Synthesis, analgesic activity and computational study of new isothiazolopyridines of Mannich base type

Article abstract of DOI:10.1016/j.farmac.2005.08.005

A series of new 4-arylpiperazine derivatives of isothiazolopyridine of Mannich base type and their non-4-arylpiperazine analogues (3 and 4) were synthesized and assayed as potential analgesic agents. Pharmacological assay demonstrated that all the compoun

Full text of DOI:10.1016/j.farmac.2005.08.005

Il Farmaco 60 (2005) 961–968
http://france.elsevier.com/direct/FARMAC/
Original article
Synthesis, analgesic activity and computational study
of new isothiazolopyridines of Mannich base type
a,
a
b
b
c
c
´
W. Malinka *, P. SwiTtek , B. Filipek , J. Sapa , A. Jezierska , A. Koll
a Department of Chemistry of Drugs, Wrocław Medical University, ul. Tamka 1, 50-137 Wrocław, Poland
b Laboratory of Pharmacological Screening, Faculty of Pharmacy, Jagiellonian University Medical College, ul. Medyczna 9, 30-688 Kraków, Poland
c Faculty of Chemistry, University of Wrocław, 14 F. Joliot-Curie, 50-383 Wrocław, Poland
Received 16 March 2005; received in revised form 16 August 2005; accepted 18 August 2005
Available online 11 October 2005
Abstract
A series of new 4-arylpiperazine derivatives of isothiazolopyridine of Mannich base type and their non-4-arylpiperazine analogues (3 and
4) were synthesized and assayed as potential analgesic agents. Pharmacological assay demonstrated that all the compounds prepared, without
exception, displayed significant activity in the mouse writhing assay. The analgesic action, expressed as ED50, was found to be 2–10 times
more potent than that of acetylsalicylic acid and 1.5–10 times weaker than that of morphine, these being used as standards. The toxicities
(LD50) of the investigated derivatives varied and ranged from 250 to 2000 mg/kg. Additionally, the computational investigations were per-
formed in order to find correlation between molecular structure and biological effects (toxicity, analgesic action) of discussed compounds.
Useful model was found for toxicity assessment.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Isothiazolopyridines; Analgesic activity; Structure–toxicity relationships
1. Introduction
It is known from literature [1–3], that benzoxazolinones
and oxazolopyridinones of Mannich base type with a phar-
macophoric 4-arylpiperazine moiety (I; Fig. 1) posses sig-
nificant analgesic activity as evidenced in the mouse during a
writhing test and very little or no antiinflammatory action in
the acute carrageenan-induced paw edema test in rats.
Fig. 1
.
In this context, recently we described the synthesis and
analgesic action of a series of related derivatives of
isothiazolo[5,4-b]pyridine of the general structure II (Fig. 1)
[4]. In our studies we revealed that the potency of the anal-
gesic effect is highly influenced by the nature of the substitu-
ent R present at the aromatic ring of the 4-arylpierazine sub-
structure of II (Fig. 1). The best analgesic results were
obtained especially with the o-chlorophenylpiperazine deriva-
tive IIa [ED50 = 16.9 mg/kg], however, the good analgesic
action of this compound was accompanied by significant tox-
icity (LD50 = 83 mg/kg). In contrast, the use of the electron-
donating o-methoxy group (IIc; Fig. 1) leads to compound
practically inactive in this test. Replacement of the
4-arylpiperazine in II with 4-benzylpiperazine or 1,2,3,4-
tetrahydroisoquinoline produces a similar effect. On the other
hand, the analgesic action was retained after replacement of
the 4-arylpiperazine pharmacophore with 4-hydroxy-4-
phenylpiperidine or 4-benzylpiperidine (III: ED50 ~ 16 mg/kg,
LD50 ~ 320 mg/kg) [4].
Due to the considerable analgesic potency exhibited by
some isothiazolopyridins of type II and III (Fig. 1), the pri-
mary purpose of the present experiments was to synthesize
and evaluate the toxicity and analgesic action of a series of
related compounds. Most of these compounds were prepared
* Corresponding author. Tel.: +48 71 7840 399; fax: +48 71 7840 392.
