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Synthesis of 1-Methyl-5-pyrazol-3- and -5-yl- and 1 2 4-triazol-3- and 5-yl-1 2 3 6-tetrahydropyridine Derivatives and Their Evaluation as Muscarinic Receptor Ligands.

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Arch. Pharm. Pharm. Med. Chem. 2003, 336, 143–154
a
b
c
d
Laboratorio di Chimica del
Farmaco,
Istituto Superiore di Sanità,
Rome, Italy
Dipartimento di Scienze
Farmacologiche e Medicina
Sperimentale,
Sezione di Anatomia
Umana, Camerino (MC),
Italy
Dipartimento di
Farmacologia delle
Sostanze Naturali e
Fisiologia Generale,
Università di Roma
“La Sapienza”, Rome, Italy
Laboratorio di Farmacologia,
Istituto Superiore di Sanità,
Rome, Italy
Synthesis of 1-Methyl-5-(pyrazol-3- and -5-yl- and
1,2,4-triazol-3- and 5-yl)-1,2,3,6-tetrahydropyridine
Derivatives and Their Evaluation as Muscarinic
Receptor Ligands
A series of 1-methyl-5-(pyrazol-3- and -5-yl- and 1,2,4-triazol-3- and 5-yl)-1,2,3,6tetrahydropyridine derivatives structurally related to arecoline were synthesized and
evaluated on M1, M2, and M3 muscarinic receptors using [3H]pirenzepine and
[3H]NMS as ligands. The binding affinity depended on the position and size of the
substituents. The most interesting compounds were further evaluated in functional
studies on isolated organs and in vivo for cholinergic side effects. Compounds 5 l
and 6 i displayed good M1 and M3 antagonistic properties in vitro and were devoid of
cholinergic side effects in vivo.
Keywords: Muscarinic receptors; Pyrazoles; Triazoles; Arecoline bioisosteres
Received: April 26, 2002 [FP693]
Introduction
Muscarinic cholinergic receptors belong to the large
family of the G protein-coupled receptors and are involved in several physiological functions such as motor
control, temperature and cardiovascular regulation,
memory and cognition in the central nervous system,
and smooth muscle contraction, glandular secretion,
and modulation of the cardiac rate and force, in the
peripheral nervous system [1].
To date, the existence of five subtypes of muscarinic receptors, partly demonstrated by using selective ligands
such as pirenzepine, has been definitively confirmed by
cloning five distinct genes [2] that encode for five glycoproteins exhibiting pharmacological properties similar to
those of the pharmacologically defined M1–M4 receptors
[3].
The number of important functions modulated by the
muscarinic receptors and their widespread central and
Correspondence: Maria Rosaria Del Giudice, Laboratorio di
Chimica del Farmaco, Istituto Superiore di Sanità, Viale Regina
Elena 299, 00161 Rome, Italy. Fax: +39 06 49387100,
e-mail: mr.delgiudice@iss.it.
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
peripheral distribution, make these proteins promising
targets for the treatment of many different pathologies
but, on the other hand, make it difficult to reach a therapeutic goal devoid of unwanted side effects.Until now the
use of muscarinic receptors ligands has been limited by
their low selectivity and, though in the last decade several agonists and antagonists are under advanced clinical
evaluation, the question of their specificity is still open
[4]. However, some M1 agonists have been evaluated in
the clinical phase for the treatment of the Alzheimer’s
disease though in most cases they show low bioavailability and poor efficacy (xanomeline and others bioisosters of arecoline) [5], while some M2 and M3 selective
antagonists have been taken into account in therapeutic
strategies for smooth muscle disorders, including urinary incontinence (darifenacin), irritable bowel syndrome (IBS), and chronic obstructive pulmonary disease [6]. It has been recently shown that M1 and M3 lung
receptor antagonists, administered by inhalation,
caused bronchodilation in the larger airways, thus proving useful in asthma therapy [7]. Besides, cholinomimetic drugs such as arecoline and oxotremorine are
under investigation for their ability to induce antinociception in experimental models of pain, as
0365-6233/03/0143 $ 17.50+.50/0
Full Paper
Maria Rosaria Del Giudicea,
Carlo Mustazzaa,
Anna Borionia,
Franco Gattaa,
Khosrow Tayebatib,
Francesco Amentab,
Paolo Tuccic,
Stefano Pierettid
Muscarinic Receptor Ligands 143
144 Del Giudice et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 143–154
the M1 and M2 receptors seem also to be involved in pain
modulation [8].
and 5-ethyl-pyrazol-3-yl derivatives 3 b, c were obtained
by reaction of diketones 2 [15] with hydrazine hydrate.
Alkylation of the pyrazole ring of 3 a–c by methyl or ethyl
iodide/sodium hydride in dimethylformamide resulted in
a mixture of the 3 g–l and 4 g–l isomers in a ratio of about
4:1. Isomeric separation was achieved by basic aluminium oxide column chromatography and the position of the
substituents was assigned by 1H-NMR studies using
NOE experiments. Irradiation of N-methyl resonance in
4 g at 3.88 ppm (δ) produced a NOE to both H-2 and H-4
pyridine protons at 8.68 and 7.72 ppm (δ), respectively,
and to H-3⬘ and H-4⬘ at 7.50 and 6.35 ppm (δ); the same
irradiation in 3 g gave a NOE only to H-4⬘ and H-5⬘ pyrazole protons at 6.56 and 7.40 ppm (δ). In addition, in
compound 4 k, when 3⬘-CH3 at 2.25 ppm (δ) was irradiated, only an intensity enhancement of H-4⬘ signal at
6.05 ppm (δ) was observed. In the other isomer 3 k, on
the contrary, irradiation of 5⬘-CH3 at 2.16 ppm (δ) afforded an Overhauser effect to H-4⬘ at 6.20 ppm (δ) and to
N-CH2-CH3 at 3.97 ppm (δ). Quaternization of the pyridine moiety with methyl iodide in acetone and reduction of
the resulting pyridinium salts with sodium borohydride in
refluxing methanol afforded the desired 1-methyl-5-(pyrazol-3- and 5-yl)-1,2,3,6-tetrahydropyridines 5 and 6.
Over the past few years the naturally occurring muscarinic agonist arecoline [9] (1-methyl-5-methoxycarbonyl-1,2,3,6-tetrahydropyridine) has been a template for
the preparation of new muscarinic ligands bearing a heterocyclic ring in substitution of the labile arecoline ester
group.Thus arecoline bioisosteres bearing a thiadiazole
[10, 11], oxadiazole [12], tetrazole, or 1,2,3-triazole [13]
ring were studied for affinities to M1-M3 receptors, while
at present the corresponding pyrazoles [14] and
[1,2,4]triazoles were only partially investigated.
On the basis of the current pharmacological demand of
selective ligands for the muscarinic receptors we synthesized a series of mono- and dialkyl-pyrazolyl- and
[1,2,4]triazolyl-1,2,3,6-tetrahydropyridine
derivatives
structurally related to arecoline (Figure 1). The new
compounds were evaluated in vitro for affinity and efficacy at M1, M2, and M3 muscarinic receptors, and in vivo for
cholinergic side effects. It is noteworthy that no arecoline
bioisosteres dialkylated on the heteroaromatic ring have
been previously reported, though alkyl substituents have
been claimed to be important for both affinity and in particular for efficacy and selectivity at muscarinic receptors
[13].
Figure 1. General structures of the synthesised compounds.
Results and discussion
Chemistry
Compounds I and II (Figure 1) were obtained following
the synthetic pathway illustrated in Figure 2.
The 3-(pyrazol-3-yl)pyridine 3 a (R1 = H) was prepared
according to the method of Plate et al. [14] with minor
modifications starting from 3-(dimethylamino)-1-(pyridin-3-yl)-2-propen-1-one 1, corresponding 5-methyl-
The synthetic routes to the corresponding 1,2,4-triazole
derivatives II are similar in many respects to ones reported above.
