Polarographic Reduction of Epimeric Haloketosteroids and Their Vinylogs

PETER KABASAKALIAN and JAMES McGLOTTEN Chemical Research and Developmenf Division, Schering Corp., Bloomfield, N. J .

A new technique has been added to the currently available spectrometric methods which differentiate between an axial and an equatorial halogen atom in rigid a-halogenated cyclo- hexanones. It also differentiates bi- sectional halogens in rigid a-halogen- ated cyclopentanones. The polaro- graphic half-wave reduction potentials of epimeric a-haloketosteroid pairs (and their vinylogs) are dependent on the configuration of the halogen atom. The thermodynamically stable isomer reduces with greater difficulty (at more negative potentials). The difference between the half-wave potentials of the epimers is greatest for fluoro compounds.

ERY FEW studies have been made V on the polarographic reduction of a-halocarbonyl compounds since Winkel and Proske (17) first examined chloro-, bromo-, and iodoacetones in 1936. The work on alicyclic a-halocarbonyls has been especially limited, being confined to a-chlorocyclohexanone (5, I S ) , a- chlorocyclopentanone (5, I S ) , and a- bromocamphor (1 5 ) .

I n steroid syntheses, a large number of a-halocarbonyl compounds occur as synthesis intermediates and final prod- ucts. These include or-iodo-, @-bromo-, a-chloro-, and a-fluoroketosteroids as well as their vinylogous r-halo-a.0- unsaturated ketosteroid counterparts.

Because of the paucity of polarographic information and the relative importance of these compounds, a systematic sur- vey of the polarographic behavior of these steroids was undertaken to deter- mine if polarography could make a sig- nificant contribution to their analytical chemistry.

As an aid to readers unfamiliar with steroid structure, Figure 1 shows the carbon skeleton of pregnane with the A and B rings fused cis and with the 4 and B rings fused trans.

EXPERIMENTAL

Apparatus. A11 the polarograms were run using the Sargent Model XXI recording Polarograph. The polarographic cell was a n H-cell (3-ml. sample volume) containing a normal calomel anode separated from the saniple compartment by a n agar plug and a fritted-glass disk. The chloride ion of the calomel cell was furnished by liM tetrabutylammo- nium chloride solution.

All p H measurements weie niaclc with the Beckman Model G p H meter.

Cell resistances (for correcting half- wave potentials) were measured with an Industrial Instrument Model R C 16 conductance bridge.

Applied potentials meie measured with a Rubicon Model B potentiometer.

Ultraviolet spectra were obtained with the Cary Model 14 recording spectrophotometer.

Materials. Commercial grade 3 8 alcohol (95y0 ethanol-57' methanol)

e

a

I

Alpha-

obtained from Publicker Industries, Inc., was used as received. The tetrabutylammonium chloride (South- western Analyt,ical Chemicals) was polarographic grade.

Xcetat'e buffer solution contained reagent grade 1Jf acetic acid-sodium acetate buffer in water.

The tetrabutylammonium chloride solution contained 0.5X tetrabutyl- ammonium chloride in wat'er.

The steroids not otherwise specified in this investigation were obtained from the Schering Laboratories.

Procedure. T h e ethanolic electrol- ysis solutions (ca. I m X ) ivere pre- pared by weighing the sample into a 10-ml. volumetric flask, dissolving i t in about 5 nil. of ethyl alcohol, then adding 1.0 nil. of the acetate buffer (or the tetrabutylanimonium chloride solution in the case of all the fluoroketosteroids). The solution was made up to 10 ml. Ivith additional ethyl alcohol (the apparent p H of the resulting solution with the acetate buffer was 6.0). then deaerated with nitro- gen for 20 minutes and polarographed. Solution temperatures were maintained constant a t 25' C. Half-wave poten- tials are reported in volts os. t'he normal calomel electrode (K.C.E.). The cur- rent constants are reported in id:'

Cm2i 3 t l ~ 6 units. Isomerizations. Ga-Bromoandrost-4-

ene-3,17-dione (AE::: 238 mp) was prepared from the 60-compound (AE::: 244 mp) by isomerization in dioxane containing 3,5y0 perchloric acid. 601- Chloro - 17a - hydroxypregn - 4 - ene- 3,20-dione JAE::: 236 mp) was obtained by isomerizing the 60-compound (AE:::

.

e

Figure 1. sectional (b) bonds

Skeletal outlines of two typical steroid structures showing spacial relationship of axial (a), equatorial (e), and bi-

I. 5a pregnane 11. 5p pregnane (C and D ring orientation ore identical toll

1440 ANALYTICAL CHEMISTRY

242 mp) in cold chloroform saturated with dry hydrogen chloride gas (1).

