Synthesis, local anaesthetic and antiarrhythmic activities of N-alkyl derivatives of proline anilides

Article abstract of DOI:10.1016/j.ejmech.2013.02.003

We describe here the design, synthesis and evaluation of in vivo local anaesthetic and antiarrhythmic activities of a series of N-alkylproline anilides. Most of the compounds demonstrated surface anaesthetic activity higher than that of lidocaine, ropivacaine and bupivacaine. We established that the local anaesthetic activity was sensitive to structural variations in the substitution pattern at the aromatic ring and the type of alkyl group at the proline nitrogen atom. Some of the prepared N-alkylproline anilides possessed significant antiarrhythmic activity with higher therapeutic indexes than the reference drugs.

Full text of DOI:10.1016/j.ejmech.2013.02.003

Accepted Manuscript
Synthesis, local anaesthetic and antiarrhythmic activities of N-alkyl derivatives of
proline anilides
Dmitrii V. Kalinin, Vladimir I. Pantsurkin, Boris Ya. Syropyatov, Svetlana A. Kalinina,
Irina P. Rudakova, Mikhail I. Vakhrin, Anton V. Dolzhenko
PII:
S0223-5234(13)00089-5
10.1016/j.ejmech.2013.02.003
EJMECH 5993
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 December 2012
Revised Date: 25 January 2013
Accepted Date: 4 February 2013
Please cite this article as: D.V. Kalinin, V.I. Pantsurkin, B.Y. Syropyatov, S.A. Kalinina, I.P. Rudakova,
M.I. Vakhrin, A.V. Dolzhenko, Synthesis, local anaesthetic and antiarrhythmic activities of N-alkyl
derivatives of proline anilides, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.02.003.
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ACCEPTED MANUSCRIPT
H
N
N
O
Local anaesthetic & antiarrythmic
less toxic than lidocaine, ropivacaine, bupivacaine and cyclomecaine

ACCEPTED MANUSCRIPT
Synthesis, local anaesthetic and antiarrhythmic activities of N-alkyl
derivatives of proline anilides
Dmitrii V. Kalinin,a Vladimir I. Pantsurkin,a Boris Ya. Syropyatov,a Svetlana A. Kalinina,a
Irina P. Rudakova,a Mikhail I. Vakhrina and Anton V. Dolzhenkob,c*
a Perm State Pharmaceutical Academy, 2 Polevaya Street, Perm 614990, Russian Federation
b Jeffrey Cheah School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon
Selatan, Bandar Sunway, Selangor 46150, Malaysia
c School of Pharmacy, Curtin Health Innovation Research Institute, Curtin University, GPO Box U1987
Perth, Western Australia 6845, Australia
Corresponding author. Tel.: +61-8-9266-3747; fax: +61-8-9266-2769.
E-mail addresses: DolzhenkoAV@gmail.com; Anton.Dolzhenko@monash.edu;
A.Dolzhenko@curtin.edu.au (A. V. Dolzhenko)

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Abstract
We describe here the design, synthesis and evaluation of in vivo local anaesthetic and
antiarrhythmic activities of a series of N-alkylproline anilides. Most of the compounds
demonstrated surface anaesthetic activity higher than that of thlidocaine, ropivacaine and
bupivacaine. We established that the local anaesthetic activity was sensitive to structural variations
in the substitution pattern at the aromatic ring and the type of alkyl group at the proline nitrogen
atom. Some of the prepared N-alkylproline anilides possessed significant antiarrhythmic activity
with higher therapeutic indexes than the reference drugs.
Keywords:
Local anesthetic
Surface anesthesia
Infiltration anesthesia
Antiarrhythmic
Proline derivative
Anilide

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1. Introduction
Local anaesthetics are drugs clinically used to produce reversible loss of sensation in a
confined area of the body. Nowadays local anaesthetics play an important role in clinical
management of acute, chronic and cancer pain [1-3]. Moreover local anaesthetics represent one of
the most frequently used in daily medical practice groups of drugs [4]. At the same time, application
of available anaesthetics was reported [5,6] to induce some severe adverse effects including life
threatening conditions, signifying therefore importance of development of new safer local
anaesthetic agents.
The first clinically available amide based local anaesthetic, lidocaine (Fig. 1), was
synthesized by Swedish chemist Nils Lofgren in 1943 [7]. This drug was characterized by fast onset
of action, strong and relatively long lasting effect, exceeded by these parameters ester based
anaesthetics of that time. Lidocaine opened a new avenue for development of local anaesthetics and
its structural features, such as tertiary amino group and arylamide moiety connected with one
carbon atom, became a classical pattern for most of the modern local anaesthetics (Fig. 1). In the
process of development of new more potent local anesthetics, N,N-dialkyl derivatives of glycine
evolved to N-alkyl substituted homoprolines and prolines. Thus Ekenstam introduced homoproline-
based mepivacaine, bupivacaine and ropivacaine (Fig. 1) in clinical practice as very potent local
anaesthetics [8-10]. At the same time effective anaesthetics, derivatives of proline, namely
cyclomecaine and pyromecaine were developed [11]. However, therapeutic application of these
drugs is limited due to their substantial general toxicity [12] and associated side effects[13,14].
Furthermore, a discovery of antiarrhythmic properties of lidocaine in the event of ventricular
fibrillation during cardiac catheterisation [15] initiated a number of studies devoted to search of new
local anaesthetics and antiarrhythmics among arylamides of α-amino acids [16,17]. It has been
established that both the anaesthetic and antiarrhythmic effects realise via the same mechanism
involving mainly blocking voltage-gated sodium [18], calcium [19] and to some extent potassium
[20] channels thus causing membrane stabilisation and therefore preventing initiation of the action
potentials. Since that time, further development of anaesthetic and antiarrhythmic drugs has been
often parallel.
Although the main trend in modern drug design and development inclines towards search for
agents highly selective against one particular target, it appears that all clinically available
anaesthetics are scarcely selective to any specific type and subtype of the ion channels. They all
have been discovered on the basis of in vitro and in vivo tests assessing changes in the neurone

