426
Action of Some Steroids on the Central Nervous System of the Mouse. 11. Pharniacologj-
The intravenous effects were inveatigatetl; 6; ui' t i l t s pregnanea had hypnotic activity. Some of the steroids produced conviil>ions. Certaiii htl.iictiire-activit- relationships were apparent ; these partly supported the concliisionr of earlier workers. One coinpound, aa-hydrosy5p-pregnane-11,20-dione 3-phosphate disodium. tried in man :is a n iirtrnvenoii. nnestheticn, prodiicaed :in 1111pleasant, paresthesia.
Under various experiiiieiital cwndit iotis soiiie steroids are aiiesthetics,1-3 arid one of theni. hydroxydione, has been used ch~iically.~This held \vas reviewed hy TTitze1,j who suniriiarized results oii 124 steroids. Later OverbeekGreported the effects of other steroids o i i the central nervous system, arid soiiie of them n.cw anesthetic. JTe present here the results of studies oii iiiicac of tlie hypnotic and other effects on the cwitral iicrvous systell1 of l(i8 steroids, 149 of which had not previously hceii tested in this way. The teriii hypnotic is prcferred to anestheti(8, berauw soiiic steroids induce light s l q in ~ iiiicc without surgicd atieithesis.
Methods C:roups of 3-10 male fawn mice (GFFatrain: body weights 16 to 22 g , ) were injected intravenoiraly with the steroids, irsiially presented a t l$>$concentration, either dissolved in water or sti+ pended in physiological Faline rontaining 0.4'; Tween 80: occasionally, when the iteroid had low tosicit higher concentrations were used. T h e suspen I)y grinding the steroids with the vehirle in a glass tissrie homogenizer. The compoiinds were administered in doses ranging from ineffective to hypnotic or lethal levels. K h e n hypnosis resiilted, the sleeping animals were plared in a cabinet maintained at 3 S o , ;ind the times bet\Feen beginning the injection and loss of t h r righting reflex ("induction time") and between 11 of the righting reflex ("deep time") were recorded. h grCJLlp mean indiiction time of 0.1 min. indicnte.; th:tt :ill the mire w ~ f r i ~ :iqleep a t the end of injection. For some sternids t h e intraveriii~rs I , l h v:tllie.< were deterrriiiied o n griJiip.G of 3-13 ri1ic.e: for otheri the anlolliit nvai1nt)lr. permitted tlie determiriation of onl>- upproximate vnlires. flJl rnch t r f their hypiioti(. F'igdiir and co-worker-3 tlcterrrii ing 1i:ilf the mire to lose i . ~ . the , dose Yteroids the their righting reflex. R e preferred t o detrrrnine the d o y e 1 ha^ indnced sleep for 25 min. This titirution FV:L- i.how1 h e c u i i s r with group mean sleep times below I5 min. some I I the ~ mice did r r o t sleep; with time3 o v e ~ niiii. t i i c wuiance rif the reqion-cJtended to t w high. The %-n>iii. -IwI)-tirne (low :uid t h e ) ('orotic sirw,id from itie i ~ i r v e iiig logarithrnh i ~ i ' t l i n r s 111 rises. For h>-pnotic+toxic at LIIJM+ tielow the 25-niiii. & ~ > 1 1 dose the in:txiniiirr niea3iir:ihle rlt'ep t inie and t h e corre>pondiny dose and indiict ion time were ti~iiallyrecorded. (St eriiidh with siic.h ICIW t h e i ~ : ~ p c ~ iindices tic SPerned trnlikply tcJ he of prartitxl 1~31nt
CNS-ACTIVESTEROIDS. I1
July 1965
