Preparation and local anaesthetic activity of benzotriazinone and benzoyltriazole derivatives

Article abstract of DOI:10.1016/S0223-5234(99)00126-9

Two sets of benzotriazinone and benzoyltriazole derivatives were prepared and tested for local anaesthetic activity in comparison with lidocaine. Several of the prepared compounds exhibited a fairly good activity comparable or superior to that of lidocaine. The presence of a benzotriazinone or a benzoyltriazole moiety as an aromatic system was quite profitable for both the intensity and duration of activity. The acute toxicity in mice of the four most potent compounds of the series was also assessed. Compound 1b, which has an anaesthetic activity comparable to that of lidocaine, was also characterized by a more favourable therapeutic index. All compounds were tested in vitro to evaluate their negative chronotropic action in isolated rat right atria.

Full text of DOI:10.1016/S0223-5234(99)00126-9

Eur. J. Med. Chem. 34 (1999) 1043−1051
© 1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
1043
Original article
Preparation and local anaesthetic activity of benzotriazinone
and benzoyltriazole derivatives
Giuseppe Caliendoa*, Ferdinando Fiorinoa, Paolo Griecoa, Elisa Perissuttia, Vincenzo Santagadaa,
Rosaria Melib, Giuseppina Mattace Rasob, Angelina Zanescoc, Gilberto De Nuccic
aDipartimento di Chimica Farmaceutica e Tossicologica, Università degli Studi di Napoli “Federico II” Via D. Montesano 49,
80131 Napoli, Italy
bDipartimento di Farmacologia Sperimentale, Università degli Studi di Napoli “Federico II” Via D. Montesano 49, 80131 Napoli, Italy
cCartesius Analytical Unit, Department of Pharmacology, Biomedical Sciences Institute, University of Sao Paulo, Sao Paulo, Brazil
(Received 19 April 1999; revised 6 July 1999; accepted 15 July 1999)
Abstract – Two sets of benzotriazinone and benzoyltriazole derivatives were prepared and tested for local anaesthetic activity in comparison
with lidocaine. Several of the prepared compounds exhibited a fairly good activity comparable or superior to that of lidocaine. The presence
of a benzotriazinone or a benzoyltriazole moiety as an aromatic system was quite profitable for both the intensity and duration of activity. The
acute toxicity in mice of the four most potent compounds of the series was also assessed. Compound 1b, which has an anaesthetic activity
comparable to that of lidocaine, was also characterized by a more favourable therapeutic index. All compounds were tested in vitro to evaluate
their negative chronotropic action in isolated rat right atria. © 1999 Éditions scientifiques et médicales Elsevier SAS
benzotriazinone / benzoyltriazole / local anaesthetic / negative chronotropic action
1. Introduction
activity and to evaluate the influence on activity by the
different aromatic systems, the benzotriazole nucleus was
replaced by a 1,2,3-benzotriazin-4(3H)-one and by a
4-benzoyl-1,2,3-triazole ring, thereby achieving general
structures 1, 2 and 3 (figure 1).
In previous articles we described the synthesis of two
sets of N-[2-(alkylamino)ethyl]benzotriazol-X-yl-acet-
amides and of N-[2-(alkylamino)ethyl]benzotriazol-X-yl-
isobutyramides designed as local anaesthetic agents [1,
2]. The compounds of two sets fulfill the requirements of
the pharmacofore scheme proposed by Löfgren [3] be-
cause they are provided with an aromatic system, an
intermediate chain and an aminic moiety ionizable under
physiological pH. They have been assayed in vivo for
their local anaesthetic activity. Some of the investigated
compounds showed anaesthetic activities comparable
with or higher than those exhibited by the reference drug
lidocaine.
The N-alkylamino groups of the intermediate acetyl-
acetamido side chain are those previously reported [1, 2]
displaying the highest anaesthetic activity: dimethyl-
amino, diethylamino, pyrrolidine and piperidine substitu-
ents.
