Synthesis and analgesic activity of some side-chain modified anpirtoline derivatives

Article abstract of DOI:10.1002/(SICI)1521-4184(20005)333:5<107::AID-ARDP107>3.0.CO;2-Y

New derivatives of anpirtoline and deazaanpirtoline modified in the side chain have been synthesized, The series includes compounds 3 with side-chains containing piperidine or pyrrolidine rings, compounds 4 containing 8- azabicyclo[3.2.1]octane moiety, an

Full text of DOI:10.1002/(SICI)1521-4184(20005)333:5<107::AID-ARDP107>3.0.CO;2-Y

Full Papers
Synthesis and Analgesic Activity of Some Side-Chain Modified
Anpirtoline Derivatives
Stanislav Rádl, Petr Hezký, Jan Proška, Lucie Hejnová, and Ivan Krejcí
Research Institute of Pharmacy and Biochemistry, Kourimska 17, 13060 Prague, Czech Republic
Key Words: Anpirtoline analogs; [(hetero)arylsulfanyl]methylpiperidines; [(hetero)arylsulfanyl]methylpyrrolidines;
[(hetero)arylmethyl]piperazines; (hetero)aroylpiperazines; analgesic activity; 5-HT receptor ligands;
1A
5-HT receptor ligands
1B
Summary
New derivatives of anpirtoline and deazaanpirtoline modified in
the side chain have been synthesized. The series includes com-
pounds 3 with side-chains containing piperidine or pyrrolidine
rings, compounds 4 containing 8-azabicyclo[3.2.1]octane moiety,
and compounds 5 having piperazine ring in their side-chains. Their
receptor binding profiles (5-HT1A, 5-HT1B) and analgesic activity
(hot plate, acetic acid induced writhing) have been studied. Opti-
mized structures (PM3-MOPAC, Alchemy 2000, Tripos Inc.) of
the synthesized compounds 3–5 were compared with that of anpir-
toline.
Introduction
In our search for centrally acting analgesics of non-opioid
type we used anpirtoline (1a), a drug developed by ASTA
[1–5]
Medica
, as a lead. In spite of its high analgesic activity,
the drug has not become commercially available, probably
due to unspecified side effects. Structural modifications of
anpirtoline would therefore appear desirable. We have modi-
fied the anpirtoline structure in several possible ways. The
initial biological evaluation of deazaanpirtoline (1b) and
several of its derivatives, including homologues 2, showed
Chemistry
Pyridine derivatives 3a, 3e, and 3g were usually prepared
by alkylation of sodium 6-chloropyridin-2-ylthiolate with the
appropriate N-methyl chloromethyl derivatives in boiling
n-propanol. The corresponding phenyl derivatives 3c and 3i
were prepared similarly from 3-chlorothiophenole and a suit-
able base. The thiophenolate anion was generated in situ by
sodium hydride or potassium carbonate. These N-methyl
derivatives 3a, 3c, 3e, 3g, and 3i were treated with ethyl
these compounds to have weaker analgesic and binding prop-
[6,7]
erties than anpirtoline
. These findings inspired us to pre-
pare a series of similar pyrrolidine and piperidine derivatives
3, containing 6-chloropyridin-2-yl or 3-chlorophenyl group
as an aromatic part and a pyrrolidine or piperidine ring as a chloroformate and the intermediate N-ethoxycarbonyl de-
rivatives were usually isolated by flash chromatography and
then used without further purification to the following hy-
drolysis yielding the corresponding N-unsubstituted com-
pounds 3b, 3d, 3f, 3h, and 3j, respectively.
cyclic amino group connected to the aromatic part by a
methylsulfanyl bridge. We also decided to prepare com-
pounds 4 containing a β-tropanyl (8-azabicyclo[3.2.1]octan-
3-yl) moiety representing, in fact, a conformation-
ally-restricted piperidine ring.
There are many piperazine derivatives with profound cen-
trally mediated analgesic activity. Therefore, we also decided
to prepare piperazine derivatives 5.
N-Methyl derivatives 4a and 4b were prepared by a modi-
[8,9]
fied literature procedure
used for stereospecific synthesis
of tropan-3β-yl ethers and thioethers from tropine methane-
[10]
sulfonate . In this reaction, the starting α-mesylate reacts
with suitable (thio)phenolate to provide stereospecifically the
corresponding β-(thio)phenoxide. In our case, we treated the
starting tropine methanesulfonate with sodium 3-chlorothio-
phenolate and sodium 6-chloropyridin-2-thiolate to obtain
compounds 4a and 4b, respectively.
This paper describes the synthesis of the above mentioned
compounds, the binding properties of the prepared com-
pounds to serotonin 5-HT and 5-HT receptors, as well as
1A
1B
their analgesic activity.