been independently reported to be a hypnotic.6 The nialeate 94, the sulfate 95, the phthalate 96, the niorpholinoacetate niethiodide 100, and the diethylaniinoacetate ethiodide 99 had no hypnotic activity; indeed 99 caused convulsions. All of the other esters of 88 were less active than their parent steroid, some markedly so. Like the three esters of 64, the acetate 89, the succinate 90, the phosphate 91, the glutarate 92, and the diglycolate 93 had longer induction times than the corresponding steroid alcohol, but the hypnotic ainino esters 97, 98, and 101 had induction times approximately equal to or shorter than that of 88. Coinpourid 101 had unusual properties. I t mas the least active for which a 25-min. sleep dose could be deterniined, yet, unlike other Tveakly active steroids, it induced hypnosis of a satisfactory quality, with smooth induction and recovery. Thus, we agree with the conclusions of E'igdor and co~ o r k e r s 3 :the 3-esters of hypnotic steroids are in general less potent than the free alcohols; the latter, especially the 3a-ols, have short induction times; esterification, other than with anlino acids, lengthens induction. They suggested that aniino acid esters of steroids niay enter the brain unhydrolyzed, adducing as evidence the high toxicity of such compounds; but 101 in our series had a low toxicity. I t niay be relevant that glycinates of antiinflaniiiiatory steroids have been reconinierided for their specific effect on the central nervous system8 The 3a-hydroxy steroid 30 and its succinate 31 had approxiniately the same hypnotic activity as each other and as their Sp-epimers 88 and 90. Seither 3ahydroxy-5a-pregnan-20-one (the 11-desoxy derivative of 30) nor 3P-hydroxy-5~-pregnane-ll,20-dione appear to have been tested for hypnotic properties, and they were unfortunately not available to us. However, we found that 103 (the 3a-methyl derivative of 30-hydroxy-5~-pregnane-l1,20-dione) was iliac tive, whereas its %epimer 102 was hypnotic, with a short induction time. Though we have not usually recorded acute toxicity results for water-insoluble steroids, it was noted that 102 was much more toxic than 103. The results with these compounds contrast with those of Langecker and Busch5 for 17 a-ethynyl-3a-niethyl-5a-androstane36,176-diol: this was a hypnotic, yet its less toxic 3epimer was a convulsant. The 36-hydroxy steroid 3 caused convulsions, and its succinate 4 was inactive; other derivatives of 3 (5-12) also had no hypnotic activity, and several of them were convulsants. The 5P-epimer of 3 (68) had no hypnotic activity in sublethal doses; yet 69, its succinate, was a fairly potent hypnotic. Similarly, although 32 was inactive, its succinate 33 was a weak hypnotic and, exceptionally, its phosphate 34 was even niore active. Compound 68 has been reported by other workers as having hypnotic proper tie^.^ We did not control the particle size of our suspensions. Though this may explain the unusual apparent production of hypnotic activity by esterification, it could not explain two other disagreements between our results and those of Laubach's group.3 First, we found the soluble succinate 69 much inore hypnotic than they did; second, they reported 4 as hypnotic, though we have found it inactive. Totwithstanding the doubt as to the properties of 4, 32, (8) J. Tamm and
K-n. Voigt, Acta Endorrznol.
S v p p l . , 64, 1 (1960).
42;
