Effect of substituting groups on the reduction of the Δ4 bond of steroids

ARCHIVES

OF

Effect

BIOCHEMISTRY

AND

of Substituting

BIOPHYSICS

292-298 (1962)

98,

Groups

on the Reduction

of the A4 Bond

of Steroids’ EDWARD From

L. RONGONE

the Division of Endocl-ine Research, Alton Ochsner Medical of Biochemistry, Tulane Univexity School Depurtment New Orleans. Louisiwna Received

July

Foundation;

and the

of Medicine,

21, 1961

We have previously reported t,hat bovine blood albumin contains an enzyme system which is capable of reducing the 4-5 double bond of several biologically active steroids in the presence of a TPNH-generating system. One hundred micrograms of steroid at pH 6.4 is incubated with 5% bovine blood albumin under nitrogen for 24 hr. The steroid is extracted from the incubation medium by solvents. The 4-5 double bond of androst4-ene-3fl, 17/%diol was not reduced by the enzyme system, suggesting the requirement of a ketone or a-hydroxyl at carbon 3 for the reduction. When androst-4-ene-17@ol, androat-4-ene-3a, 17/%diol and 2a-fluoro-androst-4-ene-3a, 17,%diol were incubated. the 4-5 double bond was not reduced. Incubation of their corresponding 3-keto dcrivatives resulted in complete reduction of the 4-5 double bond. Androst-5-em-3p, 17/Ydiol, 3,%hydroxy-pregn-5-ene-ZO-one, 3/3-hydroxy-androst-5-ene-17-one, and their corresponding 3-keto derivatives were not reduced by the enzyme system. Thus the enzyme system appears to be highly specific in that a 3-ketone is required and the 4-5 double bond is reduced while the 5-6 is not. The 5p orientation is always produced. Chromatography of the extremely crude enzyme preparat,ion on a cabunite column resulted in a 240-fold purification. Paper electrophoresis of the enzyme rrsulted in only one peak which migrated similar to y-globulins. INTRODUCTION

The presence of both a soluble A4-5/3-hydrogenase and a microsomal A4-5a-hydrogenase in male rat livers has been demonstrated by Forchielli and Dorfman (1). Some investigators (2-5) have since studied the rate of reduction of the CX,Pconjugated system of ring A in several steroids. Recently, Forchielli et al. (4) demonstrated pronounced qualitative and quantitative differences in concentration of liver A4-hydrogenases between male and female rats. Evidence was found to indicate that the livers of female rats contain the A4-5a-hydrogenase only, whereas those of male rats contain both the A4-5u-hydrogenase and the A4-5/3-hydrogenase. ‘This study was supported by a grant from dnlerican Cancer Society, Inc.

the

Meyer (5) reported the destruction of t’he conjugated system in ring A of cortisone during perfusion of the adrenal wit’h whole blood and by incubation of cortisone with whole blood, and Wotiz et al. (6) demonstrated a loss of ultraviolet absorption at 240 mp on incubation of testosterone with human serum. Previous studies with commercial preparations of bovine blood albumin demonstrated the presence of a soluble A4-5/3-hydrogenase 1.7) which is dependent on a I’Cduccd triphosphopyridine nuclcotide (TPNH) -generating system. It, was previously shown that loss of absorption at 240 mp of 1-dehydrocortisone and l-dehydrocortisol was significantly less than that of cortisone and cortisol after incubat,ion wit,11 bovine blood alburnin with the TPNH-genernt,ing system. The partial loss of absorp292

YIl~:T.\ROI,ISPII

01: RISG

tion at 240 mp of I-dehydrocortisone and I-dehydrocortisol posed the problem of the mechanism of the metabolism of ring A of the Alll-3-ketosteroids. Caspi and Pcchet I 8) tlcmonstrated both partial and complete hydrogenation of the cross-conjugated system in ring A of I-dehydrocortisol admini+ tcqwl to a female with Addison’s diwase. Their work and previous work of the author (9) suggests the prcfcrential reduction of t,he A” bond prior to the A1 hontl. Later, Vernwulcn and Caspi ( 10,1 sl~owcd that rat liver homogenates convert 1-dchydrocortisol to the natural hormone cortisol. Their work dcluonstrated a species tliffcrcnce in tlic mc+~bolism of I-dehydrocortisol. In this investigation an attempt n-as made to ascertain the effect of suhstituents at various positions on the steroid nucleus. on the rrduction of the double bond in ring A, and to tlctcrmine the specifici@ of the A+-S~liydrogenase. EXPERIMI?ST-AJ, MATERL~LS

