyclic system lead in some cases to an increase in
the CNS effects and behavioural responses which
resulted from action on different receptor systems
1) [1ꢀ3]. Initially, we found that 3-carbamoyl-2-methyl
/
derivatives IA, designed as 8-aza-analogues of piroxicam
(Fig. 1), an antiphlogistic agent, showed a similar profile
of pharmacological activity as piroxicam [2]. Further-
more, we found that replacement of the 3-carbamoyl in
IA with the acetyl(benzoyl) group and of the 2-methyl
substituent with 2-(4-phenylpiperazinylpropyl) chain
lead to pyridothiazines (IB) which exhibited high
psychopharmacological activity [3]. The compounds
strongly decreased spontaneous locomotor activity (1/
(mainly serotoninergic and dopaminergic [4ꢀ
fore, to enhance the central activity in 3-benzoyl
derivative IB (RꢁC6H5), a series of its analogues 6aꢀ
(Scheme 1) variously substituted (X"H) at the terminal
aromatic ring of the side chain were prepared (Scheme
1). Moreover, additional substituents Y were introduced
at the para position of the 3-benzoyl group of certain
compounds 6 to modify lipophilic properties of mole-
cules.
/
6]). There-
/
/l
/
40ꢀ/1/80 LD50) (sedative action), and also showed
This paper describes the synthesis and some of the
preliminary CNS pharmacology of such derivatives of
pyrido[3,2-e]-1,2-thiazine (6, Scheme 1).
Furthermore, pyridothiazines 6 were evaluated to test
their peroxyl radical scavenging activity.
* Corresponding author
E-mail address: malinka@bf.uni.wroc.pl (W. Malinka).
´
0014-827X/02/$ - see front matter # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 0 1 4 - 8 2 7 X ( 0 2 ) 0 1 2 6 7 - 3

738
W. Malinka et al. / Il Farmaco 57 (2002) 737ꢀ746
/
Free radical mediated process have been implicated in
The new final compounds 6aꢀn (Scheme 1) were
/
the pathogenesis of several diseases i.e. cancer, Alzhei-
mer’s dementia, rheumatoid arthritis [7]. Tenoxicam
(Fig. 1), which is widely employed in treatment of
rheumatic diseases because of its cyclo/lipooxygenase
inhibitory properties [8], is also a good hydroxyl,
superoxide and peroxyl radicals scavenger as it was
evidenced in cell-free experiments [9]. The probable
mechanism of inactivation of radical species is multiple
and leads, among other species, to 4-oxyradical which is
stabilized by delocalization on the b-dicarbonyl group-
ing (Fig. 2) [9].
prepared by alkylation of pyridothiazines 3 with corres-
ponding 4-(substituted-phenyl)-1-(3-chloropropyl)piper-
azines (4) or -piperidines (5). The reaction was carried
out in close analogy to the methods described in our
previous paper [3], and produced the expected com-
pounds 6 in moderate yield (40ꢀ55%). Piperazine
/
intermediates 4b, c were obtained from corresponding
commercially accessible N-arylpiperazines and 1-bro-
mo-3-chloropropane according to the method described
in Ref. [11] and our preceding paper [12]. Compound 4a
and piperidine intermediates 5a, b were prepared
analogously (exp. part).
Therefore, it was not illogical to test scavenging
activity of pyridothiazines 6 which possess similarly to
tenoxicam the b-dicarbonyl grouping partially incorpo-
rated in the 1,2-thiazine ring. For such a test, we chose
The physical data associated with the final com-
pounds 6aꢀ
/
n and intermediates (2bꢀ
/
e, 3bꢀe, 4a, 5a, b)
/
are summarized in Table 1 and exp. part, respectively. It
is noteworthy that pyridothiazines 3 and 6, as fully
enolized b-diketones, did not show the typical carbonyl
absorption, instead a broad, intense band at range
compounds 6a, 6c, 6g, 6g×/HCl, 6l and their piperidine
6m, n and heterocyclic (pyrimidine) 6o analogues
(Scheme 1). The synthesis of the last compounds was
described recently [3].
1600ꢀ
/
1560 cmꢂ1 was observed. The enolization of 3
1
was confirmed by the H NMR spectra, which showed
the presence of a peak Â16 ppm (OH enole) in addition
/
to that of sufonamide NH (9.2 ppm 3a [1]). The position
of the enol proton in 1H NMR spectra of series 6, like in
IB (Fig. 1) [3], was not established; however these
compounds, similar to 3, give intense colouration with
iron(III) chloride and in the IR (KBr) spectra a broad
band between 2750 and 2200 cmꢂ1 indicating the
presence of an enolic OH group. Because the spectral
2. Synthesis of compounds
The desired compounds of general formula 6 were
synthesized as shown in Scheme 1.
The synthesis of the key intermediates 3aꢀe was
achieved by condensation of isothiazolopyridine-1,1-
dioxide (1) [10] with v-bromoacetophenone or p-sub-
stituted v-bromoacetophenones (commercially avail-
/
data within the series of the new compounds 2bꢀ
and 6aꢀn did not show remarkable differences, they are
presented for selected derivatives in the Section 5.