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0365-6233/00/0505/0107 $17.50 +.50/0

108
Rádl, Hezký, Proška, Hejnová, and Krejcí
equivalent amounts of the reagents or high excess of
piperazine, corresponding bis-substituted piperazine 6 was
the only isolated product. When 1-ethoxycarbonylpiperazine
was used, the corresponding compound 5f was obtained. All
attempts to hydrolyze this compound to the required N-un-
substituted derivative 5h failed and only complex mixtures
were obtained. Compound 5g was finally prepared by reduc-
tion of 5e by borane formed in situ from sodium borohydride
and boron trifluoride etherate.
Results and Discussion
Scheme 1. i, chloro derivative, n-propanol, reflux; ii, chloro derivative,
DMF-1 iii, 1. ClCO2Et, toluene, 90 °C, 2. AcOH-HCl, reflux; iv, tropine
mesylate.
All the prepared compounds were evaluated for their anti-
nociceptive activity in two basic tests, the hot plate test and
the intraperitoneal writhing test, after oral application of
30 mg/kg of the tested compound. The antinociceptive activ-
Piperazine-containing derivative 5a was easily obtained as
its hydrochloride by acylation of piperazine with an equiva-
lent of 3-chlorobenzoyl chloride in acetonitrile without using
any base. Attempts to prepare similarly compound 5d using
3-chlorobenzylchloride, provided a complex mixture. How-
ever, treatment of 3-chlorobenzylchloride with 1-methyl-
piperazine or 1-ethoxycarbonylpiperazine in the presence of
triethylamine provided 5b and 5c, respectively. The hydro-
chloride of 5c was then hydrolyzed to provide 5d.
ity of anpirtoline is supposed to be mediated by 5-HT
1B
Table 1. 5-HT1A and 5-HT1B receptor binding and analgesic activity of the
prepared compounds.
——————————————————————————————
Compound
Binding studies
Analgesic activity (p.o.)
5-HT1A (%) 5-HT1B (%) Hot plate
Writhing
(%)
(%)
Preparation of the pyridine analogs 5e–5h started from
2-chloro-6-bromopyridine, which was treated with butyl-
lithium to form the corresponding lithio derivative that, upon
treatment with solid carbon dioxide, afforded moderate yields
of 6-chloropyridine-2-carboxylic acid. The acid was then
converted with thionyl chloride to the corresponding acyl
chloride. Pyridine derivative 5e was prepared from the chlo-
ride and 1-methylpiperazine. When we tried to prepare the
corresponding N-unsubstituted derivative using either
——————————————————————————————
22a)
40b)
51d)
61 e)
85
150
70
34
47
19
14
27
21
29
30
63
27
33
90
41
31
48
18
68
0
.
anpirtoline HCl
deazaanpirtoline HCl 53c)
.
.
2a maleate
82
.
2b HCl
79
34f)
.
3a Tos
66
.
3b Tos
40
60
.
3c HCl
54
n.d.
n.d.
n.d.
n.d.
67j)
n.d.
n.d.
n.d.
.
3d HCl
72
46
6
3e HCl
14g)
20h)
21i)
37
.
.
3f HCl
65
.
3g HCl
n.d.
.
3h HCl
5
.
3i HCl
82
16
.
3j HCl
76
n.d.
n.d.
.
4a HCl
102
100
25
74
37
n.d. k)
n.d. k)
n.d. k)
7
.
4b HCl
96
103
92
101
99
21
5a HCl
n.d.k)
n.d. k)
n.d. k)
38
.
.
5b 2HCl
116
89
.
5c HCl
76
.
5d 2HCl
88
85
.
5e HCl
89
94
12
n.d.
n.d.
.
5g HCl
93
96
22
——————————————————————————————
n.d. – not determined; a) – IC50 = 0.57 µM; b) – IC50 = 1.02µM; c) – IC50
=
d)
f)
1.47 µM; – IC50 = 2.45 µM; e) – IC50 = 2.25 µM; – IC50 = 0.64 µM;
h)
g)– IC50 = 0.40 µM; – IC50 = 0.42 µM; i) – IC50 = 0.40 µM; j) – IC50
=
Scheme 2. i, piperazine, CH3CN; ii, 1-methylpiperazine; iii, 1-ethoxycar-
bonylpiperazine; iv, HCl, reflux; v, NaBH4, BF3.Et2O.
1.34 µM; k) – toxic.
Arch. Pharm. Pharm. Med. Chem. 333, 107–112 (2000)

Modified Anpirtoline Derivatives
[4,5]
109
receptors
and involvement of 5-HT receptors in anal-
1A
gesic activity of some active compounds is also antici-
[11]
pated
and therefore receptor binding profiles towards
these receptor subtypes were also evaluated (Table 1).
