Indian Journal of Chemistry Vol. 38B, November 1999, pp. 1253 - 1261

Investigations on photochemical linking of steroids with amino acids: Irradiation of <1, ~-unsaturated steroidal ketones in the presence of

amino acids in aqueous medium

M P S Ishar*, N K Girdhar, K Kumar, Rama & S Kaur Department of Pharmaceutical Sciences, Guru Nanak Dev University Arnritsar 143 005, India

Received 10 August 1998; accepted 14 December 1998

Irradiation of I, 4-cholestadien-3-one I in THF-H20 solvent in the presence of amino acids (glycine/alanine) leads to dienone-phenol photo rearrangement products, 3-hydroxy-l-methyl-19-norcholesta-l , 3, 5 (10) -triene 3 (35%), 4-hydroxy-2-methyl-19-nor-cholesta-l, 3, 5 (10) -triene 4 (15%), l-hydroxy-4-methyl-19-norcholesta-l , 3, 5 (10) -triene 5 (28%) along with a novel methyl substituted-19-norcholesta-l , 3, 5 (1O)-triene 6 and a spirocycl ic-enone 7; no amino acid or sol vent addition product is obtained. Irradiation of 16-dehydropregnenolone-3p-acetate 2 under identical conditions 'in the presence of glycine affords the pregnenolone-3p-acetate 9 (25%), 16-(tetrahydrofuran-2-yl) -pregnenolone-3p-aceta te 10 and its 17-a-isomer 11 (together 35%) and 16-(glycin-2-yl) -pregnenolone-3p-acetate 12 (mixture of isomers, 25%). The results are compared and contrasted with known photochemical behaviour of steroidal enones and dienones. The studies indicate that steroidal-16-ene-20-ones can be better photoaffinity labelling agents for progestogen and possibly, adrenal steroidal receptors. Also, the photochemical additions' to l1 16-20-one steroidal systems can be exploited for the introduction of substituentslmoieties at C I6 in steroids.

Conjugated steroidal ketones have been employed for photoaffinity labelling of steroidal receptors I though no photoaddition products have been isolated or identified. It has been demonstrated that irradiation of an a, l3-unsaturated steroidal ketone in the presence of f:.5 -3-keto-steroid isomerase2 leads to de-carboxylation of an aspartate residue. A steroidal ketone-dependent photoinactivation of estrogen binding site in estrogen receptor is also reported3

. In principle, photoexcited conjugated steroidal ketones can either react with nucleophilic centres in proteins or abstract a hydrogen from an amino acid residue and a combination of radical species in latter case can also lead to cross­linking of steroids with proteins; the latter possibility has been only indirectly demonstrated on the basi s of the results of irradiation of testosterone acetate in toluene4

. We have presently investigated the photochemistry of conjugated steroidal ketones, I, 4-cholestadien-3-one 1 and 16-dehydro-pregnenolone-313-acetate 2, in the presence of amino acids, in aqueous medium. The investigations were also of interest for developing a methodology for one-step photoalkylation of steroids with amino acid residues; steroids bearing carboxy and/or amino functions have useful medicinal properties5

, and are also being exploited as vectors for anticancer molecules/moietie6

•

Results and Discussion

(A) Irradiation of f:.1 , 4-cholestadien-3-one 1 in THF -H20 solvent in the presence of glycine/alanine

Irradiation of f:.1 . q-cholestadienone 1 in the presence of glycine for 20 hr led to the products 3-7, (Scheme I). The assigned structures are based on spectroscopic data (UV, IR, IH NMR, I3C NMR and Mass); the photodienone-phenol rearrangement products of cholestadienone have been characterized for the first time.

The major product (- 35%) has been characterized as 3-hydroxy-l-methyl-19-norcholesta-l, 3, 5 (1 0) -triene 3. Its mass spectrum exhibited M+ ion at rn/z 382 and its IR spectrum displayed a band at 3460 (-OH); no carbonyl band was present in the IR spectrum. The IH NMR spectral assignments of proton resonances of ring-A and CI-methyl in 3 compared favourably with reported proton chemical shift data for I-methylestradiol, 7a, b, and related derivatives of androstane and prednisone: 7c, d. 8 The structure is corroborated by I3C NMR assignments8

. 9

of the carbons of aromatic ring [8153.84 (C3) , 140.14 (Cs), 138.74 (C I), 131.54 (C IO), 115.96 (C2) , and 113.34 (C4)] .

The second compound which was obtained in 15%

1254 INDIAN J CHEM, SEC B, NOVEMBER 1999

+CIyclne/ Alanine, hv, Pyrex

TIIF-~O, 20 hr

5

3 4

6

Scheme I

yield was assigned the structure as 4-hydroxy-2-me.thyl-19-norcholesta-l, 3, 5 (10) -triene 4. Here agairl the proton NMR assignments were aided by a comparison of the data with reported proton chemical sh ift · for 4-hydroxy-2-methyl-L1I, 3, 5 (10) -estratriene7a

The assigned structure was supported by the 13C NMR spectrum which exhibited two un substituted (CH) aromatic carbons at 0 122.64 and 114,53 indicating that these are, respectively, pgra- (C I), and ortho­(C3), to the hydroxyl substituent9

; other aromatic carbons appeared at 0 155.61 (C4), 140.63 (CIO), 137.04 (C2) , and 128.19 (C5).

