Mono- and Difluorination of Steroids at C-16 through Enamine Intermediates
(1) C. H. Kobiuson, K . I'. Briicr. 12:. 1'. Oiil f i t i i . S. Tolkdsorf, R1. Zjtrinbiiry and p. 1.. l,e,l111an,J. A n . C h e n . Soc., 82, 5 2 5 0 (19UOj. IGssnian, !I. A. S. Huffnian, a n d A i . J . JVpiss, J . O r g . C h e m . , 26, 973 ( I R I ~ I ) . (2) H. & (3) R. B. Gabbard a n d E. I'. .TrnsPn, ibid.. 23, 14OG (14.78). 76, 1918 (1953). (4) 3 . W. Hey1 a n d M. I). Herr. .I. A m . Cirem. SOC., ( 5 ) AI. D. Herr a n d F. I T . Hey1 ibid. 715, 39-17 (1953).
thc 1 1-oxo function inflnencci thc. reactivity at C-17. In the cstratrirnc scrim, 16,I (j-difliioro-n-melliox~cstra-1 ,3,>(10)trien-17-one (I) was prepared as shown (I-Y). It mas identical with material prepared by the synthetic route reported by Robinson, et d . , l 111ilizing the 16-formyl derivative for the fluorination reaction with perchloryl fluoride. Removal of the fluorine atoms Jyith zinc dust in glacial acetic acid established its relationship to the starting ketone. I n addition, a 16-monotluorinated product (IIa) was obtained which, on the basis of equilibration with its 1G-epimer6(IIb) and its
0
] HOAc
I
& OR
/
CH3O
\
IIIa, R = H b’R=Ac
(0)
0.1’. Nueiler and W. F..Jolins, .I. OTQ.Lhem., 26, 2403 (1961)
rnotlc of' Cormntjon (yrc-iimcd t u - i i ( l ( > :11i:rc.k 01 I;('iOl), h:i\ I W I I :tssigncd the 1GLY-configiiratiori. The equilibration experiments with ( H a ) and (TI\)) ~ v e i ccarricd out in a manner similar to that reported for base-catalyzed epimcr'ization of 16a-brom0-17-oxosteroids.~ Thus, the monofluoro epimers. on treatment with base, led to mixtures whose infrared spectra were essentially identical and showed good agreement with what mould he expected for a mixture of pure epimers; the total crude mixture from the equilibration of the a-epimer contained approximately 437; of the p-epimer and 43yoof the a-epimer based on calculations from the intensities of the bands at 9.94 and 10.42 microns, which are medium-strong bands due exclusively to the LY and p epimers, respectively. These experiments establish the epimeric relationship of the iwo moiiofluoro ketones, though they do riot provide any additional vvidencae for their configurations. Examination of LL nuniher oi cnhloroform spectra of other 16-halo-17-ketonesGalong with the fluoro mmpounds did not suggest any definitive relationship which could bc lmed to correlate configurations of the different halogen derivative10.95. 11 .30, and 1 1 . 5 4 (crutlc) ~B-HydroxZ.aIidrosC-jI O 3.5, 1 0 . 7 5 , en-17-one 11.52 3p-Acetoxyandrost-SI O 2.5, 10.75, en-1 7-one 11 .52 Androst-4-erie-3,l'i-dionc
1013-1luoro
1 ti, l G - l l i l l u ~ , r ~ ~
10:12, 10.7> 10.13, 10,5:3 lO.!~,I 1 . I , I O 1:j, 10 54, I O 7-1. 11.01, 11 :i1I O 2> I O tj:j, I I 22, 1 1 ,5:1
10.12, 10.23, 10 K2, 1 I ox lrB-Hydrosvandrost-4-~,ii-~~-[iii(, 11 13 a Rands noted are in thc: 1 0 11' p portion ~ i ' fingcvyriut region wtic*rcx t niost iiot alilc differences froni llic~p m n t cumpourids occur. Thc sgwtr:i \ver( ~ hands are listcd, the correspontiiiig tlctriv:i(i1,tained in chloroform. J U i ~ r cno tivcs were riot :rvailable. t L ( s
iiormal value of 5.75 p foi the unsubstituted ketone i i ihiftetl t o .i.G8 p in the monofluorofiand to 5.62 p for the difluoro compoiinds.' as previously reported.