Finally, for each compound 6 tested at the pharma-
cological screening, the log of the octanolꢀwater parti-
/
e, 3bꢀe
/
/
able) with formation of 2aꢀ
rearrangement of these compounds to the corresponding
pyridothiazines 3aꢀe. The mechanism of rearrangement
/
e and the subsequent
/
/
of isothiazolopyridine 2a to pyridothiazine 3a under
basic condition (anhydrous ethanol, NaOC2H5) has
been studied recently in our laboratory [1]. The expected
tion coefficient was calculated (log Pcalc). The
calculations of the log Pcalc values were made for the
free bases, using the CHEMPLUS program from Hyper-
cube, Inc., IBM PC version. The calculated lipophili-
cities of these compounds are shown in Table 1.
pyridothiazines 3bꢀ/e were obtained similarly.
3. Pharmacological screening and discussion
Compounds 6b, fꢀh, j, k representing a new series of
/
pyrido[3,2-e]-1,2-thiazines, were evaluated in animal
models for acute toxicity (ip) and for their influence
on the spontaneous locomotor activity, thiopental
anaesthesia, carrageenan induced edema and analgesic
activity in the ‘writing syndrome’ and ‘hot-plate’ tests.
3.1. Acute toxicity
All the investigated compounds 6b, 6fꢀh, j, k were not
/
toxic. The LD50 values of the compounds assayed after
their intraperitoneal administration were above 2000
Fig. 1.

W. Malinka et al. / Il Farmaco 57 (2002) 737ꢀ
/
746
739
Scheme 1.
and 200 mg/kg (1/10 of LD50) was taken to be the initial
dose in the further experiments.
3.3. Thiopental anaesthesia
The effect of investigated compounds on the thiopen-
tal anaesthesia is presented in Table 3. Compounds 6b,
6f, 6h, 6j and 6k, given in doses of 100 and 50 mg/kg (1/
20 and 1/40 of LD50), significantly prolonged barbitu-
3.2. Locomotor activity
The pyridothiazines 6, given intraperitoneally in doses
of 100 and 50 mg/kg (1/20 and 1/40 of LD50), decreased
locomotor activity during 30 min observation period.
The most potent effect was produced by compounds 6b,
6j and 6g, which significantly inhibited the spontaneous
locomotor activity of mice at the doses up to 50 mg/kg
(1/40 of LD50). Compounds 6h, 6k and 6f produced a
significant decrease in locomotor activity at the doses up
to 100 mg/kg (1/20 of LD50). The ED50 values and
therapeutic index for the compounds investigated are
presented in Table 2.
rate sleep in mice by 292ꢀ
/
193 and 172ꢀ129%, respec-
/
tively. Compound 6g also prolonged the time of
anaesthesia (by ca. 244%), but this effect was significant
only after dose of 100 mg/kg (1/20 of LD50).
3.4. ‘Writhing syndrome’ test in mice
The investigated compounds were tested for analgesic
activity by intraperitioneal administration in mice in
terms of the inhibition of the ‘writhing syndrome’

740
W. Malinka et al. / Il Farmaco 57 (2002) 737ꢀ746
/
Fig. 2.
induced by phenylbenzoquinone. All compounds tested
Table 1
Physical data of the compounds 6
showed strong analgesic activity in this test. The most
potent effects were produced by compounds 6b and 6j
which were significant active up to a dose of 12.5 mg/kg
(1/160 of LD50). Compounds 6h, 6f and 6g were
significantly active in the ‘writhing syndrome’ test in
mice up to a dose of 25 mg/kg (1/80 of LD50), while
compound 6k had analgesic activity in doses up to 50
mg/kg (1/40 of LD50). The ED50 values of the com-
pounds tested and standards (acetylsalicylic acid, mor-
phine) are summarized in Table 4.
Comp. M.p. (8C) (crystal
from)
Formula (M.w.)
Log Pcalc
6a
6b
6c
6d
6e
6f
123ꢀ
175ꢀ
179ꢀ
212ꢀ
182ꢀ
182ꢀ
130ꢀ
219ꢀ
(EtOHꢀ
214-216
(EtOHꢀ
190ꢀ192
(EtOHꢀacetone 1:1)
/
125 (EtOH)
177 (EtOH)
181 (EtOH)
214 (EtOH)
184 (EtOH)
184 (EtOH)
132 (EtOH)
221
C30H34N4O5S (562.68)
2.92
/
C29H31ClN4O4S (567.10) 3.69
C30H31F3N4O4S (600.65) 4.06
/
/
C31H36N4O5S (576.71)
C31H33F3N4O4S (614.68) 4.52
C31H38N4O6S (592.71) 2.67
3.39
/
/
6g
6h
/
C31H33F3N4O5S (630.68) 3.80
C30H33ClN4O5S (583.14) 3.44
/
/acetone 1:1)
6i
6j
6k
6l
C29H30Cl2N4O4S (601.54) 4.21
3.5. ‘Hot plate’ test in mice
/acetone 1:1)
/
C30H30ClF3N4O4S
(635.10)
4.57
4.48
5.12
/
All investigated compounds showed significant activ-
ity in the ‘hot plate’ test only at the dose of 200 mg/kg
(1/10 of LD50; data not shown).
205ꢀ/207 (acetone)
C29H30BrClN4O4S
(632.01)
195ꢀ/197 (EtOH)
C30H30BrF3N4O4S
(678.55)
6m
6n
6o
175ꢀ
/
177 (EtOH)
C30H31N3O4S (529.65)
C32H37N3O4S (559.72)
2.00
4.92
4.35
199ꢀ
/
201 (EtOH)
3.6. Influence on the blood pressure
Lit. [3]
All investigated compounds, injected intraperitoneally
in a single dose corresponding to ED50 in the ‘writhing
syndrome’ test did not affected the pulse rate and
arterial blood pressure in the normotensive rats (data
not shown).