RESULTS AND DISCUSSION

Saturated a-Haloketosteroids. The half-wave potentials and current con- s tants of saturated a-haloketosteroids are listed in Table I. T h e acetate buffer electrolyte system which con- sistently gave better developed and more reproducible wave forms than the tetrabutylanimonium chloride system was used for all except the fluorinated compounds. T h e tetrabutylammo- nium chloride was used for the latter compounds because of the very negative potentials involved. More polaro- graphic data for the a-bromoketosteroids than the other a-haloketosteroids were obtained as these are the most prevalent halogen compounds.

HALF-KATE P o r ~ s ~ I a L s . The nor- mally expected order of ease of reduc- tion of the halogens was found. The hdf-wave potentials increased in the following order: fluoro-, chloro-, bromo-, and iodohetosteroid. Holyever, the iodo- arid bromo- compound potentials ere too close to differentiate halogen t jpe . The poteiiti:ils for 3 given halo- gen type appeared to be a function of halogen position Eimilar to the depeiitl- ence of half-wave potential of noncon- jugated steroidal krtones (10) \\here the steric hindrance factor seemed to be operating.

CURREST Cossrams. The current constants of the single n aye compouiids except 2a:-fluorocholestan-3-one varied from 1.8 to 2.3 units, rquivalerit to the expected 2-electron reduction of the halogen. The n a i e listed for 2a:- fluorocholestan-3-one (E, 2 -2.23 i olts) is believed to be a 4-electron (curierit constant 3.7 units) reduction n a i e. The 2-electron fluorine reduction n a! e is merged with the 2-electron 3-ketone reduction wave follon-ing it. The 3- ketone in cholestan-3-one is one of the most easily reducible (Eli2 -2.28 volts; 1 2 . 3 ) steroidal ketones (10). The com- posite wave has a half-wave potential more negative than the true fluorine wave but less negative than the true ketone wave. The 9a-fluoro-11-keto- steroid and 170-fluoro-20-ketosteroid listed in Table I have half-wave poten- tials of -2.03 volts and -2.15 volts, respectively, well separated from the 11-ketone (Ell2 ca. -2.7 volts) and the 20-ketone (E112 ca. -2.4 volts) half- R ave reduction potentials (1 0).

DOUBLE WAYES. Two compounds, 2a-iodocholestan-3-one and 12a-bromo- 3a - hydroxy - 50 - pregnane - 11,20- dione acetate, gave double waves of unequal heights mhere only one wave would be expected. The sum of their current constants equaled 1.8 dnd 2.1 units, respectively, falling in the 2- electron reduction range. Although

Table I . Half-Wave Potentials and Current Constants of Saturated a-Haloketosteroids

Halogen Compound position"

2a-Iodocholestan-3-one 2a ( e )

21-Iodo-3p-hvdropregn-5-en-20-one 21 - - - acetate

2a-Bromocholestan-3-one 4a-Bromo-17a,21-dihyclroxy-5a-preg-

12a-Bromo-3a-hydroxy-jp-pregnane-

lTa-Bromo-3p-h~-droxypregn-5-en-20-

21-Bromo-3p-hydroxypregn-5-en-2O-one

nane-3,11,2O-trione 21-acetate

11,20-dione acetate

one acetate

acetate

Carbqnyl position

3

20

3 3

11

20

20

-0.02 -0.26 -0.22

-0.06 -0.04

-0.19 -0.52 -0.08

-0.21

2a-Chlorocholestan-3-one 2a (ej 3 -0.94 17a-Chloro-3P-hydroxy-5~-pregnzne- 17a ( p a ) 20 -0.93

21-Chloro-3p-hydroxypregn-5-en-2O-one 21 20 -1.25 11,20-dione

acetate 2a-Fluorocholes tan-3-oned 2a (e) 3 -2.23 9a-Fluoro-170,21-dihydroxypregn-5- g f f (3) 11 -2.03

ene-3,11,2O-trione 3-ethylene ketal- 17,20,21-bismethylene dioxalane

11,20-dione acetate

b In volts V.S. S.C.E.

