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Trimethylsilyl ethers of some 5β-pregnanes
.lS \I,T1‘1(‘4L
BIOCHEMISTRY
Trimethylsilyl
42,
382-389
Ethers
Rcwived
(1!)71)
of Some
October
5,CPregnanes
25: 1970
About 6 years ago in developing a method for the measurement of urinary cortolsl and cortolones (1)) a TMSi derivative of cortol was prepared, isolated, and characterized. As the method was extende(l to include certain other reduced metabolites of adrenocortical secretory pro(lucts, namely, C&,0, and C&,0, steroids (21, it became evident that, contrary to expectations, the TMSi ethers were rather stable in nonacidic media and could be isolated and characterized; this communication presents such data. For the preparation of the derivatives, two proccdurcs were cmployocl : 1. Reacting 2. Treating
the steroid with a mixt,ure of HMDS:TMCS: the compound with bistrimethylsilylacetamide
PY. (BSA 1.
It has been shown by Chambax and Horning (31 that the first methotl will dcrivatize hydroxyl groups at C-3, -11, -20, and -21 of a C,, steroid but not the highly hindered 17-hydroxyl, while the second, milder, procedure affords partially derivatized products in which both the 17- and ll-hydroxyl groups are free. A re-examination of the cortol-TMSi ether prepared by the first method (originally designated as a penta TMSi derivative) by techniques not availnhlc at that time shows t’he substance to be, in fact, a tetra TMSi ether in agreement with Chambaz and Horning. EXPERIMENTBL
Melting points were taken on a micro hot st,age and are corrected. NMR spectra were obtained on a Varian A-60 instrument in cleuterochloroform with tetramethylsilane as the internal standard. IR spectra ’ Abbreviations: cortol. 5~-pregnane-3ql1/3,17a,20a,21-pent~ol; p-cortol, 20,8-epimw of cortol; cortolone, 5P-pregnane-3a.l7a,2Oa 21-trtrol-11-one ; /3-eort,olone, 20/3+pimer of cortolone; TMSi, trimrthylsilyl ; HMDS, hesamethyldisilazane ; TMCS, trimrthylchlorosilane ; PY, pyridine ; BSA. bistrimethylsilylacetamide; GLC, gas-liquid chromatography: RRT, relativr retention timr (cholestane, 3.9 min) ; IR. infrared; MS. mass spectrum. 382
wc’rc cletertttined Ott :I Beckman IR-9 ~l)cctrophototnct(~r itt llotassium bromide dispersion. Mass spectrametric tneasuerments were carried out on an LKB 9000 instrunlent by direct, insertion probe; ionization was by electron impact using 70 EV elect,rotts. An ionization current of 60 ,.,,A was used and the accelcrntion voltage was 3.6 kV. The temperature of the ion chamber xas 290”. Tve are particularly grateful t,o Dr. Henry Fales of the National Heart In&it&e of the National Institutes of Health for performing these measurements. Preptrration of TllISi ethers: HSIl)bS: TN(‘S:PY method. To about 50 mg of steroid in a 100 ml round-bottotn flask was added 10 ml of HODS : TICS : PY (3 : 1: 9 1. The suspension was allowed to remain overnight at 37”, after which the excess reagents and solvent w-cre removed under vacuum at 75-80” and the residue was dried under a stream of nitrogen. The derivative was dissolrcd in pctroleutn ether and the solution was washed 3 times with 70% ethanol. After t,he solvent had been removed, the product was dissolved in a small amount of benzene and filtered through a Cclitc pat1 on a small Gntercd-glass funnel; conccnt~ratiott under nitrogen afforded the deriratirc. IzSlz : PY IjMhofZ. hpprosimately TiO mg of each steroid was dissolved in 1.5-2.0 tnl of 1)yridinc to which 20 ml of BSX was added. The solution was allowed t’o stand at, room temperature overnight. Thereafter the procedural details were identical to tliosc> dc>scribed above.
