Life Sciences Vol. 17, pp . 97-104 Printed in the U.S .A .
Pargamon Press
NEW BENZOPYRANS :
ANTICONVULSANT ACTIVITIES
Nicholas P . Plotnikoff, Harold E . Zaugg, Albert C . Peteraen, David L . Arendeen, and Richard F . Anderson . Pharmacology and Medicinal Chemistry Division, Abbott Laboratories, North Chicago, Illinois, 60064, U.S .A . (Received in final form May 22, 1975) SUMMARY Three new analogs of dimethylheptylpyran (DMHP) (SP-141, SP-143, and SP-175) were found to exhibit significant anticonvulsant activity against sudiogenic, aupramaximal electroshock, and maximal pentylenetetrazol induced seizures in mice . In rate, all three compounds were found to be more active than diphenylhydantoin in the aupramaximal electroshock test . In particular, a different profile of anticonvulsant activity was demonstrated for SP-175 compared to DMHP or delta-9-THC .
INTRODUCTION Our interest in the benzopyrans as anticonvulsants stemmed from the pioneer animal studies of Loewe and Goodman (1), and clinical studies of Davis and Ramsey (2) on the isomers of dimethylheptylpyran analog (DMHP) . Earlier O'Shaughnessy (3) had introduced marihuana to Europe to treat various central nervous system disorders including epilepsy . Thus, it became of great interest to several investigators to study the anticonvulsant activity of the constituents of marihuana, e.g ., delta-9-THC, delta-B-THC, cannabidiol, cannabinol (4-16) . Our owa interest has been to study new analogs of DMHP (Figure 1) in an attempt to find more specific anticonvulsant activity . A preliminary report on these compounds was presented recently (26) . METHODS Experimental animals, drug preparationa~ and test intervals . The test procedures were conducted with white male ARS/Sprague-Dawley strain mice, 18 to 24 grams, and male Long-Evens rata, 170 to 190 grams, unless otherwise stated . The test compounds were administered orally as a suspension in lOX olive oil and 90X Methocel (0 .5X) to ten animals per dose . All drugs were tested for activity at one, four, and twenty-four hours post drug administration in different groups of animals . Statistical analyses were carried out using simultaneous línear regression and probit techniques, Audiogenic seizures teat (mouse ) . Male O'Grady strain mice (14 to 16 grams) especially bred for susceptibility to sudiogenic seizures were used as subjects . The sudiogenic apparatus consisted of a wooden box enclosing a metal container with two doorbells attached to the upper section . After drug
98
Vol . 17, No . 1
Benzopyrans-Anticonvulsants
P2CUl6 1 6T71IC'Ni69 OP 6B112DPPM116 SP/2b .
Mborc Ilo .
C6e~1rr1 Otacrt,cima
(1)
W3 9P-104 dalta-9-76C
SP-6 DlOIP
40624
SP-141
61759
SP-143
6L634
SP-175
(1)
40566
42574
Chemistry SP-141 (ABBOTT-41759) 3-(3-Methyl-2-octyl)-1-[4-(1-piperidine)butyryloxy]-6,6,9-trimethyl-7,8, 9,10-tetrahydro-óH-dibenzo[b,d]pyran hydrochloride SP-143 (ABBOTT-41836) 3-( 3-Methyl-2-octyl)-1-[4-(4-morpholine)butyryloxy ]-6,6,9-trimethyl-7,8, 9,10-tetrahydro-óH-dibenzo[b,d] pyran hydrobromide SP-175 (ABBOTT-42574) 3-(3-Methyl-2-octyl)-1-[4-(1-homopiperidine)butyryloxy]-6,6,9-trimethyl7,8,9,10-tetrahydro-6H-dibenzo[b ,d] pyran hydrochloride (25) .
administration the animals were placed in the sudiogenic chamber and the bells activated for one minute and the animals were observed for convulsions (17) . Supramaximal electroshock test (mouse and rat) . After drug administration each animal received an electroshock (mice received 100 cps ., pulse duration 1 .0 meet ., at 140 volts for 0 .3 sec ; rata received 150 ma at 0 .2 sec .) through corneal electrodes to initiate a hindlimb tonic extension convulsion ; anti-
Vol. 17, No . 1
Henzopyrans-Anticonvulsanta
convulsant activity occurred when the above convulsion was blocked (18,
99
19) .