and 68, it is apparent that the most potent and rapidly acting coinpounds of this group are those with an unesterified 3a-hydroxyl, whatever the configuration a t C-5. 111some of this group of pregnanolone derivatives, the effects of 16-methylation have been studied; the results do not support Witzel's general conclusion that alkyl substituents diminish hypnotic activity.j The 16a-methyl derivative 72 was about half as potent as its parent steroid 64. The induction time for 72 (4.1 min.) was one of the longest we recorded for steroids with a free 3-hydroxyl group; nevertheless its slightly iiiore potent succinate 73 had a still longer induction time. 16a-;\Iethylation had different effects on the 11-oxo steroid 88. The steroid alcohol and succinate 104 arid 105 werc more potent ihari 88 and 90, although the phosphate 106 had the same hypnotic activity as 91. lGP--lIethylation of 88 (107) markedly increased its potericy to that of the niost potent steroid 64. Likewise the succinate 108 was more potent than 90, but not as potent as the succinate of 64. 16,16Dimethyl substitution on 88 and its succinate 90, yielding 110 and 111, respectively, had little effect on potency; 40, the 166-methyl derivative of 32, was, like the latter, not a hypnotic, but the succinate 41 produced ataxia, as did the 16a-methyl succinate ester 39. The 17-epinier (42) of 41 produced convulsions. Other substituents on the steroid nucleus found to result in loss of hypnotic activity were 12a-acetoxyl (compare 70 with 64; 71 with 65), 17a-hydroxyl or -acetoxyl (compare 45 and 46 with 33; 113 with 88), 17a-hydroperoxyl (compare 74 with 64; 112 with 88)) 160-hydroxyl (coinpare succinate esters 37 and 33), and lBa,l7a-epoxide (compare 38 with 33). The 20-ethylene ketal of 90 (129) wah alniost as potent a hypnotic as its parent, h i t it had an increased induction time and was about twice as toxic. -4 9(11)-ethylenic bond, present in 14 and 15, appeared to introduce weak hypnotic activity into 3, but in 77, whose saturated parent 66 was highly active, the presence of hypnotic activity was doubtful: with such toxic cornpounds it is difficult to differentiate loss of the righting reflex and the prostration that precedes death. 16,17-Unsaturation (35) abolished the hypnotic activity of 33 and reduced activity in the 16-methyl steroid 109 (compare with 107). 21-Benzylidene substitution (125) in 90 substantially increased the toxicity. Halogenation at C-21 (conipare 126 with 88; 127 and 128 with 89) either decreased or abolished hypnotic activity. The 21-hydroxyl derivatives of the pregnanolones are discussed below. Pregnanedione Derivatives.-5P-Pregnane-3,20-dione (78) was a potent hypnotic, but it had a long induction time; its 11-oxo and 11(12)-unsaturated derivatives 130 and 87 were less potent. 6a-Methylation (79) abolished the hypnotic activity of 78. saPregnane-3,20-dione (21) and its l l-oxo derivative 51 caused convulsions. The former has been previously reported to be hypnotic, with much less activity than its 5P-epimer.' a Coinpound 22, the 6a-methyl derivative of 21, and 52, the l(2)-unsaturated derivatiw of 51, were inactive. 21-Hydroxypregnanedione Derivatives.-21-Hydroxy-,56-pregnane-3,20-dione (81) had, within the
>"OO
42 IiJ
(13
>2 2 (i
44,i C'U.
300
July 1965
429
CNS-ACTIVESTEROIDS.I1 TABLE I (Continued) Induction time 25-min. (min.) a t sleep 25-min. dose, sleep dose mg./kg.
