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Blockade of adrenergic responses by halogenated phenyl-2-halogenoethyl amines
Lite Soisaoes vol. 14, pp " 2143-2150 Printed is II .8 .11.
Pargason Press
BLOCKADE OF ADRENERGIC RESPONSES BY HALOGENATED PHENYL-2-HALOGENOETHYL AMINES Vijay C. 9arsmy Department of Biochemical Pharmacology School of Pharmacy State University of New York st Buffalo Buffalo, New York 14214 (iàsceived is tiaal form 45 April 1974) The action of halogensted phenyl 2-halogenoethylamines on adrenergic C,- receptor Bites of the rat vas deferens was studied. The effect of halogen (Br) substituents in the phenyl nucleus resulted in the following differences is activity : pare > mets - para > mete > ortho . All compounds possessed a short duration of action ; recovery from adrenergic blockade was complete within 120 min. Eapoeure of tissues to phenoayben$amine, after complete recovery from adrenergic blockade by the teat compounds, did not alter the magnitude of initial blockade but sharply reduced the duration of action of phenorybenaamiae . These results are discussed in terms of a model which proposes at least 2 sites of action for 2-halogeaoethylaminee at the adrenergic a - receptors of the rat vas deferene .
2-Halogeaoethylaminee (2-HEA) have been widely studied ever since their property of irreversible blockade st adrenergic neuroeffector sites was reported for Dibenamine (1-3 ). Typically, the adrenergic blockade caused by Di benamine and related compounds is characteri$ed by slow onset of action and prolonged duration of action. The action of N, N-dimethyl-2-bromophenethylamine (DMPEA) and related compounds differ from Dibenamine and its analogs in possessing rapid onset of action and relatively short duration of action (410). Previous reports (11-13) from this laboratory provided evidence that DMPEA and other 2-HEAs act at two sites on the adrenergic poet-synaptic
sias
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Alpfra-Adrenergic Blockade by 2-HEA
Vol . 14, No . 11
surface and that the long duration of action of Dibenamine and phenoxybenzamine may be due to their interaction at sites associated with Ca++ mobilization . The present study ie a preliminary report on the adrenergic blocking properties of halogenated phenyl-2-halogenoethylamines which differ structurally from DMPEA in possessing halogen eubstituente in the phenyl ring . The results show that these compounds possess qualitatively similar properties as DMPEA in their blockade of adrenergic responses and their capacity to modify irreversible antagonism by pheaoxybeassamise . Methods Vaea deferentia from rate (Holtzmaa) weighing approximately 150 g were set up in organ bathe containing Tyrode'e solution maintained at 37oC and bubbled with a gaseous mixture of 95°Jo 02 + 5°}'o C0 2. Isotonic contractions were recorded on a smoked drum of a kymograph via levers exerting a teneion of 300 - 350 mg and possessing a magnification ration of approximately 1 :15. Tie sues were allowed to equilibrate for at least 30 min. before maximal responses to norepiaephrine (NE) were elicited . In a typical experiment, control responses to NE (10-4 M) was established followed by exposure of tissues to test compounds for 5 min. The magnitude of adrenergic blockade was determined 3 min. after the washout of the antagonist and recovery of adrenergic responses observed till they reached steady levels . In some experiments, tissues were further exposed to phenoxybenzamine (PB) for 5 min. after responses to NE had recovered to control levels, following initial exposure to test compounds. All drug solutions were made in normal saline containing 0. 05% Na metabisulfite. Solutions of halogenated phenyl-2 HEAe were placed on ice imme-
]11pha-]~dreaergia 8i^^~+~~ b9 2~~i1
Vol. 14, a~o. 11
2145
TABLE 1 ~~a-adrenergic Blockade by Halogenated Phenyl-2-Halogenoethylsmines Ar " CH Br " CH 2 N (CH3)2 " HBr
a Ar .
n
Coacea!ration
Mazimum Recovery of Response at 120 min.