Psychomotor electroshock test (mouse . After receiving drug, each animal was given an electroshock (6 cps ., pulse duration 1 .0 cosec., at 70 volts for 3 sec.) through corneal electrode to initiate a psychomotor seizure . Protection from convulsions occurred when either the clonic forepaw activity or facial clonua was blocked (19) . Pentylenetetrazol maximal seizures teat - tonic extension (mouse) . After drug administration, each animal received pentylenetetrazol at 120 mg/kg aubcutaneously to cause a tonic extension conwlaion ; anticonvulsant activity occurred when this convulsion was blocked (19) . Pentylenetetrazol minimal seizure teat (mouse) . Following drug administration each animal was given pentylenetetrazol at 85 mg/kg subcutaneously to elicit clonic convulsions ; anticonwlsant action occurred when the convulsions were blocked (18) . Rotarod teat (mouse) . Mice were trained to remain on a rotating rod (16 revolutions per minute, Stoelting Company) for a maximum period of 100 seconds for two consecutive trials, modified after Rinnard and Carr procedure (20) . After training the animals were administered drug and again tested on the rotating rod . Motor deficit (TD50) was determined by a 50% reduction in the time the animals remained on the rod as compared to control time . Inclined screen test (rat) . After drug administration each animal was placed in the center of a 14" x 16" wire mesh screen (1/2" hardware cloth) at an angle of 60 degrees for a period of 60 seconds. Motor deficit was determined by the length of time the animals remained on the inclined screen . RESULTS Audi ogenic seizures Tonic extensor component . All compounds exhibited marked activity in preventing the hind-limb extensor component of the audiogenic seizure (Table 1) . Thus, delta-9-THC was found to reduce the tonic extension component in fifty percent of the animals (ED50) at a dose of 5 mg/kg at the one hour teat period . At later times (4 and 24 hours), delta-9-THC was considerably leas active . In contrast, DMHP was found active over a prolonged period (24 hours) with ED50 values of 6 .7, 2 .9, and 15 .1 mg/kg at one, four, and 24 hours, respectively . The new compounds exhibited potent activity at both the 1 and 4 hour periods but less at the 24 hour teat time . Maximal effects were seen at the four hour teat period with SP-141 with an ED50 of 2 .8 mg/kg, SP-143 with an ED50 of 2.4 mg/kg, and SP-175 with an ED50 of 1 .1 mg/kg . By way of comparison, diphenylhydantoin was quite effective at one and four hours and less so at 24 hours, the peak effect was seen at one hour with an ED50 of 1 .7 mg/kg . Supramaximal electroshock studies. In mice the test compounds were found to be less potent than diphenylhydantoin in antagonizing the tonic extensor component of supramaximal seizures (Table 2) . Thus, delta-9-THC showed peak effects at one hour with an ED50 of 31 .9 mg/kg, while DMHP peaked at four hours with an ED50 of 57 .2 mg/kg . The new compounds (SP-141 and SP-143) showed maximal effects at four hours with ED50 values of 59 .4 and 67 .8 mg/kg, respectively . SP-175, in contrast, was effective both at one and four hour teat periods with ED50 values of 68 .3 and 75 .9 mg/kg . In rats, DMHP and the new compounds appeared to be more potent than diphenylhydantoin in preventing hindlimb extension, whereas delta-9-THC was found to be approximately equal in potency to diphenylhydantoin (Table 2) .
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Benzopyrana-Anticonvulsanta
100
TABLE 1 EFFECT OP VARIOUS BE120PY0.AN LOMPg1NUS ANO DIPH¢NVI~Uf0A1fT01N (DPN) OR AUDIOf.F11IC SEILDRES IX MICE
SP /Ib .
1 hr FDSO+ e8/kg (95S C.L .)
4 hr E050+ v¢/k8 (95f C.L .)
26 hr E050+ eR/kR -(95L C.L . _--_
SP-1D4 19-THC
5.0 2 .4-77
SP-g OIWP
6.7 2 .1-11 .0
2 .9 1.8- 4.9
15 .1 8.2-26 .7
SP-141
8.5 5.1-17.8
2.8 1 .4- b.g
32 .7 20 .0-4E .B
SP-]47
4.3 2.1- 8.0
2.4 1 .2- 5.5
19 .1 12 .5-27.0
SP-175
6.8 4.1-11 .7
1 .1 0.5- 2.4
65 .9 27 .0 - -
DPX
1.7 1 .7- 2.5
1.1 2.7- 4.4
11 .0 6.5-21.6
38 .g 29 .1-49 .3
>I00
+ Orel dosa (ng/kg) et ah3eh 5~2 of the eoivele ere protected fro. hindllob eaeaion with tao enlrle per dwe.
At the peak time of four hours, the following ED50's were observed : DMHP, 7 .5 mg/kg ; SP-141, 4 .6 mg/kg ; SP-143, 25 .2 mg/kg ; and SP-175, 7 .4 mg/kg . In contrast, diphenylhydantoin had a peak effect at one hour with an ED50 of 23 mg/kg and an ED50 of 62 mg/kg at four houra .