In addition, the 16-fluoro substitution in 17-oxosteroids results in more complex spectra in the 10-12 p region. The bands listed in Table I are those which are observed in chloroiorm solution whirh are either not present in, or :ire extremely weak absorptions in, thr parent ketone. The bands noted between 11 and 1 1 ..jp in the :mtlrostene compounds and in the estratriene-17-oncl iiot hearing :til !.-ring substituent (see Experimentd) seem, from these few examples. to be the most generally ovcwring changes ohherved as a result of a-fluorination. Changes in the shapes of b m d 111 thi, region for the 3-methoxy case suggest that fimilar nen nhsorptions have been added, though this is difficult to ascwtnin. Both 16,16-difluoroestra-l,3,5(lO)-trien-17-on(~ and the MI (m1 ketone shoxv a aeries of bands in the 11-12 p i,t.gion uhich arc of lower intensity and at slightly different positions in the unsubstitutrd ketone. Only the two strongest of these absorptioiii :ire liited i n Table I for the difluoro ketone; however, the similarity in t h e tot'il number of bands in this region for the two compounds may indicate' that the C-16 methylene and difluoromethylene groups contribut in a similar way to skeletal vibrations observed in t h k part of tlic, spectrum. Bio-assays.-Preliminary biological studies with Ihe 1ci,16dlf~uoroestratriciiesgive a range from approsimately onc-quarter (k
t o e.cliia1 the artivity of cstrorie in reducing the plasma cMrrtcrol/ phospholipid ratio in rholesterol-fed cockerels.'3 The least artivc was the 3-deoxy compound, 16,18difluoroestra-113,5(lO)-trien-17-one (see experimental section) ; the most active was 16,16-difluoro-17ocethynyl-3-methoxyestra-l,3,5( lO)-trien-l7/?-ol(IV). Feminizing activity was measured by a mouse uterine growth test.14 In this test, 113,16-difluoroestra-l,3,5(l0)-trien-l7-one has greater than 1% the estrogenicity of estrone. Of the 16,16-difluoro3-methoxyestratrienes, the 17-ethynyl derivative (IT.') is representative and has given estrogenic potencies of approximately 4% that of estrone. Robinson, et d . , l have also reported lipid shifting and feminizing activities for compounds in this series as well as for the free phenol, 16,16-difluoroestrone. In their tests, estradiol-176 wab the reference compound. In view of the ranges of activity observed due to non-parallel dose-response curves for the standard and test compounds and correcting for the difference in standards used, thc data obtained in the two laboratories seem to be in essential agrwment. Our studies on 16,16-difluoro-l7~-hydroxyandrost-4-en-3-one havc ihown that this compound is essentially inactive as an anabolic agent (rat levator anz test) and has 2.5y0the androgenic activity of testosterone propionate as measured by its effect on seminal vesicle weight in rats.'j3'6
Experimentall i 16,16-Difluoro-3-methoxyestra-1,3,5(lO)-trien-l7-one(I) and 16a-Fluoro-3methoxyestra-1,3,5(1Oj-trien-17-one(1Iaj.-Estrone methyl ether (10 g.), 15 ml. of 4-pipecoline and I 6 g. of p-toluenesulfonic acid monohydrate were placed in a flask fitted with a Dean and Stark trap (capacity 30 ml.) and a reflux condenser. The trap was filled with xylene and the reaction mixture was heated under reflux for 3l/2 hr. Then 100 ml. of dry xylene was added to the reaction mixture followed by distillation of 60 ml. of xylene over a period of 1.25 hours. During these operations the pot vapor temperature changed from 126 to 139". The reaction mixture waE rooled and 50 ml. of dry benzene and 0.5 ml. of pyridine were added. Perrhloryl fluoiide was bubbled through the reaction mixture for 20 min. while fl3) ( 1 L)
D . L. Cook, R. A. E d g r e n a n d F. J. Saunders, Endocrinology. 62, 798 (1958). R. A . Edpren, Proc. SOC.Esptl. B i d . Med.. 92, 569 (1950). F. J. Saundersand V. A. Drill, ibid.,94, 64h (19.57).