Herein we report the synthesis of a series of 1,2,3-
benzotriazinones (1a–d) and of 4-benzoyl-1,2,3-triazole
(2a–d and 3a–d) derivatives (tables I and II). The syn-
thesized compounds were first tested as local anaesthetics
with different in vivo assays and, successively consider-
ing the relationships between local anaesthetic (Na+
channel block) and antiarrhythmic activities [4, 5], all
compounds were preliminary tested to evaluate their
negative chronotropic action in rat isolated right atria.
Considering these results and the important role played
by the aromatic system in the interaction with a corre-
sponding hydrophobic region, in order to increase the
*Correspondence and reprints

1044
Figure 1. General structure of considered compounds.
Table I. Physicochemical properties of 1,2,3-benzotriazinone derivatives 1a–d.
R
Formula*
MW
Compound
M.p.
(°C)
Cryst.**
Solvent
Yield
(%)
C13H17N5O2
275.31
303.37
1a
1b
145–146
155–156
a + b
60
55
C15H21N5O2
a
C15H19N5O2
C16H21N5O2
301.35
315.38
1c
180–181
169–170
a + b
66
52
1d
a
*Satisfactory microanalyses obtained: C, H, N values are within ± 0.4% of theoretical values. **Crystallization solvents: a) diethyl ether; b)
ethyl alcohol.
2. Chemistry
tives (5) were converted into the amide derivatives (1) by
reaction in methyl alcohol solution with the appropriate
amines.
The final compounds 1a–d, reported in table I, were
purified by cromatography on a silica gel column and
further by crystallization from an appropriate solvent
(yields ranging between 52–66%).
Compounds of general formula 1 reported in figure 1
were synthesized as illustrated in figure 2. They derived
from condensation of a 1,2,3-benzotriazin-4(3H)-one ring
(4) with ethyl bromoacetate in butan-2-one in the pres-
ence of potassium carbonate. The ethyl acetate deriva-

1045
Table II. Physicochemical properties of 4-benzoyl-1,2,3-triazole derivatives 2a–d and 3a–d.
1-substituted 4-benzoyl-1,2,3-triazoles
2-substituted 4-benzoyl-1,2,3-triazoles
Compound** M.p. (°C) Yield (%)
R
Formula*
MW
Compound** M.p. (°C)
Yield (%)
C15H19N5O2
301.35
2a
2b
132–133
136–137
70
3a
3b
96–97
60
45
C17H23N5O2
329.40
40
99–100
C17H21N5O2
C18H23N5O2
327.39
341.41
2c
151–153
159–161
50
40
3c
98–99
94–95
55
50
2d
3d
*Satisfactory microanalyses obtained: C, H, N values are within ± 0.4% of theoretical values. **All compounds were crystallized by methyl
alchol/diethyl ether.
The 4-benzoyl-1,2,3-triazole derivatives (2a–d and
3a–d) were synthesized following the steps outlined in
figure 3.
The parent compound, 4-benzoyl-1,2,3-triazole (8),
was obtained in 95% yield by a modified method already
described [6, 7]. Oxidation of the phenylethynylcarbinol
(6) with chromium trioxide and concentrated sulphuric
acid produced phenyl ethynyl ketone (7) which was
successively treated with NaN3 in anhydrous dimethyl-
acetamide providing the expected compound. Reaction of
4-benzoyl-1,2,3-triazole with ethyl bromoacetate and po-
tassium carbonate in butan-2-one gave a mixture of the
triazole isomers 1- and 2- with an overall yield of
80% [8]. The two isomers were separated by column
chromatography on silica gel using diethyl ether/n-
hexane (7:3 v/v) as eluent. The faster moving 2- isomer,
ethyl 4-(benzoyl)-1,2,3-triazole-2-acetate (10), was col-
lected with a higher yield (65%) with respect to the
slower moving 1-substituted isomer (9) (35%). The ethyl
acetate derivatives (9 and 10) were converted into the
corresponding amide derivatives 2 and 3 by reaction in
methyl alcohol solution with the appropriate amines.