The main reason for this work was to check the influence
of the structural changes of anpirtoline both on the analgesic
activity and on the binding properties toward 5-HT and
1A
5-HT serotonin receptor subtypes. For this purpose, we
1B
have analyzed the optimized structures (PM3-MOPAC, Al-
chemy 2000, Tripos Inc., 1998) of the synthesized com-
pounds 3–5 and compared them with that of anpirtoline. The
series contains mostly compounds derived from anpirtoline
and deazaanpirtoline by changing the amino group-contain-
ing side chain. However, none of these changes led to com-
pounds with better antinociceptive activity. Some
compounds active as analgesics, e.g. 3f and 3g also have some
levels of binding to the tested serotonin receptors; however,
some of them, e.g. 4a, apparently do not bind to the receptors.
On the other hand, compounds 3a and 3e with strong binding
Figure 1. Superimposition of anpirtoline (bold) with compound 3f (a) and
compound 3g (b).
to the 5-HT receptor subtype do not show any analgesic
1A
activity at all. This fact can suggest that the analgesic activity
of these analogs could be mediated by a quite different mode
of action. However, some of the compounds have strong
binding to the tested serotonin receptors, especially to the
[13]
5-HT subtype. It has been shown , that both serotonin
1A
and all non-indolic serotonergic agents contain both (het)aro-
matic part and a side chain containing ionisable nitrogen
atom. The optimized structures of some of these compounds
exhibit an interesting structural similarity. Superimposition
of the PM3 optimized conformers of the individual com-
pounds 3–5 and anpirtoline was performed with the RMS fit
procedure within Alchemy 2000. All six corresponding ring
atoms of the (het)aromatic part and the side-chain nitrogen
atom were selected as atom pairs for the fitting procedure.
The best fit was found for compounds 3a–3h; the RMS
deviation values of these compounds are in the range of
0.178–0.211 L, with the exception of 3c with RMS value of
0.305 L. The superimpositions of compounds 3f
(RMS = 0.178 L) and 3g (RMS = 0.186 L) with anpirtoline
are shown in Figure 1. Sterically fixed anpirtoline and
deazaanpirtoline analogs 4a and 4b displayed neither useful
levels of analgesic activity nor binding to the tested serotonin
receptors. It could be rationalized by the fact that the fit of the
optimized structures of anpirtoline with these compounds is
not so good as that of most compounds 3 having the RMS
deviation values of 0.390 L for 4a and 0.680 L for 4b.
Figure 2. Superimposition of compound 5e (bold) with anpirtoline (a) and
mCPP (b).
in Figure 2a and Figure 2b, respectively. Evidently, further
refinement of the model is required to explain the poor
affinities of these compounds. None of the compounds 5
displayed a useful level of analgesic activity; some of them
were toxic at the tested doses.
Acknowledgment
This study was supported by the Grant Agency of the Ministry of Industry
and Trade of the Czech Republic (grant No. PP-Z1/08), and by Léciva Praha.
Experimental Part
1-Aryl- and hetaryl-piperazines are well known serotonin
ligands; 1-(3-chlorophenyl)piperazine (mCPP) being one of
[12–14]
the best known 5-HT agonists
. On the other hand, the
1
General Methods
corresponding 3-chlorobenzoyl (5a), 3-chlorobenzyl (5d), as
well as its N-methyl (5b) and N-ethoxycarbonyl (5c) deriva-
tives do not seem to possess a significant affinity for either
The melting points were determined on a Kofler block and are not cor-
rected. The 1H and 13C-NMR spectra were measured on a Bruker DPX 250
spectrometer with tetramethylsilane as an internal standard. The purity of the
substances prepared was evaluated by TLC on pre-coated silica gel 60 TLC
plates F 254 (Merck). Elemental analyses indicated by the symbols of the
elements were within 0.4% of the theoretical values.
5-HT or 5-HT sites. A similar situation is also observed
1A
1B
with their pyridine analogs, namely compounds 5e and 5g.
This situation is a little difficult to interpret based on the
simple molecular energy optimization. It is true that the
optimized structures of these compounds cannot fit with the
corresponding mCPP model. However, the fit of the pyridine
derivatives 5e–5g with optimized anpirtoline is relatively
good, having the RMS values of 0.103, 0.184, and 0.272 L.
The fit of compound 5e with anpirtoline and mCPP is shown
The following starting compounds were prepared according to the pre-
viously described methods: sodium 6-chloropyridin-2-ylthiolate[1], 2-chlo-
romethyl-1-methylpiperidine[15], 3-chloromethyl-1-methylpiperidine[16]
,
3-chloromethyl-1-methylpyrrolidine[17], tosylate of 3α-mesyloxy-8-methyl-
8-azabicyclo[3.2.1]octane (tosylate of 3α-tropinemethanesulfonate)[18], 2-
bromo-6-chloropyridine[19]
.