AND METHODS

Bovine blood albumin was l~urchasctl from the Difco Ilaboratories (Control 50. 104002) ;’ triphosIllrop>-ridine nucleot,ide (TI’Y) from the Sigma (‘hcmical Company; isocitrate from the Sutrit,ional Biochemicals Corporation. Thr stcl,oids used in thrse cspcriments wre supplied hy yariow I)ll:tl,rrln~eutical research 1al)oratoriw.” All solvr~nt~ \vew rcdistillcd. Emr-im

PURIFIC.~TIOA-

Chromatography was performed on 1500 g. cxblmitc suspended in a glass column 5 x 45 cm. Dist,illed water is run through f,hc cahmiitc unt,il a IIH of 7.0 is ntt,aincd in the clunk. The cwqmc ’ Thr bovine blood albumin is a crude preparat ion which contains several protrins as inclicxted by paper electrophoresis. One may assume that. screral enzymes, which are necessary for the rctluction of the diffrrcnt steroids, are present. Further purification an d characterization of the wtivc romponents arc now being undertaken. ” We wish to thank the following phnrmnceulic~al houses for the gcncrous supplies of strroids: Ciba Pharmaccuticsl Products. Inc.: Met&. Shari) ant1 I)ohme Research Lnborntorics : Schcring Corl)or:rtion; (;. D. Searle and Company: E. R. S(luibb :rnti Son,< : Syntrs, S. ;I,; and The T.Fl)j~h~~ con11;111y.

d OF STKROIDS

x93

(10 g.) i:: made up in distilled water at a COIKTIIt ration of 1% and percolated through the column. Fire grams of inactive material ~~3s eluted from the column with distilled tvater. One gr~am of in:wtire material xTas eluted from the column with 0.05 -11 phosphate buffer. .\ second protein frnction (100 mg.) was eluted Tvith 0.05 X phosphate buff(~l~ actiw. The buff(,r ih tlrvn whic*h was slightl! pl:~wtl in a reservoir of 200 ml. capacity. :mtl :L gratlicmt is run through ttlc reservoir with 0.1 .lI SaCI in 0.1 Jf TaH90& . This immedi:~tely cluted 8 mg. protein which was rstremely a(*1it-t> and rrsultcd in a 240-fold purification. I:urthcl vlution with 2.5 -11 K&l in 0.1 .lI SaHzPO, resultetl in another small fraction of protrin which was inactive. Paler (~le~trophoresin of xi aliquot of the active fraction displayed one pcwli whirh mot-w similar to thr y-globulin fraction of human plasma. Howver, this tloes not, signify that the c~nz~mr ia I,,ire. ISCCBSTIOSS The albumin was dissolved in txvice-redistillwl mater to give an alburnin concentration of 5R, and the pH was adjuskd to 6.4 with LV HCl.’ One milligram of TPN and 1.66 mg. trieodium i-oc,itratc, were dissolved in 1 ml. of this solution, :trItl the JIH was readjusted to 6.4. The steroid substrates (approximately 100 Fg.), dissolved in 0.03 ml. of nh~olute rthanol, wire atlded to 1 ml. of this sollltion. A1n nlicluot, of the steroid solution was :,I,I)rol)riatctly diluted with ethanol, and the c’onwnt ration wa.~ dctcrnmined by ultl,aviolet atworl)tion to obtain the exact amount of steroid atld~tl in each csperiment. The solution of steroid and albumin was incubated for 24 hr. under :I wntinuolls flow of nitrogen at, 37°C. Each experiment was carried out in triplicate with an appropriate blank. The rnzymir rcnction is not linear dllring the 24 Ix. incubation period (i).