The starting pyridine intermediate 9 a (R1 = H) was synthesized by reaction of N,N-(dimethylaminomethylene)3-pyridinecarboxamide 7 [16] with hydrazine, while the
corresponding 5-alkyltriazole derivatives 9 b,c were prepared by reaction of 3-cyanopyridine 8 with acetic or propionic hydrazide in diphenyl ether at 220 °C. It is notable
that alkylation of 3-(1H-1,2,4-triazol-3-yl)pyridines 9 a–c
carried out in the same manner as for the pyrazole derivatives, almost entirely gave the 3-(1-alkyl-1,2,4-triazol-3yl)pyridines 9 g–l. Nevertheless, in the methylation of 9 a
and 9 b the isomeric 3-(1-methyl-1,2,4-triazol-5-yl)pyridines 11 a and 11 b could be isolated in 12 and 4 % yield
respectively.We did not perform the following reaction on
9 b because of its low yield. The position of N-methyl
group in triazoles 9 g–l and 11 was ascertained by 1HNMR NOE structural assignments. Irradiation of N-methyl resonance in 11 a at 3.92 ppm (δ) gave a small NOE
to H-2 and H-4 pyridine protons at 8.83 and 7.92 ppm
(δ), respectively.The same irradiation in 9 g at 3.90 ppm
(δ) produced only a NOE to triazole H-5⬘ at 8.03 ppm (δ).
Similar behaviour was noticed when NOE experiments
were carried out on the isomers 11 b and 9h. By irradiation of N-methyl resonance in 11 b at 3.87 ppm (δ) no
NOE to 3⬘-methyl protons and a small effect to pyridine
H-2 and H-4 protons at 8.82 and 7.92 ppm (δ) were
observed, while irradiation of pyridine H-4 proton at
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 143–154
Muscarinic Receptor Ligands 145
Figure 2. Synthesis of 1-methyl-5-(pyrazol-3- and -5-yl- and 1,2,4-triazol-3- and 5-yl)-1,2,3,6-tetrahydropyridine derivatives.
146 Del Giudice et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 143–154
7.92 ppm (δ) afforded NOEs to pyridine H-5 proton at
7.36 ppm (δ) and to N-methyl protons at 3.87 ppm (δ).
On the contrary, irradiation of N-methyl resonance in 9h
at 3.82 ppm (δ) resulted only in a NOE to 5⬘-methyl protons at 2.46 ppm (δ).
In conclusion, a characteristic feature of the aromatic region in protonic spectra combined with NOE experiments allowed us to provide the correct assignation to
the isomeric structures. Quaternization of compounds 9
and 11 followed by reduction of the pyridinium salts was
carried out in the same conditions previously described
for compounds 3 and 4.
Comparison of 1H-NMR spectra of compounds 3 a–c
and 9 a–c (unsubstituted N) respectively with those of
4 g–l and 11 a, b showed that N-substitution near the pyridine ring induced an approximate 0.4 ppm (δ) shielding
of the pyridine H-2 and H-4 resonances.Such a shielding
effect was not observed in compounds 3 g–l and 9 g–l
alkylated on the “external” nitrogen atom.
Pharmacology
Acetylcholine muscarinic receptor (M1–M3) affinity of the
investigated compounds was evaluated in rat frontal cor-
Table 1. Muscarinic cholinergic receptor affinity evaluation in different rat tissues.
Compound
5g
5h
5i
5j
5k
5l
6d
6e
6f
6g
6h
6i
6j
6k
6l
10 d
10 e
10 f
10 g
10 h
10 i
10 j
10 k
10 l
12 g
Pirenzepine
Arecoline
a
b
c
X
CH3
CH3
CH3
C2H5
C2H5
C2H5
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
CH3
–
–
–
–
–
–
–
–
–
CH3
CH3
CH3
C2H5
C2H5
C2H5
–
–
–
CH3
CH3
CH3
C2H5
C2H5
C2H5
–
–
CH3
C2H5
–
CH3
C2H5
–
CH3
C2H5
–
CH3
C2H5
–
CH3
C2H5
–
CH3
C2H5
–
CH3
C2H5
–
CH3
C2H5
–
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
N
N
N
N
N
N
N
N
N
Frontal cortex
Heart
[3H]-PZ
M1 Ki (nM) ±
SEM
[3H]-NMS
M2 Ki (nM) ±
SEM
Submandibular
glands
[3H]-NMS
M3 Ki (nM) ±
SEM
IC50 > 10–5 M
4677 ± 430
1549 ± 110
IC50 > 10–5 M
IC50 > 10–5 M
810 ± 65
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
490 ± 33
1560 ± 125
IC50 > 10–5 M
3330 ± 235
5180 ± 420
IC50 > 10–5 M
IC50 > 10–5 M
1709 ± 156
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
8720 ± 660
4700 ± 285
IC50 > 10–5 M
5.62 ± 0.04
2500 ± 160
IC50 > 10–5 M
IC50 > 10–5 M
3247 ± 285
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
1129 ± 95
948 ± 77
2030 ± 192
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
2348 ± 210
1960 ± 156
1174 ± 105
5950 ± 478
9950 ± 760
IC50 > 10–5 M
500.5 ± 22
4100 ± 250
5860 ± 367
4645 ± 290
3747 ± 310
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
3680 ± 320
1083 ± 95
7900 ± 560
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
IC50 > 10–5 M
5102 ± 345
990 ± 86
4260 ± 390
4670 ± 290
1720 ± 110
IC50 > 10–5 M
125.9 ± 9
6700 ± 475
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 143–154
Muscarinic Receptor Ligands 147
tex (source of M1 receptor), heart (source of M2 receptor)
and submandibular glands (source of M3 receptor), by
radioligand binding assay. [3H]-pirenzepine and [3H]-Nmethylscopolamine were used as radioligands for M1
and M2–M3 receptors respectively (Table 1).
played higher affinity than arecoline for M1 (Ki =
1709 nM).In this series, the best M2 ligand was the monoethyl derivative 10 j, and the best M3 ligand was 10 i which
exerted seven times higher affinity than arecoline.
In the pyrazole series the unsubstituted and monoalkyl
derivatives showed low or no affinity with the exception
of the ethyl derivative 6 j which displayed a Ki lower than
arecoline for all muscarinic subtypes. The observed
higher affinity for b-substituted compounds is in accordance with similar studies on corresponding [1,2,3] triazole and tetrazole arecoline bioisosteres [13].
The introduction of a second alkyl group in the pyrazole
ring tended to raise affinity for the muscarinic receptors
and to modulate selectivity in conformity with the size
and the position of the substituents. So, significant affinities and selectivity towards M1 were exhibited by dialkylpyrazoles 5 i, l, and 6 i, all bearing an ethyl group in
the position c with Ki values ranging from 490 to
1549 nM.
M2 subtype was the preferential target for compounds
bearing an ethyl group in the position b (6 j–l) which exerted a maximum of affinity when a methyl group was
present in the position c (6 k); it is interesting to note that
6 k displayed no affinity for M1 receptors. Pyrazoles eliciting some activity on M3 seem to have strict structural requirements for the position a which must be methylated
(5 g–i), and for the position b which must be ethylated
(6 j–l), while they can have a hydrogen, a methyl, or an
ethyl group in position c. In compounds 5 g–i the order of
efficacy of substituents in the position c is H < methyl
< ethyl, while in compounds 6 j–l a maximum of affinity
was recorded when the same position was occupied by a
methyl group ( H < methyl < ethyl ). In this series the best
ligand was the 2-ethyl-3-methylpyrazole derivative 6 k
(Ki = 1083 nM).
In general, triazole derivatives showed low affinity for M1
and tended to be more selective for the M2 and M3 subtypes. The b-ethyl derivative 10 j was atypical, and dis-
In conclusion we noticed that all the more active ligands
for M1, M2, and M3 receptors (respectively 5 l, and 6 i; 6 k,
6 j and 10 j; 6 k and 10 i) had one ethyl group, while diethyl derivatives sometimes showed a certain decrease
in affinity. Accordingly, it seems that increasing the lipophilicity by increasing the number and size of substitutions results in an improvement of the affinity in conformity with the position of the substituents, unless ligands became too bulky to be accommodated by the receptors.
Arecoline bioisosteres have been reported to range in
activity from antagonism to full muscarinic agonism so
some compounds showing the most interesting affinity
(5 i, 5 l, 6 i, 6 l, and 10 i) were further evaluated in tests on
isolated organ. Functional activity was determined by
the use of the M1 receptor-mediated inhibition of neurogenic twitch contractions and inhibition of evoked noradrenaline release in rabbit vas deferens, of M2 receptor-mediated contraction of pig bladder, and of M3 receptor-mediated contraction of guinea-pig ileum longitudinal smooth muscle preparation (Table 2).The functional
activity was confirmed in vivo by using the antagonism
toward oxotremorine induced tremors, salivation, and
lacrimation (Table 3). The appearance of different sideeffects was also evaluated by using behavioural scores.
