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Two active structures for hemicholinium-like action
TOXICOLOGYANDAPPLIEDPHARMACOLOGY
Two
Active
Structures
27,676-679(1974)
for
Hemicholinium-like
ActionlJ
B. W. BLASE,T. A. LOOMIS, J. A. COLLINS AND H. Z. SOMMER Department of Pharmacology, University of Washington, Seattle, Washington, and Chemical Research Laboratory, U. S. Army, Edgewood Arsenal, Maryland Received June 4,1973; accepted August 6,1973
Two Active Structures for Hemicholinium-like action. BLASE, B. W., T. A., COLLINS, J. A., AND SOMMER, H. 2. (1973). Toxicol. Appl. Pharmacol. 27,676-679. The hemiketal structure has been reported as necessary for compounds to exhibit hemicholinium action. This report describes the hemicholinium-like effect of 2 bisquaternary compounds (Zhydroxy ethyldimethylammonio derivatives) in which the positively charged nitrogens are connected by either the phenylene diacetyl chain or by the same chain in which the keto groups have been reduced. The former compound (IIIb) exists in a cyclic hemiketal or closed ring form and the latter (IIIbOH) exists as the straight chain form. Studies on the intact rat anterior tibia1 nerve muscle preparation show that the compounds produce quantitatively similar effects and that both produce significant hemicholinium-like action. These results indicate that both the straight-chain and the hemiketal forms are active structures for hemicholinium-like action. LOOMIS,
Long and Schueler (1954) initially reported on the synthesis and toxicity of a series of bisquaternary compounds in which the nitrogens were connected by the biphenacyl group. Schueler (1955) demonstrated that those compounds which were phenacyl ethanol amines undergo cyclization resulting in dihemiketal formation, and he coined the term hemicholinium to denote such dihemiketals containing a cholinelike moiety. Schueler was concerned primarily with the effects of the hemicholiniums as respiratory depressants and he stated that only those compounds which were capable of forming the hemiketal were effective in producing respiratory depression. A more recent study of the active structure of the hemicholiniums was conducted by DiAugustine and Haarstad (1970). They prepared 4 methyl analogs of Long and Schueler’s compound known as HC-3 (or hemicholinium-3) and noted that 1 analog consisted of a mixture of the open ring and the closed ring (hemiketal form) tautomers. They separated the 2 forms and compared their activity with HC-3 on the cat nerve-gastrocnemius muscle preparation. The hemiketal analog was found to be equipotent to HC-3 but the open ring analog was only very weak in activity as compared to HC-3. These authors also concluded, as did Scheuler, that the hemiketal structure is necessary for hemicholinium-like activity and that any restraint on formation of the hemiketal leads to a marked reduction in activity. This report describes the hemicholinium-like activity of 2 bisquaternary ammonium compounds, that is, 2-hydroxyethyl dimethyl ammonio derivatives, in which the positively charged nitrogens are connected by either the phenylene diacetyl chain, or by the 1 This investigation was supported by the Army Contract DAAl5-67-C-008 and DAHC 19-72-G-008. * Presented in part at the eleventh annual Meeting of the Society of Toxicology, Virginia, March 5-9, 1972. 676 Copyright 0 1974 by Academic Press, Inc.
A11 rights of reproduction Printed in Great Britain
in any form reserved.
Williamsburg,
AGENTS
WITH
REMICHOLINIUM-LIKE
ACTION
677
same chain in which the ketonic groups have been reduced (Fig. 1). The former compound (IIIb) exists in the hemiketal or closed ring form, and the latter (IIIb-OH) exists as the straight chain form.
0,H
o/i
,, c Hz--C,$?,c--C\H;, 3 \N/8 0 0 H,C’ \ H,C-c/H, &-c/H2
,CH3 N@ ‘CH3
=b
CH3
?-I
?H
$Hs
HO-CH,-CH,-I;@-CH,-COC-CH,-N@-CH,-Cn,-OH hi,
&is mb’-OH
FIG. 1. Structures of IIIb-OH and IIIb. Both are bis compounds and are used as the dibromides. Compound IIIb exists as the cyclic dihemiketal form.