E-mail address: malinka@bf.uni.wroc.pl (W. Malinka).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2005.08.005

962
W. Malinka et al. / Il Farmaco 60 (2005) 961–968
Scheme 1
.
by incorporating the new 4-arylpiperazine or 4-arylpiperidine
moiety possessing bromo, fluoro, trifluoromethyl or nitro sub-
stituents which, as R = Cl in compound IIa, are electron-
attracting in nature (Scheme 1: 3a–c, 4a–c, 4f). Additionally,
analogues of II (Fig. 1) with the side chain, which represent a
marked departure in the fragment of 4-arylpiperazine (3d–f)
were prepared in order to extend the structure-activity (SAR)
studies. Finally, we have undertaken computational study of
the new compounds 3 and 4 to find correlation between their
biological activity and molecular structure.
carboline was synthesized by the known method [6]. The
structures of the final compounds 3 and 4 were elucidated by
IR, 1H NMR and microanalyses and all data were in accor-
dance with the assigned structures. Melting points, yields and
spectral data are shown in Section 4. The spectral data within
the series of new compounds 3 and 4 did not show remark-
able differences from those of the isothiazolopyridines of
types II and III (Fig. 1), which we described earlier [4].
Finally, all compounds 3 and 4 were subjected to screen-
ing in an animal model to test their acute toxicity and anal-
gesic action.
2. Experimental procedures
2.2. Pharmacology
2.1. Chemistry
Several structural changes were made at the 4-aryl(pipe-
razine, piperidine) moiety of isothiazolopyridines II and III
(Fig. 1) in order to improve their analgesic action and reduce
their toxicity. The biological effects of the final twelve isothia-
zolopyridines 3 and 4 were measured using standard proce-
dures (see Exp. part). The results of their bioactivity and the
reference drugs (acetylsalicylic acid and morphine) are pre-
sented at Table 1.
The new compounds 3 and 4 were synthesized as illus-
trated in Scheme 1, starting from 2H-2-hydroxymethyl-4,6-
dimethyl-3-oxo-2,3-dihydroisothiazolo[5,4-b]pyridine (2) [5].
The preparation of the final isothiazolopyridines 3 and 4
involved condensation of the substrate 2 and the appropriate
derivatives of 1-arylpiperazine, 4-arylpiperidine or 1-cyclo-
hexylpiperazine, 1,2,3,4-tetrahydro-b-carboline and N-methyl-
N′-phenylethylenediamine. The arylpiperazine(piperidine)
intermediates used are illustrated in Scheme 1.
The reactions were carried out under mild conditions as
described in our previous paper [4] and gave, in several cases,
the desired products in high yield (80%). The piperazine, pip-
eridine and N-methyl-N′-phenylethylenediamine intermedi-
ates were commercially available; 1,2,3,4-tetrahydro-b-
2.2.1. Acute toxicity
The LD50 values of the investigated compounds 3 and 4
varied and ranged from ~250 to 2000 mg/kg (Table 1). Com-
pounds 3e and 4e were not toxic (LD50 > 2000 mg/kg), and
for these derivatives 200 mg/kg (1/10 LD50) was taken as the
initial dose in the further experiments. The most toxic com-
pound was 3a (LD50 = 255 mg/kg). The toxicity of the remain-

W. Malinka et al. / Il Farmaco 60 (2005) 961–968
963
Table 1
Acute toxicity and influence of investigated compounds 3 and 4 on the “writhing syndrome” induced by phenylbenzoquinone in mice
Compound
Dose
Mean number of writhings S.E.M.