TABL¢ 2 EPFELT OF VARIOUS BENZOPY0.AN CO1Bff1UND5 AND DIPXENYLHYDANTOIN (DPH) ON 5UPRAt1AKINAL ELEL70 .05NOCK SFIZURES iN XICE AND 0.A73 !Ilce 1 hr ¢D50+ 4 hr E050 eR/k0 . nP./kg (95L (952 (95L C.L .) C.L .) SP/Nn .
SP -104 A 9-THC 9P-B OMIIP SP-lbl SP-1d7 SP-175 DPN
24 hr ED50+ ~ yI/kg C.L.) ~ --
--
Ileb 4 hr ED50+ eg/kg (952 C.L .)
71 .9 29 .1-74.5
301.5 96 .4-106 .6
dD.b 24 .1-142 .7
]10.8 270.7-587 .5
57 .2 75 .1-80.1
> 700
7 .5 7.4-11 .2
>J00
59 .4 41 .6-B1 .4
>300
4 .6 2.B- 7 .2
>J00
b7 .8 b1 .0-150 .3
" )00
25.2 5.7-52 .2
ó8 .l 61 .8 - 78 .7
75 .9 5A .6-I02
> J00
7 .4 4.2-17 .4
8.1 5.2 - 11
7.6 7.0-8 .7
> 20
~
fi2 .2 57 .0-67 .3
+Mrel dose (ag/Wt1 et vhtch 50I o( the enütele ere protected frov hindlivb tonie extenelon trlth ten enivele per dace .
Psychomotor Seizures . No significant activity was observed with the teat agents in reducing facial and forelimb clonua induced by low frequency electroshock except for alight activity with DMAP at high doses (Table 3) . Pentylenetetrazol seizures . Maximal seizures (tonic eateneion) . All of the test agents effectively reduced the incidence of tonic eateneion with the exception of delta-9-THC (Table 4) . DMHP was more effective than delta-9-THC or the new compounds SP-141 with ED50 values of 9 .5 and 10 .2 at the one and four hour test periods .
Benzopyrans-Anticonvulsants
Vol. 17, No . 1
TABLE 3 EFFECTS OF VARIOUS BENZOPYRAN COMPOUNDS AND DIPHENYLHYDANTOIN (DPH) ON PSYCHOlSYfOR ELECTROSHOCK SEIZURES IN MICE
SP/No.
SP-104 ~9-THC
4 hr ED50* mg/kg (95X C.L .)
>300
SP-8 DMfD?
SP-141
186 .3 (estimated)
SP-143
SP-175
>300
>300
>300
DHP
>240
*Oral done (mg/kg) at which 50X of the animals are protected from facial and forelimb clonus . and SP-143 were moat effective at one hour with ED50 values of 20 .2 and 31 .1 mg/kg . SP-175 was effective at both the one and four hour teats with ED50 values of 41 .8 and 38 .2 mg/kg . Minimal seizures (clonus) . No significant anticonvulsant activity with the test agents was observed against minimal (Clonus) seizures, with the exception of DMHP and SP-141 (Table 4) . TABLE 4 EFFECT OF VARIOUS BENZOPYRAN COMPOUNDS AND DIPHENYLHYDANTOIN (DPH) ON PENTYLENETETRAZOL SEIZURES IN THE MOUSE (TONIC E7CTENSION AND CLONIC SEIZURES)
SP ./No . _ SP -104 9-THC SP-8 DMfD' §P-141 SP-143 SP -175 DPH
Tonic Eateneion 1 hr ED50* 4 hr ED50* m8/k8 m8/k8 (95z C.L .) (95z C.L .)
i ~ i
300 9.5 3.5-21 .6 20 .2 10 .0-41 .0 31 .1 15 .6-62 .6 41 .8 22 .0-90.6 2.7 1 .7- 5 .9
'
> 300 10 .2 5 .3-22 . 3 54 .9 29 .4-113 52 .1 34 .5-74 . 7 38 .2 16 .2-90 .0 2 .2 1.8- 2 .7
i
Clonic Seizurea 1 hr ED50* 4 hr ED50* m8/kg m8/k8 (95X C.L .) (95X C .L .) > 300
> 300
~
100-50X
>100
~
100=70Z
>300
~
>300
>300
~ ~ ~
>100
>100
>100
>100
*Oral done (mg/kg) at which 50% of the animals are protected from hindlimb tonic extension with ten animals per dose .