17a-Fluoro-3a-hydroxy-5~-pregnane- l7a ( p a ) 20 -2.15

a = axial; b = bisectional; e = equatorial; p-a = pseudo-axial.

c I = Zd/CnLZi3t' in pa. set.' '/rnM. mg.2/3 units. Compound kindly furnished by Edward Jensen of the University of Chicago.

I C

0 . 8 1 . 0 2 . 0

1 .8 1 . 8

0 . 5 1 . 6 2 . 3

1 . 9

1 .8 2 . 0

1.8

3 . 7 2 . 3

2 . 1

Table II. Half-Wave Potentials and Current Constants o f Haloketosteroid Epimers

Compound GP-13 ro~no-~i~-h~tlrox~cliolcstan-i-one

(ia-Brorno-3p-li!.tiro~!.cholestan-i-one

1Icthyl 113-bromo-3~-hydroxy-12-0xo-

AIethyl 1 1~t-bromo-3a-hydrosy-12-oso-

16~-Bromo-:i-!~\ drox!-estra-l,R,S( 10)-

16p-Aromo-3-liytlrox~estra-1,3,5( 10)-

GP-Bromoandrost--l-eric-3,17-dione Ga-Bromoandrost-4-ene-3,1i-dione Gp-Chloro-1 ia-hydroxypregn-4-ene-R,20-

acetate

acetate

cholanute acetate"

cholanate acctnte*

trien-17-one'L

trien-1 :-onen

dione

dione Ga-Chloro-l7a-hydroxypregn-i-ene-3,20-

G@-Fluoropregn-4-ene-3,20-dione

Carbonyl position

I

- <

12

12

17

1;

3 3 3

E1 12

-0.14

-0.14 -0.33 -0 .07

-0.08

-0.16

-0.28

-0.02 -0.05 -0.74

-1.12

-1.18 -1.48 -1.46

I 1 . G

0 . 3 1 . 5 2 .2

2 . 3

2 . 3

2 . 2

2 .3 2 . 3 1 . 9

1 . 8

1 . 7 0 . 5 2 . 3

a Compounds kindly furnished by J. Fishman and T. F. Gallagher of the Sloan-Kettering Institute.

this phenomenon might be ascribed to wave-splitting, a fairly common occur- rence in organic polarography when two different reducible species can be formed because of functional group interaction with hydronium ions or buf- fer components (12, I+$), it appeared strange that this double-wave formation was not observed more frequently dur- ing the survey of the compounds in Table I if indeed it was caused by the polarographic reduction of a single halo- gen compound.

Halogen Configuration. An alter- nate explanation for the double waves noted above is possible, if one assumes t h a t .the a-haloketosteroids used were impure (contained the other halogen

epimer). a-Haloketosteroids are very difficult t o obtain pure as synthesis usually yields the kinetically con- trolled (axial-substituted) epimer as the principal product with the thermo- dynamically stable form (3) .

EPIMERIC SATURATED ~-HALOKETO- STEROIDS. Table I1 lists the polaro- graphic behavior of three saturated a- bromoketosteroid epimeric pairs. The thermodynamically more stable 6a- bromo-3~-hydroxycholestan-7-one ace- tate (2, 3) has a small prewave associ- ated with the main wave. The data listed for the 6a- and 6p-epimers would infer strongly that the small first wave of the 6a-epimer is caused by the pres- ence of a small amount of the 6p-

VOL. 34, NO. 1 1 , OCTOBER 1962 1441

Table 111. Half-Wave Potentials and Current Constants of Unsaturated Haloketosteroids

