Synthesis and Anti-Arrhythmic Activity of the Chloride and Salicylate Salts of Morpholinoacetic Acid Ortho-Toluidide

Article abstract of DOI:10.1007/s11094-017-1667-3

Acylation of ortho-toluidine hydrochloride by chloroacetylchloride followed by amination by morpholine in the presence of an excess of Et₃N produced morpholinoacetic acid ortho-toluidide and its salicylate salt. The acute toxicity and anti-arrh

Full text of DOI:10.1007/s11094-017-1667-3

DOI 10.1007/s11094-017-1667-3
Pharmaceutical Chemistry Journal, Vol. 51, No. 8, November, 2017 (Russian Original Vol. 51, No. 8, August, 2017)
SYNTHESIS AND ANTI-ARRHYTHMIC ACTIVITY
OF THE CHLORIDE AND SALICYLATE SALTS
OF MORPHOLINOACETIC ACID ORTHO-TOLUIDIDE
O. V. Gashkova,1 I. P. Rudakova,1 and B. Ya. Syropyatov1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 51, No. 9, pp. 9 – 11, September, 2017.
Original article submitted June 20, 2014.
Acylation of ortho-toluidine hydrochloride by chloroacetylchloride followed by amination by morpholine in
the presence of an excess of Et3N produced morpholinoacetic acid ortho-toluidide and its salicylate salt. The
acute toxicity and anti-arrhythmic and antifibrillatory activities of these compounds were studied. The biolog-
ical activities indicated that these compounds were promising for further studies and evaluation of their poten-
tial for implementation into medical practice.
Keywords: arylamide, acylation, morpholine, PMR spectroscopy, effective anti-arrhythmic dose, lidocaine.
Arylamides of aminocarboxylic acids are used in medi-
cal practice as anesthetics and anti-arrhythmics, e.g., lido-
caine and trimecaine [1 – 3]. However, their therapeutic ef-
fects are short-lived because of rapid hepatic destruction [4].
Therefore, compounds that retain their value and are stable
with longer anti-arrhythmic activity are highly sought among
arylamides of aminocarboxylic acids.
study the effect of the acid anion on the manifestation of bio-
logical activity (Scheme 1).
EXPERIMENTAL CHEMICAL PART
4-{[(2-Methylphenyl)carbamoyl]methyl}morpholin-4-
ium chloride (I). ortho-Toluidine (21.4 g, 0.2 mol) was
acylated by chloroacetylchloride (12.43 g, 0.11 mol). The re-
sulting precipitate of chloroacetyl o-toluidide was recrystalli-
zed from hexane (yield 27.5 g, 81.1%; mp 102 – 104°C) and
heated with morpholine (8.71 g, 0.1 mol) in the presence of
The chloride (I) and salicylate (II) salts of morpholino-
acetic acid ortho-toluidide (III) were synthesized in order to
1
Perm State Pharmaceutical Academy, Ministry of Health of Russia, 46
Krupskoi St., Perm, 614070, Russia; e-mail: rudakova.i@list.ru
an excess of Et N (10.1 g, 0.1 mol). The precipitate of
3
Ñõåìà
H
N
O
NaOH
ClCOCH2Cl
HCl
NH2
NHCOCH2Cl
NHCOCH2N
HCl
O
*
CH3
CH3
CH3
I
COOH
COOH
OH
NHCOCH2N
NHCOCH2N
OH
O *
O
+
CH3
CH3
III
II
641
0091-150X/17/5108-0641 © 2017 Springer Science+Business Media New York