The third component (28%) had same mass (M+ at m/z 382), and its IH NMR spectrum (Figure la) exhibited two doublets at 06.82 and 6.44 (J = 7.8 Hz) indicating the fresence of two vicinal aromatic hydrogens. The H NMR spectrum further exhibited an OH proton resonance at 0 4.68 (b singlet, suppressed on shaking with a drop of 0 20) and a 3H singlet at 0 2.13 which was attributed to C4-methyl. On the basis of comparison of the above spectroscopic data with those reported for I-hydroxy-4-methyl-estra-l, 3, 5 (10) -triene7a

, the structure of this compound was assigned as l-hydroxy-4-methyl-19-norcholesta-l, 3, 5 (10)-triene 5. The assigned

13 . structure was supported by C NMR spectrum which exhibited two methine carbons in the aromatic region at 0127.40 (attributed to C3, meta to hydroxyl substituent9

) and 1 12.88 (C2, ortho to hydroxyl substituent\ It may be mentioned here that Barton et at. 7r, had arrived at a l-hydroxy-4-methyl arrangement in ring-A for a photo~product of prednisone and Outler et af. 7". b, have also identified a

related phenol amongst the photoproducts of L11 , ~­testosterone-1713-acetate, Howeve~: structure 8 has been assigned for a related disubstituted-ring-A­aromatised-rroduct obtained on irradiation of prednisone I , In the latter instance lO the assignment has been based on I3C NMR chemical shift value of CI-methyl group (0 14.4) in 8 which reportedly is upfield shifted by 6.5 ppm, and hence it has been inferred that the methyl group is o-substituted to a hydroxyl function ; no such upfield shifted methyl resonance was present in the I3C NMR spectrum of 5 which a lso differea from the one reported lO for 8 in the aromatic region.

8

Compound 6 was obtained only in a mixture with 5 and the assigned expression was conjectured on the basis of IH NMR spectrum of the mixture only. The IH NMR of the mixture (Figure Ib) di splayed, besides spectral features of 5, a broad singlet at 0 7.23 (Arom.-Hs) and a singlet of comparable integrated intensity at 0 2.37 . The singlet at 0 2.37 was characteristic of a methyl substituent on an aromatic ring9

• II in 6. Appearance of a 3H singlet at 8 7.23 for aromatic-Hs was indicative of the absence of a hydrbxyl substituent on ring-A in 6. The I3C NMR

ISHAR et al.: PHOTOCHEMICAL LINKING OF STEROIDS WITH AMINO ACIDS 1255

(a)

5

8 6 4 2 o

+ 5

( b)

6

86420 Fig.l (a)lH NMR spectrumof l-hydroxy-4-meth>,l-cholestra-1 ,3,5(1 O}-triene (5) in CDCh (100MHz)

(b)lH NMR spectrum ofa mixture of(5) and (6) in CDCh (100MHz).

spectrum of the mixture is also in consonance with the assigned structure of its components.

Yet another product isolated from the reaction (~4%) exhibited the presence of an a, 13-unsaturated carbonyl function through a band in its IR spectrum at 1685 em- I which was also corroborated by its UY spectrum. It also exhibited the M+ ion in its mass spectrum at mlz 382. Its I H NMR spectrum exhibited only a IH broad singlet in the olefinic region at 85.40, a broad singlet (I H) at 2.36 and a doublet (3 H) at 2.0 I (J=I.47 Hz) . Structure of this component was assigned as a , 13-unsaturated-spirocyclic ketone 7 on the basis of coniparison of its 'H NMR spectrum with the data reported for a related spirocyclic ketone obtained from phototransformation of I, 4-androstadiene-3-one-1713- acetate7a. Comparable results were obtained on irradiation of 1 under above conditions in the presence of alanine.

(B): Irradiation of 16-dehydropregnenolone acetate (2) in THF-H20 mixture in the presence of glycine/ alanine

Irradiation of 16-dehydropregeneolone-313-acetate

2 in THF-H 20 in the presence of glycine for 16 hr led to the formation of products 9-12 (Scheme II) . Characterization of the products was again based on detailed spectroscopic analysis of the products and comparison of the spectroscopic data with those of the related steroidal systems.

Pregnenolone-313-acetate 9 (25%) was easily identified on the basis of its mp l2 lR and IH NMR spectral datal } . .It may be ment~ed here that we could find only partial 'H NMR data for 9 iIi literature 13

, therefore, its spectral data has been included. The stereochemistry at C I7 is based on IH NMR chemical shift value for 18-methyll3a.