Compounti Halogcn Chrbori> 1 position position I?',t> I

4-Rromo-1ia,2l-dih~-drox~preyna-l,4- 4 3 -1 18 1 0 diene-3,aO-dione 21-acetate -1 48 1 0

4Chloro- 17a, 2 1-dihydroxypregna - 1,4- 4 3 -1 19 1 0 diene-3,20-dione 21-acetate -1 50 0 8

4-Fluoro-17~,21-dihydroxypreg~ia-1,4- 4 3 -1 19 0 8 diene-3,11,2O-trione 9 ?

iuipurity. For the 11-bromo-12-keto- steroid listed, the more stable l l a - epimer (3) reduces a t a slightly higher negative potential. The 16a- and 16p- bromo - 3 - hydroxyestra - 1,3,5(10)- tiien-17-ones reduce in the same order of thermodynamic stability (7) as the previously mentioned epimers. Wilson and Allinger (16) have reached this con- clusion from an independent study car- ried out wit'h simple Z-halocyclohexa- nones.

STEROIDS. Table I1 also lists the polar- ographic data obtained for three pairs of 6-halo-A4-3-ketosteroids. The 6-halo- gens are act,ivated by both the allylic double bond (A4) and the 3-ketone (via the A4) to such a n ext,ent that these vinylogous compounds are more easily reduced than the vicinyl a-haloketo- steroids. This fact is not readily ap- 1)nrent for the brominated compounds as the a-bromoketosteroids are reduced a t such positive potentials. However, the reduct'ioii potential shift is quite notice- able for the chlorinated and fluorinated compounds. In all three types of halo- gen compounds the thermodynamically stable epimer has the most' negat,ive half- wave potential. The difference in half- wave potential for the brominated epi- mers is small but significant; while the difference for the chlorinated and fluori- nated compounds is substantial. -\gain there was a case of double-wave forma- tion (6p-fluoroketosteroid) where the pot,entials obtained would indicate that the minor wavc is caused by the pres- ence of the other epimer. At -1.46 volts, t'he fluorine at'orn is being reduced and not the A4-3-kctone moiety ( E I ' z ca. -l.i volts) in the tetrabut,ylainmo- ilium chloride system.

4-Hal0-A~~~-3-Ketosteroids. The thrce conipounds listed in Table I11 have approximately the same half- wave potentials and current constant's (the second wave for the fluorinated steroid is present bu t poorly defined). Obviously, these waves are not' caused by fission of a carbon-halogen bond since one would never expect t'o break n carbon-fluorine bond with the same energy t h a t is needed to break a carbon-broniinc ljond when t'hc en- vironment ,is the s:inie. The first nave 1)robably represent's a 1-clcctron (cur-

EPIMERIC GNSATURATEU HALOICETO-

reiit constant 0.8 to 1.0 unit) reduction of the A1s4-3-keto moiety (E1,* ea. - 1.20 volts) to the bimolecular pinacol (11). Thc second wave can be ac- counted for by a change in the electrode reaction to a 2-electron reduction of the 3-ketone to the 3-carbinol similar to that previously reported for prednisone (11). The 4 - halo - A I , * - 3 - ketoster- oids (and 4-halo-A4-3-ketosteroids) can be differentiated easily from B-halo-~i~ .~- 3-ketosteroids (and 6-halo-A4-3-keto- steroids).

Analytical Implications. Three spectrometric methods are currently available t'o differentiate between an axial and a n equatorial halogen atom in a-halogenat'ed cyclohexanones. The first two inethods are infrared (9) and ultraviolet (2) absorption spectrometry. These techniques are coniplement,ary; the former principally determines the prescnce of an equatorial halogen while the latter does the same for the axial halogen. The third method, optical rotatory dispersion ( d ) , is a highly re- fined extension of the ultraviolet spectro- metric method which responds only to axial halogens. Although these meth- ods work well with cyclohexanones, they are not generally applicable to a-halo- cyclopentanones (2, 4 ) . In fact, where :i biscctional bond (intermediate be- tween axial and equatorial bond) exist's as in t8hc 16a-bromo and 160-bromo derivat i \v of 3p-hydrox?i-5a-androst,an- 17-one act>tate, t8he infrared, ultraviolet', and optical rotatory dispersion mcasure- merits havc yielded identical wavelength shifts for both isomers (6, 8).