Rate of Recovery t 1/2 ( ± SEM)
(96 ± SEM) ( ± 0. 95)
21 . 05
b ( ± 1 . 5)
94 . 5
( ± 1, 9)
36 . 08
( ± 1 . 6)
1x10 -5 M
92 . 0
( ± 4, 3)
37 . 2
( ± 2. 75
15
3z10 -7 M
94 . 2
( ± 3. 3)
35 . 3
( ± 2. 1)
12
5210 -6 M
93 . 4
( ± 2. 5)
39 . 9
( ± 2. 6)
C6H5
29
1z10 -5'M
100. 8
2 Br . C6H4
12
3z10 -5 M
3 Br . C6H4
13
4 Br . C6H4 3. 4 (Br) 2C6H3
a = Concentration causing > 95°)(o blockade of adrenergic responses following a 5 min, contact period . b -- Data for DMPEA taken from Swamy and Triggle (12) . diately after preparation; PB was dissolved in warm saline (approximately 35 o C) and allowed to stand st room temperature for 15-20 min. before being placed on ice. Results Adrenergic blockade by halogenated phenyl-2-halogenoethylsmiaee . Table 1 lists some of the characteristics of adrenergic blockade caused by the teat compounds. Their potencies in antagonising (>95°}6) NE responses varied from 3 x 10 -7 M for the Qsrs compound
to 3 x 10 -5 M for the ortho
halogen compound . Recovery from blockade was essentially complete (9096 or
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2-HEA
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TABLE 2 Effect of Pretreatment by Halogenated Phenyl-2-Halogenoethylamines on the Duration of Adrenergic Blockade by Phenoxybenzamine (10-7 M/5 min. )
Recovery of Response
Compound Ar " CHBr "CHLNIv1~
n
Rate of Recovery t 1/2 (±SEM)mia.
(% ± SEM)
Ar= None
15
22 . 6
( ± 3. 98) at 240 min.
>240 min.
C6H5
17
86 . OS
( ± 2. 7)
at 120 min.
66 . 5
a ( = 3. 98)
2 Br C6H4
15
67 . 7
( ± 3. 3)
at
90 min.
68 . 5
( ± 3. 5)
3 Br C6 H4
9
77 . 2
( ± 3. 5)
at 120 min.
75 . 6
( ± 4. 6)
4 Br C6H4
8
77 . 0
( ± 5. 6)
at 120 min.
73 . 4
( = 5. 6)
3, 4 (Br) 2 C6H3
10
84 . 4
( ± 7. 1)
at 120 min.
74 . 0
( ± 3. 9)
a = Data for DMPEA taken from Swamy and Z'riggle (12) . more) in 120 min. ; no significant differences (at p = 0. 05) were observed in the rates of recovery (t 1/2 values) from blockade between the various compounds tested . The blockade and recovery of adrenergic response, se typified by the Sara -bromo phenyl substituted compound, is illustrated in Fig. 1 . Effect of pretreatment by halogenated phenyl-2-HEAs on adrenergic blockade by pheaoaybenzamine . The results obtained is these experiments are listed in Table 2. PB was added when adrenergic reeponsee had recovered completely (i . e. at 120 min. ) from initial blockade by test compounds. Exposure to 10 -7 M PB for 5 min.
Vol. 14, No . 11
Alpha-Adrsnsrgia Sioakada by ]-~11
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resulted in an essentially complete blockade (>90%) of NE reeponeee. In control tissues, blockade by PB showed its characteristically long duration ; reeponeee to NE at 240 min. was 22 . 6% ± 3 . 9 (n = 1. 5) of control responses . Pretreatment with the teat compound did not alter the magnitude of initial blockade by PB . However, a marked increase in the rate of recovery from PB-induced blockade was observed in pretreated tissues, although no significant differences (p = 0. 05) were seen between the effects of pretreatment by the various test compounds (Table 2; Fig. 1) .
h C
100 60
ô
10
30
~-~ 15
60
90
Minutes
~ 120
I 1g0
FIG. 1
Blockade and recovery of adrenergic reeponeee following treatment by : 4 Br " C6 H4 CH " Br " CH2" N (CH3)2 (3 x 10' 7 M/5 min. ),o-e , PB (10' 7 M/5 min. ) in tissues whose responses had recovered from initial blockade by psra -compound,a`i . PB (10 -7M/5 mina) in control tissues, ~-d . Figures in parenthesis refer to number of ezperimente. Discussion The anti-adrenergic activity of DMPEA has been characterized as one of prompt onset cad a brief duration of action (9-11) . The effect of halogen eub-
2148
Alpha-Adrenergic
Blockade by
2-HI-A
Vol .