Arch. Pharm. Pharm. Med. Chem. 333, 107–112 (2000)

110
Rádl, Hezký, Proška, Hejnová, and Krejcí
General Procedure for Preparation of N-Unsubstituted Derivatives 3b, 3d,
3f, 3h, and 3j by N-Demethylation
Syntheses
Ethyl chloroformate (2 mL, 20 mmol) was added to a solution of the
corresponding N-methyl derivative 3 (10 mmol) in dry toluene (50 mL) at
90 °C and the mixture was stirred at this temperature for 4 h. The insoluble
portion was removed by filtration through Celite and the filtrate was evapo-
rated under reduced pressure. The residue was dissolved in a mixture of
concentrated hydrochloric acid (20 mL) and acetic acid (20 mL) and the
mixture was refluxed for 24–48 h (TLC). Then the mixture was evaporated,
the residue dissolved in water and basified with a 20% solution of sodium
hydroxide and extracted with dichloromethane (5 × 20 mL). The extract was
dried with magnesium sulfate and the residue after evaporation was either
purified by flash chromatography and then converted into a suitable salt, or
directly converted into a salt and purified by crystallization.
General Procedure for the Preparation of Pyridine Derivatives 3a, 3e, and
3g
A mixture of sodium 6-chloropyridine-2-thiolate (3.35 g, 20 mmol), the
appropriate chloro derivative (22 mmol), and n-propanol (25 mL) was re-
fluxed for 16 h. The mixture was evaporated under reduced pressure, the
residue was treated with water (20 mL) and extracted with chloroform (5 ×
20 mL). The extract was dried with magnesium sulfate, evaporated, and the
residue was purified by flash chromatography. The pure yellowish oil was
converted to its suitable salt and crystallized. According to this method, the
following compounds were prepared:
Tosylate of 2-Chloro-6-(1-methylpiperidin-3-ylmethylsulfanyl)pyridine (3a)
Hydrochloride of 2-Chloro-6-(piperidin-3-ylmethylsulfanyl)pyridine (3b)
Yield 67%, mp 124–126 °C (ethyl acetate). 1H-NMR of the corresponding
base (CDCl3): δ= 1.15–1.50 (m, 8H, H-2′, H-4′, H-5′, H-6′), 2.86 (s, 3H,
CH3), 3.11 (Abq system, 2H, SCH2), 3.47 (m, 1H, H-3′), 6.98 (dd, J = 7.1 Hz,
J = 1.1 Hz, 1H, H-3), 7.08 (dd, J = 7.7 Hz, J = 1.0 Hz, 1H, H-5), 7.42 (t, J
= 7.7 Hz, 1H, H-4). Anal. (C19H25ClN2O3S2) C, H, Cl, N, S.
Yield 61%, mp 123–126 °C. 1H-NMR (d6-DMSO): δ = 1.02–2.79 (bm,
8H, H-2′, H-4′, H-5′, H-6′), 3.10–3.75 (bm, 4H, H-2′, H-5′), 2.85–3.39 ?(bm,
1H, H-3′), 3.12 (d, J = 6.3 Hz, 2H, SCH2), 6.97 (d, J = 7.7 Hz, 1H, H-3),
7.06 (d, J = 7.7 Hz, 1H, H-5), 7.39 (t, J = 7.7 Hz, 1H, H-4). Anal.
(C11H16Cl2N2S) C, H, Cl, N, S.
Hydrochloride of 2-Chloro-6-(1-methylpiperidin-2-ylmethylsulfanyl)-
pyridine (3e)
Hydrochloride of 3-(3-Chlorophenylsulfanylmethyl)piperidine (3d)
Yield 26%, mp 139–143 °C (ethyl acetate). 1H-NMR (CDCl3): δ = 1.15–
1.50 (m, 2H, H-5′), 1.75-2.05 (m, 2H, H-4′), 2.05-2.45 (m, 2H, H-3′),
2.80–3.05 (m, 2H, H-6′), 3.04, 3.06 (ds, 3H, CH3), 3.24 (dd, J = 13.5 Hz, J
= 9.4 Hz, 1H, SCH2), 3.52 (m, 1H, CH), 4.09 (dd, J = 13.5 Hz, J = 3.1Hz,
1H, SCH2), 7.07 (dd, J = 7.5 Hz, J = 0.7 Hz, 1H, H-3), 7.13 (dd, J = 7.5 Hz,
J = 0.7 Hz, 1H, H-5), 7.51 (t, J = 7.5 Hz, 1H, H-4). Anal. (C12H18Cl2N2S)
C, H, Cl, N, S.
Yield 43%, mp 115–118 °C (ethanol – ethyl acetate, 1:1). Anal.
(C12H17Cl2NS) C, H, Cl, N, S.