All the derivatives m-ere pure as judged by the appearance of a single peak in gas-liquid chromatography (3% SE-30 on Gas Chrom Z, 250”). TMSi ethew of cortol n,lcl p-cortol. Cortol and its 20/3-epimer each formed two TMSi derivatives deljending on the reaction conditions. Cortol-3,11,20a.21-tetra-TRW ether was crystalline (calculated for C,,HG,@,Si,: C 60.3, H 10.44, Si 17.1; found: C 60.0, H 10.24, Si 17.7) and its melting point,, 166-167”, and IR spectra agreed with those of the earlier preparation (1) ; a sharp band at 3550 cm--l is characterist’ic of hydroxyl absorption. The tetra-TMSi ether of the 20/3-epimer (RRT = 3.22) could not be induced to crystallize and showed only a very slight hydrosyl absorpt,ion at 3570 cm1 Utough elemental analysis (found: C 60.2, H 10.15, Si 18.8) and t,he tnolc(~ular n-eight determined by mass spectrometry indicated a frc,c OH groul); MS: 422 (1.0) ,? 446 10.27), 406 (0.25), 524 (0.19)) 344 (0.161. The sntall OH band might be a ronsequcnce of tlttb noncrystallinity of tltch mtbatancc attcl the att’enclant difficulty of
preparing a satisfactory dispersion in potassium hromidc for infrared analysis. Both epimcre afforded crystalline derivatives with the BSA reagent and could be recrystallized from methanol or aqueous acetone with no decomposition. Cortol-3,20a,21-tri-TMSi ether (RRT = 2.92)) 137-138” (calculated for C:,,,H,,O,Si:,: C 61.7, H 10.32, Si 14.4; found: C 61.6, H 10.28, Si 13.1) showed a broad OH absorption at 3556 cnml; MS: 452 (,l .O), 288 (0.831, 272 (0.661, 362 (0.401, 344 (0.38). The 20/3+pimcr (RRT = 3.101, XXI-1.55” (found: C 61.7, H 10.25, Si 13.0) exhibited absorption at 3530 cnr’; MS: 452 (1.0)) 362 (0.40)) 288 (0.30), 446 (0.20)) 344 (0.20). Elemental analyses and the molecular weight dctcrniinetl by 111:~s::spectromctry are in agreement with three TMSi groups. C’ortolone rind /3-cortolone. Each cpimer formccl a single 3cu,20,21-triTAN derivative on reaction with either HRII>S:TMCS:PT or BSA (RRT = 2.56, and 2.69, respectively). Both TRISi ethers were oils but gare the expected molecular ion (PII, 583) in the mass spectrum; iUS (a-epimcr) : 450 (1.0) , 360 (0.66)) 272 (0.53), 287 (0.36)) 350 (0.33’1; (/3+pimer) : 450 (1.0)) 360 (0.67)) 272 (0.54)) 390 (0.37), 287 (0.36). DCspite the interference of a high background, nl)sorption at 3500 cm’ W:W observed in each deriratkc. Tetrols: 5P-P~~eyr~nne-3~,17cu,Ooc.ll-tetr~ol(lntl the 20,8-epiwer. Each cpimer afforded a single tri-TRlSi ether with either reagent, (RRT = 2.18 and 2.15, respectively) which dicl not crystallize ant1 although no hyclrosyl absorption was dctcctetl in the infrared, ISMR showed a signal at 6 = 2.62 (20~~) and 6 = 2.50 (2Op) attributable to the 17~hydroxyl group. Major ions in MS wcrc: for 20a-epimcr-436 (1 .O), 273 (0.42)) 346 (0.32)) 282 (O.l7j, 286 (0.11‘1, and for 20/3-elki~er-436 il.Oj, 346 (0.47), 273 10.331, 282 (0.17), 286 (0.16). Each of this 5~-P~egnane-S~,11~,17~~~~Ot~-tet~ol rind the 20,8-epimer. pair formed two tlcri~ativcs, a di- and a tri-TMSi ether in analogy to cortol and p-cortol; in addition, as in the cortols, three of the TMSi ethers were crystalline ~2O~tet~rol-3,20-di-TnISi i RRT = 1.87)) 233” ; 3,11,20tri-TMSi (RRT = 1.87), 134-135” ; 20,&tctrol-3,20-di-TnlSi (RRT = 1.64’)) 182-183”) ; and the foWli, 5/3-pregnant-3cu,l lp,l70,20P-tetrol3,11,20-tri-TMSi &cr (cf. the analogous /3-cortol derivative1 (RRT = 1.89) was an oil. In these 21-methyl tetrols, a broad band at 3540 cm’ in the IR spectra in the di-TMSi ethers of both epimcrs characterized hytlroxyl absorption, while the hydrovyl absorption at 3560 en-’ in the crystalline tri-TMSi &her derivwtivcl of 5~-~~regnanc~-3~~,11~,17t~,2Q~-tctrol was a sharp peak. Major ions in &IS were: Di-TMSi ethers-2(k: 272 (LO)! 254 (0.63), 214 (0.43), 362 (0.25), 290 (0.18), 230 (0.11) ; -20,G:
P-l 1g-01 P-l lp-0 TM& P-3o1,17a,2Oa-t~iol-::,%O-di-C)TRISi P-:~a,l7~,2O~-triol-Y,2O-di-OTMSi P-:~~,17~,90a,21-tet~ul-:~,2O,2l-t~i-~~T~I~i P-3~,17a,20~,21-tet~ol-~j,20,“1-tri-oTii P-3n,ll~,17a,2Oa-tetrol-Y,20-di-OTMHi P-%X, llfl,17a,20p-tet rul-3,%0-di-OTAISi I’-:$~,1 lp,17~,2O~-tetrol-3,11,20-tri-OT~ISi P-3~,llp,17~,20/3-tetrol-3,11,2O-tri-OTMSi Cortol-3,20,21-tri-OTAlSi p-C!ortol-3,20,21-tri-OTMSi C~)~tol-~~,l1,20,21-tet~,z-OT~ISi p-Cortol-3,11,%0,21-tetra-OTMSi Cortolone-3,20,21-tri-OT&ISi i3-Cortolorle-Y,‘LO,%1-1 ri-OTMSi
0.7x4 0.750 0 667 0.700 0.716 0 7.50 0.000 0. !)50 0.884 0. MOO 0, !I50 0.984 0 0 1.5 0.900 0.684 0. 650
LS-CH, 1.17 1.08 0.000 0.900 O.Yl5 0, Y39 1.17 1 .I7 I .O7 1 (07 1.15 1.1% I .07 1 .07 1.14 1.1”
19.CHI
21-CH:,
1.11 d (J = 5 Hz) 1.12d (J = 6Hzj 1.12 d (J = 6 HE) i.12d (J = 6Hz)
l.I%diJ = BHz) 1 I 0 d (J = 6 Hz )
-..--__
:;. X3 4.02 ::.65q :;.75 :-:.SS 4.05 3 X3 4.06 ::.64 ::.74q 3.64~1 S. 70 S .64 :;.67
q (J = q (J = (J = q (J = q (J = q (J = q (J = q (J = q (J = (J = (J = q (.J = 11m q (J =
7,14 Hz)
7,U Hz ) 6,l” Hz) 9,14Hz) 7,16 Hz) 7,12 IIz) 6,12 Hz) 6,12 Hz) 6,12 Hz) 2;,13 Hz) 7,18TTz) 8,12Hz) 6,lS Hz)
W-H __--
2.6.5 2 ,6.5 2.62 2.64
17-OH
386
R.