Rotarod (mice) . Therapeutic indices (TD50/ED50) were obtained when the anticonvulsant activity of the teat agents against audiogenic seizures in mice was compared to the neurotoxicity of compounds tested on the rotarod (Table 1 and 5) . At the four hour test period, delta-9-THC, DMBP, SP-141, SP-143, SP-175, and diphenylhydantoin showed ration of 2,7,5,8,18, and 36, respectively .
102
Henzopyrana-Anticonwlsants
Vol. 17, No . 1
TABLE 5 EFFECT OF VARIOUS BENZOPYRAN COMPOUNDS AND DIPHENYLHYDANTION (DPH) ON ROTAROD PERFORMANCE IN MICE SP/No . 4 hr TD50* mg/kg (95X C.L .)
SP-104 A9-THC 82 .8 46 .6-321 .7
SP-8 DMHP 21 .0 3 .4-39.0
SP-141
SP-143
15 .7 0 .3-31.8
16 .6 7 .1-42.8
SP-175
DPH
20 .1 119 .7 11 .5-29.5 82 .3-164 .7
*Oral dose (mg/kg) at which a 50X reduction occurs in the time the animals remain on the rotating rod. Inclined screen teat (rat) . At peak time of anticonwlsant activity against supramaximal electroshock (four hours) no impairment of grip strength on the inclined screen was seen with either DZ4iP or SP-175 at doses up to 1280 mg/kg . (SP-141 and SP-143 at doses up to 640 mg/kg) . DISCUSSION The present study represents the first investigation of the profile of anticonwlsant activity of new benzopyran structures . In reviewing the anticonwlsant activity of delta-9-THC, it becomes apparent that it resembles diphenylhydantoin in profile (short duration) while DMaP appears to resemble trimethadione in profile (pentylenetetrazol antagonism) . Thus, our new analog, SP-175, may represent a therapeutic advance in providing a longer duration of action than delta-9-THC and a diphenylhydantoin-like profile . In particular, SP-175 was found to be more active than diphenylhydantoin against supramaximal electroshock in the rat . DMHP and delta-9-THC in animal and human studies has been found to exhibit significant side effects, not only in terms of neurotoxicity, but also with respect to cardiovascular parameters (22,23,24) . Most investigators have used the intravenous or intraperitoneal route of administration with delta-9-THC and DMHP (4-16) . The new compounds, particularly SP-175, appear to exhibit a low degree of neurotoxicity by the oral route, particularly in the rat . It is our hope that this new compound will be evaluated in higher species, both alone and in combination with other known anticonwlsant agents . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 .
S. Loewe, and L. S . Goodman, Fed . Proc . 6,352, (1947) . J. P . Devis, and H. H . Ramsey, Fed . Proc . _8, 284-285, (1949) . W . B. O'Shaughnesay, Trans . Med. Phys . Soc . Bengal . _71, 1838-1840. J. C. Garriott, R . B. Forney, F . W. Hughes, and A . B . Richarde, Arch . Int . Phaxmacodyn. Ther . 171, 425-434, (1968) . R. D. Sofia, T . A . Solomon, and H. Barry III, Pharmacologist, 13, 246, (1971) . D . P. Man, and P. F . Consroe, J . Int . Res. Commun . _1, 12, (1973) . W. 0. Boggan, R. A. Steels, and D. X . Freedman, Peychopharmacologia, 29, 101-106, (1973) . M. E. Corcoran, J . A. McCaughran, Jr ., and J. A. Wads, Exp. Neurol . _40, 471-483, (1973) . J. A. Wads, M. Sato, and M. E. Corcoran, Exp. Neurol . 39, 157-165, (1973) . R. Rader, Fed. Proc . _32, 756, (1973) . R. Karler, W. Cely, and S . A. Turkanis, Life Sci. _13, 1527-1531, (1973) . E . A. Carlini, J. R. Leite, M. Tannhauaer, and A. C . Bernardi, J. Phaxm. Pharmac . 25,664-665, (1973) .
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13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 .