Mean
KO.
55b 56 * 57 58 59 60 61 62 63
Steroid
Other 11-substituents 110-Hydroxy-50-pregnane-3,20-dione 1la-Hydroxy-5a-pregnane-3,20-dione 11-acetate 3p, 1lp-Dihydroxy-5a-pregnan-20-one3-hemisuccinate sodium 3p,llp,170-Trihydroxy-5a-pregnan-20-one 3-hemisuccinate sodium 50-Pregnane-36, 1lp, 20p-triol3-hemisuccinate sodium 11p-Hydroxy-5~~-pregnane-3,20-dione 9a, 1la-Epoxy-3&hydroxy-5a-pregnan-20-one 90,l 10-Epoxy-3P-hydroxy-5a-pregnan-20-one3-hemisuccinate sodium 9p,l lp-Epoxy-3p-hydroxy-50-pregnan-20-one
.Activityo
0 H 0 0 0
140
Llh, mg./kg
5.0 170 660 40
0 C C
Ca.180
0
5P-Pregnanes 11-Unsubstituted, 3-hydroxy 3a-Hydroxy-5p-pregnan-20-one (37) 3~u-Hydroxy-5p-pregnan-20-one3-acetate 30-Hydroxy-5p-pregnan-20-one 3-hemisuccinate sodium (38) 30-Hydroxy-5p-pregnan-20-one 3-phosphate disodium 3@-Hydroxy-5p-pregnan-20-one( 35) 3p-Hydroxy-5p-pregnan-20-one 3-hemisuccinate sodium (36) 30, 120-Dihydroxy-5~-pregnan-20-one12-acetate 30,12a-Dihydroxy-5p-pregnan-20-one3,12-diacetate 3~u-Hydroxy-160-methyl-5p-pregnan-2O-one 3~-Hydroxy-16a-methyl-5p-pregnan-20-one3-hemisuccinate sodium 17a-Hydroperoxy-30-hydroxy-5~-pregnan-2O-one -r 3a,21-Dihydroxy-5p-pregnan-2O-one (79) 1.3 l-Dihydroxy-5P-pregnan-20-one 2 1-acet at e 301~2 i6* 30-Hydroxy-5p-pregn-9( 11)-en-20-one 3-hemiauccinate sodium 77 11-Unsubstituted, %OXO 5p-Pregnane-3,20-dione (44) i8” 6cu-Met hyl-5p-pregnane-3,20-dione i9 1ia,21-Dihydroxy-5P-pregnane-3,20-dione 2 1-hemisuccinate sodium ( 108) 80 21-Hydroxy-5B-pregnane-3,20-dione (85) 81b 21-Hydroxy-5p-pregnane-3,2O-dione 21-acetat e 82h 21-Hydroxy-5p-pregnane-3,20-dione 21-hemisuccinate sodium (hydroxydione, 86) 830 21-Hydroxy-5p-pregnane-3,20-dione 21-phosphate disodium 84 21-Hydroxy-5P-pregnane-3,20-dione P1,P2-bis(21-pyrophosphate)disodium 85 21-Hydroxy-5P-pregnane-3,20-dione 21-benxylphosphate sodium 86 5p-Pregn-ll-ene-3,20-dione 87 11-Oxo, 3-hydroxy 3a-Hydrox~--3p-pregnane-ll,20-dione 88b 3a-Hydroxy-.5g-pregnane-l1,20-dione %acetate 89h 30-Hydroxy-50-pregnane-1 1,20-dione 3-hemisucrinat e ?odium 90 3cu-Hydrosy-5B-pregnane-l1,20-dione 3-phosphate disodium 91 6-pregnane-1 1.20-dione 3-hemigluterate sodium 92 3a-Hydroxy-6P-pregnane-l1,20-dione 3-hemidiglycolate sodium 93 30-Hydroxy-fjp-pregnane-l1,20-dione 3-hemimaleate sodium 94 3-sulfate sodium 9.i 3a-Hydroxy-5p-pregnane-ll,20-dione 3a-Hydroxy-5p-pregnane-ll,20-dione 3-hemiphthalate sodium 96 3a-Hydroxy-Sp-pregnane-ll,20-dione3-aminoacetate hydrochloride 97 3a-Hydroxy-5P-pregnane-1 I ,20-dione 3-diethylaminoacetat e hydrochloride 98 3~~Hvdroxy-.jp-pregnane-ll,20-dione 3-diethylaminoacetate ethiodide 99 3a-Hydroxy-Sp-pregnane-ll,20-dione3-morpholinoacetate methiodide 100 3a-Hydroxy-5P-pregnane-l1,20-dione3-hemi-iY-acetyl-~-glutamatesodium 101 3a-Hydroxy-3p-methy1-.iP-pregnane-llj20-dione 102 3p-Hydroxy-30-methyl-5B-pregnane-l1,20-dione 103 30-Hydroxy-16a-met hy1-5p-pregnane-11,20-dione 104b 30-Hydroxy-160-met hyl-5p-pregnane-l1,20-dione3-hemisuccinate sodium 105 3~-Hydroxy-l6a-methyl-5p-pregnane-l1,20-dione 3-phosphate disodium 106 3a-Hydroxy-16B-met hy1-5p-pregnane-l1,20-dione 107 3a-Hydroxy-16p-methyl-.ip-pregnane-ll,20-dione 3-hemisuccinate sodium 108 3a-Hydroxy-16-met hyl-5p-pregn-16-ene-11,20-dione 109 3a-Hydroxy-16, 16-dimethyl-L5P-pregnane-l1.20-dione 110 30-Hydrox~--l6,16-dimethyl-5p-pregnane-ll,20-dione 3-hemisuccinat e sodium 111 170-Hydroperoxy-3~-hydroxy-5~-pregnane-l1,20-dione 112b 64* 6200 62