After incubation, the protein was precil,itatetl I,- adding 8 vol. of hot ethanol (70°C.) to the misturc. The mixture was filtered and washed with 6 ml. of hot ethanol which was added to the filtrate. The filtrat,e was diluted with water to an ethanol concentration of 70”’ 6 and estrnctrd thrcr times with 0.5 vol. of prtrol~wm cthrr (35-50°C.). Thtl combined petroleum ether extracts ww ~ashctl once with 0.5 T-01. of 707 ethanol, and thi;; washing was added to the, main ckmol soh11ion. The ethanol was removed irk wc,(o in a f&h clvaporntor, and the aqueous Ilhnse ~vas dillltcyl with wat(,r to 15 ml. and c~stwctpd tllwc’ timw

294

KOKGONE

with 30-ml. portions of chloroform. The chloroform extract was distilled in vacua, the rc,sidue taken up in ethanol, and the amount of recovered steroid measured by ultraviolet absorption or Porter-Silber (11) or Zimmermann reactions when appropriate substrates were used. The ultraviolet absorption of the steroid recovered at 0 hr. and at the end of incubation ~-as used to calculate the per cent reduction of the a,~ conjugated group.

ANALYTICAL

PROCEDURES

The R f of the extracted steroids was determined by glass-paper chromatography (12) in chloroformformamide (1.25% formamide), benzene-ethanol (1% ethanol), and petroleum ether (100%) and hexane (100%) systems. Metabolites were isolated by chromatography of the dry chloroform extracts from the incubation media on silica gel columns. Graded mixtures of petroleum ether-chloroform and chloroform-ethanol ehlants were used in the development of the columns. The fractions that contained steroid were combined, recrystallized from acetone-hexane, and identified by glasspaper chromatography, melting points, mixed melting point,s, sulfuric acid absorption curves, and infrared spectroscopy. Melting points were taken on a KGfler micro hot stage and recorded as read. The infrared spectra were obtained from material incorporated into potassium bromide pellets on a Perkin-Elmer model 137 spectrometer. RESULTS

IDENTIFICATIOK FROM

LARGER

OF

PRODCCTS

k.4~~

ISOLATED

INCCBATIONS

Ten milligrams each of 2a-methyltestosterone, testosterone, I-dehydrocortisol, and androst-4-ene-17P-ol were incubated

Compound incubated (a)

2a-Methyltest~o~terone Testosterone

1 -Dehydrocortisol .4ndrost-4.ene-l7p-ol

Compound isolated (b)

2a-Methyltestosterone Testosterone 5a-Dihydrotestosterone 1-IIehydrocortisol Cortisol Androst-4.ene-17@-ol

separately and extracted as described. The dry chloroform residues obtained from the incubat,ion media were chromatographed on rilica gel columns and identified by the methods previously mentioned. The results are summarized in Table I. The substrates and metabolites isolat.ed from t,he incubation media were of such magnitude t,hat crystalline compounds were obtained in all instances. Recoveries of 2a-methyltestosterone and textost~erone from their respective incubations were 94 and 15% as mcasured by ultraviolet absorpt,ion. The percentage recoveries of reduced crystalline compound obt,ained from the 2u-mcthyltesterone and testosterone incubations were 0 and 735&, respectively. After repeated cryst,alliznt’ions of t,he isolated metabolite, ,5/jdihydrotestostcrone, 5.2 mg. of crystalline material was obtained; 6.8 mg. of crystallint substrate, 2a-methyltest,osterone, was isolatecl from its incubation mixture. Eighty per cent, of Porter and Silber chromogens was recovered from the l-dehydrocortisol incubation. Two milligrams of crystalline suhst.ratc 1-dehydrocortisol and 2.4 mg. of crystalline metabolite, cort,isol, were isolated and identified. Isolation of cortisol tlemonstratcd the preferential reduction of the A’ bond of dehydrocortisol by bovine blood albumin. Complete reduction of ltlehydrocortisol occurred if the incubation period was increased and additional TPN and isocitrate mere added. Seven milligrams of the crystalline sub-

M.,. of

iso I ated compound

M.p. of

Identity of isolated compound wth authentic compound

fa) + lb) Ccl

Infrared

T.

“C.

156-158 154-155 136- 138

156-158 154-155 12-1-135

238-240 218-220 160-l 63

237-239 190-207 162-l 64

+ + + + + + + + + + ~+ I + T 1T ( T

METABOIJSM

OF R.ING

&ate, androst-4-ene-17p-01, was isolated and identified from the petroleum ether washings of the androst-4-ene-17p-ol incubation. Glass-paper chromatography indicated that androst-4-ene-17/j-ol was the only steroid present in the petroleum et,her washing, and no steroids w~:crcfound in the chloroform extract of the incubation medium, thereby indicating the lack of reduction at t’he A’ bond. COMPARISOK OF REDUCTIOK OF VARIOUS STEROIDS