642
O. V. Gashkova et al.
Et NHCl was filtered off. The excess of amine was distilled
method [6]. Tests were conducted on white male mice
(18 – 24 g). The experiments used four animal groups with
two observations in each. Each pair was injected with one
dose of compound in increasing order (compound I was in-
jected at doses of 158, 200, 250, and 316 mg/kg; compound
II, 100, 126, 158, and 200 mg/kg). The number of dead mice
in each group was noted.
3
off. The resulting o-toluidide III was dissolved in Et O. The
2
solution was saturated with gaseous HCl [5]. Salt I crystal-
lized from DMF–Me CO (1:1). Yield of I, 20.1 g (85.89%),
2
mp 216 – 218°C. Next, salt I was used to isolate the free base
by treatment with base and extraction by Et O. Then, the
2
Et O was distilled off. The resulting free base III was dried
2
over CaCl .
Anti-arrhythmic activity was studied in mice using an ar-
2
4-{[(2-Methylphenyl)carbamoyl]methyl}morpholin-4-
rhythmia model induced by i.v. injection of CaCl solution
2
ium 2-methylbenzoate (II). A solution of III (2.7 g,
(3%, GUP Immunopreparat, Ufa) at a dose of 280 mg/kg
[7, 8]. The effective anti-arrhythmic doses were determined
by the Prozorovskii method [6]. Test compounds were in-
jected in increasing doses, each to two animals (compound I
was injected at doses of 5, 6.3, 7, and 10 mg/kg; compound
II, 7.9, 10, 12.6, and 15.8 mg/kg) 2 min before inducing ar-
rhythmia. Their activities were assessed from the ability to
prevent lethal disruption of cardiac rhythm.
0.01 mol) in anhydrous Et O (15 mL) at 10 – 20°C was
2
stirred for 1 h, treated with salicylic acid (1.22 g, 0.01 mol)
in anhydrous Et O (10 mL), and stirred for 30 min. The re-
2
sulting precipitate of salicylate II was filtered off, dried in
air, and recrystallized from hexane, mp 86 – 88°C, yield
3.7 g (90.68%).
Compound II was a white crystalline compound that was
soluble in H O, EtOH, MeCN, and DMF and insoluble in
2
Antifibrillatory activity was studied using an acute coro-
nary occlusion model in rats [7, 9]. This model was adequate
because it reproduced conditions similar to sudden cardiac
death. In the first step, an occluding device consisting of a
polyethylene tube with a ligature drawn through it was im-
planted into the chest cavity of rats under ether anesthesia.
The ligature was fed through the myocardium under the de-
scending coronary artery, which enabled the coronary vessels
to be occluded by tightening one of the ligature ends. Occlu-
sion was induced in awake animals 5 – 7 d after the opera-
tion. The EKG in the second standard lead was recorded dur-
ing the whole test. The frequency of fibrillation and death
was calculated. Tests were conducted on white rats of both
sexes (180 – 220 g). Each group contained 10 animals. The
reference drug was lidocaine in solution for injection (OAO
Biokhimik, Saransk). Drugs were injected i.v. 2 min before
occlusion at the effective anti-arrhythmic doses.
Me CO, hexane, and C H .
2
6
6
The structures of I and II were confirmed using PMR
spectroscopy in CDCl with HMDS internal standard
3
(BS-567A, 100 MHz).
Compound I: 2.244 (s, 3H, CH ), 2.560 – 2.851 (m, 4H,
3
N(CH CH ) O), 3.116 (s, 2H, CH ), 3.664 – 3.912 (m, 4H,
2
2 2
2
N(CH CH ) O), 6.900 – 7.976 (m, 4H, C H ), 9.089 (s, 1H,
2
2 2
6
4
-NH-), 10.601 (s, 1H, HCl).
Compound II: 2.263 (s, 3H, CH ), 2.671 – 2.755 (m, 4H,
3
N(CH CH ) O), 3.265 (s, 2H, CH ), 3.728 – 3.812 (m, 4H,
2
2 2
2
N(CH CH ) O), 6.808 – 7.986 (m, 4H, C H ), 9.210 (s, 1H,
2
2 2
6
4
-NH-), 10.045 (d, 2H, COOH, OH).
EXPERIMENTAL PHARMACOLOGICAL PART
Acute toxicity was determined using i.v. injection. The
median lethal dose was determined using the Prozorovskii
TABLE 1. Influence of Compounds on Course of Acute Coronary Occlusion in Awake Rats
Anti-arrhythmic activity
Antifibrillatory activity
frequency
Number
ED50, mg/kg, i.v.
arrhythmia
Compound
of animals
in group
of ventricular
fibrillations
at ED50, %
frequency
at ED50, %
death, %
average
confidence
intervals
parameters
Control
10
10
–
–
87.0
66.0
53.0
II
11.2
20.0
0
0
(9.2 ± 13.4)
p < 0.01
p¢ > 0.05
p < 0.01
p¢ < 0.05
p < 0.01
I
10
10
6.4
7.7
60.0
0
0
(4.9 ± 8.6)
(5.9 ± 9.4)
p > 0.05
p¢ > 0.5
p < 0.01
p¢ < 0.05
p < 0.01
Lidocaine
50.0
30.0
0
p > 0.05
p = 0.05
p < 0.01
p < 0.05 is significance of differences vs. the control; p¢ < 0.05 is significance of differences vs. lidocaine.