is
205 390 175 Ca. 140
350 465 330 325 800 >600 125
>
75
125 13 25 0.2 625 0 . 1 Ca. 40 Ca. 150 0.5 7.1 130 5.8 260 0.8 2.6 160 hllll
limits of cxpriiriieiital eiror, tho sariir hypnotic acti\ ity as the. iiiost potmt of our ~ c i i ( ~64 s. Itb iiidiit*tioii time \vas lengthy: such steroids as this and thc. 3-0~0 steroids a b o w dciiioristrate that, though ail unesterified 3-hydroxyl usually cmifers rapid induct ion. this property 14 iioi ihared hy t h c 21-hydroxyl or 3-oxo groups .4t lc,ast soiii(l of the hypnotic* ac*livitg oi :idiivc.r(>not f ~ ~ i d Coiiipourid 82, thc ?l-acelatc> of 81, was only one third ab aclivr as tlicl parent alvoliol, n hereas the su(+riiiat c (hydroxydiorie 83) was aliiiost equally active. We f(JU11d 84, the phosphate of 81, l o be aliiiost as a(*tivc and about half as toxic. as hydroxydioiie; Iniischr1r1"clairiied that the phosphate is inore active thaii the sucrinate. The bis steroid pyrophosphate 85 was alinost inactive aiid inarkedly toxic, tiit, benzyl phosphatr 86 c w i h r t l I'oIIvuI5Iol1z. 'l'he plopertleh 01 O t I l ( ' 1 ( + l ( ~ l h0 1 81 11:1\ c , j
l ) l Y > l l rc>port( d . 3 '11
110
I 1 I nnmi h t i I r f r i ktidoi i,tol 3 0 , i b 0 19i'il ,1 1 1 r n . r l 1 ~ 1 CI?ri! l i d ( 1 < d i , n l I o i i ~ 1 0 0 1
July 1965
C?\IS-i~CT1V13 STEROIDS.
11
43 1
TABLE I1 OTHERSTEROIDS LACKING HYPKOTIC ACTIVITY
143 144 145 146fl 147 148 149 150 151= 152a 1535 154
155. 156" 157" l5@ 159a 160" 161 162 163 164 165 166 167 168 a
Androstane Compounds 5a-Androstan-3~-ol3-hemisuccinatesodium 3(~'-Hydroxy-5a-androstan-ll-one3-hemisuccinate sodium 36-Hydroxy-5a-androstane-1 1,17-dione 3-hemisuccinate sodium 3P-Hydroxyandrost-5-en-17-0ne3-hemisuccinate sodium D-Homoandrostane Compounds 3p-Hydroxy-D-homo-5a-androstane-11, lia-dioneb 3~,17~-Dihydroxy-17p-methyl-D-homo-5a-androstane-11, lia-dione 3P,lia-Dihydroxy-16@,17~-dimethyl-D-homo-ja-androst-9(11)-en-17a-one 3p,l~o-Dihydroxy-16a,li~-dimethyl-l6~-flu0ro-~-homo-5a-androstane-ll~lia-dione 3-acetate 3~,17ay-Dihydroxy-17p-methyl-D-homo-5p-androstane-11,l7a-dione~ 3~,17a~-Dihydroxy-l~a~-methyl-D-honio-~~-androstane-ll,li-dioiie 3a,17a~-Dihydroxy-l7a~-methyl-D-homo-5p-andr0stane-11, 17-dione 3~,17ap-Dihydroxy-17a~-methyl-~-homo-5p-androstane-l1,1i'-dione 3-heniisuccinate sodium* Rtianic Acid Compounds Sodium 3-oxo-5a-etianate Sodium 3p,lia-dihydroxy-ll-oxo-5a-etianate Sodium 3-oxo-5P-etianate Sodium 3a, 12a-dihydroxy-5p-etianate Sodium 3-oxoeti-4-enate Sodium lia-hydroxy-3-oxoeti-4-enate Miscellaneous Compounds Estra-5,7,9-triene-3P,17P-diol 3,li-bis(hemisuccinate sodium) 17a-Methylestra-5,7,9-triene-313,178-diol 3-hemisuccinate sodium :3p-Hydroxyestra-5,7,9-trien-li-one 3-hemisuccinate sodium 16a-Acetyl-3P-hydroxy-l6p-methyl-5a-androst-9( 11)-en-li-one 3-acetate 16p-Acety1-3p-hydroxy-l6a-methyl-5a-androst-9( 11)-en-17-oneb 16~-Acetyl-3p-hydro~~-l6~-methyl-5a-androstane-ll,17-dione 3-acetate 11)-en-17-oateh Sodium 16~-acetyl-3~-hydroxy-l6~-methyl-16,17-seco-5a-androst-9( 11)-en-17-oate Methyl 16~-acetpl-3p-hydroxy-l6~-methyl-16,li-seco-5~-androst-9(
The preparation of these compounds is described in the literatrire: for the otherq, see the preceding paper.