Hydrochloride of 2-Chloro-6-(piperidin-2-ylmethylsulfanyl)pyridine (3f)
In this case, purification of the base by flash chromatography (dichlo-
romethane – methanol, 9:1) was necessary. Yield 51%, mp 141–143 °C
(ethyl acetate). Anal. (C11H16Cl2N2S) C, H, Cl, N, S.
Tosylate of 2-Chloro-6-(1-methylpyrrolidin-3-ylmethylsulfanyl)-
pyridine (3g)
Hydrochloride of 2-Chloro-6-(pyrrolidin-3-ylmethylsulfanyl)pyridine (3h)
Yield 57%, mp 202–207 °C. 1H-NMR (d6-DMSO): δ = 1.80–1.95 (m, 2H,
H-4′), 2.30 (m, 1H, H-3′), 3.10–3.75 (bm, 4H, H-2′, H-5′), 3.78 (dd, J =
13.8 Hz, J = 9.2 Hz, 1H, SCH2), 4.09 (dd, J = 13.8 Hz, J = 4.2 Hz, 1H,
SCH2), 6.95 (dd, J = 7.9 Hz, J = 0.8 Hz, 1H, H-3), 7.10 (dd, J = 7.9 Hz, J
= 0.8 Hz, 1H, H-5), 7.45 (t, J = 7.9 Hz, 1H, H-4). Anal. (C10H14Cl2N2S) C,
H, Cl, N, S.
Yield 47%, mp 113–115 °C (ethyl acetate). 1H-NMR of the corresponding
base (CDCl3): δ = 1.99–2.79 (bm, 7H, H-2′, H-3′, H-4′, H-5′), 2.36 (s, 3H,
CH3), 3.24 (d, J = 6.6 Hz, 2H, SCH2), 6.96 (dd, J = 7.8 Hz, J = 1.0 Hz, 1H,
H-3), 7.05 (dd, J = 7.8 Hz, J = 1.0 Hz, 1H, H-5), 7.38 (t, J = 7.8 Hz, 1H,
H-4). Anal. (C18H23ClN2O3S2) C, H, Cl, N, S.
General Procedure for the Preparation of 3-Chlorophenyl Derivatives
3c and 3i
Hydrochloride of 3-(3-Chlorophenylsulfanylmethyl)pyrrolidine (3j)
Yield 55%, mp 185–187 °C. 1H-NMR (d6-DMSO): δ = 1.55 = 1.72 (m,
2H, H-4), 2.25 (m, 1H, H-3), 2.65–3.10 (bm, 6H, H-2, H-5, SCH2), 7.16–7.30
(m, 4H, H-2′, H-4′, H-5′, H-6′). Anal. (C11H15Cl2NS) C, H, Cl, N, S.
Sodium hydride (0.53 g, 50% dispersion in mineral oil, 11 mmol) was
added to a solution of 3-chlorothiophenol (1.44 g, 10 mmol) in DMF (15 mL)
and the mixture was stirred at room temperature under nitrogen atmosphere
for 30 min. Then a solution of the appropriate chloro derivative (10 mmol)
in DMF (5 mL) was added and the mixture was stirred for 24 h at room
temperature. The insoluble portion was filtered off and washed with DMF
(2 mL) and the filtrate was evaporated under reduced pressure. The residue
was treated with water (20 mL) and extracted with dichloromethane (5 ×
10 mL). The extract was dried with magnesium sulfate, evaporated and the
residue was purified by flash chromatography. The pure yellowish oil was
converted to its hydrochloride and crystallized. According to this method,
the following compounds were prepared:
Hydrochloride of 3-(6-Chloropyridin-2-ylsulfanyl)-8-methyl-8-aza-
bicyclo[3.2.1]octane (4a)
A mixture of sodium 6-chloropyridine-2-thiolate (2 g, 12 mmol), tosylate
of 3α-tropinemethanesulfonate (2 g, 5.1 mmol), and ethanol (100 mL) was
refluxed under argon atmosphere for 6 h. The mixture was evaporated to
dryness, the residue was mixed with water (25 mL), extracted with dichlo-
romethane (5 × 20 mL) and the extract was dried with magnesium sulfate.
The residue after evaporation was purified by flash chromatography (dichlo-
romethane, dichloromethane – methanol, 20:1). The combinedfractions were
evaporated, dissolved in methanol and acidified with an ethereal solution of
hydrogen chloride. The formed solid was filtered off and crystallized from
ethanol to give 4a as white crystals (0.65 g, 42%), mp 256–258 °C. Mass
Hydrochloride of 3-(3-Chlorophenylsulfanylmethyl)-1-methylpiperidine
(3c)
Yield 56%, mp 107–108 °C (ethyl acetate). Anal. (C13H19Cl2NS) C, H,
Cl, N, S.