Compounds 5 i, 6 l, and 10 i showed a weak M1, M2, and
M3 activity and were not further investigated (data not reported). Compound 5 l did not antagonise ACh in the pig
bladder (4.0 × 10–7 – 10–4 M; pA2: 4.3 ± 0.8) and carbachol in the guinea-pig ileum (1 × 10–8 – 10–5 M; pA2: 5.7 ±
0.2), but it antagonised McN-A-343 in the rabbit vas deferens (1 × 10–8 – 5.0 × 10–7 M; pA2: 7.9 ± 0.2) (Table 2).
These results indicate that the compound is a M1 receptor antagonist. Compound 6 i did not antagonise ACh in
the pig bladder in the tested concentration range (4.0 ×
10–7 – 10–4 M). The compound exhibited an antagonistic
Table 2. pA2 values of 5 l and 6 i in functional assays using isolated tissuesa.
Compound
Rabbit vas deferens
(M1)
Pig urinary bladder
(M2)
Guinea-pig ileum
(M3)
5l
6i
7.9 ± 0.2
8.6 ± 0.8
4.3 ± 0.8
5.8 ± 0.8
5.7 ± 0.2
8.9 ± 0.2
a
The pA2 values, defined as –log of molar concentration of antagonist (mean with 95 % confidence limits) were calculated from Schild plots and from regression lines whose slopes were constrained to 1.00 as required for the theoretical model of competitive antagonism (mean ± S.E.M.). Values are the mean of more than 6 experiments.
148 Del Giudice et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 143–154
Table 3. ED50 values (mg/kg, ip) of atropine , 5 l and 6 i, in protecting mice from oxotremorine (0.5 mg/kg) induced tremor, salivation, and lacrimation.
Treatment
Tremor
atropine
5l
6i
4.28 (2.94–6.25)
30.50 (19.46–47.8)a
16.28 (8.19–32.3)a
a
Oxotremorine induced
Salivation
1.59 (0.77–3.24)
4.60 (1.32–16.60)
14.50 (6.85–30.69)a
Lacrimation
2.35 (0.012–466)
16.91 (8.02–35.68)
4.96 (1.66–14.74)
Significant difference vs atropine. ED50 values were obtained using at least three doses and six animal for each dose.
activity on the McN-A-343 response in the rabbit vas deferens (1 × 10–8 – 5.0 × 10–7 M; pA2: 8.6 ± 0.8) and on the
carbachol response in the guinea-pig ileum (1 × 10–8 –
10–6 M; pA2: 8.9 ± 0.2) (Table 2). These results suggest
that the compound is a M1 and M3 receptor antagonist.
Differences between affinity values estimated from
radioligand binding studies and those obtained from
functional studies were noticed. These disparities may
arise from the use of hypotonic buffers in binding studies,
while higher ionic strength buffers are used in pharmacological experiments. Furthermore, some studies
showed great differences between species and, within a
species, between districts [17].
Table 3 reported ED50 values (in mg/kg) from doseresponse studies obtained in animals treated with
oxotremorine (0.5 mg/kg, s.c) respectively after administration of atropine (1–10 mg/kg, i.p), 5 l (5–100 mg/kg,
i.p.) and 6 i (5–100 mg/kg, i.p.).
In these experimental sessions we observed only a reduction in locomotor activity and a reduction to tail pinch
response at higher doses (>200 mg/kg, i.p.). No cholinergic side-effects such as tremor, salivation, increases in
defecation and urination, lacrimation, and diarrhoea
were observed.
Statistical analysis revealed significant difference between atropine ED50 value versus 5 l and 6 i values. The
order of efficiency in preventing oxotremorine induced
tremor was atropine > 6 i > 5 l.The same effects were observed when lacrimation was considered. On the contrary, 5 l appeared more effective than 6 i to prevent oxotremorine induced salivation.
Conclusion
Most of the synthesized compounds displayed lower Ki
than arecoline for M1, M2, and M3 and their binding profile
was highly dependent on the position and size of the
substituents especially for selectivity and affinity. Com-
pounds 5 l and 6 i displayed good M1 and M3 antagonistic
behaviour in vitro. In vivo, 5 l and 6 i were devoid of
cholinergic side effects and were able to antagonise
oxotremorine induced effects. Accordingly, dialkylpyrazoles and triazoles represent valuable bioisosteres of
the arecoline for muscarinic receptors.
Experimental part
Chemistry
Melting points were measured on a Kofler hot stage apparatus
and are uncorrected. The 1H-NMR spectra were obtained on a
Gemini 200 MHz instrument; all values are reported in ppm (δ)
and standard abbreviations are used (ad = apparent doublet, at
= apparent triplet, b = broad, d = doublet, dd = doublet of doublets, dq = doublet of quadruplets, dt = doublet of triplets, m =
multiplet, q = quadruplet, t = triplet, s = singlet). 1H-NMR data
were only given for representative compounds. Column chromatographic separations were accomplished on Merck standardized aluminium oxide 90. The purity of each compound was
checked on Merck aluminium oxide 60 F254 plates and spots
were located by UV light. Sodium sulfate was used to dry organic solutions.
Analyses indicated by the symbols of the elements were within
± 0.4 % of the theoretical values.
The syntheses of 3-(dimethylamino)-1-(pyridin-3-yl)-2-propen1-one 1 [14], 1-(pyridin-3-yl)-1,3-butanedione 2 b (R1 = CH3)
[15], N,N-(dimethylamino)methylene-3-pyridinecarboxamide 7
[16] have been reported elsewhere. 3-Cyanopyridine 8 was
purchased from Aldrich Chemical Co.
1-(Pyridin-3-yl)-1,3-pentanedione 2 c (R1 = C2H5) was obtained analogously to compound 2 b starting from ethyl nicotinate and 2-butanone in 76 % yield, bp = 98–104 °C/
0.7 mm Hg). Anal. C10H11NO2 (C, H, N).
General procedure for the preparation of 3-(pyrazol-3-yl)pyridines 3 a–c
Compounds 1 or 2 (0.1 mole) were reacted with hydrazine hydrate (5.5 g, 0.11 mole) in ethanol (100 mL) at room temperature for 3 hours. The solution was concentrated under vacuum
and the residual oil was directly distilled.
Compounds 3 a–c (0.1 mole) and methyl iodide (21.3 g,
0.15 mole) in acetone (100 mL) were stirred at room tempera-
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 143–154
Muscarinic Receptor Ligands 149
ture for 6 hours. The precipitated methylpyridinium iodides
were collected by filtration, washed with acetone, then with diethyl ether, dried in vacuo, and used in the subsequent reduction without further purification.
C11H13N3 (C, H, N). Methiodide: mp 224–226 °C. Anal.
C12H16IN3 (C, H, N).
3-(1H-Pyrazol-3-yl)pyridine 3 a
This compound was obtained from 1 in 89 % yield, bp 143–
148 °C/0.01 mm Hg; mp 56–58 °C (diethyl ether); 1H-NMR
(CDCl3): δ 12.70 (bs, 1 H, NH), 9.04 (dd, 1 H, H-2, J2,4 = 2.1 Hz,
J2,5 = 0.8 Hz), 8.54 (dd, 1H, H-6, J6,4 = 1.6 Hz, J6,5 = 4.8 Hz), 8.07
(dt, 1 H, H-4, J4,2 = 2.1 Hz J4,5 = 7.9 Hz, J4,6 = 1.6 Hz), 7.63 (d,
1 H, H-5⬘, J5⬘,4⬘ = 2.4 Hz), 7.32 (dq, 1 H, H-5, J5,2 = 0.8 Hz J5,4 =
7.9 Hz, J5,6 = 4.8 Hz), 6.65 (d, 1 H, H-4⬘, J4⬘,5⬘ = 2.4 Hz). Anal.
C8H7N3 (C, H, N). Methiodide: mp 215–216 °C. Anal. C9H10IN3
(C, H, N).
3-(5-Methyl-1H-pyrazol-3-yl)pyridine 3 b
This compound was obtained from 2 b in 84% yield, mp 138–
140 °C (ethyl acetate); 1H-NMR (DMSO-d6): δ 12.40 (bs, 1 H,
NH), 8.96 (s, 1 H, H-2), 8.09 (dd, 1 H, H-4), 6.54 (s, 1 H, H-4⬘),
2.25 (s, 3 H, 5⬘-CH3). Anal. C9H9N3 (C, H, N). Methiodide: mp
285–287 °C, [lit. [18] mp 276–279 °C]. Anal. C10H12IN3 (C, H, N).