some of these and some of their 11-oxo derivatives, as both free alcohols and esters. 3a,21-Dihydroxy-5/3-pregnan-20-one (75) was almost as hypnotic as the corresponding 3-OXOand 21deoxy coinpounds 81 and 64; its induction t h e was between those of 81 and 64. The 21-acetate 76 was only half as potent as the parent steroid, but its induction time was also halved. The 3-epimer of 75, and some of its esters, have been reported as less hypnotic than 75.3 Compound 114 (the 11-oxo derivative of 75) mas inactive, like its %oxo counterpart 132. The 3-acetate of 114 (115) was weakly active, with a long induction time. The 21-acetate 116 had a short induction time and was much inore potent, not as potent as its 11-deoxy derivative 76, but niore so than its 3-oxo analog 133. The 3,21-diacetate of 114 (121) was too toxic for adequate testing; however, it was clearly hypnotic and had a long induetion time, as did the corresponding 3-succhinate 21-acetate 123. The 21-succinate 117 and the 21-phosphate 118 of 114 were weakly hypnotic and, despite their unesterified 3a-hydroxyl groups, their induction times were long. However, the more potent water-insoluble 21ester 119 had, like the 2l-acetate, a short induction time, indicating that I t must have dissolved rapidly in the bloodstream. I t is possible that 114 is too polar to pass readily into the brain, and that its 21-succinate and -phosphate have little hypnotic activity because they too are polar or because they are readily hydrolyzed in the bloodstream ; the less polar, less readily hydrolyzed 21-esters may pass as surh into the brain,
* Convulsant.
there to act by virtue of their unesterified 3a-hydroxyl groups. The 21-niethanesulfonate of 114 (120) produced only ataxia, arid the 3-acetate 21-methatiesulfotiate 122 was inactive. Thus not all the wal er-insoluble 21-esters of 114 were hypnotics. The introduction of a sulfur atom into 116 (124) coilsiderably increased its potency and gave it peculiar properties. Mice dosed with 124 appeared to experience an intense skin irritation after recovering the righting reflex : they scratched themselves vigorously, and sonie circled madly inside their cages. Some had inadequate control of their hind limbs for the first 10 min. after awakening. 3/3,21-Dihydroxy-5 a-pregnan-20-one has apparently not been tested though its 21-succinate and 3-acetate 21-succinate have been reported; we too studied the 3-succinate 21-acetate (13), m-hicah was toxic hut distinctly hypriot i ~ . ~ 3/3,2l-Dihydroxy-~cu-prcgnatie-11,20-dione has been reported's2 to be weakly hypnotic ; under our conditions its 3-succinate 21-acetate 48 arid two esters of its 17a-hydroxyl analog (49 and 50) were inactive. 11-Substitution.-Twenty-six pairs of compounds in our series illustrate the effects of 11-oxo substitution, which will now be summarized. I n three instances (1, 26; 3, 32; and 10, 37) the 11unsubstituted steroid only and in two instances (12, 44 and 21, 51) both the 11-unsubstituted arid the 11-oxo steroid were convulsant; no 11-oxo steroid caused cotivulsions unless its 11-deoxy derivative also did. In only three of the pairs (1, 26 ; 4 , 3 3 ; and 11,39) had the