(1.5) ( l t i ) T h e author wishes t o express his appreciation t o Drs. Donald L. Cook and FranriB .J. Snundere and their staffs for the observations reported in this section. (17) AI1 melting points were taken o n a Fisher-Johns melting point apparatus and are corrected. Unless otherwise stated, t h e rotations were determined a t 24 f 2 O in chloroform
and the ultraviolet spectra in methanol. The author wishes t o thank Drs. R. T. Dillon and €f. W.Sause of t h e Analytical Division of G. D . Searle & Co. for the analytical and optical data reported a n d D r . E. G. Daskalakis of the Chromatography Department for the papergranis run during t h e course of this work.
ARTHIJR 8 . GOIJIKAMP
1182
Vol 5
~.ooliiigin :in ice 1i:tth. 'l'iir, rc:siilting Y l J ~ l l t , i O i l was dilui.c:tl witli I I I ~ : ~ I cI l~i l ~~ i~- ~ I ~ ride, (:xtr:tctcd once with dil. I)otasuiuin cnrl)oiiate d u t i u n :illti the inethy1t:iii: chloride evaporated. An infrared spectrum in cliloroform had a band due to thcs difluorinated product at 5.61 p and a band at 5.74 p indicating approximately 50(,:, of unreacted estrone methyl ether. Also, a shoulder at 5.67 p suggested the presence of the 16-monofluoro derivative. The solution of the reaction products iri benzene was diluted with an equal volume of petroleum ether (b.p. 60-71') and chromatographed over i 5 0 g. of silica gel. The column was eluted successively with petroleum ether (b.p. 60-71 '), 50%>benzene-pct,roleum ether,'* 75% benzene-pet.roleum ether, benzene, and finally 57;ethyl acetate in Iienzene. The later fractions of 75yc benzene and the first 3 1. of benzene gave solid residues with melting points in the range 127-131 '. The combined fractions (2.35 g.) were recrystallized from methylene chloride-methanol t,o give 1.70 g. ( I 5 7 0 ) of 16,16-di~uoro-3-methclxyestr~-1,3,5(1~)-t,rien-l~-one~ m.p. 129-131" (report,edl 126-128'); [ a ] ~ , 166.0" (reported' +167", dioxane); A,,,:,, 277.5 Trip ( E 0):ind 286.5 mp ( E 1990); X ~ , ~ x c 5.62, '3 6.22, 6.33,6.63,10.13,10.54, : ~ ~ i 1( i0 . 2 A paper chromatogram of this materi;il showed only one componeiit. .1mi\.11. ( u r o melting point, with material of m.p. 127.5-1 2!)" obtained by 1hf: inethoc~0 1 Rot)inson, et a/.,' was 127.5--I29'. Arrtrl. C:ilcd. for C ; ~ , I - I ~ ~ FC.~ O 71.23; ~: 13, 6.92. Found: 71.44; 1 1 ,
+
('$
7.(J2.
The later fractions of henzcne gave crystalline materials with nielting points in the range CJf 137-160". ilttcmpted crystallization from ether gave 3 gel whicli lost solvent on standing, n1.p. 152-158'. Crystallization from ethanol then gave 210 mg. ( 2 % ) of 16u-fluoro-3-methoxyestra-1,3,5( I O)-trien-li-oiitj :is transparent needles, m.p. 155.5-164.5'; [ a ] u 176.5'; Am, 278 mp (e 2110) and 286.5 nip 5.68, 6.22'7, 6.33, 8.19, 8.69 and 9.94 p . ( E 1960); Anal. Calcd. for C1911?,F0,:C, 7547: H. 7.67: F, 6.28, Fonnd: C, 75.94; H. 7.62: I?; 6.30. .I paper chromatogram of this material showed only one component. Find elution .sit,h 5% ethyl acet,nte in benzene gave 5.47 g. of nnreacted estrone methyl ether.
+
Equilibration of 1601- and 16p-Fluoro-3-methoxyestra-1,3,5(lO)-trien-l7-ones. of 1601-fluoro-3-methoxyestra-1,3,5(lO)-trien-l7-one in 5 rnl. of methanol and 0.2ml. of 8.5 iV aqueous potassium hydroxide was boiled
-A solution of 24.1 mg,
on the steam plate for 3-5 min. and then stirred at room temperature overnight. The solution was then warmed to dissolve some precipitate which had formed and was transferred with ether. After extracting twice with water, the ether layer was dried and solvent removed to give 21.6 mg. of residue. An infrared spectrum of this material (3% in CHCl,) showed bands a t 9.94 and 10.42 p . The first of these is found in the 01 epimer and the second in the p epimer. Calculations from these ba~idsindicated that the mixture contained 4374 of the p epirnt,r and 48% of the 01 epimer. Equilibration starting with the p epimer was carried out using approximatel?. 15 m g . of the pure material.6*lR It was dissolved in 5 ml. of methanol on the steam (18) Removal of solvent from the second fraction (ca. 800 ml.) of 507, benzene-petroleum (.tiler gave a residue which detonated on further heating, suggesting possible formation of perchlorylated by-products. I n a run in which t h e crude product mixture was washed with sod i u m bicarbonate a n d then l N potassium hydroxide, this difficulty was not encountered. It rriny also be desirable t o wash t h e prriduct mixture with a solution of some suitable r p d u r i w
:igent.