3-(5-Ethyl-1H-pyrazol-3-yl)pyridine 3 c
This compound was obtained from 2 c in 90 % yield, mp 54–
55 °C (ethyl acetate/n-hexane); 1H-NMR (DMSO-d6): δ 12.76
(s, 1 H, NH), 8.98 (s, 1 H, H-2), 8.11 (d, 1 H, H-4), 6.57 (s, 1 H,
H-4⬘), 2.63 (q, 2 H, 5⬘-CH2CH3), 1.21 (t, 3 H, 5⬘-CH2CH3). Anal.
C10H11N3 (C, H, N). Methiodide: mp 235–237 °C. Anal.
C11H14IN3 (C, H, N).
General procedure for the alkylation of 3 a–c to give compounds 3 g–l and 4 g–l
To a stirred suspension of sodium hydride (7.5 g of 50 % oil dispersion, 0.15 mole) in anhydrous dimethylformamide
(100 mL), each compound 3 (0.15 mole) was added in several
portions. After 30 minutes’ stirring at room temperature, a solution of methyl iodide or ethyl iodide (0.15 mole) in dimethylformamide (30 mL) was added dropwise under cooling and the
reaction mixture was stirred at room temperature for 3 hours.
The inorganic salts were filtered, the filtrate evaporated to dryness, and the residue chromatographed on alumina by eluting
with an ethyl acetate/n-hexane 1:1 mixture. Compounds 4 were
eluted first followed by 3.
Quaternization was carried out as previously described.
3-(1-Methyl-1H-pyrazol-3-yl)pyridine 3 g
This compound was obtained from 3a in 72 % yield, bp 92–
97 °C/0.01 mm Hg; 1H-NMR (CDCl3): δ 8.98 (dd, 1 H, H-2),
8.06 (dt, 1 H, H-4), 7.40 (d, 1 H, H-5⬘), 6.56 (d, 1 H, H-4⬘), 3.95
(s, 3 H, N1⬘-CH3). Anal. C9H9N3 (C, H, N). Methiodide: mp 203–
205 °C. Anal. C10H12IN3 (C, H, N).
3-(1,5-Dimethyl-1H-pyrazol-3-yl)pyridine 3 h
This compound was obtained from 3 b in 68 % yield mp 90–
91 °C (ethyl acetate/n-hexane); 1H-NMR (CDCl3): δ 8.94 (dd,
1 H, H-2), 8.02 (dt, 1 H, H-4), 6.33 (s, 1 H, H-4⬘), 3.81 (s, 3 H,
N1⬘-CH3), 2.30 (s, 3 H, 5⬘-CH3). Anal. C10H11N3 (C, H, N). Methiodide: mp 235–238 °C. Anal. C11H14IN3 (C, H, N).
3-(5-Ethyl-1-methyl-1H-pyrazol-3-yl)pyridine 3 i
This compound was obtained from 3 c in 65 % yield, bp 120–
125 °C/0.01 mm Hg; 1H-NMR (CDCl3): δ 8.96 (dd, 1 H, H-2),
8.03 (dt, 1 H, H-4), 6.35 (s, 1 H, H-4⬘), 3.80 (s, 3 H, N1⬘-CH3),
2.62 (q, 2 H, 5⬘-CH2CH3), 1.33 (t, 3 H, 5⬘-CH2CH3). Anal.
3-(1-Ethyl-1H-pyrazol-3-yl)pyridine 3 j
This compound was obtained from 3a in 48 % yield, bp 115–
120 °C/0.01 mm Hg; 1H-NMR (CDCl3): δ 8.99 (d, 1 H, H-2),
8.07 (dt, 1 H, H-4), 7.43 (d, 1 H, H-5⬘), 6.56 (d, 1 H, H-4⬘), 4.22
(q, 2 H, N1⬘-CH2CH3), 1.52 (t, 3 H, N1⬘-CH2CH3). Anal.
C10H11N3 (C, H, N). Methiodide: mp 187–189 °C. Anal. C11H14IN3
(C, H, N).
3-(1-Ethyl-5-methyl-1H-pyrazol-3-yl)pyridine 3 k
This compound was obtained from 3 b in 59 % yield, bp 125–
130 °C/0.01 mm Hg; 1H-NMR (CDCl3): δ 8.88 (s, 1 H, H-2),
7.92 (dt, 1 H, H-4), 6.20 (s, 1 H, H-4⬘), 3.97 (q, 2 H, N1⬘CH2CH3), 2.16 (s, 3 H, 5⬘-CH3), 1.29 (t, 3 H, N1⬘-CH2CH3). Anal.
C11H13N3 (C, H, N). Methiodide: mp 198–200 °C. Anal. C12H16IN3
(C, H, N).
3-(1,5-Diethyl-1H-pyrazol-3-yl)pyridine 3 l
This compound was obtained from 3 c in 37 % yield, bp 120–
125 °C/0.01 mm Hg; 1H-NMR (CDCl3): δ 8.97 (d, 1 H, H-2),
8.06 (dt, 1 H, H-4), 6.35 (s, 1 H, H-4⬘), 4.12 (q, 2 H, N1⬘CH2CH3), 2.65 (q, 2 H, 5⬘-CH2CH3), 1.45 (t, 3 H, N1⬘-CH2CH3),
1.32 (t, 3 H, 5⬘-CH2CH3). Anal. C12H15N3 (C, H, N). Methiodide:
mp 237–239 °C. Anal. C13H18IN3 (C, H, N).
3-(1-Methyl-1H-pyrazol-5-yl)pyridine 4 g
This compound was obtained from 3 a in 18 % yield, bp 9095 °C/0.01 mm Hg; 1H-NMR (CDCl3): δ 8.68 (dd, 1 H, H-2),
8.63 (dd, 1H, H-6), 7.72 (dt, 1H, H-4), 7.50 (d, 1 H, H-3⬘), 7.38
(dq, 1 H, H-5), 6.35 (d, 1 H, H-4⬘), 3.88 (s, 3 H, N1⬘-CH3). Anal.
C9H9N3 (C, H, N). Methiodide: mp 193–195 °C. Anal. C10H12IN3
(C, H, N).
3-(1,3-Dimethyl-1H-pyrazol-5-yl)pyridine 4 h
This compound was obtained from 3 b in 22 % yield, mp 67–
69 °C (cyclohexane); 1H-NMR (CDCl3): δ 8.63 (d, 1 H, H-2),
7.67 (dt, 1 H, H-4), 6.10 (s, 1 H, H-4⬘) 3.77 (s, 3 H, N1⬘-CH3),
2.25 (s, 3 H, 3⬘-CH3). Anal. C10H11N3 (C, H, N). Methiodide: mp
217–219 °C. Anal. C11H14IN3 (C, H, N).
3-(3-Ethyl-1-methyl-1H-pyrazol-5-yl)pyridine 4 i
This compound was obtained from 3 c in 11 % yield, bp 110–
115 °C/0.01 mm Hg; 1H-NMR (CDCl3): δ 6.16 (s, 1 H, H-4⬘),
3.83 (s, 3 H, N1⬘-CH3), 2.66 (q, 2 H, 3⬘-CH2CH3), 1.27 (t, 3 H, 3⬘CH2CH3). Anal. C11H13N3 (C, H, N). Methiodide: mp 180–182 °C.
Anal. C12H16IN3 (C, H, N).
3-(1-Ethyl-1H-pyrazol-5-yl)pyridine 4 j
This compound was obtained from 3 a in 10 % yield, bp 100–
105 °C/0.01 mm Hg; 1H-NMR (CDCl3): δ 7.56 (d, 1 H, H-3⬘),
6.32 (d, 1 H, H-4⬘), 4.15 (q, 2 H, N1⬘-CH2CH3), 1.41 (t, 3 H, N1⬘CH2CH3). Anal. C10H11N3 (C, H, N). Methiodide: mp 154–155 °C.
Anal. C11H14IN3 (C, H, N).
3-(1-Ethyl-3-methyl-1H-pyrazol-5-yl)pyridine 4 k
This compound was obtained from 3 b in 17 % yield, bp 112–
115 °C/0.01 mm Hg; 1H-NMR (CDCl3): δ 6.05 (s, 1 H, H-4⬘),
4.02 (q, 2 H, N1⬘-CH2CH3), 2.25 (s, 3 H, 3⬘-CH3), 1.34 (t, 3 H,
N1⬘-CH2CH3). Anal. C11H13N3 (C, H, N). Methiodide: mp 188–
190 °C. Anal. C12H16IN3 (C, H, N).