METHODS The configurations of the 2 compounds were verified by IR and UV absorption data. The intact rat anterior tibia1 muscle preparation as described by Loomis et al. (1974) was used to measure hemicholinium-like activity. All injections were made iv in the external jugular vein. Compound IIIb and IIIb-OH produce qualitatively similar results on the intact anterior tibia1 muscle preparation of the rat. Figure 2 is a typical record of the effect of a series of 3 doses of IIIb-OH. Loomis et al. (1974) have shown that a hemicholinium-like effect on this test system is manifested as a slow onset, decrease in amplitude of the tetanus and the predominant effect on the form of the tetanus is impairment of the initial phase of each sustained tetanic contraction. Figure 2 shows that these characteristics of the hemicholinium-like effect are produced by IIIb-OH. Comparative activities of IIIb and IIIb-OH on the muscle preparation in paired experiments were obtained as follows : After the anesthetized (pentobarbital, 30 mg/kg ip) preparation had attained a stable base line contraction amplitude, a minimally effective dose (0.1 mg/kg) of either compound was administered. The measured effects were impairment of amplitude of the tetanus and increase in the time required for initial establishment of a new stable tetanus amplitude. Approximately 30 min after the initial dose, a second dose (0.2 mg/kg) of the same compound was administered and the muscle response was followed until a maximum depression of tetanus was attained. The time required for the onset of a new stable plateau of tetanus amplitude and the amount of depression produced by the second dose was measured. After a new stable plateau was evident, choline chloride (2 mg/kg) was administered. In order to eliminate any effect due to the anesthetic a second identical series of experiments was conducted in which the cervical spinal cord was transected in rats
678
BLASE
ET AL.
anesthetized with ether. One hour was allowed for the ether to be eliminated before proceeding with the first dose of a test compound. In order to limit the possibility of metabolic conversion of one or both of the compounds by microsomal oxidative systems, a third series of identical experiments was performed on rats that were pretreated with SKF 525A (75 mg/kg ip) 2 hr before the experiment.
.25 min
1llb‘OH 0.1 mg/Kg
60 min
4Omin
Choline 2.0 mg/Kg
111b-OH 03mg/Kg
111b-OH 0.2 mg/Kg
20 min
5min
Olllin
FIG. 2. Effect of IIIb-OH on the intact anterior tibia1 muscle of the rat. Interrupted tetanic stimuli applied to the anterior tibia1 branch of the sciatic nerve at 5O/secgiven for 0.5 set every 15 sec. Pulse duration = 5 msec; voltage = 2.5 V. Chart speed A = 0.5 mm/set, B = 5 mm/set. RESULTS
The results are summarized in Table 1. The table shows that IIIb-OH and IIIb produce quantitatively similar effects on the neuromuscular preparation, The magnitude of the effect of both compounds in the pentobarbital plus SKF 525A experiments was less TABLE 1 COMPARATIVE EFFECTS OF IIIb-OH AND IIIb ON THE RAT INTACT ANTERIOR TIBIAL MUSCLE PREPARATION’ Type of anesthesia Pentobarbital Spinal trans.
Pentobarbital + SKF 525-A
Dose bw/kg)
Time to max. effect; ratio of IIIb-OH (min): IIIb (min)
Change in amplitude of tetanus; ratio of IIIb-OH (mm): III (mm)
0.1 0.2 2.0 choline 0.1 0.2 2.0 choline 0.1 0.2 2.0 choline
1.00 1.09 1.00 No change 0.63 2.54 No change 1.00 0.84
1.00 1.08 1.00 No change 1.25 1.00 No change 1.00 1.33
’ Each datum represents the mean of 2 (paired) experiments on each of the 2 compounds.