Compounds 10 and 11 (together ~ 35~ were obtained as two mixture fractions, one of which was rich in 10 (~ 2: I) while other was rich in 11 (~ 1 :2) and both these fractions could not be resolved further ; the assigned structures are based on detailed spectroscopic analysis of their mixture . The lR spectrum of the mixture exhibited acetate carbonyl at 173 0 cm' l and C20 carbonyl band at 1708 em- I (tending to split), the latter indicating the loss of

1256 INDIAN J CHEM, SEC B, NOVEMBER 1999

4 6 2

16

~Iydn .. hv, Pyrn.

THF.HZO, " br

9

11

5'

+ +

10

+

12

Scheme II

unsaturation at C16. 17. The proton NMR revealed, besides C3-H resonance (m at 8 4.52) and C6-H resonance (doublet at 8 5.34) overlapping IT\,ultiplets at 8 3.80 and 3.65 (C2'-H and Cs'-Hs) indicating the incorporation of tetrahydrofuranyl moietyl4. Among the two singlets (together 3H) at 8 2.09 and 2.06, the one at 2.06 was attributed to CwHs in 10 and other was assigned to CwHs in 11 based on reported l3

variation of chemical shifts of CwHs in related steroidal systems as 'a function of stereochemistry at C 17 • The structural assignments were further conroborated 13 by the chemical shift value of Cls-Hs (si nglet at 8 0.58 in 10 and at 1.17 in 11). The I3C NMR spectrum of the mixture also confinmed the presence of isomeric structures through the presence of two carbonyl (C20) resonances at 8 206.68 and 206.25, two resonances at 83. nand 82.18 (C2' in isomeric structures) and two resonances at 66.00 and 65.02 (C 17 in isomeric structures); other oxygen linked carbons appeared at 863.23 (Cs') and n .28 (C3). The assigned position of tetrahydrofuranyl moiety was suggested by mechanistic considerationsl4

and was corroborated by I3C NMR chemical shift value for C 17 . The mass spectrum (M+ ion a m/z 428) and microanalytical data of the mixture were in consonance with the molecular composition. The available spectral data suggest a single geometric arrangement at C 16; an a-orientation of tetrahydro­furanyl moiety at CI6 is anticipated.

Another component isolated was tentatively

assigned the structure as 12 (20%) and was found to be a mixture of isomeric structures CIH NMR). In mass spectrum it exhibited the highest peak at rnIz 431 which corresponded to I: I addition product of 2 with glycine which was also corroborated by microanalytical data. Its IR spectrum exhibited a broad absorption at 3280 cm -I, besides carbonyl bands at 1740 (b) Int) and 1700 (sh) cm-I. The IH NMR spectrum of the mixture also indicated the absence of C 16. 17 double bond as only one olefinic-H resonance (C6-H) was located as a broad doublet at 8 5.37. The incorporation of amino acid was confinmed by the presence of two singlets at 8 11 .00 and 9.89 (together I H, carboxylic-H in isomeric structures), a 2H multiplet at 8 4.70, which also included the C)-H resonance, and another 2H broad multiplet in the region 8 3.96-3.40; cf.IS the observed IH NMR spectral pattern was suggestive of linking of amino acid residue through a C-C bond. The presence of isomeric arrangements involving C 17-side chain and possibly also the substituents at C I6 was indicated by the presence, amidst steroidal mulitplet (8 2.24-0.62) of singlets at 82.18, 2.12 (CwHs in isomeric structure) 1.20, 1.09, 0.98, o.n and 0.64 (C 18- and CwHs in isomeric structures).

Irradiation of 2 in the presence of alanine under the above conditions gave compounds 9-11 in comparable yields, beside a component (10%) whose IR bands at 3240, 1740, 1720 and 1705 cm-I and IH NMR signals at 85.63 (broad d, J= 7.5 Hz), 5.36 (d,

ISHAR e/ al. : PHOTOCHEMICAL LINKING OF STEROIDS WITH AMINO ACIDS 1257

4-H), 4.64 (m) 4.18 (m) 3.46 (m) and 2.60-0.56 (complex) indicated the incorporation of alanine residue. However, it could not be resolved sufficiently for any structural assignment.

Mechanistically, the cyclohexadienone-phenol rearrangement has been extensively investigated7, 10, 16 However, an interesting aspect of the present investigations is the non-isolation of any solvent or amino acid addition product, particularly, in the light of fact that many ionic intermediates and strained structures have been proposed as intermediates in the photochemical dienone-phenol rearrangement. This is also in contrast to the both reported photoaddition of toluene to steroidal enones4 and obtaining of water/methanoUacetic acid addition products like 13 and 14 on irradiation of cross-conju~ated-cyclic­dienones under varied conditions7b, 10, 1 , I compounds 13 and 14 have been reported to be derived from addition of components of solvent to cyclopropano­intermediate A (Scheme ill).