Penzopyrana-Anticonvulsants
103
P. F. Consroe and D . P . Man, Life Sci . _13, 429-439, (1973) . R. Rader, W. Cely, and S . A. Turkanis, Ree . Commun . Chem . Pathol . Pharmac . 7, 353-358, (1974) . G. B . Cheaher and D . M. Jackson, Psychopharmacologia, 37, 255-264, (1974) . R. Rader, W. Cely, and S . A. Turkanis, Life Sci . 15, 931-947, (1974) . N . P . Plotnikoff, D . J. Green, J. Pharmacol . Exp . Therap ., _119, 234, (1957) . E . A. Swinyard, W. C. Brown, J. Pharmacol . Exp . Therap ., _106, 319-330, (1952) . E . P. Toman, G. M. Everett, Evaluation of Drug Activities, Pharmacometrice, Vol . 1, 1974 . Published by Academic Prese . W . J. Kinnard, C. J . Carr, J. Pharm. and Exp . Therap . _121, 354 (1957) . L . H. Sternback, L. 0 . Randall, S . R . Guatafson, Psychopharmacological Agente, Vol . I. New York : Academic Pregia, 1964, p . 137-224 . H . F. Hardman, E. F . Domino, M . H. Seevers, Pharmacol. Rev ., 23, 295-315, (1971) . E . F. Domino, H. F. Hardman, M. H. Seevers, Pharmacol. Rev. 23, 317-336, (1971) . E . F. Domino, Ann. N. Y . Acad . Sci ., _191, 166-192, (1971) . R. R. Razdan, B . Zitko Terris, H. G. Pare, manuscript in preparation. (Compounds 18, 19, and 20) . N . P. Plotnikoff, H. Zaugg, A. C. Petersen, R. Anderson, D . Arendsen . 168th ACS National Meeting, Atlantic City, New Jersey, September 9-12, 1974 . ACKNOWLEDGEMENTS
We would like to express our appreciation to Dr . Harvey Rupferberg and the Anticonvulsant Advisory Committee of the National Institute of Neurological Diseases and Stroke for their encouragement of this study (supported in part by contract NO1-NS-3-2314) . The authors are indebted to Dr . R. K . Razdan, B. Zitko Terris, and H. G . Pars of The John C . Sheehan Institute for Research and Sharps Associates (SISA), Cambridge, Massachusetts, for the original chemical preparation of the three derivatives of DMfD? (25) .
Pargamon Press
NEW BENZOPYRANS :
ANTICONVULSANT ACTIVITIES
Nicholas P . Plotnikoff, Harold E . Zaugg, Albert C . Peteraen, David L . Arendeen, and Richard F . Anderson . Pharmacology and Medicinal Chemistry Division, Abbott Laboratories, North Chicago, Illinois, 60064, U.S .A . (Received in final form May 22, 1975) SUMMARY Three new analogs of dimethylheptylpyran (DMHP) (SP-141, SP-143, and SP-175) were found to exhibit significant anticonvulsant activity against sudiogenic, aupramaximal electroshock, and maximal pentylenetetrazol induced seizures in mice . In rate, all three compounds were found to be more active than diphenylhydantoin in the aupramaximal electroshock test . In particular, a different profile of anticonvulsant activity was demonstrated for SP-175 compared to DMHP or delta-9-THC .
INTRODUCTION Our interest in the benzopyrans as anticonvulsants stemmed from the pioneer animal studies of Loewe and Goodman (1), and clinical studies of Davis and Ramsey (2) on the isomers of dimethylheptylpyran analog (DMHP) . Earlier O'Shaughnessy (3) had introduced marihuana to Europe to treat various central nervous system disorders including epilepsy . Thus, it became of great interest to several investigators to study the anticonvulsant activity of the constituents of marihuana, e.g ., delta-9-THC, delta-B-THC, cannabidiol, cannabinol (4-16) . Our owa interest has been to study new analogs of DMHP (Figure 1) in an attempt to find more specific anticonvulsant activity . A preliminary report on these compounds was presented recently (26) . METHODS Experimental animals, drug preparationa~ and test intervals . The test procedures were conducted with white male ARS/Sprague-Dawley strain mice, 18 to 24 grams, and male Long-Evens rata, 170 to 190 grams, unless otherwise stated . The test compounds were administered orally as a suspension in lOX olive oil and 90X Methocel (0 .5X) to ten animals per dose . All drugs were tested for activity at one, four, and twenty-four hours post drug administration in different groups of animals . Statistical analyses were carried out using simultaneous línear regression and probit techniques, Audiogenic seizures teat (mouse ) . Male O'Grady strain mice (14 to 16 grams) especially bred for susceptibility to sudiogenic seizures were used as subjects . The sudiogenic apparatus consisted of a wooden box enclosing a metal container with two doorbells attached to the upper section . After drug
98
Vol . 17, No . 1
Benzopyrans-Anticonvulsants
P2CUl6 1 6T71IC'Ni69 OP 6B112DPPM116 SP/2b .
Mborc Ilo .