Synthesis and Anti-Arrhythmic Activity of the Chloride
643
Anti-arrhythmic activity was also studied in mice using
an arrhythmia model induced by strophanthin K (AO Galich-
farm, Lviv, solution for injection) that was injected i.v. at a
dose of 1 mg/kg 2 min after injecting the test compound at a
It was found that cardiac rhythm was disrupted in all ani-
mals after i.v. injection to white mice of strophanthin K. Le-
thal outcomes occurred in 62% of cases in the controls with
developed arrhythmia. Only 30% of animals died if com-
pound II was used. However, the decrease of this parameter
was not significant compared with the control (p > 0.05) and
lidocaine, which statistically significantly prevented animal
deaths (20%, p = 0.05). Injection of I completely prevented
development of strophanthin arrhythmia, which was 30 times
more effective than II and 20 times more effective than
dose equal to ED . The frequency of animal death was cal-
50
culated [7, 10].
Results were processed by variational statistics using the
Fisher–Student method [11].
RESULTS AND DISCUSSION
The studies showed that the synthesized compounds had
low toxicity and pronounced anti-arrhythmic activity.
lidocaine (p < 0.05).
Thus, the synthesized compounds exhibited pronounced
The LD of II was 146 mg/kg. The effective anti-arrhy-
anti-arrhythmic effects that were comparable with those of
lidocaine in various induced experimental models. The re-
sults indicated that further expanded pharmacological studies
of these compounds are warranted.
50
thmic dose calculated during the experiment for the CaCl -
2
induced cardiac rhythm disruption model in mice (ED ) was
50
11.2 mg/kg, i.e., the anti-arrhythmic index (LD /ED ) was
50
50
13.0. For I, LD was 231 mg/kg; ED , 6.4 mg/kg; which
50
50
meant that the anti-arrhythmic index was 36.1. The same in-
dex for reference drug lidocaine was 5.1.
REFERENCES
Table 1 lists data obtained from studies of the course of
acute coronary occlusion in awake rats.
1. V. S. Sklyaev, V. I. Pantsurkin, V. N. Ryazanov, et al.,
Khim.-farm. Zh., 25(9), 33 – 35 (1991); Pharm. Chem. J., 25(9),
629 – 632 (1991).
Arrhythmia was observed in control tests with i.v. injec-
tion of normal saline in 87% of tests; fibrillation, in 66% of
the animals. The outcome was lethal for most rats (53%).
The ST segment in the EKG shifted sharply, more often
above the isoelectric line even in the first seconds of the oc-
clusion. Then, the shift lessened somewhat and rose again,
after which arrhythmia developed up to fibrillation.
2. V. S. Sklyaev, V. I. Pantsurkin, V. N. Ryazanov, et al., Khim.-
farm. Zh., 26(4), 51 – 55 (1992); Pharm. Chem. J., 26(4), 338 –
343 (1992).
3. A. A. Sukhanov, V. V. Gorbunova, A. N. Gornova, and
V. A. Pantsurkin, in: Proceedings of the Jubilee Scientific-Prac-
tical Conference Dedicated to the 60th Anniversary of PSPA
“Progress of Modern Pharmaceutical Science and Education –
Practical Healthcare” [in Russian], Perm (1997), pp. 125 –
126.
Arrhythmia was observed in 50% of cases with i.v. injec-
tion of lidocaine at a dose of 7.7 mg/kg. Spikes of ventricular
fibrillation were observed in only 30% of the animals. This
was statistically significantly less than in the control tests.
More pronounced effects were found if compounds I and
4. L. N. Zhukauskaite, A. P. Tsybusov, E. I. Gendenshtein, et al.,
Khim.-farm. Zh., 18(6), 43 – 48 (1984).
5. N. I. Kudryashova, A. L. Remizov, and N. V. Khromov- Bori-
sov, Zh. Obshch. Khim., 29, 1240 – 1244 (1959).
6. V. V. Prozorovskii, M. P. Prozorovskaya, and V. M. Demchen-
ko, Farmakol. Toksikol., 41(4), 497 – 502 (1978).
7. A. N. Mironov, N. D. Bunyatyan, et al. (eds.), Handbook for
Preclinical Drug Trials [in Russian], Moscow (2012).
8. V. V. Gorbunova and N. P. Gobunov, Farmakol. Toksikol., 3,
48 – 50 (1983).
II were injected at doses equal to ED . They completely
50
protected the animals from manifestation of fibrillation. This
distinguished them statistically significantly from not only
the controls but also from the experimental effect of
lidocaine. Furthermore, arrhythmia developed in only 20%
of tests if compound II was used at a dose of 11.2 mg/kg.
This differed statistically significantly from the controls.
However, the frequency of their occurrence was 2.5 times
less than in the tests with lidocaine whereas compound I did
not prevent manifestation of arrhythmia. Lethal outcomes af-
ter coronary artery occlusion in awake rats were not recorded
in tests with compounds II and I and lidocaine injected into
the animals.
9. J. Lepran, M. Koltai, and L. Szeceres, Eur. J. Pharmacol., 69,
235 – 238 (1981).
10. N. V. Kaverina and Z. P. Senova, Guiding Methodical Mate-
rials for Experimental and Clinical Studies of New Drugs [in
Russian], Vol. 4, Moscow (1982), pp. 95 – 106.
11. V. V. Prozorovskii, Psikhofarmakol. Biol. Narkol., 7(3 – 4),
2090 – 2120 (2007).