Interestingly, whereas Kroppl6b has reported the isolation of methanol addition product 13, (R = Me) Jeger et al.7b have reported the exclusive formation of phenolic compounds on irradiation of a steroidal­dienone in methanol. A temperature dependence of overall yield of addition compounds like 13 and 14 has also been reported7b, 16b. This addition of water or acid (ace~ic acid) during phototransformation may be requiring more acidic conditions than · the ones prevalent during present investigations. It may be mentioned here that such water addition products like 13 and 14 have also been isolated on enzymatic transformations of steroidal-dienones8, along with similar phenolic substances as major products as are obtained on phototransformations. The mechanistic proposals for the enzymatic transformations do not include the intermediates like A and acid-catalysed skeletal rearrangement is postulated.

eo@~_b-=,"V.-:-,H3'-O_+ __ ~ So/lent

A

Another important feature of the present investigations is the formation of compound 6. Though it has been partially characterized as it is obatined only in mixture with 5, its formation involves reduction of carbonyl function and its eventual loss. The reduction step probably involves the electron transfer from carboxylate function of glycine to the photoexcited ketone (cf. 18). Steroidal enones have been reported to bring about decarboxylation of aspartate residues in enzymatic proteins2, and oxidative damage to protein by a photoexcited steroidal ketone has been reported to cause inactivation of esterogen binding site of esterogen receptor3. Mechanistic aspects of the ketone-sensitized decarboxylation of carboxylic acids have been delineated8b.

Mechanistically, the phototransformations leading to products 9 - 12 can be summarized as in Scheme IV. Here the compound 9 results from photoreduction of a , f3-unsaturated carbonyl system and involves H-abstraction by photoexcited carbonyl group. Though, quite often, such H-abstraction by a, f3-unsaturated-carbonyl system leads to photo­peconjugation l9, however, photoreductions of a , f3-unsaturated carbonyl compounds are also precedented20. Reductive photoadditions to a , f3-unsaturated carbonyl systems, in the present case leading to compounds pp 10-12, are generally observed under electron transfer conditions l4, 18a, 21. Recently, an electron transfer mechanism has also been proposed for photoaddition of tetrahydrofuranyl moiety to anilsI4~. Obtaining of a single stereochemical disposition at C 17 in 9 is anticipated due to pseudoequitorial orientation of side chain . Compounds 10 and 11, differing in stereochemistry at C 17, were obtained in comparable yields; stereochemistry at CI6 could not be unequivocally ascertained, though the anticipated orientation for a

~ o

13 14

R = H, C113. CH:J-c0-

Scheme III

1258 INDIAN J CHEM, SEC B, NOVEMBER 1999

1

hI' • (2 t n-7t*

~o 9

~ [c:&:~ AbS~~,ctii

I. Radical Coupling II. Tautomerization

~ 10,11,12

10, 11 :X,Y-O o

12 : X=~H

Y= NII2

Scheme IV

substituent at C 16 is a, and the obtained spectroscopic data suggest a single geometric orientation at C 16•

Component 12 was found to be a mixture of isomers involving stereochemical variations at C l7 and probably also at C16•

Present investigations have established that steroidal ring-A - dienones cannot be employed for photoaffinity labet/ing of steroidal receptors. However, photoexcited dienones can be reduced in the presence of aminoacid residues and leading to modification of receptor proteins. On the other hand, 16, 17-dehydro-20-one steroids can be quite valuable for photoaffinity labelling of both progestogen and adrenal steroidal (glucocorticoid and mineralocorticoid) receptors. The observed photochemical addition at C16 in ~16-20-one system has immense syntheti"c potential for attachment of varied substituents at C 16 and is being further investigated.

Experimental Section

General. ~l, 4-Cholestadienone 1 was prepared by dehydrogenation of ~4 -cholestenone with DDQ22 and characterized spectroscopically; ~ 4-cholestenone was prepared by Oppenauer oxidation of cholesterol23 . 16-Dehydropregnenolone-313-acetate 2 was procured

from market, recrystallised from aqueous acetone and checked spectroscopically. UV spectra were recorded on a Shimadzu 160A UV -visible spectrophotometer and IR spectra on a Shimadzu DR-800 1 FTIR

spectrophotometer. NMR spectra were recorded on JEOL-JNM-IOO FT NMR and Bruker AC-200 NMR spectrometers using CDCh as solvent with TMS as a internal standard. Mass spectra were recorded on a JEOL-JMS-D-300 mass spectrometer. Microanalyses were carried out on a Perkin-Elmer 240C elemental analyser.