ED50 (mg/kg)
LD50
Part of
LD50
mg/kg
Control
20.1 1.9
5.4 1.7*
9.9 2.5**
16.1 3.2
5.5 2.5*
9.1 3.1***
13.6 2.5
4.7 2.5*
9.8 3.1***
14.9 2.8
7.8 2.4**
10.0 1.2***
15.4 3.7
19.8 2.0
7.0 2.6*
7.2 3.2*
7.5 3.3**
10.2 3.6
20.1 1.9
4.2 1.7*
6.2 2.6**
10.0 1.9***
14.5 2.8
4.4 1.2*
9.6 3.2**
14.2 4.8
7.5 2.2*
10.2 3.1**
14.6 4.7
3a
255 (201–322)
1/40
1/80
1/160
1/40
1/80
1/160
1/40
1/80
1/160
1/40
1/80
1/160
6.4
3.4
3.2
(2.8–4.1)
1.6
3b
3c
3d
524 (392–687)
510 (401–621)
604 (524–720)
13.1
6.5
6.8
(5.7–8.2)
3.25
12.2
6.1
5.9
(4.2–8.3)
3.05
15.1
7.5
7.8
(5.6–10.9)
3.75
Control
3e
> 2000
1/40
50
4.9
1/80
25
(0.3–86.0)
1/160
1/320
12.5
6.25
Control
3f
668 (417–1069)
1/40
16.7
8.35
4.17
2.9
1/80
5.0
1/160
1/320
1/40
(2.6–9.6)
4a
4b
751(688-821)
522 (392–684)
18.8
9.4
9.9
1/80
(7.1–13.9)
1/160
1/40
4.7
13.0
6.5
6.6
1/80
(5.5–7.9)
1/160
3.25
4c
975 (842–1181)
1/40
1/80
1/160
24.4
12.2
6.1
5.9 2.1*
9.2 2.1**
12.2 6.7
19.8 2.0
5.2 3.1*
9.0 3.1***
11.4 6.8
8.0 2.2*
9.0 3.3**
12.0 5.1
20.1 1.9
7.6 2.1*
9.9 2.4***
13.8 2.1
19.2 3.2
3.2 3.2*
8.5 1.3**
11.2 2.1
1.2 0.8*
7.5 2.9**
16.2 3.5
11.6
(8.9–15.1)
Control
4d
902 (638–1658)
> 2000
1/40
1/80
1/160
1/40
1/80
1/160
50
8.4
25
(2.9–24.3)
12.5
50
4e
23.9
25
(4.0–141.5)
12.5
Control
4f
510 (401–621)
1/40
1/80
1/160
12.7
6.4
6.2
(5.2–7.4)
3.2
Control
Acetylsalicylic acid
–
–
–
–
–
–
100
50
30
10
3
39.15
(29.1–48.4)
Morphine
2.44
(1.18–5.02)
1
Each group consisted of six to eight animals. * P < 0.01; ** P < 0.02; *** P < 0.05.

964
W. Malinka et al. / Il Farmaco 60 (2005) 961–968
ing compounds fluctuated between 510 and 975 mg/kg. Toxic
doses of all the tested compounds caused sedation and depres-
sion of locomotor activity of mice.
Comparing the toxicities of the isothiazolopyridines II and
III tested previously [4] and their new analogues 3 and 4, we
can observe that, in general, the new compounds are signifi-
cantly less toxic than their precursors.
4-arylpiperazine (3) and of 4-arylpiperidine (4). For the
4-arylpiperazine series we also introduced 1,2,3,4-tetrahydro-
b-carboline-(3e) and N-methyl-N′-phenylethylenediamine-
(3f) derivatives. The 1,2,3,4-tetrahydro-b-carboline substruc-
ture can be considered as a rigid analogue of the
4-phenylpiperazine moiety, whereas the side chain of com-
pound 3f can be regarded as its extremely flexible analogue.
• In a recent report [4] we revealed that within the class of
isothiazolopyridines II (Fig. 1), the introduction of
electron-withdrawing substituents, such as o-Cl or m-CF3
to the aromatic ring of the 4-arylpiperazine moiety results
in compounds which exhibit analgesic action (IIa, b) [4].
A similar trend is observed in the series 3a–c, having
another substituents (o-F, p-NO2, m-Cl) which diminish
the electron density on the aromatic ring of the side chain.
• Another interesting finding is that replacement of the
4-arylpiperazine substructure with 1,2,3,4-tetrahydro-b-
carboline or N-methyl-N′-phenylethylenediamine (com-
pounds 3e and 3f, respectively) did not diminish analgesic
action. The above data show that the piperazine ring is not
necessary as a specific spacer for the aryl ring in the side
chain within the series II (Fig. 1) and 3.
• Taking into account the structure of isothazolopyridines I
and II (Fig. 1), we initially assumed that the presence of
the terminal aromatic ring in the side chain is an essential
requirement in eliciting analgesic action. Therefore, the
specific object for developing the structure-activity rela-
tionship was the preparation of the 4-cyclohexylpiperazine
derivative 3d, which was also introduced for pharmaco-
logical evaluation. The pharmacological study of 3d exhib-
ited, unexpectedly, that such an alicylic replacement of the
aromatic ring of the side chain results in an analgesic agent
equipotent to its 4-arylpiperazine analogues 3a–c. Hence,
these data did not support our assumption and suggested
that in the series of analgesic isothiazolopyridines of type
II and 3, even a very profound modification of the 4-aryl-
piperazine pharmacophore may be tolerated.