AGENTS WITH REMICHOLINIUM-LIKE
ACTION
679
than in the other 2 types of preparations. Only in the pentobarbital group did the 0. lmg/kg dose of either compound produce a measurable effect on tetanus amplitude. DISCUSSION
The only difference in the structures of the 2 compounds presented in this study involves the reduction of the keto groups in IIIb-OH, and this prevents hemiketal formation. The experimental results indicate that both compounds produce significant hemicholinium-like effects on the neuromuscular test system. Pretreatment of the animals with SKF 525A did not abolish the hemicholinium action of the compounds. These results do not support the conclusions of DiAugustine and Haarstad (1970) and of Schueler (1955). However, the compounds studied by those workers differed from the compounds used in the current study. In their compounds the quaternary nitrogens were separated by the diphenacyl group and the quaternary nitrogens in the current compounds were separated by a phenacyl group. Loomis et al. (1974) have shown that the acute iv lethality in mice of the diphenacyl homolog is at least IO-fold greater than that of the phenacyl homolog. In a recent report Hemsworth and Cholakis (1972) showed that the acetylated derivative of hemicholinium-3 had a hemicholinium-like effect on the rat phrenic nervediaphragm preparation. This compound is an open ring compound and the authors suggested that the hemiketal form was not necessary for hemicholinium action. REFERENCES R. P. AND HAARSTAD, V. B. (1970). The active structure of hemicholinium inhibiting the biosynthesis of acetylcholine. Biochem. Pharmacol. 19, 559-580. HEMSWORTH, B. A. AND CHOLAKIS, J. M. (1972). Mechanisms of action of a hemicholinium analogue at the neuromuscular junction. Science Communications, J. Pharm. Pharmacol., Suppl. 24, p. 147. LONG, J. P. AND SCHUELER, F. W. (1954). A new series of cholinesterase inhibitors. J. Amer.
DIAIJGUSTME,
Pharm. Ass. (Sci. Ed.) 43, 79-86.
T. A., SOMM~R, H. Z. AND COLLINS, J. A. (1974). Lethality, antiesterase and hemicholinium-like actions of a series of bisquatemary ammonium compounds. Tox. Appl.
tiMIS,
Pharmacol. 27, 666-675. SCHUELER, F. W. (1955). A new group of respiratory paralyzants. I. The hemicholiniums. J. Pharmacol. Exp. Ther. 115, 127-143.
Two
Active
Structures
27,676-679(1974)
for
Hemicholinium-like
ActionlJ
B. W. BLASE,T. A. LOOMIS, J. A. COLLINS AND H. Z. SOMMER Department of Pharmacology, University of Washington, Seattle, Washington, and Chemical Research Laboratory, U. S. Army, Edgewood Arsenal, Maryland Received June 4,1973; accepted August 6,1973
Two Active Structures for Hemicholinium-like action. BLASE, B. W., T. A., COLLINS, J. A., AND SOMMER, H. 2. (1973). Toxicol. Appl. Pharmacol. 27,676-679. The hemiketal structure has been reported as necessary for compounds to exhibit hemicholinium action. This report describes the hemicholinium-like effect of 2 bisquaternary compounds (Zhydroxy ethyldimethylammonio derivatives) in which the positively charged nitrogens are connected by either the phenylene diacetyl chain or by the same chain in which the keto groups have been reduced. The former compound (IIIb) exists in a cyclic hemiketal or closed ring form and the latter (IIIbOH) exists as the straight chain form. Studies on the intact rat anterior tibia1 nerve muscle preparation show that the compounds produce quantitatively similar effects and that both produce significant hemicholinium-like action. These results indicate that both the straight-chain and the hemiketal forms are active structures for hemicholinium-like action. LOOMIS,
Long and Schueler (1954) initially reported on the synthesis and toxicity of a series of bisquaternary compounds in which the nitrogens were connected by the biphenacyl group. Schueler (1955) demonstrated that those compounds which were phenacyl ethanol amines undergo cyclization resulting in dihemiketal formation, and he coined the term hemicholinium to denote such dihemiketals containing a cholinelike moiety. Schueler was concerned primarily with the effects of the hemicholiniums as respiratory depressants and he stated that only those compounds which were capable of forming the hemiketal were effective in producing respiratory depression. A more recent study of the active structure of the hemicholiniums was conducted by DiAugustine and Haarstad (1970). They prepared 4 methyl analogs of Long and Schueler’s compound known as HC-3 (or hemicholinium-3) and noted that 1 analog consisted of a mixture of the open ring and the closed ring (hemiketal form) tautomers. They separated the 2 forms and compared their activity with HC-3 on the cat nerve-gastrocnemius muscle preparation. The hemiketal analog was found to be equipotent to HC-3 but the open ring analog was only very weak in activity as compared to HC-3. These authors also concluded, as did Scheuler, that the hemiketal structure is necessary for hemicholinium-like activity and that any restraint on formation of the hemiketal leads to a marked reduction in activity. This report describes the hemicholinium-like activity of 2 bisquaternary ammonium compounds, that is, 2-hydroxyethyl dimethyl ammonio derivatives, in which the positively charged nitrogens are connected by either the phenylene diacetyl chain, or by the 1 This investigation was supported by the Army Contract DAAl5-67-C-008 and DAHC 19-72-G-008. * Presented in part at the eleventh annual Meeting of the Society of Toxicology, Virginia, March 5-9, 1972. 676 Copyright 0 1974 by Academic Press, Inc.