General procedure for inadiation. Steroid (500 mg) was so lubilized in 250 mL of aqueous tetrahydrofuran (THF) along with excess of amino acid (glycine or alanine, - 10 equivalents). The THF­H20 ratio (7:3 to 4: I) was adjusted so that both amino acid and steroid are solubilized and a clear solution is obtained. The irradiation was carried out in a pyrex glass immersion-we ll photoreactor employing a 125 Watt medium pressure coaxial mercury arc. Solution was purged with oxygen free nitrogen for 15 min prior to irradiation and the nitrogen was continuously bubbled through the reaction mixture during irradiation . Progress of the reaction was monitored by TLC and irradiation stopped when steroid had

ISHAR et al.: PHOTOCHEMICAL LINKING OF STEROIDS WITH AMINO ACIDS 1259

completely reacted. On evaporating THF under reduced pressure a solid (steroid) seJllU1lted out. More water (-100 mL) was added and the contents were extracted with chloroform (150 mL, twice); the residue obtained on evaporation of aqueous layer contained only amino acid. The chloroform extract was dried over anhydrous MgS04, and solvent was again removed under reduced pressure. The residue obtained was column chromatographed on silica gel to obtain various components, some of which ,were further purified by preparative TLC and their spectroscopic data were collected.

Irradiation of £\1, 4 -cholestadien-3-one 1 in the presence of Glycine. Irradiation of 1, (500 mg) in the presence of glycine (1 g) in THF-H20 for 20 hr under conditions as described above, and chromatographic resolution afforded the following products.

3-Hydroxy-1-metbyl - 19- norcbolesta-1, 3, 5 (1 O~-triene 3: A gummy material, (175 mg); Amax

(MeOH): 282 nm (E 2800); Y rnax (CCI4) : 3460, 3010, 2950, 2980, 2900, 1620, 1600, 1500, 1480, 1320, 870, 860 cm-I; IH NMR (COCh): 06.52 and 6.48 (singlets, IH each, C2-H and C4-H), 5.10 (br s, IH, suppressed on shaking with 0 20 , OH) 2.96-0.68 [complex m with singlets at 0 2.26 (CI-Me) 0.87, 0.83 and 0.68]; 13C NMR (CDCh) : 0 153 .84 (C3), 140.14 (C5) , 138.74 (CI), 131.54 (C IO) , 115.96 (C2), 113.34 (C4), 56.58, 55 .70, 46.23, 43 .53, 41.44, 40.65, 38.60, 35.60, 32.00> 29.00, 28.40,27.80, 27.20,26.21 , 25.40, 24.23,22.31 , 21.80, 21.40,18.70,12.6; MS: mJz 384

(5, ~+2) 383 (30, ~+I) 382 (100, M+) 242, 247, 207, 205, 186,174, 173, 147, 111,109,97,95,85,83, 81, 71.

4-Hydroxy-2-methyl -19-norcholesta-l, 3, 5 (10) -triene 4: A gummy material (65 mg); Amax(MeOH): 280 nm (E 2300); Vrnax (CCI4): 3485, 3010, 2975, 2950, 2850, 1610, 1590, 1500, 1460, J 320, 840 and 825 cm-I; IH NMR (COCb) : 0 6.53 (s, IH, CI-H) 6.36 (s, IH, C3-H) 4.64 (bs, IH, OH) 3.04-0.64 [complex with singlets at 0 2.20 (CrMe) O.9Q, 0.87 and 0.72] ; 13C NMR (COCb) : 0 155.61 (C4) , 140.63 (C IO) , 137.04 (C2), 128.19 (C5) , 122.64 (el ) , 114.53 (C3) , 56.71 , 55.53, 46.21,44.39, 40.87, 39.63 , 36.26, 35 .96,31.64, 29.74,29.49, 28.34, 26.73 , 26.44, 24.22, 23.95, 22.85 , 22.63, 21 .92, 18.67 and 12.66; MS : mJz 384 (6, M++2) 383 (30, ~+1) 382 (100, ~) 199, 186, 174, 163,160, 147, 71 , 70.

I-Hydroxy-4-metbyl-19-norcbolesta-1, 3, 5 (10) -triene 5: Gummy material (120 mg); Amax (MeOH) : 280 nm (E 2860): Vrnax (CCI4): 3485, 3110, 2895,

2875, 1610, 1590, 1510, 1465, 1315,816 em-I; IH NMR (CDCh): 0 6.82 (d, IH, J = 7.8 Hz, C3 -H), 6.44 (d, 1 H, J = 7.8 Hz, C2 -H) 4.68 (brs, 1 H, exchangeable with deuterium, -OH) , 2.96-0.64 [complex with singlets at 0 2.13 (C4-Me) 0.88, 0.83 and 0.69); \3C NMR (COCh) : 0 153.40 (CI), 138.82 (Cs), 128.61 (C4), 127.40 (C3), 125.16 (C IO), 112.88 (C2) , 56.62, 55.44, 44.50, 43 .00, 40.62, 39.:40, 36.80, 32.~4, 30.28, 29.61, 28.84, 27.64, 26.20, 25 .84, 23.63, 22.42, 22.19, 19.04 (C4-Me), 18.27, 12.24; MS : mJz 383 (10, ~+1) 382 (100, ~) 367, 366, '238, 199, 174, 173,160,147,125,123,122, 121 , 109,95,85, 84,83 ,. 8.1 , 71.