2.2.2. Analgesic properties
The analgesic activities of the compounds obtained in this
study were measured using the phenylbenzoquinone-induced
writhing assay and the hot plate test in mice. The data shown
in Table 1 clearly indicate that all the newly synthesized
isothiazolopyridines 3 and 4 demonstrated, without excep-
tion, strong analgesic action in the writhing assay. The ED50
values for 11 of the 12 tested compounds ranged between
3.4 and 11.6 mg/kg. The most potent effect was produced by
o-fluorophenylpiperazine-3a and the 1,2,3,4-tetrahydro-b-
carboline-3e derivatives, with values of ED50 = of 3.4 and
4.9 mg/kg, respectively. It should be noted that 3a was the
most toxic (LD50 = 255 mg/kg) while 3e the least toxic
(LD50 > 2000 mg/kg) derivative within the series of com-
pounds investigated. The weakest analgesic action was shown
by 4e with the 4-phenyltetrahydropyridine moiety
(ED50 = 23.9 mg/kg). By comparing the ED50 values of com-
pounds 3 and 4 and the reference drugs, we can see that a
similar analgesic action is observed with acetylsalicylic acid
after application at a dose of 39.15 mg/kg and with morphine
at a dose of 2.44 mg (Table 1). This indicates that the analge-
sic effects of the compounds 3 and 4 tested were ~2–10 times
more potent than that of acetylsalicylic acid and ~1.5–
10 times weaker than that of morphine.
In addition to examining analgesia with the writhing test,
compounds 3 and 4 were also evaluated with the hot plate
test. With the exception of 3b, 3e, 4a and 4c none of the
isothiazolopyridines investigated showed any activity at this
assay. However, the effect of activity at the hot plate test for
compounds 3b, 3e, 4a and 4c was observed only at the high-
est doses used, corresponding to 1/10 LD50.
The writhing test in mice has been used by many investi-
gators for measuring peripheral analgesic activity, whereas
the hot plate test is used for evaluating central analgesia [7].
In this context one can say that the isothiazolopyridines 3
and 4 possess an analgesic action similar to that of acetylsali-
cylic acid, however, elucidation of the mechanism of their
biological activity will require further investigation.
• In the series of 4-arylpiperidine derivatives 4, compounds
possessing an electron-attracting substituent on the aro-
matic ring of the 4-arylpiperidine moiety produce a more
significant analgesic effect than their unsubstituted precur-
sors (compare 4e and 4f). Introduction of a second with-
drawing substituent does not cause an increase in activity
(compare 4a and 4c).
3.2. Computational investigations
With the aim of establishing the role of the 2-substituent
as a pharmacophore which can participate in the toxic and
analgesic effects of isothiazolopyridines 3 and 4 (Scheme 1),
we undertook a computational investigations of this set of
compounds. From this study we excluded the recently
described [4] isothiazolopyridines II and III (Fig. 1), because
for these compounds and isothiazolopyridines 3 and 4 the
writhing syndrome was induced by different chemical stimu-
lants - 0.6% acetic acid for II, III and 0.02% phenylbenzo-
quinone for 3 and 4.
3. Results and discussion
3.1. SAR of isothiazolopyridines 3 and 4 (Table 1)
In spite of the limited number of compounds tested, on the
bases of the results obtained with the writhing test (Table 1),
we formulated some SAR. For SAR analyses, the new isothia-
zolopyridines were divided into two groups—derivatives of

W. Malinka et al. / Il Farmaco 60 (2005) 961–968
965
Computational investigations of correlation between bio-
logical activity and molecular structure started with deriva-
tion of descriptors values based on obtained geometries. On
the basis of descriptors space the structure–toxicity and struc-
ture–analgesic activity models were obtained. The analyzed
database contained 12 compounds so three-parameter equa-
tions were taken into account. The Fisher criterion threshold
for this number of variables and compounds is 3.71 [8]. The
equation describing structure–toxicity relationship contains
two CPSA and one topographical descriptors and is pre-
sented below:
arylpiperazine [4-cyclohexylpiperazine-(3d), 1,2,3,4-tetra-
hydro-b-carboline-(3e), N-methyl-N′-phenylethylene-
diamine-(3f)] and 4-arylpiperidine (4a–f) analogues. Phar-
macological evaluation demonstrated that all the new com-
pounds 3 and 4, without exception, possess significant anal-
gesic action in the writhing test. The analgesic action for 11 of
the 12 compounds assayed was 4–10 times higher than that
of acetylsalicylic acid and 1.5–5 times weaker than that of
morphine used for reference. The relatively weak difference
in analgesic potency of isothiazolopiyridines 3a–c with
4-arylpiperazine pharmacophore and their non-4-arylpipe-
razine analogues 3d–f suggests that a profound modification
of the 4-arylpiperazine moiety may be tolerated.As an impor-
tant result of computational analysis of isothiazolopyrid-
nines 3 and 4, the correlation between toxicity and molecular
structure was found. Much worse correlation was observed
between analgesic action and structural features. We hope that
obtained result of the computational investigations of isothia-
zolopyridines 3 and 4 may be helpful in design of novel and
more potent analgesic isothiazolopyridines of type 3 and 4
with reduced toxicity. Syntheses of such compounds are under
consideration.