3.8. Conclusion
The results of the present CNS study of pyridothia-
zines 6b, fꢀh, j, k clearly demonstrated that intraper-
/
itoneal administration of the compounds significantly
suppressed the spontaneous locomotor activity and
increased barbituric-induced sleep in the mice. These
effects suggest that new pyridothiazines assayed possess
sedative activity on the CNS of mice. Additionally,
compounds exhibit significant analgesic action deter-
mined in the phenylbenzoquinone-induced ‘writhing’
3.7. Carrageenan-induced edema
The tested compounds, given at a dose of 100 mg/kg 1
h before carrageenan injection, did not inhibit the
postcarrageenan edema of rat paw, which suggest, that
these compounds have no influence on the inflammatory
process.

W. Malinka et al. / Il Farmaco 57 (2002) 737ꢀ
/
746
741
Table 2
Influence of the investigated compounds 6 on the spontaneous locomotor activity in mice
Comp.
Dose (mg/kg)
No. of impulses9SEM after 30 min
222.4948.3
ED50 (mg/kg)
36.6
Therap. index
Control
6b
**
100
50
34.0919.1
ꢀ54.6
***
84.2914.2
(17.1ꢀ
/
78.3)
25
145.0941.0
**
6h
6j
100
50
39.4916.2
86.2921.0
37.1
ꢀ53.9
(18.5ꢀ
/
74.2)
71.6)
**
100
50
26.498.2
79.4917.4
127.0919.6
31.4
*
(13.7ꢀ
/
ꢀ63.7
ꢀ43.4
ꢀ58.1
25
*
6k
6f
100
50
74.3920.3
92.4925.1
46.1
(17.1ꢀ
/
124.5)
96.3)
57.8)
**
100
50
46.2919.0
114.0917.6
34.4
(12.3ꢀ
/
**
6g
100
50
35.6917.3
54.2920.0
24.7
*
(10.6ꢀ
/
ꢀ81.0
25
117.0916.4
Each group consisted of 6ꢀ8 animals.
/
* P B0.05.
** P B0.01
*** P B0.001.
Table 3
Influence of the investigated compounds 6 on the thiopental anaesthesia
Comp.
Dose (mg/kg)
Time of duration of anaesthesia9SEM (min.)
36.0910.1
% of control
Control
6b
****
100
50
110.0914.4
84.2914.2
205.5
133.9
16.9
***
25
42.1912.4
****
6h
6j
100
50
141.0910.2
98.0912.1
42.0911.1
291.7
172.2
16.7
***
25
***
100
50
138.0921.2
82.4918.2
51.0912.4
283.3
128.9
41.7
**
25
**
6k
6f
100
50
105.4929.1
88.9918.2
39.2917.1
192.8
146.9
8.9
*
25
***
100
50
109.4918.8
84.2921.8
44.8919.1
203.9
133.9
24.4
*
25
***
6g
100
50
124.0914.1
54.2912.5
244.4
50.5
Each group consisted of 6ꢀ8 animals.
/
* P B0.05.
** P B0.02.
*** P B0.01.
**** P B0.001.
test and weak analgesic effect in the ‘hot-plate’ test in
mice, i.e. two behaviourally different models. The
analgesic potency of the compounds in ‘writhing’ test
and 4ꢀ10 times weaker than that of morphine (Table 4).
/
The ‘writhing’ test in mice has been used by many
investigators for measuring peripheral analgesic activity,
whereas the ‘hot-plate’ test for evaluating central
was 1.5ꢀ4 times higher than that of acetylsalicylic acid
/

742
W. Malinka et al. / Il Farmaco 57 (2002) 737ꢀ746
/
analgesia [13]. Then we initially presumed that com-
pounds 6 possess strong peripheral analgesic action [50ꢀ
12.5 mg/kg (1/40ꢀ1/160 of LD50)] with partially weak
compound 6k (50 mg/kg; Xꢁ
/
m-Cl, Yꢁp-Br) in this
/
/
test, although electron-attracting X and Y substituents
in both compounds occupy the same positions at the
aromatic rings.
/
central analgesic effect, observed only at the highest
dose equivalent to 1/10 of LD50 (200 mg/kg). Therefore,
the compounds were also screened for their anti-
inflammatory activity using carrageenan-induced hind
paw edema model in mice. However, all pyridothiazines
6 were inactive at this model of inflammation. Due to
interesting analgesic properties of compounds 6, further
studies will be performed in order to define the
mechanism of their analgesic action.
4. Peroxyl radical scavenging activity of compounds 6
Compounds 6a, 6c, 6g, 6g×
/
HCl, 6lꢀo were evaluated
/
for the free peroxyl radical scavenging activity using
total peroxyl radical-trapping antioxidant potential
(TRAP) measurements. The modified Valkonen and
Kuusi method was used in this study [14]. All investi-
Lipophilicity of compounds 6b, fꢀh, j, k, expressed as
/
log Pcalc, was differentiated and ranged from 2.7 to 4.6
(Table 1). However, it is difficult to correlate these
parameters with potency of analgesic action of com-
pounds observed in the ‘writhing’ test (Table 4). For
example, the value of log Pcalc (4.57) for the most active
gated compounds and reference substances [N-acetyl-L-
cysteine (NAC), cyanidin (CC)] were evaluated in the
final concentration 0.1 mM. However, it should be
noted that for compounds 6a, 6c, 6m there are not data
on account of their not sufficient solubility in the test
system.
compound 6j (12.5 mg/kg; Xꢁ
/
m-CF3, Yꢁ/p-Cl) was
close to the value of log Pcalc
Â/4.48 of the less active
Table 4
Influence of the investigated compounds 6 on the pain reactivity in the ‘writhing syndrome’ test in mice
Comp.