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functions. This can be attributed to incomplete knowledge of actual role of each type and subtypes
of the ion channels in the activity. Therefore it seems practical to conduct search for new
anaesthetic and antiarrhythmic agents using assays based on the desirable therapeutic outcome.
In this study we focus on the compounds with the N-alkyl proline core, which seems to be
preferable over N-alkyl homoproline due to lower toxicity of the derivatives based on this scaffold
[9]. Herein we report synthesis and biological activity of N-alkyl derivatives of proline anilides,
which can be considered as structural analogues of cyclomecaine, one of the most potent modern
local anaesthetics [11,21]. Effects of two types of structural variations on the anaesthetic and
antiarrhythmic activity were explored: (1) substituents on the phenyl ring of the anilide fragments
and (2) type of alkyl group at the nitrogen atom of the proline fragment. The activity and toxicity of
the compounds were compared with structurally related cyclomecaine and widely used anaesthetics
lidocaine, bupivacaine and ropivacaine.
2. Chemistry
The synthesis of N-alkyl derivatives of proline anilides (4) for pharmacological
investigations was performed as outlined in Scheme 1. The design of the synthetic pathway allowed
exploration of substitution at the two critical for biological activity positions of the final compounds
4. The bromination of 5-chloropentanoic acid (1) in α-position using molecular bromine in the
presence of phosphorus trichloride afforded 2-bromo-5-chloropentanoic acid, which was converted
into corresponding acid chloride 2. Then arylamides of 2-bromo-5-chloropentanoic acid (3) were
prepared by treatment of 2 with various anilines in mild conditions. The treatment of 3 with primary
alkylamines under heating in toluene resulted in the intramolecular cyclization with formation of
arylamides of N-alkylprolines, which were further converted into the corresponding hydrochloric
salts 4. ortho-Toluidide of N-methylproline (4a) has been previously reported and its physico-
chemical and spectral characteristics are in agreement with the literature values [22].
In DMSO solution, most of the anilides of N-alkylproline hydrochlorides (4) appeared as a
mixture of two diastereomers due to the second stereogenic center introduced by protonation of the
proline nitrogen. The diastereomer ratio depends on the type of substituents and conditions of the
NMR experiments. Thus, only one cis-form was observed for 4a and 4m. Despite of evident
stabilization of the trans-form via intramolecular hydrogen bonding, this form was always minor
and its content in the mixture never exceeded 20%.

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3. Results and discussion
All synthesised compounds (4a-4p) were evaluated in vivo for their local anaesthetic and
antiarrhythmic activities. Initial screening of the compounds for local anaesthetic activity was
performed using a commonly used surface anaesthesia model on rabbit cornea [23]. Further
elaboration in the rat infiltration anaesthesia model [24] was carried out for the most promising
compounds.
3.1. Surface local anaesthetic activity
Surface anaesthetic activity of the compounds was evaluated in white New Zealand rabbits
using the corneal reflex test [23]. This test was used as the initial screening model due to its high
sensitivity and relative simplicity. Most of the prepared compounds were found to possess surface
anaesthetic activity in this test at 1% concentration (Table 1).
The results of structure-activity relationship analysis revealed some important features
responsible for the anaesthetic effect of the compounds. Classical substitution pattern in the
aromatic ring of local anaesthetics of the arylamide class (Fig. 1) typically include at least one
methyl group in the ortho-position (e.g. prilocaine), more often two (e.g. lidocaine, bupivacaine and
ropivacaine) and sometimes the third one in para-position (e.g. trimecaine, cyclomecaine and
pyromecaine). We also found that in our series of compounds, methyl substituent was optimal for
the activity. Even slight increase in the length of the alkyl chain from methyl (compound 4c) to
ethyl (4g) group dramatically reduced surface local anaesthetic activity. We believe that this fact
was linked with a clearly observable local irritative effect of 4g. Similarly to the previously reported
data [9, 22], we found that increase in the number of methyl groups translated into increase of
potency and duration of local anaesthetic effect. Interestingly, 2,4- and 2,6-xylidides (4d and 4e)
demonstrated almost no difference in the activity. Changing the ortho-methyl to trifluoromethyl
group or a halogen significantly decreased depth and duration of the surface anaesthesia.
Introduction of a second halogen in para-position resulted in complete loss of anaesthetic effect.
Type of the N-alkyl substituent at the pyrrolidine ring was found to be critical for the surface
local anaesthetic activity. The N-methyl and ethyl substituted 4a and 4b possessed no anaesthetic
activity in this assay. It appeared that the alkyl substituent should contain more than two carbon
atoms. Extension of the chain from n-butyl (4c) to n-pentyl (4k) and further to n-hexyl (4l) in N-

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alkyl proline ortho-toluidides led to some increase of the Regnier index and significantly
lengthened the anaesthetic activity. Making the alkyl chain even longer seemed to be unnecessary
due to local irritative effect and poor aqueous solubility, previously reported for the similar
structures with longer N-alkyl chain in the pyrrolidine ring [22]. The cyclohexyl substituted
derivatives 4n and 4o demonstrated practically the same level and duration of anaesthesia as their
corresponding n-butyl analogues 4c, e. Interestingly, despite recent reports [25-27] on high in vitro
sodium channel blocking potential of 2,6-xylidine analogue of 4m - NeP1 (Fig. 1), introduction of a
N-benzyl moiety into the pyrrolidine ring of ortho-toluidine derivative appeared to abolish
anaesthetic activity. All the above observations were found to be very close to those earlier reported
[22] for the analogous mesidides and seemed to be general for this class of local anaesthetics.
3.2. Infiltration local anaesthetic activity
The most active in the surface local anaesthesia test compounds (4d, 4l and 4o) were
subjected to the infiltration anaesthesia assay in rats [24]. The anaesthetic activity was assessed by
the onset, depth and duration of the muscle contraction blockade. The contractions were stimulated
by electrical current (0.3 ms, 50 Hz, 0.36-14.0 V). The depth of anaesthesia was expressed as a
percentage increase of the initial value of stimulation threshold. An increase of the stimulation
threshold value more than 2 times from initial values was considered as a complete (100%)
anaesthesia. To gain further understanding of a role of the second methyl group in the arylamino
moiety, ortho-toluidide derivative 4c was also tested in the assay and its effect was compared with
that of 2,6-xylidide substituted analogue 4d.
All compounds and reference drugs demonstrated fast onset of action blocking muscle
contractions within 1-2 min after administration. The duration of anaesthesia caused by the tested
compounds 4c, 4d, 4l and 4o ranged between 35.0±4.1 and 126.3±24.3 min. Similarly to the
reference drugs bupivacaine and ropivacaine, compound 4c (Fig. 2A) completely blocked the
muscle contraction at the first minute. However duration of the complete anaesthesia for 4c was
shorter (12.5±2.9 min) compare to bupivacaine and ropivacaine, which completely prevent the
stimulated muscle contraction for 102.5±37.9 and 58.8±17.5 min, respectively. The depth of
anaesthesia for compound 4c decreased sharply and the threshold values return to the normal in
35.0±4.1 min after application.