1
spectrum, m/z (%): 268 (0.5), 124 (100), 94 (9), 82 (9), 67 (17). H-NMR
(DMSO-d6): δ = 1.92–2.52 (m, 8H, CH2), 2.68 (s, 3H, CH3), 3.86 (??bs, 2H,
NCH), 4.09 (m, 1H, SCH), 7.20 (d, J = 7.8 Hz, 1H, H-3′), 7.31 (d, J =
7.88 Hz, 1H, H-5′), 7. (t, J = 7.8 Hz, 1H, H-4′). 13C-NMR (d6-DMSO) δ =
24.80 (C-6, C-7), 33.38 (CH3), 35.90 (C-2, C-4), 39.10 (C-3), 63.87 (C-1,
C-5), 120.95 (C-5′), 122.18 (C-3′), 140.89 (C-4′), 150.74 (C-6′), 159.13
(C-2′). Anal. (C13H18Cl2N2S) C, H, Cl, N, S.
Hydrochloride of 3-(3-Chlorophenylsulfanylmethyl)-1-methylpyrrolidine
(3i)
Yield 68%, mp 98–101 °C (ethyl acetate). Anal. (C12H17Cl2NS) C, H, Cl,
N, S.
Arch. Pharm. Pharm. Med. Chem. 333, 107–112 (2000)

Modified Anpirtoline Derivatives
111
Hydrochloride of 3-(3-Chlorophenylsulfanyl)-8-methyl-8-azabicyclo-
6-Chloropyridine-2-carboxylic Acid
[3.2.1]octane (4b)
n-Butyllithium (10 mL of a 2.5 M solution in hexanes, 25 mmol) was
added to diethyl ether (40 mL) under argon at –78 °C and then a solution of
2-bromo-6-chloropyridine (3.84 g, 20 mmoles) in diethyl ether (25 mL) was
added dropwise. The mixture was stirred at –78 °C for 1 h before a large
excess of solid carbon dioxide (ca. 10 g) was added. The reaction mixture
was stirred at this temperature for additional 1 h, poured into cold water and
extracted with diethyl ether. The water layer was acidified with concentrated
hydrochloric acid and then extracted with chloroform. The extract was dried
with magnesium sulfate, evaporated, and the residue was crystallized from
ethyl acetate to give 2.1 g (67%) of colorless crystals, mp 185–188 °C
(Ref. [20] gives mp 190 °C). 1H-NMR (CDCl3): δ = 3.40 (bs, 1H, COOH),
7.60 (dd, J = 0.8 Hz, J = 7.8 Hz, 1H, H-5), 8.05 (m, 2H, H-3, H-4).
Sodium hydride (1 g, 55% dispersion in mineral oil, 23 mmol) was added
to a stirred solution of 3-chlorothiophenole (3.3 g, 22.8 mmol) in DMF
(40 mL) and the mixture was stirred under nitrogen atmosphere for 1 h. Then
a solution of 3α-tropinemethanesulfonate (2.5 g, 5.1 mmol), freshly prepared
from its tosylate, in DMF (15 mL) was added and the mixture was stirred at
100 °C under nitrogen for 5 h and then left to stand overnight. The insoluble
sodium methanesulfonate was filtered off, washed with DMF (5 mL), the
filtrate was acidified with concentrated hydrochloric acid to pH 2–3 and
evaporated under reduced pressure. The residue was mixed with water
(40 mL), extracted with diethyl ether (4 × 25 mL) and then basified with
saturated potassium carbonate. The mixture was extracted with chloroform
(4 × 10 mL) and the extract was dried with magnesium sulfate. The residue
after evaporation was dissolved in methanol and acidified with an ethereal
solution of hydrogen chloride. The formed solid was filtered off and crystal-
lized from ethanol togive 4b as white crystals (1.75 g, 50%), mp 218–222 °C.
1H-NMR (d6-DMSO): δ = 1.90–2.44 (m, 8H, CH2), 2.75 (s, 3H, CH3), 3.36
(m, 1H, SCH), 3.84 (bs, 2H, NCH), 7.18–7.35 (m, 4H, H-2′,H-4′, H-5′, H-6′).
13C-NMR (DMSO-d6) δ = 24.86 (C-6, C-7), 31.00 (C-2, C-4), 31.66 (CH3),
36.24 (C-3), 39.83 (C-1, C-5), 128.11 (C-6′), 130.31 (C-4′), 130.98 (C-2′),
133.08 (C-5′), 134.22 (C-3′), 135.16 (C-1′). Anal. (C14H19Cl2NS) C, H, Cl,
N, S.
6-Chloropyridine-2-carbonyl Chloride
A mixture of 6-chloropyridine-2-carboxylic acid (3.1 g, 20 mmol) and
thionyl chloride (35 mL) was refluxed for 3 h. The reaction mixture was
evaporated to give a slightly brown semisolid residue (3.5 g, 99%) that was
used for further reaction without purification.