This study was concerned with t.he investigation of the Aa-5/3-hydrogenase specificity and the amount of reduction of three different classes of steroids, namely, corticoids, progestational agents, and androgens (testosterone and 17a-methyltcstosterone multisubstituted compounds). The results obtained from the corticoid multisubstituted compounds are summarized in Table II. Table III contains a summary of the results obtained when progesterone derivatives were studied, and Table IV the results of study of testosterone and its derivat,ives. Table V shows the results obtained when 17a-methyltest,osterone and its derivatives were investigated. In Table VI is a summary TABLE II REDI-(~TION

OF I)oL-BLE ST‘BSTITI.TED

Boxuu ok Co~~ori~vus~

Steroid

&$~~dqfed

Cart isonr 2a-Methylcortisone acetate 1-Ikhydrocortisone 16ol-Methyl-l-deh~tlrocorti-

CORTIWID-

Reduction

BE.

per cent

124 124 124 124

85 12 51 55

124 124 124 124 124 124

79 83 3 65

124 129 127

42 na 78

SOIlC

Cortisol 1LEpicortisol Be-Methylcortisol ltkkIeth~lcortiso1 2ol-Meth?-l~Bcu~fluorocortisol 2~-Methyl-9cu-flr~orocortisol acetate I-lkhydrocortisol 11-l)eoxycortisol &-Methyl-ll-deosycortisol 1~Three experiments

done in triplicate.

1

295

A OF STEROIDS T,4BLE

REDUCTION

OF %~BLE ,~I'BSTITITTEl)

III

Bolvu OE' PROGESTERONECOMPolJNIW Amount steroid

Steroid

Progesteronr 1%Norprogesterone 1LDehydroprogesterone lip-Hydrosyprogesterone IL-Hyirosyprogesterone 2cu-Methylprogesterone 201.MethJ-1-11-osyprogester

of

Reduction

WT.

per cm,

116 116 110 120 118 116 116

8T 88 89 94 92 37 0

117 116

70 28

01,e

6ol-Methylprogesterone 9ol-Fllloro-118.ol-progesterone B Three experiments

done in triplicate.

TBBLE REI)I.CTION

OF hLrBLE SUBSTITI-TED

IV

BOND OF

TESTOSTERONECOMPOCNI)S~ Amount of steroid added

Pg.

Testosterone 19.Piortest,osterone 17-I>eosytestosteronc 19.Kor-17wethinyltestosterone Testosterone phenoxy acet,at,e Testost~erone propionate 2wFlnorotest,osterone 2wPvlethyltestosterone 2w?vlethyl-19.nortestosterone 6ol-Methylt,estosteromr in-Meth~ltestosterorle 9, 11.Dehydro-~CX.lia-dmethyltestosterone A’-Dehydrotestololactone

Reduction

per cent

116 116 125 114

95 95 05 91

116

90

118 117 115 116

93 90 3 7

116 121 107

75 26 12.1

13x

86.9

n Three experiments done in triplicate.

of the results obtained in the specificit’y &dies of the A*-5p-hydrogenaxe.

6

The loss of absorption at 240 1~~~of certain steroids with the a,P conjugated system in ring B after incubation wit.11 bovine blood albumin, TPK, and isocitrate suggests

296

RONGONE TABLE

V

Steroid

s~~~~)dunad~~d

lia-Methyltestosterone ll~-Hydrox~--17ol-l1lethyltestost,erone ll@-Hydroxy-17wmethylt,estosterone 2~~,17a-IIimethyltestoster~ one 6a,l7~l~imethyltestosterone 2~,17~-l>i1net.h~l-I)~,11epoxytest,osterone 9,ll-Dehydro-2a,l7a-di~ methyltestosterone 901.Fluoro-lip-hydroxy-17amethyltest.osterone Bol-Fluoro-llp-h~tlrox~-l7~methyltestosterone propionate 9,~Flrloro-17p~hydroxy-l7aIneth?rl-i-androstene3,11-dionc

116 116

89 89

116

89

115

14

116

83

116

16

116

10

117

2-l

122

27

116

17

116

17

/a.