November, 1962
C-16 FLUORINATION OF STEROIDS
1183
plate and 0.2 ml. of 8.5 N aqueous potassium hydroxide waa added while stirring rapidly. Heating was continued for 10 min., the solution poured into ether, and the ether extracted 3 times with water. After drying, the ether waa removed to give 16.1 mg. of residue. The infrared spectrum (3% in chloroform) matched closely that of the mixture obtained from the a-epimer. 17~-Acetoxy-16,16-difluoro-3-methoxyestra-1,3,5(10)-trien (IIIb).-To a solution of 440 mg. of 16,16-difluoro-3-methoxyestra-lJ3,5(lO)-trien-l7-one in 2 ml. of 95y0 ethanol waa added a solution of 200 mg. of sodium borohydride in 1 ml. of water and 2 ml. of ethanol. After standing a t room temperature for 10 min. the reaction mixture was diluted with water and the solid filtered off. It &-as dissolved in methylene chloride, the solution dried and the solvent removed to give a glass which resisted attempts a t crystallization. The infrared spectrum in chloroform showed absorption a t 2.74 p and absence of carbonyl absorption. The 17-hydroxy compound (340 mg.) was acetylated in 5 ml. of pyridine and 4 ml. of acetic anhydride overnight at room temperature. Dilution with water gave material of m.p. 120-124" which wm recrystallized from ether-petroleum ether (b.p. 60-71 ") to give 200 mg. (4070 over-all) of 17~-acetoxy-16,16-difluoro-3methoxyestra-1,3,5(1O)-triene,m.p. 130-31"; [ a ] D 21.7'; Amax 278 mp ( E 2110) and 286 5 mp ( 'e 1870); : : A 5.71, 6.22, 6.34, 7.98, 10.28 and 10.50 p. Arkal. Calcd. for CzlHzeF20a: C, 69.21; H, 7.19. Found: C, 69.80, 69.39, H,7.04, 7.07. 16,16-Difluoro-3-methoxy-16,17-secoestra-1,3,5(10)-trien-17-oic Acid (V). A. -To a solution of 250 mg. of potassium in 5 ml. of tert-amyl alcohol was added 2.5 ml. of ether and the mixture cooled in an ice bath. There was then added 60 mg. of 16,16-difluoro-3-metho.~estra-1,3,5(10)-trien-17-one (I) and stirring was continued with cooling for 6 hr. The reaction mixture was treated with 20 ml. of ice cold saturated ammonium chloride solution and 50 ml. of benzene. The layers were separated, the benzene layer washed once with water, then dried, and solvent removed to give a paste. The paste was taken up in ether and extracted with 1 N potassium hydroxide and the extract acidified with coned. hydrochloric acid to give a solid, m.p. 174-176'. Recrystallization from etherpetroleum ether (b.p. 60-71') gave 8.6 mg. (14Oj,) with m.p. 175-178". This material waa identical with that obtained below in attempted ethynylstion, mi.;ture m.p. 174.5-178". B. A stirred solution prepared from 2 g. of potassium in 30 ml. of tert-amyl alcohol and 7 ml. of ether was cooled iu an ice bath, then saturated with acetylenr and 700 mg. of 16,16-difluoro-3-metho~estra-1,3,5(l~)-trien-l7-oiie(I) was added. Stirring and passage of acetylene was continued for 8.5 hr. Ice-rold saturated ammonium chloride solution (100 ml.) and 250 ml. of benzene were then added, the layers separated, and the benzene layer was washed twice with water. It was dried and solvents removed to give 820 mg. of a yellow crystalline solid. This was treated in ether with activated charcoal and recrystallized from ethcrpetroleum ether (b.p. 60-71') to give 510 mg. (69%) of V, m.p. 175-7.5"; [ a ] ~ 63.5'; 2.84, 5.73, 5.87, 6.22,6.33, 9.52, 9.63, 10.08, and 10.40 p. Thr infrared spectrum wm essentially identical, except for the fingerprint region, with that of the unfluorinated acid, doisynolic acid methyl ether.20
+
+
AzXci3
(19) The author is grateful to Dr. G . P. Mueller for supplies of this substance for the oquilibration experiments. (20) J. Heer and K. Miescher, Helv. Cham. Acta, 29, 1895 (1946). The author is grateful t o Dr. John Baran for a chloroform spectrum of this material.