Another gummy material (50 mg) was also obtained which was found to be a mixture of 5 with another compound (;; . IH NMR (COCh) :0 7.23 (bs, -3H, Ar-Hs in-6), 6.82 (d, IH, J= 7.8 Hz, C3-H in 5), 6.44 (d, IH, J= 7.8 Hz, C2-H in 5), 4.72 (bs, IH, -OH in 5), 3.00-0.60 [complex multiplet with singlets at 0 2.37 (3H, Me- on ring-A in 6), 2.12 (C4-Me in 5), 0.89, 0.84,0.68]; \3C NMR (CDC h) : 0 153.4, 140.0, 138.8, 135.6, 129.8, 129.2, 128.6, 128.0, 127.4, 126.2, 125.2, 112.8, 57.3, 56.6, 55.4, 45.5, 44.0, 43.0, 41.6, 40 .6, 40.0, 36.8, 32.2, 30.3, 29.6, 29.0, 28.8, 27.6, 27.2, 26.2, 25.8, 23.6, 23.1, 22.4, 22.2, 21.7, 19.8, 19.1 , 18.3,12.2.

a , J3-Unsaturated-spirocyclic-ketone 7: A semisolid (-20 mg); A.nax (Methanol) : 260 (E 2560) and 235 nm (E 4520); Vrnax (CC~) : 1685, 1654, 1600, 1480, 1460, 1420, 1'360, 1210 cm-I; IH NMR (CDCh): 05.40 (bs, 1 HO, olefinic-H) 2.66-0.64 [complex multiplet, with broad singlet at 2.36 (1H) doublet at 2.01 (3H, J= 1.47 Hz) and singlets at 1.22, 0.99, 0, 88, 0.82 and 0.68]; MS: mJz 383 (4, M+ -1), 382 (42, M+), 367 (24, ~-15), 342 (45), 339 (65), 300 (22),299 (100).

Almost comparable results were obtained when £\14-cholestadiene-3-one was irradiated under identical conditions in the presence of alanine for 20 hr.

Irradiation of 16-dehydropregnenolone-3!}-acetate 2 in the presence of glycine. Irradiation of 2 (500 mg) in the presence of glycine (1 .0 g) under conditions described above, for 16 hr and cru:omatographi'c resolution of the photo lysate afforded the following products.

Pregnenoione-3l>-acetate 9: Yield 1.~5 mg, colourless crystals (ethanol) mp 148.5°C (Iit. ' 2 mp 149-151 °C); Vrnax (KBr) : 1-730 (acetate) 1710 (C=O) 1400, 1385, 1260, 1175, 1150 cm-I; IH NMR (CDC b) : 0 5.3 7 (bd, 1 H, .1=4.4 Hz, C6-H) 4.62 (m, 1 H, C3-H) 2.64-0.64 [m with singlets at 02.12 (Cw

1260 INDIAN J CREM, SEC B, NOVEMBER 1999

Hs)i 2.03 (CH3C02-) 1.02 (C I9-Hs) and 0.63 (CwHs)]; I3C NMR (CDCh) : 0 208.43 (C20) 170.03 (02CCH3) 139.72 (C,),. 122.35 (C6) 73.73 (C3), 63.71 (C 17)

56.51,49.94,43.95,38.84,38.08,37.02,36.06,31.56, 29.68,27.75,24.52,22.87,21.41,21.05,20.73, 19.29 and 13.25; MS: mlz 314 (~-44) 298 (20, M+-60) 297,282,227,213,177,153,147,145, 109,107, 105, 97,95,85, 79, 78, 77,55 (90) 43 (100).

Mixture of 16-(tetrahydrofuran-2-yl) pregneilol­one-3~acetate 10 and its 17a isomer 11: Yield ~21 0 mg; Vrnax (CC4) : 2980, 2940, 2900, 1730, 1708 (split) 1640, 1470, 1400, 1370, 1280 and 1250 em-I; IH NMR (CDCh): 0 5.34 (bd, IH, J=4.18 Hz, C6-H) 4.52 (m, 1 H, C3-H) 3.80 and 3.65 (overlapping ms, together 3H, C2'-H and Cs'-Hs) 2.64-0.60 (complex multiplet with singlets at 02.09 (CwHs in 10) 2.06 (C2yHs in 11) 1.95 (CH3C02-) 1.17 (CwHs in 11) 0.98 (CwHs) and 0.58 (CwHs in 10)]; 13C NMR (CDCh): 0 206.68 and 206.25 (C20 in 11 and 10) 171.00 (CH3COr ) 139.55 (Cs), 122.26 (C6 in 10 and 11) 83.72 and 82.18 (C2' in isomeric structures) 72.28 (C3), 66.00, 65.02 (C 17 in isomeric structures) 63.23 (Cs') 55.75, 54.45 49.97, 44.80, 42.85, 41.00, 38.78, 38.05,36.95,36.58,32.02,31.66,30.11 , 29.58,28.80, 27.69,27.42,26.66,26.21,23.58,21.63 , 19.89,18.18, 15.75 and 12.00; MS: mlz 429 (3, M++I) 428 (10, ~) 427 (9, ~ -I) 368, 367 (100) 309, 282, 240, 239, 226,159,158,145 (97) 143,133 (95) 119 (98) 117, 107, 105 (82) 97, 93, 91,81,79,71. Anal. Calcd for C27liw0 4 : C, 75.70; H, 9.35%. Found: C, 76.04, H 9.51%.