3-(1,3-Diethyl-1H-pyrazol-5-yl)pyridine 4 l
This compound was obtained from 3 c in 9 % yield, bp 115–
118 °C/0.01 mm Hg; 1H-NMR (CDCl3): δ 6.12 (s, 1 H, H-4⬘),
150 Del Giudice et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 143–154
4.07 (q, 2 H, N1⬘-CH2CH3), 2.67 (q, 2 H, 3⬘-CH2CH3), 1.38 (t,
3 H, N1⬘-CH2CH3), 1.26 (t, 3 H, 3⬘-CH2CH3). Anal. C12H15N3 (C,
H, N). Methiodide: mp 174–175 °C. Anal. C13H18IN3 (C, H, N).
147 °C]; 1H-NMR (DMSO-d6): δ 12.60 (bs, 1 H, NH), 7.55 (bs,
1 H, H-5⬘), 6.31 (bs, 1 H, H-4⬘), ms: m/z 163 (M+), 149, 121.
Anal. C9H13N3 (C, H, N).
General procedure for the preparation of 1-methyl-5-(pyrazol3- and 5-yl)-1,2,3,6-tetrahydropyridines 5 and 6
1-Methyl-5-(5-methyl-1H-pyrazol-3-yl)-1,2,3,6-tetrahydropyridine 6 e
To a stirred suspension of each 3 or 4 methiodide (10 mmoles)
in methanol (100 mL), sodium borohydride (0.5 g, 13 mmoles)
was added in several portions at 5–10 °C. After stirring for
1 hour at room temperature, the mixture was refluxed for
1 hour. The solvent was evaporated, ice was added, and the
mixture was extracted with chloroform. The extract was dried
and evaporated to dryness to give compounds 5 and 6 which
were purified by column chromatography on alumina, using
ethyl acetate as eluent.
This compound was obtained from 3 b methiodide in 90 %
yield, mp 129–131 °C (diethyl ether) [lit. [18] mp 130.5–133 °C];
1
H-NMR (DMSO-d6): δ 12.29 (bs, 1 H, NH), 6.04 (s, 1 H, H-4⬘),
2.17 (s, 3 H, 5⬘-CH3); Anal. C10H15N3 (C, H, N).
1-Methyl-5-(1-methyl-1H-pyrazol-5-yl)-1,2,3,6-tetrahydropyridine 5 g
This compound was obtained from 4 g methiodide in 87 %
yield, dihydrochloride mp 177–179 °C (methanol/diethyl ether)
[lit. [14] maleate mp 128 °C]; 1H-NMR (DMSO-d6): δ 7.34 (d, 1 H,
H-3⬘), 6.19 (d, 1 H, H-4⬘), 6.00–5.95 (m, 1 H, H-4), 3.78 (s, 3 H,
N1⬘-CH3), 3.02–2.98 (m, 2 H, H-6), 2.46 (t, 2 H, H-2), 2.32–2.22
(overlapped s and m, 5 H, N1-CH3 and H-3). Anal. C10H17Cl2N3
· H2O (C, H, N).
5-(1,3-Dimethyl-1H-pyrazol-5-yl)-1-methyl-1,2,3,6-tetrahydropyridine 5 h
This compound was obtained from 4 h methiodide in 82 %
yield, hydrochloride mp 128–131 °C (methanol/diethyl ether);
1
H-NMR (CDCl3): δ 5.84–5.82 (overlapped s and m, 2 H, H-4⬘
and H-4), 2.35 (s, 3 H, N1-CH3), 2.16 (s, 3 H, 3⬘-CH3). Anal.
C11H18ClN3 · 2 H2O (C, H, N).
5-(5-Ethyl-1H-pyrazol-3-yl)-1-methyl-1,2,3,6-tetrahydropyridine 6 f
This compound was obtained from 3 c methiodide in 79 % yield,
hydrochloride mp 188–192 °C (methanol); 1H-NMR (CDCl3): δ
6.02 (s, 1 H, H-4⬘), 2.58 (q, 2 H, 5⬘-CH2CH3), 2.32–2.25 (m, 2 H,
H-3), 1.19 (t, 3 H, 5⬘-CH2CH3). Anal. C11H18ClN3 · 2 H2O (C, H,
N).
1-Methyl-5-(1-methyl-1H-pyrazol-3-yl)-1,2,3,6-tetrahydropyridine 6 g
This compound was obtained from 3 g methiodide in 92 %
yield, dihydrochloride mp 192-195 °C (methanol) [lit. [14]
maleate mp 141 °C]; 1H-NMR (CDCl3): δ 7.23 (d, 1 H, H-5⬘),
6.27–6.23 (overlapped s and m, 2 H, H-4⬘and H-4), 3.84 (s, 3 H,
N1⬘-CH3). Anal. C10H17Cl2N3 · 2 H2O (C, H, N).
5-(1,5-Dimethyl-1H-pyrazol-3-yl)-1-methyl-1,2,3,6-tetrahydropyridine 6 h
This compound was obtained from 3 h methiodide in 82 %
yield, dihydrochloride mp 180–182 °C (methanol); 1H-NMR
(CDCl3): δ 6.03 (s, 1 H, H-4⬘), 3.71 (s, 3 H, N1⬘-CH3), 2.21 (s,
3 H, 5⬘-CH3). Anal. C11H19Cl2N3 (C, H, N).
5-(3-Ethyl-1-methyl-1H-pyrazol-5-yl)-1-methyl-1,2,3,6-tetrahydropyridine 5 i
5-(5-Ethyl-1-methyl-1H-pyrazol-3-yl)-1-methyl-1,2,3,6-tetrahydropyridine 6 i
This compound was obtained from 4 i methiodide in 89 % yield
as a viscous oil; 1H-NMR (CDCl3): δ 5.89 (s, 1 H, H-4⬘), 3.77 (s,
3 H, N1⬘-CH3), 2.57 (q, 2 H, 3⬘-CH2CH3), 2.41–2.30 (overlapped s and m, 5 H, N1-CH3 and H-3), 1.20 (t, 3 H, 3⬘CH2CH3). Anal. C12H19N3 (C, H, N).
This compound was obtained from 3 i methiodide in 80 % yield,
hydrochloride mp 180–182 °C (methanol); 1H-NMR (CDCl3): δ
6.06 (s, 1 H, H-4⬘), 3.72 (s, 3 H, N1⬘-CH3), 2.58–2.51 (overlapped q and t, 4 H, 5⬘-CH2CH3 and H-2), 1.24 (t, 3 H, 3⬘CH2CH3). Anal. C12H20ClN3 · 2 H2O (C, H, N).
5-(1-Ethyl-1H-pyrazol-5-yl)-1-methyl-1,2,3,6-tetrahydropyridine 5 j
5-(1-Ethyl-1H-pyrazol-3-yl)-1-methyl-1,2,3,6-tetrahydropyridine 6 j
This compound was obtained from 4 j methiodide in 92 % yield
as a viscous oil; 1H-NMR (CDCl3): δ 7.41 (d, 1 H, H-3⬘), 6.06 (s,
1 H, H-4⬘,), 4.13 (q, 2 H, N1⬘-CH2CH3), 1.40 (t, 3 H, N1⬘CH2CH3). Anal. C11H17N3 (C, H, N).
This compound was obtained from 3 j methiodide in 74 % yield,
hydrochloride mp 149–151 °C (methanol/diethyl ether); 1HNMR (CDCl3): δ 7.28 (d, 1 H, H-5⬘), 6.25 (s, 1 H, H-4⬘), 4.11 (q,
2 H, N1⬘-CH2CH3), 1.43 (t, 3 H, N1⬘-CH2CH3). Anal. C11H18ClN3
(C, H, N).