Dose (mg/kg)
Mean no. of writhings9SEM
19.892.0
ED50 (mg/kg)
Control
6b
0
50
****
2.591.2
5.292.7
6.693.8
****
25
10.2
(4.3ꢀ
***
12.5
6.25
/
24.0)
14.193.1
****
6h
6j
50
25
4.792.0
5.992.2
11.594.3
**
14.8
12.5
(5.5ꢀ/40.0)
***
50
25
5.892.4
5.291.6
6.892.8
***
9.4
**
12.5
(2.8ꢀ/31.5)
6k
6f
6.25
50
13.292.4
4.692.4
12.494.3
***
25.05
25
(15.3ꢀ
/41.1)
***
50
25
7.792.4
9.292.2
*
26.4
12.5
13.292.4
(7.5ꢀ/92.9)
****
6g
50
25
4.792.3
7.793.2
***
22.2
12.5
14.793.9
(11.4ꢀ/43.3)
Control
Acetylsalicylic acid
0
19.293.2
3.291.2
8.591.3
11.292.1
****
100
50
30
**
39.15
(29.1ꢀ/48.4)
****
Morphine
10
3
1.290.8
7.592.9
**
2.44
1
16.293.5
(1.18ꢀ/5.02)
Each group consisted of 6ꢀ/8 animals.
* P B0.05.
** P B0.02.
*** P B0.01.
**** P B0.001.

W. Malinka et al. / Il Farmaco 57 (2002) 737ꢀ
/
746
743
series 6 should be directed towards syntheses of analo-
gues with reduced central activity in relation to anti-
oxidant properties. Syntheses of such compounds are
under consideration.
5. Chemical experimental section
M.p.s are uncorrected. 1H NMR spectra were ob-
tained with a Tesla spectrometer 80 MHz in CDCl3; the
chemical shifts are reported in d (ppm). IR(KBr) spectra
were recorded on Specord-75 IR spectrometer. Elemen-
tal C, H, N analyses were run on a Carlo Erba NA-1500
Fig. 3. Antioxidant capacity of NAC, compounds 6 and CC. Data
presented as means9SD.
/
The ability of pyridothiazines 6 to inhibit peroxyl
radicals generated by thermal decomposition of AAPH
is summarized in Fig. 3.
analyser, the results were within 90.4% of the values
/
calculated for the corresponding formulas. Chromato-
graphic separations were performed on a silica gel
The data from Fig. 3 furnished evidence that every
pyridothiazines 6 tested, without exception, possess
antioxidant component at their structure. The anti-
oxidant potential of these compounds is similar to that
of NAC and four times lower then cyanidin used as
standards. Based on the partial similarity of compounds
6 and tenoxicam we may assume that mechanism of
their antioxidant action may by similar to that proposed
for tenoxicam (Fig. 2).
[Kieselgel 60 (70ꢀ230 mesh), Merck] column (CC).
/
5.1. General procedure for the preparation of
2-(4-substituted-phenacyl)isothiazolopyridine-1,1-
dioxides 2bꢀe
/
A solution of 1.05g (5 mmol) of isothiazolopyridine-
1,1-dioxide (1) [10], 0.7 ml (5 mmol) of N(C2H5)3 and 5
mmol of corresponding v-bromoacetophenone (2-bro-
mo-4?-methylacetophenone for obtaining (2b), 2-bromo-
4?-methoxyacetophenone (2c), 2-bromo-4?-chloroaceto-
phenone (2d), 2,4?-dibromoacetophenone (2e)) in 7 ml of
DMF was stirred overnight at room temperature. The
mixture was then diluted with ice-water and the
separated precipitate was filtered off. The resulting
In addition the relationship between the log of the
octanolꢀwater partition coefficients (log Pcalc; Table 1)
/
and potency of antioxidant properties of compounds 6
(Fig. 3) was investigated. The compounds (bases)
exhibited a wide range value of log Pcalc (1.72ꢀ
and in general increase of lipophilicity lower their
antioxidant properties [compare 6g (log Pcalc 3.80)
and its salt 6g×HCl; 6g and 6o (log Pcalc 2.00)]. The
/4.85)
ꢁ
/
/
ꢁ
/
crude products 2bꢀ
from appropriate solvents; yield: 50ꢀ
2b: Anal. C17H16N2O4S (m.w. 344.38); m.p. 177ꢀ
/
e were purified by crystallization
same trend was observed in series of derivatives of
pyrrolo[1,2-b]pyridazine [15]. Moreover, similar effects
of antioxidant action and lipophilicity of compounds 6g,
/70%.