16-(2-Glycinyl)-pregnenolone-3-~acetate 12: Yield ~120 mg, a colourless semi-solid; vmax (CCI4):

3280 (b) 2980, 2950, 2920, 2900, 2880, 2840, 1740, 1720 (b) 1700 (sh) 1650, 1610, 1590, 1480, 1470, 1420, 1380, 1260 em-I; IH NMR (CDCI3) : 0 11.00 and 9.89 (singlets, together IH, -C02l1) 5.37 (bd, IH, C6-H) 4.70-4.25 (br multiplet, 2H) 3.96-3.40 (m, 2H) 2.76 and 2.6i (doublets, C17-H in isomeric structures) 2.28 (brd) 2.24-0.62 [complex multiplet with singlets at 82.18 and 2.12 (C21 -Hs in isomeric structures), 2.02 (CH3C02 -), 1.20, 1.09, 0.98, 0.72 and 0.64 (CIS and CwHs in isomeric structures)]; MS: mJz 431 (24, ~) 430 (95, ~-1) 368, 326, 314, 301, 300, 297, 295, 284, 281, 219, 270 (100) 269, 268, 264 (67) 256 (90) 254,253,240,238,226,225, 215,189,173,160,148, 130, 118, 106, 97 (50) 83, 69. Anal. Calcd for C2sH3705N : C, 69.61, H, 8.58, N 3.25%. Found: C, 70.04, H, 8.78, N, 3.04%.

References

(a) Simons S S (Jr) in: Affinity Labelling and Cloning of Steroid and Thyroid Hormone Receptors, · edited by R Groneymeyer (YCR, Chichester) 1988, pp 28-54. (b) Gronemeyer H in: Affinity Labelling and Cloning of Steroid and Thyroid Hormone Receptors, edited by H Groneymeyer (YCH, Chichester) 1988, pp 55-67. (c) Horwitz K B & Frances M D in: Affinity Labelling and Cloning of Steroid and ThyrOid Hormone Receptors, edited by Groneymeyer H (YCR, Chichester) 1988, pp 68-75. (d) Raynaud J P, Ojasoo T, Jauques A, Moguilewsky M & Teutsch G, Endocrinology, 1984, 533 .

2 Benisek W F, Methods Enzymol, 46, 1977, 469. 3 Katzenellenbogen J A, Johnson H J, Karlson K E & Myers H

N, Biochemistry, 13, 1974, 2986. 4 Bellus D, Keams D R & Schaffner K, Helv Chim Acta, 52,

1969, 971. 5 (a) Johnson R A, Bundy G L, Youngdale G A & Morton D R,

Chem Abstr, 108, 1988, 757141. (b) Tuba Z, Hovarth J, Lovas M, Riesz M, Szpomy L & Karapati E, Chem Abst, 108, 1987, 120143c. (c) Adeoti S B, Charpentier B, Montagnac A, Chiaroni A, Richie C & Pais j'vi , Tetrahedron, 45,1989,3713. (d) Jarreau F X & Koenig J J, Chem Abslr, 112, 1990, I 79606h. (e) MaCall J M, Jacobsen E J, Yan-Doomik F J, Palmer J R & Kames H A, Chem Abstr, 108, 1988, 6287u. (t) Porier D, Labrie C I, Meerand Y & Labrie F, J Steroid Biochem Malec Bioi, 38, 1991 , 759. (g). Mobbs B G & Johnson I E, J Steroid Biochem Malec Biol, 39, 1991 , 713. (h) Tedesco R, Katzenellenbogen J A & Napolitano A, Tetrahedron Lett, 38, 1997, 7997.

6 (a) Yoshida M, Asano M, Kaetsu I, Yamanaka E, Nakai K , Yusa H & Shida K, Chern Abstr, 105, 1986, 153396v. (b) Hatono S, Yazaki A, Yokomoto M & Hirao Y, Chem Abstr, 109, 1988, 93443d. (c) Gnewch C T & Somovsky G, Chem Rev. 97, 1997, 829.

(d) Schoenemann H, VanVliet N P, Nicolass P & Zeelan F J, Euro J Med Chem Ther, 15, 1980, 333 . (e). SchatTers E M E, Steiner N, Altmen J & Beck W, Z Naturforsch, 45b, 1990, 817. (t). Brewster M E, Druzgala P J, Anderson W R, Huang M, Bodor N & Pop E, J Pharm Sciences, 84, 1995, 38.