14, No . 11
etitution in the phenyl ring, ae seen in these experiments, reeulta in qualitatively similar activity although with minor quantitative variations . All compounds in this study caused blockade of adrenergic responses following 5 min. contact with the tissues . Halogen substitution in the phenyl nucleus resulted in clear differences in activity : pats > meta -
ra > mete > ortho . Comparison
of the potencies of the phenyl substituted compounds with DMPEA shows that only substitution in pats or mete -~ara position results in an increase of activity while the mete and ortho compounds show similar or decreased activity. The differences in activity observed in vitro generally are in good agreement with their reported relative effectiveness as antipressor agents in rate and dogs (4, 5, 14). The alterations in the duration of PB-induced blockade caused by these compounds are similar to those reported earlier for DMPEA on the rat vas deferene and rabbit aorta (11-13, 15) and consist of the conversion of the typ ically long-lasting adrenergic antagonism of PB into a relatively short one (Table 2; Fig . 1) . These reeulta can be interpreted on the basis of a model that proposes at least two sites of interaction for 2-HEA at adrenergic poetsynaptic sites (11, 12). According to thin model, the initial recovery of adrenergic response after treatment with short-acting 2-HEA represents the regeneration of eC-receptor sites and the modification of PB-induced blockade ie brought about by the fraction of 2-HEA remaining alkylated at non-receptor sites . The alkylation of non-receptor sites by 2-HEA ie also indicated by studies of uptake of labelled alkylatlng agents (16, 17) ae well as the nonspecüic nature of antagonism shown by this group of drugs. The precise nature of the non-receptor sites of Interaction is not known. Evidence obtained with calcium antagonists (12), however, suggests that the
Vol . 14, No . 11
lllpha-lldran~rgic Slockada by 2-~11
2149
long duration of action of PB sad related compounds may be due to their action st calcium mobilization sites. The relationship between the alkylating action of 2-HEA and processes of calcium mobilization is the rat vas deferene remains to be elucidated . Acknowledgement The author wishes to thank Dr . David J. Triggle for his generous güt of the teat compounds used in this study. References 1 . M. Nickereon and L. S. Goodman , J. Pharmacol. Ezper. Therap . 89, 167 - 185, 1947 . 2. D. J. Triggle, Chemical Aspects of the Autonomic Nervous System, p. 226, Academic Press, N. Y. (1965) . 3 . M. Nickereon, Pharmacol . Revs . 11, 443, 1959 . 4. G. D. P. Graham and G. W. L. James, J. Med. Chem . 3, 489 - 504, 1961 . 5. G. D. P. Graham and M. A. Karrsr, J. Med. Chem. 6, 103 - 107, 1962 . 6. N. B. Chapman and D. J. Triggle, J . Chem . Soc. 265, 1385 - 1400, 1963 . 7. G. D. P. Graham, Proa . Med. Chem. 2, 132 - 175, 1962 . 8 . D. J. Triggle, Adv. Drug Res. 2, 173 - 189, 1965 . 9. M. May, J. F. Moran, H. Kamelberg, and D. J. Triggle, Mol. Pharmacol . 3, 28 - 36, 1967 . 10 . J. F. Moran, C . R. Triggle, and D. J. Triggle, J. Phsrm. Pharmacol. 21, 38 - 46, 1969 . 11 . J . F. Moran, V. C. sWamy, and D. J. Triggle, Life Sciences 9, 1303 - 1315, 197.0 . 12 . V. C. 9wamy and D. J. Triggle, Eur. J . Pharmacol. 19, 67-78, 1972.
2150
Alpha-Adrenergic Blockade by 2-i~A
Vol . 14, No . 11
13 . S. McLean, V. C. 9wamy, D. Tomei, and D. J. Triggle, J. Mad. Ch em . 16, 54 - 57, 1973 . 14 . G. E. Garner, R. A. Woodbury, and E. E. Elko, J. Pharmacol. Exiler . Therap . 162, 38 - 48, 1962 . 15 . R. A. Janie and D. J. Triggle, Pharmacol . Ree. Commun . 3, 175 - 182, 1971 . 16 . R. D. Green, G. S. Lefever, E . M. Sheys, and M. Brietow, J. Pharmacol. Exiler . Therap. 187, 524 - 528, 1973 . 17 . J. F. Moraa, M. May, H. Kimelberg, and D. J . Triggle, Mol. Pharmacol. 3, 15 - 27, 1967 .