S. ROSENE’ELD
272 (l.O), 216 (0.42), 254 (0.35), 230 (0.28), 290 (0.17), 362 (0.14). TriTMSi ethers-20a: 272 11.0), 352 (0.98), 344 (0.33), 452 (0.21), 389 (0.13); -2Oj3: 272 (l.O), 362 10.26),480 (0.25), 388 (0.17), 344 (0.14), 452 (0.10). PTegnnne-.%,17&Oa-t,riol cmd the ?0,8-epimer. The substances formed di-TM% et,hers wit.h either reagent; the 20a-epimer Ivas crystalline, 200204” (calculated for C,,H,,O,Si,: C 67.6, H 10.72, Si 11.7; found: C 67.6, H 10.71, Pi 11.7), and the 20/3-dcrivat,ive (RRT = 1.181 was an oil. Pregnane-3~,17u,2Qa-triol-3,20-di-TMSi ether showed a narrow, intense> ba.nd at 3572 cm-l (17a-OH) in the IR while the 20P-epimer had a weak absorpt#ion at 3570 cm’; MS (20/3), 256 (l.O), 274 10.74): 252 (0.16)) 284 (0.15)) 364 (0.10). Pregnane-11,8-o/. The TMSi ether, a crystalline compound, 104-105” was prepared IHMI)S:TMS:PY method) in or&r to compare the effect of the TMSi moict’y on the angular methyl signals in the NMR spectra of a simpler compound with those of t’he derivatives of the more highly hydroxylated C,, steroids. The 19-H signal of the underivatized compound (6 = 1.17) occurred at almost the same posit’ion as the C-19 resonances of all the derivatives of the more highly substitutecl TMSi ethers bearing a free 11-hydrosy group (Table 1) and the upfirld shift 1-5 Hz) associated with the sl&Iding by the TMSi function also correxponclcd to the tetrol and pcntol derivat’ives with an ll-0-TMSi group. The 18-H rcsonancc (6 = 0.784) in pregnane-lip-ol was in accord with the value calculated from data which consider the shielding effect of the 17/3-ethyl and t,he dcshielding by the lip-hydrosy (4). The downfield shift of 7-12 Hz in t’he 1%methyl signals of the pent01 and t&o1 derivative:: with an II-hydroxy group must be a consequence of the resultant deshiclrling and shielding influences of the l7a-hydrosy and C-20-TMPi moieties, respectively.
Signals associated with the more salient fcat,ures of the TMSi ethers arc recorded in Table 1. In addition, all derivatives showed a strong signal at S = 0.117 ascribed to the protons of the TMSi moiety. hlso substances t,hat possess the ll-OH or 1 l-0-TMSi function displayed a broad multiplct at 6 = 4.23 characterist,ic of the ll,n-proton. I<‘?-Ueth !/Z. The 18-H peak in substances that. are unsuhstituted or have a ketone at C-11 was shifted only slightly from that in the unsubstituted 5P-androAanc (4) ; :ipparcntly tlicx resultant. of the iilt,eractions of the heavily suhstituted side chain is such that the C,, signal is little affected. The 11-O-TMSi suhstitucnt produced a downfield +hift# of from S-10 Hz as comparcd with the C-11 unsubstituted analogs, This shift was even
larger in the derivatives 1v11cire tllc 1l-011 is fr(~t, th:bi is, the 1 I-Tlldi frinctioil cxcrtc~tl :L blight clcsllic~ltling c~fl’wt 011 the 18-l-I. 111the c~l~irncric l)air~;, witli tlrcs cxccsl’tion of’ tllc~ c~~~~otolo~~c~.~, tll(b 18-ln(~tllyl signal iI\ the 2O,L?-(~l)iin(~rn-w slightly clo~~l~fic~l~li’rotrr tllcs 20~. I.%Mefh!//. Tlrc~ cl\Ariv:lti\-cs* po.G,wssitig 110 .s;lItwtiluc,nt at. C-11 alion-ed 19-H signals ( 6 ~: 0.900~(I.933 I tll:Lt writ virtrlally un&fted from the “naked” steroid nuc~le~k (6 = 0.925). ‘NIV 1 I/!?-li;\-~lrosy group as well as the I I-ketone exerted :L l>~rgc~cl(~~llic~ltiitlg c+fc.c~t (ca. 14 Hz downfield) whicsh was less pronounced ill tllc 11-t )-‘I’,\ISi c~~1n~~oundsnherc the TM% (Lxerted a shielding influence. Tliv c*olifigur:~tiwi at, C-20 has little effect, on the 19-H peak. ,“I-NethU1. 811 of the TRlSi ethers of the compounds unsubstituted at C-21, namc’ly, the epimeric .5/3-pregnane-3t~,l7a,20-t~riols and 5/3-pregnane3t~,llj3,17t~,2O-tetrols, show:cll an intense doublet centered at 6 = l.lOO1.115 (.J, 6 Hz) which is a consequence of coupling with the proton at C-20. C‘-NO-P~fotl. In all the derivnt’ives measured, the signal for the 20-H appeared as a quartet centered in the range 6 = 3.67-4.06; in every cast t,he 20~H (20/3-0-TMSi epimer) was downfield from the 20/3-H signals. The TMSi ether function at C-21 exertecl a shielding effect on the 20 proton, showing upfield shifts of about 11-12 Hz in the 20tu-epimers and 1622 Hz in t’he SOP-compou11ds. 17’~OH Proton. h singlet in the r:mgc’ from S = 2.18-2.65 was observed in every TMSi ether, except prcgnan-I lp-01 TAIPi, regardless of whether the 11/k?-hytlrosyl was free or silylated. The signal disappeared on equilibration with deuterium oxide, a characteristic behavior of the labile hyclrogen of a hytlrosyl group. This observation, particularly in the tleriratives prepared by the procedure in which the I l/3-hydroxyl undergoes trimet~hyleilylation, was additional evidence for the presence of the free I ‘itu-liyclrosyl group in this series. Mass
Spect,rn
The TMSi ethers prepared in this study showed complicated fragmentation patterns in mass spectroscopy (Fig. 1) ; however, for each compound the molerular ion was seen which agreed with the expected molecular weight of the derivative. Thus. under the reaction condit’ions, the 1‘ia-hydroxyl group did not form a TnISi ether (3). The mars spectra of c~onq~ounds epimcric at. C-20 wcr(~ veq similar. Bctwvccn M/e 583 ant1 Al1,‘e 272 of the epimeric cortolone-tri-TAISi ethers. ions of qua1 relative intekties wcrc present in both spectra; no compnriron was made beyond t’his.