a Three experiments

Reduction per cenl

the bovine blood albumin preparations is the AA-5p-hydrogenase. Although the presence of a h4-Ewhydrogenase is not completely ruled out, t,he isolation studies show no evidence for its presence. Several A4-$?hydrogenases capable of the hydrogenation of the A4 bond of the different steroids probably exist in bovine blood albumin, since McGuire and Tomkins work (14) indicates steroid specificity of the st’eroid A4-5phydrogenases. Interest in the metabolism of the .4’ steroids was st,imulated by isolation of Iandrostene-3,17-dione from human urine by Lieberman et al. (15). This interest was further augmented by t,hc clinical report of the activity of the two antiarthritic compounds, I-dehydrocortisone and l-dehydrocortisol by Bunim et al. (16). Later, Caspi and Pechet (8) demonstrated in human subjects the preferential reduction of the A” bond prior to the A’ bond. Vermeulen and Caspi (10) demonstrated by rat liver homogenate studies the preferential reduction of t,he A’ before the A.4 bond. In a preliminary report (17) and t,hc present study, the preferential reduction

done in triplicate.

TABLE SPECIFICITY

partial or complet,e reduction of the conjugated system. Previous work by the author (7) and the present study demonstrate that hydrogenation of the A4 bond occurs wit,hout reduct,ion of the 3-keto group. Previous work demonstrated that the addition of TPNH to the incubation media greatly increases the speed of the reduction, suggesting that the long period of time required for the reduction to occur is due to the low concentration of enzymes present (7). It was demonstrated that the reduction of the a*double bond is not due to gradual growth of bacteria ( 13). It has also been shown that after incubation of cortisone, progesterone, 4-androstenc-3,17-dione (7)) and testosterone, the only reduced metabolites isolated and identified are 5p-dihydrocortisow, 5~ - dihydroprogesteronc, 5~ - androstanc-3,17-dione, and 5p-dihydrot’cstosterone. Isolation of these four metabolites demonstrates that the A4-hydrogenase in

OF

THE

VI 5~-HYIlROGENARE”

M.

Cholesterol 5-Androstene-3(3,17&diol 5-Androstene~3-orlc~-l7~~~~14,+dimethyl 5.Pregnene-3,20-dione 5.Pregnene~38-ol-20.orie 5Androstane-3P-ol-Ii-one CAndrostene-3@, li&tliol di:tcet:tte +Androstenrm3a, 178.diol diacct at e 4-4ndroPtenem3B, li&tliol &Androstene-3a, IT~~diol ~-.4ridrosterlc-liB-01 P~~Fluoro~~-:tndrostcrle~ 301,17p-diol 2ol-Fl~~oro-4-:rndr~ster~~~3one-17p-01 a Two csperimcnts

per

100 100 100

0 0 0

100 100 100 90

0 0 0 0

90

0

95 95 95 90

0 0 0 0

117

no.1

done in t,riplicnte.

cent

METABOLISM

OF RING A OF STEROIDS

of the Al bond of 1-dehydrocortisol by a bovine blood albumin preparation has been pointed out. This work and that of Caspi and associates clearly demonstrated that there is a species difference in the met’abolism of 1-dehydrocortisol. The per cent reduction of t,he CL,~ conjugat,ed system of several steroids was inwstigated. The amount of reduction of the clouble bond appears to be dependent, on the types of changes the st,eroid nucleus undergoes and the location of these changes. In all instances, cortisol and its derirativw n-erc m&bolized more slowly than cortisone and it,s corresponding dcrivativcs. The presence of an llu-hydroxy or an Ilphvdroxy had little cffcct on the mctabolixm OF the steroids investigated. The effect of both configurat’ions appeared t,o be similar, depending on the type of compound investigated. In all the experiments the 2~ Incthyl group inhibit.ed rccluction of the U, p conjugated system of ring B t,o the greatest clxtent,. The presence of a 7a-methyl group :tlso had a great inhibit’ory cff’cct. In all compounds investigated which contained IL 6u-met,hyl group, the magnit’utlc of inhibition in the different series was similar. It is interesting to note that in tlic substrates in which loss of absorption was inhihitcd, there n-as a constituent in a 1 ,3ljosition relative to the A+ bond, c~xccpt for the 6u-met~hylated compountls. Stcrir hindrance is not considered to be of major importance in the compounds st.urlic,(l \vitli constituents at carbon 2, since 2u-fiuorotcstosterone was almost, completely r~tlucetl. The effect of the 2u-methyl group may be that it ncut,ralizcs the polarization effect of tlic carbonyl group, thereby prcrcnting reduction of t,he A~ bond (whereas the highly clcct.ronegativc fluorine atom, in the prcscncv of a 3-carbonyl, should cnhanre polarization of the 4 ,&double bond). In the cast of the AIS 4 steroids, an incubation period of 24 hr. was not sufficient for conil)lete reduction. Complete reduction of the 1’3 4 stwoids occurred by extending the incubation period and mcrcwing the :mount of TPK and isocitratc. This suggests t,lic presence of another enzyme CRpable of reducing the A1 bond of steroids.