5-(1-Ethyl-3-methyl-1H-pyrazol-5-yl)-1-methyl-1,2,3,6-tetrahydropyridine 5 k
This compound was obtained from 4 k methiodide in 81 % yield
as a viscous oil; 1H-NMR (CDCl3): δ 5.85 (s, 1 H, H-4⬘), 4.06 (q,
2 H, N1⬘-CH2CH3), 2.22 (s, 3 H, 3⬘-CH3), 1.38 (t, 3 H, N1⬘CH2CH3). Anal. C12H19N3 (C, H, N).
5-(1,3-Diethyl-1H-pyrazol-5-yl)-1-methyl-1,2,3,6-tetrahydropyridine 5 l
This compound was obtained from 4l methiodide in 88 % yield
as a viscous oil; 1H-NMR (CDCl3): δ 5.88 (s, 1 H, H-4⬘), 4.07 (q,
2 H, N1⬘-CH2CH3), 2.39 (s, 3 H, N1-CH3), 1.38 (t, 3 H, N1⬘CH2CH3), 1.21 (t, 3 H, 3⬘-CH2CH3). Anal. C13H21N3 (C, H, N).
1-Methyl-5-(1H-pyrazol-3-yl)-1,2,3,6-tetrahydropyridine 6 d
This compound was obtained from 3 a methiodide in 87 % yield,
mp 99–101 °C (ethyl acetate/n-hexane) [lit. [14] maleate mp
5-(1-Ethyl-5-methyl-1H-pyrazol-3-yl)-1-methyl-1,2,3,6-tetrahydropyridine 6 k
This compound was obtained from 3 k methiodide in 76 % yield,
hydrochloride mp 165–168 °C (methanol/diethyl ether); 1HNMR (CDCl3): δ 6.03 (s, 1 H, H-4⬘), 4.02 (q, 2 H, N1⬘-CH2CH3),
2.22 (s, 3 H, 5⬘-CH3), 1.35 (t, 3 H, N1⬘-CH2CH3). Anal.
C12H20ClN3 · 2 H2O (C, H, N).
5-(1,5-Diethyl-1H-pyrazol-3-yl)-1-methyl-1,2,3,6-tetrahydropyridine 6 l
This compound was obtained from 3 l methiodide in 79 % yield,
hydrochloride mp 144–147 °C (methanol/diethyl ether); 1HNMR (CDCl3): δ 6.05 (s, 1 H, H-4⬘), 4.02 (q, 2 H, N1⬘-CH2CH3),
2.53 (overlapped q and t, 4 H, 5⬘-CH2CH3 and H-2), 1.35 (t, 3 H,
N1⬘-CH2CH3), 1.26 (t, 3 H, 5⬘-CH2CH3). Anal. C13H22ClN3 · H2O
(C, H, N).
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 143–154
General procedure for the preparation of 3-(1-H-1,2,4-triazol3-yl)pyridines 9 a–c
Compound 9 a (R1 = H) was prepared according to a method
previously reported [16], by reaction of compound 7 with hydrazine hydrate.
Compounds 9 b (R1 = CH3) and 9 c (R1 = C2H5) were obtained
by heating in a flask fitted with a Dean-Stark trap a mixture of 3cyanopyridine 8 (10.4 g, 0.1 mole) and acethydrazide (8.9 g,
0.12 mole) to obtain 9 b, or propionic hydrazide (10.6 g,
0.12 mole) to obtain 9 c, in diphenyl ether (100 mL) at 220 °C.
The heating was maintained until the starting material had disappeared (about 2 hours).The reaction mixture was allowed to
cool to room temperature and n-hexane (300 mL) was added.
The precipitated solid was collected by filtration and thoroughly
washed with additional n-hexane. The crude product was purified by chromatography on alumina by eluting with ethyl acetate.
Quaternization was carried out as described for compounds 3.
3-(1H-1,2,4-Triazol-3-yl)pyridine 9 a
This compound was obtained from 7 in 66 % yield, mp 179–
181 °C (methanol) [lit. [16] mp 169–172 °C]; 1H-NMR (DMSOd6): δ 9.18 (dd, 1 H, H-2, J2,4 = 2.0 Hz, J2,5 = 0.8 Hz), 8.63 (dd,
1 H, H-6, J6,4 = 1.7 Hz, J6,5 = 4.8 Hz), 8.58 (s, 1 H, H-5⬘), 8.32 (dt,
1 H, H-4, J4,2 = 2.0 Hz, J4,5 = 7.8 Hz, J4,6 = 1.7 Hz), 7.51 (dd, 1 H,
H-5, J5,4 = 7.8 Hz, J5,6 = 4.8 Hz). Anal. C7H6N4 (C, H, N). Methiodide: mp 215–216 °C. Anal. C8H9IN4 (C, H, N).
3-(5-Methyl-1H-1,2,4-triazol-3-yl)pyridine 9 b
This compound was obtained from 8 in 56 % yield, mp 150–
151 °C (ethyl acetate) [lit. [19] mp 211–213 °C]; 1H-NMR (DMSO-d6): δ 13.84 (s, 1 H, NH), 9.13 (s, 1 H, H-2), 8.27 (d, 1 H,
H-4), 3.38 (s, 3 H, 5⬘-CH3). Anal. C8H8N4 (C, H, N). Methiodide:
mp 250–252 °C. Anal. C9H11IN4 (C, H, N).
3-(5-Ethyl-1H-1,2,4-triazol-3-yl)pyridine 9 c
This compound was obtained from 8 in 62 % yield, mp 96–97 °C
(diethyl ether); 1H-NMR (DMSO-d6): δ 13.90 (bs, 1 H, NH) 9.13
(d, 1 H, H-2), 8.27 (dt, 1 H, H-4), 2.75 (q, 2 H, 5⬘-CH2CH3), 1.26
(t, 3 H, 5⬘-CH2CH3). Anal. C9H10N4 (C, H, N). Methiodide: mp
229–231 °C. Anal. C10H13IN4 (C, H, N).
General procedure for the alkylation of 9 to give compounds
9 g–l and 11
Compounds 9 were alkylated with methyl iodide or ethyl iodide
according to the procedure previously described for the corresponding pyrazoles 3. Compounds 9 g–l and 11 were separated by chromatography on alumina by eluting with ethyl acetate/n-hexane 1:1 mixture. Quaternization was carried out as
previously described.
3-(1-Methyl-1H-1,2,4-triazol-3-yl)pyridine 9 g
This compound was obtained from 9a in 62 % yield, mp 58–
60 °C (diethyl ether); 1H-NMR (CDCl3): δ 9.23 (d, 1 H, H-2), 8.26
(dt, 1 H, H-4), 8.03 (s, 1 H, H-5⬘), 3.90 (s, 3 H, N1⬘-CH3). Anal.
C8H8N4 (C, H, N). Methiodide: mp 184–187 °C. Anal. C9H11IN4
(C, H, N).
3-(1,5-Dimethyl-1H-1,2,4-triazol-3-yl)pyridine 9 h
This compound was obtained from 9 b in 77% yield, mp 105–
107 °C (ethyl acetate); 1H-NMR (CDCl3): δ 9.21 (d, 1 H, H-2),
8.25 (dt, 1 H, H-4), 3.82 (s, 3 H, N1⬘-CH3), 2.46 (s, 3 H, 5⬘-CH3).
Anal. C9H10N4 (C, H, N). Methiodide: mp 268–271 °C. Anal.
C10H13IN4 (C, H, N).
Muscarinic Receptor Ligands 151
3-(5-Ethyl-1-methyl-1H-1,2,4-triazol-3-yl)pyridine 9 i
This compound was obtained from 9 c in 51 % yield, mp 85–
86 °C (diethyl ether); 1H-NMR (CDCl3): δ 9.20 (d, 1 H, H-2), 8.24
(dt, 1 H, H-4), 3.80 (s, 3 H, N1⬘-CH3), 2.74 (q, 2 H, 5⬘-CH2CH3),
1.32 (t, 3 H, 5⬘-CH2CH3). Anal. C10H12N4 (C, H, N). Methiodide:
mp 140–141 °C. Anal. C11H15IN4 (C, H, N).
3-(1-Ethyl-1H-1,2,4-triazol-3-yl)pyridine 9 j
This compound was obtained from 9 a in 73 % yield, bp 120–
125 °C/0.01 mm Hg; 1H-NMR (CDCl3): δ 9.27 (d, 1 H, H-2),
8.29 (dt, 1 H, H-4), 8.00 (s, 1 H, H-5⬘), 4.21 (q, 2 H, N1⬘CH2CH3), 1.53 (t, 3 H, N1⬘-CH2CH3). Anal. C9H10N4 (C, H, N).
Methiodide: mp 162–165 °C. Anal. C10H13IN4 (C, H, N).