388
FIG.
1. Mass
spectra
of typical
GO,-TMSi
ethers.
All compounds showed loss of 15 mass units, corresponding to a CH,, group (5,6). In addition, ions corresponding to AI-90 (loss of trimethylsilanol) and/or AI-180 (2 X 90) appeared in all spectra (5-7) and, as might be expected, ions associated with the loss of combinations of these fragments were observed, for example, PI- (90 + 15), ?II- (2 X 90 + 15)) etc. Mass spectra of compounds having the -CH (C)T,VSi)-CH, (OTMSi) and -CH(OTRISi)-CH, side chains showed certain characteristic differences (Fig. 1), e.g., the presence of XI-103 ions in t#hc former. Steroids with vicinal 0-TMSi groups (5,8) and TMSi ethers of primary alcohols 15,6) show this elimination, which has been attributed to the low of CH,O=TMSi (6). An intense X-133 ion was probably the resultant of two ionic species: M- (103 + 2 X 15) and RI- (90 + 28 + 15)) where 28 mass units represents the loss of carbons 3 and 4 after elimination of T?tlSi-OH in the A ring (5). The loss of t,no fragments, 90 and 133, probably accounted for the intense ion at M/‘e 346 (&I-223) in the 21TMSi ether derivatives while the &1/e 273 (N-296) ion couhl be ascribed to the loss of the side chain (206 mass units) and trin~ethylsilanol (90 mass units). The mass spectra of the 5 compounds with a 21-methyl group (hut not of the other compounds) had ions at N-45. It is possible that this reprosents the loss of a CH:,-CH-OH fragment concomitant with the replacement of tbc TL*ISi group from the C,, by a proton frown thr C-17hydroxyl; loss of the side chain was indicated by the fragment RI-117. In conkast to the compounds hydroxylated at C-21, the N-133 ions wcrc
of low rclativc nl~~ndancc although the M-(90 + 133) ions n-erc prewnt. Ions formed by elimination of the side chain plus one trimetliylsilanol ncre also present (M-207).
Trimethyldyl ethers of fivcb l)air hs of compountis of the 5P-lxcgnane scrie* epimeric at C-20, have been prepared, isolated, and characterized; the clbrivatives xrc stahlc in neutral media. Aksignment of configuration at, C-20 of a pair may hc made OII tklc basis of STIR spectra. Some charwtcristic fragmentat’ion patterns in the msli: c Ls spectra have been reported. ACKNOWLEDGMENTS The support and interest of Dr. T. F. Gallagher are greatly appreciated. I also wish t.o thank Mrs. Julia S. C. Liang for her invaluable help in the measurrm~nt. and interprpktion of the SMR sprctra. This investigation was supported by a grant from the American Cancrr So&t> and by grants CA-07304 from the Nat,ional Cancer Institute and RR-53 from thv Gtncral Clinical Rrsearch Centers Branch. National Institutes of Health. REFERENCE&S 1. ROBENFELD! 2. ROSENFWD. B. Lipsctt,.
3. 4. 5. 6. 7.
S.
R. S.. Steroids 4, 147 (1964). R. S.. ilL “Gas Chromatography ed.), 1,. 127. Plenum Press, New
of Strroids in Biological Fluids” (nl. York, 1965. CHASIRAZ, E. M., AA-DHORNING. E. C.. il~d. Biuchem. 30, 7 (1969). of NMR Spectra in Orgmk BHACCA, N. S., AND WILLIAMS, D. S.. “Applivation Chemistry.” Holden-Day, San Fran&co, 1964. HUHTANIEMI, I., LUUKKAINEK. T., .~ND VIHKO, R.. k/n Bndoo-itwl. 64, 273 (1970). HAMX~RSTROM. S., J. Lipid Re.s. 11, 175 (1970). EYEROTH. P.. HELLSTRBM, K.. .ASD RYHAG~. R., J. Lipid Res. 5, 245 (1964). ~~s~o.4. B. P.. GrJSTAFSSoN, J. .&., AND SJOVALI,, J., Ewopenil 1. Biochem. 4, 496 (196%.