297

The long time (18 hr.) for this enzymic reaction to be completed is probably due to the minute amount of enzyme in the crude preparation. *iddition of TPNH shortens the cnzymic time to approximately 6 hr. with the crude enzyme and to approximately 2 hr. with small amounts of the Iwified enzyme. The inability of the A%p-hydrogenaw of bovine blood albumin to rcducc cholesterol, d-anclrostene-3p, 17p-diol, 5-androstenc-3/?01-l 7-one, 5-androstene-3-one-17p-01, 4,4dimet’hyl-5-prcgncne-3,20-dione, and 5its pregnene-3P-ol-20-one demonstrates spc‘cifitity toward the ~~ bond. Lack of rctluction of the A* bond of 4nndrostcne-3,/j, 17p-dial diaretate, 4-androstew-3a, 178~dial diacctatc. and their free ulcol1ola, 2a-fluoro-4-androstene-3a, 17/3diol and 4-androstene-I 7p-01, while the corrcspontling 3-keto compounds arc strongly reduced, indicates that a 3-k&0 group is csaential for the A’ bond reduction. An explanation of the necessity of the rarbonyl group may t)e clca~w if one recalls the work of I1ZcGuire and Tomkins (18). They tlcmor&rated that’ the clrctrons of the tlout~lc bond are polarizc~l by the 3-carbony group, making t,lle C-4 relatively ncgativca to (‘-5 and permitting direct’ hydrogenation by a proton from the aqueous mctlium and of C-5 t)y a hydride from TPN. In the absence of :I 3-carbonyl, polarizat.ion of tllc 4,5ttout)lc bond would not chxist to tht, same tlrgrcc.

The author also m-ishcs lo cstcnd his th:mks to Dr. R. Brum Gnbbad for the synthesis of 3tl~osytrstosteronc and 17-deo9?-testostc~lonc, md to Bymrs Cwricrc and Lumric Sorenson for their lcchniv;ll assistnnve.

2. T~MRIYS, G. M., 1. 1j;iol. Cl/em. 225, 13 (1957). 3. BHOWS,J. H. U.. AS~IAsasos, A., E;ntloc~i,~olog!/ 62, 103 (1958).

298

RONGOSE

6. WOTIZ,

H. H., RICHTERICH-VAN BAERLE, R., ANII H. M., J. Viol. Chem. 213, 969 (1955). 7. RON~ONE, E. L., STREXGTH, D. R., Boc~~acs, B. C., ASD DOISY, E. A., J. Biol. Chcm. 225, 959 ( 1957 ) LEMOK,

8. CaSPI,

E., AZrD I'ECHET,

hl.

M.,

Al&.

Bbchem.

Riophys.68,236 (1957). 9. RONGOKE, E. L., SEGALOFF, A., FRIED, SABO, E. F.. J. Viol. Chcm. 236, 2624 10. VERMELXEK, 8., -4s~ CASPI, E., J. Viol. 233,54 (1958). 11. I’omx. C. C., .4ND SILBER, R. H., cf. niol. 185,201 (1950).

J., AXD (1961). Chem. Chem.

12. DIWKEKT, J. W., AND REISER, R., J. Am. Oil Chemist.? Sot. 33, 123 (1956). 13. Roscro.us, E. I,., PH.I). Dissrrtntion, St,. Loui: IJniv., June, 1956. 14. M&LYRE, J. S.. AND Tomxss, G. M., Awh. Hiochc~m. Biophys. 82, 477 (1959). 1s. IJEBERK~N, s., I~ORRINER, K., HILL, I+EYEH, I,. F.. .4SD RHOBDS. C. P.;

B. R., J. HiOl.

Chem.172,263 (1945). 16. B~JXIN, J. J., PECHI;T, M. M., ASD BOLLET, A. J., J. Am. MCYI. Assoc. 157, 311 (1955). 17. RONGOSE, E. I,., Fcrlcra/im Pror~. 19, 239 (1960). 18. M&VIRI:. J. s,, ASD TOXKISS, G. M., Fetlernlion Z'roc. 19, 29 (1960).