Hydrochloride of 1-(6-Chloropyridine-2-carbonyl)-4-methylpiperazine (5e)
A solution of 6-chloropyridine-2-carbonyl chloride (1.76 g, 10 mmol) in
DMF (10 mL) was added to a stirred solution of 1-methylpiperazine (2.50 g,
25 mmol) in DMF (20 mL) and the mixture was stirred at room temperature
for 3 h. The mixture was poured into water (250 mL), extracted with ethyl
acetate (4 × 25 mL), the combined extracts were washed with brine (25 mL)
and dried with magnesium sulfate. The residue after evaporation was dis-
solved in ethanol, acidified with ethanolic hydrogen chloride and evaporated.
The residue was twice crystallized from ethanol to give compound 5e as
white crystals (1.6 g, 58%), mp 192–197 °C. 1H-NMR (d6-DMSO): δ = 2.78
(s, 3H, CH3), 3.05–3.95 (bm, 8H, CH2), 7.68 (d, J = 7.6 Hz, 2H, H-3′, H-5′),
8.05 (, J = 7.6 Hz t, 1H, H-4′). Anal. (C11H15Cl2N3O) C, H, Cl, N.
Hydrochloride of 1-(3-Chlorobenzoyl)piperazine (5a)
A solution of 3-chlorobenzoyl chloride (1.75 g, 10 mmol) in acetonitrile
(5 mL) was added dropwise to a solution of piperazine (0.86 g, 10 mmol) in
acetonitrile (10 mL) at 15–20 °C. The solution was stirred for 2 h and then
the mixture was left to stand overnight in a refrigerator. The insoluble portion
was filtered off and washed with diethyl ether to give 5a (2.35 g, 89%), mp
194–196 °C. 1H-NMR (d6-DMSO): δ = 3.33 (bs, 1H, HCl), 3.41 (bs, 4H,
CH2 of piperazine), 3.64 (bs, 4H, CH2of piperazine), 7.40–7.59 (m, 4H, H-2′,
H-4′, H-5′ H-6′). Anal. (C11H14Cl2N2O) C, H, Cl, N.
Dihydrochloride of 1-(3-Chlorobenzyl)-4-methylpiperazine (5b)
Dihydrochloride of 1-(6-Chloropyridine-2-ylmethyl)-4-methylpiperazine
(5g)
3-Chlorobenzyl chloride (3.2 g, 20 mmol) was added to a stirred solution
of 1-methylpiperazine (2.0 g, 20 mmol) and triethylamine (4.0 g, 40 mmol)
in chloroform (10 mL) and the mixture was stirred at room temperature for
6 h. Water (10 mL) was added and the mixture was extracted with chloroform
(4 × 20 mL), the combined extracts were washed with water (20 mL) and
dried with magnesium sulfate. The oily residue after evaporation was dis-
solved in diethyl ether (20 mL), a small amount of insoluble material was
filtered off and the filtrate was acidified with a saturated ethereal solution of
hydrogen chloride. The precipitate formed was filtered and crystallized from
Sodium borohydride (0.58 g, 15.3 mmol) was slowly added to a solution
of a base of compound 5e (1.7 g, 7.1 mmol) in THF (18 mL) and then boron
trifluoride etherate (2.6 g, 1.8 mmol) was added dropwise. The mixture was
stirred at room temperature for 30 min and then refluxed for 3 h. The mixture
was cooled to 10 °C, further portions of sodium borohydride (0.29 g,
7.6 mmol) and boron trifluoride etherate (1.3 g, 0.9 mmol) were added, the
mixture was stirred at room temperature for 30 min and then refluxed for 4 h
and then left to stand overnight at room temperature. 5M hydrochloric acid
was slowly added to the cooled mixture that was then refluxed for 2 h. The
cold mixture was basified with a 5M solution of sodium hydroxide to
pH 10–11 and extracted with ethyl acetate. The combined extracts were
washed with brine and dried with magnesium sulfate to give an oily residue,
which was converted into the corresponding dihydrochloride. Crystallization
with isopropyl alcohol gave dihydrochloride of compound 5g (1.1 g, 49%),
1
ethanol to give dihydrochloride of 5b (3.4 g, 57%), mp 222–225 °C. H-
NMR (d6-DMSO): δ = 2.77 (s, 3H, CH3), ?3.32 (m, 8H, CH2 of piperazine),
4.10 (s, 2H, CH2), 7.42? (m, 2H, H-4′, H-6′), 7.52 (ddd, J = 8.7 Hz, J =
4.4 Hz, J = 1.5 Hz, 1H, H-5′), 7.67 (bm, 1H, H-2′). Anal. (C12H19Cl3N2) C,
H, Cl, N.