BIOCHEMISTRY
Trimethylsilyl
42,
382-389
Ethers
Rcwived
(1!)71)
of Some
October
5,CPregnanes
25: 1970
About 6 years ago in developing a method for the measurement of urinary cortolsl and cortolones (1)) a TMSi derivative of cortol was prepared, isolated, and characterized. As the method was extende(l to include certain other reduced metabolites of adrenocortical secretory pro(lucts, namely, C&,0, and C&,0, steroids (21, it became evident that, contrary to expectations, the TMSi ethers were rather stable in nonacidic media and could be isolated and characterized; this communication presents such data. For the preparation of the derivatives, two proccdurcs were cmployocl : 1. Reacting 2. Treating
the steroid with a mixt,ure of HMDS:TMCS: the compound with bistrimethylsilylacetamide
PY. (BSA 1.
It has been shown by Chambax and Horning (31 that the first methotl will dcrivatize hydroxyl groups at C-3, -11, -20, and -21 of a C,, steroid but not the highly hindered 17-hydroxyl, while the second, milder, procedure affords partially derivatized products in which both the 17- and ll-hydroxyl groups are free. A re-examination of the cortol-TMSi ether prepared by the first method (originally designated as a penta TMSi derivative) by techniques not availnhlc at that time shows t’he substance to be, in fact, a tetra TMSi ether in agreement with Chambaz and Horning. EXPERIMENTBL
Melting points were taken on a micro hot st,age and are corrected. NMR spectra were obtained on a Varian A-60 instrument in cleuterochloroform with tetramethylsilane as the internal standard. IR spectra ’ Abbreviations: cortol. 5~-pregnane-3ql1/3,17a,20a,21-pent~ol; p-cortol, 20,8-epimw of cortol; cortolone, 5P-pregnane-3a.l7a,2Oa 21-trtrol-11-one ; /3-eort,olone, 20/3+pimer of cortolone; TMSi, trimrthylsilyl ; HMDS, hesamethyldisilazane ; TMCS, trimrthylchlorosilane ; PY, pyridine ; BSA. bistrimethylsilylacetamide; GLC, gas-liquid chromatography: RRT, relativr retention timr (cholestane, 3.9 min) ; IR. infrared; MS. mass spectrum. 382
wc’rc cletertttined Ott :I Beckman IR-9 ~l)cctrophototnct(~r itt llotassium bromide dispersion. Mass spectrametric tneasuerments were carried out on an LKB 9000 instrunlent by direct, insertion probe; ionization was by electron impact using 70 EV elect,rotts. An ionization current of 60 ,.,,A was used and the accelcrntion voltage was 3.6 kV. The temperature of the ion chamber xas 290”. Tve are particularly grateful t,o Dr. Henry Fales of the National Heart In&it&e of the National Institutes of Health for performing these measurements. Preptrration of TllISi ethers: HSIl)bS: TN(‘S:PY method. To about 50 mg of steroid in a 100 ml round-bottotn flask was added 10 ml of HODS : TICS : PY (3 : 1: 9 1. The suspension was allowed to remain overnight at 37”, after which the excess reagents and solvent w-cre removed under vacuum at 75-80” and the residue was dried under a stream of nitrogen. The derivative was dissolrcd in pctroleutn ether and the solution was washed 3 times with 70% ethanol. After t,he solvent had been removed, the product was dissolved in a small amount of benzene and filtered through a Cclitc pat1 on a small Gntercd-glass funnel; conccnt~ratiott under nitrogen afforded the deriratirc. IzSlz : PY IjMhofZ. hpprosimately TiO mg of each steroid was dissolved in 1.5-2.0 tnl of 1)yridinc to which 20 ml of BSX was added. The solution was allowed t’o stand at, room temperature overnight. Thereafter the procedural details were identical to tliosc> dc>scribed above.
All the derivatives m-ere pure as judged by the appearance of a single peak in gas-liquid chromatography (3% SE-30 on Gas Chrom Z, 250”). TMSi ethew of cortol n,lcl p-cortol. Cortol and its 20/3-epimer each formed two TMSi derivatives deljending on the reaction conditions. Cortol-3,11,20a.21-tetra-TRW ether was crystalline (calculated for C,,HG,@,Si,: C 60.3, H 10.44, Si 17.1; found: C 60.0, H 10.24, Si 17.7) and its melting point,, 166-167”, and IR spectra agreed with those of the earlier preparation (1) ; a sharp band at 3550 cm--l is characterist’ic of hydroxyl absorption. The tetra-TMSi ether of the 20/3-epimer (RRT = 3.22) could not be induced to crystallize and showed only a very slight hydrosyl absorpt,ion at 3570 cm1 Utough elemental analysis (found: C 60.2, H 10.15, Si 18.8) and t,he tnolc(~ular n-eight determined by mass spectrometry indicated a frc,c OH groul); MS: 422 (1.0) ,? 446 10.27), 406 (0.25), 524 (0.19)) 344 (0.161. The sntall OH band might be a ronsequcnce of tlttb noncrystallinity of tltch mtbatancc attcl the att’enclant difficulty of