All the final products (2a–d and 3a–d) reported in
table II were further purified by crystallization from a
mixture of diethyl ether and methyl alcohol (yields
ranging between 40–70%).
The synthesized compounds listed in tables I and II
were characterized by 1H-NMR spectroscopy whose data
were fully consistent with the described structures.
Figure 2. Synthesis of 1a–d.

1046
Figure 3. Synthesis of 4-benzoyl-1,2,3-triazole de-
rivatives.
1H-NMR of compounds 2a–d and 3a–d (CDCl3)
ble IV). The ip acute toxicity and the therapeutic index of
the more active compounds were also determined (ta-
ble V). Successively the synthesized compounds were
tested in vitro to evaluate their negative chronotropic
action in rat isolated right atria (table VI). The synthe-
sized compounds were always compared for their activity
with lidocaine, taken as reference drug.
differentiated clearly between 1- and 2- substituted
4-benzoyl-1,2,3-triazole derivatives. In fact there is a
difference in chemical shift values among the protons in
the 5- position of the benzoyltriazole ring in the series of
1- and 2- isomers. The triazole proton of the 1- isomer
appears always as a singlet at lower field with respect to
the position of the same proton of analogues 2-substituted
(chemical shift values ranging between 8.45–8.70 δ and
8.20–8.35 δ, respectively) according with literature [8, 9].
Physicochemical data of all the final compounds are
summarized in tables I and II.
4. Results and discussion
Table III summarizes the results of the surface and
infiltration anaesthesia assays performed on the 1,2,3-
benzotriazinone (1a–d) and on the 4-benzoyl-1,2,3-
triazole (2a–d and 3a–d) derivatives. As for the 1,2,3-
benzotriazinone derivatives (1a–d), in both tests, the
results indicate that the structures endowed with highest
activity are 1b and 1d, with values similar to that
measured for lidocaine.
3. Pharmacology
The compounds reported in tables I and II were tested
in vivo for their anaesthetic activity by different tests:
corneal anaesthesia in the rabbit, mouse tail anaesthesia
(table III) and rat sciatic nerve block anaesthesia (ta-

1047
Table III. Rabbit corneal and mouse tail anaesthetic activities.
Table V. Acute toxicity in mouse (LD50) and therapeutic index of
selected compounds 1b, 1d, 3b and 3d.
Compound
Corneal anaesthetica Mouse tail anaestheticb
a
Com-
pound
LD50
IC50
Therapeutic
indexb
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
inactive
74.3 ± 9.3
12.1 ± 3.9
73.8 ± 8.9
inactive
4.7 ± 1.5
3.2 ± 2.1
30.1 ± 5.6
22.5 ± 4.5
105.0 ± 3.5
48.5 ± 9.1
92.3 ± 8.0
100
5.4 (± 0.38)10–2
0.69 (± 0.31)10–2
2.6 (± 0.43)10–2
1.2 (± 0.42)10–2
4.0 (± 0.36)10–2
3.0 (± 0.37)10–2
3.0 (± 0.33)10–2
3.6 (± 0.35)10–2
3.0 (± 0.29)10–2
1.4 (± 0.25)10–2
3.0 (± 0.31)10–2
1.4 (± 0.28)10–2
0.68 (± 0.39)10–2
LD50/IC50
1b
1d
3b
3d
6.09 (± 0.49)10–2
5.24 (± 0.38)10–2
8.69 (± 0.53)10–2
4.46 (± 0.35)10–2
M
M
M
M
M
0.69 (± 0.31)10–2
1.2 (± 0.42)10–2
1.4 (± 0.25)10–2
1.4 (± 0.28)10–2
0.68 (± 0.39)10–2
M
M
M
M
M
8.83
4.37
6.21
3.19
8.23
lidocaine 5.60 (± 0.39)10–2
HCl
aMolar concentration of the injected solution (v = 0.2 mL/20 g
b
body weight). Evaluated as ratio between LD50 and IC50 (mouse
tail test) expressed in mg/kg.