Hydrochloride of 1-(3-Chlorobenzyl)-4-ethoxycarbonylpiperazine (5c)
1
mp 178–181 °C. H-NMR (d6-DMSO): δ = 2.28 (s, 3H, CH3), 2.25–2.75
Using the same procedure described for the preparation of 5b with
1-ethoxycarbonylpiperazine, compound 5c was prepared as hydrochloride in
66% yield, mp 188–191 °C. 1H-NMR (d6-DMSO): δ = 1.21 (t, J = 7.1 Hz,
3H, CH3), 3.12 (m, 4H, H-3, H-5), 3.75 (m, 4H, H-2. H-6), 4.10 (q, J = 7.1 Hz,
CH2), 4.31 (bs, 2H, CH2), 7.41–7.52 (m, 2H, H-4′, H-6′), 7.65 (m, 1H, H-5′),
7.82 (m, 1H, H-2′). Anal. (C14H20Cl2N2O2) C, H, Cl, N.
(bm, 8H, CH2 of piperazine), 3.65 (m, 2H, CH2), 7.25 (d, J = 7.6 Hz, 1H,
H-3′), 7.45 (d, J = 7.6 Hz, 1H, H-5′), 7.68 (t, J = 7.6 Hz, 1H, H-4′). Anal.
(C11H18Cl3N3O) C, H, Cl, N.
Biological Experiments
Dihydrochloride of 1-(3-Chlorobenzyl)piperazine (5d)
Acetic Acid-Induced Writhing
A mixture of hydrochloride of 5c (2 g, 6.3 mmol) and concentrated
hydrochloric acid (10 mL) was refluxed for 18 h. The mixture was evaporated
to dryness, the residue was crystallized from ethanol to give dihydrochloride
of 5d (1.2 g, 67%), mp 214–217 °C. 1H-NMR (d6-DMSO): δ = 3.34 (m, 4H,
H-3, H-5), 3.45 (m, 4H, H-2. H-6), 4.30 (bs, 2H, CH2), 7.47 (m, 2H, H-4′,
H-6′), 7.63 (m, 1H, H-5′), 7.78 (m, 1H, H-2′). Anal. (C11H17Cl3N2) C, H, Cl,
N.
Writhing was induced by intraperitoneal injection of 0.2 mL of 0.7%
solution of acetic acid to male NMRI mice 30 min after the administration
of 30 mg/kg of the tested compound[21]. Writhings were counted for 20 min,
compared with the control and expressed as decrease of the stretching
movements (%). The data were statistically analyzed using the paired t-test
(p < 0.05).
Arch. Pharm. Pharm. Med. Chem. 333, 107–112 (2000)

112
Rádl, Hezký, Proška, Hejnová, and Krejcí
Hot Plate Test
[6] S. Rádl, P. Hezký, I. Krejcí, J. Proška, Collect. Czech. Chem. Commun.
1999, 64, 363–376.
The hot-plate test was used to measure the response latencies according to
the method described earlier[22], with minor modifications. All animals (male
NMRI mice) were selected on the basis of their reactivity in the model. The
selected animals were placed into a glass cylinder and the plate temperature
was maintained at 54 °C. The time necessary to induce the licking reflex of
the forepaws or jumping was recorded. The measurement was done 30 and
60 min after oral administration of 30 mg/kg of the tested compound and the
results were expressed as prolongation of the licking latencies (%). The data
were statistically analyzed using the paired t-test (p < 0.05).
[7] S. Rádl, P. Hezký, J. Proška, I. Krejcí, Arch. Pharm. Pharm. Med.
Chem.1999, 332, 13–18.
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P. Kozikowski, Ed.), Raven Press, New York 1993, pp. 167–212, and
the references cited therein.
Serotonin radioligand displacement receptor binding assays were con-
ducted in the hippocampus of the rat brain for 5-HT1A receptors, and in the
rat striatum for 5-HT1B receptors, according to the published proce-
dures[23, 24]. Adult male rats were killed by cervical dislocation and decapi-
tation. Their brains were dissected, immediately frozen, and stored at –80 °C
until needed. [3H]-8-OH-DPAT (c = 0.25 nM) and [3H]-5-HT (c = 2.00 nM)
were used for labeling of 5-HT1A and 5-HT1B receptors, respectively. The
tested compounds were used in 10–6 M concentrations. The incubation was
terminated by filtration through Whatman GF/B glass fiber filters and the
bound radioactivity retained on filters was determined by liquid scintillation
spectrometry. Generally, the binding is expressed as the retained bound
radioligand (%) after the displacement period.
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Received: September 27, 1999 [FP428a]
Arch. Pharm. Pharm. Med. Chem. 333, 107–112 (2000)