/
1
179 8C (EtOH). H NMR: 2.43 s (3H, CH3Ã
s (3H, CH3), 2.74 s (3H, CH3), 5.11 s (2H, CH2), 7.3 d
(2H, 2ArH) and 7.8 d (2H, 2ArH). Jꢁ7.8 Hz, 7.36 s
/
Ar), 2.71
6l (log Pcalc
benzylpiperidine analogue 6n (log Pcalc
ꢁ
/
3.80 and 4.85, respectively) and their 4-
4.35) demon-
ꢁ
/
/
strated that terminal arylpiperazinyl grouping of the 2-
side chain is not essential for antioxidant action in the
series of pyridothiazines type 6.
(1H, 5-Hb-pyridine). IR: 1730 and 1700 (CO).
Similar 1H NMR and IR data occur in all derivatives
of general formula 2 [2c: C17H16N2O5S (m.w. 360.38),
m.p. 206ꢀ
364.80), m.p. 229ꢀ
(m.w. 409.25), m.p. 202ꢀ
/
208 8C (MeOH); 2d: C16H13ClN2O4S (m.w.
231 8C (CHCl3); 2e: C16H13BrN2O4S
204 8C (EtOH)].
4.1. Conclusion
/
/
The results of the preliminary evaluation of pyrido-
thiazines 6 suggest that these compounds are not toxic
5.2. Rearrangement of isothiazolopyridines 2bꢀ
corresponding derivatives of 3-(4?-substituted-aroyl)-4-
hydroxypyridothiazines 3bꢀ
/
e to the
(LD50ꢀ2000 mg/kg), possess sedative influence on the
/
CNS in mice and significant peripheral analgesic activ-
ity, notably 6b and 6j (12.5 mg/kg in ‘writhing’ test).
However, the analgesic activity shown by compounds 6
in the ‘writhing’ test was not accompanied by their
inhibition of carrageenan induced endema. Moreover,
several of title pyridothiazines possess peroxyl radical
scavenging activity of equal potency to that found in
NAC. In the light of nonspecific CNS effects (analgesia,
depression of locomotor activity, prolongation of bar-
biturate induced sleeping time) further investigations in
/
e
Three millimole of corresponding isothiazolopyridine-
1,1-dioxide 2bꢀe (2b for obtaining 3b, 2c for 3c, 2d for
3d, 2d for 3d) in EtOH solution of EtONa (7.5 ml),
/
prepared from 2.3 g of Na and 100 ml of anhydrous
EtOH, was warmed up to 55ꢀ60 8C and maintained at
/
these temperatures with well stirring for 15 min. The
resulting mixture was then diluted with ice-cold water
(40 ml), filtered with charcoal and the filtrate was

744
W. Malinka et al. / Il Farmaco 57 (2002) 737ꢀ746
/
acidified with 5% aq. HCl. Precipitated product was
filtered off and purified by crystallization from EtOH to
5.5. General procedure for the preparation of
pyridothiazines 6aꢀ
/
n
give 3bꢀ
3b: Anal. C17H16N2O4S (m.w. 344.38)); m.p. 230ꢀ
2328C. 1H NMR: 2.42 s (3H, CH3Ã
Ar), 2.63 s (3H,
CH3), 2.77 s (3H, CH3), 7.27 d (2ArH) and 7.97 d
(2ArH). Jꢁ8.2 Hz, 7.28 s (1H, 6-Hb-pyridine), 16.4 br
(1H, 4-OH, enol, D2O exchangeable), position of 2-NH
signal was not established. IR: 3200 (NH), 1610ꢀ1540
/
e (yield: 45ꢀ
/
60%).
/
To the stirred mixture of 5 mmol of pyridothiazine 3
in 20 ml of anhydrous EtOH, was added 5 ml of EtOH
solution of EtONa, prepared from 2.3 g of Na and 100
ml of anhydrous EtOH. After 1 h, 5 mmol of corres-
ponding 4-(substituted-phenyl)-1-(3-chloropropyl)piper-
azine 4 or 4-substituted-1-(3-chloropropyl)piperidine 5
[3a [1] and 4-o-methoxyphenyl-1-(3-chloropropyl)piper-
azine (4a) was added for obtaining 6a; 3a and 4-m-
chlorophenyl-1-(3-chloropropyl)piperazine (4b) [11,12]
for 6b, 3a and 4-m-trifluoromethylphenyl-1-(3-chloro-
propyl)piperazine (4c) [12] for 6c; 3b and 4a for 6d; 3b
and 4c for 6e; 3c and 4a for 6f; 3c and 4c for 6g; 3d and
4a for 6h; 3d and 4b for 6i; 3d and 4c for 6j; 3e and 4b for
6k; 3e and 4c for 6l; 3a and 4-phenyl-1-(3-chloropropyl)-
1,2,3,6-terahydropyridine (5a) for 6m, 3b and 4-benzyl-
1-(3-chloropropyl)piperidine (5b) for 6n] and the reac-
tion mixture was refluxed with stirring for 15 h. Ethanol
was distilled off, the residue was treated with 50 ml of
CHCl3 and insoluble materials were filtered off. The
filtrate was then evaporated and the residue was purified
by crystallization from appropriate solvents (Table 1) to
/
/
/
[C(OH)Ä
/
CÃ
/
C(O)].
Similar 1H NMR and IR data occur in all derivatives of
general formula 3 [3c: C17H16N2O5S (m.w. 360.38), m.p.
226ꢀ
240ꢀ
242ꢀ
/
2288C; 3d: C16H13ClN2O4S (m.w. 364.80), m.p.