Pargason Press
BLOCKADE OF ADRENERGIC RESPONSES BY HALOGENATED PHENYL-2-HALOGENOETHYL AMINES Vijay C. 9arsmy Department of Biochemical Pharmacology School of Pharmacy State University of New York st Buffalo Buffalo, New York 14214 (iàsceived is tiaal form 45 April 1974) The action of halogensted phenyl 2-halogenoethylamines on adrenergic C,- receptor Bites of the rat vas deferens was studied. The effect of halogen (Br) substituents in the phenyl nucleus resulted in the following differences is activity : pare > mets - para > mete > ortho . All compounds possessed a short duration of action ; recovery from adrenergic blockade was complete within 120 min. Eapoeure of tissues to phenoayben$amine, after complete recovery from adrenergic blockade by the teat compounds, did not alter the magnitude of initial blockade but sharply reduced the duration of action of phenorybenaamiae . These results are discussed in terms of a model which proposes at least 2 sites of action for 2-halogeaoethylaminee at the adrenergic a - receptors of the rat vas deferene .
2-Halogeaoethylaminee (2-HEA) have been widely studied ever since their property of irreversible blockade st adrenergic neuroeffector sites was reported for Dibenamine (1-3 ). Typically, the adrenergic blockade caused by Di benamine and related compounds is characteri$ed by slow onset of action and prolonged duration of action. The action of N, N-dimethyl-2-bromophenethylamine (DMPEA) and related compounds differ from Dibenamine and its analogs in possessing rapid onset of action and relatively short duration of action (410). Previous reports (11-13) from this laboratory provided evidence that DMPEA and other 2-HEAs act at two sites on the adrenergic poet-synaptic
sias
2344
Alpfra-Adrenergic Blockade by 2-HEA
Vol . 14, No . 11
surface and that the long duration of action of Dibenamine and phenoxybenzamine may be due to their interaction at sites associated with Ca++ mobilization . The present study ie a preliminary report on the adrenergic blocking properties of halogenated phenyl-2-halogenoethylamines which differ structurally from DMPEA in possessing halogen eubstituente in the phenyl ring . The results show that these compounds possess qualitatively similar properties as DMPEA in their blockade of adrenergic responses and their capacity to modify irreversible antagonism by pheaoxybeassamise . Methods Vaea deferentia from rate (Holtzmaa) weighing approximately 150 g were set up in organ bathe containing Tyrode'e solution maintained at 37oC and bubbled with a gaseous mixture of 95°Jo 02 + 5°}'o C0 2. Isotonic contractions were recorded on a smoked drum of a kymograph via levers exerting a teneion of 300 - 350 mg and possessing a magnification ration of approximately 1 :15. Tie sues were allowed to equilibrate for at least 30 min. before maximal responses to norepiaephrine (NE) were elicited . In a typical experiment, control responses to NE (10-4 M) was established followed by exposure of tissues to test compounds for 5 min. The magnitude of adrenergic blockade was determined 3 min. after the washout of the antagonist and recovery of adrenergic responses observed till they reached steady levels . In some experiments, tissues were further exposed to phenoxybenzamine (PB) for 5 min. after responses to NE had recovered to control levels, following initial exposure to test compounds. All drug solutions were made in normal saline containing 0. 05% Na metabisulfite. Solutions of halogenated phenyl-2 HEAe were placed on ice imme-
]11pha-]~dreaergia 8i^^~+~~ b9 2~~i1
Vol. 14, a~o. 11
2145
TABLE 1 ~~a-adrenergic Blockade by Halogenated Phenyl-2-Halogenoethylsmines Ar " CH Br " CH 2 N (CH3)2 " HBr
a Ar .
n
Coacea!ration
Mazimum Recovery of Response at 120 min.