lidocaine HClc
aAll compounds were in aqueous solution at 2% concentration. The
values expressed as % of the anaesthetic activity of lidocaine (=
100), are means ± SE of three determinations. bIC50 values
the nature of the side chain affect surface and infiltration
activities in a similar way.
c
For all considered compounds, with respect to the
nature of the intermediate acetylacetamido side chain,
analogues with a terminal diethylamino group or a
piperidine ring displayed the highest activity in accor-
dance with previous results [1, 2]. Reducing the size from
a six to a five-membered ring resulted in an improvement
of anaesthetic activity.
In order to evaluate the duration of the local anaes-
thetic activity, additional investigations were conducted
by rat sciatic nerve block assay. In accordance with the
previous results, when a 2% solution was used, as
reported in table IV, several compounds (1b, 1d, 3a, 3b,
and 3d) exhibited a better performance in blocking the rat
sciatic nerve with respect to lidocaine (160, 160, 150, 210
and 180 min for motor activity recovering, respectively,
versus 117 min for lidocaine).
expressed as mol/L. Lidocaine hydrochloride was used for com-
parison.
As for the 4-benzoyl-1,2,3-triazole derivatives (2a–d
and 3a–d), the results indicate that the nature and the
position of the intermediate acetylacetamido side chain
on the benzoyltriazole nucleus have significant influence
on activity. In both tests the 2-substituted isomers appear
to be more active than the 1-substituted derivatives.
Compounds 3b and 3d stand out in the data set for their
high activities (these values are comparable to those
determined for lidocaine). It appears that the position and
Table IV. Duration of local anaesthetic activity in rat sciatic nerve
block.
Finally, the acute toxicity and therapeutic index of the
more active compounds in all anaesthetic tests (1b, 1d,
Compound
Durationa (min)
1%
2%
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
25 (± 10.5)
77 (± 6.8)
inactive
90 (± 3.6)
40 (± 15.0)
35 (± 11.0)
inactive
40 (± 12.0)
75 (± 7.0)
117 (± 8.0)
25 (± 12.3)
70 (± 6.6)
65 (± 10.7)
45 (± 17.0)
160 (± 18.2)
n.t.
160 (± 15.3)
72 (± 6.9)
63 (± 6.8)
n.t.
72 (± 7.0)
150 (± 17.2)
210 (± 16.9)
50 (± 6.5)
180 (± 20.3)
117 (± 11.0)
Table VI. Maximum response to different compounds in rat iso-
lated right atria compared to lidocaine.
Compound
Maximum response
(beats/min)
n
1a
1b
1c
1d
2a
2b
2c
2d
3b
–15 ± 6
–42 ± 8
–52 ± 8
–106 ± 27
–33 ± 13
–55 ± 12
–72 ± 5
–107 ± 27
–67 ± 9
6
6
6
5
6
6
6
6
6
6
6
6
lidocaine HCl
aIn vivo duration of local anaesthetic activity in rat sciatic nerve
block (each rat received 0.2 mL of 1% and 2% anaesthetic solution,
n.t. = not tested. The values are means ± SE of three determina-
tions.
3c
3d
–135 ± 31
–152 ± 12
–180 ± 35
lidocaine

1048
3b, and 3d) were determined in mice. From the corre-
sponding LD50 and LD50/IC50 values shown in table V it
is evident that 1b is characterized by the most favourable
ratio between toxicity and mouse tail anaesthetic activity
(its therapeutic index is slightly higher than that measured
for lidocaine).
These studies showed that the presence of a piperidine
ring as a terminal N-alkylamino group gives an increase
in toxicity, particularly evident on compound 3d.