3-(1-Ethyl-5-methyl-1H-1,2,4-triazol-3-yl)pyridine 9 k
This compound was obtained from 9 b in 53 % yield, mp 42–
44 ° (diethyl ether); 1H-NMR (CDCl3): δ 9.29 (d, 1 H, H-2), 8.30
(dt, 1 H, H-4), 4.15 (q, 2 H, N1⬘-CH2CH3), 2.49 (s, 3 H, 5⬘-CH3),
1.48 (t, 3 H, N1⬘-CH2CH3). Anal. C10H12N4 (C, H, N). Methiodide:
mp 235–237 °C. Anal. C11H15IN4 (C, H, N).
3-(1,5-Diethyl-1H-1,2,4-triazol-3-yl)pyridine 9 l
This compound was obtained from 9 c in 44 % yield, bp 125–
130°/0.01 mm Hg; 1H-NMR (CDCl3): δ 9.37 (d, 1 H, H-2), 8.39
(dt, 1 H, H-4), 4.24 (q, 2 H, N1⬘-CH2CH3), 2.90 (q, 2 H, 5⬘CH2CH3), 1.54 and 1.49 (dt, 6 H, N1⬘-CH2CH3 and 5⬘-CH2CH3).
Anal. C11H14N4 (C, H, N). Methiodide: mp 248–250 °C. Anal.
C12H17IN4 (C, H, N).
3-(1-Methyl-1H-1,2,4-triazol-5-yl)pyridine 11 a
This compound was obtained from 9 a in 12 % yield, mp 120–
121 °C (ethyl acetate). 1H-NMR (CDCl3): δ 8.83 (d, 1 H, H-2),
7.92 (dt, 1 H, H-4), 7.85 (s, 1 H, H-3⬘), 3.92 (s, 3 H, N1⬘-CH3).
Anal. C8H8N4 (C, H, N). Methiodide: mp 198–201 °C; Anal.
C9H11IN4 (C, H, N).
3-(1,3-Dimethyl-1H-1,2,4-triazol-5-yl)pyridine 11 b
This compound was obtained in 4 % yield, mp 87–90 °C (diethyl
ether) as by-product in the methylation of 9 b. 1H-NMR (CDCl3):
δ 8.82 (d, 1 H, H-2), 7.92 (dt, 1 H, H-4), 7.36 (dd, 1 H, H-5), 2.33
(s, 3 H, 3⬘-CH3) 3.87 (s, 3 H, N1⬘-CH3). Anal. C9H10N4 (C, H, N).
General procedure for the preparation of 1-methyl-5-(1,2,4-triazol-3- and 5-yl)-1,2,3,6-tetrahydropyridines 10 and 12
Each 9 and 11 methiodide was allowed to react with sodium
borohydride following the same procedure described for compounds 5 and 6.
1-Methyl-5-(1H-1,2,4-triazol-3-yl)-1,2,3,6-tetrahydropyridine
10 d
This compound was obtained from 9a methiodide in 90 % yield,
mp 182–184 °C (ethyl acetate); 1H-NMR (DMSO-d6): δ 14.00
(bs, 1 H, NH), 8.21 (s, 1 H, H-5⬘), 2.38–2.20 (m, 5 H, N1-CH3
and H-3). Anal. C8H12N4 (C, H, N).
1-Methyl-5-(5-methyl-1H-1,2,4-triazol-3-yl)-1,2,3,6-tetrahydropyridine 10 e
This compound was obtained from 9 b methiodide in 94 %
yield, mp 133–135°C (ethyl acetate); 1H-NMR (DMSO-d6): δ
13.00 (bs, 1 H, NH), 2.30–2.18 (m, 8 H, N1-CH3, 5⬘-CH3 and H3). Anal. C9H14N4 (C, H, N).
5-(5-Ethyl-1H-1,2,4-triazol-3-yl)-1-methyl-1,2,3,6-tetrahydropyridine 10 f
This compound was obtained from 9 c methiodide in 80 % yield,
mp 97–99 °C (ethyl acetate); 1H-NMR (CDCl3): δ 11.80 (bs, 1 H,
152 Del Giudice et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 143–154
NH), 2.66 (q, 2 H, 5⬘-CH2CH3), 2.38 (s, 3 H, N1-CH3), 1.19 (t,
3 H, 5⬘-CH2CH3). Anal. C10H16N4 · H2O (C, H, N).
40 000 g at 4 °C for 10 minutes.The pellet was resuspended in
100 volumes of the same buffer and the suspension was aliquoted. The final pellet was held at –80 °C, until use. The heart
or the submandibular glands were rinsed in an ice-cold isotonic
NaCl solution, then homogenized using an Ultraturrax for
5 seconds at maximum setting, in 2.5 mL of 50 mM Tris-HCl
buffer (pH 7.5) and enriched with 250 mM sucrose. The process was refrigerated adequately. The resulting homogenate
was diluted 8-fold with the same buffer and filtered on two layers of medical gauze.The suspension was centrifuged twice at
400 g at 4 °C for 5 minutes.The supernatant was further centrifuged at 4 °C for 10 minutes at 20 000 g.The pellet was re-suspended in 100 volumes of ice-cold 50 mM Tris-HCl buffer
(pH 7.5), and the suspension was centrifuged at 4 °C for
10 minutes at 20 000 g. The pellet was then re-suspended in
buffer without sucrose, and suspension was aliquoted.The final
pellet was kept at –80 °C, until use.
1-Methyl-5-(1-methyl-1H-1,2,4-triazol-3-yl)-1,2,3,6-tetrahydropyridine 10 g
This compound was obtained from 9 g methiodide in 72 %
yield, hydrochloride mp 226–228 °C; 1H-NMR (CDCl3): δ 7.91
(s, 1 H, H-5⬘), 3.86 (s, 3 H, N1⬘-CH3). Anal. C9H15ClN4 · H2O (C,
H, N).
5-(1,5-Dimethyl-1H-1,2,4-triazol-3-yl)-1-methyl-1,2,3,6-tetrahydropyridine 10 h
This compound was obtained from 9 h methiodide in 77 %
yield, hydrochloride mp 282–284 °C; 1H-NMR (CDCl3): 3.75 (s,
3 H, N1⬘-CH3), 2.41 (ad, 6 H, N1-CH3 and 5⬘-CH3). Anal.
C10H17ClN4 · 2 H2O (C, H, N).
5-(5-Ethyl-1-methyl-1H-1,2,4-tr iazol-3-yl)-1-methyl1,2,3,6-tetrahydropyridine 10 i
This compound was obtained from 9 i methiodide in 72 % yield,
hydrochloride mp 226–228 °C; 1H-NMR (CDCl3): δ 3.75 (s, 3 H,
N1⬘-CH3), 2.71 (q, 2 H, 5⬘-CH2CH3), 1.30 (t, 3 H, 5⬘-CH2CH3).
Anal. C11H19ClN4 · 2 H2O (C, H, N).
5-(1-Ethyl-1H-1,2,4-triazol-3-yl)-1-methyl-1,2,3,6-tetrahydropyridine 10 j
This compound was obtained from 9 j methiodide in 91 % yield,
hydrochloride mp 175–178 °C; 1H-NMR (CDCl3): δ 7.92 (s, 1 H,
H-5⬘), 4.13 (q, 2 H, N1⬘-CH2CH3), 1.47 (t, 3 H, N1⬘-CH2CH3).
Anal. C10H17ClN4 (C, H, N).
5-(1-Ethyl-5-methyl-1H-1,2,4-triazol-3-yl)-1-methyl-1,2,3,6tetrahydropyridine 10 k
This compound was obtained from 9 k methiodide in 67 % yield,
hydrochloride mp 234–236 °C; 1H-NMR (CDCl3): δ 6.72–6.64
(m, 1 H, H-4), 4.08 (q, 2 H, N1⬘-CH2CH3), 2.40 and 2.38 (ad,
6 H, N1-CH3 and 5⬘-CH3), 1.37 (t, 3 H, N1⬘-CH2CH3). Anal.
C11H19ClN4 · 2 H2O (C, H, N).
5-(1,5-Diethyl-1H-1,2,4-triazol-3-yl)-1-methyl-1,2,3,6-tetrahydropyridine 10 l
This compound was obtained from 9 l methiodide in 70 % yield,
hydrochloride mp 248–250 °C; 1H-NMR (CDCl3): δ 6.78–6.68
(m, 1 H, H-4), 4.04 (q, 2 H, N1⬘-CH2CH3), 2.68 (q, 2 H, 5⬘CH2CH3), 2.42 (s, 3 H, N1-CH3), 1.39 (dt, 6 H, N1⬘-CH2CH3 and
5⬘-CH2CH3). Anal. C12H21ClN4 · 2 H2O (C, H, N).