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Compound 4d, 2,6-xylidine analogue of 4c, with the higher surface anaesthetic activity
demonstrated only 90% increase of the threshold values in the infiltration anaesthesia assay (Fig.
2B). However duration of the anaesthetic action for 4d was longer with the maximal block lasted
for 20.0±13.5 min followed by slow recovery. No significant difference with the initial threshold
values was observed in 71.3±19.3 min after administration of 4d.
Compounds 4l and 4o were practically equipotent in terms of depth and duration of the
surface local anaesthesia, but these compounds demonstrated distinct profiles in the infiltration
anaesthesia assay (Figs. 2C and 2D). Both compounds possessed long-acting local anaesthetic
activity exceeding the effect duration of ropivacaine (80.0±15.8 min). Thus, 4l was effective for
115.0±12.2 min, while 4o induced even longer (126.3±24.3 min) infiltration anaesthesia.
Bupivacaine was found to be active for 155.0±11.5 min with 102.5±37.9 min of the complete
anaesthesia.
In general, 4d, 4l and 4o were more active than lidocaine, ropivacaine and bupivacaine in
inducing surface anaesthesia, though duration of their effects was shorter than that of bupivacaine.
At the same time, these compounds (4d, 4l and 4o) were less potent in the infiltration anaesthesia
assay than bupivacaine and seemed to be less suitable for this type of therapeutic applications.
Significantly lower toxicity of 4o compare to the reference drugs (vide infra) was one of the main
advantages making this compound a suitable candidate for further investigations.
3.3. Antiarrhythmic activity
Antiarrhythmic activity of the synthesized compounds was estimated by their ability to
prevent mortality in the event of calcium chloride induced arrhythmia in mice [28]. To exclude
contribution of the intrinsic toxicity of the compounds to the mortality, acute toxicity (LD50) [29] of
the compounds in mice was estimated prior to their testing in the antiarrhythmic assay. Effective
dose (ED50) was determined only when it was less than ½ of LD50.
In general, the compounds active in the anaesthetic tests also were found to be effective in
the antiarrhythmic assay. However, unlike the local anaesthetic activity, antiarrhythmic properties
of the compounds tolerated various types of ortho-subtituents at the phenyl ring if other positions
remained intact. Moreover, ortho-methyl substituted compounds were more toxic than their
corresponding analogues. Introduction of the second methyl group in position 4 (para) decreased
toxicity of the compounds (4e and 4o), while the same group in position 6 (ortho) significantly

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increased toxicity (4d). It seems that for cyclomecaine with the 2,4,6-trimethyl substitution, methyl
groups in positions 4 and 6 cancelled effects of each other in terms of toxicity, but appeared to be
very beneficial for the antiarrhythmic effect.
Similarly to the local anaesthetic activity, four or more carbons in the chain of N-alkyl
substituent at the pyrrolidine ring were required for the compounds to possess antiarrhythmic
properties. N-Methyl and ethyl substituted 4a and 4b were inactive at the tested doses. However, in
contrast to the local anaesthetic activity, increasing the alkyl group size did not correlate with
improvement of antiarrhythmic activity. N-Benzyl substituted 4m, which was inactive in the surface
local anaesthesia assay, appeared to be effective in the prevention of calcium chloride induced
arrhythmia in mice.
In general, six compounds (4f, 4h, 4k, 4l, 4o and 4p) exhibited better than the reference
drugs antiarrhythmic/toxicity profile. The combination of N-cyclohexyl substitution in pyrrolidine
ring and 2,4-xylidide moiety (compound 4o) were found to be optimal, balancing high potency and
relatively low toxicity. Antiarrhythmic index of this compound (7.3) significantly exceeded the
same parameter of the reference drugs. Another even less toxic candidate for further development
as an antiarrhythmic agent could be 4p with the antiarrhythmic index of 6.9.
4. Conclusion
N-Alkyl derivatives of proline anilides (4) were synthesized and proved to be a prospective
group for the search of new local anaesthetic and antiarrhythmic agents. Among the tested
compounds, N-cyclohexyl substituted proline 2,4-xylidide (4o) was the most promising compound
in terms of surface anaesthetic and antiarrhythmic actions possessing at the same time significantly
lower toxicity than the reference drugs. Toluidide 4l demonstrated similar potency in local
anaesthetic and antiarrhythmic assays together with lower than the reference drug toxicity. 2,6-
Xylidide 4d induced high level of surface anaesthesia, but was one of the most toxic in the series.
ortho-Chloroanilide 4p exhibited relatively low anaesthetic activity, but could be interesting for
further development as an antiarrhythmic agent with low toxicity.