logLD50 = –1.533 (1.052) + 0.179 (0.032) 3D Structural
Information Content (order2) – 110.955 (30.092) FPSA-
3 Fractional PPSA (PPSA-3/TMSA) + 48.029 (9.322) FNSA-
3 Fractional PNSA (PNSA-3/TMSA).
The obtained statistical parameters are: R2 = 0.826,
F = 12.62, s2 = 0.0157.
The equation is reasonable and statistically significant for
toxicity prediction for database of isothiazolopyridines 3 and
4. The comparison of experimental and calculated toxic effect,
represented by log LD50, is given in Table 2.
The data from Table 2 demonstrated that the model is able
to reproduce the toxicity quite accurately and may be useful
for further toxicity prediction for a series of similar com-
pounds.
4. Experimental
In the next step correlation between analgesic activity and
molecular structure was investigated using the same method-
ology. The obtained equation is presented as a result of the
analysis:
4.1. Chemical experimental section
logED50 = –3.804 (1.649) + 0.296 (0.103) Number of C
atoms + 0.483 (0.222) Kier Shape Index (order 3) – 0.161
(0.074) Bonding Information Content (order 0).
The obtained statistical parameters are: R2 = 0.516,
F = 2.84, s2 = 0.031.
On the basis of the Fisher criterion and low correlation
coefficient R2 value, the equation should be regarded as not
significant statistically.
Melting points are uncorrected. Proton NMR spectra were
obtained with Tesla [80 MHz] and Bruker [300 MHz] spec-
trometers [CDCl3, d (ppm)]. IR (KBr) spectra were recorded
on Specord M 80 (Carl Zeiss Jena). Elemental C, N, H analy-
ses were run on a Carlo Erba NA-1500 analyzer.All the results
of the C, N and H determinations were within 0.4% of the
values calculated for the corresponding formulae.
4.1.1. General procedure for preparation
of isothiazolopyridines 3a, 3c–f, 4a–f
3.3. Conclusion
We prepared new 4-arylpiperazine derivatives of isothiaz-
olopyridine of Mannich base type (3a–c), their non-4-
To a stirred mixture of 2.1 g (0.01 mol) of 2-hydroxy-
methylisothiazolopyridine 2 [5] in 40 ml of ethanol 0.01 mol
of an appropriate amine was added and stirring was contin-
ued for 24 h at room temp. Than the precipitated, crude prod-
uct (3c–e, 4a–e) was filtered off and crystallized with char-
coal from the appropriate solvent. In the case of compounds
3a, 3f and 4f the ethanol solution was evaporated and the
residue was crystallized from the appropriate solvent to give
pure product.
Table 2
Experimental and predicted toxicity for series of investigated compounds
Compound
Experimental
logLD50
2.407
Calculated logLD50
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
2.375
2.682
2.690
2.855
3.246
2.892
2.727
2.887
2.980
3.080
3.097
2.756
2.719
2.688
2.781
3.301
4.1.1.1. 2-[(4-o-Fluorophenylpiperazin-1-yl)methyl]-4,6-
dimethyl-3-oxo-2,3-dihydroisothiazolo[5,4-b]pyridine
(3a). Yield 1.85 g (50%) from 2 and 1-(o-fluorophenyl)-
piperazine, m.p. 85–86 °C (n-hexane). 1H NMR: 2.63 s (3H,
CH3), 2.77 s (3H, CH3), 2.85–3.00 m (4H, CHpiperazine), 3.05–
3.20 m (4H, CHpiperazine), 4.76 s (2H, CH2), 6.90–7.15 m (5H,
2.825
2.876
2.718
2.989
2.955
3.301
2.708
H
b-pyridine and 4ArH). IR: 1660 (C = O).