Rate of Recovery t 1/2 ( ± SEM)
(96 ± SEM) ( ± 0. 95)
21 . 05
b ( ± 1 . 5)
94 . 5
( ± 1, 9)
36 . 08
( ± 1 . 6)
1x10 -5 M
92 . 0
( ± 4, 3)
37 . 2
( ± 2. 75
15
3z10 -7 M
94 . 2
( ± 3. 3)
35 . 3
( ± 2. 1)
12
5210 -6 M
93 . 4
( ± 2. 5)
39 . 9
( ± 2. 6)
C6H5
29
1z10 -5'M
100. 8
2 Br . C6H4
12
3z10 -5 M
3 Br . C6H4
13
4 Br . C6H4 3. 4 (Br) 2C6H3
a = Concentration causing > 95°)(o blockade of adrenergic responses following a 5 min, contact period . b -- Data for DMPEA taken from Swamy and Triggle (12) . diately after preparation; PB was dissolved in warm saline (approximately 35 o C) and allowed to stand st room temperature for 15-20 min. before being placed on ice. Results Adrenergic blockade by halogenated phenyl-2-halogenoethylsmiaee . Table 1 lists some of the characteristics of adrenergic blockade caused by the teat compounds. Their potencies in antagonising (>95°}6) NE responses varied from 3 x 10 -7 M for the Qsrs compound
to 3 x 10 -5 M for the ortho
halogen compound . Recovery from blockade was essentially complete (9096 or
214 6
Alpha-Adrenergic Blockade by
2-HEA
Vol .
14, No . 11
TABLE 2 Effect of Pretreatment by Halogenated Phenyl-2-Halogenoethylamines on the Duration of Adrenergic Blockade by Phenoxybenzamine (10-7 M/5 min. )
Recovery of Response
Compound Ar " CHBr "CHLNIv1~
n
Rate of Recovery t 1/2 (±SEM)mia.
(% ± SEM)
Ar= None
15
22 . 6
( ± 3. 98) at 240 min.
>240 min.
C6H5
17
86 . OS
( ± 2. 7)
at 120 min.
66 . 5
a ( = 3. 98)
2 Br C6H4
15
67 . 7
( ± 3. 3)
at
90 min.
68 . 5
( ± 3. 5)
3 Br C6 H4
9
77 . 2
( ± 3. 5)
at 120 min.
75 . 6
( ± 4. 6)
4 Br C6H4
8
77 . 0
( ± 5. 6)
at 120 min.
73 . 4
( = 5. 6)
3, 4 (Br) 2 C6H3
10
84 . 4
( ± 7. 1)
at 120 min.
74 . 0
( ± 3. 9)
a = Data for DMPEA taken from Swamy and Z'riggle (12) . more) in 120 min. ; no significant differences (at p = 0. 05) were observed in the rates of recovery (t 1/2 values) from blockade between the various compounds tested . The blockade and recovery of adrenergic response, se typified by the Sara -bromo phenyl substituted compound, is illustrated in Fig. 1 . Effect of pretreatment by halogenated phenyl-2-HEAs on adrenergic blockade by pheaoaybenzamine . The results obtained is these experiments are listed in Table 2. PB was added when adrenergic reeponsee had recovered completely (i . e. at 120 min. ) from initial blockade by test compounds. Exposure to 10 -7 M PB for 5 min.
Vol. 14, No . 11
Alpha-Adrsnsrgia Sioakada by ]-~11
Z147
resulted in an essentially complete blockade (>90%) of NE reeponeee. In control tissues, blockade by PB showed its characteristically long duration ; reeponeee to NE at 240 min. was 22 . 6% ± 3 . 9 (n = 1. 5) of control responses . Pretreatment with the teat compound did not alter the magnitude of initial blockade by PB . However, a marked increase in the rate of recovery from PB-induced blockade was observed in pretreated tissues, although no significant differences (p = 0. 05) were seen between the effects of pretreatment by the various test compounds (Table 2; Fig. 1) .
h C
100 60
ô
10
30
~-~ 15
60
90
Minutes
~ 120
I 1g0
FIG. 1
Blockade and recovery of adrenergic reeponeee following treatment by : 4 Br " C6 H4 CH " Br " CH2" N (CH3)2 (3 x 10' 7 M/5 min. ),o-e , PB (10' 7 M/5 min. ) in tissues whose responses had recovered from initial blockade by psra -compound,a`i . PB (10 -7M/5 mina) in control tissues, ~-d . Figures in parenthesis refer to number of ezperimente. Discussion The anti-adrenergic activity of DMPEA has been characterized as one of prompt onset cad a brief duration of action (9-11) . The effect of halogen eub-
2148
Alpha-Adrenergic
Blockade by
2-HI-A
Vol .