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5. Experimental
5.1. Chemistry
Melting points (uncorrected) were determined on a Gallenkamp melting point apparatus in
open capillaries. The 1H NMR spectra were recorded on a Mercury-300BB spectrometer (300 MHz)
in CDCl3 and DMSO-d6 using TMS as an internal standard.
5.1.1. 2-Bromo-5-chloropentanoic acid (2)
5-Chloropentanoic acid (1, 52.2 g, 0.382 mol) and dry bromine (76.8 g, 0.480 mol) in the
presence of phosphorus trichloride (4 ml, 45.8 mmol) were heated on water bath at 80°C for 20 h,
then the heating was continued at 100°C for 2 h. After cooling to ambient temperature, the reaction
mixture was treated with 250 ml of water. The organic layer was extracted with benzene (3 times).
After drying over anhydrous MgSO4, the solvent was evaporated under reduced pressure to give
75.1 g of 2-bromo-5-chloropentanoic acid (2) as oil. 1H NMR (CDCl3), δ (ppm): 1.72-2.40 (m, 4H),
3.53 (t, 2Н, J = 5.6 Hz), 4.23 (dd, 1Н, J = 7.4, 6.5 Hz), 10.46 (br. s, 1H). Anal. Calc. for
C5H8BrClO2: C, 27.87; H, 3.74. Found: C, 27.74; H, 3.51.
5.1.2. 2-Bromo-5-chloropentanoic acid chloride (3)
2-Bromo-5-chloropentanoic acid (2) was mixed with thionyl chloride (0.870 mol, 103.6 g)
and heated under reflux in water bath for 1.5 h. The excess of thionyl chloride was removed under
1
reduced pressure affording 72.9 g of 2-bromo-5-chloropentanoic acid chloride (3). H NMR
(CDCl3), δ (ppm): 1.74-2.42 (m, 4H), 3.55 (t, 2Н, J = 6.1 Hz), 4.51 (dd, 1Н, J = 7.4, 5.6 Hz). Anal.
Calc. for C5H7BrCl2O: C, 25.67; H, 3.02. Found: C, 25.46; H, 2.94.
5.1.3. Hydrochlorides of o-toluidide of N-methylproline (4a) and o-toluidide of N-ethylproline (4b)
Solution of 2-bromo-5-chloropentanoic acid chloride (2, 23.4 g,100 mmol) in chloroform
(40 ml) was added dropwise at 0-5 °C during 45 min to the mixture of o-toluidine (10.72 g, 100
mmol) and triethylamine (10.1 g, 100 mmol) in chloroform (50 ml) and the reaction mixture was
heated under reflux for 1 h. After cooling, the mixture was treated with 3% hydrochloric acid (100
ml) and the organic layer was collected, washed with water to neutral pH and evaporated. The
residue was recrystallized from hexane to give o-toluidide of 2-bromo-5-chloropentanoic acid (3),
which was dissolved in toluene (30 mmol in 100 ml), mixed with potassium iodide (0.2 g, 1.2
mmol) and treated with dry gaseous methylamine or ethylamine via bubbling for 6 h. The

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precipitate was filtered off and the filtrate was treated with saturated K2CO3 aqueous solution (100
ml). The organic layer was collected, dried over anhydrous MgSO4 and evaporated under reduced
pressure. The residue was dissolved in dry diethyl ether and treated with gaseous HCl. The
precipitated product was filtered, washed with acetone and recrystallized from acetonitrile (for 4a)
or acetone/diethyl ether (for 4b) providing corresponding o-toluidides of N-alkylproline
hydrochlorides (4a and 4b).
5.1.3.1. o-Toluidide of N-methylproline (4a)
1
Yield 74%; mp 219-221 °C (MeCN); H NMR (DMSO-d6), δ (ppm): 1.82-2.12 (m, 2H),
2.22 (s, 3Н), 2.55-2.70 (m, 2Н), 2.85 (s, 3Н), 3.42-3.68 (m, 2Н), 4.43 (dt, 1Н, J = 7.8, 8.4 Hz),
6.98-7.38 (m, 4H, Ar), 9.85 (br. s, 1H), 10.59 (s, 1Н). Anal. Calc. for C13H19ClN2O: C, 61.29; H,
7.52; N, 11.00. Found: C, 61.13; H, 7.41; N, 10.91.
5.1.3.2. o-Toluidide of N-ethylproline (4b)
1
Yield 52%; mp 188 °C (acetone/Et2O); H NMR (DMSO-d6), δ (ppm): 1.24 (t, 3Н, J = 6.9
Hz), 1.89-2.18 (m, 2H), 2.23 (s, 3H), 2.60-2.71 (m, 2Н), 3.19-3.32 (m, 2H), 3.60-3.72 (m, 2Н), 4.48
(dt, 1Н, J = 8.1, 7.5 Hz), 4.70* (m, 1H), 7.12-7.39 (m, 4H, Ar), 9.60 (br. s,1H), 10.28* (s, 1H),
10.59 (s, 1Н), 11.99* (br. s, 1H); * - signals of the minor trans-diastereomer. Anal. Calc. for
C14H21ClN2O: C, 62.56; H, 7.88; N, 10.42. Found: C, 62.39; H, 7.72; N, 10.37.
5.1.4. Hydrochlorides of anilides of N-alkylproline (4c-4p)
Solution of 2-bromo-5-chloropentanoic acid chloride (2, 23.4 g,100 mmol) in chloroform
(40 ml) was added dropwise at 0-5 °C during 45 min to the mixture of substituted aniline (100
mmol) and triethylamine (10.1 g, 100 mmol) in chloroform (50 ml) and the reaction mixture was
heated under reflux for 1 h. After cooling, the mixture was treated with 3% hydrochloric acid (100
ml) and the organic layer was collected, washed with water to neutral pH and evaporated. The
residue was recrystallized from hexane to give anilides of 2-bromo-5-chloropentanoic acid (3),
which were dissolved in toluene (30 mmol in 100 ml), mixed with triethylamine (6.1 g , 60 mmol)
and primary alkylamine (45 mmol). Then potassium iodide (0.2 g, 1.2 mmol) was added and the
reaction mixture was heated under reflux for 25 h. After cooling, the precipitate was filtered off and
the filtrate was treated with saturated K2CO3 aqueous solution (100 ml). The organic layer was
collected, dried over anhydrous MgSO4 and evaporated under reduced pressure. The residue was
dissolved in dry diethyl ether and treated with gaseous HCl. The precipitated product was filtered,