The incthotis mentioned above are indirect as they depend principally on the effect the halogen atom has on the carbonyl function. 1)iagnosis by these individual methods usually requires a separate tletermination for the presence of t,he halogen at.oiii. The polaro- graphic method to differentiate halogen configuration is direct: both axial and equ:ttorial halogen are polarographically reducible. The tliermodynaniicaly sta- ble epimer is iiiore difficult to reduce \\hctlier the, hnlogcn is in a five- or six- membered ring in :I bisectional, pseudo- nsia,l, I)seudo-equatori:iI, axial, or equa- torial configur:ition. The relative ntnount,s of the axial and equatorial (5l)iiners can be asccrtaincd without

having p u i ~ reference coiiipounct~ avail- .ible. This method seems to be best suited to chlorine and fluorine drri \x- tives.

a-Broniiiiations and a-chlorinatioris of saturated ketosteroids can be moni- tored simply and rapidly wit? absolutely no interference from the unhalogenatcd ketone by the polarographic technique. Sonie interference will be observed for a-fluorinated ketosteroids, but even here the progrers of the reaction can be foliowed.

LITERATURE CITED

(1) &hit, C. P., Ehreristrin. 11 , J .

(2) Cookson, It. ('.> J . C ' h 2 m . Sot.. 1954,

(3) Corey, K. J., J . A m . C'hcni. S o r . 7 6 ,

Org. C'hem. 17, 15% (1952).

282.

175 (1954). (4) Djerasui, C., Osiecki, J., Riniker, 11.,

( 5 ) Elving, P. J., Van iitta, Et. F., J . Riniker, B., Ibzd . , 80, 1216 (1958).

Electrochem. Soe. 103, ti76 (1856). (t i) Fajkos, J., Collection C'zech. O h c ~ i .

( 7 ) Fishnian, J., Biggerstaff, \V. H., ,I. Ory. Commun. 20, 312 (1955).

Chem. 23. 1190 (19581. (8) Fishman, J., <)jerassi, C., b';'Jperit.nliu 16, 138 (1960).

(9) Jones, It. X., Ilauisay, 1). I., Herlirig, E ,, Dobrincr, IC., J . .4m. C'hem. Soc. 74; 2828 (1952).

1 10) Kabasakalian. P.. McGlotten. J.. ' , ~ A L . CHEM. 31,' 1091 (1959). (11) Kubasakalian, P., McGlotteIi, J , (12) Kabasakaliari, P., McGlotten, J.,

J . A m . Chem. Soc. 78, 5032 (1956).

' J . Electrochem. Sic. 105,261 (1958). (13) Pariaud, J. C., Perruche, C . , C'ompt.

k e n d . 238. 1.514 11954). (14j R u e d i , P.,' Trumpler, G., Hrlu.

(1.3 Schwabe, IC., Berg, IT., Z. Eleklro- Chim. Acta 35, 1021, 1486 (1952).

chrjn. 56, 952 (19;i2). (16) Wilson. -4. X l . , -illinger, S. I,.. J .

~ A m . Ch&. SOC. 83; 1999 (Il9til). (17) Winkel, A., Proske, G. , ('hem. I l c r .

I~ECEWED for review February 12, 1962. .lccepted May 8, 1962. Division of Ana- lytical Chemistry, 142nd Meeting .4('S, .ktlantic City, X . J., September 1062.

69, 693 (1936).

Correction

Time-of-Flig ht Mass Spec- trometry: Application to Capillary Column Gas Chromatography

In this aiticle by R. 6 . Gohlke [ . ~ A L . CHEM. 34, 1332 (1962)] on page 1333 the caljtions for Figures 2 and 3 werr reversed. Figure 2 caption should read "Bottom trace: Response of mass 43 monitor" and Figure 3 caption should read "Bottom trace : Response of inass 31 monitor."

1442 ANALYTICAL CHEMISTRY