7 (a) Dutler ,.( Ganter C, Ryf H, Ui~inger E C, Weinberg K, Schaffner K, Arigoni D & Jeger 0, Hell' Chim Acta, 45, 1962, 2346. (b) Ganter C, Utzinger E C, Schaffner K, Arigoni D, Jeger 0 , Hell' Chim Acta, 45, 1962, 2403 . (c) Barton D H R & Taylor W C, J Chern Soc, 1958, 2500. (d) Cropp P J & Erman W F, JAm Chem Soc, 85, 1963,2456.

8 (a) Greca M D, Fiorentino A, Pinto G, Pollio A & Previtera L, J Org Chem, 61,1996, 178. (b) Greca M D, Previtera L, Fiorentino A, Pinto G & Pollio A, Tetrahedron, 52, 1996, 13981.

9 Breitmaier E & Yoeller W, Carbon-13 NMR Spectroscopy, (YCH, Weinheim) 1987, pp 254-262.

10 (a) Williams J R, Moore R H, Li R & Blount J F, JAm Chem Soc, 101 , 1979,5019. (b) Williams J R, Moore R H, Li R & Weeks C M, J Org Chem, 45, 1980, 2324.

ISHAR et aJ.: PHOTOCHEMICAL LINKING OF STEROIDS WITH AMINO ACIDS 1261

II Jackman L M & Stemhell S, Applications of NMR Spectroscopy, (Pergamon, Oxford) 1969.

12 Budavari S (editor), The Merck Index, 12th edition, (Merck & Co. Inc, (New Jersy}-1996, 1328 and references cited therein .

13 (a) Plassman P S, Nelson P H, Boyle PH, Cruz A, Iriarte J, Crabbe P, Zderic J A, Edwards A J & Fried J H, J Org Chem, 34, 1969, 3779. (b) Chaudhri N K & Gut M, J Org Chem, 34, 1969, 3754. (c) Rubin M B & Bolssey E C, J Org Chern, 29,1964, 1932.

14 (a) Mariano P S, Stavinoha J & Bay E, Tetrahedron, 37, 1981,3385. (b) Janzen E J & Gerlock J L, JAm Chem Soc, 91, 1969, JI08. (c) Ishida A, Sugita D, Itoh Y & Takamuku S, JAm Chem Soc, 11 7, 1995, 11687.

15 Elad D & Sperling J, J Chem Soc (C) 1969. 1579.

16 (a) Waring A J, Zaidi J H & Pilkington J W, J Chem Soc Perkin I, 1981 , 1454. (b) Kropp P J, JAm Chem Soc, 86. 1964, 4053 . (c) Chapman 0 L, in: Advances in Photochemistry, Vol. I ., edi ted by W A Noyes (Jr). G S Hammond & J N Pitts, (John Wiley & Sons, New York) 1962, p 323 . (d) Schuster D 1 & Barringer W C, J Am Chem Soc, 93, 1971 , 731. (e) Wagner P J & Hammond G S, in Advances In

Photochemistry, Vol. 5, Edited by W A Noyes (Jr), G S Hammond & J N Pitts (John Wiley & Sons, New York) 1968, p21.

17 (a) Pete J P & Wolfhugel J L, Tetrahedron Lett, 1973, 4637. (b) Gasa S, Hamanaka N, Matsunaga S, Okuno T, Takeda N & Matsumoto T, Tetrahedron Lett, 1976, 553 .

18 (a) Mariano P S & Stavinoha J L, in: Synthetic Organic Photochemistry, edited by W M Horspool, Plenum Press, New York) 1984, p 145-257. (b) Davidson R S & Steiner P A, J Chem Soc Perkin II, 1972, 1357 and references cited therein.

19 (a) Wan C S K & Weedon A C, J Chem Soc Chem Commun, 1981 , 1235. (b) Richard R, Sauvage P & Wan C S K Weedon A C, J Org Chern, 51 , 1986, 62. (c) Duhaime R M, Lombardo D A, Skinner 1 A & Weedon A C, J Org Chem, 50, 1985, 873 . (d) Henin F, Mortezaei R, Muzart J & Pete J P, Tetrahedron Lett, 26, 1985, 4945. (e) Piva 0 , Mortezaei R, Henin F, Muzart J & Pete J P, J Am ChemSoc, 112, 1990, 9263 . (I) Deflandre A, Lheureux A, Rioual A &- Lemaire J, Can J Chem, 54, 1976, 2127.

20 (a) Muraoka 0 , Tanabe G & Igaki Y, J Chem Soc Perkin Trans I, 1997, 1669. (b) Chan A C & Schuster D I, JAm Chem Soc, 1()8, 1986, 4561.

21 Yoon U C & Mariano P S, Acc Chem Res, 25, 1992, 233. 22 Kobuta T, Yoshida K, Hayashi F & Takeda K, Chem Pharm

Bull, 13, 1965, 50. 23 Oppenauer R V, Org Syn Coli Vol. III , 1955, 207.