966
W. Malinka et al. / Il Farmaco 60 (2005) 961–968
1
4.1.1.2. 2-{[4-(5-Chloro-2-methylphenyl)piperazin-1-
yl]methyl}-4,6-dimethyl-3-oxo-2,3-dihydroisothiazolo[5,4-
b]pyridine (3c). Yield 2.2 g (55%) from 2 and 1-(5-chloro-
piperidine, m.p. 175–177 °C (ethanol). H NMR: 1.73–
2.45 m [5H, 3′ and 5′ CHpiperidine and OH (exchangeable
D2O)], 2.59 s (3H, CH3), 2.72 s (3H, CH3), 2.80–3.00 m (4H,
2′ and 6′ CHpiperidine), 4.72 s (2H, CH2), 6.93 s (1H, Hb-pyridine),
7.45 d (1H, ArH, J~9 Hz), 7.65 d (1H, ArH, J~9 Hz), 7.86 s
(1H, ArH). IR: 3100–2900 (OH), 1660 (C = O).
1
2-methylphenyl)piperazine, m.p. 139–142 °C (ethanol). H
NMR: 2.23 s (3H, CH3), 2.64 s (3H, CH3), 2.78 s (3H, CH3),
2.90–3.00 m (8H, CHpiperazine), 4.79 s (2H, CH2), 6.90–
7.20 m (4H, Hb-pyridine and 3ArH). IR: 1650 (C = O).
4.1.1.9. 2-[(4-Cyano-4-phenylpiperidin-1-yl)methyl]-4,6-
dimethyl-3-oxo-2,3-dihydroisothiazolo[5,4-b]pyridine
4.1.1.3. 2-[(4-Cyclohexylpiperazin-1-yl)methyl]-4,6-dimethyl-
3-oxo-2,3-dihydroisothiazolo[5,4-b]pyridine (3d). Yield 2.3 g
(65%) from 2 and 1-cyclohexylpiperazine, m.p. 158–160 °C
(ethanol). 1H NMR: 1.10–1.90 m (10H, CHcyclohexane), 2.15–
2.30 m (1H, CHcyclohexane), 2.59 s (1H, CH3), 2.65–2.85 m
(11H, 8Hpiperazine and CH3), 4.65 s (2H, CH2), 6.91 s (1H,
(4d). Yield 3.2
g (85%,) from 2 and 4-cyano-4-
phenylpiperidine, m.p. 177–179 °C (ethanol). 1H NMR: 2.10–
2.25 m (4H, 3′ and 5′ CHpiperidine), 2.62 s (3H, CH3), 2.71 s
(3H, CH3), 2.85–3.35 m (4H, 2′ and 6′ CHpiperidine), 4.77 s
(2H, CH2), 6.98 s (1H, Hb-pyridine), 7.30–7.60 m (5H, ArH).
IR: 2300 (CN), 1660 (C = O).
H
b-pyridine). IR: 1660 (C = O).
4.1.1.4. 2-[(1,2,3,4-Tetrahydro-b-carbolin-2-yl)methyl]-4,6-
dimethyl-3-oxo-2,3-dihydroisothiazolo[5,4-b]pyridine
(3e). Yield 3.1 g (85%), from 2 and 1,2,3,4-tetrahydro-b-
carboline [6], m.p. 154–157 °C (methanol). 1H NMR: 2.62 s
(3H, CH3), 2.79 s (3H, CH3), 2.86 t (2H, CH2, J ~6.5 Hz),
3.16 t (2H, N-CH2, J~6.5 Hz) 3.95 s (2H, N-CH2), 4.94 s
(2H, CH2), 6.93 s (1H, Hb-pyridine), 7.05–7.55 m (4H, ArH),
8.05 s [1H, NH (exchangeable D2O)]. IR: 3280 (NH), 1650
(C = O).