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washed with acetone and recrystallized from appropriate solvent providing anilides of N-
alkylproline hydrochlorides (4).
5.1.4.1. o-Toluidide of N-butylproline (4c)
1
Yield 55%; mp 202-204 °C (1,4-dioxane/toluene, 1:1); H NMR (DMSO-d6), δ (ppm) 0.85
(t, 3Н, J = 6.5 Hz), 1.22-1.38 (m, 2H), 1.48-1.80 (m, 2H), 1.92-2.10 (m, 2H), 2.16 (s, 3H), 2.56-
2.64 (m, 2Н), 3.12-3.34 (m, 2H), 3.50-3.75 (m, 2Н), 4.36-4.80 (m, 1Н), 6.95-7.34 (m, 4H, Ar), 9.50
(br. s, 1H), 10.32* (s, 1H), 10.70 (s, 1Н), 11.98* (br. s, 1H); * - signals of the minor trans-
diastereomer. Anal. Calc. for C16H25ClN2O: C, 64.74; H, 8.49; N, 9.44. Found: C, 64.60; H, 8.36;
N, 9.38.
5.1.4.2. 1,6-Xylidide of N-butylproline (4d)
1
Yield 64%; mp 244-246 °C (i-PrOH); H NMR (DMSO-d6), δ (ppm) 0.90 (t, 3Н, J = 6.9
Hz), 1.22-1.38 (m, 2H), 1.52-1.78 (m, 2H), 1.91-2.10 (m, 2H), 2.15 (s, 6H), 2.42-2.58 (m, 2Н),
3.12-3.28 (m, 2H), 3.51-3.68 (m, 2Н), 4.38-4.78 (m, 1Н), 6.90-7.15 (m, 3H, Ar), 9.45 (br. s, 1H),
10.35* (br. s, 1H), 10.70 (s, 1Н), 11.95* (br. s, 1H); * - signals of the minor trans-diastereomer.
Anal. Calc. for C17H27ClN2O: C, 65.68; H, 8.75; N, 9.01. Found: C, 65.66; H, 8.68; N, 8.94.
5.1.4.3. 1,4-Xylidide of N-butylproline (4e)
1
Yield 58%; mp 182-184 °C (1,4-dioxane/toluene, 1:1); H NMR (DMSO-d6), δ (ppm) 0.88
(t, 3Н, J = 6.5 Hz), 1.25-1.40 (m, 2H), 1.50-1.75 (m, 2H), 1.85-2.10 (m, 2H), 2.15 (s, 3H), 2.25 (s,
3H), 2.50-2.70 (m, 2Н), 3.09-3.28 (m, 2H), 3.48-3.71 (m, 2Н), 4.35-4.65 (m, 1Н), 6.89 (d, 1H, J =
7.4 Hz), 6.94 (s, 1H), 7.11 (d, 1H, J = 7.4 Hz), 9.50 (br. s, 1H), 10.05* (s, 1H), 10.65 (s, 1Н),
11.97* (br. s, 1H); * - signals of the minor trans-diastereomer. Anal. Calc. for C17H27ClN2O: C,
65.68; H, 8.75; N, 9.01. Found: C, 65.58; H, 8.64; N, 8.90.
5.1.4.4. o-Trifluoromethylanilide of N-butylproline (4f)
1
Yield 62%; mp 210-212 °C (MeCN/toluene, 1:1); H NMR (DMSO-d6), δ (ppm) 0.89 (t,
3Н, J = 6.5 Hz), 1.21-1.40 (m, 2H), 1.40-1.70 (m, 2H), 1.88-2.20 (m, 2H), 2.54-2.71 (m, 2Н), 3.16-
3.34 (m, 2H), 3.55-3.75 (m, 2Н), 4.28-4.70 (m, 1Н), 7.30-7.80 (m, 4H, Ar), 9.55 (br. s, 1H), 10.56*
(s, 1H), 10,90 (s, 1Н), 12.10* (br. s, 1H); * - signals of the minor trans-diastereomer. Anal. Calc.
for C16H22ClF3N2O: C, 54.78; H, 6.32; N, 7.99. Found: C, 54.69; H, 6.27; N, 7.91.
5.1.4.5. o-Ethylanilide of N-butylproline (4g)