In conclusion, a comparison between the pharmaco-
logical profile of benzotriazinone and benzoyltriazole
derivatives versus that of the above mentioned benzotria-
zole counterpartes [1, 2] clearly shows that the replace-
ment of the benzotriazole moiety, found in the preceeding
derivatives, by a benzotriazinone or by a benzoyltriazole
ring, seems to have a little influence on the anaesthetic
activity. These results might be ascribed to an analogue
lipophilic, steric, and electronic complementarity be-
tween the benzotriazole residue compared to the benzo-
triazinone or to the benzoyltriazole moiety.
Figure 4. Negative chronotropic response for the compounds
1a, 1b, 1c, 1d, and lidocaine in isolated right atria from rats.
Data are means ± SEM for 5–6 experiments.
As regards the results of chronotropic activity reported
in table VI, all compounds induced a negative chronotro-
pic effect in rat isolated right atria at high doses, in a
concentration range greater than µM. The intrinsic activ-
ity of different compounds in decreasing spontaneous
beating rate in isolated right atria was distinct. Lidocaine
was used as the pattern drug to compare the intrinsic
activity of all compounds; in fact it is well known that
lidocaine has local anaesthetic as well antiarrhythmic
actions
5. Experimental protocols
5.1. Chemistry
Melting points were determined on a Kofler hot stage
apparatus and are uncorrected. Structures described were
1
supported by H-NMR spectra and microanalytical data.
1H-NMR spectra were recorded on a Bruker WM 500
spectrometer using DMSO and CDCl3 as solvent; chemi-
cal shifts (δ) are reported as follows: s, singlet; d,
In series 1, compound 1d determines a larger magni-
tude of maximum response compared to drugs 1b, 1c, and
1a, whereas drug 1a was less effective in producing a
negative chronotropic response among these compounds
(figure 4 and table VI). In series 2, compound 2d had
higher intrinsic activity compared to compounds 2a, 2b,
and 2c (figure 5). The intrinsic activity of compounds 2a
and 2b were similar.
The intrinsic activity of compound 3d was the best of
all studied compounds. Compound 3c showed similar 3d
intrinsic activity even if less, whereas compound 3b
induced the lowest activity of the series (figure 6 and
table VI).
A comparison between ‘in vivo’ anaesthetic activity
and ‘in vitro’ negative chronotropic activity data dis-
played a similar pattern. In fact, clear negative chrono-
tropic action was displayed by compound 1d and 3d,
which bear a piperidine ring, but also compound 2d,
which was poorly active on anaesthetic activity, showed
an appreciable negative chronotropic action.
Figure 5. Negative chronotropic response for the compounds
2a, 2b, 2c, 2d, and lidocaine in isolated right atria from rats.
Data are means ± SEM for 6 experiments.

1049
5.1.2. General procedure for the preparation of
compounds 1a–d
To 0.01 mol of 3-carbethoxymethyl-1,2,3-benzo-
triazin-4(3H)-one (5) dissolved in anhydrous methanol
(50 mL) was added the appropriate amine (0.01 mol)
dropwise. The reaction mixture was kept under reflux
with magnetic stirring for 8–12 h and monitored by TLC
until the starting material had disappeared. After cooling
the solvent was removed under reduced pressure and the
residue was purified by silica-gel column chromatogra-
phy using mixtures of diethyl ether/methanol 9:1 v/v.
Further purification was obtained by crystallization from
the appropriate solvent (table I).
Yields ranging between 52–66%. Spectral data of title
compound 1a: 1H-NMR (CDCl3), δ = 8.33 (d, 1H, Ar-H,
J = 7.5 Hz), 8.15 (d, 1H, Ar-H, J = 7.5 Hz), 7.94 (t, 1H,
Ar-H, J = 7.5 Hz), 7.78 (t, 1H, Ar-H, J = 7.5 Hz), 5.11 (s,
2H, CH2-CO), 3.39 (t, 2H, NH-CH2, J = 7.5 Hz), 2.47 (t,
2H, CH2-N, J = 7.5 Hz) and 2.24 ppm (s, 6H, N(CH3)2).