Fotltid: C:, 67.6-1 ririal. Calcd. for C1YH&203: C, 67.44; H, 7.15; E', 11. €1, 7.42; F, 10.60. The material dissolved in di1ut.e sodium hicxrbonute solution on warming and remained in solution :it room t,emperature. Acidification of :t strongly basic solution gave material of 1n.p. 171.5-174.5"; recrystallization from methylencx chloride-hexane raised the melting point to 174-1i7.5"; misturr m.p. with material described above, 175-177.5'. 16,16-Difluoro-17~-ethyn~l-3-methoxyestr~-l,3,5(10)-trien-17B-ol (IV).-Fciiir grams of lithium was dissolved over a period of c a . 1 lir. in 1 1. of liquid ammonia. The total volumc was then brought to 1.5 1. by fiirther addition of ammoilia, acet,ylene b u b b l d through for 30 min. anti 1.35 g. of the 16,16-tlifluoro-17-ketonr (I) added. Passage of acetylene was coiitiniied for 10 min. and t,he ammonia allou-c,d t o evaporate overnight,. Ethcr (500 ni1.j and satur:tttLd ammonium chloride (I00 ml.) were added ait,ll stirring, thc lnyrrs sc,par>tted,and the ethcr solution washed threr. times with n-ttter. Drying and evaporation of the solvent, gave a residue whose infrared spectrum (chloroform) shoived approximately 36Tc conversion to the c+ynyl derivntivr; the c.:rrbonyl absorpt,ion indicated t h:Lt most of the remaining material aiis iiiirwctcd 17-ketone. This matcrial w;ls rr-trcx:rt,ed as follows. A solution of 15 8 . of lithium in 400 nil. of liquid nmmiiiii:i wviis t,rrated with acetylene for I5 min. Tht. crude product niixt,ure was thcu added in 100 nil. {Jf :inhydrous et,her over appros. 20 min. and p:tssage of ncetylene continued for 1 Iir. Ether (400 1111.) xas :idded nnd the reaction inistiire gently warmed until all of the ammonia h:td evaporated, The ether suspension was thrri heated under reflux m-ith r q i d stirring for 2.5 hr. whilfj the bubbling of acet'ylenv was cont,inued. The rwcticin niistiirr was cooled. 100 nil. of cold, saturated a n rnoniiini chloride was addrd. the h p r s separated and the ethrr solution washed twice with water. After drying: rrrnovai of solvent gave 1.05 g. of residue which was chromatographed i!vi:r 100 p. of silica gel. Elution was c-arried out with prtroleum ether (h.1). f i O - i l o : I I . ' ) , 23' twizcrit~ i 2 I.,); 50(,:;; henzene (4 i . 1 : ,or; benzene (41.) and benzenr ' I I I [ ~7;i henaenc~fractions gave 450-500 nig. of rrutle product which was recryst:illizcd fro petrcilouiii &her (b.p. GO-i1 o)-acctono t o givf! 370 mg. (%';&) of I\r which \)i%g:inl o si!ft,tYi 011 the block :it) soo TTith :]I]i);ircnt IIISS of solvrnt, finally nic~lting:it M-!)SC Air-dried inntorial cshihiti:ti l);irti:iI iiii~lfirigfrom 97 to m. 1O.i' n4th gr:idu:il rc,solidific:iticili and changr oi crystal forin, fin:Ll m.p. 121-12.1". If held at 1 LO0, the partially nielted inat~eriaI rirmplrt~rlyresolidified on scratching, m.p. 121-12Xo. When this material W:IF tlried under reduced pressure (1 lir. :it 80' then 2 hr. :it 100" 1, thert, W:IS okit:iinrd 8:10 nig. of cryst,alline mat~crinl,1ii.p. 1 .k:3--lM0(repor 2i.2' ( r q ) o r t d , 1 20", diosnrip I 2.76. 3.0'2, 0 . 2 2 , .1 v u / , Calcd. for C?,HgtIiJ)2: 1:. 1 0 , O i . I ~ t i u n t i :P', I 1I:itc~ialolitaiii(d in :in t.;irlic,r i ~ y ) c ~ r i n i t mJvhicli t 11 ~ hail hern d r I)c'trolrlum cthcr ( k i , p , 6i)biI I : i I o n ~:ind l)rt:ssurn mcltcd :it !&96": ! 6.90; E', 13.7. F'rncticins oljhined in C;OYcl ~ ~ i i z c n( 130 e m y . h;i,l infr:md spcvdrii. wliicl-r iiidi-