C6e~1rr1 Otacrt,cima
(1)
W3 9P-104 dalta-9-76C
SP-6 DlOIP
40624
SP-141
61759
SP-143
6L634
SP-175
(1)
40566
42574
Chemistry SP-141 (ABBOTT-41759) 3-(3-Methyl-2-octyl)-1-[4-(1-piperidine)butyryloxy]-6,6,9-trimethyl-7,8, 9,10-tetrahydro-óH-dibenzo[b,d]pyran hydrochloride SP-143 (ABBOTT-41836) 3-( 3-Methyl-2-octyl)-1-[4-(4-morpholine)butyryloxy ]-6,6,9-trimethyl-7,8, 9,10-tetrahydro-óH-dibenzo[b,d] pyran hydrobromide SP-175 (ABBOTT-42574) 3-(3-Methyl-2-octyl)-1-[4-(1-homopiperidine)butyryloxy]-6,6,9-trimethyl7,8,9,10-tetrahydro-6H-dibenzo[b ,d] pyran hydrochloride (25) .
administration the animals were placed in the sudiogenic chamber and the bells activated for one minute and the animals were observed for convulsions (17) . Supramaximal electroshock test (mouse and rat) . After drug administration each animal received an electroshock (mice received 100 cps ., pulse duration 1 .0 meet ., at 140 volts for 0 .3 sec ; rata received 150 ma at 0 .2 sec .) through corneal electrodes to initiate a hindlimb tonic extension convulsion ; anti-
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Henzopyrans-Anticonvulsanta
convulsant activity occurred when the above convulsion was blocked (18,
99
19) .
Psychomotor electroshock test (mouse . After receiving drug, each animal was given an electroshock (6 cps ., pulse duration 1 .0 cosec., at 70 volts for 3 sec.) through corneal electrode to initiate a psychomotor seizure . Protection from convulsions occurred when either the clonic forepaw activity or facial clonua was blocked (19) . Pentylenetetrazol maximal seizures teat - tonic extension (mouse) . After drug administration, each animal received pentylenetetrazol at 120 mg/kg aubcutaneously to cause a tonic extension conwlaion ; anticonvulsant activity occurred when this convulsion was blocked (19) . Pentylenetetrazol minimal seizure teat (mouse) . Following drug administration each animal was given pentylenetetrazol at 85 mg/kg subcutaneously to elicit clonic convulsions ; anticonwlsant action occurred when the convulsions were blocked (18) . Rotarod teat (mouse) . Mice were trained to remain on a rotating rod (16 revolutions per minute, Stoelting Company) for a maximum period of 100 seconds for two consecutive trials, modified after Rinnard and Carr procedure (20) . After training the animals were administered drug and again tested on the rotating rod . Motor deficit (TD50) was determined by a 50% reduction in the time the animals remained on the rod as compared to control time . Inclined screen test (rat) . After drug administration each animal was placed in the center of a 14" x 16" wire mesh screen (1/2" hardware cloth) at an angle of 60 degrees for a period of 60 seconds. Motor deficit was determined by the length of time the animals remained on the inclined screen . RESULTS Audi ogenic seizures Tonic extensor component . All compounds exhibited marked activity in preventing the hind-limb extensor component of the audiogenic seizure (Table 1) . Thus, delta-9-THC was found to reduce the tonic extension component in fifty percent of the animals (ED50) at a dose of 5 mg/kg at the one hour teat period . At later times (4 and 24 hours), delta-9-THC was considerably leas active . In contrast, DMHP was found active over a prolonged period (24 hours) with ED50 values of 6 .7, 2 .9, and 15 .1 mg/kg at one, four, and 24 hours, respectively . The new compounds exhibited potent activity at both the 1 and 4 hour periods but less at the 24 hour teat time . Maximal effects were seen at the four hour teat period with SP-141 with an ED50 of 2 .8 mg/kg, SP-143 with an ED50 of 2.4 mg/kg, and SP-175 with an ED50 of 1 .1 mg/kg . By way of comparison, diphenylhydantoin was quite effective at one and four hours and less so at 24 hours, the peak effect was seen at one hour with an ED50 of 1 .7 mg/kg . Supramaximal electroshock studies. In mice the test compounds were found to be less potent than diphenylhydantoin in antagonizing the tonic extensor component of supramaximal seizures (Table 2) . Thus, delta-9-THC showed peak effects at one hour with an ED50 of 31 .9 mg/kg, while DMHP peaked at four hours with an ED50 of 57 .2 mg/kg . The new compounds (SP-141 and SP-143) showed maximal effects at four hours with ED50 values of 59 .4 and 67 .8 mg/kg, respectively . SP-175, in contrast, was effective both at one and four hour teat periods with ED50 values of 68 .3 and 75 .9 mg/kg . In rats, DMHP and the new compounds appeared to be more potent than diphenylhydantoin in preventing hindlimb extension, whereas delta-9-THC was found to be approximately equal in potency to diphenylhydantoin (Table 2) .