Figure 6. Negative chronotropic response for the compounds
3b, 3c, 3d, and lidocaine in isolated right atria from rats. Data
are means ± SEM for 6 experiments.
1
Similar H-NMR data occur in all derivatives of general
formula 1.
doublet; t, triplet; m, multiplet. The spectra obtained
confirmed the proposed structures. All pure compounds
gave a satisfactory analysis (C, H, N) within ± 0.4% of
theoretical values.
Analytical TLC was performed on precoated silica-gel
(0.2 mm GF 254, E Merck); the spots were located by UV
(254 nm) light. Evaporation was performed in vacuo.
Anhydrous sodium sulfate was used as a drying agent.
Crude products were routinely passed through columns of
silica gel (0.05–0.20 mm, Carlo Erba). Analytical data,
melting points and crystallization solvents are reported in
tables I and II.
5.1.3. Phenyl ethynyl ketone (7)
A solution of chromium trioxide (0.10 mol) in water
(30 mL) and concentrated sulphuric acid (8.5 mL) was
slowly added to a stirred and cooled solution of phenyl-
ethynylcarbinol (6) (0.15 mol) in acetone (50 mL). The
reaction mixture was carried out at 4 °C under a nitrogen
atmosphere. After stirring for 7 h, water was added to
dissolve the precipitated chromium salts and the product
was extracted with chloroform. Evaporation of the or-
ganic solution gave a yellow solid which was crystallized
from aqueous methanol to give 16.6 g (85%) of 7 as pale
yellow needles. The physical data are in agreement with
those given in ref. [6].
5.1.1. 3-Carbethoxymethyl-1,2,3-benzotriazin-4(3H)-one
(5)
To
a
magnetically stirred solution of 1,2,3-
5.1.4. 4-benzoyl-1H-1,2,3-triazole (8)
benzotriazin-4(3H)-one (4) (0.01 mol) and ethyl bromo-
acetate (0.01 mol) in butan-2-one (50 mL) was added
K2CO3 (0.01 mol). The reaction mixture was heated
under reflux for 10 h and monitored by TLC. After
cooling, the butan-2-one was removed under reduced
pressure and the residue was diluted with H2O and
extracted with CHCl3. The organic layer was dried and
evaporated to dryness. The crude product 5 was purified
by crystallization from methyl alcohol/diethyl ether.
Yield 68%, m.p. 114–116 °C.1H-NMR (CDCl3) δ = 8.37
(d, 1H, Ar-H, J = 7.5 Hz), 8.20 (d, 1H, Ar-H, J = 7.5 Hz),
7.99 (t, 1H, Ar-H, J = 7.5 Hz), 7.82 (t, 1H, Ar-H), 5.19 (s,
2H, -NCH2), 4.28 (m, 2H, OCH2), and 1.30 ppm (t, 3H,
CH3).
To a stirred and heated solution of NaN3 (0.10 mol) in
anhydrous dimethylacetamide (80 mL), phenyl ethynyl
ketone 7 (0.10 mol) dissolved in anhydrous dimethylacet-
amide (80 mL) was slowly added. The reaction mixture
was kept at 100 °C for 2 h. After stirring for a further 12 h
at room temperature, evaporation of the solvent under
reduced pressure gave a liquid residue which was diluted
with water. The aqueous layer was acidified (pH = 5) with
10% HCl and extracted with ether (3 × 200 mL). The
combined organic layers were dried and evaporated to
give a solid residue which was purified by crystallization
from ethyl alcohol: 16.4 g (95%) of 8. The physical data
are in agreement with those given in ref. [7].