14, No . 11
etitution in the phenyl ring, ae seen in these experiments, reeulta in qualitatively similar activity although with minor quantitative variations . All compounds in this study caused blockade of adrenergic responses following 5 min. contact with the tissues . Halogen substitution in the phenyl nucleus resulted in clear differences in activity : pats > meta -
ra > mete > ortho . Comparison
of the potencies of the phenyl substituted compounds with DMPEA shows that only substitution in pats or mete -~ara position results in an increase of activity while the mete and ortho compounds show similar or decreased activity. The differences in activity observed in vitro generally are in good agreement with their reported relative effectiveness as antipressor agents in rate and dogs (4, 5, 14). The alterations in the duration of PB-induced blockade caused by these compounds are similar to those reported earlier for DMPEA on the rat vas deferene and rabbit aorta (11-13, 15) and consist of the conversion of the typ ically long-lasting adrenergic antagonism of PB into a relatively short one (Table 2; Fig . 1) . These reeulta can be interpreted on the basis of a model that proposes at least two sites of interaction for 2-HEA at adrenergic poetsynaptic sites (11, 12). According to thin model, the initial recovery of adrenergic response after treatment with short-acting 2-HEA represents the regeneration of eC-receptor sites and the modification of PB-induced blockade ie brought about by the fraction of 2-HEA remaining alkylated at non-receptor sites . The alkylation of non-receptor sites by 2-HEA ie also indicated by studies of uptake of labelled alkylatlng agents (16, 17) ae well as the nonspecüic nature of antagonism shown by this group of drugs. The precise nature of the non-receptor sites of Interaction is not known. Evidence obtained with calcium antagonists (12), however, suggests that the
Vol . 14, No . 11
lllpha-lldran~rgic Slockada by 2-~11
2149
long duration of action of PB sad related compounds may be due to their action st calcium mobilization sites. The relationship between the alkylating action of 2-HEA and processes of calcium mobilization is the rat vas deferene remains to be elucidated . Acknowledgement The author wishes to thank Dr . David J. Triggle for his generous güt of the teat compounds used in this study. References 1 . M. Nickereon and L. S. Goodman , J. Pharmacol. Ezper. Therap . 89, 167 - 185, 1947 . 2. D. J. Triggle, Chemical Aspects of the Autonomic Nervous System, p. 226, Academic Press, N. Y. (1965) . 3 . M. Nickereon, Pharmacol . Revs . 11, 443, 1959 . 4. G. D. P. Graham and G. W. L. James, J. Med. Chem . 3, 489 - 504, 1961 . 5. G. D. P. Graham and M. A. Karrsr, J. Med. Chem. 6, 103 - 107, 1962 . 6. N. B. Chapman and D. J. Triggle, J . Chem . Soc. 265, 1385 - 1400, 1963 . 7. G. D. P. Graham, Proa . Med. Chem. 2, 132 - 175, 1962 . 8 . D. J. Triggle, Adv. Drug Res. 2, 173 - 189, 1965 . 9. M. May, J. F. Moran, H. Kamelberg, and D. J. Triggle, Mol. Pharmacol . 3, 28 - 36, 1967 . 10 . J. F. Moran, C . R. Triggle, and D. J. Triggle, J. Phsrm. Pharmacol. 21, 38 - 46, 1969 . 11 . J . F. Moran, V. C. sWamy, and D. J. Triggle, Life Sciences 9, 1303 - 1315, 197.0 . 12 . V. C. 9wamy and D. J. Triggle, Eur. J . Pharmacol. 19, 67-78, 1972.
2150
Alpha-Adrenergic Blockade by 2-i~A
Vol . 14, No . 11
13 . S. McLean, V. C. 9wamy, D. Tomei, and D. J. Triggle, J. Mad. Ch em . 16, 54 - 57, 1973 . 14 . G. E. Garner, R. A. Woodbury, and E. E. Elko, J. Pharmacol. Exiler . Therap . 162, 38 - 48, 1962 . 15 . R. A. Janie and D. J. Triggle, Pharmacol . Ree. Commun . 3, 175 - 182, 1971 . 16 . R. D. Green, G. S. Lefever, E . M. Sheys, and M. Brietow, J. Pharmacol. Exiler . Therap. 187, 524 - 528, 1973 . 17 . J. F. Moraa, M. May, H. Kimelberg, and D. J . Triggle, Mol. Pharmacol. 3, 15 - 27, 1967 .