2428C; 3e: C16H13BrN2O4S (m.w. 409.25), m.p.
2448C].
/
/
5.3. 4-o-Methoxyphenyl-1-(3-chloropropl)piperazine
(4a)
Compound 4a was prepared in analogy to the
procedure described in Ref. [12], starting from 1.9 g
(12 mmol) of 1-bromo-3-chloropropane and 1.9 g (10
mmol) of N-(o-methoxyphenyl)piperazine. The crude
give 6 (yield: 45ꢀ
/
55%).
6d: H NMR: 1.1ꢀ1.5 m (2H, CH2CH2CH2), 1.95ꢀ
2.1 m (2H, CH2CH2CH2NÃpiperazine), 2.25ꢀ2.4 m
[4H, N(CH2)2], 2.44 s (3H, CH3Ar), 2.69 s (3H, CH3),
2.79 s (3H, CH3), 2.9ꢀ3.4 m [6H, (CH2)2NÃArꢃ2-
NCH2], 3.83 s (3H, OCH3), 6.8ꢀ7.0 m (4H, ArH), 7.2ꢀ
7.4 m (3H, 2ArHꢃHb-pyridine), 7.95ꢀ8.1 m (2H, ArH),
position of 4ÃOH (enol) signal was not established. IR:
2750ꢀ2200 (OH, enol), 1600ꢀ1560 [C(OH)ÄCÃC(O)].
6k: 1H NMR (CDCl3): 1.05ꢀ
1.7 (2H,
CH2CH2CH2), 1.9ꢀ2.4 m [6H, CH2N(CH2)2], 2.7 s
(3H, CH3), 2.78 s (3H, CH3), 2.95ꢀ3.3 m [6H, 2-
NCH2ꢃ(CH2)NÃAr], 6.7ꢀ7.35 m (5H, 4ArHꢃHb-pyr-
idine), 7.64 d (2H, ArH, Jꢁ8.6 Hz), 8.0 d (2H, ArH,
Jꢁ8.6 Hz), position of 4-OH (enol) signal was not
established, IR: 2750ꢀ2200 (OH, enol), 1600ꢀ1560
[C(OH)ÄCÃC(O)].
product was purified with CC [Rfꢁ
41ꢀ438C (65% yield; 51ꢀ52 8C (petroleum ether)].
1H NMR: 1.85ꢀ
2.2 m (2H, CH2CH2CH2), 2.5ꢀ2.8 m
[6H, CH2N(CH2)2], 3.05ꢀ3.2 m [4H, ArN(CH2)2], 3.61 t
(2H, CH2Cl, Jꢁ6.4 Hz), 3.84 s (3H, OCH3), 6.9ꢀ7.05 m
(4H, ArH).
/0.6 (EtOAc), m.p.
1
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
5.4. 4-Phenyl-1-(3-chloropropyl)-1,2,3,6-
tetrahydropyridine (5a) and 4-benzyl-1-(3-
chloropropyl)piperidine (5b)
/
m
/
/
/
/
/
/
Compounds 5a and 5b were prepared from 1-bromo-
3-chloropropane and 4-phenyl-1,2,3,6-tetrahydropyri-
dine or 4-benzylpiperidine, respectively, in analogy to
the procedure described in Ref. [12]. The crude products
/
/
/
/
/
/
1
were purified with CC (EtOAc): 5a (Rfꢁ
/
0.7, m.p. 38ꢀ
/
Similar H NMR and IR data occur in derivatives
6aꢀc, eꢀj, l.
6m: H NMR: 1.25ꢀ
2.6 m (6H, NÃCH2ꢃ2?,3?CHÃ
CH3), 2.78 s (3H, CH3), 2.9ꢀ
piperidine), 3.1ꢀ3.4 m (2H, 2Ã
(1H, 5?CHÃpiperidine), 7.25ꢀ7.65 m (9H, 8ArHꢃ
pyridine), 8.0ꢀ
(enol) signal was not established, IR: 2750ꢀ
enol), 1600ꢀ1560 [C(OH)ÄCÃC(O)].
6n: 1H NMR: 0.95ꢀ
2.0 m (7H, CH2CH2CH2ꢃ
3?,4?,5?-CHÃ
CH2N(CH2)2ꢃ
7.0ꢀ7.4 m (8H, 7ArHꢃ
ArH), position of 4-OH (enol) signal was not esta-
408C, 45% yield); 5b (Rfꢁ0.57, oil, 55% yield). 5b was
/
/
/
1
dissolved at C3H6O, acidified with HCl (saturated EtOH
solution) and the resulting precipitate was filtered off to
/
1.6 m (2H, CH2CH2CH2), 2.1ꢀ
/
/
/
/piperidine), 2.68 s (3H,
give 5b×
/
HCl [Anal. C15H22ClN×
/
HCl (m.w. 288.26), m.p.
/
3.05 m (2H, 6?CHÃ
/
162ꢀ1658C].
/
/
/
NCH2), 5.9ꢀ6.05 m
/
1
5a: Anal. C14H18ClN (m.w. 235.75). H NMR: 1.85ꢀ
/
/
/
/
Hb-
2.25 m (2H, CH2CH2CH2), 2.55ꢀ
2?,3?-CHÃpiperidine), 3.2 d (2H, 6?CHÃ
2.4 Hz), 3.65 t (2H, ClÃCH2, Jꢁ6.4 Hz), 6.06 t (1H,
5?CHÃpiperidine, Jꢁ2.4 Hz), 7.25ꢀ7.5 m (5H, ArH).