1050
5.1.5. 1-carbethoxymethyl-4-benzoyl-1H-1,2,3-triazole
(9) and 2-carbethoxymethyl-4-benzoyl-2H-1,2,3-triazole
(10)
J = 7.5 Hz), 7.62 (t, 1H, Ar-H, J = 7.5 Hz), 7.52 (t, 2H,
Ar-H, J = 7.5 Hz), 5.28 (s, 2H, CH2-CO), 3.33 (t, 2H,
NH-CH2, J = 7.5 Hz), 2.35 (t, 2H, CH2-N(CH3)2, J = 7.5
Hz) and 2.15 ppm (s, 6H, N(CH3)2). Similar H-NMR
data occur in all derivatives of the general formula 3.
1
Anydrous K2CO3 (0.04 mol) was added to a solution
of4-benzoyl-1,2,3-triazole(8)(0.04 mol)andethylbromo-
acetate (0.04 mol) in 50 mL of butan-2-one. The mixture
was refluxed for 12 h and monitored by TLC. After
cooling, the butan-2-one was removed under reduced
pressure and the residue was diluted with H2O and
extracted with CHCl3. The organic layer was washed
with 2 M NaOH, dried and evaporated to dryness.
The obtained residue, containing 1- and 2-substituted
isomers was finally fractionated by column chromatogra-
phy using diethyl ether/n-hexane, 7:3 v/v, as eluent.
Further purification of the isolated 1- and 2- isomers by
crystallization from diethyl ether gave the final products
5.2. Pharmacology
5.2.1. Corneal anaesthesia
Local anaesthetic activity was evaluated in male New
Zealand rabbits (Harlan-Nossan, Correzzana, Milan,
weighing 2.4–2.8 kg) as local surface anaesthesia [10],
by determining every 3 min the number of stimuli to the
cornea, effected rhythmically with a Frey’s horse-hair,
necessary to produce the blink reflex. If the reflex did not
occurr after 100 stimulations, anaesthesia was considered
total. At the beginning of the experiment care was taken
to ascertain that this reflex was normal in both eyes of the
rabbits. All compounds were dissolved in 0.1 N HCl and
the solution buffered to pH 6–7. The aqueous solutions
(2%) of the compounds studied were dropped onto the
conjunctival sac so that the space between the eyelids
contained a clearly visible film of solution for the set time
of 3 min. Lidocaine solution (2%) was used for compari-
son.
1
9 and 10. Characterization by H-NMR spectra showed
that the first compound to be eluted was the 2-substituted
1,2,3-triazole (compound 10, yield 65%, m.p. 55–57 °C),
whereas the 1-substituted isomer was eluted successively
(compound 9, yield 35%, m.p. 132–133 °C). 9: 1H-NMR
(DMSO), δ = 9. (s, 1H, H-triaz), 8.50 (d, 2H, Ar-H, J =
7.5 Hz), 7.68 (t, 1H, Ar-H, J = 7.5 Hz), 7.55 (t, 2H, Ar-H
J = 7.5 Hz), 5.53 (s, 2H, CH2-CO), 4.27 (q, 2H, CH2O,
1
J = 7.5Hz) and 1.28 (t, 2H, CH3, J = 7.5 Hz). 10: H-
NMR (DMSO), δ = 8 (s, 1H, H-triaz), 8.58 (d, 2H, Ar-H,
J = 7.5 Hz), 7.70 (t, 1H, Ar-H, J = 7.5 Hz), 7.58 (t, 2H,
Ar-H, J = 7.5 Hz), 5.61 (s, 2H, CH2-CO), 4.26 (q, 2H,
CH2O, J = 7.5 Hz) and 1.25 (t, 2H, CH3, J = 7.5 Hz).
5.2.2. Mouse tail anaesthesia
Male Swiss mice (Harlan-Nossan, Correzzana, Milan,
weighing 18–20 g) were used. The test was performed
according to the method of Bianchi [11] in which the
aqueous anaesthetic solution (0.1 mL) is injected sub-
cutaneously about 1 cm from the base of the tail. Fifteen
minutes after injection, the pain reflex of all injected
animals was tested by applying a small artery clip to the
zone where the compound was injected. The proportion
of animals which did not show the usual pain reflex
within 30 s was noted for each dose. Lidocaine solutions
were used for comparison. IC50 values were calculated
for each compound by probit analysis using a computer
program [12].