preparing a satisfactory dispersion in potassium hromidc for infrared analysis. Both epimcre afforded crystalline derivatives with the BSA reagent and could be recrystallized from methanol or aqueous acetone with no decomposition. Cortol-3,20a,21-tri-TMSi ether (RRT = 2.92)) 137-138” (calculated for C:,,,H,,O,Si:,: C 61.7, H 10.32, Si 14.4; found: C 61.6, H 10.28, Si 13.1) showed a broad OH absorption at 3556 cnml; MS: 452 (,l .O), 288 (0.831, 272 (0.661, 362 (0.401, 344 (0.38). The 20/3+pimcr (RRT = 3.101, XXI-1.55” (found: C 61.7, H 10.25, Si 13.0) exhibited absorption at 3530 cnr’; MS: 452 (1.0)) 362 (0.40)) 288 (0.30), 446 (0.20)) 344 (0.20). Elemental analyses and the molecular weight dctcrniinetl by 111:~s::spectromctry are in agreement with three TMSi groups. C’ortolone rind /3-cortolone. Each cpimer formccl a single 3cu,20,21-triTAN derivative on reaction with either HRII>S:TMCS:PT or BSA (RRT = 2.56, and 2.69, respectively). Both TRISi ethers were oils but gare the expected molecular ion (PII, 583) in the mass spectrum; iUS (a-epimcr) : 450 (1.0) , 360 (0.66)) 272 (0.53), 287 (0.36)) 350 (0.33’1; (/3+pimer) : 450 (1.0)) 360 (0.67)) 272 (0.54)) 390 (0.37), 287 (0.36). DCspite the interference of a high background, nl)sorption at 3500 cm’ W:W observed in each deriratkc. Tetrols: 5P-P~~eyr~nne-3~,17cu,Ooc.ll-tetr~ol(lntl the 20,8-epiwer. Each cpimer afforded a single tri-TRlSi ether with either reagent, (RRT = 2.18 and 2.15, respectively) which dicl not crystallize ant1 although no hyclrosyl absorption was dctcctetl in the infrared, ISMR showed a signal at 6 = 2.62 (20~~) and 6 = 2.50 (2Op) attributable to the 17~hydroxyl group. Major ions in MS wcrc: for 20a-epimcr-436 (1 .O), 273 (0.42)) 346 (0.32)) 282 (O.l7j, 286 (0.11‘1, and for 20/3-elki~er-436 il.Oj, 346 (0.47), 273 10.331, 282 (0.17), 286 (0.16). Each of this 5~-P~egnane-S~,11~,17~~~~Ot~-tet~ol rind the 20,8-epimer. pair formed two tlcri~ativcs, a di- and a tri-TMSi ether in analogy to cortol and p-cortol; in addition, as in the cortols, three of the TMSi ethers were crystalline ~2O~tet~rol-3,20-di-TnISi i RRT = 1.87)) 233” ; 3,11,20tri-TMSi (RRT = 1.87), 134-135” ; 20,&tctrol-3,20-di-TnlSi (RRT = 1.64’)) 182-183”) ; and the foWli, 5/3-pregnant-3cu,l lp,l70,20P-tetrol3,11,20-tri-TMSi &cr (cf. the analogous /3-cortol derivative1 (RRT = 1.89) was an oil. In these 21-methyl tetrols, a broad band at 3540 cm’ in the IR spectra in the di-TMSi ethers of both epimcrs characterized hytlroxyl absorption, while the hydrovyl absorption at 3560 en-’ in the crystalline tri-TMSi &her derivwtivcl of 5~-~~regnanc~-3~~,11~,17t~,2Q~-tctrol was a sharp peak. Major ions in &IS were: Di-TMSi ethers-2(k: 272 (LO)! 254 (0.63), 214 (0.43), 362 (0.25), 290 (0.18), 230 (0.11) ; -20,G:
P-l 1g-01 P-l lp-0 TM& P-3o1,17a,2Oa-t~iol-::,%O-di-C)TRISi P-:~a,l7~,2O~-triol-Y,2O-di-OTMSi P-:~~,17~,90a,21-tet~ul-:~,2O,2l-t~i-~~T~I~i P-3~,17a,20~,21-tet~ol-~j,20,“1-tri-oTii P-3n,ll~,17a,2Oa-tetrol-Y,20-di-OTMHi P-%X, llfl,17a,20p-tet rul-3,%0-di-OTAISi I’-:$~,1 lp,17~,2O~-tetrol-3,11,20-tri-OT~ISi P-3~,llp,17~,20/3-tetrol-3,11,2O-tri-OTMSi Cortol-3,20,21-tri-OTAlSi p-C!ortol-3,20,21-tri-OTMSi C~)~tol-~~,l1,20,21-tet~,z-OT~ISi p-Cortol-3,11,%0,21-tetra-OTMSi Cortolone-3,20,21-tri-OT&ISi i3-Cortolorle-Y,‘LO,%1-1 ri-OTMSi
0.7x4 0.750 0 667 0.700 0.716 0 7.50 0.000 0. !)50 0.884 0. MOO 0, !I50 0.984 0 0 1.5 0.900 0.684 0. 650
LS-CH, 1.17 1.08 0.000 0.900 O.Yl5 0, Y39 1.17 1 .I7 I .O7 1 (07 1.15 1.1% I .07 1 .07 1.14 1.1”
19.CHI
21-CH:,
1.11 d (J = 5 Hz) 1.12d (J = 6Hzj 1.12 d (J = 6 HE) i.12d (J = 6Hz)
l.I%diJ = BHz) 1 I 0 d (J = 6 Hz )
-..--__
:;. X3 4.02 ::.65q :;.75 :-:.SS 4.05 3 X3 4.06 ::.64 ::.74q 3.64~1 S. 70 S .64 :;.67
q (J = q (J = (J = q (J = q (J = q (J = q (J = q (J = q (J = (J = (J = q (.J = 11m q (J =
7,14 Hz)
7,U Hz ) 6,l” Hz) 9,14Hz) 7,16 Hz) 7,12 IIz) 6,12 Hz) 6,12 Hz) 6,12 Hz) 2;,13 Hz) 7,18TTz) 8,12Hz) 6,lS Hz)
W-H __--
2.6.5 2 ,6.5 2.62 2.64
17-OH
386
R.