Vol . 17, No . 1
Benzopyrana-Anticonvulsanta
100
TABLE 1 EFFECT OP VARIOUS BE120PY0.AN LOMPg1NUS ANO DIPH¢NVI~Uf0A1fT01N (DPN) OR AUDIOf.F11IC SEILDRES IX MICE
SP /Ib .
1 hr FDSO+ e8/kg (95S C.L .)
4 hr E050+ v¢/k8 (95f C.L .)
26 hr E050+ eR/kR -(95L C.L . _--_
SP-1D4 19-THC
5.0 2 .4-77
SP-g OIWP
6.7 2 .1-11 .0
2 .9 1.8- 4.9
15 .1 8.2-26 .7
SP-141
8.5 5.1-17.8
2.8 1 .4- b.g
32 .7 20 .0-4E .B
SP-]47
4.3 2.1- 8.0
2.4 1 .2- 5.5
19 .1 12 .5-27.0
SP-175
6.8 4.1-11 .7
1 .1 0.5- 2.4
65 .9 27 .0 - -
DPX
1.7 1 .7- 2.5
1.1 2.7- 4.4
11 .0 6.5-21.6
38 .g 29 .1-49 .3
>I00
+ Orel dosa (ng/kg) et ah3eh 5~2 of the eoivele ere protected fro. hindllob eaeaion with tao enlrle per dwe.
At the peak time of four hours, the following ED50's were observed : DMHP, 7 .5 mg/kg ; SP-141, 4 .6 mg/kg ; SP-143, 25 .2 mg/kg ; and SP-175, 7 .4 mg/kg . In contrast, diphenylhydantoin had a peak effect at one hour with an ED50 of 23 mg/kg and an ED50 of 62 mg/kg at four houra .
TABL¢ 2 EPFELT OF VARIOUS BENZOPY0.AN CO1Bff1UND5 AND DIPXENYLHYDANTOIN (DPH) ON 5UPRAt1AKINAL ELEL70 .05NOCK SFIZURES iN XICE AND 0.A73 !Ilce 1 hr ¢D50+ 4 hr E050 eR/k0 . nP./kg (95L (952 (95L C.L .) C.L .) SP/Nn .
SP -104 A 9-THC 9P-B OMIIP SP-lbl SP-1d7 SP-175 DPN
24 hr ED50+ ~ yI/kg C.L.) ~ --
--
Ileb 4 hr ED50+ eg/kg (952 C.L .)
71 .9 29 .1-74.5
301.5 96 .4-106 .6
dD.b 24 .1-142 .7
]10.8 270.7-587 .5
57 .2 75 .1-80.1
> 700
7 .5 7.4-11 .2
>J00
59 .4 41 .6-B1 .4
>300
4 .6 2.B- 7 .2
>J00
b7 .8 b1 .0-150 .3
" )00
25.2 5.7-52 .2
ó8 .l 61 .8 - 78 .7
75 .9 5A .6-I02
> J00
7 .4 4.2-17 .4
8.1 5.2 - 11
7.6 7.0-8 .7
> 20
~
fi2 .2 57 .0-67 .3
+Mrel dose (ag/Wt1 et vhtch 50I o( the enütele ere protected frov hindlivb tonie extenelon trlth ten enivele per dace .
Psychomotor Seizures . No significant activity was observed with the teat agents in reducing facial and forelimb clonua induced by low frequency electroshock except for alight activity with DMAP at high doses (Table 3) . Pentylenetetrazol seizures . Maximal seizures (tonic eateneion) . All of the test agents effectively reduced the incidence of tonic eateneion with the exception of delta-9-THC (Table 4) . DMHP was more effective than delta-9-THC or the new compounds SP-141 with ED50 values of 9 .5 and 10 .2 at the one and four hour test periods .
Benzopyrans-Anticonvulsants
Vol. 17, No . 1
TABLE 3 EFFECTS OF VARIOUS BENZOPYRAN COMPOUNDS AND DIPHENYLHYDANTOIN (DPH) ON PSYCHOlSYfOR ELECTROSHOCK SEIZURES IN MICE
SP/No.
SP-104 ~9-THC
4 hr ED50* mg/kg (95X C.L .)
>300
SP-8 DMfD?