5b: 1H NMR: 1.2ꢀ
2.1 m (9H, CH2CH2CH2ꢃ3?,4,?5?-
CHÃpiperidineꢃNCH2), 2.3ꢀ2.95 m [6H, 2?, 6?CHÃ
piperidineꢃArCH2], 3.55 t (2H, ClÃCH2, Jꢁ6.8 Hz),
7.0ꢀ7.4 m (5H, ArH).
/
2.77 m (6H, NCH2ꢃ
/
/8.35 (m, 2H, ArH), position of 4-OH
/
/
piperidine, Jꢁ
/
/2200 (OH,
/
/
/
/
/
/
/
/
/
/
/
/
/
piperidine), 2.35ꢀ
/
2.8 m (17H, 3ꢄ
/
CH3ꢃ
(2H, 2-NCH2),
8.2 (m, 2H,
/
/
/
/
/
/
CH2Ar), 3.0ꢀ
/
3.3 m
/
/
/
/
/
Hb-pyridine), 7.7ꢀ
/
/

W. Malinka et al. / Il Farmaco 57 (2002) 737ꢀ
/
746
745
blished, IR: 2750ꢀ
/
2200 (OH, enol), 1600ꢀ
/1560
6.6. ‘Writhing syndrome’ test in mice according to
Hendershot and Forsaith [17]
[C(OH)ÄCÃC(O)].
/
/
For compound 6g, hydrochloride was prepared by
dissolving base 6 in C3H6O, acidification with HCl
MeOH solution and filtration of the resulting precipi-
tate.
The investigated compounds were administered in-
traperitoneally in doses 50ꢀ6.25 mg/kg, corresponding
to 1/40ꢀ1/320 of LD50. Twenty-five minutes later, 0.2%
/
/
phenylbenzoquinone was injected intrapertioneally in a
constant volume of 0.25 ml. Five minutes after injection
of the irritating agent, the number of ‘writhing’ episodes
in the course of 10 min was counted. Analgesic activity
was expressed by the following formula:
6. Pharmacological experimental section
6.1. Substances used
P
of writhing incidents in experimental group
100ꢂ
P
Thiopental Natrium (HEFA-FRENON Arzneimittel,
Germany), phenylbenzoquinone (INC Pharmaceuticals,
Inc. NY), Carrageenin (Viscarin, Marine Colloids Inc.)
of writhing incidents in control group
ꢄ100ꢁ% inhibition
The ED50 values and their confidence limits were
calculated according to the method of Litchfield and
Wilcoxon [16]. Acetylsalicylic acid and morphine were
used as reference analgesics.
6.2. Animals
The experiments were carried out on male Wistar rats
(body weight 120ꢀ
(body weight 18ꢀ
constant temperature facilities exposed to 12:12 h
lightꢀdark cycle and maintained on a standard pellet
diet and tap water was given ad libitum. Control and
experimental groups consisted of 6ꢀ8 animals each. The
/
250 g) and male Albino-Swiss mice
/26 g). Animals were housed in
6.7. ‘Hot plate’ in mice according to Eddy and Leimbach
[18]
/
Animals were placed individually on the metal plate
/
heated to 55ꢀ56 8C. The time (s) of appearance of the
/
investigated compounds were administered intraperito-
neally as the suspension in 0.5% methylcellulose in
constant volume of 10 ml/kg (mice) and 1 ml/kg (rats).
The statistical significance was calculated using a one-
way ANOVA or Student’s t-test.
pain reaction (licking or jumping) was recorded by a
stop-watch. The experiments were performed 30 min
after administration of the investigated compounds.
Morphine was used as a reference analgesic.
6.8. Influence on the blood pressure
6.3. Acute toxicity
Arterial blood pressure in the common carotic artery
of normotensive anaesthetized rats was measured using
a Datamax apparatus (Columbus Instruments). The
investigated compounds were injected intraperitoneally
in dose corresponding to ED50. The influence on the
blood pressure was monitored for 1 h.
The investigated compounds were injected intraper-
itoneally in increasing doses up to 2000 mg/kg. Each
dose was given to six animals. The number of dead mice
was assessed 24 h after the injection. LD50 were
calculated according to the method of Litchfield and
Wilcoxon [16].
6.9. Carrageenan-induced edema
6.4. Locomotor activity
The method based on the assay of Winter et al. [19]
The investigated compounds were injected intraper-
was used in groups of five Wistar rats (120ꢀ150 g). Hind
/
itoneally in doses of 100ꢀ
/
25 mg/kg, equivalent to 1/80ꢀ
/
paw edema was induced by a subcutaneous injection of
0.1 ml of 1% carrageenan aqueous solution into the left
hind paw. The drugs as suspension in 0.5% methylcellu-
lose were administered 1 h before carrageenan. The
control animals received an equal volume of vehicle (10
ml/kg). The edema value was determined immediately
before and 1, 2, and 3 h after carrageenan injection.
Percent edema inhibition was calculated according to
the following formula:
1/20 of LD50. Thirty minutes later, the mice were placed
in a cage with a photocell which registered the numbers
of movements of the animals, during the first 30 min.
6.5. Thiopental anaesthesia
Thiopental in narcotic dose (55 mg/kg) was injected
intraperitoneally 30 min after administration of the
tested compounds. Duration of narcotic sleep was
counted from disappearance to return of the righting
reflex.