S. ROSENE’ELD
272 (l.O), 216 (0.42), 254 (0.35), 230 (0.28), 290 (0.17), 362 (0.14). TriTMSi ethers-20a: 272 11.0), 352 (0.98), 344 (0.33), 452 (0.21), 389 (0.13); -2Oj3: 272 (l.O), 362 10.26),480 (0.25), 388 (0.17), 344 (0.14), 452 (0.10). PTegnnne-.%,17&Oa-t,riol cmd the ?0,8-epimer. The substances formed di-TM% et,hers wit.h either reagent; the 20a-epimer Ivas crystalline, 200204” (calculated for C,,H,,O,Si,: C 67.6, H 10.72, Si 11.7; found: C 67.6, H 10.71, Pi 11.7), and the 20/3-dcrivat,ive (RRT = 1.181 was an oil. Pregnane-3~,17u,2Qa-triol-3,20-di-TMSi ether showed a narrow, intense> ba.nd at 3572 cm-l (17a-OH) in the IR while the 20P-epimer had a weak absorpt#ion at 3570 cm’; MS (20/3), 256 (l.O), 274 10.74): 252 (0.16)) 284 (0.15)) 364 (0.10). Pregnane-11,8-o/. The TMSi ether, a crystalline compound, 104-105” was prepared IHMI)S:TMS:PY method) in or&r to compare the effect of the TMSi moict’y on the angular methyl signals in the NMR spectra of a simpler compound with those of t’he derivatives of the more highly hydroxylated C,, steroids. The 19-H signal of the underivatized compound (6 = 1.17) occurred at almost the same posit’ion as the C-19 resonances of all the derivatives of the more highly substitutecl TMSi ethers bearing a free 11-hydrosy group (Table 1) and the upfirld shift 1-5 Hz) associated with the sl&Iding by the TMSi function also correxponclcd to the tetrol and pcntol derivat’ives with an ll-0-TMSi group. The 18-H rcsonancc (6 = 0.784) in pregnane-lip-ol was in accord with the value calculated from data which consider the shielding effect of the 17/3-ethyl and t,he dcshielding by the lip-hydrosy (4). The downfield shift of 7-12 Hz in t’he 1%methyl signals of the pent01 and t&o1 derivative:: with an II-hydroxy group must be a consequence of the resultant deshiclrling and shielding influences of the l7a-hydrosy and C-20-TMPi moieties, respectively.
Signals associated with the more salient fcat,ures of the TMSi ethers arc recorded in Table 1. In addition, all derivatives showed a strong signal at S = 0.117 ascribed to the protons of the TMSi moiety. hlso substances t,hat possess the ll-OH or 1 l-0-TMSi function displayed a broad multiplct at 6 = 4.23 characterist,ic of the ll,n-proton. I<‘?-Ueth !/Z. The 18-H peak in substances that. are unsuhstituted or have a ketone at C-11 was shifted only slightly from that in the unsubstituted 5P-androAanc (4) ; :ipparcntly tlicx resultant. of the iilt,eractions of the heavily suhstituted side chain is such that the C,, signal is little affected. The 11-O-TMSi suhstitucnt produced a downfield +hift# of from S-10 Hz as comparcd with the C-11 unsubstituted analogs, This shift was even
larger in the derivatives 1v11cire tllc 1l-011 is fr(~t, th:bi is, the 1 I-Tlldi frinctioil cxcrtc~tl :L blight clcsllic~ltling c~fl’wt 011 the 18-l-I. 111the c~l~irncric l)air~;, witli tlrcs cxccsl’tion of’ tllc~ c~~~~otolo~~c~.~, tll(b 18-ln(~tllyl signal iI\ the 2O,L?-(~l)iin(~rn-w slightly clo~~l~fic~l~li’rotrr tllcs 20~. I.%Mefh!//. Tlrc~ cl\Ariv:lti\-cs* po.G,wssitig 110 .s;lItwtiluc,nt at. C-11 alion-ed 19-H signals ( 6 ~: 0.900~(I.933 I tll:Lt writ virtrlally un&fted from the “naked” steroid nuc~le~k (6 = 0.925). ‘NIV 1 I/!?-li;\-~lrosy group as well as the I I-ketone exerted :L l>~rgc~cl(~~llic~ltiitlg c+fc.c~t (ca. 14 Hz downfield) whicsh was less pronounced ill tllc 11-t )-‘I’,\ISi c~~1n~~oundsnherc the TM% (Lxerted a shielding influence. Tliv c*olifigur:~tiwi at, C-20 has little effect, on the 19-H peak. ,“I-NethU1. 811 of the TRlSi ethers of the compounds unsubstituted at C-21, namc’ly, the epimeric .5/3-pregnane-3t~,l7a,20-t~riols and 5/3-pregnane3t~,llj3,17t~,2O-tetrols, show:cll an intense doublet centered at 6 = l.lOO1.115 (.J, 6 Hz) which is a consequence of coupling with the proton at C-20. C‘-NO-P~fotl. In all the derivnt’ives measured, the signal for the 20-H appeared as a quartet centered in the range 6 = 3.67-4.06; in every cast t,he 20~H (20/3-0-TMSi epimer) was downfield from the 20/3-H signals. The TMSi ether function at C-21 exertecl a shielding effect on the 20 proton, showing upfield shifts of about 11-12 Hz in the 20tu-epimers and 1622 Hz in t’he SOP-compou11ds. 17’~OH Proton. h singlet in the r:mgc’ from S = 2.18-2.65 was observed in every TMSi ether, except prcgnan-I lp-01 TAIPi, regardless of whether the 11/k?-hytlrosyl was free or silylated. The signal disappeared on equilibration with deuterium oxide, a characteristic behavior of the labile hyclrogen of a hytlrosyl group. This observation, particularly in the tleriratives prepared by the procedure in which the I l/3-hydroxyl undergoes trimet~hyleilylation, was additional evidence for the presence of the free I ‘itu-liyclrosyl group in this series. Mass
Spect,rn
The TMSi ethers prepared in this study showed complicated fragmentation patterns in mass spectroscopy (Fig. 1) ; however, for each compound the molerular ion was seen which agreed with the expected molecular weight of the derivative. Thus. under the reaction condit’ions, the 1‘ia-hydroxyl group did not form a TnISi ether (3). The mars spectra of c~onq~ounds epimcric at. C-20 wcr(~ veq similar. Bctwvccn M/e 583 ant1 Al1,‘e 272 of the epimeric cortolone-tri-TAISi ethers. ions of qua1 relative intekties wcrc present in both spectra; no compnriron was made beyond t’his.