ACCEPTED MANUSCRIPT
1
Yield 56%; mp 182-184 °C (acetone); H NMR (DMSO-d6), δ (ppm) 0.88 (t, 3Н, J = 6.5
Hz), 1.10 (t, 3H, J = 7.4 Hz), 1.25-1.30 (m, 2H), 1.40-1.70 (m, 2H), 1.85-2.20 (m, 2H), 2.45-2.75
(m, 4H), 3.10-3.29 (m, 2H), 3.50-3.70 (m, 2Н), 4.35-4.75 (m, 1Н), 7.05-7.30 (m, 4H, Ar), 9.50 (br.
s, 1H), 10.23* (s, 1H), 10.63 (s, 1Н), 12.05* (br. s, 1H); * - signals of the minor trans-diastereomer.
C17H27ClN2O: C, 65.68; H, 8.75; N, 9.01. Found: C, 65.61; H, 8.69; N, 8.95.
5.1.4.6. o-Iodoanilide of N-butylproline (4h)
Yield 66%; mp 203-205 °C (acetone/EtOH, 9:1); 1H NMR (DMSO-d6), δ (ppm) 0.89 (t, 3H,
J = 7.0 Hz), 1.16-1.33 (m, 2H), 1.52-1.68 (m, 2H), 1.93-2.17 (m, 2H), 2.42-2.68 (m, 2Н), 3.15-3.30
(m, 2H), 3.54-3.76 (m, 2Н), 4.28-4.61 (m, 1Н), 6.99 (t, 1H, J = 7.4 Hz), 7.18-7.45 (m, 2H), 7.83 (d,
1H, J = 7.4 Hz), 9.51 (br. s, 1H), 10.43* (s, 1H), 10.71 (s, 1Н), 11.98* (br. s, 1H); * - signals of the
minor trans-diastereomer. Anal. Calc. for C15H22ClIN2O: C, 44.08; H, 5.43; N, 6.85. Found: C,
43.97; H, 5.38; N, 6.78.
5.1.4.7. 2,4-Dichloroanilide of N-butylproline (4i)
1
Yield 55%; mp 197-199 °C (acetone/Et2O); H NMR (DMSO-d6), δ (ppm) 0.94 (t, 3Н, J =
7.3 Hz), 1.30-1.43 (m, 2H), 1.56-1.61 (m, 2H), 1.92-2.13 (m, 2H), 2.67-2.74 (m, 2Н), 3.15-3.30 (m,
2H), 3.65-3.75 (m, 2Н), 4.58 (dt, 1H, J = 7.2, 9.0 Hz), 4.77* (dd, 1H, J = 7.5, 4.5 Hz), 7.37-7.67 (m,
3H, Ar), 9.73 (br. s, 1H), 10.56* (s, 1H), 11.05 (s, 1Н), 12.36* (br. s, 1H); * - signals of the minor
trans-diastereomer. Anal. Calc. for C15H21Cl3N2O: C, 51.23; H, 6.02; N, 7.97. Found: C, 51.20; H,
5.97; N, 7.91.
5.1.4.8. 2,4-Dibromoanilide of N-butylproline (4j)
1
Yield 66%; mp 192-194 °C (1,4-dioxane); H NMR (DMSO-d6), δ (ppm) 0.88 (t, 3Н, J =
6.8 Hz), 1.22-1.43 (m, 2H), 1.52-1.68 (m, 2H), 1.89-2.21 (m, 2H), 2.58-2.77 (m, 2Н), 3.12-3.27 (m,
2H), 3.48-3.77 (m, 2H), 4.32-4.71 (m, 1Н), 7.38 (d, 1H, J = 7.4 Hz), 7.58 (d, 1H, J = 7.4 Hz), 7.71
(s, 1H), 9.60 (br. s, 1H), 10.60* (s, 1H), 10.90 (s, 1Н), 12.05* (br. s, 1H); * - signals of the minor
trans-diastereomer. Anal. Calc. for C15H21ClBr2N2O: C, 40.89; H, 4.80; N, 6.36. Found: C, 40.76;
H, 4.71; N, 6.27.
5.1.4.9. o-Toluidide of N-pentylproline (4k)
1
Yield 56%; mp 201-203 °C (1,4-dioxane); H NMR (DMSO-d6), δ (ppm) 0.91 (t, 3Н, J =
6.8 Hz), 1.26-1.40 (m, 4H), 1.60-1.73 (m, 2H), 1.92-2.19 (m, 2H), 2.26 (s, 3H), 2.62-2.76 (m, 2Н),
3.12-3.30 (m, 2H), 3.64-3.75 (m, 2Н), 4.62 (dt, 1Н, J = 7.8, 8.4 Hz), 7.10-7.25 (m, 3H, Ar) 7.35

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(dd, 1H, J = 7.2, 1.8 Hz), 9.64 (br. s, 1H), 10.25* (s, 1H), 10.84 (s, 1Н), 12.25* (br. s, 1H); * -
signals of the minor trans-diastereomer. Anal. Calc. for C17H27ClN2O: C, 65.68; H, 8.75; N, 9.01.
Found: C, 65.57; H, 8.66; N, 8.93.
5.1.4.10. o-Toluidide of N-hexylproline (4l)
1
Yield 56%; mp 148-150 °C (ethyl acetate); H NMR (DMSO-d6), δ (ppm) 0.89 (t, 3Н, J =
6.6 Hz), 1.25-1.40 (m, 6H), 1.58-1.72 (m, 2H), 1.92-2.20 (m, 2H), 2.25 (s, 3H), 2.60-2.75 (m, 2Н),
3.10-3.32 (m, 2H), 3.64-3.74 (m, 2Н), 4.49 (dt, 1Н, J = 8.1, 7.5 Hz), 4.67-4.73* (m, 1H), 7.10-7.39
(m, 4H, Ar), 9.57 (br. s, 1H), 10.11* (s, 1H), 10.51 (s, 1Н), 12.20* (br. s, 1H); * - signals of the
minor trans-diastereomer. Anal. Calc. for C18H29ClN2O: C, 66.54; H, 9.00; N, 8.62. Found: C,
66.41; H, 8.90; N, 8.54.
5.1.4.11. o-Toluidide of N-benzylproline (4m)
Yield 75%; mp 213-215 °C (1,4-dioxane/toluene, 1:1); 1H NMR (DMSO-d6), δ (ppm) 1.88-
2.10 (m, 2H), 2.06 (s, 3Н), 2.60-2.73 (m, 2Н), 3.55-3.70 (m, 2Н), 4.47 (dd, 2H, J = 6.9, 13.5 Hz),
4.55 (dt, 1Н, J = 6.9, 8.1 Hz), 7.01-7.22 (m, 4H, Ar), 7.41-7.60 (m, 5H, Ar), 9,85 (br. s, 1H), 10.36
(s, 1Н). Anal. Calc. for C19H23ClN2O: C, 68.97; H, 7.01; N, 8.47. Found: C, 68.86; H, 6.91; N, 8.35.
5.1.4.12. o-Toluidide of N-cyclohexylproline (4n)
Yield 56%; mp 244-246 °C (1,4-dioxane/toluene, 1:1); 1H NMR (DMSO-d6), δ (ppm) 1.18-
1.40 (m, 6Н), 1.84-1.93 (m, 2Н), 1.97-2.14 (m, 4Н), 2.24 (s, 3H), 2.54-2.60 (m, 2Н), 3.25-3.40 (m,
2Н), 3.55-3.69 (m, 1Н), 4.75 (dt, 1H, J = 7.5, 9.0 Hz), 7.13-7.39 (m, 4H), 9.36 (br. s, 1H), 10.30*
(s, 1H), 10.68 (s, 1Н), 11.93* (br. s, 1H); * - signals of the minor trans-diastereomer. Anal. Calc.
for C18H27ClN2O: C, 66.96; H, 8.43; N, 8.68. Found: C, 66.81; H, 8.32; N, 8.55.
5.1.4.13. 1,4-Xylidide of N-cyclohexylproline (4o)
1
Yield 37%; mp 248-250 °C (MeCN); H NMR (DMSO-d6), δ (ppm) 1.18-1.40 (m, 6Н),
1.85-1.92 (m, 2Н), 1.98-2.14 (m, 4Н), 2.17 (s, 3H), 2.23 (s, 3H), 2.46-2.54 (m, 2Н), 3.14-3.36 (m,
2Н), 3.42-3.63 (m, 1Н), 4.56-4.78 (m, 1Н), 6.92 (d, 1H, J = 8.4 Hz), 6.97 (s, 1H), 7.13 (d, 1H, J =
8.4 Hz), 9.20 (br. s, 1H), 10.11* (s, 1H), 10.50 (s, 1Н), 11.80* (br. s, 1H); * - signals of the minor
trans-diastereomer. Anal. Calc. for C18H27ClN2O: C, 67.74; H, 8.68; N, 8.32. Found: C, 67.68; H,
8.52; N, 8.27.
5.1.4.14. o-Chloroanilide of N-cyclohexylproline (4p)