1-Methyl-5-(1-methyl-1H-1,2,4-triazol-5-yl)-1,2,3,6-tetrahydropyridine 12 g
This compound was obtained from 11 a methiodide in 84 %
yield, hydrochloride mp 205–207 °C; 1H-NMR (CDCl3): δ 7.79
(s, 1 H, H-3⬘), 3.91 (s, 3 H, N1⬘-CH3), 2.42 (s, 3 H, N1-CH3).
Anal. C9H15ClN4 · 2 H2O (C, H, N).
Pharmacology
Radioligand binding assay
Male Wistar rats (400–450 g) were killed by decapitation and
the brain, heart, and submandibular glands were immediately
removed. The brain was dissected and homogenized in an icecold buffer at pH 7.4 containing 10 mM HEPES, 1 mM EDTA,
and 0.32 M sucrose. The homogenate was centrifuged at
400 g at 4°C for 5 minutes, the supernatant was further centrifuged at 40 000 g at 4 °C for 10 minutes. The pellet was resuspended in the same volume of 10 mM HEPES buffer at pH 7.4
containing 1 mM EDTA and centrifuged for a second time at
Membrane preparations from rat frontal cortex, heart and submandibular glands, were resuspended in 50 mM phosphate
buffer solution (PBS) at pH 7.4. Protein content was then determined by the method of Lowry [20]. The membranes (120 to
240 mg) were incubated, at 25 °C, in a final volume of 1 mL
PBS (50 mM pH 7.4), containing [3H] pirenzepine (1 nM) for
M1 receptors and [3H]N-methylscopolamine ([3H]NMS)
(0.25 nM) for M2 – M3 receptors, for 60 minutes. Drugs were
added at increasing concentration (0.1–10 000 nM). The equilibrium dissociation constants (KD) for [3H]pirenzepine and
[3H]NMS were obtained by Scatchard analysis of saturation
isotherms [21]. These experiments were performed using
0.01–3 nM [3H]pirenzepine and 0.01–2 nM [3H]NMS. Non-specific binding was defined in the presence of 1 nM atropine. Specific binding was calculated by subtracting the non specific from
the total binding. The membrane bound [3H]pirenzepine or
[3H]NMS was trapped at the end of the incubation by rapid
vacuum filtration of the incubation mixture over Whatman GF/B
glass fibre filters pre-soaked overnight in 0.1 % polyethylenimine. Filters were washed (2 × 5 mL) with an ice-cold incubation buffer and transferred to scintillation vials to which 5 mL of
scintillant was added. Radioactivity counting was made using a
liquid scintillation counter (LKB-Wallac, Fisher Scientific, Montreal, Quebec, Canada) with an efficiency of about 45 %.
The competitor dissociation constant (Ki) [22] was determined
by comparative analysis of saturation and displacement curves
obtained with membrane preparations by RADLIG analysis of
multiple data files [23].
Functional in vitro assays
Rabbit vas deferens
Vasa deferentia were isolated from New Zealand white rabbits
weighing 2.5 to 4.0 kg (Harlan, Italy). Animals were killed by a
blow on the head and bled rapidly. Each vas deferens was dissected free from surrounding tissue, divided into a prostatic
and an epididymal segment and placed in modified Krebs’solution of the following composition (mM): NaCl, 134; KCl, 3.4;
CaCl2, 2.8, KH2PO4, 1.3; NaHCO3, 16; MgSO4, 0.6; and glucose, 7.7. Experimental procedure was in accordance to Eltze
[24].
Porcine bladder
Porcine bladder strips were transferred to a jacketed tissue
bath (10 mL) and mounted between two hooks. One of the
hooks was connected to a force transducer (Type 7006, Basile,
Varese, Italy). The baths contained a temperature-controlled
(37 °C) Krebs-Henseleit solution of the following composition
(mM): NaCl, 133; KCl, 4.7; MgSO4, 1.2; CaCl2, 2.5; NaH2PO4,
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 143–154
Muscarinic Receptor Ligands 153
1.3; NaHCO3, 16.3; and glucose, 7.7. Continuous aeration with
carbogen maintained pH at 7.4. Isometric tension was recorded by Basile, Unirecord 7050 resting tension of 4 g was initially
applied to each preparation. During stabilisation (45–60 min)
the strips were repeatedly washed and the resting tension was
adjusted. When response of porcine strips to K+ were studied,
the NaCl in the Krebs solution was replaced by KCl to give a K+
concentration of 133 mM and 122.4 mM respectively. Porcine
preparations were exposed to a standard concentration of the
muscarinic receptor agonist acetylcholine (Ach) 4.4 × 10–7 M
to estabilish reproducible responses (three subsequent contractions with 10 % variation in amplitude) before a concentration-response curve to ACh (control) was generated. After exposure of the bladder strips to a fixed concentration of antagonist for 60 min [25], the concentration-response curve to ACh
was repeated in the presence of antagonist. Response were
expressed as a percentage of the maximal contractile response elicited by ACh in the control curve.The viability of bladder preparations was controlled by exposure to K+ after cumulative concentration-response curves to ACh were estabilished
(control).
15 min before the administration of 0.5 mg/kg oxotremorine s.c.
and the appearance of tremor, lacrimation and salivation was
evaluated in 1 min observation periods every 15 min for 1 h.
The incidence and severity of tremor, lacrimation and salivation
was assessed in each mouse by assigning each animal a score
from 0 to 3 corresponding to the severity of the effect. For
tremor, a score of 0 indicates no observed signs; 1, weak
tremor upon handling; 2, tremor and flexing of neck upon
handling; 3, spontaneously occurring pronounced clonic
tremor. For lacrimation score of 0 indicates no observed signs;
1, wet immediately around eye; 2 wet around eye particularly at
the inferior part; 3 wet around eye with a little drop. For salivation a score of 0 indicates no observed signs; 1, wet around the
mouth; 2, wet around mouth, throat and/or neck; 3 wet around
mouth, throat and/or neck on chest and/or belly. ED values
were obtained and analyzed as previously reported [29, 30].
Ileum guinea pig
Male guinea-pigs Harlan-Nossan, 300–500 g were used in all
experiments. Antimuscarinic potency of compounds was determined in guinea-pig isolated ileum strips according to Lambrecht group [26].
Antagonist potency evaluation
From these experiments antagonist potency was calculated
from Schild plots by extrapolation to the abscissa, which yields
pA2 estimates, defined as the –log of the molar concentration
of antagonist which reduces the effect of a concentration of
agonist to that of half the concentration [27].
In vivo experiments
Male CD-1 mice (Charles River, Italy) weighing 25–30 g were
used for all experiments. The animals were housed in colony
cages (5 mice each) under standard light (alight from
7.00 a.m. to 7.00 p.m.), temperature (22 ± 1 °C) and relative
humidity (60 % ± 10 %) conditions for at least 1 week before the
experimental sessions. Food and water were available ad libitum. All experiments were carried out according to the guidelines of the European Community Council for experimental
animal care (86/609/EEC).
On each day of testing oxotremorine and atropine (Sigma
Chemical, Italy) was freshly dissolved in 0.9 % NaCl solution for
subcutaneous (s.c.) or intraperitoneal (i.p.) administration and
injected in a volume of 5 mL/kg.Test compound solutions were
prepared by dissolving 6 i and 5 l in dimethyl sulfoxide; aliquots
of this solution were used for subsequent dilution in saline
(dimethyl sulfoxide:saline 1:4, v/v) for i.p. treatment, and were
administered immediately after sonication in a volume of
10 mL/kg.
After the treatment the animals were individually placed in a
plexiglas cage (30 × 14 × 12 cm) and the incidence and severity
of the side effects induced by test compound were evaluated
following Irwin [28].
Antagonism of oxotremorine-induced effects
Antagonism potency was determined from the protection given
by the drug from behavioural effects induced by oxotremorine
[29, 30]. Oxotremorine is able to induce tremor accompanied
by sign of parasymphatic stimulation such as salivation and
lacrimation. So, mice were treated i.p. with the test compounds
Acknowlegments
We wish to thank Dr. L. Turchetto for mass spectral data
and Mr. R. Lecce for microanalyses.
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