4.1.1.10.2-[(4-Phenyl-1,2,3,6-tetrahydropyridin-1-yl)methyl]-
4,6-dimethyl-3-oxo-2,3-dihydroisothiazolo[5,4-b]pyridine
(4e). Yield 1.75 g (50%) from 2 and 4-phenyl-1,2,3,6-
1
tetrahydropyridine, m.p. 154–156 °C (ethanol). H NMR:
2.55–2.70 m (5H, CH3 and 2CHpiperidine), 2.78 s (3H, CH3),
3.03 t (2H, CHpiperidine, J ~6 Hz), 3.49 d (2H, CH2-CH = ,
J~3 Hz), 4.85 s (2H, CH2), 6.10 t (1H, CH2-CH = , J~3 Hz),
6.94 s (1H, Hb-pyridine), 7.25–7.45 m (5H, ArH). IR: 1650
(C = O).
4.1.1.5. 2-{[N-(2-Anilinoethyl)-N-methylamino]methyl}-4,6-
dimethyl-3-oxo-2,3-dihydroisothiazolo[5,4-b]pyridine
(3f). Yield 2.7 g (80%) from 2 and N-methyl-N′-
phenylethylenediamine, m.p. 74–6 °C (n-hexane). 1H NMR:
2.54 s (3H, CH3), 2.60 s (3H, CH3), 2.78 s (3H, CH3), 2.85–
3.00 m (4H, NCH2CH2N), 4.82 s (2H, CH2), 6,99 s (1H,
4.1.1.11. 2-{[4-(p-Fluorophenyl)-1,2,3,6-tetrahydropyridin-
1-yl]methyl}-4,6-dimethyl-3-oxo-2,3-dihydroisothiazolo[5,4-
b]pyridine (4f). Yield 2.6 g (70%) from 2 and 4-(p-
fluorophenyl)-1,2,3,6-tetrahydropyridine, m.p. 106–107 °C
(n-hexane). 1H NMR: 2.55–2.70 m (5H, CH3 and
H
b-pyridine), 7.30–7.45 m (5H, ArH). IR: 3100–2800 (NH),
2CHpiperidine), 2.76 s (3H, CH3), 3.00 t (2H, CHpiperidine
,
1660 (C = O).
J ~5.5 Hz), 3.44 d (2H, CH2-CH = , J~3 Hz), 4.81 s (2H,
CH2), 6.00 t (1H, CH2-CH = , J~3 Hz), 6.90–7.40 m (5H,
4.1.1.6. 2-{[4-(p-Chlorophenyl)-4-hydroxypiperidin-1-
yl]methyl}-4,6-dimethyl-3-oxo-2,3-dihydroisothiazolo[5,4-
b]pyridine (4a). Yield 3.2 g (80%) from 2 and 4-(p-
chlorophenyl)-4-hydroxypiperidine, m.p. 178–180 °C
(toluene). 1H NMR: 1.70–2.20 m (4H, 3′ and 5′ CHpiperidine),
2.35 br [1H, OH (exchangeable D2O)], 2.58 s (3H, CH3),
2.70 s (3H, CH3), 2.80–3.05 m (4H, 2′ and 6′ CHpiperidine),
4.70 s (2H, CH2), 6,91 s (1H, Hb-pyridine), 7.20–7.50 m (4H,
ArH). IR: 3100–2930 (OH), 1670 (C = O).
Hb-pyridine and 4ArH). IR: 1660 (C = O).
4.1.2. 2-{[4-(p-Nitrophenyl)piperazin-1-yl]methyl}-4,6-
dimethyl-3-oxo-2,3-dihydroisothiazolo[5,4-b]pyridine (3b). A
mixture of 2.1 g (0.01 mol) of 2-hydroxymethyl-
isothiazolopyridine 2 [5] and 0.01 mol of 1-(p-nitro-
phenyl)piperazine in 40 ml of ethanol was refluxed with stir-
ring for 6 h. Afterwards the solvent was distilled of and the
residue was crystallized with charcoal from methanol to give
pure product.
4.1.1.7. 2-{[4-(p-Bromophenyl)-4-hydroxypiperidin-1-
yl]methyl}-4,6-dimethyl-3-oxo-2,3-dihydroisothiazolo[5,4-
b]pyridine (4b). Yield 3.1 g (70%) from 2 and 4-(p-
bromophenyl)-4-hydroxypiperidine, m.p. 183–185 °C
(ethanol). 1H NMR: 1.70–2.30 m [5H, 3′ and 5′ CHpiperidine
and OH (exchangeable D2O)], 2.59 s (3H, CH3), 2.71 s (3H,
CH3), 2.84–3.13 m (4H, 2′ and 6′ CHpiperidine), 4.70 s (2H,
CH2), 6,92 s (1H, Hb-pyridine), 7.28–7.50 m (4H, ArH). IR:
3050–2900 (OH), 1650 (C = O).