Novernher, 1962
NEUROPIIARMACOLOOICALLY ACTIVEA m s o A 4 c ~ ~ s 1 187
catcxl the presence ( J f :L r n o n d u ~ r oderivative (5.68 p ) . The material mdtt:d at 5.68, !J.Y6, 143-1'70". It was sublimed to give 74.7 mg., m.p. 140-167"; 10.52, 10.63, 10.78, 10.95,11.30, 11.54. The only absorption in the 9.9-11.6 p region of comparable intensity in the parent ketone is a t 9.94 p , though approximately the same number of extremely weak bands, slightly shifted, appear. 3~-Acetoxy-16oc-fluoroandrost-5-en-17-one (VIIb).-A solution of 5.0 mg. of 16~-fluoro-3p-hydroxyandrost-5-en-17-one in 2 drops of pyridine a i d 2 drops of acetic anhydride was allowed to stand a t room temperature overnight, then diluted with water and the precipitate collected and recrystallized from methanol t o give 1.7 mg. of VIIb, m.p. 199.5-201.5°. Material obtained by chromatography following acetylation of a crude product mixture from the fluorination of 3p-hydroxyandrost-5-en-17-onemelted a t 201-203'; mixture n1.p. 200-203". The infrared spectra (KBr pellet) were identical; [LY]D +18.8°.z2 Anal. Calcd. for C,iH29F03:C, 72.38; H, 8.39. Found: C, 72.72; 13, 8.20.
Xyaya
8. Nakanishi and E. V. Jensen [ J . Org. Chem., 27, 702 (1062)] recrntly h a w dcscribrd a 10,17-on-17-a1nide. T I I P Y ) report u1.11.205-?O(io, [,Y]I, (CIJC1)a -t 1 4 D . Tlir:ir frcea1rriliolIiad i 1 i . 1 1 . 1 % ? ~ 1 8 :[~n0 ], ~(CIIC:ld (22)
ttic preparation of this compound iising pcrchloryl flrioride and
I- 1s".
The Synthesis of Some Acidic Amino Acids Possessing Neuropharmacological Activity J. C. WATKINS Department of Physiology, John Curtin School of Medical Research, Australian National University, Canberra, A .C.T., Australia Received April 83, 1968 Several new acidic amino acids have been synthesized and tested for neuropharmacological action. N-Methyl- and N-ethyl-n-aapartic acid and D-homocysteic acid have potent excitatory actions upon mammalian and amphibian neurones.
The observation that L-aspartic and L-glutamic acids excite mammalian nerve cells and cause contraction of crust,acean led to an ext'ensive survey of the actions of related substances on mammalian and toad spinal n e u r o n e ~ . ~ -It' ~ was found that Nmethyl-DL-aspartic acid was considerably more potent, both on (1) J. Robbins, J . Physiol. (London), 148, 39 (1959). ( 2 ) 9.Van Harreveld, J . Neurochem., 3 , 300 (1959). (3) D. R. Curtis, J. W. Phillis and J. C. Watkins, J . P h y y i o l . (London). 160, 666 (1960). (4) D. R. Curtisand J. C. Watkins, J. Neurochem., 6, 117 (1960). (5) D. R. Curtis and J. C. Wrttkins, J . Fhusiol. (London), submitted for publication. ( 6 ) D. H.Curtis, J. W.Phillis and J. C. Wntlcins. Brit.J . Pharmocol., 16, 202 (1961).