A11 rights of reproduction Printed in Great Britain
in any form reserved.
Williamsburg,
AGENTS
WITH
REMICHOLINIUM-LIKE
ACTION
677
same chain in which the ketonic groups have been reduced (Fig. 1). The former compound (IIIb) exists in the hemiketal or closed ring form, and the latter (IIIb-OH) exists as the straight chain form.
0,H
o/i
,, c Hz--C,$?,c--C\H;, 3 \N/8 0 0 H,C’ \ H,C-c/H, &-c/H2
,CH3 N@ ‘CH3
=b
CH3
?-I
?H
$Hs
HO-CH,-CH,-I;@-CH,-COC-CH,-N@-CH,-Cn,-OH hi,
&is mb’-OH
FIG. 1. Structures of IIIb-OH and IIIb. Both are bis compounds and are used as the dibromides. Compound IIIb exists as the cyclic dihemiketal form.
METHODS The configurations of the 2 compounds were verified by IR and UV absorption data. The intact rat anterior tibia1 muscle preparation as described by Loomis et al. (1974) was used to measure hemicholinium-like activity. All injections were made iv in the external jugular vein. Compound IIIb and IIIb-OH produce qualitatively similar results on the intact anterior tibia1 muscle preparation of the rat. Figure 2 is a typical record of the effect of a series of 3 doses of IIIb-OH. Loomis et al. (1974) have shown that a hemicholinium-like effect on this test system is manifested as a slow onset, decrease in amplitude of the tetanus and the predominant effect on the form of the tetanus is impairment of the initial phase of each sustained tetanic contraction. Figure 2 shows that these characteristics of the hemicholinium-like effect are produced by IIIb-OH. Comparative activities of IIIb and IIIb-OH on the muscle preparation in paired experiments were obtained as follows : After the anesthetized (pentobarbital, 30 mg/kg ip) preparation had attained a stable base line contraction amplitude, a minimally effective dose (0.1 mg/kg) of either compound was administered. The measured effects were impairment of amplitude of the tetanus and increase in the time required for initial establishment of a new stable tetanus amplitude. Approximately 30 min after the initial dose, a second dose (0.2 mg/kg) of the same compound was administered and the muscle response was followed until a maximum depression of tetanus was attained. The time required for the onset of a new stable plateau of tetanus amplitude and the amount of depression produced by the second dose was measured. After a new stable plateau was evident, choline chloride (2 mg/kg) was administered. In order to eliminate any effect due to the anesthetic a second identical series of experiments was conducted in which the cervical spinal cord was transected in rats
678
BLASE
ET AL.
anesthetized with ether. One hour was allowed for the ether to be eliminated before proceeding with the first dose of a test compound. In order to limit the possibility of metabolic conversion of one or both of the compounds by microsomal oxidative systems, a third series of identical experiments was performed on rats that were pretreated with SKF 525A (75 mg/kg ip) 2 hr before the experiment.