Yield 3.2 g (80%), m.p. 201–204 °C (methanol). 1H NMR:
2.60 s (3H, CH3), 2.74 s (3H, CH3), 2.80–2.95 m (4H,
CHpiperazine), 3.38–3.60 m (4H, CHpiperazine), 4.73 s (2H, CH2),
6.75 s (1H, Hb-pyridine), 6.85–7.00 m (2H, 2′ and 6′-ArH),
8.00–8.25 m (2H, 3′ and 5′-ArH). IR: 1660 (C = O).
4.2. Pharmacological experimental section
Materials and methods.
4.1.1.8. 2-{[4-(p-Chloro-m-trifluoromethylphenyl)-4-
hydroxypiperidin-1-yl]methyl}-4,6-dimethyl-3-oxo-2,3-
dihydroisothiazolo[5,4-b]pyridine (4c). Yield 3.1 g (65%)
from 2 and 4-(p-chloro-m-trifluoromethylphenyl)-4-hydroxy-
4.2.1. Substances
Acetylsalicylic acid (Polopiryna, ZF Starogard Gdañski,
PL), morphine (Morphinum hydrochloricum, Polfa-Kutno,

W. Malinka et al. / Il Farmaco 60 (2005) 961–968
967
PL), phenylbenzoquinone (INC Pharmaceuticals, Inc., New
4.3. Computational methodology
York).
Quantum-chemical calculations were performed for a
series of investigated compounds. The Gaussian03 suite of
programs [12] was used to build the molecules and optimize
their geometry. The ab initio Hartree–Fock–Roothaan method
and 6-31G(d,p) basis set were used [13,14]. The harmonic
vibrational frequencies were calculated confirming that the
obtained geometry corresponds to the minimum on the poten-
tial energy surface (PES). In the next step the descriptors were
evaluated using computer software for calculation of molecu-
lar descriptors and derivation of QSAR/QSPR [15,16]. The
obtained descriptor space contained 86 variables divided into
five groups: constitutional, topological, topographical, geo-
metrical and CPSA (charged partial surface area), as imple-
mented in the program. In order to find correlation between
biological activity and molecular structure stepwise selec-
tion of scales for the multiple linear regression (MLR) was
used [15,16]. The correlation coefficient (R2), Fisher crite-
rion (F) and standard error of the MLR (s2) gave information
about the quality of the proposed models.
4.2.2. Animals
The experiments were carried out on male Albino-Swiss
mice (body weight 18–26 g). All of the animals were housed
at constant humidity (60%) and temperature (24–25 °C) and
kept on a 12-h light/dark cycle. Animals were fed a standard
pellet diet with free access to tap water. All procedures were
conducted according to Animal Care and Use Committee
guidelines, and approved by the Ethical Committee of Jagiel-
lonian University, Kraków.
Control and experimental groups consisted of six to eight
animals each. The investigated compounds were adminis-
tered intraperitonelly as the suspension in 0.5% methylcellu-
lose in constant volume of 10 ml/kg.
4.2.3. Statistical analysis
The statistical significance was calculated using a Stu-
dent’s t-test.
The ED50 values and their confidence limits were calcu-
lated according to the method of Litchfield and Wilcoxon [9].
Acknowledgements
4.2.4. Acute toxicity
Acute toxicity was assessed by the methods of Litchfield
and Wilcoxon [9] and presented as LD50 calculated from the
mortality of mice after 24 h.
Authors gratefully acknowledge the Wrocław Center for
Networking and Supercomputing (WCSS) and Academic
Computer Center CYFRONET-KRAKÓW (Grant KBN/SGI/
UWrocl/078/2001) for providing computer time and facili-
ties.
4.2.5. “Writhing” syndrome in mice according to
Hendershot and Forsaith [10]
Different doses of test compounds ranging from 1/320 to
1/40 LD50 i.p. were administered intraperitoneally. Twenty-
five minutes later, 0.02% phenylbenzoquinone was injected
intrapertioneally in a constant volume of 0.25 ml. Five min-
utes after injection of the irritating agent, the number of
“writhing” episodes in the course of 10 min was counted.
The analgesic effect of individual doses was expressed in per-
cent:
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