C ꢂ D
% edema inhibitionꢁ
C

746
W. Malinka et al. / Il Farmaco 57 (2002) 737ꢀ746
/
[2] T. Zawisza, W. Malinka, Synthesis and properties of 2H-4-
hydroxy-2,5,7-trimethylpyrido[3,2-e]-1,2-thiazine-1,1-dioxide-3-
C, mean value of edema volume of control animals; D,
mean value of edema volume of treated animals.
carboxamides, Farmaco 41 (1986) 892ꢀ898.
/
[3] W. Malinka, M. Sieklucka-Dziuba, G. Rajtar-Cynke, K. Bor-
owicz, Z. Kleinrok, Studies on synthesis and biological properties
of pyrazolo[4,3-c]pyrido[3,2-e]-1,2-thiazine-5,5-dioxide bearing 4-
7. Peroxyl radical scavenging activity
substituted-1-piperazinylpropyl moiety, Farmaco 49 (1994) 783ꢀ
/
792.
7.1. Substances used
[4] R.K. Raghupathi, L. Rydelek-Fitzgerald, M. Teitler, R. Glenon,
Analogues of the 5HT1A serotonin antagonist 1-(2-methoxyphe-
nyl)-4-[4-(2-phthalimido)butyl]piperazine with reduced a1-adre-
2?,7?-Dichlorodihydrofluorescein diacetate (DCFH×
/
nergic affinity, J. Med. Chem. 34 (1991) 2633ꢀ2638.
/
DA), 2,2?-diazobis(2-amidinopropane) dihydrochloride
(AAPH) and phosphate buffered saline (PBS) tablets
were obtained from Sigma (St. Louis, MO, USA) and
cyanidin chloride (CC) from Alexis Biochemicals (San
Diego, CA, USA). Other reagents being of analytical
reagent grade were used without further purification.
The Milli-Q (Millipore, Bedford, USA) water system
was used to prepare all solutions.
[5] G. Caliendo, R. Di Carlo, R. Meli, E. Perissutti, V. Santagada, C.
Slilipo, A. Vittoria, Synthesis and trazodone-like pharmacological
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Eur. J. Med. Chem. 28 (1993) 969ꢀ974.
/
[6] J.J. Kulagowski, H.B. Broughton, N.R. Curtis, I.M. Mawer, M.P.
Ridgill, R. Baker, F. Emms, S.B. Freedman, R. Marwood, Shil
Patel, Smita Patel, C.I. Regan, P.D. Leeson, 3-[(4-Chlorophe-
nyl)piperazin-1-yl]methyl]-1H-pyrrolo[2,3-d]pyridine: an antago-
nist with high affinity and selectivity for the human dopamine D4
receptor, J. Med. Chem. 39 (1996) 1941ꢀ1942.
[7] K. Kogure, K.A. Hossmann, B.K. Siesjo (Eds.), Progress in Brain
Research, vol. 96, Elsevier, Amsterdam, 1993, p. 1.
/
7.2. Apparatus
[8] J.P. Gonzalez, P.A. Todd, Tenoxicam, a preliminary review of its
pharmacodynamic and pharmacokinetic properties, and thera-
TRAP measurements were performed using DigiScan
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/
[9] R.M. Facino, M. Carini, L. Saibene, Scavenging of free radicals
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Med. Chem. 329 (1996) 457ꢀ463.
[10] T. Zawisza, W. Malinka, Synthesis and properties of some
derivatives of 2H-4,6-dimethylpyrido[3,2-e]isothiazolin-3-one-
/
Peroxyl radicals were obtained from thermal decom-
position of AAPH (final concentration, 56 mM). In the
first step carbon radicals are formed in pairs which react
rapidly with oxygen molecules to give peroxyl radicals.
Their concentration was monitored photometrically, at
1,1-dioxide, Farmaco 41 (1986) 676ꢀ683.
[11] C. Pollard, W. Lauter, N. Nuessle, Derivatives of piperazine.
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/
arylpiperazines, J. Org. Chem. 24 (1959) 764ꢀ767.
/
504 nm, measuring the conversion of DCFH×
dichlorofluorescein (DCF). Reaction was performed in
50 mM PBS solution at temperature 24 8C. DCFH×DA
/DA to
[12] W. Malinka, M. Sieklucka-Dziuba, G. Rajtar, A. Rubaj, Z.
Kleinrok, Synthesis and pharmacological screening of some N-(4-
substituted-piperazin-1-ylalkyl)-3,4-pyrroledicarboximides,
/
was dissolved firstly in DMSO followed by the same
amount of water to obtain its final concentration, 14
mM. Analysed sample (final concentration, 0.1 mM)
shifts measured S-shape kinetic curve. Results are the
delay time (measured at half time of the reaction) of
competition kinetics during which antioxidant is con-
sumed. This parameter measures the total antioxidant
reactivity and is defined as the sum, over all the
antioxidant present in the sample, of the product of
reaction rate constant and concentration. The measure-
ments of the antioxidant capacity were repeated three
times for each sample and the results were averaged and
expressed relative to the average result for the control
samples containing no sample.
Farmaco 54 (1999) 390ꢀ401.
/
[13] H.G. Vogel, W.H. Vogel, Drug Discovery and Evaluation.
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/
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/
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/
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/
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/
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