5.1.6. General procedure for the preparation of
compounds 2a–d and 3a–d
To 0.01 mol of the appropriate ethyl-4-benzoyl-1,2,3-
triazolacetate derivative (9 or 10) dissolved in anhydrous
methanol was added dropwise the appropriate amine
(0.01 mol). The reaction mixture was kept under reflux
with magnetic stirring for 8–12h and monitored by TLC,
until the starting material had disappeared. After cooling,
the solvent was removed by filtration under reduced
pressure and the residue was purified by silica gel column
chromatography using methanol as eluent. Further puri-
fication was obtained by crystallization from a mixture of
methyl alcohol/diethyl ether. Yields ranging between 40
5.2.3. Rat sciatic nerve block
This test was performed according to Al-Saadi and
Sneider [13] to determine conduction anaesthesia and its
duration. Triplicate sets of three groups of three male
Wistar rats (Harlan-Nossan, Correzzana, Milan, weighing
180–200 g) were used. Each rat received an injection
(0.2 mL) of the aqueous anaesthetic solution (1% and
2%) into the posterior side of the femur head. A positive
effect of the drug resulted in a complete loss of motor
control of the injected limb. In order to assess the
duration of the effect, the animals were observed from the
1
and 70%. Spectral data of the title compound 2a: H-
NMR (CDCl3), δ = 8 (s, 1H, H-triaz), 8.27 (d, 2H, Ar-H,
J = 7.5 Hz), 7.62 (t, 1H, Ar-H, J = 7.5 Hz), 7.51 (t, 2H,
Ar-H, J = 7.5 Hz), 5.14 (s, 2H, CH2-CO), 3.35 (t, 2H,
NH-CH2, J = 7.5 Hz), 2.40 (t, 2H, CH2-N(CH3)2, J = 7.5
1
Hz) and 2.17 ppm (s, 6H, N(CH3)2). Similar H-NMR
data occur in all derivatives of the general formula 2.
Spectral data of the title compound 3a: 1H-NMR
(CDCl3), δ = 8.31 (s, 1H, H-triaz), 8.27 (d, 2H, Ar-H,

1051
time of onset of the motor paralysis, at 10 min intervals
over time, up to the first sign of motor activity.
served in the absence of added agonist. Nonlinear regres-
sion analyses to determine the parameters Emax, log EC50
and n were done using GraphPad Prism (GraphPad
Software, San Diego, CA) with the constraint that Φ = 0.
5.2.4. Acute toxicity
The ip acute toxicity of the most active compounds 1b,
1d, 3b, and 3d was determined in male Swiss mice
(Harlan-Nossan, Correzzana, Milan, weighing 18–20 g)
7 days after treatment. LD50 values were calculated for
each compound by probit analysis using a computer
program [12].
Acknowledgements
The NMR spectral data were provided by Centro di
Ricerca Interdipartimentale di Analisi Strumentale, Uni-
versità degli Studi di Napoli “Federico II”. The assistance
of staff is gratefully appreciated.
5.2.5. Chronotropic activity: functional assay using
isolated rat right atria
This test was performed according to Hughes and
Smith [14]. Wistar rats (250–300 g) were anesthetized
with halothane and the hearts were rapidly removed and
placed in oxygenated Krebs Henseleit buffer (KHB). The
right atria were removed and mounted in a water jacketed
tissue chamber (20 mL volume) containing KHB, pH
7.3–7.5, at 37 °C and gassed with 95% O2/5% CO2. The
composition of the KHB was (mM): NaCl, 124; KCl,
4.75; MgSO4, 1.30; CaCl2, 2.25; NaHCO3, 25.0;
NaH2PO4, 0.6; dextrose, 10.0; sodium ascorbate, 0.3.
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