SP-141
186 .3 (estimated)
SP-143
SP-175
>300
>300
>300
DHP
>240
*Oral done (mg/kg) at which 50X of the animals are protected from facial and forelimb clonus . and SP-143 were moat effective at one hour with ED50 values of 20 .2 and 31 .1 mg/kg . SP-175 was effective at both the one and four hour teats with ED50 values of 41 .8 and 38 .2 mg/kg . Minimal seizures (clonus) . No significant anticonvulsant activity with the test agents was observed against minimal (Clonus) seizures, with the exception of DMHP and SP-141 (Table 4) . TABLE 4 EFFECT OF VARIOUS BENZOPYRAN COMPOUNDS AND DIPHENYLHYDANTOIN (DPH) ON PENTYLENETETRAZOL SEIZURES IN THE MOUSE (TONIC E7CTENSION AND CLONIC SEIZURES)
SP ./No . _ SP -104 9-THC SP-8 DMfD' §P-141 SP-143 SP -175 DPH
Tonic Eateneion 1 hr ED50* 4 hr ED50* m8/k8 m8/k8 (95z C.L .) (95z C.L .)
i ~ i
300 9.5 3.5-21 .6 20 .2 10 .0-41 .0 31 .1 15 .6-62 .6 41 .8 22 .0-90.6 2.7 1 .7- 5 .9
'
> 300 10 .2 5 .3-22 . 3 54 .9 29 .4-113 52 .1 34 .5-74 . 7 38 .2 16 .2-90 .0 2 .2 1.8- 2 .7
i
Clonic Seizurea 1 hr ED50* 4 hr ED50* m8/kg m8/k8 (95X C.L .) (95X C .L .) > 300
> 300
~
100-50X
>100
~
100=70Z
>300
~
>300
>300
~ ~ ~
>100
>100
>100
>100
*Oral done (mg/kg) at which 50% of the animals are protected from hindlimb tonic extension with ten animals per dose .
Rotarod (mice) . Therapeutic indices (TD50/ED50) were obtained when the anticonvulsant activity of the teat agents against audiogenic seizures in mice was compared to the neurotoxicity of compounds tested on the rotarod (Table 1 and 5) . At the four hour test period, delta-9-THC, DMBP, SP-141, SP-143, SP-175, and diphenylhydantoin showed ration of 2,7,5,8,18, and 36, respectively .
102
Henzopyrana-Anticonwlsants
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TABLE 5 EFFECT OF VARIOUS BENZOPYRAN COMPOUNDS AND DIPHENYLHYDANTION (DPH) ON ROTAROD PERFORMANCE IN MICE SP/No . 4 hr TD50* mg/kg (95X C.L .)
SP-104 A9-THC 82 .8 46 .6-321 .7
SP-8 DMHP 21 .0 3 .4-39.0
SP-141
SP-143
15 .7 0 .3-31.8
16 .6 7 .1-42.8
SP-175
DPH
20 .1 119 .7 11 .5-29.5 82 .3-164 .7
*Oral dose (mg/kg) at which a 50X reduction occurs in the time the animals remain on the rotating rod. Inclined screen teat (rat) . At peak time of anticonwlsant activity against supramaximal electroshock (four hours) no impairment of grip strength on the inclined screen was seen with either DZ4iP or SP-175 at doses up to 1280 mg/kg . (SP-141 and SP-143 at doses up to 640 mg/kg) . DISCUSSION The present study represents the first investigation of the profile of anticonwlsant activity of new benzopyran structures . In reviewing the anticonwlsant activity of delta-9-THC, it becomes apparent that it resembles diphenylhydantoin in profile (short duration) while DMaP appears to resemble trimethadione in profile (pentylenetetrazol antagonism) . Thus, our new analog, SP-175, may represent a therapeutic advance in providing a longer duration of action than delta-9-THC and a diphenylhydantoin-like profile . In particular, SP-175 was found to be more active than diphenylhydantoin against supramaximal electroshock in the rat . DMHP and delta-9-THC in animal and human studies has been found to exhibit significant side effects, not only in terms of neurotoxicity, but also with respect to cardiovascular parameters (22,23,24) . Most investigators have used the intravenous or intraperitoneal route of administration with delta-9-THC and DMHP (4-16) . The new compounds, particularly SP-175, appear to exhibit a low degree of neurotoxicity by the oral route, particularly in the rat . It is our hope that this new compound will be evaluated in higher species, both alone and in combination with other known anticonwlsant agents . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 .
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We would like to express our appreciation to Dr . Harvey Rupferberg and the Anticonvulsant Advisory Committee of the National Institute of Neurological Diseases and Stroke for their encouragement of this study (supported in part by contract NO1-NS-3-2314) . The authors are indebted to Dr . R. K . Razdan, B. Zitko Terris, and H. G . Pars of The John C . Sheehan Institute for Research and Sharps Associates (SISA), Cambridge, Massachusetts, for the original chemical preparation of the three derivatives of DMfD? (25) .