388
FIG.
1. Mass
spectra
of typical
GO,-TMSi
ethers.
All compounds showed loss of 15 mass units, corresponding to a CH,, group (5,6). In addition, ions corresponding to AI-90 (loss of trimethylsilanol) and/or AI-180 (2 X 90) appeared in all spectra (5-7) and, as might be expected, ions associated with the loss of combinations of these fragments were observed, for example, PI- (90 + 15), ?II- (2 X 90 + 15)) etc. Mass spectra of compounds having the -CH (C)T,VSi)-CH, (OTMSi) and -CH(OTRISi)-CH, side chains showed certain characteristic differences (Fig. 1), e.g., the presence of XI-103 ions in t#hc former. Steroids with vicinal 0-TMSi groups (5,8) and TMSi ethers of primary alcohols 15,6) show this elimination, which has been attributed to the low of CH,O=TMSi (6). An intense X-133 ion was probably the resultant of two ionic species: M- (103 + 2 X 15) and RI- (90 + 28 + 15)) where 28 mass units represents the loss of carbons 3 and 4 after elimination of T?tlSi-OH in the A ring (5). The loss of t,no fragments, 90 and 133, probably accounted for the intense ion at M/‘e 346 (&I-223) in the 21TMSi ether derivatives while the &1/e 273 (N-296) ion couhl be ascribed to the loss of the side chain (206 mass units) and trin~ethylsilanol (90 mass units). The mass spectra of the 5 compounds with a 21-methyl group (hut not of the other compounds) had ions at N-45. It is possible that this reprosents the loss of a CH:,-CH-OH fragment concomitant with the replacement of tbc TL*ISi group from the C,, by a proton frown thr C-17hydroxyl; loss of the side chain was indicated by the fragment RI-117. In conkast to the compounds hydroxylated at C-21, the N-133 ions wcrc
of low rclativc nl~~ndancc although the M-(90 + 133) ions n-erc prewnt. Ions formed by elimination of the side chain plus one trimetliylsilanol ncre also present (M-207).
Trimethyldyl ethers of fivcb l)air hs of compountis of the 5P-lxcgnane scrie* epimeric at C-20, have been prepared, isolated, and characterized; the clbrivatives xrc stahlc in neutral media. Aksignment of configuration at, C-20 of a pair may hc made OII tklc basis of STIR spectra. Some charwtcristic fragmentat’ion patterns in the msli: c Ls spectra have been reported. ACKNOWLEDGMENTS The support and interest of Dr. T. F. Gallagher are greatly appreciated. I also wish t.o thank Mrs. Julia S. C. Liang for her invaluable help in the measurrm~nt. and interprpktion of the SMR sprctra. This investigation was supported by a grant from the American Cancrr So&t> and by grants CA-07304 from the Nat,ional Cancer Institute and RR-53 from thv Gtncral Clinical Rrsearch Centers Branch. National Institutes of Health. REFERENCE&S 1. ROBENFELD! 2. ROSENFWD. B. Lipsctt,.
3. 4. 5. 6. 7.
S.
R. S.. Steroids 4, 147 (1964). R. S.. ilL “Gas Chromatography ed.), 1,. 127. Plenum Press, New
of Strroids in Biological Fluids” (nl. York, 1965. CHASIRAZ, E. M., AA-DHORNING. E. C.. il~d. Biuchem. 30, 7 (1969). of NMR Spectra in Orgmk BHACCA, N. S., AND WILLIAMS, D. S.. “Applivation Chemistry.” Holden-Day, San Fran&co, 1964. HUHTANIEMI, I., LUUKKAINEK. T., .~ND VIHKO, R.. k/n Bndoo-itwl. 64, 273 (1970). HAMX~RSTROM. S., J. Lipid Re.s. 11, 175 (1970). EYEROTH. P.. HELLSTRBM, K.. .ASD RYHAG~. R., J. Lipid Res. 5, 245 (1964). ~~s~o.4. B. P.. GrJSTAFSSoN, J. .&., AND SJOVALI,, J., Ewopenil 1. Biochem. 4, 496 (196%.