.25 min
1llb‘OH 0.1 mg/Kg
60 min
4Omin
Choline 2.0 mg/Kg
111b-OH 03mg/Kg
111b-OH 0.2 mg/Kg
20 min
5min
Olllin
FIG. 2. Effect of IIIb-OH on the intact anterior tibia1 muscle of the rat. Interrupted tetanic stimuli applied to the anterior tibia1 branch of the sciatic nerve at 5O/secgiven for 0.5 set every 15 sec. Pulse duration = 5 msec; voltage = 2.5 V. Chart speed A = 0.5 mm/set, B = 5 mm/set. RESULTS
The results are summarized in Table 1. The table shows that IIIb-OH and IIIb produce quantitatively similar effects on the neuromuscular preparation, The magnitude of the effect of both compounds in the pentobarbital plus SKF 525A experiments was less TABLE 1 COMPARATIVE EFFECTS OF IIIb-OH AND IIIb ON THE RAT INTACT ANTERIOR TIBIAL MUSCLE PREPARATION’ Type of anesthesia Pentobarbital Spinal trans.
Pentobarbital + SKF 525-A
Dose bw/kg)
Time to max. effect; ratio of IIIb-OH (min): IIIb (min)
Change in amplitude of tetanus; ratio of IIIb-OH (mm): III (mm)
0.1 0.2 2.0 choline 0.1 0.2 2.0 choline 0.1 0.2 2.0 choline
1.00 1.09 1.00 No change 0.63 2.54 No change 1.00 0.84
1.00 1.08 1.00 No change 1.25 1.00 No change 1.00 1.33
’ Each datum represents the mean of 2 (paired) experiments on each of the 2 compounds.
AGENTS WITH REMICHOLINIUM-LIKE
ACTION
679
than in the other 2 types of preparations. Only in the pentobarbital group did the 0. lmg/kg dose of either compound produce a measurable effect on tetanus amplitude. DISCUSSION
The only difference in the structures of the 2 compounds presented in this study involves the reduction of the keto groups in IIIb-OH, and this prevents hemiketal formation. The experimental results indicate that both compounds produce significant hemicholinium-like effects on the neuromuscular test system. Pretreatment of the animals with SKF 525A did not abolish the hemicholinium action of the compounds. These results do not support the conclusions of DiAugustine and Haarstad (1970) and of Schueler (1955). However, the compounds studied by those workers differed from the compounds used in the current study. In their compounds the quaternary nitrogens were separated by the diphenacyl group and the quaternary nitrogens in the current compounds were separated by a phenacyl group. Loomis et al. (1974) have shown that the acute iv lethality in mice of the diphenacyl homolog is at least IO-fold greater than that of the phenacyl homolog. In a recent report Hemsworth and Cholakis (1972) showed that the acetylated derivative of hemicholinium-3 had a hemicholinium-like effect on the rat phrenic nervediaphragm preparation. This compound is an open ring compound and the authors suggested that the hemiketal form was not necessary for hemicholinium action. REFERENCES R. P. AND HAARSTAD, V. B. (1970). The active structure of hemicholinium inhibiting the biosynthesis of acetylcholine. Biochem. Pharmacol. 19, 559-580. HEMSWORTH, B. A. AND CHOLAKIS, J. M. (1972). Mechanisms of action of a hemicholinium analogue at the neuromuscular junction. Science Communications, J. Pharm. Pharmacol., Suppl. 24, p. 147. LONG, J. P. AND SCHUELER, F. W. (1954). A new series of cholinesterase inhibitors. J. Amer.
DIAIJGUSTME,
Pharm. Ass. (Sci. Ed.) 43, 79-86.
T. A., SOMM~R, H. Z. AND COLLINS, J. A. (1974). Lethality, antiesterase and hemicholinium-like actions of a series of bisquatemary ammonium compounds. Tox. Appl.
tiMIS,
Pharmacol. 27, 666-675. SCHUELER, F. W. (1955). A new group of respiratory paralyzants. I. The hemicholiniums. J. Pharmacol. Exp. Ther. 115, 127-143.