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1
Yield 38%; mp 222-224 °C (MeCN); H NMR (DMSO-d6), δ (ppm) 1.18-1.42 (m, 6Н),
1.86-1.95 (m, 2Н), 2.00-2.16 (m, 4Н), 2.24 (s, 3H), 2.54-2.60 (m, 2Н), 3.25-3.40 (m, 2Н), 3.55-3.69
(m, 1Н), 4.75 (dt, 1H, J = 7.5, 9.0 Hz), 7.13-7.39 (m, 4H), 9.36 (br. s, 1H), 10.30* (s, 1H), 10.68 (s,
1Н), 11.93* (br. s, 1H); * - signals of the minor trans-diastereomer. Anal. Calc. for C17H24Cl2N2O:
C, 59.48; H, 7.05; N, 8.16. Found: C, 59.36; H, 6.94; N, 8.07.
5.2. Biological activity
5.2.1. Surface local anaesthetic activity assay
Surface anaesthetic activity was evaluated in white New Zealand rabbits using modified
Regnier corneal reflex test [23]. Briefly, aqueous solutions (1%, 0.25 ml) of compounds 4a-p or
reference drugs were instilled into the conjunctival sac of the animals during 30 sec. The tactile
stimulations of the rabbit cornea (up to 100 in a series) with the pointer were applied in 8 min after
the treatment and then in the define intervals (10, 12, 15 min and then every 5 min). The summation
of numbers of the stimulations required to cause the corneal reflex during 1 h (13 series) provided
the Regnier index. The duration of the anaesthesia was estimated as a time required for complete
restoration of the corneal reflex. Each compound was tested in 6 animals.
5.2.2. Infiltration local anaesthetic activity assay
Infiltration anaesthetic activity of the compounds was evaluated in Wistar rats using
modification of the reported method [24]. Briefly, after identification of sensitive areas on the
shaved skin of lumbar region of the rat back and determination of the electrical stimulation
(rectangular pulses, 0.36 to14.0 V, 0.3 ms, 50 Hz) threshold initiating the surface muscle
contraction, the animals were injected with 0.5% solution of the tested compounds or reference
drugs in saline (0.2 ml, intradermally and 0.2 ml, subcutaneously). The electrical stimulation
required to cause the muscle contraction was measured in 3 and 5 min after the injection; then the
measurements were continued with 5 min intervals until recovery to the initial threshold value. The
depth of anaesthesia was expressed as a percentage change in the stimulation threshold compare to
the initial value. Increase of the stimulation threshold value more than 2 times was considered as
100% anaesthesia. Each compound was evaluated on 6 rats.
5.2.3. Antiarrhythmic activity assay
Antiarrhythmic activity was evaluated in Swiss albino mice using calcium chloride-induced
arrhythmia model [28]. The compounds and reference drugs were administrated i.v. in the doses up

ACCEPTED MANUSCRIPT
to ½ LD50. After 15 min, cardiac arrhythmia was induced by i.v. injection of calcium chloride
solution (3%, 280 mg/kg). The antiarrhythmic efficacy of the compounds was expressed as mean
effective doses (ED50) preventing arrhythmia associated mortality.
5.2.4. Toxicity evaluation
The acute toxicity of the compounds was determined using albino Swiss mice of both sexes
with body weight 18-22 g. The aqueous solutions of the compounds were injected via the tail vein
in increasing doses. After the treatment, animals were observed for possible mortality cases and
behavioural changes for 72 h. LD50 values were estimated according to reported method [29].
Acknowledgments
We acknowledge financial support of Russian Ministry of Education and Science.
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(1956) 503-506.
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[14] G. Di Gregorio, J. M. Neal, R. W. Rosenquist, G. L. Weinberg, Reg. Anesth. Pain. Med. 35
(2010) 181-187.

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[15] J. L. Southworth, V. A. McKusick, E. C. Pierce, 2nd, F. L. Rawson, Jr. J. Am. Med. Assoc. 143
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[20] C. H. Kindler, M. Paul, H. Zou, C. Liu, B. D. Winegar, A. T. Gray, C. S. Yost, J. Pharmacol.
Exp. Ther. 306 (2003) 84-92.
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497-503.

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Figure captures:
Figure 1. Local anaesthetics – arylamides of amino acids
Figure 2. Infiltration anaesthesia activity of selected compounds: 4c (A), 4d (B), 4l (C) and 4o (D).
Data are mean values ± SEM
Scheme 1. Synthesis of N-alkyl derivatives of proline anilides (4a–4p)

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Table 1. Surface anaesthetic activities of the synthesized compounds 4a-4p
H
R1
N
NH+
O
Cl-
R2
Surface anaesthesia (rabbit
corneal reflex)
Compound
R1
R2
Regnier
Indexa ±SEM
NA
Duration,
min ±SEM
NA
2-CH3
2-CH3
2-CH3
CH3
4a
4b
4c
4d
4e
4f
C2H5
NA
NA
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C5H11
n-C6H13
PhCH2
1030.0±59.9
1258.0±4.8
1104.0±62.3
467.0±93.8
73.5±15.9
429.0±97.3
NA
49.4±4.5
57.5±2.1
59.0±7.8
20.4±3.6
12.7±1.4
20.3±1.1
NA
2,6-(CH3)2
2,4-(CH3)2
2-CF3
2-C2H5
2-I
4g
4h
4i
2,4-Cl2
2,4-Br2
2-CH3
NA
NA
4j
1165.6±58.3
1225.0±75.0
NA
59.8±2.8
75.7±3.1
NA
4k
4l
2-CH3
2-CH3
4m
2-CH3
2,4-(CH3)2
2-Cl
1002.3±77.0
1226.0±30.0
734.8±76.5
1300.0±0.0
46.5±2.7
73.2±3.7
4n
4o
4p
41.3±2.4
Cyclomecaine 2,4,6-(CH3)3
124.5±11.2
Lidocaine
Ropivacaine
Bupivacaine
831.7±55.6
1001.1±86.1
1218.0±29.3
37.8 ± 3.7
58.4±2.7
94.0±3.5
a
Regnier Index - indicator of the anaesthesia depth during first 60 min after the compound
application expressed as a sum of the threshold tactile stimuli needed to induce corneal reflex (13
series, up to 100 stimuli per one series) [23]

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Table 2. Antiarrhythmic activity and acute toxicity (LD50) of N-alkyl derivatives of proline anilides (4a–4p)
H
R1
Cl-
N
NH+
O
R2
Antiarrhythmic
LD50, mg/kg
ED50, mg/kg
(mice, i. v.)
Compound
R1
R2
index
(mice, i. v.)
(LD50 / ED50)
2-CH3
2-CH3
CH3
32.5 (26.0-40.0)
36.8 (26.0-50.0)
16.3 (14.0-19.0)
9.2 (6.7-12.7)
> 16.3a
> 18.4a
NA
NA
3.0
NA
NA
6.1
4.2
5.6
NA
3.5
5.9
6.3
3.3
4a
4b
4c
4d
4e
4f
C2H5
2-CH3
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C5H11
n-C6H13
PhCH2
5.4 (4.0-7.5)
> 4.6a
> 30.0a
2,6-(CH3)2
2,4-(CH3)2
2-CF3
60.0 (48.0-74.0)
35.5 (29.0-42.0)
30.0 (24.0-38.0)
41.0 (35.0-47.0)
32.5 (26.0-40.0)
239.0 (190.0-300.0)
32.5 (28.0-38.0)
35.5 (29.0-42.0)
35.5 (31.0-40.0)
5.8 (4.2-8.0)
7.1 (5.9-8.4)
7.3 (5.3-10.0)
> 16.3a
2-C2H5
2-I
4g
4h
4i
2,4-Cl2
2,4-Br2
2-CH3
69.0 (50.0-95.0)
5.5 (4.0-7.5)
5.6 (4.9-6.4)
10.9 (8.0-15.0)
4j
4k
4l
2-CH3
2-CH3
4m
2-CH3
2,4-(CH3)2
2-Cl
12.9(10.0-16.0)
65.0 (56.0-75.0)
95.0 (76.0-119.0)
15.0 (12.0-17.0)
> 6.5a
NA
7.3
6.9
5.2
4n
8.9 (7.8-10.2)
13.7 (10-19)
2.9 (2.2-3.0)
4o
4p
Cyclomecaine
2,4,6-(CH3)3
Lidocaine
Ropivacaine
Bupivacaine
39.3 (34.2-44.5)
14.1 (12.0-17.0)
8.9 (7.5-10.6)
7.7 (5.9-9.4)
3.7 (2.6-5.0)
3.5 (3.1-4.0)
5.1
3.8
2.5
a Appeared to be inactive in the dose of ½ LD50

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H
H
H
N
N
N
N
N
N
H
O
O
O
Lidocaine
Trimecaine
Prilocaine
Cl
H
H
H
N
N
N
N
H
N
O
N
O
O
Butanilicaine
N
Bupivacaine
Ropivacaine
H
H
N
N
N
O
O
Cyclomecaine
NeP1
Figure 1

ACCEPTED MANUSCRIPT
Figure 2

ACCEPTED MANUSCRIPT
O
R1
R2
Cl
Cl
OH
4a
4b
4c
2-CH3
2-CH3
2-CH3
CH3
C2H5
1
2
1) Br2, PCl3
2) SOCl2
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C5H11
n-C6H13
C6H5CH2
cyclo-C6H11
O
4d 2,6-(CH3)2
4e 2,4-(CH3)2
Cl
Br
4f
2-CF3
2-C2H5
2-I
4g
4h
4i
NH2
R1
2,4-Cl2
2,4-Br2
2-CH3
2-CH3
2-CH3
2-CH3
O
R1
4j
Cl
N
4k
4l
H
Br
3
4m
4n
4o
4p
1) R2NH2, KI
2) HCl
2,4-(CH3)2 cyclo-C6H11
2-Cl cyclo-C6H11
H
N
R1
Cl-
NH+
R2
O
4
Scheme 1

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A series of new N-alkylproline anilides was synthesized.
They demonstrated high surface and infiltration local anesthetic activity.
Significant antiarrhythmic activity was observed.
The structure-activity relationship in the series was discussed.
The lead compounds showed therapeutic indexes